Tumor

necrosis

factor

a and

lipopolysaccharide-mediated Smita Deprtment

A. Sharma,* of Pathobiology5

bovine

Timothy and

interleukin

W.J.

endothelial

Olchowy,’

Zhengang

of Rural

e,t

Department

1 a enhance Yang,*

College

of

cell injury

and

Mike

Veterinary

Medicine,

A. Breider* University

of Tennessee,

Knoxville

Abstract:

Alveolar

macrophages

(AMs)

the host response to aerogenous pulmonary fections, such as Pasteurella haemolytica-induced monia in cattle. Previous work has shown hance P. haemolytica-mediated pulmonary

important

are

in

bacterial

inpneuthat AMs enendothelial

cell (EC) damage in vitro. The purpose of this study was to determine the mechanism of AM-enhanced EC damage using an in vitro AM-EC coculture system consisting of AMs cultured on culture plate insert membranes and ECs in the underlying chamber. The addition of lipopolysaccharide (LPS) to the culture plate insert chamber resulted in EC damage indicated by 51Cr release, which was enhanced in the presence of AMs. To determine the role of AM-secreted cytokines, recombinant human interleukin 1 a (IL-i) or tumor necrosis factor a (TNF) was added to ECs simultaneously with varying concentrations of LPS. Although TNF and IL-i alone had only marginal toxic effects on ECs, the simultaneous treatment of TNF or IL-i with LPS greatly increased the LPS cytotoxic effect on ECs. In addition, IL-i receptor antagonist eliminated the IL-i enhancement of LPS-mediated EC toxicity. These results suggest that macrophage-secreted cytokines synergistically enhance LPS-mediated pulmonary EC damage.J. Leukoc. Biol. 51: 579-585; 1992.

Key

Words:

gonist . bovine

tumor

#{149} lipopolysaccharide

necrosisfactor interleukin 1 . macrophage . endothelial

.

receptor antaendotoxin

damage through the secretion of proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin 1 (IL-i) [19]. In a munine model, inhibition of TNF using anti-TNF antibodies resulted in decreased LPS-mediated effects, indicating an important role of TNF in endotoxemia. In addition, TNF and IL-i may promote intravascular coagulation through the activation of ECs, promoting polymorphonuclean neutrophil (PMN) traffic, EC-leukocyte adhesion and phagocyte-mediated EC injury [21]. Thus, it appears likely that the marked thrombosis and ischemic necrosis seen in pulmonary pasteunellosis may be due to the LPS activation of a proinflammatory and procoagulant state in the alveolar microvasculature. Cytokines in the lung provide the intercellular communication signals between the various inflammatory cells such as PMNs, AMs, and ECs; thereby regulating the degree of inflammation, the bactericidal capacity of the pulmonary defense system, and possibly the extent of EC damage. In the present study we sought to elucidate the effects of AM cytokines on the pulmonary endothelium and on endothelial cell-leukocyte interactions in mediating EC injury. By employing an in vitro model system that effectively mimics the acute inflammatory process of the bovine lung [28], we have shown that LPS-stimulated AMs can injure the pulmonary ECs through the production of proinflammatory cytokines. Our results demonstrate that IL-i and TNF can enhance mediated losis.

LPS by

P

effects and, haemolytica

thereby, amplify the in bovine pulmonary

lung injury pasteunel-

INTRODUCTION

MATERIALS The lipopolysaccharide (LPS) cell wall component of gramnegative bacteria is a major pathogenic factor and a potent stimulator ofthe acute inflammatory response [19]. The host inflammatory response to LPS is not always beneficial and can often lead to enhanced tissue damage. LPS has been associated with many of the disease symptoms of bacterial infections in both humans and animals [19]. In pulmonary diseases, LPS has dramatic effects on both the structure and function of the lungs [13], inducing inflammation as well as injuring the vascular endothelium in vivo in some animal species [i6]. LPS can also directly injure bovine endothelial cells (ECs) in vitro [4, 6, 14, 16, 23]. In bovine pneumonic pasteurellosis caused by Pasteurella haemolytica, EC injury is an important pathogenic event as characterized by the acute onset of intra-alveolar edema, hemorrhage, fibrin exudation, and capillary thrombosis. Studies from our laboratory have demonstrated that P haemolytica or its LPS component can damage bovine ECs through several different mechanisms, either directly [4] or through the bacterial stimulation of alveolar macrophages (AMs) [29]. LPS stimulation of phagocytes may lead to EC

