Diesel exhaust particles induce the over expression of tumor necrosis factor-alpha (TNF-alpha) gene in alveolar macrophages and failed to induce apoptosis through activation of nuclear factor-kappaB (NF-kappaB)

Abstract

Exposure to particulate matter (PM2.5-10), including diesel exhaust particles (DEP) has been reported to induce lung injury and exacerbation of asthma and chronic obstructive pulmonary disease. Alveolar macrophages play a major role in the lung's response to inhaled particles and therefore, are a primary target for PM2.5-10 effect. The molecular and cellular events underlying DEP-induced toxicity in the lung, however, remain unclear. To determine the effect of DEP on alveolar macrophages, RAW 264.7 cells were grown in RPMI 1640 with supplements until confluency. RAW 264.7 cultures were exposed to Hank's buffered saline solution (vehicle), vehicle containing an NF-kappaB inhibitor, BAY11-7082 (25 microM with 11/2 hr pre-incubation), or vehicle containing DEP (250 microg/ml) in the presence or absence of BAY11-7082 (25 microM with 11/2 hr pre-incubation) for 4 hr and TNF-alpha release was determined by enzyme-linked immunosorbent assay and confirmed by western blots. RAW 264.7 apoptotic response was determined by DNA fragmentation assays. U937 cells treated with campothecin (4 microg/ml x 3 hr), an apoptosis-inducing agent, were used as positive control. We report that exposure to the carbonaceous core of DEP induces significant release of TNF-alpha in a concentration-dependent fashion (31 +/- 4 pg/ml, n = 4, p = 0.08; 162 +/- 23 pg/ml, n = 4, p < 0.05; 313 +/- 31 pg/ml, n = 4, p < 0.05 at 25, 100, and 250 microg/ml, respectively). DEP exposure, however, failed to induce any apoptotic response in RAW 264.7 cells. Moreover, inhibition of NF-kappaB binding activity has resulted in DEP-induced apoptotic response in alveolar macrophages, as demonstrated by the NF-kappaB inhibitor, BAY11-7082 studies. The results of the present study indicate that DEP induce the release of TNF-alpha in alveolar macrophages, a primary target for inhaled particles effect. DEP-induced TNF-alpha gene expression is regulated at the transcriptional level by NF-kappaB. Furthermore, DEP-induced increase in NF-kappaB-DNA binding activity appears to protect against apoptosis.

Figure 1

Effect of Diesel Exhaust Particles (DEP) on Tumor Necrosis Factor-α (TNF-α) Production in Alveolar Macrophages. Confluent cultures of alveolar macrophages (RAW 264.7) were exposed to vehicle (HBSS/30 mM Hepese) alone, vehicle containing DEP at 25, 100, or 250μg/ml, or IL-1β at 0.1μg/ml for 4 hours. Following exposure, TNF-α release was measured in control and exposed cultures using mouse ELISA. Data (pg/ml) are presented as mean ± SEM (n). *Significant difference (p < 0.05) from control values.

Figure 2

Tumor Necrosis Factor-α (TNF-α) Protein Analysis by Western Blotting. Confluent monolayers of RAW 264.7 cells were treated with vehicle (HBSS/Hepes) alone, vehicle containing DEP (250 μg/ml), or IL-1β (0.1μg/ml) for 4 hours. Following the various treatments cells were harvested and lysed by sonication in lysis buffer on ice. Denatured total proteins (20 μg) of cytosolic and nuclear fractions were separated on 12% denaturing polyacrylamide gels and electrotransferred to Hybond nitrocellulose membranes. Following standard blocking and washing procedures, the membranes were incubated with polyclonal antibodies against TNF-α, and horseradish peroxidase-conjugated anti-goat IgG secondary antibodies. Detection of proteins was performed by enhanced chemiluminescence (ECL). Panel shows bands corresponding to treatment groups: 1. Control Cultures, 2. DEP-Treated Cultures, and 3. IL-1β-Treated Cultures.

Figure 3

Effect of Diesel Exhaust Particles (DEP) on Alveolar Macrophage Apoptosis. Confluent monolayers of RAW 264.7 cells were treated with vehicle (HBSS/Hepes) alone (control), vehicle containing DEP at 250 μg/ml (DEP), or IL-1β at 0.1 μg/ml for 4 hr. Following treatment, DNA was isolated and fragmentation was assessed by DNA ladder kit on agarose according to the manufacturer’s directions. U937 cells treated with campothecin, an apoptosis-inducing agent were used as positive control. Pane shows bands corresponding to treatment groups: molecular weight marker, 1; positive control, 2; control cultures, 3, 4, 5, in triplicate; DEP-treated cultures, 6, 7, 8, in triplicate; and IL-1β-treated cultures, 9, 10, 11, in triplicate.

Figure 4

Effect of Nuclear Factor-κB (NF-κB) Inhibition on Tumor Necrosis Factor-α (TNF-α) Production in Alveolar Macrophages. Monolayers of RAW 264.7 cells were pre-treated with BAY11-7082 (25μg/ml × 1 1/2 hr), an NF-κB inhibitor. Following pre-treatment, RAW 264.7 cultures were treated with vehicle (HBSS/Hepes) alone, or vehicle containing DEP (250 μg/ml × 4 hr) or IL-1β (0.1μg/ml × 4 hr), and TNF-α release in cellular supernatants were determined by enzyme-linked immunosorbent assays (ELISA). The data (pg/ml) are presented as mean ± SEM (n). *Significant inhibition (p < 0.05) from DEP-, or IL-1β-treated cultures.

Figure 5

Effect of Nuclear Factor-κB (NF-κB) Inhibition on Diesel Exhaust Particles (DEP)-induced apoptosis in Alveolar Macrophages. Monolayers of RAW 264.7 cells were pre-treated with BAY11-7082 (25 μg/ml × 1 1/2 hr), an NF-κB inhibitor. Following pre-treatment, RAW 264.7 cultures were treated with vehicle (HBSS/Hepes) alone, or vehicle containing DEP (250 μg/ml × 4 hr). Following treatment, DNA was isolated and fragmentation was assessed by DNA ladder kit on agarose according to the manufacturer’s directions. U937 cells treated with campothecin (3 μM × 4 hr), an apoptosis-inducing agent were used as positive control (positive control). Lanes are as follows: molecular weight marker, 1; positive control, 2; DEP-exposed cultures pre-treated with BAY11-7082, 3; DEP-exposed cultures, 4; control cultures, 5; and control cultures pre-treated with BAY11-7082, 6.