RESUMO
OBJECTIVES: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren's syndrome (pSS) to an H1N1 influenza vaccine. METHODS: Patients with Sjögren's syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. RESULTS: Surprisingly, treatment-naïve patients with Sjögren's syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. CONCLUSIONS: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren's syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients.
Assuntos
Anticorpos Antivirais/sangue , Linfócitos B , Citocinas/sangue , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Síndrome de Sjogren/imunologia , Antígenos CD19/análise , Antirreumáticos/farmacologia , Autoanticorpos/biossíntese , Autoantígenos/imunologia , Linfócitos B/química , Linfócitos B/fisiologia , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Expressão Gênica , Antígenos HLA-DR/análise , Herpesvirus Humano 4/imunologia , Humanos , Hidroxicloroquina/farmacologia , Imunoglobulina D/análise , Imunoglobulina G/sangue , Interferon-alfa/metabolismo , Interferon-alfa/farmacologia , Interleucina-10/farmacologia , Ativação Linfocitária , Contagem de Linfócitos , Ribonucleoproteínas/imunologia , Transdução de Sinais/genética , Síndrome de Sjogren/genética , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Transcriptoma , Vacinação , Antígeno SS-BRESUMO
Regulatory T cells (Tregs) are important for maintaining immune homeostasis, but many studies suggest that Tregs are functionally impaired in autoimmune and chronic inflammatory disorders. In addition, effector T cells may vary in sensitivity toward Treg suppression. Herein, we have studied the interplay between T effectors and Tregs in the rheumatic joint. Synovial Tregs demonstrated a high degree of FOXP3 demethylation and displayed only marginal IL-17 and virtually no IFN-γ production following in vitro stimulation, altogether indicating suppressive capacity. Still, the frequency of FOXP3 expression could not predict the degree of suppression. Instead, the inflammatory milieu in the joint, i.e. proliferative capacity of effector T cells and in situ levels of pro-inflammatory cytokines influenced Treg function. Indeed, blocking IL-6 or TNF increased the suppression by Tregs in co-cultures. Additionally, approximately 30% of the synovial FOXP3(+) T cells were Ki67(+) and hence actively dividing, but proliferation did not overlap with cytokine production, suggesting that these cells represent functional Tregs having met their cognate antigen and expanded in an attempt to alleviate joint inflammation. Overall, our data argue against a general functional deficit in joint-derived Tregs and instead emphasize the importance of the inflammatory milieu to set the threshold for immune regulation.
Assuntos
Inflamação/imunologia , Artropatias/imunologia , Doenças Reumáticas/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Antígenos CD2/imunologia , Antígenos CD2/metabolismo , Células Cultivadas , Técnicas de Cocultura , Metilação de DNA , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/imunologia , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Artropatias/metabolismo , Artropatias/patologia , Antígeno Ki-67/imunologia , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Doenças Reumáticas/metabolismo , Doenças Reumáticas/patologia , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Dermatomyositis and polymyositis are disabling rheumatic diseases characterized by an appreciable number of T cells infiltrating muscle tissue. The precise phenotype, function and specificity of these cells remain elusive. In this study, we aimed to characterize T cells in muscle tissue and circulation and to investigate their association to clinical phenotype. Twenty-four patients with dermatomyositis and 42 with polymyositis were screened for frequency of CD4+CD28(null) and CD8+CD28(null) T cells in peripheral blood by flow cytometry. Presence of these cells in inflamed muscle tissue from 13 of these patients was analyzed by three-color immunofluorescence microscopy. Effector functions, proliferation and Ag specificity were analyzed by flow cytometry after in vitro stimulation. The clinical relevance of CD28(null) T cells was analyzed by multiple regression analyses including six separate and combined disease variables. We demonstrate that muscle-infiltrating T cells are predominantly CD4+CD28(null) and CD8+CD28(null) T cells in patients with dermatomyositis and polymyositis. Muscle-infiltrating CD28(null) T cells were found already at time of diagnosis. Disease activity correlated with the frequency of CD8+ T cells in the inflamed muscles of polymyositis patients. Circulating CD4+CD28(null) and CD8+CD28(null) T cells were significantly more frequent in human CMV (HCMV) seropositive individuals, responded to HCMV Ag stimulation, and correlated with disease duration. These cells also display a proinflammatory cytokine profile, contain perforin and lack the costimulatory molecule CD28. Our observations imply that CD28(null) T cells represent clinically important effector cells in dermatomyositis and polymyositis, and that HCMV might play a role in propagating disease in a subset of patients.
