FOXP3 expression in blood, synovial fluid and synovial tissue during inflammatory arthritis and intra-articular corticosteroid treatment.

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.

Increased serum levels of B cell activating factor (BAFF) in subsets of patients with idiopathic inflammatory myopathies.

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.

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls.

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.

FOXP3 identifies regulatory CD25bright CD4+ T cells in rheumatic joints.

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.

CD28nullCD4+ T cells--characterization of an effector memory T-cell population in patients with rheumatoid arthritis.

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.

In vitro evidence that subcutaneous administration of glatiramer acetate induces hyporesponsive T cells in patients with multiple sclerosis.

Glatiramer acetate (GA; Copaxone) is a random sequence polypeptide used in the treatment of relapsing remitting multiple sclerosis (RR MS). We have recently demonstrated that prior to treatment, GA induces proliferation of resting T cells and is not cross-reactive with myelin antigens. Daily GA injections induce a significant loss of this GA responsiveness, which is associated with the induction of highly cross-reactive Th2-type T cells potentially capable of suppressing inflammatory responses. The mechanism of action by which GA induces T cell nonresponsiveness leading to T cell receptor degeneracy in patients with RR MS is unknown. Here, we examined the effects of daily GA administration on the induction of T cell hyporesponsiveness. The frequency of GA-reactive T cells in peripheral blood of seven patients with RR MS was measured by limiting dilution analysis prior to and during 6 months of treatment. In addition, a model in which GA-reactive T cells were stimulated in vitro was developed to better characterize the selection of T cell populations over time. In vivo treatment with GA induced a decrease in GA-reactive T cell frequencies and hyporesponsiveness of CD4(+) T cell reactivity to GA in vitro that was only partially reversed by the addition of IL-2. These data suggest that T cell peripheral tolerance to GA was achieved in vivo during treatment. Thus, our in vitro data suggest that the underlying changes in GA-reactive CD4(+) T cell reactivity could be explained by the induction of T cell anergy and clonal elimination.

Molecular mimicry in Lyme arthritis demonstrated at the single cell level: LFA-1 alpha L is a partial agonist for outer surface protein A-reactive T cells.

Antibiotic treatment-resistant Lyme arthritis is a chronic inflammatory joint disease that follows infection with Borrelia burgdorferi (BB:). A marked Ab and T cell response to BB: outer surface protein A (OspA) often develops during prolonged episodes of arthritis. Furthermore, cross-reaction between the bacterial OspA and human LFA-1alpha(L) at the T cell level and the inability to detect BB: in the joint implicate an autoimmune mechanism. To analyze the nature of response to OspA and LFA-1alpha(L), we used OspA-specific T cell hybrids from DR4 transgenic mice, as well as cloned human cells specific for OspA(165-184), the immunodominant epitope, from five DRB1*0401(+) patients, using OspA-MHC class II tetramers. Although OspA(165-184) stimulated nearly all OspA-specific human T cell clones tested to proliferate and secrete IFN-gamma and IL-13, LFA-1alpha(L326-345) stimulated approximately 10% of these clones to proliferate and a greater percentage to secrete IL-13. Assays with LFA- or OspA-DR4 monomers revealed that higher concentrations of LFA-DR4 were needed to stimulate dual-reactive T cell hybrids. Our analysis at the clonal level demonstrates that human LFA-1alpha(L326-345) behaves as a partial agonist, perhaps playing a role in perpetuating symptoms of arthritis.

Decreases in interleukin-4 secretion by invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells in peripheral blood of patientswith relapsing-remitting multiple sclerosis.

The cytokine profile of invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells from patients with multiple sclerosis (MS) was compared with that of healthy controls. CD4(-)CD8(-)V alpha 24(+) T cells from the peripheral blood of 12 patients with relapsing-remitting MS (RR-MS), 5 patients with progressive MS (CP-MS), and 9 control individuals were directly sorted into single wells and expanded in vitro for analysis of IL-4 and IFN-gamma secretion; 315 V alpha 24J alpha Q T cell clones were generated and their T cell receptor (TCR) sequenced. T cell functionality was determined by examining cytokine secretion upon TCR cross-linking. RR-MS patients exhibited lower frequencies of IL-4 secreting CD4(-)CD8(-)V alpha 24J alpha Q T cell clones than patients with CP-MS and controls. No differences in IFN-gamma secretion were observed between the groups. An IL-4 positive cytokine profile could be correlated to the cloning efficiency of the V alpha 24J alpha Q T cells. We conclude that alterations in cytokine secretion patterns of CD4(-)CD8(-)V alpha 24J alpha Q T cells may influence the immune system and thus contribute to relapsing-remitting MS.

Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers.

We characterized antigen-specific CD4(+) T cells in six patients with treatment-resistant Lyme arthritis, using an HLA-DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with OspA(164-175), an immunodominant epitope of Borrelia burgdorferi. Direct analysis of OspA-tetramer binding CD4(+) cells in patients expressing the HLA-DRB1*0401 allele revealed frequencies of between <0.005 and 0.1% in peripheral blood (n = 6), and between <0.005 and 3.1% in synovial fluid (n = 3). OspA-tetramer(+)CD4(+) cells were directly cloned at 1 cell per well and expanded by mitogen and IL-2 on allogeneic feeder cells. As measured by [(3)H]thymidine incorporation, 95% of 168 T cell clones from synovial fluid binding the OspA-tetramer were antigen-reactive. Clones generated from peripheral blood revealed a different pattern of responsiveness when compared with clones generated from synovial fluid, as measured by proliferation, IFN-gamma, and IL-13 secretion. These clones, selected on the basis of their peptide binding, also responded to whole protein, but with a different cytokine profile. Our studies demonstrate that MHC class II tetramers can be used in humans to directly identify, isolate, and characterize antigen-reactive T cells from an inflammatory compartment.

Fasting enhances mucosal antigen specific B cell responses in rheumatoid arthritis.

OBJECTIVE: To evaluate the influence of fasting on the antigen specific immune responsiveness in patients with rheumatoid arthritis and healthy volunteers. METHODS: Seven rheumatoid arthritis patients and 17 healthy volunteers were immunised perorally or parenterally with influenza virus vaccine after a three to six day long period of total energy deprivation (water fast). The subsequent antigen specific antibody mediated immune response was recorded in the blood at the single cell level by the ELISPOT method. RESULTS: Short term starvation induced an enhanced antigen specific mucosa derived B lymphocyte response in rheumatoid arthritis patients and healthy volunteers. In contrast, the systemic B cell responses were not significantly altered by a total energy deprivation. CONCLUSIONS: Short term starvation increases the mucosa derived B cell responsiveness, while systemic responsiveness is largely unaffected. The similar pattern of response in rheumatoid arthritis patients and healthy volunteers indicates that fasting alters the mucosal immune response independently of medical treatment.

Expression of the mucosal lymphocyte integrin alpha E beta 7 and its ligand E-cadherin in the synovium of patients with rheumatoid arthritis.

The synovial expression of the mucosal lymphocyte integrin alpha E beta 7 and its ligand E-cadherin was analysed in order to study the relationship between T lymphocytes of the gastrointestinal tract and the synovium in patients with rheumatoid arthritis (RA). Immunohistochemical evaluation of synovium revealed that the alpha E beta 7-expression was detectable in 16 of the 38 samples examined. A concomitant examination on circulating lymphocytes by flow cytometry showed that alpha E beta 7-expressing lymphocytes occur less frequently in peripheral blood (PB). In vitro culture of lymphocytes increased the alpha E beta 7-expression on synovial lymphocytes six-fold, whereas PB lymphocytes expressed a two-fold increase. The addition of PHA to the culture medium did not dramatically increase the alpha E beta 7-expression on synovial lymphocytes, in contrast to PB lymphocytes where a 24-fold increase was detected. The addition of TGF-beta 1 to the culture of PB lymphocytes increased the alpha E beta 7-expression three-fold. E-cadherin expression was found in all synovial tissues analysed by immunohistochemistry. These results demonstrate that synovial T lymphocytes have the capacity to express the 'mucosal-type' integrin alpha E beta 7, possibly due to high levels of intra-articular TGF-beta 1. This expression might be of physiological importance since E-cadherin, the ligand for alpha E beta 7, is richly expressed by synoviocytes. In addition, the results indicate that a high in vivo expression of alpha E beta 7 is suppressed in the synovial tissue by a hitherto unknown mechanism.

The gut as an inductive site for synovial and extra-articular immune responses in rheumatoid arthritis.

