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Prolactin as a modulator of B cell function: implications for SLE
Biomedicine & Pharmacotherapy 58 (2004) 310–319 www.elsevier.com/locate/biopha
Dossier: Autoimmunity and Biotherapy
Prolactin as a modulator of B cell function: implications for SLE Elena Peeva a,b, Jeganathan Venkatesh a, Daniel Michael a, Betty Diamond a,b,* a
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Rm. 405, Forch Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA b Department of Medicine, Albert Einstein College of Medicine, Rm. 405, Forch Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA Received 10 February 2004 Available online 27 April 2004
Abstract Prolactin is not only a lactigenic hormone but also an immunomodulator involved in lymphocyte survival, activation and proliferation. There is increasing data implicating prolactin in autoimmunity, and specifically in SLE. Increased serum prolactin levels have been reported in lupus patients of both genders, and have been associated with accelerated disease expression and early mortality in lupus-prone mice. Furthermore, suppression of prolactin secretion with bromocriptine provides beneficial effects in murine lupus, and perhaps in some SLE patients as well. Treatment with prolactin that causes mild to moderate hyperprolactinemia, similar to that present in SLE patients, breaks tolerance and induces a lupus-like illness in non-spontaneously autoimmune mice with a susceptible genetic background. These immuno stimulatory effects of prolactin are mediated by a decrease in negative selection and the maturation of autoreactive B cells to the follicular subset. Consistent with the fact that follicular B cells are T cell dependent, CD4+ T cells are necessary for the prolactin-mediated break down of B cell tolerance. In mice, the effects of prolactin on the immune system are genetically determined, suggesting that only a subset of SLE patients are likely to have a prolactin-responsive disease. The manipulation of serum prolactin or, even more specifically, follicular B cells that are susceptible to the immuno stimulatory effects of prolactin, may provide novel therapeutic options for those SLE patients with a prolactin-modulated disease. © 2004 Elsevier SAS. All rights reserved. Keywords: Prolactin; B cell; SLE
1. Introduction Over the past two decades, significant evidence has accumulated that the anterior pituitary hormone prolactin, well known for its lactogenic function, acts also as an important connection between the endocrine and immune systems. Prolactin is a 200 amino acid peptide hormone produced not only by the anterior pituitary, but also by many extrapituitary sites including immune cells [1]. Prolactin functions as an immunomodulator and affects apoptosis, activation and proliferation of immune cells. During the child-bearing age, females have higher serum prolactin levels than males; this difference starts in puberty as a result of increased estrogen levels. Estrogen increases prolactin secretion from the anterior pituitary by inhibition of dopaminergic suppression [2]. During pregnancy, serum prolactin increases and reaches a maximum concentration post* Corresponding author. E-mail address: [email protected] (B. Diamond). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2004.04.007
partum during lactogenesis. Prolactin levels correlate with the amount of immunoglobulin in the milk. After weaning, prolactin levels return to normal [2]. Therefore, in women prolactin plays an important role in reproductive physiology. But prolactin also exerts multiple effects on the immune system. In this article, we will review the immunomodulatory effects of prolactin and the effects of hyperprolactinemia on the expression of SLE in mice and humans. We will focus on prolactin-induced alterations in B cell maturation and repertoire selection. 2. Immunomodulatory mechanisms of prolactin It is not surprising that prolactin modulates functions of the immune system. Prolactin receptors are expressed on many cells of the immune system, including hematopoietic stem cells, T cells, B cells, monocytes, macrophages, NK cells, neutrophils and thymic epithelial cells [3–5]. Lymphocytes express fewer receptors per cell than macrophages. Among lymphocytes B cells express the highest levels of
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prolactin receptors [1,6]. The prolactin receptor is a transmembrane protein of the type I cytokine receptor family, which also includes receptors for growth hormone, erythropoietin, thrombopoietin, granulocyte/macrophage-colony stimulating factor and many interleukins [7]. Prolactin receptors bind not only lactogenic hormones (prolactin and placental lactogen), but also growth hormone [1,8] suggesting an overlap and redundancy in prolactin receptor signaling. Three isoforms of the prolactin receptor have been described in rodents, while in humans four isoforms have been observed, one of which is unable to signal, but can bind prolactin and, therefore, exerts an inhibitory function [9,10]. Prolactin binding leads to receptor dimerization which in both rodents and man, induces signaling through Janus tyrosine kinase (JAK) 2 and transcription factors Stat1, 3 and 5 [11,12] leading to activation of interferon regulating factor-1 (IRF-1) [13], inducible nitric oxide synthase and suppressors of cytokine signaling (SOCS) 2, 3 and 7 [14]. Prolactininduced activation of the Src and Tec family of tyrosine kinases, SHP-2 phosphatase, ZAP-7, MAPK signaling pathways has also been demonstrated [15]. A portion of secreted prolactin is phosphorylated [16]; since phosphorylated prolactin is reported to act as a partial agonist [17,18], the ratio between unphosphorylated and phosphorylated prolactin may be physiologically relevant. Prolactin is implicated in lymphoproliferation, cytokine production and antibody secretion [19]. Whether prolactin is important in lymphopoiesis is not clear. Administration of prolactin stimulates progenitor cells in vitro [20], and transfection of the rat prolactin receptor into a pro-B cell line leads to an expansion of B220+ pro-B cells demonstrating that prolactin may exert a direct effect on B cell development [21]. Prolactin may also function in early T cell development. For example, prolactin regulates the maturation of CD4–CD8– thymocytes to CD4+CD8+ T cells through induction of IL-2 receptor expression [22]. Additionally, it has been observed that changes in the ratio of unphosphorylated to phosphorylated prolactin affect developing cd T cells. The offspring of rats treated throughout pregnancy with a recombinant molecular mimic of phosphorylated prolactin demonstrate a lasting decrease of epidermal cd T cells [23]. Yet, both prolactin deficient [24] and prolactin receptor deficient mice [25] have normal hematopoiesis. Thus, prolactin is not essential for lymphopoiesis and it appears that other factors can compensate for its deficiency. Prolactin also affects lymphocyte survival, altering apoptotic pathways that are essential for lymphocyte homeostasis and tolerance induction. It has been shown that prolactin exerts an anti-apoptotic effect in a rat pre-T cell line by upregulation of the anti-apoptotic genes Bcl-2, Bcl-xl, X-linked inhibitor of apoptosis (XIAP) and the protooncogene Pim-1 and by down-regulation of the pro-apoptotic gene Bax [19,26–28]. It has been also demonstrated that prolactin upregulates Bcl-2 in a dose dependent manner in a human IgE secreting myeloma cell line [29]. In vitro prolactin does not induce mouse lymphocyte proliferation [30], but prolactin enhances ConA induced pro-
