C3b-induced eosinophil degranulation involves PI3-kinases and is inhibited by protein kinase C activity

Carlson M, Venge P, Lampinen M. C3b‐induced eosinophil degranulation involves PI3‐kinases and is inhibited by protein kinase C activity. APMIS 2010; 119: 119–26. Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived‐neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÖ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose‐dependent inhibition of all three granule proteins. LY294002 (10^?6 M) also inhibited the release of all proteins. Genistein (10^?6 M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration‐dependent patterns of release of all three proteins. Staurosporine (10^?7 M), GÖ6976 (10^?6 M) and PD98059 (10^?5 M) caused an increased release of the three proteins. PI3‐kinases play an important role in the C3b‐induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b‐induced degranulation.     

Serum eosinophil peroxidase (EPO) levels in asthmatic patients

Eosinophil granular proteins are a useful eosinophilic activation marker in asthmatic patients. In this study, the eosinophil peroxidase (EPO) levels were assessed in different stages of bronchial asthma, in 123 patients suffering from asthma, classified as mild (n=49), moderate (n=49), and severe (n=25), according to the International Consensus Report on Diagnosis and Treatment of Asthma, as well as in 27 healthy controls, with the aim of evaluating the importance of this protein as a severity marker in bronchial asthma, and its possible correlation with parameters such as anamnesis, respiratory function tests, and peripheral blood eosinophil count, and also with some allergologic diagnostic tests, both in vivo and in vitro. The geometric mean serum level of EPO was 9.3±11.3 ng/ml (median±SD) in controls, and 28±37.8 ng/ml in the asthmatic patients. Depending on the asthma severity, the EPO levels were 25±30.5; 29±37.1, and 41 ±47.3 ng/ml in mild, moderate, and severe asthmatics, respectively, being the significant differences between the group of patients with mild and severe asthma (P<0.001). The number of eosinophils (eos) in peripheral blood was 157±20 eos/mm³ in the controls, 334+35 eos/mm³ in mild asthmatics, 510 ±87 eos/mm³ in moderate asthmatics, and 658±72 eos/mm³ in severe asthmatics, with significant differences between all the groups (from P<0.05 to P<0.001). Both the serum levels of EPO and the number of eosinophils were greater in patients with active asthma than in patients with inactive asthma (P<0.001). Significant negative correlations (P < 0.001) were found between serum levels of EPO and FEY, (r_s= 0.30), MEF_25-75 (r_s= -0.33), and MEF₅₀ (r_s= -0.34), and a good positive correlation (r_s= 0.80, P<0.001) was found between EPO levels and the number of eosinophils in peripheral blood. We also found a significant positive correlation between eosinophil number and clinical score (r_s= 0.54, P<0.001) and between EPO levels and the mentioned score (r_s= 0.46, P<0.001).     

Interactions of eosinophil granule proteins with skin: limits of detection, persistence, and vasopermeabilization

BACKGROUND: Eosinophil granule proteins, including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO), and major basic protein (MBP), are prominently deposited in skin in several cutaneous disorders and likely contribute to disease pathology. OBJECTIVE: We sought to determine the limit of detection, persistence, and vasopermeabilization activity of the eosinophil granule proteins in skin. METHODS: The eosinophil granule proteins were injected intradermally. Their minimum detectable concentrations in human surgical waste skin and their persistence in guinea pig skin were determined by indirect immunofluorescence. Vasopermeabilization activity in the guinea pig without and with H1 antihistamine (pyrilamine maleate) pretreatment was assessed by extrusion of Evans blue dye-treated plasma. RESULTS: The lowest detectable cutaneous concentrations were 0.05 micromol/L EPO, 0.1 micromol/L MBP, 0.25 micromol/L ECP, and 1 micromol/L EDN. Granule proteins persisted in guinea pig skin in vivo for 1 week (EPO), 2 weeks (ECP), 2.5 weeks (EDN), and 6 weeks (MBP). Each of the eosinophil granule proteins increased cutaneous vasopermeability in a concentration-dependent manner. The potency of vasopermeabilization induced by each granule protein was comparable with that of histamine. Pyrilamine maleate pretreatment of guinea pigs did not alter increased vasopermeability induced by ECP and EDN but significantly inhibited that induced by EPO and MBP. CONCLUSIONS: Micromolar concentrations of eosinophil granule proteins are often deposited in skin in eosinophil-associated cutaneous disorders such as atopic dermatitis. These pathophysiologically relevant concentrations of eosinophil granule proteins cause increased cutaneous vasopermeability (both by means of histamine-independent and histamine-dependent mechanisms) and might alter cutaneous function for days to weeks.     

