Poor association between allergen-specific serum immunoglobulin E levels, skin sensitivity and basophil degranulation: a study with recombinant birch pollen allergen Bet v 1 and an immunoglobulin E detection system measuring immunoglobulin E capable of binding to FcɛRI

BACKGROUND: Results from several studies indicate that the magnitude of immediate symptoms of type I allergy caused by allergen-induced cross-linking of high-affinity Fc epsilon receptors on effector cells (mast cells and basophils) is not always associated with allergen-specific IgE levels. OBJECTIVE: To investigate the association of results from intradermal skin testing, basophil histamine release and allergen-specific IgE, IgG1-4, IgA and IgM antibody levels in a clinical study performed in birch pollen-allergic patients (n = 18). METHODS: rBet v 1-specific IgEs were measured by quantitative CAP measurements and by using purified Fc epsilon RI-derived alpha-chain to quantify IgE capable of binding to effector cells. Bet v 1-specific IgG subclasses, IgA and IgM levels were measured by ELISA, and basophil histamine release was determined in whole blood samples. Intradermal skin testing was performed with the end-point titration method. RESULTS: Our study demonstrates on the molecular level that the concentrations of allergen-specific IgE antibodies capable of binding to Fc epsilon RI and biological sensitivities are not necessarily associated. A moderate association was found between cutaneous and basophil sensitivity. CONCLUSION: Our results highlight the quantitative discrepancies and limitations of the present diagnostic tools in allergy, even when using a single allergenic molecule. The quantity of allergen-specific serum IgE is only one component of far more complex cellular systems (i.e. basophil-based tests, skin tests) used as indirect diagnostic tests for IgE-mediated allergic sensitivity.     

Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts

BACKGROUND: Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. OBJECTIVE: To assess an accurate methodology for standardization of chemically modified extracts. METHODS: GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. RESULTS: Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. CONCLUSIONS: The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.     

Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs

Allergic or pseudoallergic reactions that occur during anesthesia have been increasing for the last few years. To date, the diagnosis of allergy to muscle relaxants remains difficult. In this respect, we developed a flow cytometric method for the study of drug-induced basophil degranulation using CD63 and CCR3. Fifty patients who developed clinical features evocative of allergic reactions immediately after induction of anesthesia were included and classified into two groups. Group 1 (n = 39) comprised true allergic patients, who developed typical signs of shock associated to positive skin testing. Group 2 (n = 11) consisted of patients whose clinical history was not typical and skin testing was negative or nonconclusive. Seventeen control subjects were also studied in this report. We compared data from flow cytometry to skin tests, specific IgE, and histamine release results. Flow cytometry showed a sensitivity of 54%, while that of specific IgE was similar, at 62%. Interestingly, when considering the sensitivity of IgE + CD63 for diagnosis, we reached a sensitivity value of 80%. Of 15 negative results for specific IgE, we found 7 positive CD63 tests, while histamine release gave positive results in only 2 cases. Furthermore, the CD63 protocol showed good specificity (100%). We conclude that our flow cytometry protocol is a promising tool in allergy diagnosis since it is specific and complementary to specific IgE detection.     

Effect of nitrendipine on histamine release from human basophil leukocytes

The effect of the new calcium antagonist nitrendipine on in vitro basophil activation was evaluated in 10 subjects. The histamine release induced by calcium ionophore A23187, f-met peptide and anti-IgE was inhibited, in a dose-dependent fashion, by nitrendipine in the concentration range of 1-100 microM. The activity of this calcium antagonist seems complex and related to an interference with calcium at multiple sites. At concentrations higher than 200 microM, nitrendipine causes histamine release from basophil leukocytes. This histamine secretion is likely to be due to a cytotoxic effect, since it is associated with an increase in LDH levels in the cell supernatant.     

Inhibitory Effect of Glucocorticosteroids on Anti-IgE-Induced Histamine Release from Human Basophilic Leukocytes: Evidence for a Dual Mechanism of Action

Anti-IgE-induced histamine release from human leukocytes is inhibited when the cells before challenge are cultured overnight in the presence of glucocorticoids (GCSs). The present report suggests that the GCSs might exert their effect by at least a dual mechanism of action. Histamine release was induced by a suboptimum concentration of anti-IgE. When the release recorded in the presence of the steroid is plotted against the release recorded in its absence, the data points of several experiments fit a regression line characterized by two parameters: its slope and its intercept with the abscissa. Structure-activity examination with selected GCSs indicates that the orders of potency for affecting these two parameters are not identical. Furthermore, pulse experiments suggest that the cells require different times of contact with the steroid to express inhibition according to the two parameters. The removal of adherent cells or platelets did not markedly affect the degree of leukocyte histamine release or its inhibition by a given GCS, suggesting that the steroid interacts directly with the basophil. Finally, steroid-induced inhibition was not affected by the putative phospholipase A2-inhibitor p-bromophenacylbromide (BPB) or the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA).     

