INTRODUCTION. Hemozoin (malarial pigment) is a crystalline ferriprotoporphyrin IX polymer present in late stages of P. falciparum-parasitized RBCs, derived from incomplete digestion of host hemoglobin. Hemozoin-laden monocytes produce increased amounts of peroxidation products of polyunsaturated fatty acids (PUFAs) and stimulate generation of several cytokines. We have previously shown that in human monocytes, avidly phagocytosed hemozoin or hemozoin-containing trophozoites increased monocyte MMP-9 activity and expression by enhancing production of MMP-9-related cytokines, such as TNF-alpha and IL-1-beta. Here, the mechanism by which hemozoin enhances MMP-9 and cytokine production was studied. METHODS. Gelatin zymography with monocyte supernatants was used to assay calcium-induced protease activity; levels of soluble TNF-alpha and IL-1-beta were analyzed in cell supernatants by specific ELISA; nuclear extracts were studied by electrophoretic mobility shift assay and by western blotting to verify NF-kB nuclear translocation; phosphorylation and degradation of cytosolic IkB-alpha protein were evaluated by western blotting. RESULTS. Quercetin, artemisinin and parthenolide, molecules able to inhibit three different stages of the intracellular NF-kB signalling pathway (IkB-alpha phosphorylation and following degradation; NF-kB nuclear translocation; and p65 binding to DNA, respectively), abrogated hemozoin-dependent increase of MMP-9, TNF-alpha and IL-1-beta either in hemozoin-fed or trophozoite-fed monocytes. Moreover, 2 h and 24 h after phagocytosis the NF-kB complex was found in the nuclear fraction, and cytosolic IkB-alpha protein was phosphorylated and degraded. Otherwise, neither beta-hematin (lipid-free synthetic hemozoin) nor delipidized hemozoin did alter MMP-9, TNF-alpha and IL-1-beta production, and did induce NF-kB nuclear translocation or IkB-alpha protein degradation. In contrast, 15-HETE, a potent lipoperoxidation derivative generated by hemozoin from arachidonic acid via heme-catalysis, mimicked the hemozoin effects. Those effects were abrogated by quercetin, artemisinin and parthenolide. CONCLUSIONS. Present data suggest that hemozoin-induced upregulation of MMP-9, TNF-alpha and IL-1-beta in human monocytes may involve a NF-kB-mediated signal transduction pathway. NF-kB activation could be dependent on hemozoin-attached or hemozoin-generated lipid components. 15-HETE could be one mediator possibly responsible.
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