Stimulation of the B Cell Receptor, CD86 (B7-2), and the β₂-Adrenergic Receptor Intrinsically Modulates the Level of IgG1 and IgE Produced per B Cell

Animals

Mice were maintained in a pathogen-free facility and were used between 7 and 15 wk of age. Female BALB/c mice (H-2^d-restricted) were obtained from Harlan Sprague-Dawley (Indianapolis, IN), male and female β₂AR-deficient mice (H-2^q-restricted) (26) were generously provided by Dr. Brian Kobilka (Stanford University, Stanford, CA), and CD86-deficient mice (H-2^d-restricted) (27) were generously provided by Dr. Arlene Sharpe (Brigham and Women’s Hospital, Boston, MA). The β₂AR-deficient mouse was generated by homologous recombination resulting in the insertion of a neomycin resistance gene cassette into the fourth transmembrane domain of the β₂AR gene and has been described previously (26). The CD86-deficient mouse was generated by homologous recombination resulting in the replacement of a portion of the IgV region of the CD86 gene with a neomycin resistance gene cassette and has been described previously (27). Deficient mice were bred and housed within the pathogen-free facility. All mice were housed under 12-h light/dark cycle and provided autoclaved food pellets and water ad libitum.

Reagents

2,4,6-Trinitrobenzenesulfonic acid (TNBS), fluorescein isothiocyanate (FLU), normal rabbit gamma globulin (RGG), terbutaline, (−)-arterenol, nadolol, and the rp isomer of cAMP were purchased from Sigma (St. Louis, MO), and keyhole limpet hemocyanin (KLH) was obtained from Calbiochem (La Jolla, CA). All pharmacologic reagents were dissolved in culture medium and filter sterilized immediately before addition to cultures and tested negative 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). Recombinant mouse IL-4 was purchased from PharMingen (San Diego, CA).

Antibodies

The following Abs were used for surface staining and were purchased from PharMingen: purified and biotinylated-rat anti-mouse-CD86 (clone PO3), purified and biotinylated rat IgG2b (clone A95-1), FITC-streptavidin, and PE-streptavidin. ELISA Abs included unlabeled goat anti-mouse Ig or IgG, unlabeled and alkaline-phosphatase-conjugated rat anti-mouse IgE (clone 23G3), alkaline-phosphatase-conjugated goat anti-mouse IgM or IgG1, and streptavidin-conjugated alkaline phosphatase that were purchased from Southern Biotechnology Associates (Birmingham, AL).

Culture of Th2 cell clones

The Th2 cell clone CDC35 (RGG-specific, I-A^d-restricted) was generously provided by Dr. D. Parker (Oregon Health Sciences University, Portland, OR), maintained as described previously (24), used at least 7 days following restimulation, and was found to be Mycoplasma-free.

Isolation of Ag-specific B cells

Resting TNP-specific B cells were enriched from the spleens of unimmunized mice using a procedure described previously by Snow et al. (30) as modified by Myers et al. (28). The TNP-specific B cells recovered at the end of the procedure were cultured in complete medium in a humidified atmosphere of 5% CO₂ in air at 37°C for at least 24 h to allow for re-expression of surface-associated molecules before additional experimentation. Complete medium consisted of RPMI 1640 medium (Life Technologies, Grand Island, NY) containing 10% FBS (Life Technologies), 20 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 50 μM 2-ME. The phenotypic and functional characterization of resting splenic TNP-specific B cells has been presented previously (31). The percentage of B220⁺ B cells recovered 24 h after the isolation procedure is ∼90–95%.

Isolation of small, dense resting B cells

Small, dense resting B cells were isolated from the spleens of unimmunized mice as previously described (32, 33). The small high-density resting B cells were collected from a Percoll density gradient at the interface of the 1.082 and 1.097 g/ml layers. This population contained ∼90–95% B220⁺ B cells.

