Dermal mast cells in scleroderma: their skin density, tryptase/chymase phenotypes and degranulation

To determine the distribution, tryptase/chymase phenotypes and degranulation of mast cells (MCs) in the dermis of patients with scleroderma, we examined MC density in the skin of 22 patients with systemic sclerosis (SSc) and 11 with localized scleroderma (LSc). We used antitryptase and antichymase antibodies after Carnoy's fixation. Detailed reports of two representative patients with SSc and LSc are included. In the scleroedematous stage (grade 1) showing oedema in both papillary and reticular dermis with variable homogenization of collagen bundles in the reticular dermis, MC skin density was variable in each specimen although MC skin density, as a whole, was significantly increased as compared with normal skin ( P  < 0.05). In the sclerotic stage (grade 2) characterized by homogenization of collagen bundles in the entire dermis, MC skin density was significantly decreased as compared with normal skin ( P  < 0.005). LSc showed changes similar to those in SSc. The ratio of MC_TC cells (both tryptase- and chymase-positive MC) to MC_T cells (tryptase-positive but chymase-negative MC) was variable in SSc and LSc. MC_T cells were exclusively dominant in three patients with SSc and two with LSc. In a patient with SSc (patient 1) showing remarkable perivascular and interstitial oedema in the upper dermis, MC skin density was increased in the oedematous portion and tryptase-positive granules were distributed in extracellular locations. In another patient with LSc (patient 2), tryptase positivity increased and chymase positivity decreased in both number and intensity as the skin sclerosis progressed. MCs must have variable interactions with the lesional skin in SSc and LSc. The present study suggests that MCs are involved in the development of interstitial oedema.     

Morphea limited to the superficial reticular dermis: an underrecognized histologic phenomenon.

Morphea (localized scleroderma) is a disease of unknown etiology, presenting as circumscribed areas of indurated skin. Histologically, most cases of morphea feature thickened collagen bundles in the deep reticular dermis, sometimes also extending into the superficial dermis or into the subcutis. We present six cases of morphea in which typical histologic features were restricted to the superficial dermis and contrast these with 27 additional biopsies of conventional morphea seen during the same time period. Sections were stained for elastic fibers, and dermal dendritic cells were labeled with antibodies to CD34 and Factor XIIIa. All six cases showed thickened collagen bundles restricted to the superficial dermis, sparing the deep dermis and without associated evidence of lichen sclerosus et atrophicus (LSA). Dermal elastic fibers were not appreciably decreased in number. There was loss of CD34-positive dermal spindle cells in each of our six superficial examples of morphea, which was restricted to the area of altered collagen in four of the six cases. This report highlights the distinctly uncommon phenomenon of morphea presenting solely as alteration of the superficial reticular dermis, without features of LSA. The selective loss of CD34-labeled spindle cells may provide information regarding the role of these putative immune accessory cells in morphea. Recognition of this manifestation of morphea may be helpful diagnostically.     

Elastic fiber pattern in scleroderma/morphea

Background: Scleroderma/morphea is characterized by expansion of the dermis with thickened collagen bundles and loss of CD34⁺ dermal dendrocytes. Variable elastic fiber changes have been described, but to our knowledge, no systematic study of the elastic fiber pattern correlated with CD34 expression has been reported.
Methods: To better define the typical elastic fiber morphology, we examined seven cases of normal skin and 28 cases of scleroderma/morphea ranging from inflammatory to sclerosing stages. All but four biopsies were submitted with a clinical impression of either scleroderma or morphea. CD34 immunohistochemistry was performed on 26 biopsies with available tissue.
Results: Elastic van Gieson stain showed preservation of elastic fibers in all cases. In addition, straightening with parallel orientation and compression between thickened collagen bundles was frequently present and was graded as limited in 46% and diffuse in 54% of cases. The extent of elastic fiber alteration correlated with the degree of sclerosis. A variable loss of CD34⁺ dermal dendritic cells was seen in all cases.
Conclusion: This study confirms the preservation and frequent presence of parallel, straightened and compressed elastic fibers in scleroderma/morphea and suggests that the elastic fiber pattern, in addition to CD34 immunohistochemistry, may serve as a useful diagnostic adjunct.     

Pulsed Dye Laser in the Treatment of Localized Scleroderma and Its Effects on CD34+ and Factor XIIIa+ Cells: An Immunohistochemical Study

Background

Localized scleroderma (morphea) is characterized by hardening and thickening of the dermis due to excessive collagen deposition. A decreased number of CD34+ cells and an increased number of Factor XIIIa+ cells are seen in the affected skin. The flashlamp pulsed dye laser (FLPDL) has been used in the treatment of localized morphea with promising results.

