Increased expression of angiogenic markers in patients with seasonal allergic rhinitis

BACKGROUND: Increased vascularity due to neo-angiogenesis is an essential part of airway remodelling. Vascular endothelial growth factor (VEGF), CD34 and von Willebrand's factor (FvW) are known angiogenic markers. Angiogenesis and airway remodelling has been documented in asthma but not in allergic rhinitis. OBJECTIVE: We aimed to investigate the presence of increased angiogenesis and its relation to angiogenic molecules, namely VEGF, CD34 and FvW, in endothelial cells of nasal mucosa in patients with seasonal allergic rhinitis (SAR), using three different immunohistochemical analysis methods, namely HSCORE, microvessel density (MVD) and vascular surface density (VSD). The findings in allergic rhinitis were compared with the findings in nasal septal deviation (NSD), which is not associated with increased angiogenesis. METHODS: Twenty patients with symptomatic SAR, who were not under treatment, were enrolled in the study. Ten patients with NSD, who needed surgical therapy, served as the control group. Demographic characteristics did not differ between the two groups. Inferior turbinate biopsy was obtained from SAR patients and control patients, under local anaesthesia and during surgery respectively. All biopsies were evaluated for angiogenesis on the basis of VEGF, CD34 and FvW by two blinded histologists using three immunohistochemical analysis methods (HSCORE, MVD and VSD).Results. HSCORE, estimated on the basis of each staining technique, showed statistically significant differences among the two groups (p=0.002; p=0.045; p=0.016, respectively). Anti-CD34 and anti-VEGF showed higher MVD values in SAR when compared to the controls (p=0.038; p=0,009, respectively). No statistically significant difference was found in Anti-FvW-based MVD between SAR patients and controls (p=0.071). The measurements of VSD for FvW and VEGF from nasal biopsy specimens displayed a statistically significant difference between the two groups (p=0.004; p=0.0001, respectively). However, measurement of VSD for CD-34 was not significantly different between the groups (p=0.086). On the other hand, morphometric data obtained by all three methods did not correlated. CONCLUSION: There are a few studies that have investigated the essential role of angiogenesis in the pathogenesis of allergic rhinitis. We conclude that, increased angiogenesis may be as prominent in patients with allergic rhinitis as in patients with non-allergic nasal pathologies and may play an important role in the remodelling of nasal mucosa of subjects with SAR.     

Comparative proteomics of nasal fluid in seasonal allergic rhinitis

A comparative proteomic approach was applied to examine nasal lavage fluid (NLF) from patients with seasonal allergic rhinitis (SAR, n = 6) and healthy subjects (controls, n = 5). NLF samples were taken both before allergy (pollen) season and during season, and proteins were analyzed by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after tryptic cleavage. Twenty proteins were selected and quantified. During allergy season, the levels of six sialylated isoforms of PLUNC (palate lung nasal epithelial clone) were lower in SAR patients than controls, as were the levels of six isoforms of von Ebner's gland protein (VEGP), including a previously undescribed form with N-linked glycosylation, and of cystatin S. PLUNC is a new innate immunity protein and VEGP and cystatin S are two endogenous proteinase inhibitors. By contrast, the levels of an acidic form of alpha-1-antitrypsin were higher in SAR patients than controls. One previously unidentified NLF protein was found in all samples from the SAR patients during allergy season but not in any sample before allergy season: this protein was identified as eosinophil lysophospholipase (Charcot-Leyden crystal protein/galactin 10). MS/MS analysis of the N-terminus of the protein showed removal of Met and acetylation of Ser. Altogether, these findings illustrate the potential use of proteomics for identifying protein changes associated with allergic rhinitis and for revealing post-translational modifications of such new potential markers of allergic inflammation.     

Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines

Background

S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR).

Methods

Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC).

Results

Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC.

Conclusions

The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.     

The Impact of Allergic Rhinitis and Asthma on Human Nasal and Bronchial Epithelial Gene Expression

Background

The link between upper and lower airways in patients with both asthma and allergic rhinitis is still poorly understood. As the biological complexity of these disorders can be captured by gene expression profiling we hypothesized that the clinical expression of rhinitis and/or asthma is related to differential gene expression between upper and lower airways epithelium.

