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A Gender Gap in Autoimmunity
Caroline C. Whitacre, Stephen C. Reingold, Patricia A. O'Looney, Elizabeth Blankenhorn, Floyd Brinley, Elaine Collier, Pierre Duquette, Howard Fox, Barbara Giesser, Wendy Gilmore, Robert Lahita, J. Lee Nelson, Carol Reiss, Peter Riskind, and Rhonda Voskuhl

Supplementary Material

Sex Differences in Autoimmune Disease:
Focus on Multiple Sclerosis

The Task Force on Gender, Multiple Sclerosis and Autoimmunity

Caroline C. Whitacre (Ohio State University, Columbus, OH)
Elizabeth Blankenhorn (Allegheny University of the Health Sciences, Philadelphia, PA)
Floyd J. Brinley, Jr. (National Institutes of Health, Bethesda, MD)
Elaine Collier (National Institutes of Health, Bethesda, MD)
Pierre Duquette (Hopital Notre-Dame, Montreal Canada)
Howard Fox (Scripps Research Institute, La Jolla, CA)
Barbara Giesser (University of Arizona, Tucson, AZ)
Wendy Gilmore (University of Southern California, Los Angeles, CA)
Robert Lahita (Columbia University, New York, NY)
J. Lee Nelson (University of Washington, Seattle, WA)
Patricia A. O'Looney (National Multiple Sclerosis Society, New York NY)*
Stephen C. Reingold (National Multiple Sclerosis Society, New York, NY)
Carol S. Reiss (New York University, New York, NY)
Peter Riskind (Massachusetts General Hospital, Boston, MA)
Rhonda Voskuhl (University of California at Los Angeles, Los Angeles, CA)

I. Introduction
In autoimmune diseases, the immune system inappropriately recognizes "self," which leads to a pathologic humoral and/or cell-mediated immune reaction. In a normal, nonautoimmune state, self-reactive lymphocytes are deleted or made unresponsive to peripheral self ligands. Populations of potentially autoreactive cells can be demonstrated, yet appear to ignore their ligands. A picture of autoimmune disease is emerging wherein these autoreactive cells are activated through molecular mimicry, given that T cell receptor (TCR) interactions can be degenerate and T cells can be activated by a diversity of ligands (1, 2). There is evidence that activation of autoreactive T cells is facilitated by the induction of cytokines and the up-regulation of particular costimulatory molecules (CD80/CD86 and CD40), leading to autoimmunity.

Many autoimmune diseases are more prevalent in women than in men. This sexual dimorphism covers a broad range of autoimmune disorders, ranging from organ-specific (such as Graves' disease) to generalized [such as systemic lupus erythematosis (SLE)]. In multiple sclerosis (MS), a chronic inflammatory demyelinating disease of the central nervous system (CNS), there is a female-to-male preponderance approaching 2:1 to 3:1. The reasons for the sex bias in MS and other autoimmune diseases are unclear but may include such factors as sex-related differences in immune responsiveness, response to infection, sex steroid effects, and sex-linked genetic factors. The purpose of this article is to provide a state-of-the-field review of what is known at present about sex differences in various autoimmune diseases, with a focus on MS. Where applicable, information about SLE and rheumatoid arthritis (RA) is included. It is recognized that MS, SLE, and RA are different diseases and probably differ in etiology. However, the common link is the overwhelming prevalence of these diseases in women. Considering that each of these diseases is autoimmune, the effects of sex hormones and gender may be similar, making a comparison of these diseases useful.

II. Sex Effects on Susceptibility and Severity of Autoimmune Disease
MS is more common in women, but disease severity may be worse in men. Recent analyses of the effects of sex on MS prognosis considered mortality rates, time to requirement for assisted walking devices, and onset of progressive neurologic impairment. Analysis of a large German MS cohort showed a reduced survival rate in men versus women. The difference in mortality resembled the expected sex ratio of mortality risk, so no specific effect of sex was observed in this study (3). Similarly, a survival analysis from the date of diagnosis in Norwegian MS patients showed no effect of sex on life expectancy (4). In contrast, an increased mortality over expected levels was found in male but not in female MS patients in a population-based analysis at the Mayo Clinic (5).

Prognostic variables have been studied in MS, using longitudinal follow-up of large patient populations for 12- to 25-year intervals (6, 7). Male sex was predictive of a shorter time to reach a requirement for assisted walking devices. Men also had a higher rate of cerebellar involvement and a higher risk of primary-progressive disease, both of which are factors associated with a poorer prognosis. How can a milder disease course be reconciled with evidence that autoimmune susceptibility is higher in women? One possibility is that women with mild MS seek medical care more aggressively than comparably affected men, thereby inflating the numbers of women reaching early diagnosis. This explanation is discounted by an analysis of placebo data from recent Phase III clinical trials, which confirms that progressive disability is worse in men than in women with comparable duration of disease (8). Another possibility is that risk factors for disease severity are linked to sex. For example, the MS-associated HLA-DR2 allele is more frequent in women than in men with MS, although the specific effects on prognosis have not been fully investigated (9). An early age of onset of MS appears to confer a more benign prognosis, and there is evidence that women develop MS at an earlier age than men (10). In a Canadian MS cohort, the ratio of women to men was 3.2:1 in patients presenting before age 20, but 2:1 for the whole population (11). A third possibility is that women and younger individuals have stronger repair mechanisms that more readily effect remission and forestall progressive disease. Differences in circulating levels of progesterone or of insulin-like growth factor 1 (IGF-1) suggest an explanation for the sex and age effects on MS prognosis. Progesterone promoted remyelination in rodent sciatic nerves and increased expression of myelin proteins in oligodendrocytes in vitro (12, 13). Similarly, IGF-1 had trophic effects on myelin and attenuated experimental autoimmune encephalomyelitis (EAE), an animal model for MS. IGF-1 levels are higher in women than men, and in both sexes levels decline with age (14).

