RESUMEN
Conditional mutation of protein geranylgeranyltransferase type I (GGTase-I) in macrophages (GLC) activates Rho-GTPases and causes arthritis in mice. Knocking out Rag1 in GLC mice alleviates arthritis which indicates that lymphocytes are required for arthritis development in those mice. To study GLC dependent changes in the adaptive immunity, we isolated CD4+ T cells from GLC mice (CD4+GLCs). Spleen and joint draining lymph nodes (dLN) CD4+GLCs exhibited high expression of Cdc42 and Rac1, which repressed the caudal HOXA proteins and activated the mechanosensory complex to facilitate migration. These CDC42/RAC1 rich CD4+GLCs presented a complete signature of GARP+NRP1+IKZF2+FOXP3+ regulatory T cells (Tregs) of thymic origin. Activation of the ß-catenin/Lef1 axis promoted a pro-inflammatory Th1 phenotype of Tregs, which was strongly associated with arthritis severity. Knockout of Cdc42 in macrophages of GLC mice affected CD4+ cell biology and triggered development of non-thymic Tregs. Knockout of Rac1 and RhoA had no such effects on CD4+ cells although it alleviated arthritis in GLC mice. Disrupting macrophage and T cell interaction with CTLA4 fusion protein reduced the Th1-driven inflammation and enrichment of thymic Tregs into dLNs. Antigen challenge reinforced the CD4+GLC phenotype in non-arthritic heterozygote GLC mice and increased accumulation of Rho-GTPase expressing thymic Tregs in dLNs. Our study demonstrates an unexpected role of macrophages in stimulating the development of pro-inflammatory thymic Tregs and reveal activation of Rho-GTPases behind their arthritogenic phenotype.
Asunto(s)
Artritis , Timo , Proteínas de Unión al GTP rho , Animales , Factores de Transcripción Forkhead/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Linfocitos T Reguladores , Timo/inmunología , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6(+) germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/inmunología , Tirosina Quinasa 3 Similar a fms/fisiología , Animales , Apoptosis , Regulación de la Expresión Génica , Inmunoglobulina M/inmunología , Ligandos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/inmunología , Receptores de Interleucina-4/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
CD8+ T cells have an emerging role in RA. Resent research indicates a causal relationship between the non-exhausted state of CD8+ T cells, defined by lost function of PD-1, and development of arthritis. We investigated how smoking contributes to the non-exhausted phenotype of CD8+ T cells and cause survivin release to serum. We compared serum survivin levels between smokers and non-smokers in 252 RA and 168 healthy subjects. Nicotine effects on CD8+ T cells were studied in peripheral blood of smoking women, bone marrow of nicotine treated mice and in sorted CD8 spleen cells in vitro using flow cytometry and quantitative PCR. Smoking increased the frequency of survivin release in serum of healthy women (OR 3.64, p = 0.025) and in RA patients (OR 1.98, p = 0.039). CD8+ T cells of smokers gained a non-exhausted PD-1 deficient phenotype. Expression of the cytotoxic marker CD107 correlated to survivin levels in serum. In the experimental setting, nicotine exposure led to an accumulation of non-exhausted PD-1-IL-7R+ CD8+ T cells in the bone marrow that is abundant with survivin producing cells. The production of the cytolytic protein perforin in bone marrow correlated to serum survivin levels. In vitro stimulation of nicotinic receptors on murine CD8+ T cells induced repressive transcription factors T-bet and Blimp-1 in support of the non-exhausted phenotype. We conclude that nicotine contributes to autoimmunity by supporting the non-exhausted state of CD8+ T cells resulting in the release of survivin. This presents a new mechanism by which smoking may contribute to the pathogenesis of RA.
