Serum lysozyme: a potential marker of monocyte/macrophage activity in rheumatoid arthritis

OBJECTIVE: Estimate the contribution of monocytes/macrophages to the disease process in rheumatoid arthritis (RA), by measuring the serum levels of the leucocyte-derived granular proteins: lysozyme, myeloperoxidase (MPO), lactoferrin and human neutrophil lipocalin (HNL). METHODS: Serum levels of these granular proteins were measured in patients with RA (n=23) and in healthy controls (n=27), and in 10 patients with RA after treatment with low-dose prednisolone. The serum levels of the granular proteins were also measured before and after treatment with metyrapone, a substance that inhibits the synthesis of cortisol in the adrenals. RESULTS: The serum levels of lysozyme and MPO were elevated in patients with RA, while the concentrations of lactoferrin and HNL were similar in both groups. Prednisolone treatment decreased the serum concentration of lysozyme and MPO. Metyrapone did not influence the level of the granular proteins measured. CONCLUSIONS: The increased serum levels of lysozyme and MPO, but not of HNL and lactoferrin in RA could indicate a stimulated secretory activity of mononuclear phagocytes. The measurement of serum lysozyme, as an indicator of monocyte/macrophage activity, might be used to study disease activity in RA.     

Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis

BACKGROUND AND AIM: Accumulation and infiltration by neutrophil granulocytes is a prominent feature in the local inflammatory process in ulcerative colitis (UC). The present study was performed to evaluate human neutrophil lipocalin (HNL) as a specific neutrophil marker in the inflamed lesions of the colon and rectum in patients with colitis and proctitis. METHODS: The activity of intestinal neutrophils with respect to release of granule proteins was studied in 18 patients with UC (10 with colitis and eight with isolated proctitis) and in 18 healthy controls using perfusion fluid and biopsies from the sigmoid colon and rectum. The released amounts of the neutrophil granule proteins HNL and myeloperoxidase (MPO) were determined by radioimmunoassays, and the location of HNL and MPO in biopsies from colonic mucosa was examined by immunohistochemistry. RESULTS: Mucosal release of HNL and MPO was increased 10-55-fold in patients with colitis and proctitis compared with controls. Their bowel biopsies demonstrated that only neutrophils were stained with anti-HNL. We also found correlations between HNL and levels of granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 8 (IL-8) in perfusion fluids from the sigmoidal segments of patients with proctitis, between HNL and GM-CSF in rectal segments in patients with proctitis, and in sigmoidal segments in patients with colitis. CONCLUSION: We conclude that the increased release of HNL and MPO in colorectal perfusion fluids indicates neutrophil involvement in the local inflammatory process, and suggest that HNL may serve as a specific marker of intestinal neutrophil activation in UC. GM-CSF, and to some extent IL-8, may play a role in neutrophil accumulation and priming in this disease.     

Human neutrophil lipocalin (HNL) and myeloperoxidase (MPO). Studies of lung lavage fluid and lung tissue

Myeloperoxidase (MPO) and human neutrophil lipocalin (HNL) are proteins which are stored in neutrophil granulocytes, in the primary and secondary granules, respectively. These granules or their contents of MPO and HNL are secreted upon activation of the cells, and measurement of these soluble markers in biological fluids, such as bronchoalveolar lavage (BAL), has been proposed to mirror the degree of neutrophil activity in the tissue. We conducted a BAL study in 10 healthy volunteers, with the aim to evaluate the intra-individual variability of the concentration of HNL and MPO recovered in sequential aspirations, during a time period when the concentrations of HNL and MPO in BAL fluids were considered to have equilibrated with those in the underlying tissues. The concentrations of HNL were less variable than those of MPO (coefficients of variability 0.33 +/- 0.07 vs. 0.92 +/- 0.28; P = 0.01), suggesting HNL to be a more useful marker of neutrophil activity within the airspace. The specificity of HNL as a selective index of neutrophil cells was confirmed by means of immunohistochemical staining of uninvolved lung tissue specimens obtained from patients referred to pulmonectomy due to carcinoma. While HNL was located only to intracellular spaces of neutrophils, MPO was in addition located to other cells as well. We speculate that the dynamic changes of pressure across the membranes and flow of solutes during a lavage process might mobilize particulate matter and adherent cells, some of which may be loaded with MPO, and that this may introduce larger variability in the recovery of MPO than of HNL. We conclude that using HNL as a soluble indicator of neutrophil presence is more feasible than using MPO.     

