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INFLAMMATION AND IMMUNOPHARMACOLOGY

Effect of 2-Adrenergic Receptor Stimulation on Interleukin-18-Induced Intercellular Adhesion Molecule-1 Expression and Cytokine Production

Departments of Pharmacology (H.K.T., S.M., M.N.), Tumour Biology (H.K.T., T.M., H.I., R.T., S.S., N.T.), and Pathology (T.Y., T.A.), Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

Received August 6, 2002; accepted October 30, 2002.

	Abstract

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-Adrenergic receptor (AR) agonists have been demonstrated to modulate the production of inflammatory mediators. Recent studies implied that 2-AR agonists might be useful for chronic inflammatory diseases caused by interleukin (IL)-18. In the present study, we found that norepinephrine, epinephrine, or isoproterenol down-regulated IL-18 (100 ng/ml)-induced intercellular adhesion molecule (ICAM)-1 expression on monocytes in a dose-dependent manner (10^–8–10^–4 M), but did not effect B7.1 and B7.2 expression after 24-h incubation. The modulatory effect of these catecholamines on ICAM-1 expression was antagonized by 2-AR antagonist, but not by 1-, 2-, or 1-AR antagonist. 2-AR-selective agonists salbutanol and terbutaline down-regulated IL-18-induced ICAM-1 expression on monocytes, but 1-, 2-, or 1-AR agonist had no effect. In the same manner, salbutanol and terbutaline as well as norepinephrine, epinephrine, and isoproterenol regulated the IL-18-induced cytokine production, including IL-12, tumor necrosis factor- or interferon- through the stimulation of 2-AR. Together with the previous finding that ICAM-1/lymphocyte function-associated antigen-1 interaction plays a crucial role in the IL-18-initiated cytokine network, the present study strongly suggested that the stimulation of 2-AR inhibited the IL-18-activated cytokine cascade through the inhibitory effect on ICAM-1 expression, contributing to finding a new method for clinical treatment.

The adrenergic neurotransmitter norepinephrine is released locally from sympathetic nerve terminals in synapse-like junctions with immune cells (Ader et al., 1995). The modulation of immune function by catecholamines is pleiotypic and affects a variety of cells of the immune system, including T cells, B cells, and NK cells (Ader et al., 1995). In response to stress, norepinephrine and the related sympathetic catecholamine epinephrine are released into the blood-stream, where they alter several aspects of lymphocyte function in vitro, including inhibition of proliferation and differentiation (Bergquist et al., 1994), apoptosis (Josefsson et al., 1996), and IFN- production in Th1 cells (Sanders et al., 1997). -Adrenergic receptors (ARs) are now subdivided into three subtypes: 1, 2, and 3. Norepinephrine binds to 2-AR on the lymphocyte plasma membrane and, via the GS protein, mediates the cAMP-protein kinase A signaling cascade (Kobilka, 1992). NK cells and monocytes are very responsive to 2-AR stimulation, with regard to cAMP accumulation; however, Th and B cells showed only a modest response (Knudsen et al., 1995). These results suggested that 2-AR stimulation might play a modulatory role in immune response. In addition to well known clinical use for the treatment of asthma or heart failure, recent experiments indicated that the application of 2-AR agonists seemed to be useful for chronic inflammatory diseases (Panina-Bordignon et al., 1997), including multiple sclerosis (MS) (Makhlouf et al., 2001), rheumatoid arthritis (RA) (Malfait et al., 1999), or hepatitis (Tiegs et al., 1999), which concerned IL-18 (Saha et al., 1999; Karni et al., 2002; Yumoto et al., 2002). However, little is known about the mechanism of the effect of 2-AR agonists on these diseases.

We have reported that the interaction between monocytes and T/NK cells might play important roles in IL-18-initiated immune response (Takahashi et al., 2002). The costimulatory signals through LFA-1/ICAM-1 and CD28/B7 on NK/T cell and antigen-presenting cell surface (Greenfield et al., 1998; Kato et al., 2001) are important participants in the activation of T cells, lowering the concentration of antigen required for stimulation and promoting more sustained signaling from T-cell receptor-major histocompatibility complex recognition. The purpose of the present study was to analyze whether 2-AR stimulation induced alterations in IL-18-initiated immune response, by investigating surface-marker expression and cytokine production in human peripheral blood mononuclear cells (PBMCs).

