J Immunol 2000 Kasprowicz 680 90

of February 15, 2015.This information is current as

IgE Produced per B CellandIntrinsically Modulates the Level of IgG1

-Adrenergic Receptor2(B7-2), and the Stimulation of the B Cell Receptor, CD86

SandersAndrezj J. Chruscinski, Arlene Sharpe and Virginia M. Deborah J. Kasprowicz, Adam P. Kohm, Michael T. Berton,

http://www.jimmunol.org/content/165/2/680doi: 10.4049/jimmunol.165.2.680

2000; 165:680-690; ;J Immunol

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Stimulation of the B Cell Receptor, CD86 (B7-2), and theb2-Adrenergic Receptor Intrinsically Modulates the Level ofIgG1 and IgE Produced per B Cell1

Deborah J. Kasprowicz,* Adam P. Kohm, Michael T. Berton, Andrezj J. Chruscinski,Arlene Sharpe, and Virginia M. Sanders2*

Our findings using B cells from either wild-type, CD86-deficient, or b2-adrenergic receptor (b2AR)-deficient mice suggest threemechanisms by which the level of IgG1 and IgE production can be increased on a per cell basis. Trinitrophenyl-specific B cellsenriched from unimmunized mouse spleens were pre-exposed to Ag and/or the b2AR ligand terbutaline for 24 h before beingactivated by either a b2AR-negative Th2 cell clone or CD40 ligand/Sf9 cells and IL-4 in the presence or absence of an anti-CD86Ab. Data suggest that the first mechanism involves a B cell receptor (BCR)-dependent up-regulation of CD86 expression that, whenCD86 is stimulated, increases the amount of IgG1 and IgE produced in comparison to unstimulated cells. The second mechanisminvolves a BCR- and b2AR-dependent up-regulation of CD86 to a level higher than that induced by stimulation of either receptoralone that, when CD86 is stimulated, further increases the amount of IgG1 and IgE produced. The third mechanism is BCR-independent and involves a b2AR-dependent increase in the ability of a B cell to respond to IL-4. Flow cytometric and limitingdilution analyses suggest that the increase in IgG1 and IgE occurs independently from the isotype switching event. These findingssuggest that the BCR, the b2AR, and CD86 are involved in regulating IL-4-dependent IgG1 and IgE production. The Journal ofImmunology, 2000, 165: 680690.

T he process of Ab production requires that a series of sig-nals be delivered to the B cell. The activation signal isinitiated when Ag binds to the B cell receptor (BCR).3 Acompetence signal is delivered to the B cell when both CD40 li-gand (CD40L) and T cell-derived cytokines bind to CD40 andcytokine receptors, respectively, expressed on the B cell surface(reviewed in Refs. 1 and 2). These signals culminate in an Abresponse that is classified as either Th1-dependent when Th1 cellsinteract with B cells and secrete IFN-g to induce B cells to produceIgG2a, or Th2-dependent when Th2 cells interact with B cells andsecrete IL-4 to induce B cells to produce IgG1 and IgE (3, 4).Thus, the B cell receives signals for activation, competence, iso-type switching, and secretion by stimulation of the BCR, CD40,and cytokine receptors.

Studies have shown that the Ag-BCR interaction, the MHC classII/peptide-TCR interaction, and the CD40-CD40L interaction gen-erate intracellular signals in both T cells and B cells to regulateboth T cell and B cell function (2, 5, 6). The CD28-CD86 inter-action was thought to deliver a signal to the T cell alone (7), butrecently it was reported that stimulation of CD86 on human ton-sillar B cells can increase the level of IgG1 and IgE produced afterCD40 and IL-4R stimulation (8). Collectively, it is clear that mul-tiple immune cell-derived signals influence both T and B cell func-tion during the course of an Ab response, but much remains un-known about both the mechanism by which CD86 stimulation onB cells modulates the level of IL-4-dependent IgG1 and IgE pro-duction and the mechanisms by which the level of Ab produced bythese B cells is regulated physiologically.

While the basic immune-related mechanisms of T cell and B cellactivation and regulation have been defined, endogenous nonim-mune mechanisms that may also modulate the level of cell acti-vation and regulation remain unknown. One endogenous mecha-nism that modulates immune cell activity in vivo involves theautonomic nervous system that is comprised of the sympatheticsystem releasing the neurotransmitter norepinephrine and the para-sympathetic system releasing acetylcholine. The sympathetic (ad-renergic) system functions in a diverse fashion to induce modesteffects on cellular activity, either stimulatory or inhibitory, whilethe parasympathetic (cholinergic) system functions in a limitedfashion to conserve energy and primarily acts during periods ofminimal activity (9). In addition, all organs that are regulated bythe sympathetic nervous system are not always regulated by para-sympathetic control. For example, the kidney, spleen, and arte-rioles are regulated primarily by sympathetic input as opposed toparasympathetic input (9, 10). In particular, the spleen is regulatedalmost exclusively by sympathetic control. No definitive data existto our knowledge to show that parasympathetic regulation existsfor other lymphoid organs, including the bone marrow, lymph

Departments of *Microbiology and Immunology, and Cell Biology, Neurobiologyand Anatomy, Loyola University Medical Center, Maywood, IL 60153; Departmentof Microbiology, University of Texas Health Science Center, San Antonio, TX 78284;Department of Medicine and Molecular and Cellular Physiology, Stanford Univer-sity, Stanford, CA 94305; and Department of Pathology, Brigham and WomensHospital, Boston MA 02115Received for publication January 20, 2000. Accepted for publication April 24, 2000.The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grants AI37326(V.M.S.), AI36310 (M.T.B.), and AI38310 (A.H.S.) and American Cancer SocietyGrant IM-798 (V.M.S.). V.M.S. is the recipient of career development awards fromthe American Cancer Society (JFRA-578) and the Schweppe Foundation.2 Address correspondence and reprint requests to Dr. Virginia M. Sanders, Depart-ment of Cell Biology, Neurobiology, and Anatomy, 2160 South First Avenue, LoyolaUniversity Medical Center, Maywood, IL 60153. E-mail address: [email protected] Abbreviations used in this paper: BCR, B cell receptor; CD40L, CD40 ligand;b2AR, b2-adrenergic receptor; CD40L/Sf9 cells, CD40L-expressing baculovirus-in-fected Sf9 cells; RGG, rabbit gamma globulin; MFI, mean fluorescence intensity;TNP, trinitrophenol; ELISPOT, enzyme-linked immunospot; HEL, hen egg ly-sozyme; PKA, protein kinase A.

