The xanthine oxidase-NFAT5 pathway regulates macrophage activation and TLR-induced inflammatory arthritis.

NFAT5 (nuclear factor of activated T cells), a well-known osmoprotective factor, can be activated by isotonic stimuli such as Toll-like receptor (TLR) triggering. However, it is unclear how NFAT5 discriminates between isotonic and hypertonic stimuli to produce different functional and molecular outcomes. Here, we identified a novel XO-ROS-p38 MAPK-NFAT5 pathway (XO is xanthine oxidase, ROS is reactive oxygen species) that is activated in RAW 264.7 macrophages upon isotonic TLR stimulation. Unlike what is seen under hypertonic conditions, XO-derived ROS were selectively required for the TLR-induced NFAT5 activation and NFAT5 binding to the IL-6 promoter in RAW 264.7 macrophages under isotonic conditions. In mouse peritoneal macrophages and human macrophages, TLR ligation also induced NFAT5 activation, which was dependent on XO and p38 kinase. The involvement of XO in NFAT5 activation by TLR was confirmed in RAW 264.7 macrophages implanted in BALB/c mice. Moreover, allopurinol, an XO inhibitor, suppressed arthritis severity and decreased the expression of NFAT5 and IL-6 in splenic macrophages in C57BL/6 mice. Collectively, these data support a novel function of the XO-NFAT5 axis in macrophage activation and TLR-induced arthritis, and suggest that XO inhibitor(s) could serve as a therapeutic agent for chronic inflammatory arthritis.

p53 controls autoimmune arthritis via STAT-mediated regulation of the Th17 cell/Treg cell balance in mice.

OBJECTIVE: To investigate the connection between p53 and interleukin-17-producing Th17 cell/Treg cell balance in rheumatoid arthritis (RA). METHODS: Th17 cell and Treg cell frequencies were analyzed by flow cytometry, and cytokine levels in the supernatant were determined using enzyme-linked immunosorbent assays. The expression of transcription factors was analyzed by immunostaining and Western blotting, and the interactions between p53 and STAT-3 or STAT-5 were determined by immunoprecipitation-Western blot analysis. A p53 agonist was administered in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. RESULTS: CD4+ T cells from p53-/- mice decreased the activity of STAT-5, lowered the level of phosphorylated STAT-5, and compromised Treg cell differentiation. The protein p53 bound STAT-5 directly, and this interaction was enhanced with increasing p53 activity. Under inflammatory conditions, p53 suppressed Th17 cell differentiation and skewed T cells toward Treg cell differentiation through the activation of STAT-5 signaling cascades. In mice with CIA, injection of a p53 overexpression vector or an antagonist of Mdm2 had the effect of controlling arthritis development in vivo. The regulatory effect of p53 was recapitulated in the cells of RA patients, with more pronounced suppression due to the repressed status of p53 in RA. CONCLUSION: We demonstrated a link between p53-mediated and STAT-mediated regulation of Th17 cells/Treg cells in RA. Our results suggest that factors involved in this pathway might constitute novel therapeutic targets for the treatment of RA.

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.

Expression of aldo-keto reductase family 1 member C1 (AKR1C1) gene in porcine ovary and uterine endometrium during the estrous cycle and pregnancy.

