Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein.

The highly conserved amino acid sequence ELDKWA of HIV-1 gp41 has been inserted into Escherichia coli MalE protein which had been shown to be an adequate carrier to present foreign epitopes to the immune system. We first investigated whether eight different permissive sites of MalE are able to tolerate an insertion of 7-50 residues encoding this epitope. Secondly, antigenicity of the epitope inserted in MalE protein was estimated from monoclonal antibody 2F5 binding analysis using the BIAcore(R) technology and its immunogenicity in mice was measured as the ability of hybrid proteins to elicit antibodies against a synthetic peptide containing this epitope. This study revealed a good correlation between the antigenicity of the inserted epitope and its immunogenicity. Increasing the length of the inserted epitope, as well as inserting multicopies of this epitope increased both its antigenicity and immunogenicity. However, none of the MalE hybrid proteins tested induced anti-HIV-1 neutralizing antibodies. This study strongly suggests that the capacity of the 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented.

Fine specificity of anti-V3 antibodies induced in chimpanzees by HIV candidate vaccines.

The fine specificity of the anti-V3 antibody responses induced in chimpanzees immunized by various human immunodeficiency type 1 (HIV-1) candidate vaccines and challenged by heterologous strains of HIV-1 was analyzed by enzyme-linked immunosorbent assay (ELISA) and Pepscan epitope mapping. Two chimpanzees immunized with the recombinant canarypox virus ALVAC-HIV (vCP125) expressing gp160MN and boosted with purified gp160MN/LAI alone, then with both immunogens in combination, were not protected against challenge with HIV-1 SF2. Their sera mainly recognized one epitope of the V3 loop, located in the NH2-terminal half. By contrast, immunization of two other chimpanzees with purified gp160MN/LAI and boosting with a synthetic V3MN peptide elicited a strong anti-V3 antibody response with a broader specificity directed against multiple epitopes all along the V3 loop. These chimpanzees were protected against infection by HIV-1 SF2. However, when these two chimpanzees were challenged later with a HIV-1 clade E strain virus, they became infected. We failed to detect any reactivity with the peptide of the ectodomain of gp41 of sera harvested after immunization with the various immunogens or after challenge with HIV-1 SF2 or HIV-1 90CR402. These results demonstrated that anti-V3 antibodies with a restricted fine specificity were induced in chimpanzees immunized with gp160 purified or expressed by recombinant canarypox confirming our previous results obtained in three different species (human, guinea pig and, macaque). In contrast, a boost with the V3 peptide broadened antibody responses, suggesting that the mode of presentation of the V3 loop to the immune system strongly influences the epitope specificity of the resulting antibody response.

Restricted specificity of anti-V3 antibodies induced in humans by HIV candidate vaccines.

We analyzed the fine specificity of anti-V3 antibodies elicited in three different species (human, guinea pig, and macaque) by various HIV candidate vaccines. Following immunization with recombinant canarypox virus expressing gp160MN or with recombinant gp160MN/LAI, this antibody response was shown to be directed against the NH2-terminal region of the V3 loop. Although this response was increased by a prime-boost regimen using immunization with canarypox expressing gp160 followed by an rgp160 boost, its specificity remained restricted mainly to the recognition of this region of the V3 loop. Pepscan analysis of sera confirmed the results obtained by ELISA and allowed the definition of an immunodominant common binding site for these sera located within the sequence NKRKRIHIGPGR. In contrast to these results, a boost with the V3 peptide was shown to broaden the antibody response and pepscan analysis showed that sera from individuals boosted with the V3 synthetic peptide recognize determinants all along the V3 loop. Similar fine specificity of anti-V3 antibodies was obtained in human, guinea pig, and macaque following immunization by a prime-boost regimen using canarypox recombinants expressing gp160 or gp120 and purified rgp160. In contrast, a V3 synthetic peptide boost stimulated the production of antibodies that recognize multiple epitopes within the V3 loop. Because the induction of antibodies that recognize multiple sites in the V3 loop could be of major importance to neutralize different HIV isolates, these results may have implications for the design and selection of HIV candidate vaccines.

