Histamine content and mast cell number in tissues of mutant mice of (mi/mi) genotype

Abstract

IntroductionLoss of function mutation at loci (mi) encoding transcription factor MITF results in microphthalmia, osteopetrosis, a lack of melanocytes and a decrease in the number of mast cells [1]. The decrease in mast cell number is explained by low expression of c-kit, the receptor for major mast cell growth factor [2]. There are conflicting reports on the level of mast cell deficiency in (mi/mi) mice [2][3][4]. To verify the feasibility of using (mi/mi) mice to study mast cell function in vivo we compared mast cell number and histamine content in tissues of (mi/mi), (mi/+) and (+/+) mice. We also tested bone marrow-derived cultured mast cells (BMCMC) from (mi/mi) mice for their proliferative response to recombinant SCF. Materials and methods MiceThe (+/+), (mi/+) and (mi/mi) mice used in these studies are descendants of C57BL/6J (mi/+) breeders donated by Prof. dr W. W. Jedrzejczak, (Military School of Medicine, Warsaw). Heterozygotes (mi/+) were identified by the presence of a dorsal patch of white coat, and (mi/mi) homozygotes were identified by their white colour and microphthalmia. Cell and tissue preparationPeritoneal cells were collected by lavage with 1.5 ml of Ringer solution (pH = 7.4). Tissue samples were fixed in Carnoy's and embedded in paraffin. Mast cells in suspensions and histological sections were stained with toluidine blue [5]. Histamine determinationHistamine content in tissues and in peritoneal cells was measured using Cellex-P (Bio-Rad Laboratories, USA) chromatography, followed by spectrofluorometric detection [6,7]. Mast cell cultures and proliferation assaysBone marrow was obtained from 6 weeks old mice of (mi/mi) or (+/+) genotype and cultured for 4 weeks in the presence of 10 % FBS (fetal bovine serum) and 40 ng/ml of IL-3 [8]. BMCMC were washed and suspended in media without IL-3, supplemented with 10% FBS with or without various concentrations of recombinant murine SCF (R & D, USA). Cells were incubated on 96 well plates (4 ¥ 10 4 cells per well) for 18 h at 37°C in a CO 2 incubator. Ten mCi of [ 3 H]-Thymidine (New England Nuclear, USA) was then added to each well and the plate incubated for a further 4 h. Results and discussionHistological analysis of the lung and skin of mice expressing (mi) mutation, in the homozygotic state, showed lower numbers of mast cells in these organs when compared to the control (+/+) ( Table 1). The (mi/mi) mice had 35% and 52% of the control mast cell number in skin and in lungs, respectively. There was a decrease in the histamine content of mutant lungs, similar in magnitude to the decrease in lung mast cell number. In contrast, the histamine content of the skin from (mi/+) and (mi/mi) mice did not differ significantly from controls. Analysis of cells in peritoneal lavage fluid from (mi/mi) mice indicated the absence of toluidine blue positive mast cells and a 46 fold decrease in the total histamine content when compared to control (data not shown). We employed a thymidine incorporation assay for in vitro testing of cultured (mi/mi) genotype mast ce...