Journal article
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, Vol.141(1), pp.84-91
2011
Handle:
https://hdl.handle.net/2376/105901
PMID: 21420740
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 Lkt-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 Lkt-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 showed any difference in [Ca
2+]
i elevation when incubated with both types of Lkt preparations. Lkt did not induce any cytotoxicity or [Ca
2+]
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.
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Details
- Title
- Co-expression of ovine LPS receptor CD14 with Mannheimia haemolytica leukotoxin receptor LFA-1 or Mac-1 does not enhance leukotoxin-induced cytotoxicity
- Creators
- Rohana P Dassanayake - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USAPaulraj K Lawrence - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USADonald P Knowles - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USAWilliam C Davis - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USAWilliam J Foreyt - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USASubramaniam Srikumaran - Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA
- Publication Details
- Veterinary immunology and immunopathology, Vol.141(1), pp.84-91
- Academic Unit
- Veterinary Microbiology and Pathology, Department of
- Publisher
- Elsevier B.V
- Identifiers
- 99900546958801842
- Language
- English
- Resource Type
- Journal article