The development of endometrial hyperplasia in aged PD-1-deficient female mice
© Guo et al.; licensee BioMed Central Ltd. 2014
Received: 25 February 2014
Accepted: 15 May 2014
Published: 26 May 2014
Programmed death-1 (PD-1, Pdcd1)-deficient mice develop different types of autoimmune diseases depending on the mouse strain but its role in uterus development has not been reported.
In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in uterine tissues from aged WT mice in a 129svEv-Brd background was analyzed by immunohistochemistry and the uterine morphology between WT and PD-1-/- mice was compared by hematoxylin and eosin staining.
The aged PD-1-/- female mice in a 129svEv-Brd rather than Balb/c background develop endometrial hyperplasia. H&E staining showed an increase in the number of glands, neovascularization and an extremely large luminal cavity in aged PD-1-/- uteri. Immunohistochemical assay showed that the expression of PD-1 was observed in glandular/luminal epithelium and cells infiltrating the stroma. Fluorescent double staining demonstrated that PD-1 expresses on CD68+ macrophages, CD3+ T cells, CD16+ monocytes, CD56+ NK cells and CK-18+ epithelial cells, respectively. Additionally, PD-1 co-expresses with vascular endothelial growth factor (VEGF), and PD-1 deficiency resulted in an accumulation of glandular/luminal epithelium derived VEGF, which accelerates the expression of the proliferation-associated protein, proliferating cell nuclear antigen (PCNA), and thus potentially lead to epithelial proliferation in aged PD-1-/- uteri.
These findings showed that PD-1 deficiency augments luminal epithelial cell proliferation probably through induced VEGF secretion, suggesting PD-1 plays an important role in controlling the growth and differentiation of the uterine epithelium.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5809067461223905
KeywordsPD-1 Endometrial hyperplasia VEGF PCNA Uterus
Co-signaling by B7/CD28 family members regulates the initiation, maintenance, and termination of immune responses. Programmed death-1 (PD-1) is an inhibitory receptor expressed on activated T cells, B cells and myeloid cells . PD-1 deficiency (PD-1-/-) causes lupus-like glomerulonephritis and arthritis in C57BL/6 mice [2, 3], autoimmune dilated cardiomyopathy (DCM) and gastritis in BALB/c mice [4, 5], acute type 1 diabetes mellitus (T1DM) in nonobese diabetic (NOD) mice , and lethal myocarditis in MRL mice . In humans, polymorphisms in the PD-1 gene have been associated with susceptibility to systemic lupus erythematosus , type I diabetes , multiple sclerosis , and rheumatoid arthritis . Additionally, PD-1-/- mice in a 129svEv-Brd background were also more susceptible to the development of experimental autoimmune encephalomyelitis (EAE) . Nevertheless, the organ development regulated by PD-1 signal is still under investigation.
PD-L1 (B7-H1) and PD-L2 (B7-DC), two immunoregulatory molecules belonging to the B7 family, were identified as the ligands for PD-1 [13, 14]. The expression of PD-L1 has been detected not only in lymphoid organs but also in nonlymphoid tissues and was enhanced in several types of tumor cells under inflammation conditions, suggesting that PD-L1 might regulate lymphocyte function at sites of inflammation . The expression of PD-L2, however, was restricted in activated dendritic cells (DCs), macrophages, monocytes and T cells .
The expression, anatomic distribution and potential role for PD-1/PD-Ls in uterine development have not been investigated. We here showed that aged PD-1-deficient female mice in a 129svEv-Brd background develop endometrial hyperplasia. This effect potentially reflects the induction VEGF secretion from epithelial cells upon PD-1 signaling deficiency.
All experiments were approved and conducted in accordance with the guidelines of the Animal Care and Use Committee of the Third Military Medical University. All efforts were made to minimize animal suffering.
PD-1-deficient mice (Background: 129svEv-Brd) were kindly provided by Dr. Laura L. Carter (Inflammation Department, Wyeth Research, Cambridge, MA, USA). Prof. T. Honjo (Department of Immunology and Genomic Medicine, Kyoto University, Japan) kindly gave us the PD-1-KO-N10 mice (strain: BALB/cJ). The WT control mice were purchased from the Animal Center of Beijing University School of Medicine. All mice were maintained in micro-isolator cages and housed in the animal colony at the Animal Center, Third Military Medical University, and standard laboratory chow diet and water was supplied.
