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Activation of Antigen-Specific CD4+ Th2
Cells and B Cells In Vivo Increases
Norepinephrine Release in the Spleen and
Bone Marrow
Adam P. Kohm, Yueming Tang, Virginia M. Sanders and
Stephen B. Jones
J Immunol 2000; 165:725-733; ;
doi: 10.4049/jimmunol.165.2.725
http://www.jimmunol.org/content/165/2/725
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Copyright © 2000 by The American Association of
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References
Activation of Antigen-Specific CD4ⴙ Th2 Cells and B Cells In
Vivo Increases Norepinephrine Release in the Spleen and Bone
Marrow1
Adam P. Kohm,2* Yueming Tang,2†‡ Virginia M. Sanders,*§ and Stephen B. Jones3†‡¶
L
ymphocyte function is closely regulated by several immune system-related mechanisms, including cytokines
and cell-cell interactions that play a critical role in modulating both the intensity and the type of immune response to a
specific Ag (reviewed in Ref. 1). In addition to these immune
regulatory mechanisms, dynamic interactions that occur between
the immune system and nervous system create additional regulatory mechanisms by which the normal functioning of each system
is influenced (2– 4). Thus, lymphocyte function is closely regulated
by a combination of mechanisms associated with both the immune
and nervous systems.
Norepinephrine (NE)4 is a signaling molecule of the sympathetic nervous system that is released from sympathetic nerve terminals, which are found in all organ systems, including the primary and secondary lymphoid organs (5–7). Upon release from the
nerve terminal, NE binds to high affinity ␤2-adrenergic receptors
(␤2AR) that are expressed on various immune cell populations.
Previous studies at both protein and mRNA levels have shown that
while B cells (8) and clones of CD4⫹ Th1 cells (9, 10)5 express a
*Department of Cell Biology, Neurobiology, and Anatomy, †Department of Physiology, ‡The Burn and Shock Trauma Institute, §Department of Microbiology and
Immunology, and ¶Department of Surgery, Loyola University Medical Center, Maywood, IL 60153
Received for publication February 15, 2000. Accepted for publication April 26, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by research funds from the National Institutes of
Health (Grants MH53562 (to S.B.J.) and AI37326 (to V.M.S.)) and the American
Cancer Society (Grant RPG-96-067-04-CIM to V.M.S.).
2
A.P.K. and Y.T. contributed equally to this work.
functional ␤2AR, clones of Th2 cells do not, providing a mechanism by which NE can selectively regulate the function of specific
immune cell populations. For example, depletion of NE in scid
mice that were reconstituted with Ag-specific ␤2AR-negative Th2
cell clones and ␤2AR-positive B cells resulted in decreased serum
levels of Ag-specific IgM and IgG1, splenic follicular cell expansion, and germinal center formation (8) in response to immunization compared with reconstituted NE-intact scid mice. Hence, it
appears that NE stimulation of the ␤2AR expressed on B cells is
essential for maintaining an optimal Th2 cell-dependent Ab response in vivo.
However, for NE to influence immune cell function, it must be
released at the immediate site of action, since it is either rapidly
degraded by catechol-O-methyltransferase and monoamine oxidase, diffused into the circulation, or taken back up into the nerve
terminal following release (reviewed in Ref. 11). Therefore, if NE
is to influence the Th2-dependent Ab response in vivo, it is critical
to determine whether mechanisms exist for enhancing the normal
low level of NE release within the microenvironment in which
immune cells are responding to a soluble protein Ag. Previous
studies suggest that immune cell activation by either infectious
challenge (12–14) or SRBC immunization (15, 16) results in a
higher level of NE release in lymphoid organs. Importantly, in the
previous SRBC studies investigating NE release, the rate of NE
release was inferred from observations that SRBC administration
resulted in lower tissue NE concentrations, and this observation
could be interpreted as the result of either an enhanced level of NE
release, a suppressed level of NE production, or a suppressed level
of NE reuptake by the nerve terminal. However, Fuchs et al. (17)
reported that immunization of mice with SRBC enhanced the concentration of the dopamine metabolite, 3,4-dihyroxyphenylacetic
3
Address correspondence and reprint requests to Dr. Stephen B. Jones, The Burn and
Shock Trauma Institute, Loyola University Medical Center, 2160 South First Avenue,
Maywood, IL 60153. E-mail address: [email protected]
Abbreviations used in this paper: NE, norepinephrine; ␤2AR, ␤2-adrenergic receptor; KLH, key hole limpet hemocyanin; TNP, trinitrophenyl; FLU, fluorescein.
4
Copyright © 2000 by The American Association of Immunologists
5
A. P. Kohm, N. Morley, M. A. Swanson, and V. M. Sanders. Selective expression
of ␤2-adrenergic receptor mRNA in CD4⫹ Th1 cells, but not Th2 cells. Submitted for
publication.