Culture conditions

Resting TNP-specific or small, dense resting B cells were pre-exposed in either complete medium alone or with TNP-RGG (0.7 μg/ml) or a F(ab′)₂ rabbit anti-mouse IgM (1.0 μg/ml), respectively, in 12 × 75 mm polystyrene tubes in a final volume of 0.5 ml for 18–24 h in a humidified atmosphere of 5% CO₂ in air at 37°C. B cells were plated in quadruplicate wells in a 96-well, flat-bottom microtiter plate (no. 3596; Costar, Cambridge, MA) at 5 × 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 0.2 ml. Th2 cells were added at a one to one ratio with B cells, while one CD40L/Sf9 cell was added per 10–80 B cells. IL-4 was added to B cell cultures at a concentration ranging from 0.1 to 10 ng/ml. In some experiments, either an anti-CD86 Ab from clone PO3 (0.01–2.5 μg/ml) or a species- and isotype-matched control Ab from clone A95-1 was added to the cell cultures. All cultures were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C. Anti-CD86 Ab from both the PO3 and GL1 clones was found to have B cell stimulatory activity; however, Ab from the GL1 clone, but not the PO3 clone, cross-reacted in the IgG ELISA and, therefore, only Ab from the PO3 clone was used in the experiments described herein. Two additional species- and isotype-matched control Abs (clones R35-38 and JES6-5H4) were also used in experiments and were found to lack B cell stimulatory activity.

IgG1 or IgE ELISA

On day 8 following the addition of CD40L/Sf9 cells and cytokine, the B cell supernatant was collected and immediately frozen at −80°C until analyzed by ELISA as described in detail previously (24). A standard curve for IgG1 and IgE Ab was prepared using known quantities of the myeloma protein MOPC-21 (IgG1,κ; Sigma) or IgE-3 (IgE,κ, PharMingen). Color development was determined on a UVmax kinetic microplate reader (Molecular Devices, Palo Alto, CA) at a wavelength of 405 nm. Lower limits of detection were as follows: for IgG1, <4 ng/ml; and for IgE, <4 ng/ml.

Enzyme-linked immunospot (ELISPOT) assay

A modification of the ELISPOT assay described by Czerkinsky et al. (34) and Sedgwick and Holt (35) was used to detect individual anti-Ig-secreting cells, and the protocol used has been described in detail elsewhere (21). For the detection of the amount of Ab secreted by cells, 100 μl of a developing solution was added as described for the ELISA, and color development was determined. For the detection of the number of cells secreting Ab, 100 μl of a developing solution consisting of 2.5 mM 5-bromo-4-chloro-3-indolyl phosphate in AMP buffer and 100 μg/ml nitro blue tetrazolium in 70% dimethylformamide was added to each well and incubated at 25°C for 3–4 h. Spot-forming cells were enumerated using a dissecting microscope. The number of B cells assayed for Ab production ranged from 5 × 10³ to 2 × 10⁴ cells/well depending on the isotype analyzed, with final values normalized to Ab production by 5 × 10⁴ input B cells.

Immunofluorescence staining and flow cytometric analysis

After 24 h of pre-exposure, B cells were washed once in HBSS + 1% FBS + 0.05% sodium azide (HBSS/FBS/azide), resuspended in 0.2 ml, and analyzed for the expression of various surface markers. Cells were incubated at 4°C with primary Abs, followed by secondary Abs and two washes in HBSS/FBS/azide. Each Ab was titrated to determine optimal staining concentration for flow cytometric analysis. Cells were fixed with 1% paraformaldehyde for 12 min at 4°C followed by storage in PBS/azide until flow cytometric analysis. Cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Diego, CA) gated on all viable cells. Calibration of the FACSCalibur was manually performed daily using Rainbow Calibration Particles (Sherotech, Libertyville, IL). Data were analyzed using CellQuest software (Becton Dickinson).

Confocal microscopy and image analysis

Samples were prepared as described above for flow cytometric analysis. Cells were examined with a Zeiss (Jena, Germany) LSM510 laser scanning confocal microscope and LSM510 v.2.1 imaging software. Samples were scanned and images were collected of the middle and top planes of individual cells. A minimum of 100 cells was examined per exposure group. Image analysis was performed using NIH Image software to determine the average intensity per B cell. B cells were designated CD86⁺ cells if the overall cell intensity was higher than the background intensity.