Asunto(s)
Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Activación de Linfocitos/inmunología , Fumar , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Artritis Reumatoide/genética , Biomarcadores , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunofenotipificación , Proteínas Inhibidoras de la Apoptosis/sangre , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Nicotina/farmacología , Fenotipo , Receptor de Muerte Celular Programada 1/deficiencia , Receptor de Muerte Celular Programada 1/metabolismo , Survivin , Linfocitos T Citotóxicos/efectos de los fármacos , Adulto JovenRESUMEN
Specific serum antibodies mediating humoral immunity and autoimmunity are provided by mature plasma cells (PC) residing in the bone marrow (BM), yet their dynamics and composition are largely unclear. We here characterize distinct subsets of human PC differing by CD19 expression. Unlike CD19(+) PC, CD19(-) PC were restricted to BM, expressed predominantly IgG, and they carried a prosurvival, distinctly mature phenotype, that is, HLA-DR(low)Ki-67(-)CD95(low)CD28(+)CD56(+/-), with increased BCL2 and they resisted their mobilization from the BM after systemic vaccination. Fewer mutations within immunoglobulin VH rearrangements of CD19(-) BMPC may indicate their differentiation in early life. Their resistance to in vivo B-cell depletion, that is, their independency from supply with new plasmablasts, is consistent with long-term stability of this PC subset in the BM. Moreover, CD19(-) PC were detectable in chronically inflamed tissues and secreted autoantibodies. We propose a multilayer model of PC memory in which CD19(+) and CD19(-) PC represent dynamic and static components, respectively, permitting both adaptation and stability of humoral immune protection.
Asunto(s)
Antígenos CD19/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Inmunoglobulina G/metabolismo , Células Plasmáticas/inmunología , Suero Antilinfocítico/administración & dosificación , Células de la Médula Ósea/clasificación , Ácidos Borónicos/administración & dosificación , Bortezomib , Diferenciación Celular , Supervivencia Celular , Vacuna contra Difteria y Tétanos/administración & dosificación , Humanos , Inmunidad Humoral , Memoria Inmunológica , Inflamación/inmunología , Inflamación/patología , Depleción Linfocítica , Modelos Inmunológicos , Mutación , Fenotipo , Células Plasmáticas/clasificación , Células Plasmáticas/citología , Pirazinas/administración & dosificación , Recombinación V(D)JRESUMEN
OBJECTIVES: To evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis. METHODS: Arthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry. RESULTS: S100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3(+) Treg cells, while the number of RORγt(+) and (pTyr705)STAT3(+) cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4. CONCLUSION: The present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.
RESUMEN
T-helper cells producing interleukin (IL)-17A and IL-17F cytokines (Th17 cells) are considered the source of autoimmunity in rheumatoid arthritis (RA). In this study, we characterized specific pathogenic features of Th17 cells in RA. By using nano-string technology, we analyzed transcription of 419 genes in the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of 14 RA patients and 6 healthy controls and identified 109 genes discriminating Th17 cells of RA patients from the controls. Th17 cells of RA patients had an aggressive pathogenic profile and in addition to signature cytokines IL-17, IL-23 and IL-21, and transcriptional regulators RAR-related orphan receptor gamma of T cells (RORγt) and Janus kinase 2 (JAK2), they produced high levels of IL-23R, C-C chemokine ligand type 20 (CCL20), granulocyte-monocyte colony-stimulating factor (GM-CSF ) and transcription factor Tbet required for synovial homing. We showed that Th17 cells are enriched with Helios-producing Foxp3- and IL2RA-deficient cells, indicating altered regulatory profile. The follicular T-helper (Tfh) cells presented a functional profile of adaptor molecules, transcriptional regulator Bcl-6 and B-cell activating cytokines IL-21, IL-31 and leukemia inhibitory factor (LIF ). We observed that anti-tumor necrosis factor (TNF) treatment had a limited effect on the transcription signature of Th17 cells. Patients in remission retained the machinery of receptors (IL-23R and IL-1R1), proinflammatory cytokines (IL-17F, IL-23, IL-21 and TNF ) and adaptor molecules (C-X-C chemokine receptor 5 [CXCR5] and cytotoxic T-lymphocyte-associated protein 4 [CTLA-4]), essential for efficient transdifferentiation and accumulation of Th17 cells. This study convincingly shows that the peripheral blood CCR6(+)CXCR3(-) CD4(+) cells of RA patients harbor pathogenic subsets of Th17 and Tfh cells, which may transdifferentiate from Tregs and contribute to perpetuation of the disease.
