Mast cell chymase degrades fibrinogen and fibrin.

BACKGROUND: The accumulation of immunoreactants and fibrinoid necrosis of postcapillary vessel walls are common pathological features of cutaneous immune complex vasculitis. In more advanced lesions, these immunoreactants are subject to proteolysis. Mast cell chymase is a powerful enzyme that can degrade several substrates including the extracellular matrix. Heparin can influence the catalytic properties of chymase. OBJECTIVES: To study the effects of recombinant human (rh) chymase on fibrinogen, coagulation and fibrinolysis, and to relate these effects to the pathogenesis of vasculitis. METHODS: The colocalization of chymase and fibrin in vasculitis specimens was analysed by immunohistochemical double staining. Fibrinogen and fibrin were treated with rh-chymase and the effects were studied in vitro by sodium dodecylsulfate polyacrylamide gel electrophoresis and a variety of clotting and fibrin gel experiments. The effects of rh-chymase on vasculitis cryosections were analysed by direct immunofluorescence. RESULTS: Chymase-positive mast cells were associated with fibrin-positive vessels in vasculitis cryosections. Rh-chymase degraded the alpha-, beta- and gamma-chains of fibrinogen, while heparin enhanced the degradation of the beta-chain. Rh-chymase pretreatment of fibrinogen prolonged thrombin-induced clotting time. Fibrinogen degradation products induced by rh-chymase increased the clotting time of human plasma. Rh-chymase degraded fibrin gel prepared from fibrinogen or human plasma. Immunofluorescence staining positivity of fibrin in vasculitis cryosections decreased after pretreatment with rh-chymase for 24 h, and heparin enhanced this effect. CONCLUSIONS: Mast cell chymase may constitute a previously unrecognized endogenous anticoagulant and fibrinolytic enzyme, and may be involved in the clearance of fibrin from vessel walls in aged vasculitis lesions.

OX40 ligand and OX40 are increased in atopic dermatitis lesions but do not correlate with clinical severity.

BACKGROUND: The interaction between the OX40 ligand (OX40L) and OX40 has been suggested to have pathogenetic significance in atopic dermatitis (AD). OBJECTIVE: The purpose of this study was to investigate the expression and relevance of OX40L and OX40 in AD skin. METHODS: OX40L and OX40 were stained immunohistochemically on the cryosections of the lesional and non-lesional skin of 17 subjects with moderate-to-severe AD and of 10 patients with psoriasis vulgaris. Phorbol myristate acetate (PMA) stimulated keratinocytes and cell membrane preparations from PMA-stimulated keratinocytes or LAD-2 mast cells were incubated with peripheral blood mononuclear cells (PBMC) in the presence or absence of blocking monoclonal antibodies to OX40L, CD30L or ICAM-1. RESULTS: We show for the first time that the staining intensity of OX40L and the number of OX40(+) cells are significantly greater in the lesional dermis than in the healthy-looking dermis in AD (P < 0.001 in both comparisons) and also in psoriasis (P = 0.01 and P < 0.001 respectively), but neither molecule correlate significantly with the clinical severity of AD. Living keratinocytes and cell membranes from LAD-2 mast cells and keratinocytes increased the PBMC proliferation response. Anti-OX40L antibody inhibited, in a similar fashion as anti-ICAM-1 and anti-CD30L, PBMC proliferation induced by LAD-2 membranes, but stimulated that induced by keratinocytes. CONCLUSION: Our findings provide evidence for the involvement of OX40 and OX40L in the pathogenesis of AD though they are not specific to AD and in vitro results suggest complex interaction.

Experimentally induced psoriatic lesion associates with interleukin (IL)-6 in mast cells and appearance of dermal cells expressing IL-33 and IL-6 receptor.

