RESUMO
OBJECTIVE: Under both physiological and pathological conditions, bone volume is determined by the rate of bone formation by osteoblasts and bone resorption by osteoclasts. Excessive bone loss is a common complication of human IBD whose mechanisms are not yet completely understood. Despite the role of activated CD4(+) T cells in inflammatory bone loss, the nature of the T cell subsets involved in this process in vivo remains unknown. The aim of the present study was to identify the CD4(+) T cell subsets involved in the process of osteoclastogenesis in vivo, as well as their mechanism of action. DESIGN: CD4(+) T cells were studied in IL10-/- mice and Rag1-/- mice adoptively transferred with naive CD4(+)CD45RB(high) T cells, representing two well-characterised animal models of IBD and in patients with Crohn's disease. They were phenotypically and functionally characterised by flow cytometric and gene expression analysis, as well as in in vitro cocultures with osteoclast precursors. RESULTS: In mice, we identified bone marrow (BM) CD4(+) T cells producing interleukin (IL)-17 and tumour necrosis factor (TNF)-α as an osteoclastogenic T cell subset referred to as Th17 TNF-α(+) cells. During chronic inflammation, these cells migrate to the BM where they survive in an IL-7-dependent manner and where they promote the recruitment of inflammatory monocytes, the main osteoclast progenitors. A population equivalent to the Th17 TNF-α(+) cells was also detected in patients with Crohn's disease. CONCLUSIONS: Our results highlight the osteoclastogenic function of the Th17 TNF-α(+) cells that contribute to bone loss in vivo in IBD.
Assuntos
Doenças Ósseas/fisiopatologia , Células da Medula Óssea/fisiologia , Doenças Inflamatórias Intestinais/fisiopatologia , Osteoclastos/fisiologia , Subpopulações de Linfócitos T/fisiologia , Células Th17/fisiologia , Imunidade Adaptativa/fisiologia , Animais , Doenças Ósseas/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Doença de Crohn/imunologia , Doença de Crohn/fisiopatologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/imunologia , Interleucina-7/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoclastos/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
OBJECTIVES: Nicotinamide phosphoribosyltransferase (NAMPT)/pre-B-cell colony-enhancing factor/visfatin exerts multiple functions and has been implicated in the pathogenesis of rheumatoid arthritis. To gain insight into its role in arthritis and given that NAMPT is identified as a novel mediator of innate immunity, we addressed the function of monocyte-derived NAMPT in experimental arthritis by selective gene knockdown in inflammatory monocytes. METHODS: siRNA uptake and NAMPT expression were determined in Ly6Chigh and Ly6Clow monocyte subsets following intravenous injection of siRNA against NAMPT (siNAMPT) or non-targeting siRNA (siCT) formulated with the DMAPAP cationic liposome into mice. Mice with established collagen-induced arthritis (CIA) were treated weekly after disease onset with siNAMPT or siCT and clinical features were assessed. T-helper cell frequencies, cytokine production and percentage of IL-6-producing Ly6Chigh monocytes were analysed. Using a co-culture system consisting of purified CD14 monocytes and autologous CD4 T cells, NAMPT and cytokine production, and the percentage of IL-17-producing CD4 T cells, were determined following transfection of CD14 monocytes with siCT or siNAMPT. RESULTS: On intravenous injection, siRNA was preferentially engulfed by Ly6Chigh monocytes, and siRNA-mediated silencing of NAMPT expression in Ly6Chigh monocytes inhibited CIA progression. This effect was associated with reduced IL-6 production by Ly6Chigh monocytes, reduced proportion of Th17 cells and autoantibody titers, and decreased activation and infiltration of monocytes/macrophages and neutrophils in arthritic joints. Moreover, NAMPT-RNAi-silenced CD14 monocytes were found to reduce the percentage of IL-17-producing CD4 T cells in vitro. CONCLUSIONS: Our results show that the expression of NAMPT in Ly6Chigh monocytes promotes many downstream effects involved in inflammatory arthritis and demonstrate the utility of targeting disease-causing genes, such as NAMPT, in Ly6Chigh monocytes for therapeutic intervention in arthritis.
