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B Cell Receptor- and ₂-Adrenergic Receptor-Induced Regulation of B7-2 (CD86) Expression in B Cells¹

Adam P. Kohm^2,*, Afsaneh Mozaffarian^3,* and Virginia M. Sanders^4,*,

^* Departments of Cell Biology, Neurobiology, and Anatomy, and Microbiology and Immunology, Loyola University Stritch School of Medicine, Maywood, IL 60153

	Abstract

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The costimulatory molecule B7-2 (CD86) is expressed on the surface of APCs, including B cells. Considering the importance of B7-2 in regulating both T and B cell function, it may be important to understand the regulatory mechanisms governing its expression. We report in this study that stimulation of the B cell receptor (BCR) and/or a neurotransmitter receptor, the ₂-adrenergic receptor (₂AR), may cooperate to regulate B cell-associated B7-2 expression in vitro and in vivo. ₂AR stimulation further enhanced the level of BCR-induced B7-2 expression in B cells potentially via protein tyrosine kinase-, protein kinase A-, protein kinase C-, and mitogen-activated protein kinase-dependent mechanisms. Importantly, BCR and/or ₂AR stimulation, but not histone hyperacetylation and DNA hypomethylation alone, increased B cell-associated B7-2 expression by increasing B7-2 mRNA stability, NF-B nuclear binding, and NF-B-dependent gene transcription. Thus, this study provides additional insight into the signaling intermediates and molecular mechanisms by which stimulation of the BCR and ₂AR may regulate B cell-associated B7-2 expression.

	Introduction

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The growing B7 family of costimulatory molecules critically influences the T cell-dependent Ab response. The level of B7 expression on APCs regulates the level of T cell-dependent Ab production by the B cell, cytokine production by the T cell, and germinal center formation (reviewed in Refs. 1 and 2). For example, concurrent immunization of mice with T cell-dependent Ags and anti-B7-2 (CD86) Ab, to block the B7-2/CD28 interaction, inhibited both the level of Ag-specific Ab produced by the B cell and the level of germinal center formation in vivo (3). Also, costimulatory molecule expression may influence CD4⁺ T cell differentiation since B7-1 (CD80) or B7-2 expression on APCs may favor the formation of either a Th1 or Th2 cell-mediated Ab response, respectively (4). Thus, the level of B cell-associated B7-2 expression may exert a variety of influences on both T and B cell function.

In light of the influence of B7-2 expression on immune cell function, it may be important to understand the mechanisms regulating the expression of this and other costimulatory molecules. Presently, the exact regulatory mechanisms that govern B7-2 expression on a B cell are unknown. While B7-2 is expressed at very low levels on resting B cells (5), a number of early studies observed that B cell receptor (BCR)⁵- or LPS-induced B cell activation increased the level of B7-2 mRNA and protein expression in the B cell with peak expression at 12 and 24 h, respectively (5, 6, 7, 8, 9). Therefore, cellular activation appears to be one mechanism by which the level of B7-2 protein expression is up-regulated on the B cell surface.

Additional stimuli may also enhance the level of B7-2 expression on B cells, such as the stimulation of cytokine receptors (10, 11), complement receptors (12), MHC class II (13), and CD40 (14, 15, 16, 17). Interestingly, while the initial BCR-induced level of B7-2 expression is CD40 independent, extended expression for 48–72 h may be CD40 dependent (18). Thus, signals generated following CD40 stimulation may synergize with BCR-induced signals to maintain B7-2 expression on the B cell surface for longer periods of time. In light of the importance of B7-2 stimulation in regulating B cell and T cell function, it is not surprising that mechanisms exist to augment the level of B cell-associated B7-2 expression during the course of an immune response. However, in addition to immune cell-derived stimuli, signals originating outside the immune system may also regulate the level of B cell-associated B7-2 expression.

The sympathetic neurotransmitter norepinephrine (NE) is released from peripheral nerves during the course of a T cell-dependent Ab response in vivo to bind ₂-adrenergic receptors (₂ARs) expressed by B cells and Th1 cells, but not Th2 cells (reviewed in Refs. 19 and 20). Stimulation of the B cell-associated ₂AR in vivo has been reported to influence the level of Ab production (21, 22), cell proliferation (21), cell trafficking (23), and germinal center formation (21). In addition, ₂AR stimulation also increases the level of B7-2 expression on the B cell surface in vitro, but more importantly, concurrent stimulation of the BCR and ₂AR synergistically increases the level of B7-2 expression (24). Thus, ₂AR stimulation represents one mechanism by which signals from the nervous system may augment BCR-induced regulation of B cell function and B7-2 expression.

