HORMONES 2007, 6(1):52-61
DOI: 
Research paper
Modulation of monocytic lipopolysaccharide-induced tissue factor expression and tumor necrosis factor alpha release by estrogen and calcitriol
George Vlotides,1 Roland Gaertner,2 Matthias W.A. Angstwurm2

1Department of Internal Medicine II, Standtort Grosshadern, Ludwig-Maximilians-Universitat, Munich, 2Department of Internal Medicine, Medizinische Klinik, Standtort Innenstadt, Ludwig-Maximilians-Universitat, Munich, Germany

Abstract

OBJECTIVE: Modulatory effects of estrogens on both the immune and the coagulation system are only partially understood. In severe infections high estrogen levels have been observed both in men and postmenopausal women and are associated with increased mortality. Monocyte-derived tissue factor (TF) expression can activate the coagulation system and worsen the course of severe infection. T he aim of the current study was to evaluate the in vitro effect of estrogens on differentiation, TF expression and Tumor Necrosis Factor alpha (TNFα) release in human monocytes. DESIGN: Isolated peripheral blood monocytes, MM6- and T HP-1 cells were cultured and stimulated by lipopolysaccharides (LPS) in the presence of 17β-estradiol (E2) and/or calcitriol. Proliferative responses were evaluated by determining the proliferation rate and by cell cycle analysis. C ell surface expression of C D14 and T F was determined by flow cytometry. TNFα was determined by ELISA. RESULTS: Although calcitriol induced the expression of the differentiation marker C D14 and decreased the expression of T F in both immature monocytic cell lines and primary monocytes, the LPS stimulation of T F expression was not significantly increased in immature monocytic cells and was decreased in mature monocytes. Calcitriol-treatment increased LPS-induced TNFα release in MM6 cells but inhibited TNFα release from peripheral blood monocytes. T reatment with E2 did not alter the phenotype or cell proliferation of resting monocytic cells. However, E2-treated monocytic cells and monocytes responded to LPS by increased TF expression and decreased TNFα. CONCLUSIONS: The results suggest that estrogens may modulate T F expression and cytokine production by monocytes and may thus be involved, at least in part, in the pathophysiology of acute inflammatory processes associated with high estrogen levels.

Keywords

Calcitriol, Estrogens, Lipopolysacharides, Monocytes, Tissue factor, Tumor Necrosis Factor α


Read PDF

INTRODUCTION

The impact of gender and sex hormones on patients with severe infections is still controversial. Although there is evidence of protective effects of estrogens in critical diseases such as trauma-hem-orrhage associated sepsis,1,2 persistent elevation of estrogens during the course of severe inflammation/ sepsis3-7 may be associated with increased mortality.4,7

The biological actions of estrogens are mediated by the specific estrogen receptors (ER) ERα and ERβ, which are ligand-regulated transcription factors that influence the transcription of estrogen-responsive genes. Activated ERs regulate gene transcription through direct DNA binding to estrogen response elements (EREs) located within the regulatory regions of target genes, as well as by interacting with other transcription factors, such as NFκB, AP-1 or Sp1.8,9 Such molecular interactions of ERs with other transcription factors can regulate cytokine activity in a cell and gene-specific manner.9

In addition to interactions between E2 and cytokines at the molecular level, reciprocal interactions between the hypothalamic-pituitary-adrenal (HPA) axis, gonadal axis and immune-mediated inflammatory processes may contribute to changes in inflam-mation parameters, particularly during stress.10 E2 directly enhances corticotropin releasing hormone (CRH) gene expression and stimulates the central noradrenergic system, thereby affecting the two principal components of the stress system,10 which in turn influence the secretion of regulatory cytokines such as IL-10 and IL-1211 and modulate lymphocyte TH1/TH2 balance and/or monocyte activation.

Monocytes/macrophages are the only circulating cells that significantly synthesize Tissue Factor (TF), the main initiator of the extrinsic coagulation protease cascade.12 In various pathologic conditions, monocytes and endothelial cells may express surface TF with subsequent activation of the coagulation system.12 Indeed, TF exposure to the circulation is an important mechanism underlying the initiation of disseminated intravascular coagulation (DIC),13 a severe complication of sepsis. Although monocytes constitute targets of E2 action,14-17 little is known of the potential impact of E2 on monocyte TF expression, particularly during inflammation.

Monocytic cell lines like U-937, THP-1 or MM6 are often used as monocytic models, although they represent relatively immature cells of the monocyte-macrophage cell lineage.18 To simulate the in vivo process, monocytic cells are often treated with specific agents which block their proliferation and induce differentiation in distinct patterns.19,20 A well established differentiation inducing agent for myelomonocytic leukemia cells is the activated form of vitamin D3 (calcitriol).21,22

Upon stereo-specific ligand interaction with the vitamin D receptor (VDR) and heterodimerization of VDR with the retinoid X receptor, VDR is translocated to the nucleus. Binding of VDR to vitamin D response elements (VDRE) as well as interaction with coactivators regulate target gene expression.23 In addition to its classical target tissues, VDR is also expressed in monocytic cells24 and systemic as well as locally produced calcitriol can influence both the immune and coagulation systems.25,26

In the present study, we evaluated the differentia-tion induced by calcitriol in two human monocytic cell lines (THP-1 and MM6) as well as in freshly isolated primary monocytes. In order to understand the impact of high E2 levels on monocyte activation during inflammation, we examined the effect of E2 on monocyte TF expression and TNFα production in an in vitro sepsis model.

