And Function in T Lymphocytes Estrogen Regulates CCR Gene

Estrogen Regulates CCR Gene Expression and Function in T Lymphocytes RuRan Mo, Jun Chen, Annabelle Grolleau-Julius, Hedwig S. Murphy, Bruce C. Richardson and Raymond L. Yung This information is current as of September 27, 2021. J Immunol 2005; 174:6023-6029; ; doi: 10.4049/jimmunol.174.10.6023 http://www.jimmunol.org/content/174/10/6023 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Estrogen Regulates CCR Gene Expression and Function in T Lymphocytes1

RuRan Mo,* Jun Chen,* Annabelle Grolleau-Julius,* Hedwig S. Murphy,† Bruce C. Richardson,* and Raymond L. Yung2*

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 Downloaded from of estrogen in determining T cell chemokine response that may help explain the increased susceptibility and severity of autoimmune diseases in females. The Journal of Immunology, 2005, 174: 6023–6029.

any factors are involved in the induction of autoim- cell splenic homing is reversed when the female mice were oo- munity. The observation that many autoimmune dis- phorectomized. The gender difference in T cell homing has an eases preferentially affect females suggests that sex important in vivo consequence as oophorectomy and splenectomy M http://www.jimmunol.org/ hormones play an important role in their pathogenesis (1). Estro- completely abrogated the autoimmune phenotype. gen has been called a Janus molecule because in addition to being Chemokines are chemotactic cytokines that play a central role in a physiologic mediator it also participates in the pathogenesis of determining leukocyte trafficking to lymphoid and nonlymphoid disease processes such as atherosclerosis and autoimmune diseases tissues (11). They are classified according to the cysteine motif (2). However, the role estrogen plays in the induction of autoim- into C, CC, CXC, and CX3C chemokines. At least 19 chemokine munity is complicated and incompletely understood. For example, receptors have been identified belonging to the superfamily of G estrogen has been shown to be protective in experimental murine protein-coupled cell surface receptors. The largest group is the models of Sjogren’s syndrome (3) and autoimmune encephalitis CCR. With few exceptions, the CCR has the ability to bind to

(4). In contrast, it is well accepted that estrogen plays a detrimental multiple chemokine ligands. Because T cells display estrogen re- by guest on September 27, 2021 role in both human and murine lupus models (1). ceptors (12) and estrogen is known to have important effects on T We have previously investigated the importance of gender in cell function, it is possible that estrogen may have a direct effect on autoimmunity using a CD4ϩ T cell adoptive transfer system. In T cell homing via T cell chemokine receptor expression. In this this model, D10 cells, a cloned murine Th2 cell line derived from report we sought to determine the role of gender and estrogen on the AKR mice, were made autoreactive by drug treatment with T cell CCR expression and function. DNA demethylating agents (5–9) or by transfecting with the CD18 gene (6, 10). Syngeneic AKR mice receiving the autoreactive cells Materials and Methods developed a lupus-like illness with autoantibody production and Mice immune complex-mediated glomerulonephritis. Similar to other Young (10–12 wk) male and female C57BL/6, AKR, B10A/SgSn (B10), murine lupus models, gender plays an important role in determin- DBA/2, and BALB/c mice were obtained from The Jackson Laboratory. ing the severity of disease with females developing higher titers of Aged (18–20 mo) female mice were obtained from the National Institute autoantibodies and worse renal disease than their male counter- on Aging aged rodent colonies through Harlan Sprague Dawley. As before, parts (7). Interestingly, 2–7 times the number of autoreactive T only animals without evidence of cancer or lymphoma were used for the experiments (13). Eight- to 10-wk-old oophorectomized C57BL/6 female cells trafﬁc to the female than to the male spleen. The increased T 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 Departments of *Internal Medicine and †Pathology, University of Michigan, Ann 150–200 pg/ml) or placebo pellets (all from Innovative Research of Amer- Arbor, MI 48109 ica) were implanted under the skin on the lateral side of the neck of the aged (20–21 mo) female and oophorectomized young mice. Experiments Received for publication October 13, 2004. Accepted for publication March 3, 2005. were performed 4 wk after the pellets were implanted. The mice were main- The costs of publication of this article were defrayed in part by the payment of page tained in a pathogen-free environment provided by the Unit for Laboratory charges. This article must therefore be hereby marked advertisement in accordance Animal Medicine (University of Michigan, Ann Arbor, MI) until used. Pro- with 18 U.S.C. Section 1734 solely to indicate this fact. cedures involving the animals and their care were conducted in accordance to 1 This work was supported by U.S. Public Health Service Grants 1RO1 AG020628 the guidelines for animal treatment at the University of Michigan. and 1RO1 AI42753, American Federation for Aging Research (Paul Beeson Physi- cian Faculty Scholar Award), the University of Michigan Nathan Shock Center Grant T cell culture and isolation AG13282, and by the Geriatrics Research, Education, and Clinical Center of the Ann Arbor Department of Veterans Affairs Medical Center. D10 cells, a Th2 line originally derived from the AKR mouse strain, were 2 maintained in IL-2-containing medium and stimulated weekly with irradi- Address correspondence and reprint requests to Dr. Raymond L. Yung, Department ␮ of Internal Medicine, 5312 Cancer Center and Geriatrics Center Building, University ated syngeneic splenocytes and conalbumin (100 g/ml; Sigma-Aldrich) as of Michigan, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0940. E-mail before (5–8). Because of the concern that regular FCS has high concen- address: [email protected] tration of hormones and may affect the results, charcoal/dextran-treated

