Sex Hormones and the Genesis of Autoimmunity FREE

Autoimmune diseases encompass nearly 70 distinct clinical entities. Among the earliest and most intriguing observations is the discrepancy in the prevalence of autoimmune disease between females and males. Systemic lupus erythematosus (SLE) is among the most female dominant of all autoimmune diseases, with a lifetime female-male ratio of 9:1.^{1 - 4} The sexually dimorphic prevalence of SLE, along with the dramatic increase in disease incidence in females after puberty, the reversal of this phenomenon after menopause, and the variation in disease severity throughout the menstrual cycle and pregnancy, led to an investigation of the role of sex hormones in the manifestations of autoimmune disease.^{1 - 4}

While sex hormonal treatment of reproductive system disorders has spanned 2 millennia and become conventional practice, sex hormones have only recently been appreciated for their role in mammalian immunophysiology.^{5 - 7} This review explains the role of sex hormones in human immunology, describes the unique expression of sex hormones among patients with SLE, and outlines how an abnormal hormonal milieu contributes to the genesis of autoimmune disease.

In contrast to the nonspecific protection afforded the host by innate immunity, the acquired immune system purposefully protects the host by distinguishing “self” from “nonself.”^{3 ,8} The acquired immune system, composed almost exclusively of B and T lymphocytes, goes amiss in autoimmune disease. The acquired immune system of females differs from that of males, because estrogens stimulate immunologic processes driven by CD4⁺ T_H2 cells and B cells, whereas androgens enhance CD4⁺ T_H1 and CD8⁺ cell activity.^{5 ,9 - 13} Autoimmune diseases that are mediated by T_H2-dominant lymphocyte activity are proportionately more female dominant than those conditions that are driven by T_H1-dominant activity (Table).¹³ Estrogens and androgens and their metabolites and receptorsare all involved in immunoregulation and the development of autoreactivity.

Estrogen receptors (ERs), in addition to being present in uterine, ovarian, breast, and bone tissue, have been identified within B and T lymphocytes, macrophages, thymic stromal cells, bone marrow stromal cells, and endothelial cells.^{3 - 5} Two types of nuclear transcription-modifying ERs have been identified in humans: ER-α and ER-β. While structurally similar, ER-α and ER-β are functionally distinct.^{1 ,14 - 15} It has been suggested that variable expression of ER isoforms and distinct estrogen potency (affinity for receptor) contribute to specific cellular sensitivities to female hormones.^{16 - 18}

While originating from the same genes that encode their nuclear counterparts, membrane-associated ERs are involved in the amplification of signal transduction cascades rather than in the modification of gene transcription. These receptors, found among mature lymphocytes, explain acute physiologic changes in lymphocytes exposed to estrogen that cannot be adequately substantiated by gene regulatory mechanisms alone.^{1 ,11 ,19 - 21} Within seconds of estrogen binding to membrane ERs, T cells engaged by antigen demonstrate increased levels of intracellular calcium, an integral mediator of cytoplasmic signaling.^{1 ,20}

Prolactin receptors have also been identified within B and T lymphocytes.^{2 ,22} Activation of prolactin receptors induces lymphocyte gene transcription, T-cell proliferation, and B-cell antibody secretion.²² Prolactin expression is altered in some patients with SLE, and evidence suggests that its immunostimulatory role contributes to disease pathogenesis.^{2 ,11 ,23 - 24}

Lymphocyte maturation involves numerous, tightly coordinated events. B cells develop in the bone marrow and spleen, while T cells are made functional during passage through thymic tissue. Estrogen has specific, reproducible effects on maturing T and B lymphocytes. Estrogen causes a reduction in the number of immature thymic lymphocytes (CD4⁺/CD8⁺) and in the mass of thymic stromal tissue, a process called thymic involution, which is accelerated in healthy females during puberty and pregnancy.²⁵ While dampening lymphocyte development in the thymus, estrogen encourages hepatic T-cell lymphopoesis, where maturing cells are not subject to surveillance screens of negative selection and tolerance induction.²⁵ Estrogen also alters the comparative subsets of mature T lymphocytes to one favoring the CD4⁺/CD8⁻(T-helper)-cell phenotype.^{3 ,25} Reduction of estrogen dominance in mice models, via ovariectomy or the administration of testosterone, causes a shift in the proportion of T cells to one favoring the CD4⁻/CD8⁺ (T-suppressor)-cell phenotype.^{3 ,13 ,25}

