Endotoxic Shock Part I: A Review of Causes* Elizabeth M. Hardie, DVM, PhD, and Kris Kruse-Elliott, DVM

Endotoxic shock is a complex phenomenon resulting from systemic release of inflammatory mediators. Endotoxin interacts with inflammatory cells, platelets, and vascular endothelium. Cytokines, such as tumor necrosis factor and interleukins, and lipid mediators (platelet activating factor, thromboxane, prostacylin, leukotrienes) are released. These primary mediators act synergistically to cause many of the harmful effects associated with endotoxemia. Multiple secondary mediators are released in response to the primary mediators, compounding the damage. The end result is the species-specific clinical syndrome recognized as endotoxemia. (Journal of Veterinary Internal Medicine 1990; 4:258-266)

Structure and Sources ENDOTOXIN (lipopolysaccharide-LPS) is present in the outer cell wall of all gram-negative bacteria and has lipid, polysaccharide, and protein portions.' The most consistent feature is the lipid portion (lipid A), which is responsible for the toxic properties of the molecule. In recent years, the structure of lipid A and its nontoxic precursor, lipid X. have been determined.2The polysaccharide component of LPS determines the antigenicity of the molecule. There are two polysaccharide components: the "core" portion, which remains constant across groups of bacteria, and the "0-antigen" portion, which is unique for a given bacterial strain. The protein portion is variable, depending on the bacteria and the chemical method used to obtain the LPS. Gram-negative bacteria and LPS are normally present in the intestine.' Three mechanisms prevent systemic release of LPS. Bile acids bind LPS within the i n t e ~ t i n e , ~ the mucosal cell barrier contains bacteria and LPS within the intestine,' and the liver clears small amounts of LPS released into the portal c i r ~ u l a t i o n . ' ,High ~.~ concentrations of circulating LPS occur with obstructive jaundice, compromised intestinal mucosal integrity, and invasive gram-negative infection^.'.^.^ Patients with liver disease are more susceptible to conditions that From the Departments of Companion Animal and Special Species Medicine (Hardie). and Anatomy, Physiology and Radiology (KruseElliott). North Carolina State University. College of Veterinary Medicine. Raleigh, North Carolina. Reprint requests: Elizabeth M. Hardie, North Carolina State University, College of Veterinary Medicine, 4700 Hillsborough Street. Raleigh, NC 27606. * Part 11: A Review of Treatment will appear in the November-December 1990 issue of the Journal of Veterinary Internal Medicine.

result in endotoxemia, due t o reduced ability to clear LPS.' Effects of Endotoxin The species-specific clinical syndromes associated with endotoxemia result from activation of inflammatory mediator systems, and to a lesser extent direct cellular toxic effects of LPS.9-'2The first species studied was the dog," in which endotoxemia results in tachypnea, vomiting, mucoid bloody diarrhea, weakness, fever, and chills. Early hemodynamic changes include portal hypertension, hepatosplanchnic pooling of blood, a fall in central venous pressure, a decrease in cardiac output, and a fall in systemic blood pressure. Although pulmonary vascular resistance increases, little rise in pulmonary pressure occurs because of the low right ventricular cardiac output associated with decreased venous return. These hemodynamic changes are transitory and are followed by a period during which values return to normal levels. During the late phase of canine endotoxic shock,13 cardiac output and systemic blood pressure decrease again and pooling of blood in the gastrointestinal (GI) tract persists. Death is usually due to myocardial failure. Histopathologic changes in dogs dying of endotoxic shock include mild pulmonary edema, severe congestion and necrosis of the liver, subendocardial hemorrhage, congestion of the kidney, hemorrhage in the adrenal glands, and severe hemorrhage and sloughing of the mucosa in the GI tract. Clinical signs associated with LPS administration in horses include tachypnea, dyspnea, restlessness, pawing, shivering, abdominal cramping, a n d diarrhea.14-"

