The lupus susceptibility locus Sle1 facilitates the peripheral development and selection of anti-DNA B cells through impaired receptor editing.

Systemic lupus erythematosus is characterized by the spontaneous production of IgG autoantibodies in patients and lupus-prone mice. In this study, we investigated the effect of the Sle1 lupus susceptibility locus on the peripheral development of 56R(+) anti-DNA transgenic B cells by tracking 56R(+) B cells in mice without (B6.56R) or with (B6.Sle1.56R) the Sle1 locus. Compared with B6.56R mice, B6.Sle1.56R mice exhibited increased class-switched IgG2a anti-DNA Abs in their serum, encoded by the transgene. Interestingly, within the spleen, Sle1 facilitated the development of these cells into clusters of IgG2a class-switched B cells juxtaposed to CD4(+) T cells within extrafollicular sites. Through sequence analysis of B cell hybridomas, we also found that B cells from B6.Sle1.56R mice are inefficient at Ig H and L chain editing. Thus, the Ig H chains in Sle1.56R(+) B cells are partnered more often with cationic L chains that facilitate DNA binding. Taken together, these findings indicate that the Sle1 lupus-susceptibility locus may facilitate the emergence of anti-DNA B cells by subduing BCR revision and possibly by shaping the extrafollicular development of effector B cells, although the precise molecular mechanisms await further study.

Peritoneal catheter implantation elicits IL-10-producing immune-suppressor macrophages through a MyD88-dependent pathway.

Catheters are implanted into the peritoneal cavity during the process of peritoneal dialysis. Though these catheters may be effective and beneficial, the impact of catheters on the immune system is poorly understood. Catheters and other devices implanted in the peritoneal cavity elicit a foreign body reaction. However, the immunological consequences of this remain uncharacterized. To model this, catheters were implanted into the peritoneal cavity of healthy mice. Catheter implantation induced rapid cellular changes within the peritoneal cavity. Whereas B-cells and T-cells were reduced, catheter implantation was associated with the rapid expansion of F4/80-low-positive, CD11b-positive macrophages that elaborated IL-10, and suppressed T-cell division and Th1 skewing in co-culture assays. Peritoneal catheter elicited macrophages had increased Jmjd3 but reduced NF-κB activation, and their emergence was MyD88-dependent. Collectively, these studies indicate that foreign body implantation into the peritoneal cavity is associated with the expansion of suppressor macrophages. Whether peritoneal cavity catheter implantation may have systemic immunoregulatory roles remains to be explored.

A distinct tolerogenic subset of splenic IDO(+)CD11b(+) dendritic cells from orally tolerized mice is responsible for induction of systemic immune tolerance and suppression of collagen-induced arthritis.

In oral tolerance, locally instigated tolerance in the gut propagate to systemic tolerance. In order to investigate the mechanism, we analyzed indoleamine 2,3-dioxygenase (IDO) expression in splenic dendritic cell (DC) subsets and tested whether DCs suppress collagen-induced arthritis (CIA) by inducing regulatory T cells (Tregs). The proportion of IDO-expressing cells was higher in the CD11b(+) subset of splenic DCs from orally tolerized CIA mice. These DCs suppressed type II collagen-specific T cell proliferation and promoted Treg induction from CD4(+)CD25(-) T cells using transforming growth factor-ß. These DCs also increased the expression of cytotoxic T lymphocyte antigen-4 and programmed death-1 on Tregs. When adoptively transferred, spenic IDO-expressing CD11b(+) DCs from tolerized animals suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen. Taken together, a distinct subset of splenic IDO(+)CD11b(+)DCs is responsible for the systemic immune regulation in oral tolerance.

Transforming growth factor ß-transduced mesenchymal stem cells ameliorate experimental autoimmune arthritis through reciprocal regulation of Treg/Th17 cells and osteoclastogenesis.

