Determination of cytokine mRNA-expression in term human placenta of patients with gestational hypertension, intrauterine growth retardation and gestational diabetes mellitus using polymerase chain reaction

Summary

Objective: Our objective was to test the hypothesis, that pregnancy-related diseases are going along with changes in cytokine mRNA-expression at the placental site, either as a part of a pathological process or in connection with regulatory mechanisms induced by disturbances at the feto-maternal interface resulting from previous pathological changes – in the sense of counterregulation. Material and methods: The cytokines chosen for this investigation are known to 1.) be expressed in the human placental tissue, 2.) to be involved in immunological processes and 3.) the regulation of growth and differentiation processes of different cell types of the placenta or decidua, 4.) to play a role in the angiogenesis at the feto-placental interface and 5.) to be involved in pathological processes in other human diseases. 32 samples derived from term human placentas were examined for messenger RNA levels of interleukin 1 alpha (Il-1α), tumor necrosis factor-alpha (TNF-α), platelet derived growth factor-A chain (PDGF-A), platelet derived growth factor-B chain (PDGF-B), and platelet derived growth factor receptor (PDGF-R) using a semiquantitative reverse transcriptase (RT) polymerase chain reaction (PCR) protocol. To calibrate samples in our procedure, β-actin mRNA (messanger ribonucleid acid) known as a “house keeping” gene was proven to be constantly expressed. The sample-groups consisted of normal pregnancies (n = 8), gestational hypertension (GH, n = 7), intrauterine growth retardation (IUGR, n = 6), gestational diabetes mellitus (GDM, n = 5), and gemini (n = 3 × 2). Results: Throughout the 32 samples, a significant correlation between PDGF-A and PDGF-R expression, PDGF-A and TNF-alpha expression was stated (p = 0.007). Compared with the pattern of expression in normal placentas, placentas of growth retarded pregnancies had higher Il-1α mRNA (p = 0.016), PDGF-A (p = 0.029) and PDGF-B (p = 0.001) levels. The samples of the gestational hypertension group and placentas of patients with gestational diabetes displayed a significantly stronger PDGF-R mRNA signal (p = 0.0029 and p = 0.008). Conclusions: Though these marked differences in cytokine mRNA levels between clinical groups were statistically proven, clear correlation of these differences with clinical data was not found.

Zusammenfassung

Fragestellung: Ziel der vorliegenden Untersuchung war die Überprüfung der Hypothese, daß sich schwangerschaftsspezifische Erkrankungen in Veränderungen der Expressions-Niveaus von Zytokinen in der Plazenta, entweder als Teil pathogenetischer Prozesse oder im Sinne einer Gegenregulation, induziert durch Störungen an der feto-maternalen Einheit, als Resultat vorangegangener pathologischer Veränderungen, widerspiegeln. Material und Methoden: Für diese Untersuchungen wurden Zytokine ausgewählt, von denen bekannt ist, daß sie 1.) in der humanen Plazenta exprimiert werden, 2.) in immunologischen Prozessen mitwirken und 3.) an der Regulation von Wachstum und Differenzierung verschiedener Zelltypen in der Plazenta und Dezidua beteiligt sind, 4.) Bedeutung in der Angiogenese an der feto-maternalen Einheit haben und 5.) an pathologischen Prozessen in anderen humanen Erkrankungen beteiligt sind. Zweiunddreißig unmittelbar post partum gewonnene Plazentaproben wurden mit Hilfe einer semiquantitativen Polymerase-Kettenreaktion (PCR) auf die Expressions von Interleukin-1 alpha (IL-1α), Tumornekrosefaktor-alpha (TNF-α), platelet derived growth factor-A (PDGF-A), platelet derived growth factor-B (PDGF-B) und platelet derived growth factor-Rezeptor (PDGF-R) untersucht. Um die eingesetzte Probenmenge zu equilibrieren, wurde die β-Aktin-Expression, als „house keeping gene”, als Bezugsgröße verwendet und dessen konstante Expression nachgewiesen. Die Gruppe der untersuchten Patientinnen setzte sich aus normalen Schwangeren (n = 8), Patientinnen mit Gestationshypertonie (n = 7), intrauteriner Wachstumsretardierung des Föten (n = 6), Gestationsdiabetes (n = 5) und Zwillingen (n = 3 × 2) zusammen. Ergebnisse: In allen 32 Proben ließ sich eine signifikante Korrelation zwischen PDGF-A und PDGF-Rezeptor sowie zwischen PDGF-A und TNF-alpha (p = 0,007) feststellen. Im Vergleich mit der Normalgruppe wiesen die Plazenten von wachstumsretardierten Schwangerschaften eine stärkere Expression von IL-1α auf (p = 0,016), PDGF-A (p = 0,029) und PDGF-B (0,001). In den Plazenten von Patienten mit Gestationshypertonie und bei den Patientinnen mit Gestationsdiabetes fanden sich höhere mRNA-Spiegel für den PDGF-Rezeptor (p = 0,0029 und p = 0,008). Obwohl deutliche Unterschiede in der Zytokinexpression zwischen den klinischen Gruppen statistisch signifikant waren, konnte der Zusammenhang mit einzelnen klinischen Parametern nicht gesichert werden. Schlußfolgerung: Weitere Untersuchungen von größeren Patientenzahlen und mit verbesserten quantitativen PCR-Protokollen sind notwendig, um Korrelationen zwischen klinischem Verlauf und Zytokinexpression an der Plazenta aufzeigen zu können.

