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The Role of Endotoxin in Liver Injury
Vol. 69, N o.6
GASTHOENn:HOI.OGY 69:1346-1356,
1975 Copy right© 1975 hy The Williams & Wilkins Co.
Printed in U.S.A .
PROGRESS IN HEPATOLOGY
THE ROLE OF ENDOTOXIN IN LIVER INJURY JAMES
P.
NOLAN,
M.D.
Departm ent of M edicin e, Stat e University of N ew Y ork at Buffalo and Buffalo G eneral Hospital , Buffalo , N ew York
The liver stands as an effective barrier to the passage of bacteria and their products from the intestinal tract to the systemic circulation. The possibility that failure to normally perform the function of detoxifying endotoxin might initiate or perpetuate liver injury, or lead to systemic effects, has intrigued investigators for a number of years. While much of the evidence remains indirect, recent work suggests that the endotoxins of intestinal bacteria play a significant role in both hep.atic and extrahepatic manifestations of certain liver diseases. If bacterial products do increase the extent and contribute to the peripheral manifestations of hepatic injury, then attempts to reduce their absorption or their toxicity when absorbed, might offer therapeutic benefit. This progress report will review evidence for such a relationship, examine possible mechanisms involved, and suggest how these manifestations might be modified.
Synergism Between Endotoxin and Other Hepatotoxic Factors As early as 1941, sulfonamide drugs were noted to protect against carbon tetrachloride-induced hepatic necrosis and death, 1 and this benefit was confirmed in later studies. 2 • 3 In 1954, Luckey and his colleagues• studied the relationship between dietary liver injury and the presence of intestinal flora. Ordinary rats fed the necrogenic diet of Himsworth died of massive liver necrosis in 35 days, while germReceived December 20, 1974. Accepted April 3, 1975. This work was supported in part by United States Public Health Service Grant 5 ROl AI11552-02 .
free animals lived without any hepatic damage over the same period. These observations on germ-free rats prompted studies on the effect of antibiotic suppression of intestinal flora in dietary-induced liver disease. Gyorgy 5 found that aureomycin, streptomycin, and neomycin effectively prevented the hepatic necrosis of choline deficiency, although chloramphenicol and bacitracin did not. Studying the development of cirrhosis in rats on choline-deficient diets, Rutenburg and his colleagues 6 regularly found diffuse hepatic fibrosis within 300 days, but when absorbable antibiotics were added to the diet, cirrhosis was delayed by about 100 days. When a nonabsorable antibiotic was used, significant fibrosis was prevented for as long as 750 days, although the..antibiotics did not delay the development of a fatty liver. The authors felt the superiority of neomycin excluded a systemic effect, and concluded that the intestinal bacteria were necessary for the progression from a fatty liver to diffuse fibrosis. Salmon and Newberne 7 confirmed these results and suggested that hepatic cirrhosis in rats is not simply an effect of nutritional deficiency, but that enteric microbial agents or their toxins were essential to the progressive develop· ment of the complete chronic lesion. In seeming conflict with · these earlier observations, Levinson and Tennant 8 found that cirrhosis developed faster in germ-free animals on a choline-deficient diet. However, in an elegant and crucial study, Broitman and his co-workers 9 in 1964 demonstrated that the protection chronic neomycin administration affords against the development of dietary cirrho· sis in rats can be abolished by adding
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purified endotoxin to the drinking water during the period of the choline-deficient diet, and concluded that the absorption of intraluminal endotoxin contributed to the development of fibrosis and cirrhosis. These authors postulated that Levinson's observation that the lesion developed in germ-free animals reflected the probable presence of pyrogens in their diet. The same factors that increase sensitivity to a number of hepatic toxins, also increase sensitivity to bacterial endotoxins. Mice on inadequate nutritional regimens were found by Dubos and Schaedler' 0 to be more susceptible to the toxic effects of bacterial diseases and endotoxins than were animals with an adequate protein intake. They concluded that the nutritional state influenced the outcome of infection by modifying the host's ability to resist the toxic effects of pathogens. In a comparison of normal and cirrhotic rats, intravenously infused 131 I -labeled bacteria were cleared normally in both groups, but the capacity to destroy the bacteria was severely impaired in th(; cirrhotic animals.'' Formal and his colleagues 12 observed that prior administration of carbon tetrachloride or starvation significantly increases the lethality of a given dose of endotoxin. As an explanation for this finding, Farrar et al. 13 showed that normal rabbit liver is capable of detoxifying endotoxin in vitro whereas liver from CCL 4 treated animals is not. Examining the relationship of choline deficiency and endotoxin to the development of cirrhosis, Nolan and Ali 14 found that early in the fatty liver stage of lipotrope deprivation, the LD 50 for intraperitoneal endotoxin drops 10-fold as compared with choline-supplemented controls. More importantly, small sublethal injections of the lipopolysaccharide caused transaminase elevations and histologic evidence of liver necrosis at a dose that resulted in no such effect in rats pair fed an identical diet with choline added. They suggested that rats on a cirrhotogenic diet experience tissue necrosis from amounts of endotoxin rendered innocuous by the healthy liver. These investigators also noted a 3-fold rise in transaminase values when 0.5 ml of
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100% alcohol was administered with 1.0 mg per ml. of endotoxin by stomach gavage to animals with choline-deficient fatty livers. Such marked transaminase rises were not found when endotoxin was administered orally to rats given alcohol alone, or in those rats with only fatty livers (Ali MV, Nolan JP, unpublished data). The histologic picture of hepatic damage caused by endotoxin, well described by both light and electron microscopy, is rather nonspecific. 15 These changes include extreme vesiculation of hepatocytes with vacuolation and swelling of the endoplasmic reticulum and mitochondria and increased lipid accumulation. The Kupffer cells show an increased number of lysosomes and vacuoles. There is widening of the spaces of Disse and dilation of the bile ducts. When two spaced injections of endotoxin were given, Hirsch and his colleagues 16 demonstrated the development of fatty livers in all animals, as well as bromsulfophthalein retention and hyperlipidemia. In a provocative study , Campbell and Gilbert 17 produced giantcell transformation of the liver in both neonatal rats and rabbits by the administration of Escherichia coli endotoxin to the pregnant female . Because of the resemblance to the pathological picture seen in the human neonate, and because of the ubiquitous nature of endotoxin, they suggested this agent as one etiologic cause of this lesion. Mechanisms of Endotoxin Interaction with Other Factors Figure 1 depicts a variety of possible mechanisms that may contribute to hepatic sensitivity to endotoxins arising in the gastrointestinal tract. Altered detoxification. In liver injury, both serum and tissue factors necessary for endotoxin detoxification may be altered, making any given level of circulating endotoxin more toxic. The endotoxin may then cause tissue damage by a number of mechanisms, some direct and some mediated. The earliest hemodynamic changes seen after endotoxin administration involve evidence of vascular constriction 18 soon followed by capillary dilation. 19 Within a
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hydrate abnormalities, endotoxemia in the rhesus monkey has been shown to lead to a 2- to 4-fold increase in plasma triglycerides. 28 Serum and tissue factors also play a role IMMUN£ fACTORS HORMONAL INHU£NC£S in detoxification of endotoxin. Skarnes 29 -t GLOBULINS 4'-'' CORTICOST£ROIOS PRDT£CT studied the early response of rabbits to ESTROGENS £NHANC£ INJURY +~~:IBr~~~: ID___ __> sublethal doses of endotoxin, and demonstrated a 3-fold increase in the plasma ~ level of a heat-stable organophosphateINTESTIRAL £NDOTDXIN + ABSORPTION ~ +PRODUCTION resistant esterase associated with a strikFIG. 1. Endotoxin and liver disease. Possible meching increase in endotoxin-detoxifying caanisms for the toxic hepatic and extrahepatic effects pacity. He concluded that these esterases of endotoxin after liver injury. were a principal site of detoxification and a major defense against circulating endoshort period of time, a significant degree of toxin. Endotoxin also can activate the pooling occurs within the liver and the complement system by the alternate pathsplanchnic circulation depending on the way, and the active products generated species employed. 20 Endotoxin also in- may explain some effects such as smooth creases capillary permeability to mac- muscle constriction and increased vascular romolecules, leading to a loss of protein- permeability. 3 ° Complement is necessary rich fluid from the intravascular for endotoxin protection, and both early compartment . 21 Some of the hemodynamic and late acting components must be preschanges seen in endotoxemia may be due ent for this effect. 31 In addition, completo a direct depression of cardiac function. 22 ment may be important in the uptake of It has been suggested that endotoxin pro- endotoxin by the reticuloendothelial sysduces vascular changes by the release of a tem. 32 Thus, complement alterations in number of vasoactive substances, and that liver injury may contribute to a decreased these materials in turn promote further ability to detoxify these bacterial products. The most important tissue element in endotoxin entry into the circulation from the gut. 23 In the isolated liver preparation, protection against endotoxin is the reticu· however, endotoxin itself may exert a di- loendothelial system (RES). Beeson 33 first rect vasoconstrictive action on the portal demonstrated its importance when he showed that the phenomenon of endotoxin circulation. 24 At the subcellular level, endotoxin has tolerance could be abolished by loading the been shown to have direct metabolic ef- RES with particulate matter. Wiznitzer fects. Nicholas and his associates 25 dem- and his co-workers 34 showed that the noronstrated that endotoxemia abnormally in- mal RES rapidly removes and inactivates creases mitochondrial membrane permea- circulating endotoxin. They found, howbility for potassium and this may cause ever, that when the RES has been dam· mitochondrial swelling. When the mito- aged by a blockading agent or by shock, chondrial membrane becomes more ex~ only a small percentage of the normal tensively damaged, an inhibition of ATP removal and inactivation occurred. These production occurs. The uncoupling effect studies show that damage to the RES can on human mitochondria has been well stud- be expected to result in greatly enhanced ied by Schumer and his colleagues in human endotoxin toxicity . Other work indicates liver cells, 26 and little doubt exists that that the liver is the essential organ in endotoxin can cause hepatic cell injury and detoxification 35 and that carbon blockade death if a toxic level is achieved. Other of the (RES) in rabbits causes a significant liver effects caused by endotoxin include rise in SGOT and SGPT. 36 alterations in carbohydrate metabolism Several agents known to predispose to mediated through an initial activation of liver injury have been shown to depress adenyl cyclase. 27 As well as causing carbo- reticuloendothelial function and thus preEXIRH£PATIC £HECTS KION£Y ClOTTING H£ART f£Y£R
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dispose to endotoxin toxicity. Ali and Nolan 37 showed that the oral administration of small amounts of ethanol to rats delayed clearance of 1311-labeled microaggregated albumin by 40% over controls. These same investigators also serially investigated reticuloendothelial function in rats on a choline-deficient diet, 38 finding a significant depression of phagocytic function at the end of the first week, with a return toward normal as the cirrhotic process advanced. It was suggested that the early functional RES blockade by fatty change might contribute to the ultimate development of cirrhosis by increasing sensitivty to endotoxin. In hypotensive states a significant depression in RES function occurs. 39 In liver disease relative hypotension is often seen with decreased liver perfusion resulting. Olcay and his associates 40 found that portal vein occlusion significantly depress.e d the RES with the development of circulating endotoxemia, presumably of intestinal origin, demonstrable by the limulus assay. These studies reinforce the importance of the integrity of the RES of the liver in detoxifying endotoxins presented to it. Intestinal absorption. In addition to serum and tissue factors that may be altered in liver injury and predispose to endotoxin damage, the amount of endotoxin absorbed may be increased in certain liver diseases. Two mechanisms could be responsible, singly or in combination: increased absorption transmurally or increased production intraluminally. While there has been some dispute in the past about the absorption of endotoxin from the gut, there seems little doubt that it can and does pass through the intestinal wall. Although Sanford and Noyes 41 were unable to detect any label in the blood or tissues after feeding 51 Cr-labeled endotoxin to normal or shocked dogs, Ravin and associates 42 showed the absorption of 32Plabeled E. coli fed to both normal and shocked rabbits. They attributed the failure to show absorption with 51 Cr-labeled material to the fact that this label attaches to the lipid part of the molecule, requiring emulsification before it can be taken up.
