Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.     

Neutrophil activation in vitro

Kinetics of spontaneous and induced reactions of Nitroblue tetrazolium (NBT) restoration by neutrophils was investigated in vitro. The index of activated neutrophils (IAN), commonly characterizing cell activation degree depending on the reaction time (t), was described by the equation: IAN = [NBT0](1-exp[-k1t]) + k0t; where (NBT0) is intracellular concentration of NBT, which reflects membrane permeability, k1 and k0--speed constants of the first and zero order reactions, characterizing, respectively, stimulation and spontaneous activities of enzymatic systems involved in NBT restoration. We detected the ability of heparin (2.0-7.5 mkg/ml) or chondrotin sulphate (0.25-2.5 mkg/ml) to activate neutrophils and presumably to maintain in vivo their spontaneous activity. The values of kinetics equation parameters enable us to speculate about relative hyper-, hypo- and normal function of neutrophils, related to the functional state of the whole organism. The value of [NBT0] changes from 8 to 103 conventional units, and a high correlation exists between this index and the reaction speed constants, differing in stimulated and spontaneously activated cells. The latter are represented by neutrophils only, which, as shown before, produce active forms of oxygen, used, probably, not for phagocytosis needs. The share of such cells makes approximately 30-40% of the neutrophil recirculating pool, and positively correlates with [NBT0], (r = 0.58; P < 0.05). This is determined by a very high membrane permeability of this neutrophil subpopulation. The detected different spontaneous and stimulated ability, various membrane permeability and enzyme system activity confirm the neutrophil functional unequality, which defines their activation peculiarity.     

The phenotype of peripheral blood neutrophils during the early stage of endometrial cancer

Peripheral blood neutrophils of 123 patients in stage Ia of the endometrial cancer have been assayed. The receptor system and ability of neutrophils to form extracellular traps (NET activity) were assessed by fluorescence microscopy, and spontaneous production of IL-2, IFN-γ, matrix metalloproteinase (MMP), G-CSF was found to exist determined with enzyme-linked immunosorbent assay. Phagocytic and myeloperoxidase activity, the level of cationic proteins, and neutrophil activity were evaluated in an NBT test determining the oxygen-dependent bactericidal action of neutrophils. Topology and rigidity of neutrophil membranes were assessed using scanning probe microscopy. It was established that an increased number of neutrophils was accompanied by changes of their receptor system, aerobic and anaerobic cytotoxicity, enhanced phagocytic activity, and reduced NET activity. The secretory activity of neutrophils changed. An increased level of MMP-1, possibly by enhanced production of reactive oxygen species, reduced IL-2 level, and a drastic increase in G-CSF were observed. The architectonics of neutrophils at stage Ia of endometrial cancer was characterized by altered cell shape and granularity loss. The rigidity of the cell membrane decreased. The changes in neutrophil morphology and persistent hyperactivity suggest the existence of the equilibrium between the immune system and the tumor in stage Ia of endometrial cancer.     

Kinetics of the reaction of nitroblue tetrazolium reduction by human blood neutrophils

Kinetics of Nitro blue tetrazolium (NBT) reduction to diformasan by neutrophils was investigated using 27 samples of human blood. Analysis of alteration in the share of activated neutrophils (ANP) and activated neutrophil index (ANI) was done in relation to the reaction time. The former reaction is an irreversible reaction of zero (pseudozero) order, while the latter is an irreversible reaction of the first (pseudofirst) order. It has been found out that an induced NBT reduction occurs in parallel with a spontaneous reaction, and that neutrophils have essentially different oxidizing power. The kinetic approach enabled us to discover some indices (NBT quantity involved in the reaction, and reaction speed constant of the first order) which in different samples varied within broader limits than ANP or ANI (within the limits of an order), i.e. provided a possibility to make a more delicate analysis of processes in neutrophils.     

Functional and metabolic activity of peripheral blood neutrophils in acute viral hepatitis B and C

The functional metabolic activity of peripheral blood neutrophils in acute virus hepatitis B (VHB) and/or virus hepatitis C (VHC) was evaluated. 48 patients were examined; of these, VHB was diagnosed in 28 patients and VHC was diagnosed in 9 patients and the mixed form of virus hepatitis (VHB + VHC), in 11 patients. Determination of adhesive capacity of neutrophils, production of superoxidase anion in the nitro blue tetrazolium (NBT) test, activity of myeloperoxidase (MPO) and acidic phosphatase (AP), the amount of cation proteins (CP) was made. Most pronounced functional dysbalance of neutrophil leukocytes and considerable changes in biochemical characteristics of the activity of the infectious process in patients with the mixed form of virus hepatitis were established. These data demonstrated that in acute virus hepatitis B and C at the peak of the disease such characteristics of the functional activity of neutrophils as results of the NBT test, the activity of MPO and AP, as well as the amount of CP, were highly informative.     

