Impairment of neutrophil emigration in CD18-null mice
Barbara
Walzog1,
Karin
Scharffetter-Kochanek2, and
Peter
Gaehtgens1
1 Department of Physiology,
Freie Universität Berlin, D-14195 Berlin; and
2 Department of Dermatology,
Universität zu Köln, D-50931 Cologne, Germany
 |
ABSTRACT |
This study was undertaken to
investigate the requirement of
2-integrins (CD11/CD18) for
extravasation of neutrophils in mice. After intraperitoneal
thioglycollate injection, an in vivo model of inflammation, leukocyte
extravasation into the peritoneal cavity was studied in CD18-deficient
and wild-type control mice. Before the induction of peritonitis, total
and differential leukocyte counts in the circulation were analyzed and
found to be 10-fold elevated in CD18-deficient animals compared with
wild-type animals. This was largely due to neutrophilia, with a 30-fold
increase in neutrophil counts. In CD18-deficient animals, extravasated white blood cells in the peritoneal cavity were diminished by 46%. The neutrophil number in the peritoneal fluid was
severely reduced to 13% compared with control animals. In contrast,
the number of emigrated monocytes was enhanced and lymphocyte
emigration was not altered in the absence of CD18. The emigration
efficiency of the neutrophils, calculated as ratio of the cell number
in the lavage fluid and the circulating blood, was reduced to 0.4% in
CD18-deficient mice compared with wild-type animals. Thus efficient neutrophil emigration into the peritoneal cavity strongly required CD11/CD18.
inflammation; cell adhesion; CD18 antigen; host defense
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INTRODUCTION |
THE
2-integrin family
(CD11/CD18) of leukocyte adhesion molecules is known to play an
important role in the recruitment of leukocytes to sites of
inflammation (2). These heterodimeric molecules consist of a common
-subunit designated as CD18, which is noncovalently associated with
the
-subunits CD11a, CD11b, CD11c, or CD11d. The
2-integrins, classified by the
-subunit associated with it, form distinctive functional complexes
termed lymphocyte functions-associated antigen-1 (LFA-1) (CD11a/CD18), Mac-1 (CD11b/CD18), gp150/95 (CD11c/CD18), or CD11d/CD18.
LFA-1 and Mac-1 support the firm adhesion of leukocytes by binding to
their counterreceptors on the endothelium, the intercellular cellular
adhesion molecules 1 and 2 (5, 6, 8). This binding step is thought to
constitute a prerequisite for leukocyte emigration. Patients suffering from leukocyte adhesion deficiency type I, an
inherited defect of the CD18 gene, show severe impairment of neutrophil
recruitment to sites of infection (3). The resulting clinical
complications correspond in severity to the degree of CD18 deficiency
(1). The lack of neutrophil recruitment, i.e., the absence of an
inflammatory response despite the presence of infectious agents, was
observed in many tissues including gut and skin, suggesting that
neutrophil emigration requires CD11/CD18 in humans (9). The only organ
in which substantial neutrophil extravasation was observed in the
absence of CD18 was the lungs (9). Thus CD18-independent mechanisms
seem to allow neutrophil recruitment into bronchoalveolar space,
whereas in most microvascular beds neutrophil recruitment and host
defense mechanisms depend on CD18. In contrast, emigration of monocytes
and lymphocytes was observed in CD18-deficient patients, suggesting
that these cells can also efficiently emigrate by mechanisms
independent of CD18 (9).
Studies in a peritonitis model with Mac-1-deficient mice have shown
that neutrophil emigration is not impaired in these mice compared with
wild-type animals, suggesting that LFA-1 is sufficient to allow
emigration (10). Accordingly, blockade of LFA-1 function by monoclonal
antibodies significantly reduced neutrophil emigration in wild-type
mice (10) and LFA-1-deficient mice revealed a diminished emigration of
neutrophils in thioglycollate-induced peritonitis (14). However,
neutrophil emigration in the peritoneum was reported not to be
inhibited in CD18-deficient mice (11), a finding that is contradictory
to the paradigm of the CD18 requirement for neutrophil extravasation.
Because neutrophil emigration in humans depends on CD11/CD18, the value
of studying murine neutrophil responsiveness as an in vivo model for
human host defense would have to be reconsidered, if the underlying
molecular mechanisms differ in both species. Therefore, we
reinvestigated the requirement of
2-integrins for the recruitment
of neutrophils to sites of inflammation in mice. Leukocyte
extravasation was studied in a peritonitis model using CD18-deficient
as well as wild-type animals. To unequivocally identify the nature of
emigrated cells, neutrophils were stained for Gr-1, a marker of mature neutrophils.
