Serum-Free Serial Culture of Adult Human Keratinocytes From Suction-Blister Roof Epidermis

Coating cell culture flasks with natural extracellular matrix (ECM) enhanced the culture of adult human keratinocytes from suction-blister roof epidermis in an environment without fetal calf serum (FCS), bovine pituitary extracts or cellular feeder layers. A higher incidence of cell attachment on natural ECM was observed than on collagen and human fibronectins(HFN)-coated plastic dishes, and natural ECM was necessary for growth and proliferation of attached cells under the culture conditions used. Cells in primary culture grew to confluency on natural ECM-coated surfaces within about 14 days, and subsequent serial passage could be made up to fourth passage in collagen- and HFN-coated plastic flasks. Cultured keratinocytes in this serum-free environment formed colonies of small cuboidal, healthy cells with little keratinization or stratification and demonstrated antigenic characteristics of human basal cells

Th2 Suppressor Cells Are More Susceptible to Sphingosine Than Th1 Cells in Murine Contact Photosensitivity

Murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) is a cutaneous delayed-type hypersensitivity reaction in which both positive and negative regulatory pathways exist. The latter pathway is mediated by antigen-specific, CD4+ suppressor T cells (CPS-Ts) that are Th2 cells. We examined the effects of sphingosine and synthetic cell-permeable analogs of ceramide on the cellular kinetics of CPS-Ts and immune lymph node cells from TCSA-photosensitized mice (CPS-LNC), along with other murine T-cell populations. The addition of sphingosine at 10 or 3 μM to in vitro cultures suppressed DNA synthesis of CPS-Ts and Th2 clones, including D10 cells and 24-2 cells, but not that of CPS-LNC or Th1 clones, including 23-1-8 and 28-4 cells. This suggested that sphingosine exerts its inhibitory effects preferentially on the proliferation of Th2 cells. Although suppressing DNA synthesis, sphingosine augmented the production and mRNA expression of interleukin-4 (IL-4) and enhanced the expression of the IL-4 receptor in CPS-Ts. In addition, the ability of sphingosine to induce signal transduction of CPS-Ts was confirmed by elevation of the intracellular free Ca++ concentration. Because CPS-Ts exposed to sphingosine exhibited a lower G2M/G1 ratio than control, these seemingly ambivalent phenomena may be caused by retardation of the G1 to S phase progression, a cell-cycle dysregulation known to augment cytokine production. In contrast to sphingosine, cell-permeable ceramide did not affect the proliferation of these cells when stimulated with mitogen/antigen and did not augment IL-4 production by CPS-Ts. Our study suggests that sphingosine modifies the Th1/Th2 balance by preferentially affecting the cellular kinetics of Th2

Trps1-deficient transplanted skin gave rise to a substantial amount of hair: Trps1 is unnecessary for hair development

Trps1 is considered as an important gene involved in the interactions between the epithelial and mesenchymal cells during hair follicle morphogenesis. The number of hair follicles in Trps1 Knockout (KO) newborn mouse skin was significantly lower than that in wild-type (WT) newborn skin. To gain insight into the functional role of Trps1 in hair development, we transplanted Trps1 KO newborn mouse skin on the backs of nude mice and examined hair growth at day 42 after transplantation. Surprisingly, transplanted skin from Trps1 KO newborn mice gave rise to substantial amount of hair, although the hair was softer than that of WT mice. Histological examination revealed that the diameter of both hair follicles and hair shafts were significantly lower, whereas the density of hair follicles showed no significant difference between the Trps1 KO and WT mice. We introduce mouse hair follicles as a fascinating model to study the functions of Trps1 in mouse hair growth and pathology. This model suggests that the function of Trps1 is unnecessary for the development of normal hair follicles and hair shafts, although the loss of Trps1 affects the diameters of hair follicles and hair shaft

Immunohistochemical Analysis of CXCR4 Expression in Fibrohistiocytic Tumors

Functional chemokine receptors are expressed in many malignant tumors. These receptors promote tumor growth and metastasis in response to endogenous chemokines. We analyzed the expression of CXCR4, CCR6 and CCR7 in fibrohistiocytic tumors, including dermatofibrosarcoma protuberance (DFSP), malignant fibrous histiocytoma (MFH), dermatofibroma (DF) using immunohistochemistry. We also investigated the relationship between CXCR4 and CD34, the latter of which is an immunohistochemical marker for DFSP. We observed a higher expression of CXCR4 in DFSP and MFH as compared with DF. Interestingly, a significantly higher expression of CXCR4 was detected in relapsed DFSP than in non-relapsed DFSP, but no significant differences were detected between non-relapsed DFSP and DFSP with CD34 immunostaining. Moreover, MFH had strong immunoreactivity for CXCR4, CCR6 and CCR7. These findings suggest that the assessment of CXCR4 immunoreactivity in fibrohistiocytic tumors is a useful tool for predicting tumor aggressiveness

