IgE-mediated release of leukotriene C4, chondroitin sulfate E proteoglycan, beta-hexosaminidase, and histamine from cultured bone marrow-derived mouse mast cells.

Mouse bone marrow-derived mast cells differentiated in vitro and sensitized with monoclonal IgE respond to antigen-initiated activation with the release of histamine, beta-hexosaminidase, chondroitin sulfate E proteoglycan, and leukotriene C4 (LTC4). The chondroitin sulfate E nature of the glycosaminoglycan side chain was established by demonstrating that the chondroitinase ABC disaccharide digestion products were composed of equal quantities of 4-sulfated and 4,6-disulfated N-acetyl-galactosamine. The single immunoreactive sulfidopeptide leukotriene, released and quantitated with a class-specific antibody, was identified as LTC4 by its retention time on reverse-phase high-performance liquid chromatography and by its specific spasmogenic activity on the guinea pig ileum. The release of the preformed mediators, as well as of LTC4, was related in a dose-response fashion to the concentration of monoclonal IgE used during the sensitization step and to the concentration of specific antigen used to initiate the activation-secretion response. The optimal concentrations of IgE for sensitization and of antigen for challenge were the same for the release of preformed mediators and of LTC4. In addition, the time courses of their release were superimposable, with a plateau at 5 min after antigen challenge. The release of three preformed mediators and of LTC4 after fixation of IgE, washing of the sensitized cells, and antigen challenge unequivocally indicates a bone marrow-derived mast cell origin for these products. Linear regression analyses of the net percent release of beta-hexosaminidase to histamine and of 35S-chondroitin sulfate E to beta-hexosaminidase yielded straight lines that intersected at the origin, which indicates that the three preformed mediators are localized in the secretory granules of the bone marrow-derived mast cells. The concomitant generation of 23 ng of LTC4/10(6) sensitized bone marrow-derived mast cells represents the first example of IgE-dependent release of substantial amounts of LTC4, a component of slow reacting substance of anaphylaxis, from a mast cell population of greater than 95% purity. The IgE-dependent generation of LTC4, rather than prostaglandin D2, by the chondroitin sulfate E proteoglycan-containing bone marrow-derived mast cells contrasts with the predominant generation of prostaglandin D2 by heparin proteoglycan-containing mast cells. These differences together support the existence of two phenotypically different mast cell subclasses.

Release of a slow-reacting substance from rabbit platelets.

Washed rabbit platelets stimulated with platelet-activating factor, thrombin, or arachidonic acid, released a slow-reacting substance (SRS), whereas platelets aggregated by adenosine diphosphate did not. Production of platelet-derived SRS was neither affected by indomethacin nor aspirin but was reduced by large doses of eicosatetraynoic acid, an inhibitor of the cyclo-oxygenase and lipoxygenase. L-cysteine enhanced markedly the release of SRS from platelets. This SRS activity, which was antagonized by FPL 55712 and inactivated by arylsulfatase, followed the same elution pattern on Amberlite, silicic acid, and reverse phase high-pressure liquid chromatography columns as that described for the SRS from other origins. SRS activity released from platelets preincubated with [14C]arachidonic acid exhibited the same retention time as radioactivity in reverse phase high-pressure liquid chromatography. The release of a SRS from platelets is consistent with their implication in the pathogenesis of asthma and other lung diseases.

Effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils.

Exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) have been compared with exogenous arachidonic acid for their capacity to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human neutrophils and for their suitability as parallel substrates in this pathway. The products from specific 14C- or 3H-labeled substrates were isolated by reverse phase high performance liquid chromatography (RP-HPLC) and were identified by elution of radiolabel at the retention times of the appropriate synthetic standards. Each product was also characterized by its ultraviolet (UV) absorption spectrum, and 7-hydroxy-DCHA was defined in addition by analysis of its mass spectrum. The metabolites, 5-hydroxyeicosatetraenoic acid, leukotriene B4 (LTB4), 6-trans-LTB4 diastereoisomers, 5-hydroxyeicosapentaenoic acid, 6-trans-leukotriene B5 diastereoisomers, leukotriene B5 (LTB5), and 7-hydroxy-DCHA were quantitated by integrated UV absorbance during resolution by RP-HPLC. LTB4 and LTB5 were also quantitated by radioimmunoassay of the eluate fractions, and leukotrienes C4 and C5 (LTC4 and LTC5, respectively) were quantitated by radioimmunoassay alone. None of the unlabeled exogenous fatty acids (5-40 micrograms/ml) altered the release of radioactivity from [14C]arachidonic acid-labeled, ionophore-activated neutrophils. The metabolism of 5 and 10 micrograms/ml of exogenous EPA by ionophore-activated, [14C]arachidonic acid-labeled neutrophils not only generated 5-hydroxyeicosapentaenoic acid, 6-trans-LTB5, LTB5, and LTC5, but also stimulated the formation of 5-hydroxyeicosatetraenoic acid, 6-trans-LTB4 diastereoisomers, and LTC4 from membrane-derived arachidonic acid. In contrast, LTB4 production was diminished throughout the EPA dose-response, beginning at 5 micrograms/ml EPA and reaching 50% suppression at 10 micrograms/ml and 84% suppression at 40 micrograms/ml. The selective decrease in extracellular LTB4 concentrations in the presence of EPA was not due to a change in the kinetic appearance of LTB4 or to an increase in conversion to its omega-oxidation metabolites. DCHA was metabolized to 7-hydroxy-DCHA, did not stimulate metabolism of membrane-derived arachidonic acid, did not appreciably inhibit LTB4 formation, and was not a substrate for leukotriene formation. Incremental doses of exogenous arachidonic acid resulted in increased production of 5-hydroxyeicosatetraenoic acid and 6-trans-LTB4 by ionophore-activated, [14C]arachidonic acid-labeled neutrophils without any change in LTB4 production. 5-hydroxyeicosapentaenoic acid and 7-hydroxy DCHA were inactive as chemotactic factors whereas 5-hydroxyeicosatetraenoic acid exhibited 2% of the potency of LBT4. Thus, exogenous DCHA does not appreciably interfere with the metabolism of membrane-derived arachidonic acid by ionophore-activated, [14C]arachidonic acid-labeled neutrophils and is converted only to a monohydroxy derivative. In contrast, exogenous EPA attenuates the generation of LTB4 and is converted to LTB5, which is a weak and partial agonist as compared with LTB4.

Regulation of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin.

The regulating mechanisms of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin were investigated. The formation of PAF-acether was maximal at 5 min after stimulation and gradually decreased for up to 30 min. Thrombin induced a rapid 3-4-fold increase in the activity which was maximal by 1 min after stimulation and returned progressively to basal level within 10 min. The thrombin-induced enhancement in acetyltransferase activity was due to an increase of the Vmax of the acetylation reaction without a significant effect on the apparent Km of the enzyme for acetyl-CoA. Human endothelial cells also exhibited a basal PAF-acether acetylhydrolase activity which was not altered upon thrombin stimulation. The pretreatment with 2 mM phenylmethylsulfonyl fluoride (PMSF), a serine proteinase inhibitor reported to block the acetylhydrolase, induced about 2-times more PAF-acether production in response to 2.5 U/ml thrombin stimulation. However, this enhancement of PAF-acether formation seems to be not only due to the inhibition of the acetylhydrolase, but also to the influences on the activities of the acetyltransferase and other enzymes such as phospholipase A2. These results suggest a key role for acetyltransferase and acetylhydrolase in the regulation of PAF-acether formation and catabolism in thrombin-stimulated human endothelial cells.

Biosynthesis of platelet-activating factor (PAF-acether). V. Enhancement of acetyltransferase activity in murine peritoneal cells by calcium ionophore A23187.