Limiting dilution analysis

Various numbers of Ag alone- or Ag/terbutaline-pre-exposed TNP-specific B cells were cultured with 3–5 × 10³ irradiated (1000 rads) cells of the CDC35 Th2 clone in a volume of 10 μl/well in a Terasaki-type (Nunc, Roskilde, Denmark) microtiter plate as described in detail previously (21). Precursor frequency analysis was performed according to Poisson statistics for the calculation from 37% interpolation as described by Lefkovitz and Waldmann (36).

Statistics

Data were analyzed by a one-way ANOVA to determine whether an overall statistically significant change existed before using the two-tailed unpaired Student’s t test. Statistically significant differences were reported when the p value was <0.05.

The level of CD86 expression on Ag-pulsed B cells and the effect of CD86 stimulation on CD40L/IL-4-induced IgG1 and IgE production

Previous studies have shown that various factors including LPS (37, 38, 39), anti-Ig (40, 41, 42), and the Ag hen egg lysozyme (HEL) (42) induce an up-regulation of CD86 expression on the surface of B cells. To determine whether a hapten-carrier conjugate would enhance the level of CD86 expression on hapten-specific B cells, resting TNP-specific B cells were enriched from the spleens of unimmunized mice and incubated with either medium alone or TNP-RGG for 18–24 h before being examined by flow cytometry and confocal microscopy. As determined by flow cytometric analysis, both the percentage of cells expressing CD86 and the mean fluorescence intensity (MFI) were increased from 57% and MFI = 260 on medium alone-exposed B cells to 66% and MFI = 644 on Ag-pulsed B cells, respectively, (Fig. 1⇓A, left). Next, to verify that the Ag-specific cell isolation procedure was not influencing the level of CD86 expression that was induced by BCR stimulation of B cells, the level of CD86 expressed on small, dense resting B cells was determined after cells were cultured with a F(ab′)₂ rabbit anti-mouse IgM (RAMIgM) for 24 h. As shown in Fig. 1⇓A, right, small, dense resting B cells exposed to a F(ab′)₂ rabbit anti-mouse IgM expressed a level of CD86 that was similar to that induced by a specific Ag on Ag-specific B cells.

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FIGURE 1.

The effect of BCR stimulation on CD86 expression and anti-CD86 Ab-induced IgG1 and IgE production by B cells from wild-type and CD86-deficient mice. A, left, TNP-specific B cells (5 × 10⁵) were pre-exposed to either medium alone or TNP-RGG (0.7 μg/ml) for 24 h at 37°C. A, right, Small, dense resting B cells were cultured with either medium alone or F(ab′)₂ rabbit anti-mouse IgM (RAMIgM, 1.0 μg/ml) for 24 h at 37°C. After 24 h, CD86 expression on B cells was determined using immunofluorescence and a FACSCalibur flow cytometer. Nonspecific binding 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 × 10⁴) from CD86^+/+ (left) or CD86^−/− (right) mice were pre-exposed to either medium alone or TNP-RGG (0.7 μg/ml) for 24 h at 37°C before exposure to CD40L/Sf9 cells (1 Sf9 cell per every 10 B cells) and IL-4 (1.0 ng/ml). In addition, either a soluble anti-CD86 Ab (▪) or species- and isotype-matched control Ab (□) was added to a final concentration of 1 μg/ml. After 8 days of culture at 37°C, supernatants were collected and analyzed for IgG1 (B) and IgE (C) by ELISA. Data are presented as the percent change from the Ag alone/isotype control response ± SE from quadruplicate wells from either four (left) or two (right) separate experiments. An asterisk (∗) indicates a p value <0.05 when the comparison is within a specific pre-exposure group. Ag alone/isotype control responses for 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 CD86^−/− mice ranged from 181 to 206 ng/ml; and for IgE in CD86^−/− mice ranged from 9 to 12 ng/ml.