Objective

The purpose of this study was to evaluate the therapeutic effectiveness of the pulsed dye laser in localized scleroderma and to assess its effect on CD34+ cells, Factor XIIIa+ cells, and blood vessels.

Study Design

Thirty patients with plaque morphea were treated with a FLPDL (585 nm wavelength, 450 μs pulse duration). Fluence ranged from 7.5 to 8.5 J/cm². Sessions were performed biweekly for a maximum of 6 months. Clinical, histopathologic, and immunohistochemical assessments were performed.

Results

Patients showed varying degrees of improvement of indurated skin. There was no worsening or further improvement at the treated sites during the follow-up assessments at 3, 6, and 12 months. An increased number of CD34+ cells were found in both the upper and the lower dermis, and a decreased number of Factor XIIIa+ cells were found in the lower dermis.

Conclusion

The FLPDL is effective in the treatment of morphea, as confirmed by the changes in the pathologic tissue and levels of CD34+ and Factor XIIIa+ cells.     

Distribution of fibronectin in the skin of patients with scleroderma

The distribution of fibronectin (FN), a major glycoproteic component of extra-cellular matrix, has been studies by an indirect immunofluorescence technique in the skin of 50 normal controls and 19 sclerodermic patients. In the normal skin, FN was present mainly in the papillary dermis, as thin strips and less abundant in reticular dermis, bound to collagen bundles. In scleroderma skins, FN was increased in the deep dermis of extensive and evolutive lesions (11 cases). In an other hand, the distribution of FN was not modified in stabilized lesions (8 cases). We conclude that the detection of FN in the scleroderma skin is an useful marker of the activity of the systemic sclerosis process and we discuss the possible role of FN as a primary matrix for organization of the collagenous connective tissue during the sclerosing process.     

Increased expression of lysyl oxidase in skin with scleroderma

Summary Lysyl oxidase initiates cross-linkage of collagen and elastin by catalysing the formation of a lysine-derived aldehyde. In order to study cross-linking in scleroderma, we used monoclonal antibodies to lysyl oxidase to determine the localization of this enzyme in systemic and localized scleroderma, and compared the distributions obtained with that in normal skin. Using an indirect immunofluorescent antibody method and an avidin-biotinylated enzyme complex method. 11 cases of diffuse type of systemic scleroderma and seven cases of localized scleroderma were studied. In the oedematous stage of systemic scleroderma, intracellular and extracellular lysyl oxidase were remarkably increased in the dermis, particularly in groups around blood vessels. In the sclerotic stage of systemic scleroderma, lysyl oxidase was detected intracellularly in fibroblasts and extracellularly among collagen bundles between the lower dermis and the subcutaneous fat tissue. In localized scleroderma, a marked increase in lysyl oxidase was observed in mononudear cells and libroblasts near blood vessels in the lower dermis and in the subcutaneous fat tissue, in addition to the extracellular deposits between collagen bundles. The increase in lysyl oxidase in localized scleroderma was much more common than in the oedematous stage of systemic scleroderma. These findings indicated that intracellular and extracellular expression of lysyl oxidase expression was greater in sclerodermatous skin than in normal skin.     

Quantitative enzyme-histochemical analysis of tryptase- and chymase-containing mast cells in psoriatic skin

Summary Tryptase-containing mast cells have recently been found to be increased in the upper dermis of psoriatic lesions. In the present study, the distribution of chymaseand tryptase-containing mast cells was morphometrically analysed at different dermal levels of lesional and non-lesional psoriatic skin (12 patients) as well as normal human skin. Mast cell tryptase was identified enzyme-histochemically, using Z-Gly-Pro-Arg-MNA as the substrate. For demonstrating mast cell chymase, a simple and specific enzyme-histochemical staining method was developed, using Suc-Val-Pro-Phe-MNA as the substrate. All mast cells positive for chymase were also positive for tryptase and Giemsa stain. Although the number of tryptase-positive mast cells was slightly increased throughout the dermis of lesional psoriatic skin, this increase was most pronounced in the upper dermis immediately beneath, and in close contact with, the epidermis. In contrast, the number of chymase-positive mast cells was clearly decreased in the upper dermis of psoriatic lesions, but not in the deeper dermis, as compared with non-lesional psoriatic skin. In addition, all chymase-positive mast cells observed in the upper dermis were very weakly stained when compared with those in the deeper dermis. No differences were found between non-lesional psoriatic skin and normal skin in which the number of mast cells containing chymase was 72–73% of the number containing tryptase. The present results suggest that T mast cells particularly, containing tryptase but no chymase, proliferate in psoriatic lesions, and that the increase in tryptase activity and the decrease in chymase activitiy in the upper dermis may lead to an imbalance in the biochemical regulatory systems.     

Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization

Skin biopsies from seven patients with localized scleroderma (morphea) and from two healthy individuals were studied by in situ hybridization to localize the cells responsible for increased procollagen production. In scleroderma lesions, high levels of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNAs were detected in some but not all fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of pro alpha 1 (I) and pro alpha 1 (III) collagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens hybridized at the same time under identical conditions. Most of the scleroderma samples represented intermediate stages where the fibroblasts containing elevated levels of type I and type III procollagen mRNAs were located in the papillary and upper reticular layer of the dermis. One of the scleroderma samples from an early inflammatory stage of the disease was found to contain activated fibroblasts in all dermal layers and also in aggregates adjacent to inflammatory cell infiltrates. In situ analyses were also performed on cell cultures from affected and unaffected skin of one scleroderma patient. These experiments revealed a homogeneous population of activated fibroblasts in cultures producing high levels of collagen. The results suggest that development of fibrosis in scleroderma could evolve through activation of a certain fibroblast subpopulation. During cell culturing, however, cell selection or uncharacterized regulatory mechanisms appear to modulate the behavior of these cells with respect to collagen production.     

Increased mast cell numbers in the sclerotic skin of porphyria cutanea tarda

We quantitated numbers of mast cells in the sclerotic skin noted on the dorsa of the hands of 10 patients with porphyria cutanea tarda (PCT), and compared them with those of diffuse scleroderma and healthy controls. Mast cell counts in sclerodermoid skin of PCT patients were significantly greater than those in involved skin of 9 patients with diffuse scleroderma in its late stage and also greater than those in normal skin of 8 controls. When mast cell density was analyzed according to the depth of the dermis, an 84% increase was noted in the uppermost layer (0-0.2 mm in depth) and a 150% increase in the second uppermost layer (0.2-0.4 mm in depth) in the patients with PCT when compared with those in the corresponding sites of the controls. These results suggest a possible role of mast cells in the pathogenesis of sclerodermoid skin of PCT.     

Quantitative analysis of contact sites between mast cells and sensory nerves in cutaneous psoriasis and lichen planus based on a histochemical double staining technique

Summary The aim of the present study was to test further our previous hypothesis that the inflammatory reaction in psoriasis is neurogenic. For this purpose, contact sites between mast cells and sensory nerves were morphometrically analysed in the basement membrane zone, papillary dermis and three dermal zones of lesional/non-lesional psoriatic and lichen planus skin as well as in healthy control skin. The analyses were made on sections stained with a histochemical double stain developed for this study. With the double stain, active mast cell tryptase was stained blue enzyme histochemically, and the sensory nerves black using specific monoclonal anti-neurofilament antibodies with immunogold. In psoriatic lesions, both mast cells and mast cell — nerve contacts were markedly more frequent in the basement membrane zone and in the papillary dermis when compared with the corresponding areas in the other groups. Mast cell numbers were increased in both lesional and symptom-free skin in lichen planus, but no increase was found in the mast cell — nerve contacts. Increased contacts between mast cells and sensory nerves indicate that the elements exist for neurogenic inflammation in psoriatic lesions. These increased contacts are not due to the extensive inflammatory reaction only, because they were not observed in lichen planus lesions.     

Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma

We have established a mouse model for scleroderma induced by repeated local injections of bleomycin (BLM). Daily injection of BLM at a dose of >10 microg per ml for 4 wk induced histologic changes of dermal sclerosis, but not fibrosis, with thickened and homogenous collagen bundles and cellular infiltrates in BALB/C mice, whereas clinical signs of scleroderma were not apparent. In addition, lung fibrosis was also induced preceding the cutaneous changes. Sclerotic changes were not found in other sites of the skin distant from the injection site. Dermal sclerosis could also be induced by injecting BLM only every other day. The sclerotic changes of the dermis were sustained after ceasing BLM applications for at least 6 wk. Mast cells gradually increased in number as the sclerotic changes developed. Marked degranulation of mast cells was observed with elevated histamine release. The amount of hydroxyproline in skin was significantly increased at 4 wk of BLM treatment as compared with that in untreated or phosphate-buffered saline-treated mice. Anti-nuclear antibody was detected in serum of BLM-treated mice. Transforming growth factor-beta1 mRNA was detected at an early phase, while transforming growth factor-beta2 mRNA was strongly expressed at 4 wk when the sclerotic features were prominent. These results suggest that dermal sclerosis induced by BLM closely resembles systemic sclerosis both histologically and biochemically. Our mouse model can provide a powerful tool of inducing dermal sclerosis to examine the pathogenesis and the therapeutic approach of scleroderma.     

