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Departments of
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Cell Biology, Neurobiology, and Anatomy and
Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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AR-selective antagonist, the effect of NE on the Ab response was
shown to be mediated by the ßAR. In addition, administration of a
ß2AR-selective agonist to NE-depleted mice partially reversed the
suppressed Ab response that resulted from NE depletion. Expression of
the ß2AR on TNP-specific B cells was confirmed by radioligand
binding, immunofluorescence, and cAMP analysis. Also, while splenic
histology was comparable in NE-intact and NE-depleted mice before Ag
exposure, follicle expansion and germinal center formation were
suppressed in NE-depleted mice after Ag exposure. Taken together, these
results suggest that NE stimulation of the ß2AR expressed on B cells
is necessary for the maintenance of an optimal primary and secondary
Th2 cell-dependent Ab response in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many groups have described the innervation of both primary and
secondary lymphoid organs by sympathetic nerve fibers. These studies
demonstrate rich sympathetic innervation penetrating the parenchyma of
lymphoid organs via the vasculature that supplies the periarterial
lymphoid sheath (PALS), marginal zone, and marginal sinus (8). In
addition, electron microscopic studies reveal that sympathetic nerve
terminals are in direct apposition to T cells and interdigitating
dendritic cells (8), with this junction being 
6 nm wide compared
with a typical CNS synapse, which is 20 nm wide. Following exposure
to Ag (9, 10), LPS (11), or IL-1 (11) in vivo, the sympathetic
neurotransmitter norepinephrine (NE) is released from sympathetic nerve
terminals and is bound by the ß-adrenergic receptor (ßAR) expressed
on resident lymphocytes, which induces an increase in the intracellular
concentration of cAMP (7).
A variety of murine immune cells express the ßAR, including
macrophages, NK cells, B cells, and T cells (reviewed in 7).
Recent data show that the ß2AR is differentially expressed on both
resting and anti-CD3-activated murine CD4+ effector T
cell clones, with detectable expression on Th1 cells, but not on Th2
cells, as determined at the protein level by radioligand binding and
immunofluorescence analysis (12, 13), at the mRNA level by RT-PCR
(A. P. Kohm et al., manuscript in preparation), and at the
functional level by cAMP accumulation following receptor stimulation
with a ß2AR-selective agonist (12). Furthermore, the functional
relevance of differential ß2AR expression on Th1 and Th2 cell clones
was demonstrated by the finding that ß2ARpos Th1 clones
pre-exposed to a ß2AR-specific agonist before interaction with
nonexposed Ag-presenting B cells in vitro produced less IFN-
than
nonexposed Th1 cell controls and resulted in reduced IgG2a production
by the B cells (12). As expected, ß2ARneg Th2 clones
preexposed to a ß2AR-specific agonist before interaction with
nonexposed B cells produced the same amount of IL-4 as nonexposed Th2
clones and resulted in no change in IgG1 production by the
Ag-presenting B cells. In contrast, preliminary findings in our
laboratory suggest that stimulation of the ß2AR on B cells during Ag
processing, but before interaction with ß2ARneg Th2 cell
clones, results in an enhancement of IgG1, but not IL-4, production (D.
Kasprowicz et al., manuscript in preparation). Thus, the lack of ß2AR
expression on clones of Th2 effector cells provides a unique
opportunity to study the possible role played by this receptor that is
expressed on B cells alone in the NE-induced modulation of the Th2
cell-dependent Ab response both in vitro and in vivo.
In previous studies to determine the role of NE in modulating the Th cell-dependent Ab response in vivo, peripheral sympathetic nerve terminals containing NE were reversibly destroyed in animals by chemical sympathectomy with the neurotoxin 6-hydroxydopamine (6-OHDA) before exposure to Ag. Results from experimental model systems using 6-OHDA-treated animals have been conflicting, showing enhancement (9, 14, 15), suppression (16, 17, 18), or no change (19) in the level of Ab production and cell proliferation. One possible source of these conflicting results may be the low frequency of Ag-specific lymphocytes in previous animal model systems that makes it difficult to study the effect of NE on Ag-specific B cell interactions with Ag-specific Th2 effector cells that provide the cytokines necessary for an Ag-specific IgG response. In addition, it has been difficult in previous studies to dissect the effect of NE on CD4+ T cell differentiation into Th1 and Th2 effector cells after the first exposure to Ag from the effect of NE on fully differentiated Th1 and Th2 effector cells themselves.
