Stephen M. Spaethe

Effect of a leukotriene B4 receptor antagonist, LY293111, on allergen induced responses in asthma.

BACKGROUND: Leukotriene (LT) B4 is a potent neutrophil chemoattractant and also stimulates eosinophils in vitro, but its role in asthmatic inflammation is unknown. METHODS: The effect of the novel LTB4 receptor antagonist, LY293111, was examined using allergen challenge as a model for asthmatic inflammation in 12 atopic asthmatic subjects in a double blind placebo controlled crossover trial. Subjects with an established early (EAR) and late asthmatic response (LAR) to allergen at screening received oral LY293111 in a dose of 112 mg three times daily for seven days or placebo before further allergen challenge. Each treatment was separated by a washout period of 28 days. Individuals underwent histamine challenge one hour before and three hours after allergen challenge. Bronchoalveolar lavage (BAL) fluid was obtained at bronchoscopy 24 hours after allergen challenge. RESULTS: There was no difference in baseline lung function, EAR, LAR, or in airway responsiveness to histamine before and after allergen between placebo and LY293111. By contrast, treatment with LY293111 significantly reduced the number of neutrophils in BAL fluid expressed as both absolute cell numbers and percentage cell differential counts: absolute cell counts, median (range) 0.04 (0.02-0.15) x 10(6) after LY293111, 0.09 (0.02-0.43) x 10(6) after placebo; percentage differential cell counts 0.35 (0.1-2.0) after LY293111, 0.80 (0.1-3.6) after placebo (p < 0.05). Eosinophils, macrophages, and lymphocytes in BAL fluid did not differ between treatments. There was a significant reduction in the concentration of myeloperoxidase (MPO) with both placebo (16 (6.6) ng/ml) and LY293111 (3.5 (1.8) ng/ml) and of LTB4 (placebo 4.6 (1.2) pg/ml, LY293111 2.2 (0.2) pg/ml). Concentrations of LTC4 and interleukin 8 were reduced, although not significantly, whereas concentrations of interleukin 6, GM-CSF, and TNF-alpha were unchanged by LY293111. CONCLUSIONS: These results demonstrate an influence of LTB4 on neutrophil influx and activation in the airway following allergen challenge. Despite this anti-inflammatory effect, there was no measured physiological benefit and this questions the functional role of the neutrophil in the pathophysiology of allergen induced asthma.

Regulation of human eosinophil degranulation and activation by endogenous phospholipase A2.

The unique granular proteins of eosinophils may have a pathogenetic role in asthma and in the defense against parasitic infestations. However, the mechanisms regulating eosinophil degranulation are largely unknown. We examined the hypothesis that release of these proteins is regulated by endogenous activation of phospholipase A2. Human eosinophils (HE) were isolated from the peripheral blood of 42 subjects either by Percoll density separation or by negative-selection immunomagnetic fractionation. Eosinophil activation was initiated in vitro with 10(-6) M FMLP and 5 micrograms/ml cytochalasin B and was assessed by measurement of eosinophil peroxidase (EPO), leukotriene C4 (LTC4) and superoxide radical (.O2-) secretion. Treatment of HE with 100 microM mepacrine before activation blocked EPO release (2.0 +/- 0.2 vs 10.2 +/- 2.1% cell content for activated HE, P < 0.004, n = 9), .O2- generation (2.6 +/- 0.9 vs 44.2 +/- 10.8 nmol/ml per 10(6) HE, P < 0.002, n = 5), and LTC4 secretion (68.2 +/- 32.2 vs 1,125.2 +/- 526.8 pg/ml per 10(6) HE, P < 0.04, n = 8). Pretreatment of HE with 100 microM 4-bromophenacyl bromide before activation similarly blocked EPO release, .O2- generation and LTC4 secretion. Addition of AA to HE after treatment with 100 microM mepacrine and before subsequent activation reversed the inhibition of both EPO (10.4 +/- 2.2% with 1 microM AA vs 2.0 +/- 0.2% for mepacrine, n = 5, P < 0.02) and LTC4 secretion (695.1 +/- 412.9 with 10 microM AA vs 68.2 +/- 32.2 pg/ml per 10(6) HE for mepacrine, n = 8, P < 0.04), but did not reverse inhibition of .O2- generation by mepacrine. We demonstrate that secretion of preformed cytotoxic proteins and .O2- by eosinophils is regulated endogenously by phospholipase A2.

