Topical retinoids, such as adapalene, are an integral part of acne therapy in most regions and are considered appropriate first-line therapy by international guidelines for all cases of acne with the exception of the most severe. However, there are currently no topical retinoids available for the treatment of acne vulgaris in Japan.
To confirm efficacy and safety of adapalene gel 0.1% versus the corresponding gel vehicle in the treatment of Japanese patients with acne vulgaris for up to 12 weeks.
A total of 200 patients were randomized to receive adapalene gel 0.1%, or vehicle once-daily for 12 weeks. Percent reduction in lesion counts (total, inflammatory, and non-inflammatory) and subject satisfaction were evaluated. Safety was monitored through adverse events and laboratory tests.
Adapalene gel 0.1% produced significantly better reductions in total (P<0.0001), inflammatory (P=0.0010), and non-inflammatory lesions (P<0.0001) at endpoint (week 12, last observation carried forward) than gel vehicle, with a higher overall subject satisfaction. The primary efficacy variable, the median percent reduction of total lesion counts at endpoint, was significantly greater with adapalene gel 0.1% (63.2%) compared to that with the vehicle (36.9%) in the ITT population (P<0.0001). Significantly greater results were observed as early as week 1. Adapalene was well tolerated, with adverse events that were mostly mild-to-moderate and transient in nature.
Adapalene gel 0.1% was effective in the treatment of acne vulgaris in Japanese patients. Adapalene was safe and well tolerated, consistent with the good tolerability profile demonstrated in other patient populations.
Psoriatic skin shows markedly increased keratinocyte proliferation and altered differentiation with various abnormal signalling pathways. In the present study, we investigated the expression of mitogen-activated protein kinases in psoriatic skin. Immunohistochemical study showed increased extracellular regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) expression in the nuclei of involved epidermis. Western blot analyses confirmed the increased active phospho-ERK and phospho-JNK expression in the involved epidermis. In contrast, expression pattern of p38 was not different between the involved and uninvolved epidermis, which was confirmed by western blot analysis. These results indicate that psoriatic epidermis shows selective activation of ERK and JNK, which might be related to hyperproliferation and abnormal differentiation of psoriatic epidermis.
This study used the induction of squamous cell carcinomas on mouse skin as an experimental model to evaluate molecular and biochemical changes that contribute to the neoplastic phenotype. The study was facilitated by the development of keratinocyte cell culture assays that reproduce each stage of the carcinogenesis process, by discoveries of stage-specific genetic and epigenetic changes and by application of pharmacological and molecular tools that modify each step. An early event in the transformation of keratinocytes involves mutation and activation of the rasHa gene, producing a benign tumor. The phenotypic consequences of ras mutations are mediated by activation of the epidermal growth factor receptor (EGFR), upregulation of protein kinase C (PKC) alpha and AP-1 mediated transcriptional activity and inactivation of PKC delta through tyrosine phosphorylation. These changes in benign tumors are manifested by hyperproliferation (EGFR), aberrant expression of keratinocyte genes (PKC alpha and AP-1) and delayed terminal differentiation (PKC delta). Accumulated chromosomal abnormalities, multifocal phenotypic changes and alterations in gene expression are associated with premalignant progression. Upregulation of the fos gene and AP-1 transcriptional activity causes malignant conversion of benign keratinocytes. In the absence of c-fos, benign tumor cells fail to upregulate secreted angiogenic and proteolytic factors and this may prevent malignant conversion. These pathways provide targets for preventive strategies to interrupt the process of carcinogenesis prior to the evolution of the fully malignant tumor.
Psoriasis is a T cell-mediated inflammatory skin disease. Recent evidence suggests that activated CD4+ helper T lymphocytes of the Th1 phenotype play an important role in the pathogenesis of the disease. For psoriatic autoreactive T cells, Keratin 17 is a major target antigen and an epitope containing ALEEAN sequence has been described, but other psoriasis-related epitopes are still unknown.
To identify the HLA DR B1*04, *07-restricted T cell epitopes on Keratin 17.
HLA DRB1*04, *07-restricted T cell epitope regions on Keratin 17 were predicted based on related software and internet servers. Keratin 17 gene was amplified from psoriatic epidermis and the proteins of the predicted epitope regions were expressed, identified and purified. T cells from psoriatic patients reacted in cultivation with peptide-major histocompatibility complex (p-MHC) compound, then the level of cell proliferation and the concentration of interferon-gamma in culture supernatant were detected. After the psoriasis-related epitope regions were narrowed down, the epitopes on them were predicted further. These epitopes were then expressed and validated by T cell response in vitro.
Four epitopes--S1 (118-132), S2 (169-183), S4 (323-337) and S4 (348-362) can stimulate the proliferation and interferon-gamma production of psoriatic T cells more effectively than other epitopes (p<0.01) and react weakly with the T cells from healthy volunteers (p>0.05).
Epitopes S1 (118-132), S2 (169-183), S4 (323-337) and S4 (348-362) are immunodominant DR B1-restricted T cell epitopes for psoriasis. Among them, S1 (118-132) contains the ALEEAN sequence while the others with different amino acid sequence have not been reported before. Further studies based on these peptides would provide a more complete understanding of the immunological basis of psoriasis.
Polymyositis (PM) cause pain and weakness of muscle, even threatens patient's lives, but the etiology and pathogenesis of it remains partially understood. Previous studies have proved Cathepsin B (CB) was strongly stained in muscle tissues of PM patients. But no further studies were performed to investigate the role of CB in PM.
To investigate the protective effects of CB inhibitor CA-074Me in PM.
CB expression, inflammation and apoptosis were analyzed in muscle tissues from patients with PM. Guinea pigs were inoculated intraperitoneally with Coxsackie virus B1 (CVB1) and were then immunized with completely emulsified 0.6ml rabbit muscle homogenates in Freund's Complete Adjuvant (FCA) once a week for consecutive three weeks. The effects of CB inhibitor CA-074Me on CB expression, inflammation and apoptosis were then investigated. Inflammation was assessed by histological examination. Both immunohistochemistry and western blot were used to determine the protein expression. The mRNA levels of CB were measured by Real-Time RT-PCR. The apoptosis was determined by TUNEL assay.
In patients with PM, the protein levels of CB were significantly up-regulated in muscle tissues compared with healthy controls, which correlated with increases in inflammation score and apoptotic rate in PM patients. Consistently, the expression of CB, inflammation score, CD8(+) T-cell, CD68(+) cell, tumor necrosis factor-alpha (TNF-α) infiltration and apoptotic rate were significantly increased in the guinea-pig model of CVB1-induced polymyositis. Administration of CA-074Me reduced CB expression, decreased inflammation score and attenuated apoptosis in muscle tissues of the guinea-pig model of CVB1-induced polymyositis. The inhibitory effect of CA-074Me on apoptosis was associated with down-regulation of Bax expression and consequent increase in the ratio of Bcl-2/Bax. However, CA-074Me had effect not on CD8(+) T-cells infiltrations but on CD68(+) cells and TNF-α(+) cells infiltrations in the guinea-pig model of CVB1-induced polymyositis.
