RESUMO
BACKGROUND: CD39 and CD73 are two novel cell surface markers of CD25high Foxp3+ regulatory T-cells (Tregs). Concordant expression of these two ectoenzymes not only discriminate Tregs from other cell populations, but also generates pericellular adenosine, which has been reported to suppress proliferation of activated T effector (Teff) cells. Because it is currently unclear whether human ectoenzymes (CD39/CD73) are involved in the impaired suppressive activity of Tregs in psoriasis, we examined the frequencies and phenotypes of CD39/CD73-expressing Tregs and related receptor adenosine receptor 2A (A2A R) in peripheral blood of patients with different types of psoriasis. METHODS: Peripheral blood mononuclear cells (PMBC) were prepared from patients with three different types of psoriasis (psoriasis vulgaris, pustular psoriasis and erythrodermic psoriasis). CD4+ cells were separated from PBMC by negative selection on midiMACS columns, and the frequencies and phenotypes of CD39 and CD73 expressing Tregs, and A2A R expressing Teff were all determined by flow cytometry analysis. Blood from healthy volunteers served as controls. RESULTS: The expression of single CD73+ Tregs was markedly reduced (approximately 50%) in psoriasis vulgaris, compared to normal controls. In pustular psoriasis, the mean numbers of CD39+ Tregs and A2A R+ Teff was significantly lower than in normal controls. Among three different types of psoriasis, CD39 expression was strikingly reduced in the blood Treg population of pustular psoriasis patients. Decreased CD73+ Tregs levels were observed in psoriasis vulgaris compared to pustular psoriasis and erythrodermic psoriasis. CONCLUSIONS: The differences in the expression of CD39- and CD73- Tregs may be a factor in the pathogenesis of psoriasis.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Psoríase/imunologia , Receptores A2 de Adenosina/sangue , Linfócitos T Reguladores/metabolismo , Adulto , Contagem de Linfócito CD4 , Estudos de Casos e Controles , Feminino , Fatores de Transcrição Forkhead/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Psoríase/sangueRESUMO
Photodynamic therapy (PDT) is an emerging treatment for malignant and inflammatory dermal disorders. Photoirradiation of the silicon phthalocyanine (Pc) 4 photosensitizer with red light generates singlet oxygen and other reactive oxygen species to induce cell death. We previously reported that Pc 4-PDT elicited cell death in lymphoid-derived (Jurkat) and epithelial-derived (A431) cell lines in vitro, and furthermore that Jurkat cells were more sensitive than A431 cells to treatment. In this study, we examined the effectiveness of Pc 4-PDT on primary human CD3(+) T cells in vitro. Fluorometric analyses of lysed T cells confirmed the dose-dependent uptake of Pc 4 in non-stimulated and stimulated T cells. Flow cytometric analyses measuring annexin V and propidium iodide (PI) demonstrated a dose-dependent increase of T cell apoptosis (6.6-59.9%) at Pc 4 doses ranging from 0-300 nM. Following T cell stimulation through the T cell receptor using a combination of anti-CD3 and anti-CD28 antibodies, activated T cells exhibited increased susceptibility to Pc 4-PDT-induced apoptosis (10.6-81.2%) as determined by Pc 4 fluorescence in each cell, in both non-stimulated and stimulated T cells, Pc 4 uptake increased with Pc 4 dose up to 300 nM as assessed by flow cytometry. The mean fluorescence intensity (MFI) of Pc 4 uptake measured in stimulated T cells was significantly increased over the uptake of resting T cells at each dose of Pc 4 tested (50, 100, 150 and 300 nM, p < 0.001 between 50 and 150 nM, n = 8). Treg uptake was diminished relative to other T cells. Cutaneous T cell lymphoma (CTCL) T cells appeared to take up somewhat more Pc 4 than normal resting T cells at 100 and 150 nm Pc 4. Confocal imaging revealed that Pc 4 localized in cytoplasmic organelles, with approximately half of the Pc 4 co-localized with mitochondria in T cells. Thus, Pc 4-PDT exerts an enhanced apoptotic effect on activated CD3(+) T cells that may be exploited in targeting T cell-mediated skin diseases, such as cutaneous T cell lymphoma (CTCL) or psoriasis.
Assuntos
Indóis/farmacologia , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Linfócitos T/efeitos dos fármacos , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Autoanticorpos/administração & dosagem , Antígenos CD28/metabolismo , Relação Dose-Resposta a Droga , Humanos , Indóis/farmacocinética , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/metabolismo , Linfoma Cutâneo de Células T/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacocinética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Propídio/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
BACKGROUND: Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. RESULTS: Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). CONCLUSIONS: A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Linfócitos T Reguladores/metabolismo , Animais , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Selectina L/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologiaRESUMO
T memory/effector cells (Tmem/eff) isolated from psoriatic patients are chronically activated and poorly suppressed by regulatory T cells (Treg). The proinflammatory cytokine IL-6, which signals through Stat3, allows escape of Tmem/eff cells from Treg-mediated suppression in a murine system. We show here that IL-6 protein is markedly elevated and most highly expressed by CD31(+) endothelial cells and CD11c(+) dermal dendritic cells (DCs) in lesional psoriatic skin. We hypothesized that exposure to high IL-6 in lesional tissue may lead to the dampened Treg function observed in psoriasis patients. Indeed, we found that IL-6, but not other Stat3-activating cytokines, was necessary and sufficient to reverse human T cell suppression by Treg in an in vitro model using activated DCs as a source of IL-6. IL-6Ralpha and gp130 expression was significantly elevated in psoriatic effector T cells compared with normal controls. Overall, IL-6Ralpha expression on Treg exceeded that of effector T cells, and both populations phosphorylated Stat3 in response to IL-6. Phosphorylation of Stat3 in T cells contributes to Th17 differentiation and we identify cells within lesional tissue that coexpress CD3, IL-17, and IL-6, indicating that Th17 cells are present in vivo within the psoriatic Tmem/eff population and contribute to IL-6-mediated resistance to Treg suppression. Taken together, T lymphocytes trafficking into lesional psoriatic skin encounter high IL-6 from endothelial cells, DCs, and Th17 cells, enabling cutaneous T cell escape from Treg suppression and Th17 participation in inflammation. Targeting IL-6 signaling pathways in psoriasis may rebalance Treg/T effector activity and ameliorate disease.
Assuntos
Terapia de Imunossupressão , Interleucina-6/fisiologia , Psoríase/imunologia , Psoríase/patologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Movimento Celular/imunologia , Células Cultivadas , Derme/imunologia , Derme/metabolismo , Derme/patologia , Humanos , Interleucina-6/metabolismo , Linfócitos T Reguladores/patologiaRESUMO
Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.