Identification of collagen fibrils in scleroderma skin

Skin from early and late stages of scleroderma has been shown to contain large amounts of thin (30-40 nm diameter) collagen fibrils that may be present in bundles or intermingled with large diameter fibrils (90-120 nm). The nature of these fibrils is unknown. Skin biopsies were obtained from involved areas of nine patients with progressive systemic sclerosis (PSS), one case of generalized morphea, one case of morphea, and six normal controls. Intact skin was analyzed by immunoelectron microscopy (IEM), while extracts were subjected to sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), Western immunoblotting, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Fine fibrils 20-40 nm in diameter in the mid to lower dermis of scleroderma skin were labeled with antibodies directed against the aminopropeptide (AP) of type III procollagen. Antibodies directed against the AP of type I procollagen labelled fine fibrils in the lower dermis. Larger fibrils (80-120 nm) did not label. pN alpha 1 (III) was found to be present in both normal and scleroderma skin. Extracts of scleroderma skin contained 2.5 times the amount of pN (III) collagen and 3.0 times the amount of fibronectin as did extracts of normal skin. The data indicate that the increase in thin fibrils in scleroderma skin is most likely due to an increase in type III collagen, which retains the AP at its surface.     

Quantitation of tryptase- and chymase-containing mast cells in cutaneous lichen planus.

The distribution and density of tryptase- and chymase-positive mast cells in lesional and non-lesional cutaneous lichen planus (LP) was analysed. For this, enzyme-histochemical staining techniques and morphometrical measurements were applied. In non-lesional LP skin, chymase-positive cells (TC mast cells) showed a distribution similar to that found in both non-lesional psoriatic skin and in normal skin. Tryptase-positive cells (reflecting both T and TC mast cells), however, were increased in number in the upper dermis of non-lesional LP skin. In lesional LP skin, there were fewer chymase-positive cells in the upper dermis, whereas there were more tryptase-positive cells. In the upper dermis, no differences in the number of tryptase containing cells were detected between lesional and nonlesional LP skin. In lesions of LP and psoriasis, tryptase-positive mast cells are increased but differ in their distribution in the papillary dermis. In psoriatic lesions, tryptase-positive cells are frequently observed in epidermal contact, a feature very rarely seen in LP lesions. The present results suggest that the increased numbers of T mast cells in the upper dermis of nonlesional LP skin may be involved in initiating the LP lesion. It seems unlikely that mast cells could be responsible for the epidermal basal cell damage, though T mast cells do participate in the general inflammatory reaction.     

Two cases of scleroderma associated with vibration syndrome

Case 1, a 49-year-old male who had been engaged in repair and reclamation of automobile tires, developed symptoms of vibration syndrome (Raynaud's phenomenon, numbness of both hands, tinnitus and impaired hearing) after some 30 years' use of a grinder and impact wrench. Two years thereafter, multiple sclerodermic lesions appeared over the trunk, upper extremities, and thighs; these disappeared about 2 years later. Histologically, hyperplasia and nodular swelling of collagen bundles were present in the dermis. An immunological study showed the serum to be positive for anti-centromere antibody, but no visceral lesions were demonstrable. This case corresponded to generalized morphea. Case 2, a 53-year-old male, developed symptoms of vibration syndrome (Raynaud's phenomenon, numbness of both hands, impaired hearing and arthralgia) after 25 years' use of a jack hammer in a quarry. Thereafter, sclerodermic changes of the forearms, lower legs, face and abdomen occurred with an associated sclerodactyly. Histological examination of involved skin revealed diffuse hyperplasia and homogenization of collagen bundles throughout the entire thickness of the dermis. These findings, together with serum positivity for anti-RNP antibody and dilation of the lower portion of the esophagus, led us to a diagnosis of progressive systemic sclerosis. We inferred that the vibration syndrome in the present cases might be related etiologically to these forms of scleroderma.     