This study confirms up-regulation of CB in PM patients and demonstrates that inhibition of CB provides protective effects in a guinea pig model of CVB1-induced PM. Thus, CB will be an important therapeutic target for PM.
Human beta-defensins (hBDs) belong to a group of antimicrobial peptide that are expressed in the epithelial cells.
The present study investigated mRNA expression levels of the beta-defensins, hBD-1, -2 and -3, in human keratinocytes during differentiation in vitro.
Immortalized keratinocyte cell lines, HaCaT and PHK16-0b, were used in this study; in order to stimulate differentiation, the Ca(2+) concentration in the growth media was increased from 0.3 to 1.8 mM.
Four days after the increase, the expression levels of hBD-1 and -3 were increased in both cell lines, followed by an increase in the mRNA levels of the differentiation markers, involucrin and keratin 10. No increased expression of hBD-2 was observed.
The results indicate that keratinocyte differentiation may stimulate hBD-1 and -3 expression in stratified squamous epithelia.
Tape stripping induces transient increase in keratinocyte proliferation in vivo. The effects of tacalcitol (1,24-(R)-dihydroxyvitamin D3) ointment on the cell kinetics of pig epidermis after the tape stripping were investigated. The tacalcitol ointment (2 micrograms/g) was applied once to the back of pigs immediately after the tape stripping. The pig epidermal cell kinetics were analyzed at various times following the treatment. Tape stripping transiently increased thymidine incorporation of keratinocytes; the maximal effect was observed at 24 h. Tape stripping-induced increase in thymidine incorporation was markedly augmented by tacalcitol treatment. At 24 h following the tape stripping DNA-flow cytometry revealed an accelerated transition from G0/1 to S phase of cell cycle in tacalcitol treated epidermis. There was no significant difference, however, in mitotic counts and G2/M phase fractions between tape stripping-treated and tape stripping plus tacalcitol ointment-treated epidermis. We also measured calmodulin content of pig epidermis following the treatments. Although tape stripping slightly increased calmodulin content of pig epidermis, this was statistically not significant. Tape stripping plus tacalcitol ointment treatment resulted in a significant increase in calmodulin content at 24 h following the treatment. There was no significant difference in calmodulin content between tape stripping treated- and tape stripping plus tacalcitol-treated epidermis.
In this report, we applied the isoleucine-deprivation method to normal human keratinocytes in cultivation with serum-free MCDB153 medium. The growing cells were synchronized by transfer to MCDB153 medium prepared without isoleucine, and the degree of synchronization was analyzed by the cell cycle distribution and phosphorylation of retinoblastoma protein (pRB), one of the tumor suppressor gene products. 1,25-dihidroxyvitamin D3 (1,25(OH)2D3), a biologically active form of vitamin D3, is supposed to induce the differentiation and growth inhibition of human keratinocytes caused by cell cycle arrest. We examined its effect on cell cycle kinetics, especially progression from G1/G0 to S-phase, by using this synchronized system. We showed that 10(-6) M 1,25(OH)2D3 inhibited the progression from G1/G0 to S-phase strikingly in synchronized normal human keratinocytes.
Using fetal rat keratinizing epidermal cells (FRSK), the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on adenylate cyclase system were investigated. The beta-adrenergic adenylate cyclase response was significantly increased by the stimulation of 1 x 10(-7) M 1,25(OH)2D3. The effect was observed by 6 h and continued for at least 48 h. The 1,25(OH)2D3-induced beta-adrenergic augmentation effect was dose-dependent and the maximal response was observed at a concentration of 1 x 10(-7) M 1,25(OH)2D3. Other adenylate cyclase systems (adenosine, prostaglandin E2 and histamine) were not affected by treatment with 1,25(OH)2D3. The thymidine incorporation in FRSK cells was not significantly affected by 1,25(OH)2D3 treatment. Forskolin-induced cyclic AMP accumulation was significantly increased by 1,25(OH)2D3 treatment. Cholera toxin-induced cyclic AMP accumulation was moderately increased, but this was statistically not significant. Northern blot hybridization showed that none of the mRNAs (the beta 2-adrenergic receptor, the alpha subunits of the stimulatory or inhibitory guanine nucleotide binding proteins, Gs alpha, Gi2 alpha, Gi3 alpha) were significantly altered by 1,25(OH)2D3 treatment. We have already reported that the beta-adrenergic response was increased by dexamethasone and inhibited by retinoids in FRSK cells. The addition of both 1,25(OH)2D3 and dexamethasone to the incubation medium resulted in an additive augmentation. On the other hand, the beta-adrenergic augmentation by the 1,25(OH)2D3 treatment was suppressed by the addition of all trans-retinoic acids. Our results indicate that 1,25(OH)2D3 induces beta-adrenergic augmentation without an alternation of thymidine incorporation of FRSK cells.
The active vitamin D3 regulates proliferation and differentiation of epidermal keratinocytes. Recently topical vitamin D3, tacalcitol, calcipotriol, and maxacalcitol are widely used for psoriasis.
To examine the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on cultured normal keratinocytes (NHK) and compared its effect with those of various vitamin D3 analogues.
Cell proliferation of NHK cells was analyzed by MTS, BrdU and 3H-thymidine incorporation. The expression of involucrin, transglutaminase 1, keratin 5 and keratin 1 was investigated by western blot and PCR amplification and quantitative assay. Furthermore, we performed cornified cell envelope (CE) formation assay.
1,25(OH)2D3, tacalcitol, calcipotriol, and maxacalcitol decreased NHK cell proliferation in a concentration-dependent manner and the maximal effect was observed at 10(-7) M. There was no significant difference in the anti-proliferative effect among the active vitamin D3 analogues. The expression of involucrin and transglutaminase 1 were induced by 1,25(OH)2D3 and its analogues in mRNA and protein levels. CE formation was also induced by 1,25(OH)2D3 and its analogues. There was no significant difference in the potency among these chemicals. Keratin 5 and 1 expression was not altered by these active vitamin D3 analogues.
The present study demonstrated that active vitamin D3 analogues, tacalcitol, calcipotriol, and maxacalcitol, suppress keratinocyte proliferation and induce differentiation with similar potency.
The serum levels of calcium, inorganic phosphate, parathyroid hormone, calcitonin, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were measured in 34 patients with psoriasis vulgaris and compared with the severity of skin lesions. Severity of psoriasis was evaluated by three indices, the area-severity index (ASI), the area index (AI) and the severity index (SI), determined as the product of the area and severity, the area, and the severity of the individual skin lesions, respectively. The mean basal levels of these serum parameters were within the normal range. ASI and SI showed significant inverse correlations (r = -0.387, P less than 0.05 and r = -0.638, P less than 0.01, respectively) with the serum level of 1,25-dihydroxyvitamin D, but not with any other serum parameters, but AI was not correlated with any of these serum parameters. These data suggest that psoriatic patients are not deficient in 1,25-dihydroxyvitamin D, but that development of this skin disease may be related to a slightly decreased level of active metabolites of vitamin D or abnormalities in the responsiveness of the skin cells to them.
