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.

Induction of target cell apoptosis by channel catfish cytotoxic cells.

This study examines cytotoxic mechanisms used by channel catfish peripheral blood-derived effector cells. Transmission electron microscopy (TEM), coupled with [(3)H]thymidine DNA fragmentation (JAM) and terminal deoxynucleotidyl nick-end labeling (TUNEL) assays, provided the first evidence that catfish peripheral blood cytotoxic effectors killed allogeneic targets via an apoptotic pathway. TEM demonstrated that the effector cell population present within peripheral blood leukocytes (PBLs) was composed of agranular lymphocytes that formed conjugates with, and induced apoptosis in, allogeneic target cells. Both JAM and TUNEL assays showed that PBLs induced target cell DNA fragmentation within 1 h of coculture. In addition, fixed effectors did not induce target cell necrosis or apoptosis, and target cell lysis was completely inhibited by chelation of free Ca(2+) by EGTA. These results suggest that catfish peripheral blood-derived effector cells utilize a secretory mechanism rather than a ligand-based mechanism to trigger apoptosis.

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.

The immune microenvironment of human fracture/soft-tissue hematomas and its relationship to systemic immunity.

The immune environment of human soft-tissue injury is unstudied. We studied fracture soft-tissue hematomas (FxSTH) in 56 patients with high-energy bony fractures. FxSTH serum and mononuclear cells (MNC) as well as fracture patient plasma and blood MNC were studied. Twenty healthy controls donated plasma and MNC. Soluble tumor necrosis factor (TNF)-alpha, interleukin (IL-1 beta, IL-2, 6, 8, 10, 12, and interferon-gamma were studied by enzyme linked immunosorbent assay. Cells were studied by flow cytometry after cell-membrane stains for CD-14, TNF-alpha (mTNF), and human leukocyte antigen-DR, or intracellular stains for TNF (icTNF) and IL-10. Thirty-six patients with Injury Severity Score < 15 were analyzed further to evaluate the effects of isolated fracture on systemic immunity. Cytokines were rarely detectable in control plasma. TNF-alpha, IL-1 beta, IL-2, and interferon-gamma were rarely found in FxSTH serum or fracture patient plasma. All FxSTH sera were rich in IL-6, peaking before 48 hours (12,538 +/- 4,153 vs. 3,494 +/- 909 pg/mL, p = 0.02, U test). In Injury Severity Score < 15, IL-6 was not detectable in most early fracture patient plasma, but rose after 48 hours (p = 0.028). FxSTH serum IL-8 peaked after 48 hours (440 +/- 289 vs. 4,542 +/- 1,219 pg/mL, p = 0.006) and circulating IL-8 appeared after 72 hours. IL-6 and IL-8 showed gradients from FxSTH serum to paired PtS (p < 0.05, Wilcoxon). IL-10 was abundant (884 +/- 229 pg/mL) in FxSTH serum < 24 hours old. FxSTH serum IL-12 peaked late (3,323 +/- 799 pg/mL, day 4-7) then fell (p < 0.001, analysis of variance). Only IL-12 was higher in fracture patient plasma (1,279 +/- 602 pg/mL) than FxSTH serum (591 +/- 327 pg/mL) during the first 48 hours (p = 0.032, U test). On flow cytometry, control monocytes expressed 201 +/- 31 mTNF sites/cell, but icTNF was absent. mTNF was up-regulated after injury more in FxSTH monocytes (3,202 +/- 870 sites/cell) than peripheral blood monocytes (584 +/- 186 sites/cell) (p < 0.05 vs. peripheral blood monocytes by Wilcoxon, p < 0.001 vs. control monocytes by U test). Intracellular IL-10 was abundant in all MNC, but varied widely after injury. Fracture and peripheral blood monocytes expressed far less human leukocyte antigen-DR than control monocytes. Fractures create an inflammatory local environment. Proximal mediators are cell-associated and relatively confined to the wound, but soluble IL-6, IL-8, and IL-10 are abundant and probably exported. Systemic MNC have complex responses to local injuries. These may reflect the combined impact of multiple soluble cytokines initially generated within the wound. FxSTH appear to be a potentially important source of immunomodulatory cytokines in trauma.

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.

Induction of recurring myositis ossificans by intrathymic injection of 3-methylcholanthrene-treated cells in rats.

Studies were performed to ascertain the effects of transplantation of thymic cells exposed in vivo to 3-methylcholanthrene (3-MC) on the induction of malignancies in Copenhagen rats. Three recipient rats unexpectedly developed tumors which bore histological resemblance to myositis ossificans of humans. Specifically, histology revealed areas of peripheral ossification with the appearance of zones of primitive osteoid with a central cellular area. Other areas of the lesions were less well organized into characteristic zones or were more or less heterogeneous. The primary, as well as recurring, lesions appeared in the axilla and were well circumscribed, 24-68 g in weight and 2-7 cm in diameter. Flow cytometric analyses of DNA content indicated that these tumors contained cells with abnormal DNA characteristics as well as proliferating cells. Coupled with the observation that after excision these tumors recurred, the data suggest that these myositis ossificans lesions were malignancies.

