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Infection and Immunity, August 2002, p. 4336-4343, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4336-4343.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

F. Leite, S. O'Brien, M. J. Sylte, T. Page, D. Atapattu, and C. J. Czuprynski*

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin

Received 10 December 2001/ Returned for modification 18 January 2002/ Accepted 7 May 2002

Mannheimia (Pasteurella) haemolytica A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the ß2-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNF-{alpha}), and gamma interferon (IFN-{gamma}) on the interaction of M. haemolytica LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1ß, TNF-{alpha}, or IFN-{gamma}. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to M. haemolytica LKT.

* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706. Phone: (608) 262-8102. Fax: (608) 262-8102. E-mail: czuprync{at}svm.vetmed.wisc.edu.

Editor: R. N. Moore

Infection and Immunity, August 2002, p. 4336-4343, Vol. 70, No. 8
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.8.4336-4343.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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