ABSTRACT
OBJECTIVE: The aim of this study was to investigate pentraxin-3 (PTX3) as a potential biomarker of inflammatory activity in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) at baseline and 6 month follow-up in a longitudinal cohort. METHOD: Plasma PTX3 levels were measured in 79 newly diagnosed or relapsing AAV patients at baseline and 6 month follow-up, and in 23 healthy controls. Urinary PTX3 levels were measured in 34 of the patients. C-reactive protein (CRP), creatinine, and albuminuria were measured and the cumulative glucocorticoid dose at inclusion was calculated. The Birmingham Vasculitis Activity Score (BVAS) was assessed at baseline and follow-up. RESULTS: Plasma PTX3 levels were significantly higher at baseline than at 6 months (2.85 vs 1.23 ng/mL, p < 0.001). Plasma and urinary PTX3 levels correlated with BVAS at baseline (ρ = 0.45, p < 0.001, and ρ = 0.49, p = 0.008, respectively). A significant correlation between both plasma and urinary PTX3 levels and estimated glomerular filtration rate and albuminuria was found. However, there was no correlation between plasma and urinary PTX3 levels. At baseline, plasma and urinary PTX3 levels were significantly higher in patients with kidney involvement. PTX3 levels did not correlate with CRP, nor was there a correlation between CRP levels and BVAS at baseline. CONCLUSION: Plasma and urinary PTX3 seem to reflect disease activity in AAV better than the commonly used CRP. PTX3 may have a potential role as a biomarker in monitoring disease activity in AAV patients, particularly in patients with kidney involvement.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis , C-Reactive Protein , Humans , C-Reactive Protein/metabolism , Albuminuria , Biomarkers , Antibodies, Antineutrophil CytoplasmicABSTRACT
Arthritis is a common clinical feature of systemic lupus erythematosus (SLE) and is usually non-erosive, as opposed to rheumatoid arthritis (RA). While RA synovial pathology has been extensively studied, little is known about the pathophysiology of lupus arthritis. Here, we aimed to explore the cytokine and cellular compartments in synovial fluids of SLE patients with arthritic manifestations. Acellular synovial fluid and paired serum samples from SLE patients (n = 17) were analyzed with cytokine bead array for T helper-associated cytokines. From two SLE patients, synovial fluid mononuclear cells (SFMC) could also be captured and were analyzed by multiparameter flow cytometry to dissect T cell, B cell, monocyte and dendritic cell phenotypes. SLE-derived SFMC were further stimulated in vitro to measure their capacity for producing interferon (IFN)-γ and interleukin (IL)-17A. All patients fulfilled the ACR 1982 classification criteria for SLE. Clinical records were reviewed to exclude the presence of co-morbidities such as osteoarthritis or overlap with RA. IL-17A and IL-6 levels were high in SLE synovial fluid. A clear subset of the synovial CD4+ T cells expressed CCR6+ , a marker associated with T helper type 17 (Th17) cells. IL-17A-production was validated among CD4+ CCR6+ T cells following in-vitro stimulation. Furthermore, a strong IFN-γ production was observed in both CD4+ and CD8+ cells. Our study shows high IL-17A and IL-6 levels in synovial fluids of patients with lupus arthritis. The Th17 pathway has been implicated in several aspects of SLE disease pathogenesis and our data also point to Th17 involvement for lupus arthritis.
