By
From the * Laboratory of Signal Transduction, the Department of Radiation Oncology, the § Department of Pathology, and the
Department of Surgery, Memorial Sloan Kettering Cancer Center,
New York 10021; the ¶ Department of Pharmacology, Inflammation Research, Amgen Inc., Boulder,
Colorado 80301-2546; and the ** Department of Human Genetics, Mount Sinai School of Medicine,
New York 10029
The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ
damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)- 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-
, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-
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-
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-
. 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.
Endotoxic shock is a potentially lethal complication of
systemic infection by gram-negative bacteria (1, 2).
The toxin responsible for the induction of endotoxic shock
is the glycolipid LPS, the only lipid present in the outer
membrane of gram-negative bacteria. Release of LPS into
the circulation activates a series of tissue responses that in
their most severe forms lead to septic shock and death. Major events in the pathogenesis of the LPS syndrome include
neutrophil, monocyte, and macrophage inflammatory responses, intravascular coagulopathy resulting from activation of plasma complement and clotting cascades, endothelial cell damage, and hypotension. Death of patients results
from extensive tissue injury, multiple organ failure, and circulatory collapse.
Although a number of cytokines, including IL-1 Although TNF- Signaling through the sphingomyelin pathway is mediated via generation of ceramide, which acts as a second
messenger in stimulating a variety of cellular functions (for
review see references 17). Receptors distinct as CD28,
CD95, and the TNF- Studies on the involvement of the sphingomyelin signaling system in apoptosis revealed that several cytokines and
environmental stresses, including TNF- Definitive evidence for role of ASMase and ceramide in
signaling one form of stress-induced apoptosis was derived
from studies using genetic models of ASMase deficiency.
Santana et al. (29) reported that lymphoblasts from patients
with Niemann-Pick disease (NPD),1 an inherited deficiency
of ASMase, manifested defects in ceramide generation and
the apoptotic response to ionizing radiation. These abnormalities were reversible upon restoration of ASMase activity by retroviral transfer of human ASMase cDNA. Furthermore, ASMase knockout mice failed to generate
ceramide and develop typical apoptotic lesions in the pulmonary endothelium after exposure to total body irradiation. The apoptotic response in the thymus, however, was
preserved. The exact opposite occurred in irradiated p53
knockout mice. Whereas the thymus of the p53 knockout
mouse was protected against radiation-induced apoptosis,
the lung endothelium was not. Differences were observed
in other tissues as well. While these studies demonstrated
that radiation is capable of activating two apparently distinct and independent signaling mechanisms for induction
of apoptosis, they also suggested a specific sensitivity of endothelial cells towards the ASMase-mediated signaling system for initiating apoptosis in response to stress.
Since both TNF- LPS Treatment.
Using a 26-gauge needle, C57BL/6 mice were
injected intraperitoneally with LPS (Salmonella typhimurium; Westphal purified, Difco Laboratories, MI) resuspended in sterile water. For TNF-, IL-6,
and IL-8 are released by LPS-activated inflammatory cells
during the onset of the endotoxic response (3), mounting
evidence points to TNF-
as a primary mediator of this
event (4). Not only are substantial quantities of TNF-
rapidly released into the circulation, but intravenous injection of TNF-
produces a systemic response very similar to
LPS. Furthermore, approaches to interfere with TNF action, such as using neutralizing antibodies (4) or TNF
binding proteins (TNF-bps), abrogate experimental endotoxic shock (7). Perhaps the most compelling evidence
for a role for TNF-
is the attenuation of endotoxic shock
observed in mice lacking the 55-kD TNF receptor (12, 13).
