RESUMO
The transportation of poultry and related products for international trade contributes to transboundary pathogen spread and disease outbreaks worldwide. To prevent pathogen incursion through poultry products, many countries have regulations about animal health and poultry product quarantine. However, in Japan, animal products have been illegally introduced into the country in baggage and confiscated at the airport. Lately, the number of illegally imported poultry and the incursion risk of transboundary pathogens through poultry products have been increasing. In this study, we isolated avian influenza viruses (AIVs) from raw poultry products illegally imported to Japan by international passengers. Highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIVs were isolated from raw chicken and duck products carried by flight passengers. H5 and H9 isolates were phylogenetically closely related to viruses isolated from poultry in China, and haemagglutinin genes of H5N1 and H5N6 isolates belonged to clades 2.3.2.1c and 2.3.4.4, respectively. Experimental infections of H5 and H9 isolates in chickens and ducks demonstrated pathogenicity and tissue tropism to skeletal muscles. To prevent virus incursion by poultry products, it is important to encourage the phased cleaning based on the disease control and eradication and promote the reduction in contamination risk in animal products.
Assuntos
Aeroportos , Comércio , Vírus da Influenza A Subtipo H1N2/isolamento & purificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Vírus da Influenza A Subtipo H9N2/isolamento & purificação , Influenza Aviária/virologia , Produtos Avícolas/virologia , Viagem , Animais , Antígenos Virais/imunologia , Galinhas/virologia , China/epidemiologia , Surtos de Doenças/prevenção & controle , Surtos de Doenças/veterinária , Patos/virologia , Microbiologia de Alimentos , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H1N2/imunologia , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/imunologia , Influenza Aviária/epidemiologia , Japão , Carne/virologia , Filogenia , Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , RNA Viral/genéticaRESUMO
After the cDNA of human macrophage migration inhibitory factor (MIF) was cloned in 1989, this protein has been re-evaluated as a pro-inflammatory cytokine, pituitary hormone and glucocorticoid-induced immunoregulatory protein. We previously reported the expression of MIF in the basal cell layers of the epidermis, but its pathophysiological function in the skin has not been well understood. In this study, we examined the expression of MIF during the wound healing of rat skin injured by excision. Reverse transcription-polymerase chain reaction in combination with Southern blot analysis revealed that the increase of MIF mRNA expression was biphasic. The maximum peaks were observed at 3 and 24 h after the injury. Similarly, maximal increases of the serum MIF level were observed at 3 and 24 h after the injury. Immunohistochemical analysis at 12 h after injury demonstrated enhanced expression of MIF protein in the whole epidermal lesion of the wound tissue. By the Boyden chamber assay, we demonstrated that MIF had a chemotactic effect on freshly prepared keratinocytes from rat skin. Additionally, cultured fibroblasts from the skin wound lesion secreted a higher amount of MIF in response to lipopolysaccharide compared to those of the normal skin. Furthermore, administration of anti-MIF antibodies induced a delay of wound healing in vivo. Taken together, these results suggest that MIF contributes to the wound healing process of skin tissue.
Assuntos
Epiderme/metabolismo , Fibroblastos/metabolismo , Fatores Inibidores da Migração de Macrófagos/biossíntese , Pele/metabolismo , Cicatrização/fisiologia , Animais , Anticorpos/farmacologia , Southern Blotting , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Epiderme/lesões , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fatores Inibidores da Migração de Macrófagos/sangue , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Fatores Inibidores da Migração de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/lesões , Fatores de TempoRESUMO
The endogenous level of epidermal cyclic AMP does not remain constant but increases rapidly and transiently after removal of the tissue; this is known as the "ischemia" effect. UVB-irradiated epidermis which shows increased beta-adrenergic response revealed an increased ischemia effect, while psoriatic involved epidermis which shows decreased beta-adrenergic response revealed a decreased ischemia effect. Because of the similar rise-and-fall pattern between the ischemia effect and the beta-adrenergic response, the mechanism of the ischemia effect was investigated, especially in terms of the beta-adrenergic relationship. The ischemic rise of epidermal cyclic AMP was well preserved after 6 h pretreatment at 4 degrees C, and, following the pretreatment, the skin markedly increased its cyclic AMP level by the 37 degrees C treatment with 1 mM isobutylmethyl xanthine. The addition of propranolol or cimetidine at the time of 37 degrees C treatment (following the 4 degrees C pretreatment) had no effect on the ischemia effect; both skin groups markedly increased their cyclic AMP levels to an extent similar to that of the control skin. However, the addition of propranolol at the time of both preincubation (at 4 degrees C) and incubation (at 37 degrees C) markedly decreased the ischemic rise of cyclic AMP. Similar treatment by cimetidine had no effect on the ischemia effect. There was no significant difference in cyclic AMP phosphodiesterase activities among skin groups by propranolol or cimetidine pretreatment. These results indicate that the so-called ischemic rise of epidermal cyclic AMP is actually the beta-adrenergic adenylate cyclase-dependent process. Our results also indicate that the magnitude of the "ischemic" rise of cyclic AMP is generally parallel to the beta-adrenergic responsiveness of epidermis.
