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.     

Decreased growth inhibition by recombinant gamma interferon is associated with increased transforming growth factor-α production in keratinocytes cultured from psoriatic lesions

Keratinocytes from involved psoriatic plaques (PP), uninvolved, clinically symptomless skin of psoriatic patients (PN) and normal healthy skin (NN) have been cultured in a low calcium serum free system for multiple passages. In this way, the keratinocytes were removed from microenvironmental factors present in the skin. While the basal rate of proliferation of the PP, PN and NN keratinocytes was not different, the PP cells produced more transforming growth factor-alpha (TGF-alpha) than NN cells, and the antiproliferative response of PP cells to gamma interferon (IFN-gamma), a product of activated T lymphocytes, was reduced. We studied IFN-gamma because it can inhibit the proliferation of NN keratinocytes, induce their differentiation and the appearance of two immunoregulatory cell surface molecules, HLA-DR and intercellular adherence molecule-I (ICAM-I), and because in another epithelial cell system, epidermal growth factor (EGF) modulates IFN-gamma activity. The mean antiproliferative effects of IFN-gamma at 50,200, and 500 U/ml for the PP group (n = 10) was less compared to the NN group (n = 11); P less than 0.001, while the PN group (n = 5) had a less dramatic, but statistically significant, reduction in growth inhibition by IFN-gamma only at 200 and 500 U/ml compared to NN cells; P less than 0.05 and P less than 0.01, respectively. The amount of TGF-alpha produced and secreted by PP keratinocytes from five different individuals was significantly greater than by NN keratinocyte cultures. In addition, IFN-gamma induced TGF-alpha to a lesser extent in PP keratinocytes compared to NN keratinocyte cultures. Keratinocytes isolated from atopic dermatitis and Sézary syndrome patients were similar to NN keratinocytes. In contrast to its differential effects on TGF-alpha production and proliferation, IFN-gamma induced similar amounts of HLA-DR and ICAM-I on PP, PN and NN keratinocytes. Thus, for the PP keratinocytes, there was a dissociation between the antiproliferative and immunomodulatory effects of IFN-gamma. These results support our previous hypothesis that the hyperproliferation and altered differentiation of keratinocytes in psoriatic plaques is linked to an altered responsiveness of the keratinocytes to IFN-gamma. Moreover, these results provide an in vitro correlate of our in vivo observation of increased TGF-alpha levels in psoriatic plaques. A new pathophysiological model to understand psoriasis is proposed which integrates these observations involving IFN-gamma and TGF-alpha. This experimental approach also provides a system to dissect biochemical pathways of pathophysiological importance for keratinocyte hyperproliferation in psoriasis.     

Cyclooxygenase activity is increased in platelets from psoriatic patients

The aim of the present study was to ascertain the relationship between in vitro hyper-aggregability and alterations in arachidonic acid metabolism in platelets from psoriatic patients. We have studied the response to several concentrations of ADP, collagen, and arachidonic acid of intact platelets from psoriatic patients and normal subjects, with and without irreversible inhibition of platelet cyclooxygenase by aspirin. Apparent kinetic constants (apparent Michaelis constant [Km] and apparent maximum velocity [Vmax]) of cyclooxygenase in platelets from both controls and psoriatic patients were also studied. The maximum percentage and slope of aggregation induced by collagen or sodium arachidonate were significantly greater (p less than 0.05) in platelets from psoriasis patients, whereas lag time was significantly shorter in the psoriasis group in response to arachidonic acid only when compared to controls. Cyclooxygenase pathway blockade inhibited the response to aggregation inducers in the following order: sodium arachidonate greater than collagen greater than ADP. When platelets were pre-treated with aspirin no significant differences were observed between controls and psoriasis patients. We also found a significant increase of the apparent Vmax value (p less than 0.05) for cyclooxygenase activity in platelets from psoriatic patients in comparison with controls. Our results indicate that platelet hyperaggregation in psoriatic patients is related to enhanced cyclooxygenase activity in their platelets.     

