Neutral surface aminopeptidase activity of human tumor cell lines.

Seven human tumor cell lines were studied for their neutral surface aminopeptidase (AP) activity. The activity was shown to exist on all cell lines to varying degrees. The neutral AP activity of the cell lines had similar Km values and were affected by the same inhibitors as those reported for AP's of peripheral blood lymphocytes (PBL, Refs. 1 and 2). However, a difference was seen in the Vmax values of the various cell lines. These values were shown to correlate (r = 0.767, P less than 0.05) with cell surface area.

Expression of Duffy antigen receptor for chemokines during reticulocyte maturation: using a CD71 flow cytometric technique to identify reticulocytes.

Flow cytometric methods commonly used to identify reticulocytes are of limited usefulness in malarious areas, since RNA staining also detects plasmodia. An important antigen expressed on reticulocytes is Duffy antigen receptor for chemokines (DARC, also known as Fy), the receptor for Plasmodium vivax. An early marker for reticulocytes is CD71 (transferrin receptor). We have been interested in CD71 as an alternative marker for reticulocytes in the context of Fy expression. Flow cytometry was used to determine the expression of Fy on CD71-positive and -negative reticulocytes and to correlate serology and genotype. A reduction of 13 percent was seen in Fy6 expression between CD71-positive reticulocytes and RNA-positive reticulocytes. CD71 disappears early during reticulocyte maturation, while Fy6 expression is relatively preserved. CD71 is an alternative to staining for RNA for reticulocyte assays relating to Fy6 expression.

Cyclosporin A effects during primary and secondary activation of human umbilical cord blood T lymphocytes.

OBJECTIVE: Cyclosporin A (CsA), effective in prophylaxis and treatment of graft-vs-host disease (GVHD) after human allogeneic transplantation, blunts T-cell responses by inhibiting nuclear factor of activated T cells-1 (NFAT1) activation. This laboratory has shown that NFAT1 protein expression is severely reduced in human UCB (umbilical cord blood) T cells. Since UCB is increasingly used as a hematopoietic stem cell source in allogeneic transplantation, it is important to determine whether CsA sensitivity in UCB differs from that of adult T cells. METHODS: Surface flow cytometric analysis, intracellular cytokine staining, flow cytometric analysis of cell death, and thymidine incorporation were used in this study to determine T-cell activation and effector functions during primary and secondary stimulation in the presence of CsA. RESULTS: Although we observed differential CsA sensitivity of T-cell activation marker (CD69, CD45RO, CD25) upregulation comparing UCB and adult, we did not observe any significant difference in CsA sensitivity of T-cell effector functions. Importantly, we observed reduced IFN-gamma and TNF-alpha expression in UCB T cells both in primary and secondary stimulation, as well as increased rates of activation-induced cell death (AICD). CONCLUSION: Thus, our studies do not support the previous hypothesis that reduced GVHD observed after UCB transplantation is attributable to increased CsA sensitivity of UCB T cells. Rather, reduced UCB T-cell cytokine production and increased AICD may be important cellular mechanisms underlying these favorable rates of GVHD in UCB transplant recipients.

Synthesis of interleukin-1 alpha and beta by normal human melanocytes.

Normal human melanocytes and melanoma cells have been reported to produce several cytokines. Previously we demonstrated that neonatal human melanocyte proliferation and tyrosinase activity are inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6. We have now also shown that interleukin-1 beta induces an inhibiting effect on neonatal melanocyte tyrosinase activity with little effect on melanocyte proliferation. We investigated the ability of neonatal and adult human melanocytes to synthesize interleukin-1 alpha and beta. By immunocytochemistry, using monoclonal antibodies against interleukin-1 alpha and beta, we observed that neonatal and adult melanocytes stain positively for both cytokines. Flow-cytometric analysis revealed that the percentage of melanocytes positive for interleukin-1 alpha was always greater than that for interleukin-1 beta. The ability of neonatal and adult melanocytes to synthesize interleukin-1 alpha and beta was further confirmed using the polymerase chain reaction. These results clearly indicate that human melanocytes synthesize interleukin-1 alpha and beta, and that these cytokines may function as autocrine and/or paracrine regulators of cells in the epidermis.

Phagocytosis of opsonized fluorescent microspheres by human neutrophils. A two-color flow cytometric method for the determination of attachment and ingestion.

