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PubMed1983

Involvement of spectrin in cell-surface receptor capping in lymphocytes.

Human and mouse lymphocytes of T- and B-cell lineages express a protein (Mr, 240,000) that crossreacts with antibodies raised against chicken erythrocyte alpha-spectrin as judged by immunofluorescence, immunoprecipitation, and immunoautoradiography; by the same criteria, antibodies raised against chicken erythrocyte beta-spectrin do not react with any lymphocyte polypeptide. In all T and B cells analyzed, before surface-directed ligand challenge with concanavalin A and surface immunoglobulins the polypeptide antigenically related to erythrocyte alpha-spectrin is distributed diffusely at the plasma membrane. Upon challenge, the redistribution of this polypeptide is concurrent with that of the cell-surface receptors initially in patches and then in a cap. Immunoprecipitation of NaDodSO4-solubilized lymphocytes with erythrocyte alpha-spectrin antiserum shows that in all cases a polypeptide with the same apparent molecular weight as erythrocyte alpha-spectrin is precipitated. Variable amounts of another polypeptide (Mr, 235,000) are also coimmunoprecipitated. Immunoprecipitations and subsequent immunoautoradiography show that the lymphocyte polypeptide doublet has a composition similar to that of (brain) fodrin, a polypeptide doublet that previously has been found mainly in the cells of nervous tissue.

Cell Membrane↗
PubMed1978

Ontogeny of IgE-bearing lymphocytes in the rat.

IgE-bearing lymphocytes were detected by immunofluorescence in the spleen of neonatal Hooded Lister strain rats within 24 hr after birth. The same cells were detected in the bone marrow as early as the 4th day after birth. Both fetal spleen and liver obtained 1 day before birth contained IgM-bearing cells but no detectable IgE-bearing cells. The proportion of IgE-bearing cells in the spleen and bone marrow increased during the neonatal period and reached an adult level within 3 to 4 weeks after birth. In adult Hooded Lister rats, IgE-bearing cells were 3 to 6% of total spleen cells and 1.5 to 2.2% of bone marrow cells. Most of the IgE-bearing cells from bone marrow cells. Most of the IgE-bearing cells from both newborn and adult animals carried IgM determinants on their surface. Capping experiments showed that epsilon chain determinants and mu chain determinants belonged to separate molecules. IgG2a-bearing lymphocytes were detected in the neonatal spleen as early as the 4th day after birth, but a significant number of these cells was not detected in the bone marrow until the 4th week. In newborn spleen the percentage of IgE-IgM double bearing cells was higher than that of IgG2a-bearing cells.

Animals↗
PubMed1984

Altered surface distribution of both C3b receptors and Fc receptors on neutrophils induced by anti-C3b receptor or aggregated IgG.

Human neutrophils to which monospecific Fab' or F(ab')2 anti-C3b receptor had been bound at 0 degrees C were incubated for timed intervals at temperatures ranging from 0 degrees C to 37 degrees C, after which the cells were labeled with TRITC -conjugated second antibody. Neutrophils bearing Fab' anti-C3b receptor and incubated for up to 30 min at 37 degrees C, and cells bearing F(ab')2 anti-C3b receptor and incubated at 0 degrees C, exhibited diffusely distributed punctate clusters of receptors. Neutrophils bearing the bivalent anti-receptor and incubated at 30 degrees C or 37 degrees C for 5 min had redistributed C3b receptors into caps and patches that were associated with subplasmalemmal accumulations of myosin. The redistribution of cross-linked C3b receptors was inhibited by pretreatment of the neutrophils with either cytochalasin D or chlorpromazine. On approximately one-half of the cells demonstrating capped C3b receptors there was a corresponding redistribution of Fc receptors, as demonstrated by subsequent binding of FITC-aggregated IgG (FITC agg-IgG). In contrast, capping of C3b receptors did not alter the diffuse distribution of HLA-A on these cells. Cross-linking of Fc receptors on neutrophils by FITC agg-IgG also induced temperature-dependent capping of these receptors that was inhibited by cytochalasin D and chlorpromazine. In approximately one-half of the cells demonstrating capped Fc receptors, subsequent labeling of C3b receptors revealed a similar redistribution of these receptors. Thus, the neutrophil responds to cross-linking of either C3b receptors or Fc receptors by a cytoskeletal-dependent rearrangement of both receptors that causes their overlapping topographic distribution, demonstrating a form of cooperative interaction between these two types of receptors that are involved in the phagocytic reactions of these cells.