Objective

Defining gene expression profiles of primary nasal and bronchial epithelial cells from the same individuals and examining the impact of allergic rhinitis with and without concomitant allergic asthma on expression profiles.

Methods

This cross-sectional study included 18 subjects (6 allergic asthma and allergic rhinitis; 6 allergic rhinitis; 6 healthy controls). The estimated false discovery rate comparing 6 subjects per group was approximately 5%. RNA was extracted from isolated and cultured epithelial cells from bronchial brushings and nasal biopsies, and analyzed by microarray (Affymetrix U133+ PM Genechip Array). Data were analysed using R and Bioconductor Limma package. For gene ontology GeneSpring GX12 was used.

Results

The study was successfully completed by 17 subjects (6 allergic asthma and allergic rhinitis; 5 allergic rhinitis; 6 healthy controls). Using correction for multiple testing, 1988 genes were differentially expressed between healthy lower and upper airway epithelium, whereas in allergic rhinitis with or without asthma this was only 40 and 301 genes, respectively. Genes influenced by allergic rhinitis with or without asthma were linked to lung development, remodeling, regulation of peptidases and normal epithelial barrier functions.

Conclusions

Differences in epithelial gene expression between the upper and lower airway epithelium, as observed in healthy subjects, largely disappear in patients with allergic rhinitis with or without asthma, whilst new differences emerge. The present data identify several pathways and genes that might be potential targets for future drug development.     

Expression of Toll-like Receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis

Background

Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis.

Methods

The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively.

Results

TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis.

Conclusion

The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.     

Gender differences in inflammatory proteins and pathways in seasonal allergic rhinitis

In model organisms, thousands of genes differ in expression between females and males. It is not known if differences on a similar scale are found in humans nor how this relates to disease. However, in allergic disease gender differences in the levels of both inflammatory cells and proteins have been shown. In this study, we found lower nasal fluid allergen-specific IgE in women than men with seasonal allergic rhinitis (SAR). This led to genome-wide analyses of gene expression in allergen-challenged CD4⁺ cells from patients with SAR before and after treatment with cortisone. Before treatment, 975 genes differed in expression between women and men: 337 were higher in women. After treatment only 428 genes and one pathway differed in expression. The genes that differed in expression between women and men were over-represented in 10 pathways. Five of the pathways regulated chemotaxis. All five were less active in women. One of the pathways was induced by the eosinophilic chemokine CCL4. Analysis of nasal fluid CCL4 protein confirmed lower levels in women with seasonal allergic rhinitis, before and during the pollen season. By contrast, nasal fluid CCL3 levels did not differ between the genders. In summary, this study shows gender differences in specific inflammatory pathways and proteins in patients with seasonal allergic rhinitis. Further studies are warranted to examine if such differences have diagnostic and therapeutic implications in allergic diseases.     

应用16S rRNA测序法对过敏性鼻炎患者鼻前庭菌群组成分析

目的 探索过敏性鼻炎患者鼻前庭微生物组成与过敏性鼻炎的关系,为过敏性鼻炎的诊疗提供新思路。方法 提取细菌基因组DNA,通过16S rRNA高通量测序法分析（Illumina测序）,将测序结果与greengenes database进行比对,通过生物信息学、医学统计学分析过敏性鼻炎患者鼻前庭微生物组成与正常人的差异。结果 在门水平的相对丰度上,试验组与对照组放线菌和梭杆菌差异显著。在菌群属的研究水平上,试验组与对照组之间有明显差异的菌属有13种,包括黄单胞菌科中的一个未注释的菌属、棒状杆菌属、嗜胨菌属、厌氧球菌属、大芬戈尔德菌属、丙酸杆菌属、短杆菌属、希瓦菌属、微小单胞菌属、考克菌属、鞘脂单胞菌科中的一个未注释的菌属、嗜盐单胞菌属和叶瘤菌属。结论 过敏性鼻炎患者与健康正常人鼻前庭微生物组成存在明显的差异,进一步可深入研究鼻腔内微生物组成影响鼻炎发病的机制。     