Assuntos
Adenilil Ciclases/análise , AMP Cíclico/análise , Isquemia/metabolismo , Receptores Adrenérgicos beta/fisiologia , Pele/irrigação sanguínea , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Feminino , Humanos , Masculino , Propranolol/farmacologia , Pele/análise , Pele/efeitos da radiação , TemperaturaRESUMO
Effects of glucocorticoids on the epidermal beta-adrenergic adenylate cyclase system were investigated. Long-term incubation of pig skin slices in RPMI 1640 medium resulted in the gradual decrease in the epinephrine-induced cyclic AMP accumulations of skin. The addition of hydrocortisone (100 microM) in the incubation medium prevented this decrease, and after 24- and 48-h incubation, there was a marked difference in beta-adrenergic responsiveness between control and hydrocortisone-treated skin. The study using other steroid hormones revealed that this effect on the beta-adrenergic system was relatively specific for glucocorticoids. Hydrocortisone, prednisolone, dexamethasone, and beta-methasone-17-valerate were shown to have marked effects on the beta-adrenergic system, while androstenedione, testosterone, dihydrotestosterone, progesterone, estrone, and beta-estradiol had no effect. Cortisone and estriol were shown to have similar but weaker effects than hydrocortisone. The effect of glucocorticoids was also relatively specific to the beta-adrenergic system, since there was no significant difference in adenosine-or histamine-induced cyclic AMP accumulations of skin after long-term incubation with and without hydrocortisone. The mechanism of this glucocorticoid action does not seem to be through the simple protection of the beta-adrenergic system of the skin, since the addition of hydrocortisone in the incubation medium at 24 or 48 h incubation time, when the epinephrine-induced cyclic AMP accumulation was considerably decreased, reversed the epinephrine unresponsiveness of the skin, after the additional 24-h incubation. Furthermore, the effect of hydrocortisone was inhibited by 3 different kinds of inhibitors: (a) progesterone, an inhibitor of intracytoplasmic glucocorticoid receptor binding; (b) actinomycin D, an inhibitor of messenger RNA (mRNA) synthesis; and (c) cycloheximide, an inhibitor of protein synthesis at the translation step. These results are in accordance with the view that glucocorticoids affect the beta-adrenergic system of epidermis by a mechanism requiring mRNA and protein synthesis possibly through the intracytoplasmic glucocorticoid receptor system of epidermis.
Assuntos
Adenilil Ciclases/metabolismo , Hidrocortisona/farmacologia , Pele/enzimologia , Animais , AMP Cíclico/fisiologia , Cicloeximida/farmacologia , Epinefrina/farmacologia , Suínos , Sistema Nervoso Simpático/enzimologiaRESUMO
Although retinoids reveal various biologic and biochemical activities on epidermal keratinocytes, their effects on the epidermal cyclic AMP (cAMP) system has been less well characterized. In order to elucidate the relation between them, an in vitro pig skin-slice incubation system was employed. After a long-term (up to 24 h) incubation in vitro, control skin responded to epinephrine only slightly. The addition of Ro 10-1670, an active derivative of Ro 10-9359 (etretinate) in the incubation medium, resulted in an increase of the beta-adrenergic adenylate cyclase response of epidermis. On the other hand, histamine-induced cAMP accumulation was decreased by the retinoid treatment after long-term incubation. The augmentation of the beta-adrenergic response was observed at 1 microM concentration and the maximal effect was observed at 10 microM. There was no significant difference in cAMP phosphodiesterase activities between the control and retinoid-treated skin. The effect was also observed by the addition of all-trans-retinoic acid, retinol, and Ro 10-9359; the latter two compounds revealed much lesser effects. The addition of combinations of various drugs (Ro 10-1670 and hydrocortisone; Ro 10-1670 and colchicine) resulted in more marked (additive or synergistic) effects than the single addition of each chemical. On the other hand, the addition of Ro 10-1670 and all-trans-retinoic acid resulted in neither additive nor synergistic effect, suggesting that they probably work on the same site. Our data indicate that the epidermal beta-adrenergic adenylate cyclase response is modulated by retinoids probably as an independent mechanism stimulated by glucocorticoids or colchicine.