We report in this study two mechanisms by which BCR and ₂AR stimulation may regulate B7-2 expression on the B cell surface. While epigenetic DNA modifications alone did not influence the level of B7-2 mRNA expression, stimulation of the BCR and/or ₂AR increased both B7-2 mRNA stability and NF-B-mediated gene transcription. In support of these findings, pretreatment of cells with a NF-B inhibitor blocked BCR- and/or ₂AR-induced B7-2 protein and mRNA expression in B cells, suggesting that NF-B plays a critical role in regulating B7-2 expression in murine B cells.

	Materials and Methods

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Animals

Six-week-old female C.B-17/scid, BALB/c, C.C3H-Tlr4^Lps-d (Toll4^-/-), and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or Taconic Farms (Germantown, NY). All mice were provided autoclaved pellets and water ad libitum. Mice were permitted 2 wk to acclimate to their environment before being manipulated, and were used at 8 wk of age in all experiments. Mice were housed under a 12-h light/dark cycle in microisolater cages contained within a laminar flow system, thus maintaining a pathogen-free environment, and all experimental manipulations occurred 4 h into the light cycle.

Reagents

The following protein kinase and protein phosphatase (PP) inhibitors were purchased from Biomol (Plymouth Meeting, PA): H-89, genistein, okadaic acid, tautomycin, sangivamycin, and SB203580. The NF-B transcription factor inhibitor 1-pyrrolidinecarbodithioic acid (PDTC) was purchased from Calbiochem-Novabiochem (La Jolla, CA).

Cell transfer and immunization

Th2 cell clones and trinitrophenyl (TNP)-specific B cells were prepared and injected into each NE-intact or NE-depleted scid mouse (25) via i.v. injection, as previously described (21). Eight-week-old mice received 200 mg/kg injections (i.p.) of 6-hydroxydopamine hydrochloride (6-OHDA; Sigma-Aldrich, St. Louis, MO) in 0.5 M saline containing 1 x 10^-3 M ascorbate as an antioxidant. Mice received three injections of 6-OHDA on alternating days (days -6, -4, and -2 before cell reconstitution), while control mice received ascorbate-only injections on the same injection schedule. Two days following the last 6-OHDA injection, all animals received both D1.1 Th2 cells and TNP-specific B cells. Each cell type was prepared for adoptive transfer at 2 x 10⁶ cells in 50 µl PBS. T and B cell dilutions were prepared separately, and combined only at the time of injection. Cells were injected i.v. into the lateral tail vein in a total volume of 100 µl PBS. One week after cell reconstitution, mice received primary immunizations i.p. with 100 µg TNP/rabbit -globulin (RGG) or saline delivered in the adjuvant TiterMax Gold (CytRx, Norcross, GA).

Anti-CD43 preparation of peripheral, resting B cells

Spleen cells were isolated from either BALB/c or C.C3H-Tlr4^Lps-d mice. Ammonium chloride-treated spleen cells were incubated with rat anti-CD43 Ab (1 µg/million spleen cells; BD PharMingen, San Diego, CA) on ice for 60 min, followed by incubation with sheep anti-rat IgG Ab-coated magnetic beads (3 µl/million spleen cells; Dynal Biotech, Lake Success, NY). After incubation, resting B cells were isolated via negative selection using a magnet and used immediately in all experiments.

B cell culture conditions

A total of 5 x 10⁶ anti-CD43 isolated resting B cells was resuspended in 2 ml cRPMI containing either nothing, F(ab')₂ anti-IgM (0.1 µg/ml) to stimulate the B cell receptor, terbutaline (10^-5–10^-6 M; Sigma-Aldrich) to stimulate the ₂AR, and/or nadolol (10^-4–10^-5 M; Sigma-Aldrich), a AR antagonist. Cells were incubated for varying times at 37°C, 6% CO₂ before RNA extraction using the GlassMax Spin Column RNA Isolation System (Life Technologies, Frederick, MD). In experiments measuring the t_1/2 of B7-2 mRNA, the transcription inhibitor actinomycin-D (Sigma-Aldrich; 10 µg) was added to B cell cultures for either 0, 1.5, 3, or 6 h before mRNA isolation and real-time PCR analysis. The mRNA t_1/2 of each sample was calculated from the best-fit line of B7-2 expression at 0, 1.5, 3, and 6 h following transcription termination with actinomycin D by the following formula: mRNA t_1/2 = time T log(1/2)/ [log(expression_{time T}) - log(expression_initial)]. Immunofluorescence cell staining for B7-2 and B220 expression was performed as previously described (24).