The activity of the acetyltransferase capable of transferring the acetyl moiety of acetyl-CoA onto 2-lyso PAF-acether (1-alkyl-sn-glycero-3-phosphocholine) to form PAF-acether was compared in ionophore A23187-stimulated and in non-stimulated rat peritoneal cells. Stimulation resulted in a doubling of the acetyltransferase activity within 30 s. This effect was abolished in the presence of EDTA (1 mM) or EGTA (1 mM) and restored by addition of Ca2+ (10 mM). The specificity of acetyltransferase measured in ionophore-stimulated as well as in untreated cells is the same. In both situations we observed the same Km values for acetyl-CoA, whereas the Vmax values were different. The wide similarities of the two enzyme preparations lead us to conclude that stimulation by the ionophore involves an increase in the number of enzyme molecules rather than a change in the kinetic parameters of the acetyltransferase.

Biosynthesis of platelet-activating factor. I. Evidence for an acetyl-transferase activity in murine macrophages.

Platelet-activating factor (PAF-acether; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from murine peritoneal adherent cells by inflammatory and non-inflammatory stimuli. We have found, in extracts from these cells, an enzyme activity that synthesizes. PAF-acether from synthetic lyso-PAF-acether by transferring the acetyl moiety of acetyl-coenzyme A onto the lyso-PAF-acether molecule. The enzyme is stabilized by 1 mM dithiothreitol, is calcium-dependent, has an apparent Km of 172 microM for acetyl-CoA and is active in a 6-8 pH range. When the acetyl-CoA substrate is replaced by propionyl-CoA, an ether lipid is produced which turns out to be as potent an aggregating agent as PAF-acether. In all cases, the products of the reaction were characterized by their behaviour in platelet-aggregation tests and their high-pressure liquid chromatography (HPLC) elution profiles. The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor.

Activation of the murine monocyte/macrophage cell line, J774A1 by poly(A).poly(U): I. Binding of poly(A).poly(U) and induction of oligo-2',5'-adenylate synthetase.

Binding and internalization of the synthetic double-stranded complex poly(A).poly(U) were studied on a murine monocyte/macrophage cell line J774A1. Poly(A).poly(U) increased in a dose-dependent fashion the oligo-2',5'-adenylate synthetase demonstrating that those cells were responsive to this agonist. Binding of [32P]poly(A).[32P]poly(U) to the cells reached an apparent kinetic equilibrium within 4 h and was saturable (apparent Kd = 9.99 +/- 0.09.10(-2) g/l and Bmax 13.3 +/- 5.3.10(-3) g/l per 10(6) cells) and temperature-dependent. The binding of poly(A).poly(U) was competitively inhibited by various polynucleotides but not by other structurally unrelated compounds. Analysis of cell-associated [32P]poly(A).[32P]poly(U) demonstrated a minimal degradation of this polyribonucleotide over a 4-h incubation period. Autoradiography of cells incubated with [3H]poly(A).[3H]poly(U) revealed that poly(A).poly(U) was internalized and migrated to cell nuclei. These results suggest that poly(A).poly(U) is internalized in J774A1 cells via an endocytotic process.

Protein tyrosine kinases in activation signal of human basophils through the immunoglobulin E receptor type l.

Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.

Beta 2-adrenoceptor agonists regulate the IL-4-induced phenotypical changes and IgE-dependent functions in normal human monocytes.

The beta 2-adrenoceptor agonists salbutamol and fenoterol were tested for their regulatory effects on human monocyte phenotype and functions, either alone or in combination with interleukin-4 (IL-4). These drugs enhanced in a dose-dependent manner the IL-4-induced membrane and mRNA expression of the low-affinity receptor for immunoglobulin E (IgE) (CD23), as well as the release of its soluble form, sCD23. Salbutamol and fenoterol alone elicited expression of the monomorphic beta 2-chain (CD18) of the leukocyte functional antigen (LFA1) family. This effect appeared to be restricted to CD11b (CR3) and CD11c (gp 150-95), because CD11a (LFA-1 alpha chain) was not modified. beta 2-Adrenoceptor stimulation was also found to potentiate the effect of IL-4 on CD11b, CD11c, and CD18 expression. In contrast, these agents alone did not alter the level of major histocompatibility complex class II and CD14 antigens or modify their respective up- and down-regulation by IL-4. Ligation of CD23 on IL-4-preincubated (CD23+) monocytes with IgE/anti-IgE immune complexes induced the release of free radicals nitric oxide and of the proinflammatory mediators IL-6 and thromboxane B2 (TxB2). Addition of salbutamol, inactive alone, potentiated the generation of superoxide anion and of nitric oxide generation, as well as the production of IL-6 and TxB2 triggered by CD23 ligation. These results indicate that beta 2-adrenoceptor stimulation potentiates in vitro the IL-4-induced phenotypical and functional changes on monocytes and suggest that such an interaction could occur in IgE-dependent immune reactions.

Functional interaction between beta 2-adrenoceptor agonists and interleukin-4 in the regulation of CD23 expression and release and IgE production in human.

Normal human peripheral blood mononuclear cells (PBMC) produced IgE when stimulated with IL-4. In the present report it was shown that beta 2-adrenoceptor agonists, salbutamol and fenoterol, potentiated the IL-4-induced IgE production without significantly affecting the expression of the low affinity receptor for IgE at the cell surface of monocytes and B lymphocytes. However, beta 2-adrenoceptor agonists were shown to enhance at day 7 the IL-4-induced release of the soluble form of CD23 (sCD23) by PBMC. This effect was specific since a beta-adrenoceptor antagonist, D,L-propranolol, inhibited the IL-4-induced IgE production by these cells. Alternatively, the beta 2-adrenoceptor agonists inhibited the production by these cells of interferon-gamma (IFN-gamma) but did not affect the production of IL-4 when stimulated with phytohemagglutinin A + a phorbol ester. These data suggest that beta 2-adrenoceptor agonists influence the IL-4-induced IgE production in humans by enhancing the release of sCD23 and inhibiting the production of endogenous IFN-gamma. In addition to the effect on the IL-4-induced IgE production it was shown that beta 2-adrenoceptor agonists potentiated the effect of IL-4 on a human promonocytic cell line, U 937, by enhancing CD23 expression and release and by inducing the differentiation of these cells into monocyte-like cells. Taken together, these data indicate that beta 2-adrenoceptor agonists potentiated the effect of IL-4 and that this functional interaction is different considering the cell-lineage and the stage of differentiation of these cells.

Biologic properties of LTB4 and paf-acether in vivo in human skin.

Using an improved skin chamber technique, the consequences of prolonged contact of leukotriene B4 (LTB4) and platelet-activating factor (paf-acether) with human dermis were evaluated quantitatively and kinetically in vivo. Leukocyte chemotaxis, histoenzymologic alterations, and modifications in vascular permeability were studied in two sets of experiments. In a first set of experiments, the dose-effect relationship of LTB4 and paf-acether on leukocyte migration was studied. LTB4 (3 X 10(-8) M to 9 X 10(-7) M) in Hanks' balanced salt solution (HBSS) elicited an intense dose-dependent and time-dependent neutrophil migration. Paf-acether, at the same concentration range, induced a significant increase in cell migration only at 9 X 10(-7) M and when diluted in HBSS containing 0.25% serum albumin (HBSS-BSA). Histoenzymologic analysis demonstrated that LTB4 in vivo induced degranulation of most of the neutrophils migrating through the dermis. Paf-acether caused mild degranulation of neutrophils and induced the appearance of degranulated basophils in dermal vessels. A second set of experiments was designed to study simultaneously the modifications in vascular permeability and cell migration induced by LTB4 and paf-acether, with or without prostaglandin E2 (both at a concentration of 3 X 10(-7) M in HBSS). Since spontaneous protein diffusion in HBSS progressively declined up to a plateau reached after 20 h (1.2 +/- 0.15 mg of proteins/cm2/2 h), these experiments were carried out after a 20-h equilibration period. Leukotriene B4 induced a late and slight increase in vascular permeability. Paf-acether did so intensely and transiently. Prostaglandin E2 significantly enhanced protein diffusion and neutrophil migration induced by LTB4 and, to a lesser extent, by paf-acether. Interestingly, despite the reintroduction into the skin chambers of freshly prepared solutions containing the mediators, leukocyte migration and protein diffusion progressively decreased during the experiments. This suggests the local production of anti-inflammatory factors that inhibit local mediators and thus regulate the inflammatory response.

