Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 4845-4849, June 1992 Medical Sciences

Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor a in vitro and in vivo (cytokine inhibitors/endotoxemia/septic shock/humans/primates)

KIMBERLY J. VAN ZEE*, TADAHIKO KOHNOt, EVA FISCHER*, CRAIG S. ROCK*, LYLE L. MOLDAWER*, STEPHEN F. LOWRY*t

AND

*The Laboratory of Surgical Metabolism, New York Hospital-Cornell University Medical Center, 525 East 68th Street, F-2016, New York, NY 10021; and

tSynergen, Inc., 1885 33rd Street, Boulder, CO 80301 Communicated by Igor Tamm, January 24, 1992 (received for review November 11, 1991)

activity. This process may have additional significance in vivo, as released soluble receptors may inhibit TNFa bioactivity by binding to the molecule and preventing ligand binding to the cellular TNFR. Hence, the appearance of such extracellular soluble receptors may provide a regulatory mechanism for modulation of excessive TNFa activity arising in response to severe injury or infection.

ABSTRACT Tumor necrosis factor a (TNFa), a primary mediator of systemic responses to sepsis and infection, can be injurious to the organism when present in excessive quantities. Here we report that two types of naturally occurring soluble TNF receptors (sTNFR-I and sTNFR-II) circulate in human experimental endotoxemia and in critically ill patients and demonstrate that they neutralize TNFa-induced cytotoxicity and immunoreactivity in vitro. Utilizing immunoassays that discriminate between total sTNFR-I and sTNFR-I not bound to TNFa, we show that sTNFR-I-TNFa complexes may circulate even in the absence of detectable free TNFa. To investigate the therapeutic possibilities of sTNFR-I, recombinant protein was administered to nonhuman primates with lethal bacteremia and found to attenuate hemodynamic collapse and cytokine induction. We conclude that soluble receptors for TNFa are inducible in inflammation and circulate at levels sufficient to block the in vitro cytotoxicity associated with TNFa levels observed in nonlethal infection. Administration of sTNFR-I can prevent the adverse pathologic sequelae caused by the exaggerated TNFa production observed in lethal sepsis.

MATERIALS AND METHODS TNFa Immunoactiit. TNFa immune activity was determined by a sandwich ELISA utilizing a mouse anti-human monoclonal antibody [provided by P. Tekamp-Olsen (Chiron) and identified as 18.1.1] as the capture protein and a rabbit anti-human TNFa antiserum (rabbit no. A5293) (23). A standard curve was generated with recombinant human TNFa (human rTNFa; Synergen). The sensitivity of the assay is 34-100 pg/ml. TNFa Cytotoxicity. Cytotoxicity was assessed by using the WEHI 164 clone 13 fibroblast bioassay (17). Human rTNFa in normal human plasma was used as a standard. The sensitivity of the assay is 15-30 pg/ml. To confirm that the cytotoxicity observed was specifically due to TNFa, the bioassay was repeated with neutralizing antibodies against human rTNFa. Samples from endotoxemic volunteers and bacteremic baboons that were found to have cytotoxic activity were incubated for 30 min at room temperature with and without antibodies raised against human rTNFa. These samples were reassayed, and those incubated with the antibody were found to have no measurable cytotoxicity. Other Cytokine Assays. Interleukin-lp (IL-1ip) levels were assayed by ELISA as described (18). The sensitivity of this assay is 30 pg/ml. A B.9 hybridoma proliferation assay was used to determine IL-6 levels, with the number of units/ml being defined as the reciprocal of the sample dilution required to produce half-maximal proliferation (lower limit of detection, 2 B.9 units/ml) (19). Recombinant Soluble TNFRs (rsTNFRs). Two fragments of human TNFR-I and TNFR-II have been purified, their cDNAs cloned, and the encoded products characterized (9, 20). These soluble receptors were expressed in Escherichia coli and chromatographically purified to homogeneity. Both forms are nonglycosylated and competitively inhibit 1251_ labeled TNFa binding to cell-surface receptors on U-937 cells

Tumor necrosis factor a (TNFa) is widely appreciated as a principal mediator of systemic responses to sepsis and injury. Produced by inflammatory cells in response to diverse infectious stimuli and tissue injury, TNFa induces a cascade of endogenous mediators that direct host immunologic functions (1). While TNFa may thus serve as an essential element in host defense, the excessive tissue production of TNFa can mediate detrimental systemic effects by acutely precipitating a syndrome similar to that of septic shock (2), and lesser degrees of chronic TNFa production appear to induce anorexia and cachexia (3, 4). Thus, pathologic conditions may result from the excessive production and activity of TNFa. Naturally occurring inhibitors of TNFa activity have been identified in human urine and serum and in cell-culture systems (5-10). The isolation and characterization of these inhibitors have revealed at least two distinct species, an '=30kDa protein with the NH2-terminal sequence Asp-Ser-ValCys-Pro-Gln and an -40-kDa protein with the NH2-terminal sequence Leu-Pro-Ala-Gln-Val-Ala (7-11). These two proteins are the extracellular domains of the TNF receptors (TNFRs) types I and II (TNFR-I and TNFR-II), respectively (8, 9, 12-15), and apparently are shed from the cell surface in response to many of the same inflammatory stimuli that are known to induce TNFa production (16). The shedding of such receptors and resultant acute decrease in the number of TNFRs on the cell surface may serve to transiently desensitize cells, thereby providing a mechanism for inhibition of TNFa

