Mechanisms underlying Mannheimia haemolytica leukotoxin-induced oncosis and apoptosis of bovine alveolar macrophages

Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) binds to the bovine beta2 integrins (such as LFA-1-CD11a/CD18) and leads to subsequent cellular effects in a dose dependent manner. The objectives of this study were to delineate the mechanisms that underlie LktA-induced oncosis and apoptosis and to examine the role of LktA/LFA-1 interaction in these events. The results demonstrate that LktA-induced oncosis proceeds through a LFA-1 and caspase-1 dependent pathway referred to as 'pyrotosis', as well as through a LFA-1- and caspase-1-independent pathway. LktA-induced apoptosis in alveolar macrophages involves activation of caspase-3 and engages the extrinsic and intrinsic pathways of apoptosis, with the extrinsic pathway being dependent on LFA-1 signaling and TNFalpha.     

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

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically 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 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated.     

Monomeric expression of bovine beta2-integrin subunits reveals their role in Mannheimia haemolytica leukotoxin-induced biological effects

The ruminant-specific leukotoxin (Lkt) of Mannheimia haemolytica is the key virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies by us and others indicate that M. haemolytica Lkt binds to CD18, the beta subunit of bovine beta(2)-integrins on leukocytes, and that the species specificity of Lkt-induced effects is resident in the beta subunit CD18 and not in the alpha subunit CD11. However, Lkt also binds to the CD11a subunit of LFA-1. Furthermore, antibodies specific for CD18 or CD11a inhibit signaling events leading to elevation of intracellular [Ca(2+)], tyrosine phosphorylation of the cytosolic domain of CD18, and cytolysis of bovine leukocytes. These observations underscore the need for further investigation to identify the precise subunit of bovine LFA-1 utilized by M. haemolytica Lkt as the functional receptor. For this purpose, monomeric bovine CD18 and CD11a and heterodimeric LFA-1 were expressed in the HEK-293 cell line by transfection, and the resulting transfectants were tested for susceptibility to Lkt-induced effects. All three transfectants effectively bound Lkt. However, Lkt-induced cytolysis was observed only with transfectants expressing monomeric bovine CD18 or LFA-1. Furthermore, intracellular [Ca(2+)] elevation following exposure to Lkt, which is a marker for postbinding signaling leading to cellular activation, was seen only with transfectants expressing monomeric bovine CD18 or LFA-1. These results clearly indicate that the bovine CD18 subunit of beta(2)-integrins is the functional receptor for M. haemolytica Lkt.     

Effects of lipopolysaccharide and Mannheimia haemolytica leukotoxin on bovine lung microvascular endothelial cells and alveolar epithelial cells

Bovine respiratory disease resulting from infection with Mannheimia haemolytica commonly results in extensive vascular leakage into the alveoli. M. haemolytica produces two substances, lipopolysaccharide (LPS) and leukotoxin (LKT), that are known to be important in inducing some of the pathological changes. In the present study, we examined bovine pulmonary epithelial (BPE) cell and bovine lung microvascular endothelial cell monolayer permeability, as measured by trans-well endothelial and epithelial cell electrical resistance (TEER), after incubation with LPS, LKT, or LPS-activated neutrophils. Endothelial cell monolayers exposed to LPS exhibited significant decreases in TEER that corresponded with increased levels of proinflammatory cytokines, apoptosis, and morphological changes. In contrast, BPE cells exposed to LPS increased the levels of production of inflammatory cytokines but displayed no changes in TEER, apoptosis, or visible morphological changes. Both cell types appeared to express relatively equal levels of the LPS ligand Toll-like receptor 4. However, TEER in BPE cell monolayers was decreased when the cells were incubated with LPS-activated neutrophils. Although the incubation of BPE cells with LKT decreased TEER, this was not reduced by the incubation of LKT with a neutralizing antibody and was reversed when LKT was preincubated with the LPS-neutralizing compound polymyxin B. Because BPE cells did not express the LKT receptor CD11a/CD18, we infer that contaminating LPS was responsible for the decreased TEER. In conclusion, LPS triggered changes in endothelial cells that would be consistent with vascular leakage, but neither LPS nor LKT caused similar changes in epithelial cells, unless neutrophils were also present.     