Since Jeannin et al. (8) had reported that stimulation of CD86 on human tonsillar B cells increased the level of IL-4-dependent Ab production, we next determined if stimulation of CD86 on murine Ag-specific B cells exposed to Ag with an anti-CD86 Ab would enhance the level of IgG1 and IgE produced if cells were activated by CD40L/Sf9 cells and IL-4. The concentration of rIL-4 used was based on the level of IL-4 measured in T cell-B cell cultures (1.0 ng/ml), and the concentration of anti-CD86 Ab used (1.0 μg/ml) was based on a titration of anti-CD86 Ab for the most effective concentration to enhance the level of Ab production. B cells that were pre-exposed to Ag and cultured in the presence of an anti-CD86 Ab produced 50% more IgG1 (Fig. 1⇑B, left) and IgE (Fig. 1⇑C, left) than B cells either pre-exposed to Ag and cultured with an isotype control Ab or B cells pre-exposed to medium alone and cultured with either an anti-CD86 or isotype control Ab. These data suggest that stimulation of CD86 on Ag-exposed B cells enhances the level of IgG1 and IgE produced. To determine whether the increased level of IgG1 and IgE production was mediated through stimulation of CD86, we performed a similar experiment using B cells isolated from the spleens of unimmunized CD86-deficient mice. B cells from CD86-deficient mice pre-exposed to Ag and cultured in the presence of an anti-CD86 Ab produced a similar level of IgG1 (Fig. 1⇑B, right) and IgE (Fig. 1⇑C, right) as cells cultured with an isotype control Ab, suggesting that an intact CD86 molecule on the B cell surface is required for an anti-CD86 Ab to induce an enhancement in IgG1 and IgE. Although the anti-CD86-induced increase in IgG1 and IgE was modest, it occurred only when B cells expressed CD86 and only when B cells were pulsed with Ag before culture with CD40L/Sf9 cells, IL-4, and an anti-CD86 Ab. Taken together, these data suggest that both BCR and CD86 stimulation on the B cell are required to enhance the level of IgG1 and IgE production.

CD86 expression on B cells exposed to Ag and/or the β₂AR agonist terbutaline

We next determined if both the level of CD86 expression and the anti-CD86 Ab-induced increase in the level of IgG1 and IgE would be further modulated by stimulation of the β₂AR expressed on the Ag-specific B cell (20) during Ag pre-exposure. Previous studies have shown that stimulation of the β₂AR increases the concentration of intracellular cAMP (20) and that CD86 expression is up-regulated by increasing the concentration of intracellular cAMP in the B cell (43). We found a 10% increase in the percentage of B cells expressing a high level of CD86 when B cells were pre-exposed to Ag/terbutaline in comparison to B cells pre-exposed to Ag alone (Fig. 2⇓A, left). This increase was blocked if the βAR antagonist, nadolol, was added during pre-exposure of B cells (Fig. 2⇓A, right). The addition of a protein kinase A (PKA) inhibitor (rp cAMP) did not affect the level of CD86 expression induced by Ag alone (Fig. 2⇓B, left), but did prevent the terbutaline-induced increase in CD86 surface expression (Fig. 2⇓B, right). These findings indicate that the β₂AR-mediated increase in CD86 surface expression, above that induced by Ag alone, is mediated through activation of the cAMP-dependent PKA.

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FIGURE 2.

The effect of β₂AR stimulation on the level of CD86 surface expression by B cells from wild-type and β₂AR-deficient mice. A–C, TNP-specific B from either β₂AR^+/+ H-2^d or H-2^q or β₂AR^−/− H-2^q mice were pre-exposed to TNP-RGG (0.7 μg/ml) in either the presence or absence of terbutaline (Tb, 10⁻⁶ M), and/or nadolol (Nd, 10⁻⁵ M). After 24 h, CD86 expression on B cells was determined using immunofluorescence and a FACSCalibur flow cytometer. Nonspecific binding was determined using a species- and isotype-matched control Ab (dashed line). A left, 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 (gray shading), and Ag/Tb/Nd-exposed B cells (black shading). Data are representative of three separate experiments. B, Before B cell stimulation with Ag and Tb, B cells were incubated with a PKA inhibitor, rp cAMP (10 μM) for 30 min. Left, Ag alone-exposed B cells (gray shading), and Ag/rp cAMP-exposed B cells (black shading). Right, Ag/Tb-exposed B cells (black shading), and Ag/Tb/rp cAMP-exposed B cells (gray shading). Data are representative of two separate experiments. C, left, β₂AR^+/+ H-2^q B cells; right, β₂AR^−/− H-2^q B cells. Ag alone-exposed B cells (gray shading) or Ag/Tb-exposed B cells (black shading). Data are representative of three separate experiments.

