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Pregnancy associated hormones modulate the cytokine production but not the phenotype of PBMC-derived human dendritic cells
European Journal of Obstetrics & Gynecology and Reproductive Biology 122 (2005) 85–94 www.elsevier.com/locate/ejogrb
Pregnancy associated hormones modulate the cytokine production but not the phenotype of PBMC-derived human dendritic cells Barbara Huck, Thomas Steck, Marion Habersack, Johannes Dietl, Ulrike Ka¨mmerer * Department of Gynaecology and Obstetrics, University of Wu¨rzburg, Josef- Schneider-Str. 4, D-97080, Wu¨rzburg, Germany Received 30 January 2004; received in revised form 21 January 2005; accepted 19 February 2005
Abstract Objective: Dendritic cells (DC) play a central role in initiating and polarizing immune responses. As effects of pregnancy associated hormones on phenotype and function of DC are unknown, our objective was to test the influence of progesterone, b-estradiol and bHCG on immature (iDC) and mature (mDC) DC. Study design: DC generated from peripheral-blood-monocytes were exposed to different doses of hormones. DC phenotype was determined by FACS-analysis of surface marker expression (CD40, CD86, CD83 and HLA-DR). Modifications in the secretion of cytokines (IL12p70, IL-18, IL-10, IL-6, TNFa) and chemokines (MDC, IL-8) were analysed by ELISA. T cell stimulatory capacity of mDC was assessed by mixed lymphocyte reaction. Results: Incubation with progesterone or estradiol resulted in a significant upregulation of IL-10 production by iDC and mDC. Combinations of progesterone and bHCG or estradiol respectively induced a significant decrease in production of IL-18 by mDC. No significant changes could be observed in surface marker expression or T cell stimulatory capacity, neither in cultures of DC matured under influence of progesterone, estradiol nor bHCG. Conclusions: PBMC-derived DC seem to be relatively stable against the influence of pregnancy associated hormones apart from particular effects on cytokine production which partly could contribute to the modification of immune responses observed in normal early pregnancy. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Dendritic cells; Pregnancy; Hormones; In-vitro testing
1. Introduction Dendritic cells (DC) are specialist antigen presenting cells (APC) with a unique capacity to initiate and regulate immune responses and are considered as the most potent APC of the immune system [1]. Immature DC (iDC) are localized in peripheral tissues where they efficiently capture and process antigens. Antigen uptake and stimulation by inflammatory cytokines induce maturation of DC which is characterized by upregulation of MHC class II and costimulatory molecules like CD80 (B7-1) and CD86 (B7-2). During maturation, antigen processing DC migrate into secondary lymphoid tissues where they receive terminal maturation signals via * Corresponding author. Tel.: +49 931 201 25293; fax: +49 931 201 25406. E-mail address: [email protected] (U. Ka¨mmerer).
cytokines and CD40 triggering by activated T cells [2]. Fully matured DC (mDC) are capable to stimulate both naı¨ve and experienced T cells and are therefore essential for initiating primary immune responses. Various protocols are available to generate human dendritic cells from haematopoietic precursor cells in peripheral blood [3]. As these cells correspond morphologically, phenotypically and functionally to defined immature and mature DC they provide a suitable model system to study the multiple functions of DC. Cytokines play an important role in modulating immune responses. Several investigations have been performed to get a perspective of the cytokines expressed at the maternal–fetal interface and their regulation (for review see [4,5]). Their results propose that in successful pregnancy a local shift in the cytokine pattern from Th1 towards Th2 occurs [6–8]. The importance of altered cytokine expression is supported by the finding that women with recurrent
0301-2115/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2005.02.017
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spontaneous abortion show a different cytokine profile in the uterine compartment compared to women during normal pregnancy [5]. The development of Th1 or Th2 dominated responses depends on various factors. In classical immune responses the critical signal seems to be the cytokine microenvironment during antigen presentation by APC. Some evidence suggests that the differentiation of Th-cells into polarized Th1 or Th2 cells could be influenced by steroidal and non-steroidal hormones [6,9–11]. Since normal early pregnancy is associated with a distinct endocrine regulation at the maternal–fetal interface, it may be speculated that pregnancy related hormones do influence the maternal immune system via changing the phenotype of antigen presenting cells. The most characteristic hormone for pregnancy is human chorionic gonadotropin (bHCG) which is found in increasing doses in sera of pregnant women. With the increase in the production of bHCG during the first days of pregnancy, levels of progesterone increase as a result of the rescue of the corpus luteum. Later the placenta becomes the main source of hormones for the support of pregnancy. Other hormones contributing to the special uterine microenvironment include estradiol, glucocorticoids and relaxin among others. These hormones interact in the preparation of the endometrium for implantation and the maintenance of successful pregnancy. Several hormones have been investigated for their influence on dendritic cells [11]. There are some reports on the effects of glucocorticoids on the function and differentiation of DC [12–14]. In a recent report, Yoshimura et al. described the influence of bHCG on the maturation of lymphoid and plasmacytoid DC [15], but in general, little information is available on the influence of sex steroids and pregnancy associated hormones on the phenotype and function of DC [16]. Therefore, we examined the effects of exposure of immature DC to different doses of progesterone (Prog), Estradiol (Estr) and b-human choriongonadotropin (bHCG) prior and during their maturation into mature DC. To cover different immunomodulatory mechanisms in which DC are known to be involved, cells were tested for the production of the cytokines IL-12p70, IL-18 and IL-10 which are capable to induce differentiation of naı¨ve T helper cells into type 1 or 2 cells. In addition we determined the secretion of the chemokines MDC and IL-8 by immature and mature DC as well as TNFa and IL-6 by iDC. Cells were also tested for alterations in their surface marker expression and capacity to stimulate T cell proliferation upon hormonal stimulation.
Cells were isolated and cultured following standard procedures [3]. Briefly, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Citrate buffered buffy coats diluted with an equal volume of phosphate buffered saline (PBS) were distributed over Histopaque1-1077 (density 1.077 g/ml, Sigma, Deisenhofen, Germany) and centrifuged at 400 g for 30 min at room temperature. Cells were collected from the interface and washed in PBS. After incubation for 1 h at 4 8C with neuraminidase (Boehringer, Ingelheim, Ingelheim, Germany) treated sheep red blood cells, the cell suspension was distributed over a second Histopaque1-gradient and centrifuged as described above. Rosettes consisting of T cells and sheep red blood cells were collected separately and used for T cell preparation. The T cell depleted PBMCfraction was incubated for 1 h at 37 8C in RPMI 1640 medium (Biochrom, Berlin, Germany) with gentamycine (Biochrom) and 2% autologous plasma on cell culture petridishes (TPP, Trasadingen, Switzerland). Non adherent cells were washed free with PBS. The adherent cell fraction was used to generate monocyte-derived dendritic cells in vitro by culture in the presence of 1000 U/ml purified recombinant human GM-CSF (Leukomax1 400, Sandoz, Basel, Switzerland) and 800 U/ml IL-4 (Strathmann, Hamburg, Germany) for 7 days in RPMI 1640 (Biochrom) with 10% heat-inactivated fetal calf serum (FCS) (PAN, Aidenbach, Germany) and gentamycin 1:200 (Biochrom) (R10). On days 3 and 5 half the volume of medium was replaced with fresh medium and cytokines. In experiments with mature DC, maturation was induced on day 7 by adding a maturation cocktail consisting of 1000 U/ml purified recombinant human IL-1b (Strathmann), 1000 U/ml purified recombinant human IL-6 (Strathmann), 1000 U/ml purified recombinant human TNFa (Strathmann) and 10 8 mol/l PGE2 (Calbiochem, Bad Soden, Germany) for another three days of culture. 2.2. T cell preparation For mixed lymphocyte reaction, T cells were prepared by 0.8% NH4Cl lysis of neuraminidase treated sheep red blood cell rosettes. Remaining T cells were washed in PBS, suspended in FCS/DMSO and stored in liquid nitrogen. Purity of T cells was assured by CD3, CD4 and CD8 staining and FACS analysis to be 98%. 2.3. Cell culture with hormones
2. Material and methods 2.1. Dendritic cell preparation Buffy coats were obtained from healthy male and female human donors (University of Wu¨rzburg, Division of Immunohematology and Transfusion and Red Cross Blood Transfusion Service, Wiesenthaid, Germany).
