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.     

Altered Esophageal Histamine Receptor Expression in Eosinophilic Esophagitis (EoE): Implications on Disease Pathogenesis

Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR (H1R, H2R, H3R, and H4R) expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells in vitro. HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE (<15 eos/hpf, N = 26) and active EoE (>15 eos/hpf, N = 22) subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.     

Histamine-containing cells obtained from the nose hours after antigen challenge have functional and phenotypic characteristics of basophils.

The pattern of mediators and appearance of cells that stain with alcian blue during human experimental early and late phase allergic reactions suggest that basophils accumulate in nasal secretions within hours of local Ag stimulation. To further explore whether the histamine containing cells that enter the nose after Ag challenge are mast cells or basophils, we studied their functional and phenotypic characteristics. Approximately 24 h after intranasal Ag provocation of subjects with allergic rhinitis, nasal lavage was performed, and the cells were isolated for degranulation studies, analysis of surface Ag, and viability. The average histamine content per alcian blue staining cell was 0.78 +/- 0.2 pg (n = 7), similar to that reported for peripheral blood basophils. Nasal cells were challenged in vitro with anti-IgE, ragweed Amb a I, and FMLP and their responses were compared to those of peripheral blood basophils isolated simultaneously from the same donors. Nasal leukocytes released histamine maximally at 0.1 micrograms/ml of anti-IgE (35.8 +/- 7.8%, n = 7) and responded to FMLP (25.4 +/- 9.9%, n = 7). The response of the cells to ragweed Amb a I and anti-IgE was attenuated compared to peripheral blood basophils. Anti-IgE-induced histamine release was calcium and temperature dependent. Dual color immunofluorescence and flow cytometric analysis of the recovered nasal cells coexpressed CD18, a leukocyte marker not expressed by mast cells. The nasal cells consistently had high levels of spontaneous histamine release (19.5 +/- 2.0%, n = 22). The viability of all cells, assessed by erythrosin B dye exclusion, was 70 +/- 2% (n = 15). However, the viability of IgE-bearing cells was only 28.3 +/- 5.7% (n = 4). The characteristics of histamine release and the nature of the cellular surface markers provide functional proof that the histamine-containing cells accumulating after nasal Ag challenge are basophils and not mast cells.     

Apical CFTR Expression in Human Nasal Epithelium Correlates with Lung Disease in Cystic Fibrosis

Introduction

Although most individuals with cystic fibrosis (CF) develop progressive obstructive lung disease, disease severity is highly variable, even for individuals with similar CFTR mutations. Measurements of chloride transport as expression of CFTR function in nasal epithelial cells correlate with pulmonary function and suggest that F508del-CFTR is expressed at the apical membrane. However, an association between quantitative apical CFTR expression in nasal epithelium and CF disease severity is still missing.

Methods and Materials

Nasal epithelial cells from healthy individuals and individuals with CF between 12–18 years were obtained by nasal brushing. Apical CFTR expression was measured by confocal microscopy using CFTR mAb 596. Expression was compared between both groups and expression in CF nasal epithelial cells was associated with standardized pulmonary function (FEV₁%).

Results

The proportion of cells expressing apical CFTR in columnar epithelium is lower in CF compared to non-CF. The apical CFTR expression level was significantly correlated with FEV₁% in F508del homozygous subjects (r = 0.63, p = 0.012).

Conclusion

CFTR expression in nasal epithelial cells is lower in subjects with CF compared to healthy subjects. The proportion of cells expressing F508del-CFTR at the apical membrane is variable between subjects and is positively correlated with FEV₁% in F508del-CFTR homozygous subjects.     

Pimecrolimus Is a Potent Inhibitor of Allergic Reactions to Hymenopteran Venom Extracts and Birch Pollen Allergen In Vitro