Reverse-transcription and real-time PCR

mRNA was isolated from B cells using the GlassMax RNA isolation spin columns (Life Technologies). Before RT-PCR, all RNA samples were treated with 1 µg DNase I (Life Technologies) per 1 µg mRNA. Reverse transcription was performed on 2.5 µg DNase-treated RNA per sample using a common master mix (50 U murine leukemia virus, 2.5 µM random hexamer, 20 U RNase inhibitor, 1 mM dNTPs, 5 mM MgCl₂, 10 mM Tris-HCl, pH 8.3, and 50 mM KCl; PerkinElmer/Cetus, Norwalk, CT), and the samples were incubated at 25°C for 10 min, 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min. In addition, 2.5 µg RNA from each sample was not reverse transcribed to ensure the effectiveness of the DNase treatment. A total of 1.25 µl cDNA and gene-specific primers were added to 25 µl SYBR Green PCR Master Mix (PerkinElmer/Cetus). Amplification reaction was performed and analyzed on GeneAmp 5700 Sequence Detection System (PerkinElmer/Cetus). Primers were as follows: murine B7-2, 5'-CGAGCACTATTTGGGCACAGAG-3' and 5'-TTTCCAGAACACACACAACGGTC-3'; murine -actin, 5'-ATGGATGACGATATCGCT-3' and 5'-ATGAGGTAGTCTGTCAGGT-3'.

Histone hyperacetylation and DNA hypomethylation

For histone hyperacetylation studies, resting B cells were exposed to increasing concentrations of the histone deacetylase inhibitor butyrate (Sigma-Aldrich) for 9 h at 37°C and 6% CO₂ in cRPMI. For DNA hypomethylation studies, B cells were pretreated with the methyl transferase inhibitor 5-azacytidine (10 µM; Sigma-Aldrich) for 24 h at 37°C and 6% CO₂ in cRPMI before butyrate exposure, as described above. Concentrations of butyrate and 5-azacytidine used have previously been shown to induce significant histone hyperacetylation or DNA hypomethylation in a variety of cell types (26, 27). In some experiments, cells were first pretreated with cycloheximide (100 µg/ml).

Gel-shift assay

Nuclear extracts were generated from B cells using detergent-mediated cell lysis (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM DTT), and the nuclear pellet was resuspended in nuclear buffer (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT). The DNA-binding reaction was assembled as follows: 4 µl nuclease-free water, 2 µl gel-shift binding 5x buffer (Promega, Madison, WI), and 2 µl nuclear extract for a total volume of 9 µl. The reaction was incubated at room temperature for 10 min before the addition of -³²P-labeled consensus sequences. Consensus sequences of gel-shift probes were as follows: SP1, 5'-ATTCGATCGGGGCGGGGCGAGC-3' (27); AP1, 5'-CGCTTGATGAGTCAGCCGGAA-3' (28); NF-75 B, 5'-AFTTGAGGGGACTTTCCCAGGC-3' (29); and RNA polymerase II transcription factor (TFIID), 5'-GCAGAGCATATAAGGTGAGGTAGGA-3' (30). Following incubation at room temperature, shift products were separated by electrophoresis and visualized on Kodak Biomax MS film (Fisher, Pittsburgh, PA) using an intensifying screen-enabled film cassette (Fisher) by overnight exposure at -80°C.

Data analysis and statistics

Concentration-response data were first analyzed by a one-way ANOVA to determine whether an overall statistically significant change existed previous to using two-tailed unpaired Student’s t test. Statistically significant differences are reported when p < 0.01.

	Results

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The effect of BCR and ₂AR stimulation on the level of B cell-associated B7-2 expression in vivo and in vitro

In light of the effects of NE and ₂AR stimulation on costimulatory molecule expression in vitro, an in vivo model system was designed to test the hypothesis that NE altered the level of costimulatory molecule expression on B cells in vivo. To increase the frequency of responding Ag-specific Th and B cells in vivo, NE-intact or NE-depleted scid mice were reconstituted with RGG-specific Th2 cells and TNP-specific B cells and immunized with the cognate Ag TNP-RGG. We previously reported that immunization of reconstituted scid mice with a cognate Ag results in MHC-restricted, Ag-specific Ab production, spleen cell proliferation, and germinal center formation in vivo (21). In the current study, Ag-induced BCR stimulation increased the level of B cell-associated surface B7-2 expression in NE-depleted mice 48 h following immunization (Fig. 1A). More importantly, Ag administration to NE-intact mice further increased the level of B cell-associated B7-2 expression in comparison with NE-depleted animals, suggesting that the NE released from sympathetic nerves following Ag-induced immune cell activation (31) may enhance the level of B7-2 on the B cell surface.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. The effect of BCR and/or 2AR stimulation on B cell-associated B7-2 expression in vivo and in vitro. A, NE-depleted or NE-intact scid mice were reconstituted with a clone of RGG-specific Th2 cells and TNP-specific B cells. Forty-eight hours following immunization with TNP-RGG (100 µg), B7-2 expression was determined on B220+ splenic B cells by immunofluorescence double staining. Data are representative of four mice per treatment and two separate experiments. B and C, Resting B cells were exposed to either medium only, rabbit anti-mouse IgM (Ag, 0.1 µg/ml) to stimulate the BCR, the 2AR-selective agonist terbutaline (2AR, 10-6 M), and/or the AR-selective antagonist nadolol (Nad, 10-5 M). Some experiments were also performed using B cells isolated from 2AR-deficient mice (2AR-/-). Following 9 h of exposure at 37°C, mRNA was isolated from each group for analysis by real-time PCR. B7-2 mRNA levels were normalized to -actin mRNA and are presented as the average of four samples per treatment, the percentage of B7-2 mRNA expression in comparison with the basal level of B7-2 mRNA in B cells exposed to medium only, and are representative of three separate experiments. Significant differences from medium-only group (*) or BCR-only group (**) are marked when p < 0.01.

