Clonality of T lymphocytes expanded with IL-2 from rheumatoid arthritis peripheral blood,synovial fluid and synovial membrane

The association of rheumatoid arthritis (RA) with particular MHC class II genes suggests that autoantigen-spccific T cell clones present in joints could be central to the pathogenesis of the disease. Previous investigations on the clonal diversity of T cells infiltrating the rheumatoid synovial membrane have yielded conflicting results. With the use of Southern blot analysis, we investigated the clonalily of rheumatoid T cell lines expanded from peripheral blood, synovial fluid and synovial tissue. From peripheral blood lymphocyte (PBL) of RA patients and healthy normal controls, we also checked the consequences of two different culture conditions on the clonality of these cell lines. From control PBL, we found that in vitro non-specific expansion of non-clonal T cell populations does not create artefactual clonal selection. However, growing T cells in ritro with IL-2 seems to be able to lead to preferential expansion of cells bearing IL-2 receptor (IL-2R). We identified such in vivo activated IL-2-sensitive T cell clones frequently in RA synovial tissue (8/13) and more rarely in synovial fluid and peripheral blood (3/12). One patient presents the same T cell receptor gene rearrangements in synovial membrane of two affected joints. In RA synovial tissue, the frequency of these IL-2-responsive T cells is most prevalent among actively inflamed membranes removed early in the disease process. The role and the relevance to the disease of these I L-2-responsivc T cells remain to be elucidated.     

Evidence for enhanced interleukin 2 (IL-2) secretion and IL-2 receptor presentation by synovial fluid lymphocytes in rheumatoid arthritis.

Synovial fluid lymphocytes (SFL) and peripheral blood lymphocytes (PBL) from patients with rheumatoid arthritis (RA) and reactive oligoarthritis were investigated for activated T cells (Ia+SIg-), IL-2 receptor bearing cells (Tac+) and IL-2 production in vivo and in vitro. In contrast to negative results with blood, the synovial fluid of the arthritic joints contains considerable amounts of IL-2 activity (median: 11.8 mu/ml), elevated proportions of Ia+SIg- activated T cells (median: 12.5%) and of IL-2 receptor bearing cells (median: 2.5%). In vitro, after stimulation with several Concanavalin A (Con A) doses, SFL develop proportions of IL-2 receptor cells comparable to PBL. Furthermore, they produce higher values of IL-2 activity than comparable PBL cultures. The proportions of Ia+SIg- activated T cells increase only moderately after Con A stimulation compared to in vivo data, indicating different activated T cell subsets in the synovial fluid (Ia+SIg-, Tac+). The findings are discussed as an expression of an acute hyperactivation of lymphocytes in an inflamed joint.     

Characterization of synovial T lymphocytes in rheumatoid arthritis. I. Production of IL-2 dependent T cell clones from synovial fluid and peripheral blood.

Lymphocytes obtained from the peripheral blood (PBL) or synovial fluids (SFL) of patients with rheumatoid arthritis (RA) or other inflammatory joint diseases were compared with the PBL from normal individuals, by cloning under limiting dilution conditions in the presence of interleukin 2 (IL-2). The precursor frequency estimates of IL-2 responsive cells from these sources did not differ appreciably. However there were marked differences in the surface marker phenotypes of the clones derived from the PBL as compared to SFL. There was a predominance of OKT4-8+ cells in SFL from RA and non RA donors with inflammatory joint disease while PBL from all sources showed a marked prevalence of OKT4+8- cells. Comparison of precursor frequencies in the presence of PBL and SFL indicated that there were variations in the capacities of the SFL and PBL IL-2 dependent cells to grow on these fillers. SFL derived cells grew equally well on PBL or SFL filler, while PBL clones grew efficiently only on PBL fillers. Collectively these results indicate that there are marked differences in the surface phenotypes and growth requirements of IL-2 responsive SFL as compared to PBL.     

