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Endocrinology Vol. 141, No. 9 3225-3234
Copyright © 2000 by The Endocrine Society

ARTICLES

Vitamin D-Mediated Gene Regulation in Phenotypically Defined Human B Cell Subpopulations1

John W. Morgan, Nicola Kouttab, Dwayne Ford and Abby L. Maizel

Section of Experimental Pathology, Department of Pathology, Roger Williams Hospital, Providence, Rhode Island 02908; and Boston University, Boston, Massachusetts 02118

Address all correspondence and requests for reprints to: Dr. John W. Morgan, Department of Pathology, Roger Williams Hospital, 825 Chalkstone Avenue, Providence, Rhode Island 02908. E-mail: john_morgan{at}brown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Isolation of distinct subpopulations of density-fractionated normal human B lymphocytes reveals that the requirements for up-regulation of the vitamin D receptor (VDR) and initiation of 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25-(OH)2D3]-mediated genomic trans-activation are dependent upon the state of cellular activation. The kinetics of the response differ widely among these B cell subpopulations. However, these density-fractionated B cell subpopulations are phenotypically diverse and therefore are not representative of distinct stages of B cell maturation and differentiation. To examine the role of B cell differentiation on the induction and maintenance of biological receptivity to 1,25-(OH)2D3, we purified naive, germinal center, and memory B cells based on their expression of CD38 and CD44 surface antigens and surface Ig isotype. These phenotypically defined B cell subpopulations were all found to constitutively express VDR, and all exhibited similar activation requirements and kinetics for initiation of 1,25-(OH)2D3-mediated genomic trans-activation. Taken together, these results suggest that defined stages of differentiation in normal B cells are not significant predicators of VDR expression or receptivity to 1,25-(OH)2D3. Rather, the degree of cellular activation, regardless of maturation stage, determines whether the effects of this immunoregulatory hormone will influence a mature B lymphocyte.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACTIVE metabolite of vitamin D, 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a member of the family of lipophilic ligands that bind to specific nuclear receptors. Binding of the 1,25-(OH)2D3 ligand activates the vitamin D receptor (VDR), which facilitates its interaction with specific cis-acting regulatory vitamin D response elements (VDRE) within the promoter of the target gene.

In addition to its well defined role in calcium homeostasis and modulation of bone cell metabolic activity, 1,25-(OH)2D3 is now recognized as a potent immunoregulatory hormone (reviewed in Ref. 1). Although most cells responsive to 1,25-(OH)2D3 constitutively express VDR, lymphoid cells are unique in that in a quiescent state, these cells do not express VDR, yet require specific activation signals for its up-regulation (2, 3, 4). Given the typically subnanomolar plasma concentration of 1{alpha},25-(OH)2D3 (5), hormone availability in an effective nanomolar concentration range requires the presence of an exogenous cell source. Cells of the monocyte/macrophage lineage may provide such a source of 1,25-(OH)2D3. Reports have demonstrated that cells of this lineage can express 1{alpha}-hydroxylase (6, 7, 8, 9), the enzyme responsible for hydroxylation of the relatively inert form of vitamin D, 25-hydroxyvitamin D3, to the active metabolite, 1,25-(OH)2D3. One may envision a cognate monocyte/B cell or monocyte/T cell interaction as an event fulfilling the requirements for lymphoid cell activation, and thereby up-regulation of VDR, as well as synthesis of 1,25-(OH)2D3 in a nanomolar concentration range within a localized microenvironment (10).

We have recently reported on the kinetics of biological response to 1,25-(OH)2D3 in distinct subpopulations of normal B lymphocytes (11). Exploiting the phenomenon that the stage of lymphoid cell activation directly parallels an increase in cell volume and a decrease in their buoyant density (12), we examined the reactivity of density-fractionated B cells with 1,25-(OH)2D3 (11). Low density B cells were found to constitutively express VDR message and protein and initiate 1,25-(OH)2D3-mediated genomic trans-activation in the absence of in vitro stimulation, as determined by RT-PCR and gel shift analysis. In contrast, high density, quiescent B cells, although ultimately reactive with 1,25-(OH)2D3 subsequent to in vitro stimulation, were found to require a protracted time frame in which to up-regulate VDRE-reactive nuclear proteins and initiate 1,25-(OH)2D3-mediated genomic trans-activation.