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (MSCs) can prevent various autoimmune diseases. We examined the therapeutic potential of transforming growth factor ß (TGFß)-transduced MSCs in experimental autoimmune arthritis, using an accepted animal model of collagen-induced arthritis (CIA). METHODS: DBA/1J mice with CIA were treated with syngeneic TGFß-induced MSCs, whereas control mice received either vehicle or MSCs alone. Arthritis severity was assessed by clinical and histologic scoring. TGFß-transduced MSCs were tested for their immunosuppressive ability and differential regulation in mice with CIA. T cell responses to type II collagen were evaluated by determining proliferative capacity and cytokine levels. The effects of TGFß-transduced MSCs on osteoclast formation were analyzed in vitro and in vivo. RESULTS: Systemic infusion of syngeneic TGFß-transduced MSCs prevented arthritis development and reduced bone erosion and cartilage destruction. Treatment with TGFß-transduced MSCs potently suppressed type II collagen-specific T cell proliferation and down-regulated proinflammatory cytokine production. These therapeutic effects were associated with an increase in type II collagen-specific CD4+FoxP3+ Treg cells and inhibition of Th17 cell formation in the peritoneal cavity and spleen. Furthermore, TGFß-transduced MSCs inhibited osteoclast differentiation. CONCLUSION: TGFß-transduced MSCs suppressed the development of autoimmune arthritis and joint inflammation. These data suggest that enhancing the immunomodulatory activity of MSCs and modulating T cell-mediated immunity using gene-modified MSCs may be a gateway for new therapeutic approaches to clinical rheumatoid arthritis.

IL-23 induces receptor activator of NF-kappaB ligand expression on CD4+ T cells and promotes osteoclastogenesis in an autoimmune arthritis model.

IL-23, a clinically novel cytokine, targets CD4(+) T cells. Recent IL-1Ra(-/-) mouse studies have demonstrated that IL-23 indirectly stimulates the differentiation of osteoclast precursors by enhancing IL-17 release from CD4(+) T cells. IL-17, in turn, stimulates osteoclastogenesis in osteoclast precursor cells. In this study, we found that IL-23 up-regulates receptor activator of NF-kappaB ligand expression by CD4(+) T cells, and thus contributes to osteoclastogenesis. This indirect pathway is mediated by NF-kappaB and STAT3. We have also demonstrated that IL-23 can influence osteoclastogenesis positively under the special conditions in the IL-1-dominant milieu of IL-1Ra(-/-) mice. We propose that IL-23-enhanced osteoclastogenesis is mediated mainly by CD4(+) T cells. The results of this study show that IL-23 is a promising therapeutic target for the treatment of arthritis-associated bone destruction.

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.

Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-kappaB activation.

N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) is known to inhibit NF-kappaB activation and the expression of inflammation mediators in cultured cells. We measured the potential of TPCK to inhibit the pathogenesis of collagen-induced arthritis by blocking NF-kappaB activation. Arthritis was induced in DBA/1J mice by the injection of bovine type II collagen in adjuvant on days 0 and 14. Mice received either TPCK (3 or 10 mg/kg, i.p.) or vehicle three times a week for 3 weeks starting on day 21. TPCK moderately reduced clinical disease activity scores, whereas it markedly suppressed histological indications of joint destruction. In vitro production of tumor necrosis factor-alpha, interleukin-6, and monocyte chemotactic protein-1 from lipopolysaccharide-stimulated spleen cells was also reduced by in vivo treatment with TPCK. Proliferation of cells isolated from spleen or draining lymph nodes and production of interferon-gamma and interleukin-17 in response to stimulation with type II collagen was decreased by TPCK. Moreover, nuclear NF-kappaB activity induced by collagen immunization was significantly reduced in mice treated with TPCK. Finally, osteoclast differentiation of bone marrow cells induced by macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand was completely inhibited by TPCK. These results indicate that TPCK attenuates collagen-induced arthritis and bone erosion by suppressing NF-kappaB activation and thus expression of inflammatory and osteoclastogenic genes.

Type II collagen autoimmunity in a mouse model of human rheumatoid arthritis.

Type II collagen (CII) is expressed exclusively in the joint articular. Although the relationship between anti-CII immunity and human rheumatoid arthritis (RA) has been studied for a long time, definitive conclusions have not been reached. CII, as an autoantigen, has been studied extensively in small animal models, such as mice, and the collagen-induced arthritis (CIA) model has increased our understanding of the pathogenesis of human RA. In the present report, we summarize the available information on anti-CII immunity and discuss recent updates regarding pathogenesis in the CIA model, including the role of Th17 cells.

