The activation of human fibroblast prostaglandin E production by interleukin 1

CELLULAR

IMMUNOLOGY

110,338-349

(1987)

The Activation of Human Fibroblast Prostaglandin Production by lnterleukin 1 ROBERT C. NEWTON AND MARYANNE

E

COVINGTON

E. I. duPont de Nemours and Co., Medical Products Department, Glenolden, Pennsylvania 19036 Received June 8, 1987: acceptedAugust IO, 1987 We have examined the induction of prostaglandin E2 (PGEZ) release from fibroblasts by human interleukin 1 (IL-I). A number of fibroblast cell lines appear to respond to IL-l in a fashion similar to that seenwith synovial fibroblast cultures. Using the Gin- 1 primary Iibroblast cell line, the earliest time where a significant increase in PGE2 release can be detected is 2 hr. Thereafter PGE2 appears to increase dramatically, with levels after 5 hr increased over 50-fold above baseline. IL-l appears to directly induce the increase in PGE2 since removal of other proteins from culture medium does not affect induction. PGE2 induction by IL-1 also does not require cell proliferation. The induction appears to involve the synthesis of new protein since the enhanced releasecan be completely blocked by addition ofactinomycin D or cycloheximide. Arachidonic acid mobilization in cells does not appear to be altered following IL-I addition. However, the ability to convert arachidonic acid to PGE2 is increased following 5 hr of culture with IL-I. While increasing the release of PGEZ, the addition of phorbol esters, alone or in combination with calcium ionophores, does not mimic the protein synthesis-dependent increase seen with IL-l. Taken together these results suggestthat IL-1 induction of libroblast PGE2 involves the synthesis of new protein or proteins involved in the conversion of free arachidonic acid to PGE2. 0 1987 Academic Press,Inc.

INTRODUCTION Interleukin 1 (IL-l)’ is a protein which has been demonstrated to be important to the functions of the immune system. As such, IL- 1 has been shown to play an important role, along with antigen, in the activation of lymphocytes to become proliferating cells and to produce a number of immune components including interleukins and immunoglobulins (1,2). As well as the documented effectson the immune system, IL- 1 has also been demonstrated to have a wide range of effects on cells of other physiological systems (3). IL-l activates liver production of acute phase proteins, induces fever, alters serum metal levels, activates endothelial cells, and induces fibroblast proliferation. One of the well-documented effects of IL- 1 has been the activation of arachidonic acid metabolism (4,5). The most consistent effect is seenin the increased production of prostaglandin E. The E prostaglandins are thought to be important for their ability to suppress mitogen responses, production of T-cell lymphokines, and generation of ’ Abbreviations used: BSA, bovine serum albumin; PBS, fetal bovine serum; IL- 1, interleukin 1; PGE, prostaglandin E; PLA2, phospholipase A2; PMA, phorbol myristate acetate. 338 000%8749/87 $3.00 Copyright 0 1987 by Academic Press,Inc. All rights of reproduction in any form reserved