The role of protein kinase and PP activity in BCR- and ₂AR-induced B7-2 expression

To begin dissecting the mechanism by which stimulation of the BCR and/or ₂AR may regulate the level of B7-2 expression on B cells, we next determined whether stimulation of these receptors activated common intracellular pathways to regulate the level of B7-2 expression. For instance, activation of the nonreceptor Src family of protein tyrosine kinases (PTKs) following BCR and ₂AR stimulation has been studied extensively, but, to date, has not been determined to be important in the context of regulating the level of B7-2 expression. In addition, stimulation of either receptor has been reported to activate a number of other common intracellular signaling mediators, such as protein kinase C (PKC), protein kinase A (PKA), and the mitogen-activated protein kinase (MAPK) pathways. Therefore, to test the role of these intracellular signaling intermediates in mediating the effects of BCR and/or ₂AR stimulation on B cell-associated B7-2 expression, various inhibitors were used to block specific intermediates that are activated by stimulation of either receptor alone or both receptors at once.

Pretreatment of B cells with a PTK inhibitor (genistein), PKC inhibitor (sangavimycin), PKA inhibitor (H89), or MAPK inhibitor (SB203580) effectively blocked the BCR-induced increase in the level of B7-2 protein and mRNA expression in B cells (Fig. 2, A and B). In contrast, while pretreatment of B cells with either a PKC, PKA, or MAPK inhibitor blocked the ₂AR-induced increase in the level of B7-2 protein and mRNA (Fig. 2, C and D), pretreatment of cells with the PTK inhibitor did not effectively block ₂AR-induced B7-2 expression, suggesting that ₂AR-induced increases in B7-2 expression are not PTK dependent. Finally, pretreatment of B cells with any of the aforementioned inhibitors blocked the effects of stimulation of both receptors concurrently on B7-2 expression (Fig. 2, E and F). Taken together, these studies suggest that BCR- and/or ₂AR-induced B7-2 expression is mediated via multiple common intracellular signaling intermediates, including PTKs, PKC, PKA, and MAPKs, but that PTK-independent mechanisms may exist for ₂AR-induced B7-2 expression involving PKC, PKA, and/or MAPK activation.

View larger version (45K): [in this window] [in a new window]   FIGURE 2. The effect of protein kinase and PP inhibitors on BCR- and/or 2AR-induced B7-2 protein and mRNA expression in B cells. Resting B cells were pretreated for 30 min at 37°C with an inhibitor of the following primary targets: PTK (genistein, 40 µM), PKC (sangavimycin, 20 µM), PKA (H89, 5 µM), MAPK (SB203580, 20 µM), PP1 (tautomycin, 10 nM), PP2A (okadaic acid, 10 nM), or medium only. Following inhibitor pretreatment, resting B cells were stimulated by either their BCR and/or their 2AR before immunofluorescence staining or mRNA measurement by real-time PCR 24 or 9 h later, respectively. Data are presented as the average percentage of basal mean fluorescence intensity (MFI) or pecentage of basal mRNA expression and are representative of three separate experiments. Significant differences from basal levels of B7-2 expression are marked significant (*) when p < 0.01.

The effect of BCR and ₂AR stimulation on B7-2 mRNA stability

Since BCR and ₂AR stimulation may regulate B7-2 mRNA expression via several intracellular intermediates, we next investigated the mechanisms by which stimulation of these receptors may ultimately influence the level of B7-2 expression. There are two general mechanisms that regulate the level of mRNA expression in mammalian cells, which involve alterations in either the rate of gene transcription and/or the level of mRNA stability. For example, elevations in the level of intracellular cAMP accumulation have been shown to either increase or decrease the stability of various genes (32, 33). In addition, there are numerous other intracellular molecules that influence the level of mRNA stability, such as members of the MAPK family, including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase (34, 35, 36, 37, 38). In light of the fact that intracellular cAMP, JNK, and ERK activity are increased following either BCR (reviewed in Ref. 39) and/or ₂AR stimulation (40, 41, 42), the possibility existed that BCR and/or ₂AR stimulation augmented the level of B7-2 mRNA expression in B cells by increasing the level of B7-2 mRNA stability.

