Expression in B Cells Receptor-Induced Regulation of B7-2

The Journal of Immunology

␤ 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. Sanders4*†

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. The Journal of Immunology, 2002, 168: 6314–6322.

he growing B7 family of costimulatory molecules criti- tion increased the level of B7-2 mRNA and protein expression in cally influences the T cell-dependent Ab response. The the B cell with peak expression at 12 and 24 h, respectively (5Ð9). T level of B7 expression on APCs regulates the level of T Therefore, cellular activation appears to be one mechanism by cell-dependent Ab production by the B cell, cytokine production which the level of B7-2 protein expression is up-regulated on the by the T cell, and germinal center formation (reviewed in Refs. 1 B cell surface. and 2). For example, concurrent immunization of mice with T Additional stimuli may also enhance the level of B7-2 expres- cell-dependent Ags and anti-B7-2 (CD86) Ab, to block the B7-2/ sion on B cells, such as the stimulation of cytokine receptors (10, CD28 interaction, inhibited both the level of Ag-specific Ab pro- 11), complement receptors (12), MHC class II (13), and CD40 duced by the B cell and the level of germinal center formation in (14Ð17). Interestingly, while the initial BCR-induced level of B7-2 vivo (3). Also, costimulatory molecule expression may influence expression is CD40 independent, extended expression for 48Ð72 h ϩ CD4 T cell differentiation since B7-1 (CD80) or B7-2 expression may be CD40 dependent (18). Thus, signals generated following on APCs may favor the formation of either a Th1 or Th2 cell- CD40 stimulation may synergize with BCR-induced signals to mediated Ab response, respectively (4). Thus, the level of B cell- maintain B7-2 expression on the B cell surface for longer periods associated B7-2 expression may exert a variety of influences on of time. In light of the importance of B7-2 stimulation in regulating both T and B cell function. B cell and T cell function, it is not surprising that mechanisms exist In light of the influence of B7-2 expression on immune cell to augment the level of B cell-associated B7-2 expression during function, it may be important to understand the mechanisms reg- the course of an immune response. However, in addition to im- ulating the expression of this and other costimulatory molecules. mune cell-derived stimuli, signals originating outside the immune Presently, the exact regulatory mechanisms that govern B7-2 ex- system may also regulate the level of B cell-associated B7-2 pression on a B cell are unknown. While B7-2 is expressed at very expression. low levels on resting B cells (5), a number of early studies ob- The sympathetic neurotransmitter norepinephrine (NE) is re- 5 served that B cell receptor (BCR) - or LPS-induced B cell activa- leased from peripheral nerves during the course of a T cell-depen- ␤ ␤ dent Ab response in vivo to bind 2-adrenergic receptors ( 2ARs) *Departments of Cell Biology, Neurobiology, and Anatomy, and †Microbiology and expressed by B cells and Th1 cells, but not Th2 cells (reviewed in ␤ Immunology, Loyola University Stritch School of Medicine, Maywood, IL 60153 Refs. 19 and 20). Stimulation of the B cell-associated 2AR in Received for publication September 27, 2001. Accepted for publication April vivo has been reported to influence the level of Ab production (21, 19, 2002. 22), cell proliferation (21), cell trafficking (23), and germinal cen- The costs of publication of this article were defrayed in part by the payment of page ␤ ter formation (21). In addition, 2AR stimulation also increases the charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. level of B7-2 expression on the B cell surface in vitro, but more ␤ 1 This work was supported in part by research funds from the National Institutes of importantly, concurrent stimulation of the BCR and 2AR syner- ␤ Health AI37326 and AI47420 (to V.M.S.). gistically increases the level of B7-2 expression (24). Thus, 2AR 2 Current address: Department of Microbiology and Immunology, Northwestern Uni- stimulation represents one mechanism by which signals from the versity School of Medicine, 303 East Superior Avenue, Chicago, IL 60611. nervous system may augment BCR-induced regulation of B cell 3 Current address: Corixa Corp., 733 99th Avenue SE, Bellevue, WA 98004. function and B7-2 expression. 4 Address correspondence and reprint requests to Dr. Virginia M. Sanders at the current address: Department of Molecular Virology, Immunology and Medical Ge- netics, Ohio State University, 2194 Graves Hall, 333 West 10th Avenue, Columbus, OH 43210. E-mail address: [email protected] epinephrine; 6-OHDA, 6-hydroxydopamine hydrochloride; PDTC, 1-pyrrolidinecar- 5 ␤ ␤ Abbreviations used in this paper: BCR, B cell receptor; 2AR, 2-adrenergic re- bodithioic acid; PKA, protein kinase A; PKC, protein kinase C; PP, phosphatase; ceptor; Btk, Bruton’s tyrosine kinase; ERK, extracellular signal-regulated kinase; PTK, protein tyrosine kinase; RGG, rabbit ␥-globulin; TFIID, RNA polymerase II JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NE, nor- transcription factor; TNP, trinitrophenyl; UTR, untranslated region.

We report in this study two mechanisms by which BCR and Reverse-transcription and real-time PCR ␤ 2AR stimulation may regulate B7-2 expression on the B cell sur- mRNA was isolated from B cells using the GlassMax RNA isolation spin face. While epigenetic DNA modifications alone did not influence columns (Life Technologies). Before RT-PCR, all RNA samples were the level of B7-2 mRNA expression, stimulation of the BCR treated with 1 ␮g DNase I (Life Technologies) per 1 ␮g mRNA. Reverse and/or ␤ AR increased both B7-2 mRNA stability and NF-␬B- transcription was performed on 2.5 ␮g DNase-treated RNA per sample 2 ␮ mediated gene transcription. In support of these findings, pretreat- using a common master mix (50 U murine leukemia virus, 2.5 M random hexamer, 20 U RNase inhibitor, 1 mM dNTPs, 5 mM MgCl ,10mM ␬ ␤ 2 ment of cells with a NF- B inhibitor blocked BCR- and/or 2AR- Tris-HCl, pH 8.3, and 50 mM KCl; PerkinElmer/Cetus, Norwalk, CT), and induced B7-2 protein and mRNA expression in B cells, suggesting the samples were incubated at 25¡C for 10 min, 42¡C for 15 min, 99¡C for that NF-␬B plays a critical role in regulating B7-2 expression in 5 min, and 5¡C for 5 min. In addition, 2.5 ␮g RNA from each sample was murine B cells. not reverse transcribed to ensure the effectiveness of the DNase treatment. A total of 1.25 ␮l cDNA and gene-specific primers were added to 25 ␮l SYBR Green PCR Master Mix (PerkinElmer/Cetus). Amplification reac- Materials and Methods tion was performed and analyzed on GeneAmp 5700 Sequence Detection Animals System (PerkinElmer/Cetus). Primers were as follows: murine B7-2, 5Ј- Ј Ј Lps-d Ϫ/Ϫ CGAGCACTATTTGGGCACAGAG-3 and 5 -TTTCCAGAACACACA Six-week-old female C.B-17/scid, BALB/c, C.C3H-Tlr4 (Toll4 ), CAACGGTC-3Ј; murine ␤-actin, 5Ј-ATGGATGACGATATCGCT-3Ј and and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Har- 5Ј-ATGAGGTAGTCTGTCAGGT-3Ј. bor, ME) or Taconic Farms (Germantown, NY). All mice were provided autoclaved pellets and water ad libitum. Mice were permitted 2 wk to Histone hyperacetylation and DNA hypomethylation 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 For histone hyperacetylation studies, resting B cells were exposed to in- cycle in microisolater cages contained within a laminar flow system, thus creasing concentrations of the histone deacetylase inhibitor butyrate (Sig- maintaining a pathogen-free environment, and all experimental manipula- ma-Aldrich) for9hat37¡C and 6% CO2 in cRPMI. For DNA hypom- tions occurred ϳ4 h into the light cycle. ethylation studies, B cells were pretreated with the methyl transferase inhibitor 5-azacytidine (10 ␮M; Sigma-Aldrich) for 24 h at 37¡C and 6%