The effects of estrogen on B-cell development are significant. Estrogen reduces bone marrow stromal mass and causes rapid shuttling of B cells through intramedullary maturation in the bone marrow. These changes render the autoreactive screens of negative selection and tolerance induction less effective. Estrogen also encourages extramedullary B-cell lymphopoesis, whereby cells with potential autoreactivity completely bypass developmental deletion.^{14 - 15 ,26 - 28} Estrogen exposure measurably increases the number of B cells leaving the bone marrow that express high-affinity recognition of self-DNA as antigen.^{2 ,27}

Compared with age-matched males, females have higher immunoglobulin levels at baseline and produce more immunoglobulin in response to infection or immunization.^{3 - 5 ,7 ,12 ,14 ,16 ,26 ,29 - 31} This difference first becomes apparent during puberty and persists only throughout the female reproductive years.^{4 ,12 ,14} Interestingly, hypogonadal males with Klinefelter syndrome have immunoglobulin levels that are comparable to those of normal females, and with androgen administration, antibody levels diminish and approximate quantities seen in healthy male subjects.³²

Murine studies of antibody production among healthy males and females demonstrate that only females manifest detectable levels of anticardiolipin and anti–double-stranded DNA (dsDNA) antibodies. Also, the administration of 17β-estradiol induces sustained expression of anti-dsDNA antibodies.^{14 ,25 ,33}

Female hormones stimulate inflammatory reactions mediated by macrophages and CD4⁺ T_H2 lymphocytes that promote interactions between T and B cells. In contrast, androgens down-regulate T- and B-cell interactions and promote cell-mediated cytotoxic processes through activation of CD4⁺ T_H1 and CD8⁺ lymphocytes.^{3 - 4 ,32} Interleukin (IL)-1, an acute phase reactant secreted by macrophages that enhances clonal T-cell proliferation on antigen encounter, increases in a dose-dependent manner with macrophage exposure to estrogen.⁴ Estrogen also stimulates secretion of IL-4, -5, -6, and -10 by T_H2 lymphocytes. These cytokines are potent stimulators of B-cell proliferation, maturation into plasma cells, and synthesis of antibody. Interleukins 4, 5, 6, and 10 are expressed in greater quantity in an estrogen-dominant hormonal milieu.^{4 ,8 ,12 ,14 ,25 ,34} In contrast, androgens promote production of IL-2 by T_H1 cells. Interleukin 2 reduces T_H2 activity and stimulates CD8⁺ T cells.^{8 ,15}

Estrogen increases expression of the antiapoptotic protein Bcl-2.³⁵ It has been demonstrated that Bcl-2 blocks tolerance induction, potentiating the survival of developing autoreactive T-cell clones. T cells from patients with SLE express higher levels of Bcl-2 than do those from controls.²⁶

Patients with SLE demonstrate unique patterns of estrogen production and metabolism. Elevated aromatase enzyme activity is a common feature among patients with lupus. Aromatase is a modifier of 17-ketosteroids that converts androgens to estrogens. Aromatase, therefore, raises the ratio of estrogens to androgens.^{2 - 3 ,36 - 37} Estrogen is also an inducer of aromatase; therefore, high estrogen concentrations beget further increase in female hormones and reduction in male hormones.^{2 - 3}

