Perforation of the intestines is a feared condition in which toxic and polymicrobial contents of the bowel contaminate the abdominal cavity (1, 2). Fecal bacteria stimulate local production of various proinflammatory substances that are subsequently released into the circulation. Moreover, infective microorganisms may also directly invade the blood stream and trigger an inflammatory host response in a distant target organ. In either scenario, the lung is the most sensitive and clinically important end-organ for the inflammatory response in abdominal sepsis (3). In fact, lung injury continues to constitute a significant cause of mortality in polymicrobial sepsis despite aggressive surgical interventions and antibiotic and immunomodulating therapies (4). The multiple signaling cascades triggered by a mixed bacterial flora and their released products are complex and largely unknown (5, 6). Substantial investigative efforts have been devoted to delineate the pathophysiology of sepsis using exogenous administration of various bacterial toxins such as LPS and superantigens derived from gram-negative and gram-positive bacteria, respectively. However, administration of such toxins may not represent the pathophysiology of clinical sepsis very well, and it has been reported that different toxins activate the host immune system in a distinctly different manner. For example, LPS has been shown to be a potent activator of macrophages and stimulates TNF-α production (7, 8), whereas superantigens do not provoke clear-cut TNF-α formation and activate primarily T lymphocytes, causing FasL-dependent apoptosis (9). In contrast, the cecal ligation and puncture (CLP) model, in which the intestine is punctured and the bowel contents are allowed to contaminate the abdominal cavity, seems to be more reminiscent of the events and course in polymicrobial sepsis in terms of cytokine responses and vascular and metabolic changes (10, 11).
Convincing data have shown that septic lung injury is characterized by massive accumulation of neutrophils in the bronchoalveolar space (12-15). Pulmonary infiltration of neutrophils is a multistep process comprising initial mechanical sequestration in microvessels, followed by adhesion molecule-dependent firm adhesion to endothelial cells and transmigration through endothelial and epithelial barriers (12). Bacterial antigens provoke formation of proinflammatory substances, which in turn up-regulate endothelial cell adhesion molecules and stimulate activation of neutrophils (16). Tissue localization of neutrophils is mediated by CXC chemokines (17), including macrophage inflammatory protein 2 (MIP-2) (18) and cytokine-induced neutrophil chemoattractant (KC) (19, 20) in the mouse. Specific adhesion molecules mediate attachment between activated neutrophils and endothelial cells (13, 14). Although most relevant adhesion molecules, including P- and E-selectin and intercellular adhesion molecule 1 (ICAM-1), have been demonstrated to be up-regulated on pulmonary endothelial cells in systemic inflammation (21), the literature on the role of specific adhesion molecules in polymicrobial sepsis is incomplete and partly controversial. For example, some investigators have reported that ICAM-1 is of great importance in supporting neutrophil recruitment to the lung (14, 22), whereas others have not found such a role of ICAM-1 (23) in sepsis. Nonetheless, ICAM-1 is known to interact with members of the β-2 integrin family, including lymphocyte function antigen-1 (LFA-1; CD11a/CD18) and membrane-activated complex-1 (Mac-1; CD11b/CD18), which mediate firm leukocyte adhesion in a stimulus- and organ-dependent manner (14, 24-27). However, the potential role of LFA-1 and Mac-1 in mediating pulmonary infiltration of neutrophils in polymicrobial sepsis remains elusive.
Based on the above considerations, the aim of the present study was to define the functional role of LFA-1 and Mac-1 in mediating pulmonary neutrophil recruitment and tissue damage in polymicrobial sepsis using the CLP model in mice.
MATERIALS AND METHODS
Experiments were performed using male C57BL/6 mice weighing 20 to 25 g. All experimental procedures were performed in accordance with the legislation on the protection of animals and were approved by the Regional Ethical Committee for Animal Experimentation at Lund University, Sweden. Animals were anesthetized by administration of 7.5 mg (i.p.) ketamine hydrochloride (Hoffman-La Roche, Basel, Switzerland) and 2.5 mg (i.p.) xylazine (Janssen Pharmaceutica, Beerse, Belgium) per 100 g body weight.