Subject(s)
Arthritis/immunology , Interleukin-17/immunology , Interleukin-6/immunology , Lupus Erythematosus, Systemic/immunology , Synovial Fluid/immunology , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytokines/immunology , Female , Humans , Interferon-gamma/immunology , Male , Middle Aged , Th17 Cells/immunologyABSTRACT
Objectives: To examine whether signs of an active human cytomegalovirus (HCMV) infection are present in affected joints of patients with rheumatoid arthritis (RA).Method: Polymorphonuclear leucocytes (PMNLs) were obtained from synovial fluid (SF) of 17 RA patients and were analysed for HCMV-pp65 and HCMV-immediate early (IE) proteins using the antigenemia assay. Peripheral blood (PB) and SF obtained from these 17 patients and from 17 additional RA patients (n = 34) were tested for HCMV-IE and pp150 DNA with Taqman polymerase chain reaction. Plasma samples from the patients were analysed for HCMV-immunoglobulin M (IgM) and immunoglobulin G (IgG) by enzyme-linked immunosorbent assay and compared to 71 healthy gender-matched blood donors.Results: HCMV-pp65 protein was detected in 65% of synovial PMNL samples, but in only 18% of PMNLs from PB. In contrast, HCMV IE protein was not found in any of the analysed PMNL samples. On the DNA level, HCMV-IE and pp150 DNA was detected in SF of 13/32 (41%) and 14/23 (61%) of RA patients, respectively. HCMV-IE and pp150 DNA was also found in 24/33 (73%) and in 16/24 (67%) of PB samples obtained from RA patients, respectively. HCMV IgG seroprevalence was 76% in RA patients as well as in healthy controls, while only one RA patient was positive for specific IgM.Conclusions: HCMV pp65 antigen was found in PMNLs from SF of RA patients, indicating an active infection in the affected joint. Future studies are needed to determine whether HCMV infection can aggravate the inflammatory process in these patients.
Subject(s)
Arthritis, Rheumatoid/virology , Cytomegalovirus/isolation & purification , Neutrophils/virology , Female , Humans , Immunoglobulin G , Male , Synovial Membrane/virology , Viral Matrix ProteinsABSTRACT
Objective Rituximab-mediated late-onset neutropenia (LON) has been described in various diseases. We investigated its occurrence, consequences and contributing factors in patients with systemic lupus erythematosus (SLE). Methods Rituximab-treated patients from the Karolinska University Hospital ( n = 107) were surveyed. LON was defined as an absolute neutrophil count <1500 cells/µl, occurring four weeks to two years following rituximab treatment, or later during sustained B-cell depletion. Serum levels of B-cell-related cytokines and growth factors of the myeloid lineage were determined using enzyme-linked immunosorbent assay. Results Thirty-two patients (29.9%) developed LON after a median time of 201.5 days. Thirteen patients were admitted to the hospital; 10 due to fever. Three patients developed critical conditions. BAFF levels increased from baseline (median: 0.62 ng/ml) to the post-treatment evaluation (median: 1.16 ng/ml; p < 0.001); post-treatment levels were higher in the LON group ( p = 0.021). APRIL levels were higher in the LON group both at baseline (median: 1.54 versus 1.15 ng/ml; p = 0.027) and post-treatment (median: 2.39 versus 1.11 ng/ml; p = 0.011). IL-6 and GM-CSF levels decreased in the non-LON group ( p < 0.001), but not in LON patients. High baseline disease activity predicted LON development (OR: 4.1; 95% CI: 1.1-15.2 for SLEDAI-2K > 8). No association with neutropenia prior to rituximab treatment was documented. Conclusion Post-rituximab LON was a common complication. Although the phenomenon was predominantly self-limiting, several patients developed severe conditions. Distinct roles of BAFF and APRIL are implicated: BAFF may contribute to LON development, whereas high APRIL levels may be predictive. Rituximab-treated SLE patients should be monitored for neutrophil counts, fever and infections.
Subject(s)
Antirheumatic Agents/adverse effects , Lupus Nephritis/drug therapy , Neutropenia/chemically induced , Rituximab/adverse effects , Adult , B-Cell Activating Factor/blood , Female , Humans , Lupus Nephritis/blood , Male , Middle Aged , Retrospective Studies , Tumor Necrosis Factor Ligand Superfamily Member 13/bloodABSTRACT
BACKGROUND: Rituximab (RTX) is being used increasingly in anti-neutrophil cytoplasmatic antibody (ANCA)-associated vasculitis (AAV). Late-onset neutropenia (LON) and risks of infections have been observed following RTX therapy in rheumatological diseases including granulomatosis with polyangiitis (GPA) but data on microscopic polyangiitis (MPA) are lacking. METHOD: We studied the occurrence of LON in 59 AAV (47 GPA/12 MPA) patients treated with RTX. Patient charts were retrospectively reviewed for the occurrence of LON and clinical data were extracted and included in the analysis. RESULTS: Seven of the total 59 patients (11.9%) developed LON after a median time of 86 days (range 56-168 days) since their latest RTX treatment. Of these seven LON patients, 5/47 (10.6%) had a diagnosis of GPA and 2/12 (16.7%) of MPA. Three of the patients developed LON after the first RTX treatment and four had received repeated courses. Five LON patients developed infectious symptoms. Six of the patients were hospitalized. Retreatment with RTX was given in three cases without further LON episodes. CONCLUSIONS: LON is a potentially severe side-effect of RTX occurring in both GPA and MPA and may develop after both single and repeated treatment courses. As infections are commonly seen, the condition requires an increased awareness. No predisposing factors for LON were identified.
