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Departments of
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Microbiology and Immunology, and
Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, Maywood, IL 60153;
Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78284;
§
Department of Medicine and Molecular and Cellular Physiology, Stanford University, Stanford, CA 94305; and
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Department of Pathology, Brigham and Women’s Hospital, Boston MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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to induce B cells to produce
IgG2a, or Th2-dependent when Th2 cells interact with B cells and
secrete IL-4 to induce B cells to produce IgG1 and IgE (3, 4). Thus, the B cell receives signals for activation,
competence, isotype switching, and secretion by stimulation of the BCR,
CD40, and cytokine receptors. Studies have shown that the Ag-BCR interaction, the MHC class II/peptide-TCR interaction, and the CD40-CD40L interaction generate intracellular signals in both T cells and B cells to regulate both T cell and B cell function (2, 5, 6). The CD28-CD86 interaction was thought to deliver a signal to the T cell alone (7), but recently it was reported that stimulation of CD86 on human tonsillar B cells can increase the level of IgG1 and IgE produced after CD40 and IL-4R stimulation (8). Collectively, it is clear that multiple immune cell-derived signals influence both T and B cell function during the course of an Ab response, but much remains unknown about both the mechanism by which CD86 stimulation on B cells modulates the level of IL-4-dependent IgG1 and IgE production and the mechanisms by which the level of Ab produced by these B cells is regulated physiologically.
While the basic immune-related mechanisms of T cell and B cell activation and regulation have been defined, endogenous nonimmune mechanisms that may also modulate the level of cell activation and regulation remain unknown. One endogenous mechanism that modulates immune cell activity in vivo involves the autonomic nervous system that is comprised of the sympathetic system releasing the neurotransmitter norepinephrine and the parasympathetic system releasing acetylcholine. The sympathetic (adrenergic) system functions in a diverse fashion to induce modest effects on cellular activity, either stimulatory or inhibitory, while the parasympathetic (cholinergic) system functions in a limited fashion to conserve energy and primarily acts during periods of minimal activity (9). In addition, all organs that are regulated by the sympathetic nervous system are not always regulated by parasympathetic control. For example, the kidney, spleen, and arterioles are regulated primarily by sympathetic input as opposed to parasympathetic input (9, 10). In particular, the spleen is regulated almost exclusively by sympathetic control. No definitive data exist to our knowledge to show that parasympathetic regulation exists for other lymphoid organs, including the bone marrow, lymph nodes, and thymus. However, thymic epithelial cells have been shown to express cholinergic receptors (11, 12), but the significance of these receptors remains unknown. In contrast, there are a plethora of data to show that the neurotransmitter norepinephrine that is released from sympathetic nerve endings residing within the parenchyma of lymphoid organs (13, 14, 15) in response to Ag (16), LPS (17), or IL-1ß (18). Nerve endings containing norepinephrine directly appose lymphoid cells that express the ß2-adrenergic receptor (ß2AR) which binds norepinephrine to induce an increase in the intracellular concentration of cAMP (reviewed in Refs. 10 and 19). In vivo, norepinephrine has been shown to modulate the level of the Th cell-dependent Ab response (20), but the mechanism by which this occurs at the cellular level remains unclear. In vitro, it has been shown that norepinephrine enhances the level of Th cell-dependent IgM production through stimulation of the ß2AR (21, 22, 23), to allow for an increase in the frequency of B cells that differentiate into IgM-secreting cells (21). However, it remains unclear as to whether or not the level of IL-4-dependent IgG1 and IgE production can also be affected and whether or not the Th cell, B cell, or both cells are affected by ß2AR stimulation to induce the functional change.
