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Regulation of cytokine production in human alveolar macrophages and airway epithelial cells in response to ambient air pollution particles: Further mechanistic studies

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Abstract

In order to better understand how ambient air particulate matter (PM) affect lung health, the two main airway cell types likely to interact with inhaled particles, alveolar macrophages (AM) and airway epithelial cells have been exposed to particles in vitro and followed for endpoints of inflammation, and oxidant stress. Separation of Chapel Hill PM 10 into fine and coarse size particles revealed that the main proinflammatory response (TNF, IL-6, COX-2) in AM was driven by material present in the coarse PM, containing 90–95% of the stimulatory material in PM10. The particles did not affect expression of hemoxygenase-1 (HO-1), a sensitive marker of oxidant stress. Primary cultures of normal human bronchial epithelial cells (NHBE) also responded to the coarse fraction with higher levels of IL-8 and COX-2, than induced by fine or ultrafine PM. All size PM induced oxidant stress in NHBE, while fine PM induced the highest levels of HO-1 expression. The production of cytokines in AM by both coarse and fine particles was blocked by the toll like receptor 4 (TLR4) antagonist E5531 involved in the recognition of LPS and Gram negative bacteria. The NHBE were found to recognize coarse and fine PM through TLR2, a receptor with preference for recognition of Gram positive bacteria.

Compared to ambient PM, diesel PM induced only a minimal cytokine response in both AM and NHBE. Instead, diesel suppressed LPS-induced TNF and IL-8 release in AM. Both coarse and fine ambient air PM were also found to inhibit LPS-induced TNF release while silica, volcanic ash or carbon black had no inhibitory effect. Diesel particles did not affect cytokine mRNA induction nor protein accumulation but interfered with the release of cytokine from the cells. Ambient coarse and fine PM, on the other hand, inhibited both mRNA induction and protein production. Exposure to coarse and fine PM decreased the expression of TLR4 in the macrophages. Particle-induced decrease in TLR4 and hyporesponsiveness to LPS may be related to LPS tolerance induced by low levels of LPS.

Introduction

Epidemiological studies have associated exposure to ambient particulate matter (PM) with adverse cardiorespiratory effects (Dockery et al., 1993, Pope and Dockery, 1998, Pope et al., 1995, Schwartz et al., 1996). Proposed mechanisms behind PM-induced health effects are oxidant stress (Nel et al., 2001, Tao et al., 2003) and inflammation-associated injury (Dick et al., 2003, Ghio and Devlin, 2001). Recent in vitro studies with airway epithelial cells have demonstrated that exposure to pollution particles induces IL-8 production and various responses associated with oxidant stress, (Hetland et al., 2004, Li et al., 2002). Lung macrophages respond to PM by releasing a variety of cytokines, while showing decreased phagocytic function (Becker et al., 2003, Soukup and Becker, 2001).

Previous studies (Becker et al., 2002) have shown that ambient air pollution particles contain components which stimulate Toll like receptors 2 and 4 (TLR2 and TLR4). It was therefore proposed that components of whole Gram positive and Gram negative bacteria were associated with the particles, including the ubiquitously present lipopolysaccharide (LPS). In AM, the cytokine response to particles was inhibited by antibody to CD14 and an antagonist of TLR4, emphasizing the role of bacterial products in the response (Becker et al., 2002, Soukup and Becker, 2001). Also, airway epithelial cells have been shown to respond to various bacterial pathogens by the release of cytokines (Becker et al., 2004, Larsson et al., 1999). These cells have been shown to express several members of the TLR family of receptors (Becker et al., 2000), which are known to be involved in the recognition components of Gram-positive and -negative bacteria, viruses as well as fungal elements (Janssens and Beyaert, 2003). Therefore, epithelial cells may also respond to microbial products, other than LPS, contained in ambient air PM.

Diesel particles, which constitute the main mass of fine PM in some locations, induce negligible amounts of cytokine in AM compared to amounts induced following stimulation with ambient PM (Becker et al., 1996). Instead, the suppressive effect of diesel on cytokine production by LPS or other microbial products have been emphasized as a possible mechanism whereby diesel exerts its health effects (Amakawa et al., 2003, Yang et al., 2001). Fine pollution particles may have a similar effect to diesel since the major component of this size fraction in the area of collection (Chapel Hill, NC) is diesel dust. Experiments were conducted to compare suppressive effect of diesel, ambient fine and coarse PM, on LPS-induced cytokine production. Both PM size fractions were found to be strongly suppressive, and the effect of at least coarse PM unlikely to be related to organic compounds of diesel. Since cytokine induction by coarse PM, and fine PM, involved stimulation of TLR4, it is possible that these particles induce as state similar to well-documented LPS tolerance (Dobrovolskaia and Vogel, 2002, Nomura et al., 2000, Schwartz, 2002).

Section snippets

Particle collection

Ambient air particles: Particles were collected in Chapel Hill, North Carolina, using a ChemVol high Volume Cascade Impactor (Rupprecht and Patashnick Co. Albany, NY). Coarse PM and fine PM particles were collected for 72 h onto polyurethane foam (McMaster-Carr, Atlanta, GA), which were previously cleaned with methanol and water and dried under sterile conditions. Ultrafine particles were collected onto G5300 filters (Monandock Non-Wovens LLC, Mt. Pocono, PA).The foams/filters cut into strips

Oxidant stress

Although epidemiological studies have suggested that fine particles may be more detrimental to human health than coarse mode particles (Sarnat et al., 2001, Schwartz et al., 1996), in vitro studies (Becker et al., 2003, Hetland et al., 2004), and animal in vivo data (Dick et al., 2003, Dybing et al., 2004, Schins et al., 2004, Tao et al., 2003) have provided equivocal support for the preferential toxicity of fine particles. It was shown that coarse particles in AM and airway epithelial cells

Conclusion

Taken together, the selected experiments shown here demonstrate possible mechanisms whereby pollution particles affect lung health. If inflammation is involved as in asthma and inflammatory lung diseases, particles may exacerbate symptoms or obstruct a normal response. Proinflammatory measures appear to be the dominant response to coarse PM, which affect cytokine production and prostaglandin synthesis. This response appears to be driven by TLR2 and TLR4 stimulating compounds in both NHBE and

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