Human rheumatoid synovial fibroblasts promote osteoclastogenic activity by activating RANKL via TLR-2 and TLR-4 activation.

The interplay between the innate immune system and inflammatory bone destruction in the joints of individuals with rheumatoid arthritis (RA) remains unclear. This study was undertaken to explore the effect of toll-like receptor (TLR) signaling in fibroblast-like synoviocytes (FLS) on the expression of RANKL and induction of osteoclastogenic activity. The levels of RANKL mRNA and protein were measured using RT-PCR, real-time PCR, and immunostaining. Monocytes were cocultured with RA -FLS that had been stimulated with TLR ligands in fresh media and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. Osteoclast molecule markers were measured using real-time PCR. Expression of TLR-2 and TLR-4 was higher in RA-FLS than in OA-FLS and normal skin fibroblasts. TLR-2 and TLR-4 ligands induced RANKL expression in RA-FLS. TLR stimulation of RA-FLS also induced the production of IL-1beta and TNF-alpha to a lesser extent; however, it had no effect on IL-17 production. Inhibition of TLR induced IL-1beta production, which partially reversed the upregulation of RANKL induced by TLR ligands. RA-FLS stimulated by TLR-2 and TLR-4 ligands and cocultured with human monocytes induced high levels of expression of TRAP, RANK, cathepsin K, calcitonin receptor, and matrix metalloproteinase-9, suggesting that RA-FLS promote osteoclast differentiation. Our results suggest that the TLR signaling pathway, through TLR-2 and TLR-4, induces RANKL expression in RA-FLS and the expression of RANKL promotes the differentiation of osteoclasts in RA synovium. Targeting specific TLRs may be a promising approach to prevent inflammatory bone destruction in the pathogenesis of RA.

CD8alpha+ dendritic cells enhance the antigen-specific CD4+ T-cell response and accelerate development of collagen-induced arthritis.

To investigate the role of CD8alpha(+) DCs in the development of collagen-induced arthritis (CIA). The immunogenic properties of CD8alpha(+) and CD8alpha(-) DC subsets were investigated by mixed-lymphocyte reaction and cytokine enzyme-linked immunoassay. CII-pulsed CD8alpha(+) DCs or CD8alpha(-) DCs with CD4(+) T cells from CIA mice were adoptively transferred onto the hind footpad of DBA mice. The onset of arthritis and the arthritis index were examined for 14 weeks after adoptive transfer. Expression of MHC-II and CD80 but not CD86 and CD40 was higher in CD8alpha(+) DCs than in CD8alpha(-) DCs from the spleens of CIA mice. Culturing CD8alpha(+) DCs with CD4(+) T cells significantly increased the proliferative response of CD4(+) T cells in the presence of CII. The production of interleukin (IL)-12p70, IL-17, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha was slightly increased in CD8alpha(+) DCs than in CD8alpha(-) DCs. DBA/1 mice that were adoptively transferred with CII-pulsed CD8alpha(+) DCs and CD4(+) T cells into the footpads showed accelerated onset of CIA compared to control group. By contrast, CD8alpha(-) DCs showed a partial inhibitory effect on CIA. These findings show that CD8alpha(+) DCs accelerated the onset of CIA when aoptively transferred with CD4(+) T cells and that CD8alpha(+) DCs provoke the development of CIA probably by stimulating the immune responses of CII-reactive CD4(+) T cells and by increasing the production of inflammatory cytokines.

Cyclosporine A inhibits IL-15-induced IL-17 production in CD4+ T cells via down-regulation of PI3K/Akt and NF-kappaB.

