Aging and sympathetic modulation of immune function in Fischer 344 rats: Effects of chemical sympathectomy on primary antibody response

Journal of Neuroimmunology 165 (2005) 21 – 32 www.elsevier.com/locate/jneuroim

Aging and sympathetic modulation of immune function in Fischer 344 rats: Effects of chemical sympathectomy on primary antibody response Denise L. Bellingera,*, Suzanne Y. Stevensb, Srinivasan Thyaga Rajana, Dianne Lortonc, Kelley S. Maddend a

Department of Pathology and Human Anatomy, Loma Linda University School of Medicine, 11021 Campus Street, AH 325, Loma Linda, CA 92350, United States b Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, United States c Hoover Arthritis Center, Sun Health Research Institute, Sun City, AZ 85372, United States d Department of Psychiatry, University of Rochester School of Medicine, 300 Crittenden Blvd., Rochester, NY 14642, United States Received 5 March 2004; received in revised form 27 October 2004; accepted 25 March 2005

Abstract In aged Fischer 344 (F334) rats, sympathetic innervation of the spleen is markedly diminished compared with young rats. To determine if this diminished noradrenergic (NA) innervation maintains a functional connection with the immune system, 3- and 17-month-old male F344 rats were treated with the NA-selective neurotoxin, 6-hydroxydopamine (6-OHDA), to ablate peripheral NA nerve fibers. In sympathectomized rats immunized with keyhole limpet hemocyanin (KLH), a T-dependent protein antigen, anti-KLH IgM, IgG, IgG1, IgG2b antibody titers were increased in young and old rats 14 days after immunization compared to vehicle controls. Furthermore, the number of IgM and IgG anti-KLH antibody-secreting spleen cells was elevated 7 and 14 days post-immunization. These effects were prevented by pretreatment with desipramine, a catecholamine uptake blocker that blocks 6-OHDA uptake and subsequent sympathectomy. Chemical sympathectomy also increased KLH-induced proliferation in vitro by spleen cells from old, but not young animals. Isoproterenol (ISO), a h-adrenergic receptor agonist, elicited a rise in cAMP in spleen cells from NA-intact young and old rats, but the increase was attenuated in spleen cells from old rats. These results demonstrate that, although NA innervation in the F344 rat spleen is diminished with age, sympathetic signaling of the immune system remains intact. Thus, the SNS can inhibit antibody produced in response to a protein antigen in both young and old F344 rats. D 2005 Elsevier B.V. All rights reserved. Keywords: Aging; Neural-immune interactions; Noradrenergic; Norepinephrine; Antibody production; Humoral response

1. Introduction The sympathetic nervous system (SNS), through innervation of lymphoid tissue, provides a direct route for communication between the nervous and immune systems (reviewed in Madden and Felten, 1995; Madden, 2001, 2003). In the spleen, noradrenergic (NA) sympathetic nerves distribute along the central arterioles and their branches, and extend from these vascular plexuses into the surrounding * Corresponding author. Tel.: +1 909 558 7069; fax: +1 909 558 0432. E-mail address: [email protected] (D.L. Bellinger). 0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2005.03.022

periarteriolar lymphatic sheath, a zone where T lymphocytes predominate (Ackerman et al., 1987; Felten et al., 1987a; Williams et al., 1981; reviewed in Bellinger et al., 2001). NA nerves also course adjacent to arterioles or in nerve bundles in the marginal and parafollicular zones where macrophages and B lymphocytes reside. Norepinephrine (NE) released from these nerves diffuses through the spleen where it can interact with cells of the immune system that express adrenergic receptors. Direct ligand binding studies have demonstrated both a and/or h-adrenergic receptors on T and B lymphocytes (Amenta et al., 2002; Brodde et al., 1981; Loveland et al., 1981; Fuch et al., 1988), neutrophils

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(Dulis and Wilson, 1980; Galant and Allred, 1980, 1981), macrophages (Abrass et al., 1985; Ignatowski et al., 2000; Kang et al., 2003), and mast cells (Masini et al., 1982; Marquardt and Wasserman, 1982; Donlon et al., 1982; Undem et al., 1985). h-Adrenergic receptors on these cells are linked with adenylate cyclase and the generation of cAMP as the second messenger. Functional studies have revealed a complex role for sympathetic innervation in immune modulation (reviewed in Kohm and Sanders, 2000; Madden, 2001). Early in vitro studies suggested that h-adrenergic receptor stimulation inhibits mitogen-induced T and B cell proliferation (Watson, 1975; Goodwin et al., 1979; Johnson et al., 1981), cytolysis by cytotoxic T lymphocytes (Strom et al., 1973), IL-2stimulated proliferative responses (Beckner and Farrar, 1988) and antibody production (Watson et al., 1973; Melmon et al., 1974; Watson, 1975). However, in vivo studies employing the infusion of adrenergic agonists/ antagonists or sympathetic ablation with the selective neurotoxin, 6-hydroxydopamine (6-OHDA) indicated that the SNS may have a more complex regulatory role in immune modulation. Ablation of sympathetic innervation by treatment with 6-OHDA (chemical sympathectomy) markedly reduced cell-mediated immune responses, including delayed-type hypersensitivity and cytotoxic T lymphocyte activity (Livnat et al., 1987; Madden et al., 1989). Sympathectomy in mice reduced the number of anti-sheep red blood cell antibody-producing cells in lymph nodes and spleen (Livnat et al., 1985). More recently, Kohm and Sanders (1999) demonstrated that antibody-specific IgM and IgG responses were reduced in sympathectomized SCID mice reconstituted with h-adrenergic receptor-negative Th2 cell clones and h-adrenergic receptor-positive B cells. Furthermore, the antibody response could be partially restored by treatment of sympathectomized mice with a hadrenergic agonist. By contrast, Kruszewska et al. (1995, 1998) demonstrated increased KLH-induced interleukin (IL)-2, IL-4, and interferon (IFN)-g production by spleen cells following chemical sympathectomy in two strains of mice. Sympathectomy-induced enhancement of KLH-stimulated cytokine production appears to be mediated through effects on T cells, rather than accessory cells or antigenpresenting cells (Moynihan et al., 2004). These studies demonstrate that the effects of chemical sympathectomy on immune reactivity are dependent on multiple factors, including species and strain, and immune cell type involved in the response. Our laboratory has been interested in understanding how the relationship between the SNS and the immune system changes with advancing age. We have demonstrated a progressive age-related decline in splenic NE concentration between 17 and 27 months of age in Fischer 344 (F344) rats (approximated 25% and 60%; decrease at 17 and 27 months of age, respectively) (Felten et al., 1987b; Bellinger et al., 1987; Bellinger et al., 2001). Histochemical and immunochemical techniques revealed a greater decline in sympa-