Although considerable insight into the role of NE in the T cell-dependent Ab response was gained from these previous in vivo studies, we attempted to address some of these concerns by developing an in vivo model system in which an enriched population of resting murine TNP-specific B cells from the spleens of unimmunized mice and clones of keyhole limpet hemocyanin (KLH)-specific Th2 effector cells were adoptively transferred to scid mice previously depleted of NE by 6-OHDA. This model system enables us to determine the role that NE plays in modulating the Th2 cell-dependent IgM and IgG1 responses and to determine which AR subtype is responsible for mediating the NE-induced effect. Some of the advantages of the model system presented herein, compared with previous model systems, are that there is a high frequency of Th2 cells and B cells responding to a given Ag, the Ab response is limited to a Th2 effector cell-mediated response, the lymphocytes are not present at the time of chemical sympathectomy, and the lymphocytes are not exposed to the burst of NE that is released from nerve terminals immediately after 6-OHDA exposure. Therefore, this model system makes it possible to study the mechanism by which Th2 cells and B cells participate in a Th2 cell-dependent Ab response in the absence of AR stimulation by NE as well as after the administration of pharmacological agents that act as either agonists or antagonists at AR binding sites.
In this report we show that B lymphocytes express the ß2AR and that stimulation of this receptor by NE is essential for maintaining an optimal level of IgM and IgG1 production in reconstituted scid mice. The scid mice that were depleted of NE before their reconstitution with clones of KLH-specific Th2 cells and enriched populations of TNP-specific B cells produced less serum anti-TNP IgM and IgG1 following primary immunization with TNP-KLH than NE-intact mice. After secondary immunization these mice produced less IgG1, but control levels of IgM. In addition, although there was no effect of NE depletion on splenic histology and lymphocyte trafficking before immunization, splenic follicle expansion and germinal center formation were less evident in NE-depleted reconstituted scid mice following both primary and secondary immunization compared with those in NE-intact reconstituted scid mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six-week-old female C.B-17/ICR (H-2d,
Igh-Cb), C.B-17/ICR scid (H-2d,
Igh-Cb), BALB/c (H-2d, Igh-Ca), and
B6C3F1 (H-2b/k) mice were obtained from Harlan
Sprague-Dawley (Indianapolis, IN). All mice were provided autoclaved
pellets and water ad libitum. The scid mice received
tetracycline HCl (2 mg/ml; Pfizer, New York, NY) in their drinking
water 3 days/wk. Mice were permitted 2 wk to acclimate to their
environment before being manipulated and were used at 8 wk of age in
all experiments. The scid mice were housed under a 12-h
light, 12-h dark cycle in microisolater cages contained within a
laminar flow system thus maintaining a pathogen-free environment, and
all experimental manipulations occurred 4 h into the light cycle.
Reagents and Abs
2,4,6-Trinitrobenzenesulfonic acid was purchased from Fluka (Ronkonkoma, NY). OVA, trinitrophenyl (TNP), and fluorescein (FLU) were purchased from Sigma (St. Louis, MO), and KLH was obtained from Calbiochem (La Jolla, CA). TNP-KLH, TNP-OVA, or FLU-KLH were prepared at a haptenation ratio of 17–24 TNP or FLU molecules/KLH or OVA carrier molecule. Terbutaline, nadolol, phentolamine, and metaproterenol were purchased from Sigma. All pharmacologic agents were dissolved in sterile PBS immediately before administration in vivo. Abs used in the ELISA were purchased from PharMingen (San Diego, CA). A rabbit polyclonal IgG directed against an epitope corresponding to aa 399–418 mapping to the carboxyl terminus of the ß2AR of mouse origin and control peptide sequences (aa 399–418) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
NE depletion
Chemical sympathectomy was performed at 8 wk of age. Mice received 200 mg/kg injections (i.p.) of 6-OHDA (Sigma) in 0.5 M saline containing 1 x 10-3 M ascorbate as an antioxidant. Mice received three injections of 6-OHDA on alternating days (days -6, -4, and -2 before cell reconstitution), while control mice received ascorbate-only injections on the same injection schedule.
T cell clones
The Th2 cell clone BAC 3.2 was maintained as described previously (12). Viable cells were obtained before use by centrifugation over Lympholyte-M (Accurate, Westbury, NY) 8–14 days after Ag stimulation. Clones were maintained in IL-2-containing medium and were used at least 3 days after exposure to IL-2. BAC 3.2 was tested for the presence of Mycoplasma contamination (Life Technologies, Gaithersburg, MD) and were negative.
TNP-specific B cell preparation
The procedures for enrichment of unprimed TNP-specific B
lymphocytes from spleens of nonimmunized mice were adapted from those
described by Snow et al. (20) as modified by Myers et al. (21). All
procedures were performed at 4°C, except for RBC haptenation and
enzyme treatment at 37°C. Briefly, horse RBCs (Colorado Serum,
Denver, CO) were haptenated with 20 mg of 2,4,6-trinitrobenzenesulfonic
acid per ml of packed RBCs. Spleen cell/haptenated horse RBC
suspensions were prepared, and rosette-forming B lymphocytes were
separated by velocity and density sedimentation using a discontinuous
Percoll gradient. The lymphocyte-bound RBCs were removed by a mild
trypsin-pronase treatment, and the lymphocytes were collected over
Ficoll. The lymphocytes recovered at the end of the procedure were
incubated overnight to allow for reexpression of surface-associated
molecules before additional experimentation. Phenotypic and functional
characterization of the unprimed TNP-specific B cells have been
presented previously, and the resultant cell population contains
85–90% TNP-specific B cells (22).
