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Endocrinology Vol. 140, No. 1 381-391
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential Regulation of Gene Transcription in Subpopulations of Human B Lymphocytes by Vitamin D31

John W. Morgan, David J. Sliney, Deborah M. Morgan and Abby L. Maizel

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that freshly extirpated normal human tonsil B cells, which are phenotypically diverse, representing different stages of cellular activation and differentiation, are refractory to the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and require specific activation signals for induction of responsiveness. To determine whether these diversely activated B cell populations respond to 1,25-(OH)2D3, human tonsil B cells were density fractionated and evaluated biochemically and functionally. Low density tonsil B cells, representing the centroblastic fraction, were observed to constitutively express vitamin D receptor message and protein. In contrast, high density quiescent tonsillar B cells had no detectable vitamin D receptor message or protein and required stimulation in vitro for their up-regulation. Biological responsiveness to 1,25-(OH)2D3 was assessed by messenger RNA (mRNA) expression of the vitamin D-dependent enzyme, 25-hydroxyvitamin D3 24-hydroxylase. Low density centroblastic B cells did not require exogenous surface activation for expression of 24-hydroxylase mRNA, which was detectable after 6 h of culture in the presence of 1,25-(OH)2D3. In contrast, high density tonsil B cells required in vitro activation for induction of 24-hydroxylase mRNA, and expression was not detectable for up to 48 h of culture. These observations suggest that reactivity of normal B cell populations to vitamin D is dependent upon their specific stage of activation.


   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 also recognized as a potent immunoregulator (reviewed in Refs. 1, 2). Expression of VDR has been documented in hematopoietic cells of monocytic, T, and B cell lineages. VDR are constitutively expressed in monocytic cells, and their expression is further up-regulated by 1,25-(OH)2D3 (3, 4). Treatment of myeloid cells with 1,25-(OH)2D3 characteristically induces a more mature phenotype and enhances cellular differentiation, thereby inhibiting proliferation (5, 6, 7).

In contrast, VDR are not constitutively expressed in quiescent lymphocytes, yet upon cellular activation VDR are up-regulated (3, 8). Overall, vitamin D has been shown to exert an antiproliferative effect on T lymphocytes (9, 10, 11, 12) by repressing transcriptional activation of the interleukin-2 gene by interfering with NFATp (preexisting nuclear factor of activated T cells)/AP-1 complex formation (13). However, it has been demonstrated that up-regulation of VDR in T cells may not necessarily confer bioresponsiveness to 1,25-(OH)2D3, and that functional receptivity to the hormone is highly regulated by the mode of cellular activation. Unlike PHA-activated cells, T lymphocytes stimulated with either the anti-CD3 antibody (12, 14) or phorbol myristate acetate (12, 15) failed to respond to the antiproliferative effect of 1,25-(OH)2D3, even though VDR expression was clearly established.

Similarly, quiescent B lymphocytes do not constitutively express VDR, yet VDR are up-regulated via cellular activation in vitro (3, 8) and in vivo (16). Analogous to its effect on T lymphocytes, 1,25-(OH)2D3 exerts an antiproliferative effect on activated B lymphocytes (17, 18). Additionally, the hormone has been shown to inhibit Ig production in vitro (17, 19, 20, 21) and in vivo (22). However, the manner in which 1,25-(OH)2D3 negatively influences Ig production remains to be fully resolved, as this effect may be an indirect effect of 1,25-(OH)2D3-mediated inhibition of T cell help and/or proliferation (23, 24) or an indirect effect of hormone-mediated modulation of cytokine production by monocytes/macrophages (21, 25).

Also comparable to T lymphocytes, up-regulation of VDR in B lymphocytes may not be sufficient to confer bioresponsiveness to 1,25-(OH)2D3, as functional receptivity to the hormone has been observed to be dependent upon the mode of cellular activation. Stimulation of normal human tonsillar B lymphocytes with Epstein-Barr virus or via ligation of either the B cell receptor (BCR; membrane forms of Igs), the CD40 receptor, or the interleukin-4 (IL-4) receptor induces expression of VDR message and protein, yet the cells are refractory to many physiological effects of 1,25-(OH)2D3 (26). However, this apparent refractivity to the hormone may be overcome by combining signals derived from ligation of the BCR in conjunction with signals derived from interaction of the IL-4 receptor with its ligand (18). Activation of B cells via these two distinct signal transduction pathways renders the cells responsive to the hormone, in that 1,25-(OH)2D3 suppresses cellular proliferation and up-regulates genomic expression of 25-hydroxyvitamin D3 24-hydroxylase (18), a 1,25-(OH)2D3-dependent enzyme that catalyzes 24-hydroxylation of 25-hydroxyvitamin D3 and 1,25-(OH)2D3 (27). Paradoxically, these activation events rendering B cells biologically responsive to 1,25-(OH)2D3 are also sufficient for induction of G1 phase progression in quiescent B cell populations.