Pharmacologic modulation of picryl chloride-induced contact dermatitis in the mouse.

A biphasic response of ear swelling was observed 2 h and 24 h after application of the antigen to picryl chloride-sensitized Balb/c mice. A platelet-activating factor (PAF) antagonist, BN 52063, or the anti-inflammatory drug, betamethasone, applied topically or injected subcutaneously, inhibited in a dose-dependent fashion the antigen-induced increase in ear thickness observed after 24 h. In addition, BN 52063 and betamethasone presented a synergistic effect when administered in vivo simultaneously and subcutaneously. Indomethacin administered subcutaneously at the time of the antigen challenge significantly potentiated the early swelling phase and inhibited the late one. In contrast, the inhibitors of histamine and serotonin, ketotifen and methysergide, respectively, modulated mostly the early, and to a lower extent the late phase when administered at the time of antigen challenge. In contrast, none of these drugs inhibited the late phase reaction when administered 4 h after the antigen. A significant eosinophil and mononuclear-cell ear infiltrate was observed following topical application of the antigen, a phenomenon that was markedly reduced by either BN 52063 or betamethasone. These results demonstrate the effectiveness of PAF antagonists, either alone or in association with glucocorticosteroids, in experimental CD, the modulation of the infiltration of eosinophils and mononuclear cells possibly explaining part of the inhibitory action of these drugs.

CD23-mediated nitric oxide synthase pathway induction in human keratinocytes is inhibited by retinoic acid derivatives.

Retinoids exert various functions including anti-proliferative and anti-inflammatory effects on many cell types including keratinocytes and are widely used in skin diseases, such as psoriasis and acne. We have previously shown that human keratinocytes express low affinity immunoglobulin E receptor (FcepsilonRII/CD23) when stimulated with interleukin-4. Immunoglobulin E ligates CD23 and induces the production of nitrites (reflecting the mobilization of the nitric oxide [NO]-pathway) and tumor necrosis factor-alpha by human keratinocytes. Here, 13-cis and all-trans retinoic acid (RA) were shown to reduce the production of nitrites by immunoglobulin E-activated keratinocytes by 80% in a time- and concentration-dependent fashion. As a consequence, RA derivatives also reduced the production of tumor necrosis factor alpha by these cells by 70%. The level of inducible NO synthase activity in activated human keratinocytes was significantly decreased upon treatment of the cells with RA derivatives (inhibition by 60% of the mean inducible NO synthase activity with 13-cis RA, 2 microM). Treatment for 24 h with RA derivatives almost completely abolished transcription of inducible NO synthase-specific mRNA in activated keratinocytes. Therefore, RA derivatives downregulate tumor necrosis factor-alpha release and the NO-transduction pathway through the inhibition of inducible NO synthase transcription. Together, our data provide evidence for inhibition of the NO-pathway by 13-cis and all-trans retinoic acid on CD23-activated human keratinocytes. These data may clarify the mechanism of the anti-inflammatory activity of RA derivatives in skin diseases.

Involvement of interleukin-6 and interferon-alpha in the poly(A).Poly(U)-induced 2',5'-oligoadenylate synthetase activity in the mouse monocyte-macrophage cell line, J774A1.

