Recombinant human erythropoietin induces proliferation and Ca<Superscript>2+</Superscript>-influx in specific leukocyte subpopulations of rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) blood and head kidney cells

In mammals, erythropoietin regulates the development and differentiation of erythrocytes. Although hematopoietic cells of bony fish correspond in their ontogeneic development, morphology, and function to their mammalian counterparts, an erythropoietin (EPO)-like molecule has not been identified. In this study we present evidence for a mitogenic response of blood and head kidney leukocytes of rainbow trout after stimulation by recombinant human EPO (rhu EPO). The modulation of cellular activities is accompanied by the induction of DNA-binding activities in nuclear extracts of these cells. In addition, flow cytometric analysis of intracellular Ca²⁺ concentrations revealed a long-lasting and rhu EPO dose-dependent increase, which was shown to be abrogated by cross-aggregation of surface IgM using anti-trout-IgM monoclonal antibodies (mabs). In flow cytometric dual-labeling experiments using rhu EPO/anti-EPO antiserum and mabs specific for trout leukocyte subpopulations, it was shown that a subpopulation of trout B-cells binds rhu EPO. Moreover, in a modified Ca²⁺ activation assay, it was demonstrated that this blood B-cell subpopulation is the rhu EPO responder population. In conclusion, the data suggest the existence of EPO-binding receptors in trout that are able to trigger Ca²⁺-independent intracellular signaling in hematopoietic cells of head kidney and Ca²⁺-dependent activation of a subpopulation of B-lymphocytes.Abbreviations FSC forward scatter - IFN interferon - IL interleukin - mab monoclonal antibody - PBL peripheral blood leukocytes - PHA phythemagglutitnin - rhu EPO recombinant human erythropoietin - SSC side scatter - TNF tumor necrosis factor Communicated by G. Heldmaier     

Measuring some flounder (Platichthys flesus L.) innate immune responses to be incorporated in effect biomonitoring concepts

For an implementation of innate immune responses of flounder (Platichthys flesus) in an integrated biological effect monitoring concept, leucocytes were isolated from peripheral blood, head kidney and spleen, and analysed for their capacity to mount a respiratory burst response upon phorbol ester stimulation. Responding cells were identified by reduced nitro-blue-tetrazolium salt deposits and by dihydro-rhodamine fluorescence in light microscope and flow cytometric analysis. Responding cells were found in head kidney derived cell suspensions rather than in peripheral blood or spleen. Parallel cytometric and microscopic analysis indicated that responding cells had a granulocyte or monocyte morphology, were alpha-naphtyl-esterase or myeloperoxidase positive and in flow cytometry exhibited a characteristic forward and side scatter (FSC/SSC) pattern. These cells represented 30–40% of head kidney derived cell suspensions and only 4–5 % of peripheral blood and spleen. In order to reduce sampling effort in field studies, leucocyte cell suspensions derived from flounder head kidney could be used in respiratory burst assays without further enrichment protocols. This paper combines, for the first time, conventional and cytometric analysis of phagocytes derived from flounder peripheral blood and head kidney. Communicated by H. v. Westernhagen, A. Diamant     

Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non-pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non-pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non-pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non-pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide-related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.     

The optimal application of forward and ninety-degree light scatter in flow cytometry for the gating of mononuclear cells

Peripheral blood mononuclear cells from ten normal donors were labeled with a monoclonal antibody specific for monocytes and analyzed using a fluorescence activated cell sorter (FACS). Forward and 90 degrees light scatter parameters were studied in order to apply optimal computerized gating to identify and exclude monocytes from lymphocyte populations. An average of 9.45% versus 1.22% of cells, within chosen lymphocyte gates established by forward angle and 90 degrees scatter, respectively, were identified as monocytes. In samples from ten donors, the exclusion of monocytes from the lymphocyte population was more efficient using 90 degrees scatter than forward scatter. Simultaneous use of forward and 90 degrees scatter did not significantly improve the ability to accurately exclude monocytes, but did result in a significant increase in the improper exclusion of lymphocytes. Use of 90 degrees scatter alone, forward scatter alone, and forward and 90 degrees scatter simultaneously to identify lymphoid cells resulted in the exclusion of 12, 17, and 23% of lymphocytes from further analysis. The 90 degrees scatter alone appears to be the optimal method to eliminate monocytes electronically from mononuclear cell populations in which lymphocytes are being studied.     