Contrasting effects of gender on disease susceptibility versus severity may also exist in other autoimmune diseases. SLE is nine times more common in women than in men, but men exhibit differences in clinical presentation, with an increased prevalence of SLE-associated renal disease, vascular thrombosis, pleuropericardial disease, peripheral neuropathy, and seizures as compared to women (15). In SLE, females often present with disease symptoms during childbearing years, whereas the incidence in males tends to increase with age. Thus, women may have greater susceptibility to MS and other autoimmune diseases, in part because of more robust immune responses, yet have a better prognosis, perhaps as a result of heightened recovery mechanisms.

III. Sexual Dimorphism in the Immune Response
Data reported in the literature are often not stratified by sex, and experimental protocols frequently use a single sex of animal. However, there have been comparisons between the sexes for immune response parameters. Female mice produced more antibody and greater cell-mediated responses to immunization than males (16, 17). In humans, such differences are difficult to identify. An increased antibody response was found in girls relative to boys (18), but in adults receiving vaccines, males and females showed similar antibody responses (19).

Manipulations of sex steroid levels were performed in rodents to examine their role in immune responses. Treatment with androgens or estrogens resulted in thymic involution, whereas surgical removal of the testes or ovaries led to thymic enlargement and an increase in immature thymocytes (20). Combined estrogen and progesterone treatment of mice, mimicking pregnancy levels, resulted in lower B cell production by the bone marrow (21). During the estrous cycle, antigen presentation in both the uterus and spleen of female rats was highest at proestrus, when estrogen levels peak. Estrogen treatment of oopherectomized rats increased antigen presentation by uterine epithelial cells (22). Moreover, during the luteal phase, with high levels of progesterone, the number of dendritic cells increased in the vaginal epithelium (23).

Compared to rodents, primates have fewer reported sex-related differences in immune function, but there are two notable differences in humans. Plasma immunoglobulin M (IgM), but not IgG, levels are significantly greater in women than in men (24). Women also have an elevated CD4/CD8 T cell ratio in the peripheral blood (25) because of a greater number of CD4+ lymphocytes in women. Comparable numbers of CD8+ lymphocytes are found between the sexes.

Cytokine production was also examined for effects of sex. Female mice immunized with Bacille Calmette Guerin (BCG) showed higher serum interferon gamma (IFN-γ than males (26). Lipopolysaccharide administration produced higher levels of plasma interleukin-1 (IL-1) and IL-6 in female versus male mice, although similar tumor necrosis factor-α (TNF-α) levels were seen (27). In vitro studies showed that androgens decreased the production of IFN-γ, IL-4, and IL-5 (28), whereas estrogen enhanced IFN-γ production by murine lymphoid cells (29). Moreover, estrogen treatment of macrophages from male mice increased IL-1 secretion (30). In CD4+ T cell clones from MS patients, both IL-10 and IFN-γ production were increased in the presence of estradiol (31). Sex steroid regulation of cytokine production was also investigated in bone cells. IL-6 production by bone marrow stromal cells was inhibited by estrogen (32), and in osteoblasts, estrogen stimulated transforming growth factor-β (TGF-β production (33). Thus, the female sex and the hormone estrogen may be immunostimulatory by increasing a variety of immune interactions, including cytokine production.

IV. Sexual Dimorphism in Infectious Disease
Several viruses have been implicated as causative factors of MS (34), including coronaviruses, herpesviruses (herpes simplex virus, human herpesvirus 6, varicella-zoster, and Epstein-Barr), and endogenous retroviruses (35). The evidence for a viral etiology in MS comes from epidemiological and viral isolation studies; however, most observations have not been confirmed and the data are not compelling. Recently, evidence for molecular mimicry of myelin proteins by viral proteins has been reported (36). Molecular mimicry provides a mechanism for viral antigen activation of peripheral T cells, which induce inflammatory responses when they enter the CNS (37).

For many pathogens, particularly viruses, infection of animals reveals sex differences in disease pathogenesis. These differences depend on the type of host response and the cytokine milieu generated in response to infection. CD4+ T cell responses can be divided into T helper cell-type 1 (TH1)- and TH2-type responses. TH1 lymphocytes secrete IL-2, IFN-γ, and lymphotoxin (TNF-β) and establish a proinflammatory environment. In contrast, TH2 lymphocytes secrete IL-4, IL-5, IL-6, and IL-10 and are important for humoral immunity.

Male mice are more susceptible to lethal vesicular stomatitis virus (VSV) infection via the olfactory neuroepithelium (38). The earliest stages of disease were identical in males and females, but female mice controlled and cleared the infection (39). Similarly, in Coxsackie Type B-3 virus (CVB-3) infection, male mice are more susceptible than females. CVB-3 causes pericarditis and myocarditis, with cytotoxic T lymphocyte (CTL) activity directed against myofibrils. Increased disease was observed in pregnant or female mice treated with progesterone (40), and T cells from immune female mice inhibited the development of CTL activity by lymphocytes from infected male mice. Androgen treatment increased expression of the viral receptor on myocytes and endothelial cells and led to enhanced infection (41). Female mice made protective TH2 responses to CVB-3, whereas male mice generated TH1 responses (42).

Naive female mice are susceptible to intravaginal infection by herpes simplex virus (HSV-2) only during diestrous. Vaccination and progesterone treatment protected mice from a lethal HSV-2 infection (43). Male mice are very susceptible to HSV-1 infection through corneal scarification (44, 45), and IFN-γ is important for containing virus by this route of infection. However, male mice showed greater mortality irrespective of their genotype (wild type, IFN-γ/- or IFN-γR-/-) and exhibited greater reactivation of latent virus than did female mice (46).

