Characterization and separation of bovine lymphocyte populations

Bovine lymphocyte populations were characterized by surface markers, rosette-forming ability and behaviour towards mitogens. After pre-treatment with neuraminidase 16% of the bovine blood lymphocytes and 14% of the bovine spleen cells formed spontaneous (E) rosettes with sheep erythrocytes. About 20% EAC rosette-forming cells were detected among both cell populations. Protein A receptors were detectable among 8% of the blood lymphocytes and 26% of the spleen cells. Bovine lymphocytes responded to pokeweed mitogen (PWM), phytohemagglutinin (PHA) and concanavalin A (Con A). An enrichment of bovine B and T cells was obtained by E-rosette sedimentation (81–84% B cells) and by filtration through nylon fiber columns (51–65% T cells). The T cells obtained after nylon filtration still responded to the mitogens PHA, Con A and PWM. Enriched B-cell populations responded to bacterial lipopolysaccharide (LPS). After monocyte depletion the mitogenic response of blood lymphocytes was not influenced.     

Effect of infections with swine fever virus on immune functions II. Lymphocyte response to mitogens and enumeration of lymphocyte subpopulations

Peripheral blood and spleen lymphocytes from pigs infected with a low-virulent strain of swine fever virus (SFV) were transiently hyporesponsive to the mitogenic action of PHA, PWM and Con A. The mitogenic reactivity of lymphocytes from lymph nodes from such pigs appeared to be enhanced rather than depressed at that time. In addition, hyperresponsiveness of peripheral blood lymphocytes (PBL) to these mitogens occurred in some pigs.PBL from pigs lethally infected with virulent SFV showed a persistent depression of the response to these mitogens, whereas lymphocytes from lymph nodes had a high responding capacity.A lymphocyte response to SFV antigens could not be demonstrated in infected pigs.These SFV infections did not markedly affect the percentage of lymphocytes in the blood and most lymphoid organs rosetting with sheep red blood cells. On the other hand, surface immunoglobulin-bearing lymphocytes were markedly increased in lymph nodes from pigs exposed to virulent SFV. The sum of both lymphocyte subpopulations in the lymph nodes from these pigs often considerably exceeded the 100% value, which strongly suggests the presence of cells bearing both surface immunoglobulin and receptors for dextran-treated sheep red blood cells.Possible correlations between these findings are discussed. The results suggest that infections with SFV induce systemic alterations in the process of lymphocyte recirculation in the pig.     

Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.     

Effect of infections with swine fever virus on immune functions I. Response of lymphocytes from blood and lymphoid organs from infected and normal pigs to anti-immunoglobulin serum and protein A

Pigs exposed to a low-virulent strain of swine fever virus (SFV) developed an inapparent infection. At times when a transient leucopenia occurred, the peripheral blood lymphocytes (PBL) were unresponsive to the mitogenic stimulus of anti-immunoglobulin serum (anti-Ig) and protein A.Pigs lethally infected with a virulent SFV showed leucopenia and unresponsiveness of PBL to anti-Ig and protein A from 2 days post infection until death.This suggests a defect in B lymphocyte function in pigs infected with SFV. The unresponsiveness to anti-Ig appeared not to be caused by a reduced ability of lymphocytes to redistribute their receptors into caps, the presence of suppressor cells or absence of surface immunoglobulin bearing lymphocytes in the peripheral blood. A direct action of the virus itself also seemed unlikely.Lymphocytes from spleen reacted as PBL. However, lymph node cells did not lose their capability to respond to anti-Ig.These data suggest that a change in the migration pattern of anti-Ig responsive lymphocytes could account for the observed unresponsiveness of PBL and spleen lymphocytes to anti-Ig.     

Effect of ammonia on viability and blastogenesis of bovine lymphocytes

The effect of addition of ammonia into the tissue culture on viability and functions of bovine lymphocytes was studied. The concentrations of ammonia in the tissue cultures represented toxic, subtoxic, and normal concentrations of ammonia in the bovine blood during clinical and subclinical urea toxicosis. Lymphocytes separated from peripheral bovine blood were incubated in control medium and test medium with various concentrations of ammonia and/or PHA or Con A. Viability of the lymphocytes was measured by trypan blue exclusion test and their mitogenic reactivity by incorporation of ³H thymidine into DNA of lymphocytes. Approximately 30% bovine lymphocytes were killed by ammonia in medium during 72 hours of incubation. Ammonia also affected the response of lymphocytes to stimulation with PHA or Con A as well as mixed lymphocyte culture reaction. The mitogenic response of lymphocytes was also reduced when lymphocytes were preincubated with ammonia for even 1 hour. The mitogenic response was not restored when the number of lymphocytes preincubated with ammonia was reconstituted to the initial concentration to compensate for the killed lymphocytes before stimulation with PHA. Therefore, addition of ammonia to the culture either killed lymphocytes or permanently impaired their functions.     

