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Contractile Activity of Human Decidual Stromal Cells. II. Effect of Interleukin-10

Unidad de Inmunología (M.K., O.B., R.M.-F., I.T., A.C.A.-M., E.G.O.), Instituto de Biopatología y Medicina Regenerativa, Hospital Universitario "San Cecilio" Universidad de Granada; and Instituto de Parasitología y Biomedicina (F.M.), Consejo Superior de Investigaciones Cientificas, 18012 Granada, Spain

Address all correspondence and requests for reprints to: Dr. Enrique G. Olivares, Unidad de Inmunología, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Granada, 18012 Granada, Spain. E-mail: engarcia{at}ugr.es.

	Abstract

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Context: Human decidual stromal cells (DSC) are myofibroblast-like cells that express -smooth muscle (-SM) actin, a protein associated with cell contractility. Several lines of experimental evidence in humans and mice show that antiinflammatory cytokines favor normal pregnancy, whereas Th1 and inflammatory cytokines play a role in abortion. We previously demonstrated that IL-2, a Th1 cytokine, increased the contractility of human DSC.

Objective: We studied the effect of the antiinflammatory cytokines IL-10 and IL-4 on the contractility of DSC from first-trimester pregnancy.

Setting and Patients: We studied 10 healthy women who underwent elective vaginal termination of first-trimester pregnancy at Clínica El Sur, Málaga, and Clínica Ginegranada, Granada.

Main Outcome Measure(s): After isolation of DSC, cell contractility was measured with the collagen gel contraction assay. -SM actin was detected with Western blotting and immunofluorescence.

Results: We found that IL-10, but not IL-4, increased the volume of the collagen gel matrixes in which the cytokine-treated DSC were cultured, showing that IL-10 decreased DSC contractility. By Western blotting we demonstrated that this effect was not related to an alteration in the synthesis of -SM actin. Nevertheless, we observed by immunofluorescence microscopy that DSC treated with IL-10 exhibited stress fibers with a lower content of -SM actin than untreated control DSC.

Conclusions: IL-10 relaxes DSC by reducing the incorporation of -SM actin into their stress fibers. This relaxing activity may be of relevance for the maintenance of pregnancy.

	Introduction

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DECIDUAL STROMAL CELLS (DSC) are the main cellular component of the decidua, the maternal constituent of the maternal-fetal interface. These cells comprise a distinctive cell class whose origin and lineage remained unknown until recently. We have demonstrated that human DSC are related to bone marrow stromal precursors and that their morphology, phenotype, and functions are similar to those of myofibroblasts, fibroblastic cells with contractile activity that are involved in wound retraction (1, 2, 3). DSC express -smooth muscle (-SM) actin (1, 2, 3), a microfilament that is considered a marker of myofibroblasts (4), and are able to contract collagen gel matrixes under the effect of TGFß1 and platelet-derived growth factor (3). Furthermore, like myofibroblasts, DSC exhibit immune activities that appear to be relevant in maternal-fetal immune cross-talk (5, 6, 7, 8).

There is increasing evidence that the immune system is involved in normal pregnancy and abortion (9). In mice and humans, normal pregnancy is related to the local and peripheral production of antiinflammatory cytokines (10), whereas abortion is associated with Th1 and inflammatory cytokine production (10, 11). DSC appear to be involved in this Th1-inflammatory/antiinflammatory balance, because Th1 cytokines such as IL-2 block decidualization (12) and induce DSC contractility, and this may contribute to trophoblast expulsion during spontaneous abortion (3). Here we studied the effect of antiinflammatory cytokines, which favor normal pregnancy (10), on DSC contractility.

	Patients and Methods

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Tissues

Ten samples from elective vaginal terminations of first-trimester pregnancy (6–11 wk) from healthy patients, aged 20–30 yr, were used. We excluded women receiving any medication or with infectious, autoimmune, or other systemic or local diseases. None of the abortions was pharmacologically induced. The specimens were obtained by vaginal curettage at the Clínica El Sur, Málaga, and Clínica Ginegranada, Granada. Informed consent was obtained from each patient. This study was approved by the research and ethics committee of the Hospital Universitario de San Cecilio.

Fibroblast medium

According to the information provided by the manufacturer (Sigma-Aldrich, St. Louis, MO), fibroblast medium consists of fibroblast basal medium (a modified version of the culture medium MCDB 105) and a supplement containing fetal calf serum (FCS) (2%, final concentration) and unspecified amounts of basic fibroblast growth factor, heparin, epidermal growth factor, and hydrocortisone.

