Blocking the interleukin-1 receptor inhibits leukotriene B4 and prostaglandin E2 generation in human monocyte cultures

CELLULAR

IMMUNOLOGY

145, 199-209 (I 992)

SHORT COMMUNICATIONS Blocking the Interleukin-I Receptor Inhibits Leukotriene 84 and Prostaglandin E2 Generation in Human Monocyte Cultures PIOCONTI,MARIAR.PANARA,RENATOC.BARBACANE,FERNANDAC.PLACIDO, MAUROBONGRAZIO,MARCELLA REALE,ROYA.DEMPSEY,* AND STEFANO

FIORE

Immunology Division, Institute qf Experimental Medicine, Universiry of Chieti Medical School, Via dei Vestini, 66100 Chieti, Italy; and *Cylokine Research Laboratory, Endogen Inc., 451 D Street. Boston, Massachusetts Received May 28. 1992; accepted June 30, 1992 Interleukin-1 is a potent stimulator ofarachidonic acid (AA) metabolism and this activity could be attributed to the activation of the prostaglandin-forming enzyme cyclooxygenase or of the arachidonic-releasing enzyme phospholipase A2 or both. Prostaglandin E2 (PGE2), a cyclooxygenase product, and LTB4 (5-(S), 12-(R)-dihydroxy-6,14-cis-8, IO-trans-eicosatetraenoic acid), a lipoxygenase product, are potent mediators of inflammation. Recently a new cytokine produced by macrophages and named interleukin-1 receptor antagonist (IL-lra) (MW 22,000 Da) which specifically binds and blocks IL- 1 receptors, has proven to be a potent inflammatory inhibitor. In our studies we found that monocyte suspensions, pretreated with hrIL-lra at increasing concentrations (0.25-250 rig/ml) for 10 min and then treated with LPS in an overnight incubation inhibits, in a dose-dependent manner, the generation of LTB4 as measured by the highly sensitive radioimmunoassay method. In monocytes pretreated with hrIL-lra (250 rig/ml) for 10 min and treated with arachidonic acid ( 10m5-10-9 M) and LPS overnight, the release of LTB4 was partially inhibited when compared to hrll-lra-untreated cells. Moreover, hrIL-lra (250 rig/ml) caused a partial inhibition of monocyte LTB4 production when the cells were activated with AA (lo-’ M) and then treated with IL-l/3 (5 @ml) overnight or 24 hr incubation. In addition, human monocytes pretreated for 10 min with increasing doses of hrIL-lra (0.25-250 rig/ml) and then treated with hrIL-lcu (5 @ml) or @(5 rig/ml) for 18 hr, also resulted in the inhibition of PGE2 generation as measured by RIA when compared with hrIL-lra-untreated cells. When the cells were treated with hrIL- lra (250 rig/ml) and activated for I8 and 48 hr with increasing doses of hrIL- lb a strong inhibitory effectwas found on PGE2 production. HrIL- 1ra used at 15 rig/ml gave a partial inhibition of LTB4 generation, after LPS (I-100 rig/ml) treatment, while NDGA totally blocked the production of LTB4. Moreover, PGE2 released by macrophages activated with LPS (100 rig/ml) or hrIL-I@ (5 rig/ml) at 18 hr incubation time was strongly inhibited when hrIL-lra (250 rig/ml) was used. These data suggest that the inhibition of LTB4 and PGE2 by this new macrophagederived monokine IL- 1ra occurs through the block of the IL- 1 receptor, rather than phospholipase A2, and thus IL- 1ra may offer a potential therapeutic approach to inflammatory states. o 1992 Academic

Press, Inc.

