Evaluation of elutriation and magnetic microbead purification of canine monocytes

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody. This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.     

Separation and identification of bovine lymphocyte populations

Various methods for separation of lymphocyte populations have been modified and adapted for use in isolating and identifying bovine lymphocytes. Ficoll diatrizoate (F-D) with a specific density of 1.084 was found to be superior to those with densities of 1.080 and 1.077 which were developed originally for the mouse and human mononuclear cells, respectively. F-D with a density of 1.084 attained a lymphocyte (absolute number) recovery rate of 92% whereas those with densities of 1.080 and 1.077 yielded 81% and 71% recovery rate of lymphocytes, respectively. Subsequent separation of T lymphocytes was achieved best by nylon wool column whereas separation of B lymphocytes was attained best by complement-mediated depletion of T lymphocytes with the T lymphocyte specific monoclonal antibody (MAb), BLT-1. The former yielded 95 +/- 3% T lymphocytes with 47 +/- 9% recovery rate, and the latter gave 96 +/- 3% B lymphocytes with 71 +/- 9% recovery rate. In comparison, direct panning of F-D gradient separated mononuclear cells with goat anti-bovine IgG coated plates yielded 80% B lymphocytes with 31% recovery rate and indirect panning of MAb BLT-1 treated F-D gradient-separated mononuclear cells with goat anti-mouse IgG coated plates yielded 89% T lymphocytes with 35% recovery rate.     

Enrichment of T lymphocytes from bovine peripheral blood mononuclear cells using an immuno-affinity depletion technique ("panning")

A simple and efficient method to enrich bovine T lymphocytes from peripheral blood mononuclear cells (PBMC) by immuno-affinity depletion ("panning") has been developed. The PBMC were initially separated by density gradient centrifugation on Histopaque of density 1.077 g/ml. The T lymphocyte subset was then separated from PBMC by depletion of membrane immunoglobulin (Ig) bearing cells which had an affinity for anti-Ig antibodies bound to polystyrene tissue culture flasks. An average of 95% of the nonadherent "panned" cells were identified as T lymphocytes using a label of peanut agglutinin conjugated with fluorescein isothiocyanate (PNA-FITC). Two percent of the PNA negative cells were Ig bearing cells. The average yield was 50% of the original T lymphocytes found in the PBMC population, and the cell viability as assessed by trypan blue exclusion was greater than 95%. The separation took approximately 2 hours, and the total number of T lymphocytes recovered from 40 ml of blood was in the range of 20-40 X 10(6).     

Bovine antibody dependent cell-mediated cytotoxicity (ADCC) effector cells

ADCC effector cells from bovine blood were separated by centrifugation, adherence and rosetting techniques. Each enriched cell population, peripheral blood mononuclear cells (PBM), null lymphocytes, monocytes and neutrophils, was then examined for its capacity to mediate ADCC. Utilizing heterologous sensitizing antisera it was found that monocytes had approximately twice the ADCC activity of null lymphocytes and that neutrophils had essentially no activity. However, when homologous sensitizing antisera were used it was found that neutrophils possessed the greatest activity followed by monocytes and null cells. Results confirm the existence of an ADCC active null lymphocyte in the bovine.     

The effect of different isolation procedures on canine leucocyte populations and on lectin-induced lymphocyte proliferation

Proliferation assays performed on peripheral blood mononuclear cells (PBMC) are commonly used in experimental and clinical immunology. A prerequisite for an in vitro assay is the ability to obtain relatively pure populations of mononuclear cells from whole blood, as contaminating polymorphonuclear cells may affect the proliferation of lymphocytes. Purification of canine leucocytes from whole blood is associated with difficulties in obtaining pure lymphocytes in high yields. The aim of this study was to optimize the lymphocyte purification from canine whole blood in terms of total cell recovery and purity, while not influencing the proliferation capacity of the isolated cells. To acquire optimal isolation of canine lymphocytes several density gradient media of different densities and osmolalities were examined. For optimal phagocyte removal, pre-treatment of whole blood with carbonyl iron/arabic gum and/or adherence to fibrinogen pre-coated polystyrene tissue flasks were examined. Lectin-induced proliferation was used as measurement of cell activity of the obtained cell fractions after the different separation procedures. Canine blood pre-treated with carbonyl iron/arabic gum followed by density gradient centrifugation with medium 'G' (density: 1.079 g/cm(3), osmolality: 256 mOsm) and adherence to pre-coated polystyrene tissue flask obtained the best PBMC cultures with a median lymphocyte purity of 88% and a median yield of recovered lymphocytes of 54%. This culture also resulted in the highest proliferation and subsequently the highest stimulation index upon lectin stimulation.     