MATERIALS AND METHODS

Reagents

RPMI 1640 without phenol red, non-essential amino acids, L-glutamine and fetal calf serum (FCS) werepurchasedfromGibcoLifeTechnologies(Karls-ruhe, Germany), penicillin and streptomycin from Boehringer (Mannheim, Germany) OPI media supplement, 1α,25-dihydroxyvitamin D3, 17β-estradiol and lipopolysaccharide (LPS,Salmonella Minnesota) from Sigma Chemical Co (St. Louis, MO)and Ficoll-Paque Plus from Amersham Biosciences (Freiburg, Germany). Fluorescein (FITC)-conjugated TF monoclonal antibody (IgG1) were obtained from American Diagnostica Inc. (Pfungstadt, Germany), phycoerythrin (PE)-conjugated anti-CD14 antibody and all conjugated isotype controls (IgG1-FITC, IgG1-PE) from Coulter (Krefeld, Germany) and FcRblockingreagentfromMiltenyiBiotec(Bergisch Gladbach, Germany). CycleTESTTM PLUS DNA Reagent Kit and DNA Quality Control Particles were purchased from Becton Dickinson (Heidelberg, Germany).

Mono Mac 6 cells

Monocytic Mono Mac 6 (MM6) cells were cultured in phenol red-free RPMI 1640 medium supplemented with 2mM L-glutamine, 100U/ml penicillin, 100µg/ml streptomycin, 1mM sodium pyruvate, 1% non-essential amino acids, 1mM oxalacetic acid and 10µg/ml insulin. After the addition of supplements, the medium was ultra-filtered through a 0.2µm Me-diaKap filter (Spectrum, Huntingdon, England) followed by the addition of FCS (10%,heatinactivated). All cultures were seeded at a density of 200,000 cells/ml in 24-well plates (Costar, USA) and cultured for 2 to 3 days at 37°C in a 5% CO2 atmosphere.

THP-1 cells

VDR and ER positive monocytic THP-1 cells27,28 were cultured in the aforementioned medium and supplemented with 2mM L-glutamine and 10% heat inactivated FCS. The cells were cultured in tissue culture flasks (NUNC) for 3-4 days at 37°C in a 5% CO2 atmosphere and for experiments they were seeded at a density of 200,000 cells/ml in 24-well plates and cultured for 2 to 3 days.

Peripheral blood monocytes

Heparin (Heparin Novo) anti-coagulated peripheral blood cells were received from apparently healthy male donors, who had given informed consent after full explanation of the purpose and nature of all procedures used. PBMCs were separated by density centrifugation over Ficoll-Paque density gradient. Isolated cells from the interphase were washed twice in cold phosphate-buffered saline (PBS) and re-suspended in RPMI 1640 medium without phenol red, supplemented with 10% heat-inactivated FCS, 2mM L-glutamine, 100U/ml penicillin and 100µg/ml streptomycin.

In order to obtain a monocyte-enrichedcellpopulation (MECs), PBMCs were briefly incubated in plastic culture flasks as previously described.29 Non-adherent cells were removed by medium aspiration and adherent cells were intensively rinsed and removed with cold PBS. This procedure yielded monocytes of ~80% purity,as confirmed by flow cytometric CD14 expression. The MECs were re-suspended in fresh medium (supplemented as described above) and, in order to prevent further cell adherence and thus possible activation, they were cultured in petriperm culture plates with a hydrophobic culture surface (Vivascience, Hannover, Germany). They were then incubated for 1 to 3 days in a fully humidified air atmosphere containing 5% CO2 at 37°C.

Flow Cytometry

The cell surface expression of TF and CD14 on MM6 and THP-1 cells were determined as a single parameter measurement. A minimum of 500,000 cells diluted in 60µl PBS in Falcon 2052 tubes were incubated for 5 minutes with 20µl of FcR blocking reagent. The cells were then incubated with primary antibody(humanFITC-conjugatedanti-TF,11µg/ml, PE-conjugated anti-CD14, 25µg/ml) or isotype control for 10 minutes at room temperature. They were then washed twice in PBS and subsequently analyzed in a flow cytometer (EPICS XL, Coulter, Krefeld, Germany). The cells were gated according to their appearance in the forward scatter (FS)/side scatter (SS)-histogram in order to exclude debris and dead cells. Background staining was determined with the use of isotype control antibody and mean channel fluorescenceintensity(MCFI)/percentageofpositive cells were determined for each sample.

