American Journal of Pathology, Vol. 141, No. 5, November 1992 Copyright C American Association of Pathologists

Efficacy of Monoclonal Antibody Against Human Recombinant Tumor Necrosis Factor in E. co/i-challenged Swine Gary Jesmok, Craig Lindsey, Melinda Duerr, Michael Fournel, and Thomas Emerson, Jr. From Miles Research Center, Miles Inc., West Haven, Connecticut

Monoclonal antibody against human tumor necrosis factor a (TNFMAb) prevents death induced by intravenous gram-negative bacteria or lipopolysaccharide (LPS) in primates. Although these studies have demonstrated that TNF plays a prominent role in the development of lethal septic shock, exploration of dose-response relationships and possible mechanisms ofprotection have been limited We addressed these questions in a series of experiments conducted in E. coli-challenged pigs. First, we determined that TNFMAb neutralized the cytotoxic activity found in septic pig plasma and in culture media from pig monocytes incubated with LPS. Second, we demonstrated that pretreatment with TNF MAb promotes survival in a dose-dependent fashion, in an otherwise lethal E. coli bacteremic pig model. The results of the survival study highly correlate (r = 0.96, P < 0.01) the presence of TNF in the circulation with mortality. In an additional series of physiologic monitoring e-xperiments designed to delineate possible mechanisms of protection; the authors demonstrate that TNF MAb pretreatment abrogates the prolonged leukopenia thrombocytopenia and microvascular leakiness resulting from intravenous bacterial challenge and maintains arterial blood pressure while diminishingpulmonary edema These findings mayprovide a mechanism whereby neutralization of TNF systemically affords protection against the lethal sequelae of bacteremia (Am J Pathol 1992, 141:1197-1207)

Septic shock often develops as a consequence of overwhelming bacterial infection (sepsis). The incidence of sepsis has increased over the past decade, and this increase most likely reflects an increased number of severely ill patients who are being kept alive longer by im-

proved medical treatments and who are put at greater risk for infections by invasive medical procedures.1 The incidence of sepsis therefore will likely continue to be a major medical problem, particularly in the critical care setting. The clinical features of sepsis cover a broad spectrum, depending on the stage of the disease process.2 The development of septic shock with its attendant high mortality rate is characterized by refractory hypotension, microvascular leakiness, myocardial dysfunction, and ultimately by multi-organ failure.3 Although the pathogenesis of septic shock is exceedingly complex, it has become increasingly apparent that the host inflammatory response contributes to the eventual development of shock and multiple organ failure.4 Bacterial organisms or their products stimulate the host's immune system to elaborate a number of potent inflammatory mediators, including various cytokines, eicosanoids, complement fragments, platelet-activating factor, kinins, etc.4 These inflammatory mediators, when produced in sufficient quantities to be released into the circulation, can have profound effects on blood vessels, the heart, and circulating inflammatory cells. Recent research has demonstrated that many of the systemic effects of sepsis are mediated by cytokines.5 Cytokines are soluble proteins produced predominately by macrophages and monocytes that are beneficial in host defense when limited to discrete sites of infection. If the release is prolonged, however, or if released into the circulation in sufficient quantities, some cytokines may have deleterious consequences. Among the known cytokines, tumor necrosis factor-alpha (TNF) appears to play a significant role in the pathophysiology of sepsis and septic shock.6 Several lines of evidence support this view. Clinical studies in patients with gram-negative or gram-positive sepsis have correlated increased plasma TNF levels with a negative outcome.79 The infusion of TNF in laboratory animals'012 and humans13 mimics many metabolic and pathophysiologic derangements Accepted for publication May 13, 1992. Address reprint requests to Dr. Gary Jesmok, Miles Research Center, Miles Inc., 400 Morgan Lane, West Haven, CT 06516.

