African Dust Storms Reaching Puerto Rican Coast Stimulate the Secretion of IL-6 and IL-8 and Cause Cytotoxicity to Human Bronchial Epithelial Cells (BEAS-2B)

Abstract

African dust storm events (ADE) travel across the Atlantic Ocean (ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric particulate matter (PM) size fractions, composition and sources trigger respiratory-adverse effects to Puerto Ricans is still unclear. In the present study, we investigated the pro-inflammatory and cytotoxic effects of PM samples harvested during ADEAO (PM₁₀), ADEPRC (PM_2.5 and PM₁₀) and Non-ADE (Preand Post-ADEAO and Non-ADEPRC), using BEAS-2B cells. Endotoxins (ENX) in PM_2.5 and PM₁₀ extracts and traces of metals (TMET) in PM_2.5 extracts were also examined. IL-6 and IL-8 secretion and cytotoxicity were used as endpoints. ADEAO and ADEPRC extracts were found to be more cytotoxic than Non-ADE and ADEAO were more toxic than ADEPRC extracts. PM₁₀ extracts from ADEAO and Post-ADEAO caused significant secretion of IL-8. IL-6 and IL-8 secretion was higher following treatment with PM₁₀ and PM_2.5 ADEPRC than with Non-ADEPRC extracts. ENX levels were found to be higher in PM₁₀ ADEAO than in the rest of the samples tested. TMET levels were higher in PM_2.5 ADEPRC than in Non-ADEPRC extracts. Deferoxamine significantly reduced cytotoxicity and IL-6 and IL-8 secretion whereas Polymyxin B did not. TMET in PM_2.5 fractions is a major determinant in ADEPRC-induced toxicity and work in conjunction with ENX to cause toxicity to lung cells in vitro. ENX and TMET may be responsible, in part, for triggering PM-respiratory adverse responses in susceptible and predisposed individuals.

Keywords: BEAS-2B cells; dust storm; endotoxins; metals; particulate matter.

Figure 1

PM_2.5 and PM₁₀ monthly mean concentrations reported in 2004 by PREQB for the rural coastal region of Fajardo, Puerto Rico.

Figure 2

Cytotoxic effect of PM₁₀ extracts in BEAS-2B Cells. Cells were treated for 24 h with PM₁₀ extracts collected from the Atlantic Ocean (A: ADEAO) and Puerto Rican Coast (B: ADEPRC). LC₅₀ (lethal concentration of the PM extracts that kills 50% of the cells in 24 h) values were determined. ADEAO (LC₅₀=37 mg/ml; Post-ADEAO LC₅₀=34 mg/ml) and ADEPRC (LC₅₀=38 mg/ml; Non-ADEPRC LC₅₀=55 mg/ml) extracts were cytotoxic; however pre-ADEAO extracts were not. All extract concentrations were tested in 3 independent experiments.

Figure 2

Cytotoxic effect of PM₁₀ extracts in BEAS-2B Cells. Cells were treated for 24 h with PM₁₀ extracts collected from the Atlantic Ocean (A: ADEAO) and Puerto Rican Coast (B: ADEPRC). LC₅₀ (lethal concentration of the PM extracts that kills 50% of the cells in 24 h) values were determined. ADEAO (LC₅₀=37 mg/ml; Post-ADEAO LC₅₀=34 mg/ml) and ADEPRC (LC₅₀=38 mg/ml; Non-ADEPRC LC₅₀=55 mg/ml) extracts were cytotoxic; however pre-ADEAO extracts were not. All extract concentrations were tested in 3 independent experiments.

Figure 3

Effect of Polymyxin B (PMB, an ENX inhibitor) and PM₁₀ ADEPRC extracts on BEAS-2B cell viability. Cells were treated with PM₁₀ ADEPRC extracts in the presence (w PMB) and absence of PMB (w/o PMB) at 10 mg/ml for 24 h and cell viability was evaluated. Triton-X (+CNT) at 25 mg/ml was used as positive control. Bars represent mean cell viability ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. All samples were tested in three independent experiments; ***p < 0.001

Figure 4

Induction of IL-8 in BEAS-2B exposed to PM₁₀ extracts collected before, during and after the Atlantic Ocean dust storm (ADEAO). LPS (10 mg/ml) was used as a positive control. Bars represent mean IL-8 concentrations ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All samples were tested in three independent experiments; *p < 0.05.

