Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo

Objective. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. Methods. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. Results. IL-2—induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2—dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. Conclusion. Anti-CD5 has an inhibitory effect on in vivo—activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to “switching-off” of T cell activation in the joints.     

Mechanisms of CD23 hyperexpression on B cells from patients with rheumatoid arthritis.

OBJECTIVE: To analyze the mechanisms involved in the characteristic hyperexpression of CD23 on peripheral blood B cells from patients with rheumatoid arthritis (RA). METHODS: Peripheral blood mononuclear cells (PBMC) were obtained from patients with active disease and activated during 18 h with an anti-CD3 monoclonal antibody in the presence or absence of blocking antibodies to CD154 or CD40. PBMC were further purified by rosetting and CD23 expression was assessed on B cells by flow cytometry after double staining (CD19/CD23). Lymphocytes were also isolated from synovial fluid (SF). CD154 expression was analyzed on PB or SF CD4+ T cells after double staining (CD4/CD154) by flow cytometry at basal conditions and after different stimuli [anti-CD3 or phorbol myristic acetate (PMA) plus ionomycin]. Co-culture experiments between SF and PB cells were performed to analyze the involvement of the CD40-CD154 interaction on CD23 expression. CD154 and CD23 expression was also analyzed on synovial membrane by immunohistochemical techniques. RESULTS: A high proportion of activated CD23 B cells was detected in patients with RA. Blocking experiments with both anti-CD40 and anti-CD154 Mab showed a significant reduction in the proportion of PB B cells expressing CD23. Following activation with anti-CD3 Mab or PMA plus ionomycin, CD154 expression was mainly induced on PB CD4+ T cells. In co-culture experiments, SF T cells were more efficient than PB T cells in inducing CD40 dependent CD23 expression on PB B cells. In addition, CD4+ T cells from synovial membrane clearly expressed CD154. CONCLUSION: Our results establish a link between CD154-CD40 pathway and CD23 expression on PB B cells from patients with RA. T cells from the synovial microenvironment were active participants in this CD23 expression, presumably in the context of cell recirculation.     

High percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid

Objective. While analyzing the phenotype of the synovial fluid mononuclear cells (SFMC) clustered about dendritic cells in rheumatoid arthritis (RA) joint effusions, it was noted that most of the clustering cells were CD8+ and coexpressed Leu-7. Therefore, the present study was conducted to investigate the frequency of CD8+, Leu-7+ cells in RA SF. Methods. SFMC from 13 patients with RA and from 12 patients with non-RA inflammatory arthritides were examined for CD8 and Leu-7 expression using 2-color immunofluorescence flow cytometry. Results. RA SFMC had statistically significantly greater percentages of total CD8+ cells, total Leu-7+ cells, and CD8+, Leu-7+ cells, compared with SFMC from the non-RA patients. These RA CD8+, Leu-7+ SFMC had a distinctive electron microscopic appearance compared with CD8+, Leu-7– SFMC. When peripheral blood mononuclear cells (PBMC) from 31 RA patients (including 7 from the SFMC group) were compared with PBMC from 15 normal controls, the percentage of CD8+, Leu-7+ cells was not significantly greater in the RA patients. However, the combination of a modest increase in CD8+, Leu-7+ cells and a decrease in total CD8 cells in RA PBMC altered the composition of the RA CD8 population compared with normal PBMC, such that over 40% of RA peripheral blood CD8 cells coexpressed Leu-7. Conclusion. The increased frequency of CD8+, Leu-7+ cells in RA SFMC may arise from the fact that a high percentage of the CD8+ PBMC in RA patients are also Leu-7+. This altered composition of CD8 cells in RA SF may have a role in the pathogenesis of the disease.     

Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo.

OBJECTIVE. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. METHODS. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. RESULTS. IL-2-induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2-dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. CONCLUSION. Anti-CD5 has an inhibitory effect on in vivo-activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to "switching-off" of T cell activation in the joints.     

Interleukin-10 expression: is there a neglected contribution of CD8+ T cells in rheumatoid arthritis joints?

