Total blood eosinophils, serum eosinophil cationic protein and eosinophil protein X in childhood asthma: relation to disease status and therapy

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)     

Evidence for eosinophil activation in bronchiectasis unrelated to cystic fibrosis and bronchopulmonary aspergillosis: discrepancy between blood eosinophil counts and serum eosinophil cationic protein levels

The purpose of this prospective study was to evaluate the effect of prophylactic antibiotic treatment on postoperative antibiotic spinal wound infection after spinal surgery with instrumentation. Subjects consisted of 110 successive patients that underwent instrumented fusion with Cotrel-Dubousset (CD) or Miami Moss instrumentation. In 56 cases, the indication for surgery was painful spondylolisthesis. The remaining 54 patients were treated for idiopathic scoliosis. In total, 172 spinal procedures were performed and included in the study. Preoperative infection prophylaxis consisting of 2 g cefamandole was administered to all patients. Patients received three doses of 2 g/day cefamandole after surgery for 3 days. Follow-up ranged from 1 to 4 years. The study revealed an early infection in one (0.6%) of the 172 procedures in a patient with spondylolisthesis. A late infection occurred in one (0.6%) patient with the diagnosis of idiopathic scoliosis. In both cases, cultures were positive for Staphylococcus aureus.     

Determinants of eosinophil cationic protein in nasal lavages in children

P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, gamma) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.     

Serum eosinophil cationic protein in infants during first wheezing episode

We measured serum ECP levels in infants during first wheezing episode. Serum ECP in these infants are significantly higher than in control infants, although much higher in children with asthma. Serum ECP in these infants with high serum IgE and/or positive RAST score are higher than in infants with normal serum IgE and negative RAST score. In children with bronchial asthma serum ECP is correlated with peripheral eosinophil counts, but in infants during first wheezing episode serum ECP is often elevated not associated with increased peripheral eosinophil counts. These suggest that activated eosinophils could be responsible for bronchoconstriction in wheezing patients with atopic diathesis even in very early phase and that these eosinophilic inflammations could contribute to formation of increased airway reactivity and bronchial asthma.     

Peak expiratory flow rate (PEF) and serum eosinophil cationic protein (ECP) changes in nocturnal asthmatics

We have investigated the serum ECP and peak expiratory flow rate in 20 patients with nocturnal asthma. Changes of PEF were measured in every 2 hours around the whole day, and the blood samples were obtained at 4:00 and 16:00 to measure the serum ECP level and the peripheral Eo numbers. In addition, 10 asthmatics as well as normal subjects received methacholine challenge at 4:00 and 16:00. It was found that the PEF reached the lowest point at 4:00 and obviously less than that at 16:00 (187.50 +/- 120.31 L/min vs 313.00 +/- 108.14 L/min, P < 0.05), that the airway reactivity at 4:00 was significantly higher than at 16:00 (P < 0.05) and the difference of MCH-PC20 between the two time points was 0.34 +/- 0.31 mg/ml, that the serum ECP level at 4:00 was obviously higher than that at 16:00 (11.14 +/- 7.40 micrograms/ml vs 5.49 +/- 4.12 micrograms/ml, P < 0.05). The change rate of PEF markedly related to the change of serum ECP between the two time points (r = 0.61, P < 0.05). The findings suggested that the activation of Eo and its release of ECP might be effect of the circadian-rhythmic change of pulmonary functions in nocturnal asthma.     

Serum levels of eosinophil cationic protein and eosinophil protein x in pollen atopic patients with stable asthma and its relation with bronchial hyperresponsiveness