Histology and immunohistochemistry
Section were used to detect the indicted protein expression with using the following primary antibodies: anti-PD-L1 (2.5 μg/ml, Catalog#: AF1019, R&D Systems), anti-PD-L2 (2.5 μg/ml, Catalog#: AF1022, R&D Systems), anti-PD-1 (2 μg/ml, Catalog#: AF1021, R&D Systems) and anti-VEGF (1:100, clone: C-1, Santa Cruz). A previously published protocol for immunohistochemistry was used .
Immunofluorescent double staining
For immunofluorescent double staining, the sections were incubated with mouse monoclonal anti-PD-1 and anti-VEGF antibodies at 4°C overnight. After washing with PBS (3x5-min incubations), sections were incubated with Alexa 568-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Sections were subsequently further incubated with anti-CD3 (1:50, Abcam), anti-CD56 (1: 150, Santa Cruz), anti-CK-18 (1: 150, clone: H-80, Santa Cruz), anti-CD68 (1: 200, clone: H-255, Santa Cruz) antibodies at 4°C overnight and incubated with fluorescent isothiocyanate-conjugated goat anti-mouse IgG antibodies (Jackson ImmunoResearch) for an additional 1 h. Subsequently, the sections were incubated with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI, Sigma, CA, USA) for 10 min to stain the nuclei. Sections incubated with the appropriate isotype control primary antibodies and fluorescently labeled secondary antibodies were used as negative controls.
Cell count and statistical analysis
The proportion of PCNA-positive nuclei in the glandular epithelium was determined through image analysis of the histological sections. Photomicrographs were captured and analyzed using Image Pro-Plus 5.0 software (Media Cybernetics, Silver Spring, MD) . The number of PCNA+ nuclei per high-power field was counted. The data were analyzed using GraphPad Prism 4.03 software. An unpaired Student t test (two-tailed) was used to assess comparisons of PCNA+ nuclei between PD-1-/- and WT uteri. A p value <0.05 was considered statistically significant different.
Changes in the gross anatomy and morphology of the uteri in aged PD-1-/- female mice
PD-1 deficiency resulted in epithelial cell proliferation
Detection and localization of PD-1 and its ligands in the aged uteri
Augmented VEGF secretion in uteri from aged PD-1 deficient mice
The disruption of the PD-1 signal leads to the breakdown of peripheral tolerance and the initiation of autoimmunity like dilated cardiomyopathy. This effect is due to PD-1 negatively controls T cell receptor (TCR) signaling . Recently, it was shown that PD-1 deficiency accelerates microRNA-21 (miR-21) overexpression, thus lead to cell proliferation through the enhanced expression of programmed cell death 4 (PDCD4) . Here, we provided the first evidence that endometrial hyperplasia was developed in aged PD-1-/- female mice in a 129svEv-Brd rather than Balb/c background (Figure 1B), suggesting PD-1 plays an important role in the growth and differentiation of the uterine epithelium in129svEv-Brd mice. Our results further reflect that the development of disease mediated by PD-1 signals in animal models is strain restricted.
The expression of PD-1 has been reported on T cells, natural killer T cells, B cells and monocytes, and this expression was enhanced through stimulation with inflammatory factors, such as TNF-α and IFN-γ. However, PD-L1 and PD-L2, the two PD-1ligands, shows different expression patterns . Here, we showed that the expression of PD-1 was observed on glandular and luminal epithelium (Figure 3B) and cells infiltrated in stroma (Figure 3C). Additionally, PD-1 was also detected to be expressed on CD68+ macrophages, CD3+ T cells, CD16+ monocytes, CD56+ NK cells and CK-18+ epithelial cells, but it was absent on CD31+ endothelial cells, as detected by immunofluorescent double staining (Figure 3D). Additionally, the expression of PD-L1 and PD-L2 was also detected on luminal epithelium and infiltrating cells within the aged uterine tissues (Figure 4). Due to strong neovascularization (Figure 1F) and higher level of PCNA+ cells were seen in aged PD-1-/- uteri, suggesting strong cell proliferation is in progress in PD-1-/- uteri (Figure 2). These combine data suggest that local PD-1/PD-Ls signal probably controls glandular/luminal epithelial biofunction, like cell proliferation and neovascularization. Indeed, the expression of PD-1 on the tubular epithelium of murine Adriamycin nephropathy (AN) has been reported previously, and blockade of PD-1 worsened progressive renal histopathological and functional injury in murine AN . Taken together, our results suggested that PD-1 is not only expressed on immune cells but also on nonlymphoid tissues, and uterine local PD-1/PD-Ls signal probably directly inhibits glandular/luminal epithelial cell proliferation and neovascularization.
