Phagocytosis, bactericidal capacity, and superoxide anion (O2-) production by polymorphonuclear neutrophils from patients with diabetes mellitus

Phagocytosis, bactericidal capacity and superoxide anion production of polymorphonuclear neutrophils (PMN) were estimated in 30 patients with well-controlled insulin-dependent diabetes (IDD) and in 50 patients with non insulin-dependent diabetes (NIDD). The estimations were additionally done in 20 elderly patients without glucose intolerance. The estimations of bactericidal capacity were performed in autologous-, zymosan activated-, inactivated- and control plasma. The phagocytosis of viable staphylococci was unchanged in all evaluated groups. The bactericidal capacity in all diabetic patients was significantly reduced. It was fully correctable in patients with IDD by suspension of cells in control or zymosan activated plasma. The improvement of PMN bactericidal capacity in patients with NIDD in similar conditions was less distinct. The superoxide anion production in patients with IDD was similar to values noticed in healthy persons. Whereas, the O2- production in patients with NIDD as well as in elderly patients were significantly reduced and correlated significantly with bactericidal capacity impairment. The possible mechanism of noticed disturbances were discussed.     

Thrombin enhances opsonized zymosan-induced chemiluminescence of neutrophils

We examined the effects of alpha-thrombin (the native enzyme) on neutrophil activation as assessed by the measurement of chemiluminescence. alpha-Thrombin in physiological concentrations (10(-9)-10(-8)M) did not induce neutrophil chemiluminescence. However, when neutrophils were coincubated with opsonized zymosan and alpha-thrombin, the chemiluminescence response to opsonized zymosan was enhanced in a concentration-dependent manner. The neutrophil chemiluminescence responses to opsonized zymosan and to opsonized zymosan plus alpha-thrombin were dependent on the generation of oxygen-derived free radicals since the chemiluminescence was inhibited by superoxide dismutase. The results indicate that thrombin per se does not induce neutrophil chemiluminescence. However, thrombin enhances the chemiluminescence response to opsonized zymosan suggesting an interaction between thrombin and complement receptors in inducing neutrophil activation. The chemiluminescence response to thrombin and opsonized zymosan is the result of oxygen-derived free radicals.     

Platelet factor 4 (PF4) and heparin released platelet factor 4 (HR-PF4) in diabetes mellitus. Effect of the duration of the disease

Several investigators have reported an altered platelet function in diabetes mellitus as measured by elevated levels of platelet specific proteins platelet factor 4 (PF4) and B-thromboglobulin (BTG). We studied 20 insulin dependent (IDD), 20 non insulin dependent (NIDD) diabetic males without overt clinical symptoms of cardiovascular disorders and 30 normal controls. We evaluated PF4, BTG and heparin released platelet factor 4 (HR-PF4) as measured 2.5 minutes after a bolus injection of 5,000 I.U. of a commercial mucous heparin. The patients showed normal levels of both PF4 and BTG. Furthermore HR-PF4 failed to show statistically significant variation between patients and controls. However when the diabetics were divided on the basis of the duration of the disease, the IDD had an increased HR-PF4 mean level and the trend became statistically significant when diabetes existed more than 17 years (patients HR-PF4 149.1 ng/ml, range 17.3-194; controls HR-PF4 110.9 ng/ml range 50-160, less than p less than 0.05). NIDD failed to reveal the same pattern. Although the significance of HR-PF4 is unknown, insulin dependent diabetes mellitus after many years could cause a potentially dangerous, silent vascular damage with enhanced platelet vessel wall interaction as measured by an elevated HR-PF4.     

Effects of long‐distance running on serum opsonic activity measured by chemiluminescence

Exhaustive exercise such as long‐distance running has been shown to increase susceptibility to infection. In order to investigate whether serum opsonic activity plays a role in such conditions, we utilized luminol‐dependent and lucigenin‐dependent chemiluminescence (LmCL and LgCL). We took serum samples from 24 male marathon runners before and after running 30 km. Neutrophils were isolated from the peripheral blood of healthy volunteers. Serum opsonic activity was examined by measuring neutrophil ROS stimulated with zymosan particles opsonized by the serum samples. Immunoglobulin and complement levels in the serum were also measured. After a 30 km run, the maximum light emission was increased and the time to reach the maximum light emission was shortened significantly (p < 0.05) in LmCL. However, there were no significant changes in the immunoglobulin and complement levels. The increase of ROS production may suggest that serum opsonic activity is accelerated after running 30 km. Thus, serum opsonic activity might not play a significant role in the susceptibility to infection after long‐distance running. Copyright © 2003 John Wiley & Sons, Ltd.     

