Chemerin Regulates NK Cell Accumulation and Endothelial Cell Morphogenesis in the Decidua during Early Pregnancy

Claudia Carlino; Eleonora Trotta; Helena Stabile; Stefania Morrone; Roberta Bulla; Alessandra Soriani; Maria Luisa Iannitto; Chiara Agostinis; Carlo Mocci; Massimo Minozzi; Cesare Aragona; Giorgia Perniola; Francesco Tedesco; Silvano Sozzani; Angela Santoni; Angela Gismondi

doi:10.1210/jc.2012-1102

. 2012 Jul 12;97(10):3603–3612. doi: 10.1210/jc.2012-1102

Chemerin Regulates NK Cell Accumulation and Endothelial Cell Morphogenesis in the Decidua during Early Pregnancy

Claudia Carlino ^1,^*, Eleonora Trotta ^1,^*, Helena Stabile ¹, Stefania Morrone ¹, Roberta Bulla ¹, Alessandra Soriani ¹, Maria Luisa Iannitto ¹, Chiara Agostinis ¹, Carlo Mocci ¹, Massimo Minozzi ¹, Cesare Aragona ¹, Giorgia Perniola ¹, Francesco Tedesco ¹, Silvano Sozzani ¹, Angela Santoni ^1,^✉, Angela Gismondi ^1,^✉

PMCID: PMC3462933 PMID: 22791765

Abstract

Context:

Although decidual natural killer (NK) cell accumulation and vascular remodeling are critical steps to ensure successful pregnancy, the molecular mechanisms controlling these events are poorly defined.

Objective:

Herein we analyzed whether chemerin, a recently identified chemoattractant involved in many pathophysiological processes, could be expressed in the uterine compartment and could regulate events relevant for the good outcome of pregnancy.

Design:

Chemerin expression in human primary culture of stromal (ST) cells, extravillous trophoblast cells, and decidual endothelial cells (DEC) was analyzed by RT-PCR, ELISA, and Western blot. Migration through ST or DEC of peripheral blood and decidual (d) NK cells from pregnant women was performed using a transwell assay. A DEC capillary-like tube formation assay was used to evaluate endothelial morphogenesis.

Results:

Chemerin is differentially expressed by decidual cells during early pregnancy being present at high levels in ST and extravillous trophoblast cells but not in DEC. Notably, ST cells from pregnant women exhibit and release higher levels of chemerin as compared with ST cells from menopausal or fertile nonpregnant women. Chemerin can support peripheral blood NK cell migration through both DEC and ST cells. Although dNK cells exhibit lower chemerin receptor (CMKLR1) expression than their blood counterpart, CMKLR1 engagement on dNK cells resulted in both ERK activation and migration through decidual ST cells. Interestingly, DEC also express CMKLR1 and undergo ERK activation and capillary-like tube structure formation upon exposure to chemerin.

Conclusions:

Our data indicate that chemerin is up-regulated during decidualization and might contribute to NK cell accumulation and vascular remodeling during early pregnancy.

Chemerin, a recently identified chemoattractant belonging to the cathelicidin/cystatin protein family, is released as an inactive precursor, prochemerin, and is rapidly converted into the active form by several proteases during inflammation (1–3).

First isolated from inflamed biologic fluids, such as ovarian cancer ascites and rheumatoid arthritis synovial fluids, chemerin is expressed by other tissues, including liver, lung, lymph nodes, and biopsies from nonlesional psoriatic skin or from patients with oral lichen planus or systemic lupus erythematosus. In these injured tissues, chemerin acts as chemoattractant for innate immune cells and has been proposed as a crucial player for the initiation of immune responses (1, 4–6).

Although chemerin was discovered as chemoattractant, many evidences describe this factor as a new adipokine linked to obesity and metabolic syndrome (7, 8). Furthermore, a role for chemerin in the regulation of angiogenesis has been recently proposed based on its ability to enhance new blood vessel formation (9, 10).

Chemerin effector functions are mediated by the seven-transmembrane, G protein-coupled receptor chemokine-like receptor 1 (CMKLR1), also known as ChemR23 (1).

Among leukocytes, CMKLR1 is mainly expressed by monocytes/macrophages, immature myeloid and plasmacytoid dendritic cells (4), and natural killer (NK) cells (11). Thus, chemerin/CMKLR1 may represent an important chemotactic axis involved in the recruitment of specialized leukocyte populations during inflammation, autoimmune diseases, or tumor growth. Two other G protein-coupled receptors have been recently identified as additional chemerin receptors: G protein-coupled receptor 1, phylogenetically related to CMKLR1 but poorly expressed on leukocytes, and C-C chemokine receptor-like 2, which has been suggested to act as a concentrator of bioactive chemerin and to facilitate its presentation to CMKLR1 (12, 13).

After embryo implantation, successful pregnancy depends on crucial events including correct assembly of the maternal-fetal interface and induction of vascular remodeling. Although these events are mainly related to trophoblast functions, evidence indicating that decidual leukocytes, and in particular uterine (u) NK cells, can participate in vascular remodeling and placenta development have been provided (14–17).

Interestingly, the number of uNK cells drastically increases in early pregnancy with their frequency reaching 60–70% and in the late secretory phase of menstrual cycle. These uNK cell numerical variations have been mainly attributed to hormone-induced decidualization and to changes in chemokine expression (18–20).

Differently from their peripheral blood (pb) counterpart, human uNK cells express higher levels of CD56, lack CD16, display a unique transcriptional profile, and express a distinct pattern of adhesion molecules and chemokine receptors compared with the two major CD56^high and CD56^low pbNK cell subsets (21, 22).

Although decidual (d) NK cells are poor killers (23, 24), they release many cytokines and chemokines through which they control extravillous trophoblast (EVT) invasion and the recruitment and functions of other immune cells (14, 25). In addition, the close encirclement of spiral arteries by dNK cells together with their ability to produce angiogenic factors (i.e. vascular endothelial growth factor, placental growth factor, IL-8, and angiopoietin 2) suggest that dNK cells play a major role in the control of mucosal vascularization and placental development (14, 15, 19, 25).

The origin of dNK cells is still debated, and it is unclear whether they arise from NK cell progenitors present in the uterus or recruited from other tissues and/or from NK cells recruited from blood (26, 27).

We and others reported that trophoblast or endometrial cells can produce many chemokines acting on pbNK cells and dNK cells and that pbNK cells and dNK cells can migrate through endothelial and stromal decidual tissues using a distinct pattern of chemokines; all these events are tightly regulated by sexual hormones (14, 28–32). These findings strongly support the idea that pbNK cells can be recruited and accumulate into the uterus during early pregnancy.

Herein we analyzed whether chemerin is present in the uterine compartment and whether the chemerin/chemerin receptor axis could play a role in dNK cell accumulation and decidual vessel remodeling during early pregnancy.

Materials and Methods

Cells

Mononuclear cells from both pb and decidual samples of women undergoing elective pregnancy termination (8–12 wk of gestation), were purified as previously described (31). Highly purified pbNK and dNK cells were obtained by negative selection using FACSAria cell sorter (BD Biosciences, San José, CA) or the Miltenyi NK cell isolation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Purified pbNK cells were routinely greater than 95% CD56⁺CD16⁺CD3⁻CD9⁻CD14⁻, whereas dNK cells were greater than 90% CD56⁺CD9⁺CD16⁻CD3⁻CD14⁻ as assessed by immunocytofluorimetric analysis.

