Elsevier

Contraception

Volume 84, Issue 4, October 2011, Pages 423-435
Contraception

Original research article
Differential regulation of endogenous pro-inflammatory cytokine genes by medroxyprogesterone acetate and norethisterone acetate in cell lines of the female genital tract

https://doi.org/10.1016/j.contraception.2011.06.006Get rights and content

Abstract

Background

Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives are widely used in female reproductive therapy, but little is known about their mechanisms of action via steroid receptors in the female genital tract. MPA used as a contraceptive has been implicated in effects on local immune function. However, the relative effects of progesterone (Prog), MPA and norethisterone acetate (NET-A) on cytokine gene expression in the female genital tract are unknown.

Study Design

Using two epithelial cell lines generated from normal human vaginal (Vk2/E6E7) and ectocervical (Ect1/E6E7) cells as in vitro cell culture model systems for mucosal immunity of the female cervicovaginal environment, we investigated steroid receptor expression and activity as well as regulation of cytokine/chemokine genes by MPA and NET-A, as compared to the endogenous hormone Prog.

Results

We show that the Prog, androgen, glucocorticoid and estrogen receptors (PR, AR, GR and ER, respectively) are expressed in both the Vk2/E6E7 and Ect1/E6E7 cell lines, and that the GR and AR are transcriptionally active. This study is the first to show ligand-, promoter- and cell-specific regulation of IL-6, IL-8 and RANTES (regulated-upon-activation, normal T cell expressed and secreted) gene expression by Prog, MPA and NET-A in these cell lines. Moreover, we show that the repression of the TNF-α-induced RANTES gene by MPA in the Ect1/E6E7 cell line is mediated by the AR.

Conclusion

Collectively, these data demonstrate that cell lines from different anatomical sites of the female genital tract respond differently to Prog and the synthetic progestins, most likely due to differential actions via different steroid receptors. The results highlight the importance of choice of progestins for immune function in the cervicovaginal environment. They further suggest that choice of progestins in endocrine therapy may have implications for women's risk of susceptibility to infections due to differential actions on genes involved in inflammation and immune function.

Introduction

The mucosal surface of the lower female genital tract (cervicovaginal environment) is a complex system that provides a barrier against pathogens. Epithelial cells lining the cervicovaginal mucosa are the point of entry for many viral, bacterial and parasitic infections [1], [2]. These cells thus have features enabling them to combat infections, such as the expression of cytokines, hormone receptors and genital tract-specific defensins [3], [4], [5], [6] (and reviewed in Ref. [7]). Cervicovaginal epithelial cells constitutively express a wide variety of pro- and anti-inflammatory mediators, such as the cytokines, interleukin (IL)-1, IL-6, IL7, macrophage colony-stimulating factor, transforming growth factor beta and the chemoattractant cytokines, IL-8 and RANTES (regulated-upon-activation, normal T cell expressed and secreted), which are up-regulated in response to tumor necrosis factor (TNF)-α [1], [2], [8]. These cells thus play an important role in the innate and acquired immune systems present at the mucosal surfaces.

IL-8 and RANTES (also termed CCL5) are chemotactic cytokines, or chemokines, involved in the early inflammatory response by recruiting specific leukocytes, particularly macrophages, to sites of ongoing inflammation and injury, while IL-6 is responsible for neutrophil priming to chemotactic factors [9]. Interestingly, increased levels of the cytokines IL-1, TNF-α, IL-6 and the chemokines RANTES, macrophage inflammatory protein (MIP)-1α and MIP-1β in cervicovaginal secretions have been associated with human immunodeficiency virus (HIV)-1 infection and bacterial vaginosis [10], [11], [12]. Moreover, decreased systemic CD4+ cell counts during acute HIV infection has been associated with increased levels of IL-1β, IL-6 and IL-8 in genital tract secretions [13]. Thus, the cytokine milieu in the cervicovaginal mucosa is an important determinant of resistance and susceptibility to infections.

