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.

Accuracy and reliability of a hand-held in vivo skin indentation device to assess skin elasticity.

OBJECTIVE: The aim of this study was to evaluate the performance of a hand-held indentation device for fast and reliable determination of skin stiffness. METHODS: Device accuracy to indentation depths of 0.6 and 1.3 mm was first evaluated on plastic foam materials with mechanical properties verified by a laboratory material testing device. Subsequently, the device's sensitivity to detect age-related changes in skin stiffness was evaluated among 46 healthy women (18-79 years). Finally, the reproducibility of the method was tested with six healthy subjects. RESULTS: High correlation was detected between indentation stiffness of reference material and Young's modulus determined with mechanical testing device (0.6 mm indenter: r = 0.97, P = 0.05; 1.3 mm indenter: r = 0.98, P = 0.04). Age-related decrease of 38% in skin stiffness was observed in healthy volunteers (P < 0.05). The coefficient of variation for 0.6 and 1.3 mm indenters was 7.4% and 8.5%, respectively. No trend related to hysteresis effect was observed from repeated measurements. CONCLUSIONS: The presented indentation technique was accurate against the laboratory material testing device. Furthermore, skin changes related to ageing could be detected with the indentation technique. The new device was found to be feasible for monitoring skin stiffness in cosmetics and clinical conditions.

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.

Juvenile-onset neuronal ceroid lipofuscinosis with infantile CLN1 mutation and palmitoyl-protein thioesterase deficiency.

Accurate diagnosis, especially in progressive hereditary diseases, is essential for the treatment and genetic counseling of the patient and the family. Neuronal ceroid lipofuscinoses (NCL) are amongst the most common groups of neurodegenerative diseases. Infantile, juvenile, and adult-onset types with multiple genotype-phenotype associations have been described. A fluorimetric enzyme assay for palmitoyl protein thioesterase (PPT) from leukocytes and fibroblasts has been previously developed to confirm the diagnosis of infantile NCL. We describe a patient with juvenile-onset NCL phenotype with a new CLN1 mutation and deficient PPT activity. Over 40 different mutations have been found in patients with PPT deficiency, indicating that screening for known mutations is not an efficient way to diagnose this disorder. Therefore, PPT enzyme analysis should precede mutation analysis in suspected PPT deficiency, particularly in patients with granular osmiophilic deposits (GROD) or in patients who have negative ultrastructural data. The use of enzyme assay led to the diagnosis of this patient with juvenile-onset Finnish variant NCL with PPT deficiency, and we expect that greater awareness of the utility of the enzymatic assay may lead to identification of other similar cases awaiting a definitive diagnosis.

Purification and partial characterization of rat kidney histamine-N-methyltransferase.

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromatography and S-adenosylhomocysteine affinity chromatography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 microM parahydroxymercuric benzoate and in 10 microM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The Km values for histamine and S-adenosylmethionine were 6.0 and 7.1 microM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 microM S-adenosylhomocysteine and 100 microM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 microM caused substrate inhibition.

The allosteric effect of salt on human mast cell tryptase.

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.

Human skin tryptase: purification, partial characterization and comparison with human lung tryptase.

Human skin tryptase was isolated using stepwise low- and high-salt extraction and further purified 448-fold with 33% yield using octyl-Sepharose CL-4B hydrophobic affinity chromatography, Sephacryl S-200 gel filtration and finally octyl-Sepharose CL-4B or cellulose phosphate ion exchange chromatography. The skin tryptase, which has an apparent Mr of 120,000 by gel filtration in high-salt buffer, consisted of polypeptide chains of Mr 34,000 and 38,000 when resolved on SDS gels. Both polypeptide chains, labelled with [3H]diisopropyl fluorophosphate, indicated that they were representative of subunits and that the native proteinase was an aggregate of subunits. However, in some preparations only one band with Mr 34,000 was seen. In low-salt buffer the enzyme was labile and at least 1.4 M KCl was needed to keep the enzyme stabile when incubated at 37 degrees C for 30 min. Heparin glycosaminoglycan partially stabilized the tryptase but addition of protein (e.g. albumin, 80 micrograms/ml) to the tryptase-heparin mixture was needed to keep the enzyme stabile. Tryptases purified by exactly the same method from human lung tissue and from human skin had identical molecular size in gel filtration and in SDS-polyacrylamide gel electrophoresis. They also revealed identical enzyme kinetic parameters with several synthetic peptide substrates. The inhibition profile was identical for both enzymes, and they also crossreacted completely in immunodiffusion plates. These studies strongly indicate that mast cells found in skin as well as lung contain closely related, possible identical trypsin-like proteinases.

Effect of temporin A modifications on its cytotoxicity and antimicrobial activity.