Subject(s)
Antirheumatic Agents/adverse effects , Granulomatosis with Polyangiitis/drug therapy , Microscopic Polyangiitis/drug therapy , Neutropenia/chemically induced , Rituximab/adverse effects , Adult , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/drug therapy , Female , Humans , Male , Middle Aged , Retrospective StudiesABSTRACT
Synovial fluid from rheumatic joints displays a well-documented enrichment of forkhead box protein 3 (FoxP3)(+) regulatory T cells (tissue Tregs ). However, we have previously demonstrated that the mere frequency of FoxP3 expressing cells cannot predict suppressive function. Instead, extrinsic factors and the functional heterogeneity of FoxP3(+) Tregs complicate the picture. Here, we investigated FoxP3(+) Tregs from blood and synovial fluid of patients with rheumatic disease in relation to Helios expression by assessing phenotypes, proliferative potential and cytokine production by flow cytometry. Our aim was to investigate the discriminatory potential of Helios when studying FoxP3(+) Tregs in an inflammatory setting. We demonstrate that the majority of the synovial FoxP3(+) CD4(+) T cells in patients with inflammatory arthritis expressed Helios. Helios(+) FoxP3(+) Tregs displayed a classical Treg phenotype with regard to CD25 and cytotoxic T lymphocyte-associated antigen (CTLA)-4 expression and a demethylated Treg -specific demethylated region (TSDR). Furthermore, Helios(+) FoxP3(+) T cells were poor producers of the effector cytokines interferon (IFN)-γ and tumour necrosis factor (TNF), as well as of the anti-inflammatory cytokine interleukin (IL)-10. The less abundant Helios(-) FoxP3(+) T cell subset was also enriched significantly in the joint, displayed a overlapping phenotype to the double-positive Treg cells with regard to CTLA-4 expression, but differed by their ability to secrete IL-10, IFN-γ and TNF upon T cell receptor (TCR) cross-linking. We also demonstrate a striking enrichment of IL-1R1 expression in synovial CD4(+) T cells that was restricted to the CD25-expressing FoxP3 population, but independent of Helios. IL-1R1 expression appears to define a tissue Treg cell phenotype together with the expression of CD25, glucocorticoid-induced TNF receptor family-related gene (GITR) and CTLA-4.
Subject(s)
Arthritis, Rheumatoid/immunology , CD4-Positive T-Lymphocytes/immunology , Ikaros Transcription Factor/metabolism , Joints/immunology , Receptors, Interleukin-1 Type I/biosynthesis , Adult , Aged , Aged, 80 and over , CTLA-4 Antigen/biosynthesis , Female , Flow Cytometry , Forkhead Transcription Factors/metabolism , Humans , Interferon-gamma/metabolism , Interleukin-10/biosynthesis , Interleukin-2 Receptor alpha Subunit/biosynthesis , Male , Middle Aged , Synovial Fluid/cytology , Synovial Fluid/immunology , T-Lymphocytes, Regulatory/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. We investigated and compared a panel of polarization protocols of blood-derived monocytes to achieve a stable, optimal and effective regimen for in vitro induction of immunosuppressive human macrophages, evaluating their surface receptor expression, cytokine profile, scavenging function and ability to suppress T-cell proliferation. Importantly, we assessed the effect of copolarization or secondary pro-inflammatory stimulation of a primary anti-inflammatory activation phenotype. A combination of IL-4/IL-10/TGF-ß yielded a relatively stable and dominant immunosuppressive phenotype characterized by higher IL-10 production and down-regulated TNF-α, IL-6, CD86, CD274 and MHC II expression. Functionally, IL-4/IL-10/TGF-ß-stimulated macrophages (M2) had a potent deactivating effect on a subsequent pro-inflammatory LPS/IFNγ-activated macrophage (M1) stimulation and significantly suppressed T-cell proliferation. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti-inflammatory using this protocol. Pre-differentiation with GM-CSF or M-CSF was further demonstrated to enhance final M1/M2 activation status. Our findings indicate a robust polarization protocol for generation of specific immunosuppressive human monocyte-derived macrophages.
