Non-allergic Healthy Controls Research Articles

SLIT Improves Cedar Pollinosis by Restoring IL-10 Production from Tr1 and Monocytes~IL-10 Productivity Is Critical for Becoming Allergic~

Allergen-specific immunotherapy (SIT) is currently used for several allergic disorders and IL-10-producing regulatory T cells (Tr1) induced by SIT suppress allergic reactions. We investigated the relation between IL-10 production and acquiring allergy. A prospective study was undertaken to evaluate the effect of SIT on IL-10 production in T cells and other cell fractions in children with pollinosis. In addition, blood samples were collected from non-allergic healthy controls and patients with pollinosis to compare the levels of IL-10 production. PBMC were cultured with pollen peptides or control allergens, and the IL-10 production from monocyte and CD4 T cell was analyzed. Monocytes and CD4 T cells from SIT group of patients produced high levels of IL-10, suggesting that the induction of IL-10 is essential for inducing T cell tolerance. IL-10 production from monocytes and T cells was significantly increased in non-allergic controls compared to patients with pollinosis. This high IL-10 production was observed even when PBMC were stimulated with antigens other than pollen peptides. IL-10 is critical for induction of specific T cell tolerance, and increased production of IL-10 by monocytes and T cells during inflammatory responses or after SIT may influence effector cells in allergy. Present data implicates that the low productivity of IL-10 by monocytes and T cells is closely related with sensitivity to multiple allergens, and resistance to allergic diseases. Augmentation of constitutive IL-10 production from immune system is a potential therapeutic approach for allergic disorders.

Asthma and insulin resistance in children

Increased BMI is a risk factor for asthma in children and may be related to adipokines. Adipokines affect insulin-stimulated glucose uptake in vitro but, to date there is little evidence for such a role in vivo. We explored relationships between obesity and allergic asthma in children. Twenty-one allergic asthmatics (AA) and 10 non-allergic healthy controls, aged 6-17.9 years were studied. AA group included children with a positive mannitol challenge test, >25 ppb of exhaled nitric oxide and a positive skin prick test. BMI z-scores were calculated. Blood levels of insulin, glucose, leptin, resistin, tumour necrosis factor-alpha, IL-4, IL-5 and IL-6 were measured. Insulin resistance (IR) was estimated using the homeostasis model assessment (HOMA). There was no significant difference in BMI z-scores between AA and healthy controls (mean: 0.01 vs -0.10). However, significant differences were found in the blood levels of IL-6 (P = 0.05), IL-4 (P = 0.04), IL-5 (P = 0.01) and leptin (P = 0.02). IR was only found in the AA group (42.85%). Homeostasis model assessment insulin resistance (HOMA-IR) was significantly related to IL-6 (r = 0.44, P = 0.05) and tumour necrosis factor-alpha (r = -0.45, P = 0.05). IR was observed in AA. Our findings are suggestive of a complex interaction between the inflammatory state and adiposity, allergy and asthma.

Allergen provocation increases TH2‐cytokines and FOXP3 expression in the asthmatic lung

Allergic asthma is caused by allergen-specific IgE and T-helper cell (Th) type 2 responses towards airborne allergens. The objective of this study was to investigate local and systemic regulatory mechanisms in the early asthmatic response to bronchial allergen provocation. Birch pollen-allergic patients with mild asthma (n = 13) and healthy nonallergic controls (n = 14) were subjected to bronchoalveolar lavage (BAL) and blood sampling. On patients BAL was performed twice: without preceding provocation ('before samples') and 24 h after bronchial provocation with birch pollen allergen. Lymphocytes in BAL and peripheral blood mononuclear cells (PBMCs) were phenotyped by multi-colour flow cytometry and cytokines measured by cytometric bead array. Proliferation and secreted cytokines were analysed in allergen-stimulated PBMCs, CD25(+) depleted PBMCs and PBMCs with IL-10 neutralizing antibodies. The numbers of CD69(+) and FOXP3(+) lymphocytes were higher in BAL after compared with before allergen provocation in asthmatic patients. Moreover, allergen provocation increased expression of FOXP3 in CD4(+)CD25(bright) cells. The cytokine profile in BAL fluid from asthmatics revealed higher levels of IL-5, compared with the controls, and an increase in IL-5, IL-6, IL-9 and IL-10 after allergen provocation. Pollen allergen stimulated PBMC cultures from asthmatic patients produced elevated levels of IL-5 and IL-13 compared with the controls, which were not affected by depletion of CD25(+) cells or IL-10 neutralization. Despite an increase in CD4(+)CD25(bright) cells expressing high levels of FOXP3 in response to bronchial allergen provocation, asthmatic patients exhibit enhanced levels of Th2 cytokines in the lung, which may indicate an inability among infiltrating cells to suppress Th2 responses.

