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Originally published online as doi:10.1189/jlb.1103589 on March 12, 2004

Published online before print March 12, 2004
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(Journal of Leukocyte Biology. 2004;75:1166-1172.)
© 2004 by Society for Leukocyte Biology

Estrogen receptor-{alpha} deficiency promotes increased TNF-{alpha} secretion and bacterial killing by murine macrophages in response to microbial stimuli in vitro

K. Chad Lambert*,{dagger}, Edward M. Curran{dagger},{ddagger}, Barbara M. Judy{dagger},{ddagger}, Dennis B. Lubahn{dagger},§ and D. Mark Estes*,{dagger},{ddagger},1

* Departments of Molecular Microbiology & Immunology and
§ Biochemistry and
{dagger} Center for Phytonutrient and Phytochemical Research, University of Missouri, Columbia; and
{ddagger} University of Texas Medical Branch, Department of Pediatrics, Sealy Center for Vaccine Development, Galveston

1 Correspondence: University of Texas Medical Branch, Department of Pediatrics, 2.212 Children’s Hospital, Galveston, TX. 77554. E-mail: dmestes{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this series of studies, we determined the potential role of intracellular estrogen receptors (ER), ER{alpha} and ERß, on macrophage function in response to bacterial stimuli. The sex hormone 17ß-estradiol (E2) and ER have been shown to modulate inflammatory responses as well as T helper cell type 1 (TH1)/TH2 responses. The mechanisms E2 and its receptors use to alter these immune functions remain largely unknown. ER{alpha} and ERß possess complex actions in tissues where they are expressed. We have characterized the receptor repertoire of murine dendritic cells and thioglycollate-elicited peritoneal macrophages (PM). Both cell types express mRNA for ER{alpha}. Neither cell type expressed detectable amounts of ERß mRNA, as determined by reverse transcriptase-polymerase chain reaction using exon-specific primers spanning each of the seven intron/exon junctions. Primary macrophages from ER{alpha}- and ERß-deficient severe combined immunodeficiency mice [ER{alpha} knockout (KO) and ERßKO, respectively] were used to delineate the effects and potential mechanisms via which steroid receptors modulate macrophage function. ER{alpha}-deficient PM exposed ex vivo to lipopolysaccharide or Mycobacterium avium exhibited significant increases in tumor necrosis factor {alpha} (TNF-{alpha}) secretion as well as reduction in bacterial load when compared with wild-type (WT) PM. In contrast, ERß-deficient PM possessed no significant difference in TNF-{alpha} secretion or in bacterial load when compared with WT littermates. These studies suggest that ER{alpha}, but not ERß, modulates murine PM function.

Key Words: SERMS • inflammation • NF-{kappa}B • hormone-independent


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gender bias identified in autoimmune disease and infection in addition to mortality rates associated with sepsis are modulated in part by the sex steroid hormone 17ß-estradiol (E2) [1 2 3 4 5 6 ]. The proinflammatory cytokine, tumor necrosis factor {alpha} (TNF-{alpha}), has been shown to play a central role in these processes. This is demonstrated in part by studies conducted in TNF-{alpha}-deficient mice, which are more susceptible to infection but resistant to the effects of endotoxic shock [7 ]. Clinically, the importance of TNF-{alpha} in disease pathogenesis is demonstrated by the successful use of specific TNF-{alpha} antibodies in the treatment of rheumatoid arthritis and Crohn’s disease patients [8 , 9 ]. The ability of E2 and estrogen receptors (ER) to modulate TNF-{alpha} production, as well as effect T helper cell type 1 (TH1)/TH2 polarization and susceptibility to infection, has been well documented [10 11 12 13 14 15 16 17 ]. However, whether the precise mechanisms used by E2 to mediate these immunomodulatory effects occur as a result of its interaction with specific ER of the innate or adaptive immune systems remains largely unknown. Macrophages and dendritic cells (DC) are key players in innate immunity, which recognize pathogens via Toll-like receptors (TLRs) and ultimately direct the adaptive immune response through cytokine release and antigen presentation [18 ].

