Prostaglandins Leukotrienes and Essential Fatty Acids (1991) 44. 241-245 0 Longman Group UK Ltd 1991

Synergistic Stimulation of Amnion Cell Prostaglandin E2 Synthesis by Interleukin-1 , Tumor Necrosis Factor and Products from Activated Human Granulocytes K. Bry and M. Hallman Department of Pediatrics, University of California, Irvine, CCM Med. Surge I, Rm. 109F Irvine, California 92727, USA (Reprint requests to KB) ABSTRACT. We examined the interactions between supernatant from FMLP-activated human granulocytes, recombinant interleukm-1 (IL-l) and recombinant tumor necrosis factor (TNF) in the stimulation of prostaglandin Ez (PGEZ, production by human amnion cells. Amnion cells from elective term cesarian sections were cultured in monolayer culture. Human granulocytes were activated with FMLP and centrifuged to obtain cell-free supernatant. Amnion cells were treated with granulocyte supernatant, IL-la, IL-lfi, TNF-cq TNF-fi, or different combinations of these. Each of the stimulators alone enhanced the PGEz production 5to 27-fold. Granulocyte supernatant was synergistic with each of the cytokines. Tbe combinations of IL-la or IL-lp with either TNF-ar or TNF-p caused a synergistic stimulation of amnion cell PGEl production as well, whereas the combinations of IL-la with IL-Q or of TNF-ar with TNF-P were not synergistic. Furtbermore, granulocyte supematant was synergistic with the combination of IL-1 and TNF, resulting in a more than 1%fold stimulation of PGEz production. Indometbacin completely suppressed these effects. We propose that granulocyte products acting together with IL-l and TNF enhance PGEz synthesis during inflammation, and serve as signals for the initiation of preterm labor in the setting of i&a-amniotic infection.


lo), and IL-l (11) have each been proposed to be mediators in preterm labor. In the present study, the interactions between granulocyte supernatant, IL-l and TNF in enhancing amnion cell PGEz synthesis were examined. We propose that the synergism of granulocyte and macrophage products promotes preterm labor in the setting of inflammation.

Inflammatory reactions often result in the recruitment and activation of phagocytic cells (e.g. neutrophils and tissue macrophages) (1). We have recently shown that activated human granulocytes release a protein(s) that causes a time- and concentrationdependent stimulation of PGE:! production by human amnion cells in culture (2). Activated macrophages, on the other hand, release cytokines such as interleukin-la (IL-la), interleukin-lfi (IL-lp), tumor necrosis factor-a (TNF-ar) and tumor necrosis factor-p (TNF-P). Both IL-l and TNF-(Y activate neutropbils and stimulate prostaglandin production in many cells (3, 4), including amnion cells (5, 6). Preterm labor is often associated with intrauterine infection (7). A histological hallmark of chorioamnionitis is the presence of neutrophils in the fetal membranes. Prostaglandins produced by the fetal membranes play a central role in the initiation of labor (8, 9). Thus, neutrophil products (2), TNF (6,

MATERIALS AND METHODS Chemicals Recombinant human, TNF-(w (specific activity 4.8 x lo7 U/mg; endotoxin content less than 0.13 EU/ml), TNF-g (specific activity 2.1 x 108 U/mg; endotoxin 3.1 EU/ml), and IL-lp (specific activity 2 x lo7 U/mg; endotoxin undetectable) were generously provided by Genentech, South San Francisco, CA. Recombinant human IL-la (specific activity 3 x lo8 U/mg; endotoxin 0.5 EU/ml) was a kind gift from Hoffmann-La Roche , Nutle y , NJ. Neutralizing antibodies to TNF-or, TNF-fl, IL-la and IL-l@ were from R&D Systems, Minneapolis, MN. The PGE2 kit was from New England Nuclear, Boston, MA.