INTRODUCTION Human or murine monocyte/macrophages (M$)’ may be activated to release cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA) after stimulation ’ Abbreviations used: hrIL-lra, human recombinant interleukin- 1 receptor antagonist; IL- I@,interleukinI& AA, arachidonic acid; LPS, lipopolysaccharide; PGEZ, prostaglandin E2; LTB4, leukotriene B4; NDGA, nordihydroguaiaretic acid; MB, monocyte/macrophage; PLAZ, phospholipase A2. 199 0008-8749192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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with different substances such as LPS and certain cytokines such as interleukin- lar (IL- 1o) or IL- l/3 (l-8). IL- 1 is a monokine which, in inflammatory sites, stimulates various cell types (e.g., macrophages, fibroblasts, and endothelial cells) to release arachidonic acid products (9- 15) which contribute to augmenting inflammation. It is well known that metabolites of both cyclooxygenase and lipoxygenase mediate the inflammatory reaction. Prostaglandin E2 (PGE2), an arachidonic acid cyclooxygenase product, is a mediator of fever, augments vascular permeability and formation of edema, while the 5-lipoxygenase product 5-(S), 12-(R)-dihydroxy-6,14-c&8, IO-transeicosatetraenoic acid (LTB4) provokes chemokinesis, chemotaxis, vasoconstriction, and aggregation ( 16). Arachidonic acid is released from cell membranes by the action of phospholipase A2, is converted by the enzyme 5-lipoxygenase to SS-hydroxy-6,8truns- 11,14-cis-eicosatetraenoic acid (5-HPETE). This enzyme catalizes the conversion of 5-HETE to leukotriene A4 (LTA4) (5,6-trans-oxido-7,9-trans- 11,14-cis-eicosatetraenoic acid). Then LTA4 may be converted to two separate products: LTB4 or, conjugated with reduced glutathione, LTC4 (SS-hydroxy-6R-Sglytathionyl-7,9-trans11,14-cis-eicosatetraenoic acid). A number of studies report that IL-l is a strong inducer of arachidonic acid compounds (9) through the activation of phospholipase A2. However, little is known about the regulation of macrophage function and AA activation by IL- 1 ( 17, 18). Two biochemically different IL-l’s, rIL-lcr and rIL-10, have been cloned in both mice and humans and both stimulate target cells to release arachidonic acid products ( 19). Recently, a new cytokine called interleukin- 1 receptor antagonist (IL- 1ra) has been cloned by two independent groups (20-22). This protein has a MW of 22,000 Da, is secreted by human macrophages, is structurally related to IL-10 (26% aminoacid homology) and IL-la! (19% homology), but has no similar biological effects. IL-1 ra produced in the mouse by P388Dl monocytic cells activated with PMA (mrIL-lra) (23) is highly homologous to the human counterpart molecule. It has been recently reported that hrIL-lra is capable of inhibiting IL-la and IL16 release in macrophages, PGE2 production, and lymphocyte activation (24, 25). In contrast, hrIL- lra potentiates the stimulatory effect of IL-2 on NK cell activity, as well as hrIL- 1p activity (26), demonstrating unique cytokine properties. HrIL- 1ra also is involved in inflammatory processes since it down-regulates the acute phase response. IL- lra has proven to be a potent inhibitor of IL- l-induced synovitis and articular cartilage proteoglycan in the rabbit knee (27), inhibits endotoxin- and IL-l-induced acute inflammation, and prevents Escherichia cc&-induced shock (28). All the abovementioned effects are mediated by inflammatory mediators including arachidonic acid metabolites. Since the inflammatory activity of IL- 1 is due to its ability to increase gene expression for cyclooxygenase and lipoxygenase, augmenting new mRNA coding for phospholipase A2 (35), it is of interest to study the effects of hrIL-lra on the 5-lipoxygenase product LTB4 and the cyclooxygenase product PGE2 released by human monocyte cultures, after activation with stimuli such as IL-l and LPS. Because of the potential importance of 5-lipoxygenase and cyclooxygenase products in inflammation, in this report we examined the ability of hrIL-lra to inhibit these two products in activated human monocyte cultures. MATERIALS AND METHODS Human monocyte purijcation. Peripheral blood was obtained from healthy donors and collected in polypropylene tubes containing sufficient heparin to obtain a final

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201

concentration of 20 U/ml. Tests were performed immediately after collection. The whole blood was stratified on Ficoll-Hypaque (Eurobio Labtech, Milan, Italy) solution and centrifuged at 400g for 30 min as described (29). Mononuclear cells were recovered at the interface and erythrocytes were separated by dextran (Sigma Chemical Co., St. Louis, MO) sedimentation, and the peripheral blood mononuclear cells (PBMC) were rendered plasma free and platelet poor by washing three times with Hepes-buffered ( 10 mM) Hanks’ balanced salt solution (HBSS) and were resuspended at the desired concentration in RPM1 1640 medium (GIBCO), supplemented with 5% heat-inactivated fetal bovine serum, L-glutamine (0.3 mg/ml), and Hepes; this will be referred to as complete medium (CRPMI). Aliquots of 2.5 ml were seeded into petri dishes and were incubated at 37°C 5% C02-humidified atmosphere for 60 min. After that time, the supernatants which contained nonadherent cells were discarded and the remaining adherent monocytes (about 10% of the original population) were recovered by vigorous washings and scraping of the plates and the viability examined by trypan blue exclusion method was 92%. The isolated cells were washed and resuspended in PBS buffer and the cell suspension was adjusted to the desired concentration (5 X 1O6 cells/ml). The cells were counted in a Burker counting chamber and the cell suspension consisted of 90% monocytic leukocytes. Neutrophils and platelets were less than 1%. The culture medium contained less than 10 pg/ml of LPS, as determined by the limulus amoebocyte lysate test. The cells were then collected in a polypropylene tube (12 X 75 mm, Falcon) and were cultured in CRPMI at different periods of time. Samples were treated with LPS or IL-l and hrIL-lra at different concentrations. The controls received a vehicle at the same concentrations employed with the test agents LPS or IL- 1, after which radioimmunoassay was determined. Monocyte culture treatments. Cells were placed in test tubes (Falcon) for each experiment and were exposed only to the vehicle (heat-inactivated hrIL-lra) at the identical concentrations of LPS-treated cells, in order to determine nonspecific effects. Results are expressed in nanograms or picograms per milliliter. For each of the three donors, each test and all controls were done in triplicate. Incubations were followed by the determination of LTB4 levels. All monocyte tests were preincubated with hrILlra for 10 min at 37°C in a shaking water bath before adding LPS or IL- 1 at different additional periods of time. After the addition of LPS or IL- 1, overnight incubation at 37°C 5% CO*, viability was examined in all samples and no statistical difference was found between treated and untreated cells. Moreover, the controls with heat-inactivated hrIL- 1ra were treated at the same concentrations of the active compound, and proved to be ineffective. Human recombinant IL- lp and hrIL- 1a was kindly provided Endogen Inc. (Boston, MA); LPS was purchased from Sigma. The cytokine human recombinant IL- 1ra used in these studies was kindly provided by Dr. Robert C. Thompson, (Synergen, Boulder, CO). Radioimmunoassay of LTBI. After the purification, as previously described, monocytes were suspended in RPM1 1640 (without any serum) and dispensed into polypropylene tubes (Falcon). The cells were incubated (5 X 106/ml) with hrIL- 1ra for 10 min, either with or without LPS or IL-1 at different concentrations. Following centrifugation of the incubation mixture, the cell-free supernatants were stored at -70°C before radioimmunoassay (RIA) (30,3 1). RIA of LTB4 was determined in nanograms or picograms per milliliter. Each test and all controls were done in triplicate and RIA for LTB4 was determined in 100 /*l of sample, plus 100 ~1 [ 14, 15-3H] LTB4 (New England Nuclear, Boston, MA) plus 100 ~1 rabbit anti-LTB4 (Advanced Magnetics,

