Introduction It has been recently shown that in human monocytes haemozoin (HZ, malarial pigment) and 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis, upregulated matrix metalloproteinase-9 (MMP-9) expression through activation of the NF-kappaB pathway. The present work investigated the effects of HZ and 15-HETE on release and activity of lysozyme, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1, physiological MMP-9 inhibitor), all molecules stored in gelatinase granules of mononuclear cells. In order to verify the involvement of the NF-kappaB system, three NF-kappaB inhibitors showing antimalarial properties (quercetin, artemisin and parthenolide) were also used. Methods Human adherent monocytes isolated from peripheral blood were treated with HZ (native, delipidized or synthetic) or 15-HETE 10 microM for 2 hours, and further incubated in presence/absence of quercetin, artemisin and parthenolide for 2 hours (lysozyme activity studies) or 24 hours (TIMP-1 and MMP-9 protein release and gelatinolytic activity studies). Therefore, cell supernatants were collected. Activity of released lysozyme was evaluated by a specific spectrometric assay using suspensions of Mycrococcus Lysodeikticus. MMP-9 protein release was assayed by gelatin zymography. TIMP-1 protein release was assayed by Western blotting. Net gelatinolytic activity was measured by a fluorogenic gelatin conversion assay. Results Native HZ promoted the release of TIMP-1 and MMP-9, and enhanced either lysozyme or net gelatinolytic activity. Neither delipidized nor lipid-free synthetic HZ did reproduce the effects of native HZ, whereas 15-HETE did. Quercetin, artemisinin and parthenolide abrogated all the effects of both native HZ and 15-HETE. Discussion The present data suggest that the lipid moiety of HZ promotes the release of molecules from gelatinase granules (TIMP-1, MMP-9, lysozyme) of human monocytes. A role for 15-HETE is likely. As a consequence, lysozyme activity is increased, whereas impaired balance between upregulated TIMP-1 and MMP-9 leads to enhanced net gelatinolytic activity. All HZ/15-HETE effects appear to be NF-kappaB-dependent, and artemisinin, quercetin and parthenolide seem to be good candidate inhibitors. The present results should be put in the context of evidences showing a detrimental functional impairment of human monocytes during malaria, and might be useful in order to design future combined adjuvant therapies aimed to its prevention.
Quercetin, artemisinin and parthenolide abrogate the haemozoin- and 15-HETE-enhanced activity of enzymes released from gelatinase granules by human monocytes
PRATO, Mauro;VALENTE, Elena;ULLIERS, Daniela;KHADJAVI, AMINA;GIRIBALDI, Giuliana
2011-01-01
Abstract
Introduction It has been recently shown that in human monocytes haemozoin (HZ, malarial pigment) and 15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid (15-HETE), a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis, upregulated matrix metalloproteinase-9 (MMP-9) expression through activation of the NF-kappaB pathway. The present work investigated the effects of HZ and 15-HETE on release and activity of lysozyme, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1, physiological MMP-9 inhibitor), all molecules stored in gelatinase granules of mononuclear cells. In order to verify the involvement of the NF-kappaB system, three NF-kappaB inhibitors showing antimalarial properties (quercetin, artemisin and parthenolide) were also used. Methods Human adherent monocytes isolated from peripheral blood were treated with HZ (native, delipidized or synthetic) or 15-HETE 10 microM for 2 hours, and further incubated in presence/absence of quercetin, artemisin and parthenolide for 2 hours (lysozyme activity studies) or 24 hours (TIMP-1 and MMP-9 protein release and gelatinolytic activity studies). Therefore, cell supernatants were collected. Activity of released lysozyme was evaluated by a specific spectrometric assay using suspensions of Mycrococcus Lysodeikticus. MMP-9 protein release was assayed by gelatin zymography. TIMP-1 protein release was assayed by Western blotting. Net gelatinolytic activity was measured by a fluorogenic gelatin conversion assay. Results Native HZ promoted the release of TIMP-1 and MMP-9, and enhanced either lysozyme or net gelatinolytic activity. Neither delipidized nor lipid-free synthetic HZ did reproduce the effects of native HZ, whereas 15-HETE did. Quercetin, artemisinin and parthenolide abrogated all the effects of both native HZ and 15-HETE. Discussion The present data suggest that the lipid moiety of HZ promotes the release of molecules from gelatinase granules (TIMP-1, MMP-9, lysozyme) of human monocytes. A role for 15-HETE is likely. As a consequence, lysozyme activity is increased, whereas impaired balance between upregulated TIMP-1 and MMP-9 leads to enhanced net gelatinolytic activity. All HZ/15-HETE effects appear to be NF-kappaB-dependent, and artemisinin, quercetin and parthenolide seem to be good candidate inhibitors. The present results should be put in the context of evidences showing a detrimental functional impairment of human monocytes during malaria, and might be useful in order to design future combined adjuvant therapies aimed to its prevention.
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