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 6024 ESTROGEN IN THE PATHOGENESIS OF AUTOIMMUNE DISEASES serum (HyClone Laboratories) was used in all the in vitro experiments. (ethanol) was added to the D10 cell culture for 24 h before the 17␤-estra- Where indicated, the cells were treated with water-soluble 17␤-estradiol or diol treatment and the chemotaxis assay. progesterone (both from Sigma-Aldrich) 5 days after the last stimulation for 24 h before the assays were performed. Statistical analysis Splenic CD4ϩ T cells were isolated from male and female mice as ϩ Data were analyzed using ANOVA or Student’s t test, with Bonferroni before (13, 14). CD4 T cells were isolated by magnetic cell separation (MACS) MicroBeads technology (Miltenyi Biotec) according to the man- corrections for multiple comparisons where appropriate. Statistical signif- ϩ icance was valued at p Ͻ 0.05. ufacturer’s instructions. CD4 cells were negatively selected using a com- bination of CD8a (Ly-2), CD11b (Mac-1), and CD19 microbeads. Alter- nately, CD4 cells were positively selected using CD4 (L3T4) microbeads. Results Purity of the isolated cells was conﬁrmed by staining with the FITC-con- The female gender is associated with increased gene expression jugated anti-CD4 and control IgG2a Abs (all from BD Pharmingen) and of selected CD4ϩ T cell CCR was consistently between 94 and 99%.