Eosinophils are important effector cells in allergic inflammation described in allergic rhinitis (AR) and allergic bronchial asthma (BA). During the pollen season serum levels of eosinophil cationic protein (ECP) and eosinophil X protein/eosinophil-derived neurotoxin (EPX/EDN) are increased in BA. The aim of the present study was to evaluate the serum levels of ECP and EPC in pollen atopic patients with AR and BA during the winter. 92 patients were studied. They were divided into three groups: I 29 patients with AR, II 51 patients with BA and III 12 healthy subjects. Allergic rhinitis and bronchial asthma were diagnosed by routine clinical tests: clinical history, skin tests, total IgE and specific IgE. In addition ECP and EPX were determined in serum. All patients were asymptomatic, stable and without medical treatment. Methacholine challenge test (MCT) was performed in all patients. MCT were positive in 4 patients of group I and 45 patients of group II. ECP levels (ug/l) were: 21 (I), 24 (II) and 7 (III). EPX levels (ug/l) were 35 (I), 45 (II) and 21 (III). Statistical differences (p < 0.01) were observed both in ECP and EPX levels in patients with MCT positive in relation to patients with MCT negative, and in allergic patients (I and II) in comparison with the healthy subjects (III) (p < 0.01). ECP and EPX serum levels are increased in patients with a positive MCT in the winter, out of the pollen season, when patients are asymptomatic, stable and without treatment. This fact suggests that eosinophils play an important role in the pathogenesis of bronchial asthma.     

Eosinophil cationic protein in serum of corticosteroid-sensitive and resistant bronchial asthma

We report here the identification of the novel subunit of the mitochondrial F1F0-ATPase from Saccharomyces cerevisiae, ATPase subunit e. Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled-coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins. Estimation of its native molecular mass and ability to be co-immunoprecipitated with a subunit of the F1-ATPase, demonstrate that subunit e is a subunit of the F1F0-ATPase. Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0-ATPase. Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.     

Inhibitory effects of formoterol on platelet-activating factor induced eosinophil chemotaxis and degranulation

A new long-acting beta 2-agonist, formoterol, has been reported to have a greater efficacy and duration of action in asthmatic patients as compared to conventional beta 2-agonists. We recently demonstrated that formoterol inhibited antigen-induced late asthmatic response (LAR) and accompanying airway eosinophilia in guinea pigs. In this study, we investigated the direct effect of formoterol in vitro on human eosinophil function, focusing on platelet-activating factor (PAF)-induced eosinophil chemotaxis and eosinophil cationic protein (ECP) release. Purified normodense eosinophils were separated by discontinuous gradient from 12 mild asthmatic patients. Formoterol in concentrations of 1-100 microM significantly inhibited PAF-induced eosinophil chemotaxis in a dose-dependent manner with a concentration of drug required to produce 50% inhibition (IC50) of 10.16 microM; % inhibition: 22.9 +/- 13.0% (1 microM), 51.6 +/- 12.7% (10 microM), 75.0 +/- 11.3% (100 microM). When formyl-methionyl-leucyl-phenylamine (FMLP) was used as a chemoattractant, a similar inhibition of eosinophil chemotaxis by formoterol was observed; % inhibition: 13.1 +/- 5.0% (1 microM). 47.7 +/- 7.6% (10 microM), 65.5 +/- 16.5% (100 microM). A conventional beta 2-agonist, salbutamol, at doses to 100 microM did not show any inhibitory effects on PAF-induced eosinophil chemotaxis. Formoterol in concentrations of 1-100 microM also significantly inhibited PAF-induced ECP release from eosinophils; % inhibition: 21.7 +/- 9.0% (1 microM), 39.3 +/- 7.4% (10 microM), 39.6 +/- 8.4% (100 microM). In the presence of phosphodiesterase inhibitors, theophylline or isobutylmethyl xanthine (IBMX), the inhibition by formoterol on PAF-induced ECP release was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated serum levels of the eosinophil granule major basic protein in patients with eosinophilia