Copyright 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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nodes, and thymus. However, thymic epithelial cells have beenshown to express cholinergic receptors (11, 12), but the signifi-cance of these receptors remains unknown. In contrast, there are aplethora of data to show that the neurotransmitter norepinephrinethat is released from sympathetic nerve endings residing within theparenchyma of lymphoid organs (1315) in response to Ag (16),LPS (17), or IL-1b (18). Nerve endings containing norepinephrinedirectly appose lymphoid cells that express the b2-adrenergic re-ceptor (b2AR) which binds norepinephrine to induce an increase inthe intracellular concentration of cAMP (reviewed in Refs. 10 and19). In vivo, norepinephrine has been shown to modulate the levelof the Th cell-dependent Ab response (20), but the mechanism bywhich this occurs at the cellular level remains unclear. In vitro, ithas been shown that norepinephrine enhances the level of Th cell-dependent IgM production through stimulation of the b2AR (2123), to allow for an increase in the frequency of B cells that dif-ferentiate into IgM-secreting cells (21). However, it remainsunclear as to whether or not the level of IL-4-dependent IgG1 andIgE production can also be affected and whether or not the Th cell,B cell, or both cells are affected by b2AR stimulation to induce thefunctional change.

In the present study, we explored a unique opportunity to de-termine the mechanism by which stimulation of the b2AR on theB cell enhances IL-4-dependent Ab production by using splenicTrinitrophenyl (TNP)-specific B cells enriched from the spleens ofunimmunized mice and activated by either a clone of b2AR-neg-ative Th2 cell (24, 25) or CD40L-expressing baculovirus-infectedSf9 cells (CD40L/Sf9 cells) and IL-4. Our findings using B cellsfrom either wild-type, CD86-deficient, or b2AR-deficient micesuggest three mechanisms by which the level of IL-4-dependentIgG1 and IgE production can be enhanced on a per cell basis,independently from the isotype switching event. The followingfindings support these mechanisms and are reported in the presentstudy. First, the level of CD86 expression is increased on the B cellsurface following BCR and/or b2AR stimulation. Second, follow-ing BCR stimulation alone, CD86 becomes competent to enhancethe level of IL-4-dependent Ab production. Third, the BCR- andCD86-mediated enhancing effect on Ab production is further aug-mented by b2AR stimulation. And fourth, following b2AR stim-ulation alone, Ab production is enhanced due to an increase in theability of the B cell to respond to IL-4. These findings suggestunique mechanisms by which a combination of BCR, CD86, andb2AR stimulation enhance the ability of Ag-specific B cells toproduce IL-4-dependent IgG1 and IgE.

Materials and MethodsAnimalsMice were maintained in a pathogen-free facility and were used between 7and 15 wk of age. Female BALB/c mice (H-2d-restricted) were obtainedfrom Harlan Sprague-Dawley (Indianapolis, IN), male and female b2AR-deficient mice (H-2q-restricted) (26) were generously provided by Dr.Brian Kobilka (Stanford University, Stanford, CA), and CD86-deficientmice (H-2d-restricted) (27) were generously provided by Dr. Arlene Sharpe(Brigham and Womens Hospital, Boston, MA). The b2AR-deficientmouse was generated by homologous recombination resulting in the inser-tion of a neomycin resistance gene cassette into the fourth transmembranedomain of the b2AR gene and has been described previously (26). TheCD86-deficient mouse was generated by homologous recombination re-sulting in the replacement of a portion of the IgV region of the CD86 genewith a neomycin resistance gene cassette and has been described previ-ously (27). Deficient mice were bred and housed within the pathogen-freefacility. All mice were housed under 12-h light/dark cycle and providedautoclaved food pellets and water ad libitum.

Reagents

2,4,6-Trinitrobenzenesulfonic acid (TNBS), fluorescein isothio-cyanate (FLU), normal rabbit gamma globulin (RGG), terbutaline,(2)-arterenol, nadolol, and the rp isomer of cAMP were purchasedfrom Sigma (St. Louis, MO), and keyhole limpet hemocyanin(KLH) was obtained from Calbiochem (La Jolla, CA). All phar-macologic reagents were dissolved in culture medium and filtersterilized immediately before addition to cultures and tested neg-ative for endotoxin in the Limulus lysate assay (Sigma). The TNP-derivative of RGG (TNP-RGG) and the FLU-derivative of KLH(FLU-KLH) were prepared as described previously (28). CD40L/Sf9 cells were prepared as previously described (29). Recombinantmouse IL-4 was purchased from PharMingen (San Diego, CA).AntibodiesThe following Abs were used for surface staining and were purchased fromPharMingen: purified and biotinylated-rat anti-mouse-CD86 (clone PO3),purified and biotinylated rat IgG2b (clone A95-1), FITC-streptavidin, andPE-streptavidin. ELISA Abs included unlabeled goat anti-mouse Ig or IgG,unlabeled and alkaline-phosphatase-conjugated rat anti-mouse IgE (clone23G3), alkaline-phosphatase-conjugated goat anti-mouse IgM or IgG1, andstreptavidin-conjugated alkaline phosphatase that were purchased fromSouthern Biotechnology Associates (Birmingham, AL).Culture of Th2 cell clonesThe Th2 cell clone CDC35 (RGG-specific, I-Ad-restricted) was generouslyprovided by Dr. D. Parker (Oregon Health Sciences University, Portland,OR), maintained as described previously (24), used at least 7 days follow-ing restimulation, and was found to be Mycoplasma-free.

Isolation of Ag-specific B cellsResting TNP-specific B cells were enriched from the spleens of unimmu-nized mice using a procedure described previously by Snow et al. (30) asmodified by Myers et al. (28). The TNP-specific B cells recovered at theend of the procedure were cultured in complete medium in a humidifiedatmosphere of 5% CO2 in air at 37C for at least 24 h to allow for re-expression of surface-associated molecules before additional experimenta-tion. Complete medium consisted of RPMI 1640 medium (Life Technol-ogies, Grand Island, NY) containing 10% FBS (Life Technologies), 20 mMHEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine,and 50 mM 2-ME. The phenotypic and functional characterization of rest-ing splenic TNP-specific B cells has been presented previously (31). Thepercentage of B2201 B cells recovered 24 h after the isolation procedureis ;9095%.

Isolation of small, dense resting B cellsSmall, dense resting B cells were isolated from the spleens of unimmunizedmice as previously described (32, 33). The small high-density resting Bcells were collected from a Percoll density gradient at the interface of the1.082 and 1.097 g/ml layers. This population contained ;9095% B2201B cells.

Culture conditionsResting TNP-specific or small, dense resting B cells were pre-exposed ineither complete medium alone or with TNP-RGG (0.7 mg/ml) or a F(ab9)2rabbit anti-mouse IgM (1.0 mg/ml), respectively, in 12 3 75 mm polysty-rene tubes in a final volume of 0.5 ml for 1824 h in a humidified atmo-sphere of 5% CO2 in air at 37C. B cells were plated in quadruplicate wellsin a 96-well, flat-bottom microtiter plate (no. 3596; Costar, Cambridge,MA) at 5 3 104 cells/well. At this time, either Th2 cell clones or CD40L/Sf9 cells and murine IL-4 were added to the B cells to a final volume of 0.2ml. Th2 cells were added at a one to one ratio with B cells, while oneCD40L/Sf9 cell was added per 1080 B cells. IL-4 was added to B cellcultures at a concentration ranging from 0.1 to 10 ng/ml. In some exper-iments, either an anti-CD86 Ab from clone PO3 (0.012.5 mg/ml) or aspecies- and isotype-matched control Ab from clone A95-1 was added tothe cell cultures. All cultures were incubated in a humidified atmosphere of5% CO2 in air at 37C. Anti-CD86 Ab from both the PO3 and GL1 cloneswas found to have B cell stimulatory activity; however, Ab from the GL1clone, but not the PO3 clone, cross-reacted in the IgG ELISA and, there-fore, only Ab from the PO3 clone was used in the experiments describedherein. Two additional species- and isotype-matched control Abs (clones

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R35-38 and JES6-5H4) were also used in experiments and were found tolack B cell stimulatory activity.

