Thrombomodulin expression on dermal cells in normal and psoriatic skin

Abstract The various subsets of dermal cells with a dendritic appearance can be identified by phenotypic differences in cell markers. We report on the morphology and tissue distribution of dermal cells detected with a monoclonal antibody against thrombomodulin in histological sections of normal arm and scalp skin and psoriatic skin. Double staining with antibodies to factor XIIIa, CD34 and CD68 was also employed in scalp biopsies to elucidate the relationship between thrombomodulin⁺ dermal cells and dermal dendrocytes and macrophages described by others. Thrombomodulin⁺ dermal cells in normal arm skin had little cytoplasm with fine branched dendrites and tended to be localized just beneath the epidermis. In scalp skin these cells had longer, more numerous dendrites and were distributed in the papillae and perivascular adventitial dermis primarily in the upper and central reticular dermis. In psoriatic skin, thrombomodulin⁺ dermal cells had an increased cytoplasmic volume with stout, less branched dendrites and appeared in the papillae and among inflammatory cells. Dermal cells detectable by thrombomodulin expression were factor XIIIa^–, CD34^– and CD68^–, and seemed to represent a distinct subset of dermal cells which may function in tissue repair. However, thrombomodulin⁺ dermal cells and factor XIIIa⁺ dendrocytes were frequently seen close together and could act cooperatively to regulate extravascular thrombin homeostasis in both normal and pathological dermal environments. Received: 26 May 1997     

Cimetidine treatment for viral warts enhances IL-2 and IFN-gamma expression but not IL-18 expression in lesional skin

Cimetidine has been shown to improve various types of human neoplasms and more recently it has been shown to be effective in treating recalcitrant or multiple viral warts in some reports. However, it is not well understood why cimetidine is effective on those kinds of viral warts. We investigated 55 patients with multiple viral warts treated only with oral cimetidine for up to 4 months to examine the efficacy of treatment. The patients were divided into two groups: group A received oral cimetidine (<20 mg/kg/day) and group B received the drug (30 to 40 mg/kg/day). In addition, using real time PCR, we measured mRNA levels of the cytokines interleukin-2 (IL-2), IL-18, and interferon (IFN)-gamma taken from selected punch biopsy specimens before and during treatment. As a result, 34.5% (19/55) of the patients had a dramatic clinical improvement or complete remission (CR) of their viral warts and 23.6% (13/55) of the patients had partial responses (PR) within 4 months of cimetidine therapy. IL-2 and IFN-gamma mRNA levels were significantly increased and IL-18 mRNA levels were decreased in tissues of effectively treated viral warts. Our results show that the higher dose of oral cimetidine was more effective in treating multiple viral warts, that cimetidine activates Th1 cells to produce IL-2 and IFN-c and that their expression correlates with wart remission. These results suggest that cimetidine is an effective treatment for viral warts. In addition, based on the decrease in IL-18 mRNA elicited by the drug, IL-18 might be expressed by keratinocytes infected with HPV.     

Increased expression of adhesion receptors in both lesional and non-lesional psoriatic skin

Adhesion receptors and their ligands play a vital role in the immune system. We studied the expression of different adhesion receptors, using single- and double-staining immunohistochemical techniques, in both lesional and non-lesional skin specimens from seven psoriasis patients and in skin biopsy specimens from eight normal healthy controls. Our results showed an overall increased expression of several adhesion receptors in both lesional and non-lesional psoriatic skin. We consistently found an increased expression in particular of ICAM-1 and E-selectin on endothelial cells, and ICAM-1 on T cells and Langerhans cells. In contrast, a weak expression of VCAM-1 was found on endothelial cells and mononuclear cells in lesional psoriatic skin specimens alone. Interestingly, LFA-1 was also expressed on Langerhans cells, with a greater frequency in skin from lesional than from non-lesional sites, but was never expressed in skin from normal healthy individuals. Furthermore, significantly increased numbers of Langerhans cells and T cells with a positive reactivity for MAb HECA-452 were found in both lesional and non-lesional psoriatic skin. We hypothesize that the enhanced expression of adhesion receptors on migrating immunocompetent cells and endothelial cells of psoriatic skin in general facilitates the increased influx of activated T lymphocytes and other immunocomponent cells into the skin, and thus underscores the generalized character of the disease.     

