Original Article Erythromycin Ameliorates Bleomycin ...

Egyptian Journal of Anatomy, Jan. 2015; 38(1):13-27

Erythromycin Ameliorates Bleomycin-induced Suppressing Alveolar Macrophages Activity

Lung

Injury

by

Original Article Eyad M.T. Ali* and Ahmed A.M. Abdel Hamid

*Department of Anatomy and Embryology, Mansoura University & Department of Histology and Cell Biology, Mansoura University

ABSTRACT

Introduction: Idiopathic pulmonary fibrosis is a progressive and ultimately fatal lung disease of unknown aetiology. Human pulmonary fibrosis is characterized by alveolar epithelial cell injury, accumulation of fibroblasts and the deposition of extra-cellular matrix. Bleomycin (BLM) is an effective anti-tumor drug but must be used cautiously because of its pulmonary toxicity. BLM-induced lung injury mimics the chronic aspect of pulmonary fibrosis. Moreover, there is growing evidence that macrophages as well as fibrocytes are largely involved in disease progression by mediating fibroblast activation. There is no effective treatment for fibrosis in which excessive deposition of extra-cellular matrix such as collagen, resulting in significant morbidity and mortality. It is suggested that 14-membered ring macrolides have anti-inflammatory effects and decrease neutrophil infiltration into the airways in chronic respiratory tract diseases and may be therapeutic agents for pulmonary fibrosis. Erythromycin (EM) significantly suppressed alveolar macrophages and also lessened the collagen deposition via inhibition of the growth of fibroblasts and therefore might be useful for treatment of BLM-induced pulmonary fibrosis. Aim of the work: To investigate the anti-inflammatory and the anti-fibrotic effects of EM in an experimental model of BLM-induced lung injury in adult male albino rats and also, to find out the relation between EM and alveolar macrophage in ameliorating BLM- induced lung fibrosis. Materials and methods: Twenty adult male albino rats were used in this study. The animals were divided into three groups: Control, BLM-treated group (20 mg/kg twice weekly for 4 weeks) and BLM+EMtreated group (received EM in a dose of 12 mg/kg orally for 2 weeks before and concomitantly with BLM treatment for another 4 weeks). Rats were sacrificed after 4 weeks and both lungs were dissected. Paraffin sections were prepared and stained with Hx&E, Masson's trichrome stains for histopathological examination and anti-CD68 for detection of the alveolar macrophages. The number of positively stained cells was morphometrically estimated and statistically analyzed. Results: In BLM-treated group, the inter-alveolar septa were thick with heavy deposition of mononuclear cell inflammatory infiltrate. Moreover, collagen deposition was also observed. In EM+BLMtreated group, the degree of histopathological changes was reduced in comparison with the BLM-treated group. The number of anti-CD68 positive stained macrophages was significantly increased in the BLMtreated group in comparison with the other two groups. Key Words: erythromycin; bleomycin; alveolar macrophages; CD68; lung fibrosis. Corresponding Author: Eyad Mohamed Tolba Ali, e-mail: [email protected] Mobile: 01003060258

INTRODUCTION of usual interstitial pneumonia (ATS, 2000; Kayhan et al., 2013). The natural history of IPF is unknown and the onset of symptoms is gradual, starting usually with non-productive cough and exertional dyspnea. With involvement of larger areas of the lung, severe dyspnea at rest and signs of right heart failure develop (ATS, 2002).

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive and ultimately fatal lung disease of unknown aetiology. Its prognosis is poor and the outcome even worse than in many malignant diseases. Moreover, it is considered as one of the most frequent interstitial lung diseases and is characterized by the histological pattern

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Erythromycin Ameliorates Bleomycin-induced Lung Injury...

Although, the pathogenesis of pulmonary fibrosis and interstitial pneumonia 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 (Li et al., 2002). Human pulmonary fibrosis is characterized by alveolar epithelial cell injury, areas of type II cell hyperplasia, accumulation of fibroblasts & myofibroblasts, and the deposition of extracellular matrix proteins (Moore & Hogaboa, 2008).

