Sheep Lymphocytes Research Articles

A whole blood method for measuring mitogen-induced transformation of sheep lymphocytes

A simple and reproducible micromethod for determination of in vitro mitogenic responses of sheep peripheral blood lymphocytes is described. The test uses (i) whole blood diluted in RPMI 1640 medium to give a cell count of 0.5 x 106-1 x 106 lymphocytes/ml, (ii) mitogens in the range of 5-20 micrograms of phytohaemagglutinin/ml, 20-80 micrograms of lipopolysaccharide/ml or 20-80 microliters of poke weed mitogen/ml, and (iii) a stimulation time of 42 to 90 h. A considerable variation in mitogenic response was observed both between animals and on different occasions in the same animal. Because of the large periodic variation it was suggested that the test should be repeated using blood drawn at different times in order to determine the mitogenic response of an animal.

In vitro response of lymphocytes of normal and ovine squamous cell carcinoma-bearing sheep to phytomitogens and tumour extracts

A comparison was made of the blastogenic responses of peripheral lymphocytes (BRPL) of normal sheep with those of sheep bearing ovine squamous cell carcinoma (OSCC). In normal tumour-free sheep, BRPL to phytomitogens were found to vary in different age groups, and BRPL to OSCC extracts were found to become significantly elevated in sheep over one year old. In tumour-bearing mature sheep, BRPL to phytomitogens and to OSCC extracts decreased significantly with increasing maturity of tumours. The results are interpreted to show that increasing tumour volume, due to natural growth or enhancement, associates with increasing suppression of cell-mediated responses, and that such suppression may be a cause and a result of increased tumour volume.

Persistent lymphocytosis and virus-like particles in lymphocytes of sheep inoculated with cell-free extracts derived from ovine malignant lymphomas.

White cell counts of sheep inoculated in utero or at birth with cell-free extracts of ovine malignant lymphomas have been monitored for 5 yr. Of 28 inoculated sheep which have survived, 19 have shown persistent lymphocytosis. After 5 yr, no lymphomas had developed in the inoculated sheep. Electron-microscopic examination of short-term cultures of phytohaemaglutinin-stimulated lymphocytes from the sheep with persistent lymphocytosis has revealed virus-like particles in five of nine animals examined but none in similar cultures from 11 uninoculated sheep. The density of particles recovered from these cultures has been determined by centrifugation on caesium chloride isopycnic density as being 1.14--1.145 gm/microliter. These particles are not typical of oncornavirus as reported from sheep and cattle elsewhere and may represent a different type of virus or indeed be non-viral.

Lymph node scanning in sheep with indium-111-labeled lymphocytes

Recirculating autologous lymphocytes of sheep were labeled in vitro with indium-111 and injected intravenously. Gamma camera pictures taken 24h later revealed detectable amounts of radioactivity in lymph nodes, spleen, liver and lungs. Single lymph nodes could be identified after the intravenous injection of as few as 108 lymphocytes labeled with as little as 50 μCi indium-111. The application of this procedure to human studies is discussed.

High Oxygen Pressure Inhibits DNA Synthesis in Mitogen-Activated Lymphocytes

Peripheral sheep lymphocytes, exposed to the mitogenic lectin phytohemagglutinin do not incorporate radiolabelled thymidine in cultures with a gas phase high in oxygen. Cells kept under nitrogen incorporate thymidine to the same extent or better than cells grown under air. The inhibition is proportional to the oxygen content of the gas phase. The inhibition is relieved if oxygen is replaced by air after some time but the onset of DNA synthesis is delayed correspondingly. It is suggested that oxygen may have a regulatory function on the onset of DNA synthesis.

EAC Rosette-Forming Lymphocytes in Commercial Preparations of Sheep Erythrocytes

Abstract In vitro enumeration of human complement (C3) receptor-bearing lymphocytes is routinely performed with sheep erythrocytes (E) sensitized with 19S anti-sheep erythrocyte antibody (A) and fresh mouse complement (C), termed the EAC reagent (1). Sheep whole blood diluted with an equal volume of Alsever's solution is washed with saline and used to prepare the EAC reagent. It is assumed that the residual sheep leukocytes in the solution are rapidly lost upon storage after the blood is collected. However, here we report finding considerable numbers of residual sheep lymphocytes in sheep blood in Alsever's solution up to 30 days after storage at 4°C. In addition, one finds that about 75% of these residual sheep lymphocytes form autorosettes with the EAC reagent. Preparations of whole sheep blood diluted with an equal volume of Alsever's solution were obtained from a commercial source, and samples were assessed for the presence and viability of lymphocytes starting with the day the blood was drawn and continuing for 4 weeks.

