RESUMEN
The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant [sodium dodecyl sulfate (SDS)]. Nickel sulfate, potassium dichromate, and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration-dependent increase in chemokine (CCL20, CCL27, and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulfate and SDS was investigated in more detail: similar to chemokine secretion, no difference was observed in the time- and concentration-dependent increase in pro-inflammatory cytokine [interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha)] secretion. Maximal increase in IL-1alpha secretion occurred within 2 h after exposure to both nickel sulfate and SDS and prior to increased chemokine secretion. TNF-alpha secretion was detectable 8 h after chemical exposure. After allergen or irritant exposure, increased CCL20 and CXCL8, but not CCL27, secretion was inhibited by neutralizing human antibodies to either IL-1alpha or TNF-alpha. Our data show that alarm signals consist of primary and secondary signals. IL-1alpha and TNF-alpha are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20 and CXCL8) alarm signals. However, some chemokines, for example, CCL27 can be secreted in an IL-1alpha and TNF-alpha independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin.
Asunto(s)
Quimiocinas CC/biosíntesis , Interleucina-1/biosíntesis , Interleucina-8/biosíntesis , Proteínas Inflamatorias de Macrófagos/biosíntesis , Piel/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Alérgenos/metabolismo , Cáusticos/farmacología , Células Cultivadas , Quimiocina CCL20 , Quimiocina CCL27 , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/metabolismo , Humanos , Interleucina-1/metabolismo , Irritantes/farmacología , Queratinocitos/citología , Níquel/farmacología , Dicromato de Potasio/farmacología , Proteínas Recombinantes/química , Piel/metabolismo , Dodecil Sulfato de Sodio/farmacología , Tensoactivos/farmacología , Factores de TiempoRESUMEN
BACKGROUND: Whereas T lymphocytes are widely accepted as effector cells determining the pathogenesis of allergic contact dermatitis, contradictory results have been found regarding the roles of different T-cell subsets. The use of various experimental models, involving long-term cultured T-cell lines or clones, may explain these contradictory results. OBJECTIVE: To investigate the involvement of distinct T-cell subsets in patients with nickel contact allergy. METHODS: Different T-cell subsets were directly isolated from peripheral blood mononuclear cells (PBMCs) of nickel-allergic patients, and their proliferative capacity, type-1 or type-2 cytokine secretion [measured by interferon (IFN)-gamma or interleukin (IL)-5 release] and phenotypical marker expression were analysed after stimulation with nickel. RESULTS: Only CD4+ CLA+ CD45RO+ and not CD8+ T cells proliferate and produce both type-1 (IFN-gamma) and type-2 (IL-5) cytokines in response to nickel. Moreover, cells expressing the marker CLA in combination with CD4, CD45RO or CD69 are increased after nickel-specific stimulation. Interestingly, in addition, CD45RA+ CLA+ cells showed an increased frequency after allergen-specific stimulation. Analysis of nickel-reactive T cells for expression of distinct chemokine receptors showed that both proliferative capacity and cytokine production are restricted to subsets expressing CXCR3, CCR4 but not CCR6. Fluorescence-activated cell sorting analysis of chemokine receptors expressed on nickel-stimulated T cells confirmed these results; a subset of T cells expressing CLA and CXCR3, CCR4 and, most importantly, CCR10 increased in response to allergen, while these CLA+ nickel-reactive T cells were all negative for CCR6. CONCLUSIONS: These findings demonstrate that freshly isolated nickel-reactive T cells can be characterized as CD4+ CLA+ memory T cells which express the chemokine receptors CXCR3, CCR4 and CCR10, but not CCR6.
