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This is to certify that the dissertation entitled THE ROLE OF THE INNATE IMMUNE SYSTEM IN THE LPS-INDUCED POTENTIATION OF ALLYL ALCOHOL HEPATOTOXICITY presented by Rosie Antionette Sneed has been accepted towards fulfillment of the requirements for Ph.D. degree in Pharmacology and Tox1cology r» " “ K? Major p/pofessor / Date 3/39/00 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE UUL I am 11/00 woman-0.1.9559.“ THE ROLE OF THE INNATE IMMUNE SYSTEM IN THE LPS-INDUCED POTENTIATION OF ALLYL ALCOHOL HEPATOTOXICITY BY Rosie Antionette Sneed A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 2000 ABSTRACT THE ROLE OF THE INNATE IMMUNE SYSTEM IN THE LPS-INDUCED POTENTIATION OF ALLYL ALCOHOL HEPATOTOXICITY By Rosie Antionette Sneed Lipopolysaccharide (LPS) causes liver damage at relatively large doses in rats (>2 mg/kg). Smaller doses, however may modify the response to other hepatotoxicants. Pretreatment of rats with a small dose of LPS (100 pg/kg) significantly increased the hepatotoxicity of a subsequent, nonlethal dose of allyl alcohol (30 mg/kg). The presence of exogenous LPS accelerated the development of liver injury compared to controls and the lesions produced resembled those caused by a higher dose of allyl alcohol. Doses of LPS up to 4 orders of magnitude lower than 100 ug/kg were able to potentiate the hepatotoxicity of allyl alcohol significantly, and the presence of LPS caused a leftward shift in the dose-response curve of allyl alcohol. Allyl alcohol had to be metabolized to the aldehyde, acrolein, for the potentiation to be seen; however, LPS did not exert its effect by altering the activity of alcohol dehydrogenase. Likewise, LPS did not affect hepatocellular levels of reduced glutathione. Inhibition of either Kupffer cell function or depletion of circulating neutrophils afforded animals significant protection from LPS-induced enhancement of allyl alcohol hepatotoxicity. These data indicated that these cell populations played a significant role in this model of liver injury. Kupffer cells are key regulators of the host response to infection. They exert their effects on adjacent cells via a number of powerful biochemical mediators such as cytokines and prostaglandins. Inhibiting the synthesis of one of these mediators, tumor necrosis factor-alpha (TNF), has been shown to be protective in models of liver injury using high doses of LPS. Administration of PTX, an inhibitor of TNF synthesis, protected animals from LPS-induced potentiation of allyl alcohol hepatotoxicity, but the administration of a more specific anti-TNF serum did not. These results suggest that PTX protected animals through one of its other pharmacological properties such as increasing the intracellular levels of cyclic adenine monophosphate (cAMP) in Kupffer cells or attenuation of free radical formation. Direct exposure of cultured hepatocytes to LPS did not increase their sensitivity to allyl alcohol, and neither did direct exposure to TNF. These data suggest that neither LPS nor TNF alone was sufficient to sensitize hepatocytes to allyl alcohol. The data may also suggest that a more complex series of events occurs in vivo than simple exposure to TNF. In conclusion, LPS potentiates the hepatotoxicity of allyl alcohol, and cells of the innate immune system play a critical role in the ability of LPS to potentiate the hepatotoxicity of allyl alcohol. Copyright by Rosie Antionette Sneed 2000 To Mother and Father, the people who always encouraged me to follow my dreams. ACKNOWLEDGMENTS My achievement of the degree Doctor of Philosophy was accomplished through the generous commitment of time and resources by numerous people. I would like to thank the many people who have provided me with much needed help along the way. The first in a long line of people is Dr. Patricia Ganey, my thesis advisor and mentor. She provided the original idea for my research and guided me along the way to the final experiment. The members of my graduate committee, Dr. Robert Roth, Dr. Norbert Kaminski, and Dr. James Pestka, lent me their expertise and also provided much needed guidance. Many others provided invaluable help during my time at Michigan State University. I want to thank Ms. Patricia Lowrie of the Women’s Resource Center for her generous financial support during my time in the Department of Pharmacology and Toxicology. Dr. Margarita Contreras of Pharmacia and Upjohn was always a source of friendship and moral support. My fellow members of the Roth and Ganey laboratories, both past and present, were always ready to provide expertise, friendship and good cheer. They include Dr. Frederic Moulin, Mr. John Buchweitz, Dr. Dwayne Hill, Dr. A. Eric Schultze, Dr. Alan Brown, Dr. Paul Jean, Dr. Julia Pearson, Ms. Therese Schmidt, Mr. Shawn Kinser, Ms. Eva Barton, Dr. C. Charles vi Barton, Mr. Steve Yee, Dr. Bryan Copple, Ms. Sarah Kessel, Dr. Pat Lappin, and Dr. Marc Baillie. I want to thank the administrative staff of the Department of Pharmacology and Toxicology, Ms. Diane Hummel, Ms. Mickey Vanderlip, and Ms. Nelda Carpenter, for their help in negotiating the maze of university bureaucracy and paperwork. And last but by no means least, I want to thank the two people who provided my first opportunities to participate in basic scientific research, which in turn fueled my desire to become a research scientist. Dr. Carolyn Cousin of the University of the District of Columbia graciously allowed me to assist her with her work on Schistosoma mansoni. She was my first scientific mentor. I also have heartfelt thanks for Ms. Anita Stevenson. She was my host for one summer at Los Alamos National Laboratory. The experience that I gained from my stay there was invaluable to my career as a researcher. vii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION CHAPTER 1 GENERAL INTRODUCTION 1. A. The Innate Immune System 1. A. 1. Macrophages 1. A. 2. Kupffer Cells 1. A. 2. 1. Hepatic Architecture 1. A. 2. 2. Location and Function of Kupffer Cells 1. A. 2. 3. Interaction of Kupffer Cells and Hepatocytes 1. A. 3. Neutrophils 1. A. 4. Eosinophils and Basophils 1. A. 5. Interaction of LPS and the Innate Immune System 1. B. Lipopolysaccharide 1. B. 1. Structure 1. B. .Sources of LPS' In Host Systems 1. B. .Effects of LPS on Mammalian Physiology 1. ..3 1. Effects of LPS on the Liver 1. .3 2. Biochemical Mediators of LPS-induced Hepatotoxicity .Cellular Effects of LPS .4.1. LPS Binding Protein ..4 2. CD14 4. 3. Intracellular Mechanisms of Action 1 Summary 1. C. LPS Potentiation of Xenobiotic Hepatotoxicity 1. C. 1. Role of the Innate Immune System .Allyl Alcohol .,Structure Chemistry, and Metabolism .Pathology .Mechanisms of Toxicity ..3 1. Binding to Protein Sulfhydryl Groups ..3 2. Peroxidation of Lipid Membranes UUwN—s viii xii xiii XV A Esaaxzaamwmww 32 32 34 35 37 38 38 40 40 47 48 48 50 1. D. 4. Summary 1. E. Conclusions CHAPTER 2 CHARACTERIZATION OF LPS-INDUCED ENHANCEMENT OF ALLYL ALCOHOL HEPATOTOXICITY 2. A. Abstract 2. B. Introduction 2. C. Materials and Methods ..C 1. Materials .Animals .Treatment Protocol .Assessment of Hepatotoxicity .Determination of Factors Influencing the Metabolism of Allyl Alcohol .Determination of Hepatic Concentrations of GSH and GSSG 7. Statistical Analysis . Results ..D 1. Effect of LPS Pretreatment on Allyl Alcohol-Induced Hepatotoxicity 2. D. 2. Role of Metabolism of Allyl Alcohol 2. D. 3. Effect of LPS Treatment on Hepatocellular Concentrations of GSH and GSSG 2. E. Discussion NNNIQN 91.th O) ..C ..C ..C ..C C. C. N NON N CHAPTER 3 THE ROLE OF CELLS OF THE INNATE IMMUNE SYSTEM IN LPS- INDUCED ENHANCEMENT OF ALLYL ALCOHOL HEPATOTOXICITY 3. A. Abstract 3. B. Introduction 3. C. Materials and Methods 3. C. 1. Materials ..2 Production of Anti-Neutrophil lg ..3 Animals 4. Treatment Protocols ..C 4.1. Inactivation of Kupffer Cells .C. 4. 2. Depletion of Circulating Neutrophils .5 Immunohistochemical Staining of Neutrophils In Liver Sections 3. ..6 Assessment of Hepatotoxicity 3 7. Statistical Analysis 52 53 55 56 57 57 58 58 59 60 61 62 63 63 72 73 74 82 82 83 84 85 85 85 87 87 87 88 88 89 90 3. D. Results 3. D. 1. Protective Effect of GdCl3 3. D. 2. Protection by Depletion of Circulating Neutrophils 3. E. Discussion CHAPTER 4 ROLE OF TNF IN LPS-INDUCED POTENTIATION OF ALLYL ALCOHOL HEPATOTOXICITY . Abstract Introduction . Materials and Methods .C. 1. Materials . C. 2. Production of Anti-TNF Serum . C. 3. Animals . C. 4. Isolation of Hepatocytes . C. 5. Treatment Protocols 4. C. 5. 1. Treatment of Animals with Anti-TNF Serum 4. C. 5. 2. Treatment of Animals with Pentoxifylline . C. 6. Assessment of Hepatocyte Cytotoxicity C. 7. C. 8. as» > A-k-h-k-hop Assessment of Hepatotoxicity Determination of Activity of TNF . C. 9. Assessment of Alcohol Dehydrogenase Activity in Liver Homogenates . C. 10. Statistical Analysis . Results . D. 1. Effect of In Vitro Exposure to LPS on Allyl Alcohol- Induced Cytotoxicity in Isolated Hepatocytes . D. 2. Protection from LPS-Induced Enhancement of Allyl Alcohol Hepatotoxicity by PTX . D. 3. Lack of Effect of Inactivation of TNF on LPS-Induced Potentiation of Allyl Alcohol Hepatotoxicity . D. 4. Effect of In Vitro Exposure to TNF on Allyl Alcohol- Induced Cytotoxicity in Isolated Hepatocytes . D. 5. Allyl Alcohol-Induced Cytotoxicity in Isolated Hepatocytes from LPS-Treated Rats 4. E. Discussion 5‘ 4s 4:- 4: #U«l> S's-94> .h CHAPTER 5 SUMMARIES AND CONCLUSIONS 5. A. Characterization of the Model 5. B. The Role of the Innate Immune System 5. C. The Role of TNF 5. D. A New Hypothesis 91 91 95 101 107 107 108 109 111 111 111 112 113 114 114 115 115 116 119 119 120 120 120 121 128 134 134 135 143 143 144 145 149 150 Bibliography 1 55 xi 1.1 2.1 2.2 2.3 3.1 3.2 3.3 4.1 4.2 LIST OF TABLES Title Xenobiotics that interact with LPS to cause increased liver injury Pathological changes in the livers of rats Factors influencing or reflecting the metabolism of allyl alcohol Comparison of hepatocellular levels of reduced and oxidized glutathione in rats pretreated with saline Comparison of hepatocellular levels of reduced and oxidized glutathione in rats pretreated with GdCl3 Effect of anti-PMN lg on the numbers of circulating neutrophils Infiltration of neutrophils into hepatic sinusoids Effect of PTX on LPS-induced increase in plasma TNF activity Effect of PTX on the activity of alcohol dehydrogenase in LPS- treated rats xii page 41 67 77 78 94 96 100 125 129 1.1 1.2 1.3 2.1 2.2 2.3 2.4 3.1 3.2 4.1 4.2 4.3 LIST OF FIGURES Title Structure of LPS. Products of LPS-stimulated macrophages. Structure and hepatic metabolism of allyl alcohol. LPS enhancement of the hepatotoxicity of allyl alcohol. Time course of development of LPS-enhanced allyl alcohol hepatotoxicity. Dose-response relations for plasma ALT activity after treatment with LPS and/or allyl alcohol. Protection from LPS-induced enhancement of allyl alcohol hepatotoxicity by 4-methylpyrazole. Protection from LPS-induced potentiation of allyl alcohol hepatotoxicity by GdCI3. Protection from LPS-induced potentiation of allyl alcohol hepatotoxicity by depletion of circulating neutrophils. Dose-response for anti-TNF serum. Lack of effect of LPS exposure in Vitro on allyl alcohol cytotoxicity towards hepatic parenchymal cells. Protection by PTX from LPS enhancement of allyl alcohol hepatotoxicity. xiii page 16 23 45 65 68 75 92 97 117 122 126 4.4 4.5 4.6 5.1 5.2 Lack of protection from LPS enhancement of allyl alcohol hepatotoxicity by anti-TNF serum. Lack of effect of TNF exposure in Vitro on allyl alcohol cytotoxicity towards hepatic parenchymal cells. Cytotoxicity of allyl alcohol toward hepatocytes isolated from rats treated in vivo with LPS. Comparison of the protective effects of depletion of functional Kupffer cells versus circulating neutrophils. A hypothetical model. xiv 130 132 136 147 152 ADH ALDH AN IT ALT ANOVA AST BCA BPI CAMP CAP CD CETP C02 E. coli gm GdCI3 GSH GSSH HPLC IKB IL-10I lL-6 ip iv KC kD KDO KOH KHCO3 LBP LPS LIST OF ABBREVIATIONS allyl alcohol alcohol dehydrogenase aldehyde dehydrogenase alpha-naphthylisothiocyanate alanine aminotransferase analysis of variance aspartate aminotransferase bicinchoninic acid bactericidal/ permeability increasing protein Celsius cyclic adenine monophosphate ceramide activated kinase cluster of designation cholesterol ester transport protein carbon dioxide dexter Escherichia coli gravity gram gadolinium chloride reduced glutathione oxidized glutathione immunoglobulin high performance liquid chromatography inhibitory subunit of N kappa B interleukin 1 alpha interleukin 6 intraperitoneal intravenous Kupffer cell kilodalton 2-keto—3-deoxy—D-manno-octulosonic acid kilogram potassium hydroxide potassium bicarbonate liter LPS binding protein lipopolysaccharide molar min MAP H9 mCD14 mg ml mmol mM MTT 4-MP N NAD+ NADH ND NADPH "9 nm NO' NOS NF-KB 02 p PAF pH PMN PGD2 PGE1 PGE2 PGFm PGIZ 6-keto-PGF1a po PTX Sal sCD14 SEM TN F-a TNF-B TxAz TXBZ U minute mitogen activated protein microgram membrane-bound CD14 milligram milliliter millimole millimolar 3-(4,5-dimethylthiazol-Z-yl)-2,5-diphenyltetrazolium bromide 4-methylpyrazole chloride number oxidized nicotinamide dinucleotide reduced nicotinamide dinucleotide none detected reduced nicotinamide dinucleotide phosphate nanogram nanometer nitric oxide nitric oxide synthase nuclear factor kappa B molecular oxygen protein platelet activating factor power of hydrogen polymorphonuclear leukocyte prostaglandin D2 prostaglandin E1 prostaglandin E2 prostaglandin F1 alpha prostaglandin l 2 6-keto—prostaglandin F1 alpha per os pentoxifylline saline soluble CD14 standard error of the mean tumor necrosis factor alpha tumor necrosis factor beta thromboxane A2 thromboxane 8;; unit UV ultraviolet INTRODUCTION The innate immune system serves as the first line of defense against microbial invaders for mammalian organisms. Due to its ability to respond to biological insult without prior exposure, the innate immune system is an invaluable asset in the prevention of a bacterial infection. Individuals who have defects in this branch of the immune system are more susceptible to bacterial disease than other individuals (Anderson et al., 1985; Giger et al., 1987; Patarroyo and Makgoba, 1989). Although the activation of the innate immune system is most often beneficial to its host, it can also be detrimental. An excessive or inappropriate response of the innate immune system can result in injury to organs, derangement of blood circulation or death. Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria is an ancient signal of bacterial invasion. Cells of the innate immune system respond to this substance and activate the various host defense mechanisms. LPS is also one of the things which can trigger an overreaction from the innate immune system. Recently, activation of the innate immune system has been associated with the enhancement of adverse tissue responses to certain xenobiotic agents. Laboratory animals treated with activators of the innate immune system are more susceptible to agents such as carbon tetrachloride (Chamulitrat et al., 1994), D-galactosamine (Galanos et al., 1979), and halothane (Lind et al., 1994). It is interesting to speculate whether this also occurs in humans. If so, the state of activation of the innate immune system may explain some of the idiosyncratic responses to toxic xenobiotics observed in humans. When the innate immune system has been stimulated, an otherwise nontoxic dose of a xenobiotic agent becomes toxic. In this dissertation, the following hypothesis will be tested: Activation of the inflammatory cells of the innate immune system by lipopolysaccharide and the consequent production of mediators by these cells is important in the mechanism by which lipopolysaccharide enhances allyl alcohol- induced hepatotoxicity. In this introduction, the innate immune system, biology of lipopolysaccharide and its effects on the liver, the interaction of the innate immune system with lipopolysaccharide, the ability of lipopolysaccharide to potentiate xenobiotic hepatotoxicity, and the toxicology of allyl alcohol will be discussed. In subsequent chapters, experimental evidence supporting the above hypothesis will be presented and discussed. Chapter 1 GENERAL INTRODUCTION 1. A. The Innate Immune System The mammalian immune system is composed of two main divisions: the acquired immune system and the innate immune system. The acquired immune system must have prior exposure to pathogens before a full response is mounted. Thus it serves as a secondary, long—term line of host defense. The various populations of lymphocytes make up this branch of the immune system. In contrast, the innate immune system is designed for a rapid response to invasive agents and is nonspecific in its actions. It’s activity does not require prior exposure. The cells comprising this system are myeloid in origin, and their precursors are located in the bone marrow. Members of this branch of the immune system include macrophages, neutrophils, eosinophils, and basophils. 