Reduction in endotoxin-induced organ dysfunction and cytokine secretion by a cyclic nitrone antioxidant

Int..Z lmmunopharmac., Vol. 17, No. 7, pp. 571-580, 1995 Elsevier Science Ltd International Society for Immunopharmaeology Printed in Great Britain

Pergamon 0192-0561(95)00042-9

REDUCTION IN ENDOTOXIN-INDUCED ORGAN DYSFUNCTION AND CYTOKINE SECRETION BY A CYCLIC NITRONE ANTIOXIDANT THOMAS R. DOWNS, RICHARD C. DAGE and JOHN F. FRENCH Marion Merrell Dow Research Institute, 2110 E. Galbraith Road, Cincinnati, OH 45215, U.S.A. (Received 18 November 1994 and in final form 9 March 1995)

Abstract - - Multiple organ dysfunction (MOD) is the leading cause of mortality in septic patients with circulatory shock. Recent evidence suggests that the overproduction of the cytokine, tumor necrosis factor-or (TNF), and oxygen free radical molecules may mediate the progression of sepsis to MOD and death. In this study, we have examined the ability of MDL 101,002, a free radical scavenger, to reduce organ dysfunction and cytokine secretion induced by lipopolysaccharide (LPS) administration in rats. Treatment with MDL 101,002 (10-60 mg/kg, i.p.) 30 min prior to an LPS challenge resulted in a dose-dependent reduction in several markers indicative of organ dysfunction and mortality. MDL 101,002 markedly decreased LPS-induced liver and kidney damage as indicated by serum levels ofaspartate aminotransferase (AST) and alanine aminotransferase (ALT) or urea and creatinine, respectively. MDL 101,002 also prevented LPS-induced pulmonary edema, but did not prevent leukopenia and only partially reduced thrombocytopenia. Associated with these improvements in organ dysfunction and survival was a modest decrease in LPS-stimulated interleukin- 1tx (IL- 1o~) and interleukin- 1~l (IL- 113) secretion and a marked (>90%) inhibition of TNF secretion by MDL 101,002. The data are consistent with a role for oxygen free radicals in the development of endotoxin-induced organ dysfunction and shock and suggest that free radical scavengers could reduce the mortality consequent to sepsis by decreasing organ dysfunction, at least in part, through a reduction in free radical stimulated cytokine secretion. Keywords : sepsis, septic shock, oxygen free radicals, cytokines, organ dysfunction, free radical scavengers.

Septic shock is a systemic response that develops as or lipopolysaccharide (LPS), a major cell wall molecule the result o f severe infection. The initial symptoms o f in these bacteria, appears to be the primary activator o f sepsis encompass those usually associated with acute the systemic response. Administration of LPS to animals inflammation, including fever or hypothermia, tachypcauses the development of MOD, disseminated intranea, tachycardia, and elevated white blood cell counts, vascular coagulation (DIC), septic (endotoxic) shock, but may progress to neutropenia, thrombocytopenia, and ultimately death (Yoshikawa et al., 1994; Peristeris pulmonary edema, and falling blood pressure or shock. et al., 1992). Loss of vascular tone and/or depressed cardiac function LPS stimulates the synthesis and secretion of several appear to be the primary causes o f the severe hypoproinflammatory cytokines from monocytes and tension, which may become refractory to treatment, and macrophages, beginning with tumor necrosis factor-tx result in multiple organ dysfunction (MOD) and death (TNF), and followed by others including interleukin-1 in - 5 0 % of septic patients who develop shock (Parrillo, (IL-lct and IL-113), interleukin-6 (IL-6), interleukin-8 1990; Bone, 1991; Parker et al., 1987; Martich et al., (IL-8), and interferon-y (IFN) (Fischer et al., 1992; 1993). Pogrebniak et al., 1992; Chensue et al., 1991; Fong et Gram-negative bacteremia is the most commonly al., 1989). Administration of TNF or IL-1 ~ can individassociated infection, although Gram-positive bacteria, ually mimic many of the physiological changes observed viruses, fungi, and other microorganisms are all known in LPS-induced shock and cause tissue damage and pathogens for septic shock. Spreading of the infection death (Tracey et al., 1986; Ohlsson et al., 1990; Okucan result in invasion of the bloodstream by these sawa et al., 1988; Mallick et al., 1989). Moreover, treatmicrobes and the subsequent release of toxins. In the ment with IL-I~ receptor antagonists (Fischer et al., case of infection by Gram-negative bacteria, endotoxin 1992; Ohlsson et al., 1990; Wakabayashi et al., 1991) 571

