Toll-like receptors and chondrocytes: the lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.
OBJECTIVE
To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes.


METHODS
TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interleukin-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (35)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis.


RESULTS
TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis.


CONCLUSIONS
These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.
Bobacz
K
K
Medical University of Vienna, Vienna, Austria. klaus.bobacz@meduniwien.ac.at
Sunk
I G
IG
Hofstaetter
J G
JG
Amoyo
L
L
Toma
C D
CD
Akira
S
S
Weichhart
T
T
Saemann
M
M
Smolen
J S
JS
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D064236:Cartilage Oligomeric Matrix Protein; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000494:pharmacology; D053583:Interleukin-1beta / Q000235:genetics / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D064235:Matrilin Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000502:physiology
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D019485:Bone Morphogenetic Proteins; D064236:Cartilage Oligomeric Matrix Protein; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D053590:Interleukin 1 Receptor Antagonist Protein; D053583:Interleukin-1beta; D008070:Lipopolysaccharides; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins; D012333:RNA, Messenger; C495345:TLR4 protein, human; C577553:TSP5 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D016212:Transforming Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases