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Notizen: To study the human intestinal mast cell of children and adults, we combined a sensitive glassfibre-based histamine assay with the enzymatic and mechanical dispersion of surgical specimens or mucosal biopsies. The method yields between 1.2 × 103 to 4.6 × 103 mast cells/mg tissue constituting 1.2% to 5.3% of total cell count. The mast cell yield, however, depends on the intestinal tissue specimen used for dispersion. Aliquots containing 1500 mast cells per sample are sufficient for measuring significant amounts of histamine (± 0.15 ng histamine per sample), thus making it possible, to carry out approximately 75 tests for four mucosal biopsies of 10 mg each. The intestinal mast cell releases histamine in a dose-dependent manner on challenge with anti-IgE (6–600 U/ml), ionophore A23187 (0.25–1.0 μM), and Concanavalin A (0.7–25.0 μg/ml). The histamine release shows interindividual variation with a net histamine release between 0 to 2.5 ng/samples dependent on the secretatogue. In general, it is not necessary to passively sensitize the mast cells to obtain a sufficient histamine release response to anti-IgE challenge, indicating the presence of intact and functional cell-bound IgE. However, it is shown that four of 10 non-atopic intestinal mast cell samples could be passively sensitized with human plasma containing either mite- or grass-specific IgE without stripping off the IgE first. This indicates the presence of free and preserved Fc-receptors on the dispersed mast cells in some subjects. In addition, it is found that the phorbolester TPA increases the histamine release response to A23187 and turns anti-IgE non-responding mast cells into responding mast cells, but TPA alone at 2 to 16 ng/ml has no histamine releasing effect. In patients with anti-IgE responding mast cells no additional effect of TPA is seen. Finally, no substantial differences between mast cells of children and adults are demonstrated.

Notizen: The in vitro histamine release response of human intestinal mast cells and basophils challenged with anti-IgE, Concanavalin A, ionophore A23187 and food extracts was compared with skin prick test, RAST analysis and open food challenge. It was not possible to perform food challenge in all patients; however, seven children underwent open food challenge and in five the clinical diagnosis of “true” food allergy was confirmed. The intestinal mast cells were pooled from enzymatically dispersed duodenal biopsies obtained by duodenoscopy from 15 selected children suspected of food allergy, and five age-matched controls. In nine of 10 patients classified as “food allergic” intestinal mast cells released histamine to various food extracts in a dose-dependent fashion. From the mast cells of the nine food-allergic patients compared with non-allergics, the anti-IgE mediated mast cell histamine release was increased. Additionally, at 1000 U/ml anti-IgE the mast cell histamine release was increased compared with their corresponding basophils. However, in non-allergic subjects the histamine release of basophils was increased compared with their corresponding mast cells. Histamine release from basophils was positively correlated to the test scores of the RAST analysis, skin prick test, and food challenge. No apparent correlation between tests scores obtained from histamine release of intestinal mast cell and the other tests was demonstrated, except in children with diarrhoea as only symptom. However, the study gives evidence that duodenal mast cells actually are sensitized with specific IgE and thus may play a pathophysiological role in food hypersensitivity. In addition, the study shows that the ability of different stimuli, including food extracts, to trigger basophil histamine release does not correlate with their potency to induce histamine release from mast cells.

Notizen: Background:  Mast cells have long been recognized as the principal cell type that initiates the inflammatory response characteristic of acute allergic type 1 reactions. Our goal has been to further characterize maturation of progenitors to mast cells.Methods:  Mast cells were cultured from human cord blood derived CD133+ progenitors. Mast cell function was tested using histamine release. During differentiation mast cells surface marker expression was monitored by flow cytometry.Results:  CD133+ progenitors expressed the early haematopoietic and myeloid lineage markers CD34, CD117, CD13 and CD33. Mature mast cells expressed CD117, CD13 and CD33, and expression of the high affinity immunoglobulin E recpetor FcɛRI increased during culture. Cytokine receptors interleukin (IL)-5R, IL-3R, granulocyte-macrophage-colony stimulating factor (GM-CSF)R and IL-18R were expressed at high levels during maturation. Chemokine receptors CXCR4 and CXCR2 were highly expressed on both newly purified CD133+ cells and mature cells.Conclusion:  Human mast cells can be cultured from a CD34+/CD117+/CD13+/CD33+ progenitor cell population in cord blood that is tryptase and chymase negative. Developing and mature mast cells express a wide range of chemokine and cytokine receptors. We found high levels of expression of CD123, IL-5R and GM-CSF receptors, also found on eosinophils and basophils, and high levels of expression of the receptor for the inflammatory cytokine IL-18.

