Arachidonic acid metabolism of the murine eosinophil. II. Involvement of the lipoxygenase pathway in the response to the lymphokine eosinophil stimulation promoter

Eosinophil stimulation promoter (ESP) is a murine lymphokine that enhances the migration of eosinophils. Exogenous arachidonic acid between 0.5 and 2 micrograms/ml potentiated the activity of ESP on murine eosinophil migration, whereas such concentrations did not affect migration in the absence of ESP. Among the lipoxygenase products identified from an enriched population of murine eosinophils, leukotriene B4 (optimal activity at 100 ng/ml) and 12-HETE (optimal activity at 2 micrograms/ml) stimulated migration of these cells. Another lipoxygenase product from these cells 15-HETE inhibited ESP-induced migration; between 5 and 10 micrograms/ml 15-HETE decreased by one-half both stimulated migration and 12-HETE biosynthesis. Structurally diverse drugs at concentrations that inhibited HETE biosynthesis inhibited ESP-induced migration. The concentrations that decreased migration activity by one-half were 5 microM NDGA, 10 microM ETYA, and 150 microM BW755C. Aspirin and indomethacin at concentrations reported to inhibit prostaglandin biosynthesis did not substantially inhibit ESP activity, but concentrations of indomethacin above 20 microM caused concentration-dependent inhibition of migration. The selective lipoxygenases inhibitor 134,7,10,13-eicosatetraynoic acid was more potent than ETYA in inhibition of ESP-induced migration, and the selective cyclooxygenase inhibitor 6,9,12-octadecatriynoic acid did not effect inhibition. These results are consistent with the hypothesis that stimulation of eosinophils by the lymphokine ESP involves the generation of lipoxygenase products from arachidonic acid, which positively and negatively regulate the migratory activities of these cells.     

Prostaglandin D2 causes accumulation of eosinophils in the lumen of the dog trachea

Prostaglandin D2 (PGD2), the major product of arachidonic acid metabolism via the cyclooxygenase pathway in most mast cells, is present in the airways of atopic asthmatic patients after antigen challenge. Because eosinophilia is characteristic of asthma, we asked whether PGD2 causes eosinophils to accumulate in the airways in vivo. Using an endotracheal tube with two inflatable balloons we isolated a segment of trachea in four anesthetized mechanically ventilated dogs, and we superfused this segment with either a control solution (Hanks' balanced salt solution and antibiotics) or solution containing PGD2 (10(-6) M). Total and differential cell counts were determined at base line and every hour for 4 h during the study. PGD2 caused eosinophil accumulation in the trachea [7.0 +/- 3.4, 28.7 +/- 17.8, 33.7 +/- 13.6, and 35.4 +/- 10.7 (SD) cells/cm2 trachea after 1, 2, 3, and 4 h, respectively, P less than 0.05 vs. controls] but had no significant effect on neutrophil accumulation. The effect of PGD2 on eosinophil accumulation was significantly inhibited by the prostaglandin receptor antagonist SKF 88046 (5 mg/kg iv). We conclude that PGD2 is a selective stimulus that causes accumulation of eosinophils in the tracheal lumen of dogs in vivo.     

Glucocorticoids inhibit cytokine-mediated eosinophil survival

Glucocorticoids characteristically induce eosinopenia in vivo and are effective for treating allergic and other eosinophilic disorders. We studied the effect of glucocorticoids on cytokine-induced survival of human eosinophils in vitro. Eosinophils were purified from normal or mildly atopic volunteers by Percoll density gradient and incubated for 4 days in the presence of cytokine plus steroid. Cell viabilities were determined by staining cells with fluorescein diacetate and propidium iodide. In the absence of glucocorticoids, human rIL-5 enhanced eosinophil survival in a dose-dependent manner, from 22 fM for a minimal effect to 2200 fM for maximal effect. When eosinophils were cultured with a submaximal concentration of rIL-5 (220 fM), dexamethasone, methylprednisolone, and hydrocortisone inhibited eosinophil survival in a dose-dependent manner. Inhibition was time-dependent and required at least 2 days' exposure of eosinophils to dexamethasone. Dexamethasone, methylprednisolone, and hydrocortisone at 1000 nM inhibited survival by 88 +/- 2, 66 +/- 9 and 37 +/- 7%. In contrast, estradiol and testosterone (1000 nM) had no effect on eosinophil survival. When eosinophils were incubated with varying concentrations of human rIL-5 and 1000 nM dexamethasone, survival inhibition was reduced at higher concentrations of human rIL-5, and completely abolished by human rIL-5 23,000 fM. Human recombinant granulocyte-macrophage CSF, human rIL-3, and human rIFN-gamma also enhanced eosinophil survival in a dose-dependent manner and dexamethasone (1000 nM) strongly inhibited cell survival when submaximal concentrations of these cytokines were used. The effects of dexamethasone were reversed by higher concentrations of granulocyte-macrophage CSF (10 U/ml) and IL-3 (3 ng/ml). However, even 1000 U/ml IFN-gamma did not overcome dexamethasone inhibition, indicating a difference between the mechanism of eosinophil survival induced by IFN-gamma and other cytokines. These results suggest that glucocorticoids exert a direct, inhibitory effect on eosinophil survival, which may be important in the treatment of allergic and other eosinophilic disorders. Antagonism of this effect by higher amounts of cytokine may be a mechanism for glucocorticoid resistance.     

