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Cell Growth & Differentiation Vol. 10, 583-590, August 1999
© 1999 American Association for Cancer Research

Influence of Prolactin on the Differentiation of Mouse B-Lymphoid Precursors1

Patricia Morales, María Victoria Carretero, Haydée Geronimo, Sergio G. Copín, María Luisa Gaspar, Miguel A. R. Marcos and Jorge Martín-Pérez2

Centro de Biología Molecular Severo Ochoa, C.S.I.C., Campus Cantoblanco, 28049 Madrid [P. M., S. G. C., M. A. R. M.]; Instituto de Investigaciones Biomédicas, C.S.I.C., Arturo Duperier 4, 28029 Madrid [M. V. C., H. G., J. M-P.]; Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda 28220, Madrid [M. L. G.], Spain


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Development and activation of immune cells are submitted to hormonal influences, as illustrated by the roles of corticosteroids in thymus, pregnancy-related estrogens in B-cell development, or prolactin (PRL) on T-cell generation and function. We have analyzed the putative role of PRL in B lymphopoiesis and differentiation. We chose as an experimental model the interleukin (IL)-3 dependent BaF-3 pro-B cell line, which was transfected with the rat long form of the PRL receptor (PRL-R) and transferred from IL-3- to PRL-enriched media. When stimulated with PRL, the PRL-R transfectants underwent some changes characteristic of B-cell differentiation: (a) IL-2R {alpha} chain became positively controlled by PRL; (b) antiapoptotic Bcl-2 protein was induced by PRL in a dose-dependent manner; and (c) transcription of the pre-B cell receptor encoding the {lambda}5 gene was strongly up-regulated. We attempted to evaluate the differentiation-promoting activity of PRL in more physiological conditions, and the presence of PRL-R in bone marrow B-cell precursors was revealed. Furthermore, PRL promoted significant expansions of defined B-lineage cell populations in short-term bone marrow cell cultures. These findings suggest that PRL, in collaboration with other cytokines and hormonal influences, modulates B-cell development.


   Introduction
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 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
PRL3 is a polypeptide hormone with structural polymorphism and multiple sites of synthesis. Because PRL-R is ubiquitously expressed, PRL modulates a variety of physiological functions that go from the development of the mammary gland and lactation to immune regulation (reviewed in Ref. 1 ). The PRL-R is a member of the type I cytokine receptor superfamily, which includes those for growth hormone, IL-2 to IL-7, erythropoietin, granulocyte-colony stimulating factor, granulocyte/macrophage-colony stimulating factor, and others, and it has no intrinsic enzymatic activity (2) . Three different forms of PRL-R have been described; the short and the long forms are generated by alternative RNA splicing, and an intermediate form found in the Nb2 rat thymoma cell line is produced through a partial in-frame deletion (3) . Upon interaction with the hormone, receptor dimerization and tyrosine phosphorylation of latent signaling proteins are induced. The receptor itself, the Jak and Src families of tyrosine kinases (4 , 5) , as well as Vav and Grb2/Sos-Ras-Raf-mitogen-activated protein kinase and Jak-STAT signal transduction cascades become activated (Refs. 6, 7, 8 , and references within), leading to cellular proliferation and/or differentiation. The set of Jak-Stat activated by each cytokine could be, at least in part, responsible for their specific physiological actions (reviewed in Ref. 9 ). PRL and its receptors have been detected in mature lymphocytes and in thymocytes (reviewed in Ref. 10 ). There, PRL can induce proliferative responses, accompanied by the expression of IL-2 receptors and secretion of IL-2 and IFN-{gamma} (11 , 12) . The available information on a putative role of PRL pathways in lymphoid differentiation is limited; most of the studies have been carried out in in vitro Nb2 thymoma cells, where PRL is a mitogen inducing the expression of growth-related genes (reviewed in Ref. 10 ). PRL has been also implicited in the development and activation of other immunological cell types such as macrophages, neutrophils, and erythrocytes (10 , 13) . PRL-R was thus detected in thymocytes and thymic epithelial cells (14) . Whereas PRL had mitogenic activity in mature T cells (15) and increased the numbers of antigen-specific T cells in vivo (16) . PRL further contributed to T-cell-dependent autoimmunity in well-established murine models (17) . No PRL activity has been characterized, however, in the B-cell lineages, although the PRL-R is expressed in mature B lymphocytes (18) . PRL-controlling Pit-1/GHF-1 transcription factor has also been detected in normal BM (19) .

