Journal of Immunological Methods 232 Ž1999.

23–43
www.elsevier.nlrlocaterjim

The use of flow cytometry to measure neutrophil function

a,)
Stephan F. van Eeden , 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,
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-
oxidase

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: svaneeden@prl.pulmonary.ubc.ca 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
distinct transduction pathways may independently
regulate these processes. The test or tests selected
depend on the nature of the problem that needs to be
addressed. The measurement of physiological func-
tions such as chemotaxis, phagocytosis, bacterial
killing and apoptosis remain time consuming, but the
availability of a wide variety of fluorochromes and
flow cytometry has allowed the development of
methods that rapidly measure a variety of functions
in a single population of neutrophils.
The devastating effects of inherited abnormalities
in neutrophil function highlight the important role of
neutrophils in host defense. Clinical suspicion of
impaired neutrophil function is usually related to a
marked susceptibility to infection. In these subjects,
abnormalities in neutrophil numbers Žneutropenia. or
immune abnormalities, such as opsonic defects, are
more common and should be considered first. Flow
cytometry can assist in suggesting the diagnosis of
an inherited defect in the majority of these disorders
ŽEpling et al., 1992.. Conditions where inappropriate
neutrophil-mediated tissue injury are an important
component in the pathogenesis of the disease, such
as acute respiratory distress syndrome, ischemia
reperfusion injury and rheumatoid arthritis ŽHernan-
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
‘‘hyperfunction’’. In normal ŽTanji-Matsuba et al.,
1998. or acquired neutrophil functional abnormali-
ties, the use of flow cytometry has assisted in the
understanding of the role of neutrophils in disease
pathogenesis.
This review evaluates the various methods avail-
able for measuring neutrophil function using flow
cytometry. This powerful tool has the ability to
measure multiple parameters on individual cells at
high 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 for
neutrophil functional analysis changes their behavior.
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-
tional behavior of neutrophils as well as the number
and the affinity of their receptors can change with
the type of anticoagulant used to collect the blood
ŽRepo et al., 1995; Conklyn et al., 1996., fluctua-
tions in temperature during cell processing ŽRepo et
al., 1995; van Eeden et al., 1995; Youssef et al.,
1995., density centrifugation ŽKuijpers et al., 1991.,
hypotonic lysis of red cells ŽStyrt et al., 1988., and
endotoxin contamination of reagents or buffers
ŽHaslett et al., 1985.. All reagents used should be
pyrogen-free and of tissue culture grade and plastic
tubes should be used for processing samples. The
anticoagulant, heparin, can activate the complement
system and generate complement fragment ŽC5a. in
the plasma resulting in cell activation and calcium
chelating agents such as EDTA should be avoided
with antibodies whose binding are cation-dependent
ŽRepo et al., 1995. or when measuring functions that
are calcium-dependent. Artifactual upregulation of
sensitive adhesion receptors such as CD11b ex vivo
can be minimized by cooling blood samples on ice.
However, if samples are cooled, cell labeling and
fixation should be completed on ice because fluctua-
tions in temperature have a significant effect on the
expression of receptors such as CD11b and L-selec-
tin ŽRepo et al., 1995; van Eeden et al., 1995..
Keeping the blood samples at a constant temperature
such as room temperature or 378C Žfor cell activation
and other functional studies. following sample col-
lection is important to reduce artifactual changes in
neutrophil functional responses. Whole-blood meth-
ods combined with gating techniques to separate
leukocyte subsets or leukocyte specific surface mark-
ers can be used to measure several neutrophil func-
tions simultaneously ŽHasui et al., 1989; Haynes et
al., 1990.. If purified neutrophils are used, the inter-
pretation of sensitive surface markers such as CD11b
should be reserved ŽKuijpers et al., 1991..
3. Neutrophil functional responses
The functional response of neutrophils to an incit-
ing stimulus, such as an extravascular infection, is a
sequential multistep process Žillustrated in Fig. 1..
Factors that regulate these sequential responses are
not well-understood. One factor that could influence
these sequential responses is the level of exposure to
cell activating stimuli at each step of the cascade.
Fig. 2 shows a schematic representation of the effect
of 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 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-
sponses. It demonstrates the concept that neutrophils
respond in a graded fashion to increasing concentra-
tions of the agonist. This concept is supported by the
work of several investigators ŽPetrequin et al., 1987;
Lew, 1989; Packman and Lichtman, 1990; Fernan-
dez-Segura et al., 1995.. An increase in calcium and
F-actin assembly occurs at very low concentrations
of the agonist Ž10y9 M. in contrast to degranulation
of secondary and primary granules as wells as oxy-
gen radical production that required higher concen-
trations of fMLP Ž10y8 to 10y6 M. to elicit a
significant response. This response is also time-de-
pendent with Ca2q mobilization and F-actin assem-
bly occurring rapidly Žs. in contrast to degranulation
of primary granules and oxygen radical production
Žmin.. This organized sequential nature of the pro-
cess is important to ensure that normal host tissue is
not damaged during the process of eliminating the
invading microorganisms.
Activation of neutrophils is not an all or nothing
phenomenon and each function has its own threshold
for a response. Actin assembly in neutrophils allows
them to flatten and crawl on endothelium towards a
gradient of chemoattractant, and then migrate out of
the vasculature towards the inciting stimulus. During
chemotaxis, degranulation with the release of lysoso-
mal enzymes and oxygen radical by the neutrophils
could cause damage to normal tissues. These latter
functions are more appropriate following phago-
cytosis of microorganisms. This proposed paradigm
of concentration and time-dependent neutrophil re-
sponses protect the host from inappropriate neu-
trophil-mediated tissue damage. Although numerous
neutrophil responses occur simultaneously with cell
activation, this sequential paradigm illustrated in
Figs. 1 and 2 is a practical way to partition the
functional responses of neutrophil following cell ac-
tivation and this approach will be used to discuss
flow cytometric methods available to measure neu-
trophil function.
