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COMPLEX FUNCTION OF THE CENTRAL NERVOUS SYSTEM, SLEEP AND LOCOMOTION
1Department of Biology and Program in Neural Science, Indiana University, Bloomington, Indiana 47405; 2Department of Biology and Center for Behavioral Neuroscience, Georgia State University, Atlanta, Georgia 30303; 3Departments of Psychology and Neuroscience, Ohio State University, Columbus, 43210; and 4Department of Psychiatry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Submitted 18 April 2003 ; accepted in final form 15 June 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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- and 
-noradrenergic receptor antagonists
(phenoxybenzamine and propranolol, respectively) in vitro. Short-day animals
had increased splenic NE content compared with long-day animals. Long-day
animals had higher proliferation compared with short-day animals independent
of NE. NE (1 µM) further suppressed splenocyte proliferation in short but
not long days. Last, NE-induced suppression of proliferation in short-day
hamsters was blocked by propranolol but not phenoxybenzamine. The present
results suggest that NE plays a role in photoperiodic changes in lymphocyte
proliferation. Additionally, the data suggest that the effects of NE on
proliferation are specific to activation of -adrenergic receptors
located on splenic tissue. Collectively, these results provide further support
that photoperiodic changes in immunity are influenced by changes in SNS
activity. immune; seasonal; stress; sympathetic nervous system; catecholamines
It is well established that the sympathoadrenal system (i.e., direct sympathetic innervation of lymphoid tissue, as well as secretion of adrenal catecholamines) can have profound effects on immunity. Because of the rather extensive literature demonstrating suppression of immunity by chronic adrenal glucocorticoids (e.g., corticosterone, cortisol) (reviewed in Refs. 16, 41), sympathoadrenal contributions to immune regulation have been largely overlooked. Recent evidence, however, suggests that a wide range of environmental and intrinsic stressors can activate the sympathetic nervous system (SNS) and suppress immune function (reviewed in Refs. 32, 43). Anatomic studies also have established the direct innervation of lymphoid tissue by the SNS in a variety of species (4, 45). Norepinephrine (NE), the primary postganglionic sympathetic neurotransmitter, appears to be the predominant neurotransmitter phenotype of these neurons; NE is released from nerve terminals immediately adjacent to lymphocytes, as well as from more distant sites acting in a paracrine fashion (reviewed in Ref. 32). Moreover, NE released from these nerves can have profound effects on both humoral and cell-mediated immunity (19, 20, 33). The actions of NE on immunity appear to be direct; adrenoreceptors (AR) have been localized on lymphoid tissue (32), and NE can alter immune function in vitro (26, 33, 40).
NE exerts its effects on target cells by acting on two principal
adrenoreceptor subtypes, - and -AR. Nearly all lymphoid cells
express -AR, although evidence suggests that -AR differ in density
depending on the specific type of lymphoid cell
(43). Circulating lymphocytes
and natural killer (NK) cells also express -AR, although the number of
-AR varies during the lifespan of the cell. Other immune cells, such as
B cells, also possess low-affinity -AR. The existence of specific
subtypes of -AR on immune cells, however, remains controversial. Some
studies suggest that both 1- and 2-AR are
expressed on immune cells
(19), and there are reports of
-AR expressed on human lymphocytes. In addition, activated rodent
macrophages express 2-AR
(33); however, -AR have
not been identified in rodent lymphocytes.
