Variants of the Mucosal Mast Cell Line (RBL-2H3) Deficient in a Functional Membrane Glycoprotein

Abstract

We have isolated and characterized subpopulations of the rat mucosal mast cell line, RBL-2H3, carrying either high or low density of a glycoprotein, recently established as mast cell function-associated antigen (MAFA, ORTEGA et al., 1991), on their surface. These populations were investigated in order to better define the involvement of the MAFA in coupling the immunological stimulation of mast cells to mediator release. The MAF A density on the cell surface of the deficient subpopulation was ≤ 10–20 % that of the parental population and this phenotype was found to be stably maintained for several months. In contrast, the MAPAenriched cells had maximally twice the number of copies per cell surface than that of the parental population and this phenotype was less stable. Significantly, low copy number of MAFA on the cell's surface was accompanied by a markedly different secretory response, i.e. (i) a considerable decrease in the secretory response to the FcεRI -mediated stimulus (ii) a marked enhancement of the ionomycin induced secretion. In order to gain insight into the causes for this decrease in cellular response to the FcERImediated stimulus, we measured the amplitudes of several biochemical processes which are assigned to the stimulus-secretion coupling cascade. The FcεRI -mediated uptake of 45Ca 2+ by the MAFA-deficient cells was considerably lower than that of the parental and MAPA-enriched cells. Similarly, these cell's FcεRI-induced rise in [Ca 2+] i (both the initial transient as well as the sustained elevation), was markedly lower than that of the parental line and the MAPA-enriched cells. Moreover, the low initial transient rise in [Ca 2+] i was found to be correlated with the decrease in FcεRI-mediated IP 3 levels. We therefore examined the cell's content of the phosphatidyl-inositides hydrolyzing enzyme, phospholipase Cγ1. This was found to be similar in the parental line and in its derived subpopulations. However, PLCγ1 activation, as measured by the time course of phosphorylation of its tyrosines, showed a marked difference: while PLCγ1 tyrosine phosphorylation, in the parental cells, was only transient (detected already 1 min after antigen addition and declined afterwards to basal levels at ca. 10 min), in the MAPA-deficient cells, tyrosine phosphorylated PLCγ1 was also observed 1 min after antigen addition, yet showed no decrease with time in its phosphorylation intensity for up to 30 min. Thus, in analogy to the current mechanisms proposed for the coupling between other antigen-receptors, such as that of T cell or surface Ig on B cells, with their associated membrane components and cytosolic protein tyrosine kinases (PTKs) or phosphatases (PTPs ), it is suggested that the MAFA may serve as a regulatory element of the coupling between the FcεRI and the PTKs and/or PTPs which in turn carry on the cascade to the cell's secretory response.