In the present study, we explored a unique opportunity to determine the mechanism by which stimulation of the ß2AR on the B cell enhances IL-4-dependent Ab production by using splenic Trinitrophenyl (TNP)-specific B cells enriched from the spleens of unimmunized mice and activated by either a clone of ß2AR-negative Th2 cell (24, 25) or CD40L-expressing baculovirus-infected Sf9 cells (CD40L/Sf9 cells) and IL-4. Our findings using B cells from either wild-type, CD86-deficient, or ß2AR-deficient mice suggest three mechanisms by which the level of IL-4-dependent IgG1 and IgE production can be enhanced on a per cell basis, independently from the isotype switching event. The following findings support these mechanisms and are reported in the present study. First, the level of CD86 expression is increased on the B cell surface following BCR and/or ß2AR stimulation. Second, following BCR stimulation alone, CD86 becomes competent to enhance the level of IL-4-dependent Ab production. Third, the BCR- and CD86-mediated enhancing effect on Ab production is further augmented by ß2AR stimulation. And fourth, following ß2AR stimulation alone, Ab production is enhanced due to an increase in the ability of the B cell to respond to IL-4. These findings suggest unique mechanisms by which a combination of BCR, CD86, and ß2AR stimulation enhance the ability of Ag-specific B cells to produce IL-4-dependent IgG1 and IgE.
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Materials and Methods |
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Mice were maintained in a pathogen-free facility and were used between 7 and 15 wk of age. Female BALB/c mice (H-2d-restricted) were obtained from Harlan Sprague-Dawley (Indianapolis, IN), male and female ß2AR-deficient mice (H-2q-restricted) (26) were generously provided by Dr. Brian Kobilka (Stanford University, Stanford, CA), and CD86-deficient mice (H-2d-restricted) (27) were generously provided by Dr. Arlene Sharpe (Brigham and Women’s Hospital, Boston, MA). The ß2AR-deficient mouse was generated by homologous recombination resulting in the insertion of a neomycin resistance gene cassette into the fourth transmembrane domain of the ß2AR 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-Ad-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 x 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 x 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,
; Sigma)
or IgE-3 (IgE,, 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 x 103 to 2 x 104 cells/well depending on the isotype analyzed, with final values normalized to Ab production by 5 x 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 x 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.
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Results |
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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. 1A, 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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B, left) and IgE (Fig. 1C, 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. 1B, right) and IgE
(Fig. 1C, 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 ß2AR 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 ß2AR
expressed on the Ag-specific B cell (20) during Ag
pre-exposure. Previous studies have shown that stimulation of the
ß2AR 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. 2A, left). This
increase was blocked if the ßAR antagonist, nadolol, was added during
pre-exposure of B cells (Fig. 2A, 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. 2B, left), but did
prevent the terbutaline-induced increase in CD86 surface expression
(Fig. 2B, right). These findings indicate that the
ß2AR-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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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. 2C, left). In contrast, although
BCR stimulation alone on ß2AR-deficient B cells
up-regulated the level of CD86 surface expression, exposure to
terbutaline alone (data not shown) or Ag/terbutaline (Fig. 2C, right) did not, suggesting that stimulation
of the ß2AR 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
ß2AR-mediated effect was specific for CD86, B
cells were examined for the level of expression of other B cell surface
markers. Stimulation of the ß2AR on B cells did
not significantly change the level of expression of any other surface
markers examined, including CD80 (B7-1) (Table I).
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).
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
ß2AR on B cells and that the
ß2AR-mediated effect depends on cAMP activation
of PKA.
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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. 3A, 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. 3A) in
comparison to B cells pre-exposed to Ag and an anti-CD86 Ab alone
(Fig. 3A), suggesting that stimulation of the
ß2AR on the B cell further increased the BCR-
and CD86-mediated increase in IgG1 production. The
ß2AR-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 ßAR antagonist nadolol
(data not shown), suggesting that the terbutaline-induced effect on Ab
production was mediated through stimulation of ßAR. 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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B, stimulation of CD86 on B cells from
wild-type H-2q mice produced similar results to
those for wild-type H-2d mice described above
(Fig. 3A). Likewise, stimulation of CD86 on B cells from
ß2AR-deficient H-2q mice
pre-exposed to Ag enhanced the level of IgG1 in comparison to B cells
cultured with an isotype control Ab, suggesting that the
ß2AR was not required to induce the initial,
BCR- and CD86-mediated enhancement in IgG1. In contrast, stimulation of
CD86 on ß2AR-deficient
H-2q 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. 3C), suggesting that
the ß2AR 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
ß2AR 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 ß2AR 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
ß2AR 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
ß2AR on the B cell increased the responsiveness
of the B cell to one or both of these stimuli. First, we determined
whether ß2AR 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. 4A) produced 75–200% more
IgG1 than B cells pre-exposed to Ag alone. As shown in Fig. 4B, B cells from wild-type H-2q mice
showed a similar enhancement in IgG1 production as the B cells from
H-2d mice (Fig. 4A). In contrast, B
cells from ß2AR-deficient
H-2q mice did not display an increased ability to
respond to IL-4 following exposure to Ag/terbutaline (Fig. 4C). However, B cells that were pre-exposed to Ag alone or
Ag/terbutaline showed a similar level of IL-4R expression (Fig. 4D), 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
ß2AR on the B cell appeared to increase the
ability of the B cell to respond to IL-4, stimulation of the
ß2AR 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
ß2AR, but is independent of BCR stimulation and
does not involve an increase in the level of IL-4R surface
expression.
