INTRODUCTION: In the last decade growing evidence on the involvement of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in clinical progress towards complicated malaria (i.e. cerebral malaria, CM) has been emerging from in vivo or in vitro malaria models. In the present study, the effects of malarial pigment hemozoin (HZ) on MMP-9/TIMP-1 balance were studied in human monocytes, along with dependence on production of pro-inflammatory molecules, role of lipid moiety of HZ, involvement of NF-kappaB activation and following biological effects. METHODS: Human adherent monocytes isolated from peripheral blood were fed with HZ for 2 hours, and further incubated for 15 hours (mRNA expression studies) or 24 hours (protein release/biological effects studies). In selected experiments, unfed cells were incubated with: a) rhTNFalpha, rhIL-1beta and rhMIP-1alpha; b) lipid-free synthetic HZ, native delipidized HZ and 15-HETE. Alternatively, HZ-fed cells were treated with: a) anti-hTNFalpha, anti-hIL-1beta and anti-hMIP-1alpha blocking antibodies; b) quercetin, artemisinin or parthenolide. Therefore, MMP-9/TIMP-1 mRNA expression was measured by Real Time RT-PCR; MMP-9 protein release by gelatin zymography; TIMP-1 protein release by Western blotting; net gelatinolytic activity by fluorogenic gelatin conversion assay; cell invasion ability by Matrigel invasion assay. RESULTS: HZ enhanced MMP-9 and TIMP-1 mRNA expression and protein release, as well as net total gelatinolytic activity and cell invasiveness. HZ-dependent MMP-9/TIMP-1 enhancement was abrogated by anti-hTNFalpha, anti-hIL-1beta and anti-hMIP-1alpha blocking antibodies and was mimicked by recombinant cytokines. Lipid-free HZ did not reproduce HZ effects, whereas 15-HETE did. Quercetin, artemisinin and parthenolide, three NF-kappaB inhibitors showing anti-malaria properties, abrogated HZ effects. CONCLUSIONS: Phagocytosis of HZ by human monocytes promotes inflammation-mediated and NF-kB-dependent increased expression and release of both MMP-9 and TIMP-1; the lipid moiety of HZ appears to be relevant, and a role for 15-HETE, a potent lipoperoxidation derivative generated by HZ from arachidonic acid via heme-catalysis, is likely. Nevertheless, HZ enhances net gelatinolytic activity and cell invasion ability, suggesting that TIMP-1 enhancement is not sufficient to counterbalance MMP-9 increase, which might be detrimentally instrumental for monocyte extravasation during CM. In conclusion, a role for MMP-9 and TIMP-1 as potential markers of malaria severity, as well as the potential use of synthetic MMP inhibitors as adjuvant therapy for CM, should be carefully considered in future studies.
Malarial pigment alters MMP-9/TIMP-1 balance in human monocytes: role of cytokines, lipids and NF-kappaB
GIRIBALDI, Giuliana;VALENTE, Elena;POLIMENI, Manuela;ULLIERS, Daniela;KHADJAVI, AMINA;MANDILI, GIORGIA;PRATO, Mauro
2011-01-01
Abstract
INTRODUCTION: In the last decade growing evidence on the involvement of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in clinical progress towards complicated malaria (i.e. cerebral malaria, CM) has been emerging from in vivo or in vitro malaria models. In the present study, the effects of malarial pigment hemozoin (HZ) on MMP-9/TIMP-1 balance were studied in human monocytes, along with dependence on production of pro-inflammatory molecules, role of lipid moiety of HZ, involvement of NF-kappaB activation and following biological effects. METHODS: Human adherent monocytes isolated from peripheral blood were fed with HZ for 2 hours, and further incubated for 15 hours (mRNA expression studies) or 24 hours (protein release/biological effects studies). In selected experiments, unfed cells were incubated with: a) rhTNFalpha, rhIL-1beta and rhMIP-1alpha; b) lipid-free synthetic HZ, native delipidized HZ and 15-HETE. Alternatively, HZ-fed cells were treated with: a) anti-hTNFalpha, anti-hIL-1beta and anti-hMIP-1alpha blocking antibodies; b) quercetin, artemisinin or parthenolide. Therefore, MMP-9/TIMP-1 mRNA expression was measured by Real Time RT-PCR; MMP-9 protein release by gelatin zymography; TIMP-1 protein release by Western blotting; net gelatinolytic activity by fluorogenic gelatin conversion assay; cell invasion ability by Matrigel invasion assay. RESULTS: HZ enhanced MMP-9 and TIMP-1 mRNA expression and protein release, as well as net total gelatinolytic activity and cell invasiveness. HZ-dependent MMP-9/TIMP-1 enhancement was abrogated by anti-hTNFalpha, anti-hIL-1beta and anti-hMIP-1alpha blocking antibodies and was mimicked by recombinant cytokines. Lipid-free HZ did not reproduce HZ effects, whereas 15-HETE did. Quercetin, artemisinin and parthenolide, three NF-kappaB inhibitors showing anti-malaria properties, abrogated HZ effects. CONCLUSIONS: Phagocytosis of HZ by human monocytes promotes inflammation-mediated and NF-kB-dependent increased expression and release of both MMP-9 and TIMP-1; the lipid moiety of HZ appears to be relevant, and a role for 15-HETE, a potent lipoperoxidation derivative generated by HZ from arachidonic acid via heme-catalysis, is likely. Nevertheless, HZ enhances net gelatinolytic activity and cell invasion ability, suggesting that TIMP-1 enhancement is not sufficient to counterbalance MMP-9 increase, which might be detrimentally instrumental for monocyte extravasation during CM. In conclusion, a role for MMP-9 and TIMP-1 as potential markers of malaria severity, as well as the potential use of synthetic MMP inhibitors as adjuvant therapy for CM, should be carefully considered in future studies.
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