Asunto(s)
Antígenos CD/análisis , Dermatitis Alérgica por Contacto/inmunología , Erupciones por Medicamentos/inmunología , Níquel/efectos adversos , Receptores de Quimiocina/análisis , Subgrupos de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T , Antígenos de Neoplasias , Antígenos CD4/análisis , Estudios de Casos y Controles , Citometría de Flujo , Humanos , Memoria Inmunológica , Antígenos Comunes de Leucocito/análisis , Glicoproteínas de Membrana/análisis , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Receptores CCR10 , Receptores CCR4 , Receptores CXCR3 , Estadísticas no ParamétricasRESUMEN
BACKGROUND: Down-regulation or modulation of T cell activity by immunosuppressive drugs is an effective treatment in diseases where exaggerated T cell responses play a role. A primary effect of the anti-inflammatory drugs (AIDs) is inhibition of the synthesis of growth factors, such as IL-2, thereby down-regulating T cell proliferation. However, it is still largely unknown to what extent these AIDs are able to down-regulate specifically type-1 or type-2 T cell cytokine production, and whether they can down-modulate chemokine receptor expression, thereby preventing migration of T cells to the site of inflammation. OBJECTIVE: We investigated the suppressive effect of dermatologically used AID (cyclosporin A (CsA), lactoferrin (LF), 1 alpha, 25-dihydroxyvitamin D(3) (VD(3)), hydrocortisone (HC), di-methyl-fumarate (DMF), diclofenac (DF)) on both type-1 and type-2 T cells. Since allergic contact dermatitis is a skin disorder in which an exaggerated T cell response of both types of T cell subsets can be observed, we used this disorder as a model to study the capacity of AID to suppress type-1 or type-2 T cell responses. METHODS: Peripheral blood mononuclear cells of nickel allergic patients were cultured in the presence of allergen and increasing concentrations of AID. Proliferation was determined by measuring (3)H thymidine incorporation; chemokine receptor (CCR10, CCR4, CXCR3) expression was studied by flow cytometric analysis and IFN-gamma or IL-5 cytokine production was measured by ELISA. RESULTS: Three major patterns can be distinguished regarding the effect of AID on T cell responses. The first group, including CsA and LF, inhibited non-selectively T cell proliferation, chemokine receptor expression and cytokine production, with CsA as the most potent drug tested. A second group of AID, which included VD(3), HC and DMF, suppressed mainly type-1 T cell responses, as revealed by strong interference with IFN-gamma production and CXCR3 expression, and limited effects on either or both IL-5 and CCR4 expression. The third pattern was displayed by DF, which down-regulated IL-5 production and CCR4 expression, whereas IFN-gamma and CXCR3 were unaltered. CONCLUSIONS: Using a contact allergy model, we have demonstrated that various AIDs show distinct pharmacological profiles in that either type-1 or type-2 or both T cell responses are suppressed. These results should contribute to a more rational selection of AID in treating inflammatory skin diseases mediated by either or both of these T cell subsets.
Asunto(s)
Antiinflamatorios/uso terapéutico , Dermatitis Alérgica por Contacto/tratamiento farmacológico , Dermatitis Alérgica por Contacto/inmunología , Células TH1/inmunología , Células Th2/inmunología , Células Cultivadas , Colecalciferol/uso terapéutico , Ciclosporina/uso terapéutico , Diclofenaco/uso terapéutico , Dimetilfumarato , Citometría de Flujo , Fumaratos/uso terapéutico , Humanos , Hidrocortisona/uso terapéutico , Interleucina-12/inmunología , Interleucina-4/inmunología , Interleucina-7/inmunología , Lactoferrina/uso terapéutico , Activación de Linfocitos/efectos de los fármacos , Níquel/efectos adversos , Pruebas del ParcheRESUMEN
Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.