0022-1767/00/$02.00
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The neurotransmitter norepinephrine (NE) binds to the ␤2-adrenergic receptor (␤2AR) expressed on various immune cells to
influence cell homing, proliferation, and function. Previous reports showed that NE stimulation of the B cell ␤2AR is necessary
for the maintenance of an optimal primary and secondary Th2 cell-dependent Ab response in vivo. In the present study we
investigated the mechanism by which activation of Ag-specific CD4ⴙ Th2 cells and B cells in vivo by a soluble protein Ag increases
NE release in the spleen and bone marrow. Our model system used scid mice that were reconstituted with a clone of keyhole limpet
hemocyanin-specific Th2 cells and trinitrophenyl-specific B cells. Following immunization, the rate of NE release in the spleen and
bone marrow was determined using [3H]NE turnover analysis. Immunization of reconstituted scid mice with a cognate Ag
increased the rate of NE release in the spleen and bone marrow 18 –25 h, but not 1– 8 h, following immunization. In contrast,
immunization of mice with a noncognate Ag had no effect on the rate of NE release at any time. The cognate Ag-induced increase
in NE release was partially blocked by ganglionic blockade with chlorisondamine, suggesting a role for both pre- and postganglionic signals in regulating NE release. Thus, activation of Ag-specific Th2 cells and B cells in vivo by a soluble protein Ag
increases the rate of NE release and turnover in the spleen and bone marrow 18 –25 h after immunization. The Journal of
Immunology, 2000, 165: 725–733.
726
ACTIVATION OF Ag-SPECIFIC LYMPHOCYTES INCREASES NE RELEASE
restricted interaction between the Th2 cell and the B cell to precipitate the observed Ag-induced release of NE. Finally, since administration of the ganglionic blocker, chlorisondamine, only
partially blocked the Ag-induced release of NE, the effect of immune cell activation on the level of NE release appears to be in
part mediated by immune cell-derived factors acting on either the
CNS, postganglionic nerve, or the local nerve terminal found
within the microenvironment of the responding Th2 cells and B
cells.
Materials and Methods
Animals
Six-week-old female C.B.17/ICR scid and BALB/c mice were obtained
from Taconic Farms (Germantown, NY). All mice were provided autoclaved pellets and water ad libitum. 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 to
maintain a specific pathogen-free environment.
Reagents and Abs
Picrylsulfonic acid (2,4,6-trinitrobenzenesulfonic acid), OVA, and fluorescein (FLU) were purchased from Sigma (St. Louis, MO). KLH was obtained from Calbiochem (La Jolla, CA). TNP-KLH and FLU-OVA were
prepared at a haptenation ratio of 17–24 TNP or FLU molecules/KLH or
OVA carrier molecule.
T cell clones
The Th2 cell clone BAC 3.2 was maintained as described previously (9).
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 an exposure to IL-2. The BAC 3.2 clone was tested for the
presence of Mycoplasma contamination (Life Technologies, Gaithersburg,
MD) and was found to be 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. (26) as modified by Myers et al. (27). All procedures were
performed at 4°C, except for RBC haptenation and enzyme treatment,
which were performed at 37°C. Briefly, horse RBCs (Colorado Serum,
Denver, CO) were haptenated with 20 mg of 2,4,6-trinitrobenzene sulfonic
acid/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 Lympholyte-M (Cedarlane, Ontario, Canada). The lymphocytes recovered at the end of the
procedure were incubated overnight to allow for re-expression of surfaceassociated 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 (28).
Cell transfer and immunization
All animals received both KLH-specific BAC 3.2 Th2 cells and TNPspecific B cells. Each cell type was prepared for adoptive transfer at 2 ⫻
106 cells in 50 ␮l 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 ␮l of PBS. One week after
cell reconstitution, mice received primary immunizations i.p. with 100 ␮g
of TNP-KLH, FLU-OVA, or saline delivered in the adjuvant TiterMax
Gold (CytRx, Norcross, GA), which does not induce nonspecific inflammatory cytokine production. In addition, all mice received 25 ␮Ci of
[3H]NE (Amersham Pharmacia Biotech, Piscataway, NJ) i.p. in 200 ␮l of
saline plus 0.01% ascorbate either at the time of or 17 h following Ag
administration. In some experiments mice received an i.p. injection of the
ganglionic blocker, chlorisondamine (5 mg/kg in saline; Sigma). Spleen,
bone marrow, and heart samples were collected from the mice either 1 or
8 h following [3H]NE administration and were stored at ⫺80°C until time
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acid, in the spleen, which is a metabolic indication that the administration of SRBC enhanced NE production and release. Also, the
soluble products released during infectious challenge, e.g., IL-1,
have been shown to stimulate NE release (21), suggesting a role
for macrophage-derived products in modulating NE release in
lymphoid organs. Thus, while previous studies suggested that administration of an infectious organism, particulate Ag, or an inflammatory cytokine to mice stimulates NE release in lymphoid
organs, no studies have shown that the administration of a soluble
protein Ag plays a role in modulating NE release in the spleen or
bone marrow.
