The effects of cigarette smoke extract (CSE) on airway epithelial cells (AECs) from cystic fibrosis (CF) and non-cystic fibrosis (non-CF) individuals are not fully understood. It has been suggested that CSE modulates inflammatory cytokine release from AECs by modulating the epidermal growth factor receptor (EGFR) pathway; these pathways could reveal novel therapeutic targets. We compared the effect of CSE pre-incubation on IL-8 release from CF and non-CF bronchial epithelial cell lines, and separately, with primary nasal epithelial cells (NECs) retrieved from CF and non-CF individuals. We also determined if the EGFR pathway regulates IL-8 release by LPS or cytomix in non-CF and CF AECs at baseline and following CSE exposure.CF and non-CF cell lines, NECs derived from both CF patients (R117H heterozygous and F508del homozygous), and from healthy subjects, were cultured in the presence or absence of CSE, and subsequently exposed to inflammatory stimuli. In cell lines CSE significantly reduced IL-8 release following inflammatory challenge. Conversely, CSE pre-treatment was pro-inflammatory in primary NECs. In NECs from control subjects, CSE increased cytomix and LPS induced IL-8 release, and for the R117H heterozygous NEC cultures, CSE enhanced basal IL-8 release. Cytomix and LPS induced IL-8 release from F508del homozygous NEC cultures was further heightened following CSE pre-treatment.EGFR inhibition mitigated IL-8 release from immortalised and primary non-CF and CF AECs, suggesting that constitutive and CSE elicited IL-8 release from AECs is partly regulated via the EGFR pathway.This study demonstrates the importance of the EGFR cascade in the regulation of constitutive and CSE induced inflammatory mediator release from immortalised and primary AECs. Moreover, it clearly highlights the significance of using primary cells to confirm results obtained from immortalised cell studies, as these model systems may respond very differently to the stimuli under investigation.

Rationale Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia. Objectives We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion. Methods In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA. Results In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2μg/ml (p = 0.03) and 2μg/ml (p = 0.003) as well as mucus secretion at 2μg/ml (p = 0.04). Conclusions We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma.

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

BACKGROUND:Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs).METHODS:PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method.RESULTS:CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 μM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 μM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death.CONCLUSIONS:Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms.

In COPD inflammation driven by exposure to tobacco smoke results in impaired innate immunity in the airway and ultimately to lung injury and remodeling.To understand the biological processes involved in host interactions with cigarette derived toxins submerged epithelial cell culture is widely accepted as a model for primary human airway epithelial cell culture research.Primary nasal and bronchial epithelial cells can also be cultured in models.Air liquid interface (ALI) and submerged culture models have their individual merits, and the decision to use either technique should primarily be determined by the research hypothesis.Cigarette smoke has gaseous and particulate matter, the latter constituent primarily represented in cigarette smoke extract (CSE).Although not ideal in order to facilitate our understanding of the responses of epithelial cells to cigarette smoke, CSE still has scientific merit in airway cell biology research.Using this model, it has been possible to demonstrate differences in levels of tight junction disruption after CSE exposure along with varied vulnerability to the toxic effects of CSE in cell cultures derived from COPD and control study groups.Primary nasal epithelial cells (PNECs) have been used as an alternative to bronchial epithelial cells (PBECs).However, at least in subjects with COPD, PNECs cannot consistently substitute for PBECs.Despite having a constitutional pro-inflammatory phenotype, bronchial epithelial cells retrieved from subjects with COPD have a relatively curtailed inflammatory response to CSE exposure when compared to epithelial cells from their equivalent healthy counterparts.Furthermore, COPD epithelial cells have an increased susceptibility to undergo apoptosis, and have reduced levels of Toll-like receptor-4 expression after CSE exposure, both of which may account for the reduced inflammatory response observed in this group.The use of CSE in both submerged and ALI epithelial cultures has extended our knowledge of cell biology in COPD, and helped to unravel important pathways which may be of relevance in its pathogenesis.