The relative ability of macrophages to engulf bacteria and secrete TNF-{alpha} at sites of infection is essential to the development of an effective inflammatory response. In vitro studies suggest E2 suppresses TNF-{alpha} production by macrophages in a cell-specific manner; however, the specific ER-mediating TNF-{alpha} suppression was not addressed [11 , 15 , 19 20 21 ]. The TNF-{alpha} promoter contains binding sites for nuclear factor (NF)-{kappa}B and fos/jun complexes and both transcription factors are critical for TNF-{alpha} production [22 , 23 ]. In studies using ER{alpha}- and ERß-transfected cell lines, E2-liganded and -nonliganded ER have been reported to suppress NF-{kappa}B activity as well as modulate fos/jun complexes. However, the specific ER responsible for suppression of TNF-{alpha} varies depending on the system that was analyzed [9 , 24 25 26 27 28 ]. The existence of ER{alpha} knockout (KO) and ERßKO mice offers an approach to evaluate the roles that ER{alpha} and ERß play in a primary, immune-cell response to bacterial stimuli.

Previous studies conducted by our laboratory using ovarectomized ER{alpha}-deficient female mice have indicated that the in vitro effects of E2 on natural killer cell cytotoxicity are mediated by ERß, possibly a novel ER, but not via ER{alpha} [29 ]. To extend our study to other cell types, we examined effector activities of peritoneal macrophages (PM) from ER{alpha}KO and ERßKO severe combined immunodeficiency (SCID) male mice to delineate the role ER{alpha} and ERß have in the response to microbial stimuli. It should be pointed out that males express ER in various tissues and have low levels of circulating E2 produced by the testes and adipose tissues. ER{alpha} deficiency has been demonstrated to have a wide range of effects on male mice, ranging from decreased thymic development, altered B cell maturation, and an inability to transport functional sperm to the epididymus, which renders them incapable of reproduction [30 31 32 ]. The characterization of the ER repertoire expressed by the innate immune system is an initial step in understanding the mechanisms E2 and ER use to modulate immune function. We demonstrate that TNF-{alpha} secretion by PM from ER{alpha}-deficient mice is altered in response to lipopolysaccharide (LPS) or Mycobacterium avium. ERß deficiency appears to have no effect on the macrophage functions evaluated. Additionally, we demonstrate that ER expression of splenic DC is identical to that of macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6 SCID mice carrying the disrupted ER{alpha} and ERß coding sequences (ER+/ER–) were maintained as a breeding colony at the University of Missouri-Columbia. All experiments were conducted according to Animal Care and Use guidelines as recommended by the National Institutes of Health. Derivation of this mouse strain is described briefly. Female ER{alpha} heterozygous (ER{alpha}+/ER{alpha}–) and ERß heterozygous (ERß+/ ERß–) mice from the above colonies were crossed with male C57BL/6J scid animals obtained from the Jackson Laboratories (Bar Harbor, ME). Female ER{alpha}+/ER{alpha}–/heterozygous scid or ERß+/ ERß–/heterozygous scid offspring from these matings were again backcrossed to the male scid animals obtained from the Jackson Laboratories. Male and female ER{alpha} and ERß heterozygous offspring were screened for the presence of immunoglobulin G (IgG) antibodies in serum collected from each animal. The presence of serum IgG was measured using Micro-Ouchterlony plates (The Binding Site, San Diego, CA) with 5 µg goat anti-mouse IgG (H+L; Kirkegaard and Perry Laboratories, Gaithersburg, MD) applied to the center well and 5 µl mouse serum applied to the outer wells [33 ]. Male and female animals heterozygous for ER{alpha} or ERß and lacking serum IgG (indicative of animals homozygous for the scid mutation) were established as breeding pairs. These breeding colonies yielded wild-type (WT)/SCID, ER{alpha}KO/SCID, and ERßKO/SCID animals for experiments. Genotyping for ER{alpha} and ERß mutations were routinely performed on DNA isolated from tail snips by a multiplex polymerase chain reaction (PCR) procedure [34 ]. WT (ER+/ER+), ER{alpha}KO (ER–/ER–), and ERßKO (ER–/ER–) mice used in these experiments were littermates (LM) and/or age-matched. Mice were housed in a 12-h light/12-h dark cycle.

