BACKGROUND: We intended to confirm a decline in neutrophil function with aging and to devise a simple neutrophil function test protocol for use in a clinical setting. Reversely, the reliability of this protocol was to be verified by detectability of a decline in neutrophil function with aging. METHODS: Whole blood samples from young (thirties, N=32) and old (sixties, N=32) healthy subjects were incubated with the 7-aminoactinomycin D stained Escherichia coli. The mixture was stained by dihydrorhodamine 123 as an oxidative probe. Two kinds of fluorescence were measured by flow cytometry. RESULTS: Phagocytosis was declined with aging as indicated by a decrease in the percentage (form 28.2+/-9.5% to 21.9+/-10.9%, P<0.05) and the mean fluorescence intensity (MFI) ratio (from 1.67+/-0.27 to 1.51+/-0.27, P<0.05). Oxidative burst had a trend toward a decrease with aging, but the differences were not significant (percentage: from 35.3+/-13.2% to 32.1+/-14.1%, P=0.36; MFI ratio: from 5.26+/-3.23 to 5.08+/-3.55, P=0.84). CONCLUSIONS: The devised protocol in this study could detect a significant decline in neutrophil function with aging, and this protocol may be useful for the assessment of neutrophil function in a clinical setting.
Aging ; Escherichia coli ; Flow Cytometry ; Fluorescence ; Neutrophils* ; Phagocytosis ; Respiratory Burst

CD64 (Fc gamma RI) is the one of the three Fc gamma receptors on monocytes and represents a high affinity immunoglobin G receptor. CD64 is rapidly upregulated on monocyte in response to gamma interferone. X-linking of CD64 triggers an oxidative burst as well as antibody dependent cytotoxicity. In this experiments, peripheral blood mononuclear cells (PBMC) were separated and incubated with or without gamma interferone and PG E1. The samples were divided into four groups, the first was PBMC alone, the second was PBMC with gamma interferone 100 U/ml, the third was PBMC with gamma interferone 100 U/ml and Prostaglandin E1 1 micro M/L, and the fourth was PBMC with gamma interferone 100 U/ml and Prostaglandin E1 10 micro M /L. Flow cytometric measurements of CD64 on monocyte were performed at 0, 3, 6, and 9 hours after incubation and the mean fluorescence intensities (MFI) and the mean percentages of CD64(+) cell in monocytic gated area were obtained. After 6 hours of incubation, although there is no statistical significance, all gamma interferone added groups show the higher mean fluorescence intensity than PBMC alone group. Furthermore, at 6 and 9 hours of incubation, the mean percentages of CD64(+)cells between the PBMC with gamma interferone group and the PBMC with gamma interferone and PG E1 10 micro M/L group showed 74.83 +/- 9.72% vs. 34.07 +/-12.98%, 80.04 +/- 11.30% vs. 29.42 +/- 19.86% respectively, and there are statistical significances, p=0.05, p=0.05 respectively. It appears that PG E1 inhibits the expression of CD64 on monocyte.
Alprostadil* ; Fluorescence ; Interferons ; Monocytes* ; Receptors, IgG ; Respiratory Burst

No abstract available.
Adult* ; Fetal Blood* ; Humans ; Neprilysin* ; Neutrophils* ; Respiratory Burst*

BACKGROUND: Functional defects of neutrophils are regarded as a major factors for the susceptibility to bacterial infection in diabetes. The attribution of low antioxidant status, which is shown in uncontrolled diabetic patients, to the dysfunction in neutrophils has not been illucidated. We purposed to evaluate the correlation of respiratory burst activity in neutrophils to total antioxidant status (TAS) and lipid peroxidation. MATERIALS: Neutrophil respiratory burst activity (NRBA) before and after stimulation by PMA was measured by flowcytometry in diabetic patients with poor glucose control (n=10, HbA1C > 8.0%) and good glucose control (n=11, HbA1C < or = 6.4%) and non-diabetic subjects (n=18). TAS and lipid peroxidation assay were measured by colorimetry. RESULTS: The results of HbA1C, TAS and lipid peroxidation of diabetic patients with poor glucose control were 11.6+/-9.2%, 1.407+/-0.041 mmol/L, 2.66+/-1.84 M, and with good glucose control were 5.7+/-0.5%, 1.440+/-0.061 mmol/L, 2.64+/-1.29 M, respectively. In non-diabetic subjects were 5.2+/-0.4%, 1.464+/-0.003 mmol/L, 2.07+/-1.18 M, respectively. NRBA of diabetic patients with poor glucose control before and after stimulation by PMA was 0.858+/-0.001 and 0.897+/-0.007 of non-diabetic subjects, respectively. NRBA of diabetic patients with good glucose control before and after stimulation by PMA was 0.832+/-0.002, 0.949+/-0.002 of non-diabetic subjects, respectively. TAS of diabetic patients with poor glucose control was significantly lower than that of non-diabetic subjects (P<0.05). NRBA were significantly decreased both before and after stimulation by PMA in poor glucose control (P<0.05). TAS and NRBA after stimulation by PMA of patients with good glucose control was not statistically significant and NRBA only before stimulation by PMA was significantly decreased (P<0.05). Significant positive correlations were evident between NRBA and total antioxidant activity (r=0.507, P<0.05). CONCLUSION: Only NRBA before stimulation by PMA of diabetic patients with good glucose control was significantly decreased, but NRBA of diabetic patients with poor glucose control was significantly decreased before and after stimulation by PMA and significantly correlated with TAS.
Bacterial Infections ; Colorimetry ; Diabetes Mellitus* ; Glucose ; Humans ; Lipid Peroxidation* ; Neutrophils* ; Respiratory Burst