Asunto(s)
Quimiocinas CXC/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Quimiocina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Calcio/metabolismo , Señalización del Calcio , Células Cultivadas , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocinas CXC/química , Quimiocinas CXC/aislamiento & purificación , Quimiotaxis , Cricetinae , Humanos , Interferón gamma/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Receptores CXCR3 , Receptores de Quimiocina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Linfocitos T/citología , Linfocitos T/metabolismo , TransfecciónRESUMEN
Recruitment of activated T-cells to the skin is a common feature in a wide variety of inflammatory skin diseases. As CXCR3 activating chemokines CXCL10 (IP-10), CXCL9 (Mig), and CXCL11 (IP-9/I-TAC) specifically attract activated T-cells, this study addressed the question of whether differences in the expression of these chemokines correlate with the site and cellular composition of the skin infiltrates in different types of inflammatory skin disease. Skin biopsies from lichen planus, chronic discoid lupus erythematosus, allergic patch test reactions, psoriasis, and Jessner's lymphocytic infiltration of the skin were investigated for chemokine expression using RNA in situ hybridization, and for the expression of CXCR3 using immunohistochemistry. The results showed differential expression of CXCL10, CXCL9, and CXCL11, which correlated with differences in the localization and cellular composition of the infiltrates. Whereas CXCL10 and CXCL11 were mainly expressed by basal keratinoctyes, CXCL9 mRNA expression was located predominantly in the dermal infiltrates. Correlation with immunohistochemical data suggested that macrophages and activated keratinocytes were the main producers of these chemokines. CXCR3 was expressed by a majority of both CD4+ and CD8+ infiltrating T-cells, suggesting a functional interaction between locally produced chemokines and CXCR3-expressing T-cells. In conclusion, these findings indicate that these CXCR3 activating chemokines play a significant role in the recruitment and maintenance of T-cell infiltrates in the inflammatory skin diseases studied.
Asunto(s)
Quimiocinas CXC/metabolismo , Dermatitis/inmunología , Receptores de Quimiocina/metabolismo , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocinas CXC/genética , Expresión Génica , Antígenos HLA-DR/metabolismo , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Molécula 1 de Adhesión Intercelular/metabolismo , Liquen Plano/inmunología , Lupus Eritematoso Discoide/inmunología , Psoriasis/inmunología , ARN Mensajero/genética , Receptores CXCR3RESUMEN
BACKGROUND: The effectiveness of systemic treatment of psoriasis with fumaric acid esters has been proven, but their mode of action at the cellular and molecular level has not yet been fully elucidated. OBJECTIVES: To study the effect of dimethylfumarate (DMF) on the production of the chemokines CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11, formerly known as GROalpha, interleukin-8, Mig, IP-10 and IP-9/I-TAC, respectively, in human keratinocytes and peripheral blood mononuclear cells (PBMC). METHODS: Cultured keratinocytes were stimulated with interferon (IFN) -gamma to produce CXCL9, CXCL10 and CXCL11 and with phorbol myristate acetate to produce CXCL1 and CXCL8 in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). PBMC were stimulated with either IFN-gamma to produce CXCL9 and CXCL10 or lipopolysaccharide to produce CXCL8, in the absence and presence of DMF (5, 15 and 45 micromol L(-1)). RNA preparations from isolated keratinocytes were analysed by Northern blotting; protein production by keratinocytes and PBMC was monitored by an enzyme-linked immunosorbent assay. RESULTS: Northern blot analysis on isolated keratinocyte RNA preparations showed a dose-dependent inhibition of CXCL1, CXCL8, CXCL9, CXCL10 and CXCL11 transcription by DMF. At 45 micromol L(-1) the inhibition was almost complete. In addition, keratinocytes and PBMC showed in the presence of DMF a dose-dependent inhibition of CXCL8, CXCL9 and CXCL10 protein production. CONCLUSIONS: These results show the ability of DMF to inhibit the production of chemokines that may be critically involved in the development and perpetuation of psoriatic lesions. This might explain, at least in part, the beneficial effects of treatment with fumaric acid esters in psoriasis patients.