Cell transfer and immunization
Two days following the last 6-OHDA injection, all animals received both BAC-3.2 Th2 cells and TNP-specific B cells. Each cell type was prepared for adoptive transfer at 2 x 106 cells in 50 µl of PBS. T and B cell dilutions were prepared separately and were combined only at the time of injection. Cells were injected i.v. into the lateral tail vein in a total volume of 100 µl of PBS. One week after cell reconstitution, sympathectomized animals received primary immunizations i.p. with 100 µg of TNP-OVA, TNP-KLH, or FLU-KLH, delivered in the adjuvant TiterMax Gold (CytRx, Norcross, GA). In some experiments, mice received an i.p. injection of a ß2AR-selective agonist (5 mg/kg terbutaline or metaproterenol; Sigma), a ßAR-selective antagonist (5 mg/kg nadolol; Sigma), or saline at the time of Ag injection.
Detection of secreted Ag-specific and allotype-specific anti-TNP IgM and IgG1
Briefly, 96-well, round-bottom Immulon plates (Dynatech,
Chantilly, VA) were coated with 100 µg/ml of TNP20-OVA
overnight at 4°C, washed with PBS, and blocked with PBS/1% BSA for
1 h at 37°C. Ab-containing serum, diluted between 1/100 to
1/1000, was added to each well. Plates were incubated for 2 h at
37°C in a humidified atmosphere. Wells were washed with PBS/0.05%
Tween-20. For Ag-specific assays, a 1/2000 dilution of alkaline
phosphatase-conjugated goat anti-mouse IgM or IgG1 Ab (Southern
Biotechnology Associates, Birmingham, AL) was added to each well for
1 h at 37°C. For allotype-specific assays, a 1/1000 dilution of
biotin-conjugated goat anti-mouse IgMa (Igh-6a),
IgMb (Igh-6b), IgG1a (Igh-4a), or
IgG1b (Igh-4b) (PharMingen) was added to each well for
1 h at 37°C followed by the addition of a 1/1000 dilution of
streptavidin-alkaline phosphatase (PharMingen) to each well for 1
h at 37°C. A developing solution consisting of 1 mg/ml
p-nitrophenyl phosphate in 10 mM diethanolamine-0.5 mM
MgCl2 buffer, pH 9.4, was added. A standard curve for
IgM or IgG1 Ab was prepared using known quantities of the myeloma
protein MOPC-104E (IgM 
; Sigma) or MOPC-21 (IgG1 
; Sigma) and was
detected on a plate coated with 2 µg/ml of goat anti-mouse Ig.
Color development was determined on a UVmax kinetic microplate reader
(Molecular Devices, Menlo Park, CA) at a wavelength of 405 nm.
Tissue sample preparation for HPLC
Spleen cell suspensions were prepared from animals following sympathectomy. Cell/supernatant and capsule fractions were analyzed separately. All samples were homogenized in 0.1 M HClO4, spun at 11,000 x g, and added to extraction chambers of a centrifuge filter apparatus containing 3,4-dihydroxybenzylamine (20 ng/µl) that was used as an internal control. To perform catecholamine extraction, 50 mg of acid-washed alumina was added to each tube, the pH was adjusted to 8.3 with Tris/EDTA, and the tube was vortexed. The basic solution allows for absorption of NE onto alumina. Samples were then washed twice with distilled water, and catecholamines were extracted with 200 µl of 0.1 M HClO4. Samples were stored at -80°C until analysis by HPLC as described previously (10).
Histology and immunohistochemistry
Spleen tissue was removed from each animal and stored at -80°C until the time of analysis. For hematoxylin-eosin histology, spleens were fixed in 10% formalin, embedded in paraffin, sectioned by microtome at 8 µm, and stained with hematoxylin and eosin stains. For immunohistochemistry, frozen tissues were embedded in O.C.T. (Miles, Elkhart, IN) and sectioned at 8 µm. Sections were air-dried for 60 min, O.C.T. was removed by a 5-min incubation in 50% ethanol, and sections were fixed in cold acetone for 5 min. Following fixation, endogenous peroxidase activity was blocked by a 60-min incubation with Universal Block (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and incubation with biotinylated rat anti-mouse CD4 (clone RM4–5; PharMingen), biotinylated rat anti-mouse B220 (clone RA3-6B2; PharMingen), or peanut agglutinin (PNA)-FITC (Sigma) was performed at 37°C for 1 h in 10% BSA. Samples incubated with biotinylated primary Ab were then incubated with streptavidin-HRP (Kirkegaard & Perry Laboratories) for 30 min at room temperature and developed with cobalt-enhanced DAB (Kirkegaard & Perry Laboratories) for 10 min at room temperature. Sections incubated with PNA-FITC were washed in PBS for 10 min and coverslipped using AquaMount (Fisher, Pittsburgh, PA) with 50 mg/ml of 1,4-diazabicyclo[2.2.2]octane (DABCO, Sigma), which was used as an antifading agent for fluorescence studies. Specific staining was visualized by confocal microscopy using a Zeiss 460 confocal microscope (Zeiss, Oberkochen, Germany).
