AJP - Endo Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 292: E1410-E1417, 2007; doi:10.1152/ajpendo.00286.2006
0193-1849/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belot, M.-P.
Right arrow Articles by Machelon, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belot, M.-P.
Right arrow Articles by Machelon, V.

Progesterone reduces the migration of mast cells toward the chemokine stromal cell-derived factor-1/CXCL12 with an accompanying decrease in CXCR4 receptors

Marie-Pierre Belot,1 Latifa Abdennebi-Najar,1,2 Françoise Gaudin,1 Michèle Lieberherr,3 Véronique Godot,1 Joelle Taïeb,4 Dominique Emilie,1 and Véronique Machelon1

1Institut National de la Santé et de la Recherche Médicale, Univ Paris-Sud 11, Institut Fédératif de Recherche 13, Assistance Publique-Hôpitaux de Paris, Hôpital Antoine Béclère, Service de Microbiologie-Immunologie Biologique, Clamart; 2Institut Supérieur d'Agriculture de Beauvais, Beauvais; 3Institut National de la Recherche Agronomique, Laboratoire de Nutrition et de Sécurité alimentaire, Jouy en Josas; and 4Assistance Publique-Hôpitaux de Paris, Hôpital Antoine Béclère, Service de Biochimie, Hôpital Antoine Béclère, Clamart, France

Submitted 16 June 2006 ; accepted in final form 12 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

chemotaxis; 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 beta-actin were beta-actin-F 5'-gggtcagaaggattcctatg-3' and beta-actin-R 5'-ggtctcaaacatgatctggg-3' (214–451). The hybridization probe temperatures were 60°C for PR, 55°C for beta-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 beta-actin were those used for conventional PCR. Expression of the gene coding for CXCR4 was compared with the stable production of beta-actin mRNA. PCR was carried out with 3 mM MgCl2 (beta-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 beta-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 x 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 x 106) were sonicated on ice (4 x 5 s) at 40 Khz and stored in 1x 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-beta-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).

Flow cytometry. 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).

Calcium measurement. 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 x 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 1{alpha}-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.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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).


Figure 1
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 1. Effect of progesterone on cell migration toward CXCL12. A: CXCR4 but not CXCL12 mRNA was detected by RT-PCR in HMC-1560 cells (lane 1, positive control; lane 2, HMC-1560; lane 3, H2O) that respond to increasing CXCL12 doses from 0 to 500 ng/ml in a dose-dependent manner. B: HMC-1560 cells were incubated with 1 nM to 1 µM progesterone (P) for 24 h and without P and then allowed to migrate toward 100 ng/ml CXCL12. Controls were run in culture medium without CXCL12. The migratory response to CXCL12 was measured by the chemotactic index, which is the difference (fold) in the chemotaxis of CXCL12-treated (+) and untreated (–) samples in the absence of P. Histograms are results expressed as the mean of 3 replicates ± SE of a representative experiment of 7.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Cell migration toward CXCL12 in cells treated with 1 nM and 1 µM P and without P

 
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).


Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Effect of P on F-actin polymerization in response to CXCL12. HMC-1560 cells were incubated with 1 µM P for 24 h and without P (control); 100 ng/ml CXCL12 was then added. Results from 5 independent experiments (means ± SE) are expressed as the change over time in the fluorescence intensity in cells following the addition of CXCL12; 100% corresponds to the baseline level.

 
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).


Figure 3
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 3. Transient increase in intracellular calcium concentration ([Ca2+]i) in response to CXCL12. A: increasing (10–100 ng/ml) CXCL12 doses triggered a dose-dependent transient increase in [Ca2+]i in HMC-1560 cells. B: CXCL12 triggered a transient increase in [Ca2+]i that fell rapidly after 15 s but remained above the baseline. Cells incubated with 1 µM P for 24 h produced a smaller calcium pike. The traces are those of 1 representative of 4 independent experiments. C: histograms are results expressed as the mean ± SE of 4 independent experiments. Differences between cells treated with 1 µM P and untreated cells calculated using paired t-test were significant at P = 0.05 (*).

 
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.