Reagents
Complete Dulbecco’s minimal essential medium (cDMEM) without phenol red and containing 5% charcoal-stripped calf serum was used in all experiments [29 ]. E2 was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in 100% ethanol. Ethanol/E2 solutions were diluted 1:1000 in culture medium to achieve desired, final E2 concentrations. Control medium without E2 also received a 1:1000 dilution of 100% ethanol. The final concentration of ethanol did not detectably affect our results when compared with medium without ethanol supplementation. LPS (Escherichia coli serotype O55:B5 phenol extract by ion exchange chromatography) was purchased from Sigma Chemical Co.

Isolation and purification of PM and DC
All animals were 8–12 weeks of age. PM were harvested by peritoneal lavage [10 ml phosphate-buffered saline (PBS)] 4 days following intraperitoneal injection with 1 ml sterile 3% thioglycollate medium. Peritoneal exudate cells were centrifuged at 300 g for 10 min and washed with PBS. Cells were resuspended in cDMEM and incubated at 37°C and 5% CO2 on Falcon tissue-culture plates for 2 h. Nonadherent cells were then removed from culture dishes by washing three times with ice-cold PBS. Adherent cells were allowed to incubate overnight in E2-free medium and were washed with PBS. Culture flasks were scraped with a rubber policeman, after which resuspended macrophages were enumerated. Viability was assessed by trypan blue staining. Mouse bone marrow-derived macrophages (BMDM) were prepared by a modification of the method described by Brunt et al. [35 ]. Medium used for propagation of BMDM consisted of DMEM, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, sodium bicarbonate, 15 mM Hepes, and 20% (v/v) L cell-conditioned medium [LCCM; L-929 cell line, American Type Culture Collection (ATCC), Manassas, VA]. Femurs were removed, and bone marrow cells were flushed with 5 ml sterile PBS using a 25-gauge needle. Cells were centrifuged at 500 g for 10 min at room temperature, resuspended in DMEM and layered over 14.1% Nycoprep (Accurate Chemicals, Westbury, NY) solution, and centrifuged at 500 g for 20 min at room temperature. Cells were removed from the interface and placed in flasks for overnight incubation at 37° and 5% CO2 in media not containing LCCM (to remove residential macrophages). Nonadherent cells were seeded at a concentration of 3 x 105/cm2 with 20% LCCM medium. After 4 days of incubation, an additional 10% (v/v) LCCM was added to cultures. BMDM were removed 8 days after LCCM addition by a 5-min incubation with Dispase (1 mg/ml) and light scraping with a rubber policeman.

DC were prepared from total splenocytes by a modification of the method described by Steinman et al. [36 ]. Briefly, spleens were treated with 400 U/ml collagenase (Gibco-BRL, Grand Island, NY) in 1x balanced salt solution (BSS), and the resultant, single-cell suspension was resuspended in a 5-ml high-density bovine serum albumin gradient with 2 ml 1x BSS overlay and centrifuged at 10,000 g for 20 min. Cells obtained from the interface were washed with RPMI and incubated in a plastic dish for 90 min. Dishes were washed three times to remove nonadherent cells. Adherent cells were then incubated for an additional 18 h. At this point, the nonadherent cells (DC) were collected by washing plates three times with PBS. Cell suspensions were labeled with CD11c Microbeads and were sorted with an AutoMacs Cell Sorter (Miltenyi Biotech, Auburn, CA). Analysis by flow cytometry using CD11c phycoerythrin-labeled antibody and isotype controls revealed >96% purity of CD11c-positive DC.