Endothelial and stromal cells from decidual tissues of women undergoing elective pregnancy termination (age ranging from 20 to 35 yr) or stromal cells from endometrial tissues of menopausal (age ranging from 49 to 79 yr) or fertile (age ranging from 35 to 45 yr) women undergoing hysterectomy for leiomyomatosis in the mid proliferative and secretory phases accordingly to Noyes criteria were purified as previously described (31, 33–35). The purity of decidual endothelial cells (DEC) population was more than 98% as verified by staining with antibodies to von Willenbrand factor, CD105, VE cadherin (Dako, Milano, Italy), and CD31/platelet endothelial cell adhesion molecule-1 provided by M. R. Zocchi (San Raffaele Hospital, Milan, Italy) (Supplemental Fig. 1A). DEC were cultured using endothelial serum-free medium (SFM) (GIBCO, Carlsbad, CA) supplemented with 10% human serum, 20 ng/ml basic fibroblast growth factor, 10 ng/ml endothelial growth factor, and penicillin (50 U/ml)/streptomycin (50 μg/ml). Stromal (ST) cells were used only when they were negative for CD14, CD45 (BD Biosciences), cytokeratin 8-18, von Willenbrand factor, or CD31 and positive for α-actin, vimentin, CD13, CD10, and CD105 (Dako) (Supplemental Fig. 1B). DEC and ST cell primary cultures between the third (about 9 d culture) and sixth (about 18 d culture) passages were used.

EVT were purified from placental specimens digested with Hanks' balanced salt solution containing 0.25% trypsin and 0.2 mg/ml deoxyribonuclease (Roche, Milan, Italy) for 20 min at 37 C (33). After Percoll gradient fractionation, leukocytes were immunodepleted using anti-CD45 precoated beads (Dynal; Invitrogen, Milan, Italy), and EVT were seeded in 5 μg/cm² FN (Roche)-precoated flask, in RPMI 1640 (GIBCO) plus 10% fetal calf serum (FCS). The cells obtained were more than 95% cytokeratin 7 positive EVT (Supplemental Fig. 1C).

Informed consent was obtained from all donors providing pb and tissue specimens, and ethical approval was obtained from the Ethics Committee of University “La Sapienza” Rome, (Rome, Italy) and the Ethics Committee of Maternal-Children Hospital “Burlo Garofolo,” (Trieste, Italy).

Real-time quantitative PCR analysis

Chemerin and β-actin mRNA expression was analyzed by real-time quantitative PCR using a commercial Taqman assay reagent as previously described (31). PCR reactions were performed on ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. cDNA was amplified in triplicate with FAM-conjugated chemerin primers (Hs00161209_g1), and VIC-conjugated β-actin (4326315E) (Applied Biosystems). The relative chemerin amount of each sample, normalized with β-actin, was expressed as arbitrary units.

Enzyme-linked immunosorbent assay

Culture supernatants (100 μl) of ST cells (1.5 × 10⁵cells/well per 300 μl) from fertile nonpregnant, pregnant, and menopausal women were assayed in duplicate using the human chemerin DuoSet ELISA kit (R&D Systems, Minneapolis, MN).

Immunoprecipitation and Western blotting analysis

ST cell primary cultures and freshly isolated decidual cells were lysed for 30 min at 4 C in ice-cold lysis buffer [1% Triton X-100, 1 mm CaCl₂, 1 mm MgCl₂, 0.1% NaN₃, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 10 mm NaF, 150 mm NaCl, 10 mm iodoacetamide, 1 mm Na₃VO₄, in 50 mm Tris (pH 7.5)]. Cell lysates were immunoprecipitated with goat antihuman chemerin antibody (R&D Systems), and immunocomplexes or total cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antichemerin goat antibody.

ERK activation

pbNK and dNK cells were stimulated with chemerin or CXCL12/stromal cell-derived factor-1 (SDF-1; R&D System) or with anti-CMKLR1 or anti-CD56 monoclonal antibodies (mAb) preadsorbed in a 96-well plate for the indicated time periods at 37 C. pbNK cells pretreated with pertussis toxin (PTX) were also used. DEC were starved in human endothelial SFM for 18 h at 37 C and then stimulated with chemerin or anti-CMKLR1 crosslinked with goat antimouse secondary antibody (GAM) (Cappel Laboratories, Cooper Biomedical Inc., Malvern, PA). Stimulation was stopped by adding ice-cold lysis buffer, and cell lysates were resolved by 12.5% SDS-PAGE, transferred to nitrocellulose, and sequentially immunoblotted with an anti-phosphorylated (p) ERK mAb (Cell Signaling Technology, Inc., Danvers, MA) and an anti-ERK rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Immunofluorescence and flow cytometric analysis

CMKLR1 expression on pbNK or dNK cells was evaluated by performing a three-color immunofluorescence staining using anti-CMKLR1 (clone 1H2 or clone 4C7) (1) or affinity-purified normal mouse IgG (Sigma, St. Louis, MO) followed by fluorochrome-conjugated F(ab′)2 fragments of GAM (Cappel Laboratories, Cooper Biomedical) and then anti-CD3 and anti-CD56 fluorochrome-conjugated mAb. Cells were collected using a FACScalibur cytofluorimeter (BD Biosciences), and gated CD56⁺CD3⁻ NK cells were analyzed with CELLQuest (BD Biosciences). Peripheral blood mononuclear cell type IV collagenase treatment did not affect CMKLR1 expression on pbNK cells (data not shown).

Migration assay

Highly purified pbNK cells and dNK cells were assayed for their ability to migrate through a monolayer of DEC, or ST cells grown or not with progesterone (100 nm) in response to different concentrations of recombinant human chemerin (R&D Systems) added to the lower compartment of the Transwell migration chamber (diameter insert 6.5 mm, pore size 5 μm; Costar Corp., Cambridge, MA). After 2 h at 37 C, the number of migrated cells was evaluated by FACScalibur cytofluorimeter as previously described (36). pbNK and dNK cell migration through ST cells were also assayed using NK cells pretreated with the ERK inhibitor PD98059.

DEC capillary-like tube formation assay and DEC proliferation

To perform DEC capillary-like tube formation assay, DEC were starved in human endothelial SFM (GIBCO) plus 0.5% FCS for 12 h at 37 C and then seeded onto Matrigel (BD Biosciences) precoated wells at a density of 40 × 10³ cells/well in the absence or presence of different chemerin concentrations (10–30 nm). After 8 h incubation at 37 C, the formation of capillary-like tubes was assessed and photographed at ×10 magnification with a Leica microscope (Wetzlar, Germany) equipped with digital camera (Leica; DFC420C). DEC proliferation was assessed using ³H-thymidine incorporation assay.

Statistical analysis

A Student's t test was used for statistical evaluations.

Results

Chemerin expression and secretion by decidual ST, DEC, and EVT cells

To investigate the role of chemerin/chemerin receptor axis into the uterine compartment during early pregnancy, we first evaluated its expression on DEC, ST, and EVT cells. To this purpose, DEC, ST cell, and EVT primary cultures from first-trimester decidual tissues were assayed for the expression of chemerin mRNA by real-time quantitative PCR analysis. We found that chemerin mRNA was expressed at higher levels on both EVT and ST cells but not on DEC (Fig. 1A). In agreement with this finding, we also observed that unlike DEC, EVT and ST cells were able to secrete substantial levels of chemerin in their culture supernatants (Fig. 1B).

