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PubMed1980

Fc receptors on rabbit lymphocytes. Identification and organ distribution of rosette-forming cells; cocapping with surface immunoglobulin.

Fc receptor (FcR)-bearing cells were demonstrated using ox erythrocytes coated with homologous IgG-type antibodies (EA gamma) in rabbit peripheral blood leukocytes (PBL) and in various lymphoid organs. Discrimination of the rosette-forming cells (RFC) is carried out after prior ingestion of tetramethylrhodamine isothiocyanate-labeled latex particles and in transmission electron microscopic studies. Most of the nonlymphoid cells (5-10%) in PBL and spleen cell suspensions expose FcR. These nonlymphoid cells are almost absent in other lymphoid organs, except in bone marrow. The average percentage of cells rosetting with IgG-sensitized erythrocytes (EA gamma RFC) in lymphoid cell preparations of the various tissues was as follows: PBL 25%, bone marrow 65%, appendix 37%, spleen 40%, Peyer's patches 44%, thymus 2% and peripheral lymph node 27%. The nature of FcR-bearing PBL was further studied using F (ab')2 anti-IgM, anti-IgA or anti-T cell conjugates. About half of the population of B cells, bearing IgM or IgA express FcR. Moreover, about 80% of the RFC are found within the B cell population. Only a few T cells were found rosetting with EA gamma suggesting that most of the non-B lymphoid RFC are "null" cells. In different lymphoid organs, the percentages of EA gamma RFC and B cells are comparable but not identical A greater part of the EA gamma RFC also expresses the receptor for the third component of complement. After capping of membrane IgM determinants, FcR is located in the same cap on the majority (60%) of the FcR-positive IgM-capped cells.

Animals↗
PubMed1980

Triggering of lymphocyte capping appears not to require changes in potential or ion fluxes across the plasma membrane.

Capping induced by anti-Ig antibody on mouse spleen lymphocytes was found to proceed normally over a wide range of membrane potentials from approx. 0 to -65 mV, as estimated with fluorescent probes. The potential was manipulated by ionic substitution in the medium and/or application of gramicidin. Various agents which inhibit capping had differing effects on the membrane potential, some producing no measurable change, others depolarising the cells. In particular valinomycin (10-7 M) was found to inhibit capping in cells both slightly hyperpolarised from the normal resting potential, and fully depolarised. Valinomycin was found to deplete the lymphocytes markedly of ATP and this effect was sufficient to account for the inhibition of capping. Capping occurred in a simplified (sucrose) medium lacking Na+, K+ and Ca2+, suggesting that fluxes across the plasma membrane of these ions are not required. It is concluded that after ligand binding, some reorganisation of receptor protein at the inner face of the membrane is the sufficient stimulus for the intracellular rearrangements involved in capping.

Animals↗
PubMed1977

Ligand-independent cap formation: redistribution of surface receptors on mouse lymphocytes and thymocytes in hypertonic medium.

Most of the mobile receptors on mouse lymphocytes and thymocytes, including immunoglobulins, H-2 antigens, Thy-1.2 antigens, some concanavalin A receptors, and some antigenic determinants detected by anti-thymocyte serum, were redistributed into caps when the cells were incubated in hypertonic medium (about 600 mOsM) in the absence of ligands. The caps reverted to the original distributions if the cells were transferred again to isotonic medium. The viability of the cells was not decreased after incubation in the hypertonic medium. Ligand-independent cap formation appeared to depend upon cellular metabolism. Different species of receptors appeared to move with different mobilities during the process of ligand-independent cap formation. Most microvilli on cells showing caps in hypertonic medium were associated with the regions of the caps. These results suggest that free receptors can be induced to form caps if the receptors are allowed to interact with the machinery of cap formation under special conditions.

Animals↗
PubMed1979

Capping of exogenous Forssman glycolipid on cells.