IL-l INDUCTION

OF FIBROBLAST PGE

339

immunoglobulins by B lymphocytes (6,7). As such, PGE is thought to be a negative regulator of immune events. A potential source of PGE related to immune reactions is the production of PGE in responseto IL- 1. This induction appearsto be part of a complex responseinvolving the synthesis of numerous proteins including collagenase, granulocyte macrophage colony-stimulating factor, and plasminogen activator (5, 8, 9). The PGE2 response of fibroblasts to IL- 1 may be an attempt to inhibit or limit production of and response to IL-l. In this study we have examined various parameters of IL- 1 induction of fibroblast PGE production to elucidate potential mechanisms for this action. Our investigations have demonstrated that the induction of fibroblast PGE is not a direct mobilization of arachidonic acid but involves the synthesis of new protein(s) following stimulation by IL- 1. We have obtained evidence that the new protein is not the phospholipase which mobilizes arachidonic acid but appears to be a protein or proteins responsible for the conversion of arachidonic acid to PGE. MATERIALS AND METHODS Arachidonic acid and indomethacin were purchased from Sigma Chemical Co. (St. Louis, MO). Radioimmunoassay kits for prostaglandin E2, along with [3H]thymidine and 14C-arachidonic acid were purchased from DuPont-NEN (Boston, MA). Phorbol my&ate acetate was purchased from Consolidated Midland Corp. (Brewster, NY). The calcium ionophore ionomycin was purchased from Calbiochem-Behring (San Diego, CA). Media were prepared in the Glenolden Tissue Culture Facility and fetal bovine serum was purchased from Hyclone (Logan, UT). All tissue culture plastics were from Costar (Cambridge, MA). Recombinant IL-l alpha was purchased from Genzyme (Boston, MA) and recombinant IL- l/3 was purchased from Genzyme, Cistron Technology (Pine Brook, NJ) or prepared in our laboratory. Monocyte IL- 1(Y and -p were prepared in our laboratory using ion-exchange and gel-permeation chromatography. Cell culture. Gin- 1, a human gingival fibroblast, MRC-5, a human fetal lung fibroblast, and Detroit-55 1, a human embryonic dermal fibroblast, were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium with 10% FBS, 2 ~ML-glutamine, and 50 pg/ml gentamycin and were passagedby trypsin (GIBCO, Grand Island, NY) treatment. Gin-l cells were routinely split twice weekly and cells were used between passages15 and 25. Fibroblast activation by IL-l. For use in experiments, cells were removed from flasks by trypsinization, washed with medium, and replated at 3 X lo5 cells/ml in either 96-well plates (100 ~1 of cell suspension/well) or 24-well plates (0.5 ml of cell suspension/well). Following overnight incubation, the medium was replaced with medium containing 1 mg/ml fatty acid poor bovine serum albumin (CalbiochemBehring) and the cells were stimulated by addition of 200 units/ml IL- I. One unit of IL-I is defined as the amount of IL-1 which generates half-maximal activity in the thymocyte proliferation assay.The recombinant IL- 1 used routinely in these studies had a specific activity of 1 X 10’ units/mg. Cells were stimulated by the addition of 20 rig/ml IL- 1, final concentration. Prostagfandin assay.Supernates were collected and stored in polypropylene tubes (Sardstedt, NJ) at -20°C until assayed(within 1 week of collection). The assayused

340

NEWTON AND COVINGTON TABLE 1 Stimulation of Human Fibroblasts by Human Recombinant Interleukin ID

Cell line

N”

Control + SEM’

IL-l + SEM

Stimulation’

Gin-l Detroit 55 1 MRC-5 Synovial Fb

9 3 4 13

15s* 71 880? 50 1200 + 440 834 +- 343

13612+2496 2650+ 131 30690 k 2400 26095 + 56 11

86 3 26 31

Note. Cells were stimulated by the addition of 200 units/ml IL- 10 and supemates were sampled after 20 hr and tested for levels of PGEZ. All values are reported as picograms per milliliter of PGE2. ’ Number of independent experiments. ’ SEM, standard error of the mean. ’ IL1 stimulated PGE2 divided by control PGE2.

for detection of prostaglandin E2 is sensitive to detection of PGE2 with minimal (3.7%) cross-reaction with PGEl .

Results IL-l stimulation offibroblast PGEproduction. When fibroblasts are exposed to IL1 these cells produce relatively large amounts of prostaglandin. All of the cell lines tested responded to IL-1 addition with PGE production (Table 1). In addition, synovial fibroblast cultures also show this response to IL- 1. The differences among the three fibroblast lines appear to be in their baseline production of PGE and the degree of stimulation in response to IL- 1. Becauseof the low baseline production, and large response to IL- 1 the Gin- 1 fibroblasts were chosen for further study. Efict ofIL-l source.The responseof the Gin- 1 cells to IL- 1 from different sources was tested. As seen in Table 2, the cells appear to respond equally well to the monocyte and recombinant IL- 1 and to both (Yand p forms of IL- 1. The dose-response curves for recombinant cxand p IL- 1 did not demonstrate any significant differences TABLE 2 The Effect of Different IL- 1 Sourceson Stimulation of PGE2 Production by the Human Primary Fibroblast Line Gin- 1 Sample

PGE2 + SEM ’

No IL-l Monocyte IL-l@ Monocyte IL- 1(Y Cistron IL- l/3 Genzyme IL- I@ Genzyme IL- 101 DuPont IL-ID

446+ 154 20523 f 486 15315 f 2308 17211 + 1324 16355 + 3078 15665?2133 17354? 3716

Note, Cells were stimulated for 5 hr in the presenceof 200 units/ml IL- 1. All IL- 1 samples were standardized for activity in the thymocyte proliferation assay.All values are reported as picograms per milliliter of PGE2. Data are the summaries of three independent experiments. 0 SEM, standard error of the mean.