There are no effective treatment for fibrosis in which excessive deposition of extra-cellular matrix such as collagen, resulting in significant morbidity and mortality (Mouratis & Aidinis, 2011; Yamaguchi & Feghali-Bostwick, 2013). However, many substances were tried to reduce or prevent fibrosis. One of them is Endostatin which is a natural proteolytic fragment of collagen XVIII. The anti-fibrotic capacity was accompanied by reduced cell apoptosis and lower levels of lysyl oxidase (Yamaguchi & FeghaliBostwick, 2013). Also, angiotensin II antagonist (losartan) causes a reduction in the measures of acute lung injury. Losartan reduces renal and cardiac fibrosis in mice, but fails to significantly ameliorate bleomycin-induced pulmonary fibrosis (Keogh et al., 2005).

Bleomycin (BLM) is an effective anti-tumor drug but must be used cautiously because of its pulmonary toxicity (Culine et al., 2008). The BLM animal model remains the best available experimental tool for studying disease pathogenesis and testing of novel pharmaceutical compounds (Mouratis & Aidinis, 2011 ; Meng et al., 2013). In the early injury of the animal model, BLM promoted the development of inflammation, leading to severe pulmonary fibrosis (Shi et al., 2014). This model can be successfully established by intravenous injection or intratracheal instillation of BLM. BLM-induced lung injury mimics the chronic aspect of pulmonary fibrosis, as well as other characteristics including the presence of hyperplasic alveolar epithelial cells. Epithelial-to-mesenchymal transition seems to be a major contributor to the lung fibroblast population. Moreover, there is growing evidence that macrophages, as well as fibrocytes, are largely involved in disease progression by mediating fibroblast and myofibroblast activation (Meng et al., 2013).

In addition, both pentoxyfylline and prednisolone are known to effectively inhibit pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-alpha). However, a previous study proved pentoxyfylline to be more effective in inhibition of neutrophils and formation of reactive oxygen species in lung interstitium (Entzian et al., 1997). Furthermore , leflunomide (an immunomodulatory drug) reduces oxidative stress factors, alveolar inflammation and attenuates lung injury and fibrosis (Kayhan et al., 2013). In addition, spironolactone (Mineralocorticoid) could partially inhibit BLM-induced circulating monocytes expansion, and reduce activation of mononuclear phagocytes in alveoli (Ji et al., 2013). Macrolides have been reported to show various pharmacological activities. EM703, a new derivative of erythromycin (EM), improved BLM-induced pulmonary fibrosis in mice (Ikeda et al., 2008). BLM-induced lung fibrosis and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703 which also inhibited fibroblast proliferation and the collagen production in the lung (Li et al., 2006).

Bleomycin can directly stimulate alveolar macrophage secretion of fibroblast growth factors and monocyte chemotactic factors. This indicates that alveolar macrophages have specific, saturable, and reversible high-and lowaffinity binding sites (Denholm & Phan, 1990). However, the dose of BLM was not directly toxic to the alveolar macrophage (Lower et al., 1988). On the other hand, Mast cells are found in large numbers in lungs of patients with pulmonary fibrosis. However, the functions of Mast cells in lung fibrosis remain largely unknown (Reber et al., 2014). Pulmonary fibrosis eventually could occur due to fibroblast proliferation and collagen production in the lung and begins with alveolar inflammatory edema (Ghatak et al., 2013).

The pre-treatment with 14-membered ring macrolides (clarithromycin and roxithromycin) suppressed inflammatory cell infiltration and interstitial lung edema. These inhibitory effects were associated with a decreased number of apoptotic cells in the lungs. Pre-treatment with azithromycin (15-membered ring macrolide) was much less effective, and josamycin (16-membered ring macrolide) showed no

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Eyad M.T. Ali and Ahmed A.M. Abdel Hamid

Experimental design:

inhibitory effects. Therefore, it is suggested that 14-membered ring macrolides have antiinflammatory effects and decrease neutrophil infiltration into the airways in chronic lower respiratory tract diseases more than 15- and 16-membered ring macrolides and may be therapeutic agents for acute lung injury and pulmonary fibrosis (Kawashima et al., 2002).

