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