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At least 19 recordsLinked to original sources

PubMed1998

Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes.

To understand biological function of IL-6 in the skin in vivo, we constructed a vector that strongly expressed human IL-6 in keratinocytes and introduced it into rat keratinocytes in vivo by the naked DNA method. The overexpression of IL-6 induced macroscopic erythema and histologically evident keratinocyte proliferation and lymphocytic infiltration in the treated area of rat skin. Since previous studies using IL-6 transgenic mice have not shown skin inflammation of these mice, our result provides the first evidence that IL-6 is related to the pathogenesis of inflammatory skin diseases. ELISA suggested that a certain degree of transgenic IL-6 expression in keratinocytes was required for inducing skin inflammation. Cytokine profile in rat keratinocytes after the gene introduction was examined by reverse transcriptase-PCR assay and revealed that gene expression of rat IL-1alpha and TNF-alpha showed no marked change until 24 h, whereas that of rat IL-6 and TGF-alpha increased with time. We then introduced and expressed the IL-6 mutant genes, which were designed to behave as IL-6Ralpha antagonists, and found that their ability to induce erythema was lower than that of the wild-type gene. Furthermore, preintroduction of some mutant genes delayed the erythema induced by postintroduction of the wild-type IL-6 gene, suggesting that the mutant forms of IL-6 prevent wild-type IL-6 from binding to IL-6Ralpha. This result indicates that keratinocyte gene therapy may be possible for inflammatory skin diseases using IL-6 mutant genes.

Animals↗

PubMed1997

Measurement of anti-inflammatory effects of glucocorticoids on human keratinocytes in vitro. Comparison of normal human keratinocytes with the keratinocyte cell line HaCaT.

There are only few objective in vitro methods available for the testing of anti-inflammatory pharmaceutical products. One possibility is in the stimulation of cytokine production in cultivated human keratinocytes by UV light and the subsequent testing of suppressing activities. From the dermatological aspect the interleukins 6 and 8 are especially interesting because they are elevated in psoriatic skin. In the present work three glucocorticoids were tested in cultures of normal human keratinocytes and in the permanent keratinocyte cell line HaCaT. Both cell species produced IL-6 and IL-8 spontaneously, albeit in very small amounts. After UV irradiation the interleukin production increased in a dose dependent manner. The IL-6 and IL-8 induction could be suppressed by each of the glucocorticoids tested. The thymidine incorporation rate of the cells was not affected by the glucocorticoids indicating that the observed suppression of cytokine induction was not the result of a generalised cell damage. The response of both HaCaT keratinocytes and primary human keratinocytes to UV irradiation and glucocorticoid application was similar indicating the possible use of the generally available HaCaT cells for the pharmacological testing of anti-inflammatory activities in vitro.

Anti-Inflammatory Agents↗

PubMed1987

Surface ultrastructure of cultures of affected psoriatic keratinocytes, unaffected psoriatic keratinocytes and normal keratinocytes.

The surface structure of 11-12 days old cultures from biopsies of normal skin and uninvolved psoriatic skin was investigated by scanning electron microscopy. The keratinocytes formed flat, thin, and well-organized layers of elongated, tightly apposed cells. Newly-formed cells with few and short microplicae and microvilli were seen at the colony periphery. On the maturing cells towards the colony center, the number and size of microplicae or microvilli increased gradually. No differences were found between the structures on normal keratinocytes and keratinocytes from uninvolved psoriatic skin, but in keratinocytes from involved psoriatic skin glomerulus-like rolled microplicae occasionally were found.

Adult↗

PubMed2000

Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts.

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1alpha and interleukin-1beta are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo.

Cell Differentiation↗

PubMed1999

Ceramide potentiates, but sphingomyelin inhibits, vitamin D-induced keratinocyte differentiation: comparison between keratinocytes and HL-60 cells.

