University of Calgary

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Graduate Studies Legacy Theses

1999

Effects of azithromycin on human neutrophils

Koch, Crystal Coco

Koch, C. C. (1999). Effects of azithromycin on human neutrophils (Unpublished master's thesis).

University of Calgary, Calgary, AB. doi:10.11575/PRISM/13027

http://hdl.handle.net/1880/25122

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THE UNNERSITY OF CALGARY

Effects of Azitbromycin on Human Neutrophils

by

Crystal C . Koch

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF BIOLOGICAL SCIENCES

CALGARY, ALBERTA

JULY, 1999

O Crystal C. Koch 1999

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ABSTRACT

Pathogen virulence factors and the host inflammatory response cause tissue injury

associated with respiratory tract infections. Azithromycin has demonstrated efficacy in

treating these infections. We hypothesized that azithromycin may have anti-

inflammatory properties, perhaps through the induction of neutrophil apoptosis. This

study assessed the effects of azithromycin on human neutrophii 1) apoptosis, 2)

phagocytosis by macrophages, 3) oxidative function, and 4) interleukin-8 production in

the presence or absence of Streptococcus pneumoniae compared with penicillin,

erythromycin, dexamethasone or saline. Azithromycin significantly induced neutrophil

apoptosis but this effect was abolished in the presence of ' s pneumoniae. Macrophage

phagocytosis of azithromycin-treated neutrophils was significantly enhanced.

Azithromycin did not affect neutrophil oxidative metabolism or interleukin-8 production.

In summary, azithromycin induces neutrophil apoptosis without affecting neutrophil

function, and enhances phagocytic clearance of these cells by macrophages. These

properties may confer anti-inflammatory benefits to this antibiotic.

ACKNOWLEDGEMENTS

I would like to sincerely thank Dr. Buret for allowing me to work in his lab and

giving me the opportunity to learn many new things and make many lifelong friends.

I would also like to extend a special thanks to AIex Chin and David Esteban for

their invaluable assistance.

Additionally, I would like to thank the multitude of people who gave me help and

advice, including Carol Ann Stremick, Kathy Oke, Kevin Scott, Michaela Waiter, Wilson

Lee, Kathy Murrin, all the other members of the Biofilm Research Group, and alf of my

blood donors.

A very special thanks goes out to Michael Lang for keeping me happy and sane

while conducting this project.

Finally, I would like to thank the Natural Sciences and Engineering Research

Council of Canada, the Alberta Heritage Foundation for Medical Research and Pfizer

Canada for fhding my research.

I dedicate this to my Mom and Dad

who always support everything I do

TABLE OF CONTENTS

APPROVAL PAGE

ABSTRACT

ACKNOWLEDGEMENTS

DEDICATION

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER 1: INTRODUCTION

1.1 The Pathogenesis of Respiratory Tract Infection

1.1.1 Bacterial Virulence Factors

1 . 1 . 1 . 1 Adhesion and Uptake

1 . 1 . 1 -2 Cytotoxins

1.1.1.3 Other Factors

1.1.2 Inflammation

I . 1.2.1 Mediators

1.1.2.2 Neutrophils

1.1.2.3 Macrophages

1.2 Pneumonia

v i

iii

1.2.1 Streptococcus pneumoniae

1.3 Treatment

1.3.1 Antibiotics and Antibacterial Agents 15

1.3.1.1 Beta-lactarns I 5

1.3.1.1.1 Penicillin 16

1.3.1.1.2 Cephalosporins 16

1.3.1.2 Sulfonamides I 7

1.3.1.3 Fluoroquinolones 17

1.3.1.4 Tetracyclines 17

1.3.1 -5 Macrolides 18

1.3.1.5.1 Structure 18

1.3.1.5.2 Mode of action (antimicrobial activity) 19

1.3.1 S . 3 Resistance 19

1.3.2 Azithromycin 20

1.3 -2.1 Structure 21

1 -3 -2.2 Pharmacodynamics and Antimicrobial Activity 2 1

1.3.2.3 Pharmacokinetics 23

1 -3 -2.4 Efficacy in Animal Models 27

1.3 -2.5 Clinical Efficacy and Dosage 28

1.3.2.6 Tolerability 30

1.3.2.7 Drug Interactions 3 1

1.3.3 Anti-inflammatory Therapies 32

1.3.4 Immunomodulation by Antibiotics

1.4 Apoptosis

1.4.1 Induction

1.4.2 Neutrophil Apoptosis

1.4.3 Regulation of Apoptosis in Neutrophils

1.5 Research Hypothesis and Objectives

CHAPTER 2: METHODS AND MATERLALS

2.1 Drugs

2.2 Bacteria

2.3 Subjects

2.4 Neutrophils

2.5 Moaocytes/M~crophages

2.6 CFU Counts

2.7 Annexin-Vff ropidium Iodide Labelling

2.8 Cell Death ELISA

2.9 Macrophage Pbagocytosis

2.10 Nitro Blue Tetrazolium Assay

2.1 1 Interleukin-8 Assay

2.12 Statistical Analysis

CHAPTER 3: RESULTS viii

3.1 Cell Isolation

3.2 CFU Counts

3.3 Neutrophil Apoptosis

3.4 Macrophage Phagocytosis

3.5 Oxidative Function and IL-8 Production

CHAPTER 4: DISCUSSION

REFERENCES

APPENDIX A: Structure of Azithromycin

and Erythromycin

LIST OF FIGURES

FIGURE:

1. S. pneumonia recovery after incubation with drugs

2. Light micrographs demonstrating annexin-V/PI staining:

A) bright field; B) fluorescent apoptotic and necrotic PMNs

3. Apoptosis in PMNs treated with TNFa

4. Apoptosis in PMNs treated with drugs via annexin-V/PI:

A) without lysate; B) with S. pneumonia lysate

5. Necrosis in PMNs treated with drugs

6. Apoptosis in PMNs treated with drugs via Cell Death ELISA:

A) without S. pneumonia; B) with live S. pneumonia

7. Light micrograph of nonspecific esterase staining of mononuclear cells 62

8. Light micrograph of macrophages incubated with PMNs 64

9. Phagocytosis of azithromycin-treated PMNs by macrophages 66

10. Oxidative activity of PMNs: A) without lysate; B) with S. pneumonia lysate 68

1 1. IL-8 production by PMNs treated with drugs 70

PAGE

50

52

LIST OF ABBREVIATIONS

A R D S

CBA

CFU

ELISA

fMLP

HBSS

Ig

IL

LPS

LT

MDM

MHC

'NBT

PAF

PARP

PBS

PI

PMA

PMN(s

TGF

W a

adult respiratory distress syndrome

Columbia blood agar

colony forming unit

enzyme-linked immunosorbent assay

formyl methionine leucine phenylalanine

Hank's balanced salt solution

immunoglobulin

interleukin

1ipopoIysacc haride

leukotriene

Iscove's modified Dulbecco's medium

major histocompatibility complex

nitro-blue tetrazoliurn

platelet activating factor

pol y- ADP-ribose-pol ymerase

phosphate-buffered saline

propidium iodide

phorbol 12-myristate 1 3-acetate

polymorphonuclear neutrophil(s)

transforming growth factor

tumor necrosis factor alpha

xi

CHAPTER 1: INTRODUCTION

1.1 The Pathogenesis of Respiratory Tract Infection

The lungs are exposed to more pathogenic organisms than virtually any other

tissue [I ] . Fortunately, the lungs have many mechanisms to remove, neutralize or kill

pathogens such as mechanical, secretory and immune systems 12, 31. Mechanical

defenses include filtration by nasal vibrissae, the ciliated epithelial barrier, and the cough

reflex. The mucociliary lining constitutes the secretory defense system. If a respiratory

pathogen manages to evade these mechanisms and reaches the lungs, it will encounter

resident pulmonary alveolar macrophages and other immune components such as

polymorphonuclear neutrophils (PMNs) as a third line of defense.

Despite these defense mechanisms, bacterial colonization of the respiratory tract

is a common cause of morbidity and mortality. The pathogenesis of bacterial infection in

the respiratory tract involves both bacterial virulence factors and the host inflammatory

response [4, 51. The host inflammatory response is primarily driven by neutrophils.

Despite being necessary for host defense, there is increasing evidence of the contribution

by polymorphonuclear neutrophils to the severity of a number of inflammatory diseases

such as streptococcal pneumonia [5- 121. In such pathologic circumstances, the potential

for host tissue damage is clear: cytotoxic compounds such as elastase, hypochlorous acid,

and oxygen radicals released by neutrophils at sites of inflammation can damage

surrounding tissues, and induce necrosis in neighboring neutrophils [7, 1 1 1. Necrotic

neutrophils swell and burst as a result of metabolic collapse and failure to maintain ionic

2

homeostasis, releasing more cytotoxic compounds and pro-inflammatory mediators [I 31.

This can lead to severe host tissue damage and inflammation.

1.1.1 Bacterial Virulence Factors

Bacterial virulence factors initiate the host inflammatory response. Gram-

negative bacteria have lipopolysaccharide (endotoxin) on the outermost layer of their cell

wall that has antigenic properties and activates the inflammatory process [14]. Gram-

positive bacteria have a large peptidoglycan layer that, along with associated lipoteichoic

acids, can exert endotoxin-like effects. Virulence factors aid in bacterial colonization and

protect the microorganism from host defenses enabling bacterial invasion and infection to

occur [I 51.

1.1.1.1 Adhesion and Uptake

Adhesion of bacteria in the host localizes pathogenic bacteria to target tissues

[I 51. Adherence of bacteria to host cells and extracellular matrix components is aided by

adhesins such as pili, fimbriae, and other adhesion molecules such as M protein of

Streptococci and fibronectin-binding proteins of Streptococci and Staphylococci.

P hosphory lcholine, a cell wall component of Streptococcus pneurnoniae, binds the

platelet-activating factor receptor on activated endothelial cells leading to adherence to

and invasion of these cells. Teichoic acids aid adhesion of many Gram-positive bacteria

to host epithelium fibronectin molecules [ 161. Many pathogens produce virulence factors

(such as EspA and EspB of Escherichia coli) that activate host cell signal transduction

3

pathways leading to expression of and activation of receptors on the host cell to which

the bacteria can then bind.

Some bacteria such as Salmonelfa and Shigella enhance their uptake into

nonphagocytic cells, enabling them to enter a protected environment or allowing them to

Pass through the epithelium [IS]. Yersinia enterocolitica and Yersinia

pseudotuberculosis avoid phagocytic uptake by injecting Yop proteins into phagocytic

cells, paralyzing the actin cytoskeleton and dephosphorylating host proteins which

impairs phagocytosis. Pseudornonas aeruginosa has this same effect on phagocytic cells

via secretion of exoenzyrne S which modifies G proteins involved in actin regulation.

1.1.1.2 Cytotoxins

Bacteria produce toxins which damage host cells by interfering with

transmembrane signalling, by producing pores that alter the permeability of membranes

resulting in lysis of host cells, or by altering cytosolic components of the host ceil [is].

Some bacteria can induce apoptosis (programmed cell death) in host phagocytes, thereby

preventing bacterial clearance (15, 171. Shigella protein IpaB binds and activates

interleukin-1 p-converting enzyme (ICE), a cysteine protease which induces apoptosis

when activated via cleavage of host cell substrates [15]. Mycobacterium avium induces

apoptosis in human macrophages possibly via the generation of oxygen free radicals 1171.

Attachment of M. avium to host cell surface receptors may activate second messengers

resulting in oxidant production which could alter the redox status and activity of host

enzymes. Diphtheria toxin, Pseudomonas exotoxin A, and cholera toxin induce apoptosis

in vitro, although the mechanism differs in each case [I 51. Staphylococcus aureus toxic

4

shock syndrome toxin-1 induces T cell apoptosis and inhibits immunoglobulin G (IgG)

production. S. pneumoniae has extracellular toxins such as pneumolysin, neuraminidase,

and purpura-producing factor which kill phagocytes and interfere with chemotaxis [3,

181.

1 . I -1 -3 Other Factors

Many intracellular organisms can prevent fusion of phagosomes with lysosomes

[15]. Some intraceliular pathogens, such as Mycobacterium avium, can inhibit

acidification of the phagocytic vacuole preventing activation of Iysosomal enzymes.

Streptococcus pneumoniae and Haemophilus influenzae, two common causative agents of

pneumonia, produce proteases that digest IgA [ I 81. The thick polysaccharide capsule of

S pneurnoniae and the M protein within the cell wall of Streptococcus pyogenes help

protect these organisms from being phagocytosed by macrophages and neutrophils [3,

181.