A determination of which B lymphocyte subpopulations may be either refractory or constitutively reactive with 1,25-(OH)2D3 is confounded by the fact that peripheral lymphoid tissues, such as the tonsil, contain B cells that are highly heterogeneous with respect to their degree of activation and their stage of differentiation. To assess the role of cellular differentiation on the capacity of B cells to biologically respond to 1,25-(OH)2D3, we exploited the phenomenon that B cells characteristically undergo distinct maturational stages defined by their expression of specific surface markers (reviewed in Ref. 13). In situ, during T cell-dependent immune responses, resting naive B cells (which characteristically express a IgD+, CD38- phenotype) are activated in association with antigen-specific T cells and interdigitating dendritic cells within the extrafollicular areas of secondary lymphoid organs, such as the tonsil (14, 15). After this cognate interaction, some activated B cells migrate into B cell follicles and differentiate into IgD-, CD38+, CD77+, Ki67+ proliferating centroblasts, which form the dark zone of germinal centers where somatic mutation in IgV genes occurs (16, 17). Three types of somatically mutated B cells can be generated, including high affinity and low affinity antigen-binding subsets, in addition to autoreactive lymphocytes, which compose the basal light zone of the germinal center (18). Although the majority of centroblasts undergo apoptosis, surviving centroblasts ultimately give rise to centrocytes, which bear a characteristic IgD-, CD38+, CD77-, Ki67- phenotype (19). High affinity antigen binding centrocytes present processed antigen to antigen-specific T cells, and these activated T cells are induced to express CD40 ligand and secrete cytokines. This cognate T cell-B cell interaction results in the expansion and isotype switch of high affinity centrocytes. In the presence of prolonged CD40 ligand signaling, these centrocytes differentiate into memory B cells bearing a characteristic IgD-, CD38- phenotype. In the absence of sufficient CD40 ligand signaling, these centrocytes differentiate into CD382+, CD20- plasma cells (20, 21).

Given such a highly heterogeneous composition with respect to differentiation and maturation, it is difficult to accurately assess which of these B cell subpopulations may be constitutively reactive, inducible, or refractory to biological effects of 1,25-(OH)2D3. To address this issue and to further characterize the influence of 1,25-(OH)2D3 on B cell biology, we undertook the present study using phenotypically defined B cell subpopulations representative of distinct phases of B cell maturation. Using defined monoclonal antibodies and magnetic cell separation, normal human tonsillar B cells were fractionated into populations enriched for naive, memory, or germinal center B cells. These B cell populations were assessed for their functional capacity to up-regulate VDR and to initiate 1,25-(OH)2D3-mediated genomic trans-activation. We demonstrate that B cell reactivity with 1,25-(OH)2D3 is a characteristic universally shared by naive, germinal center, and memory B cell subpopulations, and that cellular activation, rather than differentiation, is a more stringent criterion defining their biological receptivity to 1,25-(OH)2D3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
1,25-(OH)2D3 was provided by Dr. Milan Uskokovic (Hoffmann-La Roche, Nutley, NJ). Escherichia coli-derived recombinant human interleukin-4 (IL-4) was obtained commercially (PeproTech, Inc., Rocky Hill, NJ). Rabbit antihuman {kappa} and antihuman {lambda} (anti-{kappa}/{lambda}) antibodies were obtained from DAKO Corp. (Carpinteria, CA). Antihuman {kappa} and antihuman {lambda} antibodies were covalently coupled to activated Immunobead Matrix from Irvine Scientific (Santa Ana, CA) and used at a final concentration of 10 µg/ml.

Antibodies
Antihuman CD40 mouse monoclonal antibody (22) was affinity purified in our laboratory from the G28–5 cell line, which was obtained from the American Type Culture Collection (Manassas, VA). Other monoclonal antibodies were purchased from various companies as follows: anti-IgD, anti-IgG, anti-IgA1/IgA2, anti-CD38, anti-CD39, and anti-CD44 (PharMingen, San Diego, CA); PE-conjugated anti-CD39, and PE-conjugated anti-IgD (PharMingen, San Diego, CA); fluorescein isothiocyanate (FITC)-conjugated anti-CD38, FITC-conjugated anti-CD44, and FITC-conjugated anti-IgD (PharMingen, San Diego, CA); and anti-CD77 (Biodesign International, Kennebunk, ME).

Flow cytometric analysis
Antibody binding was analyzed on a FACScan flow cytometer (Becton Dickinson and Co., San Jose, CA), as previously described (23).

