Mast cell recruitment is implicated in many physiological functions and several diseases. It depends on microenvironmental factors, including hormones. We have investigated the effect of progesterone on the migration of HMC-1560 mast cells toward CXCL12, a chemokine that controls the migration of mast cells into tissues. HMC-1560 mast cells were incubated with 1 nM to 1 μM progesterone for 24 h. Controls were run without progesterone. Cell migration toward CXCL12 was monitored with an in vitro assay, and statistical analysis of repeated experiments revealed that progesterone significantly reduced cell migration without increasing the number of apoptotic cells (P = 0.0084, n = 7). Differences between progesterone-treated and untreated cells were significant at 1 μM (P < 0.01, n = 7). Cells incubated with 1 μM progesterone showed no rearrangment of actin filaments in response to CXCL12. Progesterone also reduced the calcium response to CXCL12 and Akt phosphorylation. Cells incubated with progesterone had one-half the control concentrations of CXCR4 (mRNA, total protein, and membrane-bound protein). Progesterone also inhibited the migration of HMC-1560 cells transfected with hPR-B-pSG5 plasmid, which contained 2.5 times as much PR-B as the control. These transfected cells responded differently (P < 0.05, n = 5) from untreated cells to 1 nM progesterone. We conclude that progesterone reduces mast cell migration toward CXCL12 and that CXCR4 may be a progesterone target in mast cells.
- sex steroid
- immune system
mast cells are widely distributed throughout vascularized tissues, certain epithelia, and various mucosal sites, and their accumulation in tissues is associated with many physiological functions. They are multifunctional effector cells of the innate immune system and may also be active in adaptative immunity (13, 27), in the brain (1), in osteoclastic resorption (11), and in the reproductive tract (35, 37). Mast cells are also involved in a variety of diseases, and their presence in airway structures probably contributes to the pathology of asthma and other allergic inflammatory lung diseases (4). Mast cells are abundant at sites of endometriosis and may contribute to the fibrosis and inflammation that is characteristic of endometriosis (39). The recruitment of mast cells is also linked to the development of cancers. Many types of human cancers are infiltrated by mast cells, which migrate toward chemoattractants produced by tumor cells. Their proangiogenic action has been well documented by measuring the release of VEGF and IL-8, which help promote tumor growth and metastasis (41). Last, mastocytosis is a heterogeneous group of disorders characterized by the accumulation of mast cells in organs (7).
A reciprocal relationship between sex steroids and the immune system has been evident for several years, and there is now evidence (2, 17) indicating that ovarian hormones influence the distribution and function of mast cells. Progesterone is naturally produced by the ovary and the placenta. In addition to its key role in reproduction and during pregnancy, this hormone also controls various functions in women, including those of the immune system. Its effects are usually mediated by conventional nuclear progesterone receptors (PRs) that have been identified in the upper airways and bladder mast cells in humans (26, 47), in addition to the mammary gland, uterus, and ovary. Some studies have looked at its specific effect on mast cells. It may influence mast cell activation (26, 43), and there is evidence from in vivo studies on sheep (15) and mice (21) that it reduces the numbers of leukocytes in the endometrium; there are also fewer mast cells in the uterus of ovariectomized rats given progesterone (14). However, little is known of the mechanisms by which progesterone influences mast cell recruitment. Mast cells from the peripheral blood home in on tissues as committed progenitors and develop into mature cells in situ. The presence of mast cells in tissues is due largely to their migration within peripheral tissues in response to locally produced chemokines (4). Some reports have shown that progesterone helps decrease the production of chemokines and chemokine receptors in humans (19, 32, 34, 42, 46). Progesterone inhibits the production of CCL5 and CX3CL1, two chemokines that control the migration of mast cells in connective tissue and at sites of allergic inflammation (19, 32, 34, 46). Progesterone also reduces the densities of CCR5 and CXCR4 