Subject(s)
Immune Tolerance , Immunosuppression Therapy/methods , Macrophages/immunology , Monocytes/immunology , T-Lymphocytes/immunology , B7-2 Antigen/metabolism , B7-H1 Antigen/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Histocompatibility Antigens Class II/metabolism , Humans , Interleukin-10/immunology , Interleukin-4/immunology , Macrophage Colony-Stimulating Factor/immunology , Phenotype , Transforming Growth Factor beta/immunologyABSTRACT
Proinflammatory CD4(+) CD28(null) T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4(+) CD28(null) T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4(+) T cells. Forty-four patients with RA, displaying a distinct CD4(+) CD28(null) T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-γ, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4(+) CD28(null) T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4(+) CD28(+) T cells and produced higher levels of both IFN-γ and TNF after TCR cross-linking. CD4(+) CD28(null) T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4(+) CD28(null) cells from the blood, a significant production was observed in CD4(+) CD28(null) T cells from synovial fluid. CD4(+) CD28(null) T cells were not only found to differ from conventional CD4(+) CD28(+) T cells in the circulation, but we could also demonstrate that synovial CD4(+) CD28(null) T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28(null) population.
Subject(s)
Arthritis, Rheumatoid/immunology , CD28 Antigens/analysis , CD4-Positive T-Lymphocytes/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Cytokines/biosynthesis , DNA Methylation , Female , Humans , Interferon-gamma/genetics , Male , Middle Aged , Receptors, Chemokine/analysisABSTRACT
Many genetic variants associate with the risk of developing rheumatoid arthritis (RA); however, their functional roles are largely unknown. Here, we aimed to investigate whether the RA-associated serotonin receptor 2A (HTR2A) haplotype affects T-cell and monocyte functions. Patients with established RA (n=379) were genotyped for two single-nucleotide polymorphisms (SNPs) in the HTR2A locus, rs6314 and rs1328674, to define presence of the risk haplotype for each individual. Patients with and without the RA-associated TC haplotype were selected and T-cell and monocyte function was monitored following in vitro stimulations with staphylococcal enterotoxin B and lipopolysaccharide (LPS) using multiparameter flow cytometry. Within the cohort, 44 patients were heterozygous for the TC haplotype (11.6%) while none were homozygous. Upon stimulation, T cells from TC-carrier patients produced more proinflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-17 (IL-17) and interferon gamma (IFN-γ)) and monocytes produced higher levels of TNF-α compared with patients carrying the non-TC haplotype (P<0.05 and 0.01, respectively). Such cytokine production could be inhibited in the presence of the selective 5-HT2 receptor agonist (2,5-Dimethoxy-4-iodoamphetamine, DOI); interestingly, this effect was more pronounced in TC carriers. Our data demonstrate that association of RA with a distinct serotonin receptor haplotype has functional impact by affecting the immunological phenotype of T cells and monocytes.