Fig. 1. — Chemerin mRNA expression and chemerin secretion in DEC, EVT, and uterine ST cells from fertile, pregnant, and menopausal women. A, mRNA isolated from EVT, ST cell, or DEC primary cultures obtained from the same donor were analyzed by real-time quantitative PCR assay for the expression of chemerin. The relative chemerin amount of each sample was normalized with β-actin and expressed as arbitrary units ± sd. Results obtained from three different donors are shown. B, Culture supernatants of EVT, DEC, and ST cells (1.5 × 10⁵ cells/well per 300 μl) from pregnant women grown with medium alone were harvested after 24 h of incubation at 37 C and then tested for the presence of chemerin by ELISA. C, mRNA isolated from primary cultures of ST cells obtained from fertile nonpregnant, pregnant, and menopausal women were analyzed for the expression of chemerin by real-time quantitative PCR assay as described in A. The mean ± sd of chemerin mRNA expression obtained from different numbers (n) of donors are shown. D, Immunohistochemical analysis of first trimester decidual (A and B) and fertile nonpregnant uterine (C and D) sections with antichemerin (A and C) and control IgG1 antibodies (B and D). Original magnification, ×40 (A and B); original magnification ×20 (C and D). *, P < 0.02 and **, P < 0.01 as evaluated by performing Student's t test statistical analysis between ST cells from pregnant *vs.* fertile and menopausal women, respectively.

To investigate whether the early pregnant status could affect chemerin mRNA expression on ST cells, we performed real-time quantitative PCR analysis on ST cell primary cultures derived from fertile, pregnant, and menopausal women. We found that ST cells from pregnant women expressed significantly higher levels of chemerin mRNA with respect to ST cells from fertile and menopausal women (Fig. 1C). In addition, immunohistochemical analysis on uterine tissues from pregnant and nonpregnant women revealed a strong positive tissue staining (Fig. 1D).

Based on the evidence indicating a crucial role for sex hormones in the regulation of decidualization, we evaluated the ability of ST cell primary cultures from fertile, pregnant, and menopausal women to secrete chemerin and the effect exerted by progesterone and 17β-estradiol on this event. ST cells from pregnant women were able to release higher amounts of chemerin with respect to those from fertile or menopausal women. Moreover, exposure of uterine ST cells from fertile, pregnant, and menopausal women to progesterone and 17β-estradiol resulted in enhanced chemerin release (Fig. 2). Western blotting analysis of decidual ST cells reveals that they produce a form of chemerin migrating with a molecular weight similar to that of the human recombinant chemerin (Fig. 2, insert).

Fig. 2. — Chemerin expression and secretion by uterine ST cells. Culture supernatants of ST cells (1.5 × 10⁵ cells/well per 300 μl) from fertile nonpregnant, pregnant, and menopausal women grown with medium alone or supplemented with progesterone (100 nm) and 17β-estradiol (1 nm) were harvested after 24 h of incubation at 37 C and then tested for the presence of chemerin by ELISA. Each *circle* represents one donor and the number (n) of the donors tested for each group is shown. *Black lines* indicate the mean value of each group. *, P < 0.05, as evaluated by comparing progesterone and 17β-estradiol-treated *vs.* untreated ST cells using Student's t test statistical analysis. *Insert*, Primary cultures of ST cells (3 × 10⁶ cells) and freshly isolated decidual cells (40 × 10⁶ cells) were lysed and immunoprecipitated with goat antihuman chemerin antibody. The resulting immunocomplexes and total cell lysates (5 × 10⁵ ST cells, 3 × 10⁶ decidual cells) were resolved by 15% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antichemerin goat antibody. Fifty nanograms of recombinant human chemerin were loaded as positive control. These results represent one of three independent experiments.

These results indicate that chemerin mRNA is expressed at different levels on EVT, ST, and DEC decidual tissues and that pregnancy-associated hormones can positively modulate the expression and release of this chemoattractant.

Chemerin receptor profile on pbNK and dNK cells and chemerin ability to support pbNK and dNK cell migration through DEC and ST cells

To evaluate whether chemerin can contribute to pbNK cell recruitment in the decidua and to dNK cell positioning into the uterus during early pregnancy, pbNK cells, and their uterine counterpart were analyzed for the expression of CMKLR1 and for their ability to migrate through either DEC or ST cells in response to chemerin.

To this aim, pbNK and dNK cells were isolated from the same first-trimester pregnant woman undergoing elective pregnancy termination and assayed for CMKLR1 expression by three color immunofluorescence. As previously reported (11), we found that CMKLR1 is expressed at high levels, mainly by the CD56^low pbNK cells, whereas dNK cells exhibit only modest CMKLR1 levels (Fig. 3).

Fig. 3. — Chemerin receptor expression on pbNK and dNK cells of women in the first trimester of pregnancy. Freshly isolated pb and dNK cells from the same woman in the first trimester of pregnancy were evaluated for the expression of the chemerin receptor, CMKLR1, by a three-color immunofluorescence and cytofluorimetric analysis using a mouse mAb anti-CMKLR1. The *dot plots* shown indicate the expression of CMKLR1 on gated CD56⁺CD3⁻ NK cells. Staining with normal mouse IgG followed by fluorescein isothiocyanate-conjugated GAM antibodies was used as control. The percentage of positive cells and the mean fluorescence intensity (m) were shown. Data are representative of four independent experiments.

As shown in Fig. 4 (A and B), we observed that chemerin significantly supports pbNK cell migration through DEC and ST cells. In addition, based on our finding showing that progesterone and 17β-estradiol treatment up-regulates chemerin expression and secretion by ST cells and on previous reports indicating that NK cells exhibit enhanced adhesion and migration through pregnant tissues (31, 37, 38), we assayed the ability of pbNK cells from pregnant women to migrate in response to chemerin through progesterone cultured decidual ST cells. Progesterone treatment of ST cells did not significantly affect pbNK cell migratory behavior (Fig. 4B).

These data first demonstrate that chemerin can support pregnant pbNK cell migration through both endothelial and stromal decidual tissues and suggest that progesterone and 17β-estradiol level fluctuations occurring during early pregnancy can tightly control this process by acting on ST cells and modulating chemerin production.

To investigate the ability of chemerin to also support dNK cell migration through ST cells, freshly isolated highly purified dNK and pbNK cells from the same donor were allowed to migrate through ST cells. As shown in Fig. 4C, in accordance with CMKLR1 expression, chemerin significantly supported dNK cell migration through ST cells although at lower degree than that observed for the pb counterpart.

CMKLR1 stimulation enhances pErk activation in dNK cells

To further support the CMKLR1 functional ability on NK cells, we analyzed its capacity to activate the MAPK ERK, a signaling molecule involved in the regulation of many cell functions including migration. Thus, highly purified dNK cells were stimulated for the indicated times with CXCL12/SDF-1 or chemerin or with crosslinked mAb directed against CXCR4, or CMKLR1. Stimulation with CXCL12/SDF-1 or anti-CXCR4 mAb was used as positive control while that with anti-CD56 mAb as negative control. As shown in Fig. 5A, dNK cell stimulation with CXCL12/SDF-1 or chemerin resulted in ERK activation that was already evident at 1 min and still persisted at 7 min after stimulation. ERK activation was also observed after dNK cell stimulation for 3 min with an antibody against CXCR4 or CMKLR1. Stimulation with anti-CD56 control mAb did not significantly affect the phosphorylation status of ERK (Fig. 5B).