1. A. 1. Macrophages Macrophages are terminally differentiated mononuclear phagocytes that reside in most tissues. The cells are believed to originate mainly in the bone marrow and travel to their tissue of destination in the form of blood monocytes. Precursor populations of macrophages have also been found in other organs, and these most likely contribute to the fixed macrophage populations of the body. Macrophages exhibit both heterogeneity among tissues as well as within a given tissue. For example, in mice, the populations of macrophages within various tissues express different cell surface proteins and have different levels of biochemical activity and phagocytic ability (Unanue, 1990). In the murine spleen, two populations of macrophages can be distinguished based upon phagocytic activity and expression of MHC class II molecules (Unanue, 1990). These cells have numerous functions that protect the host from invading pathogens. They entrap and kill microorganisms and clear body fluids of soluble immune complexes. They take up and process antigen for presentation to lymphocytes. Macrophages are also highly secretory. Their products include eicosanoids, growth factors, complement proteins, nitric oxide, cytokines, reactive oxygen species, chemokines and enzymes (Decker, 1990). These products represent a mechanism by which macrophages communicate with other cells and regulate the immune response. 1. A. 2. Kupffer Cells Kupffer cells are named after their discoverer, Baron von Kupffer, and are the largest single population of macrophages in the mammalian body. These are the resident macrophages of the liver and are situated in the hepatic sinusoids. The strategic position of Kupffer cells in the hepatic sinusoids allows them to play a major role in clearing the portal blood of particulate matter, especially bacteria and bacterial products. Kupffer cells form one part of the two-part system used by the liver to detoxify and metabolize LPS (Fox et al., 1989). They perform the first part of the detoxification process by removing some of the polysaccharide moieties; the modified LPS is then passed onto hepatocytes for further metabolism. In intact animals, Kupffer cells play a major role in the liver injury caused by large doses of LPS (2 mg/kg and above). Blockade of Kupffer cell function protects the host from many of the damaging effects of endotoxemia (Marshall at al., 1987; limuro et al., 1994). The ability of LPS-stimulated Kupffer cells to affect and modify the functions of adjacent parenchymal cell populations is possibly one reason for the injurious effects produced by the resident liver macrophages. Another may be the direct toxic effects of some of the biochemical mediators produced by these cells. 1. A. 2. 1. Hepatic Architecture The liver is the largest gland in the body, with both exocrine and endocrine functions. This organ is situated between the mesenteric venous blood flow and the rest of the body, and the liver serves as the gate-keeper between the intestine and the systemic blood. The mesenteric veins combine to form the hepatic portal vein, the main blood supply to the liver. A second, much smaller blood supply comes from the hepatic artery. Histologically, the liver is composed predominantly of cuboidal epithelial cells arranged in radiating cords that are in turn arranged in laminae. The spaces between the radiating cords are called sinusoids and they converge at the central vein. The distal portion of the sinusoids originates at the portal triad, a structure composed of a branch of the portal vein, a branch of the hepatic artery, one or more bile ducts, and a lymph channel. The sinusoids can be thought of as specialized capillary beds. They are lined with fenestrated endothelial cells, Kupffer cells, lto cells, and pit cells. Blood flows from the portal triads towards the central veins. Eventually the central veins converge to form the hepatic veins that drain into the inferior vena cava. 1. A. 2. 2. Location and Function of Kupffer Cells Kupffer cells are one of the four types of hepatic sinusoidal cells. They have two origins: 1) from proliferation of existing hepatic populations and, 2) from precursors in the bone marrow (Fawcett, 1986). They are variable in shape and seem to be able to change their position within the sinusoidal space. While they are mainly found in contact with the endothelium, they may also be found within the space of Disse (Bouwens and Wisse, 1992; Wisse et al., 1996). The surface of Kupffer cells is irregular, characterized by surface folds, villous projections and vermiform bodies. A thin glycocalyx is present; most of the cell surface of Kupffer cells is in contact with the blood flowing from the portal triad. Their cytoplasm is richer in organelles than the neighboring endothelial cells. Peroxidase is present in the lumens of the endoplasmic reticulum, and peroxidase activity is a means to distinguish Kupffer cells from endothelial cells. Different populations of Kupffer cells have been identified based on size, phagocytic activity and surface antigens. Periportal Kupffer cells are larger, have more lysosomal and cathepsin G activity, and are able to phagocytose more latex particles than Kupffer cells isolated from the centrilobular or midzonal regions of the liver lobule (Sleyster and Knook, 1982). Macrophage subpopulations can be distinguished using the ED family of cell surface antigens. ED1 has been found on monocytes and all macrophage populations while EDZ has been associated with resident macrophages (Sato et al., 1998; Yamate et al., 1999). Using monoclonal antibodies to the ED1 and ED2 surface antigens, Armbrust and Ramadori (1996) distinguished between two populations in the liver. Large ED1+/ EDZ+ Kupffer cells are located mainly in the periportal and centrilobular regions. Smaller ED1+/ED2- cells are found along the midzonal region of the sinusoids. The position of Kupffer cells in the hepatic sinusoids allows them extensive access to the portal blood flow. Kupffer cells are highly phagocytic cells, and in addition to removing bacterial LPS as mentioned above, they actively remove particulate matter from blood passing over them. When laboratory animals are treated with colloidal carbon, the carbon particles are rapidly phagocytosed by the host’s macrophages, including Kupffer cells. The accumulated carbon can be seen under light microscopy (Fawcett, 1986). 1. A. 2. 3. Interaction of Kupffer Cells and Hepatocytes Kupffer cells are in contact with the other cell types of the liver and play a role in liver homeostasis in both normal and disease states. These macrophages can affect the level of protein synthesis, protein phosphorylation, and glycogenolysis in neighboring hepatocytes. For example, in a series of in Vitro studies involving hepatocyte:Kupffer cell cocultures, West and colleagues (1985, 1986, 1988) found that unstimulated Kupffer cells stimulate protein synthesis in hepatocytes as measured by 3H-leucine incorporation. In contrast to results with unstimulated Kupffer cells, when hepatocytes were cultured with Kupffer cells stimulated with LPS, protein synthesis was inhibited. The factors involved in this process were soluble because these results could be reproduced by culturing hepatocytes in medium from unstimulated Kupffer cells and LPS- or E. coli-stimulated Kupffer cells. The level of inhibition of protein synthesis was directly related to the production of nitrates and nitrites, suggesting a role for nitric oxide (NO') (Billiar et al., 1989a, Billiar et al., 1989b). Furthermore, the level of inhibition of protein synthesis was also related to the amount of L-arginine in the culture medium, and the inhibitor of nitric oxide synthesis, NGmonomethyl-L-arginine, prevented most of the observed inhibition of protein synthesis. 10 Conditioned medium from LPS-treated Kupffer cells enhanced the phosphorylation of some proteins and inhibited the phosphorylation of others in cultured hepatocytes (Castelijn et al., 1988a). These changes in phosphorylation could be reproduced with the prostaglandins PGDZ, PGE1, and PGE2, indicating that Kupffer cells use eicosanoids as a means of intercellular communication in the liver. Another example of this communication is the role of PGD2 in the increase in glucose levels seen during endotoxemia. In the isolated, perfused liver, LPS exposure produces a spike in PGDZ levels before an increase in glucose output (Castelijn et al., 1988b). This increase in glucose can be blocked with aspirin, an irreversible inhibitor of cyclooxygenase. In addition, P602 alone can stimulate glucose production in cultured hepatocytes. LPS alone could not, indicating that a cell type other than hepatocytes is producing the PGD2. Since the major eicosanoid produced by LPS-stimulated Kupffer cells is PGD2, Kupffer cells are the most likely candidate (Kuiper et al., 1988). 1. A. 3. Neutrophils Neutrophils are another cell population important in the innate immune system. They are derived from the same pluripotent stem cell as blood 11 monocytes. Mature neutrophils are released into the general circulation and have a half-life in the circulation of approximately six hours. They soon move out of the blood stream and enter the extravascular pool of neutrophils. These cells are “front line” phagocytes responsible for early defense against bacterial infection. They are the first cells to arrive at sites of inflammation and are responsible for the early killing of invading microorganisms. Neutrophil granules are storage sites for the various enzymes and mediators used by these cells as part of their role in tissue inflammation (Gallin, 1989). Even though neutrophils are largely beneficial in their activities, they can cause massive hepatocellular injury and necrosis. Large numbers of neutrophils accumulate in the sinusoids of rats treated with high doses of LPS (2 mg/kg and above) (Hewett et al., 1992). These neutrophils release proteases and reactive oxygen species into the intercellular environment for the purpose of killing invading bacteria; however, the mediators also have toxic effects on the hepatic parenchymal cells, causing cell death and injury. Reduction of circulating neutrophil populations alone or disrupting their ability to migrate into tissues can protect rats (Jaeschke et al., 1991; Hewett et al., 1992; Hewett et al., 1993) and mice (Xu et al., 1994) from the liver injury associated with exposure to high doses of LPS over 2 mg/kg, demonstrating that 12 neutrophils, like Kupffer cells, play a vital role in the liver injury associated with high blood levels of LPS. 1. A. 4. Eosinophils and Basophils Eosinophils and basophils form the balance of the innate immune system. These cells do not interact directly with LPS but are responsible for protection against parasitic infections (eosinophils) and mediating the allergic response (eosinophils and basophils) (Fawcett, 1986). 1. A. 5. Interaction of LPS and the Innate Immune System Cells of the innate immune system are very sensitive to the presence of LPS. In fact, many of the effects of LPS do not arise from direct action of LPS on the various systems of the host, but are mediated through the actions on the innate immune system. The focus of this dissertation is the role of the innate immune system in LPS effects on allyl alcohol hepatotoxicity. The previous sections provided background on the innate immune system. The next section will begin with 13 an introduction to the topic of LPS as a prelude to a discussion of the interaction of LPS with the innate immune system. 1. B. Lipopolysaccharide 1. B. 1. Structure LPS is a component of the outer membrane of Gram-negative bacteria (Anderson et al., 1985; Raetz et al., 1988; Rietschel et al., 1994). The exact structure of the molecule varies among species and strains of bacteria, but all LPS molecules share three specific characteristics. These are the O-antigen, the core, and lipid A (Figure 1.1). The most variable region of LPS is the O-antigen. The O-antigen is the outermost portion of a molecule of LPS and is in contact with the external environment. Composed of twenty to forty repeating sugar units, the O-antigen is responsible for the antigen specificity of a given LPS molecule. The core, the central region of LPS, is embedded in the outer surface of the membrane. It serves as the bridge between the O-antigen and the innermost portion, lipid A. The core contains two unusual sugars: a seven- carbon sugar (heptose), and a 2-keto-3-deoxy-D-manno-octuIosonic acid 14 (KDO). The final portion, lipid A, is the most conserved region of LPS. It is also responsible for most of the biological activity associated with LPS. The lipid A portion of LPS is fully embedded in the membrane, and its fatty acid tails oppose the fatty acid tails of the phospholipids that make up the internal side of the outer membrane lipid bilayer. LPS has been classified into three categories based on the structure of the O-antigen: smooth, semi-rough, and rough. In the smooth strains of LPS, a full complement of twenty to forty sugar residues is present in the O-antigen. The O-antigen is greatly reduced in the semi-rough strains, whereas the rough strains lack both the O-antigen and part of the core (Morrison and Ulevitch, 1978). 1. B. 2. Sources of LPS in Host Systems LPS may enter the body of the host animal by two major routes: from organisms outside of the host (exogenous) and from organisms inhabiting the intestinal tract (endogenous). Most of the toxic effects of LPS are associated with infection by Gram-negative bacteria, an exogenous source of LPS. The proliferation of bacteria and the release of their products into the host’s system lead to the clinical conditions known as septic shock, adult respiratory distress syndrome, and multiple system organ failure. 15 Figure 1.1. Structure of LPS. The typical LPS molecule has three distinct divisions. The O-antigen is the outermost portion of the molecule and faces into the environment. The middle portion is the Core and contains a unique seven-carbon sugar, KDO. Lipid A is the innermost portion of the molecule. 16 Lipopolysaccharide O-Antigen Core Lipid A Extracellular Intracellular 17 The diseases associated with bacterial infections arise from a wide variety of causes including tissue trauma, contagion, and nosocomial infections. The elderly, very young, and immunocompromised are the most vulnerable, but any individual can be affected (Durham et al., 1990). Even with intervention with antibiotics and supportive therapy, many patients diagnosed with one of the above conditions still die. This is thought to be due the enhanced release of LPS from the bacteria killed by the antibiotic and the amount of systemic damage that has occurred before the patient is treated. Endogenous LPS arises from the Gram-negative bacteria mammals normally carry in their large bowel. These microorganisms ferment ingesta, further processing it and releasing valuable nutrients. A small amount of the LPS released by the turnover of gut bacteria reaches the portal circulation and is cleared by the Kupffer cells (Jones and Summerfield, 1988; Fox et al., 1989; Nakao et al., 1994). If the gut wall is compromised by intoxication or disease, an increased amount of LPS may enter the portal circulation and overwhelm the normal hepatic clearance mechanisms. 