thetic nerve profiles (greater than 75% depletion of NA nerves at 21 and 27 months of age) of the aged spleen than indicated by neurochemical assessment alone (Felten et al., 1987b; Bellinger et al., 1987). Further, double-label immunocytochemistry demonstrated that reduced density of splenic sympathetic nerves accompanies a parallel agerelated loss in the density of T lymphocytes and ED3+ macrophages in lymphoid compartments where the nerves reside (Bellinger et al., 1992). Lastly, h-adrenergic receptor expression on splenocytes from old rats is increased (Ackerman et al., 1991), possibly reflecting a compensatory response to increased target cell sensitivity to NE. An age-related reduction in splenic innervation suggests attenuation in SNS modulation of immune responses to antigen challenge with age. Therefore, the present studies were performed to determine whether the diminished sympathetic innervation in the aged spleen is capable of modulating immune reactivity, and if so, how SNS immunomodulation in old F344 rats compares qualitatively with that of young animals. Chemical sympathectomy with 6-OHDA was used to selectively destroy sympathetic nerve terminals in the periphery of young and old rats. We report here that antibody production in response to a T-dependent antigen, KLH, is enhanced by sympathectomy in young and old F344 rats, indicating that NA sympathetic innervation in aged animals, though markedly diminished, can provide regulatory signals to the immune system.

2. Materials and methods 2.1. Experimental design Experiments were repeated two times at each time point after immunization. Young and old animals were sympathectomized in each replication to facilitate direct comparison between age groups. Because the effects of chemical sympathectomy were similar between the 2 experimental repetitions, only 1 representative experiment is shown. An n of 6 to 8 animals was used for each treatment group. 2.2. Animals Rats aged 3 or 17 months of age were used in these experiments to represent young adult animals with mature immune systems or old rats, respectively. At 17 months of age, sympathetic innervation is reduced by > 60% compared to young adult rats, as determined by fluorescence histochemistry (Felten et al., 1987b; Bellinger et al., 1987, 2001). At this age, we have evidence that certain mechanisms are up-regulated to maintain a level of sympathetic signaling in the face of this dramatic reduction in NA nerve fibers (Bellinger et al., 1992). Very old rats have a greater reduction in splenic NA innervation and NE levels, but they were not used in this study because we were concerned that sympathectomy would have no effect because there are very

D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

few intact NA nerves, and because of the higher incidence of age-related pathologies. Male F344 rats at 3 and 17 months of age (from Charles Rivers obtained through the National Institute on Aging) were housed singly in plastic cages. Animals were kept on a 12h:12h light/dark cycle, with lights on at 6:00 a.m. and off at 6:00 p.m. All experimental manipulation was done during the light period, generally between 9:00 a.m. and 12:00 p.m. Animals were allowed to acclimate to vivarium conditions for at least 1 week before experimental manipulation. Food and water were available at all times. Rats were sacrificed by decapitation without prior anesthesia. All animal protocols were approved by the animal use and care committee at the University of Rochester and adhered to NIH guidelines. Rats were examined grossly at the time of sacrifice for pathological conditions commonly seen with age, including testicular interstitial cell hyperplasia, severe chronic nephropathy (enlarged and discolored kidneys), bile duct hyperplasia (rough surface of liver), and splenomegaly (enlarged spleen resulting from lymphoma or leukemia originating in the spleen). Animals showing any grossly visible signs of these conditions were eliminated from our study. 2.3. Denervation Six-hydroxydopamine hydrobromide (6-OHDA; Research Biochemicals International, Natick, MA) was dissolved in sterile saline containing 0.1% ascorbate (an antioxidant), and injected intraperitoneally (i.p.) into 3- and 17-month-old rats every other day for 3 days. Rats received 40 mg/kg body weight 6-OHDA for the first treatment (day 5 relative to immunization), and 80 mg/kg body weight for the last 2 treatments (days 3 and 1 relative to immunization). This treatment protocol minimizes drug toxicity, optimizes NE depletion and delays NA nerve fiber regrowth (Lorton et al., 1990). Both young and old rats lose approximately 10% of their body weight following sympathectomy and do not regain pre-sympathectomy weight until 30 days after sympathectomy (Lorton et al., 1990; unpublished observation). Desipramine hydrochloride (DMI; Sigma Chemical Co., St. Louis, MO) was used in some experiments to prevent sympathetic denervation by blocking neuronal uptake of 6-OHDA. DMI was dissolved in a small volume of sterile water, and then diluted to the proper concentration with sterile saline. Young and old rats received 5 and 1 mg/kg DMI, respectively, or the corresponding vehicle 30 min before administration of 6OHDA or its corresponding vehicle. The minimum doses of DMI required to effectively prevent 6-OHDA-induced destruction of sympathetic nerves in the spleen were predetermined in each age group (data not shown). 2.4. Immunization KLH (Pacific Biomarine, Venice, CA) was diluted in sterile saline. Rats were immunized by an i.p. injection of