One complication for studies designed to examine such an effect
is that the frequency of Ag-specific Th and B cells that are able to
respond to a specific soluble protein Ag is much lower than the
frequency of cells that are able to respond to immunization with
either an infectious organism or SRBC. If the release of cytokines
from activated immune cells is important for triggering NE release
via either a CNS-mediated (20, 21) or local (22) mechanism, such
a low frequency of responding cells may restrict the level of cytokines released to such a degree that it is difficult to detect NE
release during an Ag-specific response. To increase the frequency
of responding Ag-specific Th2 and B cells, we used a model system in which scid mice (23), which normally lack T and B lymphocytes, were reconstituted with clones of KLH-specific Th2
cells and TNP-specific B cells isolated from the spleens of unimmunized mice. We have previously reported that immunization of
reconstituted scid mice with the cognate Ag TNP-KLH results in
MHC-restricted, Ag-specific Ab production, splenic follicular cell
expansion, and germinal center formation in vivo (8).
NE turnover analysis (24, 25) provides an estimate of dynamic
changes in sympathetic nerve activity that cannot be gained by the
determination of tissue NE concentration alone. This is because the
rate of NE release is balanced by the rate of NE synthesis, resulting
in constant tissue levels over a wide range of sympathetic nerve
activity (24). In the previous studies that measured the effect of
infectious challenge, particulate Ags, or inflammatory cytokines
on the NE concentration in lymphoid organs, limited information
was provided about the activity of sympathetic nerves and their
release of NE. In addition, experimental conditions that induced
detectable changes in tissue NE concentrations suggest that sympathetic nerve activity was so great that homeostatic balance was
lost, NE release outstripped synthesis, and tissue levels of NE may
have been exhausted. In addition, experimental conditions that do
not induce detectable reductions in tissue NE concentrations provide little information about the level of NE release, except that the
steady state dynamics of the nerve terminal were not interrupted.
Hence, the lack of any observed changes in the tissue concentration of NE provides no information about the level of sympathetic
nerve activity and the resulting level of NE release within the
microenvironment in which the immune cells are responding to Ag
challenge. Therefore, to more accurately measure the specific rate
of NE release in immune organs, the present study was performed
using a pulse-chase technique that measures the rate of disappearance of tissue [3H]NE over time. In addition, when experimental
conditions induced a significant change in the rate of [3H]NE release over time, the rate of NE turnover was calculated.
To investigate the role of Ag-specific Th2 cells and B cells in
evoking NE release in the spleen and bone marrow during a Th2dependent immune response, a model system was used in which
Ag-specific Th2 cells and B cells were adoptively transferred into
scid mice (8). In this report we show that activation of Ag-specific
Th2 cells and B cells by a soluble cognate Ag increases NE release
in the spleen and bone marrow by 18 h following immunization.
These results also show a critical role for an Ag-specific, MHC-
The Journal of Immunology
727
of analysis. Bone marrow samples were immediately mixed with HCl and
then stored at ⫺80°C until the time of analysis.
NE and [3H]NE analysis
Tissue samples were homogenized in 1.0 ml of cold 0.4 M perchloric acid
using a Polytron tissue homogenizer (Brinkmann Instruments, Westbury,
NY) and centrifuged. The supernatant was adjusted to pH 8.4 with 1 M Tris
buffer (pH 10) and was mixed with activated acid-washed alumina. The
alumina was washed with water, and alumina-bound NE was eluted with
0.2 M acetic acid. The concentration of NE in the elute was measured by
electrochemical detection following HPLC separation (HPLC and EC systems from BioAnalytical Systems, West Lafayette, IN). The recovery of
NE from alumina was ⬃85% efficient. Aliquots of alumina elutes (0.1 ml)
were mixed in 5.0 ml of scintillation cocktail (Bio-Safe II, RPI, Mount
Prospect, IL) and counted for 3H in a scintillation counter (LS 6500, Beckman Instruments, Fullerton, CA). The specific activity of the [3H]NE (cpm
per nanogram of NE) was calculated as the quotient of [3H]NE present in
the tissue and the total tissue NE content.
Data analysis and statistics
Results
NE release in the spleen and bone marrow following
immunization with a soluble protein Ag
FIGURE 1. The effect of immunization on the rate of NE release in scid
mice reconstituted with Ag-specific Th2 cell clones and B cells 1– 8 h
following Ag exposure. The scid mice were reconstituted with 2 ⫻ 106
cells of both KLH-specific Th2 cell clones (BAC 3.2) and TNP-specific B
cells. One week following reconstitution, mice were immunized i.p. with
either 100 ␮g of TNP-KLH or adjuvant-only (TiterMax Gold). In addition,
all mice received 25 ␮Ci of [3H]NE i.p. delivered in saline plus 0.1%
ascorbate at the time of Ag administration. One and 8 h following Ag
administration, spleen (A), bone marrow (B), and heart (C) samples were
collected for determination of NE specific activity (log cpm of [3H]NE per
nanograms of NE), and the slope of the resultant line is a direct reflection
of the rate of NE release. F, Mice receiving adjuvant only; E, mice immunized with TNP-KLH. Each point represents the mean ⫾ SEM for specific activity of organs from six mice per experiment; r values are the
calculated least square regression coefficients using raw data points for
each line. See Table I for NE tissue levels.
