- Email: [email protected]
The molecular pathogenesis of endotoxic shock and organ failure
Reviews
MOLECULAR MEDICINE TODAY, MARCH 1999
The molecular pathogenesis of endotoxic shock and organ failure Risuke Karima, Shigeru Matsumoto, Hidemitsu Higashi and Kouji Matsushima
Sepsis is still associated with a high mortality rate. Septic shock and sequential multiple organ failure have a strong correlation with poor outcome. Lipopolysaccharide (LPS) plays a pivotal role in the initiation of host responses to Gram-negative infection. A number of mediators, such as cytokines, nitric oxide and eicosanoids, are responsible for most of the manifestations caused by LPS, and circulatory failure, leukocyte-induced tissue injury and coagulation disorder appear to be critical determinants in the development of sequential organ failure. Although several anti-LPS or anti-cytokine clinical trials have been attempted, none of them has so far been successful. DESPITE recent progress in antibiotics and critical care therapy, sepsis is still associated with a high mortality rate. Septic shock and sequential multiple organ failure/dysfunction syndrome (MOF/MODS) correlate with poor outcome. Lipopolysaccharide (LPS) or endotoxin plays a pivotal role in the initiation of a variety of host responses caused by Gram-negative bacterial infection. Since the 1980s, novel insights into the molecular pathogenesis of LPS-induced shock (endotoxic shock) and organ dysfunction have been gained. The molecular cloning of proinflammatory cytokines and adhesion molecules were important steps
Risuke Karima MD Research Associate Shigeru Matsumoto MS Research Associate Hidemitsu Higashi MS Research Associate Kouji Matsushima* MD, PhD Professor of Molecular Preventive Medicine Dept of Molecular Preventive Medicine, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyou-ku, Tokyo, 113-0033, Japan. Tel: +83 3 5800 6853 Fax: +83 3 5800 6853 *e-mail: [email protected]
towards understanding the molecular mechanism of sepsis. In addition, nitric oxide (NO) has been identified as a key mediator of LPSmediated hypotension. These findings have led to clinical trials of various single agents that antagonize either LPS or sepsis-associated mediators. Unfortunately, none of the clinical trials of these agents has so far been successful. This review will summarize our current understanding of the molecular pathogenesis of endotoxic shock and endotoxin-induced MOF/MODS, as well as the clinical features of endotoxemia, with the aim of exploring the potential for an effective therapy against sepsis and endotoxemia.
Clinical features of sepsis In 1991, a new concept – systemic inflammatory response syndrome (SIRS) – was postulated to define the state of patients who exhibit a systemic response to inflammatory episodes1. SIRS is diagnosed by a combination of available clinical signs and symptoms. Nowadays, sepsis is generally defined as SIRS induced by infection. No worldwide statistics on the occurrence of sepsis are available. In the USA alone, however, it is estimated that there are 300 000–500 000 septic episodes each year, with mortality rates ranging from 20% to 40% (Ref. 2). Refractory hypotension (septic shock) is the main cause of death within a few days of the onset of sepsis. Later, MOF/MODS becomes the primary clinical problem and main cause of mortality. Once a patient develops septic shock or sequential MOF/MODS, the mortality rate increases to 60–70% (Ref. 3). Gram-negative bacteria are responsible for 45–60% of sepsis caused by bacterial infection when mixed-organism infections are included3,4. Septic shock has been reported to be a complication in 20–50% of patients with sepsis3,4. The initial cardiovascular response to sepsis is generally characterized by high cardiac output and low systemic vascular resistance. Even though cardiac output might be maintained above normal levels, impaired myocardial contractility, inadequate
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distribution of blood flow and disturbance of tissue oxygen use are common and contribute to the development of MOF/MODS is defined as a syndrome that consists of the sequential failure of two or more organ systems. The mortality of patients who have three or more types of organ failure is particularly high4. The lungs, kidneys, liver, cardiovascular system, central nervous system and coagulation system are commonly involved in septic MOF/MODS. If specific conditions are met, the lung dysfunction is termed acute lung injury or acute respiratory distress syndrome (ARDS), and the coagulatory disorder is diagnosed as disseminated intravascular coagulation (DIC). The clinical features of the dysfunction of each organ in sepsis are summarized in Table 1.
MOLECULAR MEDICINE TODAY, MARCH 1999
macrophages/monocytes and neutrophils. CD14 is a glycerophosphatidylinositol (GPI)-anchored glycoprotein that lacks a cytoplasmic portion. LPS-binding protein (LBP) might facilitate the interaction of LPS with CD14 (Ref. 5) or effect the clearance of LPS from the circulation by transferring LPS from CD14 (Fig. 1). Recent studies suggest the former because galactosamine-sensitized LBPdeficient mice were resistant to the acute lethality of LPS, but LBP deficiency did not effect the clearance of LPS (Ref. 6). In certain cell types, such as endothelial cells, that do not express CD14, soluble CD14 (sCD14) substitutes as the signaling bridge for surface recognition of the LPS–LBP complex. However, at present the molecule(s) that recognizes the complex of LPS–LBP–sCD14 has not been identified in CD142 cells. The LPS-induced signaling pathway CD14 does not have a cytoplasmic portion, but substantial eviLPS is a constituent of the cell wall of Gram-negative bacteria. The dence supports a role for unidentified signal transducing molecules major biological activities of LPS are mainly attributed to a lipid that interact with CD14 at the cell membrane5. Alternatively, binding component, termed lipid A. LPS interacts with CD14, a receptor on of LPS to CD14 might result in the rapid translocation of LPS from the cell membrane to intracellular locations, leading to LPS-induced cell activation7. Furthermore, although cellular activation by Table 1. Clinical features of organ dysfunction in sepsisa low concentrations of LPS (<1 mg ml21) requires CD14, a high concentration of LPS Organ/system Symptom can stimulate cells in a CD14-independent manner. This suggests the existence of a second molecule that recognizes LPS. Central nervous system (CNS) Reduced alertness, confusion, stupor or coma; drifting in and out of Candidates include CD11/CD18 (Ref. 8) and consciousness Toll-like receptor 2 (TLR-2)9, but the precise Cardiovascular system Decrease in bilateral ventricular contractility, although cardiac output mechanism of the CD14-independent signalcan be increased to supranormal levels ing pathway remains unclear. The molecular mechanism that transduces Decrease in systemic vascular resistance LPS receptor-mediated signals to the nucleus Lung Hypoxemia that frequently necessitates support with mechanical is not fully clarified (Fig. 2). Tyrosine kiventilation with PEEP nases, especially Hck, Fgr and Lyn, have been suggested to play an important role in Widespread, patchy or homogeneous bilateral infiltrates in chest X-ray, LPS–CD14-mediated signaling10,11. However, although radiographic abnormalities are often absent within a few days a recent study demonstrated that macroof onset phages from hck2/2fgr2/2lyn2/2 triple knockDecrease in pulmonary compliance and increased airway resistance out mice produce normal levels of proinflammatory cytokines and NO in response Liver Progressive increase in plasma bilirubin levels (often .5 mg dl21) with to LPS stimulation, indicating that these Srcmoderate increase in plasma transaminase levels (usually within family kinases are not essential for LPS21 200 u ml ) initiated signal transduction12. Involvement Reduced amino acid extraction with reduced protein synthesis of other protein kinases in the transduction of LPS-induced responses has been also sugIncreased hepatic VLDL release with reduced peripheral triglyceride gested. Such kinases include protein kinase clearance C and mitogen-activated protein kinases Impaired glucose release that often results in hypoglycemia (MAPK), such as p42 (ERK2), p44 (ERK1) and p38 (Ref. 13). LPS-induced cytokine Kidney Increase in plasma levels of BUN and creatinine with reduced GFR production requires activation of several Oliguria or anuria, although sometimes urinary volume is maintained transcription factors. Nuclear factor kB within normal or polyuremic limits (NFkB) and nuclear factor-interleukin 6 (NF-IL-6) are involved in the gene transcrip23 Coagulation system Thrombocytopenia (often ,50 000 cells mm ) tion of numerous proinflammatory cytoDecrease in plasma fibrinogen and antithrombin III levels kines, tissue factor, adhesion molecules and inducible NO synthase13. LPS activates nuIncrease in FDP and prolongation of clotting time clear translocation of NFkB by modifying its aAbbreviations: BUN, blood urea nitrogen; dl, deciliter; FDP, fibrinogen degradation products; GFR, glomerular filtration inhibitory subunit, IkB. NFkB activation rerate; PEEP, positive end-expiratory pressure; VLDL, very low density lipoprotein. quires sequential phosphorylation, ubiquitination and degradation of IkB. Recently, IkB 124
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kinases that phosphorylate N-terminal serine residues have been identified14, but activation of the IkB kinases by the stimulation of monocytes or neutrophils with LPS has not been reported.
Liver
a
LPS Gram-negative bacteria
The pathogenesis of endotoxic shock and MOF/MODS A variety of pathophysiological responses in various tissues and organ systems occur during endotoxemia. In particular, circulatory failure, leukocyte-induced tissue injury and activation of coagulation systems appear to be critical determinants in the development of sequential organ failure (Fig. 3). A number of mediators derived from host cells are responsible for most of the manifestations of endotoxemia. The proinflammatory cytokines, including tumor necrosis factor a (TNF-a), interleukin (IL) 1b, IL-6, IL-8 and IL-12, and interferon g (IFN-g), play a critical role in the inflammatory responses15. NO is now known to induce a variety of responses in addition to hypotension. Some chemokines have recently been found to be involved in the development of LPSinduced tissue injury. Lipid mediators, such as platelet activating factor (PAF), prostaglandins, thromboxanes and leukotrienes, also exert a variety of effects in endotoxemia, and the role of the inducible cyclooxygenase 2 (COX-2), which converts arachidonic acid to prostaglandins (PGs) PGE, PGF and PGI2 and thromboxanes, has been extensively investigated. Furthermore, anti-inflammatory mediators, such as IL-10 and IL-1 receptor antagonist (IL-1Ra), also contribute to the modulation of inflammatory responses in endotoxemia. Space limitations preclude a complete review of these mediators. This review will focus on recent discoveries that relate to the role of NO, leukocyte-induced tissue injury, the mechanisms of the development of DIC and immunomodulation by anti-inflammatory mediators in endotoxemia.