Subject(s)
Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Genetic Variation , Monocytes/immunology , Receptor, Serotonin, 5-HT2A/genetics , T-Lymphocytes/immunology , Adult , Aged , Aged, 80 and over , Amphetamines/pharmacology , Enterotoxins/immunology , Genotype , Haplotypes , Humans , Interferon-gamma/metabolism , Interleukin-17/metabolism , Lipopolysaccharides/immunology , Middle Aged , Polymorphism, Single Nucleotide , Serotonin Receptor Agonists/pharmacology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: It is hypothesized that the in utero environment in allergic mothers can affect the neonatal immune responses. The aim of this study was to analyse the effect of maternal allergic disease on cord blood mononuclear cell (CBMC) phenotype and proliferative responses upon allergen stimulation. METHODS: Peripheral blood mononuclear cells (PBMC) from 12 allergic and 14 nonallergic mothers and CBMC from their children were analysed. In the mothers, we determined cell proliferation, production of IL-4 and expression of FOXP3 in response to allergen stimulation. In the children, we evaluated cell proliferation and FOXP3 expression following allergen stimulation. Furthermore, expression of different homing markers on T cells and regulatory T cells and maturity of the T cells and B cell subsets were evaluated directly ex vivo. RESULTS: The timothy- and birch-allergic mothers responded with increased proliferation and/or IL-4 production towards timothy and birch extract, respectively, when compared to nonallergic mothers. This could not be explained by impairment of FOXP3(+) regulatory T cells in the allergic mothers. CBMC proliferation and FOXP3 expression in response to allergens were not affected by the allergic status of the mother. Also, phenotype of T cells, FOXP3(+) regulatory T cells and B cells was not affected by the allergic status of the mother. CONCLUSION: Our results suggest that maternal allergic disease has no effect on the neonatal response to allergens or the phenotype of neonatal lymphocytes. The factors studied here could, however, still affect later development of allergy.
Subject(s)
B-Lymphocytes/immunology , Hypersensitivity/immunology , Infant, Newborn/immunology , T-Lymphocytes/immunology , Adult , Allergens/immunology , B-Lymphocytes/cytology , Cell Proliferation , Cell Separation , Female , Fetal Blood/cytology , Fetal Blood/immunology , Flow Cytometry , Humans , Mothers , Phenotype , Pregnancy , T-Lymphocytes/cytologyABSTRACT
OBJECTIVE: To investigate serum levels of B cell activating factor (BAFF) in patients with myositis and correlate these to autoantibody profile, clinical phenotype and treatment. METHODS: BAFF levels in sera from 49 patients with dermatomyositis, 44 with polymyositis, 6 with inclusion body myositis and 30 matched controls were measured by ELISA. Specific autoantibodies were detected by line blot and western blot assays. RESULTS: Serum levels of BAFF were significantly higher in patients compared to healthy controls (p = 0.003). Patients with anti-Jo-1 autoantibodies had higher BAFF levels than control individuals (p<0.003) or patients without any specific autoantibodies (p<0.05). Patients with dermatomyositis had higher BAFF levels compared to polymyositis (p<0.05). Patients with interstitial lung disease (ILD) had higher BAFF levels than patients without ILD (p<0.05) or controls (p<0.01) but this could be explained by presence of anti-Jo-1 autoantibodies. BAFF levels correlated with serum creatine kinase (CK) (rs = 0.365, p = 0.0005) but not with C-reactive protein (CRP) levels. A negative correlation of BAFF levels with glucocorticoid daily dose for all patients (rs = -0.292, p = 0.003) and with cumulative glucocorticoid doses in early myositis cases (rs = -0.659, p<0.001) was recorded. CONCLUSION: Our finding of elevated serum levels of BAFF in patients with myositis with described phenotypes together with the correlations between levels of BAFF and CK and a negative correlation with dose of glucocorticoids, indicate that BAFF could be a potential therapeutic target in such cases.
Subject(s)
Autoantibodies/blood , B-Cell Activating Factor/blood , Myositis/blood , Adolescent , Adult , Aged , Analysis of Variance , Antibodies, Antinuclear/blood , Autoantibodies/immunology , C-Reactive Protein/analysis , Case-Control Studies , Child , Creatine Kinase/blood , Dermatomyositis/blood , Dermatomyositis/drug therapy , Dermatomyositis/immunology , Female , Glucocorticoids/therapeutic use , Humans , Male , Middle Aged , Myositis/drug therapy , Myositis/immunology , Polymyositis/blood , Polymyositis/drug therapy , Polymyositis/immunology , Statistics, Nonparametric , Tumor Necrosis Factor Ligand Superfamily Member 13/blood , Young AdultABSTRACT
BACKGROUND: Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles. OBJECTIVE: To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles. METHODS: Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, alpha-enolase peptide-1 and the C1-epitope of type II collagen (C1(III)). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed. RESULTS: 72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients' sera and in 41% for both citrullinated alpha-enolase peptide-1 and citrullinated C1(III). These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles. CONCLUSIONS: Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.
