Epidermal Growth Factor Receptor (EGFR ...

REPRODUCTION-DEVELOPMENT

Epidermal Growth Factor Receptor (EGFR) Signaling Is a Key Mediator of Hormone-Induced Leukocyte Infiltration in the Pubertal Female Mammary Gland Mark D. Aupperlee, Yong Zhao, Ying Siow Tan, Jeffrey R. Leipprandt, Jessica Bennett, Sandra Z. Haslam, and Richard C. Schwartz Breast Cancer and the Environment Research Program, Departments of Physiology (M.D.A., Y.Z., Y.S.T., J.R.L., J.B., S.Z.H.) and Microbiology and Molecular Genetics (R.C.S.), Michigan State University, East Lansing, Michigan 48824

It is well documented that macrophages and eosinophils play important roles in normal murine pubertal mammary gland development. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We show here that P and E, respectively, induce unique, but overlapping, sets of proinflammatory and angiogenic cytokines and chemokines, in the pubertal female BALB/c mammary gland, as well as induce infiltration of macrophages and eosinophils to the mammary periepithelium. This extends earlier studies showing P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium. Importantly, epidermal growth factor receptor-signaling, which is likely mediated by amphiregulin (Areg), a downstream mediator of E and P, is both necessary and sufficient for both E- and P-induced recruitment of macrophages and eosinophils to the pubertal mammary periepithelium. We further show that receptor activator of nuclear factor ␬B ligand (RANKL), although not sufficient of itself to cause macrophage and eosinophil recruitment, contributes to an optimal response to P. The potency of Areg is highlighted by the fact that it is sufficient to induce macrophage and eosinophil recruitment at levels equivalent to that induced by either E or P. Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth and its role in P-induced proliferation in the pubertal gland. (Endocrinology 155: 2301–2313, 2014)

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t is well documented that macrophages and eosinophils play important roles in normal pubertal mammary gland development in the mouse (1). The association of macrophages with the terminal end buds (TEBs) of the developing mammary gland is essential for normal ductal elongation, whereas eosinophil association with the TEBs is required for normal ductal branching. Thus, these 2 cell

types perform complimentary roles in pubertal mammary gland development. In experiments using mice carrying a null mutation in the gene encoding macrophage-colonystimulating factor, macrophages are depleted within the developing mammary gland and morphogenesis is impaired (1). Similarly, mice carrying a null nutation in the gene encoding eotaxin exhibit depletion of eosinophils

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received October 8, 2013. Accepted March 20, 2014. First Published Online April 2, 2014

Abbreviations: Areg, amphiregulin; Arg1, arginase 1; BrdU, 5-bromo-2⬘-deoxyuridine; BSA, bovine serum albumin; Ccl, chemokine (C-C motif) ligand; CD, cluster of differentiation; Cxcl, chemokine (C-X-C motif) ligand; Cx3cl1, chemokine (C-X3-C motif) ligand 1; DAPI, 4⬘,6-diamidino-2-phenylindole; E, estrogen; E2, 17-␤-estradiol; EB, end bud; EGFR, epidermal growth factor receptor; ER, E receptor; F4/80, mouse macrophage marker; Fc, fragment cystallizable region of immunoglobulin; ICI, ICI 182,780; M2, alternatively activated macrophage; NF, nuclear factor; OVX, ovariectomized; P, progesterone; PR, P receptor; qRT-PCR, quantitative RT-PCR; R5020, synthetic progestin and progesterone receptor agonist; RANKL, receptor activator of NF-␬B ligand; Ru486, mifepristone; SAA, serum amyloid A; Stat, signal transducer and activator of transcription; TEB, terminal end bud; Thbs, thrombospondin; Tnfaip, TNF, ␣-induced protein; TRAP5b,tartrate-resistant acid phosphatase 5b.

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Endocrinology, June 2014, 155(6):2301–2313

endo.endojournals.org

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Hormonal Mammary Leukocyte Infiltration

and impaired morphogenesis within the developing mammary gland (1). Mast cells have also recently been implicated in pubertal mammary gland ductal morphogenesis, with a role independent of that of macrophages (2). They are particularly associated with the TEBs, and mast cell deficiency results in reduced numbers of TEBs. Although it is accepted that estrogen (E) and progesterone (P) are key players in mammary gland development, the roles that these hormones might play in regulating the actions of leukocytes in that process is an understudied area. We previously identified receptor activator of nuclear factor (NF)-␬B ligand (RANKL) and serum amyloid A (SAA)1, SAA2, and SAA3 as proinflammatory products whose mRNA is robustly induced by R5020 (synthetic progestin and progesterone receptor agonist) in BALB/c pubertal and adult mammary epithelial organoids in vitro (3, 4). Additionally, we and others showed progestin induction of RANKL protein expression in adult mammary epithelium in vivo (5–7) and in mammary organoids in vitro (3). Consistent with the potential activity of proinflammatory factors in the recruitment of leukocytes, we demonstrated that P induces in vivo recruitment of cluster of differentiation (CD) 45-positive leukocytes to the mammary periepithelial stroma in adult mice (4). The role of hormones, P in particular, in mediating leukocyte recruitment in the mammary gland had not been previously recognized. Because leukocytes play an essential role in the development of the pubertal mammary gland, extending the study of their hormonal regulation to that life stage is particularly illuminating of the interactions between hormones and mammary gland development. E has been acknowledged as the primary hormone for pubertal ductal development (8). Although P plays a nonessential role in ductal development (9), it contributes to this process through its activity in regulating amphiregulin (Areg), a role that it shares with E (10). Our previous studies identify Areg as the key mediator of hormonally induced end bud (EB) formation and proliferation at puberty (10). The present studies were directed at examining whether the overlapping activities of P and E extend beyond the regulation of proliferation in the pubertal mammary gland and include the regulation of leukocyte recruitment that is also necessary to development of the pubertal mammary gland. Gene expression, immune, and histochemical analyses revealed that P and E can induce proinflammatory and angiogenic factors in intact mammary glands, as well as induce the recruitment of both macrophages and eosinophils to the periepithelial stroma of the developing mammary gland. The current study expands our understanding of hormonally mediated effects that promote pubertal mammary gland development to