B-cell lymphopoiesis is a complex process evolving through ordered stages of differentiation that are controlled by transcription factors, lineage-specific gene products (e.g., PBCR-encoding genes and immunoglobulin loci-derived clonotypes), stromal cell-progenitor interactions, and paracrine/endocrine factors (reviewed in Ref. 20 ). In this context, the study of pregnant mice has revealed a negative regulatory role of estrogens in BM-derived B-cell generation (21) . Androgen receptors are also expressed by immature B cells as well as by BM stromal cells, and androgens may also modulate B-cell development in male mice (22 , 23) . Also, insulin-like growth factor type I secreted by stromal cells can stimulate the differentiation to cytoplasmic µ-positive pre-B cells in short-term BM cell cultures (24) . Here we have studied the putative involvement of PRL-driven signals in mouse B-cell differentiation. We analyzed the influence of PRL in B-cell differentiation by transfecting the rat PRL-R on the IL-3-dependent BaF-3 pro-B cell line (25) . BaF-3 cells transfected with the rat PRL-R, so-called W53 cells, were selected and grown in PRL-enriched media. Several molecular changes related to B-cell differentiation in W53 cells ({lambda}5, IL-2R {alpha}, and Bcl-2) were studied, and the results revealed that PRL signaling on BaF-3 pro-B cells promoted significant features of B-cell differentiation in them. PRL-R expression was found in normal mouse BM B-cell precursors, where PRL promoted increases in defined B-lineage cell populations in short-term cultures of BM. These data suggest that PRL might influence normal B-cell differentiation in the mouse.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
IL-3-dependent BaF-3 pro-B Cells Change to PRL Dependence upon Transfection of the PRL-R Gene.
To test for a putative role of the PRL pathway on B-cell differentiation, we performed transfection experiments in the BaF-3 pro-B cell line that did not express the PRL-R (Fig. 1A)Citation . We introduced the long form of the rat PRL-R into BaF-3 cells by means of the retroviral-encoding promoter of the pBABE-Puro plasmid. After selection of transfected cells with puromycin, the W53 cells were transferred to PRL-enriched medium. Immunoprecipitation of PRL-R with the U5 mAb revealed a specific Mr 100,000 protein that was absent in BaF-3 cells transfected with the empty vector (Fig. 1A)Citation . This is in agreement with data published previously (4) . PRL-R transfection was functional in W53 cells because these cells proliferated in IL-3-deprived, PRL-enriched medium in a dose-dependent manner (Fig. 1B)Citation . In contrast, BaF-3 cells cultured under the same conditions suffered apoptosis after 12–18 h, as determined by the typical DNA fragmentation laddering pattern of this process (data not shown). We thus conclude that PRL was efficient in maintaining the survival/proliferation machinery of BaF-3 pro-B cells transfected with the PRL-R in the absence of IL-3.



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  		Fig. 1. Transfection of the rat PRL-R into BaF-3 cells. A, BaF-3 and W53 cells (107 were biosynthetically labeled with L-[35S]methionine and L-[35S]cysteine (200 µCi/ml) for 2 h. The supernatants from cell lysates were immunoprecipitated sequentially with normal mouse serum, as preimmune control, and then with the anti-PRL-R U5 mAb. The immune complexes were analyzed by SDS-9% PAGE and fluorography. Molecular markers (in thousand) are at the left, and the position of the PRL-R is indicated. B, W53 cells were seeded at 104 cells/well in 96-microwell plates in complete medium supplemented with PRL. After 48 h, cell cultures were pulsed overnight with 1 µCi of [3H]thymidine, harvested [3H]-TdR), and the incorporated radioactivity was measured in a scintillation counter (35,591 ± 818 cpm in BaF-3 cells = 100%). Bars, SD.

 
IL-2R {alpha} Chain Expression Is PRL Dependent in W53 Cells.
Among the other B-lineage-specific cell surface markers tested (CD43, BP-1, and others; data not shown), BaF-3 cells expressed moderate levels of the IL-2R {alpha} chain (CD25; Fig. 2Citation , upper histogram), probably under the control of IL-3-driven signals, as it has been shown for other hemopoietic cells (26) . When W53 cells were grown in IL-3-enriched medium, IL-2R {alpha} chain expression persisted, although at a lower frequency of cells than in the original BaF-3 cells (Fig. 2Citation , lower histogram). The IL-2R {alpha} chain expression in W53 cells is controlled by PRL in a dose-dependent manner (Fig. 2Citation , lower histograms). As discussed above, BaF-3 cells (PRL-R negative) were unable to survive when grown only on PRL. These findings suggest that PRL signals, as well as IL-3, participate in the up-regulation of the IL-2R {alpha} chain, a characteristic change of the developmental transition from B220lowCD43+ pro-B to B220highCD43- pre-B cells.



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  		Fig. 2. PRL induces the IL-2R {alpha} chain on PRL-R W53 transfected cells. Expression of B-lineage markers was analyzed by flow cytometry in BaF-3 cells and in W53 cells grown in PRL-enriched medium during 4 days. Cells (5 x 105) were incubated with biotin-conjugated PC61 mAb and then revealed with streptavidin-PE. Expression of IL-2R {alpha} chain in BaF-3 cells growth in IL-3-enriched medium (upper histogram) and in W53 cells grown in IL-3 or with titrated doses of PRL (··· 0.3, — 3, — 30 ng/ml; bottom histograms) are shown. The background signals, provided by isotype-matched irrelevant mAb in both BaF-3 and W53 cells, were adjusted during cell acquisition, and they are displayed in the shaded histograms for both cell lines. The percentages of IL-2R {alpha} chain+ cells are shown at the upper right corner of the histograms. This is one representative experiment of four independent analyses.

