More MS news articles for April 2002

Gender Issues and Multiple Sclerosis

http://current-reports.com/cr_abstract_frmst.cfm?PubID=NR02-3-2-03

Current Neurology and Neuroscience Reports 2002 2: 277-286
Rhonda R Voskuhl MD
UCLA Department of Neurology, Room A-145 710 Westwood Plaza, Reed Neurological Research Center, Los Angeles, CA, 90024, USA

Abstract

Gender-related issues in multiple sclerosis include the important and widely accepted clinical observations that men are less susceptible to the disease than women and also that disease activity in multiple sclerosis is decreased during late pregnancy. This article reviews mechanisms underlying each of these clinical observations and discusses the role of sex hormones in each. Specifically, the protective role of testosterone in young men and the protective role of the pregnancy hormone estriol in pregnant women are discussed. Rationale for novel therapies in multiple sclerosis based on the protective roles of these sex hormones is presented.

Introduction

Two gender issues in multiple sclerosis (MS) are reviewed: 1) the greater susceptibility of women as compared with men to MS; and 2) the improvement in MS disease activity during pregnancy. Mechanisms underlying each of these issues are discussed with regard to the contribution of sex hormones to disease protection and actions of sex hormoneson the immune system. Novel therapeutic approaches using sex hormones for treatment in MS to recapitulate protection observed clinically are explored.

The Greater Susceptibility of Women as Compared with Men

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) characterized by demyelination of axons in inflammatory plaques. This demyelination results acutely in conduction slowing or block with clinical deficits in the functions served by the axons. The majority of patients have either relapsing-remitting (RR) MS or secondary progressive (SP) MS. The RR phase is characterized by repeated episodes of relapses and remissions, with relapses corresponding to a flare in inflammatory CNS lesions. The RR phase is thought to be an earlier stage of disease than the SP phase because, over the course of years to decades, many RR patients eventually transition to SPMS, whereby relapses are no longer associated with full or partial recovery. In the SP phase, gradual disability accumulates with no remissions. Inflammation is more prevalent in the RR phase, and axonal pathology more predominant in the SP phase [1,2]. RRMS and SPMS are thought to be a continuum of two phases of the same disease. In contrast, primary progressive (PP) MS is thought to be a distinct entity from RRMS and SPMS. PPMS is characterized by disability accumulation from the onset. Deficits are usually referable to pathology in the spinal cord, and fewer lesions occur in the brain. Indeed, evidence suggests that PPMS differs from RRMS and SPMS pathologically, immunologically, and genetically [3]. Interestingly, as is the case with numerous other autoimmune diseases, there is a female preponderance of RRMS (female to male ratio of 3:1), whereas in PPMS, men are affected as frequently as women are [8,9]. In this review, we address mechanisms underlying the female preponderance in MS and, therefore, focus on RRMS, not PPMS.

As mentioned earlier, during reproductive ages there is a distinct female preponderance of numerous autoimmune diseases, including not only RRMS, but also rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), myasthenia gravis (MG), Sjogren's syndrome, and Hashimoto's thyroiditis. Sex hormones and/or sex-linked gene inheritance may be responsible for the enhanced susceptibility of women to these autoimmune diseases. A role for sex hormones in susceptibility to autoimmune disease is supported by observations of changes in disease symptomatology with alterations in sex hormone levels during pregnancy, menopause, or exogenous hormone administration in the form of hormone replacement therapy or oral contraceptives [10]. Further, whereas the onset of MS in women tends to be relatively early (ages 18 to 30), the onset of MS in men tends to be relatively later in life (ages 30 to 40), a time coinciding with the beginning of the decline in bioavailable testosterone in healthy men [20]. This latter observation suggests that high physiologic levels of testosterone in young men may be protective in MS.