Assuntos
Antígenos CD28 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/imunologia , Dermatomiosite/imunologia , Músculo Esquelético/imunologia , Polimiosite/imunologia , Adulto , Idoso , Antígenos CD28/biossíntese , Antígenos CD28/sangue , Antígenos CD28/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Dermatomiosite/patologia , Dermatomiosite/virologia , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Músculo Esquelético/virologia , Polimiosite/patologia , Polimiosite/virologiaRESUMO
With new treatments of inflammatory diseases targeting key inflammatory pathways follows an increased risk for infections. The aim of the present study was to identify an immunological readout where consecutive immunizations induce reproducible immune responses. Such a method could be used as a tool to assess drug-induced immunomodulation in individual patients by comparing responses to the immunizations before and after introduction of a specific treatment. Importantly, the vaccine is merely used as a model antigen and protective immunity is not the primary aim of the method. Eleven volunteers were immunized with influenza vaccine three times, four weeks apart. In order to find the optimal readout for the method, immune responses to the immunizations were measured as circulating antigen-specific B-cells, serum antibody titers and avidity, T-cell proliferation and cytokine secretion. The first exposure to the influenza vaccine induced a stronger B- and T-cell responses than the consecutive immunizations. The second and third immunizations induced comparable but lower B-cell responses as measured by ELISPOT. In summary, we have measured immune responsiveness by using repeated immunizations with influenza virus vaccine as the model antigen. The induction of comparable B-cell responses after the second and third serial immunizations provides a possibility to investigate effects on immune responsiveness by immunomodulatory drugs. The method also allows humoral memory and immune competence per se to be studied on a cellular level in different patient groups.
Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/imunologia , Fatores Imunológicos/imunologia , Memória Imunológica/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Modelos Imunológicos , Linfócitos T/imunologia , Adulto , Antígenos Virais/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunização , Técnicas Imunoenzimáticas/métodos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacologia , Vacinas contra Influenza/administração & dosagem , MasculinoRESUMO
OBJECTIVE: The cause of the exceedingly high risk of atherothrombosis in systemic lupus erythematosus (SLE) is not clear but antiphospholipid antibodies (aPL) and potentially antithrombotic annexin V have been implicated. METHODS AND RESULTS: Twenty-six women (52+/-8.2 years) with SLE and a history of cardiovascular disease (CVD) (SLE cases) were compared with 26 women with SLE but no CVD (SLE controls) and 26 healthy women (population controls). Common carotid intima-media thickness (IMT) was determined by B-mode ultrasound as a surrogate measure of atherosclerosis. Annexin V binding to human umbilical vein endothelial cells (HUVECs) as determined by flow cytometry after 24-hour culture with plasma was decreased when plasma from SLE cases was used (SLE cases versus population controls: P=0.002; SLE cases versus SLE controls P=0.02). Antibodies against cardiolipin were among IgG antibodies causing decreased binding. There was a positive association between annexin V binding and IMT (R=0.73; P<0.001) among SLE cases. Immunohistochemical analysis revealed presence of annexin V in all human atherosclerotic plaques tested, especially at sites prone to rupture. CONCLUSIONS: Decreased annexin V binding to endothelium caused by antibodies may represent a novel mechanism of atherothrombosis. We hypothesize that even though annexin V may promote plaque growth at some disease stages, it may also stabilize plaque.