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Work from this laboratory using the isolated rat gut sac demonstrates that 51 Cr does pass transmurally, about 0.5% of the endotoxin being recovered in the sac after 2 hr of incubation. 43 Furthermore, as demonstrated in the rat, endotoxin fed orally retains both its toxicity and serological activity within the gastrointestinal tract. 44 In irreversible hemorrhagic shock or traumatic shock, the absorption of endotoxin increases dramatically and is felt by some to be the cause of death in these conditions, 45 and indeed, the injection of nonabsorbable antibiotics into the intestinal tract before the production of irreversible shock prevents early death in animals. 46 The blood pressure in cirrhotics tends often to be low, conceivably promoting endotoxin absorption. The intestinal flora in patients with liver disease has been studied with conflicting results. Experimentally, deprivation of food for 48 hr in mice (a dietary stress not unusual in alcoholic patients) resulted in a marked increase of the bacterial population in the large and small intestine. 47 Furthermore, malabsorptive states are associated with significant colonization of the upper bowel, and malabsorption with stasis may occur both in the alcoholic and the cirrhotic. Martini and his colleagues 48 noted that the ileum of cirrhotic patients contained many coliform organisms and in some cases these organisms extended up to the jejunum and duodenum, areas normally sterile in noncirrhotics. Lal et al. 49 found abnormal colonization of the upper small bowel in 12 of 24 patients with biopsy-proven alcoholic cirrhosis, but found similar colonization in control patients without liver disease. Floch and his co-workers 50 showed higher numbers of anaerobes and Bacteroides in stools of cirrhotic patients. It must be concluded that increased intestinal endotoxin production and absorption in liver disease have not been firmly established, but should be investigated further. Hormonal influences. Experimentally, steroids protect the liver against hepatic damage by bacterial lipopolysaccharides. 52 • 53 Cook and DiLuzio 5 3 showed that steroid-treated rats were essentially
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refractory to the lethal effects of lead acetate and endotoxin. Since Kupffer cell function did not appear to be critical in the lead acetate-endotoxin toxicity, the authors felt the steroids helped maintain the integrity of the parenchymal cell. Janoff and his colleagues 54 found that cortisone inhibited the plasma levels of lysosomal enzymes associated with endotoxemia and attributed the protective effect of steroids to their lysosomal stabilizing action. Snyder et a!. 55 demonstrated that endotoxin and carbon tetrachloride both cause a reduction of tryptophan pyrrolase, and steroid protection against these two hepatotoxins may be related to its ability to induce this enzyme. Estrogens, in contrast, seem to impair hepatic function and to enhance susceptibility to injury by viral and toxic agents. 56 • 57 Studying the effects of estrogens and endotoxin sensitivity, Nolan and Ali 5 8 noted a profound increase in lethality to E. coli endotoxin in rats when estrogen was given 24 hr before the challenge with a 10-fold decrease in the LD 50 after this pretreatment. In contrast to this finding of enhanced susceptibility to E. coli endotoxin after estrogen administration, Trejo and his colleagues 59 found that daily injections of diethylstilbesterol in mice did not result in an enhanced sensitivity to Salmonella enteritides endotoxin. Those authors found a stimulatory effect of the diethylstilbesterol on RES function, while no such alteration was found in the rats given a variety of estrogens 24 hr before clearance studies with microaggregated albumin. A relationship between this striking enhancement in sensitivity to E. coli endotoxin and the property of estrogen to adversely affect certain liver injuries is attractive.
Extra-hepatic Effects of Endotoxin Since the liver stands between the gut and systemic circulation as a filter of bacteria and its products, it might be expected that the failure of this barrier function in liver disease would lead to the entry of these substances into the circulation. There is excellent evidence that this phenomenon does occur. Protell and his
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colleagues 60 noted agglutinins to Salmonella in 28 % of patients with chronic active hepatitis, although no evidence for actual Salmonella infection could be found. Simultaneously , a group from Denmark and Great Britain reported a significantly increased antibody titer to intestinal bacteria in patients with liver disease. 61 • 62 Triger and his colleagues 62 found that in. creased antibody levels to E. coli and Bacteroides were present frequently in chronic active hepatitis, and serial sera from patients with acute viral hepatitis showed an increased titer to E. coli that peaked 2 months after onset, before return. ing to normal. Prytz and his Danish col. leagues, 63 on the other hand, found a significantly greater titer of E. coli antibodies in patients with alcoholic cirrhosis as compared to nonalcoholic cirrhosis, and found that the incidence of antibody was high even in the fatty liver stage. It should be pointed out that it is unclear whether these elevated titers indicate specific antibody to gut-associated bacteria, or whether they represent nonspecific elevations that may be present to a number of antigens in inflammatory states. Thomas and his associates 64 noted that the distri· bution of antigen between the liver and spleen determines the magnitude of the immune response, the spleen being a larger antibody producer. They postulated that the increased production of antibody to both gut- and non gut-derived antigen re· fleeted a decreased capacity of the liver to trap antigen, which results in more reaching the spleen. Hepatorenal syndrome. It is possible that endotoxin from the gut may bypass the cirrhotic liver and produce the manifestations of the hepatorenal syndrome. There are striking similarities between the known effects of endotoxin on the renal circulation and the physiological changes seen in the renal failure of liver disease. In dogs, the effects of infusing endotoxin on the kidney are hemodynamic and not due to direct action of the lipopolysaccharide on the renal parenchyma .65 Endotoxin causes severe vasoconstriction in the renal circulation with renal ischemia, 66 and these changes can be blocked by making the
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animal nonspecifically tolerant to the toxin. 67 Similarly in the hepatorenal syndrome, the changes noted are attributable to intense vasoconstriction rather than parenchymal damage. Kew and his associates 6 8 studied renal hemodynamics in cirrhotics by the xenon washout technique and showed decreased intrarenal blood flow . The distribution to, and flow rate through the outer cortex was reduced while juxtamedullary and medullary flow was maintained. Using the same technique in cirrhotic patients with renal failure , Epstein and his colleagues 69 noted an extreme degree of renal hemodynamic instability with intermittent vasoconstriction, and postulated that the hepatorenal syndrome was secondary to active vasoconstriction by an unidentified circulating substance. In this laboratory, injections of E . coli endotoxin produced renal hemorrhage in choline-deficient rats with fatty livers at doses causing no such pat hology in controls. 14 It was postulated that these extrahepatic manifestations were due to the inability of the liver to detoxify normally innocuous, amounts of endotoxin . Wardle and Wright' 0 produced obstructive jaundice in rats by tying their common ducts, and showed that a single dose of endotoxin produced death with intravascular coagulation. The authors felt this finding explained the renal failure which occurs when biliary tract operations are complicated by bacteremia . Using a limulus lysate assay to detect circulating endotoxin in patients with acute massive hepatic necrosis, Wilkinson and his associates 71 beautifully demonstrated a close correlation between endotoxemia and the development of renal failure and intravascular coagulation . All these studies suggest that endotoxin must be a strong candidate for the unknown vasoconstrictive agent associated with renal failure in liver disease . Intravascular coagulation and fever . Other extrahepatic manifestations of liver disease may have their origin in low-grade endotoxemia . Endotoxin is known to produce intravascular coagulation, and this occurs regularly in septicemia. 72 Gans and his colleagues 73 noted that the dis-
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seminated intravascular coagulation seen in the hepatectomized animal could be delayed by the prior administration of neomycin. These investigators postulated that endotoxin from the gut that would ordinarily be detoxified by the liver was responsible for the defibrination. As cited previously, Wilkinson and his colleagues 71 supported this contention by correlating intravascular coagulation with endotoxemia in patients with hepatic necrosis . Fever of significant degree and duration has been found in a number of patients with alcoholic liver disease without any cultural evidence of infection. 74 Endotoxin injected intravenously in man produces fever of a similar nature. 75 Preliminary studies from this laboratory using the limulus test indicate a correlation between the fever of Laennec's cirrhosis and the presence of circulating endotoxin (Nolan JP, unpublished observations.). Myocardial function. Depressed cardiac function has been regularly seen in cirrhotic patients with portal hypertension, the failure often being hyperdynamic in nature .Endotoxemia has been shown experimentally to produce myocardial depression both in isolated preparations and in intact primates. 76 • 77 The data of Siegel and his colleagues 7 8 that vascular tone and cardiac output in sick cirrhotics resemble that seen in patients with septic shock raises the possibility that these cardiovascular abnormalities in liver disease may reflect the effect of circulating endotoxin on the cardiovascular system.
