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Inhibitory effect of prostacyclin (PGI2) on neutropenia induce by intravenous injection of platelet-activating-factor (PAF) in the rabbit
PROSTAGLANDINS
INHIBITORY EFFECT OF PROSTACYCLIN
(PGI2) ON NEUTROPENIA
INDUCED BY INTRAVENOUS INJECTION OF PLATELET-ACTIVATINGFACTOR
(PAF) IN THE RABBIT. G.CAMUSSI,C.TETTA and F.BUSSOLINO.
Laboratorio di Immunopatologia - Cattedra di Nefrologia Universita di Torino, Corso Polonia, 14, 10126 Torino Italy. ABSTRACT Intravenous injection into rabbits of I-0-octadecyl-2-ace tyl-sn-glyceryl-3-phosphorylcholine (synthetic Platelet-Activating Factor (PAF)) or PAF derived from rabbit base= phils caused acute thrombocytopenia and neutropenia which was consequent to the formation of intravascular polymer= phonuclear neutrophil (PMN) aggregates and to their seque stration in the microvasculature, primarily of the lung. Infusion of prostacyclin (PGI ; 10 ng/Kg/min to 50 ng/Kg/ min) inhibited in a dose-depe 4dent manner PAF-induced thrombocytopenia and neutropenia as well as the sequestra tion of PMN in the pulmonary capillary network. INTRODUCTION PLatelet-Activating Factor (PAF) derived from IgE-sensiti zed rabbit basophils (I-0-octadecyl/hexadecyl-2-acetyl-sn -glyceryl-3-phosphorylcholine)(l-3) is a phospholipid me* diator of inflammation that activates platelets causing aggregation and 'release reaction' independently from ade nosine diphosphate (ADP) and arachidonic acid-mediated pa thways (4,5).Subsequent studies have shown that, apart from basophils, PAF is synthetized and released also from PMN (6,7), platelets (5), macrophages (8) and monocytes (9). Initially regarded as a potent platelet activator, PAF has been shown to possess a wide spectrum of biologic cal activities as it is capable of inducing PMN aggrega= tion, chemotaxis, generation of superoxide and lysosomal enzyme release (7,10,11), increased vascular permeability (12) and smooth muscle contraction (13). An in vivo relea se of PAF has been documentated in IgE anaphylaxis (14), immune complex-induced neutropenia (7) and experimental acute serum sickness (15). The i-v. injection of PAF into
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rabbits induces not only thrombocytopenia but also neutro penia that has been related to massive sequestration of PMN aggregates in the capillary network, primarily of the lung (7,16). Recently, we have demonstrated that prostacyclin (PGI )is a potent inhibitor of in vitro platelet and PMN activztion induced by PAF (17,18). In the present report, we focus on the effects of PGI infusion on PAF-mediated intrava= scular alterations ana PMN sequestration in the microvascular bed in the rabbit. MATERIALS and METHODS Stimuli l-O-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine(Ba them Feinchemikalien AG,Babendorf,CH) (synthetic PAF) wasdissolved as solution stock in methanol:chloroform (2:1, v/v)(l mg/ml) and, before use, in sterile physiologic sa= line containing 0.25 % bovine serum albumin (BSA) (Behring werke AG,Marburg,W.G., fraction V)(saline-BSA), as PAF carrier. Native PAF was obtained from IgE-sensitized rab= bit basophils after antigen stimulation (horseradish pero xidase)(Sigma Chemical Co, St-Louis, MO, USA) according to Pinckard et al. (14). Briefly, PAF was extracted from cell-free supernatants with methanol (1:1.5, v/v). After centrifugation (3000 g,20 min,room temperature), the me= than01 extract was applied to a silicic acid column (Si= lit ARCC-4,Mallinkrodt,St,Louis,Mo,USA) and eluted with chloroform, acetone:methanol (l:l, v/v)and chloroform:me= than01 (1:4, v/v) which retained PAF activity (5). PAF was further purified using TLC on glass plates precoated with silica1 gel (60F254,Merck,Darmstadt GmbH, W-G.) (sol= vent system (Chloroform:methanol:water, 65:35:6, v/v)(Rf= 0,22)* PAF was characterized by the following criteria:(i) its ability to induce rabbit platelet aggregation, which was unaffected by indomethacin (1x10-5 M)(Sigma Chemical Co.) and by ADP scavengers (creatine phosphate 312 ,,g/mlcreatine phosphokinase 152.5 m/ml) (Sigma Chemical Co.) (4: 5); (ii) its physicochemical characteristics (14) and (iii) sensitivity to phospholipase A2 from porcine pancreas(0.03 mg/ml,pH 8,0,60 min;37'C)(Boehringer Mannheim GmbH, W-G.) (19). Native PAF was solubilized in saline-BSA and adju= sted at a concentration of 8,750 U.PAl?/ml. I-O-octadecylsn-glyceryl-3-phosphorylcholine (lyso-PAF), functionally inactive on rabbit platelets, was obtained from synthetic PAF after incubation with 0.03 N NaOH in methanol (14) and finally suspended in saline-BSA.