顺尔宁对变应性鼻炎患者血清肺表面活性蛋白A、D动态水平影响及近 期疗效观察

目的:探讨顺尔宁对变应性鼻炎患者体内肺表面活性蛋白A、D水平的影响及近期疗效观察。方法:纳入的变应性鼻炎患者分成单纯变应性鼻炎患者组不伴哮喘组106例,变应性鼻炎伴哮喘患者组75例,健康成人组20例作为实验对照组。连续应用顺尔宁8周后,统计患者鼻部总体症状评分,以及血清SP-A、SP-D水平的动态变化情况。结果:单纯变应性鼻炎组、变应性鼻炎伴哮喘组患者治疗4周后、8周后与治疗前比较,鼻部总体症状评分均明显降低,差异均有统计学意义(均P0.05)。治疗4周后,单纯变应性鼻炎组、变应性鼻炎伴哮喘组患者血清SP-A、D水平较治疗前,差异无统计学意义(均P0.05);治疗8周后,单纯变应性鼻炎组、变应性鼻炎伴哮喘组患者血清SP-A、D水平较治疗前明显下降(均P0.05)。结论:顺尔宁可影响变应性鼻炎患者体内SP-A、SP-D水平,改善鼻部不适的症状。     

Downregulation of peroxisome proliferator-activated receptors (PPARs) in nasal polyposis

Background

Peroxisome proliferator-activated receptor (PPAR) α, βδ and γ are nuclear receptors activated by fatty acid metabolites. An anti-inflammatory role for these receptors in airway inflammation has been suggested.

Methods

Nasal biopsies were obtained from 10 healthy volunteers and 10 patients with symptomatic allergic rhinitis. Nasal polyps were obtained from 22 patients, before and after 4 weeks of local steroid treatment (fluticasone). Real-time RT-PCR was used for mRNA quantification and immunohistochemistry for protein localization and quantification.

Results

mRNA expression of PPARα, PPARβδ, PPARγ was found in all specimens. No differences in the expression of PPARs were obtained in nasal biopsies from patients with allergic rhinitis and healthy volunteers. Nasal polyps exhibited lower levels of PPARα and PPARγ than normal nasal mucosa and these levels were, for PPARγ, further reduced following steroid treatment. PPARγ immunoreactivity was detected in the epithelium, but also found in smooth muscle of blood vessels, glandular acini and inflammatory cells. Quantitative evaluation of the epithelial immunostaining revealed no differences between nasal biopsies from patients with allergic rhinitis and healthy volunteers. In polyps, the PPARγ immunoreactivity was lower than in nasal mucosa and further decreased after steroid treatment.

Conclusion

The down-regulation of PPARγ, in nasal polyposis but not in turbinates during symptomatic seasonal rhinitis, suggests that PPARγ might be of importance in long standing inflammations.     

Psoriasin,one of several new proteins identified in nasal lavage fluid from allergic and non-allergic individuals using 2-dimensional gel electrophoresis and mass spectrometry

Background

Extravasation and luminal entry of plasma occurs continuously in the nose. This process is markedly facilitated in patients with symptomatic allergic rhinitis, resulting in an increased secretion of proteins. Identification of these proteins is an important step in the understanding of the pathological mechanisms in allergic diseases. DNA microarrays have recently made it possible to compare mRNA profiles of lavage fluids from healthy and diseased patients, whereas information on the protein level is still lacking.

Methods

Nasal lavage fluid was collected from 11 patients with symptomatic allergic rhinitis and 11 healthy volunteers. 2-dimensional gel electrophoresis was used to separate proteins in the lavage fluids. Protein spots were picked from the gels and identified using mass spectrometry and database search. Selected proteins were confirmed with western blot.

Results

61 spots were identified, of which 21 were separate proteins. 6 of these proteins (psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648) had not previously been described in nasal lavage fluids. The levels of psoriasin were markedly down-regulated in allergic individuals. Prolactin-inducible protein was also found to be down-regulated, whereas different fragments of albumin together with Ig gamma 2 chain c region, transthyretin and splice isoform 1 of Wnt-2B were up-regulated among the allergic patients.