Animals↗
PubMed1977

Age related differences in binding of concanavalin A to plasma membranes of isolated neurons.

Neurons isolated from the lateral vestibular nucleus of young adult and senescent Fischer-344 rats were incubated with fluorescamine-labelled Concanavalin A (fl-Con A) alone, or following incubation in trypsin or Vibrio cholerae neuraminidase. They were then observed and photographed. Microdensitometric analysis of fluorescence micrographs showed that senescent rat neurons were significantly more fluorescent than those from young adult rats. Additionally, either patches or caps of fl-Con A were seen on the surface of neurons from senescent rats, while most young adult rat neurons bound fl-Con A uniformly. Pretreatment with trypsin or neuraminidase had no effect on the amount of fluorescence on the surface of senescent rat neurons, and only a slight effect on the surface distribution. Trypsin and neuronal plasma membranes of young adult rats and a rearrangement of the binding pattern in the majority of neurons observed.

Aging↗
PubMed1981

Functional activities of antibodies against brain-associated T cell antigens. I. Induction of T cell proliferation.

Rabbit anti-mouse brain antiserum mediated an in vitro cooperative interaction between Ig-anti-Ig column-purified Lyt-1+, Lyt-2- T cells and spleen B cells. DNA synthesis in the B cells was inhibited with mitomycin C, and the T cell proliferative response was measured by [3H]thymidine incorporation after 24 h of culture. The T-B cell interaction was genetically unrestricted, and the accessory function of the B cell was engaged via interaction with the Fc portion of the T cell-bound anti-mouse brain antibody since the F(ab')2 fragment was inactive. The rabbit anti-mouse brain anti-serum appears to recognize a unique antigenic determinant in the T cell membrane which triggers proliferation. Although brain-absorbed, rabbit anti-mouse thymocyte antiserum contained antibodies which bound to the T cell surface, it was incapable of inducing T cell proliferation. Furthermore, the Thy-1 molecule itself did not appear to be involved because allo- and xenoantisera against the allelic determinants were inactive, and the capping of Thy-1.2 from the T cell surface did not prevent the binding of the mitogenic component in rabbit anti-mouse brain antiserum. The T cell reaction to anti-brain antiserum is unlike the antigen-specific T cell response in being genetically unrestricted. It is possible that the antibody operates via the perturbation of a T cell determinant involved in the binding of T cell growth factors, and/or reception of B cell feedback signals.

Animals↗
PubMed2001

The role of surface ig binding in the activation of human B cells by phosphorothioate oligodeoxynucleotides.

Phosphorothioate oligodeoxynucleotides (sODNs) can induce T-cell-independent polyclonal activation of human B cells by a mechanism that depends on both sequence and back-bone structure. Because matrix-bound as well as soluble sODNs are mitogenic, this stimulation may result from the engagement of surface receptor(s). In order to investigate whether surface immunoglobin (Ig) could be a receptor for sODNs, the interaction of sODNs-fluorescein isothiocyanate (FITC) with Ig-coated beads was examined. sODNs specifically bound to human IgM and IgG. Moreover, binding of sODN to human B cells induced temperature-dependent capping of bound receptors and colocalization of FITC-sODN and IgM into aggregated caps on the surface of human B cells. A role of surface Ig was furthermore shown by observations that antibody-mediated capping of B-cell surface IgM or IgD inhibited subsequent binding of sODNs and that the capacity of sODN to stimulate human B cells was blocked by excess IgM or IgG, by nonstimulatory antibodies to sIgM, as well as by a variety of negatively charged molecules. Together, these results indicate that sODNs engage surface Ig by charge-charge interactions that lead to activation of human B cells.

Adult↗
PubMed1983

Detection of human malignant melanoma antigens by immunofluorescence and autologous postimmune antimelanoma sera.

The objectives of this study are to demonstrate the specificity of sera from melanoma patients undergoing autologous immunization and to localize melanoma antigens on a cellular level. Five melanoma patients were immunized with autologous melanoma cells and Bacillus Calmette-Guerin. This immunization program was conducted at Tulane University. Indirect immunofluorescence using both viable and fixed melanoma cells was employed. Four of five postimmune sera were reactive to five of seven melanoma cell lines. Two of the four reactive antisera showed positive binding with two additional melanoma lines obtained from other laboratories. All these sera were negative against seven nonmelanoma lines. Negative controls consisted of sera from 65 nonimmunized melanoma patients and 140 nonmelanoma patients. Membrane immunofluorescence (MIF) demonstrated sequential full MIF, capping, polarization, and extrusion of antigen-antibody complexes on the cell surface. MIF inhibition showed shedding of melanoma antigens in the culture medium. Ethanol, methanol, formalin, trichloroacetic acid, and acetone yielded sharp MIF. Isopentane and isooctane gave bright cytoplasmic fluorescence. In conclusion, this study provides suggestive evidence for the existence of common melanoma antigens as defined by the postimmune antimelanoma sera. These antigens may be localized in the membrane or within the cytoplasm.