LPS stimulation of PM
PM from male mice were pooled by genotype from ER{alpha}KO mice (n=3–5) and WT LM (n=3–5), as well as ERßKO (n=3–5) mice and their WT LM (n=3–5). PM were seeded at 2 x 105 cells/well in 24-well tissue-culture plates and were incubated overnight in cDMEM. Wells were washed with PBS, and E2-containing cDMEM (E2 concentrations were 10–8 M, 10–9 M, 10–10 M, 10–11 M, 10–12 M) were added to culture wells for 24 h. After a 24-h exposure to the E2 treatments, macrophages were stimulated with LPS (100 ng/ml) for 6 h, after which time, supernatants were collected and stored at –20°C until analysis. Each treatment well was assayed in triplicate. This experiment was repeated with three different groups of animals.

TNF-{alpha} secretion in response to M. avium infection in vitro
PM pooled from ER-KO (n=3–5) male mice and WT (n=3–5) male mice were seeded at 2 x 105 cells/well in 24-well tissue-culture plates and were incubated overnight in antibiotic-free cDMEM. PM were then washed with PBS and exposed to varying molar concentrations of E2-containing cDMEM (10–8 M, 10–10 M, 10–12 M) or E2-free media for 24 h. After a 24-h exposure to various E2 treatments or without E2, macrophage monolayers were infected with smooth colony morphotype M. avium strain 49601 (ATCC) at a multiplicity of infection (MOI) of 25:1. Six hours after M. avium infection, supernatants were removed from cell-culture wells, filtered, and stored at –20°C for cytokine analysis. Each treatment was assayed in triplicate. This experiment was repeated with three different groups of animals comparing ER-KO versus WT without E2 treatment. This experiment testing E2 treatment was done with two different groups of mice.

Quantifation of intracellular M. avium
PM were plated and infected identically with the procedure used to assay TNF secretion in response to M. avium infection. To test whether there were differences in uptake of M. avium between ER{alpha}KO PM and WT PM monolayers, we sampled supernatant from each treatment well and assayed in duplicate onto Middlebrook 7H11 plates (REMEL, Lenexa, KS) 6 h postinfection. The infected PM cell cultures were then washed three times with PBS to remove extracellular bacteria, and appropriate E2-supplemented or E2-free cDMEM was added back to cell-culture wells. Forty-eight hours after infection with M. avium, supernatants were removed, and wells were washed with PBS. A 1% saponin solution was added, and cell-culture wells were scraped with a rubber policeman. Resultant lysates were plated onto Middlebrook 7H11 plates, as described previously [37 ]. M. avium plates were incubated for ~2 weeks at 37°C. Colony forming units (CFUs) were determined from each treatment well plated in duplicate. This experiment without E2 treatment was done with three different groups of mice. E2 treatment was done with two different groups of mice.

Cytokine enzyme-linked immunosorbent assay (ELISA)
A commercial TNF-{alpha} ELISA kit (R&D Systems, Minneapolis, MN) was used to measure total levels of TNF-{alpha} in culture supernatants according to the manufacturer’s protocol. Absorbance was determined at 405 nm using a SoftmaxPro 4.0 plate reader (Molecular Devices, Sunnyvale, CA), and data were analyzed with accompanying software. Cytokine concentrations were estimated by linear regression relative to the manufacturer’s standard.