receptors on activated T cells (42). CXCL12 is a chemokine produced by epithelial cells and mesothelial cells (9, 12), human trophoblast cells (44), osteoblasts (22), and tumor cells (49). CXCR4 was the first CXCL12 receptor to be described (3, 28). It occurs on circulating mast cell progenitors and on in vitro-developed and leukemic mast cells (23, 29). There is good evidence (5) that CXCR4 is implicated in mast cell recruitment to peripheral tissues. This indicates that the chemokine CXCL12 and its receptor CXCR4 are involved in the local recruitment of mast cells in various tissues under physiological and pathological conditions. Mast cells play a pertinent role in pregnancy, and the recruitment of mast cells to the reproductive tract at the beginning of gestation contributes to myometrial contractility and inadequate uterine contractility that leads to ectopic pregnancies and miscarriages (6). CXCL12 is a feature of early bone development, and the production of CXCL12 by osteoblasts is particularly important in bone physiology. CXCL12 production in the local marrow environment creates a homing gradient that favors the recruitment of mast cells (22). Mast cells accumulate in rat bone marrow after ovariectomy, concomitantly with osteoclast generation, and participate in bone resorption (25). Patients with systemic mastocytosis suffer from flushes, itching, and osteoporosis due to the abnormal accumulation of mast cells in the skin and the bone marrow; the accumulation of mast cells may result from recruitment in situ.
We have therefore investigated the role of progesterone in mast cell recruitment to the chemokine CXCL12. We used HMC-1560 cells, which respond to CXCL12 and bear PRs. We assessed the effect of progesterone on the migration of these cells in response to CXCL12 and found that CXCR4 receptors are possible targets for the hormone.
MATERIALS AND METHODS
Cell line characterization and culture.
The human mast cell line, HMC-1560, was kindly provided by Dr. M. Arock (CNRS UMR 8113, Cachan, France). This cell line is a variant subline of the HMC-1 cell line that was originally established from patients with mast cell leukemia. These nonadhering cells have a point mutation in the juxtamembrane domain of c-kit receptor, causing the replacement of the amino acid Gly-560 by Val. Point mutations in the c-kit proto-oncogene result in the spontaneous activation of c-kit in the absence of ligand. However, these cells have many of the characteristics of tissue mast cells and have been widely used in studies of human mast cell function (40). HMC-1560 cells express PR as determined by RT-PCR and Western blot. We detected a 533-bp product, with the characteristic of PR transcripts, and we revealed on the Western blotting the A isoform (94 kDa) and the B isoform (114 kDa) of the conventional PR. The cells were grown at 37°C with 5% CO2 in MEM without phenol red (Sigma-Aldrich, St-Quentin Fallavier, France) supplemented with 10% of dialyzed fetal calf serum from GIBCO-BRL (Invitrogen, Cergy Pontoise, France). The presence of progesterone in the culture medium was investigated using a competitive one-step immunoassay based on solid-phase antigen-linked technology and direct chemiluminescence detection. The method is linear from 0.05 to 60 ng/ml. The intra-assay and interassay coefficients of variation were <4%. Measurements were done in an automated multianalyte system (Advia-centaur; Bayer HealthCare Diagnostic Division). Samples were automatically diluted with the assay sample diluent before testing. No progesterone has been detected in the tested samples.
Extraction of RNA and RT-PCR procedures.
Total RNA was extracted from cultured cells using the SV total RNA isolation system (Promega France, Cergy-Pontoise, France). The mRNA was transcribed into cDNA using RT primed with random hexamers and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Charbonnières-les-bains, France). The oligonucleotides used for PCR amplification of PR were PR-F 5′-gattcagaagccagccagagc-3′ and PR-R 5′-tgcctctcgcctagttgatt-3′ (3096–3628); those of CXCR4 were CXCR4-F 5′-gaccgctacctggccatc-3′ and CXCR4-R 5′-ggcagccaacaggcgaaga-3′ (485–849); those of CXCL12 were CXCL12-F 5′-gggctcctgggttttgtatt-3′ (667–686) and CXCL12-R 5′-gtcctgagagtccttttgcg-3′ (1064–1083); those of β-actin were β-actin-F 5′-gggtcagaaggattcctatg-3′ and β-actin-R 5′-ggtctcaaacatgatctggg-3′ (214–451). The hybridization probe temperatures were 60°C for PR, 55°C for β-actin, 61°C for CXCL12, and 60°C for CXCR4.