 
PRL Increases the Amount of the Antiapoptotic Bcl-2, but not Bcl-XL, Proteins in W53 Cells.
A dominant influence of the cell survival machinery is relevant for lymphohemopoietic differentiation to proceed with efficiency (27) . Although high proliferation rates of pro-B cells expand the size of their pre-B-cell progeny, most are unsuccessful and later die, with minor populations of death-resistant pre-B cells evolving to final maturation and positive selection (28) . Bcl-2 and Bcl-XL represent intracellular membrane-bound proteins implicated in death antagonism in lymphoid lineages (29, 30, 31) . We tested whether their levels were altered by PRL-driven signals in our experimental system by undertaking intracellular immunofluorescence staining and Western blot analysis with specific mAb. Flow cytometry analysis revealed that Bcl-2 and Bcl-X proteins were constitutively expressed both in BaF-3 and in W53 cells (Fig. 3A)Citation . Bcl-2 protein levels, however, clearly increased (3–10-fold) in W53 cells at 30 ng/ml of PRL with respect to the levels obtained at 1 ng/ml. Bcl-X basal expression, in contrast, was unmodified in W53 cells between the lowest and the highest PRL dosages (Fig. 3A)Citation . These observations were confirmed by Western blot analysis (Fig. 3B)Citation . Although the Bcl-2 protein levels increased, the expression of the Bcl-X long isoform, Bcl-XL (the Mr 26,000 form of Bcl-X), remained unmodified in W53 versus BaF-3 (Fig. 3B)Citation . These results indicate that PRL increases the levels of antiapoptotic Bcl-2 protein in PRL-R-transfected BaF-3 cells, providing them with a basal requirement to stimulate their B-cell differentiation.



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  		Fig. 3. PRL-dependent up-regulation of the Bcl-2 protein. A, Bcl-2 and Bcl-X protein levels were quantified by intracellular stainings of cells incubated for 20 min on ice with the specific mAb diluted in 0.5% saponin/2% FCS/PBS and later revealed with FITC-labeled anti-hamster immunoglobulin antibody. Background signals of irrelevant mAbs were electronically adjusted during cell acquisition. —, BaF-3 + IL-3; - - -, W53 + IL-3; —, W53 + 1 ng/ml PRL; , W53 + 30 ng/ml PRL. This is one representative of three independent experiments (days 3–5 of the cell culture). B, Bcl-2 and Bcl-XL protein expression levels were determined by Western blot analysis on total cell extracts of BaF-3 cells + IL-3 and W53 cells + 30 ng/ml PRL, as described in "Materials and Methods."

 
Transcription of the pre-B Cell Receptor Encoding the {lambda}5 Gene Is Strongly Stimulated by PRL in W53 Cells.
A critical requirement for normal B-cell differentiation is the PBCR expression in the progenitors (32) . The PBCR complex is formed by the product of a fully rearranged Ig H chain bound to those of invariant {lambda}5 and VpreB genes as a surrogate of light chains (33) . {lambda}5 gene begins to be transcribed in pro-B cells, and its encoded protein is present until the stage of IgM+ B cells, where it is replaced by the products of rearranged Ig light chains (28) . We analyzed {lambda}5 gene expression in a specific, quantitative reverse transcription-PCR (34) in BaF-3 cells and in W53 cells grown in IL-3 or in PRL. As a presumably invariant gene, we also tested for ß-actin gene transcripts, and the 70Z/3 pre-B cell line was used as a positive internal control of amplification (Fig. 4Citation ; Ref. 34 ). BaF-3 pro-B cells showed very low levels of {lambda}5-specific transcripts, corresponding to their early B-lineage character (Fig. 4)Citation . In contrast, W53 cells displayed significant transcriptional up-regulation of the {lambda}5 gene when stimulated by 30 ng/ml of PRL (10–100-fold of the basal values in different experiments; P < 0.05 after applying two-tailed Student’s t analyses; Fig. 4Citation ). This shows that PRL, upon its binding to the receptor, triggers signaling pathways leading to increased gene expression of the PBCR-encoding {lambda}5 gene, a necessary requirement for normal B-cell differentiation.



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  		Fig. 4. Transcription of the {lambda}5 gene is selectively increased by PRL-induced signals. The levels of {lambda}5 transcripts were studied in both BaF-3 and W53 cel lines (grown either in IL-3 or in 30 ng/ml of PRL). Total mRNA was extracted and retrotranscribed to cDNA, and a quantitative PCR was performed to amplify a 630-bp fragment of the {lambda}5 mRNA sequence, as described (34) . A control PCR for ß-actin amplification was done to estimate the amount of cDNA in each sample. PCR products were separated by electrophoresis and transferred to Zeta-probe membranes. Signals obtained after hybridization with specific probes were quantified by densitomentry, and the ratio between {lambda}5 and ß-actin signals was calculated (bottom column graph). The later data represent means of four different experiments and are shown in a logarithmic scale; bars, SD. The 70Z/3 pre-B cell line was used as a positive control of the amplification reaction.

