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Effects of urocortin 2 and urocortin 3 on IL-10 and TNF-α expression and secretion from human trophoblast explants
Placenta 32 (2011) 969e974
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Effects of urocortin 2 and urocortin 3 on IL-10 and TNF-a expression and secretion from human trophoblast explants R. Novembri, M. Torricelli, E. Bloise, N. Conti, L.R. Galeazzi, F.M. Severi, F. Petraglia* Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico “Le Scotte”, Viale Bracci, 53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 21 September 2011
Objectives: The aim of the present study was to evaluate the effect of Ucn2 and Ucn3 on cytokine expression and secretion from placental explants. Study design: Placentas were collected from healthy pregnancies at term elective caesarean delivery and trophoblast explants were prepared and treated with Ucn2 or Ucn3 in presence/absence of the selective CRH-R2 antagonist, astressin 2b. The mRNA expression and secretion of IL-10 and TNF-a were evaluated by Real Time RT-PCR and ELISA, respectively. Main outcome measures: To evaluate the possible role of Ucn2 and Ucn3 in inflammatory pathways. Results: Ucn2 increased the mRNA expression and secretion of IL-10 and TNF-a, and Ucn3 increased the mRNA expression and secretion of IL-10, but did not modify the secretion of TNF-a. Ucn3 treatment reversed the LPS-induce increase of TNF-a expression and release, an effect blocked by astressin 2b. Ucn2 potentiated the LPS-induced increase of TNF-a expression and release, an effect reversed by astressin 2b. Conclusions: The present study showed that Ucn2 and Ucn3 differentially regulate the LPS-induced TNFa and IL-10 expression and secretion in trophoblast explants acting through CRH-R2. A pro inflammatory effect of Ucn2 and an anti-inflammatory effect of Ucn3 in placental immunomodulatory mechanisms is suggested. Published by Elsevier Ltd.
Keywords: Urocortin 2 Urocortin 3 TNF-a IL-10 Cytokines Inflammation Placenta
1. Introduction Inflammation is a critical homeostatic event which occurs in pregnant tissues (trophoblast, membranes, uterus) and has an impact in some pathological conditions as preterm delivery (PTD) and preeclampsia (PE) [1e5]. Cytokines are involved in a sequence of pro inflammatory mechanisms on uterine tissues and trophoblast vasculature promoting the development of PTD or PE [1e5]. Anti inflammatory cytokines and chemokines are also produced to counteract the inflammatory mechanism. In this context, several studies investigated the role of placental corticotrophin releasing hormone (CRH) and urocortins (Ucns) in PTD and PE suggesting their implication in the mechanisms leading these two pathologies [6e9]. Urocortins (Ucn, Ucn2 and Ucn3) share sequence homology with CRH [10e12] in detail: Ucn share 43% of homology to human CRH, Ucn2 34% and Ucn3 32% [10e12]. Despite the relative homologies in amino acid sequences, Ucns exert their effects via two known receptors CRH-type 1 (CRH-R1) and -type 2 (CRH-R2); Ucns bind these two receptors with different
affinity, and the binding activates different intracellular signaling cascades [13]. Ucn is involved in many biological functions during pregnancy [14] acting on ACTH [15], prostaglandins [16], uterine contractility [16] and as an endogenous immunomodulatory factor [17]. In our previous study we found that Ucn acts as antinflammatory agent in placental cultures stimulating IL-4 and IL-10 secretion and reversing LPS- induced TNF-a release from trophoblast cells through action on CRH-R2 receptors [17]. Ucn2 and Ucn3 represent specific ligands for CRH-R2 [13]. These neuropeptides and their receptors are expressed by trophoblast and fetal membranes [17e19] but their biological role in these tissues is unclear. However it is well known that Ucn2 stimulates estradiol secretion from trophoblast cultures [20] and both, Ucn2 and Ucn3 are activated by hypoxic events [9]. The aim of the present study was to evaluate the effect of Ucn2 and Ucn3 on cytokine expression and secretion from villous tissues. 2. Materials and methods 2.1. Samples collection
* Corresponding author. Tel.: þ39 0577 233 453; fax: þ39 0577 233 454. E-mail address: [email protected] (F. Petraglia). 0143-4004/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.placenta.2011.09.013
Placentas were collected from healthy pregnant women at term (>37 weeks of gestation), with singleton pregnancy, after uncomplicated elective caesarean
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delivery in absence of labor (n ¼ 10) in the Division of Obstetrics and Gynecology, University of Siena (Siena, Italy). The excluision criteria were fetal-maternal complications such as: thyroid disease, asthma, cardiovascular diseases, diabetes, hypertension, preeclampsia, fetal growth retardation, fetal malformation. An informed written consent from all pregnant women before inclusion in the study and an approval from the Local Human Investigation Committee were obtained. Immediately after collection, samples were put in physiological solution and processed. 2.2. Villous tissues Villous tissues were extensively washed and dissected under sterile conditions in ice-cold Hanks’ Balanced Salt Solution (HBSS) supplemented with penicillin and streptomycin. The explants (50 mg/wet weight) were placed in 24 well plastic plates and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 IU/mL penicillin and 100 mg/mL streptomycin at 37 C under 5% CO2 and 95% air. Cultured medium was replaced with fresh DMEM and the day after the tissues were treated. 