Introduction
Multiple sclerosis (MS) is a chronic T and B cell-mediated inflammatory disorder of the central nervous system (CNS) associated with progressive oligodendrocyte and neuronal loss, axonal degeneration, and demyelination [
1]. Prolactin is a pituitary hormone that stimulates milk production in mammals, and it may be implicated in the pathophysiology of MS [
2,
3]. Interestingly, MS tends to undergo remission during mid-late pregnancy, where prolactin levels are known to be at their peak during the third trimester of pregnancy [
4-
7]. Breastfeeding promotes further prolactin release, even though levels would otherwise fall post-partum [
8,
9]. Importantly, a recent meta-analysis (12 studies;
n = 1,558) found that women with MS who breastfed were almost half as likely to experience a post-partum relapse compared to women who did not [
10]. The data are even more compelling when considering only studies that examined exclusive breastfeeding [
11,
12].
In animals, our group showed that pregnant mice have an enhanced ability to remyelinate lysolecithin-induced white matter lesions [
13]. This was observed via an increase in proliferation of oligodendrocyte precursor cells, oligodendrocyte generation, expression of myelin basic protein, and in the number of myelinated axons - effects that were stimulated by prolactin infusion. Others have revealed that prolactin may possess pro-inflammatory properties. For example, there is evidence that prolactin promotes the activation of B cells cultured from the blood of MS patients [
14]. Additionally, T cell cultures treated with prolactin have an elevated T helper 1 pro-inflammatory profile and decreased suppressive function of regulatory T cells [
15]. Preclinical studies suggest that reduction of prolactin levels via treatment with dopamine D
2 agonists (bromocriptine and dihydroergocryptine) reduces severity of experimental autoimmune encephalomyelitis (EAE), an animal model of MS [
16-
19]. On the other hand, a recent study revealed that prolactin receptor- and prolactin-knockout mice develop a delayed onset EAE, compared with littermate control mice, but with full clinical severity [
20]. Importantly, no study of the direct administration of purified prolactin in EAE has been conducted to date.
The present study examined the effect of purified recombinant prolactin in EAE at different periods surrounding the onset of neurological signs and with/without concomitant interferon-β (IFN-β), a standard disease modifying therapy in MS. The addition of IFN-β was explored because of the potential pro-inflammatory properties of prolactin, making it prudent to combine its use with an immunomodulator commonly used in MS.
Methods
Recombinant murine prolactin was tested in virgin C57BL/6 female mice aged 8 to 10 weeks. EAE was induced at day 0 by 50 μg myelin oligodendrocyte glycoprotein (MOG), peptide 35-55, emulsified in Complete Freund’s Adjuvant supplemented with 4 mg/kg of mycobacterium, as described elsewhere [
21]. Mice were also administered two intraperitoneal injections of pertussis toxin (List Biological Labs, Hornby, ON, USA), on days 0 and 2. Intraperitoneal injections of 20 μg/mouse of prolactin (Harbor-UCLA, Torrance, CA, USA) were applied every morning, either for 9 (days 9 to 17) or 25 (days 9 to 33) consecutive days. Previously, we found that the same dosing regimen promoted myelin repair in virgin female mice of a similar age. In the 9-day experiment, while mice were treated from days 9 to 17, they were sacrificed on day 21. In the 25-day experiment, mice were sacrificed on the day of the last treatment itself. Day 9 was the time period when onset of clinical signs of EAE was anticipated. Groups of mice also received suboptimal doses (20,000 IU/mouse) of recombinant murine IFN-β (PBL Biomedical Laboratories, Piscataway, NJ, USA) administered subcutaneously once every 2 days, alone or in combination with prolactin. Vehicle was phosphate-buffered saline.
EAE clinical disease scores were obtained by using a 15-point scale that totals the degree of disability of the tail (scored from 0 to 2) and all four limbs (each limb scored from 0 to 3) [
22]. When the sum of scores (burden of disease) was tabulated, this represents the daily clinical score per mouse, added over the course of the experiment for that mouse.
Histological analyses were performed on spinal cord specimens retrieved at the end of treatment. The degree of inflammation and demyelination of the spinal cord was assessed through hematoxylin and eosin and luxol fast blue (H&E/LFB), as previously described [
21]. Animal studies were performed according to ethical policies outlined by the Canadian Council for Animal Care and the University of Calgary.
