Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.     

Phagocytosis of bacteria by human leukocytes measured by flow cytometry

A new method has been developed for the evaluation of the phagocytic activity of human leukocytes using fluorescently labeled bacteria and flow cytometry. By simultaneous measurement of cellular light scatter and fluorescence, extracellular bacteria, phagocytes, and nonphagocytes could be discriminated and quantified. All leukocytes assumed to be capable of phagocytosis were phagocytosing, and about 90% of these cells were polymorphonuclear neutrophilic granulocytes. Within 15 min 85% of the bacteria were phagocytosed and each phagocyte contained an average of 15-20 bacteria. The phagocytic capacity of the leukocytes from healthy individuals showed minor interindividual and day-to-day variations. This method facilitates a rapid and accurate in vitro evaluation of the phagocytic activity of human leukocytes.     

Characterization of lithium-induced enzyme release from human polymorphonuclear leukocytes

Lysozyme release from purified human polymorphonuclear leukocytes was found to be uniquely enhanced by 2.5-20 mM LiCl. This effect was dose dependent and was not detected when the media was supplemented with NaCl, KCl, MgCl2, or CaCl2. The purified isotopes of Li+, 6Li, and 7Li were equally effective in enhancing lysozyme release from the cells at 10 and 20 mM, but 6Li was more effective than 7Li at 5 mM. The enhancement of enzyme release in the presence of Li+ was comparable to the enhancement observed in the presence of N-formylmethionylleucylphenylalanine (fMLP). Addition of LiCl plus fMLP did not result in lysozyme release in excess of each stimulant alone, except when the cells were incubated with 20 mM 6Li + 10(-5) M fMLP. In addition, enzyme release induced by these two agents could be further enhanced to the same degree by addition of cytochalasin D to the incubation mixtures. While similarities between enzyme release induced by LiCl and fMLP were detected, optimal stimulation of enzyme release by Li+ was much more sensitive to inhibition by pertussis toxin than was maximal fMLP stimulation. Therefore, the intracellular events altered by Li+ and the peptide may share some metabolic steps, but they differ in their sensitivity to alterations in cAMP metabolism.     

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.     

Characterization of two gangliosides from human leukemic polymorphonuclear leukocytes.

Eight bands of gangliosides, from human polymorphonuclear leukocytes were demonstrated by thin-layer chromatography. Bands 4 and 5 were isolated and purified in sufficient amounts to allow their biochemical identification by thin-layer chromatography, gas chromatography and sequential action of glycosidases and neuraminidase. The major ganglioside was characterised as N-acetylneuraminylgalactosyl-beta-N-acetylglucosaminyl-beta-galactosyl-beta-glucosylceramide. A second ganglioside was tentatively identified as N-acetylneuraminyl-galactosyl-beta-N-acetylglucosaminyl-beta-(N-acetylneuraminyl)galactosyl-beta-glucosylceramide. Both gangliosides isolated were hydrolysed by neuraminidase. However, treatment of the intact cells with neuraminidase did not alter the ganglioside pattern.     

Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo-1 AM

The present report describes the increase in cytosolic-free calcium levels (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo-1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F-Met-Leu-Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide-angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin-1 alpha (IL-1 alpha), granulocyte-colony stimulating factor (G-CSF), nor granulocyte-macrophage-colony stimulating factor (GM-CSF), which were present in this supernatant, were able to induce a Ca2+i rise in PMN.     

Processing of staphylococcus aureus and zymosan particles by human leukocytes measured by flow cytometry

