RESUMO
Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HCT) and can present in an acute (aGVHD), a chronic lichenoid (clGVHD), and a chronic sclerotic form (csGVHD). It is unclear whether similar or different pathomechanisms lead to these distinct clinical presentations. To address this issue, we collected lesional skin biopsies from aGVHD (n = 25), clGVHD (n = 17), and csGVHD (n = 7) patients as well as serial nonlesional biopsies from HCT recipients (prior to or post-HCT) (n = 14) and subjected them to phenotypic and functional analyses. Our results revealed striking differences between aGVHD and clGVHD. In aGVHD, we found a clear predominance of T helper (Th)2 cytokines/chemokines and, surprisingly, of interleukin (IL)-22 messenger RNA as well as an increase of IL-22-producing CD4(+) T cells. Thymic stromal lymphopoietin, a cytokine skewing the immune response toward a Th2 direction, was elevated at day 20 to 30 post-HCT in the skin of patients who later developed aGVHD. In sharp contrast to aGVHD, the immune response occurring in clGVHD showed a mixed Th1/Th17 signature with upregulated Th1/Th17 cytokine/chemokine transcripts and elevated numbers of interferon-γ- and IL-17-producing CD8(+) T cells. Our findings shed new light on the T-cell responses involved in the different manifestations of cutaneous GVHD and identify molecular signatures indicating the development of the disease.
Assuntos
Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Dermatopatias/etiologia , Dermatopatias/patologia , Subpopulações de Linfócitos T/imunologia , Adulto , Biópsia , Citocinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Citocinas/metabolismo , Estudos Retrospectivos , Subpopulações de Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Chemokines are small chemotactic proteins that have a crucial role in leukocyte recruitment into tissue. Targeting these mediators has been suggested as a potential therapeutic option in inflammatory skin diseases such as psoriasis. Using quantitative RT-PCR, we found CCL7, a chemokine ligand known to interact with multiple C-C chemokine receptors, to be markedly increased in lesional psoriasis as opposed to atopic dermatitis, lichen planus, non-lesional psoriatic and normal control skin. Surprisingly, this increase in CCL7 mRNA expression exceeded that of all other chemokines investigated, and keratinocytes and dermal blood endothelial cells were identified as its likely cellular sources. In an imiquimod-induced psoriasis-like mouse model, CCL7 had a profound impact on myeloid cell inflammation as well as on the upregulation of key pro-psoriatic cytokines such as CCL20, IL-12p40 and IL-17C, while its blockade led to an increase in the antipsoriatic cytokine IL-4. In humans receiving the TNF-α-blocker infliximab, CCL7 was downregulated in lesional psoriatic skin already within 16 hours after a single intravenous infusion. These data suggest that CCL7 acts as a driver of TNF-α-dependent Th1/Th17-mediated inflammation in lesional psoriatic skin.
Assuntos
Quimiocina CCL7/metabolismo , Psoríase/etiologia , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Animais , Estudos de Casos e Controles , Quimiocina CCL7/genética , Dermatite Atópica/imunologia , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Infliximab/farmacologia , Interleucina-1beta/metabolismo , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Psoríase/imunologia , Psoríase/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Dermatopatias/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto JovemRESUMO
Although glucocorticosteroids (GCSs) have been used for many decades in transplantation and (auto)inflammatory diseases, the exact mechanisms responsible for their immunosuppressive properties are not fully understood. The purpose of this study was to characterize the effects of oral GCSs on the cutaneous immune response. We analyzed, by immunofluorescence staining and quantitative RT-PCR, residual skin biopsy material from a clinical study in which we had used oral GCS as positive control for determining the effects of candidate anti-inflammatory compounds on epicutaneous patch tests of Ni-allergic patients. Expectedly, oral GCS treatment led to a reduction of clinical symptoms and infiltrating leukocytes. Notably, we observed increased numbers of dermal FOXP3(+)CD25(+) T cells and epidermal Langerhans cells (LCs) that were associated with upregulated mRNA expression of TGF-ß in lesions of GCS-treated Ni-allergic patients. To investigate this phenomenon further, we exposed purified LCs to GCS. They exhibited, in contrast to GCS-nonexposed LCs, 1) a more immature phenotype, 2) higher intracellular amounts of TGF-ß, and 3) increased receptor activator for NF-κB expression, conditions that reportedly favor the expansion of regulatory T cells (Tregs). Indeed, we observed an enhancement of functionally suppressive FOXP3(+) T cells when CD3(+) cells were incubated with GCS-pretreated LCs. The expansion of Tregs was inhibited by TGF-ß blockage alone, and their suppressive activity was neutralized by a combination of anti-TGF-ß and anti-IL-10 Abs. Our data show that systemically applied GCSs endow LCs with Treg-promoting properties and thus shed new light on the mechanisms of GCS-mediated immunosuppression.
