- Email: [email protected]
Mechanisms of inflammatory response syndrome in sepsis
Vol. 1, No. 3 2004
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Immunological diseases MECHANISMS
Mechanisms of inflammatory response syndrome in sepsis Laszlo M. Hoesel, Peter A. Ward* Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602, USA
The systemic inflammatory response syndrome (SIRS) and sepsis are accompanied by a complex, unbalanced and often fatal activation of the immune and inflammatory systems. Although the mechanisms are not yet completely explored, a variety of infectious and noninfectious conditions can lead to hyperactive inflammatory responses at the onset of sepsis, followed by
Section Editor: Alberto Montivani – Istituto di Ricerche Farmacologiche Mario Negri, University of Milan, Italy In this review, the interplay of different elements in the pathogenesis of the systemic inflammatory response syndrome is examined. The complexity of cellular and humoral mechanisms is analyzed, with emphasis on the biphasic sequence of stimulation followed by suppression of immune reactivity. The author has made seminal contributions to the field of immunopathogenesis of sepsis.
immunoparalysis, compromised immune function and defective innate immune responses at a later stage. Introduction The systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock and multiorgan failure (MOF) are currently used to characterize the progressive stages of a very complex and therapeutically challenging disorder of the immune and inflammatory systems. Despite tremendous research efforts for over 20 years, sepsis remains the leading cause of death in intensive care units (ICUs). SIRS and sepsis occur in 750,000 patients per year in the US with a rising incidence of 1.5% per year [1]. With a mortality rate currently 30–70%, sepsis and related disorders represent a major burden to the US health care system, with costs estimated to be approximately $16.7 billion per year [2]. SIRS is a result of a systemic activation of the innate immune system regardless of cause. In contrast, sepsis, severe sepsis and septic shock are accompanied by infection and impaired organ function.
Historical aspects and definitions The sepsis syndrome, with its complexity and variability of symptoms, has resulted in difficulties in finding an accurate, *Corresponding author: (P.A. Ward) [email protected] 1740-6765/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2004.11.003
well accepted and reasonable definition. Until only 15 years ago, sepsis was defined as the systemic host response to an infection; that is, the presence of bacteria in the blood. In 1989, a simple definition of sepsis was established on the basis of clinical symptoms and a known source of infection [3]. Although many patients showed septic symptoms, however, detectable levels of bacteria in the blood were often not found. Therefore, the term ‘‘systemic inflammatory response syndrome’’ was introduced in 1992. The designation of SIRS did not require the presence of a bacterial infection [4]. Accordingly, the definitions for sepsis, severe sepsis, septic shock and MOF were established (Table 1). During the following years, however, these criteria turned out to be too non-specific to facilitate a precise and unambiguous diagnosis, stage of the disease or prognosis. Therefore, the criteria for the diagnosis of SIRS or sepsis were extended by using more clinical parameters (for a complete list of clinical parameters see [5]). In a clinical setting, physicians need a useful system to characterize patients according to the stage of their disease and their possible response to treatment as well as risks and prognosis. Therefore, the new PIRO staging system was introduced in 2002, which characterizes patients according to www.drugdiscoverytoday.com
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Table 1. Clinical definitions of sepsisa Syndrome
Clinical symptoms
SIRS
Sepsis
Two or more criteria of SIRS Documented (or suspected) infection
Severe sepsis
Sepsis associated with organ dysfunction (hypoperfusion, hypotension, lactic acidosis, oliguria or alteration of mental state)
Septic shock
Sepsis-induced hypotension (systolic blood pressure >90 mmHg or 40 mmHg below baseline) despite adequate fluid resuscitation
MODS/MOF
Altered organ function such that homeostasis needs to be maintained with intervention
Temperature >38.5 8C or <36 8C Heart rate >90 beats per minute Respiratory rate >20 breaths per minute or PaCO2 >32 mmHg White blood count >12 109/l or <4 109/l or presence of >10% immature neutrophils
Abbreviations: MODS, multiorgan dysfunction syndrome; MOF, multiorgan failure; PaCO2, arterial pressure of carbon dioxide; SIRS, systemic inflammatory response syndrome. a Taken from [4].
their predisposition (genetic factors), insult (nature and extent of infection), response (nature and magnitude of the host reaction) and organ dysfunction [5]. Although this system still requires refinement and needs to be further tested in the clinic, we encourage its further development and application in the hospital.
Current pathophysiological knowledge Systemic inflammatory response syndrome In response to a variety of infectious and noninfectious conditions, the host can react with SIRS. Among the triggers are infection, trauma, burn, ischemia and/or reperfusion, sterile inflammatory processes (e.g. pancreatitis) and extensive surgical procedures. The physiological changes that occur in patients with SIRS are summarized in Table 1. Additional biochemical indices of SIRS (e.g. serum levels of IL-6) have been studied [5] but it remains to be determined to what extent indices of the ongoing inflammatory response can be incorporated in the diagnosis of SIRS and sepsis.