Modification of Endotoxicity If endotoxin plays a role in determining the extent of liver injury caused by other agents, then attempts at lessening the effect of these gut lipopolysaccharides might benefit both the hepatic disease and its extrahepatic effects as well. Some promising work has already been accomplished in this area and table 1 lists some possible modifications that may be done. Animals can be made nonspecifically tolerant to a variety of endotoxins by giving increasingly large doses of a single endotoxin over a short period of time. 79 Jennings et al. 80 made dogs tolerant over a
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1. Modification of endotoxin toxicity in liver
diseas e
A. Increasing resistance 1. Development of nonspecific tolerance 2. Specific immunization B. Decreasing gut absorption 1. Oral nonabsorbable antibiotics 2. Cholestyramine resin 3. Immunization C. Removal of circulating endotoxin 1. Hemoperfusion with Dowex 1-X2 resin or activated charcoal
2-week to 2-month period and were able to almost completely protect against the hemodynamic and biochemical effects of a normally lethal endotoxin challenge . The mechanism for this nonspecific tolerance is not known. Endotoxin is a known potent stimulator of RES activity, 81 but also causes a marked increase in antibody formation to the endotoxin used. 80 Using a perfused rabbit liver from endotoxin-tolerant and nontolerant rabbits, Greisman and Woodward 82 concluded that tolerance to the pyrogenic activity of bacterial endotoxin is based upon increased uptake of the toxin by hepatic Kupffer cells which have become refractory to release of endogenous pyrogen. A single injection of E. coli endotoxin in an oil-water emulsion was shown to prolong survival in mice both to endotoxin challenge and to E. coli peritonitis. 83 If endotoxin plays a role in furthering liver injury, then the state of nonspecific tolerance might be expected to protect against liver injury by an unrelated toxin. Nolan and Ali 84 found such an effect in comparing the extent of carbon tetrachloride injury . Compared with controls, endotoxin-tolerant rats were significantly more resistant to the hepatotoxic effect of CC1 4 as measured by enzyme changes and amount of histological damage at the time of killing 24 and 48 hr later. Since tolerance is a short-lived phenomenon, specific immunization with endotoxin vaccines might more effectively protect animals from liver damage. Some materials may even confer protection against heterologous endotoxins. Kunin et al. 85 discovered an antigen common to all
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the enterobacteriacae that protects animals against induced infection with a variety of gram-negative bacteria. Further studies demonstrated that this common antigen induces passive immunity to heterologous endotoxins as well (Nolan JP, McDevitt JJ, unpublished data.). McCabe has used R mutants of a Salmonella to immunize mice against E. coli and Klebsiella pneumoniae, 86 and Braude and Douglas 87 were able to passively transfer immunity to heterologous endotoxins with a mutant E. coli strain, experiments proving the feasibility of cross-protection by suitable immunization. Another approach to modifying endotoxin toxicity would seek to reduce the amount of endotoxin absorbed, by shrinking the pool of gram-negative bacteria in the gut or by increasing the mucosal barrier to the lipopolysaccharide. Neomycin retards the development of choline-deficiency cirrhosis in rats, 9 but while the drug has been successfully used to reduce urease splitting bacteria in patients with hepatic encephalopathy, a controlled study on its effect in continuing liver damage has not been attempted. Nolan and Ali 43 have shown that cholestyramine not only binds bile acids and a variety of drugs, but also tightly binds E . coli endotoxin, preventing both its toxicity and absorption through the isolated rat gut. Since secretory IgA antibodies protect against enteric bacterial infections, 88 specific immunization with a heterologous endotoxin vaccine might be beneficial in decreasing endotoxin absorption. Lastly, treatment of liver failure with hemoperfusion through charged resins and charcoal has been attempted. Wilson et al. 89 prolonged survival of dogs with massive liver necrosis using Dowex-1 resins, and Gazzard and associates 90 treated some patients with massive hepatic necrosis with benefit by passing their blood through activated charcoal columns. Since both Dowex-1 resins and charcoal have been shown to efficiently remove labeled endotoxin in vitro, 91 the beneficial effects observed in liver failure may be due in part, to removal of circulating endotoxin.
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Unresolved Problems Certain theoretical difficulties are present which may not fit into the proposed role for endotoxin in perpetuating liver injury. If endotoxin regularly enters the circulation after hepatic damage, then a state of tolerance or immunity might be expected to develop, preventing or modifying the extent of further injury. Relatively little is known, however, about the status of local hepatic defense mechanisms in liver disease, and these defenses may be significantly altered. In addition, studies are needed, but not available, correlating the development of antibodies to gut bacteria and the clinical course of liver disease. It is known that nonspecific increases in RES function can occur with appropriate stimuli. Therefore, it could be argued that low-grade endotoxemia should stimulate the RES and thus result in faster removal of circulating endotoxin with some resultant protection. The RES in liver injury, however, may be incapable of normal stimulation by bacterial lipopolysaccharides. Final answers about RES function are not yet available. Lastly, while it may be possible to demonstrate bacterial antigens in liver tissue, it will be difficult to assess their pathogenic role. Until more precise methods of identifying and quantitating endotoxin in the liver are found, the evidence for its role in liver disease will remain intriguing but indirect. Summary Evidence is present that a common pathway for hepatic injury by a variety of agents may result from impairment of the liver's ability to detoxify bacterial endotoxins from the gastrointestinal tract. Many factors may structurally, metabolically, or hormonally alter the normal liver's ability to render innocuous the small amounts of lipopolysaccharide ordinarily presented to it. This impairment may accentuate existing hepatic damage by allowing toxic levels of endotoxin to develop in the liver tissue, and by allowing endotoxin entry into the systemic circulation, may
also lead to extrahepatic effects. Studies are cited that: (1) support a role for intraintestinal endotoxin in the development of experimental cirrhosis, (2) demonstrate how liver injury alters endotoxin detoxification, (3) examine the role of intestinal production and absorption of bacterial lipopolysaccharides in liver disease, and (4) point to a role for endotoxemia in extrahepatic manifestations of liver injury as well. Studies are also reviewed that suggest possible mechanisms for modifying endotoxicity in hepatic damage. REFERENCES 1. Leach BE, Forbes JC: Sulfonamide drugs as protective agents against carbon tetrachloride poisoning. Proc Soc Exp Bioi Med 48:361- 363, 1941 2. Wilson JW, Leduc EH, Arnold LE: Protective action of sulfa drugs against CCI, poisoning in mice. Fed Proc 9:349, 1950 3. Leduc EH: Sulfaguanidine protection of mouse liver from carbon tetrachloride-induced necrosis. Lab Invest 29:186-196, 1973 4. Luckey TD, Reyniers JA, Gyorgy P, et al: Germfree animals and liver necrosis. Ann N Y Acad Sci 57:932-935, 1954 5. Gyorgy P: Antibiotics and liver injury. Ann NY Acad Sci 57:925-932, 1954 6. Rutenburg AM, Sonnenblick E, Koven I, et al: The role of intestinal bacteria in the development of dietary cirrhosis in rats. J Exp Med 106:1-13, 1957 7. Salmon WD, Newberne PM: Effect of antibiotics, sulfonamides, and a nitrofuran on development of hepatic cirrhosis in choline-deficient rats. J Nutr 76:483-486, 1962 8. Levinson SM , Tennant B: Some metabolic and nutritional studies with germ-free animals. Fed Proc 22:109- 119, 1963 9. Broitman SA, Gottlieb LS, Zamcheck N: Influence of neomycin and ingested endotoxin in the pathogensis of choline deficiency cirrhosis in the adult rat . J Exp Med 119:633-641, 1964 10. Dubos RJ , Schaedler RW: Effect of nutrition on the resistance of mice to endotoxin and on the bactericidal power of their tissues. J Exp Med 110:935-950, 1959 11. Rutenburg AM, Sonnenblick E, Koven I, et al: Comparative response of normal and cirrhotic rats to intravenously injected bacteria. Proc Soc Exp Bioi Med 101:279-281, 959 12. Formal SB, Noyes HE, Schneider H: Experimental shigella infections . III. Sensitivity of normal, starved, and carbon tetrachloride treated guinea
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pigs to endotoxin. Proc Soc Exp Bioi Med 103: 415-418, 1960 13. Farra r WE, Edison M, Kant TH : Susceptibility of rabbits to pyrogenic and lethal effects of endotoxin after acute liver injury. Proc Soc Exp Bioi Med 128:7 11-71 .5, 1968 14. Nolan JP, Ali MV: Endotoxin and the liver I. Toxicity in rats with choline deficient fatty livers . Proc Soc Exp Bioi Med 129:29-3 1, 1968 1.5 . Levy E, Slusser RJ, Ruebner BH: Hepatic changes produced by a single dose of endotoxin in the mouse by electron microscopy. Am J Pathol .52:477-502, 1968 16. Hirsch RL, McKay DG, Travers RI, et al: Hyperlipidemia, fatty liver and bromsulfophtalein retention. ,J Lipid Res .5:.563-.568, 1964 17. Campbell LV, Gilbert EF: Experimental giant cell transformation in the liver induced by E. coli endotoxin. Am J Pathol 51:855-868, 1967 18. Hinshaw LB, Vick A, Wittmers LE, et a!: Changes in total peripheral resistance in endotoxic shock. Proc Soc Exp Bioi Med 108:24-27, 1961 19. Chien S , Chang C, Dellenback RJ , et al: Hemodynamic changes in endotoxin shock. Am J Physiol 210:1401-1410, 1966 20. Blattberg B, Levy MN: Early hepatic and extrahepatic pooling in response to endotoxin. Am J Physiol 219:460-463, 1970 21. Chien S, Sinclair DG, Dellenback RJ , eta!: Effect of endotoxin on capillary permeability to macromolecules. Am ,J Physiol 207:518-522, 1964 22. Alican F, Dalton ML, Hardy DJ: Experimental endotoxin shock Circulatory changes with emphasis upon cardiac function. Am J Surg 103:702-708, 1962 23. Cuevas P , Fine J: Production of fatal endotoxic shock by vasoactive substances. Gastroenterology 64:285-291, 1973 24. Nolan JP, O'Connell CJ: Vascular response in the isolated rat liver. I. Endotoxin , direct effects. J Exp Med 122:1063-1073, 1965 25. Nicholas GG , Mela LM , Miller LD: Early alterations in mitochondrial membrane transport during endotoxemia. J Surg Res 16:375-383, 1974 26. Schumer W, DasGupta TK, Moss GS, et al: Effect of endotoxemia on liver cell mitochondria in man . Ann Surg 171:875-882, 1970 27. Gimpel L, Hodgins DS , Jacobson ED : Effect of endotoxin on hepatic adenylate cyclase activity. Circ Shock 1:31-37, 1974 28. Fiser RH, Denniston JC, Beisel WR: Endotoxemia in the rhesus monkey. Alterations in host lipid and carbohydrate metabolism . Pediatr Res 8:13-17 , 1974 29. Skarnes RC: Host defense against bacterial endotoxemia . Mechanism in normal animals. J Exp Med 132:300-316, 1970 30 . Gewurz H , Snyderman R, Mergen hagen SE, et al:
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Effects of endotoxic lipopolysaccharides on the complement system. In Bacterial Endotoxins. Vol. 5. New York , Academic Press, 1971 p. 127-146 31. May JE, Kane MA, Frank MM: Host defense against bacterial endotoxemia, contribution of the early and late components of complement to detoxification. J lmmunol 109:893-895, 1972 32. Golub S, Groschel D, Nowotny A : Factors which affect the recticuloendothelial system uptake of bacterial endotoxins. J Reticuloendothel Soc 5:324-329, 1968 33. Beeson PB: Tolerance to bacterial pyrogens. II . Role of the reticuloendothelial system. J Exp Med 86:39.-44, 1947 34 . Wiznitzer T, Better N, Rachlin W , et al: In vivo detoxification of endotoxin by the reticuloendothelial system . J Exp Med 112:1157-1163, 1960 35. Farrar WE, Corwin LM: The essential role of the liver in detoxification of endotoxin. Ann N Y Acad Sci 133:668-684, 1966 36. Fisher ER, Fisher B: Hepatic damage by reticuloendothelial interference. Arch Pathol 75:191-195, 1963 37. Ali, MV , Nolan JP: Alcohol-induced depression of reticuloendothelial function in the"rat. J Lab Clin Med 70:295-301, 1967 38. Ali MV, Nolan JP: Serial assessment of reticuloendothelial function in experimentally induced nutritional cirrhosis. Lab Invest 20:184- 189, 1969 39. Hershey SG, Altura BM: Function of the reticuloendothelial system in experimental shock and combined injury . Anesthesiology 30:138-143, 1969 40. Olcay I, Kitahama A, Miller RH , et al: Reticula· endothelial dysfunction and endotoxemia follow· ing portal vein occlusion. Surgery 75:64-70, 1970 41. Sanford JP , Noyes HE: Studies on the absorption of Escherichia coli endotoxin from the gastroin· testinal tract of dogs in the pathogenesis of irreversible hemorrhagic shock. J Clin Invest 37:1425-1435, 1958 42. Rav in HA , Rowley P , Jenkins C, et al: On the absorption of bacterial endotoxin from the gas· trointestinal tract of the norma l and shocked animal. J Exp Med 112:783-792, 1960 43. Nolan JP , Ali MV: Effect of cholestyramine on endotoxin toxicity and absorption. Am J Dig Dis 17:161-166, 1972 44. Verczi I: Stability of Escherichia coli endotoxin in the rat gastrointestinal tract. J Pathol Bacterial 96:487-491, 1968 45. Woodruff, PWH, O'Carroll DI, Koizumi S, et al: Role of the intestinal flora in major trauma. J Infect Dis 128:290-294, 1973 46. Cuevas P, Fine J: Demonstration of a lethal endo· toxemia in experimental occlusion of the superior mesenteric artery. Surg Gynecol Obstet 132:8183, 1971 47. Tannock GW, Savage DC: Influences of dietary
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and environmental stress on microbial popula- 63. Prytz H, Bjornboe M, Orskov F, et al: Antibodies to Escherichia coli in alcoholic and nonalcoholic tions in the murine gastrointestinal tract. Infect patients with cirrhosis of the liver or fatty liver. lrnmun 9:591-598, 1974 Scand J Gastroenterology 4:433-438, 1973 48. Martini GA, Phear EA, Ruener B, et a!: The bacterial content of the small intestine in noma! 64. Thomas HC, McSween RNM, White RG : Hypothesis-role of the liver in controlling the immuand cirrhotic patients, relation to methionine nogenicity of commensal bacteria in the gut. toxicity. Clin Sci 16:35-51, 1957 Lancet 1:1288-1291, 1973 49 . La! D, Gorbach SL, Levitan R: Intestinal microflora in patients with alcoholic cirrhosis. Urea 65. Gillenwat er JY, Dooley ES, Frohlich ED: Effects of endotoxin in renal function and hemodynamsplitting bacteria and neomycin resistance. Gasics. Am J Physiol 205:293-297, 1963 troenterology 62:275-279, 1972 50. Floch MH, Katz J, Conn HO: Qualitative and 66. Selmyer JP, Reynolds DG, Swan KG: Renal blood flow during endotoxin shock in the subhuquantitative relationships of the fecal flora in man primate. Surg Gynecol Obstet 137:2-6, cirrhotic patients with portal systemic enceph1973 alopathy and following portacaval anastomosis. 67. Bates HM, Margolin S: Endotoxin-induced inhibiGastroenterology 59:70-75, 1970 tion of renal function in the mouse. Proc Soc Exp 51. Thomas L and Smith RT: Effect of cortisone on Bioi Med 129:642-646, 1968 response to endotoxin in mature rabbits. Proc Soc Exp Bioi Med 86:810-813, 1954 68. Kew MC, Varma RR, Williams HS, et al: Renal 52. Levitin H, Kendrick ML, Kass EH : Effect of and intrarenal blood flow in cirrhosis of the liver. route of administration on protective action of Lancet 2:504-509, 1971 corticosterone and cortisol against endotoxin. 69. Epstein M, Berk DP, Hollenberg NK, et al: Renal Proc Soc Exp Bioi Med 93:306-309, 1956 failure in the patient with cirrhosis. The role of 53. Cook JA, DiLuzio NR: Protective effect of cysactive vasoconstriction. Am J Med 49:175-185, teine a nd methylprednisolone in lead-acetate1970 endotoxin induced shock. Exp Mol Pathol 70. Wardle EN, Wright NA: Endotoxin and acute renal failure associated with obstructive jaundice. 19:127-138, 1973 54. Janoff A, Weissman G, Zweifach BW, et al: Br Med J 4:472-474, 1970 Pathogenesis of experimental shock. IV . Studies 71. Wilkinson SP, Arroyo V, Gazzard BG, et a!: Rel ationship of renal impairment and hemoron lysosomes in normal and tolerant animals subjected to lethal trauma and endotoxemia. J rhagic diathesis to endotoxernia in fulminant Exp Med 116:451-466, 1962 hepatic failure . Lancet 1:521-524, 1974 55. Snyder IS, Agarwal MK, Berry LJ: Influence of 72. Corrigan JJ, Ray WL, May N: Changes in the ca rbon tetrachloride on inducible liver enzymes blood coagulation syste m associated with sepand response to endotoxin in mice. J Bacteriol ticemia. N Eng! J Med 279:851-856, 1968 73 . Gans H, Mori K, Lindsey E , et al: Septicemia as a 94:1817-1823, 1967 56. Bearn AG, Kunkel HG, Slater RJ: The problem of manifestation of acute liver failure. Surg Gynecol chronic liver disease in young women. Am J Med Obstet 132:783-790, 1971 21:3-15, 1956 74. Tisdale WA, Klatskin G : The fever of Laennec's 57. Jones WA, Cohen RB: The effect of estrogen on cirrhosis. Yale J Bioi Med 33:94-106, 1960 the liver in murine viral hepatitis . Am J Pathol 75. Greisman SE, Hornick RB, Carozza FA, et a!: 42:237-243, 1963 The role of endotoxin during typhoid fever and 58. Nolan JP , Ali, MV: Late effect of estrogen on tularemia in man. I. Acquisition of tolerance to endotoxin response in the rat. J Clin Lab Med endotoxin. J Clin Invest 42:1064-1075, 1963 71:501-510, 1968 76. MacNicol MF, Goldberg AH, Clowes, GHA: De59. Trejo RA, Loose LD, DiLuzio NR: Influence of pression of t he isolated heart muscle by bacterial diethylstilbesterol on reticuloendothelial fun cendotoxin. J Trauma 13:554-558, 1973 tion, tissue distribution, and detoxification of S. 77. Reichgott MJ, Melmon KL, Forsyth RP, et a! : enteritides endotoxin. J Reticuloendothel Soc 11: Cardiovascular and metabolic effects of whole or 88- 97, 1972 fractionated gram-negative bacterial endotoxin in the unanesthetized rhesus monkey. Circ Res 60. Protell RL, Soloway RD , Martin WJ, et al: Anti-salmonella agglutinins in chronic active 33:346-352, 1973 liver disease . Lancet 2:330-331, 1971 78. Siegel JH, Greenspa n M, Cohen JD, et al: The 61. Bjornboe M, Prytz H, Orskov F: Antibodies to prognostic implications of altered physiology in intestinal microbes in serum of patients with operations for portal hypertension. Surg Gynecol cirrhosis of the liver. Lan cet 1:58-60, 1972 Obstet 126:249-262, 1968 62. Triger DR, Alp MH, Wright R: Bacterial and 79. Abernathy RS : Homologous and heterologous dietary antibodies in liver disea se. Lance t resistance of mice given bacterial endotoxins. J 1:60-63, 1972 Immunol 78:387-394, 1957
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80. Jennings PB, Simmons RL, Sleeman HK, et al: Hemodynamic, biochemical and coagulation alterations in endotoxin shock . Modification by induced tolerance in the dog. Ann Surg 167:204214, 1968 81. Arredondo MI, Kampschmidt RF: Effect of endotoxins on phagocytic activity of the reticuloendothelial system of the rat. Proc Soc Exp Bioi Med 112:78-81, 1963 82. Greisman SE, Woodward CL: Mechanisms of endotoxin tolerance . VII. The role of the li ver. J Immunol 105:1468-1476, 1970 83. Cuevas P , Fine J , Monaco AP : Successfu l induction of increased resistance to gram-nega tive bacteria and to endotoxin in immunosuppressed mice . Am J Surg 127:460-464, 1974 84. Nolan JP, Ali MV: Endotoxin and the li ver. II. Effect of tolerance on carbon tetrachlorideinduced injury. J Med 4:28-38, 1973 85. Kunin CM , Beard MV, Halmagy NE: Evidence for a common hapten associated with endotoxin fractions of E. coli and ot her Enterobacteriaceae . Proc Soc Exp Hiol Med Ill : 160-166, 1962
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86. McCabe WR: Immunization with R mutants ofS. Minnesota. I. Protection against challenge with heterologous gram-negative bacilli. J Immunol 108:601-610, 1972 87. Braude AI, Douglas H: Passive immunization against the local Shwartzman reaction . J Immunol 108:505-5 12, 1972 88. Fubara ES, Freter R: Protection against enteric bacteria l infection by secretory IgA antibodies. J Immunol 111: 395-403, 1973 89. Wilson RA , Hofmann, AF, Kuster GGR: Toward an artificial li ver. II. Removal of cholephilic anions from dogs with biliary obstruction by hemoperfusion through charged and uncharged resins. Gastroenterology 66:95-107, 197 4 90. Gazzard BG, Portman B, Weston MG et al: Charcoal hemoperfus ion in the treatment of fuJ. minant hepatic failure . Lancet 1:1301 - 1306, 1974 91. Nolan JP, McDevitt JJ, Goldmann, GS: Charac· teristics of endotoxin binding in vitro by charged and uncharged resin. Proc Soc Exp Bioi Med 149: 766-770, 1975
GASTHOENn:HOI.OGY 69:1346-1356,
1975 Copy right© 1975 hy The Williams & Wilkins Co.
Printed in U.S.A .