Radioligand binding assays
The iodinated ß-adrenergic antagonist pindolol ((-)-125I-IPIN) was used to assay for receptor characteristics. This ligand was purchased from New England Nuclear (Boston, MA) and has a sp. act. of 2200 Ci/mmol. IPIN is the ligand of choice for radioligand binding site analyses because of its high sp. act. of 2200 Ci/mmol and the exceptionally high affinity of the ßAR for this ligand. These properties of IPIN allow us to obtain high quality binding data from cells that are not available in large numbers and that express very low numbers of the ßAR binding site. It is important to note that the ß2AR has a very high affinity for the antagonist IPIN compared with the affinity of the receptor for the agonists terbutaline and norepinephrine. This is a characteristic of receptor antagonists in general, and it is exploited here to facilitate receptor detection as opposed to predicting cellular responsiveness to ß2AR stimulation by agonists. Cells were fixed in 3% (v/v) formalin for 10 min at room temperature and were stored in PBS/azide at 4°C until assayed. Preliminary studies showed that the kinetics of (-)-125I-IPIN binding to unfixed and fixed B cells were similar, and that binding was to a single class of saturable binding sites (data not shown). Cells were assayed using a modification of a method originally described by Williams et al. (23). Assays were conducted using 8.8 x 45-mm tubes filled with 1) 75 µl of the radioligand (-)-125I-IPIN at 1 of 12 concentrations ranging from 2–480 pM, 2) either 75 µl of 6 x 10-3 M ascorbate (in the tubes which measure total binding) or 75 µl of 6 x 10--6 M (-)-propranolol in 6 x 10-3 M ascorbate (in the tubes that measure nonspecific binding), and 3) 300 µl of the cell suspension containing 1–2 x 106 cells. The microtiter tubes were incubated for 1 h at 37°C and then filtered onto glass fiber filters using a Cell Harvester (Brandel, Gaithersburg, MD). After five washes using a cold buffer (0.45 M Tris-HCl and 0.170 M MgCl2·6H2O; pH 7.2) the samples were counted in a Beckman 5500 B gamma counter (Beckman, Palo Alto, CA) with a calculated accuracy of 87%. Both total and nonspecific binding tubes were run for each time point. Results are reported in picomols of IPIN bound. The nonspecific binding was determined in the tubes containing (-)-propranolol at a final concentration of 1 µM. Specific binding of radioligand was determined by subtracting nonspecific binding from the counts occurring in the total binding tubes for each concentration of radioligand used. Binding curve data were analyzed using the computer program LIGAND (24).
cAMP measurement
Assays were performed using B cells at a density of 1 x 106 cells/ml in serum-free HBSS containing 10 mM HEPES and 1 mM 3-isobutyl-1-methylxanthine (Sigma) to inhibit phosphodiesterase activity. After the addition of terbutaline and incubation at 37°C for 30 min, the reaction was terminated by the addition of 3 parts cold ethanol. The soluble fraction was collected by centrifugation and lyophilized. cAMP was measured using a nonisotopic immunoassay system for cAMP (Life Technologies). Results are expressed as picomols of cAMP per 106 cells.
Immunofluorescence staining
B cells were resuspended at 5 x 105 cells/ml and were fixed for 20 min at room temperature in 2% paraformaldehyde. Cells were stained using a modification (25) of a previously described procedure (26). All staining Abs and detection reagents were purchased from Santa Cruz Biotechnology. All reagents and washes were performed in the presence of 1% BSA and 0.5% saponin (Sigma), and incubations were conducted at room temperature. Cells were first incubated for 10 min in PBS/BSA/saponin and then stained for 30 min with a polyclonal rabbit anti-mouse ß2AR Ab (1 µg/ml final concentration) in the absence or the presence of a mouse ß2AR-specific peptide sequence (10 µg/ml final concentration) that corresponds to the epitope of the ß2AR recognized by the anti-mouse ß2AR Ab. Cells were washed and incubated for 30 min with optimal concentrations of a biotinylated secondary Ab in PBS/BSA/saponin followed by FITC-streptavidin. Cells were washed twice with PBS/BSA/saponin and twice with PBS/BSA without saponin to allow membrane closure. Samples were analyzed on a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA) gated on all viable cells. Calibration of the FACStar Plus was manually performed daily using Rainbow Calibration Particles (Sherotech, Libertyville, IL). Results were analyzed using LYSIS II software (Becton Dickinson).
