Possible Involvement of Intracellular Calcium-Independent Phospholipase A2 in the Release of Secretory Phospholipases from Mast Cells-Increased Expression in Ileal Mast Cells of Crohn's Disease.

Increased activity of secretory phospholipases A2 (sPLA2) type-II was previously observed in ileum of Crohn's disease (CD). Our aims were to explore the involvement of calcium-independent (i)PLA2ß in the release of sPLA2s from the human mast cell (MC) line (HMC-1) and investigate expressions of cytosolic (c)PLA2α, iPLA2ß, sPLA2-IIA and sPLA2-V in MCs of CD ileum. The release of sPLA2 was investigated in HMC-1 by immunocytochemistry and ELISA. The expression intensities of PLA2s in mucosal MCs, and the proportion of PLA2-positive MCs, were investigated in normal ileum and in ileum from patients with CD by immunohistochemistry. The calcium ionophore-stimulated release of sPLA2-IIA and sPLA2-V from HMC-1 was reduced by the iPLA2-inhibitor bromoenol lactone. All four PLA2s were detectable in mucosal MCs, both in normal ileum and in CD, but the proportion of iPLA2ß-containing mucosal MCs and the expression intensity of sPLA2-IIA was increased in CD. Results indicate that iPLA2ß is involved in the secretion of sPLA2s from HMC-1, and suggest that iPLA2ß-mediated release of sPLA2 from intestinal MCs may contribute to CD pathophysiology. Ex vivo studies on isolated mucosal mast cells are however needed to clarify the precise role of MC PLA2s in the inflammatory processes of CD.

Potential role of protease-activated receptor-2-stimulated activation of cytosolic phospholipase A(2) in intestinal myofibroblast proliferation: Implications for stricture formation in Crohn's disease.

BACKGROUND AND AIMS: Myofibroblast hyperplasia contributes to muscularis mucosae thickening and stricture formation in Crohn's disease (CD). Protease-activated receptor-2 (PAR-2) and cytosolic phospholipase A(2) (cPLA(2)) are known regulators of cell growth, but their significance in intestinal myofibroblast proliferation remain to be elucidated. The principle aims of the present study were to investigate if PAR-2 is expressed in the expanded muscularis mucosa in ileal CD specimens, if inflammatory cytokines may stimulate PAR-2 expression in intestinal myofibroblasts, and if PAR-2 and cPLA(2) may regulate intestinal myofibroblast growth. METHODS: Immunohistochemistry was used for detection of PAR-2 in ileal CD specimens. Studies on PAR-2 expression, PLA(2) activation and cell growth were performed in a human intestinal myofibroblast cell line, CCD-18Co. PAR-2 expression was investigated by RT-PCR and immunocytochemistry. PLA(2) activity was analyzed by quantification of released (14)C-arachidonic acid ((14)C-AA). Cell growth was examined by (3)H-thymidine incorporation and cell counting. RESULTS: The thickened muscularis mucosae of the CD specimens showed strong PAR-2 expression. In cultured myofibroblasts, tumor necrosis factor-α (TNF-α) up-regulated PAR-2 mRNA and protein, and potentiated PAR-2-stimulated (14)C-AA release by two known PAR-2 activators, trypsin and SLIGRL-NH(2). The release of (14)C-AA was dependent on cPLA(2). Trypsin stimulated the proliferation of serum-starved cells, and inhibition of cPLA(2) reduced normal cell growth and abolished the growth-promoting effect of trypsin. CONCLUSIONS: The results suggest that PAR-2-mediated cPLA(2) activation might be of importance in intestinal myofibroblast proliferation. The results also point to the possibility that PAR-2 up-regulation by inflammatory cytokines, like TNF-α, may modulate this effect.

Increased expression of protease-activated receptor-2 in mucosal mast cells in Crohn's ileitis.

BACKGROUND AND AIMS: Activation of protease-activated receptor-2 (PAR-2) may stimulate various events of importance in inflammatory processes, including release of inflammatory mast cell mediators. PAR-2 is frequently up-regulated during inflammatory conditions, but it is not known if the expression is altered in Crohn's disease. The aim of the present study was to investigate the ileal mucosal PAR-2 expression in Crohn's ileitis, with particular emphasis on the expression in ileal mucosal mast cells. METHODS: Surgical specimens from the distal ileum were collected from patients with Crohn's ileitis and patients with colonic cancer as controls. The overall expression of PAR-2 was investigated by Western blot, and the presence of PAR-2 expressing mucosal mast cells by immunohistochemistry and cell counting. The effect of tumor necrosis factor-α (TNF-α) on the PAR-2 expression in a human mast cell line (HMC-1) was investigated by RT-PCR and immunocytochemistry. RESULTS: In Crohn's specimens, the fraction of PAR-2-expressing mucosal mast cells was increased about 2.5 times (P<0.001; n=14) compared with specimens from control patients (n=6). No difference was found between inflamed (n=6) and uninflamed Crohn's specimens (P>0.05; n=8). Exposure to TNF-α for 48 h up-regulated PAR-2 mRNA and protein expression in the HMC-1 cell line. CONCLUSION: PAR-2 is up-regulated on ileal mucosal mast cells in Crohn's ileitis, possibly due to the action of inflammatory cytokines, such as TNF-α. This may contribute to perpetuating the inflammatory process in the intestinal mucosa in Crohn's ileitis.