the effects of chronic ethanol ingestion and smoke
TRANSCRIPT
THE EFFECTS OF CHRONIC ETHANOL INGESTION AND SMOKE
EXPOSURE ON ANTI-PNEUMOCOCCAL HOST DEFENSES
______________________________
BY
ADAM MICHAEL PITZ
______________________________
A DISSERTATION
Submitted to the Faculty of the Graduate School of Creighton University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
in the Department of Medical Microbiology and Immunology
______________________________
Omaha, December 2008
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The Effects of Chronic Ethanol (EtOH) Ingestion and Smoke Exposure on Anti-Pneumococcal Host Defenses
Adam Michael Pitz
Under the supervision of Martha J. Gentry-Nielsen
from the Department of Medical Microbiology and Immunology Creighton University
Individuals who abuse alcohol and tobacco are at increased risk for pneumonia
caused by Streptococcus pneumoniae (the pneumococcus). Alcohol and cigarette smoke
are known to exert immunosuppressive effects on pulmonary defense mechanisms that
protect the host from pneumococcal pneumonia. However, the interactive, combined
immunosuppressive effects of ethanol (EtOH) ingestion and smoke exposure remain ill
defined. To study these effects, we used a rat model that mimics chronic alcohol abuse
and smoking. Rats were exposed to smoke generated by 30 reference cigarettes for one
hour twice daily for 12 weeks or to room air (sham-exposed). During the last five weeks
of exposure, the rats were pair-fed equal volumes of liquid diet containing 0% or 36%
EtOH calories.
Nasopharyngeal colonization was unaltered by EtOH ingestion and/or smoke
exposure. The movement of pneumococci from the rats’ nasopharynx into the lungs was
slightly increased by EtOH ingestion, but EtOH did not impair ciliary beating. Smoke
exposure reduced pneumococcal movement into the lungs regardless of diet, and this was
correlated with a significant increase in ciliary beat frequency.
The effect of concurrent smoke exposure and EtOH ingestion on non-neutrophil
(PMN)-mediated killing of pneumococci was investigated using an in vivo bactericidal
assay. EtOH ingestion significantly decreased the percentage of pneumococci killed
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within the rats’ lungs. Unexpectedly, this EtOH-induced defect was counteracted by
concurrent smoke exposure, even though smoke exposure alone did not increase
intrapulmonary pneumococcal clearance. Quantification of the bactericidal proteins
lysozyme and lactoferrin in the rats’ lungs did not explain the differences in non-PMN-
mediated killing. However, EtOH-fed rats that were sham-exposed exhibited suppressed
alveolar macrophage phagocytosis of pneumococci as well as diminished opsonization of
the bacteria within their lungs. It is uncertain if smoke exposure increased macrophage
phagocytosis due to technical problems with the assay, but concurrent smoke exposure
did not exacerbate the EtOH-related decrease in opsonic protein activity.
PMN functions also were analyzed in our rat model. Using a similar in vivo
bactericidal assay, EtOH ingestion alone was shown to abolish PMN-mediated killing of
pneumococci within the lungs. Smoke exposure alone had no effect on this activity, but
the addition of smoke exposure to EtOH ingestion restored PMN killing. The differences
in bacterial killing were not due to alterations in PMN recruitment to the lungs or PMN
phagocytosis of pneumococci. EtOH ingestion alone reduced systemic levels of
cytokine-induced neutrophil chemoattractant-1 (CINC-1), which may decrease PMN
activation and help explain the absence of pulmonary killing in the EtOH-fed animals.
These results indicate that both chronic EtOH ingestion and smoke exposure
modulate anti-pneumococcal defenses, but their effects differ significantly in hosts who
either abuse alcohol or smoke and those who practice both behaviors.
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ACKNOWLEDGEMENTS
To my advisor, Dr. Martha Gentry-Nielsen, thank you for your commitment in
helping me with every aspect of my graduate career. I greatly appreciate how you took
me under your wing, pushed me to do my best and coached me to be a better scientist. I
am also grateful for your constructive feedback to improve my writing. All of my
success and achievements these last four years would not have been possible without you.
Huge thanks to Mary Snitily for training me on how to work with the rats and
perform the various animal procedures. It was a great joy and pleasure working along
side you during those long days for every single animal experiment. Thank you also for
your constant reminders to live a holistic life.
Thank you to the members of my committee: Dr. Laurel Preheim, Dr. Philip
Lister, Dr. Floyd Knoop, and Dr. Geoffrey Thiele. Thank you for attending my meetings,
reviewing my data, asking great questions, providing helpful suggestions, reading my
grants and dissertation, and your approval to receive my Ph.D.
Thanks to Dr. Greg Perry for your expertise in all of the flow cytometry analysis.
I appreciate your dedication to my project, specifically with those dreaded alveolar
macrophages.
I also would like to thank all of the undergraduate students Brock Hanisch, Ben
Beller, Meghan Lewandowski, Drew Minturn, Amanda Ross, Alicia Milliken, and Alan
Pitz for your time in smoke exposing and feeding the rats. Thank you also for getting
supplies ready for experiments, processing and transferring samples, and conducting
various assays. Without your help it would have been impossible to complete this
project.
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DEDICATION
I would like to dedicate this work to my family, who has been a constant source
of love and support. To my lovely wife Becky, thank you for your love and sacrifice
these last four years. I appreciate your bravery in helping me care for the rats. Thank
you also for all of the hours you spent listening to my seminars and reading my grants
and dissertation.
To my newborn son Noah, thanks for being such a cute baby and for going easy
on me while I finish graduate school. You provide me endless joy and love in my heart.
Thank you, Mom and Dad, for believing in me and for giving me the opportunity
to pursue my dreams. Thanks for always encouraging me to do my best. You both
sacrificed so much for me so I could receive a quality education. I believe all that you
have given me has finally paid off. Thank you also to my siblings Sara, Alan, and Lisa
for making my life fun and enjoyable.
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LIST OF ABBREVIATIONS APC – Allophycocyanin ATCC – American Type Culture Collection BAL – Bronchoalveolar lavage BALF – Bronchoalveolar lavage fluid CBF – Ciliary beat frequency CFDA/SE – Carboxyfluorescein diacetate succinimidyl ester CFU – Colony forming units CINC-1 – Cytokine-induced neutrophil chemoattractant COPD – Chronic obstructive pulmonary disease CRP – C-reactive protein ELISA – Enzyme-linked immunoabsorbent assay EtOH – Ethanol Gro-α – Growth-regulated protein alpha HBSS – Hanks Balanced Salt Solution HRP – Horseradish peroxidase IFN-γ – Interferon gamma IL-8 – Interleukin 8 LDH – Lactate dehydrogenase LPS – Lipopolysaccharide MIP-2 – Macrophage inflammatory protein PBS – Phosphate-buffered saline PBS-BSA – Phosphate-buffered saline and 0.5% bovine serum albumin PBS-FCS – Phosphate-buffered saline and 4% heat inactivated fetal calf serum PE – Phycoerythrin PKA – Protein kinase A PKC – Protein kinase C PMA – Phorbol myristate acetate PMN – Polymorphonuclear leukocyte PppA – Pneumococcal protective protein A PspA – Pneumococcal surface protein A PspC – Pneumococcal surface protein C SP-D – Surfactant protein D
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TABLE OF CONTENTS
I. INTRODUCTION…………………………………………………………………. 1
II. LITERATURE REVIEW…………………………………………………………. 3
A. Streptococcus pneumoniae and Pneumococcal Pneumonia………………. 3 B. Virulence Factors of Streptococcus pneumoniae………………………….. 17 C. Alcohol Abuse and Anti-Pneumococcal Host Defenses…………………... 21 D. Smoking and Anti-Pneumococcal Host Defenses……………………….... 28
III. SUMMARY AND RESEARCH OBJECTIVES…………………………………. 35
IV. MATERIALS AND METHODS………………………………………………… 38
A. Model of Chronic EtOH Ingestion and Smoke Exposure…………………. 38 B. Bacterial Strains…………………………………………………………… 39 C. Rat Sacrifice……………………………………………………………….. 40 D. Intranasal Infection………………………………………………………... 40 E. Culture of Nasopharynx, Trachea, and Lungs……………………………... 41 F. Intranasal Vaccination with rPppA……………………………………….... 42 G. ELISA for rPppA Antibodies…………………………………………….... 43 H. Salbutamol and Formoterol Medication…………………………………... 44 I. Ciliary Beat Frequency Analysis………………………………………….... 45 J. Transtracheal Infections……………………………………………………. 46 K. Lipopolysaccharide Instillation for PMN-mediated Assays………………. 47 L. Non-PMN-Mediated Bactericidal Assay…………………………………... 48 M. Bronchoalveolar Lavage Procedures……………………………………… 48 N. Quantification of Bactericidal Factors…………………………………….. 50 O. Quantification of Pulmonary Cell Damage………………………………... 50 P. Opsonic Deposition Assay…………………………………………………. 51 Q. C3, CRP, and SP-D ELISAs………………………………………………. 53 R. PMN-Mediated Bactericidal Assay………………………………………... 53 S. Chemokine Analysis……………………………………………………….. 54 T. PMN Phagocytosis Assay…………………………………………………. 55
U. Macrophage Phagocytosis Assay………………………………………….. 59 V. Macrophage Function Assays……………………………………………… 63 W. Intranasal Mortality Study………………………………………………… 65 X. Statistical Analysis………………………………………………………… 65
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V. RESULTS………………………………………………………………………… 67
A. Pneumococcal Colonization and Movement
from the Nasopharynx to the Lungs……………………………………… 67 B. PppA Vaccination Trial…………………………………………………... 68 C. Salbutamol Medication Trial…………………………………………….. 70 D. Formoterol Medication…………………………………………………... 71 E. Non-PMN-Mediated Killing……………………………………………… 76 F. Quantification of Bactericidal Factors……………………………………. 77
G. Macrophage Phagocytosis………………………………………………... 80 H. Additional Macrophage Functions……………………………………….. 84 I. Opsonic Deposition Assay………………………………………………… 87 J. PMN-Mediated Killing……………………………………………………. 90 K. Pulmonary and Systemic Chemokine Levels……………………………... 91 L. PMN Phagocytosis………………………………………………………… 92 M. Mortality Study…………………………………………………………… 93
VI. DISCUSSION……………………………………………………………………. 95
A. Pneumococcal Colonization and Movement Studies……………………… 95
B. Non-PMN Pulmonary Defenses…………………………………………… 100 C. PMN Functions……………………………………………………………. 111 D. Chemokine Production……………………………………………………. 115 E. Mortality Study……………………………………………………………. 120
VII. CONCLUSIONS………………………………………………………………… 123
VIII. BIBLIOGRAPHY………………………………………………………………. 126
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LIST OF TABLES 1. Smoke Exposure Reduces Pneumococcal Movement…………………………….. 68
2. rPppA Specific IgG and IgA Antibody Levels Compared to Log cfu of Nasal Washes…………………………………………… 69
3. Comparison of Macrophage Phagocytosis Values………………………………... 83
4. Chemokine Production by Macrophages Stimulated with Pneumococci or PMA……………………………………………. 87
5. Chemokine Values from Lung Homogenates and Serum…………………………. 92
6. Bacteremia and Mortality Results from an Intranasal Challenge…………………. 94
7. Summary of Conclusions………………………………………………………….. 125
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LIST OF FIGURES
1. Progression of Pneumococcal Disease…………………………………………… 4
2. Complement Pathways and Effector Functions………………………………….. 8
3. Rat Model of Chronic EtOH Ingestion and Smoke Exposure…………………… 39
4. Intranasal Infection………………………………………………………………. 41
5. Medication Device……………………………………………………………….. 45
6. Transtracheal Infection…………………………………………………………… 47
7. Lavage……………………………………………………………………………. 49
8. Lavage Fluid Collection………………………………………………………….. 49
9. Scatter Plot of S. pneumoniae……………………………………………………. 52
10. Analysis of APC Fluorescence…………………………………………………. 52
11. CFSE-Labeled Pneumococci…………………………………………………… 56
12. Determination of PMN Phagocytosis…………………………………………… 58
13. APC-Cy7-labeled S. pneumoniae………………………………………………. 60
14. Determination of Macrophage Phagocytosis…………………………………… 62
15. Syto 9-labeled S. aureus………………………………………………………… 63
16. PppA Vaccination Fails to Reduce Colonization……………………………….. 69
17. Salbutamol Medication Decreases Pneumococcal Movement in Chow-fed Rats…………………………………………………… 70
18. Formoterol Medication Reduces Pneumococcal Movement into the Lungs of Chow-fed Rats…………………………………… 72
19. Formoterol Medication Prevents Pneumococcal Movement in Sham-EtOH Rats………………………………………………… 73
20. Smoke Exposure Increases CBF Regardless of Diet and Formoterol Medication………………………………… 75
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21. Formoterol Augments CBF in Sham-EtOH Rats……………………………….. 76
22. Concurrent Smoke Exposure Negates Chronic EtOH-Induced Impairment of Non-PMN-Mediated Killing……………………………………. 77
23. No Alterations of Pulmonary Lysozyme Levels by Smoke Exposure and EtOH Ingestion………………………………………. 78
24. EtOH Ingestion Drastically Increases Pulmonary Lactoferrin Levels…………. 79
25. Cellular Release of LDH Does Not Explain the EtOH-Induced Increase in Lactoferrin……………………………………… 80
26. Smoke Exposure Enhances Macrophage Autofluorescence…………………… 81
27. EtOH Ingestion Suppresses Macrophage Phagocytosis………………………... 82
28. Flow Cytometry Results Indicate EtOH Ingestion Increases Macrophage Phagocytosis of S. aureus………………………………………… 83
29. Smoke Exposure Alone Suppresses Oxidative Burst in Macrophages Stimulated by Pneumococci…………………………………. 85
30. Smoke Exposure Hinders Oxidative Burst in PMA-Stimulated Macrophages……………………………………………… 85
31. Neither EtOH nor Smoke Significantly Alters Degranulation by Macrophages Stimulated with Pneumococci………………………………... 86
32. EtOH Ingestion Reduces C3 and SP-D Deposition on Bacteria……………….. 88
33. EtOH Ingestion Decreases C3 Basal Levels…………………………………… 89
34. Concurrent Smoke Exposure Negates EtOH-Induced Defect in PMN-Mediated Killing……………………………… 91
35. Neither EtOH Ingestion nor Smoke Exposure Affects PMN Phagocytic Activity……………………………………………………… 93
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I. INTRODUCTION
Streptococcus pneumoniae, the pneumococcus, is the major cause of community-
acquired pneumonia and bacteremia in the United States. Despite antibiotics and
vaccines, this disease still remains prevalent in several high-risk groups such as children,
the elderly, alcoholics, smokers, AIDs patients, and patients with chronic liver or lung
disease. Furthermore, the significance of this problem has risen due to the emergence of
pneumococcal resistance to multiple antibiotics. More research is needed to understand
the pathogenesis of S. pneumoniae, particularly in immunocompromised hosts, in order to
develop alternative treatments against pneumococcal infection.
Pneumococcal pneumonia begins with colonization of the nasopharynx. This
event can occur without any signs or symptoms, and colonized patients can transmit the
organism unknowingly to other hosts. Once colonization is established, pneumococci
can travel down the trachea and invade the lungs, resulting in pneumonia. The host is
equipped with several defenses to help prevent these events from happening. Mucosal
IgA antibodies aid in controlling nasopharyngeal colonization by inhibiting bacteria from
attaching to the epithelium. To prevent pulmonary invasion, the trachea is lined with the
mucociliary clearance apparatus. This consists of ciliated cells that beat in an upward
fashion to remove microorganisms trapped in mucus from the lower respiratory tract.
If S. pneumoniae breaches these upper airway defenses, it can proceed into the
lower respiratory tract. The lungs contain several extracellular and cellular defenses to
maintain a sterile environment. Among these defenses are bactericidal factors, resident
alveolar macrophages, and neutrophils (PMNs). Alveolar lining fluid that coats the lungs
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consists of several bactericidal components such as lysozyme and lactoferrin that can
quickly kill pneumococci. Alveolar macrophages are important for detecting invading
pathogens and initiating the inflammatory response. PMNs are the primary cellular
defense against S. pneumoniae. They are essential for phagocytosing and clearing
pneumococci from the lungs in order to prevent widespread dissemination of the
organisms. Few PMNs are present in healthy lungs, but they are recruited and activated
by chemokines and cytokines produced by alveolar macrophages when an infection
occurs.
Alcohol abuse is one of the most important risk factors for pneumococcal
pneumonia. Compared to non-drinkers, alcohol abusers tend to have higher morbidity
and mortality rates related to pneumonia. Alcohol impairs mucociliary clearance, making
the lungs susceptible to pneumococcal infection. Important PMN functions are also
altered by alcohol consumption, which increases the risk of developing bacteremia.
Cigarette smoke also is associated with an increased incidence and severity of
pneumococcal pneumonia. Both the number of cigarettes smoked and years of smoking
positively correlate with the risk of developing pneumococcal disease. Cigarette smoke
reduces IgA antibodies, thus promoting bacterial colonization. Smoking also damages
the mucociliary clearance apparatus and impairs PMN activity.
This thesis describes research that was performed using a rat model of separate
and combined smoke exposure and EtOH ingestion. The focus of this project was to
study chronic smoke- and EtOH-induced alterations in innate anti-pneumococcal
defenses and evaluate alternative therapies to bolster these defenses and reduce the
development of pneumococcal pneumonia.
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II. LITERATURE REVIEW
A. Streptococcus pneumoniae and Pneumococcal Pneumonia
Incidence of Disease
Pneumonia is among the top ten leading causes of death in the United States.
Every year 5 million cases of community-acquired pneumonia result in approximately 1.7
million hospitalizations and over 60,000 deaths [1-3]. The annual healthcare costs of
pneumonia cases are estimated to be $9 billion [4]. The most commonly identified cause
of community-acquired pneumonia is Streptococcus pneumoniae, or the pneumococcus
[5]. It was previously thought that pneumococcal infections could easily be conquered
with the discovery of antibiotics, but the pneumococcus continues to prevail. Since the
late 1970’s, the pneumococcus has become increasingly resistant to penicillin and many
other antimicrobial agents, and this trend is spreading rapidly throughout the world [6-8].
S. pneumoniae infections are among the leading causes of worldwide illness and
death for young children, the elderly, and people with underlying medical conditions such
as AIDS [9]. For several decades, the pneumococcus has been responsible for an
estimated two-thirds of the cases of lethal pneumonia in the United States [10]. The case-
fatality rate from uncomplicated pneumococcal pneumonia is 5-7% [9], but this rate can
be as high as 40% in the elderly and people suffering from illnesses such as chronic
obstructive pulmonary disease (COPD) or cirrhosis [10-13]. As described later,
pneumococcal polysaccharide vaccines are available for children and adults, but they are
limited by only protecting the host from serotypes included in the vaccine.
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Consequently, the mortality rate attributed to pneumococcal infections is one of the
highest of any vaccine-preventable disease [14].
Pathogenesis and Anti-Pneumococcal Host Defenses
The fundamental stages of pneumococcal disease and the host defense
mechanisms involved are depicted in Figure 1 below. The left column lists the
sequenced events of the infection from colonization to the development of fatal
bacteremia. The middle column contains the primary host defense mechanisms against
each stage of infection. This research project focused on the first three stages of this
progression, as illustrated on the right side of the diagram.
Figure 1 – Progression of Pneumococcal Disease
Bacteremia and Death
The role non-PMN-mediated and PMN-mediated killing play in eradication of pneumococcal infection from the lungs.
Extracellular Bactericidal Factors, Opsonic Proteins,
Alveolar Macrophages, Neutrophils (PMNs)
Development of
Pneumonia
Stimulation of mucociliary apparatus to prevent movement of pneumococci from nasopharynx into the lungs.
Mucociliary Clearance Apparatus
Aspiration into Lungs
Production of IgA that inhibits pneumococci from colonizing nasopharynx.
Secretory IgA
Antibodies
Nasopharyngeal Colonization
Research FocusHost DefensesBacterium
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Nasopharyngeal Colonization
Colonization of the host is the first important step in the pathogenesis of S.
pneumoniae infections. The human nasopharynx is the major reservoir for the
pneumococcus and the source of horizontal spread within the community. Pneumococcal
colonization is mainly asymptomatic with carriage rates varying from 5% to 60% among
healthy adults, depending on their living conditions [9,15,16]. Invasive disease typically
occurs when the colonizing organisms are aspirated into the lower respiratory tract, the
host is immunocompromised, or when a new or more virulent strain is acquired [17,18].
Simultaneous infection with a respiratory virus such as influenza can also greatly increase
the risk of pneumococcal disease [19,20]. For example, influenza virus infection results
in increased pneumococcal adherence to the nasal epithelium and destruction of ciliated
cells that normally help maintain lung sterility.
Mucociliary Clearance Apparatus
After nasal colonization is established, the pneumococci can be aspirated into the
airway where they can invade the lower respiratory tract. An important host defense
mechanism that protects the lungs from bacterial infection is the mucociliary clearance
apparatus. This innate defense system is dependent on mucus-producing goblet cells and
the coordinated beating of ciliated cells lining the airway lumen. The mucus entraps
aspirated microorganisms, such as pneumococci, and the cilia continuously beat in a
coordinated upward fashion to remove the mucus sheath and trapped bacteria from the
lungs. The rate at which the organisms are transported out of the airway is determined by
the frequency with which the cilia beat, also known as the ciliary beat frequency (CBF).
6
The CBF is primarily regulated by the activation of protein kinases. Ciliary beating is
increased when protein kinases A and G (PKA and PKG) are activated and decreased by
the activation of protein kinase C (PKC) [21-24].
Extracellular Bactericidal Factors
Upon entering the sterile environment of the lungs, pneumococci encounter a
variety of extracellular defenses within the alveolar lining fluid. Several of these
defenses have direct bactericidal activity that can rapidly kill the invading pneumococci
[25]. These bactericidal components include lysozyme, lactoferrin, defensins, and
surfactant.
Lysozyme and lactoferrin are the most abundant antimicrobial proteins in the
lungs [26]. Lysozyme enzymatically cleaves the peptidoglycan layer of bacterial cell
walls causing the organisms to lyse [27]. This enzyme is found in the specific granules
of neutrophils, but it is mainly produced and secreted by resident alveolar macrophages
and airway epithelial cells. Lactoferrin is an iron-binding protein that is also synthesized
by neutrophils and epithelial cells in the lungs. This extracellular protein inhibits
bacterial growth by sequestering the iron that is required for microbial metabolism. In
addition to this bacteriostatic effect, iron-free lactoferrin (apolactoferrin) exhibits direct
bactericidal activity by disrupting or possibly even penetrating the bacterial cell
membrane [28,29].
Defensins are a family of small, single-chain peptides that are secreted by airway
epithelial cells and produced by phagocytes. They have potent bactericidal activity
against both Gram-positive and Gram-negative pathogens [30]. Their antimicrobial
7
function consists of forming multimers and creating pores in the bacterial membrane
[30].
Surfactant is composed mostly of phospholipids with smaller amounts of fatty
acids and proteins. The primary function of surfactant is to prevent compression of the
lungs during respiration and reduce tension in the alveoli [31]. Besides this mechanical
function, surfactant also helps protect the lungs from infection. Surfactant long-chain
free fatty acids are the most potent anti-pneumococcal factor in the alveolar lining fluid
[32,33]. The fatty acids perform in a detergent-like manner to degrade the invading
pathogen by increasing cell membrane permeability.
