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Pharmacological Research, Vol . 25, Supplement 1, 1992
IN HUMAN MONOCYTES IL-1 STIMULATES A PHOSPHOLIPASE C ACTIVE ON PHOSPHATIDYLCHOLINE AND INACTIVE ON PHOSPHATIDYLINOSITOL
G . GALELLA, P . STEFANINI, L. MEDINI, E . TREMOLI, C . COLOMBO, and C. GALL[ Institute of Pharmacological Sciences, University of Milan, Italy
Key words : IL-1, monocytes, phospholipases, inositol phosphates, diacylglycerol
Interleukin-l (IL-1) a small polypeptide synthesized by a wide variety of cells, primarily monocytes and macrophages, is capable of initiating the synthesis of protaglandins . In turn the production of this peptide can be regulated through a self induced inhibitor, prostaglandin E 2 (PGE 2) [1], which is synthesized together with other cyclooxygenase-, lypoxygenase-derived eicosanoids, by human monocytes/ macrophages [2] . The eicosanoids synthesis occurs through the activation of cellular phospholipases and the hydrolysis of phosphatidylcholine (PC) by phospholipase A 2 (PLA2 ) is considered to be a major pathway for the formation of free arachidonic acid (AA) . Phospholipids (PL) can also be hydrolyzed by phospholipase C (PLC) and phospholipase D (PLD) to give rise to diacylglycerol (DAG) and phosphatidic acid (PA) . There are data suggesting that IL-1 activates a PLC predominantly linked to phosphatidylethanolamine (PE) [3] and that it stimulates
rapid DAG and
phosphorylcholine (pC) production from PC in the absence of PLC mediated inositol phosphate production [4] . The purpose of the present work was to study the activation of cellular phospholipases in human monocytes in response to stimulation with IL-1 and calcium ionophore A23187 . Monocytes were isolated from the blood of healthy human volunteers using standard Ficoll-Paque gradient centrifugation technique . The activation of phospholipases was monitored by measuring the products of the hydrolysis of cell PL released from cells labeled with specific precursors after 1043-6618/92/2510101-02/$03 .00/0
© 1992 The Italian Pharmacological Society
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Pharmacological Research, Vol. 25, Supplement 1, 1992
stimulation with IL-1 and the calcium ionophore A23187 . In cells labeled with [3H] myo-inositol, the
results indicate that IL-1 does not activate the formation of
inositolphosphates measured after extraction and separation by anion exghange chromatography . In cells labelled with
[3H]
AA, IL-1 causes the formation of DAG
(Fig .1,B) associated with the hydrolysis of PC (Fig .1, A) . The release of AA and the hydrolysis of PC were assessed by one and two dimensional (TLC) analysis of lipids extracted from cell preparations . Furthermore, the lack of accumulation of free AA after stimulation with IL-1 would indicate that there has been no activation of PLA2 ; the release of labeled free AA occurs instead with A23187 stimulation (Fig . 1, B) . These data suggest that, in monocytes, IL-1 does neither directly stimulate a PLA2 nor a PLC active on PI ; instead it primarily stimulates a PLC active on PC .
A
B Rad*ahvily in DAG and FFA
Radioactivity in PE
Radioactivity in PC
30
91,
O DAG 0 FFA
W
. . E,24x $
E -20%
a a
_3 20 1 c
0 Basal ~
IL1
A23187 2 min
1
Basal
ILI
A23187 2 min
A23187
IL-1 2 min
Fig . 1 Radioactivity loss by PL (A) and release of DAG and FFA (B) after stimulation in monocytes prelabelled with [3 H] AA
References 1 . Knudsen PJ, Dinarello CA, Strom TB . Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate . J Immunol 1986 ; 137 :3189-94 . 2 . Pawloski NA, Kaplan G, Hamill Al, Cohn ZA, Scott WA . Arachidonic acid metabolism by human monocytes J Exp Med 1983 ; 158:393-7. 3 . Kester M, Simonson MS, Mena P, Sedor JR . Interleukin-1 generates transmembrane signals from phospholipids through novel pathways in cultured rat mesangial cells. Clin Invest 1989 ; 83 :718-23 . 4 . Rosoff PM, Savage N, Dinarello CA . Interleukin-1 stimulates diacylglycerol production in T lymphocytes by a novel mechanism . Cell 1988 ; 54 :73-81 .
