Background
Prolactin (PRL) is a lactogenic hormone that is mainly produced by the anterior pituitary gland. PRL has multiple functions that regulate reproduction, development and growth, osmosis, metabolism of carbohydrates and lipids and the immune system. Each of these functions requires expression of the PRL receptor in different extra-pituitary regions [
1]. In the immune system, interaction between hormones and receptors activates the transcription of genes involved in different cellular functions, such as proliferation, differentiation, and cytokine production [
2‐
4]. PRL has been implicated as a modulator of both cellular and humoral immunity [
1‐
4].
Elevated serum levels of PRL have been reported in several autoimmune diseases, including multiple sclerosis [
5] and systemic lupus erythematosus (SLE) [
6‐
9], although this finding has not been reported for other diseases such as autoimmunity during chronic hepatitis C [
10]. Moreover, women with hyperprolactinaemia but without autoimmune disorders have been reported to have circulating autoantibodies [
11].
SLE is an autoimmune rheumatic disease. Serum samples from SLE patients characteristically have very strong reactivity to a broad spectrum of nuclear components, including DNA, RNA, histones, RNP, Ro, and La. These antibodies form immune complexes that are deposited in the kidneys and may cause proteinuria and kidney failure. The presence of these autoantibodies indicates abnormalities in the activation and development of B cells [
12,
13], and both B and T cells express the PRL receptor and produce and secrete PRL [
1,
14‐
16]. SLE mainly affects women of the reproductive age at a ratio of 9:1 compared to men, and this gender bias has been attributed to the immunostimulatory properties of hormones. SLE symptoms tend to start or become exacerbated during pregnancy, when serum PRL levels are high. High serum concentrations of PRL correlate with SLE activity [
6‐
8], and hyperprolactinaemic patients with antiphospholipid syndrome display significantly more serositis and peritonitis compared to healthy individuals. [
9,
17]. These findings have also been observed in the murine NZB × NZW model of lupus after the induction of hyperprolactinaemia, in which the presence of PRL correlates with the early detection of immune complexes, proteinuria, and accelerated death [
18].
MRL-MpJFas
lpr (MRL/lpr) mice have a mutation in the Fas gene and develop a disease similar to SLE, characterised by glomerulonephritis, vasculitis, splenomegaly, hypergammaglobulinemia and the production of anti-dsDNA antibodies [
19]. In this strain of mouse, eliminating B cells using an anti-CD79 antibody decreased manifestations of the SLE-like disease, demonstrating the importance of B cells in SLE physiopathology [
20,
21]. B cells start their maturation process in the bone marrow, undergoing the proB, preB and immature stages, and finish maturation in the spleen, where the transitional and mature B cell subsets can be found. These populations are distinguished by the expression of different surface molecules. Allman et al. classified transitional B cells into three types: transitional-1 (T1 [CD93
+, IgM
high, CD23
-]), transitional-2 (T2 [CD93
+, IgM
high, CD23
+]), and transitional-3 (T3 [CD93
+, IgM
low, CD23
+]), while mature B cells are classified as follicular (FO [CD93
-, CD21
int, CD23
high]) or marginal zone (MZ [CD93
-, CD21
high, CD23
-]) [
22].
The objective of this study was to determine whether different splenic B cell subsets express the PRL receptor and if the presence of PRL influences these B cells subsets and correlates with the development of lupus. We found that all B cell subsets expressed the PRL receptor but that transitional B cells displayed higher expression levels compared to mature B cells. Hyperprolactinaemia in mice susceptible to lupus accelerated the disease and increased the absolute numbers of T1 and T3 B cells but not mature B cells, suggesting that PRL participates in the early stages of splenic B cell development.
