Anti-inflammatory actions of phosphatidylinositol.

Chronic inflammatory T-cell-mediated diseases such as inflammatory bowel disease (IBD) are often treated with immunosuppressants including corticosteroids. In addition to the intended T-cell suppression, these farmacons give rise to many side effects. Recently, immunosuppressive phospholipids have been proposed as less-toxic alternatives. We aimed to investigate the immunoregulatory capacities of the naturally occurring phospholipid phosphatidylinositol (PI). Systemic PI treatment dramatically reduced disease severity and intestinal inflammation in murine 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis. Moreover, PI treatment inhibited the inflammatory T-cell response in these mice, as T cells derived from colon-draining LN of PI-treated mice secreted less IL-17 and IFN-γ upon polyclonal restimulation when compared to those of saline-treated mice. Further characterization of the suppressive capacity of PI revealed that the phospholipid suppressed Th cell differentiation in vitro irrespective of their cytokine profile by inhibiting proliferation and IL-2 release. In particular, PI diminished IL-2 mRNA expression and inhibited ERK1-, ERK-2-, p38- and JNK-phosphorylation. Crucially, PI did not ablate Treg differentiation or the antigen-presenting capacity of DCs in vitro. These data validate PI as a pluripotent inhibitor that can be applied mucosally as well as systemically. Its compelling functions render PI a promising novel physiological immune suppressant.

IL-10 signaling in dendritic cells controls IL-1ß-mediated IFNγ secretion by human CD4+ T cells: relevance to inflammatory bowel disease.

Uncontrolled interferon γ (IFNγ)-mediated T-cell responses to commensal microbiota are a driver of inflammatory bowel disease (IBD). Interleukin-10 (IL-10) is crucial for controlling these T-cell responses, but the precise mechanism of inhibition remains unclear. A better understanding of how IL-10 exerts its suppressive function may allow identification of individuals with suboptimal IL-10 function among the heterogeneous population of IBD patients. Using cells from patients with an IL10RA deficiency or STAT3 mutations, we demonstrate that IL-10 signaling in monocyte-derived dendritic cells (moDCs), but not T cells, is essential for controlling IFNγ-secreting CD4+ T cells. Deficiency in IL-10 signaling dramatically increased IL-1ß release by moDCs. IL-1ß boosted IFNγ secretion by CD4+ T cells either directly or indirectly by stimulating moDCs to secrete IL-12. As predicted a signature of IL-10 dysfunction was observed in a subgroup of pediatric IBD patients having higher IL-1ß expression in activated immune cells and macroscopically affected intestinal tissue. In agreement, reduced IL10RA expression was detected in peripheral blood mononuclear cells and a subgroup of pediatric IBD patients exhibited diminished IL-10 responsiveness. Our data unveil an important mechanism by which IL-10 controls IFNγ-secreting CD4+ T cells in humans and identifies IL-1ß as a potential classifier for a subgroup of IBD patients.

Macrophage-mediated gliadin degradation and concomitant IL-27 production drive IL-10- and IFN-γ-secreting Tr1-like-cell differentiation in a murine model for gluten tolerance.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells.

Tolerance to harmless exogenous antigens is the default immune response in the gastrointestinal tract. Although extensive studies have demonstrated the importance of the mesenteric lymph nodes (MLNs) and intestinal CD103(+) dendritic cells (DCs) in driving small intestinal tolerance to protein antigen, the structural and immunological basis of colonic tolerance remain poorly understood. We show here that the caudal and iliac lymph nodes (ILNs) are inductive sites for distal colonic immune responses and that colonic T cell-mediated tolerance induction to protein antigen is initiated in these draining lymph nodes and not in MLNs. In agreement, colonic tolerance induction was not altered by mesenteric lymphadenectomy. Despite tolerance development, CD103(+)CD11b(+) DCs, which are the major migratory DC population in the MLNs, and the tolerance-related retinoic acid-generating enzyme RALDH2 were virtually absent from the ILNs. Administration of ovalbumin (OVA) to the distal colon did increase the number of CD11c(+)MHCII(hi) migratory CD103(-)CD11b(+) and CD103(+)CD11b(-) DCs in the ILNs. Strikingly, colonic tolerance was intact in Batf3-deficient mice specifically lacking CD103(+)CD11b(-) DCs, suggesting that CD103(-) DCs in the ILNs are sufficient to drive tolerance induction after protein antigen encounter in the distal colon. Altogether, we identify different inductive sites for small intestinal and colonic T-cell responses and reveal that distinct cellular mechanisms are operative to maintain tolerance at these sites.

