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Inhibition of cPLA 2 Translocation and Leukotriene C 4 Secretion by Fluticasone Propionate in Exogenously Activated Human Eosinophils
Abstract
We examined the effect of the highly lipophilic corticosteroid, fluticasone propionate (FP), in causing (1) inhibition of nuclear translocation of cytosolic phospholipase A2 (cPLA2), and (2) blockade of leukotriene C4 (LTC4) synthesis in isolated human eosinophils in vitro. Eosinophils were isolated from peripheral blood, treated with either buffer or 10(-)10 M to 10(-)6 M FP in the presence of 10 pg/ml human recombinant interleukin-5 (rhIL-5) and activated with formyl-met-leu-phe (FMLP) + cytochalasin B (CB). At 24 h, stimulated LTC4 secretion from eosinophils was unchanged; however, when corrected for cell viability, LTC4 secretion decreased from 1,429 +/- 327 pg/10(6) cells to 762 +/- 113 pg/10(6) cells for eosinophils treated for 48 h with >/= 10(-)8 M FP (p < 0.003). FMLP/CB-stimulated translocation of cPLA2 to the nuclear envelope assessed by specific immunohistochemical staining also was blocked by FP. By contrast, membrane expression of annexin-1, which was not minimal at 30 min, was substantial at 48 h for eosinophils treated with > 10(-)10 M FP, and inhibition of LTC4 synthesis was reversed by exogenous arachidonic acid (AA). We find that FP causes a decrease in stimulated eosinophil secretion of LTC4 that is regulated by phospholipase A2 (PLA2). Inhibition of LTC4 synthesis precedes the global cytotoxic effects of FP as indicated by the simultaneous upregulation of annexin-1 expression. Inhibited stimulated secretion corresponds to inhibited translocation of cPLA2 to the nuclear envelope during cellular activation.
... Both eosinophils and neutrophils also express annexin-1 (ANXA1) [10 -12], which plays a critical role in the anti-inflammatory effects of glucocorticoids [2,3]. We have reported previously that activation of eosinophils by glucocorticoid causes translocation of ANXA1 from the cytoplasm to the outer plasma membrane of eosinophils [13]; this event blocked  2 -integrin-mediated adhesion [14]. Studies in vitro in polymorphonuclear leukocytes (PMNs) suggest that exogenous ANXA1 attenuates neutrophil migration at inflammatory sites [15]. ...
... This concentration (1 pg/ml GM-CSF) had no effect on either cell activation or adhesion in either control or experimental groups from the same subjects. For isolated eosinophil, IL-5 (10 pg/ml) was added to the culture media to maintain cell viability and similarly, had no effect on cell activation or adhesion (13). The incubation period was terminated by centrifugation at 400 g for 10 min, and the pellets were resuspended in PBS/1.0% ...
... Previous investigations have demonstrated that ANXA1 blocks eosinophil and PMN migration to the site of inflammation [27,28]. We have shown that upregulation of ANXA1 expression caused by FP blocks eosinophil synthesis of cysteinyl LTC 4 [13], affinity of  2 -integrin to ICAM-1 [14,21], and translocation of cytosolic gIVaPLA 2 to the perinuclear membrane [14,21]. Subsequent data obtained through fluorescence-activated cell sorting confirmed that FP is notably more potent and efficacious in augmenting the membrane-bound ANXA1 expression in eosinophils than in PMNs (Fig. 5). ...
We examined the effect of glucocorti- coid stimulation in blocking 2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from normal human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phos- phorylation, which are required for 2-integrin ad- hesion, were not affected by treatment with 107 M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 109 M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking 2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 syn- thesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block 2-integrin adhe- sion, a necessary step for granulocyte cell migra- tion in vivo. J. Leukoc. Biol. 83: 000-000; 2008.
... We have shown previously that short exposure (Ͻ30 min) of eosinophils to exogenous IL-5 causes cytosolic phospholipase A 2 (cPLA 2 ) phosphorylation but not translocation of cPLA 2 to the nucleus (30). This short exposure was nonetheless associated with augmented production of leukotriene C 4 (LTC 4 ) after exogenous stimulation with formyl-Met-Leu-Phe, which caused translocation of cPLA 2 to the nuclear membrane (24,30,31). ...
... Whereas these data did not establish the Th2-like cell as the cell causing direct activation of eosinophil secretion (as contrasted to augmentation of secretion), our data do not exclude the possibility that Th2 cells could trigger eosinophil secretion as well under more physiological circumstances. Although the mechanism by which augmented secretion was elicited by IL-5 and GM-CSF was not examined directly in this study, we have shown previously that rhIL-5 caused increased phosphorylation of cPLA 2 , the enzyme that hydrolyzes the nuclear membrane phospholipids causing arachidonic acid release, which is the initial step in the synthesis of cysteinyl leukotrienes (24,30,31). Because phosphorylation (30) and augmented secretion of LTC 4 occur within 30 min (24) after cell activation, we did not examine the effects of this short incubation time on mRNA expression of enzymes in the 5-lipoxygenase pathway. ...
We assessed the effect of anti-CD3-stimulated secretion of cultured human Th1- and Th2-like cells on leukotriene C(4) (LTC(4)) secretion in isolated human eosinophils. T helper (Th) cell subsets were generated from human naive CD4(+) T cells cocultured with irradiated human transformed B cells and either recombinant human interleukin (rhIL)-1beta plus rhIL-6 plus rhIL-12 for Th1-like cells or rhIL-1beta plus rhIL-6 plus rhIL-4 for Th2-like cells. Coincubation of eosinophils with 1:5 dilution of Th2-supernatant (Sup) caused an increase in LTC(4) secretion caused by 0.1 microM formyl-Met-Leu-Phe and 5 microg/ml cytochalasin B from 921 +/- 238 to 3,067 +/- 1,462 pg/10(6) eosinophils (P < 0.01). Th1-Sup at the same dilution had no augmenting effect on stimulated secretion of LTC(4) in eosinophils despite substantial concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant. Dilution of Th1-Sup caused increased LTC(4) that returned to baseline after immunoabsorption of GM-CSF, suggesting the presence of a possible inhibitory factor. We demonstrate that pretreatment of eosinophils with 1:5 dilution of Th2-Sup but not of Th1-Sup causes substantial augmentation of LTC(4) secretion in vitro and establishes that human Th2 cells cause direct augmentation of LTC(4) secretion within 15-30 min of exposure.
... 5). The current authors have previously shown that FP blocks eosinophil secretion of LTC 4 by preventing translocation of cPLA 2 to the perinuclear membrane [28]. The mechanism by which this occurs has not been further defined; however, inhibition required 48 h incubation, suggesting that FPinduced transcription is essential for the blockade of cPLA 2 migration. ...
... Finally, in preliminary experiments for this study, the authors found little efficacy of FP in concentrations comparable to those achieved in airways after inhalation. The addition of a moderate concentration SM to FP was essential to achieve substantial blockade of inhibition, and this blockade was achieved within a relatively short time course (12-24 h) in comparison with prior studies using corticosteroids alone [28]. ...
Migration of human eosinophils is regulated by integrin expression, conformational change, and activation of cytosolic phospholipase A2 (cPLA2). Corticosteroids have been shown to inhibit cPLA2 hydrolysis in human eosinophils. The objective of this study was to determine the mechanisms of fluticasone propionate (FP) alone or in combination with salmeterol (SM) in blocking adhesion mediated by b2-integrin in human eosinophils. Human eosinophils were isolated by negative magnetic selection. b2-integrin- mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min. Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (y30%) inhibited interleukin (IL)-5- induced eosinophil adhesion. Blockade of IL-5-induced eosinophil adhesion caused by 10-7 M FP at 24 h was augmented by 10-7 M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FPzSM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2. Blockade of b2-integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.
... Lipoxin A4's effect on eosinophils is thought to be mediated by the FPR2 receptor, which also transduces the antiinflammatory effect of annexin 1 (42). Both annexin 1 and FPR2 are upregulated by GCs and lead to the inhibition of AA production in a cytosolic phospholipase A2-dependent manner (43,44). Although the increased production of AA is directly associated with synthesis of proinflammatory leukotrienes, AA may also directly activate the phosphatase activity of PP5 (45). ...
The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.
... We thank S. Hart for some important observations about our paper [1], and we concur that while we have postulated that the effect obtained with fluticasone could be related to apoptosis as one possible mechanism, we have not established the initiating mechanism of the interaction between b 2adrenoceptor activation and flucticasone. In a prior publication, we used TUNEL assay [2], which showed no effect on apoptosis at 24 h for eosinophils incubated in an identical manner, as for the study in the current paper. Annexin-I synthesis increased at 24 h and further increased at 48 h in that study. ...
... Phospholipase A2 inhibition has also been identified as offtarget activity in some commonly used drugs. Non-pancreatic secretory phospholipase A2 (membrane associated) is inhibited by diclofenac [61,62], and cytosolic phospholipase A2 is inhibited by the commonly prescribed corticosteroid fluticasone propionate [63,64], as well as epirubicin [65] and niflumic acid [66]. ...
Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target ("distant off-targets"). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target ("distant off-target").
... Because LTB 4 biosynthesis by AMs in vitro can be modulated by environmental factors in vivo, such as cigarette smoking [32], smokers were excluded from the study. Glucocorticoids have been shown to inhibit LT generation through inhibition of phospholipase A 2 activity [33,34]. Chronic oral corticosteroid therapy may lead to a suppression of eicosanoid biosynthesis and could underlie the baseline reduction in LXA 4 and LTB 4 observed in the macrophages from patients with severe asthma. ...
An imbalance in the generation of pro-inflammatory leukotrienes, and counter-regulatory lipoxins is present in severe asthma. We measured leukotriene B4 (LTB4), and lipoxin A4 (LXA4) production by alveolar macrophages (AMs) and studied the impact of corticosteroids.
AMs obtained by fiberoptic bronchoscopy from 14 non-asthmatics, 12 non-severe and 11 severe asthmatics were stimulated with lipopolysaccharide (LPS,10 microg/ml) with or without dexamethasone (10(-6)M). LTB4 and LXA4 were measured by enzyme immunoassay.
LXA4 biosynthesis was decreased from severe asthma AMs compared to non-severe (p < 0.05) and normal subjects (p < 0.001). LXA4 induced by LPS was highest in normal subjects and lowest in severe asthmatics (p < 0.01). Basal levels of LTB4 were decreased in severe asthmatics compared to normal subjects (p < 0.05), but not to non-severe asthma. LPS-induced LTB4 was increased in severe asthma compared to non-severe asthma (p < 0.05). Dexamethasone inhibited LPS-induced LTB4 and LXA4, with lesser suppression of LTB4 in severe asthma patients (p < 0.05). There was a significant correlation between LPS-induced LXA4 and FEV1 (% predicted) (r(s) = 0.60; p < 0.01).
Decreased LXA4 and increased LTB4 generation plus impaired corticosteroid sensitivity of LPS-induced LTB4 but not of LXA4 support a role for AMs in establishing a pro-inflammatory balance in severe asthma.
... Application of IL-8/ CXCL8 translocated the cytosolic gIVaPLA 2 to some extent, to the nuclear component of PMNs (Figure 3b). We used FMLP as a positive control since we previously have shown that FMLP causes the translocation of cytosolic gIVaPLA 2 to nuclear membrane in eosinophils [33]. ...
Cytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of beta2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro.
Migration of PMNs caused by IL-8/CXCL8 was assessed using a transwell migration chamber. PMNs were pretreated with two structurally unrelated inhibitors of gIVaPLA2, arachidonyl trifluoromethylketone (TFMK) or pyrrophenone, prior to IL-8/CXCL8 exposure. The fraction of migrated PMNs present in the lower chamber was measured as total myeloperoxidase content. GIVaPLA2 enzyme activity was analyzed using [14C-PAPC] as specific substrate F-actin polymerization and cell structure were examined after rhodamine-phalloidin staining.
