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Cytokines and septic shock
DIAGN MICROBIOLINFECTDIS 1990;13:377-381
377
Cytokines and Septic Shock Thierry Calandra and Michel P. Glauser
INTRODUCTION Septic shock is a life-threatening complication of infection, which can be triggered by a wide spectrum of microorganisms. Bacteria, mainly Gram-negative bacilli and Gram-positive cocci, are the most frequent microbial agents isolated from patients with septic shock. However, other microorganisms such as spirochetes, rickettsia, viruses, fungi, or parasites may also occasionally cause septic shock. Most of our present knowledge on the pathogenic mechanisms involved in septic shock is derived from experimental and clinical data obtained either in animals or in humans infected with Gram-negative bacteria. With the exception of some Gram-positive infections, in most infections the pathogenic mechanisms responsible for the development of shock are either unknown or not completely understood (Sanford; 1985). In the present article we will therefore focus on Gram-negative infections and on the central role played by cytokines, especially tumor necrosis factor/cachectin (TNF), in the pathogenesis of Gramnegative septic shock. Over the past decades, the incidence of Gramnegative bacteremias has increased markedly in most medical centers (McCabe and Jackson, 1962; Kreger et al., 1980a). At theBoston City Hospital, the incidence of Gram-negative bacteremia increased from 0.9 cases per 1000 admissions in 1935 to 11.2 cases per 1000 admissions in 1972 (McGowan et al., 1975). In 1988, the incidence of Gram-negative bacteremia was 8 per 1000 admissions in our hospital. If comparable, the data from Boston in 1972 and from our institution in 1988 suggest that the incidence of Gramnegative bacteremia might have reached a plateau. One of the most striking and disappointing observations is that the mortality of these Gram-negative From the Division of InfectiousDiseases, Departmentof Internal Medicine, Centre Hospitalier UniversitaireVaudois, Lausanne, Switzerland. © 1990ElsevierSciencePublishing Co., Inc. 655 Avenue of the Americas,New York, NY 10010 0732-8893/90/$3.50
infections has been unchanged in spite of major breakthrough in both supportive care and antimicrobial therapy. Gram-negative bacteremias are still associated with a mortality of 20-35% (Felty and Keefer, 1924; Wolff and Bennett, 1974; McGowan et al., 1975; Kreger et al., 1980b; Bryan et a1.,1983). In patients developing Gram-negative septic shock, fatality ratios may be as high as 50% to 80% (Ziegler et al., 1982; Sprung et al., 1984; Bone et al., 1987; Veteran's Administration Systemic Sepsis Cooperative Study Group, 1987; Calandra et al., 1988). With the possible exception of passive immunotherapy with antibodies directed against the endotoxin core of Gram-negative bacteria (Ziegler et al., 1982), new therapeutic approaches such as treatment with corticosteriods or opiate antagonists have failed to reduce the mortality of Gram-negative bacteremia or septic shock (Sprung et al., 1984; De Maria et al., 1985; Bone et al., 1987; Veterans Administration Systemic Sepsis Cooperative Study Group, 1987).
TUMOR NECROSIS FACTOR/CACHECTIN (TNF) Most of the toxic manifestations induced by Gramnegative bacteria are triggered by endotoxin, also called lipopolysaccharide (LPS), which is a component of the outer membrane of these bacteria. Endotoxin can be released from the surface of the bacteria, either during bacterial growth or during antibiotic therapy (Shenep and Morgan, 1984). The lipid A, which is the innermost structure of endotoxin, is the moiety responsible for the toxic effects of endotoxin. In human volunteers it has been recently shown that the injection of a single intravenous bolus of Escherichi coli endotoxin reproduced the hemodynamic effects observed in patients with septic shock (Suffredini et al., 1989). A major breakthrough in our understanding of the pathophysiology of Gram-negative infections has been the recent recognition of the central role played by TNF in the pathogenesis of endotoxic shock
378
(Beutler et al., 1987 and 1988; Sherry and Cerami, 1988). TNF is a cytokine produced by activated macrophages upon exposure to endotoxin (Beutler et al., 1985c). Five lines of arguments strongly suggest that TNF is a primary mediator of the deleterious effects of endotoxin. First, in rabbits TNF was shown to be released in plasma after an intravenous challenge with E. coli LPS (Beutler et al., 1985a; Mathison et al., 1988). In a canine model of septic shock, a thrombin fibrin clot containing purified E. coli endotoxin implanted into the peritonuem or the intravenous infusion of recombinant TNF was shown to trigger cardiovascular changes similar to those observed in human septic shock (Natanson et al., 1989). Second, in rats and in dogs, the infusion of recombinant human TNF, in quantities similar to those produced endogeneously in response to endotoxin, was accompanied by hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours (Tracey et al., 1986 and 1987b). Third, mice passively immunized with polyclonal rabbit antiserum directed against murine TNF or with purified immunoglobulin were protected against the lethal effect of an intravenous injection of E. coli endotoxin (Beutler et al., 1985b). Similar results were obtained in rabbits passively immunized with a polyclonal anti-TNF antibody (Beutler et al., 1985a). Fourth, in a baboon model of septic shock, passive immunization with anti-TNF monoclonal antibodies administered 2 hr before a lethal challenge of live E. coli was shown to protect the animals from shock, organ dysfunction, persistent stress hormone release, and death (Tracey et al., 1987a). Finally, levels of immunoreactive TNF have been detected in the plasma of healthy human volunteers challenged with a single intravenous injection of 4 ng/kg of E. coli endotoxin (Hesse et al., 1988; Michie et al., 1988). In both studies the plasma concentration of TNF increased after 30-60 min and peaked at 90-120 min (358 --- 166 pg/ml and 240 _+70 pg/ml, respectively). Plasma TNF was not detectable 4-6 hr after the injection of endotoxin. The rise in TNF concentration was accompanied by chills, headache, myalgias, and nausea and by an increase in body temperature, heart rate, and stress hormones (i.e., ACTH and epinephrine) but was not associated with a drop in blood pressure. Moreover, in cancer patients the infusion of recombinant human TNF at a dose of -> 545 bLg/m2/24 hr produced peak plasma concentration of TNF, symptoms, and metabolic responses similar to those observed in volunteers challenged with endotoxin (Michie et al., 1988). Thus, in healthy humans the infusion of endotoxin was followed by a transient increase in plasma levels of TNF, which was associated with the development of the clinical signs and biochemical responses encountered in Gram-negative infections.
T. Calandra and M.P. Glauser
INTERLEUKIN-1, G A M M A INTERFERON, A N D INTERLEUKIN-6 It is obviously beyond the scope of this article to review the multiple biological properties of other mediators such as interleukin-1 (IL-1; Dinarello, 1988), gamma interferon (IFN-~/; Nathan and Yoshida, 1988), and interleukin-6 (IL-6; Wong and Clark, 1988) and to dissect out their respective roles and interplay in the pathogenesis of septic shock. Briefly, in human volunteers, concentrations of IL-1 remained at baseline values (Michie et al., 1988a and b) or increased only slightly (Hesse et al., 1988) after an intravenous injection of endotoxin or the infusion of TNF. Also, the injection of endotoxin or the infusion of TNF did not induce the release of IFN-~/in plasma. In rabbits, the infusion of a single dose of 5 ~g/kg of recombinant IL-113 was associated with the development of a transient hypotension, a decrease in systemic vascular resistance, and an increase in cardiac output and heart rate (Okusawa et al., 1988). These hemodynamic effects were prevented by ibuprofen pretreatment, indicating that it required cyclo-oxygenase products. In this model, IL-1 and TNF appeared to act synergistically, because the combination of the two cytokines was more potent than either agent alone. Thus, in rabbits, ILl was capable, like TNF, of inducing a shock state. Similar results were obtained by Waage and Espevik in a mouse model (1988).
CYTOKINES IN PATIENTS WITH SEPSIS A N D SEPTIC S H O C K In a study of the serum levels of immunoreactive TNF in 104 patients with various infectious diseases, Scuderi et al. (1986) detected elevated TNF (i.e., >39 pg/ml) in 18 of 27 (67%) patients with kala-azar, in 7 of 10 (70%) patients with malaria, and in only 2 of 20 (10%) patients with bacterial infections. None of the patients with other parasitic, viral, or fungal infections had elevated levels of serum TNF. Using a bioassay, Waage et al. (1986) detected a cytotoxic TNF activity in the serum of 3 of 23 (13%) patients with various infectious diseases but not in 20 cancer patients or in 25 controls. In two subsequent articles, the same group of investigators reported on the measurement of biologically active TNF, IL-6, and IL-1 in the serum of 79 patients with meningococcal disease (Waage et al., 1987 and 1989). TNF was detected in 10 of 11 patients who died and in only 8 of 68 survivors (p 440 U/ml survived. In this study, blood pressure, the presence of ecchymoses, and leukocyte and platelet counts were also shown to be associated with the patient's outcome (Waage et al., 1989). IL-6 was released in the serum
Cytokines and Septic Shock
of 69 patients (87%). Median serum levels of IL-6 were higher in patients with septic shock (189 ng/ml) than in patients with meningitis, bacteremia, or combined septic shock and meningitis (0.2 ng/ml in each of these groups of patients). All of the patients with IL-6 levels --<3.0 ng/ml survived. IL-6 levels were strongly associated with those of TNF (p = 0.0001). The half-lives of TNF and IL-6 were calculated to be 103 + 27 and 70 _ 11 min, respectively. Of 20 patients tested, only 3 had detectable levels of IL-1. In a group of 10 patients with meningococcal septic shock, the investigators showed that plasma levels of LPS correlated with serum levels of TNF, IL-6, IL-1, and a fatal outcome. In children with purpura fulminans, Girardin et al. (1988) measured elevated serum levels of TNF, IL-1, and IFN-~/in 86%, 21%, and 19% respectively, of the children tested on admission to the hospital. The serum concentrations of these three cytokines correlated significantly with the number of risk factors and were also significantly associated with the children's outcome. Levels of IL-1 and IFN-~ correlated with those of TNF. De Groote et al. (1989) measured plasma levels of immunoreactive TNF in 38 patients with presumed sepsis, of w h o m 14 had a Gram-negative