Human neutrophil lipocalin: a specific marker for neutrophil activation in severe Plasmodium falciparum malaria

We have earlier indicated neutrophil activation in severe malaria by measuring myeloperoxidase (MPO) and lysozyme, leukocyte granule proteins secreted by neutrophils as well as by other blood cells (monocytes/macrophages). In this study we evaluated the plasma levels of human neutrophil lipocalin (HNL), a specific neutrophil granule protein, in relation to previously reported markers MPO and lysozyme, for clinical significance in indicating severe malaria. For this purpose, plasma samples were analyzed from 65 individuals with severe malaria, mild malaria or malaria negative, all living in the Gedarif area of Sudan. The plasma levels of HNL were significantly higher in the group of patients with severe malaria as compared with the other two groups. Plasma levels of HNL correlated significantly to those of MPO and lysozyme, as well as to body temperature, degree of parasitaemia and pulse rate. These results confirm our previous findings that neutrophils are activated in-patients with severe malaria and the level of HNL is a good marker in this context.     

Increased nitric oxide in expired air in patients with Sj?gren's syndrome. BHR study group. Bronchial hyperresponsiveness.

Nitric oxide has an important role in the regulation of airway function and can have pro-inflammatory effects. Bronchial hyperresponsiveness (BHR) and respiratory symptoms are common in patients with Sj?gren's syndrome (SS). The aim of this study was to determine whether patients with SS have an increased amount of exhaled NO and whether this NO correlates with respiratory symptoms and BHR. Exhaled NO was measured in 18 patients with SS and 13 normal subjects on three different occasions with intervals of at least 3 days using a chemiluminescence method. Airway responsiveness was assessed with methacholine provocation. Serum levels of myeloperoxidase (MPO), human neutrophil lipocalin (HNL), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO) were measured. Exhaled NO was significantly higher in patients with SS than in controls (147+/-82 versus 88+/-52 nL x min(-1); mean+/-SD; p=0.041). Exhaled NO was correlated with age (partial r=0.52, p=0.006) and serum HNL (partial r=0.46, p=0.014). There were no significant correlations between exhaled NO and respiratory symptoms, BHR or serum MPO, ECP or EPO. Disease duration was negatively associated with serum MPO (r=-0.47, p=0.043). In patients with SS, a positive correlation was found between symptom score and serum ECP (partial r=0.65, p=0.003) and EPO (partial r=0.62, p=0.004) and a negative correlation with age (partial r=-0.60, p=0.005). In conclusion, elevated levels of exhaled nitric oxide in patients with Sj?gren's syndrome were demonstrated. The mechanism underlying this increase in exhaled nitric oxide in Sj?gren's syndrome is not known.     

The effect of granulocyte colony-stimulating factor (G-CSF) on the degranulation of secondary granule proteins from human neutrophils in vivo may be indirect

Granulocyte colony-stimulating factor (G-CSF) was administered at a dose of 7.5 or 10 μg/kg s.c. once daily for 6 d (days 1–6) to two groups consisting of eight and six healthy volunteers. The administration of G-CSF resulted in a rapid decrease in neutrophil counts and serum levels of the secondary granule protein, human neutrophil lipocalin (HNL) after 30 min, followed by a recovery and gradual increase within 180 min. The number of circulating neutrophils and plasma and serum levels of neutrophil secondary granule proteins were dramatically elevated on day 2 (1 d after the administration of G-CSF) and stayed so until day 7. The plasma levels of HNL and lactoferrin (LF) showed a biphasic pattern with peaks at day 2 and days 5–7, and remained highly elevated at day 12. The serum levels of HNL and LF increased rapidly (about 8-fold and 6-fold, respectively) on day 2 and stayed elevated until day 7, subsequently returning to baseline levels. At day 5, neutrophil release induced in vitro by f-MLP was significantly enhanced. The cellular contents of HNL and LF were reduced to about 50% of levels before G-CSF administration at day 5. The release of lactoferrin and HNL, but not of myeloperoxidase (MPO), was slightly enhanced after preincubation of isolated normal neutrophils with G-CSF in vitro, but no obvious release of these proteins was observed with G-CSF alone. The administration of G-CSF resulted in a dramatic increase in the alkaline phosphatase (AP) activity in the plasma membrane, with maximal activity occurring at day 5. Furthermore, during administration of G-CSF, TNF-α in plasma increased about 25-fold. TNF-α started to rise at day 2 and peaked at day 6. After discontinuation of G-CSF the levels of TNF-α gradually decreased. The elevated levels of TNF-α (tumour necrosis factor-α) were temporally correlated to the other signs of neutrophil activation. GM-CSF and IL-8, however, were not detected in plasma. Our data suggest that G-CSF affects the neutrophils not only directly but also indirectly by the induction of the production of other cytokines such as TNF-α.     