 
In parallel to this study, we also tested for putative D-JH immunoglobulin gene rearrangements with a genomic PCR assay, which simultaneously reveals the four possible DJH1–4 rearrangements (34) . In contrast with the {lambda}5 up-regulation observed, the germ-line IgH pattern of BaF-3 cells showed no modification to PRL signals (data not shown). We conclude from these data that PRL signaling positively regulates the expression of {lambda}5 in the absence of evolving immunoglobulin gene rearrangements.

PRL-R Expression Is Up-Regulated throughout Normal Mouse B-Cell Differentiation.
The findings obtained with the PRL-R-transfected BaF-3 cells could be relevant to normal B-cell development if the PRL-R was present in B-cell precursors. We decided to directly test this possibility in BM B-cell precursors. B-cell differentiation starts with a small population of B220lowCD43+ pro-B cells that express the CD43 antigen (Fig. 5Citation , window of the upper contour plot). Also, PBCR genes ({lambda}5 and VpreB) are transcribed, IgH gene loci begin the ordered process of gene rearrangement, and cells undergo {lambda}5-dependent high proliferation rates and clonal expansion. Later, B220 antigen levels increase moderately, CD43 is down-regulated, and precursors evolve to the pre-B-cell stage (Fig. 5Citation , window of the bottom contour plot), in which most cells are resting and IgH gene rearrangements are being completed, although these pre-B cells are still surface IgM-. Finally, emerging B lymphocytes express IgM before reaching their full maturation, when IgM and IgD isotypes are expressed on the cell surface (28) . We analyzed the expression of PRL-R on B220lowCD43+ pro-B cells and B220highIgM- pre-B cells, using the mouse-anti-rat PRL-R U5 mAb (cross-reactive with the mouse receptor; Refs. 14 and 35 ) and three-color flow cytometry analyses. This study revealed that the PRL-R was already expressed in a small fraction of the B220lowCD43+ pro-B-cell population. PRL-R-positive cells were predominant in the next stage of B220highIgM- pre-B cells (Fig. 5Citation , right histograms). PRL-R expression has already been described in mature B lymphocytes (18) . We did not analyze it in mature B cells because the anti-PRL-R U5 mAb was a purified mouse immunoglobulin and required a second incubation with a TC goat anti-mouse immunoglobulin, which could not discriminate between the cell surface IgM and the PRL-R signals on the same B cell. We conclude that the PRL-R is expressed in mouse B-cell progenitor populations from the earliest stages of B-lineage differentiation (pro-B cells).



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  		Fig. 5. Expression of the PRL-R in mouse BM pro-B and pre-B cells. Three-color flow cytometry analyses were performed on BM cells as described in "Materials and Methods." BM cells (5 x 105) were incubated with either FITC-anti-CD43 or FITC-anti-IgM, together with PE-anti-B220 and purified mouse anti-rat PRL-R U5 mAb. This later was revealed with TC-conjugated, goat-anti-mouse immunoglobulin. Pro-B cells were defined as B220lowCD43+, and they represented 3–4% of total BM (window of the upper control plot). Pre-B cells were included in the B220highIgM23 window (8–10% of total BM; window of the bottom contour plot). Right histograms, PRL-R expression on pro-B and pre-B cell populations. Shaded histograms, background signals of isotype-matched, irrelevant mAb. The data are representative of three independent experiments. Fluorescence signals are displayed in logarithmic scales.

 
Effects of PRL on Short-Term Cultures of BM-derived B-Cell Precursors.
Both the findings obtained in the BaF-3/W53 cell system and the detection of PRL-R in BM B-cell precursors led us to analyze the putative PRL effects on primary B-cell precursors. We then established enriched BM B-cell precursors in cultures supported by the S17 stromal cell line and IL-7, as described by other authors (36) . Titrated doses of PRL (1–1000 ng/ml) were added at the start point of cultures, and changes induced were studied 4 days later. Absolute cell recoveries of BM B-lineage cell cultures significantly increased upon addition of PRL to the medium (P < 0.05 between the basal conditions and the cultures supplied by 100 and 1000 ng/ml of PRL) in a dose-dependent manner (Fig. 6A)Citation . During the 4 days of BM cell culture, many of the predominant CD43+B220+ pro-B cells of the starting cultures ({approx}70% in different experiments) evolved to CD43-B220+ pre-B cells (contour plots, Fig. 6BCitation ), and IgM+ B cells appeared de novo (histograms, Fig. 6BCitation ). Right column graphs display the absolute cell recoveries/ml of total B220+, CD43+B220+, and B220+IgM+ cells, both in basal conditions and upon the addition of titrated doses of PRL. All of these cell subsets increased on a PRL dose-dependent basis, expansions that were more remarkable for total B220+ cells (most of them pre-B cells) and CD43+B220+ pro-B cells. IgM+ B cells underwent more moderate, although PRL dose-dependent, increases. We conclude that PRL induces selective augmentations of normal B220+ B-lineage cells established in vitro, more specifically, of CD43+B220+ pro-B cells.



