Transcriptional profiling of the acute pulmonary inflammatory response induced by LPS: role of neutrophils.

BACKGROUND: Lung cancer often develops in association with chronic pulmonary inflammatory diseases with an influx of neutrophils. More detailed information on inflammatory pathways and the role of neutrophils herein is a prerequisite for understanding the mechanism of inflammation associated cancer. METHODS: In the present study, we used microarrays in order to obtain a global view of the transcriptional responses of the lung to LPS in mice, which mimics an acute lung inflammation. To investigate the influence of neutrophils in this process, we depleted mice from circulating neutrophils by treatment with anti-PMN antibodies prior to LPS exposure. RESULTS: A total of 514 genes was greater than 1.5-fold differentially expressed in the LPS induced lung inflammation model. 394 of the 514 were up regulated genes mostly involved in cell cycle and immune/inflammation related processes, such as cytokine/chemokine activity and signalling. Down regulated genes represented nonimmune processes, such as development, metabolism and transport. Notably, the number of genes and pathways that were differentially expressed, was reduced when animals were depleted from circulating neutrophils, confirming the central role of neutrophils in the inflammatory response. Furthermore, there was a significant correlation between the differentially expressed gene list and the promutagenic DNA lesion M1dG, suggesting that it is the extent of the immune response which drives genetic instability in the inflamed lung. Several genes that were specifically regulated by the presence of activated neutrophils could be identified and these were mostly involved in interferon signalling, oxidative stress response and cell cycle progression. The latter possibly refers to a higher rate of cell turnover in the inflamed lung with neutrophils, suggesting that the neutrophil influx is associated with a higher risk for the accumulation and fixation of mutations. CONCLUSION: Gene expression profiling identified specific genes and pathways that are related to neutrophilic inflammation and could be associated to cancer development and indicate an active role of neutrophils in mediating the LPS induced inflammatory response in the mouse lung.

Lung inflammation is associated with reduced pulmonary nucleotide excision repair in vivo.

Chronic pulmonary inflammation is associated with increased lung cancer risk, but the underlying process remains unknown. Recently, we showed that activated neutrophils inhibit nucleotide excision repair (NER) in pulmonary epithelial cells in vitro via the release of myeloperoxidase (MPO). To evaluate the effect of neutrophils on NER in vivo, mice were intratracheally instilled with lipopolysaccharide (LPS) (20 microg), causing acute lung inflammation and associated neutrophil influx into the airways. Three days post-exposure, phenotypical NER capacity was assessed in lung tissue homogenate. LPS exposure inhibited pulmonary NER by approximately 50%. This finding was corroborated by down-regulation of the NER-associated genes Xpa and Xpf. To further elicit the role of neutrophils and MPO in this process, we utilized MPO-deficient mice as well as mice in which circulating neutrophils were depleted by antibody treatment. LPS-induced inhibition of pulmonary NER was not affected by either Mpo(-/-) or by depletion of circulating neutrophils. This contrasts with our previous in vitro observations, suggesting that inhibition of pulmonary NER following acute dosing with LPS is not fully mediated by neutrophils and/or MPO. In conclusion, these data show that LPS-induced pulmonary inflammation is associated with a reduction of NER function in the mouse lung.

Genotoxic effects of neutrophils and hypochlorous acid.

Chronic inflammation has been recognized as a contributing factor in the pathogenesis of lung cancer. In this process, reactive oxygen species released by neutrophils may play an important role. The aim of the present study was to investigate the capacity of the major neutrophilic oxidant hypochlorous acid (HOCl), which is formed by myeloperoxidase (MPO), to induce DNA damage and mutagenicity in lung cells. HOCl was mutagenic in lung epithelial A549 cells in vitro, showing at physiological concentrations a significant induction of mutations in the HPRT gene. We studied three major types of DNA lesions that could be relevant for this HOCl-induced mutagenicity. Single strand DNA breakage and 8-oxo-7,8-dihydro-2'-deoxyguanosine were not found to be increased following HOCl treatment. On the other hand, HOCl caused a significant increase in the formation of 3-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrimido[1,2-alpha]purin-10(3H)-one (M(1)dG), which can be formed by either malondialdehyde (MDA) or base propenals. We observed an increased MDA formation upon exposure of A549 cells to HOCl, but a role of base propenals cannot be excluded. In line with this, we observed 4-fold increased M(1)dG adduct levels in mice that were intratracheally instilled with lipopolysaccharide to induce a pulmonary inflammation with neutrophil influx. Depletion of circulating neutrophils significantly reduced pulmonary MPO activity as well as M(1)dG adducts levels, thereby providing a causal link between neutrophils/HOCl and pulmonary genotoxicity in vivo. Taken together, these data indicate that MPO catalysed formation of HOCl during lung inflammation should be considered as a significant source of neutrophil-induced genotoxicity.

Activated neutrophils inhibit nucleotide excision repair in human pulmonary epithelial cells: role of myeloperoxidase.

Neutrophils are thought to affect pulmonary carcinogenesis by promoting the metabolism of inhaled chemical carcinogens, causing enhanced formation of promutagenic DNA adducts. We hypothesized that neutrophils interfere with the removal of such DNA adducts by inhibiting nucleotide excision repair (NER) in target cells. Human alveolar epithelial cells (A549) were cocultured with activated neutrophils, and we observed a significant reduction of NER in the A549 cells, which was abrogated by addition of the myeloperoxidase (MPO) inhibitor 4-aminobenzoic acid hydrazide. The inhibitory effect of neutrophils could be mimicked by the MPO product hypochlorous acid (HOCl), which caused an acute, dose-dependent inhibition of NER in A549 cells. This was independent of cytotoxicity or ATP loss and persisted up to 24 h. These data were supported by showing that HOCl caused a delayed removal of DNA adducts in benzo[a]pyrene-diolepoxide-exposed A549 cells. The acute HOCl-induced inhibition of NER can only partly be explained by oxidative modification of repair proteins. To explain the more persistent effects of HOCl, we analyzed the expression of NER genes and found that HOCl significantly reduced XPC expression. In conclusion, these data indicate that neutrophils are potent inhibitors of nucleotide excision repair. This may provide a further biological explanation for the association between inflammation and lung cancer development.

Neutrophils and respiratory tract DNA damage and mutagenesis: a review.

Inflammation has been recognized as an important factor in cancer development. For the lung, experimental studies with rats, as well as molecular epidemiological studies in humans, have provided evidence that the influx of neutrophils into the airways may be an important process linking inflammation with carcinogenesis. Currently it is believed that the genotoxic capacity of neutrophils is a crucial aetiological factor in this carcinogenic response. In the present review we discuss two major pathways of neutrophil-induced genotoxicity: (i) induction of oxidative DNA damage through release of reactive oxygen species (ROS) and (ii) myeloperoxidase (MPO)-related metabolic activation of chemical carcinogens. So far, direct evidence for a role of neutrophils in pulmonary genotoxicity has largely been derived from in vitro studies using co-cultures of activated neutrophils and target cells. Current evidence from in vivo studies is primarily indirect and additional animal studies are needed to substantiate causality. A further challenge will be to extrapolate results from such studies to humans. Taken together, this will provide a better insight into the role of neutrophils in pulmonary carcinogenicity and may, hence, lead to novel approaches for cancer prevention strategies.