Date received 2 April 1991 Date accepted 3 July 1991 241


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Isolation and activation of granulocytes Human granulocytes were isolated from fresh buffy coats by the method of Boym (12). At least 99.0% of the isolated cells were granulocytes. The cells were suspended in Hanks’ balanced salt solution without Ca2+ and Mg2+, containing 1.5 mM Hepes, pH 7.4, in a concentration of 400 x lo6 cells per ml. Granulocytes were activated by adding 1.3 mM CaC12 (1.3 mM final concentration), MgS04 (0.8 mM) and FMLP (1 PM), and incubating the cells at 37p C for 15 min. The activation was stopped by refrigerating to 0°C. Granulocyte supernatant was obtained by centrifuging at 2000 x g for 20 min. Preparation and culture of human amnion cells Human fetal membranes were obtained from normal pregnancies at cesarian sections conducted at term prior to the onset of labor. Cells from the reflected amnion were isolated as described (13). The cells were seeded in tissue culture plates at a density ,of 1.4 X lo5 cells/cm2 and maintained at 37°C in an atmosphere of air and 5% CO2 in Eagle’s MEM with Earle’s salts supplemented with glutamine, 10% FCS, antibiotics and antimycotics. After 24 h, the wells were washed with MEM and the media were changed to the culture medium supplemented with supernatant from activated granulocytes, cytokines, or the appropriate vehicle. Amnion cells were then incubated for 36 h before the culture media were removed and centrifuged at 600 x g for 10 min to eliminate cell debris. The supernatants were stored at -70°C until assayed for PGE;!. The cells were recovered from the wells using trypsin. More than 90% of the cells excluded trypan blue.

media incubated in the same conditions but without amnion cells were used as blanks. PGE2 formation is expressed per nanogram of amnion cell DNA; DNA analysis was done as in (14). Statistics The tests used were the Friedman Wilcoxon signed-rank test.

test and the

RESULTS Effect of granulocyte supernatant and of IL-la, IL-l/$ TNF-(wor TNF-p on amnion cell PGE2 production Human amnion cells were treated with granulocyte supernatant (final concentration 100 pi/ml), IL-lo (6.8 rig/ml), IL-ll3 (5 ng/ml), TNF-(U (50 ng/ml) or TNF-0 (10 q&l) and the PGEz was measured in the media after an incubation time of 36 h. In each case, stimulation of PGE2 production by amnion cells was observed (P = 0.003 as compared to the control) (Table 1). Treatment with the combinations of granulocyte supernatant and either IL-lo, IL-ll3, TNF-(Y or TNF-6 resulted in a synergistically enhanced PGEz production (P = 0.003) (Table 1). Polyclonal antibodies to the cytokines had no effect on the basal PGE2 production by amnion cells. In each case, the cytokine antibody inhibited the stimulatory effect of the cytokine and prevented the synergism of the cytokine with granulocyte supernatant. None of the cytokine antibodies interfered


Measurement of PGEz PGE2 was measured by radioimmunoassay directly from the media. The concentrations of PGE2 in the Table 1 Synergism of granulocyte supernatant cytokines on amnion cell PGE, production

F a e

and of

3 2


Control IL-lo (6.8 rig/ml) IL-Q3 (5.0 ng/ml) TNF-or (50 ng/ml) TNF-fl (10 ng/ml)

- Granulocyte supernatant

+ Granulocyte supernatant

1.0 23.0 + 4.4 27.2 + 6.0 25.2 f 5.2 6.7 + 1.3

22.5 103.0 113.6 110.1 53.6



+ 5.4 + 14.8 f 18.8 +_20.8 + 9.2

Amnion cells were treated with or without granulocyte supernatant, concentration 100 &‘rnl, in the presence of the cytokine indicated, or vehicle (control) and the PGE, formed was assayed from the media after 36 h. PGE, was measured as pg/ng DNA. The results shown are fractions of control (=l.O) and they represent the means + SEM of 11 experiments, each done with different amnion cells and with different granulocytes.

’ 0






Final concentration of granukxyte supernatant (@/ml) Fig. 1

Effect of different concentrations of IL-16 and of granulocyte supernatant on amnion cell PGE, production. The concentration of granulocyte supematant in the media of amnion cells was varied from O-100 pi/ml in the resence of different concentrations of IL-18 (0.5 pg/ml-5 n Ppml). The PGE, released into the media was measured after an incubation time of 36 h. One representative experiment of three is shown.