Notizen: Background: IL-3 enhances basophil histamine release upon stimulation with any known secretagogue. The molecular mechanism behind this regulation is not known, although some observations suggest that IL-3 modulates the calcium part of the signal transduction mechanism. The inhibitory action of glucocorticoids on basophils can be reversed by stimulation with IL-3. Methods: Calcium-binding proteins in the basophil cell line KU812 were identified by two-dimensional gel electrophoresis, Calcium-overlay assay, N-terminal sequence analysis, and mass spectometry. The presence of the same proteins in purified human basophil leukocytes was established by comigration of KU812 and human basophil proteins on the two-dimensional gels. The expression of the calcium-binding proteins in the absence and presence of IL-3 and/or anti-IgE was determined by densitometric measurement of the spots on the two-dimensional gels. Results: Calreticulin was identified on the two-dimensional gel of KU812 proteins. A protein with exactly the same migration pattern was found on the gels of proteins from purified human basophils. Immunoblotting with a specific anti-human calreticulin antibody confirmed that this protein was calreticulin. Subsequent analysis showed that the expression of calreticulin in the basophils is upregulated twofold upon stimulation with rhIL-3, even in doses below those needed for enhancement of histamine release. Conclusions: The expression of calreticulin in human basophil leukocytes is regulated by IL-3. Calreticulin is known to modulate IP3-dependent Ca2+ influx in different cell systems, and calreticulin overexpression inhibits steroid-induced transcriptional activation. Therefore, modulation of calreticulin expression may be one mechanism by which IL-3 exerts its effects on human basophils.

Notizen: Background: Several susceptibility genes for atopy have been suggested in recent years. Few have been investigated as intensively as the interleukin-4-receptor α (IL4Rα) gene on chromosome 16. The results remain in dispute. Therefore, in a robust design, we tested for association of type I allergy to the IL4R variations I50V and Q576R, and investigated chromosome 16 for atopy candidate regions in general. Methods: We identified 100 Danish allergy sib-pair families. Five conservative phenotypes for type I allergy were defined and evaluated. The IL4R variations were genotyped in trios and evaluated by the transmission disequilibrium test (TDT). Multipoint linkage analysis and exclusion mapping were conducted with sib-pairs analyzed for 17 microsatellite markers. Results: No evidence for association or linkage to the IL4R polymorphisms was found (P values: 0.12–0.90). Linkage analysis did not support linkage of any of the phenotypes to chromosome 16. Major parts of chromosome 16 were excluded as candidate regions harboring oligogenes for type I allergy. Conclusions: We found chromosome 16 unlikely to harbor strong candidate genes for type I allergy. The role of the IL4Rα gene in the inheritance of atopy was insignificant in the Danish population. The use of conservative allergy phenotypes in the search for genes predisposing to atopic disease was discussed.

Notizen: The cellular branch of the immune defence in the newborn has been shown to differ from adults in a number of ways. This report presents new data on the functions of the histamine-sccreting cells of the newborn. Mast cells of the newborn were obtained from the human umbilical cord by enzymatic dispersion. The granules of the mast cells of the umbilical cord were found to contain both chymase and tryptase by immunohistochemical staining, and the presence of cell bound IgE on the mast cell surface was demon-strated by staining sections of umbilical cord with peeroxidase conjugated anti IgE. The enzymatic dispersion yielded 12, 660 mast cells per gram um-bilical cord (median), range 2, 500—60, 300 (n=48). The mast cells were found to constitute 3. 1 % of the total nucleated cells in the dispersate (median), range 1. 5—3. 8%. The histamine release from these cells was measured using a glass microfibre-based method. Both the umbilical cord mast cells and the cord blood basophils released histamine stimulated with anti-IgE. concanavalin A and the calcium ionophore A23187. In contrast to mast cells from adult tissue, the phorbol ester TPA was found to be an efficient secret agogue in both mast cells and basophils from the newborn. After maximal stimulation with anti-IgE and phorbol ester the quantity of histamine released per millilitre of blood was significantly higher in cord blood than in adult blood. The spontaneous histamine release from cord blood basophils was also significantly higher than from adult blood basophils. The mast cells found in the umbilical cord matrix and the cord blood basophils represent a readily available source of metabolically active histamine releasing cells for exploration of the role of histamine-secreting cells in newborn im-mune defence.

