Expression profiles of cloned channel catfish (Ictalurus punctatus) lymphoid cell lines and mixed lymphocyte cultures.

Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLCs) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date clonal catfish cell lines and MLCs have been biologically and phenotypically characterized using a variety of techniques including reverse transcription polymerase chain reaction (RT-PCR), as well as Northern and Southern blotting. To expand the molecular characterization of these cultures, microarray analysis was employed. Clonal B (3B11), macrophage (42TA), and cytotoxic T cell (TS32.15 and TS32.17) lines and MLCs were examined using a cDNA array containing approximately 2500 probes derived from EST libraries prepared from the 42TA macrophage cell line, a MLC, and 5-14-day-old catfish fry. Analysis showed that each cell line displayed a unique RNA expression profile that included a variety of immune-related genes. Pearson correlation analysis indicated that one cytotoxic T cell line (TS32.15) clustered with the MLC, whereas a second cytotoxic T cell line (TS32.17) was more closely associated with a second cluster containing B cells and macrophages. This study illustrates the utility of microarray analyses in profiling RNA expression patterns in catfish lymphoid cell lines and will serve as a platform for examining catfish immune responses following virus infection or poly [I:C] treatment.

A procedure for the isolation of highly purified populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood.

A sequential separation protocol is described which reproducibly yields biologically active populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood. The purification protocol employed T lymphocyte rosetting with unmodified sheep red blood cells, monocyte adherence to microexudate-coated flasks and anti-immunoglobulin panning of B cells. Population purity was determined by cytofluorographic light scatter analysis, expression of cell surface markers, esterase activity and mitogen responsiveness. The sequential protocol reproducibly yielded viable and functionally active cell populations of greater than 95% purity from a single starting population of mononuclear cells.

Positive selection of mouse B and T lymphocytes and analysis of isolated populations by flow cytometry.

Mouse spleen cells were separated into Ig+ and Ig- populations by positive selection using petri plates coated with rabbit anti-mouse immunoglobulin. The Ig- cells were subsequently incubated with mouse monoclonal alloantisera to Thy1.2 prior to a second positive selection. The adherent populations were characterized as B (Ig+) or T (Thy1.2+) lymphocytes on the basis of surface immunofluorescence and mitogen-induced proliferation. Analysis of the 2 isolated populations by flow cytometry showed that B and T lymphocytes could be distinguished by their forward light scatter as well as their fluorescence after incubation with fluorescein diacetate.

Effects of different protein supplements on mitogen responses of human peripheral blood lymphocytes.

We evaluated the effects of 6 supplements often used in human lymphocyte cultures, including fetal calf serum, autologous human serum, pooled human AB serum, hypogammaglobulinemic human serum, bovine serum albumin and human serum albumin. Lymphocyte proliferation of unstimulated and mitogen activated peripheral blood mononuclear cells was measured by [3H]thymidine incorporation. The response of cells stimulated with the T-cell mitogen phytohemagglutinin-P were significantly lower when cultured in bovine serum albumin supplemented media, but were otherwise not supplement dependent. In contrast, responses of cells stimulated with the B-cell mitogens Cowan I strain of S. aureus and antisera against the mu or delta chain of human immunoglobulin were significantly effected by supplement. Cultures containing fetal calf serum and bovine serum albumin had high background responses without a proportional rise in cellular proliferation when B-lymphocyte-specific mitogens were utilized. Autologous human serum and pooled human AB serum contained immunoglobulin which interacted with each of the B cell mitogens, thus limiting their usefulness as in vitro supplements. Cells grown in human serum albumin supplemented media had minimal background and high stimulated responses to B-cell mitogens. These results indicate that human serum albumin is an optimal supplement for in vitro human lymphocyte proliferative assays since it supports high stimulated cell responses with low background activity, is devoid of immunoglobulin and had minimal variability among lots.

Activation of channel catfish B cells by membrane immunoglobulin cross-linking.

