Concanavalin A-stimulated Human Lymphocytes Research Articles

C3 cleaved by membrane proteases binds to C3b acceptors expressed on concanavalin A-stimulated human lymphocytes and enhances antibody-dependent cellular cytotoxicity.

On activation of cells membrane-associated proteases--including serine esterases known to cleave the third component of complement (C3)--become expressed. In this paper it is shown that as a consequence of this enzyme activity isolated native human C3 added to concanavalin A (Con A)-activated human lymphocytes is cleaved on the surface of the blast cells. This enables the immediate fixation of nascent C3b (C3bx) through its short-lived metastable binding site to C3b acceptors (C3bA's) newly expressed on Con A-stimulated cells. Acceptor-bound C3b is detected by the immune adherence rosette formation of the C3-treated Con A blasts with the C3b receptor (C3bR)-bearing O, Rh+ erythrocytes (32 +/- 4%). The cleavage of C3 and the covalent fixation of C3b are shown to be inhibited by phenylmethylsulphonyl fluoride and methylamine, respectively. As a functional consequence of the covalent fixation of C3b to the mitogen-activated lymphocytes it is demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) of these cells against O, Rh+ erythrocytes sensitized with anti-D IgG is significantly enhanced. The C3 specificity of the process and the role of C3bR's of the target cells are proved. It is postulated that effector cell-bound C3b amplifies ADCC by improving effector cell-target cell contact.

Nuclear matrix from resting and concanavalin A-stimulated human lymphocytes

Concanavalin A-induced transformation and proliferation of human peripheral blood lymphocytes was found to be accompanied by morphological and biochemical changes of the nuclear matrix. Nuclear matrix spheres increased in size as well as in protein and RNA content. Experimental data suggesting the involvement of the nuclear matrix in DNA replication processes are also presented.

Inhibition of human lectin-dependent cell-mediated cytotoxicity, natural killer-like cytotoxicity, and cytotoxic T-lymphocyte-mediated cytolysis by xenoantisera raised against concanavalin A-stimulated human lymphocytes

This report describes the in vitro inhibition of several classes of human lymphocyte-mediated lysis by antisera from mice and rats immunized with concanavalin A-stimulated human T-enriched lymphocytes. The inhibitory antibodies in these antisera recognized molecules on the surface of cytotoxic lymphocytes, and did not bind to B-cell lines. Experiments were performed to demonstrate that these antisera were not inhibiting cytolysis by trivial mechanisms such as (i) masking of target cell antigens by anti-target cell antibodies, (ii) agglutinating effector cells, thereby preventing effector-target cell contact, or (iii) toxicity to the effector cells. Dextran dispersion experiments revealed that these antisera inhibit the post-conjugate formation phase of cytolysis mediated by cytotoxic T lymphocytes. These antisera inhibited natural killer-like cytotoxicity and lectin-dependent cell-mediated cytotoxicity as well as lysis mediated by both mixed cell culture-generated cytotoxic T lymphocytes and cytotoxic T-lymphocyte cell lines. These findings suggest that these in vitro systems of cell-mediated lysis may share a common step; however the different classes of cytolysis may have been inhibited by separate antibodies in the antisera. This rapid reproducible method of generating inhibitory antisera will facilitate the study of the molecules involved in the post-conjugate formation phase of human cytotoxic T-lymphocyte-mediated lysis.

Developmental changes in humoral suppressor activity released from concanavalin A-stimulated human lymphocytes on B cell differentiation.

Cell-free culture supernatants (Con A-activated supernatants) were obtained by incubating peripheral blood lymphocytes (PBL) from cord blood, healthy children of various ages, and healthy adults with mitogenic doses of concanavalin A (Con A) for 48 hr. It is well known that human T lymphocytes are activated by Con A to manifest suppressor function in vitro. One mechanism whereby these suppressor cells act has been shown to be by the secretion of a soluble suppressor factor. The present study has investigated the Con A-inducible suppressor cell function in cord blood, children of various ages, and adults by comparing the ability of each Con A-activated supernatant to inhibit the generation of immunoglobulin-producing cells (Ig-PC) in pokeweed mitogen- (PWM) stimulated cultures of adult PBL. Con A-activated supernatants from adults could markedly suppress the generation of Ig-PC by allogeneic as well as autologous PBL in response to PWM. Such suppression appeared to be equally effective on the generation of IG-PC of 3 major classes, IgG, IgM, and IgA. On the contrary, Con A-activated supernatants from cord blood and newborn infants showed only a negligible suppression on PWM-induced adult B cell differentiation. But the suppressor activity found in Con A-activated supernatants gradually increased with advancing age, and reached approximately to the adult level at 4 yr of age or later. The results suggest that human T lymphocytes may be relatively deficient in their Con A-induced suppressor cell function in the early period of life.