MeSH

C13.703 pregnancy complicationsA16.759 placenta

Keywords

Cytokines - gestational diabetes mellitus - gestational hypertension - interleukin-1 alpha - intrauterine growth retardation - platelet-derived growth factor - placenta - polymerase chain reaction - tumor necrosis factor-alpha

Schlüsselwörter

Zytokine - Gestationsdiabetes - Gestationshypertonie - Interleukin-1 alpha - intrauterine Wachstumsretardierung - Plazenta - PDGF - PDGF-Rezeptor - Polymerase-Ketten-Reaktion - Tumornekrose-Faktor-alpha

• References

• 1 Abbs S, Bobrow M. Analysis of quantitative PCR for the diagnosis of deletion und duplication carriers in the dystrophin gene. J Med Genet 1992; 29: 191-196
• 2 Kaufmann P. Pathology of the human placenta. 2nd Edn. Springer; New York, Heidelberg, Berlin: 1990
• 3 Boado RJ, Pardridge WM. Measurement of blood - brain barrier Glut 1 glucose transporter and β-actin mRNA by a quantitative polymerase chain reaction. J Neurochem 1994; 62: 2085-2090
• 4 Casey ML, Cox SM, Beutler B, Milewich L, MacDonald PC. Cachectin tumor necrosis factor-alpha formation in human decidua - potential role of cytokines in infection-induced preterm labor. J Clin Invest 1989; 83: 430
• 5 Chen HC, Yang Y, Hu XL, Yelavarthi KK, Fishback JL, Hunt JS. Tumor necrosis factor alpha mRNA and protein are present in human placental and uterine cells at early and late stages of gestation. Am J Pathol 1991; 139: 327
• 6 Feinberg BB, Anderson DJ, Steller MA, Fulop V, Berkowitz RS, Hill JA. Cytokine regulation of trophoblast steroidgenesis. J Clin Endocrinol Metab 1994; 78: 586-591
• 7 Halgunset J, Jonsen H, Kjollesdal AM. Cytokine levels in amniotic fluid and inflammatory changes in the placenta from normal deliveries at term. Eur J of Obstet Gynecol Reprod Biol 1994; 56: 153-160
• 8 Heinig J, Wilhelm S, Bittorf T, Müller H, Brock J, Briese V. Semiquantitative determination of IL-1α, TNF-α, PDGF-A, PDGF-B, and PDGF-Receptor in term human placenta using polymerase chain reaction (PCR). Zentralbl Gynakol 1993; 115: 317
• 9 Henco K, Heibey M. Quantitative PCR. The determination of template copy numbers by temperature gradient gel electrophoresis (TGGE). Nucleic Acids Res 1990; 18: 6733
• 10 Holmgren L, Claesson-Welsh L, Heldin Ch, Ohlsson R. The expression of PDGF-alpha and -beta receptors subpopulations of PDGF-producing cells implicates autocrine stimulatory loops in the control of the proliferation in cytotrophoblasts that have invaded the maternal endometrium. Growth factors 1992; 6: 219
• 11 Hunt JS. Immunology of pregnancy. Curr Opin Immunol 1992; 4: 591
• 12 Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols. Innis MA (Ed); Academic Press Incorp, San Diego, London: 1990
