, Volume 40, Issue 3, pp 1087–1101 | Cite as

Sex Differences in Macrophage Functions in Middle-Aged Rats: Relevance of Estradiol Level and Macrophage Estrogen Receptor Expression

  • Ivana Ćuruvija
  • Stanislava Stanojević
  • Nevena Arsenović-Ranin
  • Veljko Blagojević
  • Mirjana Dimitrijević
  • Biljana Vidić-Danković
  • Vesna Vujić


The aim of this study was to examine the influence of sex on age-related changes in phenotype and functional capacity of rat macrophages. The potential role of estradiol as a contributing factor to a sex difference in macrophage function with age was also examined. Thioglycollate-elicited peritoneal macrophages derived from the young (2 months old) and the naturally senescent intact middle-aged (16 months old) male and female rats were tested for cytokine secretion and antimicrobial activity (NO and H2O2 production and myeloperoxidase activity). Serum concentration of estradiol and the expression of estrogen receptor (ER)α and ERβ on freshly isolated peritoneal macrophages were also examined. Decreased secretion of IL-1β and IL-6 by macrophages from middle-aged compared to the young females was accompanied with the lesser density of macrophage ERα expression and the lower systemic level of estradiol, whereas the opposite was true for middle-aged male rats. Macrophages in the middle-aged females, even with the diminished circulating estradiol levels, produce increased amount of IL-6, and comparable amounts of IL-1β, TNF-α, and NO to that measured in macrophages from the middle-aged males. Age-related changes in macrophage phenotype and the antimicrobial activity were independent of macrophage ERα/ERβ expression and estradiol level in both male and female rats. Although our study suggests that the sex difference in the level of circulating estradiol may to some extent contribute to sex difference in macrophage function of middle-aged rats, it also points to more complex hormonal regulation of peritoneal macrophage activity in females.


estradiol estrogen receptor α (ERα) estrogen receptor β (ERβ) middle-aged rats thioglycollate-elicited peritoneal macrophages sex differences 



This study is supported by the Ministry of Education, Science and Technological Development Republic of Serbia, Grant No 175050. The Ministry of Education, Science and Technological Development had no role in the study design, collection, analysis and interpretation of data, writing of the report, and decision to submit the article for publication. Authors express their gratitude to Tatjana Miletić, PhD (Health & Environment Department, Molecular Diagnostics, AIT Austrian Institute of Technology GmbH), for critical reading and valuable comments.

Compliance with Ethical Standards

The experimental protocol and all procedures with animals and their care were approved by Ministry of Agriculture and Environmental Protection (license number 323-07-01577/2016-05/14, issued on 02-25-2016) and were in accordance with principles declared in Directive 2010/63/EU of the European Parliament and of the Council from 22 September 2010 on the protection of animals used for scientific purposes (revising Directive 86/609/EEC).