A radioimmunoassay was established for the human eosinophil granule major basic protein (MBP). The mean level of MBP in sera from 105 normal control patients was 454 ng/ml, whereas in a sample of 188 patients with various forms of diseases, including the hypereosinophilic syndrome, levels as high as 14,000 ng/ml were measured. Serum levels of MBP did not correlate with eosinophil counts in normal subjects, but a positive correlation was seen in patients with eosinophilia; the patients with eosinophil counts greater than 350/mm3 generally showed increased levels of MBP. Many patients with skin disease and normal eosinophil counts had elevated levels of serum MBP. Monomer MBP has a molecular weight of 9,300, but in sera of patients with eosinophilia, the MBP activity was of high molecular weight, greater than 50,000. Analyses of serum by Sephadex G-200 and by electrofocusing suggest that MBP is not simply polymerized, but rather is bound to a larger carrier molecule. Monomeric MBP can be isolated from serum by reduction of serum with dithiothreitol, alkylation with iodoacetamide, and acidification to pH 2 followed by fractionation on Sephadex G-50 at pH 2. Under these conditions, up to 80% of the MBP emerges in monomeric form. The results indicate that eosinophil granule proteins circulate in blood covalently bound to serum proteins, and that elevated concentrations of serum MBP are present in some diseases associated with eosinophilia.     

Induced sputum eosinophil cationic protein (ECP) measurement in asthma and chronic obstructive airway disease (COAD)

BACKGROUND: Induced sputum is a useful way to monitor airway inflammation in asthma, but cell counts are time-consuming and labour intensive. OBJECTIVE: The aim of this study was to evaluate a novel processing method using eosinophil cationic protein (ECP) as a biochemical marker of sputum eosinophil number and activation in subjects with asthma and other airway diseases. METHODS: Sputum was dispersed with dithiothreitol and centrifuged to yield cell free supernatant and a cell pellet. The pellet was treated with a cellular lysis buffer to release cell-associated ECP. ECP was measured in sputum supernatant and in the lysed cell pellet and was compared with sputum eosinophil counts in 31 adults with asthma, chronic obstructive airway disease (COAD), bronchiectasis and healthy controls. The ratio of supernatant to pellet ECP was evaluated as an index of eosinophil degranulation. The effect of sputum processing reagents and storage time on ECP measurement was also evaluated. RESULTS: ECP measured in the cell pellet lysate correlated closely with sputum absolute eosinophil counts across a range of subject groups (r = 0.72, P = 0.004). Sputum eosinophil counts were less well correlated with supernatant ECP levels (r = 0.54, P < 0.05). Incubation with dithiothreitol or lysis buffer did not influence ECP measurement and sputum ECP levels were stable over a 6-9 month period. Sputum supernatant and pellet lysate ECP concentrations were increased in stable asthma, asthma exacerbations and COAD/bronchiectasis (P < 0.05). The ratio of supernatant to pellet ECP was used as an index of eosinophil degranulation and found to be elevated in asthma exacerbations, COAD and bronchiectasis, but not in stable asthma. CONCLUSION: The measurement of ECP in the sputum cell pellet provides a reliable and efficient estimate of sputum eosinophil counts which can potentially be used in clinical trials and epidemiological surveys. The ECP ratio may be a useful marker of eosinophil activation, and was increased in asthma exacerbation and COAD. The increased ECP in COAD reflects a non-selective accumulation of eosinophils in this condition.     

Neisserial porins inhibit human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but prime the neutrophils to increase their oxidative burst

Porins are trimeric proteins that constitute water-filled pores that allow transmembrane diffusion of small solutes through the outer membrane layer of gram-negative bacteria. The porins are capable of inserting into the membranes of eucaryotic cells, and in the present study we have examined the in vitro effects on neutrophil functions of the following purified porins: meningococcal outer membrane protein classes 1 and 3 and gonococcal outer membrane protein 1B (P1B). The neisserial porins inhibited human neutrophil chemoattractant-induced actin polymerization and degranulation of both primary and secondary granules. The neutrophil expression of immunoglobulin G (IgG) Fc receptors II (Fc gamma RII; CDw32) and III (Fc gamma RIII; CD16), as well as the activation-dependent downregulation of Fc gamma RIII, were reduced by the meningococcal and gonococcal porins. The neisserial porins impaired the upregulation of complement receptors 1 (CD35) and 3 (CD11b) and inhibited the phagocytic capacity of neutrophils, as evaluated by the uptake of meningococci (strain 44/76) in the presence of patient serum containing known amounts of IgG against meningococcal porins. The porins also primed neutrophils to increase their intracellular hydrogen peroxide production in response to FMLP, whereas no such priming was observed if the neutrophil protein kinase C was stimulated directly with phorbol myristate acetate. The neisserial porins influenced neutrophil functions in a time- and concentration-dependent manner. The meningococcal class 1 outer membrane protein and the gonococcal P1B tended to alter neutrophil functions more than the meningococcal class 3 protein. Thus, the neisserial porins inhibited human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but primed the neutrophils to increase their oxidative burst. It remains to be determined whether these in vitro observations reflect mechanisms that may be of importance for the interaction between neutrophils and Neisseria species in vivo.     

