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Acute exacerbations of asthma represent a common clinical problem with major economic impact. Air pollutants including ozone have been shown to contribute to asthma exacerbation, but the mechanisms underlying ozone-induced asthma exacerbation are only partially understood. The present study aimed to develop a mouse model to gain insight into the development of airway hyperresponsiveness (AHR) to methacholine (MCh) in mice after exposure to both allergen and ozone. Mice were exposed for 20 min per day for 10 consecutive days to an aerosol of 1% ovalbumin (OVA) or saline followed by a single 3-h exposure to clean air or 100, 250, or 500 ppb ozone. Ozone induced AHR in mice previously exposed to OVA when compared to non-exposed (saline) control mice. After a 10-d exposure to OVA, a single exposure to a low (100 ppb) ozone concentration was sufficient to induce AHR. The AHR response was associated with goblet-cell metaplasia. Even the lowest concentration of ozone tested, 100 ppb, which may be exceeded in urban environments and in the workplace, resulted in a significant increase in AHR, most prominent 24 h after exposure in the OVA-exposed mice.

Ozone is a highly reactive gas present in urban air, which penetrates deep into the lung and causes lung injury. The alveolar epithelial cells are among the first cell barriers encountered by ozone. To define the molecular basis of the cellular response to ozone, primary cultures of rat alveolar type II and type I-like cells were exposed to 100 ppb ozone or air for 1 h. The mRNA from both phenotypes was collected at 4 and 24 h after exposure for gene expression profiling. Ozone produced extensive alterations in gene expression involved in stress and inflammatory responses, transcription factors, antioxidant defenses, extracellular matrix, fluid transport, and enzymes of lipid metabolism and cell differentiation. Real-time reverse transcription-polymerase chain reaction and Western blot analysis verified changes in mRNA and protein levels of selected genes. Besides the increased stress response, ozone exposure downregulated genes of cellular differentiation. The changes were more prominent at 4 h in the type I-like phenotype and at 24 h in the type II phenotype. The type I-like cells were more sensitive to ozone than type II cells. The genome-wide changes observed provide insight into signal pathways activated by ozone and how cellular protection mechanisms are initiated.

Ozone exposure produces acute inflammation and neutrophil influx in the distal lung. Alveolar epithelial cells cover a large surface area, secrete chemokines, and may initiate or modify the inflammatory response. The effect of ozone on chemokine production by these cells has not been defined. Isolated rat type II cells were cultured in different conditions to express the morphologic appearance and biochemical markers for the type I and the type II cell phenotypes. These cells were exposed to ozone at an air/liquid interface. The type I-like cells were more susceptible to injury than the type II cells and showed signs of injury at exposure levels of 100 ppb ozone for 60 min. Both phenotypes showed evidence of lipid peroxidation after ozone exposure as measured by 8-isoprostane production, but neither phenotype secreted increased amounts of MIP-2 (CXCL3), CINC-1 (CXCL1), or MCP-1 (CCL2) in response to ozone. Both cell phenotypes secreted MIP-2 and MCP-1 in response to IL-1beta or lipopolysaccharide, but there was no priming or synergy with ozone. It is likely that the inflammatory response to ozone in the alveolar compartment is not due to the direct effect of ozone on epithelial cells.

The common air pollutant ozone causes acute toxicity to human airways. In primary and transformed epithelial cells from all levels of human or rat airways, ozone levels relevant to air pollution (50-200 ppb) increased extracellular [ATP] within 7-30 min. A human bronchial epithelial cell line (16HBE14o(-)) that forms electrically resistant polarized monolayers had up to 10-fold greater apical than basolateral surface extracellular [ATP] within 7 min of ozone exposure. Increased extracellular [ATP] appeared due to ATP secretion or release because (1) inhibition of ectonucleotidase (cell surface enzyme(s) which degrade ATP) by ozone did not occur until >120 min of ozone exposure and (2) brefeldin A, a secretory inhibitor, eliminated elevation of extracellular [ATP] without affecting intracellular ATP. Extracellular ATP protected against ozone toxicity in a P2Y receptor-dependent manner as (1) removal of ATP and adenosine by apyrase and adenosine deaminase, respectively, potentiated ozone toxicity, (2) extracellular supplementation with ATP, a poorly hydrolyzable ATP analog ATPgammaS, or UTP inhibited apoptotic and necrotic ozone-mediated cell death, and (3) ATP-mediated protection was eliminated by P2 and P2Y receptor inhibitors suramin and Cibacron blue (reactive blue 2), respectively. The decline in glucose uptake caused by prolonged ozone exposure was prevented by supplemental extracellular ATP, an effect blocked by suramin. Further, Akt and ERK phosphorylation resulted from exposure to supplemental extracellular ATP. Thus, extracellularly released ATP signals to prevent ozone-induced death and supplementation with ATP or its analogs can augment protection, at least in part via Akt and /or ERK signaling pathways and their metabolic effects.