Preparation of tonsillar B lymphocyte subpopulations
Cells from normal human tonsils were prepared as previously described (24). Briefly, tonsils were obtained from children under age 13 yr after surgical extirpation for chronic tonsillitis. Tonsils were finely minced, and the resulting cell suspension was subjected to depletion of non-B cells by adherence to plastic and rosetting with sheep erythrocytes. Cells prepared in this manner are routinely 96% or more B lymphocytes as determined by immunofluorescence with anti-CD3, -CD19, -CD20, and -CD45 antibodies. Subpopulations of B lymphocytes were isolated by magnetic cell separation (25) (MACS; Miltenyi Biotec, Sunnyvale, CA), Percoll density gradient centrifugation (26), or a combination of the two procedures. To isolate B cell subpopulations highly enriched for naive, germinal center, and memory B cells, we exploited the observations that 1) expression of CD44 and CD38 on tonsillar B cells is mutually exclusive (27, 28, 29, 30); 2) CD38 and IgD discriminate follicular mantle (IgD+, CD38-) from germinal center (CD38+, IgD-) B cells (31, 32); and 3) CD44 expression has been reported to be low or negative on germinal center B cells (30, 33, 34, 35). Accordingly, naive B cells were obtained by depletion of CD38+, IgG+, and IgA+ B cells (30). These cells express high levels of IgD, IgM, CD44, and cytoplasmic Bcl-2 protein, and they are negative for CD38, CD77, and IgG (data not shown) (17). Germinal center B cells were obtained by depletion of CD44+ and IgD+ B cells (30). These cells express high levels of CD38, CD10, and IgG and are negative for CD44, IgD, and CD23 (data not shown) (17). Memory B cells were obtained by depletion of CD38+ and IgD+ B cells (30). These cells express high levels of IgG and CD44 and are negative for IgD, CD38, CD77, and CD23 (data not shown) (17). For purification of subpopulations by MACS, total tonsillar B cells were incubated with a murine monoclonal antibody cocktail containing either anti-CD38 and anti-IgD mAbs, anti-CD44 and anti-IgD mAbs, or anti-CD38, anti-IgG and anti-IgA monoclonal antibodies for 20 min at 10 C, and then with rat antimouse IgG1 microbeads (Miltenyi Biotec) for 15 min at 10 C. After incubation, the cells were washed in ice-cold column buffer (PBS supplemented with 0.5% BSA and 5 mM EDTA) and applied to Miltenyi VS columns on a VarioMACS magnetic separator according to the manufacturer’s directions. The negative fraction was collected, and experimental culture conditions were initiated immediately.

In some experiments naive, germinal center, and memory B cells were fractionated on discontinuous Percoll density gradients according to density, essentially as previously described (26), with the exception that Percoll mix solution was substituted for HBSS. The density of cells prepared in this manner may be categorized as high (1.094 g/ml Percoll solution), intermediate (1.089 g/ml), or low (1.082 g/ml).

Measurement of proliferation
Freshly isolated tonsillar B cell subpopulations (1 x 105 in 0.2 ml) were stimulated in 96-well round-bottomed microtiter plates containing RPMI 1640 supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, and 10% endotoxin-free heat-inactivated FCS. Twelve hours before completion of the 60-h incubation period, 1 µCi [3H]thymidine or 1 µCi [3H]uridine was added to each well. Postincubation, cells were collected onto glass-fiber filters and counted for radioactivity after being placed into a scintillation cocktail.

Cell lines
The human myelomonocytic cell line U937 (36) was obtained from the American Type Culture Collection (Manassas, VA).

Purification of RNA and RT-PCR
Total RNA was obtained using the RNeasy kit (QIAGEN, Santa Clarita, CA). RNA (5 µg) was reverse transcribed after annealing with 0.1 nM oligo(deoxythymidine) for priming of complementary DNA (cDNA) synthesis in a 20-µl reaction using the SuperScript Preamplification System (Life Technologies, Inc., Gaithersburg, MD). Primers for human VDR cDNA were: forward (sense) primer, 5'-ATG GCC ATC TGC ATC GTC TC-3' (corresponding to bases 1128–1147); and reverse (antisense) primer, 5'-GCA CCG CAC AGG CTG TCC TA-3' (corresponding to bases 1414–1433) (37). The expected length of the PCR product is 306 bp. Primers for human 24-hydroxylase cDNA were: forward (sense) primer, 5'-CGG GTG TAC CAT TTA CAA CTC GG-3' (corresponding to bases 1556–1578); and reverse (antisense) primer, 5'-CTC AAC AGG CTC ATT GTC TGT GG-3' (corresponding to bases 1850–1872). The expected length of the PCR product is 317 bp. Primers for human glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA were: forward (sense) primer, 5'-GAC ATC AAG AAG GTG GTG AAG CAG G-3' (corresponding to bases 802–826); and reverse (antisense) primer, 5'-CCT GTT GCT GTA GCC AAA TTC GTT G-3' (corresponding to bases 1002–978). The expected length of the PCR product is 201 bp. All primers were complementary to sequences that spanned intronic regions and are therefore messenger RNA (mRNA) specific. To determine that PCR contamination did not occur, a negative control of cDNA from which the reverse transcriptase enzyme had been omitted was included for each sample analysis.

A hot start technique was used in 50-µl reactions in which 2 µl reverse transcribed cDNA were amplified in 0.5-ml GeneAmp reaction tubes (Perkin-Elmer Corp., Emeryville, CA) in the presence of a 200-nM final concentration of 5'- and 3'-primers, 200 µM deoxy-NTPs, 1.5 U Taq polymerase (Promega Corp., Madison, WI), and PCR buffer containing 1.5 mM MgCl2, 15 mM (NH4)SO4, 60 mM Tris-Cl (pH 8.5; for 24-hydroxylase and GAPDH primers) or 2 mM MgCl2, 15 mM (NH4)SO4, 60 mM Tris-Cl (pH 9.5; for VDR primers). The reaction mixture was overlaid with 50 µl light mineral oil, and the amplification reaction was performed in a GeneAmp PCR System 9600 thermal cycler (Perkin-Elmer Corp.). Cycle characteristics for the VDR-, 24-hydroxylase-, and GAPDH-specific primers were 1 min at 94 C, 1 min at 55 C, and 2.5 min at 72 C.

