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β1-Adrenergic Receptors on Immune Cells Impair Innate Defenses against Listeria Rebecca T. Emeny, Donghong Gao and David A. Lawrence This information is current as of June 18, 2017. Subscription Permissions Email Alerts This article cites 53 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/178/8/4876.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References J Immunol 2007; 178:4876-4884; ; doi: 10.4049/jimmunol.178.8.4876 http://www.jimmunol.org/content/178/8/4876 The Journal of Immunology 1-Adrenergic Receptors on Immune Cells Impair Innate Defenses against Listeria Rebecca T. Emeny, Donghong Gao, and David A. Lawrence1 S tress is known to increase an organism’s susceptibility to infection and disease progression, contributing to individual morbidity and mortality. Despite the enormous impact of stress on our health (1) as well as rising health care expenditures (2), precise molecular and cellular mechanisms responsible for neuroimmunosuppression are uncertain. This study investigated the role of sympathetic nervous system modulation of murine host defenses against the well-defined intracellular pathogen Listeria monocytogenes (LM).2 Primary infection with the Gram-positive bacterium LM activates both innate and adaptive immune cells to produce cytokines required for bacterial clearance (3). Successful host resistance (measured by a decline in LM) is mediated by NK and CD8⫹ T cells (4). This decline usually begins by 3 days after infection with a relatively low dose (⬍104 CFU) of LM (5). Day 3 of infection is the time when innate immunity is initiating adaptive immune mechanisms (4, 6). Because host defense against LM infection is dependent upon the coordination of innate and adaptive cell-mediated immune responses, it is a useful infectious Laboratory of Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, New York State Department of Health, Albany, NY 12201 Received for publication September 1, 2006. Accepted for publication February 1, 2007. 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 Address correspondence and reprint requests to Dr. David A. Lawrence, Wadsworth Center, New York State Department of Health, Molecular Medicine, Empire State Plaza, C419, Albany, NY 12201. E-mail address: [email protected] 2 Abbreviations used in this paper: LM, Listeria monocytogenes; 1AR, 1-adrenoceptor; 2AR, 2-adrenoceptor; AR, -adrenoceptor; CORT, corticosterone; CR, cold restraint; DA, dopamine; DTH, delayed-type hypersensitivity; Epi, epinephrine; LLO91–99, listeriolysin O91–99; NE, norepinephrine; RT, room temperature; WT, wild type. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org model with which to analyze the influence of stress on host immunity. We have previously shown that neither cytokine profiles nor depletion of B or T cells can explain our model of stress-induced inhibition of host defenses (7, 8). In this study, we further investigated cytotoxic mechanisms that may be involved in stress-altered early host defenses, including LM-induced CD8⫹ T cell expansion, perforin expression, and anti-keyhole limpet hemocyanin (KLH) humoral and delayed-type hypersensitivity (DTH) responses. The experimental model of 1-h cold (4°C) restraint (CR) is known to elicit both physical and psychological stress (9, 10). We have used CR treatment followed by a low-dose bacterial infection in mice in an attempt to simulate a familiar human condition, namely the experience of psychological or physical stressors and exposure to common infectious agents. Psychological and physiological stresses elicit functional changes in many cell types, by modifying the supply of oxygen and metabolites required for a successful “fight or flight” response (11). In addition to controlling cardiovascular functions, energy metabolism, and thermoregulation, stress factors are known to influence immune cell functions. Numerous neuroendocrine factors, such as prostaglandins, glucocorticoids, catecholamines, and neuropeptides, have regulatory influences on host-pathogen interactions (12). It has been proposed that catecholamines provide a physiologic mechanism to prevent an overactive cell-mediated immune response, by shifting the activity of APCs and Th1 cells from a Th1-promoting to a Th2promoting response via 2-adrenoceptor (2AR) (13). However, the kinetics of stress-factor interactions with immune cells also may be critical in determining whether stress-mediated neuroimmune interactions have beneficial or detrimental consequences. We hypothesize that if immune cells encounter stress before they begin to respond to a pathogen, the immunosuppressive effects of stress-induced neuroendocrine factors can weaken host defenses and increase the pathogenic burden. It is this acute, stress-induced Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Cold restraint (CR) for 1 h elicits a psychological and physiological stress that inhibits host defenses against Listeria monocytogenes (LM). Previous analyses indicated that this inhibition is not due to depletion of B or T cells but is instead dependent on signaling through -adrenoceptors (ARs). We now show that impaired host resistance by CR cannot be accounted for by a decrease in LM-specific (listeriolysin O91–99 tetramerⴙ) effector CD8ⴙ T cells; this result is consistent with previous observations that CRinduced effects are mainly limited to early anti-LM responses. 