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Participation of histocompatibility antigens in capping of molecularly independent cell surface components by their specific antibodies.

The antibody-induced capping of several cell surface components has been investigated by immunofluorescence methods using two mouse cell lines, a parental C58 thymoma line and a mutant derived from it lacking TL and H-2 antigens. Other cell surface components were present in approximately equal amounts on the two cells. Parental cells treated with rabbit antibodies to T200, a major surface glycoprotein, rapidly formed caps containing T200, but the mutant cells similarly treated showed a uniform surface distribution of T200. On the other hand, with a secondary antibody treatment, the T200 on both cells capped equally well. When the indirect T200 caps were examined using a second immunofluorescent stain for H-2, TL, or Thy-1 antigens, it was found that on parental cells all three of these antigens were co-capped with T200; on mutant cells no staining was found for H-2 or TL, as expected, and essentially uniform distribution of Thy-1 was observed. The co-capping of H-2, TL, and Thy-1 antigens with T200 on the parent cell is remarkable, because the first three components are known to be molecularly independent in lymphocyte cell surfaces. The indirect capping of the viral glycoprotein gp 69/71 similarly induced a co-capping of H-2 and TL antigens on the parent cell. These results demonstrate that H-2 and related molecules may co-cap with a variety of independent cell surface antigens. Such co-capping of histocompatibility components could play an important role in a proposed dual recognition mechanism for cell-mediated cytotoxicity reactions and other immunologically important cell-cell interactions.

Antibodies, Neoplasm↗

Entamoeba histolytica: mechanism of surface receptor capping.

Binding of antibodies to the surface of the parasitic protozoan Entamoeba histolytica induces capping and release of antigen-antibody complexes, in a process that may be an effective means of evading the host immune response. To analyze the mechanism of capping in E. histolytica trophozoites, optimal conditions were determined for cap induction by sera from patients with amebic liver abscess, by polyclonal anti-amebic antibodies, and by concanavalin A. Cocapping of actin and myosin was documented by immunofluorescent staining of capped amebas using phalloidin, monoclonal anti-actin antibodies, and polyclonal anti-myosin antibodies. Immunoblots confirmed the presence of actin and myosin in isolated caps. These observations demonstrate that amebic cytoskeletal components participate in E. histolytica capping of surface receptors.

Actins↗

The redistribution of membrane surface immunoglobulin induces the rearrangement of some membrane integral proteins.

We investigated whether the redistribution of surface membrane receptors is associated with rearrangement of integral membrane proteins. Using a newly developed process, which combines histochemical analysis with an immunofluorescence or immuno-electron microscopy-staining technique, we studied the redistribution of two membrane-bound enzymes, 5'-nucleotidase and ATPase, on mouse splenic lymphocytes and B lymphoma cells induced by anti-mouse immunoglobulin antibodies. Labeling and capping of the membrane surface immunoglobulin induced a similar rearrangement of both 5'-nucleotidase and ATPase from uniform distribution at 4 degrees C into 'patches' and caps at 37 degrees C.

5'-Nucleotidase↗

Inhibition of antigen-induced and anti-immunoglobulin-induced capping by hydrocortisone and propranolol.

The redistribution of lymphocyte surface immunoglobulin (Ig) induced by anti-Ig antibody or bound sheep erythrocyte antigen (capping) was inhibited reversibly by hydrocortisone succinate (2 x 10(-4) M). Capping inhibition was also effected by cholesterol and progesterone, favoring the hypothesis that the effect was mediated through changes in the physical properties of the cell membrane rather than hormonal action. Capping was also inhibited by propranolol at concentrations where it may exert a local anesthetic as well as a beta-adrenergic blocking effect (2 x 10(-4) M). Since a fourfold excess of the beta agonist isoproterenol failed to restore capping in the presence of propranolol, the local anesthetic mode of action is most likely. Nitroprusside, an inhibitor of contraction in vascular smooth muscle and platelets, failed to inhibit capping.

Animals↗

Engagement of p75/AIRM1 or CD33 inhibits the proliferation of normal or leukemic myeloid cells.

P75/AIRM1 is a recently identified surface molecule that belongs to the sialoadhesin family and displays homology with the myeloid cell antigen CD33. In lymphoid cells, p75/AIRM1 is confined to natural killer cells and mediates inhibition of their cytolytic activity. In this study, we show that p75/AIRM1 is also expressed by cells of the myelomonocytic cell lineage, in which it appears at a later stage as compared with CD33. In vitro proliferation and differentiation of cord blood-derived CD34(+) cells (induced by stem cell factor and granulocyte-macrophage colony-stimulating factor) were consistently inhibited by the addition of anti-p75/AIRM1 mAb. Engagement of CD33 led to inhibition in some experiments. A sharp decrease of cell proliferation/survival was detected in all three p75/AIRM1+ chronic myeloid leukemias analyzed when cultured in the presence of either anti-p75/AIRM1 or anti-CD33 mAbs. Thus, the present study suggests that p75/AIRM1 and CD33 may play a regulatory role in normal myelopoiesis and may be viewed as suitable target molecules to counteract the proliferation/survival of chronic myeloid leukemias.