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METHODS |
Induction of peritonitis.
In all experiments, CD18-null mice (13) or wild-type control animals of
the same genetic background were used (mixed 129/Sv and C57BL/6J). Mice
were injected intraperitoneally with 2 ml of 3% thioglycollate. After
4 h, animals were killed by CO2
inhalation and injected intraperitoneally with 5 ml of PBS. All
peritoneal fluid was collected, and total leukocyte numbers were
analyzed using a Coulter Electronics counter. Differential leukocyte
counts were determined under the microscope using smears of
Hematocolor-stained cells as well as by flow cytometry (see below).
Peritoneal lavage was carefully confirmed to be negative for
erythrocytes by microscopy to exclude the possibility that leukocyte
accumulation in the peritoneal cavity was due to microbleeding, which
may occur during animal preparation. Animal experiments were subject to
institutional approval.
Flow cytometry.
Peripheral blood was collected by resection of the tip of the tail, and
aliquots of heparinized whole blood (20 µl) were diluted 1:4 with
PBS. Samples of blood and lavage fluid were stained using a
phycoerythrin-labeled rat anti-mouse CD18 antibody (clone C71/16) as
well as a FITC-labeled rat anti-mouse Gr-1 antibody (clone RB6-8C5) from PharMingen (San Diego, CA). After antibody
incubation for 1 h at 4°C in the dark, cells were washed
twice. Blood samples were treated with a fluorescence-activated cell
sorter (FACS) lysing solution according to supplier's instructions
(Becton Dickinson). In each sample,
104 cells were counted (FACScan,
Becton Dickinson) and gated off-line for granulocytes, monocytes, and
lymphocytes, using CellQuest software. Differential leukocyte numbers
were calculated from absolute leukocyte numbers and values obtained by
flow cytometry. In all experiments, >90% of cells gated for
granulocytes were neutrophils as determined by staining for Gr-1, a
marker of mature neutrophils. Statistical significance was determined
using Student's t-test where
applicable; P < 0.05 was considered
statistically significant.
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RESULTS |
Neutrophilia in CD18-deficient mice.
Leukocyte extravasation into the peritoneal cavity was studied in an in
vivo model of inflammation using wild-type
(n = 5) and CD18-deficient
(n = 5) mice. Before the induction of
peritonitis, total and differential blood leukocyte counts were
analyzed. Total leukocyte counts were ~10-fold elevated in
CD18-deficient mice (92 × 103/µl) compared with wild-type
controls (8.9 × 103/µl).
This was largely due to granulocytosis as shown in Fig. 1. The CD18-deficient mice revealed an
~30-fold increase of granulocyte counts in the circulation compared
with wild-type animals. Analysis of Hematocolor-stained leukocyte
populations under the microscope revealed that elevated granulocyte
counts were due to neutrophilia. Neutrophilia was the most pronounced
effect, but absolute monocyte counts (~11-fold) as well as lymphocyte
counts (~3-fold) were also elevated in CD18-deficient animals
compared with the wild-type control.

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Fig. 1.
Differential leukocyte counts of whole blood obtained from wild-type or
CD18-deficient (CD18 / ) mice before peritonitis. Data
represent means ± SD, n = 5. *** P < 0.05 vs. wild-type
control.
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Leukocytes of the peripheral blood were analyzed for expression of CD18
as well as Gr-1 on the cell surface, which represents a marker for
mature neutrophils (Fig. 2).
Analysis of Gr-1 expression demonstrated that both wild-type as well as
CD18-deficient neutrophils expressed high amounts of Gr-1 on their cell
surface, revealing the presence of mature neutrophils in the
circulation. For control, monocytes as well as lymphocytes were shown
to be negative for Gr-1 expression. As expected, CD18 expression was
high on wild-type leukocytes but completely absent on the surface of
cells derived from CD18-deficient animals.

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Fig. 2.
Expression of CD18 and Gr-1 on the surface of CD18-deficient and
wild-type leukocytes. Fluorescence histograms of leukocytes obtained
from whole blood before peritonitis are shown. Samples were stained
with FITC-labeled rat anti-mouse Gr-1 antibody or phycoerythrin-labeled
rat anti-mouse CD18 antibody or were left untreated for negative
control (dotted line). In each sample,
104 cells were counted and gated
off-line for granulocytes (G), monocytes (M), and lymphocytes (L).
Results are representative of 5 wild-type and 5 CD18-deficient
animals.
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Reduced emigration of CD18-deficient neutrophils.