Figure 5

Effect of Polymyxin B (PMB, an ENX inhibitor) at 10 mg/ml on the induction IL-6 (A) and IL-8 (B) in BEAS-2B exposed to PM₁₀ (ADEPRC) extracts for 24 h. Cells were treated with PM₁₀ ADEPRC extracts in the presence (w PMB) and absence of PMB (w/o PMB) and cytokine induction was measured. LPS (10 mg/ml) was used as a positive control. Bars represent mean IL-8 concentration ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All samples were tested in three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 5

Effect of Polymyxin B (PMB, an ENX inhibitor) at 10 mg/ml on the induction IL-6 (A) and IL-8 (B) in BEAS-2B exposed to PM₁₀ (ADEPRC) extracts for 24 h. Cells were treated with PM₁₀ ADEPRC extracts in the presence (w PMB) and absence of PMB (w/o PMB) and cytokine induction was measured. LPS (10 mg/ml) was used as a positive control. Bars represent mean IL-8 concentration ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All samples were tested in three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 6

Cytotoxic effect of PM_2.5 extracts in BEAS-2B Cells. Cells were treated for 24 h with PM_2.5 extracts collected in the Puerto Rican coast during the African storm (ADEPRC) and non-storm (Non-ADEPRC) season. LC₅₀ (lethal concentration of the PM extracts that kills 50% of the cells in 24 h) values were determined. ADEPRC (LC₅₀ = 122 mg/ml (extrapolated) and Non-ADEPRC (LC₅₀ = 191 mg/ml (extrapolated) were cytotoxic. All extract concentrations were tested in 3 independent experiments

Figure 7

Effect of Deferoxamine (DF, a metal chelator) and PM_2.5 ADEPRC extracts on BEAS-2B cell viability. Cells were treated with PM_2.5 extracts collected during the dust storm (ADEPRC) and non-storm (Non-ADEPRC) season in the presence (w DF) and absence of DF (w/o DF) at 50 μM for 24 h and cell viability was evaluated. Triton-X (+CNT) at 25 mg/ml was used as positive control. Bars represent mean cell viability ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All extract concentrations were tested in 3 independent experiments; ***p < 0.001, *p < 0.05.

Figure 7

Effect of Deferoxamine (DF, a metal chelator) and PM_2.5 ADEPRC extracts on BEAS-2B cell viability. Cells were treated with PM_2.5 extracts collected during the dust storm (ADEPRC) and non-storm (Non-ADEPRC) season in the presence (w DF) and absence of DF (w/o DF) at 50 μM for 24 h and cell viability was evaluated. Triton-X (+CNT) at 25 mg/ml was used as positive control. Bars represent mean cell viability ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All extract concentrations were tested in 3 independent experiments; ***p < 0.001, *p < 0.05.

Figure 8

Induction of IL-6 (a) and IL-8 (b) in BEAS-2B exposed to Puerto Rican coast (ADEPRC) PM_2.5 extracts for 24 h. LPS (10 mg/ml) was used as a positive control. Bars represent mean IL-6 and IL-8 concentrations ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All samples were tested in three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 9

Effect of Deferoxamine (DF, a metal chelator) at 50 μM on the induction IL-6 (a) and IL-8 (b) in BEAS-2B exposed to PM_2.5 (ADEPRC) extracts for 24 h. Cells were treated with PM_2.5 extracts collected during the dust storm (ADEPRC) and non-storm (Non-ADEPRC) season in the presence (w DF) and absence of DF (w/o DF) cytokine induction was measured. LPS (10 mg/ml) was used as a positive control. Bars represent mean IL-6 and IL-8 concentrations ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All samples were tested in three independent experiments; **p < 0.01, *p < 0.05.

Figure 9

Effect of Deferoxamine (DF, a metal chelator) at 50 μM on the induction IL-6 (a) and IL-8 (b) in BEAS-2B exposed to PM_2.5 (ADEPRC) extracts for 24 h. Cells were treated with PM_2.5 extracts collected during the dust storm (ADEPRC) and non-storm (Non-ADEPRC) season in the presence (w DF) and absence of DF (w/o DF) cytokine induction was measured. LPS (10 mg/ml) was used as a positive control. Bars represent mean IL-6 and IL-8 concentrations ± SEM. Stars over the bars indicate comparison of a treatment with DMSO. Stars over the line indicate a comparison between two treatments. All samples were tested in three independent experiments; **p < 0.01, *p < 0.05.

Figure 10

Schematic model explaining the movement of African dust through the Atlantic Ocean (ADEAO) reaching the Puerto Rican coast (ADEPRC) and the possible relationship between its constituents (e.g., PM_2.5-10, ENX, TMET, other PM constituents). Briefly, as African dust crosses the Atlantic Ocean, PM_2.5 and PM₁₀ distribution along with their constituents are modified. Upon arrival to the Puerto Rican coast, ENX levels are reduced whereas the levels of TMET are increased. ENX and TMET are cytotoxic to human bronchial epithelial cells, and can lead to inflammation and oxidative stress. This detrimental response may be caused due to the release of pro-inflammatory cytokines (e.g., IL-6 and IL-8), activation of nuclear receptor pathways (e.g., NF-kB, Nrf2), and the formation of reactive oxygen species (ROS). Interrogative symbols (?) represent factors involved in the signal transduction mediating cytokine secretion that are currently under study. Transient receptor potential (TRP) has also been associated with health adverse effects of ambient PM. TRP mediate calcium influx, activate signal transduction pathways (e.g., MAP kinases) and modulate MMP (matrix metalloproteases) causing lung injury. Small circles represent PM_2.5 and large circles PM₁₀; Endotoxins (ENX) are represented as red squares, traces of metals (TMET) as green triangles and other potential PM constituents as light blue stars. Cytokines are represented as dark blue and green stars.