The accumulation of platelet activating factor in the injured cornea may be interrelated with the synthesis of lipoxygenase products

The rabbit cornea accumulates platelet activating factor (PAF) three hrs after alkali burn. PAF was isolated by HPLC and assayed by platelet aggregation. This bioactivity was blocked by the PAF receptor antagonists BN 52021 and alprazolam. Added PAF increases the chemiluminescence response of the cornea in vitro and BN 52021 inhibits this effect. In vivo experiments show that the synthesis of 5-HETE and 12-HETE is inhibited by the PAF antagonist BN 52021. It is concluded that a metabolic interrelationship may exist between the PAF cycle and the lipoxygenation of arachidonic acid, and that drugs that affect these lipid mediators may modulate the inflammatory response of the anterior segment of the eye.     

Some mechanisms involved in the prostaglandin E2 immunosuppressive effect in (CBA x C57BL)F1 mice in vivo

The cellular mechanisms involved in the immunosuppressive effect of prostaglandin E2 (PGE2) multiple injections into (CBA x C57Bl)F1 mice in vivo have been studied. PGE2 injection increases the induction of specific T-suppressors. In addition, there is a decrease in macrophage phagocytic activity and in the phagocytosis index, apparently mediated by Fc gamma receptors (Fc gamma R) and not by the macrophage complement receptor (C3R). The induction of antibody synthesis by using "immune" macrophages injected into a syngeneic recipient results in considerable decrease in the accumulation of antibody-forming cells if the macrophage donor has been pretreated with exogenous PGE2 in comparison with untreated controls. These cellular mechanisms are possibly one part of the diverse way in which PGE2 exerts an immunosuppressive effect in vivo and contributes to humoral immune response suppression.     

Role of platelet-activating factor (PAF) in the initiation of the decidual reaction in the rat

Injection of PAF into the left uterine horn induced a dose-dependent decidua-like reaction in the pseudopregnant rat. This reaction was maximal when PAF was injected at Day 5 of pseudopregnancy and was blocked by the specific PAF antagonist, BN 52021. BN 52021 did not interfere with the decidual reaction induced by prostaglandin E-2 or insertion of a cotton thread in the uterine horn. In contrast, a decidua-like reaction was not evoked by the inactive lyso-PAF, demonstrating the specificity of the action of PAF. The decidua-like reaction induced by PAF involves the generation of cyclooxygenase metabolites of arachidonic acid since it was inhibited by indomethacin. The histological alterations induced by PAF were similar to those observed after embryo implantation, strengthening the postulate for a role of the autacoid in the early stages of pregnancy.     

Modulation of eosinophil recruitment in the rat by the platelet-activating factor (PAF) antagonist, BN 52021, the somatostatin analog, BIM 23014, and by cyclosporin A

The factors responsible for in vivo eosinophil recruitment are poorly defined, although T-lymphocytes appear to be involved in the etiology of eosinophilia. In order to clarify this relationship, we studied the modulation of eosinophil mobilization in the rat after immune challenge, by chronic treatment with the PAF-antagonist, BN 52021, the somatostatin analog, BIM 23014 and with Cyclosporin A (CsA). In rats made hypereosinophilic by pretreatment with cyclophosphamide or sephadex, a significant increase of the eosinophil count in blood and peritoneal fluid was induced by anaphylactic reaction. CsA totally abolished both hypereosinophilia and peritoneal eosinophil infiltration. BIM 23014 also, significantly reduced the circulating eosinophils (-68%, p less than 0.001) and cell infiltration (-86%, p less than 0.05). In contrast, BN 52021 decreased peritoneal eosinophil recruitment, while having relatively little effect on circulating cells. CsA and somatostatin are known to affect T-cell proliferation, and as T-cells are involved in the differentiation of hematopoietic cells into eosinophils, these drugs could decrease eosinophil availability for recruitment. In contrast, the PAF antagonist may act by inhibiting PAF-induced eosinophil chemotaxis, providing a more specific inhibition of this process than that exerted by CsA, BIM 23014 and other immunosuppressive agents.     