BACKGROUND: The aldo-keto reductase family 1 member C1 (AKR1C1) belongs to a superfamily of NADPH-dependent reductases that convert a wide range of substrates, including carbohydrates, steroid hormones, and endogenous prostaglandins. The 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) is a member of AKR family. The aims of this study were to determine its expression in the ovary and uterus endometrium during the estrous cycle and pregnancy. METHODS: Rapid amplification of cDNA ends (RACE) experiments were performed to obtain the 5' and 3' ends of the porcine 20 alpha-HSD cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, northern blot analysis, and western blot analysis were performed to examine the expression of porcine 20 alpha-HSD. Immunohistochemical analysis was also performed to determine the localization in the ovary. RESULTS: The porcine 20 alpha-HSD cDNA is 957 bp in length and encodes a protein of 319 amino acids. The cloned cDNA was virtually the same as the porcine AKR1C1 gene (337 amino acids) reported recently, and only differed in the C-terminal region (the AKR1C1 gene has a longer C-terminal region than our sequence). The 20 alpha-HSD gene (from now on referred to as AKR1C1) cloned in this paper encodes a deletion of 4 amino acids, compared with the C-terminal region of AKR1C1 genes from other animals. Porcine AKR1C1 mRNA was expressed on day 5, 10, 12, 15 of the cycle and 0-60 of pregnancy in the ovary. The mRNA was also specifically detected in the uterine endometrium on day 30 of pregnancy. Western blot analysis indicated that the pattern of AKR1C1 protein in the ovary during the estrous cycle and uterus during early pregnancy was similar to that of AKR1C1 mRNA expression. The recombinant protein produced in CHO cells was detected at approximately 37 kDa. Immunohistochemical analysis also revealed that pig AKR1C1 protein was localized in the large luteal cells in the early stages of the estrous cycle and before parturition. CONCLUSIONS: Our study demonstrated that AKR1C1 mRNA and protein are coordinately expressed in the luteal cell of ovary throughout the estrous cycle and in the uterus on day 30 of pregnancy. Thus, the porcine AKR1C1 gene might control important mechanisms during the estrous cycle.

Antigen-induced, tolerogenic CD11c+,CD11b+ dendritic cells are abundant in Peyer's patches during the induction of oral tolerance to type II collagen and suppress experimental collagen-induced arthritis.

OBJECTIVE: Although oral tolerance is a well-known phenomenon, the role of dendritic cells (DCs) is not well characterized. This study was conducted to better understand the differential role played by each Peyer's patch DC subset in the induction of oral tolerance to type II collagen (CII) in murine collagen-induced arthritis (CIA). METHODS: CII was fed 6 times to DBA/1 mice beginning 2 weeks before immunization, and the effect on arthritis was assessed. We compared the proportion of CD11c+,CD11b+ DCs and CD11c+,CD8alpha+ DCs in the Peyer's patches of CII-fed tolerized and phosphate buffered saline-fed nontolerized mice after the induction of CIA. The immunosuppressive properties of each DC subset were determined using fluorescence-activated cell sorter analysis for intracellular interleukin-10 (IL-10) and IL-12 and mixed lymphocyte culture. The ability of each DC subset to induce CD4+,CD25+ T regulatory cells was also examined. Mice were injected with CII-pulsed CD11c+,CD11b+ DCs isolated from Peyer's patches of tolerized mice, and the effect on CIA was examined. RESULTS: The severity of arthritis was significantly lower in tolerized mice. The proportion of CD11c+,CD11b+ DCs was increased in the Peyer's patches of tolerized mice and those DCs exhibited immunosuppressive characteristics, such as increased IL-10 production, inhibition of T cell proliferative responses to CII, and CD4+,CD25+ regulatory T cell induction. Furthermore, the CD11c+,CD11b+ DCs suppressed the severity of arthritis upon adoptive transfer. CONCLUSION: Our observations demonstrate that CD11c+,CD11b+ DCs, which are abundant in Peyer's patches during the induction of oral tolerance to CII, are crucial for the suppression of CIA and could be exploited for immunotherapy of autoimmune diseases.

Sensitization of vanilloid receptor involves an increase in the phosphorylated form of the channel.

A vanilloid receptor (VR1, now known as TRPV1) is an ion channel activated by vanilloids, including capsaicin (CAP) and resiniferatoxin (RTX), which are pungent ingredients of plants. Putative endogenous activators (anandamide and metabolites of arachidonic acid) are weak activators of VR1 compared to capsaicin and RTX, and the concentrations of the physiological condition of those activators are not sufficient to induce significant activation of VR1. One way to overcome the weak activation of endogenous activators would be the sensitization of VR1, with the phosphorylation of the channel being one possibility. The phosphorylation of VR1 by several kinases has been reported, mostly by indirect evidence. Here, using an in vivo phosphorylation method, the VR1 channel was shown to be sensitized by phosphorylation of the channel itself by multiple pathways involving PKA, PKC and acid. Also, in sensitizing VR1, BK appeared to show activation of PKC for the sensitization of VR1 by phosphorylation of the channel.

Increase of cyclooxygenase-2 expression by interleukin 15 in rheumatoid synoviocytes.