1.1.2 Inflammation

Inflammation is classified as acute on the basis of the persistence of injury,

clinical symptoms and the nature of the inflammatory response [19]. Acute inflammation

is characterized by the accumulation of fluid and plasma constituents in tissues,

intravascular stimulation of platelets, and the presence of neutrophils. Chronic

inflammation, on the other hand, is characterized by the presence of lymphocytes, plasma

cells and macrophages.

5

The hallmarks of inflammation include redness, swelling, heat, pain and loss of

function [20]. Inflammatory mediators released by cells at sites of infection cause

vasodilation and increased blood flow and, in conjunction with increased vascular

permeability, result in formation of inflammatory exudate and local edema.

The host immune system responds to bacterial products such as

lipopolysaccharide (LPS) and formyl methionine leucine phenylalanine (fMLP) and an

inflammatory response is generated [14]. A humoral response is produced that is mainly

mediated via immunoglobulin G, which opsonizes the organism [2]. In conjunction with

complement, IgG can also neutralize many viruses and toxins and lyse certain Grarn-

negative bacilli. Secretory IgA, localized at mucosal surfaces where most pathogenic

microbes gain access to the body, serves an important protective fhction [2 11. Secretory

IgM is also present in mucosal secretions but in Iower amounts than IgA. Secretory

antibodies cross-link antigens and block attachment of bacteria to mucosal cells. IgM is

the first immunoglobulin produced in response to infection (5 to 7 days after antigen

exposure) but its concentration declines rapidly and IgG and IgA become more

predominant. Previous exposure or immunization to an antigen results in a more rapid

and stronger response by lymphocytes which produce antigen-specific immunoglobulins.

A cell-mediated immune response is also elicited and alveolar macrophages are

the main effector cells. Lipopolysaccharide (endotoxin) activates macrophages,

monocytes, endothelial cells, platelets, mast cells, neutrophils, lymphocytes and

fibroblasts [14]. Alveolar macrophages process antigens that reach the lower respiratory

tract and kill many microbes [2]. As discussed further, leukocyte recruitment is mediated

by the generation of cytokines by lymphocytes, macrophages and other cells [IZ].

6

Leukocytes adhere to the endothelium and emigrate fiom the vasculature into the tissues

in response to chemotactic gradients.

Spontaneous phagocytosis can occur when neutrophils come into contact with

bacteria. Neutrophils also recognize and attach to pathogens via Fc receptors and

complement receptors on the neutrophil surface [12]. Fc receptors bind to the Fc portion

of immunoglobulin G molecules and complement receptors bind to C3b and C3bi found

on the surface of opsonized bacteria. Phagocytosis of the pathogen ensues and

microbicidal killing occurs within the phagolysosome of the neutrophil.

Mild acute idammation caused by bacteria is usually resolved when the infection

is cleared via host defense mechanisms (including the inflammatory response) or with

antibiotics [20]. In pneumonia, the lung are filled with exudate, fibrin, bacteria and

inflammatory cells. During the resolution phase of inflammation, this abnormal material

is removed (mainly via the action of macrophages), and normal structure and h c t i o n

can be restored.

1.1.2.1 Mediators

Interleukin-1 (IL-l), tumor necrosis factor a (TNFa), leukotriene Bq (LTB4), and

interleukin-8 (IL-8) are some of the major pro-inflammatory mediators involved in

inflammation. Bacterial antigens, such as LPS, as well as host immune components

stimulate the production and release of inflammatory mediators fiom many cell types

[22]. Lipopolysaccharide activates macrophages and they secrete peptide and lipid

mediators such as IL- 1, TNFa, IL-6, thromboxane, leukotrienes, and platelet activating

factor [14]. Reactions to LPS, infection and other inflammatory stimuli are mediated by

7

IL-1 and TNF [23]. JL-1 and TNF induce acute inflammation by inducing gene

expression and synthesis of proteins in other cell types found in the lungs [23]. Both IL-

la and TNFa activate neutrophils. IL-1 is produced by various cell types in response to

bacterial antigens and toxins, products of activated lymphocytes, complement and

clotting components [23]. TNFa is produced by activated monocytes and macrophages,

lymphocytes, endothelial cells and keratinocytes 1231. Neutrophils also release IL- 1,

TNFa and IL-8 [20]. IL-1 and TNF both cause an increase in IL-8 [24].

Mediators such as IL-8 and LTBJ strongly amplify inflammation. IL-8 plays an

important role in inflammation and clearance of pathogens by stimulating neutrophil

chemotaxis and degranulation, and enhancing phagocytosis of opsonized particles [25-

271. Although individual neutrophils produce only small amounts of IL-8, their large

number at sites of inflammation make neutrophils the most significant source of IL-8 in

inflammatory processes 1283. Neutrophils also release LTBJ, another potent neutrophil

chemoattractant responsible for potentiating inflammation [29].

Inflammatory mediators can be involved in the pathogenesis of many disease

states. IL-1, TNFa, IL-8, and LTB4 play a role in the pathogenesis of cystic fibrosis

patients infected with Pseudomonus aeruginosa [30-341. Increased levels of IL- 1, TNFa,

and IL-8 have been implicated in adult respiratory distress syndrome (ARDS) [35, 361.

1.1.2.2 Neutrophils

Neutrophils (polymorphonuclear leukocytes) originate and mature in bone

marrow, are then reteased into the blood where they circulate for approximately ten

hours, and can migrate fiorn the vasculature into sites of infection where they can live up

8

to three days [20, 37-39]. Neutrophils are terminally differentiated cells that retain a

smaller number of homeostatic pathways than their bone marrow precursors 1401. These

cells are highly specialized for degranulation and oxidative metabolism.

Neutrophils are the principal cell type involved in acute inflammation. These

cells are involved in phagocytic clearance of pathogens fiom infected tissues and thus are

important in the resolution of infection. They are necessary in the removal of pathogens

fiom the Lung and their influx is regulated by chemoattractants and upregulation of

adhesion molecules [7, 11, 371. In humans, neutrophils comprise approximately 65% of

circulating leukocytes but only 2% of leukocytes recovered from healthy broncho-

alveolar spaces [37]. In healthy lungs, more than 90% of lung leukocytes are alveolar

macrophages, while monocytes constitute only 5% of leukocytes in the circulation. This

distribution of leukocytes in the lung reverses upon exposure to bacterial infection.

Neutrophils carry out their anti-bacterial t'unction via oxygen-dependent or

independent mechanisms. Firstly, when neutrophils are activated by pro-inflammatory

mediators such as IL-I or TNFa, membrane enzymes including NADPH oxidase,

myeloperoxidase, and esterase are activated and result in the initiation of the respiratory

burst [ l l , 411. Reactive oxygen intermediates such as superoxide anion, hydrogen

peroxide, hypochlorous acid and hydroxyl radical are produced during the respiratory

burst of activated neutrophils. Oxygen is converted to superoxide, which is then

converted to hydrogen peroxide. Neutrophil granules contain myeloperoxidase, which

converts most of the hydrogen peroxide to hypochlorous acid if chloride is also present.

Some hydrogen peroxide is also convened to water via catalase or to hydroxyl radicals if

9

iron is present These reactive oxygen intermediates are involved in oxygen-dependent

bacterial killing-

In addition, neutrophils have intracellular granules that contain proteolytic

enzymes such as lysozyme, elastase, gelatinase, collagenase, cathepsin G, proteinase 3

and bactericidal proteins such as lacto ferrin, de fensins, bactericidaYpermeability - increasing protein, and azurocidin [ I , 1 1 1.

When neutrophils become activated during infection these enzymes, proteins and

oxidants are released into the phagosome and the extracellular environment and aid the

neutrophil in killing bacteria. Bacteria are killed by the oxidation and digestion of

membrane phospholipids and glycoproteins 1121. Some of these same compounds can

also act on host tissue components causing damage. In order to prevent such injury,

antiproteases and antioxidants are produced by the host [I 1, 411. Severe inflammatory

states overwhelm this system and cause injury to host tissues. For example, patients with

acute respiratory distress syndrome (ARDS) display abnormal antioxidant capacity in

association with severe lung damage [4 1 1. Oxygen radicals generated by neutrophils

inactivate antiproteases and W e r increase proteolytic damage to the lung. Massive

neutrophil infiltration seen in pneumonia results in the release of large quantities of

oxidants, overwhelming antioxidants and antiproteases, which in turn leads to lung injury

1.1.2.3 Macrophages

Bone marrow precursors differentiate into blood monocytes and migrate into

tissues where they develop into macrophages [42]. Local production of chemotactic

10

factors, including fMLP and the complement-derived peptide CS des arg, at the site of

infection induces the immigration of monocytes into tissues [43]. Macrophages are

important in the inflammatory process and the resolution of inflammation [44]. These

cells are major effectors of the immune response against pathogens [12, 441. Resident

alveolar macrophages are the host's first line of cellular defense against infections. Their

defensive capabilities can be overwhelmed, however, by large numbers or highly virulent

bacteria, and recruited neutrophiis help defend the host in this case [12]. Besides their

role in phagocytosis of bacteria, rnacrophages also function in antigen presentation to T

lymphocytes [453. Fragments of internalized pathogens bind to class I1 major

histocompatibility complex proteins (MHC) which are then expressed on the cell surface

[46]. Helper T cells with receptors specific for the antigen bind the MHC-antigen

complex, become activated and secrete cytokines (such as IL-4) that stimulate B cells that

have already encountered that particular antigen and display antigen fragments bound to

MHC 11. T cell receptors also bind to the antigen-MHC complex of these B cells. This

results in activation of antigen-specific B cells. Activated B cells give rise to plasma

cells that produce specific antibodies against the particular antigen. The phagocytosis of

bacteria activates macrophages, which can then recruit neutrophils to sites of infection by

secretion of neutrophil chemotactic factors such as IL-8 [2]. Macrophages are also

responsible for clearing cellular debris from inflamed sites. Macrophage phagocytosis of

dying celIs is another mechanism whereby acute inflammatory reactions during

pathological processes are physiologically controlled [47]. Indeed, macrophages have

been implicated in the regulation of neutrophil-mediated damage as they rapidly

phagocytose dead and dying neutrophils before the cell lyses and releases its cytotoxic

1 I

and pro-inflammatory compounds into the tissues, thus preventing damage to host

tissues, inflammatory reactions and autoimmune responses (autoantibodies against

nucleosomal DNA released from dead cells) [47]. Moreover, recent information suggests

that during phagocytosis of apoptotic neutrophils, in contrast to phagocytosis of

opsonized particles including bacteria, the production by macrophages of pro-

inflammatory mediators, such as IL-IP, TNFa and IL-8, is inhibited [48]. These

observations suggest that opsonin-mediated phagocytosis by macrophages activates

inflammatory processes, while vitronectin/phosphatidylserine-mediated phagocytosis

does not activate such processes. It is not yet known whether the inhibition of the

production of pro-inflammatory mediators is due to binding of apoptotic cells to receptors

on macrophages or to the uptake process. In the same study, increased production of

transforming growth factor+ 1 (TGF-P I), platelet activating factor (PAE'), and

prostaglandin E2 (PgE2) were also reported and appeared to mediate the decrease in pro-

inflammatory cytokines. The mechanism by which phagocytosis of apoptotic cells

stimulates production of TGF-B 1, PAF, and PgE2 is not known nor is the mechanism by

which these products reduce production of pro-inflammatory mediators.

1.2 Pneumonia

Pneumonia is one the most common community-acquired and nosocomial

infections, and despite improved treatment strategies, the mortality rate remains high

[49]. It is the fifth leading cause of death in the USA. In pneumonia, fibrin deposition

congests the alveoli and bronchioles of the lungs [SO, 511. Severe inflammation causes

pulmonary tissue damage that can ultimately lead to respiratory failure and death. Fever,

12

chills, headache, cough and chest pain accompany pneumonia. A number of pathogens

are known to cause pneumonia, including Streptococcus pneumoniae, Streptococcus

pyogenes, S faphylococcus aure us, HaernophiZus influenzae, Kle bsiella pneumoniae,

Escherichia coZi, Pseudomonas aeruginosa, Mycoplosma pneumoniae, Legionefla

pnettmophila, adenovirus type 111, influenza A and B viruses, rickettsiae, and fungi [50-

521. Combined infections of multiple respiratory pathogens are ofien seen 153, 541. The

most common bacterid lung infection in adults is streptococcal pneumonia caused by

SIreptococcus pneumoniae 131. Bacteria are spread via contact with an infected person.

