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Available online at www.sciencedirect.com Immunology Letters 115 (2008) 43–49 Role of ␣-adrenergic stimulus in stress-induced modulation of body temperature, blood glucose and innate immunity Mayumi Watanabe a , Chikako Tomiyama-Miyaji a,c , Eisuke Kainuma a , Masashi Inoue a , Yuh Kuwano a , Hongwei Ren a , Jiwei Shen a,b , Toru Abo a,∗ a Department of Immunology, Niigata University School of Medicine, Niigata 951-8510, Japan First Department of Surgery, Niigata University School of Medicine, Niigata 951-8510, Japan c School of Health Sciences, Faculty of Medicine, Niigata University, Niigata 951-8518, Japan b Received 31 July 2007; received in revised form 28 September 2007; accepted 28 September 2007 Available online 23 October 2007 Abstract Mice were exposed to restraint stress for 3 h. During this period, low body temperature (hypothermia, 39 ◦ C → less than 37 ◦ C) and high blood glucose levels (hyperglycemia, 150 mg/dl → up to 220 mg/dl) were simultaneously induced. Reflecting a stress-induced phenomenon, blood levels of catecholamines increased at that time. Administration of adrenaline (␣-stimulus), but neither noradrenaline (␣ but less than adrenaline) nor isoproterenol (␤), induced a similar stress-induced pattern of body temperature and blood glucose variations. This ␣-adrenergic effect was confirmed using ␣- and ␤-blockers in adrenaline-induced hypothermia and hyperglycemia. By applying this ␣-stimulus, the effect on immunoparameters was then investigated. Stress-resistant lymphocyte populations were found to be NK cells, extrathymic T cells and NKT cells, especially in the liver. Functional assays showed that both NK-cell cytotoxicity and NKT-cell cytotoxicity were augmented by ␣-stimulus. These results suggest that ␣-stimulus is one of the important factors in the stress-induced phenomenon and that it eventually produces hypothermia, hyperglycemia and innate-immunity activation seen during stress. © 2007 Elsevier B.V. All rights reserved. Keywords: Stress; Adrenergic stimuli; Hyperglyce; mia; Hypothermia; Innate immunity 1. Introduction It is widely known that stress induces immunosuppression, especially against T and B lymphocytes in the periphery, accompanied by thymic atrophy [1–3]. Such immunosuppression is mediated by not only steroid hormones but also adrenergic stimuli (i.e., catecholamines) which are secreted due to stress [4–6]. Immunomodulations, including immunopotentiaion as well as immunosuppression, by stress were also reported by many investigators [7–11]. In a recent preliminary study, we noticed that restraint stress also induced hypothermia and hyperglycemia in mice. This observation as well as the immunosuppression might be very important because many patients suffering from stress show hypothermia derived from circulation failure (i.e., due to severe constriction of vessels) and hyperglycemia (i.e., one of the causes of diabetes mellitus) [12–14]. ∗ Corresponding author. Tel.: +81 25 227 2133; fax: +81 25 227 0766. E-mail address: email@example.com (T. Abo). 0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.09.010 In light of the above-mentioned observation, in the present study, we investigated how adrenergic stimuli are associated with stress-induced responses such as the immunosuppression, hypothermia and hyperglycemia. In our previous study [15,16], we reported that innate immunity mediated by NK and NKT cells was rather augmented by restraint stress in parallel with the immunosuppression of acquired immunity mediated by T and B lymphocytes. It was therefore investigated whether adrenergic stimuli (␣- or ␤-stimulus) were able to induce a similar phenomenon. 2. Material and methods 2.1. Mice C57BL/6 (B6) mice were purchased from Charles River Japan (Yokohama, Japan). All mice were used at 8–12 weeks of age in these experiments. The mice were kept in a room under constant temperature (25 ± 2 ◦ C) and humidity (50–70%) with a 44 M. Watanabe et al. / Immunology Letters 115 (2008) 43–49 12 h light/dark cycle (light on from 8:00 to 20:00 h). They were fed under specific pathogen-free conditions in the animal facility of Niigata University (Niigata, Japan). 2.2. Restraint stress Mice were exposed to restraint stress using stainless steel mesh for 3 h (from 9:00 to 12:00 h) . All of the present experiments were done with the approval of the Animal Ethics Committee of Niigata University. 2.3. Measurement of body temperature and blood glucose Body temperature was determined by using THERMAC SENSOR (Shibaura Denki Co., Tokyo) and blood glucose was measured by Precision Xtra TM (Abott Japan Co., Ltd., Chiba, Japan). 