Increased production of interleukin-21, but not interleukin-17A, in the small intestine characterizes pediatric celiac disease.

Celiac disease (CD) is caused by inflammatory CD4(+) T-cell responses to dietary gluten. It is unclear whether interleukin (IL)-21 and IL-17A contribute to CD onset and lesion severity; therefore, we investigated IL-21 and IL-17A expression in biopsies from pediatric CD patients with different histopathological scores. High numbers of IL-21-producing cells were observed in pediatric CD lesions, even Marsh 1-2 lesions, whereas increased numbers of IL-17 secreting cells were not observed. Intraepithelial lymphocytes, CD4(+) T cells and also neutrophils secreted IL-21. Flow cytometry of lamina propria cells revealed a large population of IL-21- and interferon-γ (IFN-γ)-secreting CD3(+) T cells that did not secrete IL-17A. Adult CD patient biopsies also contained high numbers of IL-21-positive cells; however, enhanced numbers of IL-17-positive cells were observed in a small subgroup of patients with severe lesions. As duodenal tissue damage increases contact with microbe-associated molecular patterns, we hypothesized that microbial sensing by Toll-like receptors (TLRs) modulates T cell-derived cytokine secretion. Costimulation with TLR3 ligands during polyclonal T-cell activation significantly increased IL-21 secretion, whereas TLR2 ligands selectively enhanced IL-17A. These results demonstrate that an IL-17A-independent increase in IL-21 production by CD4(+) T cells is characteristic of pediatric CD. We hypothesize that incidental IL-17 secretion is caused by tissue damage rather than gluten-specific responses.

Hydrogen peroxide in exhaled air is increased in stable asthmatic children.

Exhaled air condensate provides a noninvasive means of obtaining samples from the lower respiratory tract. Hydrogen peroxide (H2O2) in exhaled air has been proposed as a marker of airway inflammation. We hypothesized that in stable asthmatic children the H2O2 concentration in exhaled air condensate may be elevated as a result of airway inflammation. In a cross-sectional study, 66 allergic asthmatic children (of whom, 41 were treated with inhaled steroids) and 21 healthy controls exhaled through a cold trap. The resulting condensate was examined fluorimetrically for the presence of H2O2. All subjects were clinically stable, nonsmokers, without infection. The median H2O2 level in the exhaled air condensate of the asthmatic patients was significantly higher than in healthy controls (0.60 and 0.15 micromol, respectively; p<0.05), largely because of high values in the stable asthmatic children who did not use anti-inflammatory treatment (0.8 micromol; p<0.01 compared to controls). We conclude that hydrogen peroxide is elevated in exhaled air condensate of children with stable asthma, and may reflect airway inflammation.

Study on the significance of bronchial hyperreactivity in the bronchus obstruction after inhalation of cat dander allergen.

The role of bronchial hyperreactivity in the process that leads to bronchial obstruction after inhalation of an allergen was investigated. In 30 asthmatic children selected because of a positive skin test to cat dander allergen, we measured the histamine threshold, the reaction after allergen inhalation, the allergen-specific IgE concentration in serum, the lowest allergen concentration to which the intracutaneous skin test was positive (skin titer), and the histamine release of leukocytes after challenge with allergen. These variables were correlated with each other. The highest correlation was found between the inhalation reaction and the combination of the histamine threshold and either the allergen-specific IgE or the skin titer. Inhalation was only positive with a decreased histamine threshold (less than or equal to 8 mg/ml). With a low histamine threshold, a positive reaction to inhalation is likely to occur at an allergen-specific IgE concentration of > or = 2 U/ml or at a skin titer of < or = 2.5 x 10(-1) micrograms/ml.

Controlled low flow off line sampling of exhaled nitric oxide in children.