Sepsis In severe cases, SIRS often progresses to sepsis. The most frequent causes of sepsis are infections because of bacterial, fungal or viral organisms, and the most common sites of infection include the lungs, abdomen and urinary tract. Interestingly, although Gram-positive bacteria were previously thought to be the primary organisms causing sepsis, there is now increasing evidence that sepsis caused by Gramnegative bacteria occurs with equal frequency [6]. Tight regulation of the immune and inflammatory systems is crucial for maintaining the balance between protective and tissue-damaging responses. SIRS and sepsis are characterized by a loss in this balance, leading to hyperactive and hypoactive immune and inflammatory responses (Fig. 1). The reason for this perturbation, however, is unknown. Early stage sepsis: the hyperdynamic response
During the onset of sepsis, the inflammatory system becomes hyperactive, involving both cellular and humoral defense 346
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mechanisms. Endothelial and epithelial cells, as well as neutrophils, macrophages and lymphocytes, produce proinflammatory mediators [e.g. tumor necrosis factor (TNF)a, interleukin (IL)-6, IL-1 and IL-8]. Simultaneously, soluble elements of the immune system become activated. The concentration of serum acute phase proteins (such as C-reactive protein) increase, and the activated cascade of the complement system leads to the production of powerful complement split products (e.g. C3a and C5a). Together, these processes cause increased production of both pro-inflammatory and anti-inflammatory cytokines and chemokines. Phagocytic cells (neutrophils and macrophages) respond to these chemoattractants by releasing granular enzymes and producing reactive oxygen species (ROS), such as H2O2, which are involved in the killing of ingested bacteria. ROS and nitric oxide (NO) are capable of causing tissue damage, increased vascular permeability and, ultimately, organ injury. To facilitate the attraction and transmigration of phagocytic cells to the site of infection, the expression of adhesion molecules on endothelial cells (e.g. ICAM-1, E-selectin) and neutrophils (e.g. CD11b/CD18) [7] increases the recruitment of leukocytes into tissues during sepsis. Furthermore, the coagulation and fibrinolytic systems become activated, which might lead to disseminated intravascular coagulopathy (DIC). In a clinical setting, the early hyperactive stage of sepsis is often referred to as the hyperdynamic phase, which is characterized by increased heart rate and cardiac output as well as increased systemic vascular resistance. Late stage sepsis: the hypodynamic phase
At a later stage of sepsis, anti-inflammatory mediators are produced, including IL-10 and IL-13, the result of which is to counteract the pro-inflammatory effects induced during the early phase. These interleukins suppress NF-kB activation, resulting in reduced gene activation and the generation of pro-inflammatory mediators [8]. The production of anti-inflammatory mediators in this setting has been referred to as the compensatory anti-inflammatory response syndrome (CARS) [9]. As sepsis progresses, neutrophils and
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Drug Discovery Today: Disease Mechanisms | Immunological diseases
Figure 1. The time-dependent inflammatory response during sepsis. The initial insult (a) activates the humoral and cellular immune systems (b) leading to a release of pro-inflammatory mediators (c) resulting in the state of a hyperactive immune system and the clinical signs of sepsis (d). Later, immune functions are compromised by release of anti-inflammatory mediators (e) leading to a hyporeactive immune system with severe sepsis or septic shock (f). PMN, polymorphonuclear neutrophil, ROS, reactive oxygen species, NO, nitric oxide.
macrophages show reduced activity for phagocytosis, bacterial killing, oxygen radical production, chemotaxis and cytokine production. The mechanisms leading to this type of immunosuppression are not yet completely understood. The shift from an inflammatory to an anti-inflammatory response, a state of anergy (loss of immune responsiveness), apoptosis-induced loss of immune cells and the immunosuppressive effects of lymphocytes undergoing apoptosis have been described and are considered to contribute to the immune suppression that occurs in patients with sepsis [10]. Taken together, various innate functions are compromised at the late stage of sepsis, leading to a hyporeactive host defense system and immunoparalysis. These changes occur during the hypodynamic phase of sepsis, which is characterized by decreased cardiac output, falling heart rate and reduced peripheral vascular resistance.
Concepts in the treatment of sepsis Despite the current aggressive management of sepsis and septic shock in ICUs, mortality remains disturbingly high. It is therefore important that more effective interventions for
the treatment of sepsis are found. Recent strategies include the neutralization of bacterial products [blockade of lipopolysaccharide (LPS)], targeting undesirable pro-inflammatory responses (blockade of TNF-a, IL-1, IL-6 and complement factors, amongst others), addition of immunostimulatory agents to restore the immune system [administration of interferon (IFN)-g, granulocyte colony-stimulating factor (G-CSF)] and correction of abnormalities in the coagulation system [administration of anti-thrombin or activated protein C (APC)].
Neutralization of bacterial products LPS, a molecule embedded in the outer membrane of Gramnegative bacteria, is a dominant activator of the innate immune system. Infusion of LPS into animals, however, does not accurately mimic the changes observed during sepsis, at least in humans. Interestingly, LPS levels in the CLP sepsis model (sepsis induced by cecal ligation and puncture) are reported to be low. Therefore, it was not surprising that administration of antibody to LPS did not improve the outcome in septic patients [11]. There is now general agreement www.drugdiscoverytoday.com
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that LPS injection might be a model for endotoxic shock but not for sepsis. Gram-positive bacteria do not have endotoxin, but peptidoglycan and lipoteichoic acid are present, both of which are pro-inflammatory agents [12]. Peptidoglycan and lipoteichoic acid can activate the innate immune system, but they are much less active than LPS and their roles in initiating sepsis remains unclear.