The transmission of and susceptibility to infections in women may be better understood if factors affecting the immune response in the vagina and cervix are more clearly defined. Research in animal models and in women indicates that local as well as regional immune responses affect the outcome of vaginal challenge with microbial pathogens (reviewed in Ref. [14]). Sex hormones are examples of factors that have been shown to influence susceptibility and disease predisposition to many genital tract infections [15]. Furthermore, there are indications that women using antibiotics, corticosteroids (immunosuppressive therapy), oral contraceptives and hormone replacement therapy (HRT) are more susceptible to fungal vaginal infections [16], [17], [18].

The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone enanthate (NET-EN) are the most widely used injectable female contraceptives, with at least 20 million current users of MPA worldwide [19]. MPA itself, rather than its metabolites, is the major progestogenic compound, while NET-EN and norethisterone acetate (NET-A) are hydrolyzed to norethisterone (NET) and its metabolites, which together have progestogenic action [20]. Both MPA and NET are also used for HRT in postmenopausal women. MPA used as contraceptive has been shown to increase HIV [21] and HSV cervical shedding in HIV-infected women [22]. Although Mostad et al. [21] did not investigate the molecular mechanism of these effects, they postulate that the effects may be mediated by factors such as direct effects on the virus, effects on local genital tract physiology or effects on immune modulation of viral replication, or a combination of these effects. Interestingly, an animal study showed that MPA increased susceptibility to vaginal simian–human immunodeficiency virus (SHIV) transmission and suppressed the antiviral cellular immune response in SHIV-infected rhesus macaques [23], indicating an immune- rather than a transmission-based mechanism. In another animal study, it was shown that MPA treatment at contraceptive doses rendered mice 100-fold more susceptible to genital HSV-2 infection compared to untreated mice [24]. In addition, the use of MPA has been associated with increased acquisition of cervical chlamydial and gonococcal infections [25]. Whether or not NET is associated with an increased risk of HIV/HSV acquisition and shedding, viral load and viral diversity remains to be determined. In this regard, two recent studies in a cohort of South African women showed no association between risk of HIV infection and NET-EN at contraceptive doses [26], [27]. However, a recent re-analysis of earlier data has shown that MPA used as an injectable contraceptive (referred to in this case as Depo-MPA or DMPA) is associated with an increase in HIV acquisition in women [28], [29].

To date, very little is known about the molecular mechanisms of action of MPA and NET on immune function, in particular the target cells, target genes and dose responses. MPA has been reported to modulate transcription of a number of genes via the glucocorticoid receptor (GR), for example, IL-2 in normal human lymphocytes [30], IL-6 and IL-8 in a mouse fibroblast cell line [31] and the nm-23 tumor suppressor gene in a breast cancer cell line [32]. In contrast, MPA's suppression of the RANTES gene in endometrial cells was progesterone receptor (PR) mediated [33]. Interestingly, NET-A is not an agonist for transactivation via the GR and only marginally (22%) transrepressed an IL-8 reporter at 10 μM [34]. Furthermore, MPA has been shown to regulate a number of genes via the PR and the androgen receptor (AR) in human breast cancer cell lines [35]. In contrast to the data available for MPA, much less is known about the biological activity of NET via the AR. However, the recent study by Sasagawa et al. [36] has now characterized both MPA and NET pharmacologically in terms of potency for transactivation via the AR. Furthermore, both MPA and NET-A have been reported to bind to the mineralocorticoid receptor (MR) with low relative affinity and do not display any agonist or antagonist properties towards the MR [37], [38]. It is thus apparent that even though MPA and NET were developed for the similarity of their biological actions to those of progesterone (Prog), mediating their effects by binding to the PR, they can also initiate a diverse range of biological effects by cross-reacting with other members of the steroid receptor family such as the GR, the AR and possibly the MR [34], [39], [40], [41], [42].

