Effects of different anti-asthmatic agents on induced sputum and eosinophil cationic protein in mild asthmatics

OBJECTIVES: Inhaled corticosteroids, leukotriene receptor antagonists, and theophylline are recommended for the treatment of mild persistent asthma. The aim of this study was to compare the changes in sputum total cell and eosinophil counts, and eosinophil cationic protein (ECP) levels in serum and sputum following treatment with leukotriene receptor antagonists, inhaled corticosteroids, and theophylline in patients with mild persistent asthma. METHODOLOGY: Total cell counts, eosinophil percentage, and ECP levels in induced sputum and serum were determined both before and after treatment. Prior to sputum induction, FEV1 and PEF values and symptom scores were recorded at baseline and after 8 weeks of treatment. After baseline measurements, the asthmatic patients (n = 30) were randomized into three groups. A total of 10 patients were treated with zafirlukast, 20 mg bd, 10 with budesonide inhaler 200 microg bd, and 10 with theophylline 200 mg bd. RESULTS: There were significant decreases in sputum total cell counts and eosinophil percentage in all treatment groups. However, the decrease in sputum eosinophil counts was more significant in the corticosteroid-treated group. Although sputum ECP levels decreased significantly in the groups treated with zafirlukast and budesonide (zafirlukast group, 580-135 microg/L, P < 0.01; budesonide group, 683-268 microg/L, P < 0.01), the decrease was not statistically significant in the theophylline-treated group (498-361 microg/L, P > 0.05). In contrast, there were no significant changes in serum ECP levels in any of the treatment groups. CONCLUSIONS: All three treatments resulted in significant decreases in sputum total cell counts and eosinophil percentage, but the decrease in sputum ECP level was only seen in the groups treated with budesonide and zafirlukast. These results suggest that although all three treatments are considered as first-line treatments in most consensuses, theophylline seems to have less of an inhibitory effect on eosinophil activation.     

Adiponectin attenuates human eosinophil adhesion and chemotaxis: implications in allergic inflammation

Abstract

Objective: Growing evidence has shown an association between obesity and asthma. Adiponectin, an adipocyte-derived cytokine, is known to have anti-inflammatory effects with reduced concentrations in obese subjects. Recent findings raised the intriguing possibility that adiponectin might play a role in allergic inflammation, although the mechanistic basis for their relationship remains unclear. The purpose of this study was to examine whether adiponectin might affect functions of eosinophils, which play an important role in the pathogenesis of asthma. Methods: Human peripheral blood eosinophils were purified to study expression of adiponectin receptors AdipoR1 and AdipoR2 using RT-PCR and flow cytometry. The effect of adiponectin on eosinophil survival was investigated using annexin V and propidium iodide staining. Eotaxin-induced cell adhesion was investigated using ICAM-1-coated plates. A Boyden chamber and real-time horizontal migration system were used for eotaxin-directed chemotaxis assay. Expression of eotaxin receptor CCR3 and intracellular calcium influx were assessed by flow cytometry. Results: AdipoR1 and AdipoR2 were expressed in human eosinophils. Adiponectin did not affect eosinophil survival or CCR3 expression; however, eotaxin-enhanced adhesion was inhibited by pretreatment with adiponectin. Adiponectin also diminished eotaxin-directed chemotactic responses by disturbing both velocity and directionality. Calcium influx in response to eotaxin was attenuated by adiponectin. Conclusions: These results indicate that adiponectin attenuates the eosinophil functions induced by eotaxin without affecting cell viability. The inhibitory effect was associated with diminished calcium signaling rather than altering of surface receptor expression. Increasing circulating adiponectin might be a novel therapeutic modality for treatment of asthma, especially in obese asthmatics.     

Effects of an eosinophil chemotaxis inhibitor,TAK-661, on antigen-induced asthmatic responses in allergic sheep

Eosinophils have been shown to play a role in allergen-induced airway responses. The aim in this study was to examine the effects of TAK-661, a newly developed product as a specific inhibitory agent of eosinophil chemotaxis, on antigen-induced asthmatic responses in allergic sheep model. Seven Ascaris-sensitive, "dual-respondent" allergic sheep were provocated by an Ascaris suum antigen or phosphate-buffered saline 2 hrs after intra-stomach administration of TAK-661 or a placebo. Pulmonary resistances were measured throughout the experiment, and airway responsiveness to methacholine, bronchoalveolar lavage (BAL), and histological examination were performed 8 hrs after the antigen challenge. Antigen provocation induced dual-phase bronchoconstriction, eosinophilia in BAL and eosinophil infiltration into the airway wall, and an increase in airway responsiveness in placebo-treated sheep. The administration of TAK-661 significantly reduced the bronchoconstriction during the late phase, along with the inhibition of eosinophilia in BAL and the eosinophil infiltration into the airway wall. TAK-661 had a tendency to reduce early-phase bronchoconstriction and airway hyperresponsiveness, but there were no significant differences. These findings suggest that the eosinophil accumulation into the airway induced by antigen provocation may contribute to the development of late-phase bronchoconstriction, however, the development of airway hyperresponsiveness during late asthmatic response may not always be due to only eosinophilic inflammation in the airway.     