Assuntos
Anexina A5/metabolismo , Arteriosclerose/etiologia , Células Endoteliais/metabolismo , Lúpus Eritematoso Sistêmico/complicações , Trombose/etiologia , Anexina A5/imunologia , Arteriosclerose/diagnóstico por imagem , Arteriosclerose/patologia , Cardiolipinas/imunologia , Cardiolipinas/metabolismo , Doenças Cardiovasculares/patologia , Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/patologia , Endotélio Vascular/patologia , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Imunoglobulina G/toxicidade , Lúpus Eritematoso Sistêmico/sangue , Pessoa de Meia-Idade , Ligação Proteica , Coloração e Rotulagem/métodos , Túnica Íntima/diagnóstico por imagem , Túnica Íntima/patologia , Túnica Média/diagnóstico por imagem , Túnica Média/patologia , Ultrassonografia , Veias Umbilicais/metabolismo , Veias Umbilicais/patologiaRESUMO
The development of rheumatoid arthritis (RA) occurs as a result of interactions between genes and environment. The most well established association with both susceptibility and severity of disease is variations in the major histocompatibility complex (MHC) class II genes. This fact constitutes evidence in favor of a contribution from specific MHC class II restricted adaptive immunity to the pathogenesis of RA. However, considerable difficulties have been encountered in identifying reactivities within the adaptive immune system that are responsible for the development of chronic arthritis in humans. In this article, the authors suggest a hypothesis for arthritis development based on their, as well as others', research. In patients with certain genetic contexts, RA can be initiated by activation of the innate immune system alone. In other patients, the adaptive immune system may be needed for the induction of disease. Additionally, the authors believe that a perpetuation to a severe chronic arthritis occurs only when both the adaptive and the innate immune systems have been recruited.
Assuntos
Artrite/imunologia , Imunidade Ativa , Imunidade Inata , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Genes MHC da Classe II , Humanos , Complexo Principal de Histocompatibilidade/genéticaRESUMO
INTRODUCTION: B cells may play an important role in promoting immune activation in the rheumatoid synovium and can produce prostaglandin E(2) (PGE(2)) when activated. In its turn, PGE(2) formed by cyclooxygenase (COX) and microsomal prostaglandin E(2) synthase 1 (MPGES1) contributes to the rheumatoid arthritis (RA) pathological process. Therapeutic depletion of B cells results in important improvement in controlling disease activity in rheumatoid patients. Therefore we investigated the expression of PGE(2) pathway enzymes in RA B cells and evaluated the effects of B cell depleting therapy on their expression in RA tissue. METHODS: B cells expressing MPGES1 and COX-2 were identified by flow cytometry in in vitro stimulated and control mononuclear cells isolated from synovial fluid and peripheral blood of RA patients. Synovial biopsies were obtained from 24 RA patients before and at two consecutive time points after rituximab therapy. Expression of MPGES1, COX-1 and COX-2, as well as interleukin (IL)-1ß and IL-6, known inducers of MPGES1, was quantified in immunostained biopsy sections using computerized image analysis. RESULTS: Expression of MPGES1 or COX-2 was significantly upregulated upon stimulation of B cells from blood and synovial fluid while control cells displayed no detectable enzymes. In synovial biopsy sections, the expression of MPGES1, COX-1 or COX-2 was resistant to rituximab therapy at 8 or 16 weeks after start of treatment. Furthermore expression of IL-1ß in the synovial tissue remained unchanged, while IL-6 tended to decrease after therapy. CONCLUSIONS: Therapy with B cell depleting agents, although efficient in achieving good clinical and radiographic response in RA patients, leaves important inflammatory pathways in the rheumatoid synovium essentially unaffected.