From these observations, it is apparent that in B lymphocytes, expression of VDR and the establishment of biological reactivity to 1,25-(OH)2D3 are separate and distinct events that are highly regulated. Elucidation 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 tissue, such as the tonsil, contains B cells that are highly heterogeneous with respect to their degree of activation and differentiation. In vivo, antigen-specific quiescent B cells are initially activated within the follicular mantle of the tonsil in association with T cells and interdigitating dendritic cells and subsequently undergo clonal expansion and differentiation (28, 29). The actively proliferating B cells of the germinal center, or centroblasts (IgD-, CD38+, CD77+, Ki67+), characteristically undergo affinity maturation associated with somatic mutation in their IgV region genes (30, 31). Surviving centroblasts, not undergoing apoptosis, ultimately give rise to centrocytes (IgD-, CD38+, CD77-, Ki67-), which are nonproliferating B cells that have isotype switched (32) and differentiated into either memory B cells (IgD-, CD38-) and/or plasma (CD382+, CD20-) cells (33, 34).

At this time it is unclear whether these diverse B cell populations are universally reactive with 1,25-(OH)2D3, and whether common signal transduction pathways provide a regulatory control for reactivity with the hormone. To resolve these questions, in the present studies we evaluated whether 1,25-(OH)2D3 can mediate genomic trans-activation in differentially activated B cell subpopulations, and whether activation in vivo modulated those minimal requirements for up-regulation of VDR and induction of hormone-mediated trans-activation. Although all tonsillar B cell fractions examined could be rendered responsive to 1,25-(OH)2D3, their in vitro activation requirements and the kinetics of their responses differed widely. These parameters were observed to vary greatly due to cellular activation events initiated before interaction with the hormone. Human B cells thus represent a unique model for evaluating components of the vitamin D response pathway not previously found in those target cell populations that constitutively express a functional receptor.


   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). Monoclonal rat anti-chicken VDR antibody (clone 9A7{gamma}) (35) was purchased commercially (Affinity BioReagents, Inc., Neshanic Station, NJ). Escherichia coli-derived recombinant human IL-4 was obtained commercially (PeproTech, Inc., Rocky Hill, NJ). Antihuman CD40 mouse monoclonal antibody (36) (mouse IgG) was affinity purified in our laboratory from the G28–5 cell line, which was obtained from the American Type Culture Collection (Manassas, VA). Rabbit antihuman {kappa} and antihuman {lambda} (anti-{kappa}/{lambda}) antibodies were obtained from Dako Corp. (Carpenteria, CA). Experiments demonstrated that ligation of the BCR with insoluble rather than soluble anti-{kappa}/{lambda} antibodies induced a stronger activation response and an enhanced reactivity to 1,25-(OH)2D3 (data not shown); therefore, experiments were performed using 10 µg/ml anti-{kappa}/{lambda} antibodies covalently coupled to a polyacrylamide matrix (Immunobead Matrix, Irvine Scientific, Santa Anna, CA).

Cell lines
The monocytic leukemia cell line U937 was obtained from American Type Culture Collection and has been described previously (37).

Isolation of tonsillar B lymphocytes from normal human tonsils was performed as previously described (26). Briefly, tonsils taken from patients during routine tonsillectomy 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 98% B lymphocytes as determined by immunofluorescence with anti-CD3, -CD19, -CD20, and -CD45 antibodies. After overnight incubation, tonsil B cells were fractionated on Percoll density gradients according to density essentially as previosuly described (38), with the exception that Percoll mix solution was substituted for Hanks’ Balanced Salt Solution. 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). Phenotypic analysis of cell surface antigens was performed on a minimum of 2 x 105 cells/evaluation, with the fluorescence intensity of the cells analyzed on a Becton Dickinson Co. FACScan (Mountain View, CA). High density tonsillar B lymphocytes are characterized phenotypically as the following: 1 ± 1% CD3+, 99 ± 2% CD19+, 7 ± 2% CD38bright+, 93 ± 2% CD38dim+, 85 ± 4% CD44+, 53 ± 6% IgD+, 15 ± 12% IgG+, and 65 ± 5% IgM+. Intermediate density cells are characterized phenotypically as the following: 0 ± 0% CD3+, 99 ± 1% CD19+, 23 ± 4% CD38bright+, 77 ± 4% CD38dim+, 66 ± 6% CD44+, 36 ± 5% IgD+, 35 ± 13% IgG+, and 54 ± 10% IgM+. Low density cells are characterized phenotypically as the following: 0 ± 0% CD3+, 100 ± 0% CD19+, 56 ± 8% CD38bright+, 44 ± 8% CD38dim+, 21 ± 2% CD44+, 8 ± 3% IgD+, 41 ± 14% IgG+, and 31 ± 12% IgM+.