Mechanisms Underlying Decreased Susceptibility of Men to Multiple Sclerosis: Lessons from Animal Models

Experiments in animal models of a variety of autoimmune diseases have demonstrated a protective effect of testosterone. In spontaneous diabetes in the NOD mouse, female mice are more severely and more frequently affected than male mice. Testosterone treatment of female mice has lowered the incidence of disease, and castration of male mice has increased the incidence of disease [26]. In thyroiditis and adjuvant-induced arthritis, castration of male mice has been shown to increase the incidence and severity of disease, whereas testosterone treatment has been shown to be protective [27,28]. In a model of Sjogren's syndrome in female MRL/Mp-lpr/lpr mice, testosterone treatment resulted in a reduction in lymphocyte infiltration into lacrimal tissue [29,30]. Finally, spontaneous SLE in NZBxNZW mice affects female mice more frequently than male mice. In this model, testosterone has also been shown to improve survival [31]. Over the past several years, experimental autoimmune encephalomyelitis (EAE), the murine model for MS, has been studied with regard to gender differences in susceptibility.

Male gender confers disease protection in both the induction and the effector phase of experimental autoimmune encephalomyelitis

Gender differences in susceptibility to EAE have been shown to be important in the effector phase of disease by transferring myelin basic protein (MBP)-specific T cells derived from females into recipients who were female or male. A more severe disease course resulted in female recipients [32]. Also, female recipients, as compared to male recipients, were more severely affected when MBP- specific T cells were derived from males [32]. Gender differences were shown to be important in the induction phase of the disease as well by comparing the ability of cells derived from female versus male donors to transfer disease. More cells were needed to transfer disease when MBP-specific cells were derived from female donors as compared with male donors. Together, these observations demonstrated that male mice, as compared with female mice, are relatively less susceptible to disease when one considers either the induction or the effector phase of disease. However, male mice are not completely protected from disease, for if high enough cell numbers are adoptively transferred, male-derived cells can indeed induce disease and male recipients can indeed manifest disease [32]. These findings in adoptive EAE using whole MBP as antigen are consistent with data showing a more severe disease course of female as compared with male SJL mice using active EAE induced with whole MBP [35], and data demonstrating that female SJL mice are more severely affected with EAE during the relapsing phase when PLP 139-151 peptide is used to induce active EAE [36].

Male and female mice differ with respect to cytokine production

Why might male SJL mice be less susceptible to EAE than female SJL mice? Previously, when sex hormones were added in vitro to immune cells they were shown to alter cytokine production. Therefore, several groups have focused on the possibility that differences in sex hormones between females and males might influence cytokine production and ultimately alter the balance between T-helper 1 (Th1)- and Th2-type lymphocyte responses. Th1 lymphocyte responses are characterized by the production of interferon γ (IFN-γ) and are preferentially selected for when T lymphocytes are stimulated in the presence of interleukin (IL)-12. Th2 lymphocyte responses are characterized by the production of IL-10, IL-4, IL-5, and IL-13 and are preferentially selected for when T lymphocytes are stimulated in the presence of IL-4. EAE is mediated by myelin protein-specific T lymphocytes of the Th1 type [37,38]. On the other hand, the Th2 cytokine IL-10 is thought to be protective in EAE. This is evidenced by observations that IL-10 has been previously shown to down-regulate Th1 responses [19], to be up-regulated during the remission phase of EAE [39], and to ameliorate EAE [40,41]. Anti-IL-10 antibody treatment exacerbates EAE [42], and IL-10 knockout mice fail to go into remission [43].

To pursue whether gender differences in EAE might be related to differences in cytokine production, myelin basic protein (MBP)-specific T-lymphocyte responses from cultured splenocytes of female and male mice with EAE were assessed for production of Th1 and Th2 cytokines. When the induction phase was studied independently of the effector phase, it was found that male responses were characterized by decreased Th1 cytokine (IL-12 and IFN-γ) production, with no difference in Th2 cytokine production [34]. On the other hand, during the effector phase, higher levels of IL-10 production in MBP-stimulated splenocytes from male (as compared with female) mice were observed, whereas there were no significant differences in IFN-γ [33]. Finally, when the active EAE model (which combines the induction and the effector phases) was used, it was found that male antigen-specific immune responses were characterized by both less Th1 cytokine (IFN-γ) and greater Th2 cytokine (IL-10 and IL-4) production [35,36].