Pimecrolimus (Elidel, SDZ ASM 981) is an anti-inflammatory and immunomodulatory 33-epichloro-derivative of macrolactam ascomycin, with low potential for affecting systemic immune responses compared with other calcineurin inhibitors, cyclosporin A and tacrolimus. Despite numerous studies focused on the mechanism of pimecrolimus action on mast cells, only the single report has addressed pimecrolimus effects on other typical FcεRI-expressing cells, the basophils. Patients allergic to birch pollen (n = 20), hymenopteran venoms (n = 23) and 10 non-allergic volunteers were examined. Primary human basophils pre-treated or not with 0.5–50 μMol pimecrolimus were exposed to various concentrations of recombinant Bet v 1a allergen, bee or wasp venom extracts and anti-IgE for 20 min, and then examined for the expression of CD45, CD193, CD203c, CD63 and CD164 using flow cytometry. The externalization of basophil activation markers (CD63 and CD164) was equally inhibited through pimecrolimus in cells activated by recombinant pollen allergen, hymenopteran venom extracts and anti-IgE. Although the individual response rate was subject to strong variation, importantly, pre-treatment with pimecrolimus lowered the number of activated basophils in response to any of the stimuli in the basophils from all patients. The inhibition was concentration-dependent; approximately half of the basophils were inhibited in the presence of 2.5 mMol pimecrolimus. Pimecrolimus is a valuable new tool for the inhibition of hyper-reactive basophils in patients with pollen allergy and a history of anaphylactic reactions to bee or wasp venoms. Further research should address short-term use of pimecrolimus in vivo in a wide spectrum of allergic diseases.     

CFTR expression analysis in human nasal epithelial cells by flow cytometry

Rationale

Unbiased approaches that study aberrant protein expression in primary airway epithelial cells at single cell level may profoundly improve diagnosis and understanding of airway diseases. We here present a flow cytometric procedure to study CFTR expression in human primary nasal epithelial cells from patients with Cystic Fibrosis (CF). Our novel approach may be important in monitoring of therapeutic responses, and better understanding of CF disease at the molecular level.

Objectives

Validation of a panel of CFTR-directed monoclonal antibodies for flow cytometry and CFTR expression analysis in nasal epithelial cells from healthy controls and CF patients.

Methods

We analyzed CFTR expression in primary nasal epithelial cells at single cell level using flow cytometry. Nasal cells were stained for pan-Cytokeratin, E cadherin, and CD45 (to discriminate epithelial cells and leukocytes) in combination with intracellular staining of CFTR. Healthy individuals and CF patients were compared.

Measurements and Main Results

We observed various cellular populations present in nasal brushings that expressed CFTR protein at different levels. Our data indicated that CF patients homozygous for F508del express varying levels of CFTR protein in nasal epithelial cells, although at a lower level than healthy controls.

Conclusion

CFTR protein is expressed in CF patients harboring F508del mutations but at lower levels than in healthy controls. Multicolor flow cytometry of nasal cells is a relatively simple procedure to analyze the composition of cellular subpopulations and protein expression at single cell level.     

Histamine releasibility and expression of Lyn and Syk kinases in Indian subjects and role of less potent IL-3 in non-releaser basophils

BACKGROUND: Allergen-mediated activation of the IgE signal pathway in basophils and mast cells leads to release of mediators in-vitro and in-vivo systems. However, basophils from 10% to 20% of the population do not release histamine and other mediators on activation of the IgE signal transduction pathway and this has been attributed to the absence of tyrosine kinases Lyn and Syk. Interestingly, when these non-releaser basophils are incubated with the IL-3, it leads to the recovery of the histamine releasibility. OBJECTIVE: To investigate histamine releasibility in the Indian population and to evaluate the role of IL-3 with reference to non-releaser phenotypes. METHODS: Peripheral blood basophils from healthy adults were purified by density gradient centrifugation and negative immuno-selection. Histamine release assay was performed fluorometrically. Assessment of Lyn and Syk expression were carried out by flow-cytometry. SNP analysis in the IL-3 gene was carried by sequencing analysis. RESULTS: Histamine release after ConA challenge varied greatly from 0% to 100% in Indian subjects. Eighteen percent subjects showed less than 5% histamine release (non-releasers). Flow-cytometric analysis revealed a significantly reduced expression of Lyn and Syk kinases in basophils (p<0.05). Histamine release also significantly correlated with expression of Lyn and Syk kinase (p<0.05). Non-releasers showed the presence of SNP at +79 (T-C), which leads to the one amino acid change at 8th position in the mature IL-3 from serine to proline. CONCLUSIONS: About 18% of the Indian subjects studied showed non-releaser phenotype and also had reduced Lyn and Syk kinase expression. Non-releasers have also shown the presence of less potent isoform of IL-3/P8, which is suspected to be responsible for the non-releaser phenotype. This needs to be extended to a larger sample size and could be a potential target for the development of therapeutics for allergic patients.     