To determine an optimal number of PCR cycles exhibiting an exponential rate of amplification, RNA extracted from U937 cells was used, as physiological effects of 1,25-(OH)2D3 on this monocytic leukemia cell line have been described previously (38, 39). U937 cells were stimulated for 24 h with 1,25-(OH)2D3 (50 nM), RNA was isolated and reverse transcribed, and multiple replicate cDNA samples were subjected to PCR amplification using oligonucleotide primers complementary to VDR, 24-hydroxylase, or GAPDH mRNA. After completion of a specific number of cycles, each sample was transferred to a 72 C water bath to permit final product extension. As PCR amplification theoretically doubles PCR product at each cycle, a plot of product vs. cycle number should lie on a straight line. A plot of the relative densities of ethidium bromide-stained PCR product was sigmoidal, with nonlinear amplification at low and high cycle numbers (data not shown). However, there was a region of linearity within each sigmoidal curve corresponding to an exponential amplification of the PCR reaction. A cycle number within this linear region was used for subsequent PCR amplifications. For determination of VDR and 24-hydroxylase message expression, PCR amplification was terminated after 27 cycles. To further control for variability in RNA extraction yield, mRNA degradation, and RT amplification efficiency, a GAPDH transcript was used as an internal standard (40); optimal PCR amplification for this reaction was determined to be at 18 cycles.

Aliquots (20 µl) of the amplified cDNA were electrophoresed on 2% agarose gels in 0.5 x TBE (Tris/borate/EDTA) running buffer containing 0.1 µg/ml ethidium bromide, and visualized under UV illumination. For improved resolution and clarity, figures depicting PCR products are derived from film negatives. Densitometric values were determined using with the Scan Analysis software program (Biosoft, Ferguson, MO), and values were normalized using the GAPDH value for each sample. The maximum signal for each experiment was then set to 100%, and all other values for the corresponding experiment were expressed accordingly. Experimental results were replicated minimally three times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Biological reactivity of magnetically fractionated B cell subpopulations
As activation signals rendering quiescent mature B lymphocytes reactive with 1,25-(OH)2D3 minimally include ligation of the B cell receptor (BCR; surface Ig) (4), this precluded our use of a positive selection technique for the isolation of phenotypically defined B cell subpopulations. However, a previously described technique of negative selection via magnetic cell separation was shown to yield B cell subpopulations of high purity (27), and cells isolated in this manner are not stimulated in vitro during the fractionation procedure due to BCR cross-linking. To determine whether these negatively selected B cell subpopulations were biologically reactive with polyclonal activators, yet were not artificially stimulated by the magnetic cell separation technique, these B cell subpopulations were assessed for their capacity to incorporate [3H]thymidine and [3H]uridine, as an indicator of de novo DNA and RNA synthesis, respectively. As depicted in Fig. 1Go, the B cell subpopulations were not artificially activated by the magnetic separation procedure (compare no activator with control). Additionally, the proliferative response profile substantiates that these B cells were indeed enriched for the target population. In agreement with previous reports (28, 41), the germinal center B cells did not respond well to signals derived from BCR ligation, yet responded optimally to ligation of the CD40 receptor. Conversely, memory and naive B cells responded vigorously to BCR ligation (30).



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  		Figure 1. B cell subpopulations are not activated by magnetic cell separation negative selection. Magnetically separated B cell subpopulations enriched for germinal center, memory, or naive B cells (1 x 105/0.2 ml) were cultured in microtiter wells in the absence or presence of insoluble anti-{kappa}/{lambda} antibodies (10 µg/ml), anti-CD40 antibodies (5 µg/ml), and/or IL-4 (10 ng/ml), as indicated. All cells were cultured for 48 h and then pulsed with 1.0 µCi/well of either [3H]thymidine (A) or [3H]uridine (B) for an additional 12 h. The control for each B cell subpopulation depicts cells pulsed for 12 h with radiolabel immediately after magnetic separation. Figures depict the mean counts per min ± SD of four independent experiments.

