Clinical immunology
Intracellular calcium ions kinetics and chemiluminescent activity in human neutrophils

Chemiluminescence of granulocytes is a renowned method for the estimation their overall activity as measured by the production of reactive oxygen species. The aim of this study was to compare chemiluminescent activity of human granulocytes to kinetics of intracellular calcium ions concentration in children with recurrent infections of respiratory tract (RRTI) and in healthy children. The studies was performed with the use of isolated peripheral blood neutrophils from 41 RRTI children (21 girls, 20 boys) 3 to 10 years old with more than five episodes of RRTI per year and from control group of 30 healthy children age and sex matched free from allergic, immune and hematological disorders. Neutrophils were activated by bacterial peptide fMLP, opsonised zymosan (OZ) and phorbol myristat acetate (PMA). Intracellular Ca concentration kinetics was assessed by flow cytometry (Coulter Epics XL) with the use of Fluo3 and Fura Red fluorescent dyes. fMLP and OZ induced Ca mobilization lasted shorter in RRTI group (p<0.05).The peak influx of free Ca and its concentration in resting state after stimulation with fMLP were lower in patients (p<0.05). In RRTI group stimulation with OZ was delayed comparing to control (p<0.01). In response to PMA free Ca concentration decreased faster. Chemiluminescent response to all the examined stimuli in children with recurrent infections was significantly lower compared to the control. Significant negative correlation was observed between intracellular calcium level and emission of light measured by chemiluminescence assay (r=–0.9643, p<0.001) after stimulation with fMLP. Intracellular calcium concentration did not correlated with chemiluminescent activity induced by OZ or PMA.Increased sensitivity to infections in RRTI children may be related to the disturbances in neutrophil activation via intracellular Ca concentration and subsequent production of free oxygen radicals. Chemiluminescent activity after stimulation with fMLP seems to be dependent on intracellular calcium ions concentration. PMA and OZ induce chemiluminescence in Ca²⁺ independent manner.