Anti-degranulation response of herbal formula in RBL-2H3 cells.

Allergic diseases not only bring serious economic burden to the patients, but also consume a lot of substantial resources of social medical systems. Thus, the prevention and treatment of allergic diseases are imperative. In this study, the anti-degranulation activity of herbal formula was evaluated using the rat basophil leukemia cells (RBL-2H3) as in vitro model. The morphological and biophysical properties of RBL-2H3 cells before and after treatment with herbal formula were also determined. Notably, the herbal formula exhibits clearly inhibited degranulation by RBL-2H3 cells in a concentration-dependent manner without cytotoxic effect. Therefore, this herbal formula can be used as an alternative and promising therapeutic agent to ameliorate allergic diseases.

Chlorogenic acid alters the biological characteristics of basophil granulocytes by affecting the fluidity of the cell membrane and triggering pseudoallergic reactions.

It is not clear whether pseudoallergic reactions are caused by similar mechanisms as type I allergic reactions. 3­Caffeoylquinic acid (chlorogenic acid) is an active ingredient in traditional Chinese medicines used for antibacterial, anti-inflammatory and cholagogic purposes. It is assumed to be the reason for the high allergic reaction rates associated with certain traditional Chinese medicine injection solutions. The aim of the present study was to investigate the possible mechanisms through which chlorogenic acid triggers pseudoallergic reactions. The fluidity of the cell membrane was investigated using fluorescence recovery after photobleaching. Western blot analysis was used to measure the phosphorylation levels of the Spleen tyrosine kinase (Syk) protein and Fluo­3/AM fluorescent probes were used to investigate the influx of calcium ions. In addition, fluorescence microscopy and phalloidin were used to determine F­actin depolymerization levels. The secretion rate of ß­hexosaminidase by RBL­2H3 cells clearly increased following treatment with chlorogenic acid and the levels of cytoskeletal disintegration were also markedly increased. Furthermore, we detected an increase in the intracellular calcium ion concentration along with distinct changes in Syk protein phosphorylation and cellular F­actin. These changes indicated that chlorogenic acid affected the restructuring of the cytoskeleton and played a role in cell degranulation. In conclusion, chlorogenic acid may lead to the aggregation of lipid rafts on the cell membrane surface by altering RBL­2H3 cell membrane fluidity, thus triggering Syk­related signal transduction and inducing a truncated type I like allergic reaction.

In vitro immunological degranulation of human basophils is modulated by lung histamine and Apis mellifica.

1. The effect of high dilutions of two homeopathic drugs Lung histamine (Lung his) and Apis mellifica (Apis mel) used for the treatment of allergic diseases has been assessed on in vitro human basophil degranulation. Experiments were conducted blind. 2. Basophil degranulation induced by 1.66 X 10(-9) M anti-IgE antibody was significantly inhibited in the presence of 5 Lung his (5th centesimal dilution of Lung his) and 15 Lung his (15th centesimal dilution of Lung his) by 28.8% and 28.6% respectively and by 65.8% in the presence of 9 Apis mel (9th centesimal dilution of Apis mel). Basophil degranulation induced by 1.66 X 10(-16) to 1.66 X 10(-18) M anti-IgE antibody was also inhibited by high dilutions of Lung his and Apis mel with an inhibition of nearly 100% with 18 Lung his (18th centesimal dilution of Lung his) and 10 Apis mel (10th centesimal dilution of Apis mel). An alternance of inhibition, inactivity and stimulation was observed when basophils were incubated in the presence of serial dilutions of Lung his and Apis mel. 3. The investigation of the clinical efficacy of high dilutions of Lung his and Apis mel should be envisaged in allergic diseases in parallel with in vitro and ex vivo biological assays.

Growth of human basophil lines derived from chronic myelocytic leukaemia cells in vitro: ultrastructure and X-ray microanalysis studies.

