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

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 ␤ 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).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-1␤), tumor necrosis factor alpha (TNF-␣), and gamma interferon (IFN-␥), can stimulate leukocyte migration and functional activation of ␤ 2 -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-1␤, TNF-␣, or IFN-␥. 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. MATERIALS AND METHODSPMN preparation. Peripheral blood ...

Characterization of Mannheimia (Pasteurella) haemolytica leukotoxin interaction with bovine alveolar macrophage β2 integrins

“…It is likely that well documented LFA-1 specific mechanisms including membrane raft formation [34], and cytokine mediated "insideout" signaling may also play a role in mediating LktA-induced biological effects. A recent report has in fact indirectly addressed this question and demonstrated that exposure of bovine neutrophils to IL-1ß, TNF-α and IFN-γ increased the expression of LFA-1 leading to enhanced LktA effects [29]. These and other reports demonstrating that diverse ligands capable of eliciting intracellular signals that enhance LFA-1 mediated LktA-effects [29][30][31] suggest that LFA-1 affinity and avidity changes occurring through "inside-out" signaling may affect overall LktA biological effects on the target cell.…”

“…A recent report has in fact indirectly addressed this question and demonstrated that exposure of bovine neutrophils to IL-1ß, TNF-α and IFN-γ increased the expression of LFA-1 leading to enhanced LktA effects [29]. These and other reports demonstrating that diverse ligands capable of eliciting intracellular signals that enhance LFA-1 mediated LktA-effects [29][30][31] suggest that LFA-1 affinity and avidity changes occurring through "inside-out" signaling may affect overall LktA biological effects on the target cell. It is very likely that binding of LktA to the I-domain of the CD11a subunit may confer, through allosteric interaction, a conformation to the CD18 subunit that is compatible for LktA signaling.…”

How Mannheimia haemolytica defeats host defence through a kiss of death mechanism

“…The latter is known to decrease host defence amongst others by diminishing the activities of T lymphocytes, B lymphocytes, monocytes and macrophages [24,67] and by interfering with the host's antigen presentation machinery to evade the host's immune response in vivo [74,134]. Moreover, it has been shown that leukocyte exposure to inflammatory cytokines released in response to BHV-1 infection (interleukin-1 beta, interleukin-8, tumour necrosis factor alpha and interferon gamma) can modulate the migration and functional activation of bovine leukocytes [26,105,106]. So, when Mannheimia haemolytica enters a BHV-1 infected lung, it encounters leukocytes whose recruitment and LFA-1 expression (and hence the leukotoxin susceptibility) are increased [26,106].…”

Tilmicosin-induced bovine neutrophil apoptosis is cell-specific and downregulates spontaneous LTB4 synthesis without increasing Fas expression

“…The pathophysiology of bovine pneumonic pasteurellosis is initiated by bacterial virulence factors and the host immune response [8,18,22,30]. During infection, Mannheimia haemolytica secretes a heatlabile leukotoxin, which stimulates polymorphonuclear neutrophilic leukocytes (PMN) to release the pro-inflammatory mediator leukotriene B 4 (LTB 4 ) and forms transmembrane pores in these cells, leading to cell death [18,34].…”

“…During infection, Mannheimia haemolytica secretes a heatlabile leukotoxin, which stimulates polymorphonuclear neutrophilic leukocytes (PMN) to release the pro-inflammatory mediator leukotriene B 4 (LTB 4 ) and forms transmembrane pores in these cells, leading to cell death [18,34]. This process is exacerbated by pro-inflammatory cytokines produced by the host [30]. The release of LTB 4 results in the recruitment of additional PMN to the lung.…”