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KLH (150 Ag/rat) 1 day (day 0) after the last 6-OHDA or vehicle injection. Animals were sacrificed 4, 7, or 14 days after immunization. 2.5. Tissue and spleen preparation After sacrifice by decapitation, trunk blood was collected and spleens were removed and weighed aseptically. A small cross-sectional piece from the middle of the spleen (20 – 50 mg) was frozen on dry ice and stored at 80 -C for neurochemical analysis of catecholamine content using high performance liquid chromatography with electrochemical detection (HPLC-LCEC). Approximately one-half of the spleen was prepared for assessing immune function. One-half of the spleen was placed into Hank’s Balanced Salt Solution (HBSS, Sigma Chemical Co.) containing sodium bicarbonate and HEPES (United States Biochemical Corp., Cleveland, OH), and dissociated using a Stomacher Lab-Blender (Tekmar Co., Cincinnati, OH). Cell suspensions were passed through fine nylon mesh (#HC3-110; Tetko, Elmsford, NY) to remove large aggregates, then centrifuged and washed once in HBSS. Erythrocytes were removed from spleen cell suspensions by layering cells over Histopaque 1077 (Sigma Chemical Co.), and centrifuged at 2500 rpm for 30 min at room temperature. Cells at the interface between the Histopaque and HBSS were removed and washed three times in HBSS. Cells were counted using a Coulter Counter (Coulter Instruments, Hialeah, FL), and then resuspended to 2  106 cells/ml for cell culture in RPMI 1640 medium supplemented with 5% fetal calf serum (Sigma Chemical Co.), 1 mM sodium pyruvate, 2 mM l-glutamate, 0.01 mM non-essential amino acids, 5  10 5 M 2-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, 24 mM sodium bicarbonate, and 10 mM HEPES (all from Life Technologies, Grand Island, NY). For the cAMP enzyme immunoassays (EIA), spleen cells were resuspended at 2  106 cells/ml in HBSS and 3-isobutyl-1-methylxanthine (IBMX; 100 AM; Calbiochem, LaJolla, CA). 2.6. Proliferation assay Cells were plated in triplicate at 2  10 5 cells per well in 96-well, flat-bottom tissue culture plates (Falcon, Becton Dickinson, Oxnard, CA) containing varying concentrations of KLH in a total volume of 200 Al. Plates were incubated at 37 -C in a 5% CO2 humidified incubator for 5 days. [ 3H]-thymidine (5.0 Ci/mmol; Amersham, Arlington Heights, IL) was added for the last 18 h of culture. Cells were harvested onto glass fiber filters (Whatman Inc, Clifton, NJ) with a semi-automated manifold harvester (Skatron Instruments Inc., Sterling, VA). Dried filters were placed in scintillation fluid (Biosafe II, Research Products International, Mount Prospect, IL), and radioactivity determined with a liquid scintillation counter (LKB, Wallac, Finland).

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D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