Associations between decay‐accelerating factor polymorphisms and allergic respiratory diseases

Allergic diseases such as asthma and allergic rhinitis are major causes of morbidity in developed countries. The pathology underlying allergic respiratory diseases is considered to be IgE-mediated type I allergy characterized by mucosal inflammation that occurs in response to allergen exposure. They are common diseases involving a complex inheritance. Complement systems are known to play an important role in allergic diseases. Decay-accelerating factor (DAF) is important for the regulation of the complement system and is a good candidate for determining the susceptibility to allergic diseases. The present study aimed to investigate whether polymorphisms in the DAF gene are associated with allergic respiratory diseases in the Japanese population. We performed mutation screenings of DAF and conducted a tag single-nucleotide polymorphisms (SNP) association analysis for 684 unrelated adult individuals with seasonal allergic rhinitis (SAR) with Japanese ceder pollen, 188 mite-sensitive adults with asthma, and 346 unrelated non-allergic healthy controls. DAF is located in the tight linkage disequilibrium (LD) block spanning 62 kb. The tag SNP analysis revealed that rs10746463 was significantly associated with SAR (P=0.00033) and mite-sensitive adult asthma (P=0.044). The rs2564978 and rs3841376 haplotypes, which are located in the promoter region of DAF, were in complete LD with rs10746463 (r2=1). Luciferase reporter assays with constructs containing the 5' flanking regions of DAF showed that the plasmid with rs2564978 C/rs3841376 deletion (the risk haplotype) had a statistically significantly lower transcriptional activity than that containing the rs2564978 T/rs3841376 insertion. Our results suggest that DAF is one of the genes involved in conferring susceptibility to allergic respiratory diseases and show that decreased levels of DAF may be associated with the enhanced specific IgE responses occurring in allergic diseases in the Japanese population.

Indices of lower airway inflammation in children monosensitized to house dust mite after nasal allergen challenge

There are few available data assessing the united airway disease and its systemic aspects in children. With this study, we aimed to investigate the inflammation markers of upper and lower airways before and after nasal allergen challenge in mite sensitive children with different clinical expression of the allergic disease. Four study groups were formed: rhinitis only, without bronchial hyper-responsiveness (R, n = 10), rhinitis with asthma (R + A, n = 22), atopic asymptomatics (AA, n = 8) and nonallergic healthy controls (C, n = 10). Blood eosinophils, nasal and sputum eosinophils, sputum eosinophil cationic protein (ECP) and cys-LTs, and serum ECP levels were measured before and 24 h after nasal allergen challenge. The groups were comparable in terms of age and gender. Cumulative symptom scores recorded during and 1 h after nasal challenge were not significantly different between patients with R, R + A and AA groups. At T(24), the children belonging to R, R + A and AA showed significant increases in nasal eosinophils (P < 0.01, P < 0.001, and P = 0.01, respectively), sputum eosinophils (P = 0.01, P < 0.001, and P < 0.05, respectively) and blood eosinophils (P < 0.01, P < 0.001, and P < 0.05, respectively). Similarly, increases in sputum ECP (P < 0.01, P < 0.001, and P = 0.07, respectively) and sputum cys-LT levels (P = 0.07, P < 0.001, and P < 0.05, respectively) were detected in children belonging to these three groups at T(24). Sputum eosinophils significantly correlated with blood eosinophils (r = 0.54, P < 0.001) and sputum ECP (r = 0.58, P < 0.001) at T(24). This study showed that nasal allergen challenge increased markers of eosinophilic inflammation in both upper and lower airways of children monosensitized to mites, even before the onset of clinical symptoms.

Immune regulation by CD4+CD25+ T cells and interleukin‐10 in birch pollen‐allergic patients and non‐allergic controls

CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.