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or passive immunization with antibodies to TNF or IFN (Tracey et al., 1986; Doherty et al., 1992) has been shown to reduce the mortality caused by LPS in animals. However, synergism between multiple cytokines and other factors probably occurs in the progression of septic shock (Okusawa et al., 1988; Doherty et al., 1992; Myers et al., 1990; Bone, 1991). Other key mediators of LPS- or TNF-induced morbidity and mortality are reactive oxygen intermediate molecules, including superoxide anions, nitric oxide, hydrogen peroxide, and hydroxyl radicals (Yoshikawa et al., 1994; Henson & Johnston, 1987; Novelli et al., 1989; Weiss, 1989; Wendel, 1991; Peristeris et al., 1992; Wright et al., 1992; Bernard & Tedgui, 1992). The tissue damage observed after exposure to oxygen radicals is comparable to that caused by LPS (Novelli et al., 1989; Manson & Hess, 1983). In addition, free radical scavengers, such as superoxide dismutase, N-acetylcysteine, and c~-phenyl N-tert-butylnitrone (PBN) reduce mortality in animal models of endotoxic shock (Pogrebniak et al., 1992; Novelli et al., 1989; Wendel, 1991; McKechnie et al., 1986; Peristeris et al., 1992). Reactive oxygen species are produced primarily by activated neutrophils after adherence at sites of infection, and their release is stimulated by several cytokines, including TNF and IL-113 (Meier et al., 1989; Jensen et al., 1992; Shau, 1988; de la Harpe & Nathan, 1989). These oxygen intermediates, in turn, up-regulate cytokine synthesis by macrophages, creating a vicious circle for the production of both of these types of inflammatory mediators (Deforge et al., 1992; Wendel, 1991; Pogrebniak et al., 1990; Jensen et al., 1992). Recently, LPS- or free radical-stimulated TNF, IL-8, and IFN production have all been shown to be greatly diminished in the presence of free radical scavengers (Pogrebniak et al., 1992; Deforge et aL, 1992; Wendel, 1991; Peristeris et al., 1992; Zhang et al., 1994). These data suggest that potent antioxidants may reduce the mortality consequent to sepsis by decreasing organ dysfunction, at least in part, through inhibition of oxygen radical-stimulated cytokine secretion. MDL 101,002, a novel cyclized variant of the free radical trapping agent PBN, with approximately ten times greater antioxidant and hydroxyl radical scavenging activity in vitro, has previously been shown to be efficacious in vivo in reducing oxidative stress and mortality in a rat model of chronic bacteremia, as well as being more effective than PBN in lowering mortality in a model of endotoxic shock in the rat (French et al., 1994). In the present study, we have examined the ability of MDL 101,002 to reduce organ dysfunction and cytokine secretion after LPS administration in rats.