Differentiation of epidermal keratinocytes and leukemia HL-60 cells induced by 1,25-dihydroxyvitamin D [1,25(OH)2D] has been reported to be mediated, at least in part, by increases in cellular ceramide levels. Ceramides produced by 1,25(OH)2D-induced sphingomyelin (SM) hydrolysis also contribute to the permeability barrier lipids in keratinocytes. Exogenously supplied SM is taken up by mammalian cells, including keratinocytes, and is incorporated into cellular pools. However, the effects of exogenously added SM on keratinocyte differentiation have not been studied. Therefore, in this study, we compared exogenously added SM with a cell-permeable ceramide for their ability to stimulate keratinocyte differentiation induced by 1,25(OH)2D. Both short-chain ceramide (C2-cer) and SM stimulated the differentiation and inhibited the proliferation of HL-60 cells. As expected, this effect was potentiated by 1,25(OH)2D. However, SM inhibited the differentiation and stimulated the proliferation of keratinocytes. While C2-cer potentiated the effects of 1,25(OH)2D, SM reversed the effects of 1,25(OH)2D on keratinocytes. The ratio of SM to ceramide was significantly different between keratinocytes and HL-60 cells. While the SM level of HL-60 cells were twice that of keratinocytes, keratinocytes contained ten times more ceramides than HL-60 cells, resulting in a ceramide/SM ratio 17 times higher in keratinocytes. Thus, we identified similarities and significant differences in the sphingolipid-mediated cell signaling pathway between keratinocytes and HL-60 cells. While SM stimulated HL-60 cell differentiation, presumably by incorporation into SMase-accessible membrane pools, it inhibited keratinocyte differentiation. In keratinocytes, SM was possibly incorporated into a different cellular pool (barrier lipid pool) or altered membrane phospholipid metabolism and membrane fluidity.

Calcitriol↗

PubMed2003

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells.

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Keratinocyte responsive element 3: analysis of a keratinocyte-specific regulatory sequence in the 230-kDa bullous pemphigoid antigen gene promoter.

The 230-kDa bullous pemphigoid antigen gene is expressed primarily, if not exclusively, in basal keratinocytes of the epidermis. Keratinocyte responsive element 3, a cis-element at position -216 to -197 of the human 230-kDa bullous pemphigoid antigen gene promoter, confers tissue-specific expression to this gene (Tamai et al: J Biol Chem 270:7609-7614, 1995). In this study, we investigated the functional characteristics of keratinocyte responsive element 3 on the 230-kDa bullous pemphigoid antigen gene core promoter by transient transfections of cultured normal human keratinocytes and normal human fibroblasts, as well as of lung carcinoma (A549), osteosarcoma (OST), and gastric adenocarcinoma (GT3TKB) cell lines. A 230-kDa bullous pemphigoid antigen gene core promoter/luciferase reporter gene plasmid construct, pBPL, was modified to develop a series of constructs (pKBPL-p4KBPL), which have insertions of one, two, three, or four tandem repeats of keratinocyte responsive element 3, and these plasmids were used in transient transfections of the cultured cells. The promoter activities of pKBPL-p4KBPL constructs, relative to pBPL, in normal human keratinocytes were 7.6-, 15.5-, 4.6-, and 2.7-fold higher, respectively, whereas no upregulatory effect by keratinocyte responsive element 3 insertion was observed in other cell lines tested. prKBPL, a plasmid constructed with keratinocyte responsive element 3 in reverse orientation, showed essentially no activity in normal human keratinocytes. Insertion of a random 20 bp sequence between keratinocyte responsive element 3 and the 230-kDa bullous pemphigoid antigen gene core promoter resulted in about 40% reduction of luciferase activity in normal human keratinocytes. These data suggest that keratinocyte responsive element 3 functions as a position-, copy number-, and orientation-dependent cis-element contributing to tissue-specific regulation of the 230-kDa bullous pemphigoid antigen gene.

Autoantigens↗

PubMed1996

The effect of IFN-gamma on healthy and psoriatic keratinocytes in a skin equivalent model is influenced by the source of the keratinocytes and by their interactions with fibroblasts.