The detection of cell surface expression of TF on MECs was performed as a dual parameter measurement. TF-FITC was detected on the first channel and CD14-PE on the second in order to specifically determine TF expression on CD14-positive cells (monocytes). Specific isotype controls-containing samples were prepared for each measurement in order to compensate spectral overlapping of fluorescence, as required by multi-parametric flow cytometric analysis.

The cells were removed from the culture plates, washed twice in cold PBS and fixed in 2ml of cold 1% formaldehyde buffer for 30 min at 4°C and then washed in cold PBS. Prior to antibody incubation (30 minutes at 4°C), the cellular pellet containing approximately 500,000 cells was re-suspended in 60µl PBS and was incubated for 5 minutes with 20µl of FcR blocking reagent as described above. The fixed cells were then washed in cold PBS and were kept at 4°C until flow cytometric analysis.

Cell viability and proliferative responses

Cell viability was determined with acridin-orange/ ethidium-bromide staining as previously described30 and cell counts were performed with a coulter-counter (Coulter, Krefeld, Germany).

For cell cycle analysis, the EPICS XL was set up using the Becton Dickinson DNA Quality Control Particles and these settings were saved to be used for the acquisition of human nuclear preparations with CycleTESTTM PLUS DNA Reagent Kit as described by the supplier.

TNFα

Determination of TNFα was performed with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Endogen, Woburn, MA) according to the manufacturer’s instructions.


Statistical analysis

Statistical analysis was performed with JMP 5.0.1 software. The data are expressed as the mean±SE of independently performed experiments. The mean of a single treatment group was compared with the mean of a single control group using the Student’s t-test and a p-value of <0.05 was considered statistically significant.

RESULTS

Effect of calcitriol on CD14 expression

MM6 cells constitutively expressed high levels of CD14.87.8±2%ofcellswereCD14positive.Culture of the cells with calcitriol (40ng/ml) for 48 hrs further up-regulated CD14 expression; 97.3±1% of cells were CD14 positive and a ~7.5-fold MCFI increase (p<0.001) was observed (Figure 1A).



Figure 1.
Effect of calcitriol on CD14 and TF expression in MM6, THP-1 and MECs. (MM6, THP-1 and MECs were treated with 40 ng/ml calcitriol for 48 hrs). A: The results represent mean values±SE of at least 3 independently performed experiments (MM6, n=6; THP-1, n=4; MECs, n=3). Significant differences vs. the respective control group are indicated with asterisks. *: P<0.05; ***: P<0.001. B: The results represent mean values ± SE of at least 3 independently performed experiments (MM6, n=9; THP-1, n=7; MECs, n=5). Significant differences vs. the respective control group are indicated with asterisks. **: P<0.01; ***: P<0.001.

THP-1 cells constitutively expressed lower levels of CD14. On average, 33.2±1% of cells were CD14 positive. Treatment with calcitriol also up-regulated CD14 expression; 94.5±1% were CD14 positive and a ~30-fold MCFI increase (p <0.001) was observed (Figure 1A).

MECs were isolated as described and cultured in the presence or absence of calcitriol for 48 hrs. Calcitriol-treatment had no effect on the overall percentage of CD14 positive cells (~20%), but in-creased CD14 MCFI ~2-fold (p-value 0.046) (Figure 1A),suggesting up-regulation of the amount of CD14 receptors in already CD14-positive cells.

Effect of E2 on CD14 expression and proliferation

Treatment of MM6 and THP-1 cells with concentrations of E2 varying from 0 to 1000 pg/ml had no effect on constitutive CD14 expression (not shown). Incubation of MM6 cells for 24 to 72 hrs with the aforementioned E2-concentrations had no signifi-cant influence on their GR. Cell cycle analysis of MM6 cells under the influence of 1000pg/ml E2 was performed after 24 and 48 hrs and no significant difference was observed compared to controls (data not shown).

Effect of calcitriol on TF expression

24.6±6% of MM6 cells were positive for TF. Treatment with calcitriol (40ng/ml) for 48 hrs decreased TF expression ~2-fold (p<0.001) (Figure 1B). 11.4±2% of THP-1 cells expressed TF and calcitriol also decreased constitutive TF expres-sion ~2-fold (p<0.001) (Figure 1B). TF expression was not detected on freshly isolated PBMCs (data not shown). However, 12.3±5% of in vitro cultured CD14-positive MECs expressed TF and calcitriol treatment decreased it ~1.7-fold (p<0.01) (Figure 1B).

LPS stimulation (1µg/ml; 5hrs) increased MM6 and THP-1 TF expression ~1.4-fold (p<0.01), and MECs TF expression ~2.4-fold (p<0.001) (Figure 2A).

Under calcitriol treatment, LPS induced MM6 and THP-1 TF expression 1.5-(p<0.001) and 1.4-fold (p<0.01), respectively (Figure 2B). No significant increase of TF expression in response to LPS was observed in calcitriol-treated MECs (Figure 2B).