We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth and differentiation of cultured human hair outer root sheath cells (ORSC) from normal subjects and patients with vitamin D-dependent rickets type II (DDR-II) with alopecia. 1,25(OH)2D3 dose-dependently suppressed the plating efficiency, clonal growth, and DNA synthesis of normal ORSC. It enhanced the cornified envelope formation and caused morphological changes in the cells. All results indicated the existence of specific receptors for 1,25(OH)2D3 in the ORSC, and suggest that 1,25(OH)2D3 is a potent inhibitor of proliferation of ORSC as well as a stimulator of terminal differentiation. However, the cells from DDR-II patients with alopecia did not respond to 1,25(OH)2D3, suggesting a lack of the specific receptors in the cells. The differences in the cellular response to the hormone between the normal ORSC and those from the patients were apparent and easily distinguishable, therefore this experiment may be a rapid and simple diagnostic test for DDR-II patients with alopecia. Large number of hairs were difficult to obtain from patients with alopecia, and we developed a new culture method to accomplish these studies from a few plucked hair follicles. Our system may be useful in the culture of ORSC from limited number of follicles, and could be utilized to analyse the cellular characteristics of ORSC in patients with hair diseases.
Previously cadmium chloride was successfully used to prevent sunburn cell (SBC) induction in mouse skin in vivo and to promote human cell survival in vitro after UVB exposure, indicating a protective effect of cadmium-induced metallothionein (MT) with the property of anti-oxidant action. Although cadmium is a potent inducer of MT, the cytotoxic metal is not available for clinical use. The aim of this study is to affirm MT gene expression by the active for of vitamin D3, 1,25-dihydroxyvitamin D3, [1,25(OH)2D3] in cultured keratinocytes and examine in vivo and in vitro the photo-protective effects of 1,25(OH)2D3. Northern hybridization with human MT-IIa cDNA showed a significant increase in the gene expression of MT in the cells treated with 1,25(OH)2D3. Intraperitoneal injection and topical application of 1,25(OH)2D3 caused a significant reduction in SBC formation in mouse skin after UVB administration. The experiment showed the existence of an optimum level of 1,25(OH)2D3 for reducing photodamage. The cells exposed to 1,25(OH)2D3 showed increased tolerance (cell survival) to UVB injury. 1,25(OH)2D3-induced MT may act as a radical scavenger in oxygen-mediated UV injury including SBC formation in the skin. These results indicate that 1,25(OH)2D3 may be practically applied to humans for the purpose of photoprotection.
The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on proliferation and collagen type I and type III production in cultured human fibroblasts was examined. Previous studies have identified receptors for the vitamin in human dermal fibroblasts, and have suggested the skin to be a target tissue. While many studies examining keratinocyte modulation by 1,25-(OH)2D3 have been undertaken, very few have been performed on dermal fibroblasts. Neonatal foreskin fibroblast cultures were examined for cell number, extracellular collagen accumulation, and collagen mRNA levels, after 5 days exposure to 1,25-(OH)2D3 at a concentration of 10(-7) M. The vitamin significantly suppressed (P < 0.01) the proliferation of fibroblasts cultured in the presence of serum. Day 5 cell culture supernatants showed a significant per cell increase in collagen type I (P < 0.05) and type III (P < 0.01) as measured by ELISA. Type I collagen production in exposed cells was 11.64 + 0.531 microgram/10(6) cell vs. 9.53 + 0.500 microgram/10(6) cells in unexposed cells. Type III collagen production was 0.601 + 0.012 microgram/10(6) cell in exposed cells and 0.247 + 0.008 microgram/10(6) cells in unexposed cells. mRNA levels were increased after a 4-day exposure to 10(-7) M 1,25-(OH)2D3 for both type I (2.5-5-fold) and type III (5.5-7.76-fold) collagen. These results suggest a novel effect of increased collagen production by dermal fibroblasts upon exposure to 1,25-(OH)2D3 that is independent of proliferation.
Vitamin D3 is a new therapeutic agent for psoriasis. Hyperproliferation of epidermis and over-secretion of IL-8 by keratinocytes are the characteristic features of psoriasis. The present study was conducted to determine whether a new vitamin D3 analogue, 22-oxacalcitriol, could be effective in inhibiting the proliferation and IL-8 secretion of normal human epidermal keratinocytes. Cell proliferation was measured colorimetrically by the MTS assay. IL-8 secretion was measured with ELISA. Proliferation of cultured normal human epidermal keratinocytes was inhibited in the presence of 1,25-dihydroxyvitamin D3 at the concentrations of 1 x 10(-8) to 1 x 10(-6) M and 22-oxacalcitriol at concentrations of more than 1 x 10(-9) M at 48 h. IL-8 secretion from normal human epidermal keratinocytes was augmented by TNF-alpha, or synergistically by TNF-alpha and IFN-gamma. 1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol inhibited cytokine-stimulated IL-8 production dose dependently after 24 h incubation without inhibition of cell proliferation. 1,25-Dihydroxyvitamin D3 and 22-oxacalcitriol are considered to inhibit the proliferation of keratinocytes directly and indirectly with inhibition of the secretion of IL-8 from keratinocytes. The inhibition of IL-8 secretion from keratinocytes by vitamin D3 could modulate the behaviour of immunocompetent cells infiltrating in the skin.
We investigated the photoprotective effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) both in vivo and in vitro, revealing its relationship with glutathione, a well-known antioxidant. We also probed into the possible mechanism of photoprotection of 1,25(OH)2D3 through immunohistochemical study for metallothionein (MT). At the same time, endogenous antioxidant effect of 1,25(OH)2D3 was examined. Survival of cultured human keratinocytes was decreased when the cells were irradiated with ultraviolet light-B (UVB) at doses above 30 mJ/cm2. But in the presence of 1,25(OH)2D3 (12 nM), the decrease of survival of keratinocytes by UVB was diminished. The formation of sunburn cells by UVB irradiation in the skin of ICR mice was inhibited by topical application of 1,25(OH)2D3, regardless of prior glutathione depletion. Immunohistochemical staining revealed that 1,25(OH)2D3 induced the expression of MT, a potent radical scavenger, mainly in the basal layer of ICR mice skin. 1,25(OH)2D3 neither inhibited peroxidation of plasma lipids nor interacted with superoxide, nor removed hydrogen peroxide as an antioxidant. These findings suggest that 1,25(OH)2D3 has photoprotective effect not related with glutathione or its endogenous antioxidant property. Rather, it could be attributed to 1,25(OH)2D3-induced MT and its capacity to prevent radical-related damage in UVB irradiation.
No previous report has investigated the involvement of glycolytic enzymes in keratinocyte migration. Fructose-1,6-bisphosphate aldolase A (ALDOA) is a glycolytic enzyme bound to the cytoskeleton by certain growth factors, which are known to enhance keratinocyte migration. We postulated that ALDOA is involved in keratinocyte migration.
To investigate the possible role of ALDOA in keratinocyte migration.
The localization of endogenous ALDOA and the actin cytoskeleton was observed by laser scanning confocal microscopy in HaCaT cells. The effects of ALDOA on lamellipodia formation and migration were evaluated using ALDOA siRNA-transfected cells. In addition, the involvement of epidermal growth factor (EGF) in ALDOA-induced events was investigated.
