Eosinophil Respiratory Burst Research Articles

Involvement of ion channels in human eosinophil respiratory burst

Background: Human eosinophils possess a variety of ion channels that play a crucial role in the regulation of cellular activity. During eosinophil respiratory burst, efflux of H+ ions through H+ channels provides an efficient mechanism of H+ extrusion and charge compensation. Interestingly, recent studies suggest that other ion channels may also be involved in this process. Objective: We sought to investigate the role of ion channels in phorbol 12-myristate 13-acetate–induced superoxide (O2•–) generation by human eosinophils. Methods: O2•– production was measured by using the superoxide dismutase–inhibitable reduction of cytochrome c. Ion channel expression and function were studied by using RT-PCR and the patch clamp technique, respectively. Results: O2•– generation was affected by several ion channel blockers, especially 4,4-diisothio-cyanostilbene-2,2′-disulfonic acid. The involvement of Cl– channels in this process was confirmed by replacement of Cl– with gluconate or other anions. The halide dependence of O2•– production could be described by the sequence Cl–≥Br–>I–, which is similar to the selectivity sequence of several members of the chloride channel (ClC) family. RT-PCR studies performed with primers for ClC-2, ClC-3, ClC-4, ClC-5, ClC-6, and the cystic fibrosis transmembrane conductance regulator showed only the expression of ClC-3. The presence of phorbol 12-myristate 13-acetate–sensitive Cl– channels in human eosinophils with biophysical properties similar to the ClC-3 channel has been studied. Conclusion: Cl– channels play an important role in the regulation of O2•– production by human eosinophils. (J Allergy Clin Immunol 2000;106:272-9.)

Effects of a Selective PDE4 Inhibitor, D-22888, on Human Airways and Eosinophils in vitro and Late Phase Allergic Pulmonary Eosinophilia in Guinea Pigs

The actions of a novel selective inhibitor of type 4 cyclic nucleotide phosphodiesterase (PDE4), D-22888, on human airway smooth muscle tone and human eosinophil respiratory burst in vitro and bronchoalveolar eosinophilia in allergen-challenged sensitized guinea pigs in vivo were assessed. D-22888 was a selective inhibitor of PDE4, exhibiting an IC50against human neutrophil PDE4 of 0.15 μm, compared to IC50values of 4.4 μmand 1.1 μmfor human platelet PDE3 and PDE5, respectively. D-22888 relaxed inherent tone in human bronchial rings in a concentration-dependent manner with an IC50of 5.0 μm(geometric mean, 95% ci 3.0–8.4 μm) and also caused a concentration-dependent inhibition of opsonized zymosan-induced superoxide anion generation by human eosinophils with an IC50of 3.1 μm(1.0–9.2 μm). Treatment of actively sensitized guinea pigs with single oral doses of D-22888 2 h before or 4 h after challenge reduced bronchoalveolar lavage (BAL) eosinophil numbers, 24 h after aerosol allergen challenge, by 48% and 73% at 10 mg/kg and 30 mg/kg, respectively, 2 h pre-challenge and 68% at 30 mg/kg 4 h post-challenge. Chronic twice-daily oral dosing with D-22888 for three days caused inhibition of 24 h post-challenge BAL eosinophilia, amounting to 88% at 30 mg/kg. These in vivo actions were comparable with those achieved with other selective PDE4 inhibitors and with the corticosteroid, dexamethasone. We conclude that D-22888 exerts actions on airway smooth muscle and eosinophil recruitment and activation that suggest that D-22888 may be a promising new drug for use in the treatment of allergic obstructive airways» diseases such as bronchial asthma.

The effect of cetirizine on the integrin-dependent respiratory burst of normodense eosinophils.

It has been proposed that cetirizine inhibits eosinophil migration and adherence. We evaluated the possible effect of cetirizine on integrin-induced eosinophil proinflammatory activation. Normodense eosinophils were triggered with monoclonal antibodies to integrins in the presence of different concentrations of certirizine. Proinflammatory activation was measured by evaluation of O2- production. Only at high concentrations (250 micrograms/ml) and in the first 15 min did certirizine significantly inhibit (p < 0.02) the eosinophil respiratory burst. No effect was shown for lower concentrations (50 and 100 micrograms/ml) or after 15 min. These data suggest that, only at very high concentrations, cetirizine may induce a transient inhibition of the integrin-induced eosinophil respiratory burst.