IgG1 or IgE ELISAOn day 8 following the addition of CD40L/Sf9 cells and cytokine, the Bcell supernatant was collected and immediately frozen at 280C until an-alyzed by ELISA as described in detail previously (24). A standard curvefor IgG1 and IgE Ab was prepared using known quantities of the myelomaprotein MOPC-21 (IgG1,k; Sigma) or IgE-3 (IgE,k, PharMingen). Colordevelopment was determined on a UVmax kinetic microplate reader (Mo-lecular Devices, Palo Alto, CA) at a wavelength of 405 nm. Lower limitsof detection were as follows: for IgG1, ,4 ng/ml; and for IgE, ,4 ng/ml.

Enzyme-linked immunospot (ELISPOT) assayA modification of the ELISPOT assay described by Czerkinsky et al. (34)and Sedgwick and Holt (35) was used to detect individual anti-Ig-secretingcells, and the protocol used has been described in detail elsewhere (21). Forthe detection of the amount of Ab secreted by cells, 100 ml of a developingsolution was added as described for the ELISA, and color development wasdetermined. For the detection of the number of cells secreting Ab, 100 mlof a developing solution consisting of 2.5 mM 5-bromo-4-chloro-3-indolylphosphate in AMP buffer and 100 mg/ml nitro blue tetrazolium in 70%dimethylformamide was added to each well and incubated at 25C for 34h. Spot-forming cells were enumerated using a dissecting microscope. Thenumber of B cells assayed for Ab production ranged from 5 3 103 to 2 3104 cells/well depending on the isotype analyzed, with final values nor-malized to Ab production by 5 3 104 input B cells.

Immunofluorescence staining and flow cytometric analysisAfter 24 h of pre-exposure, B cells were washed once in HBSS 1 1%FBS 1 0.05% sodium azide (HBSS/FBS/azide), resuspended in 0.2 ml,and analyzed for the expression of various surface markers. Cells wereincubated at 4C with primary Abs, followed by secondary Abs and twowashes in HBSS/FBS/azide. Each Ab was titrated to determine optimalstaining concentration for flow cytometric analysis. Cells were fixed with1% paraformaldehyde for 12 min at 4C followed by storage in PBS/azideuntil flow cytometric analysis. Cells were analyzed with a FACSCaliburflow cytometer (Becton Dickinson, San Diego, CA) gated on all viablecells. Calibration of the FACSCalibur was manually performed daily usingRainbow Calibration Particles (Sherotech, Libertyville, IL). Data were an-alyzed using CellQuest software (Becton Dickinson).Confocal microscopy and image analysisSamples were prepared as described above for flow cytometric analysis.Cells were examined with a Zeiss (Jena, Germany) LSM510 laser scanningconfocal microscope and LSM510 v.2.1 imaging software. Samples werescanned and images were collected of the middle and top planes of indi-vidual cells. A minimum of 100 cells was examined per exposure group.Image analysis was performed using NIH Image software to determine theaverage intensity per B cell. B cells were designated CD861 cells if theoverall cell intensity was higher than the background intensity.

Limiting dilution analysisVarious numbers of Ag alone- or Ag/terbutaline-pre-exposed TNP-specificB cells were cultured with 35 3 103 irradiated (1000 rads) cells of theCDC35 Th2 clone in a volume of 10 ml/well in a Terasaki-type (Nunc,Roskilde, Denmark) microtiter plate as described in detail previously (21).Precursor frequency analysis was performed according to Poisson statisticsfor the calculation from 37% interpolation as described by Lefkovitz andWaldmann (36).StatisticsData were analyzed by a one-way ANOVA to determine whether an over-all statistically significant change existed before using the two-tailed un-paired Students t test. Statistically significant differences were reportedwhen the p value was ,0.05.

ResultsThe level of CD86 expression on Ag-pulsed B cells and theeffect of CD86 stimulation on CD40L/IL-4-induced IgG1 andIgE productionPrevious studies have shown that various factors including LPS(3739), anti-Ig (4042), and the Ag hen egg lysozyme (HEL)

(42) induce an up-regulation of CD86 expression on the surface ofB cells. To determine whether a hapten-carrier conjugate wouldenhance the level of CD86 expression on hapten-specific B cells,resting TNP-specific B cells were enriched from the spleens ofunimmunized mice and incubated with either medium alone orTNP-RGG for 1824 h before being examined by flow cytometryand confocal microscopy. As determined by flow cytometric anal-ysis, both the percentage of cells expressing CD86 and the meanfluorescence intensity (MFI) were increased from 57% and MFI 5260 on medium alone-exposed B cells to 66% and MFI 5 644 onAg-pulsed B cells, respectively, (Fig. 1A, left). Next, to verify thatthe Ag-specific cell isolation procedure was not influencing thelevel of CD86 expression that was induced by BCR stimulation ofB cells, the level of CD86 expressed on small, dense resting B cellswas determined after cells were cultured with a F(ab9)2 rabbit anti-mouse IgM (RAMIgM) for 24 h. As shown in Fig. 1A, right, small,dense resting B cells exposed to a F(ab9)2 rabbit anti-mouse IgMexpressed a level of CD86 that was similar to that induced by aspecific Ag on Ag-specific B cells.

Since Jeannin et al. (8) had reported that stimulation of CD86 onhuman tonsillar B cells increased the level of IL-4-dependent Abproduction, we next determined if stimulation of CD86 on murineAg-specific B cells exposed to Ag with an anti-CD86 Ab wouldenhance the level of IgG1 and IgE produced if cells were activatedby CD40L/Sf9 cells and IL-4. The concentration of rIL-4 used wasbased on the level of IL-4 measured in T cell-B cell cultures (1.0ng/ml), and the concentration of anti-CD86 Ab used (1.0 mg/ml)was based on a titration of anti-CD86 Ab for the most effectiveconcentration to enhance the level of Ab production. B cells thatwere pre-exposed to Ag and cultured in the presence of an anti-CD86 Ab produced 50% more IgG1 (Fig. 1B, left) and IgE (Fig.1C, left) than B cells either pre-exposed to Ag and cultured with anisotype control Ab or B cells pre-exposed to medium alone andcultured with either an anti-CD86 or isotype control Ab. Thesedata suggest that stimulation of CD86 on Ag-exposed B cells en-hances the level of IgG1 and IgE produced. To determine whetherthe increased level of IgG1 and IgE production was mediatedthrough stimulation of CD86, we performed a similar experimentusing B cells isolated from the spleens of unimmunized CD86-deficient mice. B cells from CD86-deficient mice pre-exposed toAg and cultured in the presence of an anti-CD86 Ab produced asimilar level of IgG1 (Fig. 1B, right) and IgE (Fig. 1C, right) ascells cultured with an isotype control Ab, suggesting that an intactCD86 molecule on the B cell surface is required for an anti-CD86Ab to induce an enhancement in IgG1 and IgE. Although the anti-CD86-induced increase in IgG1 and IgE was modest, it occurredonly when B cells expressed CD86 and only when B cells werepulsed with Ag before culture with CD40L/Sf9 cells, IL-4, and ananti-CD86 Ab. Taken together, these data suggest that both BCRand CD86 stimulation on the B cell are required to enhance thelevel of IgG1 and IgE production.