Pasteurella (Mannheimia) haemolytica leukotoxin-induced cytolysis of bovine leukocytes: role of arachidonic acid and its regulation

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether AA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells were used as an experimental system to investigate the Lkt-induced [(3)H] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis toxin, inhibitors of cytosolic phospholipase A(2)(cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, Western blot analysis revealed the presence of G(i), G(s)and G(q)type G-proteins. These results demonstrate that AA metabolites from cPLA(2)activation contribute to Lkt-induced cytolysis and G(i)type G-proteins, Ca(2+)and PKC, regulate the cPLA(2)activity.     

Mannheimia haemolytica and its leukotoxin cause macrophage extracellular trap formation by bovine macrophages

Human and bovine neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of extracellular trapping and killing of pathogens. Recently, we reported that bovine neutrophils release NETs in response to the important respiratory pathogen Mannheimia haemolytica and its leukotoxin (LKT). Here, we demonstrate macrophage extracellular trap (MET) formation by bovine monocyte-derived macrophages exposed to M. haemolytica or its LKT. Both native fully active LKT and noncytolytic pro-LKT (produced by an lktC mutant of M. haemolytica) stimulated MET formation. Confocal and scanning electron microscopy revealed a network of DNA fibrils with colocalized histones in extracellular traps released from bovine macrophages. Formation of METs required NADPH oxidase activity, as previously demonstrated for NET formation. METs formed in response to LKT trapped and killed a portion of the M. haemolytica cells. Bovine alveolar macrophages, but not peripheral blood monocytes, also formed METs in response to M. haemolytica cells. MET formation was not restricted to bovine macrophages. We also observed MET formation by the mouse macrophage cell line RAW 264.7 and by human THP-1 cell-derived macrophages, in response to Escherichia coli hemolysin. The latter is a member of the repeats-in-toxin (RTX) toxin family related to the M. haemolytica leukotoxin. This study demonstrates that macrophages, like neutrophils, can form extracellular traps in response to bacterial pathogens and their exotoxins.     

Cytokine profiles following interaction between bovine alveolar macrophages and Pasteurella haemolytica.

Pasteurella haemolytica is a gram negative bacterium frequently isolated from the lungs of calves suffering from a fibrinous pneumonic condition known as shipping fever. To understand the pathogenesis of this disease, we investigated the induction of cytokin gene expression in cultures of bovine alveolar macrophages (BAM) stimulated with heat-killed P. haemolytica. Northern blot analysis of total RNA showed that P. haemolytica induced early, abundant, and consistent synthesis of IL-1, TNF-alpha, and IL-8 mRNA. Cytokine mRNAs were detected within 1 hr post-stimulation with heat-killed P. haemolytica. IL-1 and IL-8 mRNA accumulated to high levels with increase in stimulation time, whereas TNF-alpha mRNA clearly declined by 4 and 8 h post stimulation. IL-1, TNF-alpha, and IL-8 proteins were also secreted into the culture medium of BAM stimulated with heat-killed P. haemolytica. All three proteins were detected at high levels 8 and 12 h post stimulation with P. haemolytica. BAM cells treated with bovine interferon-alpha and then stimulated with P. haemolytica produced higher amounts of IL-1, IL-8 and TNF-alpha proteins compared to BAM stimulated with P. haemolytica alone. These findings demonstrate the powerful and selective induction of cytokine mRNA and protein synthesis in BAM stimulated with heat-killed P. haemolytica and may explain certain aspects of shipping fever pathogenesis.     

Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella) haemolytica leukotoxin-induced cytolysis

Leukotoxin (Lkt) secreted by Mannheimia (Pasteurella) haemolytica is an RTX toxin which is specific for ruminant leukocytes. Lkt binds to beta(2) integrins on the surface of bovine leukocytes. beta(2) integrins have a common beta subunit, CD18, that associates with three distinct alpha chains, CD11a, CD11b, and CD11c, to give rise to three different beta(2) integrins, CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), and CD11c/CD18 (CR4), respectively. Our earlier studies revealed that Lkt binds to all three beta(2) integrins, suggesting that the common beta subunit, CD18, may be the receptor for Lkt. In order to unequivocally elucidate the role of bovine CD18 as a receptor for Lkt, a murine cell line nonsusceptible to Lkt (P815) was transfected with cDNA for bovine CD18. One of the transfectants, 2B2, stably expressed bovine CD18 on the cell surface. The 2B2 transfectant was effectively lysed by Lkt in a concentration-dependent manner, whereas the P815 parent cells were not. Immunoprecipitation of cell surface proteins of 2B2 with monoclonal antibodies specific for bovine CD18 or murine CD11a suggested that bovine CD18 was expressed on the cell surface of 2B2 as a heterodimer with murine CD11a. Expression of bovine CD18 and the Lkt-induced cytotoxicity of 2B2 cells were compared with those of bovine polymorphonuclear neutrophils and lymphocytes. There was a strong correlation between cell surface expression of bovine CD18 and percent cytotoxicity induced by Lkt. These results indicate that bovine CD18 is necessary and sufficient to mediate Lkt-induced cytolysis of target cells.     

Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin

Mannheimia (Pasteurella) haemolytica leukotoxin (LktA) is the primary virulence factor contributing to the pathogenesis of lung injury in bovine pneumonic mannheimiosis (BPM), a disease which causes major economic loss to the US cattle industry annually. Recent work from our laboratory using an antibody-based approach has shown that LktA binds to bovine LFA-1 in target cells. While this study suggests that LFA-1 might be a specific receptor, it remains to be conclusively shown that LFA-1 is sufficient to induce susceptibility to LktA. It was of interest to determine if functionally active bovine LFA-1 could be reconstituted on a LFA-1 negative cell line and reconstitute susceptibility to LktA. Here, we report the successful recombinant expression of bovine LFA-1 on the cell surface of the human erythroleukemic K562 cell line. The BoLFA-1 transductant expresses bovine CD18 and CD11a as a heterodimer. We found that LktA binds to both the CD18 and CD11a subunits of BoLFA-1 cells. Exposure of BoLFA-1 cells to LktA, induced tyrosine phosphorylation of the CD18 tail, elevation of intracellular calcium, and cytolysis. This is the first report on recombinant expression of functionally active bovine LFA-1 by transduction into an LktA-non-susceptible human cell line.     

Identification of Mannheimia haemolytica adhesins involved in binding to bovine bronchial epithelial cells

Mannheimia haemolytica, a commensal organism of the upper respiratory tract in cattle, is the principal bacterial pathogen associated with the bovine respiratory disease complex. Adherence to the respiratory mucosa is a crucial event in its pathogenesis. However, the bacterial components that contribute to this process are not fully characterized. In this study, we demonstrated that M. haemolytica adhered to bovine bronchial epithelial cells (BBEC) in vitro and that adherence was inhibited by anti-M. haemolytica antibody. Western blot analysis of M. haemolytica proteins that bind to BBEC showed a dominant protein band with an apparent molecular mass of approximately 30 kDa. Peptide sequences for the 30-kDa BBEC-binding proteins, as determined by liquid chromatography-tandem mass spectrometry, matched two M. haemolytica surface proteins: heat-modifiable outer membrane protein A (OmpA) and lipoprotein 1 (Lpp1). Western blotting showed that the 30-kDa protein band is recognized by both anti-M. haemolytica OmpA and anti-Lpp1 antibodies. Furthermore, incubation with anti-OmpA and anti-Lpp1 antibodies significantly inhibited M. haemolytica binding to BBEC monolayers. In summary, these results suggest that OmpA and Lpp1 contribute to adherence of M. haemolytica to bovine respiratory epithelial cells.     