Blockade of human neutrophil activation by 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]propoxy]phenoxy]benzoic acid (LY293111), a novel leukotriene B4 receptor antagonist

Leukotriene B4 (LTB4), a naturally occurring pro-inflammatory product of arachidonic acid metabolism, has been associated with human inflammatory disease. This study compares the abilities of two LTB4 receptor antagonists, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]- propoxy]phenoxy]benzoic acid (LY293111) and 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), to displace LTB4 binding and their functional blockade of human neutrophil activation. LY293111 inhibited the binding of [3H]LTB4 with a Ki of 25 nM; SC-41930 displayed a similar potency (Ki = 17 nM). In contrast, LY293111 prevented LTB4-induced calcium mobilization with an IC50 = 20 nM, or 40 times more effectively than SC-41930 (IC50 = 808 nM). LY293111 was 300 times more potent than SC-41930 in blocking LTB4-induced CD11b up-regulation on isolated neutrophils. LY293111 also arrested LTB4-induced up-regulation of CD11b on neutrophils in whole human blood. LY293111 was not effective in blocking human neutrophil activation responses induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), platelet-activating factor (PAF), human recombinant endothelial interleukin-8 (IL-8) or human recombinant complement component 5a (C5a).

A kinetic assay for eosinophil peroxidase activity in eosinophils and eosinophil conditioned media

The activity of eosinophil peroxidase (EPO) is commonly employed as a measure of eosinophil activation in biologic fluids. Determination of product formation by this enzyme by end-point measurement may be affected profoundly by substrate concentrations, reaction time and degradation of end-product and enzyme. To determine more accurately EPO concentrations in media conditioned by isolated, purified eosinophils, we have developed a kinetic, colorimetric assay to measure EPO concentration as a function of maximum velocity of reaction (Vmax). An automated method for determining Vmax in a 96-well microplate colorimetric assay was utilized over a wide range of substrate concentrations. Concentrations greater than or equal to 3 x 10(-8) g/ml could be determined reliably with this assay. Peroxidase activity was inhibited in a concentration-dependent manner by the addition of 3-amino-1,2,4-triazole (AMT). The EPO concentration in eosinophils determined by this kinetic method was approximately 1.1 x 10(-5) g/10(6) eosinophils. Eosinophil activation with 10(-6) M f-Met-Leu-Phe (fMLP) caused substantial EPO secretion (9.0 +/- 1.7% vs. 2.9 +/- 0.6% total EPO content for control, P = 0.05) and decrease in eosinophil EPO concentration (92.3 +/- 4.2% of control, P = 0.038). Secretion was enhanced by the addition of 5 micrograms/ml cytochalasin B to 10(-6) M fMLP (25.9 +/- 12.7% total EPO content, P = 0.043 vs. control); similar decreases were noted in eosinophil EPO concentration (71.7 +/- 16.1% of control, P = 0.043). These data demonstrate that determination of EPO secretion by measurement of Vmax is a reliable, accurate method for precise quantification of this enzyme in media containing purified eosinophils or eosinophil products in the absence of other forms of peroxidase activity.

Differential effects of cyclosporine A after acute antigen challenge in sensitized cats in vivo and ex vivo