CD86 expression on B cells exposed to Ag and/or the b2ARagonist terbutalineWe next determined if both the level of CD86 expression and theanti-CD86 Ab-induced increase in the level of IgG1 and IgE wouldbe further modulated by stimulation of the b2AR expressed on theAg-specific B cell (20) during Ag pre-exposure. Previous studieshave shown that stimulation of the b2AR increases the concentra-tion of intracellular cAMP (20) and that CD86 expression is up-regulated by increasing the concentration of intracellular cAMP inthe B cell (43). We found a 10% increase in the percentage of Bcells expressing a high level of CD86 when B cells were pre-exposed to Ag/terbutaline in comparison to B cells pre-exposed to

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Ag alone (Fig. 2A, left). This increase was blocked if the bARantagonist, nadolol, was added during pre-exposure of B cells (Fig.2A, right). The addition of a protein kinase A (PKA) inhibitor (rpcAMP) did not affect the level of CD86 expression induced by Agalone (Fig. 2B, left), but did prevent the terbutaline-induced in-crease in CD86 surface expression (Fig. 2B, right). These findingsindicate that the b2AR-mediated increase in CD86 surface expres-sion, above that induced by Ag alone, is mediated through activa-tion of the cAMP-dependent PKA.

To confirm the above described findings, we used B cells en-riched from the spleens of unimmunized b2AR-deficient mice. Be-cause the b2AR-deficient mice were generated in mice of a dif-ferent haplotype (H-2q) than that used for the above describedwork (H-2d), we compared data from b2AR-deficient mice withdata generated from wild-type mice of the same haplotype. Asshown in Fig. 2C, left, BCR stimulation on B cells from wild-typemice up-regulated the level of CD86 expression above backgroundand terbutaline further increased the level of CD86 expression(Fig. 2C, left). In contrast, although BCR stimulation alone onb2AR-deficient B cells up-regulated the level of CD86 surfaceexpression, exposure to terbutaline alone (data not shown) or Ag/terbutaline (Fig. 2C, right) did not, suggesting that stimulation ofthe b2AR on B cells is responsible for the up-regulation of CD86expression on B cells above the level induced by BCR stimulationalone. To confirm that the b2AR-mediated effect was specific forCD86, B cells were examined for the level of expression of otherB cell surface markers. Stimulation of the b2AR on B cells did not

FIGURE 1. The effect of BCR stimulation on CD86 expression andanti-CD86 Ab-induced IgG1 and IgE production by B cells from wild-typeand CD86-deficient mice. A, left, TNP-specific B cells (5 3 105) werepre-exposed to either medium alone or TNP-RGG (0.7 mg/ml) for 24 h at37C. A, right, Small, dense resting B cells were cultured with either me-dium alone or F(ab9)2 rabbit anti-mouse IgM (RAMIgM, 1.0 mg/ml) for24 h at 37C. After 24 h, CD86 expression on B cells was determined usingimmunofluorescence and a FACSCalibur flow cytometer. Nonspecificbinding was determined using a species- and isotype-matched control Ab(dashed line). Gray shading, medium-alone exposed B cells; black shading,Ag-exposed B cells. B and C, Resting splenic TNP-specific B cells (5 3104) from CD861/1 (left) or CD862/2 (right) mice were pre-exposed toeither medium alone or TNP-RGG (0.7 mg/ml) for 24 h at 37C beforeexposure to CD40L/Sf9 cells (1 Sf9 cell per every 10 B cells) and IL-4 (1.0ng/ml). In addition, either a soluble anti-CD86 Ab (f) or species- andisotype-matched control Ab (M) was added to a final concentration of 1mg/ml. After 8 days of culture at 37C, supernatants were collected andanalyzed for IgG1 (B) and IgE (C) by ELISA. Data are presented as thepercent change from the Ag alone/isotype control response 6 SE fromquadruplicate wells from either four (left) or two (right) separate experi-ments. An asterisk (p) indicates a p value ,0.05 when the comparison iswithin a specific pre-exposure group. Ag alone/isotype control responsesfor IgG1 in wild-type mice ranged from 63 to 229 ng/ml; for IgE in wild-type mice ranged from 5 to 35 ng/ml; for IgG1 in CD862/2 mice rangedfrom 181 to 206 ng/ml; and for IgE in CD862/2 mice ranged from 9 to 12ng/ml.

FIGURE 2. The effect of b2AR stimulation on the level of CD86 sur-face expression by B cells from wild-type and b2AR-deficient mice. AC,TNP-specific B from either b2AR1/1 H-2d or H-2q or b2AR2/2 H-2q micewere pre-exposed to TNP-RGG (0.7 mg/ml) in either the presence or ab-sence of terbutaline (Tb, 1026 M), and/or nadolol (Nd, 1025 M). After24 h, CD86 expression on B cells was determined using immunofluores-cence and a FACSCalibur flow cytometer. Nonspecific binding was deter-mined using a species- and isotype-matched control Ab (dashed line). Aleft, Ag alone or Tb alone-exposed B cells (gray shading) and Ag/Tb-exposed B cells (black shading). A, right, Ag alone-exposed B cells (grayshading), and Ag/Tb/Nd-exposed B cells (black shading). Data are repre-sentative of three separate experiments. B, Before B cell stimulation withAg and Tb, B cells were incubated with a PKA inhibitor, rp cAMP (10mM) for 30 min. Left, Ag alone-exposed B cells (gray shading), and Ag/rpcAMP-exposed B cells (black shading). Right, Ag/Tb-exposed B cells(black shading), and Ag/Tb/rp cAMP-exposed B cells (gray shading). Dataare representative of two separate experiments. C, left, b2AR1/1 H-2q Bcells; right, b2AR2/2 H-2q B cells. Ag alone-exposed B cells (gray shad-ing) or Ag/Tb-exposed B cells (black shading). Data are representative ofthree separate experiments.


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significantly change the level of expression of any other surfacemarkers examined, including CD80 (B7-1) (Table I).

In addition to flow cytometric analysis, we quantified the levelof CD86 expression at the single cell level using confocal micros-copy and LSM510 imaging software. Images collected from Bcells pre-exposed to Ag alone, Ag/terbutaline, and Ag/terbutaline/nadolol were collected and examined for CD86 expression (TableII). Ag/terbutaline pre-exposure increased the percentage of B cellsexpressing a high level of CD86 that was blocked by culture ofcells in the presence of nadolol. Taken together, these findingssuggest that CD86 is expressed at a low level on the resting B cellsurface and is up-regulated upon stimulation of either the BCR orb2AR on B cells and that the b2AR-mediated effect depends oncAMP activation of PKA.