Staphylococcus aureus were labeled with fluorescein-isothiocyanate (FITC), stained by ethidium bromide (EB), and measured by flow cytometry (FCM). Bacteria were identified by their FITC-fluorescence and discriminated from the leukocyte cell nuclei by the much higher EB fluorescence and lower coefficient of variation of the latter. Following phagocytosis, both the bacterial FITC- and EB-fluorescence decayed. The mean FITC-fluorescence was reduced about 20% after 15 min and 30-50% after 60 min. Zymosan particles were labeled by FITC and incubated with leukocytes for 15 min. Phagocytes were sorted by FCM and the zymosan particles were liberated by sonication. Their forward angle light scatter was reduced by about 50.6 +/- 2.1% and the FITC-fluorescence by 8.7 +/- 1.0% (mean +/- SD). The reduced FITC-fluorescence and light scatter suggests degradation of proteins, and the decay of EB-fluorescence degradation of DNA, but the specificity remains to be established. By this method phagocytes from a patient with systemic lupus erythematosus seemed to have a selective defect in DNA degradation, whereas phagocytes from a patient with acute myelogenous leukemia had a low capacity to degrade bacterial proteins. This technique offers opportunities for automatic measurements of bacteria and zymosan particle degradation by phagocytes.     

Characterization of hematologic malignancies by flow cytometry

The quantitative assessment of cellular DNA and RNA content by flow cytometry to provide useful information for both diagnosis and prognosis of patients with hematologic malignancies is reviewed. While the characterization of cell surface antigens seems to be more germane to questions of the normal cell counterpart (stage) of malignant transformation and the biology of regulation of proliferation and differentiation by cell-cell contact and humoral factors, DNA-derived and RNA-derived parameters were surprisingly sensitive in the distinction of major morphologic groups, drug sensitivity and long-term prognosis. Our findings to date in the study of leukemias, lymphomas and myelomas are summarized.     

Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocysteine

Transmembrane signaling by chemoattractants in leukocytes appears to require activation of phosphoinositide metabolism with subsequent generation of the second messenger substances, inositol(1,4,5)trisphosphate and diacylglycerol. In addition, previous studies have shown that conditions which lead to an intracellular increase in S-adenosylhomocysteine (AdoHcy), a by-product and competitive inhibitor of S-adenosylmethionine-mediated methylation reactions, inhibit all chemoattractant-mediated functions of leukocytes, suggesting that AdoHcy also interferes with chemoattractant transmembrane signaling. In the present study, we determined whether AdoHcy altered the metabolism of phosphoinositides in human polymorphonuclear leukocytes. Treatment of 32P-labeled polymorphonuclear leukocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone, conditions which cause an increase in AdoHcy, produced as much as a 37% decrease in the amount of [32P]phosphatidylinositol 4-monophosphate associated with the cells. The formation of inositol bisphosphate was inhibited by as much as 45% by erythro-9-(2-hydroxy-3-nonyl)adenine, adenosine, and L-homocysteine thiolactone suggesting decreased availability of phosphatidylinositol 4-monophosphate. In support of this, AdoHcy, in concentrations ranging from 0.01 to 0.1 mM, inhibited the transfer of gamma-32P from gamma-[32P] ATP to phosphatidylinositol (PtdIns). The inhibition of PtdIns kinase was competitive with an apparent Ki for AdoHcy of 43 microM. Increased intracellular AdoHcy reduced chemoattractant-mediated increases in inositol(1,4,5)trisphosphate formation suggesting abrogation of transmembrane signaling. These findings for the first time demonstrate that AdoHcy is a competitive inhibitor of PtdIns kinase and thus a regulator of the phosphoinositide pathway.     

Characterization of bacteria by multiparameter flow cytometry

An arc-lamp based flow cytometer was used to obtain high resolution measurements of the light scattering characteristics and DNA contents of eight different bacteria. Light scatter profiles of bacteria are a useful first step when flow cytometry is used to characterize organisms. Scanning and transmission electron microscopy of the bacterial samples demonstrate that the structural basis of the light scattering profiles is not always clear, i.e. some organisms appear to have anomalous light scattering characteristics. The use of a third measurement parameter, DNA content, allowed much better discrimination of the organisms. Flow cytometry shows great promise as a method for the rapid discrimination and identification of bacterial populations.     

The omega-hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Incubations of [1-14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2-dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as omega-hydroxy-arachidonic acid and the two others were secondary products identified as omega-carboxy-arachidonic acid and 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid. Since the affinity for arachidonate of the omega-monooxygenase was quite low and the presence of LTB4 suppressed the omega-hydroxylation of arachidonate, we conclude that the known LTB4 omega-monooxygenase is responsible for the formation of omega-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.     