Pasteurella

AND METHODS haemolytica

Lipopolysaccharide

The P haemolytica LPS, extracted water method [12], was obtained ( Department of Microbiology, Knoxville). The LPS contained

Abbreviations: Dulbecco’s

AM,

modified

alveolar

Eagle’s

macrophage;

medium;

EC,

from bacteria by a phenolfrom Dr. Robert Moore University of Tennessee, 2.12 x i0 endotoxin

CI,

cytotoxic

endothelial

cell;

index; FBS,

DMEM, fetal

bovine

serum;

HBSS, Hanks’ balanced salt solution; IL-i, interleukin i; IL-ira, IL-i receptor antagonist; LPS, lipopolysaccharide; P1, post inoculation; PMN, polymorphonuclear neutrophil; rbIL-l, recombinant bovine IL-i; RGS, Ryan growth supplement; TNF, tumor necrosis factor. Reprint requests: Mike A. Breider, Department of Pathobiology, College ofVeterinary Medicine, University ofTennessee, Knoxville, TN 37996-4500. S.A. Sharma’s present address: Vanderbilt University, Division of Infectious Diseases, A33i0 Medical Center North, Nashville, TN 37232. Received July 9, 1991; accepted January 28, i992.;i04000 Journal of Leukocyte Biology Volume 51, 1992.

Journal

of Leukocyte

Biology

Volume

51,

June

1992

579

units/tg as measured with a chromogenic Limulus lysate assay (Whittaken MA Bioproducts). An Escherichia coli LPS standard was used to standardize the endotoxin assay. To neutralize the endotoxin activity of P haemolytica LPS preparation, 1000 U of polymyxin B (Sigma Chemical Co., St. Louis, MO) per ml was preincubated with LPS preparations for 60 mm at 37#{176}Cbefore application ofLPS to cell cultures.

streptomycin (pH 7.4). AMs were separated from neutrophils and choalveolar cells by density gradient centrifugation. imately 1.0 x 108 cells were resuspended in 30

Cytokines

and overlaid which was

Recombinant human IL-i a (5 x 108 U/mg) and recombinant human TNF-a (2 x i06 U/mg) were purchased from R&D systems, Minneapolis, MN. The endotoxin levels in the cytokine preparations were only 1.2 ng/tg cytokine for IL-i and 0.2 ng/tg cytokine for TNF. Recombinant bovine IL-1f3 (nbIL-1) (30,000 U/mg) was a gift from Dr. Mike Daley, American Cyanamid, Princeton, NJ, and the IL-i receptor antagonist (IL-ira) was a gift from Dr. Robert Thompson, Synengen, Boulder, CO.

Endothelial

Cell Cultures

Primary endothelial cell cultures were established from adult bovine pulmonary arteries as previously described [27]. The cells were identified as endothelial cells by typical cobblestone morphology and the presence of anti-factor VIII-related antigen as described previously [26]. The endothelial cells used in all experiments were of the same cell line and less than passage 20.