Figure 2.
Effect of calcitriol on LPS-induced TF expression in MM6, THP-1 and MECs. (MM6, THP-1 and MECs were incubated with or without 40ng/ml calcitriol for 48 hrs and treated with or without 1μg/ml LPS for the last 5 hrs). Shown is the parameter “% TF-positive cells”. 2A: LPS-induced TF expression in MM6, THP-1 and MECs without calcitriol pretreatment. The results represent mean values ± SE of at least 3 independently performed experiments (MM6, n=9; THP-1, n=7; MECs, n=6). Significant differences vs. the respective control group are indicated with asterisks. **: P<0.01; ***: P<0.001. 2B: LPS-induced TF expression in MM6, THP-1 and MECs with calcitriol pre-treatment. The results represent mean values±SE of at least 3 independently performed experiments (MM6, n=9; THP-1, n=7; MECs, n=6). Significant differences vs. the respective control group are indicated with asterisks. **: P<0.01; ***: P<0.001.

Effect of E2 on TF expression

Incubation of MM6 cells with 1000pg/ml E2 for 24 hrs had no effect on baseline TF expression (not shown). However, pre-treatment of all cell types with E2 up-regulated LPS-induced TF expression (Figure 3 and 4).



Figure 3. Dose-dependent effects of E2 on LPS-induced TF expression in MM6 (3A), THP-1 (3B) and MECs (3C) with and without calcitriol pre-treatment. MM6, THP-1 and MECs were incubated with or without 40ng/ml calcitriol for 48 hrs. 24 hrs prior to stimulation with LPS (1μg/ml for 5 hrs) they were treated with or without the indicated concentrations of E2. Shown is the parameter “% TF-positive cells”. The results represent mean values±SE of 3 independently performed experiments.





Figure 4. Effect of E2 on LPS- induced TF expression in MM6, THP-1 and MECs. MM6, THP-1 and MECs were cultured for 48 hrs in vitro and were treated in the last 24 hrs with 1000pg/ml E2. The MM6 cells were additionally treated during the whole 48 hrs with 40ng/ml calcitriol. Stimulation was performed with 1 μg/ml LPS for 5 hrs. Shown is the parameter “% TF-positive cells”. The results represent mean values±SE of 6 independently performed experiments. Significant differences vs. the respective control group (LPS+E2 vs. LPS) are indicated with asterisks. *: P<0.05.

A preliminary dose-response curve to examine the optimal E2 dose and the effect of additional treatment with calcitriol revealed that pre-incuba-tion of MM6, THP-1 and MECs with increasing E2 concentrations for 24 hrs prior to LPS stimulation (1µg/ml; 5hrs) enhanced LPS-induced TF expres-sion (Figure 3). Calcitriol-treated (40ng/ml; 48 hrs) MM6 cells showed a similar pattern of LPS-induced TF expression under E2 (Figure 3A), in contrast to THP-1 cells and MECs (Figure 3B, 3C).
In order to investigate the significance of this ef-fect, we repeated this experiment at 1000pg/ml E2,and MM6 cells were placed under calcitriol-treatment (Figure 4). E2-treatment for 24 hrs prior to stimulation significantly enhanced LPS-induced TF in MM6 cells from 20±3% to 24±2.6%, in THP-1 cells from 13.5±3.7% to 19±3.2% and in MECs from 29±4.5% to 37.7±6.1% (Figure 4).

Effect of calcitriol and E2 on TNFα production

Untreated MM6 cells produced very low levels of TNFα (1.8±4pg/ml). LPS induced TNFα 53.2-fold (95.7±5pg/ml), and pre-incubation with 500 and 1000pg/ml E2 suppressed this effect (Figure 5A).

Calcitriol-treated MM6 cells constitutively pro-duced slightly higher levels of TNFα (12.3±13.3pg/ ml)compared to base line levels. Stimulation with LPS led to a 10.9-fold increase in TNFα (133.7±10.9pg/ ml)and pre-incubation with increasing E2 concentrations suppressed LPS-induced TNFα release (Figure 5A).

TNFα in the supernatants of untreated MECs was not detectable in all experiments. After stimulation with LPS, MECs produced 485±300pg/ml TNF (Figure 5B) and treatment with increasing E2 concentrations suppressed LPS-induced TNFα ~2-fold.

Calcitriol-treated MECs produced low levels of TNFα (6.3±5.7pg/ml) and LPS stimulation did not significantly induce TNFα above baseline (Figure 5B). E2 pre-treatment had no significant effect on this low LPS-induced TNFα production under calcitriol (Figure 5B).



Figure 5.
Effect of E2 and calcitriol on TNFα production in MM6 (5A) and MECs (5B). MM6 and MECs were incubated with or without 40ng/ml calcitriol for 48hrs. 24 hrs prior to stimulation with LPS (1μg/ml for 5 hrs) the cells were treated with or without the indicated concentrations of E2. Shown are mean values±SE of 5 independently performed experiments. Significant differences are indicated. ***: P<0.001 vs. control; #: P<0.05 vs. LPS no calcitriol; Δ: P<0.05 vs. LPS calcitriol.

