Hyaluronan inhibits cytokine production by lipopolysaccharide-stimulated U937 macrophages through down-regulation of NF-kappaB via ICAM-1.
OBJECTIVE
This study investigated the inhibitory mechanism of hyaluronan (HA) on lipopolysaccharide (LPS)-stimulated production of proinflammatory cytokines in U937 macrophages.


METHODS
HA was added to U937 macrophage cultures in the presence of LPS, with or without pretreatment with anti-intercellular adhesion molecule-1 (ICAM-1) antibody. Secreted levels of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, and IL-6 were determined by enzyme-linked immunosorbent assay. The phosphorylation of nuclear factor (NF)-kappaB, IkappaBalpha, and mitogen-activated protein kinases (MAPKs) was analyzed by immunoblotting.


RESULTS
LPS stimulated production of TNFalpha, IL-1beta, and IL-6. In contrast to 800 kDa HA, 2700 kDa HA at 1 mg/ml inhibited LPS-induced cytokine production. Anti-ICAM-1 antibody blocked the effects of HA on the LPS actions on U937 cells. LPS activated NF-kappaB and MAPK pathways, whereas HA down-regulated p65 NF-kappaB and IkappaBalpha phosphorylation by LPS without affecting MAPKs. Inhibition studies revealed the requirement of NF-kappaB for LPS-stimulated cytokine production. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on phosphorylation of p65 NF-kappaB and IkappaBalpha.


CONCLUSIONS
HA of intrinsic molecular weight suppresses LPS-stimulated production of proinflammatory cytokines via ICAM-1 through down-regulation of NF-kappaB and IkappaB. Exogenous HA injected into arthritic joints could act as an anti-NF-kappaB agent by the mechanism demonstrated in the present study.
Yasuda
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Department of Sports Medicine, Faculty of Health, Budo, and Sports Studies, Tenri University, Tenri 632-0071, Japan. tadyasu@sta.tenri-u.ac.jp
D016207:Cytokines / Q000378:metabolism; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D053583:Interleukin-1beta / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D020298:U937 Cells
D016207:Cytokines; D053583:Interleukin-1beta; D015850:Interleukin-6; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D006820:Hyaluronic Acid