IL-1 INDUCTION

OF FIBROBLAST PGE

TIME

341

(Hr)

FIG. 1. Time course of the induction of PGE2 from the Gin-l fibroblast cell line by addition of 200 units/ml recombinant IL-&

in the amount of material required for either maximal or half-maximal responses (data not shown). Routinely maximum stimulation could be obtained with the addition of 20 units/ml (2 rig/ml) IL- 1 to these cells. Time course of stimulation. We examined the rate of PGE production in response to IL- 1 addition. It has been demonstrated that neutrophils are capable of detectable activation of arachidonic acid metabolism within minutes of stimulation with the synthetic peptide f-met-Zeu-phe (10). Since most previous studies of IL- 1 activation of fibroblast PGE have measured PGE levels after 24 hr, we followed the time course of Gin- 1 fibroblast PGE production. As seenin Fig. 1, no detectable increase in PGE is seen until 2-3 hr. This delay is not due to a limiting amount of IL-l since the addition of 200 units/ml IL- 1 is lo-fold higher than the level necessaryfor maximal stimulation of PGE production by these cells. The delay also cannot be attributed to a lack of sensitivity in the assayfor PGE since baseline PGE releasefrom unstimulated fibroblasts can normally be detected. Following the initial increase of PGE in the supemate after 2-3 hr there appears to be a rapid increase in production over the next few hours. The levels present after only 6 hr of culture with IL- 1 are comparable to the levels seenafter 24 hr of culture. To limit the interference of PGE metabolism in the analysis of IL-1 effects, all effects on PGE production in subsequent experiments were examined in the first 6 hr following IL- 1 addition. Efict of trypsinization of IL-1 response. IL-1 receptors have been described on a number of cells, including fibroblasts (11). Gin- 1 cells have been found to possess about 230 specific IL- 1 receptors with an affinity of 0.5 nM (R. Horuk, unpublished), Cells were examined for responsivenessto IL- 1 following trypsin treatment to determine if trypsin removed or reduced responsivenessto IL- 1. Preliminary studies indicate loss of >90% of specific IL- 1 binding following trypsinization of cells. As seenin Fig. 2A, cells are still capable of responding to IL-l immediately following trypsin treatment although at a dramatically reduced level compared with addition after allowing the cells to recover for 20 hr. Cells incubated for 0, 15, 30, 60, or 90 min following trypsin treatment before IL-l addition gave kinetic responses similar to cells immediately after trypsinization (data not shown). A significant increase in the responsewas seen only when cells were allowed to recover for at least 2 hr before IL-

342

NEWTON AND COVINGTON

Time (Hrs)

Time (Hrs)

FIG. 2. The effect of time following trypsin treatment of cells on the time course of PGE2 releaseinduced by addition of 200 units/ml recombinant IL-l& Cells were examined at 0 min (A), 1 hr (O), 2 hr (m), and 20 hr (0) following trypsin treatment. The level of PGE was measured at I-hr intervals following addition of IL-l. (A) Absolute amount of PGE2 release over the first 5 hr following IL-l addition. (B) Amount of PGE2 released, normalized against the final concentration of PGE2 found after 5 hr of incubation.

