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.     

Regulation of IL-2 production by mononuclear cells from rheumatoid arthritis synovial fluids.

Products of polyamine oxidation down-regulate IL-2 production by peripheral blood T cells. We show here that the production of IL-2 by rheumatoid arthritis synovial fluid mononuclear cells is inversely correlated with the concentrations of polyamines in these cells. In addition, the inhibition of polyamine biosynthesis or oxidation in cultures of these cells enhances their ability to produce IL-2. Our findings suggest that polyamine oxidation plays an important role in the suppression of T cell function characteristic of rheumatoid arthritis synovial fluids.     

The cytotoxic analysis of T cell receptor V delta 1+ T cell lines derived from the synovial fluid of rheumatoid arthritis patients.

We established six human T cell lines derived from rheumatoid arthritis synovial fluid (RASF). Phenotypically, T cell receptor (TCR) gamma delta T cells occupied the majority of these lines and most of them expressed the TCR V delta 1 molecule. In contrast, V delta 2+ T cells, the majority population of peripheral blood gamma delta T cells, were rarely detected in these lines. To study the immunobiological roles of RASF V delta 1+ T cells in RA development, their cytotoxic profile was studied. The results showed that these T cells selectively lysed Daudi, but not K562 cells. The cytotoxic response was MHC-unrestricted, and was inhibited by anti-CD3 MoAb. Moreover, the cold target inhibition assay showed that the cytotoxicity was competitively inhibited by autologous and allogeneic primarily cultured RA synovial cells as well as synovial sarcoma and chondrosarcoma lines. However, PBL did not inhibit this cytotoxicity. These data suggest that V delta 1+ T cells in RASF may recognize the antigen which is commonly expressed on the surface of Daudi and the cells derived from RA synovium. We can assume that the cytotoxic V delta 1+ T cells are selectively expanded in RASF, playing a significant role for the pathogenesis of certain RA cases.     

T cell receptor gene in synovial tissues of rheumatoid arthritis

Rheumatoid arthritis is a chronic and destructive autoimmune joint disease characterized by inflammation of synovial tissue of unknown aetiology. Studies on TCR genes expressed by infiltrating T cells in synovial tissues have attempted to identify mechanism and specificity of the recruitment. T cell infiltrate in rheumatoid arthritis appears to be an association of a polyclonal non specific infiltrate with dominant clones or clonotypes. T cell repertoire in synovial tissue is biased compared to peripheral blood but no TCR V gene can be identified as commonly over-used. Comparison of motifs found in the CDR3 region of dominant clones from different studies has currently failed to identified a commonly motif. The fact that a number of dominant clones or clonotypes is present in different joints and at different times of the disease suggests a selective expansion of T lymphocytes in rheumatoid arthritis synovial membrane. Further investigations are needed to characterize the specificity of these dominant clonotypes.     

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.     

Dominant clones in immortalized T-cell lines from rheumatoid arthritis synovial membranes

Transformation of human T cells by herpesvirus saimiri allows the production of an unlimited number of T cells which express a functional T-cell receptor. In this study we transformed four T-cell lines derived from rheumatoid arthritis synovial membranes. The transformed T cells were mainly CD4⁺ and expressed the phenotype of activated T cells. They were grown for more than 1 year in the absence of mitogen or feeder cells, and three of them could be maintained without exogenous LL-2. The presence of viral DNA in the transformed cells was shown by in situ hybridization with a probe from the H-DNA region of the virus. No infectious virus could be recovered from the transformed cells. The relative proportion of the 24 different Vβ families between the four transformed lines showed variations that increased with time. In the two T-cell lines transformed at an early stage of culture, the Vβ2 family was maintained at about 10%. The dominant Vβ2 clones that previously have been characterized in the patient were found in all transformed T-cell lines. We have thus shown the feasibility of obtaining transformed T cells from synovia] membranes. They contain the dominant clones that are considered of potential importance for the disease, permitting further functional studies.     

Interleukin-2 in rheumatoid arthritis: production of and response to interleukin-2 in rheumatoid synovial fluid, synovial tissue and peripheral blood.