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  		Fig. 6. Effects of PRL on short-term cultures of purified mouse B-cell precursors. BM pro-B cells were purified by a two-step separation protocol with immunomagnetic beads, which included negative selection of IgM+ and Mac-1+ cells, and positive recovery of CD43+ cells, most of them B220+ (Day 0 contour plot of B). The titrated doses of PRL shown in the figure were added to the cultures at day 0, and the induced changes were analyzed 4 days later. A, absolute cell recoveries after 4 days of culture were quantified after Trypan blue exclusion (mean of three independent cell cultures; bars, SD; *, samples significantly different from the first column, which represents cell cultures without added PRL; P < 0.05). B, display of cell populations at the startpoint and 4 days later (contour plots and histograms). The absolute cell recoveries shown in the right column histograms were calculated from the relative cell numbers obtained by flow cytometry (inside the displayed windows of the contour plots and the positive cells of the histograms) and the total cell recovery/well of one representative experiment.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The generation and cell turnover of lymphohemopoietic lineages are very active throughout the lifespan of the individual. These processes are submitted to the regulatory control of intercellular interactions and soluble factors acting in autocrine, paracrine, and endocrine manners. In the work reported here, we have analyzed the putative involvement of PRL in mouse B-cell development by transfection of the rat PRL-R in BaF-3 pro-B cells and through the study of PRL influences over short-term cultures of BM pro-B cells. When PRL-R-tranfected BaF-3 cells were studied, we found, as compared with cells transfected with the empty vector, that: (a) the expression of IL-2R {alpha} chain, a characteristic of late pre-B cells, was dependent on signals triggered by either IL-3 and/or PRL; (b) antiapoptotic Bcl-2 protein levels were increased by PRL; and (c) transcription of the PBCR-encoding {lambda}5 gene was strongly and specifically activated by PRL-dependent stimuli. We also observed that PRL-R was expressed in normal BM B-cell progenitors. PRL induced, in dose-dependent manner, the expansion of discrete B-lineage cell populations in short-term BM cultures. Collectively, these data suggest that PRL modulates the differentiation program of mouse B-cell progenitors.

Recently, genetic disruptions of both PRL-R and PRL genes in the mouse have been reported (37 , 38) . As expected, important disturbances in reproductive capacity and in mammopoiesis took place in the homozygous mice. In contrast, no drastic deficiencies were found on lymphohemopoiesis in PRL (-/-) mice (38) , although the absolute numbers of splenocytes and the levels of splenic B cells were reduced in them. Since this animal model has not revealed yet a critical requirement for PRL in lymphohemopoiesis, it is possible that the putative role of PRL in B cell development is replaced by reduntant factors. This has been shown to be the case for other gene products, as it is exemplified by members of the Src family (reviewed in Ref. 39 ).

BaF-3 pro-B cells were established in IL-3-dependent conditions, and they represent one of the earliest B-cell differentiation stages (25) , while maintaining some degree of flexibility to develop through other hemopoietic lineages. They were, however, resistant to complete B-cell differentiation associated with immunoglobulin secretion (25) . We also found that under no conditions, including in vivo transfer to SCID mice, were they able to rearrange their immunoglobulin genes, probably due to the lack of RAG-1 gene transcription (as confirmed in specific reverse transcription-PCR, data not shown). When BaF-3 cells were transfected with PRL-R, the resultant W53 cells integrated PRL signals, grew in IL-3 deprived, PRL-enriched media, and expressed parameters of B-cell differentiation. In particular, B lineage-specific {lambda}5 gene transcription was strongly up-regulated. This gene encodes part of the PBCR, it is first expressed on pro-B cells, and its deletion blocks most of B lymphopoiesis (32) . It plays a role in the clonal expansion of B-cell precursors by induction of proliferative stimuli or by activation of cell survival machinery, or both. Very little is known about {lambda}5 gene regulation, but our findings show that the signaling pathways triggered by PRL-R lead to the up-regulation of {lambda}5 gene expression in BaF-3 cells. Recently, the synergistic action of EBF and E47 transcription factors on the induction of {lambda}5 expression has been observed in BaF-3 cells (40) . {lambda}5 gene enhancer also contains binding sites for Pax-5/BSAP (41) . Future experiments will clarify whether the PRL-induced changes of {lambda}5 gene expression in W53 cells are mediated by any of these transcription factors.