Asunto(s)
Artritis Reumatoide/genética , Transdiferenciación Celular/genética , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/administración & dosificación , Adulto , Anciano , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Femenino , Humanos , Interleucina-17/inmunología , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Células Th17/patología , Transcriptoma/genética , Transcriptoma/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.
Asunto(s)
Exosomas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Línea Celular Tumoral , Electroporación , Humanos , Linfocitos/metabolismo , Monocitos/metabolismo , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
Circulating leukocytes that express selectin ligands such as the carbohydrate epitope sialyl Lewis X (sLeX) may interact with endothelial selectins, resulting in transmigration of the leukocyte across the endothelial wall to adjacent tissue. Due to the potential of selectin-ligand interactions as targets in viral pathogenesis, we aimed at determining whether herpes simplex virus type 1 (HSV1) is able to induce the appearance of sLeX at the surface of infected leukocytes. We found that HSV1 infection of a T-cell line resulted in transcriptional activation of human fucosyltransferase genes FUT3, FUT6 and FUT7, the two latter genes encoding the fucosyltransferases rate limiting for sLeX synthesis. Flow cytometry and confocal microscopy demonstrated that HSV1 infection resulted in a 2-fold rise in the proportion of sLeX-positive cells. Increased levels of FUT3, FUT6 and FUT7 RNA were detected already at 3 h post infection, and treatment with cycloheximide, a translation inhibitor, blocked a HSV1-induced increase in the expression of FUT3, FUT6 and FUT7 RNA, suggesting involvement of viral or cellular proteins. Studies with infectious viral mutants indicated that the viral immediate early (α) protein ICP0 is essential for the initiation of FUT7 though not for FUT3 or FUT6 transcription. In CD3+ cells, derived from peripheral blood mononuclear cells, HSV1 infection induced expression of FUT3, FUT5 and FUT6, whereas FUT7 was not altered. The mean sLeX fluorescence intensity of CD3+ cells was significantly higher in HSV1-infected CD3+ cells. This suggests that infected leukocytes during HSV1 viremia may express selectin ligands with possible but as yet unproven roles in viral pathogenesis.
Asunto(s)
Herpesvirus Humano 1/fisiología , Oligosacáridos/metabolismo , Linfocitos T/virología , Animales , Línea Celular , Chlorocebus aethiops , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Antígeno Lewis X/genética , Antígeno Lewis X/metabolismo , Mutación , Oligosacáridos/genética , ARN Mensajero/metabolismo , Antígeno Sialil Lewis X , Linfocitos T/inmunología , Linfocitos T/metabolismo , Iniciación de la Transcripción Genética , Activación Transcripcional , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Epstein-Barr virus (EBV) infection may initiate production of autoantibodies and development of cancer and autoimmune diseases. Here we outline phenotypic and functional changes in B cells of patients with rheumatoid arthritis (RA) related to EBV infection. The B-cell phenotype was analysed in blood and bone marrow (BM) of RA patients who had EBV transcripts in BM (EBV(+) , n = 13) and in EBV(-) (n = 22) patients with RA. The functional effect of EBV was studied in the sorted CD25(+) and CD25(-) peripheral B cells of RA patients (n = 18) and healthy controls (n = 9). Rituximab treatment results in enrichment of CD25(+) B cells in peripheral blood (PB) of EBV(+) RA patients. The CD25(+) B-cell subset displayed a more mature phenotype accumulating IgG-expressing cells. It was also enriched with CD27(+) and CD95(+) cells in PB and BM. EBV stimulation of the sorted CD25(+) B cells in vitro induced a polyclonal IgG and IgM secretion in RA patients, while CD25(+) B cells of healthy subjects did not respond to EBV stimulation. CD25(+) B cells were enriched in PB and synovial fluid of RA patients. EBV infection affects the B-cell phenotype in RA patients by increasing the CD25(+) subset and by inducing their immunoglobulin production. These findings clearly link CD25(+) B cells to the EBV-dependent sequence of reactions in the pathogenesis of RA.