Several aspects of interleukin-2 (IL-2) generation and function were studied employing mononuclear cells from synovial fluid (SF), synovial tissue (ST) and peripheral blood (PB) of patients with rheumatoid arthritis (RA). Decreased PHA stimulated IL-2 production by lymphocytes from rheumatoid ST, SF (P less than 0.02), and PB (P less than 0.01) was observed when compared to normal blood and SF of patients with gout. The proliferative response of rheumatoid lymphocyte blasts exposed to exogenous IL-2 was also defective (P less than 0.05-0.001). This defect was greater in SF than in rheumatoid PB (P less than 0.05-0.001). In addition to the proliferative response, the effect of IL-2 on interferon-gamma (IFN-gamma) production was also examined. Rheumatoid lymphocytes from both PB and SF produced less IFN-gamma after overnight treatment with IL-2 than did normal PB lymphocytes. This decreased IFN-gamma induction was discordant with the excellent enhancement by IL-2 of natural killer activity. Removal of adherent cells in synovial fluid did not correct this deficit. Abnormalities in the biology of IL-2 and IFN-gamma suggest that impaired T cell function could contribute to the immunopathogenesis of RA.     

Functionally active α2-macroglobulin and kinin release in synovial fluids of rheumatoid arthritis

The function of ₂M (₂-macroglobulin) as a proteinase inhibitor in synovial fluid of rheumatoid arthritis was investigated. Low esterase activity was found in the 19S sieve fraction of Sephadex G-200 gel filtered synovial fluid samples, comparable with that obtained with normal human plasma. The molar binding ratio of ₂M isolated from the synovial fluid and trypsin was estimated according to earlier established methods. The formation of an equimolar complex indicated that the synovial ₂M was functionally intact.Esterase activities were measured on N-tosyl-l-arginine [³H]methyl ester. Synovial fluid samples were all found to contain proteinase enzyme which did not bind to the synovial ₂M nor to the added functionally intact, immunologically pure ₂M prepared from normal human plasma.Albumin, ₁-acid glycoprotein, ₂HS-glycoprotein, ₂M and kininogen in synovial fluids were determined by single radial immunodiffusion. Only the ₂M contents were clearly lower than in normal human serum per ml.The higher than 1 ratio between the immunoreactive kininogen determined with monospecific anti-human kininogen serum and the pharmacologically active kininogen indicated that kinin release had occurred possibly in the synovial membrane. Proteinase enzymes present in the synovial fluids and unbound to ₂M caused further depletion of the active kinin segment in synovial kininogen. A model of the regulation by ₂M of the synovial kininogen-kinin system is given.     

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.     

Induction of autologous lymphocyte transformation by synovial fluids from patients with rheumatoid arthritis

Appropriately diluted synovial fluids from thirteen of eighteen patients with rheumatoid arthritis induced in vitro transformation of autologous peripheral blood lymphocytes. By contrast, no significant transformation of autologous lymphocytes was induced by ten of eleven synovial fluids from patients without rheumatoid arthritis. These studies suggest that a similar blastogenic response in vivo may perpetuate subsynovial lymphoid hyperplasia and chronic synovitis in patients with rheumatoid arthritis.     

Inhibitory effects of interleukin-10 on synovial cells of rheumatoid arthritis.

This paper describes the immunoregulatory effects of interleukin-10 (IL-10) on synovial cells in vitro. Synovial cells were cultured with IL-10 in the presence or absence of various cytokines. Following incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analysed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of IL-10 on the function of synovial cells as antigen-presenting cells (APC). Synovial cells spontaneously express several kinds of costimulatory molecule and produce various kinds of cytokines. Stimulation of synovial cells with interferon-gamma (IFN-gamma), IL-1 beta, or 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly enhanced the expression of costimulatory molecules and cytokine production of these cells. Both spontaneous and up-regulated costimulatory molecule expression and cytokine production were significantly suppressed by the addition of IL-10. Autologous T-cell proliferation was stimulated by purified protein derivative (PPD) in IFN-gamma-treated synovial cells and treatment of these synovial cells with IL-10 also suppressed T-cell proliferation. Our results suggest that IL-10 has an inhibitory effect on synovial cells and is an important immunoregulatory component of the cytokine network in rheumatoid arthritis.     

