RESUMO
The infiltration of chronic lymphocytic leukemia (CLL) cells into lymphoid organs correlates with disease severity. CXCL12 is a key chemotactic factor for the trafficking of CLL. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor and plays a role in CXCL12-mediated hematopoietic stem cell homing. We aim to explore the role of TFPI in CXCL12-mediated migration of CLL cells. In this study, plasma TFPI concentrations were measured by ELISA. CLL cells were isolated from patients and used for trans-endothelial migration (TEM) assays. Quantitative RT-PCR and Western blotting were used to detect the expression of CXCR7, CXCR4 and ß-catenin. Immunofluorescence and co-immunoprecipitation was used to detect the binding of TFPI and glypican-3 (GPC3). We found that plasma TFPI levels in CLL patients were higher than in healthy controls, particularly in the patients with advanced disease. TFPI enhanced CXCL12-mediated TEM of CLL cells by increasing the expression of the CXCL12 receptor CXCR7, but not of the CXCL12 receptor CXCR4. The effect of TFPI on TEM was abolished by the CXCR7 inhibitor, CCX771, while the CXCR4 inhibitor AMD3100 strongly increased TEM. TFPI co-localized with GPC3 on the cell surface. An antibody to GPC3, HS20, decreased CXCR7 expression and abolished the effect of TFPI on TEM. TFPI activated ß-catenin and the Wnt/ß-catenin inhibitor IWP4 repressed the effect of TFPI on CXCR7 expression and TEM. We conclude that TFPI may contribute to organ infiltration in CLL patients.
Assuntos
Quimiocina CXCL12/genética , Leucemia Linfocítica Crônica de Células B/sangue , Lipoproteínas/sangue , Receptores CXCR/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Leucêmica da Expressão Gênica/genética , Glipicanas/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Receptores CXCR4/genética , Transdução de Sinais/genética , beta Catenina/genéticaRESUMO
Enhanced blood vessel (BV) formation is thought to drive tumor growth through elevated nutrient delivery. However, this observation has overlooked potential roles for mural cells in directly affecting tumor growth independent of BV function. Here we provide clinical data correlating high percentages of mural-ß3-integrin-negative tumor BVs with increased tumor sizes but no effect on BV numbers. Mural-ß3-integrin loss also enhances tumor growth in implanted and autochthonous mouse tumor models with no detectable effects on BV numbers or function. At a molecular level, mural-cell ß3-integrin loss enhances signaling via FAK-p-HGFR-p-Akt-p-p65, driving CXCL1, CCL2, and TIMP-1 production. In particular, mural-cell-derived CCL2 stimulates tumor cell MEK1-ERK1/2-ROCK2-dependent signaling and enhances tumor cell survival and tumor growth. Overall, our data indicate that mural cells can control tumor growth via paracrine signals regulated by ß3-integrin, providing a previously unrecognized mechanism of cancer growth control.
Assuntos
Integrina beta3/metabolismo , Neoplasias/metabolismo , Carga Tumoral/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Feminino , Humanos , Masculino , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
Emerging evidence suggests that cancer cell metabolism can be regulated by cancer-associated fibroblasts (CAFs), but the mechanisms are poorly defined. Here we show that CAFs regulate malignant cell metabolism through pathways under the control of FAK. In breast and pancreatic cancer patients we find that low FAK expression, specifically in the stromal compartment, predicts reduced overall survival. In mice, depletion of FAK in a subpopulation of CAFs regulates paracrine signals that increase malignant cell glycolysis and tumour growth. Proteomic and phosphoproteomic analysis in our mouse model identifies metabolic alterations which are reflected at the transcriptomic level in patients with low stromal FAK. Mechanistically we demonstrate that FAK-depletion in CAFs increases chemokine production, which via CCR1/CCR2 on cancer cells, activate protein kinase A, leading to enhanced malignant cell glycolysis. Our data uncover mechanisms whereby stromal fibroblasts regulate cancer cell metabolism independent of genetic mutations in cancer cells.