Subject(s)
Arthritis, Rheumatoid/immunology , Autoantibodies/blood , HLA-DR Antigens/genetics , Peptides, Cyclic/immunology , Adult , Aged , Aged, 80 and over , Alleles , Arthritis, Rheumatoid/genetics , Autoantigens/immunology , Biomarkers, Tumor/immunology , Citrulline/immunology , Collagen Type II/immunology , Cross Reactions , DNA-Binding Proteins/immunology , Female , Fibrinogen/immunology , Genotype , HLA-DRB1 Chains , Humans , Immunoglobulin A/biosynthesis , Male , Middle Aged , Phosphopyruvate Hydratase/immunology , Tumor Suppressor Proteins/immunology , Young AdultABSTRACT
OBJECTIVE: To analyse the distribution of FOXP3+CD25+CD4+ regulatory T cells (Treg) in peripheral blood, synovial fluid and tissue of patients with rheumatic disease during relapse and after local treatment. METHODS: FOXP3 expression was assessed by flow cytometry, immunohistochemistry, immunofluorescence and real-time polymerase chain reaction (RT-PCR). The functional suppressive capacity of Treg was analysed after co-culture with effector CD4+CD25- T cells through assessment of proliferation and cytokine secretion. RESULTS: It was shown that FOXP3 protein and mRNA expression in synovial fluid T cells was not confined solely to CD25(bright) T cells as seen in blood, but included CD25(intermediate) and even CD25(neg) T cells. Indeed, synovial fluid CD25(high) T cells showed similar suppressive capacity as CD25(bright) T cells, indicating the presence of functional Treg in T cells with lower intensity of CD25. In synovial tissue, FOXP3+ cells were present in low numbers within T-cell infiltrates and decreased further after intra-articular glucocorticosteroid administration, in parallel with the general reduction in inflammation. CONCLUSIONS: Identification of synovial fluid FOXP3+ Treg with varying intensities of CD25 opens up possibilities for thorough characterisation of this important T-cell subset in the inflammatory compartment. However, only scarce synovial membrane expression of FOXP3 was found even in the absence of overt inflammation, suggesting that the synovial membrane is a site that would benefit therapeutically from Treg expansion.
Subject(s)
Arthritis/immunology , Forkhead Transcription Factors/biosynthesis , Synovial Membrane/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Aged , Arthritis/drug therapy , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/immunology , CD4-Positive T-Lymphocytes/immunology , Coculture Techniques , Female , Flow Cytometry/methods , Forkhead Transcription Factors/genetics , Gene Expression , Glucocorticoids/administration & dosage , Glucocorticoids/pharmacology , Humans , Injections, Intra-Articular , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-7 Receptor alpha Subunit/metabolism , Male , Middle Aged , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Synovial Fluid/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/drug effectsABSTRACT
OBJECTIVE: An association to variations in the dendritic cell immunoreceptor (DCIR) gene with rheumatoid arthritis (RA) was recently shown. However, protein expression of DCIR has so far not been assessed in a disease setting. In the present work, we aimed to determine the cellular and tissue distribution of this receptor in healthy controls and in patients with RA before and after local glucocorticoid administration. METHODS: DCIR mRNA expression was evaluated by quantitative PCR (n=3) and protein expression by flow cytometry (n=18), immunohistochemistry (n=14) and double immunofluorescence (n=5). RESULTS: DCIR protein was not detected in healthy synovia. By contrast, expression was abundant on cells from rheumatic joints in synovial fluid and in tissue. Following corticosteroid treatment this expression was downregulated. Interestingly, DCIR could be detected on natural killer (NK) cells and T cells, and CD4+ and CD8+, as well as on monocytes, B cells, dendritic cells and granulocytes. The frequency of DCIR+ T cells and the level of surface expression were increased in the rheumatic joint compared to blood. In synovial fluid the typical DCIR+ T cells were large activated cells, whereas blasted DCIR+ T cells were not detected in blood. CONCLUSIONS: We demonstrate increased protein and mRNA expression of DCIR in RA, especially in the rheumatic joint. Expression was widespread and included a subpopulation of T cells. This suggests that the inflammatory synovial environment induces DCIR expression, and this may be related to synovial T cell function. Ligation of DCIR, or lack thereof, could contribute to the chronic inflammation characterising autoimmune diseases such as RA.