A 7 to 10 day incubation period precedes the clinical manifestations of streptococcal

pneumonia (chills, fever* and pain) [55] . The bacteria invade the body through the

airways and reach the lungs where they multiply rapidly in the alveolar tissue. The

antiphagocytic capsule of S. pneumoniae prevents phagocytosis by alveolar macrophages

and other phagocytes, and the infection proceeds resulting in purulent exudate-filled

aiveoli. Neutrophil influx ensues and they accumulate in the infected alveoli and

contribute to exudate formation. This proceeds for about a week during which

anticapsular antibody is formed which usually results in recovery due to adequate

phagocytosis and killing of the organism unless the disease is too severe or the host is

immunocomprornised. Particularly virulent strains of bacteria may overwhelm host

defenses leading to respiratory failure.

Streptococcus was isolated in 55% of patients with pneumonia in one study [52].

Many normal healthy individuals carry streptococci in their upper respiratory tract,

however, only a small proportion develop streptococcal pneumonia [52]. A study by

Cherry et al. isolated both pathogenic bacteria and rhinovirus fiom 45% of patients,

13

perhaps indicating a viral infection as the initiating cause in bacterial pneumonia [53].

The large volume of mucous secretions in the nose and pharynx increase the chance of

aspiration of bacteria past the epiglottaf barrier where gravity can carry the bacteria into

the alveoli. In another study, 46% of patients had an upper respiratory tract infection

(possibly viral) in the week or so prior to the onset of bacterial pneumonia [52]. A viral

source of the initial infection was difficult to confirm in most cases since viruses may

only be shed for a few days and may not have been detectable by the time of

hospitalization for the secondary bacterial pneumonia.

The annual rate of pneurnococcal infections in the USA is approximately 500,000

cases in all age groups, but this rate can increase in certain populations [3, 551. A major

risk factor for contracting pneumonia is age; those over forty have a three to four times

greater chance than those under twenty [3]. Hospitalization also increases the risk of

contracting pneumonia [49]. Pneumonia causes 8% to 33% of all nosocomial infections

making it the third most common nosocomial infection after urinary tract and surgical

wound infections. Nosocomial infections are often caused by Gram-negative bacteria

and are fatal in more than 50% of cases. The rate of respiratory tract infection is greatest

in developing countries [56-581.

1.2.1 Streptococcus pneumoniae

Streprococcus pneumoniae is the most common causative agent of bacterial

pneumonia in humans [50]. This organism appears as a lancet-shaped Gram-positive

diplococcus that is found in the human nasopharynx and has no known environmental or

animal reservoir [3]. S. pneumoniae is encapsulated and a-hemolytic. Asymptomatic

14

carriers are often more important in spreading the disease than those with acute infection

since they are not isolated and treated [3]. A number of factors determine whether

bacterial invasion and infection will occur such as degree of challenge, virulence factor

expression, primary viral infection, host defenses and immune competence, nutritional

status, alcohol intoxication [3]. S. pneumoniae has extracellular toxins such as

pneumolysin, neurarninidase, and purpura-producing factor but, although proposed, the

exact role of these in the pathology of pneumonia remains unclear [3, 551. Hydrolytic

enzymes such as neurarninidase and IgAI protease may contribute to the colonization of

the host [3, 18, 591. Neurarninidase acts to kill phagocytes and interfere with their

cheinotaxis and the destruction of secretory IgA impairs opsonization and phagocytic

clearance of the bacteria. S. pneumoniae strains canying mutations in genes encoding for

pneurnolysin, autolysin and surface protein A demonstrate reduced virulence. Purpura-

producing factor is a glycan-teichoic acid fragment generated by hydrolysis of the cell

wall by autolysin [60]. Lipoteichoic acids aid in bacterial adhesion to host cells [16].

The primary determinant of pathogenicity is the polysaccharide capsule, which helps

protect S. pneumoniae from being phagocytosed by macrophages and neutrophils [3].

1.3 Treatment

Several options are available for the treatment of bacterial pneumonia. Besides

vaccines for the prophylaxis of pneumonia, therapeutic strategies such as antibiotics and

anti-inflammatory drugs are available 131. Drugs that modulate the immune response to

infection have been studied experimentally, and several anti-inflammatory strategies

15

focus on preventing neutrophil-mediated tissue injury. Of course, the treatment of choice

remains the use of antibiotics to eradicate the bacterial infection.

1.3.1 Antibiotics and Antibacterial Agents

There are many classes of antibiotic and/or antibacterial drugs available to treat

pneumonia including beta-lactamases, sulfonarnides, fluoroquinolones, tetracyclines, and

macrolides. The choice of drug depends on the specific organism isolated from sputum

or blood, the pharmacodynamics of the drug and its antibacterial spectrum, as well as

clinical experience and an assessment of the patient's history.

1.3.1.1 Beta-lactams

Beta-lactam antibiotics include penicillins and cephaiosporins 161 1. They contain

a beta-lactam ring with various substituents that lend different properties to the antibiotic.

Beta-lactam antibiotics bind to penicillin-binding proteins and interfere with bacterial cell

wall synthesis by inhibiting transpeptidase cross-linkage of adjacent peptidoglycans [62].

Additionally, beta-lactarns inactivate an inhibitor of an autolytic enzyme in the bacteria1

cell wall, leading to lysis of the bacterium [61]. Bacterial resistance to beta-lactams is

mainly due to the production of beta-lactamases, reduced outer membrane permeability

(decreasing drug penetration; particularly in Gram-negative organisms), or modified

penicillin-binding sites. Many strains of S. pneumoniae are resistant to p-lactams due to

modification of the penicillin-binding proteins; S. pneumoniae does not produce a P-

lactamase.

1.3.1.1. I Penicillin

Penicillin G (benzylpenicillin) has been one of the most widely used antibiotics

for treating streptococcal pneumonia, but growing bacterial resistance to penicillin has

forced the development of other drugs [SO, 511. Penicillin contains a thiazolidine ring

linked to the beta-lactam ring, which is the target of beta-lactamases and acylases fiom

penicillin-resistant organisms [62]. Penicillin G is a natural penicillin that is produced by

mold cultures. It is acid labile and bactericidal [61]. Semisynthetic penicillin derivatives

(such as nafcillin, amoxicillin, ampicillin and piperacillin) have been produced which are

acid-stable, more resistant to cleavage by beta-lactarnases, or have an enhanced spectrum

of activity against both Gram-positive and Gram-negative bacteria.

1.3.1 .1.2 Cephalosporins

Cephalosporins are derived fiom Cephalosporium h g i . They are water-soluble

and relatively acid-stable. Cephalosporins vary in their susceptibility to beta-lactamases

[6 11. Their usefulness in pneumonia may be limited since they have poor lipid solubility

which hinders their diffusion into tissues. Many semisynthetic broad-spectrum

cephalosporins, such as cefuroxirne, have been produced by varying the side chains of the

beta-lactam ring. They are effective against most Gram-positive and some Gram-

negative organisms. Resistance to this class of drugs is increasing due to both plasmid-

encoded and chromosome-encoded beta-lactamases. In fact, nearly all Gram-negative

bacteria produce a beta-lactamase that hydrolyzes cephalosporins to a greater extent than

penicillins.

17 1 -3. I -2 Sulfonamides

Sulfonamides, such as sulfadiazine and sulfamethoxazole, are derived fiom

sulfanilamide by substitution at the amide group [61]. Sulfanilimide is a smxctural

analogue of p-aminobenzoic acid that is needed for folic acid synthesis in bacteria. DNA

and RNA synthesis require folic acid. Sulfonamides compete with p-aminobenzoic acid

for the enzyme dihydro-pteroate synthetase and are bacteriostatic rather than bactericidal

since they only inhibit bacterial growth. Sulfamethoxazole, in combination with

trimethoprim (an anti biotic which acts as a folate antagonist), is called co-trimoxazole

and is effective at much lower dosages. Co-trimoxazole is sometimes used to treat

pneumonia.

1.3.1 -3 Fluoroquinolones

Fluoroquinolones, such as ciprofloxacin and norfloxacin, are synthetic antibiotics

that inhibit topoisomerase I1 (a DNA gyrase) and thus inhibit DNA transcription [61].

Although they are active against Gram-positive organisms, these antibiotics are

especially active against Gram-negative bacteria. Ciprofloxacin is used for pneumonia

caused by Haemophilus influemae. One of the new drugs of choice for pneumonia is

levofloxacin.

1 -3.1 -4 Tetracyclines

Originally, tetracyclines, such as oxytetracycline, were obtained from

Streptomyces [6 1 1. Newer tetracyclines, such as minoc ycline and tetracycline, are

semisynthetic or synthetic. They are taken up by active transport and are distributed

18

throughout the body. They act by inhibiting protein synthesis by competing with

aminoacyl tRNA for the A site on ribosomes. Tetracyclines have a very broad spectrum

of activity against both Gram-positive and Gram-negative organisms but they are only

bacteriostatic. Their usefulness is limited as many strains of bacteria have become

resistant [61]. Resistance occurs due to defective uptake by bacteria; plasmid encoded

genes lead to the development of energy-dependent efflux mechanisms which transport

the drug out of the bacterium. Some bacteria also alter the ribosome binding site so the

drug can not bind.

1.3.1.5 Macrolides

Macrolides have been used for many years for the treatment of various upper and

Iower respiratory infections [63]. The first macrolide, erythromycin, was introduced in

1952. Many derivatives of erythromycin have since been developed. One of these is

azithromycin which will be discussed in detail in a later section.

1.3.1.5.1 Structure

Structurally, macrolides consist of a lactone ring structure with attached

carbohydrate moieties. Macrolides have a variety of lactone ring structures but three

main groups are the 1 4-membered macrolides (erythromycin, clarithromycin and

roxithromycin), the 1 5-membered macrolides (azithrornycin) and the 16-membered

macrolides (josamycin, lincosamides, and streptogramins) [64]. Azithromycin is more

correctly classified as an azalide due to the presence of a nitrogen atom into the

macrolide ring.

1.3.1.5.2 Mode of action (antimicrobial activity)

Macrolides are generally considered to be bacteriostatic rather than bactericidal,

and are active against a variety of Gram-positive and Gram-negative organisms [64]. The

antibacterial activity of macrolide antibiotics is due to reversible binding of the drug to

the bacterid 50s subunit of the 70s ribosome, thus inhibiting protein synthesis within the

bacterial cell [65]. Macrolides stimulate dissociation of peptidyl tRNA from the

ribosome by a variety of mechanisms including inhibition of translocation or peptidyl

transferase reactions [66]. Some nonbacterial organisms such as Entamaeba hisrolyrica

and Toxoplasrna gondili are also affected by macrolides but the mechanism of action is

not known [64].

1.3.1 S . 3 Resistance

Cross-resistance to azithromycin and erythromycin has been shown in many

studies [64]. Enzymatic methylation of the ribosome binding site of macrotides is

postulated to be responsible for resistance of organisms to macrolides by preventing

macrolides from binding to the ribosome [67-701. The methylase modifies the 50s

ribosome subunit by ~ ~ - r n e t h ~ l a t i o n of adenine in 23s ribosomal RNA [70]. In addition

to macrolides, this also confers resistance to lincosamides and streptogramin B

antibiotics. The methylase can be constitutively produced by some bacteria such as

Staphylococcus aureus and Staphylococcus epidermidis. Subinhi bitory concentrations of

the macrolide can also induce expression of the methylase in such bacteria as

20

Streptococcus pneumoniae and S~reptococcus pyogenes. Plasrnid pE 1 94 specifies this

type of resistance to macrolides.

Another mechanism of resistance involves the presence of the plasmid pNE24,

which results in energy dependent efflux of 14- and 15-membered macrolides (but not

16-membered macrolides) from bacteria [71]. The energy dependent macrolide efflux

pump maintains intracelluiar antibiotic concentrations below those necessary for binding

of the macrolide to ribosomes. The efflux pump recognizes specific structural

components of 14- and 15-membered macrolides and is able to remove several thousand

macrolide molecules per second. Bacteria are thus able to maintain growth in the

presence of antibiotic. Plasmid pNE24 has been detected in Staphylococcus epidermidis.

Other forms of resistance to macrolide antibiotics include hydrolysis of the

macrolide ring by esterases, and phosporylation or glycosylation of the macrolide [7 11.

1.3.2 Azithromycin

Standard treatments for lower respiratory tract infections include penicillins,

cephalosporins, or erythromycin [61, 721. Other agents such as sulfonamides,

ciprofloxacin, or tetracyclines may also be prescribed. Penicillins are limited in their

usage due to patient hypersensitivity reactions and bacterial resistance, and

cep halosporins have poor activity against intracellular pathogens. Erythromycin was '

introduced over thirty years ago and has been considered the representative macrolide.

