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PubMed1980

Modulation of Epstein-Barr virus release from cells by components of normal human serum.

By filtration of normal human serum through a Sephadex G-200 column, an anti-virus release factor (AVRF), which is capable of inhibiting the release of Epstein-Barr virus (EBV) from cells cultured in vitro, was found in the fractions corresponding to IgM. Another component, antagonistic to the activity of AVRF, was found in the fractions close to those of albumin. Both AVRF and anti-AVRF were found in all sera from four EBV seropositive and three sero-negative adults tested. EBV-release inhibition by AVRF was reversible. AVRF did not neutralize virus infectivity or inhibit intracellular virus growth. Virus adsorption on to cells was not prevented by AVRF but cap formation of EBV antigens on cells was augmented by it.

Antibodies, Viral↗
PubMed1990

The lymphocyte-specific protein LSP1 is associated with the cytoskeleton and co-caps with membrane IgM.

LSP1 is a lymphocyte-specific intracellular Ca2(+)-binding protein. We found previously that a fraction of the total cellular pool of LSP1 protein accumulates at or near the cytoplasmic face of the plasma membrane. LSP1 protein was also shown to be present in the cytoplasm. Here we report that approximately 10% of the total intracellular LSP1 protein is associated with the Nonidet P-40 insoluble cytoskeleton of the mIgM+, mIgD+ B lymphoma cell line BAL17. Variation in conditions of extraction did not alter this value. To rule out the possibility that LSP1 associates with the nucleus that is also present in the detergent insoluble pellet, we prepared a separate nuclear fraction essentially free of cytoskeletal material and found only trace amounts of LSP1 protein. After accounting for yield losses during subcellular fractionation by measuring the recovery of 125I-labeled membrane IgM, or of the cytoplasmic marker enzyme lactate dehydrogenase activity, the LSP1 in membrane fractions was calculated to represent approximately 30% of the total cellular LSP1 and cytoplasmic LSP1 accounted for approximately 55% of the total. Approximately 75% of the plasma membrane LSP1 protein was soluble in 1% Nonidet P-40 containing buffer, indicating that the majority of the LSP1 in the plasma membrane fraction was distinct from the cytoskeletal LSP1 protein. The preparation of membrane fractions in the presence of 1 M NaCl, or washing of membranes in 3 M KCl did not diminish the levels of membrane LSP1. These results show the existence of three discrete intracellular LSP1 pools. Double label immunofluorescence studies showed that the peripheral ring-like distribution of LSP1 in BAL17 cells became a distinct cap upon cross-linking the mIgM. These intracellular LSP1 caps were always found to be located directly underneath the mIgM caps.

Animals↗
PubMed2001

Costimulation and endogenous MHC ligands contribute to T cell recognition.

To initiate an immune response, key receptor-ligand pairs must cluster in "immune synapses" at the T cell-antigen-presenting cell (APC) interface. We visualized the accumulation of a major histocompatibility complex (MHC) class II molecule, I-E(k), at a T cell-B cell interface and found it was dependent on both antigen recognition and costimulation. This suggests that costimulation-driven active transport of T cell surface molecules helps to drive immunological synapse formation. Although only agonist peptide-MHC class II (agonist pMHC class II) complexes can initiate T cell activation, endogenous pMHC class II complexes also appeared to accumulate. To test this directly, we labeled a "null" pMHC class II complex and found that, although it lacked major TCR contact residues, it could be driven into the synapse in a TCR-dependent manner. Thus, low-affinity ligands can contribute to synapse formation and T cell signaling.

Animals↗
PubMed1984

Immunofluorescent studies of the rat adipocyte cell surface.