IL-8/CXCL8-induced migration of PMNs was elicited in concentration- and time-dependent manner. Time-related phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus was observed for PMNs stimulated with IL-8/CXCL8 in concentration sufficient to cause upstream phosphorylation of MAPKs (ERK-1/2 and p38) and Akt/PKB. Inhibition of gIVaPLA2 corresponded to the magnitude of blockade of PMN migration. Neither AA nor LTB4 secretion was elicited following IL-8/CXCL8 activation. In unstimulated PMNs, F-actin was located diffusely in the cytosol; however, a clear polarized morphology with F-actin-rich ruffles around the edges of the cell was observed after activation with IL-8/CXCL8. Inhibition of gIVaPLA2 blocked change in cell shape and migration caused by IL-8/CXCL8 but did not cause F-actin polymerization or translocation of cytosolic F-actin to inner leaflet of the PMN membrane.
We demonstrate that IL-8/CXCL8 causes a) phosphorylation and translocation of cytosolic gIVaPLA2 to the nucleus, b) change in cell shape, c) polymerization of F-actin, and d) chemoattractant/migration of PMN in vitro. Inhibition of gIVaPLA2 blocks the deformability and subsequent migration of PMNs caused by IL-8/CXCL8. Our data suggest that activation of gIVaPLA2 is an essential step in PMN migration in vitro.
... 36 Recent evidence suggests fluticasone inhibits LTC 4 synthesis in human eosinophils in vitro by blocking translocation of cPLA 2 to the nuclear envelope. 37 Such a mechanism is consistent with dual inhibition of LTB 4 and LTC 4 synthesis reported in MNC in the present study and with suppression of prostanoid synthesis reported by others. 33 Overall, the eVects of corticosteroids on LT synthesis in vivo depend on the populations and activation status of leucocytes in the airway and other compartments, and on the balance between the induction of lipocortin expression, modulation of PLA 2 , 5-LO, and/or FLAP activity and expression, and changes in the density of membrane receptors for immunological stimuli. ...
The cysteinyl-leukotrienes (LTC(4), LTD(4), LTE(4)) are critical bronchoconstrictor and eosinophilotactic mediators in asthma while LTB(4) is a potent neutrophil chemoattractant. Glucocorticosteroids are front line anti-inflammatory treatment for asthma but the evidence that they reduce leukotriene (LT) synthesis in vivo is poor.
In a randomised, double blind, placebo controlled, crossover trial immunoassays were used to measure ex vivo synthesis of LTC(4) and LTB(4) by calcium ionophore stimulated blood leucocytes and bronchoalveolar lavage (BAL) cells of eight normal subjects and eight patients with mild allergic asthma 4-6 hours after intravenous administration of a single 100 mg dose of methylprednisolone.
Ionophore stimulated synthesis of LTC(4) (but not LTB(4)) in blood granulocytes tended to be higher in asthmatic subjects (mean 9.7 ng/10(6) cells) than in normal subjects (4.2 ng/10(6) cells; p = 0.08) and intravenous methylprednisolone reduced synthesis of LTC(4) (but not LTB(4)) to normal levels (2.9 ng/10(6) cells; 95% CI for the reduction 1.0 to 12.5 ng/10(6) cells; p = 0.03). In blood mononuclear cells methylprednisolone reduced LTC(4) synthesis in asthmatic subjects from 1.26 to 0.79 ng/10(6) cells (95% CI for the reduction 0.26 to 0.79, p = 0.014) and tended to reduce LTC(4) synthesis in normal subjects from 1.51 to 0.86 ng/10(6) cells (p = 0.08). Methylprednisolone also significantly reduced synthesis of LTB(4) in mononuclear cells from both subject groups (p = 0.014). It had no effect on LT synthesis in BAL cells from either group nor on LT levels in BAL fluid.
Intravenous methylprednisolone can reduce synthesis of leukotrienes in blood granulocytes and mononuclear cells within six hours of a single intravenous dose.
... However, it is likely that more investigations are required, perhaps on other species or with human cells in vitro, before discarding any function for ANXA1 on this leukocyte type. Interestingly, treatment of human eosinophils with the GC fluticasone produces ANXA1 externalization and this is functionally and temporally related to inhibition of phospholipase A 2 translocation into the nucleus and leukotriene release (Sano et al. 1999). Finally, the analogies between ANXA1 and lipoxin A 4 described in the neutrophil (and discussed above) together with the receptor-mediated effects of this lipid on eosinophil trafficking (Bandeira-Melo et al. 2000), imply a need for a more systematic investigation. ...
The concept of anti-inflammation is currently evolving with the definition of several endogenous inhibitory circuits that are important in the control of the host inflammatory response. Here we focus on one of these pathways, the annexin 1 (ANXA1) system. Originally identified as a 37 kDa glucocorticoid-inducible protein, ANXA1 has emerged over the last decade as an important endogenous modulator of inflammation. We review the pharmacological effects of ANXA1 on cell types involved in inflammation, from blood-borne leukocytes to resident cells. This review reveals that there is scope for more research, since most of the studies have so far focused on the effects of the protein and its peptido-mimetics on neutrophil recruitment and activation. However, many other cells central to inflammation, e.g. endothelial cells or mast cells, also express ANXA1: it is foreseen that a better definition of the role(s) of the endogenous protein in these cells will open the way to further pharmacological studies. We propose that a more systematic analysis of ANXA1 physio-pharmacology in cells involved in the host inflammatory reaction could aid in the design of novel anti-inflammatory therapeutics based on this endogenous mediator.
... Moreover, cAMP and the cAMP-responsive transcription factor cAMP response element-binding protein have been implicated in the up-regulation of annexin-1 [35]. However, the interaction of annexin-1 with cPLA 2 ␣ led to a decreased cPLA 2 ␣ phosphorylation at Ser-505 and translocation to the peri-nuclear membranes [36,37], two events that are not altered by histamine in human PMN [13]. Therefore, a role of annexin-1 in the cAMP-mediated inhibition of LT and PAF biosynthesis appears unlikely. ...
Leukotrienes (LT) and platelet-activating factor (PAF) are important lipid mediators of inflammation. We and others reported previously that autacoids such as adenosine, histamine, prostaglandin E2, and beta-adrenergic agents inhibit LT biosynthesis in activated human polymorphonuclear leukocytes (PMN). In this study, we demonstrate that CGS-21680 (a selective agonist of the adenosine A2A receptor) and histamine also potently inhibit PAF biosynthesis in agonist [formyl Met-Leu-Phe (fMLP)]- and thapsigargin-activated human PMN. The observed inhibitions of PAF biosynthesis were reversed effectively by exogenous 1-O-alkyl-lyso-sn-glyceryl-3-phosphocholine (lyso-PAF), suggesting that these effects of CGS-21680 and histamine implicate the blockade of cytosolic phospholipase A2alpha (cPLA2alpha) activity and lyso-PAF release and that the acetyl-coenzyme A/lyso-PAF acetyl transferase is not inhibited by the autacoids. Accordingly, the cPLA2alpha inhibitor pyrrophenone completely blocked PAF formation, and lyso-PAF similarly prevented this effect of pyrrophenone. The inhibitory effects of CGS-21680 and histamine on PAF biosynthesis were prevented by the protein kinase A inhibitor H-89, supporting roles for the Gs -coupled receptors A2A and H2, respectively, and cyclic adenosine monophosphate in the inhibitory mechanism. The fMLP-induced phosphorylations of p38 and extracellular signal-regulated kinase 1/2 were not altered significantly by the CGS-21680, indicating that inhibition of these kinases is not involved in the inhibitory effect of the adenosine A2A receptor ligand on LT and PAF biosynthesis. These data further emphasize the multiple and potent inhibitory effects of adenosine and histamine on leukocyte functions, in particular, on the biosynthesis of two classes of important lipid mediators and their putative regulatory roles in immune processes in health and diseases.
... Both eosinophils and neutrophils also express annexin-1 (ANXA1) [10 –12], which plays a critical role in the anti-inflammatory effects of glucocorticoids [2, 3]. We have reported previously that activation of eosinophils by glucocorticoid causes translocation of ANXA1 from the cytoplasm to the outer plasma membrane of eosinophils [13]; this event blocked 2 -integrin-mediated adhesion [14]. Studies in vitro in polymorphonuclear leukocytes (PMNs) suggest that exogenous ANXA1 attenuates neutrophil migration at inflammatory sites [15]. ...
We examined the effect of glucocorticoid stimulation in blocking beta 2-integrin adhesion of polymorphonuclear leukocytes (PMNs) isolated from human subjects. Surface expression of CD11b and ERK-1/2-mediated gIVaPLA2 phosphorylation, which are required for beta 2-integrin adhesion, were not affected by treatment with < or =10(-6) M fluticasone propionate (FP) for PMNs activated by either 10(-7) M LTB4 or 30 ng/ml TNF-alpha and caused no significant blockade of beta 2-integrin adhesion in vitro. Baseline expression of annexin-1 (ANXA1) synthesis was increased only after 10(-6) M FP for PMNs; by contrast, comparable increase in ANXA1 expression was demonstrated in human eosinophils from the same subjects with 10(-8) M FP. Viability of PMNs was verified by propidium iodide and by the persistence of beta 2-integrin adhesion in treated groups. Exogenous administration of ANXA1 mimetic peptide fragment blocked significantly and comparably the beta 2-integrin adhesion in PMNs activated by LTB4 and TNF-alpha and in eosinophils activated by IL-5. Translocation of gIVaPLA2 from the cytosol to the nucleus also was refractory for activated PMNs treated with > or =10(-7) M FP; by contrast, complete blockade of nuclear translocation of cytosolic gIVaPLA2 was effected by 10(-9) M FP in eosinophils. Our data indicate that the cell surface ANXA1 synthesis is capable of blocking beta 2-integrin adhesion in both PMNs and eosinophils. However, in contrast to eosinophils, FP does not cause either substantial ANXA1 synthesis or nuclear transport of cytosolic gIVaPLA2 in PMNs and thus does not block beta2-integrin adhesion, a necessary step for granulocyte cell migration in vivo.
Study objectives
Antileukotriene drugs are widely used in patients with bronchial asthma, but not all patients show significant clinical improvements, and no factors have been identified that are correlated with the clinical response to these drugs. This study was designed to examine the factors correlated with a response to a leukotriene receptor antagonist, pranlukast, in patients with asthma.
Design
WBC counts, IgE, and ex vivo leukotriene release from leukocytes were measured, and 31 patients with asthma were treated with pranlukast, a leukotriene receptor antagonist, for 4 weeks.
Measurements
Outcome measurements included twice-daily peak expiratory flow rate (PEFR), daytime and nocturnal symptoms, and frequency of β2-agonist use. Subjects with a reduction of > 20% in symptom scores or β2-agonist use, or an improvement of PEFR of > 10% were designated as responders; others were designated as nonresponders. Logistic regression analysis assessed the efficacy of models using various allergic markers correlated with the response to pranlukast.
Results
There were 16 responders and 15 nonresponders. The release of cysteinyl leukotrienes from the leukocytes of the responders was higher than that of the nonresponders (p < 0.05). There was a significant correlation between the clinical response and the release of cysteinyl leukotrienes, but not demographic features, WBC counts, percentage of eosinophils, or serum IgE levels (p < 0.05). Subjects with a release of cysteinyl leukotrienes of > 3,500 pg/mL were 11.0 times more likely to respond to pranlukast than those with < 3,500 pg/mL (95% confidence interval, 2.0 to 60.5).
Conclusion
Cysteinyl leukotriene release from leukocytes is correlated with leukotriene receptor antagonist response.
The effects of a variety of widely used anti-inflammatory agents (dexamethasone, indomethacin, and montelukast) as well as ubiquitous mediators of inflammation (prostaglandin E2 and nitric oxide) on the development of murine eosinophils ex vivo and in vivo have been studied over the last decade. The results indicate that developing eosinophils differ markedly in their responses to these agents from the mature forms of the same lineage, studied either in allergic human subjects or experimental animal models of allergic disease. Most strikingly, glucocorticoids strongly enhance eosinophil development, both in vitro and in vivo. The enhancing effects are also observed during stress reactions and are strictly dependent on stress-induced glucocorticoid hormone production from the adrenal glands. Some, but not all, of the developmental effects of glucocorticoids on eosinophils could be accounted for their ability to prevent generation of nitric oxide through inducible NO synthase, which leads to apoptosis through the CD95-CD95L pathway. A novel mechanism for the effects of indomethacin in upregulating the development of eosinophils has also been documented. Evidence that lineage-specific as well as stage-specific cellular response programmes determine these different outcomes is discussed, along with the perspectives for future research.
