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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: 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.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, and TNF-bp blocks, apoptosis in the endothelium of (A) intestine, lung, pericolic fat, and (B) thymus. C57BL/6 mice  were injected intraperitoneally with 90 μg of S. typhimurium LPS/25 g of  mouse body weight or diluent (PBS), and after 6 h were killed by hypercapnia asphyxiation. For studies using TNF-bp, animals were injected  with 75 μg of TNF-bp/25 g of mouse body weight or with diluent (PBS)  2 h before LPS. Tissue specimens were fixed overnight in 4% buffered  formaldehyde and apoptosis assessed as in Materials and Methods by  TUNEL assay (A) or a combination of TUNEL and immunohistochemical staining for the cell surface antigen CD31 (B). Nuclei of apoptotic  cells appear brown and granular, and in B are surrounded by a blue-black  perimeter. Normal nuclei in A stain blue and in B stain red due to hematoxylin and fast red counterstains, respectively. Original magnifications:  intestine ×400; lung, pericolic fat and thymus ×1,000. This experiment  represents one of three similar studies.
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Figure 1: LPS induces, and TNF-bp blocks, apoptosis in the endothelium of (A) intestine, lung, pericolic fat, and (B) thymus. C57BL/6 mice were injected intraperitoneally with 90 μg of S. typhimurium LPS/25 g of mouse body weight or diluent (PBS), and after 6 h were killed by hypercapnia asphyxiation. For studies using TNF-bp, animals were injected with 75 μg of TNF-bp/25 g of mouse body weight or with diluent (PBS) 2 h before LPS. Tissue specimens were fixed overnight in 4% buffered formaldehyde and apoptosis assessed as in Materials and Methods by TUNEL assay (A) or a combination of TUNEL and immunohistochemical staining for the cell surface antigen CD31 (B). Nuclei of apoptotic cells appear brown and granular, and in B are surrounded by a blue-black perimeter. Normal nuclei in A stain blue and in B stain red due to hematoxylin and fast red counterstains, respectively. Original magnifications: intestine ×400; lung, pericolic fat and thymus ×1,000. This experiment represents one of three similar studies.

Mentions: To explore whether endothelial cell apoptosis is associated with the LPS response, C57BL/6 mice were injected with 90 μg of LPS/25 g of mouse body weight and multiple tissues were evaluated for an apoptotic response using the TUNEL method. Fig. 1 shows that LPS induced an apoptotic response in microvascular endothelial cells of intestinal crypts, the lung, pericolic fat, and thymus. Crypts of the intestinal mucosa are comprised of a layer of columnar epithelial cells on the intestinal luminal surface and a central network of capillaries in the lamina propria. Intestinal crypts from sham injected animals demonstrated minimal apoptosis (Fig. 1 A, left). Apoptotic cells display an intense brown nuclear stain, whereas the nuclei of unaffected cells are visualized blue due to the hematoxylin counterstaining. LPS-injected animals, however, demonstrated diffuse endothelial apoptosis with little if any changes in the epithelial cell layer (Fig. 1 A, middle). This effect was maximal at 6 h and preceded the onset of apoptosis in the epithelial cells of the crypt, which became apparent after 8–10 h (data not shown). Similarly, the lungs of sham-treated animals displayed little apoptosis in either capillary endothelial cells or in tissue pneumocytes (Fig. 1 A, left). Substantial and selective apoptotic damage was detected, however, in the pulmonary microvascular endothelium in response to LPS injection by 6 h (Fig. 1 A, middle). In both these tissues, hematoxylin- and eosin-stained sections from LPS-treated animals revealed large numbers of endothelial cells with shrunken pyncnotic nuclei, many of which were fragmented (data not shown). These apoptotic cells appeared to be phagocytized by neighboring cells in some sections. Apoptotic damage to the endothelium of pericolic fat tissue was similarly detected by 6–8 h after LPS injection, while adipocytes and fibroblasts, seen on the periphery of Fig. 1 A, middle, were spared. This effect was also observed in mediastinal and subcutaneous fat tissue (data not shown). In all of these organs, the extent of endothelial, and the subsequent nonendothelial, tissue damage was dose-dependent, increasing from 60 to 175 μg of LPS/25 g of mouse body weight (data not shown).