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liferation of rat splenocytes in dose dependent manner [31]. Anti-prolactin antibodies inhibit ConA or phytohemagglutinin-induced proliferation of human PBMCs [32], causing an arrest of cells in the G1 phase. This inhibition is reversed by addition of purified prolactin, but not growth hormone [30]. Thus, prolactin contributes for mitogeninduced proliferation of lymphocytes in rodents and humans. Prolactin is an important regulator of cytokine production. There is some evidence that the Th1 cytokine IFNc as well as the Th2 cytokine IL-6 are upregulated by prolactin. Physiologic concentrations of prolactin stimulate normal human PBMCs to produce IFNc through activation of IRF-1. The response is dose dependent [33], suggesting that hyperprolactinemia may be responsible for increased IFNc in some individuals. Interestingly, decreased IL-2 production has been reported in hyperprolactinemic women with pituitary tumors and is corrected when normal prolactin levels are restored [34]. Prolactin causes a dose dependent increase in immunoglobulin secretion from anti-IgM and IL-2 stimulated PBMCs from healthy people [35]. In hyperprolactinemic mice, there is an increased number of antibody-secreting splenocytes, which correlates with the degree of prolactinemia, although the mechanism for this is not known. A study of a small number of lupus patients with CNS disease activity showed increased IL-6 in the cerebrospinal fluid. The increase correlated with the prolactin concentration, suggesting a possible association [36], and perhaps suggesting that prolactin may enhance antibody secretion through regulating IL-6 production, but this remains speculative. 2.1. Hyperprolactinemia in SLE patients Emerging data implicate prolactin in the pathogenesis of SLE, yet the mechanisms are not well understood. Initially, in the mid 1980s increased serum prolactin levels were described in male patients with SLE and in the early 1990s hyperprolactinemia above-expected levels were reported in pregnant lupus patients with active disease. Approximately 20% of SLE patients express some degree of hyperprolactinemia [37–40]. An association of increased prolactin levels with anti-dsDNA antibodies and with anti-Ro and anti-La antibodies has been reported [41]. However, attempts to correlate disease activity or specific organ involvement with the serum prolactin levels have led to equivocal results [42–48] suggesting that the degree of hyperprolactinemia is not a crucial factor in determining target organ susceptibility or autoantibody specificity. In the majority of lupus patients with hyperprolactinemia, the etiology of the increased prolactin levels is unknown. It was initially suggested that a G 1149T polymorphism of the prolactin gene is associated with SLE [49], but this has not been confirmed in a subsequent study [50]. In a minority of patients, hyperprolactinemia is caused by prolactinomas, medications, chronic renal insufficiency, thyroid insufficiency or hyperparathyroidism [51,52]. Some lupus patients
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have antibodies against prolactin [53], which are thought to dysregulate prolactin secretion and induce hyperprolactinemia. The immune complexes of prolactin with anti-prolactin antibodies are not hormonally active. Thus, SLE patients with prolactin-IgG complexes may have less active disease than other lupus patients with hyperprolactinemia [54]. Prolactin increases production of IgM, IgG and antidsDNA antibodies in PBMCs from SLE patients [55]. In vitro, prolactin induces increased secretion of IFN-c and IL-2 by peripheral mononuclear cells (PBMCs) from lupus patients [33] although hyperprolactinemia may be associated in vivo with reduced IL-2. Additionally, unstimulated PBMCs from lupus patients produced more prolactin than PBMCs from healthy individuals [55]. Indirect support for the role of prolactin in the pathogenesis of SLE has been provided by human studies that have investigated the therapeutic effects of prolactin inhibition. Bromocriptine is a dopamine receptor agonist that inhibits prolactin secretion. Small clinical studies showed beneficial effect of bromocriptine in the treatment of SLE patients with mild to moderately active disease [56,57]. A 6-month prospective open label trial with bromocriptine in seven lupus patients demonstrated clinical improvement in disease activity scores associated with a decrease in anti-DNA antibody titer and serum IgG levels. After the treatment, during a 5-month follow up, all patients developed increased disease activity and in two patients hyperprolactinemia corresponded with the lupus flares [56]. Another clinical study showed that in non-organ threatening lupus, the therapeutic effect of bromocriptine is comparable to that of hydroxychloroquine [57], which is a widely accepted treatment for SLE. 2.2. Hyperprolactinemia and murine lupus Studies of murine models of lupus have provided clear evidence that prolactin can exacerbate autoimmunity [40,58– 61]. Hyperprolactinemia induced by either treatment with prolactin or pituitary transplants leads to a more aggressive disease in lupus-prone mice. In female NZB/W mice, a 3–18fold increase in serum prolactin levels induced early proteinuria, elevated serum IgG and circulating immune complexes, and accelerated mortality [40,59]. Physiologic hyperprolactinemia generated by pseudopregnancy also caused worsening of disease activity [60]. In male NZB/W mice, in which the disease develops later and has a milder course, both moderate (116–239 ng/ml) and severe hyperprolactinemia (275–424 ng/ml) induced early onset of disease and premature mortality. Thus, a sustained mild to moderate increase in serum prolactin levels (100–200 ng/ml) is sufficient to accelerate disease activity and decrease longevity in NZB/W mice [40]. There was, however, no linear correlation between the degree of hyperprolactinemia and disease activity. In the same murine model of lupus, it has been shown that deleterious effects of hyperprolactinemia are not mediated via suppression of immunosuppressive androgens [60]. In MRL-lpr/fas lupus-prone mice, lactation leads to post-
partum arthritic flares [61]. Treatment with bromocriptine decreases anti-DNA titers and improves survival in the NZB/W mouse model of lupus [59,62,63]. Also, bromocriptine suppresses development of anti-DNA antibodies in MIV-7 monoclonal antibody-induced lupus in BALB/c mice [64].
3. Mechanisms of prolactin induced abrogation of B cell tolerance We have demonstrated that treatment with prolactin can induce a lupus-like serology in non-spontaneously autoimmune mice. BALB/c mice transgenic for the IgG2b heavy chain of the R4A nephritogenic anti-dsDNA antibody maintain B cell tolerance, have negligible titers of anti-DNA antibodies and no renal pathology [65]. Approximately 5% of B cells in these mice express the transgene-encoded heavy chain, while the rest express an endogenous µ heavy chain. The transgene-encoded heavy chain can pair with a variety of light chains producing antibodies with different affinities for DNA as well as antibodies that do not react with DNA. Three distinct DNA-reactive B cell populations have been described in R4A-c2b mice, one that secretes low affinity anti-DNA antibodies and two which can produce highaffinity anti-DNA antibodies [66–69]. The B cell population that secretes low affinity anti-DNA antibodies is not subject to tolerance induction; rather, it matures to immunocompetence in peripheral lymphoid organs [67]. One population of the high-affinity DNA-reactive B cells expresses germ line encoded light chains. This autoreactive B cell population is normally deleted at the immature B cell stage, but can be detected in mice transgenic for both the R4A-c2b heavy chain and bcl-2, as well as in NZB/W lupus-prone R4A-c2b mice [69]. The second B cell population that expresses antibodies with high-affinity for DNA displays somatically mutated light chains, suggesting that autoreactivity develops after an encounter with antigen and germinal center maturation. This population is anergic and cannot be activated by engagement of the B cell receptor (BCR), but can be induced to secrete antibody by stimulation with LPS [66,68]. R4Ac2b mice, because of their well-characterized B cell populations, provide an excellent model in which to study the effects of hormones on B cells. To investigate how prolactin affects survival, maturation and activation of the autoreactive B cell populations in R4Ac2b mice, we injected mice with sufficient prolactin to cause a twofold increase in serum prolactin levels (50–90 ng/ml vs. 20–35 ng/ml), similar to the modest hyperprolactinemia seen in SLE patients. Since in early murine pregnancy prolactin reaches 50 and 125 ng/ml before partuation [70,71], our prolactin-treatment remained within physiologic levels of prolactin. Because prolactin provides negative feedback for estrogen secretion, all prolactin studies were performed in ovariectomized mice. We demonstrated that mild hyperprolactinemia
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Fig. 1. (A) Anti-DNA reactivity. Fourteen R4A-c2b BALB/c mice were treated with murine prolactin (100 mg/daily) and 12 were treated with placebo (normal saline) for 4 weeks. Anti-ds DNA titers were measured before initiation of treatment and weekly thereafter for 4 weeks. At the end of the treatment, serum anti-DNA reactivity was significantly increased in prolactin-treated mice compared to placebo-treated mice (P = 0.02). (B) Immunohistochemistry of kidney sections. Mice described above were sacrificed at the end of the treatment, and their kidneys were evaluated for IgG deposition. IgG deposits were observed in prolactin-treated mice. Magnification ×10.