previously unrecognized effects of E and P on leukocyte recruitment. Importantly, this study provides mechanistic insight into the manner in which E and P effect leukocyte recruitment, identifying Areg and, to a lesser extent, RANKL as key mediators in this process.

Materials and Methods Animals Pubertal female 4-week-old BALB/c mice that had initiated estrous cycling and exhibited TEBs were ovariectomized (OVX). Recovery was allowed for 3 weeks after OVX to permit complete TEB regression before hormone treatments (11). OVX mice were injected daily for 5 days with saline control, 17-␤-estradiol (E2) (1 ␮g/injection (inj)) (Sigma), P (1 mg/inj) (Sigma), or E2⫹P (1 ␮g ⫹ 1 mg/inj, respectively). These concentrations of E2 and P are physiologically relevant and have been used in previous studies to examine the effects of E2 and P in the pubertal mammary gland (5, 10, 12). To block P receptor (PR)- or E receptor (ER)-mediated effects, antiprogestin mifepristone (RU486) (1.3 mg/inj) (Sigma) or anti-E ICI 182,780 (ICI) (1.1 ␮g/inj) (Tocris Bioscience) was coinjected with P or E, respectively. To block RANKLmediated effects, P-treated mice were coinjected with RANK fused to the fragment cystallizable region of immunoglobulin (RANK-Fc) (20 mg/kg dissolved in saline) (R&D Systems) every other day for 5 days. To block epidermal growth factor receptor (EGFR)-mediated effects, E2- and P-treated mice were given Gefitinib (Iressa; ChemieTek) (300 mg/kg dissolved in corn oil) daily for 5 days by oral gavage. To examine the effects of RANKL or Areg on the mammary gland, OVX mice received 3 daily sc injections of RANKL (3 mg dissolved in saline) (Kingfisher Biotech) or Areg (1 mg dissolved in saline) (Creative Biomart) directly into the fat pad of the inguinal mammary gland on the right side of the mouse. Contralateral inguinal mammary glands were injected with control bovine serum albumin (3 or 1 mg dissolved in saline for the RANKL or Areg experiments, respectively) (Sigma). All mice were injected with 5-bromo-2⬘-deoxyuridine (BrdU) (70 ␮g/g body weight) 2 hours before being killed. Mammary glands were formalin fixed and processed as whole mounts (13), or paraffin embedded for immunohistochemistry (14). All animal experimentation was conducted in accord with accepted standards of humane animal care and approved by the All University Committee on Animal Use and Care at Michigan State University.

Immunofluorescence Mammary gland sections (5 ␮m) were prepared and subjected to antigen retrieval and immunofluorescent staining (14). To detect macrophages, sections were incubated with rat monoclonal anti-F4/80 (a mouse macrophage marker) (1:75; AbD Serotec), followed by goat antirat antibody (Ab) conjugated to Alexa Fluor 488 (1:100; Invitrogen). To assess whether macrophages expressed arginase 1 (Arg1), consistent with an M2 (alternatively activated macrophage) phenotype, some sections were additionally incubated with goat anti-Arg1 (1:200; Santa Cruz Biotechnology, Inc), followed by rabbit antigoat Ab conjugated to Alexa Fluor 546 (1:100; Invitrogen). BrdU incorporation was detected


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by mouse monoclonal anti-BrdU (undiluted; kit from Amersham Biosciences), followed by goat antimouse Ab conjugated to Alexa Fluor 488 (1:200; Invitrogen). To detect IL-17B, sections were incubated with rabbit anti-IL-17B (1:100; Bioss), followed by goat antirabbit Ab conjugated to Alexa Fluor 488 (1:200; Invitrogen). Nuclei were counterstained with 4⬘,6-diamidino-2phenylindole (DAPI) (1:10 000; Invitrogen). Sections were visualized and images captured using a Nikon inverted epifluorescence microscope (Mager Scientific) with MetaMorph software (Molecular Devices Corp).

Histochemistry For identification of eosinophils and mast cells, deparaffinized 5-␮m sections were stained with Astra Blue/Vital New Red (15). Sections were visualized using a Nikon Eclipse 400 microscope and a SPOT RT color camera with SPOT software (Diagnostic Instruments). The number of eosinophils (Vital New Red) and mast cells (Astra Blue) is expressed as cells per structure in the mammary gland periepithelial area. For analysis of blood vessel density, deparaffinized 5-␮m sections were incubated in 2% of H2O2 in methanol/PBS (1:1 ratio) for 30 minutes followed by antigen retrieval by boiling at 95°C for 5 minutes. Sections were then treated for 2 hours with rabbit anti-CD31 (1:50 in PBS-0.5% Triton X-100, catalog number AP15436PU-N; Acris Antibodies, Inc) for endothelial cell staining and detection of blood vessels. After a brief wash, sections were incubated with secondary swine antirabbit Ab at room temperature for 30 minutes and then incubated with ABC reagent (PK-7100; Vector Laboratories, Inc) for 30 minutes. The sections were then incubated with metal enhanced 3,3⬘-diaminobenzidine substrate solution (100 ␮L of DAB substrate ⫹ 900 ␮L of stable peroxide substrate buffer; Thermo Scientific) for 7 minutes and counterstained with hematoxylin for 2 minutes. The stained sections were visualized with a Nikon Eclipse E400 light microscope. A minimum of 1000 cells was counted for each section, and a minimum of 2–3 tissue sections per animal was analyzed. Digital micrographs were captured, and the images were overlaid with grids containing 240 squares (324 ␮m2/ square). Blood vessel density is expressed as the percentage of CD31-positive squares.