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Boston, MA), plus 200 ~1 of charcoal (0.3 g charcoal + 0.03 g dextran (Sigma) in 100 ml Tris buffer: Trizma base 4.03 g/l + Trizma HC12.64 g/l + NaC19 g/l + MgS04. 7 HZ0 0.49 g/liter + CaCl* 0.022 g/liter + gelatin 1 g/liter; pH 8.6, 0.05 M. Synthetic LTB4 was a generous gift from Dr. J. Rokach, (Merck Frosst, Pointe-Claire, Quebec, Canada). Nordihydroguaiaretic acid NDGA (Sigma) was prepared in DMSO to a final concentration of 10 &f. The small amount of DMSO employed as a vehicle did not effect either cell viability or LTB4 release (45). Quantitation and characterization ofprostaglandin E2 by RIA method. After monocyte purification at 5 X lo6 cells and incubation at different periods of time ( 18 or 48 hr), the cells were centrifuged at 3000 t-pm and the cell-free supernatants were stored at -70°C before radioimmunoassay. Immunoreactive PGE2 was measured by RIA carried out in triplicate and determined in pg/ml(32, 33). Release of PGE2 was performed with a kit from New England Nuclear (kit: NEK 020). Antisera to PGE2 crossreacts with thromboxane B2 (TxB2) 0.022% and with 6-keto-PGlcr 0.01% (46). Arachidonic acid (AA) pretreatment. Human monocytes (5 X lo6 cells/ml) were preincubated for prestimulation with arachidonic acid (Sigma Co.), which was diluted in PBS and used at different concentrations ( 10-5- 1Oe9M). Control samples received PBS as a comparison to LPS, IL- 1, or AA. In these experiments, cells were preincubated with AA for 10 min, with or without hrIL- 1ra ( 10 min, preincubation), and then were treated with LPS or IL- 1. In experiments where NDGA was used DMSO (resulting a final concentration of 0.1% vol/vol) was added to the controls at the same dilutions as NDGA. Statistical analyses. Data from different experiments were combined and reported as the means ? SD. The Student’s t test for independent means was used to provide a statistical analysis (P > 0.05 was considered as not significant). RESULTS Efect of hrll-lra were pretreated for for LTB4 generation ments, the detected

on LTB4 release after treatment with LPS. Monocytic leukocytes 10 min with decreasing concentrations of hrIL- 1ra and assayed after treatment with LPS 100 rig/ml (Table 1). In these experiLTB4 was enhanced by LPS but not by hrIL- 1ra alone (used at TABLE 1

Production of LTB4 by Monocytes after Pretreatment with hrIL- I ra Treatment M$s 5 X lo6 cells Control (Nil) hrIL- 1ra (250 &ml) LPS (100 rig/ml) hrIL-lra (250 rig/ml) + LPS (100 @ml) hrIL-lra (25 rig/ml) + LPS (100 rig/ml) hrIL-lra (2.5 @ml) + LPS (100 @ml) hrIL-lra (0.25 r&ml) + LPS (100 rig/ml)

LTB4 rig/ml 0.21 + 0.29 f 19.9 * 1.1 k 3.7 + 12.3 + 15.0 f

0.09 0.07 2.0 0.8 2.0 2.9 3.4

P<

(*I 0.001 0.00 I 0.05 N.S.

Note. LTB4 was detected in human monocyte suspensions (5 X lo6 cells/ml) following the addition of increasing concentrations of hrIL-lra for 10 min and then LPS in an overnight incubation. The values represent the means + SD of triplicate determinations of three representative experiments. P values (Student’s t test) are calculated by comparing hrIL- 1ra + LPS-treated cells with LPS-treated cells alone (*).

