Kutteh et al.

mal human amniotic fluid: unique high molecular weight form of IL-II3. Immunology 1988;65:337-42. 39. Akira D, Hirano T, Taga T, Kishimoto T. Biology of multifunctional cytokines: IL-6 and related molecules (IL-I and TNF). FASEB J 1990;4:2860-7. 40. West MA, Billiar TR, Mazuski .IE, Curran RJ, Cerra FE, Simmons RL. Endotoxin mod ulation of hepatocyte secretory and cellular protein synthesis is mediated by Kupffer cells. Arch Surg 1988;123:1400-5. 41. Casteleijan E, Kuiper J, van Rooij HCG, Camps JAAM, Koster JF, van Berkel TJC. Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelialliver cells. J BioI Chern 1988;263:2699-703.

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42. Feingold KR, Serio MK, Mounzer S, Moser AH, Dinarello CA, Grunfeld C. Multiple cytokines stimulate hepatic lipid synthesis in vivo. Endocrinology 1989;125:267-74. 43. Givner ML, Jaffe RE. Cholesterol biosynthesis in human fetal liver and adrenal. Steroids 1971;18:1-10. 44. Carr BR, Simpson ER. Cholesterol synthesis in human fetal tissues . .I Clin Endocrinol Metab 1982;55:447-52. 45. Nakamura T, Arakaki R, Ichihara A. Interleukin-113 is a potent growth inhibitor of adult rat hepatocytes in primary culture. Exp Cell Res 1988; 179:488-97.

Endothelin production by amnion and its regulation by cytokines Murray D. Mitchell, DPhil, Sarah Lundin-Schiller, BA, and Samuel S. Edwin, BS Salt Lake City, Utah Cells derived from human amnion, an avascular tissue, produced significant amounts of endothelin 1 and 2, although the rate of biosynthesis was approximately one tenth of that of human umbilical vein endothelial cells. Endothelin 1 and 2 was not produced in measurable amounts by cells derived from chorion laeve or decidua. Production of endothelin 1 and 2 by amnion cells was increased by treatment with the cytokines interleukin-113, tumor necrosis factor, and interleukin-6. The biosynthesis of endothelin 1 and 2 by an avascular tissue is suggestive of a wider role for endothelins than has been recognized. (AM J OBSTET GVNECOL 1991 ;165:120-4.)

Key words: Endothelin, amnion, cytokine, avascular Endothelins are a family of small proteins that were originally isolated from the culture supernatants of porcine aortic endothelial cells.! Endothelin 1 was originally reported to be a potent vasoconstrictor, and expression of the endothelin 1 gene was found to be regulated by several vasoactive agents. Endothelins I, 2, and 3 have since been described; endothelin 1 is porcine-human endothelin and endothelin 3 is rat endothelin. 2.! These 2 I-residue peptides are formed from a preproendothelin (-200 residues) by way of a 38/39residue intermediate. I. 2 It is thought that endotheFrom the Department of Obstetrics and Gynecology, University of Utah. These studies were supported in part by National Institutes of Health grant HD 20779. Received for publication September 25, 1990; accepted January 18, 1991. Reprint requests: Murray D. Mitchell, DPhil, Department of Obstetrics and Gynecology, University of Utah Medical Center, 50 North Medical Dr., Room 2B302, Salt Lake Cit~, UT 84132. 611 128071 .

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lins act as endogenous agonists of dihydropyridinesensitive calcium channels.! Included among an increasing array of properties of endothelins are the induction of two types of contractions of the rat uterus,' activation of phospholipase A," and activation of phospholipase c. 7." Additionally, endothelins recently have been found to be synthesized not only by endothelial cells but also by kidney cells." We have recently found that endothelin I is present in amniotic fluid!" and that its concentration is elevated in amniotic liquor from women with infections of the amniotic fluid who are delivered preteI'm. This study was undertaken to evaluate potential sources of endothelin 1 within the uterus and to determine whether inflammatory mediators regulate the rate of production of this protein. Material and methods Placentas were obtained at cesarean section at term before the onset of labor. Amnion was separated manually and decidua was scraped from the maternal face

Amnion endothelin production

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Fig.!. Endothelin 1 and 2 in dilutions of culture media from amnion, chorion, and decidual cells (initial measurements are concentrates from 50 ml of conditioned media) and umbilical vein endothelial cells (initial measurements are concentrates from 10 ml of conditioned media). Circles, Amnion; triangles, chorion; asterisks, decidual cells; squares, umbilical vein endothelial cells.