The broad goal of the present study was to test the hypothesis that the SNS, via direct innervation of lymphoid tissue and concomitant release of NE, plays a role in photoperiodic changes in immune function in Siberian hamsters. Recently, direct SNS innervation of the spleen has been demonstrated in Siberian hamsters using the retrograde transneuronal tract tracer pseudorabies virus (PRV) (17). Specifically, PRV injected into the spleens of Siberian hamsters revealed infected neurons in areas traditionally implicated in SNS regulation (e.g., A5 and C1 cell groups, locus ceruleus). Labeled neurons were also identified in the paraventricular (PVN) and suprachiasmatic nuclei (SCN) of the hypothalamus (17), brain regions critical for the transduction of the photoperiodic signal (22). More recently, our laboratory has demonstrated that the SNS plays an important functional role in regulating photoperiodic changes in immune function in Siberian hamsters. Specifically, short day-housed hamsters receiving surgical denervation of the spleen displayed reduced humoral immunity compared with sham-operated hamsters; splenic denervation, however, had no effect on immunity of long-day hamsters (13). These results suggest that the SNS is associated with photoperiodic changes in immune function in Siberian hamsters. The specific aims of the present study were 1) to examine the effects of photoperiod on NE content within the spleen, 2) to test the effects of exogenous NE on lymphocyte proliferation, and 3) to identify the AR subtypes involved in mediating these potential effects.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Experiment 1: effects of photoperiod in splenic NE content. The goal of experiment 1 was to determine the effects of photoperiod on NE content within the spleens of Siberian hamsters. Hamsters (n = 40) were selected randomly and assigned to either long (LD 16:8) (n = 20) or short days (LD 8:16) (n = 20). Animals were maintained in their respective photoperiods for 10 wk. Animals were then brought one at a time into the surgery room and killed by cervical dislocation; paired testes and spleens were removed, cleaned of connective tissue, and weighed at necropsy. Spleens were then snap-frozen in liquid nitrogen to preserve catecholamine content and immediately stored at -80°C freezer until assayed. All necropsies occurred between 1000 and 1100 EST to control for circadian rhythms in lymphocyte proliferation.
Experiment 2: effects of in vitro NE on splenocyte proliferation. The goal of experiment 2 was to test the effects of exogenous NE on lymphocyte proliferation in response to mitogenic stimulation. Hamsters (n = 25) were selected randomly and assigned to either long (n = 11) or short days (n = 14) for 10 wk and were then killed by cervical dislocation between 1000 and 1100 EST. Paired testes were removed, cleaned of connective tissue, and weighed at necropsy to determine photoperiodic responsiveness. Spleens were also removed under aseptic conditions, weighed, and immediately suspended in culture medium (RPMI-1640/HEPES).
Experiment 3: effect of in vitro - and
-adrenergic antagonists on splenocyte proliferation. The goal
of experiment 3 was to determine which AR subtypes are involved in
NE-induced reductions in lymphocyte proliferation in response to mitogenic
stimulation. Hamsters (n = 26) were selected randomly and assigned to
either long (n = 10) or short days (n = 16) for 10 wk and
were then killed by cervical dislocation between 1000 and 1100 EST. Paired
testes were removed, cleaned of connective tissue, and weighed at necropsy.
Spleens were also removed under aseptic conditions, weighed, and immediately
suspended in culture medium (RPMI-1640/HEPES).
HPLC determination of catecholamine content. Splenic NE content was determined in experiment 1 by measuring NE content in the spleen by reverse-phase high-pressure liquid chromatography with electrochemical detection (HPLC-EC) according to the method of Ref. 12 after Ref. 35. Briefly, tissue was thawed, weighed, and carefully minced. A 250-mg sample was added to a microcentrifuge tube containing 790 µl of 0.2 M perchloric acid. Ten microliters of dihydroxybenzoacetic acid (DHBA; 2 µg/ml) was added to each sample and served as an internal standard. Tissue was further minced and then sonicated for 5 min on ice (5x for each sample). Catecholamines were extracted from the remaining infranatant by adding alumina (200 mg/sample), vortexing for 30 s, followed by 1 ml of 0.5 M Tris (pH = 8.6). Last, 200 µl of perchloric acid was added to elute the catecholamines. Tris buffer was aspirated using a micropipette, and the extracted samples were assayed using an ESA HPLC system with electrochemical detection (guard cell: +35 mV; cell 1: +10 mV; cell 2: -30 mV; Chelmsford, MA). The mobile phase was Cat-A-Phase II purchased from a commercial supplier (ESA). Standard solutions (50, 33, 16.5 ng/ml) were prepared from commercially supplied standard kit (ESA) and were run at the beginning, in the middle, and at the end of the sets of unknowns. Assay results were analyzed offline and expressed as nanograms per gram tissue. The sensitivity of this assay was 15 pg.