To determine whether immunization of mice with a soluble protein
Ag influences the rate of NE release from sympathetic nerve
terminals, the rate of NE release was determined in the spleen,
bone marrow, and heart following immunization of mice with a
soluble protein Ag. One week following cell reconstitution with
KLH-specific Th2 cells and TNP-specific B cells, scid mice were
immunized with TNP-KLH at time zero and administered 25 ␮Ci
of [3H]NE 1 h before tissue sample collection at the first of two
time points spanning an 8-h period. Tissue samples were collected
either at 1 and 8 h or at 18 and 24 h following immunization for
NE turnover analysis. The rate of NE release is reflected by the
slope of the line resulting from plotting the specific activity of
tissue NE ([3H]NE per picograms of NE) as a function of time
following immunization and is independent of the relative NE specific activity of each group. No significant difference was observed
in the rate of NE release (Fig. 1 and Table I) 1– 8 h following Ag
exposure in the spleen, bone marrow, and heart compared with that
Table I. The effect of immunization on the rate of NE release in scid mice reconstituted with Ag-specific
Th2 cell clones and B cells 1– 8 h following Ag exposurea
Group
n
Tissue NEb (ng/g)
Slopec
p Value of Sloped
Spleen
Adjuvant only
TNP-KLH
6
6
2130 ⫾ 366
2169 ⫾ 228
⫺0.0110
⫺0.0210
⬍0.363
Bone marrow
Adjuvant only
TNP-KLH
6
6
3.93 ⫾ 0.32
4.11 ⫾ 0.51
⫺0.0202
⫺0.0225
⬍0.705
Heart
Adjuvant only
TNP-KLH
6
6
848 ⫾ 96
813 ⫾ 77
⫺0.0186
⫺0.0119
⬍0.237
a
The experimental design is described in the legend to Fig. 1.
Tissue NE values are the mean ⫾ SEM.
Slope of the line representing the log cpm of [3H]NE/pg of tissue NE.
d
The p value of the slope in comparison to the slope of adjuvant-only controls.
b
c
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To determine NE turnover rates (rate constant ⫻ tissue NE), the specific
activity of tissue NE ([3H]NE per picograms of NE) after radiolabeled
injection was plotted as a function of time. The decay of specific activity
is a first-order function, a straight line with a negative slope, and decay
lines were calculated by the method of least squares (29). The rate constant
represents the fraction of the NE pool replaced per unit time (h⫺1). Rate
constants (k) were calculated from the slope of logarithm of the specific
activity vs time relationship (0.434 [k] ⫽ slope). Data are expressed as the
mean ⫾ SEM. Differences between the slopes of the regression lines were
tested by Student’s t test, using the pooled SE of sample regression (30).
p ⬍ 0.05 was accepted as achieving statistical significance.
728
ACTIVATION OF Ag-SPECIFIC LYMPHOCYTES INCREASES NE RELEASE
Role of Ag-specific Th2 cells and B cells in stimulating the
Ag-induced release of NE
FIGURE 2. The effect of immunization on the rate of NE release in scid
mice reconstituted with Ag-specific Th2 cell clones and B cells 18 –25 h
following Ag exposure. The scid mice were reconstituted with 2 ⫻ 106
cells of both KLH-specific Th2 cell clones (BAC 3.2) and TNP-specific B
cells. One week following reconstitution, mice were immunized i.p. with
either 100 ␮g of TNP-KLH or adjuvant only. In addition, all mice received
25 ␮Ci of [3H]NE i.p. delivered in saline plus 0.1% ascorbate 17 h following Ag administration. Eighteen and 25 h following Ag administration,
spleen (A), bone marrow (B), and heart (C) samples were collected for
determination of the rate of NE release as NE turnover rate (nanograms per
gram per hour; inset). F, Mice receiving adjuvant only; E, animals receiving the Ag TNP-KLH. Each point represents the mean ⫾ SEM for specific
activity of organs from five mice per experiment; r values are the calculated least square regression coefficients using raw data points for each line.
The p values are indicated when significant. See Table II for NE tissue
levels.
