Asthma: basic mechanisma
S77 ELISPOT EVALUATION OF PBMC RESPONSES TO STAPHYLOCOCCUS AUREUS
J. Perera, B. Green, M. Arden-Jones, P. Howarth. University of Southampton, Southampton, UK
Introduction and Objectives In treatment-resistant severe asthma there is evidence of T cell dysregulation. The basis for this is poorly understood, but bacterial superantigens may contribute, as stimulation of T cells by these molecules is poorly regulated by glucocorticosteroids and superantigens are appreciated to contribute to disease severity in other inflammatory disorders, such as atopic dermatitis and nasal polyposis. To investigate this, peripheral blood mononuclear cells (PBMCs) have been stimulated with Staphylococcus aureus enterotoxin B (SEB) along with phytohaemagglutinin (PHA), as a positive control, with ELISPOT evaluation of interferon γ (IFNγ) (T helper 1 (Th1)) and interleukin-4 (IL-4) (Th2), to assess whether there is an altered response to these T cell stimulants in severe asthma.
Methods Overnight ex vivo ELISPOTs were carried out on PBMCs stimulated by SEB (0.004–2.5 μg/ml) and PHA (1.25–10 μg/ml) from 20 subjects with severe asthma, 10 with mild asthma and 10 healthy controls. The number of IL-4- and IFNγ-producing cells was assessed and quantified as spot-forming units (SFU).
Results SEB and PHA both induced a dose-dependent increase in SFU for IL-4 and IFNγ in all subjects, with greater numbers of SFU for IFNγ than for IL-4. There was no difference in response between those with mild asthma and healthy controls, but there was evidence of increased sensitivity to stimulation in those with severe asthma, as compared with both those with mild asthma and healthy controls. This difference was most noticeable for SEB stimulation, with significantly increased numbers of IFNγ SFU in severe asthma compared with mild asthma after stimulation with SEB at 0.004 μg/ml (p<0.002), 0.02 μg/ml (p<0.0001) and 0.01 μg/ml (p<0.004), and for IL-4 SFU at 0.004 μg/ml (p<0.03) and 0.02 μg/ml (p<0.01).
Conclusions These findings indicate that these treatment-resistant subjects with severe asthma managed at step 4/5 of the BTS guidelines have increased peripheral Th1 and Th2 responses to SEB as compared with those with mild asthma and healthy controls, suggestive of an altered T cell receptor (TCR) variable beta chain (V Beta)-restricted T cell stimulatory profile. This is evident despite their current asthma medication and is also consistent with severe asthma having a significant and different systemic component from that in mild disease.
S78 INTERLEUKIN-5 INDUCES GLUCOCORTICOID RESISTANCE IN HUMAN EOSINOPHILS
S. Brode, N. Farahi, A. S. Cowburn, J. K. Juss, A. M. Condliffe, E. R. Chilvers. Respiratory Medicine Division, Department of Medicine, University of Cambridge, Cambridge, UK
Background Glucocorticoids (GCs) represent one of the most effective clinical treatments for eosinophil-mediated inflammatory diseases such as asthma and allergic rhinitis. Part of the anti-inflammatory effect of GCs has been attributed to their ability to promote eosinophil apoptosis. Cytokines such as interleukin-5 (IL-5), IL-3, eotaxin-1 and granulocyte–macrophage colony-stimulating factor (GM-CSF) increase the survival of eosinophils by inhibiting their apoptotic cell death, and expression of these cytokines is widely reported in eosinophilic inflammation.
Aim To compare the ability of a novel “high-affinity” GC, fluticasone furoate (FF) with fluticasone propionate (FP) and dexamethasone (DEX), to modulate eosinophil apoptosis and their potential to over-ride prosurvival signals from IL-5.
Methods Human eosinophils (>99% pure) and neutrophils (>97% pure) were isolated from healthy volunteers using gradient sedimentation and negative immunobead selection (eosinophils). Eosinophils were pretreated with recombinant IL-5 (0.01–1 ng/ml) for 1 h and cultured with GCs (1×10−12–1×10−6 M) for 24 h. Apoptosis was assessed by morphology and flow cytometry (annexin V, propidium iodide).
Results Co-incubation of eosinophils with DEX, FP or FF results in a concentration-dependent increase in apoptosis, with similar efficacy but markedly different potency: DEX EC50 = 2.3×10−8 M (95% CI 8.2×10−9 to 6.7×10−8 M), FP EC50 = 4.3×10−10 M (95% CI 9.6×10−11 to 1.9×10−9 M), FF EC50 = 1.4×10−10 M (95% CI 8.2×10−11 to 2.3×10−10 M). The proapoptotic effect of GCs on eosinophils was inhibited by the GC receptor antagonist RU38486 (mifepristone). Consistent with previously published data, neutrophil apoptosis was inhibited by all three GCs: DEX IC50 = 1.3×10−9 M (95% CI 1.1×10−10 to 1.6×10−8 M), FP IC50 = 4.5×10−9 M (95% CI = 7.9×10−10 to 2.6×10−8 M), FF IC50 = 1.4×10−10 M (95% CI 5.7×10−11 to 3.8×10−10 M). As expected, preincubation with IL-5 inhibited basal eosinophil apoptosis in a concentration-dependent manner (IL-5 at 0.01 ng/ml = 25.9% (95% CI 13.1% to 40.0%, p<0.05), IL-5 at 0.1 ng/ml = 60.8% (95% CI 47.4% to 74.2%, p<0.001). In the presence of DEX, FP or FF, preincubation of eosinophils with IL-5 caused a complete concentration-dependent inhibition of the proapoptotic effects of GCs without a shift of the GC concentration curves. This is indicative of a non-competitive antagonism.
Conclusions Our data demonstrate that FP and in particular FF display greater potency in inducing eosinophil apoptosis compared with DEX. However, despite this enhanced potency, physiologically relevant concentrations of IL-5 could fully over-ride the proapoptotic effects of GCs. This may play an important role in acquired GC resistance in inflammatory disease and have a significant impact on clinical management.