Ozone exposure of macrophages induces an alveolar epithelial chemokine response through IL-1alpha

Abstract

Ozone is known to produce an acute influx of neutrophils, and alveolar epithelial cells can secrete chemokines and modulate inflammatory processes. However, direct exposure of alveolar epithelial cells and macrophages to ozone (O(3)) produces little chemokine response. To determine if cell-cell interactions might be responsible, we investigated the effect of alveolar macrophage-conditioned media after ozone exposure (MO(3)CM) on alveolar epithelial cell chemokine production. Serum-free media were conditioned by exposing a rat alveolar macrophage cell line NR8383 to ozone for 1 hour. Ozone stimulated secretion of IL-1alpha, IL-1beta, and IL-18 from NR8383 cells, but there was no secretion of chemokines or TNF-alpha. Freshly isolated type II cells were cultured, so as to express the biological markers of type I cells, and these cells are referred to as type I-like cells. Type I-like cells were exposed to diluted MO(3)CM for 24 hours, and this conditioned medium stimulated secretion of cytokine-induced neutrophil chemattractant-1 (CXCL1) and monocyte chemoattractant protein-1 (CCL2). Secretion of these chemokines was inhibited by the IL-1 receptor antagonist. Although both recombinant IL-1alpha and IL-1beta stimulated alveolar epithelial cells to secrete chemokines, recombinant IL-1alpha was 100-fold more potent than IL-1beta. Furthermore, neutralizing anti-rat IL-1alpha antibodies inhibited the secretion of chemokines by alveolar epithelial cells, whereas neutralizing anti-rat IL-1beta antibodies had no effect. These observations indicate that secretion of IL-1alpha from macrophages stimulates alveolar epithelial cells to secrete chemokines that can elicit an inflammatory response.

Figure 1.

Ozone increases inflammatory cytokine secretion in NR8383 cells. NR8383 cells were cultured in 150-mm dishes and exposed to air and different concentrations of ozone for 1 hour. The media were collected immediately after exposure and the cytokines were measured by a multiplex assay as described in Materials and Methods. The results are shown as percent (%) change over control. The control values for these cytokines were (A) IL-1α, 113 pg/ml; (B) IL-1β, 26.7 pg/ml; (C) IL-18, 599 pg/ml; (D) granulocyte-macrophage colony-stimulating factor, 7.2 pg/ml; (E) IL-2, 6.2 pg/ml; and (F) IL-6, 12.7 pg/ml. The results are the means of ± SEM of three independent experiments. The asterisk signifies an increase compared to the air control (P < 0.05).

Figure 2.

NR8383 cell conditioned media increases cytokine-induced neutrophil chemoattractant (CINC-1) and monocyte chemoattractant protein (MCP-1) secretion from rat type I–like cells. Alveolar type II cells were cultured with fetal bovine serum on plastic plates to transdifferentiate into type I–like cells. Conditioned media from NR8383 cells supplemented with fresh media were added to the type I–like cells on Day 5 of culture, and the media were collected 24 hours later. Conditioned media increased the secretion of the chemokines (A) CINC-1 and (B) MCP-1, but (C) RANTES remained unchanged. The original values are shown as pg/ml. The results are the means of ± SEM of three independent experiments. * An increase compared with the untreated control; # an increase compared with air-exposed conditioned media (P < 0.05).

Figure 3.

IL-1α and IL-1β increase CINC-1 secretion from rat alveolar type I–like epithelial cells. Type I–like cells were cultured as described in Figure 2, and exogenous IL-1α, IL-1β, and IL-18 were added on Day 5 of culture. The media were collected 24 hours later. The values are shown as pg/ml. A shows the dose response of IL-1α (squares) and IL-1β (triangles), and B shows the effect of different concentrations of IL-18 and 10 ng/ml of IL-1β as a positive control.

Figure 4.

CINC-1 secretion by rat alveolar epithelial cells. Rat alveolar type I–like epithelial cells were cultured as described in Figure 2, and left untreated or pretreated with IL-1 antagonist (10 μg/ml) for 1 hour and then incubated with conditioned media. Media were collected 24 hours later and CINC-1 measured by enzyme-linked immunosorbent assay. A shows the results with NR8383-conditioned media and B shows the results with primary rat alveolar macrophage–conditioned media. The asterisk signifies inhibition compared with the respective control (P < 0.05).

Figure 5.

Macrophage IL-1α is primarily responsible for CINC-1 production from rat alveolar type I–like epithelial cells. Rat alveolar type I–like cells were cultured as described in Figure 2. Conditioned media generated from ozone-exposed macrophages were pretreated with IL-1α–selective neutralizing antibody (IL-1α NA), IL-1β NA, or control isotype match IgG at concentration of 3 μg/ml for 1 hour at room temperature before treatment of cultured cells. IL-1α NA alone as well as in combination with IL-1β NA significantly inhibited CINC-1 secretion compared with the IgG control condition (P < 0.05). In contrast, administration of IL-1β NA had no significant effect on CINC-1 secretion in these cultures.

Figure 6.

Schematic diagram of proposed mechanisms of ozone toxicity and cellular responses. Our hypothesis is that ozone induces release of IL-1α from alveolar macrophages, which in turn stimulates alveolar epithelial cells to secrete CXC chemokines, predominantly CINC-1 (CXCL-1). Consequently, the chemokines recruit the neutrophils to infiltrate the airspace.