	Materials and Methods

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Reagents and Drugs. Recombinant human IL-18 was purchased from MBL (Nagoya, Japan). Epinephrine, norepinephrine, isoproterenol, methoxamine, dobutamine, salbutamol, prazosin, terbutaline, prazosin, yohimbine, atenolol, and butoxamine were purchased from Sigma-Aldrich (St. Louis, MO). Clonidine was purchased from Boehringer Ingelheim USA (Ridgefield, CT). For flow cytometric analysis, fluorescein isothiocyanate (FITC)-conjugated mouse IgG1 mAb against ICAM-1/CD54 (6.5B5) and phycoerythrin-conjugated anti-CD3, anti-CD14, or anti-CD19 mAb were purchased from DAKO (Glostrup, Denmark). FITC-conjugated mouse IgG1 mAb against B7.1/CD80 (MAB104) was purchased from Immunotech (Marseille, France). FITC-conjugated mouse IgG1 mAb against B7.2/CD86 (2331FUN-1) was purchased from BD PharMingen (San Diego, CA). FITC-conjugated MOPC 21, an IgG1 class-matched control (CMC), was purchased from Sigma-Aldrich.

Isolation and Culture of PBMCs. Normal human PBMCs were obtained from human volunteers with their oral informed consent. We used five donors for each experiment and determined the expression of adhesion molecules and cytokine production on triplicate samples from each donor. Therefore, we analyzed totally 15 samples at least for each experiment. Samples of 20 to 50 ml of peripheral blood were withdrawn from a forearm vein. PBMCs were isolated from the buffy coat of 10 healthy volunteers by centrifugation on Ficoll-Paque (Pharmacia AB, Uppsala, Sweden) and then washed three times in RPMI 1640 medium (Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 20 mg/ml kanamycin, and 100 mg/ml streptomycin and penicillin (Sigma-Aldrich). PBMCs were suspended at a final concentration of 1 x 10⁶ cells/ml in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum. Endotoxin concentration of IL-18 solution was less than 0.02 EU/ml, which were measured by the endospecy kit (Seikagaku Kogyo, Tokyo, Japan).

Flow Cytometric Analysis. PBMCs (1 x 10⁶ cells/ml) (described under Isolation and Culture of PBMCs) were incubated with IL-18; norepinephrine; epinephrine; isoproterenol; selective 1-, 2-, 1-, and 2-AR agonists; and/or selective 1-, 2-, 1-, and 2-AR antagonists for 24 h at 37°C, 5% CO₂/air mixture under different conditions. All reagents were added to the media at the start of incubation. The cells (5 x 10⁵ cells/sample) were washed once with washing buffer (phosphate-buffered saline supplemented with 2.5% normal horse serum, 0.1% NaN₃, and 0.01 M HEPES, pH 7.3). Then, the cells were incubated with 1 µg of FITC-conjugated anti-CD54 Ab, anti-CD80 Ab, anti-CD86 Ab, or CMC, or phycoerythrin-conjugated anti-CD3 Ab, anti-CD14 Ab or anti-CD19 Ab for 20 min at 4°C. After washing, the cells were fixed with 2% paraformaldehyde and analyzed with a fluorescence-activated cell sorting Calibur (BD Biosciences, San Jose, CA), and data were processed using the CELL QUEST program (BD Biosciences). The data are expressed as the relative fluorescence intensities against CMC. The results are the means ± S.E.M. of five donors.

Cytokine Assays. PBMCs (1 x 10⁶ cells/ml) were incubated in the same conditions described in "Flow Cytometric Analysis." After culture, the cell suspensions were transferred into Eppendorf tubes and centrifuged. The cell-free supernatant fractions were assayed for IL-12 (p70), TNF-, IFN-, and IL-10 protein. The cytokines were measured using enzyme-linked immunosorbent assay (ELISA) using the multiple Abs sandwich principle (Quantikine; R & D Systems, Minneapolis, MN). The detection limits of the ELISAs for IL-12, TNF-, IFN-, and IL-10 were 10 pg/ml. The results are expressed as the mean ± S.E.M. of five donors.