Conflict of Interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Fischer, J., N. Jung, N. Robinson, and C. Lehmann. 2015. Sex differences in immune responses to infectious diseases. Infection. doi: 10.1007/s15010-015-0791-9.PubMedGoogle Scholar
  2. 2.
    Voskuhl, R.R., and S.M. Gold. 2012. Sex-related factors in multiple sclerosis susceptibility and progression. Nature Reviews Neurology. doi: 10.1038/nrneurol.2012.43.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Živković, I., B. Bufan, V. Petrušić, R. Minić, N. Arsenović-Ranin, R. Petrović, and G. Leposavić. 2015. Sexual diergism in antibody response to whole virus trivalent inactivated influenza vaccine in outbred mice. Vaccine. doi: 10.1016/j.vaccine.2015.09.006.Google Scholar
  4. 4.
    Griesbeck, M., S. Ziegler, S. Laffont, N. Smith, L. Chauveau, P. Tomezsko, A. Sharei, G. Kourjian, F. Porichis, et al. 2015. Sex differences in plasmacytoid dendritic cell levels of IRF5 drive higher IFN-α production in women. The Journal of Immunology. doi: 10.4049/jimmunol.1501684.PubMedPubMedCentralGoogle Scholar
  5. 5.
    Hewagama, A., D. Patel, S. Yarlagadda, F.M. Strickland, and B.C. Richardson. 2009. Stronger inflammatory/cytotoxic T-cell response in women identified by microarray analysis. Genes & Immunity. doi: 10.1038/gene.2009.Google Scholar
  6. 6.
    Klein, S.L., and K.L. Flanagan. 2016. Sex differences in immune responses. Nature Reviews Immunology. doi: 10.1038/nri.2016.90.PubMedGoogle Scholar
  7. 7.
    Ruggieri, A., S. Anticoli, A. D'Ambrosio, L. Giordani, and M. Viora. 2016. The influence of sex and gender on immunity, infection and vaccination. Annali dell’Istituto Superiore di Sanità. doi: 10.4415/ANN_16_02_11.Google Scholar
  8. 8.
    Mahbub, S., A.L. Brubaker, and E.J. Kovacs. 2011. Aging of the innate immune system: An update. Current Opinion in Immunology. doi: 10.2174/157339511794474181.Google Scholar
  9. 9.
    Weiskopf, D., B. Weinberger, and B. Grubeck-Loebenstein. 2009. The aging of the immune system. Transplant International. doi: 10.1111/j.1432-2277.2009.00927.x.PubMedGoogle Scholar
  10. 10.
    Linehan, E., and D.C. Fitzgerald. 2015. Ageing and the immune system: Focus on macrophages. European Journal of Microbiology and Immunology. doi: 10.1556/EUJMI-D-14-00035.PubMedPubMedCentralGoogle Scholar
  11. 11.
    Marriott, I., K.L. Bost, and Y.M. Huet-Hudson. 2006. Sexual dimorphism in expression of receptors for bacterial lipopolysaccharides in murine macrophages: A possible mechanism for gender-based differences in endotoxic shock susceptibility. Journal of Reproductive Immunology. doi: 10.1016/j.jri.2006.01.004.PubMedGoogle Scholar
  12. 12.
    Spitzer, J.A. 1999. Gender differences in some host defense mechanisms. Lupus. doi: 10.1177/096120339900800510.PubMedGoogle Scholar
  13. 13.
    Stanojević, S., I. Ćuruvija, V. Blagojević, R. Petrović, V. Vujić, and M. Dimitrijević. 2016. Strain-dependent response to stimulation in middle-aged rat macrophages: A quest after a useful indicator of healthy aging. Experimental Gerontology. doi: 10.1016/j.exger.2016.10.005.PubMedGoogle Scholar
  14. 14.
    Vermeulen, A., J.M. Kaufman, S. Goemaere, and I. van Pottelberg. 2002. Estradiol in elderly men. The Aging Male. doi: 10.1080/tam. Scholar
  15. 15.
    Laughlin, G.A., E. Barrett-Connor, D. Kritz-Silverstein, and D. von Mühlen. 2000. Hysterectomy, oophorectomy, and endogenous sex hormone levels in older women: The Rancho Bernardo Study. The Journal of Clinical Endocrinology & Metabolism. doi: 10.1210/jcem.85.2.6405.Google Scholar
  16. 16.