3 In initial experiments we determined the chemokine receptor ex- RNase protection assay (RPA) pression profile (CCR1-CCR9, CXCR2-CXCR5) of freshly iso- ϩ Quantitative measurements of T cell chemokine receptor gene expression lated CD4 T cells from young (3–4 mo) male and female were done by RPA as before using BD Pharmingen kits (13–15). Pooled C57BL/6 mice by RPA (Fig. 1). Pooled RNAs from five male and RNAs from an equal number of purified T cells from male and female mice female mice were used in each experiment to minimize individual in groups of four to six animals were used to minimize individual vari- variability and the experiments were repeated three to four times. ability. The probes were synthesized by modification of the manufacturer’s ϩ protocol. Briefly, GACU nucleotide pool and [␣-32P]UTP, RNasin, T7 The results show that CD4 T cells from female mice have sig- RNA polymerase were added to the multiprobe template set mCR-5 Ͻ nificantly higher expression (values for p 0.05) of CCR1, CCR2, Downloaded from (CCR1-CCR5), mCR-6 (CXCR2, CXCR4, and CXCX5), or a custom- CCR4, and CCR5. Increased in CCR3 was also observed in the 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 females but the results did not reach statistical significance. In and incubated at 37°C on a heat block for 30 min. Appropriate volumes of contrast, the expression levels of CCR6-CCR9 and CXCR2- EDTA, Tris-saturated phenol, chloroform to isoamyl alcohol (50:1), and CXCR5 were similar in the male and female animals. yeast tRNA were then added to the mixture, as suggested by the manufacturer. The aqueous layer was extracted by chloroform to isoamyl alco- Gender and CD4ϩ T cell chemokine receptor protein expression hol, then pelleted by adding a 1:5, 4 M ammonium acetate and ice-cold http://www.jimmunol.org/ 100% ethanol mixture. The 5 ␮g of total RNA from each T cell sample was To confirm that increased mRNA results in changes in protein used for hybridization. The protected probes were then fractionated by levels, Western blot analyses were done to determine the CCR4 electrophoresis through a 5% acrylamide gel, exposed to a phosphor and CCR5 protein expression of freshly isolated CD4ϩ T cells 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 (13–15). Briefly, proteins purified from male ϩ and female CD4 lymphocytes were resolved on 10% SDS-polyacryl- by guest on September 27, 2021 amide 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(abЈ)2 (Amersham Pharmacia Bio- tech). 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 Bio- technology) 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 ϫ 105 T cells in 100 ␮l RPMI 1640 medium supplemented with 0.5% BSA were placed in Transwell clear culture inserts with 5-␮m pores (Corning; Costar). The inserts were then placed in a 24-well tissue culture plate (Corning; Costar) containing 600 ␮l of RPMI 1640 medium containing 10% FBS for 20 h in a humidified incubator at 37°C. In the MIP-1␤ ϩ experiments exogenous IL-2 (100 ng/ml; PeproTech) was added to the FIGURE 1. Chemokine receptor gene expression of CD4 T cells from culture media 24 h beforehand (17). Cells from the top and bottom cham- male and female C57BL/6 mice. RNAs were isolated from pooled CD4 bers were then harvested and counted with a Beckman Coulter counter. For lymphocytes from five male and female mice for each experiment. Rep- D10 cells, MIP-1␣ was used as the chemoattractant. Where indicated, the resentative RPAs showing male and female CCR1-CCR5 (A), CXCR2, estrogen receptor antagonist ICI 182,780 (Tocris) or the vehicle control CXCR4, and CXCR5 (B), CCR6-CCR9 and CXCR3 (C) are shown. D, The results represent the mean ϩ SEM of four experiments and are ex- ϭ 3 Abbreviations used in this paper: RPA, RNase protection assay; SDF-1, stromal pressed relative to male mice (male 1). Data are normalized for L32 .p Ͻ 0.005 ,ءء ;p Ͻ 0.05 ,ء .cell-derived factor 1; ERE, estrogen-response element. expression. M, Male; F, female The Journal of Immunology 6025

FIGURE 4. The effect of oophorectomy and estrogen supplementation FIGURE 2. Chemokine receptor protein expression. Western blot anal- on T cell chemokine receptor expression. Placebo or 17␤-estradiol pellets yses of CCR4 and CCR5 of pooled protein isolated from young (3–4 mo) were implanted under the skin of 3-mo-old oophorectomized AKR mice. male and female C57BL/6 mice. A single band corresponding to the pro- CD4 T cells were then isolated from the mice in groups of ﬁve and their tein size of CCR4 and CCR5 was observed in each Western blot. The CCR1-CCR5 gene expressions were determined by RPA. A, RPA is show- membrane was stripped and reprobed with anti-␤-actin Abs. The results are ing the effect of in vivo estrogen supplementation on T cell CCR1-CCR5 expressed relative to the males (male ϭ 1). Loading differences were cor- expression. P, Mice receiving placebo pellets; E2, mice receiving 17␤- Downloaded from rected using the density of the ␤-actin band as before (14). There were ﬁve estradiol pellets; WT, unmanipulated age-matched wild-type female mice. animals in each experimental group. The results represent the mean Ϯ B, The results represent the mean Ϯ SEM of three experiments (total 15 SEM of six experiments (total 30 males and 30 females) for CCR4 and placebo, 15 estrogen-supplemented mice, and 15 wild-type age-matched -p Ͻ 0.001 compared with placebo im ,ءء ;p Ͻ 0.005 ,ء .(CCR3 experiments (total 15 males and 15 females) for CCR5. female mice planted mice.

