Anti-inflammatory effect of FK-506 on human skin mast cells.

FK-506 and the structurally related macrolide rapamycin are high-affinity ligands for a specific binding protein (FK-506 binding protein). We examined the effects of FK-506 and rapamycin on the release of pre-formed (histamine) and de novo synthesized inflammatory mediators (prostaglandin D2) from mast cells isolated from human skin tissue. FK-506 (0.1 to 100 nM) concentration-dependently inhibited (5 to 65%) histamine release from skin mast cells activated by anti-IgE. FK-506 was more potent in skin mast cells than in basophils (IC40 = 2.15 +/- 0.78 nM versus 5.12 +/- 1.34 nM; p < 0.001), whereas the maximal inhibitory effect was higher in basophils than in skin mast cells (88.77 +/- 2.44% versus 67.30 +/- 3.98%; p < 0.01). FK-506 had little or no inhibitory effect on histamine release from skin mast cells challenged with compound A23187 and substance P, respectively, whereas it completely suppressed A23187-induced histamine release from basophils. FK-506 (0.1 to 100 nM) also inhibited (up to 65%) the de novo synthesis of prostaglandin D2 from skin mast cells challenged with anti-IgE. Despite its structural similarity to FK-506, rapamycin (10 to 300 nM) had little or no effect on the release of histamine from skin mast cells induced by anti-IgE, A23187, and substance P. However, rapamycin competitively antagonized the inhibitory effect of FK-506 on anti-IgE-induced histamine release from skin mast cells with a dissociation constant of about 14 nM. These data indicate that FK-506, but not rapamycin, is a potent anti-inflammatory agent acting on skin mast cells presumably by binding to the FK-506 binding protein. It thus appears that binding to the FK-506 binding protein is necessary, but not sufficient, to deliver an inhibitory signal to skin mast cells.     

The effect of human anaphylatoxins and neutrophils on histamine release from isolated human skin mast cells.

The concept of mast cell heterogeneity has been studied extensively. Recently developed techniques to enzymatically disperse skin mast cells from human skin have shown that skin mast cells are somehow different from those of other organs such as lung and intestine. In this report, we have isolated and partially purified human skin mast cells from human neonatal foreskins by collagenase and hyaluronidase digestion. These mast cells are morphologically intact by histological, immunohistochemical and electron microscopic criteria. These human skin mast cells secrete histamine significantly (max. net histamine release, 20-30%) in a dose-related, temperature- and time-dependent fashion following stimulation with purified human C5a and C3a (over the ranges of 5 x 10(-8) M to 10(-7) M and 3 x 10(-7) M to 6 x 10(-6) M, respectively). On the other hand, interactions between human skin mast cells and other leukocytes have long been suspected of playing a very important role in cutaneous inflammation. Recently, a human neutrophil-derived histamine-releasing activity termed HRA-N was partially purified. HRA-N has been shown to cause human and rat basophil leukemia cells to degranulate. This study was also undertaken to assess the ability of HRA-N to directly induce histamine release from isolated human skin mast cells. HRA-N causes dose- and time-dependent histamine release as do human anaphylatoxins. These results suggest that HRA-N may lead to a better comprehension of allergic and inflammatory reactions and their modulation in the skin.     

Phenotypic evaluation of cultured human mast and basophilic cells and of normal human skin mast cells

In order to evaluate various markers for human mast cells, two human mast/basophilic cell lines (HMC-1/KU812), cultured mast cells from the peripheral blood monocytic fraction and peripheral blood monocytes were compared with mast cells in tissue sections from normal skin, using histochemistry, enzyme histochemistry and immunohistochemistry. All reagents stained normal skin mast cells, with toluidine blue, tryptase reactivity and antibodies against the FcRI and the stem cell factor receptor (c-kit) being most active. The cell lines and mast cells cultured from peripheral blood were negative for avidin, safranin and chymase, strongly positive for c-kit and variably reactive with all other reagents. All antibodies except AA1 against tryptase also stained one or several epidermal and dermal cell types or blood monocytes. Histochemical stains (toluidine blue, avidin) and reagents for the enzymes tryptase and chymase are thus specific markers for mast cells. The frequent reactivity of antibodies against mast cells with other cell types indicates interesting functional and ontogenetic relationships between these cells.     