A highly reproducible two-color fluorescence cytometric method is described for determining the amount of receptor-mediated and of non-specific phagocytosis by human neutrophils of polystyrene microspheres that have been covalently coated with C3b, iC3b, IgG, mixtures of these, BSA or human F(ab')2. The method includes a correction for externally bound particles and thus measures net phagocytosis. The method involves the incubation of neutrophils with coated green fluorescent microspheres in buffer alone or with cytochalasin D to inhibit phagocytosis followed by staining the externally bound microspheres with red fluorescent antibodies to the immobilized ligand, and determining the green and red fluorescence in a dual laser fluorescence activated flow cytometer. The red to green fluorescence ratio of the mixtures containing cytochalasin D allows for a correction of the green fluorescence due to externally bound microspheres in the mixtures not containing cytochalasin D. The corrected green intensities thus represent net phagocytosis. The specificity of receptor-mediated phagocytosis was confirmed by inhibition with fluid-phase C3b or iC3b or monoclonal antibodies to the receptors. The method can be applied to the determination of both adherent and suspension phagocytosis and can be used as a general model of the phagocytosis of bacteria by neutrophils.

Flow analysis of MHC molecules and other membrane proteins in isolated phagosomes.

A method was developed to apply flow cytometry analysis to the characterization of individual phagosomes. Macrophages were incubated with latex beads and homogenized to release the phagosomes. Intact cells and nuclei were removed by low speed centrifugation, and a crude phagosome preparation was fixed with paraformaldehyde. Distinct optical properties of latex bead phagosomes allowed their analytic isolation from other organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were established for phagosomes containing one, two and even three beads, which were sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phagosomes, but some more spacious phagosomes were also observed. Phagosomes were immunolabeled and subjected to flow analysis for MHC-I and MHC-II molecules and lysosomal membrane markers (LAMPs). The proportion of LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-II was demonstrated and remained relatively constant with time. Flow analysis of phagosomes allows the characterization and comparison of individual phagosomes, and the identification of subpopulations of phagosomes with differing membrane compositions. It also provides the advantage of analytically isolating phagosomes from other components of the cell without the need for extensive prior physical purification. Thus, it can be used to rapidly assess changes in phagosomal membrane composition as a function of phagosome maturation.

HIV type 1 Tat protein induces apoptosis and death in Jurkat cells.

Jurkat cells stably expressing high levels of the HIV-1 Tat protein were generated after transfection with an Epstein-Barr virus-based episomal replicon and selection in hygromycin B. The Jurkat Tat transfectants exhibited a longer doubling time when compared to Jurkat cells or Jurkat cells transfected with the control parent plasmid. Cell cycle analysis revealed comparable durations of each phase of the cell cycle in the Tat and control transfectants. Flow cytometric analysis using Hoechst 33342 and propidium iodide staining revealed that the Tat transfectants exhibited a higher percentage of apoptotic cells when compared to the control transfectants (29.1 +/- 3.1 vs. 11.43 +/- 3.1%). Incubation of Jurkat cells with recombinant HIV-1 Tat protein resulted in induction of apoptosis. The HIV-1 Tat protein induces apoptosis in a CD4-positive T cell line. Tat-induced programmed cell death may contribute to the lymphocyte depletion seen in persons infected with HIV-1.

Effects of combination of tumor necrosis factor alpha and chemotactic peptide, f-Met-Leu-Phe, on phagocytosis of opsonized microspheres by human neutrophils.

Pretreatment of normal, human neutrophils with 8 units/ml of TNF-alpha followed by treatment with 10(-8) M FMLP resulted in a synergistic effect of the combination of the two mediators on the enhancement of the phagocytic capacity of the cells. This enhancement of phagocytosis occurred without an additional increase in the upregulation of C3b receptors (CR1) beyond that caused by each mediator alone. Pretreatment of the cells with 8 units/ml of TNF-alpha followed by 10(-6) M FMLP resulted in an additive effect of the mediators on neutrophil phagocytosis, again without an additional up-regulation of CR1. This additive effect resulted in an increase in phagocytic capacity of the neutrophils greater than that obtained by treatment of the cells with 10(-6) M FMLP alone, which heretofore has resulted in the greatest enhancement of phagocytic capacity obtained by any pretreatment condition. These synergistic and additive effects of the combination of mediators could be of great importance in host defense against bacterial infections and have important implications regarding the mechanisms of receptor upregulation and phagocytosis.

Effects of chemotactic peptide f-Met-Leu-Phe (FMLP) on C3b receptor (CR1) expression and phagocytosis of microspheres by human neutrophils.