Quantitation and statistical analyses Macrophages, eosinophils, and mast cells were counted in the periepithelial stroma of small ducts, large ducts, and EBs, from at least 3 mice per treatment. The numbers of cells are presented per mammary gland structure (ie, large duct, small duct, EB). Large ducts were characterized by larger lumen diameter, comprising greater than 50 cells, and by an extensive extracellular matrix and fibroblasts surrounding the epithelium. Small ducts were characterized by smaller lumen diameter, comprising fewer than 50 cells, and by a limited extracellular matrix surrounding the epithelium. EBs were enlarged structures characterized by multiple epithelial cell layers, usually on one side of the structure, leading to a bulb-like appearance. Digital images of inguinal mammary gland whole mounts were analyzed with ImageJ (http://rsb.info.nih.gov/ij/download.html) for epithelial area. To analyze IL-17B fluorescence intensity, the average pixel intensity of all positively stained cells within the ductal epithelium was determined. A threshold was set to exclude background fluorescence, and images were gated to include intensity measurements


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only from positively staining epithelial cells. For all experiments, a minimum of 3 mice per treatment group was analyzed. Statistical significance was calculated by Student’s t test.

RT-PCR analyses Total RNA was extracted from mouse mammary glands by TRIzol reagent (Invitrogen) and purified using the RT2 qPCR-Grade RNA isolation kit (SABiosciences). Three micrograms of total RNA were used for cDNA synthesis using the RT2 First Strand kit (SABiosciences) following the manufacturer’s instructions. cDNAs (20 ␮L) were diluted to 150 ␮L with deionized H2O. mRNA levels were quantitated using Inflammatory Cytokines and Receptors and Angiogenesis PCR Arrays (catalog numbers PAMM-011 and PAMM-024, respectively; SABiosciences). In addition to the arrays, primers for the following selected genes were purchased from SABiosciences: Saa1 (PPM05345E), Saa3 (PPM05486E), Tnfs11 (RANKL) (PPM03047E), 18S ribosomal ribonucleic acid (RNA) (rRNA) (PPM57735E), and ribosomal protein L32 (RPL32) (PPM03300B). Primers for Areg were purchased from Applied Biosciences (Mm00437583_m1, catalog number 4331182). For quantitative RTPCR (qRT-PCR) analysis, each reaction (25 ␮L) included 12.5 ␮L of 2⫻ SABiosciences RT2 qPCR Master Mix (SYBR Green), 1 ␮L of diluted first-stand cDNA synthesis reaction, and 11.5 ␮L of deionized H2O. qRT-PCR was performed with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Inc) using the following program: step 1, 95°C for 10 minutes; step 2, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute; and step 3, dissociation curve 95°C for 1 minute, 65°C for 2 minutes (optics off), 65°C–95°C at 2°C per minute (optics on). The data were analyzed using online software from SABiosciences (http://pcrdataanalysis.sabiosciences.com/pcr/ arrayanalysis.php). For Saa1, Saa3, and RANKL, the comparative threshold cycle method was used to calculate the fold change in gene expression after normalization to values for 18S rRNA and RPL32. RNA from the inguinal mammary glands of 3 animals was analyzed from each treatment group.

Tartrate-resistant acid phosphatase 5b (TRAP5b) assay Blood was collected at the time of harvest, allowed to clot at room temperature for 5 minutes, and then centrifuged at 4000 rpm for 10 minutes. Serum was removed and stored at ⫺80°C. Serum TRAP5b was measured using a MouseTRAP assay kit (SB-TR103; Immunodiagnostic Systems, Inc) according to the manufacturer’s protocol.

Results P and E induce macrophage and eosinophil recruitment to the pubertal mammary periepthelial stroma We previously found that P induced the recruitment of CD45-positive leukocytes to the mammary periepithelial stroma of adult OVX mice (4). Because E is a key hormone directing pubertal ductal development and the role of P in pubertal leukocyte recruitment is unknown, we examined whether P, and also E, might have similar roles in leukocyte recruitment in the pubertal mammary gland.