Assuntos
Fibroblastos Associados a Câncer/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Quimiocinas/metabolismo , Feminino , Glicólise , Humanos , Masculino , Redes e Vias Metabólicas , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosfoproteínas/metabolismo , Células Estromais/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-associated macrophages (TAMs) recruited from blood monocytes are key in establishing an immunosuppressive tumor microenvironment (TME) for the support of tumor growth. We hypothesize that blocking monocyte trafficking (through the inhibition of specific chemokine receptors) into skin can positively affect tumor development. Herein, the authors examined the effects of oral administration of a small molecule inhibitor for CCR2, CCR2i, which blocks CCR2-mediated chemotaxis of monocytes in a syngeneic mouse T-cell lymphoma in skin. Following CCR2i administration, the depletion of macrophages was achieved as early as 2 days after tumor initiation in ear TME. Quantitative real-time PCR detected an increase in the levels of immune stimulatory inflammatory cytokines, for example, IFN-γ and IL-12, in CCR2i- versus vehicle-treated mice. Within 2 weeks, the tumors from the control groups attained the maximum size, whereas CCR2i-treated mice exhibited much smaller tumor sizes and weights. Immunohistochemistry revealed that CCR2i-treated tumors possessed considerably more CD8+ T cells, which demonstrated their essential role in CCR2i-induced tumor inhibition. Finally, the combination of anti-programmed cell death protein 1 with CCR2i considerably increased the efficacy of tumor eradication related to the activation of IFN-γ-producing CD8 T cells. Our findings provide strong evidence that the CCR2i, particularly in combination with an immune checkpoint inhibitor, reduces tumor growth and is a potential future treatment option for cutaneous T-cell lymphomas.
Assuntos
Linfoma Cutâneo de Células T/metabolismo , Macrófagos/metabolismo , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores CCR2/antagonistas & inibidores , Microambiente Tumoral , Administração Oral , Animais , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Inflamação , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Neoplasias/metabolismo , Neutrófilos/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores CCR2/metabolismoRESUMO
Immunotherapy directed at the PD-L1/PD-1 axis has produced treatment advances in various human cancers. Unfortunately, progress has not extended to glioblastoma (GBM), with phase III clinical trials assessing anti-PD-1 monotherapy failing to show efficacy in newly diagnosed and recurrent tumors. Myeloid-derived suppressor cells (MDSCs), a subset of immunosuppressive myeloid derived cells, are known to infiltrate the tumor microenvironment of GBM. Growing evidence suggests the CCL2-CCR2 axis is important for this process. This study evaluated the combination of PD-1 blockade and CCR2 inhibition in anti-PD-1-resistant gliomas. CCR2 deficiency unmasked an anti-PD-1 survival benefit in KR158 glioma-bearing mice. CD11b+/Ly6Chi/PD-L1+ MDSCs within established gliomas decreased with a concomitant increase in overall CCR2+ cells and MDSCs within bone marrow of CCR2-deficient mice. The CCR2 antagonist CCX872 increased median survival as a monotherapy in KR158 glioma-bearing animals and further increased median and overall survival when combined with anti-PD-1. Additionally, combination of CCX872 and anti-PD-1 prolonged median survival time in 005 GSC GBM-bearing mice. In both models, CCX872 decreased tumor associated MDSCs and increased these cells within the bone marrow. Examination of tumor-infiltrating lymphocytes revealed an elevated population, increased IFNγ expression, indicating enhanced cytolytic activity, as well as decreased expression of exhaustion markers in CD4+ and CD8+ T cells following combination treatment. These data establish that combining CCR2 and PD-1 blockade extends survival in clinically relevant murine glioma models and provides the basis on which to advance this combinatorial treatment toward early-phase human trials.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Glioma/tratamento farmacológico , Células Mieloides/metabolismo , Receptores CCR2/efeitos dos fármacos , Receptores CCR2/metabolismo , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CCL2 , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioma/patologia , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Células Supressoras Mieloides/metabolismo , Receptor de Morte Celular Programada 1 , Receptores CCR2/genética , Análise de Sobrevida , Microambiente Tumoral/efeitos dos fármacosRESUMO