Assuntos
AMP Cíclico/metabolismo , Retinoides/farmacologia , Pele/metabolismo , Adenilil Ciclases/fisiologia , Animais , Colchicina/farmacologia , Epiderme/enzimologia , Epiderme/fisiologia , Etretinato/farmacologia , Hidrocortisona/farmacologia , Pele/inervação , Suínos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Vitamina A/farmacologiaRESUMO
Pig skin was irradiated in vivo with fluorescent sunlamp tubes (peak emission at 305 nm). A significant increase in epidermal beta-adrenergic adenylate cyclase response was observed as early as 12 h following 1-2 minimum erythema doses (MEDs) UVB exposure, which lasted at least 48 h. The augmentation of adenylate cyclase response was relatively specific to the beta-adrenergic system and there was no significant difference in either adenosine- or histamine-adenylate cyclase response of epidermis. The increased beta-adrenergic adenylate cyclase response was less marked at higher doses of UVB exposure (5 MEDs); in the latter condition, a significant reduction in adenosine- or histamine-adenylate cyclase response was observed. There was no significant difference in either low- or high-Km cyclic AMP phosphodiesterase activity between control and UVB-treated skin at 1-2 MEDs. Our data indicate that the epidermal adenylate cyclase responses are affected in vivo by UVB irradiation, which might be a significant regulatory mechanism of epidermal cyclic AMP systems.
Assuntos
Adenilil Ciclases/efeitos da radiação , Epiderme/efeitos da radiação , Receptores Adrenérgicos/efeitos da radiação , Raios Ultravioleta , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/metabolismo , Relação Dose-Resposta à Radiação , Epiderme/enzimologia , Epiderme/metabolismo , Epinefrina/farmacologia , Suínos , Fatores de TempoRESUMO
Although receptor-specific refractoriness has been suggested to be one of the regulatory mechanisms of epidermal adenylate cyclase systems, its physiologic significance has been a subject of controversy because of the requirement of unusually high concentrations of agonists to induce refractoriness. In order to determine whether the epidermal adenylate cyclase system is regulated through a refractoriness mechanism by suboptimal concentrations of receptor agonists, this study was undertaken using pig skin epidermal adenylate cyclase systems. Pretreatment of pig skin with 0.1-1 microM epinephrine in vitro resulted in the reduction of the maximal epinephrine response (epinephrine-induced cyclic AMP accumulations) to various degrees without alterations in either low or high Km cyclic AMP phosphodiesterase activities. Repeated pretreatments were shown to be more effective in inducing refractoriness than a single pretreatment. Apparently there was no change in the Km value for epinephrine, suggesting that the decrease in epinephrine response represents a reduction in the number but not in the affinity of functional beta-adrenergic adenylate cyclase receptor sites. This refractoriness by low concentrations of catecholamine pretreatment was specific to the beta-adrenergic system, since there was no reduction in histamine response after the epinephrine pretreatment. These results indicate that the epidermal beta-adrenergic adenylate cyclase system is regulated by much lower concentrations of catecholamine than were previously described. It was suggested that physiologic fluctuations of plasma catecholamine levels might have a profound effect on epidermal beta-adrenergic adenylate cyclase responsiveness, resulting in the alteration of the minimal catecholamine level required for the successive activation of cyclic AMP-dependent protein kinase, which is the predominant target of cyclic AMP in epidermis.
Assuntos
Adenilil Ciclases/metabolismo , Epinefrina/farmacologia , Pele/enzimologia , Animais , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Epinefrina/administração & dosagem , Pele/efeitos dos fármacos , SuínosRESUMO
Ras oncogene expression of malignant melanoma and melanocytic nevus have been immunohistochemically analyzed on formalin-fixed and paraffin-embedded tissues from 26 melanomas and 24 melanocytic nevi with a monoclonal antibody that was generated against Harvey sarcoma virus-derived ras oncogene products (p21ras). We found distinct differences of p21ras expressions by the type of melanoma. Nodular melanoma, epithelioid cell type melanoma, and deeply invading melanoma revealed higher reactivity with anti-p21ras monoclonal antibody than the other types. The reactivity of melanomas appeared to correlate with the degree of malignancy of the melanoma. It was also demonstrated, however, that part of melanocytic nevi reacted with anti-p21ras monoclonal antibody with a relatively strong intensity. Melanocytic nevi with junctional activity and nevus cells located in the epidermis in compound nevi did not show the positive reaction in contrast to dermally located nevus cells that had relatively strong reactivity. The different p21ras expression among the type of tumors may represent the state of tumor cells differentiation with greater expression with more immaturity in the melanocyte lineage. p21ras expression does not appear to represent a marker of malignant transformation.