Mast cells are involved in the development of psoriatic lesion, but it is not known how mast cells are activated or whether mast cell cytokines are expressed during the lesion development. In this study, the Köbner reaction was induced in uninvolved psoriatic skin of 18 patients using the tape-stripping technique, and a sequence of biopsies was collected at 0 days, 2 h and 3 days or at 0 days, 1 day and 7 days for histochemical analysis. Eight patients developed the Köbner reaction verified at the follow-up visit 2-2·5 weeks later. No significant differences were observed in total tryptase(+) mast cells, psoriasis area and severity index and age/sex. Instead, the percentage of tryptase(+) mast cells showing interleukin (IL)-6 immunoreactivity was significantly higher in biopsies from Köbner-positive patients than in those from Köbner-negative patients. IL-33 is a known inducer of IL-6 in mast cells, and the number of IL-33(+) cells increased significantly in Köbner-positive dermal skin at days 3-7. The number of dermal cells with IL-6 receptor (IL-6R, CD126) also increased in Köbner-positive skin at days 3-7. Unexpectedly, the number of IL-6R(+) cells was even higher in Köbner-negative skin at days 3-7. In the chronic plaque of 10 other psoriatic patients, the numbers of IL-6(+) mast cells and dermal cells showing IL-6R were higher than those in the non-lesional skin. In conclusion, the positive Köbner reaction is associated with IL-6 in mast cells and appearance of IL-6R(+) and IL-33(+) dermal cells. This suggests that a previously unrecognized vicious circle may develop in the early psoriatic lesion.

Role of mast cells and sensory nerves in skin inflammation.

Mast cells are powerful inflammatory cells which are in close functional and anatomical association with sensory nerves in the skin. During psychological stress the neuroendocrine system and peripheral sensory nerves are activated leading to release of mediators, such as neuropeptides, neurotrophins, corticotropin-releasing hormone and a-melanocyte-stimulating hormone, which are capable of activating mast cells. On the other hand, mast cell mediators released, e.g. histamine, tryptase and nerve growth factor, can in turn excite and stimulate surrounding neuropeptide-containing C-fibers possibly resulting in feedforward loop and potentiation of neurogenic inflammation. In these mechanisms, proinflammatory cytokines and chemokines are released from mast cells. In chronic skin diseases, psoriasis, atopic dermatitis and palmoplantar pustulosis, the contacts between tryptase-positive mast cells and sensory nerves are increased in number, which provides the morphological basis for increased mast cell - sensory nerve interaction in chronically inflamed skin. Hence, in this review the current understanding of the role of cutaneous mast cells and sensory nerves and their activation in psychic stress is discussed.

The increase in tryptase- and chymase-positive mast cells is associated with partial inactivation of chymase and increase in protease inhibitors in basal cell carcinoma.

BACKGROUND: In basal cell carcinoma (BCC), mast cells accumulate in the peritumoral stroma. The serine proteinases tryptase and chymase are the major mediators in mast cell granules and they may exert their enzymatic activity in the BCC lesion by inducing matrix remodeling and epithelial cell detachment. OBJECTIVE: To analyse the numbers of mast cells showing tryptase enzyme activity, chymase enzyme activity and chymase immunoreactivity as well as the presence of chymase inhibitors alpha(1)-antichymotrypsin (alpha(1)-AC), alpha(1)-proteinase inhibitor (alpha(1)-PI) and squamous cell carcinoma antigen-2 (SCCA-2) in BCC. METHODS: Eleven biopsies were taken from the lesion and healthy-looking skin of 10 patients with superficial spreading BCC. The frozen biopsies were analysed enzyme- and immunohistochemically, and a sequential double-staining method was applied. RESULTS: In the BCC lesion, the number of mast cells with tryptase activity and chymase immunoreactivity was significantly increased by 2.2- to 2.3-fold. Practically all tryptase-immunopositive cells contained tryptase activity although occasional tryptase-immunopositive cells (about 1% of total) revealed no activity. However, the ratio of cells with chymase activity to those with chymase immunoreactivity was significantly decreased from 49 +/- 19% in the healthy skin to 33 +/- 19% in the BCC lesion. Instead, the percentage of mast cells displaying alpha(1)-AC or alpha(1)-PI immunoreactivity was significantly increased by 1.7-fold in the BCC lesion. SCCA-2 expression was strongly increased in the malignant BCC epithelium but mostly in the suprabasal layers. CONCLUSIONS: Tryptase- and chymase-positive mast cells (MC(TC)) increased in the BCC lesion. However, chymase is partially inactivated, possibly by the effective chymase inhibitors alpha(1)-AC and alpha(1)-PI. SCCA-2 increased in BCC, but was localized mostly to the suprabasal layers, and thus it seems not to be crucial in inhibiting chymase.

Mast cell tryptase and chymase in chronic leg ulcers: chymase is potentially destructive to epithelium and is controlled by proteinase inhibitors.