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In vivo experiments New Zealand White rabbits of either sex weighing from 2 to 3 Kg were used fern these studies. A saline-filled po lyethylene catheter (Braun, W.G. diameter 1.2 mm) was placed in the abdominal aorta under local anaesthesia via the femoral artery. Rabbits were either injected with synthetic PAF (1 or 3 vg/Kg body weight) or native PAF (3,100 U.PAF/Kg). PG12 sodium salt (kindly provided by Upjohn Co,Kalamazao,Mich ,,USA) was prepared at a con= centration of 500 ug/ml of Tris-HCl buffer (1x10-2 M,pH 9.3,4-C) and diluted in cold (4-C) Tris-buffered physiolo gic saline (pH 8.0) immediately before use. PGI was theii delivered at different infusion rates (IO,30 ,56 ng/Kg/mirj 10 min before and for 60 min after PAF injection. PMN and platelet counts (Malassez haemocytometer chamber:volume 1 mm3; depth 0.2 mm) were performed at different times (30 set; 1,2,3,4,5,10,30,60 min) after challenge. The folio= wing controls were performed: rabbits were injected (i) with the same volumes of saline-BSA, (ii) with 20 ug/Kg lyso-PAF or (iii) infused for 60 min with IO or 50 ng/Kg/ min of PGI2 prepared as described above. Light microscopy
studies
Kidney, spleen and liver specimens from rabbits which had died after PAF (3 pg/Kg) injection or sacrificed with i.v. overdose of sodium pentobarbital (Farmitalia,Milan,Italy) were fixed in 10% neutral buffered formalin. The respira= tory tissues (trachea, bronchi, lungs) were removed and the lungs inflated with 10% neutral buffered formalin from a 25 cm height, then immersed in 10% neutral buffe= red formalin overnight. Four micron-thick, paraffin-embed ded sections of th.e different organs were stained with he _matoxylin-eosin and periodic acid-Stiff (PAS). RESULTS Intravenous injection of synthetic PAF resulted in a dose -related fall of circulating platelets and PMN (Fig. 1). At a dose of 3 ug/Kg, synthetic PAF induced a fatal reac= tion which occurred 5-10 min after challenge (Fig. 1 A,C)_ Lower doses of PAF (1 ug/Kg) elicited an acute thrombocy= topenia (Fig. 1 B) as well as neutropenia (Fig. 1 D) that were reversible within 30 to 40 min after challenge. Simi larly acute, transient thrombocytopenia and neutropenia were observed after injection of native PAF (3,100 U.PAF/ Kg) (data not shown).. Pretreatment with PG12 (30 ng/Kg/min), infused IO min before challenge, prevented the fatal rea=
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(Fig. 1 A,C), ction induced by 3 pg/Kg of synthetic PAF the marked thrombocytopenia and neutropenia still occur= ring even at a higher infusion rate (50 ng/Kg/min). As shown in Fig. 1 B and D, 10 ng/Kg/min reduced to about 50 % the thrombocytopenia and neutropenia developping af= ter injection of 1 ug/Kg of synthetic PAF. At this rate of infusion, PGI2 completely prevented the thrombocytope= nia and neutropenia in response to native PAF (3,100 U. PAP/Kg) (data not shown). Only at a higher rate of infusion (50 ng/Kgjmin), PG12 was fully inhibitory even on the thrombocytopenia and neutropenia induced by 1 pg/Kg of syn thetic PAF. No change in the counts of circulating plate=lets and PMN was observed in rabbits injected with (i) sa= line-BSA, (ii) lyso-PAF or (iii) infused with PGI2 (from to 10 to 60 ng/Kg/min) for 60 min. Ae 50 ng/Kg/min, PG12 provoked marked hypotension. The neutropenia that occurred after challenge with synthe tic or native PAF was related to the massive sequestration of PMN in the microvasculature, primarily of the lungs (Fig. 2 A), but also present, even if to a lesser extent, in the kidneys,spleen and liver (not shown). No intravascu_ lar PMN aggregation was seen in the microvasculature in any of the above-cited organs from rabbits infused with PGI2 (50 ng/Kg/min) after challenge either with synthetic (1 Vg/Kg) or native PAF (3,100 U.PAF/Kg) (Fig_ 2 B), DISCUSSION The present study indgcates that infusion of PGI2 reduces, in a dose-dependent manner,the thrombocytopenia and the neutropenia that occur as the result of intravascular ag= gregation of platelets and PMN stimulated by PAF in the rabbit. The ability of prostaglandins to modulate both acute and chronic inflammatory responses has been well documentated in (20)s PG12 has been implicated as a chemiual mediator increasing vascular permeability (21) or as a modulator of the response of inflammatory cells (20). The effect of PG12 on inflammatory cells is related to the increase in intracellular CAMP levels (20,22)..Increases in intracellu lar CAMP inhibit not only in&tro platelet aggregation (22),but also PMN aggregation (6) and lysosomal enzyme re= lease (20).The in vitro response to PAF of platelets (17) and PMN (7), leading to their aggregation as well as to PMN chemotaxis and granule secretion, has been previously shown to be inhibited by PG12 as well as by other agents that increase intracellular CAMP levels.