Antibodies against rat adipocyte plasma membranes have been shown to mimic insulin action in isolated adipocytes (Pillion & Czech, J. biol. Chem. Vol. 253, pp. 3761 - 3764, 1978). Immunofluorescent studies with antimembrane antibodies reveal capping on the adipocyte cell surface. Dose - response studies showed that the number of fat cells with obvious caps did not increase as the concentration of antimembrane antibodies was increased, whereas stimulation of glucose oxidation was proportional to the antibody concentration. At fairly high dilutions of antiserum there was no effect on rat adipocyte glucose oxidation, but caps were still visible, suggesting that there is not a direct correlation between capping and stimulation of adipocyte metabolism. At 15 degrees C, it was found that capping was not significantly impaired, while the basal rate of glucose oxidation was reduced considerably. Both insulin and antimembrane antibodies were still able to stimulate glucose oxidation at 15 degrees C, although the maximal rate of glucose oxidation which could be achieved at this temperature was considerably lower than that observed at 37 degrees C. Fat cells that were fixed with paraformaldehyde before being exposed to antimembrane antibodies showed a similar number of caps to unfixed cells, suggesting that some fat cells had a polarized distribution of membrane antigens even before exposure to antimembrane antibodies. These results demonstrate for the first time that antibodies against the rat adipocyte plasma membrane, which are known to mimic insulin action on rat fat cells, can associate with antigens arranged non-randomly on the cell surface, but it appears unlikely that capping plays a critical role in the expression of this biological activity.

Adipose Tissue↗
PubMed1982

Redistribution of intermediate filaments during capping of lymphocyte surface molecules.

Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenström's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.

Antibodies, Monoclonal↗
PubMed1983

Redistribution of fodrin (a component of the cortical cytoplasm) accompanying capping of cell surface molecules.

Fodrin, a protein composed of two polypeptides with molecular weights of 250,000 and 240,000, is concentrated in the cortical cytoplasm of neurons, and moves down the axons by the process of axonal transport. We have used immunofluorescence techniques to determine whether fodrin antigens also move in non-neuronal cells when cell surface ligands are induced to redistribute by crosslinking them. A redistribution of fodrin antigens occurred in the following instances: (i) when 3T3 cells were incubated with concanavalin A and anti-concanavalin A, surface concanavalin A receptors formed aggregates and fodrin antigens formed corresponding intracellular aggregates; (ii) when B lymphocytes were incubated with anti-Ig, the surface Ig formed caps and fodrin antigens formed intracellular subcaps; (iii) when T lymphocytes were treated with anti-H-2 followed by a secondary antibody, the H-2 antigen formed caps and fodrin formed corresponding subcaps. These observations show that fodrin antigens can move within non-neuronal cells, as well as in axons, and that their organization can be regulated by interaction between surface proteins and environmental stimuli. They also raise the possibility that fodrin, together with other proteins that form subcaps in lymphocytes (e.g., actin, myosin, and alpha-actinin) is a component of the cellular machinery responsible for the capping process. We consider whether the similarities between the movements of fodrin in lymphocyte capping and axonal transport may indicate that certain aspects of these two processes are related.

Animals↗
PubMed1985

Functional integrity of cytokineplasts: specific chemotactic and capping responses.

Cytokineplasts (CKP) are motile, membrane-bound, anucleate, granule-poor cytoplasmic fragments that are induced from human blood polymorphonuclear leukocytes (PMN) by the brief application of heat. We examined CKP with respect to specific chemotactic and capping responses, the presence of the N-formyl-peptide chemotactide receptor, and evidence of respiratory burst activity and compared them with CB-cytoplasts, which are fragments created by the centrifugation of cytochalasin B (CB)-treated PMN at high speeds. Under agarose, CKP responded chemotactically to both N-formyl-methionyl-leucyl-phenylalanine (fmlp) and zymosan-activated serum; CB-cytoplasts responded to neither chemoattractant. Despite the functional differences, both fragments retained N-formyl-peptide receptors as measured by affinity labeling with N-formyl-norleu-leu-phe-norleu-125I-tyr-lys and autoradiography of dried SDS-PAGE gels. For studies of capping we used a murine monoclonal antibody, PMN7C3, which binds a specific, widely distributed membrane component of intact PMN, and on warming, promptly induces capping of ligand-receptor complexes. Rhodamine-conjugated PMN7C3 at 4 degrees C labeled the surface of CKP homogeneously. As the CKP warmed to 37 degrees C, label became concentrated in small fluorescent caps at the rear of migrating fragments. Although CB-cytoplasts also bound the fluorochromed antibody homogeneously in the cold, on warming they were unable to concentrate the label normally. With respect to respiratory burst activity, the situation in the two fragments was reversed: CKP did not generate superoxide anion when stimulated either with phorbol myristate acetate or with fmlp after pretreatment with CB; CB-cytoplasts, as noted earlier by other investigators, did. These two types of cytoplasts with markedly different capabilities have complementary roles in the analysis of PMN function.