RNA preparation and reverse transcriptase (RT)-PCR analysis of ER transcripts
Total RNA was extracted from the murine ovary, PM, and splenic DC cell populations using the Qiagen RNeasy mini-kit (Valencia, CA) according to the manufacturer’s protocol. Ovaries were snap-frozen in liquid nitrogen immediately upon removal. PM and DC from male and female mice were washed twice with Hanks’ BSS before RNA was extracted. Isolated, total RNA was treated with a DNase treatment and removal kit (Ambion, Austin, TX). Total RNA (1 µg) was analyzed by RT-PCR for expression of ER{alpha} and ERß using the Titan One Tube RT-PCR system (Roche, Mannheim, Germany). Total mouse ovary RNA served as a positive transcript control for ER{alpha} and ERß expression. The specific primers used for each of the eight exons spanning the seven intron/exon junctions of ERß are listed in Table 1 . For the purposes of clarity, the eight exons in mature ERß mRNA are numbered two through nine. Exon 1 of ERß is expressed in heterogeneous nuclear RNA on an unspliced transcript but not mature ERß mRNA. The following primer sequences were used to amplify ER{alpha}: sense primer, 5' GGG CTT TCC CCC AGC TCA AC 3'; antisense primer, 5' GCA CAC GGC ACA GTA GCG AG 3'. All primers were obtained from IDT (Coralville, IA). RT-PCR reaction conditions for ER{alpha} were as follows: RT at 48°C for 45 min, heat-inactivation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing 60°C for 40 s, and an elongation time of 2 min at 68°C (Gene AMP 2400 thermocycler, Perkin-Elmer, Wellesley, MA). RT-PCR reaction conditions for the ERß reactions were as follows: RT step 48°C for 45 min and heat-inactivation at 94°C for 2 min, amplification reactions for 35 cycles of denaturation at 94°C for 40 s, annealing of 51°C for 40 s, and 2 min at 68°C for elongation. Products were separated by gel electrophoresis on a 2% agarose gel, and bands were visualized by ethidium bromide staining under UV light.


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  		Table 1. RT-PCR Primer Sets for ERß

 
Statistics
Sigma Stat 3.0 (Chicago, IL) was used to estimate the significance of treatments. Each experiment with bacterial stimuli used PM pooled from ER{alpha}KO (n=3–5) and ERß KO (n=3–5) male mice or WT (n=3–5) male mice. The means and SE were obtained by pooling all values from three experiments. Two-way ANOVA analysis was performed to compare effects of E2 treatment and genotype difference. Student’s t-test was used to compare genotypes with the same E2 treatment. Significance was established at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ER repertoire of murine DC and macrophages
The presence of ER{alpha} or ERß transcripts has not been established for murine DC, and the presence of ER{alpha} mRNA and protein in murine macrophages has been demonstrated previously [21 , 38 ]. To determine the available ER repertoire in murine PM and splenic DC, total RNA was analyzed by RT-PCR. Murine PM and splenic DC expressed detectable ER{alpha} mRNA (Fig. 1 ). Initial experiments to test for the presence of ERß transcripts in J774 cells and primary murine macrophages resulted in no detectable products. As alternatively spliced forms of ERß mRNA have been described in many different tissues, we designed primers spanning the seven ERß intron/exon junctions to analyze systematically for the presence of alternatively spliced ERß transcripts expressed by PM or DC. Alternative splice sites in exons 6 and 7 have been reported for ERß, so the exons 6 and 7 primer pair was specifically designed to capture both alternatively spliced forms of the transcript [39 ]. The seven different primer pairs demonstrated expected product size in the murine-ovary control reactions. We did not detect mRNA expression for any of the ERß exons 2–9 in PM or DC (Fig. 1) .



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  		Figure 1. RT-PCR analysis for detection of ER{alpha} and ERß mRNA in murine PM and DC. Total RNA (1 µg) was used in RT-PCR reactions for ER{alpha} and ERß, and 0.5 µg total RNA was used in glyceraldehyde 3-phosphate dehydrogenase (g3pdh) controls. Total RNA isolated from the ovary served as a positive control. All amplified products gave predicted band sizes relative to known molecular weight markers. All primer pairs span intron/exon (ex) junctions for ER{alpha} and ERß. RT-PCR was performed on cells from male and female mice. The ER repertoire was identical for both sexes.

