The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

PAF antagonists inhibit monocrotaline-induced lung injury and pulmonary hypertension.

Lung platelet-activating factor (PAF) levels increased in some rats at 1-3 wk after subcutaneous injection of monocrotaline (MCT). We tested the effect of specific PAF antagonists, WEB 2086 and WEB 2170, on MCT-induced lung injury and subsequent pulmonary hypertension and right ventricular hypertrophy. Treatment with either agent decreased MCT-induced pulmonary hypertension and right ventricular hypertrophy at 3 wk after injection. Treatment with WEB 2170 reduced MCT-induced pulmonary vascular leak at 1 wk after injection, and WEB 2086-treatment exclusively during the early leak phase also decreased MCT-induced right ventricular hypertrophy at 3 wk. Treatment with WEB 2170 between the 3rd and 4th wk after MCT injection inhibited the progression of right ventricular hypertrophy at 4 wk. These results suggest that PAF contributes to the early pulmonary vascular leak, and this leak phase is important for the development of pulmonary hypertension and right ventricular hypertrophy in MCT-treated rats. Furthermore, it appears that PAF action contributes to the maintenance of a chronic inflammatory process that involves the synthesis of other lipid mediators (prostaglandins and leukotrienes) and leads to pulmonary hypertension. We conclude that PAF has a role in the MCT-induced inflammatory lung injury and pulmonary hypertension.     

Pro-inflammatory response of alveolar macrophages induced by sulphite: studies with lucigenin-dependent chemiluminescence

Alveolar macrophages (AM) were studied for their capability to release mediators involved in modulation of neutrophil (PMN) functions. Initial responses were induced by sulphite. Supernatants obtained from canine, human and rat AM pre-treated with sulphite in concentrations of 0.1–2 mmol/L enhanced the respiratory burst of canine, human and rat PMN, measured by lucigenin-dependent chemiluminescence (CL). This PMN-stimulating activity exhibited platelet-activating factor (PAF)-like properties, as indicated by desensitization of the PAF receptor, inhibition with PAF antagonists WEB 2086 and CV 3988, and the kinetic CL response like PAF after chloroform extraction of supernatants inhibitable by PAF antagonist CV 3988. These results indicate that AM are triggered by sulphite to release mediators that activate the respiratory burst of PMN, primarily via the PAF receptor. © 1998 John Wiley & Sons, Ltd.     

PAF antagonism modifies neuroprotective action of histone deacetylase and calcineurin phosphatase inhibitors in mice

To evaluate the hypothesis that platelet activating factor (PAF) antagonism may affect the functional recovery following the nerve injuries and also to evaluate the effect of PAF receptor antagonism on the neuroprotective effect of tacrolimus and sodium valproate, effect of PAF receptor antagonist, WEB2086 was evaluated in animal models of sciatic nerve crush and endothelin-1 induced focal cerebral ischemia. WEB2086, per se, while attenuating spontaneous sensory motor recovery after sciatic nerve crush, enhanced functional recovery after focal cerebral ischemia. WEB2086 also attenuated the neuroprotective effect of tacrolimus and sodium valproate subsequent to peripheral nerve injury, while it significantly improved the neuroprotective action of tacrolimus and sodium valproate following cerebral ischemia reperfusion injury. These results suggest that PAF receptor antagonists alone and in combination with tacrolimus/sodium valproate could be used in the treatment of cerebral ischemia reperfusion injuries however, their use following peripheral nerve injuries could be detrimental.     