OBJECTIVETo explore the effect of substance P (SP) on the functional proteins on plasma membrane of neutrophil (Np).

METHODThe response of Np to SP was examined by measuring the level of respiratory burst, the activities of ACP and ALP, the fluoroscopy intensity of CR3, CD45 and FM-LP.

RESULTSIt was found that SP could increase respiratory burst of Np, decrease the activity of acid phosphatase (ACP), but had no effect on alkaline phosphatase (ALP). SP could also promote the amount of CD45, complement receptor type 3 (CR3) and N-Formyl-Met-Leu-Phe (FMLP) receptors.

CONCLUSIONThe results showed that the effects of SP on functional proteins in human Np membrane were universality and diversity. It implied that SP could affect various inflammation responses in Np.

Acid Phosphatase ; metabolism ; Humans ; Membrane Proteins ; physiology ; Neutrophils ; metabolism ; Respiratory Burst ; Substance P ; pharmacology

BACKGROUNDPolymorphonuclear neutrophil (PMN), one of the most important inflammatory cells, functions throughout the initiation, progression and resolution of inflammation. This study aimed at investigating the relationship between PMN apoptosis and the lung injury after chest impact trauma.

METHODSPMNs were purified from rabbits subjected to the chest impact trauma and their apoptosis, necrosis, survival and respiratory burst were detected by flow cytometry. Meanwhile, lactate dehydrogenase and (LDH) [Ca2+]i were measured.

RESULTSThe delayed apoptosis of PMNs in bronchoalveolar lavage fluid was observed from 2 hours to 12 hours after trauma, and viable cells increased. Respiratory burst of PMNs in bronchoalveolar lavage fluid was increased significantly from 2 hours with the peak at 8 hours. Meanwhile, lactate dehydrogenase in bronchoalveolar lavage fluid was higher than that in control (P < 0.05) from 4 hours to 24 hours, and intracellular free Ca2+ in PMN was increased temporarily.

CONCLUSIONSRetention of PMN in tissues and the abnormality in apoptotic pathway inevitably generate persistent activation of PMN and excessive release of toxic substances, resulting in tissue injury. The temporary increase of intracellular free Ca2+ may be responsible for the delayed apoptosis of PMN.

Animals ; Apoptosis ; physiology ; Lung Injury ; Neutrophils ; physiology ; Rabbits ; Respiratory Burst ; physiology ; Thoracic Injuries ; complications

BACKGROUND: Bacterial infections in diabetic patients are an important cause of increased morbidity and mortality. It has been reported that bacterial infections in diabetics showed more impaired PMN functions such as reduced PMN respiratory burst and decreased microbicidal activity in inflammed tissues. Also, apoptosis(programmed cell death) is postulated to be a key mechanism for neutrophil elimination. It is very important that PMN apoptosis keeps the balance from an area of inflammation. Actuallly, as little was known about PMN apoptosis and respiratory burst in diabetes, we investigated PMN apoptosis and hydrogen peroxide production after endotoxin exposure. METHODS: Peripheral venous blood samples were collected by routine venipuncture from healthy volunteers and diabetics to harvest neutrophils. We respectively measured the PMN apoptosis, the production of hydrogen peroxide, and the cell viability. RESULTS: Normal neutrophils showed a tendency to decreased apoptosis after endotoxin treatment. In patients with diabetes, PMN apoptosis was significantly decreased compared with healthy controls. In addition, the LPS-induced neutrophils in diabetics demonstrated more decreased apoptosis. However, the production of hydrogen peroxide was not different between groups. CONCLUSION: These observations suggest that the decreased PMN apoptosis in diabetics with endotoxin exposure may also affect the increased susceptibility and severity of infections.
Apoptosis* ; Bacterial Infections ; Cell Survival ; Healthy Volunteers ; Humans ; Hydrogen Peroxide ; Inflammation ; Mortality ; Neutrophils* ; Phlebotomy ; Respiratory Burst