In addition, we have found that IL-2R {alpha} chain expression, which appears in normal BM pre-B cells after the pro-B-cell stage (20 , 28) , is positively regulated by PRL in W53 cells as well as by IL-3 in BaF-3 and in other cell lines (26) . Antiapoptotic Bcl-2, but not Bcl-XL, protein levels were also augmented by PRL in W53 cells in a dose-dependent manner. Bcl-2 shows developmental expression patterns; its up-regulation is linked to the positive selection of immature cells (30 , 42 , 43) . PRL-dependent control of Bcl-2 protein levels has also been described in rat Nb2 thymoma cells (44) . Other members of the type I cytokine receptor superfamily also influence the expression of Bcl-2 (45) and thus provide separate mitogenic and antiapoptotic signals. The observed changes suggest that PRL signals are able to provide some critical requirements for the intrinsic differentiation program of BaF-3 pro-B cells to be fully expressed, as it has been described in other systems (46, 47, 48) . The finding of PRL-R expression in normal B-cell precursors and the PRL-dependent increases of in vitro-established B-lineage cells provide evidence for the physiological relevance of this endocrine pathway as an additional support for normal B-cell differentiation. It is worth noting that some of the above differentiation changes of W53 cells in response to PRL may be interconnected in a network of genetic interactions (49) . Bcl-2 may thus respond to increased IL-2R- or {lambda}5-dependent signals (45) and vice versa; the set-up of cell survival machinery could be a basic condition for the evolution of progenitors to more differentiated stages (27) . All of these parameters of BaF-3 cell differentiation in the absence of immunoglobulin rearrangements further support the proposal of Grawunder et al. (50) about the existence of two autonomous programs of B-cell differentiation, one characterized by changes in surface receptors and growth requirements and another affecting immunoglobulin gene rearrangement. Both differentiation modules contribute to generate a functional mature B lymphocyte, and PRL may be selectively acting on the first one.

In more physiological conditions, the in vitro expansion/differentiation of normal BM pro-B cells was thus stimulated by PRL in our experiments. Other studies have shown that both estrogens (during pregnancy) and PRL (after delivery) suppress B lymphopoiesis (21) . The data described here do not necessarily diverge from the later ones (21) , because proliferation and differentiation may be two partially opposite cell fates of the same activation stimulus. As an overall conclusion, these findings obtained under in vitro conditions suggest the involvement of PRL in early stages of B-cell differentiation. The results also provide an experimental model (BaF-3/W53) with well-controlled genetic targets ({lambda}5, IL-2R {alpha}, and Bcl-2) to dissect the pathways of PRL-R signaling in lymphohemopoietic cell lineages.


   Materials and Methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Mice, Cell Cultures, and Cytokines.
The mouse IL-3-dependent BaF-3 cell line (25) was cultured in RPMI 1640 supplemented with 10% FCS (Life Technologies, Inc., Renfrewshire, United Kingdom) and 5% of WEHI-3B supernatant as a source of IL-3 (51) . BaF-3-derived W53 cell line (PRL-R transfectants) was cultured in RPMI 1640/10% FCS and different doses of ovine PRL (NIDDK-oPRL-20, 31 IU/mg; a gift of the National Hormone and Pituitary Program, Bethesda, MD).

BALB/c females, 3 months of age, maintained in the animal facilities of the CBMSO, were sacrificed for BM cell isolation. The BM was flushed out by injection of balanced salt solution 5% FCS into the femur and disrupted with a 1-ml syringe to obtain a single-cell suspension. The small subset of BM B220+CD43+ pro-B cells was purified in a two-step process: (a) staining of total BM cells with both biotinylated anti-Mac-1 and anti-IgM mAb (see "Antibodies") and indirect magnetic labeling with Streptavidin microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by their negative selection with a MiniMacs magnet (Miltenyi Biotec), and (b) staining of remaining IgM- Mac-1- cell fraction with rat anti-CD43 mAb (see "Antibodies"), labeling with goat-anti-rat microbeads (Miltenyi Biotec), and positive selection of CD43+ cells. Magnetic cell sorting protocols followed the indications of the supplier. B-cell-supporting S17 cells were grown to confluence, treated with Mitomycin C (Sigma Chemical Co., St. Louis, MO), and replated at a density of 0.5 x 106 cells/24-well plate, as described (36) . Then B220+CD43+ cells (106/ml) were added to the cultures in medium containing limiting amounts of mouse IL-7 (10 units/ml; Sigma) and titrated doses of PRL. The changes produced in the cultures were analyzed at day 4 of evolution.

Cloning and Expression of the PRL-R.
The original cDNA clone of the long form of PRL-R from rat ovary was generously provided by Dr. P. A. Kelly (INSERM, Paris, France; Ref. 52 ). Its 5' and 3' noncoding sequences were eliminated by PCR using the 5' oligonucleotide containing the starting codon (5'-CTGAAGGAAT TCATGCCATCTGCACT-3') and the 3' oligonucleotide containing the stop codon (5'-CGGGGT ACCGTCGACTCAGTGAAAGGAGTGCATGAAGC-3'). The PCR amplified product was digested with EcoRI and SalI, cloned in pBluescript (Stratagene, La Jolla, CA), and checked by sequencing. The cDNA coding region was excised from the pBluescript with EcoRI and SalI and recloned into the pBabe-Puro retroviral vector (53) . In this vector, the expression of the exogenous cDNA is driven by the long terminal repeat of the Moloney leukemia virus.