Chemotactic Factors↗
PubMed1981

Interactions between lymphocyte membrane molecules. I. Interaction between B lymphocyte surface IgM and Fc IgG receptors requires ligand occupancy of both receptors.

The independent B lymphocyte surface membrane receptors IgM and Fc IgG receptors were evaluated for interactions using immunoflourescence. Ligand [F(ab')2 anti-mu]-induced capping of surface IgM resulted in capping of Fc IgG receptors only if the latter were occupied during the capping process by: (a) soluble antigen-antibody complexes that themselves provided insufficient cross-linking to result in capping; or (b) monomeric IgG at physiologic concentrations (or less) either purified or as normal serum. Ligand-induced capping of Fc IgG receptors did not result in capping of surface IgM occupied by monomeric F(ab') anti-mu. Control experiments showed that ligand binding to or capping of only one of these two receptors has no effect on the other, and that there were no cross-reactions. The interaction appears specific in that ligand-induced capping of surface IgM did not induce capping of ligand-occupied surface IgD or I-A antigens. Thus, there appears to be a specific interaction between ligand-bound surface IgM and ligand-bound Fc IgG receptors on the B lymphocyte surface. The results also indicate that binding of monomeric IgG produces a reversible alteration in the Fc IgG receptor leading to association with ligand-bound surface IgM. Because Fc IgG receptors are continuously exposed to monomeric IgG in vivo, these results suggest that whenever surface IgM is involved in a B lymphocyte response to an immunologic stimulus, the Fc IgG receptor is also involved.

Animals↗
PubMed1983

Effects of microwaves and hyperthermia on capping of antigen-antibody complexes on the surface of normal mouse B lymphocytes.

Normal mouse B lymphocytes were tested for the ability to cap plasma membrane antigen-antibody complexes following exposure to 2.45-GHz continuous wave (CW) microwaves at power densities up to 100 mW/cm2 (45 W/kg specific absorption rate), at 37, 41, and 42.5 degrees C. After a 30-minute treatment, the irradiated cells and the nonirradiated controls were tested for capping by the direct immunofluorescence technique. First, the cells were incubated for nine minutes at 37 degrees C with fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin. After fixing and washing, the percentage of capped cells was determined under a fluorescence microscope. The results show that for the nonirradiated controls, capping is reduced from 90% at 37 degrees C, to 52% at 41 degrees C, to less than 5% for cells that were pretreated at 42.5 degrees C. There was no significant difference between the microwave-treated cells and the controls when both were maintained at the same temperature. In another experiment, there was no significant difference in the percentage of capping between controls and cells that were exposed to microwave radiation during capping, when the temperature in both preparations was kept at 38.5 degrees C. The results demonstrate that B-lymphocyte capping is sensitive to temperature in the range that is proposed for use in tumor therapy.

Animals↗
PubMed1988

Pulse profile analyses of endocytosis in capped B lymphocytes and BCL1 cells.

The effect of temperature on the kinetics of endocytosis by B lymphocytes and BCL1 cells was examined by using flow cytometry. Mouse B cells were stained with FITC-R alpha MIg and induced to undergo cap formation in the temperature-controlled sample compartment of the flow cytometer. Capping and subsequent endocytosis were measured by continually monitoring the pulse profile descriptors (width and area) of the electronic signal curves generated during flow cytometric analyses. Decreases in area values which immediately followed cap formation were shown to result from a pH-dependent quenching of fluorescence emission as the internalized FITC-R alpha MIg entered acidic subcellular compartments. Similar results were obtained with BCL1 cells, but, in addition to cap formation and acidification, cytoplasmic diffusion of the fluorescent ligand could also be discerned by flow cytometry. With either cell type the rates of cap formation and endocytosis were shown to be temperature dependent with temperature coefficient (Q10) values of 2.0-2.7. Based upon Arrhenius plots of width and area changes, activation energies for capping and endocytosis ranged from approximately 12-18 kcal/mol.