and 2 had a pneumococcal bacteremia. Only 6 of these 38 patients (16%) had detectable TNF levels. In 43 critically ill septic patients, of w h o m 11 had a Gram-positive and 9 had a Gram-negative bacteremia, Debets et al. (1989) detected plasma levels of immunoreactive TNF in only 11 patients (25%). In light of the data obtained in volunteers after an endotoxin challenge, in w h o m the TNF concentrations peaked at the onset of the clinical symptoms and signs of sepsis, one might speculate that TNF was not detected in the serum of the majority of these patients with sepsis, because serum was sampled after the short rise in TNF concentration. Most recently, Hack et al. (1989) measured plasma levels of IL-6 in 37 patients with sepsis on admission to intensive care units. IL-6 was detected in 86% of these patients. The median IL-6 levels were higher in patients with septic shock (3082 U/rnl) than in normotensive patients (38 U/ml, p<0.0001) and were also higher in nonsurvivors (2646 U/ml; range, 54150,000 U/ml) than in survivors (39 U/ml; range, <207767, p= 0.0003). The IL-6 concentrations in the 23 patients with Gram-negative infections were comparable to those in the 11 patients with Gram-positive bacteria and in the 3 patients with a mixture of the two. In this study, IL-6 levels correlated with heart rate, plasma lactate levels, platelets count, C3a and C4a levels, and inversely with Cl-inhibitor levels. Serial measurements of IL-6 obtained in nine patients showed that the plasma concentrations de-
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creased in most cases independently of the patient's outcome. As part of a double-blind trial that investigated the efficacy of two immunoglobulin G preparations for the treatment of Gram-negative septic shock (Calandra et al., 1988), we prospectively measured serum concentrations of TNF, IL-6, IL-1, IFN-o~, and IFN-~/in 70 patients with septic shock (Calandra et al., 1989 and 1990). The aim of this study was to determine the evolution and the interplay of the serum levels of these cytokines during the course of septic shock and to relate these levels with the patient's outcome. Serum was collected at study entry, at day 1, and at day 10 in surviving patients. Serum TNF, IL-1, IFN-% and IFN-~ were assayed with a competitive inhibition radioimmunoassay, whereas IL-6 was measured with a bioassay. Septic shock was due to Gram-negative bacteria in 54 patients, Gram-positive bacteria in 5 patients (7%), and yeasts in 3 patients (4%). In eight patients (11%), all cultures remained sterile. At study entry, TNF, IL-6, IL-1, IFN-o~, and IFN-~/were detected in 76%, 64%, 93%, 74%, and 23% of patients, respectively. In a univariate analysis, serum levels of TNF (p = 0.002) and ILl (p=0.05) were associated with the patient's outcome but not those of IL-6 (p = 0.08), IFN~ (p = 0.15), and IFN~ (p=0.26). However, a stepwise logistic regression analysis showed that the severity of the underlying disease (p = 0.01), the age of the patient (p=0.02), the documentation of infection (nonbacteremic infections vs. bacteremias, p = 0.03), the urine output (p=0.04), and the arterial pH (p=0.05) contributed more significantly to prediction of the patients' outcome than the serum levels of TNF (p=0.07). The median concentration of immunoreactive TNF decreased to undetectable levels (<100 Wml) after 10 days in survivors, whereas it remained elevated in nonsurvivors (305 pg/ml, p=0.002). In contrast to TNF, the concentrations of biologically active IL-6 decreased rapidly. On day 1, IL-6 was not detectable in 81% of the patients. These results showed that immunoreactive TNF, IL-1, IFN-a, and biologically active IL-6 are released in the serum of patients with septic shock due to various Gram-negative bacteria. In these patients parameters other than the absolute serum concentration of TNF contributed significantly to the prediction of the patients' outcome.
CONCLUSIONS TNF has been unequivocally shown, both in experimental animal models and in humans, to be a central mediator of the clinical and humoral manifestations of acute septic shock induced by endotoxin
380
or b y whole Gram-negative bacteria. In clinical studies TNF was detected in the blood of a small proportion of patients with Gram-negative sepsis but in the vast majority of patients with established septic shock. H o w e v e r , in patients the m a g n i t u d e and the evolution of the blood concentration of TNF differed from t h a n observed in animal models or in h u m a n volunteers after an acute challenge with either Gram-negative bacteria or endotoxin, probably reflecting differences in infectious stimuli. In addition, our results suggest that in patients with established septic shock, the absolute serum concentration of
T. Calandra and M.P. Glauser
i m m u n o r e a c t i v e TNF is not a prime indicator of the severity of the patient's condition and might be of limited value in predicting the patient's outcome. The presence of elevated concentrations of IL-1, IL6, a n d IFN-~ in the blood of patients with septic shock indicates that these cytokines are also implicated in the complex pathophysiological events occurring in these patients. Altogether, the results of these experimental and clinical studies provide n e w insights into the pathogenesis of Gram-negative infections, which will soon result in the d e v e l o p m e n t of n e w therapeutic modalities.