Selective regulation of eosinophil degranulation by interleukin 1 beta.

Recent evidence confirms that cytokines such as IL-1, IL-4, IL-5, and GM-CSF may enhance or inhibit eosinophil function. Functions that are susceptible to modulation include eosinophil-mediated antibody-dependent damage of helminthic parasites, oxidative metabolism and degranulation. We have employed IgG and IgE-coated Sepharose beads to investigate selective modulation of IgG and IgE-mediated enzyme release by IL-1 beta. Both IgG and IgE-coated beads induced release of granular enzymes beta-glucuronidase and arylsulfatase. Enzyme release from IgG-stimulated eosinophils was inhibited by preincubation with IL-1 beta (100 pg/ml, P less than or equal to 0.05). In contrast, enzyme release by IgE-stimulated eosinophils was enhanced by IL-1 beta (100 pg/ml, P less than or equal to 0.05). These studies support the hypothesis that IL-1 beta has specific selective actions on eosinophil function. Furthermore, these actions on particle-stimulated enzyme release suggest that IgG and IgE mediated processes in eosinophils are differentially regulated.     

Regulation of Ig-induced eosinophil degranulation by adenosine 3',5'-cyclic monophosphate.

We have investigated the effects of cAMP on Ig-induced human eosinophil activation. Stimulation of human normodense eosinophils with IgG- or secretory IgA (sIgA)-coated Sepharose beads induced cellular degranulation, as measured by the release of the granule protein, eosinophil-derived neurotoxin (EDN). Pretreatment with cAMP analogs (N6,O2,-dibutyryl adenosine-3,':5' cyclic monophosphate; 8-bromoadenosine 3':5' cyclic monophosphate; or N6-benzoyladenosine 3':5' cyclic monophosphate) or cAMP phosphodiesterase-inhibitors (theophylline or isobutylmethyl xanthine (IBMX] strongly inhibited Ig-induced human eosinophil degranulation. The beta-adrenoceptor agonists, isoproterenol and salbutamol, induced relatively low level increases in intracellular cAMP, and weakly suppressed EDN release induced by IgG-coated beads. However, cellular pretreatment with IBMX synergistically enhanced the inhibitory effects of isoproterenol or salbutamol on both IgG and sIgA-induced eosinophil degranulation. Similarly, PGE2 treatment increased intracellular cAMP concentrations in eosinophils and correspondingly inhibited the Ig-dependent cellular degranulation response: co-incubation with IBMX further enhanced both effects of PGE2. Finally, cholera toxin, which irreversibly activates the stimulatory guanine nucleotide-binding protein linked to adenylyl cyclase, strongly inhibited the release of EDN from IgG- or sIgA-stimulated eosinophils. The time-dependent accumulation of cAMP in cholera toxin-treated cells closely paralleled the time courses of inhibition of IgG- and sIgA-induced EDN release after toxin exposure. These data indicate that the cAMP-dependent signal transduction mechanism in eosinophils exerts a negative modulatory effect on the cellular degranulation responses induced by sIgA or IgG. The inhibitory effects of cAMP on eosinophil activation may provide an important physiologic and a clinically relevant therapeutic mechanism for limiting the release of eosinophil-derived cytotoxic proteins during certain allergic or inflammatory responses in vivo.     

Role of pertussis toxin-sensitive G proteins in stimulus-dependent human eosinophil degranulation.