Discussion
Several studies have demonstrated a role for prolactin (PRL) and B cells in the development of autoimmune diseases such as systemic lupus erythematosus (SLE) [
6‐
9,
12,
13,
18,
20,
21,
23,
24]. In this study, we evaluated how PRL affects the course of SLE development in MRL/lpr, MRL, and wild-type mice and observed how this finding correlates with changes in the different splenic B cell subsets. The MRL/lpr strain has a mutation in the Fas gene and develops a disease similar to SLE [
25]. MRL mice also exhibit autoimmune disorders despite carrying a normal Fas gene, but the symptoms manifest much later in life than in MRL/lpr mice [
19]. To our knowledge, this study is the first to address PRL receptor expression in all of the different subsets of splenic B cells.
Interestingly, expression of the PRL receptor followed a similar pattern in B cells from wild-type C57BL/6, SLE-prone MRL/lpr and MRL mice. Expression of the PRL receptor varied according to the B cell developmental stage. The highest expression of the PRL receptor was observed in the immature, transitional splenic B cells. Our findings are consistent with those reported by Morales et al. [
26], who transfected proB cells with the PRL receptor triggered their progression to the preB stage through incubation with PRL. Taken together, data from Morales et al. and our group suggest that PRL participates in early B cell differentiation. Our results showed that the pattern of PRL receptor expression levels in transitional B cells was different in C56BL/6 mice and the lupus-prone mouse strains. T2 cells showed higher expression of the PRL receptor in C7BL/6, while in lupus-prone strains the T1 cells showed higher expression. These data argue that there is altered expression of the PRL receptor in different B cells subsets during the autoimmune process. Furthermore, the differential presence of the receptor in transitional populations in C56BL/6 mice suggests a regulatory role for PRL in these cells under non-pathological conditions.
Transitional B cells are constantly testing their antigen receptors (BCR) to identify B cell clones expressing receptors with self-specificity [
27]. These clones then trigger different intrinsic mechanisms that eliminate this self-specificity. The MRL strains have a genetic background prone to develop a disease similar to SLE either earlier in life in the case of MRL/lpr mice or later in the case of MRL mice. We found that the T1 B cell subset in both of these strains had the most PRL receptor protein expression. Therefore, it is possible that increased PRL receptor expression at this stage in lupus-prone strains could promote both the rescue of autoreactive clones and B cell developmental progression, thus favouring the development of lupus symptoms. This observation is in agreement with the fact that PRL receptor signalling increases the expression of the anti-apoptotic gene Bcl-2 [
28,
29] and that T1 B cells from hyperprolactinaemic ovariectomised BALB/c mice are more resistant to apoptosis than those from PBS-treated mice [
30]. Future experiments should address the molecular role of PRL in this population and better define its contribution in autoimmune disease.
C57BL/6, MRL and MRL/lpr mice treated with metoclopramide showed a further increase in their serum PRL levels that was only accompanied by increased levels of anti-dsDNA antibodies and proteinuria in the MRL/lpr and MRL lupus-prone strains. These findings are in agreement with previous reports studying other hyperprolactinaemic and SLE models, such as reports by McMurray et al. [
18,
31] who showed hyperprolactinaemia induced by pituitary gland transplantation in NZB × NZW mice, and a report by Peeva et al. using recombinant PRL to induce hyperprolactinaemia in Sle3/5 R4A-γ2b C57BL/6 mice [
32]. Additionally, our data are consistent with several clinical trials showing that a high serum PRL level correlated with SLE disease activity [
7,
33,
34].
In our study, PRL receptor mRNA expression was increased in metoclopramide-treated 15-week-old MRL/lpr mice to a level similar to that found in untreated 25-week-old mice displaying high disease activity. This correlation between PRL receptor levels and the degree of disease suggests that PRL plays an important role in the development and exacerbation of SLE. These results also support the idea that the PRL receptor could be useful as an SLE prognostic marker.