EXPERIMENTAL PROCEDURES

Animal model

Male Sprague-Dawley rats (200-225 g), purchased from Charles River (Wilmington, MA, U.S.A.), were treated with a single dose of MDL 101,002 (10-60 mg/kg, i.p.) or saline (100 p.l/100 g body wt). Thirty minutes later, the rats were injected with endotoxin (lipopolysaccharide from E. coli, serotype O127:B8, Sigma Chemical Co., St Louis, MO, U.S.A. 10-20 mg/kg, i.p.) or saline (200 ~1/100 g body wt). No additional fluids or solutions were administered. Blood samples (250 ~tl)were collected from the tails at various times following endotoxin injection (0.5q5 h) and placed in tubes containing EDTA (1 mM final concentration) for complete blood cell counts (CBC), or allowed to clot and the serum collected for blood chemistry analysis of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine, or stored frozen until assayed for TNF and IL-lo~ or IL-I]3. In several experiments, the rats were anesthetized with sodium pentobarbital (40mg/kg, i.p.) 6 h after endotoxin injection, and the lungs were dissected and weighed. The lungs were frozen, lyophilized, and weighed again and the water content of the tissues was expressed as the difference between the wet and dry lung weights. In other studies, mortality was assessed 24 h after endotoxin administration.

C B C and blood chemistry measurements

The number ofplatelets and the total number of white blood cells in blood samples were determined on an ELT-8 automated hematology cell analyzer (Ortho Diagnostics Systems, Westwood, MA, U.S.A.) using a helium-neon gas laser optical system. Differential WBC counts were performed microscopically on blood smears after fixing and staining with Wright-Giemsa stain. Serum ALT, AST, urea, and creatinine measurements were performed on a DACOS chemistry analyzer (Coulter Electronics, Hialeath, FL, U.S.A.) using reagents from Coulter Electronics or Trace Scientific Pty (Baulkham Hills, NSW, Australia).

TNF, 1L- 1a, and IL- 11~ measurements

TNF-0~ and IL-1[3 levels were assayed in serum samples by ELISA using anti-mouse TNF-cx and antimouse IL-l~ sera (Genzyme Diagnostics, Cambridge, MA, U.S.A.) which cross-react with these proteins in the rat. The sensitivity of both assays was approximately 10-15 pg/ml. IL- 1o~was measured by RIA using an anti-

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Table 1. The effect of MDL 101,002 on LPS-induced changes in complete blood counts, serum urea, creatinine, AST, ALT, TNF, and IL-1 ct levels, and lung water weight Control (n=7) WBC (x 103/ml) Lymphocytes (× 10Vml) Neutrophils (x 10Vml) Platelets (x 103/ml) Urea (mg/dl) Creatinine (mg/dl) AST (U/l) ALT (U/I) Lung water (g) TNF (pg/ml) IL-lot (pg/ml)

14.7 ± 2.5 11.4 4- 2.1 3.2 4- 0.6 943 4- 35 13.4 4- 1.4 0.37 4- 0.03 101 4- 5" 44 -4-3 0.78 4- 0.02 <7 1133 4- 133

LPS-treated rats Vehicle MDL 101,002 ( n = 18) (n= 18) 4.4 4- 1.1" 2.0 4- 0.5* 2.4 4- 0.6* 371 4- 48* 31.1 4- 3.6" 0.49 4- 0.03* 1009 + 391" 556 + 239* 0.87 ± 0.02* 168 4- 27* 2928 ± 285*

4.2 4- 0.6* 1.7 4- 0.3* 2.4 + 0.3* 483 4- 46* 18.5 4- 2.0 ~ 0.40 4- 0.02 * 281 + 81' 127 ± 34* 0.81 + 0.0T 88 4- 15"* 2244 4- 299**

Rats were treated with MDL 101,002 (60 mg/kg, i.p.) or vehicle 30 min prior to an LPS (10 mg/kg, i.p.) challenge. Blood samples and lungs were collected 6 h post-LPS treatment. Results are shown as the mean + S.E.M. for each group. *P<0.05 compared to control group. *P<0.05 compared to LPS-vehicle group. rat IL-Itx sera (Cytokine Sciences, Boston, MA, U.S.A.), with a sensitivity o f approximately 300 pg/ml.

Data analysis The data were analyzed using one-way analysis of variance. If significant n o n - h o m o g e n e i t y o f variance was indicated, log transformation o f the data was first performed. Significant differences (P<0.05) a m o n g groups were then determined with Student-NewmanKeuls multiple comparison test. Differences in mortality between treatment groups were assessed using Fisher's exact test.