is able to break tolerance and induce a lupus-like phenotype in R4A-c2b BALB/c mice. Prolactin-treated R4A-c2b BALB/c mice developed an increased number of transgene expressing B cells and B cells that spontaneously secrete anti-dsDNA antibodies, and increased serum anti-DNA antibody titers (Fig. 1a). Furthermore, prolactin-induced antidsDNA antibodies are potentially nephritogenic as the mice develop glomerular IgG deposition (Fig. 1b). These prolactin-mediated effects are, at least in part, T cell dependent [72] as prolactin fails to induce a lupus-like illness in R4A-c2b BALB/c mice deficient in CD4+ T cells. The majority of transgene-expressing B cells are located inside the splenic follicles (Fig. 2b). Similarly, the autoreactive B cells that spontaneously secrete high-affinity anti-dsDNA antibodies have a mature follicular B cell phenotype (CD21intermediate, CD23high). These observations are consistent with the known fact that follicular B cells participate in T cell dependent responses and their activation can be blocked by CD4 T cell depletion [73], or costimulatory blockade [74,75]. Prolactin affects B cell development and maturation, causing a decrease in the percentage of immature B cells in the spleen (Fig. 3). As B cells mature from T1 to T2 stage, they are susceptible to negative selection. Normally, there are more T1 than T2 B cells in the spleen reflecting the loss of autoreactive cells occurring at the T1 to T2 transition [76,77]. In prolactin-treated mice, there is an inversion of the T1/T2 ratio with more T2 than T1 cells, suggesting that prolactin diminishes negative selection of immature B cells. Prolactin leads to an increase of all mature B cells, marginal zone and follicular B cells (Fig. 3), but as shown above, the B cells
spontaneously secreting anti-DNA antibodies in prolactintreated mice belong to the follicular subset. At the molecular level, prolactin causes upregulation of the costimulatory molecule CD40 and the anti-apoptotic protein bcl-2 in B cells. We believe that both of these molecules may play an important role in rescuing B cells from negative selection. CD40 engagement has been shown to rescue transitional B cells from BCR-mediated apoptosis [77], providing a mechanistic explanation for the increased survival of autoreactive B cells. CD40 ligation is essential for antibody production by follicular B cells [78]. Lupus-prone mice treated with anti-CD40L antibody display lower titers of anti-DNA antibody [73]. Prolactin has been shown to increase CD40 expression on dendritic cells, which may also lead to their enhanced function as an antigen presenting cells [79]. Thus, upregulation of CD40 expression may represent one mechanism by which prolactin enhances autoantibody secretion. Bcl-2 is also upregulated by prolactin in B cells. Bcl-2 is important for B cell survival at both immature and mature stages of development [80–82]. Therefore, prolactinmediated upregulation of Bcl-2 expression may represent a crucial mechanism for decreased B cell apoptosis in prolactin-treated mice and may contribute to the increased number of T2 B cells. Characterization of autoreactive B cells in prolactintreated mice was accomplished by hybridoma production. Although high-affinity DNA-reactive hybridomas cannot be generated from B cells from naive R4A c2b BALB/c mice [65], they can be obtained from hybridomas generated from B cells from prolactin-treated mice. Analysis of the light chains of the high-affinity anti-dsDNA antibody secreting
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Fig. 2. (A) c2b-Expressing B cells. Peripheral B cells from the spleen were analyzed in seven placebo and seven prolactin-treated R4A-c2b BALB/c mice. The c2b expressing B cell population was expanded in prolactin-treated mice compared to placebo-treated mice (P = 0.0l). (B) Localization of c2b expressing B cells in the spleens of placebo and prolactin-treated mice. B cells were labeled with anti-IgM staining (blue), while c2b-expressing B cells were identified with anti-c2b staining (yellow). Prolactin-treated mice displayed an increased number of c2b-expressing B cells which were localized mainly in the follicles. In placebo-treated mice, c2b-expressing B cells were localized in the red pulp and at the T cell–B cell interface.
Fig. 3. Effects of prolactin on B cell maturation. Splenocytes from five and three BALB/c mice treated with prolactin or placebo, respectively, were stained with antibodies to CD19, CD21, CD24 and CD23. The transitional T1 and T2 subsets, as well as the follicular subset, were identified with CD21 and CD24 staining, while the marginal zone B cell subset was identified by CD21 and CD23 staining. In prolactin-treated mice, immature B cells (CD24high) were decreased (P = 0.002), while the mature subsets follicular (CD21intermediate CD24low) and marginal zone (CD21high CD23low) were increased (P = 0.002 and P = 0.005, respectively).
clones revealed that autoreactive antibodies in prolactintreated mice predominantly use light chains encoded by germline genes, especially VK1A gene. This demonstrates that the autoreactive B cells rescued by prolactin are derived from the naive B cell repertoire. Thus, the DNA-reactive B cell population that is present in prolactin-treated mice would normally undergo deletion during the transitional stage of maturation in the untreated transgenic mice. Even though prolactin induces a lupus-like phenotype in R4A-c2b BALB/c mice, it does not break tolerance in R4Ac2b C57B1/6 mice. Prolactin does not cause an expansion of
transgene-expressing B cells in the spleen, an elevated number of DNA-reactive B cells, an increase in anti-DNA antibody titers or glomerular IgG deposits (Fig. 4a–c). In these mice, prolactin also does not alter B cell development and maturation (EP, VJ unpublished data). Thus, the effects of prolactin on the immune system are genetically determined and strain-specific. It is possible that there are some lupus patients with a prolactin-exacerbated disease and some with a prolactin non-responsive disease. Ultimately, we hope that an analysis of gene expression by microarray will allow us to differentiate among mouse strains and lupus patients, and identify those whose disease can be exacerbated by prolactin and might benefit from prolactin inhibition. Human and murine studies of SLE have also implicated estrogen in disease pathogenesis. However, prolactin and estrogen each break tolerance through a different mechanism. In prolactin-treated mice, the follicular B cell subset harbors the autoreactive B cells [83], while in estrogen treated mice, autoreactive B cells derive from the marginal zone B cell subset [84]. Estrogen, like prolactin, upregulates bcl-2, but also upregulates expression of CD22 and SHP-1 diminishing BCR signaling. We speculate that in estrogen treated mice, the increased CD22 and SHP-1 expression leads to a decreased BCR signal, escape from negative selection and the preferential development of marginal zone B cells [85]. The observation that distinct B cell subsets are responsible for anti-DNA antibody production in prolactin and estrogen treated mice, and that different mechanisms are involved in the breakdown of tolerance, leads to the hypothesis that specific interventions can be employed to target patients with
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Fig. 4. Effects of prolactin in C57B1/6 mice. R4A-c2b C57B16 mice treated with prolactin or placebo (10 in each group) were evaluated for anti-DNA antibody titers and renal IgG deposition. (A) Serum anti-DNA reactivity. Anti-dsDNA titers in the serum were measured before initiation of treatment and weekly for 4 weeks. No difference in anti-DNA reactivity was found between prolactin and placebo-treated mice. (B) c2b-expressing B cells. Prolactin had no effect on the number of c2b-expressing B cells. (C) Immunohistochemistry of kidney sections. Staining with anti-IgG antibody did not reveal deposits in either prolactin or placebo treated mice.
