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During asthma attacks, allergens activate sensitized basophils in the lung, thereby aggravating symptoms. Due to the paucity of basophils in bronchial lavage fluid and the lack of specific basophil detection and quantification methods, basophil-directed research in these samples was hampered in the past. This study aimed to establish and validate a flow cytometry-based basophil detection and quantification method for human basophils from bronchoalveolar lavage (BAL) and blood as a prerequisite for a better understanding of their pathogenic contribution and subtyping of asthma phenotypes. BAL and blood leukocytes from seasonal asthmatics were analyzed by flow cytometry. Chipcytometry, a highly sensitive single-cell analysis method, was used to validate the staining panel for basophils. Cell differentials of May-Grunwald-Giemsa-stained cytospins were used to compare basophil percentages. BAL basophils are identifiable as CD123(+) HLA-DR(-) CD3(-) CD14(-) CD19(-) CD20(-) CD56(-) cells in flow cytometrical analysis. Their identity was validated by Chipcytometry. CD203c was highly expressed by BAL basophils, whereas it was expressed at variable levels on blood basophils. The two quantification methods correlated, although more basophils were detected by flow cytometry. Furthermore, the increase in basophil percentages in the lung correlated with the decrease in the basophil percentages in the blood after allergen challenge. We here validated a reliable basophil quantification method, which is independent of the cell's activation and degranulation state. The results obtained with this method indicate that basophils are directly recruited from the blood circulation to the airway lumen.

BackgroundEpidemiological and experimental studies suggest that exposure to ultrafine particles (UFP) might aggravate the allergic inflammation of the lung in asthmatics.MethodsWe exposed 12 allergic asthmatics in two subgroups in a double-blinded randomized cross-over design, first to freshly generated ultrafine carbon particles (64¿g/m3; 6.1¿±¿0.4 × 105 particles/cm3 for 2h) and then to filtered air or vice versa with a 28-day recovery period in-between. Eighteen hours after each exposure, grass pollen was instilled into a lung lobe via bronchoscopy. Another 24hours later, inflammatory cells were collected by means of bronchoalveolar lavage (BAL). (Trial registration: NCT00527462)ResultsFor the entire study group, inhalation of UFP by itself had no significant effect on the allergen induced inflammatory response measured with total cell count as compared to exposure with filtered air (p¿=¿0.188). However, the subgroup of subjects, which inhaled UFP during the first exposure, exhibited a significant increase in total BAL cells (p¿=¿0.021), eosinophils (p¿=¿0.031) and monocytes (p¿=¿0.013) after filtered air exposure and subsequent allergen challenge 28days later. Additionally, the potential of BAL cells to generate oxidant radicals was significantly elevated at that time point. The subgroup that was exposed first to filtered air and 28days later to UFP did not reveal differences between sessions.ConclusionsOur data demonstrate that pre-allergen exposure to UFP had no acute effect on the allergic inflammation. However, the subgroup analysis lead to the speculation that inhaled UFP particles might have a long-term effect on the inflammatory course in asthmatic patients. This should be reconfirmed in further studies with an appropriate study design and sufficient number of subjects.

Summary In systemic lupus erythematosus (SLE) self-reactive antibodies can target the kidney (lupus nephritis) leading to functional failure and possible mortality. We report that activation of basophils by autoreactive IgE, causes their homing to lymph nodes, promoting TH2 cell differentiation, and enhancing the production of self-reactive antibodies that cause lupus-like nephritis in Lyn−/− mice. SLE patients also have elevated serum IgE, self-reactive IgE's, and activated basophils that express CD62L and the MHC Class II molecule, HLA-DR; parameters that were found to be associated with increased disease activity and active lupus nephritis. Basophils were also present in the lymph nodes and spleen of SLE patients. Thus, in Lyn−/− mice, basophils and IgE autoantibodies amplify autoantibody production that leads to lupus nephritis, and in SLE patients, the presence of IgE autoantibodies and activated basophils are factors associated with disease activity and nephritis.

Background: Epidemiological studies have shown that exposure to ambient particles is associated with increased respiratory symp-toms and exacerbations in asthmatic patients. Ultrafine particles (UFP) may contribute to these negative health effects due to their highly reactive surface area and oxidant capacity. The aim of this study was to test the hypothesis that the allergic inflammation in the lung of allergic asthmatics will be enhanced due to the pre-exposure to UFP. Methods: Fourteen mild allergic asthmatics were exposed for 2 h to a controlled atmosphere of 50 lg/m3 spark-discharge generated carbon black UFP (Ø50 nm) or clean air in a randomized, double-blind crossover design. The expositions were performed with a wash-out period of 30 days under intermittent bicycles ergometer exercise. Eighteen hour after the exposures segmental allergen challenges were performed by bronchoscopy. Forty-two h after the expositions, during a second bronchoscopy, the previously challenged segments were lavaged and BAL-cells were obtained for differentiated FACS analysis and oxygen radical generation by chemiluminescence. Results: The preexposition to ultrafine particles showed only a slight nonsignificant short term effect after 42 h on the allergic inflammation compared to the exposition with clean air. The subgroup of subjects however, which were exposed to ultrafine particles in the first period and to clean air in the second period showed after the allergen challenge in the second period a strong increase in the absolute numbers of eosinophils and oxygen radical generation by BAL-cells. Conclusion of the study: The exposure to ultrafine carbon black particles (UFP) 30 days prior to an allergen challenge had a strong pro-inflammatory effect on the allergic inflammation in allergic asthmatics.