Role of interleukin-5 in enhanced migration of eosinophils from airways of immunized guinea-pigs.

1. Platelet activating factor (PAF), leukotriene B4 (LTB4) and interleukin-5 (IL-5) are potent chemoattractants for guinea-pig eosinophils, which may be involved in eosinophil recruitment and up-regulation in allergic diseases. Eosinophils from the bronchoalveolar lavage fluid (BALF) of ovalbumin-sensitized guinea-pigs were collected 24 h after antigen provocation and migration induced by PAF, LTB4 and rhIL-5 was studied. 2. Total BALF content and distribution of eosinophils were greater in immunized, ovalbumin-challenged guinea-pigs (5.0 +/- 0.8 x 10(6)/guinea-pig; 12 +/- 1%) than in immunized, saline-challenged animals (3.0 +/- 0.7 x 10(6)/guinea-pig; 7 +/- 1%). 3. The chemoattraction of eosinophils isolated on a metrizamide gradient was studied in micro-Boyden chambers, results being expressed as the number of migrating cells (mean +/- s.e. mean). PAF and LTB4-induced migration of eosinophils from immunized and OA-challenged guinea-pigs were significantly enhanced, as compared to immunized and saline-challenged animals (170 +/- 36 vs 35 +/- 9 migrating eosinophils for 10 nM PAF; 271 +/- 60 vs 110 +/- 19 for 1 nM LTB4). 4. The IL-5 antibody TRFK-5, in vivo, reduced eosinophil recruitment in BALF of antigen-challenged immunized animals as well as the enhanced responsiveness of eosinophils from the challenged animals, suggesting a role for IL-5 in the priming of eosinophils in vivo. 5. In contrast to TRFK-5, nedocromil sodium reduced to a similar extent eosinophil, macrophage and lymphocyte recruitment into the BALF of antigen-challenged, but failed to down-regulate the enhanced responsiveness of eosinophils from the challenged animals. 6. The increased eosinophil content in lungs from antigen-challenged guinea-pigs is thus selectively reduced by the anti-IL-5 antibody, which also attenuates the concomitant enhancement of the eosinophil responsiveness, supporting the concept that IL-5 is essential for recruitment and priming of eosinophils in vivo. In contrast, nedocromil sodium reduced non-selectively the total cell recruitment to the airways,but failed to attenuate the enhanced responsiveness of those eosinophils which migrated, indicating that its effects involve a different target.

Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc gamma receptor.

Induction by gamma interferon (IFN-gamma) of the gene encoding the human high-affinity Fc gamma receptor (Fc gamma R1) in myeloid cells requires an IFN-gamma response region (GRR) and a myeloid cell-activating transcription element (MATE). GRR and MATE interact with factors to form, respectively, an IFN-gamma-activating complex (GIRE-BP), depending on the phosphorylation of the 91-kDa protein (subunit of ISGF3), and a cell-type-specific complex (MATE-BP). Although GIRE-BP is detected in cells of different origins after IFN-gamma treatment, the presence of MATE-BP was found to be restricted to B- and myeloid cell lines. Sequence analysis of a cDNA encoding a polypeptide recognizing specifically the MATE motif led to the identification of this product as the proto-oncogene PU.1/Spi-1, a transcriptional activator expressed in myeloid and B cells. Expression of this factor in nonhematopoietic cells allowed IFN-gamma-induced expression of a reporter gene under control of the GRR and MATE sequences. The presence of these motifs in other gene promoters indicates that the binding of PU.1/Spi-1 and IFN regulatory proteins to their respective motifs could be part of a general mechanism leading to cell-type-restricted and IFN-induced gene expression.

Formation of LTB4 by fMLP-stimulated alveolar macrophages accounts for eosinophil migration in vitro.

Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 +/- 11.9, number of migrating eosinophils [mean +/- SEM], n = 17) as compared to unstimulated macrophages (4.8 +/- 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 microM), a specific PAF antagonist, and with indomethacin (10 microM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 microM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 +/- 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 +/- 5.4% of desensitization toward LTB4 and 83.1 +/- 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.

Activation of guinea pig eosinophils by human recombinant IL-5. Selective priming to platelet-activating factor-acether and interference of its antagonists.

The potential role of platelet-activating factor (PAF)-acether and of IL-5 as an eosinophil-proliferating, activating, and/or recruiting mediator in asthma led us to study the effects of human (h) rIL-5 (hrIL-5) and PAF-acether, alone or combined, on isolated guinea pig eosinophils. Two populations of eosinophils were separated from peritoneal lavages of polymyxin B-treated guinea pigs upon a discontinuous metrizamide gradient: one of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 16). Chemotactic activity was evaluated on a micro-Boyden chamber, results being expressed as the number of migrating eosinophils (mean +/- SEM) at 40 microns through a cellulose nitrate filter (3 microns pore size) in the presence of the agonist or of the solvent alone. hrIL-5 dose-dependently stimulated normodense eosinophil chemotaxis, reaching a peak at 500 ng/ml (98 +/- 21 migrating eosinophils, n = 5, p less than 0.05). These eosinophils also responded to PAF-acether and to LTB4 and not to FMLP, hrTNF alpha, and LPS. Eosinophil preincubation with hrIL-5 increased significantly the migration by PAF-acether (173 +/- 23 migrating eosinophils with PAF-acether 10 nM after preincubation with hrIL-5 500 ng/ml vs 69 +/- 10 after preincubation with buffer alone, p less than 0.01) and failed to enhance migration by LTB4 or to uncover an activity for FMLP. Migration by PAF-acether was antagonized when the cells were preincubated with the antagonists BN 52021 and WEB 2086, which also inhibited migration by hrIL-5. Eosinophils were auto-desensitized by and to PAF-acether or LTB4, but were not cross-desensitized to each other. Eosinophils desensitized to PAF-acether failed to migrate with hrIL-5, but those desensitized to LTB4 responded to hrIL-5 as controls. hrIL-5 failed to induce the elevation of intracellular free calcium concentration and superoxide anion generation from basal values, whereas preincubation of eosinophils with hrIL-5 induced a significant increase in the rise in intracellular free calcium concentration and in superoxide anion generation by 10 nM PAF-acether but not by LTB4. In conclusion, the in vivo eosinophil migration in allergy may involve hrIL-5, particularly associated to PAF-acether.

LTB4, a potent chemotactic factor for purified guinea-pig eosinophils: interference of PAF-acether antagonists.

Two populations of eosinophils were separated upon a discontinuous metrizamide gradient from peritoneal lavages of polymyxin B-treated guinea-pigs. One population was of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 26). Responses to chemotactic stimuli were studied using a micro-Boyden chamber, results being expressed as the chemotactic index (CI, mean +/- S.E.M.), i.e. the ratio between the number of eosinophils migrating at 40 microns through a cellulose nitrate filter in the presence of the agonist and the number of cells migrating in the presence of the solvent alone. The normal density eosinophils responded more to LTB4 (CI = 19.4 +/- 4.6 with LTB4 10(-8) M; P less than 0.05; n = 9) than to PAF-acether (CI = 6.2 +/- 1.4 with PAF-acether 10(-8) M; P less than 0.05; n = 20). By contrast, low density eosinophils responded less intensely to LTB4 (CI = 7.6 +/- 1.8 with LTB4 10(-8) M; P less than 0.01; n = 6) and to PAF-acether (CI = 2.4 +/- 0.4 with PAF-acether 10(-8) M; P less than 0.05; n = 14). Guinea-pig eosinophils failed to migrate in response to FMLP and lyso PAF-acether.(ABSTRACT TRUNCATED AT 250 WORDS)

Cooperation between platelets and neutrophils for paf-acether (platelet-activating factor) formation.

