RESUMEN
Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- ß, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-ß compared to CD71+VISTA- erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells-compared to CD71+VISTA- and CD71+ erythroid cells from the VISTA KO mice-significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-ß in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-ß1, TGF-ß2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond.
Asunto(s)
Células Eritroides/inmunología , Proteínas de la Membrana/fisiología , Receptores de Transferrina/fisiología , Animales , Antígenos CD/fisiología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/fisiología , Células Eritroides/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Activación de Linfocitos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Receptores de Superficie Celular , Receptores de Transferrina/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
PURPOSE: Acquired resistance to immune checkpoint blockers (ICBs) is a major barrier in cancer treatment, emphasizing the need for innovative strategies. Dectin-1 (gene Clec7a) is a C-type lectin receptor best known for its ability to recognize ß-glucan-rich structures in fungal cell walls. While Dectin-1 is expressed in myeloid cells and tumor cells, its significance in cancer remains the subject of controversy. METHODS: Using Celc7a-/- mice and curdlan administration to stimulate Dectin-1 signaling, we explored its impact. VISTA KO mice were employed to assess VISTA's role, and bulk RNAseq analyzed curdlan effects on neutrophils. RESULTS: Our findings reveal myeloid cells as primary Dectin-1 expressing cells in the tumor microenvironment (TME), displaying an activated phenotype. Strong Dectin-1 co-expression/co-localization with VISTA and PD-L1 in TME myeloid cells was observed. While Dectin-1 deletion lacked protective effects, curdlan stimulation significantly curtailed B16-F10 tumor progression. RNAseq and pathway analyses supported curdlan's role in triggering a cascade of events leading to increased production of pro-inflammatory mediators, potentially resulting in the recruitment and activation of immune cells. Moreover, we identified a heterogeneous subset of Dectin-1+ effector T cells in the TME. Similar to mice, human myeloid cells are the prominent cells expressing Dectin-1 in cancer patients. CONCLUSION: Our study proposes Dectin-1 as a potential adjunctive target with ICBs, orchestrating a comprehensive engagement of innate and adaptive immune responses in melanoma. This innovative approach holds promise for overcoming acquired resistance to ICBs in cancer treatment, offering avenues for further exploration and development.
RESUMEN
The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow-derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.
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Anticuerpos Monoclonales/uso terapéutico , Arteriosclerosis/tratamiento farmacológico , Artritis Experimental/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Fisiológica , Proteínas Gestacionales/farmacología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Arteriosclerosis/inmunología , Arteriosclerosis/patología , Artritis Experimental/patología , Vasos Sanguíneos/citología , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/patología , Modelos Animales de Enfermedad , Factores de Crecimiento Endotelial/farmacología , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/tratamiento farmacológico , Isquemia/patología , Articulaciones/patología , Linfocinas/farmacología , Ratones , Ratones Desnudos , Isquemia Miocárdica/tratamiento farmacológico , Isquemia Miocárdica/patología , Isquemia Miocárdica/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , Factor de Crecimiento Placentario , Proteínas Gestacionales/genética , Proteínas Gestacionales/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/inmunología , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: In addition to activated T cells, the immune checkpoint inhibitor "V domain-containing Ig suppressor of T-cell activation" (VISTA) is expressed by myeloid cell types, including macrophages and neutrophils. The importance of VISTA expression by myeloid cells to antibody-induced arthritis and its potential for relevance in human disease was evaluated. METHODS: VISTA was immunolocalized in normal and arthritic human synovial tissue sections and synovial tissue lysates were subjected to western blot analysis. The collagen antibody-induced arthritis model (CAIA) was performed with DBA/1 J mice treated with antibodies against VISTA and with VISTA-deficient mice (V-KO). Total mRNA from arthritic joints, spleens, and cultured macrophages was analyzed with NanoString arrays. Cytokines secreted by splenic inflammatory macrophages were determined. In-vitro chemotaxis and signal transduction assays were performed with cultured macrophages. RESULTS: VISTA protein was localized to synovial membrane cells, neutrophils, and scattered cells in lymphocyte-rich foci and was detected by western blot analysis in normal synovium and synovium from rheumatoid arthritis patients. Deficiency of VISTA or treatment of mice with anti-VISTA monoclonal antibodies attenuated CAIA. Joint damage and MMP-3 expression were significantly reduced in V-KO mice. Surface expression of C5a receptor was reduced on monocytes, neutrophils, and cultured macrophages from V-KO. Upon Fc receptor engagement in vitro, gene expression by V-KO macrophages was altered profoundly compared to WT, including a significant induction of IL-1 receptor antagonist (IL1rn). CONCLUSIONS: VISTA expression supports immune-complex inflammation in CAIA and VISTA is expressed in human synovium. VISTA supports optimal responses to C5a and modulates macrophage responses to immune complexes.