from male and female C57BL/6 mice. Western blots were chosen http://www.jimmunol.org/ induced a smaller chemotaxis response (14), female CD4ϩ T cells because very few murine Abs are currently available that are suit- exhibited greater chemotaxis than male CD4ϩ T cells. This is con- able for flow cytometric study. The results confirm that the female ϩ sistent with the observed increased CCR5 gene and protein ex- gender is associated with increased CD4 T cell CCR4 and CCR5 pression in female T cells. protein expression (Fig. 2). ϩ ϩ CD4 T cell chemokine receptor expression of oophorectomized Females have increased CD4 T cell transmigration in and estrogen-treated mice response to MIP-1␤ but not SDF-1 To assess the in vivo effect of estrogen on T cell chemokine re- To determine whether the increased CCR expression in female ϩ ceptor expression, we determined the effects of oophorectomy and by guest on September 27, 2021 CD4 T cells correlates to enhanced chemokine receptor function, ϩ ϩ in vivo 17␤-estradiol supplementation on CD4 T cell chemokine the chemotactic response of freshly isolated CD4 T cells from receptor expression. The results show that freshly isolated CD4ϩ T male and female C57BL/6 mice to MIP-1␤ (ligand for CCR5) and cells from estrogen-supplemented oophorectomized AKR mice SDF-1 (ligand for CXCR4) was determined using dual chamber have significantly higher expression of CCR1-CCR5 chemokine chemotaxis assay (Fig. 3). SDF-1 induced a robust chemotaxis receptors (Fig. 4) than mice implanted with the placebo pellets. response in both male and female T cells. However, there was no However, compared with wild-type unmanipulated female mice, gender difference in the SDF-1 response, consistent with the lack estrogen supplementation only partially reversed the effect of oo- of gender effect on CXCR4 gene expression. Although MIP-1␤ phorectomy on CD4ϩ T cell chemokine receptor expression, suggesting that other gonadal factors may also play a role in the regulation of T cell chemokine receptor expression.

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 FIGURE 5. The effect of estrogen supplementation on T cell chemokine of the Transwell insert with a 5-␮m pore size membrane. The cells were receptor expression in aged female mice. A, Representative RPA of freshly harvested 20 h later and counted using a Coulter counter. The results are isolated CD4 T cells from 18-mo-old female C57BL/6 mice implanted with expressed as the transmigration index relative to the percentage of trans- placebo or 17␤-estradiol pellets. B, The results represent the mean Ϯ SEM migration of male T cells without chemokine stimulation (male ϭ 1). The of two experiments with a total of 10 placebo-implanted and 10 estrogen- results represent the mean Ϯ SEM of four and three independent experi- implanted animals. The results are expressed relative to that of the placebo- .p Ͻ 0.05 ,ءء ;p Ͻ 0.005 ,ء .(ments for MIP-1␤ and SDF-1, respectively. implanted animals (placebo ϭ 1 6026 ESTROGEN IN THE PATHOGENESIS OF AUTOIMMUNE DISEASES

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 p Ͻ ,ء .(female mice compared with male mice (male ϭ 1 .p Ͻ 0.05 ,ءءءء ;p Ͻ 0.02 ,ءءء ;p Ͻ 0.01 ,ءء ;0.005

In separate studies, we examined the effect of estrogen supple- supplementation in 21- to 22-mo-old female C57BL/6 mice. Older mentation on T cell CCR gene expression in the “natural” estro- mice were not used because they have a high incidence of malig- gen-depleted state in female aging. Because AKR mice have a very nancies. Pooled RNA from freshly isolated CD4ϩ T cells from ﬁve high incidence of lymphoma by 9–12 mo of age (18) we elected to female old C57BL/6 mice implanted with either placebo or 17␤- use C57BL/6 female mice for these experiments. Menopausal estradiol pellets was used for the RPA. Unlike young oophorec- Downloaded from changes in the C57BL/6 mice begin at the age 10–14 mo, with tomized females, only the CCR1, CCR3, and four genes were up- complete cessation of cycles occurring between 18 and 20 mo of regulated by the in vivo estrogen supplementation in aged female age (19). We therefore chose to examine the effect of estrogen mice (Fig. 5). http://www.jimmunol.org/