The animals were divided into three groups: Control (4 animals): received no treatment. BLM-treated group (8 animals): received BLM in a dose of 20 mg/kg twice weekly for 4 weeks via the intravenous injection.

Erythromycin is claimed to reduce collagen production and the mRNA levels of alpha-1 collagen in a dose-dependent manner in the normal fibroblasts (Ikeda et al., 2008). The EM significantly suppressed TNF-alpha and plateletderived growth factor (PDGF) release by alveolar macrophages and also lessened the collagen deposition (Chen et al., 1997). Co-administration of EM or clarithromycin and imatinib significantly reduced the fibrogenesis via inhibition of the growth of fibroblasts and therefore might be useful for treatment of BLM-induced pulmonary fibrosis (Azuma et al., 2007).

BLM+EM-treated group (8 animals): received EM in a dose of 12 mg/kg in 10% ethanol administrated orally for 2 weeks before and concomitantly with BLM treatment (same dose as in BLM-treated group) for another 4 weeks. Specimens’ collection and staining: Rats were sacrificed after 4 weeks under anesthesia (sodium pentobarbital, 50 mg/kg of body weight, intraperitoneal injection) and both lungs were dissected, immediately fixed by immersion in 10% buffered formalin and embedded in paraffin blocks. 5-µm thick sections were prepared and stained with Hx&E, Masson's trichrome stains for histopathological examination and anti-CD68 for detection of the alveolar macrophages. The number of positively stained cells was morphometrically estimated and statistically analyzed.

This study was undertaken to investigate the anti-inflammatory and the anti-fibrotic effects of EM in an experimental model of BLM-induced lung injury in adult male albino rats and also, to find out the relation between EM and alveolar macrophage in ameliorating BLM- induced lung fibrosis.

Immunohistochemical staining:

Materials and methods

Immunohistochemical study was performed using an avidin biotin-peroxidase technique for showing alveolar macrophages using CD68 mouse monoclonal antibody (purchased from Novocastra labs, UK, at a dilution of 1:20). This antibody was shown to react selectively with a specific cytoplasmic glycoprotein present in mononuclear phagocytes, microglia, and epidermal Langerhans cells (Elner et al., 1992). Paraffin sections of the lung were incubated with biotinylated antimouse antibody (diluted 1: 200) and the avidin biotinconjugated peroxidase complex (Vector Lab. Inc., USA). The reaction was developed with 0.05% diaminobenzidine (Dakopatts Glostrup, Denmark) as the substrate for peroxidase; finally, the slides were counterstained with Meyer’s hematoxylin (Cattoretti et al., 1993). The cytoplasmic site of the reaction stained brown whereas the nuclei appeared blue. The specificity of the immune reaction was tested by replacing the primary antiserum with phosphate-buffered saline as a negative control (Kiernan, 1999).

Animals: Twenty adult male albino rats weighing 150.9 ± 1.63 gm were used in this study. They were provided by the Center of Animal Housing and Breeding of the Faculty of Pharmacy. The animals were placed in standard plastic cages (2 animals per cage) for 8 weeks under 12/12 hours light/dark cycle with 23-25˚C room temperature. All animals received standard laboratory animal's chow and water ad labitum during the whole period of experiment. The experiment was performed according to the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC: National Academy Press, no. 85-23, revised 1996). All protocols were approved by our local committee of Animal Care and Use Committee.

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Morphometric and statistical analysis:

number of anti-CD68 positively stained alveolar macrophages (Figs.8, 9).

