TLR-2 is involved in airway epithelial cell response to air pollution particles

Abstract

Primary cultures of normal human airway epithelial cells (NHBE) respond to ambient air pollution particulate matter (PM) by increased production of the cytokine IL-8, and the induction of several oxidant stress response genes. Components of ambient air PM responsible for stimulating epithelial cells have not been conclusively identified, although metal contaminants, benzo[a]pyrene and biological matter have been implicated. Stimulation of IL-8 release from NHBE with coarse (PM_2.5–10), fine (PM_2.5), and UF particle fractions has shown that the coarse particle fraction has the greatest effect on the epithelial cells as well as alveolar macrophages (AM). Since this fraction concentrates fugitive dusts and particle-associated microbial matter, it was hypothesized that NHBE may recognize PM through microbial pattern recognition receptors TLR2 and TLR4, as has been previously shown with AM. NHBE were shown to release IL-8 when exposed to a Gram-positive environmental isolate of Staphylococcus lentus, and lower levels when exposed to Gram-negative Pseudomonas spp. Comparison of TLR2 and TLR4 mRNA expression in NHBE and AM showed that NHBE express similar levels of TLR2 mRNA as the AM, but expressed very low levels of TLR4. When NHBE were stimulated with PM_2.5–10, PM_2.5, and UF PM, in the presence or absence of inhibitors of TLR2 and TLR4 activation, a blocking antibody to TLR2 inhibited production of IL-8, while TLR4 antagonist E5531 or the LPS inhibitor Polymixin B had no effect. Furthermore, effects on expression of TLR2 and TLR4 mRNA, as well as the stress protein HSP70 was assessed in NHBE exposed to PM. TLR4 expression was increased in these cells while TLR2 mRNA levels were unchanged. Hsp70 was increased by PM_2.5–10 > PM_2.5 > UF PM suggesting the possibility of indirect activation of TLR pathway by this endogenous TLR2/4 agonist.

Introduction

Proposed mechanisms behind particulate matter (PM)-induced health effects are oxidant stress and inflammation-induced injury. Recent studies with airway epithelial cells have demonstrated that exposure to particles induces IL-8 production and various responses associated with oxidant stress or protection against oxidant injury (Fujii et al., 2001, Ghio et al., 1999). Lung macrophages respond to PM by releasing a variety of cytokines and the production of oxygen radicals (Imrich et al., 2000, Soukup and Becker, 2001), but these cells appear not experience a similar intracellular stress response (data not shown).

To define components of PM which cause health effects, PM have been separated by size depending on their source of origin. Particles smaller than 2.5 μm (fine and ultrafine PM ) are mainly of anthropogenic origin while PM between 2.5 and 10 μm (coarse PM) contain crustal materials with associated biological materials including bacteria and pollen fragments (Artinano et al., 2003, Chow et al., 2002, Wilson et al., 2002). Coarse particles induce higher levels of cytokine production in both epithelial cells and macrophages, compared to fine and UF PM (Becker and Soukup, 2003, Becker et al., 2003, Dailey and Devlin, 2003). We have previously shown that macrophage cytokine production in response to coarse PM is mediated by Toll-like receptors 2 and 4 (TLR2 and TLR4), which recognize active components concentrated in the coarse fraction (Becker et al., 2002). It was therefore proposed that products of Gram-positive and Gram-negative bacteria associated with the particles, including lipopolysaccharide (LPS), are likely to be responsible for stimulating the production of cytokines in macrophages.

However, there are many more epithelial cells in the lung than macrophages, and these cells may also be affected by inhaled particles. In many studies, epithelial cells have been shown to be unresponsive to LPS, unless LPS binding protein as well as soluble CD14 was present in the culture medium (Backhed et al., 2002, Pugin et al., 1993). On the other hand, epithelial cells have been shown to respond to various bacterial pathogens by the release of cytokines (Palfreyman et al., 1997, Ratner et al., 2001). Therefore, epithelial cells may also respond to bacterial products, other than LPS, contained in ambient PM. Indeed, airway epithelial cells have been shown to express several members of the TLR family of receptors (Becker et al., 2000), which have been shown to be involved in the recognition components of Gram-positive and -negative bacteria, viruses as well as fungal elements. Thus, these receptors may also be involved in stimulation of cytokine production by air pollution particles. Alternatively, pollution particles may induce endogenous stress proteins such as heat shock protein Hsp70 which has been shown to activate TLRs 2 and 4 (Asea et al., 2002, Vabulas et al., 2002). In this study, we investigated the role played by TLR 2 and TLR 4 in epithelial cell responses to size fractionated air pollution particles.