Statistics
Concentration-response data were first analyzed by a one-way ANOVA to determine whether an overall statistically significant change existed previous to using two-tailed unpaired Student’s t test. Statistically significant differences are reported at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the Ag specificity of the Ab response in
scid mice reconstituted with a clone of KLH-specific Th2
cells and TNP-specific B cells, mice were immunized with TNP-KLH,
TNP-OVA, FLU-KLH, or adjuvant alone and evaluated for their ability to
produce anti-TNP IgM. As shown in Fig. 1
, a significant level of anti-TNP
IgM was detected in reconstituted mice immunized with TNP-KLH. In
contrast, mice receiving either TNP-OVA or FLU-KLH produced levels of
anti-TNP IgM that were considerably lower than those in
TNP-KLH-injected mice, but were comparable to those in adjuvant-only
injected controls. Thus, these data suggest that hapten-carrier
specificity exists for the observed anti-TNP response and that
TNP-specific B cells bind TNP-KLH, process it, and present the KLH
peptide to the KLH-specific Th2 cell clone.
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). Thus,
the lack of detectable serum anti-TNP IgM in mice reconstituted
with TNP-specific B cells of different haplotypes suggests that an
MHC-restricted cognate interaction between the TNP-specific B cell and
KLH-specific Th2 cell is necessary for an appreciable level of
anti-TNP IgM to be produced.
While the scid mutation is a homozygous (-/-) mutation, it
is not absolute. "Leakiness" of the mutation is defined as the
appearance of mature T and B cells in the periphery of CB.17
scid mice in an age- and species-dependent manner (27), but this
is less common in CB.17/ICR scid mice. To determine whether
leakiness occurred in reconstituted scid mice at any time
after immunization, serum Ab was checked for allotype specificity. It
is known that B cells of BALB/c mice produce the Ab allotype
Igha, while B cells of the sibling pairs (+/-) of the
scid mouse produce the Ab allotype Ighb (28). As
shown in Fig. 2, scid mice
(-/-) reconstituted with TNP-specific B cells isolated from BALB/c
mice produced IgM and IgG1 only of the allotype Igha. In
addition, serum anti-TNP IgM was almost sixfold higher in
reconstituted scid mice than in sibling controls, suggesting
that the frequency of KLH-specific T cells and TNP-specific B cells was
greater in the reconstituted scid mice than in sibling
pairs. This result was obtained with all serum samples analyzed
regardless of mouse age. Thus, these results suggest that the donor B
cells, as opposed to leaky scid B cells, produced the serum
anti-TNP Ab in our model system.
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Administration of 6-OHDA to mice induces a long lasting, but
reversible, chemical sympathectomy that results in the depletion of
peripheral stores of NE (10, 14, 29). A previous study in rats showed
that splenic NE levels decrease 90–95% immediately following
6-OHDA exposure and return to control levels within 56 days post-6-OHDA
exposure, as determined by HPLC (29). To determine the effect of
chemical sympathectomy on splenic NE levels in our scid
model system, 8-wk-old scid mice were exposed to 6-OHDA
dissolved in an ascorbate solution for 3 alternating days 2 days before
reconstitution with a clone of KLH-specific Th2 cells and TNP-specific
B cells. Compared with ascorbate-injected controls, 95% of the NE
stores were depleted in the cellular/supernatant portion of the spleen
2 days following the last 6-OHDA injection, a time point that
corresponded to the time of cell reconstitution in the scid
model (Fig. 3A). NE remained
91% depleted in the cellular/supernatant fraction 9 days following
the last 6-OHDA injection, a time when primary immunization with
TNP-KLH was performed. Approximately 9 wk following chemical
sympathectomy (8 wk following primary immunization), a time when
secondary immunization was performed, splenic NE levels returned to
62% of the level in NE-intact control mice, but to 83% of the
level in NE-intact mice injected with the vehicle ascorbate. Thus, NE
can be depleted almost completely from the spleens of scid
mice before cell reconstitution and remain almost completely depleted
at the time of cell reconstitution and primary immunization, but NE
levels return closer to the levels in ascorbate-injected control mice
by the time of secondary immunization.