A study of porcine lymphocyte populations. II. Characterization of porcine lymphocyte populations

The percentage of E rosette forming cells amounted to 26% of the blood lymphocytes and 34% of the spleen cells in German Landrace pigs. 10% of the live lymphocytes in the peripheral blood and 22% of the spleen cells were EAC rosette forming cells. The number of E rosettes could be increased by treatment of sheep erythrocytes with neuraminidase. The number of lymphoid cells reacting with protein A in the peripheral blood and in the spleen of pigs correlated well with the number of EAC rosette forming cells. The mitogens phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) are potent stimulators of pig lymphoid cells. The mitogenic stimulation of pig lymphocytes could not be influenced significantly by the removal of phagocytic cells. By neuraminidase treatment the mitogen induced stimulation rate was decreased. For the mitogenic stimulation of porcine lymphoid cells in the presence of PHA, Con A and PWM T cells were required. Bacterial lipopolysaccharides (LPS) stimulated only B cells to a small degree.     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

Culture and Stimulation of Tammar Wallaby Lymphocytes

We describe the culture and stimulation of lymphocytes from the model marsupial, the tammar wallaby (Macropus eugenii). We also describe the capacity of tammar wallaby lymphocytes isolated from blood, spleen and lymph nodes to produce soluble immunomodulatory factors. Culture conditions were optimized for mitogen-driven stimulation using the plant lectin phytohaemagglutinin (PHA). Products secreted by stimulated cells were harvested and crudely fractionated before they were added back to freshly isolated lymphocytes. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay, both stimulatory and inhibitory bioactive factors were detected in serum-free supernatants harvested from mitogen-treated peripheral blood mononuclear cells. This paper describes the capacity of leukocytes of the tammar wallaby to respond to mitogenic stimulation and to produce soluble, low-molecular-weight bioactive molecules that possess cytokine-like activity.     

Comparison of analysis of bovine surface immunoglobulin bearing and peanut agglutinin binding lymphocytes by flow cytometry and fluorescence microscopy

Bovine peripheral blood lymphocytes were examined for their binding to anti-immunoglobulin serum, peanut agglutinin, and mu, alpha, and epsilon heavy chain specific antisera by immunofluorescence. The percentage of total lymphocytes with positive staining was determined independently by flow cytometry and fluorescence microscopy. The correlation of data from both methods was best for analysis of total surface immunoglobulin and IgM bearing cells. The percentage of lymphocytes bearing surface immunoglobulin (B cells) was determined using both whole antiserum and a F(ab')2 reagent. Quantitation by flow cytometry did not show a significant difference when the two reagents were used, whereas fluorescence microscopy revealed a significant difference (p less than .05). The mean percent of total surface immunoglobulin bearing cells was 30 +/- 3% by either method. Flow cytometry gave significantly larger values than fluorescence microscopy for samples stained with fluorescein conjugated peanut agglutinin. Peanut agglutinin binding cells comprised 70 +/- 3% by flow cytometry and 51 +/- 3% by fluorescence microscopy. Similarly, there was a significant difference between both methods when IgA bearing lymphocytes were examined. Percentages of immunoglobulin E, A, and M bearing lymphocytes as well as total B and T cells in spleen and bronchial lymph node were determined by immunofluorescence using the cytofluorograph. Peanut agglutinin binding cells were less numerous in spleen and lymph node than in peripheral blood. Immunoglobulin E bearing lymphocytes increased from 0.07% in peripheral blood to 4% in spleen and 1.9% in lymph node. In this paper we demonstrate how flow cytometry can be used to examine a large number of samples in a rapid and reproducible manner. This is the first report in which bovine lymphocytes bearing surface IgE are quantitated.     

Systemic profiles of antigen-specific lymphocytes in animals chronically exposed to staphylococcus antigen in the mammary region.

