Doxycycline attenuated lung injury by its biological effect apart from its antimicrobial function

doi:10.1016/j.pupt.2006.08.006

Pulmonary Pharmacology & Therapeutics

Volume 20, Issue 6, December 2007, Pages 669-675

https://doi.org/10.1016/j.pupt.2006.08.006 Get rights and content

Abstract

Antibiotics can have a biological effect apart from their anti-bacterial effect. We hypothesized that doxycycline could attenuate acute lung injury through its biological effect. Lipopolysaccharide or doxycycline-resistant Streptococcus pneumoniae was administered intratracheally into mice with the co-administration of doxycycline. Thereafter, the lung pathology, intraalveolar inflammatory cells, bacterial number, and matrix metalloproteinases were investigated. Matrix metalloproteinases, neutrophil migration, and alveolar destruction were induced by lipopolysaccharide. Doxycycline was thus found to improve all of these symptoms. In addition, an inhibitor of matrix metalloproteinases, CGS27023A, attenuated lipopolysaccharide-induced lung injury. Doxycycline also attenuated the lung injury induced by doxycycline-resistant S. pneumoniae and improved the mortality rate although the bacterial number in the lung did not change. Our data indicated that doxycycline could attenuate acute lung injury through a biological effect that was different from its antibiotic effect.

Introduction

Several antibiotics including macrolides are known to exert biological effects beyond their anti-bacterial activity. For instance, macrolides are known to have an inhibitory effect on inflammation by blocking IL-8 production by bronchial epithelial cells or activated neutrophils [1], and 14-member ring macrolides have been shown to suppress the transcription factors that regulate IL-8 gene expression in bronchial epithelial cells [2]. Tetracycline also has an anti-inflammatory effect via the decreased release of neutrophil chemotactic factors or reactive oxygen species [3].

Several lines of evidence have indicated the importance of matrix metalloproteineases (MMPs) in lung injury [4]. MMPs are a family of secreted proteinases that function at neutral pH and utilize the extracellular matrix as a substrate. In addition to degrading the matrix during normal tissue remodeling and repair, MMPs have also been implicated in several pathologic processes [5], [6]. MMPs consist of three groups, namelycollagenases, gelatinases, and stromelysins. Gelatinases are known to have substrate specificity for denatured collagens, basement membrane type IV collagen, and elastin. Gelatinases can digest several components of the basement membrane, including type IV collagen, heparan sulfate proteoglycan, and result in tissue damage [7].

Tetracycline inhibits the activity and expression of MMPs both in vivo and in vitro. Actually, Curci et al. demonstrated that clinical use of doxycycline reduced expression and amount of gelatinase-B (MMP-9) in aortic aneurysm. In addition, a slight reduction of activated fraction of gelatinase A (MMP-2) was observed [8]. Grenier et al. reported that doxycycline and chemically modified derivatives (CMT) inhibited collagenolytic activity of Prophyromonas gingivalis as well as conversion of pro-MMP-9 to active form. Interestingly, they documented that CMT-5, which lacks the structural elements required for cation chelation did not inhibit these activities [9]. The inhibition and down regulation of MMPs appear to be related to the cation chelating property of tetracycline. Inhibition could be reversed by adding an excess of calcium or zinc and that tetracycline derivatives lacking structural elements required for cation chelating do not inhibit MMPs.

We hypothesize that doxycycline could modulate lung injury through the protection of lung structure by biological effects, namely gelatinase inhibitor. To investigate this hypothesis, the influence of doxycycline was tested using two mouse models of lung injury. One was a lipopolysaccharide (LPS)-administration model while the other was a Streptococcus pneumoniae-inoculated model. To clarify the effect of doxycycline, we used a strain of S. pneumoniae that was resistant to doxycycline.

Section snippets

Animal model of acute lung injury

Eight week-old C57Bl/6 mice were used in this study. These mice were provided with sterile food and water in an environmentally controlled room. The intratracheal administration of a single 10 μg/kg body weight Escherichia coli LPS (Difco Laboratories, Detroit, MI) in 50 μl of sterile saline was done via a tracheotomy to the mice under nembutal anesthesia. As a LPS control, 50 μl of saline was also injected. Doxycycline was a kind gift from Pfizer Pharmaceutical Ltd. (Groton, CT). Doxycycline was

Results

A histologic assessment was performed using the lungs at 1 day after LPS administration. Lung histology revealed significant lung damage in LPS-treated mice (Fig. 1A). A morphometic study proved that LPS-induced lung injury (Lung injury score, LPS; 2.125±0.11, Saline; 0.35±0.05, p=0.001, each group consisted of four mice). Neutrophils were rarely visible in the lung tissue from control mice administered with saline intratracheally. On the other hand, a large number of inflammatory cells, mainly

Discussion

Doxycycline has been known to be a broad spectrum and effective agent against the pathogens of pneumonia [15]. Doxycycline is also known to inhibit reactive oxygen species (ROS) release, apoptosis, and decrease neutrophil chemotaxis [3], [16]. Neutrophils release ROS. High levels of ROS can result in significant damage to cell structures. Tetracycline at their sub-MIC also significantly inhibited release of ROS from human neutrophils as well as neutrophil chemotactic factors produced by

Acknowledgments

We appreciate the assistance of Dr. Brian Quinn for editing the English usage. We thank Pfizer Inc. for providing the kind gift of doxycycline and Novartis Pharmaceuticals Corporation for providing the kind gift of CGS27023A.