When motile cells are incubated with Forssman glycolipid, the antigen is incorporated into the cells' plasma membranes. If cross-linked by antibody, the patched glycolipids cap. This process is sensitive to those drugs that are known to inhibit capping of protein antigens. The results support a flow mechanism for capping.

Animals↗
PubMed1983

Capping of cholera toxin-ganglioside GM1 complexes on mouse lymphocytes is accompanied by co-capping of alpha-actinin.

We used cholera toxin, which binds exclusively and with a high affinity to the ganglioside GM1, as a probe to investigate the distribution of this glycolipid on the surface of mouse lymphocytes. When lymphocytes are incubated with cholera toxin (or its B subunit) and then sequentially with horse anti-toxin and FITC-swine anti-horse Ig at 37 degrees C, the cholera toxin-ganglioside GM1 complex is redistributed to a cap at one pole of the cell. The capping of cholera toxin-GM1 complexes is slower than the capping of surface-Ig complexes, requires two antibodies, and is inhibited at high toxin concentrations. Cholera toxin-GM1, like surface-Ig capping, is an energy-dependent process and is inhibited by sodium azide, low temperatures, or cytochalasin B, but is unaffected by demecolcine. An affinity-purified antibody against alpha-actinin was used to examine the distribution of this cytoskeletal component during the capping process. 88% of the cells that had a surface Ig cap displayed a co-cap of alpha-actinin, and 57% of the cells that had a cholera toxin-GM1 cap displayed a co-cap of alpha-actinin. Time course studies revealed similar kinetics of external ligand cap formation and the formation of alpha-actinin co-caps. We conclude that capping of a cell-surface glycolipid is associated with a reorganization of the underlying cytoskeleton. The implications of such an association are discussed in the context of current models of the mechanism of capping.

Actinin↗
PubMed1982

Lymphocyte capping: a diagnostic method in progressive muscular dystrophy?

Recent observations indicate that antibody-induced redistribution ("capping") of membrane antigens of B-lymphocytes is subnormal in patients with progressive muscular dystrophy. The present study was primarily designed to verify or refute claims that such an abnormally low capping capacity can be used to diagnose this disease group. In eight dystrophy patients we found the median capping to be reduced to 40%, while it was 52% in 27 neurological control patients, and 62% in 20 healthy blood donors. However, there was considerable overlap between the capping percentages of the three groups. Thus, our data indicate that the demonstration of reduced lymphocyte capping in the individual patient is of little, if any, diagnostic value.

Female↗
PubMedDate not supplied

Evidence from capping experiments for independence of the RT7 alloantigen and the leucocyte common antigen in the rat.

Experiments reported here demonstrate that the RT7 alloantigen and the L-C antigen are separate and distinct structures on the surface of rat lymphocytes. The distribution of the antigens in different rat strains, including the mutant WF/fz, clearly establish the RT7 antigenic system as a polymorphic diallelic system, whereas the recognized L-C antigenic determinant is monomorphic and present on the lymphocytes of all rat strains tested. These data were obtained using monoclonal antibodies to the antigens in indirect immunofluorescence experiments. The two antigens were shown to redistribute (cap) independently of one another on the surface of rat thymocytes. Cells that had been exposed to anti-L-C antibody and FITC-conjugated anti-Ig followed by anti-RT7.1 antibody and RITC-conjugated anti-Ig demonstrated FITC caps and RITC rings.

Animals↗
PubMed1980

Lectin-mediated induction of human neutrophil chemotaxis, chemokinesis, and cap formation.

