Effects of recombinant interleukin-1 beta on phospholipase A2 activity, phospholipase A2 mRNA levels, and eicosanoid formation in rabbit chondrocytes

Vol. 165, No. 3, 1989 December 29, 1989

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages

1079-1084

EFFECTSOF RECOMBINANT INTERLEUEIN-1BETA ONPHOSPHOLIPASE A2 ACTIVITY, PROSPHOLIPASE A2 mRNALEVELS, ANDEICGSANOIDFORMATIONIN RABBIT CHONDROCYTES JANET S. KERR, THERESAM. STEVENS,GARYL. DAVIS. JUDITH A. MCLAUGHLIN AND RICHARDR. HARRIS MEDICALPRODUCTS DEPARTMENT,E. I. DU PONTDE NEMOURS & CO. (INC.), EXPERIMENTALSTATION, E400/4223, P. 0. BOX 80400 WILMINGTON,DE 19880-0400 Received

October

23,

1989

We have investigated the effects of recombinant interleukin-1 beta (rIL-la) on phospholipase A2 activity (PLA2), PLA2 messenger RNA levels, and eicosanoid production in rabbit chondrocytes. Phospholipase A2 activity increased 5 fold with exposure to 1.6 x 10-llM rIL-lp for 20 hours. An mRNA specific for an extracellular PLA2 was detected by RNA blot hybridization after treatment of the cells with rIL-119. Hydroxylated derivatives of arachidonic acid, including prostaglandins, leukotriene B4, 5-hydroxyeicosa 6E, 8Z, llZ, 14Z-tetraenoic acid, 12-hydroxyeicosa 5Z, 82, lOE, 14Z-tetraenoic acid and 15-hydroxyeicosa 5Z, 82, llZ, 13E-tetraenoic acid increased in cells after rIL-lp treatment. c 1989ACddallC PreE.SS, Inc.

Interleukin-1 (IL-l), a cytokine which plays a role in inflammation, is produced by monocytes and tissue macrophages. This protein coordinates many of the activities of cells in both promoting and amplifying the inflammatory response. IL-l induces PLA2 and cyclooxygenase activities and increases prostaglandin synovial

cells

synthesis in chondrocytes,

from rheumatoid joints

(3).

fibroblasts

(l-2),

and

The purpose of this study was to

investigate both the early and late effects of rIL-119 on PLA2 activation, PLA2 mRNAlevels, and arachidonic acid metabolism in rabbit chondrocytes, in order to understand how rIL-la

affects

the inflammatory

response of these cells.

MATERIALSANDMETHODS Reagents and Chemicals. Tissue culture supplies and media were obtained from Gibco (Grand Island, NY). FBS was obtained from Hyclone Abbreviations: rIL-la: humanrecombinant interleukin-1 beta; PLA2: phospholipase A2; RIA: radioimmunoassay; HPLC: reverse phase high performance liquid chromatography; PGE2: prostaglandin E2; PGD2: prostaglandin D2; PGF2a: prostaglandin F2a; LTB4: leukotriene B4; LTC4: leukotriene Cq; LTD4: leukotriene D4; AA: arachidonic acid; PC: phosphatidylcholine; 5 BETE: 5-hydroxyeicosa-GE, 82, llZ, 14Ztetraenoic acid; 12 HETE: 12-hydroxyeicosa-5Z, 82, lOE, 14Z-tetraenoic acid; 15 HETE: 15-hydroxyeicosa-5Z, 82, llZ, 13E-tetraenoic acid; FBS: fetal bovine serum; BSA: bovine serum albumin; Northern: RNA blot hybridization.