Interaction of bacterial toxin with leukocytes measured by flow cytometry

Enterobacter cloacae toxin was purified in the form of monomer and polymer. Both forms stimulated the generation of reactive oxygen species (ROS) at sublytic concentration; the oxidative stress produced was studied by using chemiluminescence (CL). The alteration generated caused death of leukocytes, especially at high toxin concentration. Polymeric toxin produced more oxidative stress than the monomeric one. Cytometry allowed the detection of more toxin binding to neutrophils rather than to monocytes or lymphocytes. There was binding at 4°C, and the amount of toxin in the cells increased at 37°C. The interaction of toxin with leukocytes was evident even after 100°C treatment of toxin during 5 min. The incubation with 2-mercaptoethanol was not necessary for toxin binding. Received: 19 October 2001 / Accepted: 21 December 2001     

The biosynthesis of endothelin-1 by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNs) converted human big endothelin (bET; 2 microM) to an endothelin-1 (ET-1) like contractile factor, as assessed by bioassay. The generation of this ET-1 like activity was rapid (minutes), time-dependent and more pronounced in non-activated cells, suggesting a partial degradation by activated PMNs. Phosphoramidon (54 micrograms/ml) inhibited the formation of this contractile factor, whereas phenylmethylsulfonylfluoride (PMSF; 25 micrograms/ml), pepstatin A (1 microgram/ml) or epoxysuccinyl-L-leucylamido-(guanidino)butane (E-64; 10 micrograms/ml) did not. Incubations of activated PMNs with PMSF significantly potentiated the generation of ET-1 like activity and selectively inhibited the degradation of [125I]ET-1 by activated PMNs. These findings indicate that human PMNs contain and/or release neutral proteases, which can both rapidly produce and degrade ET-1, an observation which may have important (patho)physiologic implications.     

Characterization and enumeration of spontaneously proliferating human leucocytes by multiparameter flow cytometry

Spontaneously proliferating human leucocytes have been characterized and enumerated using multiparameter flow cytometry. The frequency of spontaneously proliferating cells amongst human peripheral blood mononuclear cells, was determined on the basis of BrdUrd incorporation and total DNA content in samples of cells incubated in medium without added mitogen for several days. The frequency of proliferating cells decreased from an initial level of ≈ 6 × 10^–4 to 3 × 10^–4 after 30 h of incubation, and then rose to ≈ 2 × 10^–2 after 100 h of incubation. In one reference person, the frequency showed only minor variation in this pattern over a 1.5 year interval. Simultaneous measurement of proliferation and determination of immuno-logical subclass, as indicated with Hoechst 33342 staining and surface markers, showed an over-representation of CD19-positive cells, compared with CD2-positive cells and subsets of CD2-positive cells (CD4-positives and CD8-positives). This method can be used as an indicator of exposure to agents when results from animal tests are to be compared with results from human populations. The advantages are that no cell culturing is needed to perform the test, it provides the possibility of further characterizing proliferating cells, and the rapid flow-cytometric enumeration.     

Alkaline phosphatase activity in human polymorphonuclear leukocytes

Synopsis Alkaline phosphatase has been localized ultracytochemically in PMN of man with normal and elevated levels of this enzyme. Contrary to guinea-pig PMN, no activity appears to be present in the specific granules. Instead, the plasma membrane and the membrane of the endocytic vacuoles show a strong staining. However, the demonstration of this activity depends on the preparatory procedure employed for PMN isolation. the use of dextran and Ficoll-Hypaque in the isolation procedure induces a marked increase in alkaline phosphatase staining of the PMN plasma membrane. Strongly increased activity at this site has been found in PMN from cancer patients. In most of them, additional staining has been observed in atypical vesicles and sometimes in the Golgi apparatus. These findings are discussed in the light of some previously reported controversial biochemical and cytochemical data on the distribution of alkaline phosphatase in human PMN.