It is increasingly recognized that inflammatory cytokines contribute to pulmonary tissue damage in many diseases (20
). Studies with animal models of human adult respiratory distress syndrome have helped define the contributions of inflammatory cytokines to the pathogenesis of acute lung injury (16
). These cytokines promote the production and release of additional proteins, eicosanoids, and free radicals that perpetuate the inflammatory cascade, resulting in severe lung injury (3
). There is compelling evidence that inflammatory cytokines play a central role in orchestrating the inflammatory cascade that leads to lung injury in bovine pasteurellosis (15
We recently reported that exposure of bovine PMNs to the inflammatory cytokine IL-1β resulted in increased binding of two different anti-LFA-1 MAbs, LKT binding, and cell death (25
). In the present study, we extended these observations by comparing PMNs exposed to IL-1β, TNF-α, and IFN-γ. Using an MAb (BAT75A) that recognizes LFA-1 on bovine cells, we observed that all three cytokines increased the percentage of cells that stained positive for LFA-1. IL-1β increased LFA-1 staining most rapidly (within 15 min). Likewise, LKT binding and cytotoxicity also increased within 15 min. These results suggest that IL-1β may play an important role in the early response to M. haemolytica
LKT. Although we cannot exclude the possibility that exposure to different cytokine concentrations might further alter the PMN response to LKT, we did not observe differences in the responses of PMNs that were incubated with the cytokines at concentrations of 25 to 500 ng/ml. The species of origin of the cytokines might also be a consideration, since the IL-1β and IFN-γ that we used were recombinant bovine cytokines and the TNF-α used was a recombinant human cytokine. However, we have shown previously that recombinant human TNF-α stimulates bovine PMNs in vitro (38
). Thus, we believe that our data likely reflect the general response of bovine PMNs to these cytokines. Concomitant with increased LFA-1 staining of cytokine-exposed bovine PMNs, the cells exhibited an increased ability to bind M. haemolytica
LKT and increased susceptibility to the cytotoxic effects of M. haemolytica
The results of the present study provide additional evidence that increased expression or conformational activation of LFA-1 on bovine PMNs is important for the biological effects of LKT. First, we observed a direct relationship between LFA-1 staining of bovine PMNs and LKT binding (Fig. ). Second, the cytotoxic effect of LKT was diminished when the cells were incubated with an anti-LFA-1 MAb (BAT 75A) before the LKT was added (Fig. ). Our study corroborates the results reported by other investigators, who observed reductions in LKT binding and cytotoxicity following addition of a β2
-integrin MAb (1
) and noted that PMNs from animals deficient in β2
-integrins exhibited diminished LKT binding and cytotoxicity (17
). However, we cannot exclude the possibility that there are other receptors for LKT, since addition of the anti-LFA1 MAb did not totally block LKT cytotoxicity. This was particularly true when the PMNs were incubated with IFN-γ before exposure to LKT (Fig. ).
It has been reported previously that bovine leukocytes exposed to M. haemolytica
LKT undergo apoptosis in vitro (41
). Recently, we reported that lungs from cattle experimentally infected with M. haemolytica
exhibited increased numbers of TUNEL-positive (presumably apoptotic) cells (24
). LKT-mediated apoptosis of cells responsible for defense of the lungs might increase the severity of bovine pasteurellosis. In the present study we observed that incubation with the inflammatory cytokines IL-1β, TNF-α, and IFN-γ increased LKT-mediated apoptosis of bovine PMNs, as assessed by PI staining and caspase-3 activation (Fig. ). If similar events occur in the inflammatory milieu generated by viral infection of the lung, this could reduce host defense against pasteurellosis.
There are several limitations to our study. First, we used a partially purified LKT preparation. However, we have shown previously that the binding and cytotoxicity of partially purified LKT can be blocked by the anti-LKT MAb MM601 (5
). The data suggest that the LKT, not simply contaminating LPS or other metabolic products, is required for the biological response. In the present study, we observed that heat (100°C for 15 min) destroyed the response to LKT, and pretreatment with polymyxin B did not reduce the response to LKT (data not shown). We have previously demonstrated that LKT from a noncytolytic M. haemolytica
mutant exhibits little or no binding to bovine PMNs (25
). These controls suggested that LPS contamination by itself does not account for the results obtained in our study. A second limitation is that we used only one MAb (BAT75A) to evaluate LFA-1 expression on bovine PMNs by flow cytometry. This antibody has been used by a number of investigators to detect LFA-1 expression on bovine cells, and workers in our laboratory and other workers have shown that it reduces LKT binding to bovine leukocytes and LKT cytotoxicity for these cells (17
). Using MAbs BAT75A and HI111 in Western immunoblotting provided additional evidence that bovine PMNs have increased LFA-1 expression after cytokine exposure. However, these antibodies do not provide evidence of the state of LFA-1 activation, since they recognize LFA-1 on the surfaces of stimulated and unstimulated PMNs. This is an important consideration, since lymphocytes can modulate LFA-1 binding to its ligands without altering the numbers of LFA-1 molecules on the cell surface. LFA-1 molecules can change from an inactive state to an active state in two ways: (i) by altering the affinity of the individual integrin molecule by conformational changes and (ii) by increasing the avidity because of clustering of many LFA-1 molecules in the membrane (12
). Thus, our data might reflect increased LFA-1 expression on the cell surface or an increase in the affinity or avidity of LFA-1 for both the BAT75A MAb and LKT. Finally, in our real-time PCR experiments we observed increased CD18 mRNA expression that corroborated our flow cytometry and Western immunoblot data. However, because CD18 is common to all β2
-integrins, we cannot exclude the possibility that we were detecting expression of other β2
-integrins (CD11b, CD11c, or CD11d) besides LFA-1.
In summary, the results of this study provide evidence that stimulation of bovine PMNs with the inflammatory cytokines IL-1β, TNF-α, and IFN-γ can enhance the biological response of these cells to M. haemolytica
LKT by increasing cell surface expression or conformational activation of LFA-1. We recently reported that cattle experimentally infected with bovine herpesvirus 1 had LFA-1 up-regulated on their peripheral blood leukocytes, which in turn resulted in increased LKT binding and killing of these cells (26
). If similar cytokine activation occurs during viral infection in vivo, the data might explain, in part, the virus-bacterium synergism observed in bovine pasteurellosis.