To determine whether Ag-induced activation of Th2 cells and B
cells in our model system was critical for inducing the measured
increase in NE release in the spleen and bone marrow, scid mice
were reconstituted with KLH-specific Th2 cell clones and TNPspecific B cells and immunized with the cognate Ag TNP-KLH,
the noncognate Ag FLU-OVA, or adjuvant only, using TiterMax
Gold adjuvant, which does induce nonspecific inflammatory cytokine production. As shown in Fig. 3A and Table III, immunization
of mice with FLU-OVA failed to significantly increase the rates of
NE release and NE turnover in the spleen in comparison to adjuvant-only controls. Similarly, in the bone marrow (Fig. 3B and
Table III), administration of FLU-OVA also failed to significantly
enhance the rates of NE release and NE turnover in comparison to
adjuvant-only controls. As in previous experiments, administration
of either TNP-KLH or FLU-OVA had no significant effect on the
tissue concentration of NE in the spleen, bone marrow, or heart
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in adjuvant-only controls. Interestingly, the level of NE specific
activity was significantly lower in all bone marrow samples compared with that in the heart and spleen, and this observation is
consistent with a previous report (12). However, the mechanism
responsible for this lower level of specific activity is currently
unknown, but it may involve factors such as local blood flow rates
and patterns, differences in local nerve terminal uptake mechanisms, or differences in the level of sympathetic innervation (31).
Finally, no significant differences were observed in the tissue concentration of NE between mice receiving either Ag or adjuvantonly injections (Table I). Thus, these results suggest that Ag administration did not significantly alter the rate of NE release in the
spleen, bone marrow, and heart at 1– 8 h following Ag exposure.
The possibility existed that activation of the Ag-specific Th2 and
B cell populations was responsible for altering the rate of NE release via a mechanism that was triggered at times later than 8 h
following Ag exposure. For example, APC require at least 8 –12 h
for Ag homing, uptake, processing, and presentation to a Th cell in
vivo (32–34); thus, immune cell-derived soluble products may not
be produced within 8 h of immunization. Therefore, the rate of NE
release was determined in the spleen, bone marrow, and heart
18 –25 h following immunization. As shown in Fig. 2A and Table
II, immunization of mice with the cognate Ag TNP-KLH significantly enhanced the rate of NE release in the spleen ( p ⬍ 0.01)
compared with that in adjuvant-only controls. Even though
NE specific activity was lower in mice receiving TNP-KLH, this
exerted no influence on measurement of the rate of NE release
(24, 25).
In agreement with these data, cognate Ag administration significantly enhanced the rate of NE turnover in the spleen (Fig. 2A,
inset, and Table II) and bone marrow (Fig. 2B and Table II) compared with that in adjuvant-only controls. Interestingly, Ag administration enhanced the rate of NE release in the heart to varying
levels. Finally, and most importantly with regard to previous findings, Ag administration had no significant effect on the total
amount of NE in the tissues (Table II), indicating that Ag stimulation did not impair the mechanisms responsible for maintaining
the local stores of NE within the tissue. Thus, administration of the
soluble protein Ag TNP-KLH to scid mice reconstituted with
KLH-specific Th2 cell clones and TNP-specific B cells significantly enhanced the rate of NE release and NE turnover in the
spleen and bone marrow 18 –25 h after Ag exposure.
The Journal of Immunology
729
Table II. The effect of immunization on the rate of NE release in scid mice reconstituted with Ag-specific
Th2 cell clones and B cells 18 –25 h following Ag exposurea
Group
n
Tissue NEb (ng/g)
Slopec
p Value of Sloped
Spleen
Adjuvantonly
TNP-KLH
5
5
2163 ⫾ 325
2007 ⫾ 247
⫺0.0128
⫺0.0371
⬍0.01
Bone marrow
Adjuvantonly
TNP-KLH
5
5
4.27 ⫾ 0.31
4.26 ⫾ 0.38
⫺0.0193
⫺0.0403
⬍0.01
Heart
Adjuvantonly
TNP-KLH
5
5
793 ⫾ 16
803 ⫾ 18
⫺0.0133
⫺0.0321
⬍0.057
a
The experimental design is described in the legend to Fig. 2.
b
Tissue NE values are the mean ⫾ SEM.
c
Slope of the line representing the log cpm of [3H]NE/pg of tissue NE.
d
The p value of the slope in comparison to the slope of adjuvant-only controls.
Effect of ganglionic blockade on the Ag-specific enhancement of
NE release and turnover in the spleen and bone marrow
The NE-containing sympathetic nerve terminals that innervate immune organs originate from cell bodies located in local sympathetic ganglia lying close to the spinal cord. The function of these
ganglia is to receive input signals from nerves that originate in the
CNS. Therefore, any signal from the CNS destined for delivery to
immune organs via sympathetic innervation must travel through
the local sympathetic ganglion, for example, the splanchnic ganglion for the spleen. On the other hand, it has also been proposed
that NE release from sympathetic nerve terminals may be regulated by stimulation of cytokine receptors expressed on sympathetic nerve terminals (22). In this case, NE release in the spleen
and bone marrow would be regulated locally, as opposed to regulated by signals originating from the CNS, thus eliminating the
requirement for the high levels of cytokine necessary to precipitate
alterations in CNS activity. To investigate the role of signals from
the CNS in mediating the Ag-induced increase in the rate of NE
release and turnover in the spleen and bone marrow, some mice
received injections of the ganglionic blocker chlorisondamine (5
mg/kg) at the time of [3H]NE administration to interrupt signal
transmission through sympathetic ganglia. As shown in Fig. 4A
and Table IV, administration of chlorisondamine partially blocked
the Ag-induced enhancement of the rated of NE release and NE
turnover compared with those in mice receiving TNP-KLH alone.