Neuroinflammation role on epileptogenesis has been the subject of increasing interest. Many studies showed elevation in cytokines and chemokines expression following seizures, such as, CCL2 protein (C-C motif ligand 2 chemokine) and its specific receptor, CCR2. In addition, recent studies manipulating the CCL2/CCR2 complex verified improved seizure outcome in different seizure models. In the present study, the effects of CCR2 antagonist was investigated using the pilocarpine rat model of epilepsy. Status epilepticus (SE) was induced by pilocarpine i.p. injection in adult rats. Daily oral treatment with CCR2 antagonist or vehicle was initiated 5â¯h following SE and lasted 5 or 10â¯days. Rats were euthanized 5â¯days after SE to evaluate neuronal damage and glial density or 30â¯days after SE to investigate spontaneous seizures development and seizure susceptibility to a second hit pentylenetetrazol (PTZ) test. Rats that received CCR2 antagonist presented less degenerating cells at hippocampal CA1 region. There was also a significant decrease in CA1 volume after SE that was not observed in treated rats. On the other hand, microglia cell density increased after SE regardless of CCR2 antagonist use. Treatment with CCR2 antagonist did not alter spontaneous seizure occurrence or later seizure susceptibility to PTZ in chronic rats. Additional rats were pretreated with CCR2 antagonist prior to SE induction, but this did not change SE progression. The data show that oral treatment with CCR2 antagonist is neuroprotective, but does not alter other epileptogenic factors, such as, neuroinflammation, or seizure development, after pilocarpine-induced SE in rats.
Assuntos
Quimiocina CCL2/antagonistas & inibidores , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Epilepsia/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Pilocarpina/toxicidade , Animais , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/metabolismo , Quimiocina CCL2/metabolismo , Epilepsia/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.
Assuntos
Quimiocina CCL2/biossíntese , Receptores CCR2/metabolismo , Animais , Biomarcadores , Linhagem Celular , Quimiocina CCL2/sangue , Quimiocina CCL2/genética , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica , Humanos , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Receptores CCR2/antagonistas & inibidoresRESUMO
Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare, but severe, psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of preactivated IL-36α. We found the immune cells infiltrating IL-36α-injected mouse skin to be of dramatically different composition than those infiltrating imiquimod-treated skin. The IL-36α-induced leukocyte population comprised nearly equal numbers of CD4+ αß T cells, neutrophils, and inflammatory dendritic cells, whereas the imiquimod-induced population comprised γδ T cells and neutrophils. Ligands for chemokine receptors CCR6 and CXCR2 are increased in both GPP and IL-36α-treated skin, which led us to test an optimized small-molecule antagonist (CCX624) targeting CCR6 and CXCR2 in the IL-36α model. CCX624 significantly reduced the T cell, neutrophil, and inflammatory dendritic cell infiltrates and was more effective than saturating levels of an anti-IL-17RA mAb at reducing inflammatory symptoms. These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.
Assuntos
Anti-Inflamatórios/farmacologia , Interleucina-1/toxicidade , Psoríase/imunologia , Receptores CCR6/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Animais , Interleucina-1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Psoríase/induzido quimicamente , Pele/efeitos dos fármacos , Pele/imunologiaRESUMO
Focal segmental glomerulosclerosis (FSGS) comprises a group of uncommon disorders that present with marked proteinuria, nephrotic syndrome, progressive renal failure and characteristic glomerular lesions on histopathology. The current standard of care for patients with FSGS include immunosuppressive drugs such as glucocorticoids followed by calcineurin inhibitors, if needed for intolerance or inadequate response to glucocorticoids. Renin-angiotensin-aldosterone (RAAS) blockers are also used to control proteinuria, an important signature of FSGS. Existing treatments, however, achieved only limited success. Despite best care, treatment failure is common and FSGS is causal in a significant proportion of end stage renal disease. Thus, an unmet need exists for novel disease modifying treatments for FSGS. We employed two widely-used murine models of FSGS to test the hypothesis that systemic inhibition of chemokine receptor CCR2 would have therapeutic benefit. Here we report that administration CCX872, a potent and selective small molecule antagonist of CCR2, achieved rapid and sustained attenuation of renal damage as determined by urine albumin excretion and improved histopathological outcome. Therapeutic benefit was present when CCX872 was used as a single therapy, and moreover, the combination of CCX872 and RAAS blockade was statistically more effective than RAAS blockade alone. In addition, the combination of CCR2 and RAAS blockade was equally as effective as endothelin receptor inhibition. We conclude that specific inhibition of CCR2 is effective in the Adriamycin-induced and 5/6 nephrectomy murine models of FSGS, and thus holds promise as a mechanistically distinct therapeutic addition to the treatment of human FSGS.