Soluble histocompatibility antigens in synovial fluids of patients with rheumatoid arthritis

Soluble histocompatibility antigens of the class II region have been detected in synovial fluids obtained from patients with rheumatoid arthritis. A capture immunoassay involving two monoclonal antibodies was used; interference by rheumatoid factor, which is a feature of such assays, was overcome by mild pretreatment of fluids with 2-mercaptoethanol. No HLA class II antigen could be detected in matched sera from patients, even when levels were high in synovial fluids. Released HLA-class II material was of high molecular weight (greater than 1000 kD) and was linked to HLA-class I antigen. However, no significant amounts of other common cell surface antigens were detected in the complex, suggesting a preferential release of MHC antigens from cells of the inflamed synovium. Attempts to induce production of similar material from a cell line which expresses HLA class II strongly at the cell surface, by stressing the cells in various ways did not succeed, indicating that release is an active process.     

Specific binding of immunoconglutinin in tissues and synovial fluids from patients with rheumatoid arthritis

In eluates from rheumatoid synovial tissue, immunoconglutinin was present in only one of fifteen cases. After pepsin digestion, immunoconglutinin activity was increased or revealed in almost all eluates examined. Natural antibodies, present in the corresponding sera, could not be detected in the eluates before or after pepsin digestion. Neither was any immunoconglutinin present or revealed in eluates from non-rheumatoid synovial tissue. The experiments indicated that this antibody is specifically fixed in synovial tissue of patients with rheumatoid arthritis. The immunoconglutinin activity in the eluates was probably due to an IgG antibody. Density gradient ultracentrifugation and treatment with 2-ME indicated that both IgG and IgM immunoconglutinin were present in sera from these patients.

In rheumatoid synovial fluids, immunoconglutinin was present in an amount equal to, or somewhat lower than in serum. By density gradient ultracentrifugation at pH 3·0, IgG immunoconglutinin activity seemed to be revealed or increased in most cases, indicating a fixation of this antibody also in synovial fluids.

Immunoconglutinin in human sera could be absorbed out with immune precipitates exposed to fresh human serum, and this reaction seemed to increase the complement fixing ability of the precipitates. If binding of immunoconglutinin in rheumatoid synovial tissue and synovial fluids increases complement fixation and activation in a similar way, it might be a pathogenetic factor in the rheumatoid inflammation.

     

Lack of T cell oligoclonality in enzyme-digested synovial tissue and in synovial fluid in most patients with rheumatoid arthritis.

The dominant presence of specific T-cell populations in the rheumatoid joint as detected by Southern blot analysis of T cell receptor (TCR) gene rearrangements would indicate local antigen recognition and T cell proliferation. We therefore studied TCR beta chain gene rearrangements using a C beta 2 probe in paired samples of T cell populations from synovial tissue and peripheral blood (n = 6) as well as synovial fluid (n = 16) and peripheral blood (n = 18) of patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from healthy donors (n = 7) served as a control. T cells were studied directly after isolation or after non-specific expansion with OKT3 monoclonal antibody (MoAb) and T cell growth factor (TCGF). DNA samples were digested with EcoRI and HindIII to detect rearrangements to C beta 1 and C beta 2, respectively. Extra bands were detected in all EcoRI-digested DNA samples prepared from both freshly isolated and non-specifically expanded T cell populations of patients and healthy donors, possibly representing 'common' (V-) D-J rearrangements. Dominant rearrangements were found in only two out of 16 synovial fluid T cell populations (one freshly isolated and one expanded) and not in peripheral blood or synovial tissue derived T cell populations. No extra bands were detected in HindIII-digested DNA samples. To investigate the effect of in vitro culture techniques on rearrangement patterns we studied DNA samples prepared from synovial tissue T cells obtained both by outgrowth from tissue with TCGF or by enzyme digestion and subsequent expansion either with TCGF or with OKT3 MoAb and TCGF. Whereas the latter T cell population yielded 'common' rearrangements, the former T cell populations yielded different dominant rearrangements. These data indicate that oligoclonality of the T cell populations in synovial tissue and synovial fluid of patients with RA is a rare event. The data also show the influence of in vitro culture techniques on the result of TCR gene rearrangement analysis.     

