Antigenic specificity of rheumatoid synovial fluid lymphocytes

The majority of rheumatoid arthritis (RA) synovial fluid lymphocytes (SFL) demonstrate markers that are suggestive of prior activation. While the mechanism(s) responsible is unknown, prior studies have suggested that certain Mycobacterium tuberculosis (MT) antigens may preferentially activate SFL in vitro. We therefore examined the ability of RA SFL to respond to purified protein derivative and an acetone-precipitable MT antigenic complex (AP-MT) and compared this with the responses by peripheral blood lymphocytes (PBL). The responses were contrasted with those elicited with tetanus toxoid (TT) and mitogenic anti-CD3. In patients with RA, the SF proliferative responses to both TT and anti-CD3 were reduced compared with responses by PB. In contrast, the SF response to purified protein derivative was maintained, and that to AP-MT was significantly increased, compared with PB. SF responses to AP-MT antigens were significantly greater than those to TT. The AP-MT activation of T lymphocytes from RA SF was characterized by an earlier peak proliferative response than that noted with matched PB. AP-MT responsiveness was not restricted to HLA-DR4 positive patients. These observations suggest that an epitope(s) contained within the MT complex of antigens, and enriched in the AP-MT complex, may be important in maintaining the chronic inflammation in at least some patients with RA.     

Absence of peripheral blood T cell responses to `shared epitope' containing peptides in recent onset rheumatoid arthritis

OBJECTIVES—To determine if peptides containing the `shared epitope' sequence, QKRAA, from either endogenous, HLA-DRβ1(0401), or exogenous, Escherichia coli dnaJ, sources activate T cells in recent onset rheumatoid arthritis (RA).
METHODS—Peripheral blood mononuclear cell (PBMC) proliferative and whole blood cytokine responses to shared epitope containing peptides from DRβ1(0401) and E coli dnaJ, to control peptides from DRβ1(0402) and hsp40 and to the recall antigen, tetanus toxoid, were tested in 20 untreated, recent onset RA subjects, 20 HLA, age, and sex matched healthy controls and 18 other subjects with inflammatory arthritis. PBMC proliferative responses to a second E coli dnaJ peptide (with the shared epitope at the N-terminus) and two peptides from type II collagen with high affinity for DR4(0401) were tested in a further 16 recent onset RA and 17 control subjects.
RESULTS—PBMC proliferation and whole blood interferon γ or interleukin 10 production in response to the shared epitope containing and control peptides were not different between the disease and control groups. On the other hand, compared with controls, RA subjects had significantly higher proliferation to a collagen II (aa 1307-1319) peptide, but significantly lower proliferation and interferon γ production to tetanus toxoid.
CONCLUSION—Recent onset RA subjects had no demonstrable increase in peripheral blood T cell reactivity to shared epitope containing peptides. However, a proportion had increased T cell reactivity to a peptide of similar length from a candidate RA autoantigen, collagen type II. Their impaired responses to tetanus are in keeping with evidence for general T cell hyporesponsiveness in RA.

     

Stimulatory response towards the 65 kDa heat shock protein and other mycobacterial antigens in patients with rheumatoid arthritis

Molecular mimicry between mycobacterial antigens and components of human articular cartilage has been suggested to initiate the onset of rheumatoid arthritis (RA). Therefore, the proliferative response of peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MNC) towards a variety of mycobacterial antigen preparations was tested in patients with RA and various non-RA inflammatory joint diseases. The antigen panel included a recombinant 65 kDa heat shock protein of mycobacterial origin which has recently been shown to stimulate arthritogenic rat T cell clones. In our study, no significant response towards the 65 kDa protein was observed with PBMNC of patients with or without RA. When compared to normal donors, the reactivity towards heat killed BCG M. tuberculosis and purified protein derivative was reduced in the blood of patients with RA while enhanced in the non-RA group, however these differences did not reach statistical significance. The data obtained with PBMNC were in striking contrast to the proliferative response of SFMNC. In the majority of cases both with RA and non-RA joint diseases the latter compartment was far better stimulated by mycobacterial antigens including the 65 kDa protein compared to corresponding PBMNC. However, in some cases an enhanced stimulation of SFMNC was also obtained with tetanus toxoid. Our data suggest that the enhanced reactivity of SFMNC may be a common feature of several inflammatory joint diseases and is not restricted to RA. These findings may indicate a preferential homing of antigen reactive (memory) T cells to the inflamed joints while the circulating pool of lymphocytes is depleted of this population.     