Synovial cells are potent antigen-presenting cells for superantigen,staphylococcal enterotoxin B (SEB)

There is ample evidence suggesting that superantigens may act as a triggering factor in the pathogenesis of rheumatoid arthritis (RA). We investigated whether superantigen could activate T cells in the presence of synovial cells. T cells were cultured with SEB in the presence of interferongamma (IFN-γ)-treated synovial cells. T cell proliferation and activation were assessed by ³H-thymidine incorporation and IL-2 production. The expression of HLA class II antigens and adhesion molecules on synovial ceils was detected by flow cytometer. In the presence of IFN-γ-treated synovial cells, T cells proliferated vigorously and produced IL-2 in response to SEB. A low SEB-induced T cell response was noticed in the presence of untreated synovial cells. Allogeneic as well as autologous IFN-γ-treated synovial cells markedly enhanced SEB-induced T cell proliferation. IFN-γ-treated synovial cells had increased expression of HLA class II antigens and intercellular adhesion molecule-1 (ICAM-1) adhesion molecules. MoAbs towards these antigens markedly inhibited the SEB-induced T cell response. These results indicate that activated synovial cells are potent antigen-presenting cells for SEB to T cells, and that superantigens may play a critical role in the pathogenesis of RA through activated synovial cells.     

Interleukin 2 inhibitor in synovial fluid.

Since evidence for the presence of IL-2 activity in rheumatoid synovial fluid is conflicting, we have assayed IL-2 activity in synovial fluid from patients with rheumatoid arthritis (RA) and other articular diseases (OAD). Using the IL-2-dependent murine T cell line CTLL, IL-2 activity was not demonstrable in synovial fluid tested at concentrations ranging from 50% to 0.02%. There was an inhibitory effect on IL-2 activity in the bioassay of synovial fluid from 16 of the 22 patients with RA and 15 of the 16 with OAD. This inhibitory activity was heat-labile, precipitable by ammonium sulphate, reversible with excess IL-2 and was not significantly altered by preincubation of synovial fluid with CTLL. The mean inhibitory activity of synovial fluid from patients with RA was significantly reduced in comparison with that of synovial fluid from patients with OAD. Sera also had an inhibitory effect on IL-2 activity; however sera from patients with RA were less inhibitory than control sera but were more inhibitory than sera from patients with systemic lupus erythematosus. The deficiency in synovial fluid of an inhibitor of IL-2 activity may be relevant to the pathogenesis of RA.     

Autoreactive T cells in rheumatic disease (1). Analysis of growth frequencies and autoreactivity of T cells in patients with rheumatoid arthritis and Lyme disease

A limiting dilution system was established in order to estimate frequencies of interleukin-2 (IL-2)-responsive, autoreactive and alloreactive T cells in samples of peripheral blood (PBL) and synovial fluid lymphocytes (SFL), from patients with rheumatoid arthritis (RA) and lyme disease, as well as from healthy donors and a patient with osteoarthrosis. The frequencies of IL-2-dependent T-cell colony formation were significantly higher in patients with RA and lyme disease (median: 1/287) as compared to controls (median: 1/1,313) indicating a preactivation of T cells in these patients in vivo. Autoreactivity was measured by the proliferative response of T-cell lines to autologous irradiated PBL as stimulating cells. The frequencies of autoreactive T cells in blood were significantly higher in patients (median: 1/2,615) as compared to controls (median: 1/19,607). There was no significant difference in autoreactive T-cell frequencies between the patients' SFL (median: 1/3,185) and PBL (median: 1/2,615). In every case the frequency of alloreactive T cells exceeded the frequency of autoreactive T cells. Most autoreactive T-cell lines were also alloreactive and were shown to be MHC Class II-restricted. There is evidence of a down regulation of autoreactive T cells by suppressor cells in peripheral blood in two cases with elevated autoreactive T-cell frequencies (one RA patient and one control patient suffering from a viral infection). In contrast, no suppression of autoreactive T cells was observed in the RA patients' SFL or in PBL and SFL from patients with lyme disease. These results suggest that the chronic inflammation observed in RA and lyme disease may be supported by an elevated number of autoreactive T cells in the absence of suppressive mechanisms.     