was originally defined as a cytokine
capable of inducing necrosis of tumors in vivo, recent studies suggest that in most instances TNF-
initiates an apoptotic form of cell death. In this regard, numerous studies
have linked activation of the sphingomyelin pathway to the
induction of apoptosis by TNF-
. The sphingomyelin
pathway is an ubiquitous, evolutionarily conserved signaling system analogous to the cAMP and phosphoinositide pathways. Sphingomyelin (N-acylsphingosin-1-phosphocholine) is a phospholipid preferentially concentrated in the
plasma membrane of mammalian cells (14). Sphingomyelin
catabolism occurs via the action of sphingomyelin-specific
forms of phospholipase C, termed sphingomyelinases, which
hydrolyze the phosphodiester bond of sphingomyelin,
yielding ceramide and phosphorylcholine. Several forms of
sphingomyelinase exist, distinguished by their pH optima (15). Human and murine acid sphingomyelinase (ASMase;
pH optimum 4.5-5.0) have been cloned and determined to
be the products of a conserved gene, while Mg2+-dependent or -independent neutral SMases (pH optimum 7.4)
have yet to be molecularly characterized. ASMase knockout mice retain NSMase activity, indicating that the neutral
forms are products of a distinct gene or genes (16).
, IL-1
, progesterone,
-interferon,
and glucocorticoid receptors signal via the sphingomyelin pathway after ligand binding. Thus ceramide signals pleiotropic cellular functions, including proliferation of fibroblasts, differentiation of promyelocytes, inhibition of the
respiratory burst in human neutrophils, survival of T9
glioma cells, and apoptosis, to list a few.
(20), CD95/
Fas/APO-1 (23), ionizing radiation, ultraviolet-C, heat,
and oxidative stress (26) induce rapid ceramide generation while effecting an apoptotic response. Furthermore,
cell-permeable ceramide analogues, but not analogues of
other lipid second messengers, mimicked the effect of cytokines and stress to induce apoptosis. Ceramide action was
stereospecific, as analogues of the naturally occurring dihydroceramide, failed to initiate the apoptotic program. These
studies suggested, but did not provide conclusive evidence,
that ceramide mediates cytokine- and stress-induced apoptosis.
and endothelial cell damage are critically involved in the pathogenesis of the endotoxic syndrome, we explored whether ceramide-mediated endothelial cell apoptosis plays a role in the LPS-induced response
in vivo. Genetic and pharmacologic manipulations allowed
for molecular ordering of the early and critical events in the
progression of this syndrome. The data show that injection
of LPS into C57BL/6 mice resulted in a disseminated form
of microvascular endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggested that
this cascade plays a mandatory role in the evolution of LPS-induced death.
and TNF-bp injections, mice were first anesthetized
with pentobarbital (50 mg/kg) intraperitoneally. After obtaining
adequate anesthesia, recombinant human TNF-
or TNF-bp (Amgen, Boulder, CO) was injected intravenously with a 28-gauge
needle via a retro-orbital approach. Sham-injected animals received diluent. For studies measuring survival, animals were monitored for up to 2 wk. Survival as the end point in these experiments was calculated from the time of treatment using the
product limit Kaplan-Meier method (30). Calculations of the
dose leading to 50% lethality (LD50) at a given time after LPS
treatment was performed using probit analysis. For studies evaluating histology or tissue ceramide content, mice were killed by
hypercapnia asphyxiation.
Lipid Studies. For studies measuring tissue ceramide levels, abdominal contents of killed animals were immediately exposed through a midline incision, and the gastric pylorus was identified. The duodenum was transected and the proximal 3-4 cm of small intestine were excised and placed on ice. Using a Nikon SMZ-2B dissecting microscope set at 10× magnification, the antimesenteric border of the bowel was incised, exposing the mucosal surface of the bowel. The bowel was irrigated with cold PBS and the mucosa bluntly dissected from the underlying muscularis propria with curved tissue forceps. Mucosa were homogenized in 8 vol (vol/vol) of ice-cold PBS. Homogenate (0.6 ml) was transferred to 16 × 100-mm glass tubes and lipids were extracted with 3 ml of chloroform/methanol (2:1 vol/vol). After mild alkaline hydrolysis to remove glycerophospholipids ceramide was quantified using E. coli diacylglycerol kinase (Calbiochem Novabiochem, La Jolla, CA) as described (27).
Apoptosis.