In addition, chlorisondamine partially blocked the Ag-induced enhancement of the rates of NE release and NE turnover in the bone
marrow in comparison to mice receiving TNP-KLH only. In contrast, chlorisondamine (Fig. 4C) completely blocked the cognate
Ag-induced increase in the rates of NE release and turnover (Table
IV) in the heart in comparison to adjuvant-only controls. Finally,
administration of chlorisondamine had no effect on tissue NE content (Table IV). Thus, ganglionic blockade partially prevents the
Ag-induced enhancement of the rates of NE release and NE turnover in the spleen and bone marrow.
Discussion
In this study we show that administration of a cognate soluble
protein Ag (TNP-KLH) to scid mice reconstituted with KLH-specific Th2 cell clones and TNP-specific B cells enhances the rates of
NE release and turnover in the spleen and bone marrow 18 –25 h
following immunization (Fig. 5). Interestingly, administration of a
noncognate Ag that is not recognized by either the Ag-specific Th2
cell clones or B cells used to reconstitute the scid mice in our
model system failed to enhance NE release in the spleen or bone
marrow. Finally, administration of the ganglionic blocker, chlorisondamine, partially prevented the Ag-induced activation of NE
release and turnover in the spleen and bone marrow.
Previous studies (12–14) have demonstrated that infectious
challenge enhances NE release within lymphoid organs as well as
NE levels in the circulation and suggested that macrophage-derived cytokines were involved. For example, macrophage activation is critical for successful clearance of infection, and upon activation, these cells produce significant levels of the inflammatory
Table III. Specificity of the Ag-induced increase in NE release in reconstituted scid mice 18 –25 h following Ag exposurea
Group
n
Tissue NEb (ng/g)
Slopec
p Value of Sloped
Spleen
Adjuvant only
TNP-KLH (cognate Ag)
FLU-OVA (noncognate Ag)
6
6
6
1634 ⫾ 16
1534 ⫾ 83
1677 ⫾ 43
⫺0.027
⫺0.057
⫺0.034
⬍0.004
⬍0.122
Bone marrow
Adjuvant only
TNP-KLH (cognate Ag)
FLU-OVA (noncognate Ag)
6
6
6
4.25 ⫾ 0.29
4.12 ⫾ 0.19
4.32 ⫾ 0.14
⫺0.010
⫺0.037
⫺0.011
⬍0.0001
⬍0.680
Heart
Adjuvant only
TNP-KLH (cognate Ag)
FLU-OVA (noncognate Ag)
6
6
6
893 ⫾ 52
881 ⫾ 34
907 ⫾ 47
⫺0.021
⫺0.035
⫺0.027
⬍0.310
⬍0.412
a
The experimental design is described in the legend to Fig. 3.
Tissue NE values are the mean ⫾ SEM.
Slope of the line representing the log cpm of [3H]NE/pg of tissue NE.
d
The p value of the slope in comparison to the slope of adjuvant-only controls.
b
c
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(Table III). Thus, immunization of mice with the cognate Ag TNPKLH, but not the noncognate Ag FLU-OVA, significantly enhanced the rate of NE release and NE turnover in the spleen and
bone marrow.
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ACTIVATION OF Ag-SPECIFIC LYMPHOCYTES INCREASES NE RELEASE
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FIGURE 3. Specificity of the Ag-induced increase in NE release in reconstituted scid mice18 –25 h following Ag exposure. The scid mice were
reconstituted with 2 ⫻ 106 cells of both KLH-specific Th2 cell clones
(BAC 3.2) and TNP-specific B cells. One week following reconstitution,
mice were immunized i.p. with 100 ␮g of the cognate Ag TNP-KLH, the
noncognate Ag FLU-OVA, or adjuvant only. In addition, all mice received
25 ␮Ci of [3H]NE i.p. delivered in saline plus 0.1% ascorbate 17 h following Ag administration. Eighteen and 25 h following Ag administration,
spleen (A), bone marrow (B), and heart (C) samples were collected for
determination of the rate of NE release as NE turnover rate (nanograms per
gram per hour; inset). F, Mice receiving adjuvant only; E, mice receiving
the specific Ag TNP-KLH; Œ, mice receiving the irrelevant Ag FLU-OVA.
Each point represents the mean ⫾ SEM for specific activity of organs from
six mice per experiment; r values are the calculated least square regression
coefficients using raw data points for each line. The p values are indicated
when the slope is significant. See Table III for NE tissue levels.