1 We determined the effect of cyclosporine A (CsA) treatment on mast cell degranulation and lung resistance (RL) in vivo, and tracheal smooth muscle (TSM) contraction ex vivo after antigen challenge in sensitized cats. We also determined the direct effects of addition of CsA to the tissue bath on antigen‐induced responses of TSM in vitro. 2 Cats (n=10) were sensitized by i.m. injection of Ascaris suum antigen (AA); 5 cats (CsA+) received CsA twice daily for 2 weeks before acute antigen challenge in doses sufficient to suppress interleukin‐2 secretion from feline peripheral blood mononuclear cells ex vivo. 3 Lung resistance increased comparably within 10 min of exposure to AA (P<0.03). Histamine content in bronchoalveolar lavage fluid from both groups increased comparably within 30 min of antigen challenge, from undetectable levels to 542±74 pg ml−1 post AA for CsA+ and from 74±19 pg ml−1 at baseline, to 970±180 pg ml−1 post AA CsA− (P<0.05; P=NS vs CsA+). 4 In excised TSM, active tension elicited by exposure to AA in vitro was 107±38% KCl in the CsA+ group vs 144±56% KCl in the CsA− group (P=NS). However, contraction of TSM (n=4) harvested from both groups was abolished or greatly diminished after AA challenge when tissues were pre‐incubated with 1 μM CsA in vitro (8±8% KCl, P<0.05 vs CsA+ and CsA−). This was associated with inhibited release of 5‐hydroxytryptamine into the organ bath fluid of tissues treated with CsA in vitro only. 5 We demonstrated that CsA treatment in vivo does not inhibit the early phase asthmatic response or mast cell degranulation following antigen challenge in sensitized cats. Additionally, the effects of CsA on mast cell function ex vivo do not reflect lack of effects of CsA on mast cell function in vivo in this animal model of atopic asthma.

Guinea pig whole blood 5-lipoxygenase assay : utility in the assessment of potential 5-lipoxygenase inhibitors

Guinea pigs are widely used in the study of the role of leukotrienes in airway pathophysiology. Extensive research efforts have utilized this species in the development of potential therapeutic agents associated with inhibition of leukotriene production (e.g. 5-lipoxygenase inhibitors and 5-lipoxygenase-activating protein antagonists) for the treatment of acute bronchospasm in asthma. We now report, for the first time, an ex vivo whole blood 5-lipoxygenase assay in guinea pigs which should prove useful in the future development of leukotriene biosynthesis inhibitors. Addition of 150 microM arachidonic acid (AA) to heparinized whole blood for 5 min prior to the stimulation with 20 micrograms/mL A23187 resulted in a 10-fold increase in leukotriene B4 (LTB4; 11.36 +/- 1.55 ng/mL) above basal (0.96 +/- 0.29 ng/mL) within 10 min. To further validate the utility of the assay, we utilized the 5-lipoxygenase inhibitor BW A4C. Pretreatment of guinea pig whole blood with BW A4C in vitro prior to stimulation resulted in a concentration-dependent inhibition of LTB4 production (IC50 = 229 nM), whereas thromboxane B2 (TXB2) production was unaffected. Likewise, when BW A4C was administered to guinea pigs intravenously (3 mg/kg), we observed a rapid and marked (approximately 90%) reduction in whole blood LTB4 production which returned to control (pre-drug values) by 5 hr. In contrast, TXB2 production was unaffected over the same experimental time period. In summary, we have described a whole blood assay which can discriminate 5-lipoxygenase inhibitors both in vitro and in vivo. Furthermore, this assay system will be of use in determining the potency, efficacy, selectivity, and pharmacodynamic properties of 5-lipoxygenase inhibitors in guinea pigs.

Pulmonary actions of LY255283, a leukotriene B4 receptor antagonist

The actions of LY255283, a leukotriene (LT) B4 receptor antagonist, were examined on guinea pig lung. LTB4 and LY255283 displaced [3H]LTB4 from its binding site on lung membranes with pKi values of 9.9 and 7.0, respectively. In the functional correlate of the binding studies, LY255283 competitively reduced contractile responses of lung parenchyma to LTB4 (pA2 = 7.2). LTB4 produced airway obstruction which was reduced by LY255283 administered i.v. (ED50 = 2.8 mg/kg) or orally (ED50 = 11.0 mg/kg). Contractile responses to histamine, LTD4 and the thromboxane mimetic, U46619, were not reduced by LY255283. The compound also did not inhibit cyclooxygenase or 5-lipoxygenase enzymes. We conclude that LY255283 selectively antagonized pharmacologic responses to LTB4 on lung tissue and appears to be a useful tool to investigate the role of LTB4 in pulmonary disease.