In lymphocytic choriomeningitis virus (LCMV) infection, pathology after intracerebral infection of adult mice is mediated by CD8+ major histocompatibility complex (MHC) class I-restricted T cells or by CD4+ TH1 effector cells in mice lacking MHC class I [β2 microglobulin-deficient (β2M-/-) or the H-2dm2 strain]. Female mice or castrated β2M-/- male mice treated with estrogen are more susceptible to morbidity and mortality (47). CTL activity against virally infected cells increased when estrogen was added to cell cultures (48). Vaccination of male β2M-/- mice increased IFN-γ-secreting cells and caused more inflammation and immunopathology in the CNS (49). Thus, whereas estrogen increased TH1 effectors and pathology in naive mice, vaccination that enriched TH1 responses was also correlated with LCMV disease.

There are also sex- or hormone-linked effects for other pathogens. In a primate model of acquired immunodeficiency syndrome (AIDS), female rhesus macaques treated with progesterone were more readily infected with the simian immunodeficiency virus (SIV) via the intravaginal route (50) and developed a more rapid systemic disease course. In mice infected with Theiler's murine encephalomyelitis virus (TMEV), a model for MS, male mice of two different strains were more susceptible to infection than were females (51). Treatment of male mice with androstenediol, a metabolite of dihydroepiandrosterone (DHEA), protected them from a lethal influenza infection, accompanied by increased IFN-γ secretion and lowered circulating corticosterone (52).

In summary, female mice are more likely to develop a TH1 response, and in systems where TH1 responses are beneficial (VSV, HSV, and TMEV), female mice are more resistant to disease. However, in infections where TH1 responses are deleterious (LCMV), females exhibit more pathology. Androgens can increase susceptibility to infection by upregulating viral receptors and anti-self CTL activity (CVB-3) or by enhancing the inflammatory response (influenza). Progesterone increases susceptibility to infection (in SIV, CVB-3, and HSV). At present, there are very little data on sex differences in susceptibility or severity of infections in humans.

V. Sex Hormone Regulation of the Autoimmune Response
A. Estrogens and Progesterone
The female predominance in autoimmune disease and the sexual dimorphism in immune responsiveness have stimulated interest in the role of sex steroids. Estrogens and progesterone have been studied as (i) modulators of ongoing autoimmune disease, (ii) participants in genetic susceptibility, (iii) triggers for autoimmune events, and (iv) contributors to differences in responsiveness between males and females.

There is little evidence that estrogens or progesterone are directly involved in the etiology of MS and RA. As will be discussed later, a few studies suggested that oral contraceptive use was associated with reduced disease in MS and RA. In animal models of MS, estrogen treatment resulted in disease improvement (53-55). In both rat and mouse models of EAE, oral contraceptives containing a high ratio of estrogen:progesterone or estradiol alone suppressed disease, whereas medroxyprogesterone potentiated EAE (53). Similarly, estrogen suppressed collagen-induced arthritis, but the protective effect of estrogen was potentiated by progesterone (55). Progesterone alone had no effect on disease (56). These data suggest that estrogens and progesterone may act collectively to influence autoimmune activity. Moreover, cytokine secretion by human T cells in vitro is selectively influenced by estrogens, progesterone and dexamethasone, which is consistent with an antiinflammatory influence (31, 57, 58).

Abnormalities in estrogen metabolism were reported in both males and females with SLE, with abnormal 16α-hydroxylation leading to elevated levels of the estrogen metabolites 16α-hydroxyestrone and estriol (59). Estrogen-containing oral contraceptives or hormone replacement therapy either promoted flares of existing disease or induced the onset of lupus symptoms in healthy women (59, 60). Estrogen stimulated secretion of autoantibody in SLE-resistant and SLE-prone mice in the absence of autoantigen (61). Such effects were ameliorated by treatment with the estrogen receptor modulator LY139478 (62), the estrogen antagonist tamoxifen or by antibody to estradiol (63). Collectively, these observations indicate that estrogens can trigger SLE-like autoimmune events.

There is evidence that the ability of immune cells to respond to estrogens is an inherited trait (64), which suggests that estrogen responsiveness contributes to overall genetic susceptibility to autoimmune disease. Because autoimmune diseases appear to be determined by multiple susceptibility factors, it is likely that one factor could be hormone responsiveness. Data from animal models showed that SLE-like disease was accelerated after estrogen administration in castrated male NZB/W F1 and MRL-lpr/lpr mice (65). Females exhibited higher autoantibody levels than did males regardless of hormone treatment, which suggests that hormone-independent factors also played a role (66).

Progesterone is known to inhibit mitogen-induced T cell proliferation and to promote antiinflammatory activity in vivo (67). Recently, progesterone was shown to stimulate IL-4 production (57, 58) and promote development of human TH2 cells (57) at pregnancy-associated levels. In human lymphocytes, progesterone induced the production of progesterone-induced blocking factor, which inhibited lymphocyte proliferation and natural killer cell activation and is thought to play a role in pregnancy-associated changes in immune function (68).