Experimental protocol of sepsis
Polymicrobial sepsis in mice was induced by puncture of the cecum. Animals were anesthetized, the abdomen was opened, the exposed cecum was filled with feces by milking stool backward from the ascending colon, and a ligature was placed below the ileocecal valve. The cecum was soaked with phosphate-buffered saline (PBS; pH 7.4) and was then punctured twice with a 21-gauge needle. The cecum was then returned into the peritoneal cavity, and the abdominal incision was sutured. To evaluate the functional importance of LFA-1 and Mac-1, we used a saturating dose of 4 mg/kg of monoclonal antibodies (mAbs) directed against murine CD11a (M17/184.108.40.206, rat immunoglobulin [Ig]G; Novartis Pharma AG, Preclinical Research, Basel, Switzerland), CD11b (M1/70, rat IgG2b; BD Biosciences Pharmingen, San Jose, Calif), and an isotype-matched control mAb (R3-34, IgG; BD Biosciences Pharmingen) in CLP mice. Antibodies or vehicle (100 μL PBS) was administered intravenously immediately before CLP induction. In a separate group of animals, anti-LFA-1 (4 mg/kg) and anti-Mac-1 antibodies (4 mg/kg) were given in combination before CLP. Sham mice underwent the same surgical procedures, that is, laparotomy and resuscitation, but the cecum was neither ligated nor punctured. The mice were then returned to their cages and provided food and water ad libitum. It was observed that 20% of the animals died after CLP, and this percentage was the same in all experimental groups. Animals were anesthetized 6 and 24 h after CLP induction. The left lung was ligated and excised for edema measurement. The right lung was used for collecting bronchoalveolar lavage fluid in which neutrophils were quantified in a Burker chamber. Next, the lung was perfused with PBS, and one part was fixed in formaldehyde for histology, and the remaining lung tissue was weighed, snap-frozen in liquid nitrogen, and stored at −80°C for later enzyme-linked immunosorbent assay (ELISA) and myeloperoxidase (MPO) assays as described in the next paragraphs.
Systemic leukocyte count
Blood was collected from tail vein and was mixed with Turks solution (0.2 mg gentian violet in 1 mL glacial acetic acid; 6.25% vol/vol) in a 1:20 dilution. Leukocytes were counted as monomorphonuclear (MNL) and polymorphonuclear (PMNL) leukocyte cells in a Burker chamber.
The left lung was excised, washed in PBS, gently dried using a blotting paper, and weighed. The tissue was then dried at 60°C for 72 h and reweighed. The change in the ratio of wet weight to dry weight was used as an indicator of lung edema formation.
Frozen lung tissue was thawed and homogenized in 1 mL of 0.5% hexadecyltrimethylammonium bromide. Next, the sample was freeze-thawed, after which the MPO activity of the supernatant was measured. The enzyme activity was determined spectrophotometrically as the MPO-catalyzed change in absorbance in the redox reaction of H2O2 (450 nm, with a reference filter of 540 nm; 25°C). Values were expressed as MPO units per gram of tissue.
Quantification of chemokines by ELISA
The lung sample was thawed and homogenized in PBS. MIP-2 and KC were analyzed by using double-antibody Quantikine ELISA kits (R & D Systems, Minneapolis, Minn) using recombinant murine MIP-2 and KC as standards. The minimal detectable protein concentrations are less than 0.5 pg/mL.
Lungs samples were fixed in 4% formaldehyde phosphate buffer overnight and then dehydrated and paraffin-embedded. Six-micrometer sections were stained with hematoxylin and eosin.