    Giefing-Kroll, C., P. Berger, G. Lepperdinger, and B. Grubeck-Loebenstein. 2015. How sex and age affect immune responses, susceptibility to infections, and response to vaccination. Aging Cell. doi: 10.1111/acel.12326.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Baeza, I., N.M. De Castro, L. Arranz, J. Fdez-Tresguerres, and M. De la Fuente. 2011. Ovariectomy causes immunosenescence and oxi-inflamm-ageing in peritoneal leukocytes of aged female mice similar to that in aged males. Biogerontology. doi: 10.1007/s10522-010-9317-0.PubMedGoogle Scholar
  18. 18.
    Zhao, H., Z. Tian, J. Hao, and B. Chen. 2005. Extragonadal aromatization increases with time after ovariectomy in rats. Reproductive Biology and Endocrinology. doi: 10.1186/1477-7827-3-6.Google Scholar
  19. 19.
    Dimitrijević, M., S. Stanojević, N. Kuštrimović, K. Mitić, V. Vujić, I. Aleksić, K. Radojević, and G. Leposavić. 2013. The influence of aging and estradiol to progesterone ratio on rat macrophage phenotypic profile and NO and TNF-a production. Experimental Gerontology. doi: 10.1016/j.exger.2013.07.001.PubMedGoogle Scholar
  20. 20.
    Barrat, F., B. Lesourd, H.J. Boulouis, D. Thibault, S. Vincent-Naulleau, B. Gjata, A. Louise, T. Neway, and C. Pilet. 1997. Sex and parity modulate cytokine production during murine ageing. Clinical & Experimental Immunology. doi: 10.1046/j.1365-2249.1997.4851387.x.Google Scholar
  21. 21.
    Carvalho-Freitas, M.I., J.A. Anselmo-Franci, E. Teodorov, A.G. Nasello, J. Palermo-Neto, and L.F. Felicio. 2007. Reproductive experience modifies dopaminergic function, serum levels of prolactin, and macrophage activity in female rats. Life Science. doi: 10.1016/j.lfs.2007.04.032.Google Scholar
  22. 22.
    Pick, E., and D. Mizel. 1981. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader. Journal of Immunological Methods. doi: 10.1016/0022-1759(81)90138-1.PubMedGoogle Scholar
  23. 23.
    Jr Johnston, R.B., and S. Kitagawa. 1985. Molecular basis for the enhanced respiratory burst of activated macrophages. Federation Proceedings 14: 2927–2932.Google Scholar
  24. 24.
    Choi, H.S., J.W. Kim, Y.N. Cha, and C. Kim. 2006. A quantitative nitroblue tetrazolium assay for determining intracellular superoxide anion production in phagocytic cells. Journal of Immunoassay and Immunochemistry. doi: 10.1080/15321810500403722.Google Scholar
  25. 25.
    Pick, E., J. Charon, and D. Mizel. 1981. A rapid densitometric microassay for nitroblue tetrazolium reduction and application of the microassay to macrophages. Journal of the Reticuloendothelial Society. 6: 581–593.Google Scholar
  26. 26.
    Bradley, P., D.A. Priebat, R.D. Christensen, and G. Rothstein. 1982. Measurement of cutaneous inflammation: Estimation of neutrophil content with an enzyme marker. Journal of Investigative Dermatology. doi: 10.1111/1523-1747.ep12506462.PubMedGoogle Scholar
  27. 27.
    Green, L.C., D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, and S.R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Analytical Biochemistry. doi: 10.1016/0003-2697(82)90118-X.Google Scholar
  28. 28.
    Dijkstra, C.D., E.A. Döpp, P. Joling, and G. Kraal. 1985. The heterogeneity of mononuclear phagocytes in lymphoid organs: Distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3. Immunology 54: 589–599.PubMedPubMedCentralGoogle Scholar
  29. 29.
    Thornley, T.B., Z. Fang, S. Balasubramanian, R.A. Larocca, W. Gong, S. Gupta, E. Csizmadia, N. Degauque, B.S. Kim, et al. 2014. Fragile TIM-4–expressing tissue resident macrophages are migratory and immunoregulatory. Journal of Clinical Investigation. doi: 10.1172/JCI73527.PubMedPubMedCentralGoogle Scholar
  30. 30.
    Negishi, H., Y. Ohba, H. Yanai, A. Takaoka, K. Honma, K. Yui, T. Matsuyama, T. Taniguchi, and K. Honda. 2005. Negative regulation of toll-like-receptor signaling by IRF-4. Proceedings of the National Academy of Sciences USA. doi: 10.1073/pnas.0508327102.Google Scholar
  31. 31.
