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Keywords:

  • animal models/studies (mice);
  • autoimmunity;
  • B cells;
  • dendritic cells;
  • lupus

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The lupus susceptibility interval Sle3/5 confers responsiveness to prolactin in C57BL/6 (B6) mice and hyperprolactinaemia induces a lupus-like phenotype in B6.Sel3/5 mice. In this study, the immunostimulatory effects of prolactin in B6 mice containing the Sle3 portion of the Sel3/5 interval (B6.Sle3 mice) were dissected. Because of the Sle3 interval's involvement in activation of myeloid cells, the effect of dendritic cells (DCs) from prolactin-treated B6.Sle3 mice on the phenotype of B6 mice was also evaluated. B cells from prolactin-treated B6 and B6.Sle3 mice and from B6 recipients of prolactin-modulated DCs from B6.Sle3 mice were tested for DNA-reactivity and resistance to B cell receptor (BCR)-mediated apoptosis. The expression of co-stimulatory molecules on lymphocytes and myeloid cells was also evaluated. In prolactin-treated B6.Sle3 mice, transitional type 2 B cells increased while type 1 B cells decreased as a consequence of prolactin-induced resistance to BCR-mediated apoptosis leading to the survival of DNA-reactive B cells. Follicular B cells from prolactin-treated mice expressed increased levels of CD40, B7·2 and IAb, and DCs and monocytes had higher levels of CD44 and B7·2 than placebo-treated mice. Adoptive transfer of DCs from prolactin-treated B6.Sle3 mice to B6 recipients demonstrated the intrinsic ability of prolactin-modulated DCs to induce a development of lupus-like characteristics in B6 mice. Based on these results, prolactin accelerates the breakdown of immune tolerance in B6.Sle3 mice by promoting the survival, maturation and activation of autoreactive B cells, DCs and macrophages.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Systemic lupus erythematosus (SLE), the prototypical autoimmune disease, affects predominantly women and occurs with greater frequency among African Americans, Asians and Hispanics than among Caucasians, suggesting that both gender-associated factors and genetic factors are involved in its pathogenesis. Because female preponderance is especially evident during the reproductive years, when the female-to-male ratio among SLE patients reaches a striking peak of 9:1, female sex hormones are thought, at least in part, to underlie the gender bias in lupus. About 25–30% of SLE patients have mild-to-moderately increased serum prolactin levels [1-3], with the degree of hyperprolactinaemia correlating with global lupus activity or specific organ involvement in a significant number of these patients [4-7]. A review of five published clinical studies concluded that prolactin is related to lupus activity. However, it was noted that the low statistical power of the studies prevented establishing a formal causal relationship [8]. In addition, murine studies have shown that increased serum prolactin levels accelerate disease onset and cause early mortality in lupus-prone NZB/W F1 mice [9, 10]. We have shown that mild hyperprolactinaemia similar to that seen in SLE patients can break tolerance and induce a lupus-like syndrome in strains of mice that are not predisposed to the disease [11, 12]. Sustained mild to moderately increased serum prolactin levels induce the development of lupus in mice with a BALB/c, but not in those with a C57Bl/6 (B6) genetic background. In the latter strain, the 40cM lupus-susceptibility interval Sle3/5 [13] confers responsiveness to the immunostimulatory effects of the hormone [14], indicating that the interplay between genetic and hormonal factors creates a milieu permissible to the breakdown of tolerance and development of a lupus-like phenotype. In this study, we narrow down the pool of Sle3/5 genes that are responsible for the immunostimulatory effects of prolactin by demonstrating that the lupus-susceptibility locus Sle3 alone (24cM) is sufficient to confer responsiveness to the hormone. Treatment with prolactin accelerates the onset of autoimmune characteristics in B6.Sle3 mice by affecting both lymphoid and myeloid cells. We also show that prolactin-modulated dendritic cells (DCs) from B6.Sle3 mice are sufficient to induce the development of lupus-like autoreactivity in wild-type B6 mice.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Mice

Six-week-old female B6.Sle3 mice and B6 mice were used in the experiments. B6.Sle3 mice are homozygous for a 24-cM congenic interval of the NZM2410 allele of Sle3 and were a kind gift from Dr E. Wakeland (University of Medicine, Texas Southwestern Medical Center, Dallas, TX, USA). B6.Sle3 mice were bred at the animal facility at Albert Einstein College of Medicine (Bronx, NY, USA). B6 mice were purchased from Taconic (Hudson, NY, USA).

Prolactin and granulocyte–macrophage colony-stimulating factor (GM-CSF) treatments

Prolactin

One hundred μg (in 100 μl saline solution) of prolactin from sheep pituitary gland tissue (Sigma-Aldrich, St Louis, MO, USA) or placebo (100 μl saline solution) were administered subcutaneously every day to the mice for 4 weeks. The daily treatment with 100 μg of prolactin yields a two- to threefold increase in the serum prolactin level [11]; these mild elevations in serum prolactin levels are similar to the prolactin levels observed in SLE patients with hyperprolactinaemia [1-3, 8].

GM-CSF

GM-CSF injections have been shown to moderately increase the numbers of DCs in the spleen [15]. However, as a daily dose of 1 μg GM-CSF (Gibco, Grand Island, NY, USA) per mouse injected in the presence of prolactin was not able to produce a sufficient number of DCs for adoptive transfer (5–10 × 106 DCs), we opted to implant subcutaneously GM-CSF-expressing B16-F10 melanoma cells which have been shown to have a limited effect on the immune response [16, 17]; 107 cells per mouse were injected into mice treated previously with daily injections of prolactin or saline for 2 weeks. After the implantation, the mice were treated with saline solution or prolactin for an additional 2 weeks. The percentage of DCs in the mice increased from 1 to 15% and to 30% in the placebo- and prolactin-treated mice, respectively.

