1 The role of adenosine A 3 receptors on human eosinophil degranulation and superoxide anion (O 2 7 ) release was studied in vitro using the complement fragment C5a as the main stimulus and employing a number of selective agonists and antagonists. 2 In the presence of cytochalasin B (CB), C5a induced a dose-dependent release of the granular eosinophil peroxidase (EPO), but not O 2 7, whereas in the absence of CB O 2 7 , but not EPO, was released. 3 C5a-induced EPO release was inhibited dose-dependently by the selective A 3 agonist N 6 -(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N 6 -2-(4-amino-3-iodophenyl) ethyladenosine (APNEA). The IC 50 (95% CI) for IB-MECA was 6.8 mM (3.1 ± 12.0 mM). At concentrations up to 100 mM, neither adenosine nor the selective 5 IB-MECA also signi®cantly inhibited C5a-induced O 2 7 release with IC 50 (95% CI) of 9.5 mM (4.6 ± 13.1 mM) whereas adenosine and the A 1 agonist CPA potentiated this eect at low concentrations. The potentiation appeared to be a result of their direct O 2 7 release from these cells, probably mediated via A 1 receptors. The inhibition by IB-MECA was selectively reversed by MRS 1220. 6 These results show that the A 3 receptors on human eosinophils mediate inhibition of both degranulation and O 2 7 release and suggest a therapeutic potential for A 3 agonists in diseases such as asthma in which activated eosinophils are involved.

Many mediators activate eosinophils via transduction pathways involving the enzyme phosphatidylinositol 3-kinase. The initial investigation of wortmannin, a specific inhibitor of PI3-kinase, was of its effect on human and guinea pig eosinophil superoxide (O(2)(-)) release and degranulation in vitro. Subsequently, the effect on allergen- and Sephadex-induced bronchial inflammation and airway hyperresponsiveness (AHR) in vivo in guinea pigs was investigated. Wortmannin potently inhibited complement C5a-induced O(2)(-) generation and eosinophil peroxidase (EPO) release from human eosinophils, with 50% inhibition produced by a 1-10 nM concentration. Both aerosol allergen challenge of sensitized guinea pigs and intravenous injection of Sephadex beads in normal guinea pigs caused, in 24 h, significant eosinophilia and increased EPO activity in bronchoalveolar lavage fluid (BALF) and AHR to intravenous acetylcholine and histamine. In the allergic model, intranasal pretreatment with wortmannin had no effect on BALF eosinophilia, but dose dependently inhibited BALF EPO activity. At 1 mg/kg, the drug abolished the AHR to histamine, but not acetylcholine. In the Sephadex model, the drug significantly inhibited all three parameters (eosinophilia, increased EPO activity, and AHR to both spasmogens). These results show that wortmannin is a potent inhibitor of human eosinophil degranulation and that when administered intranasally can prevent AHR in allergen-challenged guinea pigs, probably by inhibiting eosinophil degranulation, but not their accumulation in BALF. This may be relevant to the possible clinical utility of wortmannin in conditions involving eosinophilic inflammation and AHR.

1 The e ect of histamine on human eosinophil degranulation and the receptor mediating such e ect were studied in vitro using the complement C5a-mediated eosinophil peroxidase (EPO) release model. 2 Following pre-treatment with 5 mg ml 71 cytochalasin B(CB), C5a induced a concentrationdependent release of EPO from eosinophils isolated from healthy donors. 3 Histamine (0.1 ± 50 mM), but not L-histidine, inhibited concentration-dependently C5a-induced EPO release with IC 50 (95% CI) of 0.6 mM (0.3 ± 1.2 mM) and maximal inhibition of &60%. 4 A similar e ect was seen with the selective H 2 agonists dimaprit (IC 50 (95% CI)=6.9 mM (3.2 ± 10.6 mM)) and amthamine (IC 50 (95% CI)=0.4 mM (0.2 ± 0.7 mM)). Neither the selective H 1 agonist 6-(2-(4-imidazolyl)ethylamino)-N-(4-tri¯uoromethylphenyl) heptanecarboxamide(HTMT), nor the selective H 3 agonists imetit (up to 100 mM) had any signi®cant e ect. 5 The inhibition by histamine was reversed by cimetidine (0.1 ± 30 mM) and other H 2 antagonists, but not the H 1 antagonist mepyramine (1.0 ± 100 mM), nor the H 3 antagonist thioperamide (1.0 ± 100 mM). Cimetidine (1 ± 30 mM) shifted to the right the dimaprit log dose-response curve, producing a pA 2 value of 5.9 and Schild's plot slope of 0.98, thus con®rming simple competitive antagonism. 6 Histamine (10 ± 100 mM) increased intracellular level of adenosine 3',5'-cyclic monophosphate, which was completely abolished by cimetidine (30 mM), but not mepyramine or thioperamide. The cyclic AMP analogue ± dibutyryl cyclic AMP ± also inhibited degranulation (IC 50 *300 mM). The cyclic AMP phosphodiesterase(PDE) IV inhibitor rolipram (10 mM) synergistically enhanced the inhibition of EPO release by histamine. 7 These results suggest that histamine, via stimulation of H 2 receptors and a consequent elevation of intracellular levels of cyclic AMP, inhibits human eosinophil degranulation.

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Background: Drug-induced severe cutaneous adverse reactions (SCARs) are presumed T-cell-mediated hypersensitivities associated with significant morbidity and mortality. Traditional in vivo testing methods, such as patch or intradermal testing, are limited by a lack of standardization and poor sensitivity. Modern approaches to testing include measurement of IFN-g release from patient PBMCs stimulated with the suspected causative drug. Objective: We sought to improve ex vivo diagnostics for druginduced SCARs by comparing enzyme-linked immunospot (ELISpot) sensitivities and flow cytometry-based intracellular cytokine staining and determination of the cellular composition of separate samples (PBMCs or blister fluid cells [BFCs]) from the same donor.Methods: ELISpot and flow cytometry analyses of IFN-g release were performed on donor-matched PBMC and BFC samples from 4 patients with SCARs with distinct drug hypersensitivity. Results: Immune responses to suspected drugs were detected in both the PBMC and BFC samples of 2 donors (donor patient 1 in response to ceftriaxone and case patient 4 in response to oxypurinol), with BFCs eliciting stronger responses. For the other 2 donors, only BFC samples showed a response to meloxicam (case patient 2) or sulfamethoxazole and its 4-nitro metabolite (case patient 3). Consistently, flow cytometry revealed a greater proportion of IFN-g-secreting cells in the BFCs than in the PBMCs. The BFCs from case patient 3 were also enriched for memory, activation, and/or tissue recruitment markers over the PBMCs. Conclusion: Analysis of BFC samples for drug hypersensitivity diagnostics offers a higher sensitivity for detecting positive responses than does analysis of PBMC samples. This is consistent with recruitment (and enrichment) of cytokinesecreting cells with a memory/activated phenotype into blisters.