Assuntos
Albuminúria , Glomerulosclerose Segmentar e Focal , Glomérulos Renais , Receptores CCR2/antagonistas & inibidores , Sistema Renina-Angiotensina/efeitos dos fármacos , Albuminúria/tratamento farmacológico , Albuminúria/patologia , Albuminúria/urina , Animais , Linhagem Celular , Modelos Animais de Doenças , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/urina , Humanos , Glomérulos Renais/lesões , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR2/metabolismoRESUMO
Previous studies have shown that increased levels of chemokine receptor CXCR7 are associated with the increased invasiveness of prostate cancer cells. We now show that CXCR7 expression is upregulated in VCaP and C4-2B cells after enzalutamide (ENZ) treatment. ENZ treatment induced apoptosis (sub-G1) in VCaP and C4-2B cells, and this effect was further increased after combination treatment with ENZ and CCX771, a specific CXCR7 inhibitor. The levels of p-EGFR (Y1068), p-AKT (T308) and VEGFR2 were reduced after ENZ and CCX771 combination treatment compared to single agent treatment. In addition, significantly greater reductions in migration were shown after combination treatment compared to those of single agents or vehicle controls, and importantly, similar reductions in the levels of secreted VEGF were also demonstrated. Orthotopic VCaP xenograft growth and subcutaneous MDA133-4 patient-derived xenograft (PDX) tumor growth was reduced by single agent treatment, but significantly greater suppression was observed in the combination treatment group. Although overall microvessel densities in the tumor tissues were not different among the different treatment groups, a significant reduction in large blood vessels (>100 µm2 ) was observed in tumors following combination treatment. Apoptotic indices in tumor tissues were significantly increased following combination treatment compared with vehicle control-treated tumor tissues. Our results demonstrate that significant tumor suppression mediated by ENZ and CXCR7 combination treatment may be due, in part, to reductions in proangiogenic signaling and in the formation of large blood vessels in prostate cancer tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores CXCR/antagonistas & inibidores , Animais , Benzamidas , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Nitrilas , Feniltioidantoína/administração & dosagem , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/irrigação sanguínea , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores CXCR/biossíntese , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
mAbs that neutralize IL-17 or its receptor have proven efficacious in treating moderate-to-severe psoriasis, confirming IL-17 as an important driver of this disease. In mice, a rare population of T cells, γδT17 cells, appears to be a dominant source of IL-17 in experimental psoriasis. These cells traffic between lymph nodes and the skin, and are identified by their coexpression of the TCR variable regions γ4 and δ4. These cells are homologous to the Vγ9Vδ2 T cell population identified in human psoriatic plaques. In this study we report that a potent and specific small molecule antagonist of the CCR6 chemokine receptor, CCX2553, was efficacious in reducing multiple aspects of psoriasis in two different murine models of the disease. Administration of CCX2553 ameliorated skin inflammation in both the IL-23-induced ear swelling model and the topical imiquimod model, and significantly reduced the number of γδT17 cells in inflamed skin. γδT17 cells were greatly reduced in imiquimod-treated skin of CCR6-/- mice, but adoptively transferred wild-type (CCR6+/+) γδT17 cells homed normally to the skin of imiquimod-treated CCR6-/- mice. Our data suggest that γδT17 cells are completely dependent on CCR6 for homing to psoriasiform skin. Thus, CCR6 may constitute a novel target for a mechanistically distinct therapeutic approach to treating psoriasis.