Beta-adrenergic receptor function and oxygen radical production in bovine pulmonary alveolar macrophages

Reactive oxygen species production by bovine pulmonary alveolar macrophages was evaluated by a chemiluminescence assay utilizing luminol and opsonized zymosan. Incubation with dobutamine (5 x 10(-8) and 5 x 10(-7) M) or isoproterenol (5 x 10(-8) and 5 x 10(-7) M) prior to zymosan challenge significantly (p less than 0.05) increased the time for chemiluminescence to begin, and significantly decreased the level of maximum chemiluminescence. The agonists' inhibitory effects on maximum chemiluminescence were significantly reduced by pre-incubation with the appropriate antagonist (atenolol at 1 x 10(-6) M for dobutamine; and propranolol at 1 x 10(-6) M for isoproterenol). Salbutamol at 1 x 10(-6) M significantly reduced the level of maximum chemiluminescence only, but did not increase the time for chemiluminescence to begin. This effect was significantly reduced by the presence of the beta 2-antagonist ICI 118,551 at 1 x 10(-6) M. The results reveal the presence of beta 1- and beta 2-adrenoceptors on bovine pulmonary alveolar macrophages, and suggest that these receptors are important in the regulation of reactive oxygen species production by these cells.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opsonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B4 and C4. After 3-4 min, the levels of LTB4 were 93 and 35 pmol/3*10(7) cells, respectively [corrected]. These amounts were 2-4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC4 were 8 and 20 times lower than those of LTB4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

A method for the accurate measurement of opsonic activity in uterine secretions of the mare

This study was undertaken to develop a method for accurately measuring opsonic activity in the uterine secretions of the mare. Ten mares were used in the study. They ranged in ages from 6 to 19 years and were of various genital health status. Undiluted uterine secretions were collected by inserting a tampon into the uterus during estrus; serum samples were collected simultaneously Opsonic activity in the secretions and serum was analyzed in a chemiluminescence assay, in which zymosan particles were opsonized. Opsonic activity was determined as peak chemiluminescence, time to peak chemiluminescence, and total chemiluminescence (area under curve). The peak chemiluminescence was 16 to 17 times higher when uterine secretions were used for opsonization rather than when buffer was used. Compared to the opsonic activity in serum, the peak chemiluminescence was 21% (P相似文献   

Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.     

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.     

Whole blood luminol-enhanced chemiluminescence: Statistical analysis of the responses of different subjects

Whole blood luminol-enhanced chemiluminescence (CL) was studied using N-formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan particles as stimuli. The peak and the integral responses of CL were recorded. In the FMLP-induced CL the initial activation (1-minute values) was also studied, because it coincides with the extracellular production of oxy radicals. Correction factors based on neutrophil count and on haemoglobin concentration were found to decrease dispersion and shape the distributions of the CL responses close to normal in a study of 50 healthy adults. One-minute values were significantly lower in women than in men but there were no significant differences for peak or integral values between sexes. Depressed reaction is in accordance with the previous findings that phagocytic oxy radical production is depressed in female plasma. Thus, our results suggest that 1-minute value is a variable more sensitive than peak or integral value of the CL response.     

Luminol dependent chemiluminescence of quartz and zymosan stimulated neutrophils

The effect of the opsonization by zymosan and quartz particles on the chemiluminescence was investigated on human neutrophil granulocytes. Opsonization of zymosan enhanced the chemiluminescence response, while opsonized quartz inhibited the chemiluminescence reaction. Calcium ionophore A 23187 treatment did not influence the chemiluminescence of quartz but the light signal in the presence of quartz decreased rapidly. In parallel experiments the protein pattern of zymosan treated neutrophils was investigated by high resolution two-dimensional polyacrylamide gel electrophoresis.     

Measurement of whole blood phagocyte chemiluminescence in the Wistar rat

The factors influencing the rat whole blood chemiluminescence (CL): concentrations of blood, luminol, zymosan or opsonized zymosan, volume of the reaction mixture, storage time of blood samples and the presence of anticoagulants were evaluated. The CL micromethod described provides a fast and sensitive tool for the determination of metabolic activity of phagocytes in the microlitre range of rat whole blood. © 1997 John Wiley & Sons, Ltd.     