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the highest concentration 250 rig/ml), to the controls a vehicle was added at the same volume as the compounds. However, when the cells were pretreated for 10 min with hrIL-lra and then treated for 18 hr with LPS the release of LTB4 was less. This inhibition was dose dependent in accordance with increasing concentrations of hrILlra used. The cells were cultures overnight at 37°C 5% CO2 in CRPMI. Pretreatment of monocyte cultures with arachidonic acid + hrll-lra and then treated with LPS. The results in Table 2 show the amounts of LTB4 detected in human monocyte suspensions following pretreatment, or not, with hrIL-lra at 2.50 rig/ml concentration for 10 min followed by pretreatment with AA at increasing concentrations ( 10p9-10-5 M) for 10 min and then treated with LPS 100 rig/ml overnight. In these experiments, LPS strongly stimulated LTB4 production while hrIL- 1ra failed to do so. When the cells were pretreated with AA at increasing concentrations and LPS, a greater generation of LTB4 was found when compared to cells treated with LPS alone. These effects were strongly inhibited, but not totally abolished, when the cells were pretreated for 10 min, with hrIL- 1ra 250 rig/ml. Monocytes pretreated with AA ( 1O-5 M) alone show a little, if any, LTB4 release when compared to the control (Nil). Inhibition of LTB4 production by hrll-lra in human monocyte cultures stimulated with LPS. To determine the optimal concentration of LPS in stimulating LTB4 generation by the cells, in Fig. 1 we show the amount of LTB4 production by human monocytic leukocyte suspensions following the addition of increasing concentrations of LPS (0.01-100 rig/ml) in an overnight incubation. In these experiments the maximum release of LTB4 was found at LPS 100 rig/ml. However, we did not test higher concentrations on the basis of exceeding “physiologic” limits. Table 3 shows the release of LTB4 production by monocytes at 18 and 24 hr incubation after pretreatment with hrIL- 1ra (250-0.25 rig/ml), AA ( 1O-’ M) for 10 min, and then IL- I p (5 rig/ml) for the remaining incubation time. HrIL- 1ra, used at varying concentrations strongly inhibited LTB4 in all the samples stimulated with AA (lo-’

TABLE 2 Generation of LTB4 after Pretreatment of Monocytes with Arachidonic Acid and hrIL- 1ra and Treated with LPS LTB4 rig/ml Treatment M& 5 X lo6 cells Control (Nil) hrIL- I ra (250 rig/ml) AA (lo-5 M) LPS (100 rig/ml) AA (1O-9 M) + LPS (100 rig/ml) AA (lo-* M) + LPS ( 100 rig/ml) AA (lo-’ M) + LPS (100 rig/ml) AA(10~6M)fLPS(100ng/ml) AA (IO-‘M) + LPS (100 rig/ml)

- hrIL-lra (250 rig/ml) 0.1 0.2 0.4 21.4 23.1 54.3 58.9 60.2 61.4

i f ? k f f 2 f *

0.07 0.09 0.1 2.0 3.5 6.2 5.1 7.0 5.9

+ hrIL- 1ra (250 rig/ml) 8.5 f 2.1 15.7 f 3.2 19.4 r?r5.0 22.8 f 4.9 29.7 IL 6.1

Pi

0.01 0.01 0.01 0.01 0.01

Note. LTB4 was detected in human monocytes suspensions (5 X lo6 cells/ml) after preincubation or not with hrIL- 1ra (250 rig/ml) for 10 min and exogenous AA (lo-‘- I Om5M) for 10 min, before the addition of LPS 100 rig/ml overnight. The data represent the means f SD of triplicate determinations of three representative experiments. P values (Student’s t test) are calculated by comparing hrIL-lra treated cells with untreated cells.

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0.001

1

FIG. 1. Dose-dependent values of LTB4 after monocyte pretreatment with decreasing doses of LPS (loo0.01 &ml) for overnight incubation. The values represent the means + SD of triplicate determinations of three representative experiments. P values (Student’s t test) are calculated by comparing LPS-treated cells with control (untreated cells) (*). Column: A, control; B, LPS (0.01 q/ml); C, LPS (0. I rig/ml); D, LPS (I rig/ml); E, LPS (10 rig/ml); F, LPS (100 rig/ml).

M) + IL-10 (5 rig/ml). HrIL- lra (250 rig/ml) alone failed to stimulate LTB4. In these experiments, AA had a more pronounced effect on stimulating LTB4 when the cells were exposed for 18 instead of 24 hr incubations. Figure 2 shows the dose-dependent inhibition produced by hrIL- 1ra ( 15 rig/ml) in human monocytes pretreated for 10 min and then treated with LPS ( 1.O-100 rig/ml) overnight. HrIL-lra at this intermediate concentration had a significant effect only when LPS was used at lower concentrations ( lo- 1.O rig/ml). The stimulatory effect of LPS was strongly inhibited by NDGA 10 PM (column H in Fig. 2). In addition, NDGA alone had no effect on LTB4 production (column B). In column E, hrIL-lra (15 rig/ml) plus LPS (100 rig/ml) had no significant inhibitory effect respect with LPS (100 rig/ml) alone. Figure 3 shows the amounts of PGE2 (pg/ml) released by human monocytic leukocytes after addition of LPS (100 rig/ml) or hrIL-lfl (5 rig/ml) and in combination TABLE 3 Inhibition of LTB4 by hrIL-lra