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was significantly less effective for eosinophil recruitment to EBs than P and E2 together, suggesting that the effects of the 2 hormones are not entirely identical in this context. Because macrophages are associated with the tissue-remodeling processes in mammary gland development, we evaluated whether hormone-induced macrophages in the mammary periepithelium might display an M2 or alternative activation phenotype. The phenotype is associated with tissue-remodeling processes (16). To that end, we examined the proportion of F4/80positive macrophages that were also positive for Arg1, which is expressed at high levels in M2 macrophages (17). Approximately 90% of macrophages across all structures and treatments were Arg1-positive, consistent with M2 polarization (Figure 1E). P and E induce proinflammatory gene expression in intact mammary glands Having identified hormone-induced recruitment of macrophages and eosinophils, we assessed the miFigure 1. Both E2 and P regulate macrophage and eosinophil recruitment to the pubertal lieu of proinflammatory products mammary gland. Pubertal 4-week-old BALB/c mice were OVX, allowed to recover for 3 weeks, that might be directly involved in reand then treated for 5 days with vehicle control (C), E2, P, or E2⫹P. A, Immunofluorescent cruiting these cells to the mammary detection of F4/80 (green) in C and P-treated mammary gland. Nuclei were counterstained with DAPI (blue). Scale bar, 25 ␮m. B, F4/80-positive cell recruitment to the periepithelium was periepithelial stroma. We previously analyzed for large ducts, small ducts, and duct ends. The values represent the mean ⫾ SEM (n ⫽ found that the progestin (R5020) in6 animals per treatment). The number of macrophages recruited to periepithelial structures upon duced proinflammatory gene extreatment with E2, P, and E2⫹P was above C levels (*, P ⬍ .05). C, Histochemical detection of eosinophils (arrows) in C and P-treated mammary gland. Nuclei were counterstained with pression in mammary epithelial cells hematoxylin (blue). Scale bar, 50 ␮m. D, Eosinophil recruitment to the periepithelium was in vitro (4), in particular, SAA1, analyzed for large ducts, small ducts, and duct ends. The number of eosinophils recruited to SAA2, and SAA3 (Saa1, Saa2, and periepithelial structures upon treatment with E2, P, and E2⫹P was above C levels (*, P ⬍ .05). Saa3) and the NF-␬B activating cyThe number of eosinophils recruited to the periepithelium of P-treated EBs was reduced compared with E2⫹P-treated EBs (#, P ⬍ .05). The values represent the mean ⫾ SEM (n ⫽ 6 tokine, RANKL. Analysis of proinanimals per treatment). E, Macrophage analysis by dual immunofluorescent detection of F4/80 flammatory gene expression was and Arg1. The percentage of F4/80-positive cells coexpressing Arg1 is presented. The values carried out in vivo in mammary represent the mean ⫾ SEM (n ⫽ 3 animals per treatment). glands of OVX pubertal BALB/c mice that were treated for 5 days Both P and E2 treatment of OVX pubertal animals inwith saline control or hormones. RNA was isolated from duced increased numbers of macrophages (Figure 1, A and B) and eosinophils (Figure 1, C and D) in the mammary their mammary glands and analyzed by qRT-PCR. Simiperiepithelial stroma. No significant change was observed larly to the case with R5020 treatment of cultured mamfor mast cells (Supplemental Figure 1). Combined treat- mary epithelial organoids (4), P induced RANKL and ment with both P and E2 produced effects largely similar Saa1 expression in pubertal animals (Table 1). Howto that of either hormone alone. Interestingly, P treatment ever, P induced an even broader range of proinflamma-


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Table 1.


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E2 and P Regulation of Inflammation-Associated Gene Expression Known P and E Regulation

Fold Change Symbol

Description

5-d E

5-d P

Ccl1 Ccl20 Ccl3 Ccl8 Cx3cl1 Cxcl15 Cxcl5 Il17b Il8rb Tnf Areg RANKL SAA1

Chemokine (C-C motif) ligand 1 Chemokine (C-C motif) ligand 20 Chemokine (C-C motif) ligand 3 Chemokine (C-C motif) ligand 8 Chemokine (C-X3-C motif) ligand 1 Chemokine (C-X-C motif) ligand 15 Chemokine (C-X-C motif) ligand 5 IL-17B IL-8 receptor, ␤ TNF Amphiregulin Receptor activator of nuclear factor ␬ B ligand Serum amyloid A1

No change No change 2.1 5.4 No change 12 3.1 8.6 No change No change 24 No change 2.2

2.7 4.5 No change 3.3 2.9 5.7 No change 8.9 ⫺3.0 2.4 31 630 1.8

E

P

2 (56) 1 (70) 1 (71)

1 (10)

1 (10) 1 (6, 7) 1 (4)

Direction of known P and E regulation (1 or 2) and the reference; n ⫽ 3 control-treated mice, n ⫽ 3 E2-treated mice, n ⫽ 3 P-treated mice. P ⱕ .05 for all genes listed.

tory products in intact mammary glands than observed in cell culture. A different, but overlapping, set of cytokine and chemokine mRNAs was observed with E2 treatment. P and E induce angiogenesis in pubertal mammary glands Leukocyte recruitment has long been associated with angiogenesis (18), and angiogenesis accompanies mammary ductal development with capillaries in close association with epithelial ducts (19). In order to assess whether P and E might also induce factors associated with angiogenesis, we performed a qRT-PCR analysis similar to that Table 2.

for proinflammatory gene products. P and E2 induced differing, but overlapping, sets of factors associated with angiogenesis (Table 2). We also examined the effects of P and E2 on angiogenesis. Both P and E2 induced increased vascularization of the mammary gland, as evidenced by increased area of CD31 staining (Figure 2). Thus, the observed modulation of genes associated with angiogenesis appears functionally significant. Leukocyte recruitment by P and E are mediated through their cognate receptors The effects of P and E are largely mediated through their cognate receptors, respectively, PR (14) and ER␣ (8). ER␤

E2 and P regulation of Angiogenesis-Associated Gene Expression Known P and E Regulation

Fold Change Symbol

Description

5-d E

5-d P

Cxcl2 Cxcl5 F2 Hand2

Chemokine (C-X-C motif) ligand 2 Chemokine (C-X-C motif) ligand 5 Coagulation factor II Heart and neural crest derivatives expressed transcript 2 Integrin ␤ 3 Leukocyte cell-derived chemotaxin 1 Midkine Sphingosine kinase 1 Stabilin 1 T-box 1 Thrombospondin 1 Thrombospondin 2 Tissue inhibitor of metalloproteinase 1 Tumor necrosis factor TNF, ␣-induced protein 2