This study demonstrates for the first time that teleost, specifically channel catfish, B cells proliferate in response to membrane immunoglobulin (mIgM) cross-linking. An early activation event mediated by anti-IgM ligation involved a rapid increase in intracellular calcium levels similar to the situation seen in mammalian B cells. In addition, catfish B cells, like mammalian B cells, did not exhibit such calcium changes following stimulation with lipopolysaccharide. Another consequence of catfish B cell mIgM cross linking was the rapid induction of intracellular protein phosphorylation. A number of proteins were phosphorylated on tyrosine residues within minutes after anti-Ig stimulation, indicating the activation of protein tyrosine kinases similar to the situation observed in mammalian B cells. These early intracellular activation events suggest that fish B cells, like mammalian B cells, employ a conserved signal transduction system upon mIgM ligation. This ability to transduce activation signals, coupled with the fact that catfish mIgM have a very short cytoplasmic tail, implies that catfish mIgM is probably associated with accessory molecules required for signal transduction. In this regard, several of the tyrosine phosphorylated catfish proteins exhibited relative molecular weights similar to the mammalian Ig-alpha and Ig-beta/gamma accessory molecules, and may represent candidates for the putative catfish mIgM accessory molecules.

Phylogeny of lymphocyte heterogeneity. I. Membrane immunoglobulins of teleost lymphocytes.

Immunofluorescence studies of bream lymphoid tissues revealed that our 90% of the lymphocytes from blood, anterior kidney, spleen and thymus exhibited membrane immunoglobulin determinants. Furthermore, a majority of such cells were observed to undergo patching and capping when the membrane proteins were complexed with antisera to fish serum immunoglobulins. Lactoperoxidase catalysed radioiodination, detergent lysis and immunoprecipitation with appropriate antisera were employed to study the properties of this membrane immunoglobulin. Quantification, by inhibition of immunoprecipitation with serum immunoglobulin, indicated the average amount of immunoglobulin determinants for bream lymphocytes from either blood, anterior kidney, spleen or thymus to be about half that present on mouse B cells. Physiochemical characterization of labeled membrane immunoglobulin from bream lymphocytes suggested that only one class of immunoglobulin heavy chain was present and that about one-half of this material resembled the monomeric (2H-2L chain) IgM-like proteins present in bream serum. A major unanswered question raised by this study was whether or not certain of the bream membrane immunoglobulin determinants were associated with molecules that did not resemble "classical" immunoglobulins.

Effects of oleic acid on murine CD4+ T cell death and anti-CD3 or superantigen induced proliferation at low temperature.

Studies were conducted to examine the effects of low, yet physiologically relevant, temperatures on murine T lymphocyte responses. Akin to ectothermic vertebrate responses, lectin-induced murine T cell proliferation was previously shown to be ablated at temperatures 10 degrees C below optimal (i.e. 27 degrees C); responsiveness at 27 degrees C was restored by the addition of oleic acid (18:1). The aim of the present study was to address the mechanism involved in such low temperature suppression. Murine splenic CD4+ T lymphocytes stimulated with either alpha CD3 or SEB exhibited cell death, as opposed to anergy, at 27 degrees C. However proliferation was observed in the presence of 18:1. Thus low temperature-suppression of murine CD4+ T cells is also mediated through TCR and/or CD3 pathways. Additional studies examining the temporal effects of adding 18:1, as well as temperature shifts, indicated that the cell death induced by stimulation at low temperature was preventable by 18:1.

Temperature-mediated processes in teleost immunity: differential effects of in vitro and in vivo temperatures on mitogenic responses of channel catfish lymphocytes.

The in vitro mitogenic responses of channel catfish peripheral blood leucocytes to ConA and LPS were differentially affected by both in vitro and in vivo temperatures. The magnitude of the response to LPS was relatively independent of both in vitro culture temperature and in vivo acclimation temperature. The magnitude of the response to ConA was suppressed at lower in vitro temperatures although this suppression could be reduced by lower in vivo acclimation temperatures. In vitro temperature-shift experiments indicated that channel catfish PBL could respond to ConA at a lower in vitro temperature if first stimulated with ConA at a higher in vitro temperature. The converse, however was not true in that channel catfish PBL did not respond at a higher in vitro temperature after an initial stimulation with ConA at a lower in vitro temperature. This latter failure to respond could not be attributed to the induction of a suppressor cell (or factor) by exposure to ConA at a lower temperature. These studies, when coupled with other available data on channel catfish PBL subpopulations, are interpreted as supporting the hypothesis that low temperature immunosuppression in fish may result from preferential inhibitory effects on T cells rather than B cells.

