HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mo, R. Articles by Yung, R. L. Search for Related Content PubMed PubMed Citation Articles by Mo, R. Articles by Yung, R. L.

Estrogen Regulates CCR Gene Expression and Function in T Lymphocytes ¹

RuRan Mo^*, Jun Chen^*, Annabelle Grolleau-Julius^*, Hedwig S. Murphy, Bruce C. Richardson^* and Raymond L. Yung^2,*

Departments of^* Internal Medicine and Pathology, University of Michigan, Ann Arbor, MI 48109

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Estrogen has been implicated in the observed female bias in autoimmune diseases. However, the mechanisms behind this gender dimorphism are poorly defined. We have previously reported that in vivo T cell trafficking is gender- and estrogen-dependent. Chemokine receptors are critical determinants of T cell homing and immune response. In this study, we show that the female gender is associated with increased CD4⁺ T cell CCR1-CCR5 gene and protein expression in mice. The increased CCR expression correlates with enhanced in vitro chemotaxis response to MIP-1 (CCL4). In vivo treatment of young oophorectomized and postmenopausal female mice with 17-estradiol also increased CD4⁺ T cell CCR expression. Finally, 17-estradiol enhances tyrosine phosphorylation in T cells stimulated with MIP-1 in a time-dependent manner. Our results indicate an important role of estrogen in determining T cell chemokine response that may help explain the increased susceptibility and severity of autoimmune diseases in females.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Many factors are involved in the induction of autoimmunity. The observation that many autoimmune diseases preferentially affect females suggests that sex hormones play an important role in their pathogenesis (1). Estrogen has been called a Janus molecule because in addition to being a physiologic mediator it also participates in the pathogenesis of disease processes such as atherosclerosis and autoimmune diseases (2). However, the role estrogen plays in the induction of autoimmunity is complicated and incompletely understood. For example, estrogen has been shown to be protective in experimental murine models of Sjogren’s syndrome (3) and autoimmune encephalitis (4). In contrast, it is well accepted that estrogen plays a detrimental role in both human and murine lupus models (1).

We have previously investigated the importance of gender in autoimmunity using a CD4⁺ T cell adoptive transfer system. In this model, D10 cells, a cloned murine Th2 cell line derived from the AKR mice, were made autoreactive by drug treatment with DNA demethylating agents (5, 6, 7, 8, 9) or by transfecting with the CD18 gene (6, 10). Syngeneic AKR mice receiving the autoreactive cells developed a lupus-like illness with autoantibody production and immune complex-mediated glomerulonephritis. Similar to other murine lupus models, gender plays an important role in determining the severity of disease with females developing higher titers of autoantibodies and worse renal disease than their male counterparts (7). Interestingly, 2–7 times the number of autoreactive T cells traffic to the female than to the male spleen. The increased T cell splenic homing is reversed when the female mice were oophorectomized. The gender difference in T cell homing has an important in vivo consequence as oophorectomy and splenectomy completely abrogated the autoimmune phenotype.

Chemokines are chemotactic cytokines that play a central role in determining leukocyte trafficking to lymphoid and nonlymphoid tissues (11). They are classified according to the cysteine motif into C, CC, CXC, and CX3C chemokines. At least 19 chemokine receptors have been identified belonging to the superfamily of G protein-coupled cell surface receptors. The largest group is the CCR. With few exceptions, the CCR has the ability to bind to multiple chemokine ligands. Because T cells display estrogen receptors (12) and estrogen is known to have important effects on T cell function, it is possible that estrogen may have a direct effect on T cell homing via T cell chemokine receptor expression. In this report we sought to determine the role of gender and estrogen on T cell CCR expression and function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

Young (10–12 wk) male and female C57BL/6, AKR, B10A/SgSn (B10), DBA/2, and BALB/c mice were obtained from The Jackson Laboratory. Aged (18–20 mo) female mice were obtained from the National Institute on Aging aged rodent colonies through Harlan Sprague Dawley. As before, only animals without evidence of cancer or lymphoma were used for the experiments (13). Eight- to 10-wk-old oophorectomized C57BL/6 female mice were also obtained from The Jackson Laboratory. The mice were allowed to recover for at least 4 wk after the surgery before they were used. 17-estradiol (60-day release, 0.36 mg/pellet achieving blood level of 150–200 pg/ml) or placebo pellets (all from Innovative Research of America) were implanted under the skin on the lateral side of the neck of the aged (20–21 mo) female and oophorectomized young mice. Experiments were performed 4 wk after the pellets were implanted. The mice were maintained in a pathogen-free environment provided by the Unit for Laboratory Animal Medicine (University of Michigan, Ann Arbor, MI) until used. Procedures involving the animals and their care were conducted in accordance to the guidelines for animal treatment at the University of Michigan.

T cell culture and isolation

D10 cells, a Th2 line originally derived from the AKR mouse strain, were maintained in IL-2-containing medium and stimulated weekly with irradiated syngeneic splenocytes and conalbumin (100 µg/ml; Sigma-Aldrich) as before (5, 6, 7, 8). Because of the concern that regular FCS has high concentration of hormones and may affect the results, charcoal/dextran-treated serum (HyClone Laboratories) was used in all the in vitro experiments. Where indicated, the cells were treated with water-soluble 17-estradiol or progesterone (both from Sigma-Aldrich) 5 days after the last stimulation for 24 h before the assays were performed.

Splenic CD4⁺ T cells were isolated from male and female mice as before (13, 14). CD4⁺ T cells were isolated by magnetic cell separation (MACS) MicroBeads technology (Miltenyi Biotec) according to the manufacturer’s instructions. CD4⁺ cells were negatively selected using a combination of CD8a (Ly-2), CD11b (Mac-1), and CD19 microbeads. Alternately, CD4 cells were positively selected using CD4 (L3T4) microbeads. Purity of the isolated cells was confirmed by staining with the FITC-conjugated anti-CD4 and control IgG2a Abs (all from BD Pharmingen) and was consistently between 94 and 99%.