Asunto(s)
Quimiocinas CXC/biosíntesis , Fármacos Dermatológicos/farmacología , Fumaratos/farmacología , Queratinocitos/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Northern Blotting , Técnicas de Cultivo de Célula , Quimiocinas CXC/genética , Dimetilfumarato , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Queratinocitos/inmunología , Queratinocitos/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Psoriasis/tratamiento farmacológico , ARN Mensajero/genéticaRESUMEN
Differentiation between allergic and irritant contact dermatitis reactions is difficult, as both inflammatory diseases are clinically, histologically, and immunohistologically very similar. Previous studies in mice revealed that the chemokine IP-10 is exclusively expressed in allergic contact dermatitis reactions. In the present study, we investigated whether the mRNA expression of IP-10 and the related CXCR3 activating chemokines, Mig and IP-9 are also differentially expressed in human allergic contact dermatitis and irritant contact dermatitis reactions. Skin biopsies from allergic (13 cases) and sodium lauryl sulfate-induced irritant patch test reactions (13 cases), obtained 1-72 h after patch testing, were studied by means of an in situ hybridization technique. Results of chemokine mRNA expression were correlated with clinical scoring, histology, and immunohistochemical data including the proportion of inflammatory cells expressing CXCR3, the receptor for IP-10, Mig, and IP-9, and ICAM-1 and HLA-DR expression on keratinocytes. IP-10, Mig, and IP-9 mRNA were detected in seven of nine allergic contact dermatitis reactions after 24-72 h, but not in sodium lauryl sulfate-induced irritant contact dermatitis reactions. ICAM-1 expression by keratinocytes was only found in allergic contact dermatitis reactions and correlated with chemokine expression. Moreover, up to 50% of the infiltrating cells in allergic contact dermatitis expressed CXCR3, in contrast to only 20% in irritant contact dermatitis reactions. In conclusion, we have demonstrated differences in chemokine expression between allergic contact dermatitis and irritant contact dermatitis reactions, which might reflect different regulatory mechanisms operating in these diseases and may be an important clue for differentiation between allergic contact dermatitis and irritant contact dermatitis reactions.
Asunto(s)
Quimiocinas CXC/genética , Dermatitis Alérgica por Contacto/inmunología , Dermatitis Irritante/inmunología , Péptidos y Proteínas de Señalización Intercelular , Pruebas del Parche , ARN Mensajero/análisis , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Antígenos HLA-DR/análisis , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Interferón gamma/farmacología , Queratinocitos/química , Receptores CXCR3 , Receptores de Quimiocina/análisisRESUMEN
CXCL 11, encoded by the cDNA sequences designated beta-R1, H-174, or I-TAC, is a CXC chemokine ligand for CXCR3 and assumed to be involved in inflammatory diseases characterized by the presence of activated T-cells. We here describe the genomic organization (four exons interrupted by three introns of 585, 98 and 230 bp) and sequence including 960 bp from the immediate 5'-upstream region of the human CXCL 11 gene. Within the promoter region, consensus sequences for regulatory elements (ISRE, GAS, NF-kappaB) important for cytokine-induced gene transcription were identified. The effect of (pro)inflammatory cytokines on CXCL 11 mRNA expression in monocytic cell lines (THP-1, U937) and primary cultures of dermal fibroblasts and endothelial cells were examined using Northern blot analysis. For these cell types, IFN-gamma was a potent inducer of CXCL 11 transcription, which was synergistically enhanced by TNF-alpha.
Asunto(s)
Quimiocinas CXC/genética , Secuencia de Bases , Línea Celular , Quimiocina CXCL11 , Quimiocinas CXC/química , Exones , Biblioteca Genómica , Humanos , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
IFN-gamma-inducible protein-10 (IP-10) is a chemokine, which plays an important role in mediating inflammation by attracting activated T cells, and it has been demonstrated in inflammatory skin diseases and cutaneous T cell lymphomas. Keratinocytes can abundantly produce IP-10 mRNA after IFN-gamma treatment. In this study we explored possibilities to downregulate IP-10 expression using human cultured keratinocytes as a model system. Decreased IP-10 mRNA levels were found using specific inhibitors of protein kinase (PK)-C (H-7 and Calphostin C). Moreover, depletion of PK-C by pretreatment of the cells with phorbol myristate (PMA) also down-regulated IP-10 mRNA expression. In addition, elevated cAMP levels were shown to inhibit IP-10 mRNA expression as could be concluded from experiments with forskolin and W-7, substances which, directly or indirectly, raise the intracellular cAMP level. With Genistein, an inhibitor of tyrosine kinase, the IFN-gamma-induced IP-10 mRNA expression was also found to be diminished. These data suggest that inhibitors of the IP-10 mRNA expression in cultured keratinocytes may be potentially of clinical relevance to suppress inflammatory processes in the skin.