The effect of ultraviolet radiation exposure on the prevalence of mast cells in human skin

BACKGROUND: Dermal mast cells have been implicated as important effector cells in innate immunity, hypersensitivity responses and ultraviolet (UV)B-induced suppression of cell-mediated immune responses to contact allergens. Humans, like mouse strains, display variations in dermal mast cell prevalence. The factors determining these differences are yet to be fully elucidated. In mice, expression of the receptor for stem cell factor, c-kit, on dermal mast cells correlates with prevalence. OBJECTIVES: To evaluate dermal mast cell prevalence and mast cell c-kit expression in non-sun-exposed and sun-exposed skin in the same donor. METHODS: In 14 subjects, biopsies of skin (4 mm) were sampled from the skin sites of buttock, inner arm, shoulder and back of hand skin and dermal mast cell prevalence quantified. Non-sun-exposed buttock and chronically sun-exposed hand skin were evaluated for mast cell expression of c-kit and elastin content, a feature of photoageing and surrogate marker of UV exposure. RESULTS: The prevalence of dermal mast cells was significantly higher in hand skin than in the three other anatomically different skin sites. Significant correlations were observed in hand but not buttock skin between increasing dermal mast cell densities, extent of elastin content in the papillary dermis and age of the subject. Cellular expression of c-kit correlated with mast cell prevalence in hand skin. However, no relationship was observed in hand skin between c-kit expression, elastin content and age. CONCLUSIONS: The prevalence of mast cells in human skin is altered by factors that are intrinsic (mechanisms regulating c-kit expression) and extrinsic (chronic sun exposure), and the fact that the associations of mast cell prevalence with age is explained by the latter being a correlate of cumulative sun exposure.     

Retinoic acid inhibits in vitro development of mast cells but has no marked effect on mature human skin tryptase- and chymase-positive mast cells

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.     

Dendritic mast cells in prurigo nodularis skin.

Mast cells are traditionally recognized as round or oval connective tissue cells containing many specialized cytoplasmic granules. During recent years, more and more mast cell functions and properties have been clarified, and it is now evident that the mast cells are of different subtypes. The present study, utilizing chymase and tryptase immunofluorescence double-labelling and conventional electron microscopy techniques, has identified a kind of mast cells with obvious dendritic features in the lesional dermis of prurigo nodularis skin. This group of mast cell have enlarged cell bodies and contain fewer cytoplasmic granules, especially within certain dendrites. The morphological identification of such subgroups of mast cells could contribute to the understanding of mast cell heterogeneity.     

A novel in-situ-zymography technique localizes gelatinolytic activity in human skin to mast cells

Abstract: Matrix-metallo-proteinases play a key role in cutaneous tissue remodeling and wound healing, and have been implicated as the ratelimiting factor in cutaneous tumor invasion and metastasis. We here describe a novel in-situ -zymographic method, which allows to directly localize sites of gelatinolytic activity in human skin. Gelatinolysis was detected through protein-hydrolysis in a 200 μm thick polyacrylamide gel underlying tissue sections. The lysis was substrate-dependent, demonstrated time- and temperature-dependent kinetics, and was inhibited by both EDTA and 1,10-phenanthroline. Normal and diseased skin sections demonstrated multiple focal points of gelatinolysis which co-localized with individual cells. Histochemically, these were shown to represent most likely mast cells (via AS-d-chloroacetate esterase staining and metachromasia). However, immunohistochemical staining for gelatinases A and B showed no immunoreactivity patterns that corresponded to the identified foci of gelatinolysis. The reported in-situ -zymographic technique offers a decisive advantage over immunohistochemistry, since it detects only the activated and catabolically relevant proteases, and provides further evidence for a role of mast cells in extracellular matrix remodeling.     

Solar urticaria. A case with possible increase of skin mast cells.

A case of solar urticaria is described showing: (I) Action spectra for late erythema (MED), late swelling and wealing with one peak of sensitivity for erythema and wealing at 405 nm. (2) No signs of porphyria. (3) Possibly increased skin mast cells. (4) Short-lived post-irradiation fibrin deposition. (5) Haemolysis. (6) Apparent suppression of urticaria with the antihistamine Incidal.     

Ablation of human skin mast cells in situ by lysosomotropic agents

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell‐mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu‐Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.     