RNase protection assay (RPA) ³

Quantitative measurements of T cell chemokine receptor gene expression were done by RPA as before using BD Pharmingen kits (13, 14, 15). Pooled RNAs from an equal number of purified T cells from male and female mice in groups of four to six animals were used to minimize individual variability. The probes were synthesized by modification of the manufacturer’s protocol. Briefly, GACU nucleotide pool and [-³²P]UTP, RNasin, T7 RNA polymerase were added to the multiprobe template set mCR-5 (CCR1-CCR5), mCR-6 (CXCR2, CXCR4, and CXCX5), or a custom-made probe set (CCR6-CCR9, CXCR3) (BD Pharmingen) and placed on heat block at 37°C for 1 h. The reaction was terminated by adding DNase and incubated at 37°C on a heat block for 30 min. Appropriate volumes of EDTA, Tris-saturated phenol, chloroform to isoamyl alcohol (50:1), and yeast tRNA were then added to the mixture, as suggested by the manufacturer. The aqueous layer was extracted by chloroform to isoamyl alcohol, then pelleted by adding a 1:5, 4 M ammonium acetate and ice-cold 100% ethanol mixture. The 5 µg of total RNA from each T cell sample was used for hybridization. The protected probes were then fractionated by electrophoresis through a 5% acrylamide gel, exposed to a phosphor screen, and quantified by a PhosphorImager using Image Quant software (Molecular Dynamics), and the signals quantified were in the linear range.

Western immunoblotting

CCR4 and CCR5 protein expression was determined by Western blot analysis as previously described (13, 14, 15). Briefly, proteins purified from male and female CD4⁺ lymphocytes were resolved on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose-1 membrane (Invitrogen Life Technologies). The membrane was blocked in PBS containing 5% nonfat dry milk, and 0.05% Tween 20 and subsequently incubated with anti-mouse CCR4 or CCR5 (Santa Cruz Biotechnology) followed by HRP-conjugated anti-rabbit and anti-rat IgG F(ab')2 (Amersham Pharmacia Biotech). Detection was performed using the ECL system (Amersham Pharmacia Biotech). The membranes were then stripped and reprobed with anti-mouse -actin Abs (Sigma-Aldrich) to confirm equal protein loading.

For T cell tyrosine kinase signaling, D10 cells were exposed to 17-estradiol (0, 25, 250 pg/ml, and 2.5 ng/ml) for 24 h before stimulation with MIP-1 (200 ng/ml). Aliquots were taken at 0 and 30 s, and 1, 3, and 5 min and dissolved in 1% SDS, 1 mmol/L N-ethylmaleimide, 1 mmol/L EDTA, and 2 mmol/L sodium orthovanadate. The proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose-1 membrane. The membranes were then incubated with the anti-phosphotyrosine Ab 4G10 (Upstate Biotechnology) followed by a peroxidase-linked secondary Ab (16) and detected by the ECL system discussed.

In vitro chemotaxis assay

Dual chamber chemotactic assays were performed to compare the transmigration response of T cells from male and female C57BL/6 mice to the chemokines MIP-1 (100 ng/ml) and stromal cell-derived factor (SDF)-1 (CXCL12) (100 ng/ml; (PeproTech) as before (13, 14). Briefly, freshly isolated 4 x 10⁵ T cells in 100 µl RPMI 1640 medium supplemented with 0.5% BSA were placed in Transwell clear culture inserts with 5-µm pores (Corning; Costar). The inserts were then placed in a 24-well tissue culture plate (Corning; Costar) containing 600 µl of RPMI 1640 medium containing 10% FBS for 20 h in a humidified incubator at 37°C. In the MIP-1 experiments exogenous IL-2 (100 ng/ml; PeproTech) was added to the culture media 24 h beforehand (17). Cells from the top and bottom chambers were then harvested and counted with a Beckman Coulter counter. For D10 cells, MIP-1 was used as the chemoattractant. Where indicated, the estrogen receptor antagonist ICI 182,780 (Tocris) or the vehicle control (ethanol) was added to the D10 cell culture for 24 h before the 17-estradiol treatment and the chemotaxis assay.

Statistical analysis

Data were analyzed using ANOVA or Student’s t test, with Bonferroni corrections for multiple comparisons where appropriate. Statistical significance was valued at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The female gender is associated with increased gene expression of selected CD4⁺ T cell CCR

In initial experiments we determined the chemokine receptor expression profile (CCR1-CCR9, CXCR2-CXCR5) of freshly isolated CD4⁺ T cells from young (3–4 mo) male and female C57BL/6 mice by RPA (Fig. 1). Pooled RNAs from five male and female mice were used in each experiment to minimize individual variability and the experiments were repeated three to four times. The results show that CD4⁺ T cells from female mice have significantly higher expression (values for p < 0.05) of CCR1, CCR2, CCR4, and CCR5. Increased in CCR3 was also observed in the females but the results did not reach statistical significance. In contrast, the expression levels of CCR6-CCR9 and CXCR2-CXCR5 were similar in the male and female animals.

View larger version (65K): [in this window] [in a new window]   FIGURE 1. Chemokine receptor gene expression of CD4+ T cells from male and female C57BL/6 mice. RNAs were isolated from pooled CD4 lymphocytes from five male and female mice for each experiment. Representative RPAs showing male and female CCR1-CCR5 (A), CXCR2, CXCR4, and CXCR5 (B), CCR6-CCR9 and CXCR3 (C) are shown. D, The results represent the mean + SEM of four experiments and are expressed relative to male mice (male = 1). Data are normalized for L32 expression. M, Male; F, female. *, p < 0.05; **, p < 0.005.