Differential gene expression in peripheral blood CD4+ T-Cells of patients with atopic dermatitis

RATIONALE: CD4+ T-cells play a critical role in allergic diseases such as atopic dermatitis (AD) and allergic asthma (AA), both in the affected tissue as well as systemically. Our objective was to investigate the in vivo activation state of peripheral blood CD4+ T-cells from AD patients, by comparing differential gene expression profiles of unstimulated CD4+ T-cells isolated from AD and AA patients, to gene expression profiles of healthy non-allergic controls (HCs).

Dendritic cell expression of FcepsilonRI is regulated by serum IgE

Rationale Dendritic cells (DCs) are important in the initiation of Th1 and Th2 responses. In humans, the high affinity IgE receptor, FceRI, is expressed by type 1 DCs (DC1), whereas expression by DC2 cells has not been previously reported. We sought 1.) to characterize FceRI expression on DC subpopulations and 2.) to determine if DC expression of FceRI is regulated by serum IgE. Methods FceRI expression by DCs was determined by flow cytometry. Study A examined 21 non-allergic healthy controls and 23 allergic asthmatic subjects. Study B was a clinical trial of 24 subjects with ragweed allergic rhinitis who received omalizumab (anti-IgE) or placebo. Results Study A: FceRI was expressed by both DC1 and DC2 cells, and was found in both peripheral blood precursor DC (pDC) and mature tissue DC populations. FceRI expression by pDC1 and pDC2 cells was highly correlated to serum IgE and was significantly greater in subjects with allergic asthma compared to the non-atopic controls. Study B: Omalizumab treatment significantly decreased FceRI expression in both the pDC1 and pDC2 subsets (maximum decrease: 52% and 83%, respectively). The decrease in DC FceRI expression correlated with the drop in serum IgE. Conclusions FceRI is expressed by both the DC1 and DC2 subpopulations. The correlation of FceRI expression and serum IgE in Study A, and the drop in FceRI expression in Study B, support the conclusion that serum IgE is a major factor regulating DC expression of FceRI in vivo. These results suggest that FceRI expression by DC may be a useful therapeutic target.

Local production and detection of (specific) IgE in nasal B-cells and plasma cells of allergic rhinitis patients.

Allergic diseases are characterized by allergic complaints in the shock organ and specific immunoglobulin (Ig)E in serum. Literature data indicate that the nasal mucosa itself could produce at least a large part of the specific IgE in allergic rhinitis patients. In order to investigate this hypothesis, nasal mucosal biopsies from the inferior turbinate were taken from symptomatic grass pollen allergic rhinitis patients, symptomatic house dust mite allergic rhinitis patients and nonallergic healthy controls, confirmed by radioallergosorbent test and skin-prick test. Immunohistochemical double-staining was performed for B-cells (CD19) with IgE, plasma cells (CD138) with IgE and plasma cells with biotinylated allergens. Significantly more IgE-positive B-cells and IgE-positive plasma cells were found in the nasal mucosa of allergic patients than in that of nonallergic controls. Double staining with biotinylated allergens and plasma cells showed allergen-positive plasma cells in the nasal mucosa of allergic patients and no allergen-positive plasma cells in the nasal mucosa of nonallergic patients. Blocking experiments using polyclonal antibodies directed against IgE showed a significant reduction in the number of allergen-positive cells in contrast to experiments using polyclonal antibodies directed against IgG, IgA or IgM. This study describes new evidence that specific immunoglobulin E is produced locally in the nasal mucosa in patients with seasonal allergic rhinitis and perennial allergic rhinitis, but not in nonallergic controls.

The renin-angiotensin system in patients with repeated anaphylactic reactions during hymenoptera venom hyposensitization and sting challenge.

Patients with a history of anaphylactic reactions to hymenoptera venom who tolerated the hyposensitization and the sting provocation without problems (n = 10) had angiotensin I (ANG I), angiotensin II (ANG II), angiotensinogen and renin similar to the values found in healthy nonallergic controls (n = 11). In contrast, patients who repeatedly experienced anaphylactic reactions during hyposensitization and who displayed anaphylactic reactions to sting provocation with a living insect (n = 6) showed significantly lower renin (p < 0.05), angiotensinogen (p < 0.05), ANG I (p < 0.05) and ANG II (p < 0.05) plasma levels as compared to healthy nonallergic controls (n = 11). Sting provocation with a living insect induced clinical symptoms of anaphylaxis in all of the 6 patients. A decrease in ANG I was found in 4 of the patients (67%) after provocation as compared to the concentration before the sting challenge. Angiotensinogen decreased in 3 of the patients (50%) whereas renin activity decreased in 2 of the patients (29%). In all cases a decrease in ANG II was noticed (100%). It is concluded that patients at high risk of developing anaphylactic reactions possess a dysfunctional renin-angiotensin system (RAS) which is not stimulated and does not respond appropriately. These findings point to an important role of the RAS as a defense mechanism in response to anaphylactic reactions.