Assuntos
Artrite Reumatoide , Linfócitos B/enzimologia , Dinoprostona/biossíntese , Depleção Linfocítica/métodos , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Linfócitos B/patologia , Células Cultivadas , Ciclo-Oxigenase 1/análise , Ciclo-Oxigenase 2/análise , Humanos , Inflamação , Interleucina-1beta/análise , Interleucina-6/análise , Oxirredutases Intramoleculares/análise , Prostaglandina-E Sintases , Membrana Sinovial/patologiaRESUMO
Herein we investigated how rituximab-induced B cell depletion affected leukocyte subpopulations and antibody titers in SLE patients. We focused our analysis on time points related to absence and return of B cells after depletion. A correlation was found between the baseline frequency and time to repopulation; the fewer B cells initially, the longer to their return. While the few B cells remaining after treatment were of memory, double-negative (IgD-CD27-), and CD5+ phenotype, the returning B cells were mainly naïve, indicating de novo production of B cells. Serum levels of IgG and antibodies against Ro52, Ro60, La44, measles and tetanus remained unchanged, while decreases in IgM, IgE, anti-dsDNA and anti-C1q antibodies were observed. Additionally, a significant increase in activated CD4+ and CD8+ T cells, as well as CD25bright FOXP3+ regulatory T cells was observed. In conclusion, both the humoral and the cellular immune systems were affected by treatment with rituximab.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Anticorpos Monoclonais Murinos , Autoanticorpos/sangue , Linfócitos B/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Imunoglobulinas/sangue , Imunoglobulinas/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/imunologia , Depleção Linfocítica , Rituximab , Linfócitos T/efeitos dos fármacosRESUMO
OBJECTIVE: To study the effects of the antirheumatic drug sulphasalazine (SASP) on the immune system by analysing systemic and gut-associated immune responses. METHODS: A total of 23 healthy volunteers were treated with either SASP or placebo for 5 weeks in a double-blind fashion and immunised 2 weeks after the initiation of treatment. Specific immune responses were triggered by subcutaneous immunisation with tetanus toxoid and by peroral immunisation with inactivated influenza vaccine. The effects of treatment on specific immunity to tetanus and influenza were evaluated by enzyme-linked immunospot assay quantifying the number of circulating specific and total antibody-producing cells (spot-forming cells (SFC)) at 6, 8 and 10 days after immunisation. RESULTS: An immunosuppressive effect of SASP on systemic immune response was observed with a decrease in the total number of IgG-SFC, IgG anti-tetanus SFC and IgG anti-tetanus antibody levels in serum. SASP also exerted an immunosuppressive effect on the mucosa-associated immune system as seen from its down-regulating effect on the total number of circulating IgA SFC. CONCLUSIONS: These data show firstly that SASP exerts an immunosuppressive effect on defined immune responses to immunisation in vivo, and secondly that both mucosa-associated and systemic immunity are affected by SASP treatment.
Assuntos
Células Produtoras de Anticorpos/efeitos dos fármacos , Antirreumáticos/farmacologia , Imunossupressores/farmacologia , Sulfassalazina/farmacologia , Adolescente , Adulto , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Vacinas contra Influenza/imunologia , Pessoa de Meia-Idade , Toxoide Tetânico/imunologiaRESUMO
Expanded populations of CD4+ T cells lacking the co-stimulatory molecule CD28 (CD4+CD28null T cells) have been reported in several inflammatory disorders. In rheumatoid arthritis, increased frequencies of CD4+CD28null T cells in peripheral blood have previously been associated with extra-articular manifestations and human cytomegalovirus (HCMV) infection, but their presence in and contribution to joint manifestations is not clear. In the present article we investigated the distribution of CD4+CD28null T cells in the synovial membrane, synovial fluid and peripheral blood of RA patients, and analysed the association with erosive disease and anti-citrullinated protein antibodies. CD4+CD28null T cells were infrequent in the synovial membrane and synovial fluid, despite significant frequencies in the circulation. Strikingly, the dominant TCR-Vbeta subsets of CD4+CD28null T cells in peripheral blood were often absent in synovial fluid. CD4+CD28null T cells in blood and synovial fluid showed specificity for HCMV antigens, and their presence was clearly associated with HCMV seropositivity but not with anti-citrullinated protein antibodies in the serum or synovial fluid, nor with erosive disease. Together these data imply a primary role for CD4+CD28null T cells in manifestations elsewhere than in the joints of patients with HCMV-seropositive rheumatoid arthritis.