LPS LBP
HDL HDL CD14
b
LPS LBP
CD14 Neutrophil
Macrophage
c
LPS LBP
Soluble CD14
Vascular endothelial cell
Role of NO The role of NO in inducing hypotension is now well established. NO is generated by three different nitric oxide synthases (NOSs), which are divided into two major categories: constitutive NOS (cNOS) and inducible NOS (iNOS). cNOS is regulated by intracellular Ca21 and calmodulin. Two isoforms of cNOS are known, one originally isolated in neurons (nNOS) and one in endothelial cells (eNOS). Under physiological conditions, NO is released constitutively by cNOSs, contributing to the regulation of basal vascular tone, inhibition of platelet aggregation and inhibition of leukocyte adhesion to endothelium. By contrast, iNOS, which is Ca21-independent and not expressed constitutively, is induced by inflammatory stimuli, such as LPS, IL-1b, TNF-a, IFN-g and PAF, in a variety of cells, including macrophages, endothelium, smooth muscle cells, hepatocytes and cardiomyocytes. iNOS is responsible for the production of large amounts of NO during endotoxemia16. The action of NO is mediated by the activation of soluble guanylyl cyclase and the consequent increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). This increase in cGMP leads to the relaxation of vascular smooth muscle and reduced responsiveness to vasoconstrictors, thereby contributing to the development of refractory hypotension during endotoxemia. NO also has direct effects on various enzymes that contain an iron–sulphur moiety in their catalytic center. Such enzymes include ribonucleotide reductase (necessary for DNA synthesis) and several mitochondrial enzymes that are essential for survival of the cell. Furthermore, the reaction of NO with superoxide anion (O22) results
TNF-α IL-1β IL-6 IL-8 IFN-γ NO PAF Eicosanoids
Endotoxic shock
MOF/MODS
Figure 1. CD14-dependent cellular activation by lipopolysaccharide (LPS). In plasma (a) LPS binds high-density lipoprotein (HDL). (b) LPS-binding protein (LBP) transfers LPS from HDL to CD14 and facilitates the interactions of LPS with CD14 expressed on the surface of monocytes/macrophages or neutrophils. Endothelial cells and some other types of cells do not express CD14. (c) LPS stimulates these cells by binding soluble CD14. IFN-g, interferon g; IL-1, interleukin 1; MOF/MODS, multiple organ failure/multiple organ dysfunction syndrome; NO, nitric oxide; PAF, platelet-activating factor; TNF-a, tumor necrosis factor a.
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LPS
?
?
?
b
?
LPS
LBP
(High concentration)
CD14
a
GPI anchor
TLR2
?
Unidentified receptor?
? Another LPS receptor?
LPS
Plasma membrane
LPS internalization? ?
? ? ?
?
c
?
Src Lyn, Hck, Fgr
?
?
Phosphatidylinositol3-kinase
Tyrosine kinases
Intracellular Ca2+ p44 p42 p38 MAP kinases
Protein kinase A
Protein kinase C
? ?
? ROS, NO
Gene
IκB kinases α
d
IκB kinases β IκB α/β
IκB α/β
NFκB
NFκB
NF-IL-6 P
e Translocation
NFκB
some recent studies have shown that NO inhibits COX activity19. These pathophysiological roles of NO suggest that inhibiting NO production might improve the outcome of endotoxemia. However, a competitive inhibitor of NOS, NG-monomethyl-L-arginine (L-NMMA), inhibited the development of hypotension after LPS challenge but did not necessarily improve the mortality of experimental endotoxemia20. Furthermore, a Phase III multicenter clinical trial using L-NMMA was terminated because of unacceptable cardiovascular side effects. These results might be attributable to the inhibition of the beneficial roles of NO, such as maintenance of tissue perfusion and inhibition of platelet aggregation, causing impairment of splanchnic blood flow. In this respect, specific inhibition of iNOS, preserving the beneficial activities of NO produced by cNOS, might have more therapeutic potential. In support of this, iNOS-deficient mice were found to be resistant to acute lethality after LPS challenge21. In addition, the administration of selective inhibitors of iNOS showed protective effects against vascular dysfunction, organ injury and mortality in experimental endotoxemia22. However, another study reported no survival advantage of LPS-challenged iNOS-deficient mice over wild-type mice23, and deleterious effects by selective iNOS inhibition in endotoxemia have been also documented24.
Leukocyte-induced tissue injury
Since the identification of IL-8 in 1987 (Ref. 25), it has become clear that chemokines play a fundamental role in the accumulation, adhesion, migration and activation of leukocytes in the inflammatory process. IL-8 is a Figure 2. Lipopolysaccharide (LPS) signaling pathways. The precise mechanism that transduces the LPS C–x–C chemokine that has specific chemosignal to the nucleus is not well defined. (a) CD14 is anchored to the cell membrane as a glycerophosattractive effects on neutrophils and basophatidylinositol (GPI)-conjugated protein and lacks a cytoplasmic portion, suggesting the existence of phils. Anti-IL-8 antibody dramatically supunidentified transducing molecules that associate directly with CD14. At low concentrations of LPS, LPS pressed accumulation of neutrophils in the stimulates cells by binding to CD14, (b) although LPS can stimulate cells in a CD14-independent manner alveolar space and prevented acute lethality at high concentrations of LPS. (c) The important roles of tyrosine kinases and mitogen-activated protein (MAP) kinases in the LPS-induced signaling pathway have been demonstrated. Gene transcription of after systemic administration of LPS in LPS-induced inflammatory mediators requires activation of nuclear factor kB (NFkB). (d) Phosphorylation rabbits26. By contrast, C–C chemokines, of the inhibitory subunit of NFkB (IkB) by IkB kinases leads to the ubiquitination and degradation of IkB, such as monocyte chemoattractant protein resulting in (e) the translocation of NFkB to the nucleus. IL-6, interleukin 6; LBP, lipopolysaccharide-binding 1/monocyte chemotactic and activating facprotein; NO, nitric oxide; ROS, reactive oxygen species; TLR, Toll-like receptor. tor (MCP-1/MCAF) and macrophage inflammatory chemokine 1a (MIP-1a), mainly contribute to monocyte/macrophage and Tin the formation of cytotoxic oxygen radicals, such as peroxynitrite cell chemotaxis. The involvement of MIP-1a in endotoxin-induced (ONOO2), which decomposes to an intermediate with the biological tissue injury has been suggested27, whereas the neutralization of activity of the hydroxyl radical (•OH)17. Peroxynitrite is a strong and MCP-1/MCAF resulted in an increase in LPS-induced mortality28. stable oxidant that is thought to be responsible for many cytotoxic More recently, we found that administration of the monoclonal antiactions of NO via a number of independent mechanisms. In addition, body against thymus and activation-regulated cytokine (TARC) atNO has been reported to upregulate the catalytic activity of COX, tenuated LPS-induced hepatic injury and improved prognosis after which increases the production of cytotoxic eicosanoids18, although LPS challenge in Propionibacterium acnes-primed mice29. IκB α/β
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Transcriptional factors (NFκB, NF-IL-6 etc.)
LPS
Inflammatory mediators Responsiveness to vasoconstrictors Myocardial function Vascular relaxation
Leukocyte adhesion
b
Vascular injury
Fibrinolysis Anti-coagulation system Coagulation system
Leukocyte migration
Release of proteases and ROSs
Tissue edema
Microthrombosis (DIC)
a c
Endotoxic shock
Hypoperfusion
Tissue hypoxia
d Tissue destruction MOF/MODS Positive feedback
Figure 3. The development of multiple organ failure/multiple organ dysfunction syndrome (MOF/MODS) triggered by lipopolysaccharide (LPS). (a) Endotoxic shock and (b) disturbance of microcirculation by an activated coagulation system result in (c) tissue hypoperfusion and (d) subsequent tissue hypoxia. As a result of leukocyte-induced vascular injury and leukocyte transmigration through the vascular wall, tissue destructive proteases and reactive oxygen species (ROSs) are released from leukocytes. DIC, disseminated intravascular coagulation.
Rolling of neutrophils on the surface of the vessel wall and transient adhesion of neutrophils with endothelial cells is the initial step in the recruitment of leukocytes, which is mainly mediated by the selectin family of adhesion molecules. L-selectin is located on leukocytes, whereas P- and E-selectin are expressed on endothelial cells. The initial contact is followed by the firm adhesion of leukocytes with endothelial cells, which is mediated by the interaction of b1 and b2 integrins on leukocytes with their counter-receptors on endothelial cells. The b2 integrins bind to intercellular adhesion molecule 1 (ICAM-1). IL-1, TNF-a and LPS induce the expression of ICAM-1 on the surface of endothelial cells. We demonstrated that anti-ICAM-1 antibody prevented acute lethality of LPS-challenged rabbits, although the elevation of plasma TNF-a levels and decrease in the number of circulating leukocytes was not attenuated30. This observation is consistent with that in LPS-challenged ICAM-1-deficient mice31. In contrast, an antibody to CD18 not only improved the survival rate of LPSchallenged rabbits but also reduced the elevation of plasma TNF-a levels and attenuated the magnitude of LPS-induced leukocytopenia30. Once adherent to the endothelium, neutrophils transmigrate through the endothelial layer and release various proteases and reac-
tive oxygen species (ROSs). Neutrophil-elastase is a serine protease that degrades a variety of humoral and structural proteins, including transport proteins, cell receptors, membrane proteins, fibronectin, elastin and collagens. Neutrophil-elastase also degrades clotting and fibrinolysis factors, thereby contributing to the development of DIC (see below). The enhanced generation of ROSs is also responsible for tissue injury in endotoxemia. O22 is the most prominent precursor of all ROSs. The production of O22 by neutrophils and macrophages in endotoxemia is mainly attributed to membrane-associated NADPH oxidase. Most of the O22 is rapidly converted to H2O2, which can be a source of •OH and hypochlorous acid (HOCl). Recent observations have demonstrated that oxygen radicals, such as ROSs, NO and peroxynitrite, activate transcription factors, such as NFkB and activator protein 1 (AP-1)32,33, which promote gene expression of proinflammatory mediators. Thus, the positive feedback by these radicals will play an important role in amplifying the excessive inflammatory responses, suggesting that antioxidant therapy has potential therapeutic application for the modulation of proinflammatory reactions in endotoxemia. 127