No change 2.1 No change No change

⫺2.6 2.5 3.2 2.8

No change No change 3.7 2.2 2.1 ⫺2.1 2.2 2.1 3.0 No change ⫺4.8

2.1 5.0 5.1 4.4 No change ⫺2.3 5.7 No change 3.1 2.5 No change

Itgb3 Lect1 Mdk Sphk1 Stab1 Tbx1 Thbs1 Thbs2 Timp1 Tnf Tnfaip2

E

P 2 (72) 1 (76)

1 (80) 1 (82)

1 (83)

1 (87)

1 (88) 1 (90) 1 (92)

Angiogenic Effect 2 (73) 1 (74) 1 (75) 1 (77) 1 (78) 2 (79) 1 (81) 1 (84) 1 (85) 1 (86) 2 (89) 2 (89) 1 (91) 1 (93) 1 (94)

Direction (1 or 2) of known P and E regulation or effect on angiogenesis and the reference; n ⫽ 3 control-treated mice, n ⫽ 3 E2-treated mice, n ⫽ 3 P-treated mice. P ⱕ .05 for all genes listed.


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Figure 2. E2 and P increase angiogenesis in the pubertal mammary gland. Pubertal 4-week-old BALB/c mice were OVX, allowed to recover for 3 weeks, and then treated for 5 days with vehicle control (C), E2, P, or E2⫹P. Blood vessel density was measured, as described in Materials and Methods, by the area occupied by CD31-positive vessels near mammary epithelium. The values represent the mean ⫾ SEM (n ⫽ 3 animals per treatment). CD31 staining was greater in E2-, P-, and E2⫹P-treated mammary glands (*, P ⬍ .05) compared with Cs.

appears not to have a role in pubertal mammary gland development, because ER␤-deficient mice have normal mammary gland development (20). In order to confirm that hormone-induced leukocyte recruitment occurred through these classical hormone receptors, we carried out P treatment with and without the PR-binding antagonist RU486, or with E2 with and without the ER-binding antagonist ICI. RU486 effectively blocked P-stimulated macrophage and eosinophil infiltration to the periepithelial stroma surrounding large ducts, small ducts, and EBs (Figure 3, A and B). ICI was similarly effective in blocking E2-stimulated leukocyte infiltration. Therefore, we concluded that the actions of P and E2 in leukocyte recruitment in the mammary gland are mediated through their cognate receptors. Areg is an essential mediator of both P- and E-induced leukocyte infiltration in the mammary gland Both P and E contribute to ductal development and proliferation through Areg (10). Although some EGFR ligands, such as TGF␣ and heparin-binding EGF-like growth factor, have been detected in pubertal end buds (EBs), Areg is the most highly expressed EGFR ligand in ducts and EBs during puberty, and Areg is required for normal ductal morphogenesis (21). Therefore, we hypothesized that Areg might be a mediator of hormonally induced leukocyte recruitment. Stimulation of macrophage (Figure 3C) and eosinophil infiltration (Figure 3D) with either P or E2 in the presence or absence of Iressa, an antagonist of EGFR signaling, fully inhibited E2- and Pstimulated infiltration of macrophages and eosinophils and actually brought their numbers below that of control animals. This demonstrates that EGFR signaling, and likely Areg, is essential to both E- and P-stimulated leukocyte infiltration. Furthermore, Iressa fully inhibited


both E2- and P-stimulated EB development and epithelial expansion in the same animals (Figure 4, A–C). To test whether Areg treatment by itself could stimulate leukocyte infiltration to ductal structures, we stimulated macrophage (Figure 5A) and eosinophil infiltration (Figure 5B) in OVX mice with local injection of Areg or a bovine serum albumin (BSA) control into the inguinal mammary gland. Compared with control injections, Areg significantly increased numbers of macrophages and eosinophils to both small and large ducts. The contralateral control-injected gland did not exhibit increased numbers of macrophages or eosinophils, indicating that the effects of Areg are local rather than systemic. As expected, the Areg-injected glands also showed increased numbers of proliferative cells (Figure 5C). Areg was able to induce both the development of proliferating EBs (Supplemental Figure 2A) and macrophage recruitment to EBs (Supplemental Figure 2B). RANKL contributes to P-induced leukocyte infiltration in the mammary gland Most the proinflammatory products identified in hormonally treated mice are at least in part regulated by NF-␬B, which is not a primary target of EGFR signaling. We noted that 8 of the 11 hormone-induced proinflammatory cytokine and chemokine mRNAs are known targets of NF-␬B transcriptional activation (Table 1). These are chemokine (C-C motif) ligand (Ccl)1 (22), Ccl3 (23), Ccl8 (24), Ccl20 (25), chemokine (C-X3-C motif) ligand 1 (Cx3cl1) (26), chemokine (C-X-C motif) ligand (Cxcl)5 (27), Saa1 (28), and Tnf (29). Conversely, Il17b (30), Tnf (31), and RANKL (32) all mediate their effects, at least in part, through NF-␬B activation. Additionally, 5 of the hormone-induced mRNAs associated with angiogenesis are known NF-␬B targets (Table 2). These are midkine (33), thrombospondin (Thbs)1 (34), Thbs2 (35), tissue inhibitor of metalloproteinase 1 (36), and TNF␣-induced protein (Tnfaip)2 (37). Because RANKL, a potent activator of NF-␬B (38), is the most robustly induced of the P-induced cytokines that we examined (Table 1), we investigated the potential role of RANKL in P-induced leukocyte infiltration in the mammary gland. Macrophage (Figure 3E) and eosinophil (Figure 3F) infiltration was stimulated with P in the presence or absence of RANK-Fc, a decoy receptor and antagonist of RANKL signaling. RANK-Fc significantly reduced macrophage infiltration to large and small ducts (42% and 48%, respectively) but not to EBs. Eosinophil infiltration was also significantly reduced to large ducts and EBs (100% and 43%, respectively) but not to small ducts. We confirmed the activity of RANK-Fc in systemically inhibiting


