Recombinant tumor necrosis factor increases pulmonary vascular ...

Proc. Nail. Acad. Sci. USA Vol. 85, pp. 9219-9223, December 1988 Medical Sciences

Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils (lung lymph/endothelial monolayer/'"I-labeled albumin permeability/endotoxemia)

CHRISTOPHER J. HORVATH*, THOMAS J. FERRO*, GARY JESMOKt,

AND

ASRAR B. MALIK**

*Departments of Physiology and Medicine, The Albany Medical College of Union University, Albany, NY 12208; and tCetus Corporation, Emeryville, CA 94608

Communicated by Ivar Giaever, July 11, 1988

We studied the effects of intravenous infusion ABSTRACT of recombinant human tumor necrosis factor type a (rTNF-a; 12 pg/kg) on lung fluid balance in sheep prepared with chronic lung lymph fistulas. The role of neutrophils was examined in sheep made neutropenic with hydroxyurea (200 mg/kg for 4 or 5 days) before receiving rTNF-a. Infusion of rTNF-a resulted in respiratory distress and 3-fold increases in pulmonary arterial pressure and pulmonary vascular resistance within 15 m., indicating intense pulmonary vasoconstriction. Pulmonary lymph flow (i.e., net transvascular fluid filtration rate) and transvascular protein clearance rate (a measure of vascular permeability to protein) increased 2-fold within 30 min. The increased permeability was associated with leukopenia and neutropenia. The pulmonary hypertension and vasoconstriction subsided but fluid ifitration and vascular permeability continued to increase. Sheep made neutropenic had similar increases in pulmonary transvascular fluid filtration and vascular permeability. rTNF-a also produced concentrationdependent increases in permeability of 12sI-labeled albumin across ovine endothelial cell monolayers in the absence of neutrophils or other inflammatory mediators. The results indicate that rTNF-a increases pulmonary vascular permeability to protein by an effect on the endothelium.

Tumor necrosis factor type a (TNF-a) is a macrophagederived product that mediates the hemorrhagic necrosis of certain transplantable tumors in mice challenged with endotoxin (1). Macrophages also elaborate TNF-a when stimulated with endotoxin (2). This monokine is identical to cachectin (3), a protein responsible for the chronic catabolic state (cachexia) seen with some infections and cancers (4). Recent studies have proposed TNF-a as a mediator of endotoxemia (5) since TNF-a administration mimics the clinical course of endotoxemia (6, 7). Passive immunization against TNF-a reduces the mortality associated with endotoxin challenge (8, 9). Pathologic changes following TNF-a infusion include pulmonary inflammation and hemorrhage, with microaggregates of leukocytes and margination of polymorphonuclear leukocytes (neutrophils; PMNs) in the pulmonary microcirculation (7). Endothelial cells exposed to TNF-a develop cytoskeletal changes (10), procoagulant activity (11, 12), and increased adhesiveness to lymphocytes (13) and PMNs (14). TNF-a also causes endothelial cells to release interleukin 1 (15) and platelet-activating factor (16). PMNs stimulated with TNF-a display enhanced adherence (14, 17), migration (18), phagocytic and cytotoxic activities (19), respiratory burst activity (20), and degranulation (20). These effects of TNF-a on endothelial cells and on PMNs suggest that TNF-a is involved in mediating the increase in lung vascular permeability associated with endotoxemia. In the present study, we 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.

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examined whether recombinant TNF-a (rTNF-a) affected pulmonary vascular permeability by assessing alterations in pulmonary lymph flow in unanesthetized sheep following rTNF-a challenge and by determining transendothelial permeability in rTNF-a-treated endothelial monolayers. We also examined the role of PMNs in the response.

