Accessory cell function of a human colonic epithelial cell line HT-29 for bacterial superantigens

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both >90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.     

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.     

Staphylococcal enterotoxin B-induced activation and concomitant resistance to cell death in CD28-deficient HLA-DQ8 transgenic mice

HLA class II molecules present superantigens more efficiently than their murine counterpart. Therefore, transgenic mice expressing HLA-DQ8 with and without CD28 were used to address the role of CD28 in staphylococcal enterotoxin B (SEB)-driven immune responses. SEB-induced in vitro proliferation of naive DQ8.CD28(-/-) splenocytes was comparable to DQ8.CD28(+/+) cells, and was several fold higher than that of C57BL/10 and BALB/c splenocytes. SEB-activated, naive DQ8.CD28(-/-) cells in vitro produced significantly less IL-2, IL-4 and IL-10 than DQ8.CD28(+/+) cells, while IFN-gamma and IL-6 production was comparable. SEB-induced in vivo expansion of CD4(+) T cells and, to a greater extent, CD8(+) T cells was compromised in DQ8.CD28(-/-) mice, indicating that SEB-induced proliferation of CD8(+) T cells is more dependent on CD28 co-stimulation. Upon re-stimulation, SEB-primed CD28(+/+) T cells failed to proliferate but were capable of producing cytokines. Conversely, CD28(-/-) T cells were capable of proliferation, but not cytokine production. SEB-primed CD28-deficient cells produced significantly less nitric oxide when compared to CD28-sufficient cells following re-stimulation with SEB. CD28(+/+) and not CD28(-/-) mice were highly susceptible to SEB-induced lethal shock characterized by significantly elevated serum IFN-gamma. Thus, (i) efficient presentation of SEB by HLA-DQ8 circumvents co-stimulation through CD28, (ii) unique CD28-derived signals are mandatory for generation of certain effector functions, and (iii) absence of CD28-derived signals confers resistance to activation-induced cell death and protects mice from SEB-induced shock.     

Sequential production of IL-2, IFN-γ and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes

Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-γ and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-γ and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-γ, separated according to specific surface-associated IFN-γ as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-γ expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-γ-inducing monokines and limiting the pro-inflammatory effects of IFN-γ.     

Selective recruitment of CCR6-expressing cells by increased production of MIP-3 alpha in rheumatoid arthritis

Retroviral superantigens such as minor lymphocyte stimulating (Mls) antigen play an important role in the pathogenesis of acute graft-versus-host disease (GVHD). However, it remains unclear how exogenous bacterial superantigens modulate acute GVHD. In this study, we tested the effects of staphylococcal enterotoxin B (SEB) on the development of acute GVHD in a model involving the systemic transfer of parental C57Bl/6 spleen cells into BDF1 mice. SEB treatment suppressed the expansion of donor-derived T cells and blocked the decrease in the number of host cells. Impaired haematopoiesis was actually rescued by treatment with SEB. In SEB-treated mice, both spontaneous proliferation and IL-2 production in T cells were suppressed on day 2 after parental cell infusion. On day 21, the number of donor-derived CD4+ Vbeta8+ T cells markedly decreased in the spleen of SEB-treated mice. Donor-derived CD4+ T cells failed to proliferate in response to host alloantigens, and both donor- and host-derived T cells were unable to produce IL-2 in response to concanavalin A stimulation, suggesting that SEB treatment induced a general immunosuppressive state. Our results indicate that SEB treatment prevents the development of acute GVHD by leading to unresponsiveness of donor-derived T cells against host alloantigens in a Vbeta-restricted and unrestricted manner.     

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.     

Manipulation of the superantigen-induced lymphokine response. Selective induction of interleukin-10 or interferon-γ synthesis in small resting CD4+ T cells

The production of several lymphokines by freshly isolated CD4⁺ T cells has been analyzed at the single-cell level, after stimulation with staphylococcal enterotoxin B (SEB). High frequencies of cells producing interleukin-2 (IL-2) and interferon-γ (IFN-γ) were induced, but very low frequencies of CD4⁺ T cells produced IL-4, IL-5 or IL-10 in response to SEB. Exogenously added IL-4 markedly altered the lymphokine profile induced during primary SEB stimulation. IFN-γ production was reduced, while a high fraction of cells contained IL-10 and IL-4 after activation in the presence of IL-4. We further demonstrate that IL-4 and IL-10 or IFN-y production was selectively induced in resting, high-density CD4⁺ T cells during primary stimulation, by SEB + IL-4 or SEB. Under conditions where both IL-10 and IFN-γ were produced, most cells contained only one of the two lymphokines.     