FMLP caused maximal upregulation of CR1 on neutrophils at a concentration of 10(-8) M but caused maximal enhancement of CR1-dependent phagocytosis of C3b.IgG-coated microspheres only at a concentration of 10(-6) M. There were positive correlations between FMLP-mediated upregulation of CR1 and FMLP-mediated enhancement of phagocytosis (correlation coefficient = 0.73, slope = 2.2) and between FMLP-mediated upregulation of CR1 and FMLP-mediated increase in total cell-associated microspheres (correlation coefficient = 0.88, slope = 1.3). The phagocytic capacity of both untreated and 10(-6) M FMLP-treated neutrophils was completely inhibited by fluid phase C3b and partially inhibited by aggregated IgG. The data suggest that CR1 upregulation is required but is not sufficient for maximal phagocytosis by the leukocytes. The data also suggest that FMLP at the higher concentrations may impart a phagocytic function to CR1, activate other phagocytic receptors, elicit phagocytosis-inducing mediators or may elicit a separate mechanism of phagocytosis. During the study, it was observed that there was considerable individual variation among different neutrophil preparations with respect to CR1 expression and binding and phagocytic capacity.

Adhesive effect of certain cytokines and other perturbants on human neutrophils.

Pretreatment of normal human neutrophils with certain cytokines and other mediators caused some of the cells to become adhesive and stick to the plastic (polypropylene) incubation tubes during pretreatment and during the assay for phagocytosis of C3b.IgG-coated microspheres. Often as much as 40% of the cells were adherent to the tubes after the reaction. This sticking of the neutrophils to the plastic tubes was confirmed by increase in cytometer sipping time and by lactic dehydrogenase assay of the suspended cells and of the cells stuck on the sides of the empty incubation tubes. Only those perturbants that caused an up-regulation of C3b receptors (CR1, CD35) and in most cases caused an enhancement of phagocytosis mediated the adhesiveness of the cells. Unless these stuck cells were detached by vigorous flushing with cold buffer containing EDTA, many of the cells were not admitted into the cytometer for determination of the effect of the perturbants on binding and phagocytic capacity of the neutrophils. This observation could have implications regarding the possibility of subpopulations of neutrophils and differences in function of adherent cells versus cells in suspension. In the cases studied there was no appreciable difference between the total binding and phagocytic capacities of the adherent and suspended cells.

Comparison of abilities of recombinant interleukin-1 alpha and -beta and noninflammatory IL-1 beta fragment 163-171 to upregulate C3b receptors (CR1) on human neutrophils and to enhance their phagocytic capacity.

Both recombinant IL-1 alpha and -beta caused an upregulation of C3b receptors (CR1) on human neutrophils and caused a receptor-mediated enhancement of phagocytosis of C3b.IgG-coated microspheres by these leukocytes. The alpha and beta forms of the recombinant cytokine were of comparable potency regarding CR1 upregulation, although both generally had less than 25% of the potency of FMLP in this respect. Recombinant IL-1 beta was slightly more potent than the alpha form of the cytokine regarding phagocytosis of opsonized microspheres and, again, both forms were less potent than FMLP in causing an enhancement of phagocytosis by neutrophils. The synthetic noninflammatory immunostimulatory nonapeptide corresponding to residues 163-171 of IL-1 beta was completely inert with respect to upregulation of CR1 on neutrophils and the enhancement of phagocytosis by these cells. Thus this domain in the intact IL-1 beta molecule apparently is not involved in CR1 upregulation and the ensuing enhancement in phagocytosis by neutrophils, although it is apparently important in the immunostimulatory activity regarding the proliferation of lymphocytes.

A new human prostate carcinoma cell line, 22Rv1.

A cell line has been derived from a human prostatic carcinoma xenograft, CWR22R. This represents one of very few available cell lines representative of this disease. The cell line is derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft. Flow cytometric and cytogenetic analysis showed that this cell line represents one hyper DNA-diploid stem line with two clonal, evolved cytogenetic sublines. The basic karyotype is close to that of the grandparent xenograft, CWR22, and is relatively simple with 50 chromosomes. In nude mice, the line forms tumors with morphology similar to that of the xenografts, and like the parental CWR22 and CWR22R xenografts, this cell line expresses prostate specific antigen. Growth is weakly stimulated by dihydroxytestosterone and lysates are immunoreactive with androgen receptor antibody by Western blot analysis. Growth is stimulated by epidermal growth factor but is not inhibited by transforming growth factor-beta1.

Effect of antibiotics on CR1 receptor levels of human neutrophils and on the binding and phagocytosis of opsonized polystyrene microspheres by these leucocytes.

