Original contribution
Cigarette smoke triggers macrophage adhesion and activation: role of lipid peroxidation products and scavenger receptor

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Abstract

Pulmonary emphysema in chronic obstructive pulmonary disease (COPD) is characterized by the destruction of the alveolar walls leading to permanent enlargement of distal respiratory air spaces. A major causal factor is cigarette smoking, which produces conditions of chronic oxidative stress within the lungs. At a cellular level, increased macrophage accumulation and retention within the alveolar interstitial spaces is pivotal to the development of emphysema. To date it has been unclear as to the underlying mechanisms relating chronic oxidative stress to macrophage accumulation and retention. Our study was initiated to ascertain the role of modification of extracellular matrix proteins with cigarette smoke and products of lipid peroxidation on macrophage adhesion and activation. Increased numbers of macrophages were seen adhering to cigarette smoke-modified collagen IV as compared to unmodified collagen, where little or no adherent macrophages were observed. Similar observations were made when collagen was modified with either acrolein or 4-hydroxy-2-nonenal. Adhesion could be blocked with either fucoidan or a monoclonal antibody against the Type A macrophage scavenger receptor. Also, modified collagen triggered both oxidative burst and MCP-1 release in macrophages. These results, therefore, highlight a potential mechanism by which oxidative stress through the production of reactive carbonyls promotes macrophage accumulation, retention, and activation, independently of other proinflammatory stimuli. The implications of this for the development of emphysema in COPD are discussed.

Introduction

Pulmonary emphysema in chronic obstructive pulmonary disease (COPD) is characterized by the destruction of the terminal respiratory bronchiole and alveolar walls leading to permanent enlargement of the distal respiratory air spaces. This gives rise to a corresponding loss in surface area for gaseous exchange as well as loss in lung elasticity leading to peripheral airflow obstruction [1]. The main causal factor of COPD is chronic oxidative stress as a result of long-term cigarette smoking 2, 3, 4. It is this imbalance in the oxidant/antioxidant mechanism triggered by cigarette smoking that can induce tissue damage, either directly or indirectly, that has led to its proposal for playing a central role in the pathogenesis of COPD 5, 6 and, in particular, emphysema [2].

At a cellular level, however, it is the macrophage that is the principal cell type believed to be responsible for this tissue loss [7]. Indeed, there is an increase in the number of activated alveolar macrophages in the lungs of smokers [8] precisely at the sites where the earliest lesions are seen to develop [9]. Furthermore, it has been shown that there was not only a requirement for macrophage elastase in cigarette smoke-induced emphysema in mice [10] but that cell depletion studies in rats identified macrophages, rather than neutrophils, as being the critical factor in cigarette smoke-induced emphysema [11].

Cigarette smoke is not only a direct source of oxidants but also a source of highly reactive carbonyls 12, 13, the most reactive of which (and present in high concentrations) is acrolein [13]. Acrolein is a highly reactive water-soluble aldehyde and a ubiquitous environmental pollutant. Its high reactivity makes it dangerous to the cell and it is known to cause respiratory tract injury and suppression of pulmonary host defenses [14]. Moreover, acrolein can also be formed in vivo as a result of lipid peroxidation, along with numerous other reactive aldehydes such as 4-hydroxy-2-nonenal, malonlydialdehyde, and glyoxal, either from oxidants in cigarette smoke or from the release of oxidants during an inflammatory response 15, 16, 17. These reactive carbonyls can, by attacking residues such as lysine, arginine, and histidine, result in covalent protein modifications, commonly referred to as advanced glycation end products (AGE) or advanced lipoxidation end products (ALE) 12, 13, 18, 19, 20. Within this context, acrolein-derived protein modifications have been shown to be a marker for oxidative stress-derived tissue damage in chronic diseases such as atherosclerosis and Alzheimer's disease 21, 22. Furthermore, it has been demonstrated that certain AGEs and ALEs are ligands for the type A macrophage scavenger receptor (SRA). These include AGEs formed as a result of protein modification by glucose and the carbonyls malonyldialdehyde and glycolaldehyde but not glyoxal 23, 24, 25, 26. Also, oxidized LDL that contains ALEs is also a ligand for the SRA 27, 28.

There are five classes (A–E) of scavenger receptor that are structurally unrelated and were originally distinguished on the basis of their ligand-binding properties. The class A scavenger receptor (SRA) consists of four types (I, II, III, and Marco) and is principally expressed on macrophages. All four types are transmembrane homotrimeric glycoproteins with a high degree of homology, and they possess a similar ligand-binding domain within a collagen-like region of repeating gly-X-Y motifs 29, 30. The scavenger receptor superfamily has a very broad ligand-binding capability and it is because of this that the scavenger receptors are believed to play a role in innate immunity [31]. However, because of the ability of SRA to also act as an adhesion receptor for the macrophages 23, 32, 33 resulting in their tissue retention, SRA-mediated adhesion has been implicated in the pathology of atherosclerosis [23] and Alzheimer's disease [32]. Furthermore, SRA may play a role in other chronic diseases including chronic renal failure [34], diabetic complications [35], and asbestosis [36], as SRA stimulation would appear to trigger macrophage activation and the release of reactive oxygen species [32].

The underlying mechanism linking cigarette smoke to macrophage retention and accumulation in the lungs of COPD patients is still not clear. While chemokines can explain their recruitment to the sites of inflammation, it does not explain the retention of these macrophages in a chronic context at precisely the areas where the emphysematous lesions develop. Cigarette smoke and products of lipid peroxidation can account for many different carbonyls, such as crotonaldehyde, acetylaldehyde, acrolein, 4-hydoxynonenal, glycolaldehyde, and malonyldialdehyde, all of which have the potential (and in a few cases have been demonstrated) to modify proteins. Therefore, we asked whether acrolein and 4-hydoxy-2-nonenal, being either constituents of cigarette smoke and/or products of in vivo lipid peroxidation, could modify protein-causing macrophage adhesion through the SRA. Furthermore, we investigated whether this modified protein could result in macrophage retention as well as activation. Finally, we propose a mechanism, based on our in vitro findings, that could have important implications in the pathogenesis of emphysema in COPD.

Section snippets

Materials

Mouse collagen type IV was obtained from Becton Dickinson (Oxnard, CA, USA) and human collagen type IV was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Iscove's modified Dulbecco's medium (IMDM), RPMI, fetal calf serum, penicillin, streptomycin, PBS, and Hank's buffered salt solution (HBSS) were obtained from Gibco BRL (Grand Island, NY, USA). Alkaline phosphatase conjugated goat antirabbit IgG was purchased from Southern Biotechnology (Birmingham, AL, USA). Polyclonal rabbit

Protein carbonyl levels depend on concentration and frequency of exposure

The effect of protein modification by reactive carbonyls depends on temperature, concentration of reactants, time, and frequency of exposure. As temperature was kept constant at 37°C, the effect of carbonyl concentration, time, and frequency of exposure was investigated. In Fig. 1A, the effect of increasing concentration of a single dose exposure to acrolein over an 18 h period is shown. As the concentration of acrolein increased from 3 mM up to 100 mM, there was a corresponding increase in

Discussion

This study has investigated the interaction of macrophages with proteins that have been modified by highly reactive, water-soluble aldehydes that are either present in cigarette smoke or products of lipid peroxidation, such as acrolein and 4-hydroxy-2-nonenal. Both acrolein and 4-hydroxy-2-nonenal have been shown to be able to bind proteins and act as markers of oxidative stress and tissue damage in areas of chronic inflammation 20, 42. The in vitro experiments described here show for the first

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