Assuntos
Artrite Experimental/imunologia , Citocinas/imunologia , Monócitos/imunologia , Nicotinamida Fosforribosiltransferase/imunologia , Animais , Artrite Experimental/prevenção & controle , Linfócitos T CD4-Positivos/imunologia , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/genética , Inativação Gênica , Humanos , Imunomodulação/imunologia , Interleucina-6/biossíntese , Receptores de Lipopolissacarídeos/análise , Camundongos , Camundongos Endogâmicos DBA , Nicotinamida Fosforribosiltransferase/genética , RNA Interferente Pequeno/genética , Células Th17/imunologiaRESUMO
CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human ß-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.
Assuntos
Apoptose/imunologia , Velocidade do Fluxo Sanguíneo/imunologia , Quimiocina CCL20/fisiologia , Endotélio Vascular/imunologia , Mediadores da Inflamação/fisiologia , Interleucina-17/biossíntese , Linfócitos T Auxiliares-Indutores/imunologia , beta-Defensinas/fisiologia , Animais , Movimento Celular/imunologia , Células Cultivadas , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Células Epidérmicas , Epiderme/imunologia , Epiderme/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Interleucinas/biossíntese , Queratinócitos/citologia , Queratinócitos/imunologia , Queratinócitos/metabolismo , Células L , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Receptores CCR6/biossíntese , Receptores CCR6/genética , Receptores CCR6/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Interleucina 22RESUMO
The ontogenic relationship between pro-inflammatory populations of interleukin-17 (IL-17A)- and/or IL-22-producing T cells and other T-cell subsets is currently unclear in humans. To appreciate T helper cell-lineage commitment, we combined cytokine production profiles of in vitro expanded T-cell clones with T-cell receptor (TCR) clonotypic signatures. Moreover, ex vivo cytokine production profiles at the single-cell level were analyzed using an original approach based on the hierarchical cluster analysis of multiparametric flow cytometry data. These combined approaches enabled the delineation of distinct functional T-cell subsets, including Th1, Th2, Tr1, Th17 cells and a highly polyfunctional IL-22-producing T-cell population. Cluster analysis highlighted that the IL-22-producing T-cell population should be considered independently from the Th17 and Th1 subsets, although it was more closely related to the former. In parallel, we observed extensive TCRαß sharing across all five subsets defined. The strategy described here allows the objective definition of cellular subsets and an unbiased insight into their similarities. Together, our results underscore the ontogenic plasticity of CD4(+) T-cell progenitors, which can adopt a differentiation profile irrespective of antigen specificity.
Assuntos
Interleucina-17/metabolismo , Interleucinas/metabolismo , Subpopulações de Linfócitos T/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Antígenos de Diferenciação/metabolismo , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Clonais , Citometria de Fluxo , Humanos , Imunofenotipagem/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th2/citologia , Células Th2/imunologia , Interleucina 22RESUMO
Mesenchymal stem cells (MSCs) exert immunomodulatory properties via the inhibition of T cell activation and proliferation. Because of the deleterious role of Th17 cells in the pathogenesis of inflammatory disease, we investigated whether proinflammatory cytokines could modify the expression of adhesion molecules on human MSCs, thereby contributing to increased Th17 cell adhesion to MSCs and, as a consequence, modulating the function of the latter cells. IFN-gamma and TNF-alpha synergistically enhanced the expression of CD54 by MSCs, enabling the CCR6 chemokine ligand CCL20 to induce in vitro adhesion of Th17 cells to MSCs. MSCs prevented the in vitro differentiation of naive CD4(+) T cells into Th17 cells and inhibited the production of IL-17, IL-22, IFN-gamma, and TNF-alpha by fully differentiated Th17 cells; this was mediated, in part, via PGE(2), the production of which was enhanced in cocultures with Th17 cells. Moreover, MSCs induced the production of IL-10 and trimethylation of histone H3K4me3 at the promoter of the FOXP3 gene locus, whereas it suppressed trimethylation of the corresponding region in the RORC gene in Th17 cells. These epigenetic changes were associated with the induction of fork head box p3 and the acquisition by Th17 cells of the capacity to inhibit in vitro proliferative responses of activated CD4(+) T cells, which was enhanced when MSCs were preincubated with IFN-gamma and TNF-alpha. These results showed that, under inflammatory conditions, MSCs mediate the adhesion of Th17 cells via CCR6 and exert anti-inflammatory effects through the induction of a T cell regulatory phenotype in these cells.