PURPOSE: Receptors for the Fc of IgG(FcvR) and a beta2 integrin molecule, CD11c/CD18 are important in clearance of microbes by granulocytes. We performed an in vitro study on the effect of recombinant human granulocyte colony-stimulating factor(rhG-CSF), or granulocyte-macrophage colony-stimulating factor(rhGM-CSF) on the expression of Fc R II, Fc R III, CD11c and CD18 and respiratory burst function of granulocytes. METHODS: Peripheral blood was collected from six healthy adults. The isolated granulocytes were stimulated with rhG-CSF, 250mg/mL, rhGM-CSF, 100ng/mL or both of them for 1, 3, 9, 24, and 48 hours. Using flow cytometry, we compared the expression of the Fc R II, Fc R III, CD11c and CD18 of granulocytes before and after stimulation. We also the studied respiratory burst of granulocytes with chemiluminescence assay. RESULTS: Fc R II and CD18 expression were not changed significantly after stimulation. Fc R III expression was decreased significantly after stimulation with rhG-CSF, rhGM-CSF, or both of them. CD11c expression was increased initially but was found to decrease significantly after 9 hours of stimulation. Granulocytes treated with rhG-CSF, rhGM-CSF, or both displayed enhanced respiratory burst. Our results suggest that exposure of granulocyte to growth factor results in granulocyte activation. CONCLUSION: We have shown that rhG-CSF and rhGM-CSF resulted in an activation of peripheral blood granulocytes immunophenotypically and functionally.
Adult ; Antigens, CD18* ; Flow Cytometry ; Granulocytes* ; Humans* ; Luminescence ; Receptors, IgG* ; Respiratory Burst*

Treatment of rainbow trout macrophages with glycyrrhizin (GL), an aqueous extract of licorice (Glycyrrhiza glabra), enhanced their respiratory burst activity. Maximal effects were seen using concentrations of 10-100 ug/ml. GL also modulated trout lymphocytes, increasing proliferation responses to the mitogen phytohemagglutinin two-fold over a range of GL concentrations. In addition, GL elicited the release of a macrophage activating factor (MAF) kom head kidney leukocytes, as assessed by the ability of generated supernatants to increase respiratory burst activity of target macrophages. MAF activity was most apparent using 100 ug/ml GL to induce MAF release and a 48 h incubation period with the target macrophages. Finally, GL was shown to enhance the release oF MAF in response to the mitogen concanavalin A. The results suggest that GL might modulate the innate defences in fish.
Concanavalin A ; Glycyrrhiza ; Glycyrrhizic Acid* ; Head Kidney ; Leukocytes* ; Lymphocytes ; Macrophages ; Oncorhynchus mykiss* ; Oncorhynchus* ; Respiratory Burst ; Trout

Background: Recognition and ingestion of opsonized microorganisms by neutrophils induces the burst of oxidative metabolic activity. Products of the respiratory burst activity provide powerful oxygen dependent killing mechanism. Measurement of respiratory burst activity has been a major indicator of the functional capacity of neutrophils. We determined the respiratory burst activity of neutrophils in patients with pneumonia and observed the changes during the clinical course of pneumonia. Methods: The EDTA blood was drawn from 24 normal controls and same numbers of pneumonia patients. The respiratory burst activity(with the production of H2O2 which changes nonfluorescent DCF-DA to green fluorescent DCF) in the non-stimulated state and the stimulated state with fMLP and PMA of neutrophils was measured by flowcytometry at day 1, 3, 5, 7 and 9 of admission. Results: The respiratory burst activity of neutrophils was mildly increased by stimulation with fMLP. But there was no statistical significance between normal control and patients with pneumonia. The respiratory burst activity of neutrophils was markedly increased by stimulation with PMA in both groups. There was a significant difference in response to PMA between normal control and patients with pneumonia. The production of hydrogen peroxide from neutrophils was decreased during early course of pneumonia and it was recuperated gradually to normal level in 9 days. Conclusion: Hydrogen peroxide production from neutrophils was suppressed during early course of pneumonia and restored after treatment. It is suggested that the production of oxygen radical in response to PMA stimulation from each neutrophils is decreased rather than increased during the early course of pneumonia.
Eating ; Edetic Acid ; Homicide ; Humans ; Hydrogen Peroxide ; Neutrophils* ; Oxygen ; Pneumonia* ; Respiratory Burst