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In order to test whether RANKL treatment by itself could stimulate macrophage and eosinophil infiltration to ductal structures, we treated OVX mice with local injection of RANKL vs a BSA control into the inguinal mammary gland and measured macrophage (Supplemental Figure 4A) and eosinophil infiltration (Supplemental Figure 4B). No significant recruitment of macrophages or eosinophils was detected, suggesting that although RANKL is likely necessary for full leukocyte infiltration in response to P, it is not sufficient. There was also no proliferative response to local RANKL injection (Supplemental Figure 4C), consistent with the previously reported dependence of pubertal mammary proliferation on leukocyte infiltration (39). A possible role for IL-17B in P- and E-induced leukocyte infiltration in the mammary gland P induction of RANKL provides a potent activator of NF-␬B. Because RANK-Fc was not able to fully block Figure 3. Areg and RANKL are mediators of hormone-induced macrophage and eosinophil macrophage and eosinophil infiltrarecruitment to mammary epithelium. Pubertal 4-week-old BALB/c mice were OVX, allowed to recover for 3 weeks, and then treated for 5 days with vehicle control (C), E2, or P. A and B, Both tion in response to P, this suggested macrophage (A) and eosinophil (B) recruitment to mammary gland epithelial structures by E2 and the role of other factors in this proP were inhibited by E2⫹ICI and P⫹RU486. The numbers of macrophages and eosinophils cess. Furthermore, because RANKL recruited to periepithelial structures upon treatment with E2 or P were above that of other treatments (*, P ⬍ .05). C and D, Both macrophage (C) and eosinophil (D) recruitment to was not induced by E, this indicated mammary gland epithelial structures by E2 and P were inhibited by E2⫹Iressa and P⫹Iressa. E2that RANKL was certainly not meand P-induced macrophage recruitment was completely inhibited in large and small ducts by the diating leukocyte infiltration inaddition of Iressa. No EBs were detected in Iressa-treated mammary glands. The numbers of duced by E. The robust induction of macrophages or eosinophils recruited to periepithelial structures upon treatment with E2 or P were above that of other treatments (*, P ⬍ .05). E and F, Both macrophage (E) and eosinophil Il17b mRNA by both P and E2 (Ta(F) recruitment to mammary gland epithelial structures by P were inhibited by P⫹RANK-Fc. The ble 1) provides another plausible facnumbers of macrophages or eosinophils recruited to periepithelial structures upon treatment P tor for NF-␬B activation, because it were above that of C treatments (#, P ⬍ .05). P⫹RANK-Fc decreased macrophage recruitment to large and small ducts compared with P alone (*, P ⬍ .05). P⫹RANK-Fc decreased eosinophil is capable of inducing Tnf through recruitment to large ducts (*, P ⬍ .05) and EBs (**, P ⬍ .1) compared with P alone. A–F, The NF-␬B activation (30). Because Areg values represent the mean ⫾ SEM (n ⫽ 3 animals per treatment). is induced by both hormones and is likely an essential mediator of hormonally induced leukocyte infiltraRANKL activity by its suppression of serum TRAP5b levels (Supplemental Figure 3) and its inhibition of duction in the mammary gland, we tested whether EGFR sigtal and EB proliferation as measured by BrdU incorpo- naling was responsible for induction of Il17b expression. ration (10). Thus, P-induced RANKL is also a mediator Mammary gland tissue sections from animals treated with of leukocyte infiltration, in addition to Areg and pos- P and E2 in the presence or absence of Iressa (see Figure 2, sibly other P-induced factors that may contribute to this C and D) were stained for IL-17B (Figure 6A). Either P or response. E2 treatments were able to increase IL-17B expression,


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dependent cytokine expression in response to both E and P treatment.