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lends support to the importance of the macrophage in LPS-induced injury. Early phase endotoxin tolerance occurs within the first few days after injection of a sublethal dose of endotoxin. Tolerance is transient, is not 0-antigen specific, and is associated with increased production of hyporesponsive bone-marrow-derived, macrophage-progenitor cells. Decreased numbers of normally responding mature macrophages are present. On the other hand, compounds that stimulate the reticuloendothelial system (BCG, C'orvnehucteriirm purviitn. Mycobucterizm leprurmirriutn) increase sensitivity to endot~xin.'~,'~ The exact role of neutrophils, platelets, and vascular endothelial cells in the amplification of endotoxic injury is still under investigation. Whether or not direct interaction occurs between LPS and these cells varies. In In addition to causing severe hemodynamic and hissome in vitro assay systems direct interactions have been topathological changes, endotoxin administration red e m o n ~ t r a t e d . In ~ ~other , ~ ~ systems, serum or whole sults in leukopenia (followed by leukocytosis, if the animal survives 24 hours), thrombocytopenia, lactic aciblood must be added to obtain an effect, suggesting that serum factors or mediators released from other cells are dosis, hypoxemia, transient hyperglycemia followed by needed.36-37Complex interactions are known to occur hypoglycemia, and h e m o c o n ~ e n t r a t i o n . * ~Dissemi-~~ nated intravascular coagulation often O C C U T S . ~ ~ , ~ ~ between LPS and low-density lipoprotein^.^^ Depending on the species, these interactions may block or enhance cytotoxicity. Cellular Interactions Once bound to the cell, LPS may have a direct effect. such as release of a soluble m e d i a t ~ r . 'In Neutrophils, platelets, vascular endothelium, and mac~ neutrophil studies, it is more common action is to act as a "primrophages are considered to be the major cells involved in ing" agent, making the cell react much more vigorously endotoxic Each of these cell types is known to be a source of important mediators or toxic substances. to other activating agents, so that larger quantities of The interaction that initiates endotoxic injury probably oxygen radicals and lysosomal enzymes are relea~ed.'~ occurs between LPS and macrophages, resulting in the Cellular-LPS interactions thus cause both mediators and release of cytokine and lipid mediator^.^"'^ Amplificacytotoxic compounds to be released in large quantities. The contribution of the various substances to organ intion of injury is associated with sequestration of neutrophils and platelets in target organs and with vascular jury must then be determined. endothelium becoming "sticky and leaky."31 Evidence that the macrophage is the critical cell in the Mediators host response to LPS comes largely from the C3H/HeJ and C3H/HeN mouse strains.33These mice are genetiThe criteria for deciding that a particular substance is a cally identical except for one autosomal gene. The mediator of endotoxic shock are 1 ) the substance or its C3H/HeJ mouse is resistant to endotoxin, while the metabolite is present in high concentrations in plasma C3H/HeN mouse is sensitive. The resistant mouse fails or target organs during endotoxemia, 2) blocking the to produce certain macrophage cytokines in response to synthesis or receptor activity of the substance amelioendotoxemia. Compared with sensitive mice, resistant rates endotoxin damage and improves survival, and 3) mice also have decreased prostaglandin release during administration of the substance results in effects known endotoxemia. Sensitivity to endotoxin can be induced in to occur during endotoxemia. Unfortunately, much C3H/HeJ mice with sublethal irradiation followed by confusion exists due to the large number of possible transfer of bone marrow or spleen cells from C3H/HeN mediators, the complex interactions between mediators. mice. Macrophages from C3H/HeN mice produce a and the lack of specific antagonists. factor on exposure to endotoxin in vitro that is lethal to C3H/HeJ mice. The C3H/HeJ mice normally cannot be sensitized to endotoxin by galactosamine, a compound Cytokines that markedly increases sensitivity to endotoxin in normal animals. If macrophages from C3H/HeN mice are Tumor necrosis factor-cachexin (TNF) is a polypeptide transferred to the C3H/HeJ mice, they can be sensitized hormone secreted by endotoxin-activated macrophage^.^'.^* The plasma half life of T NF is short (about 6 by galactosamine. Endotoxin t ~ l e r a n c e ~is' . another ~~ phenomenon that minutes) and it rapidly binds to specific receptors in

Transient pulmonary hypertension occurs. Cardiac output and systemic blood pressure are markedly decreased. Mesenteric vasodilation is extensive. The lungs of ruminants,17 pigs," and cats'' are particularly sensitive to the effects of LPS, developing transient severe pulmonary hypertension and bronchoconstriction. Several hours later a secondary phase of respiratory failure develops, characterized by severe pulmonary edema and increasing pulmonary hypertension. Pulmonary lesions at death in horses, ruminants, pigs, and cats are usually more severe than in the dog, but GI lesions are rarely as severe.20." In some species (rat, humans), acute renal tubular necrosis is commonly found.22All species studied have evidence of severe vascular endothelial dam-

