Matt J. Sylte

Haemophilus somnus Induces Apoptosis in Bovine Endothelial Cells In Vitro

ABSTRACT Haemophilus somnus causes pneumonia, reproductive failure, infectious myocarditis, thrombotic meningoencephalitis, and other diseases in cattle. Although vasculitis is commonly seen as a result of systemic H. somnus infections, the pathogenesis of vascular damage is poorly characterized. In this study, we demonstrated that H. somnus (pathogenic isolates 649, 2336, and 8025 and asymptomatic carrier isolates 127P and 129Pt) induce apoptosis of bovine endothelial cells in a time- and dose-dependent manner, as determined by Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling, DNA fragmentation, and transmission electron microscopy. H. somnus induced endothelial cell apoptosis in as little as 1 h of incubation and did not require extracellular growth of the bacteria. Viable H. somnus organisms induced greater endothelial cell apoptosis than heat-killed organisms. Since viableH. somnus cells release membrane fibrils and blebs, which contain lipooligosaccharide (LOS) and immunoglobulin binding proteins, we examined culture filtrates for their ability to induce endothelial cell apoptosis. Culture filtrates induced similar levels of endothelial cell apoptosis, as did viable H. somnus organisms. Heat inactivation of H. somnus culture filtrates partially reduced the apoptotic effect on endothelial cells, which suggested the presence of both heat-labile and heat-stable factors. We found thatH. somnus LOS, which is heat stable, induced endothelial cell apoptosis in a time- and dose-dependent manner and was inhibited by the addition of polymyxin B. These data demonstrate that H. somnus and its LOS induce endothelial cell apoptosis, which may play a role in producing vasculitis in vivo.

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

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.

Complexities of the pathogenesis of Mannheimia haemolytica and Haemophilus somnus infections: challenges and potential opportunities for prevention?

Abstract Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral–bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the β2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1β, tumor necrosis factor-α and interferon-γ) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.

Recombinant Bovine Interleukin-1β Amplifies the Effects of Partially Purified Pasteurella haemolytica Leukotoxin 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 β2-integrins on target cells. In this study we demonstrate that exposure of bovine neutrophils to recombinant bovine interleukin-1β upregulates β2-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 β2-integrins that serve as an LKT receptor on bovine neutrophils.

Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin.

Abstract Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the β2-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the β2-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.

Stimulation of P2X receptors enhances lipooligosaccharide-mediated apoptosis of endothelial cells

Exposure of endothelial cells to lipid A‐containing molecules, such as lipopolysaccharide (LPS) or lipooligosaccharide (LOS), causes the release of purinergic compounds [e.g., adenosine 5′‐triphosphate (ATP)] and can lead to apoptosis. The P2X family of purinergic receptors (e.g., P2X7) has been reported to modulate LPS signaling events and to participate in apoptosis. We investigated the role that P2X receptors play in the apoptosis that follows exposure of bovine endothelial cells to Haemophilus somnus LOS. Addition of P2X inhibitors, such as periodate‐oxidized ATP (oATP) or pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid tetrasodium, significantly reduced LOS‐induced apoptosis. Incubation of endothelial cells with apyrase, which degrades ATP, diminished LOS‐induced apoptosis of endothelial cells. Concomitant addition of P2X agonists [e.g., 2′,3′‐(4‐benzoyl)‐benzoyl ATP or ATP] to LOS‐treated endothelial cells significantly enhanced caspase‐3 activation. The P2X antagonist oATP significantly blocked caspase‐8 but not caspase‐9 activation in LOS‐treated endothelial cells. Together, these data indicate that stimulation of P2X receptors enhances LOS‐induced apoptosis of endothelial cells, possibly as a result of endogenous release of ATP, which results in caspase‐8 activation.