Comparison of basophil histamine release, eosinophil cationic protein and non-specific airway responsiveness between mite-sensitive asthmatic and non-asthmatic children and non-allergic controls

To understand the relevance of allergy to the development of asthma in children, we examined basophil histamine release (HR) with Df antigen, blood eosinophil counts, serum eosinophil cationic protein (ECP) levels, and bronchial responsiveness to methacholine (PC20) in three groups of children, including 36 asthmatics with high RAST titre for Df (group 1), 36 non-asthmatics with similarly high RAST titre for Df (group 2) and 21 non-asthmatics with negative RAST titre for Df (group 3). The amount of Df antigen inducing 50% HR from basophils did not vary significantly between group 1 and 2 (P > 0.05), while none of the cells responded to higher concentrations of Df in group 3. The mean number of blood eosinophils and level of serum ECP were highest in group 1, and lowest in group 3, with group 2 being intermediate, and the differences were significant between all three groups (P < 0.01). The mean PC20 value was the lowest in group 1, intermediate in group 2, and the highest in group 3, and the differences were significant between all three groups (P < 0.01). While correlation studies showed that PC20 values of group 2 subjects significantly correlated with their eosinophil numbers (r = -0.48, P < 0.01) and ECP levels (r = -0.49, P < 0.01), such correlations were not found in group 1 subjects. These results suggest that the degree of the eosinophilic inflammation caused by the allergic reaction to mites is an important factor in determining the clinical expression of asthma in atopic subjects.     

Kinetics and quantitation of eosinophil and neutrophil recruitment to allergic lung inflammation in a brown Norway rat model

We quantitated neutrophil and eosinophil migration into lung parenchyma using specific peroxidase enzyme assays, and into the bronchoalveolar compartment by bronchoalveolar lavage (BALF), in sensitized brown Norway (BN), Fischer, and Lewis rats and also assessed the lungs by histopathology. Fourteen days after sensitization with ovalbumin (OA in alum [given subcutaneously] and OA with Bordetella pertussis [given intraperitoneally]), rats were challenged with an OA aerosol for 1 h. In BN rats, there was marked perivascular and peribronchial edema, focal hemorrhages, and increase in lung wet weight and BALF protein content, accompanied by neutrophilic infiltration at 3-14 h postchallenge. Few eosinophils were seen at 14 h in lung tissue or in BALF. Neutrophils peaked at 24 h in parenchyma ([94 +/- 7] x 10[6]) and in BALF ([2.7 +/- 0.4] x 10[6]) and declined rapidly thereafter. Marked eosinophil infiltration into parenchyma was apparent by 24 h. Eosinophil accumulation peaked at 48 h in parenchyma ([127 +/- 18] x 10[6]) and at 72 h in BALF ([10 +/- 2.4] x 10[6]), comprising up to 85% of lavage cells at this time. Lung eosinophilia persisted for at least 6 d with only a slow decline or clearance, not approximating baseline until day 13 after challenge. Histopathology showed peribronchial and interstitial eosinophilic pneumonia, most severe on day 3. In contrast to the BN rats, essentially no pulmonary inflammation was observed in Lewis and Fischer rats. This model in the BN rat, and the specific peroxidase assays for quantitating tissue eosinophils and neutrophils, should be useful for investigating the regulation of allergen-induced eosinophil and neutrophil migration into and clearance from the lung.     