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 10(6)/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 +/- 2.5 ng/ml paf-acether. Human washed platelets (3 x 10(8)/ml) stimulated with thrombin (1 IU/ml) formed 0.60 +/- 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 +/- 4.25 ng/ml, n = 6, P less than .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 +/- 3.81 ng/ml paf-acether vs. 5.30 +/- 2.23, P less than .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 +/- 2.26 ng/ml paf-acether vs. 5.76 +/- 0.38, P less than .05, n = 3). These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.

Pharmacological profile of 48740 R.P., a PAF-acether antagonist.

The pyrrolo-thiazole derivative 48740 R.P. inhibited the platelet-activating factor (PAF-acether)-induced aggregation of human and rabbit platelets and was poorly effective against ADP- and arachidonic acid-induced platelet aggregation. 48740 R.P. prevented the activation of guinea-pig alveolar macrophages by PAF-acether, and the PAF-acether-induced thromboxane B2 production from guinea-pig lungs. 48740 R.P. (3 mg/kg i.v.) antagonized selectively in anaesthetized guinea-pigs the bronchoconstriction due to PAF-acether without affecting that due to acetylcholine, histamine, serotonin, thromboxane A2 analogue U-46,619 and arachidonic acid. A higher dose of 48740 R.P. (10 mg/kg i.v.) was required to block the thrombocytopenia and the leucopenia induced by PAF-acether in the propranolol-treated guinea-pigs. 48740 R.P. (30 mg/kg i.v.) antagonized the PAF-acether effects when bronchoconstriction was induced by aerosolized PAF-acether. 48740 R.P. is a selective antagonist of PAF-acether under in vitro and in vivo conditions.

Platelet-leukocyte interaction: activation of rabbit platelets by FMLP-stimulated neutrophils.

1 The effect of the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) was studied on cells in whole rabbit blood or on a mixture of purified rabbit platelets and neutrophils. 2 In blood, FMLP triggered cell aggregation (measured by electrical impedance) which was dependent upon the concentration of FMLP (9.9 +/- 0.7 and 5.2 +/- 1.2 ohms at 1 and 0.01 microM FMLP respectively). This aggregation was accompanied by a strong decrease in platelet counts (54.6 +/- 6.0 and 45.6 +/- 3.8% for 1 and 0.01 microM FMLP respectively) and by a smaller decrease in neutrophil counts (25.0 +/- 1.9 and 12.9 +/- 1.7% at 1 and 0.01 microM FMLP respectively). 3 When purified platelets and neutrophils were co-incubated, the addition of 0.1 microM induced a marked aggregation (50.0 +/- 1.6 vs. 19.5 +/- 1.6% of light transmission, n = 8, P less than 0.001), ATP secretion (8.4 +/- 1.0 vs. 0.1 +/- 0.1 nmol ml-1, n = 6, P less than 0.001) and a decrease in platelet counts. FMLP induced aggregation of purified neutrophils and release of lysozyme but lacked direct platelet-stimulating effects. The release of lactate dehydrogenase, a cytoplasmic marker and lysozyme were unchanged under the interaction conditions. 4 Platelet activation was reduced by about 30% with 100 microM aspirin or indomethacin and by about 70% with 100 microM BW 755C. Two Paf-acether antagonists, BN 52021 (100 microM) and WEB 2086 (1 microM) suppressed platelet activation by 70-80%. 5 The supernatant of FMLP-stimulated neutrophils induced platelet activation only when bovine serum albumin was present. Rabbit neutrophils stimulated in the presence of serum albumin by 1 microM FMLP formed 2 nM Paf-acether of which half was released to the extracellular medium. 6 Our results indicate that the stimulation of neutrophils by FMLP induces platelet activation in whole blood and on isolated cells and that both arachidonic acid-metabolites and Paf-acether participate in platelet activation.

Exercise- and allergen-induced asthma do not change the production of Paf-acether by neutrophils and platelets.