 
The VDR is constitutively expressed in phenotypically defined B cell subpopulations
Previous experiments examining density-fractionated B lymphocytes revealed constitutive VDR expression in a low density B cell subpopulation and no detectable VDR expression in a high density B cell subpopulation (11). We questioned whether such a scenario would be mimicked in phenotypically defined B cell subpopulations. To address this issue, a semiquantitative relative RT-PCR technique was used to examine message expression (see Materials and Methods). This approach has been successfully exploited in other cells systems, particularly relating to the expression of cytokine message (42, 43) as well as VDR message (11, 37). Naive, germinal center, and memory B cells were purified from freshly extirpated tonsil specimens obtained from multiple normal individuals. Total RNA isolated from these B cell subpopulations was reverse transcribed, and PCR amplification was performed using oligonucleotide primers complementary to either VDR or GAPDH mRNA. As depicted in Fig. 2Go, varying amounts of constitutive VDR message were detected in all B cell subpopulations obtained from multiple donors (Fig. 2AGo, lanes 1–10). From these observations it is apparent that naive, germinal center, and memory B cells can express VDR mRNA, and that these B cell subpopulations may be sufficiently stimulated within the normal tonsil such that their expression of VDR is a ubiquitous phenomenon.



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  		Figure 2. The VDR is constitutively expressed in phenotypically defined B cell subpopulations. Naive, germinal center, and memory B cells were purified from tonsil specimens obtained from multiple normal individuals. Total RNA was isolated and reverse transcribed, and PCR amplification was performed. A, Amplification of a 306-bp fragment from mRNA encoding human VDR. B, Amplification of a 201-bp fragment from mRNA encoding human GAPDH. Twenty-microliter samples of the amplified cDNA were electrophoresed on 2% agarose gels in 0.5 x TBE running buffer containing 0.1 µg/ml ethidium bromide and visualized under UV illumination. Positions of molecular standards (base pairs) are indicated at the left. The data are representative of four similar experiments.

 
Induction of 1,25-(OH)2D3-mediated genomic trans-activation in phenotypically defined B cell populations
We next determined whether the constitutive expression of VDR message in naive, germinal center, and memory B cells could be modulated by ligation of either the BCR, the CD-40 receptor, and/or the IL-4 receptor. In addition, those conditions that initiated 1,25-(OH)2D3-mediated genomic trans-activation were evaluated. We chose to examine induction of message encoding 25-hydroxyvitamin D3 24-hydroxylase as an indicator of 1,25-(OH)2D3-mediated transcriptional activation. It has been shown that 1,25-(OH)2D3 exerts control of 24-hydroxylase expression at the transcriptional level (44), and that 24-hydroxylase mRNA is not detectable in nonactivated tonsil B lymphocytes, and its expression is strictly dependent upon 1,25-(OH)2D3 (4, 11). Naive B cells were cultured for 24 h in the absence or presence of IL-4, anti-{kappa}/{lambda} antibodies, anti-CD40 antibodies and/or 1,25-(OH)2D3. Total RNA was isolated and reverse transcribed, and PCR amplifications were performed using oligonucleotide primers complementary to VDR, 24-hydroxylase, or GAPDH mRNA. As depicted in Fig. 3AGo, a VDR-specific PCR product was detected in RNA derived from naive B lymphocytes regardless of exogenous stimulation in vitro or mode of in vitro stimulation. Maximal levels of VDR mRNA were induced in those cultures stimulated with IL-4 even in the absence of concomitant BCR ligation (Fig. 3AGo, lane 5). Ligation singly of either the BCR with anti-{kappa}/{lambda} antibodies (Fig. 3AGo, lane 6) or of the CD40 receptor with anti-CD40 antibodies (Fig. 3AGo, lanes 9 and 10) neither reduced nor augmented the level of constitutive VDR expression. Additionally, coligation of the IL-4 receptor and coligation of either the BCR (Fig. 3AGo, lanes 7 and 8) or the CD40 receptor (Fig. 3AGo, lanes 11 and 12) neither reduced nor augmented the level of VDR expression up-regulated by stimulation with IL-4 alone.



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  		Figure 3. Induction of 1,25-(OH)2D3-mediated genomic trans-activation in naive tonsil B cells. Naive tonsil B cells were cultured for 24 h in the absence or presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), anti-CD40 antibodies (5 µg/ml), and/or 1,25-(OH)2D3 (50 nM), as indicated. A, Amplification of a 306-bp fragment from mRNA encoding human VDR. B, Amplification of a 317-bp fragment from mRNA encoding human 24-hydroxylase. RT-PCR analysis was performed as described in Fig. 2Go. Lane 1 depicts RNA derived from freshly isolated naive tonsil B cells; lanes 2–12 depict RNA isolated from naive tonsil B cells after 24 h of culture. The quantification of each band was performed by densitometric scanning of photographic negatives. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to the GAPDH transcript density for each corresponding sample. The data are representative of three similar experiments.

 
Analysis of naive B cell-derived RNA samples using oligonucleotide primers complementary to 24-hydroxylase revealed a specific PCR product only from those cultures stimulated with 1,25-(OH)2D3 (Fig. 3BGo, lanes 3, 5, 8, 10, and 12). Maximal expression of 24-hydroxylase mRNA was observed in those cultures stimulated with IL-4 (Fig. 3BGo, lanes 5, 8, and 12); in contrast, stimulation with anti-CD40 antibodies minimally up-regulated 24-hydroxylase expression (Fig. 3BGo, lane 10).

