Abstract :
We report comparative studies on metabolic response by granulocytes from healthy subjects and Type 2 diabetic patients (T2-DM) using calorimetry and luminol-dependent chemiluminescence. Three groups of substances were used for activating or inhibiting the granulocyte reactivity: (i) opsonized zimosan particles (ZC3b), phorbol ester (PDB); (ii) cytokines (IFN-γ, GM-CSF and IL-10) and (iii) cyclic nucleotide-elevating agents (cAMP and cGMP). Results were expressed as total heat production in 1 h for calorimetric assay and relative light unit (RLU) during 60 min reaction for luminol-dependent chemiluminescence.
The results obtained with ZC3b, PDB and IFN-γ/GM-CSF showed that the metabolic response of stimulated-granulocytes were notably larger than those observed with non-stimulated cells in both techniques. The cellular metabolic response from healthy subjects and from T2-DM patients was similar. In contrast, interleukin 10 has no effect on granulocytes from T2-DM patients, but was strongly inhibitory for granulocytes from healthy subjects. The experiments with cyclic nucleotides showed an inverse metabolic response. Dibutyryl cyclic AMP inhibited both heat release and reactive oxygen species generation (ROS) in granulocytes from healthy subjects, but activated cells from T2-DM patients. 8-Br-cyclic GMP activated cells from healthy subjects and inhibited granulocytes from T2-DM patients.
Calorimetry appears to be the best technique for measuring metabolic response in stimulated-granulocytes while only the chemiluminescence assay was able to discriminate non-stimulated-granulocytes from healthy subjects and T2-DM patients. Both techniques are highly sensitive methods which could become important tools for studying signaling mechanisms in immune system cells.