hormonally exacerbated disease. For example, costimulatory blockade may provide beneficial therapeutic effect in SLE patients with a prolactin-triggered disease, while interfering with BAFF or the use of estrogen receptor modulators may be more effective in individuals with an estrogen-triggered disease. 3.1. Prolactin in estrogen-induced lupus The difference in autoreactive B cell phenotype in estrogen [84,85] and prolactin-treated mice [83] suggests that estrogen does not break tolerance by upregulating prolactin. We decided, however, to ask directly whether estrogeninduced disease could be abrogated by blocking prolactin secretion. We demonstrated that a 5-week treatment with estrogen that provides serum estrogen levels of 75– 100 pg/ml (levels found at the high end of the estrus cycle) could induce a lupus-like serology in R4A c2b BALB/c mice [86]. To evaluate whether prolactin plays a role in estrogenmediated autoimmunity, we used bromocriptine to suppress prolactin secretion. Bromocriptine, a dopamine agonist, acts as a prolactin release inhibitor that has been shown to have therapeutic effect in several autoimmune diseases, including murine lupus [56,59,62]. The major immunosuppressive activity of bromocriptine is based on its hypoprolactinemic effect [63], as prolactin can abrogate bromocriptine-induced humoral and cell-mediated immunosuppression [87], as well as bromocriptine-induced decrease in IFN c production by macrophages [88]. Treatment with bromocriptine prevented the development of elevated anti-dsDNA antibody titers (Fig. 5) and a lupus-
phenotype in estrogen-treated R4A c2b BALB/c mice [89]. Estrogen and bromocriptine-treated mice, however, displayed an expansion of transgene-expressing B cells (Fig. 6a) and upregulation of the anti-apoptotic protein Bcl-2, demonstrating that bromocriptine does not suppress estrogenmediated survival of autoreactive B cells. Consistent with this observation is the altered ratio of transitional T1 to T2 B cells present in these mice [83]. Interestingly, transgene expressing B cells which are localized in the marginal zone of the splenic follicles in estrogen treated mice [84], are distributed in the red pulp in estrogen and bromocriptine treated mice (Fig. 6b) and marginal zone B cells are not expanded in estrogen and bromocriptine treated mice (Fig. 7). Furthermore, in estrogen and bromocriptine treated mice, DNAreactive hybridomas can be generated only from LPSstimulated B cells. The light chains of these hybridomas were encoded by germline genes demonstrating that the naive DNA-reactive B cells that are rescued from deletion by estrogen [86] are still present in absence of prolactin, but in a functionally inactive state. Adoptive transfer of naïve or estrogen-exposed T cells to estrogen and bromocriptine treated mice did not lead to an expansion of the c2b expressing B cell population (Fig. 8a) or an increased number of B cells that spontaneously secrete anti-DNA antibodies or secrete antibody after stimulation with anti-CD40 antibody and IL-4 (Fig. 8b). Thus, help provided by naïve or estrogen modulated T cells, is not sufficient to induce a lupus-like phenotype in estrogen and bromocriptine treated mice. The effect of bromocriptine in abrogating the effect of estrogen is not T cell mediated.
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Fig. 5. Effects of bromocriptine on estrogen-modulated B cells. (A) Anti-DNA reactivity. R4A-c2b BALB/c mice were treated with estrogen pellets that provide 75 pg/ml estradiol, estrogen and 400 mg of bromocriptine injected intraperitoneally daily or with placebo for 6 weeks. Anti-dsDNA antibody titers were measured before initiation of treatment and weekly thereafter for 6 weeks. At the end of the treatment, serum anti-DNA reactivity was significantly increased only in estrogen-treated mice compared to estrogen and bromocriptine (P = 0.01) and placebo treated mice (P < 0.01). (B) c2b expressing B cells. Splenic B cells were evaluated for the number of c2b B expressing cells. Compared to placebo, there was an expansion of c2b expressing B cell population in both estrogen and estrogen and bromocriptine treated mice (P = 0.01 and 0.04, respectively).
Fig. 6. Localization of c2b expressing B cells in estrogen and bromocriptine treated mice. B cells and c2b-expressing B cells were labeled with anti-IgM antibody (blue) and anti-c2b antibody (yellow), respectively. In placebo-treated mice, c2b-expressing B cells were localized in the red pulp and in the T cell–B cell interface. Both estrogen and, estrogen and bromocriptine treated mice displayed an increased number of c2b-expressing B cells. However, in estrogentreated mice, c2b-expressing B cells were localized in the marginal zone, while in estrogen and bromocriptine treated mice they were confined to the red pulp and B cell–T cell interface.
4. Conclusions There is wealth of data implicating prolactin in the pathogenesis of human and murine lupus. Approximately 20% of SLE patients display some degree of hyperprolactinemia and administration of prolactin exacerbates disease activity causing early mortality in murine models of lupus. Additionally, prolactin can induce a lupus-like phenotype in nonspontaneously autoimmune mice. In mice with a susceptible genetic background, hyperprolactinemia leads to an increased survival and activation of autoreactive B cells with a subsequent increase in anti-DNA antibody titers and IgG deposits in the kidneys. Prolactin mediates these effects in part by altering B cell development, leading to decreased negative selection and an increased number of mature B cells. The follicular subset has been shown to harbor autoreactivity in prolactin-treated mice. Thus, it is not surprising that prolactin exerts its immunostimulatory effects on B cells only in presence of CD4+ T cells.
At a molecular level, prolactin upregulates expression of Bcl-2 and CD40 in B cells, causing a decrease in apoptosis and an increased susceptibility to costimulation, both of which may contribute to the survival and rescue of B cells that are signaled by antigen to undergo negative selection. Prolactin does not affect the strength of the BCR signal. Effects of prolactin on B cells are genetically determined. The fact that prolactin breaks tolerance and induces a lupus phenotype in one mouse strain but not in other, suggests that only a subset of SLE patients is likely to have a prolactinresponsive disease. Microarray studies may help distinguish between lupus patients with a prolactin-responsive disease and those with a prolactin-unresponsive disease. Given that in prolactin-induced autoimmunity, T cells play a crucial role and that follicular B cells harbor autoreactivity, therapies that interfere with costimulation may provide beneficial effect in patients with prolactin-mediated disease.
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Fig. 7. B cell subsets. To evaluate the effects of bromocriptine on estrogenmediated modulation of B cell development, splenocytes from five estrogen, three estrogen and bromocriptine and three placebo treated BALB/c mice were stained with antibodies to CD19, CD21, CD24 and CD23. Although both estrogen and, estrogen and bromocriptine treated mice displayed an inverted T1/T2 ratio, estrogen treated mice showed a significant expansion of the marginal zone (CD21high CD23low) B cells compared to estrogen and bromocriptine or placebo-treated mice (P = 0.02).
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[19] Fig. 8. Adoptive transfer of CD4+T cells from mice treated with estrogen or placebo. CD4+ T cells from mice treated with estrogen or placebo for 5 weeks (n = 5 in both groups) were transferred to mice treated with estrogen and bromocriptine for 6 weeks (n = 5). (A) Enumeration of anti-DNA antibody secreting B cells. Adoptive transfer of naïve and estrogenmodulated CD4+ T cells did not lead to increased number of autoreactive B cells that secrete anti-DNA antibodies spontaneously or after stimulation with LPS or anti-CD40/IL4 compared to a control group of estrogen and bromocriptine treated mice (n = 5). (B) Analysis of peripheral lymphocytes. Estrogen and bromocriptine treated mice, which underwent adoptive transfer of naïve or estrogen-modulated CD4+ T cells, did not develop an expansion of c2b expressing B cells compared to estrogen and bromocriptine treated mice which did not receive T cell transfer.
