Study On The Expression And Function Of Protease Activated Receptors On Mast Cells

Protease activated receptor (PAR), the new member of G-protein coupled receptor (GPCR) have been identified as receptors for serine proteases. In human and mouse, four PARs have been cloned and named as PAR-1,PAR-2,PAR-3 and PAR-4 in 1991, 1994,1997 and 1998 respectively. Among them, PAR-1 is the receptor of thrombin and trypsin, PAR-2 is the receptor of trypsin, tryptase and elastase, PAR-3 and PAR-4 are the receptors of thrombin.Mast cell has a long history of being recognized as an important mediator-secreting cell in allergic inflammation. As the primary effector cell, mast cell is actively involved in the pathogenesis of both acute and chronic allergic diseases. Upon activation, mast cell can release not only its preformed but also newly generated mediators to fulfill its biological functions. Besides histamine, heparin and proteases, mast cells can synthesize and secrete a variety of cytokines such as IL-4, IL-5 and IL-6. These cytokines have been well documented for their ability to regulate cell behavior including growth, secretion, migration in physiological and pathological conditions.Studies on the role of PARs in allergic diseases showed that PARs, especially PAR-2, played an important role in allergic response. Furthermore some allergens such as dust mites and cockroach allergens were found to have the ability to activate PAR-2. However, some controversial effects of PARs were observed from the experimental study with animal model. Some studies showed that activation of PAR-2 induced the allergic inflammatory response, while others demonstrated the protective role of PAR-2 activation in allergic disease. Thus further exploration of the role of PARs in allergic diseases might be wanted.Though the potential roles of PARs expressed by allergy-related cells such as endothelial cells, epithelial cells, neutrophils, monocytes and lymphocytes had been studied, little is known about the role of PAR expressed by mast cell, the key effector cell of allergic disease. Therefore, in this study, the function of PARs expressed by mast cell will be further explored with the potential to provide experimental data for the pathogenesis of allergic diseases.PART A:Reverse transcription polymerase chain reaction (RT-PCR), flowcytometry and immunofluorescent cell staining were used to detect expression of PARs by mast cell lines P815 and MC/9 both at mRNA level and at protein level. Results:Mast cell lines P815 and MC/9 express PAR-1, PAR-2, PAR-3 and PAR-4 both at protein level and at mRNA level. Conclusion:PAR-1,PAR-2,PAR-3 and PAR-4 were expressed on mast cells.PART B:P815 mast cells were challenged with thrombin, trypsin and tryptase with or without the corresponding inhibitor hirudin, soybean trypsin inhibitor and alpha antitrypsin and leupeptin. The supernatants were collected and analyzed by enzyme-linked immunosorbent assay(ELISA) to quantify IL-4 release from P815 cells. Results:Trypsin and tryptase induced concentration-dependent IL-4 release from P815 mast cells (P<0.05). Trypsin and tryptase induced IL-4 release from the mast cells were blocked by the corresponding inhibitors (P<0.05). Whereas thrombin failed to elicit IL-4 release from P815 mast cells(P>0.05). Conclusion:Trypsin and tryptase rather than thrombin can stimulate IL-4 release from mast cells.