Cyclosporine A (CSA) has various biological effects on T cells, including inhibition of interleukin (IL)-15-induced IL-17 production in CD4+ T cells from patients with rheumatoid arthritis (RA). However, the mechanism underlying this effect is not fully understood. Here, we tried to investigate the mechanism of CSA to inhibit IL-17 production induced by IL-15 in CD4+ T cells. Synovial fluid and serum levels of IL-15 and IL-17 were determined by ELISA. CD4+ T cells from RA patients were treated with IL-15 in the presence of CSA or several signal inhibitors. The concentration of IL-17 in culture supernatants was measured by ELISA and IL-17 mRNA expression was determined by RT-PCR. NF-kappaB binding activity for IL-17 transcription was assessed by electrophoretic mobility shift assay. IL-15 induced IL-17 production by CD4+ T cells in dose- and time-dependent manner. IL-15-stimulated IL-17 production and mRNA expression were inhibited by CSA in CD4+ T cells. Moreover PI3K/Akt inhibitor, NF-kappaB inhibitor, and FK506 significantly inhibited IL-15-induced IL-17 production in CD4+ T cells. Inhibition studies revealed the requirement of PI3K/Akt and NF-kappaB signal pathway for IL-15-induced IL-17 production. CSA down-regulated the phosphorylation of Akt and IkappaB. CSA inhibited binding of NF-kappaB to IL-17 promoter. The inhibitory effect of CSA on IL-15 induced IL-17 production partially depended on the increase in IL-10, since neutralizing anti-IL-10 antibodies were able to partially reverse this inhibition. CSA inhibits IL-17 production by CD4+ T cells and this effect is mediated by IL-15-activated NF-kappaB pathway in CD4+ T cells, which is possible mechanism of CSA in treating RA as NF-kappaB targeting strategy.

Transforming growth factor beta 1(TGF-beta1) down-regulates TNFalpha-induced RANTES production in rheumatoid synovial fibroblasts through NF-kappaB-mediated transcriptional repression.

Transforming growth factor (TGF)-beta1 is a pleiotropic cytokine with many functions, including those related to growth modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-beta1 to regulate RANTES production by activated rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were cultured in the presence of TGF-beta1 and IL-1beta, IL-15, TNFalpha, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-kappaB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-beta1 were higher in RA SF than in OA SF. TGF-beta1 dose-dependently inhibited TNFalpha-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-beta1 mimicked the effect of TGF-beta1 on TNFalpha-induced RANTES production, which was inhibited by treatment with anti-TGF-beta1 neutralizing antibody. TGF-beta1 blocked the degradation of cytosolic IkappaB-alpha and the translocation of activated NF-kappaB to the nucleus. EMSA showed that the inhibitory effect of TGF-beta1 was associated with decreased binding of NF-kappaB to the RANTES promoter. These results suggest that elevated TGF-beta1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial fibroblasts, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-beta1 regulates immune function in RA.

Interleukin-18 induces the production of vascular endothelial growth factor (VEGF) in rheumatoid arthritis synovial fibroblasts via AP-1-dependent pathways.

Interleukin-18 (IL-18) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in rheumatoid synoviocytes. In present study, we examined the effect of IL-18 on VEGF production in fibroblast-like synoviocytes (FLS) isolated from the patients with RA. FLS were prepared from the synovial tissues of patients with RA and osteoarthritis (OA) and cultured in the presence of IL-18. The production of VEGF from FLS was measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA). The VEGF messenger RNA (mRNA) expression and AP-1 binding activity of VEGF transcript were determined by reverse transcription-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assay (EMSA). IL-18 and VEGF levels of sera and synovial fluids (SF) of RA patients (n=30) were significantly higher than those of OA patients (n=20). IL-18 dose-dependently increased the production of VEGF. The effect of IL-18 on VEGF production appeared to be as potent as IL-1beta, whereas tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma showed little effects on VEGF production. AP-1-specific inhibitor Curcumin dose-dependently abrogated the effect of IL-18 on VEGF production. The VEGF enhancement of IL-18 was associated with increased AP-1 binding activity to the VEGF promoter site. These findings suggest IL-18 as an angiogenic factor in RA and down-regulation of IL-18 activity or AP-1 signal pathway can be potential therapeutic targets for RA.

Adenoviral delivery of IL-1 receptor antagonist abrogates disease activity during the development of autoimmune arthritis in IL-1 receptor antagonist-deficient mice.