Although our results showed that the T2 subset of B cells express the highest levels of the PRL receptor in the spleens of C57BL/6 mice, the absolute number of T2 cell and other B cell subsets and overall PRL receptor expression were not affected by hyperprolactinaemia, and SLE was not induced. This result differs from previous reports by Peeva et al. and Saha et al. describing decreased T1 B cell frequencies and increased T2 B cell frequencies after the induction of hyperprolactinaemia, while the MZ and FO mature B cell pools were unaffected [
30,
35]. This finding may be due to the different experimental approaches used, in that different mouse strain was used (BALB/c), BALB/c mice were ovariectomised, a procedure that eliminates oestrogen and progesterone and affects immune responses [
36,
37]. In addition, we treated mice with metoclopramide to induce the hyperprolactinaemic state, whereas Peeva and Saha used ovine PRL.
In our study, hyperprolactinaemia resulted in up-regulation of PRL receptor expression and a significant increase in the absolute numbers of T1 B cells in the MRL/lpr mice. PBS-treated mice did not show increases in the absolute number of T1 cells, despite an increase in PRL receptor expression, we believe that this is because the level of PRL receptor expression in PBS-treated mice never reached the levels found in either pharmacologically induced or age-related hyperprolactinaemic mice. Additionally, in the MRL strain, the absolute number of T1 cells only increased in hyperprolactinaemic mice, correlating with the early onset of lupus symptoms and increased PRL receptor expression. A similar observation has been noted in NK cell lines, in which high PRL receptor expression correlates with an enhanced capacity of the cells to proliferate [
38]. This finding is also in agreement with the observation that the T1 population expresses the highest level of PRL receptor expression before treatment and with other studies showing this subset to be more resistant to apoptosis in mice with hyperprolactinemia [
30]. Thus, our data and that of others [
30,
38,
39] support the importance of PRL in B cell development, in developmental associated processes such as proliferation and resistance to negative selection and in the progression of SLE. These findings highlight the idea that this disease originates at different levels, as indicated by its multifaceted nature.
We also found that hyperprolactinaemia increased the expression of the PRL receptor and, to a lesser degree, the absolute numbers of T3 B cells. T3 B cells are considered by Merrel to be a subset of anergic B cells produced by the interaction of the BCR and self-antigens and are not a part of the maturation pathway of B cells [
40,
41]. The T3 B cell subset is decreased in MRL/lpr mice, and this decrease has been proposed to be due to self-reactive clones escaping from an anergic state in this mouse strain. This diminished T3 population was described in 9-week-old mice; however, this time point is prior to disease onset, and these mice were compared to the genetically unrelated BALB/c strain [
42]. Therefore, future work should determine the effect, if any, of PRL signalling in this population and may generate interesting results.
The number of MZ B cells in MRL/lpr mice increased with age and coincided with the course of the disease, as previously reported [
43]. Higher levels of serum PRL induced a further increase in the MZ B cell population, although this change was not significant when compared to mice of the same age treated with PBS. However, this increase in the absolute number of MZ cells was statistically significant in hyperprolactinaemic-MRL mice. These results are consistent with a model in which PRL increases the expression of its receptor mainly in immature transitional B cells, leading to self-reactive cell maturation with a potential bias toward the MZ type.
Although PRL did not affect the absolute number of mature B cells or their PRL receptor expression pattern, self-reactive clones were more active in hyperprolactinaemic MRL/lpr and MRL mice as shown by an increased concentration of anti-dsDNA antibodies, specifically of the IgG isotype. It will be interesting to discern the function of PRL in the MZ and FO mature populations, as it is clear that they perform different functions. While MZ B cells are part of the innate immune system and preferentially respond to T cell-independent antigens, FO B cells perform the classic functions of adaptive immunity. MZ B cells have mainly been associated with the expression of IgM antibodies [
44,
45].
Our results show that both MRL/lpr and MRL mice develop an early onset of lupus symptoms after induction of hyperprolactinemia. Although the MRL/lpr strain has an additional mutation in Fas [
19,
46], our results also suggest that this genetic change most likely has little, if any, effect on the accelerated lupus development. It is possible that strong PRL signalling in immature T1 B cells from both mice strains could potentially trigger rescue from apoptosis induced by recognition of self-antigens and could also shape and promote their differentiation into specific mature B cell populations. The presence of self-reactive clones in MZ B cells correlates with autoimmune diseases [
47].