Targeting undesirable pro-inflammatory responses (immunosuppression) Regarding the early, hyperactive stage sepsis, elevated levels of pro-inflammatory mediators quickly became targets for clinical trials. Tumor necrosis factor a
Expression of TNF-a is increased during the very early stage of sepsis (30–90 min). Infusion of TNF-a into animals induced characteristic septic symptoms. Passive immunization to block TNF-a following LPS or Escherichia coli infusion were reported to be beneficial in animal models of septic shock [11]. Subsequent clinical trials, however, failed to demonstrate the utility of this therapy. Interleukins
Similar to TNF-a, IL-1 is a very early pro-inflammatory cytokine that simulates many of the immunopathological features of LPS-induced shock. IL-1 blockade in animal models receiving LPS therapy reduced mortality [13]. IL-6 is a cytokine that induces pro-inflammatory and anti-inflammatory effects. Blockade of IL-6 in the CLP model resulted in improved survival and reduced expression of C5aR (see below) in different organs [14]. So far, IL-6 is considered to be a biological marker of sepsis (high levels correlating with poor prognosis) rather than a potential target for blockade during sepsis therapy. Lipopolysaccharide-binding protein
LPS-binding protein (LBP) is reactive with LPS and the LPS receptor (CD14) [15]. Although LBP has the capacity to bind LPS in serum, CD14 is either membrane bound (mCD14) or present as a soluble factor in serum (sCD14). sCD14 expression is increased in septic patients [16], and the blockade of CD14 protects primates from lethal endotoxic shock [17]. Until recently, it remained unclear how binding of the LPS–LBP complex to CD14 resulted in intracellular activation. On the basis of investigations into Drosophila, a family of at least 10 TLRs was identified in humans [18]. While TLR2 is responsible for recognizing wall components of Gram-positive bacteria, TLR4 is the LPS receptor. However, the specific role of TLRs in sepsis and a possible therapeutic approach are yet to be determined. A mutant, non-functional TLR4 in mice conveys protection against the effects of endotoxemia, but in 348
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such mice there is no statistically significant protection against CLP-induced sepsis [10], questioning the role of LPS in CLP-induced sepsis. The complement system
As part of the humoral innate immune system, the complement system is a cascade of over 25 proteins. Three separate but convergent pathways of activation lead to the cleavage of C5 to form C5a and C5b, and generation of the terminal membrane attack complex, which forms pores in the membrane of bacteria, leading to their lysis. The lectin pathway, as one of the three activators of the complement cascade, becomes engaged when serum mannose-binding lectins interact with mannose-binding associated proteins and mannoselike surface structures on bacteria. The underlying mechanisms and possible implications of this pathway have been reviewed in [19]. C5a is a very potent pro-inflammatory agent. The various effects of blockade of C5a or its corresponding receptor, C5aR, have been reviewed in [1]: survival was greatly improved in the CLP model of sepsis in rats and mice, and the reduction of C5aR expression in murine blood neutrophils was reduced and preserved their pro-inflammatory capabilities. A second, recently identified C5a-binding receptor, C5L2, does not induce signaling events in cells after binding of C5a [20]; however, the role of C5L2 during sepsis remains to be determined. Therefore, the concept of blockade of C5a, C5aR and possibly C5L2, during sepsis might have therapeutic potential. The high-mobility group B1 protein
The high-mobility group B1 protein (HMGB1) is a pro-inflammatory agent produced by macrophages after LPS stimulation. It appears in plasma 8–32 h after endotoxemia and stimulates a variety of phagocytic cells (resulting in an increase in TNF-a, IL-1 and IL-6 expression). Administration of recombinant HMBG1 is lethal. Blockade of HMGB1 or antagonism with ethyl pyruvate improves survival in septic animals, making it a potentially promising candidate for treatment of sepsis [11]. Macrophage migration inhibitory factor
Macrophage migration inhibitory factor (MIF) is another proinflammatory mediator expressed during sepsis that is produced by a variety of cells (pituitary gland cells, eosinophils, renal tubular cells, lung epithelial cells and macrophages). It induces the production of various pro-inflammatory mediators in macrophages. Blockade of MIF results in improved survival after LPS challenge [21], making MIF a potentially new target for intervention in sepsis.
Addition of immunostimulatory agents (immunostimulation) Researchers have also tried to counteract the state of immunosuppression that occurs during late stage sepsis. To restore the pro-inflammatory functions of phagocytic cells (bacterial
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Drug Discovery Today: Disease Mechanisms | Immunological diseases
actions with the immune system are not yet completely understood. Although anti-thrombin, a potent anti-thrombotic and anti-inflammatory mediator, failed to improve survival, activated protein C (APC) was the first drug to be approved for treatment of patients with severe sepsis and at high risk of death.
killing and granular enzyme release), IFN-g, G-CSF and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested as possible treatments in sepsis. IFN-g, G-CSF and GM-CSF promote generation, maturation and mobilization of monocytes, neutrophils and macrophages, and result in improved survival rates in experimental models of sepsis in rodents [22,23]. However, the clinical usefulness of these interventions remains to be elucidated. Programmed cell death (apoptosis) is a naturally occurring process inducible by a variety of mediators (e.g. glucocorticoids and TNF-a). Intracellular apoptosis mediators include a family of (at least) 11 caspases, which are pro-enzymes waiting to be activated to induce cell death. During sepsis, there is evidence that lymphocytes and lymphatic tissues undergo rapid apoptosis, whereas neutrophils are reported to show delayed apoptosis [24]. Studies featuring the blockade of apoptosis (by overexpression of anti-apoptotic proteins, or use of caspase inhibitors) reported improved survival in septic animals [25,26]. Inhibition of apoptosis might become a new target for the treatment of sepsis, but results from clinical trials have yet to be published.
Other strategies for anti-inflammatory interventions of sepsis have included inhibition or antagonism of platelet-activating factor (PAF), arachidonic acid metabolites, adhesion molecules, ROS, NO, bradykinin, phosphodiesterase and C1 esterase, as reviewed elsewhere [27–29]. These trials were based on evidence that these mediators (including complement activation products) were produced during sepsis. However, new strategies failed to provide convincing evidence of efficacy. Based on the assumption that hyperglycemia was undesirable during sepsis, a clinical trial using intensive insulin therapy in an ICU environment resulted in improved survival of septic patients [30]. The underlying mechanism for this surprising effect of insulin remains unclear.
Correction of abnormalities in the coagulation system
Summary and conclusions
As described above, the coagulation system is activated during sepsis; however, the underlying mechanisms and inter-
The innate immune system responds to a variety of initiating events with a physiological defense mechanism termed SIRS.