Asunto(s)
Quimiocinas CXC/genética , AMP Cíclico/metabolismo , Queratinocitos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Células Cultivadas , Quimiocina CXCL10 , AMP Cíclico/fisiología , Humanos , Masculino , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiologíaRESUMEN
Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.
Asunto(s)
Quimiocinas CXC/metabolismo , Queratinocitos/metabolismo , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células Cultivadas , Quimiocina CXCL11 , Quimiocinas CXC/genética , Quimiocinas CXC/fisiología , Quimiotaxis , Clonación Molecular , Cricetinae , Relación Dosis-Respuesta a Droga , Humanos , Hibridación in Situ , Inflamación/metabolismo , Ligandos , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Receptores de Citocinas/genética , Linfocitos T/citologíaRESUMEN
Epidermal infiltration by neoplastic CD4+ T cells is a characteristic histologic feature of early stage mycosis fungoides, the most common type of cutaneous T cell lymphoma (CTCL). The mechanisms involved in epidermotropism are unknown. It has been suggested that the CXC chemokines IL-8 and interferon-gamma inducible protein 10 (IP-10) may play a role, but evidence that these chemokines are produced within the epidermis in epidermotropic CTCL is lacking. In this study skin biopsies from 17 CTCL patients, including 12 mycosis fungoides, four pleomorphic CTCL, and one CD8+ CTCL, were investigated for epidermal IL-8 and IP-10 mRNA expression by RNA in situ hybridization. In addition, the expression of monokine induced by gamma-interferon (Mig) mRNA, a CXC chemokine closely related to IP-10, was studied as well. The expression of IL-8 receptors A and B (CXCR1 and CXCR2, respectively) was investigated by immunohistochemistry. The results were correlated with the number and phenotype of epidermotropic T cells. Epidermal expression of IP-10 and Mig mRNA was detected in 10 of 11 and seven of 11 epidermotropic CTCL, respectively, but not in five nonepidermotropic CTCL biopsies or normal human skin. Epidermal IP-10 and Mig mRNA expression correlated with epidermal infiltration of CD4+ T cells, but not of CD8+ T cells. IL-8 mRNA was demonstrated in the epidermis of only two of 15 CTCL biopsies, and was associated, in both cases, with accumulation of neutrophils. Consistently, immunostaining of the (intraepidermal) T cells with antibodies against CXCR1 and CXCR2 was not observed. In conclusion, the results of this study indicate that IP-10, and to a lesser extent Mig, but not IL-8 is involved in the preferential infiltration of neoplastic CD4+ T cells in CTCL.