DC isolation

Spleens from prolactin- and prolactin/GM-CSF-treated B6.Sle3 mice were ballooned with a 1-ml syringe containing collagenase D solution (400 U/ml) (Roche Diagnostics, Mannheim, Germany) and ethylenediamine tetraacetic acid (EDTA) (2 mM) (Gibco) in phosphate-buffered saline (PBS). The obtained splenocytes were incubated for 30 min at 37°C. Then, DCs were purified by using anti-mouse CD11c antibody conjugated to magnetic beads (Miltenyi Biotech, Auburn, CA, USA) resuspended in buffer [0·5% bovine serum albumin (BSA); Sigma, St Louis, MO, USA] and 2 mM EDTA in PBS. Single-cell suspensions were applied onto a magnetic affinity cell sorting (MACS) column and placed in a magnetic field (MAC separator; Miltenyi Biotech). Unlabelled cells passed through the column, the column was washed and then removed from the separator and magnetically labelled cells were flushed out. The cell suspension was run again through a new column to obtain a purity of ∼100%, which was verified by flow cytometry. CD11b and CD11c staining identified myeloid DCs (CD11bhighCD11chigh), macrophages (CD11bintermediate CD11cintermediate) and neutrophils (CD11bhighCD11c).

Adoptive transfer of DCs

Five to 10 × 106 cells from placebo- or prolactin-treated B6.Sle3 mice were injected intravenously into 6-week-old B6 mice. Three injections were performed each week for 3 weeks. Splenic cells from the B6 recipients were collected 60 days after the adoptive transfer of DCs.

Phenotyping of immune cells

Identification of B cell subsets and expression of co-stimulatory and activation molecules on myeloid and lymphoid cells

Splenic cells were obtained from B6 and B6. Sle3 mice treated with prolactin or recipients of DCs from GM-CSF placebo-treated or GM-CSF prolactin-treated mice. Cells were stained on ice for 30 min with antibodies to CD19 to identify B cells, CD93 to identify transitional B cells and CD21 and CD23 to determine T1 and T2 transitional B cell subsets (CD19+ CD93+, CD21, CD23 and CD19+ CD93+, CD21+, CD23+, respectively). Staining for CD3, CD4 and CD8 was performed to identify helper and cytotoxic T cells, whereas antibodies to CD11b and CD11c were used to detect DCs (CD11bhighCD11chigh), macrophages (CD11bint CD11cint) and neutrophils CD11bhighCD11c) Also, staining for co-stimulatory molecules (CD40, CD40L and B7·2) and differentiation and activation markers (CD25, CD69 and CD44) was performed. The fluorochromes conjugated to the primary antibodies were fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll (PerCP) cyanin (Cy)5·5, PE-Cy7 and allophycocyanin (APC). Biotin-conjugated antibodies were detected by Pacific Blue-conjugated streptavidin. The cells were washed after each incubation, and then fixed in 2% paraformaldehyde. Samples were analysed in an LSR II flow cytometer (BD Bioscience, San Diego, CA, USA). All staining antibodies were purchased from BD Bioscience except CD93, which was obtained from eBioscience (San Diego, CA, USA).

Immunoassays

Apoptosis assay

Splenic cells from prolactin-treated B6.Sle3 mice and B6 recipients of DCs from B6.Sle3 mice treated with GM-CSF/prolactin and respective controls were cultured in medium containing 10 μg/ml of anti-IgM (Southern Biotech, Birmingham, AL, USA) for 16–18 h at 37°C. Then, the cells were washed and stained on ice for the surface markers CD19, CD93, CD21 and CD23 for identification of B cell subsets, as described above. After that, the cells were washed and incubated with annexin V-FITC (BD Bioscience, San Diego, CA, USA) for 15 min at room temperature. The cells were analysed by the LSR II (BD Bioscience) within an hour in the presence of TO-PRO-3 iodide, a viability probe for discrimination of necrotic cells (Invitrogen, Carlsbad, CA, USA).

Enzyme-linked immunospot (ELISPOT) assay

Splenocytes from placebo- and prolactin-treated B6.Sle3 mice and B6 recipients of DCs from placebo and prolactin-treated B6.Sle3 mice and control mice were cultured in dsDNA-coated plates for 48 h with media alone [RPMI-1640 plus 5% fetal calf serum (FCS)] or in media containing anti-CD40 (10 μg/ml; BD Bioscience) and interleukin (IL)-4 (300 U/ml; Sigma-Aldrich) or anti-CD40 and anti-mouse IgG (10 μg/ml; Jackson ImmunoResearch, West Grove, PA, USA). The cells were washed and then incubated with biotin–goat anti-mouse IgG (1 : 500) and alkaline phosphatase (AP)-streptavidin (1 : 1000; Southern Biotechnology) overnight at 4°C and 1 h at room temperature, respectively. AP substrate and 5-bromo-4-chloro-1H-indol-3-yl) dihydrogen phosphate (BCIP) (Amresco, Solon, OH, USA) were added to generate spots in a colorimetric reaction. The spots were counted under a dissecting microscope.

Kidney histology

Formalin-fixed, paraffin-embedded kidney sections from placebo- and prolactin-treated mice were deparaffinized in alcohol and then stained with biotinylated anti-mouse IgG antibody, as described previously [11] The alkaline phosphatase ABC kit (Vector Laboratories, Burlingame, CA, USA) was used to develop the slides.