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seen in acute infections. Furthermore, monoclonal antibody against human TNF (TNF MAb) has protected against the lethality associated with endotoxin,14 grampositive,15 and gram-negative16'17 bacterial challenges in nonhuman primates. Although these studies have demonstrated that TNF is intimately involved with mortality resulting from an overwhelming bacteremia, exploration of dose-response relationships and possible mechanisms of protection have been limited by the high cost of nonhuman primate experiments and lack of information concerning cross-reactivity of TNF MAb (raised against human TNF) in other species. We address these limitations in a series of experiments conducted in pigs challenged with E. coli bacteria. First, we demonstrate that TNF MAb can bind and neutralize TNF elicited in bacteremic pigs. Second, we demonstrate that TNF MAb, in a dose-dependent manner, prevented mortality after an otherwise lethal intravenous E. coli challenge. Third, we have provided evidence for possible mechanisms by which TNF MAb affords this protection.

Materials and Methods Experimental Protocols In Vitro Experiments Neutralization of TNF-dependent cytotoxic activity with TNF MAb was quantified using a modification of the TNF bioassay referenced below. Briefly, 75 IlI containing different concentrations of TNF MAb (0 to 1 ,ug/ml) were mixed with 75 ,u septic pig plasma, culture medium from LPS-stimulated pig monocytes, or rhTNF in cell culture media containing 10% fetal bovine serum at approximately 300 to 500 pg TNF cytotoxic activity/ml and incubated for 2 hours at 37°C. This mixture was added to actinomycin D-treated WEHI cells and serially diluted threefold in 96-well plates. After overnight incubation, quantitation of live cells was performed as outlined originally.18 The neutralizing ability of TNF MAb against the cytotoxicity found in culture medium from LPS-stimulated monocytes was determined only at a TNF MAb concentration of 1 ,ug/ml. The neutralizing ability of TNF MAb against the cytotoxicity found in septic pig plasma was determined at TNF doses from 0 to 1 Kg/ml. The effective dose of 50% (ED50) from these studies was calculated by linear regression analysis of the dose-response curve and represents the concentration of TNF MAb that reduced the WEHI cell cytotoxicity by 50% relative to samples without the addition of TNF MAb. In Vivo Experiments All in vivo experiments were conducted in male and female Chester White swine, mean weight, 13.7 + 0.32

kg; range, 9 to 17 kg (Microbiological Media, Concord, CA, and Gares Burrow, Grass Valley, CA). Pigs were kept in concrete floored runs at a temperature of 76 to 78°F and received food and water ad libitum.

Dose-Response Studies The pigs were fasted for 12 hours before induction of anesthesia by intramuscular injection of 8 mg/kg xylazine hydrochloride and 15 mg/kg ketamine hydrochloride. Baseline blood samples were collected aseptically by direct venipuncture of the superior vena cava. Percutaneous catheterization of an ear vein using a 24-gauge intravenous Teflon catheter (Abbocath, Abbott Hospitals Inc., North Chicago, IL) allowed intravenous delivery of 15 (n = 6), 7.5 (n = 6), 1.5 (n = 12), 0.15 (n = 6), or 0.015 (n = 6) mg/kg TNF MAb. Paired controls were infused with an equivalent volume of TNF MAb excipient (0.27 mol/l glycine and 1% maltose, n = 10) or IgG (Gamimune N, Intravenous Immune Globulin, Miles Inc., Berkeley CA, at 15 mg/kg; n = 6). The latter served as a nonspecific protein immunoglobulin control. In all animals, this was followed by intravenous infusion of E. coli, (strain 0:50 Kl, 8.9 ± 0.46 x 1 o8 CFU/kg over 5 minutes). This dose was determined from preliminary experiments that examined the dose of E. coli required to attain a LD100 within 48 hours. In these studies, doses greater than 5.0 x 108 CFU/kg resulted in uniform mortality rate. At 2 hours after challenge, unanesthetized animals were restrained, and blood samples were collected by direct venipuncture of the superior vena cava. Immediately after this blood sampling, an initial dose of 5 mg/kg gentamicin sulfate (Elkin-Sinn Inc., Cherry Hill, NJ) was given intramuscularly, followed by intramuscular injections of 3 mg/kg gentamicin twice daily through 72 hours or until death. All animals were observed without additional therapeutic support until death or for 7 days. The dose of TNF MAb required to reduce the mortality rate to 50% (ED50) was calculated by the logarithmic probit method.19