银屑病患者皮损间充质干细胞的培养鉴定及其表达HES1和CXCL6的研究

【摘要】 目的 培养鉴定银屑病患者皮损处真皮间充质干细胞（DMSC）,并研究DMSC的发状分裂相关增强子1（HES1）和趋化因子配体6（CXCL6）表达情况。方法 分离培养15例银屑病患者皮损及18例正常人皮肤的DMSC,利用流式细胞仪进行表型鉴定,实时荧光定量聚合酶链反应（RT-PCR）及免疫印迹（Western blot）检测HES1和CXCL6的mRNA及蛋白表达水平,两组间比较采用t检验。结果 银屑病组DMSC形态与正常对照组无差异,但HES1 和CXCL6基因的mRNA水平分别是对照组的3.56和3.44倍,且差异有统计学意义（P < 0.05）。在蛋白水平,银屑病组DMSC中HES1和CXCL6的表达明显高于对照组（P < 0.05）。结论 银屑病患者皮肤DMSC HES1及CXCL6表达升高可能参与银屑病的发病。     

Glycosaminoglycan content in the media of cultured dermal fibroblasts derived from burn scar and normal skin

The glycosaminoglycan (GAG) content of the extracellular matrix of burn scar in humans has been reported to differ from that of normal skin. In order to investigate whether the GAG content altered as a result of functional changes in fibroblasts, the GAG content was determined in culture media of fibroblasts derived from growing burn scar, mature scar, and normal skin tissue. No statistical differences were observed in the population doubling-times of scar and normal skin. Mature scar showed significantly higher values for all the concentrations of uronic acid, hexosamine, and sulfate measured in the glycosaminoglycan, as compared with normal skin values, and the concentrations from growing scar were slightly higher than those for normal skin. The above results may suggest an increase in glycosaminoglycan sulfate synthesis following the hyperplasia of the matrix in burn scar tissue.     

Cyclin D1, B and A expression and cell turnover in psoriatic skin lesions before and after cyclosporin treatment

BACKGROUND: Cyclosporin induces a dramatic reversal to normality in psoriatic lesions, with a reduction of inflammatory infiltrate and epidermal proliferation. It is known that the cell cycle and cell proliferation are regulated by the sequential activation of cyclin-dependent kinase/cyclin complexes. AIM: We evaluated epidermal cell turnover and thickness, as well as the expression of cyclins D1, B and A in psoriatic skin before and after therapy with cyclosporin. METHODS: Epidermal thickness, mitotic and apoptotic indices (MI, AI), as well as the percentages of epidermal cell nuclei positive for Ki-67 and cyclins D1, B and A were calculated. Cytoplasmic positivity to cyclin B was also evaluated. RESULTS: After 6 weeks of therapy, we observed a clinical improvement of the disease and normalization of the epidermis. Epidermal thickness and Ki-67-, cyclins B- and A-positive nuclei percentage were significantly higher before therapy than after (0.52 +/- 0.05 mm vs. 0.21 +/- 0.03 mm, P < 0.001; 19 vs. 2.6, 19 vs. 3, and 12 vs. 1, respectively; P < 0.0005); cytoplasmic positivity to cyclin B was slightly higher before therapy (score 3 vs. 2-3). Cyclin D1 was negative or expressed in a low percentage of nuclei in psoriasis before therapy (0.78), whereas it was always negative after therapy. MI was 0.15 before therapy, whereas mitoses were almost absent afterwards. Apoptoses were undetectable before therapy, whereas a few apoptoses were observed after treatment (AI = 0.4). CONCLUSIONS: Overexpression of cyclins B and A, rather than D1 seems to characterize psoriasis. Their evaluation could provide further insights in understanding the development of this disorder and could be used to verify the efficacy of currently used therapies as well as future ones.     

