Stimulation Of Human Peripheral Blood Research Articles

Intravenous immunoglobulin contains specific antibodies inhibitory to activation of T cells by staphylococcal toxin superantigens [see comment

Superantigens are products of bacteria with dual affinity for HLA-DR and the variable region of the beta chain of the T cell receptor, leading to the stimulation of large numbers of T cells. Because there is evidence for the involvement of superantigens in various disease conditions in which intravenous IgG (IVIgG) is used as therapy, the purpose of the present study was to determine if IVIgG contains antibodies inhibitory to T cell stimulation by the superantigens. ELISA and Western assays revealed high concentrations of antibodies in the pooled IgG against eight different staphylococcal toxin (Staph-toxin) superantigens. The IVIgG inhibited in vitro stimulation of human peripheral blood T cells by the Staph-toxins, but did not inhibit responses elicited by phytohemagglutinin or anti-CD3. Inhibition was mediated by Staph-toxin-specific antibodies as shown by affinity adsorption depletion studies. The antibodies functioned by inhibiting the binding and/or presentation of Staph-toxins by DR+ accessory cells. In conclusion, this report is the first to show that normal pooled IgG contains antibodies against a major group of the superantigens, the Staph-toxins, and that the antibodies can inhibit Staph-toxin-elicited T cell activation, suggesting a possible immunoregulatory role for the antibodies in vivo.

Inhibiting effects of serotonin antagonists on the proliferation of mercuric chloride stimulated human peripheral blood T lymphocytes.

The serotonin antagonists ketanserin and methiotepine were tested for a modulating effect on the proliferative response of human peripheral blood T lymphocytes to mercuric chloride. This response was inhibited by ketanserin at 8 x 10(-5) mol/l and by methiotepine at 8.0 x 10(-6) mol/l. There were no additive effects at these concentrations of antagonists and at 10(-5) mol/l of serotonin. Low concentrations of ketanserin eliminated the inhibiting effect of serotonin on mercuric chloride induced proliferation of T lymphocytes. It thus seems as if the inhibiting effect of serotonin on T lymphocytes is mediated by 5-HT1c or 5-HT2 receptors, while the mechanism for the intrinsic inhibiting effect of the antagonists at present is unknown.

Purification of granulocyte chemotactic peptide/interleukin‐8 reveals N‐terminal sequence heterogeneity similar to that of β‐thromboglobulin

Stimulated human peripheral blood leukocytes produce a chemotactic factor for granulocytes (granulocyte chemotactic peptide/interleukin-8; GCP/IL-8), which is structurally related to platelet-derived beta-thromboglobulin. Analytically pure CGP/IL-8 and beta-thromboglobulin could be obtained after three purification steps, comprising adsorption to silicic acid, heparin-Sepharose chromatography and ion-exchange chromatography. Although GCP/IL-8 and beta-thromboglobulin had a similar affinity for heparin, they could be separated on a cation-exchange column. Both molecules were heterogeneous in that 6-7-kDa protein doublets were detected upon SDS/PAGE. N-terminal amino acid sequence analysis revealed the presence of six immunologically related but differently truncated polypeptides of beta-thromboglobulin, of which only two corresponded to previously described forms. Similarly, apart from a major polypeptide, five minor species of GCP/IL-8 were detected that also differed by N-terminal truncation. The most processed forms of beta-thromboglobulin and GCP/IL-8 were found to have their N-terminus in that region of the primary structure where a significant similarity between the two molecules starts. GCP/IL-8 was found to be chemotactic for granulocytes with a specific activity of 10(5) units/mg, whereas none of the beta-thromboglobulin species possessed detectable chemotactic activity.

Soluble interleukin 2 receptors released from mitogen stimulated human peripheral blood lymphocytes bind interleukin 2 and inhibit IL2 dependent cell proliferation.

