Enhanced expression of human metalloelastase (MMP-12) in cutaneous granulomas and macrophage migration

Accumulation of inflammatory cells such as macrophages may lead to degeneration of connective tissue matrix in various skin diseases. Macrophage metalloelastase, is a matrix metalloproteinase (MMP-12) capable of degrading elastin as well as various basement membrane components. To investigate the role of human macrophage metalloelastase in skin, we assessed by in situ hybridization and immunohistochemistry 66 specimens representing skin diseases characterized either by changes in elastic fibers or by pronounced infiltrations of extravasating and migrating macrophages. CD68 immunostaining was performed to identify the human macrophage metalloelastase-positive cells and Weigert's Resorcin-Fuchsin staining to reveal the status of elastic fibers. We found abundant expression of human macrophage metalloelastase mRNA in macrophages in areas devoid of normal elastic fibers in granulomatous skin diseases sarcoidosis, necrobiosis lipoidica diabeticorum, and granuloma annulare. Positive cells for human macrophage metalloelastase protein could be detected in the same regions as well as positive immunostaining for urokinase plasminogen activator. Of the other matrix metalloproteinases capable of degrading elastin, 92 kDa gelatinase colocalized with human macrophage metalloelastase, while 72 kDa gelatinase was produced by surrounding fibroblast-like cells. Furthermore, human macrophage metalloelastase was expressed by macrophages in areas with disrupted basement membrane, as assessed by type IV collagen staining, in pityriasis lichenoides and dermatitis herpetiformis. Specimens of anetoderma, acrodermatitis chronica atrophicans and pseudoxanthoma elasticum showed no signal for human macrophage metalloelastase. Matrilysin was not detected in any of the samples investigated. Our study suggests that human macrophage metalloelastase may contribute to elastin degradation occurring in granulomatous skin diseases and may aid macrophage migration through the epidermal and vascular basement membranes in inflammatory disorders.     

Accumulation of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage.

Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis and several matrix metalloproteinases have previously been implicated in this process. Using immunohistochemistry and in situ hybridization, we have studied the localization of two elastolytic matrix metalloproteinases, matrilysin (matrix metalloproteinase-7) and human macrophage metalloelastase (matrix metalloproteinase-12) in solar damage. Human macrophage metalloelastase protein was detected in the superficial dermis in areas of elastotic material. Matrix metalloproteinase-7 was seen in the mid-dermis in regions with less damaged elastic fibers and morphologically better preserved collagen as well as in a band-like pattern below basal keratinocytes in eight of 18 solar elastosis. In samples taken from healthy volunteers 3 d after repeated ultraviolet A or ultraviolet B photoprovocation, occasional immunopositive cells for human macrophage metalloelastase (stromal) or matrix metalloproteinase-7 (sweat gland epithelium) were detected. In samples taken 1 d after ultraviolet B exposure, however, basal keratinocytes were matrix metalloproteinase-7 immunopositive, explaining the linear immunostaining below basal keratinocytes noted particularly in ultraviolet B treated 3 d specimens. Upregulation of metalloelastase was also demonstrated in the skin of hairless mice after repeated ultraviolet exposure. In normal skin, no staining for human macrophage metalloelastase or matrix metalloproteinase-7 was observed in association with elastin. The amount of immunoreactivity for the substrates of matrix metalloproteinase-7, versican, and tenascin, was clearly increased in solar elastosis and photoprovocated skin; versican but not tenascin was detected in the same areas as matrix metalloproteinase-7. Our results suggest that both matrix metalloproteinase-7 and -12 may contribute to remodeling of elastotic areas in sun-damaged skin.     