FIGURE 4. Effect of ganglionic blockade on Ag-specific induction of
NE release in the spleen and bone marrow. The scid mice were reconstituted with 2 ⫻ 106 cells of both KLH-specific Th2 cell clones (BAC 3.2)
and TNP-specific B cells. One week following reconstitution, mice were
immunized i.p. with either 100 ␮g of the specific Ag TNP-KLH or adjuvant only. In addition, all mice received 25 ␮Ci of [3H]NE i.p. delivered in
saline plus 0.1% ascorbate 17 h following Ag administration. Eighteen and
25 h following Ag administration, spleen (A), bone marrow (B), and heart
(C) samples were collected for determination of the rate of NE release as
NE turnover rate (nanograms per gram per hour; inset). F, Mice receiving
adjuvant only; E, mice receiving the specific Ag TNP-KLH; Œ, mice receiving both the specific Ag TNP-KLH and the ganglionic blocker, chlorisondamine. Each point represents the mean ⫾ SEM for specific activity
of organs from six mice per experiment; r values are the calculated least
square regression coefficients using raw data points for each line. The p
values are indicated when significant. See Table IV for NE tissue levels.
The Journal of Immunology
731
Table IV. Effect of ganglionic blockade on Ag-specific induction of NE release in the spleen and bone marrowa
Group
n
Tissue NEb (ng/g)
Slopec
p Value of Sloped
Spleen
Adjuvant only
TNP-KLH
TNP-KLH ⫹ chlorisondamine
6
6
6
755 ⫾ 43
715 ⫾ 78
770 ⫾ 54
⫺0.015
⫺0.054
⫺0.028
⬍0.0001
⬍0.04
Bone marrow
Adjuvant only
TNP-KLH
TNP-KLH ⫹ chlorisondamine
6
6
6
3.84 ⫾ 0.20
4.18 ⫾ 0.24
4.08 ⫾ 0.15
⫺0.014
⫺0.049
⫺0.026
⬍.0003
⬍0.08
Heart
Adjuvant only
TNP-KLH
TNP-KLH ⫹ chlorisondamine
6
6
6
810 ⫾ 46
765 ⫾ 34
762 ⫾ 48
⫺0.026
⫺0.051
⫺0.029
⬍0.001
⬍0.39
a
The experimental design is described in the legend to Fig. 4.
Tissue NE values are the mean ⫾ SEM.
Slope of the line representing the log cpm of [3H]NE/pg of tissue NE.
d
The p value of the slope in comparison to the slope of adjuvant-only controls.
b
c
FIGURE 5. Model for Ag-induced NE
release in lymphoid organs. 1, Administration of a soluble cognate Ag to scid mice
reconstituted with Ag-specific Th2 and B
cells activates Ag-specific cell populations.
2 and 3, Signals derived from activated
Th2 and/or B cells induces an increase in
NE release in the spleen (2) and bone marrow (3). 4, The Ag-induced increase in NE
release was partially blocked by a ganglionic blocker, thus suggesting that signals
originating from either the CNS or the
preganglionic nerve were in part responsible for the enhanced level of NE release.
We have previously reported (8) that NE
stimulation of the ␤2AR expressed on B
cells is necessary for the maintenance of
optimal primary and secondary Th2 celldependent Ab responses in vivo (5).
that a cognate interaction between Th2 cells and B cells is necessary for immunization-induced enhancement of NE release by a
currently undetermined mechanism.
The capacity of immune cell-derived cytokines to influence
sympathetic nerve activity was originally suggested by an early
study showing that peripheral administration of cytokines stimulates increased nerve activity in both the hypothalamus and brainstem (37). Recently, this study was further supported by the findings that cytokine receptors are present on various types of
peripheral nerves, including sensory nerves (38), sympathetic
nerves (39, 40), sympathetic ganglia (41, 42), and vagal nerve
paraganglia (4), all of which are found outside the CNS. Therefore,
the presence of cytokine receptors on peripheral nerves provides a
potential mechanism by which local immune cell-derived cytokines produced in the periphery may transmit signals to the CNS
or the peripheral nerve directly (see model in Fig. 5).
Importantly, administration of the ganglionic blocker, chlorisondamine, completely blocked any effect of Ag administration on NE
release in the heart, suggesting that the dose of chlorisondamine
used in the current studies was sufficient to block ganglionic transmission. However, chlorisondamine only partially blocked the Aginduced enhancement of NE release and turnover in the spleen and
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
cytokines IL-1␤ (18) and TNF-␣ (19). Since peripheral administration of both cytokines has also been shown to evoke CNS activation (35, 36), it is likely that such experimental results involved
macrophage activation, macrophage-derived cytokine modulation
of CNS activity, and subsequent increased sympathetic nerve activity. If such a mechanism was involved in our reconstituted scid
mouse model system, we would expect administration of a noncognate soluble protein Ag to induce alterations in the rate of NE
release in the spleen and bone marrow, since this Ag would be
processed by professional APC other than B cells. However, this
expectation was not realized, suggesting that nonspecific activation
of macrophages or other members of the innate immune system by
a soluble protein Ag was not sufficient to induce alterations in NE
release within the spleen and bone marrow when using our model
system. More importantly, Ag administration induced NE release
only when mice were challenged with the specific cognate Ag
(TNP-KLH) recognized by both the Th2 cell clones and B cells
present in our model system, suggesting that the production of
inflammatory cytokines is not essential for NE release to occur.