UM4D4+ (CDw60) T cells are compartmentalized into psoriatic skin and release lymphokines that induce a keratinocyte phenotype expressed in psoriatic lesions

UM4D4 (CDw60), the surface molecule of a novel antigen-independent T-cell activation pathway, was found to be highly expressed on lesional psoriatic T cells. To examine whether UM4D4 represents a T-cell activation pathway for psoriatic T cells, a T-cell line was initiated from an acute skin lesion and cloned by limiting dilution. Clonality was verified by analysis of T-cell receptor gene rearrangement. All T-cell clones tested, whether CD4+2H4+CD8-, CD4+2H4-CD8-, or CD4-CD8+CD11b-, expressed UM4D4 and were activated by the monoclonal antibody anti-UM4D4. Lesional psoriatic T-cell clones were heterogeneous in the degree of anti-UM4D4-induced proliferation and in their production of IL-2 and gamma-interferon. Lymphokines released by anti-UM4D4 activation were capable of inducing ICAM-1 and HLA-DR expression on cultured normal keratinocytes. Thus, the high expression of UM4D4 on T-cells in psoriatic skin provides an alternative mechanism for T-cell activation that may be operative in the psoriatic lesional milieu. Indeed, activation of lesional T-cells through the UM4D4 molecule resulted in release of lymphokines that directly induced keratinocytes to express a phenotype displayed in psoriatic skin lesions.     

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.     

Clinical improvement in chronic plaque-type psoriasis lesions after narrow-band UVB therapy is accompanied by a decrease in the expression of IFN-gamma inducers -- IL-12, IL-18 and IL-23

Type-1 cytokine-producing T cells are important in the pathogenesis of psoriasis vulgaris, for which efficient therapy is provided by means of narrow-band ultraviolet-B (NB-UVB). The expression of the type-1 cytokine interferon-gamma (IFN-gamma) is regulated by interleukin-12 (IL-12), IL-15, IL-18 and IL-23; however, not much is known about the effect of this therapy on the levels of these cytokines in lesional psoriatic skin in situ. In this study, we investigated the effects of NB-UVB therapy on the expression of IFN-gamma-inducing cytokines. Ten patients with chronic plaque-type psoriasis selected to be treated with NB-UVB therapy were recruited for these experiments and the expression of cytokines IL-12, IL-15, IL-18, IL-23 and IFN-gamma in lesional psoriatic skin before, during and after therapy was determined with the help of immunohistochemistry. Double staining was performed in order to determine the cell types expressing these cytokines. The decrease in the psoriasis area and severity index was accompanied by a significant decrease in the expression of IFN-gamma, and concomitantly, significant reduction of IFN-gamma inducers -- IL-12, IL-18 and IL-23. Thus, we concluded that the decrease of IFN-gamma expression in psoriasis lesions after NB-UVB therapy could be a result of diminished expression of IL-12, IL-18 and IL-23 in lesional skin. Therapies targeting these three cytokines should, therefore, be considered in the treatment of psoriasis.     

Exosomes derived from human dermal papilla cells promote hair growth in cultured human hair follicles and augment the hair‐inductive capacity of cultured dermal papilla spheres