Asunto(s)
Quimiocinas CXC/genética , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/farmacología , Interleucina-8/genética , Linfoma Cutáneo de Células T/metabolismo , ARN Mensajero/análisis , Neoplasias Cutáneas/metabolismo , Antígenos CD/fisiología , Quimiocina CXCL10 , Quimiocina CXCL9 , Humanos , Inmunohistoquímica , Hibridación in Situ , Receptores de Quimiocina/fisiología , Receptores de Interleucina/fisiología , Receptores de Interleucina-8A , Receptores de Interleucina-8BRESUMEN
IP-10, a member of the CXC family of chemokines, is considered to play an important role in inflammation via its T-cell chemotactic and adhesion-promoting properties. Elevated IP-10 levels in the epidermis of psoriasis, delayed-type hypersensitivity reactions, cutaneous T-cell lymphoma and fixed drug eruptions prompted us to study its expression in keratinocytes. IP-10 mRNA could be detected using the sensitive RT-PCR method, but not by Northern blotting in RNA preparations from unstimulated normal cultured keratinocytes, indicating a low steady-state level of IP-10 mRNA. Upon stimulation with IFN-gamma, IP-10 mRNA was found to accumulate in high amounts in a time- and dose-dependent manner. Superexpression was found with the combination of IFN-gamma and TNF-alpha or IL-1, although these latter cytokines by themselves did not induce accumulation of IP-10 mRNA. Nuclear run-on experiments performed to investigate the regulation of IP-10 mRNA expression, showed a very high constitutive transcriptional activity of the IP-10 gene in unstimulated keratinocytes, which was not affected by stimulation with IFN-gamma, TNF-alpha, or a combination of IFN-gamma and TNF-alpha. Protein kinase C (PKC) was shown to be involved in IP-10 mRNA expression since the PKC inhibitor H7 decreased IP-10 mRNA accumulation. A protein was isolated from culture supernatants of stimulated keratinocytes using HPLC techniques and, by sequence analysis, was found to be identical to IP-10. The dynamics of secretion of IP-10 protein as monitored by ELISA was shown to parallel the mRNA expression.
Asunto(s)
Quimiocinas CXC/genética , Queratinocitos/metabolismo , Antineoplásicos/farmacología , Quimiocina CXCL10 , Quimiocinas CXC/aislamiento & purificación , Quimiocinas CXC/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Interferón gamma/farmacología , Interleucina-1/farmacología , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Masculino , Reacción en Cadena de la Polimerasa , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/química , Piel/citología , Piel/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Recently we showed the in vivo relevance of chemokines in cases of bacterial peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients. Mesothelial cells, the most numerous cells in the peritoneal cavity, are hypothesized to function as a main source of chemokine production. We investigated the time- and dose-dependent expression patterns of four chemokines by mesothelial cells at the mRNA and protein level in response to stimulation with physiological doses of proinflammatory mediators that are present at the site of bacterial inflammation. Besides the chemokines huGRO-alpha (attractant for neutrophils), MCP-1 and RANTES (monocyte attractants), the expression and production of IP-10 was analysed. Mesothelial cells were cultured and stimulated with either IL-1beta, tumour necrosis factor-alpha (TNF-alpha) or IFN-gamma or combinations of these. The time- and dose-dependent mRNA expression of the chemokines was determined by Northern blot analysis and the protein production by ELISA. It was concluded that mesothelial cells could indeed be triggered by the mentioned stimuli to induce mRNA and protein production (huGRO-alpha and IP-10) or to augment constitutive protein production (MCP-1). However, RANTES mRNA and protein production could only be induced in some cases and only in small amounts. The chemokine response of mesothelial cells was regulated differentially, depending on the stimulus and the chemokine measured. In distinct cases, combination of the stimuli led to synergy in mRNA expression and protein production. The presented in vitro data support our hypothesis that mesothelial cells in vivo are the main source of relevant chemokines in response to proinflammatory mediators, suggesting an important role for mesothelial cells in host defence.