Nitric oxide and inflammatory response in simulated extracorporeal circulation

BACKGROUND: Simulated extracorporeal circulation (SECC) induces inflammatory reaction. Nitric oxide (NO) has pro-and anti-inflammatory properties. NO role in SECC-related inflammatory response is unclear. The aim of this study was to clarify if NO affects the foreign-surface induced leukocyte activation during SECC. METHODS: Human blood was circulated through SECC during 3 hours. Control group C was ventilated with oxygen/air mixture and the study group with oxygen/air mixture and NO. Leukocyte activation was measured as serum levels of myeloperoxidase (MPO), human neutrophil lipocalin (HNL), lactoferrin (LF), interleukin-1-beta (IL-1 beta) and interleukin-10 (IL-10). Oxygen free radical production capacity was evaluated with chemiluminescence. NO metabolites nitrite/nitrate were estimated in serum. RESULTS: Leukocyte granule release increased over time. Addition of NO significantly increased MPO, HNL and LF release. The average difference increased with SECC duration. NO addition did not significantly affect measured interleukins concentration or oxygen free radical production capacity. NO metabolites increased significantly in the NO circuits. CONCLUSIONS: Results indicate that NO addition during SECC is pro-inflammatory and has no effect on oxygen free radical production and interleukin release.     

A new method for the quantification of neutrophil and eosinophil cationic proteins in feces: establishment of normal levels and clinical application in patients with inflammatory bowel disease

OBJECTIVES: The aims of this study were 1) to develop a valid method for the measurement of the eosinophil proteins eosinophil cationic protein (ECP) and eosinophil protein X (EPX) and neutrophil proteins myeloperoxidase and human neutrophil lipocalin (HNL) in feces and 2) to investigate their potential role as disease activity markers in inflammatory bowel disease (IBD). MErHODS: Feces samples were obtained from 44 apparently healthy individuals (HIs), 18 patients with IBD (11 with ulcerative colitis [UC] and seven with Crohn's disease [CD]), and three with collagen colitis. The granulocyte markers were measured using immunoassays in supernatants from processed feces. RESULTS: ECP, myeloperoxidase, and, to a lesser degree, EPX and HNL were bound to the solid part of feces. However, feces homogenized in an extraction buffer containing the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide allowed an efficient recovery of the proteins (i.e., up to 100-fold increased levels compared to homogenization in saline). All four proteins were stable for at least 7 days at +6 degrees C and at least 3 days at +22 degrees C. The normal fecal geometric mean (95th percentile) levels of ECP, EPX, myeloperoxidase, and HNL were estimated to be, respectively, 1.69 microg/g (6.41), 0.57 microg/g (1.72), 3.54 microg/g (8.77), and 1.97 microg/g (4.91). Markedly increased feces levels of all markers (p < 0.0002), compared to HIs and CD patients, were observed in UC. However, the marker levels in CD patients were significantly increased relative to HIs (p < 0.05 to p < 0.0002). Increased levels of HNL and myeloperoxidase were also observed in the three collagen colitis patients. The discriminative capability between UC patients and HIs was somewhat superior for EPX and myeloperoxidase. CONCLUSIONS: The method described here takes into account the molecular properties of the granule proteins and the heterogeneity in feces consistency, which is a prerequisite for a valid and reproducible measurement of cationic granule proteins. We suggest that EPX and myeloperoxidase, when applied in IBD, are the best eosinophil and neutrophil markers for studying GI inflammation.     