Assuntos
Genes ras , Melanoma/genética , Nevo Pigmentado/genética , Biossíntese de Proteínas , Humanos , Imuno-Histoquímica , Melanoma/metabolismo , Nevo Pigmentado/metabolismoRESUMO
Sunburned skin is characterized by expanded numbers of macrophages (ultraviolet [UV]-MPH), and these UV-MPH differ from Langerhans cells (LC) in their abilities to initiate T-cell-mediated immune reactions. UV-MPH and LC may themselves be differentially responsive to the surrounding milieu, which may in turn modulate their immunoregulatory activity. We asked whether immunologic signal responsiveness, as assessed by cytosolic calcium mobilization, differed among normal human LC, UV-MPH, and normal blood monocytes. LC from normal skin and UV-MPH from UV-exposed skin were distinguished from keratinocytes in epidermal cell suspensions by labeling with anti-HLA-DR. Intracellular calcium content was monitored in real time with the calcium indicator, indo-1, after cross-linking Fc gamma RI, Fc gamma RII, CD11b, CD11c, or CD18 molecules, or addition of interleukin-1 alpha, IL-1 beta, interferon-gamma, bradykinin, substance P, or FMLP. Using flow cytometric analysis of cell suspensions, UV-MPH and blood monocytes were triggered by cross-linking Fc gamma RII (flux of 6.05 and 12.2, respectively). UV-MPH could also be triggered by Fc gamma RI crosslinking and FMLP (flux of 6.41 and 15.54, respectively). By contrast, none of these inflammatory stimuli could cause cytosolic calcium mobilization in normal LC (Flux of -0.2 by FcRII, and 0.18 by FMLP). Because LC calcium flux may be dependent upon extracellular attachments, LC were anchored onto fibronectin-coated coverslips and then their Fc gamma RII was crosslinked in a continuous flow chamber. However, image analysis also failed to detect calcium flux. Neither population responded to interleukin-1, interferon-gamma, bradykinin, substance P, or beta 2 integrin crosslinking. These results indicate that blood monocytes and infiltrating macrophages differ substantially from LC in their responses to immune complexes and chemoattractants. Differential responsiveness to the inflammatory milieu may influence the antigen presenting or effector capabilities of these populations.
Assuntos
Células Apresentadoras de Antígenos/fisiologia , Espaço Extracelular/fisiologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Receptores de IgG/fisiologia , Transdução de Sinais , Raios Ultravioleta , Cálcio/metabolismo , Reagentes de Ligações Cruzadas/farmacologia , Células Epidérmicas , Epiderme/efeitos da radiação , Fibronectinas/metabolismo , Corantes Fluorescentes , Fura-2 , Humanos , Membranas Intracelulares/metabolismo , Células de Langerhans/fisiologia , Macrófagos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Human epidermal cells are capable of secreting various cytokines with immunologic, inflammatory, and proliferative properties. In a previous study, by reverse transcription-polymerase chain reaction and immunohistochemical analysis, we have shown that human epidermal keratinocytes express macrophage migration inhibitory factor and identified its presence in the cytoplasm. In this study, we detected an increased serum macrophage migration inhibitory factor level by enzyme-linked immunosorbent assay after a single total-body ultraviolet B exposure in vivo, indicating that human keratinocytes respond and release this cytokine in response to ultraviolet B irradiation. Moreover, we evaluated the effect of ultraviolet B on migration inhibitory factor production in cultured human epidermal keratinocytes and epidermal sheets. The results of enzyme-linked immunosorbent assay and northern blot analyses showed that migration inhibitory factor production of cultured keratinocytes was increased by ultraviolet B exposure. During the past few years, migration inhibitory factor was found to have a variety of biologic functions, such as being essential for T cell activation and induction of inflammatory cytokines. In this context, these results should encourage further investigation on the pathophysiologic role of migration inhibitory factor in cutaneous inflammatory reactions and immune responses.