Flow cytometry measures fluorescence and light
scatter signals of suspended cells through a laser
source. Gating techniques allow the analysis of struc-
tural and functional parameters in a large population
of intact single cells excluding those that are aggre-
gated or lysed ŽDuque and Ward, 1987; Durack et
al., 1991; Carulli, 1996.. These gating techniques
could influence results because they exclude aggre-
gated cells Ždue to cell activation.. Furthermore,
gating on neutrophils includes eosinophils and re-
sults should be interpreted with caution in subjects
with high eosinophil counts. Cell specific surface
markers, such as CD32 that are not expressed by
eosinophils but by other granulocytes, can be used to
separate neutrophil and eosinophils. Flow cytometry
can also resolve the heterogeneity of a cell popula-
tion and facilitate the identification and characteriza-
tion of cell subpopulations and subcellular compart-
ments ŽConklyn et al., 1996.. It permits multipara-
metric analysis ŽOrfao and Ruiz-Arguelles, 1996;
Macey et al., 1998., particularly, intracellular and
surface-exposed antigens.
3.1. Common stimuli used to measure neutrophil
function
Chemoattractants commonly used to measure neu-
trophil functional responses are either endogenous,
such as interleukin 8 ŽIL-8., leukotriene B4 ŽLTB4.,
platelet activating factor ŽPAF., C5a, interleukin 1
ŽIL-1., and tumor necrosis factor alpha ŽTNF-a ., or
exogenous, such as the formylated peptide fMLP, a
surface receptor agonist derived from bacterial cell
products ŽElsner et al., 1992.. The phorbol esters
ŽPMA. and ionophores Žionomycin or A25187. are
agonists that do not work via receptor binding and
are useful in studying mechanisms underlying neu-
trophil activation. In addition, cytochalasins are com-
monly used to inhibit or enhance certain neutrophil
functions ŽTreves et al., 1987; Al-Mohanna et al.,
1997.. During cell activation, cell surface receptors
can be transiently desensitized when the cell is stim-
ulated by a ligand Že.g., fMLP.. Cells then become
unresponsive to the subsequent exposure to the same
ligand Žhomologous desensitization. or different lig-
ands Žheterologous desensitization. ŽSklar et al.,
1985; McColl and Naccache, 1997.. Factors that can
contribute to this desensitization after a single addi-
tion of an agonist include microaggregation of cells
ŽLofgren et al., 1993., internalization of ligand–re-
ceptor complexes, reduced receptor-associated GT-
Pase activity or increased wCa2q xi ŽVindenes and
Bjerknes, 1997.. Therefore, cells that have been
exposed to inflammatory mediators in vivo could
have an attenuated response when activated in vitro.
 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
4. Calcium mobilization
Calcium mobilization is one of the earliest events
that occur with neutrophil activation ŽFig. 2.. Upon
activation, ligands occupy the surface receptors and
elicit an immediate rise in the cytosolic free Ca2q as
a result of Ca2q release from the internal stores. This
rapid response Žs. is followed by a more sustained
Žmin. effect due to Ca2q influx from the extracellu-
lar medium ŽWhite et al., 1983; Anderson and Ma-
homed, 1997; Andersson et al., 1986.. Continuous
communication between the extracellular and intra-
cellular environment is essential for maintaining
Ca2q homeostasis and modulating PMN functional
responses ŽSklar and Oades, 1985.. Transient in-
creases in free cytosolic calcium act as a secondary
messenger for numerous neutrophil biological re-
sponses ŽBengtsson 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 Ca 2 q indicators
Numerous fluorescent dyes have been developed
as Ca2q indicators. These non-fluorescent mem-
brane-permeable esters cross the plasma membrane
of living cells and are hydrolyzed by cytoplasmic
esterases to generate a free acid fluorescent active
form of the probe that is trapped in the cytoplasm
ŽLew, 1989.. Calcium indicators are excited either at
a short wavelength of 340–380 nm Že.g., Quin-2,
Fura-2 and Indo-1. or at a long wavelength above
450 nm Že.g., Fluor-3, rhod-2, Calcium Green, Cal-
cium Crimson and Calcium Orange. ŽSimpson,
1999.. Fura-2 acetoxymethyl ester ŽFura-2rAM.
alone is considered inadequate for flow cytometric
analysis as the intracellular wCa2q xi is estimated us-
ing the ratio of absorption Žexcitation. intensity at
two different wavelengths. In contrast, Indo-1rAM
is almost non-fluorescent unless bound to calcium,
exhibits a great ability to resolve small changes in
wCa2q xi Žmicromolar level., ŽJustement et al., 1990.
and is suitable for single-cell measurements by flow
as it needs only one excitation source ŽUV light. and
two fluorescence emissions ŽMaftah et al., 1993..
However, the use of Indo-1 presents a significant
drawback because it requires UV light which is not
widely available ŽLopez et al., 1989; Novak and
27
Rabinovitch, 1994.. The long wavelength probe,
Fluor-3, shows a greater affinity for calcium than
Fura-2 or Indo-1 and is well-suited for flow cytomet-
ric analysis but unlike Fura-2 and Indo-1, it is not
amenable to ratiometric analysis. Fluor-3 causes
compartmentalization and heterogeneous loading
which could be relevant in a cell population with
variable size Žcytoplasmic volume. and granularity.