BACKGROUND: Numerous mast cells are present in chronic leg ulcers. Tryptase and chymase are the major mediators of mast cells, but their significance is mostly dependent on their activity. In addition, the proteinases may affect ulcer epithelialization. OBJECTIVES: To study levels and activity of tryptase and chymase in wash samples and biopsies from chronic leg ulcers and the possible effect of these proteinases on keratinocyte growth and adherence. METHODS: Wash samples were taken from 16 patients and a superficial shave biopsy was taken in eight of these patients; a second biopsy series was obtained from the edge of chronic venous leg ulcers (n = 6). RESULTS: Significant levels of soluble tryptase activity and histamine, but low levels of chymase activity, were measured in wash samples from chronic ulcers. No tryptase-inhibiting activity, but clear chymase-inhibiting activity, was detected in the wash samples. In superficial wound bed biopsies, relatively marked levels of chymase activity together with histamine and tryptase activity were detected. In the second biopsy series, about 80% of the mast cells belonged to the MC(TC) type (tryptase- and chymase-immunopositive). However, about 55-61% of the chymase-immunopositive cells displayed chymase activity and 64 +/- 17% of the tryptase-positive cells revealed immunoreactivity of alpha(1)-antichymotrypsin. As the activity of chymase and tryptase was detected in the ulcer base in a ratio of 1:8, a preparation containing both chymase and tryptase was partially purified from human skin yielding a similar activity ratio of 1:11-13. Treatment of fibronectin-coated plastic surfaces with this preparation decreased the adherence of cultured human keratinocytes, this effect being attributable mainly to chymase. In 2-day cultures using growth factor/serum-deficient low- or high-calcium medium, the tryptase-chymase preparation inhibited the slow growth and at higher concentrations it even induced detachment of keratinocytes. This effect was attributed to chymase, and it was partially regulated by heparin and histamine. CONCLUSIONS: Even though chymase is partially inactivated in chronic leg ulcers, accumulated mast cells in the close proximity of the epithelium edge and their chymase may impair keratinocyte adherence and migration.

Study of innervation, sensory neuropeptides, and serotonin in murine contact allergic skin.

Density of nerve fibers, axonal growth, calcitonin gene-related peptide (CGRP), and substance P, and serotonin immunoreactivity as well as concentration were all determined in a murine model of contact allergy. Female Balb/c mice were sensitized on the back with oxazolone and 6 days later challenged with the same antigen on the dorsal surface of the ears, while control mice received the vehicle only. Then, 24 hr postchallenge, one ear was processed for immunohistochemical staining, while the other was frozen and processed for gas chromatography-mass spectrometry or radioimmunoassay (RIA). Protein gene product 9.5 (PGP 9.5) positive nerve fibers showed a tendency to increase in inflamed ears versus control ears in epidermis as well as the dermis. Growth-associated protein-43 (GAP-43) positive fibers in the epidermis were increased (p < .01) in inflamed ears, compared with control ears, as was the case for the dermal fibers, indicating increased axonal growth. Total (epidermis and dermis) numbers of CGRP and substance P positive nerve fibers tended to increase in the inflamed skin in contrast to control skin. In contrast, RIA demonstrated a lower (p < .05) concentration of CGRP in the inflamed ears compared with controls and a tendency for substance P to decrease in concentration in eczematous ears versus controls. There was no difference in serotonin concentration, or in the number of serotonin positive mast cells, between the inflamed and control skin, whereas semiquantification of serotonin positive platelets showed an increase in the inflamed (+/+) compared with control ears (+). Our results indicate that 24 hr after being challenged with the antigen, at the peak of murine skin inflammation, axonal growth, sensory neuropeptides, as well as serotonin may be involved.

Release of soluble tryptase but only minor amounts of chymase activity from cutaneous mast cells.

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.

Inhibition of keratinocyte growth in cell culture and whole skin culture by mast cell mediators.

