Journal of Immunological Methods 232 Ž1999. 23–43
The use of flow cytometry to measure neutrophil function
Stephan F. van Eeden , Maria E. Klut a , Blair A.M. Walker b, James C. Hogg a
Pulmonary Research Laboratory, UniÕersity of British Columbia, St. Paul’s Hospital, 1081 Burrard Street, VancouÕer, British Columbia,
Canada V6Z 1Y6
Department of Pathology, UniÕersity of British Columbia, St. Paul’s Hospital, VancouÕer, British Columbia, Canada V6Z 1Y6
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,
F-actin assembly, adhesion, aggregation, degranulation, phagocytosis and reactive oxygen species ŽROS. 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-
1. Introduction sense the focus of infection, slow down and adhere
to the endothelium of capillaries and venules adja-
Neutrophils are important effector cells in numer-
cent to the inflammatory locus, migrate through the
ous inflammatory conditions. The relevance of neu-
vessel wall and the interstitial tissues to the infec-
trophil function is usually thought of in terms of host
tious site, phagocytose, kill and digest the invading
defense in bacterial and fungal infections ŽMalech
microorganisms. During this process, neutrophils also
and Gallin, 1987; Bainton, 1992. because of the
need to produce factors to ensure their survival in the
association between the lack of neutrophils and over-
hostile inflammatory milieu, recruit additional
whelming infection with these organisms. To fulfill
phagocytes, inactivate their own toxic products and
this defensive role, intravascular neutrophils need to
induce their own death pathway to prevent damage
to normal host tissue ŽFig. 1..
Corresponding author. Tel.: q1-604-806-8346; fax: q1-604- It may be misleading to depend on a single test to
806-8351; e-mail: firstname.lastname@example.org evaluate neutrophil function because various re-
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 1 4 8 - 9
24 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
Fig. 1. Neutrophil functional responses during recruitment to a site of bacterial infection. The broken lines represent chemoattractants
generated at the site of infection that activate the endothelium and capture the intravascular neutrophils. The neutrophils flatten and adhere
to the endothelium, migrate out of the vessel towards the infectious site, phagocytose and kill the bacteria, secrete cytokines to recruit other
inflammatory cells and eventually undergo programmed cell death Žapoptosis..
sponses can be elicited by a single stimulus, and reperfusion injury and rheumatoid arthritis ŽHernan-
distinct transduction pathways may independently dez et al., 1987; Hogg, 1987; Malech and Gallin,
regulate these processes. The test or tests selected 1987; Weiss, 1989; Horl et al., 1990; Ricevuti, 1997.,
depend on the nature of the problem that needs to be are thought to be a form of acquired neutrophil
addressed. The measurement of physiological func- ‘‘hyperfunction’’. In normal ŽTanji-Matsuba et al.,
tions such as chemotaxis, phagocytosis, bacterial 1998. or acquired neutrophil functional abnormali-
killing and apoptosis remain time consuming, but the ties, the use of flow cytometry has assisted in the
availability of a wide variety of fluorochromes and understanding of the role of neutrophils in disease
flow cytometry has allowed the development of pathogenesis.
methods that rapidly measure a variety of functions This review evaluates the various methods avail-
in a single population of neutrophils. able for measuring neutrophil function using flow
The devastating effects of inherited abnormalities cytometry. This powerful tool has the ability to
in neutrophil function highlight the important role of measure multiple parameters on individual cells at
neutrophils in host defense. Clinical suspicion of high speed. With the use of highly specific fluores-
impaired neutrophil function is usually related to a cent-conjugated monoclonal antibodies against sur-
marked susceptibility to infection. In these subjects, face and intracellular antigens as well as fluo-
abnormalities in neutrophil numbers Žneutropenia. or rochromes that change with cell activation, our un-
immune abnormalities, such as opsonic defects, are derstanding of normal and abnormal neutrophil be-
more common and should be considered first. Flow havior in disease has expanded.
cytometry can assist in suggesting the diagnosis of
an inherited defect in the majority of these disorders
ŽEpling et al., 1992.. Conditions where inappropriate 2. Sample preparation
neutrophil-mediated tissue injury are an important Collecting and manipulating blood samples for
component in the pathogenesis of the disease, such neutrophil functional analysis changes their behavior.
as acute respiratory distress syndrome, ischemia Blood should be collected at a slow rate with a large
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 25
bore needle directly into the anticoagulant. The func- tin ŽRepo et al., 1995; van Eeden et al., 1995..
tional behavior of neutrophils as well as the number Keeping the blood samples at a constant temperature
and the affinity of their receptors can change with such as room temperature or 378C Žfor cell activation
the type of anticoagulant used to collect the blood and other functional studies. following sample col-
ŽRepo et al., 1995; Conklyn et al., 1996., fluctua- lection is important to reduce artifactual changes in
tions in temperature during cell processing ŽRepo et neutrophil functional responses. Whole-blood meth-
al., 1995; van Eeden et al., 1995; Youssef et al., ods combined with gating techniques to separate
1995., density centrifugation ŽKuijpers et al., 1991., leukocyte subsets or leukocyte specific surface mark-
hypotonic lysis of red cells ŽStyrt et al., 1988., and ers can be used to measure several neutrophil func-
endotoxin contamination of reagents or buffers tions simultaneously ŽHasui et al., 1989; Haynes et
ŽHaslett et al., 1985.. All reagents used should be al., 1990.. If purified neutrophils are used, the inter-
pyrogen-free and of tissue culture grade and plastic pretation of sensitive surface markers such as CD11b
tubes should be used for processing samples. The should be reserved ŽKuijpers et al., 1991..
anticoagulant, heparin, can activate the complement
system and generate complement fragment ŽC5a. in
3. Neutrophil functional responses
the plasma resulting in cell activation and calcium
chelating agents such as EDTA should be avoided The functional response of neutrophils to an incit-
with antibodies whose binding are cation-dependent ing stimulus, such as an extravascular infection, is a
ŽRepo et al., 1995. or when measuring functions that sequential multistep process Žillustrated in Fig. 1..
are calcium-dependent. Artifactual upregulation of Factors that regulate these sequential responses are
sensitive adhesion receptors such as CD11b ex vivo not well-understood. One factor that could influence
can be minimized by cooling blood samples on ice. these sequential responses is the level of exposure to
However, if samples are cooled, cell labeling and cell activating stimuli at each step of the cascade.
fixation should be completed on ice because fluctua- Fig. 2 shows a schematic representation of the effect
tions in temperature have a significant effect on the of increasing concentrations of a receptor agonist,
expression of receptors such as CD11b and L-selec- 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 an
example and concentrations ranging from 10y1 0 to 10y6 M are shown. Maximal calcium signals and actin assembly is seen with lower
concentrations of fMLP Ž10y9 . in contrast to superoxide production that needs higher fMLP concentration Ž10y6 .. Degranulation of
secretory vesicles and gelatinase containing granules occur at low concentration of fMLP Ž10y9 . while secondary Ž10y8 . and primary
Ž10y7 . granule release need higher concentrations of fMLP.