Both events cause variability in the baseline fluores-
cence intensity and may compromise the sensitivity
and accuracy of Ca2q measurements ŽJustement et
al., 1990.. To improve quantitative flow cytometric
measurements of intracellular calcium, ratiometric
procedures are used with Fluo-3 as an adjunct to
Fura-2 ŽNovak and Rabinovitch, 1994. or SNARF-1
ŽRijkers et al., 1990..
4.2. Intracellular Ca 2 q measurements
In order to maximize calcium changes, it is im-
portant to optimize dye loading conditions for each
cell type that depends on the concentration of the
dye, the temperature and duration of the incubation,
the level of intracellular esterases and the permeabil-
ity of the plasma membrane ŽMcColl and Naccache,
1997.. Calcium levels can be measured in LRP or in
purified neutrophils suspended in a calcium-contain-
ing buffer Že.g., Hank’s balanced salt solution.. Cells
are incubated at 378C for 30 min with 2 mM of
indicator dye, washed, resuspended in the same buffer
and allowed to equilibrate for 5–15 min before
baseline measurements ŽElsner et al., 1992; McColl
and Naccache, 1997.. Although rarely needed by
hematopoietic cells, dispersing agents such as the
non-ionic detergent Pluronic F-127 or bovine serum
albumin can be used to increase the solubility of the
dye. When required, probenicid is used to inhibit
extrusion of the fluorescent dye ŽOmann 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 in
calcium measurements
Flow cytometric analysis has significant advan-
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-
geneity of a cell population and identifies and char-
acterizes subpopulations of cells ŽElsner 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
disorders such as periodontitis ŽChampagne et al.,
1998. and glycogen storage disease type 1b ŽElsner
et al., 1992.. Currently, these methods have limita-
tions due to the inability to sort cells under sterile
conditions ŽOrfao and Ruiz-Arguelles, 1996.. It is
also possible that measurements of calcium by flow
cytometry miss the initial peak since there is a time
lapse Žapproximately 15 s. between adding the ago-
nist and the actual time at which Ca2q measurements
take place. For more details on the role of Ca2q in
regulating neutrophil function, see the review of
Hallet et al. in this issue.
5. Cytoskeletal actin and cytoskeleton regulatory
proteins
Actin, a 43-kDa peptide, is a major cytoplasmic
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
activation by stimuli such as fMLP, C5a, zymosan-
activated plasma ŽZAP., 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 actin
assembly is one of the earliest and most sensitive
neutrophil functional responses ŽKelley, 1991.. Cy-
toskeletal events near the cell surface regulate func-
tional responses such as adhesion and deformability
and can provide the driving force for mobility,
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
been linked to an increased susceptibility to infection
ŽVindenes and Bjerknes, 1997. and has been shown
in newborns ŽMerry et al., 1998. as well as in
patients with thermal injury ŽVindenes and Bjerknes,
1997. and myelodysplastic syndromes ŽCarulli et al.,
1997.. Furthermore, flow cytometry has been used to
assess the role of the cytoskeleton in dialysis neu-
tropenia ŽTabor et al., 1998. and to demonstrate
neutrophil–actin dysfunction in a recessive inherited
disorder in which subjects presented with recurrent
infections ŽPackman and Lichtman, 1990..
5.1. F-actin measurements
The measurement of F-actin content by flow cy-
tometry requires a small volume of peripheral blood
Ž45 ml.. Whole-blood cells are first equilibrated at
378C and then activated with the agonist. The reac-
tion is stopped by addition of 3.0% paraformal-
dehyde. Erythrocytes are lysed and leukocytes are
permeabilized and stained with a mixture of L-a-
lysophosphatidylcholine and N-7-nitrobenz-2-oxa-
1,3-diazole-4-yl ŽNBD.-phallacidin ŽChen et al.,
1996; Klut et al., 1997.. The fluorochrome is excited
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
with quantitative assays Ži.e., gel scanning. ŽWatts
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 method
for assessing actin dynamics and allows the identifi-
cation of cell subpopulations that are less responsive
to stimulants ŽChen et al., 1996.. However, flow
cytometry does not permit discrimination between
the stable gelsolin-free and the labile gelsolin-rich
F-actin pools. Also, this approach does not sense
changes in cell shape or actin redistribution ŽWatts
and Howard, 1992; Carulli et al., 1997.. For more
details on the role of F-actin assembly in neutrophil
function, see the review of Coates et al. in this issue.
6. Adhesion and aggregation
6.1. Adhesion
The adhesion of neutrophils to endothelial cells is
a pivotal event in the process of cell migration and
subsequent tissue inflammation. This process can be
evaluated in several ways. Firstly, the number of
 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
adhesion molecules on the surface of neutrophils can
be quantitated ŽBateman et al., 1993; Repo et al.,
1993; Youssef et al., 1995. or the affinity of these
adhesion molecules for their respective ligands can
be evaluated ŽLarson and Springer, 1990.. Several
cell surface molecules that are involved in the neu-
trophil–endothelial adhesive interactions have been
identified and characterized ŽCarlos and Harlan,
1994.. The initial neutrophil–endothelial interaction
is mediated by members of the selectin family Ži.e.,
L-, E- and P-selectin. that establish a loose associa-
tion of neutrophils with the endothelium Žvon An-
drian et al., 1991; Bevilacqua, 1993.. This is fol-
lowed by firm adhesion and migration of neutrophils
involving members of the integrin family on neu-
trophils Ži.e., CD11b or MAC-1. interacting with the
immunoglobulin superfamily on endothelial cells
Ži.e., intercellular adhesion molecule 1 ŽICAM-1.
and PECAM-1. ŽSmyth et al., 1993; Carlos and
Harlan, 1994..
