Single-cell analysis of macrophage chemotactic protein-1-regulated cytosolic Ca²⁺ increase in human adherent monocytes

The increase in intracellular free Ca²⁺ ([Ca²⁺]_i) associated with interaction of monocyte chemotactic protein-1 (MCP-1) and related chemokines β with adherent human blood monocytes was investigated at the single-cell level. We used f-MLP as reference chemotactic agent. MCP-1 caused an increase in [Ca²⁺]_i in individual adherent monocytes, with 95% of cells responding to the chemokine at 20 ng/mL. Response to MCP-1 was already detectable at 1 pg/mL, whereas at least 5 ng/mL were required for significant chemotactic response. The kinetics of the increase in [Ca²⁺]_i were considerably different for MCP-1 compared with f-MLP. MCP-1 produced a slow increase of [Ca²⁺]_i that reached a plateau in 5 to 7 minutes. On the other hand, the increase of [Ca²⁺]_i induced by f-MLP appeared to be biphasic, with a fast phase peaking after 5 to 40 seconds followed by a slower wave. Blocking of Ca²⁺ channels by Ni²⁺ or Cd²⁺ and/or chelation of extracellular free Ca²⁺ considerably reduced but did not abolish response to MCP-1, had no effect on the first wave of [Ca²⁺]_i induced by f-MLP, and completely abrogated the second, slower wave. Thapsigargin, which empties intracellular Ca²⁺ stores, inhibited f-MLP-induced [Ca²⁺]_i increase but fully blocked the action of MCP-1 only when combined with Ni²⁺. Thus, increase of [Ca²⁺]_i induced by MCP-1 is apparently due to independent opening of a channel and mobilization from intracellular stores, whereas f-MLP-induced mobilization of Ca²⁺ from stores causes subsequent opening of a channel. At variance with MCP-1, the related chemokine MCP-2 induced only a low increase of [Ca²⁺]_i in about 40% of adherent monocytes. Inhibition of chemokine-induced increase of [Ca²⁺]_i by cholera or pertussis toxin indicated that MCP-1 and MCP-2 activate monocytes through different intracellular pathways. These results demonstrate at the single-cell level that the mechanisms and dynamics of increased [Ca²⁺]_i are considerably different for f-MLP and chemokines β. In addition, the [Ca²⁺]_i increase induced by the two related chemokines β MCP-1 and MCP-2 appears to be differently regulated, suggesting interaction with distinct receptors.