26 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
ŽfMLP., on the different neutrophil functional re- could influence results because they exclude aggre-
sponses. It demonstrates the concept that neutrophils gated cells Ždue to cell activation.. Furthermore,
respond in a graded fashion to increasing concentra- gating on neutrophils includes eosinophils and re-
tions of the agonist. This concept is supported by the sults should be interpreted with caution in subjects
work of several investigators ŽPetrequin et al., 1987; with high eosinophil counts. Cell specific surface
Lew, 1989; Packman and Lichtman, 1990; Fernan- markers, such as CD32 that are not expressed by
dez-Segura et al., 1995.. An increase in calcium and eosinophils but by other granulocytes, can be used to
F-actin assembly occurs at very low concentrations separate neutrophil and eosinophils. Flow cytometry
of the agonist Ž10y9 M. in contrast to degranulation can also resolve the heterogeneity of a cell popula-
of secondary and primary granules as wells as oxy- tion and facilitate the identification and characteriza-
gen radical production that required higher concen- tion of cell subpopulations and subcellular compart-
trations of fMLP Ž10y8 to 10y6 M. to elicit a ments ŽConklyn et al., 1996.. It permits multipara-
significant response. This response is also time-de- metric analysis ŽOrfao and Ruiz-Arguelles, 1996;
pendent with Ca2q mobilization and F-actin assem- Macey et al., 1998., particularly, intracellular and
bly occurring rapidly Žs. in contrast to degranulation surface-exposed antigens.
of primary granules and oxygen radical production
Žmin.. This organized sequential nature of the pro- 3.1. Common stimuli used to measure neutrophil
cess is important to ensure that normal host tissue is function
not damaged during the process of eliminating the
invading microorganisms. Chemoattractants commonly used to measure neu-
Activation of neutrophils is not an all or nothing trophil functional responses are either endogenous,
phenomenon and each function has its own threshold such as interleukin 8 ŽIL-8., leukotriene B4 ŽLTB4.,
for a response. Actin assembly in neutrophils allows platelet activating factor ŽPAF., C5a, interleukin 1
them to flatten and crawl on endothelium towards a ŽIL-1., and tumor necrosis factor alpha ŽTNF-a ., or
gradient of chemoattractant, and then migrate out of exogenous, such as the formylated peptide fMLP, a
the vasculature towards the inciting stimulus. During surface receptor agonist derived from bacterial cell
chemotaxis, degranulation with the release of lysoso- products ŽElsner et al., 1992.. The phorbol esters
mal enzymes and oxygen radical by the neutrophils ŽPMA. and ionophores Žionomycin or A25187. are
could cause damage to normal tissues. These latter agonists that do not work via receptor binding and
functions are more appropriate following phago- are useful in studying mechanisms underlying neu-
cytosis of microorganisms. This proposed paradigm trophil activation. In addition, cytochalasins are com-
of concentration and time-dependent neutrophil re- monly used to inhibit or enhance certain neutrophil
sponses protect the host from inappropriate neu- functions ŽTreves et al., 1987; Al-Mohanna et al.,
trophil-mediated tissue damage. Although numerous 1997.. During cell activation, cell surface receptors
neutrophil responses occur simultaneously with cell can be transiently desensitized when the cell is stim-
activation, this sequential paradigm illustrated in ulated by a ligand Že.g., fMLP.. Cells then become
Figs. 1 and 2 is a practical way to partition the unresponsive to the subsequent exposure to the same
functional responses of neutrophil following cell ac- ligand Žhomologous desensitization. or different lig-
tivation and this approach will be used to discuss ands Žheterologous desensitization. ŽSklar et al.,
flow cytometric methods available to measure neu- 1985; McColl and Naccache, 1997.. Factors that can
trophil function. contribute to this desensitization after a single addi-
Flow cytometry measures fluorescence and light tion of an agonist include microaggregation of cells
scatter signals of suspended cells through a laser ŽLofgren et al., 1993., internalization of ligand–re-
source. Gating techniques allow the analysis of struc- ceptor complexes, reduced receptor-associated GT-
tural and functional parameters in a large population Pase activity or increased wCa2q xi ŽVindenes and
of intact single cells excluding those that are aggre- Bjerknes, 1997.. Therefore, cells that have been
gated or lysed ŽDuque and Ward, 1987; Durack et exposed to inflammatory mediators in vivo could
al., 1991; Carulli, 1996.. These gating techniques have an attenuated response when activated in vitro.
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 27
4. Calcium mobilization Rabinovitch, 1994.. The long wavelength probe,
Fluor-3, shows a greater affinity for calcium than
Calcium mobilization is one of the earliest events Fura-2 or Indo-1 and is well-suited for flow cytomet-
that occur with neutrophil activation ŽFig. 2.. Upon ric analysis but unlike Fura-2 and Indo-1, it is not
activation, ligands occupy the surface receptors and amenable to ratiometric analysis. Fluor-3 causes
elicit an immediate rise in the cytosolic free Ca2q as compartmentalization and heterogeneous loading
a result of Ca2q release from the internal stores. This which could be relevant in a cell population with
rapid response Žs. is followed by a more sustained variable size Žcytoplasmic volume. and granularity.
Žmin. effect due to Ca2q influx from the extracellu-
Both events cause variability in the baseline fluores-
lar medium ŽWhite et al., 1983; Anderson and Ma- cence intensity and may compromise the sensitivity
homed, 1997; Andersson et al., 1986.. Continuous and accuracy of Ca2q measurements ŽJustement et
communication between the extracellular and intra- al., 1990.. To improve quantitative flow cytometric
cellular environment is essential for maintaining measurements of intracellular calcium, ratiometric
Ca2q homeostasis and modulating PMN functional procedures are used with Fluo-3 as an adjunct to
responses ŽSklar and Oades, 1985.. Transient in- Fura-2 ŽNovak and Rabinovitch, 1994. or SNARF-1
creases in free cytosolic calcium act as a secondary ŽRijkers et al., 1990..