Antibodies, Monoclonal↗

Detection and characterization of monoclonal antibodies specific to IgE receptors on human lymphocytes by flow cytometry.

BALB/c mice were immunized with human lymphoblastoid cells (RPMI 8866 cells) expressing surface receptors for IgE (Fc epsilon R). Spleen cells from animals displaying high titres of anti-Fc epsilon R antibodies were fused with HGPRT-deficient NSI myeloma cells. Anti-Fc epsilon R antibodies were identified by a flow cytometric assay based on their ability to block the binding of IgE-coated fluorescent latex particles to Fc epsilon R-positive cells. Fourteen monoclonal hybridoma cell lines secreting antibody of the required specificity were amplified in tissue culture and then grown in the peritoneal cavity of BALB/c mice in order to obtain ascitic fluids with high antibody titres. The specificity of each monoclonal antibody (Mab) to lymphocyte Fc epsilon R was shown by the following observations: (i) the intact monoclonal antibody molecule or, in some cases, its F(ab')2 fragments blocked the binding of IgE to several Fc epsilon R(+) cell lines different from that employed for the initial immunization; (ii) the Mab bound directly to all the Fc epsilon R(+) cell lines tested, but not to several Fc epsilon R(-) cells as determined by indirect immunofluorescence; (iii) the binding of Mab to Fc epsilon R(+) cells was selectively blocked by IgE, but not by the other classes of Ig; and (iv) Mab had no effect on the binding of IgG to Fc gamma R on normal human peripheral blood mononuclear cells (PBMC).

Animals↗

Relationship of microtubule organization in lymphocyte to the capping of immunoglobulin.

We have used a double fluorescence staining procedure to examine the distribution of antitubulin-staining structures in mouse splenic lymphocytes induced to patch and cap surface immunoglobulin by treatment with antiimmunoglobulin antibodies. A well organized network of fibers, extending from a single microtubule organizing center (MTOC) associated with the centriole pair, was detected by antitubulin staining at all stages of the capping process. In lymphocytes possessing as intact network, the cap was formed and internalized over the region of the cytoplasm containing the organizing center, Golgi apparatus, and most other organelles. In contrast, in lymphocytes which had been subjected to a colchicine treatment sufficient to completely disassemble networks prior to cap induction, no relationship was detected by immunofluorescence between the position of the centrioles (a marker for the MTOC) and the location of the surface cap. Electron microscopic observation of colchicine-treated cells, induced to cap by ferritin-conjugated anti-Ig, revealed extensive disorganization of cytoplasmic organelles and exclusion of organelles from the region of underlying the cap. These results indicate a role for the microtubule network of lymphocytes in maintaining cytoplasmic polarity and in specifying the site of Ig cap formation.

Animals↗

Inhibition of capping of immunoglobulin and concanavalin A receptors by cis-dichlorodiammine-platinum-(II) in mouse spleen cells.

Effects of cis-dichlorodiammineplatinum-(II) (cis-DDP), an anticancer agent, on capping of membrane receptors were investigated in mouse spleen cells in vitro, using fluorescein labeled ligands. Cap formations of surface immunoglobulin and of concanavalin A receptors were significantly reduced by cis-DDP in relation to dose, while cell viability remained unchanged. The binding of the ligands to specific receptors was not affected by cis-DDP. The results suggested that cis-DDP inhibited cap formation by blocking membrane movement of the ligand-receptor complexes.

Animals↗

Limited mobility of membrane receptors of adhered or proteose peptone-stimulated macrophages.

The mobility of the membrane receptors of macrophages, obtained from normal and proteose peptone-stimulated mice, was studied by counting the cells which form patches or caps after incubation with anti-H-2a antibody or soybean agglutinin (SBA). The test was carried out with macrophage suspension by the indirect fluorescence method. When macrophages from unstimulated donors were used, the antibodies against H-2a antigens bind at 4 degrees C to the surface of the macrophages in the form of small aggregates; only with SBA lectin a larger percentage of cells (20%) does form caps even at this low temperature. After heating the cells to 37 degrees C, a significant increase in the number of cells with receptors in caps was observed in the control mice. When macrophages from stimulated donors were used, no cap formation was observed with any of the ligands, even after a 120-minute-incubation; the ligand-receptor complexes form patches predominantly on the membrane. The pretreatment of macrophages with colchicine did not lead to a cap formation. A similar inhibition of the capping phenomenon also occurred with macrophages obtained from unstimulated mice, but tested in a monolayer.

Animals↗

Polymerization of additional actin is not required for capping of surface antigens in B-lymphocytes.

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.

Actins↗