B-150. Co-Expression of Ovine CD14 with LFA-1 or Mac-1 Does not Enhance Mannheimia haemolytica Leukotoxin-Induced Cytotoxicity

R. P. Dassanayake, P. K. Lawrence, S. Shanthalingam, W. C. Davis, W. J. Foreyt, S. Srikumaran;
Washington State Univ., Pullman, WA.

Mannheimia haemolytica produces several virulence determinants of which leukotoxin (Lkt) is the primary one that contributes to the pathogenesis of bovine and ovine pneumonic pasteurellosis. Studies in our laboratory have identified bovine and ovine CD18 as the functional receptor for Lkt. Lipopolysaccharide (LPS) complexes with Lkt resulting in increased thermal stability and enhanced cytotoxic activity of Lkt. CD14, the well-characterized LPS receptor, is involved in the initial recognition and subsequent transfer of LPS to TLR4/MD2 complex which results in the induction of secretion of pro-inflammatory cytokines. The human β2-integrin Mac-1 (CD11b/CD18) acts in concert with CD14 and TLR4 to induce the pro-inflammatory cytokine response. Therefore, we hypothesized that recombinant expression of ovine CD14 together with LFA-1 or Mac-1 would enhance the Lkt-induced cytotoxicity. Ovine cDNA for CD14 was amplified by PCR from total RNA, and cloned into mammalian expression vectors. Plasmids carrying cDNA for CD14 were transfected either into HEK-293 cells, or previously constructed HEK-293 transfectant cell-lines stably expressing ovine LFA-1 or Mac-1. Flow cytometric analysis of transfectants confirmed the cell surface expression of CD14. The transfectants expressing LFA-1 or Mac-1 alone (single transfectants), and the transfectants co-expressing CD14 and LFA-1 or Mac-1 (double transfectants), did not show any significant difference in Lkt-induced cytotoxicity or intracellular calcium elevation. In contrast, incubation of the single and double transfectants with Lkt pre-treated with polymyxin B, a compound that is known to remove LPS, resulted in reduction of cytotoxicity in all the transfectants. Lkt did not induce cytotoxicity in HEK-293 cells expressing ovine CD14 alone. Based on these findings, we conclude that expression of CD14 together with LFA-1 or Mac-1 does not enhance Lkt-induced cytotoxicity, whereas LPS enhances cytotoxicity by complexing with Lkt.