Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro.
Journal: 2002/September - Infection and Immunity
ISSN: 0019-9567
PUBMED: 12117943
Abstract:
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 beta(2)-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-1beta (IL-1beta), 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-1beta, 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.
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Infect Immun 70(8): 4336-4343

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

Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin
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: ude.csiw.demtev.mvs@cnyrpuzc.
Received 2001 Dec 10; Revised 2002 Jan 18; Accepted 2002 May 7.

Abstract

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 β2-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-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) 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-1β, TNF-α, or IFN-γ. 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.

Abstract

Mannheimia (Pasteurella) haemolytica A1 is the primary bacterial agent of bovine pneumonic pasteurellosis (shipping fever), which is characterized by acute lobar fibronecrotizing pneumonia with extensive peripheral blood neutrophil (PMN) infiltration in small airways and alveoli (4, 39, 47). Several virulence factors of M. haemolytica play an important role in the pathogenesis of pasteurellosis (7, 13). Foremost among these is a leukotoxin (LKT), whose effects are specific for ruminant leukocytes and platelets (2, 6, 9, 44). The M. haemolytica LKT is member of the repeats-in-toxin (RTX) family of gram-negative bacterial pore-forming exotoxins (46). Members of the RTX family have similar mechanisms of toxin production, secretion, and target cell intoxication (8, 45). Previously, it has been reported that other members of the RTX family bind to β2-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 β2-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).

For reasons that are not well understood, active viral infections can greatly enhance the susceptibility of cattle to M. haemolytica pneumonia (11, 28, 42, 48, 49). One mechanism that might be involved is the release of inflammatory cytokines during viral infection (33, 34). Inflammatory cytokines secreted by respiratory tract cells, such as interleukin 1 (IL-1β), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), can stimulate leukocyte migration and functional activation of β2-integrins on lung leukocytes (10, 35, 38). Once M. haemolytica infection is established in the lung, the continued release of these inflammatory cytokines could be sustained by M. haemolytica virulence factors (i.e., LKT and lipopolysaccharide [LPS]) (15, 21, 22, 30, 50, 51, 52).

PMNs are thought to contribute to the lung pathology observed in pneumonic pasteurellosis (4). PMN depletion reduces the severity of lung damage in experimentally infected cattle (4, 39). We hypothesized that inflammatory cytokines released during viral infection might increase surface expression or conformational activation of LFA-1 on bovine PMNs, thus amplifying their interaction with M. haemolytica LKT. In this study, we demonstrated increased expression of LFA-1 on bovine PMNs, as detected by flow cytometry, following incubation of PMNs with IL-1β, TNF-α, or IFN-γ. This in turn was reflected in increased LKT binding to, and cytotoxicity for, bovine PMNs. These observations suggest that the ability of inflammatory cytokines to increase surface expression or conformational activation of LFA-1 on bovine PMNs increases their interaction with M. haemolytica LKT. The outcome of the response might increase the severity of bovine pasteurellosis.

Acknowledgments

We thank Chester Thomas for his assistance with the statistical analysis. We thank Steve Giles for assistance with preparation of the illustrations. We thank Paul Lunn for his assistance with the real-time PCR.

This work was supported by funds from the Wisconsin Agricultural Experiment Station (grants142 4313 and 142 4543), the University of Wisconsin-Madison Industrial and Economic Development Research Program (grant 118 1034), the USDA National Research Initiative (grant 2000-02304), and the NIEHS Center for Developmental and Molecular Toxicology (grant P30 ES 0990). F. Leite was supported by CAPES-Ministério Da Educação, Brazil.

Acknowledgments

Notes

Editor: R. N. Moore

Notes
Editor: R. N. Moore

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