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 beta 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 beta. In addition, the [Ca2+]i increase induced by the two related chemokines beta MCP-1 and MCP-2 appears to be differently regulated, suggesting interaction with distinct receptors.
Bizzarri
C
C
Consorzio Biolaq, L'Aquila, Italy.
Bertini
R
R
Bossù
P
P
Sozzani
S
S
Mantovani
A
A
Van Damme
J
J
Tagliabue
A
A
Boraschi
D
D
D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D002478:Cells, Cultured; D018932:Chemokine CCL2; D054412:Chemokine CCL8; D002630:Chemotactic Factors / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects*; D002772:Cholera Toxin / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D018945:Monocyte Chemoattractant Proteins; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D037342:Pertussis Toxin; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
C515453:CCL8 protein, human; D018932:Chemokine CCL2; D054412:Chemokine CCL8; D002630:Chemotactic Factors; D018945:Monocyte Chemoattractant Proteins; D011994:Recombinant Proteins; D013729:Terpenes; D010566:Virulence Factors, Bordetella; D002104:Cadmium; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D009532:Nickel; D002772:Cholera Toxin; D037342:Pertussis Toxin; D002118:Calcium