The effect of b2AR and CD86 stimulation on CD40L/IL-4-induced IgG1 productionWe next determined whether the increased level of CD86 ex-pressed by Ag/terbutaline pre-exposed B cells would further in-crease the BCR- and CD86-mediated enhancement in IgG1. Asshown in Fig. 3A, B cells that were pre-exposed to Ag alone andcultured in the presence of CD40L/Sf9 cells, IL-4, and an anti-CD86 Ab produced ;50% more IgG1 than B cells pre-exposed toAg alone and cultured with an isotype control Ab. B cells pre-exposed to terbutaline alone produced ;50% more IgG1 than Bcells pre-exposed to Ag alone, but this enhancement was not mod-ulated by the presence of an anti-CD86 Ab, suggesting that theincrease in IgG1 was not due to CD86 stimulation on B cells.However, B cells that were pre-exposed to Ag/terbutaline andstimulated with an anti-CD86 Ab produced ;150% more IgG1(Fig. 3A) in comparison to B cells pre-exposed to Ag and an anti-CD86 Ab alone (Fig. 3A), suggesting that stimulation of the b2ARon the B cell further increased the BCR- and CD86-mediated in-crease in IgG1 production. The b2AR-mediated enhancement inIgG1 production, but not the BCR- and CD86-mediated enhance-ment, was prevented if B cells were pre-exposed in the presence ofthe bAR antagonist nadolol (data not shown), suggesting that theterbutaline-induced effect on Ab production was mediated through

FIGURE 3. The effect of anti-CD86 Ab on the level of IgG1 and IgE produced by Ag and/or terbutaline (Tb)-exposed B cells from wild-type andb2AR-deficient mice. AC, Resting splenic TNP-specific B cells (5 3 104) from either b2AR1/1 H-2d (A) or H-2q (B) or b2AR2/2 H-2q (C) mice werepre-exposed to either medium alone or TNP-RGG (0.7 mg/ml) for 24 h at 37C before exposure to CD40L/Sf9 cells (1 Sf9 cell per every 10 B cells) andIL-4 (1.0 ng/ml). In addition, either a soluble anti-CD86 Ab (f) or species- and isotype-matched control Ab (M) was added to a final concentration of 1mg/ml. After 8 days of culture at 37C, supernatants were collected and analyzed for IgG1 by ELISA. Although not indicated by an asterisk, all terbutalinepre-exposure conditions from b2AR1/1 mice (A and B) induced a significant change at p , 0.05 when compared with the Ag alone/isotype control Abgroup. Data are presented as the percent change from the Ag alone/isotype control Ab response 6 SE from quadruplicate wells from three to four separateexperiments. An asterisk (p) indicates a p value ,0.05 when the comparison is within a specific pre-exposure group. Ag alone/isotype control responsesfor IgG1 from b2AR1/1 H-2d mice ranged from 63 to 229 ng/ml; IgG1 from b2AR1/1 H-2q mice ranged from 4,106 to 10,302 ng/ml; and IgG1 fromb2AR2/2 H-2q mice ranged from 2,144 to 12,013 ng/ml.

Table II. CD86 expression on B cells following stimulation of the BCRand/or b2AR

Pre-exposure Conditiona % of CD861 Cellsb % of CD862 Cells

Ag 41 59Ag 1 Tb 50 50Ag 1 Tb 1 Nd 41 59

a B cells were pre-exposed to Ag, terbutaline (Tb), and/or nadolol (Nd) prior to thedetermination of CD86 expression using immunofluorescence and confocalmicroscopy.

b Data are presented as the percent of cells that were designated either CD861 ifthe overall cell intensity was higher than the background intensity or CD862 if theoverall cell intensity was equivalent to the background intensity as determined byconfocal image analysis and are representative of 100 cells examined per exposurecondition.

Table I. Surface Ag expression on B cells pre-exposed to either Agalone or Ag/terbutaline

Surface Ag

% of Cellsa Fluorescenceb

Agc Ag/Tb Ag Ag/Tb

CD19 87d 88 727 600CD21/35 16 13 759 731CD23 75 75 74 64CD40 48 50 445 288CD80 40 41 116 110IgM 82 81 2091 2000B220 91 92 1054 929I-Ad 94 94 1202 931LFA-1 79 71 33 30ICAM-1 89 92 381 509Fas (CD95) 2 2 447 320Fas ligand 96 97 33 33Thy 1.2 23 25 761 753

a The % of cells 5 the percentage of TNP-specific B cells expressing a particularsurface Ag following pre-exposure of cells to either Ag alone or Ag/terbutaline.

b Fluorescence 5 the MFI of TNP-specific B cells expressing a particular surfaceAg following pre-exposure of cells to either Ag alone or Ag/terbutaline.

c Ag, TNP-RGG; Tb, terbutaline.d The percentage of positive cells 5 specific staining minus species- and isotype-

matched control Ab staining.


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stimulation of bAR. Taken together, these data suggest that stim-ulating CD86 on B cells pre-exposed to Ag enhances the level ofIgG1 produced by these B cells, and that this response is furtherenhanced if B cells are pre-exposed to both Ag and terbutaline.

As shown in Fig. 3B, stimulation of CD86 on B cells fromwild-type H-2q mice produced similar results to those for wild-type H-2d mice described above (Fig. 3A). Likewise, stimulation ofCD86 on B cells from b2AR-deficient H-2q mice pre-exposed toAg enhanced the level of IgG1 in comparison to B cells culturedwith an isotype control Ab, suggesting that the b2AR was notrequired to induce the initial, BCR- and CD86-mediated enhance-ment in IgG1. In contrast, stimulation of CD86 on b2AR-deficientH-2q B cells pre-exposed to either terbutaline or Ag/terbutaline didnot enhance the level of IgG1 above that produced by B cellspre-exposed to Ag alone (Fig. 3C), suggesting that the b2AR isrequired to further enhance the BCR- and CD86-mediated en-hancement in IgG1 production. We found that there was no sig-nificant difference in the percentage of cells expressing surfaceIgG1 following culture of cells in the presence of an anti-CD86 orisotype control Ab, CD40L/Sf9 cells, and IL-4 (data not shown),suggesting that stimulation of CD86 was not enhancing IgG1 pro-duction by inducing more B cells to switch from IgM to IgG1production. Overall, these data suggest that stimulation of theb2AR increases the level of CD86 expression on the B cell surfaceindependently from that induced by BCR stimulation and furtherincreases the level of IgG1 produced per B cell when both CD86and the BCR are stimulated.