Induction of a pH-stable interferon in sheep lymphocytes by Mycoplasmatales virus MVL2.

The data presented in this report show that the enveloped Mycoplasmatales virus MVL2 is capable of interferon induction in sheep peripheral blood lymphocytes. The phenomenon was dose dependent, requiring approximately 10(8) plaque-forming units of virus per 2 X 10(6) lymphocytes. The interferon was stable to pH 2.0 treatment, was produced in moderately high levels (greater than 1,000 units), and met many of the criteria for classification as a type I interferon. Heat-inactivated MLV2 lost its ability to induce interferon, whereas ultraviolet-inactivated virus retained its capacity to induce levels comparable to untreated virus. Whereas the MVL2 used in these studies was contaminated with several host cell proteins as determined by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis, the contaminants probably did not play a role in the induction because isolated cell membranes or soluble cell contents from Achoplasma laidlawii are inactive as inducers. Also presented in this report is a preliminary description of the structural polypeptides of MVL2.

Behaviour of sheep-immunoglobulin-bearing and non-immunoglobulin-bearing lymphocytes isolated by nylon wool columns.

The technique of separating lymphocytes on nylon wool columns has been applied to sheep lymphocytes obtained from efferent lymph. The non-immunoglobulin-bearing (sIg-) lymphocytes which pass through the column were MLR-reactive, responsive only to T cell mitogens, and migrated in vivo like T lymphocytes described in rodents. Immunoglobulin-bearing (sIg+) lymphocytes were MLR-non-reactive, responsive to lipopolysaccharide and migrated in vivo like B lymphocytes in rodents. It is considered that the majority of sIg- lymphocytes obtained in this way are T lymphocytes and the majority of sIg+ lymphocytes are B lymphocytes.

Binding properties of radiolabeled basic protein of myelin: reassessment in relation to diagnostic immunoassays.

The binding of radioiodinated basic protein of myelin ([125I]BPM) to a sheep lymphocyte cell pellet, sheep erythrocytes, and cell-free tubes used in the assay was investigated as a possible diagnostic procedure in multiple sclerosis. [125I]BPM apparently bound to 5 x 10(6) sheep lymphocytes incrementally with no plateau, up to 300 ng, and also to sheep erythrocytes; when cells were transferred to fresh tubes, over 90% of the radioactivity remained on the original tube, regardless of the tube surface. Various substances, including BPM and unrelated basic proteins, competitively inhibited the binding of basic protein of myelin to sheep cells and assay tubes. Binding was inhibited by sera from patients with multiple sclerosis, but equally so by normal sera. The large capacity of BPM to bind nonspecifically could limit its use in the above type of binding assay and would need to be allowed for in conventional radioimmunoassays.

Studies on the lymphocytes of sheep. III. Destination of lymph-borne immunoblasts in relation to their tissue of origin.

Lymph-borne immunoblasts were labeled in vitro with 125I[]iodo-deoxy-uridine, washed and returned by intravenous injection to the sheep from which they had been collected. Twenty h later the sheep were killed and the distribution of the immunoblasts was determined by assaying the radioactivity in various organs. Immunoblasts from the efferent lymph of peripheral somatic lymph nodes (PSLN) went mainly to the spleen, lungs and other PSLN, while immunoblasts from intestinal lymph went mainly to the small gut. This ability of intestinal immunoblasts to home to the gut was demonstrated also in the sterile environment of fetuses in utero; apparently the migratory behavior of immunoblasts, like that of small lymphocytes, is not primarily "antigen-driven". A technique was devised for the collection of peripheral (i.e. afferent to the mesenteric node) intestinal lymph which was found to contain 10-20 times the numbers of small lymphocytes that occur in the peripheral lymph from other tissues. Immunoblasts from peripheral intestinal lymph also homed to the gut. The immunoglobulin content of immunoblasts was studied by making detergent extracts of lymph cells, by applying immuno-peroxidase techniques to cell films and by investigating the incorporation of 14C-labeled amino acids into immunoglobulins by immunoblasts in vitro. Immunoblasts from both somatic and intestinal lymph contained and made IgG and IgM, but many intestinal immunoblasts contained and made IgA. It is not known whether this immunoglobulin mediates the extravasation of immunoblasts into the gut. Nonetheless, there is compelling evidence that there are two major migratory pathways for lymphoid cells; one through the gut-associated lymphoid tissue and the other through the somatic-splenic lymphoid tissues.