The mechanisms by which estrogens modulate autoimmune disease are likely to reflect their ability to affect normal immune function, as well as the relative involvement of T cells, B cells, and other immune cell types in tissue damage. In addition, steroids exhibit dose-dependent biphasic effects on immune cells, whereby low concentrations facilitate, and high doses inhibit, cell-mediated immune functions (31). Thus, estrogens may act as triggers of cell-mediated autoimmune events at low concentrations and as inhibitors at higher doses. At high doses, such as those achieved during pregnancy, estrogens should be beneficial for autoimmune diseases mediated primarily by proinflammatory CD4+ T cells, including MS (69, 70) and RA (71). Because autoantibody production is central to much of the tissue damage in SLE (72), high doses of estrogens are predicted to have a deleterious effect. However, abnormal activity of B cells, T cells, and antigen-presenting cells (APCs) has been described in SLE (72). Moreover, studies of MRL-lpr/lpr mice showed that estrogens attenuated T cell-mediated periarticular inflammation, renal vasculitis, and sialoadenitis while accelerating antibody-mediated glomerulonephritis (73).

Although it is possible to classify murine immune responses into TH1 and TH2 subtypes depending on the predominant cytokine profile, this classification is not so clear-cut where human immune responses are concerned. MS and RA have been increasingly likened to TH1 responses to organ-specific antigens, whereas SLE has been described as a TH2-biased series of immune responses directed against a variety of self antigens (74). There is no good evidence to support a rigid classification of these diseases into the TH1/TH2 paradigm. Rather, certain cytokines such as IFN-γ, IL-4, or IL-10 may play a prominent role in the pathogenesis of some of these diseases, and the relative role may vary over the course of the disease.

B. Androgens
Sex differences in susceptibility to MS have been studied using the EAE model in SJL (H-2s) mice, in which females are more susceptible than males (75-77). Interestingly, this sex bias is not observed in EAE in B10.PL (H-2u) mice, where males and females are equally susceptible and males exhibit more severe disease (78). To address the mechanisms underlying the increased susceptibility of female SJL mice to EAE, the effect of sex hormones on cytokine production was examined. Hormonal influences on cytokine production can influence the balance between TH1 and TH2 responses. Therapies that shift responses toward TH2 are effective in ameliorating TH1-mediated diseases such as EAE. In male SJL mice with EAE, autoantigen specific responses were characterized by higher TH2 cytokine production (75-77). Testosterone treatment reduced the severity of EAE in females with a concommitant increase in TH2 cytokines (79).

In spontaneous diabetes in the NOD mouse, another TH1 disease, females are more susceptible than males. Increased levels of IL-4 in the pancreatic islets of male NOD mice are thought to play a role in the resistance of males (80). Testosterone treatment of female NOD mice lowered the incidence of disease, and castration of males increased disease incidence (81), which suggests that testosterone plays a protective role in TH1 diseases by promoting TH2 responses. Similarly, in thyroiditis and adjuvant arthritis, castration of males increased the incidence and severity of disease, whereas testosterone treatment was protective (82, 83). In a model of Sjogren's syndrome in female MRL/Mp-lpr/lpr mice, testosterone treatment of females reduced lymphocyte infiltration into lacrimal tissue (84). Finally, in spontaneous SLE in NZBxNZW mice, which affects females more frequently than males, testosterone improved survival (85). Thus, in models of autoimmune disease characterized by a female preponderance, male gonadal steroids (testosterone) are protective. The majority of diseases mentioned are cell mediated, except for SLE. Because testosterone is protective in each of these diseases, it is likely that (i) testosterone has immune effects in addition to affecting the TH1/TH2 balance, or (ii) testosterone may bias responses toward TH2 during some but not all experimental diseases. A differential bias of responses may be related to a biphasic dose effect (31), differences in testosterone-induced immune modulation (86), or differences in androgen metabolism (87).

Another androgen known to affect immune responses is DHEA, an adrenal steroid and precursor for sex steroids in humans. Serum levels of DHEA decrease with aging, and this androgen has been administered to correct the immune dysregulation associated with immune senescence. Decreased production of IL-2 and increased production of IFN-γ, IL-4, IL-5, IL-6, and IL-10 characterize immune senescence; DHEA treatment reversed this pattern of cytokine production (88, 89). DHEA restored the ability of aged mice to develop an appropriate antibody response upon vaccination (89, 90). How DHEA might affect autoimmune disease remains unclear. In MS, decreased IFN-γ production would be desirable, whereas increased IL-2 production and decreased IL-4 and IL-10 production would theoretically be undesirable. In one small, open-label, uncontrolled MS trial, DHEA increased subjective feelings of strength, stamina, and well-being but did not improve disability (91). However, in a double-blind controlled trial in SLE, DHEA produced an improvement in disease relative to the placebo group (92). However, agents such as cryproterone acetate, danazol, and 19-nor-testosterone were not successful in SLE because of side effects or lack of a clinical response.

C. Other Sexually Dimorphic Hormones
Three lines of evidence suggest a role for prolactin (PRL), growth hormone (GH), and IGF-1 in sex differences in autoimmune diseases: (i) these hormones have stimulatory actions on immune responses (93, 94); (ii) serum levels of PRL, GH, and IGF-1 are higher in women than in men (14, 95); and (iii) sex differences in these hormones develop with puberty, when sex differences in immune responses and risk of autoimmune diseases emerge (93). Many of the effects of GH are mediated by IGF-1, a peptide hormone secreted by the liver and also produced locally in many tissues in response to GH.

1. Prolactin
Estrogens stimulate PRL synthesis and secretion, accounting for higher levels of PRL in women than men and causing marked elevations of PRL during pregnancy (96). The importance of PRL in normal immune function is indicated by: (i) depression of cellular and humoral immune responses in rodents after hypophysectomy, (ii) restoration of immune competence by exogenous PRL and other lactogenic hormones, (iii) immunosuppression after pharmacologic inhibition of PRL secretion with the dopaminergic agonist bromocriptine, and (iv) reversal of bromocriptine immunosuppression with exogenous PRL (94, 97). Although circulating PRL is principally derived from the anterior pituitary, PRL is also synthesized and secreted by lymphocytes and therefore may act in a paracrine or autocrine manner (98). In humans, PRL receptors are found on peripheral B cells, monocytes, and 75% of peripheral T cells (99, 100). Interestingly, GH and PRL receptors (PRL-Rs) belong to the cytokine receptor superfamily (101).