The effect of b2AR stimulation on the ability of B cells torespond to IL-4Since we observed an increase in the level of IgG1 produced by Bcells pre-exposed to terbutaline alone and activated with CD40L/Sf9

cells and IL-4 that occurred independently of CD86 involvement, wewanted to determine the mechanism responsible for the b2AR alone-mediated increase in the level of Ab produced. Because B cells wereexposed to only IL-4 and CD40L/Sf9 cells, it seemed likely that stim-ulation of the b2AR on the B cell increased the responsiveness of theB cell to one or both of these stimuli. First, we determined whetherb2AR stimulation enhanced Ab production via a direct mechanismthat increased the ability of the B cell to respond to IL-4. B cellspre-exposed to either terbutaline alone (data not shown) or Ag/ter-butaline (Fig. 4A) produced 75200% more IgG1 than B cells pre-exposed to Ag alone. As shown in Fig. 4B, B cells from wild-typeH-2q mice showed a similar enhancement in IgG1 production as theB cells from H-2d mice (Fig. 4A). In contrast, B cells from b2AR-deficient H-2q mice did not display an increased ability to respond toIL-4 following exposure to Ag/terbutaline (Fig. 4C). However, B cellsthat were pre-exposed to Ag alone or Ag/terbutaline showed a similarlevel of IL-4R expression (Fig. 4D), suggesting that increased IL-4Rexpression was not allowing for the increased ability of terbutaline-exposed B cells to respond to IL-4. Also, while stimulation of theb2AR on the B cell appeared to increase the ability of the B cell torespond to IL-4, stimulation of the b2AR did not appear to increasethe ability of the B cell to respond to CD40L (data not shown). Over-all, these data suggest that the terbutaline-induced increase in the abil-ity of the B cell to respond to IL-4 is dependent on the presence of theb2AR, but is independent of BCR stimulation and does not involve anincrease in the level of IL-4R surface expression.

Th2 cell-dependent Ab production by B cells pre-exposed to Agand/or terbutalineMost experimental systems do not allow for the analysis of the abovedescribed BCR-, CD86-, and b2AR-dependent mechanisms for enhanc-ing IL-4-dependent Ab production in an Ag-specific manner. However, a

FIGURE 4. The effect of b2AR stimulationon the ability of B cells to respond to IL-4. TNP-specific B cells were enriched from b2AR1/1H-2d (A), b2AR1/1 H-2q (B), or b2AR2/2 H-2qmice (C) and pre-exposed to Ag alone (M) orAg/terbutaline (Ag/Tb) (o) as described in Fig.2. After 24 h, IL-4 was added at concentrationsranging from 0.1 to 10 ng/ml and CD40L/Sf9cells were added at a ratio of 1 CD40L/Sf9 cellper 10 B cells. After 8 days of culture, superna-tants were collected and analyzed for IgG1 (up-per) and IgE (lower) by ELISA. Data are pre-sented as the mean ng/ml for each concentrationof IL-4 6 SE from quadruplicate determinationsfrom two to four separate experiments. An as-terisk (p) indicates a p value ,0.05 in compar-ison to the Ag alone-pulsed B cells. D, B cellsfrom b2AR1/1 H-2d mice were pre-exposed asin Fig. 2. After 24 h, the level of IL-4R expres-sion was determined on TNP-exposed B cells(gray shading) and Ag/Tb-exposed B cells(black shading) using immunofluorescence and aFACSCalibur flow cytometer. Nonspecific bind-ing was determined using a species- and isotype-matched control Ab (dashed line). Data are rep-resentative of two separate experiments.


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model system is available in which Ag-specific B cells are cultured witha b2AR-negative, Ag-specific Th2 clone (24, 25). Using this model sys-tem, we can determine whether stimulation of the BCR and the b2AR onthe B cell would induce an enhancement in Th2 cell-dependent Ab pro-duction similar to the enhancement in IL-4-dependent Ab productionfound using CD40L/Sf9 cells and IL-4. As determined by a modificationof the ELISPOT assay that allows for the determination of the amount ofAb secreted by a population of B cells on a particular day of culture, Bcells pre-exposed to Ag/terbutaline before their culture with a clone ofTh2 cells produced 50200% more IgM, IgG1, and IgE (Fig. 5A), withno change in the number of Ab-secreting cell, when compared with Bcells pre-exposed to Ag alone (Fig. 5B). This enhancement was preventedby nadolol (Fig. 5A) and occurred in a terbutaline-concentration-depen-dent manner (Fig. 5C). When similar experiments were performed witha Th1 clone, we found that B cells pre-exposed to Ag/terbutaline or Agalone produced a similar level of IgM and IgG2a, suggesting that stim-ulation of the b2AR on the B cell was not enhancing Th1-dependent Abproduction (D. J. Kasprowicz and V. M Sanders, manuscript inpreparation).

Importantly, culture of Th2 cells with B cells pre-exposed toAg/terbutaline in comparison to B cells pre-exposed to Ag alonedid not affect the level of IL-4 produced by the Th2 cells, theamount of [3H]thymidine incorporated by the Th2 cells, or thenumber of Th2 cell:B cell conjugates that formed (data not shown).In addition, B cells pre-exposed to Ag/terbutaline showed a similarkinetics of Ab production to cells pre-exposed to Ag alone, sug-gesting that the b2AR-mediated enhancement in Ab productionwas not due to a shift in the timing of Ab production (data notshown). Unfortunately, it was not possible to further address theeffect of stimulating CD86 with an anti-CD86 Ab in the T cell-Bcell culture because the addition of an anti-CD86 Ab to culturesdecreased the level of cytokines produced by the T cell (data notshown and Ref. 44), suggesting that the addition of an anti-CD86Ab was blocking the CD28-CD86 interaction and preventing max-

imal activation of the T cell to secrete a required level of IL-4.Nonetheless, taken together, the above data suggest that stimula-tion of B cells through the BCR and the b2AR enhances Th2cell-dependent IgM, IgG1, and IgE production on a per cell basisby a direct effect on B cell function alone.

Limiting dilution analysis of the b2AR-induced increase in Th2cell-dependent Ab production and analysis of surface IgG1-positive B cellsTo determine whether b2AR stimulation was influencing the de-gree of isotype switching in B cells, we pre-exposed B cells to Agand/or terbutaline before culture with either a Th2 clone orCD40L/Sf9 cells and IL-4, and subsequently analyzed cells bylimiting dilution and flow cytometric analysis (45), respectively.As shown in Fig. 6A, Ag/terbutaline induced a small increase inthe frequency of IgE-secreting cells (1:2600 for Ag vs 1:1750 forAg/terbutaline), but induced no change in the frequency of eitherIgM-secreting cells (1:180) or IgG1-secreting cells (1:500) whencompared with B cells pre-exposed to Ag alone. No change inburst size was detected for any isotype of Ab measured. As shownin Fig. 6B, pre-exposure of B cells to Ag/terbutaline did not changethe percentage of surface IgG1-positive B cells generated in com-parison to B cells pre-exposed to Ag alone. Taken together withthe previously described finding using ELISPOT, pre-exposure ofB cells to Ag/terbutaline and culture with either Th2 cell clones orCD40L/Sf9 cells and IL-4 induced B cells to produce more Ab percell, as opposed to inducing an increase in either the number ofmature precursor B cells differentiating into Ab-secreting cell,burst size, or the number of cells switching isotype.