This article discusses the role of cysteinyl leukotrienes in asthma and addresses the problem of the use of leukotriene receptor antagonists (LTRA) in the treatment of asthma. The author points out that these drugs are inferior to inhaled corticosteroids and long acting beta agonists and should be used as "add on" drugs being a third choice in the management of asthma. Lack of response to LTRA has a genetic background. Side effects of LTRA therapy is discussed, particularly Churg-Strauss syndrome. According to the recent literature, LTRA monotherapy should not be applied. In general, the author suggests that there is a risk of LTRA overtreatment particularly in children.
Authors describe the clinical implications of nongenomic effects of glucocorticoids, important also for the inhaled drugs. Data suggest that these rapid actions of glucocorticoids are important in the pharmacotherapy of asthma, particularly for prevention and treatment of acute exacerbations. Their influence on remodeling is also considered.
T cells and eosinophils, which are found in close proximity in asthmatic lungs, express many surface receptors that are counterligands. These data suggest that direct interactions between these cell types could play an important role in regulating airway inflammation in asthma. We examined the effect of selective adhesion between counterligands on human eosinophils and CD4+ T cells to determine 1) the existence of specific adhesive interactions and 2) if augmented specific adhesion to CD4+ T cells also caused augmented secretion of leukotriene C4 (LTC4) from eosinophils. A new method for binding of human CD4+ T cells to microwell plates was developed, which allowed for specific quantitative assessment of eosinophil adhesion to individual CD4+ T cells in culture. Adhesion of CD4+ T cells to eosinophils was minimal in unstimulated cells but increased after activation of T cells by PMA. Augmented adhesion was regulated substantially through binding of ICAM-3 and only minimally by ICAM-1. We further evaluated whether this specific adhesion up-regulated stimulated secretion of LTC4 from eosinophils. Adhesion with CD4+ T cells augmented eosinophil secretion of LTC4 caused by FMLP plus cytochalasin. Blockade of ICAM-3, as well as ICAM-1, inhibited completely the augmented secretion of eosinophil LTC4. We demonstrate that eosinophils and CD4+ T cells are capable of ligand-specific adhesion that is mediated predominantly by ICAM-3 ligation and that this binding causes augmented eosinophil secretion.
The 85 kDa cytosolic phospholipase A2 (cPLA2) is essential for arachidonic acid release and eicosanoid production by agonists that do or do not mobilize calcium in mouse peritoneal macrophages. In other models such as LPS-stimulated human neutrophils or carbachol/neomycin-treated human astrocytoma cells, cPLA2 has been reported to be activated by mechanisms that are independent of increased levels of calcium (30,57). Results with Sf9 cells suggest that a functional C2 domain plus additional regions of cPLA2 are required to mediate arachidonic acid release in the absence of increased calcium levels. The role of phosphorylation of cPLA2 in agonist-induced arachidonic acid release has been controversial. In murine macrophages and human neutrophils, different MAPK pathways are essential for CPLA2-mediated arachidonic acid release, but they can act through mechanisms that are independent of S505 phosphorylation. However, MAPK activation is not sufficient to induce arachidonic acid release. Thus, additional unknown mechanisms are involved in regulating cPLA2-mediated arachidonic acid release by agonists that do not increase calcium levels. Alternative mechanisms that have been suggested to be involved in cPLA2 activation include guanine nucleotide-binding proteins (4,57-60), tyrosine phosphorylation of cPLA2 (61, 62) or peroxidation of membrane phospholipids (63). The roles of these and other possible mechanisms, in addition to calcium and site-specific phosphorylation, in the regulation of cPLA2 remain to be elucidated.
The use of inhaled corticosteroids compared with leukotriene modifying drugs in the treatment of persistent asthma has not been extensively studied.
To compare the efficacy and safety of a low dose of fluticasone propionate (FP) and zafirlukast in patients previously maintained on inhaled corticosteroids.
Patients (> or = 12 years old; FEV1 = 60% to 85% of predicted) with persistent asthma who were previously treated with low doses of triamcinolone acetonide (TAA) 400 to 800 microg/day or beclomethasone dipropionate (BDP) 168 to 336 microg/day were randomized to treatment with FP aerosol 88 microg BID (FP, n = 221) or zafirlukast 20 mg BID (n = 216) over 6 weeks.
Treatment with FP significantly increased the mean change at endpoint (the last post-baseline observation) in FEV1 (0.22 L versus 0.03 L, P < .001), morning PEF (17.8 versus 3.1 L/min, P = .004), evening PEF (16.7 versus 2.6 L/min, P = .002), the percentage of symptom-free days (16.2 versus 7.1%, P = .007), and the percentage of rescue-free days (23.4 versus 9.3%, P < .001), and significantly decreased rescue albuterol use (-0.66 puffs/day versus an increase of 0.27 puffs/day, P < .001) and combined symptom scores (-0.13 versus an increase of 0.08, P < .001) compared with zafirlukast. Treatment with FP maintained the percentage of awakening-free nights (-1.0 +/- 1.0); in contrast, treatment with zafirlukast reduced the percentage of awakening-free nights (-9.0 +/- 1.6, P < .001). A clinically meaningful difference (change of > or = 0.5; P < .001) was observed between FP and zafirlukast in the Asthma Quality of Life Questionnaire (AQLQ) global score and for each domain score except activity limitation (change of 0.3, P < .001). Significantly more patients in the zafirlukast group experienced an asthma exacerbation (n = 14) compared with FP-treated patients (n = 5, P = .035). Patients in the zafirlukast group were significantly more likely to be withdrawn due to lack of efficacy (P < .001).
Switching patients from low doses of inhaled corticosteroids to a lower total microgram dose of FP improves pulmonary function, asthma symptoms, and quality of life, while switching to the leukotriene receptor antagonist zafirlukast may result in worsening of asthma control. This was indicated by the significant number of zafirlukast-treated patients who were dropped from the study due to lack of efficacy within 6 weeks of discontinuing inhaled corticosteroids.
Both inhaled corticosteroids and leukotriene modifiers are used in the maintenance treatment of persistent asthma.
The goal was to compare the efficacy and safety of low-dose fluticasone propionate (FP) and montelukast as first-line maintenance therapy in symptomatic patients by using short-acting beta2-agonists alone to treat persistent asthma.
In this multicenter, randomized, double-blind, double-dummy, parallel-group study, 533 patients (>15 years old) with persistent asthma who remained symptomatic while taking short-acting beta2-agonists alone were treated with FP (88 microg [2 puffs of 44 microg] twice daily) or montelukast (10 mg once daily) for 24 weeks.
Compared with treatment with montelukast, treatment with FP resulted in significantly greater improvements at endpoint in morning predose FEV(1) (22.9% vs 14.5%, P <.001), forced midexpiratory flow (0.66 vs 0.41 L/sec, P <.001), forced vital capacity (0.42 vs 0.29 L, P =.002), morning peak expiratory flow (PEF) (68.5 vs 34.1 L/min, P <.001), and evening PEF (53.9 vs 28.7 L/min, P <.001). Similar improvements in PEF were observed in patients with milder asthma (>70%-80% predicted FEV(1)). At endpoint, FP was more effective than montelukast at decreasing rescue albuterol use (3.1 puffs/day vs 2.3 puffs/day, P <.001), asthma symptom scores (-0.85 [48.6% decrease] vs -0.60 [30.5%], P <.001), and nighttime awakenings due to asthma (-0.64 awakenings/night [62% decrease] vs -0.48 awakenings/night [47.5%], P =.023), and FP increased the percentage of symptom-free days (32.0% vs 18.4% of days, P <.001) compared with montelukast. The adverse event and asthma exacerbation profiles for FP and montelukast were similar.
Low-dose FP is more effective than montelukast as first-line maintenance therapy for patients with persistent asthma who are undertreated and remain symptomatic while taking short-acting beta2-agonists alone.
Human blood polymorphonuclear cells, which biosynthesize eicosanoids from the 5-lipoxygenase (5-LO) pathway, are likely to be involved in asthma, in which glucocorticoids represent the first line of therapy. Their effects on leukotriene release after a short course of treatment, which have been reported in several studies, are controversial.
We sought to investigate whether long-term oral glucocorticoids inhibit lipid mediators from the 5-LO pathway.
Twelve normal control subjects, 29 asthmatic subjects, and 50 glucocorticoid-dependent asthmatic subjects were included in the study. Polymorphonuclear cells were studied for endogenous and transcellular metabolism of eicosanoids.
Total leukotriene B(4) production was significantly lower in cells from glucocorticoid-dependent asthmatic subjects (mean +/- SD, 177 +/- 26 ng/10(7) cells) than in control subjects (406 +/- 27), untreated asthmatic subjects (421 +/- 34), and asthmatic subjects treated with inhaled glucocorticoids (290 +/- 56). When incubated with arachidonic acid, these polymorphonuclear cells released very low amounts of 5(S)- and 12(S)-hydroxy-eicosatetraenoic acid (HETE), whereas endogenous 15(S)-HETE was found in substantial amounts. The transformation of exogenous 15(S)-HETE into 5(S),15(S)-diHETE and lipoxins was significantly more important in untreated asthmatic subjects than in control subjects and glucocorticoid-dependent asthmatic subjects.
This study showed that long-term oral corticotherapy affects the 5-LO activity and leads to a decrease production of all metabolites in contrast to short-term or inhaled glucocorticoids. This study also questions the site of action of glucocorticoids in regulating the availability of arachidonic acid and potential eicosanoid regulation, as previously held in phospholipase A2 studies.
To compare the short-term efficacy and safety of low-dose fluticasone propionate with that of oral zafirlukast therapy for patients previously treated with beta-2-agonists alone, and to evaluate the potential therapeutic benefit of switching from zafirlukast to a low-dose inhaled corticosteroid.
This study consisted of a 4-week randomized, double-blind treatment period followed by a 4-week open-label period. Two hundred ninety-four patients > or =12 years old with asthma previously uncontrolled with beta-2-agonists alone were randomly assigned to treatment with low-dose inhaled fluticasone (88 microg twice daily) or oral zafirlukast (20 mg twice daily). After 4 weeks, all patients discontinued their double-blind therapy and received open-label fluticasone (88 microg twice daily). Outcomes included pulmonary function, asthma symptoms, albuterol use, asthma exacerbations, and adverse events.
During the double-blind treatment period, fluticasone patients had significantly greater improvements in morning peak flow (29.3 L/min vs. 18.3 L/min), percentage of symptom-free days (19.8% vs. 11.6%), and daily albuterol use (-1.8 puffs per day vs. -1.1 puffs per day) compared with zafirlukast patients (P < or =0.025, each comparison). During the open-label treatment period, patients switched from zafirlukast to fluticasone experienced additional improvements in morning peak flow (17.2 L/min), evening peak flow (13.6 L/min), and FEV(1) (0.11 liter) and daily albuterol use (-0.9 puffs daily) compared with values obtained at the end of the double-blind treatment period (P < or =0.001, each comparison).
Low-dose fluticasone was more effective than zafirlukast in improving pulmonary function and symptoms in patients with persistent asthma. In addition, switching patients from zafirlukast to fluticasone further improved clinical outcomes.
There are limited published data describing the relative efficacy of available treatment options in younger versus older patients with persistent asthma.
To compare the efficacy of fluticasone propionate (FP) and zafirlukast (Z) in younger (12 to 49 years of age) versus older (50 years and older) patients with asthma.
A retrospective analysis of five randomized, double-blind, double-dummy studies 4 to 12 weeks in duration of 1,742 patients <50 years of age and 243 patients aged 50 years or older. Interventions were inhaled fluticasone propionate (FP) 88 microg, oral Z 20 mg, or placebo twice daily.