RESULTS

Effect o f MDL 101,002 on LPS-induced changes in hematology, blood chemistry, lung edema, and serum cytokines Administration o f LPS (10 mg/kg, i.p.) induced a pronounced leukopenia (70% reduction in total white blood cell number) at 6 h (Table 1). Differential WBC counts showed that this reduction was primarily due to an 83% decrease in circulating lymphocytes, although the number o f neutrophils was also significantly lower (25%) at this time. Circulating platelets also fell to approximately 40% o f normal 6 h after endotoxin treatment. Pretreatment with MDL 101,002 (60 mg/kg, i.p.) 30 min prior to LPS injection did not prevent the LPS-induced decreases in total or differential white

blood cell counts or circulating platelet numbers (Table 1). Several markers o f the function of various organs were also evaluated after endotoxin administration with or without prophylactic treatment with M D L 101,002. Analysis o f blood chemistry demonstrated significant rises in both serum urea and creatinine levels (232% and 132% o f normal, respectively) 6 h after LPS administration (Table 1). Serum AST and ALT concentrations were markedly increased to 10-13-fold over normal levels by LPS. MDL 101,002 pretreatment (60 mg/kg) prevented the LPS-induced elevations in serum urea, creatinine, A S T and ALT (Table 1). In addition, the increase in water retention in the lung caused by endotoxin administration (112% o f normal) was eliminated by prior treatment with MDL 101,002. The serum concentrations o f several cytokines, including TNF and IL-h~ were also measured 6 h after LPS injection. Serum TNF levels, which were undetectable (<7 pg/ml) in normal rats, were 168 pg/ml at this time point after endotoxin challenge. This increase was partially attenuated by prior MDL 101,002 administration. Similar results were also observed for the LPSinduced increases in serum concentrations o f l L - 1~ with and without previous injection o f MDL 101,002 (Table 1).

Effect o f MDL 101,002 on hematology, blood chemistry, lung edema, and serum cytokine levels in normal rats Treatment o f normal rats with M D L 101,002 (60 mg/kg, i.p.) did not alter the total or differential

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Table 2. The effect of MDL 101,002 on complete blood counts, serum urea, creatinine, AST, ALT, TNF, and IL-I~x levels, and lung water weight in normal animals Control (n = 9) WBC (xl03/ml) Lymphocytes (× 1 0 V m l ) Neutrophils (xl03/ml) Platelets (× t0Vml) Urea (mg/dl) Creatinine (mg/dl) AST (U/I) ALT (U/I) Lung water (g) TNF (pg/ml) IL-lct (pg/ml)

MDL 101,002 (n = 13)

17.3 ~ 3.0 13.2 • 2.9 4.1 ± 0.6 959 ± 28 12.2 + h l 0.36 ± 0.02 105 ± 5 44 ± 2 0.84 ± 0.03 <7 1133 -- 133

15.9 ± 2.6 12.7 ± 2.6 3.1 ± 0.4 989 ± 29 10.5 ± 1.0 0.32 ± 0.02 108 :L 7 47 ± 3 0.84 ± 0.02 <7 1288 :i: 153

Rats were treated with MDL 101,002 (60 mg/kg, i.p.) or saline. Blood samples and lungs were collected 6 h later. Results are shown as the mean ± S.E.M. for each group.

WBC counts or the number o f platelets in circulation 6 h after injection (Table 2). Similarly, serum concentrations o f urea, creatinine, AST, ALT, TNF, and IL-hx, as well as lung water content were also unaffected by MDL 101,002 treatment at this dose.