Ten non-overlapping fields from the lung slides from rats of each group stained by antiCD68 were examined to estimate the mean number of alveolar macrophages in high power field (HPF x 400-magnified fields). On the other hand, lung fibrosis was examined by Masson's trichrome staining for collagen deposition in low power field (LPF x 100-magnified fields), and blue area represented collagen deposition. The extent of collagen deposition was measured by image analysis in 3 randomly selected, separate, x 100-magnified fields from each slide, and the percentage of blue area was calculated to compare the differences in collagen deposition among rats in 3 different groups. The image data were analyzed using Image J 1.47 v Software (National Institutes of Health, Bethesda, MD, United States). One-way ANOVA test with Tukey’s HSD test, P ≤ 0.001). Data were expressed as the means ± SD. Differences were regarded as non-significant (P ≥ 0.05), significant (P ≤ 0.05), or highly significant (P ≤ 0.001). All analyses in this study were implemented by SPSS 16.0 software for Windows (Chicago, IL, United States).

The lung of a BLM-treated group showed loss of lung architecture with heavy mono-nuclear inflammatory cell infiltration in the thickened inter-alveolar septa and in the connective tissue surrounding the bronchioles (Fig.10). Many macrophages and cellular debris were demonstrated within the lumen of the bronchioles (Fig.11). The inter-alveolar matrix was markedly increased compressing some of the surrounding alveoli with compensatory dilatation of others (Fig.13). In addition, fibrocytes were embedded in heavy collagen deposition (Fig.14). The interalveolar septa were thickened and contained congested blood vessels, edema and inflammatory cells infiltration (Figs.15, 16). In severe cases, intra-alveolar hemorrhage was demarcated (Fig.12). Histological examination of sections in the lung of the BLM-treated group stained with Masson's trichrome revealed increased collagen deposition in the connective tissue surrounding the bronchioles (Figs. 17, 18). Additionally, heavy peri-vascular collagen deposition extended into the interstitium between the alveoli causing fibrosing alveolitis (Figs. 19, 20). Immunohistochemical stain with anti-CD68 showed increased number of positively stained alveolar macrophages (Figs. 21, 22).

Results Histopathological changes of the lungs:

Sections in the lung of a BLM+EMtreated group showed apparently normal lung architecture with intact alveoli separated by thin inter-alveolar septa and lined by pneumocytes type I. Pneumocytes type II were noticed at some angles of the alveoli (Figs. 23, 24, 25 ). Moreover, the bronchioles appeared normal with no inflammatory cell infiltration (Fig. 24). The blood vessels were non-congested (Figs. 26, 27) with few extra-vasated leukocytes appeared in the interstitium between the alveoli (Fig.25). Sections in the lung of a BLM+EM-treated group stained with Masson's trichrome showed few collagen fibers in the connective tissue surrounding the bronchioles (Figs. 28, 29). Immunohistochemical stain with anti-CD68 showed few number of positively stained alveolar macrophages (Figs. 30, 31).

Histological examination of the lung of the control group showed normal histological structure and architecture of the lung with patent alveoli, alveolar sacs, alveolar ducts and intact bronchioles (Figs.1, 3). The alveoli appeared patent and separated from each others by thin inter-alveolar septa, their lining epithelium were formed of pneumocytes type I (squamous cells) and type II pneumocytes (cuboidal cells) (Figs.4, 5). The bronchioles were lined with simple columnar epithelium with loose connective tissue in the underlying corium. The peri-bronchiolar connective tissue contained non-congested blood vessels (Figs. 1, 2). Histological examination of sections in the lung of the control group stained with Masson's trichrome revealed minimal fine collagen fibers in the lung parenchyma around the bronchioles and the blood vessels. No collagen fibers were observed in the interstitium between the alveoli (Figs.6, 7). Staining sections in the lung of a control rat with anti-CD68 showed few

Morphometric and statistical results: The number of the anti-CD68 positively stained cells in BLM- treated group was highly

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significant increased compared with the control group. On the other hand, their number in EM + BLM-treated group was highly significant decreased in comparison with the BLM-treated group (Table 1, Fig.32). Moreover, there was a highly significant increase in the mean collagen percentage in BLMtreated group in comparison with the control and a highly significant decrease in this percentage in EM+BLM-treated group as compared with BLMtreated group (Table 2, Fig.33).