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E. Peeva et al. / Biomedicine & Pharmacotherapy 58 (2004) 310–319 [65] Offen D, Spatz L, Escowitz H, Factor S, Diamond B. Induction of tolerance to an IgG autoantibody. Proc Natl Acad Sci USA 1992;89: 8332–6. [66] Spatz L, Saenko V, Iliev A, Jones L, Geskin L, Diamond B. Light chain usage in anti-dsDNA B cell subsets: role in cell fate determination. J Exp Med 1997;185:1317–26. [67] Bynoe M, Spatz L, Diamond B. Characterization of anti-DNA B cells that escape negative selection. Eur J Immunol 1999;29:1304–13. [68] Iliev A, Spatz L, Ray S, Diamond B. Lack of allelic exclusion permits autoreactive B cells to escape deletion. J Immunol 1994;153:3551–6. [69] Kuo P, Bynoe M, Wang C, Diamond B. Bcl-2 leads to expression of anti-DNA B cells but no nephritis: a model for clinical subset. Eur J Immunol 1999;29:3168–78. [70] Fliestra R, Voogt J. Lactogenic hormones of the placenta and pituitary inhibit suckling-induced prolactin (PRL) release but not the antepartum PRL surge. Proc Soc Exp Biol Med 1997;214:258–64. [71] Freemark M, Kirk K, Pihoker C, Robertson M, Shiu R, Driscoll P. Pregnancy lactogens in the rat conceptus and fetus: circulating levels, distribution of binding, and expression of receptor messenger ribonucleic acid. Endocrinology 1993;133:1830–42. [72] Taylor S, Hickman J, Dive C. Survival signals within the tumour microenvironment suppress drug-induced apoptosis: lessons learned from B lymphomas. Endocr Relat Cancer 1999;6:21–3. [73] Wofsy D, Mayes D, Woodcock J, Seaman W. Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4: studies with soluble antigens in intact mice. J Immunol 1985;135:1698–701. [74] Mihara M, Tan I, Chuzhin Y, et al. CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J Clin Invest 2000;106:91–101. [75] Wang X, Huang W, Schiffer L, et al. Effects of anti-CD154 treatment on B cells in murine systemic lupus erythematosus. Arthritis Rheum 2003;48:495–506.
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Dossier: Autoimmunity and Biotherapy
Prolactin as a modulator of B cell function: implications for SLE Elena Peeva a,b, Jeganathan Venkatesh a, Daniel Michael a, Betty Diamond a,b,* a
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Rm. 405, Forch Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA b Department of Medicine, Albert Einstein College of Medicine, Rm. 405, Forch Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA Received 10 February 2004 Available online 27 April 2004
Abstract Prolactin is not only a lactigenic hormone but also an immunomodulator involved in lymphocyte survival, activation and proliferation. There is increasing data implicating prolactin in autoimmunity, and specifically in SLE. Increased serum prolactin levels have been reported in lupus patients of both genders, and have been associated with accelerated disease expression and early mortality in lupus-prone mice. Furthermore, suppression of prolactin secretion with bromocriptine provides beneficial effects in murine lupus, and perhaps in some SLE patients as well. Treatment with prolactin that causes mild to moderate hyperprolactinemia, similar to that present in SLE patients, breaks tolerance and induces a lupus-like illness in non-spontaneously autoimmune mice with a susceptible genetic background. These immuno stimulatory effects of prolactin are mediated by a decrease in negative selection and the maturation of autoreactive B cells to the follicular subset. Consistent with the fact that follicular B cells are T cell dependent, CD4+ T cells are necessary for the prolactin-mediated break down of B cell tolerance. In mice, the effects of prolactin on the immune system are genetically determined, suggesting that only a subset of SLE patients are likely to have a prolactin-responsive disease. The manipulation of serum prolactin or, even more specifically, follicular B cells that are susceptible to the immuno stimulatory effects of prolactin, may provide novel therapeutic options for those SLE patients with a prolactin-modulated disease. © 2004 Elsevier SAS. All rights reserved. Keywords: Prolactin; B cell; SLE
1. Introduction Over the past two decades, significant evidence has accumulated that the anterior pituitary hormone prolactin, well known for its lactogenic function, acts also as an important connection between the endocrine and immune systems. Prolactin is a 200 amino acid peptide hormone produced not only by the anterior pituitary, but also by many extrapituitary sites including immune cells [1]. Prolactin functions as an immunomodulator and affects apoptosis, activation and proliferation of immune cells. During the child-bearing age, females have higher serum prolactin levels than males; this difference starts in puberty as a result of increased estrogen levels. Estrogen increases prolactin secretion from the anterior pituitary by inhibition of dopaminergic suppression [2]. During pregnancy, serum prolactin increases and reaches a maximum concentration post* Corresponding author. E-mail address: [email protected] (B. Diamond). © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biopha.2004.04.007
partum during lactogenesis. Prolactin levels correlate with the amount of immunoglobulin in the milk. After weaning, prolactin levels return to normal [2]. Therefore, in women prolactin plays an important role in reproductive physiology. But prolactin also exerts multiple effects on the immune system. In this article, we will review the immunomodulatory effects of prolactin and the effects of hyperprolactinemia on the expression of SLE in mice and humans. We will focus on prolactin-induced alterations in B cell maturation and repertoire selection. 2. Immunomodulatory mechanisms of prolactin It is not surprising that prolactin modulates functions of the immune system. Prolactin receptors are expressed on many cells of the immune system, including hematopoietic stem cells, T cells, B cells, monocytes, macrophages, NK cells, neutrophils and thymic epithelial cells [3–5]. Lymphocytes express fewer receptors per cell than macrophages. Among lymphocytes B cells express the highest levels of
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prolactin receptors [1,6]. The prolactin receptor is a transmembrane protein of the type I cytokine receptor family, which also includes receptors for growth hormone, erythropoietin, thrombopoietin, granulocyte/macrophage-colony stimulating factor and many interleukins [7]. Prolactin receptors bind not only lactogenic hormones (prolactin and placental lactogen), but also growth hormone [1,8] suggesting an overlap and redundancy in prolactin receptor signaling. Three isoforms of the prolactin receptor have been described in rodents, while in humans four isoforms have been observed, one of which is unable to signal, but can bind prolactin and, therefore, exerts an inhibitory function [9,10]. Prolactin binding leads to receptor dimerization which in both rodents and man, induces signaling through Janus tyrosine kinase (JAK) 2 and transcription factors Stat1, 3 and 5 [11,12] leading to activation of interferon regulating factor-1 (IRF-1) [13], inducible nitric oxide synthase and suppressors of cytokine signaling (SOCS) 2, 3 and 7 [14]. Prolactininduced activation of the Src and Tec family of tyrosine kinases, SHP-2 phosphatase, ZAP-7, MAPK signaling pathways has also been demonstrated [15]. A portion of secreted prolactin is phosphorylated [16]; since phosphorylated prolactin is reported to act as a partial agonist [17,18], the ratio between unphosphorylated and phosphorylated prolactin may be physiologically relevant. Prolactin is implicated in lymphoproliferation, cytokine production and antibody secretion [19]. Whether prolactin is important in lymphopoiesis is not clear. Administration of prolactin stimulates progenitor cells in vitro [20], and transfection of the rat prolactin receptor into a pro-B cell line leads to an expansion of B220+ pro-B cells demonstrating that prolactin may exert a direct effect on B cell development [21]. Prolactin may also function in early T cell development. For example, prolactin regulates the maturation of CD4–CD8– thymocytes to CD4+CD8+ T cells through induction of IL-2 receptor expression [22]. Additionally, it has been observed that changes in the ratio of unphosphorylated to phosphorylated prolactin affect developing cd T cells. The offspring of rats treated throughout pregnancy with a recombinant molecular mimic of phosphorylated prolactin demonstrate a lasting decrease of epidermal cd T cells [23]. Yet, both prolactin deficient [24] and prolactin receptor deficient mice [25] have normal hematopoiesis. Thus, prolactin is not essential for lymphopoiesis and it appears that other factors can compensate for its deficiency. Prolactin also affects lymphocyte survival, altering apoptotic pathways that are essential for lymphocyte homeostasis and tolerance induction. It has been shown that prolactin exerts an anti-apoptotic effect in a rat pre-T cell line by upregulation of the anti-apoptotic genes Bcl-2, Bcl-xl, X-linked inhibitor of apoptosis (XIAP) and the protooncogene Pim-1 and by down-regulation of the pro-apoptotic gene Bax [19,26–28]. It has been also demonstrated that prolactin upregulates Bcl-2 in a dose dependent manner in a human IgE secreting myeloma cell line [29]. In vitro prolactin does not induce mouse lymphocyte proliferation [30], but prolactin enhances ConA induced pro-