Next, peritonitis was induced by injection of 3% thioglycollate. After
4 h, extravasated leukocytes were harvested from the peritoneal fluid,
counted, and subjected to flow cytometric analysis. As shown in Fig.
3, the majority of emigrated cells in
wild-type animals were granulocytes. In contrast, monocytes were the
dominant population of emigrated cells in CD18-deficient animals. To
confirm that neutrophil emigration was reduced in the CD18-deficient
animals, extravasated leukocytes were stained for Gr-1 as shown in Fig. 4. In the wild-type animal, ~65% of
emigrated cells showed high Gr-1 expression on the cell surface. In
contrast, only ~16% of extravasated cells were positive for high
Gr-1 expression in CD18-deficient animals, demonstrating a defect in
neutrophil extravasation.

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Fig. 3.
Leukocyte emigration in response to intraperitoneal thioglycollate
injection. Flow cytometric analysis is shown of leukocytes obtained
from peritoneal lavage 4 h after induction of peritonitis in a
wild-type and a CD18-deficient animal. In each sample,
104 cells were counted. Dot plots
of forward scatter (FSC) and sideward scatter (SSC) gated for
granulocytes, monocytes, and lymphocytes are shown. Results are
representative of 5 wild-type and 5 CD18-deficient animals.
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Fig. 4.
Expression of Gr-1 on the surface of CD18-deficient and wild-type
leukocytes emigrated in response to intraperitoneal thioglycollate
injection. Fluorescence histograms are shown of leukocytes obtained
from peritoneal lavage 4 h after induction of peritonitis in a
wild-type and a CD18-deficient animal. Samples were stained with
FITC-labeled rat anti-mouse Gr-1 antibody or were left untreated
(control). In each sample, 104
cells were counted and left ungated. Regions define cells with high
(M1) and low (M2) surface expression of Gr-1. Numbers at
top of each panel indicate cells with
high Gr-1 surface expression in percentage of total cell number.
Results are representative of 5 wild-type and 5 CD18-deficient
animals.
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The total number of emigrated leukocytes in the peritoneal fluid
revealed a marked reduction of emigration by ~46%, from 5.9 × 106 cells in wild-type animals to
3.2 × 106 cells in
CD18-deficient mice. The differential leukocyte counts are shown in
Fig. 5. In wild-type animals, the majority
(60.4%) of emigrated cells represented neutrophils (5.00 × 106), whereas monocyte and
lymphocyte emigration was poor. In contrast, only 0.66 × 106 neutrophils were
detected in the peritoneal fluid of CD18-deficient animals (20.6% of
emigrated leukocytes). On the basis of the total number of neutrophils
in the lavage, this corresponded to a reduction of neutrophil
emigration to ~13% compared with the wild-type animals. In contrast,
the number of emigrated monocytes was substantially enhanced (536%) in
CD18-deficient mice (1.93 × 106) compared with the wild-type
animals (0.36 × 106).
Lymphocyte emigration was similar in both CD18-deficient (113%) and
wild-type animals.

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Fig. 5.
Differential leukocyte numbers in the peritoneal fluid of wild-type and
CD18-deficient mice in response to intraperitoneal thioglycollate
injection. Data represent means ± SD,
n = 5. *** P < 0.05 vs. wild-type
control. ns, Not significant.
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As mentioned above, emigration in the CD18-deficient animals occurred
from a substantially larger leukocyte pool in the circulating blood
compared with wild-type animals. Therefore, a quantitative parameter of
emigration efficiency can be estimated by calculating the ratio of cell
number in the lavage fluid and the circulating blood. This revealed
that emigration efficiency of neutrophils in the CD18-deficient mice
was severely diminished to 0.4% of that in wild-type mice (100%) but
only mildly reduced for monocytes (45%) and lymphocytes (35%).
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DISCUSSION |
In this study, evidence was obtained that
2-integrins play a pivotal role
in the extravasation of neutrophils into the peritoneal fluid. With the
use of CD18-null mice, a severe reduction of neutrophil emigration was
observed in response to intraperitoneal thioglycollate injection
compared with control animals. This finding is in agreement with
previous data obtained from LFA-1-deficient mice in which the lack of
CD11a caused a marked decrease in neutrophil emigration under similar
experimental conditions (14). This is also consistent with recent data
showing a severe impairment of neutrophil emigration in response to
toxic dermatitis in CD18-null mice (13). Thus the
2-integrins seem to be required
for neutrophil emigration, which is probably a consequence of their
pivotal role for firm adhesion to the microvascular endothelium. This
is supported by the finding that the bacterial-derived tripeptide
N-formyl-Met-Leu-Phe was ineffective
in inducing leukocyte adhesion to endothelial cells in CD18-null mice,
as measured by intravital microscopy in the cremaster muscle (13). The
observed absence of substantial neutrophil recruitment is also
consistent with observations in patients suffering from leukocyte
adhesion deficiency type I. In these patients, the absence of
neutrophil emigration was observed despite the presence of
proinflammatory agents, e.g., in the gastrointestinal tract (3,
9).