Its disadvantages include need for fiequent administration, acid instability,

gastrointestinal disturbances and poor activity against Haemophilus species. This has led

to the development of new macrolides. hthromycin (CP-62,993; XZ-450) is a

21

semisynthetic macrolide antimicrobial drug of the azalide class. It is more acid-stable

than erythromycin and demonstrates improved pharmacokinetic parameters [64]. Like

other macrolides, the activity of azithromycin does, however, decrease in an acidic

environment 1731.

1.3.2.1 Structure

Azithromycin is derived from erythromycin. Its chemical structure is 9-deoxo-

9A-methyl-9A-aza-9A-homo-erythromycin A [74]. Azithromycin has a 1 5-membered

lactone ring structure with a tertiary amino group, attached to two sugars (see Appendix

A) [64]. The amino substituent and expanded ring strvcture differentiates it fiom

erythromycin (1 4-membered lactone ring). The enhanced acid-stability of azithromycin

(300-fold increase over erythromycin) is related to these structural differences [75].

1 -3 -2.2 Pharrnacodynamics and Antimicrobial Activity

The antimicrobial activity of azithromycin, like that of other macrolides, is due to

inhibition of protein synthesis fiom the drug binding to the 50s subunit of the bacterial

ribosomes [65]. Azithromycin has a similar spectrum of activity to other macrolides; it is

active against many Gram-positive and Gram-negative bacteria. In addition to its

bacteriostatic activity, azithromycin is also bactericidal against a number of bacterial

species. Bactericidal activity is a decrease in "CFU concentration of at least 3 loglo

CFU/mL or a 99.9% kill rate within 24 hours"[64]. Azithromycin i s bactericidal against

Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae,

Haernophilus influenzae, Klebsiella pneurnoniae, Borderella species, Legionella

22 pne umophila, Moraxella catarrhalis, Chlamydia trachoma t is, Chlamydia peumoniae,

Mycobacterium avium complex, and Borrelia burgdorferi [64]. Bacteria resistant to

erythromycin are also usually resistant to azithromycin.

All macrolides exhibit a postantibiotic effect (PAE) which means bacterial

regrowth is suppressed for a short period of time after exposure to an antimicrobial drug.

The PAE is important to consider because drug ievels may become lower than the

minimum inhibitory concentration (MIC) in the interval between doses. Azithromycin

has shown a postantibiotic effect of 1.7 to 3.9 hours for many respiratory tract pathogens

~761.

The d i d e azithromycin has proven to be more effective in the treatment of a

number of respiratory tract infections compared to erythromycin, although both

antibiotics have comparable antibacterid properties [77, 781. Azithrornycin has greater

activity than erythromycin against Gram-negative bacteria and this is thought to be due to

the extra positive charge from the methyl substituted nitrogen (structure-activity

relationship) [79]. It may also be due to the high binding affinity for ribosomes [80].

Azithromycin also interacts with magnesium binding sites on outer membrane

phospholipids, which serves to increase its membrane permeability and enhance its

uptake 1791. The exact mechanism of uptake, however, has not been determined but is

postulated to involve passive diffusion and lysosomal trapping [8 1, 821.

Clinically, the antimicrobial activity of azithromycin appears to be enhanced by

the ability of this compound to reach high tissue concentrations 1641. Azithromycin is

more concentrated in tissues than in blood and accumulates intracellularly within

monocytes, polymorphonuclear Ieukocytes and alveolar macrophages to concentrations

23

much greater than those in the extracellular environment [8 1-84]. This may enhance the

antimicro bid activity of azithrom ycin against intracelluiar pathogens. Azithromycin has

been shown to accumulate inside phagocytes much more effectively than erythromycin,

and is primarily located within lysosomes [8 1, 85, 861. Azithromycin does not impair the

phagocytic h c t i o n of cells and even enhances phagocytosis of S~aphyZococcus aureus at

subinhibitory concentrations [81, 871. Synergistic killing between azithromycin and

phagocytes has been demonstrated in vifro for S. aureus. L. pneumophila and H.

influenzae [84]. Azithromycin is released fiom phagocytes more slowly than

erythromycin (within 24 hours versus 3 hours, respectively) and it is suggested that

phagocytes deliver the drugs to sites of infection increasing tissue concentrations [88].

Fibroblasts also accumulate azithromycin (and release it more slowly than they release

erythromycin) and may act as a slow release reservoir of antibiotic within tissues 1891.

1 -3.2.3 Pharrnacokinetics

Animal models, healthy volunteers, and patients requiring antibiotics have been

used to investigate the pharmacokinetics of azithromycin. As discussed previously,

azithromycin is more acid-stable than erythromycin as the structural modifications block

the normal degradation pathway. At pH 2, 10% decay takes 20 minutes for azithromycin

but only 3.7 seconds for erythromycin [75].

Azithromycin is rapidly taken up from the blood into intracellular compartments

of p hagoc ytes and fibroblasts and is slowly released. Unlike other macrolides,

azithromycin achieves high concentrations in tissues, including the lungs. High tissue

concentrations are prolonged due to the long half-life of azithromycin [90]. This permits

24

a single daily dose regimen for most infections, which enhances compliance. Product

labeling states that azithromycin should be taken on an empty stomach (as food decreases

the oral bioavailability of the drug), but subsequent studies have found that food has no

effect on the bioavailability of azithromycin 191, 921. In healthy fasting volunteers, an

oral bioavailability of 37% was shown when given a single 500 mg dose (931. The low

oraI bioavailability is attributed to incomplete absorption rather than acid degradation or

extensive first-pass metabolism 1941. A 500 mg dose of azithromycin gave a mean peak

plasma or serum concentration (C,,) of 0-4 to 0.45 mg/L when measured 2.5 hours after

administration to healthy fasting volunteers [93, 951. Higher mean peak concentrations

were reported for other macrolides and were achieved in a shorter time. The mean C,,

in healthy volunteers given 500 mg twice on day 1 and once on days 2 to 5 increased

from 0.41 to 0.62 mg/L over five days 1931.

Macrolides are dicationic yet they are still lipid soluble [64]. This results in

extensive distribution in body fluids [96, 971. The volume of distribution (Vd) is the

volume of fluid required to contain the total amount of drug in the body at the same

concentration as is present in the plasma [61]. The Vd for azithromycin has been

reported to be 23 to 3 1 L k g body weight, which is greater than the total amount of body

water (0.55 Lkg body weight) indicating that the drug is stored in tissues and cells [97].

Delivery of azithromycin to tissues is accomplished via two mechanisms - direct uptake

by tissues and uptake by phagocytic cells (targeted delivery) [90]. As discussed above,

azithromycin is rapidly taken up and slowly released from many cell types leading to

high local concentrations of drug [98].

25

In patients who received a single 500 mg dose, serum concentrations declined in a

biphasic manner over three days post-administration and were between 1 and 9 mg/kg in

gynecological tissue (ovary, fallopian tube, uterus, cervix, fibroid tissue), lung, peritoneal

fluid, prostate, tonsil and urologicd tissue (testis, epididymis, vas deferens) [93, 99, 1001.

Lower concentrations (0.2 to 1 mg/kg) were found in bone, fat, gastric mucosa, and

muscle, but all concentrations were higher than those found in serum, reflecting the rapid

uptake and concentration of azithromycin in tissues versus bIood. Patients given a single

500 mg dose of azithromycin orally were also used to assess concentrations of

azithromycin in the respiratory tract [ 10 1 - 1031. Broncho-alveolar macrophages,

bronchial epithelial lining fluid, bronchial mucosa and sputum all achieved a maximum

concentration within 48 hours, while this was reached in lung tissue within 96 hours, with

concentrations ranging fiom 1.56 to 26.59 mg/L. These values represent high tissue or

fluid : senun concentration ratios (52 : 1 to 11 50 : 1). In patients, with respiratory

infection, who received 500 mg on day 1, and 250 mg on days 2 to 5, the mean peak

sputum concentration of azithromycin was 3.7 mg/L at 15 hours which represents a

sputum : serum concentration ratio of 5.8 : 1 [74].

Equilibrium dialysis at pH 7.4 and 25°C was used to determine in vitro protein

binding of [14~]-azithromycin in human senun [104]. Protein binding decreased as the

azithromycin concentration increased; binding was 50% for azithromycin concentrations

of 0.02 and 0.05 mg/L, 37% at 0.7 mgL and 7% at 1 mg/L. Erythromycin and

roxithromycin demonstrated higher binding than azithromycin (72% at 0.4 mg/L

erythromycin and 96% at 2.5 mg/L roxithromycin) [I 04, 1051. Therefore, azithromycin

has a greater proportion of drug in the free form that is then available for distribution to

26

infected tissues. Azithromycin, erythromycin, and roxithromycin are predominantly

bound to a,-acid glycoprotein as suggested by low concentrations of drug displaying

saturability 1105, 1061.

Azithrornycin is mostly eliminated in the feces via transintestinal excretion, but a

small amount leaves in the urine 1107, 1081. Azithromycin is primarily eliminated in an

unchanged form, although some biotransformation does occur. Biotransfonnation

primarily occurs via N-demethylation of the desoamine sugar or the 9a position of the

macrolide ring but hydrolysis and hydroxylation also occur [ 1 081.

Azithromycin (500 rng) showed a mean terminal elimination half-life (tlQ) fiom

serum of 9.6 to 14 hours when given orally and 35 to 40 hours when given intravenously

[95, 1041. The tin for oral azithromycin (500 mg twice on day 1 and 500 mg once on

days 2 to 5) increased from 11 to 14 hours on day 5 to 48 hours over 1 to 3 days after the

last dose and to 57 hours over 1 to 6 days after the last dose [104].

The effect of age was compared in a nonblind parallel study using healthy elderly

and young volunteers given 500 mg on day 1 and 250 mg on days 2 to 5 [109]. Mean

maximum serum concentrations (-0.4 mg/L on day 1 and -0.25 mg/L on day 5 ) and

minimum serum concentrations (0.05 mg/L) were similar for young and elderly

volunteers and there was no difference in urinary excretion. Area under the plasma

concentration-time curve (AUC) and t,, were significantly increased in the elderly. An

inverse relationship was found between creatinine clearance and AUC indicating that

dosage adjustment is not required in elderly people with mild renal impairment, only

those with more severe renal impairment. Subsequent studies have confirmed that

azithromy cin can be safely used in children of all ages [ 1 1 01.

27

Healthy volunteers and patients with mild to moderate hepatic dysfunction did not

display differences in pharmacokinetic parameters except for an increase in t , ~ (68 versus

54 hours) suggesting that dosage adjustment, when mild to moderate hepatic impairment

is present, is not necessary since azithromycin treatment is of short duration and tolerated

favorably [ 1 1 1 1.

1.3 -2.4 Efficacy in Animal Models

The efficacy of azithromycin in vivo has been assessed using animal models of

infection. Mice infected with S. pneumoniae showed a higher rate of survival when given

subcutaneous or oral azithromycin (25 - 50 mglkg) either before or after infection than

when given erythromycin [112]. Bacteria were completely cleared from lungs and blood

in mice given two subcutaneous injections of azithromycin (25 mg/kg) at 48 and 65 hours

post-infection but were not cleared in mice given erythromycin, clarithromycin,

roxithromycin or spindamycin [113]. This result was consistent with greater

concentrations of azithromycin that also remained longer in the lung. When

azithromycin (25 mg/kg) was given orally 24 hours before infection with pneumoniae,

recovery of bacteria after 6 hours of infection was reduced by greater than 99.9%

compared to untreated controls [77]. In the same study, erythromycin (100 mg/kg)

produced no difference compared to controls and roxithromycin (50 mglkg) produced a

96% reduction in bacterial recovery compared to control values.

Intraperitoneal delivery of azithrornycin (3.6 mg/kg/day) resulted in greater

therapeutic efficacy than erythromycin (96 mg/kg/day) in a lethal L. pneumophila

pneumonia model in guinea pigs. Survival rates were 100% with azithrornycin and 83%

28

with erythromycin and clearance of bacteria was complete after two days with

azithromycin whereas erythromycin did not clear the bacteria after six days 11 141. Oral

administration for two days in the same animal model resulted in 100% survival in the

azithromycin group (3.6 mg/kg once daily) and in the clarithromycin group (28.8 mgkg

twice daily). Although the higher dose of clarithromycin resulted in similar survival

rates, clarithrornycin did not eradicate bacteria fiom the lungs as did azithromycin [115].

In another study, mice infected with K pneumoniae showed a 99% reduction in bacteria

recovery from the lungs when given azithromycin (50 mg/kg/day) 24 hours before

infection compared to the untreated control [77].

Taken together, the results generated fiom these animal studies demonstrate that

the efficacy of azithromycin in vivo is greater than that of many other macrolides.