The pBabe-Puro plasmid containing the PRL-R or the empty vector (50 µg each) was transfected into BaF-3 cells (107) by electroporation at 956 µF and 350 V in a Bio-Rad apparatus (Bio-Rad, Hercules, CA). After 48 h in complete medium, transfected cultures were selected in complete medium containing 4 µg/ml of puromycin. Two weeks later, cells transfected with the PRL-R were transferred to RPMI 1640 supplemented with 10% FCS and 1 µg/ml of PRL. Subsequently, the hormone concentration was progressively reduced to a final concentration of 1 ng/ml, in which the cells have been maintained for >1 year; the resulting cell line was named W53. Cells transfected with the empty vector were maintained in complete medium containing 1 µg/ml of puromycin.

Cell Labeling, Immunoprecipitation, and Western Blot.
For biosynthetic labeling, BaF-3 and W53 cells were deprived of FCS and PRL by incubation in RPMI 1640 supplemented with 3% detoxified bovine serum albumin (BSA Fraction V; Sigma) overnight. The cells were then incubated in methionine-free DMEM (30 min at 37°C), washed, and submitted to a second incubation for 2 h in the presence of 1 ng/ml of PRL, L-[35S]methionine and L-[35S]cysteine (200 µCi/ml; Amersham, Inc.). Cells were collected by centrifugation (1500 x g for 5 min at 4°C), washed with ice-cold Tris-buffered saline [10 mM Tris-HCl (pH 7.4), 150 mM NaCl], and then solubilized in ice-cold lysis buffer [LB: 10 mM Tris-HCl (pH 7.4), 10 mM Na4P2O7 (pH 7.4), 80 mM NaCl, 50 mM NaF, 5 mM EDTA, 100 µM Na3VO4, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM 1,10-phenanthroline, and 1 mM iodoacetamide]. The cell lysates were spun (17,500 x g for 30 min at 4°C), the supernatants were transferred to new tubes, and their protein concentration was measured. These supernatants, equalized for protein concentration, were incubated for 1 h at 4°C with 30 µl of protein G-Sepharose (Sigma) and then centrifuged (3300 x g for 2 min at 4°C). The precleared supernatants were incubated under the same conditions with 30 µl of normal mouse serum Ig-protein G-Sepharose, which was bound and washed previously. The pellets obtained after this incubation were stored on ice and used as preimmune complexes. After being transferred to new tubes, the supernatants were incubated as before with 30 µl of purified mouse anti-rat PRL-R U5 mAb (5 µg; Affinity Bioreagents, Inc., Golden, CO) IgG1-protein G-Sepharose. The supernatants were discarded, and the pellets were washed three times with ice-cold LB. The preimmune and the immune complexes were extracted twice by boiling in 40 µl of 2x SDS sample buffer and resolved in a SDS-9% PAGE under reducing conditions. The biosynthetically labeled PRL-R was detected by fluorography in the gel.

For analyzing the expression of Bcl-2 and Bcl-XL from the supernatants of cell lysates by Western blot, aliquots containing 20 µg of protein were boiled in 2x SDS Laemmli sample buffer, resolved in a SDS-12% PAGE under reducing conditions, and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore Corp., Bedford, MA). Filters were blocked with 5% fat-free dried milk (Fluka Chemie AG, Buchs, Switzerland) in TTBS (Tris-buffed saline, 0.1% Tween 20), incubated with the primary antibody in blocking buffer, washed three times with TTBS, and further incubated with the suitable horseradish peroxidase-conjugated, anti-species-specific antibody (TAGO Immunologicals, Camarillo, CA). Proteins were visualized by enhanced chemiluminiscence (ECL; Amersham).

Antibodies and Flow Cytometry Analysis.
The following mAbs were purified from culture supernatants by affinity chromatography on protein G columns (Pharmacia Biotech, Uppsala, Sweden) and biotinylated by standard methods: anti-IgMa (RS3.1; Ref. 54 ), anti-Mac-1 (M1/70; Ref. 55 ), and anti-IL-2R {alpha} chain (PC61; Ref. 56 ). Phycoerythrin-conjugated anti-B220 (RA3-6B2), fluorescein-conjugated (FITC) anti-CD43 (Leukosialin, S7), purified goat-anti-mouse IgM, anti-Bcl-2, and anti-Bcl-X were purchased from Pharmingen (San Diego, CA). FITC-goat anti-hamster immunoglobulin and streptavidin-PE were obtained from Southern Biot. (Birmingham, AL). TriColor-conjugated goat-anti-mouse immunoglobulin and FITC goat-anti-rabbit immunoglobulin were from Caltag Laboratory (San Francisco, CA). For Western blot detection of Bcl-2 and Bcl-XL, the anti-Bcl-2 rabbit polyclonal antibody N-19 from Santa Cruz Biotechnology (Sana Cruz, CA) and the anti-Bcl-X mouse mAb B22620 from Transduction Laboratories (Lexington, KY) were used. Affinity-isolated, horseradish peroxidase-conjugated goat-anti-rabbit and goat-anti-mouse antibodies were from TAGO.