DiscussionThe present data suggest three mechanisms by which the amountof IgG1 and IgE produced by a B cell is enhanced on a per cell

FIGURE 5. The effect of b2AR stimulation on thelevel of IgM, IgG1, and IgE produced by B cells culturedwith a clone of Th2 cells. A and B, TNP-specific B cellswere pre-exposed to 0.7 mg/ml TNP-RGG in the presenceor absence of terbutaline (Tb, 1026 M) and/or nadolol(Nd, 1025 M). After 24 h at 37C, 5 3 104 pre-exposed Bcells were added to an equal number of irradiated (1000rads) RGG-specific Th2 cells (clone CDC35). After 7days of culture, cells were assayed by ELISPOT for theamount of Ab secreted (A) and the number of cells se-creting Ab (B). Data are presented as either the mean ng/ml 6 SE or the number of cells secreting Ab 6 SE per5 3 104 input B cells from triplicate wells from one ofthree separate experiments for IgM and IgG1 and of twoseparate experiments for IgE. C, B cells were prepared asdescribed in A, except that terbutaline was added in con-centrations ranging from 1028 to 1025 M. After 7 days ofculture, cells were assayed by ELISPOT for the amountof Ab secreted. Data are presented as the percent changefrom the Ag alone response 6 SE from triplicatewells from three separate experiments. An asterisk (p) in-dicates a p value ,0.05 in comparison to Ag alone pre-exposed B cells.


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basis, independently from the isotype switching event. The firstmechanism involves a BCR-dependent up-regulation of CD86 ex-pression that, when CD86 is stimulated, increases the amount ofIgG1 and IgE produced, in comparison to unstimulated cells. Thesecond mechanism involves a BCR- and b2AR-dependent up-reg-ulation of IgG1 to a level higher than that induced by stimulationof either receptor alone that, when CD86 is stimulated, furtherincreases the amount of IgG1 and IgE produced. The third mech-anism is BCR-independent and involves a b2AR-dependent in-crease in the ability of a B cell to respond to IL-4.

It may be argued that the modest level of increase in Ab pro-duction by b2AR-stimulated B cells has no meaningful effect invivo. However, although only a few reports exist to show a directrelationship between the level of Ab needed to provide a certainlevel of protection against a specific Ag (4649), one of thesestudies does provide compelling data supporting the relevance ofour finding (47). This study showed a correlation between Ab con-centration and opsonic titer following vaccination, such that an;2-fold increase in total Ab concentration increased the protectivetiter 3-fold, while a 3-fold increase in total Ab concentration re-sulted in a 9-fold increase in protective titer. The results from thisstudy indicate that a small increase in the level of neutralizing Abwill significantly increase protection to the host and, thus, lendrelevance to our present finding that b2AR stimulation on the Bcell induces a modest increase in the amount of Ab produced.

Although, initially, it may seem that the concentration of b2ARagonist used in the present study is higher than would be presentin vivo, several studies have addressed the concentration of nor-epinephrine present in vivo. A study by Shimizu et al. (18) used asensitive microdialysis procedure to measure basal norepinephrineconcentration released at nerve terminals in a murine spleen and

found it to be ;1.6 3 1026 M and 3.2 3 1026 M before and afterthe administration of IL-1b, respectively. In addition, a previouslypublished study in vivo from our laboratory showed that the ad-ministration of a b2AR agonist at a dose of 10 mg/kg (equivalentto ;150 mM) to immunized norepinephrine-depleted animals par-tially restored the Ab response to control levels. Finally, while theaffinity of the b2AR may be high (Kd 5 0.1 nM), immune cellsexpress fairly low numbers of the b2AR (B cell 5 ;600 receptors/cell (20), Th1 cell clones 5 ;250 receptors/cell (24), Th2 cellclones 5 0 receptors/cell) (24)). Therefore, while adrenergic re-ceptors expressed on immune cells may have a high affinity fornorepinephrine or a b2AR agonist, these receptors are expressed ata low level and may require a high concentration of norepinephrineor b2AR agonist to generate an intracellular signal. Thus, the con-centration of b2AR agonist used in the present study is within therange of the endogenous concentrations that should be presentwithin the microenvironment of a B cell responding to Ag in vivo.

Although stimulation of either CD86 or the b2AR alone mod-estly increased the level of BCR- and IL-4-dependent IgG1 andIgE produced, it is of interest that this modest increase in IgG1 andIgE was further enhanced up to 2-fold by concurrent exposure toboth stimuli. Therefore, while the generation of one signal in the Bcell may be sufficient to induce a modest change in the level of Abproduced, the generation of multiple signals may cooperate to in-duce a further amplification of the Th2-dependent Ab response.For example, norepinephrine stimulation of the b2AR on a B cellis required for Ag-specific B cells to produce normal serum lev-els of Th2 cell-dependent Ab (20). However, we also know thatwhen norepinephrine is removed in vivo, the ability of B cells toproduce Ab is never ablated completely (20, 50), and when nor-epinephrine is added in vitro, the higher amount of Ab induced is

FIGURE 6. Limiting dilution and flow cytometric analysis of B cells exposed to Ag and/or terbutaline. B cells were pre-exposed to Ag, terbutaline (Tb),and/or nadolol (Nd) as described in Fig. 2. A, B cells were plated in limiting dilution with 35 3 104 irradiated (1000 rads) Th2 cells (clone CDC35). After57 days of culture, cells were analyzed on day 5 for IgM, day 6 for IgG1, and day 7 for IgE production by ELISPOT. Precursor frequency and burst sizeanalysis were performed according to Poisson statistics as described in the Materials and Methods. Frequency values represent the mean number ofnonresponding wells with a 95% confidence limit. Burst size values are presented within parentheses as the mean number of cells produced per precursorof two to three individual experiments from groups of 60 wells. The thick line indicates the position of 37% nonresponding wells. B, Pre-exposed B cellswere plated with CD40L/Sf9 cells at ratio of 1 CD40L/Sf9 cell per 10 B cells and IL-4 at a final concentration of 1.0 ng/ml. After 6 days of culture, cellswere collected, incubated with a FITC-conjugated goat anti-mouse IgG1 Ab (black shading) or a species- and isotype-matched control Ab (gray shading),and analyzed on a FACSCalibur flow cytometer for surface IgG1 expression. B cells that were stained and examined at the initiation of culture arerepresented as resting. Data are representative of two separate experiments.


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rarely increased more than 3-fold. Thus, the immune system ap-pears to regulate intrinsically the level of Ab produced by a B cell,but this level of regulation can be further modulated by mecha-nisms normally associated with nervous system regulation of or-gan function, such as norepinephrine stimulation of the b2AR.