The presence of tryptase-positive and bikunin-negative mast cells in psoriatic skin lesions

Human mast cells are well known to produce a serine protease, tryptase, which appears to play a pathogenic role in various skin inflammations. It was previously reported that a rat homologue of bikunin may inhibit tryptase activity. Various type of cells (i.e. keratinocytes) are able to produce this protein inhibitor, it still remains unclear if bikunin is present in dermal inflammatory milieu, in which mast cells, through secretion of tryptase, play an inflammatory role. Therefore, the purpose of the present study was to exploit expression and production of bikunin in dermis and dermal constituents. We first compared the dermal mast cells in psoriatic lesions with those in lesional skin of atopic dermatitis or of chronic eczema by use of immunoelectron microscopy and immunohistochemical analyses using antibodies to bikunin and tryptase. Then, we tested what kinds of cytokines may regulate the de novo synthesis of bikunin. To do so, RNA was extracted from a human mastocytic cell line, HMC-1, reverse-transcribed, and semiquantitative RT-PCR was performed using primers specific for bikunin. With immunoelectron microscopy, bikunin was found to localize on the cell membrane, while tryptase was in the secretary granules of the mast cells. In psoriatic lesions, around 70% of dermal mast cells were positive for both tryptase and bikunin, and the remaining was mostly positive for tryptase, but the expression of bikunin was under the detection limit of the experimental setting. This observation was seen in only psoriatic lesions, even in almost cured lesions, while in atopic dermatitis or chronic eczema only mast cells doubly positive for bikuin and tryptase were seen. In HMC-1, bikunin was constitutively expressed at an mRNA level, which was upregulated by stimulation with interleukine-4, but was suppressed by interferon-γ. Bearing in mind the concept that in psoriasis local cytokine milieu is shifted toward a Th1 pattern (predominant secretion of interferon-γ), tryptase-positive, bikunin-negative mast cells may be induced.     

Mast cell tryptase and chymase in developing and mature psoriatic lesions

The number and distribution of mast cells in nonlesional and lesional skin samples from 13 psoriatic patients were analyzed enzyme- and immunohistochemically. Mast cell tryptase was stained with the sensitive substrate Z-Gly-Pro-Arg-4-methoxy-2-naphthylamide, and chymase with Suc-Val-Pro-Phe-MNA and monoclonal B7 anti-chymase antibody. In addition, healthy-looking skin from 27 psoriatic patients was tape-stripped resulting in induction of the Köbner response in 9 patients. Sequential biopsies were taken before and after (7, 14 and 21 days) tape-stripping, and both tryptase and chymase were stained enzyme-histochemically. In nonlesional psoriatic skin, 70 ± 24% (mean ± SD) of the mast cells contained chymase enzyme activity, and 78 ± 18% chymase immunoreactivity. About 10% of the chymase-immunoreactive cells lacked chymase activity. In lesional psoriatic skin, tryptase-positive cells were increased in number throughout the dermis but especially beneath the epidermis. Chymase immunoreactivity paralleled the tryptase activity, whereas chymase activity was strongly diminished both in terms of mast cell numbers and in staining intensity in the papillary dermis. The apparent inactivation of chymase may be due to the action of the chymase inhibitors, ₁-antitrypsin and ₁-antichymotrypsin, localized immunohistochemically in mast cells of lesional and nonlesional psoriatic skin. In the developing psoriatic lesion, mast cells displaying chymase activity were already 27–38% decreased in number in the upper dermis on day 7 after tape-stripping, along with the first clinical signs of psoriasis. Earliest alterations in tryptase-positive cells were observed on day 14 as increased mast cell contacts with the epidermis combined with only a slight increase in mast cell numbers in the upper dermis. During the development of a psoriatic lesion, TC mast cells (tryptase⁺, chymase⁺) increase in number in the upper dermis, but chymase becomes inactive at an early stage. The abundant presence of active trypase but inactive chymase in the upper dermis may have a potential role in psoriasis since both of these enzymes can process several biologically active peptides and proteins.     