2.7. Anti-KLH antibody enzyme-linked immunosorbent assay (ELISA) Blood was collected at the time of sacrifice and sera stored at 20 -C. Anti-KLH antibody titers in sera were determined by ELISA. Washes with phosphate-buffered saline (PBS)/Tween followed each step. Wells of 96-well microtiter plates (Immulon II, Dynex Technologies Inc., Chantilly, VA) were coated overnight at 4 -C with 10 Ag/ml of KLH in a carbonate coating buffer, pH 9.6, and blocked for 1 h with PBS containing 1% BSA at 37 -C. Experimental sera were diluted 1:25, 1:75, and 1:225 in PBS containing 1 M NaCl, 0.1% BSA and 0.05% Tween20, and then incubated for 3 h at 37 -C. The plates were incubated overnight at 4 -C with alkaline phosphatase (AP)conjugated goat anti-rat IgM or IgG (Jackson Immunoresearch, West Grove, PA), diluted 1:500 or 1:1,000, respectively, in PBS containing 0.5 M NaCl, 0.1% BSA, and 0.05% Tween-20. Rat IgG isotypes were detected using AP-conjugated goat anti-rat IgG1 (Pharmingen, San Diego, CA) and IgG2b (Pharmingen). Plates were incubated overnight at 4 -C with these antibodies at a dilution of 1:1000. After extensive washing, the substrate, p-nitrophenyl phosphate (Sigma Chemical Co.) was added to each well at 1 mg/ml in carbonate buffer, pH 9.6. Absorbance at 405 nm was measured using an automated ELISA plate reader (Bio-Tek Instruments). Sera from non-immunized rats served as a negative control. The anti-KLH IgG1 and IgG2b isotypes were analyzed to assess Th2 and Th1 activity, respectively. Based on amino acid sequence and complement fixation, rat IgG1 is equivalent to mouse IgG1 and IgG2b is equivalent to mouse IgG2a (Bruggeman, 1988). Functionally, rat IgG2b is induced by IL-12 and is preferentially elevated during a DTH response (Gracie and Bradley, 1996; Maccioni et al., 1996). 2.8. Enzyme-linked immunospot (ELISPOT) assay The ELISPOT assay was used to quantify the number of anti-KLH antibody-producing cells in the spleen. Twenty-four-well multiscreen plates (Falcon 3047; Becton Dickinson, Franklin Lakes, NJ) were coated overnight with 1.0 ml/well of 10 Ag/ml KLH in carbonate –bicarbonate coating buffer, pH 9.6 at 4 -C. Plates were washed 3 times with sterile, endotoxin-free Dulbecco’s PBS containing 0.1% Tween 20 (PBS-Tween) and blocked with 1.0 ml/ well of PBS containing 1% BSA for 1 h at 37 -C. Plates were washed twice with PBS-Tween buffer and twice with PBS, and incubated with 0.3 ml of 1 10 5 splenocytes/ml overnight at 4 -C. After 3 washes with PBS-Tween, 1.0 ml/well of either goat anti-rat IgG (1:1000; Jackson Immunoresearch Laboratories, West Grove, PA) or goatanti-rat IgM (1:500; Jackson Immunoresearch Laboratories) antibody in diluting buffer (PBS containing 0.1% BSA, 0.05% Tween, and 0.005% sodium azide) was added

to the well and the plates were incubated overnight at 4 -C. After the plates were washed 3 times with PBS-Tween, AP-conjugated rabbit anti-goat IgG antibody, diluted 1:1000 in diluting buffer (1ml/well; Vector Labs, Burlingame, CA) was added and the plates were incubated overnight at 4 -C. Plates were washed 3 times with PBSTween, and developed in the dark with the AP substrate, 6-bromo-4-chloro-3-indolyl-phosphate p-toluidine salt solution (BCIP; United States Biochemical Corp., Cleveland, OH) in a 2-amino-2-methyl-1-propanol buffer (AMP; Sigma-Aldrich Corp., St. Louis, MO) for 30 min at 37 -C. The number of KLH antibody-secreting cells per well was counted under low magnification. Negative control wells containing reagent only were included to distinguish antigen-specific from non-specific spots. Characteristic ELISPOTs were not observed in negative control wells. The number of spots derived from non-immunized spleen cells was subtracted as background from the number of spots in wells from immunized spleen cells. Data were expressed as the mean number of KLH antibody-secreting cells in duplicate wells/106 spleen cells T S.E.M. for each treatment group. 2.9. HPLC-LCEC Spleen samples were placed in 0.1 M perchloric acid (approximately 100 mg spleen/ml). The internal standard, 3,4-dihydroxybenzylamine (DHBA), was added to give a final concentration of 0.25 AM, and the sample was homogenized. The homogenates were centrifuged at 1000g for 5 min, and the resultant supernatants removed for alumina extraction. To 200 Al of supernatant, 1 ml of 0.1 M phosphate buffer (pH 7.0), 1 ml of 1.5 M Tris buffer (pH 8.6), and 50 mg of acid washed alumina were added. After vortexing, the alumina was washed 3 times in water. 200 Al of 0.1 M perchloric acid was added to the tubes containing the alumina and centrifuged at 1000g for 1 min to extract the alumina-associated NE. The eluates were stored at 80 -C until NE concentrations were measured by LCEC. Samples were loaded into a Waters 717 plus autosampler (Waters, Milford, MA) and the mobile phase was delivered at a constant flow rate of 1.0 ml/min by a Waters Model 510 pump through a C18, 5 AM, 250 mm  4.6 mm analytical column (Alltech, Deerfield, IL) placed in a column heater (35 -C). The LLC-4C amperometric detector (Bioanalytical Systems, West Lafayette, IN) potential was set at 0.85 V about an Ag – AgCl electrode and the sensitivity of the detector was maintained at 2 nA. The mobile phase was prepared by dissolving 0.1 M disodium phosphate, 0.1 M citrate, 0.1 mM EDTA, 1.4 mM octyl sodium sulfate, and 15% methanol in 1 l of nanopure water. The signal from the detector was recorded and the data were analyzed using a Waters Millennium 2010 Chromatography Manager. Recovery was determined using DHBA as an internal standard. Samples corrected for recovery were compared with external standards to determine NE concentration. Splenic