β-Adrenergic regulation of the eosinophil respiratory burst as detected by lucigenin-dependent luminescence

Because β-adrenoceptor agonists are commonly used in the treatment of disease states associated with eosinophil activation, β-adrenergic regulation of the eosinophil respiratory burst (as monitored with lucigenin-dependent luminescence) was evaluated. Normodense, nonprimed eosinophils from healthy volunteer subjects were potently inhibited by very low concentrations of isoproterenol. The inhibitory concentration of 50% for isoproterenol was approximately 2 nmol/L. The β-agonist was able to inhibit the eosinophil respiratory burst induced by receptor-mediated (chemotactic peptide) and nonreceptor-mediated (calcium ionophore and phorbol ester) stimuli. Thus β-agonist inhibition was unlikely to be isolated to an effect at the receptor or G protein linkage. To determine whether cyclic adenosine 3',5' monophosphate (cAMP) may mediate β-agonist effects, studies were performed with the type IV cAMP phosphodiesterase inhibitor Ro-201724. β-Agonist inhibition of the respiratory burst was clearly synergistic with effects of Ro-201724. We conclude that β-adrenoceptor agonists can regulate the eosinophil respiratory burst at least partially through an effect mediated by cAMP. Because regulation of the eosinophil by isoproterenol was observed at very low concentrations, these results may be relevant to pharmacologic effects of β-agonists in disease states complicated by eosinophil activation during asthma. (J A LLERGY C LIN I MMUNOL 1995;95:735-41.)

Activation of guinea pig eosinophil respiratory burst by leukotriene B4: role of protein kinase C.

Leukotriene B4 (LTB4) and the protein kinase C activator, 4-beta-phorbol dibutyrate (PDBu), both induced a pronounced and concentration-dependent stimulation of hydrogen peroxide (H2O2) generation by purified guinea pig peritoneal eosinophils in the concentration range 1 nM-1 microM. The LTB4 response was inhibited competitively by the specific LTB4 receptor antagonist, U-75302, with a KB of 25 nM, while the concentration-response curves for both stimuli were shifted rightwards (3.8-fold and 2.8-fold for LTB4 and PDBu, respectively) by the competitive protein kinase C inhibitor, 1-O-hexadecyl-2-O-methylglycerol at a concentration of 300 microM. LTB4 appears, therefore, to induce respiratory burst in eosinophils via a receptor-mediated mechanism involving protein kinase C.

The effect of anion transport inhibitors and extracellular Cl − concentration on eosinophil respiratory burst activity

Furosemide has been shown recently to protect asthmatic patients against certain bronchoconstrictor challenges. We investigated the effect of furosemide on eosinophil function. Since furosemide may be exerting its inhibitory effect on the eosinophil by inhibiting anion transport, we also assessed the effects of the anion transport inhibitors 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Furosemide, NPPB and DIDS inhibited the eosinophil respiratory burst in response to leukotriene B 4 (LTB 4) and, to a smaller extent, inhibited the response to opsonized zymosan (OZ). To assess whether the anion transport inhibitors were achieving their inhibitory effect by inhibiting an influx of Cl − ions into the eosinophil, the effect of removing extracellular Cl − on eosinophil function was determined. OZ-induced H 2O 2 production was inhibited by removing extracellular Cl − whereas the LTB 4 response was not affected by the concentration of extracellular Cl −.

Inhibition of eosinophil cyclic nucleotide PDE activity and opsonised zymosan-stimulated respiratory burst by 'type IV'-selective PDE inhibitors.

1. The cyclic nucleotide phosphodiesterase (PDE) of guinea-pig eosinophils was partially characterized and the effects of selective inhibitors of PDE isoenzymes upon opsonized zymosan (OZ)-stimulated respiratory burst were studied. 2. PDE activity in eosinophil lysates appeared to be membrane-associated, displayed substrate specificity for adenosine 3':5' cyclic monophosphate (cyclic AMP) versus guanosine 3':5' cyclic monophosphate (cyclic GMP) and was insensitive to cyclic GMP or Ca2+ and calmodulin. 3. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine caused a concentration-dependent inhibition of both OZ-stimulated hydrogen peroxide (H2O2) generation and cyclic AMP hydrolysis. The type IV-selective PDE inhibitors, rolipram and denbufylline, also inhibited H2O2 generation and cyclic AMP hydrolysis in a concentration-dependent manner whilst SK&F 94120 and Org 9935 (type III-selective) and zaprinast (type Ia or V-selective) were ineffective. 4. Dibutyryl cyclic AMP, a cell-permeable, non-hydrolysable analogue of cyclic AMP, caused a concentration-dependent inhibition of H2O2 generation stimulated by OZ. Dibutyryl cyclic GMP was ineffective. 5. It is concluded that eosinophil respiratory burst activity induced by OZ can be regulated by intracellular cyclic AMP and that the levels of cyclic AMP are controlled exclusively by a rolipram- and denbufylline-sensitive PDE isoenzyme that resembles a type IV species.