RESUMO
Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.
Assuntos
Doenças Autoimunes , Células Th17 , Humanos , Diferenciação Celular , Fenótipo , Receptores CCR6/genética , Células Th1/metabolismoRESUMO
Th17 cells, which express the chemokine receptor CCR6, are implicated in many immune-mediated disorders, such as psoriasis and multiple sclerosis. We found that expression levels of CCR6 on human effector/memory CD4(+) T cells reflect a continuum of Th17 differentiation. By evaluating the transcriptome in cells with increasing CCR6, we detected progressive upregulation of ZBTB16, which encodes the broad complex, tramtrack, bric-à-brac-zinc finger transcription factor promyelocytic leukemia zinc finger protein (PLZF). Using chromatin immunoprecipitation for modified histones, p300, and PLZF, we identified enhancer-like sites at -9/-10 and -13/-14 kb from the upstream transcription start site of CCR6 that bind PLZF in CCR6(+) cells. For Th cells from adult blood, both in the CCR6(+) memory population and in naive cells activated ex vivo, knockdown of ZBTB16 downregulated CCR6 and other Th17-associated genes. ZBTB16 and RORC (which encodes the "master regulator" RORγt) cross-regulate each other, and PLZF binds at the RORC promoter in CCR6(+) cells. In naive Th cells from cord blood, ZBTB16 expression was confined to CD161(+) cells, which are Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells, and Th17 cells could be made from luxoid mice, which harbor an inactivating mutation in Zbtb16. These studies demonstrate a role for PLZF as an activator of transcription important both for Th17 differentiation and the maintenance of the Th17 phenotype in human cells, expand the role of PLZF as a critical regulator in the human adaptive immune system, and identify a novel, essential element in a regulatory network that is of significant therapeutic interest.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Fenótipo , Receptores CCR6/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Elementos Facilitadores Genéticos , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Memória Imunológica , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Transgênicos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Ligação Proteica , Receptores CCR6/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th17/citologiaRESUMO
It was reported that host defense against pulmonary Klebsiella pneumoniae infection requires IL-22, which was proposed to be of T cell origin. Supporting a role for IL-22, we found that Il22(-/-) mice had decreased survival compared with wild-type mice after intratracheal infection with K. pneumoniae. Surprisingly, however, Rag2(-/-) mice did not differ from wild-type mice in survival or levels of IL-22 in the lungs postinfection with K. pneumoniae. In contrast, K. pneumoniae-infected Rag2(-/-)Il2rg(-/-) mice failed to produce IL-22. These data suggested a possible role for NK cells or other innate lymphoid cells in host defense and production of IL-22. Unlike NK cell-like innate lymphoid cells that produce IL-22 and display a surface phenotype of NK1.1(-)NKp46(+)CCR6(+), lung NK cells showed the conventional phenotype, NK1.1(+)NKp46(+)CCR6(-). Mice depleted of NK cells using anti-asialo GM1 showed decreased survival and higher lung bacterial counts, as well as increased dissemination of K. pneumoniae to blood and liver, compared with control-treated mice. NK cell depletion also led to decreased production of IL-22 in the lung. Within 1 d postinfection, although there was no increase in the number of lung NK cells, a subset of lung NK cells became competent to produce IL-22, and such cells were found in both wild-type and Rag2(-/-) mice. Our data suggest that, during pulmonary infection of mice with K. pneumoniae, conventional NK cells are required for optimal host defense, which includes the production of IL-22.