• 13 Kauma SW, Walsh SW, Nestler JE, Turner TT. Interleukin-1 is induced in the human placenta by endotoxin and isolation procedures for trophoblasts. J Clin Endocrinol Metab 1992; 75: 951
• 14 Klagsburn M, Folkman J. Angiogenesis in Peptide Growth Factors and their receptors (II). Sporn MB, Roberts AB (Eds); Springer, New York, Berlin, Heidelberg: 1994. 549-574
• 15 Masuhiro M, Matsuzaki M, Nishino E, Tanaguchi T, Kameda T, Li Y, Saji F, Tanizawa O. Trophoblast derived interleukin-1 (Il-1) stimulates the release of human chorionic gonadotropin by activating Il-6 and Il-6 receptor system in first trimester human trophoblast. J Clin Endocrin Metab 1991; 72: 594-601
• 16 Nestler JE. Interleukin-1 stimulates the aromatase activity of human placental Cytotrophoblasts. Endocrinology 1993; 132: 66-70
• 17 Paulesu L, King A, Loke YW, Cintorino M, Bellizzi E, Boraschi D. Immunohistochemical localization of Il-1 alpha and Il-1 beta in normal human placenta. Lymphokine Cytokine Res 1991; 10: 443-448
• 18 Rolfs A, Schuller I, Finckh U, Weber-Rolfs I. PCR: clinical diagnostics and research. Rolfs A (Ed); Springer, Berlin, Heidelberg, New York: 1992
• 19 Romero R, Mazor M, Dinarello C, Mitchell M. Natural interleukin-1 receptor antagonist blocks interleukin-1-induced prostaglandin production by human intrauterine tissues: The basis for a novel approach to the treatment of preterm labor in the setting of infection. Am J Obstet Gynecol 1992; 166: 274
• 20 Sambrock J, Fritsch EF, Maniatis T. Molecular cloning. A laboratory manual. Laboratory Press; Cold Spring Harbor: 1989
• 21 Sporn MB, Roberts AB. Peptide growth factors and their receptors. Sporn MB, Roberts AB (Eds); Springer, New York, Berlin, Heidelberg: 1991
• 22 Taylor RN, Williams LT. Developmental expression of platelet-derived growth factor and its receptor in the human placenta. Mol Endocrinol 1988; 2: 627
• 23 Taylor RN, Musci TJ, Rodgers GM, Roberts JM. Preeclamptic sera stimulate increased platelet-derived growth factor mRNA and protein expression by cultured human endothelial cells. Am J Reprod Immunol 1991; 25: 105
• 24 Vince G, Shorter S, Starkey P, Humphreys J, Clover L, Wilkins T, Sargent I, Redman C. Localization of tumor necrosis factor production in cells at the materno/fetal interface in human pregnancy. Clin Exp Immun 1992; 88: 174-180
• 25 Voigt M, Jährig K. Zur Variabilität von Perzentilwerten der Körpermaße Neugeborener. Ärztl Jugendkd 1991; 82: 139
• 26 Wang AM, Doyle M, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 1989; 86: 9717
• 27 Yang Y, Yelavarthi KK, Chen HL, Pace JL, Terranova PF, Hunt JS. Molecular, biochemical and functional characteristics of tumor necrosis factor-alpha produced by human cytotrophoblastic cells. J Immunol 1993; 150: 5615-5624