Neutrophil-associated activation markers in healthy smokers relates to a fall in DL(CO) and to emphysematous changes on high resolution CT

Smoking is a risk factor for developing chronic obstructive pulmonary disease (COPD), but there are no good indicators for early identification of subjects who will develop symptomatic COPD. The aim of this study was to investigate inflammatory mechanisms related to changes in lung function and emphysematous changes on high resolution computed tomography (HRCT) in 'healthy' smokers. Subjects were 60-year-old men from a population study. Bronchoscopy was performed in 30 smokers and 18 who had never smoked. Blood tests, lung function measurements and HRCT were carried out in 58 and 34 subjects, respectively. In comparison with never-smokers, smokers had higher levels of myeloperoxidase (MPO), human neutrophil lipocalin (HNL), eosinophil cationic protein (ECP) and lysozyme in blood, higher levels of MPO, interleukin-8 (IL-8) and HNL in bronchial lavage (BL), and of IL-8, HNL and interleukin-lbeta (IL-1beta) in bronchoalveolar lavage (BAL). Smokers also had lower levels of Clara cell protein 16 (CC-16) in blood. HNL in BL and BAL showed strong correlations to other inflammatory markers (MPO, IL-8, IL-1beta). The variations in MPO in BL were explained by variations in HNL (R2 =0.69), while these variations in BAL were explained by variations in HNL and IL-1beta (R2 = 0.76). DL(CO) was the lung function variable most closely related to MPO and IL-8 in BL and BAL and to IL-1beta in BAL. In a multiple regression analysis, MPO, IL-1beta, IL-8 and CC-16 in BL and MPO in BAL contributed to the explanation of variations in DL(CO) to 41% and 22%. respectively, independent of smoking habits. In smokers with emphysematous lesions on HRCT, HNL in BAL correlated to emphysema score (r(s) = 0.71). We conclude that 'healthy' smoking men with a near normal FEV1 show signs of inflammation in the lower airways that are related to a decrease in DL(CO) and to emphysematous lesions on HRCT. This inflammation seems to be the result of both monocyte/macrophage and neutrophil activation.     

Nitric oxide increases leukocyte granule release during simulated extracorporeal circulation

BACKGROUND: Nitric Oxide (NO) is reported to possess anti-inflammatory properties. The aim of this study was to investigate if nitric oxide affects leukocyte response during simulated extracorporeal circulation (SECC). METHODS: Human blood was circulated for 23 hours through SECC circuit. Control group C (n = 5) was ventilated with an oxygen/air mixture, and NO was added in the study group (n = 5). Leukocyte response was determined by release of myeloperoxidase (MPO) and human neutrophil lipocalin (HNL) and by oxygen free radical production, estimated using chemiluminescence. RESULTS: Addition of NO significantly increased MPO at 30 minutes and 120 minutes of SECC and HNL at 120 minutes of SECC. Oxygen free radical production in whole blood was generally not affected by NO. Similarly, no significant differences were observed between the groups with regard to the chemiluminescence in isolated granulocytes. CONCLUSIONS: Nitric oxide increased release of leukocyte granule derived proteins; MPO and HNL at an early stage of simulated extracorporeal circulation. At the same time, nitric oxide did not affect the whole blood and leukocyte capacity to produce oxygen free radicals.     

Myeloperoxidase as a marker of increasing systemic inflammation in smokers without severe airway symptoms

BACKGROUND: There is increasing evidence of systemic inflammation in patients with chronic obstructive pulmonary disease (COPD), but there is very little information on the development of systemic inflammation in smokers without severe airway symptoms. In this longitudinal study, we examined whether smokers with mild or no airway symptoms develop signs of systemic inflammation by assessing inflammatory markers in blood over a 6-year period. METHODS: Forty smokers and 28 male never-smokers were investigated in 1995 (year 0) and 6 years later (year 6). At year 6, 11 smokers had stopped smoking (quitters); these subjects were analysed as a separate group. At year 0 and 6, we measured serum levels of myeloperoxidase (MPO), lysozyme and human neutrophil lipocalin (HNL), regarded as markers of activity in neutrophils plus monocyte-lineage cells, monocyte-lineage cells only and neutrophils only. RESULTS: All systemic markers of inflammation (MPO, HNL and lysozyme) were significantly higher in smokers than in never smokers at year 6. For MPO alone, smokers only displayed a unique pattern compared with the other groups; the concentration of MPO in blood increased among smokers during the 6-year period, and this increase was statistically significant compared with that observed in never-smokers. Even though quitters did not display any clear change in MPO, we observed a statistically significant negative correlation between the change in blood MPO and the duration of smoking cessation in this group. For HNL and lysozyme, the changes over time were similar in smokers and never-smokers, with no statistically significant difference compared with quitters. CONCLUSION: This study provides evidence that male smokers without severe airway symptoms develop an increasing systemic inflammation during a 6-year period. The study forwards both direct and indirect evidence that MPO may be an early marker of this systemic inflammation. However, our study also forwards indirect evidence that ongoing tobacco smoking may "drive" the level of systemic HNL and lysozyme. The origin of the increased MPO and its value as an easily measured predictor for future COPD deserves to be further evaluated.     