of the chorion. Cells were prepared from amnion as described previously. II Chorion laeve cells were dispersed by treatment with collagenase and isolated by separation of the dispersate on a discontinuous Percoll gradient. I" Decidual cells also were prepared by collagenase dispersion and isolated on a different discontinuous Percoll gradient. 13 Endothelial cells were prepared from umbilical cords according to the method of Jaffe. 14 Cells were grown to confluence, at which time media for analyses were obtained. In most experiments culture media were used without manipulation. In one series of experiments culture media were subjected to anisotropic ultrafiltration through a filter with a molecular weight cutoff of 10,000, followed by concentration of the filtrate on a C I " Sep Pak (Millipore Corp., Milford, Mass.)." Interleukin-ll3 was purchased from Amgen Biologicals (Thousand Oaks, Calif.); tumor necrosis factor and interleukin-6 were obtained from R&D Systems (Minneapolis). Endothelin 1 and endothelin 1 and 2 were measured by means of radioimmunoassay kits from Peninsula Laboratories, Inc. (Belmont, Calif.) and Amersham Corporation (Arlington Heights, Ill.), respectively. Cellular protein was determined by the method of Lowry et al. 16 Statistical differences were assessed by Fisher's least significant difference test. Results

Measurements of endothelin 1 and 2 in serial dilutions of concentrated conditioned media clearly dem-

onstrated secretion of immunoassayable material, not only by endothelial cells (as expected) but also by amnion cells (Fig. 1). Conditioned media from chorion and decidual cells did not contain detectable amounts of endothelin 1 and 2. To establish the relative secretion rates of endothelin 1 and 2 by endothelial and amnion cells, we determined its basal rate of production by confluent monolayers from several individual tissues (Fig. 2). Endothelin 1 and 2 production by endothelial cells was approximately tenfold greater than that of amnion cells. The antiserum used in the studies at this point, although raised against endothelin 1, cross reacts 200% with endothelin 2 (Amersham Corporation). Hence we could have been measuring either or (more likely) both proteins. We deemed it important to be more specific in our measurements and to evaluate production of endothelin 1 alone. First, we compared measurements of endothelin 1 and endothelin 1 and 2 in conditioned media of amnion cells from several individual tissues under a variety of conditions (Fig. 3). The results indicated a significant correlation between measurements by each method, with endothelin I and 2 levels being greater. Although many interpretations of these data are possible, the most obvious is that amnion cells secrete both endothelin 1 and endothelin 2, with endothelin I secretion being greatest quantitatively. To evaluate regulation of endothelin 1 production alone, we used the endothelin I-specific radioimmunoassay (Peninsula Laboratories) in the next series of studies. Incubation of amnion cells with interleukin-113 and

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Mitchell, Lundin-Schiller, and Edwin

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Fig. 3. Comparison of measurements of endothelin 1 and 2 versus endothelin 1 in amnion cell culture media. Linear regression analysis with y = endothelin 1 and 2 and x = endothelin 1 gave y = Ui9x + 15; r = 0.80.

tumor necrosis factor induced significant increases in the rate of production of endothelin 1 (Fig. 4, A and B). The results from representative experiments are presented. Similar results have been obtained from experiments with cells from five individual tissues. In

contrast, incubation of amnion cells with interleukin-6 resulted in variable effects on endothelin 1 production, although the highest concentration of interleukin-6 used always stimulated endothelin 1 secretion (Fig. 4, C). The reasons for this variability are uncertain.

Amnion endothelin production

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Fig. 4. Effects of cytokines on endothelin production by amnion cells. A, Interleukin-ll3 (fL-] beta); B, Tumor necrosis factor (TNF); C, Interleukin-6 (/L-6). Control versus interleukin-ll3, p < 0.005 (all concentrations). Control versus tumor necrosis factor, p < 0.005 (all concentrations). Control versus interleukin-6, p < 0.002 at 100 ngl m!.

Similar findings were obtained with endothelin 1 and 2 measurements after cytokine stimulation of amnion cells.