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/sangre , Linfocitos B/inmunología , Transformación Celular Viral/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Herpesvirus Humano 4/inmunología , Subunidad alfa del Receptor de Interleucina-2/sangre , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/sangre , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Linfocitos B/virología , Biomarcadores/sangre , Estudios de Casos y Controles , Células Cultivadas , Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/virología , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Persona de Mediana Edad , Fenotipo , RituximabRESUMEN
Receptor for advanced glycation end products (RAGE) is a pattern recognition receptor that binds a variety of pro-inflammatory ligands. Its soluble form, sRAGE, can compete for ligand binding and thereby have an anti-inflammatory effect. We have recently reported that sRAGE also exerts pro-inflammatory and chemotactic properties suggesting a dual role for sRAGE in immune modulation. Our present aim was to analyse the immunomodulatory properties of sRAGE in vivo with respect to acquired immunity. Naive mice were treated intra-peritoneally with sRAGE and cells from peritoneal lavage, spleens and bone marrow were examined. Mice treated with sRAGE displayed an increased leucocyte count in the peritoneal cavity, enlarged spleens and increased cellularity compared with vehicle-treated animals. Furthermore, sRAGE-treated mice had a significantly increased frequency and number of CD19(+) B cells in spleen and a reduced frequency of CD19(+) B cells in bone marrow compared with controls. Functionally, splenocytes from sRAGE-treated mice showed elevated IgG production and up to a four-fold increased IgM secretion compared with control animals and produced significantly higher levels of interleukin-10, interferon-γ and interleukin-6 in response to lipopolysaccharide stimulation. Our results suggest that sRAGE has immunomodulatory properties, since intra-peritoneal administration of sRAGE into healthy mice leads to rearrangements in cellular composition in the bone marrow and spleen. Moreover, the administration of sRAGE directs B cells into the spleen and towards differentiation. Our novel findings indicate that sRAGE exerts an effect on the cells of adaptive immunity.
Asunto(s)
Antígenos CD19/metabolismo , Complejo CD3/metabolismo , Tejido Linfoide/patología , Antígeno de Macrófago-1/metabolismo , Cavidad Peritoneal/patología , Receptores Inmunológicos/administración & dosificación , Receptores Inmunológicos/metabolismo , Animales , Formación de Anticuerpos/efectos de los fármacos , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/sangre , Femenino , Inyecciones Intraperitoneales , Leucocitos/citología , Lipopolisacáridos/farmacología , Tejido Linfoide/efectos de los fármacos , Ratones , Receptor para Productos Finales de Glicación Avanzada , Solubilidad , Bazo/efectos de los fármacos , Bazo/patología , Esplenomegalia/patologíaRESUMEN
RATIONALE: The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention. OBJECTIVE: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. METHODS AND RESULTS: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2(-/-) bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2(-/-) macrophages. Further analysis in Rip2(-/-) macrophages showed that the lipid accumulation was scavenger-receptor independent and mediated by Toll-like receptor 4 (TLR4)-dependent lipid uptake. CONCLUSIONS: Our data show that lipid accumulation and inflammation are dissociated in the vessel wall in mice with Rip2(-/-) macrophages. These results for the first time identify Rip2 as a key regulator of cellular lipid metabolism and cardiovascular disease.