Stimulation of human normodense eosinophils with immobilized secretory IgA (sIgA) or IgG, or with the soluble stimulus, FMLP, triggers the exocytotic release of the granule protein, eosinophil-derived neurotoxin (EDN). In this report, we demonstrate that these stimuli also provoke an increase in phospholipase C-mediated phosphoinositide breakdown in eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) for 2 h irreversibly abolished the increases in phospholipase C activity and EDN release induced by immobilized sIgA or FMLP. In contrast, PTX treatment only transiently inhibited eosinophil activation induced by immobilized IgG. Maximal inhibition of IgG-stimulated phosphoinositide hydrolysis and EDN release occurred after 2 h of PTX pretreatment with PTX, followed by a gradual recovery of cellular responsiveness to immobilized IgG as the duration of PTX pretreatment was extended to 16 h. Activated PTX catalyzed the in vitro ADP-ribosylation of 41- and 44-kDa proteins in eosinophil membranes. A 2-h pretreatment of intact cells with PTX markedly reduced the pools of unmodified 41- and 44-kDa substrates available for subsequent ADP-ribosylation in vitro, suggesting that both proteins were substrates for PTX in intact eosinophils. Continuous exposure of eosinophils to PTX for times ranging from 2 to 15 h resulted in the gradual reappearance of unmodified 44-kDa protein, whereas the levels of unmodified 41-kDa protein were persistently reduced in PTX-treated cells. The time course of the decline and reappearance of unmodified 44-kDa substrate in PTX-treated eosinophils closely paralleled the changes in the responsiveness of these cells to immobilized IgG. These results suggest that the receptors for sIgA, FMLP, or IgG transduce activating signals for eosinophil degranulation through differential coupling to at least two PTX-sensitive G proteins.     

Effects of solubilization and vanadate/glutathione complex on inhibitor potencies against eosinophil cyclic AMP-specific phosphodiesterase.

Treatment of membranes from guinea-pig peritoneal eosinophils with deoxycholate and NaCl solubilized greater than 95% of the particulate cyclic AMP-specific phosphodiesterase (PDE IV). Solubilized PDE IV was at least 10 times more potently inhibited by selective PDE IV inhibitors (e.g. rolipram, denbufylline) than bound enzyme. Vanadate/glutathione complex (V/GSH) activated membrane-bound PDE IV and also increased potencies of these same inhibitors by at least 10-fold. Neither solubilization nor V/GSH markedly influenced the inhibitory activities of non-selective inhibitors (e.g. trequinsin, dipyridamole). Inhibitor effects on solubilized PDE IV and cyclic AMP accumulation in intact cells were strongly correlated. These results suggest a biologically important site on eosinophil PDE IV which is concealed or partially concealed in freshly prepared membranes and is exposed by solubilization or V/GSH.     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

Eosinophils as a novel cell source of prostaglandin D2: autocrine role in allergic inflammation

PGD(2) is a key mediator of allergic inflammatory diseases that is mainly synthesized by mast cells, which constitutively express high levels of the terminal enzyme involved in PGD(2) synthesis, the hematopoietic PGD synthase (H-PGDS). In this study, we investigated whether eosinophils are also able to synthesize, and therefore, supply biologically active PGD(2). PGD(2) synthesis was evaluated within human blood eosinophils, in vitro differentiated mouse eosinophils, and eosinophils infiltrating inflammatory site of mouse allergic reaction. Biological function of eosinophil-derived PGD(2) was studied by employing inhibitors of synthesis and activity. Constitutive expression of H-PGDS was found within nonstimulated human circulating eosinophils. Acute stimulation of human eosinophils with A23187 (0.1-5 μM) evoked PGD(2) synthesis, which was located at the nuclear envelope and was inhibited by pretreatment with HQL-79 (10 μM), a specific H-PGDS inhibitor. Prestimulation of human eosinophils with arachidonic acid (10 μM) or human eotaxin (6 nM) also enhanced HQL-79-sensitive PGD(2) synthesis, which, by acting on membrane-expressed specific receptors (D prostanoid receptors 1 and 2), displayed an autocrine/paracrine ability to trigger leukotriene C(4) synthesis and lipid body biogenesis, hallmark events of eosinophil activation. In vitro differentiated mouse eosinophils also synthesized paracrine/autocrine active PGD(2) in response to arachidonic acid stimulation. In vivo, at late time point of the allergic reaction, infiltrating eosinophils found at the inflammatory site appeared as an auxiliary PGD(2)-synthesizing cell population. Our findings reveal that eosinophils are indeed able to synthesize and secrete PGD(2), hence representing during allergic inflammation an extra cell source of PGD(2), which functions as an autocrine signal for eosinophil activation.     

