pii: s0022-1759(99)00148-9

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Ž . Journal of Immunological Methods 232 1999 23–43 www.elsevier.nlrlocaterjim The use of flow cytometry to measure neutrophil function Stephan F. van Eeden a, ) , Maria E. Klut a , Blair A.M. Walker b , James C. Hogg a a Pulmonary Research Laboratory, UniÕersity of British Columbia, St. Paul’s Hospital, 1081 Burrard Street, VancouÕer, British Columbia, Canada V6Z 1Y6 b Department of Pathology, UniÕersity of British Columbia, St. Paul’s Hospital, VancouÕer, British Columbia, Canada V6Z 1Y6 Abstract Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acute bacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophil function. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis of conditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia–reperfusion injury and rheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In this review, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization, Ž . F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species ROS production. The use of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majority of neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in function caused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows the simultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis provides a rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Polymorphonuclear leukocytes; F-actin; Calcium; Adhesion; Phagocytosis; Degranulation; Reactive oxygen species; Myeloper- oxidase 1. Introduction Neutrophils are important effector cells in numer- ous inflammatory conditions. The relevance of neu- trophil function is usually thought of in terms of host Ž defense in bacterial and fungal infections Malech . and Gallin, 1987; Bainton, 1992 because of the association between the lack of neutrophils and over- whelming infection with these organisms. To fulfill this defensive role, intravascular neutrophils need to ) Corresponding author. Tel.: q1-604-806-8346; fax: q1-604- 806-8351; e-mail: [email protected] sense the focus of infection, slow down and adhere to the endothelium of capillaries and venules adja- cent to the inflammatory locus, migrate through the vessel wall and the interstitial tissues to the infec- tious site, phagocytose, kill and digest the invading microorganisms. During this process, neutrophils also need to produce factors to ensure their survival in the hostile inflammatory milieu, recruit additional phagocytes, inactivate their own toxic products and induce their own death pathway to prevent damage Ž . to normal host tissue Fig. 1 . It may be misleading to depend on a single test to evaluate neutrophil function because various re- 0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0022-1759 99 00148-9

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Page 1: PII: S0022-1759(99)00148-9

Ž .Journal of Immunological Methods 232 1999 23–43www.elsevier.nlrlocaterjim

The use of flow cytometry to measure neutrophil function

Stephan F. van Eeden a,), Maria E. Klut a, Blair A.M. Walker b, James C. Hogg a

a Pulmonary Research Laboratory, UniÕersity of British Columbia, St. Paul’s Hospital, 1081 Burrard Street, VancouÕer, British Columbia,Canada V6Z 1Y6

b Department of Pathology, UniÕersity of British Columbia, St. Paul’s Hospital, VancouÕer, British Columbia, Canada V6Z 1Y6

Abstract

Neutrophils are important professional phagocytic cells that provide the host with a first line of defense against acutebacterial and fungal diseases and recurrent, severe or unusual infections are associated with inherited defects of neutrophilfunction. Furthermore, abundant evidence links inappropriate neutrophil-mediated tissue damage to the pathogenesis ofconditions such as acute respiratory distress syndrome, septicemia with multiorgan failure, ischemia–reperfusion injury andrheumatoid arthritis. Flow cytometry has been increasingly used to evaluate the functional capabilities of neutrophils. In thisreview, we discuss the use of flow cytometry to assess neutrophil functional responses including calcium mobilization,

Ž .F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species ROS production. Theuse of flow cytometry to identify neutrophil priming is also discussed. The advantage of flow cytometry is that the majorityof neutrophil functions can be measured using a small volume of whole blood that reduces artifactual changes in functioncaused by purification procedures. The advent of numerous new fluorochromes and multiparametric analysis allows thesimultaneous measurement of several neutrophil functions in the same population of cells. Flow cytometric analysis providesa rapid screen for abnormalities of neutrophil function and reflects more accurately their behavior in vivo. q 1999 ElsevierScience B.V. All rights reserved.

Keywords: Polymorphonuclear leukocytes; F-actin; Calcium; Adhesion; Phagocytosis; Degranulation; Reactive oxygen species; Myeloper-oxidase

1. Introduction

Neutrophils are important effector cells in numer-ous inflammatory conditions. The relevance of neu-trophil function is usually thought of in terms of host

Ždefense in bacterial and fungal infections Malech.and Gallin, 1987; Bainton, 1992 because of the

association between the lack of neutrophils and over-whelming infection with these organisms. To fulfillthis defensive role, intravascular neutrophils need to

) Corresponding author. Tel.: q1-604-806-8346; fax: q1-604-806-8351; e-mail: [email protected]

sense the focus of infection, slow down and adhereto the endothelium of capillaries and venules adja-cent to the inflammatory locus, migrate through thevessel wall and the interstitial tissues to the infec-tious site, phagocytose, kill and digest the invadingmicroorganisms. During this process, neutrophils alsoneed to produce factors to ensure their survival in thehostile inflammatory milieu, recruit additionalphagocytes, inactivate their own toxic products andinduce their own death pathway to prevent damage

Ž .to normal host tissue Fig. 1 .It may be misleading to depend on a single test to

evaluate neutrophil function because various re-

0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 99 00148-9

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Fig. 1. Neutrophil functional responses during recruitment to a site of bacterial infection. The broken lines represent chemoattractantsgenerated at the site of infection that activate the endothelium and capture the intravascular neutrophils. The neutrophils flatten and adhereto the endothelium, migrate out of the vessel towards the infectious site, phagocytose and kill the bacteria, secrete cytokines to recruit other

Ž .inflammatory cells and eventually undergo programmed cell death apoptosis .

sponses can be elicited by a single stimulus, anddistinct transduction pathways may independentlyregulate these processes. The test or tests selecteddepend on the nature of the problem that needs to beaddressed. The measurement of physiological func-tions such as chemotaxis, phagocytosis, bacterialkilling and apoptosis remain time consuming, but theavailability of a wide variety of fluorochromes andflow cytometry has allowed the development ofmethods that rapidly measure a variety of functionsin a single population of neutrophils.

The devastating effects of inherited abnormalitiesin neutrophil function highlight the important role ofneutrophils in host defense. Clinical suspicion ofimpaired neutrophil function is usually related to amarked susceptibility to infection. In these subjects,

Ž .abnormalities in neutrophil numbers neutropenia orimmune abnormalities, such as opsonic defects, aremore common and should be considered first. Flowcytometry can assist in suggesting the diagnosis ofan inherited defect in the majority of these disordersŽ .Epling et al., 1992 . Conditions where inappropriateneutrophil-mediated tissue injury are an importantcomponent in the pathogenesis of the disease, suchas acute respiratory distress syndrome, ischemia

Žreperfusion injury and rheumatoid arthritis Hernan-dez et al., 1987; Hogg, 1987; Malech and Gallin,

.1987; Weiss, 1989; Horl et al., 1990; Ricevuti, 1997 ,are thought to be a form of acquired neutrophil

Ž‘‘hyperfunction’’. In normal Tanji-Matsuba et al.,.1998 or acquired neutrophil functional abnormali-

ties, the use of flow cytometry has assisted in theunderstanding of the role of neutrophils in diseasepathogenesis.