Asunto(s)
Aterosclerosis/enzimología , Colesterol/metabolismo , Macrófagos Peritoneales/enzimología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Triglicéridos/metabolismo , Animales , Apolipoproteína B-100/genética , Aterosclerosis/etiología , Aterosclerosis/inmunología , Aterosclerosis/patología , Trasplante de Médula Ósea , Humanos , Inflamación , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneales/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Pinocitosis , ARN Mensajero/biosíntesis , Quimera por Radiación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Organismos Libres de Patógenos Específicos , Receptor Toll-Like 4/fisiologíaRESUMEN
The posttranslational modification of proteins has the potential to generate neoepitopes that may subsequently trigger immune responses. The carbamylation of lysine residues to form homocitrulline may be a key mechanism triggering inflammatory responses. We evaluated the role of carbamylation in triggering immune responses and report a new role for this process in the induction of arthritis. Immunization of mice with homocitrulline-containing peptides induced chemotaxis, T cell activation, and Ab production. The mice also developed erosive arthritis following intra-articular injection of peptides derived from homocitrulline and citrulline. Adoptive transfer of T and B cells from homocitrulline-immunized mice into normal recipients induced arthritis, whereas systemic injection of homocitrulline-specific Abs or intra-articular injection of homocitrulline-Ab/citrulline-peptide mixture did not. Thus, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of anti-homocitrulline Abs, is critical for the induction of autoimmune reactions against citrulline-derived peptides and provides a novel mechanism for the pathogenesis of arthritis.
Asunto(s)
Artritis Reumatoide/inmunología , Citrulina/análogos & derivados , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Traslado Adoptivo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Artritis Experimental/inmunología , Artritis Experimental/metabolismo , Artritis Reumatoide/metabolismo , Separación Celular , Citrulina/inmunología , Citrulina/metabolismo , Citocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Persona de Mediana Edad , Procesamiento Proteico-Postraduccional , Linfocitos T/metabolismoRESUMEN
BACKGROUND: Efficient host defense mechanisms are crucial for survival in sepsis and septic arthritis. S100 proteins are reported to have proinflammatory and bactericidal properties. The aim of this study was to investigate the role of S100A4 in staphylococcal arthritis. METHODS: S100A4 knockout mice (S100A4KO) and wild-type counterparts (WT) were intravenously and intra-articularly challenged with Staphylococcus aureus strain LS-1. Clinical and morphological signs of arthritis and sepsis, phagocytosis, bone mineral density (BMD), and bone metabolism were then monitored in S100A4 and WT mice. RESULTS: S100A4KO mice had a lower bacterial load in the kidneys than WT mice (P < .05) but developed more severe clinical signs of arthritis (P < .001) and had higher levels of interleukin 6 and L-selectin (P = .002). S100A4KO mice had fewer morphological signs of synovitis and cartilage/bone destruction following intra-articular instillation of bacteria. S100A4KO mice were protected from loss of BMD and had lower levels of RANKL, MMP3, and MMP9 (P < .05). S100A4 was not bactericidal in vitro. CONCLUSIONS: In staphylococcal infection, S100A4 regulates bacterial clearance as well as systemic and local inflammatory responses.
Asunto(s)
Artritis Infecciosa/patología , Enfermedades de los Cartílagos/patología , Articulación de la Rodilla/patología , Proteínas S100/deficiencia , Infecciones Estafilocócicas/patología , Sinovitis/patología , Animales , Artritis Infecciosa/metabolismo , Artritis Infecciosa/microbiología , Carga Bacteriana , Densidad Ósea , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Enfermedades de los Cartílagos/microbiología , Cartílago Articular/microbiología , Cartílago Articular/patología , Femenino , Granulocitos/metabolismo , Interleucina-6/sangre , Riñón/microbiología , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/microbiología , Selectina L/sangre , Metaloproteinasa 3 de la Matriz/sangre , Metaloproteinasa 9 de la Matriz/sangre , Ratones , Ratones Noqueados , Ligando RANK/sangre , Proteína de Unión al Calcio S100A4 , Proteínas S100/fisiología , Índice de Severidad de la Enfermedad , Infecciones Estafilocócicas/metabolismo , Sinovitis/microbiologíaRESUMEN
BACKGROUND: We have shown that approximately 30% of human peripheral blood B-cells express CD25. B cells expressing CD25 display a mature phenotype belonging to the memory B-cell population and have a better proliferative and antigen-presenting capacity. The aim of the present study was to characterize the CD25-expressing subset of B cells in human cord blood. MATERIAL AND METHODS: Mononuclear cell fraction from human cord blood (n=34) and peripheral adult blood (n=22) was sorted into CD20+CD25+ and CD20+CD25- B-cell populations. Phenotype and function of these B-cell populations were compared using flow cytometry, proliferation, cytokine production, and immunoglobulin secretion. RESULTS: CD25-expressing B cells are a limited population of cord blood mononuclear cells representing 5% of the CD20+ B cells. They are characterised by high expression of CD5 in cord blood and CD27 in adult blood. CD25-expressing B cells express a functional IL-2 receptor and high levels of CC-chemokine receptors and spontaneously produce antibodies of IgG and IgM subclass. CONCLUSIONS: CD25 expression is a common denominator of a specific immunomodulatory B-cell subset ready to proliferate upon IL-2 stimulation, possibly ready to migrate and home into the peripheral tissue for further differentiation/action.