18 1. B. 3. Effects of LPS on Mammalian Physiology LPS is a biologically active compound in mammalian systems (Raetz et al., 1988; Vogel, et. al., 1990; Raetz, et. al., 1991; Wright and Kolesnick, 1995) and may have either positive or negative responses in a given host. Several beneficial effects have been found from exposure to LPS. The oldest benefit known is the chemotherapeutic effect of bacterial products in cancer treatment. This was first noted in the late 1800’s. Researchers discovered that LPS could induce hemorrhage and necrosis in tumors (Vogel and Hogan, 1990). In time, they found the causative agent to be the cytokine, tumor necrosis factor, which is elicited by exposure to LPS (Carswell et al., 1975). Other beneficial effects of LPS include stimulation of the innate immune system to resist infection. The C3H/HeJ mouse, which is hyporesponsive to LPS, has less resistance to pathogens than the C3H strains that exhibit a normal response to LPS (O’Brien et al., 1980). Sublethal doses of LPS are also radioprotective when given 16 to 24 hours prior to exposure (Neta et al., 1986a). The protective effect of LPS was found again to be due to the induction of cytokines such as tumor necrosis factor- alpha, interleukin-1 and colony stimulating factor (Fujita et al., 1983; Neta et al., 1986a; Neta et al., 1986b). LPS has a number of detrimental effects on the host as described above. On the physiological level, LPS acts as a pyrogen. It raises the 19 normal temperature set point in the hypothalamus (Dinarello, 1983). The body raises the core temperature in response to the new set point by vasoconstriction, reduced sweating, piloerection, and shivering. LPS exposure causes hypoglycemia by inhibiting the glucocorticoid induction of phosphoenolpyruvate carboxykinase (Rippe and Berry, 1972). LPS toxicity also causes disorders of the circulatory system, notably disseminated intravascular coagulation, hypotension, and infarcts. It is the circulatory disorders that are associated with the lethal effects of LPS, and organs that have extensive vascular beds such as liver, lung, and kidney are very susceptible. The organ of interest for this dissertation is the liver. 1. B. 3. 1. Effects of LPS on the Liver Gram-negative bacteria and LPS have been shown to be hepatotoxic by several investigators (Levy, et. al., 1967; Balls, et. al., 1979; Utili, et. al., 1977; Hirata, et. 3].). Microscopic changes in the liver are seen 30 minutes after the injection of a sublethal dose of LPS in mice (100-300 pg) (Levy et al., 1967). At this time, Kupffer cells become more rounded in appearance and bulge into the hepatic sinusoids, and neutrophils begin to accumulate in the sinusoids. Neutrophils are present in the sinusoids by 60 minutes. This is accompanied by mononuclear cells and eosinophils. 20 Thrombi are also present at this time. At two hours after LPS treatment, Kupffer cells and endothelial cells are swollen. This, along with the thrombi and infiltrating leukocytes, slows and alters the pattern of blood flow through the hepatic sinusoids (McCuskey et al., 1982). The number of neutrophils in the liver continues to increase. LPS has some direct effects on hepatocytes; however, these effects (including alterations in bile and fatty acid synthesis) are not considered to be toxic (Utili et al., 1977; Feingold et al., 1992). Most of the liver injury from treatment with LPS is due indirectly to effects of LPS on the innate immune system. These various indirect effects of LPS on the liver are mediated by cytokines, free radicals, eicosanoids, and enzymes produced by cells of the innate immune system (Figure 1.2). 1. B. 3. 2. Biochemical mediators of LPS-induced hepatotoxicity Cflokines Cytokines are proteins or glycoproteins produced by cells of the immune system. They serve as a means of communication between the producing cell and the target cell, and their effects may be local or systemic. 21 Elevated levels of cytokines are found in the blood of both human patients suffering from bacterial infections and experimental animals administered LPS or Gram-negative bacteria. The plasma levels of tumor necrosis factor-alpha and interleukin-6 in patients admitted to hospitals with serious bacterial infections are elevated, and this elevation correlates with disease severity and death (Brantzaeg et al., 1991; Dofferhoff et al., 1991) Tumor necrosis factor-alpha The term “tumor necrosis factor” is used for two cytokines produced in response to inflammatory stimuli. The alpha form is released by cells of the monocyte/macrophage series and is also known as cachectin (Beutler et al., 1985; Beutler and Cerami, 1987; Bemelmans et al., 1996). The beta form is produced by lymphoid cells and is called lymphotoxin (Paul and Ruddle, 1988). Both have similar biological effects. Tumor necrosis factor-alpha (TNF) is considered to be the pivotal proinflammatory cytokine of the host response to LPS (Tracey and Cerami, 1993). It is the first cytokine detected after exposure to bacteria or endotoxin: blood concentrations peak between 90 to 120 minutes in humans (Hesse et al., 1988; Michie et al., 1988) and at 120 minutes in baboons (Creasey et al., 1991). TNF can stimulate the production of the other major cytokines of inflammation, interleukin-1 (IL-1) and interleukin-6 (IL-6) (Dinarello et al., 1986; Fong et al., 1989; Helle et al., 1991). 22 Figure 1.2. Products of LPS-stimulated macrophages. The binding of the LPS binding protein (LBP):LPS complex to the CD14 receptor activates a number of intracellular pathways, resulting in the release of a variety of biochemical mediators of inflammation. 23 LBPzLPS \ Macrophage CD14 / \ Cytokines Elcosanoids V Free Radicals 24 Inhibition of tumor necrosis factor-alpha activity, either by passive immunization with anti-tumor necrosis factor-antibodies (Beutler et al., 1985; Tracey et al., 1987) or pharmacologically with pentoxifylline (Hewett et al., 1993), protects laboratory animals from the damaging effects of both endotoxemia and bacteremia. The signs and symptoms associated with inflammation and cachexia can be mimicked by administration of exogenous TNF (Tracey et al., 1988). Interleukin-1 Interleukin-1 is a cytokine produced by a variety of cell types and tissues, including macrophages, epidermal, epithelial, lymphoid, and vascular tissues (Moore et al., 1980; Dinarello, 1988). It is the second of the proinflammatory cytokines to reach a peak in plasma after exposure to endotoxin (240-300 min in baboons; 120 minutes in humans) (Creasey et al., 1991; Zabel et al., 1989) and is found in the blood of human patients and experimental animals suffering from endotoxemia and bacteremia (van der Meer and Vogels, 1991; Brandtzaeg et al., 1991; Creasey et al., 1991) Interleukin-1 is a potent mediator of the inflammatory response. It enhances the growth of T lymphocytes, induces the expression of adhesion molecules on endothelium, elicits the release of other biochemical mediators such as prostaglandins, lL-6, TNF, and granulocyte-macrophage colony stimulating factor (Kunkel et al., 1986; 25 Durum and Oppenheim, 1989), and induces neutrophil emigration into tissues (Cybulsky et al., 1986; Movat et al., 1987). In mice, blockade of the lL-1 receptor significantly alters the inflammatory response to LPS (McIntyre et al., 1991). Neutrophil migration from the bone marrow is significantly attenuated. In addition, serum levels of hepatic acute phase proteins and lL-6 are significantly lowered in the murine model (McIntyre et al., 1991). Intefleukin-6 Following administration of LPS, blood concentrations of lL-6 peak at 120 minutes in humans (Zabel et al., 1989) and at 180 minutes in rats (Kispert 1992). lL-6 is produced by a number of cell types such as activated macrophages and T lymphocytes, endothelial cells, and fibroblasts (Durum and Oppenheim, 1989; Jirik et al., 1989; Helle et al., 1991; Gallery at al., 1992). The production of IL-6 is greatly influenced by the other two proinflammatory cytokines, TNF and lL-1. Both TNF and lL-1 have a stimulatory effect on lL-6 production (Durum and Oppenheim, 1989; Evans et al., 1992; Brouckaert et al., 1993). Interleukin-6 plays a key role in the acute phase response characteristic of endotoxemia. This cytokine is a major initiator and regulator of the hepatic synthesis of acute phase proteins such as fibrinogen, alpha-2- macroglobulin, and C-reactive protein (Zentella et al., 1991; Kispert 1992) 26 Eicosanoids The eicosanoids are a family of unsaturated, twenty-carbon fatty acid derivatives with powerful regulatory actions on host physiology. They include prostaglandins, leukotrienes, and thromboxanes. These lipids mediate a wide variety of bodily functions including the production of pain and fever, induction of blood clotting, regulation of blood pressure, induction of labor, regulation of gastric acid production and inflammation. The eicosanoids have some hormone-like properties, but unlike hormones, they generally exert their actions locally. Eicosanoids are involved in the inflammatory response. They are produced by cells of the immune system and regulate many of the activities of neighboring cells such as cytokine production (Callery et al., 1990) and glycogenolysis (Casteleijn et al.,1988c). The pattern of eicosanoid production varies among cell types. Neutrophils have a greater production of Ieukotrienes compared to macrophages, which produce more prostaglandins. Eicosanoids are produced by cells of the innate immune system in response to both normal physiologic stimuli and LPS. These compounds or their metabolites have been detected in the plasma and bile of septic patients and of experimental animals given endotoxin or othenIvise exposed to products of Gram-negative bacteria. Inhibition of one or more of the eicosanoids has been shown to increase survival in some animal 27 models. For example, the use of non-steroidal anti-Inflammatory drugs such as aspirin, naproxen, and ibruprofen increases the survival of endotoxemic or septic animals (Ball at al., 1986; Albrecht et al., 1997). Inhibitors of the 5-Iipoxygenase pathway or leukotriene receptor antagonists have also proven helpful in ameliorating the effects of endotoxic/septic shock (Keppler et al., 1987; Tiegs and Wendel, 1987; Nagai et al., 1989; Matuschak et al., 1990). The thromboxanes The thomboxanes are vasoactive eicosanoids that mediate many of the vascular events associated with endotoxic and septic shock. Thromboxane A2 (TxAz) is a potent vasoconstrictor and causes platelet aggregation. It has a very short plasma half-life (~30 seconds), and thus the stable derivative, thromboxane B2 (Tsz), is usually used an indicator of TxAz in plasma. Thromboxanes reach peak plasma concentrations rapidly after bolus iv injections of endotoxin (Ball et al., 1986). Inhibition of thromboxane synthesis or pharmacological antagonism of TxA2 increases survival in animal models in which endotoxin is given as a bolus iv injection. The prostaglandins The prostaglandins mediate a wide range of physiological functions. They play important roles in the regulation of acid production by gastric chief cells, the absorptive activities of the cells of the proximal renal 28 tubules, induction of labor, and many other physiological processes. With respect to this dissertation, prostaglandins produced by Kupffer cells in response to exposure to endotoxin will be the focus. Prostaglandins are produced by macrophages, including Kupffer cells, after treatment with endotoxin. The metabolites that have been identified are PGE1, PGEZ, PGD2, PGFZG, PGl2, and 6-keto-PGF1O, (the stable metabolite of PGIZ), (Bowers et al., 1985; Brouwer et al., 1988; Casteleijn et al.,19880; Okumura eta/.,. 1987.) Like prostaglandins in other organs of the body, the prostaglandins produced by Kupffer cells have regulatory functions after exposure to endotoxin. They regulate the production of the proinflammatory cytokines (T NF, IL-1, lL-6) (Karck et al., 1988; Gallery at al., 1990; Peters et al., 1990; Grewe et al., 1994; Roland et al., 1994) and inducible nitric oxide synthase (NOS) (Gaillard et al.,1991; Harbrecht et al., 1995), and cause the release of glucose from hepatocytes (Casteleijn et al., 1988b). The leukotrienes The presence of LPS triggers the production of leukotrienes in vivo, and the cysteinyl forms are eliminated in the bile (Hagmann et al., 1985). Leukotrienes are highly chemotactic for neutrophils and are produced by hepatic macrophages during sepsis (Doi et al., 1993). This presence of leukotrienes causes the migration of neutrophils into the liver and the associated liver injury. Rodriguez de Turco and Spitzer (1990) showed that 29 the dominant arachidonic acid metabolites produced by these infiltrating cells are leukotrienes. Additionally, there is an increase in 5-lipoxygenase activity in the liver (Kawada et al., 1992). The exact role of leukotrienes in endotoxin-induced liver injury is somewhat controversial. Inhibitors of leukotriene synthesis or leukotriene antagonists are protective in models of endotoxin-induced liver injury in vivo involving presensitization with galactosamine (Tiegs and Wendel, 1988; Keppler et al., 1987; Matuschak et al., 1990) or with Corynebacterium parvum (Nagai et al., 1989). However, in one in vivo model in which only endotoxin was administered, inhibition of 5- Iipoxygenase with zileutin was not protective (Pearson et al., 1997). These different results may indicate that the fundamental role of leukotrienes is different in the respective models. Free radicals Reactive oxygen species Cells of the myeloid lineage can generate reactive intermediates of oxygen as a means of killing invading bacteria. Molecular oxygen is converted to the superoxide anion radical. The enzyme, superoxide dismutase, then converts the superoxide anion to hydrogen peroxide, and subsequently the hydroxyl radical is produced. The production of superoxide anion and related free radicals has been linked to liver injury in a number of models. Macrophages and neutrophils 30 primed by previous exposure to endotoxin or other stimulants, such as phorbol myristate acetate, produce much higher levels of oxygen-derived free radicals than controls (Arthur et al., 1986; Arthur et al., 1988; Shiratori et al., 1988; Aida and Pabst, 1990; Bautista et al., 1990; Mayer and Spitzer, 1991). When the subsequent release of oxygen-derived free radicals is blocked by allopurinol (Arthur et al., 1985) or superoxide dismutase (Arthur et al., 1985; Shiratori et al., 1988), liver injury is prevented. E Nitric Oxide Nitric oxide (NO‘) is a free radical that is produced by most cells of the mammalian body. The physiological effects mediated by NO‘ are quite varied and are dependent on the cell producing the radical and the local environment. The enzyme that produces NO', nitric oxide synthase (NOS), has two forms, constitutive and inducible. The constitutive form of the enzyme is found in numerous cell types such as endothelial cells and neurons. Macrophages have an inducible form of NOS which becomes upregulated after exposure to LPS (Gaillard et al., 1991; Laskin et al., 1994). In both instances, the NO' is derived from L-arginine in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and nitirc oxide synthase. Macrophages use NO' as a cytotoxic molecule. It has been detected during macrophage-induced killing of a variety of targets, including tumor 31 cells, Entamoeba histolytica, Leishmania major, and bacteria (Liew et al., 1990a; Liew et al., 1990b; Lin and Chadee, 1992; Aono et al., 1994; Fukumura et al., 1996; Saito et al., 1996; Sveinbjornsson et al., 1996; Lavnialkova et al., 1997; MacMicking et al., 1997; Vouldoukis et al., 1997; Albina and Reichner, 1998). Inhibition of NO‘ synthesis prevented cytotoxicity in the above models. 1. B. 4. Cellular Effects of LPS 1. B. 4. 