The effect of cyclosporin A on proliferation and differentiation-associated antigens of normal human skin xenografted onto nude mice

Cyclosporin A (CsA) has been shown to inhibit, in vitro, the proliferation of cultured normal and neoplastic keratinocytes and to exert also in vivo an antiproliferative effect on keratinocytes of normal human skin xenografted onto nude mice. To gain further insight into the effects of CsA on human skin we investigated the immunohistochemical expression of several epidermal proliferation- and differentiation-associated antigens in the same model: six-week-old nude mice received a xenograft of full-thickness normal human skin; six animals subsequently received a daily subcutaneous injection of 50 mg/kg of CsA diluted in olive-oil while the others received an equivalent volume of olive-oil. The rate of epidermal proliferation was evaluated through a BrdU pulse-labelling technique, and was found to be decreased by 56% in the CsA-treated epidermal xenografts as compared to the controls. The xenografts were further examined for the expression of the following antigens: Epidermal Growth Factor- and Transferrin-receptors, Ki-67, 56.5 kD keratin polypeptide, Filaggrin, Involucrin, beta 2-microglobulin, Ulex Europaeus I- and Peanut-Agglutinin-binding sites. Most of these antigens were unchanged on CsA-treated human xenografts. However, the 56.5 kD keratin polypeptide which was consistently expressed by both basal and suprabasal epidermal keratinocytes in control xenografts showed a normal expression pattern (i.e. suprabasal keratinocytes only) in three out of the six CsA-treated xenografts. These results raise the possibility that, concurrently with a cytostatic effect, CsA may also affect keratinocyte differentiation and that this effect, possibly contributes in the beneficial effect of CsA in diseases of abnormal keratinization.     

Inhibitory effect of cyclosporin A on antigen and alloantigen presenting capacity of human epidermal Langerhans cells

The effect of cyclosporin A (CyA) on the capacity of human epidermal Langerhans cells (LC) to stimulate allogeneic T cells or to present antigen to autologous T cells was investigated. Preparations of LC enriched by discontinuous density gradient centrifugation were pulsed for 2 or 16 h with graded doses (5-5000 ng/ml) of CyA prior to co-culture with T cells. Pretreatment of LC with CyA resulted in a dose-dependent decrease of the functional capacity of LC to stimulate T cells. This inhibition (up to 90%), already achieved after a pulse of 2 h, was not due to a cytotoxic effect of the drug and appeared to be reversible. The possibility that CyA exerted its effect indirectly on T cells via release of CyA from LC into the supernatant during co-culture was excluded. The suppression of immunostimulatory function was a direct effect of the drug on LC. CyA did not affect the production by LC of IL-1 or prostaglandin, nor the expression of MHC class II products HLA-D and RFD1 or adhesion molecules ICAM-1 and LFA-3. These results suggest that inhibition of contact allergic skin reactions by CyA may be due in part to an impairment of the function of LC.     

Anti-inflammatory efficacy of low-dose cyclosporin A in psoriatic arthritis. A prospective multicentre study

Summary Fifty-five patients with psoriatic arthritis were treated with a low dose of cyclosporin A (CyA) (mean dose 2.7 mg/kg per day) for a period of 6 months to investigate the efficacy of CyA on disease parameters. Significant improvement in the joint complaints and inflammation parameters was observed including a decrease in the number of painful (-46%) and swollen (-45%) joints, tenderness (Ritchie Index: -50%) and degree of swelling (-46%), patient's assessment of pain (-35%), the duration of morning joint stiffness (-37%), as well as a decrease in C-reactive protein (-52%). A 50% reduction of joint complaints required a total of 24 weeks, whereas a 50% reduction of skin involvement was achieved after 5–6 weeks of treatment. Four patients left the study due to adverse events: creatinine level increase in two patients, hypertension in one patient and gastroenteritis in the fourth patient. Joint scintigraphy in 18 patients indicated an improvement or stable condition in 61% of cases after a mean follow-up of approximately 8 months. The results of this prospective study show that low-dose CyA effectively improves not only skin lesions, but also joint complaints in psoriatic arthritis.     

Mast cells and atopic dermatitis. Stereological quantification of mast cells in atopic dermatitis and normal human skin

