Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells

Abstract

Background: Cigarette smoke mediated oxidative stress and inflammatory events in the airway and alveolar epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Previously, individual cell lines were used to assess the oxidative and proinflammatory effects of cigarette smoke with confounding results. In this study, a panel of human and rodent transformed epithelial cell lines were used to determine the effects of cigarette smoke extract (CSE) on oxidative stress markers, cell toxicity and proinflammatory cytokine release and compared the effects with that of primary human small airway epithelial cells (SAEC).

Methods: Primary human SAEC, transformed human (A549, H1299, H441), and rodent (murine MLE-15, rat L2) alveolar epithelial cells were treated with different concentrations of CSE (0.2-10%) ranging from 20 min to 24 hr. Cytotoxicity was assessed by lactate dehydrogenase release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide. Glutathione concentration was measured by enzymatic recycling assay and 4-hydroxy-2-nonenal levels by using lipid peroxidation assay kit. The levels of proinflammatory cytokines (e.g. IL-8 and IL-6) were measured by ELISA. Nuclear translocation of the transcription factor, NF-kappaB was assessed by immunocytochemistry and immunoblotting.

Results: Cigarette smoke extract dose-dependently depleted glutathione concentration, increased 4-hydroxy-2-nonenal (4-HNE) levels, and caused necrosis in the transformed cell lines as well as in SAEC. None of the transformed cell lines showed any significant release of cytokines in response to CSE. CSE, however, induced IL-8 and IL-6 release in primary cell lines in a dose-dependent manner, which was associated with the nuclear translocation of NF-kappaB in SAEC.

Conclusion: This study suggests that primary, but not transformed, lung epithelial cells are an appropriate model to study the inflammatory mechanisms in response to cigarette smoke.

Figure 1

Cigarette smoke extract differentially caused cytotoxicity in a variety of alveolar epithelial cells and in primary human small airway epithelial cells. A. Various alveolar epithelial cells such as human lung cancer cells (H1299), human adenocarcinoma cells (A549), human lung epithelial cell from papillary adenocarcinoma patient (H441), rat lung epithelial cells (L2), and murine type II epithelial cells (MLE-15) were exposed to different concentrations of cigarette smoke (1R3F) extract (1.0–10.0%) for 24 hr, and % cytotoxicity induced was measured as lactate dehydrogenase release. CSE differentially induced cytotoxicity in concentration dependent manner in all the five epithelial cell lines. Amongst the five cell lines studied, H1299 cells were most resistant and MLE 15 cells were the least resistant. B. Primary human small airway epithelial cells (SAEC) were exposed to different concentrations of cigarette smoke (1R3F) extract (0.2–5.0%) for 24 hr and percentage (%) cytotoxicity induced was measured as LDH release. CSE dose-dependently induced LDH release in SAEC. Data represent mean ± SEM of 3 experiments. *p < 0.05, ^#p < 0.01, and ^§p < 0.001 compared to control group. CSE: cigarette smoke extract.

Figure 2

Cigarette smoke extract caused necrosis with no or little evidence of apoptosis in human lung cancer cells (H1299). Human lung cancer cells (H1299) were treated with media alone (control) and various concentrations of CSE; a) control, b) CSE (1.0%), c) CSE (2.5%), d) CSE (5.0%), e) CSE (7.5%), f) CSE (10%) for 24 hr. The cells were stained with ethidium bromide and acridine orange and observed under fluorescence microscopy. Living cells had normal shaped nuclei with green chromatin. Early apoptotic cells have shrunken green nuclei with chromatin condensation, whereas necrotic or late apoptotic cells had normal/condensed nuclei that were brightly stained with ethidium bromide and appeared red. Percentage of viable (white bars), apoptotic (grey bars) and necrotic/late apoptotic (black bars) determined by counting as described in Materials and Methods. Results are mean of 3 experiments ± SEM. *p < 0.05, and ^§p < 0.001 compared with control group. L = Live; A = Apoptosis; N = Necrosis.

Figure 3

Cigarette smoke extract induced necrosis with no or little evidence of apoptosis in human adenocarcinoma cells (A549). Human adenocarcinoma cells (A549) were treated with media alone (control) and various concentrations of CSE; a) control, b) CSE (1.0%), c) CSE (2.5%), d) CSE (5.0%), e) CSE (7.5%), f) CSE (10%) for 24 hr. Results are mean of 3 experiments ± SEM. ^#p < 0.01, and ^§p < 0.001 compared with control group. L = Live; A = Apoptosis; N = Necrosis.

Figure 4

Cigarette smoke extract caused necrosis with no or little evidence of apoptosis in human lung epithelial cell from papillary adenocarcinoma patient (H441). Human lung epithelial cell from papillary adenocarcinoma patient (H441) were treated with media alone (control) and various concentrations of CSE; a) control, b) CSE (1.0%), c) CSE (2.5%), d) CSE (5.0%), e) CSE (7.5%), f) CSE (10%) for 24 hr. Results are mean of 3 experiments ± SEM. ^#p < 0.01, and ^§p < 0.001 compared with control group. L = Live; A = Apoptosis; N = Necrosis.