Strong ALDOA expression was observed along the ruffling membrane and lamellipodia, and it was colocalized with the actin cytoskeleton in lamellipodia. In a scratch wound assay, the wound recovery area was significantly decreased on transfection with ALDOA siRNA. The rate of lamellipodia-forming cells also decreased. On stimulation with EGF, the wound recovery area and ALDOA and its mRNA levels increased. On the other hand, ALDOA siRNA transfection suppressed EGF-enhanced migration.
We concluded that ALDOA is involved in keratinocyte migration following the induction of lamellipodia formation, and ALDOA-related migration is enhanced by EGF.
It is well known that cyclophosphamide (Cy) treatment before sensitization paradoxically enhances rather than suppresses contact hypersensitivity (CH) reactions. In fact, Cy-treated mice developed a significant (p < 0.05) increase of the CH reactions to 2,4,6-trinitro-1-chrolobenzene (TNCB) in comparison with untreated mice.
In order to examine whether the target cells of Cy in the immuno-augmentative effect are CD25(+) CD4(+) regulatory T cells or not, we investigated effect of Cy treatment on CD25(+) CD4(+) T cells.
We examined Cy-treated CD25(+) CD4(+) T cells by flow cytometer and by inhibition assay on proliferation of CD25(-) CD4(+) T cells.
Cy treatment remarkably reduced the number and percentage of CD25(+) CD4(+) T cells in the spleen and lymph nodes 3 and 5 days later. Moreover, CD25(+) CD4(+) T cells taken from the Cy-treated mice 3 days later showed the lower suppressive activity on proliferation of CD25(-) CD4(+) T cells, as compared to that from the untreated mice. Furthermore, transfer of CD25(+) CD4(+) T cells from untreated mice resulted in a significant decrease (p < 0.05) of the CH reactions enhanced by Cy treatment.
These results indicate that enhancement of the CH reactions to TNCB by Cy treatment is attributed to the decrease in the number, percentage and the function of CD25(+) CD4(+) regulatory T cells.
The analysis of prognostic factors in Japanese cases of cutaneous T-cell lymphoma (CTCL) is scarce. Clusterin is a ubiquitous 80kDa heterodimeric glycoprotein expressed on tumor cells of systemic and primary cutaneous anaplastic large cell lymphoma. The expression of clusterin in mycosis fungoides (MF) and Sézary syndrome (SS) has only been sporadically reported in a small number of cases.
To determine the long-term prognosis of Japanese patients with MF and SS, to identify clinical and pathological factors predictive of survival, and to evaluate the prognostic significance of the International Society for Cutaneous Lymphomas (ISCL) revised staging system (2007).
We performed a retrospective cohort study of 105 Japanese patients with MF and SS managed at the Department of Dermatology Asahikawa Medical University from 1976 to 2011. Formalin-fixed, paraffin-embedded sections of MF and SS were immunostained for clusterin, CD30, and Ki-67.
No statistically significant difference in survival was found between stages IA-IIA and IIIA patients. The significant prognostic factors in the univariate analysis were higher age, TNMB classification, clinical staging, performance status, increased serum LDH level, dermal Ki-67-positive cells, and clusterin expression. In the multivariate analysis, T classification, extracutaneous disease, increased serum LDH level, clusterin expression, and performance status were the significant independent prognostic factors.
Japanese stage IIIA MF/SS patients contain a subpopulation with a favorable prognosis. The most significant prognostic factor for survival of MF and SS was the presence of extracutaneous disease. Clusterin expression was shown to be a novel unfavorable prognostic factor.
Matrix metalloproteinases (MMPs) are known as important enzymes involved in tissue metabolism. Among them, MMP-2 and MMP-9 are termed gelatinases, but their specific roles in vivo are still unknown, including their expression patterns following ultraviolet (UV) irradiation.
To elucidate the effects of UV irradiation on the skin, we analyzed the expression of MMP-2 and MMP-9 by primary human keratinocytes in culture.
We evaluated the enzymatic functions of MMP-2 and MMP-9 by gelatin-zymography, and of MMP-9 expression by immunofluorescence, using cultured keratinocytes after UV irradiation.
The secretion of MMP-2 (72 kDa) remained at low levels under all conditions examined. Although MMP-9 (92 kDa) secretion was not induced by UVA, it was stimulated by UVB irradiation in a dose-dependent manner. In addition, an enzyme-linked immunosorbent assay showed the tendency to increase for the involucrin expression following UVB exposure. Cell viability was decreased by UVB irradiation in contrast to the induction of MMP-9 and involucrin.
These results suggest that the induction of MMP-9 secretion is related to the inflammation including apoptosis of keratinocytes resulting from UVB irradiation.
Psoriatic skin lesions contain HLA-DR positive T lymphocytes, and other activation antigens, which suggest that the T cells may be producing lymphokines. Gamma interferon is produced by activated T cells, and its presence in psoriasis has been inferred by the lesional keratinocyte expression of 3 gamma interferon-inducible proteins i.e. HLA-DR, intercellular adhesion molecule-1, and gamma-IP-10. To determine whether gamma interferon is being produced directly in psoriatic lesions, punch biopsies of normal and diseased skin were separated into epidermal sheets and dermal fragments. Total cellular RNA was isolated from each epidermal and dermal compartment, and reverse transcribed followed by amplification of the resultant DNA by polymerase chain reaction. The amplification process involved the use of 5' and 3' primers for gamma interferon, and tumor necrosis factor-alpha, with beta-actin serving as a control. Gamma interferon mRNA, but not tumor necrosis factor alpha mRNA, was detectable in 4 of 5 psoriatic epidermal specimens. Neither mRNA was detectable in any normal skin dermal/epidermal specimens. Gamma interferon mRNA was also detectable in a single psoriatic dermal specimen. If reverse transcriptase was omitted, no polymerase chain reaction products were detected, indicating that the fragments detected were not derived from contaminating genomic DNA. These results indicate that gamma interferon mRNA can be extracted and successfully detected from human psoriatic lesional skin biopsies, using polymerase chain reaction technology. This molecular approach can easily be expanded to measure many other cytokines in both epidermal and dermal locations. The detection of gamma interferon in this clinical setting may be of particular pathophysiological significance because injection of gamma interferon has been reported to induce psoriatic lesions.
Cytokines play an important role in the pathogenesis of acne vulgaris together with other genetic and environmental factors.
To check for the association of TNF-α and IL-10 gene polymorphisms with the susceptibility and severity of acne in Saudi patients.
Study subjects included 166 Saudi patients (65 males, 101 females) with acne vulgaris. Their mean age±SD was 21.6±5.1 years. These cases were compared to 390 unrelated healthy controls (208 males, 182 females) with a mean age±SD of 20.1±3.3 years. Cases were sub-grouped on the basis of their severity of acne affection into mild, moderate and severe groups. For all participants, genotypic variants of the TNF-α -308 G/A and IL-10 -1082 A/G genes were determined using the real time PCR technique.