The Effects of Overexpression of Histamine Releasing Factor (HRF) in a Transgenic Mouse Model

Background

Asthma is a disease that affects all ages, races and ethnic groups. Its incidence is increasing both in Westernized countries and underdeveloped countries. It involves inflammation, genetics and environment and therefore, proteins that exacerbate the asthmatic, allergic phenotype are important. Our laboratory purified and cloned a histamine releasing factor (HRF) that was a complete stimulus for histamine and IL-4 secretion from a subpopulation of allergic donors'' basophils. Throughout the course of studying HRF, it was uncovered that HRF enhances or primes histamine release and IL-13 production from all anti-IgE antibody stimulated basophils. In order to further delineate the biology of HRF, we generated a mouse model.

Methodology/Principal Findings

We constructed an inducible transgenic mouse model with HRF targeted to lung epithelial cells, via the Clara cells. In antigen naïve mice, overproduction of HRF yielded increases in BAL macrophages and statistical increases in mRNA levels for MCP-1 in the HRF transgenic mice compared to littermate controls. In addition to demonstrating intracellular HRF in the lung epithelial cells, we have also been able to document HRF''s presence extracellularly in the BAL fluid of these transgenic mice. Furthermore, in the OVA challenged model, we show that HRF exacerbates the allergic, asthmatic responses. We found statistically significant increases in serum and BAL IgE, IL-4 protein and eosinophils in transgenic mice compared to controls.

Conclusions/Significance

This mouse model demonstrates that HRF expression enhances allergic, asthmatic inflammation and can now be used as a tool to further dissect the biology of HRF.     

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.     

Down-Regulation of FcεRI-Mediated CD63 Basophil Response during Short-Term VIT Determined Venom-Nonspecific Desensitization

Background

We recently showed a desensitization of FcεRI-mediated basophil response after short-term VIT. Our aim was to evaluate the allergen specificity of this desensitization.

Methods

In 11 Hymenoptera-venom double positive subjects, basophil threshold sensitivity (CD-sens) to anti-FcεRI, honeybee, and Vespula venom was assessed at the beginning and just before the first maintenance dose (MD) of single ultra-rush VIT. In some patients we also monitored CD-sens to rApi m 1 and/or rVes v 5 or other co-sensitizations (i.e., grass pollen). In additional 7 patients, basophils were stripped and sensitized with house dust mite (HDM) IgEs at the same time points.

Results

We demonstrated a marked reduction of CD-sens to anti-FcεRI and VIT-specific venom before the first MD in all 18 subjects included. Furthermore, in 10 out of 11 double positive subjects, a significant and comparable decrease before the first MD was also evident for non-VIT venom; this nonspecific decrease was further supported by the opposite recombinant species-specific major allergen. In one subject with additional grass pollen allergy, a decrease of CD-sens to grass allergen was also demonstrated. Similarly, in 7 cases of patients with passively HDM-sensitized basophils, a significant reduction of CD-sens was also evident to de novo sensitized HDM allergen.

Conclusions

Short-term VIT induced basophil desensitization to VIT-specific as well as to VIT-nonspecific venom. As opposed to long-term VIT, which induces venom-specific changes, the effect of short-term VIT seems to be venom-nonspecific.     

Notch-1 decreased expression contributes to leptin receptor downregulation in nasal epithelium from allergic turbinates

BackgroundAllergic rhinitis is characterized by a remodeling of nasal epithelium. Since the Notch and TGF-β signaling pathways are known to be involved in cell differentiation and remodeling processes and leptin adipokine has already been identified as a marker for homeostasis in human bronchial and nasal epithelial cells of asthmatics, roles played by these pathways have been investigated for chronic allergic rhinitis.MethodsThe leptin/leptin receptor expression has been investigated in a study with 40 biopsies from allergic (AR, n = 18) and non-allergic (C, n = 22) inferior turbinates, using immunohistochemistry, immunofluorescence staining and RT-PCR. In addition, extracts from in vitro samples prepared from primary cells of inferior turbinates as well as in vitro cultured human nasal epithelial RPMI 2650 cells (ATCC-CCL-30) were also tested for leptin expression and activation of the Notch-1 pathway.ResultsWith regards to AR, in vivo expression levels of both leptin and its receptor significantly decreased in comparison to C. Furthermore, leptin receptor mRNA was significantly reduced in AR as compared to C. Immunofluorescence showed an apparent co-expression of leptin receptor with Notch-1, which was not seen with TGF-β. In vitro, in primary turbinate epithelial cells, the expression of leptin receptor and Notch-1 significantly decreased in AR as compared to C. Moreover, in RPMI 2650 cells, leptin receptor expression was shown to be induced by Notch-1 ligand signaling.ConclusionThus, both the leptin and Notch-1 pathways appear to represent markers for epithelial homeostasis in allergic rhinitis.     