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reported to stimulate the release of proinflammatory cytokines by monocytes and endothelial cells34. This finding indicates CD14 – A 55-kDa plasma membrane glycoprotein. CD14 is anchored in the cell membrane that the activation of coagulation might also of monocytes/macrophages and neutrophils as a glycerophosphatidylinositol (GPI)-tailed contribute to the upregulation of proinflamprotein. Soluble CD14 (sCD14) acts as a co-factor of lipopolysaccharide (LPS), contributing matory responses. to the LPS-induced activation of CD142 cells. The tissue factor (TF)/factor VIIamediated pathway (extrinsic pathway), rather C–C and C–x–C chemokines – A chemoattractant chemokine is characterized by specific than the contact-system-mediated pathway structural features, especially a conserved pattern of cysteine amino acids. C–C chemokines (intrinsic pathway), is relevant in the inihave two adjacent cysteines near their N-termini; C–x–C chemokines have an intervening tiation of the LPS-induced procoagulant state amino acid between these two cysteines. (Fig. 4). LPS-induced activation of coagub2 integrins – Leukocyte membrane-bound heterodimeric glycoproteins that contain b2 sublation was completely blocked by the adminunits. Three members of the b2 integrins have been identified: CD11a/CD18 (LFA-1, aMb2), istration of monoclonal antibody against TF CD11b/CD18 (Mac-1, aLb2) and CD11c/CD18 (p150, p95, axb2). CD11a/CD18 and or factor VIIa in chimpanzees35. UpreguCD11b/CD18 account for the interaction of neutrophils and monocytes/macrophages with lation of surface expression of TF on endointercellular adhesion molecule 1 (ICAM-1) on the surface of endothelial cells. CD11b/CD18 thelial cells is the initial step that leads to exand CD11c/CD18 are candidates for the lipopolysaccharide (LPS) receptor. cessive activation of the coagulation system. Although TNF-a has been considered as a Intercellular adhesion molecule 1 (ICAM-1) – A 90–95-kDa adhesion molecule that intermain contributor to the expression of TF, acts with the integrin receptors CD11a/CD18, CD11b/CD18 and CD43 on leukocytes. ICAM-1 anti-TNF antibody failed to attenuate procoon endothelial cells is closely involved in the firm contact of leukocytes with endothelial cells agulant responses in experimental and cliniand with leukocyte migration through vessel walls. cal studies36,37. In contrast, treatment with an Lipopolysaccharide (LPS) – A constituent of the outer wall of most Gram-negative bacteria. anti-IL-6 antibody inhibited activation of coLPS is structurally and functionally composed of four distinct regions: the polymer of agulation in LPS-challenged chimpanzees36. oligosaccharides (O antigen), the outer core, the inner core (KDO) and a lipid component Supporting this observation, activation of (lipid A). Lipid A is responsible for the majority of the biological actions of LPS. the coagulation system occurred in patients with metastatic renal cell cancer who reLipopolysaccharide-binding protein (LBP) – A 65-kDa glycoprotein that is synthesized ceived recombinant human IL-6 therapy38. predominantly in the liver. In response to inflammatory stimuli, LBP behaves as an However, at present the mechanism by acute-phase reactant, transferring lipopolysaccharide (LPS) from high-density lipoprotein which IL-6 activates the coagulation system (HDL) to CD14. By binding to LBP, LPS can activate cells at low concentrations (ng ml21 remains unclear. concentrations). The anticoagulant system and the fibrinoMultiple organ failure/multiple organ dysfunction syndrome (MOF/MODS) – A syndrome lytic system are also impaired during endoconsisting of the sequential dysfunction of two or more organ systems. MOF/MODS occurs toxemia. Decreased plasma levels or diminin ~30% of patients with sepsis. ished activity of antithrombin III (AT-III) and protein C are observed during endotoxemia36. Nitric oxide synthases (NOSs) – Enzymes that catalyze conversion of the guanosine nitroProtein C is activated by thrombomodulin gen moiety of L-arginine to citrulline. In this process, nitric oxide (NO) is generated. Three (TM), and the anticoagulant activity of proisoforms of NOS have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS) and 21 tein C is dependent on the free level of its coinducible NOS (iNOS). eNOS and nNOS are constitutively expressed and regulated by Ca factor, protein S. TNF-a, neutrophil-elastase and calmodulin; whereas iNOS is Ca21-independent and induced by inflammatory stimuli in a and other proinflammatory mediators induce variety of cells, contributing to the production of large amounts of NO. downregulation of TM. Protein S binds to Systemic inflammatory response syndrome (SIRS) – A clinical inflammatory response C4b-binding protein (C4bBP) with high that is manifested by two or more of the following: temperature .38°C or ,36°C (rectal), affinity. During endotoxemia, the increased heart rate .90 beats min21, respiratory rate .20 breaths min21 or arterial partial pressure of level of C4bBP results in reduced levels of carbon dioxide (PaCO2) ,4.3 kPa (kilo pascal), white blood cell count .12 000 cells mm23 free protein S. Conversely, increased levels or ,4000 cells mm23 or 10% immature (band) forms. of tissue factor pathway inhibitor (TFPI) have been reported in sepsis39, which might Tissue factor (TF) – An integral membrane protein that triggers the extrinsic coagulation be a self-regulatory response to activation of cascade. Factor VII in the blood binds to TF and is converted to factor VIIa, resulting in the coagulation systems. Interestingly, in rabbits activation of the clotting cascade. with Gram-negative bacteremia, delayed treatment with TFPI, even 4 h after Escherichia coli challenge, reduced mortality40. Coagulation disorder (DIC) Suppression of the fibrinolytic system directly contributes to the Coagulation disorder is frequently associated with endotoxemia. persistence of intravascular clots. Decreased plasma levels of plasDisturbances of hemostatic balance lead to microvascular thrombosis minogen are observed in sepsis. Increased production of plasminoand bleeding, which will contribute to the development of gen activator inhibitor type 1 (PAI-1) and type 2 (PAI-2) might be MOF/MODS. Furthermore, thrombin and factor Xa have been involved in fibrinolytic suppression.
Glossary
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a
Prekallikrein
b Anti-coagulation system
Kinins
HMW kininogen
Kallikrein Protein C
XII
XIIa XI
LPS
TFPI
XIa
+?
– TM
AT-III
Fibrinolytic system Plasminogen
–
+ PAI-2
+ PAI-1
Urokinase
TPA Plasmin
–
–
V
Macrophage Endothelial cell
Prothrombin
Fibrinogen
Xa X
TNF-α IL-6
+
e
Thrombin
Extrinsic pathway
+ TF
Activated protein C
– Protein S
Intrinsic pathway
d VII
c
+
Clot formation
Fibrin
VIIa + Activated during endotoxemia – Suppressed during endotoxemia
Up-regulation Down-regulation
Figure 4. Disturbances of the hemostatic balance during endotoxemia. (a) Activation of the coagulation system, concomitant with suppression of (b) the anticoagulation system and (c) the fibrinolytic system leads to disseminated intravascular clot formation. The extrinsic pathway, rather than the intrinsic pathway, is relevant in the initial activation of lipopolysaccharide (LPS)-induced coagulation. (d) The upregulation of tissue factor (TF) on the surface of endothelial cells is mediated by various LPS-induced cytokines. (e) Thrombin generated in the procoagulant state stimulates the production of proinflammatory mediators, such as tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6) in macrophages and endothelial cells, resulting in the further development of inflammatory responses to endotoxemia. AT-III, antithrombin III; HMW, heavy molecular weight; PAI, plasminogen activator inhibitor; TFPI, tissue factor pathway inhibitor; TM, thrombomodulin; tPA, tissuetype plasminogen activator; uPA, urokinase-type plasminogen activator.
Anti-inflammatory responses in endotoxemia In response to the proinflammatory responses in endotoxemia, host cells produce several anti-inflammatory mediators, such as IL-4, IL-10, IL-13, transforming growth factor b (TGF-b), glucocorticoids and PGE2. These mediators suppress the synthesis and action of proinflammatory mediators. In addition, IL-1Ra and soluble receptors to proinflammatory cytokines, such as IL-1 receptor type II (sIL-1RII), soluble TNF receptor p75 (sTNFRp75) and soluble TNF receptor p55 (sTNFRp55), have anti-inflammatory effects by antagonizing or neutralizing the actions of each cytokine. IL-10, which is produced by T-helper 2 (Th2) cells, B cells and monocytes/macrophages, inhibits the production of TNF-a, IL-1b and IL-12 by activated macrophages. As a result, the function of Th1 CD41 T cells, which require stimulation by macrophage-derived proinflammatory cytokines, is also suppressed. IL-10-deficient mice41 or mice treated with a monoclonal antibody against IL-10 (Ref. 42) showed higher plasma levels of proinflammatory cytokines and increased mortality. In contrast, administration of recombinant IL-10 to LPS-challenged mice showed protective effects against proinflammatory cytokine production and lethality43. IL-1Ra inhibits the activity of IL-1 by competitively binding to IL-1 receptors, whereas IL-1Ra itself does not in-
duce any intracellular responses44. Three isoforms of IL-1Ra (16, 17 and 18 kDa) have been identified. Intraperitoneal injection of LPS resulted in higher lethality in IL-1Ra-deficient mice than in wild-type mice. Administration studies with IL-1Ra have revealed that large amounts of IL-1Ra are required to block the effects of IL-1b, perhaps because a sustained high level of IL-1Ra is required to occupy IL-1 receptors. Although sIL-1RII inhibits the activity of IL-1b in a dose-dependent manner, the relevance of sTNFRp75 and sTNFRp55 to TNF-a activity is a little more complicated. High molar ratios of receptor to ligand are necessary for both soluble TNF receptors to inhibit the activity of TNF-a. At low molar ratios, both soluble receptors act as carriers of TNF-a and enhance the activity of this cytokine45. The interactions between pro- and anti-inflammatory mediators play a crucial role in the control of adequate levels of immune activity against infection. If the balance cannot be maintained, the patient will suffer from massive proinflammatory responses or refractory immunosuppression. For example, administration of IL-4 or IL-10 increases lethality after Candida infection in mice46. More detailed information on the mechanism of the modulation between pro- and anti-inflammatory mediators will contribute to the development 129
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of novel therapeutic approaches that are designed to restore homeostasis in the immune system of patients with sepsis.
Therapeutic strategies for the treatment of sepsis Based on the molecular pathogenesis of septic shock and organ failure, clinical trials to block the effects of LPS and cytokines have been attempted. Clinical studies of two antibodies to lipid A, HA-1A and E5, demonstrated inconsistent results. Despite promising results reported by earlier studies, subsequent trials using larger numbers of patients with Gram-negative bacteremia did not support these findings47,48. Other anti-LPS strategies involve anti-CD14 antibodies, LPSbinding proteins (e.g. BPI and CAP18), and hemoperfusion with polymyxin B. Anti-cytokine strategies include the development of anti-cytokine antibodies (e.g. anti-TNF-a antibody), soluble cytokine receptors with IgG chimeric protein (e.g. sTNFRp75–IgG, sTNFRp55–IgG) and a cytokine receptor antagonist (IL-1Ra). Although some initial anti-cytokine trials suggested some benefit, more recent studies have failed to improve the overall outcome of patients with sepsis. A
Phase II trial with sTNFRp75–IgG could not improve survival rate in patients with sepsis, and higher doses of this fusion protein resulted in increased mortality49. To date, Phase III anti-cytokine clinical trials have been conducted on anti-TNF-a antibody50,51, sTNFRp55–IgG (Ref. 52) and IL-1Ra (Ref. 53). None of these trials showed a statistically significant reduction of 28-day mortality, although a significant improvement in the 3-day mortality and an increase in the 28-day survival of a subgroup of severely septic patients was observed (Table 2). Thus, so far, clinical anti-LPS or anti-cytokine trials to block the sepsis cascade have resulted in limited success. This might, in part, be the result of delays between the onset of sepsis and the beginning of treatment. The trials that target early mediators, such as LPS and TNF-a, might not be relevant because the cascade of inflammatory mediators is already under way when intervention is started. New approaches targeting the mediators of the later stage of sepsis might be more effective. Moreover, it will also be necessary to determine the optimal doses and duration of administration of these agents. New assays that can detect serum cytokine levels of a patient within a few hours are strongly desirable.
Table 2. Clinical trials with immunomodulating agents in patients with sepsis (1996–1998)a Agent
Anti-TNF-a mAb
Anti-TNF-a mAb
sTNFRp75–IgG
Phase
III (INTERSEPT)
Year
1996
III (NORASEPT II) 1998
II
1996
Number of patients
553
1879
141
Results
Ref.