The severity of several autoimmne diseases is attenuated by bromocriptine, including both the initial flares and late relapses of EAE (102, 103). Rats with EAE exhibited a threefold rise in serum PRL before the development of clinical signs. Similarly, increases in circulating PRL occur in adjuvant-immunized rats and in humans after cardiac transplantation, which suggests that immune stimulation may trigger secretion of PRL (104, 105). However, measurement of PRL levels in MS has not yielded consistent results (106, 107). Similarly, SLE patients do not show consistent elevations of circulating PRL (108, 109). Bromocriptine therapy has been used with some success in small numbers of patients with uveitis, lupus, and other autoimmune diseases, but there are no published studies of bromocriptine treatment of MS (110, 111).

2. Growth Hormone and Insulin-Like Growth Factor-1
GH is produced by lymphocytes, as demonstrated at the protein and mRNA levels (112). A functional role for lymphocyte GH is indicated by evidence that antibody to GH or antisense oligonucleotide treatment in vitro leads to inhibition of lymphocyte proliferation (113). GH receptors (GH-Rs) have been reported in human and murine lymphoid tissues, with a distribution similar to that of PRL-Rs (114, 115). GH may also have important indirect effects mediated by its trophic influence on the estrogen receptor (116). Existing evidence suggests that GH levels are not elevated in MS (117).

Some of the effects of GH are mediated by IGF-1, which is produced mainly by macrophages, and to a lesser degree by lymphocytes and other cell types (118). IGF-1 receptor expression is variably seen in T or B cells (119, 120). In vivo studies have suggested a potent stimulatory effect of IGF-1 on immunoglobulin production and lymphocyte trafficking (93). IGF-1 is recognized as a potentially useful therapeutic agent for MS, encompassing both trophic actions on oligodendrocytes and effects on immune cells. IGF-1 supports oligodendrocyte survival and myelin production in vitro and inhibits demyelination by antiserum to white matter and complement in murine spinal cord cultures (121-123). Astrocytic IGF-1 production and oligodendrocyte IGF-1 receptor expression occur in vivo during myelin regeneration after experimental demyelination (124, 125). Administration of IGF-1 to animals with EAE decreased clinical signs, reduced the number and area of demyelinating lesions, and decreased the numbers of CD4+ T cells and macrophages in the CNS (126, 127). Thus, these observations suggest a possible role for IGF-1 in recovery and repair of demyelinative injury.

D. Mechanisms of Hormone Actions
The sex steroid hormones, produced primarily in the ovary and testis, are likely to play a role in the development of autoimmune diseases. It is known that these hormones play an organizational role in brain development (128). The enzyme aromatase (cytochrome P450AR) converts testosterone to estrogen, and the regulation of aromatase activity is an important determinant in controlling the amount of estrogen, which is necessary for neural differentiation. Estrogen affects a variety of brain structures, including the basal forebrain, hippocampus, caudate putamen, midbrain raphe, and brainstem locus coeruleus (129). Male and female rats use different strategies to approach spatial navigation challenges, which are thought to be due to sex differences in hippocampal development. The possibility exists that sex hormones (testosterone, estrogen, progesterone, and/or aromatase) could play a developmental role in the immune system as well, producing sex-specific effects on the thymus and thus early affecting tolerance induction. In this regard, the effects of the steroid hormones would be more significant developmentally than at the time of disease onset.

Sex steroids, like other steroid hormones, circulate in the blood and can enter cells by diffusion. In cells expressing the specific high-affinity receptors, the hormone binding to the receptor leads to alterations in gene expression. There are distinct receptors for estrogen, androgen, and progesterone. A second estrogen receptor was recently identified (ERα and ERβ) (130). The sex steroid receptors belong to a large superfamily of nuclear receptors that include the thyroid hormone, retinoic acid, and vitamin D receptors (131). Upon ligand binding, these receptors undergo a conformational change and bind chromatin, which leads to the modulation of expression of responsive genes.

1. Immune Mechanisms
What are the mechanisms by which sex hormones affect susceptibility to autoimmune diseases? The effects of a particular hormone may be mediated directly by the hormone or indirectly through effects on other systems such as the hypothalamic-pituitary-adrenal axis. Gonadal steroids can exert either positive or negative feedback on gonadotropin-releasing hormone from the hypothalamus and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary, depending on the levels of the steroid hormone in the blood. Such feedback is important because hypothalamic/pituitary hormones modulated immune responses in castrated mice (132, 133).

Sex hormones may directly affect a variety of immune mechanisms, including the homing of lymphocytes to a target organ. T lymphocytes and the endothelial cells within the target organ must express the necessary adhesion molecules to permit entrance of the T lymphocyte (134). Thus, the effects of endogenous or exogenously administered sex hormones could relate to hormone-induced alterations in adhesion molecule expression on either autoantigen-specific T lymphocytes or on endothelial cells within the target organ. Another mechanism involves the balance between TH1 and TH2 responses during autoimmune disease. Sex hormones affect cytokine production and thus bias responses toward TH1 or TH2 depending on the particular hormone and dose (31, 57, 75, 79). Alterations in cytokine production may be induced by direct action of the hormone on the T lymphocyte or indirect action on APCs, macrophages, or B lymphocytes. Sex hormones can also affect signaling through the TCR, expression of coreceptors on T lymphocytes (CD28, CTLA-4, and CD40L), and alterations in the transcription or translation of cytokine genes.