Light scatter analysis and sorting of cells activated in mixed leukocyte culture

Cells activated in unidirectional mixed leukocyte cultures (MLC) have been analyzed on the basis of their light scatter characteristics. C57BL/6 spleen cells were cultured with irradiated (2000 rads) DBA/2 spleen cells for 5 days and the resulting suspension of activated cells was passed on a FACS II flow cytometer. Correlated parameter analysis of forward light scatter (FLS) and perpendicular light scatter (PLS) indicated that the MLC consisted of a heterogenous mixture of viable cells, dead cells, and subcellular debris. However, by appropriate gating of the FLS/PLS distribution, viable cells could be identified as a biphasic FLS histogram. Sorting and morphological analyses of these two FLS peaks demonstrated that they corresponded to almost pure populations of small lymphocytes (lower peak) and lymphoblasts (upper peak), respectively. Furthermore, when sorted cells were tested for their ability to lyse antigenically relevant (DBA/2) tumor target cells in a 51Cr release assay, lymphoblasts were found to exhibit 40-fold greater cytolytic activity (on a per-cell basis) than small lymphocytes.     

Anomalous changes in forward scatter of lymphocytes with loosely packed membranes.

BACKGROUND: Forward scatter (FSC) is generally associated with cell size and has been suggested as a way to differentiate apoptotic from viable cells. Among spleen cells cultured for 48 h, a population of cells (population B) was found to have decreased forward and increased side scatter relative to freshly purified cells (population A). Interestingly, population B was not present early in analysis; this report explores the change in FSC of population B. METHODS: Using a Coulter (Hialeah, FL) Epics Elite ESP flow cytometer, changes in forward scatter and lipid packing of spleen cells were measured. RESULTS: Over time, the FSC of unfixed cells in population B increased from that of the debris field, to reach a stable value by 30 sec (population A's FSC remained constant). When fixed, populations A and B exhibited constant FSC. Population B cells displayed altered lipid packing as reported by MC540, and the FSC changes were mimicked by Nonidet P-40 treatment of freshly purified spleen cells. CONCLUSIONS: Data emphasize the importance of delaying measurements on unfixed cells until FSC readings have stabilized, and suggest that flow cytometry may be a useful tool in studying lipid packing.     

Evaluation of flow cytometry as a method for quantification of circulating blood cell populations in salmonid fish

Flow cytometry was used to estimate the proportions of different blood cell types in brown and rainbow trout. On the basis of forward light scatter and 90° side scatter three populations were differentiated. The relative abundance of these cells correlated with that of erythrocytc (r²= 0.994), lymphocyte plus thrombocyte(r²= 0.676) and neutrophil populations (r²= 0.571) enumerated by direct microscopy. By density gradient separation of cells, cell sorting and acridine orange staining it was confirmed that these cell types could be assigned to the populations detected. Changes in blood cell populations were monitored by flow cytometry in a group of experimental fish placed under confinement stress. Flow cytometry proved to be a rapid and reliable method for monitoring cell population dynamics in fish blood.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Evidence that expression of asialo-GM1 may be associated with cell activation. Correlation of asialo-GM1 expression with increased total cellular RNA and protein content in normal thymocyte and spleen cell populations

The question of the biologic significance of asialo-GM1 (aGM1) expression by a limited number of cells including natural killer cells has been raised by the recent demonstrations that aGM1 is expressed by activated macrophages and activated T cells as well as the proliferating thymoblast and functionally mature subpopulations of thymus. The current report demonstrates that the expression of aGM1 on aGM1-negative lymphocytes can be induced by stimulation with mitogens under activating conditions. In addition, activation of the aGM1-negative tumor lines EL-4/F and P388D1/B1 under conditions that result in interleukin 2 or interleukin 1 production, respectively, also result in a significant increase in aGM1 expression by the tumor cell lines. Expression of aGM1 therefore appears to be associated with events occurring as a prelude to or during activation of effector function. Since the aGM1-negative cells do display sialylated versions of aGM1 which can be converted by neuraminidase treatment into serologically recognizable aGM1, it is suggested that the expression of aGM1 might reflect a change in the level of glycolipid sialylation rather than a change in membrane lipid composition per se. The small percentage of lymphocytes in normal, nonstimulated spleen and thymus populations which express significant levels of aGM1 were sorted and analyzed for total cellular protein and RNA. Increases in RNA synthesis and in total cellular protein and RNA above basal levels of resting lymphocytes are considered indicators of a G0----G1 transition. The aGM1-positive populations displayed a larger mean population size (as indicated both by higher forward light scatter and by higher total cellular protein content), and a higher mean population RNA content than the aGM1-negative populations. These data are discussed in the context of the hypothesis that the expression of aGM1 may be associated with early events in the activation of resting cells which prepare the cells for subsequent induction of effector function.     