Assuntos
Interleucinas/metabolismo , Células Matadoras Naturais/imunologia , Infecções por Klebsiella/imunologia , Pneumonia/imunologia , Animais , Antígenos Ly/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Interleucinas/biossíntese , Interleucinas/genética , Infecções por Klebsiella/mortalidade , Klebsiella pneumoniae/imunologia , Pulmão/citologia , Pulmão/imunologia , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Pneumonia/mortalidade , Receptores CCR6/metabolismo , Sobrevida , Interleucina 22RESUMO
CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Receptores de Quimiocinas/química , Receptores Virais/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Células Cultivadas , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Células Jurkat , Modelos Moleculares , Mutagênese , Receptores CXCR6 , Receptores de Quimiocinas/fisiologia , Receptores Virais/fisiologia , Relação Estrutura-AtividadeRESUMO
Pro-inflammatory T cells co-express multiple chemokine receptors, but the distinct functions of individual receptors on these cells are largely unknown. Human Th17 cells uniformly express the chemokine receptor CCR6, and we discovered that the subgroup of CD4+CCR6+ cells that co-express CCR2 possess a pathogenic Th17 signature, can produce inflammatory cytokines independent of TCR activation, and are unusually efficient at transendothelial migration (TEM). The ligand for CCR6, CCL20, was capable of binding to activated endothelial cells (ECs) and inducing firm arrest of CCR6+CCR2+ cells under conditions of flow - but CCR6 could not mediate TEM. By contrast, CCL2 and other ligands for CCR2, despite being secreted from both luminal and basal sides of ECs, failed to bind to the EC surfaces - and CCR2 could not mediate arrest. Nonetheless, CCR2 was required for TEM. To understand if CCR2's inability to mediate arrest was due solely to an absence of EC-bound ligands, we generated a CCL2-CXCL9 chimeric chemokine that could bind to the EC surface. Although display of CCL2 on the ECs did indeed lead to CCR2-mediated arrest of CCR6+CCR2+ cells, activating CCR2 with surface-bound CCL2 blocked TEM. We conclude that mediating arrest and TEM are mutually exclusive activities of chemokine receptors and/or their ligands that depend, respectively, on chemokines that bind to the EC luminal surfaces versus non-binding chemokines that form transendothelial gradients under conditions of flow. Our findings provide fundamental insights into mechanisms of lymphocyte extravasation and may lead to novel strategies to block or enhance their migration into tissue.
RESUMO
Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related (type 17) cells and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo . By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The CCR6 + cells' phenotypes and epigenomes are stable across cell divisions under homeostatic conditions. Nonetheless, activation in polarizing and non-polarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the continuum to yield the unusual plasticity ascribed to type 17 cells.
RESUMO
Because T cells act primarily through short-distance interactions, homing receptors can identify colocalizing cells that serve common functions. Expression patterns for multiple chemokine receptors on CD4(+) T cells from human blood suggested a hierarchy of receptors that are induced and accumulate during effector/memory cell differentiation. We characterized CD4(+)CD45RO(+) T cells based on expression of two of these receptors, CCR5 and CCR2, the principal subsets being CCR5(-)CCR2(-) (â¼70%), CCR5(+)CCR2(-) (â¼25%), and CCR5(+)CCR2(+) (â¼5%). Relationships among expression of CCR5 and CCR2 and CD62L, and the subsets' proliferation histories, suggested a pathway of progressive effector/memory differentiation from the CCR5(-)CCR2(-) to CCR5(+)CCR2(-) to CCR5(+)CCR2(+) cells. Sensitivity and rapidity of TCR-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The subsets also showed increasing responsiveness to IL-7, and the CCR5(+)CCR2(+) cells were CD127(bright) and invariably showed the greatest response to tetanus toxoid. CCR5(+)CCR2(+) cells also expressed the largest repertoire of chemokine receptors and migrated to the greatest number of chemokines. By contrast, the CCR5(+)CCR2(-) cells had the greatest percentages of regulatory T cells, activated/cycling cells, and CMV-reactive cells, and were most susceptible to apoptosis. Our results indicate that increasing memory cell differentiation can be uncoupled from susceptibility to death, and is associated with an increase in chemokine responsiveness, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells that are highly enriched in the CCR5(+)CCR2(+) subset and ideally equipped for rapid recall responses in tissue.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Memória Imunológica , Receptores CCR2/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Células Cultivadas , Humanos , Imunofenotipagem , Receptores CCR2/biossíntese , Receptores CCR5/biossíntese , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Fase de Repouso do Ciclo Celular/imunologia , Subpopulações de Linfócitos T/citologiaRESUMO
Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4(+) and CD8(+) T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα(o), a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4(+) T cells in vitro significantly increased the abundance of Gα(o) in cells stimulated under nonpolarizing or T(H)17 (but not T(H)1 or T(H)2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα(qo5), demonstrated that chemokine receptors could couple to Gα(o)-containing G proteins. We also found that Gα(i1), another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα(o) in memory (but not naive) and Gα(i1) in naive (but not memory) CD4(+) T cells inhibited chemokine-dependent migration. Moreover, although even in Gα(o)- and Gα(i1)-expressing cells mRNAs of these α-subunits were much less abundant than Gα(i2) or Gα(i3), knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα(i/o) subunits during T cell differentiation and suggest functional equivalence among Gα(i/o) subunits irrespective of their relative abundance.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Western Blotting , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Quimiocina CXCL12/metabolismo , Quimiotaxia/genética , Feminino , Sangue Fetal/citologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Recém-Nascido , Células Jurkat , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Gravidez , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR6/genética , Receptores CCR6/metabolismo , Receptores CXCR6 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3(+) and CCR4(+) cells do not express IFN-γ and/or IL-4, suggesting that the chemokine receptor genes might be inducible by mechanisms that are lineage-independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4(+) T cells by analyzing modifications of histone H3. In naïve cord-blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation-associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3(+) CCR4(-) and CXCR3(-) CCR4(+) CD4(+) T-cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high-level lineage-independent induction of CCR4 can occur following T-cell activation without accessibility-associated changes in histone H3, but that without such changes expression is transient rather than persistent.
Assuntos
Regulação da Expressão Gênica/imunologia , Histonas/imunologia , Ativação Linfocitária/imunologia , Receptores CCR4/imunologia , Células Th2/imunologia , Acetilação/efeitos dos fármacos , Butiratos/farmacologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , Interleucina-4/imunologia , Interleucina-4/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Metilação/efeitos dos fármacos , Receptores CCR4/biossíntese , Receptores CXCR3/biossíntese , Receptores CXCR3/imunologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Human MAIT cells show little expression of the selectin CD62L and the chemokine receptor CCR7, which are important for entering lymph nodes, and high expression of selectin ligands and chemokine receptors that mediate trafficking into inflamed tissue. Extravasation of leukocytes into tissue requires sequential steps including rolling, firm arrest, crawling, and transendothelial migration, and can be modeled using endothelial cell monolayers in flow chambers that approximate the sheer stress found in post-capillary venules. Using MAIT cells purified from elutriated lymphocytes by fluorescence-activated cell sorting, we have used flow chambers to demonstrate roles for individual chemokine receptors in specific steps required for extravasation. These methods provide a general way to study the molecular mechanisms underlying MAIT cell trafficking from blood into tissue.
Assuntos
Bioensaio , Movimento Celular/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Bioensaio/métodos , Biomarcadores , Movimento Celular/genética , Citometria de Fluxo , Humanos , Imunofenotipagem , Células T Invariantes Associadas à Mucosa/enzimologiaRESUMO
Many mediators and regulators of extravasation by bona fide human memory-phenotype T cells remain undefined. Mucosal-associated invariant T (MAIT) cells are innate-like, antibacterial cells that we found excelled at crossing inflamed endothelium. They displayed abundant selectin ligands, with high expression of FUT7 and ST3GAL4, and expressed CCR6, CCR5, and CCR2, which played non-redundant roles in trafficking on activated endothelial cells. MAIT cells selectively expressed CCAAT/enhancer-binding protein delta (C/EBPδ). Knockdown of C/EBPδ diminished expression of FUT7, ST3GAL4 and CCR6, decreasing MAIT cell rolling and arrest, and consequently the cells' ability to cross an endothelial monolayer in vitro and extravasate in mice. Nonetheless, knockdown of C/EBPδ did not affect CCR2, which was important for the step of transendothelial migration. Thus, MAIT cells demonstrate a program for extravasastion that includes, in part, C/EBPδ and C/EBPδ-regulated genes, and that could be used to enhance, or targeted to inhibit T cell recruitment into inflamed tissue.