Proliferative responses of T-cell lines grown from joint fluids of patients with rheumatoid arthritis and other arthritides

T-cell lines were established by culturing, in the presence of IL-2, mononuclear cells obtained from synovial fluids (SF) of most (77%) patients with rheumatoid arthritis (RA). By contrast, SF from patients with osteoarthritis and crystal synovitis rarely yielded lines. The cell lines were identified as T-cells because they express the CD3 surface antigen and either CD8 or CD4. The ability of the T-cell lines to react with putative antigens was tested using autologous Epstein-Barr virus transformed B-cells (EBV-B) as antigen presenting cells. IgG failed to stimulate proliferation of any RA patients' T-cell lines while Type II collagen stimulated T-cell lines from one RA patient and from one with osteoarthritis. Some T-cell lines (17/26) took up more tritiated thymidine after three days culture with a mixture of autologous SF and irradiated autologous EBV-B than with either alone. The effect could not be ascribed to a mitogen in the RA SF as the fluids failed to stimulate blood mononuclear cells. We consider that RA SF contain a T-cell growth factor which can only exert its effect through an antigen presenting cell (EBV-B).     

Synovial fluids from patients with rheumatoid arthritis induce the differentiation of human promyelocytic leukemia cell line HL 60]

Bone marrow abnormalities have been found to play a role in the pathogenesis of rheumatoid arthritis (RA). Recent studies have also confirmed the presence of undifferentiated hematopoietic progenitor cells as well as the expression of stem cell factor in the synovial membranes in RA. The present study investigates whether RA synovial fluids contain factors that can induce differentiation of CD 14 positive/HLA-DR positive cells from undifferentiated hematopoietic cells. Synovial fluid specimens from 18 patients with RA and from 10 control patients, including patients with osteoarthritis and Behcet's disease, were studied. Human promyelocytic leukemia cell line HL 60 (5 x 10(4)/well) were cultured in the presence or absence of the synovial fluids for 5 days, after which the expression of CD 14 and HLA-DR was examined by flow cytometry. The induction of differentiation of CD 14 positive/HLA-DR positive cells or HLA-DR positive cells from HL 60 cells was significantly enhanced more in the presence of synovial fluids from RA patients than in the presence of those of control patients. However, the sera from the RA patients could not induce the differentiation of CD 14 positive/HLA-DR positive cells or HLA-DR positive cells from HL 60 cells. Most cytokines found in RA synovial fluid could not induce the differentiation of HL 60 cells. Of note, treatment of synovial fluids with hyaluronidase significantly decreased or abrogated their capacity to induce the differentiation of HLA-DR positive cells from HL 60. There was no significant difference in the concentration of hyaluronic acid in the synovial fluid between the RA patients and the control patients. These results indicate that there are factors that can induce differentiation of HLA-DR positive cells from undifferentiated hematopoietic cells in the synovial fluid of RA. The data also suggest that such differentiation factors might be related with qualitative abnormality of hyaluronic acid.     

Role of tumor necrosis factor-α in the regulation of activated synovial T cell growth: Downregulation of synovial T cells in rheumatoid arthritis patients

To characterize the role of tumor necrosis factor (TNF)-α in regulating synovial T cell growth, cell cycle progression associated with TNF-α in mitogen-activated synovial T cells of patients with rheumatoid arthritis (RA) were analyzed. After mitogen stimulation, the majority of synovial T cells in RA patients accumulated in S-phase. Anti-human TNF-α monoclonal antibody and soluble recombinant human TNF receptor (rhTNFR) can block S-phase accumulation. Furthermore, synovial fluid (SF) from RA patients was able to inhibit the proliferation of these S-phase-accumulated T cells. These data indicate that TNF-α could regulate activated synovial T cell growth by driving them into S-phase. Combined with the activities of other components of SF, TNF-α seems to play an important role in down-regulating activated synovial T cells in RA patients. In addition, the elevated level of soluble TNFR in the SF of disease-active RA patients is believed to be associated with the promotion of synovial T cell responses.     