Immunophenotyping using gold or silver nanoparticle-polystyrene bead conjugates with multiple light scatter

BACKGROUND: The type of antibody-conjugated polystyrene (PS) latex beads for use as light scatter shift agents for targeted lymphocyte populations in whole blood has been expanded to include gold and silver nanoparticle-aminodextran-PS latex bead conjugates with antibodies. The linkers between antibody and colloidal metal were an aminotrithiol ligand or aminodextran polymer molecules. METHODS: A modified flow instrument, including forward light scatter (FS), side light scatter (SS), light scatter at other intermediate angle ranges, LMALS (10-20 degrees ) and UMALS (20-65 degrees ) was used for simultaneous bead probe measurements. A conventional flow cytometer was used in simultaneous bead-fluorescent marker experiments. RESULTS: Two mutually exclusive cell populations, CD4+ and CD8+ lymphocytes, have been simultaneously enumerated in blood by using a mixture of CD4-PS, CD8-Au-PS or CD4-Au-PS, CD8-PS beads, and one laser line, 633 nm, excitation. Similar measurements were made with mixtures of CD4-PS, CD8-Ag-PS or CD4-Ag-PS, CD8-PS beads. Also, simultaneous use of bead and fluorescent markers mixed with whole blood was demonstrated with CD4-PS beads and with the CD4-RD1/CD8-FITC dual marker. CONCLUSIONS: Enumeration of CD4 and CD8 lymphocytes in whole blood by light scatter parameters only compared well with standard analyses with fluorescent markers. In simultaneous bead-fluorescent marker labeling of lymphocytes, the labeled bead had to be mixed first with cells in whole blood.     

Effect of turpentine oil on C-reactive protein (CRP) production in rainbow trout (Oncorhynchus mykiss)

The effect of turpentine oil on C-reactive protein (CRP) production was studied in rainbow trout (Oncorhynchus mykiss). Serum CRP concentration was estimated by sandwich enzyme-linked immunosorbent assay using anti-rainbow trout CRP monoclonal antibody (mAb) AC4 and polyclonal antibody. Intracellular CRP was demonstrated by flow cytometry using anti-trout CRP mAb. Hepatocytes, head kidney macrophages, spleen lymphocytes and peripheral blood lymphocytes showed reaction against AC4, but RTG-2 fibroblastic line cells, derived from rainbow trout gonad did not. This is the first report on the detection of intracellular CRP in fish. CRP levels decreased significantly 1 day after intramuscular injection of turpentine oil and remained low for 14 days. Significant decreases in the expression of CRP in hepatocytes, head kidney macrophages and spleen lymphocytes after injection of turpentine oil were found. The reduction of serum CRP concentration after turpentine oil injection may be attributed to decreases in intracellular CRP synthesis.     

Calcium modulation and chemotactic response: divergent stimulation of neutrophil chemotaxis and cytosolic calcium response by the chemotactic peptide receptor

Stimulation of neutrophils by chemoattractants is followed by a rapid, transient rise in cytosolic calcium concentration. The role of calcium in activation of cell movement and related responses was examined by selectively chelating extracellular or both extra- and intracellular calcium. Removal of calcium from the extracellular medium did not alter the cytosolic calcium concentration (Quin 2 fluorescence, 110 to 120 nM) of unstimulated neutrophils and did not dramatically affect the rise induced by formyl peptide. Despite the intact Quin 2 response, depletion of extracellular calcium partially inhibited chemotaxis, adherence to substrate, and polarization (increased forward light scatter) in response to formyl peptide. Loading neutrophils with Quin 2 in the absence of calcium depressed cytosolic Ca2+ to 10 to 20 nM and abrogated a detectable rise with formyl peptide stimulation. Depletion of intracellular calcium further inhibited chemotaxis and polarization, although neutrophils still demonstrated significant directed migration and shape change to formyl peptide (30 to 40% of control) without an increase in Quin 2 fluorescence. Other neutrophil responses related to chemotaxis (decreased right-angle light scatter, actin polymerization) were minimally affected by depletion of calcium from either site. The data indicate that neutrophil chemotaxis and related responses to formyl peptide may be activated by intracellular signals not detectable with Quin 2.     

