Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H2O2 is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, andmore in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.     

Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.     

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

The effects of benzene exposure on apoptosis in epithelial lung cells: Localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene products

Although benzene, a well-known human carcinogen, has been shown to induce apoptosis in vitro, no studies have been carried out to confirm and characterize its role in activating apoptosis in vivo. The present study investigated the effects of benzene inhalation on the epithelial cells lining the respiratory tract including bronchioles, terminal bronchioles, respiratory bronchioles and alveoli of male Sprague-Dawley rats. Inhalation of benzene 300 ppm for 7 days induced apoptotic changes in the parenchymal components in the lung that significantly exceeded the events of programmed cell death in normal control tissues. Apoptosis was confirmed by the electrophoretic analysis of internucleosomal DNA fragmentation of benzene-exposed lung tissues, which exhibited 180–200 bp laddering subunits indicative of genomic DNA degradation. Furthermore, semi-quantitative analysis of intracellular localization of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling TUNEL) showed a significant (p < 0.001) increase in the apoptotic index calculated for bronchiolar 73.5%, terminal bronchiolar (65%), and respiratory bronchiolar 60.8% segmental epithelial components as well as alveolar (55%) epithelia. Analysis of immunohistochemical expression of apoptosis-related gene products also supported the hypothesis that benzene can induce apoptosis in chemosensitive target cells in the lung parenchyma. Quantitative immunhistochemistry showed a statistically significant increase p < 0.001 in the immunoreactive staining index for cytochrome c, Apaf-1 (apoptosis activating factor-1), DNA fragmentation factor, and representative cysteine proteases including caspase-1, caspase-2L, caspase-8 and caspase-9. Thus this is the first study of the respiratory system that demonstrates that benzene inhalation induces lung cell apoptosis as confirmed by DNA electrophoresis, in situ nick end labeling, and the upregulation of apoptosis-related gene products that facilitate caspase-cleaved enzymes which lead to cell degradation via programmed cell death. These responses may represent an important defense mechanism within the parenchymal cells of the respiratory system that reduce mutational hazard and the potential carcinogenic effects of benzene-initiated pathogenesis.     

Oxidant-induced DNA damage by quartz in alveolar epithelial cells

Respirable quartz has recently been classified as a human carcinogen. Although, studies with quartz using naked DNA as a target suggest that formation of oxyradicals by particles may play a role in the DNA-damaging properties of quartz, it is not known whether this pathway is important for DNA damage in the target cells for quartz carcinogenesis, i.e. alveolar epithelial cells. Therefore, we determined in vitro DNA damage by DQ12 quartz particles in rat and human and alveolar epithelial cells (RLE, A549) using the single cell gel electrophoresis/comet assay. The radical generation capacity of quartz was analysed by electron spin resonance (ESR) and by immunocytochemical analysis of the hydroxyl radical-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the epithelial cells. Quartz particles as well as the positive control hydrogen peroxide, caused a dose-dependent increase in DNA strand breaks in both cell lines. DNA damage by quartz was significantly reduced in the presence of the hydroxyl-radical scavengers mannitol or DMSO. The involvement of hydroxyl radicals was further established by ESR measurements and was also demonstrated by the ability of the quartz to induce formation of 8-OHdG. In conclusion, our data show that quartz elicits DNA damage in rat and human alveolar epithelial cells and indicate that these effects are driven by hydroxyl radical-generating properties of the particles.     

Activation of neutrophils by Chlamydia trachomatis-infected epithelial cells is modulated by the chlamydial plasmid

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease world-wide. Chlamydia trachomatis primarily infects epithelial cells of the genital tract but the infection may be associated with ascending infection. Infection-associated inflammation can cause tissue damage resulting in female infertility and ectopic pregnancy. The precise mechanism of inflammatory tissue damage is unclear but earlier studies implicate the chlamydial cryptic plasmid as well as responding neutrophils. We here rebuilt the interaction of Chlamydia trachomatis-infected epithelial cells and neutrophils in-vitro. During infection of human (HeLa) or mouse (oviduct) epithelial cells with Chlamydia trachomatis, a soluble factor was produced that attracted neutrophils and prolonged neutrophil survival, independently of Toll-like receptor signaling but dependent on the chlamydial plasmid. A number of cytokines, but most strongly GM-CSF, were secreted at higher amounts from cells infected with plasmid-bearing, compared to plasmid-deficient, bacteria. Blocking GM-CSF removed the secreted pro-survival activity towards neutrophils. A second, neutrophil TNF-stimulatory activity was detected in supernatants, requiring MyD88 or TRIF independently of the plasmid. The results identify two pro-inflammatory activities generated during chlamydial infection of epithelial cells and suggest that the epithelial cell, partly through the chlamydial plasmid, can initiate a myeloid immune response and inflammation.     