Conclusion

The identification of proteins in nasal lavage fluid with 2-dimensional gelelectrophoresis in combination with mass spectrometry is a novel tool to profile protein expression in allergic rhinitis and it might prove useful in the hunt for new therapeutic targets or diagnostic markers for allergic diseases. Psoriasin is a potent chemotactic factor and its down-regulation during inflammation might be of importance for the outcome of the disease.     

Clara cell protein in nasal lavage fluid and nasal nitric oxide - biomarkers with anti-inflammatory properties in allergic rhinitis

Background

Clara cell protein (CC16) is ascribed a protective and anti-inflammatory role in airway inflammation. Lower levels have been observed in asthmatic subjects as well as in subjects with intermittent allergic rhinitis than in healthy controls. Nasal nitric oxide (nNO) is present in high concentrations in the upper airways, and considered a biomarker with beneficial effects, due to inhibition of bacteria and viruses along with stimulation of ciliary motility. The aim of this study was to evaluate the presumed anti-inflammatory effects of nasal CC16 and nNO in subjects with allergic rhinitis.

Methods

The levels of CC16 in nasal lavage fluids, achieved from subjects with persistent allergic rhinitis (n = 13), intermittent allergic rhinitis in an allergen free interval (n = 5) and healthy controls (n = 7), were analyzed by Western blot. The levels of nNO were measured by the subtraction method using NIOX^?. The occurrences of effector cells in allergic inflammation, i.e. metachromatic cells (MC, mast cells and basophiles) and eosinophils (Eos) were analyzed by light microscopy in samples achieved by nasal brushing.

Results

The levels of CC16 correlated with nNO levels (r² = 0.37; p = 0.02) in allergic subjects. The levels of both biomarkers showed inverse relationships with MC occurrence, as higher levels of CC16 (p = 0.03) and nNO (p = 0.05) were found in allergic subjects with no demonstrable MC compared to the levels in subjects with demonstrable MC. Similar relationships, but not reaching significance, were observed between the CC16 and nNO levels and Eos occurrence. The levels of CC16 and nNO did not differ between the allergic and the control groups.

Conclusions

The correlation between nasal CC16 and nNO levels in patients with allergic rhinitis, along with an inverse relationship between their levels and the occurrences of MC in allergic inflammation, may indicate that both biomarkers have anti-inflammatory effects by suppression of cell recruitment. The mechanisms behind these observations warrant further analyses.     

Plasma extravasation through neuronal stimulation in human nasal mucosa in the setting of allergic rhinitis

Sanico, Alvin M., Satsuki Atsuta, David Proud, and AlkisTogias. Plasma extravasation through neuronal stimulation in humannasal mucosa in the setting of allergic rhinitis. J. Appl. Physiol. 84(2): 537-543, 1998.We havepreviously shown that capsaicin nasal challenge in subjects withallergic rhinitis produces a dose-dependent increase in the albumincontent of nasal lavage fluids. In the present set of studies, wedetermined whether this observation represents plasma extravasationthat is neuronally mediated. To evaluate whether glandular secretionscontribute to the albumin increase in nasal lavage fluids, volunteerswith allergic rhinitis were pretreated with atropine or placebo before capsaicin challenge. Atropine significantly reduced the volume ofreturned lavage fluids and their lysozyme content but increased theiralbumin and fibrinogen content. To assess the contribution of sensorynerve stimulation, subjects with allergic rhinitis were pretreated in asecond study with lidocaine or placebo before capsaicin challenge.Lidocaine significantly attenuated the capsaicin-induced increases inthe volume of nasal lavage fluids, as well as their lysozyme andalbumin content. To rule out the possibility of a direct effect oflidocaine on blood vessels rather than on nerves, healthy subjects werepretreated in a third study with lidocaine or placebo before bradykininnasal challenge. Lidocaine did not affect the bradykinin-inducedincrease in the albumin content of nasal fluids. We conclude that, inallergic rhinitis, high-dose capsaicin induces plasma extravasation inthe human nose and that this effect is neuronally mediated. Thisprovides more definitive evidence that neurogenic inflammation canoccur in vivo in the human upper airway.

