RESUMO
A monoclonal antibody was generated by immunizing rats with Langerhans cell (LC)-enriched epidermal cells obtained from BALB/c mouse earskin after epicutaneous application of the contact sensitizer 2,4-dinitrofluorobenzene (DNFB). The antibody 4F7 detects in normal mouse skin, few dermal cells showing the morphologic, phenotypic, and functional properties of accessory dendritic cells, but lacking Birbeck granules. The capacity to stimulate allogenic T cells in the mixed leucocyte reaction resembles that of freshly isolated LCs. After DNFB application, an increased number of 4F7+ dendritic cells are found in the dermis and, in addition, some labeled dendritic cells occur in the epidermis. Some of the latter cells exhibit cytoplasmic Birbeck granules. Remarkably, there is no increase of the 4F7+ cells in the regional lymph nodes after DNFB treatment. These data suggest that the 4F7 antibody labels distinct dendritic cells of the mouse skin that are involved in the mediation of contact sensitization and probably represent immature LCs.
Assuntos
Anticorpos Monoclonais/biossíntese , Células Dendríticas/imunologia , Animais , Células Dendríticas/ultraestrutura , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , RatosRESUMO
Ears of Balb/c mice were treated epicutaneously with 0.5% 2,4-dinitrofluorobenzene (DNFB) to obtain monoclonal antibodies characterizing molecules on epidermal dendritic cells that are involved in the induction and elicitation of allergic contact dermatitis. Six hours after this treatment, epidermal cells were prepared from the ear skin, and Ia-positive cells were enriched by indirect panning and injected into rats. Hybridomas were generated and supernatants were screened for antibodies on ear skin from DNFB-treated and untreated animals. A clone (4F7) was isolated and characterized by immunohistochemistry and immunoelectron microscopy on murine skin and other organs. The monoclonal antibody 4F7 (IgG1) recognized distinct dendritic cells in the dermis and very few dendritic cells in the paracortical area of the lymph nodes, the white pulp of the spleen, and the mucosa of the large intestine in normal animals. By fluorescence activated cell sorter analysis, it stained about 1.64% of the dermal and no epidermal cells in the skin of untreated animals. Approximately 50% of the dermal 4F7+ cells expressed Ia molecules on their surface. Six hours after application of 0.5% DNFB, the expression of the 4F7 antigen was strongly enhanced in vivo on dendritic cells in both the dermis and epidermis. About 15% of the epidermal dendritic cells expressing 4F7 exhibited Birbeck granules, the other Birbeck granule-negative cells resembled indeterminate dendritic cells (IDCs). The dermal and epidermal 4F7+ cells could be highly (98%) enriched with 4F7-labeled immunomagnetic particles. Transmission electron microscopic analysis of such preparations showed typical characteristics of dendritic cells with 50% or 100%, respectively, of these cells expressing Ia molecules on their cell membrane. The results suggest that the 4F7 epitope is expressed on dendritic cells related to Langerhans cells and is upregulated by an inflammatory stimulus.
Assuntos
Anticorpos Monoclonais , Células Dendríticas/imunologia , Epitopos/análise , Animais , Separação Celular , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/patologia , Dinitrofluorbenzeno , Células Epidérmicas , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Células de Langerhans/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Pele/citologiaRESUMO
Contact allergens induce several accessory signals which promote the activation of antigen-specific T cells. One of these signals is the increased expression of adhesion molecules on antigen-presenting cells and endothelial cells. Epicutaneous application of non-toxic doses of 2,4-dinitrofluorobenzene (DNFB) onto the skin of non-sensitized individuals elicited progressive staining for ICAM-1 on dermal microvascular endothelial cells. To elucidate the question of whether contact allergens can act directly on endothelial cells to elevate their expression of surface structures that bind leukocytes, confluent monolayers of human umbilical vein endothelial cells were incubated with the contact allergens NiSO4, CoSO4 or DNFB. The ICAM-1, E-selectin and HLA-DR expression were quantified by immunofluorescence flow cytometric analysis. Furthermore VCAM-1, E-selectin and ICAM-1 transcription were demonstrated by Northern blot hybridization. Constitutive ICAM-1 expression on HUVEC increased similarly to that obtained after LPS (20 micrograms/ml) stimulation after 4 and 24 hours of incubation with 1 or 2 mM NiSO4 or CoSO4, respectively. Pulse-stimulation with 100 or 500 nM DNFB resulted in a modest but significant increase of ICAM-1-positive cells. E-selectin and VCAM-1 were not expressed on untreated HUVEC; 4 to 6 hours exposure to nickel sulfate and LPS resulted in a potent induction of E-selectin and VCAM-1 expression. DNFB and PMA had no significant influence on VCAM-1 expression. None of the tested contact allergens was capable of inducing HLA-DR expression on EC at 48 to 72 hours. Enhanced expression of adhesion molecules may be an important early unspecific mechanism for induction and elicitation of a contact dermatitis.
Assuntos
Moléculas de Adesão Celular/metabolismo , Endotélio Vascular/metabolismo , Haptenos/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Selectina E , Endotélio Vascular/citologia , Antígenos HLA-DR/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , RNA Mensageiro/metabolismo , Molécula 1 de Adesão de Célula VascularRESUMO
In order to elucidate the role of keratinocytes (KCs) in the induction of contact sensitivity, we applied various contact sensitizers [2,4-dinitrofluorobenzene (DNFB), urushiol, 3-n-pentadecylcatechol (PDC), 4-ethoxymethylene-2-phenyloxazol-5-one (oxazolone)] and tolerizing compounds [2,4-dinitrothiocyanobenzene (DNTB), 5-methyl-3-n-pentadecyl-catechol (5-Me-PDC)] onto the earskin of non-sensitized Balb/c mice. In addition, we applied croton oil as a non-sensitizing, but stimulatory agent. Cytokine production was demonstrated by Northern blot hybridization of the total cellular RNA extracted from epidermal cells depleted by Langerhans cells and Thy 1+ dendritic cells using radiolabeled DNA probes encoding for the murine cytokines IL-1 alpha, -2, -3, -4, TNF alpha, IFN tau, GM-CSF and G-CSF. From all cytokines tested, TNF alpha and IL-1 alpha were markedly increased upon in vivo stimulation with contact sensitizers and also after application of croton oil. Both light and electron microscopic immunostaining with a polyclonal and monoclonal antibody demonstrated the presence of TNF alpha in the epidermis. This staining was most pronounced in KCs of the suprabasal epidermis upon application of contact sensitizers or croton oil, but not with tolerizing analogues. Using a functional assay significantly more TNF alpha was found in the supernatants of KCs treated in vitro with DNFB or LPS than with DNTB. GM-CSF was found in untreated epidermis as well as in stimulated cells. The results suggest that the sensitizing properties of contact sensitizers may partly be dependent on their ability to induce proinflammatory mediators. The induction and release of TNF alpha and IL-1 alpha in KCs by contact sensitizers may play an important role in the early response to immunogenic or inflammatory signals in vivo, whereby tolerance induction seems to be less dependent on these cytokines.