Fig. 3: A photomicrograph of a section in the lung of a control rat showing normal lung architecture with patent alveoli (arrows) , alveolar sacs (AS) and alveolar ducts (AD). Hx. & E.; × 100

Fig. 1: A photomicrograph of a section in the lung of a control rat showing normal lung architecture with patent alveoli (AV), intact bronchiole (BR) and noncongested blood vessels (BV). Hx. & E.; × 100

Fig. 4: A photomicrograph of a section in the lung of a control rat showing patent alveoli (A) lined with pneumocytes type I (arrows). Few pneumocytes type II (cuboidal in shape) appears at the angles of some alveoli (arrow heads). The alveoli are separated by thin inter-alveolar septa (thick arrows). Hx. & E.; × 400

Fig. 2: A photomicrograph of a section in the lung of a control rat showing intact bronchiole lined with simple columnar epithelium (arrows). The underlying corium contains few number of connective tissue cells (arrow heads). Non-congested blood vessels are observed in the surrounding connective tissue (thick arrow). Hx. & E.; × 400

Fig. 5: A photomicrograph of a section in the lung of a control rat showing patent alveoli (A) lined with pneumocytes type I (arrows). Few pneumocytes type II (cuboidal in shape) appears at the angles of some alveoli (arrow heads). The alveoli are separated by thin inter-alveolar septa (thick arrows). Hx. & E.; × 1000

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Fig. 9: A photomicrograph of a section in the lung of a control rat showing few number of antiCD68 immunoperoxidase positive stained alveolar macrophages (arrows). Anti-CD68 immunoperoxidase stain; × 400, Inset: × 1000

Fig.6: A photomicrograph of a section in the lung of a control rat showing minimal collagen fibers (arrows) appearing around the bronchiole (B). No collagen fibers are observed in the interstitium between the alveoli (arrow heads). Masson's trichrome; × 100

Fig. 10: A photomicrograph of a section in the lung of a BLM-treated group showing loss of lung architecture with heavy mono-nuclear inflammatory cell infiltration in the thickened inter-alveolar septa (arrows) and in the connective tissue surrounding the bronchioles (arrow heads). Hx. & E.; × 100

Fig. 7: A photomicrograph of a section of the lung of a control rat showing minimal collagen fibers (arrows) appearing around the bronchiole (B). No collagen fibers are observed in the interstitium between the alveoli (arrow heads). Masson's trichrome; × 400

Fig. 11: A photomicrograph of a section in the lung of a BLM-treated group showing heavy mono-nuclear inflammatory cell infiltration in the connective tissue surrounding the bronchioles (arrows). Cellular debris (arrow heads) and many macrophages (thick arrows) are observed within the lumen of the bronchiole. Hx. & E.; × 400

Fig. 8: A photomicrograph of a section in the lung of a control rat showing few number of antiCD68 immunoperoxidase positive stained alveolar macrophages (arrows). Anti-CD68 immunoperoxidase stain; × 100

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Fig. 15: A photomicrograph of a section in the lung of a BLM-treated group showing thickened inter-alveolar septa containing congested blood vessels (arrows), edema (arrow heads) and inflammatory cells (thick arrows). Hx. & E.; × 100

Fig. 12: A photomicrograph of a section in the lung of a BLM-treated group showing intra-alveolar hemorrhage (arrows). Hx. & E.; X100, Inset: × 400.