Isolation and culture of DSC

Decidual tissues were examined histologically to exclude the presence of infection or inflammatory infiltration. Samples of decidua from different patients were not pooled so as to avoid the induction of cytokine secretion as a result of the allogeneic reaction of leukocytes that initially contaminate DSC cultures. Tissues were thoroughly washed in PBS solution, and the decidua was carefully freed from the trophoblast. Decidual fragments were finely minced between two scalpels in a small volume of RPMI 1640 medium with 100 U/ml penicillin and 50 µg/ml gentamicin and put in a solution of 0.5% trypsin and 0.2% EDTA (Sigma-Aldrich) for 15 min at 37 C. The proteolytic reaction of trypsin was stopped by adding cold RPMI with 20% FCS (Life Technologies, Inc., Paisley, UK); the suspension was then filtered through gauze and centrifuged at 425 x g for 10 min. The supernatant was discarded and the cell pellet was suspended in RPMI and centrifuged on Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) for 20 min at 600 x g. Cells were collected from the interface, suspended in PBS, and washed. This suspension was incubated in culture flasks for 1 h in complete RPMI with 10% FCS to allow macrophages and granulocytes to adhere to the flask. The supernatant was washed and incubated in fibroblast medium with 100 U/ml penicillin and 50 µg/ml gentamicin. After overnight incubation to allow DSC to adhere to the flask, nonadherent cells in the supernatant were discarded. The remaining adherent cells were mainly DSC. Fibroblast medium was then replaced and changed twice a week, and after 2–4 wk, adherent cells covered the whole surface of the 25-cm² culture flask. Proliferating DSC overgrew other possible contaminant cells, thus further guaranteeing the purity of the cultures. Purity was further confirmed using flow cytometry to detect the coexpression of CD10 and CD13 and the lack of CD45 antigens by 95–100% of DSC (1, 2, 3, 5). In fibroblast medium, cells proliferated for 8–12 wk; during this period their antigen phenotype was stable (2).

Reagents

IL-10 and IL-4 were purchased from Sigma-Aldrich. The neutralizing antihuman IL-10 monoclonal antibody (JES3-19F1) and rat IgG2a (R35-95) were purchased from PharMingen (BD Biosciences, Erembodegem, Belgium).

Collagen gel contraction assay

Cellular collagen gel contraction assays were performed as previously described (13). A sterile solution of purified, pepsin-solubilized bovine dermal collagen (Vitrogen; Cohesion Technologies, Inc., Palo Alto, CA) was prepared according to the manufacturer’s instructions and combined with 25 x 10⁴ DSC. The collagen/cell mixture (100 µl/well) was dispensed into culture plates and allowed to polymerize at 37 C for 30 min. Immediately after polymerization, 2 ml fibroblast medium with or without the appropriate cytokine was added to each well. After incubation for 48 h, the height (l) and diameter (d) of each gel were measured with the scale of the fine adjustment knob (for l) and with the scale of the stage micrometer of the microscope (for d), and the volume (V) of each gel was calculated with the following formula: V = 1/24 x x l x (3 x d² + l²).

The mean of the measurements (n = 6) taken at each concentration point was used to estimate gel volume. The data are presented as the volume of gel of cytokine-treated DSC compared with cells cultured in the absence of cytokine.

To demonstrate the specificity of the action of IL-10, the gel contraction assay was carried out as indicated, but a neutralizing anti-IL-10 monoclonal antibody (JES3-19F1) (1, 5, or 10 µg/ml), or control rat IgG2a (10 µg/ml), was added to the wells after polymerization of the gel and incubated for 1 h before the addition of 50 ng/ml IL-10. DSC with the antibody and the cytokine were then incubated for an additional period of 48 h before measuring the heights and diameters of the gels.

Western immunoblotting

Cells were lysed with 1% Nonidet P-40 lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich), resolved by SDS-PAGE (10% polyacrylamide gels, reducing conditions), and electrotransferred to Hybond-P polyvinylidene difluoride membranes (Amersham, Little Chalfont, UK). Membranes were blocked with 5% nonfat milk and probed for 1 h at room temperature with an anti--SM actin monoclonal antibody (Sigma-Aldrich) followed by incubation for 1 h at room temperature with horseradish peroxidase-labeled goat antimouse antibody (Caltag, Burlingame, CA). The blot was developed by chemiluminescence (ECL; Amersham) and exposed to autoradiographs (Amersham). Loading controls were carried out by rehybridization of stripped membranes with an anti-ERK polyclonal antibody (anti-MAP kinase 1/2; Upstate Biotechnology, Milton Keynes, UK).