REFERENCES Beutler BA, Milsark IW, Cerami A (1985a) Cachectin/tumor necrosis factor: production, distribution and metabolic fate in vivo. J Immunol 135:3972-3977. Beutler BA, Milsark IW, Cerami A (1985b) Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science229:869871. Beutler B, Mahoney J, Le Trang N, Pekala P, Cerami A (1985c) Purification of cachectin, a lipoprotein lipasesuppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J Exp Med 161:984-995. Beutler B, Cerami A (1987) Cachectin: more than a tumor necrosis factor. N Engl J Med 316:379-385. Beutler B, Cerami A (1988) The history, properties, and biological effects of cachectin. Biochemistry 27:7575-7582. Bone RG, Fisher CJ, Clemmer TP, Slotman GJ, Metz CA, Bolk RA, and the Methylprednisolone Severe Sepsis Study Group (1987) A controlled clinical trial of highdose methylprednisolone in the treatment of severe sepsis and septic shock. N Engl J Med 317:653-658. Bryan CS, Reynolds KL, Brenner ER (1983) Analysis of 1186 episodes of Gram-negative bacteremia in non-university hospital: the effects of antimicrobial therapy. Rev Infect Dis 5:629-638. Calandra T, Glauser MP, Schellekens J, Verhoef J, and the Swiss-Dutch J5 Immunoglobulin Study Group (1988) Treatment of septic shock with human IgG antibody to Escherichia coli J5: a prospective, double-blind, randomized trial. J Infect Dis 158:312-319. Calandra T, Gerain J, Heumann D, Baumgartner JD, Glauser MP, and the Swiss-Dutch J5 Study Group (1989) Interleukin-6 in patients with septic shock: correlation with outcome and with other cytokines [abstr 6]. In the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, 17-20 September 1989, Houston, Texas. Calandra T, Baumgartner JD, Grau GE, Wu MM, Lambert PH, Schellekens J, Verhoef J, Glauser MP, and the SwissDutch J5 Immunoglobulin Study Group (1990) Prognostic values of tumor necrosis factor/cachectin, interleukin-1, alpha interferon and gamma interferon in the serum of patients with septic shock. J Infect Dis 161:982987. Debets JMH, Kampmeijer R, Van der Linden MPMH, Buurman WA, Van der Linden CJ (1989) Plasma tumor necrosis factor and mortality in critically ill septic patients. Crit Care Med 17:489-494.
De Groote MA, Martin MA, Densen P, Pfaller MA, Wenzel RP (1989) Plasma tumor necrosis factor levels in patients with presumed sepsis. JAMA 262:249-251. De Maria A, Craven DE, Heffernan JJ, McIntosh TK, Grindlinger GA, McCabe WR (1985) Naloxone versus placebo in treatment of septic shock. Lancet 1:1363-1365. Dinarello CA (1988) Cytokines: interleukin-1 and tumor necrosis factor (Cachectin). In Inflammation: Basic Principles and Clinical Correlates. Eds, JI Gallin, IM Goldstein, and R Snyderman. New York: Raven Press, pp 195208. Felty AR, Keefer CS (1924) Bacillus coli species: a clinical study of 28 cases of bloodstream infection by the colon bacillus. JAMA 82:1430-1433. Girardin E, Grau GE, Dayer JM, Roux-Lombard P, and the J5 Study Group, Lambert PH (1988) Tumor necrosis factor and interleukin-1 in the serum of children with severe infectious purpura. N Engl J Med 319:397-400. Hack CE, De Groot ER, Felt-Bersma RJF, Nuijens JH, Strack Van Schijndel RJM, Eerenberg-Belmer AJM, Thijs LG, Aarden LA (1989) Increased plasma levels of interleukin-6 in sepsis. Blood 74:1704-1710. Hesse DG, Tracey KJ, Fong Y, Manogue KR, Palladino MA, Cerami A, Shires GT, Lowry SF (1988) Cytokine appearance in human endotoxemia and primate bacteremia. Surg Gynecol Obstet 166:147-153. Kreger BE, Craven DE, Carling PC, McCabe WR (1980a) Gram-negative bacteremia. III. Reassessment of etiology, epidemiology and ecology in 612 patients. Am J Med 68:332-343. Kreger BE, Craven DE, Carling PC, McCabe WR (1980b) Gram-negative bacteremia. IV. Reevaluation of clinical features and treatment in 612 patients. Am J Med 68:344355. Mathison JC, Wolfson E, Ulevitch RJ (1988) Participation of tumor necrosis factor in the mediation of Gram-negative bacterial lipopolysaccharide-induced injury in rabbits. J Clin Invest 81:1925-1937. McCabe WR, Jackson GG (1962) Gram-negative bacteremia. I. Etiology and ecology. Arch Intern Med 110:845855. McGowan JE Jr, Barnes MW, Finland M (1975) Bacteremia at Boston City Hospital: occurrence and mortality during 12 selected years (1935-1972) with special reference to hospital-acquired cases. J Infect Dis 132:326-341. Michie HR, Manogue KR, Spriggs DR, Revhaug A, O'Dwyer