Subject(s)
Arthritis, Rheumatoid/immunology , Lectins, C-Type/biosynthesis , Membrane Glycoproteins/biosynthesis , Receptors, Immunologic/biosynthesis , Synovial Membrane/immunology , Adult , Aged , Aged, 80 and over , Arthritis, Rheumatoid/drug therapy , Dendritic Cells/immunology , Down-Regulation/drug effects , Female , Glucocorticoids/therapeutic use , Humans , Killer Cells, Natural/immunology , Lectins, C-Type/genetics , Lymphocyte Activation/immunology , Male , Membrane Glycoproteins/genetics , Middle Aged , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Synovial Fluid/immunology , T-Lymphocyte Subsets/immunology , Young AdultABSTRACT
BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.
Subject(s)
Allergens/immunology , Betula/immunology , Hypersensitivity/immunology , Interleukin-10/immunology , Pollen/immunology , T-Lymphocytes, Regulatory/immunology , Adult , Cells, Cultured , Coculture Techniques , Cytokines/immunology , Cytokines/pharmacology , Female , Homeostasis/drug effects , Homeostasis/immunology , Humans , Hypersensitivity/pathology , Immune Tolerance/drug effects , Immune Tolerance/immunology , Male , Middle Aged , T-Lymphocytes, Regulatory/pathology , Th1 Cells/immunology , Th1 Cells/pathology , Th2 Cells/immunology , Th2 Cells/pathologyABSTRACT
Regulatory T cells have recently been implicated in a number of human diseases, including rheumatoid arthritis. To investigate whether the presence of CD25+CD4+ regulatory T cells is a general finding in arthritic joints, synovial fluid of patients with different rheumatic diseases such as undifferentiated arthritides, systemic rheumatic diseases and reactive arthritis were investigated for the presence of such cells. In 95% of the patients, a higher frequency of CD25(bright)CD4+ T cells was found in synovial fluid as compared with peripheral blood. Both in vitro suppression experiments and FOXP3 mRNA analysis confirmed these cells to be natural regulatory T cells. Together with our previous data, we conclude that arthritic joints, irrespective of precise diagnosis and disease duration, are enriched with natural regulatory T cells. These results suggest that suppressor cells migrate to and/or multiply at the sites of inflammation as part of the immune responses' effort to combat injurious inflammation.
Subject(s)
Forkhead Transcription Factors/metabolism , Receptors, Interleukin-2/biosynthesis , Rheumatic Diseases/immunology , Synovial Fluid/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Adult , Aged , Aged, 80 and over , Arthritis/immunology , Arthritis/metabolism , Arthritis/pathology , Arthritis, Reactive/immunology , Arthritis, Reactive/metabolism , Arthritis, Reactive/pathology , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cell Movement/immunology , Cells, Cultured , Coculture Techniques , Female , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Humans , Male , Middle Aged , Rheumatic Diseases/metabolism , Rheumatic Diseases/pathology , Spondylitis, Ankylosing/immunology , Spondylitis, Ankylosing/metabolism , Spondylitis, Ankylosing/pathology , Synovial Fluid/cytology , Synovial Fluid/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
Myasthenia gravis (MG) is commonly regarded as the prototype of an antibody-mediated, organ-specific autoimmune disease. Antibodies against the acetylcholine receptor (AChR) on the muscle endplate trigger its typical clinical manifestations of weakness and fatiguability. T-B cell interactions are thought to play a crucial role in the pathogenesis of MG. OX40 (CD134), a costimulatory molecule that is expressed on activated CD4+ T-cells, might contribute to the development or pathogenesis of immune-mediated diseases such as rheumatoid arthritis and graft-versus-host disease. In the present study, we investigated the expression of OX40 on CD4+ T-cells from patients with MG and healthy individuals. Results from 36 MG patients and 28 healthy controls revealed that more freshly isolated CD4+ T-cells from MG patients expressed OX40 than cells from healthy individuals. High levels of antibodies against the AChR, thymic hyperplasia and onset at an early age were associated with elevated expression of OX40. Upon activation by various concentrations of anti-CD3 antibodies, CD4+ T-cells from MG patients showed a tendency toward higher levels of OX40 expression than cells from healthy individuals. Given the role of OX40 in the immune system, we conclude that OX40 might contribute to the development of MG.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Myasthenia Gravis/immunology , Receptors, Tumor Necrosis Factor/analysis , Adult , Case-Control Studies , Female , Flow Cytometry , Humans , Male , Receptors, OX40 , Statistics, NonparametricABSTRACT