PROGRESS IN HEPATOLOGY
THE ROLE OF ENDOTOXIN IN LIVER INJURY JAMES
P.
NOLAN,
M.D.
Departm ent of M edicin e, Stat e University of N ew Y ork at Buffalo and Buffalo G eneral Hospital , Buffalo , N ew York
The liver stands as an effective barrier to the passage of bacteria and their products from the intestinal tract to the systemic circulation. The possibility that failure to normally perform the function of detoxifying endotoxin might initiate or perpetuate liver injury, or lead to systemic effects, has intrigued investigators for a number of years. While much of the evidence remains indirect, recent work suggests that the endotoxins of intestinal bacteria play a significant role in both hep.atic and extrahepatic manifestations of certain liver diseases. If bacterial products do increase the extent and contribute to the peripheral manifestations of hepatic injury, then attempts to reduce their absorption or their toxicity when absorbed, might offer therapeutic benefit. This progress report will review evidence for such a relationship, examine possible mechanisms involved, and suggest how these manifestations might be modified.
Synergism Between Endotoxin and Other Hepatotoxic Factors As early as 1941, sulfonamide drugs were noted to protect against carbon tetrachloride-induced hepatic necrosis and death, 1 and this benefit was confirmed in later studies. 2 • 3 In 1954, Luckey and his colleagues• studied the relationship between dietary liver injury and the presence of intestinal flora. Ordinary rats fed the necrogenic diet of Himsworth died of massive liver necrosis in 35 days, while germReceived December 20, 1974. Accepted April 3, 1975. This work was supported in part by United States Public Health Service Grant 5 ROl AI11552-02 .
free animals lived without any hepatic damage over the same period. These observations on germ-free rats prompted studies on the effect of antibiotic suppression of intestinal flora in dietary-induced liver disease. Gyorgy 5 found that aureomycin, streptomycin, and neomycin effectively prevented the hepatic necrosis of choline deficiency, although chloramphenicol and bacitracin did not. Studying the development of cirrhosis in rats on choline-deficient diets, Rutenburg and his colleagues 6 regularly found diffuse hepatic fibrosis within 300 days, but when absorbable antibiotics were added to the diet, cirrhosis was delayed by about 100 days. When a nonabsorable antibiotic was used, significant fibrosis was prevented for as long as 750 days, although the..antibiotics did not delay the development of a fatty liver. The authors felt the superiority of neomycin excluded a systemic effect, and concluded that the intestinal bacteria were necessary for the progression from a fatty liver to diffuse fibrosis. Salmon and Newberne 7 confirmed these results and suggested that hepatic cirrhosis in rats is not simply an effect of nutritional deficiency, but that enteric microbial agents or their toxins were essential to the progressive develop· ment of the complete chronic lesion. In seeming conflict with · these earlier observations, Levinson and Tennant 8 found that cirrhosis developed faster in germ-free animals on a choline-deficient diet. However, in an elegant and crucial study, Broitman and his co-workers 9 in 1964 demonstrated that the protection chronic neomycin administration affords against the development of dietary cirrho· sis in rats can be abolished by adding
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purified endotoxin to the drinking water during the period of the choline-deficient diet, and concluded that the absorption of intraluminal endotoxin contributed to the development of fibrosis and cirrhosis. These authors postulated that Levinson's observation that the lesion developed in germ-free animals reflected the probable presence of pyrogens in their diet. The same factors that increase sensitivity to a number of hepatic toxins, also increase sensitivity to bacterial endotoxins. Mice on inadequate nutritional regimens were found by Dubos and Schaedler' 0 to be more susceptible to the toxic effects of bacterial diseases and endotoxins than were animals with an adequate protein intake. They concluded that the nutritional state influenced the outcome of infection by modifying the host's ability to resist the toxic effects of pathogens. In a comparison of normal and cirrhotic rats, intravenously infused 131 I -labeled bacteria were cleared normally in both groups, but the capacity to destroy the bacteria was severely impaired in th(; cirrhotic animals.'' Formal and his colleagues 12 observed that prior administration of carbon tetrachloride or starvation significantly increases the lethality of a given dose of endotoxin. As an explanation for this finding, Farrar et al. 13 showed that normal rabbit liver is capable of detoxifying endotoxin in vitro whereas liver from CCL 4 treated animals is not. Examining the relationship of choline deficiency and endotoxin to the development of cirrhosis, Nolan and Ali 14 found that early in the fatty liver stage of lipotrope deprivation, the LD 50 for intraperitoneal endotoxin drops 10-fold as compared with choline-supplemented controls. More importantly, small sublethal injections of the lipopolysaccharide caused transaminase elevations and histologic evidence of liver necrosis at a dose that resulted in no such effect in rats pair fed an identical diet with choline added. They suggested that rats on a cirrhotogenic diet experience tissue necrosis from amounts of endotoxin rendered innocuous by the healthy liver. These investigators also noted a 3-fold rise in transaminase values when 0.5 ml of
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100% alcohol was administered with 1.0 mg per ml. of endotoxin by stomach gavage to animals with choline-deficient fatty livers. Such marked transaminase rises were not found when endotoxin was administered orally to rats given alcohol alone, or in those rats with only fatty livers (Ali MV, Nolan JP, unpublished data). The histologic picture of hepatic damage caused by endotoxin, well described by both light and electron microscopy, is rather nonspecific. 15 These changes include extreme vesiculation of hepatocytes with vacuolation and swelling of the endoplasmic reticulum and mitochondria and increased lipid accumulation. The Kupffer cells show an increased number of lysosomes and vacuoles. There is widening of the spaces of Disse and dilation of the bile ducts. When two spaced injections of endotoxin were given, Hirsch and his colleagues 16 demonstrated the development of fatty livers in all animals, as well as bromsulfophthalein retention and hyperlipidemia. In a provocative study , Campbell and Gilbert 17 produced giantcell transformation of the liver in both neonatal rats and rabbits by the administration of Escherichia coli endotoxin to the pregnant female . Because of the resemblance to the pathological picture seen in the human neonate, and because of the ubiquitous nature of endotoxin, they suggested this agent as one etiologic cause of this lesion. Mechanisms of Endotoxin Interaction with Other Factors Figure 1 depicts a variety of possible mechanisms that may contribute to hepatic sensitivity to endotoxins arising in the gastrointestinal tract. Altered detoxification. In liver injury, both serum and tissue factors necessary for endotoxin detoxification may be altered, making any given level of circulating endotoxin more toxic. The endotoxin may then cause tissue damage by a number of mechanisms, some direct and some mediated. The earliest hemodynamic changes seen after endotoxin administration involve evidence of vascular constriction 18 soon followed by capillary dilation. 19 Within a
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hydrate abnormalities, endotoxemia in the rhesus monkey has been shown to lead to a 2- to 4-fold increase in plasma triglycerides. 28 Serum and tissue factors also play a role IMMUN£ fACTORS HORMONAL INHU£NC£S in detoxification of endotoxin. Skarnes 29 -t GLOBULINS 4'-'' CORTICOST£ROIOS PRDT£CT studied the early response of rabbits to ESTROGENS £NHANC£ INJURY +~~:IBr~~~: ID___ __> sublethal doses of endotoxin, and demonstrated a 3-fold increase in the plasma ~ level of a heat-stable organophosphateINTESTIRAL £NDOTDXIN + ABSORPTION ~ +PRODUCTION resistant esterase associated with a strikFIG. 1. Endotoxin and liver disease. Possible meching increase in endotoxin-detoxifying caanisms for the toxic hepatic and extrahepatic effects pacity. He concluded that these esterases of endotoxin after liver injury. were a principal site of detoxification and a major defense against circulating endoshort period of time, a significant degree of toxin. Endotoxin also can activate the pooling occurs within the liver and the complement system by the alternate pathsplanchnic circulation depending on the way, and the active products generated species employed. 20 Endotoxin also in- may explain some effects such as smooth creases capillary permeability to mac- muscle constriction and increased vascular romolecules, leading to a loss of protein- permeability. 3 ° Complement is necessary rich fluid from the intravascular for endotoxin protection, and both early compartment . 21 Some of the hemodynamic and late acting components must be preschanges seen in endotoxemia may be due ent for this effect. 31 In addition, completo a direct depression of cardiac function. 22 ment may be important in the uptake of It has been suggested that endotoxin pro- endotoxin by the reticuloendothelial sysduces vascular changes by the release of a tem. 32 Thus, complement alterations in number of vasoactive substances, and that liver injury may contribute to a decreased these materials in turn promote further ability to detoxify these bacterial products. The most important tissue element in endotoxin entry into the circulation from the gut. 23 In the isolated liver preparation, protection against endotoxin is the reticu· however, endotoxin itself may exert a di- loendothelial system (RES). Beeson 33 first rect vasoconstrictive action on the portal demonstrated its importance when he showed that the phenomenon of endotoxin circulation. 24 At the subcellular level, endotoxin has tolerance could be abolished by loading the been shown to have direct metabolic ef- RES with particulate matter. Wiznitzer fects. Nicholas and his associates 25 dem- and his co-workers 34 showed that the noronstrated that endotoxemia abnormally in- mal RES rapidly removes and inactivates creases mitochondrial membrane permea- circulating endotoxin. They found, howbility for potassium and this may cause ever, that when the RES has been dam· mitochondrial swelling. When the mito- aged by a blockading agent or by shock, chondrial membrane becomes more ex~ only a small percentage of the normal tensively damaged, an inhibition of ATP removal and inactivation occurred. These production occurs. The uncoupling effect studies show that damage to the RES can on human mitochondria has been well stud- be expected to result in greatly enhanced ied by Schumer and his colleagues in human endotoxin toxicity . Other work indicates liver cells, 26 and little doubt exists that that the liver is the essential organ in endotoxin can cause hepatic cell injury and detoxification 35 and that carbon blockade death if a toxic level is achieved. Other of the (RES) in rabbits causes a significant liver effects caused by endotoxin include rise in SGOT and SGPT. 36 alterations in carbohydrate metabolism Several agents known to predispose to mediated through an initial activation of liver injury have been shown to depress adenyl cyclase. 27 As well as causing carbo- reticuloendothelial function and thus preEXIRH£PATIC £HECTS KION£Y ClOTTING H£ART f£Y£R
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PROGRESS IN HEPATOLOGY
dispose to endotoxin toxicity. Ali and Nolan 37 showed that the oral administration of small amounts of ethanol to rats delayed clearance of 1311-labeled microaggregated albumin by 40% over controls. These same investigators also serially investigated reticuloendothelial function in rats on a choline-deficient diet, 38 finding a significant depression of phagocytic function at the end of the first week, with a return toward normal as the cirrhotic process advanced. It was suggested that the early functional RES blockade by fatty change might contribute to the ultimate development of cirrhosis by increasing sensitivty to endotoxin. In hypotensive states a significant depression in RES function occurs. 39 In liver disease relative hypotension is often seen with decreased liver perfusion resulting. Olcay and his associates 40 found that portal vein occlusion significantly depress.e d the RES with the development of circulating endotoxemia, presumably of intestinal origin, demonstrable by the limulus assay. These studies reinforce the importance of the integrity of the RES of the liver in detoxifying endotoxins presented to it. Intestinal absorption. In addition to serum and tissue factors that may be altered in liver injury and predispose to endotoxin damage, the amount of endotoxin absorbed may be increased in certain liver diseases. Two mechanisms could be responsible, singly or in combination: increased absorption transmurally or increased production intraluminally. While there has been some dispute in the past about the absorption of endotoxin from the gut, there seems little doubt that it can and does pass through the intestinal wall. Although Sanford and Noyes 41 were unable to detect any label in the blood or tissues after feeding 51 Cr-labeled endotoxin to normal or shocked dogs, Ravin and associates 42 showed the absorption of 32Plabeled E. coli fed to both normal and shocked rabbits. They attributed the failure to show absorption with 51 Cr-labeled material to the fact that this label attaches to the lipid part of the molecule, requiring emulsification before it can be taken up.