Methods
Mice
All studies were approved by the Animal Care Committee of Instituto Nacional de Ciencias Médicas y Nutrición "Salvador Zubiran" and Hospital de Pediatría, Centro Médico Nacional Siglo XXI IMSS, and all of the measurements taken from the mice were in accordance with approved guidelines established by Mexico (Norma Oficial Mexicana NOM-062-ZOO-1999) and the NIH Guide for the Care and Use of Laboratory Animals. The C57BL/6 mice were purchased from Harlan (Indianapolis, USA), and the MRL/MpJ FASlpr (MRL/lpr) and MRL/MpJ (MRL) mice were purchased from the Jackson Laboratory (Maine, USA). All of the mice were housed in a specific pathogen-free barrier facility and were provided sterile food and water ad libitum.
Antibodies
The following antibodies were used: anti-mouse CD21-FITC (7 G6) and CD21-APC (7 G6) were from BD Biosciences (Mountain View CA, USA); CD93-PE (AA4.1), CD23-biotinylated, IgM-APC (11/41), CD19-Cy7 (1D3), CD23-PE-Cy7 (B3B4), CD19-FITC (eBioD3), and IgM-biotinylated (11/41) were from eBioscience (San Diego CA, USA); goat anti-mouse PRL-R (E20) was from Santa Cruz Biotechnology (Santa Cruz CA, USA); and swine anti-goat-biotinylated was from Invitrogen (Carlsbad CA, USA). The biotinylated secondary antibody was detected with streptavidin-phycoerythrin-Cy5.5 from BD Biosciences (Mountain View CA, USA).
Purification of B cells
Single-cell suspensions were prepared from spleens. After red blood cell lysis with lysing buffer (Sigma Aldrich, St. Louis Missouri, USA), the cells were incubated with anti-CD43 (Ly-48) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and the B cells were isolated using negative selection with a magnetic activated cell-sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany). After depletion, > 98% of the remaining cells were CD19+ by flow cytometry.
Cell sorting
Single-cell suspensions of B cells were incubated with fluorescently labelled antibodies specific for CD19, CD93, CD21, CD23, and IgM in staining buffer (PBS with 0.5% BSA) for 20 minutes at 4°C. The cells were washed, and the B cell (CD19+) subsets were isolated according to the expression of the following surface markers: marginal zone (CD93-, CD21high, CD23-); follicular (CD93-, CD21int, CD23high); transitional-1 (CD93+, IgMhigh, CD23-); transitional-2 (CD93+, IgMhigh, CD23+); and transitional-3 (CD93+, IgMlow, CD23+). Cell sorting was performed using a FACSAria sorter with FACSDiva software (BD Bioscience). The purity of the sorted cells ranged from 95% to 98%.
Real-time PCR
Total RNA was extracted from B cells of C57BL/6 and MRL/lpr mice using TRIzol reagent (Invitrogen, Carlsbad CA, USA), according to the manufacturer's protocol, and the RNA concentration was determined using UV spectrophotometry. Next, 1 μg of total RNA was used to generate cDNA with SuperScript II reverse transcriptase (Invitrogen, Carlsbad CA, USA), according to the manufacturer's specifications. The PRL receptor cDNA was amplified by real-time PCR using a LightCycler TaqMan Master kit (Roche Diagnostic, Mannheim, Germany), according to manufacturer's specifications and using hydrolysis probes and primers designed by Roche Diagnostic. The following primers were used: PRL-R 5'-CAGTAAATGCCACGAACGAA-3' (left); PRL-R 5'-GAGGAGGCTCTGGTTCAACA-3' (right); β-actin 5'-AAGGCCAACCGTGAAAAGAT-3' (left); and β-actin 5'-GTGGTACGACCAGAGGCATAC-3' (right). The final volume of the reaction was 10 μl, and a LightCycler instrument was used to perform the PCR reaction (Roche Diagnostic). The following PCR conditions were used: 10 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C, 30 seconds at 60°C, and 1 second at 72°C and 1 cycle cooled for 30 seconds at 40°C. The samples were normalised to the β-actin gene. The relative expression of the PRL receptor was calculated using the 2ΔCT formula.