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Fig. 1: A & B: platelet counts-in rabbits injected with synthetic PAF (A: 3 Ug/Kg; B: 1 ug/Kg). Intravascular effect of PGI infusion. A: M 50 ng/Kg/min;~30 ig/Kg/min;B: w 50 ng/Kg/min; IO ng/Kg/min. C & D: PMN countsHin ra V bits injected with syn thetic PAF (C: 3 pg/Kg; D: 1 ug/Kg).IntravZ scular effect of PGI infusion. C:M 55 ng/Kg/min;M30 ng 3Kg/mini D:W 50 ng/ Kg/min;~lO ng/Kg/min. Mean -+ 1 S.D. of five experiments.
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Fig. 2 A: Intravascular PMN aggregates (arrows) in the lungs after injection of l,ug/Kg of synthetic PAF.(x 400). B: Lung vessels were congested with erythrocytes and dilated but no PMN aggregate was seen af= ter PG12 infusion (50 ng/Kg/min).(x 250).
The results of the present study show that also in vivo infused at concentrations that increase intracellu= PGI lar2c&P levels (23)exerts an inhibitory effect on cellu= lar (platelets and PMN) responses to intravenously injec= ted synthetic or native PA!?.A markedly acute thrombocyto penia and neutropenia occur after PAP challenge. These 348
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intravascular effects have been related to the massive for mation of platelet and PMN aggregates that appear to embog lize in the microvasculature of several organs, namely of the lungs (7,16). When high doses of synthetic PAF ( 3 ug/ Kg) were injected, the irreversible and fatal reaction was prevented by the infusion of 30 ng/Kg/min of PGI2. However, even with an infusion rate of 50 ng/Kg/min, the thrombocy= topenia and neutropenia were only partially reduced. Lower doses of synthetic (1 pg/Kg) or native PAF (3,100 U.PAF/Kg) induced an acute and reversible fall in circulating plate= lets and PMN which was reduced to about one half by PGI at IO ng/Kg/min. Moreover, higher infusion rates (50 ng 3Kg/ min) could fully inhibit PAF-induced intravascular effects. The evidence suggesting that PG12 infusion is capable of antagonizing the intravascular consequencies of PAF inje= ction was further strengthened by the absence of PMN aggre gates in the microvasculature, particularly of the lungs. These studies favor the hypothesis that PG12 may play a ro le in modulating the response of PMN and platelts also iii those pathological conditions in which PAF has been impli= cated, REFERENCES 1) Hanahan, D.J., C.A.Demopoulos, J.Liehr and R.N. Pinckard Identification of platelet-activating factor isolated from rabbit basophils as acetyl glyceryl ether phospho= rylcholine. J.Biol.Chem. 255:5514, 1980. and D.J.Hanahan. Plate= 2) Demopoulos, C.A., R.N.Pinzrd let-activating factor. Evidence for l-0-alkyl-2-acetylsn-glyceryl-3-phosphorylcholine as the active component (A new class oflipid chemical mediators). J.Biol.Chem. 254:9355, 1979. 3) Gveniste, J., M.Tence, P.Varenne, J.Bidault, C.Boul= let and J.Polonsky. Semi-synthese et structure proposee du facteur activant les plaquettes (P.A.F.): P.A.F.-ace ther, un alkyl analogue de la lysophosphatidylcholine.C.R.Acad.Sci.Paris (D) 289:1017, 1979. 4) Benveniste, J., P.M.Henz and C.G.Cochrane. Leukocytedependent histamine release from rabbit platelets. The role of IgE, basophils and a platelet-activating factor J.Exp.Med. 136:1356, 1972. 5) Chignard, M., J.P.Le Couedic, M.Tence, B.B.Vargaftig and J.Benveniste. The role of platelet-activating fa= ctor in platelet aggregation. Nature 279:799, 1979. 6) Lynch, J.M., G.Z.Lotner, S..Betz and P.M.Henson. The re lease of platelet-activating factor by stimulated rab= bit neutrophil. J.Iriuttunol. 123:1219, 1979.
