Estradiol and progesterone regulate the migration of mast cells from the periphery to the uterus and induce their maturation and degranulation - PubMed (original) (raw)

Estradiol and progesterone regulate the migration of mast cells from the periphery to the uterus and induce their maturation and degranulation

Federico Jensen et al. PLoS One. 2010.

Abstract

Background: Mast cells (MCs) have long been suspected as important players for implantation based on the fact that their degranulation causes the release of pivotal factors, e.g., histamine, MMPs, tryptase and VEGF, which are known to be involved in the attachment and posterior invasion of the embryo into the uterus. Moreover, MC degranulation correlates with angiogenesis during pregnancy. The number of MCs in the uterus has been shown to fluctuate during menstrual cycle in human and estrus cycle in rat and mouse indicating a hormonal influence on their recruitment from the periphery to the uterus. However, the mechanisms behind MC migration to the uterus are still unknown.

Methodology/principal findings: We first utilized migration assays to show that MCs are able to migrate to the uterus and to the fetal-maternal interface upon up-regulation of the expression of chemokine receptors by hormonal changes. By using a model of ovariectomized animals, we provide clear evidences that also in vivo, estradiol and progesterone attract MC to the uterus and further provoke their maturation and degranulation.

Conclusion/significance: We propose that estradiol and progesterone modulate the migration of MCs from the periphery to the uterus and their degranulation, which may prepare the uterus for implantation.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. MCs and human trophoblast cells strongly interact with each other.

Co-culture system between primary first trimester trophoblast cells or choriocarcinoma trophoblast cell line (JEG-3) with HMC-1 MC line (Fig. 1A–B and Fig. 1C respectively). Big adherent cells represent trophoblasts (indicated with an open arrow) whereas smaller, round cells attached to them are MCs (indicated with a closed arrow). As negative control we co-cultured a human keratinocyte cell line (HaCaT) with HMC-1 cells. Fig. 1D shows the complete absent of HMC-1 cells attached to the HaCaT cell after washing. Fig. 1E and Fig. 1G show HMC-1 CD117+ cells as stained by immunofluorescence co-cultured either with human trophoblasts or JEG-3 cells respectively. F and H pictures were done by light microscopy on the same area than Fig. E and G respectively DAPI was used as counterstaining. Fig. 1A, C, D were done with a 200× total magnification; Fig. 1B was done with a 400× total magnification and Fig. F, H were done with a 1000× total magnification under light microscopy using the Axiovision Rel 4–6 program (Zeiss AX 10 microscope). Fig. E, G were done with a 1000X total magnification by using the HXP-120 Light Source for Fluorescence Illumination and the Axiovision Rel 4–6 program (Zeiss AX 10 microscope).

Figure 2. Human MCs actively migrate towards human trophoblast cells as well as to uterine cells treated with hormones.

Using a two-chamber trans-well system, the migration of HMC-1 cells to primary first trimester trophoblasts (Fig. 2A) or JEG-3 cells (Fig. 2B) as well as to uterine cell line (AN3-CA) (Fig. 2C) upon hormonal treatment was analyzed at different time points (0, 4, 8, 24, and 48 h) by determining the relative number of HMC-1-CD117+ cells present in the lower part of the system referred to the total number of CD117+ cells in the upper and lower chambers. Spontaneous migration (ca. 5%) was subtracted for all time points. Data are representative of four experiments done in duplicates each. *:p<0.05 and **:p<0.01 as analyzed by ANOVA test followed by Tukey's test.

Figure 3. BMMCs and HMC-1 express high levels of CD117 as well as estradiol and progesterone receptors.

Dot plots of cultured BMMCs (A) or HCM-1 cells (B) stained for CD117/FceRI as and CD117 as analyzed by flow cytometry. (C) BMMCs and HCM-1 cells (D) were stained with toluidine blue and they present typical features of MCs as analyzed by light microscopy using a total augmentation of 1000 X (Zeiss AX 10/Axiovision Rel 4.6). (E) and (F) represent western blots for estrogen receptor (ERα) and beta (ERβ) while (G) represent western blot for progesterone receptor (PR), respectively for HCM-1 (a) and BMMCs cells (b). β-actin served as house keeping gene.

Figure 4. Estradiol and progesterone treatment in vitro results in the up-regulation of chemokine receptors in MCs.

A–B: Chemokine receptor expression CCR4 (A) or CCR5 (B) on HMC-1 cells treated for 1 h with different physiological concentrations of estradiol (E2). E2 significantly up-regulated the expression of CCR4 and CCR5 in HMC-1 cells. C-D: Chemokine receptor expression CCR4 (C) or CCR5 (D) on HMC-1 cells treated for 1 h with a combination of E2P4. E2P4 treatment induced a significant up-regulation of both, CCR4 and CCR5 on HMC-1. E–H: Chemokine receptor expression CCR3 (E and G) or CCR5 (F and H) on BMMCs treated for 1 h with different physiological concentrations of E2 (E and F) or progesterone (P4; G and H). Both hormones induced an up-regulation of CCR3 (E, G) and CCR5 (F, H) in BMMCs after 1 h of treatment. I–J: Chemokine receptor expression CCR3 (I) or CCR5 (J) on BMMCs treated for 1 h with E2P4. Treatment of BMMCs with a combination of both hormones induced a significant increment on the expression of CCR3 and CCR5. The data are representative of four experiments performed in duplicates or triplicates. Chemokine receptor expression was analyzed by flow cytometry and data are expressed as percentage of cells expressing the chemokine receptors analyzed as mean ± S.E.M. *:p<0.05 as analyzed by ANOVA test followed by Tukey's test (A–B, E–H). The Mann-Whitney-U test for two particular groups was used (C–D, I–J). In all cases the expression of chemokines receptors for each dose vs. control was compared. K: BMMCs migration toward CCL5 (3 hours) is expressed as percentage of cells that migrated towards the CCL5 gradient from the total amount of cells. Spontaneous migration was subtracted. The data are representative of three experiments and are expressed as means ± S.E.M. *:p<0.05, **: p<0.01 and ***: p<0.001 as analyzed by the Mann-Whitney-U test.