BACKGROUND: The aim of this study was to validate exhaled nitric oxide (eNO) values obtained with an alternative off line, single breath, low flow balloon sampling method against on line sampling according to ERS and ATS guidelines in children who could perform both methods. METHODS: One hundred and twenty seven white children of median age 14.1 years, all pupils of a secondary school, participated in the study. They performed the two different sampling techniques at three different flows of 50, 100, 150 ml/s. Additional measurements were done in random subgroups to determine the influence of the dead space air on eNO values obtained off line by excluding the first 220 ml of exhaled air. All children completed a questionnaire on respiratory and allergic disorders and underwent spirometric tests. RESULTS: The off line eNO values were significantly higher than the on line values at all flows. At 50 ml/s the geometric mean (SE) off line eNO was 18.7 (1.1) ppb and the on line eNO was 15.1 (1.1) ppb (p<0.0001). However, when dead space air was discarded, off line and on line values were similar: at 50 ml/s off line eNO was 17.7 (1.0) ppb and on line eNO 16.0 (1.2) ppb. There was a good agreement between off line eNO values without dead space air and on line eNO: for 50 ml/s the mean on/off line ratio was 0.95 (95% agreement limits 0.63 to 1.27). The off line eNO level at 50 ml/s in 80 children with negative questionnaires for asthma, rhinitis, and eczema was 13.6 (1.0) ppb compared with 33.3 (1.1) ppb in the remaining children with positive questionnaires on asthma and allergy and/or recent symptoms of cold (p<0.0001). CONCLUSIONS: In children, off line assessment of eNO using constant low flow sampling and excluding dead space air is feasible and produces similar results as on line assessment with the same exhalation flow rate. Both sampling methods are sufficiently sensitive to differentiate between groups of otherwise healthy school children with and without self-reported asthma, allergy, and/or colds. We propose that, for off line sampling, similar low flow rates should be used as are recommended for on line measurements.

Release of histamine from leucocytes and its determinants in vitro in relation to bronchial responsiveness to inhaled histamine and exercise in vivo.

The hypothesis studied is that increased responsiveness in asthma is not limited to the airways. Forty asthmatic children were analysed for their bronchial responsiveness (BR) to exercise. Twenty patients revealed bronchial obstruction after exercise while the remainder did not. These observations were compared with the responsiveness of leucocytes, which was determined by their histamine 'releasability'. Twenty healthy children served as controls. Release of histamine induced by calcium ionophore-aided calcium influx was significantly higher in both groups of asthmatics than in the healthy children (P less than 0 X 005). Similar findings were obtained by induction of microtubule aggregation due to deuterium oxide (D2O). The S-shaped dose-response relationship with D2O was shifted to the left in the patients with BR to exercise compared to patients without (P less than 0 X 025). The slope was increased in both patient groups compared with the healthy children (P less than 0 X 01). It is concluded that the mean 'releasability' of histamine release due to both stimulants correlated well (P less than 0 X 01). This suggests that the 'releasability' is determined by the responsiveness of the microtubules. This may also apply to allergen-induced histamine release, as was revealed from studies with anti-IgE. The differences in histamine release found in relation to BR due to exercise were also present if the patients were divided according to BR due to histamine. A significant relationship existed between the degree of BR to histamine and the responsiveness of the microtubules (P less than 0 X 02).

Properties of human basophils isolated by fluorescence-activated cell sorting.

The aim of the current study was twofold: (1) to consider the applicability of fluorescence-activated cell sorting (FACS) to basophil isolation from small blood volumes and (2) to compare basophils obtained from children with asthma to basophils from healthy children. With FACS, basophil suspensions were prepared with a purity of 84% (range, 75% to 95%) and a recovery of 20% (range, 15% to 30%). The purified basophils had a total histamine content of 1.6 +/- 0.12 pg per cell, not differing significantly from total histamine content observed in "total" leukocyte suspensions (1.4 +/- 0.07 pg per basophil). The same was true for IgE receptor-mediated histamine release (29 +/- 4% versus 27 +/- 4%) and for ionophore A23187-induced histamine release (41 +/- 6% versus 51 +/- 9%). Sorted basophils from subjects with asthma released more histamine after IgE receptor activation (0.67 +/- 0.09 pg per cell) than basophils from healthy children (0.40 +/- 0.04 pg per cell; p less than 0.02). Expressed as percent release, no significant difference was observed (37 +/- 3.2% versus 30 +/- 2.7%). Ionophore A23187-induced histamine release did not differ significantly between subjects with asthma and control subjects, neither expressed as picograms per cell (1.21 +/- 0.17 pg per cell versus 1.02 +/- 0.11 pg per cell) nor expressed as percent release (66 +/- 4.4% versus 74 +/- 3.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen peroxide in exhaled air of healthy children: reference values.