OBJECTIVE: To search for RA specific processes among T cell accumulation, T cell activation, or cytokine expression in CD4+ and CD8+ synovial fluid (SF) T cells. METHODS: Flow cytometry of CD4+, CD8+, CD45RA+, CD45RO+, CD69 double or triple stained peripheral blood (PB) and SF T cells. IL-2, IL-10, and IFN-gamma expression was determined in PMA + ionomycin stimulated T cells on the single cell level. Concentrations of secreted IL-2, IL-4, IL-10, and IFN-gamma were quantified in the sera and synovial fluids by enzyme linked immunosorbent assay (ELISA). RESULTS: A preferential recruitment of CD45RO+ memory T cells was found for CD4+ helper T cells, and in similar also for CD8+ suppressor T cells. An elevated CD69 expression was detected in memory, but also in CD45RA+ naive CD4+ and CD8+ SF T cells, whilst IL-2 expression was only demonstrable in a minor proportion of T cells populations. Preferential recruitment of memory T cells, but incomplete activation of naive and memory, CD4+ and CD8+ T cells were in similar found in RA and control patients. In RA but not in the control patients, a relevant proportion of CD4+ and CD8+ PB and SF T cells expressed IL-10 and IFN-gamma. High concentrations of IL-10, that were correlated with the amounts of secreted TNF-alpha, were only detected in RA joints. CONCLUSION: Memory and naive T cell state of CD4+ and CD8+ T cell accumulates in the joints, and early T cell activation occur in similar patterns in RA and control patients. High IL-10 SF concentrations in contrast, and elevated percentages of IFN-gamma and IL-10 expressing CD4+ and CD8+ T cells in the PB and SF were characteristic for RA. Here, CD8+ T cells may contribute to high IL-10 concentrations in RA joints.     

Shift toward T helper 1 cytokines by type II collagen-reactive T cells in patients with rheumatoid arthritis

OBJECTIVE: To investigate the impact of type II collagen (CII)-reactive T cells on the Th1/Th2 cytokine balance in patients with rheumatoid arthritis (RA). METHODS: T cell proliferative responses to bovine CII were examined in synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) by mixed lymphocyte culture. CII-reactive T cell lines were generated from the SFMC and PBMC. Interferon-gamma (IFNgamma), interleukin-12 (IL-12), and IL-4 were measured by enzyme-linked immunosorbent assay in the SF, sera, and culture supernatants of PBMC and SFMC that had been stimulated with CII. RESULTS: The frequency of CII-reactive T cells was higher in the PBMC from RA patients than in that from osteoarthritis patients and healthy control subjects. In RA patients, CII-reactive T cells were more prevalent in SFMC than in PBMC. The mean level of IFNgamma and the ratio of IFNgamma to IL-4 were significantly higher in the culture supernatants of T cells stimulated with CII; these differences were more prominent in SFMC. Levels of IL-12 in the culture supernatants of SFMC and PBMC stimulated with CII were significantly higher than those in unstimulated supernatants. T cell responsiveness correlated well with the level of type 1 cytokines in culture supernatants from RA T cells stimulated with CII. In the CII-reactive cell lines, the increased production of IFNgamma was consistent with clonal expansion. CONCLUSION: CII-reactive T cells are more abundant in SFMC than in PBMC and are strongly associated with a shift toward Thl cytokine in the inflamed joints of RA patients. Our results suggest that a skewing toward type 1 cytokines by CII-reactive T cells may play an important role in the chronic inflammatory process of RA.     

Proinflammatory mediator-induced reversal of CD4+,CD25+ regulatory T cell-mediated suppression in rheumatoid arthritis

OBJECTIVE: We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg-mediated suppression. METHODS: Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme-linked immunosorbent assay. Magnetically sorted CD4+,CD25- and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti-CD3 monoclonal antibody (mAb) and autologous antigen-presenting cells, in the absence or presence of anti-CD28 mAb or the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or IL-7. RESULTS: Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB-derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti-CD28 mAb to cocultures of CD4+,CD25- and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg-mediated suppression in both PB and SF. Furthermore, IL-7 and, to a limited extent, TNFalpha, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg-mediated suppression. In contrast, IL-6 did not influence Treg-mediated suppression. CONCLUSION: Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.     

Murine B7 antigen provides an efficient costimulatory signal for activation of murine T lymphocytes via the T-cell receptor/CD3 complex.