Fig. 5. — Chemerin receptor stimulation enhances pERK activation in human dNK cells. A and B, Human dNK cells (4 × 10⁵ cells) were left untreated (−) or stimulated with chemerin (10 nm) or with CXCL12/SDF-1 (250 nm) or with an antibody against CXCR4, CMKLR1 (clone 4C7), or CD56 (clone C218) precoated to a 96-well plate for the indicated time periods at 37 C. After stimulation cells were lysed and total cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and sequentially immunoblotted with anti-pERK mAb and anti-ERK1/2 rabbit Ab for loading control. C, Human pbNK cells preteated or not with PTX (200 ng/ml) were left untreated or stimulated with chemerin or anti-CMKLR1 mAb for 3 min at 37 C and then analyzed as above. These results represent one of two independent experiments. D, Highly purified pbNK cells or dNK cells from the same woman in the first trimester of pregnancy were pretreated with the ERK inhibitor (PD98059) and then assayed for their ability to migrate through a monolayer of primary decidual ST cells grown in the presence of progesterone (100 nm) in response to chemerin.

Triggering of pbNK cells with chemerin or crosslinked anti-CMKLR1 mAb also resulted in ERK activation, and interestingly, ERK activation was clearly inhibited when stimulation was performed on pbNK cells pretreated with PTX, a well-known pharmacological inhibitor of Gi proteins (Fig. 5C). Moreover, pretreatment of pbNK cells and dNK cells with the ERK inhibitor PD98059 resulted in strong inhibition of NK cell migration through ST cells (Fig. 5D).

These results demonstrate that CMKLR1 engagement on dNK cells leads to rapid ERK activation that is involved in the regulation of chemerin-supported pbNK and dNK cell migration through stromal cells.

DEC express CMKLR1 and undergo capillary-like tube formation upon chemerin stimulation

Remodeling of blood vessels is a crucial event for successful pregnancy and is a complex process regulated by many endothelial cell mitogenic signals and angiogenic factors. The recent observations that chemerin can modulate human umbilical vein endothelial cell angiogenesis (9, 10) prompted us to evaluate whether this chemoattractant could exert a similar effect in the decidua. To this purpose, we first analyzed the expression of chemerin receptor on DEC primary cultures, and we found that they are positive for CMKLR1 (Fig. 6A). In addition, receptor engagement with chemerin or specific antibody resulted in ERK activation that was already evident at 3 min after stimulation (Fig. 6B). Accordingly, chemerin promoted their ability to form capillary-like tubes on Matrigel (BD Biosciences) and to proliferate in a dose-dependent manner (Fig. 6, C and D).

Fig. 6. — DEC express CMKLR1 chemerin receptor and undergo pERK activation and capillary-like tube formation upon chemerin stimulation. A, DEC were analyzed for the expression of CMKLR1 chemerin receptor by immunofluorescence and cytofluorimetric analysis. B, DEC (2 × 10⁵ cells) were starved in human endothelial SFM for 18 h at 37 C and then left untreated (−) or stimulated with different concentrations of chemerin or with anti-CMKLR1 mAb for the indicated time periods at 37 C. After stimulation cell lysates were assayed for pERK activation as above described. C, DEC were starved in human endothelial SFM plus 0.5% FCS for 12 h at 37 C and then seeded onto Matrigel precoated wells in medium alone or in the presence of different concentrations of chemerin (10–30 nm). After 8 h incubation at 37 C, the formation of capillary-like tubes was assessed. D, Human endothelial SFM starved DEC (1 × 10⁴ cells) were seeded in triplicate onto a 96-well plate in medium alone or in the presence of different concentrations of chemerin (0.1–30 nm). After 18 or 42 h incubation at 37 C, 1 μCi of ³H-thymidine was added to each well, and the proliferation was assessed upon an additional 8 h incubation. Human endothelial SFM supplemented with 10% human serum, 20 ng/ml basic fibroblast growth factor, 10 ng/ml epithelial growth factor, and penicillin (50 U/ml)/streptomycin (50 μg/ml) was used as a positive control (Med DEC).

These results strongly suggest that chemerin participate to uterine vascular remodeling during early pregnancy.

Discussion

In this study we investigated the expression and regulation of chemerin at mRNA and protein level in decidual tissues and its involvement in pbNK cell recruitment and vascular remodeling into the uterus during early pregnancy.

We found that chemerin is expressed at mRNA level in the uterus mainly by EVT and ST cells but not by DEC. Notably, we found that uterine ST cells from pregnant women express a significantly higher amount of chemerin mRNA with respect to ST cells from fertile or menopausal women. This result, together with the ability of ST cell primary cultures from pregnant women to release higher levels of chemerin compared with ST cells from fertile nonpregnant or menopausal women, indicates that the expression and secretion of this chemoattractant can be up-modulated during the decidualization of endometrial stromal cells.

Among factors promoting endometrial decidualization and controlling the good outcome of pregnancy, a crucial role is played by sex hormones. Pregnancy-associated hormones can modulate the expression of many chemokines acting on NK cells as well as the functional adhesiveness and migratory behavior of pregnant women pbNK cells through decidual tissues (18–20, 27–32).

In agreement with these observations, we found that treatment of ST cell primary cultures from pregnant, fertile nonpregnant, or menopausal women with progesterone and 17β-estradiol results in a significant up-regulation of chemerin secretion. However, the levels of chemerin release by ST cells from fertile nonpregnant or menopausal women, even upon hormone stimulation, were lower than those from pregnant ST cells, thus suggesting that sex hormones contribute to the regulation of this chemoattractant expression.

Our findings support recent evidences indicating that chemerin serum levels increase and fluctuate during pregnancy both in humans and rodents and are even more elevated in pregnancy disorders such as preeclampsia (39–41).

The molecular events involved in the regulation of chemerin expression and secretion are still poorly defined. Increased chemerin expression has been found in psoriatic lesions, intestinal epithelial cells in response to the retinoid tazarotene (6, 42), and the bone stromal cell line ST2 after treatment with 1,25-dihydroxyvitamin D3 and dexamethasone (43). Interestingly, all of these stimuli involve nuclear receptor-initiated signaling.

Our results show that chemerin can support pregnant women pbNK cell migration through both DEC and ST cells, and notably, it can also drive dNK cell migration through ST cells, suggesting that this chemoattractant can contribute to both pbNK cell recruitment in the uterus and to finely tune dNK cell positioning into decidual specific areas. These data provide novel information and strengthen the previous observations on the ability of chemerin to function as chemoattractant for circulating NK cells (11).

Interestingly, by analyzing CMKLR1 expression, we found that although CD56^low pbNK cells from pregnant women exhibit high levels of CMKLR1, dNK cells express only low levels of this receptor. The ability of CMKLR1 to elicit dNK cell migration through ST cells in response to chemerin together with its ability to trigger ERK activation in dNK cells when engaged by the ligand or specific mAb is to the best of our knowledge the first evidence indicating that CMKLR1 is a functional receptor also for dNK cells.

The differential expression of CMKLR1 on dNK cells adds a new phenotypic difference between pbNK and dNK cells, likely associated with the different activation/differentiation state of these cell populations.

In this regard, CMKLR1 expression on NK cells, has been reported to undergo downmodulation when the CD56^lowCD16⁺ pbNK cell subset is cultured in vitro with IL-2 or IL-15 (11), and considerable evidence indicates that the pregnant uterus is a good source of cytokines acting on NK cells including IL-15 (44). Moreover, our finding (data not shown) that pbNK cells acquire a chemerin receptor profile similar to that of dNK cells when cocultured with decidual ST cells indicates that ST cells can effectively provide membrane bound and/or soluble factors that, by acting on NK cells, result in CMKLR1 down-regulation.