Currently available treatments for rheumatoid arthritis (RA) are limited in terms of their long-term effects and their abilities to control disease progression. Interleukin-1 receptor antagonist (IL-1Ra) is a natural inhibitor of the biologic actions of IL-1, which is known to promote inflammation and degeneration of the joint. In this study, we investigated whether human IL-1Ra gene transfer is effective at treating an established experimental arthritis model. A recombinant adenovirus carrying the gene that encode human hIL-1Ra and GFP (Ad.hIL-1Ra/GFP) was administered by intra-articular injection into the ankle joints of the mice with established the IL-1Ra-deficient Balb/cA mice (IL-1Ra(-/-)), which develop spontaneously chronic inflammatory arthropathy. The effects of two injections of Ad.hIL-1Ra/GFP or control virus with no inserted target gene (Ad.GFP) were compared with the effects of PBS injection with respect to the clinical characteristics of arthritis, as determined by articular index scores, histopathological and immunological assays. We further divided the outcomes of Ad.hIL-1Ra/GFP gene therapy in IL-1Ra(-/-) mice according arthritis stage; early stage and chronic stage corresponding to 8 and 15 weeks of age, respectively. Intra-articular injections of Ad.hIL-1Ra/GFP reduced arthritis severity and footpad swelling compared with control groups treated with Ad.GFP or PBS in early stage IL-1Ra(-/-) mice. Moreover, the histopathology of the ankle joints of IL-1Ra(-/-) mice treated with Ad.hIL-1Ra/GFP showed a significant decrease in synovial proliferation and inflammatory cell infiltration, and preserved proteoglycan levels in the joints of early stage IL-1Ra(-/-) mice compared with the control mice. Moreover, Ad.hIL-1Ra/GFP treated mice showed reduced levels of inflammatory T helper type 1 (Th1) driven IgG2a antibodies to collagen type II but increased levels Th2 driven IgG1 antibody. These results suggest that adenovirus-mediated gene transfer of IL-1Ra may be a promising therapeutic option in the early stage of autoimmune arthritis.

Green Tea Epigallocatechin-3-Gallate Suppresses Autoimmune Arthritis Through Indoleamine-2,3-Dioxygenase Expressing Dendritic Cells and the Nuclear Factor, Erythroid 2-Like 2 Antioxidant Pathway.

BACKGROUND: The activity of one of the major catechins in Green Tea, the polyphenol (-)-epigallocatechin-3-gallate (EGCG), has been shown to have a variety of health benefits. Recent studies suggest that EGCG can modulate both the innate and adaptive arms of the immune system. The goal of the current studies was to examine the immunomodulatory effects and mechanisms of action of EGCG on experimental arthritis in mice. METHODS: EGCG (10 mg/kg) was administered by oral gavage after CIA induction, while control mice were administered phosphate buffered saline (PBS). Disease mechanisms were studied in both groups of mice. Phenotypes were examined using repeated measure analysis of variance (ANOVA) and data from in vitro and ex vivo experiments were analyzed for significance using the Mann-Whitney U test. RESULTS: EGCG treatment ameliorated clinical symptoms and reduced histological scores in arthritic mice. Serum type-II collagen-specific immunoglobulin (Ig) IgG2a antibodies were significantly lower in EGCG-fed mice compared to PBS-treated mice. EGCG significantly suppressed T cell proliferation and relative frequencies of CD4 T cells, CD8 T cells and B cell subsets including marginal zone B cells, T1 and T2 transitional B cells, while increasing the frequency of CD4(+) Foxp3(+) regulatory T cells (Tregs) and indoleamine-2,3-dioxygenase (IDO) expression by CD11b(+) dendritic cells (DC). Splenic CD11b(+) DC from EGCG fed mice induced an increased frequency of Tregs via an IDO-dependent mechanism in in vitro cultures. Importantly, joint homogenates from EGCG-fed mice exhibited significantly increased levels of Nuclear Factor, Erythroid 2-Like 2 (Nrf-2) and Heme oxygenase-1 (HO-1) compared with PBS-fed mice. CONCLUSIONS: This is the first report of upregulation of the Nrf-2 antioxidant pathway in EGCG-mediated immunoregulation. EGCG ameliorated experimental arthritis in mice by eliciting IDO-producing DCs, increasing frequencies of T regs and inducing the activation of the Nrf-2 antioxidant pathway. It remains to be established whether EGCG is useful for the prevention and treatment of rheumatoid arthritis and other inflammatory disorders.