The present data are strictly the opposite of the results presented by
Mizgerd et al. (11) who found elevated neutrophil emigration in
response to thioglycollate-induced peritonitis in the same strain of
CD18-null mice. We have no direct evidence to demonstrate the reason
for this difference; however, varying housing or breeding conditions
between laboratories may cause some variability in animal
responsiveness. Besides this, the only explanation for this obvious
discrepancy that we have is that microbleeding in the peritoneal
cavity, which can occur during animal preparation for peritoneal
lavage, may be responsible for the observed neutrophil accumulation in
the above-mentioned study. Because leukocyte numbers in the peripheral
blood are profoundly elevated in CD18-deficient animals, contamination
of lavage fluid with peripheral blood would have a rather large impact
on the estimated leukocyte number in the lavage fluid, particularly in these animals. About 10 µl of peripheral blood of CD18-deficient mice
contain the number of neutrophils that were found within 4 h after
thioglycollate injection in the peritoneum. A 30-fold blood volume
would be required for accumulation of the same neutrophil number in the
wild-type animal via bleeding. Thus bleeding can substantially affect
the measurements of neutrophil accumulation in the peritoneal cavity,
especially in the CD18-deficient animals. In the present study, care
was taken to rule out an erroneous interpretation of the cellular
content of lavage fluid. First, the peritoneal lavage was proved to be
negative for erythrocytes by microscopy to exclude an accumulation of
neutrophils via injured vascular beds. Moreover,
neutrophils in the peritoneal fluid were not only identified by
morphology but also by Gr-1 staining, a marker for mature neutrophils.
The small number of neutrophils observed in the lavage of the CD18-null
mice may indicate that CD11/CD18-independent mechanisms exist that
allow neutrophil emigration. Although it is not clear to what degree
neutrophil emigration depends on the circulating neutrophil count, the
calculated emigration efficiency in these animals, which recognizes the
profoundly elevated neutrophil counts in the circulation, reveals that
the dimension of a CD11/CD18-independent recruitment is extremely
small. A possible candidate for CD18-independent adhesion is the
4
1-integrin
very late antigen-4 (VLA-4), which has previously been
shown to mediate firm adhesion of neutrophils to tumor necrosis
factor-
-stimulated endothelium under flow conditions (12). This
molecule also mediates monocyte emigration by binding to vascular cell
adhesion molecules (4) and may be responsible for the
profound monocyte emigration observed in CD18-deficient animals. In
CD18 hypomorphic mice, an enhanced P-selectin expression was observed
in endothelial cells subsequent to tumor necrosis factor-
administration (7), suggesting that this molecule may have a
compensatory function in leukocyte recruitment in the
absence of CD18. Although we have no evidence for
altered adhesion molecule expression in the CD18-null mice, it has to
be taken into account that the constitutive absence of CD18 may
putatively cause compensatory upregulation of other adhesion molecules
that may promote CD18-independent emigration.
Altogether, this study demonstrates that neutrophil recruitment in mice
depends on CD11/CD18 because emigration as well as emigration
efficiency of neutrophils was severely compromised in CD18-deficient
animals. In contrast, substantial monocyte and lymphocyte emigration
was detectable in the absence of CD18, demonstrating that these cells
can efficiently emigrate by employment of CD11/CD18-independent mechanisms. These mechanisms seem to be substantially less important for neutrophils, since the residual neutrophil emigration in the absence of CD18 was extremely small. Thus neutrophils, monocytes, and
lymphocytes showed different requirements for extravasation in the
peritoneal cavity.
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ACKNOWLEDGEMENTS |
We thank M. Ehrlich for excellent technical assistance.
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FOOTNOTES |
This work was supported by Deutscheforschungsgemeinschaft
(Sonderforschungsbereich 366/C3).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. Walzog, Freie
Universität Berlin, Dept. of Physiology, Arnimallee 22, D-14195
Berlin, Germany (E-mail walzog{at}zedat.fu-berlin.de).
Received 17 October 1998; accepted in final form 6 January 1999.
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