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Work from this laboratory using the isolated rat gut sac demonstrates that 51 Cr does pass transmurally, about 0.5% of the endotoxin being recovered in the sac after 2 hr of incubation. 43 Furthermore, as demonstrated in the rat, endotoxin fed orally retains both its toxicity and serological activity within the gastrointestinal tract. 44 In irreversible hemorrhagic shock or traumatic shock, the absorption of endotoxin increases dramatically and is felt by some to be the cause of death in these conditions, 45 and indeed, the injection of nonabsorbable antibiotics into the intestinal tract before the production of irreversible shock prevents early death in animals. 46 The blood pressure in cirrhotics tends often to be low, conceivably promoting endotoxin absorption. The intestinal flora in patients with liver disease has been studied with conflicting results. Experimentally, deprivation of food for 48 hr in mice (a dietary stress not unusual in alcoholic patients) resulted in a marked increase of the bacterial population in the large and small intestine. 47 Furthermore, malabsorptive states are associated with significant colonization of the upper bowel, and malabsorption with stasis may occur both in the alcoholic and the cirrhotic. Martini and his colleagues 48 noted that the ileum of cirrhotic patients contained many coliform organisms and in some cases these organisms extended up to the jejunum and duodenum, areas normally sterile in noncirrhotics. Lal et al. 49 found abnormal colonization of the upper small bowel in 12 of 24 patients with biopsy-proven alcoholic cirrhosis, but found similar colonization in control patients without liver disease. Floch and his co-workers 50 showed higher numbers of anaerobes and Bacteroides in stools of cirrhotic patients. It must be concluded that increased intestinal endotoxin production and absorption in liver disease have not been firmly established, but should be investigated further. Hormonal influences. Experimentally, steroids protect the liver against hepatic damage by bacterial lipopolysaccharides. 52 • 53 Cook and DiLuzio 5 3 showed that steroid-treated rats were essentially
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refractory to the lethal effects of lead acetate and endotoxin. Since Kupffer cell function did not appear to be critical in the lead acetate-endotoxin toxicity, the authors felt the steroids helped maintain the integrity of the parenchymal cell. Janoff and his colleagues 54 found that cortisone inhibited the plasma levels of lysosomal enzymes associated with endotoxemia and attributed the protective effect of steroids to their lysosomal stabilizing action. Snyder et a!. 55 demonstrated that endotoxin and carbon tetrachloride both cause a reduction of tryptophan pyrrolase, and steroid protection against these two hepatotoxins may be related to its ability to induce this enzyme. Estrogens, in contrast, seem to impair hepatic function and to enhance susceptibility to injury by viral and toxic agents. 56 • 57 Studying the effects of estrogens and endotoxin sensitivity, Nolan and Ali 5 8 noted a profound increase in lethality to E. coli endotoxin in rats when estrogen was given 24 hr before the challenge with a 10-fold decrease in the LD 50 after this pretreatment. In contrast to this finding of enhanced susceptibility to E. coli endotoxin after estrogen administration, Trejo and his colleagues 59 found that daily injections of diethylstilbesterol in mice did not result in an enhanced sensitivity to Salmonella enteritides endotoxin. Those authors found a stimulatory effect of the diethylstilbesterol on RES function, while no such alteration was found in the rats given a variety of estrogens 24 hr before clearance studies with microaggregated albumin. A relationship between this striking enhancement in sensitivity to E. coli endotoxin and the property of estrogen to adversely affect certain liver injuries is attractive.
Extra-hepatic Effects of Endotoxin Since the liver stands between the gut and systemic circulation as a filter of bacteria and its products, it might be expected that the failure of this barrier function in liver disease would lead to the entry of these substances into the circulation. There is excellent evidence that this phenomenon does occur. Protell and his
Vol. 69, No . 6
colleagues 60 noted agglutinins to Salmonella in 28 % of patients with chronic active hepatitis, although no evidence for actual Salmonella infection could be found. Simultaneously , a group from Denmark and Great Britain reported a significantly increased antibody titer to intestinal bacteria in patients with liver disease. 61 • 62 Triger and his colleagues 62 found that in. creased antibody levels to E. coli and Bacteroides were present frequently in chronic active hepatitis, and serial sera from patients with acute viral hepatitis showed an increased titer to E. coli that peaked 2 months after onset, before return. ing to normal. Prytz and his Danish col. leagues, 63 on the other hand, found a significantly greater titer of E. coli antibodies in patients with alcoholic cirrhosis as compared to nonalcoholic cirrhosis, and found that the incidence of antibody was high even in the fatty liver stage. It should be pointed out that it is unclear whether these elevated titers indicate specific antibody to gut-associated bacteria, or whether they represent nonspecific elevations that may be present to a number of antigens in inflammatory states. Thomas and his associates 64 noted that the distri· bution of antigen between the liver and spleen determines the magnitude of the immune response, the spleen being a larger antibody producer. They postulated that the increased production of antibody to both gut- and non gut-derived antigen re· fleeted a decreased capacity of the liver to trap antigen, which results in more reaching the spleen. Hepatorenal syndrome. It is possible that endotoxin from the gut may bypass the cirrhotic liver and produce the manifestations of the hepatorenal syndrome. There are striking similarities between the known effects of endotoxin on the renal circulation and the physiological changes seen in the renal failure of liver disease. In dogs, the effects of infusing endotoxin on the kidney are hemodynamic and not due to direct action of the lipopolysaccharide on the renal parenchyma .65 Endotoxin causes severe vasoconstriction in the renal circulation with renal ischemia, 66 and these changes can be blocked by making the
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animal nonspecifically tolerant to the toxin. 67 Similarly in the hepatorenal syndrome, the changes noted are attributable to intense vasoconstriction rather than parenchymal damage. Kew and his associates 6 8 studied renal hemodynamics in cirrhotics by the xenon washout technique and showed decreased intrarenal blood flow . The distribution to, and flow rate through the outer cortex was reduced while juxtamedullary and medullary flow was maintained. Using the same technique in cirrhotic patients with renal failure , Epstein and his colleagues 69 noted an extreme degree of renal hemodynamic instability with intermittent vasoconstriction, and postulated that the hepatorenal syndrome was secondary to active vasoconstriction by an unidentified circulating substance. In this laboratory, injections of E . coli endotoxin produced renal hemorrhage in choline-deficient rats with fatty livers at doses causing no such pat hology in controls. 14 It was postulated that these extrahepatic manifestations were due to the inability of the liver to detoxify normally innocuous, amounts of endotoxin . Wardle and Wright' 0 produced obstructive jaundice in rats by tying their common ducts, and showed that a single dose of endotoxin produced death with intravascular coagulation. The authors felt this finding explained the renal failure which occurs when biliary tract operations are complicated by bacteremia . Using a limulus lysate assay to detect circulating endotoxin in patients with acute massive hepatic necrosis, Wilkinson and his associates 71 beautifully demonstrated a close correlation between endotoxemia and the development of renal failure and intravascular coagulation . All these studies suggest that endotoxin must be a strong candidate for the unknown vasoconstrictive agent associated with renal failure in liver disease . Intravascular coagulation and fever . Other extrahepatic manifestations of liver disease may have their origin in low-grade endotoxemia . Endotoxin is known to produce intravascular coagulation, and this occurs regularly in septicemia. 72 Gans and his colleagues 73 noted that the dis-
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seminated intravascular coagulation seen in the hepatectomized animal could be delayed by the prior administration of neomycin. These investigators postulated that endotoxin from the gut that would ordinarily be detoxified by the liver was responsible for the defibrination. As cited previously, Wilkinson and his colleagues 71 supported this contention by correlating intravascular coagulation with endotoxemia in patients with hepatic necrosis . Fever of significant degree and duration has been found in a number of patients with alcoholic liver disease without any cultural evidence of infection. 74 Endotoxin injected intravenously in man produces fever of a similar nature. 75 Preliminary studies from this laboratory using the limulus test indicate a correlation between the fever of Laennec's cirrhosis and the presence of circulating endotoxin (Nolan JP, unpublished observations.). Myocardial function. Depressed cardiac function has been regularly seen in cirrhotic patients with portal hypertension, the failure often being hyperdynamic in nature .Endotoxemia has been shown experimentally to produce myocardial depression both in isolated preparations and in intact primates. 76 • 77 The data of Siegel and his colleagues 7 8 that vascular tone and cardiac output in sick cirrhotics resemble that seen in patients with septic shock raises the possibility that these cardiovascular abnormalities in liver disease may reflect the effect of circulating endotoxin on the cardiovascular system.