Effect of long-term treatment with platelet-activating factor on IL-1 and IL-2 production by rat spleen cells

To study the effect of the in vivo administration of platelet-activating factor (PAF) on cytokine production, alzet minipumps loaded with the mediator or solvent alone were connected to the jugular vein and placed under the skin of Sprague-Dawley rats. Over 7 days the animals received total doses of 0.5, 1, 4.5, 9, or 28 micrograms PAF or the solvent alone. The spleen mononuclear cells isolated from Ficoll gradients and the adherent cell fraction were separated before determination of basal and mitogen-stimulated IL-1 and IL-2 production, respectively. Adherent splenocytes from rats having received 28 micrograms PAF exhibited a decreased capability to produce IL-1, as compared to those from vehicle-treated animals. In contrast, adherent splenocytes from rats having received 9 and 4.5 micrograms PAF yielded higher amounts of released and cell-associated IL-1 activity upon LPS stimulation, as compared to those from solvent-treated animals. The PAF antagonist, BN 52021, given orally (5 mg/kg, twice a day throughout the experiments) inhibited the in vivo effect of 28 micrograms PAF. Statistically significant 144 +/- 43% (p less than 0.001, n = 5) and 73 +/- 33%, (p less than 0.01, n = 3) increases in IL-2 production were observed when whole spleen mononuclear cells from rats administered with 1 and 4.5 micrograms PAF, respectively, were stimulated with Con A. BN 52021 markedly inhibited the in vivo effect of 1 microgram PAF on the IL-2 release. Our study demonstrates that PAF can modulate immune functions in vivo and suggests that the specific PAF antagonist, BN 52021, may be used as an immunomodulatory agent.     

Effects of platelet activating factor (PAF) and its receptor antagonist BN 52021 on isolated perfused guinea-pig heart

The cardiac effects of PAF and its antagonist BN 52021 have been investigated on the isolated perfused guinea-pig heart maintained at a constant hydrostatic perfusion pressure of 80 cm water. In this model, PAF (1 x 10(-11) to 1 x 10(-7) moles) induced a dose-dependent coronary vasoconstriction, a decrease in heart rate and a fall in contractile force. BN 52021 (1 x 10(-6) to 2 x 10(-4) M) dose-dependently inhibited the vasospasm induced by PAF (1 x 10(-10) moles). BN 52021 also antagonized the decrease in coronary flow and heart rate, but not that of contractile force induced by a high dose of PAF (1 x 10(-7) moles). This dose of PAF also significantly (p less than 0.001) provoked a marked release of TxB2 but did not alter the generation of 6 Keto PGF1 alpha, PGE2 or LTC4. The PAF-induced increase in TxB2 release was completely abolished by BN 52021.     

Antagonism of PAF-induced death in mice

The ability of three platelet activating factor (PAF) antagonists, BN52021, L652, 731 and 48740RP, and the leukotriene antagonist FPL55712 to block iv PAF-induced death was tested in mice. PAF-induced sudden death was been previously characterized as a model of systemic anaphylaxis and circulatory shock related its hypotensive actions. Of the drugs, BN52021 and L652, 731 provided dose-dependent protection against PAF toxicity, whereas the others had no effect. 48740RP was, however active against PAF-induced rabbit platelet aggregation. BN52021 was inactive in three other mouse sudden death models in which arachidonic acid, U46619 or collagen combined with epinephrine is injected iv to provoke a thrombotic/ischemic sudden death. In contrast, the TXA₂ antagonist SQ29548 inhibited the acute toxicity of two of these latter challenges (arachidonic acid and thromboxane agonist U46619), but was inactive against PAF lethality.These results suggest that PAF toxicity in mice is a specific model for PAF agonism, and is not mediated by TXA₂ or peptido-leukotrienes. Further, PAF-induced mortality should be a simple and useful technique for testing potential PAF antagonists for activity by various routes of administration.     

Free Radicals Scavengers Attenuate Platelet-Activating Factor (PAF)-And Endotoxin-Induced Intestinal Myoelectric Disturbances in Rats

Pretreatment with radical scavengers significantly reduced the intestinal myoelectric disturbances following either E. coli endotoxin or platelet-activating factor (PAF) injection in the rat indicating that free radicals might be involved in the intestinal motor alterations observed in endotoxin shock and that PAF acts partially via free radical production. Moreover, dimethylsulfoxide (DMSO) was found to be more effective in inhibiting the endoxotin-induced intestinal motor alterations, than superoxide dismutase (SOD) and allopurinol. BN 52021, a specific PAF antagonist, was able to reduce the effects of endotoxin on intestinal motility, However, when BN 52021 was combined with free radical scavengers, no additive effect was observed. It is concluded that free radicals involved in endotoxin-induced intestinal motility alterations are at least in part produced in response to PAF.     

