The impact of estrogens on leukocyte function in remodeling of extracellular matrix

Background: Major reproductive events such as menstruation, ovulation, implantation and cervical ripening are characterized by an increased number of invading leukocytes in the tissues. Sex steroid hormones, particularly estrogens, play an important role in these changes. Estrogens have also been implicated in the pathogenesis of many conditions associated with leukocyte infiltration and immunological dysfunction, such as autoimmune diseases and atherosclerosis.

A rapid remodeling of the cervical connective tissue at term pregnancy, termed cervical ripening, is an important and obligatory event in parturition. Biopsies from human uterine cervix showed that leukocyte counts are dramatically increased at the time of parturition suggesting their function in cervical ripening.

The aim of this work was to investigate the relationship between leukocytes and sex steroid hormones, primarily estrogens, as they play a critical role in parturition in many animal species and are presumed to be involved in human parturition.

Results: The majority of biological effects of estrogens is mediated through two distinct receptors, estrogen receptor (ER)alpha and ERbeta. We found and characterized expression of ERs in leukocytes invading human uterine cervix at the time of parturition. The invading leukocytes in the cervix were verified as macrophages and neutrophils. The leukocytes showed positive immunostaining for ERbeta. In addition, the invading leukocytes were analyzed in terms of presence for catabolic enzymes, capable of degrading components of connective tissue. Cervical leukocytes were identified as a source of matrix metalloproteinase-2 and 9, which have collagen IV and proteoglycans as substrates. An increased level of these enzymes in the cervix was observed just prior to parturition indicating involvement in the cervical ripening.

By using samples collected from healthy female and male donors, we investigated ERs in different types of leukocytes in peripheral blood. In this report we show that leukocytes are potential target cells for estrogens as ERalpha and ERbeta were identified in mononuclear (PBMC) and polymorphonuclear (PMN) leukocytes. Interestingly, it was observed that both ERalpha and ERbeta proteins differ in size between PMN and PBMC, suggesting that the two leukocyte populations express different isoforms of ERalpha and ERbeta. These findings were confirmed by identification of multiple alternative splice variants of ER mRNAs by RT-PCR. Considering the diversity of ER isoforms in leukocyte subtypes, we conclude that the expected effect of estrogen would be highly cell-type specific.

To investigate biological roles of ERalpha and ERbeta in peripheral blood leukocytes, we studied in vivo effects of estradiol (E2), the selective ERalpha agonist PPT (4,4',4"-(4-Propyl-[lII]-pyrazole-1,3,5 -triyl)trisphenol) and the selective ERbeta agonist DPN (2,3bis(4hydroxyphenyl)-proprionitrile) on gene expression profiles in a rat animal model. This study reports the identification of genes that were commonly regulated by E2, PPT and DPN, and genes that were regulated either by an ERalpha or ERbeta agonist. 12-lipoxygenase (12-lox), fibulin1 and furin were among the regulate(] genes -and they are also involved in extracellular matrix (ECM) metabolism, therefore these genes were selected for further analysis.

In conclusion, leukocytes in tissues and peripheral blood express estrogen receptors. Estrogens produce an apparent transcriptional response in leukocytes including regulation of genes related to remodeling of ECM and inflammation. Estrogen-regulated genes in leukocytes are involved in both pro- and anti-inflammatory response, however the anti-inflammatory component is prevailing.

List of scientific papers

I. Stygar D, Wang H, Vladic YS, Ekman G, Eriksson H, Sahlin L (2001). Co-localization of oestrogen receptor beta and leukocyte markers in the human cervix. Mol Hum Reprod. 7(9): 881-6.
https://doi.org/10.1093/molehr/7.9.881

II. Stygar D, Wang H, Vladic YS, Ekman G, Eriksson H, Sahlin L (2002). Increased level of matrix metalloproteinases 2 and 9 in the ripening process of the human cervix. Biol Reprod. 67(3): 889-94.
https://doi.org/10.1095/biolreprod.102.005116

III. Stygar D, Westlund P, Eriksson H, Sahlin L (2005). Identification of wild type and variants of estrogen receptor in polymorphonuclear and mononuclear leukocytes. Clinical Endocrinology. [Accepted]
https://doi.org/10.1111/j.1365-2265.2005.02420.x

IV. Stygar D, Eriksson H, Sahlin L (2005). Investigation of biological roles of estrogen receptor alpha and beta in peripheral blood leukocytes in vivo using selective estrogen receptor agonists. [Manuscript]