Protection of T cells from activation-induced cell death by Fas+ B cells

Naive CD4+ T cells proliferate strongly in response to stimulation by superantigens such as staphylococcal enterotoxin B (SEB). However, when these same cells revert to a resting phenotype and are subjected to restimulation with either SEB or anti-CD3, the majority of these SEB-responsive cells undergo Fas ligand (FasL)-mediated activation-induced cell death (AICD). We investigated the impact of Fas expression on T cell AICD by utilizing B cell stimulators that lacked functional FasL and either expressed or did not express the Fas receptor. Our results indicate that B cells play an important role in modulating the level of T cell AICD via the Fas/FasL pathway. Activated B cells expressing high levels of Fas receptor can redirect the FasL expressed by T cells primed to undergo AICD away from the T cells and prevent the induction of AICD in these cells. Furthermore, B cells stimulated through both the CD40 receptor and membrane IgM appear to mediate a stronger protective effect on T cells by virtue of their resistance to FasL-mediated cytolysis. These observations suggest a mechanism by which normal B cell and T cell responses to foreign antigen are maintained, while responses to self antigen are not.     

Intercellular adhesion molecule-1 on synovial cells attenuated interleukin-6-induced inhibition of osteoclastogenesis induced by receptor activator for nuclear factor κB ligand

In a co-culture of osteoclast precursor cells and synovial cells, interleukin-6 (IL-6) induces osteoclast formation. In contrast, in a monoculture of osteoclast precursor cells, IL-6 directly suppresses receptor activator for nuclear factor κB ligand (RANKL)-induced differentiation of osteoclast precursor cells into osteoclasts. In the present study, we explored why the effect of IL-6 differed between the monoculture and the co-culture systems. In the monoculture, mouse osteoclast precursor cell line, RAW 264·7 (RAW) cells were cultured with soluble RANKL (sRANKL) for 24 h or 3 days. sRANKL increased both expression of osteoclastogenesis marker, tartrate-resistant acid phosphatase isoform 5b (TRAP5b) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), whereas the co-addition of IL-6 decreased them both in a dose-dependent manner. In the co-culture, RAW cells and human synovial cell line, SW982 cells were cultured with IL-6+soluble IL-6 receptor (sIL-6R) for 3 days. TRAP5b and NFATc1 expression reduced by IL-6 was increased by the addition of SW982 cells in a manner dependent upon the number of added cells. IL-6+sIL-6R treatment significantly induced RANKL production in SW982 cells, and anti-RANKL antibody inhibited IL-6+sIL-6R-induced osteoclastogenesis. SW982 cells expressed high levels of ICAM-1 originally, and ICAM-1 expression was increased significantly by IL-6+sIL-6R. Anti-ICAM-1 antibody suppressed IL-6-induced osteoclastogenesis. Finally, in the monoculture system, addition of sICAM-1 dose-dependently restored the expression of TRAP5b reduced by IL-6. Similar results were obtained when the formation of TRAP-positive multi-nuclear cells were examined using mouse bone marrow cells. In conclusion, IL-6 gave different results in the co-culture and monoculture systems because in the co-culture, ICAM-1 from the synovial cells restored osteoclastogenesis suppressed by IL-6.     

Oral administration of type-II collagen suppresses IL-17-associated RANKL expression of CD4+ T cells in collagen-induced arthritis

The receptor activator of nuclear factor kappaB ligand (RANKL) is an osteoclastogenic mediator, which is mainly expressed by stromal cells and osteoblast. However, T cells can also be an important provider for RANKL in special condition such as autoimmune arthritis. We examined the RANKL expression of hyporesponsive CD4+ T cells induced by oral feeding with type II collagen in collagen-induced arthritis (CIA) mice. The potential of RANKL expression in CD4+ T cells was downregulated in tolerance, as compared with CIA. One of possible explanations for this phenomenon is that CII-specific T cell activation was intrinsically impaired in oral tolerance, which caused suppression of RANKL expression of CD4+ T cells. We also investigated the extrinsic role of cytokine in this process. IL-17, well-known pro-inflammatory cytokine was upregulated in CIA and downregulated in tolerance. IL-17 had a potential to stimulate T cells to express RANKL in dose-dependent manner. IL-17-associated RANKL expression of CD4+ T cells was downregulated in oral tolerance, suggesting that the induction of tolerance ameliorates IL-17-induced RANKL expression of T cells in murine CIA. We also discovered that CIA - T cells could enhance osteoclastogenesis but not oral tolerance - T cells. Oral tolerance might be promising therapeutic option in viewpoints of modulating autoreactivity of CII which can induce not only IL-17 production but also RANKL expression in CD4+ T cells.     