Figure 5

Cigarette smoke extract caused necrosis with no or little evidence of apoptosis in rat lung epithelial cells (L2). Rat lung epithelial cells (L2) were treated with media alone (control) and various concentrations of CSE; a) control, b) CSE (1.0%), c) CSE (2.5%), d) CSE (5.0%), e) CSE (7.5%), f) CSE (10%) for 24 hr. Results are mean of 3 experiments ± SEM. ^§p < 0.001 compared with control group. L = Live; A = Apoptosis; N = Necrosis.

Figure 6

Cigarette smoke extract caused necrosis with no or little evidence of apoptosis in murine type II epithelial cells (MLE-15). Murine type II epithelial cells (MLE-15) were treated with media alone (control) and various concentrations of CSE; a) control, b) CSE (1.0%), c) CSE (2.5%), d) CSE (5.0%), e) CSE (7.5%), f) CSE (10%) for 24 hr. Results are mean of 3 experiments ± SEM. ^§p < 0.001 compared with control group L = Live; A = Apoptosis; N = Necrosis.

Figure 7

Cigarette smoke extract caused necrosis with no or little evidence of apoptosis in primary human small airway epithelial cells (SAEC). Primary human small airway epithelial cells (SAEC) were treated with media alone (control) and various concentrations of CSE; a) control, b) CSE (0.2%), c) CSE (0.5%), d) CSE (1.0%), e) CSE (2.5%), f) CSE (5.0%) for 24 hr. Results are mean of 3 experiments ± SEM. ^#p < 0.01, and ^§p < 0.001 compared with control group. L = Live; A = Apoptosis; N = Necrosis.

Figure 8

Cigarette smoke extract dose-dependently caused lipid peroxidation measured as 4-hydroxy-2-nonenal levels in alveolar epithelial cells as well as in primary human small airway epithelial cells. A. Transformed alveolar epithelial cells were exposed to cigarette smoke (1R3F) extract (1.0 – 5.0 %) for 24 hr and the extent of lipid peroxidation was determined by measuring 4-HNE levels. Cigarette smoke extract increased the levels of 4-HNE in all the five transformed alveolar epithelial cell lines in dose-dependent manner. However, the baseline levels of 4-HNE were varied amongst the cell lines, H1299 with lower base line levels and MLE-15 with higher baseline levels. B. Primary human small airway epithelial cells (SAEC) were exposed to cigarette smoke extract (0.2%-1.0 %) derived from 1R3F research grade cigarettes for 24 hr, and the levels of 4-HNE were measured. Cigarette smoke extract dose-dependently increased the levels of 4-HNE levels SAEC. Data represent mean ± SEM of 3 individual experiments. ^§p < 0.001 compared to control values. CSE: cigarette smoke extract.

Figure 9

Cigarette smoke extract showed differential effects on intracellular reduced glutathione levels in alveolar epithelial cells and in primary human small airway epithelial cells. A. Transformed alveolar epithelial cell lines of our interest; H1299, A549, H441, L2 and MLE-15 were treated with cigarette smoke extract (1.0–5.0%) for 24 hr. After incubation period, GSH levels were measured by the Tietze method. Although, the baseline GSH levels were varied amongst the cell lines, CSE decreased GSH levels dose-dependently at 24 hr in all five epithelial cell lines. The most resistant cell line H1299 had higher baseline GSH levels whereas the least resistant MLE-15 had lower baseline GSH levels. B. Primary human small airway epithelial cells (SAEC) were also treated with cigarette smoke extract (0.2–1.0%) for 4 and 24 hrs, and GSH levels were measured. CSE dose- dependently decreased GSH levels in SAEC at 4 hr, where as the levels were increased dose-dependently at 24 hr. Data is representative of 3 separate experiments ± SEM. *p < 0.05, ^#p < 0.01, and ^§p < 0.001 compared with corresponding control. CSE: cigarette smoke extract.

Figure 10

Cigarette smoke extract treatment caused NF-κB RelA/p65 nuclear translocation in primary human small airway epithelial cells. Primary human small airway epithelial cells were grown in 8-well chamber slides and were exposed for 20 min. to CSE (1.0%) prepared from 1R3F research grade cigarettes. TNF-α (10 ng/ml) was used as a positive. After treatment period, the cells were incubated with NF-κB RelA/p65 antibody and were visualized under fluorescent microscope. Cigarette smoke extract and TNF-α treatments caused nuclear translocation of NF-κB RelA/p65.

Figure 11

Cigarette smoke extract mediated nuclear translocation of NF-κB RelA/p65 was associated with increased nuclear levels of NF-κB RelA/p65 protein in human primary small airway epithelial cells. The primary human SAEC were treated with CSE (0.5 and 1.0%), and TNF-α (10 ng/ml) for 1 hr and nuclear proteins were isolated. Twenty microgram of nuclear protein was electrophoresed on SDS-PAGE and electroblotted onto membranes. A). Western blot showing increased nuclear levels of RelA/p65 in CSE and TNF-α treated SAEC at 1 hr. B). Nuclear protein levels of NF-κB p65 were expressed as the percentage of ratio of RelA/p65 versus actin in human SAEC. Each histogram is a representative of 3 separate experiments ± SEM. *p < 0.05, ^#p < 0.01, and ^§p < 0.001 compared with control. CSE: cigarette smoke extract.