In vivo and in vitro studies with an atypical,rhinotropic isolate of Cryptococcus neoformans

An atypical isolate of Cryptococcus neoformans was investigated because of its consistent and reproducible production of gross nasal pathology following i.v. injection in Swiss albino mice. Dose response to graded concentrations ranging from 1×l0²–1×l0⁷ cells/mouse yielded an LD₅₀ of 1.4×10³ cells/mouse for the atypical rhinotropic strain H140 which was significantly less virulent (p<0.01) than our reference strain of Cryptococcus neoformans. There was no significant difference in mortality following the injection of in vitro vs. in vivo passed inoculum. As early as two weeks after inoculation, this strain produced gross nasal enlargement to approximately 2–3 × normal dimensions with granulomatous and ulcerated lesions. The LD₆₀ resulted in the greatest percentage of nasal involvement (85%). C. neoformans was demonstrated by culture and histopathology in the noses, brains, lungs, livers and kidneys. A temperature selection was indicated by findings of a lower temperature minimum for subcultures isolated from the noses relative to those isolated from the brain, and by the fact that the most densely populated organs following intraperitoneal injection were the testes. This route of inoculation resulted in cutaneous nasal involvement in a manner analogous to that following i.v. injection. The atypical isolate was unable to assimilate trehalose or raffinose but otherwise was entirely consistent with identification as C. neoformans and produced characteristic CNS and general organ system disease in addition to the rhinotropic cutaneous manifestations. The model characterized here in normal mice may be of value in studies of fungal dermotropism.     

Cocoa polyphenols attenuate hydrogen peroxide-induced inhibition of gap-junction intercellular communication by blocking phosphorylation of connexin 43 via the MEK/ERK signaling pathway

Cocoa, a good source of dietary antioxidative polyphenols, exhibited anticarcinogenic activity in animal models, but the molecular mechanisms of the chemopreventive potential of cocoa remain unclear. Inhibition of gap-junction intercellular communication (GJIC) is strongly related to tumorigenesis. Cocoa polyphenol extracts (CPE) dose dependently attenuated hydrogen peroxide (H₂O₂)-induced inhibition of GJIC in rat liver epithelial (RLE) cells. CPE inhibited the H₂O₂-induced phosphorylation and internalization of connexin 43, which is a regulating protein of GJIC in RLE cells. The H₂O₂-induced accumulation of reactive oxygen species and activation of extracellular signal-regulated kinase were inhibited by CPE treatment. However, CPE did not block H₂O₂-induced phosphorylation of p38 mitogen-activated protein kinase. An ex vivo kinase assay demonstrated that CPE inhibited the H₂O₂-induced mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 activity in RLE cell lysates. Ex vivo pull-down assay data revealed that CPE directly bound with MEK1 to inhibit MEK1 activity. These results indicate that CPE protects against the H₂O₂-induced inhibition of GJIC through antioxidant activity and direct inhibition of MEK activity, which may contribute to its chemopreventive potential.     

Evidence that surface fibrils expressed by Haemophilus influenzae type b promote attachment to human epithelial ceils

Hemophilus influenzae type b is a Gram-negative bacillus that initiates infection by colonizing the upper respiratory tract. Previous studies have established that H. influenzae haemagglutinating pili possess adhesive properties and influence the process of colonization. Additional studies suggest the presence of a second H. influenzae adhesin distinct from haemagglulinating pili. In the present study, we examined a non-piliated H. influenzae type b strain by transmission electron microscopy and visualized occasional short, thin, surface fibrils. Subsequently, we isolated a spontaneous mutant that lacked surface fibrils and was deficient in adherence to cultured human epithelial cells. Using a cloning strategy that exploited this mutant, we isolated a fragment of DNA that promotes in vitro adherence to human epithelial cells when expressed in laboratory strains of Escherichia coli. Mutagenesis of this fragment in a series of H. influenzae type b strains resulted in loss of expression of surface fibrils and a marked decrease in attachment. Furthermore, restoration of surface fibril expression was associated with reacquisition of wild-type levels of adherence. Our results are consistent with the conclusion that H. influenzae type b surface fibrils have adhesive capacity. We speculate that these organelles facilitate colonization of the human respiratory tract.     