To confirm that increased mRNA results in changes in protein levels, Western blot analyses were done to determine the CCR4 and CCR5 protein expression of freshly isolated CD4⁺ T cells from male and female C57BL/6 mice. Western blots were chosen because very few murine Abs are currently available that are suitable for flow cytometric study. The results confirm that the female gender is associated with increased CD4⁺ T cell CCR4 and CCR5 protein expression (Fig. 2).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Chemokine receptor protein expression. Western blot analyses of CCR4 and CCR5 of pooled protein isolated from young (3–4 mo) male and female C57BL/6 mice. A single band corresponding to the protein size of CCR4 and CCR5 was observed in each Western blot. The membrane was stripped and reprobed with anti--actin Abs. The results are expressed relative to the males (male = 1). Loading differences were corrected using the density of the -actin band as before (14 ). There were five animals in each experimental group. The results represent the mean ± SEM of six experiments (total 30 males and 30 females) for CCR4 and CCR3 experiments (total 15 males and 15 females) for CCR5.

Females have increased CD4⁺ T cell transmigration in response to MIP-1 but not SDF-1

To determine whether the increased CCR expression in female CD4⁺ T cells correlates to enhanced chemokine receptor function, the chemotactic response of freshly isolated CD4⁺ T cells from male and female C57BL/6 mice to MIP-1 (ligand for CCR5) and SDF-1 (ligand for CXCR4) was determined using dual chamber chemotaxis assay (Fig. 3). SDF-1 induced a robust chemotaxis response in both male and female T cells. However, there was no gender difference in the SDF-1 response, consistent with the lack of gender effect on CXCR4 gene expression. Although MIP-1 induced a smaller chemotaxis response (14), female CD4⁺ T cells exhibited greater chemotaxis than male CD4⁺ T cells. This is consistent with the observed increased CCR5 gene and protein expression in female T cells.

View larger version (9K): [in this window] [in a new window]   FIGURE 3. Gender and T cell chemotaxis response. Male and female T cell transmigration response to MIP-1 (A) and SDF-1 (B) was compared using a Transwell system. MIP-1 (100 ng/ml) or SDF-1 (100 ng/ml) was placed in the lower chamber and allowed to equilibrate for at least 2 h. Equal numbers of freshly isolated male and female T cells (isolated from two to five animals in each group) were than placed in the upper chamber of the Transwell insert with a 5-µm pore size membrane. The cells were harvested 20 h later and counted using a Coulter counter. The results are expressed as the transmigration index relative to the percentage of transmigration of male T cells without chemokine stimulation (male = 1). The results represent the mean ± SEM of four and three independent experiments for MIP-1 and SDF-1, respectively.

To assess the in vivo effect of estrogen on T cell chemokine receptor expression, we determined the effects of oophorectomy and in vivo 17-estradiol supplementation on CD4⁺ T cell chemokine receptor expression. The results show that freshly isolated CD4⁺ T cells from estrogen-supplemented oophorectomized AKR mice have significantly higher expression of CCR1-CCR5 chemokine receptors (Fig. 4) than mice implanted with the placebo pellets. However, compared with wild-type unmanipulated female mice, estrogen supplementation only partially reversed the effect of oophorectomy on CD4⁺ T cell chemokine receptor expression, suggesting that other gonadal factors may also play a role in the regulation of T cell chemokine receptor expression.

View larger version (45K): [in this window] [in a new window]   FIGURE 4. The effect of oophorectomy and estrogen supplementation on T cell chemokine receptor expression. Placebo or 17-estradiol pellets were implanted under the skin of 3-mo-old oophorectomized AKR mice. CD4 T cells were then isolated from the mice in groups of five and their CCR1-CCR5 gene expressions were determined by RPA. A, RPA is showing the effect of in vivo estrogen supplementation on T cell CCR1-CCR5 expression. P, Mice receiving placebo pellets; E2, mice receiving 17-estradiol pellets; WT, unmanipulated age-matched wild-type female mice. B, The results represent the mean ± SEM of three experiments (total 15 placebo, 15 estrogen-supplemented mice, and 15 wild-type age-matched female mice). *, p < 0.005; **, p < 0.001 compared with placebo implanted mice.

View larger version (32K): [in this window] [in a new window]   FIGURE 5. The effect of estrogen supplementation on T cell chemokine receptor expression in aged female mice. A, Representative RPA of freshly isolated CD4 T cells from 18-mo-old female C57BL/6 mice implanted with placebo or 17-estradiol pellets. B, The results represent the mean ± SEM of two experiments with a total of 10 placebo-implanted and 10 estrogen-implanted animals. The results are expressed relative to that of the placebo-implanted animals (placebo = 1). *, p < 0.005; **, p < 0.05.

Cytokines are important regulators of chemokine receptor expression. In addition, Th1 and Th2 cells express different chemokine receptor profiles. For example, Th1 cells express high levels of CCR1, CCR5, CXCR3, CXCR6, and Th2 cells are generally CCR3- and CCR4-positive (20, 21). Mouse strain-specific differences in susceptibility to infectious agents and autoimmunity have been linked to strain bias toward either a Th1 or Th2 response (22, 23, 24, 25, 26). It has also been shown that spleen cells from different mouse strains display distinct patterns of chemokine and chemokine receptor gene expression. For example, C57BL/6 mice with a bias toward Th1 response express higher levels of CCR3 and CXCR4 than BALB/c mice, a prototypic Th2 strain (22). To exclude the possibility that the observed gender effects are specific to C57BL/6 mice, CCR1-CCR5 gene expression of freshly isolated pooled (5–15 animals in each group) CD4⁺ cells of young (12–14 wk of age) male and female AKR, B10.A2, DBA/2, and BALB/c mice were examined by RPA. The results showed considerable strain-specific differences in T cell chemokine receptor expression. However, the female mice have consistently higher CCR gene expression than their age-matched male counterparts in all the strains examined (Fig. 6).