1 was added. We believe that the increased response rate seen after allowing cells to recover for 2 hr is due to reexpression of IL- 1 surface receptors. Figure 2B represents the time course of activation when the production of PGE is normalized to the PGE levels present after 5 hr. This graph demonstrates that the rate of production is similar regardlessof the time following trypsin treatment. This indicates that the mechanism of activation is similar at all time points, varying only in intensity, which may be directly related to surface receptor number. Dependenceof responseon IL-l. Serum has been shown to contain a number of proteins which influence fibroblast growth and activity. In addition, a number of IL1 activities have been shown to depend on the presence of second signals. Because cells are often stimulated following overnight culture in medium containing serum, the impact of serum on IL- 1 stimulation was examined. Cells were incubated overnight in medium with FBS, serum-free medium and serum-free medium containing BSA. The ability to stimulate cells with IL- 1 was then examined. The results, shown in Table 3, indicate that serum components are not required for IL- 1 stimulation of fibroblasts. Since serum starvation inhibits the proliferation of fibroblasts, these results also indicate that cells do not need to be proliferating to respond to IL-l with PGE production. Indeed, treatment of cells with mitomycin C for l-2 hr prior to IL1 exposure also had no significant effect on PGE production (Table 3). Treatment with mitomycin C inhibited cell proliferation 97% while overnight culture under serum-free or BSA-added conditions inhibited proliferation 70% as measured by [3H]thymidine incorporation. Sensitivity of stimulation to inhibitors ofprotein and RNA synthesis.Becauseof the delay seenbefore PGE production in responseto IL- 1, we tested whether stimulation was a direct effect of activation of preformed cell components or involved the synthesis of new proteins. As seen in Table 4, the addition of actinomycin D, an RNA

IL-1 INDUCTION

343

OF FIBROBLAST PGE TABLE 3

Effect of Media Composition on Induction of Gin- 1 Secretion of PGE2 Induced by Recombinant IL- I fl Addition Addition

Medium”

None IL-l IL- 1 IL- 1 IL-1 IL-I

10%FBS 10%FBS 10%HS 1 mg/ml BSA 1 mg/ml HSA No protein

IL- I

10%FBS + 10 &ml

~~52 h/ml) 1472 106 25808 f 2222 21940 t 5882 28829 k 4324 28668 + 5521 13841 + 2452 Mit C

236185 754

Note. Cells were stimulated after overnight culture in the described media with 200 units/ml IL- 10 and supemates were sampled after 5 hr of culture. Results are the averagesof four experiments. ’ Media additions: FBS, fetal bovine serum; HS, human serum; BSA, fatty acid poor bovine serum albumin; HSA, human serum albumin; Mit C, mitomycin C. All additions were to Dulbecco’s modified Eagle’s medium.

synthesis inhibitor, or cycloheximide, a protein synthesis inhibitor, simultaneous with the addition of IL- 1 inhibits the increased PGE production by >90%. This inhibition is comparable to the inhibition seen with indomethacin, a cyclooxygenase inhibitor. These results were seen with all three fibroblast lines and with all of the IL- 1 forms tested, indicating a general phenomena in fibroblasts. We tested the effect ofthe timing of the addition ofthese inhibitors on PGE production in response to IL- 1. These results are shown in Fig. 3. It is evident that addition of the inhibitors at or before 3 hr following IL- 1 addition inhibits the subsequent PGE increase. However, if these inhibitors are added after 3 hr of incubation with IL- 1, there is no significant effect on PGE production. These results indicate that the protein(s) produced in responseto IL- 1 is synthesized in the 3 hr immediately following IL- 1 addition. IL-l efict on arachindonic acid metabolism. Since new protein synthesis is required for PGE production in response to IL- 1, we next attempted to determine the TABLE 4 The Effect of Various Inhibitors on IL- 1 Induction of Gin- 1 Fibroblast PGE2 Production Additions”

PGE2 (pg/ml of:SEM)

% Inhibition b

None IL- 1 IL-1 + CHX IL-1 + ActD IL-1 + Indo

103-c 40 25439 + 3 149 596+ 114 527 f 253 31-t 22

98 98 100

Note. Cells were stimulated with 200 units/ml recombinant IL&eta in the presence of inhibitors and supemates were sampled after 5 hr of incubation. Results are the averagesof three experiments. a Compounds were added simultaneous with IL-1 at concentrations of 10 &ml for cycloheximide (CHX) and actinomycin D (ActD) and at 1 X 10e5Mfor indomethacin (Indo). b Percentage inhibition of IL- I stimulation of PGE2 production.

