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