Mononuclear cells from the peripheral blood of patients with chronic myelocytic leukaemia were grown and expanded in liquid culture in the presence of Con A-conditioned medium. An accelerated development of cells of the basophilic lineage was observed and resulted in the appearance of 85% mature basophils after 14 days of incubation. Transmission electron microscopy of developing basophils showed changes in the nucleus and active granule formation in the cytoplasm. By scanning electron microscopy, the immature cells were relatively smooth in comparison with the mature basophils which showed membranous microvilli. The chemical content of the cells at different days of culture was detected by X-ray microanalysis. Immature cells were characterized by a high level of phosphorus with a low level of sulphur. As maturation progressed, the amount of phosphorus decreased, while the level of the sulphur increased, reaching its highest peak in the mature basophils. The different amount of sulphur found in the cells during the maturational process most probably represents the amount of heparin located in the cell granules. This finding may be useful for studying the influence of growth factors on the development and differentiation of human basophils.

Antigen-induced IgE-mediated degranulation of human basophils.

The events associated with antigen-induced, IgE-mediated degranulation of human basophils from allergic donors were studied ultrastructurally. Partially purified cells were examined prior to addition of antigen and after incubation with antigen or control buffer for 15 seconds to 60 minutes. Reactions were stopped instantaneously by adding fixative directly to the cell suspension. After fixation the cells were exposed to cationized ferritin as a sensitive probe for demonstrating possible continuities between the cytoplasmic granules and the cell surface. Ficoll-Hypaque-isolated basophils were of three types: Type I, cells containing basophil granules with a full complement of particles; type II, cells containing some full granules but also variable numbers of cytoplasmic vacuolar structures having the size and shape of basophilic granules but having reduced or no particle content (partially fixed or empty granules); and type III, basophils containing only empty granules. Following exposure to specific antigen, basophils of all three types underwent degranulation characterized by the fusion of the membranes bounding single basophilic granules with the plasma membrane and leading to extrusion of content. Cells in the process of degranulation (type IV and V basophils) were characterized by communications between individual granules and the cell exterior. Identification of such communications was facilitated by cationized ferritin which entered granules having open communications with the cell surface. Without this marker, the number of such communications would have been seriously underestimated, either because they were extremely narrow, tortuous, or outside the plane of section. The majority of individual basophilic granules fused singly and separately with the plasma membrane, in contrast to guinea pig basophil and rat mast cell degranulation where intercommunicating clusters of granules fused with the plasma membrane at a single point. The particle and membrane contents of extruded granules frequently remained adherent to the surface of type IV and V basophils and were not immediately solubilized. Morphologic evidence of degranulation progressed with time of exposure to antigen, exhibiting kinetics that paralleled histamine release. Cells in control incubations, and rare basophils that had been exposed to antigen E, failed to degranulate. Fully degranulated (type VI) basophils were viable cells that had a markedly irregular surface and were devoid of basophilic granules but retained the minor population of small perinuclear granules.

The antigen-induced degranulation of basophil leukocytes from atopic subjects studied by electron microscopy.

Suspensions of washed leukocytes were prepared from the blood of atopic subjects and incubated with diluent, ragweed antigen E or rye grass group I antigen. Histamine release into the suspending medium was measured and directly correlated with changes in the ultrastructure of basophil leukocytes from the same tubes. Incubation of leukocytes with either diluent or an antigen to which the donor was not hypersensitive caused no significant histamine release and the morphology of the basophils was unaltered. By contrast, incubation of leukocytes with an intigen to which the donor was hypersensitive caused substantial histamine release and characteristic morphologic changes in the basophils, many of which underwent exocytotic degranulation. Degranulated human basophils showed the following features: (1) an irregular surface, to which platelets and leukocytes were often adherent; (2) reduction in the number of basophilic granules; (3) residual granular material, both in exocytotic cavities and at the cell surface; (4) coated vesicles and cisternae of smooth endoplasmic reticulum, frequently related to the plasma membrane at sites of exocytosis; (5) thin membrane-bounded granules appeared unaltered; (6) no consistent change was observed in centrioles, microtubules, 90 A filaments, mitochondria, or multivesicular bodies, and degranulated basophils appeared ultrastructurally viable; (7) in reaction cell suspensions, nondegranulated basophils often showed a more active contour than control cells; both reacting but nondegranulated basophils and degranulated basophils sometimes showed localized filamentous webs at their periphery.