- Email: [email protected]
Fourteen-Membered Ring Macrolides Inhibit Vascular Cell Adhesion Molecule 1 Messenger RNA Induction and Leukocyte Migration
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* YingJi Li, MD, PhD; Arata Azuma, MD, PhD; Satoru Takahashi, MD, PhD; Jiro Usuki, MD, PhD; Kuniko Matsuda; Akinori Aoyama; and Shoji Kudoh, MD, PhD
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 (14MRMLs) 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. (CHEST 2002; 122:2137–2145) Key words: bleomycin; lung fibrosis; macrolide; vascular cell adhesion molecule 1 Abbreviations: AG ⫽ arabic gum; BALF ⫽ BAL fluid; cDNA ⫽ complementary DNA; G6PD ⫽ glucose-6-phosphate dehydrogenase; ICAM-1 ⫽ intercellular adhesion molecule 1; IPF ⫽ idiopathic pulmonary fibrosis; MMP ⫽ matrix metalloproteinase; 14-MRMLs ⫽ 14-membered ring macrolides; mRNA ⫽ messenger RNA; NS ⫽ normal saline solution; PCR ⫽ polymerase chain reaction; RT ⫽ reverse transcriptase; VCAM-1 ⫽ vascular cell adhesion molecule 1
www.chestjournal.org
CHEST / 122 / 6 / DECEMBER, 2002
2137
lung injury and subsequent B leomycin-induced fibrosis in animals is a widely used experimental
model of acute lung injury and fibrosis in humans.1–3 The mechanisms of interstitial pneumonia and pulmonary fibrosis are not understood in detail, but it has been found that neutrophils and the injurious substances they produce play important roles in the progression of pulmonary fibrosis.4 –7 We found that E-selectin, acting as a key molecule in the initial phase of neutrophil adhesion to vascular endothelial cells, played an essential role in the progression of pulmonary fibrosis.8 Adhesion molecules, including selectins and integrins, are required for neutrophil migration into lung tissues. The pathologic features of acute lung injury include prominent accumulation of neutrophils and macrophages in the lung parenchyma.9,10 In patients with diffuse panbronchiolitis receiving oral erythromycin, increase in number of neutrophils in BAL fluid (BALF) was significantly reduced.11 Erythromycin and other 14-membered ring macrolides (14-MRMLs) have been reported to improve the survival of patients with diffuse panbronchiolitis by several anti-inflammatory mechanisms.11,12 We previously reported that the number of neutrophils and the amount of neutrophil elastase in BAL played important roles in the acute lung injury induced by bleomycin, and that both were decreased by treatment with erythromycin.7 In this study, we examined the inhibitory effects of 14-MRMLs including erythromycin, clarithromycin, and roxithromycin on bleomycin-induced pulmonary fibrosis following acute lung injury. We further investigated the role of adhesion molecules in neutrophil adhesion to endothelial cells, subsequent migration of neutrophils into lung tissue and progression of pulmonary fibrosis, and elucidated the mechanisms by which 14-MRMLs exhibit anti-inflammatory effects and oppose the fibrosis that follows inflammation in bleomycin-challenged mice. Materials and Methods Animals Male, 7-week-old ICR mice (Nippon CLEA; Tokyo, Japan), weighing 30 g each on average, were randomly classified into groups. *From the Fourth Department of Internal Medicine (Drs. Li, Azuma, Usuki, Matsuda, Aoyama, and Kudoh), Nippon Medical School, Tokyo; and Institute of Basic Medical Sciences (Dr. Takahashi), University of Tsukuba, Ibaragi, Japan. Manuscript received September 4, 2001; revision accepted April 30, 2002. Correspondence to: Arata Azuma, MD, PhD, Fourth Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan; e-mail: [email protected] 2138
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 Industries; Tokyo, Japan]. Solutions, 0.3 mL per mouse, were orally administered by force with a microtube daily to ICR mice. Schedule and Process of Evaluation of Late-Phase Fibrosis The bleomysin-Untreated Groups included the NS-treated group (group 1), and 14-MRML alone-treated groups (erythromycin, clarithromycin, roxithromycin) [group 2]. The bleomycin-Treated Groups included the 14-MRMLuntreated group (bleomycin alone) [group 3]; 14-MRMLpretreated groups (day ⫺ 3 to day 13) [bleomycin plus pretreatment erythromycin, bleomycin plus pretreatment clarithromycin, and bleomycin plus pretreatment roxithromycin] (group 4); and 14-MRMLs-posttreated groups (day 3 to day 19) [bleomycin plus posttreatment erythromycin, bleomycin plus posttreatment clarithromycin, and bleomycin plus posttreatment roxithromycin] (group 5). NS was administered IV to bleomycin-untreated mice (day 0). Bleomycin was administered IV to bleomycin-treated mice (day 0). AG was orally administered every day to mice of groups 1 and 3 (day ⫺ 3 to day 13). 14-MRML was orally administered daily to mice of groups 2 and 4 (day ⫺ 3 to day 13) and mice of group 5 (day 3 to day 19). Mice in all groups were killed under ether anesthesia at day 28 after bleomycin or NS injection. Fibrotic foci were assessed histologically in left lung tissues, Ashcroft scores were compared among the groups, and hydroxyproline content in right lung tissues was chemically analyzed. Schedule and Evaluation of Early-Phase Inflammation NS was administered IV to NS-treated group mice (day 0). Bleomycin was administered IV to bleomycin-treated group mice (day 0). AG was orally administered daily to mice of the NS-alone and bleomycin-alone-treated groups from day ⫺ 3 until the time of death. 14-MRMLs were administered daily to the 14-MRMLpretreated groups from day 3 until the time of death. Mice of the NS-alone and bleomycin-alone groups were killed under ether anesthesia at 0, 6 h, or 12 h, or 1, 2, 3, 5, 6, 7, 9, 11, or 13 days after bleomycin or NS injection. All groups were examined for cell population in BALF 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 by reverse transcriptase (RT)-polymerase chain reaction (PCR) at 0 to 13 days after bleomycin or NS injection. Mice of the 14-MRML-pretreated groups were killed under ether anesthesia at 1 day and 9 days after bleomycin injection and underwent the same experiment. We repeated the same experiment three times in early-phase evaluation, and examined the cell population in BALF. The total number of mice was 10 in each group for each time point. RT-PCR examination was repeated three times. Histologic Analysis For histologic examination, we added 0.5 mL of 10% formalin with 10 cm H2O of pressure to the left lobe of every mouse; 10% formalin-fixed lung tissues were embedded in paraffin. Paraffin Laboratory and Animal Investigations
sections were stained with either hematoxylin-eosin or Masson trichrome stain, and systematically scanned with a light microscope using a 20 ⫻ 10 objective. We compared severities of interstitial fibrosis among the groups by the score of Ashcroft et al.13 Hydroxyproline Measurement The right lung was freeze-dried for 3 days using a Tray Dryer (FTS Systems; Stoneridge, NY) as soon as possible after the animals were killed. The total collagen content of the right lung was determined by hydroxyproline assay.14 After acid hydrolysis of the right lung with 12 N HCl at 100°C for 20 h in a sealed glass tube (Iwaki; Tokyo, Japan), hydroxyproline content was determined by high-performance liquid chromatography.
Results 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 tissue (data not shown).