View larger version (45K): [in this window] [in a new window]   FIGURE 6. Gender and strain effects on T cell CCR expression. A, Representative RPAs showing CCR1-CCR5 mRNA expression of freshly isolated CD4 cells from male and female BALB/c, DBA/2, B10, and AKR mice. B, The results represent a total of 10 male and 10 female BALB/c (with five mice in each gender in two experiments), 10 male and 10 female B10 (with five mice in each gender in two experiments), five male and five female DBA/2 (with five mice in each gender), 15 male and 15 female AKR mice (with five mice in each gender in three experiments). The results are expressed as relative chemokine receptor gene expression of CD4 T cells from female mice compared with male mice (male = 1). *, p < 0.005; **, p < 0.01; ***, p < 0.02; ****, p < 0.05.

We have previously demonstrated that D10 cells have different in vivo trafficking pattern in male and female mice (7). We therefore exposed D10 cells to physiological concentrations of 17-estradiol or progesterone for 24 h and their CCR gene expression quantitated by RPAs. As has been reported previously, D10 cells express CCR1 and CCR4, but not CCR2, CCR3, or CCR5 (27). Physiologic concentrations of 17-estradiol (28) (Fig. 7A), but not progesterone (Fig. 7B), were found to increase both CCR1 and CCR4 gene expression in D10 cells (Fig. 7, C and D). We also examined the effect of 17-estradiol on D10 cell chemotaxis response to MIP-1 (CCL3), a ligand for CCR1. Estrogen was found to increase D10 chemotaxis (Fig. 7E), and this response is reversed by pretreatment with the anti-estrogen receptor compound ICI 182,780. To determine whether estrogen affects D10 cell chemokine receptor signaling, D10 cells were treated with 250 ng/ml 17-estradiol for 24 h. The untreated and estrogen-treated T cells were then activated with 200 ng/ml MIP-1 for 30 s to 5 min. Western blot analyses were performed using the anti-phosphotyrosine Ab 4G10 (Fig. 7, F–I). As expected, very low levels of tyrosine phosphorylation were detected in resting D10 cells. In contrast, estrogen-treated D10 cells showed increased protein tyrosine phosphorylation. Further quantitative increase in tyrosine phosphorylation was detected in both estrogen-treated and untreated D10 cells when they were exposed to MIP-1 in a time-dependent manner.

View larger version (38K): [in this window] [in a new window]   FIGURE 7. The effect of estrogen and progesterone on D10 cell chemokine receptor expression and function. D10 cells express the CCR1 and CCR4 genes. Representative RPAs show the effect of 17-estradiol (A) and progesterone (B) on D10 chemokine receptor expression with 0 pg/ml (lane 1), 25 pg/ml (lane 2), 250 pg/ml (lane 3), 2.5 ng/ml (lane 4), and 25 ng/ml (lane 5). C, Relative CCR1 gene expression in estrogen and progesterone-treated D10 cells. D, Relative CCR4 gene expression in estrogen- and progesterone-treated D10 cells. The estrogen results represent the mean + SEM of six independent experiments. The progesterone results represent the mean ± SEM of three independent experiments. Data are normalized for L32 expression. E, Chemotaxis assay showing that 17-estradiol (E2, 250 pg/ml) increases D10 transmigration in response to MIP-1 (100 ng/ml). ICI 182,780 (100 nM) significantly inhibits estrogen (E2)-induced D10 cell MIP-1 transmigration response. V, Vehicle control (ethanol). Results represent mean ± SD of triplicate determinations. F, Time-dependent tyrosine phosphorylation of estrogen-treated and untreated D10 cells activated by MIP-1 (100 ng/ml). Aliquots of the cells were removed at the indicated times after they were activated by the chemokine. Western blots using the anti-phosphotyrosine Ab 4G10 were then performed. The membranes were washed and reprobed with anti--actin Abs to control for differences in gel loading. Relative density of the 48 (G), 66 (H), and 108 (I) kd bands are shown. The results are expressed relative to the value of the sample with no estrogen and no MIP-1 stimulation. The tyrosine phosphorylation results shown are representative of three independent experiments. *, p < 0.001; **, p < 0.005; ***, p < 0.025; ****, p < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Chemokine receptors play a fundamental role in lymphocyte trafficking (34). Homing chemokines expressed at sites of inflammation and specific areas of lymphoid tissue interact with the corresponding chemokine receptors on T and B lymphocytes. For example, CCR7 bearing T and B lymphocytes will respond to CCL21 in high-endothelial venules to enter into lymphoid follicles (35, 36). B cells that bear CXCR5 will then migrate further into the follicles in response to CXCL13. Although most T cells express CCR7, 5% T cell and activated T cells also bear CXCR5 (37, 38). These T cells further migrate into follicles to provide B cell help and to stimulate Ab production. Interestingly, although less well established, CCR1 and CCR3 may also participate in lymphocyte splenic homing (39). Localization of CCR2 and CCR5 at the leading edge of migrating T lymphocyte occurs during migratory response to the corresponding chemokine ligands (40). Similarly, there is a large body of literature supporting a role for CC chemokine-CCR interaction in the trafficking to and retention of leukocytes in inflamed tissues in autoimmune diseases that preferentially affect females. Interestingly, peripheral blood T cells from active lupus patients have decreased CCR2, CCR6, CXCR3, and CXCR4 (41, 42). In contrast, increased T cell CCR4 (43), CCR5 (44), and CXCR3 (45) expression has been reported in renal and skin lesions of active lupus patients, suggesting that selected T cell chemokine receptors may play a significant role in the recruitment and maintenance of T cell tissue infiltrates in this disease. Increased CCR5, CCR6, and CXCR3 expression has also been shown in the synovial tissue T cells in rheumatoid arthritis patients (46, 47, 48). The perceived critical role of chemokine receptors has led to the development of strategies to antagonize chemokine receptors such as CCR1 and CCR5 as therapy for autoimmune diseases including rheumatoid arthritis (49, 50).