2.3. Ucns treatments After 30 min of incubation in DMEM without FBS and antibiotics, the medium was changed and villous tissues were treated with: (i) vehicle e dimethyl sulphoxide (DMSO) (Euroclone, Milan, Italy) as control; (ii) Ucn2 (at 106, 107, 108 M) or iiii) Ucn3 (at 106, 107, 108 M) in presence or absence of the CRH-R2 selective antagonist, astressin 2b (Ast2b) (107 M) for 24 h. All these peptides were kindly donated by Dr W Vale e Salk Institute, La Jolla, CA, USA. The concentration used were chosen on the basis of data present in literature [17]. The study was replicated 5 times and each treatment was performed in triplicate. Ucn2 and Ucn3 were also tested in presence of an inflammatory stimulus (LPS). 2.4. LPS treatment With the purpose to investigate Ucn2 and Ucn3 in presence of a pro inflammatory stimulus, LPS from Escherichia coli serotype 0111:B4 (SigmaeAldrich) was used in three concentrations (10,100, and 1000 ng/mL) known to induce increased cytokines secretion by primary trophoblast culture cells or explants [21]. Given that LPS in the three concentrations analyzed was able to induce cytokines secretion, an intermediate concentration of LPS at 100 ng/mL LPS was chosen in the subsequent experiments. Then 30 min after LPS 100 ng/mL treatment, villous tissues were incubated with Ucn2 or Ucn3 (107 M) for 24 h. Treatments in presence of CRH-R2 selective receptor antagonist, Astressin2b (Ast2b) was performed in the same way but an initial incubation with Ast2b for 30 min was performed before Ucns treatments. Supernatants and specimens were collected and kept frozen at 20 C and at 80 in aliquots respectively until use. 2.5. RNA extraction and cDNA preparation Frozen samples were disrupted and homogenized using Mixer Mill MM 300 (Quiagen, Milan, Italy), and total RNA was extracted with RNeasy Protect Mini Kit and then treated with RNase-free DNase according to the instructions of the manufacturer (RNase protect Mini Kit Qiagen, Hilden, Germany). RNA was quantified by UV absorption (OD260) using Nanodrop (Celbio, Milan, Italy), and RNA purity was determined from the OD260:OD280. The purified RNA was stored at 80 C until cDNA preparation. About 200 ng of RNA were reverse transcribed to prepare complementary DNA (cDNA). Reversion was carried out in a reaction volume of 20 ml containing 50 mM TriseHCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 5 MM random hexamer primer, 2.7 mM deoxynucleoside triphosphate, and 10 U/Ml SuperScript II reverse transcriptase (all reagents obtained from Invitrogen Life Technologies, Milan, Italy). Negative control for each reaction consisted of retrotrascription performed in the absence of reverse transcriptase enzyme (RT) or in absence of RNA samples (H2ORT). RNA was initially denatured at 85 C for 5 min. The reaction mixture was then added, and RT was performed at 50 C for 40 min. The reaction was stopped by denaturing the enzyme at 85 C for 15 min. 1 ml of cDNA was subsequently subjected to Real-time Polymerase Chain Reaction (RT-PCR). 2.6. Real time Polymerase Chain Reaction Differences in mRNA expression of cytokines were compared by RT-PCR (TaqMan PCR, Applied Biosystems, Weiterstadt, Germany), using an Opticon 2 thermal cycler (MJ Research, Bio-Rad Laboratories, Waltham, MA). The housekeeping gene 18s (assay identification no.Hs 99999901_s1) was used as internal standard. All samples were run in triplicate on 96-well optical PCR plates (Applied Biosystems), optimized to the universal PCR protocol of the manufacturer, with a TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan probe for TNF-a (assay identification n . Hs99999043_m1), IL-10 (assay identification n Hs00961622_m1) were taken from the commercially available Assays on Demand (Applied
Biosystems). After initial denaturation for 10 min at 95 C, denaturation at the subsequent 40 cycles was performed for 15 s at 95 C, followed by primer annealing and elongation at 60 C for 1 min. The CT method was applied as a comparative method of quantification.
2.7. Cytokines assay Measurement of cytokines in cell culture supernatants was performed by ELISA using a commercially available ELISA kit in accordance with the manufacturer’s instructions. IL-10 (range: 12.5e400 pg/mL) ELISA kits was purchased (Abcam, UK). TNF-a (range: 39.0e250 pg/mL). ELISA kit was obtained (Phoenix Peptides, USA). Results are expressed as pg/mL.
2.8. Statistical analysis All data were assessed for normality of distribution using a computer programme (Prism 4; Graphpad Software, CA, USA). Where the data were normally distributed, differences among three or more groups were analyzed by ANOVA with Tukey’s Multiple Comparision Test. Two groups were analyzed using a t-test. Chisquared test was used to compare proportions. The statistical significance was achieved when P < 0.05.
3. Results 3.1. Modulatory effect of Ucn2 on placental cytokines and on LPS induced TNF-a expression and release Ucn2 at 107 and 106 M significantly increased (p < 0.01) the mRNA expression and secretion of the anti-inflammatory cytokine IL-10 (Fig. 1AeB). Such effect was counteracted by astressin 2b. Additionally, Ucn2 treatment was also able to significantly increase TNF-a expression and secretion (p < 0.001) (Fig. 1CeD) in a dose dependent fashion; also this effect was reversed in presence of Ast2b. The addition of LPS to the culture medium of villous tissues led to increased concentration of IL-10 (Fig. 2A) and TNF-a (Fig. 2B) in the supernatant at all LPS doses investigated (p < 0.001). LPS treatment led also to increased mRNA expression of IL-10 (LPS 10 ng/mL p < 0.05; 100 ng/mL p < 0.01; 1000 ng/mL p < 0.001) and TNF-a (LPS 100 ng/mL p < 0.05; 1000 ng/mL p < 0.01) in villous tissues. Ucn2 treatment modify IL-10 and TNF-a secretion and expression pattern induced by LPS; in detail Ucn2 potentiate LPS effects. IL-10 secretion and expression levels were significantly increased (Fig. 1EeF) compared to control (p < 0.001) and compared to LPS 100 ng/mL alone (p < 0.01). In presence of Ast2b these effects were reversed p < 0.01. The same trend was observed for TNF-a secretion and expression (Fig. 1GeH).