Stimulation of PGE, by IL-I, TNF and Granulocytes


No TNF-alpha 5 pa/ml 50 pa/ml 0.5 ng/ml 5 ng/ml


Table 2 Synergism of granulocyte

supematant, IL-k, IL-@,

TNF-(U and TNF-fi on PGE, production Treatment



Control IL-la IL-If.3 TNF-CY TNF-f3 IL-la + IL-lb TNF-a + TNF-0 IL-la + TNF-a IL-la + TNF-fi IL-10 + TNF-a IL-Ip + TNF-P

1.0 14.3 14.3 19.3 4.8 14.2 15.2 77.8 25.0 76.6 34.2


f 6.8 f 6.8 _+ 10.2 + 2.0 + 6.4 f 5.8 + 19.6 rt 4.9 + 25.4 + 16.2


+ + + + + + + + + +

by amnion cells PGE,

7.0 f 2.7 IL-la 56.7 + 26.0 IL-1s 57.9 t 27.4 TNF-(u 46.0 * 19.9 TNF-8 27.2 f 10.1 IL-la + IL-@, 49.5 f 19.1 TNF-(r + TNF-p 45.5 + 16.3 IL-la + TNF-a 176.6 f 17.4 IL-la + TNF+ 78.0 + 16.7 IL-Q + TNF-a 174.4 f 28.0 IL-lp + TNF-fl 144.0 f 32.2

Human granulocytes (4 x lOs/ml) were activated by FMLP (1 PM). Either vehicle (Control), granulocyte supernatant







Final concentration of granulocyte supernatant &d/ml) Fig. 2 Effect of different concentrations of TNF-or and of granulocyte supernatant on amnion cell PGE, production. The concentration of granulocyte supernatant in the media of amnion cells was increased from 0 to 100 pi/ml in the presence of different concentrations of TNF-or (5 pg/ml-50 ng/ml). The PGE, released into the media was measured after an incubation of 36 h. One representative experiment of three is shown.

with the effect of neutrophil supernatant on amnion cells. Indomethacin (0.01 pg/ml) abolished the PGE2 production irrespective of the stimulator used (data not shown). Effects of IL-1 and TNF in the presence of different concentrations of granulocyte supernatant on amnion cell PGEz production The concentration of granulocyte supernatant in the media of amnion cells was varied from O-100 pi/ml in the presence of different concentrations of IL-lp (0.05 pg/ml-O.5 E.Lg/ml)or TNF-(w (5 pg/ml-5 &ml). The stimulation of PGEz production increased with the concentration of granuiocyte supernatant (Figs 1 & 2). A synergism was observed at every concentration of granulocyte supematant and of cytokine. Stimulation of PGE2 production was evident when IL-l@ and TNF-cx concentrations exceeded 0.5 pg/ml and 50 pg/ml, respectively. The saturating concentrations of IL-l@ and‘TNF-a were 0.5 ng/ml and 5 ng/ml, respectively (Figs 1 & 2). Treatment of amnion cells with different concentra1ions of IL-la (0.07 pg/ml-0.7 &ml) or of TNF-/3 (0.01 ng/ml-1 &ml) in the presence or absence of neutrophil supernatant also resulted in a concentration-dependent synergistic stimulation of amnion cell PGE2 production. These effects were evident when the concentrations of IL-la and of TNF-j3 exceeded 0.07 pg/ml and 10 ng/ml, respectively. The saturating concentrations for IL-la and

(GS) (100 pi/ml), IL-k (6.8 ng/ml, IL-16 (5.0 &ml), TNF-ar (50 &ml), TNF-fi (10 ng/ml), or a combination of these was added to the medium of amnion cells in culture. PGE, released into the media was assayed after an incubation time of 36 h. The results are expressed as fractions of control (=l.O) and they represent the means + SEM of three experiments.

TNF-P were 70 pg/ml and 500 ng/ml, respectively (data not shown). Synergism of IL-1 with TNF. Synergism of granulocyte supernatant with the combination of IL-l and TNF The combination of either IL-la or IL-l/3 with either TNF-ar or TNF-p caused a synergistic stimulation in amnion cell PGE2 production, whereas the combinations of either TNF-CYwith TNF-/3 or IL-la with IL-16 were not synergistic. Moreover, treatment of amnion cells with granulocyte supernatant and the combination of IL-1 with TNF magnified the effect of the cytokines, resulting in a synergistically enhanced PGE2 production as compared to the effect of the cytokine combination (Table 2).

DISCUSSION The present results demonstrate for the first time that the production of PGE2 by human amnion cells is synergistically enhanced by the combination of granulocyte supernatant with either IL-1 or TNF. The magnitude of these effects depended on the concentrations of granulocyte supernatant and of cytokine. In addition, we show that IL-1 and TNF had a synergistic effect on amnion cell PGE2 production. Granulocyte, supematant amplified these responses even further, since it was synergistic with the combination of IL-1 with TNF. A lack of synergism when using the combination of IL-la with IL-@ or of TNF-a: with TNF-fi was expected, since IL-lar with IL-@ on the one hand (15), and TNF-(x with TNF-P on the other hand (16-19), share the same receptor in many cells.