Treatment of T Lymphocytes with 8-Methoxypsoralen Plus Ultraviolet A Induces Transient but Biologically Active Th1-Skewing Cytokine Production

8-Methoxypsoralen plus ultraviolet A light is suggested to shift T lymphocytes from Th2 to Th1 cells. To clarify this issue, we examined the effects of 8-methoxypsoralen/ultraviolet A on the expression/production of cytokines in peripheral blood mononuclear cells from normal subjects and a Sézary syndrome patient. 8-Methoxypsoralen/ultraviolet A augmented the expression of mRNAs for interferon-γ and interleukin-2 and reduced those for interleukin-4 and interleukin-10. It seems that this enhancement of Th1 cytokines is caused by increment of cytokine production by Th1 cells but not by conversion of Th2 cells to produce Th1 cytokines. The number of interferon-γ-secreting lymphocytes was markedly increased in 8-methoxypsoralen/ultraviolet A-treated peripheral blood mononuclear cells 20 h after treatment, whereas that of Th2 cytokine-producing cells was decreased. Accordingly, the amount of interferon-γ was elevated in culture supernatants from 8-methoxypsoralen-phototreated peripheral blood mononuclear cells, whereas interleukin-4 was significantly reduced. This enhanced production of interferon-γ, however, was found only until 3 d after 8-methoxypsoralen phototreatment and was declined by 5 d after treatment. Finally, 8-methoxypsoralen/ultraviolet A treatment of T cells regulated their ability to induce keratinocyte CD54 expression. Our results show that 8-methoxypsoralen/ultraviolet A has a transient but biologically active Th1-skewing action in human T cells, suggesting that 8-methoxypsoralen/ultraviolet A exerts a beneficial therapeutic effect on Th2-mediated or Th2-malignant diseases

The Protective Effects of Ultraviolet A1 Irradiation on Spontaneous Lupus Erythematosus-Like Skin Lesions in MRL/lpr Mice

We investigated the effects of ultraviolet A1 (UVA1) irradiation on spontaneous lupus erythematosus- (LE-) like skin lesions of MRL/lpr mice, using a disease prevention model. UVA1 irradiation significantly inhibited the development of LE-like skin lesions, without obvious changes of the disease including renal disease and serum antinuclear antibody levels. Besides the massive infiltration of mast cells in the LE-like skin lesions, in the nonlesional skins, more mast cells infiltrated in the UVA1-irradiated group compared with the nonirradiated group. Although apoptotic cells were remarkably seen in the dermis of UVA1-irradiated mice, those cells were hardly detectable in the dermis of the nonirradiated mice without skin lesions. Further analysis showed that some of those apoptotic cells were mast cells. Thus, UVA1 might exert its effects, at least in part, through the induction of the apoptosis of pathogenic mast cells. Our results supported the clinical efficacy of UVA1 irradiation for skin lesions of lupus patients

Antinuclear antibody-keratinocyte interactions in photosensitive cutaneous lupus erythematosus

Autoimmune diseases are characterized by various circulating autoantibodies, especially antinuclear antibodies (ANA). It has been a long-standing issue as to whether andlor how ANA interact with epidermal cells to produce skin lesions. Of these ANA, the anti-SS-AIR0 antibody is the most closely associated with photosensitivity in patients with systemic lupus erythematosus (SLE) and its subgroups, including subacute cutaneous lupus erythematosus (SCLE) and neonatal lupus erythematosus (NLE). SS-A/Ro antigens are present in the nucleus and cytoplasm, and interestingly, ultraviolet B (UVB) light translocates these antigens to the surface of the cultured keratinocytes. Thus, anti-SS-AIR0 antibodies in the sera can bind to the relevant antigens expressed on the UVB-irradiated keratinocyte surface, and have been speculated to be an important inducer of antibody-dependent keratinocyte damage. This interaction between the anti-SS-A/Ro antibodies and UVBirradiated keratinocytes may induce the skin lesions through a cytotoxic mechanism. This review will focus on the involvement of antibody-dependent cellular cytotoxicity in the pathogenesis of the skin lesions observed in photosensitive cutaneous lupus erythernatosus