Fluorescence-activated Cell Analysis Research Articles

Effect of diabetes mellitus on endotoxin-induced lung injury.

To examine the effects of diabetes mellitus on lipopolysaccharide (LPS)-induced pulmonary edema and alveolar neutrophil recruitment and activation. Zucker diabetic fatty rats are resistant to the effects of intratracheal LPS on the extravasation of plasma proteins into the lungs. Zucker diabetic fatty (ZDF) rats (genotype fa/fa) were used as a model of diabetes mellitus, while their normoglycemic heterozygous littermates served as controls. Lipopolysaccharide (Escherichia coli 0111: B4; 100-200 microg) or vehicle (0.25 mL of isotonic sodium chloride solution) was instilled into the airways of ZDF and control rats. Four hours later, pulmonary microvascular dysfunction was assessed by measuring the extravasation of Evans blue dye into the lung. Lipopolysaccharide-induced neutrophil recruitment was assessed by counting the number of neutrophils within the bronchoalveolar lavage fluid and measuring their expression of CD11b/CD18 by fluorescence-activated cell analysis sorting. The LPS (200 microg) induced a 32% increase in Evans blue dye extravasation into the lungs of controls (P = .008) but had no such effect in diabetic animals. Pulmonary extravasation of Evans blue dye in controls was greater than that of ZDF rats both at baseline (P = .002) and in response to 200 microg of LPS (P<.001). The LPS upregulated neutrophil CD11b/CD18 expression in diabetic and nondiabetic groups and induced a greater than 50-fold increase in the number of neutrophils within the airways of both control and diabetic groups (P<.001). Despite the recruitment of a large number of neutrophils into the lung, the LPS-induced change in pulmonary microvascular permeability in diabetic animals is substantially less than that of nondiabetic controls.

Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on the surface of anterior pituitary cells by FACS analysis. Incubation of anterior pituitary cells with dexamethasone or corticosterone (0.1 and 1.0 microM) prior to fixation and analysis produced a significant, concentration-dependent decrease in intracellular ir-LC1 and a concomitant increase in the amount of ir-LC1 detected on the surface of the cells; the effects of the two steroids were indistinguishable quantitatively. In conclusion, we report a novel method which permits (1) the detection and semi-quantitative measurement of intracellular and surface LC1 in anterior pituitary cells; and (2) the identification of the cell types in which the protein is found.

Osteoblastic differentiation and mRNA analysis of STRO-1-positive human bone marrow stromal cells using primary in vitro culture and poly (A) PCR.

Investigation of osteoblast dysfunction in osteoporosis has been hampered by a poor understanding of normal early osteoblast differentiation, due to a relative lack of markers for the earliest cells in the lineage. Attempts to identify such markers have used cultures of animal or immortalized human cells, of uncertain relevance to human biology, or heterogeneous cultures in which genetic variability precludes the isolation of stage-specific genotypic markers. Primary in vitro generation of clonal populations of human bone marrow stromal cells was used in order to overcome these problems. Fibroblast-like stromal cells were isolated from human sternal bone marrow. They showed differentiation to an osteoblastic phenotype when stimulated with dexamethasone (10(-7) M) and fluorescence activated cell analysis demonstrated immunopositivity for STRO-1 (an antibody that recognizes osteoprogenitor stem cells of the colony-forming unit-fibroblastic) in from 8 to 40 per cent of the cells, dependent on time post-harvest. Cells positive for STRO-1 were immunoselected using magnetic activated cell sorting and seeded at low density (10 cells/cm2) to produce clones. Each clone was subpassaged, osteoblastic differentiation stimulated with dexamethasone, and mRMA-extracted at time points post-stimulation (0 h and 1-14 days). A novel poly (A) reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify cDNA representative of all transcripts expressed at each time point. Differential gene expression within the amplified cDNA was assessed using 3' end cDNA probes to osteocalcin, osteopontin, and collagen type I (positive), demonstrating the acquisition of an osteoblastic phenotype. Time-specific gene products for early osteoblast differentiation have been generated from primary human cultures, utilizing very low density seeding and poly (A) RT-PCR. These products overcome the problems associated with animal, immortalized or heterogeneous culture and can be used to study normal and altered early osteoblast differentiation, indicating the possibility of using the same system to study other disease states.

Costimulation of cutaneous T-cell lymphoma cells by interleukin-7 and interleukin-2: potential autocrine or paracrine effectors in the Sézary syndrome.

