Agua Potable

Terminology for the different mast cell sensitisation methods is not universally concordant. For the purposes o f this chapter the term ‘active sensitisation’ applies to protocols that employ live animals to sensitise mast cells to specific antigens. ‘Passive sensitisation’ refers to those protocols that sensitise mast cells in vitro (i.e. without the use o f live animals).

Two o f the most commonly used protocols for active sensitisation are intra- peritoneal injections o f Bordetellapertussis (Okada et a l 1996b), and infection with the nematode Nippostrongylus brasiliensis (Peachell & Pearce, 1989a). Both produce mast cells with a good response to antigen but their limiting factors are, a) use o f expensive live animals and b) the time factor - it takes up five days for the former method and a life cycle o f six weeks for the latter. Other protocols utilise the intra- peritoneal injections o f strongly allergenic compounds such as ovalbumin

(W yczolkowska et a l 1986). But again these suffer from the drawbacks already listed. Passive sensitisation o f the immortalised mast cell line RBL-2H3 with IgE directed against dinitrophenol (anti-DNP IgE) is a relatively simple and reproducible procedure (Collins et a l 1995). Whereas that with purified rat peritoneal mast cells is notoriously difficult and unreliable (Inagaki et a l 1995). Furthermore, antigen- induced secretion from passively sensitised RPMC is generally poor (Inagaki et a l

1995). This is in contrast with reports that mouse, guinea pig and Sabre RPMC [(Desquand et a l 1989) and (R. Sagi-Eisenberg, personal communication)] can be passively sensitised with ease. The work described in previous chapters utilises RPMC purified from Sprague Dawley as the model system. In order for this work to be comparable we needed to develop an intact RPMC model that could respond to antigen. The aim is to develop a passive sensitisation model with a good, reproducible secretory response. This section describes the characterisation o f two models o f passive sensitisation.

Figure. 5.1 Secretion from passiveiy sensitised RPMC:

RPMC w ere passively sen sitised with either anti-DNP IgE (a,c,e) or IR 162 (b,d,f) a s described in m ethods (2.4c).

(a) and (b) d o s e re sp o n se for sensitising IgE: cells w ere incubated with the indicated concentrations of IgE for 28 hours a t 37 °C and then challenged for a further 20 min with 50 pg/ml of BSA-DNP or anti-IgE (1:10 dilution), respectively.

(c) and (d) d o se re sp o n se for trigger: cells w ere sensitised a s described ab o v e with the optimum concentrations of anti-DNP IgE (5 pg/ml) and IR 162 (10 pg/ml), respectively, and challenged with the indicated concentrations of BSA-DNP and anti-IgE for 20 min at 37 °C. (e) time co u rse of sensitisation: cells w ere incubated with the optimum concentrations of anti- DNP IgE ( • ) or IR 162 ( O). At th e indicated tim es of such sensitisation, cells w ere

challenged with BSA-DNP ( 50 mg/ml) and anti-IgE (1:100 dilution), respectively.

(f) pH d ep e n d en ce : cells w ere sensitised for 28 hours at 37 °C by optimum concentrations of anti-DNP IgE ( • ) or IR 162 ( O ) a t the indicated pH, and then challenged with BSA-DNP ( 50 mg/ml) and anti-IgE (1:100 dilution), respectively.

S u p ern a ta n ts w ere taken for determ ination of re le ased hexosam inidase after 20 min incubation with th e appropriate trigger. Data shown a re m ean s ± S.E. (n = 3).

% secretion % secretion % secretion o I—

r

I

This experiment was designed to characterise and compare two models o f passively sensitised mast cells. The first model uses IgE raised against the chemical antigen DNP (anti-DNP IgE) to sensitise RPMC. Cross-linking the receptor is achieved by challenge with the antigen DNP-BSA. The second model is sensitised with myeloma IgE in ascitic fluid (designated IR 162). Here cross-linking is achieved by a specific anti rat IgE.

RPMC sensitised with anti-DNP IgE had a bell shaped response curve to antigen challenge (panel a). The optimum concentration o f anti-DNP IgE for secretion was ~5 pg.ml'^ . In contrast, RPMC sensitise with IR 162 secreted in a dose-

dependent manner with an EC5 0 = 0.4 ± 0.1 pg.ml’’ (panel b). The optimum

concentration for sensitisation with IR 162 was >5 pg.m l'\

RPMC sensitised with anti-DNP IgE and IR 162 had similar secretory profiles when challenged with BSA-DNP and anti-IgE, respectively. When challenged with BSA-DNP the apparent was EC5 0 = 0.5 ± 0.2 p g .m f’ (panel c). Challenge with anti-

IgE was optimum at dilution’s > 1/100 (EC5 0 ~ 1/800) (panel d).

Both models exhibited similar sensitisation times. Both reached maximal levels o f secretion after at least 22 hours incubation with IgE (panel e). No significant deterioration in response was seen up to 48 hours incubation (not shown). Optimum pH for both models was, again, similar (pH range 13-1.5) (panel f). RPMC sensitised with anti-DNP IgE were inhibited to a greater extent at high pH compared with IR

162. Further results using Trypan Blue exclusion method found that after 48 hr incubation RPMC viability was 81.3 ± 5.8% {n =3).

From this series o f experiments I found that sensitising RPMC with IR 162 and challenging with anti-rat IgE was a more robust and reproducible system. The IR 162 passive sensitisation model was chosen for the remainder o f the intact cell studies described in this chapter.