Title: CHARACTERIZATION OF BIGHORN SHEEP (OVIS CANADENSIS) MACROPHAGES USING IMMUNOHISTOCHEMICAL, FLOW CYTOMETRIC AND FUNCTIONAL ASSAYS (POSTER PRESENTATION FOR THE 82ND CONFERENCE OF RESEARCH WORKERS IN ANIMAL DISEASE)
Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: September 21, 2001
Publication Date: N/A
Adherent peripheral blood mononuclear cells were isolated from Rocky Mountain or desert bighorn sheep (Ovis canadensis) and cultured for two weeks in glass chamber slides or in tissue culture flasks. For comparative purposes, adherent mononuclear cells isolated from domestic sheep (Ovis aries) were cultured in parallel in two experiments. Cells cultured in glass chamber slides were fixed in methanol and stained using immunohistochemical techniques for CD68, a member of the scavenger receptor family that is associated with lysosomes in macrophages and myeloid cells. Macrophages cultured in tissue culture flasks were harvested, counted and stained for subsequent flow cytometric analysis or were stimulated in vitro for 24 hr with Mannheimia (Pasteurella) haemolytica LPS. Supernatants from LPS-stimulated cultures were assayed for inducible nitric oxide (NO) activity via measurement of nitrite levels. The anti-CD68 immunoreactivity yof bighorn sheep macrophages was intense and diffuse throughout the cytoplasm. 84.23% of the cultured macrophages were CD14**+. Bighorn sheep macrophages produced significant (P<.05) levels of nitrite in response to LPS stimulation as compared to non-stimulated macrophages. The addition of an iNOS inhibitor reduced the levels of nitrite to that observed in non- stimulated cultures. In contrast, there was not a significant increase in nitrite levels following LPS stimulation of domestic sheep macrophages. The role of NO in pathogenesis is complex, having been shown to be important in clearance of certain pathogens, but responsible for tissue damage in other instances. Thus, species-specific differences in NO production may partially explain between species variance in host response to infection.