This observation is in line with accumulating evidence indicating that signals provided by decidualized ST cells including IL-15 and IL-11 critically control uNK cell development and/or functional differentiation (44, 45). In this regard, incubation of CD16^negative pbNK cells with IL-15 or coculture of pbNK cells with ST cells results in a chemokine receptor pattern similar to that of uNK cells (28, 31). Moreover, culture of CD16^positive pbNK cells with conditioned medium derived from decidual ST cell promotes the conversion of CD16^positive pbNK cells into CD16^negative cells and in the up-regulation of CD9 expression, and these effects are clearly dependent on the TGFβ released by ST cells (46).

Finally, in agreement with the recently described ability of chemerin to function as an angiogenic molecule (9, 10), we provided the first evidence indicating that DEC express the chemerin receptor CMKLR1 and that once exposed to chemerin they undergo capillary-like tube formation, thus suggesting that chemerin in the uterus can participate also to the complex events leading to decidual vessel remodeling.

As a whole, our findings indicate that chemerin production into the uterus is up-regulated during decidualization and point out a crucial role for the chemerin/CMKLR1 axis in the recruitment, accumulation, and correct positioning of dNK cells in this organ as well as in vascular remodeling during early pregnancy.

Supplementary Material

Supplemental Data

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Acknowledgments

We thank Dr. E. Masat (University of Trieste, Trieste, Italy) for the immunohistochemical analysis. A special thank you goes to all the patients who contributed to this study and to the staff of the Department of Gynecology, Sapienza University of Rome (Rome, Italy) and the Department of Reproductive and Developmental Sciences, Institute for Maternal and Child Health, “Burlo Garofolo,” University of Trieste, for their precious contribution.

This work was supported by grants from the Istituto Pasteur Fondazione Cenci Bolognetti, Regione Lombardia, MIUR-PRIN, Centro di Eccellenza BEMM, and the European NoE EMBIC within FP6 (Contract Number LSHN-CT-2004-512040).

Disclosure Summary: The authors declare no competing financial interests.

Footnotes

Abbreviations:

CMKLR1: Chemokine-like receptor 1
d: decidual
DEC: decidual endothelial cell
EVT: extravillous trophoblast
FCS: fetal calf serum
GAM: goat antimouse secondary antibody
mAb: monoclonal antibody
NK: natural killer
p: phosphorylated
pb: peripheral blood
PTX: pertussis toxin
SDF-1: stromal cell-derived factor-1
SFM: serum-free medium
ST: stromal
u: uterine.