Ultraviolet modulation of human macrophage metalloelastase in human skin in vivo

Human macrophage metalloelastase is a member of the matrix metalloproteinase family and is involved in degradation of elastin. We investigated the ultraviolet modulation of human macrophage metalloelastase in human skin in vivo. Ultraviolet induced human macrophage metalloelastase mRNA maximally (11.9-fold) within 16 h post-ultraviolet in human skin. This induction of human macrophage metalloelastase by ultraviolet was inhibited by pretreatment with the antioxidant N-acetyl cystein (20%) and vitamin E (5%) by an average of 54% and 47%, respectively, in human skin in vivo. Ultraviolet (30 mJ per cm2) and phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate, 50 nM) treatment increased expression of human macrophage metalloelastase mRNA and protein in the cultured human dermal fibroblasts, but not in the keratinocytes. Chronically sun-exposed human skin expressed significant amounts of human macrophage metalloelastase protein, which colocalized with the material of solar elastosis, whereas there was little expression in sun-protected skin of the same individuals. This study demonstrates that ultraviolet irradiation increases human macrophage metalloelastase expression in human skin in vivo, possibly in macrophages and fibroblasts, and ultraviolet-induced expression of human macrophage metalloelastase can be inhibited by antioxidant (N-acetyl cystein and vitamin E) pretreatment. Association of human macrophage metalloelastase with elastotic material suggests that it may play an important role in the development of solar elastosis, the hallmark of sun-induced damage in human skin in vivo.     

Expression of estrogen-related receptor gamma (ERRgamma) in human skin

Skin is a non-classical target for estrogens. Despite evidence showing that estrogen receptors (ER) are expressed in skin, there are still extensive gaps in our understanding of how estrogens exert their action in non-reproductive tissues. Estrogen-related receptor gamma (ERRgamma), an orphan member of the nuclear receptor superfamily, shows a strong sequence homology with estrogen receptor alpha but it does not bind estradiol. Here, for the first time, we demonstrate the expression of ERRgamma in adult human skin. ERRgamma mRNA was detected in the keratinocytes and fibroblasts of 8 female donor skins using RT-PCR. The presence of the protein was confirmed using immunohistochemistry on 11 adult human skins and Western Blotting on monolayer-cultures of fibroblasts and keratinocytes from respectively 4 and 2 donors. This study shows that ERRgamma is expressed in human skin and could intervene in a potentially new estrogen signaling pathway in the skin.     

Expression of human lymphocyte antigen (HLA)-DR on tumor cells in basal cell carcinoma

Immunohistologic studies of eight patients with basal cell carcinoma were undertaken using a series of monoclonal antibodies. In all of the patients, the majority of dermal infiltrates reacted with OKT3 and OKIa1 (HLA-DR), with a slight predominance of OKT4+ helper/inducer T cells (the mean OKT4/OKT8 ratio was 1.8). Both OKT4+ and OKT8+ cells were seen infiltrating the tumor masses. In addition, in five cases, human lymphocyte antigen (HLA)-DR was demonstrated on some tumor cells close to a vast number of HLA-DR+ infiltrates surrounding the carcinoma, but not on epidermal keratinocytes and tumor cells devoid of the HLA-DR+ infiltrates. A considerable number of OKT6+ dendritic cells were also observed surrounding the carcinoma. Staining with OKB7 and OKM1 revealed negligible reactive cells, and virtually none of the dermal infiltrates reacted with Leu-7 (HNK-1). These findings suggest that in addition to varied immunologically competent cells, expression of HLA-DR antigen on tumor cells may participate in a cellular immune reaction, a defense mechanism against tumor cell proliferation in basal cell carcinoma.     

Expression of uncoupling proteins in human skin and skin-derived cells

Uncoupling protein (UCP) is a mitochondrial membrane protein that uncouples oxidative phosphorylation. The physiological function of major isoforms of UCPs is related to the control of body temperature and reactive oxygen species production. Although skin is an important organ for heat radiation and protection against stress, the expression and function of UCPs in the skin have remained unclear. The expression of UCPs in human skin and its derived cells was researched at the mRNA and protein levels. The effects of norepinephrine (NE) and 9-cis retinoic acid (RA) on UCP expression were also investigated. The expression of UCP1 mRNA was found in the human epidermis and was upregulated in differentiated keratinocytes. UCP1 expression in keratinocytes was synergistically upregulated by NE and RA treatment. Significant expression of UCP2 and UCP3 was observed also in cultured keratinocytes and fibroblasts. By immunohistochemistry, localization of UCP1 was found in the granular layer of the epidermis, sweat glands, hair follicles, and sebaceous glands of various sites in the human body. UCP3 was widely found in the dermis. This showed that UCPs exist in human skin, with their expression being under hormonal control. These findings are in stark contrast with the well-accepted view of UCP1 expression being exclusive to brown adipose tissue.     

Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.     