OBJECTIVE: To determine the effect of interleukin 15 (IL-15) on cyclooxygenase-2 (COX-2) expression in rheumatoid synoviocytes. METHODS: Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of patients with rheumatoid arthritis (RA) and cultured in the presence of IL-15. Levels of COX-2 mRNA and protein expression were determined by reverse transcription-polymerase chain reaction and Western blot, respectively. ELISA was used to measure concentrations of IL-1beta, tumor necrosis factor-a (TNF-a), and prostaglandin E2 (PGE2) in the culture supernatants. RESULTS: IL-15 dose-dependently increased COX-2 mRNA and protein expression in FLS, but not the COX-1 mRNA level. Both IL-1beta and TNF-a upregulated COX-2 mRNA comparably to IL-15, but neither IL-2 nor interferon-g had any effect on the COX-2 mRNA level. Treatment with anti-IL-1beta or anti-TNF-a antibodies partially reduced the IL-15-stimulated COX-2 mRNA expression, suggesting that these cytokines may take part in modulating COX-2 by IL-15. Dexamethasone and pyrolidine dithiocarbamate, but not curcumin, completely blocked the IL-15-induced upregulation of COX-2 mRNA. A gel mobility shift assay revealed that nuclear factor-kB (NF-kB) was one of the major signal molecules to mediate IL-15-induced COX-2 upregulation. The increase of COX-2 by IL-15 is PGE2-dependent because exogenous PGE2 reversed the suppressive effect of NS-398, a selective COX-2 inhibitor, on COX-2 mRNA and protein expression. CONCLUSION: This study confirms the effect of IL-15 on upregulation of COX-2 in a PGE2-dependent manner. The activation of NF-kB bound to the COX-2 promoter appears to be a downstream target of IL-15 stimulation in FLS, exerted either directly or through the increase in IL-1beta and TNF-a production.

IL-17 induces production of IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts via NF-kappaB- and PI3-kinase/Akt-dependent pathways.

Recent studies of the pathogenesis of rheumatoid arthritis (RA) have revealed that both synovial fibroblasts and T cells participate in the perpetuation of joint inflammation as dynamic partners in a mutual activation feedback, via secretion of cytokines and chemokines that stimulate each other. In this study, we investigated the role of IL-17, a major Th1 cytokine produced by activated T cells, in the activation of RA synovial fibroblasts. Transcripts of IL-17R (IL-17 receptor) and IL-17RB (IL-17 receptor B) were present in fibroblast-like synoviocytes (FLS) of RA patients. IL-17R responded with increased expression upon in vitro stimulation with IL-17, while the level of IL-17RB did not change. IL-17 enhanced the production of IL-6 and IL-8 in FLS, as previously shown, but did not affect the synthesis of IL-15. IL-17 appears to be a stronger inducer of IL-6 and IL-8 than IL-15, and even exerted activation comparable to that of IL-1beta in RA FLS. IL-17-mediated induction of IL-6 and IL-8 was transduced via activation of phosphatidylinositol 3-kinase/Akt and NF-kappaB, while CD40 ligation and p38 MAPK (mitogen-activated protein kinase) are not likely to partake in the process. Together these results suggest that IL-17 is capable of more than accessory roles in the activation of RA FLS and provide grounds for targeting IL-17-associated pathways in therapeutic modulation of arthritis inflammation.

Effector function of type II collagen-stimulated T cells from rheumatoid arthritis patients: cross-talk between T cells and synovial fibroblasts.