To analyze other potential molecular mechanisms that involve in the growth and differentiation of the uterine epithelium in aged PD-1-/- mice, the secretion of VEGF, which stimulate endothelial and epithelial cell proliferation through its receptor, VEGFR, was compared in aged uteri between PD-1-/- and WT mice. An interesting finding is that glandular/luminal epithelium derived-VEGF in aged uteri from PD-1-/- mice was augmented dramatically (Figure 5C), thereby potentially promotes cell proliferation via its receptor VEGFR and thus resulted in accelerating neovascularization (Figure 1F). Additionally, VEGF in uteri was also co-expresses with PD-1 (Figure 5D), suggesting that PD-1 signaling inhibits epithelial cell proliferation potentially through a reduction of VEGF secretion, in addition to direct prevents epithelial proliferation by cross-reacts with PD-Ls. However, the expression of PTEN, a tumor suppress gene, were significantly higher in cyclical endometrium than in atypical hyperplasia and endometrioid carcinoma, indicated that PTEN involves in the pathogenesis of endometrial hyperplasia . On the other hand, CyclinD1, a cell -cycle regulator, exhibited a promising potential to predict the prognosis of patients with endometrial carcinoma . Whether the transcription of PTEN or CyclinD1 is also controlled by PD-1/PD-Ls need further investigation.
PD-1 deficiency augments luminal epithelial cell proliferation and neovascularization in aged uteri, suggesting that PD-1 plays an important role in the organization, growth and differentiation of the uterine epithelium.
This work was supported by grants from the National Natural Science Foundation of China (NSFC No. 8122223 and No. 61141012).
- Saresella M, Rainone V, Al-Daghri NM, Clerici M, Trabattoni D: The PD-1/PD-L1 pathway in human pathology. Curr Mol Med. 2012, 12 (3): 259-267. 10.2174/156652412799218903.PubMedView Article
- Nishimura H, Nose M, Hiai H, Minato N, Honjo T: Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999, 11 (2): 141-151. 10.1016/S1074-7613(00)80089-8.PubMedView Article
- Hamel KM, Cao Y, Wang Y, Rodeghero R, Kobezda T, Chen L, Finnegan A: B7-H1 expression on non-B and non-T cells promotes distinct effects on T- and B-cell responses in autoimmune arthritis. Eur J Immunol. 2010, 40 (11): 3117-3127. 10.1002/eji.201040690.PubMedPubMed CentralView Article
- Okazaki T, Tanaka Y, Nishio R, Mitsuiye T, Mizoguchi A, Wang J, Ishida M, Hiai H, Matsumori A, Minato N, Honjo T: Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice. Nat Med. 2003, 9 (12): 1477-1483. 10.1038/nm955.PubMedView Article
- Kido M, Watanabe N, Okazaki T, Akamatsu T, Tanaka J, Saga K, Nishio A, Honjo T, Chiba T: Fatal autoimmune hepatitis induced by concurrent loss of naturally arising regulatory T cells and PD-1-mediated signaling. Gastroenterology. 2008, 135 (4): 1333-1343. 10.1053/j.gastro.2008.06.042.PubMedView Article
- Filippi CM, Estes EA, Oldham JE, von Herrath MG: Immunoregulatory mechanisms triggered by viral infections protect from type 1 diabetes in mice. J Clin Invest. 2009, 119 (6): 1515-1523.PubMedPubMed Central
- Grabie N, Gotsman I, DaCosta R, Pang H, Stavrakis G, Butte MJ, Keir ME, Freeman GJ, Sharpe AH, Lichtman AH: Endothelial programmed death-1 ligand 1 (PD-L1) regulates CD8+ T-cell mediated injury in the heart. Circulation. 2007, 116 (18): 2062-2071. 10.1161/CIRCULATIONAHA.107.709360.PubMedView Article
- Kristjansdottir H, Steinsson K, Gunnarsson I, Gröndal G, Erlendsson K, Alarcón-Riquelme ME: Lower expression levels of the programmed death 1 receptor on CD4 + CD25+ T cells and correlation with the PD-1.3A genotype in patients with systemic lupus erythematosus. Arthritis Rheum. 2010, 62 (6): 1702-1711. 10.1002/art.27417.PubMedView Article