Treatment of female mice with androgens can recapitulate disease protection and alter cytokine production

In identifying a sex hormone that might be responsible for the decrease in disease severity in male SJL mice, testosterone was a leading candidate because testosterone treatment had been shown to be protective in numerous other autoimmune diseases [44], and also when male mice were castrated disease protection was lost [45,4646.

Testosterone can be converted in vivo to either dihydrotestosterone (DHT) or estradiol (E2). Thus, it remained unclear whether the protective effect of testosterone in the previous studies was due to androgen binding to androgen receptors or due to testosterone's conversion to estradiol with subsequent binding to estrogen receptors. Thus, in initial studies addressing the possible protective role of testosterone, a pure androgenic effect was maintained by using used DHT for treatment. DHT, unlike testosterone, cannot be converted to estradiol in vivo. Treatment of naive, SJL/J female mice with 90-day release DHT pellets 1 week prior to the adoptive transfer of MBP-specific T lymphocytes consistently reduced the severity of clinical EAE as compared with naive SJL/J female control mice implanted in parallel with placebo pellets (1) [33]. Interestingly, treatment of female EAE mice with a lower dose (5 mg) of DHT was not significantly different than treatment with placebo.

It was then determined whether the previously observed increased IL-10 production in males might be recapitulated when female mice were treated with DHT. Cytokine production was compared in EAE females treated with DHT versus placebo. IL-10 levels from splenocytes of DHT-treated females with EAE were significantly higher than those from placebo-treated females, whereas IFN-γ production was slightly lower [33]. Thus, both the decrease in disease severity and the increase in IL-10 production observed in males as compared with females were recapitulated in DHT-treated females as compared with placebo-treated females.

Increased interleukin-10 production during dihydrotestosterone treatment is derived primarily from CD4 T lymphocytes within spleen

Molecular mechanisms underlying how treatment with androgens caused the increase in IL-10 production from antigen-stimulated splenocytes were then addressed. Because differences in cytokine production induced by treatment with androgens should not be limited to immune responses specific for myelin proteins, gender differences in cytokine production upon stimulation with antibody to CD3 (α-CD3) were examined. Splenocytes from DHT-treated female mice produced more IL-10 and less IL-12 than those from placebo-treated female mice. IL-12 knockout mice were then used to determine whether changes in IL-10 production were mediated directly by testosterone versus indirectly by changes in IL-12. DHT treatment of female IL-12 knockout mice resulted in increased IL-10 production, thereby indicating that the testosterone--mediated increase in IL-10 production was not dependent upon a testosterone-mediated alteration in Il-12 production. In order to begin to delineate the mechanism by which DHT may be increasing IL-10 production in splenocytes, the cellular source of IL-10 was determined. At both the RNA and protein levels, IL-10 was produced primarily by CD4 T lymphocytes. CD4 T lymphocytes were then shown to express the androgen receptor, raising the possibility that testosterone could act directly on CD4 T lymphocytes to increase IL-10 production. In vitro experiments demonstrated increased IL-10 production following treatment of CD4 T lymphocytes with DHT. Thus, testosterone was shown to act directly via androgen receptors on CD4 T lymphocytes to increase IL-10 gene expression [47]. Therefore, at least one mechanism through which DHT treatment can induce an increase in IL-10 production from stimulated splenocytes is via a direct effect of DHT binding to androgen receptors expressed on CD4 T cells, with subsequent increased IL-10 production from within the same cell (2).

Could Supplemental Testosterone Treatment be Used to Treat Female Multiple Sclerosis Patients?

Because it had been shown in numerous animal models of autoimmune disease that treatment of female mice with androgens resulted in disease protection, further experiments in EAE were done to ascertain whether this might be feasible in MS.

Treatment with dihydrotestosterone after disease onset ameliorates subsequent experimental autoimmune encephalomyelitis

In the majority of EAE experiments in the literature, treatments are instituted before disease induction. This was also the case in experiments with DHT. Unfortunately, MS patients do not present for treatment before the onset of disease; they present after the onset of disease. Thus, to determine whether treatment with DHT might ameliorate EAE after disease onset, MBP-specific T cells were adoptively transferred to naive mice, then at disease onset (day 10) mice were stratified according to grade into two equal groups. One group was then treated with a 15-mg pellet of DHT and the other with placebo. By day 20 post-adoptive transfer (day 10 after DHT treatment), the disease course had become less severe in DHT-treated EAE mice as comparedwith placebo treated mice (1).

