Prolonged Tumor Necrosis Factor α Primes Fibroblast-like Synoviocytes in a Gene-Specific Manner by Altering Chromatin

Objective. During the course of rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are chronically exposed to an inflammatory milieu. The purpose of this study was to test the hypothesis that prolonged exposure of FLS to tumor necrosis factor alpha (TNF alpha) augments inflammatory responses to secondary stimuli (priming effect). Methods. FLS obtained from RA patients were exposed to TNF alpha for 3 days and were then stimulated with interferons (IFNs). Expression of IFN target genes was measured by real-time quantitative reverse transcription-polymerase chain reaction analysis and enzyme-linked immunosorbent assay. Total STAT-1 protein and IFN-mediated STAT-1 activation were evaluated by Western blotting. Total histone levels, histone acetylation, and NF-kappa B p65 and RNA polymerase II (Pol II) recruitment were measured at the CXCL10 promoter (encodes IFN gamma-inducible 10-kd protein [IP-10]) by chromatin immunoprecipitation assays. Results. Prolonged pre-exposure of FLS to TNF alpha enhanced the magnitude and extended the kinetics of CXCL10/IP-10, CXCL9, and CXCL11 production upon subsequent IFN stimulation. This phenotype was re-tained over a period of days, even after the removal of TNF alpha Prolonged TNF alpha exposure decreased histone levels, increased acetylation of the remaining histones, and heightened recruitment of NF-kappa B p65 and Pol II to the CXCL10 promoter. In parallel, an increase in intracellular STAT-1 led to amplification of IFN-induced STAT-1 activation. Conclusion. Our study reveals a novel pathogenic function of TNF alpha, namely, prolonged and gene-specific priming of FLS for enhanced transcription of inflammatory chemokine genes due to the priming of chromatin, the sustained activation of NF-kappa B, and the amplification of STAT-1 activation downstream of IFNs. These data also suggest that FLS gain an "inflammatory memory" upon prolonged exposure to TNF alpha