Hormones were added to culture on day 7. Cells (5 105) were cultured for another three days in 1 ml R10 on 24 well plates with three different concentrations of each hormone. On day 10, cells were harvested for MLR and FACS analysis. For cytokine ELISAs aliquots of supernatants were snap frozen and stored at 80 8C. 17b-estradiol and progesterone were obtained from Sigma and stored as stock solutions of 50 ng/ml
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(1.362 mol/l) in pure ethanol. These hormones were tested in concentrations of 0.1 ng/ml, 10 ng/ml and 1000 ng/ml. Furthermore, tests with combinations of 0.1 ng/ml, 10 ng/ml or 1000 ng/ml progesterone and 10 ng/ml 17b-estradiol were carried out. The highest concentration of ethanol in the final culture solution was 0.02% (v/v). bHCG (Pregnesin1 5000 U) was purchased from Serono (Unterschleißheim, Germany), dissolved in 0.9% NaCl and stored at 5000 U/ml. Experiments were carried out with concentrations of 10 mU/ ml, 50 mU/ml and 250 mU/ml of bHCG. Additionally, combinations of 0.1 ng/ml, 10 ng/ml or 1000 ng/ml progesterone with 50 mU/ml of bHCG were tested. Dexamethasone for control purposes in MLR was obtained from Sigma and stored as stock solution of 50 mM in pure ethanol. Final concentration in cell culture was 10 8 M. To take into account possible effects of the solvent, control samples incubated with the corresponding ethanol concentration were performed and all ELISA results were referenced to the corresponding control sample (see Table 1).
2.5. Flow cytometry
2.4. Mixed lymphocyte reaction
2.6. Cytokine ELISA
Three separate experiments were performed in order to determine T cell stimulating capacity of DC. On day 10, graded doses of 9000 to 333 cells per well were cocultured with 9 104 allogeneic T cells for 5 days in U-bottom 96well plates (TPP) with 200 ml of R10. To determine the proliferation activity of hormone-treated and control cells, cultures then were pulsed with 1 mCi of 3H-Thymidin (Amersham International, Arlington Heights IL, USA) per well for the final 16 h before harvesting and liquid scintillation counting. All assays were performed in triplicates.
Levels of IL-8, IL-10 and IL12(p70) produced by immature and mature cells were analysed using OptEIA ELISA-sets (Pharmingen). ELISA-sets for macrophage derived chemokine (MDC) and IL-18 were purchased from R & D Systems. In immature cells, TNFa (OptEIA, Pharmingn) and IL-6 (R & D Systems) were also measured. ELISAs were established and optimised according to the instructions of the manufacturer. For all ELISA systems, the TMB-substrate set (BD Pharmingen) was used to detect the horseradish peroxidase-reaction. Results were considered valid if optic densities gave a two standard deviation above
Cell staining for FACS analysis was performed with directly Phycoerythrin (PE)-conjugated CD83 (CoulterImmunotech, Hamburg, Germany) and HLA-DR (Cymbus Biotechnology, Chandlers Ford, UK) as well as fluoresceinisothiocyanate (FITC)-conjugated CD40 and HLA-DR (BD Pharmingen, Heidelberg, Germany) antihuman monoclonal antibodies (mAb). Directly FITC-(Pharmingen) an PE-(Cymbus Biotechnology) conjugated IgG mAb served as isotype controls. Cells resuspended in PBS with 10% human immunoglobulin (Beriglobulin1, Centeon, Marburg, Germany) were stained by incubation with fluorochrome labelled antibodies for 30 min at 4 8C and washed once in PBS. Cells were analysed on a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) using CellQuest software. Results were analysed using WinMDI software (Version 2.8, 227 Joseph Trotter, The Scripps Research Institute, La Jolla CA, USA).
Table 1 shows the p-values for the comparison of the cytokine levels in the given cultures compared to the corresponding control (1–3, column 2) Hormones
Ethanol
Immature DC
Mature DC
IL-10
IL-18
IL-8
MDC
IL-10
IL-18
IL-8
MDC
bHCG 10 mU/ml bHCG 50 mU/ml bHCG 250 mU/ml 17b-Estr. 0.1 ng/ml 17b-Estr. 10 ng/ml 17b-Estr. 1000 ng/ml Prog. 0.1 ng/ml Prog. 10 ng/ml Prog. 1000 ng/ml Prog 0.1 ng/ml + bHCG 50 mU/ml Prog 10 ng/ml + bHCG 50 mU/ml Prog 1000 ng/ml + bHCG 50 mU/ml Prog 0.1 ng/ml + Estr 10 ng/ml Prog 10 ng/ml + Estr 10 ng/ml Prog 1000 ng/ml + Estr 10 ng/ml Dexa 10 10 mol/l Dexa 10 8 mol/l Dexa 10 6 mol/l
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
n.s. n.s. n.s. 0.043 0.017 n.s. 0.035 0.004 n.s. 0.035 0.028 n.s. n.s. 0.027 n.s. n.s. 0.0001 0.043
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.015 0.005
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.005 n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s. 0.002 0.034 0.013 0.0002 0.004 0.013 0.003 n.s. n.s. 0.013 n.s. n.s. n.s. 0.049 0.002
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.005 0.002 0.007 0.023 0.013 0.019 n.s. n.s. n.s.
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. 0.023 n.s. n.s. n.s. n.s. 0.015 0.029 n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Final concentration of ethanol: (1) 0.00002%, (2) 0.0002% and (3) 0.02%.
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the mean background signal volume. For all cytokines to be tested, ELISA experiments were performed in 10–12 samples with a equal share of male and female blood donors. All assays were performed in duplicate.
referenced to their corresponding ethanol-control sample. Pvalues < 0.05 were considered as significant.
3. Results 2.7. Data analysis Due to the very high variation in the absolute cytokine production by the different patient’s DC, cytokine concentrations measured in the supernatants of untreated cells were defined as 100% and all results obtained with the ethanol-controls as well as with the hormones to be tested were calculated as per cent difference on the level of the corresponding cell culture. Since the individuals tested did not conform to a normal distribution pattern as assessed by the Shapiro–Wilkins test for normality, we used the nonparametric two-tailed Mann–Whitney U-Test for statistical analysis of relative increase/decrease of cytokine concentrations obtained under the influence of the hormones
DC from equal numbers of male and female blood donors were tested and analysed separately. No differences in cytokine or chemokine production, surface marker expression and T cell stimulation capacity could be observed between female and male cells under the influence of the tested hormones. Therefore, for evaluation of results, data of male and female donors were combined. Cytokine-ELISAs showed no significant influence of the solvent ethanol on the production of cytokines and chemokines by mature and immature DC compared to untreated cells. Surface marker expression remained unchanged and no altered capacity of T cell stimulation for mature cells cultured with the three concentrations of ethanol was found in the MLR.
Fig. 1. Comparison of the cytokine concentrations in the supernatants of the cultures with immature DC (iDC) and mature DC (mDC). Data were pooled out of all experiments including untreated cells, controls, and cells cultured with hormones. M = median of concentration (pg/ml). Level of significance as obtained by Mann–Whitney U-Test is given on top of the columns. (A): IL-10, (B): IL-8, (C): IL-18 and (D): MDC.
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Fig. 2. Results of the cytokine ELISAs of the cultures of immature DC (iDC) with the hormones in question. Left row: samples compared to the lowest concentration of ethanol (Eth; diluted 1:106). Middle row: samples compared to the median ethanol concentration (diluted 1:10.000). Right row: samples compare to the highest concentration of ethanol (diluted 1:1000). Abbreviations: Eth = ethanol; bHCG = beta HCG; Estr: estradiol; Prog = progesterone; P = progesterone, Dexa = dexamethasone. Scale: 100 = 100% of the cytokine concentration analysed in the appropriate untreated control cell culture. *p < 0.05 (Mann–Whitney U-Test). (A) IL-10; (B) IL-8; (C) IL-18; (D) MDC; (E) TNFa and (F) IL-6.