after Monocyte Treatment with Arachidonic Acid and IL-10 LTB4 pg/ml

Treatment M$JS 5 X lo6 cells

18 hr

24 hr

Control (Nil) AA (lo-’ M) hrIL-lra (250 rig/ml) IL-10 (5 q/ml) AA (IO-’ M) + IL-10 (5 rig/ml) hrIL-lra (250 rig/ml) + AA (lo-’ M) + IL-lfi hrIL-Ira (25 rig/ml) + AA (lo-‘M) + IL-l@ hrIL-lra (2.5 rig/ml) + AA (lo-’ M) + IL-10 hrIL-lra (0.25 rig/ml) + AA (lo-’ M) + IL-10

3Ok 14 81 k34 55 f 29 3450 f 320 44223 f 5210 920 rt_280 15300 + 3150 28000 f 2920 33100 -t- 4010

41 ir22 76 k 38 5Ok 18 3330 f 41 I 36564 f 6211 1600 f 840 20040 f 1900 29400 + 3010 30180 f 2800

Note. Inhibitory effect of hrIL-lra (250 rig/ml) on monocyte production of LTB4 after stimulation with AA ( 10m7M) plus IL-lp (5 rig/ml) for 18 and 24 hr. The values represent the means + SD of triplicate determinations of three representative experiments.

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8 1

(‘)

T N.S.

0 ABCDEFGH Treatment FIG. 2. Inhibitory effect of LTB4 generation by monocyte cultures after pretreatment with hrll-lra for 10 min at 15 rig/ml. The cells were activated with LPS at decreasing concentrations (100-I &ml) overnight incubation. The inhibition of LTB4 release was also found when the cells were pretreated with 10 pMNDGA overnight. The values represent the means k SD of triplicate determinations of three representative experiments. P values (Student’s I test) are calculated by comparing hrlL- lra + LPS-treated cells with LPS (100 rig/ml) alone (*). Column: A, control (Nil); B, 10 pcM NDGA; C, hrIL-lra (15 rig/ml); D hrIL-Ira (15 ng/ ml) + NDGA ( 10 pg/ml); E I, LPS (100 rig/ml); E2, LPS ( 100 rig/ml) + hrIL- 1ra ( 15 rig/ml); F 1, LPS (IO rig/ml); F2, hrIL- Ira (15 rig/ml) + LPS (10 rig/ml); G I, LPS (I rig/ml); G2, hrIL-I ra (15 rig/ml) + LPS (1 rig/ml); H, NDGA (10 gA4) + LPS (100 rig/ml).

with hrIL- 1ra (250 rig/ml) for various lengths of incubation times (30 min to 48 hr). The time course study reveals that the maximum PGE2 stimulation by hrIL-l/3 or LPS occurred after 18 hr incubation time and the pretreatment of cells for 10 min with hrIL- 1ra (250 rig/ml) strongly inhibited the release of PGE2 generation. Moreover hrIL-lra (250 rig/ml) inhibited PGE2 generation in all the tests. Based on the results obtained in Fig. 3 we incubated the cells overnight at 37°C ( 18 hr) and the values obtained are presented in Table 4, where we show the production of PGE2 in monocyte cultures treated with hrIL- ILY (5 rig/ml) or hrIL- 10 (5 rig/ml) and inhibited by the pretreatment of increasing concentrations of hrIL- 1ra (0.25-250 rig/ml). The values, expressed in picograms per milliliter, were determined by the highly sensitive RIA method. HrIL-lra at the highest concentration (250 rig/ml) alone

5000

1

Q + 4

0

O.Sh

Ih

2h

4h

la h

hrlL-1R hrlL-lra+lL-II? LPS hrlL-1 ra+LPS

48 h

Time (hours) FIG. 3. Inhibitory effect of hrll-lra (250 rig/ml) on PGE2 generation by human macrophages (5 X IO6 cells/ml) activated with hrIL-10 5 rig/ml or LPS (100 pg/ml) for different lengths of time (0.5-48 hr). The values represent the means f SD, which do not exceed 10% of triplicate determinations of three representative experiments.

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Control hrIL-lra hrIL-10 hrIL-lcu hrIL-lra hrIL-lra hrIL-lra hrIL-lra hrIL-lra hrIL-lra hrIL-lra hrIL-lra

PGE2 pg/ml overnight incubation 18 hr, 37°C

(Nil) (25 rig/ml) (5 rig/ml) (5 rig/ml) (250 rig/ml) + hrIL-l@ (5 rig/ml) (25 rig/ml) + hrIL-l/3 (5 rig/ml) (2.5 rig/ml) + hrIL-l@ (5 rig/ml) (0.25 t&ml) + hrIL-10 (5 t&ml) (250 rig/ml) + hrIL-la (5 ng/nl) (25 rig/ml) + hrIL-la (5 rig/ml) (2.5 rig/ml) + hrIL-la (5 rig/ml) (0.25 rig/ml) + hrIL-lo (5 rig/ml)

