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.

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.

Although there is extensive evidence for effects of prolactin (PRL) on the brain, knowledge about the PRL receptor (PRL-R) in the brain is limited. By using monoclonal antibodies raised against purified rat liver PRL-R, the distribution of PRL-R was investigated by immunohistochemistry in brains of the estrogen-treated ovariectomized (OVX+E) rat and the adult male rat. Immunohistochemistry was performed by using the avidin biotinylated horse radish peroxidase macromolecular complex method. In both male and OVX+E rats, strong immunostaining was detected in the choroid plexus of all cerebral ventricles. This immunostaining was localized predominately on epithelial cell membranes. In the OVX+E female rat, scattered immunoreactive perikarya were observed in the arcuate nucleus, periventricular hypothalamic nucleus, preoptic area, suprachiasmatic nucleus, and supraoptic nucleus of the hypothalamus. Immunostaining in hypothalamic nuclei was localized on neuronal cell bodies as well as on neuronal processes. In addition, there was extensive PRL-R immunoreactivity throughout the globus pallidus and ventral pallidum. Immunostaining in these striatal regions was not associated with neuronal cell bodies but appeared to be localized on processes or glial cells. In the male rat, less immunostaining was observed in the hypothalamus, and there was no immunostaining in the corpus striatum. No significant staining was observed in the cerebral cortex, thalamus, or hindbrain of either male or OVX+E rats. The implication of PRL-R existence in these brain regions remains to be investigated.

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.