Assuntos
Diferenciação Celular/imunologia , Inibidores do Crescimento/fisiologia , Interleucina-17/antagonistas & inibidores , Células-Tronco Mesenquimais/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11a/metabolismo , Antígeno CD11a/fisiologia , Antígenos CD18/metabolismo , Antígenos CD18/fisiologia , Adesão Celular/imunologia , Comunicação Celular/imunologia , Movimento Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Molécula 1 de Adesão Intercelular/biossíntese , Interferon gama/fisiologia , Interleucina-17/biossíntese , Interleucina-17/fisiologia , Células L , Ligantes , Células-Tronco Mesenquimais/patologia , Camundongos , Receptores CCR6/metabolismo , Receptores CCR6/fisiologia , Linfócitos T Reguladores/patologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells with broad immunosuppressive capacities. Recently, it has been reported that MSCs can transfer mitochondria to various cell types, including fibroblast, cancer, and endothelial cells. It has been suggested that mitochondrial transfer is associated with a physiological response to cues released by damaged cells to restore and regenerate damaged tissue. However, the role of mitochondrial transfer to immune competent cells has been poorly investigated. METHODS AND RESULTS: Here, we analyzed the capacity of MSCs from the bone marrow (BM) of healthy donors (BM-MSCs) to transfer mitochondria to primary CD4+CCR6+CD45RO+ T helper 17 (Th17) cells by confocal microscopy and fluorescent-activated cell sorting (FACS). We then evaluated the Th17 cell inflammatory phenotype and bioenergetics at 4 h and 24 h of co-culture with BM-MSCs. We found that Th17 cells can take up mitochondria from BM-MSCs already after 4 h of co-culture. Moreover, IL-17 production by Th17 cells co-cultured with BM-MSCs was significantly impaired in a contact-dependent manner. This inhibition was associated with oxygen consumption increase by Th17 cells and interconversion into T regulatory cells. Finally, by co-culturing human synovial MSCs (sMSCs) from patients with rheumatoid arthritis (RA) with Th17 cells, we found that compared with healthy BM-MSCs, mitochondrial transfer to Th17 cells was impaired in RA-sMSCs. Moreover, artificial mitochondrial transfer also significantly reduced IL-17 production by Th17 cells. CONCLUSIONS: The present study brings some insights into a novel mechanism of T cell function regulation through mitochondrial transfer from stromal stem cells. The reduced mitochondrial transfer by RA-sMSCs might contribute to the persistence of chronic inflammation in RA synovitis.
Assuntos
Células-Tronco Mesenquimais/citologia , Mitocôndrias/transplante , Células Th17/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células da Medula Óssea/citologia , Células Cultivadas , Técnicas de Cocultura , Humanos , Interleucina-17/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio , Membrana Sinovial/citologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Mesenchymal stem cells (MSC) are highly immunosuppressive cells able to reduce chronic inflammation through the active release of mediators. Recently, we showed that glucocorticoid-induced leucine zipper (Gilz) expression by MSC is involved in their therapeutic effect by promoting the generation of regulatory T cells. However, the mechanisms underlying this pivotal role of Gilz remain elusive. Methods and Results In this study, we have uncovered evidence that Gilz modulates the phenotype and function of Th1 and Th17 cells likely by upregulating the level of Activin A and NO2 secreted by MSC. Adoptive transfer experiments sustained this Gilz-dependent suppressive effect of MSC on Th1 and Th17 cell functions. In immunoregulatory MSC, obtained by priming with IFN-γ and TNF-α, Gilz was translocated to the nucleus and bound to the promoters of inos and Activin ßA to induce their expression. The increased expression of Activin A directly impacted on Th17 cells fate by repressing their differentiation program through the activation of Smad3/2 and enhancing IL-10 production. Conclusion Our results reveal how Gilz controls inos and Activin ßA gene expression to ultimately assign immunoregulatory status to MSC able to repress the pathogenic Th17 cell differentiation program and uncover Activin A as a novel mediator of MSC in this process.