In this communication the binding characteristics and possible regulatory role of sIL2R were investigated. Soluble IL2R are released or secreted in high concentrations by phytohemagglutinin (PHA) stimulated human lymphoid cells. The addition of sIL2R, purified by gel filtration chromatography, to cultures of PHA stimulated lymphoblasts resulted in a dose-dependent inhibition of [3H]TdR incorporation that could be overcome by the addition of exogenous IL2. Scatchard analysis of IL2 binding demonstrated that the presence of sIL2R did not inhibit ligand interaction with the high affinity IL2R. Immunoprecipitation studies utilizing [125I]IL2 and the non-inhibitory anti-Tac protein antibody 7G7/B6 revealed that most of the 125I-labeled IL2 migrated with a protein of approximately 45-50 kDa on SDS/PAGE. Together, these results provide evidence that the sIL2R limits the availability of free IL2 to proliferating cells and down-regulates their response without directly affecting the number or function of the cell bound high affinity IL2R.

Transcription of protooncogenes during stimulation of normal human B lymphocytes.

Protooncogenes play important roles in the regulation of growth and differentiation of normal cells. In this study we have examined the cell cycle-dependent regulation of transcription of various protooncogenes after stimulation of human peripheral blood B lymphocytes. The transcriptional rate of various genes was determined by means of a nuclear run-on assay. We found that several protooncogenes were transcriptionally activated after stimulation (myc, p53, K-ras, H-ras, sis and ets), but with different kinetics of induction. In contrast, some oncogenes, especially those encoding membrane-associated or cytoplasmatic proteins like abl, rel or mil/raf, were transcribed at a relatively constant rate during the cell cycle.

Gamma-radiation-induced chromosomal aberrations in human lymphocytes: Dose-rate effects in stimulated and non-stimulated cells

Stimulated and non-stimulated human peripheral blood lymphocytes were irradiated acutely and chronically, over 24 h. Dose-effect relationships for dicentric chromosomes were established and various models were fitted to the data. At prolonged irradiations the yield decreased in basic agreement with the linear-quadratic model of aberration induction. Dose-protraction experiments on PHA + and PHA − lymphocytes, irradiated under various conditions of oxygenation and suspension (culture medium, whole blood) showed that the rejoining time increased from about 3 h in non-stimulated cells to about 10 h after PHA stimulation, and that this retarded rejoining was most likely due to blastic transformation itself and not to other conditions of irradiation.

ISOLATION OF HUMAN LYMPHOCYTE MITOGENIC FACTOR

Stimulation of sensitized human peripheral T lymphocytes with tetanus toxoid release lymphocyte mitoqenic factor (LMF). LMP induces DNA and antibody synthesis in allogenic and autologous B lymphocytes. Initial studies showed that the biologically active fraction was precipitated by 40-60% ammonium sulfate and 33-50% ethanol. It sedimented in cesium chloride with a density of 1.250. To purify the activity, 1000ml of T cell supernate obtained from cells of a single donor were concentrated twenty fold by Amicon ultrafiltration on a xm-50 membrane. Concentrated supernate was chromatographed on Sephadex G-200. The biological activity eluted post albumin. It was electrophoresed on an LKB iso-electrofocusing column. A major peak of proliferative activity was found at pH=6.7±0.1. A second minor peak of activity was found at pH=7.2±0.1. In one instance, when the major peak was assayed for its ability to initiate de novo synthesis of IgG by tonsillar B lymphocytes, it was found to cause a twofold increase of precipitable 14C activity over control. The data suggest that stimulation of human peripheral blood T lymphocytes by antigen results in production of two factors that can induce DNA synthesis in autologous or allogenic B lymphocytes. It appears that both of these factors are of molecular weight greater than 50,000. At least one of the factors increases IgG synthesis in B cells derived from human tonsils, while the other was present in concentrations too low to study.

