JOURNAL OF CELLULAR PHYSIOLOGY 145:496500 (1990)

Tumor Necrosis Factor Enhances the Neutrophil-Dependent Increase in Endothelial Permeability LESTER S. GIBBS, LINDA LAI, AND ASRAR B. MALIK* Department of Physiology and Cell Biology, The Albany Medical College of Union University, Albany, New York 12208 We examined the effect of tumor necrosis factor a ITNF,) on the increase in pulmonary microvascular endothelial monolayer permeability induced by activated neutrophils (PMN). Layering of PMN onto endothelial monolayers followed by activation of PMN with phorbol 12-myristate 13-acetate (PMA) increased ""I-albumin clearance rate across the monolayers. Pretreatment of endothelial monolayers for 6 hr with TNF, (200 U/ml) potentiated the PMN-dependent increase in endothelial permeability, whereas 1 hr or 6 hr pretreatment of endothelial monolayers with 200 U/mland 100 U/ml, respectively, TNF, did not enhance the reponse. Adherence of PMN to the endothelial cells was increased at 1 and 6 hr after TNF, (200 U/ml) treatment, but the adherence reponse was markedly greater following 6 hr of TNF,. The TNF, treatment of endothelial cells did not enhance neutrophil activation responses to PMA. Pretreatment of PMN with IB4, a MAb to the CD18 integrin, the common P subunit of the adhesion proteins LFA-1, Mac-1, and p150,95 of PMN, reduced the increases in PMN adherence and the endothelial monolayer permeability induced by the 6 hr TNF, treatment. In contrast, pretreatment of PMN with OKM-1, a MAb to the CD11b epitope (a-subunit), had no effect on the adherence and the potentiation of the increase in permeability. The potentiation of the PMN-dependent permeability increase and enhanced endothelial adhesivity at 6 hr after TNF, priming of endothelial cells was dependent on protein synthesis. The results indicate that protein synthesis-dependent expression of an endothelial ligand for CD18 and resultant endothelial hyperadhesiveness potentiates the PMN-mediated increase in endothelial permeability after TNF,, activation of endothelial cells. The priming of endothelial cells by TNF, may be a critical step in the mediation of endothelial injury.

The involvement of polymorphonuclear leukocytes (PMN) in increasing lung microvascular permeability is well established (Flick et al., 1981b; Heflin and Brigham, 1981; Johnson and Malik, 1980). Most studies indicate that endothelial injury requires close contact of PMN with the endothelial cell surface (Shasby et al., 1983) and that oxidants and proteases released at sites of cell-cell attachment are important in the mediation of cell injury (Weiss, 1989). However, the neutrophil adhesion mechanisms mediating endothelial injury are poorly understood. Tumor necrosis factor, (TNF,), a cytokine produced by monocytes and tissue macrophages in response to endotoxin and other stimuli (Beutler et al., 1985; Neduin et al., 1985; Tracey et al., 19861, has been demonstrated to promote PMN adhesion to endothelial cells (Gamble et al., 1985). PMN adherence may occur by TNF,-mediated expression of endothelial leukocyte adhesion molecule (ELAM-1) (Bevilacqua et al., 1987) and intercellular adhesion molecule (ICAM-1)(Pober et al., 1986). The leukocyte CD18 integrin, the common P-subunit for three different PMN adhesion proteins, is a known ligand for ICAM-1 (Smith et al., 1988), and 0 1990 WILEY-LISS, INC.

may interact with ICAM-1 in processes which lead to endothelial injury. The functional implications of endothelial hyperadhesiveness mediated by cytokines in increasing endothelial ermeability following PMN activation are unclear. n the present study, we have examined the effects of TNF, priming of endothelial cells on the changes in endothelial monolayer ermeability induced by activated PMN and have ad ressed potential mechanisms of these alterations in permeability.

P

a

METHODS Reagents Purified recombinant human tumor necrosis factor, (TNF,; Cetus, Emeryville, CAI, produced by Escherichza coli after insertion of a cDNA clone encoding human TNF, was used. TNF had specific bioactivity of 24 X lo6 Units of TNF per mg of protein. A TNF unit was defined as the amount producing 50% cytotoxicity Received May 14, 1990; accepted August 11, 1990. 'To whom reprint requestdcorrespondence should be addressed.

