Kazuhito Asano

Antiasthmatic Activity of a Macrolide Antibiotic, Roxithromycin: Analysis of Possible Mechanisms in vitro and in vivo

This study was designed to examine the possible mechanisms by which macrolide antibiotics favorably influence the clinical course of asthmatic patients. In the first set of experiments, we investigated the effect of roxithromycin (RXM), a newly synthesized macrolide antibiotic, on in vitro cytokine secretion by mitogen-activated human peripheral blood leukocytes. RXM suppressed the secretion of T cell cytokine interleukins (IL) 2-4 and monocyte cytokine tumor necrosis factor alpha. This inhibitory effects on cytokine secretion was dose dependent and firstly noted at a concentration of as little as 0.5 microgram/ml which is much lower than therapeutic blood levels. In the second part of experiments, we examined the influence of RXM on cytokine appearance in mouse lung extract induced by lipopolysaccharide (LPS) inhalation and on bronchial responsiveness to methacholine in LPS-treated mice. As compared with mice pretreated with phosphate-buffered saline, RXM administered orally at a single dose of 5 mg/kg once a day for 21 days inhibited the appearance of IL-3, IL-4, IL-5, and tumor necrosis factor alpha in aqueous lung extracts. Pretreatment with RXM also decreased the bronchial responsiveness to methacholine induced by intratracheal injection of LPS. We conclude that the attenuating effect of macrolide antibiotics on asthmatic syndromes might be explained partially by their inhibitory effects on cytokine secretion from leukocytes.

Cytokine Concentrations in Sputum of Asthmatic Patients

To examine whether levels of inflammatory cytokines and eosinophil cationic protein (ECP) in the sputum reflect the severity of bronchial asthma, we measured their levels in the sputum of symptomatic and asymptomatic asthmatics. Interleukin (IL)-1 beta, IL-5, IL-6, IL-8, RANTES, tumor necrosis factor-alpha and ECP concentrations in the sputum of symptomatic patients were significantly higher than in asymptomatic subjects. These findings suggest that these inflammatory cytokines are involved in the exacerbation of asthma.

Role of endogenous interferon-γ on the enhancement of splenic NK cell activity by electroacupuncture stimulation in mice

Successive electro-acupuncture (EA) stimulation applied to bilateral anterior tibial muscles, where Zusanli (ST36) acupoints are located, once a day (30 min) for 3 successive days significantly enhanced splenic natural killer (NK) cell activity in BALB/c mice. The percentage of splenic NK cells, as measured by flow cytometry, was not affected in these mice. Interferon (IFN)-gamma level in splenic aqueous extract, prepared from the ST36 acupoint-stimulated mice, was significantly higher than that of the controls. In vivo treatment with neutralizing monoclonal antibody against mouse IFN-gamma completely abrogated the increase in splenic NK cell activity induced by ST36 acupoint stimulation. The same stimulation also significantly increased the concentration of splenic beta-endorphin, which coincided with the significant increase in splenic IFN-gamma production. Pre-administration of 10 mg/kg naloxone before initiation of EA stimulation every day reduced the enhancements of NK cell activity and IFN-gamma level. These observations strongly suggest that endogenous IFN-gamma mediates the up-regulation of NK cell activity by EA stimulation at the ST36 acupoints. Furthermore, endogenous beta-endorphin secreted by EA stimulation also plays an important role in the up-regulation of NK cell function, which may be realized through regulating IFN-gamma production.

Suppression of matrix metalloproteinase production from nasal fibroblasts by macrolide antibiotics in vitro

It is well known that low-dose and long-term administration of macrolide antibiotics favourably modify the clinical status of chronic airway inflammatory diseases. However, the therapeutic mode of action of macrolide antibiotics is not well understood. The present study aimed to examine the influence of macrolide antibiotics, roxithromycin (RXM) and josamycin (JM) on matrix metalloproteinase (MMP) production from nasal polyp fibroblasts (NPF) in vitro. NPF, at a concentration of 2.5×105 cells·mL−1, were stimulated with tumour necrosis factor (TNF)-α in the presence of various concentrations of RXM or JM for 24 h. MMP‐2 and ‐9 levels in culture supernatants were analysed by ELISA, and MMP mRNA expression was examined by RT-PCR. The influence of RXM on nuclear factor (NF)-κB and activator protein (AP)‐1 activation was also examined. Addition of RXM (but not JM) at 5.0 and 7.5 µg·mL−1 significantly suppressed the production of MMP‐2 and ‐9 from NPF induced by TNF-α stimulation. RXM also suppressed MMP mRNA expression through the inhibition of NF-κB and AP-1 activation. The present results suggest that the suppressive activity of roxithromycin on MMP‐2 and ‐9 production is, in part, responsible for the therapeutic action of macrolides on chronic airway inflammatory diseases.