Recovery of functional capacity of human granulocytes after freeze-preservation with dimethyl sulphoxide.

Huma peripheral blood leucocytes (neutrophil rich) were collected either with preservative-free heparin (PFH) or acid citrate dextrose (ACD), frozen with dimethyl sulphoxide (DMSO) at a controlled rate, stored in liquid nitrogen at ?196 °C and reconstituted in a solution containing dextran polymer 70.A battery of tests including nitroblue tetrazolium (NBT) reduction, Candida phagocytic and candidacidal capacity was used to compare anticoagulants and reconstitution methods as they affect functional capacity of freeze-thawed neutrophils during a short post-thaw period.Heparin showed an overall advantage over acid citrate dextrose. A slow titration reconstitution method did not improve cell yield or functional capacity compared with a rapid dilution method and was a more cumbersome technique. The presence of complement greatly improved the capacity of reconstituted cells to reduct NBT and synthesize formazan. Freeze-thawed cells showed a selective response to stimulation as judged by the quantitative NBT test, responding strongly to zymosan in comparison with E. coli endotoxin. Lignocaine hydrochloride added to the reconstituent medium in concentrations up to 20 mmol/l did not have additional protective effect on post-thawed leucocytes as assessed by agglutination and leucocyte yields when compared with reconstitutent solutions containing only dextran. Reducing pH did not significantly slow the rate of gelling and leuco-agglutination or improve cell yields.Using these findings to optimize conditions reconstituted neutrophils retained 31.7% of fresh NBT activity, 27.6% of fresh phagocytic, and 22.3% of fresh candidacidal capacity.     

Cytoenzymatic studies on neutrophils in workers having contact with organic solvents containing benzene, toluene and xylene

In 106 workers (47 women and 59 men) being in professional contact with organic solvents containing benzene and its homologues during 1 to 122 months the cytochemical examination of peripheral blood neutrophils has been performed. The patterns of neutrophil functional activation have been noted expressed in increased activities of acid phosphatase and beta-glucuronidase, increased NBT reduction and diminished glycogen reserves. Those changes were accompanied by diminished peroxidase and alkaline phosphatase activities. The stimulated NBT reduction, elevated in majority of workers, exhibited negative correlation with the exposure time what indicates the practical value of that test monitoring the biological effects of professional contact with the solvents.     

Functional heterogenicity of human blood neutrophils: generation of oxygen active species

Kinetics of neutrophil inactivation was investigated in vitro by Nitroblue tetrazolium (NBT) test in the process of their contact with the substrate. It has been shown that the previous thermostatation results in an exclusive inactivation of neutrophils with high reaction ability leading to their complete inactivation. Such an inactivation is a consequence of cell contacts with the substrate, whose chemical structure and physicochemical properties define the process regularities. The neutrophil inactivation is probably not a consequence of the contact itself but may follow the next scheme: stimulus (contact with substrate)--generation of reactive oxygen metabolites--inactivation. Two functional unequal classes of neutrophils were differentiated on the basis of different levels of their reactive oxygen metabolite generation, and on their ability to inactivation. In vitro cells of one of these classes actively generate reactive oxygen metabolites to be inactivated in consequence of interaction with the substrate, whereas cells of the other class produce reactive oxygen metabolites less actively and are nor inactivated. Evidently, in vivo cells of the are phagocytes and those of the latter fulfill other functions.     

Biological effects of cigarette smoking assessed with the NBT test

The NBT reduction spontaneous and stimulated test was carried out on 90 men; 30 of which had never smoked cigarettes, 28 subjects have smoked for up to 10 years and 32 subjects--since over 10 years. The investigations demonstrated show that in subjects smoking cigarettes for up to 10 years the spontaneous and stimulated ability of the neutrophils to reduce NBT was increased whereas in the subjects smoking cigarettes since over 10 years impairment of the stimulated NBT reduction was demonstrated. We've come to a conclusion that an exposure to tobacco smoke may activate oxidation-reduction processes of the neutrophils in the initial stage and impair them in longer stage of smoking.     