Asthma and leukotrienes: antileukotrienes as novel anti-asthmatic drugs

Antileukotriene drugs inhibit the formation or action of leukotrienes, which are potent lipid mediators generated from arachidonic acid in lung tissue and inflammatory cells. The leukotrienes were discovered in basic studies of arachidonic acid metabolism in leucocytes 20 years ago and were found to display a number of biological activities which may contribute to airway obstruction. Clinical studies with antileukotriene drugs have indeed demonstrated that leukotrienes are significant mediators of airway obstruction evoked by many common trigger factors in asthma. Moreover, treatment trials have established that this new class of drugs has beneficial anti-asthmatic properties, and several antileukotrienes have recently been introduced as new therapy of asthma. This communication presents an overview of the biosynthesis of leukotrienes, their biological effects and clinical effects of antileukotrienes in the treatment of asthama.     

Human eosinophil chemotaxis and selective in vivo recruitment by sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) is a sphingolipid mediator that is involved in diverse biological functions. Local administration of S1P causes inflammation coupled to a large eosinophil (EO) recruitment in the rat-paw tissue. The inflammatory response is accompanied by an increase in S1P receptors, namely S1P(1), S1P(2), S1P(3), and by an enhanced expression of CCR3, which is the main chemokine receptor known to be involved in EO function. Human EOs constitutively express S1P(1) and, at a lower extent, S1P(2), S1P(3) receptors. S1P in vitro causes cultured human EO migration and an increase in S1P receptor mRNA copies and strongly up-regulates CCR3 and RANTES (regulated on activation, normal T cell-expressed and secreted) message levels; in particular CCR3 is up-regulated 18,000-fold by S1P. A blocking anti-CCR3 Ab inhibits S1P-induced chemotaxis, implying that S1P acts as specific recruiting signal for EOs not only through its own receptors but also through CCR3. These results show that S1P is involved in EO chemotaxis and contribute to shed light on the complex mechanisms underlying EO recruitment in several diseases such as asthma and some malignancies.     

In vivo priming of platelet-activating factor-induced eosinophil chemotaxis in allergic asthmatic individuals.

The cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 are important modulators of eosinophilia and eosinophil function. Eosinophil chemotaxis is known to be particularly sensitive for cytokine priming. In the present study, we compared chemotactic responses of eosinophils derived from peripheral blood of allergic asthmatics to responses of eosinophils from peripheral blood of healthy individuals. Eosinophils from allergic asthmatics exhibited a markedly increased sensitivity in their chemotactic response toward platelet-activating factor (PAF) compared with eosinophils from normal donors. In contrast, C5a-induced eosinophil chemotaxis between both groups was similar. This in vivo-primed phenotype could be mimicked in vitro, by preincubating eosinophils from peripheral blood of healthy individuals with picomolar concentrations of either GM-CSF, IL-3, or IL-5. The chemotactic response of eosinophils derived from the circulation of allergic asthmatic patients toward GM-CSF was significantly lower compared with the response of eosinophils of healthy individuals. Our data strongly suggest that release of cytokines may be an important in vivo priming mechanism for eosinophils in the circulation of allergic asthmatic patients. Such an in vivo priming can subsequently result in selective upregulation and downregulation of chemotactic responses toward various chemoattractants release in the lung tissue.     

Modulation and induction of eosinophil chemotaxis by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5. We have investigated the modulatory role of GM-CSF and IL-3 on the platelet-activating factor (PAF)-, neutrophil-activating factor (NAF/IL-8)-, leukotriene B4 (LTB4)-, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-, and human complement factor C5a-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a chemotactic response toward PAF, LTB4, and C5a, but not to NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of GM-CSF caused a significant increase in the response toward LTB4 and induced a significant chemotactic response toward NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of IL-3 also induced a chemotactic response toward NAF/IL-8 and FMLP, and enhanced the PAF-induced chemotaxis response toward C5a was not influenced by both cytokines. Nanomolar concentrations of GM-CSF or IL-3 caused a significant inhibition of the C5a-induced chemotaxis. The LTB4-induced chemotaxis was also significantly inhibited in case of GM-CSF. At these concentrations both GM-CSF and IL-3 acted as chemotaxins for eosinophils were washed after pretreatment with GM-CSF and IL-3 the potentiation of the chemotactic response remained, whereas the inhibitory mode of action disappeared. Our data indicate that at picomolar concentrations the cytokines GM-CSF and IL-3 can modulate eosinophil chemotaxis and at nanomolar concentrations these cytokines can act as chemotaxins for eosinophils.     

