Adenosine is an extracellular signaling molecule that is generated in response to cell injury where it orchestrates tissue protection and repair. Whereas adenosine is best known for promoting anti-inflammatory activities during acute injury responses, prolonged elevations can enhance destructive tissue remodeling processes associated with chronic disease states. The generation of adenosine and the subsequent activation of the adenosine 2B receptor (A2BR) is an important processes in the regulation of both acute and chronic lung disease. The goal of this study was to examine the contribution of the A2BR in models of bleomycin-induced lung injury that exhibit varying degrees of acute and chronic injury. Intratracheal bleomycin exposure results in substantial acute lung injury followed by progressive fibrosis. In this model, genetic removal of the A2BR resulted in enhanced loss of barrier function and increased pulmonary inflammation, with few differences in indexes of pulmonary fibrosis. These results support an anti-inflammatory role for this receptor in this model of acute lung injury. In contrast, systemic exposure of mice to bleomycin resulted in modest acute lung injury together with progressive pulmonary fibrosis. In this model, the effects of A2BR removal on acute lung injury were negligible; however, there were substantial reductions in pulmonary fibrosis, supporting a profibrotic role for this receptor. A2BR-dependent regulation of IL-6 production was identified as a potential mechanism involved in the diminished pulmonary fibrosis seen in A2BR knockout mice exposed to i.p. bleomycin. These studies highlight the distinct roles of A2BR signaling during acute and chronic stages of lung injury.

The shared epitope (SE), carried by the vast majority of rheumatoid arthritis patients, is a 5-aa sequence motif in the third allelic hypervariable region of the HLA-DRβ chain. We have recently demonstrated that the SE acts as an allele-specific ligand that triggers NO-mediated pro-oxidative signaling in opposite cells. The identity of the cell surface molecule that interacts with the SE is unknown. Using affinity chromatography purification, cell-binding assays, surface plasmon resonance, and time-resolved fluorescence resonance energy transfer techniques, we have identified cell surface calreticulin (CRT) as the SE-binding molecule. SE-triggered signaling could be blocked by anti-CRT Abs or Abs against CD91 and by CRT-specific antisense or small-interfering RNA oligonucleotides. Embryonic fibroblasts from crt−/− or CD91-deficient mice failed to transduce SE-triggered signals. Exogenously added soluble CRT attached to the cell surface and restored SE-triggered signaling responsiveness in crt−/− cells. These data indicate that cell surface CRT, a known innate immunity receptor, which has been previously proposed as a culprit in autoimmunity, plays a critical role in SE-triggered signal transduction.

Objective. Susceptibility to rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles encoding a shared epitope (SE) in positions 70-74 of the HLA-DR␤ chain. The mechanistic basis for this association is unknown. Given the proposed pathogenic role of nitric oxide (NO) in RA, this study was undertaken to examine whether the SE can trigger NO signaling events.Methods. The intracellular levels of NO were measured with the fluorescent NO probe 4,5-diaminofluorescein diacetate and by the 2,3-diaminonaphthalene method. NO synthase activity was determined by measuring the rate of conversion of radioactive arginine to citrulline. Levels of cGMP were measured with a commercial enzyme-linked immunosorbent assay, and the cytolytic activity of T cells was measured using a standard 51 Cr release assay. Results. Lymphoblastoid B cell lines carrying SE-positive HLA-DR alleles displayed a higher rate of spontaneous NO production compared with SE-negative cells. L cell transfectants expressing SE-positive DR molecules on their surface also generated higher levels of NO. Tetrameric HLA-DR molecules containing a DR␤-chain encoded by the SE-positive DRB1*0401 allele stimulated fibroblast cells to produce higher levels of NO compared with cells stimulated with a control HLA-DR tetramer. Multimeric hepatitis B core proteins engineered to express region 65-79 encoded by the DRB1*0401 allele, but not the same region encoded by the control allele DRB1*0402, stimulated NO production in fibroblasts. Similarly, synthetic 15-mer peptides corresponding to the region 65-79 encoded by SEpositive alleles triggered increased NO levels when incubated with class II major histocompatibility complex-negative cells. The signaling pathway was found to involve NO synthase activation, followed by increased production of cGMP. SE-triggered increased NO levels inhibited cytolytic elimination of target cells.

Rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles that code a five-amino acid sequence motif in positions 70–74 of the HLA-DRβ chain, called the shared epitope (SE). The mechanistic basis of SE-RA association is unknown. We have recently found that the SE functions as an allele-specific signal transducing ligand that activates a nitric oxide (NO)-mediated pathway in other cells. To better understand the role of the SE in the immune system, here we have examined its effect on T cell polarization in mice. In CD11c+CD8+ dendritic cells (DCs) the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase (IDO), a key enzyme in immune tolerance and T cell regulation, while in CD11c+CD8− DCs the ligand activated robust production of IL-6. When SE-activated DCs were co-cultured with CD4+ T cells, the differentiation of Foxp3+ T regulatory (Treg) cells was suppressed, while Th17 cells were expanded. The polarizing effects could be seen with SE-positive synthetic peptides, but even more so, when the SE was in its natural tri-dimensional conformation as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted in higher abundance of Th17 cells in the draining lymph nodes and increased IL-17 production by splenocytes. Thus, we conclude that the SE acts as a potent immune-stimulatory ligand that can polarize T cell differentiation toward Th17 cells, a T cell subset that has been recently implicated in the pathogenesis of autoimmune diseases, including RA.

Objective. To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins.Methods. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RAdiscordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls.Results. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11␤-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues.Conclusion. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

BackgroundThe rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRβ chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of - RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT.Principal FindingsSurface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217–224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu217 and Glu223 - and to a lesser extent residue Asp220 - in cell-free SPR-based binding and signal transduction assays.SignificanceWe have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

a b s t r a c tOsteogenesis depends on a coordinated network of transcription factors including Sp7. Emerging evidence indicates that microRNAs (miRNAs) act as pivotal regulators in various biological processes including osteoblast proliferation and differentiation. Here, we investigated the effect of miR-145 on osteogenic differentiation. miR-145 was decreased during osteogenic differentiation, which could suppress the osteogenic differentiation of C 2 C 12 and MC3T3-E1 cells confirmed by gain-and loss-of-function experiments. Moreover, bioinformatic analysis combined with luciferase reporter assay, and Western blot validated that miR-145 negatively regulated Sp7 expression. Inhibition of Sp7 showed similar effect with miR-145 on osteogenic differentiation, whereas overexpression of Sp7 attenuated this effect. Collectively, these data indicate that miR-145 is a novel regulator of Sp7, and it suppresses the osteogenic differentiation of C 2 C 12 and MC3T3-E1 cells.

Particular alleles of human leukocyte antigen (HLA) contribute to disease susceptibility and severity in many autoimmune conditions, but the mechanisms underlying these associations are often unknown. Here, we demonstrate that the shared epitope (SE), an HLA-DRB1-coded sequence motif that is the single most significant genetic risk factor for erosive rheumatoid arthritis (RA), acts as a signal transduction ligand that potently activates osteoclastogenesis, both in vitro and in vivo. The SE enhanced the production of several pro-osteoclastogenic factors and facilitated osteoclast (OC) differentiation in mouse and human cells in vitro. Transgenic mice expressing a human HLA-DRB1 allele that code the SE motif demonstrated markedly higher propensity for osteoclastogenesis and enhanced bone degradation capacity ex vivo. In addition, the SE enhanced the differentiation of Th17 cells expressing the receptor activator for nuclear factor-κB ligand (RANKL). When the two agents were combined, IL-17 and the SE enhanced OC differentiation synergistically. When administered in vivo to mice with collagen-induced arthritis, the SE ligand significantly increased arthritis severity, synovial tissue OC abundance and bone erosion. Thus, the SE contributes to arthritis severity by activating an OC-mediated bone-destructive pathway. These findings suggest that besides determining the target specificity of autoimmune responses, HLA molecules may influence disease outcomes by shaping the pathogenic consequences of such responses.