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3.1. Different cytokine/chemokine production by iDC and mDC Levels of cytokine/chemokine concentration in the supernatants of iDC as well as mDC are shown in Fig. 1. The results of all experiments (control/hormones) were summarized to get the graph. Immature DC produce significantly higher amounts of IL-10 and IL-18 than mature DC, while the chemokines IL-8 and MDC are found in higher concentrations in the supernatants of mature DC. 3.2. Cytokine/chemokine production in dependence of the hormones The relative (compared to untreated control cells) concentration of cytokines in the culture supernatants was
analysed in comparison to the cell cultures with the related ethanol concentration Data are shown in Fig. 2A–F (immature DC) and Fig. 3A–D (mature DC), levels of significance are given in Table 1. 3.2.1. IL12p70 Levels of IL12p70 in the supernatant of immature and mature DC were below the detection range of the test in the majority of samples. Only in three samples, the values obtained by IL12p70 ELISA could be considered as valid (2 standard deviations above background), and therefore no further statistical analysis was performed. 3.2.2. IL-10 Results of the IL-10 ELISA are shown in Figs. 2B and 3B. A significant increase in IL-10 production was observed in
Fig. 3. Results of the cytokine ELISAs of the cultures of mature DC (mDC) with the hormones in question. Left row: samples compared to the lowest concentration of ethanol (Eth; diluted 1:106). Middle row: samples compared to the median ethanol concentration (diluted 1:10.000). Right row: samples compare to the highest concentration of ethanol (diluted 1:1000). Abbreviations: Eth = ethanol; bHCG = beta HCG; Estr: estradiol; Prog = progesterone; P = progesterone, Dexa = dexamethasone. Scale: 100 = 100% of the cytokine concentration analysed in the appropriate untreated control cell culture. *p < 0.05 (Mann–Whitney U-Test). (A) IL-10; (B) IL-8; (C) IL-18 and (D) MDC.
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immature DC upon culture with progesterone as well as estradiol, both at 0.1 ng/ml and 10 ng/ml. In mature DC, a significant stimulatory effect of both substances on IL-10 production could be observed at all three concentrations tested. The combination of 0.1 ng/ml progesterone and bHCG (50 U/ml) resulted in a significant increase of IL-10 levels produced by immature and mature DC. An effect of the combination of progesterone and estradiol (10 ng/ml) was seen at the lowest progesterone concentration in case of mature DC. In case of immature DC, median concentration of IL-10 increased with the combination of progesterone and estradiol. While significance for the increase was nearly missed ( p = 0.0052) in case of estradiol and progesterone at 0.1 ng/ml, with median progesterone concentration in this combination, the effect was significantly. For both maturation stages, Dexamethasone increased the IL-10 secretion into the medium at 10 8 and 10 6 mol/l.
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3.2.7. IL-6 Results of IL-6 ELISA are given in Fig. 2F. Same as with TNFa, IL-6 was used for DC maturation and therefore this cytokine was analysed in immature DC only. A significant decrease of IL-6 concentration in the supernatant was found in cultures with Dexamethasone at 10 8 M. In case of the higher concentration (10 6 M), significance for the decrease observed was nearly missed ( p = 0.059). 3.3. Phenotype of DC Hormonal influence on the phenotype of dendritic cells was tested by flow cytometry. A typical result of FACSanalysis of the corresponding untreated control cells is shown in Fig. 4. Dendritic cells cultured in medium containing IL-4 and GM-CSF exhibited an immature phenotype. Cells were CD83 /(low) and showed low expression of HLA-DR, CD40 and CD86. In the presence
3.2.3. IL-8 Results of the IL-8 ELISA are summarized in Figs. 2B and 3B. In mature DC, IL-8 production was not altered by incubation with hormones as well as with dexamethasone at all. In immature cells there was a significant decrease in IL-8 production upon culture with Dexamethasone at 10 8 and 10 6 mol/l. 3.2.4. IL-18 Results of the IL-18 ELISA are shown in Figs. 2C and 3C. Compared to the respective ethanol controls, levels of the proinflammatory cytokine IL-18 in culture supernatant were not altered in any of the cultures of immature DC. In mature DC cultures, combinations all three concentrations of progesterone with bHCG or estradiol significantly decreased the concentration of IL-18 in culture supernatant for. Progesterone alone had no significant effect. 3.2.5. MDC Results of the MDC ELISA are given in Figs. 2D and 3D. There was no significant effect on MDC concentration in cell cultures of iDC seen with all hormones tested. One exception was the significant increase of the concentration of this chemokine under the influence of progesterone (0.1 ng/ml) and estradiol (10 ng/ml). In mature DC, the combination of progesterone with bHCG at low and medium progesterone concentration, as well as estradiol at 10 ng/ml, significantly decreased the level of MDC in culture supernatant. 3.2.6. TNFa Results of the TNFa ELISA are shown in Fig. 2E. Due to the exogen TNFa used for DC maturation, this cytokine was analysed in immature DC only. In the supernatants of those cultures, the only significant increase in TNFa-concentration was obtained when immature DC were cultured in the presence of progesterone at 10 ng/ml. Significant changes in the TNF-a concentration could not be observed in any other culture experiment.
Fig. 4. Typical FACS-Analysis confirming maturation state of the DC used for the experiments. Left side: mature DC with high expression of CD40 and 86 and upregulated CD83 and HLA-DR. Right side: immature DC show a weak expression of CD40 and HLA-DR, nearly no CD83 surface expression and few CD86 on their surface.
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Fig. 5. Functional analysis of cocktail matured dendritic cells (DC) in a mixed lymphocyte reaction (MLR) with MHC-mismatched responder T lymphocytes at the indicated ratios of DC and T cells. Results are shown as the mean [3H]-thymidine uptake S.E.M. for three samples each. [3H]thymidine uptake of DC and T cell populations alone was <400 cpm and is not shown. For the MLR, the physiologically median concentrations of the appropriate hormones were chosen. In this experimental setting, only the DC cultured in medium with dexamethasone at 10 8 mol/l showed significantly reduced T stimulatory capacity.
of progesterone, estradiol or bHCG in different concentrations, no changes in CD40, HLA-DR or CD86 expression were found. Dendritic cells cultured for 3 days in the presence of the maturation cocktail showed a mature phenotype: cells are CD83+ and show increased levels of HLA-DR and CD40. The costimulatory molecule CD86 was strongly up regulated. Progesterone, estradiol and bHCG did not influence surface marker expression on mature DC, whereas for the combination of progesterone at 0.1 ng/ml with 50 mIU/ml bHCG, a weak decrease in CD83, CD40 and CD86 expression was observed (not shown). 3.4. Stimulatory capacity of DC on allogeneic T cells The MLR results are shown in Fig. 5. In the MLR experiments, mature DC generated under the influence of hormones stimulated T cell proliferation as well as corresponding mature control DC. The capacity of mature DC cultured in the presence of dexamethasone (10 8 mol/l) to stimulate T cell proliferation was seen to be very low compared to the untreated control cells.
4. Discussion In this study we investigated the influence of selected pregnancy associated hormones on the phenotype and function of PBMC derived dendritic cells. PBMC derived DC are a well defined model providing cells at high purity and similar maturation status. As DC undergo different stages of maturation during the development of an immune response, we decided to test the possible influence of hormones on both immature DC, as well as on DC undergoing maturation.