Given the importance of gender and T cell chemokine receptors in autoimmunity, it is surprising that very little is currently known about their interaction. Increased leukocyte migration into the uterus was seen in women on oral estrogen (51). Transsexual men receiving estrogens and antiandrogens were also reported to have increased T cell CCR1, CCR5, and CXCR3 expression (52). In contrast, estrogen may suppress monocytes CCR2 expression (53, 54) and inhibit monocyte migration in response to MCP-1 (55). However, the effect of gender and estrogen on T cell chemokine response has not been systematically examined.

Our results demonstrate that CCR1-CCR5 are selectively overexpressed in CD4⁺ T cells from female C57BL/6 mice. The gender difference in CCR expression is not strain-dependent, as four other mouse strains (AKR, BALB/c, B10, and DBA/2), including both Th1 and Th2 disease-prone mice, have similarly increased T lymphocyte CCR expression in females. Thus, although we found strain-dependent T cell chemokine receptor mRNA expression, the gender bias remains true in all five mouse strains. Importantly, increased CCR gene expression correlates to increased chemokine receptor protein expression and function. Quantitative increase in T cell tyrosine phosphorylation is seen in estrogen-treated T cells using the anti-phosphotyrosine Ab 4G10 in Western blot. In addition, there is further increase in tyrosine phosphorylation following MIP-1 stimulation, providing further support that estrogen increases T cell chemokine receptor function. The estrogen T cell response was further confirmed in in vivo studies, as oophorectomized mice receiving estrogen also have increased T cell CCR expression compared with mice receiving placebo. However, in vivo estrogen supplementation only partially reversed the effect of oophorectomy on T cell chemokine receptor gene expression, suggesting that additional female gonadal factors may be involved. Interestingly, estrogen supplementation affects young oophorectomized and aged female mice differently. Unlike their younger cohort, increased expression of only the CCR1, CCR3, and CCR4 but not CCR2 and CCR5 genes were observed in postmenopausal female mice receiving estrogen. The reason for this is unclear. Gender and age do not appear to affect T cell estrogen receptor expression (56). However, we have previously reported that aging is associated with increased T cell CCR expression in both humans (15) and mice (14). It is therefore possible that the observed estrogen effects may be partially masked by the influence of aging on the chemokine system. Other factors such as age and gender differences in endogenous cytokine milieu may also play a role in shaping the in vivo T cell chemokine receptor response.

Estrogen appears to have tissue-specific effects on chemokine receptor expression. This effect may help explain the differential effects of the hormone on diverse pathologic conditions. Atherosclerosis has been established as an inflammatory disease in which monocyte infiltration is critical in the formation of arterial plaques (57). The reported estrogen-induced down-regulation of CCR2 and CXCR3 expression in human and murine monocytes (53, 54, 58) suggests a mechanism through which estrogen may inhibit the local inflammatory response in this disease. In contrast, estrogen increases CCR2, CCR5, CXCR1, and CXCR4 mRNA and protein levels in endometrial epithelial cells that may determine the implantation of blastocyst (59). How estrogen regulates chemokine receptor expression is unknown. Estrogen affects gene transcription by activating its nuclear receptors via classical and nonclassical pathways (60, 61). In the classical pathway, activated estrogen receptors bind as a homodimer or heterodimer to the estrogen-response elements (EREs) in the promoter regions of estrogen-sensitive genes. Alternately, estrogen receptors interact with other transcription factors, such as steroidogenic factor-1, specific factor-1, NF-Y, and AP-1 to activate or repress transcription. Very little is known about the promoter elements of murine CCRs. A search using the Dragon ERE Finder (version 2) software (62) showed multiple potential EREs in the murine CCR5 gene. However, whether estrogen regulates T cell chemokine receptor gene expression via the classical (genomic) or nonclassical (nongenomic) pathway is unclear.

To the best of our knowledge, this is the first systematic examination of the gender difference in T cell CCR expression and function. Chemokine receptors are critical determinants of T cell lymphoid homing. The observed estrogen-dependent effects on CCR gene and protein expression, signaling, and function provide a plausible mechanism for our report showing increased T cell homing to the secondary lymphoid organ in females. The same estrogenic effects on T cell response may also have important implications in explaining the gender dimorphism observed in murine and human autoimmune diseases.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by U.S. Public Health Service Grants 1RO1 AG020628 and 1RO1 AI42753, American Federation for Aging Research (Paul Beeson Physician Faculty Scholar Award), the University of Michigan Nathan Shock Center Grant AG13282, and by the Geriatrics Research, Education, and Clinical Center of the Ann Arbor Department of Veterans Affairs Medical Center.

² Address correspondence and reprint requests to Dr. Raymond L. Yung, Department of Internal Medicine, 5312 Cancer Center and Geriatrics Center Building, University of Michigan, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940. E-mail address: ryung{at}umich.edu

³ Abbreviations used in this paper: RPA, RNase protection assay; SDF-1, stromal cell-derived factor 1; ERE, estrogen-response element.