We investigated the effect of interferon-gamma (IFN-gamma) on skin equivalents. Keratinocytes from involved and uninvolved skin from psoriatic subjects and from healthy subjects were grown on preproduced dermal equivalents (DE) containing fibroblasts from healthy skin or psoriatic lesions. Healthy keratinocytes were added when the dermal equivalents were either 22 days (DE(22)) or 37 days old (DE(37)) and psoriatic keratinocytes when the dermal equivalents were 28-52 days old (DE(28-52)). The skin equivalents were cultured for 11 days in a serum-free medium, and then with or without 500 U/ml IFN-gamma for 6 days. The expression of markers associated with differentiation and proliferation were investigated by immunohistochemistry. Differentiation was assessed by computed scores for the expression of cytokeratin 16, involucrin, filaggrin and the receptor for epidermal growth factor. The differentiating effect of IFN-gamma on healthy keratinocytes grown on DE(37) was significantly stronger than on psoriatic keratinocytes grown on DE(28-52). In healthy keratinocytes, the differentiating effect of IFN-gamma was significantly stronger in skin equivalents containing DE(37) than in those containing DE(22). The proliferation rate, i.e. the percentage of Ki-67+ keratinocytes in the basal layer, was studied in healthy keratinocytes grown on DE(22). In these cultures IFN-gamma increased the proliferation rate in the presence of psoriatic fibroblasts but not in the presence of healthy fibroblasts. HLA-DR expression was induced only in healthy keratinocytes grown on DE(22). We conclude that the influence of IFN-gamma epidermal differentiation and proliferation is influenced by the origins of both the keratinocytes and the fibroblasts. These findings suggest that interactions between keratinocytes and fibroblasts might be involved in the pathogenesis of psoriasis.

Adult↗

PubMed1992

Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure.

Animals↗

PubMed1995

Discordant expression of Bcl-x and Bcl-2 by keratinocytes in vitro and psoriatic keratinocytes in vivo.

Apoptosis is a required event in maintaining kinetic homeostasis within continually renewing tissues such as skin. However, no systematic study of the apoptotic process in epidermal keratinocytes of the skin has been performed. In this report, we examined the expression of proteins associated with promoting (Fas) or preventing (Bcl-2, Bcl-x, CD40) apoptosis in the normal, psoriatic, and malignant keratinocyte. Immunohistochemical staining and flow cytometry analysis revealed that normal cultured keratinocytes express low levels of Fas, CD40, and Bcl-x that was enhanced by cytokines including gamma-interferon (IFN-gamma) and a phorbol ester tumor promoter, TPA. Only faint Bcl-2 staining was detected in cultured keratinocytes exposed to IFN-gamma and TPA compared with the prominent expression of Bcl-x. Biopsies of normal skin, psoriatic plaques, and basal cell carcinomas were examined to extend the in vitro observations. Immunohistochemical staining revealed that while keratinocytes in normal epithelium express low to absent levels of Fas and Bcl-x, psoriatic keratinocytes expressed significantly higher levels of Fas and Bcl-x. In contrast, malignant keratinocytes in basal cell carcinomas expressed high levels of Bcl-2, but minimal Bcl-x, and no Fas. Immunoblot analysis revealed that the long form of Bcl-x (Bcl-xI), which prevents apoptosis in lymphocytes, is expressed by cultured keratinocytes and psoriatic plaque keratinocytes. We conclude that normal cytokine-activated keratinocytes can express an apoptotic (Fas) and an anti-apoptotic protein (Bcl-x). The overexpression of Bcl-x in psoriasis, or Bcl-2 in basal cell carcinomas, may contribute to the longevity of these cells by blocking the normal apoptotic process involved in the terminal differentiation program of epidermal keratinocytes.

Adult↗

PubMed1995

Keratinocytes constitutively express the Fas antigen that mediates apoptosis in IFN gamma-treated cultured keratinocytes.

The Fas antigen is a cell surface protein that can mediate apoptosis in many cell types. Although its physiological function is still unclear, recent evidence indicates that this surface molecule is involved in apoptosis in the immune system and the liver. The epidermis is an organ that undergoes terminal differentiation with the eventual death of keratinocytes, and it has been suggested that this is a specialized form of apoptosis. In the present study, we examined whether or not the Fas antigen is involved in keratinocyte apoptosis. Immunoreactivity for the Fas antigen was found throughout the epidermis in normal human skin sections and cultured normal human keratinocytes, and mRNA for the Fas antigen was found to be constitutively expressed in normal epidermis and cultured normal keratinocytes by RT-PCR analysis. To determine whether the Fas antigen in keratinocytes is functional, we used a cytotoxic monoclonal antibody (mAb) against the Fas antigen to induce apoptosis. This antibody did not induce apoptosis of cultured keratinocytes even though they expressed the Fas antigen. We then tested the ability of several cytokines (TGF beta, TNF alpha and IFN gamma) to induce Fas-mediated keratinocyte apoptosis. Only pretreatment with IFN gamma followed by the addition of the anti-Fas mAb induced apoptosis, as assessed by cell viability, morphological changes and ultrastructural characteristics, suggesting that constitutive expression of the Fas antigen is not sufficient to induce apoptosis in keratinocytes and that keratinocyte apoptosis via the Fas antigen-mediated mechanism may require the activation of keratinocytes by IFN gamma, which is thought to be produced by activated T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1991

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes.