Analysis of BALF BALF was obtained by injection of 1 mL of saline solution (three times; total, 3 mL) followed by gentle aspiration of the fluid from the right and left lungs after securing the intratracheal catheter within a main bronchus. With this catheter, recovery ratios of lavage fluids ranged from 65 to 75% and did not significantly differ among the groups. Total cell numbers in BALF were counted with a hemocytometer. For differential counts of leukocytes in BALF, cytospin (Labsystems; Tokyo, Japan) smear slides were prepared. The smears were stained with Giemsa solution (Merck; Tokyo, Japan). Differential cell counts were performed on 200 cells per smear.
Hydroxyproline Content in Lung Tissue: The concentration of hydroxyproline at day 28 after bleomycin injection was significantly higher in the bleomycin-alone group than in the NS-alone group. Of the groups pretreated with 14-MRML, hydroxyproline was significantly reduced only then in the bleomycin-alone group. Hydroxyproline was not significantly attenuated in the groups posttreated with 14-MRMLs. Comparison of hydroxyproline revealed a significant difference between pretreatment and posttreatment with 14-MRMLs (Fig 3).
Quantitative Analysis of Adhesion Molecule mRNA by RT-PCR Total RNA was extracted from each specimen of lung tissue using ISOGEN (Nippon GENE; Tokyo, Japan). Single-strand complementary DNA (cDNA) was synthesized using RT (Gibco BRL; Rockville, MD) and oligo (dT)12–18 primer. To measure gene expression, we performed the PCR method, with cDNA amplified using a TaKaRa PCR Thermal Cycler 480 and TaKaRa Ex Taq (Takara Shuzo; Otsu, Japan). For amplification of the desired cDNA, the following gene-specific primers were used15–18: E-selectin, sense (EL-11) 5⬘-gattggacactcaatggatc-3⬘, antisense (EL-9) 5⬘-cctagacgttgtaagaaggc-3⬘; P-selectin, sense 5⬘-acgagctggacggacccg-3⬘, antisense 5⬘-ggctggcactcaaatttacag-3⬘; ICAM-1, sense 5⬘-tcggaggatcacaaacgaagc3⬘, antisense 5⬘-aacataagaggctgccatcacg-3⬘; VCAM-1, sense (VC-14) 5⬘-aagcagagacttgaaatgcc-3⬘, antisense (VC-13) 5⬘-cccttgaacagatcaatctcc-3⬘. The PCR products underwent electrophoresis on 2% agarose gels, stained with ethidium bromide, and observed with ultraviolet transillumination. Intensity analysis of the bands was performed with Adobe Photoshop 5.0 (Adobe Systems; Tokyo, Japan), and expression of adhesion molecule messenger RNA was indicated by the number of white pixel areas. Glucose-6-phosphate dehydrogenase (G6PD) was measured as an internal control.19 Statistical Analysis Differences between two groups (grade of fibrosis, hydroxyproline, leukocyte counts) were tested for significance using the Mann-Whitney U test. Repeated-measures analysis of variance was used to evaluate differences in leukocyte counts among groups over time. www.chestjournal.org
Time Course of Changes in Cell Number in BALF After Treatment With Bleomycin Total cell number in BALF peaked at 6 to 13 days after bleomycin injection (Fig 4, top, A, a). The
Figure 1. Histopathologic assessment of late-phase pulmonary fibrosis in bleomycin-challenged mice; comparison of Ashcroft score. Significantly different from bleomycin alonetreated group by the Mann-Whitney U test, **p ⬍ 0.01, *p ⬍ 0.05. Significantly different from 14-MRML-pretreated groups by the Mann-Whitney U test, 夞p ⬉ 0.05. Values are means; bars ⫽ SD, n ⫽ 10. N ⫽ NS-treated group; B ⫽ bleomycin-alone-treated group; BE ⫽ bleomycin- and erythromycin-treated group; BC ⫽ bleomycin- and clarithromycintreated group; BR ⫽ bleomycin and roxithromycin-treated group. CHEST / 122 / 6 / DECEMBER, 2002
2139
Figure 3. Comparison of hydroxyproline contents. Significantly different from bleomycin alone-treated group by the MannWhitney U test, **p ⬍ 0.01; significantly different from 14MRML-pretreated groups by the Mann-Whitney U test, 多p ⬍ 0.01. Values are means; bars ⫽ SD, n ⫽ 10. HOP ⫽ hydroxyproline; DW ⫽ dry lung weight; see Figure 1 legend for expansion of abbreviations.
Effects of 14-MRMLs on Leukocyte Number in BALF 14-MRMLs significantly decreased the number of neutrophils in BALF at 1 day and 9 days after injection of bleomycin (Fig 4, bottom, B, a). The increase in number of macrophages in BALF at 9 days after bleomycin injection was significantly attenuated by erythromycin and roxithromycin, and slightly attenuated by clarithromycin (Fig 4, bottom, B, b). The number of lymphocytes in BALF at 9 days after bleomycin injection was significantly attenuated by erythromycin and clarithromycin, and slightly attenuated by roxithromycin (Fig 4, bottom, B, c). Time Course of Changes in mRNA Expression of Adhesion Molecules in Lung Tissue After Treatment With Bleomycin Figure 2. Pathologic features of lung tissues (hematoxylin-eosin, original ⫻ 200) at 28 days after treatment with bleomycin alone or in combination with clarithromycin. Top, a: treated with NS alone. Center, b: treated with bleomycin alone. Bottom, c: treated with bleomycin and pretreated with clarithromycin.
number of neutrophils in BALF peaked twice, from 12 h to 2 days and from 5 to 11 days after bleomycin injection, and returned to control level at 13 days after bleomycin injection (Fig 4, top, A, b). The number of macrophages in BALF was maximal at 6 to 11 days after bleomycin injection (Fig 4, top A, c). The number of lymphocytes in BALF was significantly increased at 9 to 13 days after bleomycin injection (Fig 4, top, A, d). 2140
E-selectin (Fig 5, top, A, a), P-selectin (top, A, b), and ICAM-1 (top, A, c) mRNAs were induced at 6 to 12 h, decreased to control levels at 1 day, and were induced for the second time 2 to 13 days after bleomycin injection. VCAM-1 mRNA (Fig 5, top, A, d) was induced 2 to 13 days after bleomycin injection. Inhibitory Effect of 14-MRML on mRNA Expression of VCAM-1 and ICAM-1 14-MRMLs clearly inhibited the induction of VCAM-1 mRNA (Fig 5, bottom, B, d) and tended to attenuate ICAM-1 mRNA expression (Fig 5, bottom, B, c) at day 9 after bleomycin. However, 14-MRMLs markedly inhibit the induction of neither E-selectin (bottom, B, a) nor P-selectin mRNAs (Fig 5, bottom, B, b). Laboratory and Animal Investigations
Figure 4. Top, A: Time course of changes in cell numbers in BALF (upper left, a: total cell number; upper right, b: No. of neutrophils; lower left, c: No. of macrophages; lower right, d: No. of lymphocytes). Significantly different from NS-treated group at each time point by the Mann-Whitney U test, 多p ⬉ 0.0004, 夞p ⬍ 0.01. Significantly different from the first time point (0 h) after bleomycin (BLM) administration by repeated-measures analysis of variance, ***p ⬍ 0.0001, **p ⬍ 0.01, *p ⬍ 0.05. Values are means; bars ⫽ SE, n ⫽ 10. Bottom, B: Effects of 14-MRMLs on leukocyte number in BALF on day 1 and day 9 after injection of bleomycin (top, a: No. of neutrophils in BALF on days 1 and 9 after injection of bleomycin; bottom left, b: No. of macrophages in BALF on day 9 after injection of bleomycin; bottom right, c: No. of lymphocytes in BALF on day 9 after injection of bleomycin). Significantly different from bleomycin alone-treated group by the Mann-Whitney U test, **p ⬍ 0.01, *p ⬍ 0.05. Values are means; bars ⫽ SE, n ⫽ 10. See Figure 1 for expansion of abbreviations. www.chestjournal.org
CHEST / 122 / 6 / DECEMBER, 2002
2141
Figure 5. Top, A: Time course of E-selectin (upper left, a), P-selectin (upper right, b), ICAM-1 (lower left, c), and VCAM-1 (lower left, d) mRNA inductions in lung tissue after bleomycin administration. Bottom, B: Effects of 14-MRMLs on expression of adhesion molecules such as E-selectin (upper left, a), P-selectin (upper right, b), ICAM-1 (lower left, c), and VCAM-1 (lower right, d) mRNA in lung tissue on day 9 after bleomycin administration. These photographs show typical results. Each density of PCR products was measured using Adobe Photoshop 5.0; G6PD was measured as an internal control. The ratio of each adhesion molecule against G6PD is shown by histogram. bp ⫽ base pair; see Figure 1 legend for expansion of abbreviations.