Prostaglandin E2 inhibits eosinophil trafficking through E-prostanoid 2 receptors

The accumulation of eosinophils in lung tissue is a hallmark of asthma, and it is believed that eosinophils play a crucial pathogenic role in allergic inflammation. Prostaglandin (PG) E(2) exerts anti-inflammatory and bronchoprotective mechanisms in asthma, but the underlying mechanisms have remained unclear. In this study we show that PGE(2) potently inhibits the chemotaxis of purified human eosinophils toward eotaxin, PGD(2), and C5a. Activated monocytes similarly attenuated eosinophil migration, and this was reversed after pretreatment of the monocytes with a cyclooxygenase inhibitor. The selective E-prostanoid (EP) 2 receptor agonist butaprost mimicked the inhibitory effect of PGE(2) on eosinophil migration, whereas an EP2 antagonist completely prevented this effect. Butaprost, and also PGE(2), inhibited the C5a-induced degranulation of eosinophils. Moreover, selective kinase inhibitors revealed that the inhibitory effect of PGE(2) on eosinophil migration depended upon activation of PI3K and protein kinase C, but not cAMP. In animal models, the EP2 agonist butaprost inhibited the rapid mobilization of eosinophils from bone marrow of the in situ perfused guinea pig hind limb and prevented the allergen-induced bronchial accumulation of eosinophils in OVA-sensitized mice. Immunostaining showed that human eosinophils express EP2 receptors and that EP2 receptor expression in the murine lungs is prominent in airway epithelium and, after allergen challenge, in peribronchial infiltrating leukocytes. In summary, these data show that EP2 receptor agonists potently inhibit eosinophil trafficking and activation and might hence be a useful therapeutic option in eosinophilic diseases.     

Glucocorticoids inhibit eosinophil responses to granulocyte-macrophage colony-stimulating factor

The role of the eosinophil as an active proinflammatory cell in asthma and other allergic disorders has been well established. Glucocorticosteroids have long been used therapeutically as antiinflammatory agents in a variety of disease states where eosinophilia is a prominent feature. Although glucocorticoids are known to reduce tissue and circulating eosinophil numbers, the mechanisms by which they do so have not been clearly elucidated. Culture of eosinophils in vascular endothelial cell supernatants (VEC SUP) induces phenotypic and functional changes and prolongs the survival of the eosinophils. The survival-promoting activity in VEC SUP was shown to be granulocyte-macrophage CSF (GM-CSF) by neutralization with specific antibody. The potent glucocorticosteroid, dexamethasone (DEX), inhibited the prolongation of eosinophil survival caused by culture in either VEC SUP or human rGM-CSF. DEX (10(-6) M) exerted a direct survival-inhibitory effect on the eosinophil by the 4th day in culture in VEC SUP. This survival-inhibitory effect was dependent on the concentration of DEX (10(-10)-10(-6) M). Other glucocorticoids, including prednisolone (10(-7), 10(-6) M) and hydrocortisone (10(-7), 10(-6) M) also inhibited survival. The rank order of potency of the steroids indicates that this effect is mediated by a glucocorticoid receptor. This conclusion is supported by the failure of the sex steroids testosterone (10(-8)-10(-6) M) or beta-estradiol (10(-6) M) to inhibit eosinophil survival in the presence of VEC SUP. The effect of glucocorticoids on eosinophils is not a simple direct toxic effect because it was reversed by higher concentrations of GM-CSF. DEX shifted the GM-CSF dose-response curve for survival approximately fivefold to the right. GM-CSF induced a shift in eosinophil buoyant density which was partially blocked by DEX. These results suggest that glucocorticoids may inhibit elements of cytokine "priming" of eosinophils and that the eosinophilopenic effects of glucocorticoids may result in part from a direct effect on the eosinophil within a regulatory system involving cytokines.     