Statistical Examination. The averages of the mean fluorescence intensity in fluorescence-activated cell sorting analysis of each experiment were tested for statistical significance using analysis of variance comparison of means. A probability value less than 0.05 was considered to be statistically significant.

	Results

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Effects of Adrenergic Receptor Agonists on IL-18-Induced ICAM-1, B7.1, and B7.2 Expression on Human Monocytes. The effects of IL-18 (100 ng/ml) and/or AR agonists (epinephrine, norepinephrine, isoproterenol, salbutamol, and terbutaline) (10^–4 M) on the changes in expression of human leukocyte antigens (ICAM-1/CD54, B7.1/CD80, and B7.2/CD86) were examined by double-stained flow cytometry using a combination of anti-CD14 (monocyte), anti-CD3 (T cell), or anti-CD19 Ab (B cell) 24 h after the start of incubation of PBMCs. IL-18 induced the expression of ICAM-1 (Fig. 1) and B7.2 (Fig. 2) on monocytes, but did not affect B7.1 expression on monocytes (Fig. 2). Although the AR agonists alone had no effect on ICAM-1 expression on monocytes, the AR agonists inhibited IL-18-induced ICAM-1 expression on monocytes (Fig. 1). These AR agonists had no effect on B7.1 and B7.2 expression on monocytes, irrespective of the presence or the absence of IL-18 (Fig. 2). The percentage of CD14- and ICAM-1-positive cell number (Fig. 1) and the cell viability (data not shown) before and after treatment with IL-18 and/or these AR agonists did not change. IL-18 and the AR agonists used had no effect on ICAM-1, B7.1, and B7.2 expression on T cells and B cells (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 1. Effects of adrenergic receptor agonists on IL-18-induced ICAM-1 expression on the surface of PBMCs. PBMCs (1 x 106 cells/ml) were cultured in medium containing IL-18 (100 ng/ml) and adrenergic receptor agonists (10–4 M) (epinephrine, norepinephrine, isoproterenol, salbutamol, and terbutaline) for 24 h. At the end of the culture, PBMCs (5 x 105 cells/ml) were subjected to double-stained-labeling flow cytometry for cell type-specific antigens, as described under Materials and Methods. The experiments were repeated 10 times using different donors. Typical results are shown.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Dose-response relationships of the effects of epinephrine, norepinephrine, and isoproterenol on IL-18-induced ICAM-1 and B7 expression on human monocytes. In the presence or absence of IL-18 (100 ng/ml), PBMCs (1 x 106 cells/ml) were incubated with increasing concentrations (0–10–4 M) of epinephrine, norepinephrine, and isoproterenol for 24 h. After incubation, PBMCs (5 x 105 cells/ml) were stained with antibodies (ICAM-1, B7.1, and B7.2) or CMC. Filled columns represent the fluorescence intensity obtained with anti-ICAM-1 Ab, anti-B7.1 Ab, or anti-B7.2 Ab after incubation in the presence or absence of epinephrine, norepinephrine, and isoproterenol, respectively. Open columns indicate the fluorescence intensity obtained with CMC after incubation in the presence of epinephrine, norepinephrine, and isoproterenol. The results are the means ± S.E.M. of five donors. #, P < 0.01; ##, P < 0.01 compared with the value in medium. *, P < 0.05; **, P < 0.01 compared with the value in the presence of IL-18 alone.

Dose-Response Relationship for the Effects of Epinephrine, Norepinephrine, and Isoproterenol on IL-18-Induced ICAM-1 and B7 Expression on Human Monocytes. We investigated the effects of different concentrations (0–10^–4 M) of epinephrine, norepinephrine, and isoproterenol on IL-18 (100 ng/ml)-induced ICAM-1, B7.1, and B7.2 expression 24 h after the start of incubation of PBMCs (Fig. 2). Epinephrine, norepinephrine, and isoproterenol (10^–8–10^–4 M) had no effect on ICAM-1, B7.1, or B7.2 expression on monocytes without IL-18 treatment. However, the same concentration range of epinephrine, norepinephrine, and isoproterenol inhibited the expression of ICAM-1 induced by IL-18 (100 ng/ml) in a concentration-dependent manner (Fig. 2). The IC₅₀ values for the inhibitory effects of epinephrine, norepinephrine, and isoproterenol on the expression of ICAM-1 induced by IL-18 were estimated to be 700, 2000, and 70 nM, respectively. All these adrenergic receptor agonists had no effect on B7.1 and B7.2 expression, even in the presence of IL-18.