Endothelial

Cell Cytotoxicity

Assay

Endothelial cell damage was quantified by a previously described 51Cr release assay [4]. Briefly, i0 cells in 0.5 ml of Dulbecco’s modified Eagle’s medium (DMEM) with 10% Ryan growth supplement (RGS) were placed into 24-well tissue culture plates. Upon reaching confluency in 24 h, the cells were labeled with 1 ml of 51Cr (10 Ci/ml of DMEM-lO% RGS; ICN Pharmaceuticals, Irvine, CA) for 24 h. The cells were then washed five times with Hanks’ balanced salt solution (HBSS) and replenished with DMEM-iO% fetal bovine serum (FBS) followed by the addition of variables. All the variables were tested in quadruplicates and experiments were repeated to confirm results. At 22 h postinoculation (P1) the plates were centrifuged and 200 tl of medium was removed and measured for Cn release with a gamma counter. The cytotoxic index (CI) for each variable was determined using the formula CI = [(A - B)/ (C - B)] x 100 where A = variable release, B = spontaneous release, and C = total release.

were pelleted at 400g for 10 mm at 4#{176}Cand washed twice with cold HBSS containing divalent cations, 25 mM HEPES, 0.1% FBS, 100 IU/ml penicillin, and 100 g/ml

on 10 ml of Histopaque then centrifuged at 400g

other ml

bnonApproxof HBSS

(Sigma Chemical Co.), for 30 mm at 22#{176}C.The

cell pellet at the bottom of the tube was composed almost entirely of granulocytes. The macrophage-rich cell layer at the interface was removed and washed twice with HBSS. After the second wash, the alveolar macrophages were resuspended in DMEM supplemented with 20% FBS, 100 IU/mL penicillin, and 100 g/ml streptomycin and checked for viability and cell yield. To establish culture well insert AM cultunes, 200 il of the AM suspension containing 2 x l0 cells/ml was plated on type IV collagen-coated 12-mm Millicell-CM 0.4-sm pore size culture plate inserts (Millipore, Bedford, MA). After i8 h of 37#{176}Cincubation, the medium was decanted and fresh medium without antibiotics was added to the cell monolayer. Incubation was continued for an additional 6 h before the EC experiments were performed.

Culture

Well Insert

Coculture

System

To model the bovine alveolar constituent components of the bovine lung, AMs were cultured in the culture well insert chambers while 51Cr-labeled ECs were added to the bottom of 24-well tissue culture plates. P haemolytica LPS was added to the AMs in the culture well insert chambers at varying concentrations ranging from 0.03 to 330 ng/ml. To determine the relative role of various cells in LPS-mediated EC damage, the following variables were used: (1) AM (insent) ‘ EC and (2) EC only. Negative controls of cells not treated with LPS were also included. The damage to EC was determined at 22 h P1 using a 51Cr release cytotoxicity assay.

Cytokine To determine EC damage,

Experimental the ECs

Protocols

effect were

of AM treated

cytokines with LPS

on at

LPS-mediated three different

70 60

x

50

0 0

Pulmonary

Alveolar

Macrophage

Procurement

C

and

U

Purification 3o Pulmonary

bovine

AMs

were

harvested

from

adult

cattle

by

bronchopulmonary lavage. Briefly, an endotracheal tube (Biovana, Gary, IN) was passed into the trachea through the oral cavity. An 8-ft-long (3 mm i.d., 8 mm o.d.) bronchoalveolan lavage catheter (Biovana) was passed through the lumen of the endotnacheal tube to the right caudal lung lobe until slight resistance occurred. Following catheter cuff inflation, 100 ml ofsterile buffered HBSS without phenol red and CA2 or Mg2 ions (pH 7.2) and containing 0.2% EDTA, 100 U/mi penicillin, 100 U/ml streptomycin, and 2.5 pg/mi fungizone was infused into the lung. Aspiration tion was done immediately. The infusion-aspiration dune was repeated until 1000 ml of lavage fluid fused. After filtering the fluid through sterile

580

Journal

of Leukocyte

Biology

Volume

of this

soluprocehad been ingauze, cells

Si, June

1992

1

330ng/ml

3.3ng/ml

units

+

pomyn Pasteurella

haemolytica

LPS (ng/ml

Fig. 1. Bovine EC cytotoxicity (51Cr release) at 22 h following treatment with varying concentrations of P haemolyiica LPS. The LPS was added to the Millicell insert and ECs were plated on the underlying bottom well. The ECs were cultured with (#{149}) or without (D) the presence of AMs. The values

are the means

and

SEM

of at least

eight

replicates.