Thus, these findings suggest that macrophage activation and inflammatory cytokine production are not responsible for the soluble
protein Ag-induced increase in sympathetic nerve activation and
732
ACTIVATION OF Ag-SPECIFIC LYMPHOCYTES INCREASES NE RELEASE
that signaling mediators derived from the nervous system regulate
immune cell function (reviewed in Ref. 46), it is not surprising that
the peripheral immune system may transmit signals to the nervous
system via local cytokine production or cell-cell interactions. By
this type of mechanism, the CNS may differentially regulate immune cell function in the periphery depending on the intensity of
the response to a specific Ag or the health of the entire body, such
as during times of stress, disease, or trauma. Such actions of the
CNS may influence the function of immune cells participating in
responses above a certain threshold of intensity, whereas local immune system or nervous system mechanisms may be sufficient for
regulating responses of lower intensity. If this should be the case,
then bidirectional communication between the immune system and
the nervous system would be critical in maintaining immune homeostasis in vivo.
Acknowledgments
We thank Joseph Podojil for critical reading of the manuscript.
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bone marrow, suggesting a role for signals originating above, as
well as at the preganglionic cell body in regulating nerve activity
in this model. One possible explanation of these results is that the
immune cell-derived signals, which are most likely cytokines, do
not act locally to enhance local NE release, but instead bind to
cytokine receptors at an unknown site before the ganglion, e.g., on
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possible that these cytokines might bind to their specific receptors
on the local nerve terminal within the splenic or bone marrow
microenvironment to initiate signals that must then be transmitted
to either the CNS or the local ganglion before another signal is
transmitted back to the local nerve terminal inducing NE release.
This possibility is supported by two of the present findings. First,
Ag administration induced a lower level of NE release in the heart
compared with that in the spleen and bone marrow. Second,
chlorisondamine completely blocked the Ag-induced alterations in
sympathetic nerve activity in the heart. Since chlorisondamine
failed to completely block the Ag-induced enhancement of sympathetic nerve activity in the spleen and bone marrow, it is possible
that local cytokine production not only serves to initiate an afferent
signal from the site of the immune response, but, in addition, modulates local nerve activity by binding to cytokine receptors on local
nerve terminals or the postganglionic cell body. Of course, it is
also possible that immune cell-nerve terminal interactions may occur after Ag exposure to induce NE release, a possibility that is
testable using our model system. Thus, while signals from the
ganglion represent a significant regulatory influence on sympathetic
nerve activity in response to a specific cognate Ag, which is blocked
by chlorisondamine, local cytokine receptor stimulation may also enhance NE release, which cannot be blocked by chlorisondamine.
Thus, there may be multiple levels of cytokine-induced regulation of
local sympathetic nerve activity and NE release within immune organs. The model system used in the present study will now help us to
dissect the multiple levels of regulation of this immune cell-modulating neurotransmitter.
Currently, it is not known whether the production of specific
cytokines by Th2 and/or B cells mediates the Ag-induced enhancement of NE release. If cytokines are involved in our model system,
the most likely source of the cytokines is the Th2 cell clone, which
characteristically produces the cytokines IL-4, IL-5, IL-6, and
IL-10 (9, 43). In light of this possibility, the current findings may
not occur in mice reconstituted with Th1 cell clones that characteristically produce IFN-␥ and IL-2 (9, 44). One possible mediator
of the Ag-induced increase in sympathetic nerve activity may be
IL-6 produced by the Th2 cell clones used in this model system.
Although IL-6 does not affect the uptake of [3H]NE into sympathetic nerve terminals (22), IL-6 does exert concentration-dependent effects on sympathetic nerve activity. For example, 1 ng/ml of
IL-6 stimulated, 10 ng/ml of IL-6 had no effect, and 100 ng/ml
IL-6 inhibited [3H]NE release from sympathetic nerve terminals
within 2 h of cytokine exposure. Thus, IL-6 may either enhance,
inhibit, or have no effect on NE release in our model system. However, if similar findings are observed in animals reconstituted with
Th1 cell clones, they would suggest that the critical signal responsible for influencing sympathetic nerve activity is produced by
both Th1 and Th2 cells, such as IL-3 (44) or TNF-␣ (45). Finally,
it is also possible that the B cell is the critical source of the immune
cell-derived cytokine signals mediating the effects of Ag administration on NE release, thus eliminating the association of a Th
cell subset specificity with the response.
The current study supports the overall hypothesis that local immune responses generate important signals that regulate nervous
system function. Since both in vitro and in vivo findings conclude
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