Purification of RNA and RT-PCR
Total RNA was obtained using Trizol (Life Technologies, Gaithersburg, MD) reagent (39). 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). Primers for human VDR messenger RNA (mRNA) 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) (40). The expected length of the PCR product is 306 bp. Primers for human 24-hydroxylase mRNA 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) mRNA 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 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, Emeryville, CA) in the presence of a 200-nM final concentration of 5'- and 3'-primers, 200 µM deoxy (d)-NTPs, 1.5 U Taq polymerase (Promega Corp., Madison, WI), and PCR buffer containing either 1.5 mM MgCl2, 15 mM (NH4)SO4, and 60 mM Tris-Cl, pH 8.5 (for 24-hydroxylase and GAPDH primers), or 2 mM MgCl2, 15 mM (NH4)SO4, and 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). 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. Aliquots (18 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.

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 previously described (41, 42). 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 (43); optimal PCR amplification for this reaction was determined to be at 18 cycles. Replicate experiments analyzing RNA extracted from tonsil B cells exhibited similar results (data not shown).

Nuclear extraction
Preparation of nuclear extracts was performed essentially as described previously (44); 0.3–1 x 108 cells were used for each nuclear extract. The protein concentration was determined by the method of Bradford (45).

Immunoblot analysis
Western analysis was performed as previously described (18).

Electrophoretic mobility shift assay (EMSA)
Gel-shift analysis was performed using a 27-mer synthetic oligonucleotide corresponding to a VDRE localized within the promoter region of the rat 24-hydroxylase gene (P450cc24 VDRE-1) (46). The sequence of this oligonucleotide is 5'-TCG AGC GGC GCC CTC ACT CAC CTC GCG-3'. This oligonucleotide and its complement were annealed, and the double-stranded oligonucleotide was labeled with [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, IL) using Klenow enzyme. Binding reactions were performed essentially as previously described (44). Briefly, 10 µg nuclear proteins were equilibrated at room temperature in a DNA-binding buffer containing 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 1% Ficoll (Mr = 400,000), and 300 ng poly(dI-dC)-poly(dI-dC). Ten femtomoles of radioactive P450cc24 oligonucleotide probe were added, and the incubation was continued for an additional 20 min. The reaction mixture was then loaded on 5% polyacrylamide gels in 1 x TBE running buffer and electrophoresed for 2 h at 200 V. Bands were visualized by autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VDR expression in density-fractionated tonsil B cells
In lymphoid cells, the extent of cellular activation directly parallels an increase in cell volume and a decrease in their buoyant density (47). Therefore, tonsil B cells were fractionated on the basis of their density using discontinuous Percoll gradients to assess the role of cellular activation, both in vivo and in vitro, as a mediator of B cell reactivity to 1,25-(OH)2D3. These B cell populations, still relatively heterogeneous with respect to phenotype (see Materials and Methods for phenotypic profile), were examined for their capacity to express the VDR. To avoid exclusion of those B cell populations that were surface IgM negative (e.g. IgG+ or IgM-/IgD+ B cells), ligation of the BCR was effected with a combination of antibodies specific for human {kappa} light chains and human {lambda} light chains. As depicted in Fig. 1Go, Western blot analysis revealed that high and intermediate density B cells did not exhibit detectable VDR protein, even after stimulation singly with 1,25-(OH)2D3, IL-4, or insoluble anti-{kappa}/{lambda} antibody (Fig. 1Go, lanes 2–5). Treatment with a combination of IL-4 and anti-{kappa}/{lambda} antibody up-regulated VDR protein expression in these B cell fractions (Fig. 1Go, lane 6), and the amount of VDR was further increased by concomitant treatment with 1,25-(OH)2D3 (Fig. 1Go, lane 7). In contrast, low density B cells (centroblasts) constitutively expressed VDR protein at low levels even in the absence of exogenous stimulation (Fig. 1Go, lane 2). VDR protein expression was significantly enhanced after stimulation with IL-4 and anti-{kappa}/{lambda} antibody (Fig. 1Go, lane 6) and by concomitant treatment with 1,25-(OH)2D3 (Fig. 1Go, lane 7).