2.4. Administration of catecholamines and adrenergic blockers administered into a mouse at a concentration of 25.0 (or 12.5 or 6.25) g/mouse. Phentolamine, ␣-blocker (Regitin, NOVARTIS Pharma Co., Ltd., Tokyo) and Propranolol, ␤-blocker (Inderal, AstraZeneca Co., Ltd., Osaka) were also used. These agents (0.2 ml) were administered i.p. into a mouse at concentrations of 6.25 g/mouse or 12.5 g/mouse. 2.5. Cell preparation Mice anaesthetized with isoflurane were sacrificed by exsanguination from the subclavian artery and vein, and the liver and spleen were removed. Hepatic lymphocytes were prepared as previously described . Briefly, the liver was pressed through 200-gauge stainless steel mesh and suspended in Eagle’s MEM medium supplemented with 5 mM HEPES and 2% FCS. After one washing, the pellet was resuspended in 35% Percoll solution containing 100 U/ml heparin and centrifuged Adrenaline (Bosmin, Daiichi Sankyo Co., Ltd., Tokyo), noradrenaline (NOR-ADRENALIN, Daiichi Sankyo Co., Ltd., Tokyo) and isoprotelenol (PROTERNOL, Nikken Chemicals, Co., Tokyo) were used. Each of these agents (0.2 ml) was i.p. Fig. 1. Time-kinetic study on body temperature and blood glucose after restraint stress. Mice were exposed to restraint stress for 3 h and were then released. Body temperature and blood glucose were measured at the indicated points of time. Three mice were used to produce the mean and one S.D. * P < 0.05. Fig. 2. Serum levels of catecholamines during restraint stress. Serum levels of adrenaline, noradrenaline and dopamine were measured at 1 and 2 h from an initial time. Three experiments were done to produce the mean and one S.D. * P < 0.05. M. Watanabe et al. / Immunology Letters 115 (2008) 43–49 at 2000 rpm (424 × g) for 15 min. The pellet was resuspended in red blood cell (RBC) lysis solution (155 mM NH4 Cl, 10 mM KHCO3 , 1 mM EDTA, and 17 mM Tris, pH 7.3) and then washed twice with the medium. Splenocytes and thymocytes were obtained by forcing the spleen and thymus, respectively, through stainless steel mesh. Splenocytes were treated with 0.2% NaCl solution to remove RBC. 2.6. Immunoﬂuorescence tests The phenotype of lymphocytes was identified by two-color immunofluorescence tests . The reagents used for this included anti-CD3 (145-2C11), anti-IL-2R␤ (TM-␤1), antiNK1.1 (PK136), anti-CD4 (RM4-5), anti-CD8␣ (53-6.7), and anti-B220 (RA3-6B2) mAbs (BD PharMingen, San Diego, CA). All mAbs were used in fluorescein isothiocyanate (FITC)and phycocerythrin (PE) conjugated-forms. To prevent nonspecific binding of mAb, anti-CD16/CD32 (2.4 G2) mAb was added before staining with labeled mAbs. The suspended lymphocytes (5 × 105 − 2 × 106 /tube) were stained with mAbs 45 and stained lymphocytes were analyzed with a FACScan (Becton–Dickinson). Dead cells were excluded by forward scatter, side scatter and propidium iodide gating. 2.7. Measurement of plasma concentration of catechalamines Plasma pooled from four mice was used to measure the concentration of adrenaline, noradrenaline and dopamine. The plasma levels of these catecholamines were analyzed by the HPLC method . 2.8. Cytotoxicity assays Cytotoxicity assay was performed as previously described . YAC-1 cells (NK cytotoxicity) and syngeneic thymocytes (NKT cytotoxicity) were used as target cells for each cytotoxicity. A low magnitude of cytotoxicity against YAC-1 cells is also mediated by NKT cells, but it is minimum. These targets were labelled with sodium [51 Cr] chromate (NEN Life Science Fig. 3. Effect of the administration of catecholamines on body temperature and blood glucose. (A) Administration of adrenaline, noradrenaline and isoproterenol, (B) dose-dependent effects of adrenaline. Mice were i.p. administered with 25.0 g of adrenaline, noradrenaline or isoproterenol in Exp. A. Body temperature and blood glucose were measured at the indicated points of time. In Exp. B, dose-dependent effects of adrenaline was examined. Three mice were used to produce the mean and one S.D. * P < 0.05. 46 M. Watanabe et al. / Immunology Letters 115 (2008) 43–49 Products, Boston, MA, USA) for 2 h and washed three times with RPMI-1640 medium. Effector cells were serially diluted and mixed with [51 Cr]-labelled target cells (1 × 104 , or 2 × 104 cells) in a 96-well U-bottomed microculture plate. The plates were centrifuged and incubated for 4 h at 37 ◦ C. At the end of the culture, 100 l supernatant was counted in a gamma counter. 2.9. Statistical analysis The difference between the values was determined by Student’s t-test and one-factor ANOVA. 