Mast cells are suggested to participate in regenerative processes, but their influence on epithelialization and wound healing has not been well studied. Since mast cells can be found in contact with epidermis in chronic inflammatory skin diseases and venous ulcers, the effect of mast cells on keratinocyte growth was studied. Keratinocytes were cultured in serum-free conditions with (complete medium) or without (basal medium) epidermal growth factor (EGF) and bovine pituitary extract (BPE) to reach subconfluence in a 24-well plate, and the cells were treated with different mast cell mediators histamine, heparin and tryptase, or lysate from HMC-1 cells, a human leukemic mast cell line. Whole skin cultures were used as a model for in vitro wounds to study the effect of mast cells on epithelial outgrowth from skin specimens. Histamine inhibited 3H-thymidine incorporation of keratinocytes dose-dependently by 29% at 1 mM, and 89% at 5 mM histamine. In whole skin culture, histamine inhibited epithelial outgrowth dose-dependently by 64% already at 0.1 mM histamine and maximally (91%) at 1 mM histamine. Heparin inhibited 3H-thymidine incorporation dose-dependently by up to 33% at 2 microg/ml in the absence, but not in the presence, of EGF/BPE. In contrast, in whole skin culture, heparin first inhibited the epithelial outgrowth by up to 27% at 2 microg/ml, but then reversed the inhibition to 30% stimulation at 200 microg/ml. Skin tryptase (0.0285 to 2.85 microg/ml) with or without heparin (0.5 to 20 microg/ml) did not affect thymidine incorporation in keratinocytes. Lysate from HMC-1 cells, but not that from control, neuroblastoma cells, inhibited 3H-thymidine incorporation in keratinocytes dose-dependently, and maximal (47%) inhibition was reached with 16,700 lysed HMC-1 cells/ml. In whole skin culture, HMC-1 lysate inhibited the epithelial outgrowth by up to 36% at 67,000 lysed cells/ml. The results show that mast cells and their mediators are inhibitory to keratinocyte 3H-thymidine incorporation and epithelial outgrowth in vitro, although, the inhibitory effect of histamine was seen at high concentrations suggesting a requirement for close morphologic vicinity of mast cells to keratinocytes. Thus, mast cells are assumed to control epidermal regeneration and to impair epithelialization of chronic ulcers.

Modulation of the immediate allergic wheal reaction in the skin by drugs inhibiting the effects of leukotriene C4 and prostaglandin D2.

OBJECTIVE: To study the effect of nordihydroguaiaretic acid (NDGA), zafirlukast and indomethacin on the size of the size of the allergic prick-test wheal. METHOD: In the first part of the study, NDGA and indomethacin, as well as the mepyramine control (10 micrograms/ml and 100 micrograms/ml), were injected intracutaneously 10 min before prick-testing with the cow dander allergen in 51 sensitised atopic subjects. In the second part, five other subjects were prick-tested with several allergens followed by administration of 40 mg zafirlukast or 100 mg indomethacin and re-prick-testing 2 h or 4 h later. RESULTS: The intracutaneous indomethacin at both concentrations enlarged the wheal by 27 +/- 50% and 29 +/- 51% (P < 0.02, n = 51), respectively. Likewise, the peroral indomethacin significantly increased the wheal area by 17 +/- 30% (P = 0.035, n = 5). Neither intracutaneous NDGA in 51 subjects nor peroral zafirlukast in 5 subjects had marked effects on the size of the prick-test wheal. As expected, mepyramine (10 micrograms/ml) decreased the wheal area by 33 +/- 32% (P < 0.001, n = 51), but 14 subjects did not show any decrease after administration of this H1-antihistamine. CONCLUSION: The prostaglandin synthesis inhibitor (indomethacin) augments the prick-test wheal, but the leukotriene synthesis inhibitor (NDGA) and leukotriene C4 antagonist (zafirlukast) have no marked effects on the size of the prick-test wheal.

The release of histamine is associated with the inactivation of mast cell chymase during immediate allergic wheal reaction in the skin.

BACKGROUND: Chymase released by mast cells can participate in the immediate allergic wheal. However, chymase may be susceptible to inactivation by protease inhibitors during degranulation. OBJECTIVE: To study the inactivation of chymase and the release of histamine in the immediate allergic wheal reaction. METHODS: Ten sensitive atopic subjects were prick-tested with the cow dander allergen, and skin biopsies were taken from the control skin and from the challenge site at 30 and 120 min. Tryptase (Tact) and chymase (Cact) activities in mast cells were measured enzyme-histochemically. Sequential double-staining was used to demonstrate the activity and immunoreactivity (Cprot) of chymase in the same mast cell as well as alpha1-proteinase inhibitor (alpha1-PI) and alpha1-antichymotrypsin (alpha1-AC) in Tact+ cells. Skin microdialysis was used to monitor histamine release after the allergen challenge for up to 120 min RESULTS: The numbers of Tact+ and Cact+ cells were already maximally decreased at 30 min by 37 +/- 17% and 61 +/- 31%, respectively (mean +/- SD, P < 0.0001). At the same time the Cact+/Cprot+ ratio decreased from 82 +/- 15% to 43 +/- 16% (P < 0.0001). The cumulative histamine release at 30 min correlated negatively with the Cact+/Tact+ (P = 0.047) and Cact+/Cprot+ (P = 0.024) ratios, but positively with the decrease in the number of Cact+ cells (P = 0.024). These data indicate that the higher the histamine release the lower the chymase activity. Also the number of Tact+ cells in the control skin correlated positively with the cumulative histamine release at 120 min (P = 0.043). In the control skin, 95 +/- 6% and 76 +/- 8% of the Tact+ cells displayed alpha1-AC and alpha1-PI, respectively. CONCLUSION: In addition to extensive degranulation of mast cells, chymase is also rapidly inactivated after the allergen challenge, possibly by pre-existing chymase inhibitors in the mast cells. This inactivation is associated with the release of histamine.