Antigens, Neoplasm↗
PubMed1993

CD4:p56lck association studied in vivo using antibody-induced capping and double indirect immunofluorescence microscopy.

Accumulating data suggest that the T-cell surface antigen CD4 transduces an independent signal during antigen-mediated T-cell activation. In vitro studies which showed that the cytoplasmic protein tyrosine kinase p56lck is present in anti-CD4 immunoprecipitates led to the model that p56lck is associated with the cytoplasmic domain of CD4. In this report we have extended these studies and examined potential CD4:p56lck associations in vivo. We show here by double immunofluorescence microscopy a specific co-distribution of p56lck with antibody-induced CD4 caps in intact cells. Murine T-cell hybridoma lines expressing mutant forms of CD4 were used to demonstrate that the 31 carboxyterminal aminoacids of its cytoplasmic domain, in particular cysteine-420 and cysteine-422, are crucial for the formation of CD4:p56lck complexes in vivo. The potential of the method applied is discussed with regard to studies of other transmembrane signalling systems involving src-like kinases.

Amino Acid Sequence↗
PubMed1988

Ligand-loaded but not free complement receptors for C3b/C4b and C3d co-cap with cross-linked B cell surface IgM and IgD.

We have performed experiments to investigate possible physical interactions between C receptors (CR) and surface Ig (sIg) on the B cell plasma membrane. These molecules were found to be independent, non-linked, B cell surface structures, because capping CR1, CR2, sIgM, or sIgD with a specific antibody did not affect the distribution of the remainder of these molecules. Both CR1 and CR2, if bound by antibodies that did not independently cap CR, however, became associated with cross-linked sIg because CR that have been bound by intact anti-CR antibodies or their Fab fragments co-capped with sIgM or sIgD that had been bound by divalent anti-IgM or anti-IgD antibody. CR1 that had bound C3b similarly co-capped with sIg when sIg was cross-linked. Ligand-bound or even cross-linked CR did not associate with non-cross-linked sIg because sIgD, bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with CR that had been cross-linked by a sandwich of mouse anti-CR antibody and goat anti-mouse Ig. Other surface molecules, such as B1 and HLA-DR Ag, when bound by specific antibodies, did not cap with cross-linked sIg, and sIgD, when bound by a univalent Fab fragment of anti-IgD antibody, did not co-cap with cross-linked sIgM. Interactions between CR and sIg were not mediated by an association with IgG FcR because co-capping of CR and sIg was observed when F(ab')2 fragments of both anti-CR and anti-Ig antibodies were used. These results demonstrate that B cell surface CR can become associated with sIg, but only if sIg is cross-linked and CR is bound by anti-CR antibody or has bound its natural ligand.

Animals↗
PubMed1997

Modulation of FSH receptor phosphorylation correlates with hormone-induced coupling to the adenylate cyclase system.

The authors have recently demonstrated that an inhibitor of protein phosphorylation, staurosporine (SSP), can dramatically enhance follicle-stimulating hormone (FSH) stimulated cyclic adenosine monophosphate (cAMP) accumulation in rat granulosa cell line (GFSHR-17) overexpressing about 20-fold FSH receptor than primary granulosa cells. Moreover, incubation with SSP can partially release the cells from FSH-induced desensitization. In this work, it was examined whether coupling of FSH receptor to the adenylate cyclase is correlated with the degree of receptor phosphorylation. Immunoprecipitation of FSH receptor after metabolic labeling of the cells with 32P-orthophosphate revealed that preincubation of the cells with SSP resulted in pronounced reduction in FSH receptor phosphorylation compared to control cells, concomitantly with a dramatic increase in FSH-stimulated cAMP accumulation. In contrast, incubation of the cells with saturating dose of FSH, which leads to uncoupling between the receptor and the adenylate cyclase, resulted in enhanced receptor phosphorylation. Moreover, cells preincubated with FSH could be released from desensitization by further incubation with SSP and a significant reduction in FSH receptor phosphorylation. Immunostaining of the cells with FSH receptor antibody reveal a homogenous distribution of the receptor on the surface of SSP-treated cells. Some aggregation of the receptor was evident in control cells that were not treated with SSP. In contrast, massive clustering and capping of the receptor molecules were observed on the surface of FSH-stimulated cells. The current data suggest that phosphorylation-dephosphorylation of the receptor molecules play an important role in the degree of coupling between the receptor and the adenylate cyclase system. Moreover, desensitization to FSH stimulation that is implicated with high degree of receptor phosphorylation may lead to aggregation of the receptor molecules on the cell surface.

