Uridine labeling of human lymphocytes: Differential uptake by T and B cells

Human peripheral blood lymphocytes were labeled with [³H]uridine and separated into immunoglobulin (Ig)-positive-enriched and Ig-negative populations by rosetting and Ficoll sedimentation. The Ig-negative (T cell-rich) fraction was found to be more heavily labeled than the B cell-enriched population, in agreement with previous results in rats. Combined autoradiography and rosetting confirmed the differential uridine labeling of T and B cells. Incorporation of cytidine and adenosine by T and B cell-enriched populations showed similar but less dramatic differential labeling.     

Flow cytometry of leukocytes after intravenous fluorescein angiography

Blood specimens were obtained for T lymphocyte analysis from patients undergoing intravenous fluorescein angiography (IVFA). Mononuclear cells were prepared by differential density centrifugation and analyzed by flow cytometry (FCM). Prior to IVFA, there was very little autofluorescence in the mononuclear cell population. After IVFA, there was an increase in the fluorescence of the mononuclear cells. Though barely visible by fluorescence microscopy, the fluorescence was uniformly distributed throughout the cytoplasm. By FCM, fluorescein was detected in 27% of the lymphocytes, 61% of the monocytes, and 75% of the granulocytes. Care must be taken when interpreting flow-cytometric lymphocyte subset data because of the increased nonspecific fluorescence of mononuclear cells in patients who have undergone IVFA.     

Human monocytes, B lymphocytes, and non-B lymphocytes each have structurally unique Fc gamma receptors

Human Fc gamma-binding macromolecules were isolated from subpopulations of mononuclear cells by repetitive affinity chromatography. Mononuclear cells, nylon wool-filtered cells, plastic-nonadherent cells, and plastic-adherent cells from normal donors were radiolabeled by using 125I and lactoperoxidase. Washed cells were solubilized in 1% NP-40 buffer containing proteinase inhibitors at 0 degrees C. Fc gamma receptors were purified on human IgG-Sepharose columns by use of the repetitive affinity chromatography procedure. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated only a 52,000 to 58,000 Mr Fc gamma receptor from nonadherent cell populations. Both rosetting and nonrosetting subpopulations of non-B lymphocytes expressed the 52,000 to 58,000 Mr receptor. The predominant Fc gamma receptor isolated from plastic-adherent cells was a 60,000 to 68,000 Mr macromolecule. Cell preparations enriched in B lymphocytes yielded prominent 43,000 Mr Fc gamma receptors. Thus human monocytes, B lymphocytes, and non-B lymphocytes each appear to have structurally distinct and unique Fc gamma receptors.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

A Method for Producing Dust-, Streak- and Hole-Free Formvar Films in Laboratories Having High Atmospheric Humidity

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Indicators of Pneumocystis carinii viability in short-term cell culture.

Growth of P. carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms. We studied three markers of cell function in P. carinii during the course of short-term cell culture, and correlated these with the number of P. carinii present in culture supernatants. The markers were glucan synthase activity, esterase activity and cell membrane integrity. The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry. The rise in P. carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity. Flow cytometry analysis of day-6 P. carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide. The demonstration of three indices of metabolic activity in an expanding P. carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P. carinii.     

Internalization and acidification of insulin by activated human lymphocytes

The binding and internalization of fluorescein isothiocyanate-conjugated insulin by nonactivated and phytohemagglutinin-activated circulating human lymphocytes was measured by flow cytometry. In confirmation of previous results, negligible binding or internalization was observed for unstimulated cells, while activated lymphocytes showed significant insulin binding. The majority of this insulin was demonstrated to be internalized via receptor-mediated endocytosis and acidified within 60 min after addition of insulin. Dual-fluorescence flow cytometry, using antibodies specific for human T cell subsets, was used to show that the expression of insulin binding sites occurs for at least some cells from both the helper/inducer and cytotoxic/suppressor T cell subsets. Insulin internalization is not an artifact of in vitro stimulation, since more than 90% of the unstimulated lymphocytes from a patient with a helper T cell leukemia are positive for insulin internalization. The usefulness of flow cytometric analysis for measuring lymphocyte activation in unstimulated populations and the therapeutic potential of the reported findings for control of lymphocyte proliferation are discussed.     