    Calippe, B., V. Douin-Echinard, L. Delpy, M. Laffargue, K. Lélu, A. Krust, B. Pipy, F. Bayard, J.F. Arnal, et al. 2010. 17Beta-estradiol promotes TLR4 triggered proinflammatory mediator production through direct estrogen receptor alpha signaling in macrophages in vivo. The Journal of Immunology. doi: 10.4049/jimmunol.0902383.PubMedGoogle Scholar
  32. 32.
    Frei, R., J. Steinle, T. Birchler, S. Loeliger, C. Roduit, D. Steinhoff, R. Seibl, K. Büchner, R. Seger, et al. 2010. MHC class II molecules enhance toll-like receptor mediated innate immune responses. PloS One. doi: 10.1371/journal.pone.0008808.PubMedPubMedCentralGoogle Scholar
  33. 33.
    Dimitrijević, M., S. Stanojević, V. Vujić, I. Aleksić, I. Pilipović, and G. Leposavić. 2014. Aging oppositely affects TNF-α and IL-10 production by macrophages from different rat strains. Biogerontology. doi: 10.1007/s10522-014-9513-4.PubMedGoogle Scholar
  34. 34.
    Simpson, E., G. Rubin, C. Clyne, K. Robertson, L. O’Donnell, S. Davis, and M. Jones. 1999. Local estrogen biosynthesis in males and females. Endocrine-Related Cancer. doi: 10.1677/erc.0.0060131.Google Scholar
  35. 35.
    Campesi, I., M. Marino, A. Montella, S. Pais, F. Franconi. 2017. Sex differences in estrogen receptor α and β levels and activation status in LPS-stimulated human macrophages Journal of Cellular Physiology. 232: 340–345. doi:  10.1002/jcp.25425
  36. 36.
    Jiang, Y., P. Gong, Z. Madak-Erdogan, T. Martin, M. Jeyakumar, K. Carlson, I. Khan, T.J. Smillie, A.G. Chittiboyina, et al. 2013. Mechanisms enforcing the estrogen receptor β selectivity of botanical estrogens. The FASEB Journal. doi: 10.1096/fj.13-234617.Google Scholar
  37. 37.
    Murphy, A.J., P.M. Guyre, C.R. Wira, and P.A. Pioli. 2009. Estradiol regulates expression of estrogen receptor ERa46 in human macrophages. PloS One. doi: 10.1371/journal.pone.0005539.Google Scholar
  38. 38.
    Matthews, J., and J.A. Gustafsson. 2003. Estrogen signaling: A subtle balance between ER alpha and ER beta. Molecular Interventions. doi: 10.1124/mi.3.5.281.PubMedGoogle Scholar
  39. 39.
    Ashcroft, G.S., J. Dodsworth, E. Van Boxtel, R.W. Tarnuzzer, M.A. Horan, G.S. Schultz, and M. Ferguson. 1997. Estrogen accelerates cutaneous wound healing associated with an increase in TGF-1 levels. Nature Medicine. doi: 10.1038/nm1197-1209.PubMedGoogle Scholar
  40. 40.
    Moestrup, S.K., and H.J. Møller. 2004. CD163: A regulated hemoglobin scavenger receptor with a role in the anti-inflammatory response. Annals of Medicine 5: 347–354.CrossRefGoogle Scholar
  41. 41.
    Geraldes, P., S. Gagnon, S. Hadjadj, Y. Merhi, M.G. Sirois, I. Cloutier, and J.F. Tanguay. 2006. Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: An ER α -mediated pathway. Cardiovascular Research. doi: 10.1016/j.cardiores.2006.05.015.PubMedGoogle Scholar
  42. 42.
    Xie, H., C. Hua, L. Sun, X. Zhao, H. Fan, H. Dou, L. Sun, and Y. Hou. 2011. 17β-estradiol induces CD40 expression in dendritic cells via MAPK signaling pathways in a minichromosome maintenance protein 6-dependent manner. Arthritis & Rheumatology. doi: 10.1002/art.30420.Google Scholar
  43. 43.
    Jackaman, C., H.G. Radley-Crabb, Z. Soffe, T. Shavlakadze, M.D. Grounds, and D.J. Nelson. 2013. Targeting macrophages rescues age-related immune deficiencies in C57BL/6J geriatric mice. Aging Cell. doi: 10.1111/acel.12062.PubMedGoogle Scholar
  44. 44.
    Straub, R.H. 2007. The complex role of estrogens in inflammation. Endocrine Reviews. doi: 10.1210/er.2007-0001.PubMedGoogle Scholar
  45. 45.
    West, P., I.E. Brodsky, C. Rahner, D.K. Woo, H. Erdjument-Bromage, O. Tempst, M.C. Walsh, Y. Choi, G.S. Shadel, and S. Ghosh. 2011. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature. doi: 10.1038/nature09973.Google Scholar
  46. 46.