When comparing cells from female and male donors there was no significant difference in cytokine and chemokine production. This ‘‘equalizing of sex’’ could possibly depend on effects of the in-vitro culture of DC with FCS containing culture medium as FCS is known to be able to modulate the degree of estradiolic action in endocrinedependent breast cancer cell lines [16]. However, the content of sex steroids in FCS and their influence on the function and phenotype of DC is still unknown. The overall production of the chemokine IL-8 analysed in the cell cultures supernatants was extremely high in mature DC compared to immature DC. MDC as well was found in significantly higher concentrations in the supernatants of mDC than in iDC. This results are concordant with the data on chemokine-RNA analysed by PCR [17] and the recently published manuscript on cytokine/chemokine profiles, demonstrating that mDC are better producers of chemoattractants than iDC [18] Other than in the latter publication, in our experiments iDC in median of all experiments secreted significantly higher amounts of IL-10 than mDC. It can be speculated that this difference is based on the different maturation protocols. Similar to IL-10, IL-18 was produced to a higher extend by iDC than by mDC. The absolute difference was not dramatic, but nevertheless statistically significant because of the high number of samples compared. To the best of our knowledge, data on the comparison of IL-18 expression by iDC and mDC were not published up to now although different expression of IL-18 in different DC populations is well known [19]. Here, we could demonstrate that in mature DC, progesterone induces a significant increase in the production of the Th2 type cytokine IL-10. This effect could indicate a possible role for progesterone in creating immunotolerance during pregnancy as IL-10 is known to influence T cells [20] as well as APC [21] in a tolerogenic way [22], with emphasis on the role of immature DC. Upon incubation with progesterone at a concentration of 1000 ng/ml, upregulation of IL-10 was not observed in immature DC. This concentration is well above the physiologic levels circulating even during late pregnancy, but was tested here to investigate effects of pharmacological doses. Combinations of progesterone and estradiol corresponding to the follicular (0.1 ng/ml progesterone and 10 ng/ml estradiol) phase of the menstrual cylce significantly increased the IL-10 concentration and decreased the IL-18 concentration in the supernatants of mature DC. In immature DC only a significant increase in IL-10 production was observed, whereas no influence of the progesterone/estradiol combination on the Th-1 cytokines analysed was observed. However, this findings could well fit with the Th2-paradigm postulated for pregnancy [23]. The progesterone/estradiol concentration corresponding to the luteal phase (10 ng/ml progesterone and 10 ng/ml estradiol) of the menstrual cycle decreased the IL-18 concentration in mature DC and decreased the IL-10 concentration in immature DC. The upregulation of the anti-inflammatory cytokine IL-10 by
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progesterone is consistent with observations made in mice, where progesterone was shown to be responsible for the suppression of Th1-type responses and development of a Th2 type response characterized by the production of Th2 type cytokine IL-4 [10]. Furthermore, in human antigenchallenged PBMC, a significant increase in IL-4 production was observed after treatment with progesterone [24]. In this study we used IL-10 and not IL-4 as representative of the Th2 type cytokine family as IL-4 was added to the culture medium and IL-4 production by DC could not be quantified in the supernatants. However, progesterone treated DC showed no alteration in their surface marker expression and in T cell stimulatory capacity. The concentration of 17b-estradiol in non pregnant woman reaches maximum levels of 0.3 ng/ml at the time of ovulation and increases to maximum levels of 10 ng/ml in pregnancy. In our in vitro experiments, concentrations of 0.1 ng/ml corresponding to the menstrual cycle and of 10 ng/ ml as found in late pregnancy showed an increasing effect on the production of IL-10 by immature and mature DC. In the presence of 1000 pg/ml of b-estradiol we found an increased IL-10 production by mature DC. T cell stimulatory capacity and surface marker expression were not altered. In has already been shown that physiological doses of estradiol exert stimulating effects on the immune system [25]. Mechanisms of action of estradiol on the immune system comprise influences on the expression of genes involved in apoptosis [22] and interaction with transcription factors like NFkB and AP-1 [24]. However data published on the effects of estradiol are inconsistent [8,24,26], possibly due to variations in experimental conditions. But there is evidence that immunomodulation by estradiol is not only cell-type and cytokine specific but also dose dependent. Estradiol shows biphasic dose effects. Low doses facilitate immune responses whereas high doses, which occur in pregnancy, suppress such responses [27]. Estradiol also might have an influence on chemoattraction as migration of macrophages into the uterus was proposed to be controlled by estradiol [28]. As chemokine with possibly important role in pregnancy, we tested MDC, which is selectively attracting Th2 polarised T cells and immature DC and seems to be responsible for the regulation of Th2 type responses [29]. While the chemokine IL-8 seems not to be influenced by the hormones tested, MDC showed an opposite regulation in the immature and mature DC tested in this study. While a slight increase (significant only in case of luteal phase corresponding progesterone/estradiol concentration) of MDC levels in immature DC seems to fit in the pattern of activation of the immune system, levels of MDC were decreased in mature DC under the influence of the hormones tested. However, this decrease was especially significant in combinations of progesterone and bHCG. Human chorionic gonadotropin (bHCG) is primarily produced during pregnancy, when serum levels often exceed 100 IU/ml. In 2003, Yoshimura et al. investigated two subsets of DC, myeloid and lymphoid ones and their reaction
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to exogenous bHCG. For both cell types, they found a dosedependent production of the cytokines IL-12 and TNFa, but at different levels. Other than in our experiments, they found no production of IL-10. Since they used differentiated cells sorted out of the peripheral blood, the cell populations are not comparable to our cells and therefore we can not compare the results [15]. We demonstrated a decrease in IL10 production by mature DC for all three bHCG concentrations tested. In general bHCG has been described to have immunosuppressive properties. In a model of murine arthritis bHCG was able to suppress the production of the proinflammatory cytokines IL-1b, IL-6 and TNFa [30], while in PBMC an inhibitory effect on IL-2 production and IL-2-receptor expression could be shown [31]. Little is known about the influence of bHCG on Th2-type cytokines. Further investigation is needed to investigate the role of bHCG in the immunological changes associated with early pregnancy. The combinations of bHCG and progesterone which correspond to the hormonal milieu during early pregnancy showed no effects on immature DC. In mature DC we observed a significant decrease in IL-18 release in all combinations of progesterone with bHCG tested. These findings are supported by the observation that surface marker expression (CD83, CD40 and CD86) in these cells was slightly, albeit insignificant, down regulated similar to the results described in the literature for dexamethasone. This observation suggests an influence of progesterone and bHCG on dendritic cell maturation or selection of less mature cells respectively. However, the T cell stimulatory capacity was not found to be altered. This is in contrast to the data presented by Yoshimura [15] demonstrating an increase in T cell stimulatory capacity of myeloid DC depending on an increasing concentration of bHCG. Since we used monocyte-derived DC and the other cells were sorted myeloid DC, this could be responsible for the differences observed. Therefore in future investigations, the different subpopulations of DC might be of interest for the described experiments. However, the combination of progesterone and bHCG seems to have a distinct influence on monocytederived mature DC by suppressing their inflammatory properties. This is supported by the observation that in mature cells stimulated with the concentration of 0.1 ng/ml progesterone with 50 mIU/ml bHCG, IL-10 production was increased. In summary our results demonstrate that there are some distinct effects of estradiol, progesterone and bHCG on the cytokine production by DC, while surface marker expression and T cell stimulatory capacity seems to be relatively stable against the influence of pregnancy- associated hormones. However it is difficult to explain the various effects by a consistent model like it has been postulated for the Th2-type cytokine environment in pregnancy. It is possible that DC are not the main targets of hormonal signalling in pregnancy. The expanding knowledge in the field of lymphocyte subsets shows that different subpopulations of DC exist, which may
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exert different stimulatory or tolerogenic functions [32–34]. Working with PBMC-derived DC we cannot speculate about the behaviour of uterine DC. It is possible that at the fetomaternal interface, other immunocompetent cell types like stromal cells or large granular lymphocytes (LGL) may be involved in the reception of hormonal signals. In conclusion, we could demonstrate that DC seem to be relatively stable against the influence of progesterone, estradiol and bHCG. Similarly, T cell stimulatory capacity and surface marker expression of DC are not altered by the culture with estradiol, progesterone and bHCG. Effects on cytokine production show a shift to higher Th2 levels. Thus further investigations on the immunological target cells in hormonal signalling during pregnancy are needed.