Specificity of Allogeneic and Xenogeneic Cell Recognition in the Fetal Lamb

Cells prepared from liver, thymus, and spleen of fetal lambs at different stages if gestation were confronted with allogeneic and xenogeneic cells in MLC. Specific elimination of the responding cells with BUdR and UV light together with a subsequent restimulation was used to study the specificity of the reaction. The response of fetal liver cells was not based on the existence of specifically recognizing cellular subpopulations; the response was concluded to be due either to stimulatory products released by the stimulating cells or to the multipotentiality of the responding cells. Specifically recognizing cells first appeared in the thymus at 58 days postconception and in the spleen at 70 days. In the response of sheep lymphocytes against allogeneic and xenogeneic (mouse, human) cells, a cross-reactivity occurred. Fetal lamb lymphocytes were also capable of recognizing intraspecies differences on the xenogeneic cells. This capacity developed simultaneously with the specific recognition of allogeneic cells. No clear difference was observed in the reactivity of fetal thymus cells and spleen cells when compared to that of adult peripheral blood lymphocytes. These findings indicate that immunologically specific recognition of foreign cells is created in the sheep during the early intrauterine development.

The binding of dehydroheliotridine to DNA and the effect of it and other compounds on repair synthesis in main and satellite band DNA

This study was aimed at elucidating the mechanisms of the preferential depression of satellite DNA synthesis by dehydroheliotridine (DHH). DHH was found to induce repair synthesis to the same extent in both main and satellite band DNA in cultured sheep lymphocytes. This was also the case with acridine orange, nitrogen mustard (HN2) and ethyl methanesulphonate (EMS). Using analytical equilibrium ultracentrifugation no difference was found between the extents of in vitro binding of DHH by main and satellite band DNA. From these results it was concluded that the depression of the synthesis of satellite DNA could not be explained by either its preferential binding of DHH or by less effective repair mechanisms. Radio labelled DHH when added to synchronized cultures of ovine kidney cells was found to be preferentially bound to the satellite DNA (one DHH molecule to 6000 nucleotides) compared with the main band DNA (one to 10 000). When 5-bromodeoxyuridine (BUdR) was added to the cultures no DHH label was found in the heavy, semiconservatively replicated DNA band. From these findings it is suggested that attack may occur during mitosis where all of the satellite DNA may be undergoing synthesis at the same time, thus explaining the increased amount of DHH bound to the satellite.

Inhibitory action of different RNA fractions on the translation of globin mRNA in vitro

Preparations of 28 S rRNA and 12 S RNA, obtained from sheep lymphocytes, were shown to inhibit the translation of globin mRNA. An inhibition by a given amount of 12 S or 28 S RNA was only obvious at saturating or near-saturating levels of globin mRNA, suggesting that the inhibitory RNAs interacted with some factor essential for protein synthesis other than mRNA. The inhibitory RNAs had no effect on the translation of the synthetic template polyuridylic acid. It is suggested therefore that the target for inhibitory RNAs might be a natural mRNA specific initiation factor.

Evidence for the presence of mRNA in the post-ribosomal cytoplasm of sheep lymphocytes

Several fractions of RNA prepared from the post-ribosomal cytosol of sheep lymphoid cells were found to include messenger-like RNA as defined by the following criteria: a, template activity, i.e. the ability to promote the incorporation of radioactive amino acids into protein in cell-free protein-synthesising systems derived from wheat embryos or ascites tumour cells; b, a low magnesium optimum (1–2.5 mM) for template activity which is characteristic of many natural mRNAs; c, sensitivity of the template response to aurintricarboxylic acid, a specific inhibitor of the initiation of protein synthesis. The lymphoid post-ribosomal RNA fractions, however, were translated less efficiently than were rabbit reticulocyte globin mRNA or tobacco mosaic viral (TMV) RNA; no explanation for this relatively poor template activity was found. The major fraction of messenger-like RNA had an average sedimentation coefficient of 12 S; this fraction directed the translation of several discrete polypeptides in the molecular weight range 10 000–25 000. On average the products of 12 S RNA-directed protein synthesis appeared lysine rich compared with TMV RNA-directed products. It is suggested that the apparent pool of uncommitted mRNA in resting lymphocytes may be utilised during the early stages of lymphocyte activation, and that the mRNAs could be stored in forms similar to those evident in other dormant tissues.