2142
Laboratory and Animal Investigations
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 with release of proteases and free-radical formation.4,7 Inhibition of production of injurious substances (eg, neutrophil elastase by ONO5046),5 other proteases by truncated secretary leukocyte protease inhibitor,9 and by ␣-1 protease inhibitor10 is a promising method for prevention of pulmonary fibrosis. The initial phase of neutrophil migration prior to activation in lung parenchyma is an important point of intervention in strategies that are effective in preventing tissue injury and subsequent fibrosis. We previously reported that E-selectin played an essential role in neutrophil adhesion to vascular endothelial cells and subsequent lung fibrosis.8 We initially investigated bleomycin-induced histopathologic changes in lung tissues. Bleomycininduced pulmonary fibrosis at day 28 was significantly inhibited by pretreatment with 14-MRMLs after bleomycin administration (Figs 1–3). The effectiveness of pretreatment with 14-MRMLs (beginning 3 days before bleomycin injection) is significantly higher than that of posttreatment with 14-MRMLs (beginning 3 days after bleomycin injection) [Figs 1–3]. The difference of potency for inhibitory effects between pretreatment and posttreatment with 14-MRML may have depended on the difference of their inhibitory effects of neutrophil recruitment in lung tissue, especially to the first peak (from 12 h to 2 days after bleomycin injection) of neutrophil migration. Development of pulmonary fibrosis is thought to include two phases: an acute inflammatory phase and a sequential fibrotic phase.22 To determine the mechanisms of the prophylactic effect of 14-MRMLs on bleomycininduced pulmonary fibrosis in mice, we further investigated the mechanisms of the early inflammatory (ie, up-stream) phase. We found two peaks of neutrophil concentration in BALF. The two peaks of neutrophil recruitment paralleled but were slightly delayed compared with induction of E-selectin, P-selectin, and ICAM-1 mRNAs. The second peak and the induction of VCAM-1 mRNA were also parallel with slight delay (Fig 4, top, A, b, and Fig 5, top, A). ␣4/1 integrin, acting as a ligand for VCAM-1, is www.chestjournal.org
believed to contribute to migration of macrophages, eosinophils, and lymphocytes into inflamed tissues.18,23 Some investigators have reported that neutrophil adhesion to vascular endothelial cells depends principally on ␣9/1-VCAM-1 interaction.24 In the setting of leukocyte-endothelial interactions, there are diverse mechanisms that modulate leukocyte integrin avidity. Crosslinking of the Eselectin and P-selectin counter-structures, CD15 (sialyl Lex), or its sialylated form on neutrophils caused activation.25 Therefore, we considered the possibility that neutrophils bound selectins in the initial phase, and that simultaneously ␣9/1 integrin was activated by selectins, with tight binding of neutrophils to VCAM-1, which might have induced the second neutrophil peak. We also considered the possibility that 14-MRMLs attenuated induction of VCAM-1 on vascular endothelial cells binding to neutrophils due to inhibition of the second neutrophil peak. We also investigated the inhibitory effects of 14MRMLs on bleomycin-induced lung injury to evaluate the early inflammatory phase in the same experimental model in mice. 14-MRMLs clearly inhibited the expression of VCAM-1 mRNA (Fig 5, bottom, B, d), and significantly inhibited the two neutrophil peaks after injection (Fig 4, bottom, B, a) in bleomycin-induced lung injury. We therefore considered the possibility that 14-MRMLs attenuated induction of VCAM-1 on vascular endothelial cells binding to neutrophils due to inhibition of the second neutrophil peak. 14-MRMLs tended to attenuate the expression of ICAM-1 mRNA (Fig 5, bottom, B, c). We therefore considered the possibility that attenuation by 14MRMLs of the expression of ICAM-1 mRNA was related to inhibition of the two peaks of neutrophil recruitment. Lung injury may have depended on macrophage antigen 1 and lymphocyte functionassociated antigen 1 integrin on neutrophils acting as ligands for ICAM-1 on vascular endothelial cells. This might be one mechanism of inhibition of neutrophil migration into the airspace by 14-MRMLs. The number of macrophages changed in parallel with slight delay for the induction of VCAM-1 mRNA in the present study (Fig 4, top, A, c, and Fig 5, top, A, d). VCAM-1 mRNA induction was mainly associated with movement of macrophages into the airspace. We considered the possibility that 14-MRMLs inhibited binding of ␣4/1 on macrophages to VCAM-1 on vascular endothelial cells due to inhibition of movement of macrophages of ligand expression, which requires further examination. These findings suggested that attenuation of inflammatory cell migration by 14-MRMLs, especially of neutrophils and macrophages, resulted in inhibiCHEST / 122 / 6 / DECEMBER, 2002
2143
tion of lung injury and subsequent fibrosis. Inhibition by 14-MRMLs of VCAM-1 mRNA expression might be one mechanism of inhibition of neutrophil and macrophage migration into the airspace. It has been reported that erythromycin inhibits chemokines including interleukin 8, which acts as a neutrophil chemoattractant, resulting in attenuation of neutrophil migration into inflamed tissues, and inhibits the expression of adhesion molecules.26 –30 In our study, 14-MRMLs may have inhibited not only VCAM-1 induction, but also that of interleukin 8 and other agents, especially at the first peak of increase in neutrophil number. It has been reported that matrix metalloproteinase (MMP) is associated with the process of pulmonary fibrosis in human and animal models, and in particular that MMP-9 correlated with neutrophil recruitment and MMP-2 with development of the fibrotic phase.31–34 Furthermore, suppression of MMP-9 activity by erythromycin is also an anti-inflammatory mechanism that inhibits the migration of inflammatory cells into inflamed tissues.35 We therefore considered the possibility that erythromycin inhibits MMPs, which would be an important mechanism of antifibrotic activity. Although neutrophils from beige mice exhibited a selective defect of release of neutrophil elastase in response to several stimuli, bleomycin-induced lung fibrosis was more prominent than that in littermates, without impairing their ability to produce superoxide anion and H2O2 in response to bleomycin.36 Neutrophil elastase promotes tissue injury,7 but other agents such as oxygen radicals might be major components in the tissue injury that occurs in response to bleomycin. No treatments that clearly improve the prognosis of IPF exist at present. New strategies for such treatment have, however, been discussed. One such strategy is to inhibit leukocyte accumulation in lung tissue.37 Antibody to CD11, CD18, or ␣9 integrin may prove useful by inhibiting neutrophil migration into lung tissue via blocking of cell-to-cell adhesion and migration.38 14-MRMLs can be expected to prevent leukocyte-mediated lung injury by mechanisms of action similar to those of antibodies to endothelial adhesion molecules. In addition, the safety and cost of 14-MRML therapy are superior to those of antibody therapy.
