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A pivotal series o f experiments were designed to ascertain whether the FceRl-mediated cell

signalling in human and mast cells was functionally comparable. The early experiments

involved the transfection o f human a units into rat cells with the aim of determining whether the human a subunit could effectively substitute for the rat a subunit. If this were possible, it would suggest that the human a unit was functionally identical to the rat a unit, and produce further evidence that might indicate that other parts of the FceRl mediated signal transduction were similar. Furthermore, it would support the argument that a study of a rat mast cell line would provide information that is comparable to the human mast cell.

2.11.7.1 Surface expression of human and rat FcsR l sub-units

Transfection of any of the individual units of rat FceRl (a, (3 or y) into COS cells was successfully achieved, as confirmed by Northern blotting. However, none was expressed on the

cell surface when transfected individually. Surface expression was only observed after

transfection of all four FceRl units, to create the aPyy tetramer (Blank et a l, 1989). By

comparison, after transfection of the human FceRl receptor units into rat mastocytoma cells, successful surface expression o f the a unit was dependent only on the presence of the y unit,

and the p unit was superfluous (Miller et a l, 1989). Transfection of the a unit together with the P unit did not produce surface expression of the a unit.

It would appear, therefore, that there is a fundamental difference in the requirements of surface expression o f the human FceRl receptor, versus the rat FceRl receptor. Whether this was reflected in differences between the human and rat mast cell signalling remained to be determined.

2.11.7.2 Transfection of FceRl a units

Despite differences in the surface expression o f the FceRl receptor, it was considered

important to elucidate whether the human a unit could substitute for the rat a unit, and visa

versa. As indicated by the previous experiments, surface expression of a alone was difficult to achieve. Efficient transfection of the a unit alone required the creation of a chimeric a unit. This was effected by substituting the transmembrane and cytoplasmic domains of the a subunit with those coding for the p55 IL-2 receptor, which had been shown previously to be expressed efficiently on cell surfaces. Transfection with the chimeric a receptor resulted in efficient cell

surface expression of a on COS cells (Hakimi et a l, 1990).

These results suggested that unknown elements in the transmembrane or cytoplasmic sequences of the a subunit prevented surface expression, a prevention that could be ablated by the presence of the y subunit. The a-transfected COS cells showed the capacity to bind both human and rat IgE, through the co-existence of both rat and human FceRl a units, although

there was variation in the affinity of binding between different isotypes o f IgE. It was

concluded that the surface binding of IgE is entirely dependent on the existence of the a subunit of the high affinity receptor.

Experiments were devised, which involved the transfection of the PS 15 rat mastocytoma cell line with a plasmid containing the cDNA sequence of the human a unit of FceRl, in combination with either, or both, of the sequences for the rat p and y units. Cells transfected with either a(human)pY(rat) or a(human) Y(rat) expressed as many as 1 x 10^ to 1.5 x 10^ receptors on their cell surface. These experiments confirmed that the human a unit could be effectively transfected into a rat mast cell line and were useful in understanding the specificity of binding. They did provide, however, no information as to the subsequent biochemical events. Neither the COS, nor the PS 15 cell line degranulates in response to FcsRl cross-linking. Downstream

events in the signalling from the human/rat receptors were, therefore, not studied (Miller et a l,

1989).

2.11.7.3 Signalling differences between human and rat mast cells

To fully elucidate the nature of the signalling events, it was necessary to study degranulation in a transfected mast cell line. Transfection experiments were performed to introduce the human a subunit o f the FceRl receptor into RBL-2H3 cells. Following successful transfection, it was observed the a subunit formed complexes with the endogenous (3 and y sub-units of the rat cells. Furthermore, the existence of the human a receptor unit provided the RBL cells with ability to respond to human stimuli.

IgE-mediated aggregation of human FceRla subunits resulted in a series o f biochemical events

that were indistinguishable from the events mediated by the rat FceRla (Gilfillan et a l, 1992).

These events included a rapid increase in the formation of inositol phosphates, an increase in intracellular calcium, selective phosphorylation of tyrosine residues, all of which ultimately

contribute to the cells’ degranulation. '

The existence of cell lines, combining characteristics of both human and rat receptors, allowed experimentation without the laborious task of extracting and purifying human cells. For example, these cell lines have been used to determine the efficacy of binding of anti-human

monoclonal antibodies to human FceRl a subunits, without the need for human cells (Lowe et

a l, 1995).

2.12 Conclusions

The results described in Chapter 2 provide evidence that the RBL-2H3 cells employed in my research were stable over the course of the experimentation. The cells routinely released measurable levels of mediators upon passive sensitisation with anti-DNP IgE, and cross-linking

with DNP-albumin. The standard experimental methodology for the IgE-mediated

degranulation of RBL-2H3 cells involved overnight incubation of 200,000 RBL-2H3 cells in a 24 well plate, to allow the cells to adhere prior to experimentation. Subsequently, the cells were exposed to 3 pg/ml of IgE, prepared in supplemented EMEM media, and incubated for a minimum o f 2 hours. The cells were washed thoroughly, exposed to DNP-albumin, at a concentration of 100 to 1000 ng/ml and then incubated for 2 to 3 hours. The release of mediators was quantitatively measured by the detection a mast cell mediator, (3- glucosaminidase. The (3-glucosaminidase assay was routinely terminated after a 30 minute incubation, after which time the OD o f the P-glucosaminide product was measured at 405 nm wavelength.

The additional observation that rat receptor units could functionally substitute for human receptor units supports the theory that RBL-2H3 cells are sufficiently comparable with normal human mast cells. RBL-2H3 cells were chosen, therefore, as the ideal method of studying Type

One Hypersensitivity reactions in an in vitro situation.

Chapter 3