This review evaluates the various methods avail-able for measuring neutrophil function using flowcytometry. This powerful tool has the ability tomeasure multiple parameters on individual cells athigh speed. With the use of highly specific fluores-cent-conjugated monoclonal antibodies against sur-face and intracellular antigens as well as fluo-rochromes that change with cell activation, our un-derstanding of normal and abnormal neutrophil be-havior in disease has expanded.

2. Sample preparation

Collecting and manipulating blood samples forneutrophil functional analysis changes their behavior.Blood should be collected at a slow rate with a large

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bore needle directly into the anticoagulant. The func-tional behavior of neutrophils as well as the numberand the affinity of their receptors can change withthe type of anticoagulant used to collect the bloodŽ .Repo et al., 1995; Conklyn et al., 1996 , fluctua-

Žtions in temperature during cell processing Repo etal., 1995; van Eeden et al., 1995; Youssef et al.,

. Ž .1995 , density centrifugation Kuijpers et al., 1991 ,Ž .hypotonic lysis of red cells Styrt et al., 1988 , and

endotoxin contamination of reagents or buffersŽ .Haslett et al., 1985 . All reagents used should bepyrogen-free and of tissue culture grade and plastictubes should be used for processing samples. Theanticoagulant, heparin, can activate the complement

Ž .system and generate complement fragment C5a inthe plasma resulting in cell activation and calciumchelating agents such as EDTA should be avoidedwith antibodies whose binding are cation-dependentŽ .Repo et al., 1995 or when measuring functions thatare calcium-dependent. Artifactual upregulation ofsensitive adhesion receptors such as CD11b ex vivocan be minimized by cooling blood samples on ice.However, if samples are cooled, cell labeling andfixation should be completed on ice because fluctua-tions in temperature have a significant effect on theexpression of receptors such as CD11b and L-selec-

Ž .tin Repo et al., 1995; van Eeden et al., 1995 .Keeping the blood samples at a constant temperature

Žsuch as room temperature or 378C for cell activation.and other functional studies following sample col-

lection is important to reduce artifactual changes inneutrophil functional responses. Whole-blood meth-ods combined with gating techniques to separateleukocyte subsets or leukocyte specific surface mark-ers can be used to measure several neutrophil func-

Žtions simultaneously Hasui et al., 1989; Haynes et.al., 1990 . If purified neutrophils are used, the inter-

pretation of sensitive surface markers such as CD11bŽ .should be reserved Kuijpers et al., 1991 .

3. Neutrophil functional responses

The functional response of neutrophils to an incit-ing stimulus, such as an extravascular infection, is a

Ž .sequential multistep process illustrated in Fig. 1 .Factors that regulate these sequential responses arenot well-understood. One factor that could influencethese sequential responses is the level of exposure tocell activating stimuli at each step of the cascade.Fig. 2 shows a schematic representation of the effectof increasing concentrations of a receptor agonist,such as N-formylmethionine-leucyl-phenylalanine

Fig. 2. A schematic illustration of the concentration-dependent neutrophil functional responses. The receptor agonist fMLP is used as anexample and concentrations ranging from 10y10 to 10y6 M are shown. Maximal calcium signals and actin assembly is seen with lower

Ž y9 . Ž y6 .concentrations of fMLP 10 in contrast to superoxide production that needs higher fMLP concentration 10 . Degranulation ofŽ y9 . Ž y8 .secretory vesicles and gelatinase containing granules occur at low concentration of fMLP 10 while secondary 10 and primary

Ž y7 .10 granule release need higher concentrations of fMLP.

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Ž .fMLP , on the different neutrophil functional re-sponses. It demonstrates the concept that neutrophilsrespond in a graded fashion to increasing concentra-tions of the agonist. This concept is supported by the

Žwork of several investigators Petrequin et al., 1987;Lew, 1989; Packman and Lichtman, 1990; Fernan-

.dez-Segura et al., 1995 . An increase in calcium andF-actin assembly occurs at very low concentrations

Ž y9 .of the agonist 10 M in contrast to degranulationof secondary and primary granules as wells as oxy-gen radical production that required higher concen-

Ž y8 y6 .trations of fMLP 10 to 10 M to elicit asignificant response. This response is also time-de-pendent with Ca2q mobilization and F-actin assem-

Ž .bly occurring rapidly s in contrast to degranulationof primary granules and oxygen radical productionŽ .min . This organized sequential nature of the pro-cess is important to ensure that normal host tissue isnot damaged during the process of eliminating theinvading microorganisms.

Activation of neutrophils is not an all or nothingphenomenon and each function has its own thresholdfor a response. Actin assembly in neutrophils allowsthem to flatten and crawl on endothelium towards agradient of chemoattractant, and then migrate out ofthe vasculature towards the inciting stimulus. Duringchemotaxis, degranulation with the release of lysoso-mal enzymes and oxygen radical by the neutrophilscould cause damage to normal tissues. These latterfunctions are more appropriate following phago-cytosis of microorganisms. This proposed paradigmof concentration and time-dependent neutrophil re-sponses protect the host from inappropriate neu-trophil-mediated tissue damage. Although numerousneutrophil responses occur simultaneously with cellactivation, this sequential paradigm illustrated inFigs. 1 and 2 is a practical way to partition thefunctional responses of neutrophil following cell ac-tivation and this approach will be used to discussflow cytometric methods available to measure neu-trophil function.

Flow cytometry measures fluorescence and lightscatter signals of suspended cells through a lasersource. Gating techniques allow the analysis of struc-tural and functional parameters in a large populationof intact single cells excluding those that are aggre-

Žgated or lysed Duque and Ward, 1987; Durack et.al., 1991; Carulli, 1996 . These gating techniques

could influence results because they exclude aggre-Ž .gated cells due to cell activation . Furthermore,

gating on neutrophils includes eosinophils and re-sults should be interpreted with caution in subjectswith high eosinophil counts. Cell specific surfacemarkers, such as CD32 that are not expressed byeosinophils but by other granulocytes, can be used toseparate neutrophil and eosinophils. Flow cytometrycan also resolve the heterogeneity of a cell popula-tion and facilitate the identification and characteriza-tion of cell subpopulations and subcellular compart-

Ž .ments Conklyn et al., 1996 . It permits multipara-Žmetric analysis Orfao and Ruiz-Arguelles, 1996;

.Macey et al., 1998 , particularly, intracellular andsurface-exposed antigens.

3.1. Common stimuli used to measure neutrophilfunction

Chemoattractants commonly used to measure neu-trophil functional responses are either endogenous,

Ž . Ž .such as interleukin 8 IL-8 , leukotriene B4 LTB4 ,Ž .platelet activating factor PAF , C5a, interleukin 1

Ž . Ž .IL-1 , and tumor necrosis factor alpha TNF-a , orexogenous, such as the formylated peptide fMLP, asurface receptor agonist derived from bacterial cell

Ž .products Elsner et al., 1992 . The phorbol estersŽ . Ž .PMA and ionophores ionomycin or A25187 areagonists that do not work via receptor binding andare useful in studying mechanisms underlying neu-trophil activation. In addition, cytochalasins are com-monly used to inhibit or enhance certain neutrophil

Žfunctions Treves et al., 1987; Al-Mohanna et al.,.1997 . During cell activation, cell surface receptors

can be transiently desensitized when the cell is stim-Ž .ulated by a ligand e.g., fMLP . Cells then become

unresponsive to the subsequent exposure to the sameŽ .ligand homologous desensitization or different lig-Ž . Žands heterologous desensitization Sklar et al.,

.1985; McColl and Naccache, 1997 . Factors that cancontribute to this desensitization after a single addi-tion of an agonist include microaggregation of cellsŽ .Lofgren et al., 1993 , internalization of ligand–re-ceptor complexes, reduced receptor-associated GT-

w 2qx ŽPase activity or increased Ca i Vindenes and.Bjerknes, 1997 . Therefore, cells that have been

exposed to inflammatory mediators in vivo couldhave an attenuated response when activated in vitro.

