INTRODUCTION

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The inflammatory response to infection appears to substantially contribute to the development of multiple organ failure (MOF) (1). As a consequence various methods of influencing the host's response to infection have been proposed and tested in experimental models or, more limitedly, in clinical trials (2). One possible approach involves acting on polymorphonuclear leukocyte (PMN) functions, because these cells can induce tissue injury and organ failure in sepsis and septic shock (4). Recruitment of PMN to inflammatory sites depends on adhesion of the cells to the endothelium, a prerequisite for extravasation. These processes rely on upregulation of adhesion molecules on PMN and endothelial cells.

B2 integrins (CD11-CD18, Leu-cam) are a family of glycoprotein heterodimers composed of a common -chain (CD18), noncovalently linked with three distinct  chains (CD11a,b,c,) (5). These molecules are functionally and quantitatively upregulated after exposure to chemotactic factors (6). Cells that express CD11-CD18 adhere strongly to endothelial cells, particularly in postcapillary venules, which express the ligand intercellular adhesion molecules ICAM-1 and ICAM-2. CD11b/CD18 also serves as a receptor (CR3) for iC3b and for factor X and fibrinogen (7). The expression of ICAM-1, but not ICAM-2, in vitro increases on endothelial cells in response to inflammatory mediators such as interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-), or lipopolysaccharides (LPS) (10). Antibodies directed against 2-integrins inhibit PMN adhesion to endothelial cells, aggregation, and other adherence-dependent functions of PMN, such as chemotaxis and phagocytosis of large particles (8).

We investigated the effects of blocking the CD11-CD18 complex by a monoclonal antibody directed against the common CD18 -chain in an experimental model of MOF. In this model in rabbits, derived from that proposed by Goris and coworkers (13) in rats, intraperitoneal injection of a sterile suspension of zymosan and mineral oil causes functional and anatomic changes in distant organs, particularly the lungs. This model mimics the respiratory distress syndrome in humans which follows abdominal inflammatory processes such as peritonitis or pancreatitis. Previous experiments (13, 14) report a mortality of approximately 40% within 24 h of the zymosan injection, mainly as a consequence of lung distress syndrome and respiratory failure.

	METHODS

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Thirty-six male New Zealand White rabbits (mean ± SD body weight 3.05 ± 0.15 kg) were used. On the day of the experiment the rabbits received diazepam 5 mg intramuscularly and were anesthetized with ketamine chloride (8 mg/kg intravenously). The abdomen was scrubbed with povidone-iodine and a 3-cm incision of the skin and muscular layer was made. Animals were injected intraperitoneally with zymosan (Zymosan A; Sigma, St. Louis, MO), 0.6 g/kg, suspended (0.1 g/ ml) by high-frequency vibration in mineral oil (Sigma). The suspension was sterilized in a water bath at 100° C for 80 min and was prepared 5 d before the experiments in order to check its sterility by culture in a growth medium. The suspension was warmed to 38° C and vibrated at high frequency for 15 min before intraperitoneal inoculation with a 14-gauge needle under direct vision. The muscular layer and skin were sutured with silk. The surgical procedure lasted approximately 15 min.

Twenty animals were treated with a murine IgG2a anti-CD18 monoclonal antibody (IB4) 10 min before zymosan. This antibody was produced as previously described (15, 16). Sixteen control animals were pretreated with nonspecific purified murine IgG (Sigma). Both groups received 1 mg antibody/kg of body weight, dissolved in phosphate-buffered saline (PBS), pH 7.4, into the ear marginal vein.

Animals were observed for up to 10 d while kept in individual cages and fed standard rabbit chow (Charles River, Como, Italy) and water. Blood samples were drawn from the central ear artery before the surgical procedure and, in surviving animals, on Days 1, 3, 7, and 10. Red and white blood cell and thrombocyte counts, plasma levels of hemoglobin, urea, creatinine, alanine and aspartate aminotransferase (ALT, AST), blood pH, PO₂, PCO₂, and plasma carbon dioxide content (HCO₃) were all measured. On Day 10, surviving animals were killed by intravenous injection of 20 ml of potassium chloride. The abdomens were opened and observed. The lungs were dissected, observed for gross anatomic changes, and fixed intratracheally with 4% formaldehyde at a pressure of 30 cm H₂O; microscopic sections were prepared and stained with hematoxylin-eosin.