For analysis of surface expression of LFA-1 and Mac-1 on circulating neutrophils, blood was collected via cardiac puncture into heparinized syringes at 16 h after CLP induction. Erythrocytes were lysed using red blood cell lysing buffer (Sigma Chemical Co., St. Louis, Mo), and the leukocytes recovered after centrifugation. Cells were incubated with anti-CD16/CD32 to block Fcγ III/II receptors and reduce nonspecific labeling for 10 min and stained at 4°C for 30 min simultaneously with phycoerythrin-conjugated anti-Gr-1 (clone RB6-8C5) and with fluorescein isothiocyanate (FITC)-conjugated anti-CD11a/anti-LFA-1 (clone M17/4) or anti-CD11b/anti-Mac-1 (clone M1/70) mAbs (all purchased from BD Biosciences Pharmingen). Flow-cytometric analysis was performed according to standard settings on a FACSort flow cytometer (Becton Dickinson, Mountain View, Calif), and a viable gate was used to exclude dead and fragmented cells.
Data are presented as mean values ± SEM. Statistical evaluations were performed using Kruskal-Wallis one-way ANOVA on ranks, followed by multiple comparisons versus control group (Dunnett method). P < 0.05 was considered significant, and n represents the number of animals.
CLP-induced expression of LFA-1 and Mac-1 on neutrophils
Flow cytometry was used to detect potential expression of LFA-1 and Mac-1 on circulating neutrophils 16 h after CLP induction. Both Mac-1 and LFA-1 were expressed on the surface of circulating neutrophils (Fig. 1, A and B). Indeed, it was found that CLP provoked increased Mac-1 (Fig. 1B) on neutrophils compared with sham-operated animals, that is, the mean fluorescence intensity values were increased from 36.3 ± 5.6 in sham to 308.5 ± 73.9 in CLP animals (Fig. 1B; P < 0.05 vs. sham; n = 4). However, LFA-1 (Fig. 1A) on neutrophils was not up-regulated compared with sham (Fig. 1A; P > 0.05 vs. sham; n = 4), and mean fluorescence intensity values for LFA-1 were 156.7 ± 23.8 and 162.3 ± 14.5 in sham and CLP animals, respectively. Thus, we next evaluated the potential role of LFA-1 and Mac-1 function in CLP-induced lung injury.
LFA-1 and Mac-1 mediate edema formation and lung injury
Cecal ligation and puncture induced significant pulmonary damage, indicated by prominent enhancement in lung edema formation (Fig. 2). Thus, it was found that the lung wet-dry ratio increased by more than 45% in polymicrobial sepsis, that is, from 4.4 ± 0.1 to 6.4 ± 0.1 (Fig. 2; P < 0.05 vs. sham; n = 5). Notably, administration of the anti-LFA-1 and anti-Mac-1 antibody decreased CLP-induced lung edema by 77% and 60%, respectively (Fig. 2; P < 0.05 vs. control ab + CLP; n = 5). The lung wet-dry ratio was reduced by 78% when anti-LFA-1 and anti-Mac-1 antibodies were given in combination before CLP (Fig. 2; P < 0.05 vs. control ab + CLP; n = 5). Moreover, histological micrographs of lung tissue of sham-operated animals revealed normal architecture (Fig. 3A), whereas CLP mice treated with vehicle (Fig. 3B) or control antibody (Fig. 3C) exhibited severe destruction of the pulmonary tissue microstructure, extensive edema of the interstitial tissue, capillary congestion, necrosis, and massive infiltration of neutrophils. Immunoneutralization of LFA-1 (Fig. 3D), Mac-1 (Fig. 3E), or both (Fig. 3F) protected the lung microarchitecture and reduced neutrophil infiltration.