Opsonic Proteins
In addition to bactericidal factors, the alveolar lining fluid also contains proteins
that opsonize or coat bacteria so they are recognized and effectively engulfed and killed
by pulmonary phagocytes. The three main pulmonary opsonic components are
complement component C3, the acute phase C-reactive protein (CRP), and surfactant
protein D (SP-D).
The complement system comprises over 30 serum and membrane proteins which,
when activated, form a cascade of reactions contributing to the elimination of invading
pathogens. Complement is a particularly important opsonin in defense against
pneumococcal infection, as people defective in complement components have a much
higher rate of developing invasive pneumococcal disease than healthy individuals [34-
36]. Complement can bind to the pneumococcus by one of three pathways as shown in
Figure 2. While these pathways are activated differently, they all converge with the
8
Figure 2 – Complement Pathways and Effector Functions
deposition of C3b, the binding portion of component C3 released after its enzymatic
cleavage, on the bacterial surface. Complement C3 is predominantly formed in the liver
by hepatocytes and secreted into the bloodstream, but bronchial epithelial cells, type II
alveolar cells, and lung fibroblasts also generate C3. Regardless of the activation
pathway, opsonization with C3b is the most important effector function of the
complement system in defense against S. pneumoniae, as this leads to phagocytosis of the
organism. The final stage of the complement cascade is the membrane attack complex,
which forms pores in the bacterial membrane, causing lysis. This complex is formed by
complement C5b, C6, C7, C8, and C9. A pore forms on the bacterial surface once C9
9
polymerizes and inserts itself through the bacterial membrane. This allows a net influx of
Na+ and water resulting in the death of the bacterial cell. However, this complex is
ineffective against Gram-positive organisms, including pneumococci, due to the thick
peptidoglycan layer of their cell wall [37].
S. pneumoniae activates complement by both the classical and alternative
pathways, but the classical pathway has been found to be the dominant complement
pathway for innate immunity to the pneumococcus [38]. This is initiated by antibodies
that bind to the surface of S. pneumoniae, primarily to the polysaccharide capsule. The
Fc portion of the antibodies binds and activates C1q, which is the first component of the
complement cascade. Teichoic acid within the pneumococcal cell wall, on the other
hand, is the main activating factor for the alternative pathway [39]. Finally, S.
pneumoniae does not efficiently activate the mannan-binding lectin pathway because
mannose binding lectins bind poorly to pneumococci [40,41].
The acute phase protein CRP also functions as an opsonin to promote
phagocytosis by interacting with CRP receptors on the surface of macrophages. CRP
binds specifically to phosphocholine, the major component of the C-polysaccharide on S.
pneumoniae [42]. CRP is produced and secreted by liver hepatocytes as well as
lymphocytes, monocytes, upper airway epithelial cells, and alveolar macrophages. The
classical pathway can also be activated by CRP by recruiting C1q to the surface of the
bacteria after it binds [43].
Besides phospholipids and free fatty acids, surfactant also consists of four distinct
proteins that have been grouped in a family called collectins. Among these is SP-D,
which is synthesized and secreted by alveolar type II cells and non-ciliated bronchial
10
cells. This protein has been shown to bind and aggregate microorganisms, activate
macrophages, and enhance phagocytosis and killing of pathogens [44-46]. SP-D knock-
out mice showed enhanced susceptibility to an intranasal inoculation of S. pneumoniae,
indicating SP-D is an important host defense against pneumococcal infection [47].
Alveolar Macrophages
Resident alveolar macrophages comprise the first line of cellular defense in the
lungs, and they are responsible for the rapid phagocytosis and killing of many bacterial
pathogens. However, several researchers have reported that alveolar macrophages do not
efficiently phagocytose and kill S. pneumoniae [48-50]. Their key defensive role during
pneumococcal infection is to coordinate the pulmonary inflammatory response. When
alveolar macrophages encounter pneumococci, they release proinflammatory cytokines
and chemokines to recruit and activate neutrophils (PMNs), which are the primary
cellular defense against pneumococci as discussed below.
In rats, the major chemokines responsible for PMN recruitment are macrophage
inflammatory protein-2 (MIP-2) and cytokine-induced PMN chemoattractant-1 (CINC-
1), the homologues to human interleukin-8 (IL-8) and growth-regulated protein alpha
(gro-α), respectively. Both chemokines act through a common receptor on PMNs called
CXCR2. These chemokines increase PMN β2-integrin adhesion molecule expression,
phagocytosis, and super oxide radical production [51-53]. Although MIP-2 and CINC-1
act on the same receptor, they have slightly different roles in PMN recruitment and
activation. MIP-2 binds with a higher affinity to CXCR2 and is capable of desensitizing
the receptor to the effects of other chemokines. Unlike MIP-2 which remains in the
11
lungs, CINC-1 can leave the alveolar space and enter the systemic circulation [54,55].
The rationale for this is CINC-1 can act upon the PMNs before they are desensitized by
MIP-2, which is the more potent chemoattractant in the lungs [54].
PMN Phagocytosis and Killing
PMNs are the major immune cells responsible for pneumococcal clearance from
the lungs. In order to effectively eradicate an infection, PMNs must be recruited to the
lungs, phagocytose the pneumococci, and kill the bacteria intracellularly. Few PMNs are
present in the uninfected lung, but following infection, proinflammatory cytokines and
chemokines, such as MIP-2 and CINC-1, are secreted by alveolar macrophages and
pulmonary epithelial cells to activate and recruit large numbers of PMNs.
Once PMNs enter the lungs, they encounter the invading pneumococci that have
been opsonized for phagocytosis. In order to bind to the opsonized bacteria, PMNs
express surface receptors that recognize either the Fc fragment of IgG or complement
component C3. Once the phagocyte binds to the pathogen, a vacuole or phagosome
forms around the bound pathogen and it becomes internalized.
Immediately after phagocytosis, PMNs destroy the engulfed organisms by
oxygen-dependent and oxygen-independent mechanisms. PMNs possess four types of
intracellular granules which contain a variety of enzymes and proteins that help kill the
phagocytosed bacteria [56]. These granules fuse with the phagosome to form the
phagolysosome and release their toxic contents into this vacuole. Along with
degranulation, the nicotinamide adenine dinucleotide phosphate oxidase system forms on
the phagolysosome membrane and converts oxygen to the superoxide radical. The
12
superoxide radical is essential for the formation of multiple bactericidal components. In
addition, its dismutation results in the production of hydrogen peroxide [57].
Myeloperoxidase and nitric oxide synthase are also involved in oxygen-dependent
killing of microorganisms. Myeloperoxidase is an enzyme that uses hydrogen peroxide
to form hypochlorous acid (HOCl), the most potent antibacterial oxidant produced by the
PMN. HOCl has been shown to impair DNA synthesis and bacterial replication [58].
Myeloperoxidase is also known to interfere with DNA synthesis and ATP synthesis
systems [58,59]. Nitric oxide synthase leads to the formation of reactive nitrogen
intermediates including nitric oxide and nitroxyl anion that destroy bacterial DNA.
Oxygen-independent killing occurs primarily through the actions of microbicidal
proteins contained within the specific granules. These non-oxidative components include
proteases, defensins, lysozyme, and lactoferrin. Their activity degrades the bacterial cell
wall and membrane, resulting in lysis and eventually death of the pathogen.
Macrophages can also undergo an oxidative burst and degranulation and execute similar
killing mechanisms as PMNs, but they lack myeloperoxidase and lactoferrin. With the
combination of reactive oxygen species and granular antimicrobial proteins, phagocytes
can effectively eradicate a wide variety of microorganisms, including S. pneumoniae
[60].
Adaptive Immune Response
Although the innate immune system is the primary host defense against
pneumococcal infections, adaptive immunity is also important against the pneumococcus.
Anti-pneumococcal antibodies provide immunity against S. pneumoniae by preventing
13
colonization and promoting pneumococcal clearance in the host. For several decades, it
was thought that the main adaptive response to pneumococcal infection was the
production of IgM and IgG antibodies that bind to the polysaccharide capsule to mediate
opsonization and enhance phagocytosis [61]. However, this idea has evolved as a result
of recent studies documenting the importance of antibodies targeting pneumococcal
proteins that are vital in pneumococcal pathogenesis [62-64]. For example, one animal
experiment showed that antibodies against pneumolysin, the multifunctional toxin
produced by S. pneumoniae, were anti-inflammatory and averted invasive disease [65].
Secretory IgA antibodies also play a role in pneumococcal resistance by providing one of
the first lines of defense against nasopharyngeal colonization [66]. These antibodies limit
colonization by binding to the surface of pneumococci and preventing attachment to the
epithelium.
Until recently, T-cells were not considered to have a major role in the adaptive
immune response against the pneumococcus. Polysaccharide capsules from many
serotypes of S. pneumoniae are considered to be T-cell independent antigens because
they express repeating epitopes that cross-link B-cell receptors and induce B-cell
activation and proliferation without T-cell help. However, T-cells are involved in
antibody responses to other pneumococcal protein antigens such as pneumolysin and
surface proteins that are important in pneumococcal immunity. T-cells also appear to
contribute to early resistance against pneumococcal infection independently of their role
in adaptive antigen-specific responses. Knock-out mice that displayed a significant
decrease in CD4+ T-cell levels had an increased susceptibility to an intranasal infection
with 100% mortality by 3 days post-infection, whereas all wild-type mice survived the
14
challenge [67]. T-cells also have been shown to migrate to infected lung tissue during
pneumococcal pneumonia by an unknown interaction with pneumolysin [68,69]. In
addition, Malley and colleagues demonstrated a crucial role for CD4+ T-cells in antibody-
independent acquired immunity to pneumococcal colonization [70]. This was shown by
immunizing antibody-deficient or CD4+ T-cell-deficient mice intranasally with an
unencapsulated pneumococcal strain followed by an intranasal challenge with type 6
pneumococci. The antibody-deficient mice had reduced pneumococcal colonization
while the CD4+ T-cell deficient mice had similar levels of pneumococcal colonization as
the unimmunized control mice. More experiments are being done to understand the exact
mechanisms of the T-cell response against S. pneumoniae.
Treatment and Prevention of Pneumococcal Disease
Since the advent of antibiotics in the 1940s, penicillin was the preferred choice for
treating pneumococcal infections until the early 1990s, when antimicrobial resistance
among clinical isolates of S. pneumoniae in the United States first emerged as a
significant problem [71]. The prevalence of resistance to penicillin has increased steadily
and is now at approximately 35% in this country [72-74]. A similar trend has been
observed for other antimicrobial classes including macrolides, clindamycin, tetracyclines,
trimethoprim-sulfamethoxazole, and chloramphenicol [75]. Multidrug resistant strains
are also on the rise; one report found that 34.6% of isolates in the U.S. are resistant to
more than one antibiotic [76]. Fluoroquinolones are the only class of antimicrobials that
has escaped the problem of emerging resistance among S. pneumoniae. However, with
the increasing use of fluoroquinolones to manage respiratory tract infections in adults and
15
the growing number of nonsusceptible isolates to these antibiotics in other countries [77],
fluoroquinolone resistance in this pathogen is expected to eventually emerge in this
country.
Despite these alarming trends, antibiotic resistance in S. pneumoniae may have
reached a plateau and started to decrease [75]. It has been speculated that several factors
may be responsible for this event. Healthcare providers are using oral antibiotics more
appropriately to treat patients with acute respiratory tract illnesses [78]. The increasing
use of the conjugated pneumococcal vaccine, which covers antibiotic-resistant serotypes,
may be impacting resistance [79-84]. Lastly, a large percentage of adults are being
vaccinated for influenza, and a lower incidence of influenza infections results in fewer
bacterial infections, including those caused by S. pneumoniae [85,86].
Vaccination is the most effective strategy in preventing pneumococcal disease.
Currently, there are two pneumococcal polysaccharide vaccines licensed in the United
States. They are a 23-valent pneumococcal capsular polysaccharide vaccine
(Pneumovax, Merk) and a 7-valent pneumococcal conjugate vaccine (Prevnar, Wyeth).
The 23-valent vaccine contains capsular material from the 23 serotypes that commonly
cause invasive disease in the United States. This vaccine is recommended for adults over
the age of 50, especially the elderly and people at high risk for infection such as patients
with cirrhosis or COPD. Several studies have found the 23-valent vaccine to be at least
partially protective and to effectively reduce the incidence and mortality of invasive
pneumococcal disease [87-90]. However, results from other studies contradict these
findings, and the extent of the vaccine’s protective effect continues to be disputed
[91,92]. A major problem of Pneumovax is that it has poor efficacy and immunogenicity
16
in certain high-risk groups, such as alcoholics, smokers, the elderly, children <2 years,
and patients with chronic liver and lung diseases [87,91,93]. The vaccine also has no
effect on pneumococcal colonization and provides protection against only 23 of the >90
pneumococcal serotypes [91,94].
In the year 2000, the pneumococcal conjugate vaccine was approved in the United
States for routine use in all children aged <5 years [95]. This vaccine contains capsular
material from 7 serotypes that cause 80% of invasive pneumococcal disease in young
children. The immunogenicity of this vaccine is effective in this age group due to the
conjugation of each polysaccharide to a non-toxic diphtheria toxin protein [96]. The
introduction of Prevnar led to substantial reductions in the incidence of invasive disease
in children [80,97-99]. Use of this vaccine reduced pneumococcal disease among
unvaccinated populations by reducing nasopharyngeal colonization and transmission of
vaccine-type pneumococci, a process known as herd immunity [80]. As mentioned
above, the conjugate vaccine also has made a strong impact in preventing infections
caused by antimicrobial-resistant strains since the included serotypes were among those
with the highest percentages of antibiotic resistance. Prevnar has even more limited
serotype coverage, and vaccinated children are now being colonized and developing
infections with non-vaccine serotypes [100-102]. Due to the drawbacks of these two
pneumococcal vaccines, more research is needed to explore other types of pneumococcal
vaccines, particularly those that target conserved proteins such as pneumolysin or
pneumococcal surface proteins [62-64,103].
17
B. Virulence Factors of Streptococcus pneumoniae
Polysaccharide Capsule
The polysaccharide capsule is considered the major virulence factor of S.
pneumoniae because unencapsulated bacteria are less invasive compared with the same
encapsulated strain [104]. For example, as little as 10 cfu of capsulated pneumococci can
cause disease in small rodents, while as many as 106 cfu of unencapsulated pneumococci
are needed to reproduce a similar disease [105]. This virulence factor constitutes the
outer most part of the pneumococcus and creates a protective shell that surrounds the
microorganism. The capsule is polymeric consisting of repeating units of
oligosaccharides. Over 90 different serotypes of S. pneumoniae have been identified
based on the structure of their capsule. Functionally, the polysaccharide capsule
modulates the interaction between the bacterium and its environment, including the
interaction and adherence to host tissues. The capsule is also known to evade
opsonization and phagocytosis [106].
Cell Wall
The pneumococcal cell wall is composed of a thick peptidoglycan layer that
surrounds the cytoplasmic membrane. Peptidoglycan is composed of repeating units of
N-acetyl muramic acid and N-acetyl glucosamine that crosslink to form glycan chains
[107]. Unlike the polysaccharide capsule, the cell wall is highly immunogenic and it has
been shown in animal models that the purified form of the pneumococcal cell wall can
reproduce symptoms of pneumococcal disease [107,108]. One of the most antigenic
components of the cell wall is teichoic acid or C-polysaccharide. Teichoic acid is
18
commonly found in Gram-positive bacteria and its immunostimulatory activity is due to
phosphocholine residues that can bind to CRP [109]. The pneumococcus utilizes teichoic
acid to anchor surface proteins such as pneumococcal surface proteins A and C [110],
which will be discussed in detail in the next section.
Pneumococcal Surface Proteins A and C
A variety of proteins expressed on the surface of S. pneumoniae contribute to its
virulence and pathogenesis within the host. The functions of these surface-exposed
proteins include interactions with host tissues or concealing the bacteria from the host’s
defense mechanisms. Some of these proteins are being used as alternative targets for
developing new vaccines. Among these proteins are pneumococcal surface proteins A
and C (PspA and PspC).
PspA is one of the major surface proteins and antigens of the pneumococcus and
is produced by all strains [111]. Antibodies raised against PspA are protective against
pneumococcal disease. In fact, mice vaccinated with PspA survived a lethal dose of S.
pneumoniae [112]. Although PspA is expressed on all pneumococcal serotypes, it is
highly variable among the different strains [113]. This allows different pneumococcal
serotypes to repeatedly infect the same host.
PspA plays a crucial role in protecting S. pneumoniae from the host complement
system. This is attributed to the electronegative charge on the surface-exposed end of the
protein which repulses binding of the complement proteins and as a consequence,
prevents complement activation [114]. By comparing a wild-type pneumococcal strain to
19
a mutant strain lacking PspA, it was demonstrated that PspA’s anti-complement activity
reduced complement-mediated clearance and phagocytosis of S. pneumoniae [115].
PspC, also known as choline binding protein A, was the first adhesion molecule to
be discovered on pneumococci. PspC aids in bacterial adherence and colonization to host
tissues and this was confirmed by studies of PspC-deficient mutant pneumococci [116].
The adhesion properties of this protein act as a physical bridge between the
pneumococcal cells and the host cells by utilizing choline of teichoic acid at one end and
the host glycoconjugates on the other end [116]. This bridging interaction seems to be
limited to cytokine-activated human cells expressing certain glycoconjugates [116]. It
has been suggested that this process might be involved in advancing the pneumococcal
disease from colonization to invasion [110].
PspC also functions in inhibiting complement. PspC has the ability to bind host
Factor H, a protein that protects host cells from complement by inhibiting the activation
of the alternative complement pathway. The binding of Factor H to a host cell promotes
degradation of C3b already deposited on the cell surface. Therefore, the ability of PspC
to bind Factor H enables the pneumococcus to evade the activation of complement by
disguising itself as a host cell [117].
The structure and sequence of PspC is very similar to PspA. Immunization
studies have reported that PspA vaccination may produce antibodies that cross-react with
PspC [115]. Mice vaccinated with PspC have been shown to be protected from
pneumococcal colonization and invasive disease [63]. However, like PspA, PspC is
highly variable among pneumococcal strains and not all pneumococci express this
surface protein [118].
20
Pneumolysin
Pneumolysin is a member of the thiol-activated cytolysins and is produced by all
clinical isolates [119]. Unlike other pneumococcal virulence factors, this protein toxin is
not surface exposed. Pneumolysin resides in the cytoplasm and is released when the
pneumococcus undergoes autolysis. However, one study has challenged this idea by
reporting that pneumolysin is also released during the exponential phase of growth [120].
Pneumolysin has several distinct functions, especially in the early stages of
pneumococcal infection. Its cytolytic activity affects all eukaryotic cells by attaching to
membrane bound cholesterol and forming pores in the lipid bilayer. The toxin damages
ciliated bronchial epithelial cells, which inhibits ciliary beating and facilitates the spread
of pneumococci into the lower respiratory tract [121]. Pneumolysin disrupts the alveolar-
capillary boundary layer providing nutrients for bacterial growth and promotes
penetration through the lung tissue into the bloodstream [122,123]. The cytotoxic effects
of pneumolysin also can directly inhibit phagocyte and immune cell function, which
leads to the suppression of the host inflammatory and immune responses. Low
concentrations of pneumolysin are able to inhibit PMN respiratory burst, chemotaxis, and
bactericidal activity [124].
In addition to cytolytic activity, pneumolysin can also activate complement.
When pneumolysin is released it can bind the Fc portion of IgG, which directly activates
the classical complement pathway [125]. This complement-activating activity of
pneumolysin consumes complement proteins and diverts complement activation away
from the bacterial surface [125]. Several studies have demonstrated that pneumolysin
21
significantly reduced opsonization of pneumococci and this subsequently results in
decreased PMN-mediated phagocytosis and killing of the organisms [126,127].
C. Alcohol Abuse and Anti-Pneumococcal Host Defenses
Incidence of Disease
Excessive use of alcohol is the third most lethal modifiable risk factor affecting
health in this country [128]. The prevalence of alcohol abuse among Americans is 17%
[129], and this has remained steady for the last fifteen years [130,131]. In the year 2000,
alcohol consumption accounted for 85,000 deaths in the U.S. [128]. Aside from its
adverse physical effects, alcohol abusers tend to have impulsive lifestyles that can lead to
risky sexual activity, injuries, and suicide [132,133]. These types of behavior also
increase the risk of developing chronic illnesses [134], such as cirrhosis of the liver, and
many types of infections [135].
Alcohol abuse is one of the most common predisposing risk factors for bacterial
pneumonia, and alcohol-abusers experience higher morbidity and mortality from
pulmonary infections than the general population [136]. Among all bacterial
pneumonias, S. pneumoniae is the most frequent pathogen in alcohol-abusing patients
[135,137-140]. These patients experience more severe clinical symptoms, require longer
hospital stays and treatment, and have slower resolution of their disease [137]. They
have an increased risk of developing pneumococcal bacteremia, which greatly increases
their mortality [141] In addition, they are more likely to have recurring episodes of
pneumonia [142]. The explanation behind these findings is that alcohol has
immunosuppressive properties that adversely affect both innate and adaptive immune
22
pulmonary host defenses. Several studies indicate ethanol (EtOH) ingestion has
detrimental effects on vital anti-pneumococcal defenses that will be further explained in
the next several sections.
Pneumococcal Colonization of Alcohol Abusers
The lifestyle of many alcohol abusers tends to increase their risk of developing
pneumococcal disease. There are no reports that link alcohol abuse to increased
pneumococcal colonization of the nasopharynx. Alcohol abuse can depress
consciousness and the cough reflex, factors that increase the risk of aspirating colonized
pneumococci into the lungs [136,143].
Impairments to Mucociliary Clearance
Chronic EtOH exposure affects CBF by blocking the activation of PKA. Studies
have shown that EtOH desensitizes the ciliary response to isoproterenol, a short-acting
β2-agonist that stimulates ciliary beating through PKA [144]. Additionally, this EtOH-
induced defect in mucociliary clearance was detected in animals that were fed EtOH for
five to six weeks [145,146]. EtOH-fed rats have decreased CBF, resulting in increased
pneumococcal movement into their lungs after an intranasal infection [145].
Defects in Extracellular Bactericidal Factors
Although no associations have been made between alcohol abuse and alterations
in levels of free pulmonary lysozyme, lactoferrin, or defensins, there is some evidence
that EtOH does affect these proteins. One study demonstrated that in vitro EtOH
23
exposure significantly reduces lysozyme secretion by human macrophages [147], and this
parallels decreased levels of lysozyme in the serum of alcoholics [148]. In another study,
acetaldehyde, the major byproduct of metabolized EtOH found in the saliva, airways and
blood, was shown to inhibit the bactericidal function of lysozyme [149]. Alcoholics
actually have higher levels of lactoferrin in their serum compared to non-drinkers [148],
because alcohol abuse increases iron concentration in the bloodstream [150,151]. This
disturbance in iron homeostasis tends to saturate lactoferrin and may limit the availability
of apolactoferrin. Chronic EtOH consumption may also modulate the defensins.
Because alcohol induces oxidative stress in the lungs, it damages pulmonary cells
including type II pneumocytes, the major cell type that generates defensins [152].