Systemic profiles of lymphocytes were assessed in goats exposed chronically with Staphylococcus antigens in the supramammary region. Animals were inoculated three times subcutaneously in the right supramammary region with heat-killed Staphylococcus aureus antigen (HKS) at 1 month intervals. Prior to immunization and 1 week following each injection, 3 and 6 day cultures of peripheral blood mononuclear cells were made to determine proliferative responses of lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). Peripheral blood lymphocytes responded significantly to both HKS and PHA in 3 day cultures after the second injection and showed peak responses after the final immunization, suggesting that repeated local injection of S. aureus antigen at the supramammary region, can induce an anamnestic response to the antigen in the peripheral blood of these animals with a concomitant increase in the responsiveness to the polyclonal mitogen, PHA. In contrast, initial antigen challenge induced little, if any, increase in responses to the specific antigen or mitogen when compared to pre-injection states. These data may also suggest that non-reactivity of peripheral blood lymphocytes to the HKS antigen immediately after the primary injection of antigen may be the result of local retention of antigen-reactive cells at the sites of infection.     

Duck lymphocytes. VI. Requirement for phagocytic and adherent cells in lymphocyte transformation.

Preparations of duck (Anas platyrhynchos) spleen and blood lymphocytes depleted of cells capable of phagocytosing carbonyl iron gave lower transformation responses to the mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), lentil lectin (LL) and phorbol ester (PMA) than intact cell preparations. When cell populations were fractionated on the basis of their adherence to plastic, it was found that the adherent cells were responsive to PHA, Con A, BSS, WGA and PMA, while the non-adherent cells responded to LL. These observations confirm the expected requirement for phagocytic accessory cells in the induction of in vitro mitogen-driven duck lymphocyte responses. The responses of plastic-adherent populations of cells to most mitogens are believed to reflect the generally close physical relationship between the adherent accessory cells and the lymphocytes, although it remains possible that duck monocytes respond to some of the mitogens employed. The data also suggest that LL stimulates a population of cells different to those responding to other mitogens.     

Characterization of monoclonal antibodies to feline T lymphocytes and their use in the analysis of lymphocyte tissue distribution in the cat.

We describe the development of three monoclonal antibodies to feline T lymphocytes. Antibody 1.572 stains 93% of feline thymocytes, 49% of lymph node, and 65% of spleen lymphocytes. Two-color analysis shows 1.572 does not stain Ig-bearing cells, and 1.572-positive lymphocytes plus Ig-positive lymphocytes make up approximately 90% of peripheral blood lymphocytes (PBL), suggesting that 1.572 is a pan-T cell marker. The other two monoclonal antibodies, 3.357 and CAT30A, stain a smaller population of thymocytes (59%) of which 40% express both antigens. The 3.357 antigen is found on 23% of lymph node and 47% of spleen lymphocytes, while the CAT30A antigen is found on 29% of lymph node and 19% of spleen lymphocytes. Two-color analysis shows that 3.357 and CAT30A stain mutually exclusive subpopulations of 1.572-positive cells. Using thymocytes as an antigen source, antibody 3.357 precipitated a molecule of 66,000 molecular weight (Mw) under nonreducing conditions and a heterodimer of 32,000 and 34,000 under reducing conditions, suggesting that 3.357 recognizes the feline CD8 homologue. Antibody CAT30A precipitated a molecule of 55,000 Mw under both reducing and nonreducing conditions, which suggests it recognizes the feline CD4 homologue. Analysis of PBL profiles of 35 normal cats using the three monoclonal antibodies indicates that the distribution of feline PBL subpopulations is similar to man, including the CAT30A:3.357 ratio (1.74), which is identical to reported CD4:CD8 ratios in man. Based on these data, the feline CD4 and CD8 homologues are similar to those reported in other species.     

In vitro stimulation of pig lymphocytes after infection and vaccination with Aujeszky's disease virus

Mitogenic and antigenic lymphocyte stimulation were examined in Aujeszky's disease virus (ADV) infected pigs and in pigs vaccinated with modified live ADV. Neither infection nor vaccination had any effect on lymphocyte responsiveness to phytohaemagglutinin (PHA), pokeweed mitogen (PWM) or concavalin A (Con A). ADV antigen-responsive lymphocytes began to appear in the peripheral blood between 7 and 14 days after inoculation and could still be demonstrated in blood and spleen of infected pigs at 174 days after infection. In vaccinated pigs, sensitized peripheral blood lymphocytes could be detected up to at least 35 days after revaccination. Pre-incubation of ADV antigen with specific antibody markedly reduced lymphocyte stimulation. Non-immunized pigs showed no lymphocyte response to ADV antigen. Infected pigs exhibited no lymphocyte reactivity against antigens of non-infected cells.     

Cell surface markers on canine lymphocytes.