Pharmacological Research, Vol . 25, Supplement 1, 1992
IN HUMAN MONOCYTES IL-1 STIMULATES A PHOSPHOLIPASE C ACTIVE ON PHOSPHATIDYLCHOLINE AND INACTIVE ON PHOSPHATIDYLINOSITOL
G . GALELLA, P . STEFANINI, L. MEDINI, E . TREMOLI, C . COLOMBO, and C. GALL[ Institute of Pharmacological Sciences, University of Milan, Italy
Key words : IL-1, monocytes, phospholipases, inositol phosphates, diacylglycerol
Interleukin-l (IL-1) a small polypeptide synthesized by a wide variety of cells, primarily monocytes and macrophages, is capable of initiating the synthesis of protaglandins . In turn the production of this peptide can be regulated through a self induced inhibitor, prostaglandin E 2 (PGE 2) [1], which is synthesized together with other cyclooxygenase-, lypoxygenase-derived eicosanoids, by human monocytes/ macrophages [2] . The eicosanoids synthesis occurs through the activation of cellular phospholipases and the hydrolysis of phosphatidylcholine (PC) by phospholipase A 2 (PLA2 ) is considered to be a major pathway for the formation of free arachidonic acid (AA) . Phospholipids (PL) can also be hydrolyzed by phospholipase C (PLC) and phospholipase D (PLD) to give rise to diacylglycerol (DAG) and phosphatidic acid (PA) . There are data suggesting that IL-1 activates a PLC predominantly linked to phosphatidylethanolamine (PE) [3] and that it stimulates
rapid DAG and
phosphorylcholine (pC) production from PC in the absence of PLC mediated inositol phosphate production [4] . The purpose of the present work was to study the activation of cellular phospholipases in human monocytes in response to stimulation with IL-1 and calcium ionophore A23187 . Monocytes were isolated from the blood of healthy human volunteers using standard Ficoll-Paque gradient centrifugation technique . The activation of phospholipases was monitored by measuring the products of the hydrolysis of cell PL released from cells labeled with specific precursors after 1043-6618/92/2510101-02/$03 .00/0
© 1992 The Italian Pharmacological Society
1 02
Pharmacological Research, Vol. 25, Supplement 1, 1992
stimulation with IL-1 and the calcium ionophore A23187 . In cells labeled with [3H] myo-inositol, the
results indicate that IL-1 does not activate the formation of
inositolphosphates measured after extraction and separation by anion exghange chromatography . In cells labelled with
[3H]
AA, IL-1 causes the formation of DAG
(Fig .1,B) associated with the hydrolysis of PC (Fig .1, A) . The release of AA and the hydrolysis of PC were assessed by one and two dimensional (TLC) analysis of lipids extracted from cell preparations . Furthermore, the lack of accumulation of free AA after stimulation with IL-1 would indicate that there has been no activation of PLA2 ; the release of labeled free AA occurs instead with A23187 stimulation (Fig . 1, B) . These data suggest that, in monocytes, IL-1 does neither directly stimulate a PLA2 nor a PLC active on PI ; instead it primarily stimulates a PLC active on PC .
A
B Rad*ahvily in DAG and FFA
Radioactivity in PE
Radioactivity in PC
30
91,
O DAG 0 FFA
W
. . E,24x $
E -20%
a a
_3 20 1 c
0 Basal ~
IL1
A23187 2 min
1
Basal
ILI
A23187 2 min
A23187
IL-1 2 min
Fig . 1 Radioactivity loss by PL (A) and release of DAG and FFA (B) after stimulation in monocytes prelabelled with [3 H] AA
References 1 . Knudsen PJ, Dinarello CA, Strom TB . Prostaglandins posttranscriptionally inhibit monocyte expression of interleukin 1 activity by increasing intracellular cyclic adenosine monophosphate . J Immunol 1986 ; 137 :3189-94 . 2 . Pawloski NA, Kaplan G, Hamill Al, Cohn ZA, Scott WA . Arachidonic acid metabolism by human monocytes J Exp Med 1983 ; 158:393-7. 3 . Kester M, Simonson MS, Mena P, Sedor JR . Interleukin-1 generates transmembrane signals from phospholipids through novel pathways in cultured rat mesangial cells. Clin Invest 1989 ; 83 :718-23 . 4 . Rosoff PM, Savage N, Dinarello CA . Interleukin-1 stimulates diacylglycerol production in T lymphocytes by a novel mechanism . Cell 1988 ; 54 :73-81 .
