Shigeru Kawara

Role and interaction of connective tissue growth factor with transforming growth factor-β in persistent fibrosis: A mouse fibrosis model

Skin fibrotic disorders are understood to develop under the influence of some growth factors, such as transforming growth factor‐beta (TGF‐β), basic fibroblast growth factor (bFGF), or connective tissue growth factor (CTGF). To establish an appropriate animal model of skin fibrosis by exogenous application of growth factors, we investigated the in vivo effects of growth factors by injecting TGF‐β, CTGF, and bFGF into the subcutaneous tissue of newborn mice. A single application of TGF‐β or bFGF resulted in the formation of transient granulated tissue that disappeared despite 7 days of consecutive injections. A single CTGF injection also caused slight granulation. However, injecting TGF‐β plus CTGF produced long‐term fibrotic tissue, which persisted for at least 14 days. Also, fibrotic tissue was observed when CTGF was injected from 4 to 7 days after TGF‐β injections for the first 1–3 days. In situ hybridization analysis revealed the expression of CTGF mRNA in the fibroblasts at least in a few fibrotic conditions. These findings suggest that either CTGF mRNA or an application of exogenous CTGF protein is required for the development of persistent fibrosis. From our study, it appears that interaction of several growth factors is required for persistent fibrotic tissue formation, with TGF‐β causing the induction and CTGF needed for maintenance of skin fibrosis. The animal model on skin fibrosis by exogenous application of growth factors developed in this study may prove useful for future studies on fibrotic disorders. J. Cell. Physiol. 181:153–159, 1999.

Connective tissue growth factor causes persistent proα2(I) collagen gene expression induced by transforming growth factor‐β in a mouse fibrosis model

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine‐rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor‐β (TGF‐β). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF‐β transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF‐β caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF‐β and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proα2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the −17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial β‐galactosidase gene. Serial injections of CTGF after TGF‐β resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF‐β alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF‐β in comparison with the injection of TGF‐β alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF‐β‐induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.

Antiepiligrin (laminin 5) cicatricial pemphigoid associated with an underlying gastric carcinoma producing laminin 5

Although bullous pemphigoid and cicatricial pemphigoid are sometimes associated with malignancy, it remains uncertain whether such an association is pathogenetically related or just a coincidence attributable to the advanced age of the patients. We report a 61‐year‐old patient with antiepiligrin (laminin 5) cicatricial pemphigoid (AeCP) associated with an advanced gastric carcinoma. The gastric carcinoma cells in this patient were shown to produce laminin 5 by immunofluorescence microscopy, and the patients serum contained autoantibodies directed against laminin 5 on immunoprecipitation. Furthermore, the blistering symptoms and the titre of antibasement membrane zone antibodies coordinately changed with the resection and subsequent relapse of the gastric cancer. These observations suggest that the gastric carcinoma producing laminin 5 may have induced the production of autoantibodies to this laminin, which were pathogenic to the skin and mucous membranes in this patient. This report demonstrates a link between this autoimmune subepithelial blistering disease and malignancy. It is of interest and potential great importance to examine other cases of AeCP for such a potential association.

Transforming growth factor-β isoforms differently stimulate proα2 (I) collagen gene expression during wound healing process in transgenic mice

The role of many growth factors and cytokines in the process of wound healing has been intensively investigated in recent two decades. Among them, transforming growth factor‐βs (TGF‐βs) are well known to have a potent stimulatory effect on collagen synthesis as shown in various in vivo experimental systems. In the present study, we examined the effects of various growth factors on the promoter activity of the proα2 (I) collagen gene (COL1A2) during the wound healing process. For this purpose, we utilized transgenic mice harboring the −17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase or a bacterial β‐galactosidase gene. These mice exhibited normal phenotypic expression and the wound healing process was not impaired. Full thickness wounds were made by punch biopsy. We examined the effects of TGF‐β1, ‐β2, ‐β3, basic fibroblast growth factor, platelet‐derived growth factor, and connective tissue growth factor by applying them locally to the open wound every 2 days. Among the growth factors examined, all of the three isoforms of TGF‐β exhibited a more potent stimulatory effect on COL1A2 promoter activity than did other factors. In addition, while TGF‐β1 and ‐β2 significantly increased the number of fibroblasts which were positive for X‐Gal staining, TGF‐β3 treatment did not change the number of β‐galactosidase expressing cells. Accumulation of collagen fibers was observed to the same extent in the mice treated with TGF‐β1 and those with TGF‐β3. These findings suggest that TGF‐β1 and ‐β3 have similar but not identical regulatory mechanisms of COL1A2 expression, and that their pathophysiological roles in wound healing might be different from each other. J. Cell. Physiol. 190: 375–381, 2002.