A strikingly similar pattern of VDR and 24-hydroxylase mRNA expression was observed from germinal center and memory B cell subpopulations (Figs. 4Go and 5Go, respectively). In each of these subpopulations, VDR-specific PCR product was detected, even in the absence of exogenous stimulation (Figs. 4AGo and 5AGo, lane 1). Maximal levels of VDR mRNA were again detected in those cultures stimulated with IL-4 (Figs. 4AGo and 5AGo, lanes 4, 5, 7, 8 11, and 12). Germinal center and memory B cell subpopulations were found to up-regulate 24-hydroxylase mRNA only in the presence of 1,25-(OH)2D3 (Figs. 4BGo and 5BGo, lanes 3, 5, 8, 10, and 12), similar to our observations with naive B cells. Taken together, these results examining the expression of the VDR and induction of 1,25-OH)2D3-mediated genomic trans-activation in naive, germinal center, and memory B cells suggest that phenotype alone (and therefore specific stage of B cell differentiation) is not a significant predicator of 1,25-(OH)2D3 responsiveness in normal B cells. Additionally, it is apparent that signals derived from ligation of the IL-4 receptor alone are sufficient to maximally up-regulate VDR in these B cell subpopulations and to initiate vitamin D-dependent 24-hydroxylase expression in the presence of 1,25-(OH)2D3.



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  		Figure 4. Induction of 1,25-(OH)2D3-mediated genomic trans-activation in germinal center tonsil B cells. Germinal center tonsil B cells were cultured for 24 h in the absence or presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), anti-CD40 antibodies (5 µg/ml), and/or 1,25-(OH)2D3 (50 nM), as indicated. A, Amplification of a 306-bp fragment from mRNA encoding human VDR. B, Amplification of a 317-bp fragment from mRNA encoding human 24-hydroxylase. RT-PCR analysis was performed as described in Fig. 2Go. Lane 1 depicts RNA derived from freshly isolated germinal center tonsil B cells; lanes 2–12 depict RNA isolated from germinal center tonsil B cells after 24 h of culture. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to the GAPDH transcript density for each corresponding sample. The data are representative of three similar experiments.

 


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  		Figure 5. Induction of 1,25-(OH)2D3-mediated genomic trans-activation in memory tonsil B cells. Memory tonsil B cells were cultured for 24 h in the absence or presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), anti-CD40 antibodies (5 µg/ml), and/or 1,25-(OH)2D3 (50 nM) as indicated. A, Amplification of a 306-bp fragment from mRNA encoding human VDR. B, Amplification of a 317-bp fragment from mRNA encoding human 24-hydroxylase. RT-PCR analysis was performed as described in Fig. 2Go. Lane 1 depicts RNA derived from freshly isolated memory tonsil B cells; lanes 2–12 depict RNA isolated from memory tonsil B cells after 24 h of culture. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to the GAPDH transcript density for each corresponding sample. The data are representative of three similar experiments.

 
Differential reactivity of density-fractionated naive, germinal center, and memory B cells to 1,25-(OH)2D3
The universal detection of constitutive VDR mRNA expression in naive, germinal center, and memory B cell subpopulations and their propensity to initiate hormone-mediated genomic trans-activation were unique and in contrast to our previous observations of density-fractionated B lymphocytes (11). We questioned whether the isolated populations of naive, germinal center, and memory B cells were heterogeneous with respect to degree of cellular activation, and whether such heterogeneity would be reflected in their expression of VDR and their reactivity to 1,25-(OH)2D3. To address this issue, we exploited the phenomenon that in lymphoid cells, the extent of cellular activation directly parallels an increase in cell volume and a decrease in their buoyant density (12). Normal tonsillar B cells were phenotypically fractionated into naive, germinal center, and memory B cell subpopulations by negative selection using magnetic beads and then immediately density fractionated on discontinuous Percoll gradients. Total RNA was extracted from the high, intermediate, and low density populations of naive, germinal, center and memory B cells either immediately or after culture stimulation for 24 h in the presence of IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3. Total RNA was reverse transcribed, and PCR amplification was performed using oligonucleotide primers complementary to VDR, 24-hydroxylase, or GAPDH mRNA. As depicted in Fig. 6AGo, significant constitutive VDR message was detected in the low density fractions of naive, germinal center, and memory B cells (Fig. 6AGo, lanes 1, 8, and 15), yet was barely detectable in the high density fractions of naive and memory B cells (Fig. 6AGo, lanes 3 and 10). Due to the activated nature (and therefore reduced density) of germinal center cells, a high density fraction of this B cell subpopulation could not be obtained. VDR message was significantly up-regulated in all density fractions of naive, germinal center, and memory B cells after culture stimulation for 24 h with IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3.



