Recombinant Bovine Interleukin-1β Amplifies the Effects of Partially PurifiedPasteurella haemolyticaLeukotoxin on Bovine Neutrophils in a β₂-Integrin-Dependent Manner

Abstract

The influx and death of polymorphonuclear leukocytes within the infected lung are hallmarks of bovine pasteurellosis. Recent reports have shown that the Pasteurella haemolytica leukotoxin (LKT) and other RTX toxins bind ␤ 2 -integrins on target cells. In this study we demonstrate that exposure of bovine neutrophils to recombinant bovine interleukin-1␤ upregulates ␤ 2 -integrins (CD11a/CD18), which in turn enhance the binding and amplify the biological effects of partially purified LKT on these cells. LKT binding and cytotoxicity were inhibited by addition of an anti-integrin antibody (CD11a/CD18). These findings help to clarify the early events that occur in bovine pasteurellosis and support the hypothesis that inflammatory mediators might increase the severity of pasteurellosis by causing upregulation of ␤ 2 -integrins that serve as an LKT receptor on bovine neutrophils.Bovine pneumonic pasteurellosis is an acute fibrinonecrotizing pleuropneumonia characterized by an influx of neutrophils into the alveoli and interlobular septa of the lung (1,25,39). Some of these infiltrating leukocytes undergo apoptosis in the lung during the infection (17). In severe cases the inflammation extends onto the pleural surface (25, 39). Although Pasteurella haemolytica produces several virulence factors that play a role in the pathogenesis of pasteurellosis, the most important is thought to be a leukotoxin (LKT), whose effects are specific for ruminant leukocytes and platelets (3, 13, 27, 28). The P. haemolytica LKT is a member of a large family of exotoxins produced by gram-negative bacteria, known as RTX toxins, that share similar synthesis and secretion systems and exhibit related biological activities (37). Previously, it has been shown that other RTX toxins bind to ␤ 2 -integrins on target cells (16). More recently, anti-␤ 2 -integrin monoclonal antibodies (MAbs) have been shown to inhibit the cytolysis by P. haemolytica LKT of BL-3 cells (a bovine B lymphoblastoid cell line) and peripheral blood neutrophils (2, 14, 18). Anti-␤ 2 -integrin MAbs have also been shown to block binding of LKT to blotted lysates of BL-3 cells and neutrophils (14,18). The mechanism by which the RTX toxins exert their lethal effect on target cells is only partially understood but is thought to involve changes in selective permeability of the cytoplasmic membrane that result in Ca 2ϩ influx and ATP efflux (23,34,36,37).The proinflammatory cytokine interleukin-1␤ (IL-1␤), which is produced by alveolar macrophages and other cells, can stimulate migration and functional activation of polymorphonuclear leukocytes (PMNs) in the lung (10). The ␤ 2 -integrins, which are found on most circulating leukocytes, play an important role in the extravasation of leukocytes during the inflammatory response (11). Studies of other inflammatory stimuli have shown that they can increase surface expression and avidity of ␤ 2 -integrins on neutrophils (24,38). In this study, we demonstrate an increase in ␤ 2 -integrin expression on bovine PMNs following their in vitro inc...

Inflammatory Cytokines Enhance the Interaction of Mannheimia haemolytica Leukotoxin with Bovine Peripheral Blood Neutrophils In Vitro

“…We recently reported that exposure of bovine PMNs to the inflammatory cytokine IL-1␤ resulted in increased binding of two different anti-LFA-1 MAbs, LKT binding, and cell death (25). In the present study, we extended these observations by FIG.…”

“…In the present study, we observed that heat (100°C for 15 min) destroyed the response to LKT, and pretreatment with polymyxin B did not reduce the response to LKT (data not shown). We have previously demonstrated that LKT from a noncytolytic M. haemolytica mutant exhibits little or no binding to bovine PMNs (25). These controls suggested that LPS contamination by itself does not account for the results obtained in our study.…”

“…A second limitation is that we used only one MAb (BAT75A) to evaluate LFA-1 expression on bovine PMNs by flow cytometry. This antibody has been used by a number of investigators to detect LFA-1 expression on bovine cells, and workers in our laboratory and other workers have shown that it reduces LKT binding to bovine leukocytes and LKT cytotoxicity for these cells (17,25,45). Using MAbs BAT75A and HI111 in Western immunoblotting provided additional evidence that bovine PMNs have increased LFA-1 expression after cytokine exposure.…”

“…LKT activity was evaluated by incubating biotinylated LKT with bovine peripheral leukocytes for 30 min at 37°C, and then the cytotoxicity was assessed by using 2,3-bis(2-methoxy-4-nitro-5-sulfenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) (Sigma, St. Louis, Mo.) as previously described (25).…”

“…Previously, it has been reported that other members of the RTX family bind to ␤ 2 -integrins on target cells (23). More recently, it has been demonstrated that M. haemolytica LKT binds to lymphocyte function-associated antigen 1 (LFA-1), a ␤ 2 -integrin (CD 11a/CD18) on bovine leukocytes (1,17,25,27). LKT binding to bovine leukocytes induces formation of pore-like structures in the plasma membrane, resulting in both activation of leukocytes and death by necrosis and apoptosis (14,18,24,29,34,40,43,45,53).…”