Marjan Luinge

Female mice are more susceptible to the development of allergic airway inflammation than male mice.

Background In humans the prevalence of asthma is higher among females than among males after puberty. The reason for this phenomenon is not clear.

The strength of the OVA-induced airway inflammation in rats is strain dependent

To examine the influence of genetics on the OVA‐induced allergic inflammatory response in lungs we compared rats that are genetically Th2‐predisposed (Brown Norway, inbred) or not genetically predisposed (Sprague Dawley, outbred). Rats were sensitized with ovalbumin (OVA) and challenged four weeks later with OVA aerosol. Eighteen hours after challenge, lung tissue was studied for evaluation of numbers of eosinophils, neutrophils, macrophages and mast cells, as well as for expression of P‐selectin, E‐selectin, intercellular adhesion molecule‐1 (ICAM‐1) and vascular cell adhesion molecule‐1 (VCAM‐1) on endothelial cells. From a separate portion of the pulmonary tissue, leucocytes were isolated to analyse numbers of IFNγ and IL‐4 producing cells (ELISPOT assay) and frequencies of T‐cell subsets and B cells. We found increased numbers of eosinophils and neutrophils in the lung, an increased number of IL‐4 producing cells in lung cell isolates and increased levels of serum (OVA‐ specific)‐IgE in both rat strains. In addition, expression of E‐selectin and ICAM‐1 was up regulated in both rat strains whereas expression of VCAM‐1 was only up regulated in the BN rat. Although the ‘allergic’ Th2 response to OVA was detectable in both rat strains, it was more pronounced in the BN rat than in the SD rat. However, the SD rat, which is not predisposed to respond in either a Th2 or Th1‐like way, appeared capable of mounting an allergic response to OVA. This suggests that other factors than genetic contribute to allergic disease.

Effects of 4 months of smoking in mice with ovalbumin-induced airway inflammation.

Background The effects of smoking on asthma pathogenesis are complex and not well studied. We have shown recently that 3 weeks of smoking attenuates ovalbumin (OVA)‐induced airway inflammation in mice and that 4–6 months of smoking induces emphysema in mice without airway inflammation. Effects of combined long‐term smoking and OVA exposure have not been investigated so far.

Distinct macrophage phenotypes in allergic and nonallergic lung inflammation

Chronic exposure to farm environments is a risk factor for nonallergic lung disease. In contrast to allergic asthma, in which type 2 helper T cell (Th2) activation is dominant, exposure to farm dust extracts (FDE) induces Th1/Th17 lung inflammation, associated with neutrophil infiltration. Macrophage influx is a common feature of both types of lung inflammation, allergic and nonallergic. However, macrophage functions and phenotypes may vary according to their polarized state, which is dependent on the cytokine environment. In this study, we aimed to characterize and quantify the lung macrophage populations in two established murine models of allergic and nonallergic lung inflammation by means of fluorescence-activated cell sorting and immunohistochemistry. We demonstrated that, whereas in allergic asthma M2-dominant macrophages predominated in the lungs, in nonallergic inflammation M1-dominant macrophages were more prevalent. This was confirmed in vitro using a macrophage cell line, where FDE exerted a direct effect on macrophages, inducing M1-dominant polarization. The polarization of macrophages diverged depending on the exposure and inflammatory status of the tissue. Interfering with this polarization could be a target for treatment of different types of lung inflammation.

Association of mast cells with lung function in chronic obstructive pulmonary disease

BackgroundIn asthma, higher chymase positive mast cell (MC-C) numbers are associated with less airway obstruction. In COPD, the distribution of MC-C and tryptase positive mast cells (MC-T) in central and peripheral airways, and their relation with lung function, is unknown. We compared MC-T and MC-C distributions in COPD and controls without airflow limitation, and determined their relation with lung function.MethodsLung tissue sections from 19 COPD patients (median [interquartile range] FEV1% predicted 56 [23–75]) and 10 controls were stained for tryptase and chymase. Numbers of MC-T and MC-C were determined in different regions of central and peripheral airways and percentage of degranulation was determined.ResultsCOPD patients had lower MC-T numbers in the subepithelial area of central airways than controls. In COPD, MC-T numbers in the airway wall and more specifically in the epithelium and subepithelial area of peripheral airways correlated positively with FEV1/VC (Spearmans rho (rs) 0.47, p = 0.05 and rs 0.48, p = 0.05, respectively); MC-C numbers in airway smooth muscle of peripheral airways correlated positively with FEV1% predicted (rs 0.57, p = 0.02). Both in COPD patients and controls the percentage of degranulated MC-T and MC-C mast cells was higher in peripheral than in central airways (all p < 0.05), but this was not different between the groups.ConclusionMore MC-T and MC-C in peripheral airways correlate with better lung function in COPD patients. It is yet to determine whether this reflects a protective association of mast cells with COPD pathogenesis, or that other explanations are to be considered.

Heme oxygenase-1 prevents smoke induced B-cell infiltrates: a role for regulatory T cells?

BackgroundSmoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD.The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.MethodsMice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.ResultsSmoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4+CD25+ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4+CD25+ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.ConclusionThese results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.