Reference values for T and B lymphocytes were determined on lymphocytes from canine thymus, spleen, lymph node, bone marrow, and peripheral blood by use of erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosette assays, plus a direct fluorescent technique for assay of surface immunoglobulins. Numbers of T lymphocytes, indicated by E rosette formation with human erythrocytes, ranged from a low of 1% in the thymus to 13% in the peripheral blood, whereas B-lymphocyte numbers ranged from 3% (thymus) to 41% (bone marrow) and from 6% (thymus) to 36% (bone marrow), as indicated by EAC rosette formation or presence of surface immunoglobulins respectively. Stimulation of peripheral blood lymphocytes with either phytohemagglutinin or concanavalin A increased the total number of E-rosetting cells two to threefold, whereas the number of EAC-rosetting cells decreased by half. Further, the percentage of cells bearing Fc receptors increased after phytohemagglutinin stimulation. These results indicate the E rosette technique can be used to identify and to monitor a population of canine T lymphocytes.     

Effect of corticosteroid on porcine leukocytes: age-related effects of corticosteroid inhibition on porcine lymphocyte responses to mitogens

The effects of prednisolone sodium succinate on the responses of porcine lymphocytes to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were investigated. Sensitivity of peripheral blood lymphocytes (PBL) to the synthetic glucocorticoid, prednisolone, was related to age of the lymphocyte donor. The greatest sensitivity was found in PBL from animals less than one week old; PBL from animals between 2 to 4 months retained some glucocorticoid sensitive cells; whereas, PBL from animals older than 6 months were exceptionally resistant to steroid. Similar age-associated sensitivities were found for lymphocytes from lymph node, spleen and thymus. Significant differential sensitivities among the various lymphoid organs were found with the thymic lymphocyte possessing the greatest sensitivity to steroid and the PBL lymph node and splenic lymphocytes possessing the highest resistance to the suppressive effects of steroid. The age related differences in sensitivity to steroid did not appear to be caused by differences in the number of steroid receptors because lymphocytes from susceptible and resistant animals had similar numbers of receptors. The results suggest that the age related sensitivity may be associated with a higher percentage of sensitive thymic-derived lymphocyte in the PBL, lymph node and spleen of the younger animals. Results of this study also suggest that the adult pig (6 months) should be classified as a steroid resistant species.     

Demonstration of cytotoxic lymphocytes to virus-infected target cells in pigs inoculated with transmissible gastroenteritis virus.

Lymphocytes, cytotoxic to virus-infected target cells, were induced in pigs orally exposed to transmissible gastroenteritis virus. They were studied and experiments were carried out by using autochthonous testicle cells as target cells to avoid genetic incompatibility of effector lymphocytes and target cells. Cytotoxic lymphocytes were demonstrated in Peyer's patches, mesenteric lymph nodes, spleen, and peripheral blood on postinoculation day (PID) 7. Cytotoxic activity of lymphocytes increased thereafter and reached the maximal amount at PID 21. Lymphocyte cytotoxicity was somewhat greater in lymphocytes of peripheral blood and spleen than in those of Peyer's patches and mesenteric lymph nodes after PID 14. On the contrary, lymphocyte reactivity to the viral antigen measured by lymphocyte proliferative assay was higher in Peyer's patch and mesenteric lymph node cells than in peripheral blood and splenic cells. Lymphocyte cytotoxicity was depressed by treating effector cells with anti-porcine thymocyte serum and complement. However, lymphocyte suspensions treated with anti-porcine thymocyte serum and complement were still cytotoxic to some extent against virus-infected target cells, although T lymphocytes were completely excluded by the treatment. This suggests that cytotoxic mechanism other than the direct action of cytotoxic T lymphocytes may be involved in the cytotoxicity assay systems used in the present studies. In experiments in which allogenic cells (testicle cells of siblings) were used together with autochthonous cells as targets, lymphocyte cytotoxicity was equally expressed against both autochthonous and allogenic target cells in 2 of 3 experiments. However, lymphocyte cytotoxicity was greater against autochthonous cells than against allogenic target cells in 1 of 3 experiments.     

Changes in subpopulations of lymphocytes in peripheral blood, and supramammary and prescapular lymph nodes of cows with mastitis and normal cows

