concanavalin-a and merocyanine-dye

Merocyanine 540 (MC 540) is a photoactive dye used to purge bone marrow of tumor cells in autologous bone marrow transplantation. The effects of MC 540 on the lymphoid components in the marrow are unknown. This study evaluates the treatment of lymphocytes by MC 540 (15 micrograms/mL) and light (70 W/m2) on: (1) phytohemagglutinin and Con A-induced proliferation; (2) allogeneic mixed lymphocyte cultures (MLC); (3) the regulation of Ig synthesis by T cells; and (4) the ability of B cells to produce polyclonal Igs as measured by an enzyme-linked immunosorbent assay-plaque assay. The results show that MC 540 and light treatment reduced Con A-stimulated T-cell proliferation greater than 50% after 30 minutes and greater than 80% after 60 minutes of MC 540-sensitized photoirradiation. Ninety minutes of MC 540 and light exposure (designated treatment) inhibited MLC greater than 90%. In polyclonal Ig synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited Epstein-Barr virus-stimulated Ig synthesis. These data show that T- and B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions.

Topics: B-Lymphocytes; Cells, Cultured; Concanavalin A; Fluorescent Dyes; Herpesvirus 4, Human; Humans; Immunoglobulins; Light; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Phytohemagglutinins; Pokeweed Mitogens; Pyrimidinones; T-Lymphocytes; T-Lymphocytes, Helper-Inducer

The fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen-induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial system.

Topics: Age Factors; Animals; Cell Differentiation; Concanavalin A; Cortisone; Humans; Lipopolysaccharides; Lymphocytes; Membrane Lipids; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Fluorescence; Pokeweed Mitogens; Pyrimidinones; Spleen; Staining and Labeling; Thymus Gland

Experiments were carried out to examine the possible physiological role of disordered membrane domains in hematopoietic cell surface differentiation. The hematopoietic stem cell line 416B has been shown to bind the dye merocyanine 540 (MC540), a fluorescent probe which may be specific for disordered regions of lipid bilayers (Williamson et al., 1981). The surface receptors for the lectins Concanavalin A (Con A) and wheat germ agglutinin (WGA) exhibit patchy distributions on the surface of 416B cells which correspond to the distribution of MC540 binding regions. Appropriate incubation of these cells with either of the two lectins results in the induced formation of a cap. The binding regions for MC540 and the receptors for the other lectin become localized to the same region of the membrane by this process. Such coordinated rearrangements of surface glycoproteins associated with disordered lipid domains may play a role in the cocapping of disparate surface molecules (Raz and Bucana, 1980) or in differentiation-related cell surface rearrangements (Schlegel et al., 1980).

Topics: Animals; Cell Line; Concanavalin A; Glycoproteins; Hematopoietic Stem Cells; Lectins; Membrane Proteins; Mice; Pyrimidinones; Receptors, Concanavalin A; Receptors, Mitogen; Wheat Germ Agglutinins