Excessive chronic alcohol ingestion suppresses the production and antimicrobial
function of surfactant. Using a rat model exposed to EtOH for six weeks, surfactant loss
was linked to EtOH-induced damage to type II pneumocytes [153], the main source of
surfactant, as well as an 80-90% reduction of glutathione in the lungs [152]. Glutathione
is a potent antioxidant synthesized primarily by the liver that is abundant in alveolar cells
and fluid. This EtOH-induced defect in surfactant production was reversed by treating
EtOH-fed rats with the glutathione precursor procysteine [153].
In contrast to chronic EtOH exposure, rats exposed to EtOH for seven to ten days
decreased the anti-pneumococcal activity of surfactant, but it was not due to a decrease in
amount of surfactant produced. Pneumococcal killing by surfactant from EtOH-fed rats
was 4 logs lower than surfactant from pair-fed control rats [33]. Unexpectedly, the robust
killing ability of free fatty acids from the surfactant of control animals was lost with the
addition of surfactant from EtOH-fed animals [33]. This suggests that EtOH ingestion
24
results in the production of an inhibitory component in the lungs that inactivates the
bactericidal function of the free fatty acids.
Alterations in Opsonic Activity
Only one published study has reported findings on the relationship between EtOH
ingestion and the opsonic activity within the lungs. Lung lavage fluid obtained from
guinea pigs after six weeks of EtOH feeding had diminished levels of surfactant with
decreased opsonic activity towards Staphylococcus aureus [154]. Serum CRP levels are
unaltered in mice that consumed EtOH for six weeks and in a sample of heavy human
drinkers [155,156], but CRP concentrations in the lungs were not reported. In contrast,
only a few reports address the effects of EtOH ingestion on host complement, and the
results are contradictory. Serum complement levels have been reported both as reduced
and increased in various studies of alcoholic patients [157,158]. Serum hemolytic
activity was decreased in acutely intoxicated patients in one study [159], but another
reported that the addition of alcohol to normal serum in vitro had no affect on this activity
[160]. A third group found no change in serum complement activity in volunteers after
8-28 days of EtOH consumption [161,162].
Meri and colleagues reported that six weeks of EtOH ingestion induced the
expression of complement components and also down-regulated the expression of
complement regulatory proteins by the liver in mice and rats [155,163]. This led to the
deposition of complement C3 and other complement components on the liver
hepatocytes. These results indicate that EtOH ingestion nonspecifically activates
25
complement, which may lead to the depletion of functional complement available for
opsonization.
Impairments in Alveolar Macrophage Function
Although alveolar macrophages inefficiently phagocytose and kill S. pneumoniae,
they still play a vital role in the pulmonary inflammatory response during pneumococcal
pneumonia. EtOH exposure has been shown to suppress a wide range of macrophage
functions. Alveolar macrophages collected from EtOH-fed rats and rat macrophages
exposed to EtOH in vitro both exhibit decreased oxidative burst when stimulated with
either Staphylococcus aureus or endotoxin [164,165]. Two weeks of EtOH feeding in
mice similarly inhibited the ex vivo production of proinflammatory cytokines by their
alveolar macrophages [166]. Release of MIP-2 and CINC-1 in the lungs of rats injected
intraperitoneally with EtOH also were inhibited after being infected with S. pneumoniae
[167]. Another published study found that peritoneal macrophages harvested from
EtOH-fed rats for 12 weeks demonstrated impaired phagocytosis of Candida albicans,
despite having larger numbers of Fc and C3b surface receptors that facilitate
phagocytosis compared to macrophages from control rats [168].
Impairments in PMN Function
PMNs are essential for effective pneumococcal clearance from the lungs. Failure
of this cellular defense can result in uncontrolled pneumococcal growth within the lungs,
leading to bacteremia and eventually death. It is well-established that EtOH exerts
various effects on the functional activity of PMNs. Circulating PMNs must first be
26
activated and recruited to the lungs by cytokines and chemokines. Rats exposed acutely
to an intraperitoneal injection of 20% EtOH and then infected intratracheally with S.
pneumoniae 30 minutes later had decreased pulmonary production of MIP-2 and CINC-1
[167,169]. This suppressed response delayed PMN recruitment, a deficiency that
persisted even when MIP-2 was administered intratracheally [169]. EtOH inhibits PMN
β2-integrin expression, which is critical for PMN adhesion to the endothelium and
migration into the infected tissue [170]. However, one week of EtOH ingestion by rats in
our laboratory consistently failed to suppress pulmonary chemokine levels or repress
PMN recruitment to their lungs [171,172].
The phagocytic activity of rat PMNs was reduced by exposure to very high EtOH
concentrations in vitro, but physiologically relevant concentrations of EtOH in the same
study failed to inhibit phagocytosis [173]. Although acute EtOH reduced the phagocytic
activity of circulating PMNs [170,174], it did not affect phagocytosis by PMNs recruited
into the lungs [171,175].
In vitro and in vivo studies in our laboratory have established that EtOH ingestion
impairs PMN-mediated killing of S. pneumoniae. PMNs isolated from the peripheral
blood of rats consuming EtOH for one week killed significantly fewer pneumococci in
vitro than PMNs isolated from the blood of pair-fed control rats [176]. This was related
to a decrease in both oxygen radical production and degranulation in PMNs from the
EtOH-fed rats [177]. These results correlate with a human study in which PMNs from
alcoholics exhibited diminished elastase activity and superoxide production [178]. The
EtOH-induced defect in PMN bactericidal activity was further confirmed in our
laboratory using an in vivo PMN killing assay in which PMNs were pre-recruited to rats’
27
lungs five hours prior to infection. Rats fed EtOH for one week had killed 20% fewer
pneumococci within their lungs than their pair-fed counterparts one hour after the
transtracheal infection [171].
Alterations in the Adaptive Immune Response
Numerous studies have shown that alcohol consumption suppresses acquired
immune defenses including both cell-mediated and humoral immunity. Chronic
alcoholics are known to be lymphopenic [179-181], and long-term alcohol feeding in
animals also resulted in decreased size and cell numbers in the thymus and spleen [182-
184]. It has been proposed that the underlying mechanism for this phenomenon is that
EtOH induces lymphocyte apoptosis [185]. In addition to the low number of lymphoid
cells, an impaired proliferation response also has been reported, signifying that EtOH-
exposed lymphocytes have a reduced capacity to undergo proliferation and differentiation
in response to an antigenic challenge [186]. This decreased response in T-cells is partly
due to impairment in antigen presentation by antigen presenting cells [187-189]. Chronic
EtOH exposure also significantly reduces the absolute number of CD4+ T-cells and
hinders T-cell recruitment to the lungs after a pulmonary infection [190].
The development of specific antibodies is important in protecting the host against
pneumococcal infection. Alcohol-induced defects of specific antibody production would
be expected to adversely affect the eradication of invading pneumococci from the airways
in patients with pneumonia [137]. Despite decreases in B-cell numbers, alcoholics with
liver disease have increased circulating levels of IgA, IgM, and IgG [158,188,191,192].
In contrast, an analysis of bronchoalveolar lavage fluid in patients with alcoholic liver
28
disease showed reduced total IgG concentrations [193]. B-cell functions were intact in
EtOH-fed animals when T-cell-independent antigens were administered [194]. This B-
cell response was also present in chronic alcoholics when vaccinated with 23-valent
pneumococcal polysaccharide vaccine [195]. However, chronic alcohol ingestion hinders
the development of specific antibodies in response to challenges with T-cell-dependent
antigens in experimental animals [186,196,197].
D. Smoking and Anti-Pneumococcal Host Defenses
Incidence of Disease
Cigarette smoking is the single most preventable cause of morbidity and mortality
in the United States [128]. One-fourth of American adults smoke cigarettes, [198] and
every year 1.5 million people in this country become daily smokers [199,200]. Although
the overall smoking rate among adults from the last four decades has decreased, there is
an alarming increase of cigarette smoking among high-school students [201,202]. In
addition, tobacco use has markedly increased worldwide since the 1980s [201]. Smoking
is responsible for 438,000 annual deaths in this country [203] and over 5 million annual
deaths worldwide [204]. The prevalence of cardiovascular diseases, lung cancer, and
microbial infections is higher in cigarette smokers than nonsmokers. Smoking is also the
strongest risk factor for developing lung carcinoma, COPD, and emphysema [205].
Clinical studies have associated smoking with an increased incidence and severity
of respiratory illnesses, including pneumococcal pneumonia [206-208]. A population-
based case-control study found smoking to be the strongest independent risk factor for
invasive pneumococcal disease among immunocompetent, non-elderly adults [209].
29
Furthermore, they found a dose response relationship between the amount of smoking
and the risk of pneumococcal pneumonia [209]. Smokers account for approximately half
of otherwise healthy adult patients with severe pneumococcal disease [210,211].
Like alcohol abuse, smoking alters both the innate and adaptive immune systems,
which is the likely cause for increased risk of pneumococcal infection and other diseases
in this population. Smoking compromises several key anti-pneumococcal defenses. It
promotes bacterial colonization of airway epithelial cells [212], damages the mucociliary
clearance apparatus [213], and increases alveolar vascular and epithelial permeability
[214,215]. The sections below will explain in more detail the alterations of host defense
mechanisms against pneumococcal pneumonia caused by the actions of cigarette smoke.
Smoking and Nasopharyngeal Colonization
Cigarette smoking increases the risk of nasopharyngeal colonization by potential
pathogens including S. pneumoniae [216-218]. Buccal epithelial cells isolated from
smokers were significantly more susceptible to pneumococcal adherence in vitro
compared to buccal cells isolated from nonsmokers [219-221]. This was also true for
cells from ex-smokers who had refrained from smoking for at least three years [212].
Furthermore, these results were reproduced when buccal cells from nonsmokers were
incubated in medium containing cigarette smoke extract before exposure to the organisms
[219]. Cigarette smokers also have lower amounts of salivary and pulmonary IgA
antibodies, which may contribute to their higher rates of pneumococcal colonization and
incidence of pneumococcal pneumonia [222,223]
30
Impairments of Mucociliary Clearance
Cigarette smoke impairs mucociliary function. When bovine bronchial epithelial
cells were exposed to cigarette smoke extract in vitro, the beat frequency of their cilia
was down-regulated in conjunction with the activation of PKC [224]. Human studies
also revealed that smoking directly damages ciliated epithelial cells, causing ciliary loss
and stasis and hindering mucociliary clearance [225-227]. These defects of the
mucociliary apparatus lead to increased pneumococcal movement from the nasopharynx
to the lungs after intranasal infection [145].
Alterations in Extracellular Bactericidal Factors
There is a paucity of studies describing the effects of smoke on the overall activity
of bactericidal factors in the lungs, but smoking has been shown to alter pulmonary levels
of antibacterial proteins. Smokers have higher lysozyme concentrations in their alveolar
lining fluid, and their alveolar macrophages have a higher lysozyme content than those of
nonsmokers [228,229]. Another study found elevated levels of lactoferrin in smokers
with chronic bronchitis, but lactoferrin was only slightly elevated in asymptomatic
smokers compared to non-smokers [230]. A proteomic analysis of human
bronchoalveolar lavage fluids (BALFs) revealed increased amounts of PMN defensins in
smokers with COPD [228]. By contrast, certain key surfactant proteins important for
lung defense such as SP-A and SP-D are significantly lower in human smokers
[231,232], and this trend is also exhibited in rats exposed to smoke for 70 weeks [233].
An in vitro study demonstrated that cigarette smoke inhibits surfactant production by type
II pneumocytes [234]. In contrast, mice smoke-exposed for 6 months have a higher
31
expression and concentration of surfactant proteins in their lungs than mice exposed to
room air [235,236].
Modifications of Opsonic Proteins
Cigarette smoke has been shown to modulate the levels of opsonic proteins. In
vitro studies reported that smoke modifies complement C3, resulting in activation of the
alternative pathway and decreased susceptibility of C3 to complement regulatory proteins
[237,238]. Smoke-induced complement activation may deplete available C3 for bacterial
opsonization, and this could account for why smokers with COPD have lower serum
levels of complement C3 [239,240]. However, similar serum concentrations of C3 were
reported for asymptomatic smokers and nonsmokers [240].
Smokers have been reported to have significantly higher levels of plasma CRP
[241-244], and there is a strong correlation between CRP levels and the number of
cigarettes smoked, pack-years of smoking, and duration of smoking [242,243]. CRP
concentrations remain elevated even after 5 years of smoking cessation, suggesting
smoke does not directly increase CRP. The elevated levels may be due to an
inflammatory stimulus such as tissue damage caused by smoking [243].
As mentioned above, smoking also decreases the production of surfactant in the
lungs. BALFs from healthy human smokers, for example, have significant reductions in
SP-D [231,232]. It therefore has been postulated that an unknown inhibitory compound
in cigarette smoke interferes with either the biosynthesis of surfactant or active transport
mechanisms involved in surfactant secretion by alveolar type II cells [234].
32
Alterations in Alveolar Macrophage Function
Several reports suggest that alveolar macrophage function may actually be
enhanced by cigarette smoke, but there are conflicting results concerning the influence of
cigarette smoke on macrophages. There is an increased number of macrophages in the
lungs of human smokers and in mice exposed to cigarette smoke for six weeks as
determined by bronchoalveolar lavage [146,245]. Macrophages from smokers also are in
a higher activation state, containing an overload of cytoplasmic material and larger
quantities of lysozyme and elastase than macrophages obtained from nonsmokers
[229,245]. However, in vitro studies have shown smoke exposure hinders human
alveolar macrophage phagocytosis and inhibits their response to endotoxin; thereby
causing them to generate fewer superoxide anions [246,247]. Yet another study reported
no smoke-induced defects in human alveolar macrophage phagocytosis [248].
Interestingly, that same study found smoking suppressed the bactericidal activity of the
macrophages even though they exhibited a robust oxidative burst. Finally, Morrison
reported no differences in IL-8 and gro-α levels in BALFs from smokers and
nonsmokers, but when alveolar macrophages from the smokers were stimulated in vitro
with endotoxin they produced more chemokines than those from nonsmokers [249].
Alterations in PMN Function
The documented effects of smoke exposure on PMN function are also
inconsistent and not well defined. Certain fractions of cigarette smoke were shown to be
potent inhibitors of PMN chemotaxis [250], but other studies report cigarette smoke
enhances this activity. This is evidenced by the fact that uninfected human smokers have
33
significantly higher concentrations of neutrophils and chemokines in their BALFs than
nonsmokers, and the increases are dose-dependent on cigarette smoke [251]. Cigarette
smoke extract stimulates human endothelial cells, lung fibroblasts, and airway epithelial
cells to release IL-8 and other PMN chemoattractants [252-254]. Furthermore, PMNs
isolated from rats that were either treated with nicotine for one week or exposed to
cigarette smoke for 15 weeks had greater chemotactic responses than those from control
rats [255]. To add to the controversy, rats exposed to smoke for 8 weeks had no
alterations in pulmonary chemokine levels or PMN recruitment to their lungs after
endotoxin was administered transtracheally [171].
The effects of smoking on PMN phagocytosis and killing of bacteria are
conflicting as well. Salivary PMNs isolated from smokers immediately after having a
cigarette had increased phagocytic activity compared to PMNs from non-smoking
controls [256]. However, this effect was specific for salivary PMNs, because there was
no difference in phagocytosis by peripheral PMNs isolated from the same subjects. Other
in vitro studies found cigarette smoke extract hinders phagocytosis by human peripheral
PMNs [257,258]. Furthermore, published studies disagree on whether cigarette smoke
inhibits [259] or stimulates [255,260] the PMN oxidative burst. One study observed a
delayed rate of bacterial clearance in smoke-exposed mice of 6-8 weeks compared to
unexposed mice infected with Pseudomonas aeruginosa, even though the smoke-exposed
animals had increased levels of proinflammatory cytokines and more PMNs in their lungs
[261]. To the contrary, smoke-exposed rats of eight weeks displayed no defects in PMN
phagocytosis and killing of S. pneumoniae within their lungs [171].
34
Impairments in Adaptive Immunity
It is well documented that chronic smoke exposure adversely affects both humoral
and cell-mediated functions of the adaptive immune response [262-264]. Cigarette
smoking in humans is known to increase the number of blood leukocytes, but at the same
time reduce their activity [265]. Nicotine has been found to be the mediating factor that
induces immunosuppression. Nicotine treatment impairs antigen receptor-mediated
signal transduction in T-cells, thus resulting in T-cell anergy [266,267]. This prevents T-
cells from responding correctly to antigens that would allow them to enter the cell cycle
and proliferate. Smoke-exposed mice therefore displayed inhibited antigen-specific T-
cell responses in their lungs [268]. Similar defects in cell signaling were also detected in
T-cells from human smokers and smoke-exposed animals [269,270].
Cigarette smoke also represses antigen-induced B-cell activation [266]. These
modulating effects on T-cells and B-cells result in defective antibody responses to both
T-cell-dependent and -independent antigens [271-274]. Several investigators have shown
that long-term smoking significantly reduces antigen-specific antibodies in humans
[222,223,275,276]. However, smokers have increased levels of autoantibodies [277,278],
which partially explains the higher incidence of certain autoimmune diseases in smokers.
35
III. SUMMARY AND RESEARCH OBJECTIVES
Pneumococcal infections still remain a significant problem throughout the world.
The majority of community-acquired pneumonia cases in the United States are caused by
S. pneumoniae. Alcohol abuse and smoking increase the incidence of respiratory tract
infections caused by the pneumococcus. Alcohol abusers have recurring bouts of
pneumococcal pneumonia, longer hospital stays, an increased risk of developing
bacteremia, and a higher mortality rate from this disease than non-alcohol abusers.
Cigarette smokers have an increased risk of being colonized by S. pneumoniae and
developing invasive pneumococcal disease. Previous research has shown EtOH ingestion
and smoke exposure deter host defenses that protect against pneumococcal pneumonia,
but few studies have evaluated the combined effects of these two insults. Between 80-
95% of alcoholics smoke cigarettes and approximately 70% of them smoke more than
one pack/day [279]. Likewise, over half of multipack/day smokers are alcohol
dependent. Therefore it is important to study the combined effects of these co-
morbidities on host immunity to pneumococcal disease. Furthermore, current therapies
are ineffective in this high risk group and new therapeutic strategies are needed to protect
this population from pneumococcal infection.
It is difficult to study the effects of alcohol and smoke in human subjects for it is
impossible to tease out the individual effects of drinking and smoking in humans that
engage in both behaviors. Additionally, it is hard to control for the amount of alcohol
that is consumed and the amount of cigarettes that are smoked as well as the duration of
each exposure. For these reasons we developed a rat model of EtOH ingestion and
36
smoke exposure that parallels a heavy human smoker and alcohol abuser [280]. The
advantages of using an animal model include controlling for the amount of alcohol
consumed, the number of cigarettes smoked, and the duration of the two insults as well as
identifying the separate and collective effects of these two behaviors.
Innate immune defenses against the pneumococcus are vital in preventing
invasive disease. Alcohol ingestion and smoke exposure impair many aspects of innate
immunity including mucociliary clearance, bactericidal factors, alveolar macrophages,
and PMNs. Little is known about the combined effects of these two behaviors on anti-
pneumococcal defenses. The purpose of this work was to utilize a rat model that
combines EtOH ingestion and smoke exposure to study their separate and combined
effects on innate defense mechanisms necessary for controlling pneumococcal disease
and then target these key defenses for therapeutic intervention. The effect of chronic
EtOH ingestion and smoke exposure on nasopharyngeal colonization and mucociliary
clearance was investigated by quantifying pneumococcal colonization and tracking
pneumococcal movement from the nasopharynx to the lungs. In addition to this study, an
intranasal vaccine containing a recently discovered pneumococcal surface protein called
pneumococcal protective protein A (PppA) was tested to reduce colonization.
Immunization with PppA was shown to be protective against several serotypes in mice
[64]. Two β2-agonists, which stimulate ciliary beating by activating PKA, also were
evaluated to up-regulate mucociliary clearance and inhibit pneumococcal invasion of the
lungs. Next, alterations of chronic EtOH consumption and smoke exposure on non-
PMN-mediated defenses were identified by an in vivo bactericidal assay, quantification of
bactericidal proteins, an opsonic protein deposition assay, and several assays measuring
37
important macrophage functions. Finally, EtOH- and smoke-induced modifications on
PMN function were analyzed by another in vivo killing assay and a phagocytosis assay.
38
IV. MATERIALS AND METHODS
A. Model of Chronic EtOH Ingestion and Smoke Exposure
Male Sprague-Dawley rats weighing 100-120 grams were used for all
experiments. Smoke exposed rats were passively exposed to smoke generated by 30
reference cigarettes (2R4F, Tobacco Health Research Institute, University of Kentucky)
in whole-body chambers (Teague Enterprises, Davis, CA) twice daily Monday through
Friday and once daily on Saturday and Sunday for 12 weeks [280]. Smoke inhalation
was quantified by measuring the total suspended particles in each chamber [281]. For the
same time periods, sham-exposed control rats housed in a separate room were placed in
similar chambers and exposed to room air.
For the first 6 weeks of smoke- and sham-exposure, the rats were housed in group
cages and fed rat chow and tap water ad libitum. After 6 weeks, the rats were placed in
single cages with their water removed and food replaced with the Lieber-Decarli liquid
control diet (Dyets, Inc., Bethlehem, PA). The rats were acclimated to the control diet for
three days. Then the rats were paired by similar weight and one rat from each pair
received an alcohol liquid diet containing 36% EtOH calories. To control for variations
of diet consumption and nutrition, the other rat was pair-fed the same amount of control
diet as his EtOH-fed mate consumed the day before. The rats consumed the indicated
diets for the last 5 weeks of concurrent smoke- and sham-exposure. This resulted in four
different treatment groups, which will be referred to as smoke-EtOHs, smoke-pairs,
sham-EtOHs, and sham-pairs. The timeline to generate rats used in this model is shown
in Figure 3.
39
Figure 3 - Rat Model of Chronic EtOH Ingestion and Smoke Exposure
36% EtOH diet ad lib Chow-feed liquid control diet (Smoke-expose) (smoke-expose) 0% EtOH pair-fed
6 weeks 3 days 5 weeks Infect
36% EtOH diet ad lib Chow-feed liquid control diet (Sham- (Sham-expose) (sham-expose) 0% EtOH pair-fed expose)
(Smoke- expose)
(Smokee-
expos ) (Smoke- expose)
B. Bacterial Strains
The serotype 3 S. pneumoniae (ATCC 6303, American Type Culture Collection,
Rockville, MD) was used for the majority of the experiments. This is a clinical isolate
that is highly encapsulated and virulent in humans and rats. In addition to S. pneumoniae
ATCC 6303, Staphylococcus aureus (ATCC 29213, American Type Culture Collection)
was used in a macrophage phagocytosis experiment. For the in vitro macrophage
function assays, an additional S. pneumoniae strain labeled as DW 3.8 was utilized. DW
3.8 is an unencapsulated serotype 3 mutant derived from a S. pneumoniae WU2 strain by
inserting transposon Tn916 into a genetic region responsible for polysaccharide capsule
formation [104].