The expression of cell adhesion molecules on the
surface of neutrophils can be measured in whole-
blood specimens. P- and E-selectin are expressed on
the endothelium after cell activation and L-selectin
ŽCD62L. is constitutively expressed on circulating
neutrophils and shed from the cell surface upon cell
activation ŽKishimoto et al., 1989.. The reduction in
surface expression of L-selectin on circulating neu-
trophils should be interpreted with caution because
active bone marrow release causes an increase in
circulating neutrophils expressing high levels of L-
selectin Žvan Eeden et al., 1995.. Studies from our
Žvan Eeden et al., 1995, 1997b,c; Nakagawa et al.,
1998. and other ŽLund-Johansen and Terstappen,
1993. laboratories have shown that this is due to the
high levels of surface expression of L-selectin on
neutrophils in the maturation pool of the bone mar-
row. Stimulation of the bone marrow is associated
with the release of these neutrophils expressing high
levels L-selectin Žvan Eeden et al., 1995; Lawrence
et al., 1996.. Furthermore, we have shown that neu-
trophils loose their surface L-selectin as they age in
the circulation Žvan Eeden et al., 1997a. and several
studies have shown that drugs such as steroids ŽBur-
ton et al., 1995; Waisman et al., 1998; Nakagawa et
al., 1999. and non-steroidal anti-inflammatory drugs
ŽDiaz-Gonzalez et al., 1995. reduce L-selectin on
circulating neutrophils. Therefore, the interpretation
29
of L-selectin expression on circulating neutrophils
should 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 mediate
cell–cell and cell–matrix adhesion and are essential
for firm adhesion of neutrophils to endothelium and
migration through vessel walls and interstitial tissue
ŽArnaout, 1990; Springer, 1990.. The b 2 integrins
CD18rCD11b are constitutively expressed on circu-
lating neutrophils and their surface expression in-
creases rapidly upon cell activation ŽKishimoto et al.,
1989; Repo et al., 1993.. This is a useful and sensi-
tive marker of cell activation in vivo in clinical
conditions such as infection, ischemia and throm-
botic events ŽHansen, 1995; Mazzone et al., 1997.. A
granulocytosis and recurrent bacterial infections in
these subjects highlight the critical importance of
this molecule in mounting an effective neutrophilic
inflammatory response in subjects with an inherited
deficiency of the b 2 integrins Žleukocyte adhesion
deficiency 1 or LAD1. ŽSpringer et al., 1984; Ander-
son and Springer, 1987.. Conformational changes in
CD18rCD11b upon cell activation alter the affinity
of the molecule for its ligands ŽLarson 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-
jects with near normal CD18 expression Ž40%–60%
levels adequate for normal function. but the clinical
phenotype of LAD1 with recurrent bacterial infec-
tions and impaired pus formation ŽKuijpers et al.,
1997; Hogg et al., 1999.. In these subjects, cell
activation did not result in CD18 activation and
high-avidity ligand binding that results in reduced
neutrophil aggregation and migration. A mutation in
the b 2-subunit conserved domain in the metal ion-
dependent adhesion site could be responsible for this
failure of the b 2 integrins to function ŽHogg et al.,
1999..
Several factors should be considered when the
CD18rCD11b adhesion function of neutrophils is
evaluated using flow cytometry. A whole-blood
method is preferred to avoid the changes induced by
neutrophil isolation procedures ŽKuijpers et al.,
1991., EDTA as an anticoagulant should be avoided
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-
bodies binding the high-affinity receptors should be
used if adhesiveness via CD18rCD11b is evaluated.
Secondly, assays of dynamic Žvon Andrian et al.,
1991. or static ŽTaylor et al., 1996. neutrophil adhe-
sion in vivo or ex vivo directly test the ability of
neutrophils to adhere to the endothelium. In the
dynamic in vivo adhesion assays, the type of leuko-
cyte that interacts with the endothelium cannot be
discriminated and can be overcome by using purified
neutrophils in an ex vivo system ŽTaylor et al., 1996;
Neelamegham et al., 1998.. These dynamic systems
largely test the functional integrity of the selectin
group of adhesion molecules ŽL-, P- and E-selectin.
and their interaction with their ligands. Flow cytome-
try has a limited role in performing these assays. The
static assays tested firm adhesion of neutrophils to
endothelial cells and evaluated largely the interaction
of the integrins ŽCD18 group of molecules. with
their counter receptors on the endothelium ŽICAM-1
and ICAM-2.. Most of these static assays used puri-
fied neutrophils incubated on cultured endothelial
monolayers ŽKorlipara et al., 1996. and used flow
cytometry to evaluate the number of neutrophils that
adhere to the endothelium with and without stimula-
tion of either the neutrophils or the endothelium.
This is accomplished by detachment of endothelial
cells from the plastic and dissociation of adherent
neutrophils by treatment with trypsin and EDTA
ŽKorlipara et al., 1997; Marshall et al., 1997.. By
gating on neutrophils and endothelial cells, the num-
ber of adherent neutrophils per endothelial cell can
be calculated. Marshall et al. Ž1997. have recently
demonstrated that this method can be modified to
use whole blood instead of purified neutrophils. This
method also allows the evaluation of the adhesion of
other leukocytes such as monocytes, eosinophils and
lymphocytes to endothelial cells. The benefit of us-
ing flow cytometry for these assays instead of direct
microscopic evaluation of cell adhesion is the ability
to evaluate the behavior of a large population of
neutrophils as well as different subpopulations of
leukocytes.