Assuntos
Movimento Celular/imunologia , Interleucina-17/imunologia , Psoríase/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores CCR6/imunologia , Pele/imunologia , Linfócitos T/imunologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Interleucina-17/genética , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Psoríase/genética , Psoríase/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores CCR6/genética , Pele/patologia , Linfócitos T/patologiaRESUMO
While it has long been established that the chemokine receptor CCR9 and its ligand CCL25 are essential for the movement of leukocytes into the small intestine and the development of small-intestinal inflammation, the role of this chemokine-receptor pair in colonic inflammation is not clear. Toward this end, we compared colonic CCL25 protein levels in healthy individuals to those in patients with ulcerative colitis. In addition, we determined the effect of CCR9 pharmacological inhibition in the mdr1a(-/-) mouse model of ulcerative colitis. Colon samples from patients with ulcerative colitis had significantly higher levels of CCL25 protein compared to healthy controls, a finding mirrored in the mdr1a(-/-) mice. In the mdr1a(-/-) mice, CCR9 antagonists significantly decreased the extent of wasting and colonic remodeling and reduced the levels of inflammatory cytokines in the colon. These findings indicate that the CCR9:CCL25 pair plays a causative role in ulcerative colitis and suggest that CCR9 antagonists will provide a therapeutic benefit in patients with colonic inflammation.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Receptores CCR/antagonistas & inibidores , Receptores CCR/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colite Ulcerativa/genética , Feminino , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Sulfonamidas/uso terapêuticoRESUMO
Topical antigen application is a focus of current vaccine research. This immunization route mimics natural antigen exposure across a barrier tissue and generates T cells imprinted for skin-selective homing. Soluble antigens introduced through this route require cross-presentation by DC to generate CD8 T cell responses. Here we have explored the relative contribution of various skin-derived DC subsets to cross-priming and skin-selective imprinting. In our model, DC acquire soluble Ag in vivo from immunized murine skin for cross-presentation to naïve CD8 T cells ex vivo. We find CD11b(+) migratory DC to be the relevant cross-priming DC in this model. Both Langerin(+) and Langerin(-) CD11b(+) migratory DC can cross-present antigen in our system, but only the Langerin+ subset can induce expression of the skin-selective addressin E-selectin ligand. Thus, the CD11b(+) Langerin(+) migratory DC population, comprised primarily of Langerhans cells, both cross-primes naïve CD8 T cells and imprints them with skin-homing capabilities.
Assuntos
Apresentação de Antígeno/imunologia , Antígeno CD11b/metabolismo , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Pele/imunologia , Animais , Antígenos/imunologia , Antígenos de Superfície/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Quimiotaxia/imunologia , Expressão Gênica , Imunização , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Lectinas de Ligação a Manose/metabolismo , Camundongos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Pele/metabolismoRESUMO
Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical "redundancy." Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, ß-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or ß-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.