Modification of Endotoxicity If endotoxin plays a role in determining the extent of liver injury caused by other agents, then attempts at lessening the effect of these gut lipopolysaccharides might benefit both the hepatic disease and its extrahepatic effects as well. Some promising work has already been accomplished in this area and table 1 lists some possible modifications that may be done. Animals can be made nonspecifically tolerant to a variety of endotoxins by giving increasingly large doses of a single endotoxin over a short period of time. 79 Jennings et al. 80 made dogs tolerant over a
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1. Modification of endotoxin toxicity in liver
diseas e
A. Increasing resistance 1. Development of nonspecific tolerance 2. Specific immunization B. Decreasing gut absorption 1. Oral nonabsorbable antibiotics 2. Cholestyramine resin 3. Immunization C. Removal of circulating endotoxin 1. Hemoperfusion with Dowex 1-X2 resin or activated charcoal
2-week to 2-month period and were able to almost completely protect against the hemodynamic and biochemical effects of a normally lethal endotoxin challenge . The mechanism for this nonspecific tolerance is not known. Endotoxin is a known potent stimulator of RES activity, 81 but also causes a marked increase in antibody formation to the endotoxin used. 80 Using a perfused rabbit liver from endotoxin-tolerant and nontolerant rabbits, Greisman and Woodward 82 concluded that tolerance to the pyrogenic activity of bacterial endotoxin is based upon increased uptake of the toxin by hepatic Kupffer cells which have become refractory to release of endogenous pyrogen. A single injection of E. coli endotoxin in an oil-water emulsion was shown to prolong survival in mice both to endotoxin challenge and to E. coli peritonitis. 83 If endotoxin plays a role in furthering liver injury, then the state of nonspecific tolerance might be expected to protect against liver injury by an unrelated toxin. Nolan and Ali 84 found such an effect in comparing the extent of carbon tetrachloride injury . Compared with controls, endotoxin-tolerant rats were significantly more resistant to the hepatotoxic effect of CC1 4 as measured by enzyme changes and amount of histological damage at the time of killing 24 and 48 hr later. Since tolerance is a short-lived phenomenon, specific immunization with endotoxin vaccines might more effectively protect animals from liver damage. Some materials may even confer protection against heterologous endotoxins. Kunin et al. 85 discovered an antigen common to all
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the enterobacteriacae that protects animals against induced infection with a variety of gram-negative bacteria. Further studies demonstrated that this common antigen induces passive immunity to heterologous endotoxins as well (Nolan JP, McDevitt JJ, unpublished data.). McCabe has used R mutants of a Salmonella to immunize mice against E. coli and Klebsiella pneumoniae, 86 and Braude and Douglas 87 were able to passively transfer immunity to heterologous endotoxins with a mutant E. coli strain, experiments proving the feasibility of cross-protection by suitable immunization. Another approach to modifying endotoxin toxicity would seek to reduce the amount of endotoxin absorbed, by shrinking the pool of gram-negative bacteria in the gut or by increasing the mucosal barrier to the lipopolysaccharide. Neomycin retards the development of choline-deficiency cirrhosis in rats, 9 but while the drug has been successfully used to reduce urease splitting bacteria in patients with hepatic encephalopathy, a controlled study on its effect in continuing liver damage has not been attempted. Nolan and Ali 43 have shown that cholestyramine not only binds bile acids and a variety of drugs, but also tightly binds E . coli endotoxin, preventing both its toxicity and absorption through the isolated rat gut. Since secretory IgA antibodies protect against enteric bacterial infections, 88 specific immunization with a heterologous endotoxin vaccine might be beneficial in decreasing endotoxin absorption. Lastly, treatment of liver failure with hemoperfusion through charged resins and charcoal has been attempted. Wilson et al. 89 prolonged survival of dogs with massive liver necrosis using Dowex-1 resins, and Gazzard and associates 90 treated some patients with massive hepatic necrosis with benefit by passing their blood through activated charcoal columns. Since both Dowex-1 resins and charcoal have been shown to efficiently remove labeled endotoxin in vitro, 91 the beneficial effects observed in liver failure may be due in part, to removal of circulating endotoxin.
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Unresolved Problems Certain theoretical difficulties are present which may not fit into the proposed role for endotoxin in perpetuating liver injury. If endotoxin regularly enters the circulation after hepatic damage, then a state of tolerance or immunity might be expected to develop, preventing or modifying the extent of further injury. Relatively little is known, however, about the status of local hepatic defense mechanisms in liver disease, and these defenses may be significantly altered. In addition, studies are needed, but not available, correlating the development of antibodies to gut bacteria and the clinical course of liver disease. It is known that nonspecific increases in RES function can occur with appropriate stimuli. Therefore, it could be argued that low-grade endotoxemia should stimulate the RES and thus result in faster removal of circulating endotoxin with some resultant protection. The RES in liver injury, however, may be incapable of normal stimulation by bacterial lipopolysaccharides. Final answers about RES function are not yet available. Lastly, while it may be possible to demonstrate bacterial antigens in liver tissue, it will be difficult to assess their pathogenic role. Until more precise methods of identifying and quantitating endotoxin in the liver are found, the evidence for its role in liver disease will remain intriguing but indirect. Summary Evidence is present that a common pathway for hepatic injury by a variety of agents may result from impairment of the liver's ability to detoxify bacterial endotoxins from the gastrointestinal tract. Many factors may structurally, metabolically, or hormonally alter the normal liver's ability to render innocuous the small amounts of lipopolysaccharide ordinarily presented to it. This impairment may accentuate existing hepatic damage by allowing toxic levels of endotoxin to develop in the liver tissue, and by allowing endotoxin entry into the systemic circulation, may
also lead to extrahepatic effects. Studies are cited that: (1) support a role for intraintestinal endotoxin in the development of experimental cirrhosis, (2) demonstrate how liver injury alters endotoxin detoxification, (3) examine the role of intestinal production and absorption of bacterial lipopolysaccharides in liver disease, and (4) point to a role for endotoxemia in extrahepatic manifestations of liver injury as well. Studies are also reviewed that suggest possible mechanisms for modifying endotoxicity in hepatic damage. REFERENCES 1. Leach BE, Forbes JC: Sulfonamide drugs as protective agents against carbon tetrachloride poisoning. Proc Soc Exp Bioi Med 48:361- 363, 1941 2. Wilson JW, Leduc EH, Arnold LE: Protective action of sulfa drugs against CCI, poisoning in mice. Fed Proc 9:349, 1950 3. Leduc EH: Sulfaguanidine protection of mouse liver from carbon tetrachloride-induced necrosis. Lab Invest 29:186-196, 1973 4. Luckey TD, Reyniers JA, Gyorgy P, et al: Germfree animals and liver necrosis. Ann N Y Acad Sci 57:932-935, 1954 5. Gyorgy P: Antibiotics and liver injury. Ann NY Acad Sci 57:925-932, 1954 6. Rutenburg AM, Sonnenblick E, Koven I, et al: The role of intestinal bacteria in the development of dietary cirrhosis in rats. J Exp Med 106:1-13, 1957 7. Salmon WD, Newberne PM: Effect of antibiotics, sulfonamides, and a nitrofuran on development of hepatic cirrhosis in choline-deficient rats. J Nutr 76:483-486, 1962 8. Levinson SM , Tennant B: Some metabolic and nutritional studies with germ-free animals. Fed Proc 22:109- 119, 1963 9. Broitman SA, Gottlieb LS, Zamcheck N: Influence of neomycin and ingested endotoxin in the pathogensis of choline deficiency cirrhosis in the adult rat . J Exp Med 119:633-641, 1964 10. Dubos RJ , Schaedler RW: Effect of nutrition on the resistance of mice to endotoxin and on the bactericidal power of their tissues. J Exp Med 110:935-950, 1959 11. Rutenburg AM, Sonnenblick E, Koven I, et al: Comparative response of normal and cirrhotic rats to intravenously injected bacteria. Proc Soc Exp Bioi Med 101:279-281, 959 12. Formal SB, Noyes HE, Schneider H: Experimental shigella infections . III. Sensitivity of normal, starved, and carbon tetrachloride treated guinea
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pigs to endotoxin. Proc Soc Exp Bioi Med 103: 415-418, 1960 13. Farra r WE, Edison M, Kant TH : Susceptibility of rabbits to pyrogenic and lethal effects of endotoxin after acute liver injury. Proc Soc Exp Bioi Med 128:7 11-71 .5, 1968 14. Nolan JP, Ali MV: Endotoxin and the liver I. Toxicity in rats with choline deficient fatty livers . Proc Soc Exp Bioi Med 129:29-3 1, 1968 1.5 . Levy E, Slusser RJ, Ruebner BH: Hepatic changes produced by a single dose of endotoxin in the mouse by electron microscopy. Am J Pathol .52:477-502, 1968 16. Hirsch RL, McKay DG, Travers RI, et al: Hyperlipidemia, fatty liver and bromsulfophtalein retention. ,J Lipid Res .5:.563-.568, 1964 17. Campbell LV, Gilbert EF: Experimental giant cell transformation in the liver induced by E. coli endotoxin. Am J Pathol 51:855-868, 1967 18. Hinshaw LB, Vick A, Wittmers LE, et a!: Changes in total peripheral resistance in endotoxic shock. Proc Soc Exp Bioi Med 108:24-27, 1961 19. Chien S , Chang C, Dellenback RJ , et al: Hemodynamic changes in endotoxin shock. Am J Physiol 210:1401-1410, 1966 20. Blattberg B, Levy MN: Early hepatic and extrahepatic pooling in response to endotoxin. Am J Physiol 219:460-463, 1970 21. Chien S, Sinclair DG, Dellenback RJ , eta!: Effect of endotoxin on capillary permeability to macromolecules. Am ,J Physiol 207:518-522, 1964 22. Alican F, Dalton ML, Hardy DJ: Experimental endotoxin shock Circulatory changes with emphasis upon cardiac function. Am J Surg 103:702-708, 1962 23. Cuevas P , Fine J: Production of fatal endotoxic shock by vasoactive substances. Gastroenterology 64:285-291, 1973 24. Nolan JP, O'Connell CJ: Vascular response in the isolated rat liver. I. Endotoxin , direct effects. J Exp Med 122:1063-1073, 1965 25. Nicholas GG , Mela LM , Miller LD: Early alterations in mitochondrial membrane transport during endotoxemia. J Surg Res 16:375-383, 1974 26. Schumer W, DasGupta TK, Moss GS, et al: Effect of endotoxemia on liver cell mitochondria in man . Ann Surg 171:875-882, 1970 27. Gimpel L, Hodgins DS , Jacobson ED : Effect of endotoxin on hepatic adenylate cyclase activity. Circ Shock 1:31-37, 1974 28. Fiser RH, Denniston JC, Beisel WR: Endotoxemia in the rhesus monkey. Alterations in host lipid and carbohydrate metabolism . Pediatr Res 8:13-17 , 1974 29. Skarnes RC: Host defense against bacterial endotoxemia . Mechanism in normal animals. J Exp Med 132:300-316, 1970 30 . Gewurz H , Snyderman R, Mergen hagen SE, et al:
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Effects of endotoxic lipopolysaccharides on the complement system. In Bacterial Endotoxins. Vol. 5. New York , Academic Press, 1971 p. 127-146 31. May JE, Kane MA, Frank MM: Host defense against bacterial endotoxemia, contribution of the early and late components of complement to detoxification. J lmmunol 109:893-895, 1972 32. Golub S, Groschel D, Nowotny A : Factors which affect the recticuloendothelial system uptake of bacterial endotoxins. J Reticuloendothel Soc 5:324-329, 1968 33. Beeson PB: Tolerance to bacterial pyrogens. II . Role of the reticuloendothelial system. J Exp Med 86:39.-44, 1947 34 . Wiznitzer T, Better N, Rachlin W , et al: In vivo detoxification of endotoxin by the reticuloendothelial system . J Exp Med 112:1157-1163, 1960 35. Farrar WE, Corwin LM: The essential role of the liver in detoxification of endotoxin. Ann N Y Acad Sci 133:668-684, 1966 36. Fisher ER, Fisher B: Hepatic damage by reticuloendothelial interference. Arch Pathol 75:191-195, 1963 37. Ali, MV , Nolan JP: Alcohol-induced depression of reticuloendothelial function in the"rat. J Lab Clin Med 70:295-301, 1967 38. Ali MV, Nolan JP: Serial assessment of reticuloendothelial function in experimentally induced nutritional cirrhosis. Lab Invest 20:184- 189, 1969 39. Hershey SG, Altura BM: Function of the reticuloendothelial system in experimental shock and combined injury . Anesthesiology 30:138-143, 1969 40. Olcay I, Kitahama A, Miller RH , et al: Reticula· endothelial dysfunction and endotoxemia follow· ing portal vein occlusion. Surgery 75:64-70, 1970 41. Sanford JP , Noyes HE: Studies on the absorption of Escherichia coli endotoxin from the gastroin· testinal tract of dogs in the pathogenesis of irreversible hemorrhagic shock. J Clin Invest 37:1425-1435, 1958 42. Rav in HA , Rowley P , Jenkins C, et al: On the absorption of bacterial endotoxin from the gas· trointestinal tract of the norma l and shocked animal. J Exp Med 112:783-792, 1960 43. Nolan JP , Ali MV: Effect of cholestyramine on endotoxin toxicity and absorption. Am J Dig Dis 17:161-166, 1972 44. Verczi I: Stability of Escherichia coli endotoxin in the rat gastrointestinal tract. J Pathol Bacterial 96:487-491, 1968 45. Woodruff, PWH, O'Carroll DI, Koizumi S, et al: Role of the intestinal flora in major trauma. J Infect Dis 128:290-294, 1973 46. Cuevas P, Fine J: Demonstration of a lethal endo· toxemia in experimental occlusion of the superior mesenteric artery. Surg Gynecol Obstet 132:8183, 1971 47. Tannock GW, Savage DC: Influences of dietary
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and environmental stress on microbial popula- 63. Prytz H, Bjornboe M, Orskov F, et al: Antibodies to Escherichia coli in alcoholic and nonalcoholic tions in the murine gastrointestinal tract. Infect patients with cirrhosis of the liver or fatty liver. lrnmun 9:591-598, 1974 Scand J Gastroenterology 4:433-438, 1973 48. Martini GA, Phear EA, Ruener B, et a!: The bacterial content of the small intestine in noma! 64. Thomas HC, McSween RNM, White RG : Hypothesis-role of the liver in controlling the immuand cirrhotic patients, relation to methionine nogenicity of commensal bacteria in the gut. toxicity. Clin Sci 16:35-51, 1957 Lancet 1:1288-1291, 1973 49 . La! D, Gorbach SL, Levitan R: Intestinal microflora in patients with alcoholic cirrhosis. Urea 65. Gillenwat er JY, Dooley ES, Frohlich ED: Effects of endotoxin in renal function and hemodynamsplitting bacteria and neomycin resistance. Gasics. Am J Physiol 205:293-297, 1963 troenterology 62:275-279, 1972 50. Floch MH, Katz J, Conn HO: Qualitative and 66. Selmyer JP, Reynolds DG, Swan KG: Renal blood flow during endotoxin shock in the subhuquantitative relationships of the fecal flora in man primate. Surg Gynecol Obstet 137:2-6, cirrhotic patients with portal systemic enceph1973 alopathy and following portacaval anastomosis. 67. Bates HM, Margolin S: Endotoxin-induced inhibiGastroenterology 59:70-75, 1970 tion of renal function in the mouse. Proc Soc Exp 51. Thomas L and Smith RT: Effect of cortisone on Bioi Med 129:642-646, 1968 response to endotoxin in mature rabbits. Proc Soc Exp Bioi Med 86:810-813, 1954 68. Kew MC, Varma RR, Williams HS, et al: Renal 52. Levitin H, Kendrick ML, Kass EH : Effect of and intrarenal blood flow in cirrhosis of the liver. route of administration on protective action of Lancet 2:504-509, 1971 corticosterone and cortisol against endotoxin. 69. Epstein M, Berk DP, Hollenberg NK, et al: Renal Proc Soc Exp Bioi Med 93:306-309, 1956 failure in the patient with cirrhosis. The role of 53. Cook JA, DiLuzio NR: Protective effect of cysactive vasoconstriction. Am J Med 49:175-185, teine a nd methylprednisolone in lead-acetate1970 endotoxin induced shock. Exp Mol Pathol 70. Wardle EN, Wright NA: Endotoxin and acute renal failure associated with obstructive jaundice. 19:127-138, 1973 54. Janoff A, Weissman G, Zweifach BW, et al: Br Med J 4:472-474, 1970 Pathogenesis of experimental shock. IV . Studies 71. Wilkinson SP, Arroyo V, Gazzard BG, et a!: Rel ationship of renal impairment and hemoron lysosomes in normal and tolerant animals subjected to lethal trauma and endotoxemia. J rhagic diathesis to endotoxernia in fulminant Exp Med 116:451-466, 1962 hepatic failure . Lancet 1:521-524, 1974 55. Snyder IS, Agarwal MK, Berry LJ: Influence of 72. Corrigan JJ, Ray WL, May N: Changes in the ca rbon tetrachloride on inducible liver enzymes blood coagulation syste m associated with sepand response to endotoxin in mice. J Bacteriol ticemia. N Eng! J Med 279:851-856, 1968 73 . Gans H, Mori K, Lindsey E , et al: Septicemia as a 94:1817-1823, 1967 56. Bearn AG, Kunkel HG, Slater RJ: The problem of manifestation of acute liver failure. Surg Gynecol chronic liver disease in young women. Am J Med Obstet 132:783-790, 1971 21:3-15, 1956 74. Tisdale WA, Klatskin G : The fever of Laennec's 57. Jones WA, Cohen RB: The effect of estrogen on cirrhosis. Yale J Bioi Med 33:94-106, 1960 the liver in murine viral hepatitis . Am J Pathol 75. Greisman SE, Hornick RB, Carozza FA, et a!: 42:237-243, 1963 The role of endotoxin during typhoid fever and 58. Nolan JP , Ali, MV: Late effect of estrogen on tularemia in man. I. Acquisition of tolerance to endotoxin response in the rat. J Clin Lab Med endotoxin. J Clin Invest 42:1064-1075, 1963 71:501-510, 1968 76. MacNicol MF, Goldberg AH, Clowes, GHA: De59. Trejo RA, Loose LD, DiLuzio NR: Influence of pression of t he isolated heart muscle by bacterial diethylstilbesterol on reticuloendothelial fun cendotoxin. J Trauma 13:554-558, 1973 tion, tissue distribution, and detoxification of S. 77. Reichgott MJ, Melmon KL, Forsyth RP, et a! : enteritides endotoxin. J Reticuloendothel Soc 11: Cardiovascular and metabolic effects of whole or 88- 97, 1972 fractionated gram-negative bacterial endotoxin in the unanesthetized rhesus monkey. Circ Res 60. Protell RL, Soloway RD , Martin WJ, et al: Anti-salmonella agglutinins in chronic active 33:346-352, 1973 liver disease . Lancet 2:330-331, 1971 78. Siegel JH, Greenspa n M, Cohen JD, et al: The 61. Bjornboe M, Prytz H, Orskov F: Antibodies to prognostic implications of altered physiology in intestinal microbes in serum of patients with operations for portal hypertension. Surg Gynecol cirrhosis of the liver. Lan cet 1:58-60, 1972 Obstet 126:249-262, 1968 62. Triger DR, Alp MH, Wright R: Bacterial and 79. Abernathy RS : Homologous and heterologous dietary antibodies in liver disea se. Lance t resistance of mice given bacterial endotoxins. J 1:60-63, 1972 Immunol 78:387-394, 1957
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80. Jennings PB, Simmons RL, Sleeman HK, et al: Hemodynamic, biochemical and coagulation alterations in endotoxin shock . Modification by induced tolerance in the dog. Ann Surg 167:204214, 1968 81. Arredondo MI, Kampschmidt RF: Effect of endotoxins on phagocytic activity of the reticuloendothelial system of the rat. Proc Soc Exp Bioi Med 112:78-81, 1963 82. Greisman SE, Woodward CL: Mechanisms of endotoxin tolerance . VII. The role of the li ver. J Immunol 105:1468-1476, 1970 83. Cuevas P , Fine J , Monaco AP : Successfu l induction of increased resistance to gram-nega tive bacteria and to endotoxin in immunosuppressed mice . Am J Surg 127:460-464, 1974 84. Nolan JP, Ali MV: Endotoxin and the li ver. II. Effect of tolerance on carbon tetrachlorideinduced injury. J Med 4:28-38, 1973 85. Kunin CM , Beard MV, Halmagy NE: Evidence for a common hapten associated with endotoxin fractions of E. coli and ot her Enterobacteriaceae . Proc Soc Exp Hiol Med Ill : 160-166, 1962
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86. McCabe WR: Immunization with R mutants ofS. Minnesota. I. Protection against challenge with heterologous gram-negative bacilli. J Immunol 108:601-610, 1972 87. Braude AI, Douglas H: Passive immunization against the local Shwartzman reaction . J Immunol 108:505-5 12, 1972 88. Fubara ES, Freter R: Protection against enteric bacteria l infection by secretory IgA antibodies. J Immunol 111: 395-403, 1973 89. Wilson RA , Hofmann, AF, Kuster GGR: Toward an artificial li ver. II. Removal of cholephilic anions from dogs with biliary obstruction by hemoperfusion through charged and uncharged resins. Gastroenterology 66:95-107, 197 4 90. Gazzard BG, Portman B, Weston MG et al: Charcoal hemoperfus ion in the treatment of fuJ. minant hepatic failure . Lancet 1:1301 - 1306, 1974 91. Nolan JP, McDevitt JJ, Goldmann, GS: Charac· teristics of endotoxin binding in vitro by charged and uncharged resin. Proc Soc Exp Bioi Med 149: 766-770, 1975