Cells were dissociated from BM or harvested from cultures, washed, and resuspended in staining solution (PBS, 2% FCS, and 0.1% sodium azide) at a density of 5 x 106/ml. For cell surface stainings, cells were incubated in optimal concentrations of either biotinylated or fluoresceinated mAb on ice for 20 min. When required, streptavidlin-PE was added after washing and incubated on ice for 20 min. Cell debris and dead cells were excluded by light scatter parameters and propidium iodide staining (50 µg/ml of propidium iodide in the staining solution). Background signals were set by incubating cells with isotype-matched irrelevant mAb. The minimum number of acquired cells/sample was always 5000 cells. Intracellular stainings were performed by incubating the cells with the purified mAb diluted in 0.5% saponin (Sigma), 2% FCS in PBS (20 min on ice). After washing, cells were incubated with either FITC-labeled goat-anti-hamster Ig or goat-anti-rabbit Ig for Bcl-2 and Bcl-X detection, respectively. For three-color stainings, 5 x 105 BM cells were incubated with either FITC-labeled anti-CD43 or goat-anti-IgM, PE-labeled anti-B220 and purified mouse-anti-rat PRL-R U5 mAb (20 min on ice). After washing, the purified antibody was revealed with TC-goat-anti-mouse immunoglobulin in a second incubation. Flow cytometry analyses were performed on an EPICS-XL flow cytometer (Coulter Corp., Hialeah, FL).

RT-PCR Analyses.
Total RNA was extracted, and equal amounts of each sample were reverse-transcribed as described (57) , by using 1 µg oligo(dT) as primer and avian myeloblastosis virus reverse transcriptase (AMV-RT; Promega, Madison, WI). For quantitative PCR amplifications of ß-actin and {lambda}5 cDNAs, 2.5 units of Taq polymerase (DNAzyme, Espoo, Finland) were added to each sample, together with the specific primers (34) . After an initial denaturation step (95°C for 5 min), ß-actin and {lambda}5 transcripts were amplified for 18 and 40 cycles, respectively, before a final 10-min incubation at 72°C. Each cycle consisted of the following steps: 1 min at 95°C, 1 min at 60°C, and 1 min at 72°C. One-fifth of the amplified products was separated electrophoretically on 2% agarose gels and then transferred to Zeta-probe membranes (Bio-Rad) after 0.4 M NaOH treatment. Hybridization of the membranes was performed at 65°C by using random priming 32P-labeled ß-actin and {lambda}5 probes that have been described elsewhere (34) . Membranes were washed, and the PCR signals were detected and quantified by densitometry in a Fujibas-1000 detector (Fuji, Tokyo, Japan).

Cell Proliferation.
Cell proliferation rates were obtained after an overnight pulse with 1 µCi of [3H]thymidine (Amersham). Cells (104) per well in triplicates were plated on 96-well, flat-bottomed plates (Nunc), cultured for 2 days, and then pulsed as above. Cells were then harvested onto glass fiber filters, and the incorporated radioactivity was quantified by a scintillation counter (Wallac LKB, Turku, Finland).

Statistical Analysis.
The two-tailed Student’s t test and Mann-Whitney rank sum test were used to compare variables between groups.


   Acknowledgments
 
We thank Dr. P. A. Kelly for the gift of the PRL-R cDNA and the National Institute of Diabetes and Digestive and Kidney Diseases and Dr. A. F. Parlow for the oPRL-20 used in these experiments. We also acknowledge C. Mark for editorial assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants PM 96-0074 and PM96-0072-002 from DGCYT and Grants SAL-AI117/96 and 07/100/96 from CAM. The CBMSO is partially founded by Fundación Ramón Areces. P. M. and M. V. C. were recipients of FPI fellowships from MEC and CAM. H. G. was supported by an ICI fellowship. Back

2 To whom requests for reprints should be addressed, at Instituto de Investigaciones Biomédicas, C.S.I.C. Arturo Duperier 4, 28029 Madrid. Phone: 34-91-585-4603; Fax: 34-91-585-4587. E-mail: jmartin{at}iib.uam.es Back

3 The abbreviations used are: PRL, prolactin; PRL-R, PRL receptor; IL, interleukin; IL-2R, IL-2 receptor; BM, bone marrow; PBCR, pre-B-cell receptor; mAb, monoclonal antibody. Back

Received for publication 2/ 1/99. Revision received 4/19/99. Accepted for publication 6/11/99.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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