Reagents CO2 in cRPMI before butyrate exposure, as described above. Concentra- tions of butyrate and 5-azacytidine used have previously been shown to The following protein kinase and protein phosphatase (PP) inhibitors were induce significant histone hyperacetylation or DNA hypomethylation in a purchased from Biomol (Plymouth Meeting, PA): H-89, genistein, okadaic ␬ variety of cell types (26, 27). In some experiments, cells were first pre- acid, tautomycin, sangivamycin, and SB203580. The NF- B transcription treated with cycloheximide (100 ␮g/ml). factor inhibitor 1-pyrrolidinecarbodithioic acid (PDTC) was purchased from Calbiochem-Novabiochem (La Jolla, CA). Gel-shift assay Cell transfer and immunization 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 Th2 cell clones and trinitrophenyl (TNP)-specific B cells were prepared 1 mM DTT), and the nuclear pellet was resuspended in nuclear buffer (20 and injected into each NE-intact or NE-depleted scid mouse (25) via i.v. mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT). injection, as previously described (21). Eight-week-old mice received 200 The DNA-binding reaction was assembled as follows: 4 ␮l nuclease-free mg/kg injections (i.p.) of 6-hydroxydopamine hydrochloride (6-OHDA; ␮ ϫ ␮ ϫ Ϫ3 water, 2 l gel-shift binding 5 buffer (Promega, Madison, WI), and 2 l Sigma-Aldrich, St. Louis, MO) in 0.5 M saline containing 1 10 M nuclear extract for a total volume of 9 ␮l. The reaction was incubated at ascorbate as an antioxidant. Mice received three injections of 6-OHDA on ␥ 32 Ϫ Ϫ Ϫ room temperature for 10 min before the addition of - P-labeled consen- alternating days (days 6, 4, and 2 before cell reconstitution), while sus sequences. Consensus sequences of gel-shift probes were as follows: control mice received ascorbate-only injections on the same injection SP1, 5Ј-ATTCGATCGGGGCGGGGCGAGC-3Ј (27); AP1, 5Ј-CGCTT schedule. Two days following the last 6-OHDA injection, all animals re- GATGAGTCAGCCGGAA-3Ј (28); NF-75 B, 5Ј-AFTTGAGGGGACTT ceived both D1.1 Th2 cells and TNP-specific B cells. Each cell type was Ј ϫ 6 ␮ TCCCAGGC-3 (29); and RNA polymerase II transcription factor (TFIID), prepared for adoptive transfer at 2 10 cells in 50 l PBS. T and B cell 5Ј-GCAGAGCATATAAGGTGAGGTAGGA-3Ј (30). Following incuba- dilutions were prepared separately, and combined only at the time of in- tion at room temperature, shift products were separated by electrophoresis jection. Cells were injected i.v. into the lateral tail vein in a total volume and visualized on Kodak Biomax MS film (Fisher, Pittsburgh, PA) using of 100 ␮l PBS. One week after cell reconstitution, mice received primary ␮ ␥ an intensifying screen-enabled film cassette (Fisher) by overnight exposure immunizations i.p. with 100 g TNP/rabbit -globulin (RGG) or saline at Ϫ80¡C. delivered in the adjuvant TiterMax Gold (CytRx, Norcross, GA). Data analysis and statistics Anti-CD43 preparation of peripheral, resting B cells Concentration-response data were first analyzed by a one-way ANOVA to Spleen cells were isolated from either BALB/c or C.C3H-Tlr4Lps-d mice. determine whether an overall statistically significant change existed pre- Ammonium chloride-treated spleen cells were incubated with rat anti- vious to using two-tailed unpaired Student’s t test. Statistically significant CD43 Ab (1 ␮g/million spleen cells; BD PharMingen, San Diego, CA) on differences are reported when p Ͻ 0.01. ice for 60 min, followed by incubation with sheep anti-rat IgG Ab-coated magnetic beads (3 ␮l/million spleen cells; Dynal Biotech, Lake Success, NY). After incubation, resting B cells were isolated via negative selection Results ␤ using a magnet and used immediately in all experiments. The effect of BCR and 2AR stimulation on the level of B cell-associated B7-2 expression in vivo and in vitro B cell culture conditions In light of the effects of NE and ␤ AR stimulation on costimula- A total of 5 ϫ 106 anti-CD43 isolated resting B cells was resuspended in 2 Ј ␮ tory molecule expression in vitro, an in vivo model system was 2 ml cRPMI containing either nothing, F(ab )2 anti-IgM (0.1 g/ml) to stimulate the B cell receptor, terbutaline (10Ϫ5Ð10Ϫ6 M; Sigma-Aldrich) to designed to test the hypothesis that NE altered the level of co- ␤ Ϫ4 Ϫ5 stimulate the 2AR, and/or nadolol (10 Ð10 M; Sigma-Aldrich), a stimulatory molecule expression on B cells in vivo. To increase the ␤ AR antagonist. Cells were incubated for varying times at 37¡C, 6% CO2 frequency of responding Ag-specific Th and B cells in vivo, NE- before RNA extraction using the GlassMax Spin Column RNA Isolation intact or NE-depleted scid mice were reconstituted with RGG- System (Life Technologies, Frederick, MD). In experiments measuring the specific Th2 cells and TNP-specific B cells and immunized with t1/2 of B7-2 mRNA, the transcription inhibitor actinomycin-D (Sigma-Ald- rich; 10 ␮g) was added to B cell cultures for either 0, 1.5, 3, or 6 h before the cognate Ag TNP-RGG. We previously reported that immuni- mRNA isolation and real-time PCR analysis. The mRNA t1/2 of each sam- zation of reconstituted scid mice with a cognate Ag results in ple was calculated from the best-fit line of B7-2 expression at 0, 1.5, 3, and MHC-restricted, Ag-specific Ab production, spleen cell prolifera- 6 h following transcription termination with actinomycin D by the following formula: mRNA t ϭ time T log(1/2)/ [log(expression ) Ϫ log(ex- tion, and germinal center formation in vivo (21). In the current 1/2 time T study, Ag-induced BCR stimulation increased the level of B pressioninitial)]. Immunofluorescence cell staining for B7-2 and B220 expression was performed as previously described (24). cell-associated surface B7-2 expression in NE-depleted mice 6316 REGULATION OF B CELL-ASSOCIATED B7-2 (CD86) EXPRESSION