Stereological quantification of mast cell numbers was applied to sections of punch biopsies from lesional and nonlesional skin of atopic dermatitis patients and skin of healthy volunteers. We also investigated whether the method of staining and/or the fixative influenced the results of the determination of the mast cell profile numbers. The punch biopsies were taken from the same four locations in both atopic dermatitis patients and normal individuals. The locations were the scalp, neck and flexure of the elbow (lesional skin), and nates (nonlesional skin). Clinical scoring was carried out at the site of each biopsy. After fixation and plastic embedding, the biopsies were cut into 2 μm serial sections. Ten sections, 30 μm apart, from each biopsy were examined and stained alternately with either toluidine blue or Giemsa stain and mast cell profile numbers were determined. The study yielded the following results: (1) in atopic dermatitis lesional skin an increased number of mast cell profiles was found as compared with nonlesional skin, (2) comparing atopic dermatitis skin with normal skin, a significantly increased number of mast cell profiles per millimetre squared was found in specimens from the neck, (3) staining with toluidine blue yielded a lower number of mast cell profiles than Giemsa staining, (4) the use of Carnoy’s fixative resulted in a lower mast cell profile count than the use of formaldehyde, and (5) there was no statistically significant correlation between the clinical score and the number of mast cell profiles per millimetre squared. Using stereological techniques, this study indicated that mast cells might participate in the inflammatory process in skin leading to atopic dermatitis. Received: 17 April 1996     

Increased numbers of mast cells in pemphigus vulgaris skin lesions. A histochemical study.

We have used a histochemical technique to study mast cells (MC) in skin biopsies of 8 patients suffering from pemphigus vulgaris (PV) and from 4 control volunteers. The MC were stained for 30 min with 0.5% toluidine blue, pH 0.5, counted and then restained for 5 days under the same conditions. This staining method allows the identification of two groups of MC, one that stains promptly (30 min) and one that stains after longer incubation times (5 days). After 30 min of staining, a slight increase was found in the number of MC in PV sections, in comparison with normal controls. However, when the 30 min stained sections were reincubated under the same conditions for 5 days, a significant increase in the number of MC in PV was found in comparison with 5-day-stained normal skin sections (p less than 0.005) and in comparison with 30-min-stained PV sections (p less than 0.005). The MC were distributed throughout the dermis and were concentrated in the upper dermis near hair follicles and vessels. The possible importance of the increased numbers of MC in PV is discussed.     

Effects of cyclosporin A on cell proliferation and collagen production by human skin fibroblasts

Cyclosporin A (CSA) is a potent immunosuppressive drug that has been used clinically for the treatment of organ rejection after transplantation as well as for patients with a wide variety of immune-mediated disorders. CSA has recently been reported to be effective in systemic sclerosis, which is a disease of the connective tissues leading to fibrosis of the skin and other involved organs. In this study, we investigated whether CSA affects the cell proliferation and collagen synthesis of human skin fibroblasts. CSA inhibited the DNA synthesis and cell growth of cultured fibroblasts at concentrations of 10(-8) M to 10(-5) M in a dose-dependent manner. The production of both collagen and non-collagenous protein at both the mRNA and protein levels was not affected by 10(-8) to 10(-6) M CSA, but was decreased in the presence of 10(-5) M CSA. These results suggest that CSA may inhibit the proliferation of fibroblasts, but not their synthesis of collagenous and non-collagenous proteins at therapeutic concentrations.     

Effects of basophil-priming and stimulating cytokines on histamine release from isolated human skin mast cells

Cell priming and stimulation of different cytokines (which include chemokines and growth factors) are typical features of human basophils. Recently, it has been shown that the macrophage chemotactic protein-1 (MCP-1), RANTES and macrophage inflammatory protein-1α (MIP-1α) are potent direct secretagogues for human basophils and that interleukin-3 (IL-3), IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF) are priming factors for subsequent potentiation of mediator release from basophils induced by different stimuli. This observation may be clinically important for the activation and recruitment of inflammatory cells in different immune responses of the skin (e.g. late-phase reactions). The aim of the present study was to investigate whether cytokines and chemokines are also capable of priming or stimulating isolated human skin mast cells (SMC). SMC were either stimulated directly with the cytokines alone or preincubated with these factors for 10 min before being activated with suboptimal concentrations of anti-IgE, A23187 or substance P. IL-3, IL-5, GM-CSF, platelet factor-4 (PF-4), IL-8, MCP-1 and MIP-1α (each at concentrations of 1 ng/ml to 1 μg/ml, log steps) did not significantly modulate histamine release from SMC induced by the three different secretagogues. RANTES exhibited a weak but significant potentiating effect on IgE-mediated activation. Stem cell factor (SCF) as a positive control was able to prime mast cell histamine release strongly. In addition, PF-4, MCP-1, RANTES and MIP-1α were incapable of inducing direct histamine release from SMC. In experiments with isolated human peripheral basophils, however, we observed potent Fc _ε RI-mediated priming effects evoked through IL-3, IL-5, and GM-CSF. We conclude that SMC derived from healthy donors are not targets of (immuno)modulatory factors that prime or stimulate basophils. Received: 21 June 1995