Immunofluorescence microscopy

DSC were plated onto slides in fibroblast medium. After 48 h, cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at 4 C, and permeabilized with cold acetone for 10 min before the addition of fluoresceinated anti--SM actin monoclonal antibody (Sigma-Aldrich) and tetramethylrhodamine-isothiocyanate-labeled phalloidin (Molecular Probes, Eugene, OR). Preparations were examined with a Leica confocal microscope (Leica Microsystems, Wetzlar, Germany).

IL-10 assay

Concentrations of IL-10 in the supernatants of the DSC cultures were determined by a commercially available enzyme immunoassay (R&D, Minneapolis, MN). The assay is sensitive to 3.9 pg/ml IL-10. Intra- and interassay coefficients of variation are less than 6.6 and less than 7.6%, respectively. The assay was performed according to the manufacturer’s instructions, and all samples were determined in duplicate.

Statistical analysis

Each experiment was made three to five times, and the figures illustrate representative results from a specific experiment (see Figs. 2–5). Gel volumes were compared with the Student’s t test. A P value of 0.05 was considered statistically significant (see Figs. 2 and 3).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effect of IL-10 and IL-4 on the volume of collagen gel matrixes containing DSC. DSC were cultured in collagen gel matrixes for 48 h in the absence of cytokines or in the presence of increasing amounts of IL-10 or IL-4. The mean number of measurements (n = 6) taken at each concentration bar was used to estimate gel volume. The data are presented as volume expressed in arbitrary units ± SD. *, P < 10–4; **, P < 10–9 vs. untreated control DSC. IL-10, but not IL-4, significantly increased the volume of the collagen gel matrixes. Results are shown from one representative experiment of five.

View larger version (21K): [in this window] [in a new window]   FIG. 3. Blocking activity of the neutralizing antihuman IL-10 monoclonal antibody (JES3-19F1) on the effect of IL-10. Decidual stromal cells were cultured in collagen gel matrixes for 48 h in the absence or in the presence of 50 ng/ml IL-10, without or with increasing amounts (in parentheses; 1, 5, or 10 µg/ml) of the anti-IL-10 antibody. Rat IgG2a (R35-95) (10 µg/ml) was used as control. The mean number of measurements (n = 6) taken at each concentration bar was used to estimate gel volume. The data are presented as volume expressed in arbitrary units ± SD. *, P < 5 x 10–3 vs. DSC incubated with IL-10 and control IgG2a. The anti-IL-10 antibody significantly reduced the increase in volume of the gel matrixes induced by IL-10. This antibody also significantly reduced the volume of the gel matrixes containing DSC cultured without IL-10 (P < 5 x 10–2; significance not shown). Results are shown from one representative experiment of three.

View larger version (16K): [in this window] [in a new window]   FIG. 4. Detection of -SM actin in DSC cultured without or with IL-10 for 48 h. Peripheral blood mononuclear cells (PBMC) and the tumor cell line JEG-3 were used as negative controls for the expression of -SM actin. MAPK ERK was used as the loading control. Results are shown from one representative experiment of three.

View larger version (40K): [in this window] [in a new window]   FIG. 5. Immunofluorescence detection of -SM actin in the cytoplasm of DSC. -SM actin was detected with a fluoresceinated monoclonal antibody. Stress fibers were detected by staining with tetramethylrhodamine-isothiocyanate-labeled phalloidin (red). A, Untreated DSC revealed the presence of -SM actin in the stress fibers (yellow, colocalization with phalloidin). B, DSC cultured with 50 ng/ml IL-10 for 48 h (B) showed diffuse staining with anti--SM actin (green) and almost no colocalization with palloidin in the stress fibers. Results are shown from one representative experiment of three.

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

DSC were cultured in collagen gel matrixes (Fig. 1). In these matrixes, the state of contraction of DSC determines gel volume (3, 13). IL-10 increased the volume in a dose-response manner, but IL-4 did not have any significant effect on the gel matrixes (Fig. 2). These results demonstrated that IL-10, but not IL-4, relaxed cultured DSC. The specificity of the effect of IL-10 on DSC was confirmed by blocking this effect with a neutralizing anti-IL-10 antibody. This antibody significantly reduced or even abrogated the increase in volume of the gel matrixes induced by IL-10, whereas the control rat IgG2a showed no blocking effect. The anti-IL-10 antibody also significantly reduced the volume of the gel matrix of DSC cultured in the absence of IL-10 (Fig. 3).

View larger version (87K): [in this window] [in a new window]   FIG. 1. Collagen gel matrix containing DSC (x40 magnification).

