Danielle Joseph

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

1 The effect of the chemotactic peptide, N‐fonnyl‐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 μm 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 μm FMLP respectively) and by a smaller decrease in neutrophil counts (25.0 ± 1.9 and 12.9 ± 1.7% at 1 and 0.01 μm FMLP respectively). 3 When purified platelets and neutrophils were co‐incubated, the addition of 0.1 μm induced a marked aggregation (50.0 ±1.6 vs. 19.5 ±1.6% of light transmission, n = 8, P < 0.001), ATP secretion (8.4 ± 1.0 vs. 0.1 ± 0.1 nmol ml−1, n = 6, P < 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 μm aspirin or indomethacin and by about 70% with 100 μm BW 755C. Two Paf‐acether antagonists, BN 52021 (100 μm) and WEB 2086 (1 μm) 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 μm 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.

Upregulation by glucocorticoids of responses to eosinopoietic cytokines in bone-marrow from normal and allergic mice

Since the production of eosinopoietic cytokines (GM‐CSF, IL‐3, IL‐5) is inhibited by glucocorticoids, while responsiveness to these cytokines is enhanced in bone‐marrow of allergic mice, we studied the ability of glucocorticoids to modulate murine bone‐marrow eosinopoiesis. Progenitor (semi‐solid) and/or precursor (liquid) cultures were established from bone‐marrow of: (a) normal mice; (b) ovalbumin‐sensitized and challenged mice or (c) dexamethasone (1–5 mg kg−1) injected mice. Cultures were established with GM‐CSF (2 ng ml−1) or IL‐5 (1 ng ml−1), respectively, alone or associated with dexamethasone, hydrocortisone or corticosterone. Total myeloid colony numbers, frequency and size of eosinophil colonies, and numbers of eosinophil‐peroxidase‐positive cells were determined at day 7. In BALB/c mice, dexamethasone (10−7 M) increased GM‐CSF‐stimulated myeloid colony formation (P=0.01), as well as the frequency (P=0.01) and size (P<0.01) of eosinophil colonies. Dexamethasone (10−7 M) alone had no effect. Dexamethasone (10−7–10−10 M) increased (P<0.002) eosinophil precursor responses to IL‐5. Potentiation by dexamethasone was still detectable: (a) on low density, immature, nonadherent BALB/c bone‐marrow cells, (b) on bone‐marrow from other strains, and (c) on cells from allergic mice. Hydrocortisone and corticosterone had similar effects. Dexamethasone administered in vivo, 24 h before bone‐marrow harvest, increased subsequent progenitor responses to GM‐CSF (P=0.001) and precursor responses to IL‐5 (P<0.001). These effects were blocked by RU 486 (20 mg kg−1, orally, 2 h before dexamethasone, or added in vitro at 10 μM, P<0.001). Glucocorticoids, acting in vivo or in vitro, through glucocorticoid receptors, enhance bone‐marrow eosinopoiesis in naïve and allergic mice.

I - Isolation and electron microscope studies of a potent platelet-aggregating glycoprotein from the venom of Crotalus durissus cascavella

A potent acid-soluble platelet-aggregating glycoprotein was purified from the venom of Crotalus durissus cascavella by molecular sieve chromatography on Sephadex G-75 and by adsorption onto Sepharose 4B gels at acidic pH. This new protein with an apparent Mr of 300,000 at acidic pH and containing a low amount of sugars is non-toxic for mice. Electron microscope studies showed that the platelet-aggregating glycoprotein appeared as regular rosettes of 150 A in diameter at acidic pH and underwent polymerization in rod-like particles in the presence of sodium chloride. This glycoprotein, probably hydrophobic, is dissociated into an active molecular form whose apparent Mr was 144,000; however, it is believed to still be a not totally dissociated molecule.

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 chemoatt‐ractants 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 × 106/guinea‐pig; 12 ± 1%) than in immunized, saline‐challenged animals (3.0 ± 0.7 × 106/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.

Structural requirements for preventing the aspirin- and the arachidonate-induced inactivation of platelet cyclo-oxygenase: additional evidence for distinct enzymatic sites.

2-Hydroxybenzoic acid (salicylic acid) prevents the inhibition by aspirin (ASA) of platelet aggregation and of the generation of thromboxane A2 from arachidonic acid (AA). We studied the ability of 2-hydroxybenzoic acid analogues to block ASA and to prevent the platelet desensitization due to a first exposure to AA. Inactivation was prevented when exposure to AA was done in the presence of reversible inhibitors of cyclo-oxygenase. Phenol, methyl salicylate and L8027 were thus strong inhibitors of AA-induced platelet activation and desensitization. The minimal structural requirement for inhibition of thromboxane A2 generation from AA was a phenol group as benzoic acid was fully inactive. 2-Hydroxybenzoic acid, and to some extent 2,6-dihydroxybenzoic acid were effective against ASA, the most active substances being methyl salicylate and L8027. The minimal structural requirement for blocking ASA was that 2-hydroxybenzoic acid, 2-methoxybenzoic acid should be devoid of activity, which highlights the fact that the hydroxyl group must be available. Our work favours the hypothesis that non-steroidal anti-inflammatory drugs react with two sites of cyclo-oxygenase, which were named the supplementary and the catalytic sites. The interaction of 2-hydroxybenzoic acid and of its analogues with the supplementary site is necessary but not sufficient for the efficacy of these compounds as cyclo-oxygenase inhibitors. The intensity of interaction with the supplementary site and the modifications of the catalytic site determine the potency of these compounds as cyclo-oxygenase inhibitors. For preventing ASA inactivation, an interaction with the supplementary sites is always necessary, but furthermore an appropriate group, preferentially in the position ortho to the hydroxyl, is needed.

