Prast&mdins 0 Longman

Leukottienes

Group UK

Ltd

and Essential 1991

Fatty

Acids

(1991) 43, 35-42

A Product of Activated Human Granulocytes Stimulates Prostaglandin Q Synthesis in Human Amnion Cells

K. Bry and M. Hallman* Department of Pediatrics, University of Helsinki, Helsinki, Finland and *Division of Neonatology, University of California, Irvine, Med. Surge I, Rm. 109F, Irvine, California 92717, USA (Reprint requests to KB) ABSTRACT

Cell-free supernatant from formylmethionyl-leucyl-phenylalanine (fMLP)-activated granulocytes causes a time- and concentration-dependent stimulation of prostaglandin E2 (PGE3 production in amnion cells. PGEz concentration in the culture medium after 36 h treatment with granulocyte supernatant (from 40 X IO6 granulocytes/ml of amnion cell medium), 1.49 f 0.71 pg/ng DNA (n = 13), was significantly higher (p = 0.0015) than in control cells (0.33 + 0.23 pg/ng DNA, n = 13). Indomethacin abolished this stimulation. Granulocyte supernatant and human epidermal growth factor (hEGF) had an additive effect on amnion cell PGEz production. Cat&se, superoxide dismutase (SOD), protease inhibitors or the platelet-activating factor (PAF) antagonist L-659,989 had no effect. Actinomycin D, cycloheximide and mepacrine reduced the PGEl production. The phospholipase AZ activity present in granulocyte supematants was resistant to heating, whereas heating decreased their PGE+timulating activity by 92%. Exogenous phospholipase A2 had no effect on PGQ synthesis. The granulocyte product could be precipitated with ammonium sulphate. On gel filtration of supernatant, two peaks of PGE+ynthesis stimulating activity were obtained (molecular weights 12 000 and 60 000). This data serve to explain the association of chorioamnionitis with preterm labor: activated granulocytes release a protein(s) that induces prostaglandin production in amnion cells, and thus promote labor.

INTRODUCTION

chorioamnion is associated with premature labor (9). Several mechanisms may be responsible for the onset of parturition in this setting. Thus, bacteria and their products may stimulate prostaglandin biosynthesis in fetal tissues (10, 11). Bacterial products may activate the host monocyte-macrophage system and cytokines released during this process may stimulate prostaglandin biosynthesis in intrauterine tissues (12, 13). On the other hand, chorioamnionitis is characterized histologically by the presence of granulocytes in the fetal membranes. The role of these cells in the initiation of labor has not previously been investigated. In this study we have examined the effect of supernatant from activated human granulocytes on PGE2 production by human amnion cells in culture.

Activated granulocytes and prostaglandins are general participants in inflammatory processes (1). Chemotaxis of granulocytes through pulmonary vascular endothelium causes endothelial production of large amounts of prostacyclin (2). In aortic endothelial cells, granulocytes or their products promote the release of prostacyclin (3). In synovial cells, granulocyte products stimulate the production of PGE2 (4). Thus, activated granulocytes may cause the production of prostaglandins in certain tissues. Prostaglandins are central to the initiation or maintenance of preterm and term labor (5, 6). An important intrauterine source of prostaglandins during labor is the amnion, which produces almost exclusively PGEz (7, 8). Inflammation of the

