Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells.
Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.
Wu
Rongqian
R
Division of Surgical Research, Department of Surgery, North Shore-Long Island Jewish Medical Center and New York University School of Medicine, 350 Community Drive, Manhasset, NY 11030, USA.
Zhou
Mian
M
Wang
Ping
P
D053607:Adrenomedullin; D000818:Animals; D002460:Cell Line; D017242:Complement Factor H / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D007728:Kupffer Cells / Q000187:drug effects / Q000276:immunology*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors; D008264:Macrophages / Q000187:drug effects / Q000276:immunology*; D010455:Peptides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics
D008070:Lipopolysaccharides; D010455:Peptides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C481978:adrenomedullin-binding protein 1, rat; D053607:Adrenomedullin; D017242:Complement Factor H