Accumulation of T cells reactive to type II collagen in synovial fluid of patients with rheumatoid arthritis

OBJECTIVE: To determine if type II collagen (CII) reactive T lymphocytes selectively accumulate in the inflamed joint of patients with rheumatoid arthritis (RA) and to study the specificity of the CII reactive cells. METHODS: Synovial fluid (SF) cells or peripheral blood lymphocytes were cultured with interleukin 2 (IL-2) for 24 h and then cultured at limiting dilution concentrations in the presence of filler cells and IL-2. The outgrowing cell lines were screened for their responses to CII. The percentages of the CII reactive T cells from SF were compared with those from peripheral blood of patients with RA. The CII reactive T cell lines were tested for their responses to different types of collagen. RESULTS: CII reactive T cell lines were identified in the SF of 3 RA patients; the frequencies were 5.0% (11/219), 3.7% (5/134), and 3.5% (3/86), respectively. In contrast, none of CII-specific T cell lines were identified in peripheral blood of the patients. The T cell lines recognized both human and bovine CII and, to a lesser extent, type I collagen. CONCLUSION: CII reactive T cells are present in high frequency in the inflamed joint of patients with RA, where they may play an important role in the pathogenesis of RA.     

Local anti—type ii collagen antibody production in rheumatoid arthritis synovial fluid

Objective. To investigate the production of type II collagen (CII) antibodies in the synovial fluid (SF) of rheumatoid arthritis (RA) patients, and to examine the HLA dependence of this local production. Methods. The ELISPOT method was used for enumerating anti-CII—reactive cells. Serologic tissue typing was performed. Results. Anti-CII—reactive cells were found in the SF of 16 of 31 patients, but not in any of the peripheral blood samples obtained in parallel. SF anti-CII antibody production showed no correlation with clinical parameters, but its frequency increased significantly with age. The IgG anti-CII response occurred exclusively in patients who were positive for HLA—DR4 and was significantly associated with DR4. Conclusion. Anti-CII production may be important in local immune complex formation. The indirect demonstration of a DR4-restricted T cell response to CII is an indication of a pathogenetic role of collagen autoimmunity in RA.     

Augmented production of chemokines by the interaction of type II collagen-reactive T cells with rheumatoid synovial fibroblasts

OBJECTIVE: To determine the impact of type II collagen (CII)-reactive T cells on the production of chemokines in the joints of patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII were assayed in synovial fluid (SF) mononuclear cells and peripheral blood mononuclear cells. CII-stimulated T cells were cocultured with fibroblast-like synoviocytes (FLS). The expression of interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 alpha (MIP-1 alpha) in the sera, SF, and supernatant of the CII-stimulated T cells and FLS coculture was measured by enzyme-linked immunosorbent assays. RESULTS: The levels of IL-8, MCP-1, and MIP-1 alpha in SF were significantly higher than those in paired sera of RA patients. IL-8, MCP-1, and MIP-1 alpha levels in SF were strongly correlated with T cell responses to CII. When FLS were cocultured with CII-stimulated T cells, the production of IL-8, MCP-1, and MIP-1 alpha was significantly increased. This increase correlated well with the T cell proliferative response to CII. Chemokine production by coculture of CII-stimulated T cells and FLS was mediated mainly by direct cell-cell contact through CD40 ligand-CD40 engagement. CONCLUSION: Our data indicate that the presence of CII-reactive T cells in RA joints can increase the production of chemokines such as IL-8, MCP-1, and MIP-1 alpha through interaction with FLS. This chemokine production is mediated by cell-cell contact, including CD40 ligand-CD40 engagement. These results suggest that CII-reactive T cells play a crucial role in the amplification and perpetuation of the inflammatory process in the rheumatoid synovium.     

Enhanced T cell proliferative response to type II collagen and synthetic peptide CII (255-274) in patients with rheumatoid arthritis.

OBJECTIVE: To determine the presence of specific immune recognition of type II collagen (CII) and its immunodominant epitope CII (255-274) in patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII and a synthetic peptide encompassing CII (255-274) in peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) from RA patients, and in PBMC from osteoarthritis (OA) patients and healthy controls were assayed by mixed lymphocyte culture. RESULTS: The stimulation index (SI) and the number of positive (SI > or = 2) T cell responses to CII were higher in RA patients (n = 106) than in OA patients (n = 26) and healthy controls (n = 34). T cell responses to CII (255-274) were also enhanced in RA patients and correlated well with those to CII. In SFMC, positive responses to CII or CII (255-274) were detected in 61.9% of 42 RA patients. T cell responses to CII in SFMC were stronger and more prevalent than peripheral responses. The SI and positive responses to CII were higher in early RA than in late RA. Levels of IgG antibodies to CII in synovial fluid inversely correlated with T cell responses to CII. CONCLUSION: T cell responses to CII or CII (255-274) were enhanced in RA, especially in early disease. Synthetic peptide CII (255-274), as well as native CII, could be recognized as immunogenic antigens by T cells, particularly in the synovial fluid. These observations suggest that CII-reactive T cells play an important role in the pathogenesis of RA. Peripheral tolerance induction using CII (255-274) might be useful in the treatment of RA.     