Apoptosis in vivo was assessed by the DNA terminal transferase nick-end translation method (also termed the
TUNEL assay), as described (31). In brief, tissue specimens were
fixed overnight in 4% buffered formaldehyde and embedded in
paraffin blocks. 5-µm-thick tissue sections, adherent to polylysine-treated slides, were deparaffinized by heating at 90°C for 10 min and then at 60°C for 5 min. Tissue-mounted slides were first
washed with 90% and then 80% ethanol (3 min each) and rehydrated. The slides were incubated in 10 mM Tris-HCl, pH 8, for
5 min, digested with 0.1% pepsin, rinsed in distilled water, and
treated with 3% H2O2 in PBS for 5 min at 22°C to inactivate endogenous peroxidase. After three washes in PBS, the slides were incubated for 15 min at 22°C in buffer (140 mM Na-cacodylate, pH 7.2, 30 mM Trizma base, 1 mM CoCl2) and then for 30 min
at 37°C in reaction mixture (0.2 U/µl terminal deoxynucleotidyl
transferase, 2 nM biotin-11-dUTP, 100 mM Na-cacodylate, pH
7.0, 0.1 mM DTT, 0.05 mg/ml bovine serum albumin, and 2.5 mM CoCl2). The reaction was stopped by transferring the slices
to a bath of 300 mM NaCl, 30 mM Na citrate for 15 min at
22°C. The slides were washed in PBS, blocked with 2% human
serum albumin in PBS for 10 min, rewashed, and then incubated
with avidin-biotin peroxidase complexes. After 30 min at 22°C,
cells were stained with the chromogen 3,3diamonobenzidine tetrachloride and counterstained with hematoxylin. Nuclei of apoptotic cells appear brown and granular, while normal nuclei stain blue.
Serum TNF- Levels.
Blood was obtained from anesthetized
mice through an abdominal incision by aspiration from the inferior vena cava using a 28-gauge needle (Becton Dickinson, Rutherford, NJ). Serum TNF-
levels were measured by ELISA according to the manufacturer's instructions (Biosource International,
Camarillo, CA).
Statistical Analysis. Statistical analysis were performed by Student's t test and Chi Square test. Differences in product limit Kaplan Meier survival curves were evaluated by the Mantel log-rank test for censored data (32).
Initial studies examined the time course and dose dependence of LPS-induced death of C57BL/6 mice. For these studies, S. typhimurium LPS or diluent were injected intraperitoneally. Death was detected as early as 16 h after a maximal dose of LPS (270 µg/25g mouse) and all of the mice were dead after 48 h. As little as 60 µg of LPS/25g mouse was effective and the LD50 was ~90 µg of LPS/25g mouse.
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).
Apoptosis was also observed in thymic tissue by 6 h after LPS injection. Apoptotic cells, as assessed by the TUNEL assay, appeared in the thymus as discrete foci manifesting a configuration reminiscent of a vascular formation (data not shown). However, the dense packing of cells within this tissue precluded histologic and morphologic identification of the apoptotic cells as endothelium. To determine whether these apoptotic cells were of endothelial origin, we developed a double staining technique. Thymic tissue, stained by the TUNEL method to detect apoptotic nuclei, were costained immunohistochemically with an antibody to the endothelial cell surface antigen CD31, also known as platelet endothelial cell adhesion molecule (PECAM)-1 (33). Normal endothelium of thymic microvessels were identified by dark blue staining of the cell membrane, whereas thymocytes lacked this stain and manifested only a light red nuclear color resulting from the use of fast red as counterstain (Fig. 1 B, left). In specimens from LPS-treated mice, apoptotic endothelial cells displayed a central brown nuclear core surrounded by a blue-black perimeter (Fig. 1 B, middle). It should be noted that the microvessels identified in the thymus are comprised of only two to four endothelial cells and thus appear smaller than those in the lung and fat, which were frequently comprised of five to eight endothelial cells. Using this double staining technique, we were able to demonstrate that virtually all of the apoptotic cells present in the thymus at 6 h after LPS stimulation represented endothelial cells in microvessels, the lumens of which were partially or completely collapsed (Fig. 1 B, middle). It should also be noted that endothelial apoptosis occurred in all of these tissues in the absence of an inflammatory response, which was subsequently detected at 10-12 h. Taken together, these studies indicate that intraperitoneal injection of LPS induces a disseminated form of endothelial apoptosis, which precedes nonendothelial parenchymal tissue damage.