Frequencies of genotypic variants of the TNF-α -308 polymorphism were significantly different in acne cases compared to controls. Further analysis showed that acne cases had significantly higher frequency of both the GG and AA homozygous forms than controls (73.8% vs. 63.6%, p=0.02, odds ratio=1.6). It was also interestingly noticed that the amount of GG homozygosity was notably higher among female cases than male ones (76.0% vs. 54.7%, p=0.006, odds ratio=2.6) whereas male cases had a higher frequency of AA and GA genotypes than female ones (9.4% and 35.9% vs. 4% and 20% respectively). Differences in the frequencies of IL-10 -1082 genotypic variants were statistically insignificant comparing cases to controls (p=0.3). On the other hand, comparing cases-subgroups in terms of the age of onset of the disease, consanguinity, family history, obesity and acne severity; no statistical significance was observed regarding frequencies of genotypic variants related to the both TNF-α -308 and IL-10 -1082 polymorphisms (>0.05).
TNF-α -308 polymorphic variants might be a predisposing factor for acne susceptibility, with no apparent relation to its severity whereas IL-10 -1082 variants showed no association with both acne susceptibility and severity.
10B1-para-boronophenylalanine (10B1-BPA), one of our boronated dopa analogues developed for thermal neutron capture therapy, has been found to have a selective affinity for malignant melanoma. We have established a method of 'in situ' detection of subcutaneous melanoma lesions, using this melanoma-seeking 10B-labeled compound. In this study, we applied an 'in situ' 10B microanalysis system via detection of the prompt gamma-ray from the 10B(n, alpha)7Li reaction triggered by irradiating the 10B-containing target with pure thermal neutrons, called prompt gamma-ray spectrometry, to hamsters bearing Greene's melanoma in subcutis and to a human patient whose occipital subcutaneous tumor was suspected of being a metastatic melanoma. In the hamsters, the time-dependent 10B dynamics showed increased 10B accumulation in melanoma, after 10B1-BPA administration, in contrast to that in non-melanoma normal skin. In the human patient, after subcutaneous injection of 10B1-BPA into perilesional sites 4 cm distant from the tumor margin, the average 10B concentration in the tumor was determined to be 24 ppm (microgram/g), in contrast to 3 ppm in skin covering the tumor and 1.1 ppm in blood, indicative of selectively high 10B1-BPA uptake by the tumor.
The loss of subcutaneous (sc) fat is associated with aging. Inflammatory cytokines, such as interleukin-1 α (IL-1α), interleukin-11 (IL-11) and tumor necrosis factor-α (TNF-α), are known to inhibit the differentiation of preadipocytes.
This study investigated the potential role of inflammatory cytokines in solar-radiation-induced facial fat loss.
Cultured fibroblasts, keratinocytes, and skin equivalents were exposed to various doses of radiation from a solar simulator. Inflammatory cytokines' mRNA production and protein secretion were examined by qRT-PCR and ELISA, respectively. In some experiments, epidermal-dermal equivalents were pretreated topically with a broad-spectrum sunscreen prior to solar simulated radiation (SSR). Human facial preadipocytes treated with recombinant IL-11 or with conditioned media from solar-irradiated equivalents were evaluated for the level of adipocyte differentiation by image analyses, Oil red O staining, and the expression of adipocyte differentiation markers.
IL-11, IL-1α, IL-6, and TNF-α protein secretion were induced from epidermal-dermal equivalents by exposure to SSR. A sunscreen prevented SSR-induced inflammatory cytokines production from such equivalents. Exposure of facial preadipocytes to conditioned medium from solar-irradiated epidermal-dermal equivalents inhibited their differentiation into mature adipocytes. Consequently, conditioned medium from sunscreen-pretreated, solar-irradiated equivalents did not inhibit differentiation of preadipocytes. A cocktail of neutralizing antibodies to IL-11, IL-1α, IL-6 and TNF-α significantly reduced the SSR-induced inhibition of preadipocyte differentiation.
These results support the hypothesis that SSR-induced inflammatory cytokine may be involved in the photoaging-induced loss of facial subcutaneous fat. Inhibition of this process, e.g. by sunscreens, might slow or prevent photoaging-induced changes in facial contouring.
Facial appearance is regarded as a typical index of ageing. However, people of the same age do not necessarily show the same degree of the facial appearance. The ageing of facial skin proceeds relatively slowly and therefore requires long-term follow-up to elucidate the mechanism of ageing changes.
The purpose of this study was to identify facial skin parameters contributing the subjective impression of the overall ageing and characterize the degree of skin ageing by a 11 year longitudinal skin monitoring.
One-hundred-eight healthy Japanese females excluded outside workers aged 5-64 at 1999, and lived in Akita, Japan till 2010 were enrolled. Facial images were collected to quantify various skin optical parameters. Skin colour, hydration and barrier function were measured with Chromameter, Corneometer and TEWAmeter, respectively. The visual evaluation of the overall facial skin ageing impression was also carried out. The skin parameters contributing visible impression of skin ageing were identified by variable importance in projection analysis, and the degree of facial skin ageing over 11 years was statistically classified by a cluster analysis.
Facial skin parameters that comprehensively influenced visible skin ageing, including hyperpigmented spots, wrinkles and texture were studied. The Skin Ageing Score calculated from these three skin factors was used to classify the subjects into a mild, age-appropriate, and severe skin ageing group. The mild skin ageing group maintained significant better both skin optical and physical conditions.
Variability and classification of the degree of facial skin ageing appearance were studied from this longitudinal research.
We have developed a method for differential detection of human papillomavirus type 6 and 11 with selective amplification of a segment of the viral DNAs by the polymerase chain reaction. The target sequence for amplification was a 134-bp (bp 206-339) in the E6 open reading frame of HPV 6 and 11 DNAs. DNA extracted from a paraffin-section or a minute fresh biopsy specimen of condyloma acuminatum was amplified and hybridized with an oligonucleotide probe specific for HPV 6 or 11 DNA. The method was applied to analyse 5 cases of genital condyloma acuminatum, and as a result, HPV 6 and 11 DNAs were detected in two and one case, respectively. This method is useful for differential diagnosis of HPV 6 and 11 infection.
Dermatologists are faced daily with the need to optimize skin repair and excise cutaneous cancers. The extracellular matrix plays a pivotal role in cellular migration, proliferation, and gene regulation during wound healing and progression of melanoma, basal cell carcinoma, and squamous cell carcinoma. Within the last few years, a new class of ligand, the matrikine or matricryptin, has been characterized as subdomains of various ECM proteins capable of signaling to the cell through receptors, such as growth factor receptors. Two classes exist: the "natural" matrikines, which signal directly from the extracellular milieu and "cryptic" matrikines (matricryptins) that require proteolytic processing to reveal the ligand or to release the ligand from its ECM protein. Unlike traditional soluble growth factors, most matrikines possess low binding affinity to their receptors and are often presented in multiple valency that likely increase avidity to receptors. The presentation of these ligands within the ECM can result in unique outcomes. The EGF-like repeats of tenascin-C and laminin-5 signal to EGFR preferentially to upregulate migration during skin repair and tumor progression. Other matrikines in collagen, elastin, decorin, and laminin-1 can promote chemotaxis, mitogenesis, and metastasis in cancers, such as melanoma. Finally, the unique properties of matrikines have been utilized in cancer therapeutics and tissue engineering. Within the next few years, the nature and function of this emerging class of matrikine ligands will have an impact on dermatology, as these proteins are altered in wound repair and skin diseases.