OBJECTIVE: To investigate the effector function exerted by type II collagen (CII)-stimulated T cells on rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and to determine their contribution to RA pathogenesis. METHODS: We used enzyme-linked immunosorbent assays to measure the levels of interleukin-15 (IL-15), tumor necrosis factor alpha (TNFalpha), and IL-18 production by FLS that were cocultured with antigen-activated T cells. Likewise, we analyzed the levels of interferon-gamma (IFN gamma) and IL-17 production by RA T cells coincubated with FLS. To investigate the cross-talk between CII-stimulated T cells and RA FLS, we examined the effect of using a transwell membrane to separate T cells and FLS in a culture chamber, as well as the effect of adding an antibody to block CD40 ligation. RESULTS: The levels of IL-15, TNF alpha, IFN gamma, and IL-17 were all significantly increased in the serum of RA patients compared with normal control serum. Among the patients, the group with a stronger T cell proliferation response to CII showed higher levels of these inflammatory mediators. When coincubated with RA FLS, these T cells induced the production of IL-15, TNF alpha, and IL-18 by FLS with an intensity that increased in proportion to the duration of CII stimulation. T cells, in turn, responded to FLS stimulation by secreting higher amounts of IL-17 and IFN gamma in coculture. Interestingly, T cells that were activated by CII for longer periods of time showed stronger induction of these cytokines. The cross-talk between T cells and FLS appeared to require direct cell-cell contact as well as CD40 ligation, at least in part. CONCLUSION: Through repeated stimulation by CII, RA synovial T cells became trained effector cells that induced the production of proinflammatory mediators by FLS, while in the process the T cells becoming more sensitized to the activation signal from FLS.

Association of homozygous deletion of the Humhv3005 and the VH3-30.3 genes with renal involvement in systemic lupus erythematosus.

To investigate whether deletion of the Humhv3005 and the homologous VH3-30.3 (both share an identical amino acid sequence) genes is associated with susceptibility and/or certain clinical manifestations of systemic lupus erythematosus (SLE), DNA from 108 Korean SLE patients and 102 healthy subjects were analysed for the status of hv3005 gene by polymerase chain reaction-enzyme-linked immunosorbent assay. This method consists of amplification of selected germline VH3 genes with biotinylated primers, efficient capture of amplicons onto streptavidin-coated wells, and quantitative typing of bound VH3 gene with diagnostic oligonucleotides. We found that deletion of the hv3005 gene (including VH3-30.3) was more frequent in SLE patients than in healthy controls (26.9 versus 11.8%, P = 0.006, odds ratio 2.75). When clinical features were examined, patients with hv3005 deletion have a higher frequency of lupus nephritis (LN) (75.9 versus 44.3% for those without, P = 0.004), and higher activity index [median (range), 6 (2-14) versus 4 (1-16) for those without, P = 0.044] when biopsy-proven LN was studied. Collectively, our data suggest that deletion of the hv3005 and the 3-30.3 genes may predispose individual SLE patients to the development of LN.

In vivo differentiation of mouse embryonic stem cells into hepatocytes.

Embryonic stem (ES) cells have been regarded as a powerful resource for cell replacement therapy. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. Another type of disease that could benefit from the availability of stem cell therapy is liver disease. However, for this potential to be realized, it is necessary to demonstrate the differentiation of ES cells into hepatocytes. To demonstrate the in vivo differentiation potential of mouse ES cells, we injected ES cells into the spleen of immunosuppressed nude mice. Histological analysis of teratomas derived from injected ES cells revealed that some areas contained typical hepatocytes arranged in a sinusoidal structure. The hepatic nature of these cells was further confirmed by showing that transcripts of liver-specific genes were present in the differentiated teratoma using reverse transcriptase-polymerase chain reaction and immunohistochemistry using several liver-specific antibodies including HEP-PAR, phenylalanine hydroxylase, and mouse N-system aminotransferase to identify the respective proteins in the differentiated hepatocytes. This is the first demonstration that mouse ES cells can differentiate in vivo into a mixed population of hepatocytes of varying maturity. This finding extends the potential use of ES cells in the cell replacement therapy by including its possible application for treating liver diseases.

Cyclosporine inhibition of vascular endothelial growth factor production in rheumatoid synovial fibroblasts.

