Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-α in macrophage cell line and rat Kupffer cells

Abstract

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-α from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-α release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-α 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-α release by 14–22% and 13–22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-α production (by 24–35%). Moreover, the upregulated TNF-α 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-α production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-α production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-α 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-α expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-α may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.