Biostatistical analysis

The evaluation of mean values, standard deviations and statistical significance (P-values) was performed by using Student's two-tailed t-test. Absolute numbers were calculated from the FACS percentage data and converted to log10 to calculate P-values by Student's two-tailed t-test.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

DNA-reactive B cells

B6.Sle3 mice have been derived from NZM2410 mice. Sle3 locus is on mid-chromosome 7, and confers the development of age-dependent low levels of anti-nuclear antibodies. To determine whether prolactin provides an additive immunostimulatory effect on B cells, young 6-week-old B6.Sle3 mice were treated with prolactin for 4 weeks. Prolactin-treated mice displayed a significantly higher number of DNA-reactive B cells upon exposure to anti-CD40 and IL-4 than did placebo-treated mice (P = 0·01) (Fig. 1a). The same prolactin treatment did not induce an increased number of DNA-reactive B cells in B6 mice [14].

figure

Figure 1. Prolactin accelerates the development of autoreactive phenotype in B6.Sle3. mice. (a) Prolactin-induced dsDNA-reactive B cells. B cells from the spleen of placebo- and prolactin-treated 2–3-month-old B6.Sle3 mice (n = 4 in each group) were tested for the secretion of antibodies to dsDNA by enzyme-linked immunospot (ELISPOT) assay. Splenic cells were cultured for 48 h in the presence of anti-CD40 antibodies and interleukin (IL)-4; a higher number of B cells, 61·6 ± 3·8 reactive B cells per 105 cells from prolactin-treated mice responded to the stimuli by secreting anti-dsDNA antibodies than the B cells from placebo-treated mice, 26·6 ± 8·8 cells per 105 cells (P = 0·01). Similar results were obtained in three other experiments. (b) Glomerular immunoglobulin (Ig)G deposition in kidneys of B6.Sle3 mice. Formalin-fixed paraffin-embedded kidney sections were evaluated by immunohistochemistry to detect IgG deposits. Mice treated with prolactin showed IgG deposits in the glomeruli, whereas placebo-treated mice did not have any IgG deposits in their kidneys. (c) Effect of prolactin on the maturation pattern of B cells and (d) representative samples depicting T1 and T2 B cell subsets fluorescence activated cell sorter (FACS) dot-plots from placebo- and prolactin-treated B6.Sle3 mice. The transitional T1 (CD19+CD93+CD21CD23) and T2 (CD19+CD93+CD21+CD23+) B cell subsets from placebo- and prolactin-treated B6.Sle3 mice (n = 5 in each group) were analysed by flow cytometry. Compared to placebo-treated mice, which had 9·25 ± 0·3 × 106 T1 and 5·1 ± 0·3 × 106 T2 B cells, prolactin-treated mice displayed fewer transitional T1 7·8 ± 0·5 × 106 (P = 0·01) and more transitional T2 B cells, 6·9 ± 0·3 × 106 (P = 0·003), which led to a lower T1/T2 ratio. Similar results were obtained in three different experiments. (e,f) Prolactin-mediated modulation of anti-IgM-induced apoptosis in transitional T1 and T2 B cell subsets. Splenocytes from B6.Sle3 mice treated with placebo or prolactin (n = 5 mice in each group) were cultured in the presence or absence of anti-IgM antibody (10μg/ml) for 16 h at 37°C, and then stained with fluorochrome-labelled antibodies to CD19, CD93, CD21 and CD23 in addition to annexin (to detect apoptosis) and TOPRO-3 (to detect necrosis). As determined by flow cytometry, upon B cell receptor (BCR) engagement, T1 cells from placebo-treated B6.Sle3 mice displayed a higher number of apoptotic cells, 5·4 ± 0·8 × 105 T1 cells in comparison to prolactin-treated mice, 1·47 ± 0·8 × 105 T1 cells (P = 0·001). There was a significant difference in apoptosis between unstimulated and anti-IgM stimulated samples (P = 0·002) in the placebo group; however, there was no significant difference in the prolactin group. A representative histograms showing annexin level differences (apoptosis) between T1 placebo- and T1 prolactin-treated mice (grey line and black line, respectively). Similar results were obtained in three separate experiments.

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Glomerulonephritis

B6.Sle3 mice treated with prolactin developed IgG deposition in the glomeruli (Fig. 1b), indicating that hyperprolactinaemia leads to the production of high-affinity nephritogenic anti-DNA antibodies. Thus, the treatment with prolactin and the lupus susceptibility locus Sle3 separately do not induce lupus in B6 mice, but together they can break B cell tolerance and induce an autoimmune phenotype.

B cell maturation during the transitional stage

Treatment with prolactin induced changes in the maturation pattern of transitional T1 (CD19+CD93+CD21CD23) and T2 (CD19+CD93+CD21+CD23+) B cells in B6.Sle3 mice. The T1 B cell subset was decreased and T2 subset was increased in prolactin-treated mice when compared to placebo-treated B6.Sle3 mice (P = 0·01 and 0·003, respectively) (Fig. 1c,d). Similarly, the T1/T2 ratio was lower in B6. Sle3 mice treated with prolactin than in the placebo-treated mice (data not shown). T1 and T2 subsets in B6.Sle3 mice were also compared to the transitional subsets in congenic wild-type B6 mice that are not affected by treatment with prolactin; there was no difference in the T1/T2 ratio (data not shown).