Superantigen and endotoxin synergize in the induction of lethal shock

Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-γ (IFN-γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D -galactosamine (D -GalN). Opposed to D -GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.     

Clonal anergy to staphylococcal enterotoxin B in vivo: selective effects on T cell subsets and lymphokines

Injection of bacterial superantigens such as staphylococcal enterotoxin B (SEB) in adult mice results in initial proliferation of SEB-responsive Vβ8⁺ T cells followed by induction of a state of non-responsiveness frequently referred to as clonal anergy. We show here that SEB-induced anergy involves selective changes in lymphokine production and that it affects CD4⁺ Vβ 8⁺ and CD8⁺ Vβ 8⁺ T cells in different fashions. Whereas both CD4⁺ Vβ 8⁺ and CD8⁺ Vβ 8⁺ cells from anergic mice exhibit strongly reduced proliferative capacity and interleukin(IL)-2 production upon restimulation with SEB either in vivo or in vitro the CD8⁺ subset from SEB-injected mice produces other lymphokines (such as interferon(IFN)-γ) at normal or slightly increased levels in response to SEB. Changes in the levels of production of IL-2 and IFN-γ protein correlated well with mRNA accumulation both in vivo and in vitro. Collectively these data suggest that superantigen-induced anergy involves selective changes in signal transduction and/or gene regulation in T lymphocytes.     

Synovial fibroblasts as target cells for staphylococcal enterotoxin-induced T-cell cytotoxicity.

Rheumatoid arthritis (RA) is a chronic autoimmune disease of unknown aetiology. Recently, superantigens have been implied in the pathogenesis of RA. Superantigens activate a large fraction of T cells leading to the production of cytokines and proliferation. In addition, superantigens direct cellular cytotoxicity towards major histocompatibility complex (MHC) class II-expressing cells. There is now increasing evidence that cytotoxic T cells may be involved in the pathogenesis of RA. In the inflamed synovia class II-positive synovial fibroblasts (SFC) are found. In the present study it was tested whether MHC class II-positive SFC serve as target cells for superantigen-induced cellular cytotoxicity. SFC were stimulated with interferon-gamma to express class II antigens, then they were cultivated in the presence of CD4-positive T cells with or without staphylococcal enterotoxins (SE). Cytotoxicity of T cells was measured as release of lactate dehydrogenase from SFC. Specific cytotoxicity was only found in the presence of class II-positive SFC depending on the dose of SE. Maximum lysis was seen after 20 hr. T-cell cytotoxicity was inhibited by antibodies to MHC class II antigens. The data suggest that class II-positive SFC not only function as accessory cells for SE-mediated T-cell proliferation and interleukin-2 production but may also be the targets of superantigen-mediated cellular cytotoxicity.     

Cartilage-reactive T cells in rheumatoid synovium

Rheumatoid arthritis (RA) is an inflammatory polyarthritis genetically linked to HLA-DR4 and related haplotypes. RA synovial tissue is characterized by T cell infiltration and activation of macrophage-like cells, strongly implicating a T cell-antigen-presenting cell (APC) interaction in RA pathogenesis. To investigate the nature of the antigens driving the T cell response, synovial tissue was obtained from a patient with chronic RA and T cells were enriched. These T cells were stimulated by endogenous APC from the same synovial tissue. The T cell lines were subsequently evaluated for responsiveness to autologous APC and cartilage antigens. Specific proliferative responses to autologous APC which were enhanced by cartilage extract were seen. Immunomagnetic bead selection and RT-PCR was used to identify TCR alphabeta pairs which appeared to respond to antigen(s) in the cartilage extract. T cell clones derived from the same joint were shown to release IL-2 in response to the cartilage extract and expressed a related TCR. With these experiments we have shown direct evidence that autoreactive T cells are found within the inflamed rheumatoid synovium and, further, that the antigens driving these T cells are cartilage derived. Since the antigens recognized by these populations of T cells are found within cartilage our data provides evidence that RA pathology could be related to a self-driven autoimmune response to cartilage proteins.     