Serum of Behqet's Disease Enhances Super oxide Production of Normal Neutrophils

Using an MCLA-dependent chemiluminescence technique, we evaluated superoxide production by neutrophils isolated from 7 patients with Behget's disease. After stimulation by phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan, neutrophils from the patients produced significantly more superoxide than those from 20 controls. Pretreatment of control neutrophils with serum prominently enhanced superoxide production, and serum from Behcet's disease patients had a significantly greater effect than that from controls. These findings suggest that serum from patients with Behget disease contains the priming factor(s) that can enhance enhanced superoxide production by neutrophils in response to stimulation.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Real-time visualization of oxyradical burst from single neutrophil by using ultrasensitive video intensifier microscopy

Dynamics of oxyradical burst from single human neutrophils stimulated by opsonized zymosan was visualized by using an ultrasensitive video intensifier microscopy in the presence of chemiluminescence probe. Luminol-dependent photonic burst activities were clearly corresponding to the distribution of zymosan-treated neutrophils. Heterogeneity of photon-bursting period and the maximum photonic intensity among reactive cells was demonstrated. This may reflect functional heterogeneity of the ability to release oxyradicals among neutrophils.     

Exposure of human neutrophils to chemotactic factors potentiates activation of the respiratory burst enzyme

NADPH oxidase activity in particulate fractions from human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan was enhanced by prior exposure of the neutrophils to chemotactic factors. Enhanced activity was seen measuring both NADPH-dependent chemiluminescence and superoxide anion production. Enhancement was observed to be both time and dose dependent with several chemotactic stimuli, including casein, N-formyl-methionyl-leucyl-phenylalanine (f-MLP), and C5a. F-MLP and C5a showed similar patterns, with peak enhancement occurring within 2 to 15 min of preincubation and lasting up to 1 hr. In contrast, enhancement of PMA-stimulated oxidase activity by casein was more gradual and sustained, lasting up to 2 hr. Fractions from cells treated only with chemotactic factors and not stimulated with PMA showed no oxidase activity. Kinetic studies of this enhanced activity show that chemotactic factors induce increases in Vmax values but do not significantly alter Km values for the oxidase. Further experiments using agents that modulate degranulation suggest that enzyme release is not involved in this enhancement. These data suggest that pretreatment with chemotactic factors results in an increase in the amount of activated oxidase in membrane fractions obtained from PMA-stimulated neutrophils. This alteration of NADPH oxidase activity provides a subcellular basis for the enhanced bactericidal activity and increased oxidative metabolism seen in neutrophils treated with chemotactic factors.     

Consumption of complement and activation of human neutrophils by an artificial immune complex: polyacrylic acid-IgG-polymer

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 micrograms/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.     

Inhibitory Effect of Bifemelane on Superoxide Generation by Activated Neutrophils Measured Using a Simple Chemiluminescence Method

We evaluated the effect of 4-(2-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane hydrochloride) on superoxide production by human neutrophils using an MCLA-dependent chemiluminescence assay. Bifemelane hydrochloride dose-dependently inhibited superoxide production by neutrophils stimulated with phorbol myristate acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-pheny-lalanine, while it had no effect on superoxide production by a hypoxanthine-xanthine oxidase system. These results indicate that bifemelane hydrochloride does not have a scavenging effect, but has an inhibitory effect on superoxide generation by neutrophils. Although this drug is commonly used for treating chronic cerebral infarction, it may also have a protective effect on acute ischemia/reperfusion injury.     

Pentoxifylline Pretreatment Enhances the Oxidative Burst Activity of Human Peripheral Blood Mononuclear Cells

The effect of pentoxifylline pretreatment on the lucigenin-augmented chemiluminescence and dismutase-inhibitable superoxide production of human neutrophils and mononuclear cells (MNCs) was studied. Pentoxifylline at 20–2,000 μg/ml enhanced the lucigenin-augmented chemiluminescence (118–165% of the control, P < 0.01) of phorbol myristate acetate (PMA)-stimulated MNC. Pentoxifylline at 20–2,000 μg/ml increased the MNC superoxide production, i.e., 142–171% of the control (P < 0.05) using PMA stimulation and 145–159% of the control (P < 0.01) using opsonized zymosan stimulation. In contrast, pentoxifylline (up to 2,000 μg/ml) did not influence the lucigenin-augmented chemiluminescence and superoxide production of human neutrophils, stimulated by either PMA or opsonized zymosan. These results suggest that pentoxifylline is an immunomodulator and may have potential usefulness in the enhancement of immune defenses in compromised hosts.