To confirm the above described findings, we used B cells enriched from the spleens of unimmunized β₂AR-deficient mice. Because the β₂AR-deficient mice were generated in mice of a different haplotype (H-2^q) than that used for the above described work (H-2^d), we compared data from β₂AR-deficient mice with data generated from wild-type mice of the same haplotype. As shown in Fig. 2⇑C, left, BCR stimulation on B cells from wild-type mice up-regulated the level of CD86 expression above background and terbutaline further increased the level of CD86 expression (Fig. 2⇑C, left). In contrast, although BCR stimulation alone on β₂AR-deficient B cells up-regulated the level of CD86 surface expression, exposure to terbutaline alone (data not shown) or Ag/terbutaline (Fig. 2⇑C, right) did not, suggesting that stimulation of the β₂AR on B cells is responsible for the up-regulation of CD86 expression on B cells above the level induced by BCR stimulation alone. To confirm that the β₂AR-mediated effect was specific for CD86, B cells were examined for the level of expression of other B cell surface markers. Stimulation of the β₂AR on B cells did not significantly change the level of expression of any other surface markers examined, including CD80 (B7-1) (Table I⇓).

In addition to flow cytometric analysis, we quantified the level of CD86 expression at the single cell level using confocal microscopy and LSM510 imaging software. Images collected from B cells pre-exposed to Ag alone, Ag/terbutaline, and Ag/terbutaline/nadolol were collected and examined for CD86 expression (Table II⇓). Ag/terbutaline pre-exposure increased the percentage of B cells expressing a high level of CD86 that was blocked by culture of cells in the presence of nadolol. Taken together, these findings suggest that CD86 is expressed at a low level on the resting B cell surface and is up-regulated upon stimulation of either the BCR or β₂AR on B cells and that the β₂AR-mediated effect depends on cAMP activation of PKA.

The effect of β₂AR and CD86 stimulation on CD40L/IL-4-induced IgG1 production

We next determined whether the increased level of CD86 expressed by Ag/terbutaline pre-exposed B cells would further increase the BCR- and CD86-mediated enhancement in IgG1. As shown in Fig. 3⇓A, B cells that were pre-exposed to Ag alone and cultured in the presence of CD40L/Sf9 cells, IL-4, and an anti-CD86 Ab produced ∼50% more IgG1 than B cells pre-exposed to Ag alone and cultured with an isotype control Ab. B cells pre-exposed to terbutaline alone produced ∼50% more IgG1 than B cells pre-exposed to Ag alone, but this enhancement was not modulated by the presence of an anti-CD86 Ab, suggesting that the increase in IgG1 was not due to CD86 stimulation on B cells. However, B cells that were pre-exposed to Ag/terbutaline and stimulated with an anti-CD86 Ab produced ∼150% more IgG1 (Fig. 3⇓A) in comparison to B cells pre-exposed to Ag and an anti-CD86 Ab alone (Fig. 3⇓A), suggesting that stimulation of the β₂AR on the B cell further increased the BCR- and CD86-mediated increase in IgG1 production. The β₂AR-mediated enhancement in IgG1 production, but not the BCR- and CD86-mediated enhancement, was prevented if B cells were pre-exposed in the presence of the βAR antagonist nadolol (data not shown), suggesting that the terbutaline-induced effect on Ab production was mediated through stimulation of βAR. Taken together, these data suggest that stimulating CD86 on B cells pre-exposed to Ag enhances the level of IgG1 produced by these B cells, and that this response is further enhanced if B cells are pre-exposed to both Ag and terbutaline.