Role of spermatozoal platelet-activating factor in fertilization

Platelet-activating factor (PAF), a potent lipid mediator of inflammation, has been shown to play a role in both the implantation and viability of mammalian embryos. We examined whether human and mouse spermatozoa release PAF during in vitro incubation and assessed the effect of exogenous PAF and the PAF receptor antagonist WEB 2086, a thieno-triazolodiazepine, on mouse in vitro fertilization (IVF) rate. PAF biological activity was detected in 11 samples of leukocyte-free, purified human spermatozoa (28 pg PAF/10(6) cells/24 hr) and 5 samples of epididymal mouse spermatozoa (7.8 pg PAF/10(6) cells/3 hr). Exogenous PAF (10(-8) and 10(-6) M) increased (p less than 0.01) the fertilization rate 2- and 3-fold, respectively of mouse oocytes by mouse epididymal spermatozoa. 10(-4) M PAF, however, reduced sperm motility and decreased (p less than 0.05) the fertilization rate. 10(-6) M WEB 2086, decreased IVF to approximately 50% of the control fertilization rate (42% vs. 89%). WEB 2086 treatment also promoted the attachment of supernumerary spermatozoa to both fertilized and unfertilized oocytes. The fertilization rate in the presence of WEB 2086 returned to control levels when zona-pellucida-free oocytes were employed, indicating that WEB 2086 did not interfere with the spermatozoal acrosome reaction. These data suggest that PAF, of spermatozoal origin, may be important in mammalian fertilization.     

WEB 2086, a platelet-activating factor antagonist, inhibits prophenoloxidase-activating system and hemocyte microaggregation reactions induced by Trypanosoma rangeli infection in Rhodnius prolixus hemolymph

The effects of the triazolodiazepine WEB 2086, a platelet-activating factor (PAF) antagonist, on hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, hemocoelic infection and mortality in fifth-instar larvae of Rhodnius prolixus inoculated with Trypanosoma rangeli were investigated. Hemocoelic injection of short T. rangeli epimastigotes (1x10(4) parasites/insect) in R. prolixus that were previously fed with blood containing 1muM of WEB 2086 resulted in (i) reduced hemocyte microaggregations as well as an attenuated proPO system in the hemolymph and (ii) greater parasitemia and mortality among the insects. In vitro assays using hemolymph from insects previously fed with blood containing WEB 2086 exhibited attenuated hemocyte microaggregations when T. rangeli was employed as the inducer of the reaction, and this effect was not counteracted by PAF treatment. In vitro assays using hemolymph from insects previously fed with blood, regardless of WEB 2086 presence increased the PO activity when incubated with the parasites. However, the PO activity was drastically inhibited when hemolymph from insects fed with blood, whether or not it contained WEB 2086, was incubated with fat body homogenates from insects fed with blood containing WEB 2086. The addition of PAF did not enhance the PO activity. These analyses did not reveal any PAF influence on WEB 2086 effects in the two defense reactions.     

Effects of platelet activating factor antagonists on arachidonic acid-induced platelet aggregation and TXA2 formation

The effects of the PAF receptor antagonists WEB 2086, WEB 2170, BN 50739 and BN 52021 on AA-induced platelet aggregation (PA) and TXA2 formation were investigated in comparison with the TXA2 synthetase inhibitor HOE 944 and the TXA2 receptor antagonist BM 13.177. All PAF antagonists tested were weak inhibitors of AA-induced PA and TXA2 formation (IC50 values between 80 and 2,737 mumol/l). HOE 944 was effective in concentrations 2-3 orders of magnitude lower than PAF antagonists in inhibiting TXA2 generation. These results imply that the inhibition of TXA2 formation is of minor relevance for the actions of the investigated PAF antagonists in AA-induced PA.     

Inhibition of immune complex-induced enteropathy by three different platelet-activating factor receptor antagonists

We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine of the Sprague-Dawley rat. In this study, we tested the effect of three different platelet-activating factor (PAF) receptor antagonists on the development of lesions. L-652, 731, a synthetic derivative of kadsurenone (at doses of 1.3–2.7 mg/kg), SRI 63–675, a substituted quinolinium compound (6.7–15 mg/kg), and WEB 2086, a thienotriazolodiazepine (5–25 mg/kg) were each capable of completely or partially inhibiting IC-induced enteropathy in the majority of animals tested. Pretreatment with WEB 2086 prevented IC-induced hemoconcentration but not neutropenia. The antagonists did not lower the level of blood complement nor interfere with the fall in complement induced by administration of IC. The ability of PAF receptor antagonists to completely or partially inhibit IC-induced small intestinal lesions suggests that endogenous PAF is a major mediator of IC-induced enteropathy.     