Assuntos
Artrite Reumatoide/sangue , Antígenos CD28/sangue , Antígenos CD4/sangue , Linfócitos Nulos/metabolismo , Linfócitos T/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/patologia , Antígenos CD28/análise , Antígenos CD4/análise , Estudos de Coortes , Feminino , Humanos , Linfócitos Nulos/química , Linfócitos Nulos/citologia , Masculino , Pessoa de Meia-Idade , Líquido Sinovial/química , Líquido Sinovial/citologia , Líquido Sinovial/metabolismo , Membrana Sinovial/química , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Linfócitos T/química , Linfócitos T/citologiaRESUMO
The objective of this study was to investigate the interaction between levels of BAFF (B-cell activation factor of the tumour necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) and B-cell frequencies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) treated with the B-cell-depleting agent rituximab. Ten patients with SLE were treated with rituximab in combination with cyclophosphamide and corticosteroids. They were followed longitudinally up to 6 months after B-cell repopulation. Nine patients with RA, resistant or intolerant to anti-TNF therapy, treated with rituximab plus methotrexate were investigated up to 6 months after treatment. The B-cell frequency was determined by flow cytometry, and serum levels of BAFF and APRIL were measured by enzyme-linked immunosorbent assays. BAFF levels rose significantly during B-cell depletion in both patient groups, and in patients with SLE the BAFF levels declined close to pre-treatment levels upon B-cell repopulation. Patients with SLE had normal levels of APRIL at baseline, and during depletion there was a significant decrease. In contrast, patients with RA had APRIL levels 10-fold higher than normal, which did not change during depletion. At baseline, correlations between levels of B cells and APRIL, and DAS28 (disease activity score using 28 joint counts) and BAFF were observed in patients with RA. In summary, increased BAFF levels were observed during absence of circulating B cells in our SLE and RA patient cohorts. In spite of the limited number of patients, our data suggest that BAFF and APRIL are differentially regulated in different autoimmune diseases and, in addition, differently affected by rituximab treatment.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Fator Ativador de Células B/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/efeitos dos fármacos , Corticosteroides/uso terapêutico , Adulto , Idoso , Anticorpos Monoclonais Murinos , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Fator Ativador de Células B/sangue , Linfócitos B/efeitos dos fármacos , Ciclofosfamida/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Rituximab , Membro 13 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangueRESUMO
Rheumatoid arthritis (RA) is a common destructive inflammatory disease that affects 0.5-1% of the population in many countries. Even though several new treatments have been introduced for patients with RA, a considerable proportion of patients do not benefit from these, and the need for alternative treatment strategies is clear. This review explores the potential for a therapy targeting the adaptive immune system by modulating co-stimulation of T cells with a CTLA4-Ig fusion protein (abatacept).
Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Doenças Autoimunes/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Abatacepte , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Murinos , Antirreumáticos/farmacologia , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Humanos , Imunoconjugados/uso terapêutico , Camundongos , Rituximab , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
BACKGROUND: Environmental factors are believed to play a role in the development of atopic allergy. This is likely to be important very early in life, at the fetal stage. The in utero environment could be affected by maternal allergy and in turn could influence the immune system of the baby. OBJECTIVE: To investigate how cord blood mononuclear blood cells (CBMCs) from children of women with and without allergy respond to microbial stimuli. METHODS: PBMCs from women with (n = 9) and without allergy (n = 10) and CBMCs from their newborn babies were stimulated in vitro with LPS and peptidoglycan. Cells were analyzed with flow cytometry for expression of CD14, Toll-like receptor (TLR)-2, and TLR4. The release of cytokines and chemokines (IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNF-alpha) and soluble CD14 into culture supernatants was measured with Cytometric Bead Array and ELISA, respectively. RESULTS: Cord blood (CB) monocytes from children with mothers with allergy had significantly lower expression of TLR2 and TLR4 compared with maternal monocytes both before and after microbial stimulation, in contrast with CB monocytes from children with mothers without allergy. Further, CBMCs from children with mothers with allergy had a lower ( P = .03) IL-6 response after stimulation with peptidoglycan than CBMCs from children with mothers without allergy. CONCLUSION: Our results imply that CB monocytes and CBMC immune responses are influenced by maternal allergy. On the basis of these findings, we speculate that monocytes from children with mothers with allergy have a reduced capacity to respond to microbial stimuli.