LFA-1 and Mac-1 mediate CLP-induced neutrophil recruitment
Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of lung injury during sepsis (14, 17). Clear-cut infiltration of neutrophils into the bronchoalveolar space was observed 6 and 24 h after CLP induction. Here, we found that neutrophil recruitment into the bronchoalveolar space increased by 12- and 73-fold at 6 and 24 h after induction of CLP, respectively (Fig. 4; P < 0.05 vs. sham; n = 5). Interestingly, administration of the anti-LFA-1 and anti-Mac-1 antibody decreased the number of pulmonary neutrophils from 18.4 ± 2.7 × 103 to 4.0 ± 1.3 × 103 (Fig. 4; P < 0.05 vs. control ab + CLP; n = 5) and 9.6 ± 2.7 × 103 (Fig. 4; P > 0.05 vs. control ab + CLP; n = 5) cells at 6 h post-CLP, which corresponds to an 86% and 52% reduction, respectively. Pretreatment with the anti-LFA-1 and anti-Mac-1 antibody or a combination of both antibodies decreased pulmonary recruitment of neutrophils 24 h after CLP induction by 90%, 65%, and 92%, respectively (Fig. 4; P < 0.05 vs. control ab + CLP; n = 5). In addition, global accumulation of neutrophils was assessed by analyzing levels of MPO in the lung. Maximum levels of MPO were found at 6 h post-CLP (not shown). It was found that MPO levels in the lung were increased by 14-fold 6 h after CLP induction (Fig. 5; P < 0.05 vs. sham; n = 5). We observed that immunoneutralization of LFA-1 and Mac-1 significantly decreased pulmonary MPO levels by more than 72% in septic mice (Fig. 5; P < 0.05 vs. control ab + CLP; n = 5).
CXC chemokine production in CLP
Numerous studies have shown that directed movement of neutrophils is mediated by CXC chemokines, including MIP-2 and KC (17-20). The lung content of CXC chemokines in sham controls was low but detectable, but CLP provoked clear-cut formation of MIP-2 and KC in the lung tissue (Fig. 6, A and B). Thus, the tissue levels of MIP-2 and KC in the lung increased from 1.7 ± 0.2 and 6.2 ± 0.5 ng/g at baseline up to 38.9 ± 5.7 and 80.8 ± 4.5 ng/g lung tissue, respectively, in septic mice at 6 h post-CLP (Fig. 6, A and B; P < 0.05 vs. sham; n = 5). At 24 h after CLP, pulmonary formation of MIP-2 and KC increased even further (Fig. 6, A and B; P < 0.05 vs. sham; n = 5). Administration of the anti-LFA-1 and anti-Mac-1 antibody as well as combined anti-LFA-1 + anti-Mac-1 antibody treatment had no effect on CXC chemokine levels in lung tissue at any point in septic animals (Fig. 6, A and B; n = 5).
A characteristic feature in early septicemia is the reduced number of circulating leukocytes (28, 29). Here, we observed that CLP caused a clear-cut leukocytopenia after 24 h (Table 1). For example, the number of neutrophils decreased by 56% 24 h after CLP induction (Table 1; P < 0.05 vs. sham; n = 5). This CLP-induced neutropenia was reversed in mice pretreated with the anti-LFA-1, anti-Mac-1 antibody and when these antibodies were given together (Table 1; P < 0.05 vs. control ab + CLP; n = 5).
New targets for treating polymicrobial sepsis are urgently needed. The present study documents key roles of LFA-1 and Mac-1 in supporting sepsis-induced lung damage. Our data show that these adhesion molecules are expressed on activated leukocytes in polymicrobial sepsis, which in turn mediate pulmonary infiltration of neutrophils. Moreover, these findings also demonstrate that functional inhibition of either LFA-1 or Mac-1 not only decreases pulmonary accumulation of neutrophils but also protects against lung edema formation and tissue destruction in polymicrobial sepsis. Considered together, these novel findings suggest that LFA-1 and Mac-1 may constitute fruitful targets in septic lung injury.