MATERIALS AND METHODS Materials

Date received 26 October 1990 Date accepted 31 December 1990

Culture media, Hanks’ balanced salt solution, fetal 35

36

Prostadandins Leukotrienes and Essential Fatty Acids

calf serum, and antibiotic/antimycotic solution (penicillin, streptomycin, fungizone) were from Gibco. Trypsin was purchased from Difco. The PGEz kit was from New England Nuclear. The Bond-Elut C-18 extraction columns were from Analytichem International. fMLP, cytochalasin B (CB), bisbenzimide, superoxide dismutase (SOD), catalase, aprotinin, pepstatin, cycloheximide, actinomycin D, phospholipase AZ (from Crotalus adamanteus venom), and mepacrine were from Sigma. 14C-arachidonate was from Amersham. Dextran T 500 and Ficoll-Paque were products of Pharmacia. Perchloric acid was from Merck, 4-methylumbelliferyl-P_D-glucuronide from KochLight, and o-dianisidine from Fluka. The PAF antagonist (+)-trans-2-(3-methoxy-5-methylsulfonyl4-propoxyphenyl)-5-(3,4,5_trimethoxyphenyl) tetrahydrofuran (L-659,989) (14) was a kind gift from Dr. John C. Chabala, PhD (Merck, Sharp and Dohme Research Laboratories, Rahway , NJ). The biosynthetic preparation of (3H)-palmitoyl phosphatidylcholine was carried out as described by Smith et al (15) using rat liver microsomes and 0.05 mM (1-3H) palmitate (New England Nuclear). Phosphatidylcholine was isolated by thin-layer chromatography followed by column chromatography. Altogether 99.2% of 3H-palmitate was associated with l-position of phosphatidylcholine. Preparation and culture of human amnion cells Human placentae were obtained from normal pregnancies at cesarian sections conducted at term prior to the onset of labor. Amnion epithelial cells were isolated as described (16). The isolated cells were seeded in tissue culture plates at a density of 1.4 x 16 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% fetal calf serum, antibiotics, and antimycotics. After 24 h, the wells were washed with MEM and the medium was changed to the culture medium supplemented with 10% (v/v) supematant from activated granulocytes or a corresponding buffer. Amnion cells were incubated for various periods of time ranging from O-48 h. At the end of the incubation the culture medium was removed, period, centrifuged at 600 g for 10 min to remove cell debris and the supematant stored at -70°C until assayed for PGE2. The adherent amnion cells were recovered from the culture wells using trypsin. More than 90% of the cells excluded trypan blue. Isolation and activation of cells Human granulocytes were isolated from fresh buffy coats by the method of Boyum (17). The granulocyte fraction contained approximately 99%

of granulocytes. Isolated cells were suspended in concentrations ranging from 50-600 X 106 cells per ml in Hanks’ balanced salt solution without Ca2+ or Mg2’, containing 15 mM HEPES, pH 7.4, and were kept on ice for a maximum of 30 min before use. Granulocytes were activated by adding Ca2+ and Mg2+ (final concentrations as in Hanks’ balanced salt solution), when indicated NLP (1 PM) and CB (O-2.5 &ml, added prior to fMLP), and incubating the cells at 37°C for 15 min. The reaction was stopped by refrigerating to -0°C. Granulocyte supernatant was obtained by centrifuging at 2000 g for 20 min. Mononuclear cells (monocytes and lymphocytes) were recovered using Ficoll gradient centrifugation (17). Monocytes were identified by nonspecific esterase staining using alpha naphthyl acetate as substrate (18). Mononuclear cells were suspended in a concentration of 400 x lo6 cells/ml in Hanks’ balanced salt solution (without Ca2+ and Mg2+). They were activated by adding Ca2+, Mg2+ and NLP (1 PM), and incubating at 37°C for 15 min. Mononuclear cell supernatant was obtained by centrifuging at 2000 g for 20 min. Measurement of PGE2 PGE2 was measured by radioimmunoassay (RIA) directly from the media. Similar results were obtained on assaying for PGE;! after the media had been acidified to pH 3.5-4.0 by addition of HCI and extracted on C-18 columns. Media with additions incubated in the same conditions but without amnion cells served as blanks. PGE2 formation is expressed per nanogram of amnion cell DNA; DNA analysis was done as described in (19). HPLC of prostaglandins After 48 h in culture, the amnion cells were washed with MEM and the medium was changed to MEM (supplemented with antibiotics and glutamine), containing 4.5% fetal calf serum and 14C-arachidonic acid (55 mCi/mmol, 0.6 &i/ml) and, in addition, either granulocyte supematant (from 30 X lo6 granulocytes/ml of amnion cell medium), or vehicle. After an incubation time of 24 h, the media were removed, acidified to pH 3.7 by 1 N HCl and loaded into C-18 extraction columns. After addition of the sample, the column was washed with Hz0 and eluted with methanol. The eluted sample was dried under nitrogen and taken up in 80 ~1 of acetonitrile followed by 160 ~1 H20. HPLC analysis of the prostaglandins was performed as in (3). One hundred twenty ~1 were injected into a Waters C 18 Bondapak column (3.9 mm X 30 cm) in a Shimazu HPLC system with a flow rate of 1 ml/min. Elution was done with a 35-85% gradient of

ActivatedGranulocytesStimulatePGE, Synthesisin Human Amnion Cells 37 acetonitrile/HzO containing 0.058% phosphoric acid, for the first 48 min, the isocratic for the next 12 min. This was followed by a 20 min reequilibration at 35% acetonitrile prior to the next injection. UV absorption was followed at 192 nm After the UV-detector, the for prostaglandins. sample stream was collected in 0.6 ml fractions and radioactivity was counted. The elution time of PGE* was established using a non-radioactive standard.