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4. Calcium mobilization

Calcium mobilization is one of the earliest eventsŽ .that occur with neutrophil activation Fig. 2 . Upon

activation, ligands occupy the surface receptors andelicit an immediate rise in the cytosolic free Ca2q asa result of Ca2q release from the internal stores. This

Ž .rapid response s is followed by a more sustainedŽ . 2qmin effect due to Ca influx from the extracellu-

Žlar medium White et al., 1983; Anderson and Ma-.homed, 1997; Andersson et al., 1986 . Continuous

communication between the extracellular and intra-cellular environment is essential for maintainingCa2q homeostasis and modulating PMN functional

Ž .responses Sklar and Oades, 1985 . Transient in-creases in free cytosolic calcium act as a secondarymessenger for numerous neutrophil biological re-

Žsponses Bengtsson et al., 1993; Sengelov et al.,1993; Sjaastad and Nelson, 1997; Suzaki et al.,1997; Bei et al., 1998; Chacon-Cruz et al., 1998;

.Majeed et al., 1998 .

4.1. Fluorescent Ca2 q indicators

Numerous fluorescent dyes have been developedas Ca2q indicators. These non-fluorescent mem-brane-permeable esters cross the plasma membraneof living cells and are hydrolyzed by cytoplasmicesterases to generate a free acid fluorescent activeform of the probe that is trapped in the cytoplasmŽ .Lew, 1989 . Calcium indicators are excited either at

Ža short wavelength of 340–380 nm e.g., Quin-2,.Fura-2 and Indo-1 or at a long wavelength above

Ž450 nm e.g., Fluor-3, rhod-2, Calcium Green, Cal-. Žcium Crimson and Calcium Orange Simpson,

. Ž .1999 . Fura-2 acetoxymethyl ester Fura-2rAMalone is considered inadequate for flow cytometric

w 2qxanalysis as the intracellular Ca i is estimated us-Ž .ing the ratio of absorption excitation intensity at

two different wavelengths. In contrast, Indo-1rAMis almost non-fluorescent unless bound to calcium,exhibits a great ability to resolve small changes inw 2qx Ž . Ž .Ca i micromolar level , Justement et al., 1990and is suitable for single-cell measurements by flow

Ž .as it needs only one excitation source UV light andŽ .two fluorescence emissions Maftah et al., 1993 .

However, the use of Indo-1 presents a significantdrawback because it requires UV light which is not

Žwidely available Lopez et al., 1989; Novak and

.Rabinovitch, 1994 . The long wavelength probe,Fluor-3, shows a greater affinity for calcium thanFura-2 or Indo-1 and is well-suited for flow cytomet-ric analysis but unlike Fura-2 and Indo-1, it is notamenable to ratiometric analysis. Fluor-3 causescompartmentalization and heterogeneous loadingwhich could be relevant in a cell population with

Ž .variable size cytoplasmic volume and granularity.Both events cause variability in the baseline fluores-cence intensity and may compromise the sensitivity

2q Žand accuracy of Ca measurements Justement et.al., 1990 . To improve quantitative flow cytometric

measurements of intracellular calcium, ratiometricprocedures are used with Fluo-3 as an adjunct to

Ž .Fura-2 Novak and Rabinovitch, 1994 or SNARF-1Ž .Rijkers et al., 1990 .

4.2. Intracellular Ca2 q measurements

In order to maximize calcium changes, it is im-portant to optimize dye loading conditions for eachcell type that depends on the concentration of thedye, the temperature and duration of the incubation,the level of intracellular esterases and the permeabil-

Žity of the plasma membrane McColl and Naccache,.1997 . Calcium levels can be measured in LRP or in

purified neutrophils suspended in a calcium-contain-Ž .ing buffer e.g., Hank’s balanced salt solution . Cells

are incubated at 378C for 30 min with 2 mM ofindicator dye, washed, resuspended in the same bufferand allowed to equilibrate for 5–15 min before

Žbaseline measurements Elsner et al., 1992; McColl.and Naccache, 1997 . Although rarely needed by

hematopoietic cells, dispersing agents such as thenon-ionic detergent Pluronic F-127 or bovine serumalbumin can be used to increase the solubility of thedye. When required, probenicid is used to inhibit

Žextrusion of the fluorescent dye Omann and Harter,.1991 . Mean fluorescence intensity is measured fol-

lowing excitation at 488 nm and emission at 520 nm,using analysis gates for neutrophils in a flow cy-tometer.

4.3. Benefits and limitations of flow cytometry incalcium measurements

Flow cytometric analysis has significant advan-tages over conventional fluorometric techniques

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Ž .Hoy, 1990; Sengelov, 1996 . It resolves the hetero-geneity of a cell population and identifies and char-

Žacterizes subpopulations of cells Elsner et al., 1992;.Pettit and Hallett, 1995 . Flow cytometric methods

have been used to investigate abnormal calcium mo-bilization in neutrophils of patients with clinical

Ždisorders such as periodontitis Champagne et al.,. Ž1998 and glycogen storage disease type 1b Elsner

.et al., 1992 . Currently, these methods have limita-tions due to the inability to sort cells under sterile

Ž .conditions Orfao and Ruiz-Arguelles, 1996 . It isalso possible that measurements of calcium by flowcytometry miss the initial peak since there is a time

Ž .lapse approximately 15 s between adding the ago-nist and the actual time at which Ca2q measurementstake place. For more details on the role of Ca2q inregulating neutrophil function, see the review ofHallet et al. in this issue.

5. Cytoskeletal actin and cytoskeleton regulatoryproteins

Actin, a 43-kDa peptide, is a major cytoplasmiccomponent of the neutrophils. It exists in two physi-

Ž .cal states, the globular monomeric actin G-actinŽ .and the filamentous polymeric actin F-actin . Upon

activation by stimuli such as fMLP, C5a, zymosan-Ž .activated plasma ZAP , multivalent IgG or LPS

ŽPackman and Lichtman, 1990; Yee and Christou,.1993; Klut et al., 1997 , G-actin changes into F-actin

and accumulates in the cell cortex. Cytoskeletal actinassembly is one of the earliest and most sensitive

Ž .neutrophil functional responses Kelley, 1991 . Cy-toskeletal events near the cell surface regulate func-tional responses such as adhesion and deformabilityand can provide the driving force for mobility,phagocytosis, granule trafficking and superoxide

Žgeneration Bengtsson et al., 1993; Fernandez-Segura.et al., 1995; Carulli et al., 1997 . The effect of

abnormal actin dynamics in neutrophil function hasbeen linked to an increased susceptibility to infectionŽ .Vindenes and Bjerknes, 1997 and has been shown

Ž .in newborns Merry et al., 1998 as well as inŽpatients with thermal injury Vindenes and Bjerknes,

. Ž1997 and myelodysplastic syndromes Carulli et al.,

.1997 . Furthermore, flow cytometry has been used toassess the role of the cytoskeleton in dialysis neu-

Ž .tropenia Tabor et al., 1998 and to demonstrateneutrophil–actin dysfunction in a recessive inheriteddisorder in which subjects presented with recurrent

Ž .infections Packman and Lichtman, 1990 .