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Artritis Reumatoide/inmunología , Antígenos B7/inmunología , Regulación de la Expresión Génica/inmunología , Macrófagos/inmunología , Animales , Complejo Antígeno-Anticuerpo/inmunología , Artritis Experimental/inmunología , Antígenos B7/deficiencia , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Membrana Sinovial/inmunologíaAsunto(s)
Linfocitos B/inmunología , Quimiocina CXCL13/inmunología , Coristoma/patología , Aparato Lagrimal/patología , Tejido Linfoide/patología , Receptor beta de Linfotoxina/inmunología , Síndrome de Sjögren/inmunología , Animales , Quimiocina CXCL13/genética , Coristoma/inmunología , Homeostasis , Humanos , Aparato Lagrimal/inmunología , Tejido Linfoide/inmunología , Receptor beta de Linfotoxina/genética , Ratones , Ratones Endogámicos NOD , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , Síndrome de Sjögren/patologíaRESUMEN
Vascular endothelial growth factor (VEGF) is a regulator of vascularization in development and is a key growth factor in tissue repair. In disease, VEGF contributes to vascularization of solid tumors and arthritic joints. This study examines the role of the mRNA-binding protein AUF1/heterogeneous nuclear ribonucleoprotein D (AUF1) in VEGF gene expression. We show that overexpression of AUF1 in mouse macrophage-like RAW-264.7 cells suppresses endogenous VEGF protein levels. To study 3' untranslated region (UTR)-mediated regulation, we introduced the 3' UTR of VEGF mRNA into a luciferase reporter gene. Coexpression of AUF1 represses VEGF-3' UTR reporter expression in RAW-264.7 cells and in mouse bone marrow-derived macrophages. The C-terminus of AUF1 contains arginine-glycine-glycine (RGG) repeat motifs that are dimethylated. Deletion of the RGG domain of AUF1 eliminated the repressive effects of AUF1. Surprisingly, expression of an AUF1-RGG peptide reduced endogenous VEGF protein levels and repressed VEGF-3' UTR reporter activity in RAW-264.7 cells. These findings demonstrate that AUF1 regulates VEGF expression, and this study identifies an RGG peptide that suppresses VEGF gene expression.
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Regiones no Traducidas 3' , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Macrófagos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Células de la Médula Ósea , Regulación de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/biosíntesis , Metilación , Ratones , Péptidos/metabolismo , Unión Proteica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Eliminación de Secuencia , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
INTRODUCTION: In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome. METHODS: Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores. RESULTS: LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01). CONCLUSIONS: Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.
