Toll-like receptor 4-dependent activation of macrophages by polysaccharide isolated from the radix of Platycodon grandiflorum

Platycodon grandiflorum, a traditional oriental herbal medicine, is known to have immunostimulatory and antitumor effects. PG, a polysaccharide isolated from P. grandiflorum, has been reported to activate macrophages and B cells. Here, we investigated the membrane receptor and intracellular signaling responsible for the activation of macrophages by PG. PG induced the production of nitric oxide (NO) and the mRNA expression of iNOS in RAW 264.7 cells. To investigate the membrane receptor involved in the activation of NO production, we examined the effect of PG on the production of NO in mouse peritoneal macrophages isolated from wild type C3H/HeN and functional Toll-like receptor 4 (TLR4)-deficient C3H/HeJ mice. PG induced NO production by macrophages isolated from C3H/HeN mice, but had no effect on NO production by macrophages isolated from C3H/HeJ mice. Moreover, monoclonal antibodies directed to TLR4 blocked PG-mediated induction of NO production. In addition, LBP and sCD14 was also found to be involved in the activation of NO production by PG. To further investigate, we examined the effect of PG on the activation of DNA binding of NF-kappaB, which is a downstream transcriptional regulator of TLR4. PG caused degradation of IkappaB and activation of DNA binding of NF-kappaB. In addition, TPCK, a specific NF-kappaB inhibitor, abolished PG-mediated induction of DNA binding of NF-kappaB, production of NO and mRNA expression of NOS, demonstrating the involvement of NF-kappaB in PG-mediated macrophage activation. Taken together, these results suggest that PG-mediated induction of NO production and NOS mRNA expression in macrophages is mediated, at least in part, by TLR4/NF-kappaB signaling pathway. (C) 2003 Elsevier B.V. All rights reserved.