Additional strategies
Table 2. Targets and related therapies during human sepsis Target Pathogenic components
Therapy
Stage of development
Advantages/disadvantages
Refs/Co
Antibodies, hemofiltration
No benefit
Low levels in septic patients
[12]
Glucocorticoids
Phase III
Low-dose with benefit
[12]
TNF-a
Antibodies, immunization
No benefit
Beneficial in animals only
[12]
IL-1, IL-6
Antibodies
No benefit
Beneficial in animals only
[12]
LBP/CD14
Anti-CD14
Animals
Protection from LPS shock
[17]
TLR (TLR4)
Possibly blockade
Animals
Role in sepsis not yet defined
Complement
C5a/C5aR-blockade
Animals
Survival *
[12]
MBL/MASP blockade
Animals
Survival *
[12]
HMBG1
Blockade, ethyl pyruvate
Animals
Survival *
[12]
MIF
Blockade
Animals
Survival *
[12]
Coagulation
AT application
No benefit
APC (drotrecogin)
Phase III
Beneficial in severe sepsis
[12], XigrisTM, Eli Lilly and Co, Indianapolis, IN
IFNg, G-CSF, GM-CSF
Application for immunostimulation
No benefit
Survival * in animals only
[29]
Apoptosis
Caspase inhibitors
Animals
Survival *
[11]
Mechanism unknown
Intensive insulin therapy
Phase III
Beneficial in sepsis
[30]
LPS (endotoxin)
Pro-inflammatory Hyper-activated responses immune system
Immunoparalysis
Other
[12]
Targets are grouped according to common mechanism. Abbreviations: APC, activated protein C; AT, anti-thrombin; C5aR, C5a receptor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HMGB1, high-mobility group B1 protein; IFN-g, interferon-g; IL, interleukin; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; MASP, mannose-binding associate proteinase; MBL, mannose-binding lectin; MIF, macrophage migration inhibitory factor; TLR, Toll-like receptor; TNF-a, tumor necrosis factor-a. www.drugdiscoverytoday.com
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If the tight regulation of the immune system during SIRS is compromised, sepsis and septic shock can develop. Patients with sepsis undergo an early hyperactive phase characterized by increased production of pro-inflammatory mediators (e.g. IL-1 and TNF-a) followed by a state of hypoactive immune status (immunoparalysis). In this later stage, secondary infections, MOF and death often occur. In addition to standard therapeutic interventions in the ICU, the blockade of proinflammatory mediators resulted in promising effects in terms of survival in animal models of sepsis. However, only three clinical trials were reported to be beneficial (according to a reduction in mortality rates) for patients with sepsis: low-dose corticoids (to suppress the hyperactive immune response), the anti-coagulant APC (to counteract the activated coagulation cascade), and insulin (to maintain a physiological blood glucose level) (see Table 2). According to the two-phase characteristics of sepsis, early application of anti-inflammatory agents and late administration of immuno-restoring substances can be beneficial. Measuring levels of circulating pro- and anti-inflammatory mediators might help to evaluate the stage of sepsis and thereby help the physician to choose the appropriate therapy. Recently investigated substances such as HMGB1, MIF and apoptosis inhibitors might be useful targets for the treatment of sepsis, but further investigation is needed. In conclusion, our knowledge of sepsis and its underlying mechanisms has grown considerably, but this has not resulted in effective therapeutic strategies for septic patients. New promising targets are currently under investigation and might facilitate the development of more powerful and effective treatments of sepsis and septic shock.
Acknowledgement Supported by the National Institute of Health (NIH) Grant GM-61656. Outstanding issues In the light of very few successful clinical trials in sepsis so far, new promising therapeutic targets are currently under investigation. According to the two-phase characteristics of sepsis, early application of antiinflammatory agents and late administration of immuno-restoring substances can be beneficial. Measuring levels of circulating pro- and antiinflammatory mediators might help to evaluate the stage of sepsis and thereby help to choose the appropriate therapy. Recently investigated substances such as HMGB1, MIF and apoptosis inhibitors, might be useful agents for the treatment of sepsis, but further investigation is needed.
References 1 Ward, P.A. (2004) The dark side of C5a in sepsis. Nat. Rev. Immunol. 4, 133–142 2 Angus, D.C. et al. (2001) Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29, 1303–1310
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3 Bone, R.C. et al. (1989) Sepsis syndrome: a valid clinical entity. Methylprednisolone Severe Sepsis Study Group. Crit. Care Med. 17, 389–393 4 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 5 Levy, M.M. et al. (2003) 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive Care Med. 29, 530–538 6 Abraham, E. et al. (1995) Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha MAb Sepsis Study Group. J. Am. Med. Assoc. 273, 934–941 7 Adams, D.H. and Shaw, S. (1994) Leucocyte–endothelial interactions and regulation of leucocyte migration. Lancet 343, 831–836 8 Riedemann, N.C. et al. (2004) Regulatory role of C5a in LPS-induced IL-6 production by neutrophils during sepsis. FASEB J. 18, 370–372 9 Bone, R.C. (1996) Sir Isaac Newton, sepsis, SIRS, and CARS. Crit. Care Med. 24, 1125–1128 10 Hotchkiss, R.S. and Karl, I.E. (2003) The pathophysiology and treatment of sepsis. N. Engl. J. Med. 348, 138–150 11 Riedemann, N.C. et al. (2003) Novel strategies for the treatment of sepsis. Nat. Med. 9, 517–524 12 Wang, J.E. et al. (2000) Peptidoglycan and lipoteichoic acid from Staphylococcus aureus induce tumor necrosis factor alpha, interleukin 6 (IL6), and IL-10 production in both T cells and monocytes in a human whole blood model. Infect. Immun. 68, 3965–3970 13 Ohlsson, K. et al. (1990) Interleukin-1 receptor antagonist reduces mortality from endotoxin shock. Nature 348, 550–552 14 Riedemann, N.C. et al. (2003) Protective effects of IL-6 blockade in sepsis are linked to reduced C5a receptor expression. J. Immunol. 170, 503–507 15 Wright, S.D. et al. (1990) CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249, 1431–1433 16 Landmann, R. et al. (1995) Increased circulating soluble CD14 is associated with high mortality in Gram-negative septic shock. J. Infect. Dis. 171, 639–644 17 Leturcq, D.J. et al. (1996) Antibodies against CD14 protect primates from endotoxin-induced shock. J. Clin. Invest. 98, 1533–1538 18 Aderem, A. and Ulevitch, R.J. (2000) Toll-like receptors in the induction of the innate immune response. Nature 406, 782–787 19 Petersen, S.V. et al. (2001) The mannan-binding lectin pathway of complement activation: biology and disease association. Mol. Immunol. 