References

1. Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, Brézillon S, Tyldesley R, Blanpain C, Detheux M, Mantovani A, Sozzani S, Vassart G, Parmentier M, Communi D. 2003. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J Exp Med 198:977–985 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Sozzani S, Vermi W, Del Prete A, Facchetti F. 2010. Trafficking properties of plasmacytoid dendritic cells in health and disease. Trends Immunol 31:270–277 [DOI] [PubMed] [Google Scholar]
3. Zabel BA, Allen SJ, Kulig P, Allen JA, Cichy J, Handel TM, Butcher EC. 2005. Chemerin activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades. J Biol Chem 280:34661–34666 [DOI] [PubMed] [Google Scholar]
4. Vermi W, Riboldi E, Wittamer V, Gentili F, Luini W, Marrelli S, Vecchi A, Franssen JD, Communi D, Massardi L, Sironi M, Mantovani A, Parmentier M, Facchetti F, Sozzani S. 2005. Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin. J Exp Med 201:509–515 [DOI] [PMC free article] [PubMed] [Google Scholar]
5. Zabel BA, Silverio AM, Butcher EC. 2005. Chemokine-like receptor 1 expression and chemerin-directed chemotaxis distinguish plasmacytoid from myeloid dendritic cells in human blood. J Immunol 174:244–251 [DOI] [PubMed] [Google Scholar]
6. Nagpal S, Patel S, Jacobe H, DiSepio D, Ghosn C, Malhotra M, Teng M, Duvic M, Chandraratna RA. 1997. Tazarotene-induced gene 2 (TIG2), a novel retinoid-responsive gene in skin. J Invest Dermatol 109:91–95 [DOI] [PubMed] [Google Scholar]
7. Bozaoglu K, Bolton K, McMillan J, Zimmet P, Jowett J, Collier G, Walder K, Segal D. 2007. Chemerin is a novel adipokine associated with obesity and metabolic syndrome. Endocrinology 148:4687–4694 [DOI] [PubMed] [Google Scholar]
8. Goralski KB, McCarthy TC, Hanniman EA, Zabel BA, Butcher EC, Parlee SD, Muruganandan S, Sinal CJ. 2007. Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. J Biol Chem 282:28175–28188 [DOI] [PubMed] [Google Scholar]
9. Kaur J, Adya R, Tan BK, Chen J, Randeva HS. 2010. Identification of chemerin receptor (ChemR23) in human endothelial cells: chemerin-induced endothelial angiogenesis. Biochem Biophys Res Commun 391:1762–1768 [DOI] [PubMed] [Google Scholar]
10. Bozaoglu K, Curran JE, Stocker CJ, Zaibi MS, Segal D, Konstantopoulos N, Morrison S, Carless M, Dyer TD, Cole SA, Goring HH, Moses EK, Walder K, Cawthorne MA, Blangero J, Jowett JB. 2010. Chemerin, a novel adipokine in the regulation of angiogenesis. J Clin Endocrinol Metab 95:2476–2485 [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Parolini S, Santoro A, Marcenaro E, Luini W, Massardi L, Facchetti F, Communi D, Parmentier M, Majorana A, Sironi M, Tabellini G, Moretta A, Sozzani S. 2007. The role of chemerin in the colocalization of NK and dendritic cell subsets into inflamed tissues. Blood 109:3625–3632 [DOI] [PubMed] [Google Scholar]
12. Barnea G, Strapps W, Herrada G, Berman Y, Ong J, Kloss B, Axel R, Lee KJ. 2008. The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci USA 105:64–69 [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Zabel BA, Nakae S, Zúñiga L, Kim JY, Ohyama T, Alt C, Pan J, Suto H, Soler D, Allen SJ, Handel TM, Song CH, Galli SJ, Butcher EC. 2008. Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis. J Exp Med 205:2207–2220 [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O. 2006. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 12:1065–1074 [DOI] [PubMed] [Google Scholar]
15. Leonard S, Murrant C, Tayade C, van den Heuvel M, Watering R, Croy BA. 2006. Mechanisms regulating immune cell contributions to spiral artery modification—facts and hypotheses—a review. Placenta 27(Suppl A):S40–S46 [DOI] [PubMed] [Google Scholar]
16. Zhang J, Chen Z, Smith GN, Croy BA. 2011. Natural killer cell-triggered vascular transformation: maternal care before birth? Cell Mol Immunol 8:1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Madeja Z, Yadi H, Apps R, Boulenouar S, Roper SJ, Gardner L, Moffett A, Colucci F, Hemberger M. 2011. Paternal MHC expression on mouse trophoblast affects uterine vascularization and fetal growth. Proc Natl Acad Sci USA 108:4012–4017 [DOI] [PMC free article] [PubMed] [Google Scholar]
18. King A. 2000. Uterine leukocytes and decidualization. Hum Reprod Update 6:28–36 [DOI] [PubMed] [Google Scholar]
19. Croy BA, van den Heuvel MJ, Borzychowski AM, Tayade C. 2006. Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones. Immunol Rev 214:161–185 [DOI] [PubMed] [Google Scholar]
20. Jones RL, Hannan NJ, Kaitu'u TJ, Zhang J, Salamonsen LA. 2004. Identification of chemokines important for leukocyte recruitment to the human endometrium at the times of embryo implantation and menstruation. J Clin Endocrinol Metab 89:6155–6167 [DOI] [PubMed] [Google Scholar]
21. Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, Schatz F, Masch R, Lockwood CJ, Schachter AD, Park PJ, Strominger JL. 2003. Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med 198:1201–1212 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Freud AG, Caligiuri MA. 2006. Human natural killer cell development. Immunol Rev 214:56–72 [DOI] [PubMed] [Google Scholar]
23. Tabiasco J, Rabot M, Aguerre-Girr M, El Costa H, Berrebi A, Parant O, Laskarin G, Juretic K, Bensussan A, Rukavina D, Le Bouteiller P. 2006. Human decidual NK cells: unique phenotype and functional properties—a review. Placenta 27(Suppl A):S34–S39 [DOI] [PubMed] [Google Scholar]
24. Kopcow HD, Allan DS, Chen X, Rybalov B, Andzelm MM, Ge B, Strominger JL. 2005. Human decidual NK cells form immature activating synapses and are not cytotoxic. Proc Natl Acad Sci USA 102:15563–15568 [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Moffett-King A. 2002. Natural killer cells and pregnancy. Nat Rev Immunol 2:656–663 [DOI] [PubMed] [Google Scholar]
26. Chantakru S, Miller C, Roach LE, Kuziel WA, Maeda N, Wang WC, Evans SS, Croy BA. 2002. Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy. J Immunol 168:22–28 [DOI] [PubMed] [Google Scholar]
27. Kitaya K, Yamaguchi T, Yasuo T, Okubo T, Honjo H. 2007. Post-ovulatory rise of endometrial CD16(−) natural killer cells: in situ proliferation of residual cells or selective recruitment from circulating peripheral blood? J Reprod Immunol 76:45–53 [DOI] [PubMed] [Google Scholar]
28. Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, Katz G, Haimov-Kochman R, Fujii N, Yagel S, Peled A, Mandelboim O. 2003. CXCL12 expression by invasive trophoblasts induces the specific migration of CD16− human natural killer cells. Blood 102:1569–1577 [DOI] [PubMed] [Google Scholar]
29. Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. 2005. Human first-trimester trophoblast cells recruit CD56brightCD16− NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. J Immunol 175:61–68 [DOI] [PubMed] [Google Scholar]
30. Sentman CL, Meadows SK, Wira CR, Eriksson M. 2004. Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone. J Immunol 173:6760–6766 [DOI] [PubMed] [Google Scholar]
31. Carlino C, Stabile H, Morrone S, Bulla R, Soriani A, Agostinis C, Bossi F, Mocci C, Sarazani F, Tedesco F, Santoni A, Gismondi A. 2008. Recruitment of circulating NK cells through decidual tissues: a possible mechanism controlling NK cell accumulation in the uterus during early pregnancy. Blood 111:3108–3115 [DOI] [PubMed] [Google Scholar]
32. Santoni A, Carlino C, Stabile H, Gismondi A. 2008. Mechanisms underlying recruitment and accumulation of decidual NK cells in uterus during pregnancy. Am J Reprod Immunol 59:417–424 [DOI] [PubMed] [Google Scholar]
33. Bulla R, Villa A, Bossi F, Cassetti A, Radillo O, Spessotto P, De Seta F, Guaschino S, Tedesco F. 2005. VE-cadherin is a critical molecule for trophoblast-endothelial cell interaction in decidual spiral arteries. Exp Cell Res 303:101–113 [DOI] [PubMed] [Google Scholar]
34. Spessotto P, Bulla R, Danussi C, Radillo O, Cervi M, Monami G, Bossi F, Tedesco F, Doliana R, Colombatti A. 2006. EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall. J Cell Sci 119:4574–4584 [DOI] [PubMed] [Google Scholar]
35. Noyes RW, Hertig AT, Rock J. 1975. Dating the endometrial biopsy. Am J Obstet Gynecol 122:262–263 [DOI] [PubMed] [Google Scholar]
36. Stabile H, Carlino C, Mazza C, Giliani S, Morrone S, Notarangelo LD, Notarangelo LD, Santoni A, Gismondi A. 2010. Impaired NK-cell migration in WAS/XLT patients: role of Cdc42/WASp pathway in the control of chemokine-induced β2 integrin high-affinity state. Blood 115:2818–2826 [DOI] [PMC free article] [PubMed] [Google Scholar]
37. van den Heuvel MJ, Horrocks J, Bashar S, Taylor S, Burke S, Hatta K, Lewis JE, Croy BA. 2005. Menstrual cycle hormones induce changes in functional interactions between lymphocytes and decidual vascular endothelial cells. J Clin Endocrinol Metab 90:2835–2842 [DOI] [PMC free article] [PubMed] [Google Scholar]
38. Chantakru S, Wang WC, van den Heuvel M, Bashar S, Simpson A, Chen Q, Croy BA, Evans SS. 2003. Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones. J Immunol 171:4011–4019 [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Stepan H, Philipp A, Roth I, Kralisch S, Jank A, Schaarschmidt W, Lössner U, Kratzsch J, Blüher M, Stumvoll M, Fasshauer M. 2011. Serum levels of the adipokine chemerin are increased in preeclampsia during and 6 months after pregnancy. Regul Pept 168:69–72 [DOI] [PubMed] [Google Scholar]
40. Duan DM, Niu JM, Lei Q, Lin XH, Chen X. 2011. Serum levels of the adipokine chemerin in preeclampsia. J Perinat Med 40:121–127 [DOI] [PubMed] [Google Scholar]
41. Garces MF, Sanchez E, Acosta BJ, Angel E, Ruíz AI, Rubio-Romero JA, Diéguez C, Nogueiras R, Caminos JE. 2012. Expression and regulation of chemerin during rat pregnancy. Placenta 33:373–378 [DOI] [PubMed] [Google Scholar]
42. Maheshwari A, Kurundkar AR, Shaik SS, Kelly DR, Hartman Y, Zhang W, Dimmitt R, Saeed S, Randolph DA, Aprahamian C, Datta G, Ohls RK. 2009. Epithelial cells in fetal intestine produce chemerin to recruit macrophages. Am J Physiol Gastrointest Liver Physiol 297:G1–G10 [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Adams AE, Abu-Amer Y, Chappel J, Stueckle S, Ross FP, Teitelbaum SL, Suva LJ. 1999. 1,25 Dihydroxyvitamin D3 and dexamethasone induce the cyclooxygenase 1 gene in osteoclast-supporting stromal cells. J Cell Biochem 74:587–595 [PubMed] [Google Scholar]
44. Verma S, Hiby SE, Loke YW, King A. 2000. Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15. Biol Reprod 62:959–968 [DOI] [PubMed] [Google Scholar]
45. Ain R, Trinh ML, Soares MJ. 2004. Interleukin-11 signaling is required for the differentiation of natural killer cells at the maternal-fetal interface. Dev Dyn 231:700–708 [DOI] [PubMed] [Google Scholar]
46. Keskin DB, Allan DS, Rybalov B, Andzelm MM, Stern JN, Kopcow HD, Koopman LA, Strominger JL. 2007. TGFβ promotes conversion of CD16+ peripheral blood NK cells into CD16− NK cells with similarities to decidual NK cells. Proc Natl Acad Sci USA 104:3378–3383 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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[B1] 1. Wittamer V, Franssen JD, Vulcano M, Mirjolet JF, Le Poul E, Migeotte I, Brézillon S, Tyldesley R, Blanpain C, Detheux M, Mantovani A, Sozzani S, Vassart G, Parmentier M, Communi D. 2003. Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J Exp Med 198:977–985 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Sozzani S, Vermi W, Del Prete A, Facchetti F. 2010. Trafficking properties of plasmacytoid dendritic cells in health and disease. Trends Immunol 31:270–277 [DOI] [PubMed] [Google Scholar]