Assuntos
Ativinas/metabolismo , Diferenciação Celular , Células-Tronco Mesenquimais/imunologia , Células Th17/imunologia , Fatores de Transcrição/metabolismo , Ativinas/genética , Animais , Células Cultivadas , Apresentação Cruzada , Interferon gama/metabolismo , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Smad/metabolismo , Células Th17/citologia , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: TNF receptor-associated syndrome (TRAPS) is a dominantly inherited autoinflammatory condition caused by mutations in the TNFRSF1A gene. The mechanism underlying the variable expressivity of the common variant R92Q (rs4149584; c.362G>A; p.Arg121Gln) is unclear and is of critical importance for patient care and genetic counseling. This study evaluated the impact of the number of R92Q mutations in two unique unrelated families. METHODS: Two patients with undefined but clear autoinflammatory symptoms were referred for genetic diagnosis. Blood samples were collected from the available family members to screen autoinflammatory genes and assess key steps of the TNFR1-mediated signaling pathway using flow cytometry and ex vivo culture. RESULTS: R92Q homozygosity was demonstrated for the two probands. In family 1, the segregation analysis revealed TRAPS-like symptoms in all carriers, with a more severe presentation in the proband, whereas in family 2, the heterozygous parents were totally asymptomatic, suggesting recessive transmission. Functional studies revealed a nonclassical pathogenesis of TRAPS in the two probands and suggested a compensatory mechanism without clear dose effect. CONCLUSION: We observed for the first time a possible clinical dose effect of R92Q. This work highlights the importance of familial studies to reconcile the contradictory reports published on the pathogenicity of this variant.
RESUMO
It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human naïve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to naïve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by naïve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.
Assuntos
Linfócitos B/efeitos dos fármacos , Imunoglobulina G/imunologia , Interleucinas/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD19/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Humanos , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/imunologia , Interleucina-10/farmacologia , Interleucina-4/farmacologia , Baço/citologia , Baço/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.
Assuntos
Linfócitos B/imunologia , Switching de Imunoglobulina , Imunoglobulina E/imunologia , Cadeias épsilon de Imunoglobulina/imunologia , Linfócitos B/citologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Células Cultivadas , Técnicas de Cultura , DNA Circular/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Sangue Fetal/citologia , Humanos , Imunoglobulina E/genética , Cadeias épsilon de Imunoglobulina/genética , Separação Imunomagnética/métodos , Ativação Linfocitária , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Baço/citologia , Baço/imunologiaRESUMO
Chemokines and their receptors play an important role in cell trafficking and recruitment. The CCR6 chemokine receptor, selectively expressed on leukocyte populations, has been shown to play a deleterious role in the pathogenesis of various chronic inflammatory diseases and, as such, may constitute a prime target in the development of immunotherapeutic treatment. However, to date no neutralizing mouse monoclonal antibodies (mAbs) specific for this chemokine receptor have been reported, whereas information on small molecules capable of interfering with the interaction of CCR6 and its ligands is scant. Here, we report the failure to generate neutralizing mouse mAbs specific for human (hu)CCR6. Immunization of mice with peptides mimicking extracellular domains, potentially involved in CCR6 function, failed to induce Abs reactive with the native receptor. Although the use of NIH-3T3 cells expressing huCCR6 resulted in the isolation of mAbs specific for this receptor, they were not able to block the interaction between huCCR6 and huCCL20. Investigation of the anti-huCCR6 mAbs generated in the present study, as well as those commercially available, show that all mAbs invariably recognize a unique, non-neutralizing, immunodominant region in the first part of its N-terminal domain. Together, these results indicate that the generation of potential neutralizing anti-huCCR6 mAbs in the mouse is unlikely to succeed and that alternative techniques, such as the use of other animal species for immunization, might constitute a better approach to generate such a potentially therapeutic tool for the treatment of inflammatory disease.