2-Adrenoceptor (2AR)ⴚ/ⴚ FVB/NJ and wild-type FVB/NJ mice had equivalent anti-LM defenses, whereas 1-adrenoceptor (1AR)ⴚ/ⴚ FVB/NJ mice had lower levels of LM even when subjected to CR treatment. Additionally, host-resistance competency of 1ARⴚ/ⴚ mice could be transferred to irradiated wild-type mice reconstituted with 1ARⴚ/ⴚ bone marrow progenitors and spleen cells, indicating that 1AR signaling on immune cells reduces anti-LM responses. 1ARⴚ/ⴚ mice had improved cellular (delayed-type hypersensitivity) responses while 2ARⴚ/ⴚ mice had improved humoral responses (IgG1, IgG2, and IgM), a result that further explains the strain differences in LM defenses. CRinduced expression of 1AR and 2AR mRNA was assessed by real-time PCR. CR treatment significantly increased AR mRNAs in Ficoll-purified and F4/80ⴙ-enhanced liver but not splenic homogenates, demonstrating an organ-specific effect of stress that alters host defenses. Finally, CR treatment induced early increases in perforin expression that may enhance immune cell apoptosis and interfere with LM clearance. In conclusion, 1AR signaling has immunomodulatory effects on early cell-mediated immune responses; a lack of 1AR signaling improves antilisterial defenses and cell-mediated immunity, in general. The Journal of Immunology, 2007, 178: 4876 – 4884. The Journal of Immunology 4877 Materials and Methods Mice, CR treatment, and LM infection All in vivo studies were performed using mice housed at the Wadsworth Center Animal Production Unit in accordance with the Institutional Animal Care and Use Committee Guidelines. The listeriolysin O91–99 (LLO91–99) (I-Ad) tetramer study used BALB/c mice from Taconic Farms. The 1AR⫺/⫺ and 2AR⫺/⫺ mice (provided by Dr. B. Kobilka, Stanford University, Palo Alto, CA) were bred on the FVB/NJ background. As previously described, both AR-deficient strains were derived by the insertion of a neomycin resistance cassette into the genetic sequence of the fourth transmembrane domain of the receptor, rendering the transcription of the complete receptor impossible (19). Strain verification studies were performed using real-time PCR and RT-PCR to confirm the lack of 1AR and 2AR transcript in both liver and spleen of 1AR⫺/⫺ and 2AR⫺/⫺mice, respectively (data not shown). Control mice were left in their original cages undisturbed, while mice subjected to CR treatment were individually restrained in well-ventilated plastic 60-ml syringes at 4°C for 1 h in the dark. CR represents a physical and a psychological stress. CR was performed between 8 and 11 a.m. on day 0; the mice were infected immediately after CR. LM was originally isolated from a meningitis patient and has been maintained as previously described (20). Mice were i.v. injected with a sublethal dose of LM (2–3 ⫻ 103 CFU/i.v. injection for FVB/NJ, and 3.5–10 ⫻ 103 CFU/i.v. for BALB/c mice) with or without CR administered before the inoculation. Except for anti-KLH Ig and DTH responses, all other measurements were obtained from different groups of mice depending upon the background strain or timing that was required for each assay. Determination of viable LM burden in liver and spleen To determine the bacterial load in mice following LM infection, we performed enumeration of viable LM as described previously (5). Briefly, mice were sacrificed by lethal CO2 anesthesia, and the spleen and liver were removed aseptically and homogenized in sterile 0.9% NaCl. Serial dilutions of organ homogenates were plated on blood-agar plates and cultured overnight for enumeration of viable LM. Bacterial burdens are expressed as number of viable LM CFU per organ. Flow cytometric analysis of T cells Spleens were removed aseptically from 6- to 8-wk-old BALB/c male mice as described above, and single-cell suspensions were prepared by grinding the tissue between two frosted microscope slides. RBCs were lysed and FIGURE 1. Listeria-specific CD8⫹ T cell expansion is unaltered by CR. Memory (CD62L⫺) CD8⫹ T cells were quantified in spleens from CRstressed and nonstressed BALB/c mice at 8 and 10 days after LM infection (i.v.; 7.3–9.5 ⫻ 103 CFU). A, Typical flow cytometric dot plots of CD8-gated lymphocytes from an uninfected mouse and from a day 8-infected mouse. The memory (CD62L⫺)/LM-specific (LLO91–99 tetramer⫹) cells were enumerated by first gating the spleen cells by light scatter and CD8 expression, as described in Materials and Methods. B, Memory (CD62L⫺)/LM-specific (LLO91–99 tetramer⫹) cells were enumerated in spleens from control and CRtreated mice. Results are representative of two repeated experiments; each bar shown is the mean (SD) for two mice. C, The kinetics of LM killing, showing the LM levels at time of CD8 analysis. cells were washed twice with PBS, and total cell numbers were determined; 1 ⫻ 106 cells/tube were used for staining. MHC class I (H2-Kd) tetramers containing an immunodominant LM epitope from the pore-forming toxin listeriolysin (LLO91–99) were used to quantify LM-specific responses; the tetramers were provided by Dr. E. G. Pamer (Sloan-Kettering Institute, New York, NY). All mAbs against cell surface molecules were purchased from BD Biosciences Pharmingen. Cells were treated