20 * 53 i 740 * 680 * 14 f 35 f 360 + 590 + 30 f 61 + 387 + 490 k

P<

0.00 I 0.00 I 0.01 N.S. 0.001 0.00 I 0.05 N.S.

9.5 21 102 (*) 110 (**) 8.2 11 54 100 9.7 16 75 104

Note. Dose-response inhibition of PGE2 released by monocytes after pretreatment for 1 hr with increasing doses of hrIL-lra (0.25-250 rig/ml) and then incubated overnight (18 hr) with hrIL-lb (5 rig/ml) or hrIL1(Y(5 rig/ml). The values represent the means + SD of triplicate determinations. P values (Student’s t test) are calculated by comparing hrIL-lra pretreated cells with untreated cells (*) (**).

failed to stimulate PGE2 generation; consequently low concentrations were not determined. The results presented in Table 5 show a dose-dependent stimulation of hrIL- lb (0. l- 10 rig/ml) at 18 and 48 hr incubations before the monocyte cultures were pretreated for 10 min with hrIL-lra (250 rig/ml). A strong inhibitory effect of the production of PGE2 was found. The maximum PGE2 release was determined at the highest concentration of hrIL- 1,0 ( 10 rig/ml) used. There was no statistical difference in the values between 18 and 48 hr incubations.

TABLE 5 Inhibitory Effect of PGE2 Released by Monocytes Stimulated with hrIL-l/3 PGE2 pg/ml Treatment 5 X lo6 cells

18 hr

48 hr

Control (Nil) hrIL-lra (250 rig/ml) hrIL- 10 (0.1 rig/ml) hrIL- 10 (1 rig/ml) hrIL- 1p ( IO rig/ml) hrIL- 1ra (250 rig/ml) + hrIL- 1p (0.1 rig/ml) hrIL-lra (250 rig/ml) + hrIL-lb (1 rig/ml) hrIL-lra (250 rig/ml) + hrIL-10 (10 &ml)

N.D. 36+ 12 430 i- 60 940 k 98 1250 f 150 12 + 8 88 f 25 220 + 74

21+7 52 -+ 20 251 f 82 870 f 79 1910 + 250 26 f 11 93 f 34 181 k 92

Note. ,Monocyte culture pretreated for 60 min with hrIL-lra (250 tug/ml) and then treated with increasing doses of hrIL-l/3 (0. I-10 @ml) for 18 and 48 hr show a strong inhibition of PGE2 generation. The values represent the means + SD of triplicate determinations.

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DISCUSSION Interleukin- 1 has a profound effect on many physiological and pathological events such as immunity and inflammation ( 12,42). Specific inhibition, via negative feedback or down-regulation of IL- 1(Yor IL- lfi may prove to have significant beneficial effects in some clinical inflammatory states. In these studies we found, for the first time, that hrIL- 1ra, a new macrophage-derived cytokine, has a potent inhibitory effect on LTB4 generation in human monocytes when they are stimulated with LPS, IL- 1LY,or IL- 1p. We also confirmed previous findings that IL-1 is a potent stimulator of PGE2 production in human monocytes (43). In addition, the inhibitory effect of PGE2 production by hrIL- 1ra in our results is in accordance with the results reported by Arend et al. (43). It has been described by many authors that IL- 1 has the capacity to activate and stimulate many different cells to produce AA oxygenation and lipoxygenation products. Moreover, both cyclooxygenase and lipoxygenase have been reported to be regulated by IL- 1 ( 1, 4, 6) and other cytokines (36). The release of PGE2 and LTB4, in general, is associated with many pathophysiological phenomena, such as hypotension, thrombocytopenia, capillary leak syndrome, leukopenia, fever, and inflammation (39). In these studies the inhibition of LTB4 produced by hrIL-lra suggests that this macrophage-derived monokine may be useful and could offer a therapeutic approach to some inflammatory diseases, such as rheumatoid arthritis and other autoimmune diseases. At the present time the mechanism of inhibition of LTB4 production by IL1ra is difficult to ascertain. LPS stimulates Mos to release IL- 1, which stimulates PGE2 and LTB4 by influencing an increase in phospholipase A2 and its mRNA, which increases approximately fivefold, as reported by Bomalasky et al. (35). The inhibitory event produced by IL- 1ra on PGE2 and LTB4 production therefore involves a receptormediated response rather than directly influencing PLA2 or its mRNA. Inhibition occurs via the block of IL- 1 receptors and not in the translocation of the 5-lipoxygenase or by inhibition of the 5-lipoxygenase enzyme expression. Other inhibitors of LTB4 that do not act on IL- 1 receptor blockade may produce increases in 15-HETE formation and decreases in 5-lipoxygenase activity or directly inhibit PLA2 formation (44). There is evidence suggesting that substances derived from the action of the enzyme 5-lipoxygenase may have a role in mediating the physiologic events of inflammation. Arachidonic acid metabolites are not stored in the cell and the cellular levels of these compounds are low. The conversion of arachidonic acid into various biologically active products is accomplished by means of two major pathways: the cyclooxygenase and the lipoxygenase pathways. LTB4 is reported to be a major mediator of leukocyte inflammation since it is capable of producing cell aggregation, lysosomal enzyme release, and chemotaxis (34). When human MB are recovered and treated with LPS in vitro, substantial amounts of prostaglandins and leukotrienes are formed as shown in this report. Activation of phospholipase A2, which liberates arachidonic acid from membrane phospholipids, is an important means of signal transduction in many systems. Functionally, IL- 1 seems to be involved with membrane perturbation and release of arachidonic acid products from different types of cells (37, 38). Since exogenously added phospholipase A2 or arachidonic acid replaces the requirement for IL-l in stimulating IL-2 production, it is possible that a lipoxygenase metabolite of arachidonic acid acts as the second messanger for IL-1 to stimulate IL-2 production. Previous reports have documented that IL-l possesses the ability to induce hemodynamic and hematologic changes typical of septic shock and these effects seem to require cyclooxygenase, since the changes are abolished by pretreatment with a cy-