[B3] 3. Zabel BA, Allen SJ, Kulig P, Allen JA, Cichy J, Handel TM, Butcher EC. 2005. Chemerin activation by serine proteases of the coagulation, fibrinolytic, and inflammatory cascades. J Biol Chem 280:34661–34666 [DOI] [PubMed] [Google Scholar]

[B4] 4. Vermi W, Riboldi E, Wittamer V, Gentili F, Luini W, Marrelli S, Vecchi A, Franssen JD, Communi D, Massardi L, Sironi M, Mantovani A, Parmentier M, Facchetti F, Sozzani S. 2005. Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin. J Exp Med 201:509–515 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Zabel BA, Silverio AM, Butcher EC. 2005. Chemokine-like receptor 1 expression and chemerin-directed chemotaxis distinguish plasmacytoid from myeloid dendritic cells in human blood. J Immunol 174:244–251 [DOI] [PubMed] [Google Scholar]

[B6] 6. Nagpal S, Patel S, Jacobe H, DiSepio D, Ghosn C, Malhotra M, Teng M, Duvic M, Chandraratna RA. 1997. Tazarotene-induced gene 2 (TIG2), a novel retinoid-responsive gene in skin. J Invest Dermatol 109:91–95 [DOI] [PubMed] [Google Scholar]

[B7] 7. Bozaoglu K, Bolton K, McMillan J, Zimmet P, Jowett J, Collier G, Walder K, Segal D. 2007. Chemerin is a novel adipokine associated with obesity and metabolic syndrome. Endocrinology 148:4687–4694 [DOI] [PubMed] [Google Scholar]

[B8] 8. Goralski KB, McCarthy TC, Hanniman EA, Zabel BA, Butcher EC, Parlee SD, Muruganandan S, Sinal CJ. 2007. Chemerin, a novel adipokine that regulates adipogenesis and adipocyte metabolism. J Biol Chem 282:28175–28188 [DOI] [PubMed] [Google Scholar]

[B9] 9. Kaur J, Adya R, Tan BK, Chen J, Randeva HS. 2010. Identification of chemerin receptor (ChemR23) in human endothelial cells: chemerin-induced endothelial angiogenesis. Biochem Biophys Res Commun 391:1762–1768 [DOI] [PubMed] [Google Scholar]

[B10] 10. Bozaoglu K, Curran JE, Stocker CJ, Zaibi MS, Segal D, Konstantopoulos N, Morrison S, Carless M, Dyer TD, Cole SA, Goring HH, Moses EK, Walder K, Cawthorne MA, Blangero J, Jowett JB. 2010. Chemerin, a novel adipokine in the regulation of angiogenesis. J Clin Endocrinol Metab 95:2476–2485 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Parolini S, Santoro A, Marcenaro E, Luini W, Massardi L, Facchetti F, Communi D, Parmentier M, Majorana A, Sironi M, Tabellini G, Moretta A, Sozzani S. 2007. The role of chemerin in the colocalization of NK and dendritic cell subsets into inflamed tissues. Blood 109:3625–3632 [DOI] [PubMed] [Google Scholar]

[B12] 12. Barnea G, Strapps W, Herrada G, Berman Y, Ong J, Kloss B, Axel R, Lee KJ. 2008. The genetic design of signaling cascades to record receptor activation. Proc Natl Acad Sci USA 105:64–69 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Zabel BA, Nakae S, Zúñiga L, Kim JY, Ohyama T, Alt C, Pan J, Suto H, Soler D, Allen SJ, Handel TM, Song CH, Galli SJ, Butcher EC. 2008. Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis. J Exp Med 205:2207–2220 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, Natanson-Yaron S, Prus D, Cohen-Daniel L, Arnon TI, Manaster I, Gazit R, Yutkin V, Benharroch D, Porgador A, Keshet E, Yagel S, Mandelboim O. 2006. Decidual NK cells regulate key developmental processes at the human fetal-maternal interface. Nat Med 12:1065–1074 [DOI] [PubMed] [Google Scholar]

[B15] 15. Leonard S, Murrant C, Tayade C, van den Heuvel M, Watering R, Croy BA. 2006. Mechanisms regulating immune cell contributions to spiral artery modification—facts and hypotheses—a review. Placenta 27(Suppl A):S40–S46 [DOI] [PubMed] [Google Scholar]

[B16] 16. Zhang J, Chen Z, Smith GN, Croy BA. 2011. Natural killer cell-triggered vascular transformation: maternal care before birth? Cell Mol Immunol 8:1–11 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Madeja Z, Yadi H, Apps R, Boulenouar S, Roper SJ, Gardner L, Moffett A, Colucci F, Hemberger M. 2011. Paternal MHC expression on mouse trophoblast affects uterine vascularization and fetal growth. Proc Natl Acad Sci USA 108:4012–4017 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. King A. 2000. Uterine leukocytes and decidualization. Hum Reprod Update 6:28–36 [DOI] [PubMed] [Google Scholar]

[B19] 19. Croy BA, van den Heuvel MJ, Borzychowski AM, Tayade C. 2006. Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones. Immunol Rev 214:161–185 [DOI] [PubMed] [Google Scholar]

[B20] 20. Jones RL, Hannan NJ, Kaitu'u TJ, Zhang J, Salamonsen LA. 2004. Identification of chemokines important for leukocyte recruitment to the human endometrium at the times of embryo implantation and menstruation. J Clin Endocrinol Metab 89:6155–6167 [DOI] [PubMed] [Google Scholar]

[B21] 21. Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, Schatz F, Masch R, Lockwood CJ, Schachter AD, Park PJ, Strominger JL. 2003. Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med 198:1201–1212 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Freud AG, Caligiuri MA. 2006. Human natural killer cell development. Immunol Rev 214:56–72 [DOI] [PubMed] [Google Scholar]

[B23] 23. Tabiasco J, Rabot M, Aguerre-Girr M, El Costa H, Berrebi A, Parant O, Laskarin G, Juretic K, Bensussan A, Rukavina D, Le Bouteiller P. 2006. Human decidual NK cells: unique phenotype and functional properties—a review. Placenta 27(Suppl A):S34–S39 [DOI] [PubMed] [Google Scholar]

[B24] 24. Kopcow HD, Allan DS, Chen X, Rybalov B, Andzelm MM, Ge B, Strominger JL. 2005. Human decidual NK cells form immature activating synapses and are not cytotoxic. Proc Natl Acad Sci USA 102:15563–15568 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Moffett-King A. 2002. Natural killer cells and pregnancy. Nat Rev Immunol 2:656–663 [DOI] [PubMed] [Google Scholar]

[B26] 26. Chantakru S, Miller C, Roach LE, Kuziel WA, Maeda N, Wang WC, Evans SS, Croy BA. 2002. Contributions from self-renewal and trafficking to the uterine NK cell population of early pregnancy. J Immunol 168:22–28 [DOI] [PubMed] [Google Scholar]