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  		Figure 1. VDR protein expression in tonsil B cells. Chemiluminescent Western blot of VDR protein expression in density-fractionated tonsil B cells. As physiological effects of 1,25-(OH)2D3 on the monocytic leukemia cell line U937 have been described previously (30 31 ), protein extracts from these cells cultured for 24 h in the presence of IL-4 (10 ng/ml) and 1,25-(OH)2D3 (10 nM) were included as a positive control. Normal tonsillar B cells were density fractionated on discontinuous Percoll gradients and cultured for 24 h in the absence or presence of recombinant IL-4 (10 ng/ml), insoluble anti-{kappa}/{lambda} antibody (10 µg/ml), and/or 1,25-(OH)2D3 (50 nM), as indicated. Postincubation, nuclear extracts were prepared, and 10 µg protein were resolved on 10% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nylon membranes and immunoblotted with an anti-VDR monoclonal antibody (9A7{gamma}). Antibody binding was detected with enhanced chemiluminescence detection reagents. The positions of molecular mass standards are indicated at the right.

 
Evaluation of 24-hydroxylase message
As VDR protein was observed to be constitutively expressed in low density B cells and could be up-regulated in intermediate and high density B cells by stimulation in vitro, we next questioned whether 1,25-(OH)2D3 could activate genomic transcription in these B cell populations. As an indicator of transcriptional activation, we chose to examine expression of message encoding 25-hydroxyvitamin D3 24-hydroxylase. It has been shown that 1,25-(OH)2D3 exerts control of 24-hydroxylase expression at the transcriptional level (48), and we have previously demonstrated that 24-hydroxylase mRNA is not detectable in nonactivated tonsil B lymphocytes, and its expression is strictly dependent upon 1,25-(OH)2D3 (18). A 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 (49, 50) as well as VDR message (40).

Low, intermediate, and high density 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 amplification was performed using oligonucleotide primers complementary to VDR, 24-hydroxylase, or GAPDH mRNA. As depicted in Fig. 2AGo, a VDR-specific PCR product was detected in RNA derived from unstimulated low density tonsil B cells (Fig. 2AGo, lane 1). Stimulation of these cells singly with IL-4 augmented the amount of VDR mRNA (Fig. 2AGo, lane 5), and maximal levels of VDR mRNA were induced in those cultures stimulated with a combination of IL-4 and anti-{kappa}/{lambda} antibodies (Fig. 2AGo, lane 8). Treatment with anti-CD40 antibodies, either singly or in combination with 1,25-(OH)2D3, neither augmented nor reduced these levels of VDR mRNA (Fig. 2AGo, lanes 3 and 4). Results obtained from these RT-PCR analyses are in agreement with those of experiments examining VDR protein expression (Fig. 1Go), in which VDR protein was detected in extracts derived from unstimulated low density tonsil B cells. Analysis of low density B cell 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. 2BGo, lanes 2, 4, 8, 10, and 12). Interestingly, stimulation of low density B cells with only 1,25-(OH)2D3 in the absence of other exogenous stimulation was sufficient for the induction of 24-hydroxylase message (Fig. 2BGo, lane 2). Maximal expression of 24-hydroxylase mRNA was observed in those cultures stimulated with 1,25-(OH)2D3 and a combination of IL-4 and BCR ligation (Fig. 2BGo, lanes 8 and 12).



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  		Figure 2. RT-PCR amplification of mRNA from in vitro stimulated low density tonsil B cells. Low density 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. 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 317-bp fragment from mRNA encoding human 24-hydroxylase. Samples (18 µl) 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. The positions of molecular standards (base pairs) are indicated at the right. 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 a constant GAPDH transcript density. Similar results were obtained from other separate experiments.

