Relationship Between Oxidative Burst Activity andCD11b Expression in Neutrophils and Monocytes From

Healthy Individuals: Effects of Race and GenderMuhammad Siddiqi,1 Zenaida C. Garcia,2 Dana S. Stein,2 Thomas N. Denny,2 and

Zoltan Spolarics1*1Department of Anatomy Cell Biology and Injury Sciences, New Jersey Medical School, Newark, New Jersey

2Department of Pediatrics, New Jersey Medical School, Newark, New Jersey

Oxidative burst activity and the expression of adhesion molecules have been used as indicators ofleukocyte activation status. The aim of the study was to delineate the relationship of oxidative burst activityand the expression of adhesion molecules in neutrophils and monocytes from a pool of healthy volunteers(n 5 96). We also tested the potential role of gender and a racial background in the individual responsedifferences. Basal and phorbol myristate acetate (PMA)-stimulated oxidative burst and CD11b expressionwere determined using dihydrorhodamine 123 and phycoerythrin (PE)-conjugated anti-CD11b monoclonalantibodies. PMA markedly increased CD11b expression and cellular oxidant content in neutrophils andmonocytes in all samples. However, the responses showed considerable variability among individuals. Apositive correlation was observed between the responsiveness of neutrophils and monocytes in their basalor PMA-stimulated CD11b expressions and PMA-stimulated oxidative burst activities. In contrast, nocorrelation was found between the level of adhesion molecule expression and cellular oxidant content inmonocytes or neutrophils either under basal or under PMA-stimulated conditions. The reactivity of oxidativeburst (i.e., PMA-stimulated over basal) was significantly lower in neutrophils from African American malescompared with cells from African American females, white females, or white males. In contrast, reactivityof monocytes was significantly elevated in white males compared with all other groups. These findingsindicate that leukocytes with a relatively high degree of adhesion molecule expression may display anaverage or decreased oxidative burst activity, and vice versa. Our findings also indicate that ethnicbackground may influence the oxidative burst activity in neutrophils and monocytes. This needs consider-ation in clinical studies utilizing healthy volunteers with mixed gender and ethnic backgrounds. Cytometry(Comm. Clin. Cytometry) 46:243–246, 2000. © 2000 Wiley-Liss, Inc.

Key terms: neutrophils; monocytes; cell activation; flow cytometry; human

Determination of oxidative burst and the expression ofadhesion molecules by flow cytometry technique havebeen widely used in the assessment of leukocyte func-tional status (1,2). It has been accepted that oxidativeburst activity and the expression of cell adhesion mole-cules parallel the activation status of neutrophils and mac-rophages. Elevated oxidative burst activity or CD11b ex-pression indicates cell activation whereas decreasedresponses reflect downregulated cellular functions. Thevalue of these tests has been well established in a varietyof disease processes. Marked differences have been ob-served in leukocyte CD11b expression (3–6) and oxida-tive burst response (7–9) between patients and healthyvolunteers. However, empirical observations have sug-gested that considerable variability exists among individ-uals in the degree of oxidative burst and CD11b expres-sion in leukocytes. It is not known whether decreased (or

augmented) responsiveness of oxidative burst is also man-ifested in a parallel decrease (or augmentation) in celladhesion molecule expression. Furthermore, whereas am-ple evidence indicates that gender and age have majorinfluences on leukocyte responses and on the host re-sponse to injury and infection (10–12), the potential in-fluence of ethnic background has not been investigated.Therefore, the aims of the study were to delineate therelationship between oxidative burst activity and expres-sion of the CD11b adhesion molecule in neutrophils andmonocytes from a representative pool of young healthy

*Correspondence to: Zoltan Spolarics, M.D., Ph.D., Department ofAnatomy, Cell Biology and Injury Sciences, UMDNJ- New Jersey MedicalSchool, 185 South Orange Avenue, Newark, NJ 07103.

E-mail: spolaric@umdnj.eduReceived 2 November 2000; Accepted 27 March 2001

Cytometry (Communications in Clinical Cytometry) 46:243–246 (2000)

© 2000 Wiley-Liss, Inc.

volunteers. Furthermore, we also aimed to delineate thepotential influence of ethnic background on individualresponse differences. Basal and phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst and CD11b ex-pression were determined in the same blood sample fromeach individual. PMA was selected as a stimulatory agentin order to minimize the effect of potential differences onthe surface receptor status among individuals. It is as-sumed that the PMA-induced response reflects the overallactivity of oxidative burst and cell adhesion expression inboth neutrophils and monocytes as it acts through thedirect activation of protein kinase C.