Fig. 13: A photomicrograph of a section in the lung of a BLM-treated group showing increased inter-alveolar matrix (arrows) compressing the surrounding alveoli (arrow heads) with compensatory dilatation of others (DA). Hx. & E.; × 100

Fig.16: A photomicrograph of a section in the lung of a BLM-treated group showing thickened inter-alveolar septa containing congested blood vessels (arrows), edema (arrow heads) and inflammatory cells (thick arrows). Hx. & E.; × 400

Fig. 17: A photomicrograph of a section in the lung of a BLM-treated group showing increased collagen deposition in the connective tissue surrounding the bronchiole (arrows), blood vessel (thick arrows) Masson's trichrome; × 100

Fig. 14: A photomicrograph of a section in the lung of a BLM-treated group showing increased inter-alveolar matrix. Fibrocytes (arrow heads) surrounding heavy collagen deposition are observed (arrows). Hx. & E.; × 400 19


Fig.18: A photomicrograph of a section in the lung of a BLM-treated group showing heavy collagen deposition in the corium of the bronchiole (arrows) and also in the surrounding connective tissue (thick arrows). Masson's trichrome; × 400

Fig. 21: A photomicrograph of a section in the lung of a BLM-treated group showing increased number of antiCD68 immunoperoxidase positive stained alveolar macrophages (arrows). Anti-CD68 immunoperoxidase stain; × 100

Fig. 19: A photomicrograph of a section in the lung of a BLM-treated group showing heavy peri-vascular collagen deposition (arrows) that extends in the interstitium between the alveoli (thick arrows). Masson's trichrome; × 100

Fig. 22: A photomicrograph of a section in the lung of a BLM-treated group showing increased number of antiCD68 immunoperoxidase positive stained alveolar macrophages (arrows). Anti-CD68 immunoperoxidase stain; × 400, Inset: × 1000

Fig. 20: A photomicrograph of a section in the lung of a BLM-treated group showing heavy collagen deposition in the interstitium between the alveoli causing fibrosing alveolitis (arrows). Masson's trichrome; × 400

Fig. 23: A photomicrograph of a section in the lung of a BLM+EM- treated group showing apparently normal lung architecture. Hx. & E.; × 100 20


Fig. 24: A photomicrograph of a section in the lung of a BLM+EM- treated group showing intact alveoli (AL) and alveolar sacs (AS) with thin inter-alveolar septa(arrows). The bronchiole (B) appears intact without inflammatory cell infiltration. Hx. & E.; × 400

Fig. 27: A photomicrograph of a section in the lung of a BLM+EM- treated group showing non-congested blood vessels (arrows). Hx. & E.; × 400

Fig. 28: A photomicrograph of a section in the lung of a BLM+EM- treated group showing few collagen fibers (arrows) in the connective tissue surrounding the bronchioles (B). Masson's trichrome; × 100

Fig. 25: A photomicrograph of a section in the lung of a BLM+EM- treated group showing intact alveoli lined with pneumocytes type I (arrows). Pneumocytes type II are noticed at some angles of the alveoli (thick arrows). Few extra-vasated leukocytes appear in the interstitium between the alveoli (arrow heads). Hx. & E.; × 1000

Fig. 29: A photomicrograph of a section in the lung of a BLM+EM- treated group showing few collagen fibers (arrows) in the connective tissue surrounding the bronchioles (B). Masson's trichrome; × 400

Fig. 26: A photomicrograph of a section in the lung of a BLM+EM- treated group showing non-congested blood vessels (arrows). Hx. & E.; × 100 21


Fig. 32: A histogram of the mean number of alveolar macrophages/ HPF in different groups.

Fig. 30: A photomicrograph of a section in the lung of a BLM+EM- treated group showing few number of anti-CD68 immunoperoxidase positive stained alveolar macrophages (arrows). Anti-CD68 immunoperoxidase stain; × 100

Table 2: The extent of collagen deposition/ LPF in different groups Group

Mean % ±SD

Control

2.57 ± 1.27

BLM-treated group EM + BLM-treated group

P value

29.62 ± 6.41

< 0.001 **

4.02 ± 2.82

< 0.001**

*P