MATERIALS AND METHODS Chronic lymph fistulas were implanted in male sheep (n = 11) weighing -25 kg (21). This procedure allows collection of blood-free pulmonary lymph after a 3- or 4-day recovery period. Hydroxyurea (HDY; Squibb) was used to deplete circulating neutrophils. HDY (5 g) was dissolved in 200 ml of O.9%o NaCl and the pH was corrected to 7.4; this solution was administered i.v. once daily for 4 or 5 days (200 mg/kg per day). Daily antibiotic treatment was continued in these animals (5 ml i.m.; Ambipen; Butler, Columbus, OH). Animals were studied within 2 days of the last HDY treatment. Awake sheep were studied while standing in a metabolic cage with free access to food and water. A 7.5-F thermodilution catheter (American Edwards Laboratories, Santa Ana, CA) was positioned in the pulmonary artery. Pulmonary artery (Pp) and pulmonary artery wedge (Ppw) pressures were determined with Statham P23Db transducers (Statham, Hato Rey, PR). Pulmonary blood flow (QL) was determined by a thermodilution technique (Cardiac Output Computer, model 9520, American Edwards Laboratories). Pulmonary vascular resistance (PVR) was calculated as (Pp - PPw)/QL. Heparinized arterial blood was used for determinations of blood gases (pH, Paco2, and PaO2) (Paco2 and Pa02 are pressure of CO2 and 02 in arterial blood) with an ABL2 Acid-Base Laboratory (Radiometer Copenhagen, MKS Scientific, Cheecktowaga, NY). Hematocrit was determined by centrifugation of microhematocrit tubes. Leukocytes were counted in a hemocytometer after staining with new methylene blue and differential counts of PMNs and mononuclear leukocytes were made on stained blood smears (Diff-Quik, American Scientific Products, McGraw Park, IL). Pulmonary lymph flow (Qlym) (a measure of net pulmonary transvascular fluid filtration rate) was determined by measuring the volume of lymph catheter effluent collected over 15- or 30-min periods. Lymph fluid was collected in plastic tubes containing 100 mg of sodium citrate (Fisher) and centrifuged to remove cells. Aliquots of lymph and plasma were stored at -70°C. Protein concentrations of paired samples of lymph (L) and plasma (P) were determined by the Abbreviations: TNF-a, tumor necrosis factor; rTNF-a, recombinant TNF-a; HDY, hydroxyurea; PMNs, neutrophils; PR, pulmonary artery pressure; P-, pulmonary artery wedge pressure; PVR, pulmonary vascular resistance; QL, pulmonary blood flow; Qjm, pulmonary lymph flow; L/P, lymph/plasma protein concentration; CL, transvascular protein clearance rate; III-albumin, III-labeled albumin; Paco2 and pressure of CO2 and 02 in arterial blood. Pao,,whom tTo reprint requests should be addressed.

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Medical Sciences: Horvath

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al.

Lowry method (22). Lymph/plasma protein concentration ratio (L/P) and pulmonary transvascular protein clearance (CL = Qlym x L/P) were calculated. Plasma concentrations of fibrinogen and fibrin degradation products were determined by the radial immunodiffusion method (23). We used purified recombinant human tumor necrosis factor (rTNF-a; Cetus), produced by Escherichia coli after insertion of a cDNA clone encoding human TNF-a (24) and with a specific bioactivity of 2.24 x 104 units of rTNF-a per ,ug of protein. A rTNF-a unit was defined as that amount producing 50% cytotoxicity of L929 cells seeded at 2 x 105 cells per ml, after an 18-hr incubation at 37°C. Endotoxin contamination was 0.058 ng of lipopolysaccharide per vial by limulus amebocyte lysate assay. Pulmonary hemodynamic parameters and Qlym were monitored to establish baseline values and rTNF-a infusion was begun when all variables had remained stable over three to five successive 30-min periods. For each sheep, rTNF-a (6.5 x 106 units of rTNF-a) was reconstituted prior to use with 1 ml of 0.01 M phosphatebuffered saline (pH 7.4). This volume was diluted into 60-ml sterile lactated Ringer's solution and delivered via the proximal infusion port (right ventricle) of the Swan-Ganz catheter over 30 min to give a total dose of 12 ,ug of rTNF-a per kg. All animals were followed until Qlym reached a steady-state value (usually 4-5 hr postinfusion). Approximately 18 hr after challenge, arterial leukocyte counts were again taken. The effect of rTNF-a on endothelial permeability was determined by the endothelial monolayer permeability system (25). Ovine pulmonary artery endothelial cells were seeded and grown to confluence for 4 days on gelatinized polycarbonate micropore membranes (diameter, 13 mm; pore size, 0.8 ,um; Nucleopore) mounted on the bottom of plastic cylinders (i.d., 11 mm; Adaps, Dedham, MA).The luminal wells (0.7-ml capacity) were suspended in larger abluminal compartments (25-ml capacity), the contents of which were stirred constantly. Both compartments were filled with Hanks' balanced salt solution (Gibco Laboratories, Chagrin Falls, OH) containing 0.5% bovine serum albumin (Sigma). The tracer 125I-labeled albumin (125I-albumin) was added to the upper well and 400-,ul samples were taken from the lower compartment every 5 min for 30 min prior to intervention, and for 30 min thereafter. Intervention consisted of placing various concentrations (0, 1, 100, 1000 units/ml) of rTNF-a into the solution bathing the endothelial monolayer in the luminal well. In some studies, a 1:10,000 dilution of murine origin monoclonal antibody against rTNF-a (obtained from