Lymphocyte proliferation. Lymphocyte proliferation in response to the T-cell mitogen, concanavalin A (Con A), was determined using a colorimetric assay based on the tetrazolium salt 3-(4,5-demethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (11). Splenocytes were separated from tissue by compressing the whole spleen between sterile frosted glass slides; separated cells were suspended in 4 ml of culture medium [RPMI-1640/HEPES supplemented with 1% penicillin (5,000 U/ml)/streptomycin (5,000 µg/ml), 1% L-glutamine (2 mM/ml), 0.1% 2-mercaptoethanol (5 x 10-2 M/ml), and 10% heat-inactivated fetal bovine serum]. Splenocyte counts and viability were determined with a hemacytometer and trypan blue exclusion. Viable cells (which exceeded 95%) were adjusted to 2 x 106 cells/ml by dilution with culture medium. In a pilot assay, Con A (Sigma Chemical, St. Louis, MO) was diluted with culture medium to concentrations of 0.6, 1.25, 2.5, 5, 10, 20 and 40 µg/ml; 50 µl of each mitogen concentration was added to the wells of the plate containing the spleen cell suspensions to yield a final volume of 150 µl/well (each in duplicate). These concentrations yielded a typical dose-response curve with proliferation absorbance values of 0.37 ± 0.02, 0.39 ± 0.26, 0.46 ± 0.03, 0.53 ± 0.07, 0.80 ± 0.20, 1.10 ± 0.12, 1.31 ± 0.17, and 0.53 ± 0.06, respectively. Based on these values, 20 µg/ml concentration of Con A was chosen as the "optimal" dose because it resulted in maximal mitogen-induced proliferation (as measured by absorbance); this Con A concentration was used in subsequent proliferation assays in experiments 2 and 3.
In experiment 2, 50-µl aliquots of each cell suspension (i.e., 100,000 cells) were added to each of three wells of sterile flat-bottom 96-well culture plates. One well then received 50 µl of 1.0 µM NE, another well received 50 µl of 10.0 nM NE, and the last well received 50 µl of culture medium. Con A was diluted with culture medium to a concentration of 20 µg/ml; 50 µl of each of the diluted mitogens was added to the wells of the plate containing the spleen cell suspensions to yield a final volume of 150 µl/well (each in duplicate).
In experiment 3, 50-µl aliquots of each cell suspension were added to each of four wells of sterile flat-bottom 96-well culture plates. One well received 50 µlofNE(1 µM) dissolved in culture media, another well received NE (1 µM) + propranolol (5 µM) in culture media, the third well received NE (1 µM) + phenoxybenzamine (5 µM), and the last well received culture media alone (no NE). In a previous pilot experiment, we demonstrated that neither propranolol nor phenoxybenzamine administered alone (i.e., without NE) has any effect on Con A-induced splenocyte proliferation in long- or short-day hamsters (Demas, unpublished data). As in experiment 2, an optimal dose of Con A (20 µg/ml) was added to all spleen cell suspensions.
The plates from both experiments 2 and 3 were incubated at 37°C with 5% CO2 for 48 h before addition of 20 µl of MTS/phenazine methosulfate (PMS) solution [Promega; 0.92 mg/ml of PMS in sterile Dulbecco's phosphate-buffered saline] per well. Plates were then incubated at 37°C with 5% CO2 for an additional 4 h. The optical density (OD) of each well was determined with a microplate reader (Bio-Rad: model 3550) equipped with a 490-nm wavelength filter. Mean OD values for each set of duplicates were used in subsequent statistical analyses. In experiment 2, dose-response curves were constructed using group means of the mean OD values at each mitogen concentration and unstimulated cultures.