B cell receptor (BCR)-mediated apoptosis of transitional T1 B cells in B6.Sle3 mice

To understand prolactin-mediated alterations of the maturation pattern of transitional B cells (decreased numbers of T1 and increased numbers of T2 B cells in B6.Sle3 mice treated with prolactin), the effect of prolactin on anti-IgM-induced apoptosis, a mechanism for negative selection of T1 B cells [18, 19] was studied. Prolactin protected T1 B cells from BCR-mediated apoptosis, but in placebo-treated mice the T1 B cell subset under the same conditions displayed a higher number of apoptotic cells (P = 0·002, Fig. 1e); a flow-cytometry histogram shows the inhibition of apoptosis of T1 B cells from prolactin-treated B6.Sle3 mice (Fig. 1f, dark line).

Activated T cells

B6.Sle 3 mice show an elevated CD4/CD8 ratio [20] and the treatment with prolactin increased this ratio further (P = 0·01, Fig. 2a). In addition, prolactin induced higher numbers of CD4 T cells expressing CD40L (P = 0·05, Fig. 2b); activated helper T cells are recognized as a major driver of autoimmunity in lupus.

figure

Figure 2. Prolactin alters CD4 : CD8 ratio and induces CD4 T cell activation. CD4 to CD8 ratio, and activation status of CD4 T cells from spleen were identified by flow cytometry in placebo- and prolactin-treated B6.Sle3 mice (n = 4 in each group). (a) CD4 : CD8 ratio. There was a significant difference (P = 0·01) in the CD4 : CD8 ratio between the two groups; placebo-treated mice had 31·13 ± 0·8 × 106 CD4 T cells in comparison to 35·2 ± 1·3 × 106 in the prolactin-treated mice (P = 0·001). (b) CD40L+CD4+ T cells. CD4+ T cells from prolactin-treated B6.Sle3 mice showed higher numbers of CD40L+ cells than did placebo-treated mice, 7·8 ± 0·8 × 105 and 4·3 ± 0·9 × 105, respectively (P = 0·05). Similar data were obtained in three separate experiments.

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Over-expression of activation and co-stimulatory molecules; CD40-, B7·2- and MHC II-expression on B cells of B6.Sle3 mice

The effect of prolactin on the expression of a variety of activation and co-stimulatory markers was compared between prolactin- and placebo-treated B6.Sle3 and B6 mice. In B6.Sle3 mice, treatment with prolactin increased the numbers of follicular (Fo) B cells expressing CD40, and B7·2 (P = 0·03) and induced an increased expression of IAb (P = 0·01) on Fo B cells; all three molecules are necessary for the B–T cell interactions, and their up-regulation may lead to enhanced antibody responses (Fig. 3a–d). However, CD44 and CD69 were not affected by prolactin in B6.Sle3 mice (data not shown).

figure

Figure 3. Prolactin-mediated immunostimulatory effects on B cells and myeloid cells. (a,b) Prolactin induces over-expression of CD40 and B7·2 on the mature follicular B cells. Splenocytes from placebo and prolactin-treated B6.Sle3 mice (n = 4 for each group) were surface-stained for CD19, CD93, CD21, CD23, B7·2, CD40, CD44, CD69 and IAb. Follicular (Fo) B cells (CD19+CD93CD21CD23+) from prolactin-treated B6.Sle3 mice showed higher numbers of cells expressing CD40 and B7·2 than the placebo group, 1·15× ± 0·7 × 106, 0·55× ± 0·7 × 106, respectively (P = 0·03). The flow cytometric histogram depicts CD40 expression [mean fluorescence intensity (MFI)] from one mouse in each group (placebo: light line; prolactin-treated mouse: dark line). (c,d) Prolactin induces expression of major histocompatibility complex (MHC) class II on B cells. Mature B cells (CD19+CD93) from prolactin-treated B6.Sle3 mice showed higher levels of IAb (MHC-class II) when compared to placebo mice (P = 0·01). The histogram represents MFI expression of IAb in a placebo mouse (light line) and a prolactin-treated mouse (dark line). The graph shows one representative experiment out of three. (e,f) Prolactin affects the phenotypes of myeloid cells in B6. Sle3 mice. Splenic cells from placebo- and prolactin-treated B6.Sle3 mice (n = 4 for placebo and n = 5 for prolactin-treated mice) were labelled with antibodies to CD11c, CD11b, IAb, CD40, CD44, CD69 and B7·2. Splenic dendritic cells (DCs) were identified as CD11chigh CD11bhigh, macrophages as CD11cint CD11bint and neutrophils as CD11cCD11bhighand then phenotyped for the expression levels of co-stimulatory, activation and differentiation markers. DCs and macrophages from prolactin-treated mice expressed higher levels of B7·2 than the DCs of placebo-treated B6.Sle3 mice (P = 0·01 for DCs and P = 0·04 for macrophages). Representative flow-cytometric plots depicting the B7·2 expression in a placebo- and prolactin-treated mice. (g,h) Treatment with prolactin increased the expression of CD44 on both DCs and macrophages (P = 0·01 for both DCs and macrophages). Representative flow-cytometric plots from mice treated with prolactin and placebo.

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Enhanced macrophage activation and maturation of DCs

B6.Sle3 mice contain more mature and activated macrophages, DCs and neutrophils than do B6 mice [21, 22]. Treatment with prolactin further increased the maturation and activation status of DCs and macrophages, as indicated by the over-expression of B7·2 (P = 0·01 and 0·04, respectively) and CD44 (P = 0·01 for both cell types) on their surface (Fig. 3e–h). Treatment with prolactin had no effect on the activation status of neutrophils in B6.Sle3 mice, nor did it affect the expression of B7·2 and CD44 on myeloid cells in B6 mice (data not shown).