Proc. Natl. Acad. Sci. USA 85

(1988)

A. Khan, Wadley Institute, Dallas, TX) was added simultaneously with 100 units of rTNF-a per ml. A 1:1000 dilution of the anti-rTNF-a antibody neutralized the L929 cytotoxic activity of rTNF-a (1000 units/ml). 1251 activity in the bottom well samples was determined with a y-counter (Auto-Gamma 5000 Series, Packard Instrument). The 1251-albumin clearance rate (a measure of endothelial permeability to albumin) was determined by least-squares linear regression for the prerTNF-a and post-rTNF-a periods (25). Data from the sheep studies were analyzed for significance (P < 0.05) of main effects and time by two-way analysis of variance corrected for repeated measurements (BMDP Statistical Softwear, Los Angeles). Comparisons between groups or of changes from baseline values were made with appropriate Bonferroni t tests corrected for repeated measurements (26). Data for leukocyte counts displayed heterogeneity of variance and required logarithmic transformation prior to conditional comparisons (26). Significance was accepted at P < 0.05. All results are presented as mean + SEM. Significance (P < 0.01) of changes in 125I-albumin clearance rates was determined by two-way analysis of variance.

RESULTS Tachypnea and dyspnea, accompanied by muscle tremors, developed within minutes after starting rTNF-a infusion. In most cases, these effects were transient, lasting 3-4 hr, and sheep appeared healthy by 24 hr, although appetite did not return until 48-72 hr. There were no significant changes in blood temperature and arterial blood gases in the rTNF-achallenged sheep (Table 1). Similarly, plasma fibrinogen and fibrin degradation product concentrations were not affected by rTNF-a infusion (Table 1). Mean baseline hematocrit values were similar in control and HDY sheep. rTNF-a infusion increased hematocrit 10.8% and 13.8% over baseline (P < 0.05) by 30 min in control and HDY groups, respectively (Table 1). Hemoconcentration persisted over all times in both groups (P < 0.05). The changes in leukocyte counts are shown in Fig. 1. The group challenged with rTNF-a had normal total leukocyte (7313 462 cells per ,ul), PMN (4022 703 cells per ,ul), and mononuclear leukocyte (3291 779 cells per ,ul) counts at baseline. By the end of rTNF-a infusion, there was a marked leukopenia (2125 357 cells per ,ul) with neutropenia (341 92 cells per ,1l) and decreased mononuclear leukocyte counts ±

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Table 1. Changes in blood temperature, arterial blood gases, plasma fibrinogen and fibrin degradation product concentrations, and hematocrit after rTNF-a challenge in control and HDY-treated sheep Time, hr Baseline Group 0.5 1.0 3.5 Control tOOC*t 39.9 ± 0.4 40.8 ± 0.2 40.2 ± 0.3 40.1 ± 0.4 HDY 40.8 ± 0.2 40.8 ± 1.0 40.9 ± 0.12 41.2 ± 0.3 Control 7.47 ± 0.02 pH 7.45 ± 0.02 7.47 ± 0.01 7.46 ± 0.01 HDY 7.44 ± 0.05 7.45 ± 0.02 7.44 ± 0.02 7.43 ± 0.03 Control 35.4 ± 1.3 36.6 ± 1.2 Paco2, mmHg 35.4 ± 0.8 35.9 ± 1.3 HDY 37.4 ± 3.3 35.2 ± 3.7 36.6 ± 1.2 38.2 ± 1.7 Control 108.6 ± 1.8 105.4 ± 2.3 Pao2, mmHg 100.6 ± 2.4 108.1 ± 2.3 HDY 108.2 ± 1.4 99.6 ± 11.5 102.7 ± 4.0 109.0 ± 4.4 Fibrinogen, mg/dl Control 336 ± 99 378 ± 117 256 ± 10 279 ± 39 HDY 392 ± 25 435 ± 47 385 ± 29 369 ± 45 Control FDP, mg/dl 5.1 ± 0.7 5.8 ± 1.1 4.2 ± 1.1 4.5 ± 0.6 HDY 4.4 ± 1.4 4.2 ± 0.5 4.3 ± 0.8 4.9 ± 1.0 Control Hematocrit, % 31.4 ± 3.6 35.0 ± 4.3t 35.8 ± 4.5t 36.2 ± 4.2t HDY 31.6 ± 3.8 34.8 ± 3.5t 33.6 ± 3.8t 36.8 ± 4.8* *Values are shown as mean ± SEM. tInfusion of rTNF-a had no significant effect on blood temperature, pH, Pacc, Pao2, plasma fibrinogen, and plasma fibrin degradation product (FDP) concentrations. *Different from baseline (P < 0.05).