An increased content of hydrogen peroxide (H2O2), a marker of inflammation, has been described in the condensate of exhaled air from adults and children with inflammatory lung disorders, including asthma. However, the normal range of [H2O2] in the exhaled air condensate from healthy children has not been established. Therefore, the aim of this study was to determine the reference range of exhaled [H2O2] in healthy school-aged children. Ninety-three healthy nonsmoking children (48 female and 45 male, mean age 10 yrs, range 8-13 yrs), with a negative history for allergy, eczema or respiratory disease and with a normal lung function, participated. Exhaled air condensate was examined fluorimetrically for the presence of H2O2. In addition, the reproducibility of [H2O2] within subjects and between days and the stability of [H2O2] during storage at -20 degrees C were assessed. The median [H2O2] in the exhaled air condensate of all children was 0.13 microM, with a 2.5-97.5% reference range of <0.01-0.48 microM. No significant difference existed between males and females. There was no correlation between exhaled [H2O2] and age or lung function. Repeated [H2O2] measurements on 2 consecutive days showed satisfactory within-subject reproducibility and [H2O2] in stored samples remained stable for at least 1 month at -20 degrees C. In conclusion, this study provides reference data for exhaled hydrogen peroxide in a large group of healthy children. The observed levels were lower than those reported previously for healthy adults and were independent of age, sex and lung function.

Hydrogen peroxide and nitric oxide in exhaled air of children with cystic fibrosis during antibiotic treatment.

Cystic fibrosis (CF) patients characteristically have severe chronic airway inflammation associated with bacterial infection. A noninvasive marker of airway inflammation could be a useful guide to treatment of CF lung disease. The aim of this study was to assess whether measurement of hydrogen peroxide (H2O2) and nitric oxide (NO) in exhaled air can serve to monitor the effect of treatment with antibiotics in CF-children with acute infective pulmonary exacerbations. Sixteen CF-patients (mean age 12.3 yrs) with exacerbation of their lung infection were treated with intravenous antibiotics in an uncontrolled study. During treatment, H2O2 in exhaled air condensate was measured twice a week. In addition, serial NO measurements were performed in nine patients. During antibiotic treatment the median H2O2 concentration in exhaled air condensate decreased significantly from 0.28 microM (range 0.07-1.20 microM) to 0.16 microM (range 0.05-0.24 microM, p=0.002) and the mean forced expiratory volume in one second significantly increased from 55% predicted to 75% pred (p=0.001). In individual subjects, changes of H2O2 and FEV1 between pairs of serial measurements correlated weakly (p=0.08). Data on exhaled NO were inconclusive; exhaled NO did not change systematically during treatment. It is concluded that cystic fibrosis patients with an acute pulmonary exacerbation have abnormally high concentrations of hydrogen peroxide, but not of nitric oxide, in exhaled air, which decrease during intravenous antibiotic treatment. Further controlled studies should establish if exhaled hydrogen peroxide, may serve as a noninvasive parameter of airway inflammation to guide antibiotic treatment in cystic fibrosis lung disease.

Activated human granulocytes contract isolated human airways.

Human granulocytes activated with serum treated zymosan (0.2 mg/ml) contract isolated human airways. The magnitude of the contraction depends on the number of granulocytes and the proportion of eosinophils among the granulocytes. The contraction is blocked by a leukotriene C4/D4 (LTC4/D4) receptor antagonist and by inhibition of lipoxygenase. This suggests that eosinophils rather than neutrophils are implicated in this response, which seems to be caused by LTC4/D4.

Additive effect of epithelial denudation and low levels of inflammatory mediators on the sensitivity of isolated human airways to methacholine.

The presence of low concentrations of histamine or the stable thromboxane analogue U46619 and the removal of the epithelium separately and addictively increase the sensitivity of isolated human airways to methacholine. This raises the possibility that these factors play a role in the pathogenesis of bronchial hyperresponsiveness to inhaled methacholine in asthma.

Bronchial responsiveness and leucocyte reactivity after influenza vaccine in asthmatic patients.

The hypothesis was studied that an increased bronchial responsiveness (BR) after viral infections in asthmatics may be related to a change in cellular functions. Histamine threshold and histamine release from leucocytes were measured in asthmatic children and healthy controls before and after administration of a live attenuated or an inactivated influenza vaccine. Live influenza vaccine did increase bronchial responsiveness (BR) to histamine in asthmatic children but not in healthy controls. The inactivated vaccine did not give rise to an increased BR. The releasability of histamine from leucocytes was not increased as a result of the immunisation.