28–30-day all-cause mortality
Other findings
No significant improvement (14.5%
More rapid reversal of shock; significant delay
reduction in the lower-dose group)
in the time of the onset of organ failure
No significant improvement (5.8%
Significant decrease in the frequency of
reduction)
coagulopathy
No significant improvement
Increase in mortality in the subgroup that
50
51
49
received higher doses sTNFRp55–IgG
IL-1Ra
III
III
1997
1997
498
696
No significant improvement (36%
Significant improvement of 28-day survival of
52
reduction)
patients with severe sepsis
No significant improvement (9%
A better resolution of organ failures
53
Improvement in lung compliance; earlier
54
reduction) Liposomal PGE1
III
1996
25 (ARDS)
No significant improvement
removal from mechanical ventilation Ibuprofen
III
1997
455
No significant improvement
Delay in the day of the onset of organ failure;
55
significant decrease in blood lactate levels Bradykinin antagonist
II
1997
504 (SIRS)
No significant improvement
Significant improvement of 28-day survival of
57
patients with Gram-negative infections Antithrombin III
a
II
1998
42
No significant improvement (39%
A better resolution of organ failures; a lower
reduction)
incidence of new organ failures
58
Abbreviations: ARDS, acute respiratory distress syndrome; IgG, immunoglobulin G; IL-1Ra, interleukin 1 receptor antagonist; INTERSEPT, International Sepsis Trial Group; mAb, monoclonal antibody; NORASEPT, North American Sepsis Trial Group; PGE1, prostaglandin E1; SIRS, systemic inflammatory response syndrome; sTNFR, soluble tumor necrosis factor receptor; TNF-a, tumor necrosis factor a.
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References
The outstanding questions
•
How is the lipopolysaccharide (LPS)–CD14 signal transduced into the cytoplasm? Is Toll-like receptor 2 (TLR-2) essential for LPS signal transduction? Do the cells have other unidentified molecules that recognize LPS? Does selective inhibition of inducible nitric oxide synthase (iNOS) provide beneficial effects during endotoxic shock? How are the chemokines and adhesion molecules involved in leukocyte-induced tissue injury during endotoxemia? Is the modulation of these mediators beneficial in preventing the development of sepsis-associated organ dysfunction? How can we attenuate the production and the cytotoxic actions of oxygen radicals, such as reactive oxygen species, nitric oxide and peroxynitrite? How can we reduce the positive feedback effects of these oxygen radicals on the production of proinflammatory mediators? How is the expression of tissue factor regulated in endotoxemia? Does interleukin 6 (IL-6) play an essential role in the upregulation of the extrinsic coagulation pathway? How can the anti-inflammatory mediators be available in the intervention of sepsis-induced proinflammatory responses without exerting immunosuppressive effects? Does combination therapy of immunomodulating agents, including anti-cytokine substances, improve the outcome of septic patients?
• • • • • •
In addition to anti-LPS or anti-cytokine agents, several strategies to interfere with the sepsis cascade have been attempted clinically. These trials include studies with PGs (e.g. liposomal PGE1)54, antiprostaglandin therapy (e.g. ibuprofen)55, bradykinin antagonists56, PAF antagonists57 and coagulation inhibitors (e.g. protein C, TFPI and AT-III)58 (Table 2). Although an initial study with PAF antagonist improved the survival rate of patients with Gram-negative sepsis57, later studies could not show a statistically significant reduction in mortality. The trials with ibuprofen did not show a significant improvement in survival either55. By contrast, Phase II trials with bradykinin antagonist or AT-III have shown promising results56,58. The trial with bradykinin antagonist showed a significant reduction in 28-day mortality of patients with Gram-negative sepsis. In addition, the trials with liposomal PGE1, protein C and TFPI are ongoing. As mentioned above, a Phase III multicenter clinical trial using L-NMMA failed because of unacceptable cardiovascular side effects. Novel selective inhibitors of iNOS might be more beneficial therapeutic choices.
Concluding remarks Sepsis is not a single disease, but a very complex condition comprising a large variety of local and systemic inflammatory responses. Therefore, we propose that a combination of several therapies or multifunctional agents, directed to various phases of sepsis or sepsisassociated molecules, might meet with more success than recent trials with single therapies. Such combined trials in sepsis, or endotoxemia, will be the subject of intense investigation over the next decade.
1 American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (1992) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis, Crit. Care Med. 20, 864–874 2 Wenzel, R. (1992) Anti-endotoxin monoclonal antibodies – a second look, New Engl. J. Med. 326, 1151–1153 3 Brun-Buisson, C. et al. (1995) Incidence, risk factors, and outcome of severe sepsis and septic shock in adults, J. Am. Med. Assoc. 274, 968–974 4 Bates, D. et al. (1997) Predicting bacteremia in patients with sepsis syndrome, J. Infect. Dis. 176, 1538–1551 5 Fenton, M. and Golenbock, D. (1998) LPS-binding proteins and receptors, J. Leukocyte Biol. 64, 25–32 6 Jack, R.S. et al. (1997) Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection, Nature 389, 742–745 7 Kitchens, R. and Munford, R. (1998) CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS, J. Immunol. 160, 1920–1928 8 Ingalls, R.R. and Golenbock, D.T. (1995) CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide, J. Exp. Med. 181, 1473–1479 9 Yang, R.B. et al. (1998) Toll-like receptor-2 mediates lipopolysaccharideinduced cellular signalling, Nature 395, 284–288 10 Geng, Y., Zhang, B. and Lotz, M. (1993) Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes, J. Immunol. 151, 6692–6700 11 Weinstein, S.L., June, C.H. and DeFranco, A.L. (1993) Lipopolysaccharideinduced protein tyrosine phosphorylation in human macrophages is mediated by CD14, J. Immunol. 151, 3829–3838 12 Meng, F. and Lowell, C.A. (1997) Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn, J. Exp. Med. 185, 1661–1670 13 Sweet, M.J. and Hume, D.A. (1996) Endotoxin signal transduction in macrophages, J. Leukocyte Biol. 60, 8–26 14 Woronicz, J., Gao, X., Cao, Z. and Rothe, M.G.D. (1997) IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK, Science 278, 866–869 15 Hack, C.E., Aarden, L.A. and Thijis, L.G. (1997) Role of cytokines in sepsis, Adv. Immunol. 66, 101–195 16 Szabo, C. (1995) Alterations in nitric oxide production in various forms of circulatory shock, New Horiz. 3, 2–32 17 Beckman, J.S. et al. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly, Am. J. Physiol. 271, C1424–1437 18 Salvemini, D. et al. (1993) Nitric oxide activates cyclooxygenase enzymes, Proc. Natl. Acad. Sci. U. S. A. 90, 7240–7244 19 Habib, A. et al. (1997) Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages, J. Immunol. 158, 3845–3851 20 Cobb, J.P. et al. (1992) NG-Amino-L-arginine, an inhibitor of nitric oxide synthase, raises vascular resistance but increases mortality rates in awake canines challenged with endotoxin, J. Exp. Med. 176, 1175–1182 21 MacMicking, J.D. (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthetase, Cell 81, 641–650 22 Szabo, C. et al. (1994) Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase, Proc. Natl. Acad. Sci. U. S. A. 91, 12472–12476 23 Laubach, V.E. (1995) Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death, Proc. Natl. Acad. Sci. U. S. A. 92, 10688–10692 24 Nathan, C. (1997) Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest. 100, 2417–2423
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25 Matsushima, K. et al. (1988) Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor, J. Exp. Med. 167, 1883–1893 26 Carvalho, G. et al. (1997) Anti-interleukin-8 monoclonal antibody reduces free radical production and improves hemodynamics and survival rate in endotoxic shock in rabbits, Surgery 122, 60–68 27 Standiford, T.J. (1995) Macrophage inflammatory protein-1 alpha mediates lung leukocyte recruitment, lung capillary leak, and early mortality in murine endotoxemia, J. Immunol. 155, 1515–1524 28 Zisman, D. et al. (1997) MCP-1 protects mice in lethal endotoxemia, J. Clin. Invest. 99, 2832–2836 29 Yoneyama, H. et al. (1998) Pivotal role of TARC, a CC chemokine, in bacterialinduced fluminant hepatic failure in mice, J. Clin. Invest. 102, 1933–1941 30 Watanabe, S. et al. (1995) Prevention of endotoxin shock by an antibody against leukocyte integrin beta 2 through inhibiting production and action of TNF, Int. Immunol. 7, 1037–1046 31 Xu, H. et al. (1994) Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice, J. Exp. Med. 180, 95–109 32 Sen, C. and Packer, L. (1996) Antioxidant and redox regulation of gene transcription, FASEB J. 10, 709–720 33 Hierholzer, C. et al. (1998) Essential role of induced nitric oxide in the initiation of the inflammatory response after hemorrhagic shock, J. Exp. Med. 187, 917–928 34 Johnson, K. et al. (1998) Potential mechanisms for a pro-inflammatory vascular cytokine response to coagulation activation, J. Immunol. 160, 5130–5135 35 Levi, M. et al. (1994) Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees, J. Clin. Invest. 93, 114–120 36 Levi, M., van der Poll, T., ten Cate, H. and van Deventer, S. (1997) The cytokinemediated imbalance between coagulant and anti-coagulant mechanisms in sepsis and endotoxaemia, Eur. J. Clin. Invest. 27, 3–9 37 Salat, C. et al. (1996) Hemostatic parameters in sepsis patients treated with anti-TNF alpha-monoclonal antibodies, Shock 6, 233–237 38 Stouthard, J. et al. (1996) Interleukin-6 stimulates coagulation, not fibrinolysis in humans, Thromb. Haemost. 76, 738–742 39 Sabharwal, A. et al. (1995) Tissue factor pathway inhibitor and von Willebrand factor antigen levels in adult respiratory distress syndrome and in a primate model of sepsis, Am. J. Respir. Crit. Care Med. 151, 758–767 40 Camerota, A. et al. (1998) Delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbits with gram-negative peritonitis, J. Infect. Dis. 177, 668–676 41 Berg, D.J. et al. (1995) Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance, J. Clin. Invest. 96, 2339–2347 42 Standiford, T.J. et al. (1995) Neutralization of IL-10 increases lethality in endotoxemia. Cooperative effects of macrophage inflammatory protein-2 and tumor necrosis factor, J. Immunol. 155, 2222–2229 43 Gerard, C. et al. (1993) Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia, J. Exp. Med. 177, 547–550 44 Arend, W.P. et al. (1998) Interleukin-1 receptor antagonist: role in biology, Annu. Rev. Immunol. 16, 27–55 45 Aderka, D. et al. (1992) Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors, J. Exp. Med. 175, 323–329 46 Tonnetti, L. et al. (1995) Interleukin-4 and -10 exacerbate candidiasis in mice, Eur. J. Immunol. 25, 1559–1565 47 The National Committee for the Evaluation of Centoxin (1994) The French National Registry of HA-1A (Centoxin) in septic shock. A cohort study of 600 patients, Arch. Intern. Med. 15, 2484–2491 48 Bone, R. et al. (1995) A second large controlled clinical study of E5, a mono-
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49
50
51
52
53
54
55
56
