Non-rosetting Lymphocytes Research Articles

Receptor Mediated Binding of the Fibrinolytic Components, Plasminogen and Urokinase, to Peripheral Blood Cells

Glu-plasminogen binds to platelets; the monocytoid line, U937, and the human fetal fibroblast line, GM1380 bind both plasminogen and its activator, urokinase. This study assesses the interaction of these fibrinolytic proteins with circulating human blood cells. Plasminogen bound minimally to red cells but bound saturably and reversibly to monocytes, granulocytes and lymphocytes with apparent Kd values of 0.9-1.4 microM. The interactions were of high capacity with 1.6 to 49 X 10(5) sites/cell and involved the lysine binding sites of plasminogen. Both T cells and non-rosetting lymphocytes and two B cell lines saturably bound plasminogen. Urokinase bound saturably to granulocytes, monocytes, non-rosetting lymphocytes and a B cell line, but minimally to T cells, platelets and red cells. Therefore, plasminogen binding sites of high capacity, of similar affinities, and with common recognition specificities are expressed by many peripheral blood cells. Urokinase receptors are also widely distributed, but less so than plasminogen binding sites. The binding of plasminogen and/or urokinase to these cells may lead to generation of cell-associated proteolytic activity which contributes to a variety of cellular functions.

DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA.

Previous studies have indicated that white blood cells possess DNA on their outer membranes. In this study we set out to determine whether exogenous DNA bound to cells in a fashion compatible with a ligand receptor union. Purified populations of white blood cells; neutrophils (polymorphonuclear leukocytes, PMN), adherent mononuclear cells (ADMC), rosetting lymphocytes (E+ cells), and nonrosetting lymphocytes (E- cells) were incubated with radiolabeled lambda phage DNA in increasing concentrations. Binding of [3H]DNA was a saturable process and was inhibited by excess cold DNA and prior trypsinization of the cells. Rate zonal density centrifugation of purified cell membrane preparations confirmed that DNA was binding to the outer cell surface. The dissociation constant for all four cell types was approximately 10(-9) M, and from 0.81 X 10(3) to 2.6 X 10(3) molecules of lambda phage DNA bound to each cell depending upon cell type. Binding was not competitively inhibited by RNA, polydeoxyadenylic acid-polydeoxythymidylic acid (poly [d(A).d(T)]), or mononucleotides. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)-separated proteins from PMN, ADMC, E+, and E- cells were electrophoretically blotted onto nitrocellulose sheets; a probe of biotin-labeled DNA indicated a single species of DNA-binding molecule migrating in a position consistent with a molecular weight of 30,000. Isotopic and immunofluorescent studies indicate that DNA is internalized and degraded to oligonucleotides; this process is inhibited by cycloheximide. These results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this apparent receptor is responsible for the endocytosis and degradation of exogenous DNA.

Monoclonal antibodies reactive with swine lymphocytes. I. Antibodies to membrane structures that define the cytolytic T lymphocyte subset in the swine.

A panel of monoclonal antibodies (mAb) with specificity for swine leukocytes was prepared by somatic cell hybridization with the use of spleen cells from mice immunized with swine thymocytes. The reactivity of two mAb (295/33 and 122/28), which both immunoprecipitated from the surface of swine leukocytes an antigen termed S-L2 with an apparent m.w. of 33 to 35 kilodaltons under reducing and 65 to 70 kilodaltons under nonreducing conditions, was investigated in detail. These mAb were reactive in indirect immunofluorescence with 50 to 60% of thymocytes, 35% of peripheral blood lymphocytes, and 55% of E rosette-positive cells; they were nonreactive with bone marrow cells, Ig+ B cells, nonrosetting lymphocytes, granulocytes, and monocytes. In functional studies, the elimination of S-L2+ cells partially reduced the proliferative response to concanavalin A and pokeweed mitogen but not to Staphylococcus aureus and lipopolysaccharide. The S-L2- subset proliferated well to alloantigens. Both cytolytic T effector cells and precursor cells carried the antigen S-L2 and could be depleted from heterogeneous cell populations by both antibodies in the presence of complement. These data suggest that the mAb 295/33 and 122/28 recognize a specific polypeptide present on the surface of swine cytolytic T cells. These antibodies will be useful in studies on the swine immune system.