Assuntos
Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Arrestinas/metabolismo , Quimiocinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Modelos Biológicos , Transdução de Sinais , beta-ArrestinasRESUMO
CCR7â»/â» mice exhibit profound anomalies in lymph node and spleen architecture, which complicates the study of CCR7-mediated T cell trafficking in vivo. To circumvent this problem, we established in vivo models in which wild-type and CCR7â»/â» populations coexist within mice possessing normal lymphoid organs and must compete for developmental niches within the tissues of these mice. Under the conditions we have created in vivo, we find the entry of memory CD4 T cells into lymph nodes from the blood to be independent of CCR7. Thus, the central memory CD4 T cells that traffic though lymph nodes, which are often defined by their expression of CCR7, do not appear to gain any competitive homing advantage by expressing this receptor. Furthermore, in contrast to cutaneous dendritic cell populations, we found that CCR7 deficiency had no appreciable effect on the exit of CD4 T cells from inflamed skin. Finally, we found that wild-type and CCR7â»/â» precursors were equally represented within the major thymic subpopulations, despite previous findings that CCR7 plays a role in seeding the thymus from bone marrow-derived T cell precursors.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Memória Imunológica/imunologia , Receptores CCR7/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Separação Celular , Citometria de Fluxo , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores CCR7/metabolismoRESUMO
A goal for developers of immunomodulatory drugs has long been a systemically administered small molecule that can selectively inhibit inflammation in specific tissues. The chemokine receptor CCR9 is an attractive target for this approach, as entry of T cells into the small intestine from blood requires interaction between CCR9 and its ligand CCL25. We have tested the ability of a small molecule CCR9 antagonist, CCX8037, to inhibit antigen-mediated T cell accumulation in the intestine. This compound prevented accumulation of gut-imprinted antigen-specific CD8 T cells within epithelium of the small intestine. Interestingly, the antagonist did not affect the robust generation of gut-imprinted CD8 T cells within mesenteric lymph nodes. To distinguish "gut-selective" from "general" T cell inhibition, we tested the drug's ability to influence accumulation of T cells within skin, a tissue in which CCR9 plays no known role, and we found no appreciable effect. This study demonstrates the feasibility of creating systemically-administered pharmaceuticals capable of tissue-selective immune modulation. This proof of concept is of utmost importance for designing effective treatments against various autoimmune disorders localized to a specific tissue.
Assuntos
Fatores Imunológicos/farmacologia , Linfócitos/metabolismo , Receptores CCR/antagonistas & inibidores , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Quimiocinas CC/metabolismo , Feminino , Citometria de Fluxo , Humanos , Linfócitos/efeitos dos fármacos , Masculino , CamundongosRESUMO
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of neoplastic disorders characterized by clonally derived and skin-homing malignant T cells that express high level of chemokine receptor CCR4, which is associated with their skin-homing capacity. CCR4 is also highly expressed on T-regulatory cells (Tregs) that can migrate to several different types of chemotactic ligand CCL17- and CCL22-secreting tumors to facilitate tumor cell evasion from immune surveillance. Thus, its high-level expression on CTCL cells and Tregs makes CCR4 a potential ideal target for antibody-based immunotherapy for CTCL and other types of solid tumors. Here, we conducted humanization and affinity optimization of a murine anti-CCR4 monoclonal antibody (mAb), mAb1567, that recognizes both the N-terminal and extracellular domains of CCR4 with high affinity and inhibits chemotaxis of CCR4(+) CTCL cells. In a mouse CTCL tumor model, mAb1567 exhibited a potent antitumor effect and in vitro mechanistic studies showed that both complement-dependent cytotoxicity (CDC) and neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC) likely mediated this effect. mAb1567 also exerts human NK cell-mediated ADCC activity in vitro. Moreover, mAb1567 also effectively inhibits chemotaxis of CD4(+)CD25(high) Tregs via CCL22 and abrogates Treg suppression activity in vitro. An affinity-optimized variant of humanized mAb1567, mAb2-3, was selected for further preclinical development based on its higher binding affinity and more potent ADCC and CDC activities. Taken together, this high-affinity humanized mAb2-3 with potent antitumor effect and a broad range of mechanisms of action may provide a novel immunotherapy for CTCL and other solid tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Linfoma Cutâneo de Células T/tratamento farmacológico , Linfoma Cutâneo de Células T/patologia , Receptores CCR4/imunologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Clonagem Molecular , Proteínas do Sistema Complemento/imunologia , Humanos , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Ligação Proteica/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Inflamed skin contains CD4 T-cell subsets that express chemokine receptors CCR4, CCR6, and/or CCR10. Prior attempts to reveal the distinct role(s) of each receptor in T-cell trafficking to skin have not produced a coherent story. Different conclusions drawn by separate research groups are difficult to reconcile because of the disparate inflammation models used. Here we directly compare CD4 T cells from wild-type, CCR4(-/-), CCR6(-/-), and CCR10(-/-) mice in parallel assays of trafficking to skin. Our models require direct competition between wild-type and receptor-deficient populations for access to inflamed cutaneous sites. Major histocompatibility complex-peptide tetramers allowed us to identify antigen-specific endogenous long-term memory CD4 T cells within skin after multiple topical immunizations. We separately analyzed cells from the dermal and epidermal layers, allowing us to assess the involvement of each receptor in trafficking between dermis and epidermis. We found that CCR4 deficiency reduces accumulation of memory CD4 T cells in skin by approximately 20-fold, but neither CCR6 nor CCR10 deficiency yielded any detectable effects. Strikingly, no differences in dermal versus epidermal localization were observed for cells lacking any of these three receptors. Our findings raise the possibility that CCR6 and CCR10 play (as yet) unknown roles in cutaneous T-cell immunology, unrelated to skin-specific trafficking.