Expression of human minor histocompatibility antigen on cultured kidney cells

Incompatibility of human minor histocompatibility (hmH) antigens can induce rejection of grafts in organ transplantation and graft-versus-host reactions in bone marrow transplantation. In spite of their importance in clinical transplantation, hmH antigens are not well studied. Previous studies have demonstrated the expression of hmH antigens on Tand B cells, hematopoietic progenitor cells and keratinocytes. We have for the first time demonstrated the expression of hmH antigens on cultured kidney cells using HLA-B35-restricted, hmH antigen-specific cytotoxic T lymphocyte (CTL) clones, which were previously established from a patient who rejected two kidneys from HLA-identical sisters. The CTL clones could not kill cultured kidney cells. Since cultured kidney cells expressed very low levels of HLA class I antigens it was thought that their failure to be killed by the CTL clones was due to lack of expression of HLA-B35 antigens. After induction of class I antigens on cultured kidney cells by interferon-y (IFN-γ), the IFN-γ-treated cultured kidney cells were killed by the CTL clones. Furthermore, we isolated hmH antigens as peptides from cultured kidney cells after treatment with IFN-γ. These results indicate that cultured kidney cells express hmH antigens when HLA class I antigen is induced by IFN-γ and hmH antigens on cultured kidney cells are recognized by T cells as peptides presented by HLA-B35 molecules.     

IL-7 promotes the survival and maturation but not differentiation of human post-thymic CD4+ T cells

This study examines the influence of IL-7 on post-thymic CD4⁺ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50 % cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-γ-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.     

Higher Bcl-2 levels decrease staphylococcal superantigen-induced apoptosis of CD4+ T cells in atopic dermatitis

BACKGROUND: Staphylococcal superantigens (SsAgs) contribute to the persistence of allergic skin inflammation in atopic dermatitis (AD). The aims of this study were to (1) determine whether there are differences between AD patients and healthy subjects in SsAg-induced caspase-3 activation and SsAg-induced changes of anti-apoptotic protein Bcl-2 and Bcl-2 mRNA levels of CD4+ T cells; (2) investigate the effect of interleukin (IL)-4 on SsAg-induced caspase-3 activation and SsAg-induced changes of Bcl-2 and Bcl-2 mRNA levels of CD4+ T cells. METHODS: Using immunofluorescence staining followed by flow cytometric analysis and real-time PCR, we analyzed peripheral blood mononuclear cells with or without staphylococcal enterotoxin B (SEB) stimulation in the presence or absence of recombinant IL-4 or anti-IL-4 neutralizing antibodies in 16 AD patients and 14 healthy subjects. RESULTS: SEB-reactive (TCRVbeta3+, Vbeta12+, and Vbeta17+) CD4+ T cells from AD patients were more resistant to SEB-induced caspase-3 activation and SEB-induced decrease of Bcl-2 and Bcl-2 mRNA than those from healthy subjects. Exogenously added IL-4 inhibited SEB-induced caspase-3 activation and SEB-induced decrease of Bcl-2 and Bcl-2 mRNA in SEB-reactive CD4+ T cells from healthy subjects. Inhibition of endogenous IL-4 by using anti-IL-4 neutralizing antibodies up-regulated SEB-induced caspase-3 activation and SEB-induced decrease of Bcl-2 and Bcl-2 mRNA in SEB-reactive CD4+ T cells from AD patients. CONCLUSIONS: Following SsAg stimulation, IL-4 produced by T cells in AD patients down-regulates SsAg-induced caspase-3 activation and apoptosis of CD4+ T cells through inhibiting the decrease of Bcl-2. This may impair deletion of SsAg-activated T cells and resolution of allergic skin inflammation.     