Prostaglandins Leukotrienes

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IL-l and TNF are synergistic in inducing the release of PGE2 in many cells, such as human skin fibroblasts (20), human (21) and rat (22) glomerular mesangial cells and Madine-Darby canine kidney (MDCK) cells (23). However, in other cells, such as synovial fibroblasts, only an additive effect was observed (24). The mechanisms by which the interactions of cytokines and granulocyte supernatant occur have not been elucidated. IL-l induces the synthesis of cyclooxygenase in fibroblasts (25) and increases intracellular phospholipase A2 in rabbit chondrocytes (26). TNF-ar has been shown to induce phospholipase A2 in endothelial cells (27). On the other hand, granulocyte supernatant only stimulates PGE2 synthesis in amnion cells if the culture medium contains nonesterified arachidonic acid; it does not stimulate the release of arachidonic acid from glycerophospholipid storage forms (Bry et al, unpublished observations). When granulocyte supernatant was subjected to gel filtration, two major fractions (with apparent molecular weights of 12 and 60 kD) of PGE2, synthesis-stimulating activity were obtained (2), the larger component having a synergistic effect with IL-l and TNF (Bry et al, unpublished observations). Macrophage products affect granulocyte function and metabolism in many ways. Thus, IL-l and TNF have important roles in the recruitment and activation of neutrophils. IL-l causes neutrophilia (4) and induces granulocyte migration into tissues (28). IL-l and TNF enhance the adherence of neutrophils to endothelial cells (29). In addition, they stimulate neutrophil respiratory burst and degranulation (30, 31). According to the present results, granulocyte product(s) may amplify some effects of macrophagederived cytokines on cells, implying cooperation between the two inflammatory cells. Increased production of prostaglandins by intrauterine tissues is central to the initiation or maintenance of human labor (8, 9). Preterm labor is often associated with subclinical intrauterine infection (7). Bacterial products may stimulate the production of PGE;! in amnion cells (32). They also induce the production of IL-l and TNF by mononuclear phagocytes (33). IL-la, IL-l/3 and TNF-(w enhance PGE2 synthesis in human amnion cells in culture (5, 6, 10). TNF also stimulates PGEz and PGF2, biosynthesis in decidual explants (34). These cytokines have therefore been suggested to have a role in infection-induced preterm labor (6, 11). We have previously proposed that granulocytes may participate in preterm labor in the setting of infection (2). According to the present results, products from activated granulocytes together with cytokines cause a large increase in amnion cell PGE;! production compared to the stimulation caused by either

cytokine or granulocyte supernatant alone. We speculate that preterm labor in the setting of inflammation is triggered by the interactions of products from mononuclear phagocytes and from granulocytes. These observations may also be of relevance regarding the roles of monocytes/macrophages and granulocytes in the regulation of prostaglandin production in other inflammatory conditions.

Acknowledgments Supported by The Sigrid Juselius Foundation, The Foundation for Maternal and Infant Care, The Foundation for Pediatric Research in Finland, and The Paivikki and Sakari Sohlberg Foundation. The buffy coats for the isolation of human granulocytes were a generous gift of the Finnish Red Cross Blood Transfusion Service.

References 1. Ward P A, Warren J S, Johnson K J. Oxvzen 2.