48 h following immunization (Fig. 1A). More importantly, Ag cell, it was important to determine whether B7-2 expression was administration to NE-intact mice further increased the level of regulated at the translational or transcriptional level. Since previ- B cell-associated B7-2 expression in comparison with NE- ous studies suggested that transcriptional mechanisms may regu- depleted animals, suggesting that the NE released from sympa- late B7-2 expression (9), the current study determined the effect of ␤ thetic nerves following Ag-induced immune cell activation (31) BCR and/or 2AR stimulation on the level of B cell-associated may enhance the level of B7-2 on the B cell surface. B7-2 protein and mRNA expression in vitro. Cells were stimulated ␤ ␤ ␤ To determine the mechanism by which 2AR stimulation by NE via their BCR (anti-Ig Ab), 2AR ( 2AR-selective agonist ter- may augment the level of BCR-induced B7-2 expression on the B butaline), or both stimuli concurrently in the presence or absence of the ␤AR-antagonist nadolol. Stimulation of either the BCR or ␤ the 2AR alone increased the level of B7-2 surface expression (Fig. 1B), but more importantly, concurrent stimulation of both B cell-associated receptors further increased the level of B7-2 expression on the B cell. Demonstrating the receptor specificity of terbutaline, either the addition of the ␤AR antagonist or the use of ␤ 2AR-negative B cells blocked any effect of terbutaline on B7-2 expression on the B cell. Similarly, stimulation of the BCR or the ␤ 2AR alone increased the level of B7-2 mRNA expression (Fig. ␤ 1C), and concurrent stimulation of the BCR and the 2AR further up-regulated the level of B7-2 mRNA expression in the B cell. Taken together, these findings suggest that stimulation of the BCR ␤ and/or the 2AR cooperates to regulate the level of B cell-associated B7-2 expression both in vivo and in vitro. 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, FIGURE 1. The effect of BCR and/or ␤ AR stimulation on B cell-as- 2 A and B). In contrast, while pretreatment of B cells with either a sociated B7-2 expression in vivo and in vitro. A, NE-depleted or NE-intact PKC, PKA, or MAPK inhibitor blocked the ␤ AR-induced in- scid mice were reconstituted with a clone of RGG-specific Th2 cells and 2 TNP-specific B cells. Forty-eight hours following immunization with TNP- crease in the level of B7-2 protein and mRNA (Fig. 2, C and D), RGG (100 ␮g), B7-2 expression was determined on B220ϩ splenic B cells pretreatment of cells with the PTK inhibitor did not effectively ␤ ␤ by immunofluorescence double staining. Data are representative of four block 2AR-induced B7-2 expression, suggesting that 2AR-in- mice per treatment and two separate experiments. B and C, Resting B cells duced increases in B7-2 expression are not PTK dependent. Fi- were exposed to either medium only, rabbit anti-mouse IgM (Ag, 0.1 ␮g/ nally, pretreatment of B cells with any of the aforementioned in- ␤ ␤ ml) to stimulate the BCR, the 2AR-selective agonist terbutaline ( 2AR, hibitors blocked the effects of stimulation of both receptors Ϫ Ϫ 10 6 M), and/or the ␤AR-selective antagonist nadolol (Nad, 10 5 M). concurrently on B7-2 expression (Fig. 2, E and F). Taken together, ␤ Some experiments were also performed using B cells isolated from 2AR- ␤ these studies suggest that BCR- and/or 2AR-induced B7-2 ex- deficient mice (␤ ARϪ/Ϫ). Following9hofexposure at 37¡C, mRNA was 2 pression is mediated via multiple common intracellular signaling isolated from each group for analysis by real-time PCR. B7-2 mRNA levels ␤ intermediates, including PTKs, PKC, PKA, and MAPKs, but that were normalized to -actin mRNA and are presented as the average of four ␤ samples per treatment, the percentage of B7-2 mRNA expression in com- PTK-independent mechanisms may exist for 2AR-induced B7-2 parison with the basal level of B7-2 mRNA in B cells exposed to medium expression involving PKC, PKA, and/or MAPK activation. ␤ only, and are representative of three separate experiments. Significant dif- Protein PP may also regulate the level of BCR- and 2AR- are marked induced signaling. Thus, if PP activity negatively influences the (ءء) or BCR-only group (ء) ferences from medium-only group when p Ͻ 0.01. level of B7-2 expression by repressing receptor signaling, then a The Journal of Immunology 6317

FIGURE 2. The effect of protein kinase and PP in- ␤ hibitors 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 ␮M), PKC (sangavimycin, 20 ␮M), PKA (H89, 5 ␮M), MAPK (SB203580, 20 ␮M), 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 measure- ment 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 Ͻ 0.01 (ء)