Histamine, tryptase, norepinephrine, angiotensinogen, angiotensin-converting enzyme, angiotensin I and II in plasma of patients with hymenoptera venom anaphylaxis.

Markers of immediate-type hypersensitivity such as histamine and tryptase were measured in the plasma of nonallergic volunteers and patients with a history of hymenoptera venom anaphylaxis. No significant differences in histamine or tryptase were found between patients and controls. Norepinephrine, an important compound involved in the control of cardiovascular functions and blood pressure, was the same in patients and nonallergic volunteers. In addition, components of the renin-angiotensin system were determined. Patients with hymenoptera venom anaphylaxis showed significantly lower plasma angiotensinogen concentrations as compared to healthy nonallergic controls (p < 0.007), whereas plasma ACE activity was the same. Likewise, the plasma levels of angiotensin I and angiotensin II were significantly reduced in patients as compared to controls (p < 0.04 and p < 0.003, respectively). These findings suggest that the renin-angiotensin system may play an important role as a counteracting factor in hymenoptera venom anaphylaxis.

Cytokine mRNA Profile and Cell Activation in Bronchoalveolar Lavage Fluid from Nonatopic Patients with Symptomatic Asthma

Recent studies have indicated that airway inflammation in atopic asthma is characterized by T-cell activation and local eosinophilia, but it is unknown whether this also applies to nonatopic asthma. In this study, the cytokine mRNA profile and activation status of inflammatory cells in bronchoalveolar lavage fluid (BALF) of eight nonallergic patients with symptomatic asthma and eight nonallergic healthy controls were compared using the message amplification phenotyping (MAPPing) with the polymerase chain reaction (PCR) procedure and immunocytochemical evaluation. Asthmatics had an increasing number of inflammatory cells in BALF, including activated eosinophils (EG2-positive) (p less than 0.001) and activated T cells (CD25-positive) (p less than 0.001). Activated T cells from five of the eight asthmatic patients and from one control subject expressed high levels of interleukin 5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF). All the asthmatic patients had increased numbers of monocytes in their BALF (p less than 0.002) and those cells invariably showed increased expression of interleukin 1 beta (IL1 beta) transcripts. In five patients they also expressed appreciable levels of IL-6 and GM-CSF mRNA. IL-5 and GM-CSF can induce local activation of eosinophils, and IL-1 beta and IL-6 are known to promote T-cell activation and proliferation. Thus, there is an increased production of cytokines with inflammatory properties in the airways of patients with nonatopic symptomatic asthma, which may contribute to the persistence of inflammation, and monocytes and activated T cells are important sources of these cytokines.

Fatty acid composition of phospholipids of plasma and of mononuclear blood cells in children with allergic asthma and the influence of glucocorticoids

Fatty acid (FA) composition of plasma phospholipids and phospholipids extracted from peripheral mononuclear white blood cells (MNC) was investigated in 11 allergic asthmatic children (age 8.9 +/- 4.6 years), in 10 age-matched non-allergic healthy controls and in 14 allergic and non-allergic children with an acute attack of asthma, who had received prednisolone medication for 2-4 days. In allergic asthmatics eicosapentaenoic acid (20:5n-3) was significantly elevated in both plasma and MNC. The relative amount of 20:5n-3 in MNC as well as in plasma correlated positively with increasing levels of total serum IgE (P less than 0.02). The pattern of the other FAs in plasma and of MNC phospholipids did not differ between allergic asthmatic and non-allergic control children. In children with an acute attack of asthma, who had been treated with glucocorticoids (2 mg prednisolone/kg body weight for 2-4 days), distinct changes of relative FA composition of phospholipids were restricted to plasma, where some very long chain FA (22:4n-6, 22:5n-6) were elevated. No significant changes in FA from MNC phospholipids could be observed after glucocorticoid treatment. These findings may indicate a possible role of 20:5n-3, the precursor of "group 3" eicosanoids, in allergic asthmatic children.