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Quimiocina CXCL13/metabolismo , Córnea/metabolismo , Aparato Lagrimal/metabolismo , Receptor beta de Linfotoxina/metabolismo , Síndrome de Sjögren/metabolismo , Adulto , Anciano , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Quimiocina CXCL13/genética , Córnea/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Femenino , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Queratoconjuntivitis Seca/tratamiento farmacológico , Queratoconjuntivitis Seca/genética , Queratoconjuntivitis Seca/metabolismo , Aparato Lagrimal/efectos de los fármacos , Receptor beta de Linfotoxina/antagonistas & inhibidores , Receptor beta de Linfotoxina/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Microscopía Fluorescente , Persona de Mediana Edad , Mucinas/genética , Mucinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Síndrome de Sjögren/tratamiento farmacológico , Síndrome de Sjögren/genética , Lágrimas/metabolismo , Vénulas/metabolismo , Vénulas/fisiologíaRESUMEN
The immunoglobulin (Ig) superfamily consists of many critical immune regulators, including the B7 family ligands and receptors. In this study, we identify a novel and structurally distinct Ig superfamily inhibitory ligand, whose extracellular domain bears homology to the B7 family ligand PD-L1. This molecule is designated V-domain Ig suppressor of T cell activation (VISTA). VISTA is primarily expressed on hematopoietic cells, and VISTA expression is highly regulated on myeloid antigen-presenting cells (APCs) and T cells. A soluble VISTA-Ig fusion protein or VISTA expression on APCs inhibits T cell proliferation and cytokine production in vitro. A VISTA-specific monoclonal antibody interferes with VISTA-induced suppression of T cell responses by VISTA-expressing APCs in vitro. Furthermore, anti-VISTA treatment exacerbates the development of the T cell-mediated autoimmune disease experimental autoimmune encephalomyelitis in mice. Finally, VISTA overexpression on tumor cells interferes with protective antitumor immunity in vivo in mice. These findings show that VISTA, a novel immunoregulatory molecule, has functional activities that are nonredundant with other Ig superfamily members and may play a role in the development of autoimmunity and immune surveillance in cancer.
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Antígenos B7/inmunología , Antígenos B7/fisiología , Inmunoglobulinas/inmunología , Linfocitos T/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/fisiología , Antígeno B7-1/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Citometría de Flujo , Regulación de la Expresión Génica , Inmunoglobulinas/fisiología , Ligandos , Activación de Linfocitos/inmunología , Activación de Linfocitos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
AIMS: Given the importance of IgG Fc receptors in immune regulation, we hypothesized that Fcg receptor type III (FcgRIII, CD16) plays an important role in atherogenesis. We therefore analysed the formation of arterial lesions in LDL receptor-deficient (LDLR(-/-)) and FcgRIII(-/-)xLDLR(-/-) double knockout mice at three different points up to 24 weeks of exposure to a high-fat diet. METHODS AND RESULTS: Analysis of Oil Red-O-stained sections revealed no difference in lesion formation between strains after 6 weeks of a high-fat diet, and a modest decrease after 14 weeks in double knockouts relative to LDLR(-/-) controls. After 24 weeks, lesion formation was decreased in the aortic root (30%) and innominate artery (50%) in FcgRIII double knockouts relative to LDLR(-/-) controls. Analysis of peripheral CD4+ T-cells by intracellular flow cytometry from double knockouts after 24 weeks of a high-fat diet revealed statistically significant increases in the percentages of cells producing interferon-gamma, interleukin (IL)-10, and IL-4 relative to controls, differences that were also observed by analyses of whole aortas for cytokine mRNA levels. As determined by flow cytometry, FcgRIII deficiency resulted in an expansion of CD4+ cells and an increase in the CD4 to CD8 ratio. Analysis of plasma anti-oxidized LDL (OxLDL) antibodies by chemiluminescent assay revealed that IgG1 and IgG2c titers to OxLDL were increased in FcgRIII (-/-)xLDLR(-/-) double knockouts relative to LDLR(-/-) controls, while total IgG levels were similar. CONCLUSION: These results reveal altered immunity in FcgRIII(-/-)xLDLR(-/-) mice and a reduction in lesion formation associated with increased production of IL-10 by an expansion of CD4+ T-cells. The reduction in lesion formation was manifest well after evidence of an immune response to OxLDL, suggesting that FcgRIII contributes to lesion progression in murine atherosclerosis.