An important question is thus whether, and to what extent, MPA, and also NET-A, regulates known pro-inflammatory mediators such as IL-6, IL-8 and RANTES, in the cervicovaginal mucosa. Understanding the mechanisms of this regulation and the receptors involved would further our understanding of differential gene regulation by different progestins and assist in the design of new progestins with fewer side effects. Investigating these mechanisms at a site relevant to infections, such as the cervicovaginal environment, is likely to be relevant to mucosal immunity. Factors that affect immunity in the cervicovaginal environment may be important determinants of transmission risk of pathogens such viruses, and understanding these factors may shed light on molecular events occurring during infections. In the light of the above, our strategy was to investigate the effects of MPA and NET-A relative to Prog on mucosal immunity in an in vitro cell culture model of the female cervicovaginal environment, by comparing their effects on the regulation of the endogenous pro-inflammatory cytokine/chemokine genes IL-6, IL-8 and RANTES. Two epithelial cell lines generated from normal human vaginal (Vk2/E6E7) and ectocervical (Ect1/E6E7) cells, immortalized by expression of the E6 and E7 genes of human papillomavirus type 16 [42], were used as model systems.

Section snippets

Inducing compounds

4-Pregnene-3,20-dione (Prog), 6α-methyl-17α-hydroxy-progesterone acetate (MPA), 17α-ethynyl-19-nortestosterone 17β-acetate (NET-A), 5α-androstan-17β-ol-3-one [dihydrotestosterone (DHT)], 11β-(4-dimethylamino)phenyl-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one [mifepristone (RU486)], 11β,16α-9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione [dexamethasone (Dex)], 11β,21-dihydroxy-3,20-dioxo-4-pregnen-18-al [aldosterone (Ald)], 17β-estra-1,3,5 (10)-triene-3,17-diol [β-estradiol

MPA and NET-A, unlike Prog, exhibit differential patterns of gene regulation on pro-inflammatory chemokines

Human Ect1/E6E7 and Vk2/E6E7 cell lines were treated for 24 h with 0.02 mcg/mL TNF-α and 1 μM Prog, MPA or NET-A, followed by real-time quantitative RT-PCR (QPCR) analysis for expression of the IL-6, IL-8 and RANTES genes, respectively. In both the Ect1/E6E7 and Vk2/E6E7 cell lines, results show that unlike Prog which up-regulates IL-6 gene expression, both MPA and NET-A have no effect (Fig. 1A and 1B). In contrast, MPA and NET-A differentially regulate both the IL-8 (Fig. 2A) and RANTES (Fig. 3

Discussion

Inflammation of the lower human female genital tract increases susceptibility to viral infections such as HIV [61], [62] and human papilloma virus [63]. In addition, excessive release of proinflammatory cytokines may alter the mucosal immune function [64], [65]. Thus, understanding factors that may influence the local mucosal immune response, such as endogenous hormones or hormonal contraception, is crucial, especially since the cervicovaginal mucosa is the primary site of HIV-1 infection

Acknowledgments

We thank Carmen Langeveldt for technical support. This work supported by grants to JPH and DA from the medical Research Council (MRC) and the National Research Foundation (NRF) in South Africa, and Stellenbosch University. Any option, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the NRF does not accept any liability in regard thereto.

References (86)

  • S. Palacios et al.

    Advances in hormone replacement therapy with drospirenone, a unique progestogen with aldosterone receptor antagonism

    Maturitas

    (2006)
  • J.M. Bentel et al.

    Androgen receptor agonist activity of the synthetic progestin, medroxyprogesterone acetate, in human breast cancer cells

    Mol Cell Endocrinol

    (1999)
  • J.P. Hapgood et al.

    Not all progestins are the same: implications for usage

    Trends Pharmacol Sci

    (2004)
  • A.O. Brinkmann et al.

    Mechanisms of androgen receptor activation and function

    J Steroid Biochem Mol Biol

    (1999)
  • P. Kastner et al.

    Transient expression of human and chicken progesterone receptors does not support alternative translational initiation from a single mRNA as the mechanism generating two receptor isoforms

    J Biol Chem

    (1990)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal Biochem

    (1976)
  • K. Gross et al.