The extent of microvascular involvement was quantified. Table 1 shows that 71% of the intestinal villae and 64-79% of the microvessels of the lung, fat and thymus displayed apoptotic damage at 6 hours after a dose of 90 µg of LPS/ 25g mouse. Similar effects were observed at 8 h after injection of 90 µg of LPS/25g mouse and with 175 µg of LPS/ 25g mouse (data not shown). It should be noted that apoptosis was detected in less than 5% of microvessels in tissues from control sham-treated animals (data not shown).
|
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.
Since TNF- is a primary mediator of the septic shock
response to LPS (4), and since ceramide has been described as a mediator of TNF-induced apoptosis in numerous cellular systems (17), we investigated the effect of
TNF-
on tissue ceramide generation, endothelial apoptosis, and survival of C57BL/6 mice. Recombinant human
TNF-
, when injected intravenously, induced time- and
dose-dependent lethality in this strain of mice. As little as 5 µg of TNF-
/25g mouse was effective and the LD50, although somewhat variable between experiments, ranged
from 25-50 µg of TNF-
/25g mouse. At a dose of 25 µg
of TNF-
/25g mouse, death occurred as early as 10 h after injection and the mean time until death in multiple experiments was 24 h (data not shown). Fig. 3 A shows that TNF-
induced time- and dose-dependent ceramide generation in
the intestinal mucosa. 25 µg of TNF-
/25g mouse stimulated an increase in ceramide content with a slightly more
rapid time course than induced by LPS. TNF-
-induced ceramide generation was detected by 0.5 h and peaked at 1.5 h (P <0.001 vs. control). As little as 2.5 µg of TNF-
/25g
mouse was effective and a maximal effect occurred with 25 µg of TNF-
/25g mouse (Fig. 3 B). TNF-
, like LPS, induced endothelial apoptosis in intestinal mucosa, lung, and
fat tissues, beginning 6 h after injection (data not shown).
These studies demonstrate that TNF-
, like LPS, induces
tissue ceramide generation followed by microvascular endothelial apoptosis and demise of the animal.
Agents that inhibit TNF- action have been shown to
prevent the endotoxic shock response in a variety of different experimental models. These include neutralizing antibodies to TNF-
(4), chimeric inhibitors comprised of
the extracellular domain of the TNF receptor fused with an
immunoglobulin heavy chain fragment (10) or as a polyethylene glycol-linked dimer (TNF-bp) (9, 11, 34), and a
TNF convertase metalloproteinase inhibitor (35), to list a
few. To evaluate whether the effect of TNF-
to induce
tissue ceramide generation and endothelial apoptosis is essential for the LPS effect, we injected TNF-bp with LPS. Fig. 4 shows that intravenous injection of TNF-bp (serum t1/2 ~30 h) abolished the effect of a maximal dose of 175 µg of
LPS/25g mouse on ceramide generation in the intestinal
mucosa. Furthermore, TNF-bp markedly attenuated LPS-induced apoptosis in the endothelium of the intestine,
lung, pericolic fat, and thymic tissue at 6 h (Fig. 1, right panels) and at 8-10 h (data not shown) after stimulation. Quantitation of apoptotic microvessels in tissues treated with
TNF-bp and LPS, demonstrated near complete protection from apoptosis in all tissues (Table 1; P <0.001 vs. LPS-treated for each tissue). These studies provide evidence that LPS-induced ceramide generation, endothelial apoptosis, and endotoxic death require TNF-
action.