DISCUSSION

The impact of gender and sex hormones on patients with severe infections is still controversial. Septic intensive care unit (ICU) patients present a ~10-fold increase of serum estrone and a ~2-fold increase of estradiol levels during the first 24 hrs after admission.4 Although this early estrogen eleva-tion may initially have a protective role, persistent elevation of estrogens during the course of severe inflammation/sepsis3-7 is associated with increased mortality.4,7 A recent study performed in elderly patients with severe infection upon admission to the ICU (208 males and 100 females) did not show gender-specific differences in demographic or infectious characteristics, but showed significantly elevated estrogen levels in non-survivors of either gender.7

Possible causes for the elevation of estrogens include adrenal dysfunction, increased aromatase activity, decreased liver metabolism and central stress-mediated HPA-gonadal axis dysregulation.

As monocyte TF is increased in septic patients31 and monocyte-derived TF is implicated in disseminated intravascular coagulation (DIC),13 a severe complication of sepsis, we sought to examine a putative role of E2 on monocyte activity and particularly TF expression.

Monocytes are the only circulating cells able to produce TF, the main initiator of the extrinsic coagulation protease cascade, and thus play a pivotal role in the regulation of physiological and pathophysiological reactions involving the coagulation system.12 In fact, monocyte TF has been reported to be increased in septic patients31 and in patients with liver cirrhosis,32 while in several in vitro studies monocytes have been induced to express TF upon stimulation.33-35

In this context, we first examined the effect of E2 on resting monocytic cells in vitro, and treated both native and calcitriol-treated MM6 and THP-1 cells with increasing concentrations of E2 for 24 hrs to determine the expression of CD14 and TF. In this experimental setup, the resting phenotype of both cell lines was not influenced by E2. An inhibitory effect of E2 on proliferation of U-937 cells has been reported, which was associated with a cell cycle arrest and accumulation in the G2/M phase.36 We treated MM6 cells with various E2 concentrations for 24-72 hrs but detected no significant effect of E2 on cell viability, cell proliferation rate and DNA phase distribution of thesecells.Thus,theseresultsdonotsuggestaroleof E2 in the regulation of phenotype or differentiation status of non-stimulated monocytic cells.

In order to examine the effect of increased concentrations of E2 on activated monocytes, as observed during sepsis in vivo, E2-pretreated monocytic cells (with and without additional calcitriol treatment) were stimulated with bacterial LPS. At rather high physiological E2 levels similar to those observed during pregnancy (500-1000pg/ml E2) and in some cases of severe inflammation in males and females, both monocytic cell lines and the primary cells responded to E2-treatment with enhanced LPS-induced TF expression (Figures 3 and 4). Thus, E2-mediated TF induction in activated monocytes and the associated procoagulant activity could contribute to the negative outcome of critically ill patients with persistently high E2 levels.

Although TNFα induces procoagulant activity in cultured human vascular endothelium37 and has been implicated in monocyte hypercoagulability,38 it is unlikely that LPS-induced TNFα release in E2-primed monocytes mediates up-regulation of TF expression, as our results showed inhibition of TNFα release in MM6 and primary monocytes (Figure 5) in accordance with previous reports.14,16,17,39

Despite seemingly contradictory data, particularly from in vitro studies, E2 is generally considered immunosuppressive with respect to secretion of cytokines such as IL-1, IL-6 and TNFα from immunocompetent cells.9 Mechanisms involved in E2-mediated inhibition of TNFα in monocytes include decreased JNK activity, which results in decreased c-Jun/Fos and JunD/Fos heterodimer binding to the AP-1 binding site in the TNFα promoter.40 An AP-1siteintheTNF-REisessentialformainlyERβ-mediated repression of promoter activity, requires activation function-2 (AF-2) and is enhanced by coactivator proteins.41

In contrast to regulation of cytokine secretion, little is known of the effect of E2 on monocyte TF expression. Twelve months (but not 3 months) of hormone replacement therapy (HRT) reduced TF activity in both unstimulated and LPS-stimulated monocytes.42 On the other hand, increased TF generation was detected in untreated and LPS-treated monocytes from women using oral contraceptives, with particular involvement of NFκB-regulated transcription,43 data analogous to the results herein presented. However, the above studies involved long-term exposure of healthy female subjects to a lower concentration of estrogens, compared to the concentrations of estrogens in septic ICU patients. Within the experimental setting of this study, the results do not suggest direct induction of TF transcription by short-term E2 exposure, as no effect was observed on baseline TF expression, in contrast to the potent calcitriol-mediated TF down-regulation. It is also unlikely that E2-mediated TF up-regulation resulted from E2-mediated enhancement of the overall reaction to endotoxin exposure, as E2 had no effect on CD14 expression and exhibited divergent effects on LPS-dependent TNFα and TF expression. The mechanisms involved in LPS-induced TF in THP-1 cells include functional interaction between c-Fos/c-Jun and c-Rel/p65 heterodimers bound to the AP-1 and κB sites within the LPS response element (LRE),44 as well as activation of MAPKs p38 and ERK.44,45 Thus, the effect of E2 on LPS-induced TF could result from E2-mediated modulation of LPS signaling.