Roles of leukotrienes in non-allergic and allergic inflammatory models

Leukotrines (LT) C₄, D₄ and ₄ show potent activities in plasma exudation and vasoconstriction, whereas LTB₄ induces chemotaxis and chemokinesis in very low concentrations (1). Thus, LTs may be involved in inflammatory reactions, particularly in allergic inflammation.The present experiments were performed to detect leukotrienes in inflammatory exudates of rat carrageenin pleurisy as a non-allergic model, compared with those of two types of allergic rat pleurisy.     

Inhibition of immune complex-induced enteropathy by three different platelet-activating factor receptor antagonists.

We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine of the Sprague-Dawley rat. In this study, we tested the effect of three different platelet-activating factor (PAF) receptor antagonists on the development of lesions. L-652,731, a synthetic derivative of kadsurenone (at doses of 1.3-2.7 mg/kg), SRI 63-675, a substituted quinolinium compound (6.7-15 mg/kg), and WEB 2086, a thienotriazolodiazepine (5-25 mg/kg) were each capable of completely or partially inhibiting IC-induced enteropathy in the majority of animals tested. Pretreatment with WEB 2086 prevented IC-induced hemoconcentration but not neutropenia. The antagonists did not lower the level of blood complement nor interfere with the fall in complement induced by administration of IC. The ability of PAF receptor antagonists to completely or partially inhibit IC-induced small intestinal lesions suggests that endogenous PAF is a major mediator of IC-induced enteropathy.     

Platelet-Activating Factor (PAF) Modulates Peritoneal Mouse Macrophage Infection by Leishmania amazonensis

The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis–macrophage infection was inhibited by PMA in all systems tested. Received: 27 September 2000 / Accepted: 15 December 2000     

A PAF antagonist blocks antigen-induced airway hyperresponsiveness and inflammation in sheep

We studied the effects of WEB-2086, a specific antagonist of platelet-activating factor (PAF), on the development of antigen-induced airway hyperresponsiveness and inflammation in sheep (n = 8). For these studies, airway responsiveness was determined from slopes of carbachol dose-response curves (DRC) performed at base line (prechallenge) and 2 h after Ascaris suum antigen challenges in the following three protocols: 1) antigen challenge alone (control trial), 2) WEB-2086 (1 mg/kg iv) given 30 min before antigen challenge (WEB pretreatment), and 3) WEB-2086 given 2 h after antigen challenge, immediately before the postchallenge DRC (WEB posttreatment). Airway inflammation was assessed by bronchoalveolar lavage (BAL) before antigen challenge and after the postchallenge DRC for each trial. A. suum challenge resulted in acute increases in specific lung resistance that were not different among the three trials. Antigen challenge (control trial) caused a 93% increase (P less than 0.05) in the slope of the carbachol DRC when compared with the prechallenge value. WEB pretreatment (1 mg/kg) reduced (P less than 0.05) this antigen-induced hyperresponsiveness, whereas pretreatment with a 3-mg/kg dose completely prevented it. WEB posttreatment was ineffective in blocking this hyperresponsiveness. BAL neutrophils increased after antigen challenge in the control trial and when WEB-2086 was given after antigen challenge (P less than 0.05). Pretreatment with WEB-2086 (1 or 3 mg/kg) prevented this neutrophilia. This study provides indirect evidence for antigen-induced PAF release in vivo and for a role of endogenous PAF in the modulation of airway responsiveness and airway inflammation after antigen-induced bronchoconstriction in sheep.     

Bone‐marrow‐derived mesenchymal stem cells inhibit gastric aspiration lung injury and inflammation in rats

