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Kupffer cells promote lead nitrate-induced hepatocyte apoptosis via oxidative stress.

Pagliara P, Carlà EC, Caforio S, Chionna A, Massa S, Abbro L, Dini L - Comp Hepatol (2003)

Bottom Line: RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate.In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3.CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Science and Biological and Environmental Technologies, University of Lecce, via per Monteroni, Lecce, Italy. luciana.dini@unile.it

ABSTRACT
BACKGROUND: Apoptosis and its modulation are crucial factors for the maintenance of liver health, allowing hepatocytes to die without provoking a potential harmful inflammatory response through a tightly controlled and regulated process. Since Kupffer cells play a key role in the maintenance of liver function, the aim of this study was to verify whether Kupffer cells are involved in the induction of liver apoptosis after i.v. injection of Pb(NO3)2 likely by secretion mechanisms. RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate. In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3. However, incubation of isolated hepatocytes and Kupffer cells and HepG2 cells with Pb(NO3)2 for 24 hours induced necrotic but not apoptotic cells. Apoptosis of hepatocytes and HepG2 cells was observed only after the addition of conditioned medium obtained from Kupffer cells cultured for 24 hours with Pb(NO3)2, thus indicating the secretion of soluble mediators of apoptosis by Kupffer cells. Apoptosis in the HepG2 cells was observed upon 24-hours incubation of HepG2 cells with 1 mM buthionine sulfoximine, a glutathione depleting agent, thus showing that there is an oxidative apoptogenic pathway in HepG2 cells. CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.

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Light micrographs of rat liver sections, (a, b) haematoxylin eosin staining and (c, d) apoptotic detection by TUNEL staining, from (a, c) animals 5 days after a single Pb(NO3)2 injection and animals 5 (b) and 3 (d) days after Pb(NO3)2 injection 24 hours GdCl3pre-treated. (a) Apoptotic hepatocytes (arrows) show round shape, condensation of chromatin and cell shrinkage. One fragmented apoptotic hepatocytes (arrowhead). (b) The GdCl3 pre-treatment reduces the hepatocytes apoptosis. In (d), the number of TUNEL-positive nuclei (arrows) in hepatic parenchyma is much lower than 5 days after a single Pb(NO3)2 injection (c, arrows). Bar = 10 μm.
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Figure 1: Light micrographs of rat liver sections, (a, b) haematoxylin eosin staining and (c, d) apoptotic detection by TUNEL staining, from (a, c) animals 5 days after a single Pb(NO3)2 injection and animals 5 (b) and 3 (d) days after Pb(NO3)2 injection 24 hours GdCl3pre-treated. (a) Apoptotic hepatocytes (arrows) show round shape, condensation of chromatin and cell shrinkage. One fragmented apoptotic hepatocytes (arrowhead). (b) The GdCl3 pre-treatment reduces the hepatocytes apoptosis. In (d), the number of TUNEL-positive nuclei (arrows) in hepatic parenchyma is much lower than 5 days after a single Pb(NO3)2 injection (c, arrows). Bar = 10 μm.

Mentions: Pb(NO3)2 administration led to time-related modifications of liver structure (hyperplasia-apoptosis), which were totally or partially abolished by pre-treatment with GdCl3 (i.v. injected 24 hours but not 2 or 4 hours before Pb(NO3)2) (Fig. 1). GdCl3 administered the same day as Pb(NO3)2, reduced the hepatocyte apoptosis only moderately, but, when administered 24 hours before Pb(NO3)2, the apoptotic index was cut by half. Morphological data are confirmed by TUNEL assay; very few TUNEL positive cells were observed in livers of animals injected with 24 hours GdCl3 pre-treatment to Pb(NO3)2 administration (Fig. 1d). A single GdCl3injection induced low rate apoptosis as well as same modifications of liver structure. In particular, an influx of monocytes, a decreased number of Kupffer cells and a minimal uptake activity for the remaining liver macrophages was observed. Liver repopulation of Kupffer cells and re-establishment of active phagocytosis was observed two days later. The time-course of GdCl3-induced Kupffer cell depletion and repopulation, (monitored by measuring colloidal carbon uptake by liver macrophages), is shown in Figure 2.