Physiologic Monitoring To assess possible mechanisms of protection afforded by TNF MAb in septic pigs, the following series of experiments were performed. Pigs were fasted for 12 hours before induction of anesthesia by intramuscular injection of 8 mg/kg xylazine hydrochloride and 15 mg/kg ketamine hydrochloride. The animals then were intubated and prepared for aseptic cannulation of the right common carotid artery and internal jugular vein. A 19-gauge vialon resin catheter (Abbocath), was secured in both the artery and vein. Anesthesia was maintained throughout

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the surgical procedure and the 5-hour experimental protocol by intravenous infusion of sodium pentobarbital (10-15 mg/kg/hour). In paired studies (six treated animals and six control animals), baseline cardiovascular monitoring and collection of blood samples was followed by an intravenous infusion of 15 mg TNF MAb/kg in one animal and an equivalent volume of its excipient in the other. In both animals, this was followed by an intravenous infusion of E. coli, 7.6 ± 1.7 x 1 08, CFU/kg. Determination of mean arterial blood pressure (MAP) was made at hourly intervals throughout the monitoring period. Arterial blood samples were collected at hourly intervals for the determination of TNF, interleukin-6 (IL-6), bacterial colony-forming units (CFU), leukocyte and platelet counts, and hematocrit. Intravenous antibiotic therapy was initiated at 3 hours after challenge, when each animal received 75 mg/kg ceftriaxone sodium Rocephin Hoffmann-La Roche Inc., Nutley, NJ). This dose of antibiotic was sufficient to remove all circulating bacterial counts with 60 minutes. Animals breathed spontaneously and were kept in sternal recumbency on heating pads throughout the monitoring protocol. At the end of the 5-hour monitoring period, animals were killed by intravenous infusion of pentobarbital sodium (Schering Corp., Kenilworth, NJ). The lungs were removed for visual inspection. The left lung was weighed and homogenized with an equal weight of distilled water for the determination wet to dry weight ratios. Because this dose of E. coli resulted in septic shock within 2 to 3 hours (systolic pressure .g and 0.27 ,ug TNF MAb/ng of porcine TNF and rhTNF, respectively. Tumor necrosis factor MAb, 1 ,ug/ml, also completely eliminated the cytotoxicity found in the culture medium of LPSstimulated pig monocytes (data not shown).

In Vivo Efficacy Once the ability of TNF MAb to neutralize porcine TNF was confirmed, studies were performed to determine the

100

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0

200 400 600 TNF MAb Concentration

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(ng/ml) Figure 1. Neutralization of cytoxicity in plasmna with a monoclonal antibody to rhTNF. *, plasma from septic pigs; A, rhTNF in 10% fetal bovine serum. Data are means ± SEM. No significant difference porcine TNF vs. rbTNF.

dose-response efficacy of TNF MAb pretreatment in E. coli challenged pigs. In paired studies, pigs were pretreated with 15 (n = 6), 7.5 (n = 6), 1.5 (n = 12), 0.15 (n = 6), 0.015 (n = 6) mg/kg TNF MAb or its excipient (n = 6) before intravenous challenge with E. coli, as described in the methods section. Because sufficient quantities of another murine monoclonal antibody were not available, human intravenous immunoglobulin (hIgG, Gamimune N) was used as a nonspecific protein control in an additional six pigs. Mortality rate in the hIgG (15 mg/kg) pretreated group was identical to the excipient pretreated group (100%), and the two groups were combined (n = 12) for comparison with TNF MAb-pretreated animals. Pretreatment with TNF MAb prevented death and detection of plasma TNF in a dose-dependent manner (Figure 2). In the control animals (excipient or hIgG), E. coli infusion resulted in the presence of 2.5 ± 0.52 ng of TNF/ml plasma at 2 hours after challenge (Figure 2B) and 100% mortality rate (Figure 2A) with a mean time to death of 16 hours (range, 3.5 to 22 hours). Plasma TNF was not detected at 2 hours after challenge in all animals pretreated with 15 to 0.15 mg/kg TNF MAb. All but one of these animals (0.15 mg/kg dose group) lived at least 7 days and were considered survivors. As the pretreatment dose of TNF MAb was reduced further, to 0.015 mg/kg, mortality rate increased to 86% and TNF was detected in the plasma of every animal, 0.49 ± 0.25 ng/ml. The mean time to death in this group was not different from that in the excipient control group (data not shown). The ED50 in this model was determined to be 50 ,ug TNF MAb/kg body weight. The detection of TNF in the plasma space at 2 hours after challenge was highly correlated with mortality rate (r = 0.96, P < 0.01). The absolute concentration of plasma TNF at 2 hours was also correlated with mortality rate but to a lesser degree, r = 0.63. The lethality of the bacterial challenge in these studies appeared to be related to overt circulatory shock (rapid breathing, pale skin tone, etc.) and severe pulmonary dysfunction