T Cells Cloned from Human Rheumatoid Synovial Membrane Functionally Represent the Th 1 Subset

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease. Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th1 subset is cytolytic and produces high levels of IFN-gamma whereas the Th2 type of T cell produces IL-4. In order to investigate whether Th1 or Th2 type cells are present in the inflammatory synovial membrane in RA, a panel of synovial membrane derived T-cell clones (n = 19) was generated and studied functionally. Anti-CD3-induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytolytic in this test. Clone cells were activated to initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN-gamma (18 out of 19 clones: over 50,000 pg/ml) whereas IL-4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL-1, IL-2 and IL-6 was variable. The functional characteristics of the clones studied indicate that they may resemble the Th1 subtype of T cells. Our data suggest a relation between Th1-type functions the chronic inflammation characteristic of RA.     

Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cells

We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system. CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells. CT however, had a differential effect on naive and activated/memory T cell subsets. Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells. IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e. also in CD45RA+ cells which had maintained proliferative responses. However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies. These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45.     

CD4+ T-cell-mediated cytotoxicity against staphylococcal enterotoxin B-pulsed synovial cells.

Apoptosis of synovial cells in rheumatoid arthritis (RA) synovium determined in vivo is suggested to counteract the overgrowth of synovium. Immunohistological examination has revealed the infiltration of activated CD4+ T cells, which express Fas ligand (FasL), in RA synovium. The presence of a putative antigen (Ag) of autoimmune disorders in a target organ may induce the activation of specific T cells in the inflammatory region such as RA synovium. We examined the possible role of CD4+ T cells activated by synovial cells in a staphylococcal enterotoxin B (SEB)-dependent manner, inducing synovial cell apoptosis. Synovial cells were cultured with or without interferon-gamma (IFN-gamma) and further incubated with CD4+ T cells in the presence of SEB. After the cocultivation, both the cytotoxicity and FasL expression of CD4+ T cells were investigated. Constitutive Fas expression was detected on both unstimulated and IFN-gamma-stimulated synovial cells. CD4+ T cells did not kill SEB-pulsed unstimulated synovial cells efficiently. In contrast, when CD4+ T cells were incubated with IFN-gamma-stimulated synovial cells with SEB whose human leucocyte antigen (HLA)-DR and -DQ expression was markedly induced, significant cytotoxicity by these cells against synovial cells was detected. The addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or human Fas chimeric protein (hFas-Fc) reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated synovial cells with SEB was significantly induced. Furthermore, the addition of mAbs against CD54, CD58 and CD106 inhibited both the cytotoxicity and FasL expression of CD4+ T cells induced by IFN-gamma-stimulated synovial cells in the presence of SEB, indicating the importance of costimulatory molecules on synovial cells in activating CD4+ T cells. Our results suggest that CD4+ T cells are activated by synovial cells by an SEB-dependent manner and express FasL, inducing Fas-mediated apoptosis of the latter cells. These phenomena may regulate the overgrowth of synovial cells in RA synovium.     

Preclinical Studies Using Immobilized OKT3 to Activate Human T Cells for Adoptive Immunotherapy: Optimal Conditions for the Proliferation and Induction of Non-MHC-Restricted Cytotoxicity

In order to obtain large numbers of T cells for adoptive immunotherapy after bone marrow transplantation (BMT), we optimized conditions for long-term proliferation of T cells that exhibit non-MHC-restricted cytotoxicity using immobilized anti-CD3 (OKT3) activation and culture in IL-2. Proliferation and cytotoxicity directed at Daudi, K562, and B cell lines were used to determine (1) the optimal concentration of IL-2 and the optimal time of exposure to immobilized OKT3 for maintaining growth and cytotoxicity, (2) the starting populations that can be used, (3) the T cell subsets that mediate cytotoxicity, and (4) the optimal medium and concentration of serum for maintaining growth and cytotoxicity. Peripheral blood lymphocytes (PBL) activated with OKT3 would proliferate and mediate cytotoxicity at IL-2 doses as low as 30 IU/ml. Increasing the IL-2 concentrations beyond 600 IU/ml did not augment the proliferative or cytotoxic responses of PBL. A 24-hr incubation on OKT3 was sufficient to activate PBL. Increasing the incubation time on OKT3 from 24 to 72 hr did not significantly enhance cytotoxicity. Comparisons between PBL and purified T cells (E-rosette) indicated that either cell population could be activated with OKT3 in the presence of IL-2 to proliferate and mediate non-MHC-restricted cytotoxicity. Purified populations of CD4⁺ or CD8⁺ T cells demonstrated equivalent proliferation and cytotoxicity when activated using IL-2 and OKT3. With equal concentrations of human or fetal bovine serum, RPMI 1640 and X-Vivo 10 were comparable for supporting proliferation and cytotoxicity. These conditions are being used to activate and expand T cells for clinical trials that involve infusing activated T cells into recipients after autologous BMT.     