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Dose-related effects o f MDL 101,002 on LPS-induced changes in hematology, blood chemistry, and survigal Administration o f LPS (10 mg/kg, i.p.) resulted in a mortality rate o f approximately 42% (58% survival) o f the rats in this study within 24 h (Fig. 1). Treatment with MDL 101,002 (10 mg/kg, i.p.) 30 min prior to LPS did not significantly improve survival (64%) compared to LPS administration alone, while treatment with 30 and 60 mg/kg MDL 101,002 increased survival to greater than 90% (Fig. 1). The changes in hematology that were observed in the initial experiments were similar in this study. LPS caused a 63% decrease in the number o f platelets in circulation 6 h after injection (Fig. 2). Pretreatment with MDL 101,002 at 10 and 30 mg/kg did not prevent the fall in platelet numbers. In these experiments, however, MDL 101,002 given at 60 mg/kg did result in a small, but statistically significant improvement in the fall in the number o f platelets in circulation (49% decrease) as compared to LPS treatment alone (Fig. 2). The total number o f white blood cells in these studies dropped to approximately 50% o f normal 6 h after endotoxin administration and was not significantly improved by prior MDL 101,002 treatment at any o f the doses tested (data not shown).

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Fig. h Dose-related effects of MDL 101,002 on survival in rats 24 h after an LPS challenge. Rats were treated with MDL 101,002 (10, 30 or 60 mg/kg, i.p., n = 22-47 rats/group, crosshatched bars) or vehicle (n = 74, solid bars) 30 min prior to an LPS challenge (10 mg/kg, i.p.). Control animals are shown in the open bars (n = 31). Results are shown as the mean for each group. **P<0.01 vs control; t~P<0.01 vs LPS-vehicle. Analysis o f blood chemistry revealed significant improvement in markers o f kidney and liver function at doses o f MDL 101,002 lower than those used in the initial studies. Serum urea concentrations rose to more

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Fig. 3. Dose-related effects of MDL 101,002 on serum urea levels in rats 6 h after an LPS challenge. See legend to Fig. 1 for other experimental details. Results are shown as the mean + S.E.M. for each group. **P<0.01 vs control; tP<0.05 vs LPS-vehicle; ttp<0.01 vs LPS-vehicle. than 2-fold normal levels 6 h after LPS injection (Fig. 3). Treatment with all doses o f M D L 101,002 significantly lowered serum urea levels c o m p a r e d to LPS administration alone, and at 30 mg/kg M D L 101,002, urea was not elevated above control. Improvements in LPS-stimulated increases in serum AST and ALT in response to MDL 101,002 treatment were again observed. LPS administration raised serum AST and

ALT levels 6-9-fold greater than normal 6 h after injection (Fig. 4, upper and lower panel, respectively). MDL 101,002 administration at 1 0 m g / k g did not attenuate the LPS-induced rises in serum AST or ALT, however, doses o f 30 and 60 mg/kg MDL 101,002 greatly reduced LPS-stimulated increases in serum AST and ALT (Fig. 4, upper and lower panel, respectively). In comparison to LPS administration alone, the serum levels o f both enzymes were not significantly elevated above control after treatment with 30 mg/kg M D L 101,002 and were only slightly elevated above normal at 60 mg/kg MDL 101,002 (Fig. 4, upper and lower panels).

Effect o f MDL 101,002 on LPS-induced stimulation o f TNF, IL-ltx and IL-I~ secretion Injection o f endotoxin (20 mg/kg, i.p.) stimulated a m a r k e d increase in the secretion o f TNF (Fig. 5). Circulating TNF concentrations rose from undetectable

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Fig. 5. Effect of MDL 101,002 on LPS-induced changes in serum TNF levels. Rats were treated with MDL 101,002 (60 mg/kg, i.p., n = 20, closed squares) or vehicle (n = 20, closed circles) 30 min prior to an LPS challenge (20 mg/kg, i.p.). Blood samples were collected at the times indicated and the serum TNF levels were measured. Each point represents the mean 4- S.E.M. for each group. **P<0.01 vs LPS-vehicle.

