Alveolar macrophage cytokine response to air pollution particles: Oxidant mechanisms
Introduction
The association between elevated concentrations of air pollution particles (≤ PM2.5) and adverse health effects is especially prominent in people with inflammatory lung diseases such as bronchitis, asthma and pneumonia (Schwartz, 1994, Goldsmith and Kobzik, 1999, Atkinson et al., 2001). In both normal and diseased lungs, the alveolar macrophage (AM) is the first line of defense against inhaled particulate matter. Since people with pre-existing respiratory illness are especially affected by high PM levels, we have postulated that AMs residing within inflamed lungs are ‘primed’ for heightened pro-inflammatory (and injurious) responses to air particles. In the initial testing of this hypothesis, priming of AMs with LPS in vitro markedly enhanced their release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF) and macrophage inflammatory protein (MIP-2) in response to suspensions of concentrated air pollution particles (CAPs) (Imrich et al., 1999). In contrast, when primed AMs were cultured with a relatively inert particle, TiO2, there was no effect on release of cytokines. Since the same AM scavenger receptors mediate initial binding of both inert (e.g. TiO2) and pathogenic (e.g., quartz, CAPs) particles (Kobzik, 1995), components unique to CAPs are most likely responsible for the enhanced cytokine release seen in primed AMs.
The question of which component(s) of particulate matter (PM) are active has been investigated using a variety of biologic outcome parameters (Tao et al., 2003). Data have been reported implicating transition metals (Ghio et al., 1999, Wilson et al., 2002), polyaromatic hydrocarbons (PAH) (Li et al., 2002a, Li et al., 2002b) and bacterial endotoxin (Becker et al., 1996, Ning et al., 2000) as bioactive components of PM. Many PM effects on normal lung cells in vitro and in vivo operate through oxidant-dependent mechanisms (Tao et al., 2003). However, much less is known about how particles mediate effects in primed or activated cells.
The studies reported here sought to directly test the hypothesis that oxidant mechanisms are necessary for the enhanced cytokine release seen in primed AMs upon culture with air pollution particles. We tested the postulate by examining the inhibitory effects of a panel of antioxidants added to the culture system, as well as the effect of exogenous oxidants produced by glucose oxidase. We also analyzed the contribution of soluble and insoluble fractions of CAPs, and the effect of metal-chelation with desferoxamine. The data support an oxidant (H2O2)-dependent mechanism for particle-mediated cytokine release by primed AMs, and identify novel differences in the bioactivity of metals (most likely iron) within soluble and insoluble fractions of air pollution particles.
Section snippets
Preparation of particle suspensions
The urban air sample SRM 1649 was collected in Washington, DC (UAP) and was purchased from the National Bureau of Standards (Washington, DC; May et al., 1992). UAP was used as a positive control particle and contains both PM2.5 and larger non-respirable particles including pollen, fungal fragments, etc. TiO2 (∼ 1 μm diameter) was generously provided by Dr. J. Brain. Both particles were suspended in analytical grade water (OPTIMA, Fisher Scientific) at 10 mg/ml and stored frozen. The CAPs samples
Antioxidants inhibit AM response to particles
Initial experiments used the positive control particle UAP to perform dose–response analysis in the presence of the antioxidants dimethylthiourea (DMTU at 2–20 mM) and N-acetyl-l-cysteine (NAC at 2–20 mM). After 3 h of LPS priming, the effect of antioxidants on AM release of TNF and nitrite, and cell death were measured. Similar to previous observations (Imrich et al., 1999, Imrich et al., 2000), the results presented in Table 1 show that in the absence of antioxidants: 1) UAP significantly
Discussion
The data presented address the role of oxidants in particle-mediated cytokine release by primed AMs. Our strategy was to use the positive control particle UAP (SRM 1649) to determine optimal treatment conditions, which were then used for study of the more relevant particles of interest, concentrated ambient particles (CAPs). The results show that, in primed AMs both UAP-mediated and CAPs-mediated TNF release are oxidant-dependent events and that H2O2 is involved. The antioxidant DMTU, a
Acknowledgments
Supported by NIH ES011903, ES00002 and US EPA Particle Center. The authors thank Dr. Jim Shine for assistance with the ferrozine assay.
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