Cytokines a n d Septic Shock
S, DinareUo CA, Cerami A, Wolff SM, Wilmore D (1988a) Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 318:1481-1486. Michie HR, Spriggs DR, Manogue KR, Sherman ML, Revhaug A, O'Dwyer ST, Arthur K, Dinarello CA, Cerami A, Wolff SM, Kufe DW, Wilmore DW (1988b) Tumor necrosis factor and endotoxin induce similar metabolic responses in human beings. Surgery 104:280-286. Natanson C, Eichenholz PW, Danner RL, Eichacker PQ, et al. (1989) Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med 169:823-832. Nathan C, Yoshida R (1988) Cytokines: interferon-7. In Inflammation: Basic Principles and Clinical Correlates. Eds. JI Gallin, IM Goldstein, and R Snyderman. New York: Raven Press, pp 229-251. Okusawa S, Gelfand JA, Ikejima T, Conolly RJ, Dinarello CA (1988) Interleukin 1 induces a shock-like state in rabbits: synergism with tumor necrosis factor and the effect of cydooxygenase inhibitors. J Clin Invest 81:11621172. Sanford JP (1985) Epidemiology and overview of the problem. In Contemporary Issues in Infectious Diseases: Septic Shock vol 4. Eds, RK Root and MA Sande. London: Churchill Livingstone, pp 1-12. Scuderi P, Sterling KE, Lam KS, Finley PR, Ryan KJ, Ray CG, Petersen E, Slymen DJ, Salmon SE (1986) Raised serum levels of tumor necrosis factor in parasitic infections. Lancet 2:1364-1365. Shenep JL, Mogan KA (1984) Kinetics of endotoxin release during antibiotic therapy for experimental Gram-negative bacterial sepsis. J Infect Dis 150:380-388. Sherry B, Cerami A (1988) CachectinYtumor necrosis factor exerts endocrine, paracrine, and autocrine control of inflammatory responses. J Cell Biol 107:1269-1277. Sprung C1, Caralis PV, Marcial EH, et al. (1984) The effects of high-dose corticosteroids in patients with septic shock: a prospective, controlled study. N Engl J Med 311:11371143. Suffredini AF, Fromm RE, Parker MM, Brenner M, et al. (1989) The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med 321:280287.
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Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey III TJ, Zentella A, Albert JD, Shires GT, Cerami A (1986) Shock and tissue injury induced by recombinant human cachectin. Science 234:470-474. Tracey KJ, Fong Y, Hesse DG, Manogue KR, Lee AT, Kuo GC, Lowry SF, Cerami A (1987) Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330:662-664. Tracey KJ, Lowry SF, Fahey II1 TJ, Albert JD, Fong Y, Hesse D, Beutler B, Manogue KR, Calvano S, Wei H, Cerami A, Shires GT (1987b) Cachectin/tumor necrosis factor induces lethal shock and stress hormone in the dog. Surg GynecoI Obstet 164:415-422. The Veterans Administration Systemic Sepsis Cooperative Study Group (1987) Effect of high-dose glucocorticoid therapy on mortality in patients with clinical signs of systemic sepsis. N Engl J Med 317:659-665. Waage A, Espevik T, Lamvik J (1986) Detection of tumour necrosis factor-like cytotoxicity in serum from patients with septicemia but not from untreated cancer patients. Scand J Immunol 24:739-743. Waage A, Halstensen A, Espevik T (1987) Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet 1:355357. Waage A, Espevik T (1988) Interleukin 1 potentiates the lethal effect of tumor necrosis factor odcachectin in mice. J Exp Med 167:1987-1992. Waage A, Braendtzaeg P, Halstensen A, Kierluf P, Espevik T (1989) The complex pattern of cytokines in serum from patients with meningococcal septic shock. J Exp Med 169:333-338. Wolff SD, Bennett JV (1974) Gram-negative rod bacteremia. N Engl J Med 291:733-734. Wong GG, Clark SC (1988) Multiple actions of interleukin 6 within a cytokine network. Immunol Today 9:137-139. Ziegler EJ, McCutchan JA, Fierer J, et al. (1982) Treatment of Gram-negative bacteremia and shock with human antiserum to a mutant of Escherichia coil N Engl J Med 307:1225-1230.