Basophil activation during pollen season in patients monosensitized to grass pollens

BACKGROUND: Basotest is a new basophil-activation test based upon the expression of CD63 (gp53) in the presence of allergens. It is an effective diagnostic test for pollen-allergic patients. However, it is not known whether Basotest results differ during the pollen season. METHODS: We examined the activation of basophils by Basotest in 13 patients sensitized only to grass pollen, before and during the pollen season, in order to assess whether Basotest could be used as a diagnostic test during the pollen season. Dose-response curves with 10-fold increasing concentrations of timothy grass pollen (10-4 to 100 AU) were carried out. RESULTS: Basophils were not activated spontaneously during the pollen season since the CD63 expression was below detectable levels before in vitro cell activation. A decreased percentage of activated basophils at the peak of activation was found in comparing the pre- and in-season tests, but all patients had a positive test. When basophil activation was at its peak, the allergen concentration was similar during the two periods. Moreover, the median area under the curve was significantly (P < 0.02) reduced during the season as compared to before the season. CONCLUSION: Basotest can therefore be used as a diagnostic test during the pollen season, but the allergen exposure needs to be characterized if quantitative studies are performed.     

Flow-Cytometric Analysis of Human Basophil Degranulation

Quantification of human basophils and their degranulation were performed using a flow-cytometric system. It was shown that the number of basophils among total leukocytes, before and after the effect of degranulating agents, can be obtained rapidly and reproducibly, following alcian blue, staining. Dose-response curves of degranulation by anti-IgE and anti-IgG₄ were studied, and it was shown that these antibodies can induce basophil degranulation in a Ca⁺⁺ dependent manner. It was also shown that degranulation is completed within 10 min.
Values obtained by flow-cytometry and microscopic evaluation, as well as comparison made between percent basophil degranulation and percent histamine release, agreed well with each other in our system. Flow-cytometric analysis of human basophil degranulation provides a new reliable method to assess in vitro the morphological basis of immediate hyper-sensitivity.     

Monomeric immunoglobulin E stabilizes FcεRIα from the human basophil cell line KU812 by protecting it from natural turnover

BACKGROUND: The high affinity IgE receptor (FcepsilonRI) on mast cells and basophils is up-regulated by its own ligand IgE; however, the mechanism is unknown. OBJECTIVE: To study the IgE-mediated effect on FcepsilonRI on basophils by using the human basophilic cell line KU812. METHODS: Expression of cell surface FcepsilonRI was assessed by flow cytometry. Western blot technique was used to illustrate tyrosine-phosphorylation and the Ca2+ level in KU812 was measured by fluorescence of Fura-2. Soluble specimens of the alpha-chain from FcepsilonRI (FcepsilonRIalpha) were obtained by lysing 107 KU812 pr. mL. FcepsilonRIalpha was detected by a sandwich immunoradiometric assay employing the IgE-binding capacity of FcepsilonRIalpha in conjunction with a monoclonal antibody. Polyclonal rabbit anti-FcepsilonRIalpha was used for detection of FcepsilonRIalpha by Western blotting. RESULTS: We found that monomeric IgE did not induce tyrosine-phosphorylation in KU812, which was the case when stimulating with IgE cross-linked by anti-IgE binding. Further, only cross-linking of IgE, but not monomeric IgE, increased the Ca2+ level. Using the immunoradiometric assay, we found a temperature dependent reduction in the amount of FcepsilonRIalpha. Samples incubated at 37 degrees C for 5 h displayed a 16-fold decrease in the FcepsilonRIalpha level compared with samples incubated at 4 degrees C. In the presence of IgE the reduction at 37 degrees C was only threefold. CONCLUSION: These results indicate that IgE does not induce intracellular signals in KU812, i.e., tyrosine-phosphorylation or Ca2+ release. Instead it appears that FcepsilonRIalpha is an unstable protein that IgE stabilizes and thereby protects from a temperature dependent turnover.     

Basophil histamine release in the diagnosis of house dust mite and dander allergy of asthmatic children

The aim of the study is to compare the glass fibre-based basophil histamine release test with skin test (Phazet), RAST (Phadebas) and bronchial provocation test in children with allergic asthma. The study comprised 68 selected children with a case history of extrinsic allergic asthma to danders (cat and dog) and house-dust mite. Skin prick test, RAST, and histamine release were performed in all children and the bronchial provocation test was used as a reference of "true allergic asthma". A total of 81 allergen bronchial challenges were performed and 44 children experienced 49 positive provocations. In 2.9% (2/68) of the children histamine release could not be performed due to technical difficulties (low histamine release with anti-IgE). Concordances in the range 76-87% were observed with no significant difference between the tests. The highest concordance (87%) was found between histamine release and bronchial provocation test followed by skin prick test vs bronchial provocation (84%) and RAST vs bronchial provocation (80%). The sensitivity and specificity were calculated for each test. All tests showed sensitivities in the range 90-94% and no significant difference between them was observed. The specificity of histamine release, skin prick test, and RAST was 0.78, 0.69, and 0.63, respectively. The specificity of histamine release was better than RAST demonstrated by 95% confidence intervals. In conclusion, it was found that the histamine release test is a convenient diagnostic method and the study indicates a diagnostic value comparable to the common diagnostic methods in clinical allergy.     

Basophil histamine release and lymphocyte proliferation tests in latex contact urticaria

Basophil histamine release and lymphocyte proliferation tests were examined with latex allergen prepared from surgical gloves in 15 patients with latex contact urticaria. The basophil histamine release test (BHRT) yielded positive results in 13/14 (93%) patients, whereas commercial latex RAST was positive in only 9/15 (60%) patients. Lymphocyte proliferation test (LPT) was positive in 3/15 (20%) patients, suggesting that cell-mediated immune reactions may also occur in latex allergy. However, patch tests to latex were negative and neither were epidermal Langerhans cells able to present latex antigen to T lymphocytes in vitro.