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Most experimental systems do not allow for the analysis of
the above described BCR-, CD86-, and
ß2AR-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
ß2AR-negative, Ag-specific Th2 clone (24, 25). Using this model system, we can determine whether
stimulation of the BCR and the ß2AR 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. 5A), with no change in the
number of Ab-secreting cell, when compared with B cells pre-exposed to
Ag alone (Fig. 5B). This enhancement was prevented by
nadolol (Fig. 5A) and occurred in a
terbutaline-concentration-dependent manner (Fig. 5C). 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
ß2AR 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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Limiting dilution analysis of the ß2AR-induced increase in Th2 cell-dependent Ab production and analysis of surface IgG1-positive B cells
To determine whether ß2AR 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. 6A, 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. 6B, 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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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It may be argued that the modest level of increase in Ab production by
ß2AR-stimulated B cells has no meaningful
effect in vivo. However, although only a few reports exist to show a
direct relationship between the level of Ab needed to provide a certain
level of protection against a specific Ag (46, 47, 48, 49), one of
these studies does provide compelling data supporting the relevance of
our finding (47). This study showed a correlation between
Ab concentration and opsonic titer following vaccination, such that an
2-fold increase in total Ab concentration increased the protective
titer 3-fold, while a 3-fold increase in total Ab concentration
resulted in a 9-fold increase in protective titer. The results from
this study indicate that a small increase in the level of neutralizing
Ab will significantly increase protection to the host and, thus, lend
relevance to our present finding that ß2AR
stimulation on the B cell induces a modest increase in the amount of Ab
produced.
Although, initially, it may seem that the concentration of
ß2AR agonist used in the present study is
higher than would be present in vivo, several studies have addressed
the concentration of norepinephrine present in vivo. A study by Shimizu
et al. (18) used a sensitive microdialysis procedure to
measure basal norepinephrine concentration released at nerve terminals
in a murine spleen and found it to be 1.6 x
10-6 M and 3.2 x
10-6 M before and after
the administration of IL-1ß, respectively. In addition, a previously
published study in vivo from our laboratory showed that the
administration of a ß2AR agonist at a dose of
10 mg/kg (equivalent to 150 µM) to immunized
norepinephrine-depleted animals partially restored the Ab response to
control levels. Finally, while the affinity of the
ß2AR may be high
(Kd = 0.1 nM), immune cells express
fairly low numbers of the ß2AR (B cell =
600 receptors/cell (20), Th1 cell clones = 250
receptors/cell (24), Th2 cell clones = 0
receptors/cell) (24)). Therefore, while adrenergic
receptors expressed on immune cells may have a high affinity for
norepinephrine or a ß2AR agonist, these
receptors are expressed at a low level and may require a high
concentration of norepinephrine or ß2AR agonist
to generate an intracellular signal. Thus, the concentration of
ß2AR agonist used in the present study is
within the range of the endogenous concentrations that should be
present within the microenvironment of a B cell responding to Ag
in vivo.