Six lectins, including concanavalin A, phytohemagglutinin P, castor bean I, wheat germ agglutinin, peanut agglutinin, and pokeweed mitogen, were studied for their ability to stimulate human neutrophil locomotion and cap formation. Five of these lectins with known monosaccharide specificities, including concanavalin A, phytohemagglutinin, P, castor bean I, wheat germ agglutinin, and peanut agglutinin, were found to stimulate human neutrophil migration in a modified Boyden assay. Pokeweed mitogen showed negligible activity in the locomotion assay as compared with other lectins. Tests were performed to determine if the observed neutrophil migration in response to lectins was directional, and it was found that concanavalin A, phytohemagglutinin P, and peanut agglutinin were both chemokinetic and chemotactic, whereas castor bean I was only chemokinetic. Wheat germ agglutinin could not be declared chemotactic or chemokinetic due to its tendency to agglutinate neutrophils. Studies with fluoresceinated lectins demonstrated that lectins which stimulate neutrophil migration also bind to neutrophil surfaces. Preincubation with specific monosaccharide ligands blocked both stimulated locomotion and fluorescence, suggesting that an available lectin-binding site was required both for lectin binding and the stimulation of migration. Additional experiments indicated that fluoresceinated concanavalin A, phytohemagglutinin P, castor bean I, wheat germ agglutinin, and peanut agglutinin all induce cap formation on the neutrophil.

Binding Sites↗
PubMed1987

Concanavalin A-induced cap formation in rat ascites hepatoma cells (AH 7974) and the interaction of cytoplasmic proteins with plasma membranes.

Concanavalin A (Con A) induced cap formation in rat ascites hepatoma cells (AH7974). In these Con A-treated cells, the association of cytoplasmic proteins with cell membranes was suggested by observing their Triton shells. The transition from G-actin to F-actin occurred in these cells. The association of membrane lipid with cytoplasmic proteins extracted from AH cells was studied by the isolation of protein-bound liposomes and phase transition release. The analysis of isolated liposomes revealed that many cytoplasmic proteins which specifically associated with liposomes were cytoskeletal elements including F-actins. The association of proteins with liposomes was affected by the lipid composition of the liposomal membrane and by the Ca2+ concentration of the incubation medium. The strong interaction of liposomal membrane with cytoplasmic proteins or isolated cytoskeletal proteins was demonstrated also by phase transition release using carboxy fluorescein-containing liposomes. These experiments showed that there was a strong affinity between lipid membrane and cytoskeletal elements including F-actins and that the amount of F-actin increased due to Con A treatment. The association of the submembranous microfilaments with the cell membrane may contribute to capping of the cells caused by Con A.

Actins↗
PubMed1981

Capping of surface immunoglobulin on rabbit and mouse lymphocytes. I. Kinetics.

The capping behaviour of surface immunoglobulin (sIg) on rabbit and mouse B lymphocytes was investigated in the direct immunofluorescence test. Rabbit B lymphocytes showed fewer caps than mouse lymphocytes. This was not due to a lower rate of cap formation, but to a smaller subpopulation of cells able to form caps. This subpopulation was age-dependent in rabbits; it increased from 25% of sIg positive cells at 2 to 3 months to 50% at 2 to 3 years of age. The capping rate was not significantly different in rabbits and C57B1 mice, but it was much higher in Swiss mice. Endocytosis did not occur at temperatures below 30 degrees C. In mouse B cells endocytosis occurred exclusively in the cap configuration. In rabbit B lymphocytes, on the other hand, two types of endocytosis were observed: first, a rather rapid endocytosis during the formation of the patch configuration, occurring on small lymphocytes, and second, a rather slow endocytosis during the cap configuration, occurring on large lymphocytes.

Age Factors↗
PubMedDate not supplied

Possible roles of compound membrane receptors in the immune system.

A compound receptor is defined as a molecular complex which attains its final, biologically active structure by rearrangement and assembly in the membrane of several structural subunits. The existence of such receptor complexes is strongly suggested by experiments which demonstrated a very close association between certain ligand (primarily insulin) receptors and the major histocompatibility complex (MHC) antigens. We present here speculations on the possible role of MHC as a structural part of a multitude of receptors with different biological functions, and we argue that recent immunological data on minor histocompatibility antigens, antigen-presenting cells and T-lymphocyte receptors fit into this hypothesis.