All bacteria strains were stored at -80oC in Todd-Hewitt broth (Becton, Dickinson
and Company, Sparks, MD) containing 10% glycerol. For the colonization, bacterial
movement, vaccination, medication, mortality, opsonic deposition, PMN phagocytosis,
and macrophage studies, the pneumococcal strains were grown to stationary phase at
40
37oC in an atmosphere of 5% CO2 in air incubator in Todd-Hewitt broth containing 5%
heat-inactivated rabbit serum. For the PMN-mediated and non-PMN-mediated killing
assays, fifteen ml of a stationary phase culture was inoculated into 75 ml of fresh Todd-
Hewitt broth containing 5% heat-inactivated rabbit serum. The resulting culture then was
grown for 4 hours at 37oC in 5% CO2 in air to mid-log phase. For each experiment, the
pneumococci were collected by centrifugation at 13,776 x g for 10 minutes. The
resulting pellets were washed twice with phosphate-buffered saline (PBS) and diluted in
PBS to the appropriate optical density at 540 nm to achieve the desired inoculum. The S.
aureus strain was grown as a lawn on sheep blood agar plates (Remel, Lenexa, KS) for
16 hours at 37oC in 5% CO2 in air. The staphylococci were collected from the plates
using a sterile swab and suspended in sterile water to an optical density of 1.0 at 540 nm.
The number of colony forming units (cfu) present in each inoculum was confirmed by
serial dilution and viable counts on sheep blood agar plates.
C. Rat Sacrifice
In all experiments, the rats were euthanized by an intraperitoneal injection of 75
mg/kg body weight of pentobarbital (Nembutal, Abbott Laboratories, Abbott Park, IL).
Once the rats lost consciousness they were exsanguinated by cardiac puncture using a 21
ga. x ¾” scalp vein set (Excel, Los Angeles, CA).
D. Intranasal Infection
Immediately after their morning smoke exposure, the rats were lightly
anesthetized with isoflurane (Midrad, Inc., Bethlehem, PA) and held in an upright
41
position. The specified inoculum of type 3 S. pneumoniae ATCC 6303 in 100 μl PBS
was deposited by a dropwise injection into the rats’ nostrils through a micropipette tip
(Figure 4). Following intranasal inoculation, the rats were held in the vertical position for
a few seconds to allow for aspiration of the fluid.
Figure 4 – Intranasal Infection
Figure 4 For intranasal infections rats were lightly anesthetized and held in a vertical position. One hundred μl of the inoculum was then deposited by a dropwise injection into both nostrils.
E. Culture of Nasopharynx, Trachea, and Lungs
One week after an intranasal infection with 1 x 106 cfu of S. pneumoniae ATCC
6303, the rats were euthanized and nasal washes were performed to quantify
pneumococcal colonization. The trachea was exposed by blunt-end dissection and the
proximal end was occluded with surgical string. A 22 ga. catheter (Becton Dickinson and
Co., Sandy, UT) then was inserted into the trachea above the occlusion and a retrograde
injection of 1 ml PBS was collected from the nares into a sterile Petri dish. The number
42
of pneumococci present in the nasal wash fluids was determined by performing standard
plate counts on blood agar plates containing 4 μg/ml of gentamycin to eliminate
contaminating normal flora organisms [282]. The trachea and lungs then were removed
en bloc and placed in a sterile Petri dish. The trachea was excised from the lungs with
sterile scissors, and the proximal end (carina) and distal end were sampled separately by
inserting an ultra-fine cotton swab (Fisher Scientific, Pittsburgh, PA) 0.5 cm into the
opening. The cotton swab was vortexed for 5 sec in a tube containing 100 μl of sterile
PBS, and plate counts were performed on the resulting suspension. The lungs then were
homogenized in a sterile tissue grinder in a total volume of 10 ml of sterile PBS and the
total number of bacteria present in the lungs was quantified by plate counting.
F. Intranasal Vaccination with rPppA Unexposed chow-fed rats were immunized intranasally with recombinant
pneumococcal protective protein A (rPppA, Wyeth Vaccine Research, Pearl River, NY)
to test its ability to reduce pneumococcal colonization as shown previously in mice [64].
The rats were vaccinated under light isoflurane anesthesia with 50 μl of PBS containing
either 20 μg rPppA or 20 μg of the irrelevant antigen keyhole limpet hemocyanin
(Calbiochem, San Diego, CA). Each antigen was combined with 5 μg of native cholera
toxin subunit B adjuvant (Sigma, St. Louis, MO). The intranasal vaccines were
administered by a dropwise instillation through a micropipette tip placed at the opening
to the nares, resulting in inhalation of the proteins. The rats were immunized three times
at two week intervals. One week after the third immunization, the rats were anesthetized
with isoflurane and a cardiac puncture was performed to collect 1 ml of blood for
measuring serum antibody titers. While the rats were under anesthesia, noninvasive nasal
43
washes were performed to determine their mucosal antibody titers. To accomplish this, a
piece of 22 ga. polyethylene tubing connected to a 1cc syringe was inserted into one
nostril and 1 ml of PBS was injected and collected from the other nostril into a sterile
Petri dish. One week later (two weeks after their last vaccination), the rats were infected
intranasally as described above with 1 x 106 cfu of S. pneumoniae ATCC 6303. They
were then euthanized 1 week post-infection and pneumococcal colonization was
quantified as described above.
G. ELISA for rPppA Antibodies
Immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody titers against
rPppA were measured in the serum and nasal washes from rats vaccinated with rPppA
using a previously described ELISA with some minor modifications [64]. For measuring
IgG, 96-well plates were coated overnight at 4oC with 100 μl/well of a 5 μg/ml solution
of rPppA in PBS. The plates were washed five times with PBS containing 0.1% Tween
20 and then blocked by the addition of 300 µl/well of 5% dry milk in PBS for one hour at
room temperature. After washing, 100 μl of rat IgG standards (0-320 ng/ml; Invitrogen,
Carlsbad, CA), or dilutions of the rats’ serum samples were added to each well and
incubated at room temperature for 1.5 hours. The plates were washed, 100 μl of
biotinylated anti-rat IgG (0.06 μg/ml, Jackson ImmunoResearch Laboratories, West
Grove, PA) was added to each well, and the plates were incubated at room temperature
for 1 hour. After washing, 100 μl of streptavidin-horseradish peroxidase (HRP) (0.1
μg/ml, Jackson ImmunoResearch Laboratories) was added to each well and incubated for
1 hour at room temperature. The plates were washed and 100 μl of 2,2'-Azinobis [3-
44
ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS) substrate (Pierce,
Rockford, IL) was added to each well. The plates were incubated for 20 min and the
reaction was then stopped by the addition of 100 μl/well of 1% sodium dodecyl sulfate
(SDS) in sterile water. Color development was quantified at 405 nm. All samples were
tested in duplicate and the results were recorded as μg/ml of specific immunoglobulin. A
similar procedure was performed to measure IgA antibodies, in which biotinylated anti-
rat IgG was replaced with biotinylated anti-rat IgA (1 μg/ml, AbD Serotec, Raleigh, NC )
Because rat IgA standards were not available, the results were recorded as endpoint titers
consisting of the reciprocal of the highest serum dilution providing an absorbance value
of 0.1.
H. Salbutamol and Formoterol Medication For experiments to examine the efficacy of β-agonist medication to protect
against pneumococcal movement from the nasopharynx to the lungs, an apparatus was
developed to facilitate delivery of the medication to the rats (Figure 5). The rats were
restrained in DecapiCones (Braintree Scientific Corp, Braintree, MA) and placed into a
series of connected PVC pipes. A nebulizer (Super SportNeb 3050SS, Medical
Industries, Adel, IA) was used to aerosolize 5 ml of 0.5% salbutamol (Nephron
Pharmaceuticals Corporation, Orlando, FL) in PBS or 48 μg of formoterol (Foradil,
Shering Corporation, Kenilworth, NJ) dissolved in 5 ml of 0.01% acetic acid. For 20
min, one of the vaporized solutions was dispersed through tubes of identical length and
diameter inserted into each rat’s DecapiCone.
Twelve hours after rats were infected intranasally with 1 x 106 cfu of S.
pneumoniae ATCC 6303, they were medicated three times a day for 1 week with
45
salbutamol or twice daily for 1 week with formoterol. Control rats were medicated at the
same time intervals in an identical apparatus with nebulized vehicle solution alone. The
rats were rotated throughout the different chambers of the appropriate apparatus to
compensate for any slight variability in the amount of drug delivered to each chamber.
After 1 week of medication, the rats were euthanized and their nasopharynx, trachea, and
lungs were cultured as described above.
Figure 5 – Medication Device
Figure 5 A medication apparatus was used to expose the rats to salbutamol and formoterol. Rats were restrained in Decapicones and placed into the PVC pipes. A nebulizer aerolized the drug and delivered it to the rats via the plastic tubes.
I. Ciliary Beat Frequency Analysis
Ciliary beat frequency was quantified in the formoterol medication study by
computerized frequency analysis at Creighton University. Tracheal rings 1-2 mm thick
were excised from each animal’s trachea. The samples were maintained at 24 ± 0.5oC on
a thermostatically controlled heated stage and digitally analyzed using the Sisson-
46
Ammons Video Analysis (SAVA) system (Ammons Engineering, Mt. Morris, MI).
Whole field analysis software (Ammons Engineering) automatically analyzed the entire
captured image of all ciliated cells in a given field. All cilia amplitudes and frequencies
were collated, mapped to the screen image, and statistically analyzed to determine the
frequency average, standard deviation, and standard error of the entire image. The
frequency of the beating cilia was sampled in at least 6 different fields, and the data from
those fields was compiled to produce a mean ciliary beat frequency in Hertz (Hz) for
each rat.
J. Transtracheal Infections
Rats were infected transtracheally with type 3 S. pneumoniae ATCC 6303 for the
non-PMN-mediated killing, PMN-mediated killing, opsonic deposition, and phagocytosis
experiments, or with S. aureus ATCC 29213 for the macrophage phagocytosis study.
Under light anesthesia with isoflurane, the rats were laid on their backs on a surgery
table. The area over the trachea was cleaned with 70% ethanol and a small incision was
made through the skin with a scalpel. The trachea was exposed by blunt-end dissection,
and a 22 ga. catheter was inserted into the trachea toward the lungs. With the rat in a
vertical position, a 1cc syringe was used to deliver the specified incoculum of bacteria
suspended in 0.3 ml PBS into the catheter. This was followed by an injection of 0.1 ml
of air to simulate aspiration (Figure 6). The catheter was then removed and the incision
was closed with two metal clips.
47
Figure 6 – Transtracheal Infection
Figure 6 Transtracheal infections were performed on anesthetized rats by making a small incision on the neck and exposing the trachea by blunt-end dissection. A catheter then was inserted into the trachea. After placing the rat in a vertical position, the inoculum was injected into the catheter followed by an injection of air.
K. Lipopolysaccharide Instillation for PMN-mediated Assays
Five hours prior to transtracheal infection for the PMN killing and phagocytosis
assays, PMNs were recruited to the rats’ lungs by transtracheally instilling
lipopolysaccharide (LPS) from Escherichia coli O26:B6 (Sigma). The rats were
anesthetized with isoflurane and a 22 ga. catheter was inserted into the trachea as
described above. Twenty μg of LPS suspended in 0.2 ml PBS was instilled into each
rats’ lungs. The incision was then closed with two metal clips.
48
L. Non
t of two control rats sacrificed immediately after infection, using the following
equati
(Mean cfu’s at time 0 – cfu’s of test rats at 1hr) / Mean cfu’s at time 0 (x 100)
cedures
Ex vivo
-PMN-Mediated Bactericidal Assay
To measure pulmonary killing of S. pneumoniae by non-PMN-mediated defenses,
including the bactericidal activity of alveolar lining fluid and macrophages, our
laboratory developed an in vivo bactericidal assay. Rats were infected transtracheally
with 1 x 106 cfu of S. pneumoniae ATCC 6303 as described previously. At one hour
post-infection, the rats were euthanized and the trachea and lungs were removed en bloc.
After detaching the trachea, the lungs were homogenized in sterile tissue grinders in a
total volume of 10 ml of PBS. Plate counts were then performed on the homogenates to
quantify the number of viable organisms remaining in the lung tissue. The percentage of
pneumococcal killing was determined by comparing the lung counts of each test rat to the
mean coun
on:
M. Bronchoalveolar Lavage Pro
Bronchoalveolar Lavage
Bronchoalveolar lavage (BAL) was performed ex vivo to collect bacteria and
pulmonary cells from the rats’ lungs for the opsonin deposition and phagocytosis assays.
In this procedure, the rats were euthanized and their lungs were perfused with 30 ml of
ice cold PBS injected into the right ventricle of the heart and drained through a 16 ga.
needle inserted into the left ventricle for the removal of peripheral blood cells. Following
perfusion, the lungs and trachea were removed en bloc, and bacteria and pulmonary cells
were washed from the lungs as described previously [283]. Briefly, 10 ml aliquots of ice
49
cold Hanks Balanced Salt Solution (HBSS) without Mg++, Ca++, and phenol red
(Gibco/Invitrogen, Carlsbad, CA) were instilled into the lungs with a 10cc syringe
through a 22 ga. catheter inserted into the trachea (Figure 7). Following each instillation,
the lavage fluid was collected by dependent drainage until a total volume of 50 ml was
covered (Figure 8).
situ
re
Figure 7 – Lavage Figure 8 – Lavage Fluid Collection
Figures 7 and 8 Ex vivo bronchoalveolar lavage was performed by extracting the trachea and lungs en bloc. Ten ml of HBSS was injected through a catheter inserted into the trachea. The lavage fluid then was collected by dependent drainage.
In Bronchoalveolar Lavage
Bronchoalveolar lavage was performed in situ to collect BAL samples from
uninfected rats for the quantification of pulmonary lysozyme, lactoferrin, lactate
dehydrogenase (LDH), and opsonins. The rats were euthanized and the trachea was
exposed as described above. A single 10 ml aliquot of ice cold PBS was repeatedly
instilled and withdrawn 5 times via a 10cc syringe attached to a 22 ga. catheter inserted
50
into the trachea. This resulted in the collection of 7-8 ml of lavage fluid which was
centrifuged at 600 x g for 10 min at 4oC to remove cells and debris. The supernatant then
was sterile filtered using a 0.22 μm filter (Millipore, Billerica, MA) and the filtrate was
stored in 1 ml aliquots at -80oC until analyzed for individual antimicrobial and opsonic
protein levels as well as total protein concentration determined by the Quick Start
radford protein assay kit (Bio-Rad Laboratories, Hercules, CA).
N. Qu
lyzed in duplicate and results were recorded as ng of
ctoferrin/mg of total protein.
O. Qu
re run in duplicate and results were
g of total protein.
B
antification of Bactericidal Factors
Bactericidal factors were quantified in lavage samples collected by in situ
bronchoalveolar lavage as described above. Lysozyme activity in the lavage samples was
quantified using the commercially available EnzChek Lysozyme Assay Kit (Molecular
Probes, Eugene, OR). Samples were assayed in duplicate and results were recorded as
activity units/mg of total protein. Lactoferrin was measured by the commercially
available Bioxytech Lactof-EIA for human lactoferrin (Oxis International, Inc., Portland,
OR). Samples were again ana
la
antification of Pulmonary Cell Damage
Lactate dehydrogenase (LDH), a protein marker for cellular damage, was
measured in lavage fluid collected from uninfected rats as described above. The LDH-
Cytotoxicity Assay Kit from Biovision Research Products (Mountain View, CA) was
utilized to measure LDH levels. All samples we
recorded as milliunits of LDH/m
51
P. Opsonic Deposition Assay
Infection and Staining of Opsonins on Pneumococci
To quantify opsonization of S. pneumoniae, our laboratory developed an assay
that measures the in vivo deposition of three opsonins on the surface of pneumococci.
Rats were infected transtracheally with 1 x 107 cfu of live S. pneumoniae ATTC 6303.
After exactly 30 min, each rat was euthanized and ex vivo bronchoalveolar lavage was
performed as described above. The rats’ pulmonary cells were collected from the lavage
fluid by centrifugation at 450 x g for 30 min. The resulting supernatant was centrifuged
at 13,776 x g for 10 min to collect pneumococci recovered from the rats’ lungs. To
remove remaining rat pulmonary cells from the bacterial pellet, the cells were lysed by
adding 10 ml of distilled water followed 10 seconds later by the addition of an equal
volume
ria
en were washed twice again and fixed in 1% formalin for flow cytometric analysis.
of double-strength PBS.
The final bacterial pellet from each rat’s lungs was washed once in PBS and
separated into three 50 μl aliquots. Each aliquot was incubated at 37oC for 30 min with 1
ml of PBS containing 10 μg/ml of biotinylated rabbit anti-rat C3 antibody (Immunology
Consultants Laboratory, Newberg, OR), rabbit anti-rat CRP antibody (Immunology
Consultants Laboratory), or mouse anti-rat SP-D antibody (BMA Biomedicals, Augst,
Switzerland). After the bacteria were washed twice, each aliquot was incubated at room
temperature for 10 min with 1 ml of 1 μg/ml of a streptavidin-conjugated
allophycocyanin (APC) fluorochrome (BD Pharmingen, San Diego, CA). The bacte
th
52
Flow Cytometric Analysis
Pneumococci isolated from the rats’ lungs and labeled with antibodies to the
various opsonins were analyzed using a FACSAria flow cytometer (Becton Dickinson,
San Jose, CA) to quantify the percentage of pneumococci with C3, CRP, or SP-D bound
to their surface. On each day of experimentation, a forward and side scatter plot of
unlabeled pneumococci grown in culture was used to set the analysis gate for
pneumococci recovered from the rats’ lungs (Figure 9). The gated bacteria then were
analyzed in the fluorescent APC channel at 660 nm to quantify C3, CRP, and SP-D
the surface of pneumococci (Figure 10). binding to
Figure 10 – Analysis of
APC Fluorescence Figure 9 – Scatter Plot of
S. pneumoniae
Forw
ard
Scat
ter
% o
f Max
Side Scatter APC
Figures 9 and 10 Bacteria were gated based on forward and side scatter in the flow cytometer. The gated bacteria then were analyzed for fluorescence to quantify opsonic protein deposition on the surface
53
Q. C3,
esults from all three ELISAs were
corded as μg of the opsonin/mg of total protein.
R. PM
CRP, and SP-D ELISAs
C3 and CRP were quantified in lung lavage samples with commercially available
rat C3 and rat CRP ELISA kits (Immunology Consultants Laboratory). SP-D in the
lavage samples was quantified by a self-developed capture ELISA. Briefly, 96-well
plates were coated overnight at room temperature with 100 μl/well of 0.5 μg/ml of anti-
rat SP-D antibody (Hycult Biotechnology, Uden, Netherlands). The plates were washed
4 times with 0.05% Tween 20 in PBS and then blocked for 1 hour at room temperature
with 200 μl/well of 1% bovine serum albumin in PBS. After the plates were washed, 100
μl/well of rat SP-D standard (0-400 ng/ml, Hycult Biotechnology) and duplicate samples
of lavage fluid diluted 1:400 in 0.05% Tween 20 with 0.1% bovine serum albumin in
PBS were added, and the plates were incubated at room temperature for 2 hours. After
washing, 100 μl/well of biotinylated anti-rat SP-D antibody (0.1 μg/ml, BMA
Biomedicals) was added and the plates were incubated for 30 min at room temperature.
The plates then were washed, 100 μl of streptavidin-HRP (Pierce) was added to each
well, and they were incubated for 30 min at room temperature. After washing, 100 μl of
tetramethylbenzidine (TMB) substrate was added to each well. After 10-20 min of
incubation at room temperature, the reaction was stopped by the addition of 100 μl/well
of 2N H2SO4 and the plates were read at 450 nm. R
re
N-Mediated Bactericidal Assay
Similar to the non-PMN-mediated bactericidal assay, this assay measures the
pulmonary killing of pneumococci in vivo after the pre-recruitment of PMNs. Five hours
54
after LPS-induced PMN recruitment, the rats were infected transtracheally with 1 x 106
cfu of S. pneumoniae ATCC 6303. Exactly one hour post-infection, the rats were
euthanized and their lungs were lavaged once in situ with 10 ml HBSS. One ml of the
collected lavage fluid was used to prepare cytospin slides (Cytospin 2, Shandon, Chesire,
UK) to determine the percentage of PMNs recruited to the rats’ lungs. Each slide was
stained with Protocol Hema-3 (Fisher Diagnostics, Middletown, VA) and two hundred
cells from each sample were counted manually in a Nikon Labopho-2 light microscope
(Melville, NY). The lungs then were removed and homogenized in a total volume of 10
ml of PBS including the remaining lavage fluid. Plate counts were performed on the
homogenates and the percentage of bacterial killing was determined as described above
r the non-PMN-mediated killing assay.
S. Ch
homogenates and serum using commercially available kits from R&D Systems
fo
emokine Analysis
Chemokine levels were measured by ELISAs in serum and lung homogenates
from each of the rats utilized in the PMN-mediated bactericidal assay. At the time of
euthanasia, blood collected from each animal by cardiac puncture was placed in a serum
separator tube. The blood was centrifuged at 1900 x g for 15 min and the serum was
frozen at -80oC in 1 ml aliquots until analyzed. After completion of the serial dilutions
for plate counts, the crude lung homogenates were centrifuged at 13,776 x g for 15 min.
The supernatants then were sterile filtered through a 0.22 μm filter to remove any
contaminating pneumococci and 1 ml aliquots were frozen at -80oC. MIP-2 levels were
measured in the lung homogenates and CINC-1 levels were measured in the lung
55
(Minneapolis, MN). All samples were analyzed in duplicate, and results were recorded
as pg of cytokine/ml of serum or homogenate.
T. PMN Phagocytosis Assay
Bacteria Staining and Infection
To quantify the phagocytic activity of recruited PMNs, a phagocytosis assay was
developed to measure the uptake of S. pneumoniae by PMNs within the rats’ lungs [171].
This assay utilized live S. pneumoniae ATCC 6303 fluorescently stained with 5-(and 6-)
carboxyfluorescein diacetate succinimidyl ester (CFDA/SE, Molecular Probes).
CFDA/SE is a non-fluorescent diacetate form of CFSE that easily passes through the
bacterial cell wall. Once inside the organism, the acetate groups are cleaved off by
cytosolic esterases and the molecule is converted to the fluorescent form of CFSE.
Pneumococci were stained by incubating them at 37oC for 30 min in the dark with 2 μM
solution of CFDA/SE. After washing twice with PBS to remove excess dye, the bacteria
were resuspended in PBS to an optical density of 1.0. The CFSE-labeled organisms
fluoresced brightly in the flow cytometer as demonstrated in Figure 11.
56
Figure 11 – CFSE-Labeled Pneumococci
Figure 11 Pneumococci were labeled with CFSE in order to quantify PMN phagocytosis by flow cytomtery. Bacteria stained with CFSE fluoresced brightly compared to unstained bacteria.
PMNs were pre-recruited to the rats’ lungs by LPS instillation as described above.
Exactly 5 hrs later, the rats were infected transtracheally with 1 x 108 cfu of CFSE-
labeled pneumococci. Exactly 15 min post-infection, each rat was euthanized and ex vivo
bronchoalveolar lavage was performed as described above. Pulmonary cells collected
from the lavage fluid were washed once with HBSS using differential centrifugation (180
x g for 10 min) to remove unassociated bacteria. Contaminating red blood cells then
were lysed by the addition of 10 ml of deionized water followed 10 seconds later by an
equal volume of double-strength PBS. The remaining cells were centrifuged and
resuspended in PBS containing 4% heat-inactivated fetal calf serum (PBS-FCS). The
57
cells were counted in a hemacytometer and the cell suspension was adjusted to a final
concentration of 2 x 107 cells/ml for antibody staining.