We demonstrate that the murine B7 (mB7) protein is a potent costimulatory molecule for the activation of resting murine CD4+ T cells through the T-cell receptor (TCR)/CD3 complex. Stable mB7-transfected Chinese hamster ovary cells, but not vector-transfected controls, synergize with anti-CD3 monoclonal antibody and Con A-induced T-cell activation, resulting ultimately in proliferation. mB7 exerted its effect by inducing production of interleukin 2 and expression of the interleukin 2 receptor. Thus, mB7 costimulates T-cell activation through the TCR/CD3 complex by positively modulating the normal pathway of T-cell expansion. In contrast to the pronounced effect of mB7 on the activation of T cells through the TCR/CD3 complex, the mB7-transfected CHO cell line costimulated T-cell activation via the glycosylphosphatidylinositol-anchored proteins Thy-1 and Ly-6A.2 only inefficiently. Finally, the combination of a calcium ionophore and mB7 is not sufficient to cause T-cell proliferation, while the combination of a calcium ionophore and phorbol 12-myristate 13-acetate (PMA) stimulates T cells efficiently. The signals that mB7 and PMA provide for murine T lymphocyte activation are therefore not interchangeable, although both costimulate activation through the TCR/CD3 complex.     

High percentage of CD8+, Leu-7+ cells in rheumatoid arthritis synovial fluid.

OBJECTIVE. While analyzing the phenotype of the synovial fluid mononuclear cells (SFMC) clustered about dendritic cells in rheumatoid arthritis (RA) joint effusions, it was noted that most of the clustering cells were CD8+ and coexpressed Leu-7. Therefore, the present study was conducted to investigate the frequency of CD8+, Leu-7+ cells in RA SF. METHODS. SFMC from 13 patients with RA and from 12 patients with non-RA inflammatory arthritides were examined for CD8 and Leu-7 expression using 2-color immunofluorescence flow cytometry. RESULTS. RA SFMC had statistically significantly greater percentages of total CD8+ cells, total Leu-7+ cells, and CD8+, Leu-7+ cells, compared with SFMC from the non-RA patients. These RA CD8+, Leu-7+ SFMC had a distinctive electron microscopic appearance compared with CD8+, Leu-7- SFMC. When peripheral blood mononuclear cells (PBMC) from 31 RA patients (including 7 from the SFMC group) were compared with PBMC from 15 normal controls, the percentage of CD8+, Leu-7+ cells was not significantly greater in the RA patients. However, the combination of a modest increase in CD8+, Leu-7+ cells and a decrease in total CD8 cells in RA PBMC altered the composition of the RA CD8 population compared with normal PBMC, such that over 40% of RA peripheral blood CD8 cells coexpressed Leu-7. CONCLUSION. The increased frequency of CD8+, Leu-7+ cells in RA SFMC may arise from the fact that a high percentage of the CD8+ PBMC in RA patients are also Leu-7+. This altered composition of CD8 cells in RA SF may have a role in the pathogenesis of the disease.     

Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patients with rheumatoid arthritis

Objective. To investigate whether T cells in the inflamed joints of patients with rheumatoid arthritis (RA) preferentially produce the T helper 1 (Th1) cytokines, interferon-γ (IFNγ) and interleukin-2 (IL-2), or the Th2 cytokine, IL-4, when compared with corresponding peripheral blood—derived T cells. Methods. Synovial fluid mononuclear cells (SFMC) and corresponding peripheral blood mononuclear cells (PBMC) from 10 patients with RA were analyzed, either directly or after in vitro stimulation, for the intracellular presence of Th1 and Th2 cytokines. The amount of secreted cytokine in the cell culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Results. IFNγ-containing cells were detected in the unstimulated SFMC, but not in the PBMC, of 3 patients with RA. Cells positive for IL-2 or IL-4 were not detected in the unstimulated samples. Following stimulation, the mean percentage of cells containing Th1 cytokines was significantly increased in the SFMC compared with the PBMC; no differences were found in the mean percentage of IL-4—containing cells. A comparable shift toward Th1 cytokines was observed when the amount of secreted cytokine was determined by ELISA. Conclusion. A shift toward T cells with a Th1 cytokine profile was observed in the joints of patients with RA. Since an imbalance between Th1 and Th2 cells is thought to be of pathogenic significance, this finding might have implications for the development of new therapies for RA.     