Hormonal effects on APCs may influence antigen presentation and costimulation. Hormone-related changes in the processing of antigens or the cell-surface expression of antigen-MHC complexes would affect the response of T lymphocytes (135). Hormone-related changes in the expression of costimulatory molecules (B7.1, B7.2, and CD40) on APCs would also influence the response of T lymphocytes (136). Sex hormones may affect cytokine production by APCs, which would affect the responding T lymphocyte, because local IL-12 or IFN-γ would favor a TH1 response, whereas local IL-10 or IL-4 would favor a TH2 response (137).

To be a target for sex steroid hormones, cells of the immune system must express the cognate receptor. However, immune cells express a relatively low level of such receptors relative to other targets. In the thymus, androgen receptors are present on immature thymocytes and progesterone receptors are present on epithelial cells; both the alpha and beta forms of the estrogen receptor are present but their cellular location is unclear (138, 139). CD8+ T cells and macrophages express estrogen receptors (140, 141). There is functional evidence that CD4+ T cells also express estrogen receptors, because estrogens regulate cytokine secretion in human CD4+ T cell clones (31) and also regulate CD4+ T cell activities in β2M-/- mice (lacking CD8+ T cells) (48). Myeloma cell lines have been found to express estrogen receptors (142), but documentation of expression by normal B cells is lacking. Endothelial cells and bone marrow stromal cells also express estrogen receptors (143, 144). Interactions between these cell types and immune cells can affect immune system development and function.

2. Hormone Action
The receptor proteins have a modular structure containing distinct conserved functional regions, including ligand binding, DNA binding, and transactivation domains. The sex steroid receptors form homodimers or heterodimers (ERα with ERβ) (145). Specific DNA response elements, capable of binding the different steroid receptor/ligand complexes, have been identified in a number of target genes, and consensus sequences have been defined, although binding of receptors to other DNA sequences is also possible. The receptors can function by interacting with other transcription factors. For example, the estrogen-ERα complex can bind the AP1 proteins Fos and Jun and enhance transcription without requiring DNA binding by the estrogen receptor (146). In contrast, the estrogen-ERβ complex inhibits transcription from an ER-dependent AP1 element (147).

The common pathway for DNA-protein interactions of these hormone-receptor complexes affects the assembly or stability of the preinitiation complex used by RNA polymerase II (148). Interactions between the steroid receptors and the preinitiation complex may be direct or indirect through coregulatory factors. Several such factors have been identified and grouped into coactivators and corepressors (149). Steroid receptor coactivator-1 (SRC-1), the best known, stimulated transactivation of all known steroid receptors (150). SRC-1 was identified as a histone acetyltransferase (HAT), interacting with another known HAT (151). Through the local acetylation of histones induced by the complex of hormone, receptor, and coactivator, remodeling of histones and chromatin templates can occur, leading to activation of the transcriptional complex (152).

E. Natural and Induced Hormonal Fluctuations
1. Endogenous and Exogenous Sex Hormones
Data on endogenous sex hormone levels in MS patients are scanty. Grinsted (106) measured hormone levels in a small sample of premenopausal women with MS and found significantly higher concentrations of prolactin, LH, FSH, and testosterone and significantly lower concentrations of estrone sulphate as compared to age- and weight-matched healthy controls. Lower testosterone levels were reported in 20 to 24% of men with MS, although the sample size was small (153).

There have been few studies on the effects of oral contraceptives (OCPs) in women with MS. Poser et al. (154) found lower levels of disability in younger women using OCPs as compared to nonusers. A prospective study of women with MS demonstrated a lower risk of developing MS in OCP users, relative to those never using OCPs, with a trend to lower risks associated with longer time of OCP use (155). There have been no therapeutic studies of OCPs in MS, although administration of OCPs has been reported to abrogate the development of EAE in susceptible rodents (53). In RA, there is an association between decreased disease incidence and OCP use (60, 156). In contrast, the use of OCPs in SLE was associated with exacerbations of disease (157, 158).

2. Effect of the Menstrual Cycle
Studies of immune function during the menstrual cycle in healthy women have yielded somewhat contradictory results. Some authors reported that immune cell responsiveness was low during the midpoint of the cycle and increased during menses (159). However, complement synthesis increased during the luteal phase of the cycle as did IL-1 levels (160). An association between MS and the menstrual cycle was examined in three reports (161-163). These studies differed in patient populations and methodology, although all used either self-reported data or questionnaires. All studies reported worsening of MS symptoms just before the onset of menses in women who noted a correlation between their menstrual cycles and neurologic status. Similarly, in RA and SLE, clinical signs were also exaggerated before the onset of menses in the postovaluatory phase (164). Other neurologic disorders, both nonimmune (migraine and epilepsy) and autoimmune (myasthenia gravis), have shown catamenial symptom fluctuation (165-167). There have been no large-scale studies of neurologic status in postmenopausal women with MS. In SLE, there is some controversy about the safety of postmenopausal use of estrogen replacement, with the majority of evidence arguing for no detrimental effects on disease.

VI. Effects of Pregnancy on Autoimmune Disease
A. Immunology of Pregnancy
The application of molecular biology to the study of pregnancy has resulted in significant recent advances. Fetal cells were found in the maternal peripheral blood in most normal pregnancies, and maternal cells were also evident in the fetal circulation in up to 42% of cord blood samples (168). Thus, the concept that the placenta barricades the genetically half-foreign child from maternal recognition must be discarded, and there is increasing evidence that the maternal immune system is immunologically aware of the fetal allograft. Recent studies have provided evidence for T and B cell recognition of paternal HLA antigens during pregnancy (169, 170).

