Glycosylation Mediates Apoptosis and Inflammation in Rheumatoid Arthritis

S128 in the control of T cell activation, is expressed on Mo and mDC in cultured peripheral blood mononuclear cells (PBMC) from healthy controls and SLE patients in remission, but not on cells from SLE patients with active disease. The current study aimed to identify the mechanism for the differential regulation of PDL1 in SLE. Using isolated PBMC from pediatric SLE patients and age-matched healthy controls, we demonstrated that PD-L1 expression on Mo significantly correlated with the level of TNFα and IL-10. PD-L1 was induced in SLE PBMC culture treated with IL-10, or TNF-α. Moreover, the defective expression of PDL1 was restored in isolated SLE Mo. This induction of PD-L1 was suppressed when isolated SLE Mo were co-cultured with autologous T cells, suggesting PD-L1 can be induced by monocyte-intrinsic signal inhibited by T cells. Likewise, PD-L1 mRNA expression was not induced in patients with active disease. Thus, regulation of PD-L1 gene expression can occur early at the mRNA level. These findings indicate that regulation of PD-L1 by cytokines, and T cells during an immune response as a potential mechanism leading to loss of peripheral tolerance to autoantigens in SLE. This study delineates the mechanism of PD-L1 dysregulation in SLE, and may lead to discovery of novel therapeutic targets for lupus treatment aimed at restoring normal expression of PD-L1. doi:10.1016/j.clim.2010.03.384

S.63. Banking RNA for Biomarker Discovery: Assessing RNA Quality and Yield for Long Term Use Mary Carmelle Philogene, Hodan Ali, Katarzyna Bourcier, Adam Asare, Noha Lim, Smita Asare, Deborah Phippard. Immune Tolerance Network, Bethesda, MD Introduction: The Immune Tolerance Network (ITN) conducts clinical trials in allergy, transplantation and autoimmune diseases with the aim of identifying biomarkers and signature(s) of tolerance induction. Over the last decade the ITN has established a rich repository of clinical samples. For mechanistic assays, such as microarray analysis, the quality of RNA is of paramount importance. Method: In this study, the effect of blood storage on RNA quality was evaluated. Whole blood collected in ABI Tempus™ tubes was stored at -80°C for 0 to 1,255 days prior to RNA extraction. RNA was extracted from 10,797 blood samples using Applied Biosystems Total RNA chemistry™. The RNA Integrity Number (RIN) and RNA yield per mL. of blood was determined for each sample and correlated with the duration of storage prior to RNA isolation. Results: Our study shows that there was no correlation between the quality of RNA (RIN value) and length of time elapsed from whole blood collection to RNA isolation (r2 = 0.001). There was also no correlation between the yield and length of time from collection (r2 = 0.001). Conclusion: These observations have implications for planning longitudinal gene expression studies. Variations can be avoided by one batch processing. In addition, considerable cost savings can be realized by only processing samples required for analysis. Finally, assuring good quality RNA will facilitate sharing of these precious resources within the scientific community. doi:10.1016/j.clim.2010.03.385

Abstracts

S.64. A Limited T Cell Receptor Repertoire with the Single Jalpha Segment is Sufficient to Cause Autoimmune Diseases Maki Nakayama, XiangLing He, Kelly Johnson, George Eisenbarth. Barbara Davis Center, University of ColoradoDenver, Aurora, CO Mice having the single Jalpha segment (TRAJ 56) show a reduction in positive selection and a possible defect in negative selection probably due to having only one chance to assemble a T cell receptor (TCR) alpha chain from one gene. We generated congenic NOD mice that have only the TRAJ 56 segment at the alpha chain Jalpha locus utilizing the speedcongenic method and analyzed BC5 and BC6 animals for the development of autoimmune diseases (all idd loci were fixed with NOD genotypes at BC3 generation). Ja56 homozygotes developed diabetes and insulin autoantibodies despite having a limited TCR alpha chain repertoire, and spleen cells responded to insulin B:9-23 peptides. In addition, the mice also had infiltration of the salivary glands. Although the number of T cells in the spleen was lower than that of wild-type mice, flow-cytometric analysis showed no skewed Vbeta usage. To investigate if the flanking regions of the alpha chain locus influence diabetes development, we also generated control congenic NOD mice that have the 129 genome at the alpha chain locus. There was no significant difference in diabetes development among homozygous, heterozygous, and wild type animals at BC5. Although the single Ja56 mice have a limited alpha chain repertoire, the repertoire is still sufficient to develop autoimmunity against islets and salivary glands. We find it remarkable how much we can limit the TCR repertoire and still produce anti-islet autoimmunity, although this would be consistent with our hypothesis of the importance of the TRAV5D-4 alpha with fixed CDR1 and CDR2 sequences. doi:10.1016/j.clim.2010.03.386