messenger for numerous neutrophil biological re-
sponses ŽBengtsson et al., 1993; Sengelov et al., 4.2. Intracellular Ca 2 q measurements
1993; Sjaastad and Nelson, 1997; Suzaki et al.,
1997; Bei et al., 1998; Chacon-Cruz et al., 1998; In order to maximize calcium changes, it is im-
Majeed et al., 1998.. portant to optimize dye loading conditions for each
4.1. Fluorescent Ca 2 q indicators cell type that depends on the concentration of the
dye, the temperature and duration of the incubation,
Numerous fluorescent dyes have been developed the level of intracellular esterases and the permeabil-
as Ca2q indicators. These non-fluorescent mem- ity of the plasma membrane ŽMcColl and Naccache,
brane-permeable esters cross the plasma membrane 1997.. Calcium levels can be measured in LRP or in
of living cells and are hydrolyzed by cytoplasmic purified neutrophils suspended in a calcium-contain-
esterases to generate a free acid fluorescent active ing buffer Že.g., Hank’s balanced salt solution.. Cells
form of the probe that is trapped in the cytoplasm are incubated at 378C for 30 min with 2 mM of
ŽLew, 1989.. Calcium indicators are excited either at indicator dye, washed, resuspended in the same buffer
a short wavelength of 340–380 nm Že.g., Quin-2, and allowed to equilibrate for 5–15 min before
Fura-2 and Indo-1. or at a long wavelength above baseline measurements ŽElsner et al., 1992; McColl
450 nm Že.g., Fluor-3, rhod-2, Calcium Green, Cal- and Naccache, 1997.. Although rarely needed by
cium Crimson and Calcium Orange. ŽSimpson, hematopoietic cells, dispersing agents such as the
1999.. Fura-2 acetoxymethyl ester ŽFura-2rAM. non-ionic detergent Pluronic F-127 or bovine serum
alone is considered inadequate for flow cytometric albumin can be used to increase the solubility of the
analysis as the intracellular wCa2q xi is estimated us- dye. When required, probenicid is used to inhibit
ing the ratio of absorption Žexcitation. intensity at extrusion of the fluorescent dye ŽOmann and Harter,
two different wavelengths. In contrast, Indo-1rAM 1991.. Mean fluorescence intensity is measured fol-
is almost non-fluorescent unless bound to calcium, lowing excitation at 488 nm and emission at 520 nm,
exhibits a great ability to resolve small changes in using analysis gates for neutrophils in a flow cy-
wCa2q xi Žmicromolar level., ŽJustement et al., 1990. tometer.
and is suitable for single-cell measurements by flow
as it needs only one excitation source ŽUV light. and 4.3. Benefits and limitations of flow cytometry in
two fluorescence emissions ŽMaftah et al., 1993.. calcium measurements
However, the use of Indo-1 presents a significant
drawback because it requires UV light which is not Flow cytometric analysis has significant advan-
widely available ŽLopez et al., 1989; Novak and tages over conventional fluorometric techniques
28 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
ŽHoy, 1990; Sengelov, 1996.. It resolves the hetero- tropenia ŽTabor et al., 1998. and to demonstrate
geneity of a cell population and identifies and char- neutrophil–actin dysfunction in a recessive inherited
acterizes subpopulations of cells ŽElsner et al., 1992; disorder in which subjects presented with recurrent
Pettit and Hallett, 1995.. Flow cytometric methods infections ŽPackman and Lichtman, 1990..
have been used to investigate abnormal calcium mo-
bilization in neutrophils of patients with clinical 5.1. F-actin measurements
disorders such as periodontitis ŽChampagne et al.,
1998. and glycogen storage disease type 1b ŽElsner The measurement of F-actin content by flow cy-
et al., 1992.. Currently, these methods have limita- tometry requires a small volume of peripheral blood
tions due to the inability to sort cells under sterile Ž45 ml.. Whole-blood cells are first equilibrated at
conditions ŽOrfao and Ruiz-Arguelles, 1996.. It is 378C and then activated with the agonist. The reac-
also possible that measurements of calcium by flow tion is stopped by addition of 3.0% paraformal-
cytometry miss the initial peak since there is a time dehyde. Erythrocytes are lysed and leukocytes are
lapse Žapproximately 15 s. between adding the ago- permeabilized and stained with a mixture of L-a-
nist and the actual time at which Ca2q measurements lysophosphatidylcholine and N-7-nitrobenz-2-oxa-
take place. For more details on the role of Ca2q in 1,3-diazole-4-yl ŽNBD.-phallacidin ŽChen et al.,
regulating neutrophil function, see the review of 1996; Klut et al., 1997.. The fluorochrome is excited
Hallet et al. in this issue. at a wavelength of 488 nm and emission is recorded
at 525 nm. F-actin levels obtained by flow cytometry
correlate well with biochemical measurements and
5. Cytoskeletal actin and cytoskeleton regulatory with quantitative assays Ži.e., gel scanning. ŽWatts
proteins and Howard, 1992; Carulli et al., 1997..
Actin, a 43-kDa peptide, is a major cytoplasmic
5.2. Benefits and limitations of flow cytometric F-
component of the neutrophils. It exists in two physi-
cal states, the globular monomeric actin ŽG-actin.
and the filamentous polymeric actin ŽF-actin.. Upon
Flow cytometry is a sensitive and reliable method
activation by stimuli such as fMLP, C5a, zymosan-
for assessing actin dynamics and allows the identifi-
activated plasma ŽZAP., multivalent IgG or LPS
ŽPackman and Lichtman, 1990; Yee and Christou, cation of cell subpopulations that are less responsive
to stimulants ŽChen et al., 1996.. However, flow
1993; Klut et al., 1997., G-actin changes into F-actin
cytometry does not permit discrimination between
and accumulates in the cell cortex. Cytoskeletal actin
the stable gelsolin-free and the labile gelsolin-rich
assembly is one of the earliest and most sensitive
F-actin pools. Also, this approach does not sense
neutrophil functional responses ŽKelley, 1991.. Cy-
changes in cell shape or actin redistribution ŽWatts
toskeletal events near the cell surface regulate func-
and Howard, 1992; Carulli et al., 1997.. For more
tional responses such as adhesion and deformability
details on the role of F-actin assembly in neutrophil
and can provide the driving force for mobility,
function, see the review of Coates et al. in this issue.
phagocytosis, granule trafficking and superoxide
generation ŽBengtsson et al., 1993; Fernandez-Segura
et al., 1995; Carulli et al., 1997.. The effect of
abnormal actin dynamics in neutrophil function has 6. Adhesion and aggregation
been linked to an increased susceptibility to infection
ŽVindenes and Bjerknes, 1997. and has been shown 6.1. Adhesion
in newborns ŽMerry et al., 1998. as well as in
patients with thermal injury ŽVindenes and Bjerknes, The adhesion of neutrophils to endothelial cells is
1997. and myelodysplastic syndromes ŽCarulli et al., a pivotal event in the process of cell migration and
1997.. Furthermore, flow cytometry has been used to subsequent tissue inflammation. This process can be
assess the role of the cytoskeleton in dialysis neu- evaluated in several ways. Firstly, the number of
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 29
adhesion molecules on the surface of neutrophils can of L-selectin expression on circulating neutrophils
be quantitated ŽBateman et al., 1993; Repo et al., should take the following variables into account:
1993; Youssef et al., 1995. or the affinity of these whether there is an active bone marrow response;
adhesion molecules for their respective ligands can intravascular cell activation; and the drugs the sub-
be evaluated ŽLarson and Springer, 1990.. Several ject is taking at the time of the study.