3.2. Modulatory effect of Ucn3 on placental cytokines and on LPS induced cytokines expression and release Ucn3 at 107 and at 106 M significantly increased the mRNA expression and secretion of IL-10 (p < 0.01 and p < 0.001 respectively) (Fig. 3AeB). Such effects were counteracted by astressin 2b (Fig. 3AeB). Conversely, Ucn3 treatment did not modify the expression and secretion of the pro-inflammatory cytokine, TNF-a, at three different concentrations (Fig. 3CeD). The effects induced by LPS alone were previously described. Ucn3 treatment was able to modify expression pattern of IL-10 induced by LPS 100 ng/mL, in detail Ucn3 significantly reduced LPS effects on IL-10 (p < 0.01, compared to LPS alone) and this effect was counteracted by the use of Ast2b (Fig. 3EeF) (p < 0.001). Ucn3 treatment was also able to modify the effect of LPS on TNF-a; in particular the presence of Ucn3 reduced TNF-a secretion and expression in villous tissues but this reduction was not significant. When Ast 2b was added to culture media, these reduction was lack and TNF-a concentration was similar to those observed with LPS only (Fig. 3GeH).
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induces IL-10 and TNF-a secretion in human placenta and we first demonstrated that while Ucn2 potentiates LPS effects, Ucn3 reduces it’s action. Several evidences indicate that cytokines play a central role in the mechanisms of inflammation/infection conditions and pregnancy is considered as a natural inflammatory state characterized by high concentration of cytokines. The anti-inflammatory cytokine
4. Discussion The present study for the first time showed that Ucn 2 and Ucn3 acting on CRH-R2 modulates IL-10 and TNF-a release from villous tissues; Ucn2 stimulates IL-10 and TNF-a while Ucn3 stimulates only IL-10 from villous tissues, suggesting a pro inflammatory role of Ucn2 in this tissue. Additionally, we also confirmed that LPS
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Fig. 1. Effect of Ucn2 on pro and anti-inflammatory cytokines. Ucn2 increased IL-10 (AeB) and TNF-a (XeD) expression and secretion. *p < 0.05; **p < 0.01; ***p < 0.001. Ucn2 potentiates the IL-10 LPS-induced expression and secretion (EeF). Ucn2 potentiates LPS effects on pro inflammatory cytokine and this effect was blocked by the addition of astressin 2b (GeH). *p < 0.05; **p < 0.01; ***p < 0.001.
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IL-10 has been considered a key cytokine for the maintenance of pregnancy since its actions are beneficial and protective [21]. It is well known that high IL-10 production in pre-labor tissues exists [18], and in vitro studies demonstrated that IL-10 inhibits cytokine and PG production by human chorion, decidual and placental cells in vitro [22] suggesting a role in the balance of pro inflammatory versus anti-inflammatory signals that orchestrate proper pregnancy outcomes [23]. On the contrary, TNF-a is involved in the pathogenesis of several gestational disease and in mechanisms leading PTD; in fact TNFa stimulates prostaglandin production by the amnion, decidua and myometrium and cervical ripening [24], additionally elevated concentrations of TNF-a are found in response to bacterial products or intra amniotic infection [25]; Our findings show that Ucn2 increases mRNA expression and secretion of TNF-a and IL-10 and also potentiates the effects mediated by LPS on these cytokines suggesting a pro-inflammatory role of Ucn2. These results fit with previous studies describing a pro inflammatory effect of Ucn2 in several human cell types. The exposure of human colonocytes to Ucn2 increases the expression of the potent chemoattractant IL-8 via nuclear factor-kappaB (NFkappaB) and mitogen-acivated protein (MAP) kinase through the CRH-R2 [26]; moreover, Ucn2 suppresses host resistance to Listeria monocytogenes infection via up-regulation of IL-10 production and finally, increased Ucn2 levels were found in immune colitis infiltrating immune cells [27] and in an animal model of gastrointestinal colitis [28]. The present study for the first time also showed that Ucn3 increased IL-10 expression and secretion and reduced LPS effects on TNF-a and IL-10 secretion suggesting that Ucn3 may acts as an antiinflammatory mediator. Our results are in agreement with previous studies demonstrating that Ucn3 exerts an anti-inflammatory function in human colon during stress conditions [29]. The fact
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that in presence of Ucn3, the LPS-induced IL-10 expression and secretion was reduced, may be explained on the basis that, under pro inflammatory conditions the presence of an exogenous antiinflammatory factor determines a cooperation with endogenous anti-inflammatory molecules acting in a synergic manner [29,30], additionally our observation confirmed previous findings describing a reduced LPS-induced IL-10 secretion in presence of an anti-inflammatory stimulus in human peripheral blood mononuclear cells [31]. Other investigations are necessary in order to better understand and clarify Ucn3 role. Our previous studies conducted in trophoblast cell cultures, showed a main anti-inflammatory role for Ucn mediated by CRHR2: our observations demonstrated that Ucn treatment induced a significant and dose dependent increase of IL-4 and IL-10, while it did not affect TNF-a secretion; additionally, Ucn was also able to reverse LPS-induced TNF-a release in this cell type, an effect that was blocked by the CRH-R2 selective antagonist, astressin 2b [17]. It is very important to consider that Ucn binds both CRH-R1 and CRHR2, even if it binds with high affinity CRH-R2 while Ucn2 and Ucn3 specifically bind only to CRH-R2 [32]. Our findings showed that astressin 2b blocks the effects of Ucn2 and Ucn3 in presence or absence of LPS, suggesting that CRH-R2 is involved in the immunomodulatory effects of Ucns in trophoblast. The evidence that Ucn3 and Ucn act as anti-inflammatory agents while Ucn2 exerts a pro inflammatory role, and the fact that these opposite effects are both mediate by the link with CRH-R2 led us to hypothesize that CRH-R2 activate different signalling pathways (pro or anti-inflammatory), depending on its ligands. Recently we demonstrated that in PTD in presence of infective/ inflammatory conditions such as chorioamnionitis and/or premature rupture of membranes, the expression level of Ucns in placental tissues was modified, in particular while Ucn2 was over expressed in these pathological conditions, Ucn and Ucn3 were
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Fig. 2. Effect of LPS on pro and anti-inflammatory cytokines. LPS increased IL-10 (A) and TNF-a (B) expression and secretion. *p < 0.05; **p < 0.01; ***p < 0.001.