Prolactin-modulated- DCs from B6.Sle3 mice exert immunostimulatory effects in B6 mice

DNA-reactive B cells

Given that the Sle3 locus generates mature DCs and that the carrying phenotype is intrinsic to APCs [22], we speculated that prolactin-modulated DCs would be able to induce a lupus-like phenotype when they are transferred adoptively in B6 mice. The B6 recipients of DCs from prolactin-treated B6.Sle3 mice developed a similar B cell phenotype to that of B6.Sle3 mice treated with prolactin. Two of the three recipients of prolactin-modulated DCs showed mild–moderate IgG deposits, and one recipient showed only mild deposits. The recipients of prolactin-modulated DCs had more DNA-reactive B cells than did the recipients of DCs from placebo-treated B6.Sle3 mice (P = 0·02) (Fig. 4a).

figure

Figure 4. (a) Dendritic cells (DCs) from prolactin-treated B 6.Sle3 mice have the capacity to induce DNA-reactive B cells in B6 mice. Splenic cells from recipients of DCs from B6.Sle3 mice treated with placebo or prolactin and granulocyte–macrophage colony-stimulating factor (GM-CSF) were cultured in the presence of anti-CD40 antibody and interleukin (IL)-4, and then incubated in the presence of immobilized dsDNA. dsDNA-reactive B cells were detected by enzyme-linked immunospot (ELISPOT) assay and the spots were counted in 105 cells under a dissecting microscope. There was a higher number of DNA-reactive B cells in prolactin-treated mice, 16·5 ± 2 cells per 105 than in mice treated with placebo, 9·6 ± 1·7 cells per 105 (P = 0·02, n = 3 in each group). (b,c) DCs from prolactin-treated B6.Sle3 mice alter the maturation pattern of B cells in B6 mice. Splenic cells from B6 mice which underwent adoptive transfer of DCs from placebo- or prolactin-treated B6.Sle3 mice were evaluated for T1 (CD19+CD93+CD21CD23) and T2 (CD19+CD93+CD21+CD23+) B cell subsets to assess the changes in the B cell maturation pattern. There was an increase in the number of T2 B cells, 8·06 ± 0·8 × 106, between the recipients of DCs from prolactin- versus placebo-treated B6.Sle3 mice, 5·2 ± 0·9 × 106 (P = 0·05), suggesting that a higher number of T1 B cells are escaping negative selection and maturing into T2 B cells (n = 3 in each group). Representative histograms depict transitional T1 and T2 subsets from a placebo- and a prolactin-treated mouse. Three experiments were performed and provided similar results to the ones shown. (d,e) DCs from B6.Sle3 mice break B cell tolerance in B6 mice by affecting the maturation pattern of transitional B cell subsets. Splenic cells cultured in medium alone or in medium containing anti-IgM antibody (10 μg/ml) were harvested and stained for cell surface markers (CD19, CD93, CD21 and CD23) to identify the B cell subsets. Annexin was added to the samples to detect apoptotic cells and TOPRO-3 was added to discriminate necrotic from apoptotic cells. The graph shows the difference in B cell receptor (BCR)-mediated apoptosis of T1 B cells between the recipients of placebo- and prolactin-modulated DCs. There was a significant difference in apoptosis in the placebo group (P = 0·004) and no significance in the prolactin group. Placebo- and prolactin-treated mice had 3·9 ± 0·8 × 105 and 1·47 ± 0·8 × 105 T1 cells, respectively, which underwent apoptosis (P = 0·02). The graph depicts a representative experiment of three performed (n = 3 in each group). The flow cytometric histogram depicts the difference in T1 B cell apoptosis from recipients of DCs from placebo- (grey line) and DCs from prolactin-treated (black line) B6.Sle3 mice.

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B cell maturation pattern

The recipients of prolactin-modulated DCs displayed an increased number of transitional T2 B cells (P = 0·05). T1 B cells were decreased in mice which received prolactin-modulated DCs when compared to the recipients of DCs from placebo-treated mice; although the difference was not significant, there was a decrease of the T1/T2 ratio in the prolactin treated DC group (Fig. 4b, c).

BCR-induced apoptosis of T1 B cells

A higher percentage of apoptotic T1 B cells was observed in the recipients of placebo DCs than in the recipients of DCs from prolactin-treated B6.Sle3 mice. The difference in apoptosis between untreated and anti-IgM stimulated T1 B cells from placebo mice was significant (P = 0·004); in contrast, the difference in the prolactin treated group was not significant (Fig. 4d,e). This observation indicates that prolactin-modulated DCs are initiating the altered B cell maturation pattern depicted by the decreased T1/T2 ratio in the B6 recipients as a consequence of the inhibition of apoptosis. Similar T1/T2 alterations were observed in B6.Sle3 mice which received a 4-week prolactin treatment, as shown in Fig. 1c,d.

B cell activation markers

The B6 recipients of DCs from prolactin-treated B6.Sle3 mice expressed higher levels of IAb on Fo B cells than the Fo B cells from recipients of placebo DCs (P = 0·01). The fact that B cells express more IAb enables them to be more efficient APCs, which may contribute to the development of a lupus-like phenotype (Fig. 5a,b). As shown previously, Sle3 DCs carry intrinsic abnormalities that induce certain autoimmune phenotypes associated with lupus [21]. DCs from prolactin-treated B6.Sle3 mice were able to transfer the prolactin-mediated lupus characteristics to B6 mice, which do not develop a lupus-like disease when treated with prolactin [11].