cell surface molecules that are involved in the neu- The integrin molecules on neutrophils mediate
trophil–endothelial adhesive interactions have been cell–cell and cell–matrix adhesion and are essential
identified and characterized ŽCarlos and Harlan, for firm adhesion of neutrophils to endothelium and
1994.. The initial neutrophil–endothelial interaction migration through vessel walls and interstitial tissue
is mediated by members of the selectin family Ži.e., ŽArnaout, 1990; Springer, 1990.. The b 2 integrins
L-, E- and P-selectin. that establish a loose associa- CD18rCD11b are constitutively expressed on circu-
tion of neutrophils with the endothelium Žvon An- lating neutrophils and their surface expression in-
drian et al., 1991; Bevilacqua, 1993.. This is fol- creases rapidly upon cell activation ŽKishimoto et al.,
lowed by firm adhesion and migration of neutrophils 1989; Repo et al., 1993.. This is a useful and sensi-
involving members of the integrin family on neu- tive marker of cell activation in vivo in clinical
trophils Ži.e., CD11b or MAC-1. interacting with the conditions such as infection, ischemia and throm-
immunoglobulin superfamily on endothelial cells botic events ŽHansen, 1995; Mazzone et al., 1997.. A
Ži.e., intercellular adhesion molecule 1 ŽICAM-1. granulocytosis and recurrent bacterial infections in
and PECAM-1. ŽSmyth et al., 1993; Carlos and these subjects highlight the critical importance of
Harlan, 1994.. this molecule in mounting an effective neutrophilic
The expression of cell adhesion molecules on the inflammatory response in subjects with an inherited
surface of neutrophils can be measured in whole- deficiency of the b 2 integrins Žleukocyte adhesion
blood specimens. P- and E-selectin are expressed on deficiency 1 or LAD1. ŽSpringer et al., 1984; Ander-
the endothelium after cell activation and L-selectin son and Springer, 1987.. Conformational changes in
ŽCD62L. is constitutively expressed on circulating CD18rCD11b upon cell activation alter the affinity
neutrophils and shed from the cell surface upon cell of the molecule for its ligands ŽLarson and Springer,
activation ŽKishimoto et al., 1989.. The reduction in 1990. and increase the adhesiveness of neutrophils.
surface expression of L-selectin on circulating neu- The importance of these changes in neutrophil func-
trophils should be interpreted with caution because tion are underlined by the recent description of sub-
active bone marrow release causes an increase in jects with near normal CD18 expression Ž40%–60%
circulating neutrophils expressing high levels of L- levels adequate for normal function. but the clinical
selectin Žvan Eeden et al., 1995.. Studies from our phenotype of LAD1 with recurrent bacterial infec-
Žvan Eeden et al., 1995, 1997b,c; Nakagawa et al., tions and impaired pus formation ŽKuijpers et al.,
1998. and other ŽLund-Johansen and Terstappen, 1997; Hogg et al., 1999.. In these subjects, cell
1993. laboratories have shown that this is due to the activation did not result in CD18 activation and
high levels of surface expression of L-selectin on high-avidity ligand binding that results in reduced
neutrophils in the maturation pool of the bone mar- neutrophil aggregation and migration. A mutation in
row. Stimulation of the bone marrow is associated the b 2-subunit conserved domain in the metal ion-
with the release of these neutrophils expressing high dependent adhesion site could be responsible for this
levels L-selectin Žvan Eeden et al., 1995; Lawrence failure of the b 2 integrins to function ŽHogg et al.,
et al., 1996.. Furthermore, we have shown that neu- 1999..
trophils loose their surface L-selectin as they age in Several factors should be considered when the
the circulation Žvan Eeden et al., 1997a. and several CD18rCD11b adhesion function of neutrophils is
studies have shown that drugs such as steroids ŽBur- evaluated using flow cytometry. A whole-blood
ton et al., 1995; Waisman et al., 1998; Nakagawa et method is preferred to avoid the changes induced by
al., 1999. and non-steroidal anti-inflammatory drugs neutrophil isolation procedures ŽKuijpers et al.,
ŽDiaz-Gonzalez et al., 1995. reduce L-selectin on 1991., EDTA as an anticoagulant should be avoided
circulating neutrophils. Therefore, the interpretation if subsequent activation of neutrophils is planned
30 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
ŽBateman et al., 1993; Hogg et al., 1999., and anti- variety of soluble stimuli by forming homo- or het-
bodies binding the high-affinity receptors should be erotypic aggregates. These intravascular aggregates
used if adhesiveness via CD18rCD11b is evaluated. contribute to the inflammatory reaction and tissue
Secondly, assays of dynamic Žvon Andrian et al., injury especially in ischemic syndromes. Activation
1991. or static ŽTaylor et al., 1996. neutrophil adhe- by chemotactic peptide induces transient aggregation
sion in vivo or ex vivo directly test the ability of both in vitro ŽSklar et al., 1985; Tandon and Dia-
neutrophils to adhere to the endothelium. In the mond, 1998. and in vivo ŽSchmid-Schonbein, 1987a;
dynamic in vivo adhesion assays, the type of leuko- Schmid-Schonbein et al., 1987b.. Light transmit-
cyte that interacts with the endothelium cannot be tance as measured by aggregometer has previously
discriminated and can be overcome by using purified been the standard method to quantitate neutrophil
neutrophils in an ex vivo system ŽTaylor et al., 1996; aggregation but provides at best a gross measure-
Neelamegham et al., 1998.. These dynamic systems ment of mostly large aggregates ŽCraddock et al.,
largely test the functional integrity of the selectin 1977; Ringertz et al., 1985.. Flow cytometric meth-
group of adhesion molecules ŽL-, P- and E-selectin. ods have the benefit of analyzing precise particle
and their interaction with their ligands. Flow cytome- size, distribution of the different particle sizes and
try has a limited role in performing these assays. The the time course of particle formation and disaggrega-
static assays tested firm adhesion of neutrophils to tion ŽSklar et al., 1985; Neelamegham et al., 1997..
endothelial cells and evaluated largely the interaction The simplest form of data acquisition is to calculate
of the integrins ŽCD18 group of molecules. with the extent of aggregation by measuring changes in
their counter receptors on the endothelium ŽICAM-1 the total number of particles per unit volume over
and ICAM-2.. Most of these static assays used puri- time. These measurements correlate well with values
fied neutrophils incubated on cultured endothelial determined by microscopy ŽRochon and Frojmovic,
monolayers ŽKorlipara et al., 1996. and used flow 1991.. Following cell activation, neutrophil aggrega-
cytometry to evaluate the number of neutrophils that tion occurs rapidly Ž; 4 s. and the subsequent rate
adhere to the endothelium with and without stimula- of forward aggregation and disaggregation depends
tion of either the neutrophils or the endothelium. on neutrophil numbers and type and the duration of
This is accomplished by detachment of endothelial the activator ŽSimon et al., 1990; Rochon and Froj-
cells from the plastic and dissociation of adherent movic, 1991..