Reduction of increased polyamine levels in psoriatic lesions by retinoid and PUVA treatments

Treatment with an oral aromatie retinoid (etretinate) and with PUVA significantly reduced the elevated levels of putrescine, spermidine and spermlne in psoriatic lesions. Both treatments also significantly reduced the spermidine/spermine ratio, which is considered to be an indicator of proliferation activity. Although both regimens produced a roughly parallel reduction of epidermal polyamines the initial fall of putrescine was much more rapid in patients receiving retinoid. This may indicate that one of the primary targets of retinoids could be ornithine deearboxylase.     

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.     

Decreased membrane-bound ATP-hydrolytic activity in psoriatic epidermis

Previous studies from our laboratory suggested that architectural alterations of the cell membranes may have a major significance in the pathogenesis of psoriasis. Consequently, the membrane-bound Mg⁺⁺ activated ATP-hydrolytic activity was investigated in normal and psoriatic epidermis under the electron microscope. The present study revealed that, in contrast to normal epidermis, only minimal ATP-hydrolytic activity is present on the cell membranes of psoriatic keratinocytes. This finding may reflect either a diminished attachment of substrate on the altered cell surface or an enzyme defect of the cell membrane. In both cases the reduced interaction between ATP and membrane-bound ATP-hydrolysing enzymes represents a functional membrane disorder of the psoriatic keratinocyte, presumably resulting in an insufficient utilization of ATP for active transport mechanisms on the cell surface.     

The activity of caspase-1 is increased in lesional psoriatic epidermis

Caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the proinflammatory cytokine IL-18, a cytokine known to play an important role in the pathogenesis of psoriasis. The purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress-induced activation of caspase-1 and IL-18. Interestingly, increased caspase-1 activity in lesional compared with non-lesional psoriatic skin was seen. In vitro experiments in cultured human keratinocytes demonstrated anisomycin-induced, p38 mitogen-activated protein kinase (p38 MAPK)-dependent increased secretion of procaspase-1 and active caspase-1. Furthermore, anisomycin increased the mRNA expression of IL-18 through a p38 MAPK-dependent but caspase-1-independent mechanism, reaching a maximum level after 12 hours of stimulation. Finally, anisomycin caused a rapid (4 hours) increase in the secretion of proIL-18 and active IL-18. Secretion of active IL-18 was mediated through a p38 MAPK/caspase-1-dependent mechanism, whereas secretion of proIL-18 was mediated by a p38 MAPK-dependent but caspase-1-independent mechanism. These data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that IL-18 secretion is regulated by a p38 MAPK/caspase-1-dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis.     

Calcipotriene-induced improvement in psoriasis is associated with reduced interleukin-8 and increased interleukin-10 levels within lesions

Calcipotriene is a synthetic analogue of 1,25-dihydroxyvitamin D₃ established to be effective topically in the treatment of psoriasis. We investigated the early cellular and immunological events induced by calcipotriene in psoriasis. Thirty patients with moderate plaque-type psoriasis were randomly assigned to receive twice daily applications of either calcipotriene ointment 0.005% or matching vehicle for 6 weeks. Skin biopsies (6 mm) were performed from designated plaques at baseline and days 3 and 7. On these days and at weeks 2, 4 and 6, complete clinical evaluations were made in a double-blind fashion. Consistent with previous studies, significant clinical improvement (P < 0.05) in psoriasis was observed in patients receiving calcipotriene vs. those receiving vehicle by day 7 for scale and erythema, and by day 14 for thickness. No significant improvement, however, was seen on day 3. None of the immunohistological markers (CD1a, CD4, CD8, ICAM-1, VCAM-1, E-selectin, HLA-DR) semiquantitatively assessed in psoriatic plaques was significantly changed by calcipotriene treatment for 7 days. In the calcipotriene-treated group, interleukin (IL)-10 levels (pg/μg of protein) increased by 57% from baseline (0.030 ± 0.006; mean ± SEM) to day 3 (0.047 ± 0.011) (P = 0.05 vs. baseline; n = 10) and remained elevated at day 7 (0.046 ± 0.012). IL-8 levels (pg/μg of protein), however, declined by 70% from baseline (0.13 ± 0.06) to day 3 (0.04 ± 0.01), and remained low at day 7 (0.03 ± 0.02) (P < 0.05 vs. baseline; n = 10). Both IL-8 and IL-10 were unaffected by vehicle treatment. Calcipotriene-induced clinical improvement of psoriasis is preceded by an increase in IL-10 and a concomitant decrease in IL-8 levels. The changes in the level of these two cytokines provide further evidence for immunological changes as a significant part of the mechanism of action of calcipotriene in psoriasis.     