Mapping of the binding site for Mannheimia haemolytica leukotoxin within bovine CD18

To map the site involved in Mannheimia haemolytica leukotoxin (LktA) binding and biological activity within bovine CD18, bovine x human CD18 chimeric constructs were generated and coexpressed with bovine CD11a in K562 cells. Studies with the chimeric leukocyte function-associated antigen 1 transductants demonstrate that the site required for LktA binding and biological effects resides within amino acid residues 500 and 600 of the extracellular region of bovine CD18.     

Mannheimia haemolytica leukotoxin-induced cytolysis of caprine (Capra hircus) leukocytes is mediated by the CD18 subunit of beta2-integrins

Mannheimiosis is the major respiratory disease among some ruminants, whereas it is not pathogenic for other mammals, an observation that has been attributed to a specific interaction between Mannheimia haemolytica leukotoxin (Lkt) and bovine or ovine CD18 subunit of lymphocyte function-associated antigen-1 (LFA-1) and Mac-1. We therefore hypothesized that Lkt utilizes CD18 as its receptor on caprine leukocytes as well. We have transiently transfected the β₂-integrins-deficient K-562 cell line with cDNAs encoding caprine CD11a and caprine CD18 to determine the susceptibility of the transfectants to Lkt-induced cytolysis. Flow cytometric analysis of the transfectants revealed surface expression of caprine LFA-1 and lysis by Lkt in a concentration-dependent manner whereas the parent cells were not. Moreover, K562 cells expressing caprine CD18 and human or bovine CD11a were also sensitive to Lkt whereas K-562 cells expressing caprine CD11a and human CD18 were not. Taken together, these results indicate that CD18 on caprine leukocytes serves as a receptor for Lkt.     

Arachidonic acid metabolism in bovine alveolar macrophages

The production of lipoxygenase metabolites of arachidonic acid was studied in bovine alveolar macrophages (BAM). Unstimulated macrophages produced small amounts of LTB₄ (0.2±0.2 ng/10⁶ BAM) but monohydroxyeicosatetraenoic acids (5t-, 12-, and 15-HETE) usually were not detectable. Both exogenous arachidonic acid and the calcium ionophore A23187 induced production of LTB₄, 5-, 12-, and 15-HETE, of which 60–80% was 5-HETE. Combined challenge of BAM with both exogenous arachidonic acid and A23187 was more effective in the production of these metabolites than with either stimulus alone. The generation of the peptidoleukotrienes LTC₄, LTD₄, and LTE₄ by BAM could not be detected under these in vitro conditions. Our results demonstrate that bovine alveolar macrophages produce similar lipoxygenase metabolites of arachidonic acid in response to A23187, as do human alveolar macrophages stimulated with the same agonist.     

Bacterial lipopolysaccharide induces release of tumor necrosis factor-alpha from bovine peripheral blood monocytes and alveolar macrophages in vitro

In this study, we demonstrate that freshly adherent bovine monocytes release tumor necrosis factor-alpha (TNF-alpha) in response to stimulation with bacterial lipopolysaccharide (LPS). TNF-alpha was detected using actinomycin D-treated WEHI-164 murine fibrosarcoma cells as targets in an 18 hr cytotoxicity assay. Doses of LPS from 20 ng/ml to 20 micrograms/ml were capable of inducing bovine TNF-alpha. The kinetics of TNF-alpha release from bovine monocytes demonstrated peak levels of cytotoxic activity at 1-3 hr post-LPS treatment, with a subsequent decline to background levels by 18 hr post-LPS treatment. A monoclonal antibody that neutralizes recombinant human TNF-alpha (rHuTNF-alpha) significantly reduced the cytotoxicity of LPS-stimulated bovine monocyte culture supernatants. Size exclusion high-performance liquid chromatography (HPLC) analysis of LPS-stimulated monocyte and alveolar macrophage culture supernatants resulted in a molecular weight elution profile similar to that of recombinant human TNF-alpha. These elution profiles are consistent with the presence of multimers of TNF-alpha. This is believed to be the first report of the in vitro production of bovine TNF-alpha.     

Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro

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.     

Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries) leukocytes is mediated by CD18, the beta subunit of beta2-integrins

Mannheimia (Pasteurella) haemolytica causes severe pneumonia in cattle, sheep and goats. Leukotoxin (Lkt) is the most important virulence determinant produced by this organism. Previously, we identified CD18, the beta subunit of beta(2)-integrins, as the receptor for Lkt on bovine leukocytes. Since Lkt is specific for leukocytes of cattle, sheep and goats, we hypothesized that Lkt utilizes CD18 as its receptor on ovine leukocytes as well. Therefore, the objective of this study was to transfect an Lkt-resistant murine cell line (P815) with cDNA encoding ovine CD18, and to determine the susceptibility of the transfectants to Lkt-induced cytolysis. cDNA for ovine CD18 cloned from polymorphonuclear leukocytes was transfected into P815 cells. Flow cytometric analysis of the transfectants revealed surface expression of ovine CD18, and Lkt binding. In a cytotoxicity assay, the transfectants were lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. Pre-incubation of Lkt with an anti-Lkt neutralizing antibody and pre-incubation of transfectants with an anti-CD18 antibody resulted in inhibition of cytolysis confirming the interaction between Lkt and CD18. Taken together, these results indicate that CD18 on ovine leukocytes serves as a receptor for Lkt, and that CD18 is sufficient to mediate Lkt-induced cytolysis of ovine leukocytes.     

Replication of five bovine respiratory viruses in cultured bovine alveolar macrophages

The replication of five bovine respiratory viruses in cultured bovine alveolar macrophages (BAM) was investigated. The bovine herpesvirus DN-599 strain did not cause cytophatic effect (CPE), extracellular virus and intracellular antigen were not demonstrated. Although a small number of bovine respiratory syncytial virions were consistently released and about 1 percent of the BAM were fluorescence antibody (FA) positive the virus caused no CPE. Infectious bovine rhinotracheitis virus caused CPE at an input multiplicity of about 0.5, and 5 percent of the BAM were FA positive. Both bovine parainfluenza-3 and bovine viral diarrhea viruses caused CPE, infective virions were released and considerable proportions of the BAM were FA positive.     

Lymphokine-mediated fusion and migration inhibition of alveolar macrophages

Guinea pigs were shown to produce a lymphokine termed macrophage fusion factor (MFF) which mediated the fusion of 70–80% of guinea pig or rabbit alveolar macrophages, but not guinea pig peritoneal macrophages. In the conventional migration inhibitory factor (MIF) assay, guinea pig aveolar macrophages were inhibited in their migration and large numbers of giant cells were present. There appeared to be a correlation between the titer of MFF and migration inhibition of alveolar macrophages but not with MIF titer as expressed on the peritoneal macrophage. Guinea pig MFF production was erratic and its absence from lymphokine supernatant fluids correlated with an absence of migration inhibitory activity for the alveolar macrophage. Guinea pig MIF production was more constant and high titers were invariably present. Rabbit crude lymphokine supernatant fluids containing MFF also inhibited the migration of their alveolar macrophages when measured at 24 and 48 hr during the MIF assay. Extensive numbers of giant cells were observed in the cell fan whenever migration inhibition was present. α-l-Fucose, which is known to block the receptor sites of MIF, failed to block giant cell formation in either the MFF or the MIF assay and also failed to block migration inhibition of the alveolar macrophages. The results suggest that lymphokines other than MIF can inhibit the migration of alveolar macrophages in the standard MIF assay and that the lymphokine responsible for migration inhibition and fusion of alveolar macrophages is the same lymphokine, MFF.