A novel monoclonal antibody to hair keratins (TYHF-1) which recognized a doublet of 43/44-kDa hair keratins in the immunoblot analysis was developed. TYHF-1 strongly reacted with the outer root sheath cells at the region of prekeratinization, the keratogeneous zone and the lower part of the isthmus portion in the indirect immunofluorescent examination. The cells of the inner root sheath and cuticle in the upper bulbar portion were also stained with this antibody. In addition, the tongue and nail matrix were also reacted with TYHF-1. However, TYHF-1 reacted with no epidermal keratins either in the indirect immunofluorescent examination or by immunoblot analysis using plantar epidermis. In addition, the cells of the eccrine and apocrine sweat glands, the epithelia of the buccal mucosa, larynx, tonsil, oesophagus, liver, intestine, gallbladder and urinary bladder demonstrated no positive reaction.
Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs), which have been evolutionarily conserved in microbes. Human melanocytes are not simply pigment-producing cells but also have the phagocytic capacity and can produce pro-inflammatory mediators. However, the mechanisms of recognition of microbes by melanocytes have not yet been fully established.
We investigated the TLRs 1-10 expression profile in human epidermal melanocytes and assessed their functions after triggering by their specific ligands.
TLRs mRNA expression was determined by RT-PCR, and the TLR protein expression was measured by flow cytometry and immunofluorescence assays. After stimulation with various TLR ligands, the production of inflammatory cytokine IL-8 and IL-6 was measured by ELISA and the mRNA for chemokine CCL2, CCL3 and CCL5 was analyzed by real-time PCR. Phosphorylation of IkappaBalpha in TLR ligands-triggered melanocytes was determined by Western blot and the nucleus translocation of NF-kappaBp65 was analyzed by immunofluorescence.
Human melanocytes constitutively expressed TLRs 1-4, 6, 7 and 9 mRNA. Ample amounts of TLRs 2-4, 7 and 9 were confirmed at protein level. Stimulation of melanocytes with TLRs ligands resulted in the release of cytokines (IL-8 and IL-6) and the mRNA accumulation of chemokines (CCL2, CCL3 and CCL5). Triggering of TLRs in melanocytes resulted in the up-regulation of phosphorylated IkappaBalpha and in the nucleus translocation of NF-kappaBp65.
Present study indicates human melanocytes express a panel of functional TLRs. The ligation of TLRs can turn these cells into active players of the skin innate immunity.
The acute skin lesions of atopic dermatitis (AD) are associated with Th2 cells; however, the chronic skin lesions of AD are associated with Th1 cells via the action of IL-12.
We evaluated the associations of single nucleotide polymorphisms (SNPs) and haplotype in the IL-12 and IL-12 receptor genes, and determined the gene-gene interactions between the SNPs of these genes and the SNPs of the IL-18 gene that we previously reported.
We genotyped 24 SNPs from 4 IL-12/IL-12R genes for 1089 case-control samples (631 AD patients and 458 normal controls). We measured the serum IL-12 concentrations in 89 individuals (79 AD patients and 10 controls) by ELISA. We analyzed the SNPs and haplotypes in each gene and also searched for the gene-gene interactions.
The rs582504 (IVS-798A/T) SNP and the haplotype TA (rs582054 and rs2243151) in the IL-12A gene, and the rs438421 (IVS12+1266T/C) SNP and the haplotype CCA (rs375947, rs438421, and rs1870063) in the IL-12RB1 gene were significantly associated with the AD phenotype. We showed that the rs438421 polymorphism in the IL-12RB1 (TT) gene and the rs2066446 polymorphism in the IL-12RB2 (AA) gene had a significant interaction to develop the ADe phenotype (allergic type of AD), and those individuals with the risk alleles, TT/AA/CC (IL-12RB1/IL-12RB2/IL-18), have more than a 10-fold increased risk to develop ADe.
This study provides evidence for a significant interaction between the IL-12RB1 and IL-12RB2 genes that contribute to a 4-fold increased risk for developing ADe. In addition to the IL-12R interaction, we suggest that the IL-18 gene can significantly interact with the IL-12R gene to develop ADe. In addition to the interaction, the SNPs and haplotypes in the IL-12A and IL-12RB1 genes are independently and significantly associated with the AD phenotype, and especially with the ADe phenotype. This data may contribute to our understanding of AD genetic interactions and account for the additional risk of certain patients to develop AD.
Acne vulgaris is a multifactorial inflammatory disease of the sebaceous follicles of the face and torso that frequently occurs in adolescence. Initially, acne starts as a non-inflammatory comedo. Subsequently, inflammatory reactions evolve to pustules, granulomas and cystic lesions. Many pathogenic mechanisms have been proposed including sebum excretion, obstruction of hair follicles, impaired keratinization of hair epithelium, bacterial overgrowth and immunological mechanisms; the role of Propionibacterium acnes (P. acnes) is particularly important. Facultative anaerobic gram-positive rods have been implicated in acne pathogenesis. However, the host immune response to P. acnes has not been as yet elucidated.
The aim of the present study is to evaluate the importance of the immune response to P. acnes and the bacteriological factor in the pathogenesis of acne.
P. acnes isolated from acne lesions and healthy volunteers skin were cultured. The peripheral blood mononuclear cells (PBMC) from acne patients or healthy volunteers were stimulated with viable P. acnes, and cytokine production was evaluated using RT-PCR and ELISA.
IFN-gamma, IL-12p40, and IL-8 mRNA and protein production were significantly increased in PBMC from acne patients compared to that from normal donors. However, different P. acnes species isolated from acne lesions or normal subjects showed no difference in cytokines production from acne patients and normal subjects PBMC.
The inflammatory response of acne appears to be attributable to P. acnes-induced host immune response rather than P. acnes strains from normal skin or acne lesions.
Psoriasis is a chronic immune-mediated skin disease, in which interleukins 12 and 23 have been postulated to play a critical role. However, the cellular source of these cytokines in psoriatic lesions are still poorly defined and their relative contribution in inducing skin inflammation has been discussed controversially.
To investigate immunoreactivity of the bioactive forms of IL-12 and IL-23 in plaque psoriasis and to characterize the dendritic cell (DC) and macrophage subsets responsible for the production of these cytokines.
Immunohistochemistry was performed on normal skin (n=11) as well as non-lesional (n=11) and lesional (n=11) skin of patients with plaque psoriasis using monoclonal antibodies targeting the bioactive forms of IL-12 (IL-12p70) and IL-23 (IL-23p19/p40) on serial cryostat sections using the alkaline phosphatase-antialkaline phosphatase. Co-localization of IL-12 and IL-23 with different dendritic cells and macrophage cell markers (CD1a, CD11c, CD14, CD32, CD68, CD163, CD208/DC-LAMP) was performed using double immunofluorescence staining.