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 2144
and macrophage migration into the airspace by 14-MRMLs, and subsequent fibrotic effects of 14MRMLs. 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 for acute exacerbation of interstitial pneumonia, acute lung injury, and prophylaxis of progression of IPF.
References 1 Adamson IYR, Bowden DH. The pathogenesis of bleomycininduced pulmonary fibrosis in mice. Am J Pathol 1974; 77:185–198 2 Aso Y, Yoneda K, Kikkawa Y. Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab Invest 1976; 35:558 –568 3 Weidner WJ, Quam DA, McClure DE, et al. Effect of acute administration of bleomycin on lung fluid balance in sheep. Exp Lung Res 1995; 21:617– 630 4 Henson PM, Johnston RB Jr. Tissue injury in inflammation, oxidants, proteinase and cationic proteins. J Clin Invest 1987; 79:669 – 674 5 Sakamaki F, Ishizaka A, Urano T, et al. Effect of specific neutrophil elastase inhibitor, ONO-5046, on endotoxininduced acute lung injury. Am J Respir Crit Care Med 1996; 153:391–397 6 Botha AJ, Moore FA, Moore EE, et al. Sequential systemic platelet-activating factor and interleukin 8 primes neutrophils in patients with trauma at risk of multiple organ failure. Br J Surg 1996; 83:1407–1412 7 Azuma A, Furuta T, Enomoto T, et al. Preventive effect of erythromycin on experimental bleomycin-induced acute lung injury in rats. Thorax 1998; 53:186 –189 8 Azuma A, Takahashi S, Nose M, et al. Role of E-selectin in bleomycin induced lung fibrosis in mice. Thorax 2000; 55: 147–152 9 Mitsuhashi H, Asano S, Nonaka T, et al. Administration of truncated secretory leukoprotease inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters. Am J Respir Crit Care Med 1996; 153:369 –374 10 Nagai A, Aoshiba K, Ishihara Y, et al. Administration of ␣1-proteinase inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters. Am Rev Respir Dis 1992; 145:651– 656 11 Oda H, Kadota J, Kohno S, et al. Erythromycin inhibits neutrophil chemotaxis in bronchoalveoli of diffuse panbronchiolitis. Chest 1994; 106:1116 –1123 12 Kudoh S, Azuma A, Yamamoto M, et al. Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. Am J Respir Crit Care Med 1998; 157:1829 –1832 13 Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988; 41:467– 470 14 Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961; 93:440 – 447 15 Araki M, Araki K, Miyazaki Y, et al. E-selectin binding promotes neutrophil activation in vivo in E-selectin transgenic mice. Biochem Biophys Res Commun 1996; 224:825– 830 16 Sanders WE, Wilson RW, Ballantyne CM, et al. Molecular Laboratory and Animal Investigations
17
18 19 20 21 22 23 24
25 26 27
28
cloning and analysis of in vivo expression of murine Pselectin. Blood 1992; 80:795– 800 Oran A, Marshall JS, Kondo S, et al. Cyclosporin inhibits intercellular adhesion molecule-1 expression and reduces mast cell numbers in the asebia mouse model of chronic skin inflammation. Br J Dermatol 1997; 136:519 –526 Araki M, Araki K, Vassalli P. Cloning and sequencing of mouse VCAM-1 cDNA. Gene 1993; 126:261–264 Manos P, Nakayama R, Holten D. Regulation of glucose-6phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes. Biochem J 1991; 276:245–250 Henson PM. Mechanisms of cellular injury in interstitial lung disease. Chest 1986; 89:108 –115 Hunninghake GW, Gadek JE, Lawley TJ, et al. Mechanisms of neutrophil accumulation in the lungs of patients with idiopathic pulmonary fibrosis. J Clin Invest 1981; 68:259 –269 Ward PA, Hunninghake GW. Lung inflammation and fibrosis. Am J Respir Crit Care Med 1998; 157:123–129 Beekhuizen H, van Furth R. Monocyte adherence to human vascular endothelium. J Leukoc Biol 1993; 54:363–378 Taooka Y, Chen J, Yednock T, et al. The integrin ␣91 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1. J Cell Biol 1999; 145:413– 420 Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994; 84:2068 –2101 Kadota J, Sakito O, Kohno S, et al. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am Rev Respir Dis 1993; 147:153–159 Sakito O, Kadota J, Kohno S, et al. Interleukin 1, tumor necrosis factor ␣, and interleukin 8 in bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis: a potential mechanism of macrolide therapy. Respiration 1996; 63:42– 48 Sugiyama Y, Yanagisawa K, Tominaga SI, et al. Effects of long-term administration of erythromycin on cytokine production in rat alveolar macrophages. Eur Respir J 1999; 14:1113–1116
www.chestjournal.org
29 Fujii T, Kadota JI, Morikawa T, et al. Inhibitory effect of erythromycin on interleukin 8 production by 1, 25-dihydroxyvitamin D3-stimulated THP-1 cells. Antimicrob Agents Chemother 1996; 40:1548 –1551 30 Oishi K, Sonoda F, Kobayashi S, et al. Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases. Infect Immun 1994; 62:4145– 4152 31 Yaguchi T, Fukuda Y, Ishizaki M, et al. Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis. Pathol Int 1998; 48:954 –963 32 Corbel M, Theret N, Caulet MS, et al. Repeated endotoxin exposure induces interstitial fibrosis associated with enhanced gelatinase (MMP-2 and MMP-9) activity. Inflamm Res 2001; 50:129 –135 33 Corbel M, Caulet MS, Germain N, et al. Inhibition of bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor batimastat. J Pathol 2001; 193: 535–545 34 Fukuda Y, Ishizaki M, Kudoh S, et al. Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinases-2 in interstitial lung diseases. Lab Invest 1998; 78:687– 698 35 Hashimoto N, Kawabe T, Hara T, et al. Effect of erythromycin on matrix metalloproteinase-9 and cell migration. J Lab Clin Med 2001; 137:176 –183 36 Phan SH, Schrier D, McGarry B, et al. Effect of the beige mutation on bleomycin-induced pulmonary fibrosis in mice. Am Rev Respir Dis 1983; 127:456 – 459 37 Hunninghake GW, Kalica AR. Approaches to the treatment of pulmonary fibrosis. Am J Respir Crit Care Med 1995; 151:915–918 38 Piguet PF, Rosen H, Vesin C, et al. Effective treatment of the pulmonary fibrosis elicited in mice by bleomycin or silica with anti-CD-11 antibodies. Am Rev Respir Dis 1993; 147: 435– 441
CHEST / 122 / 6 / DECEMBER, 2002
2145
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 (14MRMLs) 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. (CHEST 2002; 122:2137–2145) Key words: bleomycin; lung fibrosis; macrolide; vascular cell adhesion molecule 1 Abbreviations: AG ⫽ arabic gum; BALF ⫽ BAL fluid; cDNA ⫽ complementary DNA; G6PD ⫽ glucose-6-phosphate dehydrogenase; ICAM-1 ⫽ intercellular adhesion molecule 1; IPF ⫽ idiopathic pulmonary fibrosis; MMP ⫽ matrix metalloproteinase; 14-MRMLs ⫽ 14-membered ring macrolides; mRNA ⫽ messenger RNA; NS ⫽ normal saline solution; PCR ⫽ polymerase chain reaction; RT ⫽ reverse transcriptase; VCAM-1 ⫽ vascular cell adhesion molecule 1
www.chestjournal.org
CHEST / 122 / 6 / DECEMBER, 2002
2137
lung injury and subsequent B leomycin-induced fibrosis in animals is a widely used experimental