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*

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4 5 3 Time (h) Figure 3. Blood colonyforming units of E. colifollowing intravenous bacterial challenge. Data are means + SEM. Excipienttreated pigs represented by open bars and TNF MAb-treated pigs represented by closed bars. Intravenous antibiotic given at 3 hours. No significant difference TNF MAb vs. excipient-treated pigs.

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0.015 0.150 1.500 7.500 15.000 Pretreatment Dose of TNF LAb (mg/kg)

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Figure 2. Effect of TNFMAb on mortality in E. coli-challengedpigs, relationship to circulating TNF levels at 2 hours post-challenge. *P < 0.05 TNF MAb vs. excipient-treated pigs. Correlation coefficient mortality vs. plasma TNF levels, r = 0.96 (P < 0.01).

(rales with froth and bloody exudate from the airways at death).

Physiologic Monitoring Because TNF MAb pretreatment promoted survival in the acute bacteremic model, additional studies were performed to ascertain possible mechanisms of protection. These studies were conducted in anesthetized animals pretreated with 15 mg/kg TNF MAb or an equivalent volume of excipient before intravenous challenge with E. coli. Antibiotic therapy was initiated 3 hours after challenge. Colony-forming units of any type were not detectable in the blood of any animal at baseline. Intravenous challenge with E. coli resulted in the detection of CFUs in the blood that appeared to increase in concentration until the administration of 75 mg/kg ceftriaxone sodium at 3 hours post-challenge (Figure 3). The ceftriaxone sodium was very effective in sterilizing the vascular space because bacteria were not detected in the blood 60 minutes after administration. E. coli challenge resulted in a sustained decrease in mean arterial pressure (MAP), reaching a nadir of 58% of

control by 3 hours (Figure 4). Arterial pulse pressure (systolic-diastolic) also decreased (data not shown), suggesting that a decrease in cardiac output may be partly responsible for the decline in arterial pressure. In pigs treated with TNF MAb, the decline in blood pressure was abrogated and MAP was significantly greater than in untreated controls during the 1 to 5 hour time period (P < 0.05).

TNF and IL-6 Levels Tumor necrosis factor was not detectable in the plasma of any animal at baseline. After E. coli challenge, in excipient treated animals, TNF appeared as early as 30 minutes, reached peak plasma concentrations at 1.25 hours (0.8 0.13 ng/ml), and was no longer detectable in the plasma by 5 hours after challenge. Plasma TNF was not detectable at any time point in any of the animals pretreated with TNF MAb (Figure 5A). Plasma IL-6 was not detectable in any animal at baseline. In excipient treated animals, IL-6 was increased by 3 hours and continued to increase throughout the 5-hour monitoring pe±

120 r

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40

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3 4 5 Time (h) E. coli-inducedhypotension in the

Figure 4. Effect of TNFMAb on pig. Data are means + SEM. * P < 0.05 TNFMAb

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Figure 5. Effect of TNF MAb on TNF and IL-6 levels in E. colichallenged pigs. Data are means + SEM. 0, excipient treated pigs; A, TNF MAb treated pigs. *fP < 0.05 TNF MAb vs. excipient.

riod. Tumor necrosis factor MAb pretreatment significantly decreased plasma levels of IL-6 after bacterial challenge, and IL-6 levels were significantly lower in animals pretreated with TNF MAb at every point after 3 hours (Figure 5B).

