Differentiation of HL-60 promyelocytic leukemia cells monitored by flow cytometric measurement of nitro blue tetrazolium (NBT) reduction

Reduction of nitro blue tetrazolium (NBT) to insoluble blue formazan granules occurs during the stimulus-induced respiratory burst of mature granulocytes and is routinely used as an indicator of the extent of granulocytic differentiation of HL-60 acute promyelocytic leukemia cells. In the present study, the differentiation of HL-60 leukemia cells induced by dimethylsulfoxide (DMSO) or retinoic acid was monitored by flow cytometric (FCM) measurement of forward and 90 degree light scatter of NBT treated cells. Two-parameter correlated analysis permitted a distinction between cells with increased forward and decreased 90 degree light scatter (NBT-), and cells with decreased forward and increased 90 degree light scatter (NBT+). Fixation of NBT treated cells with 1% paraformaldehyde facilitated flow cytometric analysis, and allowed differences in NBT reduction to be quantitated. DMSO-induced cells expressed an all-or-none reduction of NBT to formazan, compared with retinoic acid treated cells that exhibited a graded response. Three parameter flow cytometric analysis of HL-60 leukemia cells stained with propidium iodide in combination with NBT allowed the determination of the cell cycle distribution of NBT-treated cells.     

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.     

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.     

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.     

Superoxide production from human polymorphonuclear leukocytes by human mannan-binding protein (MBP)

Mannan-binding protein (MBP) is a Ca(2+)-dependent mammalian lectin that plays an important role in innate immunity. In this study, we found that ligand-bound MBP stimulates polymorphonuclear leukocytes (PMN) to induce cell aggregation and superoxide production. The biological response of PMN to ligand-bound MBP was dose- and time-dependent. The PMN aggregation and superoxide production induced by ligand-bound MBP was blocked completely by pertussis toxin, and partially blocked by a platelet activation factor receptor antagonist, TCV-309. These findings suggest that the ligand-bound MBP stimulates PMN through a putative MBP receptor(s) on PMN.     

Interferon enhances the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMN) from healthy volunteers were tested for ADCC activity against both erythrocyte and tumor targets with and without the addition of human leukocyte interferon (IFN). It was demonstrated that IFN within 30 to 60 min enhanced the reaction in a dose-dependent manner with minimal IFN doses ranging from 1 to 100 units. Formal proof that the augmenting agent was IFN was obtained by using pure IFN proteins in combination with both mock-IFN preparations, which showed no enhancing activity, and anti-IFN antisera, which inhibited the action of the completely purified IFN proteins. In the light of data demonstrating that the IFN effect was most pronounced when the IgG antibodies in the ADCC reaction were present in suboptimal amounts, it is hypothesized that IFN may play a special role in the early nonspecific immune response against non-self antigens.     

Flow cytometric evaluation of anti-herpes drugs

A rapid means of screening drugs for toxicity and anti-herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV-1 (multiplicity of infection 1-10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4-n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.     

Separation of granule subpopulations in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes were isolated, disrupted by sonification and the nuclei and unbroken cells removed by centrifugation. The supernatant was applied on top of an optimised discontinuous Percoll gradient. After centrifugation we found nine gradient bands of distinct density. Both the nine bands and the whole fractionated gradient material were assayed for granule marker enzymes. Granule fractions of distinct density, enclosing different enzyme concentrations demonstrated the existence of granule subpopulations. There were three subpopulations of azurophil granules, about four subpopulations of specific granules, one granule fraction perhaps representing the C-particles, and a fraction of plasma membrane vesicles.     

Effects of microcystins on human polymorphonuclear leukocytes

Microcystins (MCs) are cyclic heptapeptides produced by cyanobacteria present in water contaminated reservoirs. Reported toxic effects for microcystins are liver injury and tumour promotion. In this study, we evaluated the effects of two MCs, MC-LR and [Asp(3)]-MC-LR, on human neutrophil (PMN). We observed that even at concentrations lower than that recommended by World Health Organization for chronic exposure (0.1 nM), MCs affect human PMN. Both MCs have chemotactic activity, induce the production of reactive oxygen species, and increase phagocytosis of Candida albicans. MC-LR also increased C. albicans killing. The effect of MCs on PMN provides support for a damage process mediated by PMN and oxidative stress, and may explain liver injury and tumour promotion associated to long-term MCs exposures.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Characterization of a C5a receptor on human polymorphonuclear leukocytes (PMN)

Elucidation of the interactions between C5a and granulocytes is central to understanding the role of C5a in inflammation. In this study, interactions between C5a and PMN have been studied at two levels. Binding of human C5a to intact human cells has been characterized by using the radiolabeled ligand 125I-C5a. Binding is shown to be reversible, saturable, and to reach equilibrium in 60 to 90 min at 0 degrees C. Results show high affinity C5a binding sites with Kd = 2 X 10(-9) M and a range of 50,000 to 113,000 binding sites per PMN. These values for C5a receptors are comparable with the number of fMLP and LTB4 receptors on PMN. Binding of C5a to PMN fails to reach equilibrium at 37 degrees C because there is an irreversible loss of available surface receptors caused by an active internalization of the ligand-receptor complex. Interactions between C5a and human PMN were characterized further by cross-linking experiments, with the use of ethylene glycol bis succinimidylsuccinate (EGS). Cross-linking of 125I-C5a to intact PMN followed by subcellular fractionation revealed a single radioactive band present only in the plasma membrane fraction and visualized by autoradiography. Similar experiments resulted in a covalent linkage between 125I-C5a and a component in the isolated plasma membrane of PMN. The covalent complex containing C5a and a putative receptor has been visualized by autoradiography as a single 60,000 Mr complex on SDS-PAGE. The complex is not present when experiments are performed in the presence of excess unlabeled C5a or in the absence of EGS. Therefore, the putative receptor for C5a on human neutrophils is estimated to be approximately 48,000 Mr, assuming contribution of 12,000 to 13,000 daltons by the ligand 125I-C5a.