neutrophils by treatment with trypsin and EDTA The additional benefit of flow cytometry is the
ŽKorlipara et al., 1997; Marshall et al., 1997.. By ability to simultaneously explore the mechanisms of
gating on neutrophils and endothelial cells, the num- aggregation at the level of the adhesive receptor.
ber of adherent neutrophils per endothelial cell can Activated neutrophils aggregate during shear stress
be calculated. Marshall et al. Ž1997. have recently by bonding of surface L-selectin with P-selectin
demonstrated that this method can be modified to glycoprotein ligand-1 ŽPSGP-1. ŽGuyer et al., 1996.
use whole blood instead of purified neutrophils. This followed by more stable bonding via CD18rCD11a
method also allows the evaluation of the adhesion of to ICAM-3 and CD18rCD11b to a yet a unknown
other leukocytes such as monocytes, eosinophils and ligand ŽArnaout et al., 1985; Simon et al., 1990;
lymphocytes to endothelial cells. The benefit of us- Tandon and Diamond, 1998.. The magnitude of
ing flow cytometry for these assays instead of direct these molecular interactions on cell aggregation can
microscopic evaluation of cell adhesion is the ability be evaluated by the use of blocking monoclonal
to evaluate the behavior of a large population of antibodies ŽSimon et al., 1990..
neutrophils as well as different subpopulations of Neutrophil–platelet aggregation occurs in the cir-
leukocytes. culation and may be of pathophysiological signifi-
cance in conditions such as acute myocardial in-
6.2. Aggregation farction and stroke ŽKonstantopoulos et al., 1995;
Neumann et al., 1997.. This platelet–neutrophil in-
Circulating neutrophils are found predominantly teraction propagates the inflammatory process by
as single cells in their resting state and respond to a increasing CD18rCD11b expression and enhancing
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 31
their homotypic aggregation and adhesion to en- tion and bacterial killing within the phagocytic vac-
dothelium ŽPeters et al., 1997; Konstantopoulos et uole can be measured in the same population of cells
al., 1998.. The interaction between P-selectin on ŽHasui et al., 1989; Haynes et al., 1990; Perticarari et
activated platelets with PSGP-1 on neutrophils is the al., 1991; Saresella et al., 1997.. Protein digestion is
predominant adhesive binding and can be initiated measured by fluorescence of rhodamine attached to
by hydrodynamic shear or platelet activation. Peters albumin at a high molar ratio and quenching of the
et al. Ž1997. have developed a method using small fluorescence when the protein is digested ŽHaynes et
amounts of whole blood to measure platelet–neu- al., 1990.. Using the phagocytosis of the fluo-
trophil complexes and have shown that this interac- rochrome neutral red by neutrophils, Antal and col-
tion is P-selectin and divalent cation-dependent. The leagues have shown that they can measure phago-
majority of neutrophils associated with more than cytosis after exposure to soluble activators ŽPMA
one platelet. This technique is simple and repro- and PAF., making it possible to diagnose
ducible and allows the simultaneous assessment of neutrophil-related phagocytic disorders not related to
platelet–neutrophil interaction and neutrophil activa- the production of reactive oxygen intermediates ŽAn-
tion. tal et al., 1995..
Phagocytosis studies should be done with hep-
arinized blood as EDTA and ACD capture Ca2q ions
that are essential for phagocytosis. Incubation times
7. Phagocytosis between neutrophils and particles should be opti-
mized for maximum phagocytosis because the rate
A number of workers have described methods to and extent of phagocytosis differ depending on the
quantify neutrophil phagocytic capabilities using flow type of particle used. Similarly, cell to particle ratio
cytometry ŽSteinkamp et al., 1982; Bassoe, 1984; should be optimized with a 1:10 ratio an average
Bassoe and Solberg, 1984; Stewart et al., 1986.. ratio for most standard particles.
These methods entail incubating neutrophils with
fluorescent conjugated particles, bacteria or yeast
and measuring the amount of fluorescence as an
indicator of phagocytic activity. To limit cell activa-
tion during neutrophil isolation, whole-blood meth-
ods which are rapid, objective and quantitative, have
been developed ŽHasui et al., 1989; Bohmer et al., Neutrophils ingest bacteria or other particles into
1992; White-Owen et al., 1992; Antal et al., 1995; intracellular compartments called phagosomes and
Santos et al., 1995.. Non-specific adherence of the the destruction of the infectious microorganism is
particles to neutrophils interferes with the measure- achieved by the fusion of granules with the phago-
ment of the number of particles actually internalized somes. The extracellular release of granule content
by the cells. Substances such as trypan blue or occurs concomitantly, that may result in destroying
iodoacetate that quench superficial fluorescence non-phagocyted bacteria but could also result in
without penetrating the plasma membrane have been tissue damage and amplification of the local inflam-
used to reduce this non-specific fluorescence by up matory response. The membrane of granules serves
to 95% ŽSteinkamp et al., 1982; Bassoe, 1984; as a reservoir of receptors and membrane bound
Bjerknes and Bassoe, 1984; Fattorossi et al., 1989.. proteins involved in cell adhesion ŽBainton et al.,
Alternatively, to correct for non-specific adherence 1997., signal transduction ŽRotrosen et al., 1988. and
of particles to neutrophils, a control sample incu- activation of microcidal pathways ŽBorregaard et al.,
bated at 48C, that inhibits phagocytosis but not non- 1983; Spitznagel, 1990.. The fusion of granules with
specific adherence, can be used ŽSantos et al., 1995.. the cell membrane during degranulation changes the
Using whole-blood and two-color flow cytometry, surface expression of these membrane receptors and
several workers have demonstrated that phago- this principle has been used to measure and quanti-
cytosis, reactive oxygen production, protein diges- tate neutrophil degranulation using flow cytometry.
32 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
Small secretory vesicles and granules containing
gelatinase are very sensitive to cell manipulation and
activation ŽLew, 1989. and their degranulation is
associated with the increase in surface CD35rCR1
and CD11b expression. Secondary or specific gran-
ules have different extracellular matrix receptors such
as laminin, fibronectin, vitronectin and CD18 that
binds fibrinogen and are coined ‘‘adhesomes’’
ŽSinger et al., 1989.. CD18rCD11b increase rapidly
on the neutrophil surface upon cell activation and
serves as a sensitive marker of secondary granule
release ŽKishimoto et al., 1989; Bainton et al., 1997..
Sensitive surface markers for primary or azurophilic
granule release are not available. Granule content
levels, such as myeloperoxidase, can be measured
using monoclonal antibodies ŽHewitt and Reardon,
1991; van Eeden et al., 1997b,c.. Kuijpers et al.
