A novel rapid (20-minute) IL-6 release assay using blood mononuclear cells of patients with various clinical forms of drug induced skin injuries

Abstract

Background: IL-6 is a pro-inflammatory cytokine which has many well-defined effects. Its synthesis and release from mononuclear cells of drug-sensitized patients was related before to in vitro drug-allergy diagnostics but has not yet been studied in detail.

Methods: The specific release of preformed IL-6 from peripheral blood mononuclear cells (PBMC) after 20 minutes incubation with 0.15-0.5 μM of pure drugs was measured in two groups of drug-allergy suspected donors (159) and respective controls (48). IL-6, TNF-alpha, IL-2, IL-4, IFN-gamma have been measured from cell supernatants by ELISA or by cytometric bead assay. Epicutaneous, intradermal and systemic provocation tests were performed to prove or disprove culprit substances (203 in vivo against 482 in vitro tests). T-test (paired and unpaired); chi2 contingency table; Z statistics and McNemar's test were used to evaluate results.

Results: Concanavalin A as positive control released IL-6 from PBMC in linear concentration and exponential time dependent fashion (up to 60 minutes) pointing to the existence of a preformed pool of this cytokine. Preformed IL-6 released at any of 4 standard drug dilutions tested, above 50% over their diluents' levels significantly correlated with the patients' history on drug-induced hypersensitivity symptoms and with in vivo tests. Sensitivity of 85.4% and specificity of 82.4% of the IL-6 release assay were found. The 20' drop in release of TNF-alpha had no diagnostic importance; it has accompanied increased IL-6 release. IL-2, IL-4 and IFN-gamma were undetectable in 20 minutes supernatants. IL-6 release depended on the clinical phenotype but not on the eliciting drug(s) in the molecular mass range of 76-4000 Da. Reactivity of mononuclear cells at the lowest or at multiple drug test concentrations reflected clinical severity per diagnoses and according to area of skin involvement.

Conclusion: This rapid test is applicable to detect a wide scale of drug hypersensitivity.

Keywords: Adverse drug reactions; Drug-induced skin injury; IL-6; Preformed cytokines’ release; T-lymphocytes; TNF-alpha.

Figure 1

Effect of ConA on the release of IL-6 from mononuclear cells of patients after 20-min incubation with " Test A " solution . The columns represent mean ± SD. [Brackets under abscissa indicate numbers of tests at different concentrations]. The value of 2197 ± 268 pg × 10^-6 cells^-1 was obtained at 300 μg/ml ConA. Insert: fitted time course of the mean values from 2 independent experiments using 5 μg/ml ConA on 2 non-allergic persons’ cells (red: ConA, blue: PBS).

Figure 2

Time course of drug-induced IL-6 release from PBMCs of an allergic patient (clindamycin, phenotype ANO) 6 months after the event. The drug was taken orally as monotherapy. Localized edematous rash has developed around the wrist and on dorsa of hands 30 min. after repeated intake. Results of 2 independent experiments with 6-week interval yielded 196 and 198 pg × 10^-6 cells at 0.35 μM (the points coincide).

Figure 3

IL-6 release from PBMCs upon different drug concentrations of various drugs in positively and negatively reacting groups of the cohort incubated with " Test A " medium. Positivity: >50% increase in IL-6 release at any drug concentration relative diluent control. Stars indicate statistically significant differences) between negative control samples and positively tested cases, (p < 0.05) and between negative and positive cases (p < 0.005).

Figure 4

Distribution of drugs among different pharmacological classes. a: eliciting positive IL-6 release test results (n = 43). b: tested within the control group (n=40). The numbers of individual drugs tested are higher (~70). Glibenclamide peripheral vasodilators and negative tests with acetylcystein are listed among "varia".

Figure 5

Mean cytokine releases stimulated by PHA-P (168 μg/ml) after 20-min incubation with " Test B solution " compared to controls. Non-allergic test series (n = 6) appear for both cytokines, TNFα and IL-6 in blue, allergic test series (n = 4) in light brown color.

Figure 6

TNFα and IL-6 release from PBMCs incubated with " Test B solutions " elicited by different drug concentrations measured by the CBA Th1-Th2 cytokine kit in a total of 6 negative and 4 positive assays. a: cytokine concentrations (mean +- S.E.M.); b: relative cytokine release normalized individually by their corresponding control values.