Toll-like receptors (TLRs) are involved in mediating cell activation on stimulation with microbial constituents. We investigated the role for TLRs in synovial fibroblast (SF) activation in rheumatoid arthritis (RA). We analyzed whether stimulation with interleukin-1␤ and tumor necrosis factor-␣, cytokines present in RA synovium, influences expression of TLR genes in SFs. The effects were compared with those of treatment with lipopolysaccharide and a synthetic lipopeptide (sBLP). Gene expression was examined using quantitative polymerase chain reaction. TLR2-mediated cell activation was investigated by electromobility shift assay for nuclear factor-B. To localize TLR2 expression in joint tissue sections of RA patients were stained using in situ hybridization. Expression of TLR2 in RA SFs was increased after treatment with interleukin-1␤, tumor necrosis factor-␣, lipopolysaccharide, and sBLP. Nuclear factor-B translocation in SFs was triggered by TLR2-mediated cell stimulation.

Component Affected g p9 1-phox gp91-phox gp91-phox p22-ph0~ p22-phox p47-ph0~ p67-ph0~ Cytochrome 458 Heme Spectrum

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The membrane subunit of the phagocyte NADPH oxidase, gp91 phox , possesses a H ؉ channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91 phox cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91 phox generated voltage-activated, pH-dependent, H ؉ -selective currents that were reversibly blocked by Zn 2؉ . The gp91 phox currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91 phox characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91 phox proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.The NADPH oxidase catalyzes the one-electron reduction of molecular oxygen to superoxide, the precursor of a variety of toxic oxygen radicals generated by neutrophils, eosinophils, and macrophages, to kill invading microorganisms (1, 2). This enzyme is crucial in the host defense against microbial pathogens, and patients with chronic granulomatous disease (CGD) 1 , who fail to assemble a functional oxidase, suffer from severe recurrent infections (3, 4). The NADPH oxidase is a multicomponent enzyme composed of at least three cytosolic subunits, p47 phox , p67 phox , and p40 phox , and two membrane-associated subunits, p22phox and gp91 phox (1,5). Upon stimulation, the cytosolic components associate with the membrane-bound subunits, resulting in a functional oxidase complex that transfers electrons from cytosolic NADPH to extracellular oxygen (6, 7). The transfer of electrons across the plasma membrane generates a massive depolarization, that, if uncompensated, would prevent further electron transfer and the associated production of superoxide (8,9). Because large amounts of acid equivalents are released in the cytosol during the hydrolysis of NADPH and its resynthesis by the hexose monophosphate shunt, the oxidase has been proposed to act as a H ϩ channel to preserve electroneutrality and allow the extrusion of the intracellular acid (10, 11). Accordingly, both the depolarization and the cytosolic acidification generated by the oxidase are potentiated by Zn 2ϩ , an inhibitor of H ϩ channels (9, 12). The proton channel function of gp91 phox is still debated, ...

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