Assuntos
Movimento Celular/imunologia , Epiderme/patologia , Receptores de Quimiocinas/metabolismo , Linfócitos T/patologia , Animais , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/efeitos da radiação , Movimento Celular/efeitos da radiação , Derme/efeitos dos fármacos , Derme/patologia , Derme/efeitos da radiação , Epiderme/imunologia , Epiderme/efeitos da radiação , Epitopos/imunologia , Citometria de Fluxo , Antígenos de Histocompatibilidade/imunologia , Memória Imunológica/imunologia , Memória Imunológica/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Luz Solar , Linfócitos T/imunologia , Linfócitos T/efeitos da radiaçãoRESUMO
The mechanisms that underlie the critical dendritic cell (DC) function in maintainance of peripheral immune tolerance are incompletely understood, although the ß-catenin signaling pathway is critical for this role. The molecular details by which ß-catenin signaling is regulated in DCs are unknown. Mechanical disruption of murine bone marrow-derived DC (BMDC) clusters activates DCs while maintaining their tolerogenic potential and this activation is associated with ß-catenin signaling, providing a useful model with which to explore tolerance-associated ß-catenin signaling in DCs. In this report, we demonstrate novel molecular features of the signaling events that control DC activation in response to mechanical stimulation. Non-canonical ß-catenin signaling is an essential component of this tolerogenic activation and is modulated by adhesion molecules, including integrins. This unique ß-catenin-dependent signaling pathway is constitutively active at low levels, suggesting that mechanical stimulation is not necessarily required for induction of this unique activation program. We additionally find that the immunomodulatory cytokine TGF-ß antagonizes ß-catenin in DCs, thereby selectively suppressing signaling associated with tolerogenic DC activation while having no impact on LPS-induced, ß-catenin-independent immunogenic activation. These findings provide new molecular insight into the regulation of a critical signaling pathway for DC function in peripheral immune tolerance.
Assuntos
Células Dendríticas/citologia , Fatores de Crescimento Transformadores/fisiologia , beta Catenina/fisiologia , Animais , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Patients with atopic dermatitis (AD) often suffer from food allergy and develop flares upon skin contact with food allergens. However, it is unclear whether T cells sensitized to allergens in the gut promote this skin inflammation. To address this question, we orally immunized WT mice and mice lacking the skin-homing chemokine receptor Ccr4 (Ccr4-/- mice) with OVA and then challenged them epicutaneously with antigen. Allergic skin inflammation developed in the WT mice but not in the mutants and was characterized by epidermal thickening, dermal infiltration by eosinophils and CD4+ T cells, and upregulation of Th2 cytokines. T cells purified from mesenteric lymph nodes (MLNs) of orally immunized WT mice transferred allergic skin inflammation to naive recipients cutaneously challenged with antigen, but this effect was lost in T cells purified from Ccr4-/- mice. In addition, the ability of adoptively transferred OVA-activated T cells to home to the skin following cutaneous OVA challenge was ablated in mice that lacked lymph nodes. These results indicate that cutaneous exposure to food antigens can reprogram gut-homing effector T cells in LNs to express skin-homing receptors, eliciting skin lesions upon food allergen contact in orally sensitized AD patients.