Biosynthesis of granule proteins in normal human bone marrow cells. Gelatinase is a marker of terminal neutrophil differentiation

Differentiation and maturation of myeloid cells is characterized by the sequential acquisition of two distinct cytoplasmic granule subsets, azurophil granules and specific granules. We recently showed the existence of a third granule subset, gelatinase granules. To investigate whether appearance of gelatinase granules marks a further step in maturation of myeloid cells beyond the appearance of specific granules, we sorted normal human bone marrow cells into one of three groups according to maturity by centrifugation on Percoll density gradients. The biosynthesis of myeloperoxidase (MPO) (an azurophil granule marker), lactoferrin and neutrophil gelatinase-associated lipocalin NGAL (specific granules markers) and gelatinase was then studied in each of these groups. We found that gelatinase was synthesized mainly in the group containing band cells and segmented cells. This contrasted with lactoferrin and NGAL, which were synthesized almost exclusively in the group containing myelocytes and metamyelocytes, and with MPO, which was mainly synthesized in the group containing myeloblasts and promyelocytes. Immunocytochemistry was in full agreement with the biosynthesis data, and showed that gelatinase appears in band cells, whereas NGAL and lactoferrin both appear in myelocytes. Thus, acquisition of gelatinase granules marks a step in neutrophil differentiation beyond the appearance of specific granules.     

Serum myeloperoxidase and lactoferrin in neutropenia.

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Sputum processing for evaluation of inflammatory mediators

Neutrophil-dominated inflammation is prominent in the cystic fibrosis (CF) and chronic bronchitis (CB) airways. We assessed the degree of airway inflammation by measuring the sputum concentrations of interleukin (IL)-8, myeloperoxidase (MPO), and deoxyribonucleic acid (DNA). We determined the relationship among the concentrations of these mediators and investigated methodological problems that may be responsible for reported variability in measurements. Sputa obtained from 31 patients were solubilized with phosphate-buffered saline, dithiothreitol (DTT) (0.1% or 1%), or dornase alfa (0.2 mg/mL). The sputum concentration of IL-8 and MPO was measured by enzyme-linked immunosorbent assay (ELISA), and DNA was measured using microfluorimetry. There was a significant relationship among sputum IL-8, MPO, and DNA. For MPO (means +/- SD), CF was 1,392 +/- 771 vs. CB at 75 +/- 65 mcg/mL; P < 0.0001. For IL-8: CF was 239 +/- 154 vs. CB at 121 +/- 108 ng/mL; P = 0.0002. For DNA, CF was 1.707 +/- 1.25 vs. CB at 0.184 +/- 0.272 mg/mL; P < 0.0001. The MPO concentration in CF sputum was approximately double after in vitro treatment with dornase alfa (P < 0.0001). There is a greater concentration of IL-8, MPO, and DNA in CF than in CB sputa. There is a significant relationship among these inflammatory markers in sputum. DNA polymers bind myeloperoxidase in the sputum, and we speculate that treatment with dornase alfa may remove a source of MPO inhibition.     

Serum Myeloperoxidase and Lactoferrin in Neutropenia

Radioimmunosorbent assays for determination of serum content of the neutrophil proteins myeloperoxidase and lactoferrin are described. Serial studies were performed in patients with neutropenia. In 2 cases of cyclic neutropenia the myeloperoxidase level showed slight variations within the normal range during the cycle while lactoferrin displayed a clear correlation with neutrophil counts. In 1 case with persistent agranulocytosis myeloperoxidase was normal but lactoferrin was extremely low. During the regeneration phase of drug-induced neutropenia neutrophil counts and serum lactoferrin increased in a parallel fashion. Since serum myeloperoxidase was normal during profounded neutropenia it is suggested to derive primarily from myeloperoxidase-rich granulopoietic precursor cells of the marrow. Serum lactoferrin on the other hand seems to derive from leakage of more mature granulopoietic cells of blood and marrow. Studies of neutrophil proteins of serum may aid in evaluation of neutropenic patients.     