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 by guest on September 27, 2021 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 ϩ SEM of six independent experiments. The progesterone results represent the mean Ϯ 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) signiﬁ- ␣ cantly inhibits estrogen (E2)-induced D10 cell MIP-1 transmigration response. V, Vehicle control (ethanol). Results represent mean Ϯ SD of triplicate determina- tions. F, Time-dependent tyrosine phosphorylation of estrogen-treated and untreated D10 cells activated by MIP-1␣ (100 ng/ml). Aliquots of the cells were re- moved 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 ex- ;p Ͻ 0.025 ,ءءء ;p Ͻ 0.005 ,ءء ;p Ͻ 0.001 ,ء .periments .p Ͻ 0.01 ,ءءءء The Journal of Immunology 6027

Strain effect on gender-associated changes in T cell chemokine Chemokine receptors play a fundamental role in lymphocyte receptor expression trafficking (34). Homing chemokines expressed at sites of inflam- Cytokines are important regulators of chemokine receptor expres- mation and specific areas of lymphoid tissue interact with the cor- sion. In addition, Th1 and Th2 cells express different chemokine responding chemokine receptors on T and B lymphocytes. For receptor profiles. For example, Th1 cells express high levels of example, CCR7 bearing T and B lymphocytes will respond to CCR1, CCR5, CXCR3, CXCR6, and Th2 cells are generally CCL21 in high-endothelial venules to enter into lymphoid follicles CCR3- and CCR4-positive (20, 21). Mouse strain-specific differ- (35, 36). B cells that bear CXCR5 will then migrate further into the ences in susceptibility to infectious agents and autoimmunity have follicles in response to CXCL13. Although most T cells express been linked to strain bias toward either a Th1 or Th2 response CCR7, 5% T cell and activated T cells also bear CXCR5 (37, 38). (22–26). It has also been shown that spleen cells from different These T cells further migrate into follicles to provide B cell help mouse strains display distinct patterns of chemokine and chemo- and to stimulate Ab production. Interestingly, although less well kine receptor gene expression. For example, C57BL/6 mice with a established, CCR1 and CCR3 may also participate in lymphocyte bias toward Th1 response express higher levels of CCR3 and splenic homing (39). Localization of CCR2 and CCR5 at the lead- CXCR4 than BALB/c mice, a prototypic Th2 strain (22). To ex- ing edge of migrating T lymphocyte occurs during migratory re- clude the possibility that the observed gender effects are specific to sponse to the corresponding chemokine ligands (40). Similarly, C57BL/6 mice, CCR1-CCR5 gene expression of freshly isolated there is a large body of literature supporting a role for CC che- ϩ pooled (5–15 animals in each group) CD4 cells of young (12–14 mokine-CCR interaction in the trafficking to and retention of leu- wk of age) male and female AKR, B10.A2, DBA/2, and BALB/c kocytes in inflamed tissues in autoimmune diseases that preferen- Downloaded from mice were examined by RPA. The results showed considerable tially affect females. Interestingly, peripheral blood T cells from strain-specific differences in T cell chemokine receptor expression. active lupus patients have decreased CCR2, CCR6, CXCR3, and However, the female mice have consistently higher CCR gene ex- CXCR4 (41, 42). In contrast, increased T cell CCR4 (43), CCR5 pression than their age-matched male counterparts in all the strains (44), and CXCR3 (45) expression has been reported in renal and examined (Fig. 6). skin lesions of active lupus patients, suggesting that selected T cell