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Figure 5. (a,b) Adoptive transfer of DCs from prolactin-treated B6.Sle3 mice increases the major histocompatibility complex (MHC) class II expression on B cells in B6 mice. Splenic B cells from B6.Sle3 recipients of DCs from placebo- or prolactin-treated B6.Sle3 mice (n = 3 in both groups) were assessed for the expression of several co-stimulatory and activation molecules: B7·2, CD40, CD44, CD69 and IAb. The graph depicts the IAb expression [mean fluorescence intensity (MFI)] on follicular (Fo) B cells from mice which underwent adaptive transfer with placebo- or prolactin-modulated DCs from B6.Sle3 mice; the IAb values were higher in the latter group (0·01). Similar data were obtained in two separate experiments. Histograms from one representative sample of each group are depicted: B cells from a recipient of prolactin-modulated DCs (solid black line) and B cells from recipient of placebo DC (grey line).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Twenty-five to 30% of patients with SLE have increased serum prolactin, and in a significant number of these patients prolactin levels correlate with lupus activity and/or system organ involvement [7]. These data raised interest in the role of prolactin in lupus and have led to numerous murine studies [1, 10-12, 23]. It has been shown that treatment with prolactin (100 μg/day) exerts immunostimulatory effects in BALB/c mice by altering BCR-mediated tolerance-induction during B cell maturation and, consequently, ‘rescuing’ autoreactive B cells from deletion [11]. The same prolactin treatment did not affect B cells in B6 mice [11], but the NZM240-derived Sle3/5 genetic interval provided responsiveness to prolactin in these mice. B6.Sle3/5 mice developed a lupus-like phenotype when treated with 100 μg prolactin daily for 4 weeks [14], indicating that the Sle3/5-encoding proteins confer sensitivity to the hormone. The work presented here demonstrates that a portion of the Sle3/5 interval, the lupus susceptibility locus Sle3, is sufficient for conferring responsiveness to prolactin and prolactin-modulated DCs from B6.Sle3 mice can induce the development of DNA-reactivity in B6 mice.

Using B6.Sle3 mice [20], we endeavoured to expand our evaluations of the immune effects of prolactin in order to understand more clearly their implications in the pathogenesis of lupus. The Sle3 locus induces minor immunological changes in B6 mice, such as a slightly increased CD4/CD8 ratio and activated myeloid cells. Myeloid cell hyperactivity results in the secondary activation of lymphocytes leading to an age-dependent development of anti-nuclear antibodies when B6.Sle3 mice reach 9–12 months [20-22]. The Sle3 mice require additional factors, such as the presence of the Sle1 locus for progression to full-blown lupus [24].

In the current study, prolactin is defined as an immunostimulator that exacerbates and accelerates the Sle3 phenotype towards lupus pathology when the mice are only 2·5–3 months old. A doubling of serum prolactin levels hastens the occurrence of IgG-secreting DNA-reactive B cells in young B6.Sle3 mice, which indicates that prolactin enhances the previously reported effectiveness of Sle3 in mediating Ig class-switching to IgG [24]. These IgG anti-dsDNA antibodies are pathogenic, and deposit in the kidneys of B6.Sle3 mice.

Increased prolactin levels exert a protective effect on B cell maturation from the transitional T1–T2 stage, allowing higher numbers of autoreactive B cells to escape negative selection at the T1/T2 interphase [16,17], which is a check-point for B cell tolerance induction [25-28].

Autoreactive T2 cells ‘rescued’ by prolactin in B6.Sle3 mice mature further into fully functional B cells, which differentiate into anti-dsDNA antibody-secreting plasma cells capable of initiating the development of a lupus phenotype.

Also, prolactin induced an over-expression of B7·2 and MHC class II on B cells and DCs, and also CD40 on B cells. Murine and human data indicate that DCs can provide B cells with CD40-dependent survival signals and CD40-independent proliferative signals [29], both of which could play a role in DNA reactivity observed in prolactin-treated B6.Sle3 mice. Reports of increased B7·2 expression on B cells of SLE patients [30] indicate that prolactin-induced up-regulation of B7·2 may play an important role in the acceleration of autoimmunity in the B6.Sle3 murine model.

Based on the reports that CD40-over-expressing B cells can become plasma cells in the presence of DCs without T cell intervention [31, 32], prolactin-induced up-regulation of CD40 seems to be an important contributor to the accelerated autoantibody production in B6.Sle3 mice. Thus, prolactin-mediated over-expression of CD40 on B cells with or without CD40L expression on T cells might be a contributory factor for the increased differentiation of B cells into autoantibody-secreting plasma cells [33, 34].

Nine to 12-month-old B6.Sle3 mice exhibit myeloid cell hyperactivity that is intrinsic to the Sle3 genes [22, 25]. In-vivo treatment with prolactin up-regulates the activation status of myeloid cells in 2–3-month-old B6.Sle 3 mice by increasing the expression of B7·2 and CD44 molecules on APCs. Prolactin-induced over-expression of B7·2 shows that the hormone can accelerate the maturation status of DCs and increase their co-stimulatory properties, and also enhances their capacity to adhere to the extracellular matrix components [35] in target tissues, potentially initiating the inflammatory organ-specific injuries observed in lupus [36, 37].

The adoptive transfer of DCs from prolactin-treated B6.Sle3 mice into B6 recipients induced significantly more activated MHC class II over-expressing B cells and DNA-reactive B cells than did the adoptive transfer of DCs from placebo-treated B6.Sle3 mice. A recent study reported a link between the DC hyperactivity in B6.Sle3 mice and the X chromosome-encoded gene IL-1 receptor-associated kinase-1 (IRAK-1) [38], but this association has been questioned by others [39]. It seems that because of the high linkage disequilibrium in the Xq28, both IRAK1 and methyl–cytosine–phosphate–guanosine (CpG)-binding protein 2 (MECP2) could be considered candidate genes for lupus.

