New Mediators and Novel Mechanisms in Allergic Airway Inflammation
Abstract: Hyperresponsiveness and inflammation are major pathophysiological features of both allergic rhinitis and asthma. The mechanisms behind these are not fully understood. The aims of this thesis were to investigate if cytokines involved in the allergic response can induce airway hyperreactivity by affecting the airway smooth muscle directly and to identify novel mediators involved in allergic airway inflammation. The effect of the pro-inflammatory cytokine interleukin (IL)-4 on airway smooth muscle contractions was investigated in an organ culture model. Prolonged exposure to IL-4 enhanced contractions induced by des-Arg9-bradykinin (dA-BK) and bradykinin (BK). This effect was mediated via an up-regulation of B1-receptors and through altered signaling along the mitogen-activated protein kinase (MAPK) signal transduction pathways. Thus, IL-4, besides its previously known effects on the allergic phenotype, may also exhibit direct effects on airway smooth muscle cells, contributing to the development of airway hyperreactivity. Two-dimensional gel electrophoresis (2-DE) was used in combination with mass spectrometry to identify protein patterns in nasal lavage fluid obtained from patients with allergic rhinitis and healthy controls. Six novel proteins, not previously described in the nose, were identified: S100 calcium-binding protein A7 (S100A7), Wingless-type MMTV integration site family, member 2B (WNT2B), galectin-3, alpha enolase, intersectin-2 and hypothetical protein MGC33648. Of these, S100A7 was down- and WNT2B up-regulated among patients. The previously known role of S100A7 in various forms of skin inflammation, including atopic eczema, made it of special interest to investigate its role in allergic airway inflammation. Thus, further investigations were performed on material derived from the nose, the palatine tonsils and cultured epithelial cells. In the nose, immunohistochemical staining for S100A7 was seen in the epithelium and in seromucous glands. DNA microarray analysis demonstrated increased levels of S100A7 in nasal lavage fluid cells obtained from patients with allergic rhinitis, both during season and following nasal allergen provocation. These results were confirmed in nasal biopsies using real-time PCR. In analogy, cultured nasopharyngeal epithelial cells (Detroit 562) increased their S100A7 mRNA expression after stimulation with the pro-inflammatory cytokine tumor necrosis factor (TNF)-α, an effect mediated via nuclear factor kappa B (NF-κB) induced transcription. The difference seen between mRNA and protein levels in humans might be related to disease driven consumption or alternatively to an impaired translation of mRNA into protein. In palatine tonsils, S100A7 protein was detected in the epithelium, germinal centers and T cell zones. S100A7 mRNA expression levels among allergic individuals were generally lower than in healthy control subjects. A similar picture was seen in the nose when patients sampled outside pollen season were compared to healthy controls. In addition, tonsils with bacterial infections expressed reduced mRNA levels of S100A7 compared to non-infected tonsils. The gene coding for S100A7 was resequenced in a set of allergic individuals, resulting in the identification of 13 polymorphisms. Single nucleotide polymorphism (SNP) genotyping in allergic and non-allergic individuals revealed an increased minor allele frequency (MAF) for a non-synonymous SNP (rs3014837) in allergic individuals. It was also established that the major haplotype, containing the major allele at all sites, was more common in non-allergic individuals, while the haplotype containing the minor allele at rs3014837 was equally more common among the allergic individuals. Additionally, heterozygotes at this site had significantly higher scores in skin prick tests for 9 out of 11 tested allergens, compared to homozygotes. Polymorphisms in the genes coding for four other mediator candidates identified through nasal lavage screening; Prolactin-inducible protein (PIP), WNT2B, Palate lung and nasal epithelial clone (PLUNC) and CLC (Charcot-Leyden crystal protein) were investigated for association with allergic rhinitis. All investigated SNPs in CLC were polymorphic and eight SNPs, located in the putative promoter region, yielded increased χ2-values with respect to genotype frequencies. It appeared that homozygotes for the minor alleles were more common in the allergic group, compatible with a recessive inheritance model. Furthermore, the minor alleles were all located on the same haplotype. None of the investigated SNPs in PIP, WNT2B and PLUNC were found to be associated with the disease. To summarize, results presented in this thesis demonstrate that prolonged exposure to cytokines, like IL-4, have the ability to affect airway smooth muscle directly, causing hyperreactivity. Six proteins not previously demonstrated in the nose were identified. The expression of one of these, S100A7, was found to be connected with changes caused by allergic airway inflammation. Further, genetic analysis revealed variations in the gene coding for S100A7, which appear to be associated with the occurrence of allergic rhinitis. Genetic association with allergic rhinitis was found also for another recently identified nasal protein, CLC. This renders S100A7 and CLC promising candidates for further investigations regarding their tentative role as diagnostic markers and targets for novel therapeutic strategies.
CLICK HERE TO DOWNLOAD THE WHOLE DISSERTATION. (in PDF format)