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7) Camus~i, C., C.Tetta, F.Bussolino, F.Callgaris-Cappio, R-Coda, C.Masera and G.P.Segoloni. Med'fators of immunecomplex-induced aggregation of polymorphonuclear neutro phils. II Platelet activating factor as the effector substance of immune-induced aggregation. Int.Archs Al= lergv Apol. Immunol. 64: 25,1981. 8) Mencia-Huerta,J.M.andJ.Benveniste. Platelet-activating factor and macrophages. I Evidence for the release from rat and mouse peritoneal macrophages and not from mono= cytes. Eur.J.Immunol, 9: 409, 1979. 9) Camussi G., M.AgliettaS R-Coda, F.Bussolino, W.Piacibel lo and C.Tetta. Release of platelet activating factor (PAF) and histamine. II The cellular origin of human PAF: monocytes, polymorphonuclear neutrophils and base= phils. Immunology 42: 191, 1981. lO)Shaw, J.O., R.N.Pizkard, K.S.Ferrigni, L.McManus and D.J. Hanahan. Activation of human neutrophils with 1-O hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphoryl= choline (platelet activating factor). J.Immunol. 123, 1847, 1981. ll)O'Flaherty, J.T., R.L. Wykle, C.H.Miller, J.C.Lewis, M. Waite, D.A.Bass, C.E.Mc Call and R. De Chatelet. I-O-al kyl-sn-glyceryl-3-phosphorylcholine. A novel class of neutrophil stimulants. Am.J.Pathol. 103: 70, 1981. 12)Humphrey, D.M., L.McManus, K.SatouchrD.J,Hanahan and R.N.Pinckard. Vasoactive properties of acetyl glyceryl ether phosphorylcholine and analogues. Lab.Invest. -46: 422, 1982. 13)Stimler, N.P., C.M.Bloor, T.E.Hugli, R.L.Wykle, C.E.Mc Call and J.T.O'Flaherty. Anaphylactic actions of plate let activating factor. Am.J.Pathol. 105: 64, 1981. 14)Pinckard, R.N., R.S.Farr and D.J.Hanahan. Physicochemi= Cal and functional identity of platelet activating fa= ctor (PAF) released in vivo during IgE anaphylaxis with PAF released in vitro from IgE-sensitized basophils. J.Immunol. 123: 1847, 1979. 15)Camussi G.,- C.Tetta, M.C.Deregibus, F.Bussolino, G.Sego loni and A.Vercellone. Platelet-activating factor (PAF) in experimentally-induced rabbit acute serum sickness: role of basophil-derived PAF in immune complex deposi= tion. J.Immunol. 128: 86, 1982. 16)McManus L., D.J.Hzhan, C.A.Demopoulos,and R,N.Pinckard Pathobiology of the intravenous injectionof acetyl gly= ceryl ether phosphorylcholine (AGEPC), a synthetic pla= telet-activating factor. J.Immunol. 124: 2919, 1980. 17)Bussolino, G. and G.Camussi. Effect ofprostacyclin on platelet activating factor-induced rabbit platelet ag= gregation. Prostaglandins -20: 781, 1980.
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PROSTAGLANDINS 18)Camussi G., C.Tetta, G.Segoloni, M.C.Deregibus,and F. Bussolino. Neutropenia induced by platelet-activating factor (PAF-acether) released from neutrophils: the i" hibitory effect of prostacylcin (PGI2). Agents and AC= tions 11: 550, 1981. 19)Benvenzte,J., J.P.Le Couedic, J.Polonsky and M.Ten&. Structural analysis of purified Platelet activating fa ctor by lipases. Nature 269: 170, 1977. 20)Weissman, G., J.E. Smolen and H. Korchak. Prostaglan= dins and Inflammation: receptor/cyclase coupling as an explanation of why PGE2 and PG12 inhihit functions of inflammatory cells. In Advances in Prostaglandin and Thromboxane Research (B.Samuelsson, P.W.Ramwell and R. Paoletti, Eds.), Raven Press, New York, 8: 1637, 1980. 21)Murata, S., W.C.Chang, S.Tsurufuji and ITMorita. The possible roles of prostacyclin (PGI ) and Thromboxanes in chronic inflammation. In Advance2 in Inflammation Research (G.Weissman et al., Eds), Raven Press, New York, 1: 436, 1979. 22)Tateson, J.E., S.Moncada and J.R.Vane. Effect of pro= stacyclin on cyclic AMP concentration in human plate= lets. Prostaglandins 13: 389, 1977. 23)Data, J., A.B.A. Moloz, M.M.Meinzinger and R.R.Gorman Intravenous infusion of Prostacyclin sodium in man: clL nical effects and influence on platelet adenosine di= phosphate sensitivity and adenosine 3':5'-cyclic mono= phosphate levels. Circulation 64: 3, 1981.
