Monomeric Expression of Bovine β₂-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 β subunit of bovine β₂-integrins on leukocytes, and that the species specificity of Lkt-induced effects is resident in the β subunit CD18 and not in the α 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 [Ca²⁺], 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 [Ca²⁺] 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 β₂-integrins is the functional receptor for M. haemolytica Lkt.