Variation in genes encoding eosinophil granule proteins in atopic dermatitis patients from Germany

Background  

Atopic dermatitis (AD) is believed to result from complex interactions between genetic and environmental factors. A main feature of AD as well as other allergic disorders is serum and tissue eosinophilia. Human eosinophils contain high amounts of cationic granule proteins, including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO) and major basic protein (MBP). Recently, variation in genes encoding eosinophil granule proteins has been suggested to play a role in the pathogenesis of allergic disorders. We therefore genotyped selected single nucleotide polymorphisms within the ECP, EDN, EPO and MBP genes in a cohort of 361 German AD patients and 325 healthy controls.     

Loratadine treatment of rhinitis due to pollen allergy reduces epithelial ICAM-1 expression

Background Loratadine and cetirizine are new generation antihistamines, which are clinically effective in the treatment of allergic rhinitis. Objective The aim of the study was to evaluate antiallergic activity of loratadine compared with cetirizine, over a 2 week period under natural allergen exposure, in a double-blind parallel groups, randomized, controlled trial. Methods Twenty patients, sensitized to grass and/or Parietaria pollen, were subdivided into two groups, one receiving loratadine the other cetirizine respectively. Both were dosed at 10 mg/day. Evaluated parameters were: clinical symptoms, nasal inflammatory cell (such as neutrophil, eosinophil and metachromatic cells) counts, ICAM-1 expression on nasal epithelial cells, and nasal mediators (e.g. histamine, ECP, EPO and MPO). Results Loratadine and cetirizine significantly improved symptoms (P < 0.002), significantly reduced eosinophil (P < 0.016) and metachromatic cell (P < 0.01) infiltration, levels of ECP (F < 0.002), EPO (P < 0.006) and histamine (P < 0.01) and ICAM-1 expression on nasal epithelial cells (P < 0.02). No difference was demonstrated between the two drugs. Conclusion The antiallergic activity of loratadine and cetirizine is documented by their actions on the inflammatory and clinical parameters, especially ICAM-1 modulation.     

Different airway responsiveness profiles in atopic asthma, nonatopic asthma, and Sjögren's syndrome. BHR Study Group. Bronchial hyperresponsiveness

Background : Different mechanisms may underlie bronchial hyperresponsiveness (BHR) in different diseases. The aim of this study was to investigate the bronchial responsiveness profile produced by three different challenge tests, methacholine, a direct simulus, and two indirect stimuli, adenosine 5′‐monophosphate (AMP) and cold air, in subjects with asthma and patients with Sjögren's syndrome. Methods : The study population comprised 40 adult patients with asthma, 18 subjects with Sjögren's syndrome, and 20 controls. Blood samples were collected before each challenge for measurements of serum eosinophil peroxidase (S‐EPO) and eosinophil cationic protein (S‐ECP). The investigated subjects recorded peak expiratory flow and kept a symptom diary. Results : Atopic subjects with asthma were significantly more hyperresponsive to AMP than nonatopic subjects with asthma (P=0.01) and subjects with Sjögren's syndrome (P=0.02). No difference was seen between atopic and nonatopic subjects with asthma in the case of challenges with methacholine or cold air. In atopic subjects with asthma, a significant correlation was found between challenges with methacholine and AMP (r=0.91, P=0.0001) and methacholine and cold air (r=0.83, P=0.004), but, in nonatopic subjects with asthma, no significant correlation was seen between methacholine and AMP or cold air challenges. In atopic subjects with asthma, the dose‐response slope for AMP was correlated to S‐EPO (r=?0.56; P=0.01) and S‐ECP (r=?0.51, P=0.02), while no correlation between BHR and inflammation markers was found in the two other patient groups. Conclusions : The results of this study suggest that patients with asthma and subjects with Sjögren's syndrome display different bronchial responsiveness profiles for different challenge agents. Atopic subjects with asthma are more hyperresponsive to AMP than nonatopic subjects and patients with Sjögren's syndrome. More than one challenge may be required to detect different aspects of bronchial responsiveness.     