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target tissues (mainly liver, skin, kidneys, lungs, and GI followed by hypoglycemia occur. Lesions seen at nectract)." Known action^^'.".^' of T N F include 1 ) activaropsy include pulmonary inflammation and thrombotion of neutrophil adherence, degranulation, and phagosis, acute renal tubular necrosis, and acute hemorrhagic cytosis: 2 ) alteration of vascular endothelium resulting adrenal cortical necrosis. Third, rats made tolerant to in increased procoagulant activity at the surface and LPS (by previous sublethal injection) do not produce decreased expression of thrombomodulin: 3 ) decreased TNF. Macrophages from C3H/HeJ mice do not prosynthesis of lipoprotein lipase in adipocytes and induce T N F (due to defective transcription and translacreased synthesis in other tissues; 4) increased production steps in T N F synthesis), while macrophages from tion of insulin, glucagon, and catecholamines; 5 ) inC3H/HeN mice do produce TNF. creased gluconeogenesis; 6) direct production of fever C3H/HeN mice can be protected from endotoxin by due to production of prostaglandins in the hypothalathe prior administration of dexamethasone, which inmus: 7) phospholipase A2 activation, resulting in the hibits T N F gene tran~cription.~' Once transcription and production of lipid mediators (Fig. 1); and 8) synthesis translation have begun, dexamethasone does not preand/or release of class I major histocompatibility antivent T N F production and is no longer protective. Pasgen, granulocyte-macrophage colony stimulating factor, sive immunization with antiserum (mouse model) or and interleukin-I. monoclonal antibodies (baboon model) against T N F Several lines of evidence support the contention that also prevents death due to endotoxic s h o ~ k . ~Interest"~' T N F is one of the key mediators of endotoxic s h o ~ k . ~ ' . ~ 'ingly, in people with septic meningitis, serum T N F conFirst. increased concentrations of T N F have been meacentration (measured in the admission serum sample) sured in the plasma of endotoxic animals and humans. correlates directly with m ~ r t a l i t y . ~ ' Concentrations begin to increase 15 minutes after the Increased plasma concentrations of another macroinduction of endotoxemia, reach a peak at 2 hours, and phage cytokine, interleukin-I (IL-I), occur during endoreturn to baseline values by 4 hours. Second, infusion of toxemia.43 Other sources of IL-I include gingival and T N F in doses similar to those measured during endocorneal epithelial cells, renal mesangial cells, synovial toxemia produces a lethal shock syndrome similar to lining cells, brain astrocytes, and vascular endothelium. that seen after endotoxin infusion. Hypotension, hemoRather than being produced in direct response to LPS, concentration, metabolic acidosis. and hyperglycemia IL-I is produced secondary to the production of leukotrienes or TNF.9.44-46IL-I production can be prevented by prior administration of corticosteroids or leukotriene L p s ----., synthesis inhibitors, but not by cyclooxygenase inhibii t o r ~ In . ~target ~ tissues, IL-l acts as a Ca++ ionophore, interaction with macrophages ! t causing phospholipase activation and production of I TNF I lipid mediators, the exact compounds being dependent Phospholipids j j t on the tissue.43Known actions43of IL-I include 1 ) fever P A F A Phospholipase A2 L" Ca*+ due to the production of PGE in the hypothalamus, 2 ) Arachidonic Acid increases in the number and immaturity of circulating neutrophils, 3) chemotaxis of neutrophils and T cells, 4) decreased serum iron concentrations due to lactoferrin 5 HPETE release from neutrophils, 5 ) increased synthesis of acute phase proteins, 6) increased catabolism of muscle protein, 7) lymphocyte activation, 8) activation of slowwave sleep, and 9) expression of an intracellular adheTXA2 PG!, PGE,.F,.D, sion molecule in vascular endothelium, which results in LTBI LTCd-LTD4 increased procoagulant activity and increased adhesive'4 !+.I ness of white cells. i Although bolus administration of IL-I alone to rabbits activate phospholipase A, in other cells does not reproduce all the pathophysiologic effects of endotoxemia, it does induce fever, neutropenia followed Dotted lines indicate activation of enzyme or increased production of compound. by neutrophilia, and sequestration of granulocytes in the Abbreviations: LPS=endotoxin TNF=tumor necrosis factor pulmonary microvas~ulature.~~ When given during a PAF=platelet activating factor PG=prostaglandin longer time period, it produces a respiratory distress TX=thromboxane LT4eukotriene syndrome.47C3H/HeJ mice do not produce IL-I in reIL=interleukin sponse to endotoxin, and thus do not, for instance, show (Oj,*=oxygen radical HETE=hydroxyeicosatetraenoic acid a decrease in serum iron concentration^.^^ If C3H/HeJ mice are given IL-I alone, decreased iron concentrations FIG.1. Cytokine and lipid mediators. '\\