Adenylyl Cyclases↗
PubMed1982

The relationship between endocytosis of concanavalin A and phytohaemagglutinin receptors and blast transformation, and direct identification of individual rabbit lymphocytes reactive to both mitogens.

Distribution and modulation of rabbit lymphocyte phytohaemagglutinin acceptors and concanavalin A acceptors during activation of rabbit lymphocytes have been examined by electron microscopy. Two types of cell surface acceptors have been tentatively identified, lectin binding acceptors that do not modulate, and receptors that are endocytosed when blast transformation is stimulated. All of the cells have binding acceptors for both lectins. Endocytosis correlates with early blast transformation and serves as an early marker for lymphocyte activation. When examined after 24 h of culture, those cells that undergo blast transformation contain endocytosed lectin receptors, whereas small untransformed cells do not. Capping prior to endocytosis is rarely observed. The mechanism whereby the signal for transformation is maintained after the reaction of lectin with cell surface receptors and transposed to the nucleus is not known. Although we conclude that endocytosis is an early event required for cell activation, it is possible that endocytosis is secondary to other activation events. By evaluation of sequential endocytosis, individual rabbit lymphocytes that endocytose only concanavalin A, only phytohaemagglutinin, both concanavalin A and phytohaemagglutinin, or neither lectin, have been identified.

Animals↗
PubMed1980

Independence of chicken major histocompatibility antigens and tumor-associated antigen on the surface of herpesvirus-induced lymphoma cells.

Capping of chicken major histocompatibility (MHC) antigens on normal thymus, spleen, and peripheral blood leukocytes was demonstrated, although MHC antigens appeared to be present on only 15 to 18% of normal thymus cells. MHC antigen capping also occurred on cells from a Marek's disease herpesvirus-induced transplantable lymphoma (MDCT-NYM1). Capping of a Marek's disease tumor-associated surface antigen (MATSA) could be induced on MDCT-NYM1 lymphoma cells as well as on cells of two Marek's disease in vitro lymphoblastoid cell lines (MDCC-MSB1 and MDCC-LS1). Cocapping of MHC antigens and MATSA did not occur on MDCT-NYM1 lymphoma cells. The results suggest that MHC antigens and MATSA are not structurally associated on the cell membrane.

Animals↗
PubMed1986

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice.

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.

Animals↗
PubMed1985

Differential effect of isolated placental isoferritins on in vitro T-lymphocyte function.

The effect of isoferritins isolated from human term placenta on certain T-lymphocyte parameters was studied in vitro using normal human lymphocytes. These isoferritins differed in ion exchange affinity, isoelectric point, and subunit composition. Only the acidic isoferritins caused a marked suppression of phytohaemagglutinin (PHA) blastogenesis and the most acidic isoferritin ('Acid I') was suppressive at a concentration as low as 0.25 microgram/ml. All four isoferritins suppressed concanavalin A (Con A) blastogenesis in a similar concentration dependent manner, with maximum effect at an isoferritin concentration of 1 microgram/ml. Both basic and acidic isoferritins reduced the Con-A-capping phenomenon in normal lymphocytes at concentrations higher than 0.5 microgram/ml, but at 0.25 microgram/ml only the acidic isoferritin was effective. The above findings support our previous report concerning the suppressive effect of splenic ferritin on T-lymphocyte function in vitro and indicate that acidic isoferritins, which often predominate in malignancy, demonstrate a higher degree of immunosuppressive activity. Thus, acidic isoferritins may play a role in the development of abnormal lymphocyte function encountered in certain proliferative disorders.

Concanavalin A↗
PubMed1988

Partial expression of the lpr locus in the heterozygous state: presence of autoantibodies.