Asunto(s)
Linfocitos B/inmunología , Sangre Fetal/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Fenotipo , Adulto , Anciano , Linfocitos B/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD5/metabolismo , Citocinas/biosíntesis , Femenino , Sangre Fetal/citología , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores Mensajeros de Linfocitos/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
OBJECTIVES: Viruses may contribute to RA. This prompted us to monitor viral load and response to anti-CD20 therapy in RA patients. METHODS: Blood and bone marrow from 35 RA patients were analysed for CMV, EBV, HSV-1, HSV-2, parvovirus B19 and polyomavirus using real-time PCR before and 3 months after rituximab (RTX) treatment and related to the levels of autoantibodies and B-cell depletion. Clinical response to RTX was defined as decrease in the 28-joint disease activity score (DAS-28) >1.3 at 6 months. RESULTS: Before RTX treatment, EBV was identified in 15 out of 35 patients (EBV-positive group), of which 4 expressed parvovirus. Parvovirus was further detected in eight patients (parvo-positive group). Twelve patients were negative for the analysed viruses. Following RTX, EBV was cleared, whereas parvovirus was unaffected. Eighteen patients were responders, of which 12 were EBV positive. The decrease in the DAS-28 was significantly higher in EBV-positive group compared with parvo-positive group (P = 0.002) and virus-negative patients (P = 0.04). Most of EBV-negative patients that responded to RTX (75%) required retreatment within the following 11 months compared with only 8% of responding EBV-positive patients. A decrease of RF, Ig-producing cells and CD19(+) B cells was observed following RTX but did not distinguish between viral infections. However, EBV-infected patients had significantly higher levels of Fas-expressing B cells at baseline as compared with EBV-negative groups. CONCLUSIONS: EBV and parvovirus genomes are frequently found in bone marrow of RA patients. The presence of EBV genome was associated with a better clinical response to RTX. Thus, presence of EBV genome may predict clinical response to RTX.
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Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Linfocitos B/inmunología , Médula Ósea/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Artritis Reumatoide/complicaciones , Artritis Reumatoide/inmunología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/inmunología , Femenino , Herpesvirus Humano 4/inmunología , Humanos , Masculino , Persona de Mediana Edad , Rituximab , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Rho family proteins are prenylated by geranylgeranyltransferase type I (GGTase-I), which normally target proteins to membranes for GTP-loading. However, conditional deletion of GGTase-I in mouse macrophages increases GTP-loading of Rho proteins, leading to enhanced inflammatory responses and severe rheumatoid arthritis. Here we show that heterozygous deletion of the Rho family gene Rac1, but not Rhoa and Cdc42, reverses inflammation and arthritis in GGTase-I-deficient mice. Non-prenylated Rac1 has a high affinity for the adaptor protein Ras GTPase-activating-like protein 1 (Iqgap1), which facilitates both GTP exchange and ubiquitination-mediated degradation of Rac1. Consistently, inactivating Iqgap1 normalizes Rac1 GTP-loading, and reduces inflammation and arthritis in GGTase-I-deficient mice, as well as prevents statins from increasing Rac1 GTP-loading and cytokine production in macrophages. We conclude that blocking prenylation stimulates Rac1 effector interactions and unleashes proinflammatory signaling. Our results thus suggest that prenylation normally restrains innate immune responses by preventing Rac1 effector interactions.