Expression and characterization of membrane co-factor protein (MCP) in human skin

Membrane co-factor protein (MCP; CD46) is an integral membrane protein with molecular weight (MW) of the two species of 63 kD and 55 kD, and regulates autologous complement activation, with the activity of factor I cofactor. The quantity of each species is genetically regulated, and two codominantly inherited allelic variants account for the three phenotypic patterns. By immunohistochemical study, MCP was found both in the intercellular spaces of the epidermis and on the endothelial cells in the dermis of normal human skin in vivo. The intensity of the staining pattern was higher in the basal layer than in the granular layer. By Western blot analysis with use of a monoclonal antibody, MCP in the epidermis appeared as several bands ranged from 60-50 kD, with a major band of 56 kD, which was different from those in either polymorphonuclear cells, platelets, and cultured keratinocytes. No other variants were found in the epidermis obtained from skin of 20 normal humans. Complement activation in human skin may be regulated at several steps, including DAF and HRF20, thereby protecting cells from autologous complement attack.     

Expression of the fibroblast/macrophage marker 1B10 by spindle cells in Kaposi's sarcoma lesions and by Kaposi's sarcoma-derived tumor cells

BACKGROUND: Kaposi's sarcoma (KS) is a tumor whose ontogenic origin remains a matter of contention. KS tissues are characterized by predominant expression of endothelial markers, while KS-derived cell cultures are usually characterized by expression of mesenchymal non-endothelial cell markers. AIMS: In order to clarify the ontogenic origin of KS cells, we investigated the expression of the fibroblast/macrophage marker 1B10 in KS tissues (AIDS-associated KS, n = 9; classic KS, n = 6; iatrogenic KS, n = 6) and in KS-derived cell cultures. RESULTS: 1B10 was expressed by loosely distributed spindle-shaped cells in early 'patch-stage' KS and by a variable proportion of spindle cells in late 'plaque- and nodular-stage' KS. Using immunohistochemistry and immunoblot analysis, we found that, in vitro, reactivity for 1B10 was uniformly evidenced in fibroblasts and in KS-derived spindle cell cultures, irrespective of their histological or epidemiological setting. By contrast, vascular smooth muscle cells and endothelial cells were negative for 1B10. CONCLUSIONS: These results suggest that the KS spindle cells isolated in vitro may represent a particular subpopulation of the KS spindle cell compartment.     

Codon 12 Harvey-ras mutations are rare events in non-melanoma human skin cancer

ras mutations have been reported as an early event in some human malignancies and in the mouse skin model of multistep carcinogenesis; early studies in human non-melanoma skin cancers have reported variable rates of ras mutations. A recent study, however, has reported a high frequency of activating mutations of the Harvey- ras proto-oncogene in non-melanoma skin cancers, and the site specificity of the mutation at the second position of codon 12 prompted us to re-examine the importance of Ha- ras codon 12 mutations as an early event in the development of these tumours, using a combination of PCR and restriction fragment polymorphism of codon 12 of the Ha- ras gene. Dilution experiments confirmed that the method was sensitive and capable of detecting mutations at this codon when only 4% of the total alleles are mutated. We were surprised to find no mutations in the 40 basal cell carcinomas. 12 squamous cell carcinomas and 12 cases of Bowen's disease studied. We conclude that Ha- ras codon 12 mutations are rare events in human non-melanoma skin cancer in the U.K. The marked differences in the frequency of codon 12 Ha- ras mutations in published studies may relate to either technical artefacts, or differences in the molecular epidemiology between areas of low and high sun exposure.     

Matrilysin-1 (MMP-7) and MMP-19 are expressed by Paget's cells in extramammary Paget's disease

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare malignant neoplasm of apocrine gland bearing skin characterized by intraepidermal proliferation of adenocarcinoma cells. Tumor growth depends on the ability of tumor cells to migrate by proteolysis and on angiogenesis. The matrix metalloproteinase (MMP) enzymes have been implicated in both of these processes in other types of skin cancer. METHODS: The expression of MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-19 was analyzed by immuno- histochemistry and/or in situ hybridization in 27 EMPD and five mammary PD (MMPD) specimens. The distribution of laminin-5 (LN-5) and tenascin-C, two extracellular matrix proteins associated with tumor invasion, was studied by immunohistochemistry. RESULTS: MMP-7 (matrilysin-1) and MMP-19 were the most frequently expressed MMPs in Paget's cells. Overexpression of MMP-2, MMP-9, or MMP-13, which is seen in many cancers, was not evident in EMPD. LN-5 and tenascin-C positivity did not correlate with the level of invasion. MMP-7, MMP-13, and MMP-19 were detected abundantly in MMPD, while MMP-9 was absent. CONCLUSIONS: MMP expression did not generally associate with the level of invasion of EMPD. In three samples positive for MMP-7 and four samples positive for MMP-19, an underlying carcinoma was detected, suggesting the importance of these two MMPs as predictors of secondary EMPD or the putative origin of Paget's cells from the dermal adenocarcinoma cells of apocrine duct origin.     

