Inhibition of monocyte chemotaxis to C-C chemokines by antisense oligonucleotide for cytosolic phospholipase A2.
Monocyte chemotactic protein (MCP)-1, a member of the C-C (or beta) branch of the chemokine superfamily, at chemotactic concentrations, induced a rapid release of [3H]arachidonic acid but not of [14C]oleic acid from prelabeled human monocytes. This effect was associated with an increase in the intensity of the immunoreactive band corresponding to the phosphorylated form of cytosolic phospholipase A2, (cPLA2). To address the role of cPLA2 in the induction of monocyte chemotaxis, cells were treated with a specific antisense oligonucleotide. Monocytes cultured in the presence of 10 microM antisense oligonucleotide for 48 h showed a marked decrease (57 +/- 5%; n = 4) of cPLA2 expression, as evaluated by Western blot analysis and a nearly complete inhibition (81.8 +/- 4.2%; n = 3) of [3H]arachidonic acid release in MCP-1-stimulated cells. Monocyte chemotaxis in response to MCP-l also was inhibited in a concentration-dependent manner by cPLA2 antisense oligonucleotide (IC50 = 1.9 +/- 1.1 microM; n = 3), with complete inhibition observed between 3 and 10 microM. No inhibition of chemotactic response was observed in monocytes treated with a control oligonucleotide. Monocyte migration in response to MCP-3, RANTES (regulated on activation normal T cells expressed and secreted), and MIP-1 alpha/LD78 also was inhibited (>70%) in antisense oligonucleotide-treated cells. On the contrary, the chemotactic response elicited by formyl-methionyl-leucyl-phenylalanine and C5a, two "classical" chemotactic agonists, was minimally affected (<20%) by antisense oligonucleotide treatment. These data show that cPLA2 plays a major role in [3H]arachidonic acid release by MCP-1 in human monocytes and provide direct evidence for the involvement of cPLA2 in C-C chemokine-induced monocyte chemotaxis.
Locati
M
M
Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy.
Lamorte
G
G
Luini
W
W
Introna
M
M
Bernasconi
S
S
Mantovani
A
A
Sozzani
S
S
D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D018932:Chemokine CCL2 / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects* / Q000502:physiology*; D015936:Complement C5a / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009000:Monocytes / Q000187:drug effects* / Q000502:physiology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D054467:Phospholipases A2; D010766:Phosphorylation
D018932:Chemokine CCL2; D009829:Oleic Acids; D016376:Oligonucleotides, Antisense; D016718:Arachidonic Acid; D019301:Oleic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D010741:Phospholipases A; D054467:Phospholipases A2