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B, the primary IgM response in NE-depleted mice was
suppressed 50–75% compared with that of NE-intact controls, while the
secondary IgM response returned to control levels. In contrast, both
the primary and secondary IgG1 responses were suppressed by 85% in
NE-depleted mice compared with those in NE-intact mice, but by the
second week following the secondary immunization, IgG1 production
recovered to 40% of control levels. However, it may be important to
note that while NE depletion suppressed the Ab response, it never
completely inhibited the response. IgG3 and IgG2a responses were also
detected in the sera of reconstituted scid mice, but their
levels were 200- to 300-fold lower than the levels of IgG1. In
addition, both IgA and IgE were undetectable in the serum of
reconstituted scid mice. Finally, anti-TNP IgM and IgG1
responses were either <6 µg/ml or undetectable in animals that
received adjuvant only injections (data not shown). Thus, these results
suggest that NE plays a role in maintaining an optimal level of Th2
cell-dependent IgM and IgG1 production in normal mice.
To determine whether NE depletion influenced successful lymphocyte
homing in scid mice following cell reconstitution, spleen
sections from either NE-depleted or NE-intact reconstituted
scid mice before Ag exposure were stained for Th cells and B
cells using Abs directed against the markers CD4 and B220,
respectively. One week following i.v. injection, transferred
B220+ cells (Fig. 4,
A and B) and CD4+ T cells (Fig. 4, C and D) successfully homed to and populated the
splenic white pulp of reconstituted scid mice.
B220+ cells formed tight organized coronas that were
adjacent to the less organized CD4+ T cell containing PALS.
In addition, lymphocyte homing and population of both the B
cell-containing corona and the T cell-containing PALS were equivalent
in both NE-intact (Fig. 4, A and C) and
NE-depleted (Fig. 4, B and D) mice, thus
suggesting that NE depletion did not affect the ability of the T and B
cells in our model system to successfully home to the spleen following
i.v. injection into scid mice.
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A) compared with
immunocompetent sibling pairs (Fig. 5C). In contrast,
spleens from scid mice reconstituted with a clone of Th2
cells and TNP-specific B cells had increased cellular organization into
follicles regardless of the NE status of the mice (Fig. 5, B
and E), suggesting that successful homing and cellular
compartmentalization of the transferred lymphocytes occurred similarly
in both NE-intact and NE-depleted mice. Following Ag administration,
clonal expansion and germinal center formation were evident in
NE-intact mice (Fig. 5C), but were less evident in
NE-depleted animals (Fig. 5F). Taken together, these results
suggest that NE depletion does not affect the ability of the Th2 cell
clones and B cells used in our model to successfully home to the
spleen; however, following Ag exposure, less cellular organization into
follicles was observed in NE-depleted mice compared with that in
NE-intact control mice.
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, A and
C), whereas no detectable germinal centers were observed in
NE-depleted mice (Fig. 6, B and D). However, it
is important to note that PNApos germinal center B cells
were diffusely scattered throughout the spleen in both groups,
suggesting that germinal center formation in NE-depleted mice may be
delayed. Thus, it appears that Ag-specific Th2 cells and B cells
successfully localized to the spleen in both NE-intact and NE-depleted
mice, and following Ag exposure, clonal expansion and germinal center
formation were significantly lower in NE-depleted mice compared with
those in NE-intact controls.
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All the above data collected from NE-depleted mice compared with
NE-intact mice suggested that NE played a role in maintaining an
optimal level of Th2-dependent IgM and IgG1 production in normal mice.
To determine the AR subtype responsible for mediating this effect of NE
on the in vivo immune response, the AR-selective antagonist
phentolamine and the ßAR-selective antagonist nadolol were injected
into reconstituted NE-intact scid mice at the time of Ag
exposure to block specific receptor subtypes that could potentially be
stimulated by NE. Mice receiving nadolol had lower serum titers of IgM
2 wk following Ag challenge, whereas animals receiving phentolamine had
serum titers equivalent to those of saline-injected controls (Fig. 7). Thus, these results suggest that NE
modulates the in vivo Ab response via stimulation of the ßAR as
opposed to the AR.
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A, Scatchard transformation
of saturation binding data showed that TNP-specific B cells expressed
620 binding sites/cell with an affinity (Kd)
of 0.1 nM. These values are similar to those reported for
unfractionated murine splenic B cells, but higher than those reported
for murine Th1 cell clones or splenic CD4+ T cells. To
determine whether the ßAR expressed by TNP-specific B cells was of
the ß2AR-specific subtype, TNP-specific B cells were exposed to an Ab
directed against the ß2AR-specific peptide sequence of the
cytoplasmic N-terminus of the ß2AR protein. As shown in Fig. 8B, TNP-specific B cells expressed a detectable level of the
N terminus of the ß2AR, the specificity of which was shown with
blocking peptides specific for the immunizing sequence of the ß2AR,
but not the ß1AR. To determine whether the ß2AR on TNP-specific B
cells was functional, B cells were examined for their ability to
accumulate cAMP intracellularly following exposure to the specific
ß2AR agonist terbutaline. As shown in Fig. 8C, B cells
that were exposed to increasing concentrations of terbutaline showed a
concentration-dependent increase in the intracellular cAMP
concentration. Taken together, these data suggest that an enriched
population of splenic TNP-specific B cells expresses a functional
ß2AR protein.
