Effect of Atorvastatin on PM 10 -induced Cytokine Production by Human Alveolar Macrophages and Bronchial Epithelial Cells

Abstract

Exposure to ambient air pollution particles (PM₁₀) has been associated with increased cardiovascular morbidity and mortality. Inhaled pollutants induce a pulmonary and systemic inflammatory response that is thought to exacerbate cardiovascular disease. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to have anti-inflammatory effects that could contribute to their beneficial effect in cardiovascular disease. The aim of this study is to determine the effects of statins on PM₁₀-induced cytokine production in human bronchial epithelial cells (HBECs) and alveolar macrophages (AMs). Primary HBECs and AMs are obtained from resected human lung. Cells are pretreated with different concentrations of atorvastatin for 24 hours and then exposed to 100 μg/mL urban air pollution particles (EHC-93). Cytokine levels (interleukin-1β, interleukin-8, granulocyte-macrophage colonystimulating factor, interleukin-6, and tumor necrosis factor-α) are measured at messenger RNA and protein levels using real-time polymerase chain reaction and bead-based multiplex immunoassay, respectively. PM₁₀ exposure increases production of these cytokines by both cell types. Atorvastatin attenuates PM₁₀-induced messenger RNA expression and cytokine production by AMs but not by HBECs. It is concluded that statins can modulate the PM₁₀-induced inflammatory response in the lung by reducing mediator production by AMs.

Numerous epidemiologic studies have shown that particulate matter with a diameter of less than 10 μm (PM₁₀) induces respiratory and cardiovascular disease. ^1,2 Although the mechanisms by which PM₁₀ exposure causes adverse health effects are poorly understood, PM-induced lung inflammation contributes to the disease process. ^3,4 The alveolar macrophages (AMs) and bronchial epithelial cells are pivotal cells in the pathogenesis of PM₁₀-induced lung inflammation, ³ and the mediators they produce are involved in the systemic inflammation and implicated in the accelerated atherosclerosis induced by exposure to ambient particulate matter. ⁵

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins, are used for the treatment of hyperlipidemia and have been shown to attenuate several pathogenic mechanisms in atherogenesis or thrombosis. ⁶ Recent studies have suggested that statins have pleiotropic effects such as improvement of endothelial function by endothelial nitric oxide synthase up-regulation; reduction of vascular smooth muscle cell proliferation; reduction of platelet activity; stabilization of atherosclerotic plaques; and antioxidant, anti-inflammatory, and immunomodulatory effects. ⁷ Furthermore, their anti-inflammatory effects, which are cholesterol-independent, could contribute to reducing the risk of cardiovascular events ^8,9 and the decline in lung function ^10,11 but also, in animal models, could reduce lung injury and inflammation. ^12,13

Based on this background, the present study was designed to evaluate the in vitro anti-inflammatory effects of statins on PM₁₀-induced cytokine production by human AMs and human bronchial epithelial cells (HBECs).

Methods

Urban Air Particles (PM₁₀) and Statins

PM₁₀ particles (EHC-93) were obtained from Health Canada and collected in an urban environment over Ottawa, Ontario, Canada, in 1993 using filters with a nominal cutoff of 0.3 μm, from a single-pass air filtration system of the Environmental Health Centre in Ottawa (100% outdoor air). The particles had a mean diameter of 0.8 ± 0.4 μm (mean ± SD), with 99% (in number, not mass) of particles less than 3.0 μm. Their biochemical analysis was previously described in detail elsewhere. ¹⁴ Particles were suspended in hydrocortisone-free bronchial epithelial cell growth medium (BEGM) (Clonetics, San Diego, Calif) for HBECs or RPMI-1640 medium (GIBCO BRL, Gaithersburg, Md) containing 10% fetal bovine serum (Life Technologies, Rockville, Md) for AMs at a concentration of 1 mg/mL and sonicated ¹⁹ before addition to the cells. PM₁₀ is evenly distributed over the cells; therefore, all the cells are exposed to the same dose of the PM₁₀. The endotoxin content of the PM₁₀ suspension of 100 μg/mL was 6.4 ± 1.8 EU/mL or less than 3.0 ng/mL, a dose that does not activate either AMs or lung epithelial cells to produce cytokines. ¹⁵ PM₁₀ exposure did not induce a cytotoxic effect on HBECs and AMs determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Atorvastatin and pravastatin were purchased from Toronto Research Chemicals Inc (Ontario, Canada) and Sigma Aldrich (St. Louis, Mo), respectively. Final concentrations of statins used in this study were determined by the cytotoxic effect of statins on AMs and HBECs using an MTT assay. Statins less than 10⁻⁶ M were used in the present study, because statins greater than 10⁻⁴Mand greater than 10⁻⁵ M had cytotoxic effects on HBECs and AMs, respectively (data not shown).