Figure 5. Estradiol and progesterone treatment in vitro as well as their combined application up-regulate the expression of human tryptase in HMC-1 cells.

Human mast cell tryptase expression on HMC-1 cells treated for 1 h either with estradiol (E2; A), progesterone (P4; B) or (E2 + P4; C) was analyzed by Western Blot using GAPDH as house keeping gene. Physiological concentrations of both estradiol (A) and progesterone (B) corresponding to their concentrations towards middle menstrual cycle or just after ovulation takes place respectively as well as the combination of both hormones at their most effective concentration, significantly up-regulated the expression of human tryptase on HMC-1 cells (C). Data are expressed as Tryptase/GAPDH ratio (mean ± S.E.M.) *:p<0.05 as analyzed by ANOVA test followed by Tukey's test (A–B) or The Kruskall-Wallis test followed by Mann-Whitney-U test (C). Each experiment was done in triplicate; the blots are representative from three independent experiments. Tryptase expression in HMC-1 cells after hormonal treatment was compared to the controls in each case.

Figure 6. Estradiol and progesterone as well as a estradiol-progesterone combined treatment induce in vitro HMC-1 degranulation.

Physiological concentrations of both, estradiol (E2; A) and progesterone (P4; B) significantly induced HMC-1 degranulation after 1 h of treatment as analyzed by measuring chromophore p-nitroaniline (pNA) produced after cleavage from the labeled substrate tosyl-gly-pro-lys-pNA by tryptase present in the supernatant of HCM-1 cells employing a MC-degranulation kit. (C) shows the degranulation observed after in vitro application of both, estradiol and progesterone. Positive controls included the treatment of HMC-1 cells with calcium ionophore to induce degranulation while negative controls were cells treated with calcium ionophore plus protamine inhibitor. Data are expressed as mean ± S.E.M of the optical density at 405 nm as measured in the supernatant of treated cells. *:p<0.05, **:p<0.01 and ***:p<0.001 as analyzed by ANOVA test followed by Tukey's test. Each experiment was done in triplicate.

Figure 7. Female hormones induce the migration of MCs to the uterus, their maturation and degranulation Virgin ovariectomized C57BL/6J mice were treated with estradiol (E2), progesterone (P4) or estradiol + progesterone (E2 + P4).

The number of MCs was quantified in 10 fields of uterine tissue from all animals (n = 5/group). MC visualization was performed by tolouidine blue staining and quantification was done using a total magnification of 1000 X under light microscopy using the Axiovision Rel 4–6 program (Zeiss AX 10 microscope). E2 alone or in combination with P4 induced a significantly augmentation in the number of uterine MCs as compared to the vehicle-treated animals (A). Representative pictures from uterine MCs from control (B), E2-treated (C), P4-treated (D) and E2 + P4- treated animals (E) are shown. Arrows indicate uterine MCs, which are easily distinguishable because of their granula. F-H: show the expression of MC-related genes Mcpt-1 (F), Mcpt-5 (G) and Mcpt-8 (H) in uterus of treated and control animals as analyzed by real time RT-PCR. The results are expressed as single dots showing medians. *:p<0.05 and **:p<0.01 as analyzed by Kruskall-Wallis test followed by Mann-Whitney-U test for two particular groups.

References

1. Wordinger RJ, Orr EL, Pace K, Oakford L, Morrill A. An assessment of mast-cell deficient mice (W/Wv) as a model system to study the role of histamine in implantation and deciduoma formation. J Reprod Fertil. 1985;73(2):451–6. -PubMed
1. Johnson DC, Dey SK. Role of histamine in implantation: dexamethasone inhibits estradiol-induced implantation in the rat. Biol Reprod. 1980;22(5):1136–41. -PubMed
1. Liu Z, Kilburn BA, Leach RE, Romero R, Paria BC, et al. Histamine enhances cytotrophoblast invasion by inducing intracellular calcium transients through the histamine type-1 receptor. Mol Reprod Dev. 2004;68(3):345–53. -PubMed
1. Cocchiara R, Di Trapani G, Azzolina A, Albeggiani G, Geraci D. Early embryonic histamine-releasing factor: a new model for human implantation. Hum Reprod. 1986;1(7):445–7. -PubMed
1. Fisher SJ, Leitch MS, Kantor MS, Basbaum CB, Kramer RH. Degradation of extracellular matrix by the trophoblastic cells of first-trimester human placentas. J Cell Biochem. 1985;27(1):31–41. -PubMed

Estradiol and progesterone regulate the migration of mast cells from the periphery to the uterus and induce their maturation and degranulation - PubMed (original) (raw)