The dose of dihydrotestosterone needed to ameliorate experimental autoimmune encephalomyelitis in female mice is supraphysiologic

Because it had previously found that treatment of female EAE mice with a high dose (15 mg) of DHT ameliorated EAE whereas treatment with a lower dose (5 mg) of DHT did not, the level of serum testosterone (DHT and testosterone) in females treated at each dose (5 mg and 15 mg) was ascertained and compared with levels present in serum of placebo-treated male mice [47]. As expected, levels of androgens were significantly higher in placebo-treated males as compared with placebo-treated females. Interestingly, the females treated with 5 mg of DHT had serum androgen levels that were not significantly different from the physiologic range in placebo-treated males, whereas the 15-mg DHT pellet treatment yielded levels that were higher than those in placebo-treated males. These data indicated that in order to ameliorate EAE in female mice, DHT doses that induced androgen levels significantly higher than those present normally in male mice would be required. Implications for therapy of MS were that very high doses of androgens would be needed for effective treatment of women with MS. Such high levels would likely not be well tolerated in the long term in women due to masculinizing side effects such as hirsuitism and changes in voice.

Could Supplemental Androgen Treatment be Used in Men with Multiple Sclerosis?

Dihydrotestosterone and testosterone treatment can ameliorate experimental autoimmune encephalomyelitis in young adult, hormonally intact male mice

Because high doses of DHT would not be tolerated in the majority of female patients with MS due to the masculinizing side effects, it was then determined whether supplemental testosterone treatment might be beneficial in male patients. The observation that the onset of MS in men coincided with a time when androgen levels begin to decline (ages 30 to 40) suggested that supplemental androgen treatment might be beneficial in men with MS. Young adult, hormonally intact male EAE mice were treated with DHT at a dose of 15 mg. Clinical courses were less severe in male mice treated with DHT 15-mg pellets as compared with those treated with placebo pellets [46]. Because testosterone had been used more extensively in humans than DHT, consideration of treatment of male MS patients with testosterone (as opposed to DHT) was explored. When young adult, hormonally intact male SJL mice were treated with 15-mg pellets of testosterone, clinical scores were again improved as compared with those treated in parallel with placebo [46]. In order to determine whether a lower dose of DHT or testosterone might be efficacious, EAE was then induced in a group of male mice that were treated in parallel with either 5 mg of DHT or 5 mg of testosterone. Both 5 mg of DHT and 5 mg of testosterone ameliorated EAE [46]. Together, these data indicated that supplemental treatment with either testosterone or DHT can ameliorate EAE in young adult, hormonally intact male mice when used at a wide range of doses (from physiologic replacement to supraphysiologic). Further, because treatment with physiologic doses of testosterone reduced the severity of disease in young adult, hormonally intact male mice, this indicated that the beneficial effect of supplemental testosterone treatment was not dependent upon having a low baseline testosterone level, and that hormonally intact men with MS should benefit from supplemental pharmacologic doses of testosterone.

Experimental autoimmune encephalomyelitis in C57BL/6 male mice is ameliorated by androgen treatment

Not all strains of mice demonstrate decreased disease susceptibility in males as compared with females. Unlike the SJL, the B10.PL mouse was shown to have no gender difference in disease [48], and we have found there was also no gender difference in EAE in C57BL/6 mice (Palaszynski and Voskuhl, unpublished data). The lack of a decrease in male as compared with female susceptibility in a given strain would suggest that endogenous physiologic testosterone levels might not be protective in some strains. A lack of a protective effect of endogenous testosterone in a given strain would not, however, preclude a protective effect of supplemental testosterone treatment in that given strain. Hence, it was next determined whether the protective effect of supplemental androgen treatment in young adult male mice was a phenomenon unique to the SJL strain or whether it could also be observed in myelin oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6 mice. Active EAE was induced with MOG peptide 35-55 in young adult C57BL/6 male mice treated with either 5-mg DHT pellets or placebo pellets. Treatment of young adult, hormonally intact male mice of the C57BL/6 background with a physiologic low dose (5 mg) of DHT resulted in an amelioration of disease severity (3) (Palaszynski and Voskuhl, unpublished data). These data indicated that even in strains of EAE mice not characterized by a gender difference, supplemental treatment with androgens can mediate protection. Thus, supplemental androgen treatment should be beneficial in men with MS across various genetic backgrounds.