5.1. F-actin measurements

The measurement of F-actin content by flow cy-tometry requires a small volume of peripheral bloodŽ .45 ml . Whole-blood cells are first equilibrated at378C and then activated with the agonist. The reac-tion is stopped by addition of 3.0% paraformal-dehyde. Erythrocytes are lysed and leukocytes arepermeabilized and stained with a mixture of L-a-lysophosphatidylcholine and N-7-nitrobenz-2-oxa-

Ž . Ž1,3-diazole-4-yl NBD -phallacidin Chen et al.,.1996; Klut et al., 1997 . The fluorochrome is excited

at a wavelength of 488 nm and emission is recordedat 525 nm. F-actin levels obtained by flow cytometrycorrelate well with biochemical measurements and

Ž . Žwith quantitative assays i.e., gel scanning Watts.and Howard, 1992; Carulli et al., 1997 .

5.2. Benefits and limitations of flow cytometric F-actin measurements

Flow cytometry is a sensitive and reliable methodfor assessing actin dynamics and allows the identifi-cation of cell subpopulations that are less responsive

Ž .to stimulants Chen et al., 1996 . However, flowcytometry does not permit discrimination betweenthe stable gelsolin-free and the labile gelsolin-richF-actin pools. Also, this approach does not sense

Žchanges in cell shape or actin redistribution Watts.and Howard, 1992; Carulli et al., 1997 . For more

details on the role of F-actin assembly in neutrophilfunction, see the review of Coates et al. in this issue.

6. Adhesion and aggregation

6.1. Adhesion

The adhesion of neutrophils to endothelial cells isa pivotal event in the process of cell migration andsubsequent tissue inflammation. This process can beevaluated in several ways. Firstly, the number of

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adhesion molecules on the surface of neutrophils canŽbe quantitated Bateman et al., 1993; Repo et al.,

.1993; Youssef et al., 1995 or the affinity of theseadhesion molecules for their respective ligands can

Ž .be evaluated Larson and Springer, 1990 . Severalcell surface molecules that are involved in the neu-trophil–endothelial adhesive interactions have been

Židentified and characterized Carlos and Harlan,.1994 . The initial neutrophil–endothelial interaction

Žis mediated by members of the selectin family i.e.,.L-, E- and P-selectin that establish a loose associa-

Žtion of neutrophils with the endothelium von An-.drian et al., 1991; Bevilacqua, 1993 . This is fol-

lowed by firm adhesion and migration of neutrophilsinvolving members of the integrin family on neu-

Ž .trophils i.e., CD11b or MAC-1 interacting with theimmunoglobulin superfamily on endothelial cellsŽ Ž .i.e., intercellular adhesion molecule 1 ICAM-1

. Žand PECAM-1 Smyth et al., 1993; Carlos and.Harlan, 1994 .

The expression of cell adhesion molecules on thesurface of neutrophils can be measured in whole-blood specimens. P- and E-selectin are expressed onthe endothelium after cell activation and L-selectinŽ .CD62L is constitutively expressed on circulatingneutrophils and shed from the cell surface upon cell

Ž .activation Kishimoto et al., 1989 . The reduction insurface expression of L-selectin on circulating neu-trophils should be interpreted with caution becauseactive bone marrow release causes an increase incirculating neutrophils expressing high levels of L-

Ž .selectin van Eeden et al., 1995 . Studies from ourŽvan Eeden et al., 1995, 1997b,c; Nakagawa et al.,

. Ž1998 and other Lund-Johansen and Terstappen,

.1993 laboratories have shown that this is due to thehigh levels of surface expression of L-selectin onneutrophils in the maturation pool of the bone mar-row. Stimulation of the bone marrow is associatedwith the release of these neutrophils expressing high

Žlevels L-selectin van Eeden et al., 1995; Lawrence.et al., 1996 . Furthermore, we have shown that neu-

trophils loose their surface L-selectin as they age inŽ .the circulation van Eeden et al., 1997a and several

Žstudies have shown that drugs such as steroids Bur-ton et al., 1995; Waisman et al., 1998; Nakagawa et

.al., 1999 and non-steroidal anti-inflammatory drugsŽ .Diaz-Gonzalez et al., 1995 reduce L-selectin oncirculating neutrophils. Therefore, the interpretation

of L-selectin expression on circulating neutrophilsshould take the following variables into account:whether there is an active bone marrow response;intravascular cell activation; and the drugs the sub-ject is taking at the time of the study.

The integrin molecules on neutrophils mediatecell–cell and cell–matrix adhesion and are essentialfor firm adhesion of neutrophils to endothelium andmigration through vessel walls and interstitial tissueŽ .Arnaout, 1990; Springer, 1990 . The b integrins2

CD18rCD11b are constitutively expressed on circu-lating neutrophils and their surface expression in-

Žcreases rapidly upon cell activation Kishimoto et al.,.1989; Repo et al., 1993 . This is a useful and sensi-

tive marker of cell activation in vivo in clinicalconditions such as infection, ischemia and throm-

Ž .botic events Hansen, 1995; Mazzone et al., 1997 . Agranulocytosis and recurrent bacterial infections inthese subjects highlight the critical importance ofthis molecule in mounting an effective neutrophilicinflammatory response in subjects with an inherited

Ždeficiency of the b integrins leukocyte adhesion2. Ždeficiency 1 or LAD1 Springer et al., 1984; Ander-.son and Springer, 1987 . Conformational changes in

CD18rCD11b upon cell activation alter the affinityŽof the molecule for its ligands Larson and Springer,

.1990 and increase the adhesiveness of neutrophils.The importance of these changes in neutrophil func-tion are underlined by the recent description of sub-

Žjects with near normal CD18 expression 40%–60%.levels adequate for normal function but the clinical

phenotype of LAD1 with recurrent bacterial infec-Žtions and impaired pus formation Kuijpers et al.,

.1997; Hogg et al., 1999 . In these subjects, cellactivation did not result in CD18 activation andhigh-avidity ligand binding that results in reducedneutrophil aggregation and migration. A mutation inthe b -subunit conserved domain in the metal ion-2

dependent adhesion site could be responsible for thisŽfailure of the b integrins to function Hogg et al.,2

.1999 .Several factors should be considered when the

CD18rCD11b adhesion function of neutrophils isevaluated using flow cytometry. A whole-bloodmethod is preferred to avoid the changes induced by

Žneutrophil isolation procedures Kuijpers et al.,.1991 , EDTA as an anticoagulant should be avoided

if subsequent activation of neutrophils is planned

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Ž .Bateman et al., 1993; Hogg et al., 1999 , and anti-bodies binding the high-affinity receptors should beused if adhesiveness via CD18rCD11b is evaluated.