Assuntos
Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Comunicação Celular , Células Endoteliais/fisiologia , Células T Invariantes Associadas à Mucosa/fisiologia , Animais , Movimento Celular , Células Cultivadas , Citometria de Fluxo , Fucosiltransferases/biossíntese , Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Receptores CCR2/biossíntese , Receptores CCR6/biossíntese , Sialiltransferases/biossíntese , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
[This corrects the article DOI: 10.18632/oncotarget.19945.].
RESUMO
Expression of the chemokine receptor CXCR4 by many cancers correlates with aggressive clinical behavior. As part of the initial studies in a project whose goal was to quantify CXCR4 expression on cancers non-invasively, we examined CXCR4 expression in cancer samples by immunohistochemistry using a validated anti-CXCR4 antibody. Among solid tumors, we found expression of CXCR4 on significant percentages of major types of kidney, lung, and pancreatic adenocarcinomas, and, notably, on metastases of clear cell renal cell carcinoma and squamous cell carcinoma of the lung. We found particularly high expression of CXCR4 on adrenocortical cancer (ACC) metastases. Microarrays of ACC metastases revealed correlations between expression of CXCR4 and other chemokine system genes, particularly CXCR7/ACKR3, which encodes an atypical chemokine receptor that shares a ligand, CXCL12, with CXCR4. A first-in-human study using 64Cu-plerixafor for PET in an ACC patient prior to resection of metastases showed heterogeneity among metastatic nodules and good correlations among PET SUVs, CXCR4 staining, and CXCR4 mRNA. Additionally, we were able to show that CXCR4 expression correlated with the rates of growth of the pulmonary lesions in this patient. Further studies are needed to understand better the role of CXCR4 in ACC and whether targeting it may be beneficial. In this regard, non-invasive methods for assessing CXCR4 expression, such as PET using 64Cu-plerixafor, should be important investigative tools.
RESUMO
Lymphocyte activation leads to changes in chemokine receptor expression. There are limited data, however, on how lymphocyte activators can alter chemokine signaling by affecting downstream pathways. We hypothesized that B cell-activating agents might alter chemokine responses by affecting downstream signal transducers, and that such effects might differ depending on the activator. We found that activating mouse B cells using either anti-IgM or lipopolysaccharide (LPS) increased the surface expression of CCR6 and CCR7 with large increases in chemotaxis to their cognate ligands. By contrast, while anti-IgM also led to enhanced calcium responses, LPS-treated cells showed only small changes in calcium signaling as compared with cells that were freshly isolated. Of particular interest, we found that LPS caused a reduction in the level of B-cell phospholipase C (PLC)-ß2 mRNA and protein. Data obtained using PLC-ß2(-/-) mice showed that the ß2 isoform mediates close to one-half the chemokine-induced calcium signal in resting and anti-IgM-activated B cells, and we found that calcium signals in the LPS-treated cells were boosted by increasing the level of PLC-ß2 using transfection, consistent with a functional effect of downregulating PLC-ß2. Together, our results show activator-specific effects on responses through B-cell chemokine receptors that are mediated by quantitative changes in a downstream signal-transducing protein, revealing an activity for LPS as a downregulator of PLC-ß2, and a novel mechanism for controlling chemokine-induced signals in lymphocytes.