Assuntos
Diferenciação Celular/imunologia , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Testes do Emplastro , Prednisona/farmacologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/biossíntese , Administração Oral , Adulto , Alérgenos/administração & dosagem , Alérgenos/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Método Duplo-Cego , Feminino , Humanos , Imunofenotipagem , Mediadores da Inflamação/administração & dosagem , Células de Langerhans/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Níquel/administração & dosagem , Níquel/imunologia , Testes do Emplastro/métodos , Prednisona/administração & dosagem , Reprodutibilidade dos Testes , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Adulto JovemRESUMO
Cell-mediated immunity is thought to be of critical importance in antisyphilitic host defense, but the exact mechanisms are still unknown. This fact is particularly true for HIV-infected persons with a deficit in CD4+ T-cell number. We therefore obtained lesional skin samples from HIV+ and HIV- patients with secondary syphilis at different time points of lesional age to search both for causative microorganisms and to characterize the inflammatory infiltrate. By doing so, we detected Treponema pallidum spirochetes with a much greater abundance in late lesions of HIV+ individuals compared with the HIV- cohort. The dominating inflammatory cells were T cells, macrophages, and neutrophils at all stages and plasma cells in older lesions. In HIV- persons, T cells consisted of equal numbers of CD4+ and CD8+ T-cells, whereas in HIV+ patients, the majority of T cells belonged to the CD8 lineage and produced both IFN-γ and IL-17. Regulatory T cells and Langerhans cells were reduced in these patients compared with their HIV- counterparts. Because of our observations, we propose that T cells of both the CD4 and CD8 lineage are needed for an at least partial protective antisyphilitic immunity. Compensation mechanisms in HIV+ individuals, such as an increase of Tc1/17 cells as well as a reduction in immunoregulatory Langerhans cells and T cells, apparently do not overcome the deficiencies in these patients to eliminate the spirochete.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Sífilis/imunologia , Sífilis/patologia , Adulto , Animais , Feminino , Infecções por HIV/complicações , Infecções por HIV/imunologia , Infecções por HIV/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pele/imunologia , Pele/microbiologia , Pele/patologia , Sífilis/etiologia , Treponema pallidum/imunologiaRESUMO
Artificial Toll-like receptor 7/8 (TLR7/8) ligands can endow plasmacytoid dendritic cells (pDCs) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent lytic properties. Keeping in mind that ssRNA serves as natural TLR7/8 ligand, we searched for TRAIL-expressing cells in persons infected with HIV and identified TRAIL+ pDCs in HIV-1 viremic persons, but not in nonviremic and healthy persons. TRAIL expression on pDCs was directly correlated with individual viral loads. Conversely, HIV-1 viremia was found to be associated with the up-regulation of the apoptosis-transmitting receptor TRAIL R1 on activated CD4+ T cells. As a consequence, the latter became susceptible to TRAIL-dependent pDC-mediated killing. In contrast, initiation of antiretroviral therapy led to the up-regulation of apoptosis-inhibiting TRAIL R4 on CD4+ T cells, which subsequently became resistant against pDC-mediated cellular injury. Definition of pDCs as killers of CD4+ T cells implies a new mechanism of disease progression in HIV infection.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Viremia/imunologia , Adolescente , Adulto , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/metabolismo , HIV-1/metabolismo , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , RNA Viral/imunologia , RNA Viral/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/imunologia , Receptor 8 Toll-Like/metabolismo , Regulação para Cima/imunologia , Viremia/metabolismoRESUMO
Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH(ITSN-2L)) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The "catalytically dead" DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH(ITSN-2L)-transfected cells compared with control and 1 microM jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cavéolas/metabolismo , Endocitose/fisiologia , Células Endoteliais/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/genética , Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Células Endoteliais/citologia , Ativação Enzimática/fisiologia , Humanos , Estrutura Terciária de Proteína/fisiologia , Pseudópodes/genética , Pseudópodes/metabolismo , RNA Interferente Pequeno/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/genéticaAssuntos
Linfoma Cutâneo de Células T/diagnóstico , Receptores de Antígenos de Linfócitos T gama-delta , Síndrome de Sézary/diagnóstico , Neoplasias Cutâneas/diagnóstico , Linfócitos T/imunologia , Idoso , Diagnóstico Diferencial , Humanos , Linfoma Cutâneo de Células T/imunologia , Masculino , Fenótipo , Neoplasias Cutâneas/imunologiaRESUMO
Intersectins (ITSNs) are multidomain adaptor proteins implicated in endocytosis, regulation of actin polymerization, and Ras/MAPK signaling. We have previously shown that ITSN-1s is required for caveolae fission and internalization in endothelial cells (ECs). In the present study, using small interfering RNA to knock down ITSN-1s protein expression, we demonstrate a novel role of ITSN-1s as a key antiapoptotic protein. Knockdown of ITSN-1s in ECs activated the mitochondrial pathway of apoptosis as determined by genomic DNA fragmentation, extensive mitochondrial fission, activation of the proapoptotic proteins BAK and BAX, and cytochrome c efflux from mitochondria. ITSN-1 knockdown acts as a proapoptotic signal that causes mitochondrial outer membrane permeabilization, dissipation of the mitochondrial membrane potential, and generation of reactive oxygen species. These effects were secondary to decreased activation of Erk1/2 and its direct activator MEK. Bcl-X(L) overexpression prevented BAX activation and the apoptotic ECs death induced by suppression of ITSN-1s. Our findings demonstrate a novel role of ITSN-1s as a negative regulator of the mitochondrial pathway-dependent apoptosis secondary to activation of the Erk1/2 survival signaling pathway.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Apoptose/fisiologia , Endotélio Vascular/fisiologia , Mitocôndrias/fisiologia , Sequência de Bases , Células Cultivadas , Citocromos c/metabolismo , Endotélio Vascular/citologia , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/enzimologia , RNA Interferente Pequeno , Transdução de SinaisRESUMO
We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.
Assuntos
Cavéolas/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Fusão de Membrana , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Biotina/metabolismo , Caveolina 1/antagonistas & inibidores , Caveolina 1/genética , Caveolina 1/metabolismo , Sistema Livre de Células , Endotélio Vascular/metabolismo , Humanos , Pulmão/metabolismo , Microscopia de Fluorescência , Proteínas SNARE/metabolismo , Estreptavidina/metabolismoRESUMO
Opsin, a member of the G-protein-coupled receptor family, is a polytopic membrane protein that does not encode a cleaved amino-terminal signal sequence. The amino terminus of opsin precedes the first known targeting information, suggesting that it translocates across the endoplasmic reticulum (ER) membrane after synthesis, uncoupled from translation. However, translocation across the mammalian ER is believed to be coupled to protein synthesis. In this study we show that opsin, within a range of nascent peptide lengths, targets and translocates equally efficiently co- and posttranslationally. Longer nascent opsin peptides have a lower efficiency of cotranslational translocation but an even lower efficiency of posttranslational translocation. We also show that SRP is required for both co- and posttranslational targeting.