377
Cytokines and Septic Shock Thierry Calandra and Michel P. Glauser
INTRODUCTION Septic shock is a life-threatening complication of infection, which can be triggered by a wide spectrum of microorganisms. Bacteria, mainly Gram-negative bacilli and Gram-positive cocci, are the most frequent microbial agents isolated from patients with septic shock. However, other microorganisms such as spirochetes, rickettsia, viruses, fungi, or parasites may also occasionally cause septic shock. Most of our present knowledge on the pathogenic mechanisms involved in septic shock is derived from experimental and clinical data obtained either in animals or in humans infected with Gram-negative bacteria. With the exception of some Gram-positive infections, in most infections the pathogenic mechanisms responsible for the development of shock are either unknown or not completely understood (Sanford; 1985). In the present article we will therefore focus on Gram-negative infections and on the central role played by cytokines, especially tumor necrosis factor/cachectin (TNF), in the pathogenesis of Gramnegative septic shock. Over the past decades, the incidence of Gramnegative bacteremias has increased markedly in most medical centers (McCabe and Jackson, 1962; Kreger et al., 1980a). At theBoston City Hospital, the incidence of Gram-negative bacteremia increased from 0.9 cases per 1000 admissions in 1935 to 11.2 cases per 1000 admissions in 1972 (McGowan et al., 1975). In 1988, the incidence of Gram-negative bacteremia was 8 per 1000 admissions in our hospital. If comparable, the data from Boston in 1972 and from our institution in 1988 suggest that the incidence of Gramnegative bacteremia might have reached a plateau. One of the most striking and disappointing observations is that the mortality of these Gram-negative From the Division of InfectiousDiseases, Departmentof Internal Medicine, Centre Hospitalier UniversitaireVaudois, Lausanne, Switzerland. © 1990ElsevierSciencePublishing Co., Inc. 655 Avenue of the Americas,New York, NY 10010 0732-8893/90/$3.50
infections has been unchanged in spite of major breakthrough in both supportive care and antimicrobial therapy. Gram-negative bacteremias are still associated with a mortality of 20-35% (Felty and Keefer, 1924; Wolff and Bennett, 1974; McGowan et al., 1975; Kreger et al., 1980b; Bryan et a1.,1983). In patients developing Gram-negative septic shock, fatality ratios may be as high as 50% to 80% (Ziegler et al., 1982; Sprung et al., 1984; Bone et al., 1987; Veteran's Administration Systemic Sepsis Cooperative Study Group, 1987; Calandra et al., 1988). With the possible exception of passive immunotherapy with antibodies directed against the endotoxin core of Gram-negative bacteria (Ziegler et al., 1982), new therapeutic approaches such as treatment with corticosteriods or opiate antagonists have failed to reduce the mortality of Gram-negative bacteremia or septic shock (Sprung et al., 1984; De Maria et al., 1985; Bone et al., 1987; Veterans Administration Systemic Sepsis Cooperative Study Group, 1987).
TUMOR NECROSIS FACTOR/CACHECTIN (TNF) Most of the toxic manifestations induced by Gramnegative bacteria are triggered by endotoxin, also called lipopolysaccharide (LPS), which is a component of the outer membrane of these bacteria. Endotoxin can be released from the surface of the bacteria, either during bacterial growth or during antibiotic therapy (Shenep and Morgan, 1984). The lipid A, which is the innermost structure of endotoxin, is the moiety responsible for the toxic effects of endotoxin. In human volunteers it has been recently shown that the injection of a single intravenous bolus of Escherichi coli endotoxin reproduced the hemodynamic effects observed in patients with septic shock (Suffredini et al., 1989). A major breakthrough in our understanding of the pathophysiology of Gram-negative infections has been the recent recognition of the central role played by TNF in the pathogenesis of endotoxic shock
378
(Beutler et al., 1987 and 1988; Sherry and Cerami, 1988). TNF is a cytokine produced by activated macrophages upon exposure to endotoxin (Beutler et al., 1985c). Five lines of arguments strongly suggest that TNF is a primary mediator of the deleterious effects of endotoxin. First, in rabbits TNF was shown to be released in plasma after an intravenous challenge with E. coli LPS (Beutler et al., 1985a; Mathison et al., 1988). In a canine model of septic shock, a thrombin fibrin clot containing purified E. coli endotoxin implanted into the peritonuem or the intravenous infusion of recombinant TNF was shown to trigger cardiovascular changes similar to those observed in human septic shock (Natanson et al., 1989). Second, in rats and in dogs, the infusion of recombinant human TNF, in quantities similar to those produced endogeneously in response to endotoxin, was accompanied by hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours (Tracey et al., 1986 and 1987b). Third, mice passively immunized with polyclonal rabbit antiserum directed against murine TNF or with purified immunoglobulin were protected against the lethal effect of an intravenous injection of E. coli endotoxin (Beutler et al., 1985b). Similar results were obtained in rabbits passively immunized with a polyclonal anti-TNF antibody (Beutler et al., 1985a). Fourth, in a baboon model of septic shock, passive immunization with anti-TNF monoclonal antibodies administered 2 hr before a lethal challenge of live E. coli was shown to protect the animals from shock, organ dysfunction, persistent stress hormone release, and death (Tracey et al., 1987a). Finally, levels of immunoreactive TNF have been detected in the plasma of healthy human volunteers challenged with a single intravenous injection of 4 ng/kg of E. coli endotoxin (Hesse et al., 1988; Michie et al., 1988). In both studies the plasma concentration of TNF increased after 30-60 min and peaked at 90-120 min (358 --- 166 pg/ml and 240 _+70 pg/ml, respectively). Plasma TNF was not detectable 4-6 hr after the injection of endotoxin. The rise in TNF concentration was accompanied by chills, headache, myalgias, and nausea and by an increase in body temperature, heart rate, and stress hormones (i.e., ACTH and epinephrine) but was not associated with a drop in blood pressure. Moreover, in cancer patients the infusion of recombinant human TNF at a dose of -> 545 bLg/m2/24 hr produced peak plasma concentration of TNF, symptoms, and metabolic responses similar to those observed in volunteers challenged with endotoxin (Michie et al., 1988). Thus, in healthy humans the infusion of endotoxin was followed by a transient increase in plasma levels of TNF, which was associated with the development of the clinical signs and biochemical responses encountered in Gram-negative infections.