Dermal papillae (DP) play key roles in hair growth and regeneration by regulating follicular cell activity. Owing to the established roles of exosomes (Exos) in the regulation of cell functions, we investigated whether DP‐derived Exos, especially those from three‐dimensional (3D)‐cultured DP cells, affect hair growth, cycling and regeneration. Exos derived from 3D DP (3D DP‐Exos) promoted the proliferation of DP cells and outer root sheath (ORS) cells and increased the expression of growth factors (IGF‐1, KGF and HGF) in DP cells. 3D DP‐Exo treatment also increased hair shaft elongation in cultured human hair follicles. In addition, local injections of 3D DP‐Exos induced anagen from telogen and also prolonged anagen in mice. Moreover, Exo treatment in human DP spheres augmented hair follicle neogenesis when the DP spheres were implanted with mouse epidermal cells. Similar results were obtained using Exos derived from 2D‐cultured DP cells (2D DP‐Exo). Collectively, our data strongly suggest that Exos derived from DP cells promote hair growth and hair regeneration by regulating the activity of follicular dermal and epidermal cells; accordingly, these findings have implications for the development of therapeutic strategies for hair loss.     

IL-17-positive mast cell infiltration in the lesional skin of lichen planopilaris: Possible role of mast cells in inducing inflammation and dermal fibrosis in cicatricial alopecia

Lichen planopilaris (LPP) is a primary cicatricial alopecia characterized by the infiltration of lymphocytes in the upper portion of hair follicles. Inflammation around the bulge region of hair follicles induces destruction of hair follicle stem cells and tissue fibrosis, resulting in permanent hair loss. Treatment is still challenging, and the precise pathophysiology of this disorder is unknown. To clarify the pathogenesis of LPP, we performed histological and immunohistochemical analysis on specimens obtained from LPP patients. Formalin-fixed and paraffin-embedded samples were evaluated by staining with haematoxylin and eosin (HE), toluidine blue stain, immunohistochemistry and immunofluorescence. The immunohistochemical analysis demonstrated that CD4-positive T cells preferentially infiltrated into the follicular infundibulum in the LPP lesions. Toluidine blue stain detected a large number of mast cells in the inflammatory lesions of LPP. Interestingly, immunohistochemical analysis demonstrated that the mast cells harboured IL-17A- and IL-23-producing activity and expressed the IL-23 receptor. The number of IL-17A-positive mast cells was significantly higher in the LPP lesions than in normal scalp. Moreover, the IL-17 receptor was expressed exclusively in the follicular epithelial cells in the LPP lesions. These results suggested that mast cells infiltrating hair follicles might play a role in the pathogenesis of LPP via the IL-23/IL-17 axis.     

体外培养毛囊真皮鞘细胞与皮肤成纤维细胞形态结构和bFGF、EGFR表达差异

Gharzi等[1]初步证实毛囊真皮鞘细胞参与皮肤损伤修复,但相关作用机制研究报道不多.成纤维细胞是皮肤创伤愈合主要效应细胞,毛囊真皮鞘细胞与皮肤成纤维细胞一样均属于间充质细胞.本研究旨在通过两种细胞形态结构及部分愈合相关蛋白表达差异研究,初步探讨毛囊真皮细胞在创伤愈合中的作用.     

IL-4, IL-5, TGF-beta1 and IFN-gamma mRNAs detected by a new in situ amplification system in cicatricial pemphigoid

The process that induces chronic progressive scarring in cicatricial pemphigoid (CP), a rare group of autoimmune mucocutaneous blistering diseases, is still under investigation. The tendency to heal with scar formation observed in CP could be due to the specific localization of the antigen in the basement membrane zone or could depend on the frequent recurrence of the disease in a localized area. The release of soluble fibrogenic factors by inflammatory infiltrating cells has also been considered as pathogenetically relevant. The aim of this study is to evaluate the expression of mRNA for IL-4, IL-5, TGF-beta1, IFN-gamma in patients with CP, and investigate the role of the cytokine profile as a possible cause of the clinical features and course of the disease. Fourteen patients (3 male, 11 female; age range 40-72 years) with oral (n = 10), preputial (n = 3) and cutaneous (n = 1) CP were studied. The formalin-fixed and paraffin-embedded biopsies were examined by in situ hybridization performing a new amplification system based on biotinyl-tyramide. As a control, 4 patients (2 male, 2 female; age range 58-73 years) affected by bullous pemphigoid (BP), the most common autoimmune subepidermal blistering disease, were also examined. In CP, IL-4 mRNA expression was present in 4 out of the 14 cases analysed. IL-5 was detected in 12 CP biopsies. TGF-beta1 and IFN-gamma mRNAs were identified in 9 and 11 CP cases, respectively. In BP, an IL-4 hybridization signal could not be observed in any of the cases. By contrast IL-5, TGF-beta1 and IFN-gamma mRNA analyses were positive in all four BP cases. Our results suggest the presence of a T-cell population with a mixed cytokine pattern in the cellular infiltrate of both blistering diseases, with a corresponding increase of Th2-like activity in fully developed lesions, irrespective of the different sites involved. In addition, on the basis of the constant presence of TGF-beta1 mRNA in the different lesional phases of CP, and its overlapping expression in BP, we hypothesize that the involvement of additional factors is responsible for the scarring course typical of CP.     