1079

0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Logan, UT). BSA was purchased from Sigma Chemical Company (St. Louis, MO). r-IL-l/Y was obtained from du Pont de Nemours& Co. (Inc.), (Wilmington, DE). RIA kits for PGE2, 5-HETE, 12-HETE, 15-HETE, LTB4 were purchased from Advanced Magnetics Inc. (Cambridge, MA). The cross-reactivity amongthe RIA kits were < 1% as determined by Advanced Magnetics. All standards for HPLC assays were purchased from Bio Mol (Plymouth Meeting, PA). All other reagents were reagent-grade. Chondrocyte Cultures. Articular chondrocytes from the knee joints of normal 12-week old male New Zealand white rabbits (Charles River Breedina Laboratories, Wilmington, MA) were isolated as previously described (4).Cells were incubated with 1.6 x lo-11 Y (10 U) rIL-la in media without FBS for 15 min to 20 hr. We have previously shown this concentration to be on the plateau of the dose response curve and to be non-toxic (5). The supernatants were decanted and cells were removed by scraping. Aliquots from both cells and supernatants were immediately measured for PGE2, and the remainder of the samples were then stored at -7O'C until ready for analysis. PLA2 Assay. PLA2 activity in cells or supernatants from the 24 well plates was determined using [3H] o1eic acid labeled E. coli as substrate, prepared according to Patriarca et al. (6), as modified Davidson et al. (7). PLA2 activity is expressed as nmol phospholipid (PL) hydrolyzehpz mg protein per hour. HPLCAssays. Confluent chondrocyte monolayers in 75 mmflasks were treated with rIL-l!, or medium alone. After treatment for 20 hrs aliquots of supernatants, and cells were collected, and sodium acetate added to give a final concentration of 50 mM(pH 3.5). The supernatants extracted with 6 volumes of ice-cold ethyl acetate. The samples were centrifuged at 1000 rpm for 10 min at 4°C using a Sorvall RC3 centrifuge (Du Pont, Newton, CT)! and the lower phase was re-extracted. Ethyl acetate extractions were combined, dried down under nitrogen and the samples suspended in 300 ~1 of ethanol. This method extracted more than 90% of the major cyclooxygenase and lipoxygenase products. Reverse-phase HPLCanalysis of the extracts was done using a Hewlett Packard model 109OMHPLCand a Rainin microsorb C-18 (4.6 mmx 10 cm) column (Woburn, MA). The AA metabolites were eluted using a discontinuous gradient: O-11 min H20:acetonitrile:H2P04 (70:30:0.01) and 11-22 min. methanol:H20:trifluoroacetic acid:triethylamine (400:100:0.5:0.250). The areas under each of the spectrophotometrically identified peaks were determined and were compared in relative units. RIA Analysis. Cells and supernatants from the confluent monolayers after incubation with rIL-l/? were assayed after extraction using competitive-binding radioimmunoassays for PGE2, LTB4, and 5-HETE. and purifications were carried RNAs and RNAmethods. RNAextractions out, as previously described (a), using guanidinium isothiocyanate and cesium chloride. RNAwas electrophoretically separated on 1.2% formaldehyde gels, transferred to Genescreen Plus filters (New England Nuclear, Boston, MA), and ;i;b;il;sing standard methodology (8). Total cellular RNA (1Opg) was added Placental polyadenylated RNAwas used as a positive control (9), (Clontech Palo Alto, CA); 1 pg of this RNAwas added per lane of the gel. Agarose gel purified placental PLA2 cDNA sequences (see below), nick-translated to a specific activity of 4 x 108 cpm/pg and denatured, were used at a concentration of 10 rig/ml to probe the filters. After a prehybridization step, hybridizations were performed in a solution of 5X SSC, 1% SDS, 50% formamide, 1X Denhard's solution (1X Denhard's 0.02% Ficoll, 0.02% and 0.02% BSA), 10 mg/ml denatured and sheared salmon polyvinylpyrrolidone, sperm DNA, and 10 mg/ml yeast RNA, at 42°C overnight; final washes were done in 0.2X SSC, 1% SDS, at 50°C for 30 min. HomologousRNAs were visualized by radiography using Kodak XAR-5 film. cDNA probe. A cDNAencoding a polypeptide identical to that of human synovial fluid and platelet PLA2 (10,ll) was isolated from a human placental cDNA lambda gtll library (9). The PLA2 insert, subcloned into a plasmid In a similar manner, vector, was excised and gel purified prior to labeling. a gel-purified 2 kb human beta-actin fragment was used as a probe to verify that equivalent amounts of RNAwere transferred to the filters. DNAswere 1080