radicals, inflammation, and tissue injury. -F;ee Radical Bioloev and Medicine 5: 403-40X. -> 1988. -- --Bry K, Hallm% M. A product of activated human granulocytes stimulates prostaglandin E, synthesis in human amnion cells. Prostaglandins Leukotrienes and Essential Fatty Acids. In press, 1991. Le J, Vilcek J. Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities. Laboratory Investigation 56: 234-248,1987. Dinarello C A. Cytokines: Interleukin-1 and tumor necrosis factor (cachectin). pp. 195-208 in Inflammation: Basic Principles and Clinical Correlates (J I Gallin, I M Goldstein, R Snyderman eds.) Raven Press, New York, 1988. Romero R, Durum S, Dinarello C A, Oyarzum E, Hobbins J C, Mitchell M D. Interleukin-1 stimulates prostaglandin biosynthesis by human amnion. Prostaglandins 37: 13-22, 1989. Casey M L, Cox S M, Beutler B, Milewich L. MacDonald P C. Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor. Journal of Clinical Investigation, 83: 430-436,1989. Romero R, Sirtori M, Oyarzun E. Avila C, Mazor M, Callahan R, Sabo V, Athanassiadis A P, Hobbins, J C. Infection and labor. V. Prevalence, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes. American Journal of Obstetrics and Gynecology 161: 817-824, 1989. Bleasdale J E, Johnston J M. Prostaglandins and human parturition: regulation of arachidonic acid mobilization. Reviews in Perinatal Medicine 5: 151-191,1985. Casey L M, MacDonald P C. The initiation of labor in women: regulation of phospholipid and arachidonic acid metabolism and of prostaglandin production. Seminars in Perinatology 10: 270-275, 1986. Romero R, Manozue K R. Mitchell M D, Wu Y K, Oyarzun E, Hobbins’J C, Cerami A. Infection and labor. IV. Cachectin-tumor necrosis factor in the amniotic fluid of women with intraamniotic infection and preterm labor. American Journal of Obstetrics and Gynecology 161: 336-341,1989. Romero R, Brody D T, Oyarzun E, Mazor M, Wu Y K, Hobbins J C, Durum, S K. Infection and labor. III. Interleukin-1: a signal for the onset of

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23. Leighton J D, Pfeilschifter J. Interleukin 1 and tumor necrosis factor stimulation of prostaglandin E, synthesis in MDCK cells, and potentia&n of this effect bv cvcloheximide. FEBS Letters 259: 289-29i, i!BO. 24. Godfrey R W, Johnson W J, Newman T, Hoffstein S T. Interleukin-1 and tumor necrosis factor are not synergistic for human synovial fibroblast PLA, activation and PGE, production. Prostaglandins 35: 107-114.1988. 25. Raz A, Wyche A, Siegel N, Needleman P. Regulation of fibroblast cyclooxygenase synthesis by interleukin-1. Journal of Biological Chemistry 263: 3022-3028.1988. 26. Chang J, Gilman S C, Lewis A J. Interleukin 1 activates phospholipase A, in rabbit chondrocytes: a possible signal for IL 1 action. Journal of Immunology 136: 1283-1287, 1986. 27. Clark M A, Chen M J, Crooke S T, Bomalski J S. Tumor necrosis factor (cachectin) induces phospholipase A? activity and synthesis of a phospholipase AZ-activating protein in endothelial cells. Biochemical Journal 250: 125-132, 1988. 28. Wankowicz Z, Megyeri P, Issekutz A. Synergy between tumor necrosis factor alpha and interleukin-1 in the induction of polymorphonuclear leukocyte migration during inflammation. Journal of Leukocyte Biology 43: 349-355. 1988. 29. Pohlman T H, Stanness K A, Beatty P G, Ochs H D, Harlan J M. An endothelial cell surface factor(s) induced in vitro by lipopolysaccharide, interleukin 1. and tumor necrosis factor-alpha increases neutrophil adherence by a CDwl8-dependent mechanism. Journal of Immunology 136: 4548-4553,1986. 30. Ozaki Y, Ohashi T, Kume S. Potentiation of neutrophil function by recombinant DNA-produced interleukin la. Journal of Leukocyte Biology 42: 621-627.1987. 31. Tsujimoto M, Yokota S, Vilcek J, Weissmann G. Tumor necrosis factor provokes superoxide anion . . -. . * generation from neutrophils. Biochemical Biophysical Research Communications, 137: 1094-1100.1986. 32. Lamont R F, Rose M, Elder M G. Effect of bacterial products on prostaglandin E production by amnion cells. Lancet 2: 1331-1333, 1985. 33. Beutler B, Cerami A. Cachectin and tumor necrosis factor as two sides of the same biological coin. Nature 320: 584-588, 1986. 34. Romero R, Mazor M, Wu Y K, Oyarzun E, Mitchell M D. Bacterial endotoxin and tumor necrosis factor stimulate prostaglandin production by human decidua. Prostaglandins Leukotrienes and Essential Fatty Acids 37: 183-186, 1989.


Synergistic stimulation of amnion cell prostaglandin E2 synthesis by interleukin-1, tumor necrosis factor and products from activated human granulocytes.

We examined the interactions between supernatant from FMLP-activated human granulocytes, recombinant interleukin-1 (IL-1) and recombinant tumor necros...
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