344

NEWTON AND COVINGTON

Time (Hr.9

Time (Hrs)

FIG. 3. The effect of timing ofaddition (O-5 hr relative to IL 1addition) ofeither 10&ml cycloheximide (A) or 1 &ml actinomycin D (B) on the time course of IL-l induction of PGE2 from Gin-l fibroblasts. Cells were stimulated by the addition of 200 units/ml recombinant IL-10 and the release was measured every hour over a 5-hr period. Cycloheximide or actinomycin D was added 0 (0), 2 (a), 3 (A), 4 (U), or 5 (0) hr after IL- 1. Control cultures (a) received IL- 1 alone.

general nature of the protein(s) being synthesized. We first examined the ability of IL1 to increase the mobilization of arachidonic acid in fibroblasts. This would indicate whether there is an increase in phospholipase A2 (PLA& which mobilizes arachidonic acid from cell membranes. The arachidonic acid pools in cells were labeled for either 5 or 20 hr by incubation with 14C-arachidonic acid. The cells were then washed and

1.4 I.3

,/

1.0

. .

‘S

d’ /

.8

,,f ”

.s A 2 L::.

1

3

3

4

5 Time (Hours)

FIG. 4. The mobilization of arachidonic acid from Gin- 1 fibroblasts in the presence (0) or absence (m) of 200 units/ml recombinant IL-ID. Cell pools of arachidonic acid were isotopically labeled for either 5 (A) or 20 (B) hr before induction. Total label was determined by lysis of cells at the conclusion of the experiment.

IL-l INDUCTION

OF FIBROBLAST PGE

Time

345

(Mid

FIG. 5. The ability of Gin-l fibroblasts to convert arachidonic acid to PGE2 following 5 hr of culture with 200 units/ml recombinant IL- 10. Following incubation cells were washed and free arachidonic acid was added. Samples were taken after 5 and 10 min of incubation and assayedfor PGE2 by radioimmunoassay. Samples tested include control (---), IL- 1 (-), IL- 1 with 10 &ml cycloheximide (- 1-), and IL- 1 with 1 &ml actinomycin D (. . .)-treated cultures.

IL- 1 was added. The supernate was sampled with time of incubation and the release of label was determined (Fig. 4). If IL- 1 acts to increase the level of PLA, in cells, then we should seean increase in label beginning 2-3 hr after IL- 1 addition. It is clear that the addition of IL- 1 to prelabeled cells does not significantly affect the amount of free arachidonic acid releasedby cells. There were no significant differences seenwith cells prelabeled for either time period. This also indicates that IL-l does not induce the direct mobilization of arachidonic acid as seenin chondrocytes ( 12). Cells were next incubated in the presence or absence of IL- 1. After 5 hr, the cells were washed four times with medium and fresh medium containing 1 mg/ml fatty acid poor BSA and 10 pg/ml free arachidonic acid was added to examine the ability of cells to convert the free arachidonic acid to PGE. The supernate was sampled after 5 and 10 min at 37°C and tested for PGE. In the absence of added free arachidonic acid, all of the samples produced insignificant amounts of PGE over this short time period. As seenin Fig. 5, there is a dramatic increase in the ability of IL- 1-stimulated cells to convert the free arachidonic acid to PGE. The induction of this increased ability is actinomycin D and cycloheximide sensitive. This result argues for the newly synthesized protein(s) belonging to the family of enzymes responsible for converting arachidonic acid to PGE. E$ect ofphorbol esters and calcium ionophores. Phorbol estersalone or in combination with calcium ionophores have been shown to mimic the effect of IL-1 on lymphocytes (13). We tested whether the addition of phorbol estersalone or in combi-

346

NEWTON AND COVINGTON TABLE 5 Examination of the Ability of Phorbol Ester and Ionomycin to Mimic IL- 1 Induction of Gin- 1 Fibroblast Releaseof PGE2

PGE2(pg/ml) Addition(s)”

f SEMb

None 200 units/ml IL- 1 100 tiionomycin 250 tiionomycin 500 nM ionomycin 1 rig/ml PMA 10 rig/ml PMA 100 rig/ml PMA 1 rig/ml PMA + 250 nM ionomycin 200 U/ml IL-l + 250 nM ionomycin 200 U/ml IL-1 + 1 rig/ml PMA

174k 58 I 1367 + 3445 2342 10 994 f 584 845k 216 217f 78 301a 55 25Ok 42 11127 + 2862 41343 k 8946 32600 2 3886

PGE2 + CHX ’