Effects of Adrenergic Receptor Antagonists on ICAM-1 Expression Inhibited by Epinephrine, Norepinephrine, and Isoproterenol. We investigated the blocking effects of four subtypes of AR antagonists such as prazosin (1-AR antagonist), yohimbine (2-AR antagonist), atenolol (1-AR antagonist), and butoxamine (2-AR antagonist) on the expression of ICAM-1 induced by IL-18 (100 ng/ml) in the presence epinephrine, norepinephrine, and isoproterenol (10^–5 M). Butoxamine antagonized the inhibitory effects of epinephrine, norepinephrine, and isoproterenol on ICAM-1 expression on monocytes in a concentration-dependent manner (Fig. 3). On the other hand, the same concentrations of prazosin, yohimbine, and atenolol did not produce any antagonizing action on the effects of epinephrine, norepinephrine, and isoproterenol.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Effects of adrenergic receptor antagonists on ICAM-1 expression inhibited by epinephrine, norepinephrine, and isoproterenol. In the presence of IL-18 (100 ng/ml), PBMCs (1 x 106 cells/ml) were incubated with epinephrine, norepinephrine, and isoproterenol (10–5 M) in the presence of AR antagonists (0–10–4 M) prazosin (1), yohimbine (2), atenolol (1), and butoxamine (2). After 24-h incubation, PBMCs (5 x 105 cells/ml) were stained with anti-ICAM-1 Ab or CMC. Filled columns represent the fluorescence intensity obtained with anti-ICAM-1 Ab after incubation in the presence or absence of epinephrine, norepinephrine, and isoproterenol. Open columns indicate the fluorescence intensity obtained with CMC after incubation in the presence of epinephrine, norepinephrine, and isoproterenol. The results are the means ± S.E.M. of five donors. *, P < 0.05; **, P < 0.01 compared with the value in the presence of epinephrine, norepinephrine, or isoproterenol alone.

Effects of ₂-Selective Adrenergic Receptor Agonists on IL-18-Induced ICAM-1 Expression. The effects of the four subtypes of selective AR agonists, including methoxamine (1-AR agonist), clonidine (2-AR agonist), dobutamine (1-AR agonist), and salbutamol and terbutaline (2-AR agonists) on ICAM-1 expression induced by IL-18 (100 ng/ml) were examined. Salbutamol and terbutaline mimicked the modulatory effects of isoproterenol on ICAM-1 responses (Fig. 4). The potency and efficacy of the effects of two agonists were similar to those of isoproterenol. On the contrary, methoxamine, clonidine, and dobutamine had no effect on IL-18-induced ICAM-1 expression (data not shown). Salbutamol and terbutaline did not show any influence on B7.1 and B7.2 expression. Therefore, the experiments using receptor subtype-specific antagonists and agonists strongly supported the involvement of 2-AR in the action of AR agonist on ICAM-1 expression. The IC₅₀ values for the inhibitory effect of salbutamol and terbutaline on the expression of ICAM-1 induced by IL-18 were estimated to be 20 and 10 nM, respectively. At 10^–5 and 10^–4 M, salbutamol and terbutaline, respectively, blocked the expression of ICAM-1 completely.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Effects of selective adrenergic receptor agonists on IL-18-induced ICAM-1 and B7 expression on human monocytes. PBMCs (1 x 106 cells/ml) were incubated with IL-18 (100 ng/ml) in the presence of 2-AR agonists (salbutamol and terbutaline) (0–10–4 M) for 24 h. After the treatment, PBMCs (5 x 105 cells/sample) were stained with anti-ICAM-1 Ab, anti-B7.1 Ab, anti-B7.2 Ab, or CMC. Filled columns represent the fluorescence intensity obtained with anti-ICAM-1 Ab, anti-B7.1 Ab, or anti-B7.2 Ab after incubation in the presence or absence of 2-AR agonists. Open columns indicate the fluorescence intensity obtained with CMC after incubation in the presence of 2-AR agonists. The results are the means ± S.E.M. of five donors. ##, P < 0.01 compared with the value in medium. **, P < 0.01 compared with the value in the presence of IL-18 alone.