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clonal antibody to endotoxin: results of a prospective, multicenter, randomized, controlled trial. The E5 Sepsis Study Group, Crit. Care Med. 23, 994–1006 Fisher, C.J., Jr et al. (1996) Treatment of septic shock with the tumor necrosis factor receptor:Fc fusion protein. The Soluble TNF Receptor Sepsis Study Group, New Engl. J. Med. 1996 334, 1697–1702 Cohen, J. and Carlet, J. (1996) INTERSEPT: an international, multicenter, placebo-controlled trial of monoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. International Sepsis Trial Study Group, Crit. Care Med. 24, 1431–1440 Abraham, E. et al. (1998) Double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. NORASEPT II Study Group, Lancet 351, 929–933 Abraham, E. et al. (1997) p55 tumor necrosis factor receptor fusion protein in the treatment of patients with severe sepsis and septic shock. A randomized controlled multicenter trial. Ro 45-2081 Study Group, J. Am. Med. Assoc. 277, 1531–1538 Opal, S.M. et al. (1997) Confirmatory interleukin-1 receptor antagonist trial in severe sepsis: a phase III, randomized, double-blind, placebo-controlled, multicenter trial. The Interleukin-1 Receptor Antagonist Sepsis Investigator Group, Crit. Care Med. 25, 1115–1124 Abraham, E. et al. (1996) Liposomal prostaglandin E1 in acute respiratory distress syndrome: a placebo-controlled, randomized, double-blind, multicenter clinical trial, Crit. Care Med. 24, 10–15 Bernard, G.R. et al. (1997) The effects of ibuprofen on the physiology and survival of patients with sepsis. The Ibuprofen in Sepsis Study Group, New Engl. J. Med. 336, 912–918 Fein, A.M. et al. (1997) Treatment of severe systemic inflammatory response syndrome and sepsis with a novel bradykinin antagonist, deltibant (CP-0127). Results of a randomized, double-blind, placebo-controlled trial. CP-0127 SIRS and Sepsis Study Group, J. Am. Med. Assoc. 277, 482–487 Dhainaut, J.F. et al. (1994) Platelet-activating factor receptor antagonist BN 52021 in the treatment of severe sepsis: a randomized, double-blind, placebocontrolled, multicenter clinical trial. BN 52021 Sepsis Study Group, Crit. Care Med. 22, 1720–1728 Eisele, B. et al. (1998) Antithrombin III in patients with severe sepsis. A randomized, placebo-controlled, double-blind multicenter trial plus a meta-analysis on all randomized, placebo-controlled, double-blind trials with antithrombin III in severe sepsis, Intensive Care Med. 24, 663–672
Late-breaking news Since Yang et al. reported that toll-like receptor (TLR) 2 participates in LPS-induced cellular activation (see Ref. 9 above), TLRs have been investigated as signal transducers for LPS. Kischning et al.1 report that overexpression of TLR2, but not TLR1, TLR4 or CD14, confers responsiveness to LPS in cells from a human embryonic cell line, as measured through the activation of nuclear factor kB. However, Poltorak et al.2 have found that a mutation in the murine Tlr4 gene renders mice resistant to endotoxin yet highly susceptible to Gramnegative infection. This discrepancy in the function of TLR4 is likely to be resolved in the future. 1 Kirschning, C.J. et al. (1998) Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide, J. Exp. Med. 188, 2091–2097 2 Poltorak, A. et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Science 282, 2085–2088
MOLECULAR MEDICINE TODAY, MARCH 1999
The molecular pathogenesis of endotoxic shock and organ failure Risuke Karima, Shigeru Matsumoto, Hidemitsu Higashi and Kouji Matsushima
Sepsis is still associated with a high mortality rate. Septic shock and sequential multiple organ failure have a strong correlation with poor outcome. Lipopolysaccharide (LPS) plays a pivotal role in the initiation of host responses to Gram-negative infection. A number of mediators, such as cytokines, nitric oxide and eicosanoids, are responsible for most of the manifestations caused by LPS, and circulatory failure, leukocyte-induced tissue injury and coagulation disorder appear to be critical determinants in the development of sequential organ failure. Although several anti-LPS or anti-cytokine clinical trials have been attempted, none of them has so far been successful. DESPITE recent progress in antibiotics and critical care therapy, sepsis is still associated with a high mortality rate. Septic shock and sequential multiple organ failure/dysfunction syndrome (MOF/MODS) correlate with poor outcome. Lipopolysaccharide (LPS) or endotoxin plays a pivotal role in the initiation of a variety of host responses caused by Gram-negative bacterial infection. Since the 1980s, novel insights into the molecular pathogenesis of LPS-induced shock (endotoxic shock) and organ dysfunction have been gained. The molecular cloning of proinflammatory cytokines and adhesion molecules were important steps
Risuke Karima MD Research Associate Shigeru Matsumoto MS Research Associate Hidemitsu Higashi MS Research Associate Kouji Matsushima* MD, PhD Professor of Molecular Preventive Medicine Dept of Molecular Preventive Medicine, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyou-ku, Tokyo, 113-0033, Japan. Tel: +83 3 5800 6853 Fax: +83 3 5800 6853 *e-mail: [email protected]
towards understanding the molecular mechanism of sepsis. In addition, nitric oxide (NO) has been identified as a key mediator of LPSmediated hypotension. These findings have led to clinical trials of various single agents that antagonize either LPS or sepsis-associated mediators. Unfortunately, none of the clinical trials of these agents has so far been successful. This review will summarize our current understanding of the molecular pathogenesis of endotoxic shock and endotoxin-induced MOF/MODS, as well as the clinical features of endotoxemia, with the aim of exploring the potential for an effective therapy against sepsis and endotoxemia.
Clinical features of sepsis In 1991, a new concept – systemic inflammatory response syndrome (SIRS) – was postulated to define the state of patients who exhibit a systemic response to inflammatory episodes1. SIRS is diagnosed by a combination of available clinical signs and symptoms. Nowadays, sepsis is generally defined as SIRS induced by infection. No worldwide statistics on the occurrence of sepsis are available. In the USA alone, however, it is estimated that there are 300 000–500 000 septic episodes each year, with mortality rates ranging from 20% to 40% (Ref. 2). Refractory hypotension (septic shock) is the main cause of death within a few days of the onset of sepsis. Later, MOF/MODS becomes the primary clinical problem and main cause of mortality. Once a patient develops septic shock or sequential MOF/MODS, the mortality rate increases to 60–70% (Ref. 3). Gram-negative bacteria are responsible for 45–60% of sepsis caused by bacterial infection when mixed-organism infections are included3,4. Septic shock has been reported to be a complication in 20–50% of patients with sepsis3,4. The initial cardiovascular response to sepsis is generally characterized by high cardiac output and low systemic vascular resistance. Even though cardiac output might be maintained above normal levels, impaired myocardial contractility, inadequate
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distribution of blood flow and disturbance of tissue oxygen use are common and contribute to the development of MOF/MODS is defined as a syndrome that consists of the sequential failure of two or more organ systems. The mortality of patients who have three or more types of organ failure is particularly high4. The lungs, kidneys, liver, cardiovascular system, central nervous system and coagulation system are commonly involved in septic MOF/MODS. If specific conditions are met, the lung dysfunction is termed acute lung injury or acute respiratory distress syndrome (ARDS), and the coagulatory disorder is diagnosed as disseminated intravascular coagulation (DIC). The clinical features of the dysfunction of each organ in sepsis are summarized in Table 1.
MOLECULAR MEDICINE TODAY, MARCH 1999
macrophages/monocytes and neutrophils. CD14 is a glycerophosphatidylinositol (GPI)-anchored glycoprotein that lacks a cytoplasmic portion. LPS-binding protein (LBP) might facilitate the interaction of LPS with CD14 (Ref. 5) or effect the clearance of LPS from the circulation by transferring LPS from CD14 (Fig. 1). Recent studies suggest the former because galactosamine-sensitized LBPdeficient mice were resistant to the acute lethality of LPS, but LBP deficiency did not effect the clearance of LPS (Ref. 6). In certain cell types, such as endothelial cells, that do not express CD14, soluble CD14 (sCD14) substitutes as the signaling bridge for surface recognition of the LPS–LBP complex. However, at present the molecule(s) that recognizes the complex of LPS–LBP–sCD14 has not been identified in CD142 cells. The LPS-induced signaling pathway CD14 does not have a cytoplasmic portion, but substantial eviLPS is a constituent of the cell wall of Gram-negative bacteria. The dence supports a role for unidentified signal transducing molecules major biological activities of LPS are mainly attributed to a lipid that interact with CD14 at the cell membrane5. Alternatively, binding component, termed lipid A. LPS interacts with CD14, a receptor on of LPS to CD14 might result in the rapid translocation of LPS from the cell membrane to intracellular locations, leading to LPS-induced cell activation7. Furthermore, although cellular activation by Table 1. Clinical features of organ dysfunction in sepsisa low concentrations of LPS (<1 mg ml21) requires CD14, a high concentration of LPS Organ/system Symptom can stimulate cells in a CD14-independent manner. This suggests the existence of a second molecule that recognizes LPS. Central nervous system (CNS) Reduced alertness, confusion, stupor or coma; drifting in and out of Candidates include CD11/CD18 (Ref. 8) and consciousness Toll-like receptor 2 (TLR-2)9, but the precise Cardiovascular system Decrease in bilateral ventricular contractility, although cardiac output mechanism of the CD14-independent signalcan be increased to supranormal levels ing pathway remains unclear. The molecular mechanism that transduces Decrease in systemic vascular resistance LPS receptor-mediated signals to the nucleus Lung Hypoxemia that frequently necessitates support with mechanical is not fully clarified (Fig. 2). Tyrosine kiventilation with PEEP nases, especially Hck, Fgr and Lyn, have been suggested to play an important role in Widespread, patchy or homogeneous bilateral infiltrates in chest X-ray, LPS–CD14-mediated signaling10,11. However, although radiographic abnormalities are often absent within a few days a recent study demonstrated that macroof onset phages from hck2/2fgr2/2lyn2/2 triple knockDecrease in pulmonary compliance and increased airway resistance out mice produce normal levels of proinflammatory cytokines and NO in response Liver Progressive increase in plasma bilirubin levels (often .5 mg dl21) with to LPS stimulation, indicating that these Srcmoderate increase in plasma transaminase levels (usually within family kinases are not essential for LPS21 200 u ml ) initiated signal transduction12. Involvement Reduced amino acid extraction with reduced protein synthesis of other protein kinases in the transduction of LPS-induced responses has been also sugIncreased hepatic VLDL release with reduced peripheral triglyceride gested. Such kinases include protein kinase clearance C and mitogen-activated protein kinases Impaired glucose release that often results in hypoglycemia (MAPK), such as p42 (ERK2), p44 (ERK1) and p38 (Ref. 13). LPS-induced cytokine Kidney Increase in plasma levels of BUN and creatinine with reduced GFR production requires activation of several Oliguria or anuria, although sometimes urinary volume is maintained transcription factors. Nuclear factor kB within normal or polyuremic limits (NFkB) and nuclear factor-interleukin 6 (NF-IL-6) are involved in the gene transcrip23 Coagulation system Thrombocytopenia (often ,50 000 cells mm ) tion of numerous proinflammatory cytoDecrease in plasma fibrinogen and antithrombin III levels kines, tissue factor, adhesion molecules and inducible NO synthase13. LPS activates nuIncrease in FDP and prolongation of clotting time clear translocation of NFkB by modifying its aAbbreviations: BUN, blood urea nitrogen; dl, deciliter; FDP, fibrinogen degradation products; GFR, glomerular filtration inhibitory subunit, IkB. NFkB activation rerate; PEEP, positive end-expiratory pressure; VLDL, very low density lipoprotein. quires sequential phosphorylation, ubiquitination and degradation of IkB. Recently, IkB 124
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kinases that phosphorylate N-terminal serine residues have been identified14, but activation of the IkB kinases by the stimulation of monocytes or neutrophils with LPS has not been reported.