     

Expression and localization of the thromboxane A2 receptor in human nasal mucosa

Thromboxane A2 (TXA2) has been thought a potent mediator involved in allergic rhinitis, because TXA2 was recovered from the nasal lavage fluid of allergic rhinitis patients after allergen provocation and TXA2 receptor antagonists relief nasal allergic symptoms. In order to clarify the expression of TXA2 receptor in human nasal mucosa, we investigated TXA2 receptor mRNA expression and its protein localization by polymerase chain reaction (PCR) and immunohistochemistry, respectively. Human turbinates were obtained after turbinectomy from 10 patients with nasal obstruction refractory to medical therapy. RT-PCR analysis of total RNA from nasal mucosa demonstrated the expression of TXA2 receptor alpha mRNA. The immunohistochemical studies revealed that anti-TXA2 receptor alpha antibody labeled vascular smooth muscle cells, vascular endothelial cells, epithelial cells and submucosal glands in the nasal mucosa. The results may have an important clinical implication for understanding the role of TXA2 receptor on upper airway diseases such as allergic rhinitis and non-allergic rhinitis.     

Plasma kallikrein during experimentally induced allergic rhinitis: role in kinin formation and contribution to TAME-esterase activity in nasal secretions

We have shown recently that kinins are generated during experimentally induced allergic rhinitis in man, and have demonstrated that substrates for kinin-forming enzymes are provided during the allergic response by a transudation of kininogens from plasma into nasal secretions. In light of this increased vascular permeability during the allergic reaction, we have extended our studies on the mechanisms of kinin formation to examine the potential involvement of plasma kallikrein. Allergic individuals (n = 7) and nonallergic controls (n = 7) were challenged intranasally with an allergen, and nasal lavages, obtained before and after challenge, were assayed for immunoreactive human plasma kallikrein/prekallikrein (iHPK). Post-challenge iHPK values were significantly elevated (p less than 0.01) in the allergic group (353 +/- 394 ng/ml; x +/- SD) as compared to the nonallergics (19 +/- 22 ng/ml), and correlated with increases in kinins, histamine, and N-alpha-tosyl-L-arginine methyl esterase (TAME-esterase) activity and with the onset of clinical symptoms. Gel filtration studies revealed that plasma prekallikrein is activated during the allergic response and contributes to kinin formation prior to interaction with plasma protease inhibitors. We also show that the majority of the TAME-esterase activity detected in nasal secretions during the allergic response is due to activities consistent with a plasma kallikrein/alpha 2-macroglobulin complex and with mast cell tryptase.     

Superoxide dismutase failed to attenuate allergen-induced nasal congestion in ragweed-sensitized dogs.

We hypothesized that augmentation of antioxidant defenses with exogenous superoxide dismutase (SOD), an enzyme that provides an initial defense against oxidative injury, would attenuate allergen-induced nasal congestion in the canine model of allergic rhinitis. Nasal congestion was evaluated by the measurements of nasal resistance and the volume of the nasal passage. In five nonsensitized dogs, 30,000 U of SOD from bovine erythrocytes delivered by aerosol to the nasal passages before histamine challenge reduced the histamine-induced nasal congestion. At 30 min postchallenge, nasal resistance was 1.14 +/- 0.2 cmH2O.l(-1).min(-1) in the saline pretreatment study vs. 0.36 +/- 0.02 cmH2O.l(-1).min(-1) in the SOD pretreatment study (P < 0.05), and volume of nasal passage was 10.9 +/- 0.5 cm3 vs. 17.4 +/- 1.3 cm3 (P < 0.05), respectively. In five sensitized dogs, however, neither an analogous pretreatment with SOD nor intranasal aerosolized pretreatment with 30,000 U of SOD conjugated to polyethylene glycol attenuated ragweed-induced nasal congestion. Also, systemic application of SOD did not attenuate responses to challenges with histamine and ragweed in nonsensitized and sensitized dogs, respectively. The antioxidant-induced attenuation of nasal congestion in nonsensitized dogs confirms validity of the model and indicates the involvement of free radical-mediated damage in the genesis of the histamine-induced congestion. In sensitized dogs, the data do not support the hypothesis that oxidative stress is a clinically significant component of acute ragweed-induced nasal congestion. The data do not support the use of SOD for acute protection against allergic rhinitis.