Phospholipase activity Phospholipase A2 activity was measured in 70 mM of the appropriate buffer (pH 5.5 - acetate; pH 7.4 Tris-HCl), containing 2 mM CaC12, 0.1 mM labeled phosphatidylcholine (50 000 cpm), and 30 ~1 of the granulocyte supernatant, in a volume of 0.1 ml. Phosphatidylcholine was added as a liposomal suspension, obtained by sonication. The reaction was performed at 37°C for 30 min in a shaking waterbath. The reaction was stopped by addition of 0.6 ml methanol, 1.2 ml chloroform, 0.4 ml 0.9% NaCl, followed by lipid extraction. The upper phase was re-extracted with chloroformmethanol (9:l). The combined lower phases were evaporated under N2 and developed by one-dimensional thin-layer chromatography (Silica gel H) with chloroform: methanol: water (65:25:4) using phosphatidylcholine, lysophosphatidylcholine and palmitic acid as carriers. The lipid spots, corresponding to the carriers, were identified with iodine, scraped and measured for radioactivity in Aquasol. Phospholipase A2 activity was quantitated by measuring the production of labeled lysophosphatidylcholine from labeled phosphatidylcholine, and the result was corrected for non-enzymatic hydrolysis. The activity was linear under the above assay conditions. Endogenous substrate did not contribute to the activity. In no instance was enzymatic production of 3H-palmitate found, suggesting the absence of phospholipase A1 activity. One unit (U) of enzyme activity was defined as the amount causing transformation of 1 pmol of substrate per min.

Other methods P-Glucuronidase and myeloperoxidase as described (20).

were assayed

Presentation of data The data are expressed as means + SE with the number of separate experiments given in parentheses. The tests used were the Friedman test and the Wilcoxon signed-rank test.

RESULTS Effect of granulocyte supematant on amnion cell prostagiandin production. Comparison with hEGF. Effect of heat treatment Supernatant from granulocytes (400 X lo6 cells/ml) that were activated with fMLP was studied. The amnion cells were washed and the supernatant was added to the culture medium (lo%, v/v) after the cells had been in culture for 24 h. The cumulative PGE2 release was measured after incubation for 36 h (Table 1). PGE;, release was 1.49 + 0.71 pg/ng DNA (n = 13) in supernatant-treated cells and 0.33 + 0.23 pg/ng DNA in control cells (n = 13) (p = 0.0015). As percent of control, the PGEz output in supernatant-treated cells was 1362 f 295% (n = 13). As expected (21), hEGF caused an increase in amnion cell PGEz production. PGEz production in the presence of hEGF (10 ng/ml) [2.64 + 0.91 pg/ng DNA (n = 12), or 4280 f 1265% (n = 12) of control] was significantly higher (p = 0.0037) than in the presence of granulocyte supernatant (Table 1). The effects of hEGF and granulocyte supematant were additive: in the presence of both granulocyte supernatant and hEGF, PGEZ production in amnion cells was 3.20 + 1.15 pg/ng DNA (n = 11) [or 6617.3 + 1585.6% (n = 11) of control], which is significantly greater than the effect of hEGF alone (p = 0.0033). Heat treatment of granulocyte supematants (at 74°C for 60 min) caused a decrease in their stimulatory activity on amnion cell PGE;! synthesis to 8.1 + 2.3% of the native supernatant (n = 4). In contrast, heat-treated hEGF had 87.9 f 6.3%, (n = 3) of the activity of native hEGF. PGE;-stimulating activity in lymphocytes and monocytes Monocytes

contain

cytokines

known

to activate

Table 1 Effect of granulocyte supematant

and of hEGF on amnion cell PGE, production. Granulocytes (4 x 108/ml) were activated by fMLP (1 PM). Cell-free supernatant from activated granulocytes (lo%, vol/vol), hEGF (10 ng/ml) or vehicle was added to the culture medium of amnion cells (10% of total volume) and the PGE, released was assayed after incubation for 36 h.