Assuntos
Fatores Inibidores da Migração de Macrófagos/biossíntese , Raios Ultravioleta , Sobrevivência Celular/efeitos da radiação , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Pele/citologia , Fatores de Tempo , Regulação para Cima/efeitos da radiaçãoRESUMO
Intracellular Ca++ ([Ca++]i) is one of the most important second messengers of extracellular signals that induce cellular responses. In epidermal keratinocytes, both extracellular and intracellular Ca++ are reported to be important to cell differentiation and proliferation. Several mechanisms that increase [Ca++]i have been elicited in various tissues; however, in epidermal keratinocytes they remain unknown. Thus, we investigated the [Ca++]i modulation in cultured human epidermal keratinocytes and the stimulation that increases the concentration. The [Ca++]i concentration of keratinocytes was increased immediately and transiently by epinephrine. Methoxamine hydrochloride and clonidine (alpha-1- and 2-adrenergic agonists) did not induce an increase in [Ca++]i. The beta-antagonist, propranolol, inhibited the [Ca++]i increase induced by epinephrine and salbutamol (a beta-2-agonist). These results reveal that the beta-adrenergic stimulation induces an immediate and transient [Ca++]i increase in human keratinocytes. Beta-adrenergic stimulation is known to induce adenylate cyclase activation, which results in cyclic AMP accumulation through stimulatory guanosine 5-triphosphate (GTP) binding proteins in the keratinocytes. Also, epinephrine is reported to inhibit cultured epidermal cell proliferation. The effect of epinephrine has been demonstrated by cyclic AMP accumulation; however, beta-adrenergic stimulation revealed a [Ca++]i increase in keratinocytes in our study. One of epinephrine's regulatory effects on epidermal cell proliferation is assumed to occur through the [Ca++]i increase as well.
Assuntos
Cálcio/metabolismo , Epinefrina/farmacologia , Queratinócitos/metabolismo , Metoxamina/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Células Cultivadas , Clonidina/farmacologia , Fura-2 , Humanos , Recém-Nascido , Queratinócitos/efeitos dos fármacos , Cinética , Masculino , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/fisiologia , Espectrometria de FluorescênciaRESUMO
Effects of colchicine on the epidermal adenylate cyclase systems were investigated. When pig skin (epidermis) was incubated in RPMI 1640 medium without the addition of serum, the beta-adrenergic adenylate cyclase response (epinephrine-induced cyclic AMP accumulations) gradually decreased, whereas adenosine and histamine responses remained high or increased during the long-term (up to 48 h) incubation period. The addition of colchicine (1 mumol/liter) in the incubation medium resulted in an increase in the beta-adrenergic responsiveness and a decrease in adenosine and histamine responsivenesses. The effects of colchicine were both time- and concentration-dependent; they could be observed after 9-12 h incubation, and the maximal effect was obtained at a concentration of 0.1 mumol/liter. Similar effects were observed by the addition of another microtubule-disruptive agent, vinblastine. On the other hand, cytochalasin B, which affects the microfilament system, apparently decreased the beta-adrenergic response and increased adenosine and histamine responses during the long-term incubation period. The addition of serum in the incubation medium resulted in essentially the same effect as that of colchicine; in the presence of serum, colchicine-treated skin responded much more markedly to epinephrine (and much less to adenosine and histamine) than the control skin after 24- and 48-h incubation. Previously we reported that hydrocortisone has similar potentiating effects on the beta-adrenergic system of epidermis. The comparison of the effects of both compounds revealed that colchicine had a stronger effect than hydrocortisone, and furthermore, the simultaneous addition of both compounds (colchicine and hydrocortisone) in the incubation medium resulted in the more marked increase of beta-adrenergic response than the single addition of each chemical. Our overall results, coupled with the finding that hydrocortisone has no toxic effects on the adenosine- or histamine-adenylate cyclase system of epidermis, suggest that colchicine affects epidermal adenylate cyclase systems probably through a mechanism that is independent of glucocorticoid (hydrocortisone) effect.
