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*

PURPOSE: During differentiation of HL-60 cells by all-trans retinoic acid(ATRA), we analyzed the expression of Fcr receptors and Mac-1 by molecules of equivalent soluble fluorochromes(MESF) and functional studies. METHODS: HL-60 cells were induced to differentiate by adding 1micrometer ATRA. On the 4th and 7th day after stimulation as well as before stimulation, the cells were analyzed for phenotypic and functional differentiation. Phenotypic analysis was performed by flow cytometry after staining the cells with PE-conjugated anti-human CD64(FcrRI), CD32(FcrRII), CD16(FcrRIII), CD11b, CD18. The measured fluorescent intensity was transformed into MESF. Phagocytic activity was measured by flow cytometry after incubation of the cells with fluorochrome-conjugated beads. Respiratory burst was measured by chemiluminescence assay. ADCC was measured by hemoglobin release assay. Opsonophagocytic activity was measured by fungicidal assay. Correlation between MESF of FcrR and Mac-1 and function of HL-60 was measured. RESULTS: Percent positive cells and MESF of CD11b and FcrRI increased on the 4th day and decreased on the 7th day. Percent positive cells of CD18 was 99% regardless of differentiation. But MESF of CD18 was increased on the 4th day and decreased on the 7th day. Percent positive cells of FcrRII were above 90% regardless of differentiation. MESF of FcrRII showed no significant change. FcrRIII expression was not induced. Phagocytic activity of HL-60 cells was increased twofold. Chemiluminescence of HL-60 cells was increased up to 60-fold on the 7th day. ADCC of HL-60 cells was incerased up to 2.5-fold on the 7th day. Opsonophagocytic activity increased twice on the 4th day. ADCC and opsonophagocytic activity correlates with the expression of CD11b/CD18 and FcrRII. CONCLUSION: Differentiation of HL-60 cells with ATRA induces several functional maturations until 7 days with expression of FcrR and Mac-1.
Antibody-Dependent Cell Cytotoxicity ; Flow Cytometry ; HL-60 Cells* ; Humans ; Luminescence ; Respiratory Burst ; Tretinoin*

We investigated the acute effect of recombinant human growth hormone (rhGH) on the activity of polymorphoneuclear leukocyte (PMN). We selected 6 patients of growth hormone deficient and 5 normal control children. In both groups, 0.15 IU/kg of rhGH was administered subcutaneously. The plasma growth hormone level were measured by radioimmunoassay on 0, 2, and 6 hours after administration of rhGH. To determined PMN activity, peripheral blood PMN were separated by discontinuous density-gradient centrifigation. Isolated PMN were stimulated hy fMLP and PMA and then respiratory burst activity of PMN was determined. The average growth hormone level of growth horrnone deficient and normal group were increased to the level of 41.6+/-23.7 and 96.3+/-46.5 ng/ml respectively, 2 hours after rhGH injection and decreased to the level of 18.5+/-10.6 and 42.2+/-5.5 ng/ml respectively, 6 hours after rhGH injection. Superoxide (O ) production by PMN which was stimulated by PMA was increased from 9.98+/-5.18 to 38.67+/-19.19 (x 10'cpm) after 6 hours of rhGH injection in control group children. It seemed that administration of the rhGH do not made a any effects acutely on PMN activity in growth hormone deficient group. But in a normal control children, extemal administration of rhGH acutely increased activity of PMN.
Child* ; Growth Hormone ; Human Growth Hormone* ; Humans* ; Leukocytes ; Neutrophils* ; Plasma ; Radioimmunoassay ; Respiratory Burst ; Superoxides

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

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*

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