    Tissue-specific glucocorticoid action: a family affair

    Trends Endocrinol Metab

    (2008)
  • E.W. Bergink et al.

    Binding of progestagens to receptor proteins in MCF-7 cells

    J Steroid Biochem

    (1983)
  • G.H. Deckers et al.

    Influence of the substitution of 11-methylene, delta(15), and/or 18-methyl groups in norethisterone on receptor binding, transactivation assays and biological activities in animals

    J Steroid Biochem Mol Biol

    (2000)
  • K. Ronacher et al.

    Ligand-selective transactivation and transrepression via the glucocorticoid receptor: role of cofactor interaction

    Mol Cell Endocrinol

    (2009)
  • M.L. Cho et al.

    Transforming growth factor beta 1(TGF-beta1) down-regulates TNFalpha-induced RANTES production in rheumatoid synovial fibroblasts through NF-kappaB-mediated transcriptional repression

    Immunol Lett

    (2006)
  • L. Vangelista et al.

    Rational design of novel HIV-1 entry inhibitors by RANTES engineering

    Vaccine

    (2008)
  • R.K. Mangal et al.

    Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle

    J Steroid Biochem Mol Biol

    (1997)
  • W.G. Schoonen et al.

    Hormonal properties of norethisterone, 7alpha-methyl-norethisterone and their derivatives

    J Steroid Biochem Mol Biol

    (2000)
  • Q. Zhao et al.

    Receptor density dictates the behavior of a subset of steroid ligands in glucocorticoid receptor-mediated transrepression

    Int Immunopharmacol

    (2003)
  • M. Schulz et al.

    RU486-induced glucocorticoid receptor agonism is controlled by the receptor N terminus and by corepressor binding

    J Biol Chem

    (2002)
  • G. Perez-Palacios et al.

    Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

    J Steroid Biochem

    (1983)
  • G. Perez-Palacios et al.

    Mechanism of action of contraceptive synthetic progestins

    J Steroid Biochem

    (1981)
  • D. Africander et al.

    Molecular mechanisms of steriod receptor-mediated actions by synthetic progestins used in HRT and contraception

    Steriods

    (2011)
  • R.N. Fichorova et al.

    Differential expression of immunobiological mediators by immortalized human cervical and vaginal epithelial cells

    Biol Reprod

    (1999)
  • J.E. Cummins et al.

    Biomarkers of cervicovaginal inflammation for the assessment of microbicide safety

    Sex Transm Dis

    (2009)
  • R.N. Fichorova et al.

    Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells

    Infect Immun

    (2001)
  • M.F. Press et al.

    Estrogen receptor localization in the female genital tract

    Am J Pathol

    (1986)
  • W.H. Kutteh et al.

    Mucosal immunity in the female reproductive tract: correlation of immunoglobulins, cytokines, and reproductive hormones in human cervical mucus around the time of ovulation

    AIDS Res Hum Retroviruses

    (1998)
  • A.J. Quayle et al.

    Gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract

    Am J Pathol

    (1998)
  • C.R. Wira et al.

    Innate and adaptive immunity in female genital tract: cellular responses and interactions

    Immunol Rev

    (2005)
  • J. van de Wijgert et al.

    Vaginal microbicides: moving ahead after an unexpected setback

    AIDS

    (2007)
  • R. Fichorova

    Guiding the vaginal microbicide trials with biomarkers of inflammation

    J Acquir Immune Defic Syndr

    (2004)
  • P. Borrow et al.

    Innate immunity against HIV: a priority target for HIV prevention research

    Retrovirology

    (2010)
  • F. Zara et al.

    Markers of local immunity in cervico-vaginal secretions of HIV infected women: implications for HIV shedding

    Sex Transm Infect

    (2004)
  • L.M. Bebell et al.

    Relationship between levels of inflammatory cytokines in the genital tract and CD4+ cell counts in women with acute HIV-1 infection

    J Infect Dis

    (2008)
  • E. Rakasz et al.

    Female sex hormones as regulatory factors in the vaginal immune compartment

    Int Rev Immuno

    (2002)
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