Received for publication October 13, 2004. Accepted for publication March 3, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Yung, R. L.. 1999. Mechanisms of lupus: the role of estrogens. Clin. Exp. Rheumatol. 17: 271-275.[Medline]
2. Shim, G. J., L. L. Kis, M. Warner, J.-A. Gustafsson. 2004. Autoimmune glomerulonephritis with spontaneous formation of splenic germinal centers in mice lacking the estrogen receptor gene. Proc. Natl. Acad. Sci. USA 101: 1720-1724.[Abstract/Free Full Text]
3. Ishimaru, N., R. Arakaki, M. Watanabe, M. Kobayashi, K. Miyazaki, Y. Hayashi. 2003. Development of autoimmune exocrinopathy resembling Sjogren’s syndrome in estrogen-deficient mice of healthy background. Am. J. Pathol. 163: 1481-1490.[Abstract/Free Full Text]
4. Bebo, B. F., Jr, A. Fyfe-Johnson, K. Adlard, A. G. Beam, A. A. Vandenbark, H. Offner. 2001. Low-dose estrogen therapy ameliorates experimental autoimmune encephalomyelitis in two different inbred mouse strains. J. Immunol. 166: 2080-2089.[Abstract/Free Full Text]
5. Yung, R. L., J. Quddus, C. E. Chrisp, K. J. Johnson, B. C. Richardson. 1995. Mechanisms of drug-induced lupus. I. Cloned Th2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo. J. Immunol. 154: 3025-3035.[Abstract]
6. Yung, R. L., D. Powers, K. Johnson, E. Amento, D. Carr, T. Laing, J. Yang, S. Chang, N. Hemati, B. Richardson. 1996. Mechanisms of drug-induced lupus. II. T Cells overexpressing lymphocyte function-associated antigen-1 become autoreactive and cause a lupus-like disease in syngeneic mice. J. Clin. Invest. 97: 2866-2871.[Medline]
7. Yung, R., R. Williams, K. Johnson, C. Phillips, L. Stoolman, S. Chang, B. Richardson. 1997. Mechanisms of drug-induced lupus III. Sex-specific differences in T cell homing may explain increased disease severity in female mice. Arthritis Rheum. 40: 1334-1343.[Medline]
8. Yung, R., S. Chang, N. Hemati, K. Johnson, B. Richardson. 1997. Mechanisms of drug-induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo. Arthritis Rheum. 40: 1436-1443.[Medline]
9. Yung, R., M. Kaplan, D. Ray, K. Schneider, R. R. Mo, K. Johnson, B. Richardson. 2001. Autoreactive murine Th1 and Th2 cells kill syngeneic macrophages and induce autoantibodies. Lupus 10: 539-546.[Abstract/Free Full Text]
10. Mo, R.-R., J. K. Eisenbraun, J. Sonstein, R. A. Craig, J. L. Curtis, L. M. Stoolman, R. L. Yung. 2003. CD49d overexpression and T cell autoimmunity. J. Immunol. 171: 745-753.[Abstract/Free Full Text]
11. Baggiolini, M.. 1998. Chemokines and leukocyte traffic. Nature 392: 565-568.[Medline]
12. Tornwall, J., A. B. Carey, R. I. Fox, H. S. Fox. 1999. Estrogen in autoimmunity: expression of estrogen receptors in thymic and autoimmune T cells. J. Gend. Specif. Med. 2: 33-40.[Medline]
13. Chen, J., R. Mo, P. A. Lescure, D. E. Misek, S. Hanash, R. Rochford, M. Hobbs, R. L. Yung. 2003. Aging is associated with increased T cell chemokine expression in C57BL/6 Mice. J. Gerontol. A Biol. Sci. Med. Sci 58: 975-983.
14. Mo, R. R., J. Chen, Y. Han, C. Bueno-Cannizares, D. E. Misek, P. A. Lescure, S. Hanash, R. L. Yung. 2003. T cell chemokine receptor expression in aging. J. Immunol. 170: 895-904.[Abstract/Free Full Text]
15. Yung, R. L., R. R. Mo. 2003. Aging is associated with increase human T cell CC chemokine receptor gene expression. J. Interferon Cytokine Res. 23: 575-582.[Medline]
16. Clemetson, K. J., J. M. Clemetson, A. E. I. Proudfoot, C. A. Power, M. Baggiolini, T. N. C. Wells. 2000. Functional expression of CCR1, CCR3, CCR4 and CXCR4 chemokine receptors on human platelets. Blood 96:(13): 4046-4054.[Abstract/Free Full Text]
17. Loetscher, P., M. Seitz, M. Baggiolini, B. Moser. 1996. Interleukin-2 regulates CC chemokine receptor expression and chemotactic responsiveness in T lymphocytes. J. Exp. Med. 184: 569-577.[Abstract/Free Full Text]
18. Rowe, W. P., T. Pincus. 1972. Quantitative studies of naturally occurring murine leukemia virus infection of AKR mice. J. Exp. Med. 135: 429-436.[Abstract]
19. Nelson, J. F., L. S. Felicio, P. K. Randall, C. Sims, C. E. Finch. 1982. A longitudinal study of estrous cyclicity in aging C57BL/6J mice. I. Cycle frequency, length and vaginal cytology. Biol. Reprod. 27: 327-339.[Abstract]
20. Sallusto, F., A. Lanzavecchia, C. R. Markay. 1998. Chemokines and chemokine receptors in T cell priming and Th1/Th2-mediated responses. Immunol. Today 19: 568-574.[Medline]
21. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemokine responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187: 129-134.[Abstract/Free Full Text]
22. Charles, P. C., K. S. Weber, B. Cipriani, C. F. Brosnan. 1999. Cytokine, chemokine and chemokine receptor mRNA expression in different strains of normal mice: implications for establishment of a Th1/Th2 bias. J. Neuroimmunol. 100: 64-73.[Medline]
23. Guida, J. D., G. Fejer, L. A. Pirofski, C. F. Brosnan, M. S. Horwitz. 1995. Mouse adenovirus type 1 causes a fatal hemorrhagic encephalomyelitis in adult C57BL/6 but not BALB/c mice. J. Virol. 69: 7674-7681.[Abstract]
24. Kring, S. C., C. S. King, K. R. Spindler. 1995. Susceptibility and signs associated with mouse adenovirus type 1 infection of adult outbred swiss mice. J. Virol. 69: 8084-8088.[Abstract]
25. Autenrieth, I. B., M. Beer, E. Bohn, S. H. Kaufmann, J. Heesemann. 1994. Immune responses to Yersinia enterocolitica in susceptible BALB/c and resistant C57BL/6 mice: an essential role for interferon. Infect. Immun. 62: 2590-2599.[Abstract/Free Full Text]
26. Suzuki, Y., Q. Yang, J. S. Remington. 1995. Genetic resistance against acute toxoplasmosis depends on the strain of Toxoplasma gondii. J. Parasitol. 81: 1032-1034.[Medline]
27. Murphy, H. S., Q. Sun, B. A. Murphy, R. R. Mo, J. Huo, J. Chen, S. W. Chensue, M. Adams, B. C. Richardson, R. L. Yung. 2004. Tissue-specific effect of estradiol on endothelial cell-dependent lymphocyte recruitment. Microvasc. Res. 68: 273-285.[Medline]
28. Somerville, B. W.. 1971. Daily variation in plasma levels of progesterone and estradiol throughout the menstrual cycle. Am. J. Obstet. Gynecol. 111: 419-426.[Medline]
29. Ansar Ahmed, S., M. J. Dauphinee, N. Talal. 1985. Effects of short-term administration of sex hormones on normal and autoimmune mice. J. Immunol. 134: 204-210.[Abstract]
30. Nilsson, N., H. Carlsten. 1994. Estrogen induces suppression of natural killer cell cytotoxicity and augmentation of polyclonal B cell activation. Cell Immunol. 158: 131-139.[Medline]
31. Carlsten, H., A. Tarkowski, R. Holmdahl, L. A. Nilsson. 1990. Oestrogen is a potent disease accelerator in SLE-prone MRL lpr/lpr mice. Clin. Exp. Immunol. 80: 467-473.[Medline]
32. Ansar Ahmed, S., M. J. Dauphinee, A. I. Montoya, N. Talal. 1989. Estrogen induces normal murine CD5⁺ B cells to produce autoantibodies. J. Immunol. 142: 2647-2653.[Abstract]