ŽSecondly, assays of dynamic von Andrian et al.,. Ž .1991 or static Taylor et al., 1996 neutrophil adhe-

sion in vivo or ex vivo directly test the ability ofneutrophils to adhere to the endothelium. In thedynamic in vivo adhesion assays, the type of leuko-cyte that interacts with the endothelium cannot bediscriminated and can be overcome by using purified

Žneutrophils in an ex vivo system Taylor et al., 1996;.Neelamegham et al., 1998 . These dynamic systems

largely test the functional integrity of the selectinŽ .group of adhesion molecules L-, P- and E-selectin

and their interaction with their ligands. Flow cytome-try has a limited role in performing these assays. Thestatic assays tested firm adhesion of neutrophils toendothelial cells and evaluated largely the interaction

Ž .of the integrins CD18 group of molecules withŽtheir counter receptors on the endothelium ICAM-1

.and ICAM-2 . Most of these static assays used puri-fied neutrophils incubated on cultured endothelial

Ž .monolayers Korlipara et al., 1996 and used flowcytometry to evaluate the number of neutrophils thatadhere to the endothelium with and without stimula-tion of either the neutrophils or the endothelium.This is accomplished by detachment of endothelialcells from the plastic and dissociation of adherentneutrophils by treatment with trypsin and EDTAŽ .Korlipara et al., 1997; Marshall et al., 1997 . Bygating on neutrophils and endothelial cells, the num-ber of adherent neutrophils per endothelial cell can

Ž .be calculated. Marshall et al. 1997 have recentlydemonstrated that this method can be modified touse whole blood instead of purified neutrophils. Thismethod also allows the evaluation of the adhesion ofother leukocytes such as monocytes, eosinophils andlymphocytes to endothelial cells. The benefit of us-ing flow cytometry for these assays instead of directmicroscopic evaluation of cell adhesion is the abilityto evaluate the behavior of a large population ofneutrophils as well as different subpopulations ofleukocytes.

6.2. Aggregation

Circulating neutrophils are found predominantlyas single cells in their resting state and respond to a

variety of soluble stimuli by forming homo- or het-erotypic aggregates. These intravascular aggregatescontribute to the inflammatory reaction and tissueinjury especially in ischemic syndromes. Activationby chemotactic peptide induces transient aggregation

Žboth in vitro Sklar et al., 1985; Tandon and Dia-. Žmond, 1998 and in vivo Schmid-Schonbein, 1987a;

.Schmid-Schonbein et al., 1987b . Light transmit-tance as measured by aggregometer has previouslybeen the standard method to quantitate neutrophilaggregation but provides at best a gross measure-

Žment of mostly large aggregates Craddock et al.,.1977; Ringertz et al., 1985 . Flow cytometric meth-

ods have the benefit of analyzing precise particlesize, distribution of the different particle sizes andthe time course of particle formation and disaggrega-

Ž .tion Sklar et al., 1985; Neelamegham et al., 1997 .The simplest form of data acquisition is to calculatethe extent of aggregation by measuring changes inthe total number of particles per unit volume overtime. These measurements correlate well with values

Ždetermined by microscopy Rochon and Frojmovic,.1991 . Following cell activation, neutrophil aggrega-

Ž .tion occurs rapidly ;4 s and the subsequent rateof forward aggregation and disaggregation dependson neutrophil numbers and type and the duration of

Žthe activator Simon et al., 1990; Rochon and Froj-.movic, 1991 .

The additional benefit of flow cytometry is theability to simultaneously explore the mechanisms ofaggregation at the level of the adhesive receptor.Activated neutrophils aggregate during shear stressby bonding of surface L-selectin with P-selectin

Ž . Ž .glycoprotein ligand-1 PSGP-1 Guyer et al., 1996followed by more stable bonding via CD18rCD11ato ICAM-3 and CD18rCD11b to a yet a unknown

Žligand Arnaout et al., 1985; Simon et al., 1990;.Tandon and Diamond, 1998 . The magnitude of

these molecular interactions on cell aggregation canbe evaluated by the use of blocking monoclonal

Ž .antibodies Simon et al., 1990 .Neutrophil–platelet aggregation occurs in the cir-

culation and may be of pathophysiological signifi-cance in conditions such as acute myocardial in-

Žfarction and stroke Konstantopoulos et al., 1995;.Neumann et al., 1997 . This platelet–neutrophil in-

teraction propagates the inflammatory process byincreasing CD18rCD11b expression and enhancing

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their homotypic aggregation and adhesion to en-Ždothelium Peters et al., 1997; Konstantopoulos et

.al., 1998 . The interaction between P-selectin onactivated platelets with PSGP-1 on neutrophils is thepredominant adhesive binding and can be initiatedby hydrodynamic shear or platelet activation. Peters

Ž .et al. 1997 have developed a method using smallamounts of whole blood to measure platelet–neu-trophil complexes and have shown that this interac-tion is P-selectin and divalent cation-dependent. Themajority of neutrophils associated with more thanone platelet. This technique is simple and repro-ducible and allows the simultaneous assessment ofplatelet–neutrophil interaction and neutrophil activa-tion.

7. Phagocytosis

A number of workers have described methods toquantify neutrophil phagocytic capabilities using flow

Žcytometry Steinkamp et al., 1982; Bassoe, 1984;.Bassoe and Solberg, 1984; Stewart et al., 1986 .

These methods entail incubating neutrophils withfluorescent conjugated particles, bacteria or yeastand measuring the amount of fluorescence as anindicator of phagocytic activity. To limit cell activa-tion during neutrophil isolation, whole-blood meth-ods which are rapid, objective and quantitative, have

Žbeen developed Hasui et al., 1989; Bohmer et al.,1992; White-Owen et al., 1992; Antal et al., 1995;

.Santos et al., 1995 . Non-specific adherence of theparticles to neutrophils interferes with the measure-ment of the number of particles actually internalizedby the cells. Substances such as trypan blue oriodoacetate that quench superficial fluorescencewithout penetrating the plasma membrane have beenused to reduce this non-specific fluorescence by up

Žto 95% Steinkamp et al., 1982; Bassoe, 1984;.Bjerknes and Bassoe, 1984; Fattorossi et al., 1989 .

Alternatively, to correct for non-specific adherenceof particles to neutrophils, a control sample incu-bated at 48C, that inhibits phagocytosis but not non-

Ž .specific adherence, can be used Santos et al., 1995 .Using whole-blood and two-color flow cytometry,

several workers have demonstrated that phago-cytosis, reactive oxygen production, protein diges-

tion and bacterial killing within the phagocytic vac-uole can be measured in the same population of cellsŽHasui et al., 1989; Haynes et al., 1990; Perticarari et

.al., 1991; Saresella et al., 1997 . Protein digestion ismeasured by fluorescence of rhodamine attached toalbumin at a high molar ratio and quenching of the

Žfluorescence when the protein is digested Haynes et.al., 1990 . Using the phagocytosis of the fluo-

rochrome neutral red by neutrophils, Antal and col-leagues have shown that they can measure phago-

Žcytosis after exposure to soluble activators PMA.and PAF , making it possible to diagnose

neutrophil-related phagocytic disorders not related toŽthe production of reactive oxygen intermediates An-

.tal et al., 1995 .Phagocytosis studies should be done with hep-

arinized blood as EDTA and ACD capture Ca2q ionsthat are essential for phagocytosis. Incubation timesbetween neutrophils and particles should be opti-mized for maximum phagocytosis because the rateand extent of phagocytosis differ depending on thetype of particle used. Similarly, cell to particle ratioshould be optimized with a 1:10 ratio an averageratio for most standard particles.