Liver
a
LPS Gram-negative bacteria
The pathogenesis of endotoxic shock and MOF/MODS A variety of pathophysiological responses in various tissues and organ systems occur during endotoxemia. In particular, circulatory failure, leukocyte-induced tissue injury and activation of coagulation systems appear to be critical determinants in the development of sequential organ failure (Fig. 3). A number of mediators derived from host cells are responsible for most of the manifestations of endotoxemia. The proinflammatory cytokines, including tumor necrosis factor a (TNF-a), interleukin (IL) 1b, IL-6, IL-8 and IL-12, and interferon g (IFN-g), play a critical role in the inflammatory responses15. NO is now known to induce a variety of responses in addition to hypotension. Some chemokines have recently been found to be involved in the development of LPSinduced tissue injury. Lipid mediators, such as platelet activating factor (PAF), prostaglandins, thromboxanes and leukotrienes, also exert a variety of effects in endotoxemia, and the role of the inducible cyclooxygenase 2 (COX-2), which converts arachidonic acid to prostaglandins (PGs) PGE, PGF and PGI2 and thromboxanes, has been extensively investigated. Furthermore, anti-inflammatory mediators, such as IL-10 and IL-1 receptor antagonist (IL-1Ra), also contribute to the modulation of inflammatory responses in endotoxemia. Space limitations preclude a complete review of these mediators. This review will focus on recent discoveries that relate to the role of NO, leukocyte-induced tissue injury, the mechanisms of the development of DIC and immunomodulation by anti-inflammatory mediators in endotoxemia.
LPS LBP
HDL HDL CD14
b
LPS LBP
CD14 Neutrophil
Macrophage
c
LPS LBP
Soluble CD14
Vascular endothelial cell
Role of NO The role of NO in inducing hypotension is now well established. NO is generated by three different nitric oxide synthases (NOSs), which are divided into two major categories: constitutive NOS (cNOS) and inducible NOS (iNOS). cNOS is regulated by intracellular Ca21 and calmodulin. Two isoforms of cNOS are known, one originally isolated in neurons (nNOS) and one in endothelial cells (eNOS). Under physiological conditions, NO is released constitutively by cNOSs, contributing to the regulation of basal vascular tone, inhibition of platelet aggregation and inhibition of leukocyte adhesion to endothelium. By contrast, iNOS, which is Ca21-independent and not expressed constitutively, is induced by inflammatory stimuli, such as LPS, IL-1b, TNF-a, IFN-g and PAF, in a variety of cells, including macrophages, endothelium, smooth muscle cells, hepatocytes and cardiomyocytes. iNOS is responsible for the production of large amounts of NO during endotoxemia16. The action of NO is mediated by the activation of soluble guanylyl cyclase and the consequent increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). This increase in cGMP leads to the relaxation of vascular smooth muscle and reduced responsiveness to vasoconstrictors, thereby contributing to the development of refractory hypotension during endotoxemia. NO also has direct effects on various enzymes that contain an iron–sulphur moiety in their catalytic center. Such enzymes include ribonucleotide reductase (necessary for DNA synthesis) and several mitochondrial enzymes that are essential for survival of the cell. Furthermore, the reaction of NO with superoxide anion (O22) results
TNF-α IL-1β IL-6 IL-8 IFN-γ NO PAF Eicosanoids
Endotoxic shock
MOF/MODS
Figure 1. CD14-dependent cellular activation by lipopolysaccharide (LPS). In plasma (a) LPS binds high-density lipoprotein (HDL). (b) LPS-binding protein (LBP) transfers LPS from HDL to CD14 and facilitates the interactions of LPS with CD14 expressed on the surface of monocytes/macrophages or neutrophils. Endothelial cells and some other types of cells do not express CD14. (c) LPS stimulates these cells by binding soluble CD14. IFN-g, interferon g; IL-1, interleukin 1; MOF/MODS, multiple organ failure/multiple organ dysfunction syndrome; NO, nitric oxide; PAF, platelet-activating factor; TNF-a, tumor necrosis factor a.
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LPS
?
?
?
b
?
LPS
LBP
(High concentration)
CD14
a
GPI anchor
TLR2
?
Unidentified receptor?
? Another LPS receptor?
LPS
Plasma membrane
LPS internalization? ?
? ? ?
?
c
?
Src Lyn, Hck, Fgr
?
?
Phosphatidylinositol3-kinase
Tyrosine kinases
Intracellular Ca2+ p44 p42 p38 MAP kinases
Protein kinase A
Protein kinase C
? ?
? ROS, NO
Gene
IκB kinases α
d
IκB kinases β IκB α/β
IκB α/β
NFκB
NFκB
NF-IL-6 P
e Translocation
NFκB
some recent studies have shown that NO inhibits COX activity19. These pathophysiological roles of NO suggest that inhibiting NO production might improve the outcome of endotoxemia. However, a competitive inhibitor of NOS, NG-monomethyl-L-arginine (L-NMMA), inhibited the development of hypotension after LPS challenge but did not necessarily improve the mortality of experimental endotoxemia20. Furthermore, a Phase III multicenter clinical trial using L-NMMA was terminated because of unacceptable cardiovascular side effects. These results might be attributable to the inhibition of the beneficial roles of NO, such as maintenance of tissue perfusion and inhibition of platelet aggregation, causing impairment of splanchnic blood flow. In this respect, specific inhibition of iNOS, preserving the beneficial activities of NO produced by cNOS, might have more therapeutic potential. In support of this, iNOS-deficient mice were found to be resistant to acute lethality after LPS challenge21. In addition, the administration of selective inhibitors of iNOS showed protective effects against vascular dysfunction, organ injury and mortality in experimental endotoxemia22. However, another study reported no survival advantage of LPS-challenged iNOS-deficient mice over wild-type mice23, and deleterious effects by selective iNOS inhibition in endotoxemia have been also documented24.
Leukocyte-induced tissue injury
Since the identification of IL-8 in 1987 (Ref. 25), it has become clear that chemokines play a fundamental role in the accumulation, adhesion, migration and activation of leukocytes in the inflammatory process. IL-8 is a Figure 2. Lipopolysaccharide (LPS) signaling pathways. The precise mechanism that transduces the LPS C–x–C chemokine that has specific chemosignal to the nucleus is not well defined. (a) CD14 is anchored to the cell membrane as a glycerophosattractive effects on neutrophils and basophatidylinositol (GPI)-conjugated protein and lacks a cytoplasmic portion, suggesting the existence of phils. Anti-IL-8 antibody dramatically supunidentified transducing molecules that associate directly with CD14. At low concentrations of LPS, LPS pressed accumulation of neutrophils in the stimulates cells by binding to CD14, (b) although LPS can stimulate cells in a CD14-independent manner alveolar space and prevented acute lethality at high concentrations of LPS. (c) The important roles of tyrosine kinases and mitogen-activated protein (MAP) kinases in the LPS-induced signaling pathway have been demonstrated. Gene transcription of after systemic administration of LPS in LPS-induced inflammatory mediators requires activation of nuclear factor kB (NFkB). (d) Phosphorylation rabbits26. By contrast, C–C chemokines, of the inhibitory subunit of NFkB (IkB) by IkB kinases leads to the ubiquitination and degradation of IkB, such as monocyte chemoattractant protein resulting in (e) the translocation of NFkB to the nucleus. IL-6, interleukin 6; LBP, lipopolysaccharide-binding 1/monocyte chemotactic and activating facprotein; NO, nitric oxide; ROS, reactive oxygen species; TLR, Toll-like receptor. tor (MCP-1/MCAF) and macrophage inflammatory chemokine 1a (MIP-1a), mainly contribute to monocyte/macrophage and Tin the formation of cytotoxic oxygen radicals, such as peroxynitrite cell chemotaxis. The involvement of MIP-1a in endotoxin-induced (ONOO2), which decomposes to an intermediate with the biological tissue injury has been suggested27, whereas the neutralization of activity of the hydroxyl radical (•OH)17. Peroxynitrite is a strong and MCP-1/MCAF resulted in an increase in LPS-induced mortality28. stable oxidant that is thought to be responsible for many cytotoxic More recently, we found that administration of the monoclonal antiactions of NO via a number of independent mechanisms. In addition, body against thymus and activation-regulated cytokine (TARC) atNO has been reported to upregulate the catalytic activity of COX, tenuated LPS-induced hepatic injury and improved prognosis after which increases the production of cytotoxic eicosanoids18, although LPS challenge in Propionibacterium acnes-primed mice29. IκB α/β
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Nucleus
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Transcriptional factors (NFκB, NF-IL-6 etc.)
LPS
Inflammatory mediators Responsiveness to vasoconstrictors Myocardial function Vascular relaxation
Leukocyte adhesion
b
Vascular injury
Fibrinolysis Anti-coagulation system Coagulation system
Leukocyte migration
Release of proteases and ROSs
Tissue edema
Microthrombosis (DIC)
a c
Endotoxic shock
Hypoperfusion
Tissue hypoxia
d Tissue destruction MOF/MODS Positive feedback
Figure 3. The development of multiple organ failure/multiple organ dysfunction syndrome (MOF/MODS) triggered by lipopolysaccharide (LPS). (a) Endotoxic shock and (b) disturbance of microcirculation by an activated coagulation system result in (c) tissue hypoperfusion and (d) subsequent tissue hypoxia. As a result of leukocyte-induced vascular injury and leukocyte transmigration through the vascular wall, tissue destructive proteases and reactive oxygen species (ROSs) are released from leukocytes. DIC, disseminated intravascular coagulation.
