Short-term hyperthermia prevents activation of proinflammatory genes in fibroblast-like synoviocytes by blocking the activation of the transcription factor NF-κB

Abstract

Fibroblast-like synoviocytes (FLS) play a key role in the genesis of rheumatoid arthritis (RA). FLS are among the most versatile cells with the potential to activate an array of genes that are able to initiate and propagate inflammation in RA-affected joints. Controlling activation of FLS might hold the key to restraining inflammation in RA-affected joints. In this study, we investigate the effect and mechanisms of short-term hyperthermia on a series of proinflammatory genes in FLS. In vitro experiments demonstrate that exposure of FLS to elevated temperatures for the duration of 30 min prevents activation of a series of genes with proinflammatory properties. Exposure to hyperthermia reduces IL-1beta-induced prostaglandin E2 release, suppresses activation of the adhesion molecules VCAM-1, ICAM-1, the cytokines TNFalpha, IL-1alpha, IL-1beta, IL-8 as well as COX-2 protein synthesis. Real time reverse transcriptase-polymerase chain reaction showed that hyperthermia altered gene expression at the transcriptional level. The amount and the duration of inhibition is gene-specific and lasts for up to 25 h. As to the mechanism of inhibition, electrophoretic mobility shift assay experiments demonstrated that exposure of FLS to hyperthermia prevents IL-1beta-induced NF-kappaB translocation and subsequent DNA binding. Many mechanisms have been shown to be involved in hyperthermia-mediated effects on NF-kappaB-DNA interactions. We demonstrate by Western blot experiments that in FLS, hyperthermia prevents the phosphorylation and subsequent degradation of IkappaBalpha, therefore retaining the NF-kappaB complex in the cytoplasm. Carefully controlled in vivo tests are certainly needed before one can take full advantage of those phenomena; however, the ease by which the temperature in joints can be modulated might offer an opportunity for manipulating inflammatory processes in joints by simple balneological means.

Short term hyperthermia prevents the activation of mitogen-activated protein kinase p38

“…However, like us, others also demonstrated discrepancies in the time course of HSP70 and/or HO-1 expression and the inhibition of NF-κB mediated by HT (4,6). Similar to our results 4 using FLS, it has been reported that IκB degradation in heat shocked macrophages is blocked for up to four hours (7).…”

“…As reported earlier from our laboratory, exposing FLS to HT resulted in significantly reduced mRNA and protein levels of many pro-inflammatory genes that would otherwise be highly upregulated in response to IL-1β (4). We further demonstrated by electrophoretic mobility shift assays (EMSA) and western blot experiments that HT prevented IκB degradation and nuclear translocation of NF-κB in this cell type (4).…”

Hyaluronan Production in Synoviocytes as a Consequence of Viral Infections

“…Western Blot Experiments-SDS-PAGE and Western blotting were carried out essentially as described (16). Proteins were made visible using RenaissancePlus (PerkinElmer Life Sciences) and Kodak BioMax MR films or the chemiluminescence detection device GeneGnome (Syngene, Cambridge, UK).…”

“…EMSA-Preparation of nuclear extract, execution of EMSA, and EMSA specificity control experiments have been described in detail on a number of occasions (16,19,20). In short, oligonucleotides resembling consensus binding sites for NF-B (5Ј-AGT TGA GGG GAC TTT CCC AGG C-3Ј), AP-1 (c-Jun) (5Ј-CGC TTG ATG AGT CAG CCG GAA-3Ј), and CRE (5Ј-AGA GAT TGC CTG ACG TCA GAG AGC TAG-3Ј)) were purchased from Promega (Promega, Mannheim, Germany).…”

“…Control experiments for EMSA such as supershift and competition experiments were performed as published (17). A number of quality controls, routinely performed to ensure RT-PCR specificity and linearity, have been reported elsewhere (16,17,21). Statistical analysis was done using the unpaired t test; p values Յ0.05 are considered significant.…”