Type II collagen autoimmunity in rheumatoid arthritis.

This review summarizes the autoimmune reaction to type II collagen (CII) autoimmunity with regard not only to antibody response to CII but also to the clinical significance or biological characteristics of the CII-reactive T cell, focusing on studies of human RA rather than on animal models. The authors investigated the effect of the interaction between CII-reactive T cells and fibroblast-like synoviocytes (FLSs) on the production of inflammatory cytokines. When the CII-reactive T cells were co-cultured with FLS, the production of interleukin-15 and tumor necrosis factor-alpha from FLSs were significantly increased, and this increase was clearly presented in accord with the expansion of CII-reactive T cells. In addition, the production of interferon-gamma and interleukin-17, T cell-derived cytokines, was increased by the co-incubation of CII-reactive T cells with FLSs. When FLSs were co-cultured with CII-stimulated T cells, the production of interleukin-8, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1alpha was significantly enhanced. The increased production of these chemokines was strongly correlated with an increase in T-cell response to CII. Conclusively, high reactivity to CII was frequently found in RA patients. Enhanced T-cell responses to CII were associated with increased production of proinflammatory cytokines and chemokines, which were critical for inflammatory responses in RA. Interaction of CII-reactive T cells with FLS further augmented this phenomenon. Taken together, the authors' recent studies have suggested that autoimmunity to CII could play a crucial role not only in the initiation but also in the amplification and perpetuation of the inflammatory process in RA.

Prevention of murine lupus nephritis by targeting multiple signaling axes and oxidative stress using a synthetic triterpenoid.

OBJECTIVE: Current treatment options for lupus are far from optimal. Previously, we reported that phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin, MEK-1/ERK-1,2, p38, STAT-3, STAT-5, NF-κB, multiple Bcl-2 family members, and various cell cycle molecules were overexpressed in splenic B cells in an age-dependent and gene dose-dependent manner in mouse strains with spontaneous lupus. Since the synthetic triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) has been shown to inhibit AKT, MEK-1/2, and NF-κB, and to induce caspase-mediated apoptosis, we tested the therapeutic potential of this agent in murine lupus nephritis. METHODS: The synthetic triterpenoid CDDO-Me or placebo was administered to 2-month-old B6.Sle1.Sle3 mice or MRL/lpr mice, which develop spontaneous lupus. All mice were phenotyped for disease. RESULTS: CDDO-Me-treated mice exhibited significantly reduced splenic cellularity, with decreased numbers of both CD4+ T cells and activated CD69+/CD4+ T cells compared to the placebo-treated mice. These mice also exhibited a significant reduction in serum autoantibody levels, including anti-double-stranded DNA (anti-dsDNA) and antiglomerular antibodies. Finally, CDDO-Me treatment attenuated renal disease in mice, as indicated by reduced 24-hour proteinuria, blood urea nitrogen, and glomerulonephritis. At the mechanistic level, CDDO-Me treatment dampened MEK-1/2, ERK, and STAT-3 signaling within lymphocytes and oxidative stress. Importantly, the NF-E2-related factor 2 pathway was activated after CDDO-Me treatment, indicating that CDDO-Me may modulate renal damage in lupus via the inhibition of oxidative stress. CONCLUSION: These findings underscore the importance of AKT/MEK-1/2/NF-κB signaling in engendering murine lupus. Our findings indicate that the blockade of multiple signaling nodes and oxidative stress may effectively prevent and reverse the hematologic, autoimmune, and pathologic manifestations of lupus.

Anti-nuclear antibody reactivity in lupus may be partly hard-wired into the primary B-cell repertoire.