D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

NE concentration was expressed as pmole/mg spleen wet weight T S.E.M. 2.10. Measurement of cAMP by enzyme immunoassay (EIA) To measure cAMP accumulation, spleen cells were incubated in the presence or absence of ligand (10 5 M isoproterenol (ISO) or prostaglandin E2 (PGE2)) in 1.0 ml of HBSS containing 1.0 mM IBMX (a cAMP phosphodiesterase inhibitor) at 37 -C for 10 min. The reaction was then stopped by placing the tubes on ice. Samples then were centrifuged at 4 -C for 8 min at 1000 rpm. The cells were resuspended in 0.5 ml 50 mM sodium acetate buffer. Two cycles of freezing the samples on dry ice and then placing the samples in a 100 -C water bath for 5 min were performed to lyse the spleen cells. The samples were centrifuged for 10 min at 3000 rpm to remove cellular debris, and the supernatants were collected into microfuge tubes and stored at 70 -C until cAMP analysis by EIA. EIA for cAMP determination was performed according to the manufacturer’s instructions (Amersham International, Amersham Bucks, UK). 2.11. Statistical analysis Initially, analysis of variance (ANOVA) was conducted at each time point post-immunization with experimental repetition, age, and treatment as independent variables. Serum dilution and mitogen concentration were treated as repeated measures. At each time point, experimental repetitions were analyzed separately with age and treatment as independent variables. If a significant main effect of age or age by treatment interaction was identified ( p < 0.05), the data were analyzed separately by age. Significant main

25

effects were subsequently analyzed post-hoc using Tukey– Kramer or simple effects tests. If no main effect or interaction is mentioned, no significant differences were found. Results were considered statistically significant at p < 0.05.

3. Results 3.1. NE concentration after chemical sympathectomy NA denervation following 6-OHDA treatment was verified by measuring splenic NE concentration in the hilar region of the spleen using HPLC-LCEC (Table 1). As we have reported previously (Felten et al., 1987b; Bellinger et al., 1987), splenic NE concentration in NA-intact 17-monthold rats was significantly lower than that measured in vehicle-treated young adult animals (approximately 22% lower; ANOVA, main effect of age, p < 0.05; indicated by ** in Table 1). Five to fifteen days post-sympathectomy, splenic NE concentration was reduced in the 6-OHDAtreated animals, more than 72% in both experimental repetitions, regardless of age (Table 1; ANOVA, main effect of treatment, p < 0.05). In the second experiment, additional rats were pretreated with the catecholamine uptake blocker, DMI, 30 min before administration of 6-OHDA, to prevent sympathectomy and depletion of NE. DMI pretreatment completely prevented the 6-OHDA-induced loss of splenic NE in young and old rats (Table 1). DMI treatment alone did not alter splenic NE concentration (Table 1). 3.2. Sympathectomy and anti-KLH antibody responses Four and seven days post-immunization, no significant main effects of age, treatment, or age by treatment

Table 1 Splenic NE content following chemical sympathectomya Age Experiment 1 3 months

17 months

Experiment 2 3 months

17 months

Daysb

Vehicle

6-OHDA

% Decrease

DMI/6-OHDA

DMI

5 8 15 5 8 15

5.55 T 0.55 5.78 T 0.35 5.59 T 0.18 4.50 T 0.46** 3.02 T 0.61** 4.54 T 0.23**

1.31 T 0.15* 0.66 T 0.23* 1.56 T 0.39* 1.03 T 0.20* 0.95 T 0.29* 1.19 T 0.11*

76.4% 88.6% 72.1% 77.1% 84.2% 73.8%

ND ND ND ND ND ND

ND ND ND ND ND ND

5 8 15 5 8 15

5.88 T 0.17 5.68 T 0.26 5.59 T 0.40 4.19 T 0.28** 4.23 T 0.38** 4.05 T 0.53**

0.75 T 0.26* 1.10 T 0.24* 1.26 T 0.31* 0.72 T 0.18* 0.60 T 0.14* 1.09 T 0.34*

87.2% 80.6% 77.5% 82.8% 85.8% 73.1%

ND ND 5.10 T 0.52 ND ND 5.02 T 0.55

ND ND 5.64 T 0.47 ND ND 4.55 T 0.77

a F344 rats were treated with 40 mg/kg body weight 6-OHDA on day 5, and 80 mg/kg body weight 6-OHDA on days 3, and 1. Young animals were injected with 5 mg/kg DMI and old animals were injected with 1 mg/kg DMI, as described in Materials and methods. Results are expressed as the mean pmol of splenic NE/mg wet weight T S.E.M. of 6 to 8 animals per group. n = 6 – 8 animals per treatment group. ND, not determined. b Days after the last treatment with 6-OHDA or ascorbate vehicle. * Significant compared to corresponding vehicle control group. Asterisk indicates significance at p < 0.05. ** Significant compared to 3-month-old vehicle control group. Double asterisks indicates significance at p < 0.05.