38, 133–149 20 Cain, S.A. and Monk, P.N. (2002) The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74). J. Biol. Chem. 277, 7165–7169 21 Calandra, T. et al. (2000) Protection from septic shock by neutralization of macrophage migration inhibitory factor. Nat. Med. 6, 164–170 22 Gorgen, I. et al. (1992) Granulocyte colony-stimulating factor treatment protects rodents against lipopolysaccharide-induced toxicity via suppression of systemic tumor necrosis factor-alpha. J. Immunol. 149, 918–924 23 O’Reilly, M. et al. (1992) Treatment of intra-abdominal infection with granulocyte colony-stimulating factor. J. Trauma 33, 679–682 24 Oberholzer, C. et al. (2001) Apoptosis in sepsis: a new target for therapeutic exploration. FASEB J. 15, 879–892 25 Hotchkiss, R.S. et al. (1999) Prevention of lymphocyte cell death in sepsis improves survival in mice. Proc. Natl. Acad. Sci. USA 96, 14541–14546 26 Hotchkiss, R.S. et al. (1999) Overexpression of Bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis. J. Immunol. 162, 4148–4156 27 Healy, D.P. (2002) New and emerging therapies for sepsis. Ann. Pharmacother. 36, 648–654 28 van der Poll, T. (2001) Immunotherapy of sepsis. Lancet Infect. Dis. 1, 165–174 29 Vincent, J.L. et al. (2002) Clinical trials of immunomodulatory therapies in severe sepsis and septic shock. Clin. Infect. Dis. 34, 1084–1093 30 van den Berghe, G. et al. (2001) Intensive insulin therapy in the critically ill patients. N. Engl. J. Med. 345, 1359–1367
Drug Discovery Today: Disease Mechanisms
DRUG DISCOVERY
TODAY
Editors-in-Chief Toren Finkel – National Heart, Lung and Blood Institute, National Institutes of Health, USA Tamas Bartfai – Harold L. Dorris Neurological Research Center and The Scripps Research Institute, USA
DISEASE Immunological diseases MECHANISMS
Mechanisms of inflammatory response syndrome in sepsis Laszlo M. Hoesel, Peter A. Ward* Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602, USA
The systemic inflammatory response syndrome (SIRS) and sepsis are accompanied by a complex, unbalanced and often fatal activation of the immune and inflammatory systems. Although the mechanisms are not yet completely explored, a variety of infectious and noninfectious conditions can lead to hyperactive inflammatory responses at the onset of sepsis, followed by
Section Editor: Alberto Montivani – Istituto di Ricerche Farmacologiche Mario Negri, University of Milan, Italy In this review, the interplay of different elements in the pathogenesis of the systemic inflammatory response syndrome is examined. The complexity of cellular and humoral mechanisms is analyzed, with emphasis on the biphasic sequence of stimulation followed by suppression of immune reactivity. The author has made seminal contributions to the field of immunopathogenesis of sepsis.
immunoparalysis, compromised immune function and defective innate immune responses at a later stage. Introduction The systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock and multiorgan failure (MOF) are currently used to characterize the progressive stages of a very complex and therapeutically challenging disorder of the immune and inflammatory systems. Despite tremendous research efforts for over 20 years, sepsis remains the leading cause of death in intensive care units (ICUs). SIRS and sepsis occur in 750,000 patients per year in the US with a rising incidence of 1.5% per year [1]. With a mortality rate currently 30–70%, sepsis and related disorders represent a major burden to the US health care system, with costs estimated to be approximately $16.7 billion per year [2]. SIRS is a result of a systemic activation of the innate immune system regardless of cause. In contrast, sepsis, severe sepsis and septic shock are accompanied by infection and impaired organ function.
Historical aspects and definitions The sepsis syndrome, with its complexity and variability of symptoms, has resulted in difficulties in finding an accurate, *Corresponding author: (P.A. Ward) [email protected] 1740-6765/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddmec.2004.11.003
well accepted and reasonable definition. Until only 15 years ago, sepsis was defined as the systemic host response to an infection; that is, the presence of bacteria in the blood. In 1989, a simple definition of sepsis was established on the basis of clinical symptoms and a known source of infection [3]. Although many patients showed septic symptoms, however, detectable levels of bacteria in the blood were often not found. Therefore, the term ‘‘systemic inflammatory response syndrome’’ was introduced in 1992. The designation of SIRS did not require the presence of a bacterial infection [4]. Accordingly, the definitions for sepsis, severe sepsis, septic shock and MOF were established (Table 1). During the following years, however, these criteria turned out to be too non-specific to facilitate a precise and unambiguous diagnosis, stage of the disease or prognosis. Therefore, the criteria for the diagnosis of SIRS or sepsis were extended by using more clinical parameters (for a complete list of clinical parameters see [5]). In a clinical setting, physicians need a useful system to characterize patients according to the stage of their disease and their possible response to treatment as well as risks and prognosis. Therefore, the new PIRO staging system was introduced in 2002, which characterizes patients according to www.drugdiscoverytoday.com
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Table 1. Clinical definitions of sepsisa Syndrome
Clinical symptoms
SIRS
Sepsis
Two or more criteria of SIRS Documented (or suspected) infection
Severe sepsis
Sepsis associated with organ dysfunction (hypoperfusion, hypotension, lactic acidosis, oliguria or alteration of mental state)
Septic shock
Sepsis-induced hypotension (systolic blood pressure >90 mmHg or 40 mmHg below baseline) despite adequate fluid resuscitation
MODS/MOF
Altered organ function such that homeostasis needs to be maintained with intervention
Temperature >38.5 8C or <36 8C Heart rate >90 beats per minute Respiratory rate >20 breaths per minute or PaCO2 >32 mmHg White blood count >12 109/l or <4 109/l or presence of >10% immature neutrophils
Abbreviations: MODS, multiorgan dysfunction syndrome; MOF, multiorgan failure; PaCO2, arterial pressure of carbon dioxide; SIRS, systemic inflammatory response syndrome. a Taken from [4].
their predisposition (genetic factors), insult (nature and extent of infection), response (nature and magnitude of the host reaction) and organ dysfunction [5]. Although this system still requires refinement and needs to be further tested in the clinic, we encourage its further development and application in the hospital.