叶尔羌高原鳅外周血淋巴细胞的分离

以体质健康的叶尔羌高原鳅为研究对象,分离其外周血淋巴细胞。结果表明,采用浓度为1.085g/mL的淋巴细胞分离液在2000r/min离心35min的情况下,可将叶尔羌高原鳅外周血淋巴细胞有效地分离出来。该研究不仅为叶尔羌高原鳅淋巴细胞功能研究提供了依据,还可为叶尔羌高原鳅的疾病防治、人工养殖提供重要参考。     

Natural killer (NK) activity and interferon (IFN) production by a fraction of spleen and blood lymphocytes in swine

After carbonyl iron treatment and gradient isolation, spleen and blood pig lymphocytes exhibited NK activity and produced IFN after viral induction. Removal of plastic-adherent cells, including the majority of B cells, did not change these activities. The plastic-non-adherent cells were further separated into two subsets of roughly similar size by panning using a monoclonal, anti-T, and anti-null cell antibodies (81 + cells). NK activity and IFN production were found in the 81 - cell fraction. A significantly higher proportion of null lymphocytes from blood and of splenic Fc-gamma receptor-bearing lymphocytes was also found among the 81 - cell fraction as compared to the 81 + fraction, without any change among other subsets. Similar proportions of helper (PT4+), cytotoxic (PT8+) and total T cells (MSA4+) were found among lymphocytes bound to target K562 cells and among the whole lymphocyte population. In contrast, lymphocytes that bound K562 cells demonstrated a striking increase in the proportion of Fc-gamma receptor-positive cells of high affinity. These results show that NK cells and IFN-producing cells are mainly included in the same blood and spleen fraction, and suggest that among 81 - cells only those expressing an Fc-gamma receptor of high affinity are active.     

Secretion of ovine lymphocyte suppressor factor from curetted uterine luminal cells.

The secretion of ovine lymphocyte suppressor factor from jugular vein (JV), uterine vein (UV), and curetted hemopoietic uterine luminal (UL) mononuclear cells was evaluated on d 14 of the cycle, following ovariectomy (OVX) and after 14 d of progesterone injections (OVX + P4, 1 mg/kg BW). Mononuclear cells (predominantly lymphocytes) were harvested by Ficoll-Paque and placed into culture (2.5 x 10(6).mL-1.well-1 in RPMI-1640). Cellular supernatants were obtained at 72 h and volumes (5 to 50 microL) were tested for suppression of phytohemagglutinin (PHA [.08 microgram l)-treated peripheral blood lymphocytes (1.0 x 10(5)). In a concurrent experiment, PHA-treated JV, UV, and UL cells (1.0 x 10(5)) were cultured singly and JV cells (1.0 x 10(5)) were also cocultured with each of 1.0 x 10(5), 5.0 x 10(4), and 2.5 x 10(4) UV and UL cells. The incorporation of thymidine into DNA was quantified at 60 h. For the cellular supernatant experiment, thymidine incorporation was affected by reproductive phase (P less than .036), lymphocyte source (P less than .0001), and phase x source (P less than .004). For UL cells, the degree of suppressor activity follows: d 14 greater than OVX greater than OVX+P4 (P less than .05). The UL supernatant from OVX+P4-treated ewes and supernatants of JV and UV cells, irrespective of reproductive phase, lacked suppressor activity. Sephacryl S-200 chromatography revealed that UL supernatant from d-14 ewes contained a greater than or equal to 248,000 molecular weight suppressor macromolecule. For the cellular coculture experiment, thymidine incorporation was affected by reproductive phase (P less than .05) and lymphocyte source (P less than .0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of bovine lymphocyte subpopulations by a combined bacterial adherence and fluorescent antibody technique

