Ken-Ichi Kanai

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.

Suppression of matrix metalloproteinase-9 production from neutrophils by a macrolide antibiotic, roxithromycin, in vitro

BACKGROUND Macrolide antibiotics such as erythromycin and roxithromycin (RXM) have an anti-inflammatory effect that may account for their clinical benefit in the treatment of chronic airway inflammatory diseases. However, the precise mechanism of this anti-inflammatory effect is not well understood. PURPOSE The influence of RXM on matrix metalloproteinase (MMP)-9 production from neutrophils in response to lipopolysaccharide (LPS) stimulation was examined in vitro. METHODS Neutrophils prepared from normal human peripheral blood (1 x 10(5) cells/ml) were treated with various concentrations of RXM for 1 h, and then stimulated with 1.0 microg/ml of LPS in the presence of the agent for 24 h. MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 levels in culture supernatants were examined by enzyme-linked immunosorbent assay. RESULTS Addition of RXM at more than 5.0 microg/ml into cell cultures caused significant suppression of MMP-9 production, which was increased by LPS stimulation. However, the ability of cells to produce TIMP-1 was not affected by RXM treatment, even when the cells were cultured in the presence of agent at 10.0 microg/mL We then examined the influence of RXM on transcriptional factor, nuclear factor-kappaB and activator protein (AP)-1 activation by LPS stimulation. RXM exerted suppressive action on NF-kappaB (P50 and P65) activation when the cells were cultured for 4 h at more than 5.0 microg/ml of the agent. RXM at more than 2.5 microg/ml also suppressed AP-1 (Fra 1 and Jun B) activation in 4-h cultured cells. We finally examined the influence of RXM on MMP-9 mRNA expression in neutrophils. Addition of RXM into cell cultures at more than 5.0 microg/ml caused significant inhibition of mRNA expression, which was enhanced by LPS stimulation for 12 h. CONCLUSION These results strongly suggest that RXM inhibits neutrophil transmigration into inflammatory sites and results in favorable modification of the clinical status of inflammatory diseases.

Effect of tranilast on matrix metalloproteinase production from neutrophils in-vitro.

Tranilast is an anti‐allergic agent that blocks the release of chemical mediators, such as histamine and leukotrienes from mast cells, and has been reported to suppress keloid and hypertrophic scar formation. Since matrix metalloproteinases (MMPs) play an essential role in tissue remodelling, this study was undertaken to determine whether tranilast suppresses MMP production from neutrophils after lipopolysaccharide (LPS) stimulation in‐vitro. Neutrophils from five healthy donors (1times105 cells/mL) were stimulated with 1.0 μg mL−1 LPS in the presence or absence of various concentrations of tranilast for 24 h. MMP‐7, MMP‐8, MMP‐9 and tissue inhibitor of metalloproteinase (TIMP)‐1 levels in the culture supernatants were assayed by ELISA. In addition, the influence of tranilast on MMP mRNA expression and transcriptional factor activation in cells cultured for 12 h and 4 h was also evaluated by reverse transcriptase—polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA), respectively. Tranilast inhibited MMP and TIMP‐1 production from neutrophils when cells were treated with the agent at more than 5.0times10−5 m. It also suppressed MMP mRNA expression and transcriptional factor activation induced in neutrophils by LPS stimulation. The results suggest that tranilast inhibits the formation of keloid scarring through the suppression of factors such as MMPs and TIMP, which are essential for tissue remodelling, from inflammatory cells.

Suppressive activity of macrolide antibiotics on nitric oxide production by lipopolysaccharide stimulation in mice

BACKGROUND: Low-dose and long-term administration of macrolide antibiotics into patients with chronic airway inflammatory diseases could favorably modify their clinical conditions. However, the therapeutic mode of action of macrolides is not well understood. Free oxygen radicals, including nitric oxide (NO), are well recognized as the important final effector molecules in the development and the maintenance of inflammatory diseases. PURPOSE: The influence of macrolide antibiotics on NO generation was examined in vivo. METHODS: Male ICR mice, 5 weeks of age, were orally administered with either roxithromycin, clarithromycin, azithromycin or josamycin once a day for 2-4 weeks. The mice were then injected intraperitoneally with 5.0 mg/kg lipopolysaccharide (LPS) and the plasma NO level was examined 6 h later. RESULTS: Although pre-treatment of mice with macrolide antibiotics for 2 weeks scarcely affected NO generation by LPS injection, the administration of macrolide antibiotics, except for josamycin, for 4 weeks significantly inhibited LPS-induced NO generation. The data in the present study also showed that pre-treatment of mice with macrolide antibiotics for 4 weeks significantly suppresses not only production of pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, but also inducible nitric oxide synthase mRNA expressions, which are enhanced by LPS injection. CONCLUSION: These results strongly suggest that suppressive activity of macrolide antibiotics on NO generation in response to LPS stimulation in vivo may, in part, account for the clinical efficacy of macrolides on chronic inflammatory diseases.