Pulmonary function is negatively correlated with sputum inflammatory markers and cough clearability in subjects with cystic fibrosis but not those with chronic bronchitis

BACKGROUND: Polymorphonuclear neutrophil (PMN)-dominated inflammation is prominent in the airways of subjects with cystic fibrosis (CF) and chronic bronchitis (CB). Interleukin (IL)-8, myeloperoxidase (MPO), and DNA are markers of neutrophilic inflammation. We hypothesized that sputum MPO, DNA, and IL-8 concentrations would negatively correlate with pulmonary function and sputum transportability. METHODS: We measured pulmonary function and analyzed sputum IL-8, MPO, and DNA concentrations, as well as the transport properties of sputum samples obtained from 16 subjects with CF and 15 subjects with CB. We also evaluated changes in these measurements in paired sputum samples from these subjects obtained 2 to 12 months apart. RESULTS: IL-8 and MPO concentrations in the sputum of CF subjects was inversely correlated with FEV(1) percent predicted (IL-8: r = -0.40; p = 0.003; MPO: r = -0.38; p = 0.003) and FVC percent predicted (IL-8: r = -0.4; p = 0.02; MPO: r = -0.4; p = 0.02). IL-8 and DNA concentrations were inversely correlated with sputum cough transportability (CTR) [IL-8: r = -0.4; p = 0.02; DNA: r = -0.36; p = 0.048]. Changes in DNA concentration in sputum samples from CF subjects over time were inversely correlated with changes in FEV(1) percent predicted (r = -0.58; p = 0.02), FVC percent predicted (r = -0.74; p = 0.002), and CTR (r = -0.59; p = 0.02). There was no correlation among pulmonary function, sputum properties, and inflammatory markers in the sputum from subjects with CB. CONCLUSIONS: The sputum concentrations of IL-8, MPO, and DNA appear to be closely associated with pulmonary function in subjects with CF but not in subjects with CB.     

Pleural-fluid myeloperoxidase in complicated and noncomplicated parapneumonic pleural effusions.

The diagnostic accuracy of myeloperoxidase (MPO) in pleural fluid, for differentiating between complicated and noncomplicated parapneumonic pleural effusions (PPE) evaluated prospectively. Seventy patients aged >18 yrs with PPE (36 complicated and 34 noncomplicated) were studied after admission to a tertiary referral teaching hospital. MPO concentration was measured in plasma and pleural fluid using a double-antibody competitive radioimmunoassay. The concentrations of MPO in complicated and noncomplicated PPE were compared using a Mann-Whitney U-test and multiple logistic regression models were used to predict the odds that an effusion was complicated. MPO pleural-fluid concentrations were significantly higher in complicated than in noncomplicated PPE. After excluding purulent effusions, pleural-fluid MPO was the marker that best differentiated between the two types of PPE: the area under the receiver operating characteristic curve was 0.912, the sensitivity was 87.5% and the specificity was 85.1% at a cut-point limit of 3.000 microg x L(-1). The authors concluded that the concentration of pleural-fluid myeloperoxidase helps to differentiate between nonpurulent complicated and noncomplicated parapneumonic pleural effusions.     

Effects of short-term inhaled fluticasone on oxidative burst of sputum cells in cystic fibrosis patients.