The DC-mediated elevation of the number of DNA-reactive B cells in B6 mice could be accomplished by direct interaction between DCs and B cells or indirectly by secretion of cytokines. CD40-over-expressing DCs, known for their role in the early stages of lupus [40], can help autoreactive immature B cells to escape negative selection at the T1/T2 interphase. The survival of autoreactive specificities can be facilitated by prolactin-modulated DCs presenting unprocessed autoantigen to B cells [26]. Also, prolactin-modulated DC may induce differentiation of autoreactive B cells into autoantibody-secreting plasma cells by secretion of cytokines such as IL-6, IL-12 and B lymphocyte stimulator (BlyS) [41]. However, the lower anti-DNA reactivity in B6 recipients of prolactin-modulated DCs from B6.Sle3 mice than the DNA reactivity observed in prolactin-treated B6.Sle3 mice underscores the importance of the direct effects of prolactin on autoreactive B cells such as the over-expression of CD40 [42]. Down-regulation of CD40 on B cells occurs in SLE patients after successful treatment with rituximab [43].

In conclusion, in young 2–3-month-old B6.Sle3 mice, mild hyperprolactinaemia, similar to that observed in almost 30% of SLE patients, induces a lupus-like illness characterized by activated myeloid and lymphoid cells, an increased number of DNA-reactive B cells and autoantibody deposits in the kidneys. The effects of prolactin on B cells are mediated partially by accelerated maturation of DCs which can activate the self-reactive CD40-over-expressing B cells directly or indirectly via CD40L+CD4+T cells. Increased CD40 expression on B cells seems to be a direct effect of prolactin, while the over-expression of MHC II on B cells could be induced by prolactin-modulated DCs. In addition, DCs from prolactin-treated B6.Sle3 mice have the capacity to induce autoreactivity in B6 mice, which do not respond to treatment with prolactin.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

This study was supported by the The Lupus Foundation. We would like to thank Dr E. Wakeland (University of Texas Southwestern Medical Center, Dallas) for his generous gift of the B6.Sle3 mice. We also thank Gabriel Rosenfeld and Yu Zhang for their technical help, and Joel Correa da Rosa from The Rockefeller University, CCTS, for the statistical analysis.

