Expression, regulation, and signaling of the pattern-recognition receptor nucleotide-binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts.
OBJECTIVE
Since pattern-recognition receptors (PRRs), in particular Toll-like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease-relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide-binding oligomerization domain 2 (NOD-2) in RA.


METHODS
Expression of NOD-2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD-2 in RA synovial fibroblasts (RASFs) were measured by conventional and real-time polymerase chain reaction (PCR) analyses. Levels of interleukin-6 (IL-6) and IL-8 were measured by enzyme-linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real-time PCR. NOD-2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF-kappaB was measured by electrophoretic mobility shift assay.


RESULTS
NOD-2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD-2 was detectable, but was induced by poly(I-C), lipopolysaccharide, and tumor necrosis factor alpha. After up-regulation of NOD-2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL-6 and IL-8 via p38 and NF-kappaB. Stimulation with MDP further induced the expression of MMP-1, MMP-3, and MMP-13.


CONCLUSIONS
Not only TLRs, but also the PRR NOD-2 is expressed in the synovium of RA patients, and activation of NOD-2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD-2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.
Ospelt
Caroline
C
Center of Experimental Rheumatology, University Hospital Zurich, and Zurich Center of Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
Brentano
Fabia
F
Jüngel
Astrid
A
Rengel
Yvonne
Y
Kolling
Christoph
C
Michel
Beat A
BA
Gay
Renate E
RE
Gay
Steffen
S
D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000378:metabolism / Q000494:pharmacology; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000096:biosynthesis; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051193:Toll-Like Receptors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
C508600:IL6 protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D020782:Matrix Metalloproteinases