β-Chemokines and human immunodeficiency virus type-1 proteins evoke intracellular calcium increases in human microglia

Abstract

Activation of β-chemokine receptors, co-receptors for human immunodeficiency virus type-1 (HIV-1), stimulates movement and secretion in microglia, possibly through a Ca 2+-dependent mechanism. We studied chemokine activation of Ca 2+ signaling processes in microglia. Human fetal microglia were grown in primary culture and chemokine-induced increases in intracellular calcium concentration ([Ca 2+] i) were measured in single cells using indo-1-based microfluorimetry. Application of 50 ng/ml regulated on activation, normal T expressed and secreted (RANTES; 120 s) evoked responses in 26% of the microglia (187/719 cells). [Ca 2+] i increased from a basal level of 66±6 nM to peak at 268±23 nM ( n=187). Chemokine-evoked responses rapidly desensitized as indicated by the rapid return to basal [Ca 2+] i levels in the maintained presence of RANTES. The removal of extracellular Ca 2+ or stimulation in the presence of Ni 2+ (2 mM) or La 3+ (100 μM) blocked the RANTES-elicited [Ca 2+] i increase. The l-type calcium channel antagonist nimodipine (10 μM) inhibited the RANTES-mediated increase in [Ca 2+] i by 80±16%. Thus, the RANTES-evoked calcium transient appears to result from Ca 2+ influx with little if any release from intracellular stores. Application of gp120 clade E and gp120 CM235 (50 ng/ml) neither mimicked nor antagonized the RANTES-evoked response. Application of 50 ng/ml eotaxin (120 s) evoked an increase in [Ca 2+] i in 13% of the human microglia in culture (61/469 cells). The HIV-1 regulatory protein Tat (50 ng/ml) increased the [Ca 2+] i in a subset of eotaxin-responsive cells (16/30). The l-type calcium channel antagonist nimodipine (3 μM) inhibited eotaxin- and Tat-mediated increases in [Ca 2+] i by 88±6% and 93±6%, respectively. Thus, activation of CCR3 appears to evoke Ca 2+ influx through l-type Ca 2+ channels. These results indicate that β-chemokines, RANTES and eotaxin, activate a nimodipine sensitive Ca 2+ influx pathway in human fetal microglia. HIV-1 Tat protein mimicked chemokine-mediated Ca 2+ signaling and may modulate the migratory and secretory responses of microglia.