Pristane-induced effects on cytochrome P-4501A, ornithine decarboxylase and putrescine in rats.

The effects of pristane (2,6,10,14-tetramethylpentadecane) on cytochrome P-4501A (cP4501A) activity in microsomes, as well as on ornithine decarboxylase (ODC) activity and concomitant putrescine levels were examined in Copenhagen rats. In general, pristane treatment led to increased cP4501A levels when compared to basal levels, while co-treatment with 3-methylcholanthrene (3-MC) and pristane elicited augmented cP4501A responses when compared to responses induced by 3-MC alone. Increases in both ODC activity and putrescine levels were also observed in pristane treated rats. Collectively, these results indicate that pristane influences cP4501A activity and elicits promoter-like responses as reflected in elevated ODC activity and increased amount of putrescine.

Automated immunofluorescent speciation of oral bacteria using flow cytometry.

Mixtures containing two bacterial species were analyzed using flow cytometric techniques. Light scattering characteristics of Streptococcus mutans and Actinomyces viscosus show unique profiles for pure cultures. However, the light scatter analysis of a mixture containing these two species demonstrates overlapping near the origin. Thus, light scatter analysis was not sufficient to speciate bacteria with different morphologies. Labeling of samples with species-specific immunofluorescent antibodies permitted speciation of mixtures. As the percentage of the bacterium to which the antibody is directed increased in a two-component mixture, fluorescent flow cytometric analysis showed a corresponding increase in the percentage of cells displaying fluorescent labeling. These methods could permit the rapid identification of bacteria from oral sites without culturing.

Pristane induced gene activation.

Studies were performed to examine the effects of 2,6,10,14-tetramethyl pentadecane (pristane) versus 12-O-tetradecanoylphorbol 13-acetate (TPA) on the activation of the CAT gene under the regulatory control of viral promoter/enhancer elements transfected into NIH-3T3, CV-1 and COS-7 cells. The results of these studies demonstrated that (1) pristane or TPA induced trans-activation of SV2cat, HIVcat, RSVcat and MMTVcat in cells transfected with each respective plasmid construct, (2) only pristane induced activation of pA10cat and pOSP/11 and (3) neither TPA nor pristane trans-activated pSV0cat. Furthermore, treatment with either pristane or TPA elicited changes in the morphology of each of the cell lines. Collectively these results indicate that pristane is a potent inducer of gene expression and exhibits similar characteristics as the tumor promoter, TPA.

Conformational changes in the DNA of hybridoma cells from pristane treated mice.

The effects of pristane on the DNA of hybridoma cells propagated as ascitic tumors in pristane-primed BALB/c mice were determined using flow cytometric analyses. Hybridoma cells maintained in vitro or cell isolates from solid tumors which developed in unprimed mice injected with hybridoma cells exhibited similar propidium iodide (PI) staining characteristics. In contrast, PI stained cells isolated from ascites which developed in pristane-primed mice injected with the hybridoma cells displayed significant decreases in fluorescence intensity. Diphenylamine studies and analyses of pH 10 treated cells indicated that the actual DNA content of the hybridoma cells was not altered by exposure to pristane. Furthermore, the altered staining characteristics of the ascitic tumor cells were reversible in that the fluorescence intensity after serial in vitro passage of the ascites cells was similar to that of the parent cell line which had not been exposed to pristane. In addition, there was a direct correlation between the altered PI staining characteristics and the presence of cell-associated pristane as determined by gas-liquid chromatography analyses of cell extracts. Collectively these results suggest that pristane may have a direct effect on the DNA conformation of hybridoma cells which may in turn enhance their growth as ascitic tumors. The possible role of such an altered DNA conformation in hybridoma cells on the in vivo development of ascites is discussed.

Identification of Mycoplasma gallisepticum and M. synoviae by flow cytometry.