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). In addition, serum anti-TNP IgM
was significantly increased in animals receiving chronic injections of
metaproterenol each day following NE depletion until the time of Ag
exposure. Thus, these results suggested that the Th2 cells and B cells
used to reconstitute NE-depleted scid mice remained viable,
that the B cells remained responsive to ß2AR stimulation, and that
the primary consequence of NE depletion may have been to suppress the
ability of these cells to function optimally in response to the Th
cell-dependent Ag TNP-KLH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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AR. In addition, the
suppressive effect of NE depletion on Ab production was partially
reversed by administration of a ß2AR-specific agonist to NE-depleted
mice. Although no difference was observed in lymphocyte trafficking or
cellular organization within the spleens of either NE-depleted and
NE-intact reconstituted scid mice before Ag exposure, the
extent of follicle expansion and germinal center formation was
suppressed in NE-depleted animals following Ag exposure. Taken
together, these data suggest that stimulation of the ß2AR on the B
cell maintains an optimal level of Th2 cell-dependent Ab production in
vivo. Before Ag exposure, there was no detectable effect of NE depletion on either splenic structure or the ability of lymphocytes to properly home to the spleen in reconstituted scid mice compared with that in NE-intact mice. While it is possible that some of the cells homed to the peripheral lymph nodes or mucosal lymphoid tissue, it is unlikely, since the T cell clones used in this study are L-selectin negative and are, therefore, unable to cross high endothelial venules to enter the lymph nodes following i.v. reconstitution (32). However, our finding does not support a previous report suggesting that NE depletion in mice prevents lymphocytes from homing properly and that a higher accumulation of adoptively transferred T cells was evident in the spleen and lymph nodes of mice when the T cells were pretreated with a ßAR-selective agonist (33). Thus, the effect of NE depletion on lymphocyte homing remains unclear. Also, the splenic histology of our mice showed that follicular clonal expansion and germinal center formation were less evident following Ag exposure in NE-depleted mice than in NE-intact controls. One possible explanation for this finding is that the lymphocytes present within the spleens of NE-depleted mice may have been in an unresponsive state. However, these cells were able to respond to mitogen stimulation in vitro (data not shown) and appeared to function to some degree following secondary immunization when the level of NE returned to that in controls.
It is possible that the return of IgM production to control levels after secondary immunization was due to a primary response from newly formed B cells migrating from the bone marrow of scid mice following the recovery of NE levels in the spleen. However, this possibility is unlikely, since scid mice rarely generate newly formed B cells (34), and the only Ab allotype produced in the present study was that of the donor cell type. Also, it is likely that the return of secondary IgM production to normal levels was due to the long term survival of the originally transferred donor Th2 cells and B cells within the spleen until a time when NE levels returned to normal, since it has been reported that transferred T and B cells survive indefinitely in a recipient scid mouse (35). This possibility is also supported by the present findings that serum anti-TNP IgM returned to a control level in NE-depleted scid mice following secondary exposure to Ag and that the suppressive effect of NE depletion was partially reversed by the ß2AR-selective agonist terbutaline. Thus, the production of secondary IgM and IgG1 in our model system may be due to the long term survival of the donor cells or the expansion of this original population during the primary immune response.
In contrast to the present results showing a suppressive effect of NE depletion on Th2 cell-dependent Ab production, one study by Kruszewska et al. (14) reported a strain-specific enhancement of both cytokine and Ab production in NE-depleted C57Bl/6J (Th1-slanted strain) and BALB/c (Th2-slanted strain) mice. These studies showed that 6-OHDA treatment of mice enhanced primary serum anti-KLH IgM and IgG in KLH-primed C57Bl/6J mice, while NE depletion increased only IgG1 in KLH-primed BALB/c mice. Thus, the effects of NE depletion by 6-OHDA on immune cell function are varied depending on the target organ cells studied, the stimulus used for cell activation, the mouse strain used for study, whether a primary or a secondary response is being measured, and whether the response being measured is generated in vivo or in vitro.