Asunto(s)
Quimiocina CCL2/biosíntesis , Quimiocina CCL5/biosíntesis , Quimiocinas CXC/biosíntesis , Factores Quimiotácticos/biosíntesis , Células Epiteliales/metabolismo , Sustancias de Crecimiento/biosíntesis , Péptidos y Proteínas de Señalización Intercelular , Células Cultivadas , Quimiocina CXCL1 , Quimiocina CXCL10 , Células Epiteliales/efectos de los fármacos , Humanos , Interferón gamma/metabolismo , Interferón gamma/farmacología , Interleucina-1/farmacología , Epiplón , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The expression of IL-8 in psoriasis has been clearly shown with the use of immunocytochemical, RT-PCR and in situ hybridization methods. The presence of its ligand, the IL-8 receptor, has been demonstrated by the RT-PCR technique. We report here a study of the expression of both IL-8 type A and B receptors by immunohistochemical techniques, using one polyclonal and four monoclonal antibodies. By this technique, we found that the neutrophilic granulocytes express the IL-8 type A receptor, whereas the IL-8 type B receptor was present on the keratinocytes. The type B receptor on the keratinocytes was localized in the suprabasal layers of the epidermis. Following therapy, the expression of the IL-8 type B receptor on the keratinocytes was reduced. This could suggest that IL-8 in psoriasis is involved in the disturbed differentiation rather than in proliferation, probably via an autocrine loop.
Asunto(s)
Antígenos CD/análisis , Psoriasis/metabolismo , Receptores de Interleucina/análisis , Animales , Humanos , Inmunohistoquímica , Ratones , Psoriasis/terapia , Conejos , Receptores de Interleucina-8A , Piel/químicaRESUMEN
BACKGROUND AND DESIGN: From previous studies, we concluded that the fluorescence overlay antigen mapping (FOAM) technique could be of value to the differential diagnosis of the acquired subepidermal bullous skin disorders, bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA). In these diseases, ultrastructural identification of the site of skin-bound IgG deposits at the epidermal basement membrane zone (EBMZ) may be essential to the correct diagnosis. Since ultrastructural studies are more expensive, time-consuming, and less widely available than immunofluorescence, we addressed the question of whether the FOAM technique can reliably identify the site of IgG deposits at the EBMZ, and distinguish BP from EBA. For this purpose, the technique was applied to perilesional skin from seven patients with BP and six with EBA, using computer-aided imaging of red-stained type VII collagen and green-stained IgG, according to previous findings. RESULTS: Digitized multicolor FOAM images of perilesional skin from patients with BP showed nonoverlap band patterns of green-stained lamina lucida IgG deposits (ultrastructurally proven) and red-stained type VII collagen. By contrast, FOAM images of EBA skin typically showed overlap patterns of green-stained sublamina densa IgG deposits and red-stained type VII collagen. These findings were observed also in skin tissue stored in Michel's transport medium or stored frozen for 15 years. CONCLUSIONS: The computer-aided FOAM technique may have great potential in distinguishing between IgG deposits above (BP) and just below (EBA) the lamina densa of the EBMZ in skin tissue. The technique is not as simple as saline-split skin methodology but offers more flexibility, and it certainly is quicker and less expensive than electron microscopy. Furthermore, the use of digitized fluorescence images offers improved possibilities for evaluating the various "linear" patterns of immune reactant deposition at the EBMZ in subepidermal bullous autoimmune skin diseases.
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Epidermólisis Ampollosa Adquirida/inmunología , Inmunoglobulina G/aislamiento & purificación , Penfigoide Ampolloso/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Membrana Basal/inmunología , Colágeno/inmunología , Diagnóstico Diferencial , Epidermólisis Ampollosa Adquirida/diagnóstico , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Masculino , Penfigoide Ampolloso/diagnósticoRESUMEN
We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.