1 -3.2.5 Clinical Efficacy and Dosage

The clinical efficacy of azithromycin has been demonstrated in the treatment of

lower and upper respiratory tract infections, skin and soft tissue infections,

urethritis/cervicitis (1 g over 1 to 3 days), early Lyme disease and M. avium complex

infections (500 mg/day for 10 to 30 days) [64]. The efficacy of azithromycin is

correlated with high tissue concentrations. Typical treatment regimens are a single or

divided 500 mg dose on day one followed by either 250 mg/day for four more days or

500 mg/day for three more days. In both regimens, the azithromycin concentrations in

respiratory tract tissues (> 2 mg/L) are above the minimum inhibitory concentrations

(MIC) for key pathogens (Haemophilus injluenrae, azithromycin MICgO of 0.5-2.0 mg/L;

Streptococcus pyogenes, azithromycin MICgO of 0.12 mgk; Streptococcus pneumoniae,

29

MormceZZa catarrhalis, Mycoplasma pneumoniae, ChZamydia pneumoniae, Legionella

pneumophila, all with an azithromycin MICgO of I 2.0 rng/L) for up to ten days following

treatment [98]. Small children should be given 10 mgkg on day one and 5 mg/kg on

days two to five and children weighing over 45 kg should be given the adult dosage via

either regimen.

Standard regimens of azithromycin have been shown to have comparable efficacy

compared to erythromycin, amoxicillin, cefaclor, amoxicillin.clavulanate, roxithromycin,

and clarithromycin in the treatment of tower respiratory tract infections, although all the

other drugs required more frequent and prolonged regimens than azithromycin to achieve

this same eficacy [72, 1161. This suggests that azithromycin has a more potent clinical

effect than the other drugs to which it was compared. In a study of patients with upper or

lower respiratory tract infection given azithromycin (500 mg on day one and 250 mg on

days 2 to 5), a 95% clinical cure rate and a 91% clearance of sputum bacteria was

demonstrated [117]. A 9 1% clinical cure rate was recorded for patients with acute

bacterial pneumonia given azithromycin (500 mg/day for 3 days) 11181. Studies have

found that azithromycin is more effective than erythromycin in eradicating H. injluenzae

[74]. Clinical cure rates for azithromycin in comparative studies of lower respiratory

tract infections ranged from 36% to 80% and clinical success rates (cure + improvement)

ranged from 92% to 100% [64]. Azithromycin was better tolerated and showed a lower

incidence of adverse effects compared to roxithromycin 172, 1 191. Upper respiratory

tract infections treated with azithromycin (500 mg on day 1 and 250 muday on days 2-5)

showed clinical cure rates of 98% and a 96% clearance of bacteria [117]. Clinical cure

rates for azithromycin ranged from 74% to 95% and clinical success rates ranged fiom

30

98% to 100Y0 in comparative studies on the eficacy of azithromycin in upper respiratory

tract infections [64]. Eradication rates for bacteria were greater than 89% and the clinical

eficacy of azithromycin was demonstrated to be equivalent to the other antibiotics tested.

Children given a totai of 30 mgkg of azithromycin over three or five days demonstrated

clinical cure rates of 78% to 95% for those with acute otitis media and 86% to 93% in

those with pharyngitis and/or tonsillitis [64]. The clinical cure rate of azithromycin was

greater but clinical success rates were not different than those of amoxiciilin in children

with acute otitis media [120].

Overall, clinical efficacy and pharmacological studies suggest that azithromycin

displays comparable or greater efficacy in eradicating certain infections compared to

other antibiotics, and that azithromycin requires a shorter and less frequent dosage

schedule.

1.3.2.6 Tolerability

The overall adverse effect rate is 12% for azithromycin with the most frequent

being gastrointestinal disturbances (9.6%) [diarrhea (2.6%), nausea (2.6%) and

abdominal pain (2.5%)] and central and peripheral nervous system effects such as

headaches and dizziness (1 -3%) [121]. This rate is lower than those seen for

arnoxicillin/probenecid (54.5%), cefaclor (1 6.7%), doxycycline (1 5.8%) and

erythromycin (20.4%). A study comparing the numbers of discontinuations of therapy

demonstrated that azithromycin had a lower percentage of discontinuations of therapy

compared to all the other drugs tested except for doxycycline [121]. There were no

neurological, audiometric or ophthalmoscopic abnormalities detected nor did

3 1

phospholipidosis occur in this study. Patients with hypersensitivity to other macrolides

should not be given azithromycin [121]. The adverse effects of azithromycin are similar

in young and elderly people 112 I].

1 -3 2 . 7 Drug Interactions

Drug metabolism in the body involves enzyme systems such as the mixed

h c t i o n oxidase system [61]. The enzyme cytochrorne P450 is one of the key enzymes

involved in this system. Hydroxylation by P450 often produces a reactive intermediate.

Some drugs can induce this enzyme which can iead to an increase or a decrease in the

metabolism of a second drug. Unlike erythromycin and many other macrolides,

azithromycin does not induce or inhibit cytochrome P450 IIIA enzymes [l22]. In part

because of this, no pharmacokinetic interactions have been found for patients

concomitantly receiving theophylline, cydosporine, warfarin, cimetidine, carbarnazepine,

methyl prednisolone, terfenadine, oral contraceptives or zidovudine [ 1 2 1, 123- 1251.

Azithromycin should be taken 1 hour before or 2 hours after antacids containing

aluminum and magnesium as this has been shown to reduce the peak serum

concentrations but not the total absorption of azithromycin [104, 1 2 11. Interactions

between azithromycin and either ergotamine or digoxin have not been investigated but

since erythromycin csuses ischemia and peripheral vasospasm when given with

ergotarnine and erythromycin causes increased serum digoxin levels, co-administration is

not recommended for ergotamine and azithrornycin and digoxin levels must be monitored

[641.

1.3.3 Anti-inflammatory Therapies

Anti-inflammatories can firnction to reduce the inflammation associated with

pneumonia and prevent host-mediated damage but they do not eradicate the source of the

infection. A number of anti-inflammatory strategies aim at down-regulating the

neutrophil response to infection and inflammation. One example of anti-inflammatory

drugs are the glucocorticoids that inhibit neutrophil emigration out of the vasculature and

into sites of inflammation by decreasing neutrophil adherence to endotheliurn, inhibiting

chemotaxis and inhibiting production of neutrophil chemotactic factors [126]. Inhibition

of phospholipase A2 by glucocorticoids prevents the production of arachidonic acid

derivatives such as leukotriene B4 that attract and activate neutrophils.

The anti-inflammatory glucocorticoid dexarnethasone inhibits neutrophil

superoxide generation, release of Iactoferrin and lysozyme, emigration, chemotaxis, and

production of LTB4 and IL-1 [127, 1281. Dexamethasone induces apoptosis of rat

thymocytes, however, it inhibits apoptosis in human neutrophils [129, 1301.

Dexamethasone decreases IL-8 production by macrophages pretreated with

dexamethasone before exposure to lipopolysaccharide [ 1 3 1 1.

1.3.4 Immunomodulation by Antibiotics

Respiratory tract infections are commonly treated with macrolide antibiotics.

Besides having antibacterial effects, many macrolides have been postulated to have anti-

inflammatory properties that may enhance their efficacies in reducing illness.

Azithromycin has proven to be more effective in the treatment of a number of respiratory

33

tract infections compared to erythromycin, another macrolide with comparable

antibacterial properties [64].

A number of reports have suggested that macrolides, such as erythromycin,

roxithromycin, and clarithromycin, have anti-inflammatory effects [7, 10, 28, 132- 1361.

Azithromycin may also have anti-inflammatory properties. Macrolides may exert this

effect by modulating the production of cytokines including IL- 1, IL-6, IL-8 and TNFa 17,

28, 132-1 391. Macrolides may also alter fhctions of inflammatory cells 120, 140-1421.

Some macrolides, such as azithromycin, roxithromycin, and clarithromycin, inhibit the

respiratory burst of neutrophils [143-1471. It was recently demonstrated that induction of

bovine neutrophil apoptosis by the veterinary macrolide tilmicosin may be associated

with anti-inflammatory benefits [ 1 481. Erythromycin, roxithromycin, clarithrornycin, and

midecarnycin have also been demonstrated to induce neutrophil apoptosis [149].

Apoptotic neutrophils, d i k e necrotic neutrophils, remain intact and are removed from

tissues by phagocytes, thus preventing the release of pro-inflammatory products in siru

148, 1501. The induction of apoptosis by azithromycin has not been previously studied

and its biological significance has yet to be uncovered.

1.4 Apoptosis

Cells can die via one of two forms of cell death, necrosis or apoptosis. Necrotic

cell death results in release of intracellular contents leading to inflammation and

extensive tissue damage [47].

Apoptosis, or programmed cell death, is a distinct form of cell death, both

morphologically and biochemically. In apoptosis, the cell actively participates in its own

34

destruction. Apoptosis is characterized by a number of features: the membrane of an

apoptotic celI remains intact, the cytoplasm and the nucleus becomes condensed, and the

DNA is cleaved into mono- and oligo-nucleosomes, which are commonly used as

indicators of apoptosis [150, 1511. In vivo, apoptosis is rapidly followed by uptake of

apoptotic cells into phagocytic cells [48]. This process is involved in the regulation of

cell turnover, tissue remodeling, regulation of the immune response, and resolution of

inflammation [48, 1 521.

1.4.1 Induction

Apoptosis can be induced by the binding of an appropriate ligand to either a

membrane receptor or a cytoplasmic receptor, which activates a second messenger

system [I 73. Various signals can induce apoptosis, such as the activation of receptors for

Fas ligand or TNF, glucocorticoid treatment, UV irradiation, and serum or growth factor

withdrawal (such as IL-3 or platelet-derived growth factor) [ I 48, 1 53 - 1 5 71. Although the

stimuli may vary, one mechanism consistently implicated in apoptosis is the activation of

a cascade of cytosolic proteases called caspases that initiate the changes associated with

apoptosis [Mi]. In human cells, many substrates for caspases have been identified as

DNA repair enzymes @oly-ADP-ribose-polymerase [PARP], and DNA-protein kinase),

homeostatic proteins (protein kinase C, D4-GDI, and sterol regulatory element binding

proteins), and structural proteins (nuclear lamins A and B, fodrin, and the nuclear mitotic

apparatus protein PUMA]) [40]. It is not known exactly how cleavage of these and other

substrates leads to the apoptotic phenotype except that multiple homeostatic pathways are

interrupted [40].

1.4.2 Neutrophil Apoptosis

Accumulation and bursting of necrotic neutrophils at the site of inflammation

contribute to the propagation of inflammation during infectious pneumonia and other

respiratory tract infections. Neutrophils recruited to sites of infection can undergo either

necrosis or apoptosis (programmed cell death). Cytotoxic compounds such as elastase,

hypoch1orous acid, and oxygen radicals reieased by necrotic neutrophils recruited to sites

of infection can damage surrounding tissues, and induce necrosis in neighbouring

neutrophils [7, 1591. Neutrophils injured in this fashion further release cytotoxic

compounds and pro-inflammatory mediators [13]. In contrast, apoptotic neutrophils may

be eliminated without spilling pro-inflammatory and cytotoxic products in siru [48, 1501.

Apoptosis is a less harmful process than necrosis since the membrane of an apoptotic

neutrophil remains intact. In addition, apoptotic cells are more rapidly phagocytosed by

macrophages, through the use of various surface-signaling molecules, including the

exposure of phosphatidylserine on the outer leaflet of the cell membrane 1160, 1611.

Thus, apoptosis is a more desirable form of cell death since the preservation of membrane

integrity and rapid clearance of apoptotic cells from the site of infection minimize host

tissue damage and inflammation [132, 162, 1631. This could be important in the

resolution of lung inflammation resulting fiom diseases such as streptococcal pneumonia.

1.4.3 Regulation of Apoptosis in Neutrophils

Activation of caspases (interleukin l Ptonverting enzyme homologues) initiates

apoptosis [40]. After isolation fiom blood, neutrophils undergo spontaneous apoptosis,

36

and thus neutrophil age is one factor involved in inducing apoptosis 1401. Forty-five

percent of neutrophils incubated for 24 hours are apoptotic. Caspase-3 is the primary

caspase involved in the initiation of the apoptotic cascade [40]. Active caspase-3 is

detected in apoptotic neutrophils but not in fieshly isolated neutrophils. Neutrophils

contain a number of caspase substrates including D4-GDI, gelsolin, lamin B, and fodrin

[40]. D4-GDI is a negative regulator of Rho GTPases, which may regulate the

cytoskeletal and membrane alterations accompanying cell death [164, 1651. Cleaved D4-

GDI is unable to bind GTPases thus allowing these GTPases to exert their effects.