Antigen-recall assay
Mice were sacrificed, and lymph nodes were removed on day 10 after MOG immunization. Lymph nodes (LNs) were dissociated, and single cell suspensions were collected in phosphate-buffered saline. LN cells were plated in round-bottom plates at a density of 250,000 cells/100 μl in RPMI containing 1% mouse serum (Invitrogen, Carlsbad, CA, USA). The LN populations served to provide T cells for subsequent antigen-recall proliferation. Antigen-presenting cells (APCs) were harvested from spleens taken from non-immunized animals. Spleens were dissociated and subjected to red blood cell lysis. Cells were re-suspended in RPMI containing 1% mouse serum. APCs were irradiated at room temperature with a Gamma Cell 1000 (Nordion International Inc., Vancouver, Canada) using Cs-137 for 14 min at 3,000 rad, which was then followed by incubation of the APC cells with 20 μg/ml MOG 35-55 for 30 min at 4°C. The APCs (250,000 APCs/100 μl) were then added to the LN cells described above for a total of 500,000 cells/well. Recombinant mouse prolactin was added (1, 10, and 30 nM) and incubated for 3 days. The doses were chosen from previous literature that had suggested a pro-inflammatory role for prolactin in vitro on T lymphocytes. 3H-thymidine was added 18 h before the harvesting and collection of cells for measurement of thymidine uptake. Cells were pulsed with 1 μCi of [H3] thymidine (Perkin Elmer, Waltham, MA, USA) to determine the proliferative state of the cultures. After incubation, cells were harvested using a PHD cell harvester (Brandel Inc., Gaithersburg, MD, USA) and [H3] thymidine incorporation was determined by using a Beckman LS3801 scintillation counter (Beckman Coulter, Mississauga, Canada).
Statistics
Statistical analysis was performed using SPSS Statistics v.22.0 (IBM Corporation, Armonk, NY, USA, 2013). Statistical differences between groups were evaluated using a non-parametric Kruskal-Wallis analysis. Multiple comparisons were performed using the Mann-Whitney U test. In all tests, P ≤ 0.05 was considered statistically significant.
Discussion
Previous work has demonstrated that the hormone prolactin promotes the proliferation of oligodendrocyte precursor cells and remyelination in the lysolecithin model of demyelination in mice [
13]. That study also showed that white matter changes occurred in the maternal CNS, and these changes were initiated during early pregnancy and include increases in proliferation of oligodendrocyte precursor cells, oligodendrocyte generation, expression of myelin basic protein, and, ultimately, an increase in the number of myelinated axons. In addition, we found that prolactin signaling was necessary and sufficient for the regenerative effects of pregnancy and that prolactin treatment of virgin mice enhanced their remyelinating capacity following demyelination [
13]. Pro-regenerative effects were also observed by Möderscheim
et al. [
23] wherein prolactin exerted trophic and pro-proliferative effects on glia.
The pro-remyelinating capacity of prolactin suggests its utility in demyelinating conditions such as MS, except that this must be weighed against reports that prolactin can have pro-inflammatory roles. A pro-inflammatory role for prolactin has been reported for T lymphocytes previously by stimulating splenocytes
in vitro using concanavalin A [
17,
18]. These cells displayed lymphoproliferation in a dose-dependent manner that could be antagonized with the use of corticosteroids with lymphoproliferation observed at concentrations of 5 nM but not 1 nM prolactin in culture [
24]. Additionally, the proliferation of splenocytes and thymocytes stimulated with anti-CD3 was further promoted in the presence of 10 nM ovine prolactin as assessed by thymidine uptake [
25]. In both cases, the antigen non-specific response was measured and these effects were mimicked by the addition of growth hormone suggesting that prolactin may act similarly to this hormone as a mitogen for cell proliferation. It remains to be shown whether prolactin plays a role in stimulating memory or recall responses. Here, the mitogenic effect of prolactin seen previously with anti-CD3 and concanavalin A was replicated in a MOG peptide-specific recall assay, suggesting that prolactin may be pro-proliferative when present during antigen-recall in an ongoing immune response.