Senile Cardiac Amyloidosis: Evidence of Two Different Amyloid Substances in the Ageing Heart

Lymphocytes were eluted from the synovial tissue of seventeen patients with rheumatoid arthritis (RA) and one with ankylosing spondylitis. In eight of these patients immunoglobulin production by synovial lymphocytes in the presence and absence of pokeweed mitogen was studied. In nine patients T lymphocytes were isolated from the eluted cells, and the T helper and suppressor cell functions were evaluated in an allogeneic co-culture system. Peripheral blood lymphocytes (PBL) from twenty-eight normal donors were also studied for comparison. Immunoglobulin produced by synovial lymphocytes was higher than in PBL of normal donors. However, the stimulation index of synovial tissue lymphocytes was lower. Most of the normal donors had suppressor cell activity in their peripheral blood, whereas in synovial tissue lymphocytes a statistically significant number of patients did not have any suppressor cell activity. In contrast, the synovial tissue lymphocytes showed helper activity not differing significantly from that of the T lymphocytes from peripheral blood of normal individuals.     

HLA-DR4Dw4-restricted T cell recognition of self antigen(s) in the rheumatoid synovial compartment

The pathogenesis of joint destruction in rheumatoid arthritis remains ill defined, although it is thought to be the result of tissue damage mediated by T cells. This prompted us to isolate and characterize in vivo activated T cells from rheumatoid arthritis synovial fluid in an attempt to determine their specificity. Heterogeneous synovial fluid cells, containing both adherent and non-adherent cell types, were recovered from joint aspirates and cultured in the presence of IL-2. After 2 weeks, the non-adherent cells were phenotyped as CD3-positive and TCR alpha beta-positive T cells. Polyclonal T cell lines were derived from four rheumatoid arthritis patients; of these, two proliferated, in a dose-dependent manner to only autologous synovial fluid in the presence of autologous or DR4Dw4 histocompatible antigen presenting cells. T cell proliferation to the synovial fluid could be inhibited by monomorphic anti-HLA-DR monoclonal antibody, but not by anti-DQ or anti-class I antibodies. T cell clones were established by limiting dilution of a synovial T cell line in the presence of autologous synovial fluid and DR4Dw4 histocompatible accessory cells. Examination of the antigen specificity of these T cell clones demonstrated that they were reactive with a component of synovial fluid. The results of these experiments suggest the presence of an MHC class II-restricted antigen in the rheumatoid arthritis synovial compartment that induces proliferation of in vivo activated T cells.     

Cytokines in synovial fluid. I. The presence of biologically active and immunoreactive IL-1.

Using a sensitive IL-1-dependent T cell clone we estimated interleukin 1 (IL-1)-like activity in a variety of synovial fluids (SF). Although we demonstrate that SF contains potent inhibitors of endogenous and exogenous IL-1 we were able to estimate that samples from rheumatoid arthritis, osteoarthritis and other arthritides contain the equivalent of tens or hundreds of picograms of IL-1 per millilitre, when compared to a standard IL-1 preparation. Although there was some correlation between these results and those obtained using a radio-immunoassay (RIA) for IL-1 beta, the RIA indicated that IL-1 was present in the nanogram range. The results suggest that the importance of inhibitors of IL-1 in SF merits particular attention and that it is also important to consider the bioactivity of cytokines which may be detected by physical methods.     

Complement activating properties of complexes containing rheumatoid factor in synovial fluids and sera from patients with rheumatoid arthritis.