To examine interleukin-7 (IL7)- and interleukin-2 (IL2)-induced proliferation of Sézary lymphoma cells and to consider if an autocrine or paracrine growth-stimulatory circuit involving IL7 exists in the Sézary syndrome (SS). Fresh Sézary lymphoma cells were maintained in short-term culture in the presence of cytokines, and growth was measured by incorporation of (3H)-thymidine (TdR). Expression of IL7 and IL7 and IL2 receptors (IL7-R and IL2-R, respectively) was assessed by polymerase chain amplification of first-strand complementary DNA (RT-PCR), by affinity cross-linking of radioactive iodine-125-IL7, and by dual-color fluorescence-activated cell analysis. IL-7 production was measured by immunoassay. Sézary lymphoma cells from seven patients showed synergistic (five of seven) or additive (two of seven) proliferation when cultured in the presence of IL2 and IL7, as compared with culture with either cytokine alone. Two patients with evidence of synergistic stimulation of [3H]-TdR incorporation showed IL7-R gene expression by RT-PCR and IL7 affinity cross-linking. Incubation of all seven patients' cells with IL7 induced coexpression to varying degrees of IL7-R and IL2-R. Sézary lymphoma cells from at least three of five patients studied expressed IL7 mRNA, and skin from three of five patients studied, as well as normal skin, expressed IL7 mRNA by RT-PCR. Sézary lymphoma cells respond by proliferation to IL7 plus IL2, and in some instances produce IL7. Therapeutic maneuvers should be pursued to take advantage of this potential autocrine or paracrine growth-stimulatory mechanism.

Correlation of VLA-4 integrin expression with metastatic potential in various human tumour cell lines

Abstract This investigation has focused on whether a number of molecular species, which have recently been recognised as components of cell attachment receptors utilised in recirculatory leukocyte traffic, are expressed on metastatic tumour cell populations. This has been studied on live cultured metastatic and non-metastatic tumour cell lines as well as on histological sections of frozen tissue from primary tumours and metastases which they formed after inoculation into nude mice. Here we report data we have obtained using immunofluorescence microscopy, fluorescence activated cell analysis, immunocytochemistry and pathological investigation of tumour behaviour in vivo, which converge to indicate that expression of the integrin molecule VLA-4 is positively associated with the execution of the metastatic process. This molecule is known to be a receptor for at least two ligands, namely the inducible endothelial adhesion molecule VCAM-1 and the extracellular matrix component fibronectin, and is thought to be mechanistically important in the attachment and diapodesis of lymphocytes. The present findings, indicating differential expression of this molecule on metastatic cell populations relative to non-metastatic cell populations, support and extend recent reports from other laboratories, of the presence of various leukocyte adhesion receptors on metastatic tumour cells. This accumulating evidence suggests that inappropriate expression of one or more of these surface adhesion molecules in tumour cell lineages may endow the progeny of the affected clones with some of the properties needed for metastatic behaviour. The total information so far assembled by various groups also provides some early clues suggesting that the types of molecules expressed may be related to the histogenetic origin of the tumour and its pattern of metastatic spread. At this stage there is insufficient information to comment on whether expression of any of the leukocyte adhesion molecules so far identified is essential for metastasis, or whether their deployment on the tumour cell surface is just facilitatory. However, the body of information now available provides a strong incentive to study whether interference with the functions of such molecular species could impede or reduce the geometric progression of the disease.

Identification of müllerian inhibiting substance specific binding in human cell lines.

The receptor for Müllerian Inhibiting Substance (MIS), a gonadal glycoprotein hormone, has not been previously identified. Plasma membranes from MIS-sensitive human tumor cell lines (HTB-111, endometrial carcinoma; and A-431, vulvar squamous carcinoma) were detergent extracted and incubated with 125I-labeled MIS anti-idiotypic antibody, or radioiodinated human recombinant MIS (125I rhMIS), with and without unlabeled competitors. 125I anti-idiotypic MIS antibody bound to HTB-111 membrane extracts was displaceable by unlabeled anti-idiotypic antibody, but not by anti-isotypic antibody prior to cross-linking. Specific binding of the anti-idiotypic MIS antibody to endometrial carcinoma cells was verified using fluorescence activated cell analysis and fluoresceinated antibody. Furthermore, unlabeled anti-idiotypic MIS antibody competed for 125I rhMIS binding to A-431 vulvar carcinoma membranes. The labeled anti-idiotypic MIS antibody binding complex could be separated from 32P labeled EGF receptor in the A-431 membranes, indicating that EGF, a natural inhibitor of MIS activity, and MIS itself bind to different receptors. These studies demonstrate a specific, displaceable binder for MIS in the plasmalemmae of two human tumor lines. Purification of this cell surface receptor protein will be greatly aided by using the MIS anti-idiotypic antibody.

Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.

Fluorescence-activated cell analysis of P-fimbriae receptor accessibility on uroepithelial cells of patients with renal scarring.