Immunoreactivity for IL-12 and IL-23 was significantly enhanced in lesional psoriatic skin as compared to non-lesional and normal skin. No difference was observed between IL-12 and IL-23 immunoreactivity in any skin types. Both IL-12 and IL-23 immunoreactivity was readily detected mainly in CD11c+, CD14+, CD32+, CD68+ and some CD163+, DC-LAMP+ cells. IL-12 and occasionally IL-23 were also found in some CD1a+ dendritic cells. In addition, an enhanced expression mainly of IL-23 was observed in keratinocytes.
Bioactive forms of IL-12 and IL-23 are highly expressed in various DC and macrophage subsets and their marked in situ production suggest that both cytokines have crucial pathogenic role in psoriasis.
Recent studies have shown increased T-helper (Th) 2 cytokine expression and decreased IFN-gamma expression in peripheral blood from patients with atopic dermatitis (AD). In the present study, we examined the cytokine expression in peripheral blood mononuclear cells in patients with AD and tested how the cytokine profile correlated with the patients' age, severity and laboratory findings. Peripheral blood mononuclear cells obtained from 20 patients with AD were stimulated with phorbol 12-myristate 13-acetate and ionomycin, and were examined for the frequencies of CD4+ cells expressing IL-4, IL-13 and IFN-gamma at the single cell level using intracellular cytokine staining and flow cytometry. There was a significant positive correlation between IL-4 and IL-13 expression in CD4+ cells. Expression levels of both IL-4 and IL-13 in CD4+ cells were significantly correlated with peripheral blood eosinophilia. These findings suggest that CD4+ cells producing IL-4 and IL-13 play important roles in the pathogenesis of AD. IFN-gamma expression did not correlate with either IL-4 or IL-13 expression, or with blood eosinophilia. We also measured serum levels of IL-13 in 144 patients with AD using enzyme-linked immunosorbent assay, and found 16 patients with detectable levels of serum IL-13. IL-13+ patients had significantly higher serum IgE levels than IL-13- patients, suggesting a direct association between serum levels of IL-13 and IgE. It was also shown that the IL-13+ group was significantly younger in age than the IL-13- group, although the implication of this finding is not clear at present. The sum of these findings suggested that the predominance of Th2 cells and the consequent overexpression of IL-4 and IL-13 in peripheral blood may be deeply involved in the pathogenetic process of AD.
Stasis dermatitis is a common disorder, which is a consequence of impaired venous drainage of the legs. It is characterized histologically by proliferation of small blood vessels in the papillary dermis. This neovascularization may lead occasionally to the formation of discrete papules due to inflammatory processes. In order to evaluate the role of matrix metalloproteinases (MMPs) in the acute phase of chronic venous insufficiency, we examined the production of MMP-1, -2, -13 and tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in lesional skin of stasis dermatitis. A total of 19 patients affected by stasis dermatitis were included in this experimental study. Polymerase chain reaction, western blot and immunohistochemical studies on tissue specimen were performed. In lesional skin of stasis dermatitis, there was elevated gene expression and immunoreactivity for MMP-1, -2 and -13 in comparison to healthy controls. In contrast, genexpression and immunoreactivity for TIMP-1 and -2 were diminished in stasis dermatitis in comparison with healthy controls. Overexpression and production of MMP-1, -2 and -13 without inhibitory effects could be the result of cytokine mediated induction. Matrix metalloproteinases (MMPs) may play an important role in the remodeling of lesional skin in stasis dermatitis.
Interleukin (IL)-13 is a pleiotropic cytokine, which shares many biological functions with IL-4. The receptor subunits of IL-13 consist of IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2. The regulatory mechanisms of the IL-13Ralpha expression in the keratinocytes of certain skin disease have not been known.
To clear the roles of IL-13 and the regulatory mechanisms of its receptor in atopic dermatitis (AD) and psoriasis.
The expression of IL-13Ralpha1 in the skin of AD and psoriasis was investigated by immunohistochemistry. The regulation of IL-13Ralpha mRNA in the skin and human primary keratinocyte (HPK) was investigated by quantitative PCR. The secretion of IL-6 and RANTES from HPK was measured by ELISA.
The expression of IL-13Ralpha1 was more prominent on the suprabasal keratinocytes in the skin of AD and striking increase of staining was observed on all layers of keratinocyte in the skin of psoriasis. The mRNA of IL-13Ralpha1, but not of IL-13Ralpha2 was overexpressed in both skin of AD and psoriasis. In vitro experiment using HPK demonstrated that IFN-gamma, IL-13 but not IL-4 could up-regulate the mRNA expression of IL-13Ralpha1. In contrast, IL-13Ralpha2 mRNA expression was up-regulated by IFN-gamma plus IL-4. Furthermore, the stimulation of HPK with IFN-gamma plus IL-13 and/or IL-4 resulted in significant enhancement of IL-6 and RANTES secretion.
These findings indicate that IL-4 and IL-13 have different regulatory effects on the expression of IL-13Ralpha1 and alpha2, and the overexpression of IL-13Ralpha1 may play some roles in the pathogenesis of chronic stage of AD or psoriasis.
Interleukin (IL)-13 plays an important role in the induction of immunoglobulin E (IgE) and in the pathogenesis of atopic dermatitis (AD). We investigated the allele and genotype frequencies of three IL-13 single nucleotide polymorphisms (SNPs) (A704C and C1103T in the promoter region and G4257A in exon 4) in Japanese patients with AD. For A704C and C1103T SNPs, there were no significant differences in allele or genotype frequencies between AD patients and controls. For G4257A SNP, A allele was significantly increased in AD patients (39.5%) compared with controls (29.4%) (P = 0.016). The same proportion of each genotype and allele was observed in the patient subgroup with and without asthma. Serum IgE levels and peripheral eosinophil counts were not significantly different among genotypes in G4257A SNP. There was also no significant difference in allele or genotype frequencies between AD patients with mild disease and those with severe disease, between those with family history of AD and those without it, or between those with family history of atopic disorders and those without it. This result suggests that 4257A allele is associated with susceptibility to AD and that it may function in the pathogenesis of AD itself, presumably by other mechanisms than inducing IgE production.
We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+-switch from low (0.07 mM) to normal (1.87 mM) concentration in culture medium, which were also linked to activation of protein kinase C (PKC), lead to phosphorylation of 180 kDa-bullous pemphigoid antigen (BPAG) 2, but not of 230 kDa-BPAG1, and possibly to its disassembly from hemidesmosomes in a human squamous cell carcinoma cell line (DJM-1). In this study, we examined the effects of TPA and Ca2+-switch on intracellular localization of BPAG1 by immuno-blotting and immuno-fluorescence microscopy with monoclonal antibodies to the antigen after sub-cellular fractionation. In DJM-1 cells cultured in low Ca2+ medium, BPAG1 was detected as phosphate buffered saline-soluble (cytosolic), Triton X-100 soluble (roughly membrane-associated) and Triton X-100 insoluble (cytoskeleton-bound) forms, whereas in normal Ca2+-grown cells only as cytosolic and cytoskeleton-bound forms. In normal Ca2+-cultured cells, TPA (50 nM) caused a complete translocation of BPAG1 from cytosol to membrane fractions within 10 min, that was inhibited by pretreatment with H7 (a selective PKC inhibitor) at 40 microM. After 30 min and 4 h of TPA-treatment, BPAG1 was exclusively detected in cytoskeleton fractions. Morphologically, immuno-fluorescence microscopy showed that treatment caused a marked reduction of BPAG1 from the cytoplasm and generated a linear pattern at cell-cell contacts, suggesting translocation of BPAG1 from the cytosol to the plasma membrane. In contrast, the Ca2+-switch from low to normal caused a prominent increase of BPAG1, both in cytosolic and membrane-associated forms after 4 h, that was inhibited both with H7 and cycloheximide (an inhibitor of protein synthesis) at 70 microM, suggesting a role for PKC and BPAG1 synthesis in these Ca2+-induced effects. These results suggest that TPA and Ca2+-switch induced BPAG1 translocation to membrane fractions possibly mediated by PKC-activation. Furthermore, whereas TPA affects the redistribution of BPAG1 among their pools without inducing their synthesis, Ca2+-switch induces both membrane translocation and synthesis of BPAG1, suggesting involvement of signaling other than PKC pathways in control of BPAG1 synthesis.