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Inmunidad Innata/genética , Prenilación de Proteína , Transducción de Señal/genética , Proteína de Unión al GTP rac1/genética , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Animales , Citocinas/metabolismo , Macrófagos/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica , Células RAW 264.7 , Proteína de Unión al GTP rac1/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Uric acid, the naturally occurring degradation product of purine metabolism, is a danger signal, driving maturation of dendritic cells. It is well known that uric acid crystals display potent proinflammatory properties--the cause of gout--whereas the biological properties of soluble uric acid are less well documented. We have demonstrated previously that nucleic acids of endogenous and exogenous origin display proinflammatory properties. The aim of the present study was to assess the impact of soluble uric acid on in vivo inflammatory responses. Mice were administered with uric acid suspension in saline or saline alone prior to induction of neutrophil-mediated inflammation, delayed-type hypersensitivity, histamin-induced edema (measure of vasodilation capacity), as well as double-stranded (ds)RNA-triggered arthritis. Frequency and severity of arthritis were decreased significantly in mice exposed to dsRNA and simultaneously treated with uric acid as compared with saline-treated controls. Also, granulocyte-mediated inflammatory response and vasodilation capacity were reduced significantly in mice treated with uric acid as compared with their control group. The data suggest that down-regulation of inflammation was mediated by skewing the inflammatory response from the peripheral sites to the peritoneal cavity and down-regulating vasodilatatory capacity and thereby affecting leukocyte migration. In contrast, the T cell-mediated delayed-type hypersensitivity reaction was not affected significantly in mice exposed to uric acid. These findings demonstrate that uric acid displays a potent, distant anti-inflammatory effect in vivo. This property seems to be mediated by down-regulation of neutrophil influx to the site of inflammatory insult.
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Artritis Experimental/inmunología , Regulación hacia Abajo/inmunología , Inmunosupresores/inmunología , Ácidos Nucleicos/inmunología , ARN Bicatenario/inmunología , Ácido Úrico/inmunología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Edema/inducido químicamente , Edema/inmunología , Edema/fisiopatología , Femenino , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/fisiopatología , Inmunosupresores/metabolismo , Inmunosupresores/farmacología , Mediadores de Inflamación/efectos adversos , Mediadores de Inflamación/inmunología , Articulaciones/efectos de los fármacos , Articulaciones/inmunología , Articulaciones/fisiopatología , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Ácidos Nucleicos/metabolismo , ARN Bicatenario/efectos adversos , Ácido Úrico/metabolismo , Ácido Úrico/farmacología , Vasodilatación/efectos de los fármacos , Vasodilatación/inmunologíaRESUMEN
Continuous recruitment of neutrophils into the inflamed gastric mucosal tissue is a hallmark of Helicobacter pylori infection in humans. In this study, we examined the ability of H. pylori to induce transendothelial migration of neutrophils using a transwell system consisting of a cultured monolayer of human endothelial cells as barrier between two chambers. We showed for the first time that live H. pylori, but not formalin-killed bacteria, induced a significantly increased transendothelial migration of neutrophils. H. pylori conditioned culture medium also induced significantly increased transendothelial migration, whereas heat-inactivated culture filtrates had no effect, suggesting that the chemotactic factor was proteinaceous. Depletion of H. pylori-neutrophil activating protein (HP-NAP) from the culture filtrates resulted in significant reduction of the transmigration. Culture filtrates from isogenic HP-NAP deficient mutant bacteria also induced significantly less neutrophil migration than culture filtrates obtained from wild-type bacteria. HP-NAP did not induce endothelial cell activation, suggesting that HP-NAP acts directly on the neutrophils. In conclusion, our results demonstrate that secreted HP-NAP is one of the factors resulting in H. pylori induced neutrophil transendothelial migration. We propose that HP-NAP contributes to the continuous recruitment of neutrophils to the gastric mucosa of H. pylori infected individuals.