Dose-Response Relationship for the Effect of Epinephrine, Norepinephrine, and Isoproterenol on IL-18-Induced Cytokine Production in PBMCs. In the following experiments, we investigated the effect of epinephrine, norepinephrine, and isoproterenol (0–10^–4 M) on IL-18 (100 ng/ml)-induced cytokine (IL-12, TNF-, IFN-, and IL-10) production in PBMCs (Fig. 5). In the presence of IL-18, epinephrine, norepinephrine, and isoproterenol inhibited the production of IL-12, TNF-, and IFN- in a concentration-dependent manner, whereas these agonists increased the production of IL-10. The IC₅₀ values for the inhibitory effect of epinephrine, norepinephrine, and isoproterenol on IL-18-induced IL-12 production were estimated to be 500, 500, and 50 nM, respectively, when the inhibitory effects were assumed to be maximal at 10^–4 M.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Dose-response relationship for the effect of epinephrine, norepinephrine, and isoproterenol on IL-18-induced cytokine production in PBMCs. PBMCs (1 x 106 cells/ml) were incubated with increasing concentrations (0–10–4 M) of epinephrine, norepinephrine, and isoproterenol in the presence of IL-18 (100 ng/ml) for 24 h and the levels of IL-12, TNF-, IFN-, and IL-10 in the conditioned media were determined by ELISA, as described under Materials and Methods. The results are the means ± S.E.M. of five donors. *, P < 0.05; **, P < 0.01 compared with the value of IL-18 alone.

Effects of Adrenergic Receptor Antagonists on Modulatory Effects of Epinephrine, Norepinephrine, and Isoproterenol on IL-18-Induced Cytokine Production. AR antagonists prazosin (1), yohimbine (2), atenolol (1), and butoxamine (2) (0–10^–4 M) were added to the culture medium in the presence of epinephrine, norepinephrine, or isoproterenol (10^–5 M each). 2-AR antagonist butoxamine antagonized the inhibitory effects of epinephrine, norepinephrine, and isoproterenol on IL-18 (100 ng/ml)-induced cytokine (IL-12, TNF-, IFN-, and IL-10) production in a concentration-dependent manner, whereas prazosin, yohimbine, and atenolol had no effect on cytokine responses modulated by AR agonists (Fig. 6).

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effects of adrenergic receptor antagonists on IL-18-induced cytokine production inhibited by epinephrine, norepinephrine, and isoproterenol. PBMCs (1 x 106 cells/ml) were incubated with IL-18 (100 ng/ml) and epinephrine, norepinephrine, and isoproterenol (10–5 M) in the presence of AR antagonists (0–10–4 M). At the end of the culture, the concentrations of IL-12, TNF-, IFN-, and IL-10 in the conditioned media were determined using an ELISA assay kit, as described under Materials and Methods. The results are the means ± S.E.M. of five donors. *, P < 0.05; **, P < 0.01 compared with the value in the presence of epinephrine, norepinephrine, or isoproterenol alone. Where error bars are not shown, they were smaller than the symbol.

Effects of Selective Adrenergic Receptor Agonists on IL-18-Induced Cytokine Production. The effects of AR agonists methoxamine (1), clonidine (2), dobutamine (1), and salbutamol and terbutaline (2) (0–10^–4 M) were determined. 2-AR agonists salbutamol and terbutaline mimicked the modulatory effects of isoproterenol on IL-18 (100 ng/ml)-induced cytokine production (Fig. 7). The potency and efficacy of 2-AR agonists were similar to those of isoproterenol in the four cytokine responses. On the contrary, methoxamine, clonidine, and dobutamine had no effect on IL-18-induced cytokine production (data not shown). Thus, the experiments using receptor subtype-specific antagonists and agonists strongly suggested that the stimulation of 2-AR inhibited IL-18-induced cytokine production. The IC₅₀ values for the inhibitory effect of salbutamol or terbutaline on IL-18-induced IL-12 production were estimated both be 50 nM.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Effects of selective adrenergic receptor agonists on IL-18-induced cytokine production in PBMCs. PBMCs (1 x 106 cells/ml) were incubated with IL-18 (100 ng/ml) in the presence of 2-AR agonists (salbutamol and terbutaline) (0–10–4 M). At the end of the culture, the concentrations of IL-12, TNF-, IFN-, and IL-10 in the conditioned media were determined using an ELISA assay kit, as described under Materials and Methods. The results are the means ± S.E.M. of five donors. *, P < 0.05; **, P < 0.01 compared with the value in the presence of IL-18 alone.