1. LPS Binding Protein The mammalian body has evolved a system by which LPS is transferred from the general circulation to cells of the innate immune system. Several proteins that can bind LPS are present in blood and greatly enhance the movement of small amounts of circulating LPS to monocytes, macrophages, and neutrophils. One of the most studied of these proteins is LPS-binding protein. LPS binding protein (LBP) is an acute phase protein synthesized by hepatocytes (Ramadori et al.,1990). Trace amounts are normally found in the bloodstream of healthy individuals (0.5ug/ml), and the concentration 32 rises as high as 50 pg/ml 24 hours after the induction of an acute phase response (Tobias et al., 1986, 1988). Structurally, LBP is a 60-kD glycoprotein. It has sequence homology with bactericidal/permeability increasing protein (BPI; 69% amino acid identity with human BPI) and cholesterol ester transport protein (CETP; 23% amino acid identity) (Schumann et al., 1990). This molecule has a high affinity site for binding lipid A (Schumann et al., 1990) and can bind both rough and smooth forms of LPS (Tobias et al., 1989). LBP serves as an opsonizing protein, mediating the attachment of LPS-coated particles and intact Gram- negative bacteria to macrophages (Schumann et al., 1990). The combined presence of LBP and LPS significantly enhances the production of TNF by rabbit peritoneal macrophages as compared to LPS alone. Addition of LBP to peritoneal macrophages exposed to LPS accelerates the expression of TNF (4.5 hours versus 8 hours) and increases the stability of TNF mRNA (>5 hour versus 1 hour) (Heumann et al., 1992; Mathison et al., 1992). Immunodepletion of LBP inhibits production of TNF in whole blood and in preparations of monocytes, indicating that this protein is a key element in the innate immune system response to LPS (Schumann et al., 1990; Heumann et al., 1992). 33 1. B. 4. 2. CD14 CD14 was first recognized as a marker for monocytes and macrophages. Now investigators realize that this cell surface molecule is the cellular receptor for complexes of LPS and LBP (Kirkland et al., 1989; Wright et al., 1990). CD14 is a 55 kD glycoprotein attached to the cell membrane by a phosphatidylinositol glycan anchor and is mobile in the plane of the membrane (Haziot et al., 1988; Simmons et al., 1989). CD14 allows macrophages, monocytes and neutrophils to respond to very low circulating concentrations of LPS:LBP complexes (Wright et al., 1990; Haziot et al., 1993b). LPS alone is able to bind to the CD14 molecule; however, this occurs at much higher concentrations of LPS than in the presence of LBP (Hailman et al., 1994). Additionally, a soluble form of CD14 (sCD14) is present in blood and acts as the equivalent of the membrane-bound CD14 (mCD14) for cells which do not have mCD14 (e.g. endothelial cells and astrocytes) (Bazil et al., 1989; Pugin et al., 1993; Haziot et al., 1993a; Goldblum et al., 1994; Tobias et al.,1995; Jack at al., 1995; Goldenbock et al., 1995). The soluble CD14 appears to originate from CD14 molecules shed from the surface of circulating monocytes (Bufler et al., 1995). 34 1. B. 4. 3. Intracellular Mechanisms of Action Many of the biological effects of LPS have been described above. Here, the cellular basis of some of these effects will be discussed. LPS induces rapid protein tyrosine phosphorylation (4-5 minutes) in both murine peritoneal macrophages and RAW 264.7 cells, and this phosphorylation results in the release of arachidonic acid metabolites (Weinstein et al., 1991). Inhibition of protein tyrosine phosphorylation by herbimycin A causes a dose-dependent decrease in the concentrations of arachidonic acid metabolites produced by RAW 264.7 cells (Weinstein et al., 1991). LPS also induces tyrosine phosphorylation of at least two mitogen-activated protein (MAP) kinases (Weinstein et al., 1992). These data correlate with another set of experiments in which mice are protected from lethal endotoxemia by administration of tyrphostins, compounds that inhibit protein tyrosine phosphorylation (Novogrodsky et al., 1994). In recent years, experimental evidence indicates that LPS can activate monocytes and macrophages via the sphingomyelin pathway. The lipid sphingomyelin is concentrated in the outer leaflet of the plasma membrane of mammalian cells (Kolesnick and Golde, 1994). Ceramide is the fatty acid in an amide linkage at position two, and hydrolysis of the phosphodiester bond by sphingomyelinase yields free ceramide and phosphocholine (Kolesnick and Hermer, 1991). It is possible that free 35 ceramide interacts with ceramide activated protein (CAP) kinase, a proline- directed protein kinase which may phosphorylate p42 MAP kinase (Raines et al., 1993; Joseph et al., 1993; Liu et al., 1994). In addition to activating a portion of the MAP kinase system, ceramide may also induce the degradation of inhibitory KB (IKB), the cellular inhibitor of nuclear factor- 10.01 pg/kg. Histological analysis of livers revealed prominent Kupffer cells in sinusoids of rats treated with doses of LPS >5 ug/kg and edema and mild neutrophilic infiltration at doses of 10—50 pg/kg LPS. A limited number of animals was pretreated with 500 pg/kg LPS 64 Figure 2.1. LPS enhancement of the hepatotoxicity of allyl alcohol. Animals were treated with LPS (100 ug/kg iv) or vehicle two hours before administration of allyl alcohol (30 mg/kg ip) or vehicle. ALT (A) and AST (B) activities in plasma were measured as markers of liver toxicity 18 hrs after treatment with allyl alcohol. Data are expressed as mean 1 SEM. a, significantly different from respective value in the absence of allyl alcohol. b, significantly different from respective value in the absence of LPS. N = 5 -13. 65 Plasma ALT Activity (Units/L) Plasma AST Activity (Units/L) 2500 2000 1500 1000 500 4000 3000 2000 1000 T I I I [II] Safine IIII LPS a,b I 13 ' 1 — SAL 66 Pathological changes in the livers of rats Table 2.1 TREATMENT HEPATOCYTIC PERIPORTAL HEPATIC SINUSOIDAL NECROSIS EDEMA PARENCHYMAL INFLAMMATORY INFLAMMATORY INFILTRATE INFILTRATE Sal/Sal 010 019 010 010 LPS/Sal 0.1 1 0.09 0.3 1 0.2 0.1 1 0.09 0.1 1 0.09 Sal/AA 1.2 1 0.3 1.2 1 0.3 1.2 1 0.3 1.2 1 0.3 LPS/AA 2.9 1 0.3 2 1 0 2.2;1).1 2 + 0 ‘ Rats were treated as described in Materials and Methods. Liver sections were taken, fixed, and lesions were evaluated. Microscopic lesions were assigned a grade using the following scale: 0, no lesions; 1, mild; 2, moderate; 3, marked; 4, severe. See text for detailed description of lesions. N: 67 4 for Sal/Sal group; 9—10 for all other groups. Figure 2.2. Time course of development of LPS-enhanced allyl alcohol hepatotoxicity. Animals were treated as described in the legend to Figure 1, and plasma ALT activity was measured at the indicated times. a, significantly different from respective value in the absence of allyl alcohol. b, significantly different from respective value in the absence of LPS. N = 5 -14. 68 Plasma ALT Activity (Units/L) 4000 3000 2000 1 000 O Sal/Sal 8;? v LPS/Sal I Sal/AA O LPS/AA a_,_b _L 8,? a,b 8,. OJ 4 4 4 2 4 6 12 18 24 Hours after allyl alcohol 69 Figure 2.3. Dose-response relations for plasma ALT activity after treatment with LPS and/or allyl alcohol. A) Animals were treated with the indicated doses of LPS and with either 30 mg/kg of allyl alcohol or vehicle. B) Animals were treated with the indicated doses of allyl alcohol and with either 100 ug/kg of LPS or vehicle. Plasma ALT activity was measured eighteen hours after allyl alcohol administration. Data are expressed as mean 1 SEM. a, significantly different from respective value in the absence of allyl alcohol. b, significantly different from respective value in the absence of LPS. N = 3-14. 70 Plasma ALT Activity (Units/L) 4°00 ’——O——_Allyl Alcohol A I Saline 3000 - 2000 r 1000 '- OPI— I 4 WI // / / ‘ ' ‘ 0 0.01 0.1 1 10 100 LPS(ug/kg) 2000 O LPS B I Saline 1500 - a,b 1000— a,b 500- oi #— ’l 0.0 10.0 20.0 30.0 Allyl Alcohol (mg/kg) 71 prior to administration of allyl alcohol: most (> 75%) rats in that group died within 18 hrs. The dose-response relationship for allyl alcohol is shown in Figure 2.3.B. In the absence of LPS, no significant increase in plasma ALT activity was seen at doses of allyl alcohol ranging from 0-30 mg/kg. LPS (100 ug/kg) cotreatment increased plasma ALT activity at doses of 20 and 30 mg/kg allyl alcohol. Significant mortality occurred at allyl alcohol doses greater than 30 mg/kg. 2. D. 2. Role of Metabolism of Allyl Alcohol ALT activity was low in plasma of rats treated only with saline or LPS and was not affected by administration of the ADH inhibitor, 4-MP (Figure 2.4). In allyl alcohol-treated rats pretreated with LPS, ALT activity in plasma was markedly increased. This increase was abolished in rats pretreated with 4-MP. ALT activity in rats treated with allyl alcohol only was also decreased by pretreatment with 4-MP. Allyl alcohol requires bioactivation by ADH to acrolein, a reactive aldehyde that binds rapidly to glutathione and other sulfhydryl-containing molecules (Beauchamp, et al., 1985; Serafini-Cessi, 1971; Reid, 1972; Butterworth et al., 1978; Patel et al., 1980; Ohno et al., 1985). Allyl 72 alcohol is metabolized rapidly after administration in vivo as evidenced by a dramatic decrease in hepatic reduced glutathione within 12-20 minutes (Penttila, 1987; Pompella et al., 1988). Accordingly, the activity of ADH in liver cytosolic fractions was examined 2 hours after administration of LPS, the time at which allyl alcohol was given in experiments described above. ADH activity in livers from LPS-treated rats was not significantly different from activity in cytosolic fractions from control rats (Table 2.2). Perfusion of isolated livers with allyl alcohol caused an increase in fluorescence of NADH. This was used as an indirect measure of oxidation of allyl alcohol to acrolein (allyl alcohol + NAD+ yielded acrolein and NADH). LPS pretreatment did not affect the increase in fluorescence of NADH upon perfusion of allyl alcohol into isolated livers. 2. D. 3. Effect of LPS Treatment on Hepatocellular Concentrations of GSH and GSSG To assess GSH status in the liver prior to administration of allyl alcohol, the concentrations of both GSH and GSSG were determined 1.5 hr after administration of LPS or saline vehicle. The concentration of GSH was not different in livers of LPS-treated and control rats (Table 2.3). Similarly, 73 administration of LPS to rats did not affect the concentration of GSSG in the liver. 2. E. Discussion Pretreatment of rats with a nontoxic dose of LPS (100 ug/kg) significantly increased liver damage, as measured by both biochemical assays and pathological examination in rats exposed to allyl alcohol. A significant increase in plasma ALT activity was detected as early as two hours after allyl alcohol treatment. This biochemical marker of liver damage continued to increase through 6 hrs and then declined, indicating that injury occurred within the first 6 hrs after administration of allyl alcohol. The decrease in ALT activity after 6 hrs may be due to clearance of this enzyme from the circulating blood. In contrast to the situation in LPS- pretreated rats, plasma ALT activity in rats treated only with allyl alcohol did not increase significantly over a 24 hour period. The results of histological evaluation were consistent with changes in aminotransferase activities. Based on the histopathologic lesions, it appears that LPS enhanced the toxic effects of allyl alcohol, rather than 74 Figure 2.4. Protection from LPS-induced enhancement of allyl alcohol hepatotoxicity by 4-methylpyrazole. Animals were treated with 4-MP (77 mg/kg ip) 15 minutes before administration of allyl alcohol. The remainder of the experimental protocol was the same as in Figure 1 except that animals were killed 6 hours after allyl alcohol administration. Data are expressed as mean 1 SEM. a, significantly different from respective value in the absence of allyl alcohol. b, significantly different from respective value in the absence of LPS. 0, significantly different from respective value in the absence of 4-MP. N = 4-8. 75 Plasma ALT Activity (Units/L) 3000 2500 2000 1 500 1 000 500 1 I I :1 Saline a,b - 4-Methylpyrazole C r—-——_.=-__ C L— Sal LPS Sal LPS Saline Allyl Alcohol 76 Table 2.2 Factors influencing or reflecting the metabolism of allyl alcohol TreatmentA Alcohol DehydrogenaseB FluorescenceC (mM/min/gm of liver) (% increase from basefine) Saline 0.19 1 0.01 13.51 7.3 LPS 0.24 1 0.05 14.51 3.5 A, Rats were treated with LPS (100 ug/kg) or saline two hours prior to measurements. B, Activity of alcohol dehydrogenase was measured in cytosolic fractions prepared from liver (N = 6). C, Livers were isolated and perfused in a nonrecirculating system. Fluorescence of NADH from the surface of the liver was monitored using a light guide (366 nm excitation, 450 nm emission). Increase in fluorescence during perfusion with allyl alcohol (100 pM) was expressed as a percentage of baseline fluorescence (n = 3). Statistically significant differences between the saline and LPS exposure groups were not seen for either parameter. 77 Table 2.3 Comparison of hepatocellular levels of reduced and oxidized glutathione in rats pretreated with saline Treatment GSH GSSG pM/g liver pM/g liver Sal 6.6 + 0.2 0.42 + 0.02 LPS 5.7 + 0.5 0.48 + 0.09 Female, Sprague—Dawley rats received saline 24 hours before receiving either LPS (4 mg/kg) or saline. Animals were killed 1.5 hours after LPS or saline exposure, and liver GSH and GSSG concentrations were determined as described in Materials and Methods. N = 4-7. No significant differences were observed. 78 allyl alcohol increasing LPS-induced liver damage. Hepatic lesions caused by administration of LPS at doses larger than those used in these studies are confined to midzonal areas of the lobule and have as a prominent feature neutrophils in those areas (Jaeschke et al., 1991; Hewett et al., 1992; Pearson et al., 1995). On the other hand, injury caused by exposure to allyl alcohol alone at doses larger than those used in these studies produces predominantly periportal hepatic lesions (Thorgeirsson et al., 1976; Gumucio et al., 1978). The lesions observed in livers of rats cotreated with small doses of LPS and allyl alcohol in these studies were predominantly periportal, suggesting a histological picture more like allyl alcohol than LPS. The inflammatory infiltrate was moderate and may either be a response to LPS or to hepatic injury. Intravenous doses of LPS exceeding 1 mg/kg are required to produce liver injury in Sprague-Dawley rats (Hewett et al., 1992). Doses of LPS 4 orders of magnitude smaller than this significantly potentiated the hepatotoxicity of allyl alcohol. This effect of LPS to enhance the hepatotoxicity of allyl alcohol does not appear to be the result of increased production of acrolein, the reactive metabolite of allyl alcohol. There was no significant difference between the activities of ADH in liver cytosolic fractions from LPS- and vehicle-treated rats. Moreover, LPS did not affect the rate of conversion of allyl alcohol into acrolein in isolated, perfused livers. As with hepatotoxicity caused by allyl alcohol alone, bioactivation to 79 acrolein was necessary for LPS-induced potentiation of injury: inhibition of acrolein production with 4-MP prevented toxicity in cotreated rats. Acrolein readily forms adducts with GSH, causing a loss of hepatocellular GSH without a concomitant increase in GSSG (Silva and O’Brien, 1989; Dogterom et al., 1989b; Rikans et al., 1994). This loss occurs very rapidly, e.g., within 20 minutes after administration of allyl alcohol (Penttila et al.,1987; Pompella et al.,1988). GSH serves a protective role against allyl alcohol toxicity, and agents which diminish hepatocellular GSH increase toxicity of allyl alcohol (Maellaro et al., 1990). The possibility existed that LPS augmented the response to allyl alcohol by diminishing the availability of hepatocellular GSH; however, this does not appear to be the case because LPS pretreatment did not affect the hepatic concentrations of either GSH or GSSG. LPS has been shown to potentiate the hepatotoxicity of other compounds in experimental animals. Administration of exogenous LPS at doses similar to those used in this study (i.e., > 0.05 mg/kg) increases the hepatotoxicity of carbon tetrachloride (Chamulitrat et al., 1994), galactosamine (Galanos et al., 1979), and halothane (Lind et al., 1984). Endogenous LPS has been associated with the hepatotoxicity of galactosamine and alpha-naphthylisothiocyanate (Calcamuggi et al., 1992; Czaja et al., 1994). For example, Czaja and coworkers (1994) prevented galactosamine-induced hepatotoxicity by administration of an 80 antibody to LPS. The mechanism by which LPS enhances hepatotoxicity of these xenobiotics is unknown. In summary, LPS is able to potentiate the hepatotoxicity of allyl alcohol and is able to do so at remarkably small doses. Allyl alcohol must be converted into the reactive metabolite acrolein for the potentiation to occur. LPS does not affect the hepatic metabolism of allyl alcohol into acrolein nor does it affect the protective mechanism for removal of acrolein. At present, the mechanism by which LPS can enhance the potentiation of allyl alcohol hepatotoxicity is unknown. In the following chapter, we will discuss the role of the innate immune system in this model of liver injury. 