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
]. 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
] 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
] 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
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
]. 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
]. 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
] 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
]. 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
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
], 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