33. Talal, N., S. Ansar Ahmed. 1988. Sex hormones, CD5⁺ (Lyl⁺) B-cells, and autoimmune diseases. Isr. J. Med. Sci. 24: 725-728.[Medline]
34. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, E. C. Butcher. 1998. Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279: 381-384.[Abstract/Free Full Text]
35. Campbell, J. J., E. P. Bowman, K. Murphy, K. R. Youngman, M. A. Siani, D. A. Thompson, L. Wu, A. Zlotnik, E. C. Butcher. 1998. 6-C-kine (SLC), a lymphocyte adhesion-triggering chemokine expressed by high endothelium, is an agonist for the MIP-3 receptor CCR7. J. Cell Biol. 141: 1053-1059.[Abstract/Free Full Text]
36. Muller, G., U. E. Hopken, M. Lipp. 2003. The impact of CCR7 and CXCR5 on lymphoid organ development and systemic immunity. Immunol. Rev. 195: 117-135.[Medline]
37. Glabinski, A. R., B. Bielecki, S. O’Bryant, K. Selmaj, R. M. Ransohoff. 2002. Experimental autoimmune encephalomyelitis: CC chemokine receptor expression by trafficking cells. J. Autoimmun. 19: 175-181.[Medline]
38. Schaerli, P., K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, B. Moser. 2000. CXC chemokine receptor 5 expression defines follicular homing T cells with B cell helper function. J. Exp. Med. 192: 1553-1562.[Abstract/Free Full Text]
39. Breitfeld, D., L. Ohl, E. Kremmer, J. Ellwart, F. Sallusto, M. Lipp, R. Forster. 2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J. Exp. Med. 192: 1545-1552.[Abstract/Free Full Text]
40. Nieto, M., J. M. Frade, D. Sancho, M. Mellado, C. Martinez-A, F. Sanchez-Madrid. 1997. Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis. J. Exp. Med. 186: 153-158.[Abstract/Free Full Text]
41. Eriksson, C., K. Eneslatt, J. Ivanoff, S. Rantapaa-Dahlqvist, K. G. Sundqvist. 2003. Abnormal expression of chemokine receptors on T-cells from patients with systemic lupus erythematosus. Lupus 12: 766-774.[Abstract/Free Full Text]
42. Amoura, Z., C. Combadiere, S. Faure, C. Parizot, M. Miyara, D. Raphael, P. Ghillani, P. Debre, J. C. Piette, G. Gorochov. 2003. Roles of CCR2 and CXCR3 in the T cell-mediated response occurring during lupus flares. Arthritis Rheum. 48: 3487-3496.[Medline]
43. Yamada, M., H. Yagita, H. Inoue, T. Takanashi, H. Matsuda, E. Munechika, Y. Kanamaru, I. Shirato, Y. Tomino, K. Matushima, et al 2002. Selective accumulation of CCR4⁺ T lymphocytes into renal tissue of patients with lupus nephritis. Arthritis Rheum. 46: 735-740.[Medline]
44. Vielhauer, V., H. J. Anders, G. Perez de Lema, B. Luckow, D. Schlondorff, M. Mack. 2003. Phenotyping renal leukocyte subsets by four-color flow cytometry: characterization of chemokine receptor expression. Nephron Exp. Nephrol. 93: e63.[Medline]
45. Flier, J., D.M. Boorsma, P.J. van Beek, C. Nieboer, T.J. Stoof, R. Willemze, C.P. Tensen. 2001. Differential expression of CXCR3 targeting chemokines CXCL10, CXCL9, and CXCL11 in different types of skin inflammation. J. Pathol. 194: 398-405.[Medline]
46. Loetscher, P., B. Moser. 2002. Homing chemokines in rheumatoid arthritis. Arthritis Res. 4: 233-236.[Medline]
47. Nissinen, R., M. Leirisalo-Repo, M. Tiittanen, H. Julkunen, H. Hirvonen, T. Palosuo, O. Vaarala. 2003. CCR3, CCR5, interleukin 4, and interferon- expression on synovial and peripheral T cells and monocytes in patients with rheumatoid arthritis. J. Rheumatol. 30: 1928-1934.[Medline]
48. Szekanecz, Z., J. Kim, A. E. Koch. 2003. Chemokines and chemokine receptors in rheumatoid arthritis. Semin. Immunol. 15: 15-21.[Medline]
49. Gladue, R. P., S. H. Zwillich, A. T. Clucas, M. F. Brown. 2004. CCR1 antagonists for the treatment of autoimmune diseases. Curr. Opin. Investig. Drugs 5: 499-504.[Medline]
50. Shadidi, K. R.. 2004. New drug targets in rheumatoid arthritis: focus on chemokines. BioDrugs 18: 181-187.[Medline]
51. DeLoia, J. A., A. M. Stewart-Akers, J. Brekosky, C. J. Kubik. 2002. Effects of exogenous estrogen on uterine leukocyte recruitment. Fertil. Steril. 77: 548-554.[Medline]
52. Giltay, E. J., J. C. Fonk, B. M. von Blomberg, H. A. Drexhage, C. Schalkwijk, L. J. Gooren. 2000. In vivo effects of sex steroids on lymphocyte responsiveness and immunoglobulin levels in humans. J. Clin. Endocrinol. Metab. 85: 1648-1657.[Abstract/Free Full Text]
53. Han, K. H., K. O. Han, S. R. Green, O. Quehenberger. 1999. Expression of the monocyte chemoattractant protein-1 receptor CCR2 is increased in hypercholesterolemia: differential effects of plasma lipoproteins on monocyte function. J. Lipid Res. 40: 1053-1063.[Abstract/Free Full Text]
54. Janis, K., J. Hoeltke, M. Nazareth, P. Fanti, K. Poppenberg, S. M. Aronica. 2004. Estrogen decreases expression of chemokine receptors, and suppresses chemokine bioactivity in murine monocytes. Am. J. Reprod. Immunol. 51: 22-31.
55. Okada, M., A. Suzuki, K. Mizuno, Y. Asada, Y. Ino, T. Kuwayama, K. Tamakoshi, S. Mizutani, Y. Tomoda. 1997. Effects of 17 -estradiol and progesterone on migration of human monocytic THP-1 cells stimulated by minimally oxidized low-density lipoprotein in vitro. Cardiovasc. Res. 34: 529-535.[Abstract/Free Full Text]
56. Kohen, F., L. Abel, A. Sharp, Y. Amir-Zaltsman, D. Somjen, S. Luria, G. Mor, A. Knyszynski, H. Thole, A. Globerson. 1998. Estrogen-receptor expression and function in thymocytes in relation to gender and age. Dev. Immunol. 5: 277-285.[Medline]
57. Nabel, E. G.. 2003. Cardiovascular disease. N. Engl. J. Med. 349: 60-72.[Free Full Text]
58. Han, K. H., K. O. Han, S. R. Green, O. Quehenberger. 1999. Expression of the monocyte chemoattractant protein-1 receptor CCR2 is increased in hypercholesterolemia: differential effects of plasma lipoproteins on monocyte function. J. Lipid Res. 40: 1053-1063.
59. Dominguez, F., A. Galan, J. J. Martin, J. Remohi, A. Pellicer, C. Simon. 2003. Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst. Mol. Hum. Reprod. 9: 189-198.[Abstract/Free Full Text]
60. Tsai, M. J., B. W. O’Malley. 1994. Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63: 451-486.[Medline]
61. Lopez, D., M. D. Sanchez, W. Shea-Eaton, M. P. McLean. 2002. Estrogen activates the high-density lipoprotein receptor gene via binding to estrogen response elements and interaction with sterol regulatory element binding protein-1A. Endocrinology 143: 2155-2168.[Abstract/Free Full Text]
62. Bajic, V. B., S. L. Tan, A. Chong, S. Tang, A. Strom, J. A. Gustafsson, C. Y. Lin, E. T. Liu. 2003. Dragon ERE Finder version 2: a tool for accurate detection and analysis of estrogen response elements in vertebrate genomes. Nucleic Acids Res. 31: 3605-3607.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. H. Windahl, M. K. Lagerquist, N. Andersson, C. Jochems, A. Kallkopf, C. Hakansson, J. Inzunza, J.-A. Gustafsson, P. T. van der Saag, H. Carlsten, et al. Identification of Target Cells for the Genomic Effects of Estrogens in Bone Endocrinology, December 1, 2007; 148(12): 5688 - 5695. [Abstract] [Full Text] [PDF]