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can be demonstrated. It will require detailed investigations to determine which other effects of endotoxin are directly attributable to IL-I. For example, it is known that fever in endotoxemia is due to both to a direct effect of T N F on the hypothalamus. and to induction of IL-I release by TNF.45It is not known whether the increased adhesiveness of vascular endothelium is due to independent direct actions of T N F and IL-I, or whether T N F is acting through IL-I release.

Lipid M d i a t o r s Platelet activating factor ( PAF)4X.4y is a phosphorylcholine compound that is synthesized in activated cells (mainly leukocytes, platelets, and endothelial cells) in the presence of calcium (Fig. 2). The precursor compound, alkyl-acyl GPC, is present in the cell membrane and is also an important "storage" site of arachidonic acid. Phospholipase A2 converts alkyl-acyl GPC into lyso-PAF (which is inactive) by release of a fatty acid (i.e., arachidonic acid). Lyso-PAF is converted to PAFacether, the active form, by acetyl transferase. PAFacether is released into the plasma. Both intra- and extracellularly, PAF-acether is rapidly inactivated by conversion back to lyso-PAF by acetyl hydrolase. Known actions of PAF-acether include I ) platelet activation and aggregation; 2) neutrophil activation, aggregation, and chemotaxis; 3 ) increased activity of plasma proteases; 4)

Lps

[ransferase

(

P A r a c h i d o n i c Acid

Lyso - PAF

acether Intracellular

ace01 hydrolase

PAF + albumin carrier

/

Lyso PAF (inactive)

Dotted line indicates activation of enzyme Abbreviations.

LPS=endotoxin PAF=Platelet Activaung Factor CoA=Coenzyme A GPC= 1-n-alkyl-Z-arachidonuylm-glycero-3-phosphocholine

FIG.2. Platelet activating factor.

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vasodilation, with resulting severe systemic hypotension; 5 ) vasoconstriction in selected vascular beds. such as the heart and lungs: 6) increased vascular permeability, with resulting hemoconcentration; and 7) severe GI ulceration. PAF has been demonstrated in the plasma during endotoxemia, and is produced in vifro by LPS-activated peritoneal and spleen ~ e l l s . ~ ' PAF-induced ~~') shock in the dog and rat is similar to endotoxic shock. Administration of PAF-receptor antagonists before endotoxic shock results in prevention of immediate hypotension. hyperglycemia, and lactic acidosis. In rats, PAF antagonists prevent LPS-induced GI ulceration, while co-administration of PAF and LPS in doses that are nontoxic results in severe ulceration. Once arachidonic acid has been released from membrane phospholipids by the action of phospholipase A?. it is converted into leukotrienes (5-lipoxygenase pathway) or prostaglandins (cyclooxygenase pathway) (Fig. l)." Both oxygenases require the presence of activating hydroperoxides, which are supplied by activated phagocytes or generated by later steps in the prostaglandin and leukotriene pathways." The constant production of oxygen radicals has been shown to cause irreversible destruction of cyclooxygenase after processing of several thousand molecules of arachidonic acid. Whether the same is true of the lipoxygenases is not known. Increased plasma concentrations of prostaglandins have been demonstrated in virtually every model of endotoxemia in which they have been measured." Increases in thromboxane, a potent vasoconstrictor and initiator of platelet aggregation, occur early in endotoxemia and correlate well with the initial development of pulmonary hypertensi~n.'~.''Increases in prostacyclin (PGI'), a vasodilatory compound that prevents platelet aggregation, occur more gradually and are correlated with the development of systemic hypotension.5' Increases in prostaglandin F'n, a vasoconstrictive prostaglandin, and prostaglandin E, a prostaglandin whose action depends on the specific vascular bed, correlate less well with specific pathophysiologic event^.",'^ The acute organ-specific vasoconstrictive events associated with endotoxin administration can be blocked with cyclooxygenase blockers".'6.'x or thromboxane synthesis inhibit01-s.~~ Cyclooxygenase inhibitors delay o r prevent the development of systemic hypotension. 15.16.56.57 Other more variable effects of cyclooxygenase blockers include increased cardiac output, maintenance of blood glucose concentrations, and prevention of metabolic a c i d o s i ~ . ' ~ ' l ~ In . I ~all . ~ ' models cyclooxygenase blockers delay death, and in a few studies improved long-term survival has been documented.57,5x There are four important leukotriene corn pound^^^: leukotriene (LT) B4, and the sulfidopeptideleukotrienes