Reduced Expression of CD27 by Collagenase Treatment: Implications for Interpreting B Cell Data in Tissues

Surface markers have been used to identify distinct cell subpopulations and to delineate various stages of maturation or activation of lymphocytes. In particular CD27 is used for delineation of naïve and memory B cell populations, and is readily detected by flow cytometry. We here used flow cytometry to examine the expression of CD27 on lymphocytes isolated from various tissues of rhesus macaques, and found its expression was consistently low to absent on intestinal cell suspensions. However, immunohistochemistry revealed abundant CD27+ cells in intestinal tissue sections. Further investigation showed the marked loss of CD27 expression on processed intestinal cells was due to collagenase digestion of intestinal tissues, yet CD27 expression was recoverable within hours of cell isolation. By combining confocal microscopy, we confirmed that only a fraction of B cells express CD27, in contrast to expression on all T cells from tissues examined including the gut. Taken together, our results suggest that CD27 may be a memory marker for B cells, but not for T cells, since essentially all CD3 T cells expressed CD27. In summary, it is important to consider the influence of isolation procedures on cell surface expression of phenotypic markers, especially when examining tissue-resident lymphocytes by flow cytometry.     

Flow cytometry as a method for assaying the biological activity of phytotoxins

A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat (Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.     

Permeabilization of lymphocytes with polyethylene glycol 1000. Discrimination of permeabilized cells by flow cytometry

The toxicity of polyethylene glycol 1000 (PEG) used similarly as in cell hybridization experiments, has been studied by flow cytometry, measuring the light scattering and fluorescence distributions of PEG-treated human lymphocytes stained with propidium iodide, fluorescein diacetate and acridine orange (AO). The sensitivity of these tests to detect permeabilized, or potentially dead cells, was equal. In addition, PEG proved to interfere with AO staining most likely through the inhibition of its binding to nucleic acids. The decrease of AO fluorescence in cells killed by PEG was unexpected since intercalation of propidium iodide was the same as in alcohol fixed cells. Permeabilization of cells by PEG appears to be an all-or-none phenomenon, accompanied by entrance of PEG into the cells. The findings are described in the context of a review of the currently used flow cytometric techniques to discriminate viable and lethally affected cells; also, the problems of interpretation are discussed.     

Flow system fluorescence polarization measurements on fluorescein diacetate-stained EL4 cells.

We have adapted a multiparameter cell sorter to measure the distribution of fluorescence polarization in cell populations. Measurements carried out on EL4 cells show that the percent polarization of fluorescein fluorescence decreases with increasing fluorescence intensity. This inverse relationship between polarization and intensity is shown both within the cell population and by the average values of the two quantities during both the increase and decrease of fluorescence intensity. The quantitative relation between intensity and polarization is different in hypertonic than in isotonic media. These results suggest that polarization measurements carried out at a fixed time after incubation of cells with fluorescein diacetate, which is converted to fluorescein within the cells, may depend in part on the rate of fluorescein accumulation, and that agents that have been reported to change the polarization of fluorescein in living cells may do so by changing the kinetics of fluorescein accumulation.     

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.     

细胞计数板在荧光显微镜观察B细胞吞噬现象的应用探讨

目的:基于细胞计数板建立一种简单、快速使用免疫荧光显微镜观察B淋巴细胞吞噬卡介苗(BCG)现象的新方法,对即将进行流式细胞检测的样品进行质控,提高流式细胞术检测吞噬率的稳定性,同时为流式细胞仪检测吞噬率提供镜下依据。方法:B细胞与FITC标记的BCG共培养24 h后,PE anti-human CD19抗体直接标记细胞膜,应用细胞计数板在荧光显微镜下观察B细胞吞噬现象,流式细胞仪检测吞噬率。结果:应用细胞计数板在荧光镜下可观察到B细胞与BCG的荧光标记及B细胞与BCG共标记现象,证实B细胞可吞噬BCG,流式细胞仪检测结果显示吞噬率为13.9%。结论:应用细胞计数板在荧光镜下可观察B细胞吞噬现象,且操作简便快速,能对流式细胞检测的样品进行质控,并提供镜下依据。     

Identification of inflammatory cells in bovine milk by flow cytometry

Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.     