Pyelonephritogenic Escherichia coli possess P-fimbriae that bind to uroepithelial cells by recognizing a receptor containing the alpha-D-Galp-(1-4)-beta-D-Galp carbohydrate structure. The accessibility of P-fimbriae receptors was determined using uroepithelial cells from 19 female patients with verified renal scarring and a history of febrile urinary tract infection, and 13 healthy controls. The binding of fluorescein isothiocyanate labeled P-fimbriated Escherichia coli to a large number of uroepithelial cells from each individual was studied using the method of fluorescence-activated cell sorting analysis, which allows examination of a large number of cells and selection of cells of a given size. The uroepithelial cells from the patients exhibited a significantly higher binding capacity to P-fimbriated Escherichia coli than did uroepithelial cells from healthy controls (p less than 0.01). The determination of P-fimbriae receptor accessibility on uroepithelial cells may be useful for detecting risk groups among patients with recurrent urinary tract infections.

Density and localization of P-fimbriae-specific receptors on mammalian cells: fluorescence-activated cell analysis.

More than 95% of acute non-obstructive pyelonephritis cases in children are caused by P-fimbriated Escherichia coli. Uroepithelial cells from individuals prone to urinary tract infections bind such bacteria more avidly than do cells from healthy controls. A high density of receptors for P-fimbriae on cells of the urinary tract and kidney may thus be an important host factor in the acquisition of upper urinary tract infections. In this communication we are describing methods to determine the densities and localization of P-fimbriae receptors on mammalian cells. To determine the receptor density on cells in suspension, the cells were incubated with fluorescein-labelled P-fimbriated E. coli. They were then subjected to fluorescence-activated cell sorting (FACS) analysis which monitors both the size and the relative fluorescence of each cell. FACS analysis proved versatile for rapid and specific analyses of P-fimbriae receptor densities on large numbers of any chosen type of cell. The fluorescein-labelled P-fimbriated E. coli were also useful in localizing P-fimbriae receptors in tissues. These techniques are not limited to the assessment of P-fimbriae receptor densities and tissue localization but open up new aspects for studies of the specificities involved in the interactions between other microorganisms (e. g. parasites, bacteria and virus) and their host cells.

Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.

The synthesis of a novel class of reagents for fluorescence analyses of molecules and cells is reported. These compounds consist of a highly fluorescent phycobiliprotein conjugated to a molecule having biological specificity. Phycoerythrin-immunoglobulin, phycoerythrin-protein A, and phycoerythrin-avidin conjugates were prepared. These conjugates bind specifically to beads containing a covalently attached target molecule and render them highly fluorescent. Femtomole (10(-15) mole) quantities of phycoerythrin conjugates can be detected because of the high extinction coefficient (epsilon M = 2.4 x 10(6) cm-1 M-1 for 2.4 x 10(5) daltons) and high fluorescence quantum yield (Q = 0.8) of the phycobiliprotein moiety. An important feature of these conjugates is that they emit in the orange-red spectral region, where background fluorescence is less than at shorter wavelengths. Phycoerythrin conjugates are well-suited for two-color flow cytofluorimetric analyses employing a single excitation line. The distributions of Leu antigens (also called OKT antigens) on the surface of T-lymphocytes were analyzed using fluoresceinated antibody as the green-fluorescent stain and biotinylated antibody counter-stained with phycoerythrin-avidin as the red one. This one-laser two-color analysis showed that cells express Leu-3a and Leu-3b or neither antigen. In contrast, the distributions of Leu-2a (a marker of suppressor and cytotoxic T-cells) and Leu-3a (a marker of helper and inducer T-cells) are mutually exclusive. These studies show that phycobiliprotein conjugates can be applied to fluorescence-activated cell sorting and analysis, fluorescence microscopy, and fluorescence immunoassay.

Cholera-toxin binding to cells of developing chick retina analyzed by fluorescence-activated cell sorting

The occurrence of gangliosides on nerve cells of the developing retina was studied by fluorescence-activated cell analysis and sorting, using fluorescent cholera toxin as marker. This toxin binds to G M1; neuraminidase converts several other gangliosides into G M1. Without pretreatment by this enzyme weak binding of toxin is detected at later stages of development, whereas pretreatment leads to considerable toxin binding at earlier stages. The number of cells binding toxin as well as the amount bound per cell increase with developmental age of the retina. Cells binding a given amount of toxin vary strongly in size. Cell sorting was used to separate postmitotic cells from proliferating cells. Proliferating cells have little binding capacity, while post-mitotic cells bind relatively large amounts of toxin. Localization of gangliosides which bind toxin in the developing retina was studied in cryostat sections. At an early stage (day 6) toxin binding is localized in the inner layer of the developing retina which contains the ganglion and other postmitotic cells, but is not found in the outer layer of matrix cells. At later stages complex staining patterns evolve with binding predominantly in the nerve fiber layers.