	Discussion

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All lymphoid organs contain the termini of neurons from the sympathetic division of the autonomic nervous system (Felten et al., 1987). The stimulations of these neurons induce micromolar quantities of norepinephrine into sites that are rich in T lymphocytes and macrophages (Felten et al., 1987). We have reported that anti-ICAM-1 Ab, anti-LFA-1 Ab, and anti-B7.2 Ab prevented IL-18-induced production of IL-12, IFN-, and TNF- in PBMCs, suggesting that the regulation of IL-18-induced cytokine production might depend on the cell-cell interaction through ICAM-1/LFA-1 and B7.2/CD28 on monocytes and T/NK cells (Takahashi et al., 2002). In the present study, we found that the endogenous catecholamines epinephrine and norepinephrine down-regulated IL-18-induced production of IL-12, TNF-, and IFN- as well as ICAM-1 expression on the cell surface of monocytes, but did not affect B7.2 expression (Figs. 2 and 5). The down-regulation of ICAM-1 should be strong enough to impair T-cell stimulation induced by monocyte-T-cell interactions during antigen presentation. These results implied that the immune response by catecholamines epinephrine and norepinephrine might effect through the expression of ICAM-1.

To investigate the receptor subtypes involved in the action of epinephrine and norepinephrine, we used subtype-selective AR antagonists and agonists. The effects of epinephrine, norepinephrine, and isoproterenol on ICAM-1 expression and cytokine production induced by IL-18 were blocked by 2-AR antagonist butoxamine but not by 1-(prazosin), 2-(yohimbine), and 1 (atenolol)-AR antagonist (Figs. 3 and 6). Selective 2-AR agonists salbutamol and terbutaline were potent inhibitors of IL-18-induced ICAM-1 expression and cytokine production in human PBMCs (Figs. 4 and 7); however, 1-(methoxamine), 2-(clonidine), or 1 (dobutamine)-AR-selective agonist had no effect (data not shown). Therefore, these findings indicated that sole 2-ARs were involved in epinephrine-, norepinephrine-, and isoproterenol-initiated down-regulation of IL-18-induced cytokine production as well as ICAM-1 expression elicited by IL-18. Another -AR subtype, 3-AR, has 49 and 51% overall homology at the amino acid level with 2- and 1-AR in humans, respectively (Emorine et al., 1989; Granneman and Lahners, 1994). The affinity of norepinephrine and epinephrine for human 3-AR expressed on Chinese hamster ovary cells was reported to be 20- and 5-fold higher than that of isoproterenol, respectively (Isogawa et al., 2002). On the other hand, it is well known that isoproterenol is more potent than norepinephrine and epinephrine at inducing 2-AR-mediated effects. The relative potency of the effects of isoproterenol and endogenous catecholamines on cytokine production and ICAM-1 expression excluded the possibility of a major role of 3-AR in these responses. Recent studies reported that 2-AR stimulation increased B7.2 expression in mouse B-cell and mouse B-lymphoma cell line (Kasprowicz et al., 2000; Kohm et al., 2002). In the present study, 2-AR stimulation did not effect on the expression of B7 on both unstimulated and IL-18-stimulated B cell (data not shown). This discrepancy may be ascribed to the species difference between human and mouse B cell. Further studies are necessary to clarify this point.