It is known that certain strains of bacteria bind selectively to subpopulations of human peripheral blood lymphocytes. We have developed a technique which used the specificity of bacterial binding concurrently with fluorescent antibody staining methods to identify 5 B-cell and 5 T-cell subpopulations of bovine lymphocytes. In addition, greater than 95% of the peripheral blood lymphocytes could be positively identified as being either T-cells or B cells. Using ethidium bromide-stained bacteria and lymphocytes in combination with fluorescent antibody staining to detect surface immunoglobulins or T-cell antigens, the method provided a simple yet highly specific technique for the enumeration of both B and T cells in 1 preparation of peripheral blood lymphocytes. The use of bacterial rosetting with fluorescent antibody staining was found to be easier and more reliable than the methods currently used to identify bovine B- and T-lymphocyte subpopulations.     

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.     

B and T lymphocytes in the bovine mammary gland: Rosette formation and mitogen response

Characterization of lymphocyte subpopulations in the bovine mammary gland was accomplished using cells obtained from dry secretions. Correlation of cell surface properties with functional capacity was attempted by assaying the ability to form erythrocyte-antibody (EA) rosettes, erythrocyte-antibody-complement (EAC) rosettes, and sheep erythrocyte (E) rosettes and the ability to respond to phytohemagglutinin (PHA), Concanavalin A (Con A), and pokeweed mitogen (PWM) in the lymphocyte stimulation test. Results were compared with those obtained for peripheral blood lymphocytes (PBL) from the same animals. Mammary gland lymphocytes (MGL) formed significantly fewer (p < .01) EA and EAC rosettes, but significantly greater (p < .01) E rosettes compared to PBL. MGL were significantly less responsive (p < .05) to mitogens than were PBL. MGL contained a large proportion of T lymphocytes, which do not respond to T lymphocyte mitogens in culture.     

Enumeration of T and B lymphocytes in bovine leukemia virus-infected cattle, using monoclonal antibodies

Monoclonal antibodies and microfluorimetry were used to determine the absolute number of B and T lymphocytes in the blood of bovine leukemia virus (BLV)-infected cows. The blood lymphocyte populations from BLV-infected cows were significantly higher than those from BLV-negative cows. The increase in the lymphocyte population in 3 BLV-infected nonlymphocytotic cows was attributed to a significant increase in the number of T lymphocytes; in 3 BLV-infected persistently lymphocytotic cows, the increase was attributed to a significant increase in the number of B and T lymphocytes. One persistently lymphocytotic cow had a high lymphocyte count, and lymphocytes from this cow contained cells that appeared to stain with markers specific for bovine B and T lymphocytes. We concluded that infection of cattle with the B-cell lymphotropic retrovirus, BLV, not only affected B cells, but also T cells.     

Subpopulations of bovine lymphocytes separated by rosetting techniques

A combination of E-rosetting techniques with both neuraminidase- and AET-treated sheep erythrocytes (RBC) was used to enumerate subsets of bovine T lymphocytes. Direct (anti-Ig) rosetting procedures were used to enumerate B lymphocytes bearing surface immunoglobulin (Ig). Approximately 10% of bovine peripheral blood lymphocytes formed rosettes only with neuraminidase-treated RBC; 20% formed rosettes with either neuraminidase- or AET-treated RBC; 30% formed rosettes only with AET-treated RBC; 25% possessed surface Ig, as shown by rosette formation with anti-Ig-coupled RBC; and the remaining 15% lacked both E receptors and surface Ig. These five populations were physically separated by centrifugation on Ficoll-Diatrizoate and recovered for functional analysis. The procedures reported here should be useful for the identification of lymphocyte populations responsible for recognition, effector, and cooperative functions in the bovine immune system.     

Evaluation of the heterophil/lymphocyte ratio as a measure of stress in chickens

The number of lymphocytes in chicken blood samples decreased and the number of heterophils increased in response to stressors and to increasing levels of corticosterone in the chicken feed. The ratio of heterophils to lymphocytes was less variable than the number of heterophil or lymphocyte cells, and the range of values for this ratio was greater than the range of values for heterophils and lymphocytes among control and experimental groups. The heterophil/lymphocyte ratio appears to be a more reliable indicator of levels of corticosterone in the feed and to social stress than were the plasma corticosteroid levels.     