Several mechanisms can be involved in counter active effects between E2 and calcitirol. E2-mediated VDR up-regulation has been observed in several tissues, including colon/duodenum,46 liver,47 osteoblast-like48 and breast cancer cells.49 In breast cancer cells, direct ERα and ERβ-mediated activation of human VDR promoter activity has been reported.50 On the other hand, calcitriol down-regulates ER abundance and suppresses E2 actions in MCF-7 cells51 which contain a potential VDRE within the ER promoter.52 In addition to direct transcriptional regulation of VDR by E2 and vice versa, stimulatory or inhibitory effects can also be the result of interaction with the signaling of other molecules, such as suppression of calcitriol-induced differentiation of primary bone marrow-derived hematopoietic precursors into osteoclast-like cells by E2-mediated inhibition of IL-6 signaling.53

The counteracting effect of calcitriol on E2-mediated regulation of LPS-induced TF and TNFα in primary monocytes observed in our study is most probably the result of calcitriol-mediated Toll-like receptor down-regulation and decline in LPS responsiveness, supported by the absence of a counter acting effect of calcitriol on E2-mediated up-regulation of LPS-induced TF and TNFα in MM6 cells.

These results suggest that particularly during inflammation, high estrogen levels may modulate monocyte TF expression and thus be involved, at least in part, in the pathophysiology of sepsis syn-drome.Targeting E2-mediated monocyte TF expression in a subset of septic patients with high estrogen levels could decrease procoagulant activity and associated intravascular thrombosis. However, whether monocytic TF induced by other inflammatory stimuli is also modulated by estrogens and the underlying molecular mechanisms still need to be elucidated.

In summary, although calcitriol induces the expression of the monocyte differentiation marker CD14 and decreases the expression of TF, the LPS response with respect to TF expression is not significantly increased in immature monocytic cells and is decreased in mature monocytes. Treatment with E2 does not alter the phenotype or proliferation of resting monocytic cells. However, E2-treated monocytic cells and primary monocytes respond with increased TF expression but decreased TNFα production to stimulation with LPS.


ACKNOWLEDGMENT

We gratefully acknowledge the skillful technical support provided by Petra Rank.