Recent studies have reported that IL-18 plays a crucial role in various pathological conditions, including MS (Karni et al., 2002), RA (Karni et al., 2002), acute hepatitis/fulminant hepatic failure (Yumoto et al., 2002) and graft-versus-host disease (Reddy et al., 2001). Plasma levels of IL-12 were higher in MS patients than in healthy controls and the enhanced activation of cell-mediated immunity by IL-12 seems to constitute one of the pathophysiological features of MS (Makhlouf et al., 2001). In fact, anti-IL-12 Ab was effective on an animal model of human MS (Ichikawa et al., 2000). In a prospective open-label study, the effect of a 2-week-long oral salbutamol treatment significantly decreased IL-12 production in monocytes and dendritic cells derived from MS patients that lasted up to 1 week after treatment interruption (Makhlouf et al., 2001). Thus, salbutamol has immunomodulatory properties both in vivo and in vitro and may be beneficial in the treatment of MS. The therapeutic potential of salbutamol was also explored in collagen-induced RA mice (Malfait et al., 1999). In peritoneal macrophages and synovial cells from the RA mice, salbutamol reduced TNF- and/or IL-12 release in a dose-dependent manner (Malfait et al., 1998). Because the collagen-induced RA mice produced increased levels of IL-18 followed by IL-12 production (Leung et al., 2000), it is likely that IL-12 production in this model was dependent on the interaction of ICAM-1/LFA-1 between monocytes/macrophages and T/NK cells, as observed in our previous in vitro study (Takahashi et al., 2002). IL-12 knockout mice or mice that have been treated with neutralizing anti-IL-12 Abs before onset of disease develop little or no RA (Malfait et al., 1998). Therefore, salbutamol could improve the IL-18-initiated cytokine network activation in collagen-induced RA. In the mouse models of TNF--dependent liver injury induced by either concanavalin A or a combination of D-galactosamine and staphylococcal enterotoxin B, salbutamol prevented liver injury with a significant reduction in TNF- production (Tiegs et al., 1999). Thus, strategies stimulating 2-AR might be of benefit in immune-mediated liver disease. The stimulation of 2-AR leads to significantly enhanced cardiac function by elevating the basal rate of cardiac contraction in transplanted hearts (Edelberg et al., 1998). It has been reported that specific tolerance to murine cardiac allografts can be induced by a short-term administration of mAbs to ICAM-1 and LFA-1 (Isobe et al., 1992). The inhibitory effect of 2-AR agonists on IL-18-initiated ICAM-1 expression suggested that 2-AR agonists could regulate immune response in allo-rejection through the regulation of expression of these adhesion molecules. Together with these results, 2-adrenergic compounds have important immunological effects that should be taken into consideration in the treatment of other IL-18-initiated diseases. Recent studies have reported that IL-18 and 2-agonists enhance IgE and Th2 cytokine production and that these mediators exacerbate extrinsic, IgE-dependent asthma (Fedyk et al., 1996). The present results suggested that 2-AR stimulation shifts the Th1/Th2 balance to Th2 dominance resulting from the regulation of ICAM-1 expression. Hence, 2-AR agonists, despite the symptomatic rescue in asthma due to dramatic direct action on bronchial smooth muscle cells, should not be used indiscriminately as long-term therapeutic agents.

In conclusion, the present study suggested that the activity of sympatho-adrenomedullary system could influence the immune system, resulting in the regulation of cell-to-cell interaction through the control of adhesion molecule ICAM-1. The fact that 2-AR activation causes inhibition of ICAM-1 expression can explain the many kinds of effects of 2-AR agonists thus far reported on immune cells.

	Acknowledgements

 
We thank Miyuki Shiotani and Yuki Onoda for excellent technical assistance.

	Footnotes

 
This study was supported in part by a grant from Japan Society for Promotion of Science (BSAR-521/0003815; to M.N.), grants for promotion of research from Okayama University (21, to M.N.; 26, to T.A.), and a grant from the Okayama Medical Foundation (to H.K.T.).

DOI: 10.1124/jpet.102.042622.

ABBREVIATIONS: IL, interleukin; Th, T helper; NK, natural killer; IFN, interferon; ICAM, intercellular adhesion molecule; AR, adrenergic receptor; MS, multiple sclerosis; RA, rheumatoid arthritis; LFA, lymphocyte function-associated antigen; PBMC, peripheral blood mononuclear cell; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; CMC, class-matched control; Ab, antibody; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay.

Address correspondence to: Dr. Masahiro Nishibori, Department of Pharmacology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: mbori{at}md.okayamau.ac.jp

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