Ž1991. propose that one of the tetraspanins, CD63,
which is a lysosomal membrane protein, can be used
as a marker of primary granule release in vivo and in
Fig. 3. ROS produced by activated neutrophils. Membrane-associ-
vitro. Expression of this molecule on senescent neu- ated NADPH-oxidase produce superoxide and hydrogen peroxide
trophils is very low and an increase during cell upon activation. During the oxidative burst, the fluorochromes
activation is associated with a decrease in neutrophil 2 ,7-dichlorofluorescein ŽDCFH. and dihydrorhodamine 123 are
myeloperoxidase activity. The reliability of this sur- oxidated mainly by H 2 O 2 to either green or red fluorescence,
face marker to measure degranulation of primary
granules still needs to be established.
ucts ŽMcCord and Fridovich, 1969; Babior et al.,
1970; Boveris et al., 1977; Roots and Metcalf, 1977..
9. Oxygen radical production
The addition of superoxide dismutase or catalase in
parallel samples provides specificity to the assays for
Central to antimicrobial activity of neutrophils is superoxide anion or H 2 O 2 , respectively. The major
the oxidative burst that generates reactive oxygen disadvantages of these assay methods are that cells
species ŽROS. through the NADPH oxidase system. must be separated from red blood cells and plasma
The best demonstration of the essential role of this is and that relatively large numbers of neutrophils are
the consequences of a lack of ROS generation seen needed Žapproximately 0.5 millionrexperimental
in patients with chronic granulomatous disease condition.. In these assays, the measured response is
ŽCGD.. These patients have defects in their NADPH of a population and individual response cannot be
oxidase pathway, are unable to generate superoxide assessed.
anions necessary for the formation of a number of In 1983, a method was developed to measure
ROS ŽFig. 3. and have recurrent life threatening ‘‘oxidant product formation’’ by flow cytometry us-
bacterial infections from birth ŽGallin, 1992.. ing 2X ,7X-dichlorofluorescein ŽDCF. ŽBass et al.,
In assessing the oxidative burst in neutrophils, the 1983.. In this method, neutrophils are loaded with
most widely used standard methods measure the the diacetate form of 2X ,7-dichlorofluorescein
extracellular release of superoxide anion ŽOy. or 2
ŽDCFH., which is transported across the plasma
hydrogen peroxide ŽH 2 O 2 . by their ability to reduce membrane. In the cytoplasm, the acetate groups are
or oxidize substrates to colored or fluorescent prod- removed by esterases concentrating the polar non-
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 33
fluorescent DCFH in the cytoplasm. During the ox- tions this assumption is valid. However, H 2 O 2 and
idative burst, DCFH is converted to DCF through its precursor, superoxide anion, can cross intact cell
oxidation mainly by H 2 O 2 . Dihydrorhodamine 123 membranes and diffuse to adjacent cells. This was
is used in a similar way resulting in red fluorescence demonstrated by mixing DCFH-loaded neutrophils
ŽEmmendorffer et al., 1990; Rothe et al., 1991; from normal individuals and from patients with CGD
Vowells et al., 1995.. Strong non-physiologic stimuli Žincapable of a respiratory burst.. In this mixture, the
such as PMA and calcium ionophores cause a sus- oxidant products from the cells with a respiratory
tained respiratory burst mirrored by high levels of burst were capable of oxidizing DCFH in non-re-
fluorescence in this assay. More physiologic stimuli sponding bystanders ŽBass et al., 1983.. This is of
produce much smaller and variable responses. little concern for most applications but may be a
The use of flow cytometry to measure the produc- confounding feature in experiments using mixed cell
tion of H 2 O 2 offers an easy semi-quantitative tech- populations. It should be remembered that eosinophils
nique that is applicable to a few thousand unpurified are capable of an oxidative burst, although normally
cells. In contrast to the standard assay mentioned they represent a small fraction of cells in the
previously, the flow cytometric assay can be per- ‘‘granulocyte’’ window. Finally, it should be remem-
formed in mixed cell samples Žwhole blood. where bered that neutrophil activation results in aggregation
neutrophil responses can be determined using gating of neutrophils with other neutrophils and other cells
techniques ŽRichardson et al., 1998.. The small num- especially platelets. These larger aggregates may not
ber of unpurified cells necessary for the flow cytom- be seen in the gates set for single neutrophil and may
etry assay allows studies on small sample volumes exclude a population of activated neutrophils from
such as neonatal blood or from other small animals. the analysis.
In clinical laboratories, the availability and familiar- More recently, flow cytometric assays combining
ity with flow cytometry make these flow cytometry phagocytes and oxidative burst have been developed
assays very attractive in screening patients for CGD using labeled bacteria or immune complexes ŽHasui
ŽEmmendorffer et al., 1990; Roesler et al., 1990.. et al., 1989; Haynes et al., 1990.. The advantage of
these stimuli is that they are more physiologic and
the intensity of the fluorescence is much higher than
9.1. Benefits and limitations of flow cytometry that achieved with DCFH-loaded cells. But the re-
sponse to these particulate fluorochromes is more
The limits of flow cytometry in measuring H 2 O 2 complex. The oxidative burst and release of H 2 O 2
production need to be recognized. Quantitative mea- can occur on contact without uptake of the particle.
surements are difficult. Variation in loading and Phagocytosis concentrates these particles in phago-
hydrolysis of the acetate from DCFH can alter the somes allowing more efficient delivery of the H 2 O 2
relative fluorescence seen. Quantifying the number to the stimulus and fluorochrome. Thus the intensity
of moles of fluorescein per cell is cumbersome and of the signal from each neutrophil is dependent on
most studies report only relative fluorescence. Intra- particulate adherence and uptake as well as the respi-
cellular elements such as catalases or peroxidases ratory burst.
Žespecially myeloperoxidase. significantly affect the
oxidant reaction. The addition of 5 mM azide en-
hances the oxidation of DCFH possibly by inactivat-
ing intracellular enzymes like MPO which reduces
the available H 2 O 2 through their enzymatic reaction
Že.g., the conversion of H 2 O 2 to HOCl. ŽBass et al.,
1983; Smith and Wiedemann, 1993.. Apoptosis or programmed cell death is a well-de-
The assumption in the flow cytometric assessment fined mode of cell demise and are brought about by
of the respiratory burst is that changes in fluores- a diverse array of physiological and non-physio-
cence on stimulation are indicative of the production logical stimuli. It can be initiated through a geneti-
of H 2 O 2 in individual neutrophils. For most applica- cally defined pathway or by intra- or extracellular
34 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
stimuli such as cytokines, hormones, endogenous monly used methods will be discussed. The ability of
proteins, oxidative stress and hypoxia. Apoptosis is flow cytometry to assess apoptosis in large numbers
an important process in normal neutrophil physiol- of cells and simultaneously evaluate subpopulations
ogy because of the extend of neutrophil turnover within a larger cell population are some of the
Ž1–2.5 billion kgy1 dayy1 . and their short life span benefits of using flow cytometry over more conven-
Ž6–10 h. in the circulation ŽMaloney and Patt, 1968; tional methods such as morphology and gel elec-
McAfee and Thakur, 1976.. The changes in aging trophoresis. Because all the methods described below
PMN which allow recognition by macrophages in have shortcomings, it is imperative that at least one
the liver and spleen to remove them from the circula- additional independent method is used to confirm
tion or sites of extravasation are a field of active results.
research ŽSavill et al., 1989; Savill et al., 1992..