with FcR-blocking buffer containing 20 g/ml streptavidin and anti-mouse CD16/CD32 FcR (1 g/tube) in 50 l of staining buffer (0.5% BSA and 0.02% NaN3 in PBS (pH 7.5)) for 20 min on ice. Further staining with PE-MHC class I tetramer (LLO91–99), allophycocyanin-anti-mouse-CD62L, and FITC-anti-mouseCD8 was performed for 60 min on ice. Enumeration of LM-specific CD8⫹ populations was accomplished using TruCount tubes (BD Biosciences Pharmingen). Regulatory T cell subsets following LM infection with or without CR-treatment were analyzed using a combination of FITC-CD4/ PE-GITR/allophycocyanin-CD25 Abs. The acquisition and analysis of all Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 immunosuppression before immune activation that is the focus of our current studies. Cell surface dopaminergic and adrenergic receptors, which are expressed on immune cells (12), bind the catecholamines dopamine (DA), epinephrine (Epi), and norepinephrine (NE). Pharmacologic blocking studies provided the first evidence for an immunomodulatory role of 1-adrenoceptor (1ARs) in host immunity (7). Specifically, peripheral administration of atenolol (a 1AR-specific antagonist commercially available as Tenormin) blocked the CR-induced delay in bacterial clearance whereas ␣AR- or 2AR-specific antagonists (phentolamine and ICI118,551, respectively) did not. It is well documented that 2AR is ubiquitously expressed on Th0, Th1, and B cells; 2AR is down-regulated on Th2 cells by histone deacetylation (14), thereby implicating this receptor in the regulation of humoral and Th1 functions by catecholamines (15). Depending upon the activation state and developmental stage of the Th cell, the 2AR signal can enhance (16, 17) or suppress (18) IFN-␥ production. Despite a well-established role of sympathetic neuroimmune modulation and despite also the widespread clinical use of “ blockers” and synthetic catecholamines, the immunologic relevance of 1ARs has been largely unexplored. The current in vivo studies used wild-type (WT) mice (either BALB/c or FVB/NJ) and FVB/NJ mutant strains with a deficiency of 1AR or 2AR (1AR⫺/⫺ and 2AR⫺/⫺, respectively) to investigate the role of stress-induced -adrenoceptors (ARs) on host immunity, and to evaluate overall in vivo immunity to KLH. Based on previous studies, our a priori hypothesis was that CR stress-induced 1AR signaling impairs host defenses against a low-dose LM infection by altering innate immune mechanisms. 4878 1ARs ALTER HOST IMMUNITY FIGURE 2. CR-induced modulation of 1AR⫺/⫺, 2AR⫺/⫺, and FVB/NJ (WT) host defenses against LM. These three strains (n ⫽ 5–11/ treatment/strain) were assessed for viable LM in the liver (A) and spleen (B) at 3 days after infection with 2.5– 3.0 ⫻ 103 CFU immediately after CR treatment. The ⴱ indicates a significant difference (⬍0.05) compared with the FVB/NJ LM-infected, nonstressed control. Perforin expression by Western blot analysis Protein was isolated from frozen livers and spleens of 2- to 3-mo-old male BALB/c mice and 1AR⫺/⫺ male and female mice were treated with or without CR and infected with 3.6 ⫻ 103 LM by homogenization of a portion of each organ in 0.5 ml of mammalian protein extraction reagent (Pierce) with protease inhibitor mixture (Sigma-Aldrich). Lysed homogenates were centrifuged for 30 min at 15,000 ⫻ g. Protein concentrations were measured using the BCA protein assay kit (Pierce), and 100 g of protein from each sample was diluted with one-half volume of sample buffer containing 30% (v/v) glycerol, 10% (v/v) 2-ME, and 0.25% (w/v) bromophenol blue in 62.5 mM Tris-HCl buffer (pH 6.8). SDS-PAGE was performed in 4 –20% gradient separating gels; the gels were then placed upon nitrocellulose paper and subjected to blot transfer. The blotted proteins were blocked with 5% fish gelatin in PBS 0.01% NaN3 for 1 h at room temperature (RT) and washed in TBS-T (25 mM Tris-HCl, 125 mM NaCl, and 1.0% Tween 20 (pH 8.0)). Blots were incubated with mAb to mouse -actin (Sigma-Aldrich) and either rabbit anti-rat perforin (catalog no. CPP100; Cell Sciences) or rabbit anti-mouse granzyme B (catalog no. RB9015-PO; NeoMarkers) in blocking buffer overnight at 4°C. Blots were washed twice in TBS-T and then incubated with HRP-conjugated secondary Abs (goat anti-rabbit IgG for perforin and granzyme and goat antimouse IgG, for actin; Sigma-Aldrich) for 2 h at RT with rocking. Blots were washed three times for 20 min each in TBS-T and then developed with Super Signal/chemiluminescent substrate (Pierce) for 5 min and assayed with a LAS-1000plus (Fuji). Splenocyte and liver preparation for RNA isolation Male BALB/c mice were either CR-treated or left undisturbed in their home cage. Immediately after CO2 administration, mice were perfused through the right ventricle with 30 ml of PBS, followed by 10 ml of digestion buffer (0.05 M TES (C6H15NO6S) and 0.36 M CaCl (pH 7.5)). Livers and spleens were placed into digestion buffer containing collagenase IV (200 g/ml; Sigma-Aldrich) and DNase I (50 g/ml; Sigma-Aldrich) and were coarsely chopped with a sterile razor blade before 37°C incubation for 20 min. Splenocytes were then homogenized as described above, whereas livers were pipetted through a Teflon mesh. Single-cell suspensions were washed twice in PBS and then layered over mouse Ficoll (Ficoll:metrizoate 12:5, density 1.090 g/ml) (21) and centrifuged for 15 min at RT at 1300 ⫻ g. The total Ficoll-purified cells were counted and pelleted for immediate isolation of total RNA. F4/80⫹ Kupffer cells were isolated from liver homogenates following the above procedure with slight modifications. After single-cell suspensions had been obtained by passage through a Teflon mesh, liver homogenates were centrifuged (110 ⫻ g for 2 min at 4°C) to remove hepatocytes (22). F4/80⫹ cells were enriched from the single-cell suspension by use of a SpinSep mouse enrichment mixture, SpinSep mouse dense particles, and density gradient centrifugation cell separation procedures per the manufacturer’s protocol (StemCell Technologies). RNA isolation from immune cells and AR mRNA determination by TaqMan real-time PCR Cell pellets containing 5 ⫻ 105–1 ⫻ 106 Ficoll-purified liver or spleen cells or F4/80-enriched cells from liver homogenates were resuspended in 50 l FIGURE 3. ARs increase in liver but not spleen immediately following CR stress. RNA was isolated from Ficoll-purified BALB/c liver (A) and spleen (C) homogenates, and expression of ARs was quantified by real-time TaqMan PCR (n ⫽ 5 and 4 for nonstressed, control and CR groups, respectively). F4/80⫹cells (B) were enhanced from liver homogenates obtained from CR-treated and control mice (n ⫽ 3). The ⴱ indicates significant differences (p ⬍ 0.05) measured between stressed and control mice. The # indicates significant differences (p ⬍ 0.005) between 1AR and 2AR expression in the spleen, irrespective of CR treatment. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 specimens were performed on a BD FACSCalibur using CellQuest software at the Wadsworth Center Flow Cytometry Core. The Journal of Immunology 4879 Table I. Serum corticosterone at baseline and following CR treatmenta BALB/c FVB/NJ 1AR⫺/⫺ 2AR⫺/⫺ Baseline Serum CORT (pg/ml)b Average CORT Increase in CR as % Controlc 269.7 ⫾ 163.4 272.0 ⫾ 85.0 221.0 ⫾ 52.1 283.9 ⫾ 97.6 208.8 155.2 130.9 128.8 n ⫽ 4 – 8 animals tested/strain. No significant differences found between strains. c Significant increases in CR vs control observed for all strains ( p ⬍ 0.05). a b Hemopoietic ablation and lymphocyte reconstitution of recipient mice FVB/NJ WT- and AR-deficient mice that were used as recipient hosts for lymphocyte reconstitution experiments received lethal 137Cs irradiation of 10 Gy (dose rate 2.5 Gy/min). On the following day, a mixture of 1 ⫻ 106 bone marrow progenitor cells and 10 ⫻ 106 splenocytes was injected i.v. in 200 l through the tail vein as described previously (23). Transplant recipients were housed in pathogen-free facilities and given neomycin (1 mg/ml) in drinking water for the first 2 wk. After 6 – 8 wk total, CR administration, LM infection, and subsequent immunologic analysis of host immune responses were performed as described above. Splenocyte and bone marrow isolation Spleens were aseptically harvested from euthanized FVB/NJ WT- and AR-deficient donor mice into a sterile petri dish containing Dulbecco’s PBS (without calcium and magnesium; Sigma-Aldrich) on ice. Inside a biosafety hood, spleens were transferred into another sterile petri dish with 5 ml of Dulbecco’s PBS. Each spleen was homogenized between two frosted microscope slides (Erie Scientific), and the cell suspension was transferred into a 15-ml polypropylene tube with a Pasteur capillary pipette. The cell suspension was allowed to settle for 3 min at RT for separation of cellular debris, and the single-cell suspension in the supernatant was then transferred to a new tube. Following centrifugation at 200 ⫻ g for 10 min, RBCs were eliminated by use of lysing buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2 EDTA (pH 7.2–7.4)). After lysis (5 min at RT), the cell suspension was centrifuged again, and the cell pellet was resuspended in 10 ml of DPBS; 20 l of this cell suspension was taken for determination of total cell numbers (Coulter Counter). After centrifugation, the cell pellet was resuspended to the appropriate concentration in sterile PBS. Bone marrow progenitors were isolated as described previously (24). KLH immunization Female WT (FVB/NJ), 1AR⫺/⫺, and 2AR⫺/⫺ mice (age 2 mo) were immunized with 100 g of KLH (Calbiochem) plus TiterMax Gold adjuvant in 200 l of saline at days 1 and 28. Corticosterone (CORT) measurement Serum preparation The concentrations of serum CORT at baseline and immediately following 1-hr CR treatment were determined by enzyme immunoassay with CORT EIA Ab (Assay Designs). The sensitivity of the CORT EIA was 32 pg/ml. Peripheral blood was obtained by retro-orbital phlebotomy, into 1.7-ml Eppendorf tubes. After it had been allowed to clot overnight at 4°C, serum was collected following centrifugation. FIGURE 4. CR stress increases perforin protein. Protein was obtained from livers (A) and spleens (B) of stressed (n ⫽ 3) or nonstressed, control BALB/c mice (n ⫽ 4) in the absence of LM and from mice infected with LM with or without prior stress treatment (n ⫽ 3 for all time points examined following LM infection (4, 24, and 48 h), except LM infected, nonstressed, control 48 h, n ⫽ 2). The ⴱ indicates significant increases (p ⬍ 0.05) in CR-treated mice compared with the control group at that time point. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 of PBS and lysed with RLT/2-ME buffer from Qiagen. Cell lysates were homogenized with the Shredder Spin Columns provide by Qiagen, and RNA was isolated using the Qiagen RNA capture minicolumns, according to the manufacturer’s protocol for isolation of RNA from cultured cells. Isolated RNA was resuspended in 60 l of RNase-free water and quantified using a Beckman DU 640 spectrophotometer. After dilution of each RNA preparation to a working concentration of 0.1 g/l, cDNA was prepared from 1 g of total RNA using a High-Capacity cDNA Archive kit from Applied Biosystems. Following the synthesis reaction, an aliquot of 100 l of PCR-grade water was added to each tube, and 5 l was used for amplification of murine 1AR, 2AR, and GAPDH using TaqMan Gene Expression Assay kits from Applied Biosystems. Amplifications were conducted in quadruplicate in a 7500 Real Time PCR instrument (Applied Biosystems). Amplification conditions used were specified by the manufacturer as follows: 2 min at 50°C, then 10 min at 95°C, then 40 cycles of 15 s at 95°C, and 1 min at 60°C. Data for the amplification plot was collected during the 60°C step. Relative quantitation results were measured using the comparative cycle threshold method, whereby the amplification of the gene of interest is normalized to amplification of the gene encoding GAPDH, measured from the same cDNA synthesis sample. Four APC lines, RAW 264.7, PMJ2-PC, N9, and A20, were also used for RNA isolation and subsequent AR amplification procedures. 1ARs ALTER HOST IMMUNITY 4880 Table II. Western blot analysis of perforina in 1AR⫺/⫺ mice Control CR Baseline (0 h) 24 h after LM Infection 70.7 ⫾ 28.3 82.0 ⫾ 42.1 58.1 ⫾ 18.3 57.3 ⫾ 8.9 a Perforin protein normalized to actin, n ⫽ 4/group; no significant differences were detected between control and CR-treated mice. ELISA for IgG isotype and IgM IgG isotype and IgM were measured by a standard ELISA, as described previously (25). DTH assay Statistical analysis Data from two or more independent experiments were combined and differences between two experimental groups (where n ⱖ 3) were determined using the t test SigmaStat (Jandel Scientific). Effects of CR stress and 1AR or 2AR deficiency on host immunity were considered significant only if the p value was ⬍0.05. Results Ag-specific expansion is not altered by CR stress The in vivo expansion of CD8⫹ Ag-specific lymphocytes of control or CR-treated BALB/c mice was measured using the immunodominant LLO91–99 tetramer. Tetramer-positive cells were detected by day 8 after LM-infection (Fig. 1A), but no differences were detected between CR-treated and control mice (Fig. 1B). Stress-related changes in the percentage of either CD4⫹/CD25⫹ or CD4⫹/CD25⫹/GITR⫹ splenic subsets were not observed at 24 h after LM infection (data not shown). FIGURE 5. Adoptive transfer of 1AR⫺/⫺, 2AR⫺/⫺, or FVB/NJ (WT) bone marrow progenitor cells and splenocytes into lethally irradiated WT, 1AR⫺/⫺, or 2AR⫺/⫺ recipient mice. Six weeks after reconstitution, WT (A and D), 1AR⫺/⫺ (B and E), or 2AR⫺/⫺ (C and F) host mice (n ⫽ 3–5 for all groups, except n ⫽ 2 for 1AR⫺/⫺ donor cells into 2AR⫺/⫺ hosts, and n ⫽ 2 for 2AR⫺/⫺ donor cells into 1AR⫺/⫺ hosts) were LM infected (3 ⫻ 103) and 3 days later spleens (top panels, A–C) and livers (bottom panels, D–F) were quantified for LM (CFU/organ). The ⴱ indicates significant differences (p ⬍ 0.05) compared with the WT host reconstituted with WT immune cells. ⴱⴱ, Differences in LM burden compared with the 1AR⫺/⫺ host reconstituted with 1AR⫺/⫺ immune cells (p ⫽ 0.057). The immunosuppressive effect of CR previously reported for BALB/c mice (5, 7, 8, 26) was also observed in WT FVB/NJ mice by day 3 of a low-dose LM infection. However, because FVB/N mice were more sensitive than BALB/c mice to LM, we had to use a lower LM inoculum (⬃3 ⫻ 103 CFU). Liver colonization was significantly increased in CR-treated WT mice compared with control WT mice (Fig. 2A; p ⫽ 0.037). CR did not suppress host resistance of 1AR⫺/⫺ mice. Relative to WT or 2AR⫺/⫺ mice, 1AR⫺/⫺ mice had improved host defenses against LM; CR did not impair their resistance in livers (Fig. 2A) or spleens (Fig. 2B). Splenic and overall body weights did not differ statistically between the LM-infected mice treated with CR and those not subjected to CR (data not shown). CR stress increases AR mRNA in immunocytes of liver but not spleen Because 1AR and 2AR appear to have substantially different effects on host defenses against LM, it was critical that we assess the expression of these receptors by immune cells. We prepared immune cells from both liver and spleen homogenates of BALB/c mice and analyzed them for mRNA expression of 1AR and 2AR transcripts (Fig. 3). Immediately following CR treatment and in the absence of LM infection, significant increases in 1AR and 2AR expression were measured in livers (Fig. 3A; p ⫽ 0.04 and p ⫽ 0.004, respectively). We further investigated the expression of 1AR and 2AR on the Kupffer (F4/80⫹) cells of livers obtained from stressed and control mice (Fig. 3B). Although both receptor subtypes were identified by real-time TaqMan PCR analysis, stress treatment did not significantly alter the expression levels. Splenocytes expressed greater levels of 2AR than 1AR (Fig. 3C; p ⬍ 