Elevated phosphorylase kinase activity in psoriatic epidermis: correlation with increased phosphorylation and psoriatic activity

Summary To determine whether abnormal activity of a calmodulin-containing enzyme which catalyses phosphorylation reactions may play a pathogenetic role in psoriasis, the presence and activity of phosphorylase kinase (PK) in human epidermis were determined in patients with untreated/active psoriasis (n =10), treated/resolving psoriasis (n= 10), and non-psoriatic controls (n= 10). Biopsies were taken from involved and uninvolved skin for PK, organic phosphorus, and inorganic phosphate estimation, and light and electron microscopy. The enzyme was present in involved and uninvolved skin of every patient in the study. PK activity (units/mg protein) was significantly higher in active psoriasis than in resolving psoriasis and controls. PK activity correlated directly with organic phosphorus levels, and inversely with the extent of cellular glycogenolysis measured by the depletion of glycogen granules within the keratinocytes. The study demonstrates that PK is present in both psoriatic and normal epidermis, with significantly higher levels in active psoriasis. Furthermore, higher levels of PK activity, glycogenolysis and phosphorylation are associated with increased clinical psoriatic activity. We conclude that PK, a calmodulin-containing enzyme, is involved in regulating calcium-dependent phosphorylation events in human epidermis, and disturbance of its activity may play a key role in the clinical manifestations of psoriasis.     

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.     

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.     

银屑病瘙痒皮损中肥大细胞的数量与分布研究

目的探讨肥大细胞在银屑病瘙痒症状发生中的作用。方法应用免疫组化技术对30例患者(无瘙痒12例,瘙痒18例)皮损区、非皮损区以及2例正常人皮肤组织中肥大细胞数量进行检测。结果银屑病伴有瘙痒症状的患者皮损中肥大细胞数量明显增多(Z=-2.732,P<0.05)。结论皮损中肥大细胞的数量变化与银屑病的瘙痒症状具有相关性。     

Immunohistochemical analysis of sensory nerves and neuropeptides,and their contacts with mast cells in developing and mature psoriatic lesions

The distribution of the neuropeptides substance P (SP), vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) was studied immunohistochemically in psoriatic skin during the Koebner response (6 h, 2 days, 7 days, 14 days, 21 days), and in mature psoriatic plaques, of 37 psoriatic patients. The morphological association of sensory nerves, SP and VIP with papillary mast cells was also monitored. The nerves containing SP, VIP or CGRP were very scanty in control skin, and in non-lesional and Koebner-negative psoriatic skin. The first psoriatic lesions were seen 7 days after tape stripping the symptomless psoriatic skin. SP- and VIP-containing nerves were slightly increased in Koebner-positive specimens, but the increase was very prominent in dermal papillae of mature psoriatic plaques. In the plaques, nerve-mast cell contacts were significantly increased (p<0.001) compared with non-lesional psoriatic skin. Only SP-positive fibres were detected in the epidermis and in contact with papillary mast cells. VIP was mainly located around capillaries where SP was also found. No change was noted in CGRP-positive fibres between lesional and non-lesional specimens. The appearance of SP and VIP in the capillary walls is morphological evidence for their function as vasodilators in psoriatic lesion. A slight increase in SP- and VIP-positive fibres in Koebner-positive specimens suggests that these neuropeptides may participate in the inflammatory reaction at an early stage. Their prominence in mature psoriatic plaques in turn indicates a role for them in the maintenance of psoriatic lesions. Morphological contacts between mast cells and SP-containing nerves give further evidence to the view that SP is capable of amplifying the inflammatory reaction also through the axon-reflex mechanism.Part of this work was presented at the meeting of the European Society for Dermatological Research, London, UK, 4–7 April 1992