Effect of cyclosporin A and the platelet-activating factor (PAF) antagonist, BN 52021, on PAF- and antigen- induced bronchoconstriction in the guinea-pig

The effects of the PAF antagonist, BN 52021, and cyclosporin A (CsA), either alone or in combination, on PAF- and antigen- induced bronchoconstriction were investigated in control and passively sensitized guinea-pigs, respectively. Although single administration of CsA alone has no effect on the PAF-induced bronchoconstriction, a marked inhibition of this phenomenon is observed when the drug is given along with an inactive dose of BN 52021. This effect of the association of the two drugs on the bronchoconstriction is also related to an action on the PAF-induced alterations in the number of leukocytes and platelets. In addition, administration of CsA for 48 hrs, which alone does not influence PAF-induced bronchoconstriction, markedly increases the inhibition evoked by BN 52021. Although bolus administration of CsA has no effect on the antigen -induced bronchoconstriction, a marked inhibition of this phenomenon is observed when the drug is given for 2 days. This inhibition by CsA is not further enhanced when the animals are also treated with BN 52021. These results strengthen the hypothesis that PAF and the immune system are involved in the regulation of bronchopulmonary reactions.     

Trypanosoma gambiense: immunosuppression and polyclonal B-cell activation in mice

The relationship between the suppression of antibody response and polyclonal B-cell activation was studied in mice treated with a cell homogenate of Trypanosoma gambiense. The cell homogenate injection in mice caused a progressive increase in splenic background plaque-forming cell response to sheep erythrocyte. In the mice with markedly increased background plaque-forming cell response, the different reactivity in the primary antibody response to sheep erythrocytes was observed between the intraperitoneal and intravenous immunization with sheep erythrocytes. The intraperitoneal immunization of mice with sheep erythrocytes strongly suppressed the antibody response, while the intravenous immunization with sheep erythrocytes led to an enhancement of the antibody response. The intraperitoneal injection of silica particles, a toxic agent to macrophages, 30 min before intraperitoneal immunization with sheep erythrocytes abolished the suppression of the antibody response completely. In addition, restoration of the suppressed antibody response was found in mice immunized intraperitoneally with a high dose of sheep erythrocytes. It appears that the suppression of antibody response is not attributable to polyclonal B-cell activation, and is associated with the elevation of the phagocytic activity of peritoneal macrophages.     

Release of eicosanoids in rat peritoneal cavity stimulated with platelet-activating factor (PAF). Effect of the PAF-antagonist BN-52021

Platelet activating factor (PAF; 10 micrograms) was injected in the peritoneal cavity of rats in the absence or presence of the PAF antagonist BN-52021 (5 mg/kg). Thirty min later, the peritoneal cavity was washed with 3 ml of saline, the fluid was collected and the concentrations of selected eicosanoids were measured using novel enzyme immunoassays. PAF increased by 2.9, 2.8 and 1.7 fold the levels of thromboxane B2, prostaglandin E2 and leukotriene B4 respectively in the peritoneal fluid. The stimulatory effects of PAF was reduced by 42, 51, and 86% for thromboxane B2, prostaglandin E2 and leukotriene B4 respectively by the specific PAF antagonist. These results confirm the presence of specific PAF receptors in tissues and/or cells of rat peritoneal cavity and underline the complex interactions between PAF and eicosanoids.     