An increase in TH2 immunity and a shift away from TH1 responses occurs during pregnancy, promoting successful fetal growth and development (171, 172). Proinflammatory cytokines such as IFN-γ, TNF-α, and IL-1 are deleterious to pregnancy (171-174). A protective systemic TH1 response to Leishmania infection in pregnant C57BL/6 mice significantly increased fetal loss either by preventing implantation or interfering with placentation, whereas a TH2-like environment during pregnancy impaired resistance and resulted in more severe disease (175, 176). Peripheral blood leukocytes collected during pregnancy in humans showed increased levels of IL-4 and IL-10 and decreased secretion of IL-2 and IFN-γ, as compared with cells from nonpregnant women (177).

Changes in immune function during pregnancy are likely to be effected by the dramatic hormonal changes and may involve a variety of immunoregulatory factors, including cytokines, pregnancy-associated glycoproteins, alpha-fetoprotein, placental suppressor factor, and trophoblast cell-derived factor (172, 174). Alpha-fetoprotein (178) and IFN-τ (179) were shown to suppress EAE. In vitro studies of cytokine secretion by antigen-specific T cell lines and clones suggested that pregnancy-associated concentrations of estrogens, progesterone, and glucocorticoids may collectively promote an antiinflammatory environment (31, 57, 58).

B. Effect of Pregnancy on Maternal Disease Activity and the Fetus
Pregnancy results in a change in disease activity for several of the autoimmune diseases. In MS, disease amelioration accompanies pregnancy, with a lower exacerbation rate during the 9-month period (157, 180-182). Disease activity as measured by MRI decreased during pregnancy, with a return to prepregnancy levels immediately postpartum (183). In RA, pregnancy-induced changes can be dramatic, with complete remission of all signs and symptoms beginning early and continuing throughout the course of gestation (184). In contrast, women with SLE do not improve (185), and many often have exacerbated symptoms during pregnancy (173, 186), although this point is controversial. In myasthenia gravis, about one-third of patients improve, one-third worsen, and one-third show no change in disease status (187).

To test whether fetal-maternal HLA disparity might be associated with pregnancy-induced remission of RA, HLA antigens in women with RA and their children were determined (188). The results showed that the children of pregnancies characterized by RA amelioration were more often HLA-disparate at the class II loci DRb1, DQa1, and DQb1 than were the children of active disease pregnancies. These results suggest that exposure to fetal paternally inherited class II antigens results in modulation of the maternal immune system, contributing to amelioration of the mother's RA. In experimental models of RA and MS, antibodies to MHC antigens abrogated disease (189, 190). Moreover, improvement in RA was reported after administration of preparations containing alloantibodies to HLA class II antigens eluted from pooled human placentas (191).

Because antibody-mediated immunity is enhanced during pregnancy, it is anticipated that SLE would worsen, and some studies have demonstrated disease worsening during pregnancy. However, the percentage of patients reporting a flare of their disease varies from 8 to 74%, and some studies question whether pregnancy causes SLE to worsen (185). Variability may reflect a dependence on disease activity immediately before conception; thus, if a patient has active disease, it will tend to remain active during gestation; and if disease is quiescent, it will tend to remain quiescent during pregnancy. Using a murine model of SLE, it was shown that proinflammatory cytokines (IL-2 and IFN-γ) dominated in early stages of disease, and TH2 cytokines (IL-4 and IL-10) were prevalent in later stages (192). Thus, the effects of pregnancy on SLE are debated and may depend on the pattern of cytokine secretion and disease activity at the time of conception.

Although the signs and symptoms of MS and RA decrease during pregnancy, the postpartum period is often associated with disease recurrence and relapse (180-182, 184, 193). In MS, the risk of postpartum relapses was estimated at 20 to 40% (182), and breastfeeding was not associated with increased relapses of disease. Furthermore, pregnancy in MS was not associated with increased long-term disability (180, 194-196); and in women who became pregnant after the onset of MS, there was a decreased risk of a progressive disease course (197). In RA, by the end of the fourth month postpartum, 98% of women experienced disease recurrence (198), which was not associated with breastfeeding (184). The mechanisms by which autoimmune disease activity is promoted in the postpartum period are unknown. It is possible that the processes involved in parturition may actively promote proinflammatory activity or that proinflammatory activity is due to an abrupt loss of antiinflammatory influences. It is also possible that hormones elevated during the postpartum period, such as prolactin, may promote disease activity as discussed above.

In MS, there is no indication that the disease results in any adverse effect on the fetus, because no increase in spontaneous abortions or congenital anomalies was observed (199). The same is true for women with RA (184). In contrast, women with SLE showed both an increased rate of spontaneous abortion and of offspring affected by neonatal lupus and/or congenital heart block, although the latter conditions are uncommon (200).

C. Effect of Pregnancy on Susceptibility to Autoimmune Disease
Analogies between MS and RA also extend to the effect of pregnancy on disease susceptibility. A lower incidence of MS in parous as compared to nulliparous women was reported (197), with higher parity being suggestive of lower incidence (155). A protective effect of pregnancy on susceptibility to RA was also reported (201). However, pregnancy appears to afford no protection against the development of SLE (202), but a definitive study is lacking.

An observation that has potential significance for increased susceptibility to some autoimmune diseases in women is the long-term persistence of fetal cells after the completion of pregnancy. Recently, fetal CD34+CD38+ immune progenitor cells were found in the circulation of normal women as long as 27 years after pregnancy completion (203). As noted above, there is bidirectional traffic of cells during pregnancy, and the hypothesis has been proposed that nonhost cells are involved in the pathogenesis of some autoimmune diseases. In the first study to examine this question, evidence for fetal cells was found more frequently and was quantitatively greater in women with the autoimmune disease scleroderma than in control group women (204).