Paf-acether, whose role has been suggested in asthma, is a mediator released by stimulated neutrophils, platelets and other cells. Neutrophils and platelets are activated in vivo during exercise or allergen-induced asthma. Upon in vitro stimulation, macrophages from mice treated with an inflammatory stimulus, such as thioglycoccollate, release less paf-acether than macrophages from non-treated mice. We hypothesized that upon in vitro activation platelets and neutrophils should produce less paf-acether after exercise- or allergen-induced asthma. To test this hypothesis, we measured the production of paf-acether by neutrophils and platelets obtained before, 15 and 75 min after exercise in seven normal subjects and five asthmatic subjects with exercise-induced asthma, and in five other asthmatic subjects after specific challenge with Dermatophagoides Pteronyssinus. Purified neutrophils and washed platelets were incubated independently for 10 min at 37 degrees C with no specific activator, with a platelet activator (thrombin, 1 IU.ml-1), a neutrophil activator (opsonized zymosan, 1 mg.ml-1), and both together. We found no significant difference between asthmatic and normal subjects in the amount of paf-acether synthesized by platelets or neutrophils and no fall in the production of paf-acether after exercise- or allergen-induced asthma. However, our method may lack sensitivity in detecting partial activation of these cells and is based on the assumption that changes in peripheral blood cells are representative of changes of these cells in lungs.

Structure-activity relationship in PAF-acether. 3. Hydrophobic contribution to agonistic activity.

The synthesis of some selected PAF-acether homologues with an alkoxy-chain length from C1 to C20 in position 1 is described. All agonist activities are closely correlated among themselves and with the calculated fatty-chain hydrophobicity. After a discussion on recent published results and comparison with our data, we conclude that the ether oxide function is absolutely essential at the glycerol 1-position for potent agonist activity and that potency correlates well with hydrophobicity parameters. We indicate the importance of steric and configurational constraints.

Effects of PAF-acether and structural analogues on platelet activation and bronchoconstriction in guinea-pigs.

PAF-acether (platelet-activating factor) (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine) induces platelet-dependent bronchoconstriction in the guinea-pig which correlates with its in vivo thrombocytopenic effect. We investigated the influence of modifications of the polar head group in position 3 of the glycerol skeleton of PAF-acether on guinea-pig platelet activation and bronchoconstriction. PAF-acether itself induced concentration-dependent platelet activation (EC50 for aggregation = 0.41 nM and EC20 for secretion of ATP = 0.56 nM). The 3-phosphoryl-N-methyl-morpholino ethanol analogue was slightly more active than PAF-acether and the 3-phosphoryl-N-methyl-piperidinium ethanol, 3-phopshoryl-(N-methyl-piperidino-3') methanol and 3-phosphoryl-(N-methyl-hydroxy-4') piperidine analogues were equieffective to PAF-acether in activating platelets. The 3-phosphoryl-piperidino ethanol analogue was 8 times less active than PAF-acether; the 3-phosphoryl-morpholino ethanol analogue and the 1-O-octadecyl-2-O-acetyl-3-O-[trimethyl-ammonio)-propyl) glycerol were inactive up to 1 microM. Our data show that the choline head group is not a compulsory requirement for activity. When injected i.v. to the propranolol-treated guinea-pigs, the platelet-activating analogues also induced bronchoconstriction. Two PAF-acether antagonists, compounds 48740 RP and BN 52021, inhibited PAF-acether-induced platelet activation when added to PRP at the final concentration of 0.1 mM (aggregation inhibited by 91 +/- 4 and by 94 +/- 3% respect.; secretion inhibited by 80 +/- 12 and 79 +/- 10% respectively, mean +/- S.E.M., n = 4). Both antagonists also suppressed platelet activation and in vivo bronchoconstriction, thrombocytopenia, leukopenia and hypotension induced by PAF-acether and the various analogues. Our results indicate that PAF-acether and the analogues studied trigger platelet activation and the consequent bronchoconstriction through mechanisms which share sensitivity to same antagonists.