[B27] 27. Kitaya K, Yamaguchi T, Yasuo T, Okubo T, Honjo H. 2007. Post-ovulatory rise of endometrial CD16(−) natural killer cells: in situ proliferation of residual cells or selective recruitment from circulating peripheral blood? J Reprod Immunol 76:45–53 [DOI] [PubMed] [Google Scholar]

[B28] 28. Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, Katz G, Haimov-Kochman R, Fujii N, Yagel S, Peled A, Mandelboim O. 2003. CXCL12 expression by invasive trophoblasts induces the specific migration of CD16− human natural killer cells. Blood 102:1569–1577 [DOI] [PubMed] [Google Scholar]

[B29] 29. Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. 2005. Human first-trimester trophoblast cells recruit CD56brightCD16− NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1. J Immunol 175:61–68 [DOI] [PubMed] [Google Scholar]

[B30] 30. Sentman CL, Meadows SK, Wira CR, Eriksson M. 2004. Recruitment of uterine NK cells: induction of CXC chemokine ligands 10 and 11 in human endometrium by estradiol and progesterone. J Immunol 173:6760–6766 [DOI] [PubMed] [Google Scholar]

[B31] 31. Carlino C, Stabile H, Morrone S, Bulla R, Soriani A, Agostinis C, Bossi F, Mocci C, Sarazani F, Tedesco F, Santoni A, Gismondi A. 2008. Recruitment of circulating NK cells through decidual tissues: a possible mechanism controlling NK cell accumulation in the uterus during early pregnancy. Blood 111:3108–3115 [DOI] [PubMed] [Google Scholar]

[B32] 32. Santoni A, Carlino C, Stabile H, Gismondi A. 2008. Mechanisms underlying recruitment and accumulation of decidual NK cells in uterus during pregnancy. Am J Reprod Immunol 59:417–424 [DOI] [PubMed] [Google Scholar]

[B33] 33. Bulla R, Villa A, Bossi F, Cassetti A, Radillo O, Spessotto P, De Seta F, Guaschino S, Tedesco F. 2005. VE-cadherin is a critical molecule for trophoblast-endothelial cell interaction in decidual spiral arteries. Exp Cell Res 303:101–113 [DOI] [PubMed] [Google Scholar]

[B34] 34. Spessotto P, Bulla R, Danussi C, Radillo O, Cervi M, Monami G, Bossi F, Tedesco F, Doliana R, Colombatti A. 2006. EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall. J Cell Sci 119:4574–4584 [DOI] [PubMed] [Google Scholar]

[B35] 35. Noyes RW, Hertig AT, Rock J. 1975. Dating the endometrial biopsy. Am J Obstet Gynecol 122:262–263 [DOI] [PubMed] [Google Scholar]

[B36] 36. Stabile H, Carlino C, Mazza C, Giliani S, Morrone S, Notarangelo LD, Notarangelo LD, Santoni A, Gismondi A. 2010. Impaired NK-cell migration in WAS/XLT patients: role of Cdc42/WASp pathway in the control of chemokine-induced β2 integrin high-affinity state. Blood 115:2818–2826 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37. van den Heuvel MJ, Horrocks J, Bashar S, Taylor S, Burke S, Hatta K, Lewis JE, Croy BA. 2005. Menstrual cycle hormones induce changes in functional interactions between lymphocytes and decidual vascular endothelial cells. J Clin Endocrinol Metab 90:2835–2842 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38. Chantakru S, Wang WC, van den Heuvel M, Bashar S, Simpson A, Chen Q, Croy BA, Evans SS. 2003. Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones. J Immunol 171:4011–4019 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39. Stepan H, Philipp A, Roth I, Kralisch S, Jank A, Schaarschmidt W, Lössner U, Kratzsch J, Blüher M, Stumvoll M, Fasshauer M. 2011. Serum levels of the adipokine chemerin are increased in preeclampsia during and 6 months after pregnancy. Regul Pept 168:69–72 [DOI] [PubMed] [Google Scholar]

[B40] 40. Duan DM, Niu JM, Lei Q, Lin XH, Chen X. 2011. Serum levels of the adipokine chemerin in preeclampsia. J Perinat Med 40:121–127 [DOI] [PubMed] [Google Scholar]

[B41] 41. Garces MF, Sanchez E, Acosta BJ, Angel E, Ruíz AI, Rubio-Romero JA, Diéguez C, Nogueiras R, Caminos JE. 2012. Expression and regulation of chemerin during rat pregnancy. Placenta 33:373–378 [DOI] [PubMed] [Google Scholar]

[B42] 42. Maheshwari A, Kurundkar AR, Shaik SS, Kelly DR, Hartman Y, Zhang W, Dimmitt R, Saeed S, Randolph DA, Aprahamian C, Datta G, Ohls RK. 2009. Epithelial cells in fetal intestine produce chemerin to recruit macrophages. Am J Physiol Gastrointest Liver Physiol 297:G1–G10 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43. Adams AE, Abu-Amer Y, Chappel J, Stueckle S, Ross FP, Teitelbaum SL, Suva LJ. 1999. 1,25 Dihydroxyvitamin D3 and dexamethasone induce the cyclooxygenase 1 gene in osteoclast-supporting stromal cells. J Cell Biochem 74:587–595 [PubMed] [Google Scholar]

[B44] 44. Verma S, Hiby SE, Loke YW, King A. 2000. Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15. Biol Reprod 62:959–968 [DOI] [PubMed] [Google Scholar]

[B45] 45. Ain R, Trinh ML, Soares MJ. 2004. Interleukin-11 signaling is required for the differentiation of natural killer cells at the maternal-fetal interface. Dev Dyn 231:700–708 [DOI] [PubMed] [Google Scholar]

[B46] 46. Keskin DB, Allan DS, Rybalov B, Andzelm MM, Stern JN, Kopcow HD, Koopman LA, Strominger JL. 2007. TGFβ promotes conversion of CD16+ peripheral blood NK cells into CD16− NK cells with similarities to decidual NK cells. Proc Natl Acad Sci USA 104:3378–3383 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Chemerin Regulates NK Cell Accumulation and Endothelial Cell Morphogenesis in the Decidua during Early Pregnancy

Claudia Carlino

Eleonora Trotta

Helena Stabile

Stefania Morrone

Roberta Bulla

Alessandra Soriani

Maria Luisa Iannitto

Chiara Agostinis

Carlo Mocci

Massimo Minozzi

Cesare Aragona

Giorgia Perniola

Francesco Tedesco

Silvano Sozzani

Angela Santoni

Angela Gismondi

Abstract

Context:

Objective:

Design:

Results:

Conclusions:

Materials and Methods

Cells

Real-time quantitative PCR analysis

Enzyme-linked immunosorbent assay

Immunoprecipitation and Western blotting analysis

ERK activation

Immunofluorescence and flow cytometric analysis

Migration assay

DEC capillary-like tube formation assay and DEC proliferation

Statistical analysis

Results

Chemerin expression and secretion by decidual ST, DEC, and EVT cells

Fig. 1.

Fig. 2.

Chemerin receptor profile on pbNK and dNK cells and chemerin ability to support pbNK and dNK cell migration through DEC and ST cells

Fig. 3.

Fig. 4.

CMKLR1 stimulation enhances pErk activation in dNK cells

Fig. 5.

DEC express CMKLR1 and undergo capillary-like tube formation upon chemerin stimulation

Fig. 6.

Discussion

Supplementary Material

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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