PP inhibitor should allow for the overexpression of B7-2. The two such as members of the MAPK family, including c-Jun N-terminal serine/threonine-sensitive PP inhibitors used in this study were kinase (JNK), extracellular signal-regulated kinase (ERK), and tautomycin, which inhibits primarily PP1, and okadaic acid, which p38 kinase (34Ð38). In light of the fact that intracellular cAMP, inhibits primarily PP2A. Inhibition of PP1 augmented the level of JNK, and ERK activity are increased following either BCR (re- ␤ BCR-induced B7-2 expression, whereas inhibition of PP2A viewed in Ref. 39) and/or 2AR stimulation (40Ð42), the possi- ␤ blocked the effects of BCR stimulation on B7-2 protein and mRNA bility existed that BCR and/or 2AR stimulation augmented the expression (Fig. 2, A and B). In contrast, inhibition of PP1 blocked level of B7-2 mRNA expression in B cells by increasing the level ␤ 2AR-induced B7-2 expression, whereas inhibition of PP2A in- of B7-2 mRNA stability. ␤ creased the level of 2AR-induced B7-2 protein and mRNA ex- Stimulation of the BCR by anti-Ig Ab induced detectable B7-2 pression (Fig. 2, C and D). Therefore, the effects of the PP1 and mRNA expression with a t1/2 that varied from 1 to 8 h (Fig. 3). ␤ PP2A inhibitors appear to be receptor specific. These findings sug- Similarly, 2AR stimulation alone also induced detectable B7-2 ␤ gest that while stimulation of either the BCR or 2AR produces mRNA expression with a t1/2 that varied from 1 to 12 h. Interest- similar elevations in B7-2 expression, distinct intermediates may ingly, the levels of B7-2 mRNA expression and stability resulting ␤ critically influence these signal transduction pathways via either from either BCR or 2AR stimulation alone were similar at all direct or indirect mechanisms. Surprisingly, pretreatment of B time points, except between 12 and 18 h following exposure, when cells with either PP inhibitor blocked the effects of concurrent the level of B7-2 mRNA stability was significantly higher in ␤ stimulation of both receptors on the level of B7-2 expression (Fig. 2AR-stimulated B cells in comparison with BCR-stimulated ␤ 2, E and F). Taken together, these data suggest that a number of cells. Finally, concurrent stimulation of both the BCR and 2AR protein kinases and PP may mediate the effects of BCR and/or significantly enhanced the t1/2 of B7-2 mRNA at all times, except ␤ 2AR stimulation on B7-2 expression in B cells. 1Ð6 h following exposure, in comparison with stimulation of the BCR alone. Thus, these studies suggested that ␤ AR stimulation ␤ 2 The effect of BCR and 2AR stimulation on B7-2 mRNA stability up-regulates the level of BCR-induced B7-2 expression, in part, by ␤ Since BCR and 2AR stimulation may regulate B7-2 mRNA ex- increasing B7-2 mRNA stability in B cells. pression via several intracellular intermediates, we next investigated the mechanisms by which stimulation of these receptors may The role of histone acetylation and DNA methylation in ultimately influence the level of B7-2 expression. There are two regulating B7-2 mRNA expression in resting B cells general mechanisms that regulate the level of mRNA expression in In a quiescent state, DNA is hypermethylated and tightly packed mammalian cells, which involve alterations in either the rate of into tertiary and quaternary structures by its association with hy- gene transcription and/or the level of mRNA stability. For exam- poacetylated histones. In many cases, the first requirement for gene ple, elevations in the level of intracellular cAMP accumulation transcription is increased locus accessibility and/or the removal of have been shown to either increase or decrease the stability of inhibitory methylation. Therefore, the current study tested the role various genes (32, 33). In addition, there are numerous other in- of epigenetic DNA modifications in regulating the level of B7-2 tracellular molecules that influence the level of mRNA stability, mRNA expression. 6318 REGULATION OF B CELL-ASSOCIATED B7-2 (CD86) EXPRESSION

␤ 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, or6hinthepresence of actinomycin-D. Data are presented as the average of four samples per treatment and are representative of three separate ex- -or BCR (ء) periments. Signiﬁcant differences from medium-only group .are marked when p Ͻ 0.01 (ءء) only group

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 rest- FIGURE 4. The effect of histone hyperacetylation and DNA hypom- ing B cells. To test this hypothesis, unstimulated B cells were ethylation on B7-2 mRNA expression in resting B cells. A, B cells were exposed to an increasing concentration of the histone deacetylase inhibitor exposed to increasing concentrations of the histone deacetylase butyrate for 9 h before mRNA isolated and analysis by real-time PCR. B, inhibitor butyrate for 9 h before measuring the level of B7-2 B cells were first preexposed to the methyltransferase inhibitor 5-azacyti- mRNA expression by semiquantitative real-time PCR. Butyrate- dine (10 ␮M) for 24 h before exposure to butyrate. mRNA levels were mediated histone hyperacetylation did not increase the level of normalized to ␤-actin mRNA and are presented as the percentage of B7-2 ␤ B7-2 mRNA expression in unstimulated B cells (Fig. 4A). How- or 2AR mRNA expression in comparison with the basal level of mRNA ever, since the possibility existed that the concentrations of in B cells exposed to medium only. Data are representative of three sep- butyrate used in these studies did not result in histone hyperacety- arate experiments. Significant differences from basal levels of B7-2 ex- .when p Ͻ 0.01 (ء) pression are marked significant ␤ lation 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 unstimu- ␤ lated B cells in a dose-dependent manner, thus ensuring that bu- investigated the possibility that BCR and/or 2AR stimulation in- tyrate induced histone hyperacetylation in this study, but did not creased transcription factor synthesis and binding to the B7-2 pro- increase the level of B7-2 mRNA expression in these same cells. moter to alter gene expression. To test this hypothesis, resting B Next, it was possible that histone hyperacetylation failed to in- cells were pretreated with the protein synthesis inhibitor cyclohex- ␤ crease the level of B7-2 mRNA expression in unstimulated B cells imide before BCR and/or 2AR stimulation. Cycloheximide pre- ␤ due to the presence of inhibitory methylation in the B7-2 promoter. treatment did not block BCR- and 2AR-induced increases in B Therefore, if inhibitory DNA methylation blocked histone hyper- cell-associated B7-2 mRNA (Fig. 5A), suggesting that de novo acetylation from increasing the level of B7-2 mRNA expression, transcription factor synthesis was not necessary for receptor-in- then the removal of endogenous DNA methylation before butyrate duced alterations in B7-2 mRNA expression. Cycloheximide treat- treatment should allow for butyrate-induced B7-2 mRNA expres- ment also failed to block increases in B7-2 expression following ␤ sion. To test this possibility, resting B cells were exposed to the stimulation of either the BCR or 2AR alone (data not shown). methyltransferase inhibitor 5-azacytidine for 24 h before the ad- However, these findings did not rule out transcription factor-de- dition of butyrate. Butyrate exposure failed to increase the level of pendent regulation of B7-2 mRNA expression, since various tran- B7-2 gene transcription from hypomethylation DNA (Fig. 4B). scription factors, such as NF-␬B, are produced and stored in the ␤ Importantly, butyrate further increased the level of 2AR mRNA cytoplasm before phosphorylation-dependent activation (43). ␤ expression in cells pretreated with 5-azacytidine in comparison If stimulation of the BCR and/or 2AR increased the level of with cells containing normal levels of DNA methylation. Thus, transcription factor-dependent B7-2 gene transcription, then recep- these studies suggested that epigenetic DNA modifications were tor stimulation should increase the nuclear binding of transcription not sufficient to increase the level of B7-2 mRNA expression in factors relevant to B7-2 gene regulation. However, since the mu- resting B cells. rine B7-2 promoter has not been described, the current studies measured the nuclear binding of various transcription factors ␤ The effect of 2AR stimulation on the nuclear binding of known to be activated following the stimulation of the BCR and transcription factors ␤ ␬ 2AR, i.e., AP-1, NF- B, Sp1, and TFIID (44Ð48). Since epigenetic DNA modifications were unsuccessful in regu- Stimulation of the BCR slightly increased the level of AP-1 lating B7-2 mRNA expression in B cells, the current study next nuclear binding (Fig. 5B), which is in agreement with previous The Journal of Immunology 6319

␤ Ϫ/Ϫ 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 ␮g) ␤ 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 are marked when p Ͻ 0.01. B, The effect of BCR (ءء) or BCR-only group (ء) of two separate experiments. Signiﬁcant differences from medium-only group ␤ ␤ 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.