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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 and β₂AR-deficient mice. A–C, Resting splenic TNP-specific B cells (5 × 10⁴) from either β₂AR^+/+ H-2^d (A) or H-2^q (B) or β₂AR^−/− H-2^q (C) mice were pre-exposed to either medium alone or TNP-RGG (0.7 μg/ml) for 24 h at 37°C before exposure to CD40L/Sf9 cells (1 Sf9 cell per every 10 B cells) and IL-4 (1.0 ng/ml). In addition, either a soluble anti-CD86 Ab (▪) or species- and isotype-matched control Ab (□) was added to a final concentration of 1 μg/ml. After 8 days of culture at 37°C, supernatants were collected and analyzed for IgG1 by ELISA. Although not indicated by an asterisk, all terbutaline pre-exposure conditions from β₂AR^+/+ mice (A and B) induced a significant change at p < 0.05 when compared with the Ag alone/isotype control Ab group. Data are presented as the percent change from the Ag alone/isotype control Ab response ± SE from quadruplicate wells from three to four separate experiments. An asterisk (∗) indicates a p value <0.05 when the comparison is within a specific pre-exposure group. Ag alone/isotype control responses for IgG1 from β₂AR^+/+ H-2^d mice ranged from 63 to 229 ng/ml; IgG1 from β₂AR^+/+ H-2^q mice ranged from 4,106 to 10,302 ng/ml; and IgG1 from β₂AR^−/− H-2^q mice ranged from 2,144 to 12,013 ng/ml.

As shown in Fig. 3⇑B, stimulation of CD86 on B cells from wild-type H-2^q mice produced similar results to those for wild-type H-2^d mice described above (Fig. 3⇑A). Likewise, stimulation of CD86 on B cells from β₂AR-deficient H-2^q mice pre-exposed to Ag enhanced the level of IgG1 in comparison to B cells cultured with an isotype control Ab, suggesting that the β₂AR was not required to induce the initial, BCR- and CD86-mediated enhancement in IgG1. In contrast, stimulation of CD86 on β₂AR-deficient H-2^q B cells pre-exposed to either terbutaline or Ag/terbutaline did not enhance the level of IgG1 above that produced by B cells pre-exposed to Ag alone (Fig. 3⇑C), suggesting that the β₂AR is required to further enhance the BCR- and CD86-mediated enhancement in IgG1 production. We found that there was no significant difference in the percentage of cells expressing surface IgG1 following culture of cells in the presence of an anti-CD86 or isotype control Ab, CD40L/Sf9 cells, and IL-4 (data not shown), suggesting that stimulation of CD86 was not enhancing IgG1 production by inducing more B cells to switch from IgM to IgG1 production. Overall, these data suggest that stimulation of the β₂AR increases the level of CD86 expression on the B cell surface independently from that induced by BCR stimulation and further increases the level of IgG1 produced per B cell when both CD86 and the BCR are stimulated.

The effect of β₂AR stimulation on the ability of B cells to respond to IL-4

Since we observed an increase in the level of IgG1 produced by B cells pre-exposed to terbutaline alone and activated with CD40L/Sf9 cells and IL-4 that occurred independently of CD86 involvement, we wanted to determine the mechanism responsible for the β₂AR alone-mediated increase in the level of Ab produced. Because B cells were exposed to only IL-4 and CD40L/Sf9 cells, it seemed likely that stimulation of the β₂AR on the B cell increased the responsiveness of the B cell to one or both of these stimuli. First, we determined whether β₂AR stimulation enhanced Ab production via a direct mechanism that increased the ability of the B cell to respond to IL-4. B cells pre-exposed to either terbutaline alone (data not shown) or Ag/terbutaline (Fig. 4⇓A) produced 75–200% more IgG1 than B cells pre-exposed to Ag alone. As shown in Fig. 4⇓B, B cells from wild-type H-2^q mice showed a similar enhancement in IgG1 production as the B cells from H-2^d mice (Fig. 4⇓A). In contrast, B cells from β₂AR-deficient H-2^q mice did not display an increased ability to respond to IL-4 following exposure to Ag/terbutaline (Fig. 4⇓C). However, B cells that were pre-exposed to Ag alone or Ag/terbutaline showed a similar level of IL-4R expression (Fig. 4⇓D), suggesting that increased IL-4R expression was not allowing for the increased ability of terbutaline-exposed B cells to respond to IL-4. Also, while stimulation of the β₂AR on the B cell appeared to increase the ability of the B cell to respond to IL-4, stimulation of the β₂AR did not appear to increase the ability of the B cell to respond to CD40L (data not shown). Overall, these data suggest that the terbutaline-induced increase in the ability of the B cell to respond to IL-4 is dependent on the presence of the β₂AR, but is independent of BCR stimulation and does not involve an increase in the level of IL-4R surface expression.