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Autoantígenos/sangre , Immunoblotting , Síndromes Paraneoplásicos/sangre , Síndromes Paraneoplásicos/etiología , Pénfigo/sangre , Pénfigo/etiología , Baculoviridae/química , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Queratinocitos/química , Queratinocitos/citología , Linfoma no Hodgkin/inmunología , Membrana Mucosa/inmunología , Síndromes Paraneoplásicos/inmunología , Pénfigo/inmunología , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Piel/inmunología , Proteínas Virales/metabolismoRESUMEN
Calcitriol has recently been shown to be effective against psoriasis. However, its mode of action is not exactly known. The present study focused on the influence of calcitriol on growth, differentiation, chemokine mRNA and ICAM-1 mRNA expression of keratinocytes (KC) and on the binding of T-cells to keratinocytes. In vitro studies showed that calcitriol has a strong anti-proliferative effect and induces terminal differentiation. gamma-IP-10 and ICAM-1 mRNA were induced by gamma-IFN, an induction not influenced by calcitriol. Moreover, the functional expression of ICAM-1 on the KC cell surface as measured by a cell adhesion assay, was not influenced either. IL-8 and huGRO mRNAs were constitutively produced in KC, as was demonstrated after incubation with cycloheximide. Up-regulation of both IL-8 and huGRO mRNA by IL-1 alpha was also not affected by calcitriol. It is concluded that calcitriol has a strong antiproliferative activity and does not interfere with KC responsiveness to gamma-IFN and IL-alpha induced chemokine expression or with the adhesion of T-cells to keratinocytes.
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Calcitriol/farmacología , Queratinocitos/efectos de los fármacos , Linfocitos T/fisiología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Factores Quimiotácticos/genética , Citocinas/genética , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/fisiología , ARN Mensajero/metabolismoRESUMEN
HuGRO, IL-8 and gamma-IP-10 belong to a recently described superfamily of genes encoding a group of cytokines with inflammatory, growth regulating and/or leukocyte chemotactic properties (chemokines). We studied huGRO, IL-8 and gamma-IP-10 gene expression in unstimulated and stimulated (TNF alpha, INF gamma, TNF alpha + IFN gamma, IL-1 beta, PMA and LPS) normal human keratinocytes by Northern blot analysis. The mRNA for none of the three chemokines was detectable in unstimulated keratinocytes, but considerably elevated levels of huGRO and IL-8 mRNA, but not of gamma-IP-10 mRNA, were found in the presence of cycloheximide, indicating that huGRO and IL-8 mRNA, but not gamma-IP-10 mRNA, are constitutively produced. gamma-IP-10 mRNA was exclusively induced by IFN gamma, with a strong and transient rise between 8 and 18 h, and superinduced by the combination of IFN gamma and TNF alpha, indicating marked synergism. Both huGRO and IL-8 mRNA were induced by TNF alpha and PMA (a strong and transient rise between 2 and 8 h), but not by IFN gamma or LPS. The combination of TNF alpha and IFN gamma did not show a synergistic effect. In addition, IL-1 beta transiently upregulated huGRO mRNA but failed to induce IL-8 mRNA. Using specific oligonucleotides for alpha, beta and gamma huGRO, TNF alpha was found to induce all three forms, alpha and beta to an equal extent and gamma to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)
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Quimiocinas CXC , Factores Quimiotácticos/genética , Citocinas/genética , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/farmacología , Interleucina-8/genética , Queratinocitos/metabolismo , ARN Mensajero/análisis , Células Cultivadas , Quimiocina CXCL1 , Quimiocina CXCL10 , Quimiocina CXCL2 , Expresión Génica , HumanosRESUMEN
The pathogenetic mechanisms involved in the development of drug-induced erythema multiforme (EM) are still largely unknown. The observation that epidermal keratinocytes (KC) in EM express intercellular adhesion molecule-1 (ICAM-1) points to a putative role for T-cell/KC adhesion in the pathogenesis of EM. In this study, the binding of peripheral blood mononuclear leucocytes (PBML) from a patient with carbamazepine-induced EM and of normal control PBML to autologous and heterologous KC was investigated, using two different binding assays. Patient PBML obtained at the time of disease (t0) showed an increased binding to ICAM-1-positive heterologous KC, which could be inhibited completely by anti-LFA-1. Adhesion of patient PBML-t0 to autologous KC, and to carbamazepine-pretreated heterologous KC in sections of skin biopsies, was also increased, but was found to be only partially LFA-1-dependent. These findings support the view that PBML/KC adherence plays an important role in the pathogenesis of this drug-induced EM.