␤ findings (48). In contrast, 2AR stimulation significantly increased PDTC pretreatment lowered B7-2 expression below the low basal the level of AP-1 nuclear binding. Importantly, concurrent stimu- level normally observed in B cells following stimulation of either ␤ lation of both the BCR and the 2AR did not increase the level of receptor alone or both receptors, thus suggesting that PDTC not AP-1 nuclear binding significantly higher than levels induced by only blocked the mechanisms responsible for the up-regulation of ␤ ␤ 2AR stimulation alone. Next, the effect of BCR and/or 2AR B7-2, but in addition, blocked a mechanism responsible for the low stimulation on the levels of NF-␬B, Sp1, and TFIID nuclear bind- basal levels of B7-2 expressed in unstimulated B cells. Therefore, ing was measured (Fig. 5B). Similar to studies measuring AP-1 these studies suggest that NF-␬B activation, nuclear translocation, ␤ nuclear binding, stimulation of the BCR increased the level of and DNA binding are essential for BCR- and/or 2AR-induced NF-␬B nuclear binding, which is in agreement with previous find- enhancements in B cell-associated B7-2 expression. ␤ ings (47, 49). However, stimulation of the 2AR alone induced In light of the current gel-shift data showing that concurrent ␬ ␤ higher levels of NF- B nuclear binding in comparison with BCR stimulation of the BCR and the 2AR induces higher levels of stimulation alone. More importantly, concurrent stimulation of NF-␬B nuclear binding in comparison with either receptor alone, both receptors resulted in a significantly higher level of NF-␬B the effect of a lower concentration of PDTC was determined on ␤ nuclear binding, and similar findings were observed with Sp1 and BCR- and 2AR-induced B7-2 expression. If stimulation of both TFIID. Thus, these findings suggested that the stimulation of the receptors induces higher levels of nuclear NF-␬B nuclear binding, ␤ BCR and the 2AR concurrently more significantly increased the in comparison with stimulation of either receptor alone, then pre- level of NF-␬B, Sp1, and TFIID nuclear binding. treatment of B cells with a lower concentration of PDTC should block the effects of low-level NF-␬B induction on B7-2 mRNA ␬ ␤ The role of NF- B in mediating the effects of BCR and/or 2AR expression, but not higher levels of NF-␬B activity induced by stimulation on the level of B7-2 mRNA expression in B cells stimulation of both receptors. To test this possibility, B cells were Currently, the mouse B7-2 promoter has not been characterized. pretreated with a lower concentration of PDTC. At this lower con- However, the human B7-2 promoter has been described (50), pro- centration, PDTC completely blocked the increase in B7-2 mRNA ␤ viding insight into the possible structure and regulatory mecha- expression induced by stimulation of either the BCR or the 2AR nisms associated with the murine B7-2 promoter. For example, it alone (Fig. 6C). However, this concentration of PDTC did not has now been reported that the human B7-2 promoter contains two efficiently block the increase in B7-2 mRNA expression following possible NF-␬B binding sites (50), one of which is critical for concurrent stimulation of both receptors. Thus, these data suggest ␤ BCR-induced B7-2 promoter activity. Since the findings of the that concurrent stimulation of the 2AR and the BCR induces ␤ ␬ current study and others (45) suggested that 2AR stimulation in- higher levels of NF- B nuclear binding, in comparison with the creased the level of NF-␬B activity, the possibility existed that level of NF-␬B activity resulting from the stimulation of either ␤ ␬ 2AR stimulation increased the level of BCR-dependent B7-2 receptor alone, and that NF- B nuclear binding critically regulates mRNA expression by increasing the level of NF-␬B-dependent the level of B7-2 expression in B cells. gene transcription. ␤ ␬ If 2AR stimulation increased NF- B-dependent B7-2 gene Discussion transcription, then inhibition of NF-␬B nuclear translocation and The importance of B7-2 expression in regulating T cell and B cell DNA binding should block this effect. To test this hypothesis, rest- function is demonstrated by studies disrupting the B7-2/CD28 in- ing B cells were pretreated with an inhibitor of NF-␬B nuclear teraction (reviewed in Refs. 51 and 52). In addition, recent data ␤ translocation, PDTC. As seen in Fig. 6, A and B, 2AR stimulation suggest that B7-2 cross-linking may initiate signals directly into augmented the level of BCR-induced B7-2 protein and mRNA the B cell to influence the level of BCR-dependent Ab production expression in the absence of PDTC as in previous experiments. (24, 53). In light of this, it is important to understand the mecha- However, PDTC pretreatment completely blocked BCR- and/or nisms that regulate the level of B7-2 expression on the B cell. In ␤ 2AR-induced increases in B7-2 protein and mRNA. In addition, the current study, we report two molecular mechanisms by which 6320 REGULATION OF B CELL-ASSOCIATED B7-2 (CD86) EXPRESSION

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. How- ever, 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 con- tribute 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 ␮M) 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. Sim- ilarly, 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 FIGURE 6. The effect of an NF-␬B inhibitor on the BCR- and ␤ AR- 2 of MAPK activation that eventually results in augmented B7-2 induced B7-2 mRNA expression in B cells. Before stimulation, resting B cells were exposed to either 100 ␮M(A and B)or50nM(C) of the NF-␬B expression on the B cell. However, inhibition of PKA activity also ␤ inhibitor PDTC for 30 min. B cells were then stimulated by either their blocked BCR- and/or 2AR-induced B7-2 expression, suggesting ␤ that additional signaling intermediates in addition to the Btk/PKC/ BCR and/or 2ARfor24or9hat37¡C before flow cytometry (A)or real-time PCR (B and C). B7-2 mRNA levels were normalized to ␤-actin MAPK pathway may be critical to regulate the level of B7-2 ex- mRNA and are presented as the percentage of B7-2 mRNA expression in pression in B cells. Importantly, while the current findings suggest comparison with the basal level of B7-2 mRNA in B cells exposed to a role for multiple protein kinases and PP in regulating B7-2 ex- medium only. Data are representative of three separate experiments. Sig- pression in B cells, it is possible that the inhibitors used in this ءء ء nificant differences from medium-only group ( ) or BCR-only group ( ) study exerted nonspecific effects, and future studies are necessary Ͻ are marked when p 0.01. 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 ␤ stimulation of the BCR and/or 2AR may cooperate to up-regulate gene expression is locus accessibility. In the current study, B cell- the level of B7-2 surface protein and mRNA expression in B cells, associated B7-2 mRNA expression was not solely mediated by i.e., increased mRNA stability and NF-␬B-dependent gene tran- gene locus accessibility, since chemical-induced histone hyper- scription. Importantly, the concurrent stimulation of both receptors acetylation by the deacetylase-inhibitor butyrate and/or DNA hy- results in an additive enhancement in the level of B7-2 expression pomethylation by the methyltransferase-inhibitor 5-azacytidine on the B cell, and this cooperative effort between the BCR and failed to increase the low level of basal mRNA expression in rest- ␤ 2AR may be one mechanism by which signals originating from ing B cells. These findings suggest additional components of the the immune and nervous system synergize to regulate immune cell transcriptional apparatus, e.g., required transcription factors, which function. are not expressed in the nucleus of resting B cells, are necessary to B7-2 expression may be essential to the generation of normal transcribe the B7-2 gene. In contrast, histone hyperacetylation levels of Ab production and germinal center formation in vivo alone increased the level of B7-2 expression in human myeloid (reviewed in Ref. 54). For example, disruption of the B7-2/CD28 leukemia cells (56), suggesting that histone acetylation may exert interaction that occurs between a T cell and a B cell responding to species-, cell type-, and/or cellular activation-dependent effects of The Journal of Immunology 6321