Cleavage and consequent activation of gelsolin, an actin-severing protein, results in a

reduction in F-actin content and a disruption of cell structure leading to apoptosis [166].

Gelsolin cleavage also contributes to nuclear fragmentation. Lamin B is a nuclear matrix

protein which when cleaved leads to the chromatin condensation characteristic of

apoptosis [ 1671. Proteolytic processes during apoptosis make the surface of neutrophils

pro-phagocytic and more readily identifiable by macrophages. Fodrin is a cytoskeletal

component and its cleavage may be involved in membrane blebbing of apoptotic cells

[168]. Fodrin cleavage has been linked to the exposure of phosphatidylserine on the

outer membrane, which appears to be one mechanism by which phagocytic cells

recognize and clear apoptotic neutrophils [169]. Phosphatidylserine, normally localized

to the inner half o f the bilayer of the cell membrane of neutrophils, is translocated to the

outer half of the bilayer through the activation of scramblases and the downregulation of

aminophospholipid translocase (which normally reinternalizes any phosphatidylserine

that reaches the outer membrane) [161]. The receptor, 61D3, on macrophages may be

involved in phosphatidylserine-dependent phagocytosis [ I 701. Uptake of apoptotic cells

37

also occurs via the vitronectin receptor and CD36 on macrophages, which interact with

neutrophils via thrombospondin and an as yet unidentified molecule on the surface of

apoptotic neutrophils, which does not appear to be phosphatidylserine [47, 1 70, 1 7 13.

1 .S Research Hypothesis and Objectives

While the clinical effectiveness of azithromycin may be due in part to its

pharmacokinetic properties, it may have anti-inflammatory properties in addition to its

antibacterial effects. It is my hypothesis that azithromycin may induce apoptosis in

human neutrophils more effectively than other antibiotics and hence help the host to

dispose of these cells without perpetuating local inflammation via enhanced macrophage

cIearance of apoptotic neutrophils. The aim of this study was to determine the effects of

azithromycin on human neutrophil apoptosis, oxidative function and IL-8 production, in

the presence or absence of S. pneurnoniae.

Specific objectives of this study are:

1. To assess the induction of neutrophil apoptosis by azithromycin in comparison to

other drugs, in the presence or absence of Srreptococcus pneumoniae.

2. To investigate the effect of azithromycin on the phagocytosis of neutrophils by

macrophages.

3. To determine the effects of azithromycin on neutrophil fhction (oxidative

metabolism, IL-8 production) in comparison to other drugs, in the pcn ,,sence or

absence of Srreptococcus pneurnoniae.

38

CHAPTER 2: METHODS AND MATERIALS

2.1 Drugs

Azithromycin (Pfizer Canada Inc., Montreal, PQ, Canada), penicillin-G (Sigma

Diagnostics, St. Louis, MO, USA), and erythromycin (Sigma) were dissolved in pyrogen-

free phosphate buffered saline (PBS, pH 7.1) and diluted to a concentration of 0.05

@mL. Dexamethasone (Schering Canada Inc., Pointe-Claire, PQ, Canada) was diluted

in PBS (pH 7.1) to a concentration of 0.02 &mL.

2.2 Bacteria

Sfreptococcus pneumoniae 14259 (human clinical isolate obtained from Dr. R. R.

Read, Foothills Hospital, Calgary, AB, Canada) was grown overnight on Columbia blood

agar (CBA) plates at 37°C. Bacterial cells were collected, suspended in PBS (pH 7.1),

and diluted to approximately 1.5 x lo8 bacterialml using a 0.5 McFarland standard

(Dalynn Laboratory Products, Calgary, AB, Canada). The final concentration of viable

bacteria (- 1 x 10' CFU/mL) was calculated via colony forming unit (CFU) counts of

serial dilutions on CBA after a 24 h incubation at 37OC. Bacteria were used either live or

as sonicates (4 h, on ice, 85% duty cycle, output control setting 4, W-350 sonifiera,

Branson Ultrasonics Corporation, Danbury, CT, USA).

2.3 Subjects

Male and female subjects were recruited from volunteers at the University of

Calgary. Only those not taking medications were included in these studies. Throughout

39

this study more than 10 different donors were used and at least three different donors

were used to obtain data for the individual experiments.

2.4 Neutrophils

Blood was collected by venipuncture from healthy human volunteers and

collected in sodium heparin vacutaine? tubes (Becton Dickinson, Franklin Lakes, NJ,

USA). Neutrophils were purified by density gradient centrifugation using HistopaqueTM

1 11911 077 (Sigma). Whole blood was layered onto a gradient of Histopaquem 1077 (3

mL) that was underlayered with Histopaquem 1 1 19 (3 mL). AAer centrifugation

( I SOOxg, 30 min, Beckman J-6B centrifuge, Beckrnan Instruments, Pa10 Alto, CA, USA),

the mononuclear cell layer was removed and discarded. The neutrophil layer was

removed, washed in 10 mL of lx Hanks' balanced salt solution without calcium and

magnesium (HBSS, Gibco BRL, Life Technologies Inc., Grand Island, NY, USA), and

centrifbged (1 500xg, 1 0 min). Contaminating erythrocytes were lysed by resuspending

the pellet in 1 mL of sterile distilled water for 30 seconds, and osmolarity was restored by

adding 1 mL of 2 x HBSS (Gibco BRL). AAer washing in 10 mL of lx HBSS, purified

cells were resuspended in either HEPES-buffered RPMI 1630 (Sigma) supplemented

with 0.05pg/mL L-glutarnine, ca2+/Mg2+-free PBS (pH 7.1) (for NBT assay) or 1 x HBSS

(for macrophage phagocytosis) to a final concentration of 10' to 1 o6 cells/ml depending

on the experiment. Cells were counted in a haemacytometer (Spencer Bright-Line

Improved Neubauer, American Optical Corp., Buffalo, NY, USA), and viability was

assessed by 0.1 % trypan blue (Gibco BRL) exclusion. The cell suspension (1 00 pL) was

40

centrifuged (20xg, 10 min) onto a microscope slide with a cytospin (Shandon Southern

Producers Ltd., Cheshire, England), fixed and stained with Diff Quick (Baxter Healthcare

Corp., Miami, FL, USA) and neutrophil purity was assessed by direct microscopy

differential leukocyte counts. Neutrophils were incubated (37"C, 5% C 0 2 ) for various

times with PBS (pH 7.1) (controls), azithromycin (0.05 pg/mL), penicillin (0.05 pg/mL),

erythromycin (0.05 pg/mL), dexamethasone (0.02 pg/mL), or tumor necrosis factor-a

(TNFa, R&D Systems, Minneapolis, MN, USA, 5 ng/mL), with or without live

Streptococcus pneumoniae or lysate fiom S. pneumoniae.

2.5 Monocytes/Macrophages

Human peripheral blood monocytes can be cultured in vifro. They become

activated both by the isolation process fiom whole blood and by attachment to tissue

culture plastic surface 11721. This adherence in the presence of serum mimics the

immigration of monocytes through the vasculature into the site of infection and primes

the monocyte for inflammatory responses [173]. The activated monocytes differentiate

into macrophages when serum is present in the medium [174]. As the monocytes

differentiate they increase in size and phagocytic activity, and many biochemical and

morphological changes occur [172]. Human leukocytes contain esterases which

hydrolyze aliphatic and aromatic esters [45, 1751. Macrophages contain a nonspecific

esterase that can be detected by addition of the substrate a-naphthyl acetate coupled to a

diazonium salt, followed by counter-staining with methyl green. Hydrolysis of the

substrate produces an insoluble coloured azo dye. Macrophages show a diffuse

41

r e a r o w n staining of the cytoplasm with a green nucleus (positive for nonspecific

esterase), while monocytes, which produce very littie nonspecific esterase, appear green

(negative for esterase).

Blood was collected by venipuncture from healthy human volunteers and

collected in Vacutainea C P V cell preparation tubes with sodium citrate (Becton

Dickinson). Monocytes were purified by density centrifbgation (2000xg, 30 min). The

mononuclear cell layer was removed, washed in 1 x HBSS (1 0 mL), and centrifuged

(300xg, 15 min). The pellet was resuspended in 1 x HBSS (10 mL) and centrifuged

(3OOxg, 10 min). The pellet was resuspended in Iscove's modified Dulbecco's medium

(MDM) (Gibco BRL) to a final concentration of 6 x lo6 cells/mL. Cells were counted in

a haemacytometer, and viability was assessed by trypan blue (0.1%) exclusion.

Monocytes (1 mL) were incubated (37"C, 5% CO2) in 24-well tissue culture plates

(Costar, Cambridge, MA, USA) for 60 min after which non-adherent cells (mainly

lymphocytes) were removed by washing 3 times with l x HBSS (37OC, 1 mL) [176].

Monocyte purity was assessed by staining cytospin preparations with Diff Quick and

direct microscopy differential leukocyte counts. Monocytes were cultured in 1 mL

Iscove's MDM containing 10% fetal bovine serum (Sigma) and were allowed to

differentiate into macrophages for seven days. The media was replaced every 2-3 days

by aspiration.

Macrophage differentiation was ascertained by mature cell morphology (size and

shape changes) after being stained with Diff Quick and by a positive reaction for

nonspecific esterase. Nonspecific esterase staining was done as previously described

42

[45]. Briefly, after cells were fixed, they were incubated with esterase substrate solution

containing a-naphthyl acetate (Sigma) and hexazoatized pararosalanine (Sigma) for 45

min at 37°C. Slides were counter-stained for 2 min with 1% methyl green and examined

by light microscopy.

2.6 CFU Counts

S. pneumoniae was incubated with PMNs and drugs (37"C, 5% COz) at a ratio of

10:l for 2 h and 6 h, plated on CBA plates after serial dilutions, and colonies were

counted after 24 h incubation at 37°C.

2.7 Annexin-VIPropidium Iodide Labelling

Neutrophil apoptosis and necrosis were assessed by double staining with FITC-

conjugated mexin-V and propidium iodide (PI) using an Annexin-V FLUOS kit

(Boehringer-Mannheim GmbH, Germany), according to manufacturer's instructions.

Annexin binds with high affinity to phosphatidylserine which is translocated from the

inner half of the bilayer to the outer half of the membrane bilayer during both apoptosis

and necrosis 11 591. Necrotic ceils are differentiated by staining with propidium iodide.

After 1 hour co-incubation with or without drugs and with or without bacterial lysate (4: 1

bacteria to cell ratio), PMNs (5.5 x lo6 cells/mL) were centrifuged (Baxter Canlab

Biofuge A, Heraeus Sepatech GmbH. Germany) (200xg, 5 min), then washed with I mL

PBS (pH 7.1). Cells were centrihged again, resuspended in 100 pL of FITC-conjugated

annexin-V and propidiurn iodide staining solution, and incubated for IS minutes in the

43

dark. Samples were then centrihged (200xg, 5 min) and resuspended in PBS (pH 7.1),

and a wet mount of 15 pL was observed by epifluorescent microscopy (450 m and 535

nm) (Leitz Aristoplan, Germany) at 400x magnification and the percentages of apoptotic

and necrotic cells were determined from ten different fields per preparation. The high

and low counts were discarded and the other 8 were averaged.

As a positive control for the annexin-V/PI labeIling system, PMNs were incubated

with 5 ng/mL (approximately 100 U/mL) recombinant human TNFa in PBS (pH 7.1)

with 0.1% bovine serum albumin for 1, 2, and 3 hours. TNFa at this concentration is

known to induce neutrophil apoptosis [177].

2.8 Cell Death ELISA

Neutrophil apoptosis was also assayed via ELISA. Neutrophils (lo5 cells/mL)

were co-incubated for 6 hours with or without drugs and with or without live bacteria

(10: 1 bacteria to cell ratio) and stored at -70°C for later analysis. Samples were assayed

for apoptosis with a Cell Death Detection ELISA kit (Boehringer Mannheim), which

detects the histone region (H 1, H2A, H2B, H3, and H4) of mono-nucleosomes and oligo-

nucleosomes that are formed during apoptosis. The plates were read at 405 nrn

(THERMOmaxTM microplate reader, Molecular Devices Corp., Menlo Park, CA, USA) at

intervals for 58 min, starting 1 rnin after addition of substrate. Results were expressed as

the ratio of the sample absorbance versus the mean absorbance of the vehicle-treated

control.

44

2.9 Macrophage Phagocytosis

PMNs (6 x 1 o6 cells/mL) were incubated for 1 hour with or without azithromycin

and then centrifuged (minimum speed, 5 min, microcentrifuge). The pellet was washed

by resuspending in 1 x HBSS (1 mL) and centrifuged (minimum speed, 5 min). This

pellet was resuspended in l x HBSS to the original ceH volume of 300 pL. The PMNs

were acided to the wells containing 7 day old cultured macrophages and incubated for 1

hour (37"C, 5% C02). PMNs were obtained from the same donor as described above.