It is also important to note that previous NE depletion studies in vivo used immunocompetent mice with intact mechanisms of precursor development and differentiation to study the role of NE depletion on immune cell function. Thus, since NE and ß2AR stimulation have been reported to influence hemopoiesis and modulate the numbers of immune cell progenitors produced in the bone marrow (36), it is possible that NE depletion may remove the ability of this neurotransmitter to influence hemopoiesis in the bone marrow of immunocompetent mice and contribute to the previously discussed conflicting findings. In addition, intracellular cAMP levels have been shown to influence the path of CD4+ T cell differentiation into Th1 and Th2 effector cells (37), suggesting that NE and/or ß2AR stimulation may affect the composition of the effector cell population that predominates in NE-depleted immunocompetent mice. Therefore, any conclusions drawn from a study addressing the effects of NE on a resultant immune response in intact animals must also take into consideration the effects of ßAR stimulation on both the developmental and effector stages of the response. We have attempted to address some of these possibilities by establishing a model system in which the mouse possesses a high frequency of Ag-specific T and B cell effector populations that cannot be supplemented by newly formed lymphocytes. Thus, our model system eliminates the possibility that the effect of NE depletion on Ab production is due to an effect on lymphocyte development or differentiation and thereby permits study of the role of NE in modulating a Th2-dependent Ab response to a soluble protein Ag when there is a high frequency of both ß2ARneg Th2 cells and ß2ARpos B cells responding to Ag in a hapten-, carrier-, and MHC-restricted manner.
One concern with all in vivo investigations is the contribution of stress and glucocorticoids to the response. While 6-OHDA does not cross the blood-brain barrier in adult mice to enter the CNS, Callahan et al. (38) showed that 6-OHDA administration to mice increased both Fos protein and corticotropin-releasing factor expression on cells residing within the hypothalamus. This increase in Fos protein within the hypothalamus is believed to have resulted from the peripheral nerve terminal damage induced by 6-OHDA, which, by some mechanism, stimulated the hypothalamic-pituitary-adrenal axis to release glucocorticoids from the adrenal cortex. Therefore, a decreased Ab response in vivo following 6-OHDA-induced depletion of NE may be due to the immunosuppressive effects of glucocorticoids and not to a loss of ß2AR stimulation by NE. However, another study showed that administration of the glucocorticoid receptor antagonist RU486 in vivo was unable to prevent the effects induced by 6-OHDA (39). Therefore, although 6-OHDA administration may enhance Fos protein and corticotropin-releasing factor expression within the hypothalamus, it appears unlikely that a glucocorticoid effect was responsible for the observed immunosuppressive effects of NE depletion in the present study. However, this possibility will be tested in future studies using our model system.
If stimulation of the ß2AR by NE is necessary to maintain optimal Th2 cell-dependent Ab production in vivo, as suggested by the present study and others (12, 13), and if ß2AR stimulation leads to an increase in the intracellular concentration of cAMP within the lymphocytes responsible for Th cell-dependent Ab production, then the influence of cAMP accumulation on the ability of lymphocytes to function during a response to Ag needs to be understood. It is possible that the lack of NE stimulation during the primary response to Ag results in a loss of a cAMP-induced effect in the B cell that may remain with the cell until the time of secondary Ag exposure. Such an effect may include the loss of a cAMP-induced effect to either modulate the level of expression of a cell surface molecule on the B cell that is important for optimal B cell function or alter the level of responsiveness of the B cell to cytokines. For example, in vitro stimulation of B cells by either dibutyryl cAMP (40, 41), a ß2AR-selective agonist (D. Kasprowicz et al., manuscript in preparation), or NE (D. Kasprowicz et al., manuscript in preparation) enhanced the level of expression of the costimulatory molecule B7-2 on the B cell. Thus, B7-2 expression may be lower on the B cells of mice depleted of NE, since these B cells would not receive a cAMP signal via ß2AR stimulation to induce optimal B7-2 expression. As a consequence, it is possible that the lack of enhancement of B7-2 expression on the B cell may hinder optimal B cell function, such as Ab production (D. Kasprowicz et al., manuscript in preparation) and optimal germinal center formation (42). Also, the responsiveness of the B cell to IL-4 may be directly affected by cAMP elevation in the B cell, since there is an apparent role for cAMP in enhancing the level of B cell responsiveness to the Th2 cell-derived cytokine IL-4 (43, 44). Since IL-4 stimulation is necessary for up-regulation of MHC class II on B cells (45, 46), B cell proliferation (47, 48), and B cell switch to IgG Ab production (49, 50, 51, 52), it is possible that depletion of NE in vivo will remove a signal that is necessary to generate cAMP in a B cell to increase responsiveness to IL-4. Therefore, the possibility exists that the levels of both B7-2 expression and IL-4 responsiveness by the B cell are affected by NE depletion, and this possibility may partially explain why follicle expansion, Ab production by primary B cells, and germinal center formation were less evident after Ag exposure in NE-depleted mice compared with those in NE-intact controls.
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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, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153. E-mail address:
3 Abbreviations used in this paper: AR, adrenergic receptor; PALS, periarterial lymphoid sheath; NE, norepinephrine; 6-OHDA, 6-hydroxydopamine; KLH, keyhole limpet hemocyanin; TNP, trinitrophenyl; FLU, fluorescein; PNA, peanut agglutinin; IPIN, iodopindolol.
Received for publication October 19, 1998. Accepted for publication February 16, 1999.
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