IFN-γ directly inhibits TNF-α-induced osteoclastogenesis in vitro and in vivo and induces apoptosis mediated by Fas/Fas ligand interactions

Cytokines secreted by T cells play a pivotal role in inflammatory bone destruction. Tumor necrosis factor-α (TNF-α) is a major proinflammatory cytokine produced by macrophages following T cell activation, and directly promotes osteoclast differentiation resulting in accelerated bone resorption. Interferon-γ (IFN-γ) attenuates RANKL-initiated cellular signals through osteoclast formation and counterbalances aberrant bone resorption. With respect to this crosstalk during osteoclastogenesis, the direct interruption of IFN-γ in TNF-α-induced osteoclast formation still requires elucidation. We have demonstrated that IFN-γ directly inhibits osteoclastogenesis induced by TNF-α stimulation and accelerates apoptosis mediated by Fas/Fas ligand signals. There were a decreased number of osteoclasts and reduced mRNA levels encoding Nfatc1 in cultured bone marrow macrophages. Apoptotic responses of cultured cells were observed, with accelerated nuclear fragmentation in osteoclast precursor cells and increased FasL mRNA levels in bone marrow cells stimulated with TNF-α evident. IFN-γ reduced the level of osteoclastogenesis in response to TNF-α treatment in vivo. IFN-γ inhibited TNF-α-induced osteoclastogenesis in mice with T cells that had been exposed to anti-CD4 and -CD8 antibodies. These results provide evidence that IFN-γ directly inhibits osteoclastogenesis and induces cells apoptosis by Fas/FasL signals, leading to the indirect regulation of bone resorption, which is required for protective roles in bone destruction at an inflammation site.     

Expression of membrane-bound and soluble receptor activator of NF-kappaB ligand (RANKL) in human T cells

The receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK are critical regulators for immune responses as well as bone remodeling. RANKL is a type II transmembrane protein that has two forms-a membrane-anchored protein and a secreted protein. In this report, we demonstrate for the first time the kinetical expression of two forms of RANKL in human T cells using two monoclonal antibodies (mAbs) against human RANKL, which we newly derived. Freshly isolated T cells rarely expressed mRANKL, while the activation of T cells induced a substantial but minimal level of mRANKL as well as the accumulation of considerable amounts of sRANKL. The addition of the metalloprotease inhibitor KB-R8301 efficiently suppressed the release of sRANKL from activated T cells or RANKL-transfectants, and reciprocally enhanced the mRANKL expression. The membrane form of RANKL was also expressed on the infiltrating T cells in the rheumatoid synovial fluid and in the gingival tissues of patients with periodontitis. Our results demonstrate that the expression of mRANKL on T cells is strictly limited, and the majority of RANKL protein produced by T cells may be active in the soluble form after shedding. The mAbs that were derived in this study may be useful for investigating the regulation and function of RANKL in immune responses and bone remodeling.     

小分子肽对成骨前体细胞MC3T3-E1骨保护素和核转录因子κB受体活化因子配体表达的影响

背景：体内实验显示,小分子肽能明显增加去卵巢大鼠的骨钙含量,使其骨密度增加,能很好地预防骨质疏松。同时体外实验显示,小分子肽能促进小鼠成骨细胞和成骨前体细胞MC3T3-E1增殖、分化、矿化,并且可能是通过抑制核转录因子 p50和p65的表达来起作用。而小分子肽对骨保护素/核转录因子κB受体活化因子配体的影响尚不明确。 目的：观察小分子肽对MC3T3-E1在增殖、分化、矿化过程中骨保护素和RANKL表达的影响。 方法：以体积分数10%胎牛血清的DMEM培养液为空白对照组,50,100 mg/L质量浓度小分子肽作用小鼠成骨前体细胞MC3T3-E1,分别于作用3,6,12,18,24,30 d后,收集细胞提取蛋白,Western Blot检测骨保护素和核转录因子κB受体活化因子配体蛋白的表达。 结果与结论：50,100 mg/L小分子肽作用MC3T3-E1后能明显促进作用骨保护素的表达(P < 0.01),而对核转录因子κB受体活化因子配体无明显影响。小分子肽作用后MC3T3-E1中骨保护素/核转录因子κB受体活化因子配体的比值要明显高于空白对照组(P < 0.01)。因此,认为小分子肽可以通过增加骨保护素的表达来影响骨保护素/核转录因子κB受体活化因子配体系统,间接地抑制破骨细胞的数量和功能。     