MATERIALS AND METHODSFlow cytometry analyses were carried out in a central-

ized laboratory using a FACScan flow cytometer equippedwith a 488-nm laser, 530/30 and 585/42-nm band passfilters, and a 650-nm long pass filter (Becton Dickinson[BD] Biosciences, San Jose, CA). Instrument calibrationwas performed daily employing Calibrite Beads (BD Bio-sciences). For each of the assays, target channel valueswere initially set using Sphero beads (Spherotech, Lib-ertville, IL). The channel value used was the mean of thethird brightest peak on the logarithmic scale. These targetchannel values were adhered to throughout the study.

This study was approved by the Institutional ReviewBoard of the New Jersey Medical School (Newark, NJ).Informed consent was obtained from all individuals whoparticipated in the study. Healthy volunteers werescreened for glucose 6 phosphate dehydrogenase defi-ciency (G6PD) as described earlier (13). G6PD deficiencyis a common genetic polymorphism, with a particularlyhigh frequency in the African American and Hispanicpopulations (14). Volunteers with G6PD deficiency wereexcluded from the study due to the potential effects of thedeficiency on leukocyte responses.

Initiation of cell incubations and acquisitions were car-ried out in a time-scattered fashion and incubation timeswere strictly adhered to throughout the study. Cell-asso-ciated oxidant content was measured by using dihydror-hodamine 123 (DHR; Molecular Probes, Eugene, OR) aspreviously described (1). Blood was collected in tubescontaining EDTA as anticoagulant. Whole blood wastreated with prewarmed hypotonic ammonium chloridesolution. Cells were sedimented and washed in Hanksbalanced salt solution (HBSS) containing 5 mM glucose.Isolated white blood cells were incubated in the presenceof 1 mM DHR for 10 min, followed by incubation with10–90 ng/ml PMA or vehicle. Cell-associated fluorescencewas measured 20 min later using the 530/30-nm filter.

Determination of CD11b expression on neutrophils andmonocytes was performed as follows (2): 0.1 ml wholeblood was incubated at 37°C for 15 min in the presence orabsence of 90 ng/ml PMA. Cells were incubated for 15min at room temperature in the dark in the presence ofphycoerythrin (PE)-conjugated monoclonal antibodiesagainst CD11b. PE-conjugated nonspecific IgG served as acontrol in parallel incubations. After lysing the red bloodcells, white blood cells were suspended in 2 ml HBSS and

washed once. Cells were fixed with 0.3 ml 1% formalde-hyde and cell-associated fluorescence was measured usingthe 582/42-nm filter.

The mean of the relative fluorescence intensity (RFI)from logarithmically amplified data expressed as linearvalue was recorded and compared.

Statistical Analysis

Statistical calculations were performed using JMP soft-ware (SAS Institute, Cary, NC). Differences in multiplecomparisons were tested by analysis of variance (ANOVA)followed by the Tukey-Kramer test. Statistically significantdifferences were concluded at P , 0.05.

RESULTS AND DISCUSSIONFigure 1 depicts the mean response of CD11b expres-

sion in neutrophils and monocytes from 96 individuals.PMA stimulation increased CD11b expression by an aver-age of 20 and 8.5-fold in neutrophils and monocytes,respectively (top panels). PMA administration resulted in adose-dependent increase in cellular oxidant content inthese cells (lower panels). The employed maximally stim-ulating PMA dose (90 ng/ml) increased the basal oxidantcontent by approximately 120-fold in neutrophils and7-fold in monocytes.

Figure 2 indicates that basal or PMA-stimulated levels ofoxidants and CD11b expression show considerable indi-vidual variability in neutrophils as well as in monocytes.Also, there was no correlation between the degree of

FIG. 1. CD11b expression and oxidative burst in healthy volunteers.Cell-associated fluorescence was determined in neutrophils and mono-cytes using PE-conjugated CD11b antibodies or isotypic IgG (top pan-els). Cell-associated oxidants (lower panels) were determined by usingDHR as described in the Materials and Methods section. Mean RFI 6SEM, n 5 96.

244 SIDDIQI ET AL.

CD11b expression and cellular oxidant content underbasal (Fig. 2A,B) or PMA-stimulated (Fig. 2C,D) conditions.These observations indicate that, in a particular individual,a relatively high responsiveness of cell adhesion moleculeexpression may be accompanied by a relatively normal oreven decreased responsiveness of oxidative burst activity,and vice versa.