Lactoferrin binds to cell membrane DNA. Association of surface DNA with an enriched population of B cells and monocytes.

The binding of human 125I-labeled lactoferrin (LF) to a population of adherent mononuclear cells (ADMC) and nonrosetting lymphocytes (E-) was abolished by prior treatment of the cells with deoxyribonuclease (DNase), but not ribonuclease (RNase). When DNase-treated ADMC were incubated with exogenous DNA, the binding of 125I-LF was restored. Enzymatic digestion with other enzymes, trypsin, phospholipase D, and neuraminidase, did not significantly influence 125I-LF binding. Saturable binding of LF at 0 degrees C was demonstrated for both E- and ADMC, with equilibrium dissociated constants of 0.76 x 10(-6) M and 1.8 x 10(-6) M, respectively. E- cells bound 2.5 x 10(7) and ADMC bound 3.3 x 10(7) molecules of Lf at saturation. Cell membranes were isolated from ADMC, E- and E+ and reacted with 125I-labeled LF; significant binding was only seen with ADMC and E-. Prior treatment of the membranes with DNase abolished the binding. Immunofluorescence studies indicated that a population of ADMC and E-, but not E+, exhibited a peripheral staining pattern for LF. Prior treatment of ADMC and E- with DNase abolished the surface immunofluorescence. This study provides evidence that cell membrane DNA acts as a binding site for exogenous LF. This is a novel role for DNA that has not been previously reported. Furthermore, it points to a basic difference between E+ cells vs. ADMC and E- cells in respect to their possession of cell surface DNA.

Two monoclonal antibodies identifying a subset of human peripheral mononuclear cells with natural killer cell activity.

Two monoclonal antibodies (H25 and H366) raised against the cultured T lymphoid cell line HSB-2 were shown to define a subset of peripheral mononuclear cells with natural killer and killer cell activity. The two antibodies show similar tissue distributions. Radioimmune trace binding assay shows that H25 and H366 react with all T cell lines tested and with the monocyte line U937, but weakly or not at all with cell lines of B, myeloid or erythroid origin. Both antibodies react with about 10% of E rosette-forming cells and a proportion of nonrosetting lymphocytes and monocytes. They do not react with B lymphocytes, granulocytes, red cells or platelets. A proportion of thymocytes and low numbers of tonsil and spleen mononuclear cells are positive. H25+ and H366+ lymphocytes are medium-sized cells with abundant granular cytoplasm, and most carry Fc receptors for IgG. H25 and H366 immunoprecipitate two polypeptide chains of 96 and 53kDa from surface-labeled HSB-2 cells.

In vitro modulation of cell-mediated immunity by prostaglandin E2. II. Enhancement of spontaneous plaque-forming cell generation

The effect of the pretreatment of human peripheral blood lymphocytes by Prostaglandin E2 (dissolved in medium) on the spontaneous Plaque-Forming Cell generation has been evaluated. A significant enhancement of immunoglobulin production, markedly increased by the addition of further PGE2 to the pretreated cells, has been demonstrated. Experiments carried out with indomethacin have shown an inhibition of plaque formation, thus indicating that the content of endogenous prostaglandin E2 may play an important role in the enhancement of antibody synthesis, previously described. Results obtained with populations of rosetting and non-rosetting lymphocytes pointed out that non-rosetting cells are exclusively responsive to prostaglandin treatment.

