Characterization Of Chemokine Responses In Purified Human Basophils

We have studied how allergens interact in their effect on PBMC's from Asthmatics with mite sensitisation (MAA, n=8), Asthmatics with sensitisation to both cat and mite (MCAA, n=8), compared to control subjects (Nonatopic, NN, n=8).Cells were cultured at 1 x 105 (proliferation) and 5 × 106 (cytokine) cells/well in Aim V serum-free medium and stimulated with PHA (ll.tg/ml), dust mite (30~tg/ml) and Cat extracts (30,000SQU/ml), dust mite and Cat combined or negative control (Aim V).Proliferation was assessed by 3H-thymidine uptake on day 6 (ll.tCi well).IL-4, IL-5, IFN-~' and TGF-I] mRNA expression was quantified by Taqman Real-Time RT-PCR (3 days PHA, 6 days for other stimulants).RNA was extracted by Trizol LSTM/acid-phenol chloroform and mRNA expressed in orders of magnitude less or greater than the negative control.AA PBMC proliferated more than NN after stimulation with mite extract only: [MAA: median: 34.1 (range: 1.61-81.97];[MCAA: 39.96 (18.23-176.42)];[NN: 16.37 (2.97-27.68)];[Mann-Whitney, p<0.05 for both].HDM+Cat costimulation produced a greater effect in PBMC proliferation across all 3 clinical groups, compared to the sum of their separate effects: [HDM+Cat co-stimulation: 71.53 (6.52-206.34),Sum (HDM and Cat): 43.49 (4.86-191.09),p=0.009]; in the NN group alone: [HDM+Cat co-stimulation: 61.5 (6.52-206.34),Sum (HDM and Cat): 31.96(8.64-44.36),p=0.01].IL-4 mRNA expression in PBMC was significantly higher post HDM+Cat costimulation for MAA [4.28 (0.77-57.4)] and MCAA [1.58 (0.51-5.74)]than NN [0.57(0.12-4.65)] (p=0.029 and 0.038 respectively); and post cat stimulation for MAA than 2); 0.16 (0.05-1.01); p=0.021].IL-5 mRNA expression was significantly higher post HDM+Cat costimulation for ] and than )] (p=0.001 and 0.007 respectively); post cat stimulation for MAA [3.04 (1.17-57.6)]and ] than NN [0.62 (0.01-66.1)] (p=0.029 and 0.010); post HDM stimulation for MCAA than ; 17 (0.02-48.8); p=0.007]; and post PHA stimulation for MCAA than ; p=0.038].IFN-7 mRNA was incrementally decreased by costimulation in the MAA group: [HDM+Cat co-stimulation: 8.07 (2.2-15.5),Sum (HDM and Cat): 42.13 (7.56-102.36),p=0.015] and TGF-13 mRNA expression in the MCAA group: [HDM+Cat co-stimulation: 1.76 (0.84-3.34),Sum (HDM and Cat): 3.49 (1.45-8.34),p=0.021].In conclusion: (a) Both proliferative responses and cytokine mRNA expression are significantly different in AA versus NN.(b) Co-stimulation by allergens (HDM+Cat) appears to have a positive synergistic effect on PBMC proliferation in both AA and NN, but down-regulates IFN-'/and TGF-I] mRNA expression only in AA.