Fig. 2. Phase-contrast micrographs of bovine EC cytotoxicity 22 h following addition ofcontrol media (A), 3.3 ng/ml P ho.emolylica LPS with AMs in culture well inserts (B), 3.3 ng/ml LPS only (C), 70 U/ml recombinant human TNF-a (D), 10 U/ml recombinant human IL-ia (IL-i) (E), 25 U/ml IL-i + 0.03 ng/ml LPS (F), and 0.03 ng/ml LPS (G).

Sharma

ci al.

Cytokine-mediated

endothelial

cell

injury

581

concentrations, absence of (25 U/well) responses mine the recombinant recombinant determine

0.03, 3.3, and 330 ng/ml, in the presence or individual cytokines, recombinant human IL-i a or recombinant human TNF-a (50 U/well). Dose of cytokines on ECs were also evaluated to detersensitivity and optimum concentration of the cytokines. Additional experiments with bovine IL-i/S were done in the presence of IL-ira to whether enhanced LPS-mediated EC toxicity was

the

of IL-i

result

Morphological At 5 and Olympus

specific

activity

on

A 18 16 14

ECs.

.1

12

.

10

Observations

22 h P1, the K2 inverted

cell monolayers phase-contrast

were observed microscope.

with

an 4 2

RESULTS 2

Alveolar Macrophage and Lipopolysaccharide Interactive Effects on Endothelial Cells

Effect of Tumor Necrosis

The observation that hanced LPS-mediated that LPS-tneated AMs

LPS activation of macrophages enendothelial cell damage suggested may secrete cytokines that have direct

TNF-treated (Fig. 2d) and IL-i-treated (Fig. 2e) ECs, which were characterized by a fibroblastoid shape. Because some synergism between IL-i and TNF in inducing acute inflammation and Schwartzman-like reactions in rabbits in vivo has been reported [20], we tested the effects of different combinations of IL-i and TNF on the EC monolayens. Although there did not appear to be significant synergism between these cytokines in mediating EC toxicity at both low and high cytokine doses, a slight additive effect

582

Journal

(Fig. 4). produced either TNF

The addition slightly higher on IL-i alone.

of Leukocyte

Biology

20 18 16

01 I’)

12 0

10 8

. .

-i--

i5

1&

IL-I (units/mi) Fig. with and

3. Bovine recombinant SEM.

EC

cytotoxicity human TNF-a

(51Cr release) (A) or IL-ia

at 22 h following treatment (B). Values represent means

Factor and lnterleukin-i

toxic effects on ECs. We therefore added TNF and IL-i at various concentrations to EC monolayens. Both TNF and IL-i preparations had low toxic effects on the ECs as mdicated by low rates of 51Cr release (Fig. 3a and b). The CI observed at the highest TNF concentration (70 U) was 15, and a CI of 4 was evident at 100 U of IL-i. Morphological observations at 22 h P1 were consistent with activation of both

was observed combination tamed with

70

(unl)

B

To determine whether AMs can modulate EC injury mediated by purified P /zaeinolytica LPS, varying concentrations of LPS from 0.03 to 330 ng/ml were added to the culture well insert chamber in the presence or absence of AMs. LPStreated AMs significantly enhanced EC toxicity at all the LPS concentrations tested as indicated by 51Cn release and morphologic changes. By 22 h P1, 0.03 ng/ml LPS produced degenerative changes in the EC monolayers cocultured with AMs characterized by cell contracture and detachment of occasional cells and a CI of 19 (Fig. 1). At LPS concentrations of 3.3 and 330 ng/ml the CI was in the range 61-65 ( Fig. 1). In contrast to the normal monolayer appearance ( Fig. 2a), these LPS concentrations produced almost complete loss of EC monolayer confluence and numerous detached, shrunken cells (Fig. 2b). Less severe morphologic changes were apparent in the ECs treated with 3.3 and 330 ng/ml LPS in the absence of AMs (Fig. 2c), and the CI was much less in ECs without AMs than in ECs with AMs (36-43 versus 61-65). As expected, addition of polymyxin B to the P haemolytica LPS almost completely eliminated LPSmediated EC damage as indicated by CI values of only 2 and 3 in the EC and AM-EC groups, respectively (Fig. 1).