PI3-Kinase Regulates Eosinophil and Neutrophil Degranulation in Patients with Allergic Rhinitis and Allergic Asthma Irrespective of Allergen Challenge Model

The PI3K pathway plays a major role in many vital cell processes. Our primary aim was to investigate signalling through PI3K for in vitro degranulation from allergen-primed eosinophils and neutrophils in allergic rhinitis and allergic asthma after seasonal and experimental allergen challenge. Nine patients with allergic rhinitis, eight with allergic asthma and four controls were studied during birch pollen season and after nasal and bronchial allergen challenge. Primed blood eosinophils and neutrophils were stimulated for in vitro degranulation with C3b-coated Sephadex particles, after prior incubation with Wortmannin, a PI3K inhibitor. The released amounts of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) were measured by radioimmunoassay. Wortmannin (10⁻⁶ to 10⁻⁹ M) inhibited ECP, EPO and MPO release in a dose-dependent manner in allergic rhinitis and allergic asthma in all three allergen challenge models. Inhibition of ECP release tended to be lower in the asthmatics in all allergen challenge models, statistically significant compared to the controls during season for 10⁻⁸ M Wortmannin (p = 0.01). A clear propensity towards less inhibition in the rhinitic patients was seen after nasal and bronchial challenge compared to seasonal exposure, significant for ECP (10⁻⁸ M Wortmannin; p = 0.034 and 0.002, respectively). Signalling through PI3K is clearly involved in ECP, EPO and MPO release in allergic rhinitis and allergic asthma irrespective of allergen challenge model. Allergic asthma demonstrated less inhibition of ECP release via PI3K during pollen season, indicating that other pathways play a greater role in eosinophil degranulation in allergic asthma than allergic rhinitis.     

Eosinophil markers in seasonal allergic rhinitis

Background The purpose was to study activation markers of the eosinophil granulocytes in seasonal allergic rhinitis, and the impact of topical steroid therapy thereupon.
Methods Sixty-three rhinitis patients with monoallergy to grass were examined before and at peak pollen season. Blood eosinophil count, eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO) in serum and nasal lavage fluid were measured. During the season, patients were randomized to treatment with intranasal fluticasone propionate 0.1 mg o.d. ( n =26), 0.2 mg o.d. ( n =25), or placebo (n = 12). Six healthy persons served as controls.
Results During the season, all parameters, except nasal lavage ECP, increased in the placebo group (P<0.001 – P<0.05). Significant differences were seen between the steroid grotips and the placebo group for all parameters (P<0.001–F<0.05). Higher eosinophil count (P<0.05), serum EPO (F<0.02), and nasal lavage EPO (P<0.05) were found in patients before season than in controls. The following winter, 44 patients returned for repeated measurement. Lower levels of nasal lavage EPO were observed for patients than levels at the beginning of the season (P<0.0001).
Conclusions Intranasal fluticasone propionate reduced inflammation of the nasal mucosa, demonstrated locally by nasal lavage ECP and EPO, and systemically by blood eosinophils, serum ECP, and serum EPO. EPO seemed more sensitive than ECP as indicator of allergic inflammation. EPO demonstrated some perennial eosinophil activity in hay fever patients, increasing locally during spring.     

Granulocyte proteins in serum in childhood asthma: relation to spirometry and therapy