81 Chapter 3 THE ROLE OF CELLS OF THE INNATE IMMUNE SYSTEM IN LPS- INDUCED ENHANCEMENT OF ALLYL ALCOHOL HEPATOXICITY 82 3. A. Abstract We have shown previously that small doses of LPS are able to enhance the hepatotoxicity of allyl alcohol in rats. Due to the important role of cells of the innate immune system (i.e. Kupffer cells and neutrophils) in mediating the hepatotoxicity of large doses of LPS, we examined the role of these cell populations in the model of low dose LPS enhancement of the hepatotoxicity of allyl alcohol. Male, Sprague-Dawley rats were treated with gadolinum chloride (GdCl3) (10 mg/kg, iv) or saline vehicle 22 hours prior to treatment with LPS (100 ug/kg, iv) or saline vehicle followed by allyl alcohol (30 mg/kg, ip) or saline vehicle. Gadolinium chloride inactivates Kupffer cells. The inactivation of Kupffer cell function provided the rats significant protection from the ability of LPS to potentiate the hepatotoxicity of allyl alcohol as measured by both ALT activity and histopathological examination. In a separate series of experiments, circulating neutrophils were eliminated with a polyclonal anti-neutrophil lg (1 ml/dose) administered 16 and 4 hours prior to treatment with LPS. The rats received LPS and allyl alcohol as described above. Removal of neutrophils from the model also significantly protected animals from LPS- enhanced allyl alcohol hepatotoxicity. These data suggest that both Kupffer cell-dependent and neutrophil-dependent mechanisms are 83 involved in this model and that each of these cell populations has a distinct contribution to the ability of LPS to potentiate the hepatotoxicity of allyl alcohol. 3. B. Introduction LPS, a component of the outer leaflet of the cell membrane of Gram- negative bacteria, is a powerful stimulant for the innate immune system. It elicits a number of biochemical events such as the release of proteases, free radicals, eicosanoids, cytokines, etc., which are ideally present to protect the host from bacterial infection but may actually cause tissue damage to the host. LPS may also be Involved in another source of damage to a host mammal: acting synergistically with a xenobiotic to produce more damage to the host than either toxicant alone. In Chapter 2, results of experiments in which a nontoxic dose of LPS enhanced liver damage from a subtoxic dose of allyl alcohol were presented. In this chapter, the role of two important cell types of the innate immune system, Kupffer cells and neutrophils will be examined. The hypothesis to be addressed is as follows: removal of functional populations of either Kupffer cells or neutrophils protects rats from the synergistic effects of LPS and allyl alcohol. 84 3. C. Materials and Methods 3. C. 1. Materials Lipopolysaccharide (Escherichia coli, serotype 0128:812, specific activity 12 x 107 endotoxin units/mg for Kupffer cell inactivation studies and 7 x 107 endotoxin units/mg for neutrophil depletion studies) and Sigma Diagnostics Kits No. 59 UV and No. 58 UV for determination of activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) respectively, were purchased from Sigma Chemical Company (St. Louis, MO). Allyl alcohol and GdCla were purchased from Aldrich Chemical Company (St. Louis, MO). Sterile saline was obtained from Abbott Labs (Abbott Park, IL). The Vectastain ABC-AP and Vector Red kits used for the immunohistochemical staining of neutrophils were purchased from Vector Laboratories (Burlingame, CA). 3. C. 2. Production of Anti-Neutrophil lg The immunoglobulin G fraction of rabbit blood serum, both anti- neutrophil and control, was produced in our laboratory using New Zealand 85 White and nonpedigreed rabbits respectively (Hewett et al., 1992). Glycogen-elicited, peritoneal neutrophils were obtained from Sprague- Dawley rats. These were washed and homogenized using a Dounce homogenizer. For initial injections, the neutrophil homogenate was mixed (1:1) with Freund’s complete adjuvant. Rabbits were tranquilized with acepromazine and restrained. The skin on the back of the neck area was disinfected, and the neutrophil/adjuvant solution (0.1 ml) was injected subcutaneously into six areas on either side of the spine. The initial injection was followed by boost injections every two weeks. The boost injections were identical to the initial injection except that Freund’s incomplete adjuvant was used in place of Freund’s complete adjuvant. Blood was collected from rabbits (ear artery) on weeks in which the animals did not receive boost injections. Blood was also collected from na'I've rabbits for production of control lg. The blood was allowed to clot at room temperature for collection of serum. The lg fraction of the serum was prepared by ammonium sulfate precipitation followed by extensive dialysis to remove excess ammonium sulfate. The neutrophil-depleting activity of the lgG fraction was determined by peripheral white cell counts of blood smears from rats given various doses of lgG. The dose of 1 ml/rat resulted in 97% reduction in the absolute numbers of circulating neutrophils compared to controls. 86 3. C. 3. Animals Male, Sprague-Dawley rats [CD-CrlzCD-(SD)BR VAF/Plus]; Charles River, Portage, MI) weighing 200-300 g were used in all studies except for the determination of GSH levels. Female, Sprague—Dawley rats were used for those experiments. The animals were allowed food (Rodent Chow, Teklad, Madison, WI) and water ad Iibitum. They were maintained on a 12-hour light and dark cycle under conditions of controlled temperature and humidity. 3. C. 4 Treatment Protocols 3. C. 4. 1. Inactivation of Kupffer Cells In experiments to examine the role of Kupffer cells, animals were pretreated with GdCl3 (10 mg/kg iv) or vehicle 22 hours before administration of LPS or its vehicle. Animals were given 100 pg/kg iv of LPS or an equivalent volume of saline vehicle 2 hours before treatment with allyl alcohol (30 mg/kg ip). The experimental protocol was a 2 x 2 x 2 design. Treatment of rats with GdCI3 using this dosing regimen resulted in 87 inhibition of Kupffer cell phagocytic activity as assessed by clearance of colloidal carbon from blood (Husztik et al., 1980; Roland et al., 1993; Ganey and Schultze, 1995). Animals were killed 18 hours after treatment with allyl alcohol or its saline vehicle. 3. C. 4. 2. Depletion of Circulating Neutrophils Rats received anti-neutrophil lg (1 ml/dose) 16 and 4 hours before injection with LPS. Animals were given 100 ug/kg iv of LPS or an equivalent volume of saline vehicle 2 hours before treatment with allyl alcohol (30 mg/kg ip). The experimental protocol was a 2 x 2 x 2 design. Animals were killed 6 hours after treatment with allyl alcohol or saline control. The extent of neutrophil depletion was determined by differential counts of stained, peripheral blood smears. 3. C. 5. Immunohistochemical Staining of Neutrophils in Liver Sections In order to determine the effect of the anti-neutrophil lg on the accumulation of neutrophils into the liver, the liver was removed intact. 88 Samples taken for microscopic examination were preserved in ten percent buffered formalin. Tissue sections (6 microns thick) were fixed to glass slides and deparaffinized using a reverse xylene gradient to properly hydrate the tissue. The deparaffinized sections were then treated with proteinase K (Sigma, St Louis, MO). A polyclonal rabbit lg fraction was used as the primary antibody and a biotinylated anti-rabbit lg was used as the secondary antibody (Vector Laboratories, Burlingame, CA). A substrate was then added to the tissues to produce a red precipitate (Vector Red) on the antibody-labeled neutrophils. The tissues were counterstained with hematoxylin (Gill No.3) (Sigma, St Louis, MO). The number of neutrophils was counted in thirty high-power fields per slide (ten fields per tissue section). 3. C. 6. Assessment of Hepatotoxicity Rats were anesthetized with sodium pentobarbital (50 mg/kg ip), and blood was collected from the abdominal aorta into syringes containing 3.8% sodium citrate (final concentration 0.38%). Activities of ALT and AST were determined in plasma using Sigma Diagnostics Kits No. 59 UV and No. 58 UV, respectively. The liver was removed intact. Samples taken for microscopic examination were preserved in Histochoice fixative (Amresco, 89 Solon, OH) or 10% buffered formalin. Tissue sections were processed for light microscopy, cut at 6 microns, stained with hematoxylin and eosin and evaluated for lesion severity. 3. C. 7. Statistical Analysis Data are expressed as means 1 SEM. For all results presented, N represents the number of individual animals. Homogeneous data were analyzed by two-way analysis of variance (ANOVA). Individual means were compared using Tukey’s omega test. When variances were not homogenous, data were analyzed using the KruskaI-Wallis ANOVA on ranks for more than two groups. In the latter case, Dunn’s or Tukey’s test was used to assess significance. The criterion for statistical significance was p _<_ 0.05. 90 3. D. Results 3. D. 1. Protective Effect of GdCI3. To test the hypothesis that LPS activation of Kupffer cells is important in the enhanced response to allyl alcohol, rats were treated with GdCI3 before exposure to LPS. Plasma ALT activity was low in animals exposed to saline, LPS or allyl alcohol alone and was unaffected by pretreatment with GdCI3 (Figure 3.1.A). Plasma ALT activity was significantly elevated in animals cotreated with LPS and allyl alcohol, and GdCl3 pretreatment prevented this increase. Similar results were observed for activity of AST in plasma (Figure 3.1.B) GdCl3 did not alter the hepatocellular concentration of GSH or GSSG (Table 3.1) as compared to the concentrations of GSH and GSSG in rats pretreated with saline (Table 2.2). Consistent with the histopathological observations described in Chapter 2, examination of liver sections from rats cotreated with LPS and allyl alcohol revealed a necrotizing, coagulative hepatitis. In most cases the lesions were not confined to the periportal region but had spread to other regions of the lobule, becoming panlobular. The lesions observed in livers of cotreated rats pretreated with GdCl3 were qualitatively similar to 91 Figure 3.1. Protection from LPS-induced potentiation of allyl alcohol hepatotoxicity by GdCI3. Animals were treated with GdCI3 (10 mg/kg iv) 22 hours before administration of LPS. Animals were treated with LPS (100 ug/kg iv) or vehicle two hours before administration of allyl alcohol (30 mg/kg ip) or vehicle. (A) ALT and (B) AST activities in plasma were measured as markers of liver toxicity 18 hrs after treatment with allyl alcohol. Data are expressed as mean 1 SEM. a, significantly different from respective value in the absence of allyl alcohol. b, significantly different from respective value in the absence of LPS. 0, significantly different from respective value in the absence of GdCl3. N = 4-12. 92 2500 - - Sal A 2000 - ‘33:] 6°03 .1 \ 3 v I— 1500 . _l < (D E 1000 - (I) L“ CL 500 - o 3000 — - Sal A 2500 GdCla d a 2000 — [..— (D < 1500 4 (D E L.“ 1000 - Q 500 — 0 .. Sal LPS Sal 93 Sal a,b LPS Allyl Alcohol I76 'spoutew pue slepetew uI pequosep se pequIIetep eIeM suonenueouoo 9339 pue H89 1911" pue ‘eJnsodxe eunes JO Sd'l Jaye smou 9'1 paupj eJeM slewguv 'euues .IO (Bx/6w 17) Sd'l Jeuue Bug/119091 eIOjeq smou 173 eunes peAIeoeI $18.1 Aewea-enfimds ‘9|Bu18:j 170'0 7- 970 9'0 1 9'9 Sd'l 800 1 [170 7'0 1 9'2 IBS JeAu B/wri IeAII B/wd OSSE) HSE) tuewteeu HOPE) 1411M peteeJteId 8181 u! euogutemjfi pezngxo pue peonpeJ jO S|9A9| JB|n||90019d3u to uosuedwog VS qu81 those seen in cotreated animals pretreated with saline; however, the extent of the necrosis was less severe in nature. GdCl3 did not affect the inflammatory infiltrate observed in livers of cotreated rats. Livers from rats treated with allyl alcohol in the absence of LPS were also less severely damaged. Sections of livers from rats treated with saline or LPS only were histologically normal with no evidence of hepatocellular necrosis or edema. 3. D. 2. Protection by Depletion of Circulating Neutrophils To ascertain that the anti-PMN lg had successfully depleted animals of circulating neutrophils, differential leukocyte counts were performed on blood smears from all animals. The number of circulating neutrophils was significantly lower in those animals pretreated with anti-PMN lg compared to animals pretreated with control lg, indicating that the circulating population of neutrophils was reduced by the anti-PMN lg (Table 3.2). Depletion of the circulating neutrophil pool by an anti-neutrophil IgG fraction afforded protection to rats treated with both LPS and allyl alcohol (Figure 3.2). Plasma ALT activity was significantly lower in cotreated animals pretreated with the anti-neutrophil lg compared to those pretreated with the control lg fraction. In rats treated with allyl alcohol alone, there was no significant difference in the plasma ALT activity between groups 95 Table 3.2 Numbers of circulating leukocytes in control rats versus rats treated with anti-PMN lgG (cells per cubic millimeter). TOTAL WHITE NEUTROPHILS MONOCYTES LYMPHOCYTES CELL COUNT C/Sal/AA 9400 i 1754 1472 i 483 1948 i 299 5232 :|_- 1432 P/Sal/AA 3167 i 547 15 i 103 245 _t 563 2906 i 497 C/LPS/AA 6720 i 820 2084 i 378 1415 j; 175 3413 i 479 P/LPS/AA 4440 i 1307 36 1 24a 141 1 30a 4344 i 1265 Rats were treated with anti-PMN lg (1 mI/animal) or control lg 16 and 4 hours prior to administration of LPS (100 ug/kg). Rats were treated with allyl alcohol (30 mg/kg ip) 2 hrs after LPS (100 ug/kg iv). a, significantly different from respective control lg group 96 Figure 3.2 Protection from LPS-induced potentiation of allyl alcohol hepatotoxicity by depletion of circulating neutrophils. Animals were treated with an anti-PMN lgG fraction or a control lgG fraction (1 ml/animal iv) 16 and 4 hours prior to administration of LPS. Animals were treated with LPS (100 ug/kg iv) or vehicle two hours before administration of allyl alcohol (30 mg/kg ip) or vehicle. Plasma ALT activity was measured 6 hours after allyl alcohol treatment. Data are expressed as mean 1; SEM. a, significantly different from respective value in the absence of LPS. b, significantly different from respective value in the presence of anti-PMN lgG. N = 2-12. 97 Plasma ALT (U/L) 5000 - — Control lgG fraction [:23 Anti-PMN lgG fraction 4000 -+ 3000 . 2000 - 1000 ~ Sal LPS Sal LPS Saline Allyl Alcohol 98 that received the anti-neutrophil lg fraction or control lg. These data indicate that lg alone does not affect the baseline hepatotoxicity of allyl alcohol. Examination of liver sections by light microscopy revealed liver lesions similar to those described above. In animals cotreated with LPS and allyl alcohol, there was extensive coagulative necrosis of the hepatic parenchyma which ranged from lesions confined to the periportal regions of the liver lobules to lesions encompassing several adjacent lobules. A prominent inflammatory infiltrate consisting primarily of neutrophils was present in the necrotic sinusoids of those cotreated animals that had been pretreated with control lg. In contrast, cotreated animals pretreated with anti-PMN lg had markedly fewer neutrophils present in the necrotic areas. There was no accumulation of inflammatory cells in areas of healthy tissue in either group. Hepatic lesions were also noted in animals treated with allyl alcohol only. These lesions were similar to those observed in cotreated animals but they tended to be less severe. The infiltration of neutrophils into the livers of animals was also quantified. There was a significant decrease in the number of neutrophils present in the liver lesions of cotreated animals pretreated with the anti- PMN lg as compared to the liver lesions of cotreated animals pretreated with control lg (Table 3.3). 