Stimulation of the BCR by anti-Ig Ab induced detectable B7-2 mRNA expression with a t_1/2 that varied from 1 to 8 h (Fig. 3). Similarly, ₂AR stimulation alone also induced detectable B7-2 mRNA expression with a t_1/2 that varied from 1 to 12 h. Interestingly, the levels of B7-2 mRNA expression and stability resulting from either BCR or ₂AR stimulation alone were similar at all time points, except between 12 and 18 h following exposure, when the level of B7-2 mRNA stability was significantly higher in ₂AR-stimulated B cells in comparison with BCR-stimulated cells. Finally, concurrent stimulation of both the BCR and ₂AR significantly enhanced the t_1/2 of B7-2 mRNA at all times, except 1–6 h following exposure, in comparison with stimulation of the BCR alone. Thus, these studies suggested that ₂AR stimulation up-regulates the level of BCR-induced B7-2 expression, in part, by increasing B7-2 mRNA stability in B cells.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. The effect of BCR and 2AR stimulation on B7-2 mRNA stability. B cells were stimulated by either their BCR and/or 2AR for either 1, 6, 12, or 18 h at 37°C and then incubated for an additional 1.5, 3, or 6 h in the presence of actinomycin-D. Data are presented as the average of four samples per treatment and are representative of three separate experiments. Significant differences from medium-only group (*) or BCR-only group (**) are marked when p < 0.01.

In a quiescent state, DNA is hypermethylated and tightly packed into tertiary and quaternary structures by its association with hypoacetylated histones. In many cases, the first requirement for gene transcription is increased locus accessibility and/or the removal of inhibitory methylation. Therefore, the current study tested the role of epigenetic DNA modifications in regulating the level of B7-2 mRNA expression.

If B7-2 promoter accessibility is a critical factor regulating the level of B7-2 mRNA expression in B cells, then histone hyperacetylation and the resulting dissociation of the DNA from histones should increase the level of B7-2 mRNA expression in resting B cells. To test this hypothesis, unstimulated B cells were exposed to increasing concentrations of the histone deacetylase inhibitor butyrate for 9 h before measuring the level of B7-2 mRNA expression by semiquantitative real-time PCR. Butyrate-mediated histone hyperacetylation did not increase the level of B7-2 mRNA expression in unstimulated B cells (Fig. 4A). However, since the possibility existed that the concentrations of butyrate used in these studies did not result in histone hyperacetylation in unstimulated B cells, ₂AR mRNA expression was measured in these same cells as a positive internal control. Histone hyperacetylation increased ₂AR mRNA expression in unstimulated B cells in a dose-dependent manner, thus ensuring that butyrate induced histone hyperacetylation in this study, but did not increase the level of B7-2 mRNA expression in these same cells.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. The effect of histone hyperacetylation and DNA hypomethylation on B7-2 mRNA expression in resting B cells. A, B cells were exposed to an increasing concentration of the histone deacetylase inhibitor butyrate for 9 h before mRNA isolated and analysis by real-time PCR. B, B cells were first preexposed to the methyltransferase inhibitor 5-azacytidine (10 µM) for 24 h before exposure to butyrate. mRNA levels were normalized to -actin mRNA and are presented as the percentage of B7-2 or 2AR mRNA expression in comparison with the basal level of mRNA in B cells exposed to medium only. Data are representative of three separate experiments. Significant differences from basal levels of B7-2 expression are marked significant (*) when p < 0.01.

The effect of ₂AR stimulation on the nuclear binding of transcription factors

Since epigenetic DNA modifications were unsuccessful in regulating B7-2 mRNA expression in B cells, the current study next investigated the possibility that BCR and/or ₂AR stimulation increased transcription factor synthesis and binding to the B7-2 promoter to alter gene expression. To test this hypothesis, resting B cells were pretreated with the protein synthesis inhibitor cycloheximide before BCR and/or ₂AR stimulation. Cycloheximide pretreatment did not block BCR- and ₂AR-induced increases in B cell-associated B7-2 mRNA (Fig. 5A), suggesting that de novo transcription factor synthesis was not necessary for receptor-induced alterations in B7-2 mRNA expression. Cycloheximide treatment also failed to block increases in B7-2 expression following stimulation of either the BCR or ₂AR alone (data not shown). However, these findings did not rule out transcription factor-dependent regulation of B7-2 mRNA expression, since various transcription factors, such as NF-B, are produced and stored in the cytoplasm before phosphorylation-dependent activation (43).