The relationship between complexes containing rheumatoid factor and complexes activating complement was examined in synovial fluids and sera from patients with rheumatoid arthritis (RA). In each case this was performed by quantifying the amount of rheumatoid factor bound by solid phase Fab'2 anti-C3 and/or solid phase conglutinin. Both anti-C3 coated and conglutinin coated microtitre plates bound high levels of complexes containing rheumatoid factor from sera of RA patients with vasculitis. Unexpectedly, these complexes were detected in synovial fluids from only a minority of RA patients with synovitis. However, RA synovial fluids did contain other complexes as shown by the presence of complement consuming activity, C1q binding material and immunoglobulin attaching to conglutinin. It is considered that in RA synovial fluids the complexes containing RF and those activating complement are not necessarily the same whilst in vasculitic sera the complexes containing rheumatoid factor also activate complement.     

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid.

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti-CD3 and lectin-induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non-cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector-to-target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non-cytotoxic (less than 20% lysis at an effector-to-target cell ratio of 10:1). The induction of cytotoxicity in the anti-CD3-driven system was shown to be dependent on the dose of anti-CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non-RA synovial fluids. Both anti-CD3 and lectin-dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.     

Interleukin-15 induces interleukin-17 production by synovial T cell lines from patients with rheumatoid arthritis

IL-17-producing T cells (Th17 cells) are believed to contribute to local inflammation and joint damage in rheumatoid arthritis (RA). Limited data exist on Th17 cells located within the inflamed synovial tissue (ST) of patients with RA. Here, we aimed to generate polyclonal T cell lines (TCLs) from the RA ST and assess their cytokine production, including the effects of exogenous IL-15 on IL-17 production in vitro. For five patients with RA, polyclonal TCLs were established from ST obtained by joint surgery. Synovial TCLs were expanded and stimulated by anti-CD3/CD28 microbeads and exogenous cytokines. Cytokine production was assessed by culture supernatant analyses and intracellular flow cytometry, and TCLs were sorted based on their surface expression of CCR6. In addition to IL-17, we detected IL-6, IL-10, IFN-γ and TNF-α in the synovial TCL culture supernatants. Exogenous IL-15 increased the production of IL-17 as well as the other cytokines except IFN-γ. For IL-17, this effect was more pronounced after prolonged culture times. Intracellular flow cytometry confirmed the presence of IL-17+ and IL-17+ IFN-γ+ CD4+ T cells in the TCLs. IL-17+ and IL-17+ IFN-γ+ T cells were enriched in the CD4+ CCR6+ population. In conclusion, Th17 cells can be detected after polyclonal expansion and stimulation of RA synovial TCLs generated by joint surgery. The Th17 cells from the RA ST were enriched in the CD4+ CCR6+ population, and they were sensitive to exogenous IL-15. Th17 cells present within the synovial compartment may contribute to the RA pathogenesis and local joint damage.     

In vivo activated T cells in rheumatoid synovitis. Analysis of Th1- and Th2-type cytokine production at clonal level in different stages of disease

T-cell cytokines play a crucial role in the pathogenesis and progression of rheumatoid arthritis (RA). Their detection in the joint, however, is impaired by the complex network present in the synovium. Although many synovial T cells show signs of previous activation, only a few express interleukin (IL)-2 receptor, marker of recent activation. The aim of this study was to analyse the cytokine production by in vivo activated (IL-2R +) T cells from RA at different stages of the disease. For this purpose, T cells were isolated from peripheral blood and synovial fluid of four patients with active RA, two at the onset of the disease, one in the early phase during treatment, one in long-lasting chronic phase. One patient was studied at the onset of the disease and 52 months later. Cells were initially expanded with a low dose of IL-2, cloned and analysed for cytokine production. The results showed a strong predominance of T helper (Th) 1 clones in the blood and a slight prevalence of Th0 clones in the joint of all the four patients. Interferon-gamma and IL-2 production was higher in the long-lasting RA, whereas IL-4 synthesis was prevalent in early RA. Enrichment in IL-10-producing clones was present only in the joint of the untreated patients. The longitudinal study confirmed the differences in cytokine production between early and late phases of disease. These data confirm that RA is mainly a Th1-driven condition. However, in vivo activated synovial T cells produce also Th2-type anti-inflammatory cytokines, such as IL-4 and IL-10. The synthesis of both cytokines is a feature of the very early phase of RA, although the selective recruitment of IL-10-producing T cells is quickly lost.