T. Calandra and M.P. Glauser
INTERLEUKIN-1, G A M M A INTERFERON, A N D INTERLEUKIN-6 It is obviously beyond the scope of this article to review the multiple biological properties of other mediators such as interleukin-1 (IL-1; Dinarello, 1988), gamma interferon (IFN-~/; Nathan and Yoshida, 1988), and interleukin-6 (IL-6; Wong and Clark, 1988) and to dissect out their respective roles and interplay in the pathogenesis of septic shock. Briefly, in human volunteers, concentrations of IL-1 remained at baseline values (Michie et al., 1988a and b) or increased only slightly (Hesse et al., 1988) after an intravenous injection of endotoxin or the infusion of TNF. Also, the injection of endotoxin or the infusion of TNF did not induce the release of IFN-~/in plasma. In rabbits, the infusion of a single dose of 5 ~g/kg of recombinant IL-113 was associated with the development of a transient hypotension, a decrease in systemic vascular resistance, and an increase in cardiac output and heart rate (Okusawa et al., 1988). These hemodynamic effects were prevented by ibuprofen pretreatment, indicating that it required cyclo-oxygenase products. In this model, IL-1 and TNF appeared to act synergistically, because the combination of the two cytokines was more potent than either agent alone. Thus, in rabbits, ILl was capable, like TNF, of inducing a shock state. Similar results were obtained by Waage and Espevik in a mouse model (1988).
CYTOKINES IN PATIENTS WITH SEPSIS A N D SEPTIC S H O C K In a study of the serum levels of immunoreactive TNF in 104 patients with various infectious diseases, Scuderi et al. (1986) detected elevated TNF (i.e., >39 pg/ml) in 18 of 27 (67%) patients with kala-azar, in 7 of 10 (70%) patients with malaria, and in only 2 of 20 (10%) patients with bacterial infections. None of the patients with other parasitic, viral, or fungal infections had elevated levels of serum TNF. Using a bioassay, Waage et al. (1986) detected a cytotoxic TNF activity in the serum of 3 of 23 (13%) patients with various infectious diseases but not in 20 cancer patients or in 25 controls. In two subsequent articles, the same group of investigators reported on the measurement of biologically active TNF, IL-6, and IL-1 in the serum of 79 patients with meningococcal disease (Waage et al., 1987 and 1989). TNF was detected in 10 of 11 patients who died and in only 8 of 68 survivors (p
Cytokines and Septic Shock
of 69 patients (87%). Median serum levels of IL-6 were higher in patients with septic shock (189 ng/ml) than in patients with meningitis, bacteremia, or combined septic shock and meningitis (0.2 ng/ml in each of these groups of patients). All of the patients with IL-6 levels --<3.0 ng/ml survived. IL-6 levels were strongly associated with those of TNF (p = 0.0001). The half-lives of TNF and IL-6 were calculated to be 103 + 27 and 70 _ 11 min, respectively. Of 20 patients tested, only 3 had detectable levels of IL-1. In a group of 10 patients with meningococcal septic shock, the investigators showed that plasma levels of LPS correlated with serum levels of TNF, IL-6, IL-1, and a fatal outcome. In children with purpura fulminans, Girardin et al. (1988) measured elevated serum levels of TNF, IL-1, and IFN-~/in 86%, 21%, and 19% respectively, of the children tested on admission to the hospital. The serum concentrations of these three cytokines correlated significantly with the number of risk factors and were also significantly associated with the children's outcome. Levels of IL-1 and IFN-~ correlated with those of TNF. De Groote et al. (1989) measured plasma levels of immunoreactive TNF in 38 patients with presumed sepsis, of w h o m 14 had a Gram-negative and 2 had a pneumococcal bacteremia. Only 6 of these 38 patients (16%) had detectable TNF levels. In 43 critically ill septic patients, of w h o m 11 had a Gram-positive and 9 had a Gram-negative bacteremia, Debets et al. (1989) detected plasma levels of immunoreactive TNF in only 11 patients (25%). In light of the data obtained in volunteers after an endotoxin challenge, in w h o m the TNF concentrations peaked at the onset of the clinical symptoms and signs of sepsis, one might speculate that TNF was not detected in the serum of the majority of these patients with sepsis, because serum was sampled after the short rise in TNF concentration. Most recently, Hack et al. (1989) measured plasma levels of IL-6 in 37 patients with sepsis on admission to intensive care units. IL-6 was detected in 86% of these patients. The median IL-6 levels were higher in patients with septic shock (3082 U/rnl) than in normotensive patients (38 U/ml, p<0.0001) and were also higher in nonsurvivors (2646 U/ml; range, 54150,000 U/ml) than in survivors (39 U/ml; range, <207767, p= 0.0003). The IL-6 concentrations in the 23 patients with Gram-negative infections were comparable to those in the 11 patients with Gram-positive bacteria and in the 3 patients with a mixture of the two. In this study, IL-6 levels correlated with heart rate, plasma lactate levels, platelets count, C3a and C4a levels, and inversely with Cl-inhibitor levels. Serial measurements of IL-6 obtained in nine patients showed that the plasma concentrations de-