6.2. Aggregation
Circulating neutrophils are found predominantly
as single cells in their resting state and respond to a
variety of soluble stimuli by forming homo- or het-
erotypic aggregates. These intravascular aggregates
contribute to the inflammatory reaction and tissue
injury especially in ischemic syndromes. Activation
by chemotactic peptide induces transient aggregation
both in vitro ŽSklar et al., 1985; Tandon and Dia-
mond, 1998. and in vivo ŽSchmid-Schonbein, 1987a;
Schmid-Schonbein et al., 1987b.. Light transmit-
tance as measured by aggregometer has previously
been the standard method to quantitate neutrophil
aggregation but provides at best a gross measure-
ment of mostly large aggregates ŽCraddock et al.,
1977; Ringertz et al., 1985.. Flow cytometric meth-
ods have the benefit of analyzing precise particle
size, distribution of the different particle sizes and
the time course of particle formation and disaggrega-
tion ŽSklar et al., 1985; Neelamegham et al., 1997..
The simplest form of data acquisition is to calculate
the extent of aggregation by measuring changes in
the total number of particles per unit volume over
time. These measurements correlate well with values
determined by microscopy ŽRochon and Frojmovic,
1991.. Following cell activation, neutrophil aggrega-
tion occurs rapidly Ž; 4 s. and the subsequent rate
of forward aggregation and disaggregation depends
on neutrophil numbers and type and the duration of
the activator ŽSimon et al., 1990; Rochon and Froj-
movic, 1991..
The additional benefit of flow cytometry is the
ability to simultaneously explore the mechanisms of
aggregation at the level of the adhesive receptor.
Activated neutrophils aggregate during shear stress
by bonding of surface L-selectin with P-selectin
glycoprotein ligand-1 ŽPSGP-1. ŽGuyer et al., 1996.
followed by more stable bonding via CD18rCD11a
to ICAM-3 and CD18rCD11b to a yet a unknown
ligand ŽArnaout et al., 1985; Simon et al., 1990;
Tandon and Diamond, 1998.. The magnitude of
these molecular interactions on cell aggregation can
be evaluated by the use of blocking monoclonal
antibodies ŽSimon 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-
farction and stroke ŽKonstantopoulos et al., 1995;
Neumann et al., 1997.. This platelet–neutrophil in-
teraction propagates the inflammatory process by
increasing CD18rCD11b expression and enhancing
 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
their homotypic aggregation and adhesion to en-
dothelium ŽPeters et al., 1997; Konstantopoulos et
al., 1998.. The interaction between P-selectin on
activated platelets with PSGP-1 on neutrophils is the
predominant adhesive binding and can be initiated
by hydrodynamic shear or platelet activation. Peters
et al. Ž1997. have developed a method using small
amounts of whole blood to measure platelet–neu-
trophil complexes and have shown that this interac-
tion is P-selectin and divalent cation-dependent. The
majority of neutrophils associated with more than
one platelet. This technique is simple and repro-
ducible and allows the simultaneous assessment of
platelet–neutrophil interaction and neutrophil activa-
tion.
7. Phagocytosis
A number of workers have described methods to
quantify neutrophil phagocytic capabilities using flow
cytometry ŽSteinkamp et al., 1982; Bassoe, 1984;
Bassoe and Solberg, 1984; Stewart et al., 1986..
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.,
1992; White-Owen et al., 1992; Antal et al., 1995;
Santos et al., 1995.. Non-specific adherence of the
particles to neutrophils interferes with the measure-
ment of the number of particles actually internalized
by the cells. Substances such as trypan blue or
iodoacetate that quench superficial fluorescence
without penetrating the plasma membrane have been
used to reduce this non-specific fluorescence by up
to 95% ŽSteinkamp et al., 1982; Bassoe, 1984;
Bjerknes and Bassoe, 1984; Fattorossi et al., 1989..
Alternatively, to correct for non-specific adherence
of particles to neutrophils, a control sample incu-
bated at 48C, that inhibits phagocytosis but not non-
specific adherence, can be used ŽSantos et al., 1995..
Using whole-blood and two-color flow cytometry,
several workers have demonstrated that phago-
cytosis, reactive oxygen production, protein diges-
31
tion and bacterial killing within the phagocytic vac-
uole can be measured in the same population of cells
ŽHasui et al., 1989; Haynes et al., 1990; Perticarari et
al., 1991; Saresella et al., 1997.. Protein digestion is
measured by fluorescence of rhodamine attached to
albumin at a high molar ratio and quenching of the
fluorescence when the protein is digested ŽHaynes 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-
cytosis after exposure to soluble activators ŽPMA
and PAF., making it possible to diagnose
neutrophil-related phagocytic disorders not related to
the production of reactive oxygen intermediates ŽAn-
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
between neutrophils and particles should be opti-
mized for maximum phagocytosis because the rate
and extent of phagocytosis differ depending on the
type of particle used. Similarly, cell to particle ratio
should be optimized with a 1:10 ratio an average
ratio for most standard particles.