Control Granulocyte supematant hEGF Granulocyte supernatant and hEGF

PGE, production (p&g DNA/36 h) + SE (n)

PGE, production (% of control) + SE (n)

0.33 + 0.23 (13)

100

1.49 * 0.71 (13) 2.64 + 0.91 (12)

1362 f 295 (13) 4280 + 1265 (12)

3.20 + 1.15 (11)

6617 f 1.586 (11)

38

Prostaglandins Leukotrienes

and Essential Fatty Acids

PGEz production in amnion cells (12, 13). Human mononuclear cells (lymphocytes and monocytes) were isolated and activated by fMLP in the same way as the granulocytes. The PGE2 stimulating activity in the mononuclear cell supematants was 263.3 + 75.9% (n = 6) of the activity in the granulocyte fraction. However, due to the low number of mononuclear cells (approximately 0.7%) in the granulocyte fraction, the activity of the mononuclear cells explained 1.43 ? 0.69% (n = 6) of the PGEZ-stimulating activity of the granulocyte fraction. HPLC of prostaglandins, released from amnion cells To show that the product that was measured in the RIA assay corresponded to PGE2 and to show directly that stimulation by granulocyte supernatant caused increased release of this prostaglandin, HPLC was used to analyze the amnion cell media as described in Materials and Methods. In the of supernatant from fMLP-activated presence granulocytes (from 30 x lo6 cells/ml of medium), the PGE* released into the medium during an incubation time of 24 h was 250% compared with the control, as measured by RIA. HPLC analysis showed that the radioactivity in the peak corresponding to PGE2 was increased by 2.3-fold in cells treated with granulocyte supernatant as compared with control cells.

the addition of either granulocyte supematant (from 20 X lo6 granulocytes per ml of amnion cell medium) or vehicle to the medium (Fig. 1). The granulocytes were activated by fMLP. The difference between PGEz synthesis by neutrophilsupernatant treated cells and by control cells was detectable after 12 h of incubation, but became more evident after 24 h (Fig. 1). Dependence on the number of granulocytes The stimulation of PGEz production in human amnion cells depended on the concentration of granulocytes used (Fig. 2). There was a linear dependence between PGE;! release and the number of granulocytes activated by fMLP (1 PM) (R* = 0.971) as well as by fMLP (1 PM) in the presence of CB (1 pg/ml) (R* = 0.993). A saturating concentration of granulocytes was not reached. Granulocyte supematants contained very little PGE2 (less than 1 pg/106 cells), irrespective of the granulocyte activation procedure.

Dependence on the incubation time PGEz concentration in the medium of amnion cells was measured at regular time intervals after Next,

on 0

1 200

I 400

, 600

Granulocyte concentration (x10 (exp 6) /ml) Fig. 2 Release of PGE, from amnion cells as a function of granulocyte concentration. Granulocytes in various concentrations (range O-6 x lo8 per ml) were activated by n fMLP (final concentration 1 PM) or 0 fMLP (1 PM) with CB (1 pg/ml). Granulocyte supernatant was added to amnion cell culture medium (10% of total volume) and the PGE, released was assayed after incubation for 36 h. PGE, is expressed in percent of controls. The results shown represent the means f S.E. of three experiments. Each experimental condition was repeated twice within each experiment.

Effect of cytochalasin 0

12

24

36

46

Time (h) Fig. 1

Release of PGE, from amnion cells as a function of time. Granulocvtes (200 x lO”/ml) were activated bv fMLP (1 PM) for 15 &in at 37°C. A’mnion cells were incubated in the presence of cell-free supematant from activated granulocytes (lo%, vol:vol) n or vehicle 0. PGE, was measured after the indicated incubation times. The results shown are the means + SE of four experiments. Each experimental condition was repeated twice within each experiment.

In the presence of CB, granulocyte degranulation, as measured by the release of myeloperoxidase and P-glucuronidase, was enhanced. Amounts of /3glucuronidase and of myeloperoxidase released were linear functions of granulocyte concentration (data not shown). When exposed to supernatant from granulocytes stimulated with fMLP in the presence of CB, amnion cells produced less PGEz than when treated

Activated Granulocytes Stimulate PGE, Synthesis in Human Amnioa Cells

with supernatant from granulocytes stimulated with NLP alone (Fig. 2). PGEz release in the absence of CB was 182% (n = 6)ofthatinthepresenceofCB. To test whether CB affects the PGEz production by amnion cells, different amounts of CB were added to the culture medium of amnion cells. CB tended to reduce the effect of granulocyte supernatant on PGEz production in amnion cells (Table 2). In the absence of granulocyte supernatant, PGEz release was low, but was inhibited by CB (Table 2). Table 2 Amnion cell PGE, production as a function of CB concentration. Supematant from fMLP-activated granulocytes (from 50 granulocytes/amnion cell) or vehicle was added to the amnion cell medium in addition to various concentrations of CB. The PGE, released was measured after 36 h. The results are expressed as percent of control (PGE, production in cells without granulocyte supematant and without CB). They represent the means + SE of three experiments.