␤ B7-2 expression. Interestingly, 2AR-induced B7-2 expression oc- hibit the Th2 cell-dependent Ab response in vivo (21) by decreas- curred in the absence of protein synthesis. In light of these find- ing the level of Ag-specific Ab production, cell proliferation, and ings, the transcription factors regulating B7-2 mRNA expression germinal center formation in vivo to similar levels induced by may be present, but sequestered in an inactivated state either in the blockade of the B7-2/CD28 interaction (3). In light of these find- nucleus or in the cytoplasm of resting B cells. ings, regulation of B7-2 expression may be one mechanism by Since NF-␬B binding to consensus sequences in the human which signals from the nervous and immune systems cooperate to B7-2 promoter may be critical to regulating B7-2 gene transcrip- regulate the level of the normal immune response in vivo. tion, as suggested by reporter assays (50), then NF-␬B nuclear translocation may be essential to B7-2 gene transcription in murine Acknowledgments ␤ ␬ B cells following either BCR and/or 2AR stimulation. NF- B We thank Dr. Kimberly Foreman for use of the GeneAmp 5700 Sequence was originally characterized as a heterodimeric complex of the p50 Detection System. and p65 subunits (57), and NF-␬B is normally sequestered in the cytoplasm by formation of a NF-␬B/I-␬B complex. NF-␬B trans- References locates to the nucleus following the PKC-mediated phosphoryla- 1. June, C. H., J. A. Bluestone, L. M. Nadler, and C. B. Thompson. 1994. The B7 ␬ and CD28 receptor families. Immunol. Today 15:321. tion and disassociation of the I- B suppressor subunit in a protein 2. McAdam, A. J., A. N. Schweitzer, and A. H. Sharpe. 1998. The role of B7 synthesis-independent manner (43). The current studies strongly co-stimulation in activation and differentiation of CD4ϩ and CD8ϩ T cells. Im- suggest a critical role for NF-␬B-dependent gene transcription in munol. Rev. 165:231. ␤ 3. Han, S., K. Hathcock, B. Zheng, T. B. Kepler, R. Hodes, and G. Kelsoe. 1995. mediating the effects of BCR- and 2AR-induced B7-2 mRNA Cellular interactions in germinal centers: roles of CD40 ligand and B7-2 in es- expression in B cells, since the stimulation of the BCR and/or tablished germinal centers. J. Immunol. 155:556. ␤ AR increased B7-2 mRNA expression in B cells in a protein 4. Lenschow, D. J., S. C. Ho, H. Sattar, L. Rhee, G. Gray, N. Nabavi, K. C. Herold, 2 and J. A. Bluestone. 1995. Differential effects of anti-B7-1 and anti-B7-2 mono- synthesis-independent manner, increased the nuclear binding of clonal antibody treatment on the development of diabetes in the nonobese dia- NF-␬B, and in a NF-␬B inhibitor-sensitive manner. However, until betic mouse. J. Exp. Med. 181:1145. 5. Lenschow, D. J., G. H. T. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, the murine B7-2 promoter is further cloned and characterized, the G. S. Gray, J. Miller, and J. A. Bluestone. 1993. Expression and functional sig- exact role of NF-␬B in regulating B7-2 expression cannot be nificance of an additional ligand for CTLA-4. Proc. Natl. Acad. Sci. USA 90: determined. 11054. ␤ 6. Freedman, A. S., G. Freeman, J. C. Horowitz, J. Daley, and L. M. Nadler. 1987. Importantly, BCR and 2AR stimulation increased B7-2 mRNA B7, a B cell-restricted antigen that identifies preactivated B cells. J. Immunol. stability in the current study, but the mechanisms regulating B7-2 139:3260. mRNA stability in this model system are unknown. mRNA stabil- 7. Freeman, G. J., J. G. Gribben, V. A. Boussiotis, J. W. Ng, V. A. J. Restivo, L. A. Lombard, G. S. Gray, and L. M. Nadler. 1993. Cloning of B7-2: a CTLA-4 ity is often regulated by trans-acting factors binding cis-acting counter-receptor that costimulates human T cell proliferation. Science 262:909. elements within the 3Ј or 5Ј untranslated region (UTR) of mRNA. 8. Boussiotis, V. A., G. J. Freeman, J. G. Gribben, and L. M. Nadler. 1993. Acti- vated human B lymphocytes express three CTLA-4 binding counter-receptors Trans-acting factors include subunits of RNA polymerase and which costimulate T cell activation. Proc. Natl. Acad. Sci. USA 90:11059. transcription factors, whereas cis-acting elements are regulatory 9. Lenschow, D. J., A. I. Sperling, M. P. Cooke, G. Freeman, L. Rhee, D. C. Decker, DNA sequences contained in the UTR region of mRNA containing G. Gray, L. M. Nadler, C. C. Goodnow, and J. A. Bluestone. 1994. Differential up-regulation of B7-1 and B7-2 costimulatory molecules after Ig receptor en- consensus sequences for transcription factor binding. The activity gagement by antigen. J. Immunol. 