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liferation of rat splenocytes in dose dependent manner [31]. Anti-prolactin antibodies inhibit ConA or phytohemagglutinin-induced proliferation of human PBMCs [32], causing an arrest of cells in the G1 phase. This inhibition is reversed by addition of purified prolactin, but not growth hormone [30]. Thus, prolactin contributes for mitogeninduced proliferation of lymphocytes in rodents and humans. Prolactin is an important regulator of cytokine production. There is some evidence that the Th1 cytokine IFNc as well as the Th2 cytokine IL-6 are upregulated by prolactin. Physiologic concentrations of prolactin stimulate normal human PBMCs to produce IFNc through activation of IRF-1. The response is dose dependent [33], suggesting that hyperprolactinemia may be responsible for increased IFNc in some individuals. Interestingly, decreased IL-2 production has been reported in hyperprolactinemic women with pituitary tumors and is corrected when normal prolactin levels are restored [34]. Prolactin causes a dose dependent increase in immunoglobulin secretion from anti-IgM and IL-2 stimulated PBMCs from healthy people [35]. In hyperprolactinemic mice, there is an increased number of antibody-secreting splenocytes, which correlates with the degree of prolactinemia, although the mechanism for this is not known. A study of a small number of lupus patients with CNS disease activity showed increased IL-6 in the cerebrospinal fluid. The increase correlated with the prolactin concentration, suggesting a possible association [36], and perhaps suggesting that prolactin may enhance antibody secretion through regulating IL-6 production, but this remains speculative. 2.1. Hyperprolactinemia in SLE patients Emerging data implicate prolactin in the pathogenesis of SLE, yet the mechanisms are not well understood. Initially, in the mid 1980s increased serum prolactin levels were described in male patients with SLE and in the early 1990s hyperprolactinemia above-expected levels were reported in pregnant lupus patients with active disease. Approximately 20% of SLE patients express some degree of hyperprolactinemia [37–40]. An association of increased prolactin levels with anti-dsDNA antibodies and with anti-Ro and anti-La antibodies has been reported [41]. However, attempts to correlate disease activity or specific organ involvement with the serum prolactin levels have led to equivocal results [42–48] suggesting that the degree of hyperprolactinemia is not a crucial factor in determining target organ susceptibility or autoantibody specificity. In the majority of lupus patients with hyperprolactinemia, the etiology of the increased prolactin levels is unknown. It was initially suggested that a G 1149T polymorphism of the prolactin gene is associated with SLE [49], but this has not been confirmed in a subsequent study [50]. In a minority of patients, hyperprolactinemia is caused by prolactinomas, medications, chronic renal insufficiency, thyroid insufficiency or hyperparathyroidism [51,52]. Some lupus patients
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have antibodies against prolactin [53], which are thought to dysregulate prolactin secretion and induce hyperprolactinemia. The immune complexes of prolactin with anti-prolactin antibodies are not hormonally active. Thus, SLE patients with prolactin-IgG complexes may have less active disease than other lupus patients with hyperprolactinemia [54]. Prolactin increases production of IgM, IgG and antidsDNA antibodies in PBMCs from SLE patients [55]. In vitro, prolactin induces increased secretion of IFN-c and IL-2 by peripheral mononuclear cells (PBMCs) from lupus patients [33] although hyperprolactinemia may be associated in vivo with reduced IL-2. Additionally, unstimulated PBMCs from lupus patients produced more prolactin than PBMCs from healthy individuals [55]. Indirect support for the role of prolactin in the pathogenesis of SLE has been provided by human studies that have investigated the therapeutic effects of prolactin inhibition. Bromocriptine is a dopamine receptor agonist that inhibits prolactin secretion. Small clinical studies showed beneficial effect of bromocriptine in the treatment of SLE patients with mild to moderately active disease [56,57]. A 6-month prospective open label trial with bromocriptine in seven lupus patients demonstrated clinical improvement in disease activity scores associated with a decrease in anti-DNA antibody titer and serum IgG levels. After the treatment, during a 5-month follow up, all patients developed increased disease activity and in two patients hyperprolactinemia corresponded with the lupus flares [56]. Another clinical study showed that in non-organ threatening lupus, the therapeutic effect of bromocriptine is comparable to that of hydroxychloroquine [57], which is a widely accepted treatment for SLE. 2.2. Hyperprolactinemia and murine lupus Studies of murine models of lupus have provided clear evidence that prolactin can exacerbate autoimmunity [40,58– 61]. Hyperprolactinemia induced by either treatment with prolactin or pituitary transplants leads to a more aggressive disease in lupus-prone mice. In female NZB/W mice, a 3–18fold increase in serum prolactin levels induced early proteinuria, elevated serum IgG and circulating immune complexes, and accelerated mortality [40,59]. Physiologic hyperprolactinemia generated by pseudopregnancy also caused worsening of disease activity [60]. In male NZB/W mice, in which the disease develops later and has a milder course, both moderate (116–239 ng/ml) and severe hyperprolactinemia (275–424 ng/ml) induced early onset of disease and premature mortality. Thus, a sustained mild to moderate increase in serum prolactin levels (100–200 ng/ml) is sufficient to accelerate disease activity and decrease longevity in NZB/W mice [40]. There was, however, no linear correlation between the degree of hyperprolactinemia and disease activity. In the same murine model of lupus, it has been shown that deleterious effects of hyperprolactinemia are not mediated via suppression of immunosuppressive androgens [60]. In MRL-lpr/fas lupus-prone mice, lactation leads to post-
partum arthritic flares [61]. Treatment with bromocriptine decreases anti-DNA titers and improves survival in the NZB/W mouse model of lupus [59,62,63]. Also, bromocriptine suppresses development of anti-DNA antibodies in MIV-7 monoclonal antibody-induced lupus in BALB/c mice [64].
3. Mechanisms of prolactin induced abrogation of B cell tolerance We have demonstrated that treatment with prolactin can induce a lupus-like serology in non-spontaneously autoimmune mice. BALB/c mice transgenic for the IgG2b heavy chain of the R4A nephritogenic anti-dsDNA antibody maintain B cell tolerance, have negligible titers of anti-DNA antibodies and no renal pathology [65]. Approximately 5% of B cells in these mice express the transgene-encoded heavy chain, while the rest express an endogenous µ heavy chain. The transgene-encoded heavy chain can pair with a variety of light chains producing antibodies with different affinities for DNA as well as antibodies that do not react with DNA. Three distinct DNA-reactive B cell populations have been described in R4A-c2b mice, one that secretes low affinity anti-DNA antibodies and two which can produce highaffinity anti-DNA antibodies [66–69]. The B cell population that secretes low affinity anti-DNA antibodies is not subject to tolerance induction; rather, it matures to immunocompetence in peripheral lymphoid organs [67]. One population of the high-affinity DNA-reactive B cells expresses germ line encoded light chains. This autoreactive B cell population is normally deleted at the immature B cell stage, but can be detected in mice transgenic for both the R4A-c2b heavy chain and bcl-2, as well as in NZB/W lupus-prone R4A-c2b mice [69]. The second B cell population that expresses antibodies with high-affinity for DNA displays somatically mutated light chains, suggesting that autoreactivity develops after an encounter with antigen and germinal center maturation. This population is anergic and cannot be activated by engagement of the B cell receptor (BCR), but can be induced to secrete antibody by stimulation with LPS [66,68]. R4Ac2b mice, because of their well-characterized B cell populations, provide an excellent model in which to study the effects of hormones on B cells. To investigate how prolactin affects survival, maturation and activation of the autoreactive B cell populations in R4Ac2b mice, we injected mice with sufficient prolactin to cause a twofold increase in serum prolactin levels (50–90 ng/ml vs. 20–35 ng/ml), similar to the modest hyperprolactinemia seen in SLE patients. Since in early murine pregnancy prolactin reaches 50 and 125 ng/ml before partuation [70,71], our prolactin-treatment remained within physiologic levels of prolactin. Because prolactin provides negative feedback for estrogen secretion, all prolactin studies were performed in ovariectomized mice. We demonstrated that mild hyperprolactinemia
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Fig. 1. (A) Anti-DNA reactivity. Fourteen R4A-c2b BALB/c mice were treated with murine prolactin (100 mg/daily) and 12 were treated with placebo (normal saline) for 4 weeks. Anti-ds DNA titers were measured before initiation of treatment and weekly thereafter for 4 weeks. At the end of the treatment, serum anti-DNA reactivity was significantly increased in prolactin-treated mice compared to placebo-treated mice (P = 0.02). (B) Immunohistochemistry of kidney sections. Mice described above were sacrificed at the end of the treatment, and their kidneys were evaluated for IgG deposition. IgG deposits were observed in prolactin-treated mice. Magnification ×10.