Characterization of specific binding sites for PAF in the iris and ciliary body of rabbit

The protective effect exerted by BN 52021 a specific PAF-receptor antagonist in experimentally induced ocular inflammatory disorders led us to investigate the possible presence of specific receptors for PAF in rabbit iris and ciliary body. Two classes of PAF binding sites were found in isolated iris and ciliary process of pigmented rabbit eyes: a high affinity site Kd1 congruent to 4.9 +/- 0.47 nM, Bmax1 congruent to 3.17 +/- 0.50 pmoles/mg protein, a low affinity sites Kd2 congruent to 11.6 +/- 0.33 nM, Bmax2 congruent to 12.46 +/- 2.3 pmoles/mg protein for iris. The specific binding was not affected by lyso-PAF the biologically inactive precursor and metabolite of PAF, up to 10(-6) M; inhibition by unlabelled PAF demonstrated a biphasic curve partially antagonized by BN 52021. The present results demonstrate the presence of specific binding sites for PAF in rabbit eyes which could mediate the action of this mediator in eye inflammatory processes and explain the protective effect observed with BN 52021.     

Failure of platelet-activating factor (PAF-acether) to induce decidualization in mice and failure of antagonists of PAF to inhibit implantation

The possible role of platelet-activating factor (PAF) in the uterine responses associated with implantation was investigated. Attempts to trigger a decidual cell response in the uteri of hormonally sensitized, ovariectomized mice by instilling PAF-acether (1-1000 ng) intraluminally were unsuccessful. The effect of PAF antagonists on implantation was investigated in females ovariectomized on Day 3 of pregnancy and treated with progesterone. Implantation was induced in these females by injection of 10 ng oestradiol-17 beta on Day 8. Hourly intraperitoneal injections of three PAF antagonists (WEB 2086, CV 3988 and BN 52021 at doses of 1.2-1.4 mg/kg) given over a 24-h period starting 1 h before the injection of oestradiol-17 beta had no significant effect on the occurrence of implantation sites. Intraluminal injection of WEB 2086 (15 micrograms) or BN 52021 (5 micrograms) either 3 h before or 6 h after the nidatory oestradiol also had no significant inhibitory effect on implantation. SRI 63-441 given once daily over the first 4 days of pregnancy at a dose of 40 micrograms/30 g body weight had no inhibitory effect on the establishment of pregnancy. These results are not consistent with a critical role for PAF in implantation in mice.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Pharmacologic profile of BN 50739, a new PAF antagonist in vitro and in vivo

The effect of a new PAF antagonist BN 50739 was studied on PAF-induced [3H]-serotonin release from washed rabbit platelets in vitro and on PAF-induced hypotension in vivo. BN 50739 competitively inhibited PAF-induced [3H]-serotonin release from the platelets in a dose-dependent manner. In the presence of 4, 10 and 50 nM of BN 50739, the concentration of PAF inducing 50% maximal [3H]-serotonin release from the platelets (EC50) increased from 2.15 nM to 5.10, 45.10 and 900 nM, respectively. The IC50 of BN 50739 for PAF (10 nM) induced [3H]-serotonin release was 3.67 nM. Under the same experimental condition, the IC50s of BN 50726, BN 50730, BN 50741, WEB 2086, SRI 63-441 and BN 52021 were 5.40, 4.61, 6.88, 5.98, 40.90 nM and 14.90 microM, respectively. PAF-induced hypotension in conscious rats was also inhibited dose-dependently by i.p. pretreatment of BN 50739 (3 and 10 mg/kg). PAF-induced hypotension was diminished both in magnitude and duration in rats pretreated with BN 50739. These data taken together indicate that BN 50739 is a most potent PAF antagonist in vitro and in vivo.     

Effect of Platelet-activating Factor on in vitro and in vivo Interleukin-6 Production

The aim of the present study was to investigate the possible effect of platelet-activating factor (PAF), by comparison with interleukin-1beta and polyriboinositic/polyribocytidylic (poly I-C) acid, on IL-6 production by L 929 mouse fibroblasts. At concentrations above 1 muM PAF, the production of IL-6 by mouse fibroblasts was enhanced in a dose dependent fashion. At 5 muM PAF, the peak increase (60.1 +/- 19.4 U/ml) was similar to that induced by 50 mug/ml poly I-C (60.0 +/- 35.0 U/ml) and higher than the one evoked by 100 U/ml IL-1beta (3.8 +/- 1.8 U/ml). The increase of 11-6 activity induced by 5 muM PAF was maximal after a 22 h incubation period with L 929 cells. Lyso-PAF (5 muM) also increased IL-6 activity from fibroblasts to a similar extent compared with 5 muM PAF. In addition, the IL-6 activity induced by 5 muM PAF was still observed when the specific PAF antagonist, BN 52021 (10 muM), was added to the incubation medium of L 929 cells. The result suggests that the production of IL-6 by L 929 cells evoked by PAF in vitro is not receptor mediated. The in vivo effect of PAF on IL-6 production was also investigated in the rat. Two hours after intravenous injection of PAF (2 to 4 mug/kg), a dramatic increase of IL-6 activity in rat serum was observed, this effect being dose dependent. The increase of IL-6 induced by 3 mug/kg PAF was not observed when the animals were treated with the PAF antagonist, BN 52021 (1 to 60 mg/kg0. These results demonstrate that PAF modulates IL-6 production and that the in vivo effect is receptor mediated.     