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.

Differential effects of TPA and pristane on gene expression and transformation in mouse epidermal cells.

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) or 2,6,10,14-tetramethylpentadecane (pristane) on gene expression and transformation were examined using two clones (P+, TPA transformation sensitive and P-, TPA resistant) of the mouse epidermal cell line JB6. Results from transformation studies indicated pristane was more efficient, i.e., lower concentrations were required to elicit an equivalent response, in transforming the P+, but not the P-, clone of JB6 compared to TPA. Furthermore, results from these studies demonstrated either TPA or pristane was effective in the transactivation of the chloramphenicol acetyltransferase gene under the regulatory control of most viral promoter/enhancer elements transfected into the P+, but not the P-, clone of JB6. However, if a consensus cAMP response element was linked to the simian virus 40 early promoter, pristane activation was observed in both P+ and P- cells. The differential effects of these two compounds suggest that while they have similar characteristics, they may utilize different pathways to elicit their effects.

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.

Cellular pathway(s) of antigen processing and presentation in fish APC: endosomal involvement and cell-free antigen presentation.

Studies were conducted to address further the role(s) of antigen processing and presentation in the induction of immune responses in a phylogenetically lower vertebrate, specifically a teleost, the channel catfish. In particular, studies were aimed at determining the subcellular compartments involved in antigen degradation by channel catfish antigen-presenting cells (APC) as well as ascertaining the reexpression of immunogenic peptides on the surfaces of APC. The results showed that exogenous protein antigens were actively endocytosed by APC as detected by flow cytometry. Use of radiolabeled antigen and subcellular fractionation protocols also showed that antigen localized in endosomes/lysosomes. Furthermore, there was an apparent redistribution of antigen between these organelles and the plasma membrane during the course of antigen pulsing. Functional assays for the induction of in vitro antigen-specific proliferation of immune catfish peripheral blood leukocytes (PBL) showed that membrane preparations from antigen-pulsed autologous APC were highly stimulatory. The magnitude of responses elicited with such membrane preparations was very similar to that of PBL cultures stimulated with native antigen-pulsed and fixed intact APC or prefixed intact APC incubated with a peptide fragment of the nominal antigen. Current data further corroborate our previous findings that steps akin to antigen processing and presentation are clearly important in the induction of immune responses in lower vertebrates like fish, in a manner similar to that seen in mammalian systems. Consequently, it would appear that many immune functions among the diverse taxa of vertebrates are remarkably conserved.

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.

Thymic tumors induced by 3-methylcholanthrene treatment of rat Peyer's patches.

Studies were performed to characterize thymic tumors which were induced after a single injection of 500 microgram or 3-methylcholanthrene (3-MC) into surgically exposed Peyer's patches (PP) of Copenhagen rats. Detailed gross, histological, and morphological analyses revealed thymic tumors differing in size and weight (1 to greater than 8 g) with distorted architecture and infiltration by lymphocytes and epithelial cells in varying proportions. Approximately 25% of the rats with thymic tumors exhibited abnormal spleens, whereas 66% developed low grade leukemias. A majority of the thymic tumors contained cells which exhibited (1) phenotypic markers characteristic of normal thymocytes, (2) abnormal DNA, and (3) increased percentages in S + G2 phases of the cell cycle. Further studies of tumor cell isolates demonstrated an increased frequency of colony formation on soft agar, as well as the ability to elicit thymic tumors upon transplantation. Collectively these studies describe chemically induced thymic lymphomas.

The role of the Peyer's patch in the carcinogenesis of lymphoid cells.

The dose response to 3-methylcholanthrene (3-MC), the promoter effects of 2,6,10,14-tetramethylpentadecane (pristane) and the target-organ specificity in the preferential induction of B-lymphoid malignancies versus thymic tumors were examined. Lymphoid malignancies were induced in approximately 30% of the Copenhagen rats treated with injections in Peyer's patches (PP) of low, intermediate or high doses of 3-MC. A low dose of 3-MC induced B-lymphocytic leukemias or B lymphomas, whereas thymic tumors were detected in rats treated with high doses. Co-treatment of rats with pristane and 3-MC resulted in increased incidences and decreased latency of the lymphoid malignancies observed, suggesting that pristane acts as a tumor promoter. To address the possible role of PP in the induction events, PP were surgically removed after 3-MC treatment and the remaining small intestine anastomosed. Thymic tumors, but no B-lymphoid malignancies, were observed, indicating that the PP environment was important in the induction of the B-lymphoid malignancies. Radiotracer studies also revealed that appreciable amounts of 3-MC were disseminated to the thymus within 24 hr after treatment of PP with a high dose of 3-MC. Furthermore, direct intrathymic injection of the thymus with 3-MC resulted in the development of thymic tumors only. These results support the hypothesis that the PP has an important role in early events in the carcinogenesis of B lymphocytes and in the dissemination of 3-MC to the thymus.

