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Irradiation leads to apoptosis of Kupffer cells by a Hsp27-dependant pathway followed by release of TNF-alpha.

Tello K, Christiansen H, Gürleyen H, Dudas J, Rave-Fränk M, Hess CF, Ramadori G, Saile B - Radiat Environ Biophys (2008)

Bottom Line: The pathomechanisms underlying this phenomenon, however, remained to be elucidated.However, normalization of the TNF-alpha concentration with respect to cell number revealed that TNF-alpha concentration per attached cell remained constant during the observation period.We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075, Göttingen, Germany.

ABSTRACT
In a previous publication, we were able to show that irradiation of Kupffer cells, the liver resident macrophages, leads to an increased TNF-alpha concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of Kupffer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-alpha concentration in cell lysates of Kupffer cells attached to the culture plate decreased. However, normalization of the TNF-alpha concentration with respect to cell number revealed that TNF-alpha concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated Kupffer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in Kupffer cells was shown to prevent the effect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-alpha concentration in the Kupffer cell medium. We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of Kupffer cells leads to an increase of TNF-alpha concentration in the culture medium which may be due to cell death rather than active release or synthesis.

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a Survival of Kupffer cells irradiated with 2 and 8 Gy (Trypan blue exclusion). The values presented are means of seven independent Kupffer cell isolations. b Apoptosis of Kupffer cells at different time points after irradiation with 8 Gy. For detection of apoptosis the AnnexinV/PI method was used, where cells that bind AnnexinV but do not incorporate propidiumiodide are regarded as apoptotic. Necrotic cells (Annexin V positive and propidiumiodide positive) were always less than 3%. Values presented are means ± SD of seven independent Kupffer cell isolations. Level of significance of irradiated Kupffer cells compared to the respective sham-irradiated cultures: *P < 0.05; **P < 0.01. c Apoptosis detection using the TUNEL method. Panels presented show Kupffer cells 6 and 48 h after irradiation at 8 Gy or sham irradiation. Arrows show examples for apoptotic cells. Consistent data could be found in seven independent experiments derived from seven different Kupffer cell isolations
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Fig1: a Survival of Kupffer cells irradiated with 2 and 8 Gy (Trypan blue exclusion). The values presented are means of seven independent Kupffer cell isolations. b Apoptosis of Kupffer cells at different time points after irradiation with 8 Gy. For detection of apoptosis the AnnexinV/PI method was used, where cells that bind AnnexinV but do not incorporate propidiumiodide are regarded as apoptotic. Necrotic cells (Annexin V positive and propidiumiodide positive) were always less than 3%. Values presented are means ± SD of seven independent Kupffer cell isolations. Level of significance of irradiated Kupffer cells compared to the respective sham-irradiated cultures: *P < 0.05; **P < 0.01. c Apoptosis detection using the TUNEL method. Panels presented show Kupffer cells 6 and 48 h after irradiation at 8 Gy or sham irradiation. Arrows show examples for apoptotic cells. Consistent data could be found in seven independent experiments derived from seven different Kupffer cell isolations

Mentions: Single-dose administration of 8 Gy leads to a substantial decrease of living cells, beginning 4 h after irradiation and even more enhanced at 48 h (Fig. 1a). In contrast, a single dose of 2 Gy did not lead to statistically significant alteration of the portion of living cells when compared to sham-irradiated Kupffer cells within a time window of 48 h.Fig. 1


Irradiation leads to apoptosis of Kupffer cells by a Hsp27-dependant pathway followed by release of TNF-alpha.

Tello K, Christiansen H, Gürleyen H, Dudas J, Rave-Fränk M, Hess CF, Ramadori G, Saile B - Radiat Environ Biophys (2008)

a Survival of Kupffer cells irradiated with 2 and 8 Gy (Trypan blue exclusion). The values presented are means of seven independent Kupffer cell isolations. b Apoptosis of Kupffer cells at different time points after irradiation with 8 Gy. For detection of apoptosis the AnnexinV/PI method was used, where cells that bind AnnexinV but do not incorporate propidiumiodide are regarded as apoptotic. Necrotic cells (Annexin V positive and propidiumiodide positive) were always less than 3%. Values presented are means ± SD of seven independent Kupffer cell isolations. Level of significance of irradiated Kupffer cells compared to the respective sham-irradiated cultures: *P < 0.05; **P < 0.01. c Apoptosis detection using the TUNEL method. Panels presented show Kupffer cells 6 and 48 h after irradiation at 8 Gy or sham irradiation. Arrows show examples for apoptotic cells. Consistent data could be found in seven independent experiments derived from seven different Kupffer cell isolations
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Related In: Results  -  Collection

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Fig1: a Survival of Kupffer cells irradiated with 2 and 8 Gy (Trypan blue exclusion). The values presented are means of seven independent Kupffer cell isolations. b Apoptosis of Kupffer cells at different time points after irradiation with 8 Gy. For detection of apoptosis the AnnexinV/PI method was used, where cells that bind AnnexinV but do not incorporate propidiumiodide are regarded as apoptotic. Necrotic cells (Annexin V positive and propidiumiodide positive) were always less than 3%. Values presented are means ± SD of seven independent Kupffer cell isolations. Level of significance of irradiated Kupffer cells compared to the respective sham-irradiated cultures: *P < 0.05; **P < 0.01. c Apoptosis detection using the TUNEL method. Panels presented show Kupffer cells 6 and 48 h after irradiation at 8 Gy or sham irradiation. Arrows show examples for apoptotic cells. Consistent data could be found in seven independent experiments derived from seven different Kupffer cell isolations
Mentions: Single-dose administration of 8 Gy leads to a substantial decrease of living cells, beginning 4 h after irradiation and even more enhanced at 48 h (Fig. 1a). In contrast, a single dose of 2 Gy did not lead to statistically significant alteration of the portion of living cells when compared to sham-irradiated Kupffer cells within a time window of 48 h.Fig. 1

Bottom Line: The pathomechanisms underlying this phenomenon, however, remained to be elucidated.However, normalization of the TNF-alpha concentration with respect to cell number revealed that TNF-alpha concentration per attached cell remained constant during the observation period.We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Robert-Koch-Strasse 40, 37075, Göttingen, Germany.

ABSTRACT
In a previous publication, we were able to show that irradiation of Kupffer cells, the liver resident macrophages, leads to an increased TNF-alpha concentration in the culture medium. The pathomechanisms underlying this phenomenon, however, remained to be elucidated. Here, we show that following irradiation of Kupffer cells, the apoptosis rate increased drastically within 48 h. At the same time, the total TNF-alpha concentration in cell lysates of Kupffer cells attached to the culture plate decreased. However, normalization of the TNF-alpha concentration with respect to cell number revealed that TNF-alpha concentration per attached cell remained constant during the observation period. Western blot analysis showed that heat shock protein 27 (Hsp27) is strongly downregulated and bax is upregulated in irradiated Kupffer cells as compared to sham-irradiated cells. Overexpression of Hsp27 in Kupffer cells was shown to prevent the effect of irradiation on bax expression, apoptosis and, at the same time, on increase of TNF-alpha concentration in the Kupffer cell medium. We conclude that irradiation of Kupffer cells leads to apoptosis because of downregulation of Hsp27 and consecutive upregulation of bax expression. Furthermore, we suggest that apoptosis of Kupffer cells leads to an increase of TNF-alpha concentration in the culture medium which may be due to cell death rather than active release or synthesis.

Show MeSH
Related in: MedlinePlus