				F. J. Martinez, J. L. Curtis, F. Sciurba, J. Mumford, N. D. Giardino, G. Weinmann, E. Kazerooni, S. Murray, G. J. Criner, D. D. Sin, et al. Sex Differences in Severe Pulmonary Emphysema Am. J. Respir. Crit. Care Med., August 1, 2007; 176(3): 243 - 252. [Abstract] [Full Text] [PDF]

				A. J. Lengi, R. A. Phillips, E. Karpuzoglu, and S. A. Ahmed Estrogen selectively regulates chemokines in murine splenocytes J. Leukoc. Biol., April 1, 2007; 81(4): 1065 - 1074. [Abstract] [Full Text] [PDF]

				A. Cernetich, L. S. Garver, A. E. Jedlicka, P. W. Klein, N. Kumar, A. L. Scott, and S. L. Klein Involvement of gonadal steroids and gamma interferon in sex differences in response to blood-stage malaria infection. Infect. Immun., June 1, 2006; 74(6): 3190 - 3203. [Abstract] [Full Text] [PDF]

				X. Jiang, B. A. Orr, D. M. Kranz, and D. J. Shapiro Estrogen Induction of the Granzyme B Inhibitor, Proteinase Inhibitor 9, Protects Cells against Apoptosis Mediated by Cytotoxic T Lymphocytes and Natural Killer Cells Endocrinology, March 1, 2006; 147(3): 1419 - 1426. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mo, R. Articles by Yung, R. L. Search for Related Content PubMed PubMed Citation Articles by Mo, R. Articles by Yung, R. L.