99 Table 3.3 Infiltration of neutrophils into hepatic sinusoids (number of cells per 30 high power fields) Control lg Fraction Anti-PMN lg Fraction Sal/Sal 6 1 2 7 1 4 LPS/Sal 72 i 9 12 1 3 Sal/Allyl Alcohol 115 1- 44 24 1 9a LPS/Allyl Alcohol 276 i 63 19 i 6a Rats were treated with anti-PMN lgG (1 ml/animal) 16 and 4 hours prior to administration of LPS (100 ug/kg). Liver sections (6 microns thick) were immunostained for neutrophils. a, significantly different from values in the absence of neutrophil depletion. 100 3. E. Discussion Pretreatment of rats with nontoxic doses of LPS (100 pg/kg) significantly increased liver damage in rats exposed to a nonlethal dose of allyl alcohol as measured by both biochemical assays and pathological examination. This increase in liver injury was significantly attenuated by either inactivation of Kupffer cell phagocytosis by GdCl3 or depletion of circulating neutrophils with an anti-neutrophil lg fraction. These data strongly suggest that both of these cellular components of the innate immune system play important roles in the ability of small amounts of LPS to potentiate allyl alcohol hepatotoxicity. In mammals, a small amount of LPS normally escapes the intestinal tract and enters the portal circulation (Jones and Summerfield, 1988; Fox et al., 1989; Nakao et al., 1994). This LPS is removed by Kupffer cells before it can reach the general circulation and potentially elicit a systemic inflammatory response. Kupffer cells and hepatocytes are believed to work together in the chemical detoxification of LPS (Fox et al., 1989). Kupffer cells perform the first part of the detoxification process by removing some of the polysaccharide moieties; the modified LPS is then passed onto hepatocytes for further metabolism. 101 Despite the fact that Kupffer cells serve this beneficial role in removal of LPS, inhibition of Kupffer cell function affords protection from the lethal effects and liver injury caused by large doses of LPS (limuro et al.,, 1994; Pearson et al., 1996; Brown et al., 1997). This apparent paradox may be explained in part by activation of Kupffer cells under conditions in which there are large concentrations of LPS in the portal circulation which overload the detoxification mechanism of Kupffer cells; for example, during overgrowth of gram-negative bacteria or after intravenous administration of LPS (Biliar et al., 1988). In response to activation by LPS, Kupffer cells release a number of chemical mediators of inflammation including cytokines, prostaglandins, leukotrienes, reactive oxygen species (ROS), platelet activating factor, and nitric oxide (Van Bossuyt et al., 1988; Van Bossuyt and Wisse, 1988; Decker, 1990; Spolarics et al., 1993; Portoles et al., 1994; Brouwer et al., 1995). These mediators may have adverse effects on neighboring cell types and contribute to the development of tissue injury. Thus, the mechanism by which inhibition of Kupffer cell function protects animals from adverse effects of LPS may involve attenuated release of potentially toxic mediators. Indeed, although inhibition of Kupffer cell activity with GdCl3 decreased liver injury in response to large doses of LPS, hepatic accumulation of neutrophils was not attenuated (Brown et al., 1997) and accumulation of platelets was decreased only slightly (Pearson et al., 1996). These results suggest that 102 Kupffer cells are not responsible for signaling the initiation of inflammation in the liver during hepatic injury caused by large doses of LPS, but rather contribute to subsequent events such as release of soluble mediators that lead ultimately to tissue damage. Results similar to those in animals exposed to large doses of LPS were observed in rats cotreated with small doses of LPS and allyl alcohol: GdCla decreased the severity of hepatic necrosis but did not alter the inflammatory infiltrate. Although Kupffer cell activation was not measured directly, at the doses of LPS used in these studies TNF activity in plasma is increased (see Chapter 4). Kupffer cells are hypothesized to be the primary source of this TNF, although this hypothesis has been questioned. Furthermore, histological analysis revealed unusually prominent Kupffer cells in the sinusoids of rats treated with small doses of LPS, consistent with modification of these cells. Thus, low doses of LPS may increase susceptibility of hepatic parenchymal cells to injury from allyl alcohol through some of the same, Kupffer cell-dependent events that ultimately lead to liver injury at larger doses of LPS. It is likely that the protective effect of GdCl3 on LPS enhancement of allyl alcohol hepatotoxicity is mediated through abrogation of LPS effects rather than allyl alcohol effects. In support of this, it has been demonstrated that treatment with GdCl3 does not affect activity of ADH (Przybocki et al., 1992), ruling out decreased bioactivation of allyl alcohol 103 as a mechanism by which GdCl3 affords protection. Furthermore, inhibition of Kupffer cell function did not prevent the hepatotoxicity of allyl alcohol in the absence of LPS in our studies (Ganey and Schultze, 1995). These results suggest that, although functional Kupffer cells are not required for liver damage from larger doses of allyl alcohol, LPS-induced augmentation of allyl alcohol hepatotoxicity occurs through a Kupffer cell-dependent mechanism. Another suggestion from this study is that the activation of Kupffer cells may be important in their augmentation of the hepatotoxicity of allyl alcohol. These data are consistent with the findings of other researchers using different hepatotoxicants. For example, activation of Kupffer cells with vitamin A increased the hepatotoxicity of carbon tetrachloride (ElSisi et al., 1993a; ElSisi et al., 1993b; ElSisi et al., 1993c; Rosengren et al., 1995). Release of reactive oxygen species by vitamin A-stimulated Kupffer cells is believed to be a contributing factor in this increased liver damage (ElSisi et al., 1993a). Inflammatory mediators are likely to be important in the ability of LPS-stimulated Kupffer cells to potentiate the hepatotoxicity of allyl alcohol, and in Chapter 4 we begin to explore this possibility. In addition to Kupffer cells, the other major cell type of innate immunity, the neutrophil, is implicated in the mechanism by which LPS enhances allyl alcohol hepatotoxicity. Neutrophils play a central role in the acute 104 inflammatory response. These cells are among the first to arrive at a focus of infection and begin killing bacteria by phagocytosis and release of free radicals and proteases. A certain amount of tissue damage occurs during this process but is usually repaired during the healing phase of inflammation. As mentioned in Chapter 1, neutrophils are a critical component of liver injury induced by large doses of LPS. Large numbers of neutrophils accumulate in the liver sinusoids and release mediators of inflammation which are toxic to adjacent hepatocytes. Removal of neutrophils with an anti-neutrophil antibody (Hewett et al., 1992; Sato et al., 1993) or prevention of neutrophil migration with an antibody to leukocyte adhesion molecules (Jaeschke et al., 1991) protected animals from the toxic effects of LPS, confirming the important role neutrophils play in organ damage during exposure to large doses of LPS. A marked feature of the histopathology from the initial studies of the LPS/allyl alcohol model presented in Chapter 2 was the presence of large numbers of neutrophils in the damaged and necrotic sinusoids. The exact role of the neutrophils was unclear: were they attracted by the damaged tissue or did they have an active role in causing the damage? Depletion of circulating neutrophils in cotretreated animals reduced ALT release and liver injury as seen histologically to a level that was significantly different from that of cotreated controls. These results suggest that neutrophils 105 attracted into the sinusoids by LPS contribute to the liver injury when LPS potentiates the hepatotoxicity of allyl alcohol. However, neutrophils were seen in the necrotic hepatic sinusoids of rats treated with allyl alcohol alone. The role of these cells is unclear. Work by Ganey and Schulze (1995) has demonstrated that neutrophil depletion did not protect rats from a toxic dose of allyl alcohol. In summary, LPS appears to potentiate the hepatotoxicity of allyl alcohol by activating the host’s innate immune system, specifically Kupffer cells and neutrophils. These cells are the source of many powerful biochemical mediators during inflammation and it is possible that some of these affect adjacent hepatic parenchymal cells, making them more sensitive to the toxic properties of allyl alcohol given alone. One possible mediator will be explored in Chapter 4. 106 Chapter 4 PENTOXIFYLLINE ATTENUATES BACTERIAL LlPOPOLYSACCHARIDE-INDUCED ENHANCEMENT OF ALLYL ALCOHOL HEPATOTOXICITY 107 4. A. Abstract Small amounts of exogenous lipopolysaccharide (LPS) (10 ng/kg-1OO ug/kg) enhance the hepatotoxicity of allyl alcohol in male, Sprague-Dawley rats. This augmentation of allyl alcohol hepatotoxicity appears to be linked to Kupffer cell function, but the mechanism of Kupffer cell involvement is unknown. Since Kupffer cells produce tumor necrosis factor-alpha (TNF) upon exposure to LPS and this cytokine has been implicated in liver injury from large doses of LPS, we tested the hypothesis that TNF contributes to LPS enhancement of allyl alcohol hepatotoxicity. Rats were treated with LPS (10-100 ug/kg iv) 2 hours before allyl alcohol (30 mg/kg ip). Cotreatment with LPS and allyl alcohol caused liver injury as assessed by an increase in activity of alanine aminotransferase in plasma. Treatment with LPS caused an increase in plasma TNF concentration which was prevented by administration of either pentoxifylline (PTX) (100 mg/kg iv) or anti-TNF serum (1ml/rat iv) one hour prior to LPS. However, only PTX protected rats from LPS-induced enhancement of allyl alcohol hepatotoxicity; anti-TNF serum had no effect. Exposure of cultured hepatocytes to LPS (1-10 ug/ml) or to TNF (15-150 ng/ml) for 2 hours did not increase the cytotoxicity of allyl alcohol (0.01-200 uM). These data suggest that neither LPS nor TNF alone is sufficient to increase the sensitivity of isolated hepatocytes to allyl alcohol. Furthermore, 108 hepatocytes isolated from rats treated 2 hours earlier with LPS (i.e. hepatocytes which were exposed in vivo to TNF and other inflammatory mediators) were no more sensitive to allyl alcohol-induced cytotoxicity than hepatocytes from nai've rats. These data suggest that circulating TNF is not involved in the mechanism by which LPS enhances hepatotoxicity of allyl alcohol and that the protective effect of PTX may be due to another of its biological effects. 4. B. Introduction We have recently demonstrated that the hepatotoxicity of allyl alcohol is enhanced by pretreatment with quite small doses of LPS and that this augmented response is prevented by inhibition of the function of another cellular mediator of inflammation, Kupffer cells (Sneed et al., 1997). These results indicate that properly functioning Kupffer cells are important in the mechanism of LPS-induced enhancement of allyl alcohol hepatotoxicity and evoke interest in whether inflammatory mediators released by these cells participate in augmenting toxicity. Kupffer cells are the resident macrophages of the hepatic sinusoids (Jones and Summerfield, 1988; Bouwens and Wisse, 1992; Wisse et al., 1996) and have a major role in clearing the hepatic portal blood of 109 intestinally derived LPS (Fox et al., 1989; Toth et al., 1992). These macrophages respond to LPS with production of mediators such as cytokines (e.g. tumor necrosis factor-alpha (TN F), interleukin-1, interleukin- 6), reactive oxygen species, and prostaglandins (Decker et al., 1990). Kupffer cells play a critical role in liver injury from large doses of LPS as evidenced by the observation that inhibition of their function with gadolinium chloride (GdCl3) affords protection (limuro et al., 1994; Pearson et al., 1996; Brown et al., 1997). Cytokines are also essential to LPS-induced responses. For example, inhibition of TNF synthesis or activity attenuates liver injury and lethality in baboons (Tracey et al., 1987), mice (Beutler et al., 1985) and rats (Hewett et al., 1993). These results indicate that TNF is important in the pathogenesis of tissue injury from large doses of LPS and raise the possibility that TNF may be a factor in the ability of LPS to enhance the hepatotoxicity of xenobiotics. The present study was undertaken to test the hypothesis that TNF participates in the potentiation of allyl alcohol hepatotoxicity by LPS. Two approaches were taken to inhibit the effects of TNF in animals treated with LPS and allyl alcohol: pentoxifylline (PTX) was given to inhibit synthesis of TNF, and an antiserum directed against TNF was administered to neutralize TNF activity. 110 4. C. Materials and Methods 4. C. 1. Materials Lip0polysaccharide (Escherichia coli, serotype 0128:812. Specific activity 1 x 107 endotoxin units/mg for the in Vitro studies and 7 x 107 endotoxin units/mg for the in vivo studies), and Sigma Diagnostics Kit No. 59 UV for determination of activity of alanine aminotransferase (ALT) were purchased from Sigma Chemical Company (St. Louis, MO). Collagenase and pentoxifylline were obtained from Sigma Chemical Company (St. Louis, MO). Allyl alcohol was purchased from Aldrich Chemical Company (St. Louis, MO). Williams’ medium E was purchased from Gibco (Gaithersburg, MD). 4. C. 2. Production of Anti-TNF Serum The rabbit blood serum, both anti-TNF and control, was produced in our laboratory using New Zealand White and nonpedigree rabbits respectively (Hewett et al., 1992). Recombinant murine TNF (50 pl) (R & D Systems, Minneapolis, MN) was compounded with 1 ml of saline and 1 ml of 111 Freund’s complete adjuvant. Rabbits were tranquilized with acepromazine and restrained. The skin on the back of the neck area was disinfected, and 30 pl of the TNF/adjuvant solution was injected intradermally into six areas on either side of the spine. A separate injection (0.5 ml) was given intraperitoneally. The initial injection was followed by boost injections 14 and 28 days later. The boost injections were identical to the initial injection except that Freund’s incomplete adjuvant was used in place of Freund’s complete adjuvant. Blood was drawn from the ear artery or vein at two week intervals after the last boost series. The blood was allowed to clot for two hours at room temperature then overnight at 4°C. Serum was harvested and tested for anti-TNF titer using the WEHI subclone 13 bioasaay (Espevik and Nissen- Meyer, 1986; Eskandari et al., 1990). The serum was stored at —20°C. 4. C. 3. Animals Male, Sprague-Dawley rats [CD-Crl:CD-(SD)BR VAF/Plus]; Charles River, Portage, MI) weighing 200-300 gm were used in all studies. The animals were allowed food (Rodent Chow, Teklad, Madison, WI) and water ad Iibitum. They were maintained on a 12-hour light and dark cycle under conditions of controlled temperature and humidity. 112 4. C. 4. Isolation of Hepatocytes Hepatocytes were isolated by collagenase digestion (Seglen, 1973; Klaunig et al., 1981), placed in Williams’ medium E supplemented with 10% fetal calf serum and 0.1% gentamicin. Hepatocytes from isolations with a cell viability of 90% or greater were plated in 6-weIl primaria plates (Falcon Laboratories) at a density of 5 x 105 cells per well. In some experiments, hepatocytes were obtained from the livers of rats treated 2 hours earlier with LPS (4 mg/kg iv). The hepatocytes were allowed to stabilize in culture for 3 hours, the medium was removed, and the cells were washed once with Williams’ medium E supplemented only with 0.1% gentamicin. A final volume of 2 ml per well of the latter medium was used in the remainder of the study. Allyl alcohol was added to the hepatocyte cultures at the concentrations of O, 0.1, 1, 25, 30, 50, 60, 100, or 200 mM as indicated in the Figures and Results. Hepatocyte injury was assessed 90 or 180 minutes after addition of allyl alcohol. In studies in which hepatocyte cultures were incubated with LPS or TNF, these agents were added 2 hours prior to treatment with allyl alcohol. 