D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

interaction were detected in serum anti-KLH IgM or IgG (data not shown). Analysis of the IgM and IgG antibody response 14 days after immunization revealed no main effect of age or age by treatment interaction (Fig. 1), but a main effect of treatment was detected. Sympathetic denervation significantly enhanced serum IgM (Fig. 1A, B) and IgG (Fig. 1C, D) anti-KLH responses in young (Fig. 1A, C) and old (Fig. 1B, D) animals (ANOVA, IgM: main effect of treatment; p < 0.0003; IgG: main effect of treatment, p < 0.05). Furthermore, DMI treatment prior to 6-OHDA administrations completely blocked the 6-OHDA-induced increase in IgM and IgG antibody responses (Fig. 1A – D); treatment with DMI alone had no effect (Fig. 1A –D). Further analysis of the IgG response to KLH revealed that IgG1 (Fig. 2A, B) and IgG2b (Fig. 2C, D) were elevated significantly in both young (Fig. 2A, C) and old (Fig. 2B, D) denervated rats (ANOVA, IgG1: main effect of treatment, p < 0.0001; IgG2b: main effect of treatment,

A.

p < 0.0001). Sympathectomy-induced enhancement of serum IgG1 and IgG2b titers were blocked by pretreatment with DMI (Fig. 2A –D), and DMI alone had no effect on serum IgG1 and IgG2b levels (Fig. 2A –D) in young and old animals. 3.3. Sympathectomy and anti-KLH antibody-secreting cells Anti-KLH-specific antibody secreting spleen cells from young and old rats were enumerated by ELISPOT. B cells secreting IgM specific for KLH predominated in the spleens of sympathectomized young and old rats 7 days after challenge with KLH (Fig. 3), but by day 14 the number of IgG-secreting B cells increased relative to IgM-secreting cells (Fig. 4). Seven (Fig. 3) and fourteen (Fig. 4) days after immunization, sympathectomy significantly ( p < 0.05) increased the number of KLH-specific IgM- and IgGsecreting cells in both young and old immunized rats

B.

IgM

IgM

1.500

*

1.000 0.500 0.000

1.500 1.000

0.500 0.000

1:50

1:150

1:450

1:50

Serum Dilution

1.500 1.000 0.500 0.000

17-mo-old

2.000

*

*

2.000

*

*

3-mo-old

*

IgG 2.500

*

Optical Density (A405 nm)

1:150 1:450 Serum Dilution

D.

IgG

Optical Density (A405 nm)

C. 2.500

17-mo-old

*

3-mo-old

Optical Density (A405 nm)

2.000

*

Optical Density (A405 nm)

2.000

*

26

1.500 1.000 0.500 0.000

1:50

1:150

1:450

1:50

Serum Dilution

1:150

1:450

Serum Dilution Vehicle

DMI/6-OHDA

6-OHDA

DMI

Fig. 1. Serum IgM (A, B) and IgG (C, D) anti-KLH antibody responses in 3-month-old (A, C) and 17-month-old (B, D) F344 rats 14 days after immunization with KLH. Animals (n = 6 – 8 per treatment per age) were immunized 1 day after the last 6-OHDA or vehicle injection with 150 Ag KLH and bled 14 days later. Other animals were treated with DMI before 6-OHDA or vehicle administration (n = 6 – 8 per treatment per age). Asterisks (*) indicate significant differences between 6-OHDA-treated rats and all control groups (Tukey – Kramer post-hoc analysis, p < 0.05). The results from this experiment are representative of the repeat experiment at this time point.

D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

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Fig. 2. Serum IgG1 (A, B) and IgG2b (C, D) anti-KLH antibody responses in 3-month-old (A, C) and 17-month-old (B, D) F344 rats 14 days after immunization with KLH. Animals (n = 6 – 8 per treatment per age) were immunized 1 day after the last 6-OHDA or vehicle injection with 150 Ag KLH and bled 14 days later. Other animals were treated with DMI before DMI before 6-OHDA or vehicle administration (n = 6 – 8 per treatment per age). Asterisks (*) indicate significant differences between 6-OHDA-treated rats and all control groups (Tukey – Kramer post-hoc analysis, p < 0.05). Similar results were obtained from the repeat experiment.

compared with immunized-vehicle controls. As found with anti-KLH serum antibody titer, no age-related change in antibody-secreting cells was detected with age. Although there was an age-related trend towards reduced numbers of KLH-specific IgM- and IgG-secreting cells in sympathectomized rats at 7 and 14 days post-immunization, this difference was not statistically significant (Figs. 3 and 4; p > 0.05). 3.4. Sympathectomy and KLH-induced proliferation To determine if responsiveness to antigen was altered in sympathectomized animals, spleen cell KLH-induced proliferation was measured in vitro. Four and seven days after immunization, sympathectomy did not alter antigen-specific proliferation, regardless of age (data not shown). Fourteen days after immunization, ANOVA revealed a significant main effect of age ( p < 0.0001), with KLH-induced spleen cell proliferation significantly reduced in old rats (Fig. 5B) compared to young (Fig. 5A). Simple effects analysis of the

age  treatment interaction ( p < 0.01) confirmed that the effect of treatment was present in the old ( p < 0.05), but not young animals. Sympathectomy increased KLH-induced proliferation in old rats to levels comparable to the young animals (Fig. 5B). In the old animals, post-hoc analysis revealed that the 6-OHDA group was significantly different from all other treatment groups (Fig. 5B; p < 0.05). Therefore, DMI pretreatment completely prevented the sympathectomy-induced increase in KLH-induced proliferation observed in old rats. To determine if the 6-OHDA induced changes in the proportions of splenic T and B lymphocytes, CD5+ T cells and sIgM+ B cells were quantified by flow cytometry. There was no effect of age or treatment on the proportion of splenic CD5+ T cells or sIgM+ B cells 4, 7, or 14 days after immunization (data not shown). Chemical sympathectomy had no effect on total spleen weights from immunized young and old rats (data not shown), suggesting that the total number of splenic T and B lymphocytes was not altered by 6-OHDA treatment.