 
ER{alpha} deficiency alone alters TNF-{alpha} secretion by PM in response to LPS
E2 has been shown to negatively regulate transcription of TNF-{alpha} in various systems. We sought to determine whether specific ER were involved in this process. PM were treated with various concentrations of E2 or no E2 for 24 h. Cells were stimulated with LPS (100 ng/ml) for an additional 6 h. PM from ER{alpha}KO mice secreted significantly more TNF-{alpha} than PM from their WT LM (P< 0.02; 1800–1200 pg/ml, respectively) in response to LPS stimulation (Fig. 2 ). However, E2 treatment had no effect on TNF-{alpha} secretion by WT or ER{alpha}KO PM in response to LPS. TNF-{alpha} secretion, in response to LPS, remained unaffected in ERß-deficient PM compared with WT LM PM (data not shown). Additionally, we treated BMDM in vitro with E2 and did not observe an effect of E2 treatment on TNF-{alpha} secretion in response to LPS (data not shown). The fact that E2 treatment did not affect TNF-{alpha} secretion in response to LPS by PM, combined with the increased ability of ER{alpha}KO PM to secrete TNF-{alpha}, suggest that nonliganded ER{alpha} suppressed TNF-{alpha} secretion in WT PM.



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  		Figure 2. ER{alpha} deficiency results in increased TNF-{alpha} secretion in response to LPS. PM from ER{alpha}KO male mice (n=3–5) and their WT male LM (n=3–5) were pretreated for 24 h with various concentrations of E2. Macrophages were then stimulated with LPS (100 ng/ml) for an additional 6 h, and supernatants were analyzed for TNF-{alpha} by ELISA. Macrophages without LPS stimulation expressed lower-than-detectable levels of TNF-{alpha} (<32 pg/ml). Error bars represent the SEM for three independent experiments. Student’s t-test (*, P<0.02) comparisons were done for each treatment group comparing ER{alpha}KO with WT PM.

 
ER{alpha} deficiency results in increased TNF-{alpha} secretion by PM in response to M. avium
We next assessed the effect of ER{alpha} and ERß deficiency on the ability of PM to secrete TNF-{alpha} in response to M. avium challenge. Although LPS is known to signal primarily through activation of TLR4, mycobacterium cell-wall components such as lipoarabinomannan are known, at least in part, to signal through TLR2 [40 ]. PM cell cultures were infected with M. avium (MOI 25:1), and supernatants were removed 6 h postinfection and assayed for secreted cytokine. Once again, ER{alpha}-deficient PM exhibited a significant increase in TNF-{alpha} secretion (P<0.02); however, ERß deficiency in PM did not have a significant effect on TNF-{alpha} secretion in response to M. avium (Fig. 3 ). Treatment with various E2 concentrations (data not shown) did not affect the ability of WT or ER-deficient PM to secrete TNF-{alpha} in response to M. avium challenge. ER{alpha} deficiency had a significant effect on the ability of PM to secrete TNF-{alpha} in response to M. avium, and ERß deficiency had no significant effect.



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  		Figure 3. TNF-{alpha} secretion by PM from ER-KO male (n=3–5) and WT LM male mice (n=3–5) in response to M. avium infection in vitro. Supernatants were assayed by ELISA for TNF-{alpha} at 6 h following infection. ER{alpha}-deficient PM secrete significantly more TNF-{alpha} versus WT PM (*, P>0.02). PM from ERßKO male mice versus WT LM exhibit no significant difference in TNF-{alpha} secretion. Error bars represent SEM for three different experiments.