8. Degranulation

Neutrophils ingest bacteria or other particles intointracellular compartments called phagosomes andthe destruction of the infectious microorganism isachieved by the fusion of granules with the phago-somes. The extracellular release of granule contentoccurs concomitantly, that may result in destroyingnon-phagocyted bacteria but could also result intissue damage and amplification of the local inflam-matory response. The membrane of granules servesas a reservoir of receptors and membrane bound

Žproteins involved in cell adhesion Bainton et al.,. Ž .1997 , signal transduction Rotrosen et al., 1988 and

Žactivation of microcidal pathways Borregaard et al.,.1983; Spitznagel, 1990 . The fusion of granules with

the cell membrane during degranulation changes thesurface expression of these membrane receptors andthis principle has been used to measure and quanti-tate neutrophil degranulation using flow cytometry.

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Small secretory vesicles and granules containinggelatinase are very sensitive to cell manipulation and

Ž .activation Lew, 1989 and their degranulation isassociated with the increase in surface CD35rCR1and CD11b expression. Secondary or specific gran-ules have different extracellular matrix receptors suchas laminin, fibronectin, vitronectin and CD18 thatbinds fibrinogen and are coined ‘‘adhesomes’’Ž .Singer et al., 1989 . CD18rCD11b increase rapidlyon the neutrophil surface upon cell activation andserves as a sensitive marker of secondary granule

Ž .release Kishimoto et al., 1989; Bainton et al., 1997 .Sensitive surface markers for primary or azurophilicgranule release are not available. Granule contentlevels, such as myeloperoxidase, can be measured

Žusing monoclonal antibodies Hewitt and Reardon,.1991; van Eeden et al., 1997b,c . Kuijpers et al.

Ž .1991 propose that one of the tetraspanins, CD63,which is a lysosomal membrane protein, can be usedas a marker of primary granule release in vivo and invitro. Expression of this molecule on senescent neu-trophils is very low and an increase during cellactivation is associated with a decrease in neutrophilmyeloperoxidase activity. The reliability of this sur-face marker to measure degranulation of primarygranules still needs to be established.

9. Oxygen radical production

Central to antimicrobial activity of neutrophils isthe oxidative burst that generates reactive oxygen

Ž .species ROS through the NADPH oxidase system.The best demonstration of the essential role of this isthe consequences of a lack of ROS generation seenin patients with chronic granulomatous diseaseŽ .CGD . These patients have defects in their NADPHoxidase pathway, are unable to generate superoxideanions necessary for the formation of a number of

Ž .ROS Fig. 3 and have recurrent life threateningŽ .bacterial infections from birth Gallin, 1992 .

In assessing the oxidative burst in neutrophils, themost widely used standard methods measure the

Ž y.extracellular release of superoxide anion O or2Ž .hydrogen peroxide H O by their ability to reduce2 2

or oxidize substrates to colored or fluorescent prod-

Fig. 3. ROS produced by activated neutrophils. Membrane-associ-ated NADPH-oxidase produce superoxide and hydrogen peroxideupon activation. During the oxidative burst, the fluorochromes

X Ž .2 ,7-dichlorofluorescein DCFH and dihydrorhodamine 123 areoxidated mainly by H O to either green or red fluorescence,2 2

respectively.

Žucts McCord and Fridovich, 1969; Babior et al.,.1970; Boveris et al., 1977; Roots and Metcalf, 1977 .

The addition of superoxide dismutase or catalase inparallel samples provides specificity to the assays forsuperoxide anion or H O , respectively. The major2 2

disadvantages of these assay methods are that cellsmust be separated from red blood cells and plasmaand that relatively large numbers of neutrophils are

Žneeded approximately 0.5 millionrexperimental.condition . In these assays, the measured response is

of a population and individual response cannot beassessed.

In 1983, a method was developed to measure‘‘oxidant product formation’’ by flow cytometry us-

X X Ž . Žing 2 ,7 -dichlorofluorescein DCF Bass et al.,.1983 . In this method, neutrophils are loaded with

the diacetate form of 2X,7-dichlorofluoresceinŽ .DCFH , which is transported across the plasmamembrane. In the cytoplasm, the acetate groups areremoved by esterases concentrating the polar non-

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fluorescent DCFH in the cytoplasm. During the ox-idative burst, DCFH is converted to DCF throughoxidation mainly by H O . Dihydrorhodamine 1232 2

is used in a similar way resulting in red fluorescenceŽEmmendorffer et al., 1990; Rothe et al., 1991;

.Vowells et al., 1995 . Strong non-physiologic stimulisuch as PMA and calcium ionophores cause a sus-tained respiratory burst mirrored by high levels offluorescence in this assay. More physiologic stimuliproduce much smaller and variable responses.

The use of flow cytometry to measure the produc-tion of H O offers an easy semi-quantitative tech-2 2

nique that is applicable to a few thousand unpurifiedcells. In contrast to the standard assay mentionedpreviously, the flow cytometric assay can be per-

Ž .formed in mixed cell samples whole blood whereneutrophil responses can be determined using gating

Ž .techniques Richardson et al., 1998 . The small num-ber of unpurified cells necessary for the flow cytom-etry assay allows studies on small sample volumessuch as neonatal blood or from other small animals.In clinical laboratories, the availability and familiar-ity with flow cytometry make these flow cytometryassays very attractive in screening patients for CGDŽ .Emmendorffer et al., 1990; Roesler et al., 1990 .

9.1. Benefits and limitations of flow cytometry

The limits of flow cytometry in measuring H O2 2

production need to be recognized. Quantitative mea-surements are difficult. Variation in loading andhydrolysis of the acetate from DCFH can alter therelative fluorescence seen. Quantifying the numberof moles of fluorescein per cell is cumbersome andmost studies report only relative fluorescence. Intra-cellular elements such as catalases or peroxidasesŽ .especially myeloperoxidase significantly affect theoxidant reaction. The addition of 5 mM azide en-hances the oxidation of DCFH possibly by inactivat-ing intracellular enzymes like MPO which reducesthe available H O through their enzymatic reaction2 2Ž . Že.g., the conversion of H O to HOCl Bass et al.,2 2

.1983; Smith and Wiedemann, 1993 .The assumption in the flow cytometric assessment

of the respiratory burst is that changes in fluores-cence on stimulation are indicative of the productionof H O in individual neutrophils. For most applica-2 2

tions this assumption is valid. However, H O and2 2

its precursor, superoxide anion, can cross intact cellmembranes and diffuse to adjacent cells. This wasdemonstrated by mixing DCFH-loaded neutrophilsfrom normal individuals and from patients with CGDŽ .incapable of a respiratory burst . In this mixture, theoxidant products from the cells with a respiratoryburst were capable of oxidizing DCFH in non-re-

Ž .sponding bystanders Bass et al., 1983 . This is oflittle concern for most applications but may be aconfounding feature in experiments using mixed cellpopulations. It should be remembered that eosinophilsare capable of an oxidative burst, although normallythey represent a small fraction of cells in the‘‘granulocyte’’ window. Finally, it should be remem-bered that neutrophil activation results in aggregationof neutrophils with other neutrophils and other cellsespecially platelets. These larger aggregates may notbe seen in the gates set for single neutrophil and mayexclude a population of activated neutrophils fromthe analysis.