Induction of hyperprolactinaemia
Groups of 14 female, 9-week-old C57BL/6 and MRL/lpr mice and 7 female MRL mice were given a daily subcutaneous injection of 100 μg of metoclopramide (Sigma Aldrich, St Louis MO, USA) in 100 μl of PBS for six weeks. A matched control group (C57BL/6, MRL and MRL/lpr) received only PBS (100 μl) over the same period. Urinary protein levels were assessed semiquantitatively using reagent strips for urinalysis (Uri-Quick Stanbio Laboratory, Kendall TX, USA). Serum samples were obtained at the beginning and at the end of the experiments, between 08:00 and 11:00 hours, and kept at -35°C until assayed for prolactin and anti-DNA antibodies.
Prolactin assessment
Serum levels of prolactin were detected using ELISA. The 96-well maxisorp plates (Nunc, Rochester NY, USA) were coated overnight with 100 μl of 4 μg/ml anti-mouse prolactin monoclonal antibody (clone 207518, R&D Systems, Minneapolis MN, USA) in PBS at 4°C, blocked with 2% bovine serum albumin (Invitrogen, Carlsbad CA, USA), and incubated with the serum sample (1:10) overnight at 4°C. Recombinant mouse prolactin (National Hormone and Peptide Program, NIH, donated by AF Parlow) was used as a standard. The plates were then incubated with 0.1 μg/ml biotinylated anti-prolactin antibody (R & D Systems, Minneapolis MN, USA), avidin-alkaline phosphatase (Zymed Laboratories, San Francisco CA, USA) and 5-bromo-4-chloro-3 indolyl phosphate (Sigma-Aldrich, St Louis MO, USA) as a substrate, according to the manufacturer's instructions. The OD was monitored at 405 nm using a Dynatech MR5000 ELISA reader.
Determination of anti-DNA antibodies
Anti-dsDNA antibody serum concentrations were detected using ELISA. A 96-well maxisorp plate (Nunc, Rochester NY, USA) was coated with 100 μl of 2.5 μg/ml calf thymus dsDNA (Sigma Aldrich, St Louis MO, USA) in bicarbonate buffer overnight at 4°C and was blocked with 2% bovine serum albumin (Invitrogen, Carlsbad CA, USA). The plates were then incubated for 1 h at 37°C with serum (1:50) or the anti-dsDNA antibody standard (clone 16-13, Chemicon International, Billerica MA, USA), followed by rabbit anti-mouse IgG, IgG1, IgG2a or anti-mouse IgM conjugated to alkaline phosphatase (Zymed Laboratories, San Francisco CA, USA) and substrate ([5-bromo-4-chloro-3- indolyl phosphate; Sigma-Aldrich, St Louis MO, USA]). The OD was monitored at 405 nm using a Dynatech MR5000 ELISA reader.
Cell surface staining and flow cytometry
Splenocytes were incubated with fluorescently labelled antibodies for 20 minutes at 4°C in staining buffer (PBS with 0.5% BSA and 0.01% sodium azide). The cells were then washed and fixed in 2% paraformaldehyde (Sigma Aldrich, St Louis MO, USA). The data were acquired using a FACSAria flow cytometer (BD Bioscience) and analysed with FlowJo software (Tree Star, Ashland OR, USA).
Statistical analysis
The data were analysed with standard statistical tests (mean value, SD, Student's t test, and ANOVA), and the results are expressed as the mean ± SD. The level of significance was set at p ≤ 0.05. All calculations were performed using SPSS 15 software.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YLS, MVLH, EMD and LCH performed and analysed the experiments, FBF and EMFP interpreted the results and prepared the manuscript preparation, ETC and RHG performed the mouse experiments, LAP performed the cell sorting, AKCR contributed to the experimental design, interpretation of results, and manuscript preparation. All of the authors read and approved the final manuscript.