CD4+ T cells lacking the costimulatory molecule CD28 have been described both in elderly individuals and in chronic inflammatory disorders, one being rheumatoid arthritis (RA). We, in this study, provide a comprehensive characterization of cell surface markers on and function of such CD28nullCD4+ T cells, as well as correlations with clinical parameters. We conclude that of all surface markers associated with these cells, only CD57 and CD11b are expressed on the majority of them. This CD28null population occurred in one-third of patients with RA and was independent of clinical characteristics. The population was persistent and expanded in peripheral blood, but was excluded from the joint in most patients. Functionally, CD28nullCD4+ T cells were potent effector memory cells with regard to their proliferation and cytokine-secretion profiles. This capacity correlated with a hitherto unpublished surface phenotype, the cells being uniformly CCR7- and CD43high. Moreover, a new terminally differentiated CD45RA+CCR7- population of CD4+ T cells was identified. We would like to suggest that in the unbalanced immune system of patients with autoimmune disease and chronic infection an expanded CD28nullCD4+ T-cell population able to secrete high levels of cytokines is likely to contribute to disease manifestations.
Subject(s)
Arthritis, Rheumatoid/immunology , CD28 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Adult , Aged , Arthritis, Rheumatoid/pathology , CD4-Positive T-Lymphocytes/cytology , Female , Flow Cytometry , HLA-DR Antigens/immunology , Humans , Immunophenotyping , Male , Middle Aged , Receptors, Antigen, T-Cell/immunology , T-Lymphocyte Subsets/immunologyABSTRACT
Transfer of CD4+ T cells to immune-deficient mice in the absence of the CD25+ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice. Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen. These cells resemble CD4+CD25+ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens. Development of colitis is dependent on accumulation of activated CD134L+ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4+CD25+ cells, indicating that regulatory T cells may control DC activation in vivo. Whilst inhibition of T-cell activation in vitro by CD4+CD25+ cells does not involve interleukin-10 and transforming growth factor-beta, these cytokines are required for the suppression of colitis. It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression. Recently, we identified CD4+CD25+ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.
Subject(s)
Antigens, CD , Colitis/immunology , Colitis/pathology , Inflammatory Bowel Diseases/immunology , Inflammatory Bowel Diseases/pathology , T-Lymphocyte Subsets/immunology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Antigens, Differentiation/immunology , Bacteria/pathogenicity , Dendritic Cells/immunology , Humans , Interleukin-10/immunology , Leukocyte Common Antigens/immunology , Membrane Glycoproteins , NAD+ Nucleosidase/immunology , Receptors, Interleukin-2/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Transforming Growth Factor beta/immunologyABSTRACT
Transfer of CD45RB(high) CD4+ T cells to immune-deficient mice in the absence of regulatory T cells leads to a Th1-mediated colitis. In this study, we show that intestinal inflammation is characterized by a 15-fold increase in the number of CD134L+ (OX40L+)-activated DC in the mesenteric lymph nodes (MLNs) compared with BALB/c mice. This was important functionally, as administration of an anti-CD134L mAb inhibited the proliferation of T cells in the MLNs as well as their expression of the gut-homing integrin alpha(4)beta(7). Most importantly, the anti-CD134L mAb completely blocked development of colitis. Surprisingly, CD134L was found to be expressed by a proportion of dendritic cells (DC) in the MLNs of unreconstituted SCID mice, suggesting that CD134L can be induced on DC in the absence of T cell-derived signals. These results indicate that some DC in the MLNs of SCID mice express an activated phenotype and that CD134L expression by these cells is involved in the development of colitis induced by T cell transfer. Accumulation of CD134L+ DC was inhibited by cotransfer of regulatory T cells, suggesting that inhibition of the accumulation of activated DC is one mechanism by which these cells prevent immune pathology.