113 4. C. 5. Treatment Protocols 4. C. 5. 1. Treatment of Animals with Anti-TNF Serum In preliminary experiments to determine the effective dose of serum, a limited number of rats were pretreated intravenously with 0, 0.25, 0.50, or 1.0 ml of anti-TNF serum diluted 1:1 with saline 1 hour before treatment with LPS (100 ug/kg iv) (Hewett et al., 1993). Ninety minutes after administration of LPS animals were anesthetized with pentobarbital (50 mg/kg ip) and blood was drawn from the abdominal aorta into syringes containing 3.8% sodium citrate (final concentration 0.38%) allyl alcohol (30 mg/kg) or sterile saline was injected intraperitoneally. The activity of TNF was determined using the WEHI subclone 13 bioassay (Figure 4.1). Administration of either 0.25 or 0.5 ml of anti-TNF serum neutralized TNF to undetectable levels in 3 out of 4 rats; however, I ml of serum effectively eliminated TNF activity in all rats tested. This dose was chosen for the remaining studies. 114 4. C. 5. 2. Treatment of Animals with Pentoxifylline Rats were treated intravenously with PTX (100 mg/kg) or with an equivalent volume of sterile saline (Abbott Laboratory, Abbott Park, IL) 1 hour prior to treatment with LPS (100 ug/kg). This treatment protocol for PTX has been shown to prevent the LPS-induced rise in plasma TNF activity (Hewett et al., 1993). Two hours after administration of LPS, allyl alcohol or its sterile saline vehicle was injected intraperitoneally. Liver injury was assessed six hours later. 4. C. 6. Assessment of Hepatocyte Cytotoxicity Cytotoxicity was assessed from release of ALT into the medium. The medium was collected from the wells, and 2 ml of 1% TritonX-100 were added to each well and allowed to remain for at least 5 minutes at room temperature. The wells were scraped thoroughly with a rubber policeman to remove all cells, and the resulting solution was sonicated to disperse subcellular components. All samples (both medium and Iysate) were centrifuged for 10 minutes at 600 x g. The activity of ALT in all cell-free supernatant fluids was determined with Sigma Diagnostics Kit No. 59-UV. The activity in the medium (i.e., ALT released) was expressed as a 115 percentage of the total (medium plus Iysate) activity (Ganey et al., 1994; Ho et al., 1996). 4. C. 7. Assessment of Hepatotoxicity. Rats were anesthetized with sodium pentobarbital (50 mg/kg ip). and blood was collected from the abdominal aorta into syringes containing sodium citrate (final concentration, 0.38%). ALT activity was determined in plasma. 4. C. 8. Determination of Activity of TNF The activity of TNF in the blood of rats was determined 90 minutes after administration of LPS or its saline vehicle. Rats were anesthetized with sodium pentobarbital (50 mg/kg ip), and blood was collected from the abdominal aorta into syringes containing sodium citrate (final concentration, 0.38%). Plasma was collected, diluted serially and 116 Figure 4.1. Dose-response for anti-TNF serum. In order to determine the amount of anti-TNF serum needed to completely neutralize circulating levels of plasma TNF, rats were treated with either 0.25, 0.50, or 1.0 ml of anti-TNF serum diluted 1:1 in saline or a saline control. The animals were given LPS (100 ug/kg) one hour later. Plasma was collected 90 minutes after injection with LPS, and activity of TNF was determined as described in Materials and Methods. N = 4. 117 TNF (pg/ml) 7000 — c 6000 — 5000 ~ 4000 — 0 o 3000 — // / 0 — 0 III 4“ WV 0 0.25 0.50 1.00 Dose of anti-TNF Serum (ml) 118 incubated for 22 hours in the presence of the TNF-sensitive fibrosarcoma cell line, WEHI 164 clone 13 (Espevik and Nissen-Meyer, 1986; Eskandari et al., 1990). The extent of cell lysis was measured with 3-(4,5- dimethylthiazoI-Z-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) using a Bio-Tek plate reader, and the amount of TNF was calculated from a standard curve using a serial dilution of a recombinant murine TNF standard (R & D Systems, Minneapolis, MN). 4. C. 9. Assessment of Alcohol Dehydrogenase Activity in Liver Homogenates Rats were treated with PTX (100 mg/kg) or saline vehicle 90 minutes prior to treatment with LPS (10 ug/kg). Two hours after treatment with LPS or saline vehicle, animals were killed. The liver was removed and homogenized in a solution of 0.05 M HEPES (pH 8.4) and 0.33 mM dithiothreitol. The homogenate was centrifuged at 100,000 x g for 45 min. The supernatant fluid was collected, and activity of ADH was measured spectrophometrically (366 nm) by monitoring the reduction of nicotinamide adenine dinucleotide (NAD) using ethanol as a substrate (Krebs et al., 1969). Protein concentration in the supernatant was determined using the bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL). 119 4. C. 10. Statistical Analysis Data are expressed as means 1 S.E.M. For all results presented, N represents the number of individual animals used for in vivo or in Vitro studies. Homogenous data were analyzed by one-way or two-way analysis of variance (ANOVA). Individual means were compared using Tukey’s omega test. When variances were not homogenous, data were analyzed using Kruskal-Wallis ANOVA on ranks, and Dunn’s test was used to assess significance. Data expressed as percentages were transformed by the arc sine square root method prior to analysis. The criterion for statistical significance was p 5 0.05. 4. D. Results 4. D. 1. Effect of In Vitro Exposure to LPS on Allyl Alcohol-Induced Cytotoxicity in Isolated Hepatotcytes To test whether LPS has direct effects on hepatocytes that contribute to enhancement of allyl alcohol toxicity, isolated hepatocytes were pretreated with LPS for 2 hours, then exposed to allyl alcohol (Figure 4.2). This 120 experimental design was selected to mimic the dosing regimen for LPS and allyl alcohol that results in LPS enhancement of allyl alcohol toxicity in vivo (Sneed et al., 1997). In hepatocytes not exposed to LPS, allyl alcohol caused a concentration-related increase in ALT release at 90 minutes; significant differences were observed at concentrations of allyl alcohol _>_ 100 pM. In hepatocytes exposed to LPS, a significant rise in ALT release was also seen at concentrations of allyl alcohol 3 100 uM, and there were no significant differences in ALT release ‘ among groups at any concentration of allyl alcohol. LPS was not cytotoxic in the absence of allyl alcohol (Figure 4.2). Similar experiments were performed in which cytotoxicity was assessed at 180 minutes after addition of allyl alcohol. No further increase in toxicity was observed at this time, and no differences were observed among the LPS-treated groups (data not shown). 4. D. 2. Protection from LPS-Induced Enhancement of Allyl Alcohol Hepatotoxicity by PTX PTX decreases the synthesis of TNF at the mRNA level (Dezube et al., 1993). Control animals treated with saline only or with PTX and saline only did not have detectable plasma activity of TNF (Table 4.1). TNF 121 Figure 4.2. Lack of effect of LPS exposure in Vitro on allyl alcohol cytotoxicity towards hepatic parenchymal cells. Rat hepatocytes were cultured in medium containing 0, 1, or 10 ug/ml LPS for 2 hours. Allyl alcohol was added at the indicated concentrations, and cytotoxicity was assessed 90 minutes later from release of ALT activity into the medium. Data are expressed as mean i S.E.M. a, all values at these points, irrespective of LPS concentration, are significantly different from respective controls not exposed to allyl alcohol. N=5. 122 ALT Released (% of Total) 100 - (D O l (I) O l 70— 60— 50— 40— 30— 20— 10‘ + 0 ug/ml LPS _l— 1 ug/ml LPS -—-*- 10 ug/ml LPS l 30 60 Allyl Alcohol mm 123 100 200 activity was increased significantly in the plasma of animals treated 90 minutes earlier with LPS. Pretreatment with PTX significantly reduced the circulating activity of TNF. Plasma ALT activity was low in rats in the saline/saline or LPS/saline groups irrespective of PTX pretreatment (Figure 4.3). In animals that received the vehicle for PTX and then were treated with saline and allyl alcohol there was an increase in plasma ALT activity, but this increase was not statistically significant. Animals treated with the vehicle for PTX and then cotreated with LPS and allyl alcohol had significantly elevated plasma ALT activity. Pretreatment with PTX attenuated the increase in plasma ALT activity in cotreated animals. Allyl alcohol hepatotoxicity requires bioactivation by ADH to acrolein. Accordingly, we examined the effect of PTX on the activity of ADH in the livers of rats pretreated with LPS to determine if PTX afforded protection by inhibition of the bioactivation of allyl alcohol. As shown in Table 4.2, pretreatment with PTX did not affect the activity of ADH in rat liver. 124 Table 4.1 Effect of PTX on the LPS-induced increase in plasma TNF activity TNF (ng/ml) Treatment Saline PTX Saline ND ND LPS 15.0 1 3.2 0.04 _4; 0.013 Rats were treated with PTX (100 mg/kg, iv) or saline vehicle one hour before treatment with LPS (0.1 mg/kg, iv). The activity of TNF was measured in plasma collected 90 minutes after LPS treatment. a, significantly different from respective value in the absence of PTX. ND, Not Detected. 125 Figure 4.3. Protection by PTX from LPS-enhancement of allyl alcohol hepatotoxicity. Animals were pretreated with PTX (100 mg/kg) 1 hour prior to treatment with LPS (100 ug/kg). Allyl alcohol (30 mg/kg) was given 2 hours after LPS; liver damage was measured 6 hours after allyl alcohol treatment. Data are expressed as mean 1 S.E.M. a, significantly different from values in absence of LPS . b, significantly different from respective value in the absence of PTX. N = 6. 126 Plasma ALT (Units/L) 2500 F o :1 Saline P t ' I' 2000 _ - enoxrfyllne 1500 — 1000 — I b 500 — 0 Mi Sal LPS Sal LPS Saline Allyl Alcohol 127 4. D. 3. Lack of Effect of Inactivation of TNF on LPS-Induced Potentiation of Allyl Alcohol Hepatotoxicity To verify that reduction of circulating TNF protects rats from LPS- potentiated allyl alcohol hepatotoxicity, animals were treated with an anti- serum specific for TNF prior to treatment with LPS. In preliminary studies, the efficacy of the anti-TNF antibody was determined. The administration of 1 ml of anti-TNF serum prevented the rise in plasma TNF such that TNF activity in all samples was below the limit of detection. There was no significant elevation in plasma ALT activity in animals in the saline/saline, LPS/saline or saline/allyl alcohol groups irrespective of pretreatment with control or anti-TNF serum (Figure 4.4). Plasma ALT activity was significantly elevated in animals cotreated with LPS and allyl alcohol compared to animals treated with LPS alone or allyl alcohol alone. There was no significant difference in ALT activity between cotreated animals pretreated with control serum and anti-TNF serum. 128 Table 4.2 Effect of PTX on the activity of alcohol dehydrogenase in LPS-treated rats ALCOHOL DEHYDROGENASE ACTIVITY (umol/min/g of liver) Treatment Saline PTX Saline 7.32 1 1.96 7.44 1 2.31 LPS 7.31 + 2.19 8.071 0.54 Rats were treated with PTX (100 mg/kg, iv) or saline vehicle one hour before treatment with LPS (0.1 mg/kg, iv). The activity of alcohol dehydrogenase was measured in homogenates collected 90 minutes after LPS treatment. There were no significant differences in the activity of hepatic alcohol dehydrogenase among any of the treatment groups. 129 Figure 4.4. Lack of protection from LPS enhancement of allyl alcohol hepatotoxicity by anti-TNF serum. Animals were pretreated with control or anti-TNF serum 1 hour prior to treatment with LPS (10 pg/kg). Allyl alcohol (30 mg/kg) was given 2 hours after LPS; liver damage was measured 6 hours after allyl alcohol treatment. Data are expressed as mean 1 S.E.M. a, significantly different from respective value in absence of LPS. b, significantly different from respective values in the absence of allyl alcohol. N = 3-13. 130 Plasma ALT (Unitle) 2000 1500 1000 500 :1 Control Serum - Anti-TNF Serum Sal LPS Saline 131 a,b a,b Sal LPS Allyl Alcohol Figure 4.5. Lack of effect of TNF exposure in Vitro on allyl alcohol cytotoxicity towards hepatic parenchymal cells. Rat hepatocytes were cultured in medium containing 0, 15, or 150 ng/ml TNF for 2 hours. Allyl alcohol was added at the indicated concentrations, and cytotoxicity was assessed 90 minutes later. Data are expressed as mean 1 S.E.M. a, all values at these points, irrespective of TNF concentration, are significantly different from respective controls not exposed to allyl alcohol. N = 3-5. 132 ALT Released (% of Total) 100 - (D O I 80- 70— —-O— 0 ng/ml TNF —l— 15 ng/ml TNF +- 150 ng/ml TNF ‘3 Allyl Alcohol (0M) 133 4. D. 4. Effect of In Vitro Exposure to TNF on Allyl Alcohol-Induced Cytotoxicity in Isolated Hepatocytes To test whether TNF alone can enhance the hepatotoxicity of allyl alcohol in isolated hepatocytes, cells were pretreated with TNF for 2 hours before exposure to allyl alcohol. Two concentrations of TNF were used: 15 ng/ml to replicate the TNF activity found in peripheral plasma of rats treated with LPS (Table 4.1) and 150 ng/ml to estimate a greater TNF activity potentially found in the liver sinusoids after treatment with LPS . As in experiments depicted in Figure 1, allyl alcohol caused a concentration- dependent increase in release of ALT (Figure 4.5). Cytotoxicity of allyl alcohol was unaffected by pretreatment with TNF. 4. D. 5. Allyl Alcohol-Induced Cytotoxicity in Isolated Hepatocytes from LPS-Treated Rats TNF reaches a maximal concentration in plasma 90 minutes after treatment with LPS in vivo. To examine whether exposure in vivo to TNF increased sensitivity of hepatocytes to allyl alcohol, hepatocytes were isolated from rats treated with LPS 2 hours earlier and were exposed to ally alcohol as described in Figure 4.1. Allyl alcohol caused a concentration-dependent increase in ALT release in hepatocytes isolated 134 from LPS-treated rats (Fig 4.6); however there was no significant difference in allyl alcohol-induced toxicity. 4. E. Discussion We have reported previously that very small amounts (10 ng/kg — 100 ug/kg) of LPS potentiate the hepatotoxicity of allyl alcohol (Sneed et al., 1997), and the studies presented here were performed to begin to explore the mechanism of potentiation. Hepatic injury resulting from exposure to relatively large doses of LPS is dependent upon several factors. These factors include, but are not limited to, the release of inflammatory mediators by activated macrophages and the influx of inflammatory cells into the liver. Blockade or inhibition of any one of these factors prevents the hepatic injury associated with large doses of LPS (Tracey et al., 1987; Jaescshke et al., 1991; Sato et al., 1993; Chang et al., 1993; Hewett et al.,1993; Imuro et al., 1994). Although LPS damages the liver through indirect means via inflammatory cells and soluble mediators, direct effects of LPS on hepatocytes have been reported. For example, LPS decreases bile formation (Utili et al., 1977) and increases fatty acid synthesis in the liver (Feingold et al., 1992). It is unlikely that the direct effects of LPS 135 Figure 4.6. Cytotoxicity of allyl alcohol toward hepatocytes isolated from rats treated in vivo with LPS. Rats were treated with LPS (4 mg/kg) 2 hours prior to hepatocyte isolation. The indicated concentrations of allyl alcohol were added to the culture medium, and cytotoxicity was measured 90 minutes later. For reference, allyl alcohol cytotoxicity in hepatocytes from na‘ive rats is presented. Data are expressed as mean 1 S.E.M. a, significantly different from control not exposed to allyl alcohol. N = 5. 