RESUMO
The CD45 tyrosine phosphatase plays an important role in regulating T lymphocyte activation in vertebrate species. In this study we describe some molecular and functional features of the CD45 receptor molecule from the European sea bass Dicentrarchus labrax. Following immunization with fixed sea bass thymocytes, we obtained a murine monoclonal antibody (mAb) able to stain fish leucocytes both alive, by immunofluorescence of thymus and mucosal tissues, and fixed, by in situ immunohistochemistry of tissue sections. The selected IgG(2) mAb (DLT22) was able to recognise by western blots polypeptides mainly at 180 kDa and 130 kDa in thymus, spleen, intestine and gill leucocyte. Accordingly, a 130 kDa polypeptide immunoprecipitated with DLT22 from thymocytes and analysed by nano-RP-HPLC-ESI-MS/MS, gave peptide sequences homologous to Fugu CD45, that were employed for the homology cloning of a partial sea bass CD45 cDNA sequence. This cDNA sequence was employed to measure by quantitative PCR the transcription of the CD45 gene both in unstimulated and in in vitro stimulated leucocytes, showing that the gene transcription was specifically modulated by LPS, ConA, PHA, IL-1, and poly I:C. When splenocytes were stimulated in vitro with ConA and PHA, a cell proliferation paralleled by an increase of DLT22-positive leucocytes was also observed. These data indicate that the DLT22 mAb recognizes a putative CD45 molecule in sea bass, documenting the presence of CD45-like developing lymphocytes in thymus and CD45-associated functional stages of lymphocytes in this species, thus dating back to teleost fish the functional activities of these cell populations in vertebrates.