Rolling of neutrophils on the surface of the vessel wall and transient adhesion of neutrophils with endothelial cells is the initial step in the recruitment of leukocytes, which is mainly mediated by the selectin family of adhesion molecules. L-selectin is located on leukocytes, whereas P- and E-selectin are expressed on endothelial cells. The initial contact is followed by the firm adhesion of leukocytes with endothelial cells, which is mediated by the interaction of b1 and b2 integrins on leukocytes with their counter-receptors on endothelial cells. The b2 integrins bind to intercellular adhesion molecule 1 (ICAM-1). IL-1, TNF-a and LPS induce the expression of ICAM-1 on the surface of endothelial cells. We demonstrated that anti-ICAM-1 antibody prevented acute lethality of LPS-challenged rabbits, although the elevation of plasma TNF-a levels and decrease in the number of circulating leukocytes was not attenuated30. This observation is consistent with that in LPS-challenged ICAM-1-deficient mice31. In contrast, an antibody to CD18 not only improved the survival rate of LPSchallenged rabbits but also reduced the elevation of plasma TNF-a levels and attenuated the magnitude of LPS-induced leukocytopenia30. Once adherent to the endothelium, neutrophils transmigrate through the endothelial layer and release various proteases and reac-
tive oxygen species (ROSs). Neutrophil-elastase is a serine protease that degrades a variety of humoral and structural proteins, including transport proteins, cell receptors, membrane proteins, fibronectin, elastin and collagens. Neutrophil-elastase also degrades clotting and fibrinolysis factors, thereby contributing to the development of DIC (see below). The enhanced generation of ROSs is also responsible for tissue injury in endotoxemia. O22 is the most prominent precursor of all ROSs. The production of O22 by neutrophils and macrophages in endotoxemia is mainly attributed to membrane-associated NADPH oxidase. Most of the O22 is rapidly converted to H2O2, which can be a source of •OH and hypochlorous acid (HOCl). Recent observations have demonstrated that oxygen radicals, such as ROSs, NO and peroxynitrite, activate transcription factors, such as NFkB and activator protein 1 (AP-1)32,33, which promote gene expression of proinflammatory mediators. Thus, the positive feedback by these radicals will play an important role in amplifying the excessive inflammatory responses, suggesting that antioxidant therapy has potential therapeutic application for the modulation of proinflammatory reactions in endotoxemia. 127
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reported to stimulate the release of proinflammatory cytokines by monocytes and endothelial cells34. This finding indicates CD14 – A 55-kDa plasma membrane glycoprotein. CD14 is anchored in the cell membrane that the activation of coagulation might also of monocytes/macrophages and neutrophils as a glycerophosphatidylinositol (GPI)-tailed contribute to the upregulation of proinflamprotein. Soluble CD14 (sCD14) acts as a co-factor of lipopolysaccharide (LPS), contributing matory responses. to the LPS-induced activation of CD142 cells. The tissue factor (TF)/factor VIIamediated pathway (extrinsic pathway), rather C–C and C–x–C chemokines – A chemoattractant chemokine is characterized by specific than the contact-system-mediated pathway structural features, especially a conserved pattern of cysteine amino acids. C–C chemokines (intrinsic pathway), is relevant in the inihave two adjacent cysteines near their N-termini; C–x–C chemokines have an intervening tiation of the LPS-induced procoagulant state amino acid between these two cysteines. (Fig. 4). LPS-induced activation of coagub2 integrins – Leukocyte membrane-bound heterodimeric glycoproteins that contain b2 sublation was completely blocked by the adminunits. Three members of the b2 integrins have been identified: CD11a/CD18 (LFA-1, aMb2), istration of monoclonal antibody against TF CD11b/CD18 (Mac-1, aLb2) and CD11c/CD18 (p150, p95, axb2). CD11a/CD18 and or factor VIIa in chimpanzees35. UpreguCD11b/CD18 account for the interaction of neutrophils and monocytes/macrophages with lation of surface expression of TF on endointercellular adhesion molecule 1 (ICAM-1) on the surface of endothelial cells. CD11b/CD18 thelial cells is the initial step that leads to exand CD11c/CD18 are candidates for the lipopolysaccharide (LPS) receptor. cessive activation of the coagulation system. Although TNF-a has been considered as a Intercellular adhesion molecule 1 (ICAM-1) – A 90–95-kDa adhesion molecule that intermain contributor to the expression of TF, acts with the integrin receptors CD11a/CD18, CD11b/CD18 and CD43 on leukocytes. ICAM-1 anti-TNF antibody failed to attenuate procoon endothelial cells is closely involved in the firm contact of leukocytes with endothelial cells agulant responses in experimental and cliniand with leukocyte migration through vessel walls. cal studies36,37. In contrast, treatment with an Lipopolysaccharide (LPS) – A constituent of the outer wall of most Gram-negative bacteria. anti-IL-6 antibody inhibited activation of coLPS is structurally and functionally composed of four distinct regions: the polymer of agulation in LPS-challenged chimpanzees36. oligosaccharides (O antigen), the outer core, the inner core (KDO) and a lipid component Supporting this observation, activation of (lipid A). Lipid A is responsible for the majority of the biological actions of LPS. the coagulation system occurred in patients with metastatic renal cell cancer who reLipopolysaccharide-binding protein (LBP) – A 65-kDa glycoprotein that is synthesized ceived recombinant human IL-6 therapy38. predominantly in the liver. In response to inflammatory stimuli, LBP behaves as an However, at present the mechanism by acute-phase reactant, transferring lipopolysaccharide (LPS) from high-density lipoprotein which IL-6 activates the coagulation system (HDL) to CD14. By binding to LBP, LPS can activate cells at low concentrations (ng ml21 remains unclear. concentrations). The anticoagulant system and the fibrinoMultiple organ failure/multiple organ dysfunction syndrome (MOF/MODS) – A syndrome lytic system are also impaired during endoconsisting of the sequential dysfunction of two or more organ systems. MOF/MODS occurs toxemia. Decreased plasma levels or diminin ~30% of patients with sepsis. ished activity of antithrombin III (AT-III) and protein C are observed during endotoxemia36. Nitric oxide synthases (NOSs) – Enzymes that catalyze conversion of the guanosine nitroProtein C is activated by thrombomodulin gen moiety of L-arginine to citrulline. In this process, nitric oxide (NO) is generated. Three (TM), and the anticoagulant activity of proisoforms of NOS have been identified: endothelial NOS (eNOS), neuronal NOS (nNOS) and 21 tein C is dependent on the free level of its coinducible NOS (iNOS). eNOS and nNOS are constitutively expressed and regulated by Ca factor, protein S. TNF-a, neutrophil-elastase and calmodulin; whereas iNOS is Ca21-independent and induced by inflammatory stimuli in a and other proinflammatory mediators induce variety of cells, contributing to the production of large amounts of NO. downregulation of TM. Protein S binds to Systemic inflammatory response syndrome (SIRS) – A clinical inflammatory response C4b-binding protein (C4bBP) with high that is manifested by two or more of the following: temperature .38°C or ,36°C (rectal), affinity. During endotoxemia, the increased heart rate .90 beats min21, respiratory rate .20 breaths min21 or arterial partial pressure of level of C4bBP results in reduced levels of carbon dioxide (PaCO2) ,4.3 kPa (kilo pascal), white blood cell count .12 000 cells mm23 free protein S. Conversely, increased levels or ,4000 cells mm23 or 10% immature (band) forms. of tissue factor pathway inhibitor (TFPI) have been reported in sepsis39, which might Tissue factor (TF) – An integral membrane protein that triggers the extrinsic coagulation be a self-regulatory response to activation of cascade. Factor VII in the blood binds to TF and is converted to factor VIIa, resulting in the coagulation systems. Interestingly, in rabbits activation of the clotting cascade. with Gram-negative bacteremia, delayed treatment with TFPI, even 4 h after Escherichia coli challenge, reduced mortality40. Coagulation disorder (DIC) Suppression of the fibrinolytic system directly contributes to the Coagulation disorder is frequently associated with endotoxemia. persistence of intravascular clots. Decreased plasma levels of plasDisturbances of hemostatic balance lead to microvascular thrombosis minogen are observed in sepsis. Increased production of plasminoand bleeding, which will contribute to the development of gen activator inhibitor type 1 (PAI-1) and type 2 (PAI-2) might be MOF/MODS. Furthermore, thrombin and factor Xa have been involved in fibrinolytic suppression.
Glossary
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a
Prekallikrein
b Anti-coagulation system
Kinins
HMW kininogen
Kallikrein Protein C
XII
XIIa XI
LPS
TFPI
XIa
+?
– TM
AT-III
Fibrinolytic system Plasminogen
–
+ PAI-2
+ PAI-1
Urokinase
TPA Plasmin
–
–
V
Macrophage Endothelial cell
Prothrombin
Fibrinogen
Xa X
TNF-α IL-6
+
e
Thrombin
Extrinsic pathway
+ TF
Activated protein C
– Protein S
Intrinsic pathway
d VII
c
+
Clot formation
Fibrin
VIIa + Activated during endotoxemia – Suppressed during endotoxemia
Up-regulation Down-regulation
Figure 4. Disturbances of the hemostatic balance during endotoxemia. (a) Activation of the coagulation system, concomitant with suppression of (b) the anticoagulation system and (c) the fibrinolytic system leads to disseminated intravascular clot formation. The extrinsic pathway, rather than the intrinsic pathway, is relevant in the initial activation of lipopolysaccharide (LPS)-induced coagulation. (d) The upregulation of tissue factor (TF) on the surface of endothelial cells is mediated by various LPS-induced cytokines. (e) Thrombin generated in the procoagulant state stimulates the production of proinflammatory mediators, such as tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6) in macrophages and endothelial cells, resulting in the further development of inflammatory responses to endotoxemia. AT-III, antithrombin III; HMW, heavy molecular weight; PAI, plasminogen activator inhibitor; TFPI, tissue factor pathway inhibitor; TM, thrombomodulin; tPA, tissuetype plasminogen activator; uPA, urokinase-type plasminogen activator.
Anti-inflammatory responses in endotoxemia In response to the proinflammatory responses in endotoxemia, host cells produce several anti-inflammatory mediators, such as IL-4, IL-10, IL-13, transforming growth factor b (TGF-b), glucocorticoids and PGE2. These mediators suppress the synthesis and action of proinflammatory mediators. In addition, IL-1Ra and soluble receptors to proinflammatory cytokines, such as IL-1 receptor type II (sIL-1RII), soluble TNF receptor p75 (sTNFRp75) and soluble TNF receptor p55 (sTNFRp55), have anti-inflammatory effects by antagonizing or neutralizing the actions of each cytokine. IL-10, which is produced by T-helper 2 (Th2) cells, B cells and monocytes/macrophages, inhibits the production of TNF-a, IL-1b and IL-12 by activated macrophages. As a result, the function of Th1 CD41 T cells, which require stimulation by macrophage-derived proinflammatory cytokines, is also suppressed. IL-10-deficient mice41 or mice treated with a monoclonal antibody against IL-10 (Ref. 42) showed higher plasma levels of proinflammatory cytokines and increased mortality. In contrast, administration of recombinant IL-10 to LPS-challenged mice showed protective effects against proinflammatory cytokine production and lethality43. IL-1Ra inhibits the activity of IL-1 by competitively binding to IL-1 receptors, whereas IL-1Ra itself does not in-
duce any intracellular responses44. Three isoforms of IL-1Ra (16, 17 and 18 kDa) have been identified. Intraperitoneal injection of LPS resulted in higher lethality in IL-1Ra-deficient mice than in wild-type mice. Administration studies with IL-1Ra have revealed that large amounts of IL-1Ra are required to block the effects of IL-1b, perhaps because a sustained high level of IL-1Ra is required to occupy IL-1 receptors. Although sIL-1RII inhibits the activity of IL-1b in a dose-dependent manner, the relevance of sTNFRp75 and sTNFRp55 to TNF-a activity is a little more complicated. High molar ratios of receptor to ligand are necessary for both soluble TNF receptors to inhibit the activity of TNF-a. At low molar ratios, both soluble receptors act as carriers of TNF-a and enhance the activity of this cytokine45. The interactions between pro- and anti-inflammatory mediators play a crucial role in the control of adequate levels of immune activity against infection. If the balance cannot be maintained, the patient will suffer from massive proinflammatory responses or refractory immunosuppression. For example, administration of IL-4 or IL-10 increases lethality after Candida infection in mice46. More detailed information on the mechanism of the modulation between pro- and anti-inflammatory mediators will contribute to the development 129
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of novel therapeutic approaches that are designed to restore homeostasis in the immune system of patients with sepsis.
