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Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK, Schuchman EH, Fuks Z, Kolesnick R - J. Exp. Med. (1997)

Bottom Line: TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses.Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha.These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

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LPS induces rapid ceramide generation in the mucosa of the  intestine. These studies were performed as in Fig. 1 except mice were  killed at the indicated times. The intestinal mucosa was dissected as described in Materials and Methods. Mucosal specimens were homogenized  in 8 vol (vol/vol) of ice-cold PBS and lipids were extracted with 2 ml of  chloroform:methanol (2:1, vol/vol)/400 μl of homogenate. After mild alkaline hydrolysis to remove glycerophospholipids, ceramide was quantified using E. coli diacylglycerol kinase (Calbiochem Novabiochem) as described (27), and results normalized for protein content. The data (mean  ± SEM) represent triplicate determinations from two mice per point  from two experiments for time course (A), and from one representative of  two experiments for dose dependence (B).
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Figure 2: LPS induces rapid ceramide generation in the mucosa of the intestine. These studies were performed as in Fig. 1 except mice were killed at the indicated times. The intestinal mucosa was dissected as described in Materials and Methods. Mucosal specimens were homogenized in 8 vol (vol/vol) of ice-cold PBS and lipids were extracted with 2 ml of chloroform:methanol (2:1, vol/vol)/400 μl of homogenate. After mild alkaline hydrolysis to remove glycerophospholipids, ceramide was quantified using E. coli diacylglycerol kinase (Calbiochem Novabiochem) as described (27), and results normalized for protein content. The data (mean ± SEM) represent triplicate determinations from two mice per point from two experiments for time course (A), and from one representative of two experiments for dose dependence (B).

Mentions: To determine whether ceramide generation plays a role in LPS-induced apoptosis, C57BL/6 mice were treated with 175 μg of LPS/25g mouse and at various periods of time thereafter, the intestinal mucosa was dissected away from the muscularis layer. Ceramide content of the intestinal mucosa significantly increased from a basal level of 1,200 pmol/mg tissue by 1 h after LPS injection and peaked at twofold by 2 h (P <0.001 vs. control; Fig. 2 A). As little as 60 μg/25g mouse was effective and a maximal effect occurred with 175 μg/25g mouse (Fig. 2 B). Similar ceramide elevation was detected in the lung of C57BL/6 mice within the first hour after LPS injection (n = 3; data not shown). In contrast, the level of the lipid second messenger 1,2-diacylglycerol was not elevated (data not shown). These studies demonstrate that ceramide generation precedes the apoptotic response.


Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK, Schuchman EH, Fuks Z, Kolesnick R - J. Exp. Med. (1997)

LPS induces rapid ceramide generation in the mucosa of the  intestine. These studies were performed as in Fig. 1 except mice were  killed at the indicated times. The intestinal mucosa was dissected as described in Materials and Methods. Mucosal specimens were homogenized  in 8 vol (vol/vol) of ice-cold PBS and lipids were extracted with 2 ml of  chloroform:methanol (2:1, vol/vol)/400 μl of homogenate. After mild alkaline hydrolysis to remove glycerophospholipids, ceramide was quantified using E. coli diacylglycerol kinase (Calbiochem Novabiochem) as described (27), and results normalized for protein content. The data (mean  ± SEM) represent triplicate determinations from two mice per point  from two experiments for time course (A), and from one representative of  two experiments for dose dependence (B).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199151&req=5

Figure 2: LPS induces rapid ceramide generation in the mucosa of the intestine. These studies were performed as in Fig. 1 except mice were killed at the indicated times. The intestinal mucosa was dissected as described in Materials and Methods. Mucosal specimens were homogenized in 8 vol (vol/vol) of ice-cold PBS and lipids were extracted with 2 ml of chloroform:methanol (2:1, vol/vol)/400 μl of homogenate. After mild alkaline hydrolysis to remove glycerophospholipids, ceramide was quantified using E. coli diacylglycerol kinase (Calbiochem Novabiochem) as described (27), and results normalized for protein content. The data (mean ± SEM) represent triplicate determinations from two mice per point from two experiments for time course (A), and from one representative of two experiments for dose dependence (B).
Mentions: To determine whether ceramide generation plays a role in LPS-induced apoptosis, C57BL/6 mice were treated with 175 μg of LPS/25g mouse and at various periods of time thereafter, the intestinal mucosa was dissected away from the muscularis layer. Ceramide content of the intestinal mucosa significantly increased from a basal level of 1,200 pmol/mg tissue by 1 h after LPS injection and peaked at twofold by 2 h (P <0.001 vs. control; Fig. 2 A). As little as 60 μg/25g mouse was effective and a maximal effect occurred with 175 μg/25g mouse (Fig. 2 B). Similar ceramide elevation was detected in the lung of C57BL/6 mice within the first hour after LPS injection (n = 3; data not shown). In contrast, the level of the lipid second messenger 1,2-diacylglycerol was not elevated (data not shown). These studies demonstrate that ceramide generation precedes the apoptotic response.

Bottom Line: TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses.Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha.These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

Show MeSH
Related in: MedlinePlus