NKG2A is a marker for acquisition of regulatory function by human CD8+ T cells activated with anti-CD3 antibody

Treatment with anti-CD3 mAb modulates immune responses that cause type 1 diabetes and other diseases. CD8+ Tregs can be induced in vitro and in vivo by mAb. However, 1/3 of patients do not respond to drug therapy and in an equal proportion, anti-CD3 mAb does not induce Tregs in vitro. The acquisition of CD8+ Treg activity is a function of the CD8+ cells and not the targets in the assay. To identify markers to differentiate responses of CD8+ Tregs, we analyzed genes differentially expressed in CD8+ T cells of non-responders compared with responders, and found that an inhibitory receptor NKG2A (CD159a) was highly expressed in cells from all non-responders tested. Application of a mAb agonistic to NKG2A during in vitro CD8+ Treg induction by anti-CD3 prevented induction of CD8+ Tregs. CD8+ T cells that are TNFR2+ but NKG2A- are the most potently induced Tregs. The level of NKG2A expression on resting CD8+ T cells inversely correlated with acquisition of regulatory function when activated. We suggest that the induction of human CD8+ Tregs by anti-CD3 mAb is controlled by a negative signaling through NKG2A, and that NKG2A may serve as a negative marker of human CD8+ Tregs.     

Human Th1 responses driven by IL-12 are associated with enhanced expression of CD40 ligand

IL- 12 is the prominent inducer of Th1 responses in humans and in the mouse. CD40 ligand (CD40L) plays important roles in regulation of immune responses, including T cell-dependent activation of B cells and cytokine production by monocytes and dendritic cells. The present study examined the influences of IL-12 on the CD40L expression of activated human CD4⁺ T cells. IL-12 enhanced CD40L expression on CD4⁺ T cells stimulated with immobilized anti-CD3 in the complete absence of accessory cells, whereas IL-4 and IL-10 decreased it. Exogenous interferon-gamma (IFN-γ) did not increase CD40L expression on immobilized anti-CD3 stimulated CD4⁺ T cells at any time up to 168 h of culture. The IL-12-induced enhancement of CD40L expression on anti-CD3 activated CD4⁺ T cells was not influenced in the presence of a metalloproteinase inhibitor KB8301, which up-regulated CD40L expression by preventing the processing of membrane-bound CD40L, or B cells, which down-regulated CD40L expression by receptor-mediated endocytosis. These results indicate that IL-12 enhances the CD40L expression of activated CD4⁺ T cells independently of the IFN-γ production. The data thus suggest that Th1 responses induced by IL-12 might play an important role in the regulation of humoral immune responses through up-regulated CD40L expression.     

cAMP inhibits the OKT3-induced increase in cytoplasmic free calcium in the Jurkat T cell line: the degree of inhibition correlates inversely with the amount of CD3 binding ligand used

We have investigated the effect of cAMP concentration on the CD3-mediated rise in intracellular Ca2+ induced by anti-CD3 monoclonal antibody in the human T cell leukemia line Jurkat. Forskolin, prostaglandin E2 (PGE2) and dibutyryl cAMP (db-cAMP) were used to increase intracellular cAMP concentrations. Treatment of Jurkat cells with forskolin or db-cAMP for 3 h inhibited the subsequent rise in intracellular Ca2+ concentration induced by an optimally mitogenic dose of 100 ng/ml of the anti-CD3 OKT3, whereas PGE2 counteracted the Ca2+ rise only marginally. The inhibitory effect of forskolin and PGE2 on the Ca2+ signal correlated with their abilities to induce increased cAMP levels in Jurkat cells. The suppression of the Ca2+ response was dependent on the concentration of the cAMP-elevating agent and the time of pre-incubation with the drug. The cAMP-mediated inhibition of the Ca2+ response diminished or disappeared when increasing concentrations of OKT3 were used to stimulate the rise in intracellular Ca2+ concentration. The results indicate that cAMP participates in the regulation of T lymphocyte activation at the level of signal transduction. Our findings, which stress the crucial role of the concentration of the ligand used to trigger Ca2+ responses, provide an explanation to previous contradictory reports on the impact of cAMP on the signal transduction in T cells.     

