Nonbiological therapeutics are frequently used for the treatment of patients with rheumatoid arthritis (RA). Because the mechanisms of action of these therapeutics are unclear, the authors aimed to elucidate the molecular effects of typical antirheumatic drugs on the expression profile of RA-related genes expressed in activated synovial fibroblasts. For reasons of standardization and comparability, immortalized synovial fibroblasts derived from RA (RASF) and normal donors (NDSF) were treated with methotrexate, prednisolone, or diclofenac and used for gene expression profiling with oligonucleotide microarrays. The cytotoxicity of the antirheumatic drugs was tested in different concentrations by MTS tetrazolium assay. Genes that were differentially expressed in RASF compared to NDSF and reverted by treatment with antirheumatic drugs were verified by semiquantitative polymerase chain reaction and by chemiluminescent enzyme immunoassay. Treatment with methotrexate resulted in the reversion of the RA-related expression profile of genes associated with growth and apoptosis including insulin-like growth factor binding protein 3, retinoic acid induced 3, and caveolin 2 as well as in the re-expression of the cell adhesion molecule integrin α6. Prednisolone reverted the RA-related profile of genes that are known from inflammation and suppressed interleukins 1β and 8. Low or high doses of diclofenac had no effect on the expression profile of genes related to RA in synovial fibroblasts. These data give the first insight into the mechanisms of action of common antirheumatic drugs used for the treatment of arthritides. Synovial fibroblasts reflect the disease-related pathophysiology and are useful tools for screening putative antirheumatic compounds. (Journal of Biomolecular Screening 2007:328-340)

Background La/SSB is a phosphoprotein that associates with various small RNA molecules. It has been found that the primary phosphorylation site of the molecule during various physiological processes is in Ser366. Objectives To determine whether the phosphorylation state of Ser366 could affect the antigenicity and the recognition of the protein by antibodies from patients with primary Sjögren's syndrome (pSS). Methods Peptides 349-368aa and phos349-368aa (with the Ser366 residue phosphorylated) were synthesized. Sera with anti-La specificity from 30 patients with pSS and sera from 19 normal individuals were examined against the two synthetic peptides in ELISA. The antibody specificity against the epitopes was tested with homologous and heterologous inhibition assays. Results Of pSS sera 23% reacted against the 349-368aa peptide. Sera binding to unphosphorylated peptide reacted also with phos349-368aa. Although the same sera gave a positive reaction against both peptides, the optical density values received from antibodies to phos349-368aa were higher, indicating a higher concentration or stronger affinity. When phos349-368aa was used as soluble inhibitor, in homologous inhibition the reactivity was almost completely abolished (92%). In contrast, when the unphosphorylated peptide was used as inhibitor, the reactivity of sera against phos349-368aa was only partially reduced (35%), indicating that sera from these patients possess two distinct groups of antibodies: one against the unphosphorylated and one against the phosphorylated epitope. Conclusion The phosphorylation of the serine366 residue resulted in a significant increase in antibody binding on epitope 349-368aa of La/SSB. These observations might explain the increased antigenicity of La/SSB autoantigen in various pathological situations in which phosphorylation may occur. 2 Surface-bound immune complexes containing antibodies to collagen type II induce production of TNF-α α, IL-1β β and IL-8 from monocytes via Fcγ γRII