Antibody Staining of Pulmonary Cells
Pulmonary cells from each rat’s lavage sample were stained in 96-well U-bottom
microplates with fluorochrome-tagged antibodies for flow cytometric analysis. Fifty μl
of the cell suspension were incubated for 30 min in the dark on ice with 50 μl of a
monoclonal antibody cocktail containing 0.25 μg of phycoerythrin (PE)-conjugated anti-
rat granulocyte antibody RP-1 (BD Pharmingen) and 2 μg of the biotinylated anti-rat
monocyte antibody 1C7 (BD Pharmingen). The cells then were centrifuged at 180 x g for
4 min and washed twice with PBS-FCS. One-hundred μl of PBS-FCS containing 1 μg of
streptavidin-APC was added and the cells were incubated for an additional 5 min in the
dark on ice. After two more washes in PBS-FCS, the cells were resuspended in PBS
containing 1% formalin for flow cytometric analysis. Negative control samples for cell
staining consisted of unstained cells from each rat and cells stained with streptavidin-
APC only.
Flow Cytometric Analysis
Three-color flow cytometric analysis was performed on cells from each rat using
a FACSAria flow cytometer with dual laser excitation (488 nm and 633 nm). For each
day the assay was run, a control rat infected with non-fluorescent pneumococci was
included to exclude autofluorescence in the CFSE channel of the RP-1 positive PMNs.
Based on scatter characteristics that included neutrophils and macrophages, a minimum
58
of 10,000 cells were analyzed in the PE (575 nm channel) for PMNs and the APC (660
nm channel) for macrophages. The percentage of RP-1 positive cells fluorescing more
bright in the CFSE (530 nm) channel than the PMNs from the control rat was used to
determine the percentage of PMNs with associated bacteria (Figure 12). The frequency
of RP-1 positive PMNs and the percentage of PMNs containing associated fluorescent
bacteria were determined for each sample using FlowJo Software (Tree Star, Ashland,
OR). To determine the relative number of fluorescent pneumococci engulfed by each
PMN, a phagocytic index was calculated by multiplying the percentage of PMNs that
contained fluorescent pneumococci by their mean fluorescent intensity.
Figure 12 – Determination of PMN Phagocytosis
Test Rat Infected with CFSE-Labeled Bacteria
Control Rat Infected with Unlabeled Bacteria
Figures 12 To determine bacterial uptake gated PMNs from test rats were analyzed for CFSE fluorescence and compared to PMNs from a control rat infected with unlabeled bacteria.
59
U. Macrophage Phagocytosis Assay
Bacterial Staining and Infection
To evaluate the ability of macrophages to phagocytose bacteria within the lung,
an assay was adapted from the PMN phagocytosis assay to measure the in vivo uptake of
bacteria by macrophages within the lungs. This assay used fluorescently-labeled S.
pneumoniae ATCC 6303. To override the problem of macrophage autofluorescence, S.
pneumoniae was labeled with the far-red fluorochrome APC-Cy7 (BD Pharmingen)
which fluoresced brighter than the autofluorescence of the macrophages (Figure 13). The
pneumococci were incubated for 30 min at room temperature with 0.5% rabbit antiserum
to type 3 pneumococcal polysaccharide (Statens Seruminstitut, Copenhagen, Denmark).
After the bacteria were washed twice with PBS, they were incubated for 30 min at room
temperature with 1 μg/ml of a biotinylated anti-rabbit immunoglobulin antibody (BD
Pharmingen). After washing, the pneumococci then were incubated for an additional 10
min at room temperature with 1 μg/ml of a streptavidin-APC-Cy7 conjugate, washed
again, and resuspended in PBS to an optical density of 1.0.
60
Figure 13 – APC-Cy7-labeled S. pneumoniae
% o
f Max
APC-Cy7
Macs
S. pneumoniae
Figure 13 APC-Cy7 caused bacteria to fluoresce brighter than macrophages.
Rats were infected transtracheally with 1 x 108 cfu of S. pneumoniae. At exactly
15 min post-infection, each rat was euthanized and an ex vivo bronchoalveolar lavage was
performed. Pulmonary cells were collected and washed as described above. Cells then
were resuspended in PBS with 0.5% bovine serum albumin (PBS-BSA) and the
macrophages were separated from PMNs by magnetic cell sorting as described below.
Magnetic Cell Sorting
Macrophages could not be labeled with the biotinylated 1C7 antibody due to the
possibility of streptavidin-APC binding to pneumococci since they also were labeled with
a biotinylated antibody. As an alternative, macrophages were separated from
contaminating PMNs by magnetic cell sorting [48]. For 15 min, cells were incubated on
ice with 1 ml of PBS-BSA containing 4 μg of biotinylated anti-rat granulocyte antibody
61
(22262D, BD Pharmingen). The cells were centrifuged at 180 x g for 6 min and washed
twice with PBS-BSA. The final cell pellets were resuspended in 90 μl of PBS-BSA, to
which were added 10 μl of streptavidin-coated MACS super-paramagnetic microbead
suspension (Miltenyi Biotec Inc., Sunnyvale, CA). After incubation for an additional 15
min on ice, the cells were washed and resuspended in 0.5 ml of PBS-BSA. The cell
suspension then was loaded onto a MiniMACS separation column suspended in a
magnetic field according to the manufacturer’s directions (Miltenyi Biotec Inc.).
Unlabeled macrophages that traveled through the column were collected, washed, and
resuspended in PBS containing 1% formalin for flow cytometric analysis. This method
resulted in > 95% purity.
Flow Cytometric Analysis
Similar to the PMN phagocytosis assay, the FACSAria flow cytometer was used
to analyze the fluorescence of macrophages from each rat. A control rat infected with
non-fluorescent bacteria was included each day the assay was performed to determine the
extent of the macrophage autofluorescence in the APC-Cy7 channel detecting the
bacteria. Macrophages isolated by magnetic cell sorting were gated based on forward
and side scatter and analyzed in the APC-Cy7 (780 nm) channel for phagocytosis of
pneumococci. The percentage of macrophages fluorescing more bright in the APC-Cy7
channel than macrophages from the control rat infected with unlabeled bacteria was
determined as the percentage of macrophages with associated bacteria (Figure 14).
FlowJo Software was used to analyze the flow cytometric results, and the phagocytic
index was calculated for each sample as described for the PMN phagocytosis assay.
62
Figure 14 – Determination of Macrophage Phagocytosis
Bacteria Fluorescence
Test Rat Infected with APC-Cy7-labeled Bacteria
Control Rat Infected with Unlabeled Bacteria
Figure 14 To determine macrophage phagocytosis, macrophages gated by forward and side scatter were analyzed for bacterial fluorescence and compared to macrophages from a control rat infected with unlabeled bacteria.
Bacteria Fluorescence
Adaptation of Phagocytosis Assay
The macrophage phagocytosis assay was repeated using S. aureus ATCC 29213.
S. aureus was labeled with the bright green DNA dye Syto 9 (Molecular Probes) which
also fluoresced brighter than the autofluorescent macrophages (Figure 15). S. aureus was
stained by incubating at room temperature for 15 min in the dark with 10 nM Syto 9 in
sterile water. The bacteria were washed twice to remove excess dye and resuspended in
sterile water to an optical density of 1.0. The rats were infected, euthanized, and their
pulmonary cells were collected as described above. The cells from each rat were
resuspended in PBS-FCS, stained with RP-1 and 1C7 antibodies, and analyzed in the PE
(PMNs) and APC (macrophages) channels as described in the PMN phagocytosis assay.
The APC positive cells were then analyzed in the Syto 9 (530 nm) channel for uptake of
63
staphylococci and the percentage of macrophages with associated bacteria was
determined as described in the previous section.
Figure 15 – Syto 9-labeled S. aureus
Macs S. aureus
% o
f Max
Syto 9 Figure 15 Syto 9 caused bacteria to fluoresce brighter than macrophages.
Microscopic Analysis of Macrophages
Cytospin slides prepared from lavage samples from each rat infected with S.
aureus were stained as described above and examined in a light microscope. Two
hundred cells from each sample were counted to verify the percentage of macrophages
determined by flow cytometry to have phagocytosed staphylococci.
V. Macrophage Function Assays
Three assays were performed to measure oxidative burst, degranulation, and
chemokine release by macrophages stimulated in vitro with either S. pneumoniae or
phorbol myristate acetate (PMA). Macrophages were harvested from uninfected rats’
64
lungs by ex vivo bronchoalveolar lavage as described above. After washing the
pulmonary cells and lysing any contaminating erythrocytes, macrophages from each rat
were counted in a hemacytometer.
To measure oxidative burst, a chemiluminescence assay was performed. One x
105 macrophages seeded in each well of a 96-well plate were incubated at room
temperature in 200 μl of HBSS (Gibco/Invitrogen) containing Mg++, Ca++, 5 mM
glucose, and 50 μM Luminol salt (Sigma). The macrophages in individual wells were
exposed to either 5 x 106 cfu of DW 3.8 S. pneumoniae preopsonized with normal rat
serum or 1 μg/ml phorbol myristate acetate (PMA) (Sigma) plus 0.5 μg/ml ionomycin
(Sigma). Chemiluminescence emitted from the oxidized Luminol was measured using a
Victor3 1420 Multilabel Counter (Perkin Elmer, Waltham, MA). The plate was read at
time zero and every 10 min up to 1 hour. Each sample was run in duplicate and the
results were recorded as light units and compared to unstimulated cells.
To measure degranulation and chemokine release, 5 x 105 macrophages in 1 ml of
complete RPMI media (Gibco/Invitrogen) supplemented with 50 μM 2-mercaptoethanol
were aliquoted into wells of a 24-well cell culture plate. The plate was incubated for 1
hour at 37oC to allow macrophage adherence. After removing the supernatant, 1 ml of
fresh RPMI media was added to each well that contained 0.5 μg/ml recombinant rat IFN-
γ (eBioscience, San Diego, CA) and either 2.5 x 107 cfu of preopsonized DW 3.8 S.
pneumoniae or 1 μg/ml PMA plus 0.5 μg/ml ionomycin. The cells were incubated at
37oC for 1 hour, after which 250 μl of supernatant was collected to measure lysozyme.
The remaining supernatant was collected after 5 hours for chemokine analysis. All
65
samples were stored at -80oC until analyzed for lysozyme, MIP-2, and CINC-1 using
commercially available kits as described above.
W. Intranasal Mortality Study
A mortality study was conducted following an intranasal infection with 1 x 108 of
S. pneumoniae ATCC 6303. Mortality was assessed for 9 days post-infection.
Temperature transponders (BioMedic Data Systems, Seaford, DE) were implanted
subcutaneously in the top of the rats’ heads at the time of infection. Temperatures then
were recorded twice daily and the rats were observed for abnormal movement, ruffling of
the fur, and/or bleeding from the eyes or nose. On days 2 and 5 post-infection, 50 μl of
blood was collected by aseptic puncture of the rats’ foot veins [284] and standard plate
counts were performed for quantification of bacteremia. If a rat’s temperature dropped
below 35oC, it was euthanized and was counted as having succumbed to the infection.
X. Statistical Analysis
The mortality data were analyzed using Fisher’s Exact test. All other data were
tested for normality and equal variance before statistical tests were performed. To
compare smoke-exposed vs. sham-exposed rats or vaccinated/medicated vs. control
groups in the vaccination and medication trials, the Students t-test was utilized for normal
data or the Mann-Witney Rank Sum Test for non-normal data. Paired t-tests were used to
compare EtOH-fed rats to their pair-fed controls within the same exposure group. Two
Way ANOVA was utilized with the Holm-Sidak method for pair-wise comparisons to
determine differences among exposure, diet, and the combination of the two factors. In
66
the PppA vaccination trial, Spearman’s rank order correlation was used to correlate anti-
PppA antibody levels and the log cfu recovered from each rat’s nasal passages.
Spearman’s correlation test also was used in the formoterol medication study to correlate
ciliary beat frequency with the number of organisms recovered from the lung
homogenates of each rat. For all statistical tests, a p-value <0.05 was considered
significant.
67
V. RESULTS
A. Pneumococcal Colonization and Movement from the Nasopharynx to the Lungs After 7 weeks of smoke or sham exposure plus 5 weeks of concurrent exposure
and pair-feeding, 6-8 rats per group were infected intranasally with S. pneumoniae ATCC
6303. One week post-infection, the rats were euthanized and the numbers of
pneumococci were quantified in their nasopharynx, trachea, carina, and lung tissue. The
plate count data from rats in this study are summarized in Table 1. All means of the log
include rats that were negative for pneumococci. There were no smoke- and/or EtOH-
induced alterations in colonization, since 100% of the rats remained colonized in the
nasopharynx with similar numbers of organisms one week after infection. EtOH
ingestion alone resulted in slightly more pneumococci remaining in the rats’ lungs. A
larger percentage of sham-EtOH rats had positive carina and lung cultures and with a
higher number of pneumococci than their sham-pair counterparts. Smoke exposure alone
greatly reduced the number of pneumococci remaining in the rats’ lungs, even in the
presence of EtOH ingestion. Regardless of diet, the percentage of smoke-exposed rats
with pneumococci in their tracheas and carinas was significantly lower than the sham-
exposed rats (p = 0.003 and 0.021, respectively). Smoke-exposed rats also had
significantly fewer organisms in their tracheas and carinas (p < 0.001 and p = 0.009,
respectively), as well as fewer organisms in their lungs compared to the sham-exposed
animals (p = 0.05).
68
Table 1 – Smoke Exposure Reduces Pneumococcal Movement
Smoke-EtOH n=7
Smoke-Pair n=8
Sham-EtOH n=6
Sham-Pair n=8
% Positive
Log cfu/ml
% Positive
Log cfu/ml
% Positive
Log cfu/ml
% Positive
Log cfu/ml
Nasal Wash 100 4.5 100 4.3 100 4.1 100 4.0
Trachea 29 0.8 25 0.9 83 2.6 88 3.5
Carina 14 0.6 13 0.2 67 2.2 50 1.8
Lungs 14 0.7 13 0.5 67 1.8 38 1.2
B. PppA Vaccination Trial
To determine if antibodies to PppA could reduce pneumococcal colonization of
the nasopharynx, a preliminary vaccination trial was conducted in which unexposed,
chow-fed rats were immunized intranasally with rPppA. Two weeks after their third
vaccination, the rats were infected intranasally with type 3 S. pneumoniae. At one week
post-infection, the rats were euthanized and pneumococcal colonization of the
nasopharynx was quantified. Figure 16 depicts the log cfu from nasal washes of
vaccinated and control rats. Intranasal immunizations with rPppA failed to reduce
pneumococcal colonization of the nasopharynx, as all of the rats remained colonized 1
week after infection. Although there were two vaccinated rats that had a 2-3 log lower
cfu count in their nasal washes, the mean log cfu of the vaccinated group was similar to
that of the control group.
The rPppA ELISAs showed the vaccinated rats had a higher concentration of anti-
PppA IgG antibodies in their serum and a higher anti-PppA IgA antibody titer in their
nasal washes compared to the control rats (Table 2). However, these heightened antibody
69
levels were not effective in reducing nasopharyngeal colonization since there were no
strong inverse correlations between anti-PppA IgG or anti-PppA IgA levels and the log
cfu recovered from the nasopharynx (-0.212 and -0.266, respectively).
Figure 16 – PppA Vaccination Fails to Reduce Colonization
Vaccinated Control0
1
2
3
4
5
Log
cfu/
ml N
asal
Was
h
Table 2 – rPppA Specific IgG and IgA Antibody Levels Compared to Log cfu of Nasal Washes
Serum IgG
(μg/ml) Mucosal IgA
titer Log cfu/ml nasal wash
Vaccinated Rats 182 5 3.2 Control Rats 30 <2 3.8
Figure 16 Pneumococcal colonization of the nasopharynx was quantified by performing plate counts on retrograde nasal washes from vaccinated and control rats. n=5-6
70
C. Salbutamol Medication Trial
To determine if salbutamol, a short-acting β2-agonist that increases ciliary
beating, could hinder pneumococcal movement into the lungs after an intranasal
infection, unexposed chow-fed rats were again used in a preliminary medication trial.
Twelve hours after the rats were infected intranasally with S. pneumoniae, they were
medicated three times a day for one week with salbutamol or PBS. At the end of
medication, the rats were euthanized and their nasopharynx, trachea, carina, and lungs
were cultured to quantify the number of pneumococci. Figure 17 below depicts the log
cfu at the various locations of each rat.
Figure 17 – Salbutamol Medication Decreases Pneumococcal Movement in Chow-fed Rats
0
1
2
3
4
5
Nasal Wash Trachea Carina Lungs
ControlSalbutamol
Log
cfu
Figure 17 Animals were medicated with salbutamol or PBS three times a day for one week after an intranasal infection with S. pneumoniae. Pneumococcal colonization and movement then were quantified by plate counts of nasal washes, tracheas, carinas, and lung homogenates. n=4-5
71
Medication with aerosolized salbutamol somewhat reduced pneumococcal
movement from the nasopharynx into the lower respiratory tract. As expected,
salbutamol exposure did not alter colonization, since all the animals had similar numbers
of pneumococci in their nasal washes. However, the majority of the medicated rats had
no organisms in their tracheas, and the one medicated rat with pneumococci in its trachea
had a 1.5 log lower cfu count than the positive control rats. No organisms were detected
in the carina and lungs of the medicated group, whereas one control animal remained
positive for pneumococci in those two samples.
D. Formoterol Medication
The longer-acting β2-agonist formoterol also was pretested for its ability to reduce
pneumococcal movement from the nasopharynx to the lungs of unexposed chow-fed rats.
Starting 12 hours after being infected intranasally with S. pneumoniae, the rats were
medicated with formoterol or vehicle solution alone every 12 hours for 1 week. The rats
then were euthanized and pneumococcal colonization and movement were quantified.
The log cfu from each sample for the medicated and control rats are shown in Figure 18
below.
72
Figure 18 – Formoterol Medication Reduces Pneumococcal Movement into the Lungs of Chow-fed Rats
0
1
2
3
4
5
6
Nasal Wash Trachea Carina Lungs
ControlFormoterol
Log
cfu
Figure 18 Log cfu of nasal washes, tracheas, carinas, and lung homogenates from rats medicated twice daily for one week with formoterol or vehicle solution alone after an intranasal infection with S. pneumoniae. n=5
As shown before, all the animals in this study remained colonized with a similar
number of organisms one week after infection followed by medication. Formoterol was
more effective than salbutamol in reducing pneumococcal movement into the lower
respiratory tract. All except one medicated rat were negative for pneumococci in their
trachea, carina, and lungs, while 4 of the 5 control rats had pneumococci in all three
cultures.
Due to the success of formoterol in this trial, it was chosen to test further in the
chronic EtOH ingestion and smoke exposure model. Following 12 weeks of smoke- or
sham-exposure, with the last five weeks including concurrent pair-feeding, rats were
infected and medicated with formoterol twice daily for one week as described above.
73
During their medication, the rats continued to be smoke- or sham-exposed and fed their
liquid diets. At the end of medication, the rats were euthanized and pneumococci were
quantified throughout their respiratory tracts. Figure 19 displays the log cfu of nasal
washes, trachea, carina, and lungs for the individual rats in each treatment and medication
group.
Figure 19 – Formoterol Medication Prevents Pneumococcal Movement in Sham-EtOH Rats
B. Trachea A. Nasal Wash
0
1
2
3
4
5
6
EtOH + + - - + + - -Formoterol + - + - + - + -
Sham Smoke
Log
cfu
0
1
2
3
4
5
6
EtOH + + - - + + - -Formoterol + - + - + - + -
Sham Smoke
Log
cfu
0
1
2
3
4
5
6
EtOH + + - - + + - -Formoterol + - + - + - + -
Sham Smoke
Log
cfu
C. Carina D. Lungs
0
1
2
3
4
5
6
EtOH + + - - + + - -Formoterol + - + - + - + -
Sham Smoke
Log
cfu
Figure 19 Rats were infected intranasally with S. pneumoniae and medicated twice daily for one week with formoterol or vehicle solution. Plate counts then were performed on nasal washes (A), tracheas (B), carinas (C), and lung homogenates (D). n=5-8
74
As seen with salbutamol, formoterol medication failed to alter pneumococcal
colonization of the nasopharynx in any of the treatment groups. Unexpectedly,
pneumococcal numbers in the lungs was not reduced by formoterol in the sham-pair
group as it was in chow-fed animals. The percentages of medicated and unmedicated
sham-pair rats positive for pneumococci in their trachea, carina, and lungs were alike
along with similar cfu counts. The only group in which formoterol medication appeared
to be successful in preventing pneumococcal movement into the lower respiratory tract
was the sham-EtOHs. Pneumococci were undetected in either the carina or the lungs of
each of the medicated sham-EtOH rats, whereas 66% of the unmedicated rats in that
same treatment group had positive carina and lung cultures. Interestingly, smoke
exposure itself increased the ability of the rats to maintain sterility in their lungs, but
formoterol had no effect on pneumococcal movement in smoke-exposed rats. Regardless
of medication and diet, the majority of the smoke-exposed rats had no detectable
organisms in their lower respiratory tract.
In addition to tracking pneumococcal movement into the lungs, baseline ciliary
beat frequency (CBF) was analyzed in rats from each medicated and treatment group.
Following euthanasia, tracheal rings from each rat were excised and transported to
Creighton University to be analyzed by the SAVA system. Smoke exposure significantly
augmented the CBF (Figure 20). This was true for all smoke-exposed vs. sham-exposed
rats regardless of diet and formoterol medication.
75
Figure 20 – Smoke Exposure Increases CBF Regardless of Diet and Formoterol Medication
Medicated Unmedicated All Animals0
5
10
15
SmokeSham
p < 0.001p = 0.009 p < 0.001C
BF
(Hz)
± SE
M
Figure 20 Baseline CBF of smoke-exposed vs. sham-exposed rats for formoterol medicated (n=10-14), unmedicated (n=13-15), and all animals combined (n=25-27).
There were no statistical differences in CBF between medicated and unmedicated
rats within any of the treatment groups except for the sham-EtOHs, in which formoterol
medication significantly increased baseline CBF (Figure 21). There also was a strong
inverse relationship between CBF and the log cfu recovered from the lung homogenates
of sham-EtOH rats, with a correlation coefficient of -0.642 (p = 0.029).
76
Figure 21 – Formoterol Augments CBF in Sham-EtOH Rats
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
5
10
15
FormoterolUnmedicated
p = 0.005C
BF
(Hz)
± SE
M
Figure 21 Baseline CBF of tracheal ring explants was analyzed by the SAVA system. Measurements were recorded on a thermostatically controlled microscope. n=5-8
E. Non-PMN-Mediated Killing To investigate the individual and combined effects of chronic EtOH ingestion and
smoke exposure on pre-PMN recruitment defenses in the lungs, an in vivo bactericidal
assay was conducted. At exactly one hour after rats were infected transtracheally with S.
pneumoniae, they were euthanized and their lungs homogenized. Plate counts were
performed on the lung homogenates to determine the percentage of bacteria killed within
each rat’s lungs (Figure 22). Five weeks of EtOH ingestion alone significantly
suppressed bacterial killing, such that the mean percentage of killing for the sham-EtOHs
was significantly lower than that for the sham-pairs (5% vs. 31%, p = 0.001). Smoke
exposure alone had no effect on early pre-PMN killing (26% vs. 31% for sham-pairs, p =
0.65). Unexpectedly, however, the addition of concurrent smoke exposure to EtOH
77
ingestion significantly increased, rather than decreased, this non-PMN-mediated
pulmonary killing (23% vs. 5% for sham-EtOHs, p = 0.016). These results indicate that
smoke exposure may actually negate rather than exacerbate EtOH-induced defects on
non-PMN-mediated pulmonary host defenses.
