| Asthma is a common disease, it affect about 10% of population in the world. The incidence of asthma is different in different country and district. The mechanism of asthma is the result of many genes working with environment. The nature theory about asthma is I type allergy till now, however, IgE dependent mast cells activation is just the core of I type allergy theory, and it also is what we want to study in our research.The mast cell has long been considered as one of the important cells in asthma. Indeed, elevated numbers of mast cells and levels of histamine and tryptase were observed in the bronchoalveolar lavage fluids of asthmatics compared with non - asthmatics, and these were associated with increased airway obstruction, indicating further that mast cell activation could be a critical step in the pathogenesis of asthma. Mast cell degranulation was also observed under light - or electro - microscopy in the BALF from the patients with asthma following local allergen challenge.However, information on the functional properties of mast cells from BALF is relatively limited. As such issue is crucial for understanding mast cells in asthmatic airways the mast cells from asthmatic BALF were challenged in the current study and their functional properties were evaluated.MATERIAL AND METHODSMaterial : The following compounds were purchased from Sigma ( Poole, Dorset, UK) : calcium ionophore A23187, Theophylline, Adenosine (9 - b -D - Ribofuranosyladenine) , NECA(5'- N - ethylcarboxamidoadenosine) , colla-genase (type I) , hyaluronidase (type I) , BSA ( fraction V) , penicillin and streptomycin, MEM containing 25 mM HEPES, sheep anti - mouse immuno-globulins, extr - Avidin peroxidase and o - phenylene diamine ( OPD ). Goatanti - human IgE ( inactivated) and histamine enzyme immunoassay kit were from Serotec (Kidlington, Oxford, UK).Subjects : The study included 29 mild asthmatics, and all of them were stable at the time of study.Preparation of BAL cells and challenge : BALF was obtained with an 0-lympus BF fibreoptic bronchoscope and was filtered through a sterile nylon gauze. Following washing with MEM (containing 2% FCS) twice cells were re-suspended into complete HBSS ( HBSS with 1. 8 mM CaCl2 and 0. 5 niM MgCl2 ). After taking 10 ul out for cell counting, cells were added to tubes containing anti - IgE, CI, adenosine, NEC A or buffer alone or anti - IgE + adeno-sine, anti - IgE + theophylline, adenosine + theophlline 100 ul per tube and incubated at 37 ℃ for 20 min. The reactions were stopped by placing the tubes on ice and adding 100 ul cold incomplete HBSS (HBSS without 1.8 mM CaCl2 and 0.5 mM MgCl2). Soon after the tubes were centrifuged at 2000 rpm, 4 ℃ for 7 min, cell supernatants were collected into Eppendorfs and stored at - 20 ℃.Histamine measurement ; The histamine chromogenic assay was employed to detect histamine released from BALF mast cells. The assay was performed according to the procedures supplied by manufacturer. In brief, 100 ul of histamine standards, control or samples were mixed with 50 ul acylation solution in a 96 - well plate. Following 60 min incubation at room temperature, 50 ul of the acylated standards, control or samples were pipetted into an antibody - coated plate and mixed with 200 ul of enzymatic conjugate at 4℃ for 18 hours. The reactions were visualized by addition of substrate supplied by manufacturer and the plate was read at 410 nm on a plate reader. The histamine fluorometric assay was used to measure histamine in supernatants of dispersed mast cells and was performed as follows, a glass fibre - based, fluorometric assay was employed to determine histamine levels in supernatants. The procedure is involving in a glass -fiber matrix (Lundbeck Diagnostics, Copenhagen, Denmark) that selectively binds histamine. The histamine is detected by adding o - phthaldialdehyde ( OPD) to the glass fibre plate and read on a spectrophotofluorometer ( Perkin -Elmer LS 2, Denmark). Histamine release was expressed as a percentage of total cellular... |
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