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos Imunodominantes/imunologia , Receptores CCR6/imunologia , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular , Quimiocina CCL20/imunologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Receptores CCR6/administração & dosagem , Proteínas Recombinantes/imunologiaRESUMO
Functional analysis of T lymphocytes requires in vitro stimulation of these cells under experimental conditions that mimic as closely as possible physiological in vivo stimulation and that involve antigen/T cell receptor (TCR)-mediated activation. Because of the low frequency of antigen-specific T cells in human clinical samples, stimulation with a combination of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) is a preferred method. Interaction of these mAbs with their ligand results in modulation of the mAb-ligand complex from the cell surface. However, as a result of incomplete modulation, CD3/CD28 mAb complexes often remain at the cell surface, thereby precluding subsequent indirect immunofluorescence and flow cytometry analysis using mouse immunoglobulin (Ig)-specific antibodies. This is of importance in situations in which no specific fluorochrome-conjugated mAbs are available, such as in screening procedures of Ig-containing hybridoma culture supernatants. We propose here the use of CD3/CD28 mAbs, linked to magnetic beads allowing standardization of the activation conditions, optimal activation of T cells and complete modulation of antigen-antibody complexes from the cell surface.
Assuntos
ADP-Ribosil Ciclase/imunologia , Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Complexo CD3/imunologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Imunofenotipagem/métodos , Ativação Linfocitária , Linfócitos T/imunologia , ADP-Ribosil Ciclase 1 , Humanos , Magnetismo , Glicoproteínas de MembranaRESUMO
Carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a cell-surface glycoprotein, belonging to the carcinoembryonic antigen family, expressed by human neutrophils, epithelial cells, activated T and NK cells. CEACAM1 is expressed as a cell-surface molecule with different isoforms or can be secreted as a soluble protein. Here, we show that keratinocytes in the outer epidermal layer of psoriatic skin express CEACAM1, unlike those in healthy skin or in cutaneous lesions of patients with atopic or nummular dermatitis. Stimulation of primary human keratinocytes or in vitro reconstituted epidermis with culture supernatants of activated psoriatic lesion-infiltrating T cells, IFN-gamma or oncostatin M, but not IL-17, induced the expression of transcripts for the CEACAM1-long and -short isoforms and cell-surface CEACAM1, whereas soluble CEACAM1 was not produced. The uppermost layers of the epidermis in psoriatic lesions also contain neutrophils, a cell type with inflammatory and antimicrobial properties. Coculture of CEACAM1-expressing keratinocytes or CHO transfectants with neutrophils delayed spontaneous apoptosis of the latter cells. These results show that cytokine-induced cell-surface expression of CEACAM1 by keratinocytes in the context of a psoriatic environment might contribute to the persistence of neutrophils and thus to ongoing inflammation and the decreased propensity for skin infection, typical for patients with psoriasis.
Assuntos
Antígenos CD/metabolismo , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular/metabolismo , Queratinócitos/metabolismo , Neutrófilos/metabolismo , Psoríase/metabolismo , Pele/patologia , Animais , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oncostatina M/metabolismo , Linfócitos T/metabolismoRESUMO
Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.
