Toru Fujiu

Fenoterol inhibits superoxide anion generation by human polymorphonuclear leukocytes via β2-adrenoceptor-dependent and -independent mechanisms

BACKGROUND Beta2-adrenoceptor agonists, used widely as bronchodilator in treating bronchial asthma, may have anti-inflammatory activity. OBJECTIVE We examined whether various widely prescribed beta2-adrenoceptor agonists differ in anti-inflammatory mechanisms. METHODS We investigated effects of these drugs on superoxide anion generation by stimulated human polymorphonuclear leukocytes in vitro using chemiluminescence. RESULTS At high concentrations, fenoterol significantly inhibited both N-formylmethionyl-leucyl-phenylalanine- and phorbol myristate acetate-induced superoxide generation by neutrophils. In contrast, salbutamol or procaterol partially inhibited generation with the former stimulus but not the latter. Inhibition by salbutamol or procaterol was completely reversed by either propranolol, a nonselective beta-adrenoceptor antagonist, or ICI-118551, a beta2-adrenoceptor-selective antagonist. In contrast, the effect of fenoterol at concentrations exceeding 10(-6) M against superoxide generation with the former stimulus was only partially reversed by antagonists, and the effect of high concentrations of fenoterol against generation with the latter stimulus was not reversed. No drugs scavenged superoxide at the highest concentration used (10(-5) M). CONCLUSIONS Fenoterol at high concentrations has an inhibitory effect on superoxide generation that includes a component not mediated via beta2-adrenoceptors. Direct inhibition at or downstream from protein kinase C may be involved.

Inhibition of human eosinophil activation by a cysteinyl leukotriene receptor antagonist (pranlukast; ONO-1078).

Eosinophils produce cysteinyl leukotrienes such as leukotriene C4 and D4 upon stimulation by platelet-activating factor or other mediators, and these cells themselves express cysteinyl leukotriene receptors. Pranlukast, a compound developed in Japan, antagonizes cysteinyl leukotriene receptors and inhibits contraction of airway smooth muscle, microvascular leakage into airways, and eosinophil infiltration. This agent can decrease symptoms of bronchial asthma, but its specific influences on effector functions of eosinophils important to the pathogenesis and exacerbation of asthma remain unknown. In the present study, we investigated the effect of pranlukast on human eosinophil functions. Eosinophils obtained from peripheral blood of normal volunteers were stimulated by platelet-activating factor, leukotriene D4, or phorbol ester. Superoxide anion generation was measured by reduction of cytochrome c. Expression of αMβ2 was analyzed by flow cytometry. To evaluate eosinophil degranulation, eosinophil protein X, a toxic granule constituent, was measured by radioimmunoassay in sample supernatants. Pranlukast partially inhibited major eosinophil effector functions of superoxide anion generation and degranulation induced by platelet-activating factor, although at concentrations tested pranlukast failed to significantly reduce platelet-activating factor- induced αMβ2 expression. Pranlukast completely inhibited leukotriene D4-induced superoxide generation and αMβ2 expression. In contrast, pranlukast at 10−6 M did not affect phorbol ester-induced superoxide generation at 120 minutes, degranulation, or αMβ2 expression. The results suggested that inhibition by pranlukast of platelet-activating, factor-induced eosinophil effector functions such as superoxide generation and degranulation might result at least partly from antagonism of autocrine mechanisms involving cysteinyl leukotrienes produced in response to platelet-activating factor.

Inhibition by fenoterol of human eosinophil functions including β2‐adrenoceptor‐independent actions

Agonists at β2 adrenoceptors are used widely as bronchodilators in treating bronchial asthma. These agents also may have important anti‐inflammatory effects on eosinophils in asthma. We examined whether widely prescribed β2‐adrenoceptor agonists differ in ability to suppress stimulus‐induced eosinophil effector functions such as superoxide anion (O2–) generation and degranulation. To examine involvement of cellular adhesion in such responses, we also investigated effects of β2 agonists on cellular adhesion and on CD11b expression by human eosinophils. O2– was measured using chemiluminescence. Eosinophil degranulation and adhesion were assessed by a radioimmunoassay for eosinophil protein X (EPX). CD11b expression was measured by flow cytometry. Fenoterol inhibited platelet‐activating factor (PAF)‐induced O2– generation by eosinophils significantly more than salbutamol or procaterol. Fenoterol partially inhibited PAF‐induced degranulation by eosinophils similarly to salbutamol or procaterol. Fenoterol inhibited phorbol myristate acetate (PMA)‐induced O2– generation and degranulation by eosinophils, while salbutamol or procaterol did not. Fenoterol inhibition of PMA‐induced O2– generation was not reversed by ICI‐118551, a selective β2‐adrenoceptor antagonist. Fenoterol, but not salbutamol or procaterol, significantly inhibited PAF‐induced eosinophil adhesion. Fenoterol inhibited O2– generation and degranulation more effectively than salbutamol or procaterol; these effects may include a component involving cellular adhesion. Inhibition also might include a component not mediated via β2 adrenoceptors.