OBJECTIVE: To determine the antiangiogenic effect of cyclosporin A (CSA) in rheumatoid arthritis (RA). METHODS: We investigated the effect of CSA on the production of vascular endothelial growth factor (VEGF) by rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and cultured in the presence of CSA. The production of VEGF by FLS was measured in culture supernatants by enzyme-linked immunosorbent assay. The VEGF messenger RNA (mRNA) expression and activator protein 1 (AP-1) binding activity for VEGF transcription were determined by polymerase chain reaction and electrophoretic mobility shift assay, respectively. RESULTS: CSA dose-dependently inhibited both constitutive and transforming growth factor beta-induced VEGF production at the protein and mRNA levels. The suppressive action of CSA on VEGF synthesis was calcineurin dependent, as evidenced by a comparable inhibition by FK-506. Agonists of cAMP, 3-isobutyl-1-methylxanthine and N-2-O-dibutyryl-cAMP, mimicked the effect of CSA on VEGF production, while a cAMP antagonist, 2',3'-dideoxyadenosine, abrogated the effect of CSA. A gel mobility shift assay showed that the inhibitory effect of CSA was associated with decreased AP-1 binding activity to the VEGF promoter, in a cAMP-dependent manner. CONCLUSION: CSA may exert an antiangiogenic effect by inhibiting AP-1-mediated VEGF expression in rheumatoid synovial fibroblasts.

Suppression of collagen-induced arthritis by single administration of poly(lactic-co-glycolic acid) nanoparticles entrapping type II collagen: a novel treatment strategy for induction of oral tolerance.

OBJECTIVE: Poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer, is a carrier for drug delivery systems. This study was undertaken to investigate the tolerogenic effect of single administration of PLGA entrapping type II collagen (CII) on the development of collagen-induced arthritis (CIA). METHODS: The biophysical properties of PLGA nanoparticles entrapping CII (PLGA-CII) were investigated by in vitro release testing of CII, immunohistochemistry analysis, and electron microscopy. PLGA-CII was fed singly to animals 14 days before immunization, and the effect on joint inflammation was assessed. Circulating IgG anti-CII antibodies and T cell responses to CII in draining lymph nodes were assayed by enzyme-linked immunosorbent assay and (3)H-thymidine incorporation assay, respectively. The expression of messenger RNA (mRNA) for transforming growth factor beta (TGFbeta) and tumor necrosis factor alpha (TNFalpha) was determined by reverse transcriptase-polymerase chain reaction. RESULTS: The in vitro release test showed that CII was slowly discharged from PLGA-CII over a period of a month. After single administration of PLGA-CII, numerous particles approximately 300 nm in size were detectable in Peyer's patches, by electron microscopy and immunohistochemical staining for CII, 14 days after the original feeding. Mice fed a single dose of PLGA containing 40 microg of CII had significantly reduced values for incidence and severity of arthritis, serum IgG anti-CII antibodies, and CII-specific T cell proliferation as compared with mice fed solvent alone, those fed 6 doses of 20 microg CII alone, and those fed a single dose of PLGA alone. PLGA-CII was also able to suppress CIA after disease onset. Moreover, PLGA-CII-fed mice showed a higher level of TGFbeta mRNA expression in Peyer's patches, but a lower level of TNFalpha mRNA expression in draining lymph nodes, compared with the other groups of mice. CONCLUSION: Our data show that PLGA may serve as a powerful vehicle to promote the tolerance effect of oral CII and that single administration of PLGA-CII may hold promise as a new treatment strategy in rheumatoid arthritis.

The MHC class I homolog of human cytomegalovirus is resistant to down-regulation mediated by the unique short region protein (US)2, US3, US6, and US11 gene products.

Human CMV encodes four unique short region proteins (US), US2, US3, US6, and US11, each independently sufficient for causing the down-regulation of MHC class I molecules on the cell surface. This down-regulation allows infected cells to evade recognition by cytotoxic T cells but leaves them susceptible to NK cells, which lyse cells that lack class I molecules. Another human CMV-encoded protein, unique long region protein 18 (UL18), is an MHC class I homolog that might provide a mechanism for inhibiting the NK cell response. The sequence similarities between MHC class I molecules and UL18 along with the ability of UL18 to form trimeric complexes with beta(2)-microglobulin and peptides led to the hypothesis that if the US and UL18 gene products coexist temporally during infection, the US proteins might down-regulate UL18 molecules, similar to their action on MHC class I molecules. We show here that temporal expression of US and UL18 genes partially overlaps during infection. However, unlike MHC class I molecules, the MHC class I homolog, UL18, is fully resistant to the down-regulation associated with the US2, US3, US6, and US11 gene products. The specific effect of US proteins on MHC class I molecules, but not on UL18, represents another example of how viral proteins have evolved to evade immune surveillance, avoiding fratricide by specifically targeting host proteins.