Figure 4
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 4. Effect of P on Akt phosphorylation induced by CXCL12. A: HMC-1560 cells were incubated with 1 µM wortmannin, a phosphatidylinositol 3-kinase inhibitor, and with 20 µM PD-98059, a MAPK/ERK kinase inhibitor, for 20 min and then allowed to migrate toward 100 ng/ml CXCL12 for 3 h. Results are means ± SE of triplicates of 1 experiment. They reveal a significant inhibitory effect of wortmannin at P < 0.001 (***), whereas PD-98059 has no effect on the migratory response of HMC-1560 cells. B: HMC-1560 cells were incubated for 24 h with or without 1 µM P and then with 100 ng/ml CXCL12 for 5, 10, and 15 min. Equivalent amounts of protein (60 µg) were separated by SDS-PAGE (8% resolving gel) and probed with antibodies against phosphorylated Akt (Ser473) and total Akt. Representative immunoblot of 3 independent cultures.

 
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).


Figure 5
View larger version (33K):
[in this window]
[in a new window]

  		Fig. 5. Effect of P on CXCR4 expression. A: HMC-1560 cells were incubated with or without 1 µM P for 24 h. The amount of mRNA was measured by real-time PCR using primers specific for CXCR4. Histograms represent means ± SE of 4 experiments. **P < 0.01, paired t-test. B: CXCR4 immunoblot revealing a 40-kDa band for CXCR4. The intensity of the CXCR4 signal in cells incubated for 24 h with 1 µM P was lower than in controls without P. Data are those of 1 representative experiment. Histograms are the results of 3 independent experiments calculated as %control (100%, without P). *Differences were significant at P < 0.05 using the paired t-test. C: fluorescence histogram of cell surface CXCR4 measured by flow cytometry. HMC-1560 cells were incubated with (dashed line) or without (solid line) 1 µM P for 24 h. The isotype control is shown in gray. Histograms are the mean fluorescence intensities of P-treated cells and untreated cells (control); results are means ± SE of 5 independent experiments. Differences calculated by paired t-test were significant at P < 0.05.

 
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).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Measurement of apoptosis in P-treated cells. Apoptotic cells were detected by flow cytometry using the TACS Annexin V-FITC apoptosis detection kit. Cells were incubated without P (culture medium) or with 1 µM P for 48 h. Cells treated with 100 nM dexamethasone (Dex) were used as a positive control of apoptosis according to the manufacturer's instructions. Each subpopulation of cells was defined as viable cells (annexin V–/PI–; bottom left), early apoptotic cells (annexin V+/PI–; bottom right), late apoptotic cells (annexin V+/PI+; top right), and dead cells (PI+; top left). Results were those of 1 representative experiment.

 
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).


Figure 7
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 7. Effect of P on P receptor (PR)-B-overexpressing cells. A: the amount of PR-A and PR-B measured by Western blot in HMC-1560 (lane 1) and in HMC-1560 cells transiently transfected with pSG5-hPR-B (lane 2). B: HMC-1560 cells transfected with pSG5-hPR-B were incubated with 1 nM to 1 µM P for 24 h and without P and then allowed to migrate toward 100 ng/ml CXCL12. Controls were run in culture medium without CXCL12. The migratory response to CXCL12 was measured by the chemotactic index, which is the difference (fold) in the chemotaxis of CXCL12-treated (+) and untreated (–) samples in the absence of P. Histograms are results expressed as the mean of 3 replicates ± SE of a representative experiment out of 3.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Cell migration toward CXCL12 in pSG5-hPR-B-transfected cells treated with 1 nM and 1 µM P and without P

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
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).