IFN-gamma-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL

The current study explored our hypothesis that IFN-gamma-producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN-gamma-producing T cells (IFN-gamma(+) T cells) and IFN-gamma-non-producing T cells (IFN-gamma(-) T cells). IFN-gamma(+) T cells were cultured with human monocytes in the presence of macrophage-CSF alone. The concentration of soluble receptor activator of NF-kappaB ligand (RANKL) and IFN-gamma, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non-steroidal anti-inflammatory drugs alone, CD4+ T cells expressing both IFN-gamma and RANKL were detected by flow cytometry. Surprisingly, IFN-gamma(+) T cells, but not IFN-gamma(-) T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti-IFN-gamma antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN-gamma(+) T cells. The ratio of CD4+ T cells expressing both IFN-gamma and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN-gamma(+) human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.     

IL-27 suppresses RANKL expression in CD4+ T cells in part through STAT3

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.     

Activated T lymphocytes suppress osteoclastogenesis by diverting early monocyte/macrophage progenitor lineage commitment towards dendritic cell differentiation through down-regulation of receptor activator of nuclear factor-kappaB and c-Fos

Activated T lymphocytes either stimulate or inhibit osteoclastogenesis from haematopoietic progenitors in different experimental models. To address this controversy, we used several modes of T lymphocyte activation in osteoclast differentiation--mitogen-pulse, anti-CD3/CD28 stimulation and in vivo and in vitro alloactivation. Osteoclast-like cells were generated from non-adherent immature haematopoietic monocyte/macrophage progenitors in murine bone-marrow in the presence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and monocyte-macrophage colony-stimulating factor (M-CSF). All modes of in vivo and in vitro T lymphocyte activation and both CD4(+) and CD8(+) subpopulations produced similar inhibitory effects on osteoclastogenesis paralleled by enhanced dendritic cell (DC) differentiation. Osteoclast-inhibitory effect was associated with T lymphocyte activation and not proliferation, and could be replaced by their culture supernatants. The stage of osteoclast differentiation was crucial for the inhibitory action of activated T lymphocytes on osteoclastogenesis, because the suppressive effect was visible only on early osteoclast progenitors but not on committed osteoclasts. Inhibition was associated specifically with increased granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by the mechanism of progenitor commitment toward lineages other than osteoclast because activated T lymphocytes down-regulated RANK, CD115, c-Fos and calcitonin receptor expression, and increased differentiation towards CD11c-positive DC. An activated T lymphocyte inhibitory role in osteoclastogenesis, confirmed in vitro and in vivo, mediated through GM-CSF release, may be used to counteract activated bone resorption mediated by T lymphocyte-derived cytokines in inflammatory and immune disorders. We also demonstrated the importance of alloactivation in osteoclast differentiation and the ability of cyclosporin A to abrogate T lymphocyte inhibition of osteoclastogenesis, thereby confirming the functional link between alloreaction and bone metabolism.     

Expression of osteoprotegerin and RANK ligand in breast cancer bone metastasis

Bone destruction is primarily mediated by osteoclastic bone resorption, and cancer cells stimulate the formation and activation of osteoclasts next to metastatic foci. Accumulating evidences indicate that receptor activator of NF-kB ligand (RANKL) is the ultimate extracellular mediator that stimulates osteoclast differentiation into mature osteoclasts. In contrast, osteoprotegerin (OPG) inhibits osteoclast development. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MDA-MB-231, were directly co-cultured with ST2, MC3T3-E1, or with primary mouse calvarial cells. Osteoclast-like cells and tartarate resistant acid phosphatase (TRAP) activities were then quantitated. We examined these cell lines and samples from breast cancer by RT-PCR for the expressions of OPG and RANKL mRNA. Compared to controls, co-culture of MDA-MB-231 cells with stromal or osteoblastic cells induced an increase in number of osteoclasts and TRAP activities. MDA-MB-231 cells alone or breast cancer samples did not express RANKL mRNA. However, co-culture of these cancer cells with stromal or osteoblastic cells induced RANKL mRNA expression and decreased OPG mRNA expression. These experiments demonstrate that direct interactions between breast cancer and stromal or osteoblastic cells induce osteoclastogenesis in vitro through modulating RANKL expression.     