model of acute lung injury and fibrosis in humans.1–3 The mechanisms of interstitial pneumonia and pulmonary fibrosis are not understood in detail, but it has been found that neutrophils and the injurious substances they produce play important roles in the progression of pulmonary fibrosis.4 –7 We found that E-selectin, acting as a key molecule in the initial phase of neutrophil adhesion to vascular endothelial cells, played an essential role in the progression of pulmonary fibrosis.8 Adhesion molecules, including selectins and integrins, are required for neutrophil migration into lung tissues. The pathologic features of acute lung injury include prominent accumulation of neutrophils and macrophages in the lung parenchyma.9,10 In patients with diffuse panbronchiolitis receiving oral erythromycin, increase in number of neutrophils in BAL fluid (BALF) was significantly reduced.11 Erythromycin and other 14-membered ring macrolides (14-MRMLs) have been reported to improve the survival of patients with diffuse panbronchiolitis by several anti-inflammatory mechanisms.11,12 We previously reported that the number of neutrophils and the amount of neutrophil elastase in BAL played important roles in the acute lung injury induced by bleomycin, and that both were decreased by treatment with erythromycin.7 In this study, we examined the inhibitory effects of 14-MRMLs including erythromycin, clarithromycin, and roxithromycin on bleomycin-induced pulmonary fibrosis following acute lung injury. We further investigated the role of adhesion molecules in neutrophil adhesion to endothelial cells, subsequent migration of neutrophils into lung tissue and progression of pulmonary fibrosis, and elucidated the mechanisms by which 14-MRMLs exhibit anti-inflammatory effects and oppose the fibrosis that follows inflammation in bleomycin-challenged mice. Materials and Methods Animals Male, 7-week-old ICR mice (Nippon CLEA; Tokyo, Japan), weighing 30 g each on average, were randomly classified into groups. *From the Fourth Department of Internal Medicine (Drs. Li, Azuma, Usuki, Matsuda, Aoyama, and Kudoh), Nippon Medical School, Tokyo; and Institute of Basic Medical Sciences (Dr. Takahashi), University of Tsukuba, Ibaragi, Japan. Manuscript received September 4, 2001; revision accepted April 30, 2002. Correspondence to: Arata Azuma, MD, PhD, Fourth Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, Japan; e-mail: [email protected] 2138
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 Industries; Tokyo, Japan]. Solutions, 0.3 mL per mouse, were orally administered by force with a microtube daily to ICR mice. Schedule and Process of Evaluation of Late-Phase Fibrosis The bleomysin-Untreated Groups included the NS-treated group (group 1), and 14-MRML alone-treated groups (erythromycin, clarithromycin, roxithromycin) [group 2]. The bleomycin-Treated Groups included the 14-MRMLuntreated group (bleomycin alone) [group 3]; 14-MRMLpretreated groups (day ⫺ 3 to day 13) [bleomycin plus pretreatment erythromycin, bleomycin plus pretreatment clarithromycin, and bleomycin plus pretreatment roxithromycin] (group 4); and 14-MRMLs-posttreated groups (day 3 to day 19) [bleomycin plus posttreatment erythromycin, bleomycin plus posttreatment clarithromycin, and bleomycin plus posttreatment roxithromycin] (group 5). NS was administered IV to bleomycin-untreated mice (day 0). Bleomycin was administered IV to bleomycin-treated mice (day 0). AG was orally administered every day to mice of groups 1 and 3 (day ⫺ 3 to day 13). 14-MRML was orally administered daily to mice of groups 2 and 4 (day ⫺ 3 to day 13) and mice of group 5 (day 3 to day 19). Mice in all groups were killed under ether anesthesia at day 28 after bleomycin or NS injection. Fibrotic foci were assessed histologically in left lung tissues, Ashcroft scores were compared among the groups, and hydroxyproline content in right lung tissues was chemically analyzed. Schedule and Evaluation of Early-Phase Inflammation NS was administered IV to NS-treated group mice (day 0). Bleomycin was administered IV to bleomycin-treated group mice (day 0). AG was orally administered daily to mice of the NS-alone and bleomycin-alone-treated groups from day ⫺ 3 until the time of death. 14-MRMLs were administered daily to the 14-MRMLpretreated groups from day 3 until the time of death. Mice of the NS-alone and bleomycin-alone groups were killed under ether anesthesia at 0, 6 h, or 12 h, or 1, 2, 3, 5, 6, 7, 9, 11, or 13 days after bleomycin or NS injection. All groups were examined for cell population in BALF 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 by reverse transcriptase (RT)-polymerase chain reaction (PCR) at 0 to 13 days after bleomycin or NS injection. Mice of the 14-MRML-pretreated groups were killed under ether anesthesia at 1 day and 9 days after bleomycin injection and underwent the same experiment. We repeated the same experiment three times in early-phase evaluation, and examined the cell population in BALF. The total number of mice was 10 in each group for each time point. RT-PCR examination was repeated three times. Histologic Analysis For histologic examination, we added 0.5 mL of 10% formalin with 10 cm H2O of pressure to the left lobe of every mouse; 10% formalin-fixed lung tissues were embedded in paraffin. Paraffin Laboratory and Animal Investigations
sections were stained with either hematoxylin-eosin or Masson trichrome stain, and systematically scanned with a light microscope using a 20 ⫻ 10 objective. We compared severities of interstitial fibrosis among the groups by the score of Ashcroft et al.13 Hydroxyproline Measurement The right lung was freeze-dried for 3 days using a Tray Dryer (FTS Systems; Stoneridge, NY) as soon as possible after the animals were killed. The total collagen content of the right lung was determined by hydroxyproline assay.14 After acid hydrolysis of the right lung with 12 N HCl at 100°C for 20 h in a sealed glass tube (Iwaki; Tokyo, Japan), hydroxyproline content was determined by high-performance liquid chromatography.
Results 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 tissue (data not shown).
Analysis of BALF BALF was obtained by injection of 1 mL of saline solution (three times; total, 3 mL) followed by gentle aspiration of the fluid from the right and left lungs after securing the intratracheal catheter within a main bronchus. With this catheter, recovery ratios of lavage fluids ranged from 65 to 75% and did not significantly differ among the groups. Total cell numbers in BALF were counted with a hemocytometer. For differential counts of leukocytes in BALF, cytospin (Labsystems; Tokyo, Japan) smear slides were prepared. The smears were stained with Giemsa solution (Merck; Tokyo, Japan). Differential cell counts were performed on 200 cells per smear.