Evidence for altered density characteristics of the peripheral blood lymphocytes in kwashiorkor

Peripheral blood lymphocytes obtained from Ficoll-Hypaque gradient separation from 15 kwashiorkor children and 14 controls were separated into fractions by centrifuging (900 × g, 10C) through discontinuous bovine serum, albumin gradients of 17, 20, 24, 28, 30, and 36 percent bovine serum albumin solutions. The top two fractions were pooled and designated fraction A and the remaining fractions were designated B, C, and D, respectively. Lymphocytes from kwashiorkor showed a marked alteration in density distribution in that a vast majority of them (57%) settled to the lowermost fraction (D) as against 17% in controls. Using nonimmune sheep erythrocyte adherence (erythrocyte-rosettes) as a T-cell marker and erythrocyte antibody complement rosettes specific for C3 receptor as a B-cell marker it is shown that, in addition to a reduction in the proportion of erythrocyte-rosetting lymphocytes there is a marked increase in their density. There is no significant alteration either in the proportion or in the density characteristics of the erythrocyte antibody complement-rosetting lymphocytes. The vast majority of nonrosetting lymphocytes (“null cells”) also settled in the fraction with the highest density. The findings warrant further studies on the density characteristics of lymphocytes and their characterization using a wider range of surface markers in kwashiorkor, particularly after nutritional status of the children had returned to normal.

A pen smear technique for assays of rosette-forming lymphocytes

A simple technique is described for the preparation of permanent stained smears of human E-rosetted lymphocytes. Smears of the cells suspended in foetal calf serum were drawn as thin strips on slides with a pen nib. Such smears, after Romanowsky staining, and incorporating latex-particle phagocytosis as a marker for non-lymphoid cells, show excellent rosette preservation and permit easy distinction and counting of rosetting lymphocytes, non-rosetting lymphocytes, monocytes and granulocytes. The percentages of rosette-forming lymphocytes estimated on such smears correspond closely to those obtained from counts made on viable cell suspensions.

Cytochemical reactions of normal and neoplastic lymphocytes.

Rosetting and non-rosetting lymphocytes collected from normal individuals were stained for the presence of beta-glucuronidase, periodic-acid Schiff activity, gamma glutamyl transpeptidase, acid phosphatase, and alpha-naphthyl butyrate esterase. Lymphocytes which formed rosettes with sheep erythrocytes and non-rosette forming lymphocytes contained cytochemical reaction products for all five stains. Beta-glucuronidase (P less than 0-02) and acid phosphatase (P less than 0-01) were more frequently found in rosette forming lymphocytes. However, non-rosetting cells were more frequently periodic-acid Schiff positive (P less than 0-001). Gamma-glutamyl transpeptidase and alpha-naphthyl butyrate esterase were present equally in rosette and non-rosette forming lymphocytes. In addition, 33 non-Hodgkin's lymphomas were studied for cell surface markers and cytochemical reactions. In 17 of 19 B cell lymphomas, there was a paucity of lymphocytes containing beta-glucuronidase. However, in three of four T cell proliferations, there were numerous lymphoid cells positive for beta-glucuronidase. The periodic-acid Schiff and acid phosphatase reactions varied greatly within B, T, and null cell lymphomas and thus were of little diagnostic value in determining the cell of origin of these neoplastic lymphoid cells.

Production of osteoclast-activating factor by normal human peripheral blood rosetting and nonrosetting lymphocytes.

This study has tested the ability of human mononuclear cells and the subpopulations of lymphocytes that rosette (E-RFC) or do not rosette (non-E-RFC) with sheep red blood cells to synthesize DNA and to produce a mediator with bone resorbing activity (osteoclast-activating factor, OAF) in response to stimulation with phytohemagglutinin (PHA). Mononuclear cells were stimulated by PHA both to synthesize DNA and to produce a factor that caused bone resorption. E-RFC enriched for T lymphocytes and depleted of macrophages synthesized considerable DNA in response to stimulation with PHA, but were unable to produce significant bone resorbing activity in tissue culture unless macrophages were re-added to the E-RFC. In contrast, mononuclear cells depleted of E-RFC (non-E-RFC enriched for B cells) did not synthesize DNA in response to stimulation with PHA, but did produce a mediator that caused significant bone resorption in tissue culture. In most of the experiments, unstimulated non-E-RFC produced as much OAF as the PHA-stimulated non-E-RFC. These results suggest that both activated T lymphocytes (E-RFC) and B lymphocytes (non-E-RFC) can produce OAF.

