Flow cytometric investigation of neutrophil activation pathways by n-formyl-Met-Leu-Phe and phorbol myristate acetate

Summary— Recent evidence suggests that multiple pathways exist in PMN activation and that specific leukocyte response may be due to the activation of a particular signaling pathway. Using flow cytometry, PMN activation pathways were studied through the parallel comparison of n-formyl-Met-Leu-Phe (fMLP)- and phorbol-12-myristate-13-acetate (PMA)-induced stimulation and by simultaneous assays for CD11b expression and morphology. The maximal CD11b expression was higher with PMA than with fMLP, suggesting different activation pathways. Under these experimental conditions, a morphological response to fMLP was not observed. However, significant shape change was detected in PMA treated samples and was suppressed by either the removal of extracellular calcium or staurosporine at the concentrations above 14.5 μM. Calcium ionophore induced a similar light scattering pattern to that by PMA and enhanced CD11b expression, both of which were not inhibitable by staurosporine. These observations, for the first time, indicated that Ca²⁺ was a mediator in activation processes and that the treatment of PMN with PMA resulted in Ca²⁺ influx.     

Flow cytometric measurements of neutrophil functions: the dependence on the stimulus to cell ratio

Phagocytosis and antimicrobial killing of neutrophils has been quantitatively determined as a function of the stimulus (Candida albicans) to cell ratio R using two donor collectives containing a total of 115 blood samples. Analysis of the collectives in two different laboratories according to the same flow cytometric protocol for simultaneous measurement of neutrophil functions did not produce statistically significant differences. The number of phagocytosing leukocytes as well as that of killed fungi per leukocyte depends strongly on R. While each phagocytosing neutrophil kills one fungus at low values of R, each neutrophil kills on average 2.5 fungi for large R.     

Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

Flow cytometric measurements of cytoplasmic calcium changes in human platelets

Thrombin-induced stimulation of human platelets is accompanied by a dramatic increase in cytoplasmic calcium concentrations followed by a slow decrease. These changes are very rapid, are maximal by 10-15 s, and can be detected with probes such as Indo-1. Suspension studies using spectrofluorometry, which reflect a value which is the average of 3 x 10(7) cells per ml, indicate a thrombin dose-dependent increase in cytoplasmic calcium at doses up to 0.025 units per ml. We show here, using flow cytometry, that at less than half-saturating thrombin doses only subpopulations of platelets rather than the entire sample are responding. The extent of these responses, however, still depends on thrombin concentration. When the thrombin doses are between half and fully saturating, one subpopulation responds fully (i.e., its extent of increase in cytoplasmic calcium concentration, [Ca++]in is 100% of that seen at saturating thrombin concentrations) while the remaining platelets respond partially or not at all. There is thus evidence of positive cooperativity leading to disproportionate thrombin receptor occupancy on different subpopulations when platelets are subjected to subsaturating doses of thrombin. The existence of responding subpopulations may explain how the reported multiple stimulations of the same suspension of platelets at low thrombin doses occur.     

Sequential phospholipase activation in the stimulation of the neutrophil NADPH oxidase

Abstract Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 min after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by luminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca²⁺ and phospholipase A₂ activity, but not the activities of phospholipase D or D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.     

Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Flow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Sz?llosi et al., Cytometry, 5:210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes. This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.     

Application of the CYTOMIC 12 flow cytometric compact analyzer for automatic kinetic measurements

Flow cytometry, usually applied to cells which have time independent features, can also be used for kinetic experiments where the change of cell populations with time is investigated. Dedicated time sequencing programs written in Assembler and incorporated in the CYTOMIC 12 analyzer (4) are described. A sequence of 64 one parameter histograms can be automatically acquired and immediately displayed as a pseudo-two-parameter histogram. The acquisition time for each of the subsequent histograms can be selected between 1 and 32 seconds. Kinetics lasting up to 34 minutes are resolved into 64 time intervals. Two parameter kinetics can be resolved into 12 32 X 32 channel, two parameter histograms which are displayed and evaluated immediately on the analyzer screen in groups of 4 without using complicated list mode procedures. The standard CYTOMIC 12 software can be applied for processing and printing of the sequence distribution curves.     