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creased in most cases independently of the patient's outcome. As part of a double-blind trial that investigated the efficacy of two immunoglobulin G preparations for the treatment of Gram-negative septic shock (Calandra et al., 1988), we prospectively measured serum concentrations of TNF, IL-6, IL-1, IFN-o~, and IFN-~/in 70 patients with septic shock (Calandra et al., 1989 and 1990). The aim of this study was to determine the evolution and the interplay of the serum levels of these cytokines during the course of septic shock and to relate these levels with the patient's outcome. Serum was collected at study entry, at day 1, and at day 10 in surviving patients. Serum TNF, IL-1, IFN-% and IFN-~ were assayed with a competitive inhibition radioimmunoassay, whereas IL-6 was measured with a bioassay. Septic shock was due to Gram-negative bacteria in 54 patients, Gram-positive bacteria in 5 patients (7%), and yeasts in 3 patients (4%). In eight patients (11%), all cultures remained sterile. At study entry, TNF, IL-6, IL-1, IFN-o~, and IFN-~/were detected in 76%, 64%, 93%, 74%, and 23% of patients, respectively. In a univariate analysis, serum levels of TNF (p = 0.002) and ILl (p=0.05) were associated with the patient's outcome but not those of IL-6 (p = 0.08), IFN~ (p = 0.15), and IFN~ (p=0.26). However, a stepwise logistic regression analysis showed that the severity of the underlying disease (p = 0.01), the age of the patient (p=0.02), the documentation of infection (nonbacteremic infections vs. bacteremias, p = 0.03), the urine output (p=0.04), and the arterial pH (p=0.05) contributed more significantly to prediction of the patients' outcome than the serum levels of TNF (p=0.07). The median concentration of immunoreactive TNF decreased to undetectable levels (<100 Wml) after 10 days in survivors, whereas it remained elevated in nonsurvivors (305 pg/ml, p=0.002). In contrast to TNF, the concentrations of biologically active IL-6 decreased rapidly. On day 1, IL-6 was not detectable in 81% of the patients. These results showed that immunoreactive TNF, IL-1, IFN-a, and biologically active IL-6 are released in the serum of patients with septic shock due to various Gram-negative bacteria. In these patients parameters other than the absolute serum concentration of TNF contributed significantly to the prediction of the patients' outcome.
CONCLUSIONS TNF has been unequivocally shown, both in experimental animal models and in humans, to be a central mediator of the clinical and humoral manifestations of acute septic shock induced by endotoxin
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or b y whole Gram-negative bacteria. In clinical studies TNF was detected in the blood of a small proportion of patients with Gram-negative sepsis but in the vast majority of patients with established septic shock. H o w e v e r , in patients the m a g n i t u d e and the evolution of the blood concentration of TNF differed from t h a n observed in animal models or in h u m a n volunteers after an acute challenge with either Gram-negative bacteria or endotoxin, probably reflecting differences in infectious stimuli. In addition, our results suggest that in patients with established septic shock, the absolute serum concentration of
T. Calandra and M.P. Glauser
i m m u n o r e a c t i v e TNF is not a prime indicator of the severity of the patient's condition and might be of limited value in predicting the patient's outcome. The presence of elevated concentrations of IL-1, IL6, a n d IFN-~ in the blood of patients with septic shock indicates that these cytokines are also implicated in the complex pathophysiological events occurring in these patients. Altogether, the results of these experimental and clinical studies provide n e w insights into the pathogenesis of Gram-negative infections, which will soon result in the d e v e l o p m e n t of n e w therapeutic modalities.
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