In addition to elevated estrogen-androgen ratios, patients with SLE metabolize estrogens in patterns that are distinct from those observed in unaffected controls. Cytochrome p450 isoenzymes CYP1B1 and CYP1A1 convert estradiol to the biologic effectors 16α-hydroxyestrone and 2α-hydroxyestrone, respectively. 16α-Hydroxyestrone is among the most biologically active serum estrogens with potent immunomodulatory effects. It activates B and T cells, induces transcription, and promotes cell division. In contrast, the 2α-hydroxy product has little biologic effect.^{36 ,38} Patients with SLE have increased activity of CYP1B1, with preferential hydroxylation of estradiol to the more “feminizing” 16α-hydroxy metabolite.^{10 - 11 ,23 ,30 ,36} The altered conversion results in a 20-fold increase in the fraction of high-potency to low-potency estrogens in patients with SLE when compared with healthy controls.³⁶

In addition to having high quantities of a biologically potent estrogen, males and females with SLE have decreased availability of testosterone, dihydrotestosterone, dehydroepiandrosterone (DHEA), and DHEA sulfate.^{10 - 11 ,14 ,23 ,36} To underscore the relationship between sex hormones and immune function, it is worthwhile to mention that this observation is not a unilateral phenomenon. Not only do patients with SLE have decreased accessible androgens, but the reverse is also true. Androgen-deficient males with Klinefelter syndrome have a higher incidence of T_H2-driven autoimmune diseases (including SLE) than do healthy males.^{4 ,25 ,29}

While not a universal finding, higher prolactin levels are associated with the presence of high anti-DNA titers and an accelerated autoimmune disease course among females with SLE.^{2 ,11 ,23 - 24} In many female patients with SLE, pregnancy and the postpartum period adversely affect disease activity.²² While it may appear that elevated estrogen levels explain pregnancy-related increases in SLE disease severity, the continuation of disease exacerbation into the postpartum period (after estrogen levels have returned to normal) is better substantiated by the persistence of increased prolactin.^{2 ,22}

Autoimmune diseases are frequently associated with an imbalance in the relative activities of T-helper-cell subsets (Table). Lupus is characterized by an overactive CD4⁺ T_H2 response, a relative depression in CD4⁺ T_H1 activity, and an absolute decrease in CD8⁺ suppressor T cells. These deviations cause relatively unopposed stimulation of B-cell activity, changes fundamental to disease processes in which humoral immunity is primarily pathogenic.

In healthy controls, estradiol causes an increase in total B-cell IgG synthesis in the absence of altering B-cell proliferation or anti-dsDNA antibody expression.³³ Among patients with SLE, estradiol increases total IgG synthesis and induces polyclonal expansion of cell lines that produce IgG, including autoreactive anti-dsDNA types. Testosterone suppresses total immunoglobulin production, including the anti-dsDNA variety.^{23 ,33}

Models of antibody production in lupus-prone mice demonstrate the following:

When compared with healthy subjects, patients with SLE demonstrate distinct patterns of cytokine secretion that result from their unique expression of sex hormones. Patients with SLE have increased levels of IL-6 and IL-10, products of T_H2 lymphocytes and macrophages, which can be directly correlated with clinical disease severity.^{14 ,34} Patients with lupus also have an inherent defect in interferon gamma production. Interferon gamma is a product of T_H1 and CD8⁺ cells that has a role in depressing T_H2 activity. Under normal circumstances, increased estrogen leads to an increase in interferon gamma concentration, presumably as a mechanism of feedback inhibition to prevent unopposed stimulation of T_H2 cells. In patients with SLE, the estrogen-associated increase in interferon gamma secretion is absent.^{8 ,14 ,39}

Extracellular stimuli are translated into intranuclear effector mechanisms through signal transduction cascades. In T lymphocytes, antigen binding to the T-cell receptor increases tyrosine kinase and phospholipase C activity. Intracellular calcium is generated in this process, calcineurin is activated, which dephosphorylates the nuclear factor of activated T cells, and nuclear transcription ensues (FigureA).¹ In healthy cells, estrogen increases the generation of intracellular calcium among T cells engaged by antigen. However, in patients with SLE, estrogen-induced increases in intracellular calcium levels are substantially greater than those in healthy subjects when both are stimulated by an equivalent antigen burden.^{20 - 21} Also, calcineurin messenger RNA expression is greater in patients with SLE than in healthy controls. Effectively, these changes allow lupus T cells to be “primed” for enhanced cytokine expression and efficient T-and B-cell interactions^{1 ,21 ,40} (FigureB).