8. Degranulation
Neutrophils ingest bacteria or other particles into
intracellular compartments called phagosomes and
the destruction of the infectious microorganism is
achieved by the fusion of granules with the phago-
somes. The extracellular release of granule content
occurs concomitantly, that may result in destroying
non-phagocyted bacteria but could also result in
tissue damage and amplification of the local inflam-
matory response. The membrane of granules serves
as a reservoir of receptors and membrane bound
proteins involved in cell adhesion ŽBainton et al.,
1997., signal transduction ŽRotrosen et al., 1988. and
activation of microcidal pathways ŽBorregaard et al.,
1983; Spitznagel, 1990.. The fusion of granules with
the cell membrane during degranulation changes the
surface expression of these membrane receptors and
this principle has been used to measure and quanti-
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
X
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,
respectively.
face marker to measure degranulation of primary
granules still needs to be established.

ucts ŽMcCord and Fridovich, 1969; Babior et al.,

9. Oxygen radical production
Central to antimicrobial activity of neutrophils is
the oxidative burst that generates reactive oxygen
species ŽROS. through the NADPH oxidase system.
The best demonstration of the essential role of this is
the consequences of a lack of ROS generation seen
in patients with chronic granulomatous disease
ŽCGD.. These patients have defects in their NADPH
oxidase pathway, are unable to generate superoxide
anions necessary for the formation of a number of
ROS ŽFig. 3. and have recurrent life threatening
bacterial infections from birth ŽGallin, 1992..
In assessing the oxidative burst in neutrophils, the
most widely used standard methods measure the
extracellular release of superoxide anion ŽOy 2
. or
hydrogen peroxide H 2 O 2 by their ability to reduce
Ž .
or oxidize substrates to colored or fluorescent prod-
1970; Boveris et al., 1977; Roots and Metcalf, 1977..
The addition of superoxide dismutase or catalase in
parallel samples provides specificity to the assays for
superoxide anion or H 2 O 2 , respectively. The major
disadvantages of these assay methods are that cells
must be separated from red blood cells and plasma
and that relatively large numbers of neutrophils are
needed Žapproximately 0.5 millionrexperimental
condition.. In these assays, the measured response is
of a population and individual response cannot be
assessed.
In 1983, a method was developed to measure
‘‘oxidant product formation’’ by flow cytometry us-
ing 2X ,7X-dichlorofluorescein ŽDCF. ŽBass et al.,
1983.. In this method, neutrophils are loaded with
the diacetate form of 2X ,7-dichlorofluorescein
ŽDCFH., which is transported across the plasma
membrane. In the cytoplasm, the acetate groups are
removed by esterases concentrating the polar non-
 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
fluorescent DCFH in the cytoplasm. During the ox-
idative burst, DCFH is converted to DCF through
oxidation mainly by H 2 O 2 . Dihydrorhodamine 123
is used in a similar way resulting in red fluorescence
ŽEmmendorffer et al., 1990; Rothe et al., 1991;
Vowells et al., 1995.. Strong non-physiologic stimuli
such as PMA and calcium ionophores cause a sus-
tained respiratory burst mirrored by high levels of
fluorescence in this assay. More physiologic stimuli
produce much smaller and variable responses.
The use of flow cytometry to measure the produc-
tion of H 2 O 2 offers an easy semi-quantitative tech-
nique that is applicable to a few thousand unpurified
cells. In contrast to the standard assay mentioned
previously, the flow cytometric assay can be per-
formed in mixed cell samples Žwhole blood. where
neutrophil responses can be determined using gating
techniques ŽRichardson et al., 1998.. The small num-
ber of unpurified cells necessary for the flow cytom-
etry assay allows studies on small sample volumes
such as neonatal blood or from other small animals.
In clinical laboratories, the availability and familiar-
ity with flow cytometry make these flow cytometry
assays very attractive in screening patients for CGD
ŽEmmendorffer et al., 1990; Roesler et al., 1990..
9.1. Benefits and limitations of flow cytometry
The limits of flow cytometry in measuring H 2 O 2
production need to be recognized. Quantitative mea-
surements are difficult. Variation in loading and
hydrolysis of the acetate from DCFH can alter the
relative fluorescence seen. Quantifying the number
of moles of fluorescein per cell is cumbersome and
most studies report only relative fluorescence. Intra-
cellular elements such as catalases or peroxidases
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..
The assumption in the flow cytometric assessment
of the respiratory burst is that changes in fluores-
cence on stimulation are indicative of the production
of H 2 O 2 in individual neutrophils. For most applica-
33
tions this assumption is valid. However, H 2 O 2 and
its precursor, superoxide anion, can cross intact cell
membranes and diffuse to adjacent cells. This was
demonstrated by mixing DCFH-loaded neutrophils
from normal individuals and from patients with CGD
Žincapable of a respiratory burst.. In this mixture, the
oxidant products from the cells with a respiratory
burst were capable of oxidizing DCFH in non-re-
sponding bystanders ŽBass et al., 1983.. This is of
little concern for most applications but may be a
confounding feature in experiments using mixed cell
populations. It should be remembered that eosinophils
are capable of an oxidative burst, although normally
they represent a small fraction of cells in the
‘‘granulocyte’’ window. Finally, it should be remem-
bered that neutrophil activation results in aggregation
of neutrophils with other neutrophils and other cells
especially platelets. These larger aggregates may not
be seen in the gates set for single neutrophil and may
exclude a population of activated neutrophils from
the analysis.
More recently, flow cytometric assays combining
phagocytes and oxidative burst have been developed
using labeled bacteria or immune complexes ŽHasui
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
that achieved with DCFH-loaded cells. But the re-
sponse to these particulate fluorochromes is more
complex. The oxidative burst and release of H 2 O 2
can occur on contact without uptake of the particle.