The synthetic polyribonucleotide poly(A).poly(U) induces 2',5'-oligoadenylate synthetase activity in the murine macrophage cell line J774A1. The possible role of several cytokines involved in macrophage activation (i.e., IL-1, IL-6, TNF, and IFN) was examined in the present study. It was first demonstrated that among the anticytokine antibodies, only monoclonal antibodies directed against IL-6 inhibited the induction of 2',5'-oligoadenylate synthetase by poly(A).poly(U) in a dose-dependent manner. Moreover, it was established that poly(A).poly(U) elicited IL-6 production in J774A1 cells in a time-and dose-dependent manner. Consequently, the effect of IL-6 on 2',5'-oligoadenylate synthetase activity was studied. IL-6 either alone or in combination with IL-1 and TNF did not induce 2',5'-oligoadenylate synthetase activity. IL-6 did not potentiate IFN-gamma-induced 2'-5'-oligoadenylate synthetase activity. In contrast, addition of IL-6 to the incubation medium potentiated the stimulation of 2'-5'-oligoadenylate synthetase activity by IFN-alpha. These results suggest that IL-6 is a necessary but not sufficient factor in the induction of 2'-5'-oligoadenylate synthetase activity in the J774A1 cell line by poly(A).poly(U).

Comparison of the effects of intra-arterial and aerosol administration of endothelin-1 (ET-1) in the guinea-pig isolated lung.

1. Intra-arterial injection of endothelin-1 (ET-1, 400 pmol; 1 microgram) in guinea-pig isolated perfused lungs, induced increases in pulmonary inflation pressure (PIP) and perfusion pressure (PPP), associated with oedema formation and thromboxane B2 (TxB2) release but not with the generation of sulphidopeptide leukotrienes or release of histamine. In contrast, aerosol administration of ET-1 (3, 6, 10 micrograms ml-1, for 2 min) evoked a dose-dependent increase in PIP, without significant changes in PPP, oedema formation or TxB2 release. 2. Addition of indomethacin (5 microM) or BW 755C (10 or 100 microM), but not nordihydroguaiaretic acid (NDGA, 50 microM) or FPL 55712 (10 microM), to the perfusion medium led to a significant inhibition of the increases in PIP and PPP, TxB2 release and oedema formation evoked by intra-arterial injection of 400 pmol ET-1. In contrast, indomethacin (5 microM), BW 755C (100 microM) or FPL 55712 (10 microM), added to the perfusion medium 10 min prior to challenge, did not affect the increase in PIP induced by a 2-min aerosol of a solution of ET-1 10 micrograms ml-1. 3. In vivo aerosol administration of indomethacin (100 mg ml-1, for 20 min) to non-anaesthetized guinea-pigs, 15 min before lung removal, did not modify the bronchopulmonary response evoked in isolated perfused lungs by an aerosol of ET-1 10 micrograms ml-1. However, under the same experimental conditions, indomethacin significantly inhibited TxB2 release evoked by aerosolized arachidonic acid (2 mg ml-1). 4. In conclusion, the present study shows that when injected by the intra-arterial route, ET-1 effects are mediated primarily via the generation of cyclo-oxygenase metabolites of arachidonic acid, whereas when the aerosol route is used, the peptide appears to act on airway smooth muscle cells, through an indomethacin-insensitive process which may involve some other, as yet unidentified, mediator(s).

Platelet-activating factor (PAF) and PAF antagonists in asthma.