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)

Pristane induced changes in rat lymphocyte membrane fluidity.

The ability of pristane (2,6,10,14-tetramethylpentadecane) to act as a membrane perturbant was examined. Data obtained from rats treated with pristane by either intraperitoneal injection or the diet indicated there were significant increases over normal in the amount of pristane in lymphoid cells; 50-89% was incorporated into the plasma membranes. Fluorescence polarization analyses, using 1,6-diphenyl-1,3,5-hexatriene, of normal plasma membrane isolates demonstrated that splenic and Peyer's patch lymphocytic membranes were more viscous than those of the thymus, mesenteric lymph nodes or peripheral blood. Studies to assess the effects of pristane on membrane viscosity demonstrated that there were significant differences in the viscosities of plasma membrane isolates from lymphocytes of normal versus pristane treated rats. The observed changes were dependent on route of administration, length of exposure and the lymphoid organ examined.

Dietary effects of pristane on rat lymphoid tissues.

Studies were conducted to assess the normal tissue-associated levels of pristane (2,6,10,14,-tetramethylpentadecane) in Copenhagen rats during ontogeny and adult life and to address whether or not dietary pristane can be adsorbed from the gut and disseminated throughout the body. During the course of this study the possible effects of dietary pristane on chromatin conformation of lymphoid cells were also examined by flow cytometry. The data indicated that 1) pristane crossed the placenta and accumulated in fetal tissues, 2) neonates were exposed to pristane via the colostrum, 3) there were significant increases in the amount of tissue-associated pristane in young adults and subsequent redistribution of the pristane to the muscle and adipose tissues in older rats and 4) after dietary exposure, significantly elevated levels of pristane were associated with the tissues and concomitant changes in chromatin conformation were observed. Collectively, these results suggest that pristane was adsorbed from dietary sources, disseminated to the tissues and exerted a transient, yet marked effect on chromatin of lymphoid cells in rats.

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.

Pulse profile analyses of endocytosis in capped B lymphocytes and BCL1 cells.

The effect of temperature on the kinetics of endocytosis by B lymphocytes and BCL1 cells was examined by using flow cytometry. Mouse B cells were stained with FITC-R alpha MIg and induced to undergo cap formation in the temperature-controlled sample compartment of the flow cytometer. Capping and subsequent endocytosis were measured by continually monitoring the pulse profile descriptors (width and area) of the electronic signal curves generated during flow cytometric analyses. Decreases in area values which immediately followed cap formation were shown to result from a pH-dependent quenching of fluorescence emission as the internalized FITC-R alpha MIg entered acidic subcellular compartments. Similar results were obtained with BCL1 cells, but, in addition to cap formation and acidification, cytoplasmic diffusion of the fluorescent ligand could also be discerned by flow cytometry. With either cell type the rates of cap formation and endocytosis were shown to be temperature dependent with temperature coefficient (Q10) values of 2.0-2.7. Based upon Arrhenius plots of width and area changes, activation energies for capping and endocytosis ranged from approximately 12-18 kcal/mol.

Temperature mediated processes in teleost immunity: differential abilities of channel catfish T and B lymphocytes to cap membrane antigen.

1. The effect of both in vivo acclimation temperature and in vitro assay temperatures on channel catfish T and B lymphocyte membrane antigen (mAg) capping were investigated to determine if capping might be the temperature sensitive step involved in the low temperature immunosuppression of channel catfish T cell responses. 2. Flow cytometry was used to monitor the kinetics of capping induced by a mouse monoclonal antibody (mAb 11G3) specific for a common antigenic determinant present on channel catfish T and B cells. Results indicated that the kinetics of mAg capping were dependent on in vitro assay and in vivo acclimation temperatures and the length of time of in vivo acclimation. 3. T cells from fish appropriately acclimated to 27 degrees C cap mAg more efficiently at low assay temperatures than do B cells. 4. Activation energies were 32 and 47 kcal/mol for B and T cells, respectively, from fish acclimated to 17 degrees C for 3 weeks, but were significantly lower (14 and 22 kcal/mol, respectively) after acclimation for 5 weeks. 5. In summary, it appears that after appropriate in vivo acclimation, channel catfish T cells are better able to cap mAg at low assay temperatures than are B cells. These results suggest that mAg capping is not the low temperature sensitive step involved in T cell immunosuppression in channel catfish.

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.