Phorbol ester treatment inhibits phosphatidylinositol 3-kinase activation by, and association with, CD28, a T-lymphocyte surface receptor.

CD28 is a costimulatory receptor found on the surface of most T lymphocytes. Engagement of CD28 induces interleukin 2 (IL-2) production and cell proliferation when combined with an additional signal such as treatment with phorbol ester, an activator of protein kinase C. Recent studies have established that after CD28 ligation, the cytoplasmic domain of CD28 can bind to the 85-kDa subunit of phosphatidylinositol 3-kinase (PI3 kinase). There is a concomitant increase in PI3 lipid kinase activity that may be important in CD28 signaling. Despite the requirement of phorbol 12-myristate 13-acetate (PMA) for effector function, we have found, however, that treatment of Jurkat T cells with the phorbol ester PMA dramatically inhibits (i) the association of PI3 kinase with CD28, (ii) the ability of p85 PI3 kinase to be immunoprecipitated by anti-phosphotyrosine antibodies, and (iii) the induction of PI3 kinase activity after stimulation of the cells with the anti-CD28 monoclonal antibody 9.3. These changes occur within minutes of PMA treatment and are persistent. In addition, we have found that wortmannin, a potent inhibitor of PI3 kinase, does not interfere with the induction of IL-2 after stimulation of Jurkat T cells with anti-CD28 monoclonal antibody and PMA. We conclude that PI3 kinase activity may not be required for CD28-dependent IL-2 production from Jurkat T cells in the presence of PMA.     

In vitro apoptosis and expression of apoptosis-related molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases

Objective. To analyze factors related to apoptosis in systemic lupus erythematosus (SLE) peripheral blood mononuclear cells (PBMC) and to compare the findings in SLE PBMC with those in normal donor PBMC or PBMC from patients with other autoimmune diseases. Methods. PBMC from normal healthy donors or patients with SLE, mixed connective tissue disease (MCTD), rheumatoid arthritis (RA), or various vasculitides were isolated. The percentage of apoptosis after activation through different signaling pathways was quantified using propidium iodide staining. Protein expression of Fas/APO-1 or bcl-2, and messenger RNA (mRNA) expression of bcl-2, bcl-x_L, bax, bak, Fas/APO-1, Fas ligand (Fas-L), c-myc, mad, or max were determined. Results. We confirmed previous findings of increased numbers of apoptotic cells in SLE PBMC compared with normal donor cells after in vitro incubation. After activation of PBMC with CD28 monoclonal antibody plus phorbol myristate acetate (CD28 MAb/PMA), staphylococcal enterotoxin B (SEB), or phytohemagglutinin (PHA), the percentage of apoptotic cells was unchanged (SEB) or diminished (CD28 MAb/PMA, PHA) in SLE cells, and the difference between normal donor and SLE cells was less pronounced. On the mRNA level, expression of apoptosis-related gene products did not differ between SLE cells and normal donor cells. Expression of Fas/APO-1 protein was increased in freshly isolated SLE T lymphocytes compared with normal donor T lymphocytes, whereas bcl-2 protein was up-regulated after a 3-day culture period. Cellular activation further increased bcl-2 protein levels, eliminating differences between normal donors and SLE patients. In RA cells, the percentage of apoptosis was similar to that in normal donor PBMC, whereas results using cells from patients with other autoimmune diseases (MCTD, Wegener's granulomatosis, Takayasu arteritis, polyarteritis nodosa) were comparable with those found using SLE PBMC. Addition of growth factors such as interleukin-2 (IL-2), IL-4, or IL-15 to culture medium decreased the percentage of in vitro apoptosis in both normal donor and SLE cells. Conclusion. Based on these data, we conclude that accelerated in vitro apoptosis and increased Fas/APO-1 and bcl-2 protein expression in SLE are nonspecific for the disease, and might be explained at least in part by the increased in vivo activation levels of PBMC from patients with SLE, MCTD, or autoimmune vasculitides combined with in vitro incubation under “noninflammatory” conditions and growth factor withdrawal.     