S.65. Glycosylation Mediates Apoptosis and Inflammation in Rheumatoid Arthritis Jun Li, Hui-Chen Hsu, PingAr Yang, Qi Wu, John Mountz. University of Alabama at Birmingham, Birmingham, AL Rheumatoid Arthritis (RA) is one of the most common inflammatory diseases and aberrant apoptosis of synovial fibroblasts (SF) is linked to the pathogenesis. Glycosylation has been shown to regulate apoptotic sensitivity in cancers. The role of glycosylation in RA was investigated in this study. Real-time PCR indicated that between RA synovial tissues and osteoarthritis (OA) synovial tissues (N=14 each), there was no difference in the expression of death receptor related molecules, including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), its receptors DR4, DR5, DR6, DcR1, DcR2, and regulators of apoptosis FLIPL and cIAPs. No difference of N-acetyl galactosamine transferases (GALNTs) was found. However, there was a higher level of α1-2-Fucosyltransferase (Fut1, P=0.016), α1-3/1-4-Fucosyltransferase (Fut3, P=0.018) and α1-3-Fucosyltransferase (Fut6, P=0.038), but not α1-6-Fucosyltransferase (Fut8, P=0.910) in RA compared to OA synovial tissues. Interestingly,

Abstracts a strong positive correlation between TNFα and Fut1, Fut3 and Fut6 was identified. Fut1 also correlated with Il17a. Preincubation of RASFs with 2-Deoxy-D-Galactose (15 mM/ ml), a FUT1 inhibitor, decreased the killing sensitivity of TRA8 which is an anti-human DR5 antibody. This effect can be reversed by Fucose (15 mM). Similar results were also found on human monocyte/macrophage cell lines, HL60 and THP-1 with and without differentiation. Our studies suggest that fucosylation is strongly associated with altered apoptosis and inflammation in RA effector cells. Modifying fucosylation might be a novel therapeutic strategy for RA. doi:10.1016/j.clim.2010.03.387

S.66. Intestinal Lamina Propria-associated Factors Regulate Effector CD4+ T Cell Proliferation, Cytokine Release and Apoptosis Kayci Huff, Travis Hull, Lesley Smythies, Phillip Smith. University of Alabama at Birmingham, Birmingham, AL Extracellular matrix (stromal) factors in the normal intestinal lamina propria regulate innate and adaptive immune cell responses to microorganisms and immunostimulatory antigens, at least in part, through the down-regulatory cytokine TGF-β. Here we show that Crohn's disease mucosa, which is characterized by the loss of tolerance, contained equivalent levels of stromal TGF-β (pN0.05), but significantly elevated levels of IL-6 and IL-1 (pb0.03) compared to the stroma of normal intestinal mucosa. Antibody neutralization experiments showed that the TGF-β in normal intestinal stroma inhibited CD4+ T-cell proliferation and IFN-γ production. In contrast, Crohn's disease stromal IL-6 (augmented by IL-1) promoted T-cell proliferation (3-fold; pb0.05), and IL-1 (but not IL-6) promoted IFN-γ release (3-fold; pb0.05), despite the presence of TGF-β. Importantly, intestinal stromal factors in Crohn's mucosa, but not normal mucosa, also protected T-cells from apoptosis (7.8 + 0.5% vs 18.3 + 1.5% annexin V-positive, respectively, pb0.01). Furthermore Crohn's stromal factors induced the T-cells to phosphorylate STAT3, suggesting that STAT3 signaling is involved in the blockade of TGF-β-mediated down-regulation and protection against apoptosis in effector T-cells. These findings implicate an important role for the intestinal lamina propria stroma in the regulation of mucosal effector T-cell responses in healthy and Crohn's disease mucosa. doi:10.1016/j.clim.2010.03.388