 
RNA derived from intermediate density B cells and subjected to relative RT-PCR analysis revealed a different pattern of message expression for VDR and 24-hydroxylase. VDR-specific PCR product was not detected from unstimulated intermediate density B cell cultures (Fig. 3AGo, lane 1), and addition of 1,25-(OH)2D3 minimally up-regulated VDR message (Fig. 3AGo, lane 2). However, addition of IL-4 maximally augmented VDR message expression even in the absence of 1,25-(OH)2D3 and/or BCR ligation (Fig. 3AGo, lane 5). Furthermore, maximal levels of VDR mRNA expression were detected only in those cultures costimulated with IL-4 regardless of surface antigen ligation. Interestingly, RNA derived from intermediate density B cell cultures exhibited minimally detectable levels of 24-hydroxylase message, regardless of polyclonal B cell activator combination or exogenous addition of 1,25-(OH)2D3 (Fig. 3BGo). 24-Hydroxylase-specific PCR product was detectable only in those cultures treated with a combination of IL-4, anti-{kappa}/{lambda} antibodies and 1,25-(OH)2D3 (Fig. 3BGo, lanes 8 and 12).



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  		Figure 3. RT-PCR amplification of mRNA from in vitro stimulated intermediate density tonsil B cells. Intermediate density 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. RT-PCR analysis was performed as described in Fig. 2Go. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to a constant GAPDH transcript density. Similar results were obtained from other separate experiments.

 
Relative RT-PCR analysis of RNA derived from high density tonsil B cells provided results similar to those obtained from intermediate density B cell cultures. VDR-specific PCR product was not detectable from either unstimulated or 1,25-(OH)2D3-treated high density B cells (Fig. 4AGo, lanes 1 and 2), and maximal levels of VDR mRNA were detected only in those cultures stimulated with a combination of IL-4 and anti-{kappa}/{lambda} antibodies (Fig. 4AGo, lanes 7, 8, 11, and 12). In contrast to results obtained from low and intermediate density B cells, high density B cells evaluated after 24 h of culture did not yield detectable levels of 24-hydroxylase-specific PCR product regardless of the polyclonal activator combination or exogenous addition of 1,25-(OH)2D3 (Fig. 4BGo).



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  		Figure 4. RT-PCR amplification of mRNA from in vitro stimulated high density tonsil B cells. High density 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. RT-PCR analysis was performed as described in Fig. 2Go. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to a constant GAPDH transcript density. Similar results were obtained from other separate experiments.

 
Kinetics of VDR and 24-hydroxylase message appearance in density-fractionated tonsil B cells
Experiments examining the expression of VDR and 24-hydroxylase message in density-fractionated B cells ( Figs. 2–4GoGoGo) demonstrated that all populations analyzed were capable of up-regulating VDR mRNA, correlating well with results obtained from the VDR Western blot analysis (Fig. 1Go). Furthermore, exogenous addition of only 1,25-(OH)2D3 up-regulated 24-hydroxylase mRNA in low density B cells (although this level of expression was enhanced by concomitant ligation of BCR and IL-4 receptors). However, although significant VDR message and protein could be up-regulated in high density tonsil B cells, this population apparently failed to up-regulate 24-hydroxylase mRNA. It may be hypothesized that the quiescent, high density B cells 1) are refractory to 1,25-(OH)2D3-mediated transcriptional activation (minimally at the level of the 24-hydroxylase gene), 2) require additional and/or different activation signals to effect 1,25-(OH)2D3-dependent 24-hydroxylase genomic transcription, or 3) require a protracted activation phase either before or after exposure to 1,25-(OH)2D3. To address this issue, a kinetic analysis was performed examining VDR and 24-hydroxylase message expression in density-fractionated tonsil B cells. As depicted in Fig. 5AGo, low density B cells expressed significant VDR message at the time of Percoll density fractionation (Fig. 5AGo, lane 1), and the level of message increased as a function of time after in vitro stimulation. PCR amplification indicated maximal levels of VDR mRNA after approximately 8 h of culture (Fig. 5AGo, lane 5). mRNA encoding 24-hydroxylase was detected after approximately 6 h of culture in the presence of IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3 (Fig. 5BGo, lane 4), with maximal amounts observed after approximately 18 h of culture (Fig. 5BGo, lane 8). In contrast, freshly isolated high density tonsil B cells did not exhibit detectable VDR message (Fig. 6AGo, lane 1), yet significant levels were observed after approximately 9 h of culture in the presence of IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3 (data not shown). High density B cells exhibited maximal amounts of VDR mRNA after 24 h of culture (Fig. 6AGo, lane 2), and this level was maintained for up to 84 h of culture (Fig. 6AGo, lane 11). mRNA encoding 24-hydroxylase was not detectable until approximately 48 h of culture with IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3 (Fig. 6BGo, lane 5), and expression of this mRNA species was maintained for up to 84 h of culture (Fig. 6BGo, lane 11). A kinetic analysis of 24-hydroxylase mRNA appearance in intermediate density B cells indicated that expression is rapidly up-regulated after a 24-h period, and maximal amounts can be detected after approximately 30 h of stimulation with IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3 (data not shown).



