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* From the Fourth Department of Internal Medicine, Nippon Medical School, Bunkyo-ku, Tokyo, Japan.
Correspondence to: Arata Azuma, MD, PhD, Fourth Department of Internal Medicine, Nippon Medical School, 1–1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan; e-mail: azuma/med4{at}nms.ac.jp
It has been reported1 2 3 4 that neutrophils and the injurious substances associated with them play an important role in the progression of lung fibrosis induced by bleomycin (BLM). We have reported5 further that E-selectin plays an essential role in the neutrophil adhesion to endothelial cells, the subsequent migration of neutrophils into lung tissue, and the progression of lung fibrosis. A new structural macrolide, macrosphelide-A, which is an inhibitor for sialyl-LeX-dependent adhesion of HL60 cells against vascular endothelial cells, successfully inhibits BLM-induced lung fibrosis.6 Fourteen-membered ring macrolides (14-MRMLs) have been reported7 to improve the survival times of patients with diffuse panbronchiolitis owing to several anti-inflammatory mechanisms. In the present study, we investigated the mechanisms of the preventive effect of 14-MRML (ie, erythromycin [EM], clarithromycin [CAM], and roxithromycin [RXM]) in mice with BLM-induced lung fibrosis.
Materials and Methods
BLM (Nippon Kayaku Co; Tokyo, Japan) was administered to ICR male mice (n = 10) IV at a dosage of 100 mg/kg (day 0). Control mice (n = 10) received saline solution (0.3 mL). 14-MRML suspended in gum arabic solution, EM (50 mg/kg), RXM (10 mg/kg), and CAM (8.9 mg/kg) were orally instilled every day from day -3 to day 28 after the BLM injection. At 4 weeks after BLM injection, fibrotic foci were observed pathologically. The Ashcroft score was compared among mice in each group, and the hydroxyproline content in right lung tissues was chemically analyzed. For the evaluation of early-phase inflammation, 14-MRML was administered daily until the mice were killed. Using left lung tissues, cell populations taken from BAL fluid were examined. Further, we investigated messenger RNA induction of adhesion molecules (CD62E, CD62P, and VCAM vascular cell adhesion molecule [VCAM]-1) in lung tissues by reverse transcriptase-polymerase chain reaction.8 9 10 These parameters also were examined in groups of mice that received not only BLM but also daily 14-MRML (day -3 to 7).
Results
In ICR mice with BLM-induced lung fibrosis, fibrotic foci were significantly attenuated in the BLM plus 14-MRML groups. Ashcroft scores also paralleled pathologic features. Hydroxyproline levels in lung tissues also were attenuated in the BLM plus 14-MRML groups. BLM maximally increased the number of neutrophils in BAL fluid at peaks of 12 to 24 h and 144 to 168 h after BLM injection. 14-MRML inhibited neutrophil infiltration into the airway at both 24 h and 144 h after BLM injection (Fig 1 ). E-selectin and P-selectin messenger RNA maximally induced a peak at 6 to 24 h and at 24 to 48 h, respectively.5 The levels of VCAM-1 messenger RNA, with maximal induction at 6 to 24 h, and neutrophil migration into the airway ran parallel after BLM administration. 14-MRML significantly inhibited the induction of VCAM-1 messenger RNA but, remarkably, not that of E-selectin messenger RNA, causing the inhibition of neutrophil migration into the airspace and finally attenuating the degree of fibrosis in the lung. CAM was more effective than EM or RXM (Fig 1) .
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Although the mechanism of pulmonary fibrosis in BLM-treated
mice remains unclear, many researchers recognize that
neutrophil-mediated lung injury is necessary for the initiation and/or
propagation of the fibrogenic process.11
12
13
Neutrophil
adhesion to vascular endothelial cells must be an important process in
cell-mediated lung injury with the release of proteases and free
radical formation. We actually reported5
that E-selectin
played an essential role in neutrophil adhesion to vascular endothelial
cells and in subsequent lung fibrosis. We investigated the inhibitory
effect of 14-MRMLs on a well-designed experimental mouse model for the
evaluation of lung fibrosis. 14-MRML, especially CAM rather than EM and
RXM, obviously attenuated the expression of VCAM-1 messenger RNA during
the early phase of BLM-induced lung injury that led the inhibition of
neutrophil migration into the airway. The inhibition of
4/ß1-integrin as a
ligand for VCAM-1 also caused the attenuation of neutrophil migration
and subsequent fibrosis, which was reported by Giri et al at the
2000 American Thoracic Society meeting in Toronto, Canada.
Conclusion
14-MRML successfully inhibited the induction of VCAM-1 messenger RNA. This attenuated migration of neutrophils into the airway resulted in the prevention of BLM-induced lung fibrosis. We hope that stopping the adhesion of neutrophils to endothelial cells will be a future strategy for the prevention of BLM-induced lung injury and fibrosis, as well as for the inhibition of selectin. 14-MRMLs are promising agents for the prevention of disease progression.
Footnotes
Abbreviations: BLM = bleomycin; CAM = clarithromycin; EM = erythromycin; 14-MRML = 14-membered ring macrolide; RXM = roxithromycin; VCAM = vascular cell adhesion molecule
References
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