CALCIUM MOBILIZATION AND EXPRESION OF TRPM2 AND CRAC CHANNELS ARE ALTERED IN HUMAN NEUTROPHILS WITH AGING

Abstract

Abstract Elderly susceptibility to infectious diseases may be a result of a decline in the function of neutrophils. We analyzed the number and percentage of neutrophils in peripheral blood of healthy elderly (60–85 years old) and young adults (20–35 years old), as well as the effect of aging on store-operated Ca+2 entry (SOCE), Ca+2 mobilization towards 100 nM or 1μM fMLP, 300 nM IL-8, 10 nM C5a, and 25 μg/ml LPS, by flow cytometry; and its relation to the expression of CD88, CXCR2, FPLR1, TRL4 and TRPM2 and CRAC channels. To examine Ca+2 mobilization, cells were loaded with 1 μM Fluo-4-AM. Baseline Ca+2 level was recorded for 60 s before the addition of the stimulus; response was recorded up to 10 min. Subcellular expression of CRAC and TRPM2 was investigated by confocal microscopy and their mRNA quantified by real time RT-PCR. Finally, migration towards 10 nM fMLP, C5a or IL-8 was measured by transwell chemotaxis assay. The results showed increased numbers and percentage of neutrophils in elderly, mainly in females. However, these cells presented lower Ca+2 entry in response to C5a, IL-8 and fMLP, and significantly lower SOCE. We did not find difference in the Ca+2 response to LPS or in the expression of the receptors of the stimuli (CD88, CXCR2, FPLR1 and TRL4). A punctuated intracellular pattern of TRPM2 and CRAC was observed in the neutrophils. TRPM2 was located in late endosomes and presented changes in its distribution with age. It was also detected a decrease in the mRNA of the TRPM2 and CRAC channels, mainly in women. Finally, we found that neutrophils from elderly migrated less towards fMLP, IL-8 and C5a. Our data suggest that the mechanisms that underlie the decrease on neutrophil functions with aging might be alterations in Ca+2 signals.