Hydroxyproline Content in Lung Tissue: The concentration of hydroxyproline at day 28 after bleomycin injection was significantly higher in the bleomycin-alone group than in the NS-alone group. Of the groups pretreated with 14-MRML, hydroxyproline was significantly reduced only then in the bleomycin-alone group. Hydroxyproline was not significantly attenuated in the groups posttreated with 14-MRMLs. Comparison of hydroxyproline revealed a significant difference between pretreatment and posttreatment with 14-MRMLs (Fig 3).
Quantitative Analysis of Adhesion Molecule mRNA by RT-PCR Total RNA was extracted from each specimen of lung tissue using ISOGEN (Nippon GENE; Tokyo, Japan). Single-strand complementary DNA (cDNA) was synthesized using RT (Gibco BRL; Rockville, MD) and oligo (dT)12–18 primer. To measure gene expression, we performed the PCR method, with cDNA amplified using a TaKaRa PCR Thermal Cycler 480 and TaKaRa Ex Taq (Takara Shuzo; Otsu, Japan). For amplification of the desired cDNA, the following gene-specific primers were used15–18: E-selectin, sense (EL-11) 5⬘-gattggacactcaatggatc-3⬘, antisense (EL-9) 5⬘-cctagacgttgtaagaaggc-3⬘; P-selectin, sense 5⬘-acgagctggacggacccg-3⬘, antisense 5⬘-ggctggcactcaaatttacag-3⬘; ICAM-1, sense 5⬘-tcggaggatcacaaacgaagc3⬘, antisense 5⬘-aacataagaggctgccatcacg-3⬘; VCAM-1, sense (VC-14) 5⬘-aagcagagacttgaaatgcc-3⬘, antisense (VC-13) 5⬘-cccttgaacagatcaatctcc-3⬘. The PCR products underwent electrophoresis on 2% agarose gels, stained with ethidium bromide, and observed with ultraviolet transillumination. Intensity analysis of the bands was performed with Adobe Photoshop 5.0 (Adobe Systems; Tokyo, Japan), and expression of adhesion molecule messenger RNA was indicated by the number of white pixel areas. Glucose-6-phosphate dehydrogenase (G6PD) was measured as an internal control.19 Statistical Analysis Differences between two groups (grade of fibrosis, hydroxyproline, leukocyte counts) were tested for significance using the Mann-Whitney U test. Repeated-measures analysis of variance was used to evaluate differences in leukocyte counts among groups over time. www.chestjournal.org
Time Course of Changes in Cell Number in BALF After Treatment With Bleomycin Total cell number in BALF peaked at 6 to 13 days after bleomycin injection (Fig 4, top, A, a). The
Figure 1. Histopathologic assessment of late-phase pulmonary fibrosis in bleomycin-challenged mice; comparison of Ashcroft score. Significantly different from bleomycin alonetreated group by the Mann-Whitney U test, **p ⬍ 0.01, *p ⬍ 0.05. Significantly different from 14-MRML-pretreated groups by the Mann-Whitney U test, 夞p ⬉ 0.05. Values are means; bars ⫽ SD, n ⫽ 10. N ⫽ NS-treated group; B ⫽ bleomycin-alone-treated group; BE ⫽ bleomycin- and erythromycin-treated group; BC ⫽ bleomycin- and clarithromycintreated group; BR ⫽ bleomycin and roxithromycin-treated group. CHEST / 122 / 6 / DECEMBER, 2002
2139
Figure 3. Comparison of hydroxyproline contents. Significantly different from bleomycin alone-treated group by the MannWhitney U test, **p ⬍ 0.01; significantly different from 14MRML-pretreated groups by the Mann-Whitney U test, 多p ⬍ 0.01. Values are means; bars ⫽ SD, n ⫽ 10. HOP ⫽ hydroxyproline; DW ⫽ dry lung weight; see Figure 1 legend for expansion of abbreviations.
Effects of 14-MRMLs on Leukocyte Number in BALF 14-MRMLs significantly decreased the number of neutrophils in BALF at 1 day and 9 days after injection of bleomycin (Fig 4, bottom, B, a). The increase in number of macrophages in BALF at 9 days after bleomycin injection was significantly attenuated by erythromycin and roxithromycin, and slightly attenuated by clarithromycin (Fig 4, bottom, B, b). The number of lymphocytes in BALF at 9 days after bleomycin injection was significantly attenuated by erythromycin and clarithromycin, and slightly attenuated by roxithromycin (Fig 4, bottom, B, c). Time Course of Changes in mRNA Expression of Adhesion Molecules in Lung Tissue After Treatment With Bleomycin Figure 2. Pathologic features of lung tissues (hematoxylin-eosin, original ⫻ 200) at 28 days after treatment with bleomycin alone or in combination with clarithromycin. Top, a: treated with NS alone. Center, b: treated with bleomycin alone. Bottom, c: treated with bleomycin and pretreated with clarithromycin.
number of neutrophils in BALF peaked twice, from 12 h to 2 days and from 5 to 11 days after bleomycin injection, and returned to control level at 13 days after bleomycin injection (Fig 4, top, A, b). The number of macrophages in BALF was maximal at 6 to 11 days after bleomycin injection (Fig 4, top A, c). The number of lymphocytes in BALF was significantly increased at 9 to 13 days after bleomycin injection (Fig 4, top, A, d). 2140
E-selectin (Fig 5, top, A, a), P-selectin (top, A, b), and ICAM-1 (top, A, c) mRNAs were induced at 6 to 12 h, decreased to control levels at 1 day, and were induced for the second time 2 to 13 days after bleomycin injection. VCAM-1 mRNA (Fig 5, top, A, d) was induced 2 to 13 days after bleomycin injection. Inhibitory Effect of 14-MRML on mRNA Expression of VCAM-1 and ICAM-1 14-MRMLs clearly inhibited the induction of VCAM-1 mRNA (Fig 5, bottom, B, d) and tended to attenuate ICAM-1 mRNA expression (Fig 5, bottom, B, c) at day 9 after bleomycin. However, 14-MRMLs markedly inhibit the induction of neither E-selectin (bottom, B, a) nor P-selectin mRNAs (Fig 5, bottom, B, b). Laboratory and Animal Investigations
Figure 4. Top, A: Time course of changes in cell numbers in BALF (upper left, a: total cell number; upper right, b: No. of neutrophils; lower left, c: No. of macrophages; lower right, d: No. of lymphocytes). Significantly different from NS-treated group at each time point by the Mann-Whitney U test, 多p ⬉ 0.0004, 夞p ⬍ 0.01. Significantly different from the first time point (0 h) after bleomycin (BLM) administration by repeated-measures analysis of variance, ***p ⬍ 0.0001, **p ⬍ 0.01, *p ⬍ 0.05. Values are means; bars ⫽ SE, n ⫽ 10. Bottom, B: Effects of 14-MRMLs on leukocyte number in BALF on day 1 and day 9 after injection of bleomycin (top, a: No. of neutrophils in BALF on days 1 and 9 after injection of bleomycin; bottom left, b: No. of macrophages in BALF on day 9 after injection of bleomycin; bottom right, c: No. of lymphocytes in BALF on day 9 after injection of bleomycin). Significantly different from bleomycin alone-treated group by the Mann-Whitney U test, **p ⬍ 0.01, *p ⬍ 0.05. Values are means; bars ⫽ SE, n ⫽ 10. See Figure 1 for expansion of abbreviations. www.chestjournal.org
CHEST / 122 / 6 / DECEMBER, 2002
2141
Figure 5. Top, A: Time course of E-selectin (upper left, a), P-selectin (upper right, b), ICAM-1 (lower left, c), and VCAM-1 (lower left, d) mRNA inductions in lung tissue after bleomycin administration. Bottom, B: Effects of 14-MRMLs on expression of adhesion molecules such as E-selectin (upper left, a), P-selectin (upper right, b), ICAM-1 (lower left, c), and VCAM-1 (lower right, d) mRNA in lung tissue on day 9 after bleomycin administration. These photographs show typical results. Each density of PCR products was measured using Adobe Photoshop 5.0; G6PD was measured as an internal control. The ratio of each adhesion molecule against G6PD is shown by histogram. bp ⫽ base pair; see Figure 1 legend for expansion of abbreviations.