Effects of stem cell factor (SCF) on human marrow neutrophil, neutrophil/macrophage mixed, macrophage and eosinophil progenitor cell growth

The effects of stem cell factor (SCF) on the subpopulations of granulocyte/macrophage colony-forming units (CFU-GM) were examined. Hematopoietic progenitor cells were enriched from normal adult bone marrow specimens by immunomagnetic beads using an anti-CD34 antibody and lineage marker antibodies for positive selection and negative selection, respectively. SCF enabled neutrophil and neutrophil/macrophage mixed progenitors to respond to granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3) and to develop the colony and further cluster formation. The neutrophil colonies stimulated by GM-CSF or IL-3 consisted mainly of immature cells, while the colonies stimulated by granulocyte colony-stimulating factor (G-CSF) consisted of mature neutrophils irrespective of the addition of SCF. In macrophage and eosinophil lineages, SCF augmented the colony formation in the presence of GM-CSF or IL-3, whereas the enhancement of total progenitor cell growth (colonies plus clusters) was not so marked as compared with the neutrophil lineage. Time-course observation revealed that SCF could stimulate macrophage and eosinophil progenitors to form colonies rapidly. These findings indicate that SCF acts on late myeloid progenitor cells in manners different from the lineages of commitment.     

Characterization of the protein and glycan moieties in different forms of bovine lactoferrin

The BrCN cleavage of lactoferrin-a or -b (LF-a or LF-b) led to the observation of four fragments by SDS-PAGE, whose molecular masses were 77, 58, 52, and 30 kDas, or 74, 54, 47, and 30 kDas, respectively. N-Terminal amino acid sequence analyses show that the sequences of 58, 52, and 30 kDa fragments (residues 64-471, 130-471, and 472-689) of LF-a coincide with those of the 54, 47, and 30 kDa fragments of LF-b. respectively. All these fragments, which were positive by PAS staining, were not stained after being treated with glycopeptidase F. This treatment changed the 58 and 52 kDa fragments of LF-a to the 54 and 47 kDa fragments, respectively, whose molecular masses were the same as those of the treated fragments of LF-b. The 58 and 52 kDa fragments of LF-a bound to the lectin, Ricinus communis agglutinin, while the 54 and 47 kDa fragments of LF-b hardly bound to it.     

Eosinophilic cationic protein in chronic eosinophilic pneumonia and eosinophilic granuloma

We measured eosinophilic cationic protein (ECP) concentrations in the circulation and bronchoalveolar lavage (BAL) fluids from patients with chronic eosinophilic pneumonia, patients with eosinophilic granuloma, and normal control subjects. Significantly increased ECP concentrations were found in the circulation of patients with chronic eosinophilic pneumonia and with eosinophilic granuloma compared with those found in control subjects. The ECP concentrations were well correlated to eosinophil counts in the circulation of patients with chronic eosinophilic pneumonia, while they were not in patients with eosinophilic granuloma. Chronic eosinophilic pneumonia patients had prominently increased ECP concentrations in BAL fluids compared with those found in control subjects, while eosinophilic granuloma patients did not. Those concentrations in chronic eosinophilic pneumonia patients were well correlated to eosinophil counts in the BAL fluid. Corticosteroid therapy remarkably decreased circulating ECP concentrations in three patients with chronic eosinophilic pneumonia, but it had no significant effects in two patients with eosinophilic granuloma. Measurement of ECP concentrations seems to be useful to evaluate the disease activity of chronic eosinophilic pneumonia.     

Cytolysis and piecemeal degranulation as distinct modes of activation of airway mucosal eosinophils

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a noninitiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 +/- 0.50 vs. 2.64 +/- 0.20, p < or = 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer.     

3-Morpholino-sydnonimine-induced suppression of human neutrophil degranulation is not mediated by cyclic GMP, nitric oxide or peroxynitrite: inhibition of the increase in intracellular free calcium concentration by N-morpholino-iminoacetonitrile, a metabolite of 3-morpholino-sydnonimine

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.