Induction of IL-2 and lymphokine activated killer cells in the cat

We have described the use of a cloned murine IL-2-dependent T-cell line to directly measure feline IL-2. Concanavalin A stimulated feline peripheral blood lymphocytes produced an IL-2-rich supernatant that supported the growth of this murine IL-2-dependent T-cell line. In addition to producing IL-2, Con A stimulated killer cells in PBL were cytotoxic for the FeLV transformed tumor cell line FL74. Incubating feline PBL with a cocktail of the calcium ionophore A23187 and phorbol ester also led to the generation of cytotoxic cells as well as the production of high levels of IL-2. Finally, IL-2-rich supernatant was able to stimulate cytotoxic activity in PBL from normal cats.     

Bovine interleukin 2: biochemical and biological characterization

Interleukin 2 (IL-2), secreted by bovine peripheral blood mononuclear cells (PBL) on stimulation with concanavalin A (Con A), was purified and characterized by different chromatographic and electrophoretic techniques. The ability of IL-2 to support proliferation of Con A-stimulated bovine lymphoblasts was used to assay and quantitate IL-2 activity. Bovine IL-2 having an apparent MW of 27,000 eluted from a gel-filtration column; from an anion exchange column peak activity was detected at 190 mM NaCl. Binding of bovine IL-2 to phenyl-Sepharose gel and elution with 35-60% ethanediol indicated its hydrophobic nature. Studies on cross-species reactivity revealed that both buffalo and goat lymphocytes respond to cattle IL-2 and detected 35% of activity from a standard cattle IL-2 preparation. Sheep lymphocyte response to cattle IL-2 was negligible.     

Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens

The impact of live and killed Salmonella vaccines on cell-mediated immunity (CMI) was investigated in 18- and 32-week-old White Leghorn chickens, by assessing splenic lymphocyte proliferation, expression of IL-2 mRNA in concanavalin A (Con A) stimulated cells and flow cytometric analysis of cell subpopulations. Con A and Salmonella enteritidis (SE) flagella induced proliferation of splenocytes were enhanced in the 18- and 32-week-old chickens treated with live vaccine, compared to the corresponding control chickens. Among the killed vaccine treated birds, Con A-mediated response was higher in the 18-week-old chickens compared to the corresponding control birds. Increased proliferation was accompanied by increased CD4 and reduced CD8 and gammadelta T-lymphocytes in the 18-week-old live vaccine treated chickens. Relative expression of IL-2 mRNA in Con A-stimulated splenocytes from 18-week-old birds was not affected by vaccine treatment. Overall, live vaccine was more effective in increasing the lymphocyte proliferation to Con A as well as SE antigen. This enhanced CMI may prove beneficial in protecting chickens against SE infection.     

Characterization of surface interleukin-2 receptor expression on gated populations of peripheral blood mononuclear cells from manatees, Trichechus manatus latirostris

An in vitro system to determine surface interleukin-2 receptor (IL-2R) expression on mitogen-stimulated peripheral blood mononuclear cells (PBMC) from free-ranging manatees, Trichechus manatus latirostris was developed. Human recombinant IL-2, conjugated with a fluorescein dye was used in conjunction with flow cytometric analysis to determine changes in surface expression of IL-2R at sequential times over a 48-h period of in vitro stimulation. Surface expression of IL-2R was detected on manatee PBMC, which also cross-reacted with an anti-feline pan T-cell marker. An expression index (EI) was calculated by comparing mitogen-activated and non-activated PBMC. Based on side- and forward-scatter properties, flow cytometric analysis showed an increase in the number of larger, more granular "lymphoblasts" following concanavalin A (Con A) stimulation. The appearance of lymphoblasts was correlated with an increase in their surface expression of IL-2 receptors. Surface IL-2R expression, in Con A-stimulated PBMC, was detected at 16 h, peaked at 24-36 h, and began to decrease by 48 h. Characterization of the IL-2R expression should provide additional information on the health status of manatees, and the effect of their sub lethal exposure to brevetoxin.     