is able to break tolerance and induce a lupus-like phenotype in R4A-c2b BALB/c mice. Prolactin-treated R4A-c2b BALB/c mice developed an increased number of transgene expressing B cells and B cells that spontaneously secrete anti-dsDNA antibodies, and increased serum anti-DNA antibody titers (Fig. 1a). Furthermore, prolactin-induced antidsDNA antibodies are potentially nephritogenic as the mice develop glomerular IgG deposition (Fig. 1b). These prolactin-mediated effects are, at least in part, T cell dependent [72] as prolactin fails to induce a lupus-like illness in R4A-c2b BALB/c mice deficient in CD4+ T cells. The majority of transgene-expressing B cells are located inside the splenic follicles (Fig. 2b). Similarly, the autoreactive B cells that spontaneously secrete high-affinity anti-dsDNA antibodies have a mature follicular B cell phenotype (CD21intermediate, CD23high). These observations are consistent with the known fact that follicular B cells participate in T cell dependent responses and their activation can be blocked by CD4 T cell depletion [73], or costimulatory blockade [74,75]. Prolactin affects B cell development and maturation, causing a decrease in the percentage of immature B cells in the spleen (Fig. 3). As B cells mature from T1 to T2 stage, they are susceptible to negative selection. Normally, there are more T1 than T2 B cells in the spleen reflecting the loss of autoreactive cells occurring at the T1 to T2 transition [76,77]. In prolactin-treated mice, there is an inversion of the T1/T2 ratio with more T2 than T1 cells, suggesting that prolactin diminishes negative selection of immature B cells. Prolactin leads to an increase of all mature B cells, marginal zone and follicular B cells (Fig. 3), but as shown above, the B cells
spontaneously secreting anti-DNA antibodies in prolactintreated mice belong to the follicular subset. At the molecular level, prolactin causes upregulation of the costimulatory molecule CD40 and the anti-apoptotic protein bcl-2 in B cells. We believe that both of these molecules may play an important role in rescuing B cells from negative selection. CD40 engagement has been shown to rescue transitional B cells from BCR-mediated apoptosis [77], providing a mechanistic explanation for the increased survival of autoreactive B cells. CD40 ligation is essential for antibody production by follicular B cells [78]. Lupus-prone mice treated with anti-CD40L antibody display lower titers of anti-DNA antibody [73]. Prolactin has been shown to increase CD40 expression on dendritic cells, which may also lead to their enhanced function as an antigen presenting cells [79]. Thus, upregulation of CD40 expression may represent one mechanism by which prolactin enhances autoantibody secretion. Bcl-2 is also upregulated by prolactin in B cells. Bcl-2 is important for B cell survival at both immature and mature stages of development [80–82]. Therefore, prolactinmediated upregulation of Bcl-2 expression may represent a crucial mechanism for decreased B cell apoptosis in prolactin-treated mice and may contribute to the increased number of T2 B cells. Characterization of autoreactive B cells in prolactintreated mice was accomplished by hybridoma production. Although high-affinity DNA-reactive hybridomas cannot be generated from B cells from naive R4A c2b BALB/c mice [65], they can be obtained from hybridomas generated from B cells from prolactin-treated mice. Analysis of the light chains of the high-affinity anti-dsDNA antibody secreting
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Fig. 2. (A) c2b-Expressing B cells. Peripheral B cells from the spleen were analyzed in seven placebo and seven prolactin-treated R4A-c2b BALB/c mice. The c2b expressing B cell population was expanded in prolactin-treated mice compared to placebo-treated mice (P = 0.0l). (B) Localization of c2b expressing B cells in the spleens of placebo and prolactin-treated mice. B cells were labeled with anti-IgM staining (blue), while c2b-expressing B cells were identified with anti-c2b staining (yellow). Prolactin-treated mice displayed an increased number of c2b-expressing B cells which were localized mainly in the follicles. In placebo-treated mice, c2b-expressing B cells were localized in the red pulp and at the T cell–B cell interface.
Fig. 3. Effects of prolactin on B cell maturation. Splenocytes from five and three BALB/c mice treated with prolactin or placebo, respectively, were stained with antibodies to CD19, CD21, CD24 and CD23. The transitional T1 and T2 subsets, as well as the follicular subset, were identified with CD21 and CD24 staining, while the marginal zone B cell subset was identified by CD21 and CD23 staining. In prolactin-treated mice, immature B cells (CD24high) were decreased (P = 0.002), while the mature subsets follicular (CD21intermediate CD24low) and marginal zone (CD21high CD23low) were increased (P = 0.002 and P = 0.005, respectively).
clones revealed that autoreactive antibodies in prolactintreated mice predominantly use light chains encoded by germline genes, especially VK1A gene. This demonstrates that the autoreactive B cells rescued by prolactin are derived from the naive B cell repertoire. Thus, the DNA-reactive B cell population that is present in prolactin-treated mice would normally undergo deletion during the transitional stage of maturation in the untreated transgenic mice. Even though prolactin induces a lupus-like phenotype in R4A-c2b BALB/c mice, it does not break tolerance in R4Ac2b C57B1/6 mice. Prolactin does not cause an expansion of
transgene-expressing B cells in the spleen, an elevated number of DNA-reactive B cells, an increase in anti-DNA antibody titers or glomerular IgG deposits (Fig. 4a–c). In these mice, prolactin also does not alter B cell development and maturation (EP, VJ unpublished data). Thus, the effects of prolactin on the immune system are genetically determined and strain-specific. It is possible that there are some lupus patients with a prolactin-exacerbated disease and some with a prolactin non-responsive disease. Ultimately, we hope that an analysis of gene expression by microarray will allow us to differentiate among mouse strains and lupus patients, and identify those whose disease can be exacerbated by prolactin and might benefit from prolactin inhibition. Human and murine studies of SLE have also implicated estrogen in disease pathogenesis. However, prolactin and estrogen each break tolerance through a different mechanism. In prolactin-treated mice, the follicular B cell subset harbors the autoreactive B cells [83], while in estrogen treated mice, autoreactive B cells derive from the marginal zone B cell subset [84]. Estrogen, like prolactin, upregulates bcl-2, but also upregulates expression of CD22 and SHP-1 diminishing BCR signaling. We speculate that in estrogen treated mice, the increased CD22 and SHP-1 expression leads to a decreased BCR signal, escape from negative selection and the preferential development of marginal zone B cells [85]. The observation that distinct B cell subsets are responsible for anti-DNA antibody production in prolactin and estrogen treated mice, and that different mechanisms are involved in the breakdown of tolerance, leads to the hypothesis that specific interventions can be employed to target patients with
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Fig. 4. Effects of prolactin in C57B1/6 mice. R4A-c2b C57B16 mice treated with prolactin or placebo (10 in each group) were evaluated for anti-DNA antibody titers and renal IgG deposition. (A) Serum anti-DNA reactivity. Anti-dsDNA titers in the serum were measured before initiation of treatment and weekly for 4 weeks. No difference in anti-DNA reactivity was found between prolactin and placebo-treated mice. (B) c2b-expressing B cells. Prolactin had no effect on the number of c2b-expressing B cells. (C) Immunohistochemistry of kidney sections. Staining with anti-IgG antibody did not reveal deposits in either prolactin or placebo treated mice.