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LTC4, LTD4, and LTE4 (collectively known as slow reacting substance of anaphylasis [SRS-A]). Cells known to produce leukotrienes include leukocytes, macrophages, mast cells, connective tissue cells, smooth muscle cells (especially vascular smooth muscle), and lung parenchymal ~ e l l s . ~ ’LTB4 - ~ ~ causes adhesion of neutrophils in the postcapillary venules, as well as neutrophil chemotaxis, enzyme release, and oxygen radical generation. SRS-A causes intense pulmonary and coronary vasoconstriction, bronchoconstriction, increased permeability of the postcapillary venules, and contraction of the smooth muscle of stomach and ileum. The known actions of leukotrienes make them good candidates for mediators of endotoxic The best evidence that they are important is that a number of lipoxygenase/cyclooxygenase blockers, specific 5-lipoxygenase blockers, or specific leukotriene antagonists have protective actions in endotoxemia models. These actions are different from those seen with cyclooxygenase blockers. Unfortunately, many of these blocking agents are less specific than was originally thought, which weakens much of this evidence. For example, the ability of corticosteroids (which induce production of a phospholipase A2 inhibitor) to prevent actions not blocked by cyclooxygenase inhibitors was originally used as evidence for the importance of leukotrienes. It is now recognized that corticosteroids block PAF, TNF, and other important macrophage products. In vitro LPS causes the release of LTC4 from mouse peritoneal macro phage^^^ and horse n e u t r ~ p h i l s ,but ~~ high doses may be required. Leukotrienes are metabolized by the liver and excreted in bile.66 Transient increases in the metabolite n-acetyl-LTE4 have been measured in the bile of endotoxic rats.67 The HETE (hydroxyeicosatetraenoic acid) metabolites (Fig. I ) are increased in the lung lymph of endotoxic sheep.64Leukotrienes are difficult to isolate from tissues and plasma, but sensitive assays failed to demonstrate increases in sulfidopeptide leukotrienes from plasma and bronchoalveolar lavage fluid in a well-characterized porcine endotoxemia On the other hand, large increases in LTB4, 5-HETE, 12-HETE, and 15-HETE have been documented in the same model indicating activation of lipoxygenase pathways.6’ More work on direct measurement of leukotrienes in endotoxemia needs to be completed before the exact compounds involved and their role is understood. Other Mdiators Endotoxic shock causes a massive increase in plasma beta-endorphin concentrations, well beyond the response seen for a corresponding level of hypotension in hemorrhagic shock.70Whether these compounds are important mediators of hypotension and bradycardia in

Journal of Veterinary Internal Medicine

endotoxic shock is a highly debated issue.7’Major points of evidence that they are important are the facts that naloxone and thyrotropin-releasing hormone (TRH) improve cardiovascular status in e n d o t ~ x e m i a The .~~ problem is that, at the doses used to treat shock, naloxone and TRH have a number of actions, only one of which is antagonism of opiate receptor^.^' Current research is focused on determining the mechanism of protection by naloxone and TRH. The role of histamine in endotoxemia is also debated.74 Increased plasma concentrations of histamine have been measured in endotoxic animals and systemic histamine administration causes many of the same early cardiovascular changes as endotoxin. Antihistamines and histamine depletion prevent the early hypotensive response to LPS. H1 and H2 blockers, given separately or together, improve survival in endotoxic rats and mice. The problem is that the efficacy of these compounds as histamine receptor blockers bears no relation t o their efficacy in the treatment of endotoxemia. Whether these compounds have other effects that are responsible for their ability to improve survival is not known. Serotonin (S H T ), released from platelets, acts as a vasoconstrictive, bronchoconstrictive, and platelet aggregating agent. It has mainly been considered to have a role in endotoxin-induced pulmonary fai1u1-e.~~ In sheep, increased concentrations of 5-HT are found in lung lymph 3 to 5 hours after endotoxin administration, at a time corresponding with the development of late hypoxemia and pulmonary hypertension. Pretreatment of endotoxic pigs or sheep with 5-HT antagonists attenuates the development of the late (2-5 hour) phase, but not the early (