Assuntos
Doenças Autoimunes/imunologia , Citocinas/análise , Inflamação/imunologia , Interleucina-17/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Doenças Autoimunes/metabolismo , Diferenciação Celular , Citocinas/imunologia , Perfilação da Expressão Gênica , Humanos , Inflamação/metabolismo , Interleucina-17/imunologia , Queratinócitos/citologia , Ativação Linfocitária , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Receptores CCR6/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Linfócitos T Auxiliares-Indutores/fisiologia , Células Th1/fisiologiaRESUMO
Certain individuals are resistant to HIV-1 infection, despite repeated exposure to the virus. Although protection against HIV-1 infection in a small proportion of Caucasian individuals is associated with mutant alleles of the CCR5 HIV-1 coreceptor, the molecular mechanism underlying resistance in repeatedly HIV-1-exposed, uninfected individuals (EU) is unclear. In this study, we performed complementary transcriptome and proteome analyses on peripheral blood T cells, and plasma or serum from EU, their HIV-1-infected sexual partners, and healthy controls, all expressing wild-type CCR5. We report that activated T cells from EU overproduce several proteins involved in the innate immunity response, principally those including high levels of peroxiredoxin II, a NK-enhancing factor possessing strong anti-HIV activity, and IL-22, a cytokine involved in the production of acute-phase proteins such as the acute-phase serum amyloid A (A-SAA). Cell supernatants and serum levels of these proteins were up-regulated in EU. Moreover, a specific biomarker for EU detected in plasma was identified as an 8.6-kDa A-SAA cleavage product. Incubation of in vitro-generated myeloid immature dendritic cells with A-SAA resulted in CCR5 phosphorylation, down-regulation of CCR5 expression, and strongly decreased susceptibility of these cells to in vitro infection with a primary HIV-1 isolate. Taken together, these results suggest new correlates of EU protection and identify a cascade involving IL-22 and the acute phase protein pathway that is associated with innate host resistance to HIV infection.
Assuntos
Infecções por HIV/imunologia , HIV-1 , Interleucinas/metabolismo , Proteína Amiloide A Sérica/metabolismo , Linfócitos T/imunologia , Proteínas de Fase Aguda/análise , Proteínas de Fase Aguda/metabolismo , Biomarcadores/sangue , Células Dendríticas/imunologia , Feminino , Perfilação da Expressão Gênica , Infecções por HIV/genética , Humanos , Imunidade Inata/genética , Interleucinas/sangue , Interleucinas/genética , Ativação Linfocitária , Masculino , Células Mieloides/imunologia , Peroxidases/sangue , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas , Fosforilação , Proteoma/análise , Proteoma/genética , Proteoma/metabolismo , Receptores CCR5/metabolismo , Proteína Amiloide A Sérica/análise , Transcrição Gênica , Regulação para Cima , Interleucina 22RESUMO
Cutaneous inflammatory diseases such as psoriasis vulgaris and atopic dermatitis are associated with altered keratinocyte function, as well as with a particular cytokine production profile of skin-infiltrating T lymphocytes. In this study we show that normal human epidermal keratinocytes express a functional type II oncostatin-M (OSM) receptor (OSMR) consisting of the gp130 and OSMRbeta components, but not the type I OSMR. The type II OSMR is expressed in skin lesions from both psoriatic patients and those with atopic dermatitis. Its ligand, OSM, induces via the recruitment of the STAT3 and MAP kinase pathways a gene expression profile in primary keratinocytes and in a reconstituted epidermis that is characteristic of proinflammatory and innate immune responses. Moreover, OSM is a potent stimulator of keratinocyte migration in vitro and increases the thickness of a reconstituted epidermis. OSM transcripts are enhanced in both psoriatic and atopic dermatitic skin as compared with healthy skin and mirror the enhanced production of OSM by T cells isolated from diseased lesions. Results from a microarray analysis comparing the gene-modulating effects of OSM with those of 33 different cytokines indicate that OSM is a potent keratinocyte activator similar to TNF-alpha, IL-1, IL-17, and IL-22 and that it acts in synergy with the latter cytokines in the induction of S100A7 and beta-defensin 2 expression, characteristic of psoriatic skin. Taken together, these results demonstrate that OSM and its receptor play an important role in cutaneous inflammatory responses in general and that the specific effects of OSM are associated with distinct inflammatory diseases depending on the cytokine environment.
