Localization of protease-activated receptors-1 and -2 in human mast cells: indications for an amplified mast cell degranulation cascade.

Protease-activated receptors (PARs) belong to a family of G-coupled seven transmembrane receptors that are activated by a proteolytic cleavage of their N-termini. Recent studies suggest the involvement of protease-activated receptors-1 and -2 (PAR-1, PAR-2) activators in mast cell degranulation in various physiological and pathophysiological processes in inflammatory responses. Although PAR-1 and PAR-2 activating proteases, thrombin and tryptase, have been associated with mast cell activation, PAR-1 and PAR-2 have not been localized within these cells. We describe here the localization of PAR-1 and PAR-2 in mast cells from various normal human tissues using immunohistochemical and double immunofluorescence techniques. The presence of these receptors on the membrane may explain the actions of accessible extracellular thrombin and tryptase for mast cell activation. In addition to the membrane labeling, these receptors are also localized on the membrane of the intracellular tryptase-positive granules, which may function to sustain further mast cell degranulation upon exocytosis. The localization of these two receptors in mast cells suggests a novel mechanism for controlling mast cell activation through regulation of PAR-1 and PAR-2.

A combined histochemical and double immunohistochemical labeling protocol for simultaneous evaluation of four cellular markers in restenotic arteries.

We designed an effective quadruple staining protocol that combines histochemistry (HC) and double-labeling immunohistochemistry (IHC: IHC) to stain simultaneously several different morphological features and cell types in vascular lesions. Morphometric image analysis to quantitate vascular wall thickening, lumen area, and proliferating smooth muscle cells on consecutive serial sections is adequate, but morphometric precision and dependable cellular characterization and co-localization could be obtained if analyses are performed on one tissue section. The development of a neointima in the rat carotid artery was induced by angioplasty with a balloon catheter. Tissues were stained for elastin by a modified van Gieson method, then processed for double-labeling IHC:IHC for proliferating cell nuclear antigen and smooth muscle actin followed by hematoxylin staining. The four resulting tissue stains labeled elastin filaments black, proliferating nuclei brown, smooth muscle actin red and nonproliferating nuclei blue. Our staining protocol improved the descriptive and quantitative analysis of relation between smooth muscle cell proliferation and protein expression. Also, neointimal thickening could be measured to analyze its relation to cellular proliferation. Providing one slide with four stains maximizes the information from a single slice of tissue, reduces slide preparation and analysis time, and overcomes the restriction of tissue sample availability. This technique can be applied to a wide spectrum of morphologic and morphometric studies.