Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella (Mannheimia) haemolytica leukotoxin

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharid e (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have ch aracterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammat ory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Si nce AMs are exposed to both of these bacterial virulence factors during dis ease, previous studies may have underestimated the possibility of functiona l interactions between Lkt and LPS. The purpose of this study was to charac terize the effect of simultaneous exposure to both Lkt and LPS on AM cytoly sis and proinflammatory cytokine expression. Using cellular leakage of lact ate dehydrogenase as an indirect measure of cytolysis, we studied AM respon ses to Lkt alone, LPS alone and LMS-LPS. We found that 80-200 pg/ml LPS, wh ich does not itself cause cytolysis, synergisticaliy enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRN A, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression indu ced by Lkt+LPS differed from those induced by each agent separately. Finall y, the WEHI 164 (clone 13) bioassay was used to show that LM/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These resu lts provide direct evidence of synergism between Lkt and LPS in AM cytolysi s and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated. (C) 2001 Academic Pre ss.