Current pathophysiological knowledge Systemic inflammatory response syndrome In response to a variety of infectious and noninfectious conditions, the host can react with SIRS. Among the triggers are infection, trauma, burn, ischemia and/or reperfusion, sterile inflammatory processes (e.g. pancreatitis) and extensive surgical procedures. The physiological changes that occur in patients with SIRS are summarized in Table 1. Additional biochemical indices of SIRS (e.g. serum levels of IL-6) have been studied [5] but it remains to be determined to what extent indices of the ongoing inflammatory response can be incorporated in the diagnosis of SIRS and sepsis.
Sepsis In severe cases, SIRS often progresses to sepsis. The most frequent causes of sepsis are infections because of bacterial, fungal or viral organisms, and the most common sites of infection include the lungs, abdomen and urinary tract. Interestingly, although Gram-positive bacteria were previously thought to be the primary organisms causing sepsis, there is now increasing evidence that sepsis caused by Gramnegative bacteria occurs with equal frequency [6]. Tight regulation of the immune and inflammatory systems is crucial for maintaining the balance between protective and tissue-damaging responses. SIRS and sepsis are characterized by a loss in this balance, leading to hyperactive and hypoactive immune and inflammatory responses (Fig. 1). The reason for this perturbation, however, is unknown. Early stage sepsis: the hyperdynamic response
During the onset of sepsis, the inflammatory system becomes hyperactive, involving both cellular and humoral defense 346
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mechanisms. Endothelial and epithelial cells, as well as neutrophils, macrophages and lymphocytes, produce proinflammatory mediators [e.g. tumor necrosis factor (TNF)a, interleukin (IL)-6, IL-1 and IL-8]. Simultaneously, soluble elements of the immune system become activated. The concentration of serum acute phase proteins (such as C-reactive protein) increase, and the activated cascade of the complement system leads to the production of powerful complement split products (e.g. C3a and C5a). Together, these processes cause increased production of both pro-inflammatory and anti-inflammatory cytokines and chemokines. Phagocytic cells (neutrophils and macrophages) respond to these chemoattractants by releasing granular enzymes and producing reactive oxygen species (ROS), such as H2O2, which are involved in the killing of ingested bacteria. ROS and nitric oxide (NO) are capable of causing tissue damage, increased vascular permeability and, ultimately, organ injury. To facilitate the attraction and transmigration of phagocytic cells to the site of infection, the expression of adhesion molecules on endothelial cells (e.g. ICAM-1, E-selectin) and neutrophils (e.g. CD11b/CD18) [7] increases the recruitment of leukocytes into tissues during sepsis. Furthermore, the coagulation and fibrinolytic systems become activated, which might lead to disseminated intravascular coagulopathy (DIC). In a clinical setting, the early hyperactive stage of sepsis is often referred to as the hyperdynamic phase, which is characterized by increased heart rate and cardiac output as well as increased systemic vascular resistance. Late stage sepsis: the hypodynamic phase
At a later stage of sepsis, anti-inflammatory mediators are produced, including IL-10 and IL-13, the result of which is to counteract the pro-inflammatory effects induced during the early phase. These interleukins suppress NF-kB activation, resulting in reduced gene activation and the generation of pro-inflammatory mediators [8]. The production of anti-inflammatory mediators in this setting has been referred to as the compensatory anti-inflammatory response syndrome (CARS) [9]. As sepsis progresses, neutrophils and
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Figure 1. The time-dependent inflammatory response during sepsis. The initial insult (a) activates the humoral and cellular immune systems (b) leading to a release of pro-inflammatory mediators (c) resulting in the state of a hyperactive immune system and the clinical signs of sepsis (d). Later, immune functions are compromised by release of anti-inflammatory mediators (e) leading to a hyporeactive immune system with severe sepsis or septic shock (f). PMN, polymorphonuclear neutrophil, ROS, reactive oxygen species, NO, nitric oxide.
macrophages show reduced activity for phagocytosis, bacterial killing, oxygen radical production, chemotaxis and cytokine production. The mechanisms leading to this type of immunosuppression are not yet completely understood. The shift from an inflammatory to an anti-inflammatory response, a state of anergy (loss of immune responsiveness), apoptosis-induced loss of immune cells and the immunosuppressive effects of lymphocytes undergoing apoptosis have been described and are considered to contribute to the immune suppression that occurs in patients with sepsis [10]. Taken together, various innate functions are compromised at the late stage of sepsis, leading to a hyporeactive host defense system and immunoparalysis. These changes occur during the hypodynamic phase of sepsis, which is characterized by decreased cardiac output, falling heart rate and reduced peripheral vascular resistance.
Concepts in the treatment of sepsis Despite the current aggressive management of sepsis and septic shock in ICUs, mortality remains disturbingly high. It is therefore important that more effective interventions for
the treatment of sepsis are found. Recent strategies include the neutralization of bacterial products [blockade of lipopolysaccharide (LPS)], targeting undesirable pro-inflammatory responses (blockade of TNF-a, IL-1, IL-6 and complement factors, amongst others), addition of immunostimulatory agents to restore the immune system [administration of interferon (IFN)-g, granulocyte colony-stimulating factor (G-CSF)] and correction of abnormalities in the coagulation system [administration of anti-thrombin or activated protein C (APC)].