TNF,, AND PMN-MEDIATED ENDOTHELIAL INJURY

497

of L929 cells seeded at 2 x lo5 cells per ml after an 18 taken from the lower compartment every 5 min for 1hr. hr incubation at 37°C. Endotoxin contamination was Studies were made using endothelial cells treated with 0.02 ng per ml by limulus amebocyte lysate assay. HBSS (controls) for 1or 6 hr and with cells treated with Endothelial cells were treated with TNF, dissolved in TNF, (100 or 200 U/ml) for 1 or 6 hr. With each phosphate-buffered saline (PBS) at a concentration of treatment, fresh medium, which contained the PMN, 100 and 200 U/ml. Monoclonal antibodies (MAb) IB4 was added to the monolayer. and OKM-1 were gifts from Dr. Samuel Wright, RockIn some experiments, cycloheximide (10 pg/ml) was efeller University, New York, NY. added to monolayers 30 min prior to the application of TNF. This concentration of cycloheximide was shown to Isolation of neutrophils inhibit protein synthesis based on 3H-leucine incorpoBlood from healthy human donors was collected into ration. heparinized syringes (100 U heparidlo ml blood). The Neutrophil adherence assay method of neutrophil isolation involved layering 7 ml 51Cr-labeledPMN (2 x lo6 cells/ml) were distributed blood onto 4 ml of neutrophil isolation medium (Los Alamos Diagnostics, Los Alamos, NM) and centrifug- at 1 ml per well over the confluent BPMVE cells. The ing the suspension at 250g for 5 min (Ferrante and cells were incubated (37"C, 5% C02, 98% humidity) for Thong, 1986). Distilled water (5 ml) was added to the 1hr in HBSS on the 1or 6 hr TNF,-treated endothelial PMN suspension and the mixture was shaken for 20 sec monolayers. In each case, TNF, was removed prior to to lyse erythrocytes. The osmolarity of the solution was applyin the PMN. At the end of each incubation, the restored with 15 ml of 1.2%saline. The number of PMN endothe ial monolayers were gently washed three in the isolate was determined using a hemacytometer. times with PBS to remove nonadherent PMN. The endothelial monolayers were kept overnight under 1ml Pulmonary microvessel endothelial cells of 1N NaOH at 4°C. The cell lysates were scra ed, Bovine pulmonary microvascular endothelial cells collected in polypropylene test tubes, and counte for (BPMVE) were isolated using the technique of Del radioactivity in a Tm Analytical Gamma Counter. Vecchio et al. (un ublished data). Briefly, tissue from Neutrophil superoxide anion and elastase the periphery of ovine lung was minced, exposed to generation collagenase, filtered, centrifuged, and resuspended in PMN were applied to monolayers of BPMVE cells at culture media. After several days' incubation, colonies a ratio of 1 O : l and stimulated by the addition of PMA were selected based on uniform morphology and isolated with a cloning ring. The cells were confirmed to (6 x lop8 M). Production of superoxide anion was debe endothelial in origin by the presence of factor termined by the reduction of ferricytochrome C (Type VIII-related anti en and incorporation of acylated 111,horse heart, Sigma) (Perlman et al., 1987).Elastase LDL. These BPMbE cells have a characteristic glyco- release was determined by the method of Gilboa et al. protein profile different from the mainstem pulmonary (1989). artery endothelial cells (Del Vecchio et al., unpublished Statistics data). Differences between two group means were compared by Student's t:test (Sokal and Rohlf, 1981). Multigroup Endothelial monolayer permeability comparisons were carried out by one-way analysis of BPMVE cells were seeded and grown to confluence variance. If statistically significant differences were for 4-6 days on gelatinized polycarbonate micropore revealed, the group means were compared by the membranes (diameter, 13 mm; pore size, 0.8 bm; Numultiple comparison procecleopore) mounted on the bottom of lastic cylinders Student-Newman-Keuls dure (Steel and Torrie, 1980). (inner diameter 11 mm; Adaps, De ham, MA). The RESULTS luminal well (0.7 ml capacity) was suspended in a Effect of PMN activation on endothelial well-stirred larger abluminal compartment (25 ml capermeability to '251-albumin pacity). Both compartments were filled with Hanks' balanced salt solution (HBSS,Gibco Laboratories, ChaThe addition of PMA (6 x 10-'M) to PMN layered on grin Falls, OH) containing 0.5%bovine serum albumin the endothelial monolayer resulted in activation of (Sigma Chemical Co., St. Louis, MO). The tracer 1251- PMN. Superoxide anion generation increased from the labeled albumin was added to the upper well. Endo- control value of 6.7 ? 0.5 nmoles 02-/106 PMNIGO rnin thelial permeability to lZ5Iwas determined using the with the addition of HBSS to 64.5 ? 0.6 nmoles Ozp/106 endothelial monolayer permeability system described PMN/6O min with the addition of PMA. The addition of by Cooper et al. (1987). PMA also increased the 1251-albuminclearance rate Freshly isolated PMN were layered onto endothelial across the monolayer from the basal control values monolavers at a ratio of 1O:l PMN:endothelial cell and (Fin. 1).In contrast. addinn PMN alone or PMA alone allowed to settle for 10 min. At that time, 50 pl of a to &e monolayers did not s&pificantly increase perme6 x lo-' M solution of phorbol12-myristate 13-acetate ability (Fig. 1). (PMA,Sigma) in PBS (pH 7.4) was added to the luminal Effect of TNF, priming of endothelial well. The sampling from the abluminal well was begun monolayers on PMN-dependent increases 60-90 min after the addition of PMA, since preliminary in permeability data indicated that the increase in monolayer permeTNF, (200 U/ml) was added to endothelial monolayability required at least this period of time following PMN activation with PMA. Samples (400 pl) were ers for 1or 6 hr, followed by removal of residual TNF,

Fi

cf

;

B

498

GIBBS ET AL. 0.12

e 5

1

*

.E > a

I

Group

Y

d .

a

0.08-

Y

C

z

Y

i2 C

-E a

TABLE 1. Effect of 1or 6 hr TNF,, treatment 5200 u/ML) of endothelial monolayers on PMN-dependentincrease in l2 I-albumin clearance rate induced with activation of the layered PMN with PMA (6 x 10-'M)' '%I-Albumin clearance rate (pl/min)

1 hr HBSS

0.20 +_ 0.029

1 hr TNF 6 hr HBSS 6 hr TNF

+

0.17 f 0.032 0.19 0.016 0.29 f 0.034*

'Data represent the mean f SE (n = 5 to 9 per group). *Significantly different from group that received 6 hr HBSS.

0.04-

Y) r( N

O A Basal

PMN (101)

PMA

PMN

+

PMA

Fig. 1. Effect of activation of PMN with PMA (6 x 10-'MM) on the clearance rate of '*Talbumin across endothelial monolayers. The ratio 1 O : l indicates the ratio of neutrophil-to-endothelialcells used in the study. Each value represents the mean ? SE (n = 4 to 6 per group). "Different from basal, PMN, and PMA clearance values (P