Background Measurement of markers of eosinophil activation in asthmatics provides information indicative of ongoing inflammatory processes in the airways. Objectives This study was eonducted to determine the correlations between serum markers of allergie inflammation with spirometry parameters in asthmatic children in different treatment groups. Methods Blood eosinophils. serum levels of eosinophil cationic protein (ECP). eosinophil protein X (EPX), myeloperoxidase (MPO) and tryptase were measured simultaneously with serial measurements of FEV₁/FVC, FEF_25–75 and FEF in 60 children with acute asthma on admission and after 2, 14, 30 and 60 days. Group A received bronchodilators only (n= 20). group B received sodium cromoglycate (SCG) (n= 20) and group C received oral and/or inhaled corticosteroids (n= 20). Results Oral steroid treatment (2 mg /kg/day). given at the onset of the asthma attack, resulted in significant reduction in the ECP and EPX levels in all the children. However, these reduced ECP and EPX levels were not sustained in the children, even in those who continued on maintenance steroid treatment. Significant, but inconsistent, correlations between ECP, EPX with total eosinophil count, percentage eosinophils and spirometry parameters were observed at the different time-points. Tryptase levels were normal in all subjects. There were no significant correlations between myeloperoxidase levels and the spirometry parameters or eosinophil parameters. Serial monitoring of ECP and EPX levels was found to be of some use in predicting clinical outcome in certain steroid-dependent asthmatics (group C) but of no value in the mild asthmatics (group A). Conclusion While elevation of ECP, EPX and MPO in the serum of childhood asthmatics suggests ongoing inflammation and may inversely correlate with spirometry parameters in some patients, the relationship between these markers and airway function is not a simple one.     

Major basic protein, but not eosinophil cationic protein or eosinophil protein X, is related to atopy in cystic fibrosis.

Increased eosinophil granule proteins have been described in serum and sputum samples of patients with cystic fibrosis (CF). It has been assumed that eosinophil degranulation is enhanced in atopic subjects - as in asthmatics. Since in CF no differences in eosinophil cationic protein (ECP), eosinophil protein X (EPX), and eosinophil peroxidase between atopic and nonatopic subjects have been detected, we investigated whether major basic protein (MBP) is increased in serum and sputum samples derived from atopic (n = 14) compared with nonatopic CF subjects (n = 26). In CF patients, high mean serum (sputum) levels of ECP 29.7 microg/l (2.7 mg/l), EPX 53.7 microg/l (7.9 mg/l), and MBP 984.6 microg/l but low sputum MBP levels (57.4 microg/l) were measured. In addition, in serum and in sputum samples, a significant correlation between MBP and ECP (P<0.03 and P<0.0001, respectively) or EPX (P<0.05 and P<0.0004, respectively) was detected. By subdivision of the patients into allergic and nonallergic subjects, significant differences were found for serum MBP values only(mean 1382.2 microg/l vs. 770.5 microg/l; P<0.0001), but not for ECP or EPX serum levels or for eosinophil proteins in sputum. Although no differences between atopic and nonatopic CF patients in ECP and EPX were found, serum MBP levels were higher in patients sensitized to inhalant allergens than in nonsensitized subjects. These results indicate differential release of eosinophil granule proteins in peripheral blood from eosinophils, and they also indicate that MBP in serum likely is to be a better discriminator of atopy in CF.     

Serum eosinophil granule proteins predict asthma risk in allergic rhinitis

Background: Allergic rhinitis is a common disease, in which some patients will deteriorate or develop asthma. It is important to characterize these patients, thereby offering the possibility for prevention. This study evaluated eosinophil parameters as potential indicators of deteriorating allergic airway disease. Methods: The subjects of the study included all patients who suffered seasonal allergic rhinitis and had participated in a study 6 years earlier, in which blood eosinophils, serum eosinophil cationic protein (ECP) serum eosinophil peroxidase (EPO), nasal lavage ECP and nasal lavage EPO levels were measured. Patients in the present study were interviewed on occurrence of rhinitis symptoms during the last season, rhinitis outside season, asthma‐like symptoms and asthma diagnosis, and were skin‐prick tested for common aeroallergens. Eosinophil parameters from the study 6 years earlier were then tested for the ability to predict occurrence of new allergies, worsening of rhinitis and occurrence of asthma. Results: Forty‐four patients participated in the study. In four patients seasonal rhinitis symptoms had deteriorated, 10 had experienced perennial rhinitis symptoms, 14 reported asthma‐like symptoms and seven had been diagnosed with asthma. Thirteen had developed additional sensitization. Patients developing asthma‐like symptoms compared with patients with no such symptoms had significantly higher serum ECP (16.7 μg/l vs 8.2 μg/l; P ≤ 0.01) and serum EPO (17.9 μg/l vs 8.8 μg/l; P ≤ 0.05). Results were similar, considering patients diagnosed with asthma. Blood eosinophils and nasal lavage parameters were not related to development of asthma and asthma‐like symptoms. No eosinophil parameter was related to deterioration of rhinitis or additional sensitization. Conclusion: Serum ECP and EPO in patients with seasonal rhinitis demonstrated a high predictive ability for later development of asthma.     