Kupffer cells promote lead nitrate-induced hepatocyte apoptosis via oxidative stress.

Pagliara P, Carlà EC, Caforio S, Chionna A, Massa S, Abbro L, Dini L - Comp Hepatol (2003)

Light micrographs of rat liver sections, (a, b) haematoxylin eosin staining and (c, d) apoptotic detection by TUNEL staining, from (a, c) animals 5 days after a single Pb(NO3)2 injection and animals 5 (b) and 3 (d) days after Pb(NO3)2 injection 24 hours GdCl3pre-treated. (a) Apoptotic hepatocytes (arrows) show round shape, condensation of chromatin and cell shrinkage. One fragmented apoptotic hepatocytes (arrowhead). (b) The GdCl3 pre-treatment reduces the hepatocytes apoptosis. In (d), the number of TUNEL-positive nuclei (arrows) in hepatic parenchyma is much lower than 5 days after a single Pb(NO3)2 injection (c, arrows). Bar = 10 μm.
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Related In: Results  -  Collection

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Figure 1: Light micrographs of rat liver sections, (a, b) haematoxylin eosin staining and (c, d) apoptotic detection by TUNEL staining, from (a, c) animals 5 days after a single Pb(NO3)2 injection and animals 5 (b) and 3 (d) days after Pb(NO3)2 injection 24 hours GdCl3pre-treated. (a) Apoptotic hepatocytes (arrows) show round shape, condensation of chromatin and cell shrinkage. One fragmented apoptotic hepatocytes (arrowhead). (b) The GdCl3 pre-treatment reduces the hepatocytes apoptosis. In (d), the number of TUNEL-positive nuclei (arrows) in hepatic parenchyma is much lower than 5 days after a single Pb(NO3)2 injection (c, arrows). Bar = 10 μm.
Mentions: Pb(NO3)2 administration led to time-related modifications of liver structure (hyperplasia-apoptosis), which were totally or partially abolished by pre-treatment with GdCl3 (i.v. injected 24 hours but not 2 or 4 hours before Pb(NO3)2) (Fig. 1). GdCl3 administered the same day as Pb(NO3)2, reduced the hepatocyte apoptosis only moderately, but, when administered 24 hours before Pb(NO3)2, the apoptotic index was cut by half. Morphological data are confirmed by TUNEL assay; very few TUNEL positive cells were observed in livers of animals injected with 24 hours GdCl3 pre-treatment to Pb(NO3)2 administration (Fig. 1d). A single GdCl3injection induced low rate apoptosis as well as same modifications of liver structure. In particular, an influx of monocytes, a decreased number of Kupffer cells and a minimal uptake activity for the remaining liver macrophages was observed. Liver repopulation of Kupffer cells and re-establishment of active phagocytosis was observed two days later. The time-course of GdCl3-induced Kupffer cell depletion and repopulation, (monitored by measuring colloidal carbon uptake by liver macrophages), is shown in Figure 2.

Bottom Line: RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate.In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3.CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Science and Biological and Environmental Technologies, University of Lecce, via per Monteroni, Lecce, Italy. luciana.dini@unile.it

ABSTRACT
BACKGROUND: Apoptosis and its modulation are crucial factors for the maintenance of liver health, allowing hepatocytes to die without provoking a potential harmful inflammatory response through a tightly controlled and regulated process. Since Kupffer cells play a key role in the maintenance of liver function, the aim of this study was to verify whether Kupffer cells are involved in the induction of liver apoptosis after i.v. injection of Pb(NO3)2 likely by secretion mechanisms. RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate. In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3. However, incubation of isolated hepatocytes and Kupffer cells and HepG2 cells with Pb(NO3)2 for 24 hours induced necrotic but not apoptotic cells. Apoptosis of hepatocytes and HepG2 cells was observed only after the addition of conditioned medium obtained from Kupffer cells cultured for 24 hours with Pb(NO3)2, thus indicating the secretion of soluble mediators of apoptosis by Kupffer cells. Apoptosis in the HepG2 cells was observed upon 24-hours incubation of HepG2 cells with 1 mM buthionine sulfoximine, a glutathione depleting agent, thus showing that there is an oxidative apoptogenic pathway in HepG2 cells. CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.

No MeSH data available.


Related in: MedlinePlus