Direct

50 ThF

of

IL-i CI

Volume

and values

TNF than

51, June

in ob-

1992

Synergism

Between

LPS and Cytokines

To determine whether cytokines enhanced LPS-mediated EC toxicity, EC monolayers were treated with various concentrations of LPS (0.03 to 330 ng/well) and simultaneously with either TNF (50 U/well) or IL-i (25 U/well). The CI was greatly increased following addition of either cytokine at all LPS concentrations (Fig. 5a and b). At LPS (0.03 ng) the CI increased from 2 to ded. IL-i also greatly increased the CI but than TNF. This increased toxicity was decreased EC confluence and numerous (Fig. 2f), compared to ECs treated with (Fig.

the lowest dose of 35 with TNF adto a lesser degree also evidenced by degenerative cells only 0.03 ng LPS

2g).

Endothelial cell monolayers were also treated with rbIL-if3 (100 ng/ml), IL-ira (1000 ng/ml), on combinations of both in the presence of varying concentrations of P /zaemolytica LPS. As shown in Figure 6, rbIL-i significantly enhanced LPS-mediated EC toxicity at LPS concentrations of 6 and 200 ng/ml. However, the presence of IL-ira effectively eliminated this IL-i-enhanced EC toxicity. The IL-ira also significantly decreased EC toxicity in the presence of 200 ng/ml LPS.

not by binding to the IL-i molecule [3]. Several studies have demonstrated an in vivo protective effect of IL-ira in expenimental endotoxemia [31, 32]. In our study IL-ira also diminished EC toxicity mediated by LPS in the absence of exogenous IL-i. This suggests that IL-ira either inhibited LPS binding on the ECs or inhibited the autocnine influence of EC-generated IL-I. Both bovine and human ECs treated with LPS have the capacity to secrete IL-i, which could have an

a Fig. with (b), and

4. Bovine

e

C

EC cytotoxicity

(5tCr

release)

at 22 h following

treatment

1 U/ml recombinant human IL-ia (a), 2 U/ml recombinant TNF-a 1 U/ml IL-i + 2 U/ml TNF (c), 25 U/mI IL-i (d), 50 U/ml TNF (e), 25 U/mI IL-i + 50 U/mI TNF. Values represent means and SEM.

autocnine influence on the ECs [9, 17, 18]. It has been shown that LPS, IL-i, and TNF act via a common pathway of activation mediated by protein kinase C in human endothelial cells [10]. Lethal EC injury by these agents is dependent on de novo RNA on protein synthesis, demonstrated by inhibitor studies [24]. It is possible that cytokines may augment LPS toxicity in bovine ECs by stimulating similar activation pathways. Multiple intraceilular pathways are involved in TNF-mediated cellular injury on cytotoxicity, such as G protein-coupled activation of phospholipases, generation of free radicals, and damage to DNA by endonucleases. TNF binding to a cell surface receptor not only initiates the transcription of protective genes but also may initiate cellular damage, as determined by the metabolic state of the cell [7]. The mechanism by which LPS directly damages bovine ECs awaits clarification, although several