Assuntos
Dermatite/imunologia , Queratinócitos/imunologia , Oncostatina M/fisiologia , Receptores Tipo II de Oncostatina M/fisiologia , Linfócitos T/imunologia , Movimento Celular , Células Cultivadas , Dermatite/genética , Dermatite/patologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata/genética , Queratinócitos/química , Queratinócitos/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oncostatina M/metabolismo , Oncostatina M/farmacologia , Receptores Tipo II de Oncostatina M/análise , Receptores Tipo II de Oncostatina M/metabolismo , Fator de Transcrição STAT3/metabolismo , Pele/imunologia , Pele/patologiaRESUMO
BACKGROUND: Results from a transcriptome analysis of human CD4+ T regulatory type 1 (Tr1) clones have indicated that transcripts for the integrins CD18 and CD49b are overexpressed in these cells. The aim of this study was to investigate whether the presence of T cells concomitantly expressing these molecules could be detected in asthmatic patients and represent Tr1 cells. METHODS: Expression of CD18 and CD49b was analyzed by flow cytometry on peripheral blood mononuclear cells from asthmatic patients of various severity and healthy subjects. The cytokine production profile of purified CD4+ CD18(high) CD49b+ T cells was analyzed by ELISA. The effect of glucocorticoid treatment on the expression of CD18 and CD49b was determined. RESULTS: The frequency of peripheral blood CD18(high) CD49b+ T cells was significantly elevated in severe asthmatic patients, as compared with both mild asthmatic and healthy donors, and was diminished in asthmatic patients with a controlled status of the disease. Neither short-course oral glucocorticoid treatment of asthmatic patients ex vivo, nor culture of their peripheral blood mononuclear cells with dexamethasone in vitro, increased the frequency of CD18(high) CD49b+ T cells, indicating that their presence seems to be independent from recent anti-inflammatory treatment. However, purified CD4+ CD18(high) CD49b+ T cells from these patients, in contrast to those from healthy donors, lacked the production of the immunosuppressive cytokine interleukin-10. CONCLUSION: In contrast to healthy donors, peripheral blood CD18(high) CD49b+ T cells of asthmatic patients do not fulfill the phenotypic criteria of Tr1 cells. Nevertheless, the presence of elevated numbers of peripheral blood CD18(high) CD49b+ T cells is characteristic for patients with severe and uncontrolled asthma.
Assuntos
Asma/sangue , Asma/imunologia , Antígenos CD18/imunologia , Integrina alfa2/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Asma/tratamento farmacológico , Antígenos CD18/biossíntese , Dexametasona/farmacologia , Feminino , Citometria de Fluxo , Volume Expiratório Forçado , Glucocorticoides/uso terapêutico , Humanos , Imunofenotipagem , Integrina alfa2/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/citologiaRESUMO
Solar ultraviolet (UV) radiation has hazardous effects on human health that are, in part, associated with its immunosuppressive effects via the induction of interleukin (IL)-10 production. Although IL-10 is produced by both T helper type 2 (Th2) cells and T-regulatory type 1 (Tr1) cells, the relative contribution of either subset in UV radiation-induced immunosuppression has not been established. Here, we show that T cells isolated from non-treated allergic contact dermatitis (ACD) reactions, 48 h following nickel challenge and propagated for 7-10 days in the presence of IL-2, were mainly CD4(+) and produced IL-10, but little interferon-gamma. A single sub-erythematous solar-simulated radiation (SSR) prior to antigen challenge exposure resulted in a clinical attenuation of the intensity of ACD reactions which was associated with a significant increase in both the magnitude of IL-10 production by skin-infiltrating T cells and the frequency of IL-10-producing Tr1 cells. Skin-infiltrating T cells in SSR-exposed, as well as non-exposed, ACD reactions showed a perturbed T-cell receptor (TCR)-Vbeta repertoire, without overexpression of a particular TCR-Vbeta gene product, indicating the presence of high frequencies of nickel non-specific T cells in ACD reactions. These results show that a single sub-erythematous SSR induces immunosuppression via the cutaneous infiltration of IL-10-producing Tr1, and to a lesser extent, Th2 cells.