Bovine T lymphocytes. I. Generation and maintenance of an interleukin-2-dependent, cytotoxic T-lymphocyte cell line.

Primary and secondary bovine allogeneic mixed leucocyte cultures were examined for the generation of antigen-specific cytotoxic leucocytes. While optimal generation of murine and human cytotoxic T lymphocytes typically requires 4-8 days, alloantigen-specific cytotoxic bovine leucocytes were demonstrated consistently only after prolonged incubation periods, optimally found to be about 15 days. Restimulation of long-term bovine mixed leucocyte cultures with the original stimulator population revealed responder cells demonstrating augmented alloantigen-specific lytic activity. When placed into human recombinant interleukin-2, responder cells expanded and required passaging every 3-4 days. The same was not true of cells placed into interleukin-2-free medium. Cells cultured in interleukin-2-containing medium retained alloantigen specificity after 10 weeks of culture. Moreover, they continued to display total dependence on human, simian or bovine interleukin-2 for growth.     

Analysis of the IgG subclass production from rheumatoid arthritis synovial cell cultures.

In man there are four subclasses of IgG which differ from each other with respect to their biological properties. Some evidence suggests that the production of IgG3 is unusually high in rheumatoid synovia. In this study secretion of IgG subclasses by synovial lymphocytes in vitro was measured using sensitive subclass-specific ELISAs. It was found that, in both synovial membrane- and synovial fluid-derived cell cultures, the general pattern of IgG subclass secretion was IgG1 greater than 2 greater than 3 greater than or equal to 4, and that, in most cultures, IgG3 was a minor subclass accounting, on average, for only 8% of the total IgG. This was similar to the percentage of this subclass in normal human serum and in culture supernatants from the patients' peripheral blood lymphocytes.     

Enhanced oxidative response of polymorphonuclear leukocytes from synovial fluids of patients with rheumatoid arthritis

Polymorphonuclear leukocytes (PMN) isolated from the synovial fluid (SF) of patients with rheumatoid arthritis (RA) exhibited an enhanced oxidative response to heat aggregated IgG (Hagg) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) as compared with either autologous or normal blood PMN. Normal blood PMN pretreated with synovial fluid showed a significantly increased response to FMLP which was unaffected by prior dialysis of SF. The degree of enhancement produced varied between SF's and was dependent on the period for which cells were incubated and on the concentration of SF used. In contrast there was no enhancement of the oxidative response when Hagg was used as a stimulus. Indeed, SF produced a dose dependent inhibition of Hagg stimulated superoxide production. These observations suggest that SF's from patients with RA contain factors which both enhance and inhibit the oxidative response of PMN depending on the subsequent stimulus.     

Clonal analysis of T cell infiltrates in synovial tissue of patients with rheumatoid arthritis

To investigate the possibility of the presence of disease-relevant, antigen-specific immune reactions in rheumatoid arthritis (RA), clonal diversity of the T cells in the synovial tissue was examined. T cells were directly cloned by in vitro stimulation with phytohemagglutin and interleukin 2 from both the peripheral blood and inflammatory synovial tissue. Their clonotypes were defined by analyzing rearrangement patterns of T cell receptor (TCR) beta and gamma chain genes using Southern blotting. In total, 111 clones from the synovial tissue (four patients) and 45 from the peripheral blood (one patient) were studied. Although most of the clones were unique in their TCR gene rearrangement patterns, 2 clones from the synovial tissue of one patient had identical patterns. These 2 clones were CD3+, 4-, 8+. Since phenotypic analysis of 82 clones from the synovial tissues revealed that CD8+ T cell clones were less frequent (24%) than CD4+ clones, the clonal identity observed here in 2 clones may not be negligible. Furthermore, 1 CD8+ clone from the peripheral blood of the same patient also had the same clonotype. These results may suggest selective trafficking or proliferation of CD4-, CD8+ T cells in RA synovial tissue.