Effects and interaction studies of triflusal and other salicylic derivatives on cyclooxygenase in rats

Triflusal (TR) is a new salicylic acid derivative used clinically as an antiplatelet drug. Both aspirin (ASA) and TR inhibit platelet cyclooxygenase but the effects of these drugs are different. TR (0.5-2 mM) strongly inhibited platelet aggregation and malondialdehyde formation induced by arachidonic acid. The IC50 was 0.8 mM for TR and less than 0.1 mM for ASA. Deacetylated compounds, salicylic acid (SA) and HTB (the main metabolite of TR) were apparently competitive and reversible inhibitors of cyclooxygenase and HTB was 15 times more potent than SA. They did, however, partially prevent the inhibitory effects of ASA and TR in vitro. A similar effect was observed ex vivo in rats treated with HTB (100 mg/k i.p.) before TR or ASA (20 and 5 mg/kg i.v., respectively). Moreover, TR at 10 and 20 mg/kg i.v., inhibited thromboxane production by more than 50% while its effect on vascular cyclooxygenase was negligible. These findings indicated that TR is a weaker inhibitor of cyclooxygenase than ASA, and that HTB interferes with the effect of TR and ASA, despite the fact that HTB is a more potent reversible inhibitor than SA with probably a higher affinity for this enzyme.     

In vivo airway eosinophil accumulation does not enhance antigen- or propranolol-induced bronchoconstriction in guinea pigs

BACKGROUND: Chronic airway eosinophil accumulation is characteristic of asthma. However, it remains unclear whether airway eosinophils enhance or reduce release of chemical mediators and/or action of the released mediators in the airways in vivo, because previous investigators have indicated that eosinophil-derived factors such as histaminase and arylsulfatase may alter the allergic reaction by metabolizing chemical mediators. Recently, we have developed a guinea pig model of propranolol-induced bronchoconstriction (PIB), which is mediated by lipid mediators such as thromboxane A2 (TxA2), cysteinyl leukotrienes (cLTs) and platelet activation factor (PAF). This study was conducted to explain the influence of airway eosinophil accumulation on antigen-induced bronchoconstriction and the following PIB, both of which are mediated by lipid mediators. METHODS: Guinea pigs were transnasally treated with 75 microg/kg of polymyxin-B or vehicle twice a week for a total of 3 weeks. Guinea pigs were anesthetized and treated with diphenhydramine hydrochloride, and then artificially ventilated 24 h after the last administration of polymyxin-B or vehicle followed by passive sensitization. Propranolol at a concentration of 10 mg/ml was inhaled 20 min after an aerosolized antigen challenge. RESULTS: The proportion of eosinophils in bronchoalveolar lavage fluid obtained 15 min after the propranolol inhalation was significantly increased in guinea pigs treated with polymyxin-B compared with the vehicle. The polymyxin-B treatment did not affect antigen-induced bronchoconstriction or the following PIB. CONCLUSIONS: We conclude that eosinophils accumulated in the airways by polymyxin-B does not affect release of chemical mediators induced by antigen or propranolol inhalation, or action of released mediators in vivo.     

Regulatory effects of eotaxin,eotaxin-2, and eotaxin-3 on eosinophil degranulation and superoxide anion generation

Eosinophilic leukocytes have been implicated as primary effector cells in inflammatory and allergic diseases. When activated by cytokines, human eosinophils secrete and produce a variety of proinflammatory or tissue damaging substances. Although well known for their chemoattractant effects, little is known about the precise contribution of the eosinophil-selective chemokines, eotaxin, eotaxin-2, and eotaxin-3 to the effector functions of eosinophils. This forms the central focus of these investigations for which clone 15-HL-60 human eosinophilic cells were used as the in vitro model. Investigation results suggest that all three subtypes of eotaxin directly stimulate eosinophil superoxide anion generation that is inhibited by neutralizing eotaxin antibody or pretreatment of cells with the receptor antibody anti-CCR3. Pretreatment or co-treatment with each of the eotaxins augmented phorbol myristate-induced superoxide generation. Concentration-dependent degranulation of eosinophil peroxidase was noted for all three chemokines, and potentiation of calcium ionophore-induced degranulation was observed with eotaxin pretreatments. Results of interleukin-5 pretreatment studies suggest that the eotaxin chemokines may act cooperatively to enhance effector functions of eosinophils. Collectively, the present studies have advanced knowledge of the eotaxin family of chemokines to include eosinophil priming and modulation of eosinophil activation and secretion effector functions.     

Identification of lipoxygenase products from arachidonic acid metabolism in stimulated murine eosinophils

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A4, and the other included products derived from the 14,15-oxido analog of leukotriene A4 (14,15-leukotriene A4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipoxygenase products which modulate the migratory activity of the cells.     