More recently, flow cytometric assays combiningphagocytes and oxidative burst have been developed

Žusing labeled bacteria or immune complexes Hasui.et al., 1989; Haynes et al., 1990 . The advantage of

these stimuli is that they are more physiologic andthe intensity of the fluorescence is much higher thanthat achieved with DCFH-loaded cells. But the re-sponse to these particulate fluorochromes is morecomplex. The oxidative burst and release of H O2 2

can occur on contact without uptake of the particle.Phagocytosis concentrates these particles in phago-somes allowing more efficient delivery of the H O2 2

to the stimulus and fluorochrome. Thus the intensityof the signal from each neutrophil is dependent onparticulate adherence and uptake as well as the respi-ratory burst.

10. Apoptosis

Apoptosis or programmed cell death is a well-de-fined mode of cell demise and are brought about bya diverse array of physiological and non-physio-logical stimuli. It can be initiated through a geneti-cally defined pathway or by intra- or extracellular

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stimuli such as cytokines, hormones, endogenousproteins, oxidative stress and hypoxia. Apoptosis isan important process in normal neutrophil physiol-ogy because of the extend of neutrophil turnoverŽ y1 y1.1–2.5 billion kg day and their short life spanŽ . Ž6–10 h in the circulation Maloney and Patt, 1968;

.McAfee and Thakur, 1976 . The changes in agingPMN which allow recognition by macrophages inthe liver and spleen to remove them from the circula-tion or sites of extravasation are a field of active

Ž .research Savill et al., 1989; Savill et al., 1992 .Ž .Studies from our laboratory Bicknell et al., 1994

showed that apoptosis of neutrophils occurs mainlyin the spleen and it is possible that the senescentcells are recognized and removed in these organs ina manner similar to that occurring at inflammatorysites. Aging neutrophils in vitro develop morphologi-cal and biochemical features typical of apoptosis

Žwithout the addition of external stimuli Whyte et al.,.1993a,b and we have shown similar events occur in

Žcirculating neutrophils as they age in vivo Matsuba.et al., 1997 . This process is characterized by DNA

cleavage by calcium-dependent endogenous endonu-cleases capable of cleaving chromatin at internucleo-

Ž .somal sites Arends et al., 1990 .There is a 10–100 fold increase in neutrophil

Žturnover during acute infection McAfee and Thakur,.1976 and these neutrophils meet their fate in the

tissue, as there is little evidence that they return tothe blood from the inflamed site. The survival ofneutrophils can be prolonged by cytokines present at

Žthe inflammatory site Brach et al., 1992; Colotta et.al., 1992 . The suppressed apoptosis in neutrophils

during acute infectious or inflammatory conditionshas been postulated to be important in the pathogen-esis of neutrophil-mediated diseases such as is-chemia reperfusion injury, acute respiratory distress

Žsyndrome, sepsis and multiorgan failure Jones and.Morgan, 1995; Liles and Klebanoff, 1995 .

11. Detection of apoptosis by flow cytometry

Several flow cytometric methods have been de-scribed to detect and quantitate apoptosis in neu-trophils and in this short synopsis the more com-

monly used methods will be discussed. The ability offlow cytometry to assess apoptosis in large numbersof cells and simultaneously evaluate subpopulationswithin a larger cell population are some of thebenefits of using flow cytometry over more conven-tional methods such as morphology and gel elec-trophoresis. Because all the methods described belowhave shortcomings, it is imperative that at least oneadditional independent method is used to confirmresults.

11.1. Size and granularity

Flow cytometry allows the detection of the light-scattering properties of neutrophils and quantitate the

Ž . Žcell size forward scatter and granularity side scat-. Žter . Apoptotic neutrophils loose water reduced for-

. Žward scatter , become less granular reduced side.scatter and these changes in their light scattering

properties can be use to identify apoptotic cellsŽ .Gorman et al., 1997 . These changes are non-specificand can not distinguish between apoptotic andnecrotic cells. Both neutrophil size and granularitycan also change with cell activation. Therefore, theseparameters of neutrophil apoptosis are not useful asthe only discriminator for apoptosis because of thepotential heterogeneity of most neutrophil popula-tions.

11.2. Fluorescent dyes

The DNA-binding dye propidium iodide can beuse to reveal the amount of DNA in each cell andthis assay make use of the fact that apoptotic cells

Žcontain subdiploid amounts of DNA Mesner and.Kaufman, 1997 . This technique has disadvantages

since it does not distinguish between apoptotic andnecrotic cells and requires additional techniques toconfirm that the subdiploid cell population is trulyapoptotic. The presence of multiple apoptotic bodiesmay result in overestimation of the number of cellsthat are apoptotic, conversely, using gating tech-niques to exclude these bodies may underestimatethe number of apoptotic cells.

ŽOther vital dyes such as acridine orange green. Žfluorescence and ethidium bromide orange fluores-

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.cence rely on the differential uptake of these dyesby normal healthy, apoptotic and necrotic cells. Us-ing ethidium bromide cells can be graded as negativeŽ . Ž . Ž .healthy , weakly apoptotic or strongly necroticpositive. Similar results are obtained with com-pounds such as Hoechst 33342 or 7-aminoacti-

Žnomycin D Gong et al., 1994; O’Brien and Bolton,.1995 but labeling with these dyes must be done at

48C. The benefits of these dyes are that large popula-tions of cells can be screened and these dyes can becombined with other fluorochromes to identify apop-

Ž .tosis in subset of cells O’Brien and Bolton, 1995 .One potential disadvantage of this approach is thelack of good studies to verify the specificity of thesemethods. Furthermore, cell membrane integrity ismaintained in the early stage of apoptosis but is lostwith advanced apoptosis and these techniques mayunderestimate the actual number of apoptotic cells ifall strongly stained cells are exclude from the analy-sis.

11.3. Surface markers of apoptosis

Annexin V, a polypeptide that binds strongly andspecifically to cell surface phosphatidylserine is a

Žsensitive measurement of early apoptosis Koopman.et al., 1994; Martin et al., 1995 . Phosphatidylserine

is present exclusively in the inner layer of the cellmembrane of healthy cells and becomes accessibleon the surface of cells undergoing apoptosis. This isthe basis for detecting apoptotic neutrophils usingfluorescent labeled Annexin V. Cells are labeledfollowing fixation with a non-permeabilizing fixativeŽ .e.g., formaldehyde or without fixation. Once theplasma membrane integrity is lost, cells will becomeAnnexin V positive and accordingly, this techniqueis not suitable for specimens contaminated withnecrotic cells or cells that have been permeabilized.