View larger version (23K): [in this window] [in a new window]   FIGURE 5. The role of transcription factors on BCR- and/or 2AR-induced B7-2 mRNA expression. A, B cells isolated from the spleens of Toll4-/- mice by anti-CD43 Ab negative selection were pretreated with increasing concentrations of the protein synthesis inhibitor cycloheximide (CHX, 100 µg) for 30 min. Following pretreatment, cells were stimulated by either their BCR and/or 2AR for 9 h before real-time PCR analysis. Data are presented as the average SYBR Green intensity of four samples per treatment, normalized in respect to -actin mRNA expression in the same cells, and are representative of two separate experiments. Significant differences from medium-only group (*) or BCR-only group (**) are marked when p < 0.01. B, The effect of BCR and 2AR stimulation on the level of nuclear binding of transcription factors. B cells were stimulated by either their BCR and/or 2AR for 4 h before nuclear extract generation and gel-shift analysis. Data are representative of three separate experiments.

Stimulation of the BCR slightly increased the level of AP-1 nuclear binding (Fig. 5B), which is in agreement with previous findings (48). In contrast, ₂AR stimulation significantly increased the level of AP-1 nuclear binding. Importantly, concurrent stimulation of both the BCR and the ₂AR did not increase the level of AP-1 nuclear binding significantly higher than levels induced by ₂AR stimulation alone. Next, the effect of BCR and/or ₂AR stimulation on the levels of NF-B, Sp1, and TFIID nuclear binding was measured (Fig. 5B). Similar to studies measuring AP-1 nuclear binding, stimulation of the BCR increased the level of NF-B nuclear binding, which is in agreement with previous findings (47, 49). However, stimulation of the ₂AR alone induced higher levels of NF-B nuclear binding in comparison with BCR stimulation alone. More importantly, concurrent stimulation of both receptors resulted in a significantly higher level of NF-B nuclear binding, and similar findings were observed with Sp1 and TFIID. Thus, these findings suggested that the stimulation of the BCR and the ₂AR concurrently more significantly increased the level of NF-B, Sp1, and TFIID nuclear binding.

The role of NF-B in mediating the effects of BCR and/or ₂AR stimulation on the level of B7-2 mRNA expression in B cells

Currently, the mouse B7-2 promoter has not been characterized. However, the human B7-2 promoter has been described (50), providing insight into the possible structure and regulatory mechanisms associated with the murine B7-2 promoter. For example, it has now been reported that the human B7-2 promoter contains two possible NF-B binding sites (50), one of which is critical for BCR-induced B7-2 promoter activity. Since the findings of the current study and others (45) suggested that ₂AR stimulation increased the level of NF-B activity, the possibility existed that ₂AR stimulation increased the level of BCR-dependent B7-2 mRNA expression by increasing the level of NF-B-dependent gene transcription.

If ₂AR stimulation increased NF-B-dependent B7-2 gene transcription, then inhibition of NF-B nuclear translocation and DNA binding should block this effect. To test this hypothesis, resting B cells were pretreated with an inhibitor of NF-B nuclear translocation, PDTC. As seen in Fig. 6, A and B, ₂AR stimulation augmented the level of BCR-induced B7-2 protein and mRNA expression in the absence of PDTC as in previous experiments. However, PDTC pretreatment completely blocked BCR- and/or ₂AR-induced increases in B7-2 protein and mRNA. In addition, PDTC pretreatment lowered B7-2 expression below the low basal level normally observed in B cells following stimulation of either receptor alone or both receptors, thus suggesting that PDTC not only blocked the mechanisms responsible for the up-regulation of B7-2, but in addition, blocked a mechanism responsible for the low basal levels of B7-2 expressed in unstimulated B cells. Therefore, these studies suggest that NF-B activation, nuclear translocation, and DNA binding are essential for BCR- and/or ₂AR-induced enhancements in B cell-associated B7-2 expression.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. The effect of an NF-B inhibitor on the BCR- and 2AR-induced B7-2 mRNA expression in B cells. Before stimulation, resting B cells were exposed to either 100 µM (A and B) or 50 nM (C) of the NF-B inhibitor PDTC for 30 min. B cells were then stimulated by either their BCR and/or 2AR for 24 or 9 h at 37°C before flow cytometry (A) or real-time PCR (B and C). B7-2 mRNA levels were normalized to -actin mRNA and are presented as the percentage of B7-2 mRNA expression in comparison with the basal level of B7-2 mRNA in B cells exposed to medium only. Data are representative of three separate experiments. Significant differences from medium-only group (*) or BCR-only group (**) are marked when p < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The importance of B7-2 expression in regulating T cell and B cell function is demonstrated by studies disrupting the B7-2/CD28 interaction (reviewed in Refs. 51 and 52). In addition, recent data suggest that B7-2 cross-linking may initiate signals directly into the B cell to influence the level of BCR-dependent Ab production (24, 53). In light of this, it is important to understand the mechanisms that regulate the level of B7-2 expression on the B cell. In the current study, we report two molecular mechanisms by which stimulation of the BCR and/or ₂AR may cooperate to up-regulate the level of B7-2 surface protein and mRNA expression in B cells, i.e., increased mRNA stability and NF-B-dependent gene transcription. Importantly, the concurrent stimulation of both receptors results in an additive enhancement in the level of B7-2 expression on the B cell, and this cooperative effort between the BCR and ₂AR may be one mechanism by which signals originating from the immune and nervous system synergize to regulate immune cell function.