Real-time quantitative PCR was run on a Light Cycler apparatus (Roche Diagnostics, Mannheim, Germany) using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics). The primers for CXCR4 and β-actin were those used for conventional PCR. Expression of the gene coding for CXCR4 was compared with the stable production of β-actin mRNA. PCR was carried out with 3 mM MgCl2 (β-actin) or 4 mM MgCl2 (CXCR4), 0.5 μM of each primer, enzyme mix, 5 μl of template cDNA with a serial dilution range, and H2O added to a final volume of 15 μl. The hybridization step was performed at 58°C for 5 s for β-actin and at 62°C for 5 s for CXCR4. The experiments that were retained did not exceed 0.05 in differences in amplification efficiency between two gene expressions.
Transfer of progesterone receptor gene.
The B isoform of the human PR hPR-B cloned into the pSG5 expression vector was a generous gift from Dr. H. Loosfelt (Kremlin-Bicêtre Hospital, Paris, France), and the A isoform of the human PR hPRA cloned into the pSTC expression vector was a generous gift from Dr. F. Claessens (University of Leuven, Leuven, Belgium). pSG5 (Stratagene) plasmid was used as control. HMC-1560 cells were transfected using the nucleofection technology (Amaxa, Cologne, Germany). Cells were resuspended in 100 μl of solution from nucleofector kit V, following the Amaxa guidelines for cell line transfection. Briefly, 100 μl of 2 × 106 cell suspension mixed with 2 μg of pSG5-hPR-B, 2 μg hPRA-pSTC, or 2 μg of pmax green fluorscent protein (GFP) was transferred to the provided cuvette and nucleofected with an Amaxa Nucleofector apparatus (Amaxa). Cells were transfected using the T-20 pulsing parameter and were immediately transferred into wells containing 1 ml of 37°C prewarmed culture medium in 12-well plates. Nucleofection efficiency was assessed with a GFP reporter vector provided in the Amaxa kit. GFP expression in HMC-1560 cells was determined by FACS analysis at 4 and 16 h after transfection using FACSCan (BD Biosciences). Results are shown as percentage of cells expressing GFP and the mean fluorescence intensity (MFI) of expression. Transfected cells were incubated for 24 h with 1 nM to 1 μM progesterone at 24 h after transfection.
Protein extraction and Western blot analysis.
Cells (1 × 106) were sonicated on ice (4 × 5 s) at 40 Khz and stored in 1× Laemmli buffer (Bio-Rad, Ivry, France). Cell lysates (60 μg protein) were separated by 6–12% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond-ECL; Amersham Pharmacia Biotech Europe). The proteins were probed with anti-CXCR4 antibody (Affinity BioReagents; Interchim, Montluçon, France), antiprogesterone receptor, anti-β-actin antibodies (Santa Cruz Biotechnology, Tebu, Le Perray-en-Yvelines, France), antiphospho-Akt (Ser473), and anti-Akt (Cell Signaling Technology, Ozyme, Saint Quentin-en- Yvelines, France). Primary antibodies were visualized with peroxidase-conjugated anti-rabbit or anti-goat IgG (Santa Cruz Biotechnology) followed by chemiluminescence using the enhanced chemiluminescence kit (Amersham, Saclay, France). Exposed films were scanned on an Imstar densitometer, and the intensity of the signals was measured using FotoLook SA 2.0 software (AGFA-Gevaert, Munich, Germany).