Treatment with FP resulted in significantly greater improvements than Z in all efficacy measurements (except for nighttime awakenings) regardless of age. In older patients, treatment with FP significantly increased pulmonary function compared with Z: FEV (FP= +0.19 L; placebo = -0.34 L; Z = -0.06 L); AM peak expiratory flow rate [PEFR] (FP = +25 L/minute; placebo = -18 L/minute; Z = +4 L/minute); PM PEFR (FP = +24 L/minute; placebo = -24 L/minute; Z = +5 L/minute; P < or = 0.023; for all comparisons). Compared with Z, treatment with FP in older patients also resulted in significantly greater increases in the percentage of symptom-free days (25% vs 13%) and rescue-free days (35% vs 17%); and significantly greater reductions in albuterol use (-1.6 vs -0.3 puffs/day) and the percentage of patients with exacerbations (2.7% vs 14.3%; P < or = 0.031).
Regardless of age, treatment with FP in patients with asthma significantly improved pulmonary function and overall asthma control. In contrast, treatment with Z in older patients with asthma resulted in small improvements in asthma symptoms, whereas lung function improved minimally or not at all, and exacerbations increased. These data suggest that FP effectively controls inflammation in older patients, whereas Z may mask inflammation and may not provide the level of bronchodilatory or anti-inflammatory activity needed for effective asthma control in older patients.
To compare the efficacy and safety of fluticasone propionate and zafirlukast in patients with relatively stable persistent asthma who were previously treated with inhaled corticosteroids and short-acting beta(2)-agonists.A total of 440 patients (> or =12 years of age) previously treated with inhaled corticosteroids (beclomethasone dipropionate or triamcinolone acetonide) and short-acting beta(2)-agonists were included in this randomized double-blind study. After an 8-day run-in period, patients were treated with fluticasone (88 microg) or zafirlukast (20 mg) twice daily for 6 weeks. Outcome measures included pulmonary function (forced expiratory volume in 1 second [FEV(1)], peak expiratory flow [peak flow]), albuterol use, asthma symptoms, withdrawals due to lack of efficacy, and asthma exacerbations. Patients treated with fluticasone (n = 224) experienced greater mean increases in FEV(1) (0.24 L vs. 0.08 L, P <0.001), morning peak flow (30 L/min vs. 6 L/min, P <0.001), and evening peak flow (23 L/min vs. 5 L/min, P <0.001) during the study than did those treated with zafirlukast (n = 216). Fluticasone-treated patients had significantly greater increases in the mean percentages of symptom-free days (22% vs. 8%, P <0.001), rescue-free days (23% vs. 10%, P = 0.002), nights with uninterrupted sleep (<1% vs. -5%, P = 0.006), and fewer asthma exacerbations (1% vs. 6%, P = 0.005). Fewer fluticasone-treated patients were withdrawn due to lack of efficacy (2% vs. 13%, P <0.001).Inhaled fluticasone was more effective than zafirlukast in maintaining or improving asthma control in patients with relatively stable asthma who were switched from low-dose inhaled corticosteroids.
Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 (ANXA1) (previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels.
We have investigated, for the first time, ANXA1 ultrastructural expression in rat eosinophils and compared it with that of extravasated neutrophils. The effect of inflammation (carrageenin peritonitis) was also monitored.
Electron microscopy was used to define the sub-cellular localisation of ANXA1 in rat eosinophils and neutrophils extravasated in the mesenteric tissue. A pair of antibodies raised against the ANXA1 N-terminus (i.e. able to recognise intact ANXA1, termed LCPS1) or the whole protein (termed LCS3) was used to perform the ultrastructural analysis.
The majority of ANXA1 was localised in the eosinophil cytosol (approximately 60%) and nucleus (30-40%), whereas a small percentage was found on the plasma membrane (< 10%). Within the cytosol, the protein was equally distributed in the matrix and in the granules, including those containing the typical crystalloid. The two anti-ANXA1 antibodies gave similar results, with the exception that LCPS1 gave a lower degree of immunoreactivity in the plasma membrane. Inflammation (i.e. carrageenin injection) produced a modest increase in eosinophil-associated ANXA1 reactivity (significant only in the cytoplasm compartment). Extravasated neutrophils, used for comparative purposes, displayed a much higher degree of immunoreactivity for the protein.
We describe for the first time ANXA1 distribution in rat eosinophil by ultrastructural analysis, and report a different protein mobilisation from extravasated neutrophils, at least in this acute model of peritonitis.
We previously reported that exogenously added human group V phospholipase A2 (hVPLA2) could elicit leukotriene B4 biosynthesis in human neutrophils through the activation of group IVA phospholipase A2 (cPLA2) (Kim, Y. J., Kim, K. P., Han, S. K., Munoz, N. M., Zhu, X., Sano, H., Leff, A. R., and Cho, W. (2002) J. Biol. Chem. 277, 36479-36488). In this study, we determined the functional significance and mechanism of the exogenous hVPLA2-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nm exogenous hVPLA2 was able to elicit the significant release of AA and LTC4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA2 also augmented the release of AA and LTC4 from eosinophils activated with formyl-Met-Leu-Phe + cytochalasin B. A cellular fluorescent PLA2 assay showed that hVPLA2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA2 induced neither the increase in intracellular calcium concentration nor cPLA2 phosphorylation; consequently, cPLA2 activity was not affected by hVPLA2. Pharmacological inhibition of cPLA2 and the hVPLA2-induced activation of eosinophils derived from the cPLA2-deficient mouse corroborated that hVPLA2 mediates the release of AA and leukotriene in a cPLA2-independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA2 activation.
Background Airway inflammation in asthma is associated with cysteinyl leukotriene and prostaglandin D2 production. Measurement of urinary metabolites of these eicosanoids may be useful for monitoring asthma patients. However, the influence of asthma phenotype and severity on basal urinary excretion of these metabolites is unknown.
Objective To compare urinary leukotriene (LT)E4 and 9α, 11β-prostaglandin (PG)F2 concentrations in large groups of mild, moderate and severe asthmatic patients and healthy control subjects.
Methods Asthma severity, treatment and aspirin sensitivity were assessed by questionnaire in 168 asthmatic patients. Basal LTE4 and 9α, 11β-PGF2 concentrations were measured in urine samples from these patients and from 175 control subjects using enzyme immunoassays.
Results Urinary LTE4 was correlated with 9α, 11β-PGF2 in both control subjects and asthmatic patients (P<0.002). Median LTE4 and 9α, 11β-PGF2 concentrations in patients with severe asthma were significantly reduced compared with mild asthmatic patients (P<0.05 and <0.001, respectively). Urinary 9α, 11β-PGF2, but not LTE4 was lower in asthmatic patients using inhaled corticosteroids (P<0.02). Multiple regression analysis indicated that urinary 9α, 11β-PGF2 concentration was negatively correlated with asthma severity (P=0.003) and also with % predicted FEV1 (forced expiratory volume in 1 s) (P=0.005).
Conclusions Baseline urinary LTE4 and 9α, 11β-PGF2 concentrations are of limited value in discriminating between patients with different severities of asthma. Reduced urinary LTE4 and 9α, 11β-PGF2 in patients with severe asthma suggest that direct or indirect effects of high-dose corticosteroid therapy combined with other factors associated with severe asthma may influence eicosanoid production. However, the negative association of urinary 9α, 11β-PGF2 with lung function suggests an adverse effect of chronic PGD2 production on lung function in asthma, irrespective of severity.
Migration of human eosinophils is regulated by integrin expression, conformational change, and activation of cytosolic phospholipase A2 (cPLA2). Corticosteroids have been shown to inhibit cPLA2 hydrolysis in human eosinophils. The objective of this study was to determine the mechanisms of fluticasone propionate (FP) alone or in combination with salmeterol (SM) in blocking adhesion mediated by beta 2-integrin in human eosinophils. Human eosinophils were isolated by negative magnetic selection. beta 2-integrin-mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min. Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (approximately 30%) inhibited interleukin (IL)-5-induced eosinophil adhesion. Blockade of IL-5-induced eosinophil adhesion caused by 10(-7) M FP at 24 h was augmented by 10(-7) M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FP + SM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2. Blockade of beta 2-integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.
Clinicians and scientists should work together, as the understanding of molecular mechanisms of diseases may help clinicians to improve their clinical practice. This is clearly exemplified by the studies on clinical and molecular interactions between long-acting inhaled β2‐agonists (LABA) and glucocorticoids in the treatment of asthma and chronic obstructive pulmonary disease. Indeed, the possibility of obtaining a drug synergism combining in a single inhaler, LABA and inhaled glucocorticoids, in the regular long-term treatment of moderate-to-severe persistent asthma in adults has intrigued many clinicians and scientists in the last few years.
Inhaled β2-agonists are the most effective bronchodilators, and inhaled glucocorticoids the most effective anti-inflammatory drugs currently available for the treatment of asthma in adults. Since 1994 1 many controlled clinical trials have conclusively demonstrated that the inhalation of a combination of low-dose glucocorticoids together with a LABA has at least the same efficacy as doubling the dose of the inhaled glucocorticoids in the long-term treatment of moderate-to-severe persistent asthma in adults 2.
Conventional wisdom had been that glucorticosteroids and LABAs act through separate and distinct pathways. Studies on the efficacy of inhaled glucocorticoids in the treatment of asthma have demonstrated that they have a relatively flat dose/response curve with most of the benefit obtained at the lowest doses 3, 4. The flatness of the dose/response means that the inflammation is suppressed by a relatively low dose of glucocorticoids. The addition of β2‐agonists may exert some additional actions on the airways that is complementary to the effect of glucocorticoids.
In vitro and in vivo data …
Two double-blind, randomized, placebo-controlled, parallel group safety and efficacy studies included evaluation of the hypothalamic-pituitary-adrenal (HPA)-axis effects of concurrent treatment with intranasal and orally inhaled fluticasone propionate (FP). In the first study, patients with asthma who were > or =12 years of age were assigned randomly to receive twice-daily doses (either 88 or 220 microg) of orally inhaled FP delivered from a metered-dose inhaler (MDI). In the second study, patients were assigned randomly to receive either orally inhaled FP 250 microg or orally inhaled FP 250 microg/salmeterol 50 microg delivered via the Diskus device. In both studies, patients with rhinitis were allowed to continue the use of intranasal FP at their usual dosing. Treatment periods were 26 weeks and 12 weeks for the MDI and Diskus studies, respectively. HPA-axis effects were assessed using response to short cosyntropin stimulation testing. The number and percentage of patients with an abnormal cortisol response, defined as a morning plasma cortisol of <5 microg/dL, a poststimulation peak of <18 microg/dL, or a poststimulation rise of <7 microg/dL, were summarized in two subgroups: patients who used intranasal FP and those who did not. The concurrent administration of intranasal FP and orally inhaled FP via an MDI or Diskus or via Diskus with salmeterol was not associated with HPA-axis effects compared with orally inhaled FP alone. The results of these two studies suggest that concurrent use of intranasal FP with orally inhaled FP administered via MDI or Diskus for treatment of comorbid rhinitis and asthma does not increase the risk of HPA-axis abnormalities.
Glucocorticoids attenuate the population of eosinophils and T lymphocytes in asthmatic airways. The decrease in airway eosinophilia is caused both by accelerated cell death and by induction of blockade of integrin adhesion. In this study, we examined the hypothesis that annexin 1 surface expression, which is upregulated by the glucocorticoid receptor, prevents integrin adhesion essential to cell migration by blocking intracellular translocation of cytosolic group IV phospholipase A2 (cPLA2).
To examine the relationship of the glucocorticoid on annexin 1 expression and the effect of blockade of annexin 1 activity on adhesion of human eosinophils in vitro. To determine the relationship between annexin 1surface expression and nuclear membrane translocation of cPLA2.
Eosinophils isolated from human peripheral blood were pretreated with fluticasone propionate (FP), and beta2-integrin adhesion was measured after stimulation with IL-5 or eotaxin. Effects of FP on cPLA2 expression, phosphorylation, and translocation were determined. The role of annexin 1 was examined by using annexin 1 blocking antibody and/or mimetic peptides.
