Cytometry (Communications in Clinical Cytometry) 46:243246 (2000)

Relationship Between Oxidative Burst Activity and

CD11b Expression in Neutrophils and Monocytes From
Healthy Individuals: Effects of Race and Gender
Muhammad Siddiqi,1 Zenaida C. Garcia,2 Dana S. Stein,2 Thomas N. Denny,2 and
Zoltan Spolarics1*
1

Department of Anatomy Cell Biology and Injury Sciences, New Jersey Medical School, Newark, New Jersey
2
Department of Pediatrics, New Jersey Medical School, Newark, New Jersey

Oxidative burst activity and the expression of adhesion molecules have been used as indicators of
leukocyte activation status. The aim of the study was to delineate the relationship of oxidative burst activity
and the expression of adhesion molecules in neutrophils and monocytes from a pool of healthy volunteers
(n 96). We also tested the potential role of gender and a racial background in the individual response
differences. Basal and phorbol myristate acetate (PMA)-stimulated oxidative burst and CD11b expression
were determined using dihydrorhodamine 123 and phycoerythrin (PE)-conjugated anti-CD11b monoclonal
antibodies. PMA markedly increased CD11b expression and cellular oxidant content in neutrophils and
monocytes in all samples. However, the responses showed considerable variability among individuals. A
positive correlation was observed between the responsiveness of neutrophils and monocytes in their basal
or PMA-stimulated CD11b expressions and PMA-stimulated oxidative burst activities. In contrast, no
correlation was found between the level of adhesion molecule expression and cellular oxidant content in
monocytes or neutrophils either under basal or under PMA-stimulated conditions. The reactivity of oxidative
burst (i.e., PMA-stimulated over basal) was significantly lower in neutrophils from African American males
compared with cells from African American females, white females, or white males. In contrast, reactivity
of monocytes was significantly elevated in white males compared with all other groups. These findings
indicate that leukocytes with a relatively high degree of adhesion molecule expression may display an
average or decreased oxidative burst activity, and vice versa. Our findings also indicate that ethnic
background may influence the oxidative burst activity in neutrophils and monocytes. This needs consideration in clinical studies utilizing healthy volunteers with mixed gender and ethnic backgrounds. Cytometry
(Comm. Clin. Cytometry) 46:243246, 2000. 2000 Wiley-Liss, Inc.
Key terms: neutrophils; monocytes; cell activation; flow cytometry; human

Determination of oxidative burst and the expression of

adhesion molecules by flow cytometry technique have
been widely used in the assessment of leukocyte functional status (1,2). It has been accepted that oxidative
burst activity and the expression of cell adhesion molecules parallel the activation status of neutrophils and macrophages. Elevated oxidative burst activity or CD11b expression indicates cell activation whereas decreased
responses reflect downregulated cellular functions. The
value of these tests has been well established in a variety
of disease processes. Marked differences have been observed in leukocyte CD11b expression (3 6) and oxidative burst response (79) between patients and healthy
volunteers. However, empirical observations have suggested that considerable variability exists among individuals in the degree of oxidative burst and CD11b expression in leukocytes. It is not known whether decreased (or

2000 Wiley-Liss, Inc.

augmented) responsiveness of oxidative burst is also manifested in a parallel decrease (or augmentation) in cell
adhesion molecule expression. Furthermore, whereas ample evidence indicates that gender and age have major
influences on leukocyte responses and on the host response to injury and infection (10 12), the potential influence of ethnic background has not been investigated.
Therefore, the aims of the study were to delineate the
relationship between oxidative burst activity and expression of the CD11b adhesion molecule in neutrophils and
monocytes from a representative pool of young healthy
*Correspondence to: Zoltan Spolarics, M.D., Ph.D., Department of
Anatomy, Cell Biology and Injury Sciences, UMDNJ- New Jersey Medical
School, 185 South Orange Avenue, Newark, NJ 07103.
E-mail: spolaric@umdnj.edu
Received 2 November 2000; Accepted 27 March 2001

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SIDDIQI ET AL.