A one‐step procedure for separating mouse T and B lymphocytes

AbstractA procedure is described for both depleting and obtaining in pure form Ig‐bearing cells from mouse lymphoid populations. The Ig‐bearing cells are identified by rosetting and the rosettes separated from the nonrosetting lymphocytes by centrifugation on Isopaque/Ficoll. The rosettes sink with the red cells and the nonrosetting lymphocytes float. The procedure is > 99.5 % efficient at depleting mouse lymphoid populations of Ig‐rosetting cells and is also > 97 % efficient at removing the Ig‐bearing cells detected by radioautography. Furthermore, after lysis of the red cells by hypotonic shock the Ig‐bearing cells are recovered in a highly pure form. The total recovery of white cells and rosettes applied is > 85 %.This procedure was shown to produce a functional separation of T and B lymphocytes. The cell population depleted of Ig‐rosettes behaved as a pure T cell preparation. It lacked precursors of antibody‐forming cells, but contained virtually all of the Θ‐positive lymphocytes, the bulk of the helper cells detected in two in vitro hapten carrier antibody responses and all the cells which responded and produced cytotoxic cells in MLC. In contrast, the preparation of Ig rosettes expressed B cell properties. This population contained all of the antibody forming cell precursors, few helper cells and Θ‐positive lymphocytes and no MLC‐responding cells. However, there was some evidence that a small subpopulation of T cells exists which possesses surface Ig.The separation system was used to formally demonstrate that carrier primed T cells collaborate with hapten primed B cells to generate an anti‐hapten antibody response to a hapten‐carrier conjugate. It was also established that in MLC responder B cells in no way collaborate with responder T cells to generate cytotoxic cells.

The lymphocyte surface. II. Separation of Fc receptor, C'3 receptor and surface immunoglobulin-bearing lymphocytes.

A one-step separation procedure is described for both depleting and obtaining in pure form Fc receptor (FcRL), C'3 receptor (CRL) and surface immunoglobulin bearing (IgL) lymphocytes from rat lymphoid populations. The method is a modification of the Bӧyum (1968) technique for separating lymphocytes from whole blood by sedimentation on Ficoll/Isopaque, and is based on the fact that when a lymphocyte forms a rosette with sensitized erythrocytes it will sediment with the red cells rather than float with the non-rosetting lymphocytes. The technique is > 99.5% efficient at depleting thoracic duct lymphocytes (TDL) of FcRL, CRL and IgL and these subpopulations can be recovered 93-98% pure. The total recovery of lymphocytes applied is usually > 90% and the separated lymphocytes are > 95% viable. This technique allowed the cellular distribution of Fc receptors, C'3 receptors and surface Ig to be determined. It was found that (a) Almost all CRL carry surface Ig, although a very small sub-population of CRL (0.2-0.8%) which lacks surface Ig could regularly be detected. (b) A substantial proportion of IgL (12-25%) lacks C'3 receptors. (c) IgL and CRL which lack Fc receptors are more frequent in spleen and lymph nodes than in TDL. The proportion of this subpopulation increases in TDL after prolonged thoracic duct drainage. (d) Some FcRL exist which lack both C'3 receptors and surface Ig. These cells are more evident in TDL after prolonged thoracic duct drainage and in lymph nodes (20-30% of FcRL) than in early TDL or spleen (5-10% of FcRL). (e) The thymus contains very few FcRL, CRL or IgL. (f) A large population of lymphocytes exists in B rats (32-42% of TDL) which is killed by an anti-B serum but which lacks surface Ig. These cells are much less frequent in normal TDL ( < 5%) and probably also lack Fc and C'3 receptors. (g) Large lymphocytes probably shed their Fc and C'3 receptors, but retain their surface Ig, during S-phase. (h) Studies on a secondary anti-DNP response showed that a substantial proportion of direct and indirect plaque forming cells (PFC) in the spleen express Fc receptors, whereas only indirect PFC carry C'3 receptors. Virtually all PFC ( > 98%) possess surface Ig.