LPS-binding protein (LBP) is a central mediator that transfers LPS to CD14 to initiate TLR4-mediated proinflammatory response. However, a possibility of another LPS transfer molecule has been suggested because LBP-deficient mice showed almost normal inflammatory response after LPS injection. In this study, we describe the novel finding that high mobility group box 1 protein (HMGB1) recently identified as a mediator of sepsis has a function of LPS transfer for a proinflammatory response. We used ELISA and surface plasmon resonance to show that HMGB1 binds LPS in a concentration-dependent manner and that the binding is stronger to lipid A moiety than to the polysaccharide moiety of LPS. This binding was inhibited by LBP and polymyxin B. Using native PAGE and fluorescence-based LPS transfer analyses, we show that HMGB1 can catalytically disaggregate and transfer LPS to both soluble CD14 protein and to human PBMCs in a dose-dependent manner. However, this effect was dramatically reduced to the baseline level when HMGB1 was heat inactivated. Furthermore, a mixture of HMGB1 and LPS treatment results in a higher increase in TNF-α production in human PBMCs and peripheral blood monocytes than LPS or HMGB1 treatment alone or their summation. Thus, we propose that HMGB1 plays an important role in Gram-negative sepsis by catalyzing movement of LPS monomers from LPS aggregates to CD14 to initiate a TLR4-mediated proinflammatory response.

High-mobility group box 1 protein (HMGB1) has been studied as a key mediator of inflammatory diseases, including sepsis. Regulating secretion is important in the control of HMGB1-mediated inflammation. Previously, it was shown that HMGB1 needs to be phosphorylated for secretion. In this study, we show that HMGB1 is phosphorylated by the classical protein kinase C (cPKC) and is secreted by a calcium-dependent mechanism. For this study, RAW264.7 cells and human peripheral blood monocytes were treated with PI3K inhibitors wortmannin, LY294002, and ZSTK474, resulting in inhibition of LPS-stimulated HMGB1 secretion, whereas inhibitors of NF-κB and MAPKs p38 and ERK showed no inhibition. Akt inhibitor IV and mammalian target of rapamycin inhibitor rapamycin did not inhibit HMGB1 secretion. However, the PKC inhibitors Gö6983 (broad-spectrum PKC), Gö6976 (cPKC), and Ro-31-7549 (cPKC) and phosphoinositide-dependent kinase 1 inhibitor, which results in protein kinase C (PKC) inhibition, inhibited LPS-stimulated HMGB1 secretion. PKC activators, PMA and bryostatin-1, enhanced HMGB1 secretion. In an in vitro kinase assay, HMGB1 was phosphorylated by recombinant cPKC and by purified nuclear cPKC from LPS-stimulated RAW264.7 cells, but not by casein kinase II or cdc2. HMGB1 secretion was also induced by the calcium ionophore A23187 and inhibited by the Ca2+ chelators BAPTA-AM and EGTA. These findings support a role for Ca2+-dependent PKC in HMGB1 secretion. Thus, we propose that cPKC is an effector kinase of HMGB1 phosphorylation in LPS-stimulated monocytes and PI3K-phosphoinositide-dependent kinase 1 may act in concert to control HMGB1 secretion independent of the NF-κB, p38, and ERK pathways.

We found that absence of osteopontin (OPN) in immunocompromised Rag2−/− mice, which lack T and B cells, made the mice extremely susceptible to an opportunistic fungus Pneumocystis, although immunocompetent OPN-deficient mice could clear Pneumocystis as well as wild-type mice. OPN has been studied as an extracellular protein, and the role of an intracellular isoform of osteopontin (iOPN) is still largely unknown. Here, we elucidated the mechanism by which iOPN was involved in anti-fungal innate immunity. First, iOPN was essential for cluster formation of fungal receptors that detect Pneumocystis; including dectin-1, TLR2, and mannose receptor. Second, iOPN played a role as an adaptor molecule in TLR2 and dectin-1 signaling pathways, and mediated ERK activation and cytokine production by zymosan, which simultaneously activates TLR2 and dectin-1 pathways. Third, iOPN enhanced phagocytosis and clearance of Pneumocystis. Our study here suggests the critical involvement of iOPN in anti-fungal innate immunity.

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