CXCR4 surface antigens were stained by incubating cells for 30 min at 4°C with PE-conjugated monoclonal against CXCR4 (R&D Systems, Abingdon, UK). The Fc receptors on human cells were blocked by incubating cells for 20 min with 10% normal human serum. The cells were washed twice with PBS. PE-conjugated mouse IgG1 isotype was used as a control. Fluorescence emission was measured using Becton-Dickinson FACScan. Data were analyzed with CellQuest software (Becton-Dickinson Immunocytometry Systems).
The cells (500,000 cells in 6 ml) were loaded by incubation for 10 min at room temperature with 2 μM fura 2-AM (Calbiochem France Biochem, Meudon, France) dissolved in Hanks'-HEPES buffer, pH 7.4 (137 mM NaCl, 5.6 mM KCl, 0.441 mM KH2PO4, 0.442 mM Na2HPO4, 0.885 mM MgSO4.7 H2O, 27.7 mM glucose, 1.25 mM CaCl2, and 25 mM HEPES). They were washed three times and transferred to a quartz cuvette that was placed in a temperature-controlled (37°C) Hitachi F-2500 spectrofluorometer (Sciencetec, Les Ulis, France). Recombinant human CXCL12 (rhCXCL12), a gift from F. Arenzana (Institut Pasteur, France), was added directly to the cuvette under continuous stirring. The intracellular calcium concentration ([Ca2+]i) response, in terms of fura 2 fluorescence, was measured using an emission wavelength of 510 nm and excitation wavelengths of 340 and 380 nm and calibrated as previously described (45).
F-actin polymerization assay.
Changes in G- and F-actin were examined by the use of flow cytometric analysis employing FITC-labeled phalloidin, a fluorescent probe that binds specifically to F-actin, and experiments were performed as previously described (48). Briefly, cells (6 × 106 cells/ml) were incubated at 37°C in RPMI medium containing 20 mM HEPES with or without CXCL12 (100 ng/ml). Aliquots (100 μl) of cell suspension were removed every 15 s and added to 400 μl 10−7 M FITC-labeled phalloidin (Sigma), 0.12 mg/ml 1α-lysophosphatidylcholine, and 4.5% formaldehyde in PBS. The fixed cells were analyzed by flow cytometry. Mean fluorescence was measured by FACscan (Becton-Dickinson).
Transwell migration assays.
Migration assays were performed using the Transwell system (Corning Costar, Brumath, France), as previously described (12). Cells were placed in the upper chamber of an 8-μm-pore Costar Transwell (12-well plate format), and CXCL12 (100 ng/ml) was added to chemotaxis buffer in the lower chamber. Cells were allowed to migrate for 3 h, recovered from the lower chamber, and counted using a FACScan.
Annexin V staining.
HMC-1560 cells were stained with annexin V using the TACS Annexin V-FITC apoptosis detection kit (R&D Systems Europe, Lille, France) according to the manufacterer's instructions. Briefly, cells were washed in PBS and incubated in the dark for 15 min at room temperature in 100 μl of binding buffer (100 mM HEPES, pH 7.4, 1.5 M NaCl, 50 mM KCl, 10 mM MgCl2, 18 mM CaCl2) containing 10 μl propidium iodide and 1 μl of annexin V conjugated to FITC and then analyzed on a flow cytometer FACScan. Cells treated with 100 nM dexamethasone were used as a positive control according to manufacturer's instructions.
Data analysis and statistics.
Arithmetic means, median, standard deviation, standard error, and statistical analyses were done using PRISM softwares. To confirm reproducibility of results, four to seven independent experiments were performed and analyzed. Multiple comparisons between groups were made using the nonparametric Friedman test for repeated experiments followed by Dunn's multiple comparison test. Statistical significance was defined as a two-tailed P value of <0.05. Data presented in the figures are means ± SE. Paired groups were compared using the paired t-test.
Effect of progesterone on cell migration toward CXCL12.