An early effect of acute plasma volume expansion in humans on serum erythropoietin concentration

The effect of acute plasma volume change in humans on serum erythropoietin [EPO]_s, plasma active renin [REN] and plasma aldosterone [ALDO] concentrations was examined. Plasma volume (PV) expansion was induced by intravenous infusion of 150 ml (30g) of plasma albumin and 500 ml of physiological saline. The [EPO]_s decreased by 14.3% (corrected values for PV expansion) and remained decreased for 5 h. The [REN] was decreased by more than 25% during the day of the experiment and [ALDO] by more than 60%. Only a weak positive correlation was found between [EPO]_s and [REN] (r = 0.35;P < 0.05) but a lack of correlation between changes in PV and [EPO]_s as well as between [EPO]_s and [ALDO] was seen. We postulated that in healthy men an acute PV expansion by 10% to 17.5% would not appear to promote stimulation of EPO synthesis for at least 11 h. Since a weak positive correlation was observed between [EPO]_s and [REN] and a lack of correlation between [EPO]_s and [ALDO], it would seem that there is no direct link between [REN] and [ALDO] and erythropoietin synthesis in healthy subjects.A preliminary report on the topic was presented at the 33rd German National Congress on Sportsmedicine, Padeborn, Germany     

Eosinophil granule proteins in peripheral blood granulocytes.

Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in lysates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (mean +/- SEM) in ng/10(6) eosinophils (and in nM/10(6) eosinophils) were: MBP, 8,982 +/- 611 (641.6); EDN, 3,283 +/- 116 (178.4); ECP, 5,269 +/- 283 (250.9); and EPO, 12,174 +/- 859 (171.5). Basophils from a normal person contained (in ng/10(6) cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/10(6) cells) MBP, 3 +/- 0.5; EDN, 72 +/- 9; and ECP, 50 +/- 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.     

The effect of short and long duration exercise on serum erythropoietin concentrations

Summary The effects of short and long duration exercise on serum erythropoietin concentrations [EPO]_s were studied in seven male cross-country skiers of national team standard and eight male marathon runners, respectively. The short duration exercise was performed as 60 min of cycling at an intensity of 80%–95% of maximal heart rate. Arterial blood oxygen saturations monitored by pulse-oximetry remained unchanged throughout exercise. The partial pressure of O₂ at which haemoglobin was half-saturated with O₂ calculated from forearm venous blood gas tension and blood O₂ saturation, and the erythrocyte 2,3-diphosphoglycerate did not change significantly during the exercise. Blood lactate concentrations were increased at the end of exercise [from 1.3 (SEM 0.1) to 3.6 (SEM 0.3) mmol · 1^–1]. The [EPO]_s determined (by enzyme-linked immunosorbent assay) pre-exercise, 5 min, 6 h, 19 h, and 30 h after the exercise were unchanged [from 16.1 (SEM 2.6) to 19.1 (SEM 3.2), 17.9 (SEM 3.0), 17.0 (SEM 2.5), and 18.6 (SEM 2.9) U·l^–1, respectively]. The [EPO]_s were not correlated to the earlier parameters. The long duration exercise consisted of habitual training, a 3 week break from training followed by 2 and 4 weeks of re-training. The [EPO]_s, body fat (BF), and serum free-testosterone concentrations determined at the end of each period remained unchanged. The maximal oxygen uptakes were decreased after the break from training and increased during retraining (P=0.04). Body mass (m _b) increased after the break in training (P=0.02). The [EPO]_s were correlated to BF,r=0.42,P=0.02;m _b,r=0.45,P=0.01; and free-testosterone concentrations,r=0.44,P=0.01. Thus, short and long-duration exercise had no direct influence on [EPO]_s; but relationships among [EPO]_s, free-testosterone concentrations and body composition were noted.     