Antigen-presenting human T cells and antigen-presenting B cells induce a similar cytokine profile in specific T cell clones

One of the factors that may influence the cytokine secretion profile of a T cell is the antigen-presenting cell (APC). Since activated human T cells have been described to express major histocompatibility complex (MHC) class II molecules as well as costimulatory molecules for T cell activation, like e.g. ICAM-1, LFA-3 and B7, they might play a role as APC and be involved in the regulation of T-T cell interactions. To define further the role of T cells as APC we tested their capacity to induce proliferation and cytokine production in peptide- or allospecific T cell clones and compared it with conventional APC, like B lymphoblasts (B-LCL) or HTLV-1 - transformed T cells, or with non-classical APC, like activated keratinocytes or eosinophils. CD4⁺, DP-restricted T cell clones specific for a tetanus toxin peptide (amino acids 947-967) and CD4⁺, DR-restricted allospecific Tcell clones produced interleukin (IL)-2, IL-4, tumor necrosis factor-α and interferon-γ (IFN-γ) after phorbol 12-myristate 13-acetate and ionomycin stimulation and a more restricted cytokine pattern after antigen stimulation. Dose-response curves revealed that the antigen-presenting capacity of activated, MHC class II⁺, B7⁺ T cells was comparable to the one of B-LCL. Both APC induced the same cytokine profile in the T cell clones despite a weaker proliferative response with T cells as APC. Suboptimal stimulations resulted in a lower IFN-γ/IL-4 ratio. Cytokine-treated, MHC class II⁺ keratinocytes and eosinophils differed in the expression of adhesion molecules and their capacity to restimulate T cell clones. The strongly ICAM-1-positive keratinocytes induced rather high cytokine levels. In contrast, eosinophils, which express only low densities of MHC class II and no or only low levels of adhesion molecules (B7, ICAM-1 and LFA3), provided a reduced signal resulting in a diminished IFN-γ/IL-4 ratio. We conclude that non-classical APC differ in their capacity to restimulate T cell clones, whereby the intensity of MHC class II and adhesion molecules (B7, ICAM-1) expressed seems to determine the efficacy of this presentation.     

Superantigens augment antigen-specific Th1 responses by inducing IL-12 production in macrophages.

Superantigens (SAg) are microbial proteins that mediate antigen-presenting cell (APC)-T cell interaction by cross-linking MHC class II molecules with subsets of TcRVbeta. SAgs are implicated in the pathogenesis of several infectious, inflammatory, and autoimmune diseases. In this study, we examined the influence of SEB on interleukin-12 (IL-12) production and the activation of antigen-specific Th1 responses. Addition of SEB augmented the antigen-induced proliferation of HS-17, a murine MBPp91-103 peptide-specific TcRVbeta6+ CD4+ Th1 clone. SEB augments HS-17 T cell proliferation through its interaction with IA(S) molecules on macrophages, but not with the TcRVbeta6 on HS-17 cells. On binding to IA(S), SEB induces IL-12 production in macrophages, which in turn augments antigen-induced proliferation of HS-17 T cells. Treatment with anti-IA(S) nmAb 10-3.6 inhibited the antigen- and SEB-induced IL-12 production and T cell proliferation. These results suggest that SAgs augment antigen-specific T cell responses by inducing IL-12 production in macrophages.     

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V_β8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V_β8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing “anergy” and apoptotic cell death of V_β8⁺ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V_β8⁺ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)α chains in vivo, although SEB induces IL-2R α in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V_β8⁺ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.     

Anti-Vbeta8 antibodies induce and maintain staphylococcal enterotoxin B-triggered Vbeta8+ T cell anergy

