Funding sources: Supported by the Wright Foundation.Conflicts of interest: Dr Wysong serves as an unpaid scientific advisor for Castle Biosciences. Ms Lobl, Drs Clarey and Higgins, and Ms Thieman have no conflicts of interest to declare.IRB approval status: All studies were approved by the instituutional review board.

The complement factor C3 and chymase released from tryptase(+), chymase(+) mast cells may be involved in the pathogenesis of cutaneous leukocytoclastic vasculitis. To study whether mast cells contain C3 in vasculitis and whether chymase interacts with C3, cryosections from vasculitis biopsies were double-stained histochemically for C3c in tryptase(+) mast cells, as well as for chymase and vessel wall C3c, or they were treated with 5 µg/ml rh-chymase for 24 h followed by immunofluorescence (IF) analysis of C3c, IgG, IgM and IgA. The effect of rh-chymase on purified human C3, C3a and IgG was studied using SDS-PAGE electrophoresis and LAD2 mast cell cultures. The results show that 34.2 ± 17.9, 37.4 ± 15.5 and 43.4 ± 18.6 % (mean ± SD) of the mast cells express C3c immunoreactivity in the healthy skin, initial petechial (IP) and palpable purpura (PP) lesions, respectively. About 9.4-12.1 % of the chymase(+) mast cells were in apparent contact with C3c(+) vessels in IP and PP. The treatment of cryosections with rh-chymase decreased the IF staining of C3c, but not that of immunoglobulins. In SDS-PAGE, 1-10 µg/ml rh-chymase degraded the alpha- and beta-chains of C3, but did not degrade IgG. Unexpectedly, the rh-chymase treatment of C3 produced fragments that resulted in the release of tryptase and histamine from LAD2 cells. However, rh-chymase degraded C3a and consequently inhibited C3a activity on LAD2. In conclusion, mast cells can be one source for C3 in the early and late phases of vasculitis pathogenesis. However, rh-chymase degraded native C3, vessel wall C3c, and biologically active C3a. Therefore, chymase may control C3-related pathology.

In animal models of vasculitis, mast cells are essential in the pathogenesis, but their involvement in human skin vasculitis is obscure. Because tryptase and chymase are potent serine proteinases in the secretory granules of mast cells, the purpose was to examine the number of mast cells expressing tryptase and chymase during the progress of cutaneous vasculitis. These numbers were correlated with the appearance of immunoreactants (C3c, fibrin, IgM, IgA and IgG) in vessel walls. For this, skin biopsies were taken from the healthy-looking skin, initial petechial lesion (IP), and palpable purpura (PP) of the leg of patients with leukocytoclastic vasculitis (n = 10). The frozen biopsies were analysed using enzyme- and immunohistochemistry and direct immunofluorescence (IF) staining. The results show that there are no marked changes in the numbers of mast cells expressing chymase or tryptase proteins. Instead, chymase enzyme activity decreased, but the score of α1-antichymotrypsin staining increased, during the progress of vasculitis. The IF positivity of fibrin correlated positively with chymase activity (p = 0.01) and the ratio of chymase activity to tryptase protein in IP (p = 0.03), as well as with mast cells showing tryptase (p = 0.03) and chymase (p = 0.01) proteins in PP. The IF positivity of C3c correlated with the ratio of chymase activity to tryptase protein in IP (p = 0.01). In conclusion, chymase is partially inactivated in vasculitis possibly due to α1-antichymotrypsin. Several positive correlations between chymase and fibrin and/or C3c in IP or PP suggest that this enzyme is involved in the deposition of immunoreactants in the vessel wall.