PMN infiltration of each animal lung was quantified by morphometry. Three hematoxylin-eosin-stained lung sections of each animal were photographed and reproduced at ×2,500. A Weibel's M 168 grid (17) was superimposed four times to the photographs excluding those areas with large pulmonary vessels. PMN volume density (Vv) was calculated as the ratio between the hits falling within a PMN (P) and those falling within lung tissue (Pt) according to the equation: Vv = P/Pt. For the stereological assessment (Vv) the limit of 672 points was chosen in order to obtain an intra- and interobserver coefficient of variation within 10% (18).

Lipid peroxides were measured by estimation from malonyldialdehyde (MDA). MDA was measured as thiobarbituric acid activity using the method of Okhawa and coworkers (19), as follows. A lung specimen of approximately 0.4 g was homogenized in PBS using a homogenizer. A sample of the homogenate was mixed with sodium dodecyl sulfate (8.1%), acetic acid (20%), and thiobarbituric acid (0.8%). The mixture was heated to 100° C for 60 min, then cooled, and n-butanol/pyridine (15:1) was added as a chromogen extractor. The solution was centrifuged and MDA was detected in the supernatant by absorbance at 532 nm. The MDA levels were expressed as pmol/ mg of protein content of the homogenate, the latter determined by Lowry's method (20). In the same homogenate -tocopherol (vitamin E) was estimated by the method described by Burton and coworkers (21). A sample of homogenate was mixed with sodium dodecyl sulfate (0.1 M), ethanol, and n-heptane. After centrifugation at 4,000 rpm for 10 min, -tocopherol was measured in the supernatant using high- pressure liquid chromatography (Perkin Elmer, Norwalk, CT) with fluorescence at 325 nm, and excitation at 295 nm. Vitamin E was expressed as ng/mg of protein.

Statistical Analysis

Survival data were analyzed by plotting survival curves for treated and control animals and comparing them by log rank test. Data in the text, figures, and tables are means ± standard deviation (SD).

The significance of the differences between the means at different times in each group was analyzed by one-way analysis of variance (ANOVA) and, when appropriate, the Scheffé test was used to compare pairs of means. The significance of the differences between means for the two groups at different times was analyzed by Student's t test. Because repeated measures were performed for each parameter, the levels of significance were corrected according to Bonferroni (22). The level of statistical significance was set at p < 0.05.

	RESULTS

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Three of 20 (15%) anti-CD18-treated rabbits and nine of the 16 control animals (56%) died within 24 h of intraperitoneal administration of zymosan. By the end of the tenth day mortality was 30% and 75%, respectively, in the two groups. The log rank test showed significant differences between survival rate curves (Figure 1).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Survival rate curves for rabbits treated with monoclonal anti-CD18 antibody or nonspecific rat IgG. Survival was significantly higher in the first group (log-rank test, p < 0.05).

Figure 2 shows white blood cell (WBC) counts and platelet counts during the observation period. Control animals still alive on Day 1 were significantly leukopenic (p < 0.05 versus baseline). In those still alive on Day 10 the number of leukocytes had returned to close to baseline. Anti-CD18-treated animals had a slight, though not significant, elevation of circulating leukocytes on Day 1 and the WBC count on Day 1 was significantly higher than control animals (Figure 2, top panel ). The number of platelets did not vary significantly from baseline, although it was higher in anti-CD18-treated rabbits than in the control group throughout the observation period. The difference was significant on Day 10 (Figure 2, bottom panel ).

View larger version (25K): [in this window] [in a new window]   Figure 2.   WBC (top panel ) and platelet (bottom panel ) counts in rabbits treated with monoclonal anti-CD18 antibody or nonspecific rat IgG. Significant differences are indicated (Student's t test). WBC counts on Day 1 in nonspecific rat IgG-treated animals were significantly lower than at baseline (Scheffé test, p < 0.05).