When monoclonal ANAs and non-ANAs generated from a genetically simplified mouse model of lupus, B6.Sle1, were recently compared, the ANAs exhibited three sequence motifs in their immunoglobulin heavy chains, including increased cationicity in CDR3 ("motif A"), reduced anionicity in CDR2 ("motif B") and increased aspartate at H50 ("motif C"). The present study was designed to elucidate the extent to which these ANA-associated sequence motifs might be hard-wired into the primary B-cell repertoire in lupus. The immunoglobulin heavy chain sequence of total splenic B-cells, follicular B-cells and marginal zone B-cells from B6.Sle1 congenic mice and C57BL/6 controls were amplified by single-cell PCR and compared. Analysis of the primary immunoglobulin heavy chain repertoire indicated that the first two sequence motifs "A" and "B" were already encoded in the naïve repertoire of B6.Sle1(z) mice, whereas the third motif "C" was introduced in part by somatic mutation. Site-directed mutagenesis confirmed that non-anionic CDR2 and cationic CDR3 residues in the immunoglobulin heavy chain facilitated nuclear antigen binding in concert, whereas aspartate at H50 strongly vetoed DNA-binding, while preserving nucleosome reactivity. Hence, anti-nuclear antibodies appear to arise as a consequence of two distinct processes-genetically programmed selection of specific CDR charge motifs into the primary immunoglobulin repertoire, with secondary contribution from somatic mutation. Polymorphisms in the lupus susceptibility gene Ly108 that impair central B-cell tolerance may be mechanistically responsible for these early repertoire differences in lupus.

Bone marrow T cells are superior to splenic T cells to induce chimeric conversion after non-myeloablative bone marrow transplantation.

BACKGROUND/AIMS: The bone marrow functions not only as the primary B-lymphocyte-producing organ but also as a secondary lymphoid organ for CD4 and CD8 cell responses and a site of preferential homing and persistence for memory T cells. Bone marrow T (BM-T) cells are distinguished from peripheral blood T cells by surface phenotype, cytokine secretion profile, and immune functions. In this study, we evaluated the alloreactive potential of donor lymphocyte infusion (DLI) using BM-T cells in mixed chimerism compared to that using spleen T (SP-T) cells. METHODS: Cells were prepared using established procedures. BM-T cells were obtained as a by-product of T-cell depletion in BM grafting and then cryopreserved for subsequent DLI. We performed DLI using BM-T cells in allogeneic mixed chimera mice on post-BMT day 21. RESULTS: When the same dose of T cells, 5-10x10(5) (Thy1.2+), fractionated from BM and spleen were administered into mixed chimeras, the BM-T group showed complete chimeric conversion, with self-limited graft-versus-host disease (GVHD) and no pathological changes. However, the SP-T group showed persistent mixed chimerism, with pathological signs of GVHD in the liver and intestine. CONCLUSIONS: Our results suggest that DLI using BM-T cells, even in small numbers, is more potent at inducing chimeric conversion in mixed chimerism than DLI using SP-T cells. Further study is needed to determine whether cryopreserved BM-T cells are an effective cell source for DLI to consolidate donor-dominant chimerism in clinical practice without concerns about GVHD.

Murine thymic stromal lymphopoietin promotes the differentiation of regulatory T cells from thymic CD4(+)CD8(-)CD25(-) naïve cells in a dendritic cell-independent manner.

Human thymic stromal lymphopoietin (TSLP) activates dendritic cells (DCs), which promote the proliferation and differentiation of CD4+ T cells. However, murine TSLP (mTSLP) can act directly on CD4+ T cells and bring about their differentiation. We studied the role of mTSLP in the generation of CD4+CD25+FoxP3+ T cells from thymocytes. mTSLP promoted the differentiation of CD4+ single-positive thymocytes into CD4+CD25+FoxP3+ T cells. Although we cannot exclude an effect of TSLP mediated through DCs due to co-stimulatory effects, mTSLP appears to act directly on thymocytes. T-cell receptor and TSLP receptor signaling act synergistically on thymocytes to generate CD4+CD25+FoxP3+ T cells. mTSLP may play an important role in maintaining immune tolerance by promoting the conversion of thymocytes into natural regulatory T cells via escape from negative selection.