Effects of beta-adrenoceptor blocking drugs on human sigmoid colonic motility

Effects of propranolol and metoprolol on sigmoid colonic motility were studied in 12 healthy volunteers in a double-blind randomized fashion. Colonic pressure was recorded 15-18 cm from anus and contractile activity quantified for periods of 25 min. On separate days propranolol, metoprolol, and placebo, respectively, was administered intravenously preceded by a control period. After propranolol, 10 mg intravenously, pressure activity increased significantly from 3.8 +/- 1.1 (SEM) kPa X min (28 +/- 8 mm Hg X min) to 5.9 +/- 1.0 kPa X min (44 +/- 8 mm Hg X min) (P less than 0.001). Also, after propranolol, 5 mg intravenously, the pressure activity was increased (P less than 0.05). After metoprolol, 10 mg intravenously, contractile activity increased from 4.3 +/- 0.9 kPa X min (32 +/- 7 mm Hg X min) to 6.1 +/- 1.0 kPa X min (46 +/- 8 mm Hg X min) (P less than 0.01). The two drugs caused equipotent reduction of heart rate. After placebo, no effect on sigmoid pressure or heart rate was observed. The study shows that unselective (propranolol) and beta 1-selective (metoprolol) beta-blocking drugs enhance distal colonic pressure in man. Colonic motility seems to be under sympathetic beta-adrenergic influence even under fairly unstrained conditions.     

Effects of narcotic analgesic drugs on human Oddi＇s sphincter motility

AIM: To assess the effects of intramuscular analgesics(morphine, Ap-237, pethidine and tramadol) on humanOddi‘s sphincter motility with choledochoscope manometry.METHODS: A total of 70 patients having T tubes aftercholecystectomy and choledochotomy were assessed bycholedochoscope manometry. They were randomly dividedinto morphine group, Ap-237 group, pethidine group andtramadol group. Basal pressure of Oddi‘s sphincter (BPOS),amplitude of phasic contractions (SOCA), frequency ofphasic contractions (SOF), duration of phasic contractions(SOD), duodenal pressure (DP) and common bile ductpressure (CBDP) were scored and analyzed. All narcoticanalgesic drugs were administered intramuscularly.RESULTS: Levels of BPOS, SOCA and SOF were increasedafter injection of morphine and Ap-237 (P&lt;0.05), level of CBDPwas increased from 4.97+3.87 mmHg to 8.62+7.43 mmHg(10 min later) and 7.32+5.95 mmHg (20 min later) afterinjection of morphine (P&lt;0.01). No apparent changeoccurred after intramuscular injection of pethidine. Level ofBPOS was increased from 7.01+5.50 mmHg to 2.87+2.78 mmHg10 min after injection of tramadol and SOCA was decreasedfrom 63.34+35.29 mmHg to 45.90+27.86 mmHg (10 minlater, P&lt;0.05) and 35.97+24.30 (20 min later, P&lt;0.01) afteradministration of tramadol.CONCLUSION: All these findings indicate that Oddi‘ssphincter manometry via choledochoscope is a practicaland new way to study the dynamics of Oddi‘ s sphincter.The regular dose of morphine and Ap-237 could increaseBPOS, SOF and SOCA. Morphine could increase the levelof CBDP, demonstrating an excitatory effect on the sphincterof Oddi. Pethidine had no effect on Oddi‘s sphincter motility.Tramadol shows an inhibitory effect on the motility of thesphincter of Oddi and decreases levels of BPOS and SOCA.     

Effects of calcium-regulating hormones and drugs on human monocyte chemiluminescence

Chemiluminescence (CL) associated with phagocytosing monocytes has been used as an index of the oxygen dependent metabolic activity of these cells. Because of the relationships between monocytes and cells involved in bone resorption, we studied the effects on human monocyte CL of hormones and drugs active in mineral metabolism. Two-hour preincubations of monocytes with human PTH-(1-34), bovine PTH, or prostaglandin E2 caused significant decreases in peak CL during phagocytosis stimulated by the addition of latex particles. Similar studies with dichloromethylene diphosphonate, ethane hydroxydiphosphonate, or salmon calcitonin (sCT) caused significant increases in CL, whereas preincubation with human CT at the same molar concentration did not. CL was also decreased after preincubations with methylprednisolone or bacterial endotoxin. The effect of bovine PTH was dose dependent to concentrations as low as 10 ng/ml, was not fully present after a shorter 1-min preincubation with the hormone, and differed in an otherwise identical system using polymorphonuclear leukocytes instead of monocytes. The production of hydrogen peroxide by phagocytosing monocytes was also significantly affected by each of the drugs and hormones. The direction and magnitude of these changes were similar to those in CL experiments, except for the effects of sCT. These studies relate the oxygen-dependent function of phagocytes to mediators of bone resorption and provide a new system for studying the effects of hormones and drugs on the cellular elements of bone and blood.     