Statistical analyses. Differences in testes masses in all three experiments and between splenic NE content in experiment 1 were assessed by separate independent Student's t-tests. Differences in splenocyte proliferation in experiments 2 and 3 were assessed via a two-way (photoperiod x drug) between-groups ANOVA. Post hoc comparisons between pairwise means were conducted using Tukey's honestly significant difference tests when the overall ANOVAs were significant. All statistical tests were conducted using SigmaStat Statistical software (San Rafael, CA). In all cases, differences between group means were considered statistically significant if P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Experiment 2. As in experiment 1, short day-housed hamsters had significantly smaller paired testes masses compared with long-day animals (P < 0.05; data not shown). Short day-housed hamsters had significantly reduced splenocyte proliferation in response to Con A compared with long-day hamsters (P < 0.05; Fig. 2). In addition, in vitro NE (1 µM) but not NE (10 nM) significantly reduced splenocyte proliferation in short-day hamsters (P < 0.05); neither concentration of NE had an effect on splenocyte proliferation in long-day hamsters (P > 0.05; Fig. 2). There were no differences in splenic masses between long- and short-day hamsters (P > 0.05).
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Experiment 3. As in experiment 1, short day-housed hamsters had significantly smaller paired testes masses compared with long-day animals (P < 0.05; data not shown). Short day-housed hamsters had significantly reduced splenocyte proliferation in response to Con A compared with long-day hamsters, and in vitro NE significantly reduced splenocyte proliferation in short-day hamsters (P < 0.05; Fig. 3). In addition, NE-induced suppression of proliferation in short-day hamsters was blocked by the presence of in vitro propranolol, but not phenoxybenzamine (P < 0.05; Fig. 3). There were no differences in splenic mass between long- and short-day hamsters.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-AR; pharmacological blockade of
-AR, but not -AR, attenuated NE-induced suppression of splenocyte
proliferation. Interestingly, short days also increased splenocyte
proliferation in the absence of NE in culture, suggesting that photoperiodic
changes in immune cell proliferation are not completely regulated by changes
in SNS activity. Collectively, the results of the present study are consistent
with the idea that there are photoperiodic changes in immunity and suggest
that changes in SNS activity (and subsequent NE release) may coordinate, at
least in part, photoperiodic changes in lymphocyte proliferation. Furthermore,
these effects appear to be mediated directly through interactions with
-AR within lymphoid tissue. These results confirm and extend the results
of previous research suggesting an important role for the SNS, and
specifically -AR, in the regulation of cell-mediated immunity
(23,
43). The presence of a
photoperiodic effect on splenocyte proliferation independent of NE, however,
suggests that NE is not absolutely required. Thus, although NE, likely acting
through -AR directly on splenocytes, can modulate photoperiodic changes
in lymphocyte proliferation, factors other than the SNS (e.g., melatonin,
steroid hormones) may also play an important role in mediating photoperiodic
changes in lymphocyte proliferation. Ongoing studies are aimed at elucidating
these specific factors. An increasing amount of evidence supports an important role of the SNS in the regulation of immunity (20, 32, 33). For example, an inhibitory role of the SNS in the regulation of immune function has been suggested in several species after denervation of a variety of lymphoid tissue (32). Specifically, global chemical denervation of the peripheral nervous system of adult animals via systemic injections of 6-hydroxydopamine (6-OHDA) enhances antibody responses to T-independent antigens but has no effect on T-independent antibody production (36). In contrast, it has also been reported that 6-OHDA-induced denervation reduces antibody responses to T-dependent antigens in adult rats (21, 28), whereas surgical denervation of the splenic nerve enhances antibody production in neonatal animals (6, 47). More recently, it has been demonstrated that chemical denervation results in a modest increase in anti-KLH antibody production in young rats, but a marked increase in antibodies in older animals (4). These results suggest that the effects of the SNS on immune function depend on several factors, including the age of the animals and the type of denervation, as well as the type of immune response measured.