C-reactive protein antigenicity on the surface of human peripheral blood lymphocytes. Characterization of lymphocytes reactive with anti-neo-CRP

Previously, we have shown that antibodies specific for C-reactive protein determinants, not present on the native molecule, termed neo-CRP, also react with a significant percentage of PBL. In the present study, cells were evaluated by flow cytometry using alpha-neo-CRP antisera and mAb specific for lymphocyte subsets. With use of either monocyte-depleted PBL or Percoll-enriched large granular lymphocytes, we observed an overlap between cells reactive with alpha-neo-CRP and cells bearing the surface markers CD16, CD11b, Leu-7, and/or Leu-19, which are expressed on NK cells. In addition, we showed co-expression of the neo-CRP antigen with CD19, CD20, and HLA-DR, cell surface markers which are expressed on B lymphocytes. The major proportion of CD3+ cells failed to exhibit co-expression of neo-CRP. Single parameter flow cytometric analyses demonstrated that cells reactive with alpha-neo-CRP exhibited a bimodal staining pattern based on fluorescence intensity: high intensity neo-CRPbright and low intensity neo-CRPdim. Two-color analysis revealed that neo-CRPbright cells co-expressed CD19, CD20, and HLA-DR, whereas neo-CRPdim cells co-expressed CD16, CD11b, Leu-7, and Leu-19. Anti-neo-CRP also reacted with PBL obtained from patients with CD16+ lymphoproliferative disorders and from patients with chronic lymphocytic leukemia of B cell origin, but not with cells from patients with T cell or myeloid leukemias. The alpha-neo-CRP cells from patients with NK cell expansions showed dim fluorescence, whereas patients with B cell expansions showed bright fluorescence, consistent with the staining patterns observed with normal PBL. In addition, cell lines of T cell, B cell, NK cell, myeloid, and erythroid origin were evaluated for reactivity with alpha-neo-CRP. The cloned NK cell line NK 3.3 reacted as neo-CRPdim, but the B cell lines BL41, BL41/95, T1, T2, and CESS all reacted as neo-CRPbright. The cell lines K562, Molt-4, Hut-78, HL-60, U-937, and THP-1-0, which lack characteristic NK and B cell markers, did not react with alpha-neo-CRP. Additional study of the two-color histograms revealed a distinct diagonal staining pattern that was observed only when cells were co-stained with alpha-neo-CRP and either alpha-CD16 (alpha-Fc gamma RIII) or antibody IV3 (alpha-CDw32; alpha-Fc gamma RII). This finding suggests a 1:1 relationship between Fc gamma R on both NK and B cells and determinants recognized by alpha-neo-CRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multiparameter flow cytometric approach for simultaneous evaluation of T lymphocyte-endothelial cell interactions.

Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.     

Human T cell subpopulations defined by a monoclonal antibody. I. A small subset is responsible for proliferation to allogeneic cells or to soluble antigens and for helper activity for B cell differentiation

A monoclonal antibody (5/9) was obtained that reacts with the majority of alloactivated human T cells but only with 15 to 20% peripheral T cells. The populations reactive with the 5/9 antibody were separated from the remaining T cells by rosetting techniques using 5/9-coated ORBC. 5/9+ and 5/9- populations were analyzed for different in vitro activities. The helper activity of PWM- driven B cell differentiation appeared to be restricted and highly enriched in the 5/9+ population. In addition, 5/9+ cells contained all the cells capable of proliferation to tetanus toxoid and allogeneic cells. Thus, in vitro activities commonly used for evaluation of T cell function seem to be confined to a small fraction of peripheral T cells.     

Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate

Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a γ-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes.     

Intracellular pH-determination by fluorescence measurements.

A method was developed to determine the intracellular pH (pHi) of individual cells by use of fluorescence measurements. The method is based on the observation that the fluorescence excitation spectrum of fluorescein is pH-dependent. Fluorescence excitation spectra from individual rat bone marrow cells treated with fluorescein diacetate (FDA) were compared with those of fluorescein solutions of known pH values. Cells which were suspended in media of pH between 4.0 and 8.1 with high to normal buffering capacities had pHi values equal to those of the media. Cells suspended in media with low buffering capacities maintained a pH,i of 6.7 +/- 0.2. Preliminary results indicated that the pHi of individual cells may also be determined by using flow cytometry.     

Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester

This protocol outlines the carboxyfluorescein diacetate succinimidyl ester (CFSE) method for following the proliferation of human lymphocytes in vitro and mouse lymphocytes both in vitro and in vivo. The method relies on the ability of CFSE to covalently label long-lived intracellular molecules with the highly fluorescent dye, carboxyfluorescein. Following each cell division, the equal distribution of these fluorescent molecules to progeny cells results in a halving of the fluorescence of daughter cells. The CFSE labeling protocol described, which typically takes <1 h to perform, allows the detection of up to eight cell divisions before CFSE fluorescence is decreased to the background fluorescence of unlabeled cells. Protocols are outlined for labeling large and small numbers of human and mouse lymphocytes, labeling conditions being identified that minimize CFSE toxicity but maximize the number of cell divisions detected. An important feature of the technique is that division-dependent changes in the expression of cell-surface markers and intracellular proteins are easily quantified by flow cytometry.