Figure 22 – Concurrent Smoke Exposure Negates Chronic EtOH-Induced Impairment of Non-PMN-Mediated Killing
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
10
20
30
40p = 0.001
p = 0.016
Perc
enta
ge o
f Kill
ing±
SEM
Figure 22 An in vivo bactericidal assay was performed to determine the effect of smoke exposure ± EtOH ingestion on non-PMN-mediated killing. Rats were infected transtracheally with live pneumococci. The lungs were removed and homogenized at one hour post-infection. Plate counts were performed to determine the percentage of pneumococci killed within the lungs during the one hour experimental infection. n=10-11
F. Quantification of Bactericidal Factors
Two key bactericidal proteins were quantified to determine whether the
modulations on non-PMN-mediated killing were due to alterations in the rats’ pulmonary
levels of those bactericidal factors. Lysozyme activity in lavage fluid collected from
uninfected rats from each treatment group is shown in Figure 23. Although smoke
78
exposure alone slightly increased this activity, there were no significant differences
among the mean levels of the four groups.
Figure 23 – No Alterations of Pulmonary Lysozyme Levels by Smoke Exposure and EtOH Ingestion
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
5000
10000
15000
20000Ly
sozy
me
(U/m
g pr
otei
n)±
SEM
Figure 23 Lysozyme concentrations were quantified in lavage fluid from uninfected rats. n=7
The second bactericidal protein examined in lavage fluid was lactoferrin.
Surprisingly, the animals receiving EtOH alone had significantly higher concentrations of
lactoferrin in their lungs compared to sham-pairs (p < 0.001) as shown in Figure 24.
Smoke exposure alone had no effect, but concurrent smoke exposure resulted in the loss
of this EtOH-induced increase in lactoferrin (p < 0.001).
79
Figure 24 – EtOH Ingestion Drastically Increases Pulmonary Lactoferrin Levels
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
20
40
60
80
100p < 0.001 p < 0.001
Lact
ofer
rin (n
g/m
g pr
otei
n)±
SEM
Figure 24 Quantification of lactoferrin in lavage fluid from uninfected rats. n=7-8
To determine whether EtOH ingestion alone so dramatically increased lactoferrin
levels due to release from intracellular pools, cellular damage was assessed in the lungs
by quantifying lactate dehydrogenase (LDH) in the lavage fluid (Figure 25). This was
not the case, as the results from the LDH assay were inversely related to the lavage fluid
lactoferrin levels. The mean concentration of LDH was significantly lower, rather than
higher, for the sham-EtOHs vs. the sham-pairs (p = 0.02) and the smoke-EtOHs (p =
0.004).
80
Figure 25 – Cellular Release of LDH Does Not Explain the EtOH-Induced Increase in Lactoferrin
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
50
100
150
200
p = 0.02
p = 0.004
LDH
(mU
/mg
prot
ein)
± SE
M
Figure 25 Quantification of LDH in lavage fluid from uninfected rats. n=7-8
G. Macrophage Phagocytosis
To evaluate the ability of macrophages to phagocytose pneumococci in the lungs
after chronic EtOH ingestion and smoke exposure, a novel flow cytometric assay was
developed to measure the in vivo uptake of bacteria by alveolar macrophages. In this
assay, macrophages collected from the rats’ lungs 15 minutes after infection with
fluorescently labeled pneumococci were analyzed by flow cytometry for uptake of the
fluorescent bacteria. Macrophages from smoke-exposed animals in both feeding groups
had a greatly increased fluorescent intensity than macrophages from sham-exposed
animals (Figure 26). However, this was shown to be due to a smoke-induced increase in
macrophage autofluorescence since the fluorescent intensity was similar to that of
macrophages collected from an uninfected smoke-exposed rat.
81
Figure 26 – Smoke Exposure Enhances Macrophage Autofluorescence
Bacterial Fluorescence
Forw
ard
Scat
ter
Infected Smoke-exposed Rat
Infected Sham-exposed Rat
Figure 26 Flow cytometry was used to compare the MFI of macrophages from a sham- and smoke-exposed rat infected with fluorescent pneumococci and an uninfected smoke-exposed rat.
Uninfected Smoke-exposed Rat
Although this shift in fluorescent intensity precluded use of the assay in smoke-
exposed rats, data from the sham-exposed animals proved interesting. Consistent with
the literature, few macrophages were positive for fluorescent bacteria in either group of
sham-exposed animals (Figure 27A). However, macrophages from the sham-EtOH rats
phagocytosed significantly fewer pneumococci than their pair-fed counterparts, as
determined by a significant increase in their mean fluorescent intensity (MFI) (Figure
27B; p < 0.001) and their phagocytic index (Figure 27C; p = 0.02).
82
Figure 27 – EtOH Ingestion Suppresses Macrophage Phagocytosis
Sham-EtOH Sham-Pair0
1
2
3
4
5
Perc
enta
ge o
f Pha
gocy
tosi
ngM
acro
phag
es±
SEM
Sham-EtOH Sham-Pair0
250
500
750
1000
1250
1500p < 0.001
Fluo
resc
ent I
nten
sity
± SE
MSham-EtOH Sham-Pair
0
1000
2000
3000
4000
5000
6000p = 0.02
Phag
ocyt
ic In
dex±
SEM
A B C
Figure 27 A phagocytosis assay was performed to determine the effect of smoke exposure and EtOH ingestion on macrophage uptake of pneumococci in vivo. Rats were infected with fluorescent pneumococci. Fifteen minutes after infection BAL was performed. Flow cytometry was used to determine the percentage of macrophages that had phagocytosed pneumococci (A) and the MFI (B). Phagocytic index was calculated by multiplying the percentage of macrophages containing bacteria by their MFI (C). n=8
To confirm that EtOH ingestion suppresses macrophage phagocytosis of a Gram-
positive bacterium more readily taken up by macrophages, the assay was repeated in
EtOH-fed and pair-fed sham-exposed rats using fluorescently labeled S. aureus. Results
of that assay indicated that EtOH ingestion significantly increased rather than reduced
macrophage phagocytosis (Figure 28). However, when the percentage of macrophages
containing bacteria was determined by manual counts of cytospin slides from each animal
by two independent observers, the means for the two feeding groups were similar.
Furthermore, the flow cytometry data did not correlate with the values determined by
light microscopy for both the sham-EtOH (-0.086, p = 0.92) and sham-pair (0.029, p =
1.0) rats (Table 3).
83
Figure 28 – Flow Cytometry Results Indicate EtOH Ingestion Increases Macrophage Phagocytosis of S. aureus
Figure 28 To confirm EtOH ingestion decreases macrophage phagocytosis, the phagocytosis assay was repeated in sham-exposed rats infected with fluorescent staphylococci. n=7-8
Sham-EtOH Sham-Pair0
5
10
15
20
25
30
35p = 0.031
Perc
enta
ge o
f Pha
gocy
tosi
ngM
acro
phag
es±
SEM
Table 3 – Comparison of Macrophage Phagocytosis Values
Rat Flow
Cytometry Manual Counts Rat
Flow Cytometry
Manual Counts
1 53 4 1 3 22
2 3 20 2 5 11 3 6 15 3 29 13
4 22 16 4 9 16 5 50 28 5 18 32
6 39 36 6 7 14 Avg. 24 20 Avg. 7 18
Percentages of Macrophages from Sham-EtOHs Containing S. aureus
Percentages of Macrophages from Sham-Pairs Containing S. aureus
84
H. Additional Macrophage Functions
Macrophages isolated from rats in the four treatment groups then were stimulated
with either unencapsulated S. pneumoniae or phorbol myristate acetate (PMA) to
determine the effects of smoke and/or EtOH on several of their functions. A
chemiluminescence assay was performed to quantify oxidative burst. Regardless of
smoke or sham exposure, macrophages from EtOH-fed rats produced more oxygen
radicals than pair-fed rats at both 10 and 20 minutes after being stimulated by
pneumococci (Figure 29). The differences in oxidative burst between all EtOH-fed and
all pair-fed animals at 10 and 20 minutes after stimulation were statistically significant (p
= 0.023 and p = 0.032, respectively). After 20 minutes, the oxidative burst continued to
rise at a similar rate and then plateaued at 40-50 minutes in all treatment groups except
the smoke-pairs. Smoke exposure alone suppressed the oxidative response such that
smoke-pairs produced the lowest amounts of reactive oxygen species from 30 to 60
minutes after stimulation. PMA stimulated a more rapid and robust oxidative burst than
did pneumococci in macrophages from all groups of rats. Disregarding the effect of diet,
smoke exposure hindered the oxidative response by PMA-stimulated cells, such that
macrophages from smoke-exposed rats produced fewer oxygen radicals than those from
sham-exposed rats for all time points (Figure 30).
85
Figure 29 – Smoke Exposure Alone Suppresses Oxidative Burst
in Macrophages Stimulated by Pneumococci
0 10 20 30 40 50 600
50
100
150
200
250
300
350 Smoke-EtOH
Sham-Pair
Smoke-PairSham-EtOH
Time (min.)
Che
milu
min
esce
nce
(ligh
t uni
ts)±
SEM
Figure 29 A chemiluminescence assay was utilized to identify the effects of smoke exposure and EtOH ingestion on oxidative burst in macrophages. Macrophages incubated with unencapsulated pneumococci were analyzed in a luminometer every 10 minutes up to one hour. n=5-8
Figure 30 – Smoke Exposure Hinders Oxidative Burst in PMA-Stimulated Macrophages
0 10 20 30 40 50 600
100200300400500600700800 Smoke
Sham
Time (min.)
Che
milu
min
esce
nce
(ligh
t uni
ts)±
SEM
p = 0.039
p = 0.038p = 0.048
p = 0.042 p = 0.023
Figure 30 The same chemiluminescence assay was used to measure oxidative burst in macrophages stimulated with PMA. Macrophages from smoke-exposed animals produced significantly fewer oxygen radicals than from sham-exposed animals. n=12-15
86
Degranulation was measured by quantifying lysozyme release one hour after
macrophage stimulation. When pneumococci were used as the stimulus, there were no
statistical differences in lysozyme release among the four treatment groups, even though
the lysozyme level was twice as high for sham-EtOH rats (Figure 31). PMA stimulation
produced no detectable levels of lysozyme release by alveolar macrophages from any of
the rats (data not shown).
Figure 31 – Neither EtOH nor Smoke Significantly Alters Degranulation by Macrophages Stimulated with Pneumococci
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
1020304050607080
Lyso
zym
e (U
/ml)±
SEM
Figure 31 Lysozyme was quantified in media from macrophages stimulated in vitro with pneumococci for one hour. n=6-7
Macrophage chemokine release was evaluated by measuring CINC-1 and MIP-2
levels in the media five hours after stimulation (Table 4). When stimulated by
pneumococci, macrophages from both smoke- and sham-exposed rats that consumed the
EtOH diet released less of both chemokines than their pair-fed counterparts. Smoke
exposure made no difference in CINC-1 and MIP-2 levels when comparing smoke-pairs
and sham-pairs.
87
When macrophages were stimulated with PMA, EtOH ingestion alone resulted in
the release of significantly higher amounts of CINC-1 (p = 0.04). However, even though
smoke exposure alone did not affect CINC-1 production, the addition of smoke to EtOH
ingestion reversed the EtOH effect. The smoke-EtOH rats therefore released CINC-1
levels comparable to those of the control group receiving neither smoke nor EtOH.
Neither EtOH ingestion nor smoke exposure alone significantly modified macrophage
release of MIP-2, but when administered in combination, they resulted in a >2-fold
reduction in MIP-2 release than that for any of the other treatment groups.
Table 4 – Chemokine Production by Macrophages Stimulated with Pneumococci or PMA
Pneumococci PMA CINC-1 pg/ml
± SEMMIP-2 pg/ml
± SEMCINC-1 pg/ml
± SEMMIP-2 pg/ml
± SEMSmoke-EtOH 39 ± 20 44 ± 16 13 ± 6 57 ± 15
Smoke-Pair 119 ± 61 173 ± 89 26 ± 12 132 ± 46 Sham-EtOH 19 ± 18 12 ± 12 52 ± 16a,b 141 ± 60
Sham-Pair 92 ± 91 186 ± 186 18 ± 6 135 ± 52 n=4-8 aSham-EtOH vs. Sham-pair, p = 0.04. bSham-EtOH vs. Smoke-EtOH, p = 0.017.
I. Opsonic Deposition Assay
An in vivo assay was used to quantify the deposition of the three major opsonic
proteins on the surface of S. pneumoniae to determine if opsonization of intrapulmonary
bacteria is modified by chronic EtOH ingestion and/or smoke exposure. Pneumococci
recovered from the rats’ lungs were labeled with fluorochrome-tagged antibodies specific
for rat complement C3, CRP, or SP-D, and analyzed by flow cytometry. The results in
88
Figure 32 show that in the absence of smoke exposure, significantly fewer pneumococci
were coated with either C3 or SP-D within the lungs of rats consuming EtOH than in
their pair-fed controls (*p = 0.017 and **p = 0.01, respectively). CRP binding also was
somewhat lower in sham-EtOHs than sham-pairs, but the difference did not reach
statistical significance (p = 0.09). Smoke exposure did not alter opsonization when
administered alone, and it did not exacerbate the EtOH-induced decrease in opsonin
deposition, such that there were no differences in C3, CRP, or SP-D binding between
EtOH-fed and pair-fed rats that were smoke-exposed.
Figure 32 – EtOH Ingestion Reduces C3 and SP-D Deposition on Bacteria
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
10
20
30
40CRPSP-D
C3
*
*
**
**
Perc
enta
ge o
f Ops
oniz
edPn
eum
ococ
ci±
SEM
Figure 32 Mean percentage of bacteria with bound C3, CRP, or SP-D isolated from the lungs of rats. Values were quantified by transtracheally infecting rats and lavaging the organisms from their lungs 30 minutes later. Flow cytometry then was used to determine the percent of bacteria bound by each opsonin. C3 and SP-D values for sham-EtOH rats significantly lower than that for sham-pair rats (*p = 0.017 and **p = 0.01, respectively). n=7-8
89
To some extent the results of the opsonin deposition assay correlated well with
levels of the same three opsonic proteins measured by ELISAs in lavage fluid of
uninfected rats from each treatment group. Pulmonary C3 levels were significantly
decreased in EtOH-fed compared to pair-fed rats, whether or not they had been exposed
to smoke (Figure 33; *p = 0.026 and **p = 0.004). There was no significant EtOH-
induced decrease in the levels of SP-D. CRP levels were similar for all of the treatment
groups.
Figure 33 – EtOH Ingestion Decreases C3 Basal Levels
Smoke EtOH Smoke Pair Sham EtOH Sham Pair0
2
4
6
8
CRPSP-D
200
400
600
800 C3
**
**
**
Ops
onic
Pro
tein
Lev
els±
SEM
( μg/
mg
prot
ein)
Figure 33 Opsonic protein levels were quantified in lavage samples from
uninfected rats by ELISAs. C3 values for both EtOH-fed groups were significantly lower than their pair-fed counterparts (*p = 0.026 for smoke-exposed; **p = 0.004 for sham-exposed). n=6-8
90
J. PMN-Mediated Killing
The PMN-mediated killing assay was conducted to confirm that chronic, like
acute EtOH ingestion with or without concurrent smoke exposure impairs PMN killing
ability. At exactly five hours after PMNs were recruited to the rats’ lungs by LPS
instillation plus one hour after the rats were infected transtracheally with S. pneumoniae,
they were euthanized and plate counts of their lung homogenates were compared to those
of control rats sacrificed immediately after infection. This was done to determine the
percentage of bacteria killed when PMNs were present in each rat’s lungs (Figure 34).
Chronic EtOH ingestion dramatically impaired this PMN-mediated pneumococcal killing
(p = 0.01), such that the majority of sham-EtOHs (6 of 7 rats) had pneumococcal growth
in their lungs shown as negative killing. Smoke exposure did not exacerbate, but rather
abolished this detrimental effect of EtOH. Smoke-EtOHs killed significantly more
pneumococci in their lungs than sham-EtOHs (p = 0.026), making their PMN bactericidal
activity similar to that of rats that had not been exposed to either insult. Based on manual
counts of cytospin slides, neither smoke exposure nor EtOH ingestion significantly
altered PMN recruitment to the lungs, as all four rat groups averaged 85-90% of the
pulmonary cells in their bronchoalveolar lavage as PMNs.
91
Figure 34 – Concurrent Smoke Exposure Negates EtOH-Induced Defect in PMN-Mediated Killing
Smoke EtOH Smoke Pair Sham EtOH Sham Pair-40-30-20-10
010203040 p = 0.026 p = 0.01
Perc
enta
ge o
f Kill
ing
Figure 34 To determine the effect of smoke exposure and EtOH ingestion on PMN killing ability, an in vivo bactericidal assay was performed after LPS-induced PMN recruitment. Rats were infected transtracheally with pneumococci and the percentage of bacterial killing was determined one hour later by performing plate counts on lung homgenates. n=7-8
K. Pulmonary and Systemic Chemokine Levels
Pulmonary chemokine levels were measured by ELISA in the lung homogenates
from rats used in the PMN-mediated bactericidal assay (Table 5). Serum CINC-1, but
not MIP-2, values also were measured from these same rats because CINC-1, as opposed
to MIP-2, leaves the lung tissue where it is produced and enters into the systemic
circulation [55]. All four treatment groups had similar concentrations of MIP-2 and
CINC-1 in their lung homogenates. EtOH ingestion in the absence of smoke exposure
slightly decreased CINC-1 serum levels, but the decrease was not statistically significant
(p = 0.25). Once again, the EtOH-induced decrease was not seen when the rats were also
92
smoke-exposed even though smoke exposure alone did not increase serum CINC-1 levels
in pair-fed rats.
Table 5 – Chemokine Values from Lung Homogenates and Serum
Pulmonary MIP-2 (pg/ml)
Pulmonary CINC-1 (pg/ml)
Serum CINC-1 (pg/ml)
Smoke-EtOH 4309 6219 1558 Smoke-Pair 3813 5509 1551
Sham-EtOH 4788 5993 944 Sham-Pair 4155 6176 1334
L. PMN Phagocytosis
To determine if smoke- and/or EtOH-induced modifications in PMN phagocytosis
are responsible for the differences in intrapulmonary killing in the presence of PMNs, a
phagocytosis assay was performed to quantify the in vivo uptake of S. pneumoniae by
PMNs pre-recruited into the rats’ lungs for five hours. Rats infected with CFSE-labeled
pneumococci were sacrificed exactly 15 minutes later and PMNs in their bronchoalveolar
lavage fluid were identified with fluorescent antibodies and analyzed by flow cytometry.
The percentage of phagocytosing PMNs was unchanged by EtOH-ingestion and/or smoke
exposure (Figure 35A). However, EtOH ingestion without smoke exposure significantly
decreased the relative number of organisms being ingested by PMNs as indicated by the
decreased MFI in Figure 35B. This EtOH-induced decrease did not affect the phagocytic
index for this was not statistically different compared to the sham-pairs (Figure 35C). No
EtOH effect was detected among the smoke-exposed animals, and there were no
93
significant differences in either the MFI or the phagocytic index for the smoke-exposed
rats vs. their sham-exposed counterparts.
Figure 35 – Neither EtOH Ingestion nor Smoke Exposure Affects PMN Phagocytic Activity
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
10
20
30
40
50
60
70
80
% o
f Pha
gocy
tosi
ng P
MN
s±
SEM
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
1000
2000
3000p = 0.004
Fluo
resc
ent
Inte
nsity
± S
EM
Smoke-EtOH Smoke-Pair Sham-EtOH Sham-Pair0
50000
100000
150000
200000
Phag
ocyt
ic In
dex±
SEM
C
A B
Figure 35 A PMN phagocytosis assay was performed to determine the effect of smoke exposure and EtOH ingestion on PMN uptake of pneumococci. After LPS-induced PMN recruitment rats were infected with fluorescent pneumococci. BAL was then performed on each rat at 15 minutes post-infection. Flow cytometry was used to determine the percentage of PMNs that had phagocytosed labeled bacteria (A) and the MFI (B). The phagocytic index was calculated by multiplying the percentage of PMNs containing bacteria by their MFI (C). n=6-8
M. Mortality Study An intranasal mortality study was conducted on 8 rats from each treatment group.
Each rat was infected intranasally with a lethal dose of S. pneumoniae and then
94
monitored for 9 days. Bacteremia was quantified by plate counts on each rats’ blood
sample taken on days 2 and 5 post-infection. Table 6 shows the bacteremia and mortality
results for each treatment group.
Table 6 – Bacteremia and Mortality Results from an Intranasal Challenge
Bacteremia Day 2 Bacteremia Day 5 Mortality Smoke-EtOH 1/8 (13%) 0/4 (0%) 4/8 (50%)
Smoke-Pair 0/8 (0%) 0/8 (0%) 0/8 (0%) Sham-EtOH 2/8 (25%) 0/6 (0%) 4/8 (50%)
Sham-Pair 3/8 (38%) 1/5 (20%) 4/8 (50%)
All rats that developed bacteremia eventually succumbed to the infection. All but
one of them that had bacteremia on day 2 died from the infection before blood samples
were taken again on day 5, and no other rats developed positive blood cultures on day 5.
Bacteremia was not detected in any of the surviving rats, but it was also undetected on
days 2 and 5 in half of the rats that died. Sham-exposed rats consuming the EtOH diet
had a similar bacteremia rate as their pair-fed controls on day 2 of infection. Only one of
the 16 smoke-exposed rats had organisms in its blood on day 2. The mortality rate was
50% for all treatment groups except for the smoke-pairs. Unexpectedly, this group never
developed bacteremia and all the rats survived the intranasal challenge.
95
VI. DISCUSSION
A. Pneumococcal Colonization and Movement Studies
The pneumococcal infection process begins with adherence of the bacteria to
epithelial cells of the nasopharynx. One potential reason smokers and alcohol abusers
have a higher incidence of developing pneumococcal pneumonia is an increase in
colonization. Smoking promotes pneumococcal binding to buccal epithelial cells and
reduces mucosal IgA levels, resulting in a higher risk of being colonized by S.
pneumoniae [219-223]. No studies have elucidated the effects of alcohol on
nasopharyngeal colonization by pneumococci, but alcohol ingestion may further increase
this occurrence. It was shown by flow cytometric analysis that mice ingesting EtOH for
two weeks had reduced numbers of CD4+ T-cells in the spleen and thymus [183] and this
was linked to EtOH inducing apoptosis by culturing murine thymocytes in the presence
of EtOH [184]. A decrease in CD4+ T-cells may enhance pneumococcal colonization of
the nasopharynx for it was recently reported that mice deficient in CD4+ T-cells had
decreased immunity to pneumococcal colonization when challenged intranasally [70].