Assuntos
Hipersensibilidade/complicações , Leucócitos Mononucleares/imunologia , Exposição Materna , Complicações na Gravidez/imunologia , Adulto , Células Cultivadas , Feminino , Sangue Fetal , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Interleucina-6/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/microbiologia , Receptores de Lipopolissacarídeos/biossíntese , Lipopolissacarídeos , Masculino , Glicoproteínas de Membrana/biossíntese , Peptidoglicano , Gravidez , Complicações na Gravidez/sangue , Receptores de Superfície Celular/biossíntese , Fatores de Risco , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4(+) but similar numbers of CD8(+) lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4(+) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4(+) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4(+) T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4(+) T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4(+) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Movimento Celular/imunologia , Mucosa Gástrica/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/imunologia , Adulto , Linfócitos T CD4-Positivos/microbiologia , Agregação Celular/imunologia , Quimiocinas/biossíntese , Feminino , Mucosa Gástrica/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Receptores CCR3 , Receptores CCR5/biossíntese , Receptores de Quimiocinas/biossíntese , Receptores de Retorno de Linfócitos/biossínteseRESUMO
OBJECTIVE: Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF) antagonists is highly effective, but their mechanisms of action are not completely clear. Since anti-TNF therapy induces a decrease in synovial cellularity, this study focused on the modulation of RA synovial apoptosis following treatment with either soluble TNF receptor (etanercept) or TNF chimeric monoclonal antibody (infliximab). METHODS: Apoptosis (TUNEL and active caspase 3 staining) and cell surface markers were evaluated by immunohistochemistry in synovial biopsy samples obtained before and after 8 weeks of treatment with etanercept (12 patients) or infliximab (9 patients). We also determined by flow cytometry the in vitro effect of etanercept and infliximab on apoptosis of RA mononuclear cells derived from the synovial fluid (SF) and peripheral blood (PB). RESULTS: Eight weeks of treatment with etanercept and with infliximab significantly increased synovial apoptosis. This change was accompanied by a significant decrease in the synovial monocyte/macrophage population. The decrease in lymphocyte numbers did not reach statistical significance. In vitro, 24 hours of incubation with either etanercept or infliximab induced apoptosis of the SF monocyte/macrophage population. PB monocyte/macrophages were less susceptible to anti-TNF-mediated apoptosis. No changes in the rate of apoptosis were observed in the lymphocyte population derived from either SF or PB. CONCLUSION: In RA patients, both etanercept and infliximab are able to induce cell type-specific apoptosis in the monocyte/macrophage population. This suggests a potential pathway that would account for the diminished synovial inflammation and the decreased numbers of synovial macrophages evident after TNF blockade.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Apoptose , Artrite Reumatoide/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Articulações/fisiopatologia , Macrófagos/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/uso terapêutico , Inibidores do Fator de Necrose Tumoral , Adulto , Idoso , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Etanercepte , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Infliximab , Articulações/patologia , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Líquido Sinovial , Membrana Sinovial/patologia , Membrana Sinovial/fisiopatologia , Resultado do TratamentoRESUMO
OBJECTIVE: Microsomal prostaglandin E synthase 1 (mPGES-1) is up-regulated in experimental arthritis and markedly expressed in synovial tissue biopsy samples from patients with rheumatoid arthritis (RA). This study was carried out to determine the effects of tumor necrosis factor (TNF) blockers and glucocorticoids on mPGES-1 and cyclooxygenase (COX) expression, as well as biosynthesis of PGE(2) in rheumatoid joints. METHODS: In vitro effects of TNF blockers and dexamethasone on the PGE(2) biosynthetic pathway were examined in RA synovial fluid mononuclear cells (SFMCs) by flow cytometry. PGE(2) levels in culture supernatants were measured by enzyme immunoassay. Expression of enzymes responsible for PGE(2) synthesis ex vivo was evaluated by immunohistochemistry in synovial biopsy samples obtained from 18 patients before and after treatment with TNF blockers and from 16 patients before and after intraarticular treatment with glucocorticoids. Double immunofluorescence was performed using antibodies against mPGES-1, COX-1, COX-2, and CD163. RESULTS: Double immunofluorescence revealed that mPGES-1 and COX-2 were colocalized in SFMCs as well as in RA synovial tissue cells. The addition of either TNF blockers or dexamethasone suppressed lipopolysaccharide-induced mPGES-1 and COX-2 expression in synovial fluid monocyte/macrophages in vitro and decreased the production of PGE(2). Intraarticular treatment with glucocorticoids significantly reduced both mPGES-1 and COX-2 expression in arthritic synovial tissue ex vivo. The number of COX-1-expressing cells in synovial tissue was also significantly decreased by glucocorticoid treatment. In contrast, neither mPGES-1 nor COX-2 expression in synovial tissue was significantly suppressed by anti-TNF therapy. CONCLUSION: These data are the first to demonstrate the effects of antirheumatic treatments on mPGES-1 expression in RA and suggest that the inhibition of PGE(2) biosynthesis, preferably by targeting mPGES-1, might complement anti-TNF treatment for optimal antiinflammatory results in RA.