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Arterias/patología , Interleucina-10/fisiología , Receptores de IgG/fisiología , Animales , Aterosclerosis/patología , Autoanticuerpos/sangre , Linfocitos T CD4-Positivos/inmunología , Citocinas/biosíntesis , Femenino , Inmunidad , Interferón gamma/fisiología , Lípidos/sangre , Lipoproteínas LDL/inmunología , Masculino , Ratones , Receptores de LDL/deficiencia , Receptores de LDL/fisiologíaRESUMEN
INTRODUCTION: The lymphotoxin-beta receptor (LTbetaR) pathway is important in the development and maintenance of lymphoid structures. Blocking this pathway has proven beneficial in murine models of autoimmune diseases such as diabetes and rheumatoid arthritis. The aim of this study was to determine the effects of LTbetaR pathway blockade on Sjögren syndrome (SS)-like salivary gland disease in non-obese diabetic (NOD) mice. METHODS: The course of SS-like disease was followed in NOD mice that were given lymphotoxin-beta receptor-immunoglobulin fusion protein (LTbetaR-Ig) starting at 9 weeks of age. Treatment was given as a single weekly dose for 3, 7, or 10 weeks. Age-matched NOD mice treated with mouse monoclonal IgG1, or not treated at all, were used as controls. The severity of inflammation, cellular composition, and lymphoid neogenesis in the submandibular glands were determined by immunohistochemistry. Mandibular lymph nodes were also studied. Saliva flow rates were measured, and saliva was analyzed by a multiplex cytokine assay. The salivary glands were analyzed for CXCL13, CCL19, and CCL21 gene expression by quantitative polymerase chain reaction. RESULTS: Treatment with LTbetaR-Ig prevented the increase in size and number of focal infiltrates normally observed in this SS-like disease. Compared with the controls, the submandibular glands of LTbetaR-Ig-treated mice had fewer and smaller T- and B-cell zones and fewer high endothelial venules per given salivary gland area. Follicular dendritic cell networks were lost in LTbetaR-Ig-treated mice. CCL19 expression was also dramatically inhibited in the salivary gland infiltrates. Draining lymph nodes showed more gradual changes after LTbetaR-Ig treatment. Saliva flow was partially restored in mice treated with 10 LTbetaR-Ig weekly injections, and the saliva cytokine profile of these mice resembled that of mice in the pre-disease state. CONCLUSIONS: Our findings show that blocking the LTbetaR pathway results in ablation of the lymphoid organization in the NOD salivary glands and thus an improvement in salivary gland function.
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Receptor beta de Linfotoxina/inmunología , Glándulas Salivales/inmunología , Glándulas Salivales/patología , Transducción de Señal/inmunología , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Animales , Quimiocina CCL19/biosíntesis , Quimiocina CCL21/biosíntesis , Quimiocina CXCL13/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Expresión Génica , Inmunohistoquímica , Receptor beta de Linfotoxina/antagonistas & inhibidores , Ratones , Ratones Endogámicos NOD , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Stat5 proteins are critical signaling molecules activated by many cytokines. Within the immune system, Stat5 plays important roles related to the development of thymocytes and proliferation of T cells. Stat5 has been implicated in malignant transformation, and moreover, the activated tyrosine phosphorylated form of Stat5 is frequently observed in human lymphomas. We previously demonstrated the oncogenic potential of Stat5, with thymic lymphoblastic lymphomas developing in a significant proportion of transgenic (TG) mice overexpressing Stat5a or Stat5b in lymphocytes. In addition, immunization or expression of a T-cell receptor (TCR) transgene augmented the rate of tumor formation. Here, we investigate the mechanism of Stat5-mediated lymphomagenesis by exploring the contributions of major histocompatibility complex (MHC)/TCR and pre-TCR signals. We present data demonstrating that Stat5b TG mice unexpectedly develop CD8(+) lymphoma even in the absence of either pre-TCR signaling or normal thymic selection. Indeed, acceleration of Stat5b transgene-mediated lymphoma occurred on TCRalpha(-/-) and pre-TCRalpha(-/-) backgrounds. In light of these data, we propose a model in which alterations in T-cell development at the double-negative/double-positive (DN/DP) stages cooperate with cytokine-mediated pathways in immature thymocytes to give rise to lymphoblastic T-cell lymphomas in Stat5b TG mice.