We therefore hypothesized that smoke exposure would increase pneumococcal
colonization and chronic EtOH ingestion would exacerbate this problem.
When pneumococci were quantified in the nasal washes of rats infected
intranasally, neither smoke exposure nor EtOH ingestion increased nasopharyngeal
colonization. These results did not coincide with in vitro studies that found increased
bacterial adherence to buccal epithelial cells from smokers and cultured cells exposed to
cigarette smoke extract [219]. These results also do not correlate with several other
96
studies that detected higher rates of pneumococcal colonization in the nasopharynx of
smokers vs. nonsmokers [216-218]. The human studies are different from our current
experiment where they only determined the percentage of nasopharyngeal cultures that
were positive or negative for pneumococci. These studies did not quantify colonization
in human subjects at a certain time after inhaling S. pneumoniae. The inoculum used to
infect the rats was too concentrated to detect any smoke- and EtOH-induced increases in
colonization because all of the sham-pair control rats remained colonized. The amount of
pneumococci the rats inhaled is more than what people are normally exposed to in the
natural environment.
Despite not detecting any smoke- or EtOH-induced differences in colonization, it
was important to evaluate potential therapies to reduce nasal carriage of pneumococci
thereby reducing pneumococcal access to the lungs of all patients. Therapies to protect
adults from pneumococcal colonization are nonexistent, and the heptavalent vaccine for
children is limited by only reducing carriage rates for seven of the >90 different serotypes
[100-102]. Several pneumococcal proteins, including pneumococcal surface proteins A
and C (PspA and PspC), have been tested as potential vaccine antigens. Mice immunized
with PspA or PspC were partially protected against pneumococcal colonization, leading
to reduced development of pneumonia and bacteremia [62,63,282,285]. However, an
effective pneumococcal protein vaccine must provide protection against numerous
serotypes. The structure of PspA is highly variable among different pneumococcal
strains [113], and PspC is found on only 75% of pneumococci [118]. Therefore, like the
capsular polysaccharide vaccines, immunizing with these alternative protein targets
elicits limited protection.
97
A newly recognized surface-exposed pneumococcal protein called pneumococcal
protective protein A (PppA) may have great potential as a vaccine candidate. Intranasal
vaccination with recombinant PppA (rPppA) induced both local and systemic IgG and
IgA anti-PppA antibody responses [64]. Dr. Bruce Green, who first discovered the
protein, showed PppA is conserved among many different pneumococcal serotypes, and
immunizing mice intranasally with the rPppA reduced nasopharyngeal carriage of a
number of different strains [64]. In light of the success of Green’s experiment, we
obtained rPppA from him, predicting that intranasal immunization with the protein would
reduce pneumococcal colonization in our rats. Before the rPppA vaccine was used in
smoke- and EtOH-exposed rats, however, it was evaluated in unexposed control rats.
After three immunizations, pneumococcal colonization was measured after an intranasal
infection. Unlike what occurred in the mouse study, the rPppA vaccine failed to
significantly reduce pneumococcal colonization in rats. This could be explained by the
fact that although the majority of vaccinated rats had higher IgG and IgA titers than
unvaccinated rats, these antibody responses were not nearly as robust as what Green
reported in vaccinated mice [64]. Dr. Green suggested this might be due to instability of
the rPppA protein (personal communication). We intended to repeat the study using
freshly prepared rPppA, but Dr. Green has decided not to purify and pursue the protein
further.
Once colonization has been established, pneumococcal movement from the
nasopharynx to the lungs is required for development of pneumonia. Alcohol and
smoking both cause defects in mucociliary clearance, allowing organisms to enter the
sterile environment of the lungs. Alcohol consumption blunts the ciliary response,
98
decreases the gag reflex, and increases the risk of aspiration [136,143,144,286]. Smoking
is also harmful to mucociliary clearance by damaging ciliated cells and down-regulating
the CBF [224-227]. We therefore hypothesized both insults would aid in pneumococcal
invasion of the lungs.
To determine if chronic EtOH ingestion and/or smoke exposure increased
pneumococcal movement into our rats’ lungs, S. pneumoniae was tracked from the
nasopharynx to the lungs after an intranasal inoculation. EtOH ingestion alone slightly
increased pneumococcal movement to the lungs. This is consistent with a previous study
that quantified pneumococcal movement into the lungs of chronic EtOH-fed rats four
hours after an intranasal infection [145].
Contradictory to those previous findings and to our hypothesis, smoke exposure
alone appeared to inhibit rather than exacerbate pneumococcal entry into the lower
respiratory tract. This is evidenced by fewer positive cultures of the trachea, carina, and
lungs as well as lower mean numbers of organisms at each location in smoke-exposed
rats. Smoke exposure also reversed any EtOH-induced defect present in the sham-
exposed group, for EtOH-fed and pair-fed rats exposed to smoke had nearly identical
counts. The reason fewer pneumococci were seen in the airways and lungs of smoke-
exposed animals is smoke exposure dramatically increased their CBF regardless of EtOH
ingestion.
The results from the smoke-exposed animals are quite interesting, considering
that smoke exposure has been reported to decrease CBF through the activation of PKC
[224]. However, previous research in our laboratory suggested that smoke-exposed rats
had a higher baseline CBF than sham-exposed rats [145]. Yet this same experiment also
99
showed that concurrent smoke exposure exacerbated the effects of EtOH, causing
increased pneumococcal movement to the lungs four hours after an intranasal infection
[145]. The increased movement by both insults was not detected in the present
experiment at one week post-infection. The different concentrations of bacteria used to
infect the rats and the length of time the experiments were conducted after infection
might account for these conflicting results. The inoculum from the previous study was
two logs higher than our inoculum and bacteria in the lungs at four hours after infection
may be due to drainage from the nasopharynx. Whereas at one week post-infection this
initial drainage is no longer present and differences in pneumococcal infection of the
lungs may be due to alterations in pulmonary killing as demonstrated.
Two therapies to reduce the number of colonized pneumococci from entering the
lungs were tested. Salbutamol and formoterol are β2-agonists currently used as
bronchodilators in patients who suffer from asthma or COPD. β2-agonists are also
known to stimulate ciliary beating by activating PKA. We hypothesized that β2-agonist
medication would enhance mucociliary clearance in all treatment groups and perhaps
negate the EtOH-induced increase in pneumococcal movement into the lungs.
Salbutamol and formoterol were tested first in normal control rats infected intranasally
and medicated for one week by inhalation. Both β2-agonists were efficient in reducing
pneumococcal movement into the lower respiratory tract. Formoterol was chosen to treat
smoke- and EtOH-exposed rats, since it is a longer-acting β2-agonist that required less
frequent administration and it is now more commonly used in patients than the shorter-
acting salbutamol.
100
As hypothesized, formoterol prevented pneumococcal penetration into the lungs
of all EtOH-ingesting rats, but it did not alter the bacterial counts in smoke-exposed
animals. The majority of smoke-exposed rats had little bacterial movement through their
airways whether or not they received the β2-agonist. This is similar to the results from
the initial movement study discussed earlier. Interestingly, the beneficial effect of
formoterol in the normal control rats was not seen in the sham-pair animals. Stress
induced by pair-feeding may have interfered with the response to the medication. To
verify if this is true, further testing should be done in rats consuming unlimited quantities
of the liquid control diet.
Formoterol medication significantly increased the CBF in the sham-EtOH rats,
which correlates with the decreased pneumococcal movement to their lungs. However, a
formoterol up-regulation of CBF in EtOH-fed animals contradicts a previous study that
used the same rat model and showed chronic EtOH exposure desensitizes ciliated cells,
preventing their stimulation by the β2-agonist isoproterenol [145]. This difference may
be due to the previous study being conducted in vitro rather than in vivo. In the previous
study, ciliated cells were stimulated once in vitro and then the change in CBF was
analyzed whereas our current study exposed the ciliated cells to formoterol for one week
in vivo and then measured the baseline CBF.
B. Non-PMN Pulmonary Defenses
Before the arrival of recruited PMNs, a significant number of pneumococci that
reach the lungs are rapidly killed by various pulmonary defenses. Prominent among
these defenses are the alveolar lining fluid and resident alveolar macrophages. Alveolar
101
lining fluid is known to exhibit bactericidal properties consisting of lysozyme, lactoferrin,
defensins, and surfactant. This fluid also contains several opsonic proteins including
complement C3, CRP, and SP-D that coat the bacteria and promote phagocytosis.
Alveolar macrophages coordinate the inflammatory response by releasing cytokines and
chemokines to recruit and activate PMNs. Although not nearly as efficient as PMNs,
alveolar macrophages also can phagocytose pneumococci and eliminate the pathogen
through their production of an oxidative burst and degradative enzymes. Numerous
studies have shown that these various host defenses are affected by chronic EtOH or
smoke exposure. However, no in vivo studies have been performed previously to
determine the combined effects of smoke and EtOH on these pulmonary defenses in
relation to pneumococcal clearance from the lungs.
We hypothesized that both smoke and EtOH would impair non-PMN-mediated
killing, and that both insults together would decrease this host defense even further.
Using an in vivo bactericidal assay we demonstrated that half of the sham-EtOH rats had
bacterial growth in their lungs after one hour of a transtracheal infection. This severe
EtOH-induced decrease in pneumococcal clearance from the lungs corroborates previous
studies that showed EtOH inhibits the bactericidal activities of lysozyme and surfactant
[33,149]. In addition, it supports in vitro studies showing EtOH suppresses macrophage
phagocytosis and oxidative burst [164,165,168].
Contrary to our hypothesis, concomitant exposure of the rats to cigarette smoke
negated, rather than exacerbated, the EtOH-induced defect in pulmonary pneumococcal
killing. Smoke-exposed EtOH-fed rats killed similar numbers of pneumococci in their
lungs as their smoke-exposed pair-fed controls, and this activity was no different than
102
that in the sham-pair controls. Human smokers are known to have higher levels of
lysozyme, lactoferrin, and defensins in their lungs than nonsmokers [228-230], and this
could potentially contribute to the counteractive effect of smoke exposure on the EtOH-
induced defect in bactericidal activity within the lungs. However, smoke hinders
surfactant production [234] and free fatty acids in surfactant are thought to be its most
potent bactericidal factors against pneumococci [33]. Even though the effects of smoke
on macrophages is still being debated, some studies have shown smoke enhances
macrophage activity [146,245]. If so, the beneficial effects of smoke on macrophages
may out-weigh its negative effects on other pulmonary defenses and, therefore, overcome
the EtOH-induced defect on non-PMN-mediated killing.
Due to these initial results on the effects of EtOH ingestion and smoke exposure
on non-PMN-mediated killing, we had to reformulate our hypothesis to be: the
detrimental effects of chronic EtOH ingestion on innate pulmonary anti-pneumococcal
defenses are reduced by concurrent smoke exposure. To elucidate the reason for this
phenomenon, we next analyzed bactericidal factors, alveolar macrophage activity, and
opsonic proteins in smoke-exposed ± EtOH-fed rats. Lysozyme and lactoferrin, the two
most abundant pulmonary bactericidal proteins, were quantified in the airways of
uninfected animals. Although acetaldehyde from metabolized EtOH has been shown to
partially inhibit lysozyme activity [149] whereas smoking increases lysozyme
concentrations in the lungs [228,229], no differences in lysozyme activity were detected
among the four rat treatment groups. Lactoferrin concentrations however, were
dramatically increased in sham-exposed rats consuming the EtOH diet. As seen for the
non-PMN-mediated killing assay, concurrent smoke exposure negated this EtOH effect
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since smoke-EtOH rats had similar concentrations of lactoferrin in their lungs as their
smoke-pair counterparts and the sham-pair controls. One caveat for this assay is the
lactoferrin kit was not able to quantify apolactoferrin separately from lactoferrin, making
it difficult to relate this data to the pneumococcal killing assay. Another limitation for
this kit is it quantifies human lactoferrin, but all the rat samples were analyzed using this
same kit. Unfortunately, no lactoferrin kits that measure rat lactoferrin exist.
To determine if EtOH ingestion causes damage to pulmonary epithelial cells,
resulting in their spontaneous release of lactoferrin, LDH levels were measured. LDH is
an intracellular enzyme found in most cells and is commonly used as a marker for
cellular damage. Surprisingly, the LDH results were actually the reverse of those from
the lactoferrin data, in that sham-EtOH rats had significantly lower rather than higher
levels of LDH than either the sham-pair or smoke-EtOH rats. While intriguing, these
results suggest that cellular damage was not responsible for the EtOH-induced increase in
lactoferrin levels. EtOH ingestion is known to interfere with iron homeostasis, leading to
increased iron concentrations that could explain the rise in lactoferrin [150,151]. The
increased availability of free iron could decrease apolactoferrin levels and therefore
reduce the bactericidal activity of this anti-pneumococcal protein in the lungs.
Furthermore, altered iron homeostasis also may contribute to the bacterial growth that
occurred in the EtOH-fed rats’ lungs. More studies need to be performed to quantify
apolactoferrin and iron concentrations in the lungs to elucidate this interesting
phenomenon. Although the results of our assays of pulmonary levels of lysozyme or
lactoferrin could not fully explain the differences in non-PMN-mediated pneumococcal
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killing among the treatment groups, it is possible there are alterations of other bactericidal
factors such as defensins and surfactant fatty acids by smoke and alcohol.
Alveolar macrophages are the first immune cells to encounter S. pneumoniae
when it invades the lungs. In vitro studies found chronic EtOH ingestion and smoke
exposure decrease macrophage phagocytosis separately [168,246,247], although another
study reported that smoke has no effect at all on this macrophage activity [248].
Nonetheless, no studies have reported the effects of smoke and alcohol on macrophage
phagocytosis in vivo. Based on the results from the non-PMN-mediated killing assay, we
hypothesized that EtOH decreases macrophage phagocytosis and that smoke exposure
up-regulates this activity by activating the macrophages, thereby helping to negate the
EtOH defect. To measure the EtOH- and smoke-induced effects on macrophage
phagocytosis, we developed a novel assay to quantify in vivo bacterial uptake by alveolar
macrophages.
Within the sham-exposed rat groups, very little phagocytosis of pneumococci was
detected, agreeing with the scientific literature that macrophages have difficulty taking up
serotype 3 S. pneumoniae due to their heavy polysaccharide capsule. Although little
phagocytosis occurred, EtOH ingestion significantly hindered this macrophage function,
as evidenced by both a low number of bacterial organisms within each macrophage and a
low overall phagocytic index in the sham-EtOH rats. Suppression of macrophage
phagocytosis in the EtOH-fed rats no doubt contributes to the decreased non-PMN-
mediated pneumococcal killing within their lungs.
Macrophage phagocytosis in the smoke-exposed animals as measured by flow
cytometry was 16x higher than that in the sham-exposed animals and much higher than
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we expected. Smoke exposure greatly increased the percentage of cells fluorescing
brightly in the bacteria channel to an average of 80%. However, based on the
fluorescence of macrophages from an uninfected smoke-exposed rat, this increase was
shown to be due to enhanced autofluorescence rather than actual bacterial uptake.
Alveolar macrophages from human smokers and smoke-exposed rats also have been
shown in the literature to emit augmented autofluorescence [287,288]. This result
precluded us from using our assay to accurately measure the effects of smoke exposure
on macrophage phagocytosis in the rats’ lungs. Additional experiments are needed to
overcome this problem, either by labeling the bacteria with an even brighter fluorescent
stain or quenching the macrophage autofluorescence without affecting their uptake or
quenching the fluorescence of the bacteria.
Because macrophage phagocytosis of pneumococci was so limited in the sham-
exposed rats, we elected to further confirm this EtOH-induced defect in sham-EtOH and
sham-pair rats using fluorescently labeled S. aureus, that are phagocytosed much more
efficiently than pneumococci. S. aureus was labeled with Syto 9 which is an
exceptionally bright green DNA stain that causes the bacteria to fluoresce brighter than
the autofluorescent macrophages. APC-Cy7 was replaced with Syto 9 because it does not
require a specific biotinylated antibody to effectively label bacteria, and we were unable
to find such an antibody that would bind to our staphylococcal strain.
The percentage of macrophages phagocytosing S. aureus was much higher in both
feeding groups than what was determined in the previous experiment for S. pneumoniae.
Contradicting our previous results, however, the flow cytometric results showed that
chronic EtOH ingestion increased instead of decreased macrophage phagocytosis of
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staphylococci. This was not confirmed by the manual counts of cytospin slides, as they
showed no difference in phagocytosis between the two feeding groups. Flow cytometry
results from more than half of the rats in each group did not correlate with the manual
counts by light microscopy. It is not completely understood why these results occurred.
Macrophages exhibit the highest autofluorescence at the same wavelength as the Syto 9
fluorescence, so autofluorescence may have contributed to the overestimations by flow
cytometry. The underestimations by flow cytometry may be due to the sensitivity of Syto
9 to acidic conditions. Bacteria stained with Syto 9 and incubated in buffers with a pH
<5 for 15 minutes resulted in the decrease of Syto 9 fluorescence. It is known within
phagocytic cells that phagosomes become acidic (pH 3.5-4.0) upon phagosome/lysosome
fusion to help eliminate bacteria. Most of the underestimations by flow cytometry
occurred in the sham-pair rats, which suggests that EtOH ingestion may either suppress
phagosome/lysosome fusion or acidification of the phagocytic vacuole.
In addition to phagocytosis, two bactericidal mechanisms in macrophages isolated
from the lungs of smoke-exposed ± EtOH-fed rats were analyzed. In vitro assays were
performed to measure smoke- and EtOH-induced alterations in oxidative burst and
degranulation in macrophages stimulated with either unencapsulated pneumococci or
PMA. Unencapsulated pneumococci were used because macrophages can phagocytose
this mutant strain much better and produce a greater response than with type 3
pneumococci in vitro. PMA was utilized because it is known to stimulate macrophages,
but unlike bacteria, this non-particulate chemical can freely diffuse into the cell
independent of receptor-mediated phagocytosis.
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EtOH ingestion alone up-regulated the oxidative burst in macrophages stimulated
by either S. pneumoniae or PMA. This increased response eventually leveled off and
oxygen radical production became similar to that for macrophages from sham-pair
controls. These results are contrary to those from previous studies showing alcohol
inhibits the oxidative burst in macrophages stimulated with endotoxin, heat-killed S.
aureus, or PMA [164,165]. These differences may result from the different models and
methods used to measure oxidative burst. One study gave rats a continuous intravenous
infusion of EtOH for seven hours or pair-fed rats for 12-14 weeks. Then the alveolar
macrophages were isolated at three hours after the rats received an intravenous injection
of endotoxin [164]. The other study isolated alveolar macrophages from rats that were
pair-fed for one to four weeks or exposed normal rat alveolar macrophages to various
concentrations of EtOH in culture [165]. Both studies quantified the release of certain
oxygen radical species, such as superoxide anion and nitric oxide, using specific assays to
measure each product. In our experiment we measured total oxygen radical production
within the macrophages using a chemiluminescence assay. Finally, the published studies
measured oxidative burst at one hour to 24 hours after the cells were stimulated, while
our assay measured this activity every 10 minutes up to one hour after stimulation.
A similar trend also occurred with EtOH ingestion in conjunction with smoke
exposure, where smoke-EtOH rats had a more robust early oxidative burst than smoke-
pair rats, but this only occurred when the cells were stimulated with pneumococci. PMA-
stimulated macrophages from smoke-EtOH rats, on the other hand, produced fewer
oxygen radicals than those from sham-EtOH rats. This is likely due to a smoke-induced
decrease in oxidative burst since macrophages from smoke-exposed animals in general
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produced lower amounts of oxygen radicals than the sham-exposed animals in response
to PMA.
Smoke exposure alone delayed the oxidative burst in macrophages stimulated
with pneumococci. This was similar to another study in which macrophages isolated
from human smokers had a suppressed superoxide anion response to endotoxin [289].
However, other studies reported that alveolar macrophages from human smokers exhibit
an increased respiratory burst. Those authors utilized a 20 minute assay without
stimulation [290] and after four days of incubation with endotoxin [248]. Again, as with
the alcohol studies, comparison of these earlier smoking studies with our results is
difficult because of methodological differences.
Along with oxygen radical production, delivery of the lysosomal granule contents
to the target organisms is a major mechanism of macrophage bactericidal activity.
Lysozyme is commonly assayed as a marker for degranulation, because it is present in
the specific granules. Chronic EtOH ingestion and smoke exposure in our rat model did
not cause any significant alterations in lysozyme release in response to pneumococci.
Although macrophages from human smokers contain higher amounts of lysozyme [229],
macrophages from smoke-exposed rats released slightly less lysozyme than macrophages
from sham-exposed rats. Degranulation, as measured by lysozyme release, did not occur
in macrophages from any of the four treatment groups when stimulated by PMA. It is not
clear why this occurred, because PMA is known to cause degranulation in PMNs after 20
minutes [177]. Macrophage degranulation may not be as sensitive to non-receptor-
mediated stimulation, and this process may take longer than one hour.
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The results from the oxidative burst and degranulation experiments do not provide
an explanation for the smoke- and EtOH-induced alterations of non-PMN-mediated
killing. Comparisons between these ex vivo studies and the in vivo bactericidal assay are
complicated, as macrophages may act differently within their native environment than in
vitro. However, it is also difficult to identify alterations in alveolar macrophages without
isolating them from the lungs. Additional experiments are required to identify the effects
of smoke and EtOH on macrophages at the sub-cellular level such as cell signaling, toll-
like receptor expression, and phagolysosome formation.
Opsonization is an important process for eliminating infection by facilitating
phagocytosis of the microorganisms. From the macrophage phagocytosis assay, EtOH
ingestion alone suppressed phagocytosis of pneumococci in the rats’ lungs. To determine
if this is due to impaired opsonization, an assay was performed to measure the in vivo
deposition of complement C3, CRP, and SP-D on pneumococci. Smoke-exposed animals
also were included in this study to determine if concurrent smoke exposure counteracts
the effect of EtOH.
No reported studies have determined the effects of EtOH ingestion on the opsonic
activity of C3 and CRP, and a review of the literature indicates no associations have been
made between smoking and this activity in the lungs. Only one study has reported that
chronic EtOH exposure decreases opsonization by surfactant [154]. In our assay, chronic
EtOH ingestion alone significantly decreased the opsonic deposition of both C3 and SP-D
on pneumococci, as well as slightly reduced in CRP opsonization. These results could
help explain the EtOH-induced suppression of macrophage phagocytosis of
pneumococci. Smoke exposure did not significantly alter the binding of any of the
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opsonic proteins to the bacteria, but within the smoke-exposed rats the EtOH-induced
defect was not apparent. This was depicted by smoke-EtOH rats and their pair-fed
counterparts having similar percentages of pneumococci opsonized by each opsonin.
Smoke and EtOH are known to modify the levels of these opsonic proteins. To
clarify if the differences in deposition were due to altered baseline levels of opsonic
proteins in the lungs, each opsonin was quantified in uninfected rats. Significantly lower
levels of C3 were detected in the sham-EtOH rats which correlates with their reduction in
C3 opsonization. Alcohol has been shown to activate complement through the alternative
pathway, increase the deposition of C3 in the liver, and reduce C3 levels in serum
[155,157,163]. This nonspecific activation and deposition also may be occurring in the
rats’ lungs, resulting in impaired bacterial opsonization. Smoking also causes similar
activation of C3 [237-240], but smoke exposure alone did not alter baseline C3 levels in
the lungs of our rats. Within the smoke-exposed group, C3 was significantly decreased in
the EtOH-fed rats compared to their pair-fed counterparts. However, this decrease was
apparently not great enough to negatively affect C3 opsonization of pneumococci.