An improved method for the purification of blood cells of turkeys

Methods were developed to purify monocytes, heterophils, or erythrocytes from the whole blood of turkeys. After the bulk of thrombocytes was removed by centrifugation, blood was fractionated over a Ficoll-Hypaque discontinuous gradient. Five fractions were harvested separately, and two were further purified by attachment to a plastic surface and/or by lysis of erythrocytes. The monocyte fraction contained 41% +/- 1% monocytes, and the heterophil fraction contained 96% +/- 1% heterophils. The erythrocyte fraction showed a purity of 99.4% +/- 0.3%. These methods would be useful for various in vitro studies that require purified populations of blood cells.     

Effects of alpha-ketoisocaproate on adrenocorticotropin-induced suppression of lymphocyte function in sheep.

Previous studies of the amino acid analogue, alpha-ketoisocaproate (KIC), indicate that it can stimulate lymphocyte blastogenesis and antibody responses of sheep. To determine whether KIC could overcome the effects of adrenocorticotropic hormone (ACTH)-induced lymphocyte suppression, 24 lambs were fed a control diet, a diet supplemented with 0.05% KIC, or a diet supplemented with 0.05% of the parent amino acid leucine. Immune status was monitored by determining lymphocyte blastogenic responsiveness to phytohemagglutinin-P (PHA), concanavalin A (conA), and pokeweed mitogen (PWM) and percentages of T-cell subsets in the blood, using monoclonal antibodies and a flow cytometer. Serum cortisol, insulin, and glucagon concentrations also were determined. After 60 days of consuming the respective diet, lambs were administered either saline solution or ACTH (100 IU) twice daily for 3 consecutive days. Administration of ACTH increased serum cortisol and insulin concentrations; however, no effects were seen for serum glucagon concentration. Compared with saline administration, ACTH administration significantly (P less than 0.05) suppressed mitogen-stimulated lymphocyte blastogenesis by approximately 50%, regardless of the mitogen used, and significantly (P less than 0.01) decreased the percentage of circulating T lymphocytes and decreased (P less than 0.01) the ratio of T4 to T8 cells. Lambs fed KIC had greater PHA- and conA-stimulated blastogenic responses and significantly (P less than 0.05) increased ratio of T4 to T8 cells in the blood, compared with lambs fed the leucine-supplemented diet or the control diet and given corresponding injections. These data indicate that ACTH decreased in vitro lymphocyte blastogenesis and altered the subset ratios of blood lymphocytes in sheep. These changes were partially prevented by feeding KIC.     

Guinea pig erythrocyte rosette formation as a nonspecific cell surface receptor assay in the cat

The specificity of guinea pig erythrocyte (GPE) rosettes for feline peripheral blood lymphocytes was studied. Of the GPE rosette-positive cells from peripheral blood, 54% were monocytes, 29% were granulocytes, and only 17% were lymphocytes. Results were similar for rosettes incubated at 4 C and those incubated at 37 C. Mononuclear cells separated with polyvinylpyrrolidone-coated silica formed fewer monocyte rosettes (49%) and more granulocyte rosettes (34%) than did cells separated with sodium diatrizoate-Ficoll (60% monocyte rosettes and 18% granulocyte rosettes), whereas the percentage of lymphocyte rosettes was similar for both media. Mononuclear cells suspended in Eagle's minimum essential medium had a higher percentage of monocyte rosettes (75%) and a lower percentage of granulocyte rosettes (12%) than did cells suspended in RPMI 1640 medium (59% monocyte rosettes and 27% granulocyte rosettes). The percentage of lymphocyte rosettes was similar in the 2 media. Two sequential 45-minute plastic adherent cell depletions decreased monocyte rosettes to 51% and increased lymphocyte rosettes to 23% compared with 63% monocyte rosettes and 12% lymphocyte rosettes before adherent cell depletion. The granulocyte rosettes were unchanged by plastic adherent cell depletion. The percentage of rosette-positive cells (9%) was not significantly affected by incubation at 4 C or 37 C, cell separation with polyvinylpyrrolidone-coated silica or lymphocyte separation medium, or suspension in Eagle's minimum essential medium or RPMI 1640 medium. Plastic adherent cell depletion decreased the percentage of rosette-positive cells. Feline thymocytes were 38% to 80% GPE rosette-positive and a feline leukemia virus-infected lymphoblastic cell line (F422) was 88% GPE rosette-positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of sheep and goat lymphocyte proliferation by sheep, goat, and sheep x goat hybrid trophoblast tissue cultures.