2142
Laboratory and Animal Investigations
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 with release of proteases and free-radical formation.4,7 Inhibition of production of injurious substances (eg, neutrophil elastase by ONO5046),5 other proteases by truncated secretary leukocyte protease inhibitor,9 and by ␣-1 protease inhibitor10 is a promising method for prevention of pulmonary fibrosis. The initial phase of neutrophil migration prior to activation in lung parenchyma is an important point of intervention in strategies that are effective in preventing tissue injury and subsequent fibrosis. We previously reported that E-selectin played an essential role in neutrophil adhesion to vascular endothelial cells and subsequent lung fibrosis.8 We initially investigated bleomycin-induced histopathologic changes in lung tissues. Bleomycininduced pulmonary fibrosis at day 28 was significantly inhibited by pretreatment with 14-MRMLs after bleomycin administration (Figs 1–3). The effectiveness of pretreatment with 14-MRMLs (beginning 3 days before bleomycin injection) is significantly higher than that of posttreatment with 14-MRMLs (beginning 3 days after bleomycin injection) [Figs 1–3]. The difference of potency for inhibitory effects between pretreatment and posttreatment with 14-MRML may have depended on the difference of their inhibitory effects of neutrophil recruitment in lung tissue, especially to the first peak (from 12 h to 2 days after bleomycin injection) of neutrophil migration. Development of pulmonary fibrosis is thought to include two phases: an acute inflammatory phase and a sequential fibrotic phase.22 To determine the mechanisms of the prophylactic effect of 14-MRMLs on bleomycininduced pulmonary fibrosis in mice, we further investigated the mechanisms of the early inflammatory (ie, up-stream) phase. We found two peaks of neutrophil concentration in BALF. The two peaks of neutrophil recruitment paralleled but were slightly delayed compared with induction of E-selectin, P-selectin, and ICAM-1 mRNAs. The second peak and the induction of VCAM-1 mRNA were also parallel with slight delay (Fig 4, top, A, b, and Fig 5, top, A). ␣4/1 integrin, acting as a ligand for VCAM-1, is www.chestjournal.org
believed to contribute to migration of macrophages, eosinophils, and lymphocytes into inflamed tissues.18,23 Some investigators have reported that neutrophil adhesion to vascular endothelial cells depends principally on ␣9/1-VCAM-1 interaction.24 In the setting of leukocyte-endothelial interactions, there are diverse mechanisms that modulate leukocyte integrin avidity. Crosslinking of the Eselectin and P-selectin counter-structures, CD15 (sialyl Lex), or its sialylated form on neutrophils caused activation.25 Therefore, we considered the possibility that neutrophils bound selectins in the initial phase, and that simultaneously ␣9/1 integrin was activated by selectins, with tight binding of neutrophils to VCAM-1, which might have induced the second neutrophil peak. We also considered the possibility that 14-MRMLs attenuated induction of VCAM-1 on vascular endothelial cells binding to neutrophils due to inhibition of the second neutrophil peak. We also investigated the inhibitory effects of 14MRMLs on bleomycin-induced lung injury to evaluate the early inflammatory phase in the same experimental model in mice. 14-MRMLs clearly inhibited the expression of VCAM-1 mRNA (Fig 5, bottom, B, d), and significantly inhibited the two neutrophil peaks after injection (Fig 4, bottom, B, a) in bleomycin-induced lung injury. We therefore considered the possibility that 14-MRMLs attenuated induction of VCAM-1 on vascular endothelial cells binding to neutrophils due to inhibition of the second neutrophil peak. 14-MRMLs tended to attenuate the expression of ICAM-1 mRNA (Fig 5, bottom, B, c). We therefore considered the possibility that attenuation by 14MRMLs of the expression of ICAM-1 mRNA was related to inhibition of the two peaks of neutrophil recruitment. Lung injury may have depended on macrophage antigen 1 and lymphocyte functionassociated antigen 1 integrin on neutrophils acting as ligands for ICAM-1 on vascular endothelial cells. This might be one mechanism of inhibition of neutrophil migration into the airspace by 14-MRMLs. The number of macrophages changed in parallel with slight delay for the induction of VCAM-1 mRNA in the present study (Fig 4, top, A, c, and Fig 5, top, A, d). VCAM-1 mRNA induction was mainly associated with movement of macrophages into the airspace. We considered the possibility that 14-MRMLs inhibited binding of ␣4/1 on macrophages to VCAM-1 on vascular endothelial cells due to inhibition of movement of macrophages of ligand expression, which requires further examination. These findings suggested that attenuation of inflammatory cell migration by 14-MRMLs, especially of neutrophils and macrophages, resulted in inhibiCHEST / 122 / 6 / DECEMBER, 2002
2143
tion of lung injury and subsequent fibrosis. Inhibition by 14-MRMLs of VCAM-1 mRNA expression might be one mechanism of inhibition of neutrophil and macrophage migration into the airspace. It has been reported that erythromycin inhibits chemokines including interleukin 8, which acts as a neutrophil chemoattractant, resulting in attenuation of neutrophil migration into inflamed tissues, and inhibits the expression of adhesion molecules.26 –30 In our study, 14-MRMLs may have inhibited not only VCAM-1 induction, but also that of interleukin 8 and other agents, especially at the first peak of increase in neutrophil number. It has been reported that matrix metalloproteinase (MMP) is associated with the process of pulmonary fibrosis in human and animal models, and in particular that MMP-9 correlated with neutrophil recruitment and MMP-2 with development of the fibrotic phase.31–34 Furthermore, suppression of MMP-9 activity by erythromycin is also an anti-inflammatory mechanism that inhibits the migration of inflammatory cells into inflamed tissues.35 We therefore considered the possibility that erythromycin inhibits MMPs, which would be an important mechanism of antifibrotic activity. Although neutrophils from beige mice exhibited a selective defect of release of neutrophil elastase in response to several stimuli, bleomycin-induced lung fibrosis was more prominent than that in littermates, without impairing their ability to produce superoxide anion and H2O2 in response to bleomycin.36 Neutrophil elastase promotes tissue injury,7 but other agents such as oxygen radicals might be major components in the tissue injury that occurs in response to bleomycin. No treatments that clearly improve the prognosis of IPF exist at present. New strategies for such treatment have, however, been discussed. One such strategy is to inhibit leukocyte accumulation in lung tissue.37 Antibody to CD11, CD18, or ␣9 integrin may prove useful by inhibiting neutrophil migration into lung tissue via blocking of cell-to-cell adhesion and migration.38 14-MRMLs can be expected to prevent leukocyte-mediated lung injury by mechanisms of action similar to those of antibodies to endothelial adhesion molecules. In addition, the safety and cost of 14-MRML therapy are superior to those of antibody therapy.