A dopaminergic pathway in the hypothalamus-pituitary axis controls the production of prolactin. Treatment with D
2 agonists lowers prolactin levels, and a number of studies have reported beneficial effects of prolactin suppression in EAE. In one study, bromocriptine given 1 week before immunization significantly decreased serum prolactin levels, and this was accompanied by an inhibition of disease progression in acute EAE [
16]. In that study, immunocompetence of bromocriptine-treated animals was restored by additional treatment with either prolactin or growth hormone. A similar study by Riskind
et al. [
17] revealed that induction of acute EAE resulted in a threefold rise in prolactin levels on day 4 after immunization and maintained elevated levels on day 10 before the onset of neurological signs. Bromocriptine significantly reduced the rise in prolactin levels and inhibited disease progression when initiated 1 week after immunization and also in late disease. Another report administered bromocriptine after the onset of clinical signs in acute as well as in chronic relapsing EAE [
19]. Their results revealed that bromocriptine suppressed prolactin levels and reduced the severity and duration of clinical signs in acute EAE and the duration of the second attack in chronic EAE. Finally, there is evidence that dihydroergocryptine induced a large reduction of prolactin levels accompanied by a significant improvement in neurological signs of acute EAE when given 2 days before immunization [
18]. Taken together, these studies suggest that reduction of prolactin levels by selective D
2 agonists is effective at reducing disease severity in acute and chronic EAE, supporting a pro-inflammatory effect of prolactin. However, a small clinical trial (
n = 18) did not find a benefit of bromocriptine in RRMS and progressive MS patients [
26]. More recent literature suggests that dopamine may be directly linked to immunomodulation - for example, by inhibiting activated T cell function, modulating Tregs, and altering B cell function [
27]. Thus, suppression of prolactin in the aforementioned studies may not be the primary mechanism via which dopamine agonists reduce EAE severity. Taken together, it is currently not clear whether suppression of physiologic levels of prolactin via therapy with D
2 agonists would benefit patients with MS.
A recent study revealed that prolactin receptor- and prolactin-knockout mice develop a delayed onset EAE, compared with littermate control mice, but with full clinical severity [
20]. Because prolactin receptor knockouts have been shown to be hyperprolactinemic, these data suggest that neither high nor low prolactin levels significantly impact EAE. The data are correspondent with previous results showing that prolactin receptor-knockout mice develop normal immune function [
28,
29].
In view of the uncertainty of the impact of prolactin in MS, we have reviewed the literature and concluded that there is no compelling argument against the use of prolactin in MS [
3]. In support, post-partum data of breast-feeding where prolactin levels are expected to be high have indicated that there may be a protective effect of breast-feeding against MS relapses [
10-
12].
Nonetheless, the use of prolactin in MS must be approached cautiously, and it would be prudent to combine its use with an immunomodulator commonly used in the condition. In the current study, we have used purified recombinant prolactin in EAE. Our data shows that recombinant prolactin-alone had no effect on EAE clinical scores during treatment for 9 or 25 days. Indeed, prolactin did not exacerbate EAE - as would be predicted based on its pro-inflammatory properties - but may have exerted further beneficial properties not apparent in the overwhelming inflammatory and potentially pro-proliferative mechanisms of EAE. With this in mind, we examined the effect of prolactin in the presence of suboptimal doses of the therapeutic immunomodulator IFN-β. Our data suggests that the mixture of these two therapies given over a protracted period after disease onset provides a benefit not observed with either of the single treatment therapies. In addition, observation of spinal cord histopathology of these mice suggests that combination therapy decreased the overall disease burden as reflected by inflammatory infiltrates. Consequently, it is possible that IFN-β counteracted a pro-inflammatory effect of prolactin, thus allowing for the putative remyelinating properties of prolactin to take effect - as previously shown by our group in the lysolecithin model [
13].
We were unable to determine whether the improvement in clinical scores in response to combined prolactin and IFN-β treatment could be attributed to remyelination. The EAE model is notoriously difficult to evaluate remyelination, as lesions appear at unpredictable locations, and it is therefore not trivial to address the evolution of demyelination and its subsequent repair. Thus, while the histological analyses (Figures
2 and
4) show reduced inflammation and demyelination in response to combined prolactin and IFN-β treatment, to what extent remyelination has occurred remains unclear.
In conclusion, we sought to determine whether prolactin could alter the course of EAE, either when used alone, or in combination with IFN-β. We found that the combination of prolactin and IFN-β during the 9-day treatment period resulted in a greater amelioration of clinical signs and histological scores of EAE compared to either treatment alone. The combination therapy also resulted in significantly less inflammatory infiltrates with prolonged 9- to 33-day treatment. Consequently, in light of the seemingly beneficial effects of breast-feeding on MS symptomatology, and our promising results in the lysolecithin and EAE models, future trials of prolactin in MS may be warranted. Either prolactin itself, or medications that elevate prolactin levels, may be considered.
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Competing interests
Sam Weiss served on the board of Stem Cell Network. All other authors declare that they have no competing interests.
Authors’ contributions
SZ and VWY were involved in the data analysis and drafting of the manuscript. VWY and SW were involved in the research design. TJ was involved in the research design and experimental performances. LM and SW discussed the results and edited part of the manuscript. All authors read and approve the final manuscript.