Fourteen antibiotics were studied for their effects on the following properties or functions of normal, human neutrophils: (1) the expression of CR1 receptors; (2) the total binding of C3b.IgG-coated polystyrene microspheres in the presence of cytochalasin D to inhibit phagocytosis; (3) the net phagocytosis of the opsonized microspheres; (4) the residual, external binding after phagocytosis. Fluorescence, flow cytometric methods were used to determine binding and phagocytosis of the model target particles. Only nafcillin, a penicillin, caused a decrease (33 per cent) in phagocytic capacity of the neutrophils at a physiologically significant dose (serum level); the antifungal antibiotic, amphotericin B, caused a 30 per cent increase in phagocytic capacity. These small changes in neutrophil phagocytic capacity may not be physiologically significant. There were no significant differences in the four measured parameters caused by other antibiotics tested at physiologically significant doses.

Combination simvastatin and metformin induces G1-phase cell cycle arrest and Ripk1- and Ripk3-dependent necrosis in C4-2B osseous metastatic castration-resistant prostate cancer cells.

Castration-resistant prostate cancer (CRPC) cells acquire resistance to chemotherapy and apoptosis, in part, due to enhanced aerobic glycolysis and biomass production, known as the Warburg effect. We previously demonstrated that combination simvastatin (SIM) and metformin (MET) ameliorates critical Warburg effect-related metabolic aberrations of C4-2B cells, synergistically and significantly decreases CRPC cell viability and metastatic properties, with minimal effect on normal prostate epithelial cells, and inhibits primary prostate tumor growth, metastasis, and biochemical failure in an orthotopic model of metastatic CRPC, more effectively than docetaxel chemotherapy. Several modes of cell death activated by individual treatment of SIM or MET have been reported; however, the cell death process induced by combination SIM and MET treatment in metastatic CRPC cells remains unknown. This must be determined prior to advancing combination SIM and MET to clinical trial for metastatic CRPC. Treatment of C4-2B cells with combination 4 µM SIM and 2 mM MET (SIM+MET) led to significant G1-phase cell cycle arrest and decrease in the percentage of DNA-replicating cells in the S-phase by 24 h; arrest was sustained throughout the 96-h treatment. SIM+MET treatment led to enhanced autophagic flux in C4-2B cells by 72-96 h, ascertained by increased LC3B-II (further enhanced with lysosomal inhibitor chloroquine) and reduced Sequestosome-1 protein expression, significantly increased percentage of acidic vesicular organelle-positive cells, and increased autophagic structure accumulation assessed by transmission electron microscopy. Chloroquine, however, could not rescue CRPC cell viability, eliminating autophagic cell death; rather, autophagy was upregulated by C4-2B cells in attempt to withstand chemotherapy. Instead, SIM+MET treatment led to Ripk1- and Ripk3-dependent necrosis by 48-96 h, determined by propidium iodide-Annexin V flow cytometry, increase in Ripk1 and Ripk3 protein expression, necrosome formation, HMGB-1 extracellular release, and necrotic induction and viability rescue with necrostatin-1 and Ripk3-targeting siRNA. The necrosis-inducing capacity of SIM+MET may make these drugs a highly-effective treatment for apoptosis- and chemotherapy-resistant metastatic CRPC cells.

Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis.

A simple flow cytometric method (FCM) for measuring phagocytosis of Staphylococcus aureus by human neutrophils (polymorphonuclear leukocytes [PMNs]) is described. This assay utilizes 100 microliters of EDTA-anticoagulated whole blood and a simplified method of fluorescently labeling bacteria. A commercially available whole-blood lysing reagent allows for the removal of erythrocytes and the exclusion of external free or adherent bacteria. Phagocytized bacteria are unaffected by this reagent, so PMNs containing internalized bacteria can be easily identified by FCM. Advantages of this method include the following: (i) small sample size, (ii) no requirement for PMN separation, (iii) rapid reliable method of labeling the bacteria, (iv) ability to distinguish between adherent bacteria and those which are actually internalized, (v) avoidance of vital dyes as quenching agents, and (vi) ability to fix cells and store for future FCM analysis.

Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data.

BACKGROUND: Flow cytometry of immunofluorescence and DNA content provides measures of cell-cycle-related gene expression (protein and/or epitope levels) for asynchronously growing cells. From these data, time-related expression through S phase can be directly measured. However, for G1, G2, and M phases, this information is unavailable. We present an objective method to model G1 and G2 kinetic expression from an estimate of a minimum biological unit of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. METHODS: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B1) distribution of p105-positive cells was used to model the B1 distribution of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. RESULTS: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mitotic levels were equivalent to the highest G2 levels. G1 modeling of B1 was less certain than that of G2 due to low levels of expression but demonstrated general feasibility. CONCLUSIONS: By this method, the upper and lower bounds of cyclin B1 expression could be estimated and kinetic expression through G1, G2, and M modeled. Together with direct measurements in S phase, expression of B1 throughout the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions.