Reappraisal of in situ immunophenotypic analysis of psoriasis skin: interaction of activated HLA-DR+ immunocompetent cells and endothelial cells is a major feature of psoriatic lesions

Psoriasis is an inflammatory skin disease of unknown aetiology. Many observations indicate that T cells play an important role in the pathogenesis of the disease. Upregulation of MHC class-II molecules on immunocompetent cells, endothelial cells and keratinocytes on lesional psoriatic skin has been regarded as a hallmark of the disease. However, there is some controversy in the literature regarding the cell types expressing class-II molecules and there is limited information about the presence of immune cells other than T cells and antigen presenting cells in the cellular infiltrates of psoriatic skin. We therefore reinvestigated the subject using immunocytochemical single and multiple staining techniques. In agreement with earlier reports, our studies showed that the cellular infiltrates in lesional skin consist largely of HLA-DR⁺/IL-2R⁺ T cells, HLA-DR⁺/CD1a⁺ Langerhans cells, and HLA-DR⁺/CD68⁺ macrophages. We found increased HLA-DR expression mostly on immunocompetent cells and endothelial cells, but no prominent HLA-DR expression on keratinocytes in lesional psoriatic skin. Upregulation of HLA-DR on endothelial cells and in mononuclear infiltrates was also evident in the non-lesional skin of psoriatic patients as compared with normal controls. B cells and natural killer cells were also found in the cellular infiltrates in lesional psoriatic skin. In spite of the presence of a large amount of activated T cells in the epidermis, we found that HLA-DR expression on keratinocytes was not a major feature of psoriatic skin.     

Proliferative responses of peripheral blood mononuclear cells from psoriatic patients to T lymphocyte-stimulating cytokines (IL-2, IL-3, IL-4, and granulocyte-macrophage colony-stimulating factor) and OK-432

Summary We have examined the effects of four well-characterized cytokines with T lymphocyte-stimulatory activity, i.e., interleukin (IL)-2, IL-3, IL-4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) on [³H]-thymidine incorporation in peripheral blood mononuclear cells (PBMs) obtained from patients with psoriasis. Under the influence of these cytokines, the incorporation of [³H]-thymidine into the PBMs was not different between psoriatic patients and healthy controls either in culture supplemented with pooled AB serum or with autologous serum. However, a potent immunopotentiator OK-432, which is a lyophilized preparation of penicillin-treated low virulence Su-strain of Streptococcus pyogenes group A3, induced significantly less incorporation of [³H]-thymidine into the PBMs from psoriatic patients than those from healthy controls [in both the culture supplemented with pooled AB serum (p<0.01) and that with autologous serum (p<0.01)]. This reduction in thymidine uptake was closely related to the disease activity as well as to the extent of skin lesions of the psoriatic patients. The defective immune response of PBMs from psoriatic patients to OK-432 probably reflects an abnormality at the level of interaction between monocytes and T cells or at the subsequent production and release of cytokines by them.