The wells were washed three times with 1 x HBSS (37OC, 1 mL) and stained with Diff

Quick. Three counts of 100 cells were done on each well to determine the number of

macrophages containing phagocytosed PMNs. A total of 2400 macrophages were

counted. Only macrophages that had completely surrounded a PMN were counted.

2.10 Nitro Blue Tetrazolium (NBT) Assay

The oxidative function of PMNs (lo6 cells/mL) co-incubated for 1 hour with or

without drugs and with or without bacterial lysate (10:l bacteria to cell ratio) was

assessed by NBT reduction. Cells were incubated in chamber slides (Nalge Nunc

International, Naperville, IL, USA) for 20 minutes with nitro blue tetrazoliwn (NBT, 2

mg/mL, Sigma) in the presence or absence of 2 pg/mL phorbol 12-myristate 13-acetate

(PMA) (Sigma). Cells were fixed with methanol and counterstained with safianin (1%).

The percentage of oxidatively active cells with blue formazan crystals in their cytoplasm

was calculated following direct microscopic examination and counting under oil

immersion ( 1 000x) with a light microscope (Zeiss, Germany).

45

2.1 1 Interleukin-8 Assay

PMNs (2x10' cells/mL) were incubated for 2 hours with drugs, then washed in

PBS (pH 7.1 ) and incubated (24 h, 3 7OC, 5% C02) with or without S. pneumoniae lysate

(10:l bacteria to cell ratio). Cells were then centrifbged (800xg, 10 min) and the

supernatant collected. IL-8 production was assessed by a Quantikinem human IL-8

sandwich enzyme immunoassay (R&D Systems) according to the manufacturer's

instructions. This assay is specific for natural and recombinant human IL-8. No

significant cross-reactivity or interference has been observed for other human or mouse

interleukins or other factors tested. The sensitivity of this kit is 10 pg/rnL.

2.12 Statistical Analysis

Results were expressed as mean + SEM and compared by one-way analysis of

variance (ANOVA) followed by Tukey's test for multiple comparison except for the

macrophage phagocytosis results which were analyzed using Fisher's Exact test with a 2-

sided P value and a chi-square approximation. P<0.05 was considered significant.

CHAPTER 3: RESULTS

3.1 Cell Isolation

The success of the cell isolation procedures was determined via examination of

the viability and purity of the resultant populations. The viability of purified PMNs

incubated in PBS (pH 7.1), HBSS, or RPMI 1640 was >95%, and cell purity was >98%.

The purity of the isolated mononuclear cells was >98% and viability was >97%.

Monocytes accounted for -40% of the mononuclear cells. Contaminating cells in the

purified monocytes were mainly Lymphocytes and the occasional neutrophil, but due to

the short lifespan of neutrophils none remained after the third day of incubation [44].

Contaminating lymphocytes in the monocyte preparation were removed by washing

following the initial 1 hour incubation in the well plates, as lymphocytes do not adhere to

the wells [ I 761.

3.2 CFU Counts

CFU counts were done to determine whether the antibacterial activity of the

antibiotics was altered by the presence of neutrophils and whether S. pneumoniae was

susceptible to the antibiotics tested. After 6 hours of co-incubation with PMNs,

azithromycin, penicillin, and erythromycin significantly decreased S. pneumoniae

numbers cor~pared to controls (Figure I). At 6 hours, bacterial numbers in preparations

containing azithromycin were significantly lower than those exposed to other antibiotics.

Incubation with dexamethasone did not affect bacterial numbers.

3.3 Neutrophil Apoptosis

To assess the anti-inflammatory potential of azithromycin, I looked at the

induction of apoptosis in neutrophils. To determine whether azithromycin induces

neutrophil apoptosis two systems were used - annexin-V/PI labelling and Cell Death

Detection ELISA. The annexin-VPI fluorescent labelling system allowed discrimination

of apoptotic fiom necrotic cells (Figure 2A and Figure 28). TNFa induced neutrophil

apoptosis in a time-dependent fashion (Figure 3). In the absence of S. pneumoniae lysate,

azithromycin induced neutrophil apoptosis at levels similar to those seen in cells

incubated with TNFa for 3 h (Figure 4A). Cells incubated with penicillin, erythromycin,

or dexarnethasone were not different fiom vehicle controls. Addition of bacterial lysate

inhibited the induction of apoptosis by azithromycin (Figure 4B).

In an attempt to explain this inhibition of apoptosis by bacterial lysate, cell

necrosis was assessed to see if neutrophil necrosis was higher in the samples containing

lysate. As shown in Figure 5, S. pneumoniae did not afCect neutrophil necrosis. In

addition, neutrophil recovery was not significantly different between cells incubated with

saline ( 7 . 4 3 ~ 1 o6 f 4.61 x 10' cells/mL), azithromycin (6 .06~ 1 o6 f 4.8 1 x 10' cells/ml), S.

pneumoniae (6 .88~ lo6 f 7 . 0 4 ~ I 0' cellslml) or azithromycin and S. pneumoniae together

( 7 . 1 5 ~ 1 o6 i 3.3 1 x 10' cells/mL).

The induction of neutrophil apoptosis by azithromycin was also detected by

ELISA at 6 h in the absence of S. pneumoniae (Figure 6A). The colorimetric reaction

was rapid enough to be detected after 1 minute of incubation with reagent. As was

observed with annexin-V labelling, co-incubation with S. pneimoniae abolished this

48

effect (Figure 6B). Incubation of neutrophils with penicillin, erythromycin, or

dexamethasone did not affect neutrophil apoptosis in the presence or absence of S.

pneumoniae.

3.4 Macrophage Phagocytosis

Apoptotic neutrophils are rapidly phagocytosed in vivo by phagocytes, a process

that disposes of these cells without perpetuating Iocal inflammation. To determine

whether the increased number of apoptotic cells seen in azithromycin-treated neutrophil

populations contributed to an increased uptake of these cells by macrophages, I used

monocyte-derived macrophages. Monocyte differentiation into macrophages was

determined using two techniques: mature cell morphology and nonspecific esterase

staining. On day 0, all cells looked like typicai monocytes (cell size Iess than 13 pm, a

round nucleus, and a thin ring of cytoplasm). Cell morphology (cell size greater than 13

pm, a bean shaped nucleus, and much larger cytoplasmic area) revealed differentiation of

70% on day 2, 96% on day 5, and 98% on day 7. Nonspecific esterase staining showed

differentiation of 79% on day 2, 92% on day 5, and 98% on day 7 (Figure 7). These

measures indicate that a large population of differentiated macrophages was present on

day seven

Macrophages which had ingested neutrophils were identified via tight microscopy

(Figure 8). Macrophage phagocytosis of neutrophils after 1 hour was found to be

significantly increased for azithromycin-treated PMNs compared to control PMNs

(Figure 9).

49

3.5 Oxidative Function and LC-8 Production

To determine whether the induction of apoptosis by azithromycin affects the

function of neutrophils, I looked at oxidative function and 1L-8 production. The ability to

produce reactive oxygen intermediates for microbial killing is an essential function of

neutrophils. None of the drugs affected oxidative h c t i o n in resting (not exposed to

PMA) or stimulated (exposed to PMA) neutrophil populations (Figure IOA). Similarly,

the drugs did not affect oxidative function when co-incubated with bacterial lysate

(Figure 10B).

Neutrophils produce chemotactic IL-8 in order to recruit more neutrophils to sites

of infection. Exposure of neutrophils to S. pneumoniae lysate promoted synthesis of IL-8

(Figure 1 1). IL-8 secretion was not affected by any of the antibiotics in the presence or in

the absence of S. pneumoniae lysate compared to controls (Figure 11). In contrast,

dexamethasone significantly inhibited S. pneumoniae lysate-induced IL-8 synthesis.

Figure 1. S. pneumoniae recovery (loglo CFU) after 2 h and 6 h co-incubation of PMNs

with azithromycin (---.em- -*), penicillin (- -@ - -), erythromycin (- X - -) and

dexamethasone (- -A-). Antibiotic treatments are significantly different from

control (-*-) and dexarnethasone at both times (n=5; experiment was repeated 3

times). * PcO.05 compared with control. A Pc0.05 azithromycin compared with all

groups.

1 2 3 4 5 6 7 Time (h)

Figure 2. Light micrographs (400x) of PMNs stained with the annexin-V/propidiurn

iodide system. A) Bright field micrograph illustrating several PMNs, and B)

fluorescence micrograph of the same field showing an apoptotic PMN (arrow) and two

necrotic PMNs (arrowheads); viable neutrophils seen on the bright field micrograph were

not labelled.

Figure 3. Apoptosis in PMNs treated with 5 ng/mL TNFa for 1 , 2, and 3 h (n=4;

experiment was repeated 2 times), determined by annexin-VIP1 labelling. The bar

labelled control represents the mean of the control values (n=6) from 1, 2, and 3 h. *

P<0.05 compared with control.

CONTROL TNF I H TNF2H TNF3H SAMPLE

Figure 4. Apoptosis in neutrophils determined by annexin-VffI labelling after 1 h

incubation with azithromycin (Azi), penicillin (Pen), erythromycin (Ery), or

dexamethasone (Dex). A) Without lysate, B) with S. pneumoniae lysate (4 CFU:I

PMN). TNFa sample is the 3 h incubation value from Figure 2. * P<0.05 compared

with control. ** Pc0.05 compared with control and other drugs (n25; experiment was

repeated 5 times).

TNFa CONT AZI PEN ERY DEX

Figure 5. PMN necrosis in samples incubated with azithromycin (Azi), penicillin (Pen),

erythromycin (Ery), or dexamethasone (Dex) and with (black bars) or without (white

bars) S. pneumoniae Iysate determined by annexin-V/PI labelling. No significant

difference between any samples (n25; experiment was repeated 5 times).

CONT AZI PEN ERY D M CONT AZI PEN E R Y DEX

Figure 6. Apoptosis detected by Cell Death ELISA, afier 6 h incubation. A) Without S.

pneurnoniae, B) with live S. pneurnoniae. Absorbance readings were plotted as a ratio of

the sampIe absorbance to the control, which was set at 1 . Azithromycin (-==C--• ==),

penicillin (- -0 - -), erythromycin (- =X- -) and dexamethasone (- -A-),

control (-*-). *Pc0.05 compared with control and dexamethasone (n 2 5;

experiment was repeated 4 times).

Figure 7. Light micrograph (400~) of monocytes!macrophages stained for nonspecific

esterase. Positively stained cells (monocyte-derived macrophages) appear red and

negatively stained cells (undifferentiated monocytes) appear green.

Figure 8. Light micrograph (400~) of macrophages incubated with PMNs for one hour.

A macrophage that has phagocytosed a neutrophil (arrow) and a non-phagocytosed PMN

(arrowhead) are seen.

Figure 9. Phagocytosis of azithromycin-treated PMNs by macrophages afier 1 h,

following a 1 h incubation of PMNs with azithromycin or HBSS (control). *P<0.05

versus control (experiment was repeated 3 times; a total of 2400 macrophages were

counted). No error bars are shown as values are discrete numbers and not a mean.

control azithromycin

Sample

Figure 10. Oxidative activity determined by the percentage of resting (white bars) and

PMA-stimulated (black bars) PMNs able to reduce NBT. Samples incubated 1 h with

azithrornycin (Azi), penicillin (Pen), erythromycin (Ery), or dexamethasone (Dex) and

A) without S. pneumoniae Iysate or B) with S. pneumoniae lysate. In A) and B), all

stimulated groups are significantly different from a11 resting groups. No significant

difference between drug-treated and control samples within any group (n=5; experiment

was repeated 3 times).

CONT AZl PEN ERY DEX C O W AZI PEN ERY DU(

CONT AZI PEN ERY DEX CONT AZI PEN ERY DEX

Figure 1 1 . IL-8 production by PMNs incubated 24 h with (black bars) or without (white

bars) S. pneumoniae lysate following a 2 h incubation with azithromycin (Azi), penicillin

(Pen). erythromycin (Ery), or dexamethasone (Dex). No significant difference was

observed between antibiotic-treated and control samples within groups treated with or

without lysate (1124; experiment was repeated 2 times). *P<0.05 versus sham-treated

control.