% Inhibitiond

440+

178

98

663+

358

40

29Ok

57

9

4060 f 2875 1533 + 528 530+ 235

65 97 99

Note. Cells were stimulated with various combinations of IL-I, phorbol my&ate acetate, and ionomycin, and supemates were sampled after 5 hr. Data are the results of two separate experiments. ’ Additions were made at the start of cell culture. b SEM, standard error of the mean. ’ PGE2 levels determined following incubation of cultures with specified additions and 10 &ml cycloheximide. d Percentage inhibition of stimulated PGE2 release.

nation with the calcium-specific ionophore ionomycin could mimic the effectsof IL1 on fibroblast PGE production. As seenin Table 5, the addition of phorbol estersin combination with ionomycin could enhance the production of PGE in the absence of IL- 1. In addition, these compounds synergized with IL- 1 in enhancing PGE production. Studies have demonstrated that activation of protein kinase c by phorbol esters and mobilization of calcium using calcium ionophores can directly affect the activity of phospholipase A2 (14). To determine if the stimulation was a mimic of IL- 1 or a direct effect on phospholipase A2, the sensitivity of this stimulation to inhibition by RNA and protein synthesis inhibition was tested. As seen in Table 5, the enhanced stimulation with this combination of compounds is relatively insensitive to cycloheximide inhibition. In addition, the synergy of either compound with IL- 1 remains sensitive to inhibition. When added together, these compounds also demonstrate a significant increase in PGE levels within I hour following addition which is different from the time course of the IL- 1 induction (data not shown). Therefore the enhanced activity demonstrated with PMA and ionomycin is not a direct mimic of the effects seenwith IL- 1 but is probably due to an effect on activation of phospholipaseA2. DISCUSSION Prostaglandins have been shown to be potent modulators of a number of physiological reactions. Among the effects are the suppression of a number of in vitro immune reactions (6,7). Prostaglandins have also been demonstrated to be involved in a number of the described biological responsesto IL- 1. IL- 1 induces fever through a

IL-I INDUCTION

OF FIBROBLAST PGE

347

PGE-dependent mechanism (15) and the IL- 1 induction of plasminogen activator production by fibroblasts is dependent on PGE (9). A recent hypothesis was proposed in which all IL- 1 effects involved the mobilization of arachidonic acid leading to the production of various metabolites yielding the biological effects seen in different cell types ( 16). We have confirmed and extended previous observations that IL- 1 activates arachidonic acid metabolism in fibroblasts resulting in an increased production of PGE (4, 5). The different cell lines examined varied widely in their baseline releaseof PGE as well as the degree of increase in PGE production in response to IL- 1 (Table 1). These parameters are probably related to the number and affinity of IL-l receptors, the available pool of arachidonic acid, and the levels of the enzymes which metabolize free arachidonic acid. There appears to be no difference in the response to different forms or sources of IL-l (Table 2). In addition, the dose-response curves for the various IL- 1 forms are similar when activities of the different IL- 1 samples are standardized using the thymocyte comitogenic assay(data not shown). Since most studies of IL-l activation of PGE production measure levels of PGE after 24-48 hr, we examined the time course of the response. Fibroblasts begin to produce detectable increasesin PGE 2 hr following IL- 1 addition followed by a rapid increase over the next few hours. This is in direct contrast to the results seenwith IL1 stimulation of chondrocytes ( 12) where PGE2 release can be detected within 1 hr of IL-1 addition. In chondrocytes, IL-l appears to act through the mobilization of arachidonic acid. The 2-hr delay seen with fibroblasts indicates another mechanism of activation. It is interesting that that 2-hr lag correlates with the recent hypothesis proposed by Mizel et al. ( 17) that internalization and possible nuclear reception are components of the activation processin lymphocytes and fibroblasts. Also notable from the time course studies is that little increase is seen between 6 and 24 hr following IL- 1 addition. This implies either that there is a feedback inhibition of further PGE2 production, that the IL- 1 response lessensin responseto receptor down regulation or that there is increased metabolism of IL-l in the culture. To lessen the effect of these possible mechanisms, most studies were conducted in the first 6 hr following IL- 1 addition. To confirm that the IL- 1 activation is a result of binding to receptors, surface receptors were stripped with trypsin so that over 90% of specific binding was lost. It was found that cells were still capable of responding to IL- 1 immediately following trypsinization (Fig. 2A). This is probably due to residual IL- 1 receptors since there is no lengthening of the 2-hr lag seenbefore PGE begins to increase. Cells begin to recover from the trypsin treatment after 2 hr, probably through the synthesis of new surface receptors for IL- 1. Importantly, the rate of the response over the first 5 hr following IL- 1 addition is the same, varying only in intensity (Fig. 2B). This may indicate that the intensity of the IL- 1 response is directly related to the receptor number expressed on the cell surface. The activation of PGE production appears to occur with only IL-l stimulation. Fibroblasts also do not need to be in a state of active proliferation to produce PGE in response to IL- 1. The addition of the fibroblast mitogenic protein platelet-derived growth factor has no effect on IL- 1 stimulation of PGE under any of these conditions (data not shown). This is not to conclude that IL- 1 is the only activator of fibroblast PGE since tumor necrosis factor (Y( 18) and substance P ( 19) have been demonstrated to also produce this effect. It will be interesting to test whether these effects occur through a mechanism similar to that seenhere for IL- 1.