Methyl-vitamin B12 blocks the CD28 co-stimulatory pathway in human T cells and its possible therapeutic application for T cell-mediated diseases,including rheumatoid arthritis

Abstract

The effects of metyl-vitamin B12 have been examined on human T cell activation induced by stimulation of T cell receptor (TCR)-CD3 and an accessory molecule, CD28. When T cells in the presence of 10% mitomycin-treated non-T cells were stimulated with anti-CD3 mAb at the optimal concentration, methyl-B12 did not inhibit T cell proliferation. However, when T cells were stimulated with the suboptimal concentration of anti-CD3 and anti-CD28 mAbs, methyl-B12 exhibited potent inhibition of T cell proliferation. Methyl-B12 did not affect cell surface expression of the CD3 and CD28 molecules of T cells. Methyl-B12 inhibited a 29 kDa protein tyrosine phosphorylation that was specifically induced by anti-CD3 and anti-CD28 mAbs. Similarly, T cell proliferation of patients with rheumatoid arthritis (RA), which is representative of T cell-mediated disease was inhibited by methyl-B12, when T cells were stimulated by anti-CD3 and anti-CD28 mAbs. These results suggest that methyl-B12 modulates lymphocyte function through blockade of the CD28 signaling pathway and that methyl-B12 may have T cell inhibitory activity that is applicable for treating patients with RA.     

Methyl-vitamin B12 blocks the CD28 co-stimulatory pathway in human T cells and its possible therapeutic application for T cell-mediated diseases, including rheumatoid arthritis

The effects of metyl-vitamin B12 have been examined on human T cell activation induced by stimulation of T cell receptor (TCR)-CD3 and an accessory molecule, CD28. When T cells in the presence of 10% mitomycin-treated non-T cells were stimulated with anti-CD3 mAb at the optimal concentration, methyl-B12 did not inhibit T cell proliferation. However, when T cells were stimulated with the suboptimal concentration of anti-CD3 and anti-CD28 mAbs, methyl-B12 exhibited potent inhibition of T cell proliferation. Methyl-B12 did not affect cell surface expression of the CD3 and CD28 molecules of T cells. Methyl-B12 inhibited a 29 kDa protein tyrosine phosphorylation that was specifically induced by anti-CD3 and anti-CD28 mAbs. Similarly, T cell proliferation of patients with rheumatoid arthritis (RA), which is representative of T cell-mediated disease was inhibited by methyl-B12, when T cells were stimulated by anti-CD3 and anti-CD28 mAbs. These results suggest that methyl-B12 modulates lymphocyte function through blockade of the CD28 signaling pathway and that methyl-B12 may have T cell inhibitory activity that is applicable for treating patients with RA.     

Cd28 expression on t cell subsets in vivo and cd28-mediated t cell response in vitro in patients with rheumatoid arthritis

Objective. In view of the critical importance of the CD28–CD80 (B7/BB1) costimulatory pathway in antigen-specific T cell activation and clonal expansion, we examined CD28 surface molecule expression in vivo, and T cell receptor/CD3-mediated and B7/BB1-costimulated T cell proliferation in vitro, in rheumatoid arthritis (RA). Methods. Two-color immunofluorescence analyses of peripheral blood and synovial fluid–derived T cells, as well as ³H-thymidine incorporation assays, were performed. Results. In the peripheral blood of 31 patients with active, untreated RA, a mean of 91% (range 48–100%) of CD4+ and 46% (range 13–82%) of CD8+ T cell subsets were CD28+, which was not significantly lower than normal. Although an overall decrease in the number of T cells was not observed, the numbers of CD28+CD8+ T cells were significantly lower in RA patients (mean 233/μl, versus 292/μl in controls), and this decrease was more pronounced in patients with severe disease (mean 172/μl). CD28 expression on peripheral CD8+ T cells in RA patients, but not in healthy individuals, correlated inversely with T cell activation as assessed by HLA–DR antigen expression. In contrast to the peripheral blood, RA synovial fluid T cells were almost exclusively CD28+, suggesting that migration of CD28+CD8+ T cells to active sites of inflammation may occur. In vitro proliferative responses of peripheral blood T cells to B7/BB1 costimulation in the presence of mitogenic doses of anti-CD3 monoclonal antibody were identical in patients with RA and healthy individuals. Conclusion. Functionally intact CD28+ T cells may contribute to the abnormal immunoregulation and joint inflammation in RA.