REFERENCES
1. Angele MK, Frantz MC, Chaudry IH, 2006 Gender and sex hormones influence the response to trauma and sepsis: potential therapeutic approaches. Clinics 61: 479-488.
2. Choudhry MA, Bland KI, Chaudry IH, 2006 Gender and susceptibility to sepsis following trauma. Endocr Metab Immune Disord Drug Targets 6: 127-135.
3. Christeff N, Benassayag C, Carli-Vielle C, Carli A, Nunez EA, 1988 Elevated oestrogen and reduced testosterone levels in the serum of male septic shock patients.J Steroid Biochem 29: 435-440.
4. Christeff N, Carli A, Benassayag C, Bleichner G, Vaxelaire JF, Nunez EA, 1992 Relationship between changes in serum estrone levels and outcome in human males with septic shock. Circ Shock 36: 249-255.
5. Sharma AC, Bosmann HB, Motew SJ, Hales KH, Hales DB, Ferguson JL, 1996 Steroid hormone alterations following induction of chronic intraperitoneal sepsis in male rats. Shock 6: 150-154.
6. Fourrier F, Jallot A, Leclerc L, et al, 1994 Sex steroid hormones in circulatory shock, sepsis syndrome, and septic shock. Circ Shock 43: 171-178.
7. Angstwurm MW, Gaertner R, Schopohl J, 2005 Outcome in elderly patients with severe infection is influenced by sex hormones but not gender. Crit Care Med 33: 27862793.
8. Nilsson S, Makela S, Treuter E, et al, 2001 Mechanisms of estrogen action. Physiol Rev 81: 1535-1565.
9. Pfeilschifter J, Koditz R, Pfohl M, Schatz H, 2002 Changes in proinflammatory cytokine activity after menopause. Endocr Rev 23: 90-119. 10. Chrousos GP, Torpy DJ, Gold PW, 1998 Interactions between the hypothalamic-pituitary-adrenal axis and the female reproductive system: clinical implications. Ann Intern Med 129: 229-240.
11. Elenkov IJ, Papanicolaou DA, Wilder RL, Chrousos GP, 1996 Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production: clinical implications. Proc Assoc Am Physicians 108: 374-381.
12. Lwaleed BA, Bass PS, Cooper AJ, 2001 The biology and tumour-related properties of monocyte tissue factor. J Pathol 193: 3-12.
13. Osterud B, Bjorklid E, 2001 The tissue factor pathway in disseminated intravascular coagulation. Semin Thromb Hemost 27: 605-617.
14. Deshpande R, Khalili H, Pergolizzi RG, Michael SD, Chang MD, 1997 Estradiol down-regulates LPS-induced cytokine production and NFkB activation in murine macrophages. Am J Reprod Immunol 38: 46-54.
15. Li ZG, Danis VA, Brooks PM, 1993 Effect of gonadal steroids on the production of IL-1 and IL-6 by blood mononuclear cells in vitro. Clin Exp Rheumatol 11: 157-162.
16. Shanker G, Sorci-Thomas M, Adams MR, 1994 Estrogen modulates the expression of tumor necrosis factor alpha mRNA in phorbol ester-stimulated human monocytic THP-1 cells. Lymphokine Cytokine Res 13: 377-382.
17. Shanker G, Sorci-Thomas M, Register TC, Adams MR, 1994 The inducible expression of THP-1 cell interleukin-1 mRNA: effects of estrogen on differential response to phorbol ester and lipopolysaccharide. Lymphokine Cytokine Res 13: 1-7.
18. Abrink M, Gobl AE, Huang R, Nilsson K, Hellman L, 1994 Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage. Leukemia 8: 1579-1584.
19. Schwende H, Fitzke E, Ambs P, Dieter P, 1996 Differences in the state of differentiation of THP-1 cells induced by phorbol ester and 1,25-dihydroxyvitamin D3. J Leukoc Biol 59: 555-561.
20. Ziegler-Heitbrock HW, Schraut W, Wendelgass P, et al, 1994 Distinct patterns of differentiation induced in the monocytic cell line Mono Mac 6. J Leukoc Biol 55: 73-80.
21. Frankenberger M, Hofmann B, Emmerich B, Nerl C, Schwendener RA, Ziegler-Heitbrock HW, 1997 Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells. Br J Haematol 98: 186-194.
22. Kido S, Inoue D, Hiura K, Javier W, Ito Y, Matsumoto T, 2003 Expression of RANK is dependent upon differentiation into the macrophage/osteoclast lineage: induction by 1alpha,25-dihydroxyvitamin D3 and TPA in a human myelomonocytic cell line, HL60. Bone 32: 621-629.
23. Christakos S, Dhawan P, Liu Y, Peng X, Porta A, 2003 New insights into the mechanisms of vitamin D action. J Cell Biochem 88: 695-705.
24. Koyama T, Shibakura M, Ohsawa M, Kamiyama R, Hirosawa S, 1998 Anticoagulant effects of 1alpha,25-dihydroxyvitamin D3 on human myelogenous leukemia cells and monocytes. Blood 92: 160-167.
25. Hayes CE, Nashold FE, Spach KM, Pedersen LB, 2003 The immunological functions of the vitamin D endocrine system. Cell Mol Biol (Noisy-le-Grand) 49: 277-300.
26. Aihara K, Azuma H, Akaike M, et al, 2004 Disruption of nuclear vitamin D receptor gene causes enhanced thrombogenicity in mice. J Biol Chem 279: 35798-35802.
27. Pramanik R, Asplin JR, Lindeman C, Favus MJ, Bai S, Coe FL, 2004 Lipopolysaccharide negatively modulates vitamin D action by down-regulatingexpression of vitamin D-induced VDR in human monocytic THP-1 cells. Cell Immunol 232: 137-143.
28. Cutolo M, Carruba G, Villaggio B, et al, 2001 Phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) up-regulates the expression of estrogen receptors in human THP-1 leukemia cells. J Cell Biochem 83: 390-400.