Animals↗
PubMed1977

Mechanism of lymphocyte activation: the binding of phytohemagglutinin to the lymphocyte surface.

The dynamics of phytohemagglutinin (PHA)-lymphocyte interaction was studied using 125I-labeled PHA (leucoagglutinin) and pig mesenteric lymph node lymphocytes that had been depleted of erythrocytes, dead cells, adherent cells and immunoglobulin-bearing cells. Evidence was obtained that PHA stimulated the majority of the lymphocytes to transform. Binding of PHA at 37 degrees C was fairly rapid (rate constant for association: 2.6 X 10(5) M-1 sec-1), saturable, reversible and specifically inhibited by N-acetylgalactosamine (Kdiss: 3 X 10(-4) M) and unlabeled PHA. A Scatchard plot was curvilinear and gave evidence for 3.6 X 10(5) binding sites per cell comprising 8.7% of high affinity sites (Kdiss: 3.7 X 10(-9) M) and 91.3% of lower affinity (Kdiss: 1.4 X 10(-7) M). About 20% of the sites were occupied under culture conditions giving maximal transformation. Alternative explanations for the curvilinear plot included negative cooperative interactions and/or increase in affinity through multivalent interaction. Negative cooperativity was supported by the demonstration that free PHA promoted the dissociation of bound PHA. Binding was not affected by metabolic inhibitors, and binding to purified lymphocyte plasma membrane resembled that to whole cells. These results suggested that PHA binding to whole lymphocytes was not grossly influenced by "capping", endocytosis and shedding.

Acetylgalactosamine↗
PubMed1978

The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections.

Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 1 micrometer thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F-actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.

Actins↗
PubMed1982

Kinetic evidence for a common mechanism of capping on lymphocytes.

1. Differences in the rates at which ligands cap various receptors on the same cells, and their sensitivity to various drugs, have been interpreted as evidence that there are distinct mechanisms for ;fast' and ;slow' cap formation. We have examined the factors which determine the rate of cap formation of three receptors on mouse splenic lymphocytes or thymocytes, and compared the effects of cytochalasin B or colchicine under conditions where the different receptors cap at similar rates. 2. When surface immunoglobulin, concanavalin A receptors, or theta antigen are induced to cap at their maximal rates by appropriate concentrations of one or more cross-linking ligands, the half-time for maximal capping of each receptor population is between 1.5 and 3.0min at 37 degrees C. Slower rates of cap formation are obtained by using non-optimal concentrations of the cross-linking ligands. 3. When the three receptors were induced to cap at similar rates (either maximal or slower), 10mum-cytochalasin B caused a similar decrease in the rate of cap formation for each receptor, without affecting the eventual extent of capping. At comparable capping rates on control cells, colchicine (10mum) increased the rate of cap formation for surface immunoglobulin and concanavalin A receptors to a similar extent, without affecting the eventual extent of cap formation. In contrast, colchicine had no detectable effect on the capping of theta antigen. 4. From these results, we conclude that there are no intrinsic differences in the rates at which different receptors can be induced to cap that can be used to diagnose differences in their mechanisms of cap formation. The observation that ligand concentration and the drugs acting on the cytoskeleton generally affect the rate but not the extent of cap formation accounts for the wide variation in reported effects of the drugs on cap formation measured at fixed times. The receptor-specific effect of colchicine on surface immunoglobulin and concanavalin A receptors, but not theta antigen, is not readily compatible with models of cap formation which depend on lipid or membrane flow.

Animals↗
PubMed1981

Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.

Colchicine induces the clustering of at least three different T-lymphoma surface antigens (T200, Thy-1, and gp 69/71) into a cap structure in the absence of any external ligand. In addition, colchicine induces the intracellular accumulation of actin and myosin directly beneath the surface cap structure. We have discovered that myosin molecules (both heavy and light chains) are closely associated with the plasma membrane of T-lymphoma cells. Most importantly, we have found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of colchicine-induced capped cells. It is proposed that myosin light chain is directly involved in the activation of membrane-associated actomyosin required for the collection of surface proteins into a cap structure (analogous to muscle cell sliding filament contraction).