    Gantner, B.N., R.M. Simmons, S.J. Canavera, S. Akira, and D.M. Underhill. 2003. Collaborative induction of inflammatory responses by dectin-1 and toll-like receptor 2. The Journal of Experimental Medicine. doi: 10.1084/jem.20021787.PubMedPubMedCentralGoogle Scholar
  47. 47.
    Ozinsky, A., D.M. Underhill, J.D. Fontenot, A.M. Hajjar, K.D. Smith, C.B. Wilson, L. Schroeder, and A. Aderem. 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proceedings of the National Academy of Sciences. doi: 10.1073/pnas.250476497.Google Scholar
  48. 48.
    Rodrigues, M.R., D. Rodriguez, M. Russo, and A. Campa. 2002. Macrophage activation includes high intracellular myeloperoxidase activity. Biochemical and Biophysical Research Communications. doi: 10.1006/bbrc.2002.6724.PubMedGoogle Scholar
  49. 49.
    Abu-Soud, H.M., and S.L. Hazen. 2000. Nitric oxide is a physiological substrate for mammalian peroxidases. The Journal of Biological Chemistry. doi: 10.1074/jbc.M002579200.Google Scholar
  50. 50.
    Shirato, K., and K. Imaizumi. 2015. Mechanisms underlying the suppression of inflammatory responses in peritoneal macrophages of middle-aged mice. In Physical activity, exercise, sedentary behavior and health, eds. Kazuyuki Kanosue, Satomi Oshima, Zhen-Bo Cao, and Koichiro Oka, 193–202. Tokyo: Springer. doi: 10.1007/978-4-431-55333-5_16.
  51. 51.
    Dieter, P. 1992. Relationship between intracellular pH changes, activation of protein kinase C and NADPHoxidase in macrophages. FEBS Letters. doi: 10.1016/0014-5793(92)80012-6.Google Scholar
  52. 52.
    Porto, M.L., B.P. Rodrigues, T.N. Menezes, S.L. Ceschim, D.E. Casarini, A.L. Gava, T.M.C. Pereira, E.C. Vasquez, B.P. Campagnaro, and S.S. Meyrelles. 2015. Reactive oxygen species contribute to dysfunction of bone marrow hematopoietic stem cells in aged C57BL/6 J mice. Journal of Biomedical Science. doi: 10.1186/s12929-015-0201-8.PubMedPubMedCentralGoogle Scholar
  53. 53.
    Kovats, S. 2015. Estrogen receptors regulate innate immune cells and signaling pathways. Cellular Immunology. doi: 10.1016/j.cellimm.2015.01.018.PubMedPubMedCentralGoogle Scholar
  54. 54.
    Linehan, E., Y. Dombrowski, R. Snoddy, P.G. Fallon, A. Kissenpfennig, and D.C. Fitzgerald. 2014. Aging impairs peritoneal but not bone marrow-derived macrophage phagocytosis. Aging Cell. doi: 10.1111/acel.12223.PubMedPubMedCentralGoogle Scholar
  55. 55.
    Gaytan, F., J. Aceitero, C. Bellido, J.E. Sanchez-Criado, and E. Aguilar. 1991. Estrous cycle-related changes in mast cell numbers in several ovarian compartments in the rat. Biology of Reproduction. doi: 10.1095/biolreprod45.1.27.PubMedGoogle Scholar
  56. 56.
    Baird, D.T., P.E. Burger, G.D. Heavon-Jones, and R.J. Scaramuzzi. 1974. The site of secretion of androstenedione in non-pregnant women. Journal of Endocrinology. 63: 201–212.CrossRefPubMedGoogle Scholar
  57. 57.
    Thijssen, J.H., M.A. Wiegerninck, G.H. Donker, and J. Poortman. 1984. Uptake and metabolism of oestriol in human target tissues. Journal of Steroid Biochemistry. 20: 955–958.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Ivana Ćuruvija
    • 1
  • Stanislava Stanojević
    • 1
  • Nevena Arsenović-Ranin
    • 2
  • Veljko Blagojević
    • 1
  • Mirjana Dimitrijević
    • 3
  • Biljana Vidić-Danković
    • 1
  • Vesna Vujić
    • 4
  1. 1.Immunology Research Centre “Branislav Janković”Institute of Virology, Vaccines and Sera “Torlak”BelgradeSerbia
  2. 2.Department of Microbiology and Immunology, Faculty of PharmacyUniversity of BelgradeBelgradeSerbia
  3. 3.Department of Immunology, Institute for Biological Research “Siniša Stanković”University of BelgradeBelgradeSerbia
  4. 4.Department of Chemistry, Faculty of MedicineUniversity of BelgradeBelgradeSerbia

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