Differential cytokine gene expression in CD4+ and CD8+ T cell subsets of calves

The distinct patterns of cytokine expression in CD4+ and CD8+ T cells are well understood in mice and humans. However, little information is available about cytokine expression in bovine CD4+ and CD8+ T cells. In this study, mRNA expression of 19 different cytokines was analyzed in CD4+ and CD8+ T cells of calves with or without Concanavalin A (Con A) stimulation. CD4+ and CD8+ T cell populations were enriched to 98% purity by positive selection using magnetic cell sorting (MACS). CD4+ T cells spontaneously expressed the mRNAs of interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-6, IL-7, IL-8, IL-10, IL-18, IFN-gamma, TNF-alpha, TNF-beta and TGF-beta, and augmented the mRNA expression of IL-10, IFN-gamma and TNF-beta after Con A stimulation. The mRNAs of IL-3, IL-4, IL-5, IL-13 and GM-CSF were newly expressed in Con A-stimulated CD4+ T cells. CD8+ T cells displayed spontaneous mRNA expression of IL-6, IL-18, TNF-alpha, TNF-beta and TGF-beta, and newly expressed the mRNA of IL-2, IL-7, interferon-gamma (IFN-gamma) and GM-CSF after Con A stimulation. It was found that CD4+ T cells expressed the mRNA of 17 cytokines except for IL-12 and IL-15, while CD8+ T cells expressed only the mRNA of 9 cytokines after Con A stimulation. The profile of cytokine mRNA expression was substantially different in the CD4+ and CD8+ T cells of calves, indicating that CD4+ T cells can be distinguished from CD8+ T cells by the cytokine gene expression of IL-1alpha, IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-10 and IL-13. Differential cytokine expression between CD4+ and CD8+ T cells serve to interpret an individual function of T cell subsets in the immune system of calves.     

Characterization of a monoclonal antibody identifying a CD45RA antigen on feline leukocytes

The antibody produced by a murine hybridoma obtained from the fusion of SP2/0 plasmacytoma cells with splenocytes of a mouse immunized with feline bone marrow was found to react with 60% of bone marrow cells and 80% of peripheral blood leukocytes (PBL); reactivity in the latter tissue was restricted almost entirely to mononuclear cells. Two-color FACScan analyses of this antibody with mAbs specific for feline lymphocytes revealed positive and negative populations of CD4 and CD8 cells. The reactivity for CD4 and CD8 cells was animal age dependent, binding to a higher percentage of the cells in young (2-9 months) versus older animals (> 4 years). In a mitogen driven assay for IgG production by PBL the addition of this antibody to the cultures enhanced the suppressor activity of CD8 cells, a function attributed to activation of a CD4 suppressor-inducer population; removal of CD8 cells negated any induction of suppression. Mild papain digestion of bone marrow and PBL completely removed the antigen detected by this antibody while not affecting reactivity of a pan-T antibody. Western blot analysis showed binding of the antibody to polypeptides of approximately 200 kDa on feline bone marrow and PBL. The data suggest that this mAb is identifying the feline homologue of the leukocyte common antigen of cells with a functional specificity characteristic of a CD45RA isoform.     

Altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus.

Expression of CD4, CD8, IL-2 receptor alpha chain (IL-2R alpha), and MHC class II (MHC-II) on peripheral blood mononuclear cells were examined in cats infected with feline immunodeficiency virus (FIV). CD4/CD8 T cell ratio in FIV-infected cats was slightly decreased, as compared with that in specific-pathogen-free (SPF) cats. However, there was no statistical differences between them. The number of circulating IL-2R alpha+ cells in FIV-infected cats was higher than that in healthy cats, whereas induction of IL-2R alpha expression by concanavalin A (Con A) stimulation was depressed in FIV-infected cats. By using two-color cytofluorometry, Con A-induced enhancement of IL-2R alpha expression was found to be reduced in both CD4+ and CD8+ populations in PBMC from FIV-infected cats. The circulating MHC-II+ cells were also increased in FIV-infected cats. Furthermore, the induction of IL-2R alpha expression on PBMC after Con A-stimulation significantly depressed by FIV inoculation in vitro. These results suggest that FIV activates PBMC in vivo via direct and/or indirect mechanisms, leading to the unresponsive state of T cells to further stimuli in vitro.     

HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6

The most common viral disease of cats worldwide is the infection with feline herpesvirus 1 (FeHV-1). This infection may be followed by Herpetic stromal keratitis (HSK), which is supposed to have an immunopathological basis. Experiments using herpes simplex viruses (HSV) in mouse models indicated that HSK may be treated by topical application of the interleukin 10 (IL-10) gene. The objective of this study was the construction of human herpes simplex virus type 1 (HSV-1)-based amplicon vectors expressing feline interleukin genes and delivery of these genes into cells of feline origin. HSV-1-based amplicon vectors encoding either the enhanced green fluorescent protein, the feline IL-6 or the feline IL-10 under control of the HSV-1 immediate-early 4/5 promotor were constructed, packaged into amplicon particles, transduced into feline cells, and tested for RNA synthesis and biological activity. Feline cells were successfully transduced by HSV-1-based amplicon particles and RNA specific for the transgene was detected already at 2h post transduction, with a maximum at 24h. The recombinant feline IL-10 was functionally active as demonstrated by the reduction of both IL-12 p40 and interferon-gamma-mRNA production in Pansorbin stimulated feline peripheral mononuclear cells. Similarly, the recombinant feline IL-6, which was secreted into the supernatant of transduced cells, was able to support the growth of the IL-6-dependent murine B cell hybridoma 7TD1. HSV-1-based amplicon particles are able to transduce cells of feline origin with genes encoding biologically functional feline IL-10 or IL-6. It will be of high interest to study the effects of these tools in vivo.     

