cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract

Background Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, up regulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhale d allergen. Objectives The aim of this study was to characterize the sequence and distr ibution of sheep tryptase(s), to validate the sheep model of allergic lung disease. Methods Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tr yptase for partial purification and characterization of the protein. The di stribution of tryptase in skin, lung and gut was determined by immunohistoc hemistry, and compared with the distribution of sheep mast cell proteinase- 1 (sMCP-1). Results Two highly similar cDNA sequences encoding sheep tryptase were foun d, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep trypt ase from lung and gut extracts was achieved using heparin-Sepharose affinit y chromatography. Rabbit antihuman skin tryptase antiserum recognized the p utative sheep tryptase on Western blot (approximate M-r 32-34 000) and para formaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were det ected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detecte d by a specific monoclonal antibody, was present in all intestinal and gast ric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all der mal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. Conclusion In view of the structural and functional similarities of sheep a nd human tryptases, and their similarity in tissue distribution in normal s heep, the sheep lung appears to be a good model for in vivo studies relatin g to human tryptase.