Editor: BrendanWhittle Received: 7-28-82 Accepted: l-7-83
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1983 VOL. 25 NO. 3
351
INHIBITORY EFFECT OF PROSTACYCLIN
(PGI2) ON NEUTROPENIA
INDUCED BY INTRAVENOUS INJECTION OF PLATELET-ACTIVATINGFACTOR
(PAF) IN THE RABBIT. G.CAMUSSI,C.TETTA and F.BUSSOLINO.
Laboratorio di Immunopatologia - Cattedra di Nefrologia Universita di Torino, Corso Polonia, 14, 10126 Torino Italy. ABSTRACT Intravenous injection into rabbits of I-0-octadecyl-2-ace tyl-sn-glyceryl-3-phosphorylcholine (synthetic Platelet-Activating Factor (PAF)) or PAF derived from rabbit base= phils caused acute thrombocytopenia and neutropenia which was consequent to the formation of intravascular polymer= phonuclear neutrophil (PMN) aggregates and to their seque stration in the microvasculature, primarily of the lung. Infusion of prostacyclin (PGI ; 10 ng/Kg/min to 50 ng/Kg/ min) inhibited in a dose-depe 4dent manner PAF-induced thrombocytopenia and neutropenia as well as the sequestra tion of PMN in the pulmonary capillary network. INTRODUCTION PLatelet-Activating Factor (PAF) derived from IgE-sensiti zed rabbit basophils (I-0-octadecyl/hexadecyl-2-acetyl-sn -glyceryl-3-phosphorylcholine)(l-3) is a phospholipid me* diator of inflammation that activates platelets causing aggregation and 'release reaction' independently from ade nosine diphosphate (ADP) and arachidonic acid-mediated pa thways (4,5).Subsequent studies have shown that, apart from basophils, PAF is synthetized and released also from PMN (6,7), platelets (5), macrophages (8) and monocytes (9). Initially regarded as a potent platelet activator, PAF has been shown to possess a wide spectrum of biologic cal activities as it is capable of inducing PMN aggrega= tion, chemotaxis, generation of superoxide and lysosomal enzyme release (7,10,11), increased vascular permeability (12) and smooth muscle contraction (13). An in vivo relea se of PAF has been documentated in IgE anaphylaxis (14), immune complex-induced neutropenia (7) and experimental acute serum sickness (15). The i-v. injection of PAF into
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rabbits induces not only thrombocytopenia but also neutro penia that has been related to massive sequestration of PMN aggregates in the capillary network, primarily of the lung (7,16). Recently, we have demonstrated that prostacyclin (PGI )is a potent inhibitor of in vitro platelet and PMN activztion induced by PAF (17,18). In the present report, we focus on the effects of PGI infusion on PAF-mediated intrava= scular alterations ana PMN sequestration in the microvascular bed in the rabbit. MATERIALS and METHODS Stimuli l-O-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine(Ba them Feinchemikalien AG,Babendorf,CH) (synthetic PAF) wasdissolved as solution stock in methanol:chloroform (2:1, v/v)(l mg/ml) and, before use, in sterile physiologic sa= line containing 0.25 % bovine serum albumin (BSA) (Behring werke AG,Marburg,W.G., fraction V)(saline-BSA), as PAF carrier. Native PAF was obtained from IgE-sensitized rab= bit basophils after antigen stimulation (horseradish pero xidase)(Sigma Chemical Co, St-Louis, MO, USA) according to Pinckard et al. (14). Briefly, PAF was extracted from cell-free supernatants with methanol (1:1.5, v/v). After centrifugation (3000 g,20 min,room temperature), the me= than01 extract was applied to a silicic acid column (Si= lit ARCC-4,Mallinkrodt,St,Louis,Mo,USA) and eluted with chloroform, acetone:methanol (l:l, v/v)and chloroform:me= than01 (1:4, v/v) which retained PAF activity (5). PAF was further purified using TLC on glass plates precoated with silica1 gel (60F254,Merck,Darmstadt GmbH, W-G.) (sol= vent system (Chloroform:methanol:water, 65:35:6, v/v)(Rf= 0,22)* PAF was characterized by the following criteria:(i) its ability to induce rabbit platelet aggregation, which was unaffected by indomethacin (1x10-5 M)(Sigma Chemical Co.) and by ADP scavengers (creatine phosphate 312 ,,g/mlcreatine phosphokinase 152.5 m/ml) (Sigma Chemical Co.) (4: 5); (ii) its physicochemical characteristics (14) and (iii) sensitivity to phospholipase A2 from porcine pancreas(0.03 mg/ml,pH 8,0,60 min;37'C)(Boehringer Mannheim GmbH, W-G.) (19). Native PAF was solubilized in saline-BSA and adju= sted at a concentration of 8,750 U.PAl?/ml. I-O-octadecylsn-glyceryl-3-phosphorylcholine (lyso-PAF), functionally inactive on rabbit platelets, was obtained from synthetic PAF after incubation with 0.03 N NaOH in methanol (14) and finally suspended in saline-BSA.