B7-2 expression may be essential to the generation of normal levels of Ab production and germinal center formation in vivo (reviewed in Ref. 54). For example, disruption of the B7-2/CD28 interaction that occurs between a T cell and a B cell responding to a T cell-dependent Ag with exogenous anti-B7-2 Ab inhibited both the level of Ab production and the level of germinal center formation in vivo (3). Findings from the current study suggest that NE stimulates the ₂AR to increase the level of BCR-induced B7-2 expression on B cells in vivo, and we have previously reported that stimulation of the ₂AR increases the level of BCR-induced B7-2 expression on B cell in vitro (24). Finally, NE depletion in vivo decreases the level of Ag-specific IgG1 production, cell proliferation, and germinal center formation (21). Taken together, these findings suggest that NE may stimulate the B cell-associated ₂AR to regulate B7-2 expression, B cell function, and the Th cell-dependent Ab response in vivo.

Currently, little is known about the cellular and molecular mechanisms regulating the level of B7-2 expression on the B cell. However, additional insight into the mechanisms controlling B7-2 expression may be gained by examining the role of various intracellular signaling intermediates in receptor-induced alterations of B7-2 expression. For example, increases in intracellular cAMP and the activity of Bruton’s tyrosine kinase (Btk) may contribute to receptor-induced increases in B7-2 expression. Using CTLA-4Ig to measure the expression of both B7-1 and B7-2, the exogenous addition of dibutyryl cAMP increased the level of B7-1 and B7-2 expression on the surface of a B lymphoma in a concentration-dependent manner (55). More specifically, later studies reported that exogenous dibutyryl cAMP (300 µM) increased the level of CD40-induced B7-2 expression on B cells (14), suggesting that in addition to cellular activation, elevations in intracellular cAMP may enhance B7-2 expression on the B cell surface. Similarly, the CD40-mediated up-regulation of B7-2 expression was decreased in Btk-deficient mice (14). Thus, intracellular elevations in cAMP and Btk activity may be involved in CD40-induced regulation of B7-2 expression.

In the current study, inhibition of PKC or MAPK activity blocked BCR- and/or ₂AR-induced B7-2 expression on B cells. In light of the fact that stimulation of both receptors alone may lead to the initiation of the Btk/PKC/MAPK signaling cascade, findings from these studies suggest that MAPK activation may be required for BCR- or ₂AR-induced B7-2 expression, and that concurrent stimulation of both receptors may lead to higher levels of MAPK activation that eventually results in augmented B7-2 expression on the B cell. However, inhibition of PKA activity also blocked BCR- and/or ₂AR-induced B7-2 expression, suggesting that additional signaling intermediates in addition to the Btk/PKC/MAPK pathway may be critical to regulate the level of B7-2 expression in B cells. Importantly, while the current findings suggest a role for multiple protein kinases and PP in regulating B7-2 expression in B cells, it is possible that the inhibitors used in this study exerted nonspecific effects, and future studies are necessary to further define the specific role of each of these signaling mediators in regulating B cell B7-2 expression.

As previously discussed, a critical factor regulating the level of gene expression is locus accessibility. In the current study, B cell-associated B7-2 mRNA expression was not solely mediated by gene locus accessibility, since chemical-induced histone hyperacetylation by the deacetylase-inhibitor butyrate and/or DNA hypomethylation by the methyltransferase-inhibitor 5-azacytidine failed to increase the low level of basal mRNA expression in resting B cells. These findings suggest additional components of the transcriptional apparatus, e.g., required transcription factors, which are not expressed in the nucleus of resting B cells, are necessary to transcribe the B7-2 gene. In contrast, histone hyperacetylation alone increased the level of B7-2 expression in human myeloid leukemia cells (56), suggesting that histone acetylation may exert species-, cell type-, and/or cellular activation-dependent effects of B7-2 expression. Interestingly, ₂AR-induced B7-2 expression occurred in the absence of protein synthesis. In light of these findings, the transcription factors regulating B7-2 mRNA expression may be present, but sequestered in an inactivated state either in the nucleus or in the cytoplasm of resting B cells.