Acknowledgements We thank E. Kampgen for helpful discussions and M. Kapp for excellent technical assistance
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Pregnancy associated hormones modulate the cytokine production but not the phenotype of PBMC-derived human dendritic cells Barbara Huck, Thomas Steck, Marion Habersack, Johannes Dietl, Ulrike Ka¨mmerer * Department of Gynaecology and Obstetrics, University of Wu¨rzburg, Josef- Schneider-Str. 4, D-97080, Wu¨rzburg, Germany Received 30 January 2004; received in revised form 21 January 2005; accepted 19 February 2005
Abstract Objective: Dendritic cells (DC) play a central role in initiating and polarizing immune responses. As effects of pregnancy associated hormones on phenotype and function of DC are unknown, our objective was to test the influence of progesterone, b-estradiol and bHCG on immature (iDC) and mature (mDC) DC. Study design: DC generated from peripheral-blood-monocytes were exposed to different doses of hormones. DC phenotype was determined by FACS-analysis of surface marker expression (CD40, CD86, CD83 and HLA-DR). Modifications in the secretion of cytokines (IL12p70, IL-18, IL-10, IL-6, TNFa) and chemokines (MDC, IL-8) were analysed by ELISA. T cell stimulatory capacity of mDC was assessed by mixed lymphocyte reaction. Results: Incubation with progesterone or estradiol resulted in a significant upregulation of IL-10 production by iDC and mDC. Combinations of progesterone and bHCG or estradiol respectively induced a significant decrease in production of IL-18 by mDC. No significant changes could be observed in surface marker expression or T cell stimulatory capacity, neither in cultures of DC matured under influence of progesterone, estradiol nor bHCG. Conclusions: PBMC-derived DC seem to be relatively stable against the influence of pregnancy associated hormones apart from particular effects on cytokine production which partly could contribute to the modification of immune responses observed in normal early pregnancy. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Dendritic cells; Pregnancy; Hormones; In-vitro testing
1. Introduction Dendritic cells (DC) are specialist antigen presenting cells (APC) with a unique capacity to initiate and regulate immune responses and are considered as the most potent APC of the immune system [1]. Immature DC (iDC) are localized in peripheral tissues where they efficiently capture and process antigens. Antigen uptake and stimulation by inflammatory cytokines induce maturation of DC which is characterized by upregulation of MHC class II and costimulatory molecules like CD80 (B7-1) and CD86 (B7-2). During maturation, antigen processing DC migrate into secondary lymphoid tissues where they receive terminal maturation signals via * Corresponding author. Tel.: +49 931 201 25293; fax: +49 931 201 25406. E-mail address: [email protected] (U. Ka¨mmerer).
cytokines and CD40 triggering by activated T cells [2]. Fully matured DC (mDC) are capable to stimulate both naı¨ve and experienced T cells and are therefore essential for initiating primary immune responses. Various protocols are available to generate human dendritic cells from haematopoietic precursor cells in peripheral blood [3]. As these cells correspond morphologically, phenotypically and functionally to defined immature and mature DC they provide a suitable model system to study the multiple functions of DC. Cytokines play an important role in modulating immune responses. Several investigations have been performed to get a perspective of the cytokines expressed at the maternal–fetal interface and their regulation (for review see [4,5]). Their results propose that in successful pregnancy a local shift in the cytokine pattern from Th1 towards Th2 occurs [6–8]. The importance of altered cytokine expression is supported by the finding that women with recurrent
0301-2115/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2005.02.017
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spontaneous abortion show a different cytokine profile in the uterine compartment compared to women during normal pregnancy [5]. The development of Th1 or Th2 dominated responses depends on various factors. In classical immune responses the critical signal seems to be the cytokine microenvironment during antigen presentation by APC. Some evidence suggests that the differentiation of Th-cells into polarized Th1 or Th2 cells could be influenced by steroidal and non-steroidal hormones [6,9–11]. Since normal early pregnancy is associated with a distinct endocrine regulation at the maternal–fetal interface, it may be speculated that pregnancy related hormones do influence the maternal immune system via changing the phenotype of antigen presenting cells. The most characteristic hormone for pregnancy is human chorionic gonadotropin (bHCG) which is found in increasing doses in sera of pregnant women. With the increase in the production of bHCG during the first days of pregnancy, levels of progesterone increase as a result of the rescue of the corpus luteum. Later the placenta becomes the main source of hormones for the support of pregnancy. Other hormones contributing to the special uterine microenvironment include estradiol, glucocorticoids and relaxin among others. These hormones interact in the preparation of the endometrium for implantation and the maintenance of successful pregnancy. Several hormones have been investigated for their influence on dendritic cells [11]. There are some reports on the effects of glucocorticoids on the function and differentiation of DC [12–14]. In a recent report, Yoshimura et al. described the influence of bHCG on the maturation of lymphoid and plasmacytoid DC [15], but in general, little information is available on the influence of sex steroids and pregnancy associated hormones on the phenotype and function of DC [16]. Therefore, we examined the effects of exposure of immature DC to different doses of progesterone (Prog), Estradiol (Estr) and b-human choriongonadotropin (bHCG) prior and during their maturation into mature DC. To cover different immunomodulatory mechanisms in which DC are known to be involved, cells were tested for the production of the cytokines IL-12p70, IL-18 and IL-10 which are capable to induce differentiation of naı¨ve T helper cells into type 1 or 2 cells. In addition we determined the secretion of the chemokines MDC and IL-8 by immature and mature DC as well as TNFa and IL-6 by iDC. Cells were also tested for alterations in their surface marker expression and capacity to stimulate T cell proliferation upon hormonal stimulation.
Cells were isolated and cultured following standard procedures [3]. Briefly, peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Citrate buffered buffy coats diluted with an equal volume of phosphate buffered saline (PBS) were distributed over Histopaque1-1077 (density 1.077 g/ml, Sigma, Deisenhofen, Germany) and centrifuged at 400 g for 30 min at room temperature. Cells were collected from the interface and washed in PBS. After incubation for 1 h at 4 8C with neuraminidase (Boehringer, Ingelheim, Ingelheim, Germany) treated sheep red blood cells, the cell suspension was distributed over a second Histopaque1-gradient and centrifuged as described above. Rosettes consisting of T cells and sheep red blood cells were collected separately and used for T cell preparation. The T cell depleted PBMCfraction was incubated for 1 h at 37 8C in RPMI 1640 medium (Biochrom, Berlin, Germany) with gentamycine (Biochrom) and 2% autologous plasma on cell culture petridishes (TPP, Trasadingen, Switzerland). Non adherent cells were washed free with PBS. The adherent cell fraction was used to generate monocyte-derived dendritic cells in vitro by culture in the presence of 1000 U/ml purified recombinant human GM-CSF (Leukomax1 400, Sandoz, Basel, Switzerland) and 800 U/ml IL-4 (Strathmann, Hamburg, Germany) for 7 days in RPMI 1640 (Biochrom) with 10% heat-inactivated fetal calf serum (FCS) (PAN, Aidenbach, Germany) and gentamycin 1:200 (Biochrom) (R10). On days 3 and 5 half the volume of medium was replaced with fresh medium and cytokines. In experiments with mature DC, maturation was induced on day 7 by adding a maturation cocktail consisting of 1000 U/ml purified recombinant human IL-1b (Strathmann), 1000 U/ml purified recombinant human IL-6 (Strathmann), 1000 U/ml purified recombinant human TNFa (Strathmann) and 10 8 mol/l PGE2 (Calbiochem, Bad Soden, Germany) for another three days of culture. 2.2. T cell preparation For mixed lymphocyte reaction, T cells were prepared by 0.8% NH4Cl lysis of neuraminidase treated sheep red blood cell rosettes. Remaining T cells were washed in PBS, suspended in FCS/DMSO and stored in liquid nitrogen. Purity of T cells was assured by CD3, CD4 and CD8 staining and FACS analysis to be 98%. 2.3. Cell culture with hormones
2. Material and methods 2.1. Dendritic cell preparation Buffy coats were obtained from healthy male and female human donors (University of Wu¨rzburg, Division of Immunohematology and Transfusion and Red Cross Blood Transfusion Service, Wiesenthaid, Germany).