D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

3.5. b-Adrenergic receptor agonist stimulated cAMP generation in young and old splenocytes To determine the ability of splenocyte h-adrenergic receptors to respond to NE released from sympathetic nerves, spleen cells from young and old F344 rats were incubated in the presence or absence of the h-adrenergic receptor agonist, isoproterenol (ISO) (10 5 M) or prostaglandin E2 (PGE2) (10 5 M), a ligand that also stimulates cAMP production via G-protein coupled receptors. Addition of either ISO or PGE2 resulted in a significant increase in intracellular cAMP concentration in both young and old splenocytes compared with no drug

A. 30 Antibody-Secreting Cells/106 Spleen Cells

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40 Fig. 4. The number of KLH-specific IgM- and IgG-secreting (A and B, respectively) spleen cells in 3- and 17-month-old F344 rats 14 days after immunization with KLH. Animals (n = 6 – 8 per treatment per age) were immunized 1 day after the last 6-OHDA or vehicle injection with 150 mg KLH and spleen cells were prepared for ELISPOT assay 14 days later. Asterisks (*) indicate age-related differences (p < 0.05) in the number of antibody-secreting cells.

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Fig. 3. The number of KLH-specific IgM- and IgG-secreting (A and B, respectively) spleen cells in 3- and 17-month-old F344 rats 7 days after immunization with KLH. Animals (n = 6 – 8 per treatment per age) were immunized 1 day after the last 6-OHDA or vehicle injection with 150 mg KLH and spleen cells were prepared for ELISPOT assays 7 days later. Asterisks (*) indicate significant differences between young and old treatment groups (p < 0.05).

treatment (Fig. 6). ISO- or PGE2-stimulated cAMP production was significantly attenuated ( p < 0.05) in spleen cells from old compared to young F344 rats (17 and 3 months of age, respectively) (Fig. 6).

4. Discussion The results presented here demonstrate that the diminished splenic NA innervation observed in aged F344 rats retains the capacity to modulate immune reactivity. If splenocytes in the aged rat were no longer receiving signals from sympathetic nerve terminals, then removal of these

D.L. Bellinger et al. / Journal of Neuroimmunology 165 (2005) 21 – 32

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Fig. 5. Spleen cell KLH-induced proliferation in young (A) and old (B) F344 rats 14 days after immunization. Spleen cells were cultured with varying concentrations of KLH for 5 days. Pound signs in B indicate significant differences between young and old rats (ANOVA, p < 0.0001; Tukey – Kramer post-hoc analysis, p < 0.05). (B) Asterisks (*) indicate significant differences between 6-OHDA-treated and vehicle-treated rats. Similar results were obtained in the second experiment. n = 6 – 8 rats per treatment group.

nerve fibers by sympathectomy would have no impact on immune reactivity. Instead, in the absence of NA nerve fibers, antibody production and the number of antibodysecreting cells were increased following immunization of young and old F344 rats with the protein antigen, KLH. Pretreatment with the catecholamine uptake blocker, DMI, prevented the 6-OHDA-induced decrease in splenic NE content, and completely prevented the sympathectomyinduced alterations in immune reactivity in young and old rats. Therefore, the effects of chemical sympathectomy on immune function were dependent on sympathetic denervation, and did not result from toxicity that may be associated with 6-OHDA, or its metabolic by-products, interacting with cells of the immune system. Previous studies have reported that chemical sympathectomy-induced changes in the antibody response to KLH is glucocorticoid-independent (Kruszewska et al., 1998; Moynihan et al., 2004), despite

sympathectomy-induced (Kruszewska et al., 1998; Callahan et al., 1998; Moynihan et al., 2004) and KLH-induced (Fleshner et al., 2001) hypothalamic-pituitary-adrenal cortical activation. These results suggest that the SNS can inhibit antibody production in response to a protein antigen in young and old F344 rats. In this study, an age-related reduction in KLH-induced proliferation in vitro was observed in the absence of ageassociated changes in antibody production. Few reports have examined antigen-specific responses in aged rats. However, Massari et al. (2002) reported similar age-related effects in Wistar rats with reduced antigen-specific proliferation in vitro and no change in antibody production in vivo following primary immunization. In the present study, antigen-specific proliferation was elevated by sympathectomy only in the old animals, suggesting a connection between NA innervation and lymphocyte proliferation that is revealed in the aged microenvironment. The increase in KLH-stimulated proliferation in vitro in old sympathectomized rats is indicative of unchecked expansion of antigenspecific lymphocytes. Several possible mechanisms may account for such unregulated proliferation. For example, sympathectomy may prevent activation-induced apoptosis, which is elevated in aged rat T cells (Pahlavani and Vargas, 2001). If h-adrenergic receptor stimulation inhibits or limits lymphocyte proliferation, then aged animals may exhibit disinhibition of a mechanism to regulate lymphocyte expansion, as suggested by the attenuated response to ISO (Fig. 6). The results demonstrate that NA innervation can play an inhibitory role in antigen-induced proliferation in the 15

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Fig. 6. Measurement of intracellular cAMP concentrations in spleen cells from sympathetically intact 3- or 17-month-old F344 rats. Spleen cells were incubated in the presence or absence of either isoproterenol (10 5 M) or prostaglandin E2 (10 5 M) for 10 min, then lysed and cAMP was extracted for quantitation by EIA. Data represent an n of 6 – 8 rats per treatment group, and expressed as mean pmol of cAMP concentration per 106 spleen cells.