Assuntos
Colchicina/farmacologia , AMP Cíclico/biossíntese , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Adenosina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Citocalasina B/farmacologia , Epinefrina/farmacologia , Histamina/farmacologia , Hidrocortisona/farmacologia , Suínos , Teofilina/farmacologia , Vimblastina/farmacologiaRESUMO
The expression of nPKC eta, a Ca(++)-independent isoform of protein kinase C in normal human skin, and skin from patients with psoriasis, squamous cell carcinoma, basal cell epithelioma, nevus pigmentosus, and seborrheic keratosis, were examined by immunohistochemical staining using a polyclonal antibody raised against a synthetic peptide at a diverse region of the nPKC eta molecule. In normal epidermis, the strongest staining was observed in the uppermost granular layer with no staining of the spinous or basal layers. The inner layer of the intra-epidermal eccrine duct was also strongly stained. Weak staining was observed in several layers of the outer root sheath of the follicular infundibulum. No staining was detected in the inner root sheath of the hair follicles, hair matrix, sebaceous gland, eccrine gland, intradermal eccrine duct, arrectores pilorum, melanocytes, Langerhans cells, fibroblasts, or blood vessels. In psoriatic skin, stained keratinocytes were distributed in the suprabasal layers with the most being observed in the uppermost layer and the least in layers closed to the basal layer. In squamous cell carcinoma, weak staining was observed in the keratotic cells around horny pearls. In the basal cell epithelioma and nevus pigmentosus, the cells were not stained, whereas in seborrheic keratosis, cells that stained were located in the granular layer. We conclude from the evidence presented above that nPKC eta is expressed in close association with epidermal differentiation in normal skin and skin diseases.
Assuntos
Isoenzimas/análise , Proteína Quinase C/análise , Dermatopatias/enzimologia , Pele/enzimologia , Adulto , Carcinoma Basocelular/enzimologia , Carcinoma de Células Escamosas/enzimologia , Diferenciação Celular/fisiologia , Feminino , Humanos , Ceratose Seborreica/enzimologia , Masculino , Pessoa de Meia-Idade , Nevo Pigmentado/enzimologia , Psoríase/enzimologia , Pele/citologia , Dermatopatias/patologia , Neoplasias Cutâneas/enzimologiaRESUMO
A new disease characterized by the presence of in vivo bound and/or circulating IgA anti-intercellular (IC) antibodies has recently been identified. We propose the term intercellular IgA vesiculopustular dermatosis (IAVPD) for this entity, which seems to be divided clinicopathologically into at least two distinct subtypes: intraepidermal neutrophilic IgA dermatosis (IEN type) and subcorneal pustular dermatosis-like cases (SPD type). Using immunoblot technique, we examined the antigen substances for the IgA anti-IC antibodies in the sera from one Japanese patient with IEN type of IAVPD and three Japanese patients with SPD type. A serum from a patient with IEN type reacted exclusively with a 120-kD protein in both the normal human skin extract and the bovine desmosome sample. Sera from three patients with SPD type reacted specifically with a doublet of 115-kD and 105-kD proteins, which appeared to be identical to desmocollins I and II, well known desmosomal core proteins, in the bovine desmosome sample. IgA antibody from our patients with IAVPD bound to neither pemphigus vulgaris antigen nor pemphigus foliaceus antigen. From these results, we suggest that IAVPD is different from pemphigus and is heterogeneous in terms of the antigens to which IgA autoantibodies bind.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/análise , Imunoglobulina A/imunologia , Dermatopatias Vesiculobolhosas/imunologia , Criança , Humanos , ImmunoblottingRESUMO
Epidermal Langerhans cell heterogeneity is poorly understood with regard to phenotypic characteristics, such as the expression of human leukocyte antigen (HLA)-DR, integrin, and Fc receptor molecules, as well as functional characteristics, such as the ability to process and present antigens or produce cytokines during various phases of immigration and maturation. Technical limitations of Langerhans cell number have limited functional assays on putative Langerhans cell subsets in in vivo epidermis. Therefore, we used flow cytometry for simultaneous phenotypic and functional assessment at the single-cell level within the Langerhans cell population. Freshly isolated human epidermal cell suspensions were stained with a battery of monoclonal antibodies, including anti-HLA-DR, -CD1a, -CD1c, -CD11c, -Fc gamma RII, and -Fc epsilon RI. Two distinct Langerhans cell subsets were identified by their different levels of HLA-DR expression. The DRHi subset expressed higher amounts of CD11c and exhibited greater cytoplasmic complexity and higher baseline calcium than the DRLo subset (p < or = 0.03 for each). Some subjects also expressed high levels of Fc epsilon RI in the DRHi, CD11cHi subset. To determine whether these phenotypic subsets may exhibit differential signal-transduction functional properties, Langerhans cells were partially enriched over Ficoll-Hypaque and their cytosolic mobilization after the addition of ionomycin was analyzed using the calcium indicator, indo-1, in conjunction with quantitative analysis of HLA-DR expression. By this real-time flow cytometric analysis, a new subpopulation was revealed within the DRLo Langerhans cell subset. This subset increased its cytosolic calcium concentration much more than the other two subsets (change in indo-1 blue:violet emission ratio of 37.33 +/- 2.34 in the Hi Flux DRLo subset versus 13.23 +/- 0.29 in the Lo Flux DRLo subset, and versus 7.6 +/- 2.99 in the Lo Flux DRHi subset). These data indicate that functional, as well as phenotypic, subsets of Langerhans cells exist within normal human epidermis. Their responses to physiologic stimuli may relate to maturational stage or the level of in vivo activation.
