BACKGROUND Suppressive activity of macrolide antibiotics ... their clinical conditions. However,

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  • BACKGROUND: Low-dose and long-term administrationof macrolide antibiotics into patients with chronicairway inflammatory diseases could favorably modifytheir clinical conditions. However, the therapeuticmode of action of macrolides is not well understood.Free oxygen radicals, including nitric oxide (NO), arewell recognized as the important final effector mole-cules in the development and the maintenance ofinflammatory diseases.Purpose: The influence of macrolide antibiotics onNO generation was examined in vivo .Methods: Male ICR mice, 5 weeks of age, were orallyadministered with either roxithromycin, clarithro-mycin, azithromycin or josamycin once a day for 2/4weeks. The mice were then injected intraperitoneallywith 5.0 mg/kg lipopolysaccharide (LPS) and theplasma NO level was examined 6 h later.Results: Although pre-treatment of mice with macro-lide antibiotics for 2 weeks scarcely affected NOgeneration by LPS injection, the administration ofmacrolide antibiotics, except for josamycin, for 4weeks significantly inhibited LPS-induced NO genera-tion. The data in the present study also showed thatpre-treatment of mice with macrolide antibiotics for4 weeks significantly suppresses not only productionof pro-inflammatory cytokines interleukin-1b, inter-leukin-6, and tumor necrosis factor-a, but also in-ducible nitric oxide synthase mRNA expressions,which are enhanced by LPS injection.Conclusion: These results strongly suggest that sup-pressive activity of macrolide antibiotics on NOgeneration in response to LPS stimulation in vivomay, in part, account for the clinical efficacy ofmacrolides on chronic inflammatory diseases.

    Key words: Macrolide antibiotics, Nitric oxide, Mouse,Inducible nitric oxide synthase mRNA, Pro-inflammatorycytokine, Suppression, In vivo

    Mediators of Inflammation, 12(4), 195/202 (August 2003)

    Suppressive activity of macrolideantibiotics on nitric oxideproduction by lipopolysaccharidestimulation in mice

    Hajime Terao1, Kazuhito Asano2, Ken-ichi Kanai1,

    Yoshiyuki Kyo1, So Watanabe1, Tadashi Hisamitsu2

    and Harumi Suzaki1, CA

    1Department of Otolaryngology and 2Department ofPhysiology, School of Medicine, Showa University, 1-5-8 Hatanodai, Shinagawa-Ku, Tokyo 142-8666, Japan

    CACorresponding AuthorTel: /81 3 3784 8676Fax: /81 3 3784 3757E-mail: h-suzaki@mve.biglobe.ne.jp

    Introduction

    Low-dose and long-term administration of macrolide

    antibiotics, so-called macrolide therapy, is reported

    to be effective in the treatment and the management

    of chronic inflammatory diseases, such as diffuse

    panbronchiolitis and chronic sinusitis.1,2 It is also

    observed that administration of azithromycin (AZM),

    a newly developed 15-membered macrolide antibio-

    tic, could modify the clinical condition of cystic

    fibrosis.3,4 Although the precise mechanisms of the

    macrolide therapy are not well understood, there is

    circumstantial evidence that the therapeutic mode of

    macrolides is due in part to their anti-inflammatory,

    rather than antibacterial, effects.2,5,6

    It is reported that an accumulation of both

    neutrophils and macrophages in the airway is an

    important feature of the diseases, which is normal-

    ized after appropriate macrolide therapies.7 It is also

    recognized that, in response to antigenic stimulation,these inflammatory cells can secret not only inflam-matory cytokines, but also free oxygen radicals suchas O2

    and H2O2.8,9 Our previous work clearly shows

    the suppressive activity of macrolide antibiotics oninflammatory cytokine production from inflammatorycells, including T cells, mast cells and macrophagesin vitro and in vivo .5,8,10 However, there is littleinformation about the influence of macrolide anti-biotics on free oxygen radical generation frominflammatory cells.9

    Nitric oxide (NO) is recognized as one of theimportant regulators of many cell and tissue func-tions. It is also accepted NO is produced from varioustypes of cells and tissues (e.g. skeletal muscle,epithelium and fibroblasts) in response to inflamma-tory stimuli.11 Although NO is generally believed tobe a short-lived gaseous free radical, it reactsextremely rapidly with superoxide and producesthe very reactive and toxic peroxinitrite, which

    Research Communication

    ISSN 0962-9351 print/ISSN 1466-1861 online/03/40195-08 2003 Taylor & Francis LtdDOI: 10.1080/09629350310001599620

    195

  • subsequently decomposes into additional highlyreactive intermediates.11,12 Clinical evidence hasclearly shown that NO concentration in exhaled airis increased in patients with airway inflammations,including rhinitis, as compared with normal sub-jects.13,14 Recently, nitrogen derived from oxidantswas found to play a role in airway diseases as one ofthe final effector molecules,15 suggesting the possi-bility of an important role for peroxynitrite ininflammatory airway diseases. Recent in vitro studiesclearly showed that macrolide antibiotics, such aserythromycin and roxithromycin (RXM), significantlysuppress NO generation from both macrophages andnasal fibroblasts in response to inflammatory sti-muli.16,17 These reports may suggest that macrolideantibiotics exert an attenuating effect on chronicinflammatory diseases through suppression of NOgeneration. However, before drawing the conclusionthat this suppressive activity of macrolide antibioticsunderlies one of the therapeutic modes of action ofagents on the diseases, it is necessary to examinewhether macrolide antibiotics could also suppressNO generation in vivo . In the present study, there-fore, we examined the influence of macrolide anti-biotics on NO generation in vivo using anexperimental mouse model.