Hematology Figure 6A illustrates the profound leukopenia that characterizes the acute inflammatory reaction induced by intravenous E. coli challenge. Leukocytes (primarily neutrophils) decline precipitously by 1 hour and remain at very low levels (10% of control) through 5 hours. A much different leukocyte profile is observed in the TNF MAbtreated pigs. Although leukocytes also decrease substantially at 1 hour, the leukopenia is transient, and levels return to baseline by 4 to 5 hours. E. coli infusion also resulted in a progressive decrease in circulating platelet levels (Figure 6B). Pretreatment with TNF MAb significantly diminished the thrombocytopenia at 3 to 5 hours.

The hematocrit profile in pigs subjected to E. coli challenge is illustrated in Figure 7. In both TNF MAb treated

4

6

Time (h)

Figure 6. Effect of TNF MAb on E. coli-induced leukopenia and thrombocytopenia in the pig. Data are means t SEM. *P < 0.05 TNF MAb vs. excipient.

and untreated pigs, the hematocrit increases very early (1 hour), most likely as a result of splenic contraction and the release of stored red blood cells. In the untreated group, however, there is a gradual increase in the hematocrit after 2 hours that continues to rise through 5 hours. In marked contrast, in pigs treated with TNF MAb, the hematocrit, after increasing early, quickly stabilized and remained unchanged for the duration of the experiment. Other indices of increased microvascular permeability were also evident. A significant increase in lung wet/dry weight ratio (W-D/D) was noted in excipient treated pigs versus TNF MAb treated pigs (Table 1). 40

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Figure 7. Effect of TNF MAb on E. coli-induced hemoconcentration in the pig. Data are means SEM. *P < 0.05 TNFMAb-treated pigs vs. excipient.

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Table 1. Effect of TNF MAb on Lung

Wet/TDy Ratios in

E. coli-challengedPigs

W-D/D

Groups -TNF MAb +TNF MAb

5.5 ± 0.3 4.6 ± 0.2*

* P < 0.05 TNF MAb vs. excipient. Values are means ± SEM.

A further conformation of incre ased microvascular leakiness was apparent in an additiional series of experiments designed to maintain arteria pressure above the shock levels (>90 mm Hg systolic), which result from the E. coli infusion, by the intravenous> infusion of isotonic saline. This treatment regimen was chosen because it is standard procedure for patients in the early stages of sepsis. Over the 5-hour monitoring period, excipient pretreated animals required an averac;e of 2030 ± 332 ml (149 ml/kg) of fluid and the TNF MAib pretreated animals required 208 138 ml (14 ml/kg) to maintain systolic arterial pressure above 90 mm Hg (FFigure 8). Aggressive fluid resuscitation in excipient trealted animals was not necessary until 90 minutes after chzallenge. ±

Discussion Gram-negative bacteremia in pigs induces an acute intravascular inflammatory reaction c:haracterized by profound leukopenia, thrombocytopeni a, cardiovascular deterioration, increased microvasculau permeability, multipie organ failure, and death.22 /Although somewhat artificial, live E. coli challenge in piglIscan mimic many of the early pathophysiologic changes, that occur in clinical sepsis,1 and the pathogenic mecha nisms of tissue injury may be similar to those that play a role in more clinically relevant situations.3 It is well known tlhat species vary considerably in their sensitivity to E. co)1i or LPS.23 Pigs and ruminants (eg, sheep, calves, gocats) are exceedingly 2400 r

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excipient.