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levels (<10 pg/ml) to more than 400 pg/ml at 30 min. TNF levels peaked at 17,500 pg/ml at 60 min, then declined, but remained elevated (200 pg/ml) 6 h after LPS injection (Fig. 5). Pretreatment with 60 mg/ml MDL 101,002 prior to LPS injection resulted in a comparable time course for the TNF response, but a significantly lower serum TNF level at 30 min (122 pg/ml), and markedly lower peak concentration o f TNF at 6 0 m i n ( 1 0 8 5 p g / m l ) . Serum TNF levels at 2 h in L P S / M D L 101,002-treated animals remained m u c h lower (10-fold less) than in rats treated with LPS alone (Fig. 5). Circulating I L - I ~ concentrations in response to endotoxin were not significantly elevated until 2 h after LPS injection (Fig. 6, upper panel). By this time, serum IL-I[~ levels had doubled from 23 pg/ml to 50 pg/ml and remained elevated (45 pg/ml) 6 h after endotoxin challenge. MDL 101,002 treatment did not lower serum IL-1[~ concentrations 2 h after LPS, but IL-113 levels were significantly decreased at 6 h. Serum IL-ltx levels in LPS treated rats showed a biphasic pattern, with an initial peak at 30 min, followed by a decline to normal levels at 60 min, and then elevated concentrations for at least 6 h after LPS administration (Fig. 6, lower panel). MDL 101,002 treatment prevented the initial increase in serum IL-lot levels at 30 min, but did not significantly lower serum IL-let concentrations at 6 h after LPS.

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Fig. 6. Effect ofMDL 101,002 on LPS-induced changes in serum IL-I [3 (upper panel) and IL-Itx (lower panel) levels. Rats were treated with MDL 101,002 (60 mg/kg, i.p., n = 20, closed squares) or vehicle (n = 20, closed circles) 30 min prior to an LPS challenge (20 mg,/kg, i.p.). Blood samples were collected at the times indicated and the serum IL-I ~ and IL-I~ levels were measured. Each point represents the mean ± S.E.M. for each group. *P<0.05 vs LPS-vehicle; **P
DISCUSSION

Multiple organ dysfunction is commonly associated with sepsis and is one of the leading causes o f death in septic patients who develop circulatory shock (Parrillo, 1990; Bone, 1991; Parker et al., 1987). A similar association has been reported in the pathogenesis o f adult respiratory distress syndrome (ARDS) where an initial lung injury, often found in conjunction with sepsis, leads to MOD and death (Dorinsky & Gadek, 1989; Bernard, 1991). Functional disorders in the lungs, liver, intestines, heart, and kidneys are the most frequently observed in patients and in animal models of