Immunosuppressive activity of conditioned medium from cultured ovine, caprine, and hybrid trophoblast tissue was examined. Conceptuses were obtained from naturally mated donor ewes and does at d 20 of gestation and trophoblast tissue was cultured for 24 h in medium supplemented with 15% calf serum and 1% antibiotic/antimycotic. Conditioned medium was added to pokeweed mitogen-stimulated sheep and goat lymphocyte cultures. Quantification of [3H]thymidine uptake by cells was used to measure lymphocyte proliferation. Ovine, caprine, and hybrid conditioned medium effectively suppressed sheep and goat lymphocyte proliferation (P less than .01). There were no differences (P greater than .05) between the immunosuppressive activity of the three tissue types on either sheep or goat lymphocytes. For all treatment groups, sheep lymphocytes were suppressed more than goat lymphocytes (P less than .05). These results indicate that, at d 20 of gestation, sheep x goat hybrid trophoblast tissue is capable of suppressing pokeweed mitogen-stimulated lymphocyte proliferation.     

Distribution of T and B lymphocytes in mammary dry secretions, colostrum and blood of adult dairy cattle

The distribution of lymphocyte subpopulations from dry secretions, colostrum and blood from 10 healthy adult Hostein-Fresian cows was studied using the TH21A and B26A mouse monoclonal antibodies (MAb) to adult bovine B and T lymphocytes, respectively. The mammary gland lymphocytes (MGL) were isolated from composite sample of all four quarters by density centrifugation over discontinuous gradient of ficoll-diatrizoate. The peripheral blood lymphocytes (PBL) were purified using the ficoll-thrombin method. Isolated PBL and MGL were analyzed using the two fluorochromes method (TFM) and laser flow cytometry (LFC). The mean viability of isolated PBL and MGL from dry secretions and colostrum after the TFM and LFC were 92.4% +/- 3.2%, 91.4% +/- 6.0% and 87.1% +/- 6.1%, respectively. There was a good correlation between the two MAbs and the percentage of surface immunoglobulin (SIg) positive cells in the peripheral blood using the TFM. The PBL yielded a mean percentage of 21.2% B cells, 66.4% T cells and 9.4% "Null cells" (TH21A+; SIg-). The TFM on MGL from dry secretions and colostrum indicated two distinct patterns (group I and II) of SIg and reactivity to MAb markers (p less than 0.001). The MGL data included in group I and group II were gathered from both colostral and dry secretions. In comparison to the distribution of lymphocyte subsets within peripheral blood the mean percentages of B cells, T cells and "Null cells" in the mammary gland were respectively, 2.8%, 88.1% and 5.4% for group I and 3.5%, 89.0% and 15.1% for group II. In the mammary secretions, the use of SIg alone was not considered to be a good marker for B cells; in four animals a mean percentage of 15.6% (13.9/89.0 X 100) of the mammary gland T lymphocytes were also SIg+. Of the TH21A+ MGL, only 18.8% were SIg+ in group II compared with 34.1% for MGL from group I and 69.3% for the PBL. Marked differences in cell size distribution and cell surface antigen density were found when PBL and MGL from dry secretions were compared by LFC using the B26A MAb. The results of this study demonstrate a difference in the percentages of peripheral blood and mammary gland B and T lymphocytes and confirm previous findings in which the T lymphocytes were found to represent the major subpopulation of lymphocytes in bovine mammary secretions. This may represent an essential event in the adoptive transfer of cellular immunity through the colostrum in cattle.     

Separation of equine bronchopulmonary lavage cells by density gradient centrifugation and expression of procoagulant activity in unpurified cells and cell subpopulations

Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease. The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses. Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions. Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide. Macrophages were enriched in subpopulations of low density. Neutrophils could not be purified by density gradient centrifugation using either gradient medium. PCAs of cell subpopulations were plotted against their respective macrophage, neutrophil, and lymphocyte content. PCA was positively correlated with macrophage content (P less than 0.001) and negatively correlated with neutrophil (P less than 0.02) and lymphocyte (P less than 0.001) content. Therefore, PCA of equine lung cells most likely originates from macrophages as shown in other species. The density shift of lung neutrophils requires further investigation.