Transient activation of the acetyltransferase necessary for paf-acether biosynthesis in thrombin-activated platelets.

Thrombin-activated platelets formed paf-acether (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine), a molecule susceptible to play a role in haemostasis and thrombosis, and its deacetylated analogue, lyso paf-acether, a biologically inactive molecule. We presently show the presence in human and rabbit platelet lysates of an acetyltransferase which transfers the acetyl moiety of acetyl-coenzyme A (acetyl-CoA) onto synthetic lyso paf-acether, yielding the fully active paf-acether molecule. Under our optimal standard conditions, 0.36 +/- 0.23 nmol paf-acether/10 min/mg proteins was formed by the acetyltransferase from resting human platelets. Upon thrombin stimulation, the acetyltransferase activity doubled within 30 s, reaching a maximum at 2 min (1.17 +/- 0.31 nmol paf-acether/10 min/mg proteins) and decreased progressively. Similar results were obtained using rabbit platelets. In addition we demonstrated that the activation and deactivation of the acetyltransferase correlated with the kinetics of paf-acether formation by thrombin-activated rabbit platelets. It is hypothesized that this enzyme may represent one of the regulating mechanism of paf-acether formation by platelets.

[A rapid method: the 100-hue test]. / Méthode rapide--test 100 hue.

A new and fast method for scoring and assessing the 100-Hue test (Farnsworth-Munsell) is reported. This method allows to simultaneously transcribe graphically the responses of the patient. This method reduces drastically the duration of examination and calculation, without modification of the principle of the 100-Hue Test. The qualitative and graphic validity, the statistical validity are analysed from 103 pathological subjects and 53 normal subjects. The two methods (Farnsworth-Kinnear and fast method) are examined comparatively.

Paf-acether formation and arachidonic acid freeing from platelet ether-linked glyceryl-phosphorylcholine.

Ether-linked glyceryl-phosphorylcholines (GPC) are potential precursors for paf-acether biosynthesis. We show that such phospholipids are present in rabbit platelets in sufficient amounts to insure lyso paf-acether and paf-acether formation. Indeed, upon stimulation, platelets formed lyso paf-acether and paf-acether in amounts representing 10% and 0.2% of the total amounts of ether-linked GPC. Following preincubation with [3H]paf-acether, ether-linked GPC labelled at position 1 of the glycerol were detected in platelets. They were hydrolysed, and gave birth to lyso paf-acether upon platelet activation. Platelet ether-linked GPC labelled at position 2 with [3H]arachidonic acid (AA) were also hydrolyzed under the same experimental conditions. These data indicate that in rabbit platelets these phospholipids are precursors for paf-acether and AA, two mediators of platelet activation.

Effect of the Ca2+ antagonist nifedipine on the release of platelet-activating factor (PAF-acether), slow-reacting substance and beta-glucuronidase from human neutrophils.

Ca2+ antagonists such as nifedipine (Nif) inhibit processes that depend on extracellular Ca2+ in many muscular and secretory cells. The effect of Nif on mediator release and Ca2+ uptake by human polymorphonuclear neutrophils (PMN) has been investigated. Nif caused a concentration-dependent inhibition of the Ca2+ ionophore-induced release of platelet-activating factor (PAF-acether), slow-reacting substance (SRS) and to a lesser degree beta-glucuronidase (beta-glu). Nif inhibited the synthesis of PAF-acether rather than its release. Increasing Ca2+ concentration in the bathing medium from 1.3 to 2.8 mM completely reversed the effect of Nif on PAF-acether secretion. Nif at 1 and 5 microM reduced PMN45Ca2+ uptake induced by the Ca2+ ionophore A 23187. These results indicate that the inhibition by Nif of mediator release depends probably on the Ca2+-antagonistic property of the drug. A preliminary ex vivo study suggests that this inhibitory effect on neutrophil functions exists during therapeutic use.