Assuntos
Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Regulação para Baixo/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fosfolipase C beta/metabolismo , Receptores de Quimiocinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Quimiocinas/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Imunoglobulina M/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Modelos Imunológicos , Fosfolipase C beta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Fosfolipases Tipo C/metabolismoRESUMO
Psoriasis is a common immune-mediated chronic inflammatory skin disorder, but the mechanisms of pathogenesis are still poorly understood. IL-23 is expressed in psoriatic skin, and IL-23 injection produces IL-22-dependent psoriasiform changes in mouse skin. Th17 cells produce IL-22 and display CCR6, the CCL20 receptor; CCR6+ T cells and CCL20 are abundant in psoriatic skin. We investigated a possible role for CCR6 in recruiting Th17 cells and producing psoriasiform pathology by injecting IL-23 into the skin of WT and Ccr6-/- mice. Unlike for WT mice, IL-23-injected ears of Ccr6-/- mice showed neither substantial epidermal/dermal changes nor increased Il22 mRNA expression. However, injection of IL-22 yielded equivalent psoriasiform changes in WT and Ccr6-/- mice. Surprisingly, IL-23-injected ears of WT and Ccr6-/- mice contained similar numbers of Th cells able to make IL-17A and/or IL-22. Furthermore, in ears of Rag1-/- mice, IL-23 initially induced skin changes and levels of Il22 mRNA that were indistinguishable from WT mice, revealing at least one non-T cell source for IL-22. We conclude that CCR6 is essential in a model of IL-23-induced, IL-22-mediated dermatitis, which develops in sequential T cell-independent and T cell-dependent phases. These findings reveal an expanded role for CCR6 in IL-23-related responses and identify CCR6 as a potential therapeutic target in psoriasis.
Assuntos
Interleucina-23/toxicidade , Psoríase/etiologia , Receptores CCR6/fisiologia , Animais , Linfócitos T CD4-Positivos/fisiologia , Células Dendríticas/fisiologia , Proteínas de Homeodomínio/fisiologia , Interleucina-17/biossíntese , Interleucinas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
Some pathways of T cell differentiation are associated with characteristic patterns of chemokine receptor expression. A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL-17 cytokines and whose differentiation requires the nuclear receptor, RORgammat. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL-17 production for human T cells. Activating cord blood (naive) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9, and CXCR6. Despite these data, we found no strong correlation between the production of IL-17 and expression of CCR9 or CXCR6. By contrast, our analyses revealed that virtually all IL-17-producing CD4+ T cells, either made in our in vitro cultures or found in peripheral blood, expressed CCR6, a receptor found on approximately 50% of CD4+ memory PBL. Compared with CD4+CD45RO+CCR6- cells, CD4+CD45RO+CCR6+ cells contained at least 100-fold more IL-17A mRNA and secreted 100-fold more IL-17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORgammat. CCR6 was likewise expressed on all IL-17-producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL-17-producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL-17-related immune responses and possible targets for preventing inflammatory injury.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-17/metabolismo , Receptores CCR6/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/citologia , Linhagem da Célula , Células Cultivadas , Sangue Fetal/imunologia , Humanos , Memória Imunológica , Interleucina-17/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Receptores CCR/metabolismo , Receptores CXCR6 , Receptores de Quimiocinas/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Receptores Virais/metabolismo , Subpopulações de Linfócitos T/citologia , Regulação para CimaRESUMO
The pathways for differentiation of human CD4(+) T cells into functionally distinct subsets of memory cells in vivo are unknown. The identification of these subsets and pathways has clear implications for the design of vaccines and immune-targeted therapies. Here, we show that populations of apparently naive CD4(+) T cells express the chemokine receptors CXCR3 or CCR4 and demonstrate patterns of gene expression and functional responses characteristic of memory cells. The proliferation history and T cell receptor repertoire of these chemokine-receptor(+) cells suggest that they are very early memory CD4(+) T cells that have "rested down" before acquiring the phenotypes described for "central" or "effector" memory T cells. In addition, the chemokine-receptor(+) "naive" populations contain Th1 and Th2 cells, respectively, demonstrating that Th1/Th2 differentiation can occur very early in vivo in the absence of markers conventionally associated with memory cells. We localized ligands for CXCR3 and CCR4 to separate foci in T cell zones of tonsil, suggesting that the chemokine-receptor(+) subsets may be recruited and contribute to segregated, polarized microenvironments within lymphoid organs. Importantly, our data suggest that CD4(+) T cells do not differentiate according to a simple schema from naive --> CD45RO(+) noneffector/central memory --> effector/effector memory cells. Rather, developmental pathways branch early on to yield effector/memory populations that are highly heterogeneous and multifunctional and have the potential to become stable resting cells.