Suppressive activity of a macrolide antibiotic, roxithromycin, on pro-inflammatory cytokine production in vitro and in vivo

This study was designed to examine the influence of a macrolide antibiotic, roxithromycin (RXM), on the production of pro-inflammatory cytokines, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. In the first experiments, we examined the effect of RXM on in vitro cytokine production from lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes. The monocytes were cultured in the presence of various doses of the agent. After 24 h, the culture supernatants were obtained and assayed for IL-1beta and TNF-alpha contents by enzyme-linked immunosorbent assay. RXM suppressed the in vitro production of IL-1beta and TNF-alpha in response to LPS stimulation. This was dose dependent and first noted at a concentration of as little as 0.05 microg/ml, which is much lower than therapeutic blood levels. In the second part of the experiments, we examined the influence of RXM on the appearance of IL-1beta and TNF-alpha in mouse lung extract induced by LPS inhalation. RXM was administered orally into BALB/c mice at a single dose of 2.5 mg/kg once a day for 5-12 weeks. These mice were then instilled with LPS into the trachea and examined for the presence of cytokines in aqueous lung extracts. Pretreatment of mice with RXM for 5 weeks did not influence of the appearance of both IL-1beta and TNF-alpha in aqueous lung extracts. However, pretreatment for more than 7 weeks dramatically suppressed the cytokine appearance in the extracts.

Anti-HIV (human immunodeficiency virus) activity of sulfated paramylon

Sulfated derivatives of paramylon, a water-insoluble (1-3)-beta-D-glucan from Euglena gracilis, significantly inhibited the cytopathic effect of human immunodeficiency virus (HIV-1, HIV-2) and the expression of HIV antigen in cultured MT-4, MOLT-4 cells and human peripheral blood mononuclear cells. Native paramylon, N,N-dimethylaminoethyl paramylon, N,N-diethylaminoethyl paramylon, 2-hydroxy-3-trimethylammoniopropyl paramylon chloride, and carboxymethyl paramylon had little or no anti-HIV activity. The anti-HIV activity of the sulfated paramylon derivatives depended on the number of sulfate groups, and the molecular weight. Paramylon sulfate significantly inhibited HIV-1 binding to MT-4 cells. The anti-coagulant activity of the sulfated paramylon derivatives also depended on the number of sulfate groups, but was generally lower than that of dextran sulfate. The results point to the potential of paramylon sulfate in the treatment of HIV infection.

Suppressive effects of Tripterygium wilfordii Hook f., a traditional Chinese medicine, on collagen arthritis in mice.

The effect of chloroform extract of Tripterygium wilfordii Hook f. (TWH extract), a traditional immunosuppressive Chinese herb, on type II collagen (C II)-induced arthritis (CIA) in DBA/1J mice was studied. In the first set of experiments, we examined the effect of TWH extract on cellular immune responses to C II. As compared with mice treated with saline, TWH extract administered orally at doses of more than 400 microg kg(-1) once a day for 14 days inhibited the ability of inguinal lymph node cells to produce T cell cytokines interleukin-2 and interferon-gamma when the cells were obtained from mice 21 days after immunization and cultured in vitro with C II. Treatment with TWH extract also inhibited production of macrophage cytokines interleukin-1beta and tumor necrosis factor-alpha in response to in vitro stimulation of lymph node cells with C II. In the second part of the experiment, we evaluated the influence of TWH extract on the incidence and development of arthritis in murine CIA. Mice were immunized twice at a 3-week interval with bovine C II, with TWH extract being given orally once a day for 14 days with four different regimens. A 14-day course of TWH extract treatment at a daily dose of 400 microg kg(-1), which began on the day of the first C II immunization, suppressed the development of arthritis, as well as antibody production and delayed-type hypersensitivity to C II. Treatment with TWH extract, which started on the same day as the booster immunization, also resulted in inhibition of development of arthritis and of immune responses to C II. On the other hand, therapeutic administration with TWH extract did not affect the clinical course of the disease and the immune response to C II.