Fluticasone propionate decreased stimulated eosinophil adhesion and caused 4-fold increase in annexin 1 expression on the plasma membrane. Inhibition of adhesion by FP was blocked with annexin 1 blocking antibody. Annexin 1 N-terminal mimetic peptide also blocked beta2-integrin adhesion. Translocation of cPLA2 to the nuclear membrane was significantly blocked by incubation with FP. Blockade was reversed with annexin 1 blocking antibody.
Blockade of beta2-integrin adhesion by glucocorticoid is regulated by annexin 1, which blocks cPLA2 translocation to nuclear membrane.
Apoptosis may be important in limiting airway eosinophilia. Treatment with leukotriene antagonists decreases the number of eosinophils in both peripheral blood and sputum.
To assess the effect of montelukast on eosinophil apoptosis in a group of patients with mild persistent asthma (MPA) and to compare this effect with the apoptotic effect of fluticasone propionate (FP).
Randomly selected patients with MPA (n = 22) who had not taken anti-inflammatory therapy within the preceding 12 months were included in the study. The sputum induction procedure was performed and the patients were divided into two groups: group 1 (n = 10) received FP 250 microg/day and group 2 (n = 22) received montelukast 10 mg/day orally for 4 weeks. Sputum induction was repeated after the treatment period. The resulting cytospin slides were stained by Wright's stain and morphologic changes in apoptotic eosinophils were assessed by the use of light microscopy by two blinded expert pathologists. Serum soluble Fas ligand (sFasL) concentrations were measured by an ELISA method at baseline and after treatment in both groups, as well as in a group of healthy subjects.
In within-group comparisons, the apoptotic ratio (AR) increased at the end of the study period in group 1 (p = 0.05). In the group treated with FP the ratio of sputum eosinophils significantly decreased (p = 0.02), and the AR significantly increased (p < 0.005). No differences were found in the two study groups in serum sFasL levels at the end of the treatment period compared with baseline values (p > 0.05).
Our findings demonstrate that 4 weeks' treatment with a CysLT receptor antagonist (montelukast) resulted in an increase in eosinophil apoptosis comparable to that produced by FP, suggesting that induction of apoptosis may be a potential mechanism for the mode of action of CysLT receptor antagonists in asthma.
Asthmatic inflammation involves the recruitment and activation of inflammatory cells, and changes in the structural cells of the lung and asthma are characterized by an increased expression of components of the inflammatory cascade including cytokines, chemokines, growth factors, enzymes, receptors and adhesion molecules. The increased expression of these proteins seen in asthma is generally the result of enhanced gene transcription, since many of the genes are not expressed in normal cells but are induced in a cell-specific manner during the inflammatory process. There is clear evidence that neutrophils, long thought of as being transcriptionally inert, can respond to stimuli to induce inflammatory genes. Glucocorticoids are very effective in controlling the inflammation seen in asthmatic airways. Beyond their recognized actions on eosinophil and neutrophil apoptosis, glucocorticoids have profound effects on the chemotaxis, activation and release of mediators from granulocytes (eosinophils, neutrophils and basophils). Few mechanistic studies are available in these cells, but it appears that in granulocytes, glucocorticoids target the same signaling pathways, such as nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), that are important in other cells. We summarize these known mechanisms at the end of this review.
Therapies currently used to reduce exacerbations of chronic obstructive pulmonary disease (COPD) are compounds used almost entirely for asthma therapy. A notable exception is tiotropium, a long-acting parasympatholytic agent. This compound and its precursor, iprotropium, are only occasionally used for asthma therapy. Likewise, leukotriene-modifying drugs are used occasionally for the treatment of COPD. In neither circumstance is there agency-approved indication for these particular cross-over therapies, but the use of long-acting beta(2)-adrenergic compounds and high-solubility inhaled steroids is a mainstay for therapy in both asthma and COPD. Similarly, theophylline, although less often used for either process, is therapeutically applicable to both asthma and COPD. Although overlap syndromes point to the occurrence of a common pathway in some cases, the inflammatory process for asthma and chronic obstructive pulmonary disease (COPD) differs substantially in most cases. Hence, the application of therapies designed to relax airway smooth muscle and ameliorate asthmatic inflammation lacks a therapeutic rationale for a disease characterized by predominant neutrophilic inflammation occurring in the small airways and alveoli. By definition, COPD is poorly reversible airflow obstruction; hence, the use of drugs designed to relax airway smooth muscle is somewhat counterintuitive and does not address the pathophysiological process of the disease.
In asthma, activation and recruitment of eosinophils to the bronchial mucosa amplifies many cellular functions. The blood eosinophil count and the number of hypodense eosinophils increase with asthma severity. Eosinophils produce numerous proinflammatory mediators in response to a variety of agonists, notably the peptido-leukotriene (LT) C4, a potent bronchoconstrictor. In this study, we have evaluated blood eosinophil LTC4 release and its modulation by cytokines in normal individuals and in subjects with asthma of various severities: mild (beta 2-agonist on demand); moderate (inhaled steroids on a regular basis); and severe (inhaled and oral steroids on a regular basis). Eosinophils were isolated using a modified Percoll gradient technique, which recovers both hypodense and normodense eosinophils in a global cell population. Eosinophils released detectable amounts of LTC4 only in the presence of the stimulus (calcium ionophore A23187, 2 microM). The ionophore-induced LTC4 release was greater in moderate asthmatics (mean +/- SEM 5.7 +/- 1.3 pg x 10(3)/250,000 eosinophils) than in normal individuals (1.6 +/- 0.4 pg x 10(3)/250,000 eosinophils), mild asthmatics (1.8 +/- 0.3 pg x 10(3)/250,000 eosinophils) and severe asthmatics (2.0 +/- 0.3 pg x 10(3)/250,000 eosinophils). Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) amplified the ionophore-induced LTC4 release in the four groups from 1.9 to 2.6 and 1.9 to 2.8 fold, respectively. Interleukin-3 (IL-3) did not increase LTC4 production except by the eosinophils of the severe asthmatics whose ionophore-induced LTC4 production was enhanced by 1.9 fold. These data demonstrate that the asthmatic bronchial inflammatory process may modify blood eosinophil LTC4 release and its modulation by cytokines according to asthma severity and treatment.
While considerable progress has been made in understanding the events by which eosinophils accumulate in various pathophysiological conditions, the mechanisms controlling the resolution of eosinophilic inflammation are poorly understood. In the present study, we demonstrate that lung eosinophils obtained by bronchoalveolar lavage (BAL) after aerosol allergen provocation of immunized mice expressed the Fas receptor. Stimulation of purified eosinophils in vitro with a monoclonal anti-Fas mAb (1 ng-1 microg/ml) induced a dose/time dependent loss of cell viability from 24-72 h. Measurement of DNA fragmentation with propidium iodide confirmed that anti-Fas induced eosinophil death by apoptosis. While incubation with IL-3, IL-5, or GM-CSF prevented spontaneous apoptosis, these factors failed to prevent anti-Fas induced apoptosis. Administration of anti-Fas mAb to the lungs after the induction of a lung eosinophilia increased the number of peroxidase positive macrophages in BAL fluid 4-12 h later which was followed by a marked reduction in the number of eosinophils in the airways. Importantly, Fas-mediated resolution of eosinophilic inflammation occurred in the absence of any overt secondary inflammatory changes in the lungs. We speculate that defects in this pathway may at least in part explain the chronic eosinophilic inflammation often observed in the lungs of asthmatic individuals.
Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M.(1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel.
Eosinophil adhesion and degranulation appear to be associated with cell death. Eosinophils bound avidly and degranulated with secretory immunoglobulin A (sIgA)- and IgG-coupled Sepharose 4B beads but bound poorly and did not degranulate with ovalbumin beads. Through the use of dye staining, we found that about 50% of the bound eosinophils were dead by 4 h, regardless of the protein coating. Colchicine and reduced calcium concentration inhibited binding to beads and eosinophil degranulation in a concentration-dependent manner but did not decrease the percentages of dead bound eosinophils. Electron microscopy showed that eosinophils bound to and spread over bead surfaces. Typical granule exocytosis with release of membrane-free granules occurred in about 20% of bound eosinophils. Eosinophil degeneration and lysis with release of membrane-coated granules occurred in about 50% of bound eosinophils; often only membrane-bound granules were present. Therefore, bound eosinophils degranulate both by classical exocytosis and by release after cytolytic degeneration. By increasing the numbers of bound cells, both IgG and sIgA increase the numbers of dying cells.
Theophylline, in addition to its bronchodilator effect, is reported to have an antiinflammatory action that may account for its clinical effectiveness in the reduction of inflammatory cells in the airway. In bronchial asthma, such inflammatory cytokines as GM-CSF and IL-5 are upregulated and have been proposed to cause granulocyte infiltration (neutrophils and eosinophils) in the airway by inhibition of granulocyte apoptosis. We examined the abilities of theophylline to counteract the prolongation of human granulocyte survival caused by cytokines. Theophylline was shown to shorten granulocyte survival in a dose-dependent manner. Upon incubation with a therapeutical concentration of theophylline (0.1 mM; 18 microg/ml), percentages of GM-CSF (10 ng/ml)-induced delayed apoptosis increased from 18+/-2% to 38+/-3% (p < 0.02) in neutrophils and from 21+/-2% to 35+/-2% (p < 0.02; 24-h incubation) in eosinophils. The percentage of IL-5 (5 ng/ml)-induced delayed eosinophil apoptosis also increased from 22+/-4% to 33+/-2% (P < 0. 05). In contrast, cyclic AMP (cAMP)-increasing agents (3-isobutylmethylxanthine, dibutyryl cAMP, and rolipram) inhibited granulocyte apoptosis in the control and anti-Fas antibody-treated cells. In eosinophils, the expression of bcl-2 protein decreased after incubation with theophylline. These findings suggest that theophylline accelerates granulocyte apoptosis, which may play an essential role in inflammation, and controls granulocyte longevity regardless of the elevation of intracellular cAMP levels.
Glucocorticoids have long been used as the most potent drugs in the treatment of bronchial asthma. Data reported recently have led to the proposal that theophylline and macrolides have antiinflammatory effects.
We examined the abilities of theophylline, glucocorticoids, and macrolides to counteract the prolongation of eosinophil survival caused by IL-5.
Purified guinea pig eosinophils were cultured in the presence or absence of human IL-5 and with or without the aforementioned drugs at various concentrations. The percentage of cells alive after 3 days in culture was determined.
Aminophylline (AM), methylprednisolone (MP), erythromycin (EM), and clarithromycin (CAM) suppressed the IL-5 induced prolongation of eosinophil survival in a dose-dependent manner. The effects of these drugs on eosinophil survival were significantly greater at low concentrations of IL-5 than at high concentrations of IL-5. When eosinophils were cultured in the presence of IL-5 (1 ng/ml) with physiologic concentrations of MP (10(-6) mol/L), AM (10(-4) mol/L), and either EM or CAM (both 10 micrograms/ml), the effect of IL-5 was almost completely abolished, and the morphologic changes in eosinophils observed by electron microscopy were consistent with apoptosis. DNA extracted from eosinophils cultured with IL-5 and each of the drugs was definitely fragmented.
One mechanism of the effectiveness of these drugs is induction of eosinophil apoptosis. Some combination of these drugs may be useful in the treatment of bronchial asthma.
Dexamethasone has been shown to inhibit opiate withdrawal, in an in vitro model. In this respect, we suggested that dexamethasone could reduce opiate withdrawal by blocking the release of prostaglandins' precursor, arachidonic acid through protein synthesis dependent-mechanisms (1). Since arachidonic acid is released by the enzyme phospholipase A2 (PLA2) in the present paper we evaluate whether dexamethasone effect may by related to inhibition of PLA2 activity. Therefore, the effect of a neutralizing anti-lipocortin-1 antibody and a polyclonal anti-type II extracellular phospholipase A2 antibody on the opiate withdrawal in vitro was considered. Following a 4 min in vitro exposure to morphine a strong contracture of guinea-pig isolated ileum was observed after the addition of naloxone. Dexamethasone at concentration of 5x10−5 M reduces of 50% morphine withdrawal and the polyclonal anti-type II extracellular PLA2 antibody (in a dilution 1:1000) mimicked dexamethasone inhibitory effect. Incubation of the ileum preparation with a neutralizing anti-lipocortin-1 antibody (at a dilution of 1:10.000) 30 min before dexamethasone reverted the steroid effects. These results suggest that dexamethasone inhibition of opiate withdrawal is due to extracellular type II PLA2 inhibition through lipocortin-1.