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Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunología , Animales , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/fisiología , Transformación Celular Neoplásica/inmunología , Células Asesinas Naturales/patología , Células Asesinas Naturales/fisiología , Complejo Mayor de Histocompatibilidad/fisiología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/patología , Linfocitos T/fisiología , Transgenes/fisiologíaRESUMEN
OBJECTIVE: Receptor activator of nuclear factor-kappaB ligand (RANKL) promotes osteoclast differentiation from monocyte precursors by inducing a cohort of genes, including tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). A family of synthetic triterpenoids with antiinflammatory and pro-apoptotic properties was described to modulate differentiation in monocytic cell lineages. We therefore investigated the ability of the potent and bioavailable synthetic triterpenoid TP-222 to inhibit RANKL-induced osteoclast formation and MMP-9 expression from monocytic precursor cells. METHODS: Osteoclast formation was assayed by staining for TRAP-positive multinucleated cells. MMP-9 expression was measured by quantitative RT-PCR, Western blot, immunohistochemistry, and gel zymography. In vivo effects of TP-222 were assessed by daily intraperitoneal injection of 4-week-old mice for 7 days followed by measurement of osteoclast number and MMP-9 expression at the cartilage/bone junction of the epiphyseal growth plate. RESULTS: RANKL promoted and TP-222 (300 nM) inhibited osteoclast formation in cultures of RAW264.7 cells or bone marrow-derived monocytes. RANKL also induced MMP-9 expression in RAW264.7 cells and this was reduced by concurrent or subsequent addition of TP-222. TP-222 treatment significantly reduced the mean number of osteoclasts present at the cartilage/bone interface compared to vehicle-injected control mice. Morphometric analyses of tissue sections showed that TP-222 treatment reduced the amount of immunoreactive MMP-9 present in both mononucleated pre-osteoclasts and osteoclasts. CONCLUSION: Our data demonstrate that TP-222 inhibits osteoclast formation and MMP-9 expression in vitro and in vivo, and suggest that triterpenoids may be useful compounds for modulating bone resorption diseases.
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Metaloproteinasa 9 de la Matriz/metabolismo , Osteoclastos/efectos de los fármacos , Ligando RANK/antagonistas & inhibidores , Triterpenos/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/enzimología , Recuento de Células , Línea Celular , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inyecciones Intraperitoneales , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Osteoclastos/enzimología , Osteogénesis/efectos de los fármacos , ARN Mensajero/metabolismoRESUMEN
The lymphotoxin axis is important for the maintenance of several specialized lymphoid microenvironments in secondary lymphoid tissue. Lymphoid-tissue architecture is highly plastic and requires continual homeostatic signaling to maintain its basal functional state. The cellularity of lymph nodes in adult mice was reduced by systemic blockade of lymphotoxin-beta receptor (LTbeta R) signaling with a soluble decoy receptor both in resting and reactive settings. This reduction in cellularity resulted from greatly impaired lymphocyte entry into lymph nodes due to decreased levels of peripheral lymph node addressing (PNAd) and MAdCAM on high endothelial venules (HEV). LTbeta R signaling was required to maintain normal levels of RNA expression of MAdCAM, and also of PNAd by regulating the expression of key enzymes and scaffold proteins required for its assembly. Thus, the homeostatic maintenance of functional HEV status in adult mice relies largely on LTbeta R signaling.