 
ER{alpha} deficiency enhances the ability of PM to control intracellular growth of M. avium
Given that ER{alpha} deficiency increased TNF-{alpha} production after M. avium exposure, we next assessed the ability of macrophages from ER{alpha}- and ERß-deficient mice to engulf and kill bacteria. PM monolayers were infected at a MOI of 25:1. Bacterial numbers isolated from ER{alpha}-deficient PM were significantly decreased (P<0.03) compared with PM of WT LM at the same MOI (Fig. 4a ). E2 treatments (data not shown) did not affect the number of viable bacteria present after 48 h, which correlates with previous reports that E2 treatment in vitro did not affect intracellular M. avium survival in PM. To test whether the differences of bacterial numbers were a result of uptake of M. avium, supernatants from infected monolayers were plated at 6 h postinfection and showed no statistical difference in M. avium CFUs (Fig. 4b) . These results indicate that uptake of bacteria was similar for ER{alpha}-deficient PM and their WT LM. Preliminary experiments with MOI of 100:1 also demonstrated that ER{alpha} deficiency decreases viable intracellular bacteria (data not shown).



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  		Figure 4. ER{alpha}-deficient PM are more effective than WT PM in controlling M. avium infection in vitro and permit an equal number of bacteria to infect monolayers. PM pooled from ER-KO male mice (n=3–5) and their respective WT LM (n=3–5) were plated and infected with a MOI of 25:1 M. avium strain 49601. (a) Intracellular M. avium CFUs 48 h postinfection from ER{alpha}- and ERß-deficient PM and WT LM PM. (b) Extracellular M. avium CFUs 6 h postinfection from wells of ER{alpha}-deficient and WT PM monolayers. Error bars represent SEM CFU from duplicate plates for three different experiments. *, P < 0.05, as demonstrated by Student’s t-test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate ER{alpha} deficiency but not ERß deficiency leads to increased TNF-{alpha} secretion by murine PM in response to two different TLR stimuli. Furthermore, increased secretion of TNF-{alpha} by ER{alpha}-deficient PM correlated with reduced numbers of viable, intracellular M. avium when challenged ex vivo. ERß deficiency did not affect the ability of PM to secrete TNF-{alpha} in response to LPS or M. avium. Additionally, ERß deficiency did not affect M. avium survival. Using ER-deficient mice, Vegeto et al. [41 ] demonstrated that E2 responds through an ER{alpha}-mediated pathway and not through ERß, resulting in suppression of neuroinflammatory mediators and neural injury caused by brain macrophages. Our investigation is the first to demonstrate that ER{alpha} deficiency and not ERß deficiency results in increased TNF-{alpha} secretion in conjunction with decreased intracellular bacterial numbers in murine macrophages, irrespective of E2 concentration.

The effects of ligand-bound ER in the modulation of inflammatory cytokines are well known. The current use of selective ER modulators in breast cancer and their potential therapeutic use in inflammatory disease states emphasize the need to define the ER repertoire of immune cells. The expression of ER{alpha} in macrophages is well documented; however, the expression of ERß has not been fully ascertained and may vary by species and stage of cell maturation [27 , 38 , 42 43 44 45 46 ]. Reports of ERß mRNA expression in macrophages are conflicting, as in the case of the RAW 264.7 cell line. Srivastava et al. [27 ] demonstrated expression of ERß mRNA in RAW 264.7 cells; however, Benten et al. [38 ] were unable to detect ERß transcript with two sets of primers in the RAW 264.7 cell line. We were unable to detect mRNA expression from any of the ERß exons 2–9 in murine PM or DC, but we were able to detect ER{alpha} transcripts in both cell types. These data, combined with the observation that ERß-deficient PM responded in a similar manner to WT PM, strongly suggest that these cells do not express ERß and that ER{alpha} modulates PM function.