Assuntos
Anticorpos Monoclonais/farmacologia , Antirreumáticos/farmacologia , Glucocorticoides/farmacologia , Imunoglobulina G/farmacologia , Oxirredutases Intramoleculares/biossíntese , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Biomarcadores/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Quimioterapia Combinada , Etanercepte , Feminino , Glucocorticoides/administração & dosagem , Glucocorticoides/uso terapêutico , Humanos , Imunoglobulina G/uso terapêutico , Infliximab , Injeções Intra-Articulares , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Prostaglandina-E Sintases , Receptores do Fator de Necrose Tumoral/uso terapêutico , Líquido Sinovial/citologia , Líquido Sinovial/efeitos dos fármacos , Líquido Sinovial/metabolismo , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/enzimologia , Membrana Sinovial/patologiaRESUMO
A panel of CD4 T-cell clones was isolated from synovial fluid by single cell flow cytometry from a patient with treatment-resistant Lyme arthritis using a DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with outer surface protein A (OspA) peptide164-175, an immunodominant epitope of Borrelia burgdorferi. Sequencing of the T-cell receptors of the OspA reactive clones showed significant skewing of the T-cell receptor repertoire. Of the 101 T-cell clones sequenced, 81 possessed TCR beta chains that were present in at least one other clone isolated. Complete sequencing of both alpha and beta chains of a subset of clones showed that at least two distinct T-cell clones were expanded in vivo. Binding studies using a panel of Ala-substituted peptide ligands were performed to determine potential MHC binding sites of the OspAp164-175 to DRB1*0401. In addition, T-cell clones were tested functionally for their reactivity to the wild-type peptide as well as to altered peptide ligands (APLs) and peptide libraries based on the OspA epitope in order to determine the TCR contact residues and the stringency in T cell recognition. We are among the first to define the characteristics of TCR usage of T cells isolated from an inflamed immune compartment in an individual with an autoimmune disease potentially triggered by a microbial antigen.
Assuntos
Antígenos de Superfície/química , Proteínas da Membrana Bacteriana Externa/química , Linfócitos T CD4-Positivos/imunologia , Lipoproteínas/química , Doença de Lyme/imunologia , Sequência de Aminoácidos , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas , Células Clonais , Antígenos HLA-DR/metabolismo , Cadeias HLA-DRB1 , Humanos , Lipoproteínas/genética , Lipoproteínas/imunologia , Ativação Linfocitária , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Líquido Sinovial/química , Líquido Sinovial/citologia , Líquido Sinovial/imunologiaRESUMO
CD25+CD4+ regulatory T cells participate in the regulation of immune responses. We recently demonstrated the presence of CD25brightCD4+ regulatory T cells with a capacity to control T cell proliferation in the joints of patients with rheumatoid arthritis. Here, we investigate a possible accumulation of these regulatory T cells in the inflamed joint of different rheumatic diseases including rheumatoid arthritis. The studies are also extended to analyze whether cytokine production can be suppressed by the regulatory T cells. Synovial fluid and peripheral blood samples were obtained during relapse from 36 patients with spondyloarthropathies, 21 adults with juvenile idiopathic arthritis and 135 patients with rheumatoid arthritis, and the frequency of CD25brightCD4+ T cells was determined. Of 192 patients, 182 demonstrated a higher frequency of CD25brightCD4+ T cells in synovial fluid than in peripheral blood. In comparison with healthy subjects, the patients had significantly fewer CD25brightCD4+ T cells in peripheral blood. For functional studies, synovial fluid cells from eight patients were sorted by flow cytometry, and the suppressive capacity of the CD25brightCD4+ T cells was determined in in vitro cocultures. The CD25brightCD4+ T cells suppressed the production of both type 1 and 2 cytokines including interleukin-17, as well as proliferation, independently of diagnosis. Thus, irrespective of the inflammatory joint disease investigated, CD25brightCD4+ T cells were reduced in peripheral blood and enriched in the joint, suggesting an active recruitment of regulatory T cells to the affected joint. Their capacity to suppress both proliferation and cytokine secretion might contribute to a dampening of local inflammatory processes.