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, and TNF-bp blocks, apoptosis in the endothelium of (A) intestine, lung, pericolic fat, and (B) thymus. C57BL/6 mice  were injected intraperitoneally with 90 μg of S. typhimurium LPS/25 g of  mouse body weight or diluent (PBS), and after 6 h were killed by hypercapnia asphyxiation. For studies using TNF-bp, animals were injected  with 75 μg of TNF-bp/25 g of mouse body weight or with diluent (PBS)  2 h before LPS. Tissue specimens were fixed overnight in 4% buffered  formaldehyde and apoptosis assessed as in Materials and Methods by  TUNEL assay (A) or a combination of TUNEL and immunohistochemical staining for the cell surface antigen CD31 (B). Nuclei of apoptotic  cells appear brown and granular, and in B are surrounded by a blue-black  perimeter. Normal nuclei in A stain blue and in B stain red due to hematoxylin and fast red counterstains, respectively. Original magnifications:  intestine ×400; lung, pericolic fat and thymus ×1,000. This experiment  represents one of three similar studies.
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Related In: Results  -  Collection

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Figure 1: LPS induces, and TNF-bp blocks, apoptosis in the endothelium of (A) intestine, lung, pericolic fat, and (B) thymus. C57BL/6 mice were injected intraperitoneally with 90 μg of S. typhimurium LPS/25 g of mouse body weight or diluent (PBS), and after 6 h were killed by hypercapnia asphyxiation. For studies using TNF-bp, animals were injected with 75 μg of TNF-bp/25 g of mouse body weight or with diluent (PBS) 2 h before LPS. Tissue specimens were fixed overnight in 4% buffered formaldehyde and apoptosis assessed as in Materials and Methods by TUNEL assay (A) or a combination of TUNEL and immunohistochemical staining for the cell surface antigen CD31 (B). Nuclei of apoptotic cells appear brown and granular, and in B are surrounded by a blue-black perimeter. Normal nuclei in A stain blue and in B stain red due to hematoxylin and fast red counterstains, respectively. Original magnifications: intestine ×400; lung, pericolic fat and thymus ×1,000. This experiment represents one of three similar studies.
Mentions: To explore whether endothelial cell apoptosis is associated with the LPS response, C57BL/6 mice were injected with 90 μg of LPS/25 g of mouse body weight and multiple tissues were evaluated for an apoptotic response using the TUNEL method. Fig. 1 shows that LPS induced an apoptotic response in microvascular endothelial cells of intestinal crypts, the lung, pericolic fat, and thymus. Crypts of the intestinal mucosa are comprised of a layer of columnar epithelial cells on the intestinal luminal surface and a central network of capillaries in the lamina propria. Intestinal crypts from sham injected animals demonstrated minimal apoptosis (Fig. 1 A, left). Apoptotic cells display an intense brown nuclear stain, whereas the nuclei of unaffected cells are visualized blue due to the hematoxylin counterstaining. LPS-injected animals, however, demonstrated diffuse endothelial apoptosis with little if any changes in the epithelial cell layer (Fig. 1 A, middle). This effect was maximal at 6 h and preceded the onset of apoptosis in the epithelial cells of the crypt, which became apparent after 8–10 h (data not shown). Similarly, the lungs of sham-treated animals displayed little apoptosis in either capillary endothelial cells or in tissue pneumocytes (Fig. 1 A, left). Substantial and selective apoptotic damage was detected, however, in the pulmonary microvascular endothelium in response to LPS injection by 6 h (Fig. 1 A, middle). In both these tissues, hematoxylin- and eosin-stained sections from LPS-treated animals revealed large numbers of endothelial cells with shrunken pyncnotic nuclei, many of which were fragmented (data not shown). These apoptotic cells appeared to be phagocytized by neighboring cells in some sections. Apoptotic damage to the endothelium of pericolic fat tissue was similarly detected by 6–8 h after LPS injection, while adipocytes and fibroblasts, seen on the periphery of Fig. 1 A, middle, were spared. This effect was also observed in mediastinal and subcutaneous fat tissue (data not shown). In all of these organs, the extent of endothelial, and the subsequent nonendothelial, tissue damage was dose-dependent, increasing from 60 to 175 μg of LPS/25 g of mouse body weight (data not shown).

Bottom Line: 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.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