Cell Culture and Treatment of the Cells

Bronchial tissue and bronchoalveolar lavage (BAL) fluid were obtained from 10 patients who underwent lobectomy or pneumonectomy for small peripheral nodules at St. Paul’s Hospital, Vancouver. Informed consent was obtained from all subjects, and these studies were approved by the Human Ethics Committee of the University of British Columbia. All subjects were current smokers and were asked to abstain from smoking for 6 weeks prior to the operation. Their mean age was 67.2 years (range, 56–74 years) (6 women and 4 men). Both messenger RNA (mRNA) and cytokine production in AMs of 5 patients were analyzed, and both mRNA and cytokine production in HBECs of the other 5 patients were analyzed. Primary HBECs were isolated from these bronchial tissues according to a previously described procedure. ^15,16 Primary HBECs were isolated from 1-cm-long excised human bronchial tissue that was incubated at 4°C for 24 hours with 0.1% protease (type 14; Sigma) solution prepared in BEGM containing Fungizone (1 μg/mL; GIBCO BRL). The harvested cells were prepared and cultured until 80% to 90% confluent, as described. Then the cells were trypsinized and seeded on 6-well plates (Falcon; Becton Dickinson Ltd, Mississauga, ON, Canada) and cultured in BEGM. The purity and identity of the cells were checked morphologically using light microscopy. HBECs were passaged at 80% to 90% confluence, and all experiments were performed at either the third or fourth passage when hydrocortisone was removed from the culture medium 24 hours before treatment and during the time of the study. After 24 hours, cells were incubated in 2 mL of BEGM alone as control or BEGM with 100 μg/mL of PM₁₀ (EHC-93) suspension prepared as outlined above with or without different concentrations of statins.

Human AMs were harvested from BAL fluid obtained from lung segments or lobes that were free of tumor using a method previously described in detail. ^15,17 The BAL fluid cells were more than 90% viable (trypan blue exclusion method) and consisted of 90% to 95% AMs (as assessed by Wright-Giemsa stain) and less than 2% neutrophils. AMs (1.0 × 10⁷) were suspended in RPMI-1640, placed in 6-well plates, and allowed to adhere to the plastic plate for 30 minutes in a humidified incubator with 5% CO ² at 37°C. The nonadherent cells were then removed by rinsing twice with RPMI-1640, and adherent cells (>98% AMs as assessed by Wright-Giemsa stain) were incubated in 2 mL of RPMI-1640 alone as control or RPMI-1640 with 100 μg/mL of PM₁₀ (EHC-93) suspension with or without different concentrations of statins.

RNA Extraction and Reverse-Transcriptase Polymerase Chain Reaction

Total RNA was isolated from the cells using the Qiagen RNeasy Mini Kit (Qiagen, Ontario, Canada) according to the manufacturer’s instructions and included DNase I (Qiagen) treatment to remove contaminating genomic DNA. Isolated total RNA was analyzed on GeneQuant Pro (Biochrom Ltd, Cambridge, UK). Five micrograms of total RNA was reverse-transcribed with SuperScript™ II first-strand synthesis kit (Invitrogen, Carlsbad, Calif) according to the manufacturer’s instructions. Ten-microliter polymerase chain reactions (PCR) were prepared in 384-well optical reaction plates (ABI Prism™, Applied Biosystems, Foster City, Calif) using TaqMan™ Universal PCR Master Mix (Applied Biosystems). Human β-actin was used as an internal control. The TaqMan probes and primers of human interleukin (IL)-1β (assay ID: Hs00174097_m1), IL-8 (assay ID: Hs00174103_m1), granulocyte-macrophage colony-stimulating factor [GM-CSF] (assay ID: Hs01587813_g1), IL-6 (assay ID: Hs99999032_m1), tumor necrosis factor (TNF)-α (assay ID: Hs99999043_m1), and β-actin (assay ID: Hs99999903_m1) were purchased from Applied Biosystems. Using the ABI Prism® 7900 (Applied Biosystems), samples were heated to 95°C for 10minutes before 40 cycles at 95°C for 15 seconds and 60°C for 1 minute per the manufacturer’s instructions. Real-time data were analyzed using the comparative C_T method, where C_T is the cycle number at which the fluorescence reading is first recorded above background levels. ¹⁸ The comparative C_T method is similar to the standard curve method, except that it uses the arithmetic formula 2^−ΔΔCT to achieve relative quantification. A prior validation experiment demonstrated that amplification efficiencies of the primer/probe sets of the target genes and β-actin were approximately equal and efficiencies were within 0.1. Each sample was assayed in triplicate.