OBJECTIVES: To analyse the immunological interactions between the gut lymphoid tissue, synovium, and peripheral blood compartments in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS: Patients with RA and AS and healthy controls were orally or parenterally immunised with an influenza virus vaccine. Antigen-specific antibody responses were measured at the single cell level by ELISPOT assay using lymphocytes isolated from peripheral blood and from enzymatically dispersed synovial tissues. RESULTS: Both oral and parenteral immunisations induced antigen-specific antibody-secreting cells in the synovial tissue of patients with RA. Parenterally immunised patients with RA showed significantly decreased antigen-specific antibody responses in peripheral blood compared with patients with AS and with healthy controls. In contrast, oral vaccination evoked comparable peripheral blood antibody responses in all three study groups. CONCLUSIONS: Despite a decreased immune responsiveness in the systemic compartment, the functional status of the gut-associated lymphoid tissue in patients with RA is intact. In addition, there is evidence that the lymphocytes in the inflamed joints are accessible for signals both from the systemic and mucosal compartments. The findings of immunological 'cross-talk' are relevant to future vaccination and tolerization procedures in patients with RA.

Expression of decay-accelerating factor on synovial lining cells in inflammatory and degenerative arthritides.

The decay-accelerating factor (DAF) is a complement regulatory cell surface protein that protects cells from complement-mediated lysis. We analysed synovial tissue biopsies from patients with chronic arthritides for the presence of DAF using immunohistochemistry. DAF was expressed in the synovial lining cell layer both in rheumatoid arthritis (RA) and in osteoarthritis (OA). DAF was also on vascular endothelial cells of synovial tissue. A significant correlation was found between the expression of DAF and of HLA-DR in the lining layer, suggesting that DAF may be induced during a local inflammatory response. In addition, C5b-9 terminal complement complexes were found in several DAF-positive cases, suggesting that complement activation might, in itself, induce DAF expression. We propose that the occurrence of DAF may represent a physiological mechanism for local complement regulation in synovial tissue.

Intra-articular immunization induces strong systemic immune response in humans.

There is no information available about immunological interactions between the synovial tissue compartment and systemic immunity in health and in disease. The aim of the present study was to evaluate effects of intra-articular immunization on the systemic immune responses in humans. Control subjects were immunized with the same dose of immunogen subcutaneously. Peripheral blood lymphocytes were analysed by spot-ELISA with respect to numbers of immunoglobulin-producing cells and antigen-specific antibody-secreting cells before and 1 week after immunization. Serum and salivary antibody levels were measured by an ELISA before and 14 days after the antigenic exposure. In addition, serum levels of interleukin-6 (IL-6) were analysed before and after immunization. The results indicate that the influenza virus antigen deposited in the joint space induces strong systemic antibody response of IgG, IgA and IgM classes. This response is significantly higher (P less than 0.05) than that of control subjects immunized subcutaneously. In contrast, no significant differences were detected between intra-articularly and subcutaneously immunized subjects with respect to mucosal immune responses. Increased serum levels of IL-6 were observed 1-2 weeks after the vaccination in both experimental groups. We conclude that human joints possess very efficient antigen-presenting properties enhancing systemic B cell reactivity.

Frequency and phenotypic feature of autoantibody-producing cell precursors in the preclinical stage of murine lupus.

The present study addresses the question of whether there is a difference in the frequencies of autoantibody-producing B-cell precursors in healthy compared with lupus-prone mouse strains. Spleen mononuclear cells (MNC) from 4-week-old (i.e. at the preclinical stage of lupus) mice were activated in vitro for 3 and 6 days with lipopolysaccharides (LPS) and the numbers of IgG, IgA and IgM autoantibody-producing cells were analysed by the ELISPOT assay. The results indicate a high frequency of IgM autoantibody-secreting cells after both 3 and 6 days in vitro stimulation. In spite of high frequencies of IgG-producing cells appearing late during the course of LPS stimulation, no IgG or IgA autoantibody producing cells were detected. No significant differences in the autoantibody repertoire were noted between healthy and lupus-prone mice, indicating that independent of the genetic background the immune system has the capacity to react with autoantibody production. Phenotypic analysis of LPS-induced, IgM-secreting B cells showed clearly that the majority of them were surface IgM+, CD5+ but Thy-1-.