The mitogenicity of dextrans for B lymphocytes in man

Branched dextran B1355 (57% α1–6, 35% α1–3, and 8% α1–2 α1–4 linkages) induced in vitro stimulation of human peripheral blood and spleen lymphocytes. However, linear dextran B512 (96% α1–6 and 4% α1–3 linkages) and its depolymerised fractions irrespective of molecular weight (from 1 × 10 4 – 2 × 10 6) were not mitogenic for human lymphocytes from spleen or peripheral blood. This difference in response was not due to the mitogenicity of the α1–3 epitope, as α1–3 glucan itself was not stimulatory. The incidence and magnitude of the response to dextran B1355 were increased in patients with gingival or periodontal disease compared with subjects with clinically healthy gingiva. B-cell mitogens stimulate human peripheral blood lymphocytes from control subjects in the following order of effectiveness: dextran sulphate (100% responders), levan (87% responders), and dextran B1355 (62% responders).

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. III. Species specificity of lymphocyte target cell recognition in vitro

Cytotoxic effector lymphocytes were produced by stimulation of human peripheral blood or mouse spleen lymphocytes with PPD, PHA or PWM in vitro. The specificity of the lymphocyte target cell interaction was studied in vitro. The specificity of the lymphocyte target cell interaction was studied by adsorption of effector cells on various target cell monolayers. The cytotoxic activity of human lymphocytes against 51Cr-labeled human or monkey target cells was reduced by prior incubation on primate monolayers while it was much less affected by incubation on rodent monolayers. Conversely the cytotoxicity of mouse lymphocytes against mouse L cells was strongly reduced by absorption on mouse or rat monolayers but significantly less by that on human monolayers. This suggests that this species-specific cytotoxicity reflects recognition by activated lymphocytes of some common surface structures present only on cells of the species of the effector cell donor, or on cells from phylogenetically closely related species. Lymphocyte-mediated cytotoxicity against xenogeneic target cells was studied after stimulation with PWM. The capacity of human lymphocytes to kill 51Cr-labeled mouse cells was reduced by adsorption on either rodent or primate monolayers. Conversely, prior incubation of mouse lymphocytes on either human or mouse monolayers led to inhibition of 51Cr release from labeled Chang cells. These results suggest that the mitogen-activated effector cells which are cytotoxic for more distantly related xenogeneic target cells have receptors for structures which are common for these cells and for target cells of the species of the lymphocyte donors.

Carbohydrate metabolism in PHA stimulated human peripheral blood lymphocytes.

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-14C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can be easily quantitated by an enzymatic assay.

Proliferation of Stimulated Human Peripheral Blood Lymphocytes: Preferential Incorporation of Concanavalin a by Stimulated Cells and Mitogenic Activity

Abstract Binding of concanavalin A (conA) to human peripheral blood lymphocytes or to lymphoblasts is inhibited by α-D-mannopyranosides or α-D-glucopyranosides. After 1 hr incubation of cells with con A over 80% of the bound con A can be washed off with methyl-α-D-mannoside; after 24 hr only about 25% of the cell associated con A can be removed by competitor. This sequestration is temperature dependent. Cells which have been treated with con A for 20 hr still have as many combining sites for freshly added con A as do non-pretreated cells, even though about 75% of the previously added con A remains associated with the cells. Con A stimulated cells can be separated from non-stimulated cells on Ficoll gradients. Irreversibly bound con A is preferentially accumulated in the stimulated cells. However, this irreversibly bound con A does not stimulate. We suggest that irreversibly bound con A is inside the cell and that internalization is a result of the early activation of energy generating reactions by the mitogenic stimulus. The stimulatory effect depends on reactions at the cell surface.

Immunologically stimulated human peripheral blood lymphocytes in vitro. I. Morphological changes.

Immunologically stimulated human peripheral blood lymphocytes in vitro. I. Morphological changes.

Immunologically stimulated human peripheral blood lymphocytes in vitro. II. Mixed lymphocyte cultures with related and unrelated donors.

Immunologically stimulated human peripheral blood lymphocytes in vitro. II. Mixed lymphocyte cultures with related and unrelated donors.