Generalized morphea with blisters. A case report

A patient suffering from generalized morphea developed blisters in the morpheic plaques on her buttocks. The plaques had an increased concentration of serum aminoterminal propertice of type III procollagen, an echo response and thickened skin on ultrasound scanning, and compact bundles of collagen fibrils with bimodal distribution of the diameters. The blisters appeared as an echo-free band in the subepidermal zone by ultrasound scanning. Electron microscopy revealed blisters in the upper papillary dermis, surrounded by degraded collagen fibrils.     

Medium-dose UVA1 phototherapy in localized scleroderma and its effect in CD34-positive dendritic cells

BACKGROUND: UVA1 radiation seems to be effective in morphea. CD34+ dendritic cells are significantly decreased in lesional skin of morphea patients. OBJECTIVE: We evaluated the therapeutic effectiveness of medium-dose UVA1 phototherapy in localized scleroderma and its effect in the number of dermal CD34+ dendritic cells in skin biopsy specimens of these patients. METHOD: Patients were irradiated with UVA1 (30 J/cm(2)) 30 times. Dermal CD34+ dendritic cells were counted before and after therapy. RESULTS: There was clinical improvement after UVA1 irradiation. Dermal CD34+ dendritic cells significantly increased after UVA1 irradiation. CONCLUSION: Medium-dose UVA1 therapy is effective in the treatment of localized morphea. Effectiveness is associated with an increase in the number of CD34+ dendritic cells in the dermis.     

Dermal Changes in Osteoporosis Following Prolonged Treatment with Human Growth Hormone

Five patients with osteoporosis were treated with human growth hormone (hGH) for a year and the changes in their skin were studied by light and electron microscopy. The abnormally thin skin of osteoporosis appeared to change towards normal after treatment with hGH. There was a consistent proliferation of blood vessels, and increased number of mast cells and fibrocytes. The collagen bundles and elastic tissue fibers appeared hyperplastic and more horizontally oriented. The fine, vertical elastic fibrils of the papillary dermis had appeared decreased before treatment, but seemed to be restored to their normal configuration after treatment. Since there was no evidence of stimulation of hair, sebum, or melanin such as occurs in acromegaly, it is suggested that the scope of the direct action of hGH on the skin is limited to mesenchymal structures.     

Histologic Observations of Pleomorphic, Variably Acid-fast Bacteria in Scleroderma, Morphea, and Lichen Sclerosus et Atrophicus

ABSTRACT: Variably acid-fast cocoid forms, suggestive of cell wall deficient forms of mycobacteria, were observed in the dermis in microscopic sections of skin from six patients with generalized scleroderma, 10 patients with localized scleroderma (morphea), and four patients with lichen sclerosus et atrophicus(LSA). These cocoid forms werde found within the collagen bundles, around the adnexae (Hair shafts, pilosebaceous units, eccrine glands), and less commonly around the blood vessels and nerves. These cocoid forms may be related to cocci and also to granular cocoid elements of corynebacteria-like coccobacilli, which, on occasion, can be cultured from the skin of these three diseases. The findings in this study support the three-decade old hypothesis concerning the constant association of pleomorphic acid-fast bacteria with scleroderma. The study also suggests that closely related diseases, such as morphea and LSA, are also associated with the presence of similar appearing microbes.     

Morphea--a tick transmitted borreliosis of the skin? A contribution to the pathogenesis of circumscript scleroderma

We report on a 34-year-old patient suffering from erythema chronicum migrans, who developed clinically and histologically typical morphea, which was confined to the area previously involved by the erythema migrans. The patient's serum antibody level against borrelia burgdorferi spirochetes was significantly elevated. By means of the silver impregnation technique, we were able to identify spirochetal organisms both in the lower dermis and within the septa of the subcutaneous fatty tissue. In frozen sections, spirochetes were demonstrated by the immunoperoxidase method using specific anti-spirochetal antibodies. For a better assessment of the composition of the inflammatory infiltrate in morphea, we applied a panel of monoclonal antibodies to a sensitive two-stage immunoperoxidase technique. Helper-inducer T-cells and a large number of suppressor-cytotoxic T-cells were observed both in the perivascular infiltrate and between collagen fibers in close proximity to the HLA-DR reactive fibroblasts. A large number of mast cells were seen in the dermal infiltrate. The detection of spirochetal organisms in histological sections as well as the demonstration of closely associated helper-inducer T-cells, macrophages, and activated fibroblasts in the dermis strongly suggest that a cell-mediated immune response against borrelia may be the dominant pathogenetic event in this variant of scleroderma. Activated T-lymphocytes and various factors secreted either by activated lymphocytes or mast cells may cause proliferation of fibroblasts, which can lead to increased collagen synthesis and dermal fibrosis.