Lymphocyte proliferation response during Eimeria tenella infection assessed by a new, reliable, nonradioactive colorimetric assay

The application of a tetrazolium salt, WST-8,2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt to the lymphocyte proliferation assay in the chicken system was evaluated. Proliferation of concanavalin (Con A)-induced splenic lymphocytes and peripheral blood lymphocytes (PBL) was evaluated with WST-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Coefficients of correlation (r) between these two reagents were 0.98 and 0.97 in splenic lymphocytes and PBL, respectively. In general, the sensitivity of the WST-8 assay was significantly higher than that of the MTT assay, and the standard deviations of the WST-8 assay were significantly lower than those of the MTT assay. The WST-8 assay was fast and highly reproducible and provided a good indication of mitogen-induced proliferation of spleen cells induced by Con A. With the use of the WST-8 assay, splenic mitogenic response of chickens infected with Eimeria decreased transiently at 7 days but increased significantly at 10 days after primary infection compared with that of uninfected chickens. Additionally, the measurement of interleukin (IL)-2 production with WST-8 was highly reproducible and showed a significant increase in IL-2 production upon stimulation of Eimeria tenella-immune spleen cells with Con A. After E. tenella infection, splenic IL-2 production increased significantly at 7 days post-primary and at 2 days post-secondary infection. The WST-8 assay is fast, simple, and more reproducible and sensitive than the MTT assay. This study demonstrates the effectiveness of the WST-8 assay to assess cell-mediated immune response of chickens in normal and disease states.     

Persistent upregulation of MHC class II antigen expression on T-lymphocytes from cats experimentally infected with feline immunodeficiency virus.

A significant elevation in the percentage of CD4+ and CD8+ T-lymphocytes expressing major histocompatibility complex (MHC) Class II antigens was observed in the blood of cats shortly after they were experimentally infected with feline immunodeficiency virus (FIV). In addition to an increase in the relative proportion of T-lymphocytes expressing Class II antigens, there was an increase in the density of Class II antigens on the cell surface. These elevations were still evident at the completion of the 5 month study. A second group of cats that had been infected with FIV for almost 5 years, and with either normal or abnormally low levels of CD4+ T-lymphocytes, had similar elevations in MHC II expression, suggesting that such abnormalities are lifelong. Cats with chronic (2 year) feline leukemia virus (FeLV) infection or dual FIV/FeLV infections also showed similar alterations in MHC II expression on CD4+ and CD8+ T-lymphocytes, suggesting that these alterations were not FIV specific. Feline T-lymphocytes expressed more MHC II antigen and interleukin-2 (IL-2) receptor following stimulation in vitro with conconavalin A and IL-2, demonstrating that feline T-lymphocytes respond to activation signals in a manner similar to T-lymphocytes of other species. However, changes in MHC II expression on T-cells of FIV infected cats were not explainable by viral induced T-cell activation alone, because FIV infected cats with elevated MHC II expression did not have coincident elevations in IL-2 receptor expression.     

Effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats

The effect of recombinant human interferon-alpha (rHuIFN-alpha) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. Pre-incubation of isolated feline peripheral blood lymphocytes (PBL) in vitro with either 10(4) or 10(3) International Units (U) of rHuIFN-alpha for 24 h significantly suppressed (P less than 0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Lower doses of IFN (range, 10-10(-3) U/ml) neither suppressed nor enhanced mitogenesis. In the absence of phytomitogens, incubation of PBL with 10(4) - 10(2) U (P less than 0.001) or 10 U (P less than 0.05) of rHuIFN-alpha/ml resulted in a significant decrease in incorporation of [methyl-3H] thymidine into newly synthesized cellular DNA. Cultures of PBL exposed continuously for 4 days to rHuIFN-alpha doses of 10(4) U/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of IFN apparently were independent of any direct cytotoxic effects. To investigate the dose-response effects of rHuIFN-alpha in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose (10(6) U/kg), moderate dose (10(4) U/kg), or a relatively low dose (10(2) U/kg) of rHuIFN-alpha. Cats inoculated with 10(6) U of rHuIFN-alpha/kg had significantly reduced (P = 0.037) blastogenic responses to Con a at 24 h postinoculation compared to preinoculation values; mean PWM responses were also decreased, but this effect was not statistically significant. In contrast, inoculation of cats with either 10(4) or 10(2) U of rHuIFN-alpha/kg significantly enhanced (P = 0.05 or 0.008, respectively) Con A-induced blastogenesis and had no discernible effect on PWM responses. These findings suggest that very high doses of rHuIFN-alpha given parenterally may be associated with suppression of certain T-cell responses in cats; conversely, much lower doses may be immunoenhancing.     