It is interesting that we did not observe suppression of macrophage function as a result of the presence of varying E2 concentrations, although ER{alpha} deficiency brought about a significant increase in TNF-{alpha} secretion and lower numbers of viable bacteria. Studies demonstrating the ability of E2 to suppress macrophage’s secretion of TNF-{alpha} in response to LPS appear to be cell- and species-specific [13 , 15 , 16 , 19 20 21 ]. In contrast to our findings, PM from male rats have been shown to possess suppressed TNF-{alpha} secretion to LPS stimuli when treated with E2 in vitro [47 ]. Murine splenic macrophages treated with E2 have also been shown to have suppressed TNF-{alpha} secretion. However, RAW 264.7 macrophage cell line’s secretion of TNF-{alpha} was not to be affected by E2 treatment [19 , 21 ].

Our data suggest that ER{alpha} is functioning in a hormone-independent manner and suppresses the ability of macrophages to secrete TNF-{alpha} and kill intracellular bacteria. It is plausible that phosphorylation of nonliganded ER{alpha} may have led to the suppression of TNF-{alpha} secretion and M. avium killing. Quaedackers et al. [28 ] reported that nonliganded ER{alpha} suppressed NF-{kappa}B activity in the tranfected osteoblastic U2-O2 cell line [28 ]. The evidence that ER suppress NF-{kappa}B activity has been established, but the mechanisms of ER-mediated suppression are often debated [25 , 48 49 50 ]. Mitogen-activated protein kinases (MAPKs) as well as phosphatidylinositol 3-kinase/AKT pathways have been shown to phosphorylate ER{alpha} and promote hormone-independent activity of the receptor. [31 , 51 52 53 ]. There is also evidence that p38 and c-jun NH2-terminal kinase (JNK) MAPK pathways phosphorylate ER and regulate their activity [54 , 55 ]. The microbial stimuli used here are potent activators of p38 and JNK kinases, which may lead to ER{alpha} phosphorylation and the subsequent ability of this receptor to suppress NF-{kappa}B activity. For optimal expression at the TNF-{alpha} promoter, the transactivators NF-{kappa}B and c-fos/c-jun complex are needed to bind the promoter and activate transcription. As mentioned earlier, ER have been shown to inhibit the activity of both.

Alternatively, ER{alpha} may play a role in the development of functionally mature macrophages. Experiments with ER{alpha}-deficient male mice show that ER{alpha} is necessary for normal development of the thymus and thymocytes [30 ]. Thurmond et al. used ER{alpha}-deficient male mice chimeras to show ER{alpha} was needed for normal numbers of B cells to reach maturity [31 ]. Although change in function of these lymphocytes as a result of ER{alpha} deficiency has not been reported in these systems, it appears that E2 and ER play important roles in lymphopoiesis in the bone marrow [42 ]. Relatively little is known about E2 and ER repertoire effects on myeloid cell development. The potential effects ER{alpha} may exert on macrophage development could be attributed to direct action on the developing cell or by indirect action through regulation of other hormones or nervous tissue, potentially acting on the development of macrophages.

In summary, we have demonstrated that neither murine DC nor PM expresses detectable ERß transcript, but both cell types express ER{alpha} mRNA. The use of ER{alpha}- and ERß-deficient mice enabled us to determine the specific roles ER{alpha} and ERß play in response to two bacterial stimuli. Our data show ER{alpha} deficiency but not ERß deficiency in PM leads to increased TNF-{alpha} secretion and reduced intracellular M. avium bacterial load, regardless of E2 treatment. These data suggest that in vitro-nonliganded ER{alpha}, present in PM, is capable of suppressing cytokine secretion as well as bacterial killing by PM in vitro.


   ACKNOWLEDGEMENTS
 
The University of Missouri Center for Phytonutrient and Phytochemical Research, Grant NIEHS P01 ES10535, supported this work. We sincerely thank Leslie Newton and Pete Ansell, Ph.D., for their generous assistance with the handling of mice and design of exon-specific primers. We give a special thanks to Joel A. Miller, M.S., for his valuable insights regarding the manuscript.

Received November 25, 2003; revised January 27, 2004; accepted February 9, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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