Temporin A (TA), a short alpha-helical antimicrobial peptide isolated from the skin of the frog Rana temporaria, is effective against a broad spectrum of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium strains. TA interacts directly with the cell membrane of the microorganism and it has been reported to be non-toxic to erythrocytes at concentrations that are antimicrobial. Less is known about the effects on the viability and growth of nucleated eukaryotic cells. In this study we have tested antibacterial and growth-inhibitory properties of TA, its dimeric analogue (TAd), and all-L (TAL L512) and all-D (TAD L512) enantiomeric derivatives of modified TA towards S. aureus and cultured human keratinocytes, respectively. All molecules were antibacterial at concentrations from 1.5 microM to 10 microM. In keratinocyte cultures, TAD L512, as well as TAd, showed cytotoxicity. The original TA and TAL L512 did not affect the viability of the cells at their bacteriolytic concentrations. The growth of keratinocytes in low- and high-calcium media was only slightly inhibited by temporins at concentrations which were antibacterial to S. aureus. This suggests that original TA and its modification, TAL L512, are promising molecules against multiresistant bacterial infections.

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.

Demonstration of tryptase in bovine cutaneous and tumor mast cells.

We examined three tissue samples from each of four cows with non-lesional skin, tissue samples from a cow with multiple cutaneous mast cell tumors, and samples from another cow in which mast cells were infiltrating multiple lymphosarcomas of the skin, for the presence of tryptase and chymase by enzyme cytochemical and immunohistological methods. The enzyme activities of tryptase and chymase were tested using N-carbobenzoxy-glycilglycil-L-arginine-2-naphthylamide (Z-Gly-Gly-Arg-NA) and naphthol-AS-D-chloroacetate (N-AS-D-CA) as substrates, respectively. Tryptase reactivity could be demonstrated in frozen and Carnoy-fixed paraffin sections. Chymase reactivity was seen in neither frozen nor paraffin sections of formalin- or Carnoy-fixed skin tissues. Antibody linkage with a polyclonal rabbit anti-human skin tryptase antibody was highly specific in bovine normal cutaneous, infiltrating, and tumor mast cells. More than 90% of the tumor mast cells were distinctly tryptase-positive. With alcian blue, only slightly more than 10% of the mast cells stained clearly positive and with methylene blue hardly any staining of mast cell granules could be demonstrated. No antibody labeling of mast cell granules in any of the tissue sections was detected by the use of rabbit anti-dog chymase antiserum. These results indicate that there is a striking antigenic similarity of bovine tryptase to its canine and human equivalents. The demonstration of tryptase is an important tool in confirming the diagnosis of undifferentiated mast cell tumors. In contrast to other species, chymase appears to be completely absent in bovine skin mast cells.

Optimization of histamine radio enzyme assay with purified histamine-N-methyltransferase.

The radio enzyme assay for histamine based on the transmethylation with purified histamine-N-methyltransferase and utilizing [3H-methyl]-S-adenosylmethionine as the methyl donor has been optimized to measure low histamine concentrations, for example in plasma. The pH-optimum for the assay is pH 8.3 in Tris-glycine buffer at 20 degrees C. An incubation time of 90 min is necessary using an enzyme concentration of 5.8 micrograms/ml. EDTA and dithiothreitol were included in the assay to keep the histamine-N-methyltransferase active as agents that oxidize -SH groups were found to be inhibitory to the reaction. The present assay is sensitive to about 0.5 nmol/l of histamine in a sample volume of 50 microliter (about 3 pg/sample).

Effect of drugs on histamine radio-enzyme assay.

The effect of over 200 drugs and other compounds on histamine radio-enzyme assay was studied. Some muscle relaxants (e.g. alcuronium), some sympathomimetics (e.g. dopamine, isoxsuprine, tyramine and possibly phenylethylamine), antimalarial drugs, procaine, procainamide, Berenil and serotonin were potent compounds to interfere with this assay. In some special cases still potentially inhibitory drugs seemed to be some muscle relaxants (e.g. vecuronium, pancuronium and tubocurarine), antidepressants, antihistamines (e.g. cimetidine, ranitidine and diphenhydramine), chinidin, disopyramide, tolazoline and salazosulfapyridine.

Mast cells of psoriatic and atopic dermatitis skin are positive for TNF-alpha and their degranulation is associated with expression of ICAM-1 in the epidermis.