Flow cytometric analysis of the kinetic effects of cisplatin on lung cancer cells

The effects of cisplatin on the cell cycle and DNA synthesis of human lung adenocarcinoma cell line PC-9 were examined by flow cytometry. The cellular DNA content and the bromodeoxyuridine (BrdUrd) incorporation rate were measured simultaneously using a monoclonal anti-BrdUrd antibody. Following exposure to cisplatin (1.0 micrograms/ml) for 1 and 24 hr, the bivariate DNA/BrdUrd distributions revealed a delayed S-phase transit and an accumulation of cells in the G2M phase. The BrdUrd-linked green fluorescence intensity continued to decrease with the lapse of time. However, early- and mid-S-phase cells soon recovered DNA synthesis activity, and the former showed higher activity than the control cells. These findings suggested the vigorous DNA synthesis of cells in early S phase. However, for quantitative analysis of chemotherapeutic effects, some problems remained to be resolved regarding the condition for DNA denaturation and its alteration by the agents.     

Flow cytometric estimation of transmembrane potential of macrophages--a comparison with microelectrode measurements

Potential-dependent accumulation of the lipophilic cationic dye 3,3' dihexyloxacarbocyanine (DiOC6(3)) in macrophages has been investigated. Resulting fluorescence of cells was measured by flow cytometry. Alterations of membrane potential of macrophages were induced by ionophore treatment (valinomycin and gramicidin) in a dose-dependent (10(-5) M-10(-7) M) and time-dependent (0 min-45 min) manner. Resulting changes in relative fluorescence intensity were compared with changes of transmembrane potential measured by intracellular recordings obtained by applying glass microelectrodes. The comparative studies offer the possibility to calibrate the flow cytometric estimate of membrane potential of suspended cells. Equilibration of dye partition between cells and surrounding medium is strictly potential-dependent at dye concentrations between 5 X 10(-8) M and 10(-7) M and within an incubation interval from 10 min up to 30 min after addition of dye. Conclusions are drawn concerning the field of application of the optical method. Dynamics of electrical processes following ionophore treatment are discussed in terms of molecular mechanisms of altered ionic transport.     

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

BACKGROUND: The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. METHODS: To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. RESULTS: The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. CONCLUSIONS: The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.     

Flow cytometric analysis of circadian changes in platelet activation using anti-GMP-140 monoclonal antibody.

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-alpha-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

Selective phospholipase C activation.

Phospholipase C is a family of cellular proteins believed to play a significant role in the intracellular signaling mechanisms utilized by diverse hormones. One class of hormones, polypeptide growth factors, elicits its influence on cellular function through stimulation of cell surface receptor tyrosine kinase activity. Certain growth factors appear to stimulate cellular phospholipase C activity by selective, receptor-mediated tyrosine phosphorylation of the phospholipase C-gamma 1 isozyme. While the role of phospholipase C activity in growth factor regulation of cell proliferation remains to be clarified, the selective growth factor-stimulated tyrosine phosphorylation and activation of phospholipase C-gamma 1 is an interesting example of enzyme-substrate interaction at the crossroads of two important intracellular signaling pathways.     

Flow cytometric analysis of porcine preadipocytes.

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal-vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD-1 cells surface antigen was greater in cultures supplemented with 2% pig serum and 10 nM dexamethasone than in other media. In cultures, GH, TNF-alpha and TGF-beta each inhibited lipid deposition, whereas TNF-alpha and TGF-beta, but not GH, inhibited AD-1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF-alpha and TGF-beta was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.     

Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil.

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.     

Flow cytometric measurements and electron microscopy of cell surface glycosaminoglycans using Acridine Orange

Summary Cell surface glycosaminoglycans (GAGs) were measured, after various treatments, by their binding to Acridine Organge using flow cytometry. Using a critical electrolyte concentration and combining it with specific degradation of individual GAG elements, it was found possible to differentiate between GAG components. The technique was adapted for electron microscopy level to reveal characteristics of membrane-associated GAG. By this means, the cell membrane of the human leukaemic cell line K562 was shown to contain a large amount of GAG; 75% of it was highly sulphated GAG, mostly heparan sulphate. This component was evenly distributed in the outer plasma membrane layer. In the presence of other GAGs, the appearance of complex proteoglycan granules was detected.