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)

Murine myeloid progenitor responses to GM-CSF and eosinophil precursor responses to IL-5 represent distinct targets for downmodulation by prostaglandin E2

Because Prostaglandin E2 (PGE2) and dibutiryl cyclic AMP (dbcAMP) modulate the production and effects of haemopoietic cytokines in allergy, we examined their ability to modulate responses of myeloid progenitors to GM‐CSF, and of eosinophil precursors to IL‐5. The ability of PGE2, dbcAMP, rolipram, forskolin, dbcGMP and PGD2, to modulate the responses to GM‐CSF and IL‐5 in colony formation (progenitor) and eosinophil differentiation (precursor) assays using bone‐marrow from nonsensitized or from intranasally‐challenged, ovalbumin‐sensitized mice of five strains was studied. PGE2 (10−7 M) inhibited GM‐CSF‐stimulated colony formation in bone‐marrow from BP‐2 mice. This effect was duplicated by dbcAMP (0.3–1×10−6 M), Rolipram (10−5 M) and forskolin (3×10−5 M), but not Prostaglandin D2 (10−6 M). Inhibition affected similarly all myeloid colony types. Progenitors from sensitized and challenged BP‐2 mice were also inhibited by PGE2 and cyclic AMP. PGE2 inhibited progenitors from C57BL/10, CBA/J and A/J, but not BALB/c mice. However, BALB/c progenitors were sensitive to dbcAMP and Forskolin (10−4 M). In contrast, in precursor assays, PGE2 (10−7–10−9 M) blocked responses to IL‐5 in bone‐marrow from BP‐2 and BALB/c mice, either naïve or sensitized and challenged, to a similar extent. PGD2 (10−6 M) was ineffective, as was PGE2 (10−7 M), if added after 48 h of culture. In conclusion, PGE2 inhibits the responses of bone‐marrow myeloid progenitors to GM‐CSF and of eosinophil precursors to IL‐5, in naïve or ovalbumin sensitized and challenged mice. These effects are duplicated by cyclic AMP‐elevating agents. In the BALB/c strain, the resistance of progenitors, but not precursors, to PGE2 inhibition, indicates these developmental stages are separate targets for PGE2 modulation.

II―subunit structure of a potent platelet-activating glycoprotein isolated from the venom of Crotalus durissus cascavella

The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acid-soluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, alpha and beta, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the alpha and beta subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an alpha 3 beta 3 complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets were the tetrameric and dimeric states of the molecule respectively.

Ectopic lung transplantation induces the accumulation of eosinophil progenitors in the recipients' lungs through an allergen- and interleukin-5-dependent mechanism.

Background Airway challenge of ovalbumin‐sensitized mice induces intrapulmonary accumulation of eosinophil progenitors.

Allergenic sensitization prevents upregulation of haemopoiesis by cyclo-oxygenase inhibitors in mice.

We evaluated whether immunization affects bone‐marrow responses to indomethacin, because allergenic sensitization and challenge upregulate responses to haemopoietic cytokines (including IL‐5‐driven eosinopoiesis) in murine bone‐marrow, while indomethacin upregulates haemopoiesis and protects bone‐marrow from radiation damage. Progenitor (semi‐solid) and/or precursor (liquid) cultures were established from bone‐marrow of: (a) normal mice; (b) ovalbumin‐sensitized mice, with or without intranasal challenge. Cultures were established with GM‐CSF (2 ng ml−1) or IL‐5 (1 ng ml−1), respectively, alone or associated with indomethacin (10−7 – 10−11 M) or aspirin (10−7 – 10−8 M). Total myeloid colony numbers and numbers of eosinophil‐peroxidase‐positive cells were determined at day 7. In naïve BALB/c mice, indomethacin (10−7 – 10−9 M) increased GM‐CSF‐stimulated myeloid colony formation (P=0.003 and P=0.009, respectively). In contrast, it had no effect on bone‐marrow of ovalbumin‐sensitized and challenged mice. Indomethacin (10−7 – 10−9 M) also increased eosinophil precursor responses to IL‐5 in bone‐marrow of naïve (P<0.001 and P=0.002 respectively), but not sensitized‐challenged mice. Aspirin (10−7 M) had similar effects, equally abolished by sensitization. Enhancement of haemopoiesis by indomethacin required adherent cells from naïve bone‐marrow. Nonadherent cells responded to IL‐5 but not to indomethacin. Indomethacin was effective on bone‐marrow from sham‐sensitized, ovalbumin‐challenged, but not from sensitized, saline‐challenged mice. Plasma transfer from immune mice abolished eosinophil precursor responses to indomethacin in bone‐marrow of naïve recipients. This was not prevented by previous removal of antibody from immune plasma. COX inhibitors enhance haemopoiesis in naïve but not allergic mice. Responsiveness to indomethacin can be abolished either by active sensitization or by immune plasma transfer. Specific antibody is not involved.