Studies on the lymphocytes of sheep. II. Some properties of cells in various compartments of the recirculating lymphocyte pool.

The proportion of cells bearing surface immunoglobulin (Ig) and the normal lymphocyte transfer (NLT) reactivity of mononuclear cells obtained from the blood, afferent lymph, and efferent lymph from both somatic and intestinal nodes of sheep were measured. The proportion of Ig-positive lymphocytes was approximately 30% in blood and efferent lymph, but only about 10% in afferent lymph. The proportion of unlabeled lymphocytes (T cells) was about the same in all the preparations, as afferent lymph contains 15--20% macrophages. Despite this apparent uniformity in the T cell content, the NLT reactivity of efferent lymph was 3 to 5 times higher than that of blood or afferent lymph. No humoral factors could be detected by incubating cells in the various plasmas in vitro before injection. No inhibitory cells could be detected when mixtures of cell populations were used to initiate lesions. We suggest that lymphocytes in efferent lymph are more reactive than others because they are "groomed" by macrophages during their recirculatory passage through the substance of the node.

Studies on the lymphocytes of sheep. I. Recirculation of lymphocytes through peripheral lymph nodes and tissues

Recirculating lymphocytes were collected from the efferent ducts of peripheral somatic nodes and incubated in vitro with 51Cr. After washing, the labeled cells were returned to the sheep by i.v. injection. The partitioning of the labeled cells between blood and efferent (intermediate) lymph, or between efferent and afferent (peripheral) lymph was monitored for the next 24 h by assaying the radioactivity in appropriate samples of cells. The labeled lymphocytes took several hours to migrate through the nodes so that they equilibrated between blood and lymph in about 10 h. This pattern was not altered grossly during the inductive phase of an immune response. The small numbers of lymphocytes which migrated through peripheral tissues entered the lymph at least quickly as those that migrated through lymph nodes.

Heterogeneity of Sheep Small Lymphocyte Populations in RNA Labelling

The labelling pattern of 3H-labelled uridine incorporation into RNA was studied by autoradiography in small lymphocytes obtained from the efferent lymph of normal sheep lymph nodes. Heavily labelled small lym-phocytes were always observed in the sheep lymphocyte population. They constituted 3.6 % of the total population. Antigen-stimulation did not alter the number of heavily labelled small lymphocytes. The majority of small lympho-cytes contained only one or two nucleoli, but 5 to 8 percent of the population possessed more than five nucleoli. The possibility was considered that the heterogeneity of small lymphocytes in RNA labelling may be associated with the development of nucleoli and that the heavily labelled small lymphocytes were engaged in particular functions being actively pursued in the efferent lymph.

Cytotoxic mechanisms detected in vitro following sheep renal allografts.

Cytotoxic effects of lymph and blood components, removed from sheep following renal allografting were determined by in vitro assay using 51Cr labelled donor sheep lymphocytes as target cells. Lytic antibodies were detected in lymph efferent from the node draining the graft site and at lower concentrations in blood serum and in lymph leaving the graft. The cytotoxic antibodies had both complement-dependent and leucocyte-dependent functions; the complement-dependent antibodies were fractionated and shown to be of IgM and IgG1 subclasses. Cytotoxic cells were found in lymph leaving the graft but not in blood or in lymph efferent from the draining node; those detected in the renal lymph were non-specific in action and their appearance correlated with increased numbers of macrophages.

Lack of T-cell immune abnormalities in peripheral blood lymphocytes in patients with Graves' disease or hypothyroidism.

Lymphocytes capable of forming nonimmune (E) and immune (EAC) rosettes with sheep erythrocytes and lymphocyte reactivity to phytohemagglutinin were determined in normal subjects, in patients with hyperthyroidism due to Graves' disease and in patients with hypothyroidism with and without goiter. Neither patient group differed from the normal subjects either in regard to lymphocyte count, the proportion of thymus dependent (t) lymphocytes forming nonimmune or bone marrow-dependnet (B) lymphocytes forming immune rosettes or lymphocyte reactivity to phytohemagglutinin. These results fail to provide evidence that these disorders are associated with an immune abnormality with increased or abnormal T cells in peripheral blood.

Effects of mitogens on sodium-potassium transport, 3H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na +) potassium (K +) transport, 3H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K + pump influx, while the cardiac glycoside-insensitive K + leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K + pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na + efflux in human lymphocytes. Apparently these mitogens activate the Na +K + pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na +K +-ATPase. From rate studies of 3H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na +K + pumps.