Correlation between IgA antibody and eosinophil cationic protein levels in induced sputum from asthmatic patients

Background Eosinophils are known to be main effector cells in allergic inflammation and IgA antibody has been shown to be a potent stimulus for eosinophil degranulation in in vitro conditions. Objective To evaluate the possible role of IgA antibodies on eosinophil degranulation in lower respiratory mucosa of asthmatics, we tried to find a correlation between total IgA and eosinophil cationic protein (ECP) levels in induced sputum from asthmatics. Methods We measured total IgA and albumin levels by nephelometry, and eosinophil cationic protein levels by Pharmacia CAP system in induced sputum from 23 atopic asthmatics and 12 healthy controls. Results IgA and albumin levels in induced sputum from asthmatics with sputum eosinophilia (sputum eosinophil count 5% of 200 counted non-squamous cells) were significantly higher (P < 0.05) than those from controls. However, IgA and albumin levels in induced sputum from asthmatics without sputum eosinophilia were not significantly different with those from controls (P > 0.05). In induced sputum from asthmatics, ECP levels were significantly correlated with albumin (r= 0.44, P= 0.04) and IgA levels (r= 0.67, P= 0.002). ECP/albumin ratio was also significantly correlated with IgA/albumin ratio (r= 0.61, P= 0.004). Conclusion Our results support the hypothesis that IgA antibodies in tracheobronchial secretion may be involved in eosinophil degranulation in asthma, and further study is needed to prove this hypothesis.     

Allergic rhinitis to grass pollen: Measurement of inflammatory mediators of mast cell and eosinophils in native nasal fluid lavage and in serum out of and during pollen season

Background: In allergic rhinitis, mast cells, activated by cross-linking of allergen to mast cell–bound specific IgE, release both vasoactive mediators related to the early nasal symptoms and chemotactic mediators that attract inflammatory cells, such as eosinophils, related to the late-phase response. Objective: We have analyzed, during and out of pollen season, in blood and nasal fluid from patients allergic to grass pollen, histamine and tryptase to monitor the early phase markers and eosinophil and eosinophil cationic protein (ECP) to monitor the late phase. Methods: Twenty patients were enrolled in the study. As a control, we studied 10 nonatopic subjects. Mediators and eosinophils were assessed in blood and nasal fluid. Histamine was tested only in nasal fluid. Results: During pollen season, tryptase but not histamine increased in nasal fluids from patients (2.96 vs 0.22 U/ml, p = 0.001) and correlated with symptom scores (r_s = 0.63, p = 0.003). Tryptase was not detected in serum. Eosinophils increased in nasal cytology (17.0% vs 2.0%, p = 0.001) and in the blood (26.5 vs 12.7 × 10⁶ L, p = 0.001) from patients, but they did not correlate with symptom scores. ECP increased only in the nasal lavage (16.33 vs 1.30 ng/ml, p = 0.001) and correlated with symptom scores (r_s = 0.53, p = 0.016). Conclusions: Both ECP and tryptase increase in nasal secretion in natural disease. Therefore the measurement of tryptase and ECP levels in nasal fluid might be a useful clinical test for monitoring disease activity and the effects of therapeutic agents. (J Allergy Clin Immunol 1997;100:832-7.)     

The human eosinophil in inflammation

Active research during recent years has clearly shown that the eosinophil is a potent inflammatory cell taking active part in almost all kinds of inflammatory processes. The activity of the human eosinophil is mediated by the secretion of four well characterized cytotoxic proteins, ECP, EPO, EPX/EDN and MBP in addition to lipid mediators such as LTC₄ and PAF and toxic oygen metabolites. The cytotoxic potential of the eosinophil has been demonstrated in a number of diseases with a close association of eosinophil accumulation with secretion of granule proteins and tissue injury. Also the measurements of the proteins in various body fluids have provided evidence for the active participation of eosinophils in a number of diseases such as asthma, ulcerative colitis, rheumatoid arthritis, psoriasis to mention a few. The identification of the principles that attract eosinophils to the sites of inflammation must be a major goal in our attempts to control the activity of this potents cell.     

