Tryptase and chymase are the major serine proteinases of skin mast cells but their biologic significance depends on their activity. In this study, we demonstrate the release of soluble activity of tryptase, but not markedly that of chymase, into skin blister fluids induced by freezing with liquid nitrogen as well as into supernatant during incubation of 8 whole skin specimens with compound 48/80 for up to 2 days followed by sonication. Incubation of 3 other skin specimens in compound 48/80 for up to 2 days revealed that the number of mast cells displaying tryptase activity decreased significantly on day 2, and the number of mast cells showing chymase activity (but not those showing chymase immunoreactivity) decreased significantly on day 1 but not thereafter on day 2. The results of 3 skin organ cultures for up to 14 days showed steady decrease in the number of tryptase-positive cells but persistence of mast cells containing chymase activity. Chymase in solution was sensitively inhibited by 0.01 mg/ml alpha1-antichymotrypsin but higher concentrations (0.3-3.0 mg/ml) were needed for inhibiting chymase on skin sections. In conclusion, after mast cell degranulation tryptase activity is substantially solubilized and it may potentially affect both local and distant skin structures. Instead, chymase is partially inactivated and the remaining chymase activity persists at the site of degranulation having only local effects.

Mast cells accumulate and persist predominantly in the upper dermis of the skin but the mechanism for this is obscure. The skin is normally exposed to external air, which is essential for the maturation of the epidermis and probably also the dermis. In order to clarify the importance of air exposure on dermal mast cells, skin organ culture at the air-liquid interface (ALI) and submerged (SM) in medium (10% fetal calf serum and Dulbecco's modification of Eagle's medium) was used to study changes in tryptase-, chymase- and Kit-positive mast cell numbers during cultivation for up to 14 days. In addition, possible apoptosis (TACS TdT in situ apoptosis detection method) in chymase-positive mast cells was studied during the culture. In the less-physiologic SM culture, the number of Kit-positive mast cells decreased rapidly on day 1-2 and tryptase-positive cells decreased markedly on day 14. This decrease in mast cell numbers can be explained by the finding that a rapid increase in the apoptosis index of mast cells was induced on day 1-2. In contrast, in the more physiologic ALI culture, the number of Kit-positive cells was sustained over 1-2 days but then decreased on day 7. In addition, tryptase-positive cells decreased steadily in number but not to the same extent as those in the SM culture. Moreover, the increase in the apoptosis index of mast cells was delayed until day 7 in the ALI culture. Addition of exogenous stem cell factor (up to 200 ng/ml) to the SM culture could not prevent the decay in tryptase- and chymase-positive cells. However, stem cell factor reduced significantly the number of Kit-positive cells already on day 2 indicating that the cells had responded. Addition of histamine (0.25 or 1 mM) or tumor necrosis factor-alpha (500 or 2000 U/ml) caused a decrease in the number of tryptase- and Kit-positive cells in the SM culture. In conclusion, a novel finding was that air exposure in the ALI culture markedly delayed the rapid apoptosis and subsequent decrease in mast cell numbers noted to occur in the SM culture. Stem cell factor could not prevent the rapid decrease in mast cell numbers. Histamine and tumor necrosis factor-alpha are possible factors promoting the decline in mast cells.

Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02-200 microg/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 microg/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-alpha is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2-20 microg/ml augmented or even potentiated this growth-inhibitory effect of TNF-alpha. The association of TNF-alpha with heparin was shown by demonstrating that TNF-alpha bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-alpha-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 microg/ml inhibited significantly by 12-13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 microg/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-alpha.

Stem cell factor plays a key role in the development of human mast cells via interaction with Kit receptor. We and other groups have previously shown that a number of cytokines can regulate the stem-cell-factor-dependent development of mast cells in vitro. In this study we investigated the effect of retinoic acid on human mast cells in vitro and in vivo. Retinoids are known to have strong modulatory effects on hematopoietic differentiation. We found that all-trans-retinoic acid, at concentrations as low as 1 nM, inhibits the stem-cell-factor-dependent differentiation of mast cells in vitro. This effect of retinoic acid was found to be on progenitor cells, whereas more mature mast cells were less affected. The use of specific agonists binding either to the RAR or the RXR nuclear receptors indicated involvement of both the RAR/RXR and RXR/RXR pathways in inhibiting mast cell differentiation. In contrast to the effects on mast cell progenitors, retinoic acid had no effect on the number of mature mast cells in skin organ cultures. Furthermore, topical treatment of normal skin with a retinoic-acid-containing cream caused an increase in the number of tryptase-positive mast cells, whereas the numbers of the major cutaneous mast cell type, tryptase- and chymase-positive mast cells, remained unaffected. Our results suggest that retinoic acid suppresses commitment of progenitor cells into the mast cell lineage and/or acts on early mast cell progenitors, whereas mature cutaneous mast cells are less susceptible to retinoic acid.

In this study, quantitative digital image analysis was utilized to measure the optical density of immunostains of involucrin at different depths in the epidermis to obtain reliable ordinal-scaled interpretations of the staining intensity. The distribution of involucrin within the epidermis was investigated in air-liquid interface and submerged skin organ cultures at different time-points. A greyscale calibration procedure to standardize the optical units was used. By the 2nd day of culture, staining of involucrin had shifted markedly towards the mid or basal epidermis. Air-liquid interface cultures showed a less intensive shift than the submerged cultures. Up to the 7th day, involucrin staining remained in the upper epidermis in the air-liquid interface cultures, though weak staining was already observed in the basal epidermis. The results suggest that air-liquid interface conditions maintained physiological conditions better than submerged conditions which result in cultures that may have to increase their involucrin synthesis to improve the barrier function against the surrounding liquid during culture. Alternatively, changes in involucrin synthesis could reflect disturbed homeostasis. Concentrating measurements on certain cell layers might give more detailed information about changes in involucrin expression. Although the detection method was used to study the histochemistry of skin, it could easily be applied to other tissues as well.