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [ 3H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K + transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K +, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K + transport, controlling mitosis at distinct phases of the cell cycle.

Release of endogenous pyrogen-activating factor from concanavalin A-stimulated human lymphocytes.

Evidence is presented that the mitogen concanavalin A stimulates human lymphocytes to produce a nonpyrogenic lymphokine (LK) that is capable of activating human monocytes but not granulocytes to produce endogenous pyrogen (EP) in vitro. The potency of this preparation should facilitate further studies to purify and characterize this agent and determine its relation to other know LK. It seems likely that this factor, which we have called EP-activating factor (EPAF), plays a significant role in the development of fever in states of delayed hypersensitivity in man.

Pulse fluorimetry of [formula omitted] in concanavalin A-stimulated human lymphocytes

Human peripheral lymphocytes were cultured with a fluorescent probe, N-(1- pyrenesulfonyl)dipalmitoyl- l-α- phosphatidylethanolamine , and with concanavalin A. Fluorescence microscopic observations revealed that in lymphoblasts, pyrenesulfonyl dye was distributed mainly in vacuoles whereas in normal cells cultured without concanavalin A the dye was distributed exclusively in plasma membranes. The fluorescence spectra of the pyrenesulfonyl group incorporated into the cells exhibited two emission maxima, band A (monomer fluorescence of the pyrenesulfonyl group at about 400 nm) and band B (dimer fluorescence of the dye at about 500 nm). The values of the fluorescence lifetime measured at bands A and B indicated that in the absence of concanavalin A, the environment surrounding the pyrenesulfonyl group at the lipid/water interface became more hydrophilic with cultivation time. Concanavalin A made the environment of the interface more hydrophobic than that of lymphocytes cultured without concanavalin A. Fluorescence polarization measured at band A revealed that the mobility of pyrenesulfonyl monomers at the aqueous interface of the membranes was reduced upon concanavalin A stimulation.

Colchicine-Sensitive Structures and Lymphocyte Activation

The possible modulatory role of microtubules or closely related colchicine-sensitive structures in the response of human lymphocytes to mitogenic lectins was investigated. Colchicine (0.1 to 10 muM) AND VINBLASTINE (0.1 TO 10 MUM) inhibited early [14C]-aminoisobutyric acid and late [3H]-thymidine uptake in phytohemagglutinin-and concanavalin A-stimulated human lymphocytes but failed to alter 45Ca uptake. Lumicolchicine, an inactive congener of colchicine, was ineffective in all three systems. Both microtubular agents accentuated and prolonged the early cyclic AMP response to lectin. Little or no alteration in cyclic AMP levels was seen with colchicine or vinblastine alone or in combination with PGE (10MUM) or epinephrine (1muM) suggesting that the effect on cyclic AMP metabolism is largely selective for lectin stimulation. Neither microtunular agent altered 125I-concanacalin A binding. Since the inhibition of DNA synthesis was throughout the culture period and early aminoisobutyric acid uptake is affected, it appears that these agents are acting on an early event, or events, in the activation sequence. Although the mechanism of the inhibition is not known, the effect of colchicine and vinblastine in prolonging the cyclic AMP response to lectin may be involved. Alternatively, alterations in microtubules assembly may exert effects on membrane architecture interfering with propagation of the stimulus from the membrane to the cell interior.

Partial Characterization of Leukocyte Inhibitory Factor by Concanavalin A-Stimulated Human Lymphocytes (LIFCon A)

Abstract Leukocyte inhibitory factor (LIF) obtained from normal human lymphocytes stimulated by Concanavalin A was characterized by Sephadex gel filtration, disc electrophoresis, isopycnic centrifugation, enzymatic treatment and by immunoabsorbent studies. LIF activity was found after Sephadex G-100 gel filtration in a fraction containing molecules having the size of albumin (68,000 daltons). This is the same region where LIF activity derived from antigen stimulated lymphocytes has previously been found to elute. On disc gel electrophoresis at pH 9.1, peak LIF activity was eluted from the acrylamide gel fraction containing molecules that migrate with albumin. In isopycnic centrifugation studies, human LIF was shown to have a buoyant density similar to that of pure protein. Human LIF was inactivated by treatment with chymotrypsin but not by neuraminidase treatment. LIF activity could not be removed by incubating the material on columns containing Sepharose 6b coupled with anti-human serum albumin or anti-Fab. The characteristics of human LIF and migration inhibitory factor were compared.