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Artropatias/patologia , Articulações/patologia , Receptores de Interleucina-2/metabolismo , Doenças Reumáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Juvenil/patologia , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/fisiologia , Doença Crônica , Feminino , Humanos , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Espondiloartropatias/patologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/fisiologiaRESUMO
In the homeostasis of the immune system regulatory cells play a major role. Removal of one group of regulatory cells, the CD25(+)CD4(+) T cells, leads to autoimmune manifestations in experimental animal models, and reintroduction of this population prevents disease. This study addresses the role of such regulatory T cells in humans with an autoimmune disease, where we demonstrate the presence of CD25(bright)CD4(+) T cells in the target organ of patients with active rheumatoid arthritis. The patients displayed an enrichment of CD25(bright)CD4(+) T cells in synovial fluid as compared to peripheral blood. These cells are functional regulatory cells, as they were able to suppress in vitro proliferation of autologous T cells, both from synovial and peripheral blood origin. Although the frequency of CD25(bright)CD4(+) T cells varied between patients, it was found to be constant over time in any one joint during each relapse. Numbers were also comparable in two inflamed knee joints of one and the same patient, emphasizing the symmetry of the disease. In summary, it is striking that in addition to all activated, potentially pathological T cells the synovial fluid from RA patients also contains CD25-expressing CD4(+) T cells with a regulatory capacity.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Receptores de Interleucina-2/metabolismo , Adulto , Idoso , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/citologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Articulações/imunologia , Articulações/patologia , Pessoa de Meia-Idade , Líquido Sinovial/imunologiaRESUMO
OBJECTIVE: To investigate whether CpG oligodeoxynucleotides (ODNs) can induce or accelerate arthritis in rats. METHODS: The CpG-induced response was studied by recording joint inflammation, cell activation in draining lymph nodes, and levels of the acute-phase reactant alpha(1)-acid glycoprotein (AGP) in sera. The role of T cells was investigated by in vivo administration of monoclonal antibodies specific for the T cell receptor alpha/beta (TCRalpha/beta), followed by analysis of cell phenotypes by flow cytometry. RESULTS: One intradermal injection of CpG ODN emulsified with Freund's incomplete adjuvant (IFA) induced arthritis in LEW and LEW.1AV1 rats, while the control ODN sequence without CpG motifs or IFA alone did not trigger disease. The CpG/IFA and control-ODN/IFA injections induced lymphoplasia as well as elevated levels of interleukin-1beta and interferon-gamma messenger RNA in lymph nodes. The arthritis was preceded by elevated levels of AGP in serum. In vivo administration of anti-TCRalpha/beta antibodies after disease induction caused decreased expression of the TCR-CD3 complex on circulating T cells and ameliorated the arthritis. CONCLUSION: We demonstrated that injection with immunostimulatory CpG, both in phosphorothioate-modified and native forms, can induce a T cell-dependent joint-specific inflammation in LEW and LEW.1AV1 rat strains. This arthritis is preceded by signs of activation of the innate immune system. Since unmethylated CG dinucleotides are common in bacterial DNA but rare in mammalian DNA, our results indicate that exposure to bacterial DNA during infection may contribute to arthritis induction by amplifying the innate immune response.