VII. Genetic Factors that Control MS and EAE
In autoimmune disorders, the influence of genetic factors is suggested by the increased prevalence of the disease in families; twin studies demonstrate higher concordance rates in monozygotic than in dizygotic pairs (205). In MS, a comparison of the frequency of disease in biological and nonbiological relatives showed that familial clustering has a genetic basis (205, 206).

For autoimmune disorders, identification of the genes responsible for disease is complicated because such genes are expected to have small additive effects. In MS, the identification of susceptibility genes is hindered by the heterogeneity of the population and the disease, given the wide variations in age of onset, disease course, and severity. Three groups have published results of whole-genome exclusion mapping, using similar methodologies but different sets of markers and different MS families, and the combined results have recently been reviewed (207). The groups have, however, identified different linkages or loci. For example, Ebers et al. (208), studying a Canadian population, identified five loci with maximum lod scores (MLS) of >1 on chromosomes 2, 3, 5, 11, and X, but not on 6p. The strongest association was for marker D5S406 (MLS 4.24). The MS Genetics Group, studying MS patients in the United States (209), identified 19 regions potentially harboring MS susceptibility genes, including the MHC region on 6p. Sawcer et al. (210) identified two principal regions on chromosomes 17q22 and 6p21 (MHC) in a British population. Using a candidate gene approach in Finnish patients (211), a putative vulnerability locus maps to 5p14-p12, in a region syntenic to one (Eae2) (212) of three previously identified murine loci. In addition, Epplen et al. (213), studying a German cohort, found associations with HLA antigens and TCR-β gene polymorphisms, using a candidate gene approach. The only regions that are well-supported in most studies are the MHC region on chromosome 6p21 and a region on chromosome 19q (207). In type I diabetes, two genome scans have been completed. The conclusion is that a MHC-linked locus (IDDM1) is probably the major genetic factor in disease incidence, and a number of other genes with lesser effects also contribute (214). This conclusion is similar to that from the NOD mouse.

Animal models have been helpful to the MS gene mapping efforts because: (i) EAE is under complex genetic control, (ii) some regions of the mouse genome showing linkage to EAE are syntenic to human MS loci, (iii) several EAE genes are sexually dimorphic, and (iv) EAE loci may be modified by the MHC haplotype of the segregating progeny. Like the human mapping studies, the strongest EAE linkage in rodents is to the MHC (215-218), which is thought to reflect differences in the class II region. Efforts are underway to identify non-MHC loci that modify the incidence or severity of EAE. Certain candidate genes with known impact on immune function (such as TCRs, cytokines, and immunoglobulins) have been loosely associated with EAE (216, 219).

Candidate genes may be the same for different autoimmune diseases, because mouse EAE studies have identified regions of the genome that are important in the regulation of other autoimmune diseases, such as diabetes or autoimmune orchitis (220). In contrast, genes that are unique to a disease could determine the susceptibility of the target organ to autoimmune response. In the case of the demyelinating diseases, such genes might control various aspects of remyelination or CNS resilience to autoimmune attack. It is therefore important to specifically compare EAE-modifying loci with those involved in the demyelination due to encephalitis viruses (221).

Finally, almost all autoimmune disorders are more frequent in women than in men. No definite sex chromosome site has yet been conclusively identified, although a modest effect of the X chromosome has been observed (212). In MS and SLE, some HLA class II alleles have been reported to be associated with disease more frequently in women than in men, which could involve epistasis between X, or Y, and autosomal chromosomes (11). A clear direction for future studies is to examine the data sets generated in both mouse and human genetic studies to determine the relative impact of susceptibility genes in males versus. females. Toward this end, it has been observed that F2 crosses involving SJL/J mice, known to exhibit sexual dimorphism for EAE susceptibility at young ages (75-77), also displayed a gender bias when the analyses of the phenotypes were stratified by sex (216). This and related approaches will probably contribute significantly to the understanding of how genes function in males and females to create an environment conducive to the development of autoimmune diseases.

VIII. Summary
Autoimmune diseases are more prevalent in women than in men. In this review, we examined the underlying reasons for that sex bias, focusing primarily on MS with consideration of RA and SLE. Although MS is more common in women and may appear earlier, there is evidence that disease severity is worse in men. In general, immune responses are more robust in women, and the hormone estrogen appears to be immunostimulatory, increasing TH1 cytokine production. It follows that females are more resistant to infectious diseases that require a TH1 cytokine response for protection. These findings contrast with observations that OCP use in MS and estrogen administration in EAE are associated with reduced disease. However, a biphasic dose effect has been ascribed to estrogen, with low doses facilitating and higher doses inhibiting immune cell functions. Other sexually dimorphic hormones, such as prolactin, GH, and IGF-1 may play a role in the enhanced immune responses in females. Testosterone, on the other hand, exerts a protective effect in many experimental autoimmune diseases mediated by either TH1 or TH2 responses. Sex hormone effects may be mediated directly or indirectly by affecting lymphocyte homing, TH1/TH2 balance, or antigen presentation. Some autoimmune diseases such as MS and RA improve during pregnancy, with postpartum flares of disease activity occurring. Genetic studies of MS and EAE have identified several different loci that are putatively associated with disease susceptibility, with the MHC region as the major genetic determinant. Thus, the basis for sex differences in autoimmune disease is most likely multifactorial, with contributions being made by genetics, hormones, immune bias, and the environment. Pressing research questions in the area of sex differences in autoimmune disease are included in a companion paper (222).

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*To whom correspondence should be addressed at the National Multiple Sclerosis Society, 733 Third Avenue, New York, NY 10017, USA. E-mail: patricia.olooney{at}nmss.org