Suppressive activity of fexofenadine hydrochloride on metalloproteinase production from nasal fibroblasts in vitro

Background Allergic rhinitis (AR) is an inflammatory disease characterized by nasal wall remodelling with intense infiltration of eosinophils and mast cells/basophils. Matrix metalloproteinases (MMPs), MMP‐2 and MMP‐9, are the major proteolytic enzymes that induce airway remodelling. These enzymes are also important in the migration of inflammatory cells through basement membrane components.

Suppressive activity of tiotropium bromide on matrix metalloproteinase production from lung fibroblasts in vitro

Background Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling with an accumulation of inflammatory cells. There is also increasing evidence that metalloproteinases (MMPs) may contribute to the pathogenesis of COPD, but the influence of agents that used for the treatment of COPD is not well understood. Objective We evaluated whether tiotropium bromide hydrate (TBH), a M3 muscarinic receptor antagonist, could inhibit MMP production from lung fibroblasts (LFs) in response to tumor necrosis factor (TNF)-α stimulation. Methods LFs were established from normal lung tissues taken from patients with lung tumors. LFs (5 × 105 cells/ml) were stimulated with TNF-α in the presence of various concentrations of TBH. After 24 h, culture supernatants were obtained and assayed for the levels of MMPs and tissue inhibitor of metalloproteinases (TIMPs) by ELISA. The influence of TBH on mRNA expression of MMPs and TIMPs in 4 h-cultured cells was also examined by real-time RT-PCR. Furthermore, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) in LFs treated with TBH for 4 h was examined by ELISA. Results TBH at more than 15 pg/ml inhibited the production of MMP-2 from LFs after TNF-α stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected by TBH through the suppression of both mRNA expression and transcription factor, NF-κB, activation in LFs induced by TNF-α stimulation. Conclusion These results suggest that the attenuating effect of TBH on MMP-2 production from LFs induced by inflammatory stimulation may be additional beneficial therapeutic effects not directly relating to its bronchodilatory effects.

Suppression of Matrix Metalloproteinase Production in Nasal Fibroblasts by Tranilast, an Antiallergic Agent, In Vitro

Allergic rhinitis is an inflammatory disease characterized by nasal wall remodeling with intense infiltration of eosinophils and mast cells/basophils. Matrix metalloproteinases (MMPs), MMP-2 and MMP-9, are the major proteolytic enzymes that induce airway remodeling. These enzymes are also important in the migration of inflammatory cells through basement membrane components. We evaluated whether tranilast (TR) could inhibit MMP production from nasal fibroblasts in response to tumor necrosis factor-α (TNF-α) stimulation in vitro. Nasal fibroblasts (NF) were established from nasal polyp tissues taken from patients with allergic rhinitis. NF (2 × 105 cells/mL) were stimulated with TNF-α in the presence of various concentrations of TR. After 24 hours, the culture supernatants were obtained and assayed for MMP-2, MMP-9, TIMP-1, and TIMP-2 levels by ELISA. The influence of TR on mRNA expression of MMPs and TIMPs in cells cultured for 12 hours was also evaluated by RT-PCR. TR at more than 5 × 10−5 M inhibited the production of MMP-2 and MMP-9 from NF in response to TNF-α stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected. TR also inhibited MMP mRNA expression in NF after TNF-α stimulation. The present data suggest that the attenuating effect of TR on MMP-2 and MMP-9 production from NF induced by inflammatory stimulation may underlie the therapeutic mode of action of the agent in patients with allergic diseases, including allergic rhinitis.