Female hormones promote humoral autoreactivity. Sex hormone expression is different in patients with SLE than in healthy controls. High concentrations of biologically potent estrogens cause patients with SLE to circulate (1) more self-reactive lymphocytes that bypassed developmental deletion, (2) imbalanced proportions of lymphocytes favoring humoral responsiveness, and (3) lymphocytes that are primed to react. However, propensity for disease and manifestation of disease differ, because without an inciting factor, autoreactive potential is just that, potential. Patients with lupus are immunoreactive to self, not only for the reasons previously stated but also because their lymphocytes are exposed to and stimulated by intracellular self-components. Errant apoptosis permits exposure of self-antigens in patients with SLE.

In multicellular organisms, homeostatic growth regulation involves delicately balanced methods of cellular proliferation and cellular destruction. Cell survival is achieved by the elaboration of growth factors, proto-oncogenes, and antiapoptotic factors. Regulated cell death is accomplished by endonuclease degradation of DNA, whereby apoptotic cells remain intact while they are dying. Apoptotic cells are opsonized by complement and subsequently cleared by phagocytes.^{8 ,36 ,41 - 43} If clearance of apoptotic material is not expeditious, apoptotic cells become necrotic, with dissolution of the cellular membrane and release of previously sequestered intracellular contents. Self-components, such as cell membrane phospholipids, dsDNA, ribonucleoproteins, nucleolar proteins, and histones, are then available for immunosurveillance.^{41 - 45}

Patients with SLE have dysregulation of both apoptosis and apoptotic cell clearance. Estrogen increases monocyte Fas ligand expression and increases rates of monocyte apoptosis.⁴⁶ Also, viable phagocytes from patients with lupus are less adept at clearing dying cells than are those from healthy controls.⁴⁷ In patients with lupus, an abundance of apoptotic material (secondary to rapid apoptotic rates) that cannot be readily cleared (owing to ineffective phagocyte function) accumulates. Cellular necrosis ensues and previously sequestered self-products become available for immunosurveillance. Among patients with SLE, phagocyte clearance of apoptotic cellular debris is negatively correlated with patient assessment of disease activity, serum levels of anti-dsDNA antibodies, and the erythrocyte sedimentation rate, whereas clearance capacity is positively correlated with serum levels of C3, C4, albumin, hemoglobin, and white blood cells.⁴⁷

Cross-incubation studies demonstrate that increased monocyte apoptotic rates and decreased phagocytic cell capacity are not inherent cellular defects in patients with lupus. Monocytes extracted from patients with lupus and exposed to serum samples from healthy controls regain normal function. Moreover, cells from control subjects that are incubated with lupus serum have higher rates of apoptosis and reduced phagocytic capacity.^{43 ,47} In patients with lupus, errant apoptosis and apoptotic cell clearance result from exposure to component(s) of lupus serum. Suggested factors include autoantibodies, immunoregulatory cytokines, and sex hormones.^{43 - 47}

Pharmacologic hormone manipulation in patients with lupus is targeted toward normalizing the high effective ratio of female-male sex hormones. Interventions include the use of antigonadotropic drugs, ER antagonists, androgens, and antagonists of prolactin secretion. In patients with SLE, the gonadotropin-inhibitor cyproterone acetate has been shown to reduce clinical disease activity in parallel with reducing circulating estrogen levels.⁵ Estrogen blockade with tamoxifen, in murine models of lupus, improves disease severity by interfering with estrogen binding to lymphocyte nuclear ERs.⁵ The administration of DHEA and DHEA sulfate to patients with moderately active SLE disease leads to clinical improvement, as ascertained by a decreased frequency of disease symptoms or flares,^{14 ,30 ,48 - 49} decreases in corticosteroid requirements, up-regulation of IL-2 (a T_H1 cytokine), down-regulation of IL-6 and IL-10 (T_H2 cytokines),³² and reduced serum titers of anti-DNA antibodies.^{11 ,23} Over the past decade, 5 controlled trials have investigated DHEA for its disease-modifying potential in females with lupus. Administration of 200 mg of DHEA for 7 to 12 months reduced corticosteroid requirements and the frequency of disease flares but resulted in acne and hirsutism in 35% and 10% of patients, respectively.⁴⁹ The weaker androgens, danazol and 19-nortestosterone, have been less well evaluated but may also prove to be beneficial.⁴ The dopamine agonist bromocriptine prevents the secretion of prolactin by cells of the anterior pituitary gland. In murine models of SLE, bromocriptine diminishes activation of autoreactive B cells and reduces autoantibody production in direct relationship to reduction in prolactin levels.²