Role of Ca2+ in prostaglandin E2-induced T-lymphocyte proliferative suppression in sepsis.

Prostaglandin E2 (PGE2) has been known to modulate immune responses by inhibiting T-cell activation following hemorrhagic and traumatic injury. Recently, we documented a sepsis-related depression in concanavalin A (ConA)-induced T-cell proliferation and intracellular Ca2+ (Ca2+i) mobilization. The present study evaluated the potential role of PGE2 in the sepsis-related attenuation in Ca2+ signaling and proliferation in T cells. Sepsis was induced in rats by implanting into their abdomen fecal pellets containing Escherichia coli (150 CFU) and Bacteroides fragilis (10(4) CFU). A group of rats implanted with septic pellets were treated with indomethacin at three consecutive time points. Levels of PGE2 in blood were measured with a radioimmunoassay kit. ConA-induced [Ca2+]i mobilization in T cells obtained from indomethacin-treated and untreated rats was measured with Fura-2 and microfluorometry. We observed a 10-fold increase in PGE2 levels in the circulation of septic rats compared with levels in rats implanted with bacterium-free sterilized pellets. The proliferative response and Ca2+i mobilization were significantly depressed in T cells obtained from septic rats 48 h after implantations compared with those in rats implanted with sterile pellets. However, treatment of rats with the cyclooxygenase inhibitor indomethacin prevented the sepsis-related depression in ConA-induced T-cell Ca2+i mobilization as well as proliferation. Further, incubation of T cells from nonimplanted control rats with PGE2 resulted in a substantial depression in both T-cell proliferation and Ca2+i mobilization. The restoration of T-cell proliferation and Ca2+ signaling after indomethacin treatment of septic rats and the depression in the mitogen responsiveness in T cells previously exposed to PGE2 suggest that the PGE2 does play a significant role in the modulation of T-cell responses in septic rats and that such PGE2-induced suppression in T-cell activation is likely due to an attenuation in Ca2+ signaling.     

Diminished forkhead box P3/CD25 double-positive T regulatory cells are associated with the increased nuclear factor-kappaB ligand (RANKL+) T cells in bone resorption lesion of periodontal disease

Periodontal disease involves multi-bacterial infections accompanied by inflammatory bone resorption lesions. The abundant T and B lymphocyte infiltrates are the major sources of the osteoclast differentiation factor, receptor activator for nuclear factor-kappaB ligand (RANKL) which, in turn, contributes to the development of bone resorption in periodontal disease. In the present study, we found that the concentrations of RANKL and regulatory T cell (T(reg))-associated cytokine, interleukin (IL)-10, in the periodontal tissue homogenates were correlated negatively, whereas RANKL and proinflammatory cytokine, IL-1beta, showed positive correlation. Also, according to the fluorescent-immunohistochemistry, the frequency of forkhead box P3 (FoxP3)/CD25 double-positive cells was diminished strikingly in the bone resorption lesion of periodontal disease compared to healthy gingival tissue, while CD25 or FoxP3 single positive cells were still observed in lesions where abundant RANKL+ lymphocytes were present. Very importantly, few or no expressions of FoxP3 by the RANKL+ lymphocytes were observed in the diseased periodontal tissues. Finally, IL-10 suppressed both soluble RANKL (sRANKL) and membrane RANKL (mRANKL) expression by peripheral blood mononuclear cells (PBMC) activated in vitro in a bacterial antigen-specific manner. Taken together, these results suggested that FoxP3/CD25 double-positive T(reg) cells may play a role in the down-regulation of RANKL expression by activated lymphocytes in periodontal diseased tissues. This leads to the conclusion that the phenomenon of diminished CD25+FoxP3+ T(reg) cells appears to be associated with the increased RANKL+ T cells in the bone resorption lesion of periodontal disease.