Hormones were added to culture on day 7. Cells (5 105) were cultured for another three days in 1 ml R10 on 24 well plates with three different concentrations of each hormone. On day 10, cells were harvested for MLR and FACS analysis. For cytokine ELISAs aliquots of supernatants were snap frozen and stored at 80 8C. 17b-estradiol and progesterone were obtained from Sigma and stored as stock solutions of 50 ng/ml
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(1.362 mol/l) in pure ethanol. These hormones were tested in concentrations of 0.1 ng/ml, 10 ng/ml and 1000 ng/ml. Furthermore, tests with combinations of 0.1 ng/ml, 10 ng/ml or 1000 ng/ml progesterone and 10 ng/ml 17b-estradiol were carried out. The highest concentration of ethanol in the final culture solution was 0.02% (v/v). bHCG (Pregnesin1 5000 U) was purchased from Serono (Unterschleißheim, Germany), dissolved in 0.9% NaCl and stored at 5000 U/ml. Experiments were carried out with concentrations of 10 mU/ ml, 50 mU/ml and 250 mU/ml of bHCG. Additionally, combinations of 0.1 ng/ml, 10 ng/ml or 1000 ng/ml progesterone with 50 mU/ml of bHCG were tested. Dexamethasone for control purposes in MLR was obtained from Sigma and stored as stock solution of 50 mM in pure ethanol. Final concentration in cell culture was 10 8 M. To take into account possible effects of the solvent, control samples incubated with the corresponding ethanol concentration were performed and all ELISA results were referenced to the corresponding control sample (see Table 1).
2.5. Flow cytometry
2.4. Mixed lymphocyte reaction
2.6. Cytokine ELISA
Three separate experiments were performed in order to determine T cell stimulating capacity of DC. On day 10, graded doses of 9000 to 333 cells per well were cocultured with 9 104 allogeneic T cells for 5 days in U-bottom 96well plates (TPP) with 200 ml of R10. To determine the proliferation activity of hormone-treated and control cells, cultures then were pulsed with 1 mCi of 3H-Thymidin (Amersham International, Arlington Heights IL, USA) per well for the final 16 h before harvesting and liquid scintillation counting. All assays were performed in triplicates.
Levels of IL-8, IL-10 and IL12(p70) produced by immature and mature cells were analysed using OptEIA ELISA-sets (Pharmingen). ELISA-sets for macrophage derived chemokine (MDC) and IL-18 were purchased from R & D Systems. In immature cells, TNFa (OptEIA, Pharmingn) and IL-6 (R & D Systems) were also measured. ELISAs were established and optimised according to the instructions of the manufacturer. For all ELISA systems, the TMB-substrate set (BD Pharmingen) was used to detect the horseradish peroxidase-reaction. Results were considered valid if optic densities gave a two standard deviation above
Cell staining for FACS analysis was performed with directly Phycoerythrin (PE)-conjugated CD83 (CoulterImmunotech, Hamburg, Germany) and HLA-DR (Cymbus Biotechnology, Chandlers Ford, UK) as well as fluoresceinisothiocyanate (FITC)-conjugated CD40 and HLA-DR (BD Pharmingen, Heidelberg, Germany) antihuman monoclonal antibodies (mAb). Directly FITC-(Pharmingen) an PE-(Cymbus Biotechnology) conjugated IgG mAb served as isotype controls. Cells resuspended in PBS with 10% human immunoglobulin (Beriglobulin1, Centeon, Marburg, Germany) were stained by incubation with fluorochrome labelled antibodies for 30 min at 4 8C and washed once in PBS. Cells were analysed on a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) using CellQuest software. Results were analysed using WinMDI software (Version 2.8, 227 Joseph Trotter, The Scripps Research Institute, La Jolla CA, USA).
Table 1 shows the p-values for the comparison of the cytokine levels in the given cultures compared to the corresponding control (1–3, column 2) Hormones
Ethanol
Immature DC
Mature DC
IL-10
IL-18
IL-8
MDC
IL-10
IL-18
IL-8
MDC
bHCG 10 mU/ml bHCG 50 mU/ml bHCG 250 mU/ml 17b-Estr. 0.1 ng/ml 17b-Estr. 10 ng/ml 17b-Estr. 1000 ng/ml Prog. 0.1 ng/ml Prog. 10 ng/ml Prog. 1000 ng/ml Prog 0.1 ng/ml + bHCG 50 mU/ml Prog 10 ng/ml + bHCG 50 mU/ml Prog 1000 ng/ml + bHCG 50 mU/ml Prog 0.1 ng/ml + Estr 10 ng/ml Prog 10 ng/ml + Estr 10 ng/ml Prog 1000 ng/ml + Estr 10 ng/ml Dexa 10 10 mol/l Dexa 10 8 mol/l Dexa 10 6 mol/l
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
n.s. n.s. n.s. 0.043 0.017 n.s. 0.035 0.004 n.s. 0.035 0.028 n.s. n.s. 0.027 n.s. n.s. 0.0001 0.043
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.015 0.005
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.005 n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s. 0.002 0.034 0.013 0.0002 0.004 0.013 0.003 n.s. n.s. 0.013 n.s. n.s. n.s. 0.049 0.002
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.005 0.002 0.007 0.023 0.013 0.019 n.s. n.s. n.s.
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. 0.023 n.s. n.s. n.s. n.s. 0.015 0.029 n.s. n.s. n.s. n.s. n.s. n.s. n.s.
Final concentration of ethanol: (1) 0.00002%, (2) 0.0002% and (3) 0.02%.
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the mean background signal volume. For all cytokines to be tested, ELISA experiments were performed in 10–12 samples with a equal share of male and female blood donors. All assays were performed in duplicate.
referenced to their corresponding ethanol-control sample. Pvalues < 0.05 were considered as significant.
3. Results 2.7. Data analysis Due to the very high variation in the absolute cytokine production by the different patient’s DC, cytokine concentrations measured in the supernatants of untreated cells were defined as 100% and all results obtained with the ethanol-controls as well as with the hormones to be tested were calculated as per cent difference on the level of the corresponding cell culture. Since the individuals tested did not conform to a normal distribution pattern as assessed by the Shapiro–Wilkins test for normality, we used the nonparametric two-tailed Mann–Whitney U-Test for statistical analysis of relative increase/decrease of cytokine concentrations obtained under the influence of the hormones
DC from equal numbers of male and female blood donors were tested and analysed separately. No differences in cytokine or chemokine production, surface marker expression and T cell stimulation capacity could be observed between female and male cells under the influence of the tested hormones. Therefore, for evaluation of results, data of male and female donors were combined. Cytokine-ELISAs showed no significant influence of the solvent ethanol on the production of cytokines and chemokines by mature and immature DC compared to untreated cells. Surface marker expression remained unchanged and no altered capacity of T cell stimulation for mature cells cultured with the three concentrations of ethanol was found in the MLR.
Fig. 1. Comparison of the cytokine concentrations in the supernatants of the cultures with immature DC (iDC) and mature DC (mDC). Data were pooled out of all experiments including untreated cells, controls, and cells cultured with hormones. M = median of concentration (pg/ml). Level of significance as obtained by Mann–Whitney U-Test is given on top of the columns. (A): IL-10, (B): IL-8, (C): IL-18 and (D): MDC.
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Fig. 2. Results of the cytokine ELISAs of the cultures of immature DC (iDC) with the hormones in question. Left row: samples compared to the lowest concentration of ethanol (Eth; diluted 1:106). Middle row: samples compared to the median ethanol concentration (diluted 1:10.000). Right row: samples compare to the highest concentration of ethanol (diluted 1:1000). Abbreviations: Eth = ethanol; bHCG = beta HCG; Estr: estradiol; Prog = progesterone; P = progesterone, Dexa = dexamethasone. Scale: 100 = 100% of the cytokine concentration analysed in the appropriate untreated control cell culture. *p < 0.05 (Mann–Whitney U-Test). (A) IL-10; (B) IL-8; (C) IL-18; (D) MDC; (E) TNFa and (F) IL-6.