volunteers. Furthermore, we also aimed to delineate the

potential influence of ethnic background on individual
response differences. Basal and phorbol 12-myristate 13acetate (PMA)-stimulated oxidative burst and CD11b expression were determined in the same blood sample from
each individual. PMA was selected as a stimulatory agent
in order to minimize the effect of potential differences on
the surface receptor status among individuals. It is assumed that the PMA-induced response reflects the overall
activity of oxidative burst and cell adhesion expression in
both neutrophils and monocytes as it acts through the
direct activation of protein kinase C.
MATERIALS AND METHODS
Flow cytometry analyses were carried out in a centralized laboratory using a FACScan flow cytometer equipped
with a 488-nm laser, 530/30 and 585/42-nm band pass
filters, and a 650-nm long pass filter (Becton Dickinson
[BD] Biosciences, San Jose, CA). Instrument calibration
was performed daily employing Calibrite Beads (BD Biosciences). For each of the assays, target channel values
were initially set using Sphero beads (Spherotech, Libertville, IL). The channel value used was the mean of the
third brightest peak on the logarithmic scale. These target
channel values were adhered to throughout the study.
This study was approved by the Institutional Review
Board of the New Jersey Medical School (Newark, NJ).
Informed consent was obtained from all individuals who
participated in the study. Healthy volunteers were
screened for glucose 6 phosphate dehydrogenase deficiency (G6PD) as described earlier (13). G6PD deficiency
is a common genetic polymorphism, with a particularly
high frequency in the African American and Hispanic
populations (14). Volunteers with G6PD deficiency were
excluded from the study due to the potential effects of the
deficiency on leukocyte responses.
Initiation of cell incubations and acquisitions were carried out in a time-scattered fashion and incubation times
were strictly adhered to throughout the study. Cell-associated oxidant content was measured by using dihydrorhodamine 123 (DHR; Molecular Probes, Eugene, OR) as
previously described (1). Blood was collected in tubes
containing EDTA as anticoagulant. Whole blood was
treated with prewarmed hypotonic ammonium chloride
solution. Cells were sedimented and washed in Hanks
balanced salt solution (HBSS) containing 5 mM glucose.
Isolated white blood cells were incubated in the presence
of 1 M DHR for 10 min, followed by incubation with
10 90 ng/ml PMA or vehicle. Cell-associated fluorescence
was measured 20 min later using the 530/30-nm filter.
Determination of CD11b expression on neutrophils and
monocytes was performed as follows (2): 0.1 ml whole
blood was incubated at 37C for 15 min in the presence or
absence of 90 ng/ml PMA. Cells were incubated for 15
min at room temperature in the dark in the presence of
phycoerythrin (PE)-conjugated monoclonal antibodies
against CD11b. PE-conjugated nonspecific IgG served as a
control in parallel incubations. After lysing the red blood
cells, white blood cells were suspended in 2 ml HBSS and

FIG. 1. CD11b expression and oxidative burst in healthy volunteers.

Cell-associated fluorescence was determined in neutrophils and monocytes using PE-conjugated CD11b antibodies or isotypic IgG (top panels). Cell-associated oxidants (lower panels) were determined by using
DHR as described in the Materials and Methods section. Mean RFI
SEM, n 96.

washed once. Cells were fixed with 0.3 ml 1% formaldehyde and cell-associated fluorescence was measured using
the 582/42-nm filter.
The mean of the relative fluorescence intensity (RFI)
from logarithmically amplified data expressed as linear
value was recorded and compared.
Statistical Analysis
Statistical calculations were performed using JMP software (SAS Institute, Cary, NC). Differences in multiple
comparisons were tested by analysis of variance (ANOVA)
followed by the Tukey-Kramer test. Statistically significant
differences were concluded at P 0.05.
RESULTS AND DISCUSSION
Figure 1 depicts the mean response of CD11b expression in neutrophils and monocytes from 96 individuals.
PMA stimulation increased CD11b expression by an average of 20 and 8.5-fold in neutrophils and monocytes,
respectively (top panels). PMA administration resulted in a
dose-dependent increase in cellular oxidant content in
these cells (lower panels). The employed maximally stimulating PMA dose (90 ng/ml) increased the basal oxidant
content by approximately 120-fold in neutrophils and
7-fold in monocytes.
Figure 2 indicates that basal or PMA-stimulated levels of
oxidants and CD11b expression show considerable individual variability in neutrophils as well as in monocytes.
Also, there was no correlation between the degree of

OXIDATIVE BURST AND CD11B EXPRESSION

245

FIG. 2. Correlation between oxidative metabolism and CD11b expression in monocytes and neutrophils. AD: Relationships between oxidant
content and CD11b expression in neutrophils (A,C) and monocytes (B,D)
under basal (A,B) or PMA-stimulated (C,D) conditions. EH: Relationship
between monocyte and neutrophil responses testing CD11b expression
(E,F) or cellular oxidant content (G,H) under basal (E,G) or PMA-stimulated (F,H) conditions (N 96). Statistically significant correlations are
shown when present.