Anti-allergic activity of roxithromycin: Inhibition of interleukin-5 production from mouse T lymphocytes

This study was designed to evaluate the effects of roxithromycin (RXM), a newly synthesized macrolide antibiotic on allergic responses in mice. RXM was orally administered into BALB/c mice once a day for 42 days in a single dose of 5 mg/kg body weight. Spleen cells (Sp C) collected from mice on day 7, 14, 28 and 42 post-RXM administration showed higher blastic activity of lymphocytes than those from control. The activity peaked on the 7th day, then gradually decreased, and returned to the control level by the 42nd day. Production of cytokines, IL-2 and IL-5, by Sp C in response to concanavalin A stimulation was also examined in the course of RXM administration. The capacity of Sp C to produce IL-2 was enhanced by oral administration of RXM for 28 days. However, a long-term (for 42 days) administration inhibited it. On the other hand, the capacity of of Sp C to produce IL-5 was strongly inhibited by oral administration of RXM; the titer of IL-5 was similar to that obtained in cultures of Sp C from control mice. These results strongly suggest that oral administration of RXM inhibits the function of Th2-type helper T lymphocytes and that a long-term administration of RXM may be beneficial in asthma and allergy.

Influences of roxithromycin on cell-mediated immune responses

We investigated the effects of roxithromycin (RXM), a synthesized macrolide antibiotic on murine cellular immune responses by examining the in vitro proliferative response of lymphocytes, interleukin 1 (IL-1) production and interleukin 2 (IL-2) production. RXM was orally administered to BALB/c mice at a dose of 5 mg/kg once a day for 42 days. Spontaneous blastic activity of lymphocytes prepared from mice administered with RXM for 7 days was higher than those from control mice. The activity peaked at the 14th day, and then decreased gradually to control levels by the 42nd day. Time kinetics of lymphocyte blastogenesis to concanavalin A showed a pattern similar to that observed in spontaneous blastic activity. Oral administration of RXM also influenced cytokine production; short-term (for 14 days) administration of RXM enhanced both IL-1 and IL-2 production but long-term (for 42 days) administration inhibited them.

Tiotropium bromide inhibits TGF-β-induced MMP production from lung fibroblasts by interfering with Smad and MAPK pathways in vitro.

Background: Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and structural alterations (ie, tissue remodeling) throughout the conducting airways, parenchyma, and pulmonary vasculature. Matrix metalloproteinases (MMPs) are extracellular degrading enzymes that play a critical role in inflammatory cell infiltration and tissue remodeling, but the influence of the agents that are used for the treatment of COPD on the production of MMPs is not well understood. Purpose: The present study aimed to examine the influence of tiotropium bromide hydrate (TBH) on the production of MMPs from lung fibroblasts (LFs) induced by transforming growth factor (TGF)-β in vitro. Methods: LFs, at a concentration of 5 × 105 cells·mL−1, were stimulated with TGF-β in the presence of various concentrations of TBH. MMP-1 and MMP-2 levels in culture supernatants were examined by enzyme-linked immunosorbent assay (ELISA), and MMP messenger ribonucleic acid (mRNA) expression was examined by real-time polymerase chain reaction (RT-PCR). The influence of TBH on TGF-β signaling pathways was also analyzed by examining Smad activation and signaling protein phosphorylation by ELISA. Results: TBH at more than 15 pg·mL−1 inhibited the production of MMP-1 and MMP-2, but not tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2, from LFs, after TGF-β stimulation. TBH also suppressed MMP mRNA expression through the inhibition of Smad activation and signaling protein, extracellular-signal-regulated kinase (ERK) 1 and 2, and c-Jun N-terminal kinase (JNK), phosphorylation. Conclusion: These results may suggest that TBH suppresses MMP production from LFs, through interference of TGF-β-mediated signaling pathways and results in favorable modification of the clinical status of COPD.