Fluorokine MAP Assay

IL-1β, IL-8, GM-CSF, IL-6, and TNF-α levels in supernatants from HBECs and AMs were measured using the specific Fluorokine MultiAnalyte Profiling (MAP) kits together with the human MAP base kit A (R&D Systems, Minneapolis, Minn; Biomedica, Vienna, Austria) following the manufacturers’ instructions. The supernatant samples were collected from the same samples analyzed for mRNA expression. After a final wash step, the beads were resuspended in buffer and read on the Luminex 100 Analyzer (Biomedica) to determine the concentration of these cytokines. Each sample was assayed in triplicate. Data analysis was performed using the Luminex 100 IS software version 2.3. The minimum detectable concentration was 0.27 pg/mL (IL-1β), 0.39 pg/mL (IL-8), 1.05 pg/mL (GM-CSF), 0.36 pg/mL (IL-6), and 0.47 pg/mL (TNF-α).

Statistical Analysis

Data are expressed as mean values ± SE. For realtime PCR and fluorokine MAP assay, differences between matched pairs (control vs PM₁₀-treated) were compared by Wilcoxon’s signed ranked test. Differences between multiple groups were compared by 1-way analysis of variance. The post hoc test for multiple comparisons was Dunnett’s test. Significance was assumed at P < .05.

Results

Cytokine mRNA Expressions Induced by PM₁₀

In HBECs, mRNA expression of IL-1β, IL-8, GM-CSF, IL-6, and TNF-α was significantly increased after 8 hours of incubation with 100 μg/mL PM₁₀ compared with control (medium alone) (Figure 1A). Also, in AM, PM₁₀ significantly increased cytokine mRNA expression after 2 hours of incubation with 100 μg/mL PM₁₀ (Figures 1B and 1C).

Effect of Atorvastatin on Cytokine mRNA Expression Induced by PM₁₀

To assess the effect of atorvastatin on cytokine mRNA expression induced by PM₁₀, cells were treated with different concentrations of atorvastatin for 24 hours followed by 100 μg/mL PM₁₀ exposure for 8 hours in HBECs (Figure 1A) and for 2 hours in AMs (Figure 1B). Atorvastatin at 10⁻⁸ M attenuated PM₁₀-induced cytokine mRNA expression in AMs but not in HBECs. Higher doses (atorvastatin >10⁻⁶ M) augmented PM₁₀-induced cytokine mRNA expression in both HBECs and AMs (data not shown).

Effect of Pravastatin on Cytokine mRNA Expression Induced by PM₁₀

To confirm that the effect of atorvastatin on PM₁₀-induced cytokine mRNA expression in AMs is a class effect, AMs were treated with pravastatin for 24 hours followed by 100 μg/mL PM₁₀ exposure for 2 hours (Figure 1C). As with atorvastatin, pravastatin attenuated the PM₁₀-induced mRNA expression of all 5 cytokines.

Effect of Atorvastatin on Cytokine Secretion Induced by PM₁₀

We next assessed cytokine protein production by HBECs (Figure 2A) and AMs (Figure 2B) treated with 10⁻⁸ M atorvastatin for 24 hours followed by PM₁₀ (100 μg/mL) exposure for 24 and 8 hours, respectively. With the exception of IL-6 in HBEC, PM₁₀ treatment increased cytokine secretion in both cells. Atorvastatin attenuated PM₁₀-induced IL-1β, IL-8, GM-CSF, IL-6, and TNF-α secretion by AMs but not by HBECs.