Influence of levamisole on the capacity of neutrophil granulocytes for ingestion and for the reduction of nitrobluetetrazolium

With the aid of a modification of the NBT test it has been shown in in vitro and in vivo studies that levamisole does not affect the NBT reduction capacity of neutrophil granulocytes. The increased NBT positivity of these cells under the influence of levamisol is due to stimulation of ingestion.     

The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L.

The phagocytosis of neutrophils and serum lysozyme activity were investigated in carp experimentally infected with Pseudomonas alcaligenes and Aeromonas punctata. The total leucocyte numbers, relative leucocyte counts, nitroblue tetrazolium (NBT) reduction, NBT test rate, lysozyme activity and lysozyme index were examined on days 7, 14 and 21 after injection. On days 7 and 14 there was a significant increase in total leucocyte numbers and serum lysozyme activity, but a decrease in the NBT test rate and lysozyme index. NBT reduction was unchanged compared with the control group. On day 21 the total leucocyte numbers and lysozyme activity had declined and were less than control values, but there was a significant increase of the NBT test rate, NBT reduction and lysozyme index.     

Unique human neutrophil populations are defined by monoclonal antibody ED12F8C10.

A mouse IgG1 monoclonal antibody ED12F8C10 (C10) binds a constant percentage of peripheral blood neutrophils in the same individual when studied over time, defining a distinct subset of neutrophils in all normal individuals studied to date. Bone marrow studies confirm that the heterogeneity is present to the same degree at all stages of neutrophil development from the myelocyte to the mature neutrophil. Neither in vivo nor in vitro activation of neutrophils explains or significantly alters the relative percentages of C10-positive and -negative neutrophils in the same individual. With both activation and exudation, however, expression of the C10-defined epitope increases in intensity in the C10 binding subpopulation. Studies of NBT reduction, phagocytosis, adherence, light scattering characteristics, and monoclonal antibody surface binding have failed to demonstrate physical or functional differences between the C10-defined populations. We examined C10 binding in patients with different defects of phagocyte function. In two patients with neutrophil-specific granule deficiency, less than 1% of the neutrophils were found to be C10 positive, while neutrophils from a patient with idiopathic leukemoid reaction and recurrent infections demonstrated greater than 99% C10 binding. Although the present study does not delineate the physiologic significance of C10 binding heterogeneity, it firmly supports the concept of neutrophil heterogeneity at the level of surface antigen expression.     

NO signaling in exercise training-induced anti-apoptotic effects in human neutrophils

Short-lived neutrophils play a predominant role in innate immunity, the effects of exercise training on neutrophil survival is unclear. In this study, we investigated the underlying mechanisms of training effects on human neutrophil apoptosis. Healthy male subjects were trained on a cycling ergometer for 8 weeks and followed by 4 weeks of detraining. Blood neutrophils were collected before exercise, after training, and after detraining. Comparing with pre-exercise specimens, neutrophils collected after training showed reduced apoptosis rate, which partially returned after detraining. Various intracellular proteins, including iNOS, Mcl-1, A1, Grp78, and IL-8, were upregulated by training, and they remained high after detraining. Upregulated iNOS was closely correlated with these anti-apoptotic molecules in neutrophils. Furthermore, the possible mechanism by which iNOS suppressed apoptosis was explored. Neutrophil apoptosis was accelerated by blocking and retarded by stimulating the endogenous iNOS activity. As an anti-apoptosis mediator of NO signaling, the Mcl-1 level dropped by depletion of the major NO downstream molecule cGMP and such loss of Mcl-1 was avoidable when supplying exogenous NO. Upon activation of NO-cGMP signaling, neutrophils held increased Mcl-1 expression and delayed apoptosis. Collectively, our results suggested that exercise training may retard neutrophil apoptosis by upregulating the iNOS-NO-cGMP-Mcl-1 pathway.     

IL-4 increases surfactant and regulates metabolism in vivo

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.     

Increased nitroblue tetrazolium dye reduction by human neutrophils stimulated by interferon in vitro

Human leukocyte interferon enhanced nitroblue tetrazolium dye (NBT) reduction by human neutrophils (PMNs). Increase in NBT reduction paralleled increase in interferon dose. When human leukocyte interferon was heated to 60 C or 80 C for 30 min, both the antiviral activity and the effect on NBT reduction decreased. Human leukocyte interferon neutralized with anti-human leukocyte interferon serum showed no effect on NBT reduction. A human fibroblast interferon preparation also enhanced NBT reduction. The species dependency of interferon was shown in NBT reduction as well as in antiviral activity.     