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In vivo experiments New Zealand White rabbits of either sex weighing from 2 to 3 Kg were used fern these studies. A saline-filled po lyethylene catheter (Braun, W.G. diameter 1.2 mm) was placed in the abdominal aorta under local anaesthesia via the femoral artery. Rabbits were either injected with synthetic PAF (1 or 3 vg/Kg body weight) or native PAF (3,100 U.PAF/Kg). PG12 sodium salt (kindly provided by Upjohn Co,Kalamazao,Mich ,,USA) was prepared at a con= centration of 500 ug/ml of Tris-HCl buffer (1x10-2 M,pH 9.3,4-C) and diluted in cold (4-C) Tris-buffered physiolo gic saline (pH 8.0) immediately before use. PGI was theii delivered at different infusion rates (IO,30 ,56 ng/Kg/mirj 10 min before and for 60 min after PAF injection. PMN and platelet counts (Malassez haemocytometer chamber:volume 1 mm3; depth 0.2 mm) were performed at different times (30 set; 1,2,3,4,5,10,30,60 min) after challenge. The folio= wing controls were performed: rabbits were injected (i) with the same volumes of saline-BSA, (ii) with 20 ug/Kg lyso-PAF or (iii) infused for 60 min with IO or 50 ng/Kg/ min of PGI2 prepared as described above. Light microscopy
studies
Kidney, spleen and liver specimens from rabbits which had died after PAF (3 pg/Kg) injection or sacrificed with i.v. overdose of sodium pentobarbital (Farmitalia,Milan,Italy) were fixed in 10% neutral buffered formalin. The respira= tory tissues (trachea, bronchi, lungs) were removed and the lungs inflated with 10% neutral buffered formalin from a 25 cm height, then immersed in 10% neutral buffe= red formalin overnight. Four micron-thick, paraffin-embed ded sections of th.e different organs were stained with he _matoxylin-eosin and periodic acid-Stiff (PAS). RESULTS Intravenous injection of synthetic PAF resulted in a dose -related fall of circulating platelets and PMN (Fig. 1). At a dose of 3 ug/Kg, synthetic PAF induced a fatal reac= tion which occurred 5-10 min after challenge (Fig. 1 A,C)_ Lower doses of PAF (1 ug/Kg) elicited an acute thrombocy= topenia (Fig. 1 B) as well as neutropenia (Fig. 1 D) that were reversible within 30 to 40 min after challenge. Simi larly acute, transient thrombocytopenia and neutropenia were observed after injection of native PAF (3,100 U.PAF/ Kg) (data not shown).. Pretreatment with PG12 (30 ng/Kg/min), infused IO min before challenge, prevented the fatal rea=
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(Fig. 1 A,C), ction induced by 3 pg/Kg of synthetic PAF the marked thrombocytopenia and neutropenia still occur= ring even at a higher infusion rate (50 ng/Kg/min). As shown in Fig. 1 B and D, 10 ng/Kg/min reduced to about 50 % the thrombocytopenia and neutropenia developping af= ter injection of 1 ug/Kg of synthetic PAF. At this rate of infusion, PGI2 completely prevented the thrombocytope= nia and neutropenia in response to native PAF (3,100 U. PAP/Kg) (data not shown). Only at a higher rate of infusion (50 ng/Kgjmin), PG12 was fully inhibitory even on the thrombocytopenia and neutropenia induced by 1 pg/Kg of syn thetic PAF. No change in the counts of circulating plate=lets and PMN was observed in rabbits injected with (i) sa= line-BSA, (ii) lyso-PAF or (iii) infused with PGI2 (from to 10 to 60 ng/Kg/min) for 60 min. Ae 50 ng/Kg/min, PG12 provoked marked hypotension. The neutropenia that occurred after challenge with synthe tic or native PAF was related to the massive sequestration of PMN in the microvasculature, primarily of the lungs (Fig. 2 A), but also present, even if to a lesser extent, in the kidneys,spleen and liver (not shown). No intravascu_ lar PMN aggregation was seen in the microvasculature in any of the above-cited organs from rabbits infused with PGI2 (50 ng/Kg/min) after challenge either with synthetic (1 Vg/Kg) or native PAF (3,100 U.PAF/Kg) (Fig_ 2 B), DISCUSSION The present study indgcates that infusion of PGI2 reduces, in a dose-dependent manner,the thrombocytopenia and the neutropenia that occur as the result of intravascular ag= gregation of platelets and PMN stimulated by PAF in the rabbit. The ability of prostaglandins to modulate both acute and chronic inflammatory responses has been well documentated in (20)s PG12 has been implicated as a chemiual mediator increasing vascular permeability (21) or as a modulator of the response of inflammatory cells (20). The effect of PG12 on inflammatory cells is related to the increase in intracellular CAMP levels (20,22)..Increases in intracellu lar CAMP inhibit not only in&tro platelet aggregation (22),but also PMN aggregation (6) and lysosomal enzyme re= lease (20).The in vitro response to PAF of platelets (17) and PMN (7), leading to their aggregation as well as to PMN chemotaxis and granule secretion, has been previously shown to be inhibited by PG12 as well as by other agents that increase intracellular CAMP levels.
