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Title: Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity

Author
item DASSANAYAKE, ROHANA - Washington State University
item LAWRENCE, PAULRAJ - Washington State University
item Knowles Jr, Donald
item DAVIS, WILLIAM - Washington State University
item FOREYT, WILLIAM - Washington State University
item SRIKUMARAN, SUBRAMANIAM - Washington State University

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2011
Publication Date: 5/15/2011
Citation: Dassanayake, R.P., Lawrence, P.K., Knowles Jr, D.P., Davis, W.C., Foreyt, W.J., Srikumaran, S. 2011. Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity. Veterinary Immunology and Immunopathology. 141(1-2):84-91.

Interpretive Summary: Mannheimia (Pasteurella) haemolytica is a bacterium that causes pneumonia in cattle, goats, domestic sheep (DS, Ovis aries) and bighorn sheep (BHS, Ovis canadensis). However, BHS are much more susceptible to M. haemolytica-caused pneumonia than other some other ruminants. The reason(s) for this susceptibility is/are not known. This bacterium produces several virulence determinants (which contribute to disease/pneumonia), of which leukotoxin (Lkt) and LPS are the major ones that contribute to the pathogenesis of pneumonia. This study was designed to explore the interactions of certain host and bacterial components to understand how their interactions contribute to the disease process. The derived data support the conclusion that expression of CD14 together with host components LFA-1 or Mac-1 does not enhance Lkt-induced cytotoxicity, whereas LPS enhances cytotoxicity by complexing with Lkt. These findings further support studies to understand the susceptibility of bighorn sheep to pneumonia caused by Mannheimia haemolytica.

Technical Abstract: Leukotoxin (Lkt) and LPS are the major virulence determinants of Mannheimia haemolytica that contribute to the pathogenesis of bovine and ovine pneumonic pasteurellosis. We have previously identified bovine and ovine CD18 as the functional receptor for Lkt. LPS complexes with Lkt resulting in increased thermal stability and enhanced cytotoxic activity of Lkt. Cellular recognition of LPS involves several different molecules including CD14. We hypothesized that expression of ovine CD14 together with LFA-1 or Mac-1 would enhance Lkt8 induced cytotoxicity. Ovine cDNA for CD14 was amplified by PCR and cloned into mammalian expression vectors. The 1122 bp cDNAs for bighorn sheep (BHS) and domestic sheep (DS) CD14 encode 373 amino acids which exhibit 99% identity with each other. Ovine CD14 plasmids were transfected either into HEK-293 cells, or previous HEK-293 transfectants 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 and the transfectants co-expressing CD14 with LFA-1 or Mac-1 did not show any significant difference in Lkt15 induced cytotoxicity when incubated with LPS complexed Lkt. In contrast, incubation of the LFA-1 or Mac-1 and LFA-1/CD14 or Mac-1/CD14 transfectants with Lkt which lacks LPS, resulted in reduced cytotoxicity. None of the above transfectants show any difference in [Ca2+17 ]i elevation when incubated with both types of Lkt preparations. Lkt did not induce any cytotoxicity or [Ca2+19 ]i elevation in ovine CD14 transfectants or parent HEK-293 cells. 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.