Since NF-B binding to consensus sequences in the human B7-2 promoter may be critical to regulating B7-2 gene transcription, as suggested by reporter assays (50), then NF-B nuclear translocation may be essential to B7-2 gene transcription in murine B cells following either BCR and/or ₂AR stimulation. NF-B was originally characterized as a heterodimeric complex of the p50 and p65 subunits (57), and NF-B is normally sequestered in the cytoplasm by formation of a NF-B/I-B complex. NF-B translocates to the nucleus following the PKC-mediated phosphorylation and disassociation of the I-B suppressor subunit in a protein synthesis-independent manner (43). The current studies strongly suggest a critical role for NF-B-dependent gene transcription in mediating the effects of BCR- and ₂AR-induced B7-2 mRNA expression in B cells, since the stimulation of the BCR and/or ₂AR increased B7-2 mRNA expression in B cells in a protein synthesis-independent manner, increased the nuclear binding of NF-B, and in a NF-B inhibitor-sensitive manner. However, until the murine B7-2 promoter is further cloned and characterized, the exact role of NF-B in regulating B7-2 expression cannot be determined.

Importantly, BCR and ₂AR stimulation increased B7-2 mRNA stability in the current study, but the mechanisms regulating B7-2 mRNA stability in this model system are unknown. mRNA stability is often regulated by trans-acting factors binding cis-acting elements within the 3' or 5' untranslated region (UTR) of mRNA. Trans-acting factors include subunits of RNA polymerase and transcription factors, whereas cis-acting elements are regulatory DNA sequences contained in the UTR region of mRNA containing consensus sequences for transcription factor binding. The activity at cis-acting elements can influence mRNA stability. For example, the cis-acting A + U-rich element found in lymphokines, such as GM-CSF, targets the mRNA for degradation in the cytoplasm (58). Importantly, increased mRNA stability of other genes, such as Nramp1, has been reported to be PKC dependent and may be the direct result of PKC-mediated phosphorylation and activation of trans-acting factors or the activation of other signaling cascades such as MAPK (35). In our model system, the up-regulation of B7-2 mRNA expression was both PKC and NF-B dependent. Thus, in addition to increasing the rate of B7-2 gene transcription, ₂AR-induced PKC activity may also induce additional trans-acting factors to stabilize B7-2 mRNA. In support of this, others have reported JNK- and p38-mediated mRNA stabilization of IL-2, IL-3, IL-6, and IL-8 mRNA (34, 36, 37) and ERK-mediated stabilization of macrophage-inflammatory protein-2 mRNA (38). Since ₂AR stimulation may also induce JNK activation (42), ₂AR-induced B7-2 mRNA stability may be mediated via PKC- and/or JNK-dependent mechanisms. Since the 5'-UTR and 3'-UTR of the murine B7-2 gene are still unknown, current studies cannot determine whether JNK binding to the B7-2 gene regulates mRNA stability.

The current findings suggest that stimulation of the BCR or ₂AR alone may up-regulate the level of B7-2 expression on a B cell via increased NF-B-mediated gene transcription and increased B7-2 mRNA stability, thus providing additional insight into the mechanisms by which B7-2 expression is regulated on the B cell surface. Additionally, concurrent stimulation of both receptors further enhanced the level of B cell-associated B7-2 expression in vitro and in vivo. Importantly, past studies report that NE depletion and the lack of B cell-associated ₂AR stimulation inhibit the Th2 cell-dependent Ab response in vivo (21) by decreasing the level of Ag-specific Ab production, cell proliferation, and germinal center formation in vivo to similar levels induced by blockade of the B7-2/CD28 interaction (3). In light of these findings, regulation of B7-2 expression may be one mechanism by which signals from the nervous and immune systems cooperate to regulate the level of the normal immune response in vivo.

	Acknowledgments

 
We thank Dr. Kimberly Foreman for use of the GeneAmp 5700 Sequence Detection System.

	Footnotes

 
¹ This work was supported in part by research funds from the National Institutes of Health AI37326 and AI47420 (to V.M.S.).

² Current address: Department of Microbiology and Immunology, Northwestern University School of Medicine, 303 East Superior Avenue, Chicago, IL 60611.

³ Current address: Corixa Corp., 733 99th Avenue SE, Bellevue, WA 98004.

⁴ Address correspondence and reprint requests to Dr. Virginia M. Sanders at the current address: Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, 2194 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210. E-mail address: sanders.302{at}osu.edu

⁵ Abbreviations used in this paper: BCR, B cell receptor; ₂AR, ₂-adrenergic receptor; Btk, Bruton’s tyrosine kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NE, norepinephrine; 6-OHDA, 6-hydroxydopamine hydrochloride; PDTC, 1-pyrrolidinecarbodithioic acid; PKA, protein kinase A; PKC, protein kinase C; PP, phosphatase; PTK, protein tyrosine kinase; RGG, rabbit -globulin; TFIID, RNA polymerase II transcription factor; TNP, trinitrophenyl; UTR, untranslated region.

Received for publication September 27, 2001. Accepted for publication April 19, 2002.

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