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Fig. 1: A & B: platelet counts-in rabbits injected with synthetic PAF (A: 3 Ug/Kg; B: 1 ug/Kg). Intravascular effect of PGI infusion. A: M 50 ng/Kg/min;~30 ig/Kg/min;B: w 50 ng/Kg/min; IO ng/Kg/min. C & D: PMN countsHin ra V bits injected with syn thetic PAF (C: 3 pg/Kg; D: 1 ug/Kg).IntravZ scular effect of PGI infusion. C:M 55 ng/Kg/min;M30 ng 3Kg/mini D:W 50 ng/ Kg/min;~lO ng/Kg/min. Mean -+ 1 S.D. of five experiments.
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Fig. 2 A: Intravascular PMN aggregates (arrows) in the lungs after injection of l,ug/Kg of synthetic PAF.(x 400). B: Lung vessels were congested with erythrocytes and dilated but no PMN aggregate was seen af= ter PG12 infusion (50 ng/Kg/min).(x 250).
The results of the present study show that also in vivo infused at concentrations that increase intracellu= PGI lar2c&P levels (23)exerts an inhibitory effect on cellu= lar (platelets and PMN) responses to intravenously injec= ted synthetic or native PA!?.A markedly acute thrombocyto penia and neutropenia occur after PAP challenge. These 348
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intravascular effects have been related to the massive for mation of platelet and PMN aggregates that appear to embog lize in the microvasculature of several organs, namely of the lungs (7,16). When high doses of synthetic PAF ( 3 ug/ Kg) were injected, the irreversible and fatal reaction was prevented by the infusion of 30 ng/Kg/min of PGI2. However, even with an infusion rate of 50 ng/Kg/min, the thrombocy= topenia and neutropenia were only partially reduced. Lower doses of synthetic (1 pg/Kg) or native PAF (3,100 U.PAF/Kg) induced an acute and reversible fall in circulating plate= lets and PMN which was reduced to about one half by PGI at IO ng/Kg/min. Moreover, higher infusion rates (50 ng 3Kg/ min) could fully inhibit PAF-induced intravascular effects. The evidence suggesting that PG12 infusion is capable of antagonizing the intravascular consequencies of PAF inje= ction was further strengthened by the absence of PMN aggre gates in the microvasculature, particularly of the lungs. These studies favor the hypothesis that PG12 may play a ro le in modulating the response of PMN and platelts also iii those pathological conditions in which PAF has been impli= cated, REFERENCES 1) Hanahan, D.J., C.A.Demopoulos, J.Liehr and R.N. Pinckard Identification of platelet-activating factor isolated from rabbit basophils as acetyl glyceryl ether phospho= rylcholine. J.Biol.Chem. 255:5514, 1980. and D.J.Hanahan. Plate= 2) Demopoulos, C.A., R.N.Pinzrd let-activating factor. Evidence for l-0-alkyl-2-acetylsn-glyceryl-3-phosphorylcholine as the active component (A new class oflipid chemical mediators). J.Biol.Chem. 254:9355, 1979. 3) Gveniste, J., M.Tence, P.Varenne, J.Bidault, C.Boul= let and J.Polonsky. Semi-synthese et structure proposee du facteur activant les plaquettes (P.A.F.): P.A.F.-ace ther, un alkyl analogue de la lysophosphatidylcholine.C.R.Acad.Sci.Paris (D) 289:1017, 1979. 4) Benveniste, J., P.M.Henz and C.G.Cochrane. Leukocytedependent histamine release from rabbit platelets. The role of IgE, basophils and a platelet-activating factor J.Exp.Med. 136:1356, 1972. 5) Chignard, M., J.P.Le Couedic, M.Tence, B.B.Vargaftig and J.Benveniste. The role of platelet-activating fa= ctor in platelet aggregation. Nature 279:799, 1979. 6) Lynch, J.M., G.Z.Lotner, S..Betz and P.M.Henson. The re lease of platelet-activating factor by stimulated rab= bit neutrophil. J.Iriuttunol. 123:1219, 1979.