Oxidative metabolism of the human eosinophil

We have compared the oxidative metabolism of human eosinophils (80%-90% purity) to that of neutrophils. Hexose monophosphate (HMP) shunt activity of eosinophils was higher than that of neutrophils under either resting or phagocytizing conditions. Eosinophil HMP shunt activity also was stimulated by phorbol myristate acetate, a membrane- active agent. Eosinophils showed a marked incorporation of 125I into trichloroacetic acid-insoluble material under resting conditions, which increased markedly during phagocytosis. Eosinophils likewise showed a greater reduction of nitroblue tetrazolium dye during phagocytosis than did neutrophils. Measurement of other parameters of oxidative metabolism indicated that eosinophils generated superoxide anion following phagocytosis and also elicited a burst of chemiluminescence similar to that observed during phagocytosis by neutrophils. Measurement of NADPH oxidase activity demonstrated that this enzyme was 3-6 times more active in fractions isolated from eosinophils than in corresponding fractions isolated from neutrophils; this was observed over a range of substrate concentrations. The eosinophil enzyme sedimented differently than the neutrophil enzyme with differential centrifugation; neither showed sedimentation characteristics of peroxidase. These data indicate that eosinophils possess a similar, although in some ways more potent, oxidative burst than neutrophils and are consistent with a role for NADPH oxidase in the initiation of that burst.     

Effects of human mast cell tryptase and eosinophil granule proteins on the kinetics of blood clotting

Hypereosinophilic syndromes are often associated with thrombosis through unclear mechanisms, and mastocytosis has been associated with a variety of bleeding disorders. The present studies were aimed at defining the roles and interactions of eosinophil and mast cell constituents on the kinetics of blood clotting as measured by thromboelastograms. Eosinophil granule proteins and purified eosinophil peroxidase markedly reduced the anticoagulant properties of the mast cell tryptase/heparin complex. Moreover, eosinophil peroxidase by itself functioned as a powerful procoagulant and also inhibited the anticoagulant actions of heparin in a chromogenic assay for antithrombin III/factor Xa activity. The anticoagulant activity of the tryptase/heparin complex was attributable exclusively to the associated heparin and not to the intrinsic enzymatic activity of tryptase. Eosinophil granule proteins also strongly inhibited the enzymatic activity of tryptase in the presence of hydrogen peroxide, thus implicating a critical role for eosinophil peroxidase. We conclude that eosinophil granule proteins and eosinophil peroxidase both function as powerful procoagulants and also inhibit the anticoagulant and enzymatic activities of mast cell tryptase. The present results thus provide a mechanistic rationale for the well-established link between certain eosinophilic inflammatory disorders and hypercoagulant states. They also suggest that eosinophils may play an important role in neutralizing the anticoagulant activity of mast cell tryptase/heparin in various diseases.     

Modulatory effects of N-acetyl-L-cysteine on human eosinophil apoptosis.

Eosinophils are oxidant-sensitive cells considered relevant in allergic inflammation. The present study aimed to examine the effects of the antioxidant N-acetyl-L-cysteine (NAC) on constitutive and cytokine-delayed apoptosis in human isolated eosinophils. Human eosinophils were purified from the blood of healthy donors by a magnetic separation system. Apoptosis and cellular glutathione were assessed by cytofluorometric analysis and nuclear factor (NF)-kappaB binding activity assessed by electrophoresis mobility shift assay. The rate of spontaneous apoptosis of human eosinophils after 24 h culture, as assessed by annexin-V-positive staining, was mean+/-sem 48.2+/-1.4%, n = 5. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng.mL(-1)) decreased apoptosis to 19.4+/-1.8%, n = 5. NAC (5 mM) inhibited spontaneous apoptosis (33.6+/-2.7%, n = 5) but augmented apoptosis in the presence of GM-CSF (30.9+/-1.5%, n = 5). NAC (5 mM) also increased the rate of apoptosis in the presence of tumour necrosis factor (TNF)-alpha (10 ng.mL(-1)) and interleukin-5 (5 ng.mL(-1)). NAC (5 mM) increased eosinophil glutathione content. The increase in eosinophil NF-kappaB binding activity induced by GM-CSF and TNF-alpha was suppressed by NAC. In conclusion, N-acetylcysteine modulates eosinophil apoptosis by inhibiting constitutive apoptosis but reversing the survival effect produced by inflammatory cytokines in human eosinophils.