Studies from our laboratory ŽBicknell et al., 1994.
showed that apoptosis of neutrophils occurs mainly 11.1. Size and granularity
in the spleen and it is possible that the senescent
cells are recognized and removed in these organs in Flow cytometry allows the detection of the light-
a manner similar to that occurring at inflammatory scattering properties of neutrophils and quantitate the
sites. Aging neutrophils in vitro develop morphologi- cell size Žforward scatter. and granularity Žside scat-
cal and biochemical features typical of apoptosis ter.. Apoptotic neutrophils loose water Žreduced for-
without the addition of external stimuli ŽWhyte et al., ward scatter., become less granular Žreduced side
1993a,b. and we have shown similar events occur in scatter. and these changes in their light scattering
circulating neutrophils as they age in vivo ŽMatsuba properties can be use to identify apoptotic cells
et al., 1997.. This process is characterized by DNA ŽGorman et al., 1997.. These changes are non-specific
cleavage by calcium-dependent endogenous endonu- and can not distinguish between apoptotic and
cleases capable of cleaving chromatin at internucleo- necrotic cells. Both neutrophil size and granularity
somal sites ŽArends et al., 1990.. can also change with cell activation. Therefore, these
There is a 10–100 fold increase in neutrophil parameters of neutrophil apoptosis are not useful as
turnover during acute infection ŽMcAfee and Thakur, the only discriminator for apoptosis because of the
1976. and these neutrophils meet their fate in the potential heterogeneity of most neutrophil popula-
tissue, as there is little evidence that they return to tions.
the blood from the inflamed site. The survival of
neutrophils can be prolonged by cytokines present at
the inflammatory site ŽBrach et al., 1992; Colotta et 11.2. Fluorescent dyes
al., 1992.. The suppressed apoptosis in neutrophils
during acute infectious or inflammatory conditions The DNA-binding dye propidium iodide can be
has been postulated to be important in the pathogen- use to reveal the amount of DNA in each cell and
esis of neutrophil-mediated diseases such as is- this assay make use of the fact that apoptotic cells
chemia reperfusion injury, acute respiratory distress contain subdiploid amounts of DNA ŽMesner and
syndrome, sepsis and multiorgan failure ŽJones and Kaufman, 1997.. This technique has disadvantages
Morgan, 1995; Liles and Klebanoff, 1995.. since it does not distinguish between apoptotic and
necrotic cells and requires additional techniques to
confirm that the subdiploid cell population is truly
apoptotic. The presence of multiple apoptotic bodies
may result in overestimation of the number of cells
11. Detection of apoptosis by flow cytometry
that are apoptotic, conversely, using gating tech-
niques to exclude these bodies may underestimate
Several flow cytometric methods have been de- the number of apoptotic cells.
scribed to detect and quantitate apoptosis in neu- Other vital dyes such as acridine orange Žgreen
trophils and in this short synopsis the more com- fluorescence. and ethidium bromide Žorange fluores-
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 35
cence. rely on the differential uptake of these dyes free 3X-ends produced by endonuclease action during
by normal healthy, apoptotic and necrotic cells. Us- apoptosis ŽGavrieli et al., 1992.. Briefly, neutrophils
ing ethidium bromide cells can be graded as negative are fixed, permeabilized and incubated with biotinyl-
Žhealthy., weakly Žapoptotic. or strongly Žnecrotic. ated deoxyuridine triphosphate in the presence of
positive. Similar results are obtained with com- TdT and Co 2q. After washing, cells are incubated
pounds such as Hoechst 33342 or 7-aminoacti- with fluorescent labeled streptavidin that binds to the
nomycin D ŽGong et al., 1994; O’Brien and Bolton, biotin. One of the benefits of this technique is that it
1995. but labeling with these dyes must be done at can be combined with fluorescent microscopic analy-
48C. The benefits of these dyes are that large popula- sis and compared with morphological changes of
tions of cells can be screened and these dyes can be DNA fragmentation in individual cells. The disad-
combined with other fluorochromes to identify apop- vantage of this method is that it could detect nicks in
tosis in subset of cells ŽO’Brien and Bolton, 1995.. DNA due to the presence of other enzymes such as
One potential disadvantage of this approach is the topoisomase that are not necessarily associated with
lack of good studies to verify the specificity of these apoptosis ŽFroelich-Ammon et al., 1995.. In addi-
methods. Furthermore, cell membrane integrity is tion, necrotic cells can stain with TUNEL because
maintained in the early stage of apoptosis but is lost random DNA degradation occurring during necrosis
with advanced apoptosis and these techniques may also generates ends with 3X OH groups ŽGrasl-Kraupp
underestimate the actual number of apoptotic cells if et al., 1995.. Therefore, alternative techniques needs
all strongly stained cells are exclude from the analy- to be combined with TUNEL to confirm that cells
sis. are undergoing apoptosis rather than necrosis.
The measurement of changes in mitochondrial
11.3. Surface markers of apoptosis transmembrane potential ŽZamzami et al., 1995., ac-
tivation of various proteases such as the IL-1b con-
verting enzyme ŽICE. family ŽTakahashi et al., 1996.
Annexin V, a polypeptide that binds strongly and
and bax the proapoptotic member of the bcl-2 fam-
specifically to cell surface phosphatidylserine is a
ily of apoptosis regulators ŽMiyashita and Reed,
sensitive measurement of early apoptosis ŽKoopman
1995. are all indicative of apoptosis and can be
et al., 1994; Martin et al., 1995.. Phosphatidylserine
measured using flow cytometry. Flow cytometry al-
is present exclusively in the inner layer of the cell
lows the rapid detection of apoptotic neutrophils by
membrane of healthy cells and becomes accessible
permitting the measurement of a variety of parame-
on the surface of cells undergoing apoptosis. This is
ters that alter during the onset and progression of
the basis for detecting apoptotic neutrophils using
apoptosis. It has significantly aided our understand-
fluorescent labeled Annexin V. Cells are labeled
ing of neutrophil apoptosis and its potential role in
following fixation with a non-permeabilizing fixative
Že.g., formaldehyde. or without fixation. Once the the pathogenesis of neutrophil-induced inflammatory
plasma membrane integrity is lost, cells will become
Annexin V positive and accordingly, this technique
is not suitable for specimens contaminated with
necrotic cells or cells that have been permeabilized. 12. Priming of neutrophils
11.4. Terminal deoxyribonucleotidyl transferase- Priming of neutrophils is one of the pivotal pro-
mediated dUTP nick-end labeling (TUNEL) cesses that regulate their functional responses ŽCon-
dliffe et al., 1998a.. Priming refers to the process
The TUNEL technique is a sensitive method to where the response of neutrophils to an activating
detect apoptosis in neutrophils using flow cytometry. stimulus is enhanced by prior exposure to small
The principle of these techniques is that in the ‘‘non-activating’’ concentrations of this or another
presence of a divalent cation, terminal deoxyribonu- stimulus ŽGuthrie et al., 1984.. Guthrie et al. de-
cleotidyl transferase ŽTdT. will add nucleosides to scribed up to 20-fold enhancement of superoxide
36 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
production by neutrophils by prior exposure to a the real magnitude of intravascular neutrophil prim-
priming agent. In the strictest sense the priming ing.