39

Table 3 Effect of inhibitors of prostaglandin and of protein synthesis, of protease inhibitors, PAF antagonist and oxygen radical scavengers on amnion cell PGE, production. Amnion cells were treated with the indicated concentrations of a protease inhibitor, of the PAF antagonist L-659989, of oxygen radical scavengers or of protein synthesis inhibitors either in the presence or in the absence of granulocyte supematant (from 40 X lo6 cells/ml medium). After incubation for 36 h, the PGE, released was assayed. The results are expressed as percent of control + SE. Granulocyte supematant

Vehicle

Control

100

100

Cyclooxygenase inhibitor Indomethacin (0.01 &ml)

< 1

i C Y, Lam K W, Yam L T. Esterases in human eucocytes. Journal of Histochemistr$ and zytochemistry 21, 1-12, 1973. 19. Shapiro D L, Schrier B K. Cell cultures of fetal rat Irain. Growth and marker enzyme development. Experimental Cell Research 77: 239-247, 1973. 20. Dewald B, Baggiolini M. Methods for assessing :xocytosis by aeutrophil leukocytes. Methods in Enzymology 132: 267-277, 1986. 21. Mitchell M D. Eoidermal growth factor actions on srachidonic acid metabolisk in human amnion :ells. Biochimica et Biophysics Acta 928: 240-242, 1987. 22. Walsh C E, Dechatelet L R, Chilton F H, Wykle R L, Waite M. Mechanisms of arachidonic acid release in human polymorphonuclear leukocytes. Biochimica et Biophysics Acta 750, 32-40, 1983. 23. Meade C J, Turner G, Bateman P E. The role of phosphoinositides and their breakdown @oducts in A 23187-induced release of arachidonic &id from rabbit polymorphonuclear leucocytes. Biochemical Journal 238,425-436, 1986. 24. LeRoy E C, Ager A, Gordon J L. Effects of neutrophil elastase and other proteases on porcine aortic endothelial prostaglandin I, production, adenine nucleotide release, and response to vasoactive agents. Journal of Clinical Investigation 74, 1003-1010, 1984. 25. Harlan J M, Callahan K S. Role of hydrogen peroxide in the neutrophil-mediated release of prostacyclin from cultured endothelial cells. Journal of Clinical Investigation 74: 442-448, 1984. 26. Marshall P J, Lands W E M. In vitro formation ot activators for prostaglandin synthesis by neutrophils and macrophages from humans and guinea pigs. Journal of Lab Clin Med 108, 525-534, 1986. 27. Sisson J H, Prescott S M, McIntyre T M, Zimmerman G A. Production of platelet-activating factor by stimulated human polymorphonuclear leucocytes. Correlation of synthesis with release, functional events, and leukotriene B, metabolism. Journal of Immunology 138: 3918-3926, 1987. 28. Lvnch J M. Henson P M The intracellular retention of newly synthesized platelet-activating factor. Journal of Immunology 137: 2653-2661, 1986. 29. Billah M M, DiRenzo G C, Ban C, Truong C T, Hoffman D R, Anceschi M M, Bleasdale J E, Johnston J M. Platelet-activating factor metabolism in human amnion and the responses of this tissue to extracellular platelet-activating factor. Prostaglandins 30: 841-850, 1985. 30. Casey M L, Mitchell M D, MacDonald P C. Epidermal growth factor-stimulated prostaglandin E, production in human amnion cells: specificity and nonesterified arachidonic acid dependency. Molecular and Cellular Endocrinology 53: 169-176, 1987. 31. Balsinde J, Diez E, Schuller A, Mollinedo F. Phospholipase A, activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization. Journal of Biological Chemistry 263: 1929-1936, 1988. 32. Casey M L, Korte K, MacDonald P C: @idermal growth factor stimulation of prostaglandin E, biosynthesis in amnion cells. Induction of orostaglandin H, synthase. Journal of Biological ‘Chem&y 263: j&46-7654, 1988. 33. McCrea J M, Robinson P and Gerrard J M. Meoacrine (auinacrine) inhibition of thrombin-inh;ced platdlet responses can be overcome by lysophosphatidic acid. Biochimica et Biophysics Acta 842: 189-194, 1985. 34. Bejar R, Curbelo V, Davis C, Gluck L. Premature labor. II. Bacterial sources of phospholipase. Obstetrics and Gynecology 57, 479-482, 1981.

A product of activated human granulocytes stimulates prostaglandin E2 synthesis in human amnion cells.

Cell-free supernatant from formylmethionyl-leucyl-phenylalanine (fMLP)-activated granulocytes causes a time- and concentration-dependent stimulation o...
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