To determine whether ceramide generation is necessary
for progression of the endotoxic syndrome, we treated
wild-type and ASMase knockout mice with 175 µg of
LPS/25g mouse. LPS-induced elevation of serum TNF-
was unaffected in the ASMase knockout mice, increasing
to a maximum of 12 ± 3 ng/ml at 1.5 h after LPS injection. These data indicate that monocyte/macrophage activation is normal in the ASMase mouse. However, ASMase
knockout mice were defective in LPS-induced ceramide
generation and endothelial apoptosis. In contrast to the
twofold maximal ceramide elevation observed in the intestines of wild-type animals 2 h after 175 µg of LPS/25 g
mouse, in the ASMase knockout mouse the ceramide level
did not increase significantly and after 2 h was only 1.27 ± 0.18-fold of control (mean ± SD; 4 mice/group). Furthermore, upon evaluation of 150 intestinal villae for apoptotic
microvessels at 6 h after injection of 175 µg of LPS/25g
mouse, 118 (79%) were positive in the wild-type mice,
while only 17 (11%) were positive in the ASMase knockout mice (P <0.001 vs. LPS-treated wild type; 6 mice/ group). Upon evaluation of 150 capillaries in lungs from
the same animals, 109 (72%) demonstrated apoptotic damage in the wild type animal, whereas only 15 (10%) were
apoptotic in the ASMase knockout mice (P <0.001 vs.
LPS-treated wild type). Apoptosis progressed with time in
LPS-treated ASMase knockout mice, reaching a maximum at 8 h when 33 of 150 (22%) intestinal villae, and 29 of 150 (19%) lung microvessels, were positive (P <0.001 vs. LPS-treated wild-type mice tissues in which 70-80% of microvessels display apoptosis at 8 h). Furthermore, ASMase
knockout mice were protected against LPS-induced death
(P = 0.05 vs. LPS-treated wild type; Fig. 5). These studies
suggest that LPS-induced apoptosis, like radiation-induced apoptosis, requires a functional sphingomyelin pathway.
To provide additional support for the notion that endothelial damage is essential for evolution of the endotoxic
response, C57BL/6 mice were treated concomitantly with
LPS and bFGF. Prior studies from our laboratory showed
that bFGF protected endothelium in vitro and in vivo from
radiation-induced apoptosis (31). In vivo, intravenously injected bFGF has been shown to be retained within blood
vessels, apparently bound to the heparan sulfate proteoglycan coating the vascular surface of the endothelium and its
basement membrane (31). Consequently, intravenously injected bFGF served as a selective endothelial survival factor,
preventing radiation-induced apoptosis and lethal radiation
pneumonitis (36). Fig. 6 A shows that bFGF abrogated
LPS-induced apoptosis in the endothelium of the intestine
and lung of C57BL/6 mice. Fig. 6 B demonstrates that intravenous bFGF, when injected concomitantly with a dose
of 175 µg of LPS/25g mouse, provided protection from the lethal effects of LPS (P <0.001 vs. untreated). bFGF
also rescued C57BL/6 mice from maximal doses of 270 and
350 µg of LPS/25g mouse, although the protection was
not as complete (data not shown). Additional studies delineated the site of bFGF action. Table 2 shows that while
TNF- was not detected in the serum of sham- or bFGF-injected animals, 175 µg of LPS/25g mouse induced an elevation of serum TNF-
to a maximum of 4.2 ± 0.9 ng/
ml. The LPS-induced elevation of serum TNF-
was not
blocked by bFGF. In contrast, bFGF prevented the elevation of tissue ceramide in response to LPS. These studies
indicate that intravenous bFGF does not affect cell types
that generate TNF-
in response to LPS (i.e., monocytes and macrophages), but specifically targets endothelial cells
and the ceramide response to TNF stimulation. These data
also substantiate endothelial damage as mandatory for LPS-induced death, and define inhibition of TNF signaling as
the mechanism of the protective effect of bFGF on endothelium.