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aging rat spleen, but the functional significance of this in vitro observation is unknown. Massari et al. (2002) demonstrated that the age-related decrease in antigenspecific proliferation in vitro was not associated with agerelated alterations in ex vivo effector function (cytotoxic T lymphocyte activity) or alterations in the secondary antibody response in aged animals. These results suggest that the Frecall_ response to antigen in vivo, as measured by antibody production, is not significantly affected by age in these rats. Our hypothesis that NA sympathetic innervation in the aged F344 rat spleen retains some signaling capacity was confirmed by measuring cAMP induction in spleen cells from NA-intact young and old rats. It is tempting to link the attenuated ISO-induced cAMP response to diminished splenic NA innervation in the aged spleen, but PGE2 also elicited a reduced cAMP response in aged lymphocytes, demonstrating that h2-adrenergic receptors are not the only G-protein coupled receptor exhibiting deficient signaling capacity with age. This suggests that a broader mechanism is responsible for reduced G-protein receptor signaling in lymphocytes with age. Functionally, this reduced signaling capacity may lead to less efficient compensation for the agerelated loss of NA innervation or destruction of NA nerves following sympathectomy in aged animals compared to young animals. As discussed above, the elevated KLHinduced proliferation observed with spleen cells from aged, sympathectomized rats may be a consequence of an inability to compensate for the loss of NA innervation as efficiently as young rats do. Another explanation to consider for interpreting our data is that increased adrenal medullary catecholamine production after sympathectomy (Mueller et al., 1969) may in part compensate for sympathectomy-mediated effects on in vivo proliferation (Madden et al., 1994). However, other investigators have not found an increase in circulating epinephrine after chemical sympathectomy (Ilcol et al., 2002; Molina, 2001; Eldrup and Richter, 2000). Furthermore, plasma NE concentrations after sympathectomy were not significantly different from levels measured after sympathectomy combined with demedullation (Eldrup and Richter, 2000). Finally, we did not observe a change in young or old splenic epinephrine concentration, an organ that filters the blood (data not shown). It is interesting that the increase in serum antibody levels in young and old animals reported here is directionally opposite to the reports of sympathectomy-induced decreases in cell-mediated and primary antibody responses in young mice (Livnat et al., 1985; Madden et al., 1989). Similarly, Kohm and Sanders (1999) reported that sympathetic ablation with 6-OHDA suppressed antibody production in severe combined immunodeficient (SCID) mice reconstituted with an antigen-specific Th2 cell line and antigen-specific B cells. This effect was partially reversed by treatment with NE. On the other hand, Kruszewska et al. (1995) demonstrated a sympathectomy-induced elevation in anti-KLH IgG1 and IgG2a

antibody production in a Th1-predominant strain, C57Bl/6, an effect that was not apparent in BALB/c mice, a Th2 predominant strain. In both strains of mice, antigeninduced Th1 and Th2 cytokine production in vitro were enhanced following sympathectomy, providing no evidence for selective regulation of Th1 versus Th2 dominance by the SNS (Kruszewska et al., 1995, 1998). In the present report, we found no evidence for dominance of one Th response over the other, as assessed by IgG isotype. Sympathectomy increased both IgG1 and IgG2b isotypes, the rat equivalents of mouse IgG1 and IgG2a, respectively (Bruggeman, 1988; Gracie and Bradley, 1996; Maccioni et al., 1996). However, spleen cells from rats immunized with KLH in the absence of adjuvant do not produce cytokines in response to KLH in vitro, so we were unable to assess cytokine production in these animals (unpublished observations). In summary, these results reveal that lymphocytes from aged F344 rats retain the capacity to receive signals from sympathetic NA nerves in the spleen. Sympathectomy increased the primary antibody response in young and old rats suggesting that an intact SNS inhibits antibody production in rats independent of age. Interestingly, no sympathectomy-induced changes in antibody production were observed in aged mice, where splenic NA innervation is largely intact (Bellinger et al., manuscript in preparation). Although the biological relevance of the differences in the response to sympathectomy between aged mice and rats is not known, we speculate that differences in the signaling capacity of sympathetic innervation may contribute to individual differences in immune reactivity that occur with age in human populations, for example in response to vaccination (Webster, 2000). The nature of age-related changes in sympathetic NA innervation may be an influential factor in determining susceptibility to infectious disease and other pathologies of the immune system with age. Acknowledgements The authors thank John Housel, Charlie Richardson, Angela Tong, Heather Frazer, and Than Nguyen for their excellent technical assistance. We also thank David L. Felten for helpful discussion. This work was supported by R29 MH47783, R01 NS44302, and a grant from the Markey Charitable Trust. References Abrass, C.K., O’Connor, S.W., Scarpace, P.J., Abrass, I.B., 1985. Characterization of the h-adrenergic receptor of the rat peritoneal macrophage. J. Immunol. 135, 1338 – 1341. Ackerman, K.D., Felten, S.Y., Bellinger, D.L., Livnat, S., Felten, D.L., 1987. Noradrenergic sympathetic innervation of spleen and lymph nodes in relation to specific cellular compartments. Prog. Immunol. 6, 558 – 600.

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