ovalbumin and Leukemia--Basophilic--Acute

RBL-2H3 mediator release assay, developed for specific IgE screening studies, was not as sensitive as passive cutaneous anaphylaxis (PCA) assay in the polyclonal antibody detection. In the present investigation, the detection sensitivity of RBL-2H3 assay was elevated by modifying the experiment protocols from choosing the proper releasing medium and optimizing the sensitization manner. The polyclonal antibody was generated from Brown Norway (BN) rats exposed to Ovalbumin (OVA). In contrast to Tyrode buffer A, RBL-2H3 cells cultured in DMEM had a lower spontaneous secretion and a higher response to antigen stimulation, both of which could help to increase the detection sensitivity. The rat sera used in the sensitization process should be diluted appropriately to avoid the proliferation-promoting effect on RBL-2H3 cells. The results of the kinetics of sensitization showed that prolonging the sensitization time and then reculturing the cells in IgE free medium for a further 24 h after the removal of rat sera could reach a marked increase in the degree of sensitization. The highest anti-OVA antibody titer detected by the modified RBL-2H3 assay was 4096, while PCA assay was 1024. These data provide evidence that the modified RBL-2H3 mediator release assay has a promising prospect in the determination of the biologic activity of polyclonal antibody.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Immunoglobulin E; Leukemia, Basophilic, Acute; Ovalbumin; Passive Cutaneous Anaphylaxis; Rats; Rats, Inbred BN

The anti-inflammatory effects of low-molecular weight chitosan oligosaccharides (LM-COS) prepared from high-molecular weight chitosan by enzymatic digestion were investigated against allergic reaction and allergic asthma in vivo and in vitro. Allergic asthma is an inflammatory disease of the airways associated with enhanced degranulation and cytokine generation. The LM-COS (<1 kDa), consisting of glucosamine (GlcN)(n), n=3-5, were capable of inhibiting both antigen-stimulated degranulation and cytokine generation in rat basophilic leukemia RBL-2H3 cells. The protective effect of LM-COS against ovalbumin (OVA)-induced lung inflammation in asthma model mice was also examined. Oral administration of LM-COS (16 mg/kg body weight/day) resulted in a significant reduction in both mRNA and protein levels of interleukin (IL)-4, IL-5, IL-13, tumor necrosis factor (TNF)-α in the lung tissue and bronchoalveolar lavage fluid (BALF); The protein levels of IL-4, IL-13 and TNF-α in BALF were decreased by 5.8-fold, 3.0-fold and 9.9-fold, respectively, compared to those in the OVA-sensitized/challenged asthma control group. These results suggest that the oral administration of LM-COS is effective in alleviating the allergic inflammation in vivo and thus can be a good source material for the development of a potent therapeutic agent against mast cell-mediated allergic inflammatory responses and airway inflammation in allergic inflammatory diseases, including asthma.

Topics: Animals; Anti-Inflammatory Agents; Antigens; Asthma; Bronchoalveolar Lavage Fluid; Cell Line, Tumor; Chitosan; Disease Models, Animal; Female; Glucosamine; Hypersensitivity; Immunoglobulin E; Interleukins; Leukemia, Basophilic, Acute; Lung; Mice; Mice, Inbred BALB C; Molecular Weight; Oligosaccharides; Ovalbumin; Tumor Necrosis Factor-alpha

Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of beta-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1--2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.

Topics: Acetylcysteine; Animals; Azo Compounds; Blotting, Northern; Blotting, Western; Cell Degranulation; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluoresceins; Fluorescence; Free Radical Scavengers; Heme Oxygenase-1; Leukemia, Basophilic, Acute; Metalloporphyrins; Ovalbumin; Rats; Rats, Inbred BN; Reactive Oxygen Species; RNA, Messenger; Up-Regulation

Rat basophil leukemia (RBL) cells were sensitised with varying proportions of monoclonal IgE anti-ovalbumin (OVA) and anti-DNP antibodies, and serotonin release was measured after challenge with aggregated OVA or dinitrophenylated human serum albumin (DNP-HSA). Highly aggregated OVA was shown to provoke the degranulation of RBL cells that had been sensitised with an IgE preparation containing 2% IgE anti-OVA antibodies. Highly substituted DNP32-HSA induced degranulation of RBL cells sensitised with just 0.5% antigen-specific IgE. When cells were sensitised with high percentages of specific IgE, maximum degranulation was seen at concentrations of 2 micrograms/ml (aggregated OVA) and 50 ng/ml (DNP-HSA), while moderate degranulation was still seen at antigen concentrations as low as 50 and 2 ng/ml, respectively. Low-molecular weight aggregates of OVA and low-valency DNP4-HSA only stimulated degranulation when high percentages of RBL Fc epsilon receptor were occupied by antigen-specific IgE. The sensitising abilities of two anti-DNP monoclonal antibodies of differing affinities were compared. When challenged with low-valency antigen, only cells sensitised with the higher-affinity monoclonal antibody exhibited moderate levels of degranulation. Degranulation required exposure to high antigen challenge doses (5 micrograms/ml). Cells sensitised with either monoclonal antibody responded strongly when challenged with a wide range of concentrations (1-250 ng/ml) of high-valency DNP32-HSA, although greater sensitivity was always seen with the higher-affinity antibody. These results suggest that antigen valency is a critical parameter for mast cell function, and that low-affinity antibody may be capable of sensitising mast cells to high-valency antigen.

Topics: Animals; Antibody Affinity; Antigens; Cell Degranulation; Immunoglobulin E; Leukemia, Basophilic, Acute; Mast Cells; Ovalbumin; Rats; Serotonin