Mannheimia haemolytica leukotoxin activates a nonreceptor tyrosine kinase signaling cascade in bovine leukocytes, which induces biological effects

The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine ly mphocyte function-associated antigen I (LFA-1) and induces biological effec ts in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting a ny effects. These findings suggest that the specificity of LktA effects mus t entail both binding to LFA-1 and activation of signaling pathways which a re present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA -1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to dete rmine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to e xamine whether LktA-induced NRTK activation is target cell specific, and (i ii) to determine whether LktA-induced NRTK activation is required for biolo gical effects. We used a biologically inactive mutant leukotoxin (Delta Lkt A) for comparison with LktA. Our results indicate that LktA induces tyrosin e phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The Delta LktA mutant does not induce TP of the CD18 tail, albeit binding to bo vine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhi bitor, herbimycin A; a phosphatidylinositol 3 ' -kinase (PI3-kinase) inhibi tor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-depend ent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but no t porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+] i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus , our data represent the first evidence that binding of LktA to bovine LFA- 1 induces a species-specific NRTK signaling cascade involving PI3-kinase an d Src kinases and that this signaling cascade is required for LktA-induced biological effects.