Neutralization of bacterial products LPS, a molecule embedded in the outer membrane of Gramnegative bacteria, is a dominant activator of the innate immune system. Infusion of LPS into animals, however, does not accurately mimic the changes observed during sepsis, at least in humans. Interestingly, LPS levels in the CLP sepsis model (sepsis induced by cecal ligation and puncture) are reported to be low. Therefore, it was not surprising that administration of antibody to LPS did not improve the outcome in septic patients [11]. There is now general agreement www.drugdiscoverytoday.com
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that LPS injection might be a model for endotoxic shock but not for sepsis. Gram-positive bacteria do not have endotoxin, but peptidoglycan and lipoteichoic acid are present, both of which are pro-inflammatory agents [12]. Peptidoglycan and lipoteichoic acid can activate the innate immune system, but they are much less active than LPS and their roles in initiating sepsis remains unclear.
Targeting undesirable pro-inflammatory responses (immunosuppression) Regarding the early, hyperactive stage sepsis, elevated levels of pro-inflammatory mediators quickly became targets for clinical trials. Tumor necrosis factor a
Expression of TNF-a is increased during the very early stage of sepsis (30–90 min). Infusion of TNF-a into animals induced characteristic septic symptoms. Passive immunization to block TNF-a following LPS or Escherichia coli infusion were reported to be beneficial in animal models of septic shock [11]. Subsequent clinical trials, however, failed to demonstrate the utility of this therapy. Interleukins
Similar to TNF-a, IL-1 is a very early pro-inflammatory cytokine that simulates many of the immunopathological features of LPS-induced shock. IL-1 blockade in animal models receiving LPS therapy reduced mortality [13]. IL-6 is a cytokine that induces pro-inflammatory and anti-inflammatory effects. Blockade of IL-6 in the CLP model resulted in improved survival and reduced expression of C5aR (see below) in different organs [14]. So far, IL-6 is considered to be a biological marker of sepsis (high levels correlating with poor prognosis) rather than a potential target for blockade during sepsis therapy. Lipopolysaccharide-binding protein
LPS-binding protein (LBP) is reactive with LPS and the LPS receptor (CD14) [15]. Although LBP has the capacity to bind LPS in serum, CD14 is either membrane bound (mCD14) or present as a soluble factor in serum (sCD14). sCD14 expression is increased in septic patients [16], and the blockade of CD14 protects primates from lethal endotoxic shock [17]. Until recently, it remained unclear how binding of the LPS–LBP complex to CD14 resulted in intracellular activation. On the basis of investigations into Drosophila, a family of at least 10 TLRs was identified in humans [18]. While TLR2 is responsible for recognizing wall components of Gram-positive bacteria, TLR4 is the LPS receptor. However, the specific role of TLRs in sepsis and a possible therapeutic approach are yet to be determined. A mutant, non-functional TLR4 in mice conveys protection against the effects of endotoxemia, but in 348
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such mice there is no statistically significant protection against CLP-induced sepsis [10], questioning the role of LPS in CLP-induced sepsis. The complement system
As part of the humoral innate immune system, the complement system is a cascade of over 25 proteins. Three separate but convergent pathways of activation lead to the cleavage of C5 to form C5a and C5b, and generation of the terminal membrane attack complex, which forms pores in the membrane of bacteria, leading to their lysis. The lectin pathway, as one of the three activators of the complement cascade, becomes engaged when serum mannose-binding lectins interact with mannose-binding associated proteins and mannoselike surface structures on bacteria. The underlying mechanisms and possible implications of this pathway have been reviewed in [19]. C5a is a very potent pro-inflammatory agent. The various effects of blockade of C5a or its corresponding receptor, C5aR, have been reviewed in [1]: survival was greatly improved in the CLP model of sepsis in rats and mice, and the reduction of C5aR expression in murine blood neutrophils was reduced and preserved their pro-inflammatory capabilities. A second, recently identified C5a-binding receptor, C5L2, does not induce signaling events in cells after binding of C5a [20]; however, the role of C5L2 during sepsis remains to be determined. Therefore, the concept of blockade of C5a, C5aR and possibly C5L2, during sepsis might have therapeutic potential. The high-mobility group B1 protein
The high-mobility group B1 protein (HMGB1) is a pro-inflammatory agent produced by macrophages after LPS stimulation. It appears in plasma 8–32 h after endotoxemia and stimulates a variety of phagocytic cells (resulting in an increase in TNF-a, IL-1 and IL-6 expression). Administration of recombinant HMBG1 is lethal. Blockade of HMGB1 or antagonism with ethyl pyruvate improves survival in septic animals, making it a potentially promising candidate for treatment of sepsis [11]. Macrophage migration inhibitory factor
Macrophage migration inhibitory factor (MIF) is another proinflammatory mediator expressed during sepsis that is produced by a variety of cells (pituitary gland cells, eosinophils, renal tubular cells, lung epithelial cells and macrophages). It induces the production of various pro-inflammatory mediators in macrophages. Blockade of MIF results in improved survival after LPS challenge [21], making MIF a potentially new target for intervention in sepsis.