The mechanism involved in the maintenance of staphylococcal enterotoxin B (SEB)-induced T cell anergy is poorly understood. We demonstrated earlier that B cells play an important role in the maintenance of SEB-induced T cell anergy in vivo and in vitro. Here, we demonstrate that B cells are not essential in SEB-induced T cell activation, but are important for the maintenance of T cell memory phenotype and anergy in vivo. Studying the activated B cell repertoire, we observe that SEB treatment increases serum anti-Vbeta8 antibody titer as detected by enzyme-linked immunosorbent assay using soluble Vbeta8 chains as antigens, and by staining of a Vbeta8-expressing thymoma. These antibodies disappear gradually after immunization with SEB, whereas the capacity of the T cells to respond to SEB in vitro is restored. Anti-Vbeta8 monoclonal antibody treatment causes Vbeta8+ T cell unresponsiveness to SEB in vitro (anergy), without affecting CD4Vbeta8+ T cell frequency. Together, these results suggest a new mechanism to explain the maintenance of SEB-induced T cell anergy, which is dependent on B cells and on anti-Vbeta8 antibody that specifically interacts with Vbeta8+ T cells.     

Inhibitory effect of clarithromycin on costimulatory molecule expression and cytokine production by synovial fibroblast-like cells

This study was undertaken to investigate the immunomodulatory effect of clarithromycin against synovial fibroblast-like cells (synoviocytes). Synovial tissue obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients was enzymatically digested to separate synoviocytes. The synoviocytes were cultured with or without cytokines in the presence of various concentrations of clarithromycin. The expression of costimulatory molecules was examined on the surface of the synoviocytes, using specific MoAbs and flow cytometry. The production of cytokines by synoviocytes was also measured using an immunoenzymatic assay. Finally, autologous T cells were stimulated by interferon-gamma (IFN-γ)-treated synoviocytes in response to purified protein derivative (PPD). In some experiments, MoAbs specific for costimulatory molecules or clarithromycin were added and ³H-thymidine incorporation was counted. Intercellular adhesion molecule-1 (ICAM-1), LFA-3 and vascular cell adhesion molecule-1 (VCAM-1) were detected on the surface of both RA and OA synoviocytes. However, ICAM-2, B7–1 and B7–2 were not detected, and cytokines failed to induce these molecules. Both spontaneous and up-regulated expression of ICAM-1, LFA-3 and VCAM-1 by IFN-γ, IL-1β or 12-o-tetradecanoyl phorbol 13-acetate (TPA) were markedly suppressed by clarithromycin in a dose-dependent manner at concentrations between 0.1 and 10 μg/ml. The production of IL-1β, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-1α and tumour necrosis factor-alpha (TNF-α) by synoviocytes was detected. Clarithromycin significantly suppressed the production of these cytokines, but did not enhance IL-10 production. Finally, autologous T cells were stimulated by IFN-γ-treated synoviocytes in response to PPD. As clarithromycin suppressed HLA-DR and costimulatory molecule expression was enhanced by IFN-γ, autologous T cell proliferation was markedly inhibited by clarithromycin. Clarithromycin has a considerable immunosuppressive effect on synoviocytes by inhibiting costimulatory molecule expression, cytokine production and antigen-specific T cell proliferation induced by synoviocytes.     

Interleukin-15 up-regulates the expression of CD154 on synovial fluid T cells

To investigate the role of the CD40-CD154 interaction in rheumatoid arthritis (RA), we analysed the expression of CD154 on CD3+ and CD4+ T cells in synovial fluid (SF) from patients with RA and in peripheral blood (PB) from patients and normal controls. As interleukin (IL)-15 is a potent activator of synovial T cells we wanted to study whether IL-15 also regulated the expression of CD154 on these T cells. Freshly isolated synovial T cells did not express significant levels of CD154, as evaluated using flow cytometry, whereas the expression of CD86 and human leucocyte antigen (HLA)-DR was significantly elevated on SF T cells when compared with PB T cells from patients or controls. Synovial T cells could up-regulate their CD154 expression following activation with phorbol 12-myristate 13-acetate (PMA) + ionomycin or anti-CD3 + anti-CD28 monoclonal antibodies (mAbs), but the maximal level of expression remained lower than in control T cells. IL-15 significantly increased the expression of CD154 on SF and PB T cells from patients, whereas IL-2 had minimal effects. Furthermore, IL-15 induced extensive proliferation in SF T cells. Our results show that SF T cells up-regulate the expression of CD154 in the presence of IL-15, a cytokine present in the synovium of patients with RA. These results further emphasize the role of IL-15 in the pathogenesis of RA.