   ACKNOWLEDGMENTS
 
We thank Dr Roman Krzysiek, INSERM U-764, for helpful discussion. The English text was edited by Dr. Owen Parkes.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Machelon INSERM-U764, 32 rue des Carnets, Clamart, F-92140 France (e-mail: veronique.machelon{at}u-psud.fr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Asarian L, Yousefzadeh E, Silverman AJ, Silver R. Stimuli from conspecifics influence brain mast cell population in male rats. Horm Behav 42: 1–12, 2002.[CrossRef][Medline]
  2. Beagley KW, Gockel CM. Regulation of innate and adaptive immunity by the female sex hormones oestradiol and progesterone. FEMS Immunol Med Microbiol 38: 13–22, 2003.[CrossRef][ISI][Medline]
  3. Bleul CC, Farzan M, Choe H, Parolin C, Clark-Lewis I, Sodroski J, Springer TA. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382: 829–833, 1996.[CrossRef][Medline]
  4. Brightling CE, Bradding P, Pavord ID, Wardlaw AJ. New insights into the role of the mast cell in asthma. Clin Exp Allergy 33: 550–556, 2003.[CrossRef][ISI][Medline]
  5. Brightling CE, Kaur D, Berger P, Morgan AJ, Wardlaw AJ, Bradding P. Differential expression of CCR3 and CXCR3 by human lung and bone marrow-derived mast cells: implications for tissue mast cell migration. J Leukoc Biol 77: 759–766, 2005.[Abstract/Free Full Text]
  6. Bulletti C, de Ziegler D. Uterine contractility and embryo implantation. Curr Opin Obstet Gynecol 17: 265–276, 2005.[ISI][Medline]
  7. Castells MC. Mastocytosis: classification, diagnosis, and clinical presentation. Allergy Asthma Proc 25: 33–36, 2004.[ISI][Medline]
  8. Chodniewicz D, Alteraifi AM, Zhelev DV. Experimental evidence for the limiting role of enzymatic reactions in chemoattractant-induced pseudopod extension in human neutrophils. J Biol Chem 279: 24460–24466, 2004.[Abstract/Free Full Text]
  9. Coulomb-L'Hermin A, Amara A, Schiff C, Durand-Gasselin I, Foussat A, Delaunay T, Chaouat G, Capron F, Ledee N, Galanaud P, Arenzana-Seisdedos F, Emilie D. Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells. Proc Natl Acad Sci USA 96: 8585–8590, 1999.[Abstract/Free Full Text]
  10. Floridi F, Trettel F, Di Bartolomeo S, Ciotti MT, Limatola C. Signalling pathways involved in the chemotactic activity of CXCL12 in cultured rat cerebellar neurons and CHP100 neuroepithelioma cells. J Neuroimmunol 135: 38–46, 2003.[CrossRef][ISI][Medline]
  11. Fouilloux I, Duplan MB, Baroukh B, Cherruau M, Saffar JL, Lesclous P. Mast cell activation and degranulation occur early during induction of periosteal bone resorption. Bone 38: 59–66, 2006.[Medline]
  12. Foussat A, Balabanian K, Amara A, Bouchet-Delbos L, Durand-Gasselin I, Baleux F, Couderc J, Galanaud P, Emilie D. Production of stromal cell-derived factor 1 by mesothelial cells and effects of this chemokine on peritoneal B lymphocytes. Eur J Immunol 31: 350–359, 2001.[CrossRef][ISI][Medline]
  13. Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, Tsai M. Mast cells as "tunable" effector and immunoregulatory cells: recent advances. Annu Rev Immunol 23: 749–786, 2005.[CrossRef][ISI][Medline]
  14. Gibbons AF, Chang MC. Number of mast cells in the rat uterus with special reference to its relation to hormonal treatment and decidual response. Biol Reprod 6: 193–203, 1972.[Abstract]
  15. Gottshall SL, Hansen PJ. Regulation of leucocyte subpopulations in the sheep endometrium by progesterone. Immunology 76: 636–641, 1992.[ISI][Medline]
  16. Graham JD, Clarke CL. Expression and transcriptional activity of progesterone receptor A and progesterone receptor B in mammalian cells. Breast Cancer Res 4: 187–190, 2002.[CrossRef][ISI][Medline]
  17. Grossman CJ. Regulation of the immune system by sex steroids. Endocr Rev 5: 435–455, 1984.[ISI][Medline]