A simple method is described for the isolation of human peripheral blood eosinophils by an immunomagnetic procedure. Superparamagnetic particles were coupled to a monoclonal antibody against CD16, a molecule present on neutrophils but not on eosinophils. A peripheral blood granulocyte preparation, containing neutrophils and eosinophils, was incubated with these anti-CD16 particles. In the magnetic field of a permanent magnet, magnetically labelled neutrophils were then retained on columns with a ferromagnetic matrix. By this negative selection procedure, eosinophils of 99.5% purity were obtained from normal individuals and 99.6% purity from patients with eczema. A comparison was made between the immunomagnetic method and eosinophil isolation after N-formyl-methionyl-leucyl-phenyl-alanine treatment. Even in the case of individuals with very low eosinophil counts, the immunomagnetic method permits the efficient isolation of highly purified and functionally active eosinophils.
The leukotrienes are known bronchoactive agonists with potential proinflammatory effects that may be involved in mediating airway hyperresponsiveness. We investigated the effects of zileuton, an inhibitor of 5-lipoxygenase (5-LO), on airway responsiveness to cold, dry air in patients with moderate asthma. A group of 10 asthmatic patients underwent cold, dry air hyperventilation challenge; challenges were performed before drug treatment and 1 to 10 d after the completion of treatment with study drugs. The cold air minute ventilation required to cause a 15% decrease in FEV1 (PD15 VE) increased by 58% compared with the response before treatment, 1 to 10 d after the completion of 13 wk of treatment with zileuton. The geometric mean (geometric mean/SEM and geometric mean x SEM) PD15 VE increased from 24.5 (20.4, 29.5) L/min to 38.8 (34.7, 43.7) L/min (p = 0.01). Zileuton treatment inhibited 5-LO as measured ex vivo by ionophore-stimulated LTB4 levels in whole blood. In four of seven subjects, LTB4 levels before zileuton ingestion fell from 110.88 +/- 25.42 to 5.40 +/- 1.95 ng/ml 2 h post-zileuton dosing (p = 0.02, pre- versus 2 h postzileuton ingestion). Consistent with the short half-life of zileuton, 6 h postzileuton dosing the ionophore-stimulated, LTB4 levels in whole blood had increased to 89.68 +/- 35.54 ng/ml (p = 0.41, pre- versus 6 h postzileuton ingestion). Based on the first-order kinetics of zileuton, its effect on 5-LO activity should have been dissipated less than 16 h postingestion. Thus, chronic zileuton treatment decreased airway hyperresponsiveness as determined by reactivity to cold, dry air.(ABSTRACT TRUNCATED AT 250 WORDS)
Fluticasone propionate (FP) is a novel androstane glucocorticoid with potent anti-inflammatory activity which has been effectively used, intranasally, as therapy for seasonal and allergic perennial rhinitis. When taken by the inhaled route, FP has shown significant therapeutic efficacy in the management of asthma. Fluticasone propionate is a highly lipophilic molecule with good uptake, binding and retention characteristics in human lung tissue. Fluticasone propionate has high glucocorticoid receptor selectivity and affinity, demonstrating rapid receptor association and slow receptor dissociation. In vitro, FP has been shown to potently inhibit T lymphocyte proliferation, cytokine generation, tumour necrosis factor alpha (TNF-alpha)-induced adhesion molecule expression, interleukin-5-induced eosinophilia, mucosal oedema and toluene 2,4-diisocyanate-induced mast cell proliferation, while promoting secretory leucocyte protease inhibitor production and eosinophil apoptosis. In human studies, FP has demonstrated marked vasoconstrictor potency in normal subjects and inhibited antigen-induced mucosal platelet activating factor/eicosanoid production, T lymphocytes and CD25+ cells in patients with rhinitis. Biopsy data from mild asthmatics demonstrate FP-associated reduction in CD3, CD4, CD8 and CD25 cells, with an accompanying reduction in eosinophil and mast cell markers. Clinical studies have evaluated lung function, bronchial reactivity, exacerbation rates and oral corticosteroid-sparing effect. Results show that FP has at least twice the clinical potency of beclomethasone dipropionate and budesonide. This appears to be achieved without an accompanying increase in systemic effects, suggesting a therapeutic index which may be higher than other currently available inhaled corticosteroids.
Airflow obstruction can have a circadian pattern with nocturnal worsening. Airway inflammation is a cardinal feature of asthma, and it has been shown to increase at night in association with the decline in pulmonary function. Although the mechanisms regulating enhanced airway inflammation in asthma at night have yet to be ascertained, we hypothesized that circadian variation in cytokine expression or production is an important factor in the development of nocturnal airflow limitation. To investigate this possibility, spirometry and bronchoscopy were performed; the bronchoalveolar lavage (BAL) fluid obtained at 4:00 A.M. and at 4:00 P.M. were measured for IL-1 beta in asthmatics with (n = 5) and without (n = 9) nocturnal asthma. In addition, the activity of IL-3, IL-5, and GM-CSF was measured using a biologic assay (eosinophil survival-enhancing activity). BAL fluid concentrations of IL-1 beta were significantly greater at 4:00 A.M. than at 4:00 P.M. (1.14 +/- 0.6 versus 0.7 +/- 0.6 pg/ml; p = 0.05) in asthmatics with nocturnal airflow obstruction. Moreover, IL-1 beta levels at 4:00 A.M. tended to be higher in subjects with nocturnal asthma than in those without nighttime airflow reduction (1.14 +/- 0.6 versus 0.3 +/- 0.4 pg/ml; p = 0.1). On the other hand, eosinophil survival-enhancing activity in BAL fluid, which is usually associated with IL-3, IL-5, or GM-CSF, was not detected in relationship to nocturnal asthma. Because IL-1 beta can activate air-space cells, particularly alveolar macrophages, we propose that an increased release of this cytokine is a significant contributor to nocturnal airway inflammation and obstruction in asthma.
Eosinophils (EOS) are specifically recruited to sites of allergic inflammation and parasitic infection, while neutrophil (NEUT) influx predominates in bacterial infections. This biologic selectivity suggests that granulocytes may respond differently to inflammatory mediators such as granulocyte-macrophage-CSF (GM-CSF). To establish the mechanisms of the response of granulocytes to GM-CSF, isolated human peripheral blood EOS and NEUT were obtained from allergic rhinitis patients and incubated in vitro with this cytokine. Incubation with GM-CSF (10 or 100 pM) significantly enhanced FMLP-stimulated EOS superoxide anion (O2-) generation, LTC4 release, and adhesion to tissue culture plates. Both GM-CSF-enhanced EOS adhesion and O2- generation were inhibited by an anti-beta 2 (CD18) Ab suggesting that this beta 2 integrin was associated with increased cell function. In contrast, FMLP + cytochalasin B were required to demonstrate GM-CSF enhancement of NEUT O2- and LTB4 generation; GM-CSF had no effect on NEUT adhesion. Furthermore, GM-CSF augmentation of FMLP + cytochalasin B-activated NEUT O2- generation was not affected by anti-beta 2 Ab but was blocked by a 5-lipoxygenase-activating protein antagonist, BAY x 1005. Finally, 0.1 to 10 nM LTB4 mimicked the GM-CSF-priming effect on NEUT O2- generation, thus suggesting that the augmented NEUT respiratory burst was the result of LTB4 production and its effect on the NEUT. These data demonstrate that GM-CSF promotes EOS and NEUT O2- generation to FMLP via distinct mechanisms: enhanced adhesion of EOS vs autocrine-priming by enhanced LTB4 generation of NEUT.
Eosinophils were isolated by the three methods of CD16-negative depletion: 1) magnetic beads, 2) fluorescence-activated cell sorter (FACS), and 3) complement reaction. Their purity, yield, and viability were compared. The second procedure produced well purity and viability (94.65 +/- 1.51% and 94.98 +/- 1.40%, respectively) but low yield of eosinophils (65.47 +/- 2.47%). The viability of cells obtained by the third procedure was not efficient (80.83 +/- 2.85%), while the purity and the yield were efficient (96.23 +/- 1.09% and 90.75 +/- 1.72%, respectively). In conclusion, the magnetic beads method (purity: 98.02 +/- 0.45%, yield: 91.05 +/- 2.43%, viability: 97.57 +/- 0.37%) was the most advantageous of these three procedures. Moreover, in the functional assay, radical oxygen products from eosinophils isolated by the procedure with complement reaction were less than with the magnetic beads or FACS procedures.
A hemocytometer-based trypan blue dye exclusion cell quantitation and viability assay was compared with a similar assay using simultaneous fluorometric staining with fluorescein diacetate and propidium iodide. Viable and nonviable cell densities were measured, and culture viability was calculated both during the normal growth cycle of a murine hybridoma and in response to the application of millimolar concentrations of either tert-butyl hydroperoxide or ferrous iron. During the early phase of rapid hybridoma cell growth, assay-based differences in viable cell density were not significant. As the culture aged, the trypan blue dye exclusion assay significantly overestimated cell viability, thereby underestimating nonviable cell density and yielding an erroneous estimation of the overall viability of the culture. Because of its lack of ambiguity in the identification of stained, nonviable cells and its resulting increased accuracy in the estimation of culture viability, the fluorometric assay was considered a better choice for the evaluation of cell viability.
Studies in asthma with systemic corticosteroids given at 3:00 PM have shown a superior therapeutic benefit compared with dosing at other time points.
The study was designed to compare beneficial and systemic effects of 800 micrograms of inhaled triamcinolone once daily at 3:00 PM (QD group) versus 200 micrograms conventional four times a day dosing (QID group).
Efficacy outcome measures included forced expiratory volume in 1 second (FEV1), peak expiratory flow rates, bronchial responsiveness, and use of beta-agonist. Systemic effects were blood eosinophil and cortisol levels, 24-hour urinary cortisol, and evaluation for oral candidiasis and dysphonia.
The baseline FEV1 was comparable in the two groups: QD = 67% +/- 2% and QID = 66% +/- 2% of predicted value. After 4 weeks of treatment, FEV1 increased similarly in the QD group to 77% +/- 4% and in the QID group to 74% +/- 4% of predicted value. Likewise, the improvement in morning and evening peak expiratory flow rates was not significantly different between the groups. Both QD and QID groups experienced comparable daily decrements in beta-agonist use. The systemic responses to the two regimens as assessed by eosinophil count, morning serum cortisol, and 24-hour urinary cortisol were also comparable.
The single daily administration of inhaled triamcinolone at 3:00 PM has no increased systemic effects and produces similar improvement in efficacy variables. A dosing strategy based on once daily dosing should increase compliance of inhaled steroid use in the clinical setting.
We studied the effects of 5-lipoxygenase inhibition with A63162 and cyclooxygenase inhibition with indomethacin (INDO) on 1) eosinophil chemotaxis and 2) airway narrowing caused by 10(-6) M formyl-Met-Leu-Phe (fMLP) in tracheal explants from guinea pigs. Airway narrowing was assessed by calibrated micrometry, and eosinophil migration from the lamina propria was expressed as number of eosinophils contained per 1 cm tracheal segment. After 120 min, treatment with fMLP caused an increase in luminal eosinophils from 6,804 +/- 1,786 to 303,347 +/- 75,609 cells (P < 0.001); airway diameter narrowed by 20.4 +/- 1.4%. In six preparations, A63162 inhibited airway narrowing caused by fMLP by 54.9 +/- 6.1%; INDO had a similar effect on airway diameter. However, maximal inhibition of eosinophil migration was greater after 10(-6) M A63162 (38,393 +/- 7,434 cells; P < 0.001 vs. fMLP alone) than after treatment with 10(-5) M INDO (123,547 +/- 19,499 cells; P < 0.05). We demonstrate a method that permits simultaneous measurements of eosinophil migration and airway smooth muscle contraction in a guinea pig tracheal explant preparation. Our data suggest that eosinophil chemotaxis and changes in internal airway diameter are caused by activation of both 5-lipoxygenase and cyclooxygenase pathways and that cell migration is independent of the physical consequences of airway smooth muscle contraction.