HMC-1560 cells bore functional CXCR4 receptors and did not produce CXCL12 (no PCR signal for CXCL12); exogenous CXCL12 (10 to 500 ng/ml) stimulated the dose-dependent chemotaxis of HMC-1560 mast cells, with a maximal effect at 100–300 ng/ml (Fig. 1A). We therefore explored in the following experiments the impact of progesterone on cell migration toward 100 ng/ml CXCL12. Controls were run in the culture medium without CXCL12. The chemotactic index is the difference (fold) in the chemotaxis of CXCL12-treated (+) and untreated (−) samples in the absence of progesterone. The culture medium contained no trace of progesterone (see materials and methods). Progesterone had no significant effect on the chemokinesis of mast cells when there was no CXCL12 in the lower chamber. No effect was detected in cultures containing 1% ethanol, the progesterone vehicle. Cells were incubated with 1 nM to 1 μM progesterone for 24 h before migration assay (Fig. 1B). Incubation with progesterone reduced cell migration to around 30% of control. This effect was repeated in seven independent experiments and demonstrated a mean decrease at 58% of control at 1 μM and at 76% of control at 1 nM (Table 1). Statistics for repeated experiments revealed a significant effect of progesterone (P = 0.0084, n = 7).
Effect of progesterone on CXCL12-mediated F-actin polymerization.
Adding CXCL12 to HMC-1560 cells caused the rapid accumulation of microfilaments actin (F-actin), followed by their depolymerization as quantified by F-actin polymerization assay. Cells were incubated for 24 h with 1 μM progesterone and then treated with CXCL12. Controls were cells cultured without progesterone. Results are expressed as the time-dependent change in fluorescence intensity following the addition of chemokine. A rapid increase in fluorescence intensity in control cells indicated that they responded to CXCL12 by actin polymerization. Cells incubated with 1 μM progesterone showed no rearrangment of actin filaments following the addition of CXCL12 (Fig. 2).
Effect of progesterone on the calcium response to CXCL12.
CXCL12 triggered a transient increase in [Ca2+]i in HMC-1560 cells that fell rapidly after 15 s but remained above the base line. The CXCL12 effect on [Ca2+]i was dose dependent (30–100 ng/ml), with maximal effect at 100 ng/ml (Fig. 3A). This dose increased the [Ca2+]i from its basal level of 147 ± 36 nM (n = 10) to 236 ± 23 nM (n = 10). The [Ca2+]i peak produced by 100 ng/ml CXCL12 was lower in cells incubated for 24 h with 1 μM progesterone (Fig. 3B). This effect was repeated in four independent experiments, demonstrating that progesterone significantly decreased the calcium response of HMC-1560 cells (P < 0.05; Fig. 3C).
Akt phosphorylation in response to CXCL12 is reduced in cells treated with progesterone.
The phosphatidylinositol PI 3-kinase (PI3K) inhibitor wortmannin (1 μM) abolished the migration of HMC-1560 cells toward CXCL12, whereas the MAPK/ERK kinase (MEK) inhibitor PD-98059 (20 μM) did not (Fig. 4A). Akt is a serine/threonine kinase that is involved in the signaling pathway downstream of PI3K, a key biochemical signal for cell migration in response to chemokines (38). CXCL12 (100 ng/ml) caused the phosphorylation of Akt in HMC-1560 cells as early as 5 min and had no effect on total Akt. Incubating cells for 24 h with 1 μM progesterone blocked the CXCL12-induced phosphorylation of Akt (Fig. 4B). This effect was repeated in three independent experiments.
Effect of progesterone on CXCR4 expression.
CXCR4 mRNA was detected in HMC-1560 mast cells by RT-PCR; it gave a 365-bp PCR product. The identity of the CXCR4 PCR product was confirmed by restriction enzyme digestion using BamH1. The amount of CXCR4 mRNA was measured using real-time PCR. Four independent experiments demonstrated a significant decrease in mRNA CXCR4 in cells incubated with 1 μM progesterone for 24 h (P < 0.05, paired t-test; Fig. 5A). Western blots probed with anti-CXCR4 antibody showed a twofold decreased intensity of the 40-kDa band (Fig. 5B), as resulted from three independent experiments. We also measured membrane CXCR4 by flow cytometry (Fig. 5C). The fluorescence histogram of anti-CXCR4-stained cells that had been incubated with 1 μM progesterone was different from that of untreated cells. The histogram shows the MFI of CXCR4-stained cells. The results are those of one representative experiment. The inhibition was ∼40% of control and was demonstrated as significant from five independent experiments (P < 0.05, paired t-test).