Notizen: Background Atopy is closely associated with the cellular T helper type-2 (Th2) phenotype, that is dominated by the pleiotrophic cytokine IL-4. The cellular source of IL-4 has yet to be determined, although basophils have been proposed. Eosinophils and mast cells are likely contenders investigated here, and the eosinophil-like leukaemia line AML14.3D10 is compared to eosinophils as an in vitro culturable model for eosinophils. Lectins can cross-link-specific surface glycoproteins and are found in the ingested (processed foods) and inhaled (airborne pollen grains) human environment. Therefore it is of interest to determine whether lectins can elicit the release of IL-4 from Th2-associated granulocytes other than basophils.Method This study investigated the ability of eosinophils, AML14.3D10 and mast cells to secrete preformed IL-4 in response to stimulation with lectins, and explored molecular mechanisms underlying the interaction.Results Purified eosinophils and basophils, and cultured mast cells and AML14.3D10 cells were incubated with 1 µm lectin. Agglutination was scored by microscopy. IL-4 secretion was measured by enayme-linked immunosorbent assay. Biotinylated lectins were used to determine binding to cells by flow cytometry and in lectin blots of sodium dodecyl sulphate (SDS) gels.Discussion Purified human eosinophils, AML14.3D10 cells and cultured mast cells secrete IL-4 with a pattern similar to that found in basophils when stimulated with a panel of reactive and unreactive lectins. The lectin SNA induces IL-4 secretion from mast cells and basophils, but not from eosinophils or AML14.3D10. Eosinophils appear to secrete only pre-formed IL-4, whereas mast cells may synthesize IL-4 on ligation with the lectin LCA. Lectins that agglutinate the granulocytes investigated do not necessarily induce secretion of IL-4. Lectins that elicit secretion of IL-4 bind more to eosinophils than unreactive lectins as determined by flow cytometry and lectin blotting of SDS gels.Conclusion As granulocytes with functions related to that of basophils, eosinophils, AML14.3D10 and cultured mast cells respond to stimulation with lectins similarly to basophils. This emphasizes the possibility that eosinophils and mast cells may be linked in their cellular heritage as the cellular partners, and lectins as ligands, may contribute to the maintenance of a Th2-favoured microenvironment that is thought to underlie the allergic march.

Notizen: Histamine release (HR) from washed human blood cells after challenge with the excretory-secretory antigens (ES) of the parasitic nematodes Toxocara canis and Ascaris suum was studied, employing a recently developed microfibre–based assay combined with hyperosmolar release media for maximal sensitivity.Blood samples were obtained from 30 patients suspected of parasite infection and 11 healthy volunteers serving as controls. Specific antibodies of IgE, IgG1 and IgG4 subclasses were determined by ELISA.Virtually no HR could be provoked by ES in the 11 controls. In contrast, HR was seen in 16 patients after challenge with T. canis ES and in II patients with A. suum ES. In the majority of these. HR was detectable after challenge with ES protein concentrations of less than 1 ng/ml. and the maximal HR obtained with ES was greater than that seen with optimal concentrations of anti-IgE.The HR after ES antigen challenge correla ed with the amount of specific IgE in patient plasma, and coincided with the presence of specific IgGi and IgG4.

Notizen: Cord blood samples were collected from a birth cohort of 2631 infants to elucidate the association between genetic and environmental factors and fetal production of IgE. The cord blood IgE values were treated both as a continuous and as a dichotomous variable in the statistical analyses. Multivariate analysis was used to control for confounding factors. Infants with single and biparental atopic heritage had higher IgE concentrations in cord plasma than children of parents without atopy. Multiple logistic regression analysis revealed a significant association to maternal allergic eczema or perennial rhinitis. The cord blood IgE concentration varied with month of birth with peaks in late autumn. This seasonal variation was not related to parental atopic disease. Boys had significantly higher levels of IgE and more often elevated IgE values (≥0.5 kU/1) than girls. Alcohol and caffeine consumption by the mothers during pregnancy were both significantly associated with elevated IgE concentration. There was also a relation between mothers prepregnant weight and elevated CB-IgE levels. No significant association was observed between maternal smoking and cord plasma IgE levels. The fact that many factors presumably not related to child allergy seem to influence the regulation of fetal IgE production, could explain the questionable value of cord blood IgE in predicting allergy in childhood.