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.

Antibodies↗

PubMed1993

Leukotriene B4 formation during human neutrophil keratinocyte interactions: evidence for transformation of leukotriene A4 by putative keratinocyte leukotriene A4 hydrolase.

In the present study, keratinocytes were coincubated with human neutrophils to determine whether or not an increase in leukotriene B4 formation can occur. Human keratinocytes used were cultured in serum-free, low-calcium medium, whereas neutrophils were purified from heparinized venous blood. After coincubations, formation of leukotriene B4 was determined by reversed-phase high-performance liquid chromatography, coupled with its characteristic UV scan. Confirmation and quantification was by radioimmunoassay. Our data revealed that incubations of keratinocytes (1.5 x 10(6)) alone stimulated with calcium ionophore resulted in no detectable amounts of leukotriene B4. In contrast, incubations of neutrophils (5 x 10(6)) alone resulted in the generation of 62.2 +/- 8.5 ng of LTB4. Coincubations of the neutrophils with keratinocytes (ratio 3:1) resulted in a 56-163% increase in leukotriene B4 formation. To delineate the source of the newly formed leukotriene B4, incubations of keratinocytes with leukotriene A4 revealed that keratinocytes can transform leukotriene A4 into leukotriene B4. These latter findings indicate that although keratinocytes cannot directly metabolize arachidonic acid into leukotriene B4 via the 5-lipoxygenase enzyme, they can transform neutrophil-derived leukotriene A4 into leukotriene B4, thus indicating the possible existence of a putative keratinocyte-leukotriene A4 hydrolase. It is therefore reasonable to speculate that the keratinocytes possess the capacity to generate leukotriene B4 in the epidermis when provided leukotriene A4 and thereby can amplify the inflammatory processes occurring during neutrophil exocytosis. These findings indicate that transcellular metabolism of arachidonic acid metabolites in the epidermis by keratinocytes and neutrophils may contribute to the high levels of leukotriene B4 in lesional skin of inflammatory skin diseases.

Arachidonic Acid↗

PubMed1991

55-kd tumor necrosis factor receptor is expressed by human keratinocytes and plays a pivotal role in regulation of human keratinocyte ICAM-1 expression.

Tumor necrosis factor alpha (TNF alpha) is a potent modulator of human keratinocyte intercellular adhesion molecule-1 (ICAM-1) expression. TNF alpha is known to exert its biologic effects by binding to specific cell-surface receptors. Two distinct TNF binding molecules, the 55-kd and the 75-kd TNF receptor (TNFR) recently have been found to be expressed by human cells. These two receptor types are independently regulated and differ markedly in their intracellular regions, indicating functional dichotomy. In order to gain further insight into the mechanisms underlying ICAM-1 regulation in human keratinocytes, in the present study, the receptor molecules mediating TNF alpha induced ICAM-1 upregulation in human keratinocytes was defined. Human keratinocyte TNFR expression was assessed using monoclonal antibodies that specifically recognize the 55-kd or the 75-kd TNFR. Using FACS analysis, normal (HNK) as well as transformed (KB) human keratinocytes were found to react with anti-55-kd TNFR, but not anti-75-kd TNFR antibodies. These immunofluorescence data were confirmed by Northern blot analysis revealing clearly detectable amounts of mRNA specific for the 55-kd TNFR in KB cells. Incubation of human keratinocytes with anti-55-kd TNFR antibodies at 37 degrees C for 24 h increased ICAM-1 expression in a TNF alpha-like fashion. Moreover, the well known synergistic effect of IFN gamma plus TNF alpha on keratinocyte ICAM-1 induction could be mimicked by stimulation of cells with IFN gamma plus anti-55-kd TNFR antibodies. Synergistic ICAM-1 induction was not associated with increased expression of the 55-kd TNFR in IFN gamma-stimulated human keratinocytes. These studies indicate that human keratinocytes express the 55-kd TNF receptor and that this surface molecule may play an important role in regulation of human keratinocyte ICAM-1 expression.

Binding Sites, Antibody↗

PubMed2000

Growth-regulated peptide-alpha (GRO-alpha) production by oral keratinocytes: a comparison with skin keratinocytes.