Assuntos
Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Expressão Gênica/imunologia , Memória Imunológica/imunologia , Subpopulações de Linfócitos T/citologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Hibridização In Situ , Ligantes , Tonsila Palatina/citologia , Receptores CCR4 , Receptores CXCR3 , Receptores de Quimiocinas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de TetradecanoilforbolRESUMO
The chemokine stromal-derived factor-1 (SDF-1), which is constitutively expressed in most tissues as SDF-1alpha and SDF-1beta resulting from alternative gene splicing, regulates hematopoiesis, lymphocyte homing, B-lineage cell growth, and angiogenesis. Because SDF-1alpha and SDF-1beta are constitutively and ubiquitously expressed, their degradation must serve an important regulatory role. Here we show that SDF-1alpha and SDF-1beta are secreted as full-length molecules. When exposed to human serum, full-length SDF-1alpha (1-68) undergoes processing first at the COOH terminus to produce SDF-1alpha 1-67 and then at the NH2 terminus to produce SDF-1alpha 3-67. By contrast, full-length SDF-1beta (1-72) is processed only at the NH2 terminus to produce SDF-1beta 3-72. CD26/dipeptidyl peptidase is responsible for serum cleavage of SDF-1alpha and SDF-1beta at the NH2 terminus. Serum processing of SDF-1alpha at the COOH terminus, which has not been previously reported, reduces the ability of the polypeptide to bind to heparin and to cells and to stimulate B-cell proliferation and chemotaxis. The additional processing at the NH2 terminus renders both forms of SDF-1 unable to bind to heparin and to activate cells. The differential processing of SDF-1alpha and SDF-1beta provides biologic significance to the existence of 2 splice forms of the chemokine and adds a tool to precisely regulate SDF-1's biologic activity by changes in specific activity.
Assuntos
Quimiocinas CXC/genética , Citocinas/genética , Regulação da Expressão Gênica/imunologia , Processamento Alternativo , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/sangue , Quimiocinas CXC/imunologia , Citocinas/sangue , Citocinas/imunologia , Endotélio Vascular/imunologia , Citometria de Fluxo , Variação Genética , Humanos , Cinética , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Deleção de Sequência , Espectrometria de Massas por Ionização por Electrospray , Veias UmbilicaisRESUMO
Monokine induced by IFN-gamma (Mig; CXC chemokine ligand 9) is an IFN-gamma-inducible CXC chemokine that signals through the receptor CXCR3 and is known to function as a chemotactic factor for human T cells, particularly following T cell activation. The mig gene can be induced in multiple cell types and organs, and Mig has been shown to contribute to T cell infiltration into immune/inflammatory reactions in peripheral tissues in mice. We have investigated the expression and activities of Mig and CXCR3 in mouse cells and the role of Mig in models of host defense in mice. Murine (Mu)Mig functioned as a chemotactic factor for resting memory and activated T cells, both CD4(+) and CD8(+), and responsiveness to MuMig correlated with surface expression of MuCXCR3. Using mig(-/-) mice, we found that MuMig was not necessary for survival after infections with a number of intracellular pathogens. Surprisingly, however, we found that mig(-/-) mice showed reductions of 50-75% in Abs produced against the intracellular bacterium Francisella tularensis live vaccine strain. Furthermore, we found that MuMig induced both calcium signals and chemotaxis in activated B cells, and that B cell activation induced expression of MuCXCR3. In addition, IFN-gamma induced the expression of mumig in APCs, including CD8 alpha(+) and CD8 alpha(-) dendritic cells. Together, our data suggest that Mig and CXCR3 may be important not only to recruit T cells to peripheral inflammatory sites, but also in some cases to maximize interactions among activated T cells, B cells, and dendritic cells within lymphoid organs to provide optimal humoral responses to pathogens.