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Phagocytosis of haemozoin (malarial pigment) enhances metalloproteinase-9 activity in human adherent monocytes: role of IL-1beta and 15-HETE.

Prato M, Gallo V, Giribaldi G, Arese P - Malar. J. (2008)

Bottom Line: The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ) and trophozoites on production of IL-1beta and MMP-9 activity and expression.The second aim was to find out which component of HZ was responsible for the effects.Results may clarify aspects of cerebral malaria pathogenesis, since MMP-9, a metalloproteinase able to disrupt the basal lamina is possibly involved in generation of hallmarks of cerebral malaria, such as blood-brain barrier endothelium dysfunction, localized haemorrhages and extravasation of phagocytic cells and parasitized RBCs into brain tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino Medical School, Torino, Italy. paolo.arese@unito.it.

ABSTRACT

Background: It has been shown previously that human monocytes fed with haemozoin (HZ) or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9) enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ) and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects.

Methods: Native HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free), delipidized HZ, beta-haematin (lipid-free synthetic HZ), trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting.

Results: Monocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants) and protein/mRNA expression (in cell lysates) of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid) a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator possibly responsible for increase of both IL-1beta production and MMP-9 activity.

Conclusion: Results indicate that specific lipoperoxide derivatives generated by HZ may play a role in modulating production of IL-1beta and MMP-9 expression and activity in HZ/trophozoite-fed human monocytes. Results may clarify aspects of cerebral malaria pathogenesis, since MMP-9, a metalloproteinase able to disrupt the basal lamina is possibly involved in generation of hallmarks of cerebral malaria, such as blood-brain barrier endothelium dysfunction, localized haemorrhages and extravasation of phagocytic cells and parasitized RBCs into brain tissues.

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IL-1beta production and MMP-9 enzyme activity (in cell supernatants) in human adherent monocytes unfed or fed with HZ, delipidized HZ or beta-haematin. Human adherent monocytes were unfed or fed with HZ, delipidized HZ (D-HZ) and beta-haematin. Panel A: IL-1beta production. After 3 h phagocytosis and a further incubation during 48 h, IL1-beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of four independent experiments). Data were analysed for significance by Student's t-test and differences between delipidized HZ or beta-haematin against unfed controls were not significant. Panel B: Gelatin zymography and densitometric quantification of MMP-9 enzyme activity. After 3 h phagocytosis and a further incubation during 48 h, cell supernatants were separated by PAGE and MMP-9 enzyme activity measured by gelatin zymography and densitometric quantification (see legend to Figure 2 for details). The 83-kDa negative band in the gel corresponds to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data (Panel A, panel B) were analysed for significance by Student's t-test. Significance of differences (column/lane numbers). HZ-fed(2) vs unfed(1)/D-HZ-(3)/beta-haematin(4)-fed monocytes, p < 0.01; unfed(1) vs D-HZ(2)/beta-haematin(4)-fed monocytes, n.s.
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Figure 3: IL-1beta production and MMP-9 enzyme activity (in cell supernatants) in human adherent monocytes unfed or fed with HZ, delipidized HZ or beta-haematin. Human adherent monocytes were unfed or fed with HZ, delipidized HZ (D-HZ) and beta-haematin. Panel A: IL-1beta production. After 3 h phagocytosis and a further incubation during 48 h, IL1-beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of four independent experiments). Data were analysed for significance by Student's t-test and differences between delipidized HZ or beta-haematin against unfed controls were not significant. Panel B: Gelatin zymography and densitometric quantification of MMP-9 enzyme activity. After 3 h phagocytosis and a further incubation during 48 h, cell supernatants were separated by PAGE and MMP-9 enzyme activity measured by gelatin zymography and densitometric quantification (see legend to Figure 2 for details). The 83-kDa negative band in the gel corresponds to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data (Panel A, panel B) were analysed for significance by Student's t-test. Significance of differences (column/lane numbers). HZ-fed(2) vs unfed(1)/D-HZ-(3)/beta-haematin(4)-fed monocytes, p < 0.01; unfed(1) vs D-HZ(2)/beta-haematin(4)-fed monocytes, n.s.

Mentions: Previous work has shown that PUFAs stably adherent to the crystalline poly-haem core of native HZ are transformed by non-enzymatic haem catalysis into a number of potent lipoperoxidation derivatives [5]. To ascertain whether lipids were involved in HZ-elicited activation of MMP-9, lipid-free beta-haematin (synthetic HZ) and delipidized native HZ were fed to adherent monocytes. After phagocytosis, monocytes were further incubated for 48 hours and cell supernatants analysed by ELISA for IL-1beta production and MMP-9 activity. Beta-haematin and delipidized HZ were unable to enhance IL-1beta production (Figure 3, panel A) and stimulate MMP-9 activity (Figure 3, panel B).


Phagocytosis of haemozoin (malarial pigment) enhances metalloproteinase-9 activity in human adherent monocytes: role of IL-1beta and 15-HETE.