Autoantibodies directed against the protease inhibitor calpastatin in psoriasis

Psoriasis is believed to be a T cell‐mediated autoimmune disease, but also exhibits autoantibody production. Calpastatin is an endogenous inhibitor of calpain, a ubiquitous protease that regulates inflammatory processes. Anti‐calpastatin autoantibody was first identified as an autoantibody specific to rheumatoid arthritis, but has been also detected in other autoimmune diseases. In this study, we examined the presence and levels of anti‐calpastatin antibody in 77 psoriasis patients by enzyme‐linked immunosorbent assay. Compared with normal controls, psoriasis patients exhibited significantly elevated IgG anti‐calpastatin antibody levels that were similar to those found in rheumatoid arthritis patients. Remarkably, IgG anti‐calpastatin autoantibody in sera from psoriasis patients inhibited calpastatin activity. Calpain II expression was up‐regulated in psoriasis skin lesions compared with normal skin while calpastatin expression was normal. The results of this study reveal the presence of anti‐calpastatin autoantibody in psoriasis.

High frequency of DNA aneuploidy detected by DNA flow cytometry in Bowen's disease

To detect DNA aneuploidy in Bowens disease, we investigated DNA flow cytometric analysis. Single cell suspensions were prepared from 18 fresh samples histopathologically diagnosed as solitary Bowens disease and analyzed by DNA flow cytometry. In 16 (89%) of 18 lesions, DNA aneuploidy was demonstrated with a single aneuploid peak. DNA indices ranged from 1.29 to 1.74. The incidence of DNA aneuploidy in Bowens disease is higher than those of cutaneous squamous cell carcinoma, which was 25-80% in the previous reports. Therefore, in Bowens disease. DNA aneuploidy may not imply a good marker for characteristics of non-melanoma skin cancer. A single aneuploid peak commonly observed in Bowens disease suggests that this disease consists of the monoclonal proliferation of keratinocytes containing abnormal DNA content.

Tape stripping induces marked epidermal proliferation and altered TGF-α expression in non-lesional psoriatic skin

Psoriasis is a chronic inflammatory disorder characterized by epidermal hyperproliferation. Although recent evidence suggests that T cell activation is a primary trigger for psoriasis lesions, there may be alterations in the keratinocyte growth regulatory pathways which induce epidermal hyperproliferation in psoriatic patients. To test this hypothesis, we investigated the proliferative activity of epidermal keratinocytes 48 h after tape stripping, one of the standard mechanical ways to stimulate the epidermis, in 20 psoriasis patients and in 18 controls. Epidermal cell kinetics were assessed with DNA flow cytometry, the mitotic index, bromodeoxyuridine incorporation, Ki-67 antigen expression and DNA polymerase alpha expression. The expression of TGF-alpha and EGF receptors, critical mediators of keratinocyte proliferation, were also investigated immunohistochemically. The results of multiparameter assays showed that the baseline proliferative activity in uninvolved skin was the same in psoriasis patients and normal controls. After tape stripping, although both psoriasis patients and the normal controls showed significant increases in epidermal cell proliferation, the values of all the parameters investigated were significantly greater in the psoriasis patients than in the normal controls. EGF receptors were overexpressed in basal and suprabasal keratinocytes after tape stripping in both the psoriasis patients and the normal controls. In contrast, overexpression of TGF-alpha was only observed in the patients with psoriasis, which may explain their increased proliferative response to trauma.