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  		Figure 5. Kinetics of VDR and 24-hydroxylase message appearance in low density tonsil B cells. Low density tonsil B cells were cultured in the presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), and 1,25-(OH)2D3 (50 nM). At the indicated time intervals, cell aliquots were removed, and total RNA was isolated. RT-PCR analysis was performed as described in Fig. 2Go. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to a constant GAPDH transcript density. Similar results were obtained from other separate experiments.

 


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  		Figure 6. Kinetics of VDR and 24-hydroxylase message appearance in high density tonsil B cells. High density tonsil B cells were cultured in the presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), and 1,25-(OH)2D3 (50 nM). At the indicated time intervals, cell aliquots were removed, and total RNA was isolated. RT-PCR analysis was performed as described in Fig. 2Go. Plots depict relative amounts of VDR and 24-hydroxylase transcript as normalized to a constant GAPDH transcript density. Similar results were obtained from other separate experiments.

 
Activation events before 1,25-(OH)2D3 exposure are rate limiting in high density tonsil B cells
The data indicated that when activation signals mediated by IL-4 receptor and BCR ligation were specifically provided for a protracted time frame, high density B cells were fully capable of up-regulating 24-hydroxylase genomic transcription in a hormone-dependent fashion. However, the time frame required for induction of 24-hydroxylase message in these quiescent B cell populations was significantly greater (42 h) than that observed in low density B cells and greater (24 h) than that in intermediate density B cells. To address this issue, we next evaluated whether the rate-limiting factor for up-regulation of 24-hydroxylase message correlated with events before or after 1,25-(OH)2D3 exposure. High density tonsil B cells were stimulated with a combination of IL-4 and anti-{kappa}/{lambda} antibodies for 48 h, and subsequently 1,25-(OH)2D3 was added to the B cell cultures. At intervals after the addition of 1,25-(OH)2D3, cell aliquots were removed, and RNA was isolated. After a 48-h activation phase in vitro, in the absence of exogenously added 1,25-(OH)2D3, significant levels of VDR message were detected (Fig. 7Go, lane 2), yet 24-hydroxylase mRNA was not detected (Fig. 7Go, lane 8), confirming that this enzyme is up-regulated in B cells in a hormone-dependent fashion. However, after a 48-h activation phase in vitro, culture maintenance in the presence of 1,25-(OH)2D3 for as little as 6 h up-regulated 24-hydroxylase message in these high density B cells (Fig. 7Go, lane 9). Taken together, the results indicate that the apparent temporal lag of 1,25-(OH)2D3-mediated 24-hydroxylase gene transcription in high density B cells correlates with events occurring before cellular interaction with 1,25-(OH)2D3.



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  		Figure 7. Kinetics of VDR and 24-hydroxylase message appearance in high density tonsil B cells preactivated in vitro. High density tonsil B cells were cultured in the presence of IL-4 (10 ng/ml) and anti-{kappa}/{lambda} antibodies (10 µg/ml) for 48 h. Cultures were then incubated in the presence of 50 nM 1,25-(OH)2D3, and at the indicated time intervals, cell aliquots were removed, and total RNA was isolated. RT-PCR analysis was performed as described in Fig. 2Go. Plots depict relative amounts of VDR and 24-hydroxylase transcript normalized to a constant GAPDH transcript density. -, RT-PCR analysis of RNA isolated from high density B cells before culture initiation (i.e. freshly isolated high density B cells).