Separation of cell subpopulations from embryonic chick neural retina with fluorescence-activated cell sorting

Fluorescence-activated cell analysis and sorting was used to characterized and separate cell subpopulations from the developing chick retina according to size, stage in the cell cycle and location in the tissue. It was shown that uptake of rhodamine B isothiocyanate (RITC) protein stain, light-scattering at large angles, and the time of flight of cells through the lase beam all correlate with cell size. Vital staining of DNA by H33342 permits cell separation according to phase in the cell cycle (G 1, S, G 2 + M). Further, a method was developed for separating postmitotic cells, based upon the quenching of H33342 fluorescence by the incorporation of 5-bromo-2′-deoxyuridine (BrdU) into DNA. Quantitative data reflect the decrease of DNA-synthesizing cells and the increase of postmitotic celss as the development of the retina proceeds. Isolation of cells on the basis of their location in the whole tissue is achieved by exposing the desired surface (e.g. the inner (concave) surface of the retina, which is rich in ganglion cells) to a suspension of Fluorecamine, which stains only cell layers close to the surface. Sorting out the fluorescing cells yields a high proportion of cells with the appearance, size and postmitotic characteristics of ganglion cells.

Effects of cytotoxic monoclonal antibody specific for T200 glycoprotein on functional lymphoid cell populations

A monoclonal antibody against T200 glycoprotein is selectively cytotoxic for thymocytes and mature thymus-dependent (T) lymphocytes. All T-cell functions assayed, cell-mediated cytotoxicity, helper cell activity, and proliferation in response to T-cell mitogens or allogeneic cells were abolished by prior treatment of spleen cells with anti-T200 antibodies and complement. In contrast, thymus-independent (B) cell responses to lipopolysaccharide (LPS) and other B-cell mitogens were unaffected. Although treatment of spleen cells with anti-T200 antibodies and complement markedly reduced their capacity to mount an in vitro antibody response to sheep red blood cells (SRBC), responsiveness could be restored by the addition of SRBC-primed T-helper cells. Treatment of bone marrow cells with anti-T200 antibodies and complement did not eliminate either in vivo colony-forming units-spleen (CFU-S) or prothymocytes. It is concluded that T lymphocytes become sensitive to complement-mediated lysis by anti-T200 antibodies as a consequence of cell-surface modifications occurring shortly before or just after their entry into the thymus. In contrast to Thy-1 antigen, the selective killing by antibody against T200 glycoprotein cannot be readily accounted for by quantitative differences in the expression of T200 glycoprotein on the cell surface. Fluorescence-activated cell analysis showed that T200 glycoprotein was expressed in similar amount on the majority of all thymocytes, spleen, and bone marrow cells.

A means for orienting flat cells in flow systems

Flattened cells, such as red blood cells, epithelial cells, and sperm of many species, cause problems for fluorescence-activated cell analysis and sorting machines because the flow systems of such devices are unable to control the orientation of these cells as they flow past the detectors. For this reason, the fluorescence or scattered light measurements for identical cells may vary greatly. A flow geometry is here described that orients flat cells in a coaxial flow system so that each cell presents the same aspect to the observation device. A wedge-shaped exit on the sample injection tube in a coaxial flow system is sufficient to produce the desired orientation effect when used with low sample flow rates. Data is presented showing the effect of orientation of fixed chicken erythrocytes on histograms of small forward-angle light-scattering measurements.

Myiasis of Palpebral Conjunctiva

Natural killer T cells are an immunoregulatory population of lymphocytes that plays a critical role in controlling the adaptive immune system and contributes to the regulation of autoimmune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the nonobese diabetic (NOD) mouse strain, a well-validated model of type 1 diabetes and systemic lupus erythematosus. In this study, we report the results of a genetic linkage analysis of the genes controlling NKT cell numbers in a first backcross (BC1) from C57BL/6 to NOD.<i>Nkrp1</i>^<i>b</i> mice. The numbers of thymic NKT cells of 320 BC1 mice were determined by fluorescence-activated cell analysis using anti-TCR Ab and CD1/α-galactosylceramide tetramer. Tail DNA of 138 female BC1 mice was analyzed for PCR product length polymorphisms at 181 simple sequence repeats, providing greater than 90% coverage of the autosomal genome with an average marker separation of 8 cM. Two loci exhibiting significant linkage to NKT cell numbers were identified; the most significant (<i>Nkt1</i>) was on distal chromosome 1, in the same region as the NOD mouse lupus susceptibility gene <i>Babs2/Bana3</i>. The second most significant locus (<i>Nkt2</i>) mapped to the same region as <i>Idd13</i>, a NOD-derived diabetes susceptibility gene on chromosome 2.