348

NEWTON

AND

COVINGTON

Unlike the activation of arachidonic acid metabolism seen in neutrophils, PGE production by fibroblasts demonstrates a delayed time course (Fig. 1). This delay cannot be overcome by the addition of increasing amounts of IL- 1 (data not shown). The delay appears to be attributable to the dependence of increased PGE production on the synthesis of a new protein(s) in response to IL-l activation (Table 4). The addition of cycloheximide or actinomycin D at levels capable of inhibiting >90% of protein or RNA synthesis, respectively, can completely block IL- 1 induction of PGE. This effect has been seen in every fibroblast cell line examined to date. This effect is in contrast to the induction of neutrophil PGE releasewhich occurs independent of protein synthesis (10). Timing the addition of these inhibitors indicates that the greatest effect is seen when addition occurs in the 3 hr following IL-1 (Fig. 3). This would correlate with the onset of the large increases in PGE2 production seen after 3 hr. Together with the 2-hr lag in the time course of induction, these data indicate the synthesis of a protein(s) 2-3 hr following IL-1 addition. This result is similar to the data seen with IL-1 induction of platelet-activating factor release by endothelial cells (20). In contrast to the results seen with chondrocytes (12), we have obtained evidence that there is no demonstratable increase in phospholipase activity (Fig. 4). Rather, we have been able to demonstrate an enhanced ability of IL-l-activated fibroblasts to convert free arachidonic acid to PGE. The mechanism for this conversion involves an enzyme complex including the fatty acid cyclooxygenase system. The IL- 1 activation seen could therefore be due to an increase in the rate-limiting member of this series. Alternately, IL- 1 could be affecting the synthesis of a protein which regulates these enzymes. We are currently determining which of these mechanisms are active in this system. We have also demonstrated that the effects of IL-l cannot be replaced by the actions of phorbol esters, either alone or in combination with calcium ionophores. When added alone, PMA or ionomycin induces minimal increases in PGE release. The increase is relatively insensitive to inhibition by cycloheximide. The combination of PMA with ionomycin induces a 64-fold increase in PGE which is partially inhibitable by cycloheximide, although the degree of inhibition is far less than that seenwith IL- 1. That inhibition of the PMA + ionomycin response is seen at all may indicate that the inducible protein(s) is constantly being synthesized and degraded by these cells. Together with other data presented here, these results would indicate that IL1 synergizes with PMA or ionomycin by increasing the level of cyclooxygense enzymes which are capable of rapidly converting arachidonic acid, mobilized by PMA or ionomycin activated PLA2, into PGE. This indicates that the activation of protein kinase c, alone or in combination with calcium mobilization, is not the only signal delivered by IL-l in the activation of fibroblasts. This does not rule out these signals being part of a more complex pattern of activation of fibroblasts following IL1 addition. ACKNOWLEDGMENTS We gratefully acknowledge the expert technical assistance of Andrea Daulerio. We also thank Dr. David Goddard of Albert Einstein Medical Center, Philadelphia, Pennsylvania, for providing synovial fibroblast cultures and Dr. James Huang of DuPont for providing Escherichiu coli expressing IL- 18. Thanks also go to Lois Boulden for assistance in manuscript preparation.

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