Gastric aspiration lung injury is one of the most common clinical events. This study investigated the effects of bone‐marrow‐derived mesenchymal stem cells (BMSCs) on combined acid plus small non‐acidified particle (CASP)‐induced aspiration lung injury. Enhanced green fluorescent protein (EGFP⁺) or EGFP^? BMSCs or 15d‐PGJ₂ were injected via the tail vein into rats immediately after CASP‐induced aspiration lung injury. Pathological changes in lung tissues, blood gas analysis, the wet/dry weight ratio (W/D) of the lung, levels of total proteins and number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) were determined. The cytokine levels were measured using ELISA. Protein expression was determined by Western blot. Bone‐marrow‐derived mesenchymal stem cells treatment significantly reduced alveolar oedema, exudation and lung inflammation; increased the arterial partial pressure of oxygen; and decreased the W/D of the lung, the levels of total proteins and the number of total cells and neutrophils in BALF in the rats with CASP‐induced lung injury. Bone‐marrow‐derived mesenchymal stem cells treatment decreased the levels of tumour necrosis factor‐α and Cytokine‐induced neutrophil chemoattractant (CINC)‐1 and the expression of p‐p65 and increased the levels of interleukin‐10 and 15d‐PGJ₂ and the expression of peroxisome proliferator‐activated receptor (PPAR)‐γ in the lung tissue in CASP‐induced rats. Tumour necrosis factor‐α stimulated BMSCs to secrete 15d‐PGJ₂. A tracking experiment showed that EGFP⁺ BMSCs were able to migrate to local lung tissues. Treatment with 15d‐PGJ₂ also significantly inhibited CASP‐induced lung inflammation and the production of pro‐inflammatory cytokines. Our results show that BMSCs can protect lung tissues from gastric aspiration injury and inhibit lung inflammation in rats. A beneficial effect might be achieved through BMSC‐derived 15d‐PGJ₂ activation of the PPAR‐γ receptor, reducing the production of proinflammatory cytokines.     

Control of local blood flow in pulmonary inflammation: role for neutrophils, PAF, and thromboxane

The intrapulmonary instillation of C5a results in a local inflammatory response that, in this site, is accompanied by a decrease in local blood flow. Reversal of this decrease by vasodilators or the thromboxane synthesis inhibitor dazmegral has been shown to result in enhanced lung inflammation. In the present study the mechanisms underlying the decrease in flow in pulmonary inflammation were investigated in the rabbit in vivo and in the isolated blood-perfused rabbit lung. In vivo, the decrease in local blood flow was shown to be dependent on circulating neutrophils. In the isolated blood-perfused lung, inflammation induced by airway instillation of C5a was similar histologically to that seen in vivo and was also accompanied by a decrease in local blood flow. The decrease in blood flow appeared to require circulating neutrophils and was prevented by dazmegral and the platelet-activating factor (PAF) antagonists WEB 2086 and L-659,989. Furthermore, no decrease occurred in aspirin-treated lungs perfused with normal blood, suggesting that the source of thromboxane was lung rather than circulating cells. The decrease in blood flow in inflammation did not appear to be a consequence of hypoxic vasoconstriction. Inflammation in the guinea pig lung was also accompanied by a decrease in local blood flow and was also prevented by dazmegral and PAF antagonists. We conclude that local inflammation in the lung is accompanied by a decrease in blood flow that involves neutrophils and the lipid mediators PAF and thromboxane. We suggest that this form of negative feedback by the neutrophil serves to control the inflammatory response.     

Leukotriene receptor antagonists,LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors

In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB₄ (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A₂ (sPLA₂-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA₂-IB triggered the influx of Ca²⁺ into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB₄ before neuronal cell death. Neither administration of LTB₄ nor cysteinyl LTs such as LTC₄, LTD₄ and LTE₄ killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA₂-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca²⁺ into neurons but also rescues neurons from the sPLA₂-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA₂-IB-induced Ca²⁺ influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.     

Role of the p38 mitogen‐activated protein kinase signaling pathway in estrogen‐mediated protection following flap ischemia‐reperfusion injury

Ischemia‐reperfusion (I/R) injury often occurs during skin flap transplantation and results in tissue damage. Although estrogen treatment significantly alleviates this I/R injury‐induced damage, the detailed molecular mechanism is not clear. In this study, a superficial epigastric artery flap I/R injury model was created in adult Wistar rats. Severe necrosis was observed in skin tissue after I/R injury. Histological examination of skin tissue revealed that I/R injury damages skin structure and results in neutrophil infiltration. Inflammation‐related parameters, including neutrophil count, tumor necrosis factor‐α, and interleukin‐10 levels, were increased due to I/R injury. These pathological phenomena were reduced by estradiol treatment. Further investigation found that I/R injury triggers the p38 mitogen‐activated protein kinase (p38‐MAPK) pathway. The expression levels of p38‐MAPK and phosphorylated p38‐MAPK were increased after I/R injury. Estradiol increased the expression level of MAPK phosphatase‐2, a putative phosphatase of p38, and reduced the levels of p38‐MAPK and phosphorylated p38‐MAPK. These results suggest that estradiol can improve skin flap survival, possibly by inhibiting neutrophil infiltration and the expression of p38‐MAPK. This study provides an explanation for how estrogen alleviates I/R injury‐induced damage that occurs during skin flap transplantation. In a rat pathological model, I/R injury leads to skin necrosis, skin structure damage, neutrophil infiltration, and inflammatory cytokine secretion, which are probably downstream effects of activation of the p38‐MAPK pathway. On the other hand, estradiol treatment triggers the expression of MAPK phosphatase‐2, a putative phosphatase of p38‐MAPK, and reduced all examined pathological phenomena. Therefore, estrogen may reduce the deleterious effect of I/R injury on skin flap transplantation through modulating the p38‐MAPK pathway.     