hormonally exacerbated disease. For example, costimulatory blockade may provide beneficial therapeutic effect in SLE patients with a prolactin-triggered disease, while interfering with BAFF or the use of estrogen receptor modulators may be more effective in individuals with an estrogen-triggered disease. 3.1. Prolactin in estrogen-induced lupus The difference in autoreactive B cell phenotype in estrogen [84,85] and prolactin-treated mice [83] suggests that estrogen does not break tolerance by upregulating prolactin. We decided, however, to ask directly whether estrogeninduced disease could be abrogated by blocking prolactin secretion. We demonstrated that a 5-week treatment with estrogen that provides serum estrogen levels of 75– 100 pg/ml (levels found at the high end of the estrus cycle) could induce a lupus-like serology in R4A c2b BALB/c mice [86]. To evaluate whether prolactin plays a role in estrogenmediated autoimmunity, we used bromocriptine to suppress prolactin secretion. Bromocriptine, a dopamine agonist, acts as a prolactin release inhibitor that has been shown to have therapeutic effect in several autoimmune diseases, including murine lupus [56,59,62]. The major immunosuppressive activity of bromocriptine is based on its hypoprolactinemic effect [63], as prolactin can abrogate bromocriptine-induced humoral and cell-mediated immunosuppression [87], as well as bromocriptine-induced decrease in IFN c production by macrophages [88]. Treatment with bromocriptine prevented the development of elevated anti-dsDNA antibody titers (Fig. 5) and a lupus-
phenotype in estrogen-treated R4A c2b BALB/c mice [89]. Estrogen and bromocriptine-treated mice, however, displayed an expansion of transgene-expressing B cells (Fig. 6a) and upregulation of the anti-apoptotic protein Bcl-2, demonstrating that bromocriptine does not suppress estrogenmediated survival of autoreactive B cells. Consistent with this observation is the altered ratio of transitional T1 to T2 B cells present in these mice [83]. Interestingly, transgene expressing B cells which are localized in the marginal zone of the splenic follicles in estrogen treated mice [84], are distributed in the red pulp in estrogen and bromocriptine treated mice (Fig. 6b) and marginal zone B cells are not expanded in estrogen and bromocriptine treated mice (Fig. 7). Furthermore, in estrogen and bromocriptine treated mice, DNAreactive hybridomas can be generated only from LPSstimulated B cells. The light chains of these hybridomas were encoded by germline genes demonstrating that the naive DNA-reactive B cells that are rescued from deletion by estrogen [86] are still present in absence of prolactin, but in a functionally inactive state. Adoptive transfer of naïve or estrogen-exposed T cells to estrogen and bromocriptine treated mice did not lead to an expansion of the c2b expressing B cell population (Fig. 8a) or an increased number of B cells that spontaneously secrete anti-DNA antibodies or secrete antibody after stimulation with anti-CD40 antibody and IL-4 (Fig. 8b). Thus, help provided by naïve or estrogen modulated T cells, is not sufficient to induce a lupus-like phenotype in estrogen and bromocriptine treated mice. The effect of bromocriptine in abrogating the effect of estrogen is not T cell mediated.
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Fig. 5. Effects of bromocriptine on estrogen-modulated B cells. (A) Anti-DNA reactivity. R4A-c2b BALB/c mice were treated with estrogen pellets that provide 75 pg/ml estradiol, estrogen and 400 mg of bromocriptine injected intraperitoneally daily or with placebo for 6 weeks. Anti-dsDNA antibody titers were measured before initiation of treatment and weekly thereafter for 6 weeks. At the end of the treatment, serum anti-DNA reactivity was significantly increased only in estrogen-treated mice compared to estrogen and bromocriptine (P = 0.01) and placebo treated mice (P < 0.01). (B) c2b expressing B cells. Splenic B cells were evaluated for the number of c2b B expressing cells. Compared to placebo, there was an expansion of c2b expressing B cell population in both estrogen and estrogen and bromocriptine treated mice (P = 0.01 and 0.04, respectively).
Fig. 6. Localization of c2b expressing B cells in estrogen and bromocriptine treated mice. B cells and c2b-expressing B cells were labeled with anti-IgM antibody (blue) and anti-c2b antibody (yellow), respectively. In placebo-treated mice, c2b-expressing B cells were localized in the red pulp and in the T cell–B cell interface. Both estrogen and, estrogen and bromocriptine treated mice displayed an increased number of c2b-expressing B cells. However, in estrogentreated mice, c2b-expressing B cells were localized in the marginal zone, while in estrogen and bromocriptine treated mice they were confined to the red pulp and B cell–T cell interface.
4. Conclusions There is wealth of data implicating prolactin in the pathogenesis of human and murine lupus. Approximately 20% of SLE patients display some degree of hyperprolactinemia and administration of prolactin exacerbates disease activity causing early mortality in murine models of lupus. Additionally, prolactin can induce a lupus-like phenotype in nonspontaneously autoimmune mice. In mice with a susceptible genetic background, hyperprolactinemia leads to an increased survival and activation of autoreactive B cells with a subsequent increase in anti-DNA antibody titers and IgG deposits in the kidneys. Prolactin mediates these effects in part by altering B cell development, leading to decreased negative selection and an increased number of mature B cells. The follicular subset has been shown to harbor autoreactivity in prolactin-treated mice. Thus, it is not surprising that prolactin exerts its immunostimulatory effects on B cells only in presence of CD4+ T cells.
At a molecular level, prolactin upregulates expression of Bcl-2 and CD40 in B cells, causing a decrease in apoptosis and an increased susceptibility to costimulation, both of which may contribute to the survival and rescue of B cells that are signaled by antigen to undergo negative selection. Prolactin does not affect the strength of the BCR signal. Effects of prolactin on B cells are genetically determined. The fact that prolactin breaks tolerance and induces a lupus phenotype in one mouse strain but not in other, suggests that only a subset of SLE patients is likely to have a prolactinresponsive disease. Microarray studies may help distinguish between lupus patients with a prolactin-responsive disease and those with a prolactin-unresponsive disease. Given that in prolactin-induced autoimmunity, T cells play a crucial role and that follicular B cells harbor autoreactivity, therapies that interfere with costimulation may provide beneficial effect in patients with prolactin-mediated disease.
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Fig. 7. B cell subsets. To evaluate the effects of bromocriptine on estrogenmediated modulation of B cell development, splenocytes from five estrogen, three estrogen and bromocriptine and three placebo treated BALB/c mice were stained with antibodies to CD19, CD21, CD24 and CD23. Although both estrogen and, estrogen and bromocriptine treated mice displayed an inverted T1/T2 ratio, estrogen treated mice showed a significant expansion of the marginal zone (CD21high CD23low) B cells compared to estrogen and bromocriptine or placebo-treated mice (P = 0.02).
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[19] Fig. 8. Adoptive transfer of CD4+T cells from mice treated with estrogen or placebo. CD4+ T cells from mice treated with estrogen or placebo for 5 weeks (n = 5 in both groups) were transferred to mice treated with estrogen and bromocriptine for 6 weeks (n = 5). (A) Enumeration of anti-DNA antibody secreting B cells. Adoptive transfer of naïve and estrogenmodulated CD4+ T cells did not lead to increased number of autoreactive B cells that secrete anti-DNA antibodies spontaneously or after stimulation with LPS or anti-CD40/IL4 compared to a control group of estrogen and bromocriptine treated mice (n = 5). (B) Analysis of peripheral lymphocytes. Estrogen and bromocriptine treated mice, which underwent adoptive transfer of naïve or estrogen-modulated CD4+ T cells, did not develop an expansion of c2b expressing B cells compared to estrogen and bromocriptine treated mice which did not receive T cell transfer.
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