Interleukin-4-positive mast cells are highly associated with the extent of immediate allergic wheal reaction in the skin.

BACKGROUND: In addition to histamine, mast cells contain other potent mediators which can contribute to the allergic wheal reaction in the skin. METHODS: To study the association of tryptase-, chymase-, and interleukin-4 (IL-4)-positive mast cells with the size of the prick-test wheal reaction, 50 sensitive atopic subjects were prick-tested with the cow-dander allergen on the forearm skin, and the wheal area was measured. A corresponding site of intact healthy-looking skin was biopsied and examined enzyme-histochemically for tryptase and chymase. A double-staining method was used to demonstrate the immunoreactivity of IL-4 and chymase inhibitors (alpha1-proteinase inhibitor and alpha1-antichymotrypsin) in mast cells. The levels of total and cow-specific immunoglobulin E (IgE) were measured in serum. RESULTS: The number of tryptase- and chymase-positive mast cells or those containing chymase inhibitors revealed no correlation with the wheal reaction. In contrast, both the percentage and the number of IL-4-positive mast cells showed significant positive correlation with the wheal size per se (P<0.0001), as well as with the ratio of the wheal size by cow allergen to that by histamine control (P<0.003). In addition, tryptase-, chymase-, and IL-4-positive mast cells correlated with total IgE, but not with specific IgE, levels, and they showed no relation to the clinical manifestation of atopic disease, asthma or atopic dermatitis. CONCLUSIONS: The novel finding was that IL-4-positive, but not tryptase- and chymase-positive, mast cells are intimately associated with the extent of the prick-test wheal.

Bee venom induces high histamine or high leukotriene C4 release in skin of sensitized beekeepers.

Histamine is the principal mediator released in the skin during immediate bee venom allergy but the significance of cysteinyl leukotrienes in these reactions is not known. We measured skin histamine and cysteinyl leukotriene release induced by bee venom in six sensitized beekeepers with the skin microdialysis technique. The skin was dialyzed for 2 h after skin prick test with bee venom, and the release of histamine and leukotriene C4 (LTC4) into the microdialysis fractions was measured. Leukotriene E4 (LTE) and methylhistamine excretion into the urine was assayed and whole blood histamine release test was performed. The release of histamine in the skin was variable: either high delayed, high immediate and delayed, weak release or no marked release. The histamine releasability in the skin correlated with that in whole blood. The three subjects with low histamine release exhibited high LTC4 release in the skin as well as high LTE4 excretion into the urine. Thus, the histamine and LTC4 releases were inversely associated with each other. These differences may explain the variation in the clinical reaction by bee stings in sensitized beekeepers.

Release of histamine and leukotriene C4 in immediate allergic wheal reaction as measured with the microdialysis technique.

Other mediators as well as histamine can contribute to the allergic wheal reaction. In this study, the microdialysis technique was used to monitor the release of histamine, leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in prick-test wheal reactions induced by cow dander allergen. Of 31 atopic subjects, 25 showed detectable histamine release that correlated significantly with the number of tryptase-positive mast cells and serum cow-specific IgE but not with the wheal size. Detectable LTC4 release was shown by 16 of 18 subjects, but PGD2 release was shown by only 7 of 17 subjects, and neither mediator was associated with tryptase-positive mast cells, IgE levels or wheal size. An inverse association between histamine release and LTC4 release in these 18 subjects was found rather than a direct correlation. With advancing age of the subject histamine release (n = 31) tended to decrease, although insignificantly, but LTC4 release (n = 18) and sensitivity to histamine prick increased significantly, which seemed to parallel the changes in the wheal size induced by cow allergen. In conclusion, the results showed that the release of histamine, LTC4 or PGD2 alone cannot explain the extent of the wheal reaction. In addition, the amount of histamine released was not related to the amount of LTC4 released, but rather an inverse association existed between these mediators.