Assuntos
Células de Langerhans/classificação , Células de Langerhans/fisiologia , Adulto , Antígenos CD/análise , Antígenos CD1 , Antígenos CD18 , Cálcio/metabolismo , Citosol/metabolismo , Antígenos HLA-DR/análise , Humanos , Integrinas/análise , Células de Langerhans/química , Fenótipo , Receptores Fc/análise , Pele/ultraestruturaRESUMO
In order to elucidate a part of calmodulin actions in the hyperproliferative state in human epidermis, calmodulin activities in the psoriatic and in the normal human epidermis were determined using calmodulin-deficient phosphodiesterase from bovine heart and purified pig skin epidermal calmodulin as a standard. Skin samples were obtained from 11 normal healthy controls and from both the uninvolved and involved regions of 8 nonconsanguineous psoriatic patients. Pure epidermal samples, prepared by the microdissection method, were used for calmodulin assays. Normal human epidermis contained 270 +/- 13 ng/mg dry weight, whereas calmodulin activities were significantly increased in psoriatic epidermis, 412 +/- 29 ng/mg dry weight for the uninvolved epidermis and 747 +/- 46 ng/mg dry weight for the involved epidermis, respectively. These results suggest that calmodulin may play an important role in cell proliferation in human epidermis.
Assuntos
Calmodulina/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Adolescente , Adulto , Idoso , Calmodulina/fisiologia , Divisão Celular/efeitos dos fármacos , Criança , Humanos , Pessoa de Meia-Idade , Diester Fosfórico HidrolasesRESUMO
Although it has been reported that adenosine has an inhibitory effect on keratinocyte proliferation at both G2 and S phases of the cell cycle, its relation to cyclic AMP formation through the adenylate cyclase system has been less well characterized. In order to determine the precise mechanism of the adenosine effect, another physiologic adenine nucleoside, 2'-deoxyadenosine was employed. 2'-Deoxyadenosine was shown to be remarkably different from adenosine in its ability to stimulate the epidermal adenylate cyclase; whereas adenosine markedly increased cyclic AMP levels of pig epidermis, deoxyadenosine had a much weaker effect on the cyclic AMP levels of the skin. Using several parameters of cell proliferation, comparison was made between the effects of these two compounds. Pig keratinocyte explant culture system was employed for the measurement of outgrowth and mitosis. Mitosis was determined after 72-h incubation (to monitor the overall cell proliferation inhibition) and 4-h incubation (to monitor G2 phase inhibition) with the chemicals. Pig skin keratome slice system was employed for [3H]thymidine uptake measurement. Both adenosine and deoxyadenosine were shown to have marked inhibitory effects on keratinocyte out-growth, [3H]thymidine uptake, and keratinocyte mitosis. The effects of deoxyadenosine on outgrowth and [3H]thymidine uptake were greater than that of adenosine. The inhibitory effect of adenosine and deoxyadenosine on mitosis were about the same in both 4-h and 72-h incubation systems. Thus deoxyadenosine, which is a much weaker stimulator of epidermal adenylate cyclase, was also shown to be as potent an inhibitor of keratinocyte proliferation as adenosine. These results further substantiate the view that cyclic AMP elevating agents (such as adenosine and deoxyadenosine) might not necessarily reveal their inhibitory effects on keratinocyte proliferation through their effects of cyclic AMP formation.