153:1990. at cis-acting elements can influence mRNA stability. For example, 10. Stack, R. M., D. J. Lenschow, G. S. Gray, J. A. Bluestone, and F. W. Fitch. 1994. the cis-acting A ϩ U-rich element found in lymphokines, such as IL-4 treatment of small splenic B cells induces costimulatory molecules B7-1 and B7-2. J. Immunol. 152:5723. GM-CSF, targets the mRNA for degradation in the cytoplasm (58). 11. Valle, A., J. P. Aubry, I. Durand, and J. Banchereau. 1991. IL-4 and IL-2 up- Importantly, increased mRNA stability of other genes, such as regulate the expression of antigen B7, the B cell counterstructure to T cell CD28: an amplification mechanism for T-B cell interactions. Int. Immunol. 3:229. Nramp1, has been reported to be PKC dependent and may be the 12. Kozono, Y., R. Abe, H. Kozono, R. G. Kelly, T. Azuma, and V. M. Holers. 1998. direct result of PKC-mediated phosphorylation and activation of Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on trans-acting factors or the activation of other signaling cascades murine splenic B cells. J. Immunol. 160:1565. 13. Koulova, L., E. A. Clark, G. Shu, and B. Dupont. 1991. The CD28 ligand B7/ such as MAPK (35). In our model system, the up-regulation of BB1 provides costimulatory signal for alloactivation of CD4ϩ T cells. J. Exp. B7-2 mRNA expression was both PKC and NF-␬B dependent. Med. 173:759. Thus, in addition to increasing the rate of B7-2 gene transcription, 14. Goldstein, M. D., M. A. Debenedette, D. Hollenbaugh, and T. H. Watts. 1996. ␤ Induction of costimulatory molecules B7-1 and B7-2 in murine B cells: the 2AR-induced PKC activity may also induce additional trans-act- CBA/N mouse reveals a role for Bruton’s tyrosine kinase in CD40-mediated B7 ing factors to stabilize B7-2 mRNA. In support of this, others have induction. Mol. Immunol. 33:541. 15. Lalmanach-Girard, A. C., T. C. Chiles, D. C. Parker, and T. L. Rothstein. 1993. reported JNK- and p38-mediated mRNA stabilization of IL-2, T cell-dependent induction of NF-␬B in B cells. J. Exp. Med. 177:1215. IL-3, IL-6, and IL-8 mRNA (34, 36, 37) and ERK-mediated sta- 16. Berberich, I., G. L. Shu, and E. A. Clark. 1994. Cross-linking CD40 on B cells bilization of macrophage-inflammatory protein-2 mRNA (38). rapidly activates nuclear factor-␬B. J. Immunol. 153:4357. ␤ 17. Bajpai, U. D., K. Zhang, M. Teutsch, R. Sen, and H. H. Wortis. 2000. Bruton’s Since 2AR stimulation may also induce JNK activation (42), tyrosine kinase links the B cell receptor to nuclear factor ␬B activation. J. Exp. ␤ 2AR-induced B7-2 mRNA stability may be mediated via PKC- Med. 191:1735. and/or JNK-dependent mechanisms. Since the 5Ј-UTR and 18. Evans, D. E., M. W. Munks, J. M. Purkerson, and D. C. Parker. 2000. Resting B lymphocytes as APC for naive T lymphocytes: dependence on CD40 ligand/ 3Ј-UTR of the murine B7-2 gene are still unknown, current studies CD40. J. Immunol. 164:688. cannot determine whether JNK binding to the B7-2 gene regulates 19. Kohm, A. P., and V. M. Sanders. 2000. Norepinephrine: a messenger from the brain to the immune system. Immunol. Today 21:539. mRNA stability. 20. Sanders, V. M., D. J. Kasprowicz, A. P. Kohm, and M. A. Swanson. 2001. The current findings suggest that stimulation of the BCR or Neurotransmitter Receptors on Lymphocytes and Other Lymphoid Cells, Vol. 1. ␤ Academic Press, San Diego. 2AR alone may up-regulate the level of B7-2 expression on a B ␬ 21. Kohm, A. P., and V. M. Sanders. 1999. Suppression of antigen-specific Th2 cell via increased NF- B-mediated gene transcription and in- cell-dependent IgM and IgG1 production following norepinephrine depletion in creased B7-2 mRNA stability, thus providing additional insight vivo. J. Immunol. 162:5299. into the mechanisms by which B7-2 expression is regulated on the 22. Kruszewska, B., S. Y. Felten, and J. A. Moynihan. 1995. Alterations in cytokine and antibody production following chemical sympathectomy in two strains of B cell surface. Additionally, concurrent stimulation of both recep- mice. J. Immunol. 155:4613. tors further enhanced the level of B cell-associated B7-2 expres- 23. Carlson, S. L., S. Fox, and K. M. Abell. 1997. Catecholamine modulation of lymphocyte homing to lymphoid tissues. Brain Behav. Immun. 11:307. sion in vitro and in vivo. Importantly, past studies report that NE 24. Kasprowicz, D. J., A. P. Kohm, M. T. Berton, A. J. Chruscinski, A. H. Sharpe, ␤ depletion and the lack of B cell-associated 2AR stimulation in- and V. M. Sanders. 2000. Stimulation of the B cell receptor, CD86 (B7-2) and the 6322 REGULATION OF B CELL-ASSOCIATED B7-2 (CD86) EXPRESSION

