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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was undertaken to investigate the requirement of beta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE beta 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 beta -subunit designated as CD18, which is noncovalently associated with the alpha -subunits CD11a, CD11b, CD11c, or CD11d. The beta 2-integrins, classified by the alpha -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 beta 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.


    METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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.

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.

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.

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.

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%).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, evidence was obtained that beta 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 beta 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 alpha 4beta 1-integrin very late antigen-4 (VLA-4), which has previously been shown to mediate firm adhesion of neutrophils to tumor necrosis factor-alpha -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-alpha 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.


    ACKNOWLEDGEMENTS

We thank M. Ehrlich for excellent technical assistance.


    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.


    REFERENCES
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 276(5):G1125-G1130
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society