Agents and Actions, vol. 34, 1/2 (1991)

0065-4299/91/020113-04 $1.50+0.20/0 9 1991 Birkh/iuserVerlag, Basel

Phospholipase A2 (PLA2) activity in bovine pulmonary artery endothelial cells R. Goodman, T.M. Stevens, L.R. Mantegna, P.R. Kidd, R.R. Harris and J.S. Kerr The Du Pont Merck PharmaceuticalCompany, Wilmington, DE 19898, USA

Abstract

We have investigated the role of recombinant human interleukin-l]~ (rIL-1]~) and recombinant human tumor necrosis factor ~ (rTNF-c~) on PLAz activity, protein synthesis and eicosanoid production in bovine puhnonary artery endothelial cells. Cellular PLA 2 activity increased 4-fold and production of PGE 2 increased 3-fold at 1 - 2 hrs in the presence of 10 units/ml rIL-lfl. PLA2 activity increased 3-fold at 30 min and PGE 2 production increased 2-fold with 5 x 10-9 M rTNF-a. The data show that endothelial cells respond more rapidly to rIL-lfl ( 2 - 6 hr) and rTNF-a (30 min) than do chondrocytes and synovial cells (6-16 hrs), suggesting endothelial cells may play a primary role in initiating the inflammatory response.

Introduction

Cytokines play a significant role in the inflammatory response. Interleukin-1 (IL-1) and tumor necrosis factor (TNF) are two polypeptide cytokines produced by monocytes and tissue macrophages. IL-l and T N F have been shown to increase phospholipase A2 (PLAz) activity in rabbit chondrocytes and human synovial cells leading to the production of eicosanoids, in particular, the generation of prostaglandins E2 (PGEz) [1-3]. T N F also has been shown to stimulate bone resorption and collagenase production [4]. Endothelial cells, unlike chondrocytes, have specific receptors for both T N F - e and TNF-fi [5]. Binding of lymphocytes to endothelial cells and the movement of lymphocytes toward sites of inflammation is enhanced by IL-1 and T N F [6]. The strategic position of endothelial cells between blood and tissues makes them a good model for the study of the role of the vascular in the initiation and maintenance of inflammation. The purpose of

this study was to investigate the effects of rlL-lfl and rTNF-~ on PLA 2 activity and PGE 2 production of bovine pulmonary artery endothelial cells.

Materials and methods

Bovine pulmonary artery endothelial cells [(CPAE) American Type Culture Collection, Rockville, Maryland] were seeded at 2 x 10 s cells/ well in 12-well plates with 1 : 1 Dulbecco's Modified Eagle's Medium and H a m ' s (F-12) medium with 10% fbtal bovine serum (FBS) and grown to confluency at 37 ~ in the presence of 5% COz in air. Media were then changed to H a m ' s (F-12) medium containing rIL-lfl (The Du Pont Merck Pharmaceutical Co., Inc., Glenolden, PA) (0-1000 units/ml) or rTNF-c~ (Genzyme Corp., Boston, MA) (5 x 10 -8 to 1 x 10 -11 M). After incubation with rIL-lfl or rTNF-~ culture media were decanted and cells harvested by scraping.

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Agents and Actions, vol. 34, 1/2 (1991)

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Agents and Actions, vol. 34, 1/2 (1991)

PLA2 activity was measured in cell homogenates and culture media by using heat inactivated 3Holeic acid labeled E. coli as substrate, according to Patriarca et al. [7] as modified by Davidson et al. [8]. Data were expressed as nmol phospholipid hydrolyzed per mg protein per hour. PGE 2 production in culture media was measured using 3(H)-RIA kits (New England Nuclear, Boston, MA); data were expressed in ng/ml. To determine the effects on protein synthesis, CPAE's were treated with actinomycin D (ACTD) (Sigma Chemical Co., St. Louis, MO) (15 ~tg/ml) or cycloheximide (CHX) (Sigma Chemical Co., St. Louis, MO) (150~tg/ml) in conjunction with rIL-lfl (2 hrs) or rTNF-a (16 hrs). Cell homogenates and culture media were then assayed for PLA z activity. Results

The effects on PLA 2 activity from CPAE's treated with rIL-lfl were investigated (Fig. 1). Cell-associated PLA z activity increased in the presence of rIL-lfl and rTNF-a: PLA2 activity increased from 481 (control) to 1220 nmol/mg/hr as rIL-lfl increased from 0 to 1000 units/ml (20 hrs). There was an increase to 1500 nmol/mg/hr after 6 hrs with 10 g/m1 rIL-lfl. PGE 2 production also increased from 0.83 + 0.07 ng/ml (controls) to 2.24 +_0.03 ng/ ml after 2 hrs with 10 ~/ml rIL-lfl and then the production reached a plateau. PLAz activity remained constant in the presence of CHX (947 umol/ mg/hr control vs 908 nmol/mg/hr IL-I + C H X ) while ACTD blocked the increase of PLA 2 activity of cells treated with 10 g/ml rIL-lfl (404 nmol/mg/ hr, rlL-lfl + ACTD). In the presence of rTNF-~, PLA 2 activity increased from 481 nmol/mg/hr to 1105 nmol/mg/hr as rTNF-c~ was increased from l x l 0 -~1 to 1 x 10 -8 M (p