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(Yoshikawa et al., 1994; Novelli et al., 1989; Gerdin & Haglund, 1993). LPS activation of the complement system causes the adherence and degranulation of 1993). The results of these studies demonstrate that neutrophils at the site of infection, resulting in the MDL 101,002, a cyclized variant of the free radical spin release of free radicals and various enzymes (Parrillo, trapping agent, PBN, with approximately ten times 1990; Henson & Johnston, 1987; Weiss, 1989). Oxygen greater antioxidant and hydroxyl radical scavenging free radicals are also produced during the transient activity than PBN in vitro (French et al., 1994), reduced episodes of tissue ischemia and disruption in normal organ dysfunction and mortality caused by endotoxin. metabolism commonly seen in the events leading to Pretreatment with MDL 101,002 markedly decreased septic shock (Wendel, 1991; Gerdin & Haglund, 1993). LPS-induced liver and kidney damage, as assessed by Increased free radical production may exceed local serum AST and ALT or urea and creatinine levels, cellular dismutase and antioxidant capacity and allow respectively. In addition, MDL 101,002 prevented the radicals to produce tissue damage (Wendel, 1991; endotoxin-induced pulmonary edema. Decreased serum Gerdin & Haglund, 1993). cytokine levels were associated with the improvements Other evidence suggests a causative interrelationship in organ dysfunction markers and survival. TNF secre- between cytokines and reactive oxygen molecules tion in response to an LPS challenge was markedly leading to their overproduction during the progression inhibited by >90%, while LPS-induced IL- 1o~and IL- 113 of sepsis to MOD and death. TNF and IL-1 [3 stimulate serum levels were also significantly reduced, but only the production of oxygen free radicals by neutrophils at one time point each by MDL 101,002 treatment. (Meier et al., 1989; Jensen et al., 1992; Shau, 1988; de The results of numerous studies suggest that over- la Harpe & Nathan, 1989), which, in turn, up-regulate production of cytokines and oxygen free radicals, which cytokine synthesis by macrophages (Deforge et al., are involved in normal, localized immune defenses, lead 1992; Wendel, 1991; Pogrebniak et al., 1990; Jensen et to the development of MOD and septic shock. Toxins al., 1992). This reciprocating relationship may result released during severe infection stimulate the production in an uncontrolled circle for the overproduction of these of various cytokines including TNF, IL-lot and IL-11] agents. The release of TNF induced by LPS was markedin a characteristic cascade-like pattern (Fischer et al., ly diminished by treatment with MDL 101,002 and was 1992; Pogrebniak et al., 1992; Chensue et al., 1991; associated with a decreased incidence of organ dysfuncFong et al., 1989), as well as increase the release of tion and improved survival in this study. Serum TNF oxygen-derived free radical molecules (Yoshikawa et levels peaked at 1 h after LPS administration and were al., 1994; N o v e l l i e t a l . , 1989; M e i e r e t a l . , 1989; French reduced >90% by MDL 101,002. Other investigators et al., 1994). TNF, IL-I~, and oxygen radical molecules have also previously shown that various types of free can each individually cause tissue damage similar to radical scavengers decrease TNF production in response that seen in response to LPS (Tracey et al., 1986; to LPS. N-acetylcysteine (a precursor in the synthesis Ohlsson et al., 1990; Okusawa et al., 1988; Novelli et ofglutathione), superoxide dismutase (which dismutates al., 1989; Mallick et al., 1989; Manson & Hess, 1983). the superoxide anion), and PBN all greatly reduced Administration of IL-I~ receptor antagonists (Fischer serum TNF and TNF mRNA levels in dogs, rats, and et al., 1992; Ohlsson et al., 1990; Wakabayashi et al., mice from 30 min to 6 h after LPS administration 1991) or antibodies to TNF or IFN (Tracey et al., 1986; (Pogrebniak et al., 1992; Wendel, 1991; Peristeris et Doherty et al., 1992) reduce the mortality caused by al., 1992; Zhang et al., 1994). The importance of TNF administration of bacteria or LPS to animals. Further- in the development of endotoxin-mediated organ dysmore, antioxidants which act by trapping or metabo- function was also recently shown in a rat model of lizing free radicals, or inhibiting the enzymes involved ARDS, where intratracheal administration of LPS initiin free radical production, also improve survival in ates lung damage and leads to MOD. Inhibition of TNF animal models of septic shock and chronic bacteremia release with rolipram, a specific phosphodiesterase IV (Pogrebniak et al., 1992; Novelli et al., 1989; Wendel, inhibitor, reduced lung, liver, and kidney damage 1991; McKechnie et al., 1986; Peristeris et al., 1992; (Turner et al., 1993). Improved survival has also been French et al., 1994). shown in all of these studies in which TNF release was A key role for reactive oxygen molecules as medi- blocked or greatly reduced (Pogrebniak et al., 1992; ators of endotoxin-induced shock and tissue damage has Wendel, 1991 ; Peristeris et al., 1992; Zhang et al., 1994; been demonstrated by many investigators. An increase Turner et al., 1993). in LPS-stimulated lipid peroxidation which leads to TNF is initially synthesized by activated macroincreased vascular permeability due to endothelial cell phages as a 26,000 mot. wt protein (pro-TNF) which damage occurs after the release of oxygen radicals includes an unusually long (76 amino acids in the MOD and ARDS in conjunction with sepsis (Yoshikawa

et al., 1994; Turner et al., 1993; Dorinsky & Gadek, 1989; Left et al., 1994; Bernard, 1991; Martich et al.,