Assuntos
Adenosina/farmacologia , Divisão Celular/efeitos dos fármacos , AMP Cíclico/biossíntese , Desoxiadenosinas/farmacologia , Células Epidérmicas , Animais , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , SuínosRESUMO
The presence and tissue localization of macrophage migration inhibitory factor (MIF) in human skin were examined. Reverse transcription-polymerase chain reaction analysis revealed that MIF mRNA was expressed in both surgically obtained normal human epidermis and primary cultured human keratinocytes. The expression of MIF was further confirmed by Western blot analysis, which demonstrated a single band at about 12.5 kDa using a polyclonal antibody against human recombinant MIF. Immunohistochemical studies showed that MIF existed in human epidermis, especially in the basal layer. The pathophysiological role of MIF in human skin remains undefined; however, the present results indicate that MIF may play an important role in immunity, inflammation and cellular differentiation of epidermal cells.
Assuntos
Queratinócitos/citologia , Queratinócitos/metabolismo , Fatores Inibidores da Migração de Macrófagos/análise , Fatores Inibidores da Migração de Macrófagos/biossíntese , Pele/citologia , Animais , Sequência de Bases , Western Blotting , Diferenciação Celular , Células Cultivadas , Primers do DNA , Células Epidérmicas , Epiderme/metabolismo , Expressão Gênica , Humanos , Imunidade , Imuno-Histoquímica , Inflamação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Coelhos/imunologia , Valores de Referência , Pele/metabolismoRESUMO
Monocyte chemoattractant protein (MCP)-1 is a chemotactic cytokine for monocytes, memoryT cells and dendritic cells (DC). However, the precise role of MCP-1 in a variety of immunological responses remains unclear. In the present study, we analyzed contact hypersensitivity (CHS) using human MCP-1 transgenic mice (hMCP-1Tgm) that constitutively produce high levels of hMCP-1 in the sera. Following 2,4-dinitrofluorobenzene (DNFB) sensitization, enhancement of CHS was demonstrated in Tgm as compared with that in non-Tgm. Anti-hMCP-1 antibodies significantly inhibited the CHS in Tgm. A prominent accumulation of B7-1+I-Ad+ Langerhans' cells (LC) bearing haptens was detected in draining lymph nodes (DLN) of Tgm 24 h after DNFB or fluorescein isothiocyanate (FITC) sensitization. Similar results were obtained with BALB/c mice administrated recombinant (r) hMCP-1. Langerhans' cells (LC) in the epidermal sheets of Tgm increased in size and expressed high levels of I-Ad and B7-1 12 h after FITC application compared with those of non-Tgm. After 18 h, the number of LC in the epidermis was reduced in Tgm. It was also shown that the B7-1 expression on LC of BALB/c mice was augmented after culture with rhMCP-1. These findings demonstrate that MCP-1 not only accelerates LC migration from epidermis into the DLN after sensitization with haptens but also up-regulates the I-Ad and B7-1 expressions, which results in the enhanced T cell activation and CHS.
Assuntos
Quimiocina CCL2/imunologia , Dermatite de Contato/imunologia , Animais , Antígeno B7-1/imunologia , Divisão Celular , Movimento Celular , Células Cultivadas , Quimiocina CCL2/genética , Células Dendríticas/imunologia , Dinitrofluorbenzeno/imunologia , Dinitrofluorbenzeno/farmacologia , Haptenos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Queratinócitos/citologia , Queratinócitos/imunologia , Cinética , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Monócitos/imunologia , Linfócitos T/citologia , Regulação para CimaRESUMO
Histamine is present in the epidermis in intracellular and extracellular area and is released from mast cells and keratinocytes in the early stage of inflammation of the skin. Such release may contribute to common itching or intensify the inflammatory responses. Histamine binds to its receptors and participate in regulation of the inflammatory responses by acting on endothelial cells, nerve endings, lymphocytes, monocytes, and leukocytes. Histamine has direct effects on keratinocytes as well. Histamine modulates the proliferation of keratinocytes. The binding of histamine to the receptor on keratinocyte membrane induces activation of adenylate cyclase and phospholipase C through GTP binding protein. We previously reported that histamine induces transient increase in intracellular Ca2+ in cultured normal human epidermal keratinocytes (NHEK) and normal epidermis. H1 and H2 histamine receptors are widely distributed in many tissues and cells. In this study, we investigated which types of histamine receptors are related to the increase in intracellular Ca2+ by histamine stimulation in cultured human epidermal keratinocytes. NHEK were cultured in serum-free KGM medium. With H1 antihistamines, mepyramine and diphenhydramine, histamine responses were moderately but not statistically significantly inhibited. With H2 antihistamine, cimetidine, histamine response was significantly inhibited. Epinephrine response was not affected by these antihistamines. Thus, it is considered that H2 antihistamines specifically block histamine-mediated increase in intracellular Ca2+ of cultured normal human keratinocytes.