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.

Ozone (O3) can induce airway hyperresponsiveness (AHR) and neutrophilic inflammation. We evaluated the role of complement in development of AHR and inflammation after acute O3 exposure in mice. Mice were exposed to O3 at 2 ppm for 3 hours, and airway responsiveness to methacholine was measured 8 hours after O3 exposure. Complement was depleted or inhibited by intraperitoneal injection of cobra venom factor (CVF) or complement receptor-related gene y (Crry)-Ig, a potent C3 convertase inhibitor; neutrophils were depleted using an antineutrophil monoclonal antibody. CVF attenuated the development of AHR by O3. Administration of Crry-Ig also prevented the development of AHR. Bronchoalveolar lavage (BAL) fluid neutrophilia after O3 exposure was significantly decreased by administration of either CVF or Crry-Ig. Increased BAL fluid total protein after O3 exposure was lowered by depletion or inhibition of complement. In contrast to the effects of complement inhibition or depletion, depletion of BAL neutrophil counts by more than 90% with the monoclonal antibody did not affect the development of AHR after O3 exposure. These data indicated that activation of the complement system follows acute O3 exposure and is important to the development of AHR and airway neutrophilia. However, this neutrophil response does not appear necessary for the development of AHR.

Cell lines derived from non-small cell carcinomas of the lung (NSCLC) often express a variety of polypeptide growth factors able to activate receptors that encode tyrosine kinases. These receptors initiate numerous biological responses, including cell proliferation, and constitutive activation can result in cellular transformation. Consequently, it is possible that the aberrant growth properties of NSCLC cell lines is due at least in part to the action of mutated or perpetually activated receptor tyrosine kinases (RTK)s. As a first step in the process of testing this hypothesis we set out to examine the different RTKs expressed in NSCLC cell lines. A small group of NSCLC cell lines was screened for the expression of RTKs using a reverse transcriptase PCR approach with nested degenerate primers. We found four different RTKs expressed: the fibroblast growth factor receptor (FGFR) type III, the FGFR type IV, the platelet-derived growth factor beta receptor (beta PDGFR) and Flk-1/KDR, one of the receptors that binds vascular endothelial growth factor (VEGF). Since Flk-1/KDR and the beta PDGFR are not usually expressed in epithelial cells, we verified that they are expressed in NSCLC by Northern and Western blot analysis. To assess the incidence of expression of these RTKs in NSCLC cell lines a larger panel of cell lines was examined by Western blot analysis. The beta PDGFR was expressed in 30% (3/10) of the cell lines examined, while Flk-1/KDR was expressed in 10% (1/10). We also examined the expression of ligands for these RTKs. PDGF-A, PDGF-B and VEGF were expressed in 89%, 0% and 78%, respectively. While the high incidence of ligand expression made it likely that receptor and growth factor would be coexpressed, we found that in most instances this was not the case. Furthermore, in the cell lines in which the RTK and its ligand were coexpressed, we were unable to detect a functional autocrine loop. These studies indicate that while NSCLC cell lines aberrantly express various RTKs and growth factors, this does not always result in the establishment of an autocrine loop. These findings suggest that growth factors such as PDGF and VEGF act in a paracrine manner to contribute to the growth, survival and angiogenic program of a lung tumor.