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clooxygenase inhibitor (39). Arachidonic acid metabolism may participate in the biological activity of IL-l since prostaglandins and leukotrienes have been reported to exert a wide variety of regulatory effects on immunofunction and inflammation. Moreover, the most accredited mechanism of action of IL-l in inflammation is its capacity to activate MB by specifically binding to the IL-l receptor and releasing arachidonic acid metabolites (40). In our study, the addition of LPS or IL-la or p to Me, cultures caused an increase in the levels of LTB4 and PGE2. In both cases, the pretreatment of human monocytes with increasing doses of hrIL-lra inhibited the generation of LTB4 and PGE2 in a dose-dependent manner. Cells preincubated with IL-lra and arachidonic acid and then treated with LPS produced a significant partial inhibition of LTB4 compared to cells not pretreated with hrIL- 1ra. Henderson et al. reported that intravenous injection of hrIL- 1ra into rabbits after injection of hrIL- 1 not only inhibits the entry of leukotrienes into the synovial lining but also blocks the ability of IL-l to cause loss of proteoglycan from articular cartilage (27). Belavoine et al. reported that prostaglandin E2 and collagenase production by fibroblast and synovial cells is regulated by interleukin-1 and its inhibitor, which has been recently termed IL-l receptor antagonist (40). Wakabayashi et al. found that hrIL-lra injected in the rabbit 15 min before Escherichia coli infusion prevents leukopenia, hypotention, and death, all symptoms occurring in septic shock, a condition mediated by arachidonic acid metabolites (4 1). Our’irbsetiations demonstrate further the anti-inflammatory potential of hrIL- 1ra and extend the range of activities of this new monokine to LTB4 generation in monocytic leukocytes (42). In our model, the data suggest that the blockade of LTB4 and PGE2 by hrIL- 1ra may be of potential therapeutic significance in inflammatory and immunological diseases. Long-term clinical studies using IL- 1ra should be conducted to evaluate hrIL-1 ra’s effect on inflammatory states. However, more studies on the release of the lipoxygenase and cyclooxygenase precursor metabolites after treatment of IL- 1 are undergoing in our laboratory to clarify the effects of hrIL- 1ra on the spec: ificity of inhibition. ACKNOWLEDGMENT This work was supported by the Italian Ministry of University, Scientific Research and Technology (4060%) 1990. We gratefully acknowledge the AVIS blood donor association of Pescara, Italy, for providing whole blood.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Williams, J. D., Robin, J.-L., Lewis, R. A., Lee, T. H., and Austen, K. F., J. Immunol. 136, 642, 1986. Durum, S. K., Schmidt, J. A., and Oppenheim, J. J., Annu. Rev. Immunol. 3, 263, 1985. Rankin, J. A., Sylvester, I., Smith, S., Yoshimura, T., and Leonard, E. J., J. Clin. Invest. 86, 1556, 1990. Doerfler, M. E., Danner, R. L., Shelhamer, J. H., and Parrillo, J. E., J. Clin. Invest. 83, 970, 1989. Conti, P., Reale, M., Barbacane, R. C., Bongrazio, M., Panara, M. R., Fiore, S., Dempsey, R., and Borish, L., Cytokine 2, 142, 1990. Williams, J. D., Czop, J. K., and Austen, K. F., J. Immunol. 132, 3034, 1984. Wingfield, P., Payton, M., Tavernier, J., Barnes, M., Shaw, A., Rose, K., Simona, M. G., Demczuk, S., and Dayer, J. M., Eur. J. Biochem. 136,2492, 1986. Pawlowski, N. A., Kaplan, G., Hamill, A. L., Cohn, C. A., and Scott, W. A., J. Exp. Med. 158, 393, 1983. Marusic, A., Kalinowski, L. F., Harrison, J. R., Centrella, M., Raisz, L. G., and Lorenzo, J. A., J. Immunol. l&&2633, 1991. Dinarello, C. A., Bull. Inst. Pasteur 85, 267, 1987.