Decreased expression levels of L‐selectin on subsets of leucocytes and increased serum L‐selectin in severe psoriasis

L‐selectin is a leucocyte adhesion molecule involved in leucocyte interactions with vascular endothelial cells. Following leucocyte activation L‐selectin is endoproteolytically released from the cell surface. To assess whether psoriasis vulgaris results in systemic leucocyte activation, we examined expression levels of L‐selectin on subsets of peripheral blood leucocytes from patients with psoriasis (n = 25) and normal control subjects. Serum levels of soluble L‐selectin were quantified by ELISA in patients with psoriasis (n = 75), pustulosis palmaris et plantaris, and contact dermatitis, as well as normal control subjects. Psoriasis severity was evaluated by psoriasis area and severity index (PASI). L‐selectin expression levels on CD4+ T cells, B cells, monocytes, and neutrophils from patients with severe‐type psoriasis (PASI ≥ 15) was significantly decreased compared with leucocytes from normal control subjects. Furthermore, L‐selectin expression on CD4+ T cells showed good inverse correlation with PASI scores. Monocyte L‐selectin expression was restored when the skin lesions of psoriasis were remitted. The frequencies of L‐selectin+ CD4+ T cells or L‐selectin+ CD8+ T cells from patients with psoriasis were almost normal. Serum L‐selectin levels in patients with severe‐type psoriasis were significantly higher than those in normal control subjects. These results suggest that subsets of leucocytes may be activated in psoriasis, and that L‐selectin expression levels on some leucocyte subsets, especially CD4+ T cells, tend to correlate with disease severity of psoriasis.

Pigmented Dermatofibrosarcoma protuberans (Bednář Tumor) Occurring in a Japanese Infant

A case of pigmented dermatofibrosarcoma protuberans (Bednář tumor) occurring in a Japanese infant is reported. A 6-month-old girl developed a 16 × 10 mm erythematous tumor with a slight elevation on the lower back at 1 month of age. Histological examination revealed proliferation of spindle-shaped, fibroblast-like cells in the myxoid interstitium. Subsequently the tumor grew gradually to a red-purplish tumor measuring 45 × 36 mm. The second biopsy specimen presented hypercellular proliferation composed of spindle-shaped cells showing slight nuclear atypia and a characteristic storiform pattern, with scattered slender dendritic cells containing a large amount of brown pigment. Immunohistochemical studies of the second biopsy specimen showed that the spindle-shaped tumor cells were positive for vimentin and CD34 and negative for factor XIIIa. The number of CD34-reactive spindle-shaped tumor cells increased in the second biopsy specimen compared with the first biopsy.

Ultraviolet Light Exposure Suppresses Contact Hypersensitivity by Abrogating Endothelial Intercellular Adhesion Molecule-1 Up-Regulation at the Elicitation Site

Hapten sensitization through UV-exposed skin induces systemic immune suppression, which is experimentally demonstrated by inhibition of contact hypersensitivity (CHS). Although this UV-induced effect has been shown to be mediated by inhibition of the afferent phase of the CHS, the UV effects on the efferent (elicitation) phase remain unknown. In this study, UV effects on endothelial ICAM-1 expression at elicitation sites were first examined. Mice were sensitized by hapten application onto UV-exposed back skin, and ears were challenged 5 days later. ICAM-1 up-regulation at nonirradiated elicitation sites following hapten challenge was eliminated by UV exposure on sensitization sites distant from elicitation sites. To assess whether loss of the ICAM-1 up-regulation at elicitation sites contributed to UV-induced immunosuppression, we examined CHS responses in UV-exposed ICAM-1-deficient (ICAM-1−/−) mice that genetically lacked the ICAM-1 up-regulation. ICAM-1−/− mice exhibited reduced CHS responses without UV exposure, but UV exposure did not further reduce CHS responses in ICAM-1−/− mice. Furthermore, ICAM-1 deficiency did not affect the afferent limb, because ICAM-1−/− mice had normal generation of hapten-specific suppressor and effector T cells. This UV-induced immunosuppression was associated with a lack of TNF-α production after Ag challenge at elicitation sites. Local TNF-α injection before elicitation abrogated the UV-induced CHS inhibition with increased endothelial ICAM-1 expression. TNF-α production at elicitation sites was down-regulated by IL-10, a possible mediator produced by hapten-specific suppressor T cells that are generated by UV exposure. These results indicate that UV exposure inhibits CHS by abrogating up-regulation of endothelial ICAM-1 expression after Ag challenge at elicitation sites.