Prato M, Gallo V, Giribaldi G, Arese P - Malar. J. (2008)

IL-1beta production and MMP-9 enzyme activity (in cell supernatants) in human adherent monocytes unfed or fed with HZ, delipidized HZ or beta-haematin. Human adherent monocytes were unfed or fed with HZ, delipidized HZ (D-HZ) and beta-haematin. Panel A: IL-1beta production. After 3 h phagocytosis and a further incubation during 48 h, IL1-beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of four independent experiments). Data were analysed for significance by Student's t-test and differences between delipidized HZ or beta-haematin against unfed controls were not significant. Panel B: Gelatin zymography and densitometric quantification of MMP-9 enzyme activity. After 3 h phagocytosis and a further incubation during 48 h, cell supernatants were separated by PAGE and MMP-9 enzyme activity measured by gelatin zymography and densitometric quantification (see legend to Figure 2 for details). The 83-kDa negative band in the gel corresponds to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data (Panel A, panel B) were analysed for significance by Student's t-test. Significance of differences (column/lane numbers). HZ-fed(2) vs unfed(1)/D-HZ-(3)/beta-haematin(4)-fed monocytes, p < 0.01; unfed(1) vs D-HZ(2)/beta-haematin(4)-fed monocytes, n.s.
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Figure 3: IL-1beta production and MMP-9 enzyme activity (in cell supernatants) in human adherent monocytes unfed or fed with HZ, delipidized HZ or beta-haematin. Human adherent monocytes were unfed or fed with HZ, delipidized HZ (D-HZ) and beta-haematin. Panel A: IL-1beta production. After 3 h phagocytosis and a further incubation during 48 h, IL1-beta levels were measured by ELISA in cell supernatants. Data are given as ng IL-1beta/ml supernatant (mean values ± SD of four independent experiments). Data were analysed for significance by Student's t-test and differences between delipidized HZ or beta-haematin against unfed controls were not significant. Panel B: Gelatin zymography and densitometric quantification of MMP-9 enzyme activity. After 3 h phagocytosis and a further incubation during 48 h, cell supernatants were separated by PAGE and MMP-9 enzyme activity measured by gelatin zymography and densitometric quantification (see legend to Figure 2 for details). The 83-kDa negative band in the gel corresponds to MMP-9 enzyme activity. Data are given as arbitrary densitometric units (mean values ± SD of four independent experiments). Data (Panel A, panel B) were analysed for significance by Student's t-test. Significance of differences (column/lane numbers). HZ-fed(2) vs unfed(1)/D-HZ-(3)/beta-haematin(4)-fed monocytes, p < 0.01; unfed(1) vs D-HZ(2)/beta-haematin(4)-fed monocytes, n.s.
Mentions: Previous work has shown that PUFAs stably adherent to the crystalline poly-haem core of native HZ are transformed by non-enzymatic haem catalysis into a number of potent lipoperoxidation derivatives [5]. To ascertain whether lipids were involved in HZ-elicited activation of MMP-9, lipid-free beta-haematin (synthetic HZ) and delipidized native HZ were fed to adherent monocytes. After phagocytosis, monocytes were further incubated for 48 hours and cell supernatants analysed by ELISA for IL-1beta production and MMP-9 activity. Beta-haematin and delipidized HZ were unable to enhance IL-1beta production (Figure 3, panel A) and stimulate MMP-9 activity (Figure 3, panel B).

Bottom Line: The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ) and trophozoites on production of IL-1beta and MMP-9 activity and expression.The second aim was to find out which component of HZ was responsible for the effects.Results may clarify aspects of cerebral malaria pathogenesis, since MMP-9, a metalloproteinase able to disrupt the basal lamina is possibly involved in generation of hallmarks of cerebral malaria, such as blood-brain barrier endothelium dysfunction, localized haemorrhages and extravasation of phagocytic cells and parasitized RBCs into brain tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino Medical School, Torino, Italy. paolo.arese@unito.it.

ABSTRACT

Background: It has been shown previously that human monocytes fed with haemozoin (HZ) or trophozoite-parasitized RBCs displayed increased matrix metalloproteinase-9 (MMP-9) enzyme activity and protein/mRNA expression and increased TNF production, and showed higher matrix invasion ability. The present study utilized the same experimental model to analyse the effect of phagocytosis of: HZ, delipidized HZ, beta-haematin (lipid-free synthetic HZ) and trophozoites on production of IL-1beta and MMP-9 activity and expression. The second aim was to find out which component of HZ was responsible for the effects.

Methods: Native HZ freshly isolated from Plasmodium falciparum (Palo Alto strain, Mycoplasma-free), delipidized HZ, beta-haematin (lipid-free synthetic HZ), trophozoites and control meals such as opsonized non-parasitized RBCs and inert latex particles, were fed to human monocytes. The production of IL-1beta by differently fed monocytes, in presence or absence of specific MMP-9 inhibitor or anti-hIL-1beta antibodies, was quantified in supernatants by ELISA. Expression of IL-1beta was analysed by quantitative real-time RT-PCR. MMP-9 activity and protein expression were quantified by gelatin zymography and Western blotting.

Results: Monocytes fed with HZ or trophozoite-parasitized RBCs generated increased amounts of IL-1beta and enhanced enzyme activity (in cell supernatants) and protein/mRNA expression (in cell lysates) of monocyte MMP-9. The latter appears to be causally related to enhanced IL-1beta production, as enhancement of both expression and enzyme activity were abrogated by anti-hIL-1beta Abs. Upregulation of IL-1beta and MMP-9 were absent in monocytes fed with beta-haematin or delipidized HZ, indicating a role for HZ-attached or HZ-generated lipid components. 15-HETE (15(S,R)-hydroxy-6,8,11,13-eicosatetraenoic acid) a potent lipoperoxidation derivative generated by HZ from arachidonic acid via haem-catalysis was identified as one mediator possibly responsible for increase of both IL-1beta production and MMP-9 activity.

Conclusion: Results indicate that specific lipoperoxide derivatives generated by HZ may play a role in modulating production of IL-1beta and MMP-9 expression and activity in HZ/trophozoite-fed human monocytes. Results may clarify aspects of cerebral malaria pathogenesis, since MMP-9, a metalloproteinase able to disrupt the basal lamina is possibly involved in generation of hallmarks of cerebral malaria, such as blood-brain barrier endothelium dysfunction, localized haemorrhages and extravasation of phagocytic cells and parasitized RBCs into brain tissues.

Show MeSH
Related in: MedlinePlus