Biphasic culture of rat lung slices for pharmacotoxicological evaluation of complex atmospheres

The relevance to the in vivo situation of in vitro toxicity studies of complex atmospheres has frequently been limited by the procedures used for the exposure of the biological samples. We have evaluated from on-road measurements the size distribution pattern and the subsequent respiratory tract deposition rates of particulate matter from urban atmospheric aerosols, which are in the range of 110 and 3 pg/cm² per min for tracheobronchial and alveolar areas, respectively. Continuous flow-through rotating chambers and a specific design for exhaust sampling and dilution with controlled adjustment of pO₂ and pCO₂ to 20% and 5%, respectively, have been developed to expose biphasic air/liquid organotypic cultures of rat lung slices to continuous flows of diluted exhausts from diesel engines with preservation of the physicochemical properties of the exhaust. The size distribution of the particulate matter and the bioavailability of pollutants were preserved, thus allowing us to closely mimic in vitro the in vivo atmosphere/tissue interactions that occur mainly through diffusion mechanisms. The toxicity response profile has been assessed in terms of tissue viability, oxidative stress, DNA injury, and the early phase of inflammatory reaction. Exhaust filtration, addition to fuel of rapeseed methyl ester, and preincubation of lung tissue with soy isoflavones modulated the toxicity response profile of exhausts. The importance of preserving both particulate matter size distribution and adsorbed pollutant bioavailability, which could not be ascertained using more classical in vitro approaches, is discussed and should be considered a prerequisite for further developments of in vitro studies to modelize in vivo inhalation of complex atmospheres. This revised version was published online in August 2006 with corrections to the Cover Date.     

Neutrophils cause oxidative DNA damage in alveolar epithelial cells.

Inflammation has been recognized as a contributing factor in the pathogenesis of some cancers. In the lung, inflammation is characterized by an influx of polymorphonuclear leukocytes (PMN) that release a variety of reactive oxygen species (ROS). The aim of the present study was to investigate the direct effect of PMN on oxidative DNA damage in lung target cells. Therefore, rat alveolar epithelial cells (RLE) were coincubated with PMN or hydrogen peroxide. Known to be correlated with the incidence of cancer, 7-hydro-8-oxo-2'deoxyguanosine (8-oxodG) was used as an effect marker for oxidative damage. Viability of the RLE, when coincubated with PMN, decreased to 43%, dependent on the ratio between PMN and RLE. After washing off PMN, 8-oxodG levels were significantly increased in RLE, but the highest levels were observed in the washed off PMN fraction. In addition, to avoid washing off procedures, immunohistochemical analysis was used to measure the 8-oxodG levels specifically in the RLE and similar results were obtained. In addition, inhibitor experiments showed that antioxidants ameliorated oxidative DNA damage. Our data provide evidence that ROS released by PMN as well as H2O2, cause oxidative DNA damage in epithelial cells.     

Defining a pro‐inflammatory neutrophil phenotype in response to schistosome eggs

Neutrophils contribute to the pathological processes of a number of inflammatory disorders, including rheumatoid arthritis, sepsis and cystic fibrosis. Neutrophils also play prominent roles in schistosomiasis japonica liver fibrosis, being central mediators of inflammation following granuloma formation. In this study, we investigated the interaction between Schistosoma japonicum eggs and neutrophils, and the effect of eggs on the inflammatory phenotype of neutrophils. Our results showed significant upregulated expression of pro‐inflammatory cytokines (IL‐1α, IL‐1β and IL‐8) and chemokines (CCL3, CCL4 and CXCL2) in neutrophils after 4 h in vitro stimulation with S. japonicum eggs. Furthermore, mitochondrial DNA was released by stimulated neutrophils, and induced the production of matrix metalloproteinase 9 (MMP‐9), a protease involved in inflammation and associated tissue destruction. We also found that intact live eggs and isolated soluble egg antigen (SEA) triggered the release of neutrophil extracellular traps (NETs), but, unlike those reported in bacterial or fungal infection, NETs did not kill schistosome eggs in vitro. Together these show that S. japonicum eggs can induce the inflammatory phenotype of neutrophils, and further our understanding of the host–parasite interplay that takes place within the in vivo microenvironment of schistosome‐induced granuloma. These findings represent novel findings in a metazoan parasite, and confirm characteristics of NETs that have until now, only been observed in response to protozoan pathogens.     