Inhibition of bovine T lymphocyte responses by extracts of the stomach worm Ostertagia ostertagi

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.     

Optimal conditions for in vitro blastogenesis of feline peripheral blood lymphocytes

A microculture technique was developed for the in vitro blastogenesis of feline lymphocytes. Blastogenesis of ficoll-diatriazoate gradient separated mononuclear cell, washed blood and whole blood were compared. In general the whole blood cultures yielded higher stimulation indices (SI) than the washed blood or separated mononuclear cell cultures.The effect of several variables on the stimulation of lymphocyte cultures was examined. A cell concentration of 3 × 10⁵ cells/well and a 1:20 dilution of washed and unwashed whole blood gave optimal stimulation with concanavalin A (Con A). Phytohaemagglutinin-P (PHA-P) did not give significant levels of stimulation. Inactivated fetal calf serum (FCS) at levels of 2.5% (for washed blood) and 5% (for separated mononuclear cell and whole blood) gave highest SI. Supplementation with FCS was preferable to autologous, homologous or horse sera for all cultures. Optimal SI was obtained in all cultures incubated for 3 days and labelled with 1 μCi tritiated thymidine (³H-TdR) for the last 16 hours. The highest SI were in the range of 70 to 105 (18,764 to 42,681 counts per minute (CPM) for separated mononuclear cell culture, 100 to 165 (28,403 to 45,334 CPM) for washed blood culture and 105 to 186 (41,076 to 69,999 CPM) for whole blood culture.     

In vitro effects of prepartum concentrations of oestradiol-17 beta on cell-mediated immunity and phagocytosis by porcine leukocytes

The in vitro effect of oestradiol-17 beta (Oe2) on cell-mediated immunity (CMI) and phagocytosis by leukocytes obtained from ten and eight sows, respectively, was studied. The concentrations of Oe2 used were 300 pmol/l and 3000 pmol/l, to reflect a low and a high prepartum concentration of the hormone. The CMI was measured by lymphocyte proliferation (LP) tests performed on whole blood and purified mononuclear cells (MNC) and by estimation of the production of interleukin-2 (IL-2) in the latter type of culture. In the whole blood LP test, the mitotic response to concanavalin A (Con A) was increased by 29% (P less than or equal to 0.05) when the Oe2 concentration was 300 pmol/l. The higher concentration of Oe2 (3000 pmol/l) increased the amount of IL-2 in supernatants from Con A-stimulated MNC by 17% (P less than or equal to 0.05). The phagocytic function of polymorphonuclear leukocytes was determined in a chemiluminescence assay. In this assay, the maximal rate of phagocytosis was reduced by 12% (P less than or equal to 0.01) and the area under the curve by 10% (P less than or equal to 0.05) at an Oe2 concentration of 3000 pmol/l. The results show that prepartum concentrations of Oe2 can promote CMI in vitro, but weaken phagocytosis by porcine leukocytes.     

Mitogen-induced transformation of sheep and goat peripheral blood lymphocytes in vitro: The effects of varying culture conditions and the choice of an optimum technique

Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H³]-thymidine ([H³]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 10⁵ viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H³]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.     

Development of an in vitro model of feline cartilage degradation

Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1α, or IL-1β. Media samples and digested cartilage explants were analysed for glycosaminoglycan (GAG) and collagen content. The combination of IL-1β and OSM, both at 20 ng/ml, was able to promote GAG release to the greatest extent at 14 days. At 28 days, all groups showed relatively high release of GAG. At 14 days, only IL-1β and OSM in combination were associated with a statistically significant increase in collagen release over and above control tissue. IL-1β dose-response studies showed that an IL-1β dose of 10 ng/ml promotes a statistically significant increase in GAG breakdown when used with OSM, and higher doses of IL-1β did not result in significantly greater response. The model demonstrated both GAG and collagen degradation and will be of use for further understanding of feline cartilage metabolism and for screening of potential structure-modifying agents to be used in cats.     

An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.