 
VDRE-reactive nuclear proteins in density-fractionated tonsil B lymphocytes
As the kinetics of 24-hydroxylase message induction varied widely in density-fractionated B cells, we questioned whether this temporal discrepancy could be detected at the level of nuclear protein(s) reactive with the DNA response element. We have previously examined this parameter in unfractionated tonsil B cells by means of an electrophoretic mobility shift assay (EMSA) (18), using as probe an oligonucleotide (P450cc24 VDRE-1) corresponding to a VDRE localized within the promoter region of the 24-hydroxylase gene (46, 51, 52, 53). This VDRE is composed of a single canonical VDRE consisting of two imperfect direct repeat 6-bp half-sites separated by a 3-bp nucleotide spacer (54). It has been shown that this direct repeat 6-bp motif, which exhibits sequence similarity to VDREs found in the osteocalcin (55, 56) and osteopontin (57) genes, can bind to a heterodimer consisting of VDR and retinoid X receptor-ß (RXRß) (46). As physiological effects of 1{alpha},25-(OH)2D3 on the monocytic leukemia cell line U937 have been described (41, 42), nuclear extracts from these cells were included as a positive control. As shown in Fig. 8Go, U937 cells up-regulated two shifted complexes: a dominant, more rapidly migrating complex (Fig. 8Go, band B, lane 2) and a less intense, more slowly migrating complex (Fig. 8Go, band A, lane 2). Binding was specific, as coincubation in the presence of a 50-fold molar excess of unlabeled probe blocked formation of the two retarded DNA:protein complexes (data not shown) (18). We have previously shown that band A was reactive with an anti-RXRß antibody (yet not with either anti-RXR{alpha} antibodies, anti-RXR{gamma} antibodies, or control preimmune sera), and that band B was reactive with both the anti-VDR and anti-RXRß antibodies (18).



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  		Figure 8. VDRE-reactive nuclear proteins in density-fractionated tonsil B lymphocytes. Density-fractionated tonsil B cells were cultured for the indicated time intervals in the presence of IL-4 (10 ng/ml), anti-{kappa}/{lambda} antibodies (10 µg/ml), and 1,25-(OH)2D3 (50 nM). Postincubation, nuclear extracts were prepared, and EMSA was performed using as the probe a 32P end-labeled oligonucleotide corresponding to a VDRE localized within the promoter of the rat 24-hydroxylase gene (P450cc24). Shifted bands representing DNA:protein complexes are indicated by arrows. Lane 1 contains free probe. Lane 2 contains nuclear extract derived from U937 cells cultured for 24 h in the presence of 1,25-(OH)2D3 (50 nM).

 
Nuclear extracts were prepared from density-fractionated tonsil B cells that were cultured in the presence of IL-4, anti-{kappa}/{lambda} antibodies, and 1,25-(OH)2D3 for varying time intervals. Extracts derived from high density B cells cultured for 24 h exhibited a weakly detectable single bandshift pattern corresponding to band B (Fig. 8Go, lane 3). This retarded protein profile intensified after 48 h of culture (Fig. 8Go, lane 6) and further intensified after 72 h of culture (Fig. 8Go, lane 9). These results agree with RT-PCR analysis of 24-hydroxylase message expression in high density B cells (Fig. 4Go), in that 24-hydroxylase specific message was not detected until approximately 48 h of culture. A different electrophoretic profile was detected from nuclear extracts of intermediate density B cells. After culture of these cells for 24 h, two shifted complexes were detected (Fig. 8Go, lane 4), corresponding in electrophoretic mobility to those complexes observed in extracts derived from U937 cells (Fig. 8Go, lane 2). The retarded protein complex diminished in intensity after the cells were cultured for 48 h (Fig. 8Go, lane 7) and was barely detectable after 72 h of culture (Fig. 8Go, lane 10). In contrast, nuclear extracts derived from low density tonsil B cells cultured for 24 h exhibited a single shifted band corresponding in electrophoretic mobility to complex B (Fig. 8Go, lane 5). This complex was significantly diminished in intensity after 48 h of culture. After 72 h of culture, low density tonsil B cells were predominantly nonviable, and nuclear extracts could not be obtained. Taken together, these observations indicate that in normal human B cells, the kinetics of appearance of nuclear proteins reactive with VDRE are significantly influenced by the specific stage of B cell activation.


   Discussion
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 Discussion
References
 
The described studies were initiated to address 1) whether distinct B cell subpopulations representative of different stages of cellular activation up-regulate VDR expression and 1,25-(OH)2D3-mediated genomic trans-activation differently, and 2) whether activation in vivo modulated those minimal requirements for VDR up-regulation and genomic trans-activation. Evidence has been presented demonstrating that the activation requirements rendering tonsillar B cells responsive to 1,25-(OH)2D3 and the kinetics of the response are widely different among B cell subpopulations. Low density, centroblastic tonsillar B cells were observed to constitutively express VDR message and protein. In contrast, VDR protein and message were not detectable in high density B cells, and as such these cells were apparently refractory to 1,25-(OH)2D3. These observations are in agreement with earlier studies of Provvedini et al. (16) and suggest that in normal B lymphocytes there is a direct correlation of stage of cellular activation and expression of VDR (1). In this context, the expression of the VDR may be considered an activation marker in B lymphocytes (1).

