Drenaje y Alcantarillado

Although RPMC passively sensitised with IR 162 produce a good secretory response when challenged (-40-50% ) in occasional experiments (~ 1 in 6 ). the

response was poor ( < 25%). Furthermore secretion elicited by this method was certainly not as robust as that induced by polycationic compounds. It has been found that RPMC obtained from non-barrier housed rats, a significant number o f FceRI were occupied by non-specific endogenous IgE molecules as a result o f exposure to animal house pathogens (Chen & Enerback, 1995). It is likely then that this is also the case with RPMC obtained from Sprague-Dawaley rats. Although, polyclonal rat anti-IgE should be able to cross-link all surface IgE molecules regardless o f specificity, it is has been suggested that the endogenous FcERI-IgE complexes are unable to achieve correct conformational orientation to allow cross-linking (Stump et a l 1988). By

removing this endogenous IgE from RPMC and replacing it with IR 162 (myeloma IgE) we hoped to achieve more consistent and higher levels o f secretion. In order to achieve this I have utilised lactic acid treated RPMC

Lactic acid (LA) has been reported to reversibly dissociate IgE from human basophils without compromising cellular activity (Pruzansky et al. 1983). The mechanism by which it achieves this is not known, however, it is believed to involve the disruption o f bonds between IgE and FceRI (Vally et al. 1995b). I have used FITC-conjugated goat anti-IgE (Fc) and flow cytometry to evaluate the occupancy o f FceRI by IgE in both actively and passively sensitised cells, both before and after LA treatment. Receptor occupancy has been related to the secretory competence o f these cells.

a) b ) 120 % ₁₀₀ o ro Q. 3 80 8 o 5 60 LL 0) 40 ro E 20 0 c) 200 ü 150 o 100 50 at 28 hrs w/o addition at 28 hrs 100 _{+ IR 162} + LA + L A /IR 162 80 60 40 20 0 1 hr 2 8 hr 28 hr d) + anti-IgE + 48/80 100 80 60 40 20 0 1 hr 28 hr 28 hr + anti-IgE + 48/8 0

Figure 5.3 LA removes endogenous IgE but com prom ises secretion

Ig receptor occupancy (a,c) and secretory responsiveness (b,d) w ere examined in actively (a,b), or passively (c,d) sensitised RPMCs. W here indicated, cells w ere pre-treated with lactic acid before sensitisation (+LA). Cells w ere incubated for the indicated time (i.e. 28 hrs or 1 hr) at 37°C without any addition (w/o addition) or with with 60 pg.ml'^ IR 162 (+IR 162). Lactic acid treated cells (0.2 mM LA, 2 min at 30°C, pH 3.4, followed by a w ash and a 20 min recovery period) w ere either incubated without any addition (closed bars, +LA) or with 60 pg.mM IR 162 (+LA/IR 162).

(a, c) Cells w ere then incubated with FITC-conjugated goat anti-IgE (Fc) (1:80 dilution) for 60 min at 37°C, and Ig receptor occupancy w as evaluated by flow cytometry a s above. The fluorescence value obtained from control cells (w/o addition) w as taken a s 100%. Results shown are m eans ± S.E. {n = 3).

(b, d) Cells w ere then challenged with goat anti-rat IgE (1/100 dilution) or with compound 48/80 (10 pg.m M 'as indicated. S upernatants w ere taken for determination of released hexosam inidase. Basal levels of secretion w ere 5-10 %.

Experiment shown is representative of two different experiments.

Actively sensitised RPMC were obtained from rats infected with the parasite

Nippostrongolyodis. As expected, these cells have most o f their high affinity Fee

receptors occupied since addition o f exogenous IgE (IR 162) did not significantly enhance binding o f FITC- anti-IgE (Fig. 5.3a). Upon treatment with LA, this

endogenous IgE is dissociated as shown by the almost complete absence o f FITC- anti- IgE binding. After addition o f IR 162 to LA-treated cells, most o f the receptors are again occupied (Fig. 5.3 a).

Fig. 5.3 c shows results obtained with passively sensitised RPMC. In control (unsensitised) cells, about 40-50% o f FcsRI seem to be occupied by endogenous IgE. In this case, addition o f exogenous IgE (IR 162) resulted in approximately twofold increase in FITC- anti-IgE binding. Again, this endogenous IgE was almost totally removed by LA treatment. Addition o f IR 162 to LA treated cells resulted in FcsRI occupancy similar to that obtained with untreated cells.

When challenged with anti-rat -IgE, actively sensitised RPMCs respond without any requirements for sensitisation, secreting -7 0 % o f their total

hexosaminidase content (Fig. 5.3 b, w/o addition). Incubation o f actively sensitised cells with IR 162 (1 or 28 hrs) did not improve their secretory responsiveness. Treatment with LA abolishes secretion. This effect is not reversed by incubation o f LA-treated cells with IR 162 (for 1 or 28 hrs) although the receptors are now occupied to the same extent as those in untreated cells (Fig. 5.3a). In contrast, all cells were able to respond to compound 48/80, indicating that LA treatment does not interfere with the secretory mechanism but with the receptor.

Unsensitised RPMCs do not secrete when challenged with anti-IgE 1 hr after their purification (Fig. 5.3 d). Small response to anti IgE ( - 20%) was apparent after 28 hrs incubation in the supplemented DMEM, in agreement with previous

observations that autocrine (and possibly paracrine) signalling/conditioning is involved (Coleman et al. 1993). Passive sensitisation for 28 hrs improved secretory competence 2-3 times; 1 hr sensitisation was ineffective. Again, cells treated with LA were unresponsive even after sensitisation. All cells were ftilly competent to respond to compound 48/80.

In conclusion, results presented in 5.2 and 5.3 show that occupancy o f FcsRI does not determine secretory competence: but both the type o f IgE and the state o f the receptor are important factors.