Differences in WBC and platelet counts were not a consequence of changes in hematocrit which in fact was similar in the two groups before zymosan (40 ± 3% in treated rabbits and 39 ± 7% in controls), dropped to 33 ± 3% and 34 ± 2%, respectively, by the third day, and did not significantly change afterwards. There were no changes in renal function and transaminases (AST and ALT) in either group.

Table 1 shows blood gas analysis data for both groups. Control animals showed a significant decrease of pH on Day 1 compared with before zymosan, but there was no significant difference in pH, PCO₂, and HCO₃ between the two groups. Figure 3 shows PO₂ in the two groups. PO₂ significantly decreased in control rabbits within the first 24 h and was still lower than baseline values on Day 3 (p < 0.05). PO₂ was significantly lower in control animals than in anti-CD18-treated animals on Days 1, 3, and 10. 

View larger version (24K): [in this window] [in a new window]   Figure 3.   PO2 in rabbits treated with monoclonal anti-CD18 antibody or nonspecific rat IgG. Significant differences are indicated (Student's t test). PO2 in nonspecific rat IgG-treated animals was significantly lower on Days 1 and 3 than at baseline (Scheffé test, p < 0.05).

Control rabbits showed changes to the lung anatomy. Grossly, the lungs appeared edematous with areas of hemorrhage, atelectasis, and consolidation. Light microscopy showed interstitial and intra-alveolar edema and granulocyte (and eosinophil) infiltration of interalveolar septa and interstitial spaces, entrapment of red blood cells, platelets, and PMN in blood vessels. The lungs of anti-CD18-treated animals were fairly well conserved, with no or only mild inflammation visible by light microscopy (Figure 4).

View larger version (159K): [in this window] [in a new window]   Figure 4.   Histologic appearance of the lungs of rabbits treated with monoclonal antibody anti-CD18 or with nonspecific rat IgG (hematoxylin-eosin stain). (A) Section of the lung of a nonspecific IgG-treated rabbit (original magnification ×94) showing severe interstitial edema and neutrophil infiltration. (B) Section of the lung of a nonspecific IgG-treated rabbit (original magnification ×94) showing an area of PMN infiltration together with relatively conserved parenchyma. (C ) Section of the lung of a nonspecific IgG-treated rabbit (original magnification ×188) showing an area of PMN (and eosinophil) accumulation. (D, E, F ): Section of the lung of anti-CD-18-treated rabbits (original magnification ×94) showing no or mild interstitial edema or PMN infiltration.

Table 2 shows the results of the morphometric analysis of the lungs of the two groups of animals. Vv was constantly lower in the anti-CD18-treated animals. The mean of the two groups was significantly different (p < 0.001). In the animals surviving on Day 1 there was a highly significant inverse correlation between the PO₂ measured on Day 1 (r = 0.613; p < 0.001) and PMN volume density (Figure 5). The same was true also when PMN volume density was correlated with the last measure of PO₂ obtained in each animal (r = 0.696; p < 0.001).

View larger version (17K): [in this window] [in a new window]   Figure 5.   Correlation between PMN volume density (Vv) and the PO2 measure on Day 1. Statistical significance is indicated.

Figure 6 shows mean tissue levels (± SD) of MDA and -tocopherol in lung specimens from animals from both groups killed on Day 10. MDA was significantly higher and -tocopherol significantly lower in lung homogenates from control rabbits than treated animals.

View larger version (15K): [in this window] [in a new window]   Figure 6.   MDA (pM/mg of protein) and vitamin E (ng/mg of protein) in lung homogenates from animals treated with anti-CD18 monoclonal antibody (open bars) (n = 14) or nonspecific rat IgG (filled bars) (n = 4) killed on Day 10. Significant differences are indicated (Student's t test).

	DISCUSSION

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In the present study we have shown that pretreatment of rabbits with a monoclonal antibody directed to the CD18 -chain of the CD11-CD18 complex prevents lung damage and reduces mortality after zymosan-induced peritonitis.