|
The present studies define a set of early biochemical and
biological responses to LPS using a standard model of endotoxic shock. Fig. 7 orders these events. Within 1 h of intraperitoneal injection of LPS, elevation of tissue ceramide
content was detected in the intestinal mucosa and lung. Although our evidence supports endothelium as the primary
source of the increase in ceramide, it remains formally possible that cells other than endothelium contribute to the ceramide elevation. Ceramide elevation appeared dependent
on TNF action since TNF mimicked the LPS effect, and TNF-bp blocked the LPS-induced increase in tissue ceramide. Elevation of ceramide preceded the appearance of a
generalized form of apoptosis, expressed initially in the microvascular endothelium of a variety of organs, beginning
at 6 h after LPS injection. Both ceramide elevation and endothelial apoptosis preceded damage to nonendothelial parenchymal tissue and the death of the animal, which became apparent at 16 h after a dose of 175 µg LPS/25g
mouse. Endothelial apoptosis appeared mandatory for the
progression of the endotoxic response, since intravenous
injection of bFGF, which specifically protects the endothelium against stress-induced apoptosis, prevented death. Furthermore, ceramide appeared to be a key intracellular
mediator of this response, as the ASMase knockout mouse,
which is defective in ceramide generation but not in TNF-
production, exhibited decreased endothelial apoptosis and
death. Nevertheless, additional models of endotoxic shock
must be studied before it can be concluded that these observations are representative of this process in general, as
significant species differences have been observed (3).
Previous studies reported that LPS induces endothelial
damage in vivo and under some conditions in vitro. Microvascular injury occurs in numerous tissues during sepsis,
including the lung, gut and liver, and this event has been
generally considered an important element in the pathogenesis of the septic shock syndrome (1). The mechanism
of microvascular injury and its relevance to the evolution of
the septic shock syndrome have been a subject of substantial debate. Disseminated intravascular thrombosis, extensive endothelial necrosis, and humoral microvascular dysfunction have all been ascribed a role as mediating vascular
collapse (1). Generalized endothelial apoptosis has not hitherto been reported, although apoptosis of liver endothelium ex vivo was recognized subsequent to induction of
TNF- on Kupfer cells by LPS (37). The large majority of
studies reported that LPS did not induce apoptosis in primary cultures of endothelial cells (37) unless a second
stress such as heat shock or cycloheximide was applied subsequently (39, 40). However, one group has argued that LPS can induce direct DNA damage leading to apoptosis in
primary cultures of sheep pulmonary endothelial cells (41, 42).
In the present studies, endothelial apoptosis appeared to be preferentially increased in tissues known to play prominent roles in the pathogenesis of endotoxic shock. In this regard, the microvascular endothelium of the bowel and lung were markedly affected. However, even the endothelium of tissues which play no overt role in the endotoxic response, such as the pericolic fat and the thymus, seemed to be affected. Endothelial damage preceded nonendothelial damage suggesting that loss of vascular integrity may play a role in the parenchymal tissue damage and the multi-organ failure that characterizes the endotoxic syndrome. It is reasonable to suggest that the generalized nature of the apoptotic response in the microvascular endothelium may account, in part, for the circulatory failure that is a major factor in the progression of the endotoxic response. Whether endothelial apoptosis in the lung is the critical lesion leading to the asphyxiation that results in the ultimate demise of affected mice (5) cannot be ascertained from the present information.
The critical role of endothelial cell apoptosis in the pathogenesis of endotoxic shock is similar to its role in the evolution of the inflammatory phase of radiation-induced pneumonitis. As in the case of the LPS response, microvascular endothelial apoptosis preceded the expression of other histopathological manifestations of lethal radiation-induced pneumonitis, and intravenous injections of bFGF abrogated the evolution of pneumonitis and death after whole lung irradiation (31, 36). Furthermore, both LPS- and radiation-induced endothelial apoptosis in vivo appeared initiated by activation of ASMase. Thus, the sphingomyelin pathway may integrate diverse responses to signal death in stressed endothelial cells. Consistent with this hypothesis, the prevention of ceramide generation by bFGF suggests that the anti-apoptotic survival function of bFGF may be mediated, in part, via this mechanism.
The present investigations establish a role for TNF- in
LPS-induced generation of ceramide and apoptosis in vivo.
