Get access

Evaluation of Cytokine Production from Peripheral Blood Mononuclear Cells of Type 1 Diabetic Patients

Importance of the Methodologic Approach


Address for correspondence: Prof. Milton C. Foss, Ribeirão Preto Medical School, São Paulo University, Internal Medicine, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, BR 14049-900. Voice: +55 16 36022467; fax +55 16 3636695.


This study aims to evaluate the production of cytokines, tumor necrosis factor (TNF), and interleukin 10 (IL-10) in peripheral blood mononuclear cells (PBMCs) from type 1 diabetic (T1D) patients by means of intracellular staining, flow cytometry, and ELISA and to correlate it with inadequate (IN) and adequate (A) metabolic controls. We studied 28 patients with T1D and 20 healthy individuals (C) paired by sex and age. T1D patients were divided in patients with IN and A metabolic control. PBMC cultures were stimulated with LPS to evaluate TNF or were stimulated with PMA/ionomycin or concanavalin A to evaluate IL-10. The TNF levels in supernatant of stimulated cultures, evaluated by ELISA, of diabetic patients were similar to those of healthy individuals, although the percentage of CD 33+ cells that were positive for TNF was higher in the T1D IN group compared to the T1D A group (P= 0.01). Similarly, the IL-10 levels evaluated by ELISA in stimulated cultures of T1D patients were not different from those in the control group; moreover, the percentage of CD3+ cells positive for intracellular IL-10 were higher in the T1D IN group compared to C groups (P= 0.007). The increased levels of cytokines in T1D IN diabetic patients, with reduction in the A group, suggests that hyperglycemia stimulates an inflammatory state that can result in a deficient immune cellular response. The data suggest that assessment by intracellular staining seems to be more accurate than the ELISA technique in evaluating diabetic patients.