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Diferenciación Celular , Células Endoteliales/citología , Células Endoteliales/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Animales , Antígenos de Superficie/metabolismo , Movimiento Celular , Proliferación Celular , Homeostasis , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Receptor beta de Linfotoxina , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/deficiencia , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismoRESUMEN
OBJECTIVE: The zinc-finger protein tristetraprolin (TTP) has been demonstrated to regulate tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) instability in murine macrophages. We sought to develop a model system to characterize the effects of human TTP (hTTP) on TNFalpha 3'-untranslated region (3'-UTR)-mediated expression. We also generated a specific polyclonal antibody against hTTP that enabled the examination of the subcellular distribution of hTTP and its RNA binding in vivo. METHODS: Transfection of reporter gene constructs were used to functionally characterize the role of hTTP in regulating TNFalpha expression in a 3'-UTR-dependent manner. An immunoprecipitation reverse transcription-polymerase chain reaction technique, immunoblotting, immunocytochemistry, and sucrose density fractionation were used to identify and localize hTTP. RESULTS: We found that hTTP interacted with human TNFalpha mRNA in the cytoplasm. The presence of the TNFalpha 3'-UTR was sufficient to confer binding by TTP in vivo. This interaction resulted in reduced luciferase reporter gene activity in a TNFalpha 3'-UTR adenine-uridine-rich element (ARE)-dependent manner. Immunoblotting and immunocytochemistry indicated that endogenous and transfected hTTP localized to the cytoplasm. Results of sucrose density fractionation studies were consistent with a polysomal location of hTTP. In rheumatoid synovium, hTTP expression was restricted to cells in the synovial lining layers. CONCLUSION: Through the development of an antiserum specific for hTTP, we have been able to demonstrate that hTTP binds specifically to the TNFalpha 3'-UTR and reduces reporter gene expression in an ARE-specific manner. These studies establish that hTTP is likely to function in a similar, if not identical manner, in the posttranscriptional regulation of TNFalpha. Understanding the posttranscriptional regulation of TNFalpha biosynthesis is important for the development of novel treatment strategies in rheumatoid arthritis.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Factor de Necrosis Tumoral alfa/genética , Regiones no Traducidas 3'/fisiología , Especificidad de Anticuerpos , Artritis Reumatoide/inmunología , Secuencia de Bases , Línea Celular , Expresión Génica/fisiología , Humanos , Proteínas Inmediatas-Precoces/análisis , Riñón/citología , Luciferasas/genética , Datos de Secuencia Molecular , Células Mieloides/química , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/análisis , Transfección , Tristetraprolina , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
A lymphotoxin-beta (LTbeta) receptor-Ig fusion protein (LTbetaR-Ig) was used to evaluate the importance of the lymphotoxin/LIGHT axis in the development and perpetuation of arthritis. Prophylactic treatment with the inhibitor protein LTbetaR-Ig blocked the induction of collagen-induced arthritis in mice and adjuvant arthritis in Lewis rats. Treatment of mice with established collagen-induced arthritis reduced the severity of arthritic symptoms and joint tissue damage. However, in a passive model of anti-collagen Ab-triggered arthritis, joint inflammation was not affected by LTbetaR-Ig treatment precluding LT/LIGHT involvement in the very terminal immune complex/complement/FcR-mediated effector phase. Collagen-II and Mycobacterium-specific T cell responses were not impaired, yet there was evidence that the overall response to the mycobacterium was blunted. Serum titers of anti-collagen-II Abs were reduced especially during the late phase of disease. Treatment with LTbetaR-Ig ablated follicular dendritic cell networks in the draining lymph nodes, suggesting that impaired class switching and affinity maturation may have led to a decreased level of pathological autoantibodies. These data are consistent with a model in which the LT/LIGHT axis controls microenvironments in the draining lymph nodes. These environments are critical in shaping the adjuvant-driven initiating events that impact the subsequent quality of the anti-collagen response in the later phases. Consequently, blockade of the LT/LIGHT axis may represent a novel approach to the treatment of autoimmune diseases such as rheumatoid arthritis that involve both T cell and Ab components.