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Medical Sciences: Horvath et al.

Proc. Natl. Acad. Sci. USA 85 (1988) 45

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FIG. 1. Arterial total leukocyte, PMN, and mononuclear leukocyte counts in control and HDY-treated sheep after intravenous infusion of rTNF-a (stippled area). Values are shown as mean SEM.

(1784 + 370 cells per Al) (P < 0.05). Total leukocyte count increased steadily to baseline values by 4 hr, due primarily to an increase in PMNs. At 18 hr, neutrophilia and increased mononuclear leukocyte numbers (10,526 263 and 4537 1032 cells per 1.l, respectively) were evident. The HDYtreated group had lower total leukocyte (1350 197 cells per pl) and PMN (145 83 cells per pl) counts than the control group at baseline and at all other times (P < 0.05). The decreased leukocyte count was primarily the result of HDYinduced neutropenia. The pulmonary hemodynamic data are shown in Fig. 2. rTNF-a infusion produced a peak Pr response within 15 min after initiation of infusion in both control (38.2 3.9 mmHg) and HDY (36.3 3.2 mmHg) groups. However, by 30 min Ppr in control sheep decreased to 24.2 1.2 mmHg, remaining elevated above baseline until 4 hr (P < 0.05). In contrast, Pru in HDY sheep returned to baseline values by 30 min, remained lower than in control sheep until 1.5 hr (P < 0.05), then increased to values similar to those in control sheep. Piw- and QL did not change significantly over time and were not different between groups. PVR was increased 157% over mean baseline (2.11 0.10 mmHg liter-1-mind) values in control sheep by 15 min (5.42 1.07 mmHg'liter-1-min'), but was only 46% greater at 30 min, and remained elevated for the duration of the study (P < 0.05). In HDY sheep, at 15 min the PVR was increased 123% over baseline values (from 2.13 0.13 to 4.76 0.03 mmHg-liter-1 min1), but PVR returned to baseline values from 30 min to 1.5 hr. This return of PVR to baseline was significantly different from control sheep (P < 0.05); however, by 2.5 hr PVR had increased over baseline (P < 0.05) to the same level as the control group. ±

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FIG. 2. PjW, AP, QL, and PVR in control and HDY-treated sheep after intravenous infusion of rTNF-a (stippled area). Values are shown as mean ± SEM.

Alterations in pulmonary lymph parameters are shown in Fig. 3. The changes in Qiym were similar in the two groups. By 30 min, rTNF-a infusion increased Qlym over baseline values by 97% and 113%, respectively, in control (10.0 0.9 ml/hr) and HDY (11.2 + 2.6 ml/hr) groups (P < 0.05). Olym continued to increase in control and HDY sheep to reach steady-state values of 14.3 ± 1.0 ml/hr and 15.7 3.4 ml/hr at 3.5 hr. The L/P ratios remained at or above baseline values in both groups. L/P ratio increased (P < 0.05) over mean baseline values at 2 and 4 hr in control sheep and from 1.5 to 3.5 hr in HDY sheep. CL for both groups behaved in a similar fashion as 01y, increasing after rTNF-a infusion (P < 0.05). By 30 min, rTNF-a produced a 110% increase over baseline CL in control sheep (6.3 0.4 ml/hr) and a 148% increase in HDY sheep (6.7 2.1 ml/hr). The increases in 1)ym and CL returned to baseline by 18 hr post-rTNF-a challenge. rTNF-a caused an increase in the clearance rate of 1251_ albumin across sheep pulmonary endothelial monolayers (Table 2). Compared to controls, 1251-albumin permeability increased with increasing concentrations of rTNF-a until a concentration of 100 units/ml (74.6% 7.8% increase; P 0.01). 1251-albumin permeability plateaued between concentrations of 100 units/ml and 1000 units/ml. A 1:10,000 dilution of anti-rTNF-a monoclonal antibody prevented the increase in permeability of 100 units of rTNF-a per ml (Table 2). ±

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