The animal model we used was originally proposed by Goris and coworkers (13) to demonstrate that inflammation in the abdomen (zymosan-induced peritonitis) can cause microscopic and functional alterations in distant organs such as spleen, liver and, particularly, the lungs. According to Goris and coworkers, changes in different organs occur primarily as a consequence of chemical peritonitis, in the absence of overt infection, as they were reproducible in germ-free rats. However, a bacterial contribution cannot be excluded, because bacterial translocation from the gut to the abdomen was demonstrated in some of the wild-type animals as a consequence of increased permeability of the intestinal wall induced by inflammation. This model mimics clinical situations such as peritonitis or pancreatitis, in which the effects of inflammation and infection cannot be easily disentangled; these diseases are frequently complicated by acute lung insufficiency or MOF (23, 24).

Various laboratories have helped characterize this model in detail. Mortality was reproducible: approximately 40% of animals of different species (rabbits or rats) died within 24 h of intraperitoneal inoculum of zymosan (13, 14). Respiratory changes have been extensively studied: hypoxemia was observed during the first 24 to 48 h after challenge with zymosan. Lung pathology has been studied by light and electron microscopy: typical changes have been observed such as PMN infiltration and interstitial and alveolar edema, resembling acute respiratory distress syndrome (ARDS) in humans (13, 14, 25). Our data clearly indicate that inhibition of CD11-CD18 is beneficial in this particular model of respiratory distress syndrome. The animals treated with the anti-CD18 antibody did not have leukopenia. Lung morphometry showed that there are invariably less PMNs in these animals than in the control rabbits. Although we did not distinguish between intra- and extravascular PMNs, the increased number of PMNs observed in the lungs of control rabbits cannot be due to differences in the number of circulating leukocytes between the two groups; this in fact was similar in the two groups before zymosan injection and lower in nonspecific IgG-treated animals afterwards. The anti-CD18 antibody, therefore, reduced PMN emigration in the lungs (i.e., adhesion to endotheliun and vascular transmigration). As a consequence of the reduction of PMN infiltration, fewer toxic oxygen-free radicals are released by PMN in the lung. This is borne out by the reduced formation of one of the by-products of peroxidation of unsaturated fatty acids, MDA, and by the limited consumption of the antioxidant -tocopherol in lung tissue. As alveolocapillary membranes are spared, gas exchange is preserved and hypoxemia does not arise. These effects on the lungs, and possibly other, not demonstrated, beneficial effects of the monoclonal antibody in other organs, meant that anti-CD18-treated animals had significantly lower mortality.

Our data are in agreement with the role of PMN in the development of respiratory distress syndrome and multiple organ dysfunction which emerges from clinical studies. PMN infiltration in bronchoalveolar lavage (BAL) or in lung specimens from patients with respiratory distress syndrome has been widely observed (26). Transient leukopenia has also been reported in patients with sepsis who developed respiratory failure, suggesting that disappearance of PMN from the bloodstream and entrapment of the cells in lung capillaries is an early event of ARDS (27). Studies in hemodialysis patients further suggested that PMN, stimulated by chemotactic substances such as anaphylatoxin C5a and C5a des-Arg, change their morphology and can stick to the lung endothelial cells (28).

The protective effects of a specific monoclonal anti-CD18 antibody in this particular model of respiratory distress syndrome induced by peritonitis indicate that upregulation of CD11-CD18 by inflammatory stimuli has a role in PMN infiltration of the lungs. In this experimental model many putative molecules are released which can induce the expression or change the conformation of CD11-CD18 complex on the PMN surface. Zymosan injected in the peritoneum activates the alternative pathway of the complement system and generates chemotactic anaphylatoxin C5a (29). Bacterial translocation from the gut to the peritoneum may help spread bacterial wall fragments with chemotactic activity, as well as endotoxins (1, 13). It is also highly conceivable that inflammatory cytokines such as IL-1 and TNF- are released in this model.