Epinastine Hydrochloride Antagonism against Interleukin-4-Mediated T Cell Cytokine Imbalance in vitro

Background: Interleukin (IL)-4 is well accepted to be a cytokine that plays many roles in the regulation of immune responses. Although the primary pharmacological target of antihistamines has been regarded as the histamine H1 receptor, there is little information about the influence of antihistamines on IL-4-mediated immune responses. The present study was undertaken to examine whether H1 receptor antagonists could modulate IL-4-mediated immune responses in vitro. Methods: CD4+ T cells from normal human peripheral blood (1 × 106 cells/ml) were incubated with various concentrations of epinastine hydrochloride (EP) or chlorpheniramine (CH) for 30 min and then stimulated with 10.0 ng/ml IL-4. After 24 h, culture supernatants were collected and assayed for IL-5, IL-6, IL-13 and interferon-γ by ELISA. The influence of EP on transcription factor activation and mRNA expression for cytokines was also examined. Results: Addition of EP into cell cultures at more than 20.0 ng/ml significantly suppressed the production of IL-5, IL-6 and IL-13, which were increased by IL-4 stimulation. EP at more than 20.0 ng/ml also suppresses nuclear factor-ĸB activation, signal transducers and activators of transcription 6 phosphorylation and mRNA expression, which were upregulated by IL-4 stimulation. However, the ability of CD4+ T cells to produce interferon-γ was decreased by IL-4 stimulation, which was dramatically restored by treatment with EP at more than 15.0 ng/ml. On the other hand, CH, a first-generation H1 receptor antagonist, could not inhibit cytokine production from CD4+ T cells in response to IL-4 stimulation, even when 90.0 ng/ml of the agent was added to cell cultures. Conclusion: The present results strongly suggest that EP, a second-generation H1 receptor antagonist, interferes with IL-4-activated signaling in CD4+ T cells and results in favorable modification of the allergic disease state or conditions.

Induction of apoptosis in nasal polyp fibroblasts by glucocorticoids in vitro

Objective—To examine the possible mechanisms underlying the therapeutic mode of action of glucocorticoids (GCs) in nasal polyposis. Material and Methods—The effects of GCs on nasal polyps were firstly evaluated by examining the growth of fibroblasts derived from 10 nasal polyps in vitro. Subsequently, the ability of GCs to induce apoptotic cell death in fibroblasts was examined. Results—Addition of betamethasone 21-phosphate (BET) at a concentration of > 1 × 10−3 M to cell cultures inhibited cell growth in all cases examined. BET and dexamethasone 21-phosphate, but not testosterone or estradiol, caused apoptotic cell death in 2/10 nasal polyp fibroblasts, as assessed by agarose gel electrophoresis, when the cells were cultured with the agents for >96 h. The minimum concentration of agent needed to cause apoptosis was 1 × 10−3 M, which is half of the recommended therapeutic dose. Conclusion—The present findings suggest that topical application of GCs in nasal polyposis patients suppresses proliferation of fibroblasts in polyps and results in favorable modification of the clinical status of these patients.

Suppressive Activity of Fexofenadine Hydrochloride on Thymus- and Activation-Regulated Chemokine Production from Human Peripheral Blood Leukocytes in Response to Antigenic Stimulation in vitro

Background: Thymus- and activation-regulated chemokine (TARC) is accepted as being an important molecule in the development and maintenance of allergic diseases. However, there is little information about the influence of antiallergic agents on TARC production after allergen challenge. The aim of this study is to examine the influence of fexofenadine hydrochloride (FEX), an H1-receptor antagonist, on TARC production from human peripheral blood leukocytes (PBL) using an in vitro cell culture technique. Methods: PBL prepared from donors with pollinosis were cultured with either Japanese cedar pollen allergen, Cry j 1, or interleukin (IL)-4 in the presence of various doses of FEX for 6 days. Levels of TARC and the T cell cytokines IL-4 and interferon (IFN)-γ in culture supernatants were examined by ELISA. Results: FEX did not affect PBL proliferation induced by Cry j 1 stimulation, even when 500 ng/ml of the agent, twice the therapeutic blood levels, was added to cell cultures as assessed by measuring 3H-thymidine incorporation into DNA. On the other hand, FEX at 250 ng/ml (but not 125 ng/ml), similar to therapeutic blood levels, significantly inhibited the ability of PBL to produce IL-4 (but not IFN-γ), which was enhanced by Cry j 1 stimulation. FEX at concentrations of more than 250 ng/ml also exerted a suppressive effect on TARC production from PBL in response to Cry j 1 and IL-4 stimulation in vitro. Conclusion: This inhibitory action of FEX may be partially responsible for the attenuating effect of the agent on allergic diseases.