Chest
Volume 122, Issue 6, December 2002, Pages 2137-2145
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Laboratory and Animal Investigations
Fourteen-Membered Ring Macrolides Inhibit Vascular Cell Adhesion Molecule 1 Messenger RNA Induction and Leukocyte Migration: Role in Preventing Lung Injury and Fibrosis in Bleomycin-Challenged Mice*

https://doi.org/10.1378/chest.122.6.2137Get rights and content

Background and objective

Although the pathogenesis of interstitial pneumonia and pulmonary fibrosis are not well understood, it has been reported that inflammatory cells, especially neutrophils, and the injurious substances produced by them play important roles in the progression of interstitial pneumonia and subsequent fibrosis. Erythromycin and other 14-membered ring macrolides (14-MRMLs) have been reported to improve the survival of patients with diffuse panbronchiolitis by antineutrophil and several other anti-inflammatory mechanisms. The present study was undertaken to investigate the effects of 14-MRMLs on an experimental model of bleomycin-induced acute lung injury and subsequent fibrosis in mice.

Methods

Bleomycin was administered IV to ICR mice. At 28 days after bleomycin injection, fibrotic foci were histologically observed in left lung tissues, and hydroxyproline content in right lung tissues was chemically analyzed. The inhibitory effects of 14-MRMLs were assessed by overall comparison between control (normal saline solution [NS] alone), untreated (bleomycin alone), and treated (bleomycin plus 14-MRMLs) groups. For evaluation of early-phase inflammation, cell populations in BAL fluid and induction of messenger RNA (mRNA) of adhesion molecules (E-selectin, P-selectin, intercellular adhesion molecule 1 [ICAM-1], and vascular cell adhesion molecule 1 [VCAM-1]) in lung tissues were examined at 0 to 13 days after bleomycin treatment. These parameters were also compared with those for the control (NS alone), 14-MRML untreated (bleomycin alone), and 14-MRML pretreated (bleomycin plus 14-MRML pretreated) groups.

Results

Bleomycin-induced pulmonary fibrosis was inhibited by erythromycin and other 14-MRMLs on day 28 after bleomycin injection in ICR mice, especially those pretreated with 14-MRMLs. Hydroxyproline content in lung tissues was also decreased in the 14-MRML-pretreated groups. The number of neutrophils in BAL fluid significantly increased, with two peaks at 1 day and 9 days (from 6 to 11 days) after bleomycin administration. 14-MRMLs significantly inhibited both peaks of neutrophil infiltration into the airspace. Changes in mRNA expression of adhesion molecules (E-selectin, P-selectin, ICAM-1, VCAM-1) were associated with leukocyte migration into the airspace. 14-MRMLs clearly inhibited the induction of VCAM-1 mRNA, and tended to attenuate that of ICAM-1 mRNA, but inhibited the induction of neither E-selectin mRNA nor P-selectin mRNA.

Conclusion

These findings indicate that attenuation of inflammatory cell migration into the airspace by 14-MRMLs, especially of neutrophils and macrophages, resulted in inhibition of lung injury and subsequent fibrosis. 14-MRMLs clearly attenuated the expression of VCAM-1 mRNA during the early phase of bleomycin-induced lung injury, and this might be one mechanism of inhibition of neutrophil and macrophage migration into the airspace by 14-MRMLs. This may be one mechanism of the anti-inflammatory and antifibrotic effects of 14-MRMLs. These findings suggest that prophylactic administration of 14-MRMLs may be clinically efficacious in preventing acute exacerbation of interstitial pneumonia and acute lung injury.

Section snippets

Animals

Male, 7-week-old ICR mice (Nippon CLEA; Tokyo, Japan), weighing 30 g each on average, were randomly classified into groups.

Materials

Bleomycin (Nippon Kayaku; Tokyo, Japan) was dissolved in normal saline solution (NS) and administered IV to ICR mice at a dosage of 100 mg/kg (0.3 mL per mouse). Erythromycin, 50 mg/kg (Dainabott; Osaka, Japan); clarithromycin, 8.9 mg/kg (Dainabott); and roxithromycin, 10 mg/kg (Aventis Pharma; Tokyo, Japan) were suspended in 5% arabic gum (AG) [Wako Pure Chemical

Histopathologic Assessment of Late-Phase Pulmonary Fibrosis

Bleomycin-induced lung fibrosis was significantly inhibited by treatment with erythromycin, clarithromycin, and pretreatment with roxithromycin at day 28 after bleomycin injection in ICR mice. Comparison of Ashcroft scores revealed significant difference between pretreatment and posttreatment with 14-MRMLs (Fig 1). A typical picture of attenuation of fibrosis is shown in Figure 2. Administration of 14-MRMLs alone resulted in no remarkable changes in results of histopathologic assessment of lung

Discussion

Idiopathic pulmonary fibrosis (IPF) is the prototype of many other interstitial lung disorders in which parenchymal inflammation is a typical features; such disorders number > 100. Although the pathogenesis of pulmonary fibrosis remains unclear, many investigators have found that neutrophil-mediated lung injury occurs prior to initiation and progression of the fibrogenic process.20,21 Neutrophil adhesion to vascular endothelial cells is an important process in cell-mediated lung injury, along

Conclusion

We conclude that 14-MRMLs successfully inhibited the induction of VCAM-1 mRNA during the early phase of bleomycin-induced lung injury. This might be one mechanism of inhibition of neutrophil and macrophage migration into the airspace by 14-MRMLs, and subsequent fibrotic effects of 14-MRMLs. Inhibition of adhesion of leukocytes to endothelial cells may be useful for prevention of leukocyte-mediated lung injury and subsequent fibrosis, including IPF in humans. 14-MRMLs are thus promising agents

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