DISCUSSION We reported previously that bovine AMs contribute injury in an in vitro model of pulmonary pasteunellosis In this study we have attempted to further define role in the disease we demonstrated

to EC [29]. the AM

process. By employing a cocultune system, that P haemolytica LPS-mediated EC toxicity is due both to direct LPS effects and to LPS activation of AMs and subsequent release of proinflammatory cytokines, presumably IL-i and TNF. These results compare favorably with those of another study which showed that bacterial LPS induces release of TNF from bovine AMs and monocytes in vitro [1]. Increased serum concentrations of TNF in neonatal calves have also been observed following LPS administration [2]. Furthermore, TNF or IL-i has been shown to mimic the entire spectrum of LPS toxicity in vivo in some animal species [ii, 22, 30]. The adoptive transfer of LPS sensitivity by LPS-sensitive C3H/HeN mouse macrophages into LPS-nonsensitive C3H/HeJ mice has been reported, further supporting the role of the macrophage in mediating LPS toxicity in vivo [5]. In vitro experiments have shown that tissue factor expression of human ECs stimulated by LPS is enhanced when ECs are cocultured with human monocytes [33]. This enhanced tissue factor expression is dramatically reduced with anti-IL-i antibodies [33], demonstrating the importance of IL-i in monocyte-EC interaction. The apparent synergism between nonlethal doses of P haemolytica LPS and either IL-i or TNF in mediating EC injury in vitro suggests that our observed AM amplification of LPS effects is due to release of IL-i and TNF from AMs. A similan synergism was previously shown in an in vivo study of a munine model. TNF greatly enhanced the effects of bacterial products such as heat-killed Corynebacterium parvum, Mycoplosma sp.-infected lysates, or LPS in causing hemorrhagic necrosis and lethal shock [25]. In our system the ability of IL-i to mimic TNF in augmenting LPS-mediated EC injury is compatible with the known overlapping effects of these cytokines in a number of animal models [8]. The importance of IL-i enhancement of LPS-mediated EC damage was further supported by the results using IL-ira. The IL-ira inhibits the biological activity of IL-i on mammalian cells by binding to the cellular IL-i receptor and

A

90 80 70

n

I,

0.

-

‘I

33 LPS (n)

B

3.3

0.90

LPS (nmi) Fig. 5. Bovine EC cytotoxicity with P haemolylica LPS (#{149}) and (A) (U) or 25 U/mI recombinant means and SEM.

Sharina

ci at

Cytokine-mediated

(51Cr release) either 50 U/mi human IL-ia

endothelial

at 22 h following treatment recombinant human TNF-a (B) (U). Values represent the

cell

injury

583

6. Harlan,

J.M.,

Schwartz,

Harker,

S.M.,

bovine

and

endothelial

7. Larrick,

J.W.,

necrosis 8. Last-Barney,

factor-alpha. K.,

V.J.

and

and

MA.,

IL-i.

P., Ordovas,

CA.

, Faanas,

overlapping

10.

Magnuson, kinase C:

j

Immunol.

J.M.,

D.K., a potential

endotoxin, 216,

0.0

0.2

200

6.0

Fig.

6. Bovine EC cytotoxicity (51Cr release) at 22 h following treatment varying concentrations of P haemolylica LPS in the presence of control medium (A), 1000 ng/ml recombinant IL-ira (#{149}), 100 ng/ml rbIL-l (#{149}), or a combination of rbIL-l and IL-ira (#{149}).Values represent the mean and SEM.

12.

Auger,

D.N.,

A.Y.

Falk,

KR.,

2363, Meyrick,

Robbins,

implicated From

[15]. this study

toxic

oxygen oxidases,

xanthine

it is apparent

that

nary pasteurellosis is enhanced Further work is needed to define

EC

damage

14.

in pulmo-

by AM-secreted cytokines. molecular mechanisms

15.

of

this synergism, although it is likely that a common mediator at the second-messenger level may be involved. Understanding the synergism between LPS and TNF on IL-i at the molecular level should provide new approaches to therapeutic intervention.

B.,

Brigham,

K.L.

We thank Dr. Robert Moore for P haemolytica LPS, Sanmishtha Kumar for technical assistance, and Jan Grady for manuscript preparation. This work was supported by U.S. Department of Agriculture Special Grant 87-CRSR-2-3i25, the University of Tennessee Center of Excellence in Livestock Disease, and the University of Tennessee Agnicultural Experiment Station.

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