Cellular adhesion is required for effector functions of human eosinophils via G-protein coupled receptors

BACKGROUND Eosinophils play an important role in the pathogenesis of allergic diseases. Chemoattractants, including platelet-activating factor (PAF) and complement component 5a (C5a), induce eosinophil infiltration and promote eosinophil effector functions. OBJECTIVE To compare eosinophil degranulation and superoxide anion (O2-) generation induced by various chemoattractants, and to elucidate the role of cellular adhesion on these effector functions. METHODS Human eosinophils were stimulated with PAF, C5a, eotaxin, or leukotriene B4 (LTB4). O2- generation was assayed by a chemiluminescence method using a Cypridina luciferin analog as the amplifier. Degranulation and adhesion were measured by quantitating eosinophil protein X by radioimmunoassay. Expression of CD11b on eosinophils was measured by flow cytometry. RESULTS PAF and C5a induced significant degranulation and O2- generation from eosinophils. In contrast, the potency of eotaxin or LTB4 for these functions was much less. PAF and C5a also significantly enhanced eosinophil adhesion, whereas eotaxin and LTB4 did not. CD11b expression on eosinophils was enhanced by all four stimulants, and the order of potency to induce CD11b expression was C5a > PAF > eotaxin > LTB4. CONCLUSIONS The potency of PAF and C5a for inducing effector function in eosinophils was greater than that of eotaxin or LTB4. The magnitude of the effector function was consistent with the degree of eosinophil adherence induced by each stimulant. These results suggest that effector functions of eosinophils which are mediated through G-protein coupled receptors are dependent on cellular adhesion.

Distinct isoforms of protein kinase C are involved in human eosinophil functions induced by platelet-activating factor

Background: Platelet-activating factor (PAF) is a potent stimulator of eosinophils. Recently, treatment with a protein kinase C (PKC) inhibitor which generally inhibits PKC isoforms has been shown to modulate several eosinophil functions in distinct manners, in that PKC inhibition enhanced CD11b expression and cellular adhesion, but inhibited superoxide generation and degranulation in PAF-stimulated human eosinophils. These results suggested that distinct PKC isoforms were likely to be involved in each eosinophil function induced by PAF. We have therefore investigated whether or not the PKC isoforms involved in PAF-induced CD11b expression and superoxide generation were different. Methods: Human eosinophils prepared from healthy volunteers were treated with PKC inhibitors, bis-indolylmaleimide I (BisI; a general PKC inhibitor), myristoylated PKC inhibitor peptide (myr-ψPKC; a PKCα, β and δ inhibitor) and rottlerin (a PKCδ inhibitor), followed by stimulation with PAF. CD11b expression was determined using flow cytometry and superoxide generation was evaluated using a cytochrome c reduction assay. Results: BisI treatment led to enhancement of PAF-induced CD11b expression, while myr-ψPKC and rottlerin did not. In contrast, PAF-induced superoxide generation was inhibited by treatment with BisI, myr-ψPKC and rottlerin. Conclusions: PKCα, β and δ are not involved in PAF-induced CD11b expression, but PKCδ is involved in the PAF-induced activation of superoxide anion generation.

Oral iron supplementation in preterm infants treated with erythropoietin

Background : It is not known whether a moderate dose of oral iron supplementation would further enhance erythropoiesis in recombinant human erythropoietin (EPO)‐treated very low‐birthweight (VLBW) infants.

Problems with using total serum bilirubin as a criterion for phototherapy in extremely low-birthweight infants

Despite the early use of phototherapy and exchange transfusion in premature infants based on total serum bilirubin (TSB), the reemergence of kernicterus has been reported. The aim of this study was to assess the validity of using TSB as the criterion for phototherapy in extremely low‐birthweight infants (ELBWI).

Feeding interval and postprandial intestinal blood flow in premature infants

The feeding interval is an important factor in enteral feeding of premature infants. We investigated postprandial intestinal blood flow in stable very‐low‐birthweight infants fed at 2‐h and 3‐h intervals.

Proinflammatory Cytokinemia Associated with Transient Myeloproliferative Disorder in Down Syndrome

A transient myeloproliferative disorder (TMD) occurs in 10% of the infants with Down syndrome. While most cases resolve within a few months, in 20% of them TMDs are life-threatening or fatal. We encountered 4 patients with TMD, including 1 patient who died of liver failure and disseminated intravascular coagulation. Suspecting involvement of proinflammatory cytokines, we serially assayed them in patients’ sera. Cytokines were significantly more abundant in patients than in controls. Interleukins 1 and 2, tumor necrosis factor alpha, interferon gamma, and granulocyte-macrophage colony-stimulating factor were greatly increased, especially in the infant who died. Sustained cytokinemia is likely to participate in TMD pathophysiology, and very high serum concentrations might predict a poor outcome.