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. 2010 Nov 23:7:56.
doi: 10.1186/1476-9255-7-56.

Suppression of nitric oxide production from nasal fibroblasts by metabolized clarithromycin in vitro

Ayako Furuya  1 , Kazuhito Asano, Naruo Shoji, Kojiro Hirano, Taisuke Hamasaki, Harumi Suzaki
Affiliations

Suppression of nitric oxide production from nasal fibroblasts by metabolized clarithromycin in vitro

Ayako Furuya et al. J Inflamm (Lond). .

Abstract

Background: Low-dose and long-term administration of 14-membered macrolide antibiotics, so called macrolide therapy, has been reported to favorably modify the clinical conditions of chronic airway diseases. Since there is growing evidence that macrolide antibiotic-resistant bacteria's spreaders in the populations received macrolide therapy, it is strongly desired to develop macrolide antibiotics, which showed only anti-inflammatory action. The present study was designed to examine the influence of clarithromycin (CAM) and its metabolized materials, M-1, M-4 and M-5, on free radical generation from nasal polyp fibroblasts (NPFs) through the choice of nitric oxide (NO), which is one of important effector molecule in the development of airway inflammatory disease in vitro.

Methods: NPFs (5 × 105 cells/ml) were stimulated with 1.0 μg/ml lipopolysaccharide (LPS) in the presence of agents for 24 hours. NO levels in culture supernatants were examined by the Griess method. We also examined the influence of agents on the phosphorylation of MAPKs, NF-κB activation, iNOS mRNA expression and iNOS production in NPFs cultured for 2, 4, 8, and 12 hours, respectively.

Results: The addition of CAM (> 0.4 μg/ml) and M-4 (> 0.04 μg/ml) could suppress NO production from NPFs after LPS stimulation through the suppression of iNOS mRNA expression and NF-κB activation. CAM and M-4 also suppressed phosphorylation of MAPKs, ERK and p38 MAPK, but not JNK, which are increased LPS stimulation. On the other hand, M-1 and M-5 could not inhibit the NO generation, even when 0.1 μg/ml of the agent was added to cell cultures.

Conclusion: The present results may suggest that M-4 will be a good candidate for the agent in the treatment of chronic airway inflammatory diseases, since M-4 did not have antimicribiological effects on gram positive and negative bacteria.

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Figures

Figure 1
Figure 1
Influence of LPS stimulation on NO production from NPFs. NPFs at a concentration of 5 × 105 cells were stimulated with various concentrations of LPS. After 24 hours, culture supernatants were obtained and assayed for NO (NO2-/NO3-) levels by the Griess method. Data are the mean ± SE of five different subjects. LPS, lipopolysaccharide; NO, nitric oxide; NPFs, nasal polyp fibroblasts. NS, not significant (P > 0.05).
Figure 2
Figure 2
Influence of CAM on NO production from NPFs in response to LPS stimulation. NPFs at a concentration of 5 × 105 cell/ml were stimulated with 1.0 μg/ml LPS in the presence of various concentrations of CAM. After 24 hours, culture supernatants were obtained and NO (NO2-/NO3-) levels were assayed by the Griess method. Data are the mean ± SE of five different subjects. CAM, clarithromycin; NO, nitric oxide; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide.
Figure 3
Figure 3
Influence of metabolized clarithromycin, M-1 (A), M-4 (B) and M-5 (C) on NO production from NPFs in response to LPS stimulation. NPFs at a concentration of 5 × 105 cell/ml were stimulated with 1.0 μg/ml LPS in the presence of various concentrations of the agents. After 24 hours, culture supernatants were obtained and NO (NO2-/NO3-) levels were assayed by the Griess method. Data are the mean ± SE of five different subjects. NO, nitric oxide; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide; NS, not significant (P > 0.05).
Figure 4
Figure 4
Influence of CAM (A) and M-4 (B) on cell proliferation induced by LPS stimulation. NPFs at a concentration of 1 × 105 cells/ml were stimulated 1.0 μg/ml LPS in the presence of various concentrations of CAM and M-4. After 48 hours, cell proliferation was examined by ELISA. Data are the mean OD at 450 nm ± SE of five different subjects. LPS, lipopolysaccharide; NPFs, nasal polyp fibroblasts; CAM, clarithromycin; CAM, clarithromycin; ELISA, enzyme-linked immunosorbent assay; OD, optical density; NS, not significant (P > 0.05).
Figure 5
Figure 5
Influence of CAM (A) and M-4 (B) on iNOS production in NPFs after LPS stimulation. NPFs at a concentration of 5 × 105 cells/ml were stimulated 1.0 μg/ml LPS in the presence of various concentrations of CAM and M-4. After 12 hours, NPFs were collected and iNOS levels were assayed by ELISA. Data are the mean ± SE of five different subjects. iNOS, inducible nitric synthase; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide; CAM, clarithromycin; ELISA, enzyme-linked immunosorbent assay.
Figure 6
Figure 6
Influence of clarithromycin (A) and M-4 (B) on iNOS mRNA expression in NPFs after LPS stimulation. NPFs at a concentration of 5 × 105 cells/ml were stimulated 1.0 μg/ml LPS in the presence of various concentrations of CAM and M-4. After 8 hours, Poly A+ was obtained from NPFs and iNOS mRNA levels were assayed by ELISA. Data are the mean ± SE of five different subjects. iNOS, inducible nitric synthase; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide; CAM, clarithromycin; ELISA, enzyme-linked immunosorbent assay.
Figure 7
Figure 7
Influence of clarithromycin (A) and M-4 (B) on NF-κB activation in NPFs after LPS stimulation. NPFs at a concentration of 5 × 105 cells/ml were stimulated 1.0 μg/ml LPS in the presence of various concentrations of clarithromycin (CAM) and M-4. After 4 hours, nuclear extracts were obtained from NPFs and NF-κB p50 activity was assayed by ELISA. Data are the mean ± SE of five different subjects. NF-κB, nuclear factor-κB; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide; CAM, clarithromycin; ELISA, enzyme-linked immunosorbent assay.
Figure 8
Figure 8
Influence of clarithromycin on MAPKs activation in NPFs after LPS stimulation. NPFs at a concentration of 5 × 105 cells/ml were stimulated 1.0 μg/ml LPS in the presence of various concentrations of CAM. After 4 hours, MAPKs activation was assayed by ELISA. Data are the mean ± SE of five different subjects. A, p38 MAPK; B, ERK1/2; C, JNK. MAPKs, mitogen-activated kinases; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide; CAM, clarithromycin; NS, not significant (P > 0.05).
Figure 9
Figure 9
Influence of metabolized clarithromycin, M-4, on MAPKs activation in NPFs after LPS stimulation. NPFs at a concentration of 5 × 105 cells/ml were stimulated 1.0 μg/ml LPS in the presence of various concentrations of M-4. After 4 hours, MAPKs activation was assayed by ELISA. Data are the mean ± SE of five different subjects. A, p38 MAPK; B, ERK1/2; C, JNK. MAPKs, mitogen-activated kinases; NPFs, nasal polyp fibroblasts; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; NS, not significant (P > 0.05).
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