The effects of cytokines, platelet activating factor, and arachidonate metabolites on C3b receptor (CR1, CD35) expression and phagocytosis by neutrophils.

The cytokines tumor necrosis factor alpha (TNF alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and interleukin 1 (IL 1) all caused an upregulation of C3b receptors (CR1) on neutrophils that ranged from around 76% (G-CSF and IL 1) to 93% (TNF alpha and GM-CSF) of the upregulation obtained by pretreatment of the neutrophils with the chemotactic peptide FMLP. However, only TNF alpha and G-CSF caused a significant increase in phagocytosis of opsonized microspheres. Platelet derived growth factor, interleukin 2, and transforming growth factor beta had no effect on either of these parameters. The mediators platelet activating factor (PAF) and leukotriene B4 (LTB4) both caused a large upregulation of CR1 (93% and 80%, respectively, of the FMLP-mediated value); however, only PAF caused a significant enhancement of phagocytosis by the neutrophils. Prostaglandin E2 and thromboxane B2 had no effect on these parameters. Considerable individual variation was observed among some of the untreated and mediator-treated neutrophil preparations regarding CR1 expression and phagocytosis. The upregulation of CR1 and associated increase in phagocytic capacity of neutrophils caused by certain cytokines and other mediators may be important in host defense. Also the lack of enhancement of phagocytosis accompanying an upregulation of CR1 is unusual and may have important implications regarding the cellular mechanisms of phagocytosis by neutrophils.

Isolation of Pneumocystis carinii cysts by flow cytometry.

Pneumocystis carinii cysts were separated as enriched populations from suspensions of lung homogenate obtained from infected rats containing all developmental stages of this organism. Isolation of cyst populations was achieved by incubating filtered lung homogenate with the fluorescent phospholipid analog 1-palmitoyl-2-C6-NBD-phosphatidylcholine. Whereas cysts did not fluorescence as a result of outer-wall restraint of lipid integration, trophozoites and young intermediate stages readily incorporated the fluorescently labeled lipid analog into their outer membrane. The two distinct labeling patterns displayed by cysts and other developmental phases of P. carinii constitute a novel, easy, and reproducible means of isolating cysts from infected lung homogenate by flow cytometry.

Differential expression of the duffy antigen receptor for chemokines according to RBC age and FY genotype.

BACKGROUND: The Duffy (Fy) blood group (also known as Duffy antigen receptor for chemokines, or DARC) may be involved in regulation of the level of circulating proinflammatory chemokines, and it is an obligatory receptor on RBCs for the human malaria parasite Plasmodium vivax. STUDY DESIGN AND METHODS: Because quantification of Fy expression by using RBCs of various ages will not detect acute changes associated with inflammatory states, and because P. vivax exclusively invades reticulocytes, a flow cytometric method was developed to measure the level of surface expression of Fy. Reticulocytes and mature RBCs from persons with different genotypes (GATA-1 T-->C promoter mutation at nt -46; FY*A and FY*B in the ORF) were used. RESULTS: Expression of the Fy6 epitope, which is required for P. vivax invasion, was 49 +/- 19 percent higher on reticulocytes than on mature RBCs, regardless of donor genotype (p<0.0001). Fy6 levels were approximately 50 percent lower in persons who were heterozygous for the GATA-1 promoter mutation and were significantly lower on reticulocytes and mature RBCs of the FY*B/FY*B genotype than on those of the FY*A/FY*A or FY*A/FY*B genotype. CONCLUSION: Fy has greater expression on reticulocytes than on mature RBCs in flow cytometry. This method may be useful in further studies of this antigen, such as characterization of reticulocytes and RBC phenotypes across populations, in response to chemokine regulation, and in the context of susceptibility to P. vivax and other parasites.

Synthesis of two biologically active fluorescent probes of thymopentin.

This study reports on the synthesis of two fluorescent analogues of thymopentin (TP-5; Arg-Lys-Asp-Val-Tyr). A fluorescein isothiocyanate labeled analogue (FITC-TP-5) and a stilbene isothiocyanate labeled analogue (SITS-TP-5) were extensively purified by ion-exchange and gel filtration chromatography. Characterization of the coupling site through amino acid analysis, dansylation and N-terminal cleavage of the fluorescent amino acid yielded results which indicated that both were mono-labeled analogues derivatized at the N-terminal. These analogues were shown to be TP-5-like in nature by their ability to induce the expression of the Thy 1.2 surface marker on nude mouse prothymocytes in both in vivo and in vitro assays. In addition, these analogues were able to inhibit the specific binding of radiolabeled TP-5 to human lymphocytes. Initial studies describing the interaction of FITC-TP-5 with human lymphocytes are shown.