␤ ␤ 2-adrenergic receptor intrinsically modulates the level of IgG1 produced per B cologic stimulation. I. Lymphocyte responsiveness to adrenergic agents. cell. J. Immunol. 165:680. J. Allergy Clin. Immunol. 62:349. 25. Martino, A., J. H. Holmes, J. D. Lord, J. J. Moon, and B. H. Nelson. 2001. Stat5 41. Bishopric, N. H., H. J. Cohen, and R. J. Lefkowitz. 1980. ␤-adrenergic receptors and Sp1 regulate transcription of the cyclin D2 gene in response to IL-2. J. Im- in lymphocyte subpopulations. J. Allergy Clin. Immunol. 65:29. munol. 166:1723. 42. Jo, H., K. Sipos, Y. M. Go, R. Law, J. Rong, and J. M. McDonald. 1997. Dif- 26. Buscail, L., P. Robberecht, P. DeNeef, D. N. Bui, R. Hooghe, and J. Christophe. ferential effect of shear stress on extracellular signal-regulated kinase and N- ␤ ␤ ␥ 1990. Divergent regulation of 2-adrenoceptors and adenylate cyclase in the terminal Jun kinase in endothelial cells: Gi2- and G / -dependent signaling path- CycϪ mouse T lymphoma cell line TL2-9. Immunobiology 181:51. ways. J. Biol. Chem. 272:1395. 27. Kassis, S., M. Sullivan, and P. H. Fishman. 1988. Modulation of the ␤-adrenergic 43. Shirakawa, F., and S. B. Mizel. 1989. In vitro activation and nuclear translocation receptor-coupled adenylate cyclase by chemical inducers of differentiation: ef- of NF-␬B catalyzed by cyclic AMP-dependent protein kinase and protein kinase fects on ␤ receptors and the inhibitory regulatory protein Gi. J. Recept. Res. C. Mol. Cell. Biol. 9:2424. 8:627. 44. Wagner, J. P., F. J. Seidler, F. H. Schachat, and T. A. Slotkin. 1994. ␤ Adrenergic 28. Gonzalez, E., C. Punzon, M. Gonzalez, and M. Fresno. 2001. HIV-1 Tat inhibits control of c-fos protooncogene expression in developing rat brain regions. IL-2 gene transcription through qualitative and quantitative alterations of the J. Pharmacol. Exp. Ther. 269:1292. cooperative Rel/AP1 complex bound to the CD28RE/AP1 composite element of 45. Takemoto, Y., M. Yoshiyama, K. Takeuchi, T. Omura, R. Komatsu, Y. Izumi, ␬ the IL-2 promoter. J. Immunol. 166:4560. S. Kim, and J. Yoshikawa. 1999. Increased JNK, AP-1 and NF- B DNA binding 29. Smith, C., E. Andreakos, J. B. Crawley, F. M. Brennan, M. Feldmann, and activities in isoproterenol-induced cardiac remodeling. J. Mol. Cell. Cardiol. 31: B. M. Foxwell. 2001. NF-␬B-inducing kinase is dispensable for activation of 2017. NF-␬Bininflammatory settings but essential for lymphotoxin ␤ receptor activa- 46. Saadane, N., L. Alpert, and L. E. Chalifour. 2000. Altered molecular response to tion of NF-␬B in primary human fibroblasts. J. Immunol. 167:5895. adrenoreceptor-induced cardiac hypertrophy in Egr-1-deficient mice. 30. Carter, A. B., K. L. Knudtson, M. M. Monick, and G. W. Hunninghake. 1999. Am. J. Physiol. Heart Circ. Physiol. 278:H796. The p38 mitogen-activated protein kinase is required for NF-␬B-dependent gene 47. Healy, J. I., R. E. Dolmetsch, L. A. Timmerman, J. G. Cyster, M. L. Thomas, expression: the role of TATA-binding protein (TBP). J. Biol. Chem. 274:30858. G. R. Crabtree, R. S. Lewis, and C. C. Goodnow. 1997. Different nuclear signals 31. Kohm, A. P., Y. Tang, V. M. Sanders, and S. B. Jones. 2000. Activation of are activated by the B cell receptor during positive versus negative signaling. ϩ Immunity 6:419. antigen-specific CD4 Th2 cells in vivo increases norepinephrine release in the spleen and bone marrow. J. Immunol. 165:725. 48. Huo, L., and T. L. Rothstein. 1995. Receptor-specific induction of individual AP-1 components in B lymphocytes. J. Immunol. 154:3300. 32. Sinn, P. L., and C. D. Sigmund. 1999. Human renin mRNA stability is increased 49. Rooney, J. W., P. M. Dubois, and C. H. Sibley. 1991. Cross-linking of surface in response to cAMP in Calu-6 cells. Hypertension 33:900. IgM activates NF-␬B in B lymphocyte. Eur. J. Immunol. 21:2993. 33. Oddis, C. V., R. L. Simmons, B. G. Hattler, and M. S. Finkel. 1995. cAMP 50. Li, J., Z. Liu, S. Jiang, R. Cortesini, S. Lederman, and N. Suciu-Foca. 1999. T enhances inducible nitric oxide synthase mRNA stability in cardiac myocytes. suppressor lymphocytes inhibit NF-␬B-mediated transcription of CD86 gene in Am. J. Physiol. 269:H2044. APC. J. Immunol. 163:6386. 34. Winzen, R., M. Kracht, B. Ritter, A. Wilhelm, C. Y. Chen, A. B. Shyu, 51. Lu, P., Y. L. Wang, and P. S. Linsley. 1997. Regulation of self-tolerance by M. Muller, M. Gaestel, K. Resch, and H. Holtmann. 1999. The p38 MAP kinase CD80/CD86 interactions. Curr. Opin. Immunol. 9:858. pathway signals for cytokine-induced mRNA stabilization via MAP kinase-acti- 52. Judge, T. A., A. Tang, and L. A. Turka. 1996. Immunosuppression through block- vated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. ade of CD28:B7-mediated costimulatory signals. Immunol. Res. 15:38. 18:4969. 53. Jeannin, P., Y. Delneste, S. Lecoanet-Henchoz, J.-F. Gauchat, J. Ellis, and J.-Y. 35. Lafuse, W. P., G. R. Alvarez, and B. S. Zwilling. 2000. Regulation of nramp1 Bonnefoy. 1997. CD86 (B7-2) on human B cells: a functional role in proliferation mRNA stability by oxidants and protein kinase C in RAW264.7 macrophages and selective differentiation into IgE- and IgG4-producing cells. J. Biol. Chem. expressing Nramp1Gly169. Biochem. J. 351:687. 272:15613. 36. Ming, X. F., M. Kaiser, and C. Moroni. 1998. c-jun N-terminal kinase is involved 54. Greenfield, E. A., K. A. Nguyen, and V. K. Kuchroo. 1998. CD28/B7 costimu- in AUUUA-mediated interleukin-3 mRNA turnover in mast cells. EMBO J. 17: lation: a review. Crit. Rev. Immunol. 18:389. 6039. 55. Watts, T. H., N. Alaverdi, W. F. Wade, and P. S. Linsley. 1993. Induction of 37. Chen, C. Y., F. Del Gatto-Konczak, Z. Wu, and M. Karin. 1998. Stabilization of costimulatory molecule B7 in M12 B lymphomas by cAMP or MHC-restricted T interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science 280: cell interaction. J. Immunol. 150:2192. 1945. 56. Maeda, T., M. Towatari, H. Kosugi, and H. Saito. 2000. Up-regulation of co- 38. Xiao, Y. Q., K. Someya, H. Morita, K. Takahashi, and K. Ohuchi. 1999. In- stimulatory/adhesion molecules by histone deacetylase inhibitors in acute my- volvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced eloid leukemia cells. Blood 96:3847. production of macrophage inflammatory protein-2 in rat peritoneal neutrophils. 57. Lenardo, M., A. K. Rustgi, A. R. Schievella, and R. Bernards. 1989. Suppression Biochim. Biophys. Acta 1450:155. of MHC class I gene expression by N-myc through enhancer inactivation. EMBO 39. Campbell, K. S. 1999. Signal transduction from the B cell antigen-receptor. Curr. J. 8:3351. Opin. Immunol. 11:256. 58. Shaw, G., and R. Kamen. 1986. A conserved AU sequence from the 3Ј untrans- 40. Galant, S. P., S. B. Underwood, T. C. Lundak, C. C. Groncy, and lated region of GM-CSF mRNA mediates selective mRNA degradation. Cell D. I. Mouratides. 1978. Heterogeneity of lymphocyte subpopulations to pharma- 46:659.