Anomalous behaviour of forward and perpendicular light scattering of a cyanobacterium owing to intracellular gas vacuoles

Extinction, absorption, and forward and perpendicular light scatter of the blue-green alga Microcystis aeruginosa with different amounts of intracellular gas vacuoles were determined. The amount of gas vacuoles in the cells was controlled by application of pressure. The presence of the gas vacuoles caused a tenfold increase in perpendicular light scatter, and a fivefold decrease in forward light scatter as measured by flow cytometry. Chlorophyll fluorescence showed a 16% decrease. The presence of gas vacuoles did not affect the size of the algae. The absorption spectrum of Microcystis aeruginosa was slightly raised but practically not distorted by the gas vacuoles. The attenuation spectrum, a measure for light extinction by the algal cells, was significantly distorted. The increase of perpendicular light scatter intensity of the cells is a direct consequence of the relatively high scatter of each vacuole, whereas the forward light scatter decrease is attributed to a lower phase-shift factor rho of the whole cells, caused by the intact gas vacuoles.     

Different expression patterns of TRP genes in murine B and T lymphocytes

A prolonged increase in the intracellular calcium concentration ([Ca2+]i) is essential for lymphocyte activation that includes cell proliferation and differentiation. This increase in [Ca2+]i results from Ca2+ release from the intracellular store and the subsequent Ca2+ influx from the extracellular environment via calcium channels located on the plasma membrane. Although transient receptor potential (TRP) channels have been reported to play important roles in the [Ca2+]i increase in lymphocytes, the function of these channels in lymphocyte activation remains unknown. Here, we report the comprehensive expression profile of TRP channel gene families including TRPC, TRPV, and TRPM in the murine immune system. RT-PCR analysis revealed different expression patterns of the TRP channel genes in B and T lymphocytes isolated from the spleen. Therefore, our results provide an appropriate reference of TRP gene expression in murine lymphocytes.     

Separation of leukemic myeloblasts and splenic lymphocytes based upon differences in light scatter profile.

Cell sorting based upon differences in light scatter profile was carried out to sort cells which differ in size. This technique permitted the acquisition of subpopulations of murine spleen cells enriched for leukemic myeloblasts or lymphocytes. Morphologic assessment of the sorted populations was confirmed by malignancy assay $\text{in}$$\text{vivo}$. The same technique has been applied to human leukemic cells and significant enrichment for proliferating and quiescent cells was accomplished.     

Evidence that TRH controls prolactin release from rat lactotrophs by stimulating a calcium influx

Prolactin (PRL) release and intracellular free calcium concentration [Ca²⁺]_i were measured in two populations of normal rat lactotrophs (light and heavy fractions) in culture. Spontaneous PRL release of heavy fraction cells was more sensitive to dihydropyridines (DHPs; Bay K 8644 and nifedipine) when compared to the light fraction lactotrophs. The stimulatory effect of thyrotropin-releasing hormone (TRH) on PRL release from heavy fraction cells was inhibited by Cd²⁺ and mimicked by Bay K 8644. Indo-1 experiments revealed that TRH-increased [Ca²⁺]_i was reversibly inhibited by Cd²⁺. In a Ca²⁺-free EGTA-containing medium, TRH did not modify [Ca²⁺]_i.Abbreviations [Ca²⁺]_i intracellular free calcium concentration - DA dopamine - DHP dihydropyridine(s) - DMEM Dulbecco's Modified Eagle's Medium - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PRL prolactin - RIA radioimmunoassay - TRH thyrotropin-releasing hormone - VGCC voltage-gated calcium channel     

Morphological and flow cytometric characterization of leukocytes from the notothenioid teleosts Dissostichus eleginoides, Notothenia coriiceps, and Trematomus hansoni

To date, the adaptive immune response of the notothenioid fishes of the Sub- and High-Antarctic has received little attention. Here we characterize leukocyte preparations derived from head (pronephric) kidney, spleen, and intestine of three notothenioid species, Dissostichus eleginoides, Notothenia coriiceps, and Trematomus hansoni. Cells were collected from head kidney, spleen, and intestine, and were fixed in paraformaldehyde and analyzed by flow cytometry and optical microscopy. The three species displayed typical leukocyte populations composed of lymphocytes, granulocytes, monocytes/macrophages and thrombocytes with a peculiar organ distribution and peculiar flow cytometric patterns. This work represents the first characterization of cells involved in immune reactions in the investigated species.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.