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FIGURE 4.

The effect of β₂AR stimulation on the ability of B cells to respond to IL-4. TNP-specific B cells were enriched from β₂AR^+/+ H-2^d (A), β₂AR^+/+ H-2^q (B), or β₂AR^−/− H-2^q mice (C) and pre-exposed to Ag alone (□) or Ag/terbutaline (Ag/Tb) (▨) as described in Fig. 2⇑. After 24 h, IL-4 was added at concentrations ranging from 0.1 to 10 ng/ml and CD40L/Sf9 cells were added at a ratio of 1 CD40L/Sf9 cell per 10 B cells. After 8 days of culture, supernatants were collected and analyzed for IgG1 (upper) and IgE (lower) by ELISA. Data are presented as the mean ng/ml for each concentration of IL-4 ± SE from quadruplicate determinations from two to four separate experiments. An asterisk (∗) indicates a p value <0.05 in comparison to the Ag alone-pulsed B cells. D, B cells from β₂AR^+/+ H-2^d mice were pre-exposed as in Fig. 2⇑. After 24 h, the level of IL-4R expression was determined on TNP-exposed B cells (gray shading) and Ag/Tb-exposed B cells (black shading) using immunofluorescence and a FACSCalibur flow cytometer. Nonspecific binding was determined using a species- and isotype-matched control Ab (dashed line). Data are representative of two separate experiments.

Th2 cell-dependent Ab production by B cells pre-exposed to Ag and/or terbutaline

Most experimental systems do not allow for the analysis of the above described BCR-, CD86-, and β₂AR-dependent mechanisms for enhancing IL-4-dependent Ab production in an Ag-specific manner. However, a model system is available in which Ag-specific B cells are cultured with a β₂AR-negative, Ag-specific Th2 clone (24, 25). Using this model system, we can determine whether stimulation of the BCR and the β₂AR on the B cell would induce an enhancement in Th2 cell-dependent Ab production similar to the enhancement in IL-4-dependent Ab production found using CD40L/Sf9 cells and IL-4. As determined by a modification of the ELISPOT assay that allows for the determination of the amount of Ab secreted by a population of B cells on a particular day of culture, B cells pre-exposed to Ag/terbutaline before their culture with a clone of Th2 cells produced 50–200% more IgM, IgG1, and IgE (Fig. 5⇓A), with no change in the number of Ab-secreting cell, when compared with B cells pre-exposed to Ag alone (Fig. 5⇓B). This enhancement was prevented by nadolol (Fig. 5⇓A) and occurred in a terbutaline-concentration-dependent manner (Fig. 5⇓C). When similar experiments were performed with a Th1 clone, we found that B cells pre-exposed to Ag/terbutaline or Ag alone produced a similar level of IgM and IgG2a, suggesting that stimulation of the β₂AR on the B cell was not enhancing Th1-dependent Ab production (D. J. Kasprowicz and V. M Sanders, manuscript in preparation).

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FIGURE 5.