Together, the above results serve as a basis for a preliminary trial of supplemental testosterone treatment in men with RRMS.

The Improvement in Multiple Sclerosis Disease Activity During Pregnancy

A second major gender-related issue in MS is that MS disease activity is improved during pregnancy. Decades of observations have revealed that MS improves with late pregnancy [10,49,50]. There was a period of relative "safety" with regard to relapses during pregnancy followed by a period of increased relapses postpartum. Other studies found that in addition to having a decrease in disease activity in patients with established MS, the risk of developing the first episode of MS was decreased during pregnancy as compared with nonpregnant states [51]. The most definitive study of the effect of pregnancy and the post-partum period involved a study in which relapse rates were determined in 254 women with MS during 269 pregnancies and for up to 1 year after delivery. Relapse rates were significantly reduced during the third trimester as compared with the year prior to pregnancy. Rates then increased during the first 3 months postpartum before returning to prepregnancy rates. No significant changes were observed between relapse rates in the first and second trimester as compared with the year prior to pregnancy [52]. Also, a small study of two patients who underwent serial cerebral magnetic resonance imaging (MRI) scans during pregnancy and postpartum demonstrated that there was a decrease in T2-lesion number during the second half of pregnancy and a return to prepregnancy levels in the first months postpartum [53]. Together, these reports demonstrated that the latter part of pregnancy is associated with a significant reduction in disease activity.

Mechanisms underlying the improvement in multiple sclerosis disease activity during pregnancy

Although an improvement in disease activity during pregnancy in MS could be due to an effect of pregnancy factors on either the CNS or on the immune system, the fact that pregnancy was known to be associated with an improvement in other autoimmune diseases, such as rheumatoid arthritis [11,12,54,55], suggested that the effect of pregnancy on MS disease activity was mediated, at least in part, through effects on the immune system. Pregnancy is a challenge for the immune system. From the mother's standpoint, the fetus is an allograft because it harbors antigens inherited by the father. It is evolutionarily advantageous for the mother to transiently suppress cytotoxic, cell-mediated, Th1-type immune responses involved in fetal rejection during pregnancy. However, not all immune responses should be suppressed because humoral, Th2-type immunity is needed for passive transfer of antibodies to the fetus. Indeed, a shift in immune responses with a down-regulation of Th1 and an up-regulation of Th2 has been shown to be necessary for fetal survival [19,56]. This shift in immune responses from Th1 to Th2 occurs both locally at the maternal fetal interface [19,61,62] as well as systemically [57,59,63]. The physiologic importance of this shift in the systemic immune response has been demonstrated by a significant alteration in the response to systemic infection in mice during pregnancy [66]. It thus became highly plausible that the systemic shift away from Th1 and toward Th2 during pregnancy might underlie the improvement in Th1-mediated autoimmune diseases, such as MS and RA, during this time.