Parental risk factors and anorectal malformations: systematic review and meta-analysis

Background

Anderson's disease (AD) or chylomicron retention disease (CMRD) is a very rare hereditary lipid malabsorption syndrome. In order to discover novel mutations in the SAR1B gene and to evaluate the expression, as compared to healthy subjects, of the Sar1 gene and protein paralogues in the intestine, we investigated three previously undescribed individuals with the disease.

Methods

The SAR1B, SAR1A and PCSK9 genes were sequenced. The expression of the SAR1B and SAR1A genes in intestinal biopsies of both normal individuals and patients was measured by RTqPCR. Immunohistochemistry using antibodies to recombinant Sar1 protein was used to evaluate the expression and localization of the Sar1 paralogues in the duodenal biopsies.

Results

Two patients had a novel SAR1B mutation (p.Asp48ThrfsX17). The third patient, who had a previously described SAR1B mutation (p.Leu28ArgfsX7), also had a p.Leu21dup variant of the PCSK9 gene. The expression of the SAR1B gene in duodenal biopsies from an AD/CMRD patient was significantly decreased whereas the expression of the SAR1A gene was significantly increased, as compared to healthy individuals. The Sar1 proteins were present in decreased amounts in enterocytes in duodenal biopsies from the patients as compared to those from healthy subjects.

Conclusions

Although the proteins encoded by the SAR1A and SAR1B genes are 90% identical, the increased expression of the SAR1A gene in AD/CMRD does not appear to compensate for the lack of the SAR1B protein. The PCSK9 variant, although reported to be associated with low levels of cholesterol, does not appear to exert any additional effect in this patient. The results provide further insight into the tissue-specific nature of AD/CMRD.     

Neutrophils and mast cells. Comparison of neutrophil-derived histamine-releasing activity with other histamine-releasing factors

Human neutrophil-derived histamine-releasing activity (HRA-N) was partially purified and found to contain a heat-stable 1400 to 2300-Da fraction which caused human basophils and rat basophil leukemia cells (RBL) to degranulate. The capacity of HRA-N to activate basophils was not related to the gender or atopic status of the basophil donor, but was related to anti-IgE responsiveness. Several lines of evidence suggest that HRA-N and anti-IgE induce histamine release through distinctly different mechanisms: 1) the time course of HRA-N- and anti-IgE-induced RBL histamine release are different; 2) HRA-N causes histamine release from RBL with and without surface-bound IgE; 3) lactic acid stripping of IgE from human basophils reduces anti-IgE-induced histamine release, but has no consistent effect on HRA-N-induced histamine release; and 4) passive sensitization of lactic acid-stripped basophils with IgE restores anti-IgE-induced histamine release but not HRA-N-induced histamine release. Several histamine-releasing factors (HRF) were compared with HRA-N. Human nasal HRF (HRF-NW, crude and partially purified fractions of 15 to 30, 3.5 to 9, and less than 3.5 kDa), like HRA-N, caused equal histamine release from both native and IgE-sensitized RBL. However, only the 15- to 30-kDa fraction caused histamine release from human basophils in the doses tested. Mononuclear cell HRF (HRF-M, crude and a partially purified 25 kDa Mr fraction) and platelet HRF (HRF-P, crude preparation) failed to cause histamine release from either native or IgE-sensitized RBL but caused 30 +/- 5.5% and 20 +/- 10% net histamine release from human basophils, respectively. HRA-N and HRF-NW were both stable to boiling. These data, taken together, suggest that the capacity of HRA-N to induce RBL and human basophil histamine release and of HRF-NW to stimulate RBL histamine release is independent of IgE. The data further suggest that HRA-N and HRF-NW can be distinguished by size, and that they both differ from mononuclear cell HRF and platelet HRF. Thus, it appears that inflammatory cells generate a family of distinct HRF.     

Nontypeable Haemophilus influenzae Induces Sustained Lung Oxidative Stress and Protease Expression

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.