Discussion This study examined the roles of P and E in inducing leukocyte infiltration in the pubertal mammary gland that is concomitant with hormonally induced proliferation and ductal outgrowth. It is well established that inflammation-like processes are required for tissue remodeling and angiogenesis, which are essential for normal mammary gland development (39). We now show that P and E, respectively, induce unique, but overlapping, sets of proinflammatory cytokines and chemokines, in the pubertal mammary gland, as well as induce the infiltration of macrophages and eosinophils to the mammary periepithelium. This extends our earlier studies that showed P induction of proinflammatory products in pubertal and adult mammary epithelial organoids and P-induced in vivo infiltration of leukocytes to the adult mammary periepithelium (4). Importantly, we show that EGFR signaling is both necessary and sufficient for both EFigure 4. Iressa inhibits both E2- and P-induced EB formation and epithelial outgrowth. Pubertal and P-induced recruitment of mac4-week-old BALB/c were OVX, allowed to recover for 3 weeks, and then treated for 5 days with rophages and eosinophils to the vehicle control (C), E2 ⫾ Iressa (300 mg/kg), or P ⫾ Iressa. A, Whole mounts showing effect of treatment on EB formation after 5 days of C, E2, E2⫹Iressa, P, or P⫹Iressa. Note the absence of mammary periepithelium in puberEBs in Iressa-treated mammary glands. Scale bar, 2 mm. B, EB quantitation from C and treated tal mice, as well hormonally induced whole mounts. Values represent the mean ⫾ SEM number of EBs in an inguinal mammary gland EB development and epithelial ex(n ⫽ 4 mice). E2 and P increased the average EB number (*, P ⬍ .05), and this increase was pansion. This linkage of leukocyte inhibited by the addition of Iressa. C, Epithelial area quantitation from C and treated whole mounts. Values represent the mean ⫾ SEM epithelial area in a pair of inguinal mammary glands recruitment and epithelial develop(n ⫽ 4 mice). E2 and P increased the average epithelial area (*, P ⬍ .05), and this increase was ment to EGFR signaling is consistent inhibited by the addition of Iressa. with the established role of macrophages and eosinophils in mammary and Iressa was able to fully block this increase. In order to gland morphogenesis (1). Because Areg is the most highly directly test whether Areg treatment by itself might induce expressed EGFR ligand in ducts and EBs during puberty, IL-17B expression, mammary gland tissue sections from and Areg is required for normal ductal morphogenesis animals subjected to local injection of Areg (see Figure 5) (21), it is likely that Areg is responsible for this in vivo were stained for IL-17B (Figure 6B). Areg was able to requirement for EGFR signaling. Both E (40) and P (41) increase IL-17B expression in a dosage-dependent fashion can up-regulate Areg expression. The potency of Areg is to levels equivalent to those seen after 3 days of P treat- highlighted by the fact that it is sufficient of itself to induce ment. Thus, it is plausible that Il17b is a downstream tar- macrophage and eosinophil recruitment at levels equivaget of Areg that participates in the induction of NF-␬B- lent to that induced by either E or P. There is precedent for


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Figure 5. Areg increased macrophage and eosinophil recruitment and mammary epithelial cell proliferation. Pubertal 4-week-old BALB/c mice were OVX, allowed to recover for 3 weeks, and then treated for 3 days with bovine serum albumin (BSA) control (C) or Areg. A and B, Both macrophage (A) and eosinophil recruitment (B) were significantly increased by Areg over BSA Cs across all mammary epithelial structures (*, P ⬍ .05). C, The percentage of BrdU⫹ cells was increased by Areg over BSA Cs across all mammary epithelial structures (*, P ⬍ .05). A–C, The values represent the mean ⫾ SEM (n ⫽ 3 animals per treatment).

Areg-induced production of proinflammatory factors in rheumatoid arthritis (42). Our finding of a dominant role for Areg in hormonally induced leukocyte recruitment to the pubertal mammary gland parallels its dominance in regulating ductal outgrowth (21) and parallels its role in P-induced proliferation in the pubertal gland (10). RANKL, although not sufficient of itself to cause recruitment of macrophages and eosinophils, likely contributes to an optimal response to P, because RANK-Fc significantly blocks P’s activity in leukocyte recruitment. This is consistent with data presented here showing that P induces a proinflammatory expression profile with elements unique from those induced by E. The basis for this difference may be the ability of RANKL to activate the noncanonical pathway for NF-␬B activation, which targets some genes distinct from those activated by the canonical


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NF-␬B pathway (43). One possible explanation for the fact that the effects of RANK-Fc antagonism are more significant than the inductive effects of RANKL alone is that although leukocyte recruitment may require robust activation of NF-␬B, this alone may not be sufficient. Signaling through EGFR is known to activate signal transducer and activator of transcription (Stat) 5 (44) and Stat3 (45), both important regulators of mammary gland development (46). Ductal development is decreased in Stat5 knockout animals (47), and, because leukocyte recruitment plays a critical role in ductal development, we speculate that Stat5 may be involved in the process of leukocyte recruitment. Furthermore, some proinflammatory cytokines similarly require 2 pathways of transcriptional activation for expression. For instance, Ccl20, which is induced by P in our studies, displays synergistic enhancement of expression by Stat3 and NF-␬B (48). Ccl3, induced by E in our studies, is regulated by NF-␬B but also has a putative Stat5 binding site (49). RANKL is required for the development of side branches leading to the formation of lobulo-alveolar structures during pregnancy (50), and its targeted expression to mammary epithelial cells in PR-deficient mice is sufficient to induce branching and alveologenesis (51). Our finding that antagonism of the P-specific induction of RANKL has less profound effects than antagonism of Areg signaling is consistent with the reduced ability of the pubertal gland to develop side branches in response to P treatment (10). Indeed, treatment with RANK-Fc only partially suppressed P-induced leukocyte recruitment and proliferation. It may well be that Areg-induced expression of IL-17B, an inducer of proinflammatory cytokines in fibroblasts and macrophages, and presumed NF-␬B activator (30), supports Areg induction of proinflammatory cytokines in the absence of RANKL activity. We found that both E and P robustly induce IL-17B expression, probably through Areg. Interestingly, RANK-Fc treatment displayed a more complete suppression of P-induced eosinophil recruitment and proliferation in large ducts. This suggests that RANKL may play a greater role in large ducts than other structures, and this is consistent with its role in side-branching off of large ducts (52). A greater role in large ducts is also consistent with the observation of a higher level of RANKL expression in pubertal ducts (10). It will be interesting to examine whether the dominance of Areg over RANKL in supporting leukocyte infiltration in the pubertal mammary gland is reversed in adult animals, where side-branching and alveologenesis are critical glandular events during pregnancy. In the case of Areg, where inflammation-like effects are closely associated with proliferation, it may also be useful to examine whether inflammation-like effects and proliferation might be disso-