The present findings suggest a role for both BCR and b2ARstimulation in modulating the level of CD86 expressed on the Bcell surface. Using flow cytometry and confocal microscopy, weshow that the expression of CD86 is up-regulated on murine Bcells pulsed with either a specific Ag or terbutaline, findings thatare in agreement with other studies using either HEL-transgenicsplenic B cells stimulated with HEL (42) or splenic B cells stim-ulated with the cAMP analogue dibutyryl cAMP (dbcAMP) (43).However, the present study goes on to show that CD86 expressionis further increased by stimulation of both the BCR by Ag and theb2AR by terbutaline. In contrast, neither Ag or terbutaline in-creased the level of expression of the other B7 molecule, CD80,unlike the study using splenic B cells and dbcAMP in which CD80expression was enhanced (43). However, similar to our finding,HEL stimulation of HEL-transgenic B cells did not modulateCD80 expression (42), suggesting that other mechanisms thanthose used by either the BCR or the b2AR may be required toinduce CD80 expression on a B cell. These findings also suggestthat following both BCR and b2AR stimulation, a higher level ofCD86 would be available on the B cell surface at the initiation ofan Ab response, perhaps to ensure optimal B cell activation duringan Ag-specific response in vivo. Such an event is likely given thatthe amount and turnover of norepinephrine in the spleen increasesafter Ag administration (16, 60).

When CD40L/Sf9 cells and IL-4 were used to activate B cells,stimulation of CD86 on either medium or terbutaline alone-ex-posed B cells induced no change in the level of Ab produced incomparison to stimulation of CD86 on either Ag- or Ag/terbutal-ine-exposed B cells, which induced an increase in the level of IgG1produced. This finding suggests that stimulation of the BCR affectsthe ability of B cells to signal through CD86 differently from Bcells that are not stimulated through the BCR. For example, arecent report showed that CD86 stimulation on human tonsillar Bcells exposed to IL-4 and an anti-CD40 Ab increased the level ofIgG4 (human equivalent of IgG1) and IgE produced (8). Althoughit initially appeared in this study that stimulation of the BCR wasnot required for the CD86 signal to enhance IgG1 and IgE pro-duction, it is possible that human tonsillar B cells represent pre-viously activated cells that have already received a signal throughthe BCR (51). Thus, the findings from both studies may indicatethat stimulation of the BCR is necessary for expression of theCD86 signal in an IL-4-dependent Ab response. Therefore, a func-tional consequence of BCR stimulation, in addition to up-regulat-ing CD86 expression, may be to allow for the generation of asignal through CD86 to increase the level of IL-4-dependent IgG1and IgE produced per B cell. These findings have suggested to usthat a similar mechanism may explain not only the normal amountof Ab produced in a Th2-dependent response in the absence ofb2AR stimulation, but also the increased level of Ab producedfollowing exposure of these B cells to a b2AR agonist.

While we attempted to address the effect of CD86 stimulation onB cells in a T cell-B cell culture system, we were concerned thatthe decreased IL-4 production (data not shown and Ref. 44) andCD40L expression (2) resulting from a lack of CD28 stimulationwould adversely affect the level of Ab produced and confound anyresults using the anti-CD86 Ab to directly stimulate the B cell.However, using the CD40L/Sf9 cell and IL-4 culture system, we

were able to show that culture of Ag-pulsed B cells from CD86-deficient mice with an anti-CD86 Ab, in comparison to B cellsfrom wild-type mice, failed to enhance the level of IL-4-dependentIgG1 and IgE produced, suggesting that the CD86 molecule isrequired for an anti-CD86 Ab to enhance IL-4-dependent Ab pro-duction. Although little is known about the signaling mechanismused by the CD86 molecule, sequence analysis of the CD86 pro-tein shows that the cytoplasmic domain of CD86 contains threepotential sites for tyrosine phosphorylation (52). Therefore, withsuch limited information about the CD86 molecule, it is difficult topropose either an intracellular signaling pathway used by CD86, amechanism by which CD86 may signal, or a mechanism by whichthe CD86 signal may influence the IL-4 signaling pathway. Incontrast to the b2AR- and CD86-mediated enhancement in theTh2-dependent Ab response, B cells pre-exposed to Ag/terbutalineand cultured with a Th1 clone produced a similar level of Th1-dependent Ab as B cells pre-exposed to Ag alone. In addition,stimulation of CD86 on B cells undergoing a Th1-dependent Abresponse did not enhance the level of either IgM or IgG2a pro-duced (D. J. Kasprowicz et al., manuscript in preparation), sug-gesting a specificity of both the b2AR- and CD86-mediated signalsfor the Th2-dependent Ab response. These findings suggest thatstimulation of CD86 on B cells in the presence of Ag and IL-4 mayprovide a mechanism for amplification of a component of the IL-4signaling pathway specifically to increase the level of IgG1 andIgE produced by an individual B cell. In a physiological sense, thismechanism may partially explain the slant of humoral immunitytoward a Th2- or IL-4-dependent response (reviewed in Refs. 53and 54), a response which the present data indicate can be mod-ulated by CD86 stimulation to provide a higher level ofprotective Ab.

The third mechanism by which IgG1 and IgE production wasenhanced involved a BCR-independent, b2AR-dependent increasein the ability of the B cell to respond to IL-4. Since it has beenreported that either IL-4 or Th2 cells induce B cells to switch to theproduction of either IgG1 and IgE (3, 29, 55, 56), it was importantto determine whether the mechanism by which b2AR stimulationenhances the level of IgG1 and IgE was similar to the switchingeffect typically induced by IL-4. The present experimental designused both flow cytometric and limiting dilution analysis to showthat b2AR stimulation on the B cell affected the amount of IL-4-dependent Ab secreted per cell, as opposed to increasing the num-ber of cells switching to IgG1 or IgE production. In addition, thiseffect required b2AR expression on B cells, as indicated by a fail-ure of terbutaline to induce the increase in Ab when cells fromb2AR-deficient mice were used. This effect of b2AR stimulationon the B cell also involved an increase in the ability of the B cellto respond to IL-4, without involving an increase in the level ofIL-4R expression on the B cell. These findings support the hy-pothesis that stimulation of the b2AR on the B cell influences astage of Ab production that occurs after the switching mechanismhas been activated. Further support for this hypothesis comes froma recent report showing that IL-4-exposed human B cells trans-formed with the EBV produced a higher level of Ab on a per cellbasis (IgM, IgG, and IgA) than transformed B cells that were notexposed to IL-4 (57), suggesting that IL-4 could influence a stageof Ab production other than isotype switching. In addition, otherstudies showed that stimulation of human PBMC with a b2ARagonist in the presence of IL-4 induced a higher level of IgE pro-duction via a mechanism that involved increased cell responsive-ness to IL-4, but not increased isotype switching (58, 59). There-fore, these findings suggest that the b2AR-induced effect on the Bcell will influence only those cells that switch isotype in responseto IL-4, possibly explaining the IL-4-dependency of the b2AR-


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induced effect. However, no reports exist at present to show a rolefor cAMP in modulating either the level of IL-4R expression, thedownstream signaling events associated with the IL-4R, or thelevel of IL-4R recycling.

AcknowledgmentsWe thank Michelle Swanson and Afsaneh Mozaffarian for critical readingof the manuscript, Andy Torres for technical assistance, Patricia Simms forflow cytometry assistance, and Linda Fox for confocal microscopyassistance.

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