Figure 2E–H depict the relationship between the re-sponsiveness of neutrophils and monocytes within anindividual in the investigated group. A positive correlationwas observed between neutrophil and monocyte re-sponses testing basal (Fig. 2E) or PMA-stimulated (Fig. 2F)CD11b expression or after PMA-stimulated oxidative burstactivity (Fig. 2H). These data indicate that an augmentedresponsiveness of monocyte oxidative metabolism or celladhesion property is accompanied by a similarly elevatedresponsiveness of neutrophils in a particular individual.

The same set of data was also analyzed to test thepotential role of gender together with a racial background

in the observed individual differences in neutrophil andmonocyte responses. The analyzed pool of individualscomprised 19 African American females (AAF), 19 whitefemales (WF), 32 African American males (AAM), and 26white males (WM). Further subdivision of ethnic groupswas not feasible due to the low number of observations.

PMA-stimulated oxidative burst was significantly lowerin neutrophils (Fig. 3A) and monocytes (Fig. 3B) fromAAM compared with AAF, WF, and WM. The reactivity ofoxidative burst (i.e., PMA-stimulated over basal) in neutro-

FIG. 2. Correlation between oxidative metabolism and CD11b expres-sion in monocytes and neutrophils. A–D: Relationships between oxidantcontent and CD11b expression in neutrophils (A,C) and monocytes (B,D)under basal (A,B) or PMA-stimulated (C,D) conditions. E–H: Relationshipbetween monocyte and neutrophil responses testing CD11b expression(E,F) or cellular oxidant content (G,H) under basal (E,G) or PMA-stimu-lated (F,H) conditions (N 5 96). Statistically significant correlations areshown when present.

FIG. 3. Effect of ethnic background and gender on the oxidative burstresponse in neutrophils and monocytes. Following PMA stimulation,oxidant levels in neutrophils (top panels) or monocytes (bottom panels)were compared in cells from AAM, AAF, WM, and WF. Insets depictvalues calculated as PMA stimulated over basal levels of oxidants (i.e.,reactivity or fold increase). Stars indicate statistically significant differ-ences compared with all the other groups. “&” depicts statisticallysignificant difference compared with WF.

245OXIDATIVE BURST AND CD11B EXPRESSION

phils from AAM individuals was also markedly decreasedcompared with cells from all the other groups (inset, Fig.3A). In contrast, the reactivity of monocytes was signifi-cantly greater in WM compared with cells from the othergroups (inset, Fig. 3B). No statistically significant differ-ences were observed in basal or PMA-stimulated CD11bexpression among the investigated gender or racialgroups. The employed pool of healthy volunteers wasrelatively young (mean age 38.4 6 0.8; range 22–58) andage showed no statistically significant correlation withoxidative burst activity or cell adhesion molecule expres-sion.

Interaction between leukocytes and endothelial cellsduring neutrophil or monocyte adhesion and transmigra-tion triggers oxidative burst. Oxidative burst has beenshown to play an important role in phagocyte-mediatedendothelial injury and subsequent tissue damage in a va-riety of disease processes (15–18). Elevated expression ofcell adhesion molecules results in an augmented recruit-ment of leukocytes to injury sites or target organs. Thedegree of phagocyte-mediated tissue damage is dependenton a balance between phagocyte activation (e.g., degreeof cell recruitment, release of reactive oxidant or nitrogenspecies or proteinases) and the activity of compensatorydefensive mechanisms in the target cells and organs (e.g.,antioxidant status, presence of anti-proteinases; 15,19). Adivergence between oxidative burst activity and the re-sponsiveness of cell adhesion expression in a particularindividual may represent a “built in” protective mecha-nism against phagocyte-mediated tissue injury underpathological conditions. Namely, an elevated responsive-ness of cell adhesion property will not necessarily beprone to phagocyte-mediated tissue injury unless this con-dition is accompanied by an increased responsiveness ofoxidative burst activity, and vice versa.

Our observations also indicate that, in addition to theknown effects of gender and age (10–12,20,21), a racialbackground may also have an important influence in de-termining the oxidative burst activity in neutrophils andmonocytes. It remains to be elucidated if the observedindividual characteristics or the gender and race-depen-dent differences in oxidative burst activity have an impacton the inflammatory response under pathological condi-tions. Because the representation of minority patients or aparticular gender may be disproportionally high in certainhospitals (trauma centers, inner city hospitals), our studysuggests the importance of race and gender-matched se-lection of healthy volunteer controls for clinical investiga-tions utilizing these flow cytometry assays.

ACKNOWLEDGMENTSThis work was supported by NIH grants, NIGMS

GM55005 (Z.S.) and 1 S10 RR 14753 (T.M.D.)

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