Perforation of the gastrointestinal tract and leakage of bowel contents into the abdominal cavity are common and serious conditions encountered in surgical patients. Despite modern treatment modalities, the mortality of these patients remains high in intensive care units (4). Thus, there is an urgent need to develop more specific and effective therapies for patients with systemic sepsis. The most vulnerable organ in polymicrobial sepsis is the lung. The present study demonstrates that LFA-1 and Mac-1 may constitute specific targets to protect against pulmonary damage in abdominal sepsis. Thus, we observed that LFA-1 and Mac-1 were expressed on circulating neutrophils after intestinal puncture, which made us hypothesize that these adhesion molecules may be involved in the pathophysiology of septic lung injury. Interestingly, we found that inhibition of LFA-1 and Mac-1 reduced neutrophil infiltration in the lung by more than 52% in polymicrobial sepsis, suggesting that both LFA-1 and Mac-1 support adhesive interactions between circulating leukocytes and lung microvascular endothelial cells in sepsis. Previous works have reported conflicting data on the individual role of LFA-1 and Mac-1 in specific models of inflammation (30-32). However, taking more recent studies into consideration, it is comprehendible that both of these adhesion molecules may cooperate for optimal recruitment of inflammatory cells. For example, a recent study reported that LFA-1 may initiate first stable contact, and that Mac-1 establishes more sustainable adhesion onto the endothelium of inflamed organs (33). Nonetheless, we observed that inhibition of LFA-1 and Mac-1 not only attenuated pulmonary infiltration of neutrophils but also reduced edema formation by more than 60% in the lung. In addition, immunoneutralization prevented widespread tissue damage in the lungs of CLP mice. Considered together, these findings suggest a link between LFA-1- and Mac-1-dependent accumulation of neutrophils on one hand and edema formation and tissue injury in the lung on the other. Thus, our data indicate that blocking the function of LFA-1 or Mac-1 may be of beneficial value in septic lung injury. Interestingly, we found that combined treatment with both anti-LFA-1 and anti-Mac-1 antibodies had no additional inhibitory effect on CLP-induced neutrophil recruitment. Notably, this observation is in line with the study by Hentzen et al. (33) showing that LFA-1 and Mac-1 cooperate in a sequential manner, and that both of these adhesion molecules may be necessary for optimal adhesion onto the endothelium, and that inhibition of either one would be sufficient to decrease neutrophil recruitment. In this context, it should be mentioned that one of the prominent features in clinical responses to sepsis is an early drop in the numbers of blood neutrophils (28, 29), which was also observed herein and culminated 24 h after CLP initiation. Interestingly, we observed that inhibition of both LFA-1 and Mac-1 protected septic animals against this neutropenia, supporting the concept that inhibition of these β-2 integrins ameliorates systemic effects of sepsis. Indeed, this preservation of circulating neutrophils exerted by inhibition of LFA-1 and Mac-1 may help maintain effective immune function in abdominal sepsis.
Numerous studies have shown that septic conditions induce a complex inflammatory reaction in peripheral tissues, including the lung, after only a few hours (15, 34). In the lung, this reaction involves rapid production and release of cytokines and CXC chemokines by alveolar macrophages and epithelial cells (15). In line with this view, we found in the present experimental model of abdominal sepsis that intestinal puncture caused significant elevation of pulmonary production of the CXC chemokines, MIP-2, and KC, which are murine homologs of human growth-related oncogene chemokines (18). Because these CXC chemokines have been shown to attract neutrophils in particular (18, 19), they are considered to be important mediators of several pathological processes such as septic lung injury (17), glomerulonephritis (35), bacterial meningitis (36), and endotoxemic liver injury (37). However, we found here that inhibition of LFA-1 and Mac-1 had no effect on CXC chemokine production in the lungs of septic animals, suggesting that the reduced leukocyte infiltration in the lung of CLP mice treated with anti-LFA-1 and anti-Mac-1 antibodies is not likely related to local changes in CXC chemokine production. In this context, it should be mentioned that one limitation of this study may be that mortality was not used as an end point, but instead, lung injury was the focus of this particular study.
In conclusion, this study demonstrates that LFA-1 and Mac-1 play important roles in polymicrobial sepsis by supporting pulmonary infiltration of neutrophils. Moreover, our data show not only that inhibition of LFA-1 or Mac-1 reduces neutrophil recruitment but also protects against sepsis-induced edema formation and tissue damage in the lung. Thus, on the basis of these novel findings, we propose that targeting LFA-1 and Mac-1 alone or in combination may be useful to protect against lung injury in polymicrobial sepsis.
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