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strongly down regulated [32]. Moreover, previous studies showed different biological effects of Ucn2 and Ucn3 in trophoblast cells: Ucn2 stimulated aromatization of androgen into estradiol output in a time and dose dependent manner increasing estradiol secretion and cytochrome P450 aromatase mRNA levels and protein expression from cultured human trophoblast cells [20,33]. Under hypoxic conditions, the expression of Ucn2 and Ucn3 is increased
suggesting that both peptides may reflect a response to the oxidative stress [9]. On the basis of the present findings and previous studies, we demonstrate that Ucn, Ucn2 and Ucn3 act as immunomodulatory agents in placental cells and this fact led us to speculate that these neuropepides could exert a key role in some gestational disease, in particular at early preterm delivery (PTD).
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Fig. 3. Effect of Ucn3 on pro and anti-inflammatory cytokines. Ucn3 increased expression and secretion of IL-10 (AeB) while they are no able to affect TNF-a (CeD). *p < 0.05; **p < 0.01; ***p < 0.001. Ucn3 reversed both the anti-inflammatory IL-10 (EeF) and the pro inflammatory TNF- a expression and secretion (GeH). The addition of astressin 2b reversed these effects. *p < 0.05; **p < 0.01; ***p < 0.001.
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5. Conclusions In conclusion, the present study showed that Ucns, regulate in a different manner the LPS-induced TNF-a and IL-10 expression and secretion acting through CRH-R2 receptor in villous tissues, supporting that urocortins are immunomodulatory peptides which may be involved in the regulation of placental inflammatory mechanisms and that the signalling pathway activated by CRH-R2 is ligand specific. References [1] Gargano JW, Holzman C, Senagore P, Thorsen P, Skogstrand K, Hougaard DM, et al. Mid-pregnancy circulating cytokine levels, histologic chorioamnionitis and spontaneous preterm birth. J Reprod Immunol 2008;79:100e10. [2] Grigsby PL, Novy MJ, Waldorf KM, Sadowsky DW, Gravett MG. Choriodecidual inflammation: a harbinger of the preterm labor syndrome. Reprod Sci 2010; 17:85e94. [3] Romero R, Erez O, Espinoza J. Intrauterine infection, preterm labor, and cytokines. J Soc Gynecol Investig 2005;12:463e5. [4] von Dadelszen P, Magee LA. Could an infectious trigger explain the differential maternal response to the shared placental pathology of preeclampsia and normotensive intrauterine growth restriction? Acta Obstet Gynecol Scand 2002;81:642e8. [5] Redman CW, Sargent IL. Pre-eclampsia, the placenta and the maternal systemic inflammatory response e a review. Placenta 2003;24:S21e7. [6] Smith R, Nicholson RC. Corticotrophin releasing hormone and the timing of birth. Front Biosci 2007;1(12):912e8. [7] Florio P, Torricelli M, Galleri L, De Falco G, Leucci E, Calonaci G, et al. High fetal urocortin levels at term and preterm labor. J Clin Endocrinol Metab 2005;90: 5361e5. [8] Florio P, Torricelli M, De Falco G, Leucci E, Giovannelli A, Gazzolo D, et al. High maternal and fetal plasma urocortin levels in pregnancies complicated by hypertension. J Hypertens 2006;24:1831e40. [9] Imperatore A, Rolfo A, Petraglia F, Challis JR, Caniggia I. Hypoxia and preeclampsia: increased expression of urocortin 2 and urocortin 3. Reprod Sci 2010;17:833e43. [10] Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, et al. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 1995;378:287e92. [11] Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, et al. Urocortina II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci U S A 2001;98:2843e8. [12] Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, et al. Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci U S A 2001;98:7570e5. [13] Aguilera G, Nikodemova M, Wynn PC, Catt KJ. Corticotropin releasing hormone receptors: two decades later. Peptides 2004;25:319e29. [14] Florio P, Vale W, Petraglia F. Urocortins in human reproduction. Peptides 2004;25:1751e7. [15] Petraglia F, Sawchenko PE, Rivier J, Vale W. Evidence for local stimulation of ACTH secretion by corticotropin-releasing factor in human placenta. Nature 1987;328:717e9.