Pregnancy factors as candidates for the improvement in disease activity

Numerous factors, including sex hormones, cortisol, vitamin D, α-feto protein, early pregnancy factor, human chorionic gonadotropin, and pregnancy-specific glycoproteins have been identified in sera during pregnancy and have been shown to be immunosuppressive either in cultures of immune cells in vitro or in animal models in vivo. Hence, numerous factors have been proposed as possibly contributing to disease protection during pregnancy. The two key issues that should be considered when one weighs the possibility of whether a candidate factor is or is not likely to be responsible for the decrease in disease relapse in MS include the following: 1) is the factor increased early or late during pregnancy? and 2) are doses of the factors used in the in vitro and in vivo models to demonstrate an immunosuppressive effect associated with levels of the factor that are similar to what occurs during natural pregnancy? Regarding the first issue, if a factor is increased only transiently very early during pregnancy, then it seems unlikely that it would be responsible for disease activity reduction in the third trimester followed by postpartum relapse. On the other hand, if a factor gradually increases in concentration to peak in the last trimester followed by a precipitous drop postpartum, then it seems more likely that this factor might be responsible for alterations in the disease course. Regarding the second issue, if the factor is immunosuppressive only at a dose that is much higher than what occurs during pregnancy, then it would be unlikely that the factor is responsible for disease amelioration during pregnancy. On the other hand, if the factor is immunosuppressive at doses that are similar to those which occur during pregnancy, then it is likely that the factor may be responsible, at least in part, for the immunosuppression and disease-activity alteration during pregnancy.

Pregnancy Hormones as Candidate Factors for the Improvement in Disease Activity During Pregnancy

For many of the proposed factors that are candidates for mediating the disease improvement during pregnancy, these two key issues above have not been fully addressed; therefore, it becomes difficult to ascertain what the contribution of each factor might be to the improvement in diseaseactivity in MS during pregnancy. On the other hand, these issues have been addressed in the case of pregnancy hormones as discussed in the following text.

Pregnancy hormones (estrogens and progesterone) in animal models of multiple sclerosis and rheumatoid arthritis

It was shown over a decade ago that pregnancy was protective in EAE in guinea pigs, rats, and rabbits [10]. More recently, it was shown that relapsing-remitting EAE in SJL mice improved during late pregnancy [46]. Because estrogens and progesterone increase progressively during pregnancy to the highest levels in the third trimester, these hormones were candidates for possibly mediating a protective effect. Initially it was shown that treatment of B10.RIII mice with either estriol or estradiol delayed the onset of actively induced EAE [67] and collagen-induced arthritis [67]. Importantly, a decreased incidence of disease occurred when estriol was used at doses to induce serum levels that were physiologic with pregnancy. On the other hand, estradiol needed to be used at doses several-fold higher than what would occur naturally during pregnancy in order to induce the same degree of disease protection as estriol [67]. Studies in the relapsing-remitting model of EAE in SJL mice initially focused on investigations of estriol as the estrogen of pregnancy that might underlie disease protection. It was found that SJL mice treated with estriol had less severe disease as compared with those implanted with placebo pellets when identical MBP-specific cells were adoptively transferred (4) [70]. Various doses of estriol were used. There tended to be greater disease protection when higher doses were used. However, a dose as low as one that would yield a serum level of estriol in the blood that approximated that observed during natural pregnancy was indeed capable of ameliorating disease. In contrast, there was no protection observed when various doses of progesterone were used, ranging from pregnancy doses to greater than pregnancy doses. The lack of a protective effect in EAE when progesterone alone was used had been previously observed in collagen--induced arthritis [71]. Further, it was then shown that treatment with estriol could result in significantly decreased disease severity, even if it was administered after the onset of clinical signs of EAE (4) [70].

Pathologic studies of EAE mice treated with estriol revealed decreased inflammation and demyelination in spinal cord sections as compared with placebo-treated mice [70]. As discussed previously, a systemic shift in immune responses from Th1 to Th2 had been described during pregnancy [19,44,66]. Therefore, it was next determined whether treatment of nonpregnant mice with estriol could recapitulate some of the immune changes of pregnancy [70]. It was found that mice with EAE who had been treated with estriol had increased MBP-specific immunoglobulin (Ig)G1 antibodies in serum as compared with mice with EAE who had been treated with placebo. It was also found that splenocytes from estriol-treated EAE mice had greater IL-10 production upon stimulation with MBP than those from placebo-treated mice. Finally, it was demonstrated that the cellular source of the IL-10 production in splenocytes of estriol-treated EAE mice was primarily the T-cell population [70]. This increase in IL-10 production by T lymphocytes within spleen upon in vivo estriol treatment was consistent with the increase in IL-10 production that had been observed by others when human T lymphocytes were treated in vitro with pregnancy doses of estrogens [72,73]. Thus, it appeared that estriol did indeed recapitulate, at least in part, some of the immune changes of pregnancy.