sensitive, and relatively small doses induce marked effects on cardiopulmonary function, whereas dogs, rodents, and certain nonhuman primates are relatively insensitive.24 Irrespective of the species, however, the pathophysiologic manifestations of sepsis include severe inflammatory injury, ie, loss of microvascular integrity and barrier function, extravasation of plasma protein, hypovolemic shock, and death. In the experiments described herein, we were primarily interested in the role of TNF in the early pathophysiologic events associated with acute bacteremia in pigs. We have shown that a murine monoclonal antibody directed against rhTNF (BAY X1351) neutralizes the cytotoxic activity found in septic pig plasma toward WEHI clone 13 cells. The neutralization profile of TNF MAb against porcine TNF was not significantly different from that against rhTNF and may likely be due to the significant sequence homology between rhTNF and porcine TNF.25 Prompted by these in vitro observations, we evaluated the effect of TNF MAb in the E. coli challenged pig. Without further treatment, this challenge proved to be 100% lethal, with a mean time to death of 16 hours. We pretreated pigs intravenously with 15.0 to 0.015 mg/kg TNF MAb immediately before bacterial challenge and demonstrated that if circulating TNF was neutralized, mortality rate was significantly reduced. Tumor necrosis factor MAb therapy has been previously shown to prevent or significantly reduce mortality rate in baboons after LD100 challenge with E. coli.16,17 Although the pig and baboon models share some similarities, ie, LD100 challenge dose, antibiotics given, similar mean time to death, restricted fluid resuscitation, doses of TNF MAb required to promote survival differ significantly in these two species. Hinshaw et al17 reported that 15 mg/kg TNF MAb prevented death after E. coli challenge in the baboon (at lower doses, mortality rate was significantly increased, personal communication). In contrast, in the E. coli challenged pig, 1.5 mg/kg TNF MAb completely prevented death, and 0.15 mg/kg TNF MAb reduced the mortality rate from 100% to 17% (P < 0.05). Based on the many similarities between these two models, including similar neutralizing ability of TNF MAb (Bayer Xl 351) to neutralize baboon and porcine TNF, one possible reason for differences in the dose of TNF MAb required to prevent death may be related to the amount of intravascular TNF detected in each of these species subsequent to bacterial challenge (in the absence of TNF MAb). Peak plasma TNF levels ranged between 82 and 213 ng/ml in the baboon,17 but were only 0.68 to 7.5 ng/ml (mean of 2.5 + 0.52 ng/ml) in the pigs. The reason for the differences in circulating TNF levels is unclear, although the dose of E. coli required to achieve lethality was also lower in the pig.

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In vitro neutralization of septic pig plasma allowed calculation of the dose of TNF MAb that would be required to neutralize 50% of the cytotoxicity of porcine TNF (ED50). Under these conditions, approximately 260 ng TNF MAb is required to neutralize 1 ng TNF. Using this neutralizing ratio, a calculation of the theoretical dose of TNF MAb necessary to neutralize intravascular TNF after E. coli challenge in the pig can be made. Total intravascular TNF after bacterial challenge in the pig can be estimated by assuming that plasma TNF is in equilibrium (production and clearance rates are equal) and that the plasma volume of the pig is 50 ml/kg. Total intravascular TNF is then approximated by the area under the curve defined in Figure 5A. If this value, 6.28 ng/ml/hour, is multiplied by 260 (approximate amount of TNF MAb necessary to neutralize 1 ng TNF), then the dose of TNF MAb necessary to neutralize total estimated intravascular TNF is 78.5 ,ug TNF MAb/kg. Half of this value is then equivalent to the theoretical dose of TNF MAb necessary to reduce plasma TNF by 50%. This value, 39.2 ,ug TNF MAb/kg, compares favorably with the calculated dose of TNF MAb necessary to reduce mortality rate by 50%, ED50 = 50 ,ug TNF MAb/kg (determined from doseresponse studies). Although these calculations are subject to criticism, that a calculated dose of TNF MAb based only on neutralization of intravascular TNF can approximate the dose of TNF MAb necessary to reduce mortality rate implicates circulating TNF as at least predictive and possibly responsible for subsequent deaths in this acute bacteremic model. It would also suggest that neutralization of circulating TNF, by an antibody that is mostly retained within the vascular space, may offer therapeutic advantages over inhibition of TNF synthesis or neutralization at extravascular sites. Although these results demonstrate that plasma TNF in the septic pig is highly correlated with ultimate death, the value of circulating TNF as a predictive marker of outcome in patients is not clear. In experimental animal studies, after intravenous bacteria or endotoxin infusion, plasma TNF has been shown to peak at approximately 90 minutes after challenge and disappear rapidly from the circulation.1826 In contrast, the sepsis syndrome in humans results in a plasma TNF profile very different from that seen after acute intravenous challenge in laboratory animals. Plasma TNF concentrations in septic patients79 27 are similar to those we report for septic pigs; however, unlike the pig model we describe, TNF levels in patients were associated with multiple peaks and valleys and can be sustained for days.9 Although several clinical studies have shown a positive correlation between elevated levels of plasma TNF and ultimate death,8927 a significant fraction of septic patients do not have detectable levels of TNF in the plasma.8'27 It is not known, however, at what stage of the disease process these samples