Growth regulated peptide (GRO-alpha) is chemotactic for neutrophils. It also stimulates keratinocyte proliferation and migration, and angiogenesis in cutaneous wound healing. We compared GRO-alpha production by normal human skin and oral keratinocytes, and the effects of cytokine stimulation. Resting keratinocytes produced little, if any, GRO-alpha. TNF-alpha induced a large increase in GRO-alpha mRNA and protein production in both cell types (P<0.001). However, the response of oral keratinocytes was significantly higher (P<0.01). Oral, but not skin, keratinocytes also produced significant amounts of GRO-alpha in response to IL-1 alpha (P<0.005) and IL-4 (P<0.01) stimulation. Indeed, there was an additive effect on GRO-alpha production when oral keratinocytes were stimulated with combinations of TNF-alpha and IL-1 alpha or TNF-alpha and IL-4. Neither cell type responded to interferon gamma. Keratinocyte GRO-alpha production may help selectively recruit neutrophils in mucocutaneous inflammatory diseases, and differences in production by skin and oral keratinocyte could explain the different presentation of these diseases at the two sites. The increased GRO-alpha responsiveness of oral keratinocytes may also help explain the excellent wound healing properties of oral mucosa.

Biopsy↗

PubMed2006

Hyaluronic acid inhibits the adherence and growth of monolayer keratinocytes but does not affect the growth of keratinocyte epithelium.

Hyaluronic acid (HA) is involved in epidermal biology but evidence for its functional significance is sparse. In this study, low-calcium monolayer and high-calcium epithelium cultures of human keratinocytes were used to study the effect of up to four different HA preparations on keratinocyte growth and on the adherence of proliferating keratinocytes onto the plastic surface coated with different matrix proteins. In suboptimally growing monolayer culture, up to 1,000 microg/ml rooster comb HA and streptococcus equi HA inhibited keratinocyte growth. Instead, all HA preparations tested did not affect the growth and migration of keratinocyte epithelium using optimal or suboptimal growth conditions. In the cell adherence assays, up to 1,000 microg/ml rooster comb HA and streptococcus equi HA inhibited the keratinocyte adherence onto the fibronectin- and collagen-coated substratum. In contrast to other HA preparations, HA from human umbilical cord did not affect the growth of monolayer keratinocytes and it increased markedly the cell adherence onto the collagen-coated substratum. This increase, however, can be attributed to chonroitin sulphate proteoglycan contaminant present in this HA preparation. In conclusion, HA can inhibit the growth and adherence of proliferating monolayer keratinocytes, but it has no apparent effect on the growth and migration of keratinocyte epithelium.

Adjuvants, Immunologic↗

PubMed2003

Keratinocyte growth inhibition by high-dose epidermal growth factor is mediated by transforming growth factor beta autoinduction: a negative feedback mechanism for keratinocyte growth.

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

Activin Receptors, Type I↗

PubMed2005

Less keratinocyte-derived factors related to more keratinocyte apoptosis in depigmented than normally pigmented suction-blistered epidermis may cause passive melanocyte death in vitiligo.

Stem cell factor (SCF) of keratinocyte origin regulates melanocyte growth and survival. Deprivation of survival factors causes the apoptosis of melanocytes. Vitiligo often develops following physical trauma, even if this is minor. The exact mechanism of the Koebner phenomenon in vitiligo is unclear. Apoptosis of keratinocytes, which occurs more in depigmented suction-blistered epidermis than in the normally pigmented counterpart, could reduce levels of keratinocyte-derived factors such as SCF and basic fibroblast growth factor (bFGF). Levels of SCF expression were examined in the depigmented and normally pigmented paired epidermis of 19 patients with vitiligo, and bFGF expression in six patients. The expression of SCF (p<0.001) and bFGF was usually reduced in the depigmented compared with the normally pigmented epidermis. Apoptosis of cultured normal human keratinocytes, which was induced by staurosporine, resulted in a concentration-dependent decrease in levels of SCF mRNA and protein. Normal human melanocytes proliferated more in medium containing SCF or keratinocyte (XB-2) feeder than in medium with neither. Deprivation of SCF or keratinocyte feeder in the culture medium induced a marked decrease in melanocytes as a result of apoptosis. Therefore, lower expression of keratinocyte-derived factors, including SCF, in vitiliginous keratinocytes, which could result from keratinocyte apoptosis, might be responsible for passive melanocyte death and may explain the Koebner phenomenon.

Adolescent↗