Phagocytosis concentrates these particles in phago-
somes allowing more efficient delivery of the H 2 O 2
to the stimulus and fluorochrome. Thus the intensity
of the signal from each neutrophil is dependent on
particulate 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 by
a diverse array of physiological and non-physio-
logical stimuli. It can be initiated through a geneti-
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
proteins, oxidative stress and hypoxia. Apoptosis is
an important process in normal neutrophil physiol-
ogy because of the extend of neutrophil turnover
Ž1–2.5 billion kgy1 dayy1 . and their short life span
Ž6–10 h. in the circulation ŽMaloney and Patt, 1968;
McAfee and Thakur, 1976.. The changes in aging
PMN which allow recognition by macrophages in
the liver and spleen to remove them from the circula-
tion or sites of extravasation are a field of active
research ŽSavill et al., 1989; Savill et al., 1992..
Studies from our laboratory ŽBicknell et al., 1994.
showed that apoptosis of neutrophils occurs mainly
in the spleen and it is possible that the senescent
cells are recognized and removed in these organs in
a manner similar to that occurring at inflammatory
sites. Aging neutrophils in vitro develop morphologi-
cal and biochemical features typical of apoptosis
without the addition of external stimuli ŽWhyte et al.,
1993a,b. and we have shown similar events occur in
circulating neutrophils as they age in vivo ŽMatsuba
et al., 1997.. This process is characterized by DNA
cleavage by calcium-dependent endogenous endonu-
cleases capable of cleaving chromatin at internucleo-
somal sites ŽArends et al., 1990..
There is a 10–100 fold increase in neutrophil
turnover during acute infection ŽMcAfee and Thakur,
1976. and these neutrophils meet their fate in the
tissue, as there is little evidence that they return to
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
al., 1992.. The suppressed apoptosis in neutrophils
during acute infectious or inflammatory conditions
has been postulated to be important in the pathogen-
esis of neutrophil-mediated diseases such as is-
chemia reperfusion injury, acute respiratory distress
syndrome, sepsis and multiorgan failure ŽJones 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 of
flow cytometry to assess apoptosis in large numbers
of cells and simultaneously evaluate subpopulations
within a larger cell population are some of the
benefits of using flow cytometry over more conven-
tional methods such as morphology and gel elec-
trophoresis. Because all the methods described below
have shortcomings, it is imperative that at least one
additional independent method is used to confirm
results.
11.1. Size and granularity
Flow cytometry allows the detection of the light-
scattering properties of neutrophils and quantitate the
cell size Žforward scatter. and granularity Žside scat-
ter.. Apoptotic neutrophils loose water Žreduced for-
ward scatter., become less granular Žreduced side
scatter. and these changes in their light scattering
properties can be use to identify apoptotic cells
ŽGorman et al., 1997.. These changes are non-specific
and can not distinguish between apoptotic and
necrotic cells. Both neutrophil size and granularity
can also change with cell activation. Therefore, these
parameters of neutrophil apoptosis are not useful as
the only discriminator for apoptosis because of the
potential heterogeneity of most neutrophil popula-
tions.
11.2. Fluorescent dyes
The DNA-binding dye propidium iodide can be
use to reveal the amount of DNA in each cell and
this assay make use of the fact that apoptotic cells
contain subdiploid amounts of DNA ŽMesner and
Kaufman, 1997.. This technique has disadvantages
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
that are apoptotic, conversely, using gating tech-
niques to exclude these bodies may underestimate
the number of apoptotic cells.
Other vital dyes such as acridine orange Žgreen
fluorescence. and ethidium bromide Žorange fluores-
 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
cence. rely on the differential uptake of these dyes
by normal healthy, apoptotic and necrotic cells. Us-
ing ethidium bromide cells can be graded as negative
Žhealthy., weakly Žapoptotic. or strongly Žnecrotic.
positive. Similar results are obtained with com-
pounds such as Hoechst 33342 or 7-aminoacti-
nomycin D ŽGong 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 be
combined with other fluorochromes to identify apop-
tosis in subset of cells ŽO’Brien and Bolton, 1995..
One potential disadvantage of this approach is the
lack of good studies to verify the specificity of these
methods. Furthermore, cell membrane integrity is
maintained in the early stage of apoptosis but is lost
with advanced apoptosis and these techniques may
underestimate the actual number of apoptotic cells if
all strongly stained cells are exclude from the analy-
sis.
11.3. Surface markers of apoptosis
Annexin V, a polypeptide that binds strongly and
specifically to cell surface phosphatidylserine is a
sensitive measurement of early apoptosis ŽKoopman
et al., 1994; Martin et al., 1995.. Phosphatidylserine
is present exclusively in the inner layer of the cell
membrane of healthy cells and becomes accessible
on the surface of cells undergoing apoptosis. This is
the basis for detecting apoptotic neutrophils using
fluorescent labeled Annexin V. Cells are labeled
following fixation with a non-permeabilizing fixative
Že.g., formaldehyde. or without fixation. Once the
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.
11.4. Terminal deoxyribonucleotidyl transferase-
mediated dUTP nick-end labeling (TUNEL)
The TUNEL technique is a sensitive method to
detect apoptosis in neutrophils using flow cytometry.