response is not associated with a functional response Inflammatory mediators generated locally at the
but a variety of biochemical events ŽWalker and site of inflammation serve to upregulate the func-
Ward, 1992.. Enhancement of agonist-induced de- tional responses of neutrophils sequestered and ex-
granulation ŽFittschen et al., 1988. and generation of travasated into the tissues. Cross-linking of neu-
lipid mediators such as LTB4 and PAF ŽDoerfler et trophil adhesion receptors is a priming stimulus
al., 1989. or other cytokines ŽZallen et al., 1999. has ŽWaddell et al., 1994; Liles et al., 1995.. The process
been described. A variety of substances, both physio- of extravasation of neutrophils itself results in in-
logical and pharmacological, have been shown to crease stimulus-induced oxidant release and other
prime neutrophils ŽWalker and Ward, 1992; Condliffe phenotypic changes such as changes in their expres-
et al., 1998b., and many have biological relevance in sion of surface adhesion molecules. This needs to be
vivo because they are released in response to infec- taken into account when extravasated neutrophils
tion, hemorrhage and trauma. Priming agents such as from areas such as the alveolar space or peritoneal
endotoxin and cytokines, notably TNF-a, IL-6 and cavity are harvested for analysis.
IL-8, are detectable in the blood and are associated Flow cytometry has been used extensively to
with a poor outcome in subjects with septic shock, identify and quantify neutrophil priming. Activation
ARDS and multiorgan failure ŽParsons et al., 1989; of the NADPH oxidase enzyme system with the
Pinsky et al., 1993.. Smedley et al. Ž1986. have production of superoxide is the hallmark of neu-
shown that neutrophils activated with fMLP, C5a trophil priming. This is the most primable neutrophil
and LPS cause minimal damage to endothelial cells function with the potential to increase superoxide
in vitro but if cells were first primed with LPS and production up to 20-fold. However, other changes in
then stimulated with fMLP or C5a, extensive damage neutrophils following priming are shape changes and
to endothelial cells ensued. stiffening ŽHaslett et al., 1985., changes in the ex-
Work from our laboratory has shown that the pression of adhesion molecules with an increase in
process of sequestration of neutrophils may also CD11b and a decrease in L-selectin ŽCondliffe et al.,
result in priming ŽKitagawa et al., 1997.. Because 1996., degranulation of primary and secondary gran-
neutrophils are larger than the majority of pulmonary ules ŽDoerfler et al., 1994. and a delay in apoptosis
capillary segments they have to pass through, these ŽLee et al., 1989.. Lastly, sample preparation for
cells have to deform to cross the pulmonary vascular flow cytometry and trace amounts of endotoxin in
bed ŽDoerschuk et al., 1993; Wiggs et al., 1994.. solutions used for cell isolation could prime neu-
Using flow cytometry, we have shown that passive trophils ŽHaslett et al., 1985.. Because many of these
deformation of neutrophils by filtration through 5-mm neutrophil functions can be measured in whole blood,
pores Žsimilar to deformation in the pulmonary capil- flow cytometry is the method of choice to evaluate
lary bed. causes enhanced CD18rCD11b expression neutrophil priming. It is vital to realize that neu-
following maximal stimulation with fMLP. The trophils are not terminally differentiated cells and
priming effect of this deformation was comparable to that their functional responses can be up- or down-
using fMLP as a priming agent. Neutrophils exposed regulated both in vivo and in vitro.
to priming concentrations of inflammatory mediators
undergo shape changes as a result of modification of
their cytoskeletal actin, a key event that makes neu-
trophils less deformable ŽWorthen et al., 1989. and
increases their sequestration in the pulmonary capil-
lary bed ŽDoerschuk et al., 1989.. Prolonged reten-
tion of these primed neutrophils in the lung increases Our understanding of the basic functions of neu-
the risk of neutrophil-mediated tissue damage. trophils relies heavily on in vitro observations with
Therefore, in conditions such as septicemia, measur- extrapolation to in vivo events. However, it is re-
ing priming in circulating neutrophils underestimates markably easy to disturb normal neutrophil function
S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43 37
by removing them from the circulation. The use of surface glycoprotein in the granulocytopenia of hemodialysis.
flow cytometry has advanced our ability to measure N. Engl. J. Med. 312, 457.
Babior, B.M., Kipnes, R.S., Curnutte, J.J., 1970. Biological de-
and quantify neutrophil functions. Using whole-blood fense mechanism. The production by leukocytes of superoxide
methods with minimal manipulation of cells gives a a potential bactericidal agent. J. Clin. Invest. 52, 791.
more accurate measure of neutrophil behavior in Bainton, D.F., 1992. Development biology of neutrophils and
vivo. The ability of flow cytometry to measure the eosinophils. In: Gallin, J.I., Goldstein, I.M., Snyderman, R.
ŽEds.., Inflammation: Basic Principles and Clinical Correlates,
function of a single cell or large numbers of cells
Chap. 17, 2nd edn. Raven Press, New York, p. 303.
and the potential to measure multiple functions si- Bainton, D.F., Miller, L.J., Kishimoto, T.K., Springer, T.A., 1997.
multaneously by using multiple fluorochromes has Leukocyte adhesion receptors are stored in peroxidase-nega-
improved our ability to study the function of a tive granules of human neutrophils. J. Exp. Med. 166, 1641.
complex cell in health and disease. The application Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C.,
of flow cytometry to measure neutrophil function is Thomas, M., 1983. Flow cytometric studies of oxidative prod-
uct formation by neutrophils: a graded response to membrane
progressively expanding in basic and clinical sci- stimulation. J. Immunol. 130, 1910.
ences. Bassoe, C.F., 1984. Processing of staphylococcus aureus and
zymosan particles by human leukocytes measured by flow
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Bassoe, C.F., Solberg, C.O., 1984. Phagocytosis of Staphylococ-
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This work was supported by the BC Lung Associ- Bateman, J., Parida, S.K., Nash, G.B., 1993. Neutrophil assay for
clinical studies. Cell Biochem. Funct. 11, 87.
ation and the Medical Research Council of Canada
Bei, L., Hu, T., Qian, Z.M., Shen, X., 1998. Extracellular Ca2q
grant a4219. regulates the respiratory burst of human neutrophils. Biochem.
Biophys. Acta 1404, 475.
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