PAF is produced by and activates inflammatory cells, such as monocytes/macrophages, mast cells, platelets, neutrophils, eosinophils and endothelial cells. Its ability to imitate anaphylaxis, inducing for instance bronchoconstriction (BC) in guinea-pigs, and its identification (and/or that of lyso-PAF) in exudates from shocked lungs, led to the hypothesis that PAF is involved in immediate hypersensitivity. Recent results of Bachelet et al. show that PAF reduces the increased cyclic AMP content of guinea-pig alveolar cell population exposed to PGE2, salbutamol or isoprenaline, which agrees with its hypothesized stimulating role in conditions where increased cyclic AMP may reduce mediator release. PAF antagonists are usually selected with in vitro platelet tests and their in vivo activity is characterized in normal animals. Recent data of Pretolani et al. demonstrate nevertheless that the antagonists may lose part of their ability to inhibit PAF itself if tested on lungs from actively sensitized guinea-pigs. These lungs differ from those of passively sensitized or of naive animals in that they become hyper-responsive to mediators (PAF, leukotriene D4 [LTD4], histamine, arachidonate [AA]): BC and formation of thromboxane A2 are enhanced, and histamine is released dose-dependently under conditions where it is absent from perfusates from LTD4, AA or PAF-stimulated naive lungs. Peripheral inflammatory cells (basophils, eosinophils, monocytes) are possibly recruited into the lungs of the actively sensitized animals sometime during the second and/or third week of sensitization, and provide a new target which may account for the enhanced lung responsiveness. Ultra-structural studies of Lellouch-Tubiana et al. (abstract in this meeting) support this concept. Neither the primary target nor the chemotactic substance responsible for the reported modifications are identified, but recent data of Bachelet et al, showing that alveolar populations from actively sensitized guinea-pigs are less responsive to the cyclic AMP stimulating effects of PGE2, salbutamol or isoprenaline suggest the existence of a cell defect which may be important for the triggering of allergen-induced BC and cell recruitment. Our present concept involves a "pre-inflamed" lung in actively sensitized guinea-pigs and in human asthmatics, a stand-by process following sensitization and which is revealed following the activation of a target cell. This may be the alveolar macrophage which releases substances (PAF, TXA2, IL1) likely to start BC and protracted cell recruitment and activation.

Phosphoramidon potentiates the endothelin-1-induced bronchopulmonary response in guinea-pigs.

We investigated the effect of the endopeptidase-inhibitor, phosphoramidon, on the bronchopulmonary response induced by endothelin-1 in vivo or in isolated perfused lungs. In vivo aerosol administration of 1 or 3 ?g/ml endothelin-1 for 2 min provoked no significant bronchopulmonary response. When awake animals were pretreated by an aerosol of phosphoramidon (0.1 mM, for 15 min), the bronchopulmonary response induced by 1 and 3 ?g/ml endothelin-1 was markedly enhanced. In isolated guinea-pig lungs, aerosol administration of endothelin-1 (3 ?g/ml, for 2 min) evoked a low increase in pulmonary inflation pressure. Treatment of awake animals with an aerosol of phosphoramidon before lung recollection led to a significant potentiation of the endothelin-1-induced increase in pulmonary inflation pressure. These results demonstrate that phosphoramidon potentiates the in vivo and in vitro bronchopulmonary response evoked by low doses of endothelin-1 and suggest that endopeptidase-like enzymes present in the airway tissue modulate the effect of the peptide.

Bronchopulmonary and pressor effects of big-endothelin-1 in the guinea-pig.

Injection of 1 nmol/kg Big-endothelin-1 (ET-1) into anaesthetized and ventilated guinea-pigs did not evoke significant changes in pulmonary inflation pressure and mean arterial blood pressure. In contrast, injection of 1 nmol/kg ET-1 induced marked and rapid bronchoconstrictor and pressor responses. When administered at a dose of 10 nmol/kg, Big-ET-1 induced marked long-lasting changes in pulmonary inflation pressure and mean arterial blood pressure developing slowly as compared to those evoked by ET-1. Furthermore, these increases reached maximal values by 20 min for pulmonary inflation pressure and 45 min for mean arterial blood pressure after injection of the peptide. When Big-ET-1 was incubated with ?-chymotrypsin [45 min at 37 degrees C, enzyme : substrate ratio (wt/wt) : 0.5%] and injected into guinea-pigs at a dose of 1 nmol/kg, marked bronchoconstrictor and pressor responses were observed, developing with the same kinetics as those evoked by ET-1. The extent of the pressor response was similar and the bronchoconstriction was slightly lower than those evoked upon injection of 1 nmol/kg ET-1 treated or not with ?-chymotrypsin. The present results indicate that Big-ET exhibits moderate, if any, direct bronchoconstrictor and pressor activities in the guinea-pig. The slow metabolism of Big-ET-1 in an active form probably explains its long-lasting effects at a dose of 10 nmol/kg. This is indirectly confirmed by the in vitro treatment of Big-ET-1 with ?-chymotrypsin which converts the peptide into an active form.