Animals↗
PubMed1980

Model for capping derived from inhibition of surface receptor capping by free fatty acids.

When low concentrations (2-5 mole %) of cis unsaturated free fatty acids (group A) are intercalated into lymphocyte plasma membrane, capping is inhibited. No effect is seen with trans unsaturated or saturated fatty acids (group B). The capping inhibition is reversible with increasing doses of extracellular calcium. Fluorescence photobleaching recovery has shown that the group A free fatty acids do not inhibit the receptor immobilization associated with patch formation, but inhibit the final energy-dependent movement of the patched receptors into a cap. We have also shown that the group A free fatty acids cause a shift in membrane-bound calcium to the lipid phase from probable protein-associated sites. We have incorporated these findings into a model for capping and membrane-cytoskeletal interactions.

Animals↗
PubMed1991

Effects of anti-immunoglobulin antibodies, interleukin-4 and second messenger agonists on B cells from neonatal mice.

Crosslinking of surface Ig receptors on mature B cells with mitogenic anti-Ig antibodies stimulates phosphoinositide breakdown with subsequent activation of protein kinase C (PKC) and elevation of [Ca2+]i leading to B-cell activation. This response can be mimicked using second messenger agonists such as phorbol 12,13-dibutyrate (PDB) plus a Ca2+ ionophore. Furthermore, interleukin-4 (IL-4) synergizes with sub-mitogenic concentrations of anti-Ig (or with PDB) to activate adult B cells. In contrast anti-Ig does not activate neonatal B cells but rather desensitizes or kills them. The nature of the signals involved in these effects on neonatal B cells is poorly understood. Here we have investigated the proliferative responses of small, resting B cells from 1-, 4-, 8-, and 12-week-old mice stimulated with combinations of anti-Ig, PDB, ionomycin and IL-4. We find that B cells from 8- and 12-week-old mice show an adult pattern of reactivity. B cells from 4-week-old mice respond to high doses of anti-Ig, anti-Ig + IL-4 and also to PDB + ionomycin + IL-4, but not to PDB + ionomycin alone. Neonatal (1-week-old) B cells respond only to the combination of PDB, ionomycin and IL-4. Most strikingly, pre-incubation of neonatal cells with anti-Ig completely abrogates this response, whilst IL-4 renders them refractory to such anti-Ig mediated inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Distribution and redistribution of pancreatic islet cell surface antigen reactive with islet cell surface antibody in the rat.

Antigens on the rat pancreatic islet cell surface were redistributed into patch and cap formation when the cells were incubated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, in tissue culture medium 199 for 24 h, before addition of rat pancreatic islet cell surface antibody. In contrast, if the cells were cultured in tissue culture medium 199 supplemented with glucose (5.5 or 16.7mmol/l) and 10% heat-inactivated fetal calf serum without 3-isobutyl-1-methylxanthine, cap formation was not detectable. These results suggest that mobile antigen on the surface of pancreatic B cells can be induced to aggregate into patch and cap formations during conditions of increased cellular metabolism.

1-Methyl-3-isobutylxanthine↗
PubMed1981

Spontaneous and drug induced concanavalin A capping of neutrophils from human infants and their mothers.

We investigated concanavalin A capping that occurred either spontaneously (neutrophils incubated only with buffer) or was drug induced (neutrophils with colchicine or diamide) using neutrophils obtained from the blood of newborn infants and their mothers. A greater proportion of infant and maternal neutrophils than controls formed caps spontaneously (P less than 0.01). The percent of capped neutrophils (mean +/- S.E.) for 24 infants and their mothers was 23 +/- 3 and 39 +/- 4, respectively, versus 13 +/- 2 for 26 controls. Spontaneous capping was significantly decreased when neutrophils were incubated with catalase and superoxide dismutase to suggest that it was related, at least in part, to oxidation. Drug-induced capping of infant and maternal neutrophils was decreased when compared to controls (P less than 0.05). Colchicine increased capping of control neutrophils 11.76-fold above the spontaneous value, whereas capping of