Therapeutic strategies for the treatment of sepsis Based on the molecular pathogenesis of septic shock and organ failure, clinical trials to block the effects of LPS and cytokines have been attempted. Clinical studies of two antibodies to lipid A, HA-1A and E5, demonstrated inconsistent results. Despite promising results reported by earlier studies, subsequent trials using larger numbers of patients with Gram-negative bacteremia did not support these findings47,48. Other anti-LPS strategies involve anti-CD14 antibodies, LPSbinding proteins (e.g. BPI and CAP18), and hemoperfusion with polymyxin B. Anti-cytokine strategies include the development of anti-cytokine antibodies (e.g. anti-TNF-a antibody), soluble cytokine receptors with IgG chimeric protein (e.g. sTNFRp75–IgG, sTNFRp55–IgG) and a cytokine receptor antagonist (IL-1Ra). Although some initial anti-cytokine trials suggested some benefit, more recent studies have failed to improve the overall outcome of patients with sepsis. A
Phase II trial with sTNFRp75–IgG could not improve survival rate in patients with sepsis, and higher doses of this fusion protein resulted in increased mortality49. To date, Phase III anti-cytokine clinical trials have been conducted on anti-TNF-a antibody50,51, sTNFRp55–IgG (Ref. 52) and IL-1Ra (Ref. 53). None of these trials showed a statistically significant reduction of 28-day mortality, although a significant improvement in the 3-day mortality and an increase in the 28-day survival of a subgroup of severely septic patients was observed (Table 2). Thus, so far, clinical anti-LPS or anti-cytokine trials to block the sepsis cascade have resulted in limited success. This might, in part, be the result of delays between the onset of sepsis and the beginning of treatment. The trials that target early mediators, such as LPS and TNF-a, might not be relevant because the cascade of inflammatory mediators is already under way when intervention is started. New approaches targeting the mediators of the later stage of sepsis might be more effective. Moreover, it will also be necessary to determine the optimal doses and duration of administration of these agents. New assays that can detect serum cytokine levels of a patient within a few hours are strongly desirable.
Table 2. Clinical trials with immunomodulating agents in patients with sepsis (1996–1998)a Agent
Anti-TNF-a mAb
Anti-TNF-a mAb
sTNFRp75–IgG
Phase
III (INTERSEPT)
Year
1996
III (NORASEPT II) 1998
II
1996
Number of patients
553
1879
141
Results
Ref.
28–30-day all-cause mortality
Other findings
No significant improvement (14.5%
More rapid reversal of shock; significant delay
reduction in the lower-dose group)
in the time of the onset of organ failure
No significant improvement (5.8%
Significant decrease in the frequency of
reduction)
coagulopathy
No significant improvement
Increase in mortality in the subgroup that
50
51
49
received higher doses sTNFRp55–IgG
IL-1Ra
III
III
1997
1997
498
696
No significant improvement (36%
Significant improvement of 28-day survival of
52
reduction)
patients with severe sepsis
No significant improvement (9%
A better resolution of organ failures
53
Improvement in lung compliance; earlier
54
reduction) Liposomal PGE1
III
1996
25 (ARDS)
No significant improvement
removal from mechanical ventilation Ibuprofen
III
1997
455
No significant improvement
Delay in the day of the onset of organ failure;
55
significant decrease in blood lactate levels Bradykinin antagonist
II
1997
504 (SIRS)
No significant improvement
Significant improvement of 28-day survival of
57
patients with Gram-negative infections Antithrombin III
a
II
1998
42
No significant improvement (39%
A better resolution of organ failures; a lower
reduction)
incidence of new organ failures
58
Abbreviations: ARDS, acute respiratory distress syndrome; IgG, immunoglobulin G; IL-1Ra, interleukin 1 receptor antagonist; INTERSEPT, International Sepsis Trial Group; mAb, monoclonal antibody; NORASEPT, North American Sepsis Trial Group; PGE1, prostaglandin E1; SIRS, systemic inflammatory response syndrome; sTNFR, soluble tumor necrosis factor receptor; TNF-a, tumor necrosis factor a.
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References
The outstanding questions
•
How is the lipopolysaccharide (LPS)–CD14 signal transduced into the cytoplasm? Is Toll-like receptor 2 (TLR-2) essential for LPS signal transduction? Do the cells have other unidentified molecules that recognize LPS? Does selective inhibition of inducible nitric oxide synthase (iNOS) provide beneficial effects during endotoxic shock? How are the chemokines and adhesion molecules involved in leukocyte-induced tissue injury during endotoxemia? Is the modulation of these mediators beneficial in preventing the development of sepsis-associated organ dysfunction? How can we attenuate the production and the cytotoxic actions of oxygen radicals, such as reactive oxygen species, nitric oxide and peroxynitrite? How can we reduce the positive feedback effects of these oxygen radicals on the production of proinflammatory mediators? How is the expression of tissue factor regulated in endotoxemia? Does interleukin 6 (IL-6) play an essential role in the upregulation of the extrinsic coagulation pathway? How can the anti-inflammatory mediators be available in the intervention of sepsis-induced proinflammatory responses without exerting immunosuppressive effects? Does combination therapy of immunomodulating agents, including anti-cytokine substances, improve the outcome of septic patients?
• • • • • •
In addition to anti-LPS or anti-cytokine agents, several strategies to interfere with the sepsis cascade have been attempted clinically. These trials include studies with PGs (e.g. liposomal PGE1)54, antiprostaglandin therapy (e.g. ibuprofen)55, bradykinin antagonists56, PAF antagonists57 and coagulation inhibitors (e.g. protein C, TFPI and AT-III)58 (Table 2). Although an initial study with PAF antagonist improved the survival rate of patients with Gram-negative sepsis57, later studies could not show a statistically significant reduction in mortality. The trials with ibuprofen did not show a significant improvement in survival either55. By contrast, Phase II trials with bradykinin antagonist or AT-III have shown promising results56,58. The trial with bradykinin antagonist showed a significant reduction in 28-day mortality of patients with Gram-negative sepsis. In addition, the trials with liposomal PGE1, protein C and TFPI are ongoing. As mentioned above, a Phase III multicenter clinical trial using L-NMMA failed because of unacceptable cardiovascular side effects. Novel selective inhibitors of iNOS might be more beneficial therapeutic choices.
Concluding remarks Sepsis is not a single disease, but a very complex condition comprising a large variety of local and systemic inflammatory responses. Therefore, we propose that a combination of several therapies or multifunctional agents, directed to various phases of sepsis or sepsisassociated molecules, might meet with more success than recent trials with single therapies. Such combined trials in sepsis, or endotoxemia, will be the subject of intense investigation over the next decade.
1 American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (1992) Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis, Crit. Care Med. 20, 864–874 2 Wenzel, R. (1992) Anti-endotoxin monoclonal antibodies – a second look, New Engl. J. Med. 326, 1151–1153 3 Brun-Buisson, C. et al. (1995) Incidence, risk factors, and outcome of severe sepsis and septic shock in adults, J. Am. Med. Assoc. 274, 968–974 4 Bates, D. et al. (1997) Predicting bacteremia in patients with sepsis syndrome, J. Infect. Dis. 176, 1538–1551 5 Fenton, M. and Golenbock, D. (1998) LPS-binding proteins and receptors, J. Leukocyte Biol. 64, 25–32 6 Jack, R.S. et al. (1997) Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection, Nature 389, 742–745 7 Kitchens, R. and Munford, R. (1998) CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS, J. Immunol. 160, 1920–1928 8 Ingalls, R.R. and Golenbock, D.T. (1995) CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide, J. Exp. Med. 181, 1473–1479 9 Yang, R.B. et al. (1998) Toll-like receptor-2 mediates lipopolysaccharideinduced cellular signalling, Nature 395, 284–288 10 Geng, Y., Zhang, B. and Lotz, M. (1993) Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes, J. Immunol. 151, 6692–6700 11 Weinstein, S.L., June, C.H. and DeFranco, A.L. (1993) Lipopolysaccharideinduced protein tyrosine phosphorylation in human macrophages is mediated by CD14, J. Immunol. 151, 3829–3838 12 Meng, F. and Lowell, C.A. (1997) Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn, J. Exp. Med. 185, 1661–1670 13 Sweet, M.J. and Hume, D.A. (1996) Endotoxin signal transduction in macrophages, J. Leukocyte Biol. 60, 8–26 14 Woronicz, J., Gao, X., Cao, Z. and Rothe, M.G.D. (1997) IkappaB kinase-beta: NF-kappaB activation and complex formation with IkappaB kinase-alpha and NIK, Science 278, 866–869 15 Hack, C.E., Aarden, L.A. and Thijis, L.G. (1997) Role of cytokines in sepsis, Adv. Immunol. 66, 101–195 16 Szabo, C. (1995) Alterations in nitric oxide production in various forms of circulatory shock, New Horiz. 3, 2–32 17 Beckman, J.S. et al. (1996) Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly, Am. J. Physiol. 271, C1424–1437 18 Salvemini, D. et al. (1993) Nitric oxide activates cyclooxygenase enzymes, Proc. Natl. Acad. Sci. U. S. A. 90, 7240–7244 19 Habib, A. et al. (1997) Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages, J. Immunol. 158, 3845–3851 20 Cobb, J.P. et al. (1992) NG-Amino-L-arginine, an inhibitor of nitric oxide synthase, raises vascular resistance but increases mortality rates in awake canines challenged with endotoxin, J. Exp. Med. 176, 1175–1182 21 MacMicking, J.D. (1995) Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthetase, Cell 81, 641–650 22 Szabo, C. et al. (1994) Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase, Proc. Natl. Acad. Sci. U. S. A. 91, 12472–12476 23 Laubach, V.E. (1995) Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death, Proc. Natl. Acad. Sci. U. S. A. 92, 10688–10692 24 Nathan, C. (1997) Inducible nitric oxide synthase: what difference does it make? J. Clin. Invest. 100, 2417–2423
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Late-breaking news Since Yang et al. reported that toll-like receptor (TLR) 2 participates in LPS-induced cellular activation (see Ref. 9 above), TLRs have been investigated as signal transducers for LPS. Kischning et al.1 report that overexpression of TLR2, but not TLR1, TLR4 or CD14, confers responsiveness to LPS in cells from a human embryonic cell line, as measured through the activation of nuclear factor kB. However, Poltorak et al.2 have found that a mutation in the murine Tlr4 gene renders mice resistant to endotoxin yet highly susceptible to Gramnegative infection. This discrepancy in the function of TLR4 is likely to be resolved in the future. 1 Kirschning, C.J. et al. (1998) Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide, J. Exp. Med. 188, 2091–2097 2 Poltorak, A. et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene, Science 282, 2085–2088