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are uniquely induced by P or E. We found that P was significantly less effective than E in inducing eosinophil recruitment to the vicinity of EBs, and this is consistent with the 2 hormones inducing nonidentical sets of proinflammatory factors. For instance, Ccl3 has chemotactic activity toward eosinophils (65) and is induced by E rather than P. Among the P-induced mRNAs whose expression is associated with angiogenesis, Cxcl5, coagulation factor II, integrin ␤ 3, leukocyte cellFigure 6. IL-17B is regulated by E2 and P through Areg. A, Pubertal 4-week-old BALB/c mice derived chemotaxin 1, stabilin 1, Twere OVX, allowed to recover for 3 weeks, and then treated for 5 days with vehicle control (C), box 1, Thbs2, and Tnfaip2 were not E2, E2⫹Iressa, P, or P⫹Iressa. A, IL-17B was measured in mammary epithelium by previously known to be P or E reguimmunofluorescence, as intensity above background. The values represent the mean ⫾ SEM (n ⫽ 3 animals per treatment). Five-day E2 and P treatment increased IL-17B compared with C (*, P ⬍ lated. Most of these observed .05). E2⫹Iressa inhibited E2-induced increases in IL-17B (**, P ⬍ .05). B and C, Pubertal 4-weekchanges in expression have been asold BALB/c mice were OVX, allowed to recover for 3 weeks, and then treated for 3 days with sociated in the literature with proanBSA C, 1 or 3 ␮g of Areg, or P. B, Immunofluorescent detection of IL-17B (green). White arrows mark examples of cytoplasmic IL-17B expression colocalizing to DAPI staining in mammary giogenic effects. Consistent with the epithelial cells. Scale bar, 25 ␮m. C, IL-17B was measured in mammary epithelium by observation of a largely proangioimmunofluorescence, as intensity above background. The values represent the mean ⫾ SEM genic expression profile, we found (n ⫽ 3 animals per treatment). Three-day P treatment (*, P ⬍ .05) and 3-day 5-␮g Areg treatment (#, P ⬍ .05) increased IL-17B compared with C treatment. that both P and E increased vascularization of the mammary gland. These studies expand our understanding of hormonally ciated by antagonism of fibroblast growth factor 2 and fibroblast growth factor 7, which have been implicated as mediated effects that promote pubertal mammary gland downstream proliferative effectors of EGFR signaling in development to previously unrecognized effects of E and P on leukocyte recruitment. We provide insight into the the mammary gland (53). Treatment with P and E each induce a number of pro- mechanisms linking P and E signaling and the inflammainflammatory cytokines and chemokines that plausibly tion-like and angiogenic processes essential to normal support the recruitment of macrophages and eosinophils. mammary gland development. However, in the context of Although Areg has been directly implicated in chemotaxis mammary carcinogenesis, these same processes can also of mesothelioma cells (54) and bone marrow mesenchy- promote tumor development. Animal models of breast cancer have demonstrated that inflammatory processes conmal progenitors (55), it is likely that the role of Areg in tribute to tumor proliferation and metastasis (66). It is leukocyte recruitment is largely indirect and through the known that long-term exposure to P alone (67), or in comparticipation of EGFR signaling and EGFR-dependent inbination with E, promotes the development of mammary duction of IL-17B in the regulation of these proinflammacancer in rodents (68). It is noteworthy that most macrotory cytokines and chemokines. Among the P-induced phages recruited to the mammary periepithelium in response mRNAs whose expression is associated with inflammato P and E express Arg1, consistent with an M2 or alternative tion, Ccl1, Ccl20, Cxcl15, Il17b, and Tnf have not previ- activation phenotype. This phenotype is not only associated ously been reported to be P regulated. None of the genes with tissue-remodeling processes (16) but also tumor profound to be up-regulated by E here are known to be E gression (69). Thus, the hormonally induced inflammationregulated. However, opposite to our results, Ccl3 was re- like response in the mammary gland presents a plausible ported to be down-regulated by E in murine mammary mechanism underlying the promotional effects of hormones tissue (56). Among the inflammation-associated mRNAs in mammary tumorigenesis that may be at play in addition to identified as being induced by either P or E, Ccl1 (57), Ccl3 the proliferative effects of E and P. (58), Ccl8 (59), Cx3cl1 (60, 61), RANKL (62), Saa1 (63), and Tnf (64) have all been shown to have chemotactic Acknowledgments activity toward monocytes, macrophages, and/or eosinophils. Although the spectrum of cytokines and chemo- We thank Regina Irwin in the laboratory of Dr Laura McCabe kines induced by P and E, respectively, overlap, some for performing the TRAP5b assays.


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Address all correspondence and requests for reprints to: Richard C. Schwartz, PhD, Department of Microbiology and Molecular Genetics, Michigan State University, 567 Wilson Road, Room 2209, East Lansing Michigan 48824. E-mail: [email protected]; or Sandra Z. Haslam, PhD, Department of Physiology, Michigan State University, 567 Wilson Road, Room 2201, East Lansing MI 48824. E-mail: [email protected]. This work was supported by the Breast Cancer and the Environment Research Program award 1UO1ESO19434 and the Breast Cancer and the Environment Research Centers award 1U01ES12800 from the National Institute of Environmental Health Sciences and the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. This work was also supported by the Avon Foundation, Ladies Auxiliary to the Veterans of Foreign Wars, and the Helen L. Kay Charitable Trust. Disclosure Summary: The authors have nothing to disclose.

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