Intracellular expression and serum levels of eosinophil peroxidase (EPO) and eosinophil cationic protein in asthmatic children

BACKGROUND: Eosinophils are involved in the chronic inflammatory response in asthma and their basic proteins are thought to play a major pathophysiological role in this process. While serum levels of basic proteins have been used to monitor the ongoing allergic disease, little is known about the intracellular expression of these proteins in clinical situations. OBJECTIVE: The aim of the study was to determine the intracellular expression of eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) in asthmatic children and control subjects and relate it to serum levels of both proteins, lung function tests and immunoglobulin (Ig)E levels. METHODS: Serum ECP and EPO concentrations were determined by immunoassays in 13 asthmatic children (mean age: 9 +/- 1 years, mean FEV1: 92 +/- 10% predicted, geometric mean PC20 histamine 0.5 mg/mL) and 10 age-matched, healthy control subjects. A flow cytometric single cell assay was employed to detect intracellular ECP and EPO in peripheral blood eosinophils. RESULTS: While serum concentrations of both ECP (asthma: median 15.0 microg/L [range 3.6-57.7] vs control: 5.9 microg/L [2.7-9.1]; P = 0.02) and EPO (22.9 microg/L [5.2-82.5] vs 7. 2 microg/L [2.5-12.7]; P = 0.008) were significantly elevated in asthmatics, the intracellular expression of ECP and EPO (measured as mean fluorescence intensity) was decreased (EG1: 55.3 [17.7-120.8] vs 100.3 [46.5-264.4]; P = 0.01; EG2: 80.2 [24.1-135.3] vs 133.7 [32. 1-244.9]; P = 0.04 and EPO: 49.7 [23.1-155.8] vs 94.9 [28.8-115.2]; P = 0.03). In asthmatics there was a significant correlation of FEV1 with intracellular ECP and of bronchial hyperresponsiveness with serum EPO and ECP. Furthermore, total IgE levels were positively correlated with serum EPO only. CONCLUSION: We conclude that in asthmatics the intracellular content of ECP and EPO in peripheral eosinophils is reduced possibly due to degranulation. Epitope masking in activated eosinophils or a shift to early bone marrow-derived progenitors with less granule proteins are further possible explanations.     

The molecular biology of eosinophil granule proteins.

Here, we briefly review the molecular biology of the human eosinophil granule proteins, major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). The nucleotide sequence of MBP cDNA indicates that MBP is translated as a 25.2-kilodalton preproprotein; the mpb gene consists of 6 exons and 5 introns spanning 3.3 kilobases (kb). The approximately 2.1-kb nucleotide sequence of EPO cDNA corresponds to a prosequence, light chain and heavy chain in that order; similarities to other peroxidases suggest the existence of a multigene family. EDN and ECP cDNAs and genes are remarkably similar throughout, suggesting a relatively recent divergence. Promoter regions of the 4 genes show interesting differences and similarities which may be related to differential gene regulation.     

Increased eosinophil activity in acute Plasmodium falciparum infection—association with cerebral malaria

To assess the eosinophil response to Plasmodium falciparum infection a cohort of initially parasite-free Ghanaian children was followed for 3 months. Seven of nine children who acquired an asymptomatic P. falciparum infection showed increase in eosinophil counts, while a decrease was found in seven of nine children with symptomatic malaria, and no change was observed in 14 children who remained parasite-free. In a hospital-based study, paediatric patients with cerebral malaria (CM), severe anaemia (SA), or uncomplicated malaria (UM) had uniformly low eosinophil counts during the acute illness followed by eosinophilia 30 days after cure. Plasma levels of eosinophil cationic protein (ECP) and eosinophil protein X (EPX) were measured as indicators of eosinophil activation. In spite of the low eosinophil counts, ECP levels were increased on day 0 and significantly higher in patients with CM (geometric mean (95% confidence interval) 8.5 ng/ml (6.8–10.7 ng/ml)) than in SA (4.7 ng/ml (3.0–7.5 ng/ml)) and UM patients (4.3 ng/ml (3.6–5.3 ng/ml), P < 0.001). A similar pattern was found for EPX. It thus appears that the low eosinophil counts may be due to tissue sequestration and destruction rather than decreased production. The plasma levels of the granule proteins correlated with levels of tumour necrosis factor and soluble IL-2 receptor, implicating inflammatory responses and T cell activation as causes of the eosinophil activation. By contrast, the eosinophil induction did not appear to be part of a Th2-like response. Eosinophil granule proteins may be important in both control of malaria infection and the pathogenesis of severe malaria.