    Materials and methods

    Mouse

    Specific pathogen free ICR mice were purchasedfrom Charles River Japan Inc. (Atsugi, Japan). Afterarrival at our university, they were kept in filter (0.2mm)-barriered cages, and provided with autoclavedfood and tap water ad libitum throughout theexperiments to prevent un-wanted microbiologicalinfection. The rats were all male and 5 weeks of ageat the start of experiments. All animal experimentalprocedures were approved by the Animal Care andUse Committee of Showa University and were carriedout in accordance with the guidelines of the Physio-logical Society of Japan.

    Antibiotics

    RXM was kindly donated by Aventis PharmaceuticalCo. Ltd (Tokyo, Japan) as a water-insoluble, pre-servative-free pure powder. This agent was wellmixed with 5% tragacanthogum solution at 20.0mg/ml and diluted with normal saline to give aconcentration of 2.0 mg/ml. The mice were given 2.5mg/kg of RXM, one-half of the recommended humantherapeutic dose, once a day for 2/4 weeks via astomach tube. Clarithromycin (CAM) was also a giftfrom Abott Japan Co. Ltd. (Tokyo, Japan) as apreservative-free pure powder. CAM, dissolved in

    normal saline as in the case of RXM, was orallyadministered into mice at a dose of 7.0 mg/kg, one-half of the recommended human therapeutic dose,once a day for 4 weeks. Josamycin (JM) waspurchased from SIGMA Chemicals (St Louis, MO,USA) and dissolved in normal saline as was RXM. JMwas also administered orally into mice at a dose of20.0 mg/kg, the recommended human therapeuticdose, once a day for 4 weeks. AZM was extractedfrom ZITHROMAC dry syrup (Pfizer Co. Ltd., Tokyo,Japan) for pediatoric use, which contains 100.0 mg ofAZM, Yellow 5 and sodium lauryl sulfate. AZM wasdissolved by suspending dry syrup in 5.0 ml ofethanol, and then homogenized with a sonic dis-membrator for 5 min at 48C, and centrifuged at 5000rpm for 15 min at 48C. The supernatants werecollected and the ethanol was evaporated. Theresultant powder was dissolved in saline as RXMadministered orally into mice at a dose of 8 mg/kgonce a day for 4 weeks. In all cases, the volumeadministered did not exceed 0.2 ml, and control micereceived 5% tragacanthogum solution alone.

    Lipopolysaccharide treatment

    Mice were injected intraperitoneally with lipopoly-saccharide (LPS) derived from Escherichia coli(SIGMA Chemicals) in a volume of 0.5 ml.

    Preparation of plasma

    Blood (1.0 ml) was obtained from cardiac puncture inthe presence of 0.1 ml of heparin, and then centri-fuged at 3000 rpm for 10 min at 48C. Plasma wasobtained and stored at /408C until used.

    Assay for NO (NO2 /NO3

    )

    To remove proteins from plasma, samples werefiltered through centrifugal ultrafiltration devices at5000 x g for 30 min at 48C (Centricon YM-10; cut-offmolecular weight, 10,000; Millipore Corp., Bedford,MA, USA). The NO levels in samples were examinedby a NO assay kit that contained sufficient reagents(DojinDo, Co., Ltd, Kumamoto, Japan).

    Assay for cytokines

    Interleukin (IL)-1b, IL-6 and tumor necrosis factor-a(TNF-a) levels in plasma were examined usingcommercially available mouse cytokine enzyme-linked immunosorbent assay kits (R & D Corp.,Minneapolis, MN, USA). The sensitivity of these kitsfor IL-1b, IL-6, and TNF-a was 3.0 pg/ml, 7.0 pg/ml,and 2.0 pg/ml, respectively.

    H. Terao et al.

    196 Mediators of Inflammation Vol 12 2003

  • Assay for inducible nitric oxide synthase mRNAexpression

    The lungs were obtained from mice after decapita-tion, and homogenized with a glass homogenizer at48C for 60 sec. Poly A mRNA was separated fromhomogenates with oligo(dT)-coated magnetic mi-crobeads (Milteny Biotec, Bergische Gladbach, Ger-many). Inducible nitric oxide synthase (iNOS) mRNAwas examined with mouse iNOS mRNA enzyme-linked immunosorbent assay test kits (R & D Corp.)according to the recommended manufacturers pro-tocol.

    Statistical analysis

    Data were analyzed with analysis of variance fol-lowed by Fishers PLSD test. p B/0.05 was consideredsignificant.

    Results

    Influence of macrolide antibiotics on NOproduction by LP