Toll-like Receptor 4-Dependent Adjuvant Activity of Kakkon-to Extract Exists in the High Molecular Weight Polysaccharide Fraction

Abstract

Kakkon-to, a traditional herbal medicine (Kampo formula), has been used historically in China and Japan for the treatment of infectious diseases such as influenza and the common cold. However, the biological mechanism of its therapeutic action has not yet been elucidated. In this study, we investigated the immunological function of Kakkon-to and found that the high molecular weight fraction of the extract activated macrophages in vitro. This fraction was found to be composed primarily of saccharides and in vitro intensively stimulated mouse peritoneal macrophages that produce Th1 inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), interferon-γ (IFN-γ), and interleukin-6 (IL-6). The fraction did not activate macrophages from C3H/HeJ lacking Toll-like receptor 4 (TLR4) or MyD88-deficient mice, indicating that macrophage activation by the fraction was mediated by TLR4. The route of administration of the fraction into mice regulated the kinetics of TNFα production in immune organs. Intravenous administration induced TNFα production in the four target organs of spleen, liver, lung, and Peyer's patch; however, the most abundant production occurred in the liver and peaked at 30–60 min post administration. Peritoneal administration induced similar kinetics but the most abundant production occurred in the spleen. In contrast, oral administration induced TNFα production in the liver, lung, and Peyer's patch, but not in the spleen. Although liver and lung are TNFα-abundant organs, production peaks in these organs occurred later than in Peyer's patch. We also found that the fraction induced antibody production as an adjuvant against a specific antigen [ovalbumin (OVA)] when administered simultaneously and subcutaneously in a dose-dependent manner. Interestingly, the fraction induced IgG-class antibody in response to low doses of the antigen, which induced only IgM-class antibody when administered alone, suggesting that the fraction induces a class switch of immunoglobulin as an adjuvant in vivo. The high molecular weight fraction of Kakkon-to extract could be applicable as a potent immunostimulating drug and adjuvant.