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3.1. Different cytokine/chemokine production by iDC and mDC Levels of cytokine/chemokine concentration in the supernatants of iDC as well as mDC are shown in Fig. 1. The results of all experiments (control/hormones) were summarized to get the graph. Immature DC produce significantly higher amounts of IL-10 and IL-18 than mature DC, while the chemokines IL-8 and MDC are found in higher concentrations in the supernatants of mature DC. 3.2. Cytokine/chemokine production in dependence of the hormones The relative (compared to untreated control cells) concentration of cytokines in the culture supernatants was
analysed in comparison to the cell cultures with the related ethanol concentration Data are shown in Fig. 2A–F (immature DC) and Fig. 3A–D (mature DC), levels of significance are given in Table 1. 3.2.1. IL12p70 Levels of IL12p70 in the supernatant of immature and mature DC were below the detection range of the test in the majority of samples. Only in three samples, the values obtained by IL12p70 ELISA could be considered as valid (2 standard deviations above background), and therefore no further statistical analysis was performed. 3.2.2. IL-10 Results of the IL-10 ELISA are shown in Figs. 2B and 3B. A significant increase in IL-10 production was observed in
Fig. 3. Results of the cytokine ELISAs of the cultures of mature DC (mDC) with the hormones in question. Left row: samples compared to the lowest concentration of ethanol (Eth; diluted 1:106). Middle row: samples compared to the median ethanol concentration (diluted 1:10.000). Right row: samples compare to the highest concentration of ethanol (diluted 1:1000). Abbreviations: Eth = ethanol; bHCG = beta HCG; Estr: estradiol; Prog = progesterone; P = progesterone, Dexa = dexamethasone. Scale: 100 = 100% of the cytokine concentration analysed in the appropriate untreated control cell culture. *p < 0.05 (Mann–Whitney U-Test). (A) IL-10; (B) IL-8; (C) IL-18 and (D) MDC.
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immature DC upon culture with progesterone as well as estradiol, both at 0.1 ng/ml and 10 ng/ml. In mature DC, a significant stimulatory effect of both substances on IL-10 production could be observed at all three concentrations tested. The combination of 0.1 ng/ml progesterone and bHCG (50 U/ml) resulted in a significant increase of IL-10 levels produced by immature and mature DC. An effect of the combination of progesterone and estradiol (10 ng/ml) was seen at the lowest progesterone concentration in case of mature DC. In case of immature DC, median concentration of IL-10 increased with the combination of progesterone and estradiol. While significance for the increase was nearly missed ( p = 0.0052) in case of estradiol and progesterone at 0.1 ng/ml, with median progesterone concentration in this combination, the effect was significantly. For both maturation stages, Dexamethasone increased the IL-10 secretion into the medium at 10 8 and 10 6 mol/l.
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3.2.7. IL-6 Results of IL-6 ELISA are given in Fig. 2F. Same as with TNFa, IL-6 was used for DC maturation and therefore this cytokine was analysed in immature DC only. A significant decrease of IL-6 concentration in the supernatant was found in cultures with Dexamethasone at 10 8 M. In case of the higher concentration (10 6 M), significance for the decrease observed was nearly missed ( p = 0.059). 3.3. Phenotype of DC Hormonal influence on the phenotype of dendritic cells was tested by flow cytometry. A typical result of FACSanalysis of the corresponding untreated control cells is shown in Fig. 4. Dendritic cells cultured in medium containing IL-4 and GM-CSF exhibited an immature phenotype. Cells were CD83 /(low) and showed low expression of HLA-DR, CD40 and CD86. In the presence
3.2.3. IL-8 Results of the IL-8 ELISA are summarized in Figs. 2B and 3B. In mature DC, IL-8 production was not altered by incubation with hormones as well as with dexamethasone at all. In immature cells there was a significant decrease in IL-8 production upon culture with Dexamethasone at 10 8 and 10 6 mol/l. 3.2.4. IL-18 Results of the IL-18 ELISA are shown in Figs. 2C and 3C. Compared to the respective ethanol controls, levels of the proinflammatory cytokine IL-18 in culture supernatant were not altered in any of the cultures of immature DC. In mature DC cultures, combinations all three concentrations of progesterone with bHCG or estradiol significantly decreased the concentration of IL-18 in culture supernatant for. Progesterone alone had no significant effect. 3.2.5. MDC Results of the MDC ELISA are given in Figs. 2D and 3D. There was no significant effect on MDC concentration in cell cultures of iDC seen with all hormones tested. One exception was the significant increase of the concentration of this chemokine under the influence of progesterone (0.1 ng/ml) and estradiol (10 ng/ml). In mature DC, the combination of progesterone with bHCG at low and medium progesterone concentration, as well as estradiol at 10 ng/ml, significantly decreased the level of MDC in culture supernatant. 3.2.6. TNFa Results of the TNFa ELISA are shown in Fig. 2E. Due to the exogen TNFa used for DC maturation, this cytokine was analysed in immature DC only. In the supernatants of those cultures, the only significant increase in TNFa-concentration was obtained when immature DC were cultured in the presence of progesterone at 10 ng/ml. Significant changes in the TNF-a concentration could not be observed in any other culture experiment.
Fig. 4. Typical FACS-Analysis confirming maturation state of the DC used for the experiments. Left side: mature DC with high expression of CD40 and 86 and upregulated CD83 and HLA-DR. Right side: immature DC show a weak expression of CD40 and HLA-DR, nearly no CD83 surface expression and few CD86 on their surface.
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Fig. 5. Functional analysis of cocktail matured dendritic cells (DC) in a mixed lymphocyte reaction (MLR) with MHC-mismatched responder T lymphocytes at the indicated ratios of DC and T cells. Results are shown as the mean [3H]-thymidine uptake S.E.M. for three samples each. [3H]thymidine uptake of DC and T cell populations alone was <400 cpm and is not shown. For the MLR, the physiologically median concentrations of the appropriate hormones were chosen. In this experimental setting, only the DC cultured in medium with dexamethasone at 10 8 mol/l showed significantly reduced T stimulatory capacity.
of progesterone, estradiol or bHCG in different concentrations, no changes in CD40, HLA-DR or CD86 expression were found. Dendritic cells cultured for 3 days in the presence of the maturation cocktail showed a mature phenotype: cells are CD83+ and show increased levels of HLA-DR and CD40. The costimulatory molecule CD86 was strongly up regulated. Progesterone, estradiol and bHCG did not influence surface marker expression on mature DC, whereas for the combination of progesterone at 0.1 ng/ml with 50 mIU/ml bHCG, a weak decrease in CD83, CD40 and CD86 expression was observed (not shown). 3.4. Stimulatory capacity of DC on allogeneic T cells The MLR results are shown in Fig. 5. In the MLR experiments, mature DC generated under the influence of hormones stimulated T cell proliferation as well as corresponding mature control DC. The capacity of mature DC cultured in the presence of dexamethasone (10 8 mol/l) to stimulate T cell proliferation was seen to be very low compared to the untreated control cells.
4. Discussion In this study we investigated the influence of selected pregnancy associated hormones on the phenotype and function of PBMC derived dendritic cells. PBMC derived DC are a well defined model providing cells at high purity and similar maturation status. As DC undergo different stages of maturation during the development of an immune response, we decided to test the possible influence of hormones on both immature DC, as well as on DC undergoing maturation.