Assuntos
Dermatite de Contato/metabolismo , Eritema/metabolismo , Interleucina-10/biossíntese , Luz Solar , Linfócitos T/metabolismo , Raios Ultravioleta , Adulto , Linfócitos T CD4-Positivos/metabolismo , Humanos , Interferon gama/metabolismo , Pessoa de Meia-Idade , Níquel , Radiometria , Células Th2 , Fatores de TempoRESUMO
IL-21 is a cytokine produced by CD4+ T cells that has been reported to regulate human, as well as, mouse T and NK cell function and to inhibit Ag-induced IgE production by mouse B cells. In the present study, we show that human rIL-21 strongly enhances IgE production by both CD19+ CD27- naive, and CD19+ CD27+ memory B cells, stimulated with anti-CD40 mAb and rIL-4 and that it promotes the proliferative responses of these cells. However, rIL-21 does not significantly affect anti-CD40 mAb and rIL-4-induced Cepsilon promoter activation in a gene reporter assay, nor germline Cepsilon mRNA expression in purified human spleen or peripheral blood B cells. In contrast, rIL-21 inhibits rIL-4-induced IgE production in cultures of PBMC or total splenocytes by an IFN-gamma-dependent mechanism. The presence of a polymorphism (T-83C), in donors heterozygous for this mutation was found to be associated not only with lower rIL-21-induced IFN-gamma production levels, but also with a lower sensitivity to the inhibitory effects of IL-21 on the production of IgE, compared with those in donors expressing the wild-type IL-21R. Taken together, these results show that IL-21 differentially regulates IL-4-induced human IgE production, via its growth- and differentiation-promoting capacities on isotype-, including IgE-, committed B cells, as well as via its ability to induce IFN-gamma production, most likely by T and NK cells, whereas the outcome of these IL-21-mediated effects is dependent on the presence of a polymorphism in the IL-21R.
Assuntos
Imunoglobulina E/biossíntese , Interferon gama/fisiologia , Interleucinas/fisiologia , Polimorfismo Genético , Receptores de Interleucina-21/genética , Linfócitos B/citologia , Linfócitos B/imunologia , Proliferação de Células , Células Cultivadas , Heterozigoto , Humanos , Memória Imunológica , Interferon gama/biossíntese , Interleucina-4/farmacologia , Leucócitos/citologia , Ativação Linfocitária/imunologia , Baço/citologiaRESUMO
Human somatic cells have a limited life span in vitro. Upon aging and with each cell division, shortening of telomeres occurs, which eventually will lead to cell cycle arrest. Ectopic hTERT expression has been shown to extend the life span of human T cells by preventing this telomere erosion. In the present study, we have shown that ectopic hTERT expression extends the life span of CD4+ T helper type 1 or 2 and regulatory T-cell clones and affected neither the in vitro cytokine production profile nor their specificity for antigen. In mixed cell cultures, ectopic hTERT-expressing clones were found to expand in greater numbers than untransduced cells of the same replicative age. This ectopic hTERT-induced growth advantage was not due to an enhanced cell division rate or number of divisions following T-cell receptor-mediated activation, as determined in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling experiments. Moreover, the susceptibility to activation-induced cell death of both cell types was similar. However, cultures of resting hTERT-transduced T cells contained higher frequencies of Bcl-2-expressing cells and lower active caspase-3-expressing cells, compared with wild-type cells. Furthermore, hTERT-transduced cells were more resistant to oxidative stress, which causes preferential DNA damage in telomeres. Taken together, these results show that ectopic hTERT expression not only protects proliferating T cells from replicative senescence but also confers resistance to apoptosis induced by oxidative stress.