Morphometric analysis of peripheral blood and synovial fluid lymphocytes of patients with rheumatic disease

A morphometric analysis of lymphocytes from the peripheral blood and the synovial fluid (SF) of patients with rheumatoid arthritis (RA) was based on a determination of the nuclear contour index (NCI = perimeter divided by the square root of the area of the nucleus). Use of this method showed that a particular type of lymphocyte, the cerebriform mononuclear cell (CMC), occurred in higher percentages in the SF than in the peripheral blood of patients with RA. The mean NCI of the lymphocytes (non-CMC) was also higher in the SF. These findings indicate that lymphocytes in the inflammatory compartment are morphologically altered, compared to the corresponding cells in the peripheral blood of the same patients, the change probably expressing an alteration in functional status.     

CD4+ lymphocyte function with early human immunodeficiency virus infection.

The pathogenesis of cellular immune deficiency following human immunodeficiency virus (HIV) infection could result from quantitative and/or qualitative dysfunction of the CD4+ lymphocyte population. To better characterize the T-cell response to soluble antigen with HIV infection, we have isolated peripheral blood lymphocytes and purified populations of CD4+ lymphocytes from healthy HIV antibody-positive subjects, patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC), and healthy HIV antibody-negative controls. T-lymphocyte function was determined by proliferative response to lectin (phytohemagglutinin), phorbol 12-myristate 13-acetate (PMA), calcium ionophore, purified recombinant HIV envelope gp120, tetanus toxoid antigen, and tetanus toxoid antigen in the presence of recombinant gp120 or purified recombinant soluble CD4. PBLs and CD4+ lymphocytes from asymptomatic HIV-infected subjects responded equally well to lectin, PMA, and/or calcium ionophore and to tetanus toxoid as cells from uninfected control subjects. The cells that proliferated in response to a soluble antigenic stimulus did not respond to gp120. Cells from subjects with ARC had a selective antigen recognition defect independent of the number of CD4+ lymphocytes. Recombinant gp120 inhibited CD4+ lymphocyte proliferation to antigenic stimulus by 30-40%. Recombinant soluble CD4, a proposed therapeutic for HIV, had no effect on T-cell response to antigen. A selective antigen recognition response was not compromised early in HIV infection but was compromised in subjects with ARC. Inhibition of proliferation to tetanus toxoid by gp120 suggests that HIV may affect major histocompatibility complex II restricted antigen recognition independent of CD4+ cell loss.     

Enhancement of antigen-induced T-cell proliferation by soluble CD26/dipeptidyl peptidase IV.

The addition of a soluble recombinant CD26 (sCD26) enhanced proliferation of peripheral blood lymphocytes induced by the recall antigen tetanus toxoid. sCD26 itself did not provide a mitogenic signal and did not augment the proliferative response of T cells to other mitogenic stimuli such as phytohemagglutinin and anti-CD3. Dipeptidyl peptidase IV-negative sCD26 did not have this enhancement effect, implying a requirement for enzyme activity. It was found that there exists a large variation in the levels of human plasma sCD26/dipeptidyl peptidase IV in vivo which may regulate T-cell activity. Peripheral blood lymphocytes from individuals whose plasma sCD26 was high and responded strongly to tetanus toxoid stimulation were insensitive to the enhancing effects of exogenously added sCD26. This suggests that plasma sCD26 had modulated the responsiveness of T cells of these individuals in vivo and that the endogenous plasma sCD26 regulates immune responses by allowing antigen-specific T cells to exert a maximal response to their specific antigen.     

A study of the effect of rheumatoid synovial fluid on proliferation and IL-2 production by total mononuclear cells and purified CD4+ cells of synovial fluid and peripheral blood

T cells from synovial fluid (SF) of rheumatoid arthritis (RA) patients have previously been shown to proliferate less after mitogenic stimulation and produce less interleukin 2 (IL-2) than normal T cells. To test whether SF is responsible for the reduced T-cell responses, we studied the effect of inflammatory SF on peripheral blood (PB) RA and normal mononuclear cells (MNC) and CD4+ T cells and on RA SF MNC and CD4+ cells in vitro. Most rheumatoid SF present in concentrations of 50% and 5% during in vitro stimulation increased mitogen-induced IL-2 production and proliferative response by normal PB and RA MNC and CD4+ cells. Other rheumatoid SF samples did not influence the T cell responses, while only a few samples had an inhibitory effect. The results indicate that SF contain both stimulatory and inhibitory factors and that the resultant effect on T cells may depend on the net effect of these. The results do not support the hypothesis that the apparently impaired function of SF T cells is due to contact with SF.     