More recent studies confirmed that not only estriol, but also estradiol, could ameliorate EAE if given in sustained doses that are sufficiently high [46,74]. Although it is clear that very high doses of estradiol are protective in EAE, it has not yet been clearly established whether low estradiol doses are protective. Some reports have found that ovariectomy of female mice makes EAE severity worse [75], whereas others have found that ovariectomy does not have a significant effect on disease [46]. Thus, it is controversial whether low levels of endogenous estradiol have a significant influence on EAE. Levels of estrogen that are lower than that which occurs during pregnancy, such as levels induced by doses used in oral contraceptives or hormone replacement therapy, may or may not be high enough to be protective in MS. It is not surprising that past use of oral contraceptives in healthy women would have no effect on subsequent risk to develop MS, because one would not anticipate that the effect of treatment on the immune system would be permanent [76]. This data in and of itself would not exclude the possibility that the use of oral contraceptives could have a temporary protective effect on disease in women with MS during use. However, in a very large study it was found that the incidence rates for MS in current users were not decreased as compared with never-users [77]. This latter observation would suggest that the estrogens in oral contraceptives are not of sufficient type or dose to ameliorate the immunopathogenesis of MS even temporarily during intercurrent use. This conclusion is supported by disappointing results in studies of hormone replacement therapy and effects on disease activity in rheumatoid arthritis [13]. It is likely that a sustained level of a sufficient dose of an estrogen, creating an estrogen profile similar to that of late pregnancy, will be necessary to ameliorate disease activity in MS and RA.

Pregnancy doses of estriol used in a pilot clinical trial in multiple sclerosis

Observations in animal models of Th1-mediated auto-immune diseases had indicated that estriol was a strong candidate sex hormone for mediating disease protection during pregnancy [67,70,74]. Thus, estriol was administered in the form of a pilot clinical trial to women with MS in an attempt to recapitulate this protective effect on disease [78]. A crossover study was used whereby patients were followed for 6 months pretreatment to establish baseline disease activity that included cerebral MRI every month and neurologic exam every 3 months. The patients were then treated with oral estriol (8 mg/d) for 6 months with the same parameters being followed, then observed for 6 more months in the post-treatment period. There were six RR patients and four SP patients who finished the entire 18-month study period. The oral estriol dose resulted in serum estriol levels that approximated levels observed in untreated, healthy control women who were 6 months pregnant. As had been previously observed when estriol was given for hormone replacement therapy [79,80], treatment was very well tolerated, with only menstrual cycle abnormalities. Interestingly, a significant decrease in the delayed-type hypersensitivity response (to the recall antigen tetanus) was observed at the end of the 6-month treatment period as compared with the pretreatment period. Further, levels of IFN-γ were found to be significantly lower in unstimulated peripheral blood mononuclear cells by reverse transcriptase-polymerase chain reaction testing at the end of the 6-month treatment period as compared with baseline pretreatment in the RR patients. On serial MRIs, the RR patients demonstrated a significant reduction in gadolinium-enhancing lesions during treatment as compared with pretreatment, whereas the SP patients demonstrated no significant change. Importantly, gadolinium-enhancing disease activity gradually returned to baseline in the post-treatment period. Finally, treatment with estriol was reinstituted in the RR patients in an extension phase of the study, and again enhancing lesions were decreased. As expected, relapse rates and disability were unaffected in this trial of very short duration [78]. Based on the results of this pilot trial, a double-blind, placebo-controlled trial of oral estriol in RRMS is planned (Voskuhl, unpublished data).

Conclusions

There are two gender issues in multiple sclerosis in which a mechanistic understanding could potentially lead to a novel therapy in MS. First, young males are relatively protected from disease as compared with older males and females. This appears to be due at least in part to a protective effect of testosterone. Thus, supplemental testosterone therapy should be considered as a possible therapy for men with MS. Second, there is a decrease in relapse rates in women during the third trimester of pregnancy. High levels of the pregnancy hormone estriol appear to contribute to this disease protection during this time. Thus, treatment with estriol to establish a level physiologic with pregnancy should be considered for women with relapsing-remitting MS.

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