Actions of mast cell tryptase on myofibroblasts : implications for inflammation and tissue modelling in the human lung

Abstract

Tryptase, the major secretory product of the mast cell is emerging as a key mediator that can modulate cell function through the activation of protease activated receptor (PAR)-2, a G protein coupled receptor.  The aim of these studies has been to investigate the ability of tryptase and other agonists of PAR-2 to alter myofibroblast and fibroblast function and to act as stimuli for inflammation and tissue remodelling in the lung.

Expression of mRNA for PAR-2 was detected in both fibroblasts and myofibroblasts by PCR, and the presence of this receptor on the cell membrane and within the cytoplasm was indicated by immunocytochemistry with specific monoclonal antibody P2A.  Functional PAR-2 on these cells was demonstrated by the ability of a peptide agonist to induce calcium flux, though surprisingly this effect was not induced by tryptase.  Mitogenic responses were stimulated with tryptase in fibroblasts and myofibroblasts, but cell proliferation was not observed under the conditions used.  Pre-incubation of tryptase with protease inhibitors, or heat inactivation of the enzyme did not consistently inhibit the actions on cells, but responses similar to those with tryptase were observed following stimulation with peptide agonists of PAR-2.

The synthesis and release of IL-6, IL-8 and GM-CSF by fibroblasts and myofibroblasts was indicated by quantitative Taqman PCR and ELISA techniques, though there was considerable heterogeneity between cell preparations in patterns of cytokine generation.  Tryptase induced increased IL-8 release from myofibroblasts (but not from fibroblasts), and there was no overall alteration in IL-6 release, or in expression mRNA for any of the three cytokines examined.  The PAR-2 agonist peptide induced increased release of IL-6 from myofibroblasts, but otherwise was found to have little effect on cytokine generation by either cell type.

Incubation of fibroblasts and myofibroblasts with tryptase provoked increased release of collagens as determined by measurement of [³H]-proline.  PCR studies suggested that collagen type III rather than type I was generated by these cells.  Matrix metalloproteinase (MMP)-2 release in both the pro and active forms was stimulated by tryptase.  A similar pattern of MMP-2 release in response to tryptase was seen with myofibroblasts.  The peptide agonist of PAR-2 failed to induce collagen synthesis in fibroblasts and myofibroblasts, and although it induced increased release of the pro-form of MMP-2 from fibroblasts, induction of the active form was not observed.

Tryptase being present in substantial quantities in the asthmatic airways could modulate myofibroblast and fibroblast behaviour, and represent an important stimulus for inflammation and tissue remodelling. However, these studies indicate that there is no simple relationship between tryptase-induced alterations in cell function and the activation of PAR-2.  It seems likely that tryptase may stimulate non-PAR-2 mediated processes as well as those in which this receptor is involved.

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