Development of cell lines as analogues of tissue cells

The research performed for this thesis endeavoured to elucidate the existence and role of GPl- PLD in mast cell physiology. Clearly, the aforementioned heterogeneity of mast cells presents a challenge to the interpretation of the data. The possibility that an artificial cell line might provide less heterogeneity than the human tissue mast cells, was evaluated. A number of different mast cell lines exist, including human mast cell lines, mouse mastocytomas and rat basophil leukaemia cell lines. For example, the human mast cell line, HMC-1, was derived

from a patient with mast cell leukemia (Butterfield et a l, 1990). Solid tumors of these cells

were serially passed in mice using 5-azacytidine transformed cells.

This cell line was shown to display a phenotype similar to the MCt (Section 2.2.4.3.2), as

judged by the expression of surface antigens, and the cytochemical analysis (Nilsson et a l,

1994). Both heparin and chondroitin sulfate were found to be present in approximately equal amounts. The HMC-1 cell line, however, lacked surface expression of the high-affinity IgE receptor. Analysis of the mRNA from these cells showed an absence of both alpha- and beta- chain mRNA, although the gamma-chain of the IgE-receptor complex was strongly detected. Despite the lack of high affinity receptor, HMC-1 has been successfully employed in a number

of different experimental studies, including receptor-expression (Sillaber et a l, 1997) and

cytokine metabolism studies (Macchia et al, 1995).

The choice of the RBL-2H3 cell line, in preference to a human mast cell line, was based on the pilot experiments that were performed in collaboration with Dr Alasdair Gilfillan, Hoffman-la- Roche. These experiments, which are discussed in greater detail in Chapter 3, involved the analysis o f the role of GPI-PLD in mast cell function. The experiments were performed using RBL-2H3 cells, and demonstrated that GPI-PLD might be involved in the Type One

hypersensitivity reaction (Lin et a l, 1995). It seemed of importance, therefore, to pursue the

role of GPI-PLD in mast cell function using the RBL-2H3 cell line. The development and the characteristics of the RBL-2H3 cell line are discussed in greater detail in Section 2.11.

2.5 Experimental aims

The experimental aim of the research performed for this thesis was to determine the existence, and role of GPI-PLD in the function of mast cells. Most importantly, to determine whether GPI-PLD was a participant in the signalling machinery activated upon IgE-mediated stimulation of mast cells.

The reasons for the selection of the RBL-2H3 cell line have already been discussed. Prior to any research into GPI-PLD, however, it was essential to establish a consistent culture of the RBL-2H3 cells, and to verify the cells’ characteristics, particularly in relation to their IgE mediated degranulation. This is a complex process, which involves the specific interaction of a number of components. It was essential to establish a consistent methodology for the IgE- dependent stimulation of the RBL-2H3 cells and to accurately measure the quantities of mediators released as a result of the cells’ degranulation.

2.6 Culture of RBL-2H3 cells

The culture of RBL-2H3 cells was dependent on supplemented culture medium and growth in a

humidified, carbon dioxide-enriched incubator. In addition, the RBL-2H3 cells required

specific culture flasks and plates to permit attachment o f the cells. Culture o f the RBL-2H3 cells was significantly impeded if the cells were maintained as a suspension, rather than as a confluent monolayer.

2.7 Non-IgE-mediated Degranulation of RBL-2H3 cells

2.7.1 Lysis, using Triton X-100

The selection of Triton X-100 as a positive control was used as a measure o f the complete mediator content of the RBL-2H3 cells. The mechanism by which Triton X-100 releases mediators from cells is discussed in greater detail in Section 2.11.4.1.

2.7.2 In vitro degranulation, using Calcium lonophore A23187

The primary experimental aim of the research described in this Chapter was to establish a consistent method for the IgE-dependent degranulation of RBL-2H3 cells. It was also of importance, however, to establish an IgE-independent method for cell degranulation.

Experiments performed in later Chapters required the manipulation of the cells’ culture environment, and determination of whether the IgE mediated degranulation of the cells was affected by this manipulation. The inclusion of a positive control in these experiments, in the form of an IgE-independent method of cell stimulation was also essential. If a reduction in the quantity of mediators released by IgE-dependent route was accompanied by a reduction in the IgE-independent route, it could be concluded that the manipulation of the cells’ environment was non-specific. That is, the manipulation affected the complete functioning of the RBL-2H3 cells, rather than the IgE system specifically.

The calcium ionophore, A23187, was selected as an IgE independent positive control, to permit the selective release of mediators from the mast cells by an IgE-independent route. A23187

was isolated from Streptomyces chartreusensis and belongs to the family o f monocarboxylic

antibiotics (Chaney et a i, 1974). It was shown to release mediators from partially purified rat

mast cells (Caswell et a l, 1972). The mechanism by which A23187 releases mediators from

cells is given in greater detail in the Section 2.11.4.2.4.