SHORT COMMUNICATIONS

209

11. Conti, P., Reale, M., Fiore, S.,Cancelli, A., Angeletti, P. U., and Dinarello, C. A., Scund. J. Rheumatol. 75, 318, 1988. 12. Dubois, C. M., Ruscetti, F. W., Keller, J. R., Oppenheim, J. J., Hestdal, K., Chizzonite, R., and Neta, R., Blood 78,2841, 1991. 13. Wakabayashi, G., Gelfand, J. A., Jung, W. K., Connolly, R. J., Burke, J. F., and Dinarello, C. A., J. Clin. Invest. 87, 1925, 1991. 14. Conti, P., Cifone, M. G., Reale, M., Fieschi, C., and Dinarello, C. A., Prostaglundins 32, 112, 1986. 15. Censini, D., Bartalini, M., Tagliabue, A., and Boraschi, D., Lymphokine Res. 8, 107, 1989. 16. Dinarello, C. A., Cannon, J. G., Mier, J. W., Bernheim, H. A., et al., J. Clin. Invest. 77, 1734, 1988. 17. Lohmann-Matthes, M. L., Curr. Sci. 2, 33, 1989. 18. Johnston, R. B., N. Engl. J. Med. 318, 747, 1988. 19. Dinarello, C. A., Adv. Immunol. 44, 153, 1989. 20. Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin, F. G., et al., Nature 343, 336, 1990. 2 I. Eisenberg, S. P., Evans, R. J., Arend, W. P., Verde&r, E., et al., Nature 343, 341, 1990. 22. Carter, D. B., Deibel, M. R., Dunn, C. J., Tomich, C. S. C., Laborde, A. L., et al., Nature 344, 633, 1990. 23. Zahedi, K., Seldin, M. F., Ritz, M., Ezekowitz, R. A. B., and Whitehead, A. S., J. Immunol. 143; 4228, 1991. 24. Conti, P., Reale, M., Barbacane, R. C., Panara, M. R., Bongrazio, M., Dempsey, R. A., and Dinarello, C. A., Immunol. Lett. 28, 19, 1991. 25. Conti, P., Reale, M., Panara, M. R., Barbacane, R. C., Bongrazio, M., and Dempsey, R. A., FEBS Lett. 286,137,1991.

Conti, P., Dempsey, R. A., Reale, M., Barbacane, R. C., Panara, M. R., Bongrazio, M., and Mier, J. W., Immunology 73,450, 1991. 27. Henderson, B., Thompson, R. C., Hardingham, T., and Lewthwaite, J., Cytokine 3, 246, 1991. 28. Ulich, T. R., Yin, S., Guo, K., del Castillo, J., Eisenberg, S. P., and Thompson, R. C., Am. J. Pathol. 26.

138,521,

1991.

29. 30. 31. 32. 33. 34.

Boyum, A., Stand. J. Clin. Lab. Invest. 21, 77, 1968. Salmon, J. A., Simmons, P. M., and Palmer, R. M. J., Prostaglandins 12, 929, 1982. Lewis, R. A., Mencia-Huerta, J. M., and Soberman, R. J., Proc. Nutl. Acad. Sci. USA 79, 7904, 1982. Zijlastra, F. J., and Vincent, J. E., J. Chromat. 311, 39, 1984. Elliott, G. R., and Van de Meent, L., Int. J. Immunoputhol. Phurmucol. 4, 67, I99 I, Ford-Hutchinson, A. W., Bray, M. A., Doig, M. V., Shipley, M. E., and Smith, M. J. H., Nature 286,

35.

Bomalaski, J. S., Steiner, M. R., Simon, P. C., and Clark, M. A., J. Immunol. 148, 155, 1992. Fiore, S., and Serhan, C. N., J. Exp. Med. 172, 145 I, 1990. Ehas, J. A., Gustilio, K., Baeder, W., and Freundlich, F., J. Immunol. 138, 3812, 1987. Dinarello, C. A., and Bernheim, H. A., J. Neurochem. 37, 702, 1981. Okusawa, S., Gelfand, J. A., Ikejima, T., Connolly, R. J., and Dinarello, C. A., J. Clin. Invest. 81, 1162,

264, 1980.

36. 37. 38. 39.

1988.

Balavoine, J. F., De Rochemonteix, B., Williamson, K., Seckinger, P., Cruchaud, A., and Dayer, J. M., J. Chin. Invest. 78, 1120, 1986. 41. Wakabayashi, G., Gelfand, J. A., Burke, J. F., Thompson, R. C., and Dinarello, C. A., FASEB J. 5, 40.

338,1991. 42.

43. 44. 45. 46.

Conti, P., and Dempsey, R. A., Int. J. Immunoputhol. Phurmucol. 3, 1, 1990. Arend, W. P., Welgus, H. G., Thompson, R. C., and Eisenberg, S. P., J. Clin. Invest. 85, 1694, 1990. Vanderhock, J. Y., Karmin, H. T., and Ekborg, S. L., J. Biol. Chem. 260, 14482, 1985. Salmon, J. A., Simmons, P. M., and Palmer, R. M. J., Prostaglandins 24, 225, 1982. Granstrom, E., Kindahl, H., and Samuelsson, B., Prostaglundins 12, 929, 1976.