Actins↗
PubMed1989

Cigarette smoke impairment of human lymphocyte function by inhibition of transglutaminase.

The in-vitro and in-vivo effects of cigarette smoke were studied in human peripheral blood lymphocytes by applying a method for the capping of beta 2-microglobulin- or phytohaemagglutinin (PHA)-stimulated lymphocyte transformation (measured as (3H)thymidine incorporation) involving the transglutaminase pseudosubstrate monodansylthiacadaverine (MDTC), whose presence resulted in significantly reduced capping and (3H)thymidine incorporation in a concentration-dependent manner. The addition of dimethyl sulphoxide-soluble particles from cigarette smoke to lymphocytes in vitro significantly reduced the capping ability and the PHA-induced (3H)thymidine incorporation. Whereas no significant change in MDTC-dependent capping inhibition was seen in lymphocytes from smokers after 10 d abstinence from smoking. there was a marked decrease in (3H)thymidine incorporation in lymphocytes from smokers after smoking three cigarettes following 10 h abstinence. The tentative conclusion is that exposure to cigarette smoke, or smoke extract, impairs MDTC-dependent capping inhibition and PHA-stimulated lymphocyte transformation by transglutaminase inhibition.

Adult↗
PubMed1980

Enumeration and characterization of bovine blood, spleen and lymph node cells containing immunoglobulins.

In this study, the average percentage of bovine spleen and lymph node cells with surface immunoglobulin (S-Ig) was found to be 19.62 and 23.18%, respectively. The average percentage of these cells with cytoplasmic immunoglobulin (C-Ig) was 14.46 and 17.21%. Only the percentage of cells with S-Ig showed a strong correlation between the spleen and the lymph node. Also, several methods of removing S-Ig from bovine peripheral blood mononuclear cells were investigated. It was found that the loss of passively bound S-Ig by warm washing was minimal, and that the anti-Ig-treated mononuclear blood cells would lose their caps after 24 h of incubation at 37 degrees C, but not after 45 min of incubation, although 95% of the cells with S-Ig were capped within 15 min.

Animals↗
PubMed1990

Interactions between a lymphoma membrane-associated guanosine 5'-triphosphate-binding protein and the cytoskeleton during receptor patching and capping.

In this study we have used several complementary techniques to isolate and characterize a lymphoma membrane-associated 41-kDa protein that shares a number of structural and functional similarities with the alpha i subunit of the guanosine 5'-triphosphate (GTP)-binding protein (e.g., Gi alpha-like protein). In addition, using permeabilized lymphoma cells, we have found that: 1) GTP or GTP-tau-S augments, and pertussis toxin inhibits, phospholipase C (PLC) activity and receptor capping; and 2) the addition of lymphoma 41-kDa Gi alpha-like protein stimulates PLC activity and receptor patching/capping, and reverses the inhibitory effect of pertussis toxin on both activity and receptor patching/capping. Additional cytochemical and biochemical data indicate that the lymphoma 41-kDa protein is closely associated with several cytoskeletal proteins (e.g., actin, myosin, and fodrin) all of which colocalize under receptor cap structures. Furthermore, both the 41-kDa-mediated phospholipase C activity and receptor patching/capping are inhibited by cytochalasin D (a microfilament disrupting drug) and W-7 drug (a calmodulin inhibitor). Together, these data provide strong evidence for a functional association between the lymphoma membrane cytoskeleton and the 41-kDa (Gi alpha-like) protein. Specifically, this association appears to be required for the activation of phospholipase C that results in inositol triphosphate production, subsequent internal Ca2+ release, and finally surface receptor patching and capping.

Animals↗