The principle of these techniques is that in the
presence of a divalent cation, terminal deoxyribonu-
cleotidyl transferase ŽTdT. will add nucleosides to
35
free 3X-ends produced by endonuclease action during
apoptosis ŽGavrieli et al., 1992.. Briefly, neutrophils
are fixed, permeabilized and incubated with biotinyl-
ated deoxyuridine triphosphate in the presence of
TdT and Co 2q. After washing, cells are incubated
with fluorescent labeled streptavidin that binds to the
biotin. One of the benefits of this technique is that it
can be combined with fluorescent microscopic analy-
sis and compared with morphological changes of
DNA fragmentation in individual cells. The disad-
vantage of this method is that it could detect nicks in
DNA due to the presence of other enzymes such as
topoisomase that are not necessarily associated with
apoptosis ŽFroelich-Ammon et al., 1995.. In addi-
tion, necrotic cells can stain with TUNEL because
random DNA degradation occurring during necrosis
also generates ends with 3X OH groups ŽGrasl-Kraupp
et al., 1995.. Therefore, alternative techniques needs
to be combined with TUNEL to confirm that cells
are undergoing apoptosis rather than necrosis.
The measurement of changes in mitochondrial
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.
and bax the proapoptotic member of the bcl-2 fam-
ily of apoptosis regulators ŽMiyashita and Reed,
1995. are all indicative of apoptosis and can be
measured using flow cytometry. Flow cytometry al-
lows the rapid detection of apoptotic neutrophils by
permitting the measurement of a variety of parame-
ters that alter during the onset and progression of
apoptosis. It has significantly aided our understand-
ing of neutrophil apoptosis and its potential role in
the pathogenesis of neutrophil-induced inflammatory
conditions.
12. Priming of neutrophils
Priming of neutrophils is one of the pivotal pro-
cesses that regulate their functional responses ŽCon-
dliffe et al., 1998a.. Priming refers to the process
where the response of neutrophils to an activating
stimulus is enhanced by prior exposure to small
‘‘non-activating’’ concentrations of this or another
stimulus ŽGuthrie et al., 1984.. Guthrie et al. de-
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
priming agent. In the strictest sense the priming
response is not associated with a functional response
but a variety of biochemical events ŽWalker and
Ward, 1992.. Enhancement of agonist-induced de-
granulation ŽFittschen et al., 1988. and generation of
lipid mediators such as LTB4 and PAF ŽDoerfler et
al., 1989. or other cytokines ŽZallen et al., 1999. has
been described. A variety of substances, both physio-
logical and pharmacological, have been shown to
prime neutrophils ŽWalker 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 as
endotoxin and cytokines, notably TNF-a, IL-6 and
IL-8, are detectable in the blood and are associated
with a poor outcome in subjects with septic shock,
ARDS and multiorgan failure ŽParsons et al., 1989;
Pinsky et al., 1993.. Smedley et al. Ž1986. have
shown that neutrophils activated with fMLP, C5a
and LPS cause minimal damage to endothelial cells
in vitro but if cells were first primed with LPS and
then stimulated with fMLP or C5a, extensive damage
to endothelial cells ensued.
Work from our laboratory has shown that the
process of sequestration of neutrophils may also
result in priming ŽKitagawa et al., 1997.. Because
neutrophils are larger than the majority of pulmonary
capillary segments they have to pass through, these
cells have to deform to cross the pulmonary vascular
bed ŽDoerschuk et al., 1993; Wiggs et al., 1994..
Using flow cytometry, we have shown that passive
deformation of neutrophils by filtration through 5-mm
pores Žsimilar to deformation in the pulmonary capil-
lary bed. causes enhanced CD18rCD11b expression
following maximal stimulation with fMLP. The
priming effect of this deformation was comparable to
using fMLP as a priming agent. Neutrophils exposed
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
the 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 the
site 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 stimulus
ŽWaddell et al., 1994; Liles et al., 1995.. The process
of extravasation of neutrophils itself results in in-
crease stimulus-induced oxidant release and other
phenotypic changes such as changes in their expres-
sion of surface adhesion molecules. This needs to be
taken into account when extravasated neutrophils
from areas such as the alveolar space or peritoneal
cavity are harvested for analysis.
Flow cytometry has been used extensively to
identify and quantify neutrophil priming. Activation
of the NADPH oxidase enzyme system with the
production of superoxide is the hallmark of neu-
trophil priming. This is the most primable neutrophil
function with the potential to increase superoxide
production up to 20-fold. However, other changes in
neutrophils following priming are shape changes and
stiffening ŽHaslett et al., 1985., changes in the ex-
pression of adhesion molecules with an increase in
CD11b and a decrease in L-selectin ŽCondliffe et al.,
1996., degranulation of primary and secondary gran-
ules ŽDoerfler et al., 1994. and a delay in apoptosis
ŽLee et al., 1989.. Lastly, sample preparation for
flow cytometry and trace amounts of endotoxin in
solutions used for cell isolation could prime neu-
trophils ŽHaslett et al., 1985.. Because many of these
neutrophil functions can be measured in whole blood,
flow cytometry is the method of choice to evaluate
neutrophil priming. It is vital to realize that neu-
trophils are not terminally differentiated cells and
that 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 with
extrapolation to in vivo events. However, it is re-
markably easy to disturb normal neutrophil function
 S.F. Õan Eeden et al.r Journal of Immunological Methods 232 (1999) 23–43
by removing them from the circulation. The use of
flow cytometry has advanced our ability to measure
and quantify neutrophil functions. Using whole-blood
methods with minimal manipulation of cells gives a
more accurate measure of neutrophil behavior in
vivo. The ability of flow cytometry to measure the
function of a single cell or large numbers of cells
and the potential to measure multiple functions si-
multaneously by using multiple fluorochromes has
improved our ability to study the function of a
complex cell in health and disease. The application
of flow cytometry to measure neutrophil function is
progressively expanding in basic and clinical sci-
ences.
Acknowledgements
This work was supported by the BC Lung Associ-
ation and the Medical Research Council of Canada
grant a4219.
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