Generation and functional characterization of bovine bone marrow-derived macrophages

Abstract

A culture system for bovine bone marrow derived macrophages (BBMM) was devised, starting with bone marrow cells from tibiae of calf fetuses of over 6 months gestation. Tibiae removed aseptically were sawed open, and the bone marrow was collected, washed and placed in hydrophobic (teflon) containers, thereby allowing the propagation and differentiation of macrophage lineage cells. All other cell types were negatively selected for. This results in enriched macrophage populations between 12 and 18 days of culture which were suitable for use in functional studies. The culture system was strictly serum-dependent but independent of the addition of conditioned medium containing macrophage colony-stimulating factor. The cells were mature macrophages by morphological and histochemical criteria. They expressed CD14 and CD11b, two typical macrophage markers. They were positive for CD18 and CD36 and bound monomeric murine IgG2a, indicating that a high-affinity Fc receptor for this isotype is expressed by bovine macrophages. The cells ingested erythrocytes opsonized by rabbit IgG, human IgG, bovine IgG1 and IgG2. Upon triggering with opsonized zymosan or phorbol 12-myristate 13-acetate, reactive oxygen species were generated. Upon triggering with lipopolysaccharide, BBMM expressed enhanced amounts of procoagulant activity and secreted cytokines, such as tumor necrosis factor and transforming-growth-factor-beta. These criteria identify the cultured cells as resting macrophages.