chemokine receptors may play a significant role in the recruitment http://www.jimmunol.org/ Estrogen enhances D10 cell CCR expression and function and maintenance of T cell tissue infiltrates in this disease. In- We have previously demonstrated that D10 cells have different in creased CCR5, CCR6, and CXCR3 expression has also been vivo trafficking pattern in male and female mice (7). We therefore shown in the synovial tissue T cells in rheumatoid arthritis patients exposed D10 cells to physiological concentrations of 17␤-estradiol (46–48). The perceived critical role of chemokine receptors has or progesterone for 24 h and their CCR gene expression quanti- led to the development of strategies to antagonize chemokine re- tated by RPAs. As has been reported previously, D10 cells express ceptors such as CCR1 and CCR5 as therapy for autoimmune dis- CCR1 and CCR4, but not CCR2, CCR3, or CCR5 (27). Physio- eases including rheumatoid arthritis (49, 50). logic concentrations of 17␤-estradiol (28) (Fig. 7A), but not pro- Given the importance of gender and T cell chemokine receptors gesterone (Fig. 7B), were found to increase both CCR1 and CCR4 in autoimmunity, it is surprising that very little is currently known by guest on September 27, 2021 gene expression in D10 cells (Fig. 7, C and D). We also examined about their interaction. Increased leukocyte migration into the the effect of 17␤-estradiol on D10 cell chemotaxis response to uterus was seen in women on oral estrogen (51). Transsexual men MIP-1␣ (CCL3), a ligand for CCR1. Estrogen was found to in- receiving estrogens and antiandrogens were also reported to have crease D10 chemotaxis (Fig. 7E), and this response is reversed by increased T cell CCR1, CCR5, and CXCR3 expression (52). In pretreatment with the anti-estrogen receptor compound ICI contrast, estrogen may suppress monocytes CCR2 expression (53, 182,780. To determine whether estrogen affects D10 cell chemo- 54) and inhibit monocyte migration in response to MCP-1 (55). kine receptor signaling, D10 cells were treated with 250 ng/ml However, the effect of gender and estrogen on T cell chemokine 17␤-estradiol for 24 h. The untreated and estrogen-treated T cells response has not been systematically examined. were then activated with 200 ng/ml MIP-1␣ for 30 s to 5 min. Our results demonstrate that CCR1-CCR5 are selectively over- Western blot analyses were performed using the anti- expressed in CD4ϩ T cells from female C57BL/6 mice. The gen- phosphotyrosine Ab 4G10 (Fig. 7, F–I). As expected, very low der difference in CCR expression is not strain-dependent, as four levels of tyrosine phosphorylation were detected in resting D10 other mouse strains (AKR, BALB/c, B10, and DBA/2), including cells. In contrast, estrogen-treated D10 cells showed increased pro- both Th1 and Th2 disease-prone mice, have similarly increased T tein tyrosine phosphorylation. Further quantitative increase in ty- lymphocyte CCR expression in females. Thus, although we found rosine phosphorylation was detected in both estrogen-treated and strain-dependent T cell chemokine receptor mRNA expression, the ␣ untreated D10 cells when they were exposed to MIP-1 in a time- gender bias remains true in all five mouse strains. Importantly, dependent manner. increased CCR gene expression correlates to increased chemokine receptor protein expression and function. Quantitative increase in Discussion T cell tyrosine phosphorylation is seen in estrogen-treated T cells A number of mechanisms explaining the gender dichotomy in lu- using the anti-phosphotyrosine Ab 4G10 in Western blot. In ad- pus have been proposed (29–33). We have previously shown that dition, there is further increase in tyrosine phosphorylation follow- adoptive transfer of autoreactive T cells causes a more severe lu- ing MIP-1␣ stimulation, providing further support that estrogen pus-like disease in female than male AKR mice (7). We further increases T cell chemokine receptor function. The estrogen T cell established that in vivo T cell trafficking is dependent on the gen- response was further confirmed in in vivo studies, as oophorecto- der and the gonadal status of the host animals, with increased mized mice receiving estrogen also have increased T cell CCR splenic homing in female mice that decreases following oophorec- expression compared with mice receiving placebo. However, in tomy to the level seen in intact male animals. However, the mech- vivo estrogen supplementation only partially reversed the effect of anisms for the estrogen effect on T cell trafficking have not been oophorectomy on T cell chemokine receptor gene expression, sug- elucidated. gesting that additional female gonadal factors may be involved. 6028 ESTROGEN IN THE PATHOGENESIS OF AUTOIMMUNE DISEASES

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