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  		Figure 6. Differential reactivity of density-fractionated naive, germinal center, and memory B cells to 1,25-(OH)2D3. Naive, germinal center, and memory B cells were purified, and Percoll density fractions were obtained from each subpopulation. Total RNA was extracted from high (H), intermediate (I), and low (L) density populations of naive, germinal center, and memory B cells either immediately (0 h) or after culture stimulation for 24 h in the presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), and 1,25-(OH)2D3 (50 nM). A, Amplification of a 306-bp fragment from mRNA encoding human VDR. B, Amplification of a 317-bp fragment from mRNA encoding human 24-hydroxylase. RT-PCR analysis was performed as described in Fig. 2Go. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to the GAPDH transcript density for each corresponding sample. The data are representative of three similar experiments.

 
Examination of these density-fractionated naive, germinal center, and memory B cells for 1,25-(OH)2D3-mediated induction of 24-hydroxylase message revealed a unique response profile. The low density fraction derived from naive, germinal center, and memory B cells up-regulated significant 24-hydroxylase mRNA (Fig. 6BGo, lanes 4, 11, and 17). However, this mRNA was minimally detected from the high density fraction of memory B cells (Fig. 6BGo, lane 13), and was not detected in the high density fraction of naive B cells (Fig. 6BGo, lane 6). Taken together, these results demonstrate that expression of VDR and the initiation of 1,25-(OH)2D3-mediated genomic trans-activation are solely dependent upon the degree of cellular activation of naive, germinal center, and memory B cell subpopulations.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The described studies address whether phenotypically defined B cell subpopulations representative of distinct stages of B cell differentiation can up-regulate VDR expression and initiate 1,25-(OH)2D3-mediated genomic trans-activation. Evidence has been presented demonstrating that phenotypic profile, and therefore stage of B cell differentiation, is not a significant indicator of the propensity of B cells to biologically respond to 1,25-(OH)2D3. Phenotypically fractionated populations highly enriched for naive, germinal center, and memory B cells were all found to be receptive to biological effects of 1,25-(OH)2D3. It was routinely observed that these B cell subpopulations constitutively expressed VDR message and protein (data not shown) and were fully capable of initiating 1,25-(OH)2D3-mediated genomic trans-activation, even in the absence of exogenous stimulation. These observations are a logical extension of a previous report demonstrating the capacity of freshly isolated normal human B cells to constitutively express VDR (45). However, the B cell cultures used in the report of Provvedini et al. were not fractionated with respect to phenotype and were therefore highly heterogeneous with respect to stage of differentiation.

The similar kinetic response of phenotypically defined B cell subpopulations is in contrast to our previous report examining the influence of cellular activation on B cell reactivity with 1,25-(OH)2D3 (11). Therein we demonstrated that activation requirements rendering tonsillar B cells responsive to 1,25-(OH)2D3, and the kinetics of the response, are widely different among density-fractionated normal B cell subpopulations. Similar to phenotypically defined B cell subpopulations, low density B cells were observed to constitutively express VDR message and protein and could rapidly up-regulate VDRE-reactive nuclear proteins and initiate 1,25-(OH)2D3-dependent genomic trans-activation even in the absence of exogenous simulation. In contrast, high density B lymphocyte subpopulations did not express detectable VDR message or protein, yet required specific cellular activation signals initiating its up-regulation. Furthermore, the high density B cell subpopulations required a protracted activation phase for the initiation of 1,25-(OH)2D3-dependent genomic trans-activation. This temporal lag of hormone-mediated genomic transcription is attributed to events occurring before cellular interaction with 1,25-(OH)2D3.

Density fractionation of naive, germinal center, and memory B cells revealed that these subpopulations were indeed heterogeneous with respect to density and therefore degree of cellular activation. It may be argued that the phenotypically defined B cells isolated do not accurately represent these B cell subpopulations in situ, as the techniques used for cell fractionation and isolation may have artificially activated these B cells. To address this issue, we used a negative selection procedure, such that the cells to be analyzed were not those B cell subpopulations that reacted with either antibodies or magnetic microbeads during fractionation. Furthermore, an examination of de novo DNA and RNA synthesis, as determined by incorporation of [3H]thymidine and [3H]uridine revealed that the B cell subpopulations were not artificially activated by the selection procedure, yet could vigorously respond to stimulation in vitro.