There have been several reports about Th1/Th2 imbalances in atopic dermatitis (AD), but there have been few precise investigations about the differences between Th1 and Th2 cytokine secretion patterns of peripheral blood mononuclear cells (PBMCs) in such patients. We cultured PBMCs, taken from AD patients and healthy subjects, with dust mite extract (DME) and measured subsequent immunoreactive interferon (IFN)-gamma (Th1 cytokine), interleukin (IL)-4, IL-5, and IL-13 (Th2 cytokines) levels in the supernatants by ELISA assays. There is a difference between IL-4 and IL-13 secretion patterns by DME-stimulated PBMCs in AD subjects; immunoreactive IL-4 levels were detectable maximally within 24-h cultures, while IL-13 levels increased time-dependently within 7-day cultures. IL-13 levels were significantly elevated in AD subjects compared to healthy subjects, while IFN-gamma levels did not significantly differ between the two groups. IL-13 levels were significantly higher in AD patients who had high levels (>100 U/ml) of Dermatophagoides pteronyssinus-specific IgE (Dp-IgE) than in those AD patients who had low levels (<10 U/ml) of Dp-IgE. Tacrolimus (FK-506), at a concentration of 10(-8) M, significantly inhibited DME-induced IL-13 production from PBMCs. These findings suggest that IL-13 produced by Th2 cells are involved in IgE overproduction in AD subjects.
Skin is one of the main target organs in lupus erythematosus and in some circumstances, skin lesions precede systemic manifestations. Previous studies have demonstrated that Ro/SSA antigen antibody might be involved in the pathogenesis of lupus erythematosus. The present study was performed to investigate the factors regulating expression of Ro/SSA antigens on the cell surface of keratinocytes. Cultured normal human keratinocytes were treated with 50-200 mJ/cm(2) of ultraviolet B (UVB) irradiation, 10(-9) to 10(-5) mol/l of 17beta-estradiol, 5-10 microg/l of phorbol 12-myristate 13-acetate (PMA), and 42 and 45 degrees C heat shock, respectively. The Ro/SSA antigen expressions were determined by indirect immunofluorescence. The results showed that keratinocytes receiving UVB irradiation expressed Ro/SSA antigen on cell membranes in a dose-dependent fashion. 17beta-estradiol treatment also induced Ro/SSA antigen expression dose-dependently. Keratinocyte expression of Ro/SSA antigens was also induced by heat shock stimulation. The 45 degrees C heat shock showed a stronger effect than 42 degrees C heat shock. Keratinocytes incubated for 24 h after heat shock had more antigen-expressing cells than those incubated for 6 h after heat shock. PMA at 5 and 10 microg/l also strongly induced Ro/SSA antigen expression. These results suggest that Ro/SSA antigen expression can be regulated by many factors and that protein kinase C signal transduction pathway might be involved in this process.
Macrophage-derived chemokine (MDC) is a Th2 type chemokine and its receptor CC chemokine receptor 4 (CCR4) is preferentially expressed on Th2 cells. Recent reports demonstrated that MDC is expressed not only by macrophages, dendritic cells and lymphocytes, but also by cultured human keratinocytes (KCs). However, the regulation of MDC production in KCs by various cytokines has not been well documented.
In this study, we investigated how Th1/Th2 cytokines regulate MDC production in a human KC cell line, HaCaT cells.
HaCaT cells were cultured with or without various cytokines for 24 h and RT-PCR was performed using these cells to evaluate MDC mRNA levels. ELISA was carried out using supernatant of HaCaT cells to calculate secreted MDC protein levels.
MDC mRNA was weakly expressed in HaCaT cells, and upon stimulation with TNF-alpha or IFN-gamma, MDC expression was strongly upregulated. The supernatant MDC levels when stimulated with TNF-alpha or IFN-gamma were significantly higher than those without stimulation, and were synergistically increased when stimulated with a combination of TNF-alpha and IFN-gamma. Both interleukin-4 (IL-4) and IL-13 inhibited TNF-alpha and IFN-gamma enhanced MDC production in HaCaT cells in a dose-dependent manner.
Th2-type cytokines IL-4 and IL-13 downregulate the production of MDC, a Th2 type chemokine, by KCs. This may partially contribute to maintaining Th1/Th2 balance in inflammatory skin diseases like atopic dermatitis.
Evidence suggests anti-inflammatory effects of glucosamine (GS) on inflammatory diseases. COX-2 is an enzyme to produce prostaglandins. MMPs are the family of matrix metalloproteinases degradable of ECM. Excess expression of COX-2 or MMPs involves in skin inflammation.
We evaluated whether GS-HCl modulates expression of COX-2 and/or MMPs by IL-1beta or PMA in human skin fibroblasts (HSF) or keratinocytes (HaCaT).
HSF or HaCaT cells were exposed to IL-1beta or PMA without or with GS-HCl. COX-2 or MMPs protein and mRNA expression, respectively, were analyzed by Western blot and RT-PCR. MTS assay was utilized to assess the cytotoxicity of GS-HCl on HSF cells.
In HSF cells, IL-1beta treatment induced COX-2 and MMP-13 expressions in association with activation of ERKs, p38 MAPK, JNKs, and NF-kappaB. PMA treatment also induced COX-2 and MMP-13 expressions in association with p38 MAPK activation. Of interest, treatment with GS-HCl (10mM) led to blockage of p38 MAPK activation, accumulation of 66kDa COX-2 protein variant (without affecting COX-2 mRNA expression), and transcriptional down-regulation of MMP-13 in the IL-1beta- or PMA-treated HSF cells. Distinctly, pharmacological inhibition of p38 MAPK with SB203580 was associated with transcriptional down-regulation of COX-2 and MMP-13 in the IL-1beta- or PMA-treated HSF cells. In addition, the GS-HCl-mediated COX-2 protein modification was observed in both endogenous and PMA-induced COX-2 in HaCaT cells.
GS-HCl differentially down-regulates COX-2 and MMP-13 expression in the IL-1beta- or PMA-treated human skin fibroblasts via the p38 MAPK-independent COX-2 translational inhibition and the p38 MAPK-dependent MMP-13 transcriptional suppression, respectively.