The SNS is also involved in lymphocyte trafficking and circulation, with different lymphocyte populations having differing sensitivities to catecholamines (19). For example, NK cells and granulocyte circulation are both increased by catecholamines, whereas B and T cell numbers remain relatively unaffected (5, 44). In addition, 6-OHDA-induced depletion of NE increases mitogen-induced proliferation of T, but not B cells (31). A previous report, however, has demonstrated reduced Con A-induced splenocyte proliferation after NE depletion via 6-OHDA in vivo (33). Sympathectomy can exert differential effects on mitogen-induced splenocyte proliferation, with reduced proliferation in sympathectomized animals in the presence of low doses of Con A, but increased proliferation at high doses (1). Furthermore, the effects of sympathectomy on mitogen-induced splenocyte proliferation appear to be strain dependent (31); thus differences between species are likely as well. Last, as discussed above, systemic 6-OHDA results in a global SNS depletion of NE; thus it is unclear whether the decreased proliferation after 6-OHDA-induced sympathectomy is due to a direct effect of NE removal on splenocytes or, rather, an indirect effect on other immunological factors (e.g., cytokines).
The concentrations of NE used in the present study were chosen because these doses have been shown previously to result in reliable inhibition of cellular proliferation (25). The use of a high physiological dose allowed us to be confident that we would not exclude potentially subtle effects of NE on proliferation, while at the same time it enabled us to assess a meaningful, biologically relevant physiological response. Given that the effects of NE on lymphocyte proliferation in short-day hamsters occurred at high but not low levels of NE, however, it may be suggested that this effect is an artifact of potentially pharmacological levels of the neurotransmitter. Although we cannot totally rule out this possibility, we believe it is unlikely because the relatively high effective doses of NE used in these experiments are within the range of NE concentrations typically experienced within the local environment surrounding peripheral tissue (25). Regardless of whether the NE concentrations used in these studies are physiological or pharmacological, however, the results of the present demonstrate that splenocytes from short-day hamsters are more responsive to NE in vitro than long-day splenocytes.
Consistent with previous studies, the results of the present study suggest
that the reduction in splenocyte proliferation in response to NE in vitro is a
result of -AR activity. For example, stimulation of -AR can
inhibit both mitogen- or anti-CD3 antibody-induced T cell proliferation
(9,
23). Several subtypes of
-AR have now been identified, including 1-,
2-, and 3-AR subtypes
(19). Based on radioligand
binding studies, mature lymphocytes appear to express predominantly
2-AR (27),
and this receptor subtype plays a primary functional role in regulating immune
responses (reviewed in Ref.
42). For example, nonspecific
-AR agonists and selective 2-but not
1-agonists reduce interleukin-2 and 
-interferon
production, as well as mitogen-induced lymphocyte proliferation
(42). Although the present
results suggest a role of -AR in regulating NE-induced suppression of
lymphocyte proliferation, the use of the nonspecific -AR antagonist
propranolol in the present study prevents us from determining the specific
receptor subtype involved. Studies remain to be done that utilize
subtype-specific antagonists and agonists and that will allow us to address
these questions.
The results of the present study suggest that the SNS, and specifically NE,
plays an important role in photoperiodic changes in lymphocyte proliferation.
Furthermore, these results demonstrate that short days increase NE content
within the spleen compared with long days. These data suggest that maintenance
in short winterlike days increases SNS outflow to peripheral lymphoid tissues
such as the spleen, supporting previous findings of short day-induced changes
in SNS outflow to peripheral tissues in Siberian
(49) and Syrian hamsters
(46). The latter finding is
particularly interesting in that SNS activity is decreased in short compared
with long days in Syrian hamsters, a finding consistent with increased immune
function reported in short days for this species. Thus it is possible that the
differential effects of short days on SNS outflow may underlie the differences
in photoperiodic effects on immunity reported for these hamster species,
although this hypothesis requires testing. In the present study, increased SNS
activity and concomitant increases in NE release in short days appear to act
directly on -AR on splenocytes to modulate lymphocyte proliferation.
These data provide novel and important insights into the neuroendocrine
mechanisms underlying environmentally induced fluctuations in lymphocyte
proliferation and likely immune function.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-adrenergic receptor
density and the humoral immune response in vivo and in vitro. Cell
Immunol 117:
339-351, 1988.[ISI][Medline]
-adrenergic mechanisms. J
Immunol 156:
93-99, 1996.[Abstract]
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