136 ALT Released (% of Total) 100 - + Naive Rats —I— LPS-treated rats a 80 — 60 — 40 — 20 — 0 l I //// I l I I .o 10 50 100 200 Allyl Alcohol (0M) 137 contribute to the enhancement of hepatotoxicity of allyl alcohol because allyl alcohol-induced cytotoxicity in isolated hepatocytes was not altered by pretreatment of cells with LPS (Figure 4.1). Thus, these results support the hypothesis that factors other than LPS alone are responsible for the enhancement of hepatotoxicity seen in vivo. This hypothesis is consistent with results of studies in which inhibition of Kupffer cell function prevented enhancement of allyl alcohol hepatotoxicity by LPS (Sneed et al., 1997). One of the inflammatory mediators produced by LPS-activated Kupffer cells is the proinflammatory cytokine, TNF, which plays a critical role in liver injury from large doses of LPS (Beutler et al., 1985; Tracey et al., 1987; Hewett et al., 1993). Accordingly, we examined the role of TNF in LPS potentiation of allyl alcohol hepatotoxicity. The methylxanthine, PTX, inhibits the synthesis of TNF (Zabel et al., 1989; Han et al., 1990; Doherty et al., 1991; Noel et al., 1990; Dezube et al., 1993; Semmler et al., 1993; Zabel et al., 1993), and results presented here (Table 4.1) confirm this. Furthermore, administration of PTX prior to LPS treatment protected animals from the enhanced hepatotoxicity of allyl alcohol. These data suggested that TNF may be involved in the mechanism by which LPS augments the hepatotoxicity of allyl alcohol. PTX has multiple pharmacological effects, however; therefore a more specific approach, neutralization of TNF with an anti-TNF serum, was used to test further whether inhibition of TNF afforded protection. The anti-TNF 138 serum did not diminish LPS enhancement of allyl alcohol hepatotoxicity despite complete neutralization of circulating TNF activity. Similar protection by PTX and lack of protection by anti-serum-induced neutralization of TNF have been observed in a rat model of intestinal injury induced by nonsteroldal anti-inflammatory drugs (Reuter and Wallace, 1999) and in a model of bacteria-induced lung injury in rabbits (Miyazaki et al., 1999). One explanation for the disparate results observed with PTX and anti- TNF serum in these studies is that since PTX inhibits synthesis of TNF, it affords a more complete blockade of TNF action in the liver, whereas TNF is still produced by Kupffer cells after treatment with anti-TNF serum and can act locally before neutralization by the anti-serum. An alternative explanation is that TNF is not involved in the mechanism by which LPS enhances the hepatotoxicity of allyl alcohol. This explanation is supported by results obtained from in Vitro experiments presented here. Exposure of isolated hepatocytes to TNF did not alter the cytotoxic response to allyl alcohol (Figure 4.4), indicating that direct effects of TNF on hepatocytes are not sufficient to increase sensitivity to allyl alcohol. Others have also shown that TNF alone is not cytotoxic to isolated hepatocytes, but that cell damage requires the addition of other cytokines or induction of oxidative stress in the cells (Adamson and Billings, 1992; Sieg and Billings, 1997). 139 Experiments were performed using hepatocytes isolated from rats treated two hours earlier with LPS. Since TNF activity in plasma reaches a peak 90 minutes after administration of LPS, these hepatocytes were exposed to TNF in vivo. Despite this exposure to TNF and other mediators evoked by treatment with LPS, allyl alcohol was neither more potent nor more toxic in these cells. Maximal cytotoxicity was observed at the same concentration of allyl alcohol (100 uM) in both cell populations, and the concentration of allyl alcohol required to achieve half-maximal cytotoxicity was greater, not smaller, in hepatocytes from LPS-treated rats compared to those from na'l've rats. One possible explanation for these results is that the procedure used to isolate hepatocytes could select cells that are resistant to the effects of LPS and the mediators elicited by LPS. Alternatively, these results suggest that exposure in vivo to LPS-induced mediators for up to two hours is not sufficient to increase sensitivity of hepatocytes toward allyl alcohol. It is possible that interactions among hepatocytes and nonparenchymal cells in the intact liver may be needed at times beyond 2 hours, or augmentation of toxicity may only be observed under conditions of continuous exposure to LPS-induced soluble mediators. Another possibility is that the presence of a mediator that becomes involved at times beyond 2 hours after exposure to LPS may be required for enhanced hepatotoxicity. For example, thrombin is necessary for liver injury from larger doses of LPS, and protection is afforded when 140 the action of thrombin is blocked up to 2 hours after administration of LPS (Moulin et al., 1996). If TNF is not involved in the mechanism by which LPS enhances allyl alcohol hepatotoxicity, then the protective effect produced by PTX is due to one or more of the other pharmacological properties of this drug. The phosphodiesterase properties of PTX cause increases in intracellular levels of cyclic adenosine monophosphate (CAMP), and this increase in cAMP may inhibit macrophage function (Taffet et al., 1989). PTX also improves blood flow in tissues (Ward and Clissold, 1987). This effect of PTX has been demonstrated to be protective in one model of sepsis in which high mortality was associated with hemodynamic shock (Yang et al., 1999). In addition PTX can reduce the levels of toxic free radicals. PTX attenuates the expression of inducible nitric oxide synthase (Wu et al., 1999) and decreases the respiratory burst of neutrophils (Kowalski et al., 1999). A combination of the above factors may be involved in the ability of PTX to protect animals from LPS-enhanced allyl alcohol hepatotoxicity. In summary, inflammatory mediators may participate in the ability of LPS to enhance the hepatotoxicity of certain xenobiotics. In LPS-induced enhancement of allyl alcohol hepatotoxicity however, circulating TNF does not appear to play a major role, although the possibility of autocrine or paracrine hepatic effects of TNF cannot be completely ruled out. The drug 141 PTX protects animals from this enhancement and may do so by affecting the responses of Kupffer cells to the presence of LPS. 142 Chapter 5 SUMMARIES AND CONCLUSIONS 143 5. A. Characterization of the Model Very small, nontoxic doses of exogenous LPS can significantly potentiate the liver injury observed in rats exposed to allyl alcohol. Furthermore, this potentiation occurs at doses of allyl alcohol which are nontoxic or only mildly so. Histologically, the lesions produced are much more allyl alcohol-like than LPS-like in appearance, suggesting that the hepatotoxicity of allyl alcohol is potentiated, not that of LPS. Based upon the observations presented in Chapter 2, LPS accelerates the development of liver damage from allyl alcohol. Pretreatment of rats with LPS reduces the time for significant liver injury to occur. Normally allyl alcohol produces hepatic lesions at 18 to 24 hours. In the presence of LPS, lesions are apparent as early as 2 hours. LPS also makes the lesions more severe: animals cotreated with LPS and 30 mg/kg of allyl alcohol have hepatic lesions that resemble those of animals treated with 40 mg/kg of allyl alcohol. Allyl alcohol must be bioactivated to the aldehyde acrolein for liver damage to occur. This is also true of LPS-induced potentiation of allyl alcohol. However, LPS has no effects on the actual metabolism of allyl alcohol. LPS does not affect the hepatocellular concentration of reduced glutathione. 144 5. B. The Role of the Innate Immune System LPS is a potent stimulant for the innate immune system. Under normal conditions the stimulation provided by LPS is just adequate to elicit an immediate response from the innate immune system towards invading bacteria. However, sometimes, the components of the innate immune system will overrespond to the stimulus provided by LPS and, in turn, damage the body that they are defending. The two primary divisions of the innate immune system, macrophages and neutrophils, have prominent roles in high dose models of LPS hepatotoxicity. Disabling either cell population will protect a host from the toxic effects of endotoxemia. Recent research has also suggested that these cell populations participate in the toxic mechanisms of certain xenobiotics. Depletion of circulating neutrophils protects animals from the liver injury produced by ANIT, while inactivation of Kupffer cells protects rats from the hepatotoxicity of ethyl alcohol (Dahm et al., 1991; Adachi et aL,1994) Based on the data presented in Chapter 3, the current state of one major population of innate immune system cells, Kupffer cells, was crucial to the ability of allyl alcohol to produce significant hepatotoxicity. Stimulation of these cells with LPS prior to exposure to allyl alcohol resulted in a significant increase in liver injury compared to no prior 145 stimulation. Disabling Kupffer cells protected experimental animals from enhanced hepatotoxicity of allyl alcohol even if they were pretreated with LPS. These data fulfilled two of Koch’s postulates concerning the involvement of agents in a disease process. The role of neutrophils was not as clear. Early in the development of this model, large numbers of neutrophils were present in the necrotic areas of the hepatic sinsoids. Their contribution to the injury was unclear: were they active participants or were they there in response to tissue necrosis. Depletion of circulating neutrophils provided significant protection from the ability of LPS to potentiate the hepatotoxicity of allyl alcohol; however, unlike in the case of GdCl3, removal of neutrophils did not totally eliminate the increase in liver injury seen in LPS-potentiated allyl alcohol hepatotoxicity. A significant difference in plasma ALT activities still existed between neutrophil—depleted animals from the LPS/allyl alcohol group and animals from the saline/allyl alcohol group. These data suggested that neutrophils did not act independently of Kupffer cells but most likely acted in concert with them to potentiate the hepatotoxicity seen when animals were given both LPS and allyl alcohol (Figure 5.1). While the data from Chapter 3 demonstrate the important role of both Kupffer cells and neutrophils in LPS-enhanced hepatotoxicity of allyl alcohol, they do not indicate the relative contribution of each cell population to the experimental model. Inhibition of either leg of the innate 146 Figure 5.1. Comparison of the protective effects of depletion of functional Kupffer cells versus circulating neutrophils. Rats were depleted of either functional Kupffer cells or circulating neutrophils prior to treatment with LPS (100 pg/kg) or saline control. Two hours later, animals were treated with allyl alcohol (30 mg/kg) or saline control. LPS potentiated the hepatotoxicity of allyl alcohol in control animals in both studies while depletion of functional Kupffer cells or circulating neutrophils provided significant protection from liver injury. Exclusivedepletion of neutrophils, however, did not provide the same degree of protection as did depletion of Kupffer cells. The level of hepatotoxicity in neutrophil-depleted, cotreated animals was significantly higher than in the same group in the absence of LPS. a, significantly different from value in the absence of LPS. b, significantly different from value in the presence of functional Kupffer cells or circulating neutrophils. c, significantly different value in absence of LPS. 147 4000 - - Control/LPS/Allyl Alcohol CZ] KC or PMN removed/LPSlAllyl Alcohol - KC or PMN removed/Sal/Allyl Alcohol 3 a 3000 l 2000 l 1 000 KC PMN 148 immune system is equally protective even though inactivation of Kupffer cells provided more absolute protection than did depletion of neutrophils. The latter observation may indicate that neutrophils work in concert with Kupffer cells to produce the liver injury associated with sequential administration of LPS and allyl alcohol. Such an observation may serve as a starting point for other research questions such as how do Kupffer cells and neutrophils interact to produce the LPS-induced potentiation of allyl alcohol hepatotoxicity. 5. C. The Role of TNF LPS does not directly make hepatocytes more sensitive to the toxic effects of allyl alcohol. Simply exposing cultured hepatocytes to LPS has none of the effects seen in intact animals. As has already been established, participation of Kupffer cells and neutrophils is crucial to the ability of LPS to potentiate the hepatotoxicity of allyl alcohol. A logical supposition would be that these cells produce a factor or factors that make hepatocytes more sensitive to the toxic effects of allyl alcohol. As described in the Chapter 1, both Kupffer cells and neutrophils produce a number of biochemical mediators in response to exposure to LPS and some of these mediators play an important role in the liver injury 149 seen in endotoxemia. It is quite possible that a secretory product of these cells has a deleterious effect on adjacent hepatocytes. One of these mediators is the cytokine TNF. Inhibition of TNF activity in intact animals protects them from the organ damage and death associated with endotoxemia and bacteremia. In the current model of LPS-induced enhancement of allyl alcohol toxicity, pretreatment of rats with PTX, a known inhibitor of TNF synthesis, is protective. However, inhibition of TNF activity with an anti-TNF serum is not. These data may indicate that TNF is not involved and they may indicate that PTX provides protection via another mechanism such as affecting cyclic adenosine monophosphate levels in Kupffer cells or inhibiting the respiratory burst in neutrophils. Adding TNF directly to cultured hepatocytes does not affect their sensitivity to allyl alcohol. Similarly, coculturing them with LPS-stimulated peritoneal macrophages or RAW cells has no effect. Based on the results described above, the complex cellular interactions found in the intact animal are missing from cell culture situations. 5. D. A New Hypothesis In the work presented in this dissertation, we have established that very small doses of LPS can potentiate the hepatotoxicity of allyl alcohol. We 150 have also proven our original hypothesis that cells of the innate immune system are crucial elements in this model of liver injury. Our experimental data correlate quite well with a growing body of research linking exposure to LPS to the augmentation of the hepatotoxicity of other biologically-based toxicants or synthetic xenobiotic agents. Even though these compounds vary in their mechanism of hepatotoxicity, coexposure to LPS is the common factor. The data generated for this dissertation has answered the original hypothesis, but they have in turn generated a new hypothesis and a new set of questions to be solved. A new hypothesis might be the following: After exposure to LPS, Kupffer cells and neutrophils release biochemical mediators that make hepatocytes more sensitive to the toxic properties of allyl alcohol (Figure 5.2). The identity of the mediator needs to discovered as well as how it exerts its effect on hepatocytes to make them more sensitive. The data from this dissertation has also generated a number a new questions. One interesting aspect of this model is the inability to reproduce it in in Vitro culture systems. The simple presence of LPS does not make hepatocytes more sensitive to allyl alcohol. Questions arising from this finding might be: What are the cell types crucial to the mechanism? Is there something in the environment of the sinusoid that is missing from the in Vitro system? Finding the answer to these and related 151 Figure 5. 2. A hypothetical model. Kupffer cells and neutrophils release inflammatory mediators in the presence of LPS. When these mediators reach hepatocytes, they alter the physiology of the parenchymal cells such that the hepatocytes are more susceptible to injury by allyl alcohol. 152 LPS:LBP / \. @0 / Free radicals Cytokines Eicosanoids 153 Allyl Alcohol I ~ Hepatocyte Enhanced Cytotoxicity questions will provide valuable insight into the workings of the ability of LPS to potentiate allyl alcohol hepatotoxicity. The knowledge gained from solving the mechanism behind this model may prove useful in understanding other models in which the interaction of LPS and a second agent results in enhanced liver injury. This knowledge may also provide insight into some of the variable and idiosyncratic responses of individual humans to various toxicants. The current state of their innate immune system may be a very important factor in how that person will respond to a given toxicant. 154 BIBLIOGRAPHY 155 Bibliography Adachi, Y., Bradford, B. U., Gao, W., Bojes, H. K., Thurman, R. G. (1994). 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