Abbreviations: TNF, tumor necrosis factor; rTNFa, recombinant TNFa; TNFR, TNF receptor; sTNFR-I and sTNFR-II, soluble TNFR types I and II; rsTNFR-I and rsTNFR-II, recombinant human sTNFR types I and II; NYH-CUMC, New York Hospital-Cornell University Medical Center; IL-1p and IL-6, interleukins 1,B and 6; APACHE, acute physiology and chronic health evaluation. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4845

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Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Van Zee et al.

with the same binding capacity as naturally occurring inhibitors (9, 20). sTNFR Assays. Concentrations of sTNFR-I and sTNFR-II were determined by ELISA. To determine the total concentration of sTNFR-I or sTNFR-II, an affinity-purified polyclonal antibody was utilized as the capture protein. To determine the quantity of sTNFR-I not bound to TNF, an ELISA with human rTNFa as the capture protein was used. This latter ELISA takes advantage of the fact that sTNFRI-TNF complexes do not bind to TNFa-coated polyvinyl chloride plates, whereas free, unbound sTNFR-I does. Rabbit polyclonal antisera raised against human rsTNFR-I (rabbit no. 8081) and rsTNFR-II (rabbit no. 2138) were purified by affinity chromatography using Affi-Gel 10 (Bio-Rad) columns coupled with rsTNFR-I or rsTNFR-II, respectively. Aliquots of the affinity-purified antibodies were biotinylated with NHS-LC-biotin (Pierce) according to the manufacturer's specification and were used as the second antibody in the ELISAs. The concentration of sTNFR was calculated according to a standard curve generated with human rsTNFR-I or rsTNFR-II. The sensitivity of the assays is 0.2 ng/ml. In Vitro Neutralization of Endogenous TNFa by Soluble Receptors. rsTNFR-I or rsTNFR-II was added to plasma obtained from volunteers 1.5 hr after administration of 20 units of endotoxin per kg of body weight (see "Human Studies" below). Endogenous sTNFR-I and sTNFR-II levels (determined by ELISA) were less than 5 ng/ml. Plasma samples from different individuals were allowed to incubate at room temperature for 30 min with increasing quantities of soluble receptors (5-50 ,ug/ml) and then assayed in triplicate for TNFa cytotoxicity by using the WEHI 164 clone 13 fibroblast bioassay. A standard curve in normal human plasma was generated with human rTNFa and used to equate cytotoxicity of the samples with concentrations of TNFa. In Vitro Neutralization of Human rTNFa by Soluble Receptors. rsTNFR-I and rsTNFR-II (5 ng/ml to 5 ,ug/ml) were added to normal human plasma to which 1.5 ng of human rTNFa had been added per ml. The mixtures were allowed to incubate at room temperature for 30 min and then assayed for TNFa cytotoxicity by WEHI bioassay and TNFa immunoreactivity by ELISA. All samples were assayed in triplicate and compared with those of the control samples (normal human plasma with 1.5 ng of added human rTNFa per ml). Human Studies. Adult male volunteers were admitted to the Clinical Research Center of New York Hospital-Cornell University Medical Center (NYH-CUMC) after screening by history, physical examination, and biochemical and hematological profiles to exclude those with preexisting disease. Informed written consent was obtained under guidelines approved by the Institutional Review Board of NYH-CUMC. At t = 0, 20 units of national reference endotoxin (E. coli 0113, lot EC-5; Bureau of Biologics, Food and Drug Administration, Bethesda, MD) per ml was administered i.v. Heparinized arterial blood samples were obtained prior to endotoxin administration (baseline) and at intervals thereafter. After obtaining informed consent from the patient or family members, and following guidelines of the Institutional Review Board of NYH-CUMC, we obtained blood samples from 12 critically ill patients repeatedly during their clinical course (total number of samples = 56). The patients had sustained a major traumatic injury or undergone a major surgical procedure, and each experienced episodes of hemorrhagic shock and/or sepsis. To quantify the severity of illness in these patients, a revised acute physiology and chronic health evaluation (APACHE II) score (21) was calculated for each patient. The APACHE II scores in the 12 patients ranged from 11 (5% predicted mortality) to 31 (70%o predicted mortality) with a mean score of 19 ± 2 (mean +

SEM, 20% predicted mortality).