References (31)

J. Curci et al.
Preoperative treatment with doxycycline reduces aortic wall expression and activation of matrix metalloproteinases in patients with abdominal aortic aneurysms
J Vasc Surg
(2000)
H. Iwasaki et al.
Doxycycline induces apoptosis by way of caspase-3 activation with inhibition of matrix metalloproteinase in human T-lymphoblastic leukemia CCRF-CEM cells
J Lab Clin Med
(2002)
A.C. Windsor et al.
Acute lung injury: what have we learned from animal models?
Am J Med Sci
(1993)
K. Oishi 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)
S. Abe et al.
Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding sites in human bronchial epithelial cells
Am J Respir Cell Mol Biol
(2001)
A. Jain et al.
Anti-inflammatory effects of erythromycin and tetracycline on Propionibacterium acnes induced production of chemotactic factors and reactive oxygen species by human neutrophils
Dermatol Online J
(2002)
J.J. Atkinson et al.
Matrix metalloproteinase-9 in lung remodeling
Am J Respir Cell Mol Biol
(2003)
G. Murphy et al.
The matrix metalloproteinases and their inhibitors
Am J Respir Cell Mol Biol
(1992)
T.D. Tetley
New perspectives on basic mechanisms in lung disease. 6. Proteinase imbalance: its role in lung disease
Thorax
(1993)
R.H. Simon et al.
Type IV collagen production by rat pulmonary alveolar epithelial cells
Am J Respir Cell Mol Biol
(1993)

D. Grenier et al.

Inhibition of proteolytic, serpinolytic, and progelatinase-b activation activities of periodontopathogens by doxycycline and the non-antimicrobial chemically modified tetracycline derivatives

J Periodontol

(2002)

M. Fujita et al.

Doxycycline attenuated pulmonary fibrosis induced by bleomycin in mice

Antimicrob Agents Chemother

(2006)

Performance standards for antimicrobial susceptibility testing, 14th informational supplement

(2003)

K. Ahamed et al.

Deficiency in type 1 insulin-like growth factor receptor in mice protects against oxygen-induced lung injury

Respir Res

(2005)

M. Fujita et al.

Overexpression of tumor necrosis factor-α produces an increase in lung volumes and pulmonary hypertension

Am J Physiol Lung Cell Mol Physiol

(2001)

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CGS27023A can be dissolved in water. An amount of 0.2 mg/ml of CGS27023A in drinking water was given every day as previously reported [21]: (i) CGS27023A had been orally administered from one day before bleomycin administration to 28 days after administration. The approximate daily doses consisted of 1.2 mg/body weight per mouse.
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Doxycycline attenuated lung injury by its biological effect apart from its antimicrobial function

Abstract

Introduction

Section snippets

Animal model of acute lung injury

Results

Discussion

Acknowledgments

J Vasc Surg

J Lab Clin Med

Am J Med Sci

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

Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding sites in human bronchial epithelial cells

Am J Respir Cell Mol Biol

Anti-inflammatory effects of erythromycin and tetracycline on Propionibacterium acnes induced production of chemotactic factors and reactive oxygen species by human neutrophils

Dermatol Online J

Matrix metalloproteinase-9 in lung remodeling

Am J Respir Cell Mol Biol

The matrix metalloproteinases and their inhibitors

Am J Respir Cell Mol Biol

New perspectives on basic mechanisms in lung disease. 6. Proteinase imbalance: its role in lung disease

Thorax

Type IV collagen production by rat pulmonary alveolar epithelial cells

Am J Respir Cell Mol Biol

Inhibition of proteolytic, serpinolytic, and progelatinase-b activation activities of periodontopathogens by doxycycline and the non-antimicrobial chemically modified tetracycline derivatives

J Periodontol

Doxycycline attenuated pulmonary fibrosis induced by bleomycin in mice

Antimicrob Agents Chemother

Performance standards for antimicrobial susceptibility testing, 14th informational supplement

Deficiency in type 1 insulin-like growth factor receptor in mice protects against oxygen-induced lung injury

Respir Res

Overexpression of tumor necrosis factor-α produces an increase in lung volumes and pulmonary hypertension

Am J Physiol Lung Cell Mol Physiol