Differentiation of human mast cells from bone-marrow and cord-blood progenitor cells by factors produced by a mouse stromal cell line.

Human bone-marrow or cord-blood progenitors (i.e., CD34+ cells) are easily purified by immunological methods and can be cultured on normal human-bone-marrow stromal cells for limited periods of time. Under these culture conditions, the number of progenitors declines in a few weeks and these cells disappear completely in less than 8 weeks. This fact suggests that this culture system is deprived of growth factor(s) able to support the self-renewal of stem cells. We have developed the culture of immunomagnetically purified human-bone-marrow- or cord-blood-derived CD34+ cells on a supportive mouse lipoblastic stromal cell line, MS-5. The long-term survival of clonogenic cells was analyzed in these cultures and compared with the results obtained by culture on human-bone-marrow stromal cells. The results demonstrated that only coculture of CD34+ cells on MS-5 layers allows the survival of clonogenic progenitors for at least 12 weeks. Cytospin smears were regularly performed and cell morphology was examined after classical staining methods (i.e., M.G.G. and toluidine blue staining). Histologic analysis demonstrated the growth of mast-cell-like metachromatic cells after the second week of incubation on MS-5 layer. The highest percentage of these cells was observed after 8 weeks, and averaged about 30 percent for cord-blood cells and 70 percent for bone-marrow cells. To further confirm the nature of the metachromatic cells obtained under this culture condition, immunohistochemical staining of tryptase was performed on the same samples. The results demonstrated similar percentages of tryptase+ cells and of metachromatic elements. Measurement of cellular histamine demonstrated that culture of CD34+ cells on MS-5 monolayers induced the formation and increase of this mediator. To determine whether the contact between MS-5 layers and CD34+ cells was an absolute requirement for the development of mast cells, CD34+ cells were cultured in the presence of MS-5 conditioned medium. This condition allowed the development of similar percentage of mast cells when compared with the coculture experiments, indicating that a soluble factor was involved in mast cell differentiation. Whatever the soluble factor(s) responsible for this mast cell growth activity, our culture system allows us to obtain significant amounts of highly enriched normal human mast cell populations useful for further studies on the reactivity of this cell subset.

Effect of the lipidic lipidosterolic extract of Serenoa repens (Permixon) on the ionophore A23187-stimulated production of leukotriene B4 (LTB4) from human polymorphonuclear neutrophils.

Although the lipidic extract of Serenoa repens (LESSr, Permixon, Sereprostat) is widely used in patients suffering from benign prostatic hypertrophy (BPH), its mechanism of action is not fully elucidated. It has been demonstrated that infiltration of the prostate by inflammatory cells is one of the aetiologic factors involved in the development of BPH. These inflammatory cell types, such as polymorphonuclear neutrophils (PMNs), produce chemotactic mediators and contribute to the development of the disease. Among the chemotactic factors generated by inflammatory cell types, the derivatives of arachidonic acid have been extensively studied. For instance, leukotriene (LT) B4 is one of the most potent chemotactic factors for PMNs and also exhibits a wide range of biological activities. In order to investigate the potential action of LESSr on arachidonate metabolism, and particularly on the synthesis of LTB4, the effect of this extract on the in vitro synthesis of LT by human PMNs stimulated with the calcium ionophore A23187 was investigated. LESSr significantly inhibits the production of 5-lipoxygenase metabolites (5-HETE, 20-COOH LTB4, LTB4 and 20-OH LTB4) at concentrations as low as 5 microg/ml. Such an effect of LESSr was also observed in the presence of exogenous arachidonic acid (20 microg/ml) and when f-MLP was used as the agonist, suggesting that inhibition of LTB4 production by the extract was unrelated to phospholipase A2 blockade and independent of the stimulating agent. The capability of LESSr to antagonize 5-lipoxygenase metabolites production may contribute, at least partly, to the understanding of its therapeutic activity on the inflammatory component of BPH.