Immunofluorescence and flow cytometric methods were examined to detect and distinguish Mycoplasma gallisepticum and M. synoviae. The procedure employed 24-hr broth cultures of each organism, direct immunofluorescence staining with either homologous or heterologous antiserum, and analyses by flow cytometry. The organisms were distinguishable on the basis of fluorescent profiles when stained with the appropriate antiserum.

Assessment of the lymphocyte response to silicone.

The biocompatibility of silicone is once again the focus of increased interest. Long considered inert, silicone has now been reported to be responsible for macrophage inhibition in rats and to possibly cause adjuvant disease in humans, and the related compound silica has elicited an antibody response in mice. The present study evaluates lymphocytic response to silicone as expressed by the demonstration of immunologic memory, or changes in specific lymphocyte subpopulations. Thirty-six female Lewis rats (250 gm body weight) were used as test animals. Group 1 (n = 12) was injected subcutaneously with 2.5 ml Freund's Complete Adjuvant (FCA) alone. Group 2 (n = 12) was injected with 2.5 ml FCA sonicated with silicone gel. Group 3 (n = 6) was injected with 2.5 ml FCA, and at 4 weeks, gel-filled silicone implants were placed subcutaneously. Group 4 (n = 6) was injected with 2.5 ml FCA sonicated with silicone gel, and gel-filled silicone implants were placed at 4 weeks. An additional group of six rats (group 5) served as control for the experimental animals, and a group of four rats (group 6) served as naive control. Groups 1 and 2 were sacrificed at 4 weeks, and splenic lymphocytes were obtained for lymphocyte transformation assays performed against silicone. Assays also were run with the addition of the known mitogens Con A, PHA, LPS, and pokeweed. Cytofluorographic analysis of pan-T, T-helper, T-suppressor, and B-cell populations was performed. Groups 3, 4, 5, and 6 were harvested at 8 months, and splenic lymphocytes were subjected to lymphocyte transformation assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the Peyer's patch in carcinogenesis. I. The adsorption from the gut and retention of 3-methylcholanthrene by Peyer's patches.

Radiotracer methods were used to determine the distribution of 3-methylcholanthrene (3-MC) within the lymphoid organs of rats following i.g. intubation, i.l. injection into the small intestine, i.v. injection or direct injection of the Peyer's patches with 3-[6-14C]methylcholanthrene (14C-MC). The data indicate that the gut-associated Peyer's patches and mesenteric lymph nodes were exposed to higher amounts of orally administered 14C-MC than any of the other lymphoid organs. Whereas the Peyer's patches exhibited the highest sp. act. for longer periods of time when low amounts of 14C-MC were administered, the sp. act. of the mesenteric lymph node were greater when rats were intubated with higher amounts of 14C-MC. Furthermore, the Peyer's patches were exposed to higher amounts of possible metabolites of 14C-MC. Injection of 14C-MC into the small intestinal lumen resulted in increased ratios of the Peyer's patch sp. act. to mesenteric lymph node sp. act., indicating that by-passing the stomach altered the distribution patterns. Data from rats injected i.v. with 14C-MC demonstrated that mesenteric lymph nodes but not Peyer's patches adsorbed and retained 14C-MC from the blood and indicated that the 14C-MC associated with Peyer's patches of i.g. treated rats was adsorbed from the gut rather than from the blood. Results obtained from rats which were exposed to 3-MC by directly injecting Peyer's patches with 14C-MC also indicated that the Peyer's patches were able to retain 3-MC once localized within this lymphoid organ, to metabolize the 3-MC and to possibly excrete the polycyclic aromatic hydrocarbon into the small intestine. Collectively the data indicate that Peyer's patches have an important role in the adsorption from the gut and subsequent retention of 3-MC and hence may be a likely target organ for lymphoid carcinogenesis following oral exposure to carcinogenic polycyclic aromatic hydrocarbons.

Pristane induced effects on chromatin of rat lymphoid cells.

The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane-treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane-treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane-induced changes in the DNA staining characteristics of lymphocytes demonstrated that 1) there were no decreases in the amount of DNA present in the nuclei, 2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and 3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear-associated histone proteins.