Regulatory T cell activity specific to human type II and III collagens in rheumatoid arthritis.

Fifty-one patients with rheumatoid arthritis (RA) were examined for their immune response potential to human collagen type II and III. It was found that T cells of 57% of patients with RA proliferated to collagen type III whereas only 27% of T cells of patients with osteoarthritis (OA) and healthy controls responded to this antigen by proliferation (p less than 0.04). A lower percentage (38%) of patients with RA had proliferative responses to collagen type II in comparison to 17% of responders in healthy controls. The capability to produce T cell helper factors specific to collagen type III was found to be significantly higher in patients treated with nonsteroidal antiinflammatory drugs (NSAID) (60%) in comparison to patients with OA (16%) and healthy controls (13%). Immunoregulatory drugs affected the specific T helper function in response to collagen type III but did not change the proliferative responses to collagen type II and III in patients with RA. HLA analyses revealed a significant difference in the frequency of HLA-DRw10 between our sample of patients with RA and healthy controls.     

Systemic and local expression of perforin in lymphocyte subsets in acute and chronic rheumatoid arthritis

OBJECTIVE: To investigate the role of the cytolytic action mediated by perforin in the course of rheumatoid arthritis (RA), we studied the immunophenotypic characteristics of lymphocytes containing perforin in peripheral blood (systemic level), in synovial fluid (SF), and in the synovial membrane (local level) in patients during the acute or chronic phase of RA. Cells from patients with osteoarthritis were used as controls. METHODS: Flow cytometry was used for simultaneous detection of intracellular (perforin) and cell surface antigens. Mean fluorescence intensity (MFI) was a measure of the mean perforin content per cell. Immunocytochemical staining was used to visualize perforin in the cytoplasmic compartment of cells. RESULTS: In acute RA highly significant changes in perforin expression were found in all compartments (peripheral blood, SF, and synovial membrane): (1) increase of percentage of total perforin positive cells; (2) increase of both subsets of cytolytic cells, T (CD8+P+) and NK (CD56+P+) cells; (3) increase in the frequency of perforin positive cells in CD8+ and CD56+ cell populations; and (4) the highest content of perforin/cell (MFI values) in all compartments, except in the synovial membrane. CONCLUSION: Perforin positive cells may participate in the acute phase of RA by maintaining and perpetuating inflammation and contributing to tissue destruction.     

Inhibitor of interleukin-2 synthesis and response in rheumatoid synovial fluid

We studied the effects of a factor present in rheumatoid arthritis (RA) synovial fluid (SF) on interleukin-2 (IL-2)-dependent cell proliferation and on the production of IL-2 by mitogen-stimulated peripheral blood mononuclear cells. RA SF suppressed the responsiveness of a mouse T cell line (HT-2) to IL-2, indicating that it contained an inhibitor of the IL-2 response. When RA SF was fractionated by Sephadex G-200 gel filtration, the inhibitory activity was detected mainly in fractions with a molecular weight of approximately 150,000, but was also found in a 15-19-kd fraction. Removal of IgG from the 150-kd fraction, by means of an anti-IgG affinity column, did not reduce the activity of the fraction, nor was activity found in the eluted IgG. The inhibitory fractions reduced mouse thymocyte proliferative responses to IL-1 in the presence of phytohemagglutinin, and reduced the production of IL-2 by human peripheral blood mononuclear cells, but did not inhibit IL-1-induced human foreskin fibroblast proliferation; this suggests that the factor was not an IL-1 inhibitor. The inhibitory activity of the RA SF factor was blocked by an antibody against an inhibitor of IL-2 that was purified from a culture of the human monocytic leukemia cell line, THP-1. This finding also supports the conclusion that RA SF contains an IL-2 inhibitory factor. The observed inhibition of both IL-2 synthesis and IL-2 response suggests that the target of the inhibition was the T lymphocyte.     