Another approach to altering sex hormone ratios in patients with lupus is through nonpharmacologic dietary manipulation. Dietary interventions can improve the elevated estrogen-androgen ratios and the expression of biologically potent estrogens in patients with SLE. Plant lignans, members of the phytoestrogen class, are found in rich quantity in flax seeds. These phytoestrogens have great structural similarity to estradiol. However, contrary to estradiol's induction of aromatase (which promotes conversion of androgens into estrogens), lignans inhibit the aromatase enzyme, thus reducing circulating estrogen levels.³⁸

Metabolism of estrone and estradiol to hydroxylated end products is also a modifiable process. The ratio of high- to low-potency estrogens is 20-fold higher among patients with SLE than among healthy controls. CYP1A1 hydroxylates estrogens into biologic effectors of low potency (2-hydroxyestrone). Greater CYP1A1 activity results in less available estradiol for metabolism into high-potency forms (16α-hydroxyestrone). CYP1A1 is an inducible enzyme.³⁸ Enzyme induction occurs in the presence of indole-3-carbinol, an organic compound found in cruciferous vegetables and omega-3 fatty acids. Notably, these 2 dietary factors have proved to be preventive in the development of ER-positive adenocarcinoma of the breast, another condition with SLE-like ratios of high- to low-potency estrogens.^{30 ,38} Indole-3-carbinol is currently under investigation for its disease-modifying potential in SLE. Dietary approaches to therapy are attractive because they are inexpensive, readily available, and reliably safe.

Rather than altering sex hormone expression, another therapeutic approach is to target aberrant, hormone-dependent signal transduction cascades in lymphocytes of patients with lupus. Increased calcineurin messenger RNA expression in females with lupus results directly from estrogen binding to T-cell ERs.¹ Calcineurin is an essential part of the T-cell signal transduction cascade, allowing the nuclear factor of activated T cells to enter the nucleus and induce transcription of cytokine genes. Treatment of lupus-prone mice with the calcineurin antagonist tacrolimus reduces T-cell gene transcription, decreases the severity of disease, and prolongs survival.¹ Calcineurin is dependent on calcium as a cofactor. Without adequate intracellular calcium, calcineurin activity is blunted. Intracellular calcium levels increase when antigen-engaged T cells are exposed to estrogen and increase more in patients with lupus than in healthy controls.^{1 ,20} The calcium channel blocker nifedipine reduces intracellular calcium levels in T cells from murine models of SLE.²⁰

While clinicians and scientists may be far from preventing or eradicating autoimmune disorders, awareness of disease complexity is evolving rapidly. Sex hormones affect the function of the mammalian immune system and make undeniable contributions to the sexually dimorphic expression of autoimmune disease. Appreciation of the molecular mechanisms involved in autoimmune disease pathogenesis is essential to a better understanding of immune dysregulation and its management.

Correspondence: Lindsay S. Ackerman, MD, 2168 E LaVieve Ln, Tempe, AZ 85284 (lindsay_ackerman@hotmail.com).

Financial Disclosure: None.

Accepted for Publication: January 5, 2006.

Acknowledgment: I thank Donna Holland, MD, for her critical content review of the manuscript, Mara Haseltine, MD, for her help in editing the manuscript, and Michelle Silver for technical support and for her help with formatting the Figure.