Effect of interferon on the increased reduction of nitroblue tetrazolium (NBT)]

Human leukocyte interferon (HL-IF) enhanced the NBT reduction of human peripheral neutrophil in vitro. Dose relation between IF activity and the NBT reduction was recognized. Heat-inactivated HL-IF, HL-IF neutralized by anti-IF serum or heterologous IF could not increase the NBT reduction.     

New informative index of the functional status of polymorphonuclear neutrophils

A new test was applied to study the function of polymorphonuclear neutrophils according to their ability to form oreols in a hypertonic medium. During inhibition of neutrophil function in vivo (desympathetization, tumor growth), oreol formation increased. Stimulation of neutrophils in vitro by adding noradrenaline to the incubation medium led to a decrease in their oreol-forming ability. The data obtained enabled the authors to offer the test for detecting the functional activity of polynuclear cells.     

Nitroblue tetrazolium reduction by human blood neutrophils. I. The influence of pH

Activity of blood neutrophils of 14 healthy volunteers (8 women, 6 men, aged from 21 to 37 years) was investigated in the Nitroblue Tetrazolium (NBT) reduction reaction at pH 7.0, 7.3, and 7.6. The dependence of the percentage of NBT-positive neutrophils on the reaction time was described by a kinetic equation with constants k1 and k2, characterizing, accordingly, speeds of NBT absorption and reduction by neutrophils. A strong positive correlation (r = 0.99; P < 0.0001), was found between k1 and k2, which pointed to dependence of the speed of NBT restoring by neutrophils on the speed of its entry to the cell. Both the constants and speeds of successive process stages have maximum values at pH 7.3 (corresponding to physiological value of blood pH), and decrease at medium acidification or alkalization. The k1/k2 ratio does not depend on the time (0-6 h) or pH (7.0-7.6) of blood incubation before the reaction. The most active neutrophils (potential pH agocytes) have a lesser membrane permeability than do neutrophils of another class, and their activity more increases at medium acidification in vitro and, probably, in vivo. The activation of neutrophils leads to an increase in medium pH that alongside with an increase in the number of active potential phagocytes, evidently has a physiological meaning.     

Endotoxins of enteric pathogens are chemotactic factors for human neutrophils

Early activation of human peripheral blood polymorphonuclear neutrophils is characterized by their morphological changes from spherical to polarized shapes. The endotoxins from enteric pathogens (S. dysenteriae type 1, V. cholerae Inaba 569B, S. typhimurium, and K. pneumoniae) were assessed by their ability to induce morphological polarization of the neutrophils as measures of early activation. Phagocytic activity, adhesion, chemokinetic locomotion, and nitroblue tetrazolium (NBT) dye-reduction ability measured the later activation of the cells. Neutrophils showed distinct morphological polarization in suspension over a wide range of concentrations of these endotoxins when were compared with those that were induced by the standard chemotactic factor, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). It was discovered that all of the endotoxins induced locomotor responses in neutrophils in suspension that were dose- and time-dependent. The optimum concentration for the endotoxins of S. dysenteriae, V. cholerae, and K. pneumoniae was 1 mg/ml in which 71, 69, and 66% of the neutrophils were polarized. However, the S. typhimurium dose was 2 mg/ml in which 50% of the cells responded. Neutrophils that were stimulated with endotoxins also showed increased random locomotion (p<0.005) through cellulose nitrate filters, but an enhanced adhesion of the cells to glass surfaces (p<0.03). These are important functions of these cells to reach and phagocytose damaged cells, as well as invading microorganisms. Interestingly, the endotoxins had a highly-significant inhibitory effect upon the proportions of neutrophils phagocytosing opsonized yeast (p<0.01) with a small number of yeast that were engulfed by the cells (p<0.02). Further, endotoxin-treated cells showed an enhanced ability to reduce NBT dye (p<0.03). Therefore, we concluded that endotoxins of enteric pathogens are neutrophil chemotactic factors.     

Intrapopulational heterogeneity of human neutrophils based on chemokinetic and respiratory burst reactions

The chemokinetic test (ameboid motility) and enhancement of oxygen-dependent metabolism of neutrophils (the NBT test) were considered in human blood stimulated with Staph. aureus allergen. There were three variants of stimulated cells: 1) neutrophils developing ameboid motility (13,5 +/- 1,7% cells), 2) neutrophils with activated oxygen-dependent metabolism (11.5 +/- 0.6%), 3) neutrophils positive in both the tests (2.6 +/- 0.8%). Unstimulated cells accounted for 71.6 +/- 4.1%. Considerable differences were recorded for each variant. The data obtained are regarded as evidence of dissimilar capability of neutrophils of the realization of effector functions.