Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation

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.

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.

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.

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).

Figure 3

TNF-α induces ceramide generation in intestinal mucosa. (A) Time course of the effect of 25 μg of recombinant human TNF-α/ 25g mouse; (B) dose response at 2 h. These studies were performed as in Fig. 2 except C₅₇BL/6 mice were injected with TNF-α retro-orbitally. The data (mean ± SEM) represent triplicate determinations from two mice per point from one representative of three experiments for A and one representative of four experiments for B.

Figure 4

TNF-bp blocks LPS-induced ceramide generation. For these studies, animals were injected with TNF-bp and LPS as described in Fig. 1, and ceramide levels determined as in Fig. 2. These data (mean ± SEM) represent triplicate determinations from two mice per point from two experiments.

Figure 5

Acid SMase knockout mice are defective in LPS-induced death. Actuarial (Kaplan-Meier) survival curves of wild-type and ASMase knockout mice injected intraperitoneally with 175 μg of LPS/25g mouse. The number in parenthesis indicates the number of animals in each group.

Figure 6

Basic FGF blocks LPS-induced endothelial apoptosis and animal death. (A) C₅₇BL/ 6 mice injected intravenously with 800 ng bFGF 30 min before, and 5 min, 1 and 2 h after, an intraperitoneal injection of 175 μg of LPS/25g mouse. Endothelial apoptosis was assessed as in Fig. 1 by TUNEL assay. (B) Actuarial (Kaplan-Meier) survival curves of C₅₇BL/6 treated as in A. The number in parenthesis indicates the number of animals in each group.

Figure 6

Basic FGF blocks LPS-induced endothelial apoptosis and animal death. (A) C₅₇BL/ 6 mice injected intravenously with 800 ng bFGF 30 min before, and 5 min, 1 and 2 h after, an intraperitoneal injection of 175 μg of LPS/25g mouse. Endothelial apoptosis was assessed as in Fig. 1 by TUNEL assay. (B) Actuarial (Kaplan-Meier) survival curves of C₅₇BL/6 treated as in A. The number in parenthesis indicates the number of animals in each group.

Figure 7

Proposed schema for progression of the endotoxic response. LPS, released by gram negative bacteria, interacts with inflammatory cells leading to generation of TNF-α and other cytokines. TNF-α, acting upon endothelium, stimulates sphingomyelin hydrolysis to ceramide via an ASMase. Apoptosis of the endothelium ensues, which can be blocked by bFGF via inhibition of ceramide generation. We further propose that endothelial apoptosis results in generalized microvascular dysfunction sufficient to compromise the circulation to major organs, leading to nonendothelial tissue damage, circulatory collapse, and death.