Although stimulation of either CD86 or the ß2AR alone modestly increased the level of BCR- and IL-4-dependent IgG1 and IgE produced, it is of interest that this modest increase in IgG1 and IgE was further enhanced up to 2-fold by concurrent exposure to both stimuli. Therefore, while the generation of one signal in the B cell may be sufficient to induce a modest change in the level of Ab produced, the generation of multiple signals may cooperate to induce a further amplification of the Th2-dependent Ab response. For example, norepinephrine stimulation of the ß2AR on a B cell is required for Ag-specific B cells to produce "normal" serum levels of Th2 cell-dependent Ab (20). However, we also know that when norepinephrine is removed in vivo, the ability of B cells to produce Ab is never ablated completely (20, 50), and when norepinephrine is added in vitro, the higher amount of Ab induced is rarely increased more than 3-fold. Thus, the immune system appears to regulate intrinsically the level of Ab produced by a B cell, but this level of regulation can be further modulated by mechanisms normally associated with nervous system regulation of organ function, such as norepinephrine stimulation of the ß2AR.
The present findings suggest a role for both BCR and ß2AR stimulation in modulating the level of CD86 expressed on the B cell surface. Using flow cytometry and confocal microscopy, we show that the expression of CD86 is up-regulated on murine B cells pulsed with either a specific Ag or terbutaline, findings that are in agreement with other studies using either HEL-transgenic splenic B cells stimulated with HEL (42) or splenic B cells stimulated with the cAMP analogue dibutyryl cAMP (dbcAMP) (43). However, the present study goes on to show that CD86 expression is further increased by stimulation of both the BCR by Ag and the ß2AR by terbutaline. In contrast, neither Ag or terbutaline increased the level of expression of the other B7 molecule, CD80, unlike the study using splenic B cells and dbcAMP in which CD80 expression was enhanced (43). However, similar to our finding, HEL stimulation of HEL-transgenic B cells did not modulate CD80 expression (42), suggesting that other mechanisms than those used by either the BCR or the ß2AR may be required to induce CD80 expression on a B cell. These findings also suggest that following both BCR and ß2AR stimulation, a higher level of CD86 would be available on the B cell surface at the initiation of an Ab response, perhaps to ensure optimal B cell activation during an Ag-specific response in vivo. Such an event is likely given that the amount and turnover of norepinephrine in the spleen increases after 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-exposed B cells induced no change in the level of Ab produced in comparison to stimulation of CD86 on either Ag- or Ag/terbutaline-exposed B cells, which induced an increase in the level of IgG1 produced. This finding suggests that stimulation of the BCR affects the ability of B cells to signal through CD86 differently from B cells that are not stimulated through the BCR. For example, a recent report showed that CD86 stimulation on human tonsillar B cells exposed to IL-4 and an anti-CD40 Ab increased the level of IgG4 (human equivalent of IgG1) and IgE produced (8). Although it initially appeared in this study that stimulation of the BCR was not required for the CD86 signal to enhance IgG1 and IgE production, it is possible that human tonsillar B cells represent previously activated cells that have already received a signal through the BCR (51). Thus, the findings from both studies may indicate that stimulation of the BCR is necessary for expression of the CD86 signal in an IL-4-dependent Ab response. Therefore, a functional consequence of BCR stimulation, in addition to up-regulating CD86 expression, may be to allow for the generation of a signal through CD86 to increase the level of IL-4-dependent IgG1 and IgE produced per B cell. These findings have suggested to us that a similar mechanism may explain not only the normal amount of Ab produced in a Th2-dependent response in the absence of ß2AR stimulation, but also the increased level of Ab produced following exposure of these B cells to a ß2AR agonist.