Inhaled corticosteroids have been proposed to decrease pulmonary inflammation in cystic fibrosis (CF). In this study the effects of therapy with inhaled fluticasone on clinical and sputum outcomes (leukocyte count, activity of myeloperoxidase, superoxide anion release) in adult CF patients were investigated in an open label design. Twenty-six stable patients (median+/-SD forced expiratory volume in one second (FEV1) 58.1+/-19.9% pred.) were randomly assigned to the study group (500 microg b.i.d., for three weeks) or the control group (n=14; nonsteroid medication). Sputum samples were obtained during inhalation of hypertonic saline (3%, 20 min), which was found not to alter the investigated sputum parameters. No significant changes in clinical parameters, sputum leukocyte count, activity of myeloperoxidase, and baseline superoxide anion release where observed following therapy. Surprisingly, stimulated superoxide anion release increased significantly after therapy (34.1+/-17.7 versus 25.2+/-17.4 nmol x hr(-1) x 10(6) cells, p<0.03) and exceeded spontaneous variability of this parameter (p=0.02 versus control group). In conclusion, in adult cystic fibrosis patients short-term fluticasone therapy had no evident effect on clinical and sputum parameters. Further investigations are necessary to evaluate whether the observed up-regulation of oxidative capacity of inflammatory cells is of concern or benefit in these patients.     

Effect of clarithromycin on airway obstruction and inflammatory markers in induced sputum in cystic fibrosis: a pilot study.

To determine whether macrolide antibiotics improve pulmonary function and decrease airway inflammation in cystic fibrosis (CF), we treated 10 patients (females; aged 19-26 years, all colonized with P. aeruginosa, none with atypical Mycobacteria) with 3 weeks of placebo, followed by 6 weeks of clarithromycin (500 mg BID) in a single-blind prospective study. We also determined the safety of sputum induction and the reproducibility of assessing inflammatory markers in induced sputum. Subjects performed spirometry and underwent sputum induction (12-min inhalation of 3% saline) at 3-week intervals. We found that sputum induction was well-tolerated. We also found that the reproducibility was high for neutrophil (PMN) number (R = 0.87, P = 0.009), interleukin (IL)-8 (R = 0.73, P < 0.05, free neutrophil elastase (NE) (R = 0.82, P < 0.05), and myeloperoxidase (MPO) levels (R = 0.86, P < 0.05), but was less so for tumor necrosis factor (TNF)-alpha (R = -0.15, P = 0.7). We found no significant difference in pulmonary function after 6 weeks of treatment with clarithromycin (FEV(1) (% predicted) (mean +/- SEM), 2.2 +/- 0.9 (60 +/- 24%) vs. 2.3 +/- 1 (61 +/- 29%)), and no significant differences in any of the inflammatory indices measured. The median (and range) values before and after treatment for indices of airway inflammation in the induced sputum samples were: for PMNs, 8 (1-326) and 21 (0.2 -175) x 10(6) cells/mL sputum; for IL-8, 156 (24-656) and 202 (16-680) ng/mL; for free NE, 260 (31-1,264) and 237 (49-1,048) microg/mL; for TNF-alpha, 20 (7-128) and 35 (17-87) pg/mL; and for MPO, 169 (13-960) and 195 (14-816) microg/mL. We conclude that clarithromycin is not uniformly effective in improving airway obstruction or in decreasing airway inflammation in patients with CF.     

Identification of intracellular markers in induced sputum and bronchoalveolar lavage samples in patients with respiratory disorders and healthy persons

Induced sputum and bronchoalveolar lavage (BAL) are widely used for retrieving cells and soluble materials for studies of airway inflammation. Centrifuged cell samples are suitable for immunochemical identification of cellular products. The aim was to determine the optimal fixation procedure to detect intracellular antigens in situ. In immunocytochemistry, an appropriate choice of fixation method is a prerequisite for identification of cells and, consequently, for reliability results. We compared eight fixation and permeabilization methods to detect intracellular antigens in cytocentrifuged cell samples. Four granular proteins specific to eosinophils (eosinophil cationic protein, ECP; eosinophil peroxidase, EPO) and neutrophils (human neutrophil lipocalin, HNL; myeloperoxidase, MPO) were the antigens studied. We found that the organic solvents often used in immunocytochemistry are unsuitable fixatives for detection of these intracellular low-molecular-weight proteins. Treatment with crosslinking fixatives alone resulted in incomplete penetration of antibodies into the cell interiors. Best results were obtained using a commercial reagent Ortho PermeaFix (OPF) for flow cytometry. With this, fixation and permeabilization take place simultaneously OPF-treated cells retained their structural characteristics, and the antibodies studied penetrated both cellular and granule membranes. With OPF treatment, ECP EPO, HNL, and MPO were fixed on their places in granules, and their antigenicity was retained. Correct identification of intracellular proteins is important in characterization of the respiratory inflammatory response in clinical work and research.