- Ni R, Ihara K, Miyako K, Kuromaru R, Inuo M, Kohno H, Hara T: PD-1 gene haplotype is associated with the development of type 1 diabetes mellitus in Japanese children. Hum Genet. 2007, 121 (2): 223-232. 10.1007/s00439-006-0309-8.PubMedView Article
- Kroner A, Mehling M, Hemmer B, Rieckmann P, Toyka KV, Mäurer M, Wiendl H: A PD-1 polymorphism is associated with disease progression in multiple sclerosis. Ann Neurol. 2005, 58 (1): 50-57. 10.1002/ana.20514.PubMedView Article
- Kong EK, Prokunina-Olsson L, Wong WH, Lau CS, Chan TM, Alarcón-Riquelme M, Lau YL: A new haplotype of PDCD1 is associated with rheumatoid arthritis in Hong Kong Chinese. Arthritis Rheum. 2005, 52 (4): 1058-1062. 10.1002/art.20966.PubMedView Article
- Carter LL, Leach MW, Azoitei ML, Cui J, Pelker JW, Jussif J, Benoit S, Ireland G, Luxenberg D, Askew GR, Milarski KL, Groves C, Brown T, Carito BA, Percival K, Carreno BM, Collins M, Marusic S: PD-1/PD-L1, but not PD-1/PD-L2, interactions regulate the severity of experimental autoimmune encephalomyelitis. J Neuroimmunol. 2007, 182 (1–2): 124-134.PubMedView Article
- Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, Fitz LJ, Malenkovich N, Okazaki T, Byrne MC, Horton HF, Fouser L, Carter L, Ling V, Bowman MR, Carreno BM, Collins M, Wood CR, Honjo T: Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2002, 192 (7): 1027-1034.View Article
- Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I, Iwai Y, Long AJ, Brown JA, Nunes R, Greenfield EA, Bourque K, Boussiotis VA, Carter LL, Carreno BM, Malenkovich N, Nishimura H, Okazaki T, Honjo T, Sharpe AH, Freeman GJ: PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol. 2001, 2 (3): 261-268. 10.1038/85330.PubMedView Article
- Greaves P, Gribben JG: The role of B7 family molecules in hematologic malignancy. Blood. 2013, 121 (5): 734-744. 10.1182/blood-2012-10-385591.PubMedPubMed CentralView Article
- Rozali EN, Hato SV, Robinson BW, Lake RA, Lesterhuis WJ: Programmed death ligand 2 in cancer-induced immune suppression. Clin Dev Immunol. 2012, 2012: 656340-PubMedPubMed CentralView Article
- Cao D, Xu H, Guo G, Ruan Z, Fei L, Xie Z, Wu Y, Chen Y: Intrahepatic expression of programmed death-1 and its ligands in patients with HBV-related acute-on-chronic liver failure. Inflammation. 2013, 36 (1): 110-120. 10.1007/s10753-012-9525-7.PubMedView Article
- Guo S, Yang C, Mei F, Wu S, Luo N, Fei L, Chen Y, Wu Y: Down-regulation of Z39Ig on macrophages by IFN-gamma in patients with chronic HBV infection. Clin Immunol. 2010, 136 (2): 282-291. 10.1016/j.clim.2010.03.007.PubMedView Article
- Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL: SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J Immunol. 2004, 173 (2): 945-954. 10.4049/jimmunol.173.2.945.PubMedView Article
- Iliopoulos D, Kavousanaki M, Ioannou M, Boumpas D, Verginis P: The negative costimulatory molecule PD-1 modulates the balance between immunity and tolerance via miR-21. Eur J Immunol. 2011, 41 (6): 1754-1763. 10.1002/eji.201040646.PubMedView Article
- Chen L, Flies DB: Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013, 13 (4): 227-242. 10.1038/nri3405.PubMedPubMed CentralView Article
- Qin XH, Lee VW, Wang YP, Zheng GP, Wang Y, Alexander SI, Harris DC: A protective role for programmed death 1 in progression of murine adriamycin nephropathy. Kidney Int. 2006, 70 (7): 1244-1250. 10.1038/sj.ki.5000345.PubMedView Article
- Sarmadi S, Izadi-Mood N, Sotoudeh K, Tavangar SM: Altered PTEN expression; a diagnostic marker for differentiating normal, hyperplastic and neoplastic endometrium. Diagn Pathol. 2009, 4: 41-10.1186/1746-1596-4-41.PubMedPubMed CentralView Article
- Liang S, Mu K, Wang Y, Zhou Z, Zhang J, Sheng Y, Zhang T: CyclinD1, a prominent prognostic marker for endometrial diseases. Diagn Pathol. 2013, 8: 138-10.1186/1746-1596-8-138.PubMedPubMed CentralView Article
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.