For in vitro studies of airway pathophysiology, primary epithelial cells have many advantages over immortalised cell lines. Nasal epithelial cells are easier to obtain than bronchial epithelial cells and can be used as an alternative for in vitro studies. Our objective was to compare nasal and bronchial epithelial cells from subjects with COPD to establish if these cells respond similarly to pro-inflammatory stimuli. Cell cultures from paired nasal and bronchial brushings (21 subjects) were incubated with cigarette smoke extract (CSE) prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 (TLR-4) message and expression by RT-PCR and FACS respectively. IL-8 release correlated significantly between the two cell types. IL-6 secretion was significantly less from bronchial compared to nasal epithelial cells and secreted concentrations did not correlate. A 4 h CSE incubation was immunosuppressive for both nasal and bronchial cells, however prolonged incubation for 24 h was pro-inflammatory solely for the nasal cells. CSE reduced TLR-4 expression in bronchial cells only after 24 h, and was without effect on mRNA expression. In subjects with COPD, nasal epithelial cells cannot substitute for in vitro bronchial epithelial cells in airway inflammation studies.

We hypothesised that primary bronchial epithelial cells (PBECs) from subjects with chronic obstructive pulmonary disease (COPD) respond differently toPseudomonas aeruginosalipopolysaccharide (LPS) after cigarette smoke extract (CSE) exposure than PBECs obtained from smokers without airflow obstruction and nonsmokers.PBECs from 16 COPD subjects, 10 smokers without airflow obstruction and nine nonsmokers were cultured at air–liquid interface. Cultures were incubated with CSE prior to stimulation withP. aeruginosaLPS. Interleukin (IL)-6 and IL-8 were measured by ELISA and Toll-like receptor (TLR)-4 expression by fluorescence-activated cell sorter. Activation of nuclear factor (NF)-κB was determined by Western blotting and ELISA, and MAPK and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labelling methods.Constitutive release of IL-8 and IL-6 was greatest from the COPD cultures. However, CSE pretreatment followed byP. aeruginosaLPS stimulation reduced IL-8 release from COPD PBECs, but increased it from cells of smokers without airflow obstruction and nonsmokers. TLR-4 expression, MAPK and NF-κB activation in COPD cultures were reduced after CSE treatment, but not in the smokers without airflow obstruction or nonsmoker groups, which was associated with increased apoptosis.CSE attenuates inflammatory responses to LPS in cells from people with COPD but not those from nonsmoking individuals and those who smoke without airflow obstruction.

Few studies compare the function of primary bronchial (PBEC) and nasal (PNEC) epithelial cells. Our aim was to compare the responses of paired PNEC and PBEC cultures to LPS stimulation and any modulatory effects of exposure to cigarette smoke extract (CSE). Cells, from subjects with COPD, were obtained by nasal or bronchial brushing and used at passage 3. They were stimulated for 24 h with LPS [0–25 μg/ml] ± pre-treatment with CSE. CSE was prepared by combusting a 12 mg tar Marlboro cigarette through 25 ml of media. Supernatants were collected and IL-8 and IL-6 measured by ELISA. The localization of TLR-4 was established by FACS. For the PNEC cultures, a brief incubation with CSE (4h) significantly inhibited LPS-induced IL-6 and IL-8 release (IL-8: 24h treatment with 25 μg/ml LPS alone 5457±424 pg/ml and with 4h CSE pre-treatment 3772±432 pg/ml). A more prolonged incubation with CSE (24h) was pro-inflammatory (IL-8: 25 μg/ml LPS alone 5485±562 pg/ml and with 24h CSE pre-treatment 7757±449 pg/ml). Although a brief incubation with CSE resulted in a lower percentage of surface and intracellular TLR4, a prolonged incubation was without effect. In contrast, both a brief and a prolonged exposure of PBEC cultures to CSE reduced LPS induced IL-8 release (IL-8: 24h treatment with 25 μg/ml LPS alone 5107±797 pg/ml, with 4h CSE pre-treatment 3345±650 pg/ml, and with 24h CSE pre-treatment 3010±328 pg/ml), and both lead to a reduced percentage of surface and intracellular TLR4. There was minimal IL-6 release from the PBEC cultures. In conclusion, our data indicate that PNEC cultures are not a suitable surrogate for PBEC cultures in terms of their response to CSE/LPS combination treatment.

BACKGROUND:A seventy nine year old lady presented with acute bilateral foot drop and paraesthesia of her lower limbs as a presenting feature of Wegener's Granulomatosis (WG).CASE PRESENTATION:There was no evidence of pulmonary involvement and her renal function was normal. WG can masquerade as very diverse pathology. It is recognised that neuropathy can occur early and often in the absence of more classical pulmonary and renal findings, often resulting in a delay in diagnosis.CONCLUSION:Anti-neutrophil cytoplasmic antibody (ANCA) testing was particularly useful in this case permitting early diagnosis.