[16] Petraglia F, Florio P, Benedetto C, Marozio L, Di Blasio AM, Ticconi C, et al. Urocortin stimulates placental adrenocorticotropin and prostaglandin release and myometrial contractility in vitro. J Clin Endocrinol Metab 1999;84: 1420e3. [17] Torricelli M, Voltolini C, Bloise E, Biliotti G, Giovannelli A, De Bonis M, et al. Urocortin increases IL-4 and IL-10 secretion and reverses LPS-induced TNFalpha release from human trophoblast primary cells. Am J Reprod Immunol 2009;62:224e31. [18] Petraglia F, Florio P, Gallo R, Simoncini T, Saviozzi M, Di Blasio AM, et al. Human placenta and fetal membranes express human urocortin mRNA and peptide. J Clin Endocrinol Metab 1996;81:3807e10. [19] Imperatore A, Florio P, Torres PB, Torricelli M, Galleri L, Toti P, et al. Urocortin 2 and urocortin 3 are expressed by the human placenta, deciduas, and fetal membranes. Am J Obstet Gynecol 2006;195:288e95. [20] Imperatore A, Li W, Petraglia F, Challis JR. Urocortin 2 stimulates estradiol secretion from cultured human placental cells: an effect mediated by the type 2 corticotrophin-releasing hormone (CRH) receptor. Reprod Sci 2009;16: 551e8. [21] Dong M, He J, Wang Z, Xie X, Wang H. Placental imbalance of Th1- and Th2type cytokines in preeclampsia. Acta Obstet Gynecol Scand 2005;84:788e93. [22] Fortunato SJ, Menon R, Lombardi SJ. Interleukin-10 and transforming growth factor-beta inhibit amniochorion tumor necrosis factor-alpha production by contrasting mechanisms of action: therapeutic implications in prematurity. Am J Obstet Gynecol 1997;177:803e9. [23] Thaxton JE, Sharma S. Interleukin-10: a multi-faceted agent of pregnancy. Am J Reprod Immunol 2010;63:482e91. [24] Casey ML, Cox SM, Beutler B, Milewich L, MacDonald PC. Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infectioninduced preterm labor. J Clin Invest 1989;83:430e6. [25] Romero R, Mazor M, Manogue K, Oyarzun E, Cerami A. Human decidua: a source of cachectin-tumor necrosis factor. Eur J Obstet Gynecol Reprod Biol 1991;41:123e7. [26] Moss AC, Anton P, Savidge T, Newman P, Cheifetz AS, Gay J, et al. Urocortin II mediates pro-inflammatory effects in human colonocytes via corticotropinreleasing hormone receptor 2alpha. Gut 2007;56:1210e7. [27] Kokkotou E, Torres D, Moss AC, O’Brien M, Grigoriadis DE, Karalis K, et al. Corticotropin-releasing hormone receptor 2-deficient mice have reduced intestinal inflammatory responses. J Immunol 2006;1(177):3355e61. [28] Chang J, Hoy JJ, Idumalla PS, Clifton MS, Pecoraro NC, Bhargava A. Urocortin 2 expression in the rat gastrointestinal tract under basal conditions and in chemical colitis. Peptides 2007;28:1453e60. [29] Saruta M, Takahashi K, Suzuki T, Fukuda T, Torii A, Sasano H. Urocortin 3/ stresscopin in human colon: possible modulators of gastrointestinal function during stressful conditions. Peptides 2005;26:1196e206. [30] Elenkov IJ, Chrousos GP. Stress hormones, pro-inflammatory and antiinflammatory cytokines, and autoimmunity. Ann N Y Acad Sci 2002;966: 290e303. [31] Dong H, Rowland I, Tuohy KM, Thomas LV, Yaqoob P. Selective effects of Lactobacillus casei shirota on T cell activation, natural killer cell activity and cytokine production. Clin Exp Immunol; 2010. [32] Torricelli Michela, Novembri Romina, Bloise Enrrico, De Bonis Maria, Challis John R, Petraglia Felice. Changes in placental CRH, urocortins, and CRHreceptors mRNA expression associated with preterm delivery and chorioamnionitis. J Clinical Endocrinology Metab; 2011. [33] Pepels PP, Spaanderman ME, Hermus AR, Lotgering FK, Sweep CG. Placental urocortin-2 and -3: endocrine or paracrine functioning during healthy pregnancy? Placenta 2010;31:475e81.
Contents lists available at SciVerse ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Effects of urocortin 2 and urocortin 3 on IL-10 and TNF-a expression and secretion from human trophoblast explants R. Novembri, M. Torricelli, E. Bloise, N. Conti, L.R. Galeazzi, F.M. Severi, F. Petraglia* Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Policlinico “Le Scotte”, Viale Bracci, 53100 Siena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 21 September 2011
Objectives: The aim of the present study was to evaluate the effect of Ucn2 and Ucn3 on cytokine expression and secretion from placental explants. Study design: Placentas were collected from healthy pregnancies at term elective caesarean delivery and trophoblast explants were prepared and treated with Ucn2 or Ucn3 in presence/absence of the selective CRH-R2 antagonist, astressin 2b. The mRNA expression and secretion of IL-10 and TNF-a were evaluated by Real Time RT-PCR and ELISA, respectively. Main outcome measures: To evaluate the possible role of Ucn2 and Ucn3 in inflammatory pathways. Results: Ucn2 increased the mRNA expression and secretion of IL-10 and TNF-a, and Ucn3 increased the mRNA expression and secretion of IL-10, but did not modify the secretion of TNF-a. Ucn3 treatment reversed the LPS-induce increase of TNF-a expression and release, an effect blocked by astressin 2b. Ucn2 potentiated the LPS-induced increase of TNF-a expression and release, an effect reversed by astressin 2b. Conclusions: The present study showed that Ucn2 and Ucn3 differentially regulate the LPS-induced TNFa and IL-10 expression and secretion in trophoblast explants acting through CRH-R2. A pro inflammatory effect of Ucn2 and an anti-inflammatory effect of Ucn3 in placental immunomodulatory mechanisms is suggested. Published by Elsevier Ltd.