When examined for their capacity to up-regulate VDR expression and initiate 1,25-(OH)2D3-mediated genomic trans-activation, these density-fractionated B cell populations demonstrated a reactivity profile strikingly similar to that observed in our earlier report (11). Constitutive VDR expression was detected from each phenotypically defined population in the low density fraction, substantiating our detection of constitutive VDR expression in naive, germinal center, and memory B cells. In contrast, little or no VDR was detected in the high density fraction of the relatively quiescent naive and memory B cells. The high density fraction of germinal center B cells could not be analyzed, as very few of these cells were obtained, consistent with the blastogenic nature of this stage of B cell maturation (46). After a 24-h in vitro activation phase in the presence of 1,25-(OH)2D3, VDR message was up-regulated in all density fractions of naive, germinal center, and memory B cells; however, significantly less VDR message was induced from the more quiescent, high density naive B cell subpopulation. Taken together, these observations demonstrate that in human B lymphocytes, the expression of VDR closely parallels the degree of cellular activation, yet it is not correlated with their specific phenotypic profile. In this context, expression of the VDR may be considered an activation marker in B lymphocytes (47).

Additionally revealing were experiments examining initiation of 1,25-(OH)2D3-mediated genomic trans-activation in density-fractionated naive, germinal center, and memory B cells. As expected, 24-hydroxylase message was not detected in any freshly isolated density fractions of naive, germinal center, and memory B cells. After a 24-h in vitro activation phase in the presence of 1,25-(OH)2D3, 24-hydroxylase message was detected in all three of the phenotypically defined B cell subpopulations in the low and intermediate density fractions. Significantly less 24-hydroxylase message was found in the high density fraction of memory B cells, and 24-hydroxylase message was not detected in the high density fraction of naive B cells. Of interest is the observation that although high density naive B cells up-regulate significant VDR expression, 1,25-(OH)2D3-mediated genomic trans-activation is not detected in these B cell subpopulations within a 24-h time period. Extended culture revealed that 24-hydroxylase message was up-regulated in these B cell subpopulations after approximately 48 h of culture (data not shown). These results mimic our previous study (11), in which phenotypically heterogeneous high density B cells required approximately 48 h for hormone-dependent up-regulation of 24-hydroxylase mRNA.

The protracted time interval required for high density B lymphocytes (regardless of their surface phenotype) to initiate transcription of 1,25-(OH)2D3-responsive genes is a conundrum that remains unresolved from these studies. Although we chose to examine up-regulation of 24-hydroxylase message expression primarily as an indicator of 1,25-(OH)2D3-mediated transcriptional activation, it is noteworthy that this enzyme is critical for initiation of a catabolic pathway eliminating 1,25-(OH)2D3 from the target cell. Induction of 24-hydroxylase activity ensures a limited amount of ligand and a limited time frame for the effects of intranuclear 1,25-(OH)2D3 (48). Currently it is unclear whether the extended exposure of high density B lymphocytes to the biologically active form of vitamin D [1,25-(OH)2D3] serves a biological necessity unique to these relatively quiescent cells or is of little consequence to the cell.

It has been well documented that a functional VDR:ligand complex is comprised of a heterodimer consisting of the VDR and the retinoid X receptor (RXR) (49, 50, 51). Preliminary observations examining the expression of RXR isoforms (RXR-{alpha}, RXR-ß, and RXR-{gamma}) in both phenotypically defined B lymphocytes and density-fractionated B lymphocytes have provided evidence implicating this auxiliary nuclear receptor as a central mediator of B lymphocyte reactivity with 1,25-(OH)2D3. Naive, germinal center, and memory B cells were found to express message for RXR-{alpha} and RXR-ß, but not for RXR-{gamma} (our manuscript in preparation). Message encoding RXR-ß was observed to be constitutively expressed regardless of B cell phenotypic profile (i.e. stage of maturation) or density (i.e. degree of activation). As such, although RXR-ß may be a significant participatory component of a functional VDR complex in human B lymphocytes (4), it is difficult to ascribe RXR-ß as a limiting heterodimeric partner with VDR. In contrast, we observed that message encoding RXR-{alpha} is minimally detected in quiescent, high density B lymphocytes, yet is up-regulated in these cells after activation in vitro. Furthermore, the kinetic profile of RXR-{alpha} message expression in these activated B cells is very similar to that observed for the expression of 24-hydroxylase mRNA in these cells (our manuscript in preparation). Taken together, it may be speculated that RXR-{alpha} is a limiting component in quiescent, high density B lymphocytes, and as such may modulate the establishment of a functional VDR:RXR nuclear complex, thereby providing an additional level of regulatory control mediating 1,25-(OH)2D3-dependent genomic trans-activation in normal B lymphocytes. A precedent for multiple RXR isoforms contributing to the establishment of functional VDR:RXR complexes has been demonstrated (52). The contribution of these auxiliary nuclear receptors as modulators of B cell receptivity to 1,25-(OH)2D3 is the subject of current studies.


   Acknowledgments
 
We acknowledge the excellent technical assistance of Mr. David J. Sliney.


   Footnotes
 
1 This work was supported in part by NIH Grant R29-DK-49649 and funds from the Department of Pathology Teaching and Research Foundation, Roger Williams Medical Center (Providence, RI). Back

Received March 16, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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