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.     

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.     

Characterization of eosinophil cell activation by peptides. Differential effects of substance P, melittin, and FMET-Leu-Phe.

The ability of three different peptides, substance P (SP), FMLP and melittin, to activate eosinophils purified from the peritoneal cavity of human serum-treated guinea pigs was investigated. Degranulation (eosinophil peroxidase, EPO), oxidative burst (O2-), [Ca2+]i mobilization, and arachidonic acid metabolism (thromboxane B2, TXB2) were used as indices of eosinophil activation. SP (100 nM to 100 microM), FMLP (1 to 100 microM) and melittin (10 nM to 100 microM) induced EPO release but only FMLP (1 to 100 microM) led to an elevation of [Ca2+]i. The melittin- and SP-induced EPO secretion occurred at both cytotoxic and noncytotoxic concentrations as assessed by lactate dehydrogenase release. In addition, the effect of SP was not inhibited by the SP analogue (D-Pro4, D-Trp7,9,10)SP(4-11) and SP failed to promote the generation and subsequent release of TXA2. In contrast, FMLP (10 to 100 microM) stimulated the release of TXB2 from guinea pig eosinophils that was selectively inhibited by pretreatment of the cells with BOC-FMLP. On an equimolar basis (1 microM), melittin was approximately fivefold more active at promoting TXB2 release than FMLP. The results indicate that eosinophils respond to the three peptides, SP, melittin, and FMLP in differential fashion. We conclude that activation of guinea pig eosinophils by FMLP is likely to be receptor-mediated whereas the actions of SP and melittin may act through nonspecific peptide-membrane phospholipid interactions.     

Relative effects of flurbiprofen on platelet 12-hydroxy-eicosatetraenoic acid and thromboxane A2 production: influence on collagen-induced platelet aggregation and adhesion

Flurbiprofen has been shown to inhibit cyclo-oxygenase metabolism of arachidonic acid to thromboxane A2 (TxA2), resulting in the inhibition of platelet aggregation. Recently, our laboratory reported that the "irreversible" phase of platelet aggregation and adhesion were regulated, in part, by the lipoxygenase metabolism of arachidonic acid to 12-hydroxy-eicosatetraenoic acid (12-HETE) in platelets, and that selective inhibition of one enzyme i.e. either cyclo-oxygenase or lipoxygenase, resulted in paradoxical effects on the metabolism of arachidonic acid and platelet response related to the other pathway. Therefore, we performed experiments to assess the relative effects of flurbiprofen on TxA2 and 12-HETE synthesis, and on collagen-induced platelet aggregation and platelet adhesion to collagen-coated surfaces. "Irreversible" collagen-induced platelet aggregation was only partially inhibited by pre-incubation with 1 x 10(-6) M flurbiprofen, while TxA2 production was elevated and 12-HETE production was maximally inhibited in these platelets. At this concentration of flurbiprofen (1 x 10(-6)M), collagen-induced platelet adhesion was also reduced by 50%. At higher concentrations of flurbiprofen, both platelet aggregation and adhesion were further reduced, with a corresponding inhibition of TxA2 production. Thus it appears that the lipoxygenase pathway of arachidonic acid metabolism in platelets is not only inhibited by flurbiprofen, but is more sensitive to inhibition by flurbiprofen than the cyclo-oxygenase pathway. This differential effect of flurbiprofen on arachidonic acid metabolism in the platelet is related to differential effects on platelet function.     

Blockade of JAK2 by tyrphostin AG-490 inhibits antigen-induced eosinophil recruitment into the mouse airways

We studied the effect of tyrphostin AG-490, a specific Janus kinase 2 (JAK2) inhibitor, on antigen-induced eosinophil recruitment into the airways of sensitized mice and on IL-5-induced chemokinesis and adhesiveness of eosinophils. The in vivo administration of AG-490 prevented antigen-induced eosinophil infiltration in the airways of sensitized mice in a dose-dependent manner. However, the administration of AG-490 did not affect antigen-induced IL-5 production in the airways nor in vitro antigen-induced IL-5 production and T cell proliferation of spleen cells. Furthermore, AG-490 inhibited IL-5-induced chemokinesis and beta1-integrin adhesiveness of eosinophils in vitro. Because antigen-induced eosinophil recruitment into the airways is mediated by IL-5, these results indicate that JAK2 activation is critical for antigen-induced, IL-5-dependent mobilization of eosinophils into the tissue.