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

Activation of β-chemokine receptors, co-receptors for human immunodeficiency virus type-1 (HIV-1), stimulates movement and secretion in microglia, possibly through a Ca²⁺-dependent mechanism. We studied chemokine activation of Ca²⁺ signaling processes in microglia. Human fetal microglia were grown in primary culture and chemokine-induced increases in intracellular calcium concentration ([Ca²⁺](i)) were measured in single cells using indo-1-based microfluorimetry. Application of 50ng/ml regulated on activation, normal T expressed and secreted (RANTES; 120s) evoked responses in 26% of the microglia (187/719 cells). [Ca²⁺](i) increased from a basal level of 66±6nM to peak at 268±23nM (n=187). Chemokine-evoked responses rapidly desensitized as indicated by the rapid return to basal [Ca²⁺](i) levels in the maintained presence of RANTES. The removal of extracellular Ca²⁺ or stimulation in the presence of Ni²⁺ (2mM) or La³⁺ (100μM) blocked the RANTES-elicited [Ca²⁺](i) increase. The L-type calcium channel antagonist nimodipine (10μM) inhibited the RANTES-mediated increase in [Ca²⁺](i) by 80±16%. Thus, the RANTES-evoked calcium transient appears to result from Ca²⁺ influx with little if any release from intracellular stores. Application of gp120(clade) (E) and gp120(CM235) (50ng/ml) neither mimicked nor antagonized the RANTES-evoked response. Application of 50ng/ml eotaxin (120s) evoked an increase in [Ca²⁺](i) in 13% of the human microglia in culture (61/469 cells). The HIV-1 regulatory protein Tat (50ng/ml) increased the [Ca²⁺](i) in a subset of eotaxin-responsive cells (16/30). The L-type calcium channel antagonist nimodipine (3μM) inhibited eotaxin- and Tat-mediated increases in [Ca²⁺](i) by 88±6% and 93±6%, respectively. Thus, activation of CCR3 appears to evoke Ca²⁺ influx through L-type Ca²⁺ channels.These results indicate that β-chemokines, RANTES and eotaxin, activate a nimodipine sensitive Ca²⁺ influx pathway in human fetal microglia. HIV-1 Tat protein mimicked chemokine-mediated Ca²⁺ signaling and may modulate the migratory and secretory responses of microglia. Copyright (C) 2000 IBRO.