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 2144
and macrophage migration into the airspace by 14-MRMLs, and subsequent fibrotic effects of 14MRMLs. 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 for acute exacerbation of interstitial pneumonia, acute lung injury, and prophylaxis of progression of IPF.
References 1 Adamson IYR, Bowden DH. The pathogenesis of bleomycininduced pulmonary fibrosis in mice. Am J Pathol 1974; 77:185–198 2 Aso Y, Yoneda K, Kikkawa Y. Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice. Lab Invest 1976; 35:558 –568 3 Weidner WJ, Quam DA, McClure DE, et al. Effect of acute administration of bleomycin on lung fluid balance in sheep. Exp Lung Res 1995; 21:617– 630 4 Henson PM, Johnston RB Jr. Tissue injury in inflammation, oxidants, proteinase and cationic proteins. J Clin Invest 1987; 79:669 – 674 5 Sakamaki F, Ishizaka A, Urano T, et al. Effect of specific neutrophil elastase inhibitor, ONO-5046, on endotoxininduced acute lung injury. Am J Respir Crit Care Med 1996; 153:391–397 6 Botha AJ, Moore FA, Moore EE, et al. Sequential systemic platelet-activating factor and interleukin 8 primes neutrophils in patients with trauma at risk of multiple organ failure. Br J Surg 1996; 83:1407–1412 7 Azuma A, Furuta T, Enomoto T, et al. Preventive effect of erythromycin on experimental bleomycin-induced acute lung injury in rats. Thorax 1998; 53:186 –189 8 Azuma A, Takahashi S, Nose M, et al. Role of E-selectin in bleomycin induced lung fibrosis in mice. Thorax 2000; 55: 147–152 9 Mitsuhashi H, Asano S, Nonaka T, et al. Administration of truncated secretory leukoprotease inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters. Am J Respir Crit Care Med 1996; 153:369 –374 10 Nagai A, Aoshiba K, Ishihara Y, et al. Administration of ␣1-proteinase inhibitor ameliorates bleomycin-induced pulmonary fibrosis in hamsters. Am Rev Respir Dis 1992; 145:651– 656 11 Oda H, Kadota J, Kohno S, et al. Erythromycin inhibits neutrophil chemotaxis in bronchoalveoli of diffuse panbronchiolitis. Chest 1994; 106:1116 –1123 12 Kudoh S, Azuma A, Yamamoto M, et al. Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. Am J Respir Crit Care Med 1998; 157:1829 –1832 13 Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988; 41:467– 470 14 Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961; 93:440 – 447 15 Araki M, Araki K, Miyazaki Y, et al. E-selectin binding promotes neutrophil activation in vivo in E-selectin transgenic mice. Biochem Biophys Res Commun 1996; 224:825– 830 16 Sanders WE, Wilson RW, Ballantyne CM, et al. Molecular Laboratory and Animal Investigations
17
18 19 20 21 22 23 24
25 26 27
28
cloning and analysis of in vivo expression of murine Pselectin. Blood 1992; 80:795– 800 Oran A, Marshall JS, Kondo S, et al. Cyclosporin inhibits intercellular adhesion molecule-1 expression and reduces mast cell numbers in the asebia mouse model of chronic skin inflammation. Br J Dermatol 1997; 136:519 –526 Araki M, Araki K, Vassalli P. Cloning and sequencing of mouse VCAM-1 cDNA. Gene 1993; 126:261–264 Manos P, Nakayama R, Holten D. Regulation of glucose-6phosphate dehydrogenase synthesis and mRNA abundance in cultured rat hepatocytes. Biochem J 1991; 276:245–250 Henson PM. Mechanisms of cellular injury in interstitial lung disease. Chest 1986; 89:108 –115 Hunninghake GW, Gadek JE, Lawley TJ, et al. Mechanisms of neutrophil accumulation in the lungs of patients with idiopathic pulmonary fibrosis. J Clin Invest 1981; 68:259 –269 Ward PA, Hunninghake GW. Lung inflammation and fibrosis. Am J Respir Crit Care Med 1998; 157:123–129 Beekhuizen H, van Furth R. Monocyte adherence to human vascular endothelium. J Leukoc Biol 1993; 54:363–378 Taooka Y, Chen J, Yednock T, et al. The integrin ␣91 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1. J Cell Biol 1999; 145:413– 420 Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994; 84:2068 –2101 Kadota J, Sakito O, Kohno S, et al. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am Rev Respir Dis 1993; 147:153–159 Sakito O, Kadota J, Kohno S, et al. Interleukin 1, tumor necrosis factor ␣, and interleukin 8 in bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis: a potential mechanism of macrolide therapy. Respiration 1996; 63:42– 48 Sugiyama Y, Yanagisawa K, Tominaga SI, et al. Effects of long-term administration of erythromycin on cytokine production in rat alveolar macrophages. Eur Respir J 1999; 14:1113–1116
www.chestjournal.org
29 Fujii T, Kadota JI, Morikawa T, et al. Inhibitory effect of erythromycin on interleukin 8 production by 1, 25-dihydroxyvitamin D3-stimulated THP-1 cells. Antimicrob Agents Chemother 1996; 40:1548 –1551 30 Oishi K, Sonoda F, Kobayashi S, et al. Role of interleukin-8 (IL-8) and an inhibitory effect of erythromycin on IL-8 release in the airways of patients with chronic airway diseases. Infect Immun 1994; 62:4145– 4152 31 Yaguchi T, Fukuda Y, Ishizaki M, et al. Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis. Pathol Int 1998; 48:954 –963 32 Corbel M, Theret N, Caulet MS, et al. Repeated endotoxin exposure induces interstitial fibrosis associated with enhanced gelatinase (MMP-2 and MMP-9) activity. Inflamm Res 2001; 50:129 –135 33 Corbel M, Caulet MS, Germain N, et al. Inhibition of bleomycin-induced pulmonary fibrosis in mice by the matrix metalloproteinase inhibitor batimastat. J Pathol 2001; 193: 535–545 34 Fukuda Y, Ishizaki M, Kudoh S, et al. Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinases-2 in interstitial lung diseases. Lab Invest 1998; 78:687– 698 35 Hashimoto N, Kawabe T, Hara T, et al. Effect of erythromycin on matrix metalloproteinase-9 and cell migration. J Lab Clin Med 2001; 137:176 –183 36 Phan SH, Schrier D, McGarry B, et al. Effect of the beige mutation on bleomycin-induced pulmonary fibrosis in mice. Am Rev Respir Dis 1983; 127:456 – 459 37 Hunninghake GW, Kalica AR. Approaches to the treatment of pulmonary fibrosis. Am J Respir Crit Care Med 1995; 151:915–918 38 Piguet PF, Rosen H, Vesin C, et al. Effective treatment of the pulmonary fibrosis elicited in mice by bleomycin or silica with anti-CD-11 antibodies. Am Rev Respir Dis 1993; 147: 435– 441
CHEST / 122 / 6 / DECEMBER, 2002
2145