were taken. Thus, the predictive value of plasma TNF for outcome in clinical sepsis remains controversial. Several studies have suggested that mediators with longer circulating half-lives, such as IL-6 or IL-1, that are at least partially induced by TNF, may be better predictors of outcome in the clinical setting.28' 9 The experiments reported herein support this concept. Interleukin-6 levels in excipient treated animals were significantly elevated above control by 3 hours after challenge and continued to increase throughout the monitoring period (Figure SB). These levels were significantly reduced in animals pretreated with TNF MAb, as was shown in septic baboons by Fong et al.30 Because these and other studies16'17 have demonstrated that TNF MAb promotes survival in an otherwise lethal bacteremic model, we sought to delineate possible mechanisms whereby this protection is afforded. We measured mean arterial pressure (MAP) as a gross index of circulatory function and sequentially sampled arterial blood to assess leukocyte and platelet levels as indices of pathologic cellular-endothelial interactions. We measured arterial hematocrit, lung wet/dry ratios, and isotonic fluid requirements as indicators of endothelial barrier dysfunction. The results of these studies suggest several mechanisms whereby neutralization of plasma TNF may provide protection against the lethal sequelae of bacteremia. First, neutralization of circulating TNF attenuated the decrease in MAP resulting from E. coli challenge. The fall in arterial blood pressure in untreated pigs is indicative of circulatory shock (MAP < 70 mmHg or systolic pressure < 90 mmHg). Whether the decline in blood pressure is a direct effect of TNF or an indirect effect induced through release of other mediators6 is not known. It is known that hypotension is a major dose-limiting side effect associated with TNF infusion in humans when TNF is employed as an antineoplastic agent.31 In experimental animals, TNF has been shown to cause peripheral vasodilation, perhaps by the generation of nitric oxide32 and myocardial dysfunction.33 Whether TNF is mediating the decline in arterial pressure through an effect on the myocardium or the peripheral vasculature cannot be determined from this study; nevertheless, TNF MAb treatment abrogates the development of circulatory shock in this model. Damage to the endothelium appears to play a pivotal role in the development of refractory septic shock.1 We have several indirect indications that the microvascular endothelial barrier has been compromised after E. coli challenge, eg, increased hemoconcentration, increased lung wet/dry ratio, and increased fluid requirements necessary to maintain arterial pressure above shock levels. The pathophysiologic mechanism whereby this lesion is induced during sepsis is perhaps an aberrant extension of the known biologic effects of TNF on leukocytes and