S.67. Regulatory Natural Antibodies to Apoptotic Cell Membrane Determinants That Suppress Inflammatory Arthritis Inhibit Dendritic Cell Inflammatory Responses by Induction of a Dual Specificity Phosphatase Caroline Grönwall, Jaya Vas, Yifang Chen, Gregg Silverman. University of California-San Diego, La Jolla, CA We have recently demonstrated that natural antibodies (NAbs) to apoptotic-cell membrane (ACM) determinants,

S129 which distinguish cells undergoing cell death from healthy cells, form complexes that have potent properties to suppress in vitro and in vivo inflammatory responses mediated by a range of MyD88- and TRIF-dependent TLRs. Anti-ACM monoclonal Ab infusions also inhibited arthritis mediated by IgG anti-collagen II antibodies and development of collagen-induced arthritis. Furthermore, in dendritic cells (DCs), anti-ACM inhibited LPS-induced secretion of IL-6, TNFα, IL-12p70, MCP-1, IL-10 and others, but without induction of TGFβ. To understand the pathways responsible for these immunomodulatory properties, we investigated the MAPK system that plays central roles in TLR responses. By immunoblot, anti-ACM NAb significantly inhibited LPSmediated phosphorylation of cytoplasmic P38 MAPK and downstream nuclear transcription factor, ELK, without inhibition of JNK or ERK1/2. We also found similarities in the downstream anti-inflammatory signaling of the anti-ACM NAb with those of the glucocorticoid, dexamethasone. By flow cytometric, immunoblot, and transcript analyses, both anti-ACM antibodies and dexamethasone induced transactivation of the dual specificity phosphatase, MAPK phosphatase-1, MKP-1 (also termed DUSP-1), that peaked rapidly, while control MKP-5 was not induced. For the anti-ACM NAb, these effects were also antigen-inhibitable, and dependent on recruitment to immune complexes of C1q or MBL. Studies of DCs from knock-out mice confirmed the MKP-1 dependence of the MAPK inhibitory properties of anti-ACM antibodies. Our studies therefore demonstrate that regulatory anti-ACM NAbs can suppress activating P38 MAPK responses via key inhibitory signal transduction pathways that overlap with those of glucocorticoids. doi:10.1016/j.clim.2010.03.389

S.68. Identification of an IL-27/osteopontin Axis in Dendritic Cells and its Modulation by IFN-γ Limits IL-17 Mediated Autoimmune Inflammation Murugaiyan Gopal, Akanksha Mittal, Howard Weiner. Brigham & Women's Hospital, Boston, MA Dendritic cells (DCs) play a central role in determining the induction of T cell responses. IL-27 production by DCs favors induction of IL-10 producing regulatory T cells whereas osteopontin (OPN) promotes pathogenic IL-17 T cell responses. The regulatory mechanisms in DCs that control these two cells types are not understood well. Here, we show that IFN-γ induces IL-27 while inhibiting OPN expression in DCs both in vitro and in vivo and that engagement of IFNγR expressed by DCs leads to suppression of IL-17 production while inducing IL-10 from T cells. DCs modified by IFN-γ acquire IL-27-dependent regulatory function, promote IL-10mediated T cell tolerance and suppress autoimmune inflammation. Thus, our results identify a previously unknown pathway by which IFN-γ limits IL-17 mediated autoimmune inflammation through differential regulation of OPN and IL27 expression in DCs.

doi:10.1016/j.clim.2010.03.390