Localization and enhanced mRNA expression of the orphan chemokine receptor L-CCR in the lung in a murine model of ovalbumin-induced airway inflammation

Various CC chemokine receptors are expressed on effector cells in allergic inflammation and their distinct expression pattern may dictate, to a large extent, the migration of inflammatory cells to sites of airway inflammation. The lipopolysaccharide (LPS)-inducible CC chemokine receptor (L-CCR) is an orphan chemokine receptor that has previously been identified in the murine macrophage cell line RAW 264.7 and in murine brain glial cells. In this study we investigated the induction and localization of L-CCR mRNA expression in mouse lung after ovalbumin (OVA)-induced airway inflammation. Both RT-PCR experiments and in situ hybridization (ISH) experiments in whole lung sections revealed a rapid upregulation of L-CCR mRNA expression as early as 1 hr and 3 hr after OVA challenge. Expression was found predominantly in MAC3+ macrophages and in bronchial epithelium, as shown by ISH and immunohistochemistry (IHC). We demonstrated that L-CCR mRNA expression is strongly upregulated in mouse lung after OVA challenge and is localized in macrophages and bronchial epithelium. Regarding the likely role of L-CCR as a chemokine receptor with the putative ligand monocyte chemotactic protein-1 (MCP-1, CCL2), this receptor may have an important function in the early phase of airway inflammation.

Susceptibility to chronic mucus hypersecretion, a genome wide association study.

Background Chronic mucus hypersecretion (CMH) is associated with an increased frequency of respiratory infections, excess lung function decline, and increased hospitalisation and mortality rates in the general population. It is associated with smoking, but it is unknown why only a minority of smokers develops CMH. A plausible explanation for this phenomenon is a predisposing genetic constitution. Therefore, we performed a genome wide association (GWA) study of CMH in Caucasian populations. Methods GWA analysis was performed in the NELSON-study using the Illumina 610 array, followed by replication and meta-analysis in 11 additional cohorts. In total 2,704 subjects with, and 7,624 subjects without CMH were included, all current or former heavy smokers (≥20 pack-years). Additional studies were performed to test the functional relevance of the most significant single nucleotide polymorphism (SNP). Results A strong association with CMH, consistent across all cohorts, was observed with rs6577641 (p = 4.25×10−6, OR = 1.17), located in intron 9 of the special AT-rich sequence-binding protein 1 locus (SATB1) on chromosome 3. The risk allele (G) was associated with higher mRNA expression of SATB1 (4.3×10−9) in lung tissue. Presence of CMH was associated with increased SATB1 mRNA expression in bronchial biopsies from COPD patients. SATB1 expression was induced during differentiation of primary human bronchial epithelial cells in culture. Conclusions Our findings, that SNP rs6577641 is associated with CMH in multiple cohorts and is a cis-eQTL for SATB1, together with our additional observation that SATB1 expression increases during epithelial differentiation provide suggestive evidence that SATB1 is a gene that affects CMH.

Shifted T-cell polarisation after agricultural dust exposure in mice and men

Rationale A low prevalence of asthma and atopy has been shown in farmers and agricultural workers. However, in these workers, a higher prevalence of respiratory symptoms has been reported, in which T helper 1 (Th1) and/or Th17 responses may play a role. Aim We investigated the effect of exposure to dust extracts (DEs) from different farms on airway inflammation and T-cell polarisation in a mouse model and assessed T-cell polarisation in agricultural workers from the same farms. Methods DEs were prepared from settled dust collected at cattle and pig farms and bulb and onion industries. Mice were exposed to phosphate-buffered saline (PBS), DEs, house dust mite (HDM) or HDM+DE via nasal instillation, four times per week during 5 weeks. Hyperresponsiveness, airway inflammation, IgE levels and T-cell polarisation were assessed. Th-cell and T cytotoxic (Tc)-cell subsets were investigated in peripheral blood samples from 33 agricultural workers and 9 non-exposed controls. Results DEs induced interleukin(IL)-17, IL-1β and IL-6 in mouse lung homogenates. DE-exposed mice had more mixed inflammatory infiltrates in the lungs, and more neutrophils compared with PBS-exposed mice. DEs protected against the HDM-induced Th2 response and methacholine hyperresponsiveness. Interestingly, occupationally exposed humans had higher frequencies of Th cells spontaneously expressing IL-17 and interferon γ compared with controls. Conclusion Chronic exposure to different types of farm dust induces a Th/Tc-17 inflammatory response in mice and agricultural workers. This may contribute to the low prevalence of Th2-related diseases but may constitute a risk for other chronic respiratory diseases.

Nitrogen Dioxide Exposure Attenuates Cigarette Smoke-Induced Cytokine Production in Mice

Cigarette smoke is the most important cause for the development of chronic obstructive pulmonary disease (COPD). Since only a minority of smokers and some nonsmokers develop COPD, other factors must be involved as well. NO2 is an important air pollutant associated with respiratory symptoms in humans and emphysema development in animal models. We hypothesized that combined exposure to NO2 and cigarette smoke will enhance pulmonary inflammation and emphysema development. Mice were exposed to 20 ppm NO2 for 17 h/day, to 24 puffs of cigarette smoke 2 times per day, to their combination, or to control air for 5 days/wk during 4 wk. Following the last NO2 exposure and within 24 h after the last smoke exposure the mice were sacrificed. Lungs were removed and analyzed for several inflammatory parameters and emphysema. Cigarette smoke exposure increased eosinophil numbers and levels of tumor necrosis factor (TNF)-α, KC, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6. NO2 exposure increased goblet cells, eosinophils, and the levels of IL-6, while it decreased the levels of IL-10. Four weeks of NO2, cigarette smoke, or their combination was not sufficient to induce significant emphysema, nor did it lead to increased numbers of lymphocytes, neutrophils, or macrophages in lung tissue. Instead, NO2 exposure attenuated the smoke-induced increases in levels of TNF-α, KC, and MCP-1. These dampening effects of NO2 may be due to modulating effects of NO2 on cytokine production by macrophages and epithelial cells, which have been reported earlier. The next step is to translate these findings of combined, controlled exposure in animals to the human situation.