Cloning and expression analysis of grouper (Epinephelus coioides) M-CSFR gene post Cryptocaryon irritans infection and distribution of M-CSFR+ cells

Abstract

The M-CSF/M-CSFR system plays a central role in the cell survival, proliferation, differentiation and maturation of the monocyte/macrophage lineage. In present study, we cloned the sequence of the M-CSFR cDNA from the orange-spotted grouper (Epinephelus coioides). Sequence analysis reveals that ten cysteines in the extracellular immunoglobulin-like (Ig-like) domains of EcM-CSFR are conserved in fish and mammals, its nine possible N-glycosylation sites are conserved in fish but not mammals, 7 of 8 identified mammal M-CSFR intracellular autophosphorylation tyrosine sites was found in EcM-CSFR. Real-time PCR showed that the constitutive expression level of EcM-CSFR was the highest in the spleen, less in the gill, kidney, head kidney and liver, least in the blood, skin, gut and thymus. A rabbit anti-EcM-CSFR polyclonal antibody against the recombinant EcM-CSFR extracellular domain was developed and it was efficient in labeling the monocytes and macrophages isolated from the head kidney. Immunochemistry analysis showed that M-CSFR+ cells located in all tested paraffin-embedded tissues and M-CSFR+ cell centres with the characteristic of melano-macrophage centres(MMCs) was found in the spleen, head kidney, kidney, gut and liver. All these results indicate the widespread distribution of macrophages in grouper tissues and its importance in fish immune system. In Crytocaryon irritans infected grouper, EcM-CSFR was transient up-regulated and rapidly down-regulated in skin, gill, head kidney and spleen. The possible activation mechanism of macrophage via EcM-CSFR signal transduction in the fish anti-C. irritans infection was discussed.