Disclosure

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors affirm that they have no actual or potential conflicts of interest to disclose.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
  • 1
    McMurray RW. Prolactin and systemic lupus erythematosus. Ann Med Interne (Paris) 1996; 147:253258.
  • 2
    Allen SH, Sharp GC, Wang G et al. Prolactin levels and antinuclear antibody profiles in women tested for connective tissue disease. Lupus 1996; 5:3037.
  • 3
    Neidhart M. Elevated serum prolactin or elevated prolactin/cortisol ratios are associated with autoimmune processes in systemic lupus erythematosus and other connective tissue diseases. Br J Rheumatol 1996; 23:476481.
  • 4
    Miranda JM, Priet RE, Panigua R et al. Clinical significance of serum and urine prolactin levels in lupus glomerulonephritis. Lupus 1998; 7:387391.
  • 5
    Orbach H, Zandman-Goddard G, Boaz M et al. Prolactin and autoimmunity: hyperprolactinemia correlates with serositis and anemia in SLE patients. Clin Rev Allergy Immunol 2012; 42:189198.
  • 6
    Shelly S, Boaz M, Orbach H. Prolactin and autoimmunity. Autoimmun Rev 2012; 11:A465470.
  • 7
    Karimifar M, Tahmasebi A, Bonakdar ZS, Purajam S. Correlation of serum prolactin levels and disease activity in systematic lupus erythematosus. Rheumatol Int 2011; doi:10.1007/s00296-011-2211-5.
  • 8
    Blanco-Fabela F, Quintal-Alvarez G, Leaños-Miranda A. Association between prolactin and disease activity in systemic lupus erythematosus. Influence of statistical power. J Rheumatol 1999; 26:5559.
  • 9
    Saha S, Tieng A, Pepeljugoski KP, Zandamn-Goddard G, Peeva E. Prolactin, systemic lupus erythematosus, and autoreactive B cell: lessons learn from murine models. Clin Rev Allergy Immunol 2011; 40:815.
  • 10
    McMurray R, Keisler D, Izui S, Walker S. Hyperprolactinemia in male NZB/NZW (B/W) F1 mice: accelerated autoimmune disease with normal circulating testosterone. Clin Immunol Immunopathol 1994; 71:338343.
  • 11
    Peeva E, Michael D, Cleary J, Rice J, Chen X, Diamond B. Prolactin modulates the naïve B cell repertoire. J Clin Invest 2003; 11:275283.
  • 12
    Saha S, Gonzalez J, Rosenfeld G, Keiser H, Peeva E. Prolactin alters the mechanisms of B cell tolerance induction. Arthritis Rheum 2009; 60:17431752.
  • 13
    Morel L, Mohan C, Yu Y et al. Multiplex inheritance of component phenotypes in a murine model of lupus. Mamm Genome 1999; 10:176181.
  • 14
    Peeva E, Gonzalez J, Hicks R, Diamond B. Lupus susceptibility interval Sle3/5 confers responsiveness to prolactin in C57BL/6 Mice. J Immunol 2006; 177:14011405.
  • 15
    Maraskovsky E, Brasel K, Teepe M et al. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J Exp Med 1996; 84:19531962.
  • 16
    Dranoff G, Jaffee E, Lazenby A et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte–macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor. Proc Natl Acad Sci USA 1993; 90:35393543.
  • 17
    Van Elsas A, Hurwitz AA, Allison J. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-production vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med 1999; 190:355363.
  • 18
    Su TT, Rawlings DJ. Transitional B lymphocyte subsets operate as distinct checkpoints in mature splenic B cell development. J Immunol 2002; 168:21012110.
  • 19
    Vossenkamper A, Luttalo PM, Spencer J. Translational Mini-Review Series on B cell subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies. Clin Exp Immunol 2012; 167:714.
  • 20
    Mohan C, Yu Y, Morel L, Yang P, Wakeland EK. Genetic dissection of Sle pathogenesis: Sle3 on murine chromosome 7 impacts T cell activation, differentiation, and cell death. J Immunol 1999; 162:64926502.
  • 21
    Sobel ES, Morel L, Baert R, Mohan C, Schiffenbauer J, Wakeland EK. Genetic dissection of systemic lupus erythematosus pathogenesis: evidence for functional expression of Sle3/5 by Non-T Cells. J Immunol 2002; 69:40254032.
  • 22
    Zhu J, Liu XB, Xie C et al. T cell hyperactivity in lupus as a consequence of hyperstimulatory antigen-presenting cells. J Clin Invest 2005; 115:18691878.
  • 23
    Peeva E, Grimaldi C, Spatz L, Diamond B. Bromocriptine restores tolerance in estrogen-treated mice. J Clin Invest 2000; 106:13731379.
  • 24
    Liu K, Li QZ, Yu Y et al. Sle3 and Sle5 can independently couple with Sle1 to mediate severe lupus nephritis. Genes Immun 2007; 8:634645.
  • 25
    Wardemann H, Yurasov S, Schaefer A, Young JW, Meffre E, Nussenzweig MC. Predominant autoantibody production by early human B cell precursors. Science 2003; 301:13741377.
  • 26
    Grimaldi CM, Hicks R, Betty Diamond B. B cell selection and susceptibility to autoimmunity. J Immunol 2005; 174:17751781.
  • 27
    Ding C, Yan J. Regulation of autoreactive B cells: checkpoints and activation. Arch Immunol Ther Exp (Warsz) 2007; 55:8389.
  • 28
    Von Boehmer H, Melchers F. Check points in lymphocytes development and autoimmunune disease. Nat Immunol 2010; 11:1420.
  • 29
    Wykes M, Macpherson G. Dendritic cell–B-cell interaction: dendritic cells provide B cells with CD40-independent proliferation signals and CD40-dependent survival signals. Immunology 2000; 100:13.
  • 30
    Nagafuchi H, Shimoyama Y, Kashiwakura J, Takeno M, Sakane T, Suzuki N. Preferential expression of B7.2 (CD86), but not B7.1 (CD80), on B cells induced by CD40/CD40L interaction is essential for anti-DNA autoantibody production in patients with systemic lupus erythematosus. Clin Exp Rheumatol 2003; 21:7177.
  • 31
    Caux C, Massacrier C, Vanbervliet B et al. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte–macrophage colony-stimulating factor plus tumor necrosis factor-α. II. Functional analysis. Blood 1997; 90:14581470.
  • 32
    Dubois B, Massacrier C, Vanbervliet B et al. Critical role of IL-12 in dendritic cell-induced differentiation of naive B lymphocytes. J Immunol 1998; 161:22232231.
  • 33
    Hostmann A, Jacobi AM, Mei H, Hiepe F, Dörner T. Peripheral B cell abnormalities and disease activity in systemic lupus erythematosus. Lupus 2008; 17:10641069.
  • 34
    Dörner T, Lipsky PE. Correlation of circulating CD27high plasma cells and disease activity in systemic lupus erythematosus. Lupus 2004; 13:283289.
  • 35
    Vigetti D, Genasetti A, Karousou E, Viola M, Moretto P, Clerici M. Proinflammatory cytokines induce hyaluronan synthesis and monocyte adhesion in human endothelial cells through hyaluronan synthase 2 (HAS2) and the nuclear factor-κB (NF-κB) pathway. J. Chem Biol 2010; 285:2463924645.
  • 36
    Tang T, Rosenkranz A, Assmann K et al. A role for Mac-1 (CD11b/CD1 in FcgR interactions in vivo: Mac-1 deficiency abrogates sustained neutrophil adhesion and proteinuria in Fc-dependent anti-glomerular basement membrane nephritis. J Exp Med 1997; 186:18531863.
  • 37
    Westerhuis R, van Straaten SC, van Dixhoorn M et al. Distinctive roles of neutrophils and monocytes in anti-thy-1 nephritis. Am J Pathol 2000; 156:303310.
  • 38
    Jacob CO, Zhu J, Armstrong DL et al. Identification of IRAK1 as a risk gene with critical role in the pathogenesis of systemic lupus erythematosus. Proc Natl Acad Sci USA 2009; 106:62566261.
  • 39
    Sawalha AH. Xq28 and lupus: IRAK1 or MECP2? Proc Natl Acad Sci USA 2009; 106:E62.
  • 40
    Colonna L, Dinnall JA, Shivers DK, Frisoni L, Caricchio R, Gallucci S. Abnormal costimulatory phenotype and function of dendritic cells before and after the onset of severe murine lupus. Arthritis Res Ther 2006; 8:111.
  • 41
    Jego G, Pascual V, Palucka AK, Banchereau J. Dendritic cells control B cell growth and differentiation. Curr Dir Autoimmun 2005; 8:124139.
  • 42
    Kaneko Y, Hirose S, Abe M, Yagita H, Okumura K, Shirai T. CD40-mediated stimulation of B1 and B2 cells: implication in autoantibody production in murine lupus. Eur J Immunol 1996; 26:30613065.
  • 43
    Tokunaga M, Fujii K, Saito K et al. Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab. Rheumatology (Oxf) 2005; 44:176182.