Keywords: Urocortin 2 Urocortin 3 TNF-a IL-10 Cytokines Inflammation Placenta
1. Introduction Inflammation is a critical homeostatic event which occurs in pregnant tissues (trophoblast, membranes, uterus) and has an impact in some pathological conditions as preterm delivery (PTD) and preeclampsia (PE) [1e5]. Cytokines are involved in a sequence of pro inflammatory mechanisms on uterine tissues and trophoblast vasculature promoting the development of PTD or PE [1e5]. Anti inflammatory cytokines and chemokines are also produced to counteract the inflammatory mechanism. In this context, several studies investigated the role of placental corticotrophin releasing hormone (CRH) and urocortins (Ucns) in PTD and PE suggesting their implication in the mechanisms leading these two pathologies [6e9]. Urocortins (Ucn, Ucn2 and Ucn3) share sequence homology with CRH [10e12] in detail: Ucn share 43% of homology to human CRH, Ucn2 34% and Ucn3 32% [10e12]. Despite the relative homologies in amino acid sequences, Ucns exert their effects via two known receptors CRH-type 1 (CRH-R1) and -type 2 (CRH-R2); Ucns bind these two receptors with different
affinity, and the binding activates different intracellular signaling cascades [13]. Ucn is involved in many biological functions during pregnancy [14] acting on ACTH [15], prostaglandins [16], uterine contractility [16] and as an endogenous immunomodulatory factor [17]. In our previous study we found that Ucn acts as antinflammatory agent in placental cultures stimulating IL-4 and IL-10 secretion and reversing LPS- induced TNF-a release from trophoblast cells through action on CRH-R2 receptors [17]. Ucn2 and Ucn3 represent specific ligands for CRH-R2 [13]. These neuropeptides and their receptors are expressed by trophoblast and fetal membranes [17e19] but their biological role in these tissues is unclear. However it is well known that Ucn2 stimulates estradiol secretion from trophoblast cultures [20] and both, Ucn2 and Ucn3 are activated by hypoxic events [9]. The aim of the present study was to evaluate the effect of Ucn2 and Ucn3 on cytokine expression and secretion from villous tissues. 2. Materials and methods 2.1. Samples collection
* Corresponding author. Tel.: þ39 0577 233 453; fax: þ39 0577 233 454. E-mail address: [email protected] (F. Petraglia). 0143-4004/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.placenta.2011.09.013
Placentas were collected from healthy pregnant women at term (>37 weeks of gestation), with singleton pregnancy, after uncomplicated elective caesarean
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delivery in absence of labor (n ¼ 10) in the Division of Obstetrics and Gynecology, University of Siena (Siena, Italy). The excluision criteria were fetal-maternal complications such as: thyroid disease, asthma, cardiovascular diseases, diabetes, hypertension, preeclampsia, fetal growth retardation, fetal malformation. An informed written consent from all pregnant women before inclusion in the study and an approval from the Local Human Investigation Committee were obtained. Immediately after collection, samples were put in physiological solution and processed. 2.2. Villous tissues Villous tissues were extensively washed and dissected under sterile conditions in ice-cold Hanks’ Balanced Salt Solution (HBSS) supplemented with penicillin and streptomycin. The explants (50 mg/wet weight) were placed in 24 well plastic plates and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 IU/mL penicillin and 100 mg/mL streptomycin at 37 C under 5% CO2 and 95% air. Cultured medium was replaced with fresh DMEM and the day after the tissues were treated. 2.3. Ucns treatments After 30 min of incubation in DMEM without FBS and antibiotics, the medium was changed and villous tissues were treated with: (i) vehicle e dimethyl sulphoxide (DMSO) (Euroclone, Milan, Italy) as control; (ii) Ucn2 (at 106, 107, 108 M) or iiii) Ucn3 (at 106, 107, 108 M) in presence or absence of the CRH-R2 selective antagonist, astressin 2b (Ast2b) (107 M) for 24 h. All these peptides were kindly donated by Dr W Vale e Salk Institute, La Jolla, CA, USA. The concentration used were chosen on the basis of data present in literature [17]. The study was replicated 5 times and each treatment was performed in triplicate. Ucn2 and Ucn3 were also tested in presence of an inflammatory stimulus (LPS). 2.4. LPS treatment With the purpose to investigate Ucn2 and Ucn3 in presence of a pro inflammatory stimulus, LPS from Escherichia coli serotype 0111:B4 (SigmaeAldrich) was used in three concentrations (10,100, and 1000 ng/mL) known to induce increased cytokines secretion by primary trophoblast culture cells or explants [21]. Given that LPS in the three concentrations analyzed was able to induce cytokines secretion, an intermediate concentration of LPS at 100 ng/mL LPS was chosen in the subsequent experiments. Then 30 min after LPS 100 ng/mL treatment, villous tissues were incubated with Ucn2 or Ucn3 (107 M) for 24 h. Treatments in presence of CRH-R2 selective receptor antagonist, Astressin2b (Ast2b) was performed in the same way but an initial incubation with Ast2b for 30 min was performed before Ucns treatments. Supernatants and specimens were collected and kept frozen at 20 C and at 80 in aliquots respectively until use. 2.5. RNA extraction and cDNA preparation Frozen samples were disrupted and homogenized using Mixer Mill MM 300 (Quiagen, Milan, Italy), and total RNA was extracted with RNeasy Protect Mini Kit and then treated with RNase-free DNase according to the instructions of the manufacturer (RNase protect Mini Kit Qiagen, Hilden, Germany). RNA was quantified by UV absorption (OD260) using Nanodrop (Celbio, Milan, Italy), and RNA purity was determined from the OD260:OD280. The purified RNA was stored at 80 C until cDNA preparation. About 200 ng of RNA were reverse transcribed to prepare complementary DNA (cDNA). Reversion was carried out in a reaction volume of 20 ml containing 50 mM TriseHCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 5 MM random hexamer primer, 2.7 mM deoxynucleoside triphosphate, and 10 U/Ml SuperScript II reverse transcriptase (all reagents obtained from Invitrogen Life Technologies, Milan, Italy). Negative control for each reaction consisted of retrotrascription performed in the absence of reverse transcriptase enzyme (RT) or in absence of RNA samples (H2ORT). RNA was initially denatured at 85 C for 5 min. The reaction mixture was then added, and RT was performed at 50 C for 40 min. The reaction was stopped by denaturing the enzyme at 85 C for 15 min. 1 ml of cDNA was subsequently subjected to Real-time Polymerase Chain Reaction (RT-PCR). 2.6. Real time Polymerase Chain Reaction Differences in mRNA expression of cytokines were compared by RT-PCR (TaqMan PCR, Applied Biosystems, Weiterstadt, Germany), using an Opticon 2 thermal cycler (MJ Research, Bio-Rad Laboratories, Waltham, MA). The housekeeping gene 18s (assay identification no.Hs 99999901_s1) was used as internal standard. All samples were run in triplicate on 96-well optical PCR plates (Applied Biosystems), optimized to the universal PCR protocol of the manufacturer, with a TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan probe for TNF-a (assay identification n . Hs99999043_m1), IL-10 (assay identification n Hs00961622_m1) were taken from the commercially available Assays on Demand (Applied
Biosystems). After initial denaturation for 10 min at 95 C, denaturation at the subsequent 40 cycles was performed for 15 s at 95 C, followed by primer annealing and elongation at 60 C for 1 min. The CT method was applied as a comparative method of quantification.