Differences in SP-D deposition on the pneumococci were not explained by
changes in basal levels of the protein, for sham-EtOHs, if anything, had even higher
amounts of SP-D as their sham-pair controls. These results indicate that EtOH may
suppress SP-D opsonization by another mechanism, such as altering surfactant protein
function. Finally, minimal CRP concentrations were quantified in the lungs of any of the
rats and there were no smoke- or EtOH-induced effects on these levels. This explains
why fewer pneumococci were opsonized by CRP in all treatment groups.
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The EtOH-induced defects in C3 and SP-D deposition on pneumococci help
explain the decreased macrophage phagocytosis as well as decreased pneumococcal
clearance from the lungs of rats treated with EtOH alone. Although smoke exposure
alone did not affect opsonization, the EtOH-induced defect in opsonic activity was
undetected with concurrent smoke exposure even though EtOH ingestion still reduced the
C3 baseline levels in smoke-exposed rats. Further experiments are needed to determine if
chronic EtOH ingestion and smoke exposure alter other early complement proteins
needed for opsonic deposition such as C2, C4, or Factor B and how these results relate to
intrapulmonary clearance of pneumococci.
C. PMN Functions
PMNs are the primary cellular defense against S. pneumoniae because they can
effectively phagocytose and kill these microorganisms. Several studies have shown that
acute and chronic EtOH ingestion have different effects on PMN function. One week of
EtOH ingestion increases PMN recruitment, but decreases phagocytosis and bactericidal
activity while five weeks of EtOH ingestion does not alter PMN recruitment and
phagocytosis, but it continues to impair PMN-mediated killing. Previous work in the
laboratory showed that peripheral PMNs from short-term EtOH-fed rats were defective in
killing certain strains of pneumococci, but not others [176,177]. Using a more acute
exposure model in our laboratory, PMNs pre-recruited to the lungs of rats consuming
EtOH for one week were deficient in their ability to kill type 3 pneumococci [171]. This
same study also demonstrated that 8 weeks of concurrent smoke exposure prevented this
EtOH-induced decrease from occurring. These results from the one-week model led to
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our hypothesis that chronic EtOH ingestion would reduce PMN-mediated killing in the
lungs and concurrent smoke exposure would negate this EtOH defect.
To examine the effects of chronic EtOH ingestion and smoke exposure on PMN-
mediated killing, we utilized the same in vivo bactericidal assay that was used in the one-
week model. This assay specifically measures PMN-mediated killing of S. pneumoniae
in the lungs because the administration of LPS to recruit the PMNs inactivates
extracellular anti-pneumococcal factors [171]. Similar to the results from the non-PMN-
mediated killing assay, EtOH ingestion alone significantly diminished PMN killing
within the lungs as compared to the sham-pair rats. The severe detrimental effect of
EtOH on PMN killing allowed pneumococci to grow within the lungs one hour after a
transtracheal infection. This result is associated with previous research that showed
PMNs from EtOH-fed rats exhibited decreased bactericidal activity in vitro against
pneumococci [176,177]. As hypothesized, the EtOH-induced defect in PMN-mediated
killing did not occur when the rats were treated by concurrent smoke exposure. The
smoke-EtOH rats killed significantly more pneumococci than their sham-exposed
counterparts. In fact, smoke exposure in conjunction with EtOH ingestion restored
killing to the level of the sham-pair rats. However, consistent with the non-PMN-
mediated killing assay, smoke exposure alone did not increase pneumococcal killing
above the level reported in the sham-pair controls.
PMN recruitment and phagocytosis then were assessed in the rats’ lungs to
determine whether a smoke-induced increase in PMN numbers or uptake of pneumococci
could explain the restoration of PMN killing in the presence of EtOH. It has been
described in the literature that chronic EtOH ingestion causes neutropenia which is
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amplified during infection [291,292]. This was explained by EtOH down-regulating the
expression of β2-integrin which allows PMNs to bind to the endothelial wall and migrate
to the site of infection [170]. Cigarette smoking, on the other hand, is known to increase
pulmonary PMN numbers. Smoke exposure increases the production of PMNs in the
bone marrow and decreases their transit time, resulting in higher numbers of immature
PMNs circulating in the periphery [293]. Cigarette smoke stimulates alveolar
macrophages to release cytokines and chemokines leading to recruitment of PMNs to the
lungs in the absence of infection. Finally, smokers suffering from COPD are repeatedly
colonized by respiratory pathogens that result in a constant state of inflammation in the
lungs [294]. Based on these reports and the results from the PMN-mediating killing
assay, we hypothesized that EtOH ingestion would impair PMN recruitment and
concurrent smoke exposure would correct this defect by increasing the number of PMNs
recruited to the lungs.
PMN recruitment was evaluated using manual cytospin counts of lavage fluid
from the same rats used in the PMN-mediated killing assay. There were no smoke-
and/or EtOH-induced differences in the percentage of cells that were PMNs. This result
corroborates the earlier study in our laboratory that evaluated PMN recruitment in the
one-week exposure model [171]. LPS is a potent stimulator of alveolar macrophages,
causing them to produce proinflammatory cytokines and chemokines including MIP-2
and CINC-1. One possible reason no differences in PMN recruitment were measured
between the treatment groups was that the overwhelming stimulatory effects of LPS may
have masked any subtle smoke- and EtOH-induced changes in PMN recruitment.
Whether or not this is true, the decrease in PMN killing by EtOH ingestion and
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restoration of killing by concurrent smoke exposure do not appear to be due to alterations
in PMN recruitment to the lungs.
PMNs recruited to the lungs are essentially useless if they fail to effectively
phagocytose invading pneumococci. Our laboratory showed previously that peripheral
blood PMNs isolated from rats exposed to EtOH for one week did not exhibit decreased
phagocytosis of several different pneumococcal strains [177]. Physiologically relevant
concentrations of EtOH also did not impair the phagocytic ability of cultured PMNs in
vitro [173]. In contrast, other studies reported acute EtOH exposure suppresses PMN
phagocytosis [170,174]. Cigarette smoke has been shown to have both stimulatory and
inhibitory effects on PMN phagocytic activity [256]. Based on the results from our in
vivo PMN-mediated killing assay, we hypothesized chronic EtOH ingestion would impair
PMN phagocytosis and concurrent smoke exposure would reverse this EtOH defect by
enhancing phagocytic activity.
To determine if smoke- and EtOH-induced alterations in PMN phagocytosis are
responsible for the differences in PMN-mediated killing, a PMN phagocytosis assay was
used to measure the in vivo uptake of pneumococci by pre-recruited PMNs. This is the
same assay that was developed previously in our laboratory to identify the acute effects
of smoke and EtOH exposure on PMN phagocytosis [171]. EtOH ingestion alone failed
to reduce the percentage of phagocytosing PMNs, but it significantly reduced the amount
of organisms taken up by each cell as compared to sham-pair rats. However, we believe
this decrease is not biologically relevant for it did not affect the overall amount of
phagocytosed organisms as indicated by the phagocytic index, and this is analogous to
previous results from in vitro and in vivo studies. Smoke exposure with or without EtOH
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ingestion did not alter the phagocytic activity of PMNs. This result is not surprising since
published studies have reported no differences in phagocytosis by peripheral PMNs from
human smokers [256] and PMNs pre-recruited to the lungs of rats exposed to smoke for 8
weeks [171].
It is still unclear how the addition of smoke exposure was able to restore the
EtOH-induce impairments in killing, particularly when there was no increase in killing in
the animals exposed to smoke alone. Further experiments are needed to identify the
exact mechanisms of chronic EtOH ingestion and smoke exposure on PMN killing.
Functional assays must be performed to measure the ability of PMNs to undergo
respiratory burst and degranulation. The surface expression of the CXCR2 receptor in
PMNs also needs to be assessed due to its importance in priming and activating the cell
through the interaction with MIP-2 and CINC-1.
D. Chemokine Production
The release of chemokines by alveolar macrophages is important for the
activation and recruitment of PMNs during a pulmonary infection. Acute EtOH exposure
in rats has been shown to inhibit their ability to produce MIP-2 and CINC-1 in their lungs
after an intratracheal inoculation of S. pneumoniae [167,169]. The effect of smoke
exposure is just the opposite, where human smokers have higher concentrations of
chemokines, including IL-8 and gro-α, in their lungs [249]. Cigarette smoke is also
known to stimulate a variety of pulmonary cells to release PMN chemoattractants such as
granulocyte colony-stimulating factor and leukotrienes [252-254]. Previous research in
our laboratory found no effect of smoke or EtOH alone on pulmonary chemokine levels
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in our short-term model after a transtracheal administration of endotoxin followed by a
transtracheal infection of pneumococci [171]. However, the combination of smoke and
EtOH significantly increased the production of MIP-2 and CINC-1 in the lungs.
Furthermore, EtOH ingestion alone reduced CINC-1 levels in the serum, but this was
normalized by the addition of smoke. The results from this previous experiment helped
formulate our hypothesis that impairments in PMN killing by chronic EtOH ingestion are
due to a reduction in serum CINC-1, a defect that is corrected by concurrent smoke
exposure through the augmentation of pulmonary chemokine production and restoration
of normal CINC-1 levels in serum.
To analyze the effects of chronic EtOH ingestion and smoke exposure on
chemokine production in response to pneumococcal infection, MIP-2 was measured in
the lung homogenates and CINC-1 was measured in both the lung homogenates and
serum from rats that were used in the PMN-mediated killing assay. Chemokine
production in the lungs was not significantly altered by smoke or EtOH alone. The
combination of the two insults also failed to increase pulmonary chemokine levels, unlike
what was detected in the short-term model [171]. EtOH ingestion alone appeared to
reduce CINC-1 in the serum and concurrent smoke exposure negated this EtOH defect,
even though smoke exposure alone did not increase the amount of CINC-1 in the serum.
Although these effects of smoke and EtOH on serum CINC-1 were not statistically
significant, they did parallel previous results from our short-term model [171] and
mirrored the results from the PMN-mediated killing assay.
The EtOH-induced defect in PMN-mediated killing was not related to decreased
pulmonary levels of MIP-2 and CINC-1. However, this defect may be associated with
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the reduction in serum CINC-1. Pulmonary CINC-1 is known to leave the alveolar space
and enter the systemic circulation to pre-activate PMNs for phagocytosis and killing
[54,55]. The exact mechanism of CINC-1 movement out of the lungs is unknown, but it
has been shown to be an active, one-directional process [54]. The decrease in serum
CINC-1 by EtOH ingestion may be a result of impaired movement of pulmonary CINC-1
from the lungs to the circulation. Somehow, concurrent smoke exposure compensates for
this EtOH-induced impairment by allowing sufficient amounts of CINC-1 to enter the
bloodstream for PMN priming. To determine if restoration of serum CINC-1 in sham-
EtOH rats will restore PMN-mediated killing of pneumococci in the lungs, the PMN-
mediated killing assay would need to be repeated in sham-EtOH rats that receive an
intravenous injection of CINC-1.
Although no differences in pulmonary chemokine levels were detected in the rats
that received LPS, we further evaluated the ability of alveolar macrophages to release
chemokines ex vivo in the presence of pneumococci. Earlier research has reported that
macrophages from rats consuming EtOH for two weeks had decreased production of
proinflammatory cytokines in response to LPS [166]. On the other hand, macrophages
from human smokers stimulated by endotoxin secreted higher amounts of IL-8 and gro-α
than macrophages from nonsmokers. Given the results of the pulmonary and systemic
cytokine levels, we predicted chronic EtOH ingestion would decrease chemokine
production by alveolar macrophages and concurrent smoke would abolish this deficit by
up-regulating the release of chemokines.
MIP-2 and CINC-1 were quantified in media from isolated alveolar macrophages
stimulated by unencapsulated pneumococci or PMA. When incubated with
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pneumococci, macrophages from sham-EtOH rats released reduced amounts of MIP-2
and CINC-1 than macrophages from the sham-pairs. This effect of chronic EtOH
ingestion correlates with previous studies demonstrating that EtOH-treated rats produce
decreased amounts of chemokines in their lungs when infected with S. pneumoniae
[167,169]. Smoke exposure alone did not increase chemokine production.
Unexpectedly, concurrent smoke exposure was unsuccessful in negating the EtOH-
induced impairment in chemokine production. The levels of MIP-2 and CINC-1 released
by smoke-EtOH rat macrophages were less than those from animals exposed to smoke
alone. These results are different from the chemokine levels measured in lung
homogenates from rats used in the PMN-mediated killing assay, indicating the
differences in chemokine production by alveolar macrophages are not responsible for the
results of the PMN-mediated killing assay.
No significant alterations in MIP-2 production were detected in PMA-stimulated
macrophages from each of the four treatment groups, although EtOH ingestion with
smoke exposure reduced it by greater than half. However, EtOH ingestion alone
significantly increased CINC-1 production such that macrophages from sham-EtOH rats
secreted higher amounts of CINC-1 than macrophages from their pair-fed counterparts.
Smoke exposure did not decrease CINC-1 release, but concurrent smoke exposure
reduced this EtOH-induced increase in CINC-1 back to a similar level measured in the
sham-pair controls.
The smoke- and EtOH-induced differences in chemokine production between
pneumococcal and PMA stimulation is that these two stimulators act on the macrophages
through separate pathways. Pneumococci activate macrophages through receptor-
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mediated recognition and phagocytosis, while PMA activation is receptor-independent.
Two key receptors expressed on the surface of alveolar macrophages that recognize S.
pneumoniae are Toll-like receptors 2 and 4 (TLR2 and TLR4). The EtOH-induced
decrease in chemokine production in the presence of pneumococci may be due to
alterations in the functions of these receptors. Several studies have shown that EtOH
exposure in vitro and in vivo impairs TLR4 activation in monocytes and macrophages by
inhibiting the phosphorylation of downstream signaling proteins such as mitogen-
activated protein kinases [295-297]. Two of these same studies did not detect an EtOH
effect on TLR2 signaling [295,297], however, one study reported that acute EtOH
exposure in mice down-regulated the TLR2-mediated inflammatory response in alveolar
macrophages [298].
Cigarette smoke modulates the expression and function of TLR2 and TLR4.
Alveolar macrophages from human smokers had decreased expression of TLR2 on their
surface and failed to up-regulate TLR2 expression when stimulated with LPS in contrast
to macrophages from nonsmokers [299]. Another study found a dose-dependent down
regulation in TLR4 messenger RNA and protein expression in human airway epithelial
cells exposed to cigarette smoke extract [300]. Cell activation also was suppressed in
alveolar macrophages from human smokers stimulated with TLR2 and TLR4 agonists,
resulting in the reduction of chemokine secretion including IL-8 [301]. These smoke-
induced defects were not apparent in our assay, for smoke exposure alone did not alter
chemokine production in macrophages stimulated with pneumococci. Additional
experiments are required to further understand these results by identifying the effects of
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smoke and EtOH on expression and function of TLR2 and TLR4 in alveolar
macrophages from our rat model.
It is unclear why differences between MIP-2 and CINC-1 production occurred in
response to PMA. Although alveolar macrophages from sham-EtOH rats secreted
significantly higher amounts of CINC-1, it is difficult to believe EtOH ingestion up-
regulated CINC-1 production when there was such a wide range of variability among the
four treatment groups. Considering these cells were isolated out of the rats’ lungs
without additional exposure to EtOH and/or smoke, the results from this assay may not
accurately represent what occurs within the lungs of the rats.
E. Mortality Study
There is a strong agreement throughout the literature that alcohol abuse and
chronic smoking increase the severity and mortality of pneumococcal pneumonia. Both
of these behaviors suppress or even damage key innate and adaptive anti-pneumococcal
defenses. However, no studies have identified the outcome of pneumococcal disease in
the chronically drinking and smoking host. We hypothesized that EtOH ingestion with
smoke exposure would further increase the mortality rate of pneumococcal pneumonia
than either insult alone.
A mortality trial was conducted to identify the effects of chronic EtOH ingestion
with or without smoke exposure on the host in response to a lethal, intranasal challenge
of S. pneumoniae. The mortality rate was unchanged by chronic EtOH ingestion where
the same number of sham-exposed rats from each diet group succumbed to the infection.
Concurrent smoke exposure did not exacerbate the mortality rate as we predicted. In fact,
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mortality among the smoke-EtOH rats was no greater than for both sham-exposed
groups. The results from the smoke-EtOHs and sham-pairs are consistent with the trend
that is present in our previous experiments involving the host defenses of the lungs.
There were no significant differences in the bacteremia rates among these three treatment
groups, and all rats that developed bacteremia eventually died within the next 2-3 days.
Interestingly, smoke exposure alone did not compromise the rats’ immunity and actually
protected the animals from developing bacteremia and death.
Performing an intranasal mortality study includes all of the important defense
mechanisms that are involved at the different stages of pneumococcal pathogenesis.
Several studies, including our own experiments, have shown the defects of EtOH
ingestion on these key host defenses including mucociliary clearance, PMN function, and
chemokine production. Based on our results that show chronic EtOH ingestion promotes
pneumococcal invasion, decreases non-PMN- and PMN-mediated killing, impairs
opsonization, and suppresses macrophage phagocytosis and chemokine production, one
would expect the sham-EtOH rats to have a worse outcome than their pair-fed
counterparts. It is possible pair-feeding induces stress in animals which may alter their
immune response to infection. Stress increases the production of the hormone
corticosterone which is known to promote an anti-inflammatory response. Previous work
in the laboratory showed sham-pair rats have similar concentrations of corticosterone as
the sham-EtOH rats.
Smoke-EtOH rats have significantly higher corticosterone levels than sham-
EtOHs and smoke-pairs. This excessive amount of corticosterone in the smoke-EtOH
group did not increase their mortality rate to pneumococcal infection, but it may cause a
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similar end-result as the sham-EtOH rats. Among the four different treatment groups,
smoke-pairs had the lowest concentrations of corticosterone. In addition to low
corticosterone, smoke reduced pneumococcal movement to the lungs and did not affect
any of the other host defenses that were evaluated. This could be a potential reason why
all the smoke-pair rats survived the lethal infection.
Why did smoke exposure in conjunction with EtOH ingestion not perform in the
same manner? Besides up-regulating corticosterone levels, EtOH ingestion with smoke
exposure inhibited chemokine production from alveolar macrophages when they were
exposed to pneumococci. Hindering this activity could essentially shut down PMN
recruitment and activation. Defects in PMN function were not detected in smoke-EtOH
rats when they were stimulated with LPS. However, these defects may appear during a
normal pneumococcal infection without the presence of endotoxin from Gram-negative
bacteria. Other host defenses that are susceptible to the actions of smoke and EtOH, but
have not been evaluated yet, also may have contributed to the mortality results.
Additional studies are needed to further understand these findings from the mortality
trial. Such studies include examining PMN function and chemokine production in the
lungs infected with S. pneumoniae without LPS stimulation or instilled with the major
Gram-positive cell wall component lipoteichoic acid.
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VII. CONCLUSIONS
The studies described in this thesis have analyzed the effects of chronic EtOH
ingestion and smoke exposure on various host defenses against the pneumococcus which
work to prevent pneumococcal dissemination throughout the host. In addition,
alternative therapies have been evaluated for preventing pneumococcal pneumonia in the
smoking and drinking host. The consequences of alcohol abuse and smoke exposure on
anti-pneumococcal defenses are conflicting in the literature, and comparisons of these
studies are complicated due to the use of different models and methods. This research
was conducted using a well-established rat model of chronic EtOH ingestion and smoke
exposure, which standardizes the approach of studying these dual insults in relation to the
immune system.
The experiments were designed to mimic a natural pneumococcal infection. The
first stage of pneumococcal pathogenesis is colonization of the nasopharynx.
Nasopharyngeal colonization was unaltered by EtOH ingestion and smoke exposure.
After colonization, pneumococci must evade the mucociliary clearance apparatus to reach
the lungs. EtOH slightly increased pneumococcal movement to the lower respiratory
tract, but did not impair ciliary beating. Surprisingly, smoke exposure enhanced
mucociliary clearance resulting in a reduction of pneumococcal infection of the lungs.
Once the pneumococci reach the lungs, they encounter a variety of non-PMN defenses
including bactericidal factors and alveolar macrophages. EtOH ingestion suppressed
non-PMN-mediated killing within the lungs while concurrent smoke exposure restored
killing, even though smoke alone did not increase this activity. Although the effects of
124
EtOH and smoke on pulmonary concentrations of lysozyme and lactoferrin did not
explain the differences in non-PMN-mediated killing, the EtOH-induced defect on
pulmonary clearance was found to be associated with decreased opsonization and
macrophage phagocytosis of pneumococci. Concurrent smoke exposure did not
exacerbate the EtOH defect on opsonization, which may also be true for macrophage
phagocytosis, but this remains unknown due to the inability of our assay to accurately
measure macrophage phagocytosis in smoke-exposed animals. During a pulmonary
infection, alveolar macrophages produce cytokines and chemokines to activate PMNs and
recruit them to the lungs. Neither EtOH ingestion nor smoke exposure affected
chemokine production in the lungs, which relates to their lack of effect on PMN
recruitment and phagocytosis of pneumococci. Despite the fact that PMN recruitment
and phagocytosis were unaltered, EtOH ingestion abolished PMN-mediated killing of
pneumococci within the lungs. Once again, this EtOH-induced defect was corrected by
concurrent smoke exposure.
Pneumococci must follow a step-wise progression and evade various host
defenses in order to cause pneumonia and bacteremia. This provides several host defense
mechanisms that can be targeted for therapeutic intervention. An intranasal vaccine
containing a conserved pneumococcal surface protein was tested to reduce
nasopharyngeal colonization. The vaccine slightly increased antibody levels in the
mucosa and serum, but did not protect against pneumococcal colonization. Salbutamol
and formoterol inhalation also were evaluated to increase ciliary beating and reduce
pneumococcal invasion into the lungs. Both β2-agonists were effective in decreasing
pneumococcal movement to the lungs in unexposed, control rats. Formoterol also
125
prevented pneumococcal infection of the lungs in rats exposed to EtOH alone, but the
protection did not extend to any of the other rat treatment groups.
These studies show that EtOH ingestion and smoke exposure have differential
effects on a range of host defenses against the pneumococcus as summarized in Table 7.
More research is warranted to describe the effects of chronic EtOH ingestion and
concurrent smoke exposure on host defense strategies against the pneumococcus.
Understanding the pathogenesis of S. pneumoniae will lead to the development of
improved therapies to prevent pneumococcal infection and reduce overall mortality from
pneumococcal pneumonia. Immunity to other pathogens such as Haemophilus
influenzae, Mycobacterium tuberculosis, and influenza virus also needs to be examined in
the presence of EtOH and smoke. Our rat model would be an effective tool for
identifying the separate and combined effects of these morbidities in association with a
variety of infectious diseases.
Table 7 – Summary of Conclusions
EtOH Alone Smoke Alone EtOH with Smoke
Mucociliary Clearance —
Pulmonary Killing — Lost EtOH Defect
Bactericidal Factors — Lost EtOH Effect
Macrophage Phagocytosis ??? ???
Opsonization — Lost EtOH Defect
126
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