  18. Guzeloglu-Kayisli O, Kayisli UA, Al-Rejjal R, Zheng W, Luleci G, Arici A. Regulation of PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression by estradiol and progesterone in human endometrium. J Clin Endocrinol Metab 88: 5017–5026, 2003.[Abstract/Free Full Text]
  19. Hannan NJ, Jones RL, Critchley HO, Kovacs GJ, Rogers PA, Affandi B, Salamonsen LA. Coexpression of fractalkine and its receptor in normal human endometrium and in endometrium from users of progestin-only contraception supports a role for fractalkine in leukocyte recruitment and endometrial remodeling. J Clin Endocrinol Metab 89: 6119–6129, 2004.[Abstract/Free Full Text]
  20. Henschler R, Piiper A, Bistrian R, Mobest D. SDF-1alpha-induced intracellular calcium transient involves Rho GTPase signalling and is required for migration of hematopoietic progenitor cells. Biochem Biophys Res Commun 311: 1067–1071, 2003.[CrossRef][ISI][Medline]
  21. Hunt JS, Miller L, Platt JS. Hormonal regulation of uterine macrophages. Dev Immunol 6: 105–110, 1998.[Medline]
  22. Jung Y, Wang J, Schneider A, Sun YX, Koh-Paige AJ, Osman NI, McCauley LK, Taichman RS. Regulation of SDF-1 (CXCL12) production by osteoblasts; a possible mechanism for stem cell homing. Bone 38: 497–508, 2006.[Medline]
  23. Juremalm M, Hjertson M, Olsson N, Harvima I, Nilsson K, Nilsson G. The chemokine receptor CXCR4 is expressed within the mast cell lineage and its ligand stromal cell-derived factor-1alpha acts as a mast cell chemotaxin. Eur J Immunol 30: 3614–3622, 2000.[CrossRef][ISI][Medline]
  24. Lazarini F, Casanova P, Tham TN, De Clercq E, Arenzana-Seisdedos F, Baleux F, Dubois-Dalcq M. Differential signalling of the chemokine receptor CXCR4 by stromal cell-derived factor 1 and the HIV glycoprotein in rat neurons and astrocytes. Eur J Neurosci 12: 117–125, 2000.[CrossRef][ISI][Medline]
  25. Lesclous P, Guez D, Baroukh B, Vignery A, Saffar JL. Histamine participates in the early phase of trabecular bone loss in ovariectomized rats. Bone 34: 91–99, 2004.[Medline]
  26. Letourneau R, Pang X, Sant GR, Theoharides TC. Intragranular activation of bladder mast cells and their association with nerve processes in interstitial cystitis. Br J Urol 77: 41–54, 1996.[ISI][Medline]
  27. Marshall JS, Jawdat DM. Mast cells in innate immunity. J Allergy Clin Immunol 114: 21–27, 2004.[CrossRef][ISI][Medline]
  28. Oberlin E, Amara A, Bachelerie F, Bessia C, Virelizier JL, Arenzana-Seisdedos F, Schwartz O, Heard JM, Clark-Lewis I, Legler DF, Loetscher M, Baggiolini M, Moser B. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature 382: 833–835, 1996.[CrossRef][Medline]
  29. Ochi H, Hirani WM, Yuan Q, Friend DS, Austen KF, Boyce JA. T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro. J Exp Med 190: 267–280, 1999.[Abstract/Free Full Text]
  30. Odemis V, Moepps B, Gierschik P, Engele J. Interleukin-6 and cAMP induce stromal cell-derived factor-1 chemotaxis in astroglia by up-regulating CXCR4 cell surface expression. Implications for brain inflammation. J Biol Chem 277: 39801–39808, 2002.[Abstract/Free Full Text]
  31. Okabe S, Fukuda S, Broxmeyer HE. Src kinase, but not the src kinase family member p56lck, mediates stromal cell-derived factor 1alpha/CXCL12-induced chemotaxis of a T cell line. J Hematother Stem Cell Res 11: 923–928, 2002.[CrossRef][ISI][Medline]
  32. Papadopoulos EJ, Fitzhugh DJ, Tkaczyk C, Gilfillan AM, Sassetti C, Metcalfe DD, Hwang ST. Mast cells migrate, but do not degranulate, in response to fractalkine, a membrane-bound chemokine expressed constitutively in diverse cells of the skin. Eur J Immunol 30: 2355–2361, 2000.[CrossRef][ISI][Medline]
  33. Ramhorst R, Patel R, Corigliano A, Etchepareborda JJ, Fainboim L, Schust D. Induction of maternal tolerance to fetal alloantigens by RANTES production. Am J Reprod Immunol 56: 302–311, 2006.[CrossRef][ISI][Medline]