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labeled using [32P]dCTP (New England Nuclear, Boston, MA) and a commercial nick-translation kit (Promega, Madison, WI). RESULTS We investigated

the kinetics

of appearance of PLA2 activity

and the

relative amounts of AA metabolite formation in cells following stimulation with 10 U rIL-la (Table 1). Specific activity of cell-associated PLA2 increased from 27 nmol PL hydrolyzed/mg 208 * 36 nmol PL hydrolyzed/mg

protein/hr

protein/hr

at time 0 to

at 20 hr (p < 0.05).

the AA products in the supernatants within

the first

In general,

hr were variable,

particularly PGD2/PGE2/PGF2and the RETE's (data not shown). However, in the cells at 30 min the PG's were elevated, and at 60 min the ElETE's were maximal. At 20 hr, cellular

AA and LTB4 levels were maximal, which coincided with the

maximal PLA2 activity. The relative peak area for AA was 112 f 47 (Table 1) and for LTB4 was 95 2 17, which were significantly elevated above controls and all

other time points

12-RETE's were verified hydroxylated

(p < 0.05).

The presence of PGE2, LTB4 and 5 and

by RIA.

These data demonstrate that low levels of

derivatives

of AA are produced by rabbit

chondrocytes.

20 hr of r&la exposure were AA products measured consistently supernatants, when AA, LTB4 and RETE's increased above control

Not until

in the levels (data

not shown). After

20 hr incubation

homologous with human synovial

with rIL-lp, fluid

PLA2 sequences was apparent (Fig. Table

HPLC

Identification

of

the presence of an mRNAhighly

1

AA Metabolites

Following

rIL-18

Stimulation

Cells Treatment

and

Time

PLA21 Activity

PGW PGE2l PGF2n

AA

BETES (5,12,15)

Controls 0 20 hr

3 2

18+ 262

8 8

26k 6 31 + 12

202 5 65 f 28

33k 7 322 8 392 9 208 + 362

12+ 12t

6 7

14* 2 150 2 72 102 3 55 + 43

60 f 27 332 4 79 f 30 262 5

272 30?

w (1.6 15 30 60 20

x lo-1lM) min min min hr

Area = Mean of the lnmol PL hydrolyzed/mg 2P < 0.05 compared

relative with

area protein/hr controls

11;

? 472

k SEM and

all

1081

(n=3-9). other

time

points

(n=5).

l),

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1.4 1.3 0.8

0.2 Fivure 1 RNA blot hybridization identifies homologous RNA in rIL-l/V-treated rabbit chondrocytes. 10 pg of total rabbit (Rb.) chondrocyte RNA fractionated on 1.2% agarosefformaldehyde gels and transferred to membrane filters were probed with a cDNA from human placenta (9) encoding the "synovial fluid" PLA2 (10,ll). Under stringent washing conditions, a highly homologous RNA of approximately 900 bases appeared after 24 hr of recombinant human IL-l treatment at 10 p rIL-$7. This band migrated in the gel to an identical location as the positive control RNA (1 pg of polyandenylated human placental RNA). Untreated chondrocytes (w/o IL-) were unreactive with the probe. Values (in kilobases) for the molecular weight standards (MW STD), visualized after ethidium bromide staining the gel, appear in the left margin; the point of migration of 18s ribosomal RNA is identified (18s).

whereas it was not measurable in controls.