Most of the above molecules increase PMN adhesion to endothelial cell monolayers, which can be prevented by monoclonal antibodies against the CD11-CD18 complex, particularly against CD11b, CD11c, and the CD18 chain (10, 16, 30, 31). The same molecules cause leukopenia when injected intravenously in animals (32, 33). LPS-induced neutropenia can be prevented by pretreatment with anti-CD11-CD18 antibody (34). Barton and coworkers (35) reported that anti-CD18 and anti-CD11b, as well as anti-ICAM-1, but not anti-CD11a antibodies, reduced neutrophil migration in rabbit lungs after phorbol ester-induced inflammation. However, the antibody we used did not block the transient neutropenia after intravenous injection of the chemotactic substance FMLP or C5a (16). This suggests that mechanisms more complex than simple generation of chemotactic substances such as C5a are responsible for the leukopenia observed in the control animals. Chemoattractants act on PMN by increasing their stiffness through a CD11-CD18-independent change in the assembly of actin filaments (36).

It is also probably worth mentioning that IL-1 and TNF- induce the expression of ICAM-1 in different human cell lines (11). If this is also true for endothelial cells, IL-1 and TNF- could potentially induce the expression of both CD11-CD18 and its ligand.

Pretreatment with anti-CD11-CD18 antibody has proved beneficial in a variety of experimental models in which PMN have a potential role in the tissue damage (e.g., ischemia-reperfusion injury). Monoclonal antibody against the common -chain of the anti-CD11-CD18 complex prevented intestinal mucosal injury and vascular permeability and reduced mortality after hemorrhagic shock and resuscitation in rabbits (37) and primates (38), and reduced the myocardial infarction area after induction of regional myocardial ischemia in dogs (39). More relevant to this study, blocking the CD11-CD18 complex has proved beneficial in animal models reproducing the clinical scenario of severe abdominal sepsis, septic shock, and MOF. Inoue and coworkers (40) reported that a monoclonal antibody against CD18 attenuated the severity of acute lung injury in rats with experimental acute pancreatitits. Treatment with a monoclonal antibody against an epitope of CD11b prevented hypoxemia and increased early survival in dogs challenged with TNF- (41). Using the same antibody Walsh and coworkers (42) observed less severe neutropenia and alveolocapillary membrane injury after injection of Pseudomonas aeruginosa in pigs. A CD11-CD18-dependent neutropenia follows fecal peritonitis in pigs (43).

Our data confirm and extend these observations, showing that blocking PMN adherence by inhibiting the CD11-CD18 complex is effective in an animal model which is closer to the clinical situation. These findings suggest a possible therapeutic use of anti-CD18 antibodies in human infection or inflammation, particularly after recent clinical studies have shown that in human sepsis and ARDS there is indeed an upregulation of CD11-CD18 molecules on PMNs as well as of its counterpart ICAM-1 on endothelial cells (44) and that this is quantitatively correlated with IL-6 and TNF- levels and with organ failure scores (47).

Further studies, however, are certainly needed to verify whether this approach is feasible in humans. First, experiments in CD18-deficient mice seem to suggest that there are other CD18-independent pathways of PMN recruitment to the sites of inflammation (48). Moreover, blocking PMN adherence may have negative effects on bacterial phagocytosis and clearance of endotoxins. Several studies addressed this issue specifically, with conflicting results. Milewski and coworkers (49) reported that the anti-CD18 antibody did not increase morbidity (wound infections and abdominal abscesses) or mortality in a model of abdominal sepsis. Lyden and coworkers (50) showed that anti-CD18 injection during fluid resuscitation in swine after cecum ligation and incision and 35% hemorrhage did not increase vulnerability to endogenous pathogens. On the other hand, Eichacker and coworkers (51) and Mercer-Jones and coworkers (52) reported that treatment with a monoclonal antibody against CD18 worsened endotoxemia in two different models of peritonitis. Eichacker and coworkers (51) reported that this was associated with worsening of hemodynamics and shortened survival. The same group reported also that using anti-CD11b in a model of pneumonia in rats was associated with reduced survival, despite a protective effect on the lungs (53). We cannot comment whether blockade of the CD11-CD18 complex in our experiments had a deleterious effect on abdominal infection or on hemodynamics; this seems, however, very unlikely in light of our mortality data.

In conclusion, in this rabbit model in which peritonitis is complicated by acute respiratory failure, the anti-CD18 antibody, which blocks PMN adherence, reduced changes in distant organs such as the lungs, prevented respiratory failure, and significantly reduced mortality. In selected situations, therefore, it may be useful to act on PMN adherence as a potential therapeutic approach.