Inhibition by BN 52021 (ginkgolide B) of the binding of [3H]-platelet-activating factor to human neutrophil granulocytes

The inhibitory effect of BN 52021, a specific antagonist of platelet-activating factor (PAF) on PAF-induced activation of human polymorphonuclear granulocytes (PMNL) and on the binding of [3H]-PAF to neutrophils were examined. BN 52021 over the range of 10(-9)-10(-4) M inhibited PAF-induced degranulation and superoxide production of PMNLs in a dose-dependent manner with Kd values of 0.6 +/- 0.1 x 10(-6) M and 0.4 +/- 0.1 x 10(-6) M, respectively. BN 52021 (up to 1 mM) did not show any agonistic activity and it did not affect neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine or leukotriene B4. The Ki value of BN 52021 for the specific binding of [3H]-PAF to neutrophils was 1.3 +/- 0.5 x 10(-6) M versus a Ki of 1.1 +/- 0.3 x 10(-7) M for PAF itself. BN 52021 did not affect metabolism of PAF by PMNL. These studies indicate that BN 52021 inhibits neutrophil responses to PAF by inhibiting binding of PAF to its specific PMNL receptor.     

Involvement of platelet-activating factor (PAF) in endotoxin-induced intestinal motor disturbances in rats

Intestinal myoelectrical activity was investigated in conscious fasted rats chronically implanted with Nichrome electrodes in the duodeno-jejunum. Motility of the small intestine was characterized by the presence of migrating myoelectric complex (MMC) occurring regularly at 16.2 +/- 5.8 minute intervals. Intravenous administration of endotoxin (E. coli S.0111:B4) at a dose of 50 micrograms/kg increased the interval between MMC to 112.6 +/- 26.8 min, the duration of these effects being dose-related between 10 to 100 micrograms/kg. Such a typical myoelectrical alteration, corresponding to rapidly propagated groups of spike bursts, was mimicked by the IP administration of PAF at doses of 10 to 50 micrograms/kg. Previous administration of BN 52021, a specific PAF antagonist at a dose of 50 mg/kg abolished the motor alterations induced by IP injection of PAF (25 micrograms/kg) and significantly (p less than 0.01) reduced by 61.2% those induced by IV endotoxin (50 micrograms/kg). Indomethacin (10 mg/kg IP) as well as SC 19220 (5 mg/kg IV), a PGE2 antagonist, injected prior to endotoxin (50 micrograms/kg IV) or PAF (25 micrograms/kg IP) also reduced significantly (p less than 0.01) the duration of MMC inhibition. It is concluded that endogenous release of PAF is partly responsible for the intestinal motor alterations induced by endotoxin; these effects, strongly reduced after treatment with BN 52021, are also mediated through the release of prostaglandins.     

Increased levels of platelet-activating factor in blood following intestinal ischemia in the dog

The possible role of platelet-activating factor (PAF) in superior mesenteric artery occlusion induced circulatory collapse was studied in anesthetized dogs. PAF was measured by platelet aggregation assay. Identity of PAF-like product in blood was ascertained by thin layer chromatography, high pressure liquid chromatography and alkaline treatment. Low amount of PAF was detected in the mesenteric blood under normal conditions, during reperfusion PAF levels were significantly higher. Pretreatment of the animals with BN 52021, a specific PAF receptor antagonist abolished the fall in mean arterial pressure and the rise in hematocrit due to ischemia/reperfusion. These findings suggest that PAF may play an important role in mesenteric ischemia-induced circulatory collapse.