CD11b expression and cellular oxidant content under

basal (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 molecule
expression may be accompanied by a relatively normal or
even decreased responsiveness of oxidative burst activity,
and vice versa.
Figure 2EH depict the relationship between the responsiveness of neutrophils and monocytes within an
individual in the investigated group. A positive correlation
was observed between neutrophil and monocyte responses testing basal (Fig. 2E) or PMA-stimulated (Fig. 2F)
CD11b expression or after PMA-stimulated oxidative burst
activity (Fig. 2H). These data indicate that an augmented
responsiveness of monocyte oxidative metabolism or cell
adhesion property is accompanied by a similarly elevated
responsiveness of neutrophils in a particular individual.
The same set of data was also analyzed to test the
potential role of gender together with a racial background

FIG. 3. Effect of ethnic background and gender on the oxidative burst

response 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 depict
values calculated as PMA stimulated over basal levels of oxidants (i.e.,
reactivity or fold increase). Stars indicate statistically significant differences compared with all the other groups. & depicts statistically
significant difference compared with WF.

in the observed individual differences in neutrophil and

monocyte responses. The analyzed pool of individuals
comprised 19 African American females (AAF), 19 white
females (WF), 32 African American males (AAM), and 26
white males (WM). Further subdivision of ethnic groups
was not feasible due to the low number of observations.
PMA-stimulated oxidative burst was significantly lower
in neutrophils (Fig. 3A) and monocytes (Fig. 3B) from
AAM compared with AAF, WF, and WM. The reactivity of
oxidative burst (i.e., PMA-stimulated over basal) in neutro-

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SIDDIQI ET AL.

phils from AAM individuals was also markedly decreased

compared with cells from all the other groups (inset, Fig.
3A). In contrast, the reactivity of monocytes was significantly greater in WM compared with cells from the other
groups (inset, Fig. 3B). No statistically significant differences were observed in basal or PMA-stimulated CD11b
expression among the investigated gender or racial
groups. The employed pool of healthy volunteers was
relatively young (mean age 38.4 0.8; range 2258) and
age showed no statistically significant correlation with
oxidative burst activity or cell adhesion molecule expression.
Interaction between leukocytes and endothelial cells
during neutrophil or monocyte adhesion and transmigration triggers oxidative burst. Oxidative burst has been
shown to play an important role in phagocyte-mediated
endothelial injury and subsequent tissue damage in a variety of disease processes (1518). Elevated expression of
cell adhesion molecules results in an augmented recruitment of leukocytes to injury sites or target organs. The
degree of phagocyte-mediated tissue damage is dependent
on a balance between phagocyte activation (e.g., degree
of cell recruitment, release of reactive oxidant or nitrogen
species or proteinases) and the activity of compensatory
defensive mechanisms in the target cells and organs (e.g.,
antioxidant status, presence of anti-proteinases; 15,19). A
divergence between oxidative burst activity and the responsiveness of cell adhesion expression in a particular
individual may represent a built in protective mechanism against phagocyte-mediated tissue injury under
pathological conditions. Namely, an elevated responsiveness of cell adhesion property will not necessarily be
prone to phagocyte-mediated tissue injury unless this condition is accompanied by an increased responsiveness of
oxidative burst activity, and vice versa.
Our observations also indicate that, in addition to the
known effects of gender and age (10 12,20,21), a racial
background may also have an important influence in determining the oxidative burst activity in neutrophils and
monocytes. It remains to be elucidated if the observed
individual characteristics or the gender and race-dependent differences in oxidative burst activity have an impact
on the inflammatory response under pathological conditions. Because the representation of minority patients or a
particular gender may be disproportionally high in certain
hospitals (trauma centers, inner city hospitals), our study
suggests the importance of race and gender-matched selection of healthy volunteer controls for clinical investigations utilizing these flow cytometry assays.
ACKNOWLEDGMENTS
This work was supported by NIH grants, NIGMS
GM55005 (Z.S.) and 1 S10 RR 14753 (T.M.D.)

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