Alterations in mast cells showing tryptase and chymase activity in epithelializating and chronic wounds.

Mast cells can be found in contact with epidermis in certain circumstances; especially in chronic inflammatory skin diseases and chronic ulcers, but the significance of this association is obscure. In this study, the association of mast cells with wound healing was studied by counting mast cells in the wound edges at different stages after wounding the donor site skin for pinch-grafting. Chronic venous leg ulcers were biopsed for comparison. Tryptase- and chymase-positive mast cells were stained enzyme-histochemically for active proteinases. Both the number of tryptase-positive, i.e. total mast cells, and chymase-positive mast cells decreased during wound healing, but only the change in chymase-positive mast cells was statistically significant (P< or =0.03) the maximal decrease being 63% on day 7. No mast cells could be found in the vicinity of epithelialization margin. In venous leg ulcers, significantly more mast cells were present in the perilesional skin near the epithelium margin than in the wound bed (P=0.03), and mast cells were also seen in close contact with the basement membrane. Immunoreactivity for IL-4 and TNF-alpha in mast cells was studied to see if either of these molecules was associated with wound healing. In normally healing wounds, only a minority of mast cells were immunoreactive for these cytokines and no change in positive mast cell numbers could be seen during wound healing. In chronic wounds, IL-4 was absent in mast cells, and TNF-alpha positive mast cells were present only in perilesional skin and in small numbers. These results show that mast cells especially chymase-positive - decrease in number and can not be found in the epithelialization zone in normal wound healing, whereas tryptase-positive mast cells are associated with delayed wound healing and epithelialization in chronic wounds. Thus it seems, that mast cells attempt to control hyperproliferation of epidermis in chronic wounds.

Focal dermal-epidermal separation and fibronectin cleavage in basement membrane by human mast cell tryptase.

Mast cell proteases are believed to participate in the basement membrane destruction in blistering diseases. Thus, normal human skin specimens were incubated with purified human skin tryptase or compound 48/80 (a mast cell degranulator) for up to 24 h. Thereafter, the specimens were studied immunohistochemically. Tryptase caused, in the presence and absence of 1,10-phenanthroline, focal dermal-epidermal separation above laminin and almost complete disappearance of the staining of the extra domain A region of cellular fibronectin in and beneath the basement membrane. The immunopositivity of the cell-binding region of fibronectin, laminin, and collagens IV and VII, however, was unaltered. Compound 48/80 induced almost complete dermal-epidermal separation above intact laminin and only focal reduction in the extra domain A region of cellular fibronectin staining. These alterations by compound 48/80 were prevented partially by Nalpha-p-tosyl-L-lysine chloromethyl ketone or 1,10-phenanthroline alone but completely when both inhibitors were present suggesting the involvement of tryptic serine proteinases, probably also tryptase, and metalloproteinases. Preventive effect of N-tosyl-L-phenylalanine chloromethyl ketone was weak suggesting minor function of chymotryptic serine proteinases. When tryptase was incubated with heparin and pure plasma fibronectin, an abrupt decrease in the adherence of cultured keratinocytes on to plastic surface coated with these substances and a gradual plasma fibronectin cleavage to 173, 161, and 28 kDa fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis were found. In conclusion, tryptase can cause focal dermal-epidermal separation above laminin in skin specimens but it is not known to what extent the decreased keratinocyte adherence in vitro and fibronectin cleavage are related to this dermal-epidermal separation.

Mast cells in developing subepidermal bullous diseases: emphasis on tryptase, chymase and protease inhibitors.