While we attempted to address the effect of CD86 stimulation on B cells in a T cell-B cell culture system, we were concerned that the decreased IL-4 production (data not shown and Ref. 44) and CD40L expression (2) resulting from a lack of CD28 stimulation would adversely affect the level of Ab produced and confound any results 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 cells from wild-type mice, failed to enhance the level of IL-4-dependent IgG1 and IgE produced, suggesting that the CD86 molecule is required for an anti-CD86 Ab to enhance IL-4-dependent Ab production. Although little is known about the signaling mechanism used by the CD86 molecule, sequence analysis of the CD86 protein shows that the cytoplasmic domain of CD86 contains three potential sites for tyrosine phosphorylation (52). Therefore, with such limited information about the CD86 molecule, it is difficult to propose either an intracellular signaling pathway used by CD86, a mechanism by which CD86 may signal, or a mechanism by which the CD86 signal may influence the IL-4 signaling pathway. In contrast to the ß2AR- and CD86-mediated enhancement in the Th2-dependent Ab response, B cells pre-exposed to Ag/terbutaline and 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 Ab response did not enhance the level of either IgM or IgG2a produced (D. J. Kasprowicz et al., manuscript in preparation), suggesting a specificity of both the ß2AR- and CD86-mediated signals for the Th2-dependent Ab response. These findings suggest that stimulation of CD86 on B cells in the presence of Ag and IL-4 may provide a mechanism for amplification of a component of the IL-4 signaling pathway specifically to increase the level of IgG1 and IgE produced by an individual B cell. In a physiological sense, this mechanism may partially explain the slant of humoral immunity toward a Th2- or IL-4-dependent response (reviewed in Refs. 53 and 54), a response which the present data indicate can be modulated by CD86 stimulation to provide a higher level of protective Ab.
The third mechanism by which IgG1 and IgE production was enhanced involved a BCR-independent, ß2AR-dependent increase in the ability of the B cell to respond to IL-4. Since it has been reported that either IL-4 or Th2 cells induce B cells to switch to the production of either IgG1 and IgE (3, 29, 55, 56), it was important to determine whether the mechanism by which ß2AR stimulation enhances the level of IgG1 and IgE was similar to the switching effect typically induced by IL-4. The present experimental design used both flow cytometric and limiting dilution analysis to show that ß2AR stimulation on the B cell affected the amount of IL-4-dependent Ab secreted per cell, as opposed to increasing the number of cells switching to IgG1 or IgE production. In addition, this effect required ß2AR expression on B cells, as indicated by a failure of terbutaline to induce the increase in Ab when cells from ß2AR-deficient mice were used. This effect of ß2AR stimulation on the B cell also involved an increase in the ability of the B cell to respond to IL-4, without involving an increase in the level of IL-4R expression on the B cell. These findings support the hypothesis that stimulation of the ß2AR on the B cell influences a stage of Ab production that occurs after the switching mechanism has been activated. Further support for this hypothesis comes from a recent report showing that IL-4-exposed human B cells transformed with the EBV produced a higher level of Ab on a per cell basis (IgM, IgG, and IgA) than transformed B cells that were not exposed to IL-4 (57), suggesting that IL-4 could influence a stage of Ab production other than isotype switching. In addition, other studies showed that stimulation of human PBMC with a ß2AR agonist in the presence of IL-4 induced a higher level of IgE production via a mechanism that involved increased cell responsiveness to IL-4, but not increased isotype switching (58, 59). Therefore, these findings suggest that the ß2AR-induced effect on the B cell will influence only those cells that switch isotype in response to IL-4, possibly explaining the IL-4-dependency of the ß2AR-induced effect. However, no reports exist at present to show a role for cAMP in modulating either the level of IL-4R expression, the downstream signaling events associated with the IL-4R, or the level of IL-4R recycling.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Virginia M. Sanders, Department of Cell Biology, Neurobiology, and Anatomy, 2160 South First Avenue, Loyola University Medical Center, Maywood, IL 60153.
3 Abbreviations used in this paper: BCR, B cell receptor; CD40L, CD40 ligand; ß2AR, ß2-adrenergic receptor; CD40L/Sf9 cells, CD40L-expressing baculovirus-infected Sf9 cells; RGG, rabbit gamma globulin; MFI, mean fluorescence intensity; TNP, trinitrophenol; ELISPOT, enzyme-linked immunospot; HEL, hen egg lysozyme; PKA, protein kinase A.
Received for publication January 20, 2000. Accepted for publication April 24, 2000.
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subunit mRNA expression in human thymus: augmented expression in myasthenia gravis and upregulation by interferon-. Clin. Immunol. 91:170.[Medline]
-1 and 
Ig gene expression in murine B cells: interleukin 4 and the CD40 ligand-CD40 interaction provide distinct but synergistic signals. J. Immunol. 155:5637.[Abstract]
: Role of B7-2 in the activation of naive and memory CD4+ T cells. Eur. J. Immunol. 25:1992.[Medline]
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) 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.
) as described in Fig. 2