2.7. Cytokines assay Measurement of cytokines in cell culture supernatants was performed by ELISA using a commercially available ELISA kit in accordance with the manufacturer’s instructions. IL-10 (range: 12.5e400 pg/mL) ELISA kits was purchased (Abcam, UK). TNF-a (range: 39.0e250 pg/mL). ELISA kit was obtained (Phoenix Peptides, USA). Results are expressed as pg/mL.
2.8. Statistical analysis All data were assessed for normality of distribution using a computer programme (Prism 4; Graphpad Software, CA, USA). Where the data were normally distributed, differences among three or more groups were analyzed by ANOVA with Tukey’s Multiple Comparision Test. Two groups were analyzed using a t-test. Chisquared test was used to compare proportions. The statistical significance was achieved when P < 0.05.
3. Results 3.1. Modulatory effect of Ucn2 on placental cytokines and on LPS induced TNF-a expression and release Ucn2 at 107 and 106 M significantly increased (p < 0.01) the mRNA expression and secretion of the anti-inflammatory cytokine IL-10 (Fig. 1AeB). Such effect was counteracted by astressin 2b. Additionally, Ucn2 treatment was also able to significantly increase TNF-a expression and secretion (p < 0.001) (Fig. 1CeD) in a dose dependent fashion; also this effect was reversed in presence of Ast2b. The addition of LPS to the culture medium of villous tissues led to increased concentration of IL-10 (Fig. 2A) and TNF-a (Fig. 2B) in the supernatant at all LPS doses investigated (p < 0.001). LPS treatment led also to increased mRNA expression of IL-10 (LPS 10 ng/mL p < 0.05; 100 ng/mL p < 0.01; 1000 ng/mL p < 0.001) and TNF-a (LPS 100 ng/mL p < 0.05; 1000 ng/mL p < 0.01) in villous tissues. Ucn2 treatment modify IL-10 and TNF-a secretion and expression pattern induced by LPS; in detail Ucn2 potentiate LPS effects. IL-10 secretion and expression levels were significantly increased (Fig. 1EeF) compared to control (p < 0.001) and compared to LPS 100 ng/mL alone (p < 0.01). In presence of Ast2b these effects were reversed p < 0.01. The same trend was observed for TNF-a secretion and expression (Fig. 1GeH).
3.2. Modulatory effect of Ucn3 on placental cytokines and on LPS induced cytokines expression and release Ucn3 at 107 and at 106 M significantly increased the mRNA expression and secretion of IL-10 (p < 0.01 and p < 0.001 respectively) (Fig. 3AeB). Such effects were counteracted by astressin 2b (Fig. 3AeB). Conversely, Ucn3 treatment did not modify the expression and secretion of the pro-inflammatory cytokine, TNF-a, at three different concentrations (Fig. 3CeD). The effects induced by LPS alone were previously described. Ucn3 treatment was able to modify expression pattern of IL-10 induced by LPS 100 ng/mL, in detail Ucn3 significantly reduced LPS effects on IL-10 (p < 0.01, compared to LPS alone) and this effect was counteracted by the use of Ast2b (Fig. 3EeF) (p < 0.001). Ucn3 treatment was also able to modify the effect of LPS on TNF-a; in particular the presence of Ucn3 reduced TNF-a secretion and expression in villous tissues but this reduction was not significant. When Ast 2b was added to culture media, these reduction was lack and TNF-a concentration was similar to those observed with LPS only (Fig. 3GeH).