Rapid method for automated on-line extraction and fractionation of plasma leukotrienes and 12-hydroxy-5,8,10,14-eicosatetraenoic acids by reversed-phase high-performance liquid chromatography

A method is described for automated on-line extraction and fractionation of plasma leukotrienes (LTs) and (5Z,8Z,10E,14Z)-(12S)-hydroxy-5,8,10,14-eicosate traenoic acid [12(S)-HETE] by reversed-phase high-performance liquid chromatography (RP-HPLC). This method was utilized to assess the accuracy of leukotriene B4 (LTB4) and leukotriene C4 (LTC4) determinations obtained by direct immunoassay of guinea pig plasma. Plasma LTB4 levels were significantly higher (p less than 0.01) and plasma LTC4 levels were unchanged when immunoassays were performed post versus pre RP-HPLC fractionation. Rapid separation, high recovery and baseline separation of LTB4, LTC4 and 12(S)-HETE, and minimal hardware requirements clearly demonstrate the general utility of this method.

Contraction of guinea pig inferior vena cava by eicosanoids

SummaryGuinea pig inferior vena cava contracted in response to leukotriene (LT)C4, LTD4, LTE4 U46619, phenylephrine, histamine, and KCl. Although LTC4, LTD4, and U46619 were the most potent agonists, active tension generated by these eicosanoids was only about half that of histamine or KCl. LTE4 and phenylephrine were marginally active. Biochemical analysis showed vena cava able to convert about 23% LTC4 to LTD4 and LTE4 in 45 min. Pretreatment with acivicin prevented this by abrogating conversion of LTC4 to LTD4. A subthreshold concentration of LTE4 reduced responses to LTC4 and LTD4. LY171883 and WY-48252 competitively antagonized LTD4-induced contractions of vena cava. In contrast, these antagonists blocked contractions to LTC4 in a biphasic manner. Lower segments of the LTC4 concentration-response curves were less affected than the upper portion suggesting the possibility of 2 LTC4 receptor subtypes. Our results indicate that LTE4 is a weak or partial agonist in this tissue and furthermore they suggest a lack of high affinity receptors for LTE4 favoring LTC4 and LTD4. Indomethacin did not influence contractions to the leukotrienes or histamine. However, the response to U46619 was greatly enhanced suggesting release of a vasodilator prostaglandin as part of the overall response of the vena cava to the thromboxane A2 mimetic.

Contributory role of lung pleura to release of anaphylactic mediators from guinea pig lung in response to ovalbumin or A23187

Previous findings revealed greater contractile responses of guinea pig lung pleural surface strips to antigen or A23187 challenge than denuded lung parenchymal strips (lung strip devoid of any pleura). Moreover, we have identified a high density of mast cells distributed throughout the lung pleura. The present study examined mediators released from guinea pig lung pleural surface and denuded lung parenchyma fragments in response to immunologic challenge with ovalbumin (OA) or non-immunologic challenge with the ionophore A23187. Histamine levels were measured radioenzymatically; leukotrienes (LTs), prostaglandins (PGs) and thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), were quantitated using an enzyme immunoassay. Histamine release reached a maximal level 3-5 min after OA challenge, whereas A23187-induced histamine release increased gradually in a time-dependent manner. Similar kinetics were observed in the release of LTs, PGs and TXA2. Pleural surface released a substantially (P < 0.05) greater amount of histamine to both challenges than denuded parenchyma. Moreover, histamine content in pleural surface was significantly (P < 0.05) higher than in denuded parenchyma. Pleural surface also released considerably (P < 0.05) more LTB4, LTC4, and LTE4 in response to OA and A23187 than denuded parenchyma. In contrast, pleural surface and denuded parenchyma released equivalent amounts of PGD2, PGE2, PGF2 alpha, and TXA2 in response to both challenges. The rank order of leukotriene release was LTC4 > LTE4 > LTB4, whereas that of prostanoid release was TXA2 >> PGD2 > or = PGF2 alpha >> PGE2. We conclude that pleural surface is the major source of histamine and leukotrienes released from guinea pig lung in vitro in response to OA and A23187, whereas both pleural surface and denuded parenchyma participate to the same extent in prostaglandin and TXA2 production after such challenges.