Cont Azi Pen Ery D e w Cont Ati Pen Ery Dex

CHAPTER 4: DISCUSSION

My study investigated the effects of azithromycin on human circulating

pol ymorphonuclear neutrophils. My results indicate that azithromycin induces neutrophil

apoptosis without affecting the oxidative metabolism or IL-8 production of these cells in

vib-o (Figures 4A, 6A, 10, and I 1) Exposure of neutrophils to azithromycin significantly

increased in vitro phagocytic uptake of these cells by macrophages (Figure 9). Co-

incubation of neutrophils with S. pneumoniae inhibited the induction of apoptosis by

azithromycin (Figure 4B, and 68).

Co-incubation of live S. pneumoniae with antibiotics (azithromycin,

erythromycin, and penicillin) and neutrophils confirmed that the antibacterial effects of

these antibiotics are not altered by the presence of neutrophils, and that azithromycin

exhibits effective antibacterial properties (Figure 1). After six hours of incubation

azithromycin was more effective in eradicating S. pneumoniae than the other antibiotics.

My observations are consistent with those published in other reports [77, 781.

Previous studies have suggested that macrolides have anti-inflammatory effects,

but the mechanisms of this action remain unclear 17, 10, 28, 132-1361. A number of

these studies have focused on the effects of macrolides on cytokine production [7, 28,

132-1391. Other studies have suggested that macroiides modulate the hc t i ons of

polymorphonuclear leukocytes, macrophages, and lymphocytes, as well as altering the

functions of airway secretory cells and epithelial cells [lo, 140-1421. Although still

controversial, findings from these previous studies indicate that erythromycin,

roxithromycin and clarithromycin may reduce the production of pro-inflammatory IL- 1,

73

IL-6, IL-8 and TNF. In addition, erythromycin derivatives, including azithromycin,

roxithromycin and clarithromycin, are known to inhibit the oxidative response of

neutrophils in a time and concentration dependent fashion [I 43- 1471. My results indicate

that after two hour exposure to azithromycin, the release of IL-8 by a population of

human neutrophils remains unchanged, despite the commitment of some cells to undergo

programmed cell death (Figure 11). Moreover, results from my study indicate that one

hour exposure to azithromycin is insufficient to reduce oxidative metabolism of human

neutrophils (Figure 10). Taken together, these observations indicate that the effects of

azithromycin on IL-8 production as well as oxidative metabolism are time and

concentration dependent (as the other studies used different drug concentrations and

exposure times than my study).

Dexamethasone is known to reduce IL-8 production by LPS-treated human

peripheral blood monocytes and alveolar macrophages [131]. In my study,

dexarnethasone significantly inhibited Srreprococcus pneumoniae lysate-induced IL-8

synthesis by neutrophils (Figure 11). In vivo, a reduction in IL-8 could be an important

strategy to reduce the inflammatory lung injury caused by neutrophils. IL-8 is a major

chemotactic cytokine for neutrophils, and macrophages and monocytes are the major

producers of this cytokine during the inflammatory response 1281. The large numbers of

neutrophils at sites of inflammation, however, make these cells an additional important

contributor of IL-8 [28, 1781. The results of my study, however, indicate that the eficacy

of azithromycin is not dependent on the reduction of pro-inflammatory IL-8 (Figure I 1).

Accumulation and necrosis of neutrophils in tissues contribute to the propagation

of inflammation in a number of inflammatory diseases, including infectious pneumonia

74

[I, 11, 551. In contrast, apoptotic neutrophils may be eliminated without spilling pro-

inflammatory products in siru. A recent report indicates that erythromycin,

roxithrom ycin, clari throm ycin, and midecarn ycin shorten neutrophil survival by

accelerating apoptosis 11491. The biological significance of this observation has yet to be

uncovered. It is shown in my study that after one hour exposure to azithromycin,

apoptosis may be detected in human neutrophils via annexin-V labelling of exposed

phosphatidylserine (Figure 4A). Additionaily, neutrophils exposed to azithrornycin for

one hour exhibited an increased level of phagocytic uptake by human monocyte-derived

macrophages (Figure 9). Only activated macrophages are able to phagocytose apoptotic

neutrophils. Human peripheral blood monocytes cultured in vitro become activated both

by the isolation process from whole blood and by attachment to tissue culture plastic

s u ~ a c e s in the presence of serum [172]. The adherence mimics the immigration of

monocytes through the vasculature into the site of infection and primes the monocyte for

inflammatory responses [173]. Macrophages use phosphatidylserine to recognize and

phagocytose certain populations of apoptotic cells. The macrophage receptor, 61D3,

binds to phosphatidylserine on apoptotic cells [170]. Additionally, the vitronectin

receptor and CD36 on macrophages interact with apoptotic neutrophils via

thrombospondin and a surface molecule (which does not appear to be phosphatidylserine)

[47, 170, 1711. The results of my study suggest that apoptotic neutrophils may also use

the p hosphatidy lserine-dependent mechanism for uptake into macrophages since both

phosphatidylserine exposure (which annexin-V binds) and phagocytic uptake of

neutrophils by macrophages were increased in azithromycin-treated neutrophils.

75

I hypothesize that azithromycin may induce apoptosis in human neutrophils more

effectively than other antibiotics and hence help the host dispose of these cells without

perpetuating local inflammation. Consistent with this hypothesis, it was recently shown

that tilmicosin, a macrolide used in the treatment of bovine pneumonic pasteurellosis, has

anti-inflammatory benefits which are associated with neutrophil apoptosis and inhibition

of local leukotriene B4 release [148]. In addition, it is well established in the literature

that physiological removal of apoptotic neutrophils in the inflamed lung in vivo promotes

the resolution of inflammation [179, 1801. Neutrophil influx must be limited and

neutrophils that are no longer necessary must be removed in order for the resolution of

inflammation to occur [179]. Macrophage clearance of apoptotic neutrophils prevents

tissue destruction by the cytotoxic intracellular contents of neutrophils.

Annexin-V labelling detects the exposure of phosphatidylserine on the outer

leaflet of the cell membrane, an early event in cell death [159, 1811. In this system,

apoptosis is distinguished fiom necrosis by propidium iodide labelling, which enters only

cells with a compromised membrane. The experimental assessment of apoptosis was

validated by using TNFa as a positive control (Figure 3) [177]. My results indicate that

human neutrophils exposed to azithromycin for one hour in vitro were committed to

apoptotic death, while erythromycin, penicillin and dexamethasone showed no induction

of apoptosis in neutrophils (Figure 4A). In addition, the formation of mono- and oligo-

nucleosomes, a late event in apoptotic cell death, was detected in neutrophils via ELISA

after six hours of incubation with azithromycin (Figure 6A).

In another study, erythromycin was shown to induce apoptosis in neutrophils in

virro [149]. The fact that this was not observed in my study may be explained by the

76

different incubation times and antibiotic concentrations; the previous study assessed the

induction of neutrophil apoptosis afier twenty-four hours of incubation, and used a

minimum concentration of 1 pg/mL [149]. My findings suggest that azithromycin

induces neutrophil apoptosis more quickly and at lower drug concentrations than

erythromycin. Neutrophils have a higher affinity for azithromycin than erythromycin.

Additional studies will help assess whether this difference in affinity accounts for the

more rapid induction of neutrophil apoptosis by lower concentrations of azithromycin

compared to erythromycin 1641. The anti-inflammatory glucocorticoid dexamethasone

has been reported to inhibit neutrophil apoptosis [130], but this effect was not seen in my

study. Again, as inhibition of apoptosis in human PMNs was observed after twenty-four

hours of exposure to dexarnethasone, the six hour incubation used in my study may not

have been sufficient to significantly inhibit apoptosis [130]. Regardless, my study

demonstrates that azithrornycin induces apoptosis in human neutrophils to levels greater

than those observed for other drugs and that azithromycin-treated neutrophils show a

greater uptake by macrophages (Figures 4A, 6A, and 9). The induction of apoptosis may

be responsible for the increased phagocytic uptake of azithromycin-treated neutrophils

via recognition of apoptotic cells by a phosphatidylserine-dependent mechanism. I

postulate that this mechanism may at least in part contribute to the clinical efficacy of this

macrolide in the treatment of infectious pneumonia by limiting the ability of neutrophils

to damage surrdunding tissues and preventing further amplification of inflammation.

The mechanism by which azithrom ycin indwes apoptosis in neutrophils remains

unknown and is an area that requires W e r study. It may be that azithromycin binds to

and activates receptors such as the Fas receptor or the TNF receptor that have been

77

implicated in the induction of the apoptotic cascade of events [153, 154, 1821.

Alternatively, azithromycin may enter neutrophils and induce apoptosis by activating a

nuclear or cytosolic receptor. It has been suggested that the loss of lipid asymmetry is

linked to activation of the Fas system [161]. The mechanism by which the enzymes

involved in phosphatidylserine translocation become activated or inhibited is not known

but has been postulated to involve increased cytosolic calcium concentrations in

erythrocytes and platelets [183]. Alternatively, the mechanism by which TNFa results in

neutrophil apoptosis is postdated to involve the generation of reactive oxygen

intermediates [ 1 841.

The effect of azithromycin on other cell types involved in the mediation of

inflammatory lung injury is another area that may give additional information about the

clinical efficacy of azithromycin in resolving pulmonary infection.

My results suggest that the apoptosis-inducing effect of azithromycin is abolished

when neutrophils are co-incubated with Streptococczrs pneumoniae lysate (Figures 4B,

and 6B). Levels of necrosis were not different between samples with or without lysate,

indicating that the low apoptotic level was not due to an increase in necrosis (Figure 5).

The mechanisms whereby S. pneumoniae products may reverse or inhibit the apoptotic

signal remain to be investigated. In one study, the LPS of Gram-negative bacteria has

been shown to inhibit TNFa-induced apoptosis and spontaneous apoptosis in neutrophils

in virro, possibly by inducing synthesis of proteins which are involved in regulating the

apoptotic pathway or by production of cytokines such as IL-I P and TNFa [185]. TNFa

(in concentrations under 0.1 U/mL), and IL-1P inhibit apoptosis. In another study,

Staphylococcal enterotoxins (superantigens) have been shown to increase production of

78

LTBJ, and LTB4 is able to delay neutrophil apoptosis [186]. Thus, superantigen may act

indirectly to inhibit neutrophil apoptosis. In vivo, superantigen may also inhibit

neutrophil apoptosis through its effects on cytokine production by other immune cells

such as monocytes and lymphocytes [186]. It remains to be shown whether other Gram-

positive bacteria have components that also inhibit apoptosis, and whether this effect may

contribute to the difficulty in treating infections caused by these organisms. Further

studies are also needed to assess whether the inhibition of azithromycin-induced

apoptosis observed in my study occurs in the presence of other bacterial species,

including Haernophilus injluemae, another respiratory tract pathogen which azithromycin

is more effective against than S. pneurnoniae [64]. A paper recently suggested that

macrolides might significantly reduce the inflammatory injury associated with the

presence of H. influenzae in the lower respiratory tract, via unknown mechanisms [134].

In summary, azithrornycin induces apoptosis in human neutrophils more

effectively than erythromycin or penicillin (Figures 4A and 6A). Phagocytic uptake of

azithromycin-treated neutrophils by macrophages is enhanced (Figure 9), which in turn

may confer a significant anti-inflammatory benefit to this drug. The oxidative function of

resting or stimdated neutrophils and the production of IL-8 remain unchanged (Figures

10 and l l ) , indicating that these functions are unaffected in the early stages of

azithromycin-induced apoptosis. I postulate that the clinical efficacy of azithromycin in

the treatment of lower respiratory tract infections is due, at least in part, to committing

neutrophils to apoptotic death, hence reducing the amount of cytotoxic and pro-

inflammatory compounds released by these cells in the infected lung. Whether inhibition

79

of neutrophil apoptosis by S. pneumoniae contributes to the difficulty in treating

infections caused by this pathogen requires further investigation.

Results from my study open the way to a number of directions for future research.

A summary of these include:

1 ) Exploring the mechanism by which azithromycin induces apoptosis in neutrophils.

2) Determining if the higher affinity of azithrornycin for neutrophils is responsible for

inducing apoptosis more rapidly and at lower drug concentrations than erythromycin.

3) Examining the effect of azithromycin on other inflammatory cells.

4) Determining whether azithromycin-induced neutrophil apoptosis and enhanced

phagocytosis by macrophages occur in vivo.

5) Exploring the biological significance and clinical importance of azithromycin-

induced apoptosis in v iva

6) Elucidating the mechanism by which Streptococcus pneumoniae reverses or inhibits

the induction of apoptosis by azithromycin and determining whether this inhibition

occurs in the presence of other species of bacteria.

7) Determining whether the inhibition of apoptosis by Streptococcus pneumoniae

contributes to the difficulty in treating streptococcal pneumonia.

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APPENDIX A: Structure of Azithrornvcin and Ewthromvcin

Azithromycin

Erythromycin

(Adapted fiom reference 64)