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induces IL-10 and TNF-a secretion in human placenta and we first demonstrated that while Ucn2 potentiates LPS effects, Ucn3 reduces it’s action. Several evidences indicate that cytokines play a central role in the mechanisms of inflammation/infection conditions and pregnancy is considered as a natural inflammatory state characterized by high concentration of cytokines. The anti-inflammatory cytokine
4. Discussion The present study for the first time showed that Ucn 2 and Ucn3 acting on CRH-R2 modulates IL-10 and TNF-a release from villous tissues; Ucn2 stimulates IL-10 and TNF-a while Ucn3 stimulates only IL-10 from villous tissues, suggesting a pro inflammatory role of Ucn2 in this tissue. Additionally, we also confirmed that LPS
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Fig. 1. Effect of Ucn2 on pro and anti-inflammatory cytokines. Ucn2 increased IL-10 (AeB) and TNF-a (XeD) expression and secretion. *p < 0.05; **p < 0.01; ***p < 0.001. Ucn2 potentiates the IL-10 LPS-induced expression and secretion (EeF). Ucn2 potentiates LPS effects on pro inflammatory cytokine and this effect was blocked by the addition of astressin 2b (GeH). *p < 0.05; **p < 0.01; ***p < 0.001.
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IL-10 has been considered a key cytokine for the maintenance of pregnancy since its actions are beneficial and protective [21]. It is well known that high IL-10 production in pre-labor tissues exists [18], and in vitro studies demonstrated that IL-10 inhibits cytokine and PG production by human chorion, decidual and placental cells in vitro [22] suggesting a role in the balance of pro inflammatory versus anti-inflammatory signals that orchestrate proper pregnancy outcomes [23]. On the contrary, TNF-a is involved in the pathogenesis of several gestational disease and in mechanisms leading PTD; in fact TNFa stimulates prostaglandin production by the amnion, decidua and myometrium and cervical ripening [24], additionally elevated concentrations of TNF-a are found in response to bacterial products or intra amniotic infection [25]; Our findings show that Ucn2 increases mRNA expression and secretion of TNF-a and IL-10 and also potentiates the effects mediated by LPS on these cytokines suggesting a pro-inflammatory role of Ucn2. These results fit with previous studies describing a pro inflammatory effect of Ucn2 in several human cell types. The exposure of human colonocytes to Ucn2 increases the expression of the potent chemoattractant IL-8 via nuclear factor-kappaB (NFkappaB) and mitogen-acivated protein (MAP) kinase through the CRH-R2 [26]; moreover, Ucn2 suppresses host resistance to Listeria monocytogenes infection via up-regulation of IL-10 production and finally, increased Ucn2 levels were found in immune colitis infiltrating immune cells [27] and in an animal model of gastrointestinal colitis [28]. The present study for the first time also showed that Ucn3 increased IL-10 expression and secretion and reduced LPS effects on TNF-a and IL-10 secretion suggesting that Ucn3 may acts as an antiinflammatory mediator. Our results are in agreement with previous studies demonstrating that Ucn3 exerts an anti-inflammatory function in human colon during stress conditions [29]. The fact
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that in presence of Ucn3, the LPS-induced IL-10 expression and secretion was reduced, may be explained on the basis that, under pro inflammatory conditions the presence of an exogenous antiinflammatory factor determines a cooperation with endogenous anti-inflammatory molecules acting in a synergic manner [29,30], additionally our observation confirmed previous findings describing a reduced LPS-induced IL-10 secretion in presence of an anti-inflammatory stimulus in human peripheral blood mononuclear cells [31]. Other investigations are necessary in order to better understand and clarify Ucn3 role. Our previous studies conducted in trophoblast cell cultures, showed a main anti-inflammatory role for Ucn mediated by CRHR2: our observations demonstrated that Ucn treatment induced a significant and dose dependent increase of IL-4 and IL-10, while it did not affect TNF-a secretion; additionally, Ucn was also able to reverse LPS-induced TNF-a release in this cell type, an effect that was blocked by the CRH-R2 selective antagonist, astressin 2b [17]. It is very important to consider that Ucn binds both CRH-R1 and CRHR2, even if it binds with high affinity CRH-R2 while Ucn2 and Ucn3 specifically bind only to CRH-R2 [32]. Our findings showed that astressin 2b blocks the effects of Ucn2 and Ucn3 in presence or absence of LPS, suggesting that CRH-R2 is involved in the immunomodulatory effects of Ucns in trophoblast. The evidence that Ucn3 and Ucn act as anti-inflammatory agents while Ucn2 exerts a pro inflammatory role, and the fact that these opposite effects are both mediate by the link with CRH-R2 led us to hypothesize that CRH-R2 activate different signalling pathways (pro or anti-inflammatory), depending on its ligands. Recently we demonstrated that in PTD in presence of infective/ inflammatory conditions such as chorioamnionitis and/or premature rupture of membranes, the expression level of Ucns in placental tissues was modified, in particular while Ucn2 was over expressed in these pathological conditions, Ucn and Ucn3 were
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strongly down regulated [32]. Moreover, previous studies showed different biological effects of Ucn2 and Ucn3 in trophoblast cells: Ucn2 stimulated aromatization of androgen into estradiol output in a time and dose dependent manner increasing estradiol secretion and cytochrome P450 aromatase mRNA levels and protein expression from cultured human trophoblast cells [20,33]. Under hypoxic conditions, the expression of Ucn2 and Ucn3 is increased
suggesting that both peptides may reflect a response to the oxidative stress [9]. On the basis of the present findings and previous studies, we demonstrate that Ucn, Ucn2 and Ucn3 act as immunomodulatory agents in placental cells and this fact led us to speculate that these neuropepides could exert a key role in some gestational disease, in particular at early preterm delivery (PTD).
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Fig. 3. Effect of Ucn3 on pro and anti-inflammatory cytokines. Ucn3 increased expression and secretion of IL-10 (AeB) while they are no able to affect TNF-a (CeD). *p < 0.05; **p < 0.01; ***p < 0.001. Ucn3 reversed both the anti-inflammatory IL-10 (EeF) and the pro inflammatory TNF- a expression and secretion (GeH). The addition of astressin 2b reversed these effects. *p < 0.05; **p < 0.01; ***p < 0.001.
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