We also examined whether the decrease in CXCR4 was associated with an increased number of apoptotic and dead cells by using annexin V propidium iodide double staining. We did not detect any significant increase in apoptotic and dead cells in progesterone-treated cells vs. untreated cells (Fig. 6).
Effect of progesterone in cells overexpressing PR.
We performed a set of five independent experiments with HMC-1560 cells transfected with pSG5-hPR-B plasmid. In transfected cells the amount of PR-B was 2.5-fold higher than in untransfected cells (Fig. 7A). Cells were incubated with 1 nM to 1 μM progesterone for 24 h before migration assay. Incubation with progesterone reduced cell migration of transfected cells in the five independent experiments (Fig. 7B). Statistics for repeated experiments revealed a significant effect of progesterone at 1 nM (P = 0.0394, n = 5; Table 2). Further experiments were run with with HMC-1560 cells transfected with pSCT-hPR-A plasmid. These cells contained twice as much PR-A as controls (untransfected cells). Transfected cells did not respond differently from untransfected cells to 1 μM and to 1 nM progesterone (data not shown).
There is much evidence that ovarian hormones influence the function of mast cells (14, 15, 21, 26, 43). However, the role of steroid hormones on their tissue recruitment still remains poorly defined. Our results demonstrate that progesterone reduces the migration of human HMC-1560 mast cells toward CXCL12, and these findings bring new insights on hormone control of mast cell recruitment in tissues.
We found that HMC-1560 cell migration was reduced after exposure to 1 nM to 1 μM progesterone for 24 h. The inhibitory effect was visible at 1 nM and at 1 μM. At 1 μM, a dose usually considered as supraphysiological, differences were statistically significant and the effects more reproducible than at 1 nM, a dose usually considered as physiological. The high concentration of progesterone (1 μM), which was needed to detect a significant inhibitory effect of progesterone on HMC-1560 cell migration, was probably due to the small amount of PR produced by these cells. That was suggested by some further experiments that were driven in cells transfected with pSG5-hPR-B plasmid. In these cells the amount of PR-B, which is often considered as the active PR isoform (16), was 2.5-fold increased; the inhibitory effect of 1 nM progesterone on migration toward CXCL12 was more reproducible, and differences with untreated cells were statistically significant. In contrast with the data obtained from PR-B overexpressing cells, the migratory response of PR-A overexpressing cells was not significantly different from untransfected cells. Hence, the action of physiological doses of progesterone may depend on the amount of PR-B in mast cells. Indeed, we are aware that HMC-1560 cells are far from in situ mast cells, and the true amount of PR-B in mast cells in humans remains widely unknown and uneasy to assess. Furthermore, PR expression is controlled by its hormone ligand itself, and the amount of PR in mast cells may depend on the serum level of progesterone and thus might be sex dependent.
Transitory transfected cells are not the best-fitted model to investigate the cellular and molecular mechanisms of progesterone action. Thus we run all of the following experiments with HMC-1560 cells that were treated with 1 μM progesterone, the only dose that prompted reproducible effects in cells that did not overexpress PR-B. Migratory responses are associated with cytoskeletal reorganization, and changes in cell shape are crucial in cell motility. Formation and depolarization of actin microfilaments were clearly shown in HMC-1560 mast cells in response to CXCL12, and F-actin polymerization in response to CXCL12 was abolished in cells treated with progesterone. The effect of progesterone on actin polymerisation probably contributed to decrease cell migration, but it was not associated with a complete inhibition of migration. We cannot exclude that the stimulated cytoskeleton rearrangement may also be controlled by distinct pathways of F-actin polymerization (8).