Role of macrophage migration inhibitory factor (MIF) in the skin

Macrophage migration inhibitory factor (MIF) functions as a pleiotropic protein, participating in both inflammation and immune responses. MIF was originally discovered as a lymphokine involved in delayed type hypersensitivity and various macrophage functions, including phagocytosis, and tumor surveillance. Recently, MIF has been re-evaluated as a pro-inflammatory cytokine and identified as a pituitary-derived hormone, potentiating endotoxemia. MIF is ubiquitously expressed in various tissues, including the skin. Clinical evidence of increased MIF expression in inflammatory diseases supports this potential role of MIF in inflammation. In addition to its role in inflammation, MIF has been shown to exhibit growth-promoting activity, with anti-MIF antibodies effectively suppressing tumor growth and tumor-associated angiogenesis. This review presents the latest findings on the roles of MIF in the skin with regard to inflammation, the immune response, skin disease, tumorigenesis and cutaneous wound healing, and discusses its potential functions in various pathophysiological states.     

Expression of matrix metalloproteinases (MMP-2, MMP-3, and MMP-9) and fibronectin in lichen planus

BACKGROUND: Keratinocyte damage and lichenoid-interface reaction are the two major pathologic findings in lichen planus (LP). Matrix metalloproteinases (MMPs) are proteinases that participate in extracellular matrix (ECM) degradation and may play an important role in basal membrane (BM) damage in LP. Fibronectin (FN) mediates a variety of cellular interactions with ECM and plays important roles in cell adhesion, migration, growth and differentiation. OBJECTIVE: To determine MMP-2, MMP-3, MMP-9 and FN expressions in LP and discuss the possible associations. MATERIALS AND METHODS: Skin biopsy samples of 55 patients with LP and 11 normal skin were investigated. Five discoid lupus erythematosus (DLE) and 5 chronic dermatitis (CD) samples were also examined for comparison. Immunochemical stainings were performed for MMP-2, MMP-3, MMP-9 and fibronectin. RESULTS: Weak or absent expressions of MMP-2 and MMP-3 in epidermis; and dense MMP-9 expression in dermal inflammatory infiltrate cells were detected in LP. FN expression was lost in epidermal basal layer and papillary dermis. CONCLUSION: Loss of MMP-2, MMP-3 and FN in LP can be explained with the destruction of the epidermal basal layer. Similar expressions of MMP-2 and MMP-3 both in LP and DLE implied that these MMPs may be involved in the pathogenesis of interface dermatitis.     

Collagenase-3 (MMP-13) enhances remodeling of three-dimensional collagen and promotes survival of human skin fibroblasts

Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of remodeling and contraction of 3D collagen. Fibroblasts expressing MMP-13 in 3D collagen possessed altered filamentous actin morphology with patch-like actin distribution in cell extensions. The expression of MMP-13 promotes survival and proliferation of fibroblasts in floating collagen gel, and results in activation of Akt and extracellular signal-regulated kinase-1/2 by these cells. The results provide evidence for a novel role for human MMP-13 in regulating dermal fibroblast survival, proliferation, and interaction in 3D collagen, which may be an important survival mechanism for fibroblasts in chronic skin ulcers and contribute to scarless healing of adult gingival and fetal skin wounds.     

雌二醇对体外培养的人皮肤成纤维细胞基质金属蛋白酶-1基因表达的影响

绝经后女性皮肤老化的加速使人们很早就注意到了雌激素在皮肤老化中的作用.研究显示,在光老化和正常皮肤中,局部使用雌激素可以增加皮肤组织中胶原的含量,改善皮肤的厚度和弹性,本试验通过观察雌二醇对体外培养的人皮肤成纤维细胞基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)mRNA水平的影响,探讨雌二醇减缓皮肤老化的机制.