B6 mice heterozygous at the lpr locus (B6 +/lpr and B6 lpr/+) were compared with lpr homozygous mice (B6 lpr/lpr) and control mice (B6 +/+) for levels of serum immunoglobulin (Ig), presence of autoantibodies and rate of B-cell membrane immunoglobulin (mIg) capping. The total serum Ig levels in B6 +/lpr and B6 lpr/+ mice remained much below the high titres found in B6 lpr/lpr mice, and were close to the titres found in B6 +/+ mice. However, the presence of anti-single-stranded (ss) DNA antibodies and of anti-nuclear antibodies (ANA) was detected in most B6 +/lpr and B6 lpr/+ mice, although less frequently and in lower titres than in B6 lpr/lpr mice. The rate of mIg capping was higher in B6 +/lpr and B6 lpr/+ mice than in B6 +/+ mice, but the acceleration of the capping process remained inferior to the one found in B6 lpr/lpr mice. Therefore, the lpr locus is not totally recessive: some B-cell hyperactivity is expressed in the heterozygous state. This is in contrast with its lack of expression at the level of lympho-proliferation of the lpr-characteristic T-cell subset: none of the lpr heterozygous B6 mice displayed detectable lymphadenopathy.

Aging↗
PubMed1994

Antibody-induced changes on rabbit sperm surface inhibit gamete interaction.

Interaction of specific ligands with cell surface molecules may induce reorganization of surface components. A monoclonal antibody (B-12) against sperm surface antigens of 40kDa size induced molecules on the plasma membrane overlying the acrosome of rabbit sperm to cluster in small aggregates at 0 degree C (patching). At an elevated temperature of 37 degrees C these clusters of antigen antibody complexes collected into a large aggregate on one pole of the cell forming a cap (capping). This cap-like structure showed a reduction in size over a period of time and eventually disappeared from the sperm surface. Inhibition of capping by sodium azide indicated that it is an energy-dependent process. Patching of antigens did not require energy. Involvement of sperm head cytoskeleton in the process of capping was evident from potentiation of cap formation by cytoskeleton disrupting agents like cytochalasin B and D. Patching of antigen antibody complexes was not affected by either of the agents. The loss of antigen antibody complexes from sperm surface was mainly due to shedding of the complexes in the surrounding media. Sperm with patches of antigen antibody complexes did not adhere to oocytes. Sperm from the group where a majority of the sperm were denuded of the antigen antibody complexes also did not bind to oocytes.

Acrosome↗
PubMed1979

Hairy cell leukaemia: surface markers and functional capacities of the leukaemic cells analysed in eight patients.

In eight patients with hairy cell leukaemia (HCL) peripheral blood cells and in two patients also spleen cells were analysed for surface markers and functional capacities. Only cells containing the tartrate resistant isoenzyme 5 of the acid phosphatase were considered. Hairy cells (HC) of all patients were found to adhere spontaneously to glass and plastic surfaces and to spread after adherence like monocytes. They ingested latex particles of more than 1 micron diameter, but, in contrast to monocytes, did not phagocytose erythrocytes sensitized either by IgM or by IgG antibodies. HC of all patients bore Fc-receptors with a high binding affinity for aggregated IgG. Using 125 I-labelled F(ab')2-fragments of monospecific antibodies in autoradiography, only one light chain type was detected on HC of individual patients. In four patients mu- and delta-chains were simultaneously expressed on HC, whereas in two patients only gamma-chains and in one case only mu-chains were observed on HC. One patient showed a combination of gamma- and delta-chains on his HC. A great variation in density of surface immunoglobulings of HC was observed within individual patients. After removal by capping, surface immunoglobulin reappeared on HC during cell culture, but more slowly than on normal B-lymphocytes. As shown in two patients by internal labelling, HC secreted immunoglobulin light chains, but no heavy chains. On the basis of these findings the classification of HC as belonging to the B-cell lineage, rather than to the monocytic lineage, seems to be justified.

Adult↗
PubMed1981

The effect of oxidant injury on the lymphocyte membrane and functions.

We have studied the effect of oxidant stress on the lymphocyte membrane and lymphocyte functions. Lymphocyte cultures were incubated with xanthine oxidase and xanthine, an enzyme system known to generate several highly reactive oxygen compounds. We demonstrated that these lymphocytes were viable after exposure to an in vitro oxidant stress. However, there was a marked reduction in their ability to bind SRBCs and to form caps after Con A stimulation. These lymphocytes also demonstrated a delay in PHA-induced LBT, with maximal response occurring at 5 days instead of 3 days. Catalase, a hydrogen peroxide scavenger, protected lymphocytes from this injury, implicating hydrogen peroxide as the causative agent. Another lymphocyte membrane-related function, the ability to stimulate or respond in MLC, was not impaired after oxidant injury. These results demonstrate that after in vitro oxidant injury, lymphocytes may have alterations in the cell membrane and impaired function.

B-Lymphocytes↗