Addition of immunostimulatory agents (immunostimulation) Researchers have also tried to counteract the state of immunosuppression that occurs during late stage sepsis. To restore the pro-inflammatory functions of phagocytic cells (bacterial
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actions with the immune system are not yet completely understood. Although anti-thrombin, a potent anti-thrombotic and anti-inflammatory mediator, failed to improve survival, activated protein C (APC) was the first drug to be approved for treatment of patients with severe sepsis and at high risk of death.
killing and granular enzyme release), IFN-g, G-CSF and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested as possible treatments in sepsis. IFN-g, G-CSF and GM-CSF promote generation, maturation and mobilization of monocytes, neutrophils and macrophages, and result in improved survival rates in experimental models of sepsis in rodents [22,23]. However, the clinical usefulness of these interventions remains to be elucidated. Programmed cell death (apoptosis) is a naturally occurring process inducible by a variety of mediators (e.g. glucocorticoids and TNF-a). Intracellular apoptosis mediators include a family of (at least) 11 caspases, which are pro-enzymes waiting to be activated to induce cell death. During sepsis, there is evidence that lymphocytes and lymphatic tissues undergo rapid apoptosis, whereas neutrophils are reported to show delayed apoptosis [24]. Studies featuring the blockade of apoptosis (by overexpression of anti-apoptotic proteins, or use of caspase inhibitors) reported improved survival in septic animals [25,26]. Inhibition of apoptosis might become a new target for the treatment of sepsis, but results from clinical trials have yet to be published.
Other strategies for anti-inflammatory interventions of sepsis have included inhibition or antagonism of platelet-activating factor (PAF), arachidonic acid metabolites, adhesion molecules, ROS, NO, bradykinin, phosphodiesterase and C1 esterase, as reviewed elsewhere [27–29]. These trials were based on evidence that these mediators (including complement activation products) were produced during sepsis. However, new strategies failed to provide convincing evidence of efficacy. Based on the assumption that hyperglycemia was undesirable during sepsis, a clinical trial using intensive insulin therapy in an ICU environment resulted in improved survival of septic patients [30]. The underlying mechanism for this surprising effect of insulin remains unclear.
Correction of abnormalities in the coagulation system
Summary and conclusions
As described above, the coagulation system is activated during sepsis; however, the underlying mechanisms and inter-
The innate immune system responds to a variety of initiating events with a physiological defense mechanism termed SIRS.
Additional strategies
Table 2. Targets and related therapies during human sepsis Target Pathogenic components
Therapy
Stage of development
Advantages/disadvantages
Refs/Co
Antibodies, hemofiltration
No benefit
Low levels in septic patients
[12]
Glucocorticoids
Phase III
Low-dose with benefit
[12]
TNF-a
Antibodies, immunization
No benefit
Beneficial in animals only
[12]
IL-1, IL-6
Antibodies
No benefit
Beneficial in animals only
[12]
LBP/CD14
Anti-CD14
Animals
Protection from LPS shock
[17]
TLR (TLR4)
Possibly blockade
Animals
Role in sepsis not yet defined
Complement
C5a/C5aR-blockade
Animals
Survival *
[12]
MBL/MASP blockade
Animals
Survival *
[12]
HMBG1
Blockade, ethyl pyruvate
Animals
Survival *
[12]
MIF
Blockade
Animals
Survival *
[12]
Coagulation
AT application
No benefit
APC (drotrecogin)
Phase III
Beneficial in severe sepsis
[12], XigrisTM, Eli Lilly and Co, Indianapolis, IN
IFNg, G-CSF, GM-CSF
Application for immunostimulation
No benefit
Survival * in animals only
[29]
Apoptosis
Caspase inhibitors
Animals
Survival *
[11]
Mechanism unknown
Intensive insulin therapy
Phase III
Beneficial in sepsis
[30]
LPS (endotoxin)
Pro-inflammatory Hyper-activated responses immune system
Immunoparalysis
Other
[12]
Targets are grouped according to common mechanism. Abbreviations: APC, activated protein C; AT, anti-thrombin; C5aR, C5a receptor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; HMGB1, high-mobility group B1 protein; IFN-g, interferon-g; IL, interleukin; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; MASP, mannose-binding associate proteinase; MBL, mannose-binding lectin; MIF, macrophage migration inhibitory factor; TLR, Toll-like receptor; TNF-a, tumor necrosis factor-a. www.drugdiscoverytoday.com
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If the tight regulation of the immune system during SIRS is compromised, sepsis and septic shock can develop. Patients with sepsis undergo an early hyperactive phase characterized by increased production of pro-inflammatory mediators (e.g. IL-1 and TNF-a) followed by a state of hypoactive immune status (immunoparalysis). In this later stage, secondary infections, MOF and death often occur. In addition to standard therapeutic interventions in the ICU, the blockade of proinflammatory mediators resulted in promising effects in terms of survival in animal models of sepsis. However, only three clinical trials were reported to be beneficial (according to a reduction in mortality rates) for patients with sepsis: low-dose corticoids (to suppress the hyperactive immune response), the anti-coagulant APC (to counteract the activated coagulation cascade), and insulin (to maintain a physiological blood glucose level) (see Table 2). According to the two-phase characteristics of sepsis, early application of anti-inflammatory agents and late administration of immuno-restoring substances can be beneficial. Measuring levels of circulating pro- and anti-inflammatory mediators might help to evaluate the stage of sepsis and thereby help the physician to choose the appropriate therapy. Recently investigated substances such as HMGB1, MIF and apoptosis inhibitors might be useful targets for the treatment of sepsis, but further investigation is needed. In conclusion, our knowledge of sepsis and its underlying mechanisms has grown considerably, but this has not resulted in effective therapeutic strategies for septic patients. New promising targets are currently under investigation and might facilitate the development of more powerful and effective treatments of sepsis and septic shock.
Acknowledgement Supported by the National Institute of Health (NIH) Grant GM-61656. Outstanding issues In the light of very few successful clinical trials in sepsis so far, new promising therapeutic targets are currently under investigation. According to the two-phase characteristics of sepsis, early application of antiinflammatory agents and late administration of immuno-restoring substances can be beneficial. Measuring levels of circulating pro- and antiinflammatory mediators might help to evaluate the stage of sepsis and thereby help to choose the appropriate therapy. Recently investigated substances such as HMGB1, MIF and apoptosis inhibitors, might be useful agents for the treatment of sepsis, but further investigation is needed.
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