The effect of β₂AR stimulation on the level of IgM, IgG1, and IgE produced by B cells cultured with a clone of Th2 cells. A and B, TNP-specific B cells were pre-exposed to 0.7 μg/ml TNP-RGG in the presence or absence of terbutaline (Tb, 10⁻⁶ M) and/or nadolol (Nd, 10⁻⁵ M). After 24 h at 37°C, 5 × 10⁴ pre-exposed B cells were added to an equal number of irradiated (1000 rads) RGG-specific Th2 cells (clone CDC35). After 7 days of culture, cells were assayed by ELISPOT for the amount of Ab secreted (A) and the number of cells secreting Ab (B). Data are presented as either the mean ng/ml ± SE or the number of cells secreting Ab ± SE per 5 × 10⁴ input B cells from triplicate wells from one of three separate experiments for IgM and IgG1 and of two separate experiments for IgE. C, B cells were prepared as described in A, except that terbutaline was added in concentrations ranging from 10⁻⁸ to 10⁻⁵ M. After 7 days of culture, cells were assayed by ELISPOT for the amount of Ab secreted. Data are presented as the percent change from the Ag alone response ± SE from triplicate wells from three separate experiments. An asterisk (∗) indicates a p value <0.05 in comparison to Ag alone pre-exposed B cells.

Importantly, culture of Th2 cells with B cells pre-exposed to Ag/terbutaline in comparison to B cells pre-exposed to Ag alone did not affect the level of IL-4 produced by the Th2 cells, the amount of [³H]thymidine incorporated by the Th2 cells, or the number of Th2 cell:B cell conjugates that formed (data not shown). In addition, B cells pre-exposed to Ag/terbutaline showed a similar kinetics of Ab production to cells pre-exposed to Ag alone, suggesting that the β₂AR-mediated enhancement in Ab production was not due to a shift in the timing of Ab production (data not shown). Unfortunately, it was not possible to further address the effect of stimulating CD86 with an anti-CD86 Ab in the T cell-B cell culture because the addition of an anti-CD86 Ab to cultures decreased the level of cytokines produced by the T cell (data not shown and Ref. 44), suggesting that the addition of an anti-CD86 Ab was blocking the CD28-CD86 interaction and preventing maximal activation of the T cell to secrete a required level of IL-4. Nonetheless, taken together, the above data suggest that stimulation of B cells through the BCR and the β₂AR enhances Th2 cell-dependent IgM, IgG1, and IgE production on a per cell basis by a direct effect on B cell function alone.

Limiting dilution analysis of the β₂AR-induced increase in Th2 cell-dependent Ab production and analysis of surface IgG1-positive B cells

To determine whether β₂AR stimulation was influencing the degree of isotype switching in B cells, we pre-exposed B cells to Ag and/or terbutaline before culture with either a Th2 clone or CD40L/Sf9 cells and IL-4, and subsequently analyzed cells by limiting dilution and flow cytometric analysis (45), respectively. As shown in Fig. 6⇓A, Ag/terbutaline induced a small increase in the frequency of IgE-secreting cells (1:2600 for Ag vs 1:1750 for Ag/terbutaline), but induced no change in the frequency of either IgM-secreting cells (1:180) or IgG1-secreting cells (1:500) when compared with B cells pre-exposed to Ag alone. No change in burst size was detected for any isotype of Ab measured. As shown in Fig. 6⇓B, pre-exposure of B cells to Ag/terbutaline did not change the percentage of surface IgG1-positive B cells generated in comparison to B cells pre-exposed to Ag alone. Taken together with the previously described finding using ELISPOT, pre-exposure of B cells to Ag/terbutaline and culture with either Th2 cell clones or CD40L/Sf9 cells and IL-4 induced B cells to produce more Ab per cell, as opposed to inducing an increase in either the number of mature precursor B cells differentiating into Ab-secreting cell, burst size, or the number of cells switching isotype.

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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 3–5 × 10⁴ irradiated (1000 rads) Th2 cells (clone CDC35). After 5–7 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 size analysis were performed according to Poisson statistics as described in the Materials and Methods. Frequency values represent the mean number of nonresponding wells with a 95% confidence limit. Burst size values are presented within parentheses as the mean number of cells produced per precursor of two to three individual experiments from groups of 60 wells. The thick line indicates the position of 37% nonresponding wells. B, Pre-exposed B cells were 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, cells were 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 are represented as “resting.” Data are representative of two separate experiments.