  34. Romagnani P, De Paulis A, Beltrame C, Annunziato F, Dente V, Maggi E, Romagnani S, Marone G. Tryptase-chymase double-positive human mast cells express the eotaxin receptor CCR3 and are attracted by CCR3-binding chemokines. Am J Pathol 155: 1195–1204, 1999.[Abstract/Free Full Text]
  35. Salamonsen LA, Zhang J, Brasted M. Leukocyte networks and human endometrial remodelling. J Reprod Immunol 57: 95–108, 2002.[CrossRef][ISI][Medline]
  36. Sentman CL, Meadows SK, Wira CR, Eriksson M. 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, 2004.[Abstract/Free Full Text]
  37. Sivridis E, Giatromanolaki A, Agnantis N, Anastasiadis P. Mast cell distribution and density in the normal uterus—metachromatic staining using lectins. Eur J Obstet Gynecol Reprod Biol 98: 109–113, 2001.[CrossRef][ISI][Medline]
  38. Sotsios Y, Ward SG. Phosphoinositide 3-kinase: a key biochemical signal for cell migration in response to chemokines. Immunol Rev 177: 217–235, 2000.[CrossRef][ISI][Medline]
  39. Sugamata M, Ihara T, Uchiide I. Increase of activated mast cells in human endometriosis. Am J Reprod Immunol 53: 120–125, 2005.[CrossRef][ISI][Medline]
  40. Sundstrom M, Vliagoftis H, Karlberg P, Butterfield JH, Nilsson K, Metcalfe DD, Nilsson G. Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene. Immunology 108: 89–97, 2003.[CrossRef][ISI][Medline]
  41. Theoharides TC, Conti P. Mast cells: the Jekyll and Hyde of tumor growth. Trends Immunol 25: 235–241, 2004.[CrossRef][ISI][Medline]
  42. Vassiliadou N, Tucker L, Anderson DJ. Progesterone-induced inhibition of chemokine receptor expression on peripheral blood mononuclear cells correlates with reduced HIV-1 infectability in vitro. J Immunol 162: 7510–7518, 1999.[Abstract/Free Full Text]
  43. Vliagoftis H, Dimitriadou V, Theoharides TC. Progesterone triggers selective mast cell secretion of 5-hydroxytryptamine. Int Arch Allergy Appl Immunol 93: 113–119, 1990.[ISI][Medline]
  44. Wu X, Li DJ, Yuan MM, Zhu Y, Wang MY. The expression of CXCR4/CXCL12 in first-trimester human trophoblast cells. Biol Reprod 70: 1877–1885, 2004.[Abstract/Free Full Text]
  45. Zagar Y, Chaumaz G, Lieberherr M. Signaling cross-talk from Gbeta4 subunit to Elk-1 in the rapid action of androgens. J Biol Chem 279: 2403–2413, 2004.[Abstract/Free Full Text]
  46. Zhao D, Lebovic DI, Taylor RN. Long-term progestin treatment inhibits RANTES (regulated on activation, normal T cell expressed and secreted) gene expression in human endometrial stromal cells. J Clin Endocrinol Metab 87: 2514–2519, 2002.[Abstract/Free Full Text]
  47. Zhao XJ, McKerr G, Dong Z, Higgins CA, Carson J, Yang ZQ, Hannigan BM. Expression of oestrogen and progesterone receptors by mast cells alone, but not lymphocytes, macrophages or other immune cells in human upper airways. Thorax 56: 205–211, 2001.[Abstract/Free Full Text]
  48. Zou W, Foussat A, Houhou S, Durand-Gasselin I, Dulioust A, Bouchet L, Galanaud P, Levy Y, Emilie D. Acute upregulation of CCR-5 expression by CD4+ T lymphocytes in HIV-infected patients treated with interleukin-2. ANRS 048 IL-2 Study Group. AIDS 13: 455–463, 1999.[CrossRef][ISI][Medline]
  49. Zou W, Machelon V, Coulomb-L'Hermin A, Borvak J, Nome F, Isaeva T, Wei S, Krzysiek R, Durand-Gasselin I, Gordon A, Pustilnik T, Curiel DT, Galanaud P, Capron F, Emilie D, Curiel TJ. Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat Med 7: 1339–1346, 2001.[CrossRef][ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belot, M.-P.
Right arrow Articles by Machelon, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belot, M.-P.
Right arrow Articles by Machelon, V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.