The pharmacological profile of ovalbumin-induced paw oedema in rats

Rats are commonly used in anaphylaxis models, mainly in intestinal anaphylaxis. Hypersensitivity mechanisms are complex and they are not clearly defined. Ovalbumin (OVA) is commonly used for studies on the hypersensitivity mechanism. However, the potential pro-inflammatory mediators induced by this antigen in the model of paw oedema in immunized rats are still not completely understood. This work examines the pharmacological modulation of several mediators involved in rat hind paw immune oedema induced by OVA. Wistar rats were previously immunized (14-18 days) with OVA (30 microg, intraperitoneally) or sham-sensitized with aluminum hydroxide (control). The paw volumes were measured before the antigenic stimuli and 1, 2, 3 and 4 h after the intraplantar injection of OVA (10 microg/paw). Subcutaneous injection of dexamethasone, diphenhydramine, cyproheptadine, chlorpromazine or methysergide significantly inhibited (p < 0.05) the allergic paw oedema. The dual inhibitor of cyclooxygenase and lipoxygenase (NDGA), the cyclooxygenase inhibitor (indomethacin), the lipoxygenase inhibitor (MK-886), the PAF antagonist (WEB 2086), the mast cell stabilizer (ketotifen), and the anti-histamine (meclizine) did not inhibit the immune oedema. In addition, thalidomide and pentoxifylline (anti-tumour necrosis factor drugs) were ineffective against OVA-induced oedema. The fact that indomethacin, MK-886, NDGA and WEB 2086 are unable to inhibit this allergic oedema indicates that the dexamethasone action seems not to be via phospholipase A2, but possibly due to the synthesis and/or the inhibitory activity of cytokines. The paw oedema inhibition by diphenhydramine, but not by meclizine, may suggest a different mechanism, which is independent of the effect of histamine. These data indicate that allergic oedema is more sensitive to anti-serotonin drugs, mainly anti-5-HT2, suggesting that the principal mediator of this inflammatory response is serotonin.     

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.     

Neutrophil migration across monolayers of cytokine-prestimulated endothelial cells: a role for platelet-activating factor and IL-8

In a previous study we observed that neutrophils respond with a rapid rise in [Ca2+]i during adherence to cytokine-activated endothelial cells (EC), caused by EC membrane-associated platelet-activating factor (PAF). In the present study, we investigated whether this form of PAF was important in neutrophil adherence and migration across monolayers of rIL-1 beta- or rTNF alpha-prestimulated EC. PAF receptor antagonists prevented neutrophil migration across cytokine-pretreated EC by approximately 60% (P less than 0.005) without interfering with the process of adherence. The antagonists WEB 2086 and L-652,731 had no effect on neutrophil migration across resting EC induced by formylmethionyl-leucyl-phenylalanine (FMLP). A murine anti-IL-8 antiserum was found to also partially inhibit the neutrophil transmigration across cytokine-activated EC. When the anti-IL-8 antiserum was used in combination with a PAF receptor antagonist, neutrophil migration across cytokine-pretreated monolayers of EC was completely prevented. During transmigration, LAM-1 and CD44 on the neutrophils were down-modulated; both WEB 2086 and anti-IL-8 antiserum partially prevented this down-modulation caused by cytokine-prestimulated EC. Our results indicate that human neutrophils are activated and guided by EC-associated PAF and EC-derived IL-8 during the in vitro diapedesis in between cytokine-stimulated EC.