IL-10 decreases the presence of -SM actin in DSC stress fibers

The relaxing effect of IL-10 on DSC did not depend on inhibition of -SM actin synthesis, because Western blot results showed that IL-10 did not affect the amount of -SM actin in these cells (Fig. 4). Nevertheless, we observed by immunofluorescence microscopy that IL-10 decreased the presence of -SM actin in the stress fibers (Fig. 5). This effect appears to be related to earlier findings in myofibroblasts, in which an increase in contractility was related to the incorporation of -SM actin in stress fibers (14, 15). Thus, the contraction or relaxation of myofibroblasts or myofibroblast-like cells appears to be associated with the increase or decrease, respectively, in the incorporation of -SM actin in the stress fibers.

Cultured DSC secrete IL-10

We detected IL-10 in supernatants collected from the culture medium of all the different DSC lines (Fig. 6). This showed that our cultured DSC secreted IL-10. This endogenous IL-10 may contribute to the relaxation of DSC. The fact that the anti-IL-10 antibody added to DSC cultured without exogenous IL-10 significantly reduced the volume of the gel matrixes suggests that the antibody blocked the effect of endogenous IL-10 (Fig. 3).

View larger version (12K): [in this window] [in a new window]   FIG. 6. Concentrations of IL-10 in the supernatants of six different DSC lines.

	Discussion

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The expression of -SM actin by DSC and their ability to contract led us to conclude that these cells were a form of myofibroblast (1, 3). The contractility of myofibroblasts appears to be related to the incorporation of -SM actin in stress fibers (15). In this connection, the relaxing effect of IL-10 (Fig. 2) seems to be related to a decrease in -SM actin in stress fibers of DSC (Fig. 5). Progesterone, a hormone that favors pregnancy, up-regulates the production of Th2 cytokines by lymphocytes (19) and inhibits the production of Th1 cytokines (20). It also differentiates DSC to a progestation status and blocks their immune activities (16, 17). In fact, we have observed that progesterone also relaxes DSC cultured in collagen cell matrixes (in preparation), although this may be an indirect effect through the induction of IL-10 secretion by DSC (21).

The trophoblast is the main source of IL-10 in the maternal-fetal interface (22), although decidual leukocytes (23, 24) or DSC (21) can also produce this cytokine, and in our system, DSC secreted IL-10 in the culture medium (Fig. 6). Therefore, this cytokine may have a paracrine/autocrine effect on the relaxation of DSC. IL-10 has been shown to restore pregnancy in mice programmed to acquire inflammatory immunity as a result of intrinsic immune polarization (25) and also to prevent lipopolysaccharide-induced preterm delivery (26). In humans, a deficiency of placental IL-10 occurs in preeclampsia (27), and a decrease of decidual IL-10 was observed in missed abortion (28). This cytokine increases the resistance of trophoblast cells to Fas-mediated apoptosis (29) and induces the trophoblast expression of human leukocyte antigen G, a major histocompatibility complex Ib molecule involved in maternal tolerance to the fetus (30). Our results, together with these findings, show that IL-10 is a relevant molecule in the mechanism of maternal-fetal tolerance. Nevertheless, observations in pregnant IL-10 null mutant (IL-10–/–) mice showed that this molecule is a determinant of the growth trajectory of the progeny in utero and after birth, rather than of the success of pregnancy (31). Like IL-10, the null mutation of human leukocyte antigen G, a molecule that also appears to be essential for maternal-fetal tolerance in humans, was associated with successful pregnancies (32). These apparent contradictions may be explained if we take into account the high rate of redundancy in the actions of immune system molecules. On the other hand, reproduction is such an important function for the survival of the species that it may not depend on a single immunological molecule; many molecules may redundantly contribute to this function. One of these molecules is probably IL-10.

	Acknowledgments

 
We are grateful to Dr. S. Jordán from the Clínica el Sur (Málaga) and Dr. F. Garcia Gallego from the Clínica Ginegranada (Granada) for providing us with decidual specimens. We thank K. Shashok for improving our use of English in the manuscript.

	Footnotes

 
This work was funded by the following grants from the Ministerio de Ciencia y Tecnología, Spain: SAF2003-06799, SAS PFI 3/02, and SAS 195/2002.

First Published Online August 16, 2005

Abbreviations: DSC, Decidual stromal cells; FCS, fetal calf serum; -SM, -smooth muscle.

Received January 11, 2005.

Accepted August 5, 2005.

	References

Top Abstract Introduction Patients and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kimatrai, M. Articles by Olivares, E. G. Search for Related Content PubMed PubMed Citation Articles by Kimatrai, M. Articles by Olivares, E. G. Pubmed/NCBI databases Substance via MeSH Related Collections Female Endocrinology