When comparing cells from female and male donors there was no significant difference in cytokine and chemokine production. This ‘‘equalizing of sex’’ could possibly depend on effects of the in-vitro culture of DC with FCS containing culture medium as FCS is known to be able to modulate the degree of estradiolic action in endocrinedependent breast cancer cell lines [16]. However, the content of sex steroids in FCS and their influence on the function and phenotype of DC is still unknown. The overall production of the chemokine IL-8 analysed in the cell cultures supernatants was extremely high in mature DC compared to immature DC. MDC as well was found in significantly higher concentrations in the supernatants of mDC than in iDC. This results are concordant with the data on chemokine-RNA analysed by PCR [17] and the recently published manuscript on cytokine/chemokine profiles, demonstrating that mDC are better producers of chemoattractants than iDC [18] Other than in the latter publication, in our experiments iDC in median of all experiments secreted significantly higher amounts of IL-10 than mDC. It can be speculated that this difference is based on the different maturation protocols. Similar to IL-10, IL-18 was produced to a higher extend by iDC than by mDC. The absolute difference was not dramatic, but nevertheless statistically significant because of the high number of samples compared. To the best of our knowledge, data on the comparison of IL-18 expression by iDC and mDC were not published up to now although different expression of IL-18 in different DC populations is well known [19]. Here, we could demonstrate that in mature DC, progesterone induces a significant increase in the production of the Th2 type cytokine IL-10. This effect could indicate a possible role for progesterone in creating immunotolerance during pregnancy as IL-10 is known to influence T cells [20] as well as APC [21] in a tolerogenic way [22], with emphasis on the role of immature DC. Upon incubation with progesterone at a concentration of 1000 ng/ml, upregulation of IL-10 was not observed in immature DC. This concentration is well above the physiologic levels circulating even during late pregnancy, but was tested here to investigate effects of pharmacological doses. Combinations of progesterone and estradiol corresponding to the follicular (0.1 ng/ml progesterone and 10 ng/ml estradiol) phase of the menstrual cylce significantly increased the IL-10 concentration and decreased the IL-18 concentration in the supernatants of mature DC. In immature DC only a significant increase in IL-10 production was observed, whereas no influence of the progesterone/estradiol combination on the Th-1 cytokines analysed was observed. However, this findings could well fit with the Th2-paradigm postulated for pregnancy [23]. The progesterone/estradiol concentration corresponding to the luteal phase (10 ng/ml progesterone and 10 ng/ml estradiol) of the menstrual cycle decreased the IL-18 concentration in mature DC and decreased the IL-10 concentration in immature DC. The upregulation of the anti-inflammatory cytokine IL-10 by
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progesterone is consistent with observations made in mice, where progesterone was shown to be responsible for the suppression of Th1-type responses and development of a Th2 type response characterized by the production of Th2 type cytokine IL-4 [10]. Furthermore, in human antigenchallenged PBMC, a significant increase in IL-4 production was observed after treatment with progesterone [24]. In this study we used IL-10 and not IL-4 as representative of the Th2 type cytokine family as IL-4 was added to the culture medium and IL-4 production by DC could not be quantified in the supernatants. However, progesterone treated DC showed no alteration in their surface marker expression and in T cell stimulatory capacity. The concentration of 17b-estradiol in non pregnant woman reaches maximum levels of 0.3 ng/ml at the time of ovulation and increases to maximum levels of 10 ng/ml in pregnancy. In our in vitro experiments, concentrations of 0.1 ng/ml corresponding to the menstrual cycle and of 10 ng/ ml as found in late pregnancy showed an increasing effect on the production of IL-10 by immature and mature DC. In the presence of 1000 pg/ml of b-estradiol we found an increased IL-10 production by mature DC. T cell stimulatory capacity and surface marker expression were not altered. In has already been shown that physiological doses of estradiol exert stimulating effects on the immune system [25]. Mechanisms of action of estradiol on the immune system comprise influences on the expression of genes involved in apoptosis [22] and interaction with transcription factors like NFkB and AP-1 [24]. However data published on the effects of estradiol are inconsistent [8,24,26], possibly due to variations in experimental conditions. But there is evidence that immunomodulation by estradiol is not only cell-type and cytokine specific but also dose dependent. Estradiol shows biphasic dose effects. Low doses facilitate immune responses whereas high doses, which occur in pregnancy, suppress such responses [27]. Estradiol also might have an influence on chemoattraction as migration of macrophages into the uterus was proposed to be controlled by estradiol [28]. As chemokine with possibly important role in pregnancy, we tested MDC, which is selectively attracting Th2 polarised T cells and immature DC and seems to be responsible for the regulation of Th2 type responses [29]. While the chemokine IL-8 seems not to be influenced by the hormones tested, MDC showed an opposite regulation in the immature and mature DC tested in this study. While a slight increase (significant only in case of luteal phase corresponding progesterone/estradiol concentration) of MDC levels in immature DC seems to fit in the pattern of activation of the immune system, levels of MDC were decreased in mature DC under the influence of the hormones tested. However, this decrease was especially significant in combinations of progesterone and bHCG. Human chorionic gonadotropin (bHCG) is primarily produced during pregnancy, when serum levels often exceed 100 IU/ml. In 2003, Yoshimura et al. investigated two subsets of DC, myeloid and lymphoid ones and their reaction
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to exogenous bHCG. For both cell types, they found a dosedependent production of the cytokines IL-12 and TNFa, but at different levels. Other than in our experiments, they found no production of IL-10. Since they used differentiated cells sorted out of the peripheral blood, the cell populations are not comparable to our cells and therefore we can not compare the results [15]. We demonstrated a decrease in IL10 production by mature DC for all three bHCG concentrations tested. In general bHCG has been described to have immunosuppressive properties. In a model of murine arthritis bHCG was able to suppress the production of the proinflammatory cytokines IL-1b, IL-6 and TNFa [30], while in PBMC an inhibitory effect on IL-2 production and IL-2-receptor expression could be shown [31]. Little is known about the influence of bHCG on Th2-type cytokines. Further investigation is needed to investigate the role of bHCG in the immunological changes associated with early pregnancy. The combinations of bHCG and progesterone which correspond to the hormonal milieu during early pregnancy showed no effects on immature DC. In mature DC we observed a significant decrease in IL-18 release in all combinations of progesterone with bHCG tested. These findings are supported by the observation that surface marker expression (CD83, CD40 and CD86) in these cells was slightly, albeit insignificant, down regulated similar to the results described in the literature for dexamethasone. This observation suggests an influence of progesterone and bHCG on dendritic cell maturation or selection of less mature cells respectively. However, the T cell stimulatory capacity was not found to be altered. This is in contrast to the data presented by Yoshimura [15] demonstrating an increase in T cell stimulatory capacity of myeloid DC depending on an increasing concentration of bHCG. Since we used monocyte-derived DC and the other cells were sorted myeloid DC, this could be responsible for the differences observed. Therefore in future investigations, the different subpopulations of DC might be of interest for the described experiments. However, the combination of progesterone and bHCG seems to have a distinct influence on monocytederived mature DC by suppressing their inflammatory properties. This is supported by the observation that in mature cells stimulated with the concentration of 0.1 ng/ml progesterone with 50 mIU/ml bHCG, IL-10 production was increased. In summary our results demonstrate that there are some distinct effects of estradiol, progesterone and bHCG on the cytokine production by DC, while surface marker expression and T cell stimulatory capacity seems to be relatively stable against the influence of pregnancy- associated hormones. However it is difficult to explain the various effects by a consistent model like it has been postulated for the Th2-type cytokine environment in pregnancy. It is possible that DC are not the main targets of hormonal signalling in pregnancy. The expanding knowledge in the field of lymphocyte subsets shows that different subpopulations of DC exist, which may
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exert different stimulatory or tolerogenic functions [32–34]. Working with PBMC-derived DC we cannot speculate about the behaviour of uterine DC. It is possible that at the fetomaternal interface, other immunocompetent cell types like stromal cells or large granular lymphocytes (LGL) may be involved in the reception of hormonal signals. In conclusion, we could demonstrate that DC seem to be relatively stable against the influence of progesterone, estradiol and bHCG. Similarly, T cell stimulatory capacity and surface marker expression of DC are not altered by the culture with estradiol, progesterone and bHCG. Effects on cytokine production show a shift to higher Th2 levels. Thus further investigations on the immunological target cells in hormonal signalling during pregnancy are needed.
Acknowledgements We thank E. Kampgen for helpful discussions and M. Kapp for excellent technical assistance
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