In Vivo Adinistration of sTNFR-I. The experimental protocol was approved by the Institutional Animal Care and Use Committee at NYH-CUMC, and is similar to that described (22). After an overnight fast, six male or female Papio anubis baboons were anesthetized with ketamine (10 mg/kg, i.m.), and anesthesia was maintained thereafter by administration of sodium pentobarbital (5 mg/kg per hr, i.v.). All animals were then administered 1011 live E. coli 086:B7 as an i.v. infusion over 30 min. Animals were studied in pairs (1 treatment, 1 control) to negate any variation in the E. coli preparation. Beginning simultaneously with the E. coli infusion, treatment animals (n = 3) received a 3-hr primed continuous i.v. infusion of sTNFR-I (3 mg/kg priming dose, followed by 4.5 mg/kg per hr of rsTNFR-I). Control animals received an isovolemic infusion of human serum albumin (3 mg/kg priming dose followed by 4.5 mg/kg per hr). All animals received a maintenance (3 ml/kg per hr) i.v. fluid (155 mM NaCl) infusion. In addition, animals were resuscitated with 10 ml of fluid per kg of weight i.v. every 15 min if judged to be hemodynamically unstable by investigators blinded to treatment group (hemodynamic instability was defined as meeting two or more of the following criteria: mean arterial blood pressure < 70%6 of baseline, heart rate > 130%o of baseline, pulmonary capillary wedge pressure < 2 mmHg, and urine output < 1 ml/kg/hr). Blood samples were obtained prior to E. coli administration (baseline) and at intervals thereafter. Statistics. All values are expressed as mean + SEM unless otherwise noted.

RESULTS Two of five healthy adult male volunteers had detectable baseline levels of both sTNFRs (0.4 and 1.0 ng of sTNFR-I per ml; 0.8 and 1.2 ng of sTNFR-II per ml) (Fig. 1). Plasma samples from these five volunteers were also examined at intervals following endotoxin administration. Like TNFa, the appearance of circulating sTNFR-I and sTNFR-II species was found to be monophasic, with peak levels occurring after that of TNFa and detectable levels persisting several hours beyond the period in which circulating TNFa was detectable by ELISA (Fig. 2). Concentrations of sTNFR-I and sTNFR-II peaked at 3.7 ± 1.6 ng/ml and 1.4 ± 0.2 ng/ml, respectively, at 2 to 3 hr after the administration of endotoxin and remained above baseline levels for up to 24 hr. To evaluate further the presence of sTNFRs in human disease, the plasma of 12 critically ill patients was assayed at *

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FIG. 1. sTNFR-I and sTNFR-II concentrations in normal volunteers, volunteers after endotoxin administration, and critically ill patients. The maximal concentration of sTNFR-I and sTNFR-II for each volunteer and each concentration measured in the critically ill patients are shown. The median value-for each of the groups is represented by a horizontal bar.

Medical Sciences: Van Zee

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FIG. 2. Appearance of TNFa and sTNFRs in the circulation of healthy volunteers administered endotoxin [lipopolysaccharide (LPS)]. Concentrations (mean ± SEM) of circulating TNFa (o), sTNFR-I (e), and sTNFR-II (v) are shown at baseline and at intervals after endotoxin administration (indicated by arrow).

various points during their clinical course, resulting in 2-12 samples for each patient (mean + SD = 5 + 3). TNFa was detected by ELISA in only 37% ofthe 56 samples and by WEHI bioassay in only 20%. In contrast, sTNFR-I and sTNFR-II were detected in 94% and 89%o of samples, respectively, and a positive correlation between sTNFR-I and sTNFR-II levels was evident (n = 56, R = 0.48, P < 0.001). There was no statistically significant correlation between sTNFR levels and APACHE II scores, although there was a trend toward higher sTNFR-II levels in patients with higher APACHE II scores (R = 0.26, P = 0.06) and higher sTNFR-I levels in nonsurvivors (P = 0.06; two-sided t test). The concentrations of sTNFR-I and sTNFR-II observed in these critically ill patients were similar to the peak levels observed during experimental endotoxemia (Fig. 1). To determine whether the concentrations of sTNFRs found in the circulation are capable of neutralizing endogenous TNFa, plasma samples from volunteers administered endotoxin were incubated with rsTNFR-I and rsTNFR-II at S ng/ml to 50 ;Lg/ml. The addition of S ng of rsTNFR-I per ml reduced endogenous TNFa bioactivity (calculated to be 585 pg/ml by WEHI) by 66% (Table 1). In contrast, 500 ng of rsTNFR-II per ml was required to neutralize >50% of endogenous TNFa cytotoxicity. These findings show that the addition of physiologic concentrations of rsTNFR-I to plasma will significantly neutralize the cytotoxicity of endogenous levels of TNFa and suggest that the higher levels of sTNFR-I observed circulating in critically ill patients (5-10 ng/ml) are sufficient to at least partially attenuate the biological responses to TNFa production. The same concentration of rsTNFR-II is inadequate to block TNFa cytotoxicity. To evaluate the quantities of soluble receptors required to neutralize lethal concentrations of TNFa, plasma samples containing 1.5 ng of human rTNFa per ml were coincubated with 5-5000 ng of rsTNFR-I and rsTNFR-II per ml. This concentration of human rTNFa is roughly 2-3 times the peak concenTable 1. Neutralization of endogenous TNFa cytotoxicity by sTNFR-I and sTNFR-II Added sTNFR,

ng/ml 0 5 50 500

5,000 50,000

Calculated TNFa, pg/ml sTNFR-II

sTNFR-I 585 201 167