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Proteínas Bacterianas/metabolismo , Quimiotaxis de Leucocito/efectos de los fármacos , Células Endoteliales/inmunología , Helicobacter pylori/inmunología , Neutrófilos/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Células Cultivadas , Factores Quimiotácticos/genética , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Humanos , Activación Neutrófila/inmunologíaRESUMEN
INTRODUCTION: Alternative splicing distinguishes normal and pathologic cells. High levels of oncoprotein survivin recognise patients with severe rheumatoid arthritis (RA). Here, we assess clinical relevance of alternative splicing of survivin in leukocytes of peripheral blood (PBMC) and bone marrow (BM) in RA patients. METHOD: Transcription of survivin wild-type (survivin-WT), survivin-2B and survivin-ΔEx3 was measured in 67 randomly selected RA patients and in 23 patients before and after B cell depletion with rituximab. Analysis was done in relation to disease activity, anti-rheumatic treatment and serum levels of rheumatoid factor (RF) and survivin. RESULTS: Survivin-WT was the dominant splice variant equally expressed in T and B cells, while survivin-2B and survivin-ΔEx3 were higher in B cells. High disease activity (DAS28>5.1) was associated with an excess of survivin-WT and low ratios between survivin-2B/WT (p=0.035) and survivin-ΔEx3/WT in PBMC. Depletion of B cells by rituximab caused a decrease in survivin-WT (p=0.005) in PBMC, increasing the ratio between survivin-2B/WT (p=0.009) and survivin-ΔEx3/WT (p=0.001) in BM. This increase in survivin-2B/WT was associated with reduction in CD19+ BM cells (r=0.929, p=0.007), RF (IgM, r=0.857, p=0.024; IgA, r=0.739, p=0.021), and DAS28 (0.636, p=0.054). The increase in survivin-ΔEx3 in BM was associated with a reduction of CD19+ BM cells (r=0.714, p=0.058) and DAS28 (r=0.648, p=0.049), while survivin-ΔEx3/WT was associated with RF (IgG, r=0.882, p=0.016). CONCLUSION: This study demonstrates that the suppressed diversity of survivin splicing in leukocytes may attribute to adverse self-recognition in RA. Depletion of autoantibody producing B cells improves the balance of survivin splicing.
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Empalme Alternativo/fisiología , Artritis Reumatoide/sangre , Artritis Reumatoide/genética , Proteínas Inhibidoras de la Apoptosis/sangre , Proteínas Inhibidoras de la Apoptosis/genética , Adulto , Anciano , Artritis Reumatoide/diagnóstico , Autoanticuerpos/sangre , Autoanticuerpos/genética , Biomarcadores/sangre , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , SurvivinRESUMEN
Follicular T helper (Tfh) cells are recognized by the expression of CXCR5 and the transcriptional regulator Bcl-6. Tfh cells control B cell maturation and antibody production, and if deregulated, may lead to autoimmunity. Here, we study the role of the proto-oncogene survivin in the formation of Tfh cells. We show that blood Tfh cells of patients with the autoimmune condition rheumatoid arthritis, have intracellular expression of survivin. Survivin was co-localized with Bcl-6 in the nuclei of CXCR5+CD4 lymphocytes and was immunoprecipitated with the Bcl-6 responsive element of the target genes. Inhibition of survivin in arthritic mice led to the reduction of CXCR5+ Tfh cells and to low production of autoantibodies. Exposure to survivin activated STAT3 and induced enrichment of PD-1+Bcl-6+ subset within Tfh cells. Collectively, our study demonstrates that survivin belongs to the Tfh cell phenotype and ensures their optimal function by regulating transcriptional activity of Bcl-6.