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the endothelium.3 The adherence of peripheral blood leukocytes to the endothelial lining of blood vessels (principally postcapillary venules) is one of the major hallmarks of the inflammatory reaction.35 The adhesive interaction between the leukocyte and endothelium is a critical event in host response to infection, but inappropriate control of this interaction may contribute to tissue injury during bacteremia.36 Activated leukocytes produce many products that can damage endothelial cells, including toxic oxygen radicals, arachidonate metabolites, platelet-activating factor, and multiple proteases that can degrade elastin, collagen, and basement membranes.36 Errant activation of these phagocytic cells or abnormal leukocyteendothelial interaction may result in tissue injury during inflammatory states like sepsis. It has become apparent that TNF plays a major role in modulating changes on both the leukocyte and endothelial cells, which may lead to the development of inappropriate leukocyteendothelium interaction and microvascular injury under certain conditions.5'6 Tumor necrosis factor enhances the adhesiveness of neutrophils by upregulating cell surface glycoprotein receptors (CD1 1 b/CD1 8).6,37 Tumor necrosis factor increases cytotoxicity, enhances production of superoxide anion and H202, and stimulates degranulation and the release of a variety of proteolytic enzymes.3840 Tumor necrosis factor primes neutrophils, causing an increased production Of 02 on stimulation.41 Recent experiments have demonstrated that neutrophil activation by TNF is greatly enhanced when neutrophils are adherent to biologic surfaces.42 Tumor necrosis factor also has dramatic effects on the "stickiness" of the endothelium for leukocytes. On exposure to TNF, endothelial cells become markedly adhesive for neutrophils, eosinophils, basophils, and monocytes.43 Tumor necrosis factor exposure induces ELAM-144 and ICAM-145 on endothelial cells. In addition to increased adhesion of both the leukocyte and endothelial surface, TNF promotes endothelial cell production of leukocyte-activating factors (TNF itself activates adherent neutrophils).42 Tumor necrosis factor causes endothelial cells to synthesize platelet-activating factor46 and IL-8,47 a novel neutrophil chemoattractant and activator. Tumor necrosis factor also causes endothelial cells to express and secrete IL148 and (IL-6).49 Acute intravenous bacterial challenge in pigs is characterized by prolonged leukopenia (Figure 6A). Tumor necrosis factor plays a major role in mediating this effect because TNF MAb treatment attenuates the leukopenia. We hypothesize that it is the hyperadherent state of both the leukocyte and endothelium induced by plasma TNF and the concomitant activation of the adherent leukocytes that are directly responsible for endothelial injury. In the current model, we postulate that the hemoconcentra-

tion, increased lung wet/dry ratio, and massive fluid requirements are a manifestation of this deleterious process. Tumor necrosis factor also has profound effects on endothelial hemostatic properties. Tumor necrosis factor exposure causes endothelial cells to synthesize and express tissue factor on their surface and causes a loss of thrombomodulin expression,'0 increases plasminogen activator inhibitor type 1,51 and induces PAF synthesis.46 These endothelial alterations and the induced release of platelet-activating factor promote a shift to a state conducive to coagulation and possibly microthrombosis. These effects of TNF may play a major role in the induction of disseminated intravascular coagulation associated with severe infections52 and may be responsible for the hemorrhagic necrosis of tumors in experimental animals. The thrombocytopenia observed in these experiments (Figure 6B) may reflect microthrombi formation mediated by TNF-induced procoagulant alterations because TNF MAb diminishes the decline in platelets counts. The thrombocytopenia also may result from endothelial injury. In summary, neutralization of circulating TNF promoted survival in an otherwise lethal E. coli bacteremic pig model. Neutralization of plasma TNF attenuated the leukopenia, thrombocytopenia, and severe microvascular leakage induced by bacterial challenge and maintained arterial blood pressure. These findings may suggest a mechanism whereby neutralization of circulating TNF provides protection against the lethal inflammatory sequelae of sepsis.

Acknowledgments The authors thank Ms. Keri Sweet, Ms. Cecilia Valdez, and Mr. Thomas Thompson for technical assistance, Dr. A. A. Fowler for (in part) plasma from septic pigs, Dr. R. Hector and Ms. Alena Edwards for bacterial inoculum preparation, and Ms. Stacey Lawn and Ms. Deborah Smith for manuscript preparation.

References 1. Bone RC: The pathogenesis of sepsis. Ann Intern Med 1991, 115:457-469 2. Bone RC: Sepsis, the sepsis syndrome, multiorgan failure: A plea for comparable definitions. Ann Intern Med 1991, 114: 332-333 3. Parrillo JE, Parker MM, Natanson C, Suffredini AF, Danner RL, Cunnion RE, Ognibene FP: Septic shock in humans: Advances in understanding of pathogenesis, cardiovascular dysfunction and therapy. Ann Intern Med 1990,113:227242 4. Dunn DL: Role of endotoxin and host cytokines in septic shock. Chest 1991, 100(3):164s-1 68s

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