11.4. Terminal deoxyribonucleotidyl transferase-( )mediated dUTP nick-end labeling TUNEL

The TUNEL technique is a sensitive method todetect apoptosis in neutrophils using flow cytometry.The principle of these techniques is that in thepresence of a divalent cation, terminal deoxyribonu-

Ž .cleotidyl transferase TdT will add nucleosides to

free 3X-ends produced by endonuclease action duringŽ .apoptosis Gavrieli et al., 1992 . Briefly, neutrophils

are fixed, permeabilized and incubated with biotinyl-ated deoxyuridine triphosphate in the presence ofTdT and Co2q. After washing, cells are incubatedwith fluorescent labeled streptavidin that binds to thebiotin. One of the benefits of this technique is that itcan be combined with fluorescent microscopic analy-sis and compared with morphological changes ofDNA fragmentation in individual cells. The disad-vantage of this method is that it could detect nicks inDNA due to the presence of other enzymes such astopoisomase that are not necessarily associated with

Ž .apoptosis Froelich-Ammon et al., 1995 . In addi-tion, necrotic cells can stain with TUNEL becauserandom DNA degradation occurring during necrosis

X Žalso generates ends with 3 OH groups Grasl-Kraupp.et al., 1995 . Therefore, alternative techniques needs

to be combined with TUNEL to confirm that cellsare undergoing apoptosis rather than necrosis.

The measurement of changes in mitochondrialŽ .transmembrane potential Zamzami et al., 1995 , ac-

tivation of various proteases such as the IL-1b con-Ž . Ž .verting enzyme ICE family Takahashi et al., 1996

and bax the proapoptotic member of the bcl-2 fam-Žily of apoptosis regulators Miyashita and Reed,

.1995 are all indicative of apoptosis and can bemeasured using flow cytometry. Flow cytometry al-lows the rapid detection of apoptotic neutrophils bypermitting the measurement of a variety of parame-ters that alter during the onset and progression ofapoptosis. It has significantly aided our understand-ing of neutrophil apoptosis and its potential role inthe pathogenesis of neutrophil-induced inflammatoryconditions.

12. Priming of neutrophils

Priming of neutrophils is one of the pivotal pro-Žcesses that regulate their functional responses Con-

.dliffe et al., 1998a . Priming refers to the processwhere the response of neutrophils to an activatingstimulus is enhanced by prior exposure to small‘‘non-activating’’ concentrations of this or another

Ž .stimulus Guthrie et al., 1984 . Guthrie et al. de-scribed up to 20-fold enhancement of superoxide

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production by neutrophils by prior exposure to apriming agent. In the strictest sense the primingresponse is not associated with a functional response

Žbut a variety of biochemical events Walker and.Ward, 1992 . Enhancement of agonist-induced de-Ž .granulation Fittschen et al., 1988 and generation of

Žlipid mediators such as LTB4 and PAF Doerfler et. Ž .al., 1989 or other cytokines Zallen et al., 1999 has

been described. A variety of substances, both physio-logical and pharmacological, have been shown to

Žprime neutrophils Walker and Ward, 1992; Condliffe.et al., 1998b , and many have biological relevance in

vivo because they are released in response to infec-tion, hemorrhage and trauma. Priming agents such asendotoxin and cytokines, notably TNF-a , IL-6 andIL-8, are detectable in the blood and are associatedwith a poor outcome in subjects with septic shock,

ŽARDS and multiorgan failure Parsons et al., 1989;. Ž .Pinsky et al., 1993 . Smedley et al. 1986 have

shown that neutrophils activated with fMLP, C5aand LPS cause minimal damage to endothelial cellsin vitro but if cells were first primed with LPS andthen stimulated with fMLP or C5a, extensive damageto endothelial cells ensued.

Work from our laboratory has shown that theprocess of sequestration of neutrophils may also

Ž .result in priming Kitagawa et al., 1997 . Becauseneutrophils are larger than the majority of pulmonarycapillary segments they have to pass through, thesecells have to deform to cross the pulmonary vascular

Ž .bed Doerschuk et al., 1993; Wiggs et al., 1994 .Using flow cytometry, we have shown that passivedeformation of neutrophils by filtration through 5-mm

Žpores similar to deformation in the pulmonary capil-.lary bed causes enhanced CD18rCD11b expression

following maximal stimulation with fMLP. Thepriming effect of this deformation was comparable tousing fMLP as a priming agent. Neutrophils exposedto priming concentrations of inflammatory mediatorsundergo shape changes as a result of modification oftheir cytoskeletal actin, a key event that makes neu-

Ž .trophils less deformable Worthen et al., 1989 andincreases their sequestration in the pulmonary capil-

Ž .lary bed Doerschuk et al., 1989 . Prolonged reten-tion of these primed neutrophils in the lung increasesthe risk of neutrophil-mediated tissue damage.Therefore, in conditions such as septicemia, measur-ing priming in circulating neutrophils underestimates

the real magnitude of intravascular neutrophil prim-ing.

Inflammatory mediators generated locally at thesite of inflammation serve to upregulate the func-tional responses of neutrophils sequestered and ex-travasated into the tissues. Cross-linking of neu-trophil adhesion receptors is a priming stimulusŽ .Waddell et al., 1994; Liles et al., 1995 . The processof extravasation of neutrophils itself results in in-crease stimulus-induced oxidant release and otherphenotypic changes such as changes in their expres-sion of surface adhesion molecules. This needs to betaken into account when extravasated neutrophilsfrom areas such as the alveolar space or peritonealcavity are harvested for analysis.

Flow cytometry has been used extensively toidentify and quantify neutrophil priming. Activationof the NADPH oxidase enzyme system with theproduction of superoxide is the hallmark of neu-trophil priming. This is the most primable neutrophilfunction with the potential to increase superoxideproduction up to 20-fold. However, other changes inneutrophils following priming are shape changes and

Ž .stiffening Haslett et al., 1985 , changes in the ex-pression of adhesion molecules with an increase in

ŽCD11b and a decrease in L-selectin Condliffe et al.,.1996 , degranulation of primary and secondary gran-Ž .ules Doerfler et al., 1994 and a delay in apoptosis

Ž .Lee et al., 1989 . Lastly, sample preparation forflow cytometry and trace amounts of endotoxin insolutions used for cell isolation could prime neu-

Ž .trophils Haslett et al., 1985 . Because many of theseneutrophil functions can be measured in whole blood,flow cytometry is the method of choice to evaluateneutrophil priming. It is vital to realize that neu-trophils are not terminally differentiated cells andthat their functional responses can be up- or down-regulated both in vivo and in vitro.

13. Conclusions

Our understanding of the basic functions of neu-trophils relies heavily on in vitro observations withextrapolation to in vivo events. However, it is re-markably easy to disturb normal neutrophil function

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by removing them from the circulation. The use offlow cytometry has advanced our ability to measureand quantify neutrophil functions. Using whole-bloodmethods with minimal manipulation of cells gives amore accurate measure of neutrophil behavior invivo. The ability of flow cytometry to measure thefunction of a single cell or large numbers of cellsand the potential to measure multiple functions si-multaneously by using multiple fluorochromes hasimproved our ability to study the function of acomplex cell in health and disease. The applicationof flow cytometry to measure neutrophil function isprogressively expanding in basic and clinical sci-ences.

Acknowledgements

This work was supported by the BC Lung Associ-ation and the Medical Research Council of Canadagrant a4219.

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