The immune response to alkaline phosphatase and other immunogens in patients with primary biliary cirrhosis

BACKGROUND/AIMS: Primary biliary cirrhosis is a potentially lethal hepatobiliary disorder in which 90% of the patients are women. Histologically, the disease is characterized by a progressive destruction of intrahepatic bile ducts by autoreactive T lymphocytes. Although the underlying etiology remains unknown, potential hypotheses must take into account; a) the predilection of the disease for women of childbearing age, b) the frequent coexistence of bone and intestinal involvement, and c) the high prevalence of autoantibodies directed towards intracellular enzymes. With these considerations in mind, we hypothesized that exposure to P-ALP (placental alkaline phosphatase) during pregnancy results in autoreactivity directed towards all human tissues harboring the ALP enzyme (liver, bone and intestine) in genetically predisposed individuals. METHODOLOGY: To test this hypothesis, we stimulated peripheral blood mononuclear cells of primary biliary cirrhosis patients (n = 17) cholestatic liver disease controls (n = 6) and healthy controls (n = 14) with P-ALP, polyclonal activators (phytohemagglutinin [PHA], anti-CD3) and recall antigens (tetanus toxoid, streptokinase). We then determined their proliferative and cytokine responses by 3H-thymidine incorporation and ELISA assays for Il-10, IL-6, tumor necrosis factor-alpha and interferon-gamma, respectively. RESULTS: The results of the study revealed that the proliferative response to P-ALP was similar in peripheral blood mononuclear cells from primary biliary cirrhosis patients, cholestatic and healthy controls. Although the proliferative responses to PHA (P < 0.001) and anti-CD3 (P < 0.001) were decreased in peripheral blood mononuclear cells from primary biliary cirrhosis patients when compared to both control groups, responses to the recall antigens; tetanus toxoid and streptokinase were similar in the three groups. Cytokine production following exposure to P-ALP, polyclonal activators or recall antigens in peripheral blood mononuclear cells from primary biliary cirrhosis patients was similar to that of cholestatic and healthy controls. CONCLUSIONS: The results of the above experiments suggest that P-ALP is unlikely to be the target autoantigen in primary biliary cirrhosis. The results also support the findings of other investigators that primary biliary cirrhosis patients have suppressed proliferative responses to polyclonal stimulation.     

Evidence for activation of platelets in the synovial fluid from patients with rheumatoid arthritis

Summary Platelets were isolated by gel filtration from paired samples of peripheral blood and synovial fluid (SF) aspirated from inflamed knee joints from 20 adult patients with rheumatoid arthritis (RA) as well as from peripheral blood obtained from 20 healthy subjects. The platelets from the three different sources were investigated for quantitative differences in the number of two distinct types of intracellular storage organelles using immunofluorescence staining for platelet factor 4 (PF4) and labelling with the fluorescent substance mepacrine (MC). The number of PF4-stained organelles per cell was the same in the peripheral normal and RA platelets. This number was distinctly lower in the SF platelets. The peripheral and SF platelets from the RA patients had the same number of MC-labelled organelles. This number was distinctly lower than in the normal cells. The results suggest that the peripheral RA platelets had been activated to liberate serotonin and other substances from one type of organelles, and that the SF platelets had been activated to an additional liberation of PF4 from another such type. Liberated PF4, serotonin, and other substances from SF platelets may, in several ways, contribute to the inflammatory responses of RA.     

Spontaneous and induced immunoglobulin secretion by synovial fluid B lymphocytes in rheumatoid arthritis.

The functional properties of B lymphocytes in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were analysed by means of a reverse haemolytic plaque forming cell (PFC) assay. SF mononuclear cells spontaneously secreted IgG, but little IgM or IgA. The SF cells failed to respond to the polyclonal B cell activators pokeweed mitogen (PWM) and Epstein-Barr virus. However, SF B cells cocultured with autologous T lymphocytes from the blood and stimulated with PWM secreted IgG but little IgM or IgA. The PFC responses of blood B cells cocultured with autologous SF T cells in the presence of PWM were low; irradiation of the T cells increased the blood B lymphocyte responses, but the differences were not statistically significant. It is concluded that suppressor SF T cells may be partly responsible for the poor response of SF B cells to PWM.     

CTLA-4IG suppresses reactive oxygen species by preventing synovial adherent cell-induced inactivation of Rap1, a Ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells

OBJECTIVE: Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes. METHODS: ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS-dependent fluorescence of 6-carboxy-2',7'-dichlorofluorescein. Rap1 GTPase activation was assessed by activation-specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by 3H-thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA-4Ig fusion proteins. Short- and long-term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies. RESULTS: T lymphocyte ROS production and Rap1 inactivation were mediated by cell-cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4-Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti-CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production. CONCLUSION: Cell-cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4-Ig fusion protein.