Comment Our results indicate that endothelin 1 and probably endothelin 2 are synthesized and secreted by avascular amnion. However, it will be important to determine that these proteins have bioactivity, as well as immunoreactivity, and that they are produced by fresh amnion tissue, as well as by cells derived from the tissue. Nevertheless, the data suggest that endothelin production may be more widespread than previously thought. Endothelin production by some cells and tissues may be

missed as a result of difficulties in the assay procedure. In our original studies l7 we were often misled by significant matrix effects of different culture media in the assay. Careful attention to the control of these effects was necessary to permit the correct determination of endothelin production by amnion cells. The increase in amnion endothelin 1 production induced by cytokines is consistent with our finding of raised amniotic fluid levels of endothelin 1 and 2 in pregnancies complicated by intrauterine infections.lo Moreover, these findings are also consistent with the increase in endothelin messenger ribonucleic acid expression in amnion cells induced by interleukin-l. IH What role(s) could amnion-derived endothelin 1 serve

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during pregnancy? Endothelin 1 has been shown to be potent in both elevating cytoplasmic calcium concentrations and increasing myosin phosphorylation in human myometrium. l9 Hence, if endothelin secreted by amnion could reach the myometrium, it could have a significant effect on contractile function. More speculatively, it is possible that endothelin participates in the mechanisms governing amniotic fluid volume. Overall, therefore, our finding of endothelin 1 production by amnion may be of great significance to our future understanding of certain physiologic changes with pregnancy and parturition in women. REFERENCES 1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411-5. 2. Itoh y, Yanagisawa M, Ohkubo S, et al. Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin. FEBS Lett 1988;231 :440-4. 3. Yanagisawa M, Inoue A, Ishikawa T, et al. Primary structure, synthesis, and biological activity of rat endothelin, an endothelin-derived vasoconstrictor peptide. Proc Nat! Acad Sci USA 1988;85:6964-7. 4. Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Nat! Acad Sci USA 1989;86:2863-7. 5. Kozuka M, Ito T, Hirose S, Takahashi K, Hagiwara H. Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels. Biochem Biophys Res Commun 1989;159:317-23. 6. Resink TJ, Scott-Burden T, Buhler FR. Activation of phospholipase A~ by endothelin in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1989; 158:279-86. 7. Resink TJ, Scott-Burden T, Buhler FR. Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1988; 157: 1%0-8.

July 1991 Am J Obstet Gynecol

8. Sugiura M, Inagami T, Hare GMT, JohnsJA. Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem Biophys Res Commun 1989;158:170-6. 9. Kosaka T, Suzuki N, Matsumoto H, et al. Synthesis of the vasoconstrictor peptide endothelin in kidney cells. FEBS Lett 1989;249:42-6. 10. Romero R, Avila C, Mitchell MD. Endothelin-l in human parturition [Abstract]. In: Proceedings of the thirtyseventh annual meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24, 1990. St. Louis: Society for Gynecologic Investigation, 1990: 351. 11. OkitaJR, Sagawa N, Casey ML, Snyder JM. A comparison of human amnion tissue and amnion cells in primary culture by morphological and biochemical criteria. In Vitro 1983;19:117-26. 12. Gibb W, Riopel L, Lavoie JC. Primary culture of cells from human chorion laeve: steroid metabolism and properties of cells grown in defined media supplemented with 0.1 % or 10% fetal calf serum. J Clin Endocrinol Metab 1986;62: 1124-9. 13. Devlin EE, Arabian A, Glorieux F, Marner O. In vitro metabolism of 25-hydroxycholecalciferol by isolated cells from human decidua. J Clin Endocrinol Metab 1985; 60:880-5. 14. Jaffe EA. Biology of endothelial cells. Boston: Martinus Nijhoff, 1984. 15. Koyama H, Nishzawa Y, Morii H, Tabata T, Inoue T, Yamaji T. Plasma endothelin levels in patients with uraemia. Lancet 1989:991-2. 16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. J Bioi Chern 1951; 193:265-75. 17. Mitchell MD, Lundin-Schiller S, Edwin S. Actions of endothelin-l on prostaglandin production by gestational tissues [Abstract]. In: Proceedings of the thirty-seventh annual meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 21-24,1990. St. Louis: Society for Gynecologic Investigation, 1990: 171. 18. Sunnergren KP, Word RA, SambrookJF, MacDonald PC, Casey ML. Expression and regulation of endothelin precursor mRNA in avascular human amnion. Mol Cell EndocrinoI1990;68:R7-RI4. 19. Word RA, Kamm KE, Stull JT, Casey ML. Endothelin increases cytoplasmic calcium and myosin phosphorylation in human myometrium. AM J OBSTET GYNECOL 1990; 162: 1103-8.

Endothelin production by amnion and its regulation by cytokines.

Cells derived from human amnion, an avascular tissue, produced significant amounts of endothelin 1 and 2, although the rate of biosynthesis was approx...
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