We assessed the effect of activated eosinophils isolated from human peripheral blood in causing contraction of explanted human bronchi in vitro. Sixty-three epithelium-intact fifth generation airway sections were obtained from 16 subjects undergoing lung resection for carcinoma. Eosinophils were isolated by negative immunoselection, and activation with 10(-7) M platelet-activating factor (PAF) was confirmed by measurements of eosinophil peroxidase (EPO) secretion and superoxide (O2-.) generation. EPO secretion increased from 68.6 +/- 13.4 ng/10(6) cells to 420 +/- 125 ng/10(6) cells after activation with PAF (P < 0.05). Similarly, PAF-induced O2-. generation increased from 15.3 +/- 4.64 nmol cytochrome c reduced/10(5) cells to 44.2 +/- 8.50 nmol cytochrome c reduced/10(5) cells (P < 0.05). Cells were instilled into an isolated airway pouch preparation, and, 60 min later, airway contractile responses were determined by optical micrometry as percent decrease in lumenal diameter (%decrease) and percent increase in wall thickness (%increase) using a calibrated magnifying lens. Treatment with either vehicle, PAF alone, or untreated eosinophils had no effect on airway caliber or thickness. PAF-activated cells caused a 30.5 +/- 1.52% decrease in airway caliber (P < 0.001 vs. untreated cells) and a 36.6 +/- 2.54% increase in wall thickness (P < 0.001 vs. untreated cells). Preincubation with A63162, a 5-lipoxygenase inhibitor, caused concentration-dependent inhibition of airway narrowing. After 10(-5) M A63162, decrease in airway diameter caused by PAF was 8.00 +/- 0.10% vs. 30.5 +/- 1.52% for PAF alone (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Blood eosinophils, and serum levels of the eosinophil proteins, eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured in childhood asthma. Seventeen patients mean age 11.9 years who were symptomatic with asthma, were enrolled in a study examining the eosinophil counts and eosinophil proteins at the onset of study and after treatment in relation to changes in their baseline forced expiratory volume at 1 second (FEV1) and % predicted FEV1. The patients with symptomatic asthma were compared with 17 patients mean age 12.0 years with asymptomatic asthma maintained on daily inhaled steroid and 13 patients, mean age 12.0 years, without asthma but with urticaria who served as non-asthma controls. Patients with symptomatic asthma did not have significantly higher initial eosinophil counts compared with those with asymptomatic asthma (0.43 x 10(9)/l vs 0.26 x 10(9)/l, P = 0.09) but had higher serum ECP levels (28.9 micrograms/l vs 18.5 micrograms/l). Both asthma patient groups had significantly higher serum ECP levels (P < 0.01) than the controls (9.8 micrograms/l). After therapy consisting of increased dose of inhaled steroids and/or oral steroids, patients in the symptomatic asthma group demonstrated a significant rise in FEV1 (1.67 l/sec at Visit 1 vs 2.08 l/sec at Visit 2, P < 0.001). A similar rise was seen for % predicted FEV1.(ABSTRACT TRUNCATED AT 250 WORDS)
The inhibitory effect of beta-2 adrenergic receptor stimulation on leukotriene C4 (LTC4) secretion and eosinophil peroxidase (EPO) release caused by exogenous activation with 10(-8) to 10(-6) M formyl-met-leu-phe (fMLP) + 5 micrograms/ml of cytochalasin B (Cyto B) in purified human peripheral blood eosinophils was studied. Cells from normal subjects were isolated by negative immunoselection and remained > or = 98% viable as determined by trypan blue exclusion. Duplicate aliquots of eosinophils (10(5) cells/intervention) were activated with 1) fMLP + Cyto B alone, 2) fMLP + Cyto B after pretreatment with 10(-8) M albuterol, 3) 10(-8) M albuterol + fMLP + Cyto B after pretreatment with 10(-8) M propranolol or 4) vehicle control. After incubation, the supernatants were tested for concentration of LTC4 and EPO. Concentration-related release of EPO was demonstrated for 10(-8) M fMLP + 5 micrograms/ml of Cyto B to 10(-6) M fMLP + 5 micrograms/ml of Cyto B, and the greatest concentration of fMLP was used in all subsequent studies. FMLP + Cyto B caused substantial LTC4 secretion in eosinophils (300 +/- 83.0 pg/ml) as compared to sham-activated eosinophils (3.3 +/- 1.9 pg/ml; P < .02). Similarly, maximum EPO release increased from 277 +/- 17.8 to 3956 +/- 1230 ng/10(6) cells (P < .02) after activation with fMLP + Cyto B. Treatment with albuterol decreased markedly both LTC4 secretion to 144 +/- 54.0 pg/ml (P < .05 vs. fMLP + Cyto B-activated eosinophils) and EPO release to 1993 +/- 368 ng/10(6) cells (P < .05 vs. fMLP + Cyto B-activated eosinophils).(ABSTRACT TRUNCATED AT 250 WORDS)
We assessed the effects of cultured human promyelocytic leukemia (HL-60) cells and polymorphonuclear leukocytes (neutrophils) isolated from peripheral human blood on tracheal smooth muscle responsiveness in 40 male Hartley guinea pigs. Undifferentiated HL-60 cells (16-25 passages) were activated in vitro by incubation with 1 microM f-Met-Leu-Phe (fMLP), and force of contraction was measured isometrically using an in situ preparation of tracheal smooth muscle. Increasing concentrations of acetylcholine (ACh; 10(-10) to 10(-6) mol/cm2 tracheal surface) were applied topically to the epithelial surface pretreated with 4 x 10(6) fMLP-activated HL-60 cells, 4 x 10(6) fMLP-activated neutrophils, 4 x 10(6) sham-activated HL-60 cells, fMLP+vehicle, or vehicle control. Topical application of fMLP-activated HL-60 cells caused a maximum active tension (AT) of 1.13 +/- 0.2 g/cm after 5 min; fMLP-activated neutrophils, sham-activated HL-60 cells, or fMLP+vehicle had no effect. The fMLP-activated HL-60 cells also caused substantial augmentation of tracheal contraction to ACh (P < 0.05 vs. sham-activated cells for all concentrations > 10(-9) mol/cm2). Although fMLP treatment caused 247 +/- 28% increase from baseline level in O2-. production, neither direct contraction nor augmentation of muscarinic stimulation was demonstrated after topical application of 4 x 10(6) neutrophils. In 12 other preparations, fMLP-activated HL-60 cells were pretreated with either 10 microM indomethacin (Indo) or 100 microM A63162, a 5-lipoxygenase inhibitor. Pretreatment with Indo caused complete blockade of direct tracheal contraction and 88 +/- 13% blockade of muscarinic augmentation; there was no effect after A63162.(ABSTRACT TRUNCATED AT 250 WORDS)
Using serial bronchoalveolar lavage (BAL), we have studied changes in the airway inflammatory cell populations in 20 asthmatic patients, before and after treatment with inhaled beclomethasone dipropionate (BDP), 2,000 micrograms daily in an uncontrolled study. There was a significant improvement in asthma severity, as measured by symptom score and airways responsiveness, and there were significant reductions in the total BAL eosinophil, epithelial cell and mast cell counts, with a significant increase in the percentage BAL lymphocyte count. No significant correlations were found between the changes in airway inflammatory cell numbers and the reduction in asthma severity. In contrast, the fall in ROS generation by the pulmonary macrophage and granulocyte populations was nonsignificant, but the improvement in airways responsiveness was positively correlated to the reduction in the unstimulated pulmonary macrophage activity. Although these data are uncontrolled, the results are compatible with previous studies in suggesting an effect of steroids on the eosinophil, mast cell and epithelial cell in asthmatic airways. They also highlight the probable importance of the luminal lymphocyte population and pulmonary macrophage activation within the asthmatic airway, the beneficial modulatory effect of inhaled BDP treatment upon them, and the relative steroid-resistance of pulmonary inflammatory cell activity.
Ser-228 has been shown to be essential for the catalytic activity of the human cytosolic phospholipase A2 (cPLA2). However, its involvement in catalysis has not yet been demonstrated. Using site-directed mutagenesis, active-site directed irreversible inhibitors, and the novel fluorogenic substrate 7-hydroxycoumarinyl gamma-linolenate, evidence is presented to show that the hydroxyl group of Ser-228 is the catalytic nucleophile of cPLA2. Replacement of Ser-228 by Ala, Cys, or Thr resulted in the inability of these mutants to mediate calcium ionophore induced PGE2 production in COS-7 cells cotransfected with the cPLA2 mutants and cyclooxygenase-1. Cell lysates from these transfected cells also had undetectable levels of cPLA2 phospholipid hydrolyase activity as did the affinity column purified S228A and S228C cPLA2 mutants overexpressed in insect cells. The loss in activity was not due to the inability of the mutant enzymes to translocate to the substrate lipid interface since the purified S228C cPLA2 mutant, like the wild type, translocated to the phospholipid membrane in the presence of calcium as judged by fluorescence energy transfer. However, when an activated substrate, 7-hydroxycoumarinyl gamma-linolenate (pKa approximately 7.8 for its leaving group) was used as substrate, there was a significant level of 7-hydroxycoumarin esterase (7-HCEase) activity (about 1% of wild type) associated with the purified S228CC cPLA2 mutant. The S228A cPLA2 mutant was catalytically inactive. Contrary to wild type cPLA2, the 7-HCEase activity of the thio-cPLA2 was not titrated by the irreversible active-site-directed inhibitor methyl arachidonyl fluorophosphonate, but rather titrated by one equivalent of arachidonyl bromomethyl ketone, an arachidonyl binding site directed sulfhydryl reagent. These results are compatible with the hydroxyl of Ser-228 being the catalytic nucleophile of cPLA2 and that cysteine can replace serine as the nucleophile, resulting ina thiol-cPLA2 with significantly reduced catalytic power.
Eosinophils and neutrophils are closely related, terminally differentiated cells that in vitro undergo constitutive cell death by apoptosis. The onset of apoptosis in both cell types can be delayed by hemopoietins and inflammatory mediators. Although there have been a number of reports demonstrating that glucocorticoids (in particular dexamethasone) antagonize the eosinophil life-prolonging effects of hemopoietins, direct effects of dexamethasone on eosinophil apoptosis have not been documented. In this study we examined the direct effects of glucocorticoids on eosinophil and neutrophil apoptosis in light of their common therapeutic use as anti-inflammatory and anti-allergic/hypereosinophilic agents. We found that treatment with dexamethasone induced eosinophil apoptosis. In contrast, dexamethasone was a potent inhibitor of neutrophil apoptosis. The effect of dexamethasone on both cell types was mediated through the glucocorticoid receptor, i.e., it was abolished by the glucocorticoid receptor antagonist RU38486. This is the first description of an agent that promotes eosinophil apoptosis while inhibiting neutrophil apoptosis, and thus presents a novel approach to the study of control of apoptosis in these closely related cell types as well as increases our understanding of the clinical action of glucocorticoids in inflammation.
Asthma is accompanied by the accumulation of potentially damaging eosinophils within inflamed airways. How eosinophils may be removed from the airways is not clear. The phagocytic removal of eosinophils in vitro requires that they undergo apoptosis, a form of cell death. We postulated that eosinophil apoptosis may occur in vivo, promoting the removal of airway eosinophils and the resolution of inflammation in asthma. We examined eosinophil apoptosis in sputum samples obtained from 11 subjects during an asthma exacerbation and 2 wk after corticosteroid treatment of the exacerbation. Airway function improved following corticosteroid treatment, and eosinophilic inflammation subsided, with significant decreases occurring in the number of airway eosinophils and the percentage of activated eosinophils. The proportion of apoptotic airway eosinophils increased significantly following corticosteroid treatment, and eosinophil products were apparent within macrophages. Our findings indicate that eosinophil apoptosis is clinically relevant in asthma. Apoptosis may represent a mechanism that promotes the resolution of eosinophilic inflammation in asthma.