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human) signal peptide (Kreigler et al., 1988; Kim et al., 1993). Pro-TNF is then incorporated into the cell membrane through the binding of the N-terminal of the peptide which acts as a membrane anchoring region. Finally, enzymatic cleavage of membrane-bound proTNF results in the release of the mature, 17,000 mol. wt circulating form of TNF. Although this mode of secretion differs from that for most secretory proteins, similar secretory pathways have also been described for several other cytokines and growth factors (Kreigler et al., 1988; Kim et at., 1993). A pro-TNF cleaving enzyme has recently been found in macrophages as a membrane-associated protein, and LPS-stimulated TNF release in vitro could be blocked by serine protease inhibitors (Kim et al., 1993; Scuderi, 1989; Scuderi et al., 1989), and more recently, by metalloendopeptidase inhibitors (McGeehan et al., 1994; Mohler et al., 1994). Since serine protease inhibitors, such as ct~ proteinase inhibitor (ctlPl), can be inactivated by oxidation of the methionine residue by superoxide anions (Johnson & Travis, 1979), Wendel has proposed that reactive oxygen radicals produced during septic shock oxidize and inactivate ot~Pl (Wendel, 1991). Loss of this tonic inhibition of tx~PI increases serine protease activity in monocytes and neutrophils leading to cleavage of membrane-bound pro-TNF and increased circulating levels of TNF. This concept is supported by studies in which treatment with ct~Pl protected animals from LPS-, but not TNF-induced shock and death (Wendel, 1991). The mechanism by which free radical scavengers, such as MDL 101,002, reduced endotoxin-mediated TNF secretion in this study may be related to protection of protease inhibition through trapping of reactive oxygen radicals. In agreement with previous studies, circulating IL- 1ct and IL-1[~ levels were highest between 2 and 6 h after LPS challenge When TNF levels had begun to decline (Fischer et al., 1992; Chensue et al., 1991; Fong et al., 1989). Evidence that TNF stimulates IL-1 synthesis has been reported by several groups (Fong et al., 1989; Pober et al., 1986), although others have also reported nearly complete inhibition of TNF release with little or no effect on IL-1 production (Peristeris et al., 1992;

Turner et al., 1993). In. this study, the suppression of LPS-induced TNF secretion by MDL 101,002 was >90% at 1 and 2 h after LPS, while the IL-ltx and IL-I~ levels in the MDL 101,002 treated animals were only modestly reduced at one time point each. Therefore, it is unclear whether free radicals play a significant role in IL-lct and IL-115 production following endotoxin administration. MDL 101,002 did not prevent endotoxin-induced leukopenia, and only partially reduced the thrombocytopenia caused by LPS. The partial inhibition ofthrombocytopenia despite a >90% reduction in LPS-stimulated TNF secretion is in agreement with the results of Turner et al. (1993). In their study, LPS-induced TNF secretion was completely inhibited by a phosphodiesterase IV inhibitor and a small reduction in thrombocytopenia, similar in magnitude to that seen in our study, was observed. In addition, administration of IL-1 receptor antagonists prevented leukopenia and thrombocytopenia in response to IL-11~-induced shock (Ohlsson et al., 1990), but had no effect or only partially prevented these losses in response to bacterial infusion (Fischer et al., 1992; Wakabayashi et al., 1991), suggesting that these responses are not only mediated by TNF and/or IL-1 ~. In summary, we have shown that MDL 101,002, a free radical spin trap, markedly decreases organ dysfunction and mortality caused by LPS in a model of endotoxemia. The decrease in markers of organ dysfunction by MDL 101,002 was associated with a modest reduction in LPS-stimulated IL-Ict and IL-II3 secretion and a marked (>90%) inhibition of TNF secretion. These results are consistent with a role for oxygen free radicals in the pathogenesis of endotoxininduced organ dysfunction and shock. Moreover, they provide evidence of a link between the free radical scavenger-mediated reduction in cytokine secretion and the decrease in tissue damage and mortality in an animal model of endotoxemia.

Acknowledgement - - The authorswish to thank Cindy Wallace

for her excellenttechnicalassistancein the blood chemistryand blood count analysis.

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