Increased levels of prolactin receptor expression correlate with the early onset of lupus symptoms and increased numbers of transitional-1 B cells after prolactin treatment

Abstract

Background: Prolactin is secreted from the pituitary gland and other organs, as well as by cells such as lymphocytes. Prolactin has an immunostimulatory effect and is associated with autoimmune diseases that are characterised by abnormal B cell activation, such as systemic lupus erythematosus (SLE). Our aim was to determine if different splenic B cell subsets express the prolactin receptor and if the presence of prolactin influences these B cell subsets and correlates with development of lupus.

Results: Using real-time PCR and flow cytometry, we found that different subsets of immature (transitional) and mature (follicular, marginal zone) B cells express different levels of the prolactin receptor and are differentially affected by hyperprolactinaemia. We found that transitional B cells express the prolactin receptor at higher levels compared to mature B cells in C57BL/6 mice and the lupus-prone MRL/lpr and MRL mouse strains. Transitional-1 (T1) B cells showed a higher level of prolactin receptor expression in both MRL/lpr and MRL mice compared to C57BL/6 mice. Hyperprolactinaemia was induced using metoclopramide, which resulted in the development of early symptoms of SLE. We found that T1 B cells are the main targets of prolactin and that prolactin augments the absolute number of T1 B cells, which reflects the finding that this B cell subpopulation expresses the highest level of the prolactin receptor.

Conclusions: We found that all B cell subsets express the prolactin receptor but that transitional B cells showed the highest prolactin receptor expression levels. Hyperprolactinaemia in mice susceptible to lupus accelerated the disease and increased the absolute numbers of T1 and T3 B cells but not of mature B cells, suggesting a primary effect of prolactin on the early stages of B cell maturation in the spleen and a role of prolactin in B cell differentiation, contributing to SLE onset.

Figure 1

Purification of B cell subsets by flow cytometry. B cells were purified by negative selection from 9-week-old mice. Splenic cells were incubated with different antibodies (anti-CD19, anti-CD93, anti-CD21, anti-CD23, and anti-IgM), and the different B cell subsets were purified by flow cytometry as detailed in the Methods section. The purity of the collected populations oscillated between 95% and 98%. A representative example of the purified B cells from wild-type C57BL/6 mice is shown.

Figure 2

Expression of prolactin receptor in different subsets of splenic B cells from C57BL/6 mice. The different B cell subsets from 9-week-old C57BL/6 mice were purified by flow cytometry in four independent experiments using eight mice per experiment. Using real-time PCR, PRL receptor (PRL-R) mRNA expression was determined in the following different subsets of splenic B cells: A) Follicular (FO), marginal zone (MZ), and transitional (T). The protein expression levels of the PRL receptor were determined for ten mice of each strain by flow cytometry, and the mean fluorescence intensity (MFI) is shown in the following B cell subsets: B) Follicular; marginal zone; and transitional. C) Histograms of PRL receptor expression in the different subsets of B cells are shown. D) Transitional 1, 2 and 3. The asterisks denote statistical significance with the p value shown.

Figure 3

Expression of prolactin receptor in C57BL/6 wild-type, MRL and MRL/lpr lupus-prone mice. Protein expression of the PRL receptor (PRL-R) was determined by flow cytometry (MFI) from 14 C57BL/6 and MRL/lpr mice and 7 MRL mice. Splenocytes were marked with anti-CD19, anti-CD93, anti-IgM, anti-CD21, anti-CD23, and goat anti-PRL receptor A) splenic B cells; B) Follicular (FO), marginal zone (MZ), transitional B cells; and C) Transitional 1, 2 and 3. Asterisks denote statistical significance with the p value shown.

Figure 4

SLE manifestations in mice with hyperprolactinaemia. Fourteen C57BL/6 and MRL/lpr mice, and seven MRL mice at 9 weeks of age were treated for 6 weeks with metoclopramide (100 μg/100 μl) or PBS (100 μl). At the end of the treatment, the following measurements were taken: A) PRL concentration by ELISA; B) level of proteinuria using reactive strips; C) anti-dsDNA IgG antibodies; and D) anti-dsDNA antibodies of the IgG1 and IgG2a isotypes in the MRL and MLR/lpr mice by ELISA. The asterisks denote statistical significance between groups with the p value shown. HyperPRL = Hyperprolactinaemic

Figure 5

Prolactin receptor expression in MRL/lpr mice at different ages. Ten C57BL/6 and ten MRL/lpr mice were treated with metoclopramide (100 μg/100 μl) for 6 weeks [hyperprolactinaemia (HyperPRL)] or PBS (control). Splenic B cells were purified using negative selection from treated and untreated mice: at 9 and 25 weeks of age (untreated) and 15 weeks of age, mice were treated with metoclopramide or PBS (expression of the PRL receptor was determined by real-time PCR [mRNA]). The asterisks denote statistical significance between populations with the p value shown.

Figure 6

Prolactin receptor expression after the induction of hyperprolactinaemia. The levels of PRL receptor protein in transitional (T1, T2, and T3) and mature (FO and MZ) B cells were determined by flow cytometry (MFI). At the end of the treatment, spleen cells were marked with anti-CD19, anti-CD93, anti-IgM, anti-CD21, anti-CD23, and goat anti-PRL receptor. A) C57BL/6 mice; B) MRL mice and C) MRL/lpr mice. The asterisks denote statistical significance between populations with the p value shown.

Figure 7

Absolute numbers of mature B cells after the induction of hyperprolactinaemia. Nine-week-old mice were treated with metoclopramide (100 μg/100 μl) [hyperprolactinaemia (HyperPRL)] or with PBS (100 μl) for 6 weeks, with fourteen mice per condition in the C57BL/6 and MRL/lpr groups, and seven mice per condition in the MRL group. At the end of this treatment, splenocytes were marked with antibodies against CD19, CD93, CD21, IgM and CD23, and the absolute number of each population was calculated. A) The absolute number of splenic B cells. B) Representative dot plot of the mature MZ and FO B cell subsets. C) Graph of the absolute number of MZ B cells. D) Graph of the absolute number of FO B cells. The asterisks denote statistical significance between populations with the p value shown.

Figure 8

Absolute number of immature B cells after the induction of hyperprolactinaemia. Nine-week-old mice were treated with metoclopramide (100 μg/100 μl) [hyperprolactinaemia (HyperPRL)] or PBS (100 μl) for 6 weeks, with fourteen mice per condition in the C57BL/6 and MRL/lpr groups, and seven mice per condition in the MRL group. At the end of the treatment, splenocytes were marked with antibodies against CD19, CD93, CD23, CD21 and IgM. A) Representative dot plot of the transitional B cell subset. B) Graph of the absolute numbers of transitional-1 B cells (T1). C) Graph of the absolute numbers of transitional-2 B cells (T2). D) Graph of the absolute numbers of transitional-3 B cells (T3). The asterisks denote statistical significance between populations with the p value shown.