29. Martin U, Bock D, Arseniev L, et al, 1997 The human C3a receptor is expressed on neutrophils and monocytes, but not on B or T lymphocytes. J Exp Med 186: 199-207.
30. Hunold W, Wirtz M, Kreiner C, Greite JH, Kaden P, 1991 [Lens epithelium necrosis factor for prevention lens opacity]. Fortschr Ophthalmol 88: 386-389.
31. Vickers J, Russwurm S, Dohrn B, et al, 1998 Monocyte tissue factor (CD142) and Mac-1 (CD11b) are increased in septic patients. Thromb Haemost 79: 1219-1220.
32. Saliola M, Lorenzet R, Ferro D, et al, 1998 Enhanced expression of monocyte tissue factor in patients with liver cirrhosis. Gut 43: 428-432.
33. Luther T, Flossel C, Hietschhold V, Koslowski R, Muller M, 1990 Flow cytometric analysis of tissue factor (TF) expression on stimulated monocytes-comparison to procoagulant activity of mononuclear blood cells. Blut 61: 375-378.
34. van der Logt CP, Dirven RJ, Reitsma PH, Bertina RM, 1994 Expression of tissue factor and tissue factor pathway inhibitor in monocytes in response to bacterial lipopolysaccharide and phorbolester. Blood Coagul Fibrinolysis 5: 211-220.
35. Henriksson CE, Klingenberg O, Ovstebo R, Joo GB, Westvik AB, Kierulf P, 2005 Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis. Thromb Haemost 94: 1236-1244.
36. Mossuz P, Cousin F, Castinel A, et al, 1998 Effects of two sex steroids (17beta estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937. Leuk Res 22: 1063-1072.
37. Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Cotran RS, Gimbrone MAJr, 1986 Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci USA 83: 4533-4537.
38. Spillert CR, Sun S, Ponnudurai R, Miller MA, Lazaro EJ, 1995 Tumor necrosis factor-induced necrosis: a monocyte-mediated hypercoagulable effect. J Natl Med Assoc 87: 508-509.
39. Salem ML, Matsuzaki G, Madkour GA, Nomoto K, 1999 Beta-estradiol-induced decrease in IL-12 and TNF-al-pha expression suppresses macrophage functions in the course of Listeria monocytogenes infection in mice. Int J Immunopharmacol 21: 481-497.
40. Srivastava S, Weitzmann MN, Cenci S, Ross FP, Adler S, PacificiR, 1999 Estrogen decreases TNF gene expression by blocking JNK activity and the resulting production of c-Jun and JunD. J Clin Invest 104: 503-513.
41. An J, Ribeiro RC, Webb P, et al, 1999 Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators. Proc Natl Acad Sci USA 96: 15161-15166.
42. Aune B, Oian P, Omsjo I, Osterud B, 1995 Hormone replacement therapy reduces the reactivity of mono-cytes and platelets in whole blood-a beneficial effect on atherogenesis and thrombus formation? Am J Obstet Gynecol 173: 1816-1820.
43. Holschermann H, Terhalle HM, Zakel U, et al, 1999 Monocyte tissue factor expression is enhanced in women whosmokeanduseoralcontraceptives.ThrombHaemost 82: 1614-1620.
44. Oeth P, Parry GC, Mackman N, 1997 Regulation of the tissue factor gene in human monocytic cells. Role of AP-1, NF-kappa B/Rel, and Sp1 proteins in uninduced and lipopolysaccharide-induced expression. Arterioscler Thromb Vasc Biol 17: 365-374.
45. Chu AJ, Wang ZG, Walton MA, Seto A, 2001 Involvement of MAPK activation inbacterial endotoxin-inducible tissue factor upregulation in human monocytic THP-1 cells. J Surg Res 101: 85-90.
46. Liel Y, Shany S, Smirnoff P, Schwartz B, 1999 Estrogen increases 1,25-dihydroxyvitamin D receptors expression and bioresponse in the rat duodenal mucosa.Endocrinology 140: 280-285.
47. Duncan WE, Glass AR, Wray HL,1991 Estrogen regula-tion of the nuclear 1,25-dihydroxyvitamin D3 receptor in rat liver and kidney. Endocrinology 129: 2318-2324.
48. Liel Y, Kraus S, Levy J, Shany S, 1992 Evidence that estrogens modulate activity and increase the number of 1,25-dihydroxyvitamin D receptors in osteoblast-like cells (ROS 17/2.8). Endocrinology 130: 2597-2601.
49. Escaleira MT, Sonohara S, Brentani MM, 1993 Sex steroids induced up-regulation of 1,25-(OH)2 vitamin D3 receptors in T 47D breast cancer cells. J Steroid Biochem Mol Biol 45: 257-263.
50. Byrne IM, Flanagan L, Tenniswood MP, Welsh J, 2000 Identification of a hormone-responsive promoter im-mediately upstream of exon 1c in the human vitamin D receptor gene. Endocrinology 141: 2829-2836.
51. Swami S, Krishnan AV, Feldman D, 2000 1alpha,25-Dihydroxyvitamin D3 down-regulates estrogen receptor abundance and suppresses estrogen actions in MCF-7 human breast cancer cells. Clin Cancer Res 6: 3371-3379.
52. Stoica A, Saceda M, Fakhro A, Solomon HB, Fenster BD, Martin MB, 1999 Regulation of estrogen receptor-alpha gene expression by 1, 25-dihydroxyvitamin D in MCF-7 cells. J Cell Biochem 75: 640-651.
53. Schiller C, Gruber R, Redlich K, et al, 1997 17 Beta-estradiol antagonizes effects of 1 alpha, 25-dihydroxyvitamin D3 on interleukin-6 production and osteoclast-like cell formation in mouse bone marrow primary cultures. Endocrinology 138: 4567-4571.

Address for correspondence:
George Vlotides, MD, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Los Angeles, CA 90048,
Tel: (310) 423 7669, Fax: (310) 423 0119, E-mail: George.Vlotides@cshs.org

Received 11-09-06, Revised 20-10-06, Accepted 15-11-06

FEATURED ARTICLES

NEWSLETTER