The release of cytokines from cutaneous cells may be of major importance in the initiation and development of many inflammatory skin disorders. For example, tumor necrosis factor-alpha (TNF-alpha), which in healthy skin is found preformed only in mast cells, is able to induce the expression of several adhesion molecules including intercellular adhesion molecule-1 (ICAM-1). Increased expression of ICAM-1 occurs in keratinocytes in lesional skin of psoriasis and atopic dermatitis (AD) and it is considered to be an important initiator of leucocyte/keratinocyte interactions in skin inflammation. We counted the mast cells showing TNF-alpha immunoreactivity using a double-staining method in nonlesional and lesional skin sections from 12 patients with AD and 12 patients with psoriasis. The percentage of TNF-alpha+ mast cells in lesional and nonlesional AD skin was 36 +/- 22% and 21 +/- 15% (P < 0.018, paired t-test), respectively, and in psoriatic skin was 16 +/- 25% and 15 +/- 15%, respectively (P < 0.89, paired t-test). We also cultured whole skin biopsies taken from the healthy-looking skin of psoriatic and AD patients in the presence of mast cell degranulator compound 48/80, which resulted in focal expression of ICAM-1 in the epidermis. In cultured keratinocytes, both histamine and an extract of a human mast-cell line (HMC-1) induced ICAM-1 immunostaining only in occasional cells, but the combination of histamine and the HMC-1 extract resulted in intense ICAM-1 staining in numerous cells. This enhancement of ICAM-1 staining was abolished by preincubation of the HMC-1 extract with anti-TNF-alpha antibody. These results suggest that the degranulation of mast cells induces the expression of ICAM-1 in keratinocytes probably via TNF-alpha and histamine.

Mast cell proteinases and cytokines in skin inflammation.

The role of mast cells in provoking immediate-type hypersensitivity reactions is well established, but their involvement in chronic inflammation and immune reactions is not so clear. Mast cells synthesize and secrete large amounts of active proteinases, including tryptase, chymase, carboxypeptidase and cathepsin G, which can rapidly process numerous biologically active peptides and proteins or their precursors. Furthermore, mast cells are able to produce a variety of cytokines such as interleukin-4 (IL-4), IL-5, IL-6, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) which are known to be intensively involved in modulating and directing inflammatory responses in the skin. In this review, the role of mast cell proteinases and cytokines in skin inflammation is discussed.

Enzyme- and immunohistochemical localization of mast cell tryptase in psoriatic skin.

The localization of mast cell tryptase in involved and noninvolved skin sections from 12 psoriatic patients was investigated using both enzyme- and immunohistochemical staining techniques. Each involved skin section contained an increased number of tryptase-positive mast cells in the superficial dermis as compared with corresponding noninvolved skin sections. The substrate-hydrolyzing and inhibition properties for tryptase activity in involved and non-involved psoriatic skin sections were identical with each other as well as with those previously described in normal human skin or mastocytoma skin sections. In four patients, epidermal enzyme activity was observed, but only in the involved skin. None of the uninvolved sections showed tryptase activity in the epidermis. This activity was not inhibited by alpha 1-antitrypsin, and after removing the enzyme-histochemical stain with Tween 20, positive results obtained with tryptase-specific antibody were found in the same locations. In addition, tryptase-positive cells were detected in close contact to lesional epidermis, but without epidermal staining in four patients. In the epidermis, the positive staining was granular, and active tryptase was detected as far as the stratum corneum. This study is the first description of the presence of active mast cell tryptase in psoriatic epidermis, where this enzyme may have a role in increased cell division.

Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin.

Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzamidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-anti-trypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15, 30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefulness of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of multiple forms of tryptase from human mast cells.

Mast cell tryptase purified from human adult skin (AS), adult lung (AL) and newborn foreskin (NS) with a monoclonal antitryptase B2 immunoaffinity Sepharose column was further fractionated by HPLC using a Mono-S cation exchange column at pH 6.5. Tryptases exhibited two clearly separated major fractions, both of which also revealed at least two overlapping peaks. Native tryptase molecules from skin consisted of two diffuse protein bands in SDS-PAGE at about 31 and 35 kDa, whereas those from lung usually exhibited a predominant diffuse band at about 29 kDa. The forms of tryptases separated by Mono-S HPLC gave a different banding pattern in SDS-PAGE. Tryptase from NS exhibited chromatographic peaks that each showed Mr values approximately 1-3 kDa higher than those of tryptase from AS. By gel filtration, the Mr values for native major fractions of tryptases derived from AS and AL were 178 kDa and 141 kDa, respectively. After carbohydrate removal by glycanase, the observed differences in Mr values in SDS-PAGE reduced to two similar sharp bands of Mr approximately 28 kDa and 30 kDa for all tryptase preparations. AS and AL tryptases and their subfractions exhibited similar enzyme kinetic values and similar immunoreactivities in a tryptase immunoassay. Inactivation rates at physiologic ionic strength were similar for both AL and AS tryptases. The results show the enzymatic and antigenic similarity between lung and skin tryptases, and suggest that tryptase is stored mainly as beta-tryptase in human mast cells. Tryptase immunoassay measures similarly both lung and skin tryptases and, thus, this assay is suitable for detection of mast cell activation, in contrast to assays for other proteinases of mast cells, e.g. chymase, cathepsin G and carboxypeptidase, that are present in MC(TC) cells mainly in skin only.