Direct immunofluorescence (IF) and indirect IF techniques were employed to analyze the distribution of B and T lymphocyte populations in peripheral blood, and in supramammary (draining), and prescapular (non-draining) lymph nodes of cows with mastitis and normal cows. In the peripheral blood there was a significant decrease in the percent and absolute number of B lymphocytes in mastitic cows (n = 29; 17.1 +/- 10.2%; 3.4 +/- 2.7 X 10(5) cells/ml) as compared to normal cows (n = 38; 25.2 +/- 7.8%; 9.3 +/- 5.4 X 10(5) cells/ml). The percent T lymphocyte count in mastitic cows (71.2 +/- 7.1%) was slightly increased over that of normals (65.8 +/- 7.2%), although the absolute number of T lymphocytes was decreased in mastitic cows (1.49 +/- 0.91 X 10(6) cells/ml vs. 2.47 +/- 1.28 X 10(6) cells/ml). In the prescapular lymph node the percent of B lymphocytes, but not T or "null lymphocytes", decreased significantly in mastitic cows as compared to that of normals. The decrease, i.e. 32%, paralleled the 32.1% decrease found in peripheral blood B lymphocytes. In contrast, in the supramammary lymph node of mastitic cows, the percent B lymphocytes increased over that of normals (35.1 +/- 2.0% vs. 20.4 +/- 9.4%), whereas the percent T lymphocytes decreased to 54.5 +/- 2.8% compared to 70.7 +/- 3.5% in normal cows. There was no significant change in percent "null lymphocytes". The weight of prescapular lymph nodes did not change in mastitic cows when compared to that of normals. As a result, the estimated number of B lymphocytes, but not of T and "null lymphocytes", decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of bovine luteal oxytocin secretion in vitro by a phorbol ester and calcium ionophore

Experiments were conducted to examine the in vitro effects of a phorbol ester and a calcium ionophore on bovine luteal oxytocin (OT) secretion and synthesis and progesterone secretion. Corpora lutea removed from beef heifers on d 8 of an estrous cycle were sliced and incubated for 2 h with .81 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), 1.62 nM TPA or .3 microM calcium ionophore A23187. Both concentrations of TPA increased (P less than .01) OT secretion (ng.g-1.2 h-1; control, 407.1; .81 nM TPA, 494.7; 1.62 nM TPA, 528.1; SE = 21.2). Increased secretion of OT was accompanied by a corresponding increase (P less than .02) in synthesis of the hormone (ng.g-1.2 h-1; control, 368.5; .81 nM TPA, 427.6; 1.62 nM TPA, 492.1; SE = 25.7). Phorbol ester also induced (P less than .025) progesterone secretion (ng.g-1.2 h-1; control, 1,056.2 vs .81 nM TPA, 1,333.3; SE = 86.4). Calcium ionophore increased (P less than .01) OT secretion (ng.g-1.2 h-1; control, 248.9 vs A23187, 327.4; SE = 16) and there was a trend (P = .09) toward increased synthesis of OT in response to the ionophore (control, 124.4 vs A23187, 165.6; SE = 16.4). Because TPA can activate protein kinase C and A23187 increases intracellular calcium, these intracellular constituents probably are involved in promoting secretion of OT and progesterone.     

In vitro evaluation of porcine lymphocyte response to phytohaemagglutinin using a modified “whole blood” technique

The purpose of these investigations was to develop a modified whole blood technique for measuring quantitatively the responsiveness of pig peripheral blood lymphocytes to phytohaemagglutinin (PHA) in vitro. Washed blood cells from a fixed volume of blood were suspended in culture medium supplemented with foetal calf serum and stimulated with a pure mitogenic PHA preparation. The stimulation was measured by the incorporation of ³H-thymidine.Data are presented to show the effect of different variables on the culture system. The transformation response was measured at different levels of PHA and the reproducibility of the different responses from repeated investigations was used to evaluate the usefulness of the test.The best reproducibilities of the stimulation response were found at the high PHA concentrations.Also the calculated PHA concentration giving maximum stimulation response had a relatively high reproducibility and indicates that this value is a convenient and reliable alternative measure of the functional capacity of the lymphocytes.     

Effects of ionizing radiation on in vitro proliferative responses of porcine peripheral blood lymphocytes

The radiosensitivity of $\text{in vitro}$ proliferative responses of porcine peripheral blood lymphocytes (PBL) was assessed. PBL were stimulated by Con-A, PHA, culture supernates from mitogen-stimulated porcine lymphocytes, or in the case of antigen-primed swine, specific antigens. The resulting levels of proliferation were assessed by a determination of the level of incorporation of tritiated thymidine $\text{in vitro}$, and in some cases by the presence of blast cells in the cultures. Porcine PBL were found to be more radioresistant than either mouse PBL or mouse spleen cells. Irradiation levels of greater than 3000 rads were necessary to arrest Con-A or PHA-induced proliferative responses. Proliferation induced by lymphokines in the form of supernates from mitogen-stimulated lymphocyte cultures was arrested in PBL that had received 3000 rads prior to culture. Antigen-induced proliferative responses in primed porcine PBL populations were the most radiosensitive, in that a previous irradiation with 500 rads was sufficient to completely abolish a secondary $\text{in vitro}$ proliferative response.