Discussion

In the present study, we demonstrated that atorvastatin decreased PM₁₀-induced IL-1β, IL-8, GM-CSF, IL-6, and TNF-α mRNA expression and production by AMs but not by bronchial epithelial cells. Because pravastatin similarly attenuated this mRNA expression, our results most likely reflect a statin class effect.

Mechanistic studies by others and by our own laboratory support the hypothesis that inhalation of small ambient particulate matter causes lung inflammation, driving a systemic inflammation that affects blood vessels. ^19–22 Particulate matter exposure also causes progression of atherosclerosis in both animal models and humans and precipitates acute cardiac events, such as ischemic and arrhythmic cardiac disease, heart failure, and stroke. ^2,22–24

Recent studies have identified anti-inflammatory actions of statins in cardiovascular disease, ⁹ and this is an evolving field of investigation. Our novel results add to the understanding of the anti-inflammatory nature of statins by showing that they reduce proinflammatory mediators produced by lung macrophages. Of these mediators, IL-1β, IL-6, and TNF-α are well-known proinflammatory markers that induce C-reactive protein (CRP) and fibrinogen production by hepatocytes. ²⁵ Circulating CRP is elevated with PM₁₀ exposure, ²⁶ and high levels of CRP are a strong predictor for future cardiovascular risk in healthy men and women. ²⁷ Furthermore, recent studies showed that CRP is not only a marker of vascular disease but also a participant in atherogenesis. ²⁸ It was also demonstrated that treatment of hypercholesterolemic patients with atorvastatin led to a decrease in IL-1β, IL-6, and TNF-α as well as CRP in serum. ²⁹ Taken together with these findings, our results suggest that atorvastatin could potentially attenuate the PM₁₀-induced cardiovascular disease by reducing proinflammatory mediator production in AMs.

Several studies have demonstrated that statins reduce lung injury and inflammation in animal models. ^12,13 In addition, studies in humans have shown that statins could reduce the decline in lung function in smokers ^10,11 and the mortality of chronic obstructive pulmonary disease, which could be affected by PM₁₀. ³⁰ These reports suggest that statins could have a therapeutic effect on several inflammatory pulmonary diseases including bronchial asthma, pulmonary hypertension, idiopathic pulmonary fibrosis, acute lung injury, and community-acquired pneumonia, and as a consequence of lung transplantation. ³¹ The mediators analyzed in our study are key “acute response” proinflammatory mediators that are elevated in many of these inflammatory lung diseases, and the anti-inflammatory effects of atorvastatin on alveolar macrophages may reduce this lung inflammation. Because a close link between lung inflammation and cardiovascular events exists, ³² this effect of statins could translate into additional therapeutic benefits for cardiovascular diseases.

Interestingly, we demonstrated that although PM₁₀ increased expression of cytokine mRNAs in both HBECs and AMs, the corresponding protein expression was suppressed by atorvastatin only in AMs. A possible explanation for this difference between HBECs and AMs may lie in the effect that statins have on the signaling molecules used by these 2 cells types in responding to particulate stimuli. AMs appear to signal through Toll-like receptor (TLR) 4 whereas HBECs do so through TLR2, ³ and statins lower the expression of TLR4 in monocytes ^13,33 and in the lung. ¹³ Although statins also reduce TLR2 expression in monocytes, ³⁴ whether they affect TLR2 in HBECs has not been investigated. Taken together, these results suggest that statins reduce the inflammatory response of AM by lowering the expression of a receptor involved in signaling PM₁₀ exposure. A second mechanism by which atorvastatin could decrease PM₁₀-induced cytokine production by AMs is by inhibiting the phagocytic capacity of these cells. Such a mechanism is supported by Loike et al, ³⁵ who reported that statins blocked phagocytosis by cultured human monocytes and mouse peritoneal macrophages.

Atorvastatin decreased PM₁₀-induced inflammatory cytokines by alveolar macrophages but not by bronchial epithelial cells. This result indicates that statins could reduce the risk of PM₁₀-induced cardiopulmonary diseases through inhibition of inflammatory mediator production by AMs.