0.005); stress treatment did not significantly alter the expression of either receptor. Although radioisotope-binding assays have provided evidence for ARs on monocyte-derived cells, we used real time PCR to confirm expression of 1AR and 2AR in two cell lines of monocyte origin Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 DTH assays were performed 21 days after the last immunization with KLH. After measurement of the thickness of the left and right footpad of each mouse, KLH (100 g/25 l saline) was s.c. injected into the right footpad and saline only was injected into the left footpad. The DTH response was measured 24 h later with a Spi dial thickness gauge. The response was defined as the difference between the right and left footpad swellings. Mice lacking 1AR are more resistant to CR-induced immunosuppression than are WT or 2AR⫺/⫺ mice The Journal of Immunology 4881 (RAW 264.7 and PMJ2-PC), a microglial cell line (N9), and a B cell line (A20) (data not shown). Serum CORT is increased following CR treatment Although CORT is known to play a significant role in the stress response, both WT- and AR-deficient strains appear to have equally robust increases in CORT following CR treatment (Table I). Furthermore, our previous research using pharmacological blocking agents had suggested a protective role for CORT in this model of stress and infection rather than an immunosuppressive one (5). Perforin expression in liver is increased following CR treatment Suppression of host immunity is associated with 1AR⫹ immune cells AR-competent immune cells were reconstituted in AR-deficient, irradiated mice, and AR-deficient immune cells were reconstituted in AR-competent, irradiated mice to aid us in assessing the effects of AR nonimmune and immune cells on immune defenses after CR. WT (FVB/NJ) recipient mice reconstituted with 1AR⫺/⫺ immune cells had a statistically significant decrease (by ⬃3-fold) in bacterial colonization in the liver, relative to mice reconstituted with WT immune cells (Fig. 5D; p ⫽ 0.03). The transfer of 2AR⫺/⫺ cells into WT mice produced a nonsignificant decrease in liver colonization. The transfer of WT immune cells into 1AR⫺/⫺ hosts or 2AR⫺/⫺ immune cells into 2AR⫺/⫺ hosts gave rise to a greater bacterial burden in liver than did the transfer of 1AR⫺/⫺ immune cells into 1AR⫺/⫺ hosts (Fig. 5, E and F; p ⫽ 0.05). 1AR⫺/⫺ mice reconstituted with their own immune cells had significantly lower bacterial colonization of the liver than did control mice reconstituted with their own immune cells (Fig. 5, D and E; p ⫽ 0.04). 2AR⫺/⫺ hosts reconstituted with either WT or 1AR⫺/⫺ immune cells had lower bacterial loads in their livers and spleens on day 3 of the infection than did the same 2AR⫺/⫺ hosts reconstituted with 2AR⫺/⫺ donor cells, but these effects were not statistically significant. The absence of 1AR enhances cell-mediated immunity, whereas 2AR deficiency improves humoral responses The production of Abs specific for KLH was measured in 1AR⫺/⫺, 2AR⫺/⫺, and WT (FVB/NJ) mice (Fig. 6, A–C). IgG subtypes and IgM were highest in serum from 2AR⫺/⫺ mice and lowest in 1AR⫺/⫺ mice; the single exception was IgG1 for which WT mice had the lowest production. The differences in production FIGURE 6. Anti-KLH humoral and cellular responses measured in 1AR⫺/⫺(n ⫽ 5), 2AR⫺/⫺(n ⫽ 7), or FVB/NJ (WT, n ⫽ 9) mice; bars indicate SE. Female mice were injected with 100 g of KLH in TiterMax adjuvant, and retro-orbital blood samples were obtained for evaluation of KLH-specific Ab responses (A–C) at the end of week 1 (IgM) and week 2 (IgG1 and IgG2a). Ab data are presented as optical densities standardized to an internal assay control. DTH responses (D) were measured 3 wk after a secondary immunization given at week 4. E, The ratio of humoral to cell-mediated responses as the product of the summed Ab response divided by the DTH response. The ⴱ indicates a significant difference (p ⬍ 0.05) compared with WT control. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 The expression of the cytotoxic protein perforin was analyzed in livers and spleens of BALB/c mice (Fig. 4) and 1AR⫺/⫺ mice (Table II). Early changes in perforin expression were evident in livers of CR-treated BALB/c mice (Fig. 4A). The greatest increases in perforin were observed immediately after stress treatment, and 4 h after LM infection ( p ⬍ 0.05). No changes in perforin expression were observed in the spleen (Fig. 4B), nor were differences in granzyme B expression detected in either liver or spleen (data not shown). No significant increases were observed in liver homogenates of 1AR⫺/⫺ mice immediately following CR treatment or 24 h after LM infection (Table II). 4882 of IgG1 between 2AR⫺/⫺ and WT mice were statistically significant (Fig. 6A). In contrast to humoral immunity, the DTH response in 1AR⫺/⫺ mice was more robust than the DTH response in either WT or 2AR⫺/⫺ mice (Fig. 6D). The ratio of humoral to cell-mediated responses was significantly higher in 2AR⫺/⫺ mice than in WT mice (Fig. 6E). Discussion cells to NK cell perforin-dependent killing (33). Thus, we suggest that the early CR-induced increase of the cytotoxic perforin molecule plays an important role in our experimental model of neuroimmunomodulation by increasing apoptosis, which consequently increases susceptibility to infection. LM infection is known to induce lymphocyte apoptosis through the immunodominant LLO protein (34), and type 1 IFNs increase the susceptibility of lymphocytes to apoptosis (35). LM-induced apoptosis is thought to attenuate innate antilisterial responses because the engulfment of apoptotic lymphocytes by macrophages favors IL-10 production (36). It was recently shown that NE also can induce apoptosis of lymphoid cells (37), and our previous studies have demonstrated a stress-induced increase in TNF-␣ production in the liver between days 1 and 3 of an LM infection (26); such an increase could confer increased susceptibility of lymphocytes to apoptosis. Therefore, a possible explanation for the observed stress-induced immunosuppression of host defenses is that stressed mice display premature release of cytotoxic factors, and these factors increase lymphocyte apoptosis, thereby overwhelming the innate mechanisms required for both bacterial clearance and activation of optimal innate and adaptive responses. Perforininduced death of LM-infected cells does not influence total bacterial burden (38). Therefore, our observation that LM burdens are similar in both stressed and control animals until day 2–3 of the infection is consistent with a possible role of perforin-induced apoptosis early in the infection. Although differences in regulatory T cell subsets were not detected at 24 h after LM infection, it is possible that other CD4⫹ T cell subsets (i.e., CXCR3⫹) induce apoptosis in the liver via mechanisms not involving proinflammatory cytokines (39). Additionally, the stress-induced increase in AR receptor expression in the liver, specifically 1AR signaling on F4/80⫹ cells, implicates catecholamine-induced changes in monocytes. It is known that catecholamines alter macrophage redox state (40 – 42) and early inflammatory cytokine profiles (43– 45). Although our research aim has been to elucidate stress-induced mechanisms of immunosuppression, the improved host resistance that we observe in 1AR⫺/⫺ infected mice, relative to WT (FVB/ NJ)-infected mice, exemplifies the importance of sympathetic nervous system activities on host-pathogen interactions. The role of sympathetic neuronal activity in regulation of host susceptibility to infectious organisms, in particular LM, is well documented (46). Along with our laboratory, others have shown that early host defenses against LM are enhanced by treatment with the neurotoxicant 6-hydroxydopamine (6-OHDA). 6-OHDA selectively and temporarily ablates peripheral dopaminergic nerves, thereby depleting peripheral tissues of catecholamines because DA is the precursor to NE. In these studies, LM colonization of livers and spleens in sympathectomized mice was significantly reduced at days 3–5 after infection, as compared with the colonization of control mice with innervated organs (5, 47– 49). Additionally, peripheral administration of 6-OHDA blocked stress-impaired immunity in spleen but not in liver (5), a pattern that differs from the complete abrogation of stress-induced immunosuppression in both organs of 1AR⫺/⫺ mice. It is possible that the livers of 6-OHDAtreated mice are still susceptible to stress-induced release of Epi from the adrenal glands, and circulating Epi could influence liver defenses through functional 1AR signaling. The enhanced host resistance of mice with denervated peripheral organs had been correlated with increased numbers of splenic neutrophils (48) and activated peritoneal macrophages (49) during the first 3 days of an i.p. infection. However, between days 5 and 7 of LM infection, 6-OHDA treatment decreased splenic leukocyte Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Although there have been numerous reports that 2AR-signaling modulates immunity, this is the first report to describe 1AR inhibition of a DTH response and cell-mediated host immune defenses. Additionally, we have shown that CR treatment inhibits host defenses against LM in mice with functional 1AR signaling (WT FVB/NJ and 2AR⫺/⫺ mice), whereas 1AR⫺/⫺ mice are not immunologically impaired by the CR treatment. Previous studies have demonstrated normal immune functions in 2AR⫺/⫺ mice (27). Our results agree with these findings in that WT and 2AR⫺/⫺ mice coped similarly with Listeria infection. Moreover, both WT and 2AR⫺/⫺ mice were immunocompromised by CR treatment. The present data demonstrating improved host defenses against LM in the absence of 1AR corroborate the findings of previous pharmacological studies in which 1AR was identified as a stress-signaling molecule that weakens host resistance (5, 7, 8, 26). The CR-induced increase in bacterial colonization of WT and mutant FVB/NJ mice and mRNA expression of ARs in the liver, but not in the spleen of BALB/c mice, also delineates the existence of stress-induced responses that are organ specific. Based on the lack of CR-induced inhibition of host immunity in CD4⫺/⫺ BALB/c mice (8), it had been suggested that CD4⫹ T cells, possibly regulatory T cells, or downstream CD8⫹ T cell effectors are involved in the stress-induced immunosuppression. However, CR-induced early changes in the number or phenotype of peripheral lymphocytes have not been detected (8). In this study, we show that CR does not alter the expansion of Ag-specific CD8⫹ lymphocytes of LM-infected BALB/c mice, indicating that CR-treatment does not interfere with the priming of LM-specific effectors, which occurs within the first 12 h of infection (28). The observed LM-specific CD8 responses further confirm the previous suggestion (5) that CR-treatment inhibits defenses only early (days 2–3) after infection. The investigation of LM-specific CD8⫹ T cells in AR-deficient mice was not performed because the LLO91–99-specific class I (H2d) tetramers do not match the MHC of the FVB/NJ strain (H2q). Additionally, an increase in the number of regulatory T cells does not appear to be able to account for the inhibition of host defenses against LM. Because an increase in perforin expression would likely be perceived as beneficial for handling early primary LM infection (29), we were surprised that CR treatment actually increased perforin expression in the liver of BALB/c mice. This stress-induced increase of perforin was still seen at 4 h after LM infection of the CR-treated mice but declined by 24 h. Stress-mediated alteration of NK activities in mice has been previously reported; the duration and type of stress treatment had differential effects on the immunologic outcome in that 1 h of hyperthermic treatment (30) or the stress treatment of electric foot shock for 1 h daily for 3 days (31) both decreased perforin. NK perforin-dependent cytotoxic activities (YAC-1 cytotoxicity) of mononuclear liver preparations were also diminished 24 h after Epi injection (20 g i.p.); however, NKT cell Fas-dependent killing of syngeneic thymocytes was increased (32). Interestingly, Oya et al. (32) found a significant 50% decrease in T cells (NK1⫺CD3high) 24 h after a 12-h restraint stress treatment, the decrease was posited to be due to stress-induced apoptotic mechanisms. In another study, stress (H2O2, heat, or high-density growth) increased the susceptibility of activated T 1ARs ALTER HOST IMMUNITY The Journal of Immunology Acknowledgment We thank the Immunology Core of Wadsworth Center for their assistance with the flow cytometry. Disclosures The authors have no financial conflict of interest. References 1. McEwen, B. S., and E. Steller. 1993. Stress and the individual: mechanisms leading to disease. Arch. Int. Med. 1153: 2093–2101. 2. Thorpe, K. E. 2005. 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Theses studies suggest that peripheral innervations suppress early innate mechanisms, but not adaptive responses, during an infection; this conclusion is in agreement with our quantification of LM-specific CD8⫹ cells. Because the 1AR⫺/⫺ mice used in the current study can synthesize catecholamines both centrally and peripherally but lack the capacity to signal NE and Epi via 1AR, an assessment of the role of CNS innervation is important in an understanding of the implications of our results. Interestingly, peripheral administration of 6-OHDA does not affect the CNS because this molecule cannot pass the blood-brain barrier (52). However, central ablation of dopaminergic neurons by intrastriatal injection of 6-OHDA (51), bilateral injection of 6-OHDA into the lateral ventricles (53), or genetic removal of DA -hydroxylase (an enzyme required for the production of DA) (54) impaired peripheral host immunity. In other words, the elimination of CNS neurons that release DA (51, 53) or the prevention of DA synthesis by dopaminergic neurons (54) is immunosuppressive, yet the loss of DA neurons in the periphery is immunoenhancing, as noted above. How the elimination of catecholamine signaling in the brain functions to suppress peripheral immune activity is as yet unresolved, but it likely involves inappropriate hypothalamic-pituitary-adrenal axis activation (51) and dysregulation of peripheral catecholamines essential for immune homeostasis (53). Based on the hemopoietic reconstitution experiments, we infer that the presence of 1AR or 2AR expression in the brain, or lack thereof, does not detectably affect peripheral immunity against LM. Although central release and signaling of DA are critical factors in maintaining immunocompetence, signaling of NE and/or Epi through the 1AR in peripheral organs is immunosuppressive. Improved DTH responses in 1AR⫺/⫺ mice suggest the existence of cell-mediated specific mechanisms of immunosuppression by 1AR signaling; when these are absent, antilisterial defenses are improved. We observed significant increases in Ag-specific humoral responses of 2AR⫺/⫺ mice relative to WT mice, following immunization with KLH, whereas previous studies showed equivalent humoral responses produced in vivo by WT FVB/NJ and 2AR⫺/⫺ mice after immunization with the trinitrophenyl hapten conjugated to KLH (27). The discrepancy between our results and theirs may be a function of differing numbers of naive and/or memory KLH-specific or hapten-specific T and B cells in the two studies. In any case, expression of 1ARs and 2ARs clearly has multiple influences on immunity. In summary, our study corroborates previously published pharmacological evidence for a 1AR-associated mechanism of immunosuppression. 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Selective activation of adrenergic 1 receptors induces heme oxygenase-1 production in RAW264.7 cells. FEBS Lett. 579: 5494 –5500. 1ARs ALTER HOST IMMUNITY