Natural‐host animal models indicate functional interchangeability between the filamentous haemagglutinins of Bordetella pertussis and Bordetella bronchiseptica and reveal a role for the mature C‐terminal domain,but not the RGD motif,during infection

Bacteria of the Bordetella genus cause respiratory tract infections. Both broad host range (e.g. Bordetella bronchiseptica) and human‐adapted (e.g. Bordetella pertussis) strains produce a surface‐exposed and secreted protein called filamentous haemagglutinin (FHA) that functions in adherence and immunomodulation. Previous studies using B. pertussis and cultured mammalian cells identified several FHA domains with potential roles in host cell interactions, including an Arg‐Gly‐Asp (RGD) triplet that was reported to bind integrins on epithelial cells and monocytes to activate host signalling pathways. We show here that, in contrast to our previous report, the fhaB genes of B. pertussis and B. bronchiseptica are functionally interchangeable, at least with regard to the various in vitro and in vivo assays investigated. This result is significant because it indicates that information obtained studying FHA using B. bronchiseptica and natural‐host animal models should apply to B. pertussis FHA as well. We also show that the C‐terminus of mature FHA, which we name the MCD, mediates adherence to epithelial and macrophage‐like cells and is required for colonization of the rat respiratory tract and modulation of the inflammatory response in mouse lungs. We could not, however, detect a role for the RGD in any of these processes.     

Convergent sets of data from in vivo and in vitro methods point to an active role of Hsp60 in chronic obstructive pulmonary disease pathogenesis

Background

It is increasingly clear that some heat shock proteins (Hsps) play a role in inflammation. Here, we report results showing participation of Hsp60 in the pathogenesis of chronic obstructive pulmonary diseases (COPD), as indicated by data from both in vivo and in vitro analyses.

Methods and Results

Bronchial biopsies from patients with stable COPD, smoker controls with normal lung function, and non-smoker controls were studied. We quantified by immunohistochemistry levels of Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and HSF-1, along with levels of inflammatory markers. Hsp10, Hsp40, and Hsp60 were increased during progression of disease. We found also a positive correlation between the number of neutrophils and Hsp60 levels. Double-immunostaining showed that Hsp60-positive neutrophils were significantly increased in COPD patients. We then investigated in vitro the effect on Hsp60 expression in bronchial epithelial cells (16HBE) caused by oxidative stress, a hallmark of COPD mucosa, which we induced with H₂O₂. This stressor determined increased levels of Hsp60 through a gene up-regulation mechanism involving NFkB-p65. Release of Hsp60 in the extracellular medium by the bronchial epithelial cells was also increased after H₂O₂ treatment in the absence of cell death.

Conclusions

This is the first report clearly pointing to participation of Hsps, particularly Hsp60, in COPD pathogenesis. Hsp60 induction by NFkB-p65 and its release by epithelial cells after oxidative stress can have a role in maintaining inflammation, e.g., by stimulating neutrophils activity. The data open new scenarios that might help in designing efficacious anti-inflammatory therapies centered on Hsp60 and applicable to COPD.     

Reactive Nitrogen Species Mediate DNA Damage in Helicobacter pylori-Infected Gastric Mucosa

Background:  Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can play an important role in cellular injury and carcinogenesis of gastric epithelial cells infected with Helicobacter pylori . 8-OH-deoxy guanosine (8-OHdG) and 8-nitroguanine (8-NG) are markers for ROS- and RNS-mediated DNA oxidation, respectively. In this study, RNS-mediated DNA damage in gastric mucosa was observed directly using a newly developed antibody to 8-NG to clarify how H. pylori infection causes nitrative DNA damage to gastric epithelial cells.
Methods:  Immunohistochemistry with anti-8-OHdG and anti-8-NG antibodies was performed on gastric tissue samples from 45 patients (25 men and 20 women) with H. pylori -positive gastritis and 19 patients (11 men and 8 women) exhibiting successful H. pylori eradication. Histologic factors for gastric mucosal inflammation were graded according to the guidelines of the Updated Sydney system.
Results:  In corpus mucosa, 8-OHdG and 8-NG production were significantly associated with the degree of glandular atrophy, infiltration of chronic inflammatory cells and intestinal metaplasia in the glandular epithelial cells. Successful H. pylori eradication resulted in a significant reduction of chronic inflammatory cell infiltration and neutrophilic activity. Mean 8-OHdG production was lower after H. pylori eradication in both corpus and antral mucosa ( p  = .022 and .049, respectively). However, the reduction in 8-NG exhibited was more pronounced than the reduction of 8-OhdG ( p  = .004 and .007, respectively).
Conclusions:  Helicobacter pylori infection can induce inflammatory cells infiltration, which evokes DNA damage of gastric epithelial cells through ROS and RNS production. 8-NG might be a more sensitive biomarker than 8-OHdG for H. pylori -induced DNA damage in gastric mucosa.     

Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition

Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRas^V12. Live imaging revealed that expression of HRas^V12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRas^V12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRas^V12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRas^V12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.