Recent studies have clarified the mechanism by which the
55-kD TNF receptor signals the apoptotic response (43-
52). This receptor contains a carboxy-terminal death domain which appears to be required for transmission of the
apoptotic signal. Binding of TNF-
to the receptor triggers
formation of a multiprotein complex in which cytoplasmic
proteins and the receptor interact through their respective death domain motifs. Upon TNF stimulation, the receptor
death domain binds to the death domain of a cytoplasmic
protein known as TRADD (TNF receptor 1-associated
death domain), which in turn binds the death domain of
another cytoplasmic protein, termed FADD/MORT-1.
The latter protein also contains a death effector domain
(DED) motif, which binds the DED of the ICE/Ced-3
protease FLICE/MACH-1 (Caspase 8). It has been suggested that activation of FLICE/MACH-1 initiates activation of a cascade of caspases, which serves as the effector
system for the apoptotic destruction of the cell.
This model suggests that ligand binding to the TNF receptor is capable of activating the final death effector pathway without apparent involvement of lipid second messengers. However, several recent studies demonstrated a role
for ceramide in TNF-induced cell death, in some systems.
In this regard, activation of the death domain system of the
55-kD TNF and CD95 receptors has been shown to couple to ASMase (24, 53). This notion was based on the observation that mutations in these receptor death domains that abolished apoptosis also abolished ceramide generation.
Furthermore, dominant negative FADD/MORT-1 blocked
ceramide generation and apoptosis in BJAB B lymphoma cells,
but not apoptosis induced by ceramide analogues. Whether
ASMase activation might couple to FLICE/MACH-1 activation is presently uncertain. However, Pronk et al. (54),
using peptide inhibitors of ICE/Ced-3 proteases, molecularly ordered ceramide generation downstream of an undefined CPP32-like protease during REAPER-induced apoptosis in Drosophila Schneider L2 cells. In the present
studies, TNF appeared essential for ceramide generation
during the evolution of the endotoxic syndrome. Whether
the TNF receptor death domain adaptor protein system is
involved in LPS-induced ceramide generation via TNF- in vivo, remains uncertain.
Although the present studies define ceramide as critical
for the induction of endothelial apoptosis by LPS, its precise role in signaling apoptosis is as yet unknown. Kroemer
and coworkers (55) have provided evidence that ceramide
acts upstream of mitochondria to initiate apoptosis. Their
investigations suggested that ceramide, once generated, signals mitochondrial membrane permeability transition (MPT),
a committed step in the apoptotic process. MPT may signal apoptosis via release of an apoptosis-initiating factor, a Z-VAD- but not DEVD-inhibitable ICE-like protease (55). Consistent with this paradigm, Pastroini et al. (56) showed that
TNF-- and ceramide-stimulated MPT was not inhibited
by the protein synthesis inhibitor cycloheximide. Alternatively, ceramide-initiated MPT may involve the release of
cytochrome C from mitochondria and activation of a
CPP32-like protease (Caspase 3) (57). Either scenario is
consistent with the inhibition of ceramide-mediated apoptosis by Bcl-2 that has been reported in numerous systems (55, 60). Whether ceramide-mediated mitochondrial
damage is linked to the SAPK/JNK signaling system, also
reported to be critically involved in TNF-mediated apoptosis
in endothelial cells (26), remains unknown.
The present investigations enhance the understanding of the mechanism of the endotoxic syndrome, defining upstream elements of the LPS signaling system and their molecular ordering, as well as the early tissue responses that trigger its pathogenesis. The identification of biochemical pathways that signal pro- and anti-apoptosis during the LPS response, and the characterization of the primary tissue target for the endotoxic syndrome, provide a molecular and cellular context for testable experimental hypotheses, and a basis for developing strategies for pharmacologic intervention, with potential for clinical application. In particular, the ability of bFGF to inhibit ceramide generation suggests that treatment with bFGF may affect the progression of the LPS syndrome in gram-negative septicemia with evidence of rising serum TNF, or in patients already manifesting symptoms of septic shock.
Received for publication 27 June 1997 and in revised form 23 September 1997.
1 Abbreviations used in this paper: MPT, mitochondrial membrane permeability transition; NPD, Niemann-Pick disease; PECAM, platelet endothelial cell adhesion molecule-1.This work was supported by grant CA52462 to Z. Fuks from the National Institutes of Health.
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