Cytosolic phospholipase A2 (cPLA2) is a good candidate for mediating the agonist-stimulated release of arachidonic acid (AA) from membrane phospholipids. This enzyme undergoes a Ca(2+)-dependent translocation from the cytosol to a membrane site in a variety of cell types, and this site has recently been identified as the nuclear envelope in leucocytes. The functional correlate of this finding has not yet been established. The present study was therefore undertaken to determine whether translocation of cPLA2 to the nuclear envelope was associated with localized phospholipid hydrolysis at this site. Rat alveolar epithelial cells, previously shown to contain cPLA2, were prelabelled with [3H]AA and stimulated with the model agonist, ionophore A23187. Ionophore-induced AA release exhibited characteristics typical of a cPLA2-mediated response, in that it was Ca(2+)-dependent, sn-2 AA-selective, and inhibited by arachidonyl trifluoromethyl ketone. As determined by indirect immunofluorescence microscopic analysis as well as subcellular fractionation with immunoblotting, ionophore treatment resulted in a translocation of cPLA2 protein from the cytoplasm to the nuclear envelope. To determine whether the nuclear membrane was indeed the source of released AA, prelabelled cells were incubated in the presence or absence of A23187, after which the phospholipid radioactivity was quantified in nuclear and non-nuclear membrane fractions. [3H]AA was distributed in both nuclear and non-nuclear membrane phospholipids. Following A23187 stimulation, the loss of [3H]AA from nuclear membrane phospholipids accounted for 88.1 +/- 5.8% of the total loss from phospholipids and for 92.9 +/- 2.3% of the total [3H]AA released into the medium. These results demonstrate for the first time that agonist-stimulated translocation of cPLA2 to the nuclear envelope is associated with phospholipid hydrolysis which is preferentially localized to that site.
The leukotrienes (LTs), a family of inflammatory mediators arising from the metabolism of arachidonic acid via the 5-lipoxygenase pathway, are prominently implicated in the pathobiology of asthma. Two classes of LTs, the cysteinyl LTs (LTC4, LTD4, and LTE4) and the dihydroxy-LT (LTB4) have been identified, with each class acting via distinct receptors. Inhibition of LT-mediated inflammation can be achieved by either interruption of 5-lipoxygenase action, thereby preventing formation of the LTs, or inhibition at specific LT receptor sites in the airway. Both the 5-lipoxygenase inhibitors and the cysLT receptor antagonists have thus far demonstrated the capacity to improve pulmonary function and reduce symptoms in clinical models of asthma, such as exercise-, aspirin-, or antigen-induced bronchoconstriction, and to improve pulmonary function in patients with mild-to-moderate, chronic stable asthma. The LTs are therefore critical effector molecules in some patients with asthma and important targets in the pharmacologic management of this disease.
We studied the effects of the 5-lipoxygenase inhibition and sulfidopeptidyl leukotriene receptor antagonism on lumenal chemotaxis of eosinophils in 124 guinea pig tracheal explant preparations from 62 animals. Cell migration was assessed histologically and by differential cell count, and airway narrowing was measured by calibrated micrometry. Intralumenal instillation of the chemotaxin, formyl-met-leu-phe (FMLP) caused migration of 163,509 +/- 18,103 eosinophils/cm segment (eos/cm) versus 15,443 +/- 3,557 eos/cm for segments receiving vehicle only (p < 0.001). Coincubation of FMLP with zileuton, a selective inhibitor of 5-lipoxygenase, caused a concentration-related inhibition of eosinophil migration. At 10(-10) M zileuton, cell migration caused by FMLP was decreased by 57% and nearly complete reduction to 17,200 +/- 3,620 eos/cm resulted after 10(-6) M zileuton (p < 0.001 versus FMLP). Lumenal narrowing caused by FMLP (15.3 +/- 3.4%) was attenuated maximally to 1.15 +/- 2.51% after 10(-8) M zileuton (p < 0.02). In 36 preparations, concentration of leukotriene B4 (LTB4) was measured in treated tracheal perfusate. LTB4 secretion caused by FMLP was 6.4 +/- 0.48 pg/ml versus 3.32 +/- 0.89 pg/ml for buffer control at 5 min (p < 0.02) and was undetectable 120 min after activation with FMLP. Blockade of LTB4-receptor with the selective antagonist, LTB4 dimethyl amide, caused > 90% inhibition of eosinophil migration (p < 0.001). Comparable results were obtained with zafirlukast, an LTD4-receptor antagonist. Our data demonstrate that both LTB4 and LTD4 facilitate eosinophil migration from lamina propria to lumen caused by the chemotaxin, FMLP, and that LTB4-induced eosinophil migration is accompanied by initial lumenal secretion of LTB4.
The mode of action of corticosteroids, important drugs in the treatment of inflammatory disease, is not yet fully understood. Corticosteroids are known to inhibit phospholipase A2 in unprimed eosinophils and basophils, preventing leukotriene synthesis, but their effect on cells that are already primed is unknown.
As inflammatory cells from atopic subjects are often primed in vivo, we studied the effects of two potent corticosteroids on basophil sulfidoleukotriene production in peripheral blood mixed leukocytes (PBML) from in-season and out-of-season atopic individuals.
Cells were incubated for 24 hours with mometasone furoate or beclomethasone dipropionate, primed with IL-3, stimulated with calcium ionophore, buffer, allergen or anti-IgE, and leukotriene production was quantified.
Peripheral blood mononuclear leukocytes from five of ten donors (in season) produced elevated sulfidoleukotrienes without a stimulus; cells from seven donors responded to anti-IgE by increased sulfidoleukotrienes. Neither steroid consistently affected sulfidoleukotriene production in anti-IgE-stimulated cells which were releasing sulfidoleukotrienes in the absence of a stimulant. In comparison, sulfidoleukotriene production was significantly reduced by 0.01 to 10 nM beclomethasone dipropionate or mometasone furoate when the cells were primed with IL-3 after exposure to the drug and stimulated with calcium ionophore or allergen, but no dose-relationship was apparent. Leukotriene production by PBML in response to anti-IgE was potently inhibited by all concentrations of mometasone furoate (0.01 nM to 1 microM) with an inhibitory concentration50 of less than 0.01 nM. Beclomethasone dipropionate inhibited sulfidoleukotriene production in this group (inhibitory concentration50 6 nM) in a dose-dependent manner.
Sulfidoleukotriene production and, conceivably, priming may be more effectively inhibited by mometasone furoate than beclomethasone dipropionate.
The cysteinyl leukotrienes (LTC4, LTD4 and LTE4) have been implicated in the pathogenesis of allergen-induced airway responses. The effects of pretreatment with BAYx 1005, an inhibitor of leukotriene biosynthesis via antagonism of 5-lipoxygenase activating protein, on allergen-induced early and late asthmatic responses has been evaluated.
Eight atopic subjects with mild asthma participated in a two period, double blind, placebo controlled, cross-over trial. Subjects were selected on the basis of a forced expiratory volume in one second (FEV1) of > 70% predicted, a methacholine provocative concentration causing a 20% fall in FEV1 (PC20) of < 32 mg/ ml, a documented allergen-induced early response (EAR, > 15% fall in FEV1 0-1 hour after allergen inhalation) and late response (LAR, > 15% fall in FEV1 3-7 hours after allergen inhalation), and allergen-induced airway hyperresponsiveness (at least a doubling dose reduction in the methacholine PC20 30 hours after allergen inhalation). During the treatment periods subjects received BAYx 1005 (500 mg twice daily) or placebo for 3.5 days; treatment periods were separated by at least two weeks. On the third day of treatment, two hours after administration of medication, subjects performed an allergen inhalation challenge and FEV1 was measured for seven hours.
Treatment with BAYx 1005 attenuated the magnitude of both the allergen-induced early and late asthmatic responses. The mean (SE) maximal fall in FEV1 during the EAR was 26.6 (3.3)% during placebo treatment and 11.4 (3.3)% during treatment with BAYx 1005 (mean difference 15.2 (95% confidence interval (CI) 9.4 to 21.00) with a mean protection afforded by BAYx 1005 of 57.1%. The mean (SE) maximal fall in FEV1 during the LAR was 19.8 (5.7)% during placebo treatment and 10.7 (4.4)% during BAYx 1005 treatment (mean difference 9.2 (95% CI 1.4 to 17.0) with a mean protection afforded by BAYx 1005 of 46.0%. The area under the time response curve (AUC0-3) was also reduced after treatment with BAYx 1005 compared with placebo by 86.5%.h (mean difference 26.3 (95% CI 17.1 to 38.5)) and the AUC3-7 by 59.6%.h (mean difference 26.9 (95% CI-3.8 to 57.6)).
These results show that antagonism of 5-lipoxygenase activating protein can attenuate allergen-induced bronchoconstrictor responses and support an important role for the cysteinyl leukotrienes in mediating these asthmatic responses.
The present studies were undertaken to characterize the potential role of eosinophil programmed cell death (PCD) in atopic diseases. Peripheral blood eosinophil PCD was found to be delayed in inhalant allergy (p < 0.05) and delayed to an even greater extent in atopic dermatitis (AD) (p < 0.0001) when compared to nonatopic subjects. There was no difference in the occurrence of PCD between the extrinsic and the intrinsic type of AD, pointing to a secondary role of specific sensitization. Blockade of eosinophil PCD was not responsible for peripheral blood eosinophilia, because we found no obvious relationship of eosinophil survival to blood eosinophil count. Eosinophil supernatants of more patients with AD than of patients with inhalant allergy dose-dependently inhibited PCD in nonatopic eosinophils, and it was shown that this effect was possibly due to autocrine production of granulocyte-macrophage-colony stimulating factor, probably IL-5. Eosinophil expression of CD95 (Fas antigen) did not change over time in culture and was not modulated by cytokines prolonging eosinophil survival. In contrast, IL-3, IL-5, and granulocyte-macrophage-colony stimulating factor caused an upregulated expression of CD69. However, in AD, CD69 on eosinophils was upregulated without the need of exogenous growth factor or factors over time in culture, thus confirming an autocrine production of proeosinophilic cytokines. In conclusion, our data clearly indicate that eosinophil PCD is markedly delayed in the so-called atopic diseases irrespective of allergen sensitization and suggest that this effect is mediated by the autocrine production of growth factors by eosinophils.
We studied the effect of R-, S- and R,S-albuterol in inhibiting the eosinophil peroxidase (EPO) secretion caused by 10(-10) to 10(-6) M formyl-met-leu-phe + 5 micrograms/ml cytochalasin B (FMLP/CB) in non-allergic and allergic subjects. Total RAST score obtained for allergic subjects was 4.12 +/- 0.21 vs 0.36 +/- 0.17 for non-allergic subjects (P < 0.0001). Stimulated EPO secretion was comparable in allergic [2,051 +/- 567 ng/10(6) eosinophils (eos)] and non-allergic subjects [2,337 +/- 488 ng/10(6) eos (P = NS)]. At all concentrations used, both R- and R,S-enantiomers caused comparable (27-32%) inhibition of FMLP/CB stimulated secretion of EPO in allergic and non-allergic subjects. Pretreatment with S-albuterol caused no augmentation of EPO secretion in either allergic (115 +/- 34.6%) or non-allergic subjects (114 +/- 23.7%) subjects, and there was no significant difference in secretion caused by FMLP/CB alone in either experimental group. Similar results were obtained for subjects stratified according to serum IgE concentration. Our data demonstrate that both R- and R,S-albuterol are equivalently effective in inhibiting stimulated secretion of EPO in both normal and allergic subjects and that there is no paradoxical augmenting effect of S-albuterol in stimulated eosinophil secretion.