The CXCL12-induced chemotactic response of HMC-1560 cells was accompanied by an intracellular calcium peak, which is a key biochemical signal for cell migration in response to chemokines (20). The PI3K inhibitor wortmannin (1 μM) blocked Akt phosphorylation and inhibited the migration of HMC-1560 cells. In contrast, although the MAPK pathway, including the p42/44 MAPK, has been implicated in the CXCL12-mediated change in the migration of some cell types (10, 24, 31), the MEK inhibitor PD-98059 (20 μM) did not block the migration of HMC-1560 cells toward CXCL12. Thus it appears that the PI3K/Akt pathway was the main pathway implicated in HMC-1560 cell migration in response to CXCL12. Indeed, CXCL12 exposure induced Akt phosphorylation. In contrast, in progesterone-treated cells the calcium response to CXCL12 was reduced and the CXCL12-induced Akt phosphorylation abolished. These data reveal a discrepancy between the calcium response, which was only reduced, and the Akt phosphorylation, which was completely abolished. Thus we suggest that progesterone should further act on phosphatase and tensin homolog deleted on chromosome 10, which inhibits the PI3K/Akt signaling pathway and is also one of the signaling proteins that progesterone may directly upregulate (18). Indeed, further evidence will be needed to conclude.
The chemotactic effect of CXCL12 on target cells may also depend on the amount of CXCR4 at the cell surface (30). We measured CXCR4 expression in cells treated with progesterone and found that the amounts of CXCR4 (mRNA, total protein) were two- to threefold reduced in cells incubated with progesterone. In parallel, the amount of membrane-bound protein measured by flow cytometry was about 40% of control. One can assume that less CXCR4 leads to weaker CXCL12-mediated signaling and changes in the responsiveness of cells to CXCL12 in terms of migration. Although we cannot determine whether the decrease in CXCR4 is due to the direct action of progesterone on the transcription of the CXCR4 gene or to an indirect consequence of progesterone action, our results reveal the inhibition of CXCR4 expression as a possible mechanism to explain progesterone's migration-inhibitory effect. It remains that the recruitment of mast cells in tissues also depends on the local production of a variety of chemoattractants (5) so that a decrease in the numbers of CXCR4 molecules in mast cells may have only a small impact on mast cell recruitment in certain tissues.
Few reports have examined the effects of progesterone on chemokine receptor expression in humans. Sentman et al. (36) found that progesterone induced CXCL10 and CXCL11 expression in endometrium. Vassiliadou et al. (42) demonstrated that progesterone reduced the density of CCR5 receptor on activated T cells and reduced the induction of CXCR4 in T lymphocytes following IL-2 treatment. Progesterone upregulates RANTES production in CD4 and CD8 endometrial lymphocytes, which prompts a decrease in CXCR4 mRNA (33). The present report is the first study that reveals that progesterone reduces the amount of mRNA and protein CXCR4 in mast cells. However, HMC-1560 cells may exhibit different chemokine receptor profiles, and human mast cells from various tissues show significant heterogeneity, and indeed we have no evidence that progesterone reduces CXCR4 expression in mast cells in vivo.
A variety of processes depend on mast cell infiltration, including the deposition of fibrous tissue and angiogenesis, and mast cell recruitment in tissues is associated with many physiological processes. Their increase in tissues also occurs in allergy and in diseases such as endometriosis, parasite infestations, tumor development, and mastocytosis. Our findings provide new evidence showing how progesterone acts in mast cells and that CXCR4 receptors are possible targets for progesterone in these cells and, hence, in the control of mast cell recruitment. Our present findings further suggest that sex and postmenopausal state might influence mast cell recruitment into tissues.
This work was funded in part by a grant from The Association Française pour les Initiatives de Recherche sur le Mastocyre et les Martocytose (AFIRMM).
We thank Dr Roman Krzysiek, INSERM U-764, for helpful discussion. The English text was edited by Dr. Owen Parkes.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by American Physiological Society