This correlates with the observed increase in PLA2 activity. The data shown in Table 2 demonstrate that in the presence of rIL-la plus actinomycin D (a protein synthesis inhibitor of transcription)

protein

or cycloheximide

translation),

(an inhibitor

PLA2 activity

of the initiation

remained at control

levels.

of cytosolic These data

suggest that rIL-l/J acts to induce the synthesis of new PLA2 protein, than affecting PLA2 activity alone.

rather

DISCUSSION In the present study we demonstrated that after

incubation

with

rIL-$9, cellular eicosanoid production significantly increased in rabbit chondrocytes at 15-60 min. After incubation with r-IL-lp for 20 hr the increase in cell-associated AA and enzyme activity correlated with an appearance of an mRNAreactive with a humanPLA2 probe. This is the first report that demonstrates an early increase in PG-production and 1082

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

2

Cell-Associated PLA2 Activity in the Presence of Protein Inhibitors After 20 hr Incubation with rIL-la Treatment

PLA2 Activity hydrolyzed/tag

(nmol/PL

Control

192

rIL-l/?l

n=3. *P < 0.05 compared with lrIL-lb=1.6 x lo-11 M.

for

17?

D + rIL-la1

Cycloheximide (150 pglml)

level

protein/hr)

5

220 3 18*

Actinomycin (15 ~glml)

+ rIL-lpl

cell

PLA2 and eicosanoid

3

22 + 15

after

values.

and at 20 hr correlates

HETE formation,

Synthesis

production

PLA2 enzyme activity, under

the

message

same experimental

conditions. We and others (1,5,12,13) increasing

levels

chondrocytes. PLA2 activity

of rIL-la

have previously

increase PLA2 activity

demonstrated that associated with

Chang -et al. (1) reported an increased cell-associated after 6 hr of exposure to 5 U/ml IL-la, and Chin 8t Lin (14)

reported that at approximately

the same time (7 hr) PGE2production

increased. Our findings extend these data by showing that both prostanoids and leukotrienes were generated by rIL-lp stimulation. is the first the early

report

of prostanoids and EIETEproduction

This

by these cells

at

time points.

which is five

Chang -et al. (1) used 108 U/mg (5 U/ml) rIL-lp, times the level of rIL-la we used (0.2 x 108 U/mg; 10 U/ml)

and may explain the earlier increase in PLA2 activity since we did not observe an increase in PLA2 activity

(6 hr vs 20 hr), at 6 hr (data not

shown). rIL-$9

The prostanoid and EETE data suggest that upon stimulation with there was a transient increase in PG's, followed by HJSTE's, and

then at 20 hr a more prolonged increase in AA which probably was the result of the increased PLA2 activity. The lack of a sustained PG and HETEresponse could be related to either increased metabolite breakdown or product inhibition of the cyclooxygenase or lipoxygenase enzymes. Further these data suggest that the cell-associated AA metabolites may be involved in, or the result of early signalling within the chondrocyte as a result of stimulation with rIL-lg. It should be noted that we found significant increases in PG's in the supernatants at 20 hr of r-IL-la 1083

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

incubation, but no such increase in leukotrienes or HETE's, suggesting that after 20 hr of stimulation the cells may begin to release prostaglandins.

Further, these observations suggest that the supernatants do not accurately reflect the cellular levels of these

metabolites. We selected 20 hr of stimulation with rIL-la to investigate the relationship between the changes in arachidonic acid

closely

metabolism, PLA2 activity

and PLA2 mRNAlevels.

At this time point, PLA2 increased in concert with increased mRNAand AA, PGA2/PGE2/PGF2,

activity

and LTB4 production

in these cells

PLA2 biosynthesis, PLA2 activity chondrocyte PLA2 may be similar (15), in rat platelets peritoneal (18) -

more

Its

fluid

suggesting

a

possible link between

and eicosanoid production. The to that found in human synovial fluid

both membrane-boundand secreted (16), and in

of casein-treated

characterization

rats (16) or glycogen-treated

awaits its

rabbits

isolation.

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