A prerequisite of 1,25-(OH)2D3-mediated genomic trans-activation is the ability of the cell to up-regulate nuclear proteins interactive with DNA response elements localized within the promoter regions of target genes. Interestingly, the constitutive VDR expression in low density centroblastic B cells was observed to be functional; exposure of these cells to 1,25-(OH)2D3, even in the absence of exogenous stimulation, rapidly up-regulated VDRE-reactive nuclear proteins and 24-hydroxylase mRNA. Message encoding 24-hydroxylase was detected in low density B cells as early as 6 h after culture initiation [i.e. exposure to 1,25-(OH)2D3]. As indicated by our previous studies (18, 26), transcriptional activation of the 24-hydroxylase gene in B cells is strictly dependent upon VDR:ligand interaction; in the absence of either component, 24-hydroxylase message or function is not detectable. These observations were confirmed in the current study, in that 24-hydroxylase message was detectable by RT-PCR only in those B cell cultures that concurrently expressed VDR in the presence of exogenously added 1,25-(OH)2D3.

In contrast, quiescent, high density B cells were found to not constitutively express VDR message or protein. As such, these B cell populations were apparently refractory to 1,25-(OH)2D3, in that up-regulation of VDRE-reactive nuclear proteins and 24-hydroxylase mRNA was not readily detectable. However, when provided with a prolonged activation phase of approximately 48 h, hormone-dependent transcriptional activation was detectable after concomitant ligation of the BCR and the IL-4 receptor, indicating that these B cell populations can also functionally respond to 1,25-(OH)2D3. Compared with the kinetic profile observed from low density B lymphocytes, this apparent temporal lag of hormone-mediated genomic transcription in high density B cells cannot be attributed solely to the rate of vitamin D receptor up-regulation, in that VDR mRNA was detectable after approximately 9 h of culture, and VDR protein was detectable within 12 h of culture (data not shown). Rather, experiments examining initiation of 24-hydroxylase genomic transcription in high density B cells preactivated in vitro indicated that this delay correlated with events occurring before cellular interaction with 1,25-(OH)2D3. We observed that when high density B cells were preactivated for 48 h in the absence of hormone and subsequently exposed to 1,25-(OH)2D3, 24-hydroxylase mRNA was detectable after only an additional 6-h culture period. From these results it is apparent that most, if not all, normal B cells are biologically responsive to 1,25-(OH)2D3 after their attainment of a specific level of cellular activation.

Previous experimentation has partially characterized the B cell-derived nuclear proteins reactive with the P450cc24 synthetic VDRE as including VDR and RXRß (18). It is puzzling that activated high density B cells exhibit minimal VDRE-reactive complex formation within a 24-h time frame, given that VDR (Fig. 6AGo, lane 2) and RXR-ß message (data not shown) are clearly expressed in these cells. Several scenarios may be envisioned providing a rationale for this apparent delay in VDRE-reactive complex formation and subsequent genomic trans-activation. Evidence has accrued indicating that the trans-activational capacity of steroid receptors may be further regulated by a direct interaction with components of the transcriptional preinitiation complex (58, 59, 60) and by recruitment of repressors and/or coactivators to the receptor-DNA complex (61, 62, 63, 64). Although a direct interaction between VDR and repressor moieties has not been demonstrated, such a corepressor network has been identified as a participatory component of retinoid-mediated trans-activation systems (65, 66). Conversely, to achieve full trans-activation capacity, DNA-bound VDR may require interaction with specific coactivators. Germane to this contention is the recent report demonstrating that the human steroid receptor coactivator-1 interacts with the VDR in a ligand-dependent manner (67). It is tempting to speculate that such a coactivator network may not be constitutively expressed in quiescent high density B cells given the relatively low transcriptional activity of these cells. However, this hypothesis remains to be experimentally determined, as the presence and functional significance of such a coactivator network has yet to be demonstrated in human lymphoid cells.

In summary, the data we have presented demonstrate that differentially activated populations of normal human B cells regulate vitamin D-mediated genomic trans-activation in a unique fashion, highly dependent upon the activation stage of the cell. As such, these natural targets of vitamin D should provide an excellent model system for both an elucidation of the requisite nuclear proteins establishing a functional complex and delineation of the physiological effects of this hormone on B cell biology.


   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. Back

Received June 17, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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J. W. Morgan, N. Kouttab, D. Ford, and A. L. Maizel
Vitamin D-Mediated Gene Regulation in Phenotypically Defined Human B Cell Subpopulations
Endocrinology, September 1, 2000; 141(9): 3225 - 3234.
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