The possible involvement of mast cell tryptase and chymase in subepidermal bullous diseases was studied enzyme-histochemically in specimens from erythematous and vesicular skin and from non-involved skin of patients with dermatitis herpetiformis, bullous pemphigoid, erythema multiforme, infective bullous eruption and linear IgA dermatosis. Patients with pemphigus were biopsied for comparison. The immunoreactivity of chymase inhibitors, alpha1-proteinase inhibitor (alpha1-PI) and alpha1-antichymotrypsin (alpha1-AC), in mast cells was demonstrated using the sequential double staining method. Tryptase-positive mast cells were unchanged or only slightly increased in number in erythematous lesions and slightly decreased in blistering skin compared with healthy-looking skin. Only occasionally were mast cells seen in apparent contact with the basement membrane zone. Chymase-positive mast cells and the chymase/tryptase ratio steadily decreased during the development of the lesions in each subepidermal bullous disease. The percentage of alpha1-PI+ and/or alpha1-AC+ mast cells increased simultaneously, which could explain the disappearance of chymase activity. Similar results were obtained regardless of the bullous disease. The results were also similar in pemphigus, which is an intraepidermal bullous disease. In conclusion, these results show significant alterations in mast cell chymase and protease inhibitors in a range of different bullous diseases, suggesting mast cell involvement. The apparent inactivation of chymase could be due to the action of chymase inhibitors detected in numerous mast cells. However, these alterations probably reflect general inflammation rather than a specific reaction in a certain bullous disease.

Suction blister formation in skin after acute and repeated mast cell degranulation.

Mast cells and their proteases are thought to participate in the development of skin blisters in various pathological conditions. In this study, suction blistering was used as an experimental model to evaluate the significance of mast cells in blister formation after pre-treatment of normal skin with intradermal injections of 100 microg/ml compound 48/80 (a mast cell degranulator) or with 0.1% capsaicin cream. Tryptic and chymotryptic enzyme activities in blister fluids were measured with sensitive p-nitroanilide substrates. Repeated injections of compound 48/80 once a day on 3 or 5 consecutive days or capsaicin applications 3 times a day for 7 or 10 days were used to induce mast cell degranulation and inflammation in normal skin. Both treatments ultimately led to decreased wheal and erythema reactions before suction blistering, but neither treatment affected the size or formation rate of suction blisters. No suction blister fluids had detectable levels of chymotryptic activity, but blister fluids from bullous pemphigoid, herpes zoster and insect bullous eruption, used as the control, revealed clear chymotryptic activity. In addition, tryptic activity in suction blister fluids was not significantly altered after compound 48/80 and capsaicin pre-treatments. However, if the wheal reaction was induced immediately before suction blistering, a significantly increased rate in blister formation together with increased tryptic activity was found, but, unexpectedly, no chymotryptic activity could be detected in blister fluids. The results show that repeated mast cell degranulation in normal skin has no effect on the formation rate of suction blisters, which developed more rapidly on acutely whealing skin. This is probably due to skin oedema rather than mast cell proteases, since no chymotryptic activity was detected in suction blisters where tryptic activity exhibited high individual variation.

Cultured allogeneic skin cells are effective in the treatment of chronic diabetic leg and foot ulcers.

Diabetic ulcers on the lower extremities present a difficult treatment problem, and some ulcers respond poorly to conventional topical and cast treatment. The purpose of this study was to assess the effect of cultured allogeneic keratinocyte epithelium and fibroblast-gelatin sponge on the healing of chronic, refractory diabetic leg and foot ulcers. Non-diabetic chronic leg ulcers were treated for comparison. This open study comprised 22 patients with type I or type II diabetes and 16 patients with leg or ankle ulcers of different aetiologies. A total of 26 diabetic and 25 non-diabetic ulcers were treated mainly with keratinocyte epithelium and/or fibroblast-gelatin sponge once weekly until complete healing or until no further healing could be observed despite several repeated treatments. The duration of diabetic ulcers was 10.3+/-15.8 (mean+/-SD) months and the size 3.1+/-6.6 cm2. The diabetic ulcers were located in the heel (7), toe (7), sole (5), leg (6) and Achilles (1). The mean duration of non-diabetic ulcers was 6.8+/-6.0 months and the size 10.5+/-11.8 cm2. A total of 12+/-11 skin cell transplantations were performed for the diabetic ulcers. All but 1 diabetic ulcer healed during the study. The time for 50% reduction in ulcer area was 32+/-32 days, but 99+/-110 days were needed for complete ulcer closure. The longer the ulcer had existed the longer was the healing time. Heel ulcers showed significantly slower healing response than leg, sole and toe ulcers. Preliminary results suggest that both keratinocytes and fibroblasts are equally effective in the healing process. The time required for healing of the diabetic ulcers did not differ markedly from that of the non-diabetic ulcers. The results suggest that cultured allogeneic skin cells used once weekly are effective in the treatment of recalcitrant diabetic ulcers.