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7) Camus~i, C., C.Tetta, F.Bussolino, F.Callgaris-Cappio, R-Coda, C.Masera and G.P.Segoloni. Med'fators of immunecomplex-induced aggregation of polymorphonuclear neutro phils. II Platelet activating factor as the effector substance of immune-induced aggregation. Int.Archs Al= lergv Apol. Immunol. 64: 25,1981. 8) Mencia-Huerta,J.M.andJ.Benveniste. Platelet-activating factor and macrophages. I Evidence for the release from rat and mouse peritoneal macrophages and not from mono= cytes. Eur.J.Immunol, 9: 409, 1979. 9) Camussi G., M.AgliettaS R-Coda, F.Bussolino, W.Piacibel lo and C.Tetta. Release of platelet activating factor (PAF) and histamine. II The cellular origin of human PAF: monocytes, polymorphonuclear neutrophils and base= phils. Immunology 42: 191, 1981. lO)Shaw, J.O., R.N.Pizkard, K.S.Ferrigni, L.McManus and D.J. Hanahan. Activation of human neutrophils with 1-O hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphoryl= choline (platelet activating factor). J.Immunol. 123, 1847, 1981. ll)O'Flaherty, J.T., R.L. Wykle, C.H.Miller, J.C.Lewis, M. Waite, D.A.Bass, C.E.Mc Call and R. De Chatelet. I-O-al kyl-sn-glyceryl-3-phosphorylcholine. A novel class of neutrophil stimulants. Am.J.Pathol. 103: 70, 1981. 12)Humphrey, D.M., L.McManus, K.SatouchrD.J,Hanahan and R.N.Pinckard. Vasoactive properties of acetyl glyceryl ether phosphorylcholine and analogues. Lab.Invest. -46: 422, 1982. 13)Stimler, N.P., C.M.Bloor, T.E.Hugli, R.L.Wykle, C.E.Mc Call and J.T.O'Flaherty. Anaphylactic actions of plate let activating factor. Am.J.Pathol. 105: 64, 1981. 14)Pinckard, R.N., R.S.Farr and D.J.Hanahan. Physicochemi= Cal and functional identity of platelet activating fa= ctor (PAF) released in vivo during IgE anaphylaxis with PAF released in vitro from IgE-sensitized basophils. J.Immunol. 123: 1847, 1979. 15)Camussi G.,- C.Tetta, M.C.Deregibus, F.Bussolino, G.Sego loni and A.Vercellone. Platelet-activating factor (PAF) in experimentally-induced rabbit acute serum sickness: role of basophil-derived PAF in immune complex deposi= tion. J.Immunol. 128: 86, 1982. 16)McManus L., D.J.Hzhan, C.A.Demopoulos,and R,N.Pinckard Pathobiology of the intravenous injectionof acetyl gly= ceryl ether phosphorylcholine (AGEPC), a synthetic pla= telet-activating factor. J.Immunol. 124: 2919, 1980. 17)Bussolino, G. and G.Camussi. Effect ofprostacyclin on platelet activating factor-induced rabbit platelet ag= gregation. Prostaglandins -20: 781, 1980.
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PROSTAGLANDINS 18)Camussi G., C.Tetta, G.Segoloni, M.C.Deregibus,and F. Bussolino. Neutropenia induced by platelet-activating factor (PAF-acether) released from neutrophils: the i" hibitory effect of prostacylcin (PGI2). Agents and AC= tions 11: 550, 1981. 19)Benvenzte,J., J.P.Le Couedic, J.Polonsky and M.Ten&. Structural analysis of purified Platelet activating fa ctor by lipases. Nature 269: 170, 1977. 20)Weissman, G., J.E. Smolen and H. Korchak. Prostaglan= dins and Inflammation: receptor/cyclase coupling as an explanation of why PGE2 and PG12 inhihit functions of inflammatory cells. In Advances in Prostaglandin and Thromboxane Research (B.Samuelsson, P.W.Ramwell and R. Paoletti, Eds.), Raven Press, New York, 8: 1637, 1980. 21)Murata, S., W.C.Chang, S.Tsurufuji and ITMorita. The possible roles of prostacyclin (PGI ) and Thromboxanes in chronic inflammation. In Advance2 in Inflammation Research (G.Weissman et al., Eds), Raven Press, New York, 1: 436, 1979. 22)Tateson, J.E., S.Moncada and J.R.Vane. Effect of pro= stacyclin on cyclic AMP concentration in human plate= lets. Prostaglandins 13: 389, 1977. 23)Data, J., A.B.A. Moloz, M.M.Meinzinger and R.R.Gorman Intravenous infusion of Prostacyclin sodium in man: clL nical effects and influence on platelet adenosine di= phosphate sensitivity and adenosine 3':5'-cyclic mono= phosphate levels. Circulation 64: 3, 1981.
Editor: BrendanWhittle Received: 7-28-82 Accepted: l-7-83
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