3. Results 3.1. Stress-induced responses Mice were exposed to restraint stress for 3 h and were then released. Body temperature and blood glucose were exam- ined at each point of time (Fig. 1). Primarily, control mice had a high body temperature of around 38.9 ± 0.5 ◦ C, whereas mice exposed to stress showed decreased levels of body temperature between 36.0 and 37.0 ◦ C (P < 0.05). After release from stress, such body temperature returned to the control level. In the case of blood glucose, control mice had a level of 150 mg/dl. However, mice exposed to stress showed elevated levels of blood glucose (220–240 mg/dl). These levels returned to the control level after the release from stress. In an acute response to stress , plasma levels of catecholamines are known to vary. Therefore, the plasma levels of catecholamines were examined before and during stress (Fig. 2). The plasma levels of adrenaline, noradrenaline and dopamine were all elevated after stress (1 or 2 h, P < 0.05). This raises the possibility that some catecholamines may be associated with the observed stress-induced responses. Fig. 4. Effect of ␣- or ␤-blockers on adrenaline-induced hypothermia and hyperglycemia. ␣- and ␤-blockers was subcutaneously administered at the same time when adrenaline was administered. Three mice were used to produce the mean and one S.D. * P < 0.05. M. Watanabe et al. / Immunology Letters 115 (2008) 43–49 47 3.2. α-Adrenergic stimulus We examined which types of catechalamines were able to induce both hypothermia and hyperglycemia seen in restraint stress (Fig. 3A). In preliminary experiments, the maximum doses of adrenaline (␣-stimulus), noradrenaline (␣ but less than adrenaline) and isoproterenol (␤) were determined to avoid the death or unhealthy conditions of mice. Adrenaline, but neither noradrenaline nor isoproterenol, was found to simultaneously induce hypothermia and hyperglycemia. In Fig. 3B, dosedependent curve of adrenaline (25.0, 12.5 and 6.25 g/mouse) was shown. Prominent effects of hypothermia and hyperglycemia were obtained at the dose of 25.0 g/mouse. 3.3. Use of α- and β-blockers To further confirm that ␣-adrenergic stimulus was responsible for the present phenomena, ␣-blocker (phentolamine) and ␤-blocker (propranolol) were used in the experiments of adrenaline-induced hypothermia and hyperglycemia (Fig. 4). Adrenaline (25.0 g/mouse) was administered intraperitoneally while ␣- or ␤-blocker (6.25 g or 12.5 g/mouse) was administered intraperitoneally 10 min before adrenaline injection. In a preliminary experiment, ␣-blocker alone or ␤-blocker alone did not induce any effects on body temperature and blood glucose at the applied doses (data not shown). It was found that ␣-blocker suppressed partially hypothermia, especially at late phase (2–3 h). More prominently, ␤-blocker enhanced hypothermia induced by adrenaline. It is conceivable that ␤-adrenergic stimulus had a potential to suppress hypothermia. In the case of blood glucose, ␣-blocker had a potential to suppress hyperglycemia but ␤-blocker did not such a prominent potential at the both doses. 3.4. Immunomodulation by α-adrenergic stimulus We previously reported that restraint stress in mice induced the suppression of acquired immunity but inversely augmented the innate immunity [5,6]. It was therefore investigated whether an ␣-stimulus was able to induce a phenomenon similar to that of restraint stress (Fig. 5). The number of lymphocytes in the liver and thymus decreased whereas that in the spleen remained unchanged. To identify the distribution of lymphocyte subsets in various organs, immunofluorescence tests were conducted (Fig. 6). Two-color staining for CD3 and IL-2R␤ (or NK1.1 or B220) was conduced in the liver and spleen. The most prominent change was in NK cells, extrathymic T cells and NKT cells in the liver. At 2–6 h, the proportions of IL-2R␤+ (or NK1.1+ ) NK cells (e.g., 8.4 → 12.1%, 4 h), IL-2R␤+ CD3int extrathymic T cells (e.g., 14.1 → 34.5%, 4 h) and NK1.1+ CD3int NKT cells (e.g., 10.0 → 28.8%, 4 h) were found to increase. A peak time of elevation was seen at 4 h. B220+ B cells and IL2R␤− CD3high T cells remained unchanged in proportion by stress. Two-color staining for CD4 and CD8 in the thymus showed that the ␣-stimulus slightly reduced the proportion of CD4+ 8+ double-positive cells (e.g., 90.4 → 80.2%, 2 h) (bottom of Fig. 5. Number of lymphocytes after the administration of adrenaline. Lymphocytes were isolated from the liver, spleen and thymus. Three mice were used to produce the mean and one S.D. * P < 0.05. Fig. 6). In other words, the ␣-stimulus applied here as stress was not as severe as restraint stress. Since the proportion of NK cells and NKT cells increased in the liver by the ␣-stimulus, it was examined whether there were any accompanying functional activities (Fig. 7). In this experiment, lymphocytes were isolated from both the liver and spleen. In the liver, but not the spleen, NK activity against YAC1 cells and NKT activity against syngeneic thymocytes were found to be augmented by the ␣-stimulus (P < 0.05). 4. Discussion In the present study, we demonstrated that an ␣-adrenergic stimulus induced hypothermia, hyperglycemia and innateimmunity activation in mice, a phenomenon resembling stress-induced responses. In the case of restraint stress, it has been reported that the secretion of catecholamines, steroid hormones and inflammatory cytokines simultaneously occurs [4–6]. Among these humoral factors, attention was focused on catecholamines in this study. Since restraint stress was confirmed to induce adrenaline, noradrenaline and dopamine, we then examined which types of adrenergic stimuli could induce similar stress-associated responses. We administrated adrenaline 48 M. Watanabe et al. / Immunology Letters 115 (2008) 43–49 Fig. 7. Cytotoxicity assays. NK cytotoxicity against YAC-1 cells and NKT cytotoxicity against syngeneic thymocytes were examined at the indicated effector to target (E/T) ratios. Three experiments were done to produce the mean and one S.D. * P < 0.05. Fig. 6. Two-color staining of lymphocytes in the liver and spleen after the administration of adrenaline. Lymphocytes were isolated at the indicated points of time and immunofluorescence tests were conducted to identify lymphocyte subsets. Two-color staining of lymphocytes for CD3 and IL-2R␤ (or NK1.1 or B220) and that of thymocytes for CD4 and CD8 were conducted. Representative results of three experiments are depicted. Numbers in the figure represent the percentages of fluorescence-positive cells in corresponding areas. (␣-stimulus), nonadrenaline (␣ but less than adrenaline) and isoproterenol (␤-). The administration of ␣-stimulant but not that of ␤-stimulant seemed to induce stress-associated responses, especially with regard to body temperature, blood glucose and immune function. The association of ␣-adrenergic effect with hypothermia and hyperglycemia was confirmed using ␣- and ␤-blockers. Adrenaline-induced hypothermia was partially suppressed by ␣blockers but that was enhanced by ␤-blocker. Some suppressive effects of ␤-stimulus seemed to be included in adrenergic stimuli. In the case of adrenaline-induced hyperglycemia, ␣-blocker, but not ␤-blocker, was effective to eliminate this effect. Many studies on stress to date have mainly dealt with immunosuppressive responses, including the immunosuppression of T and B lymphocytes in the periphery and in acute thymic atrophy [1–3]. In this case, steroid hormones play an important role in stress-associated immunosuppression. On the other hand, the ␣-adrenergic stimulus resulted in only mild immunosuppression, with a decreased number of lymphocytes in the liver and thymus. Moreover, the effect of the ␣-stimulus on the decrease in the number of double-positive CD4+ 8+ cells was limited (the bottom of Fig. 5). These results suggest that immunosuppressive responses may be the result of multiple effects of humoral factors. In contrast to the immunosuppression of T and B cells during stress, the innate immunity mediated by NK cells (NK1.1+ CD3− ), extrathymic T cells (IL-2R␤+ CD3int ) and NKT cells (NK1.1+ CD3int ) was augmented inversely. This response was also reproduced by the administration of adrenaline (␣stimulus). The functional activation by the ␣-stimulus was confirmed in NK and NKT cytotoxicities. In this study, NK cytotoxicity was determined using YAC-1 cells whereas NKT cytotoxicity was investigated using syngeneic thymocytes (i.e., autologous cytotoxicity) . Primarily, sympathetic nerve stimulation increases body metabolism and results in the elevation of body temperature, whereas parasympathetic nerve stimulation inversely affects body metabolism and results in the decrease of body temperature [21–23]. However, severe sympathetic nerve stimulation, such as restraint stress in the present study, induces hypothermia, possibly due to the constriction of vessels (i.e., circulation failure). Mild or severe sympathetic nerve stimulation consistently leads to hyperglycemia [24,25]. There have been many reports that stress has a potential to induce hypothermia and hyperglycemia [12–14]. An ␣-adrenergic stimulus by the administration of adrenaline induced a similar pattern of hypothermia and hyperglycemia. At that time, the M. Watanabe et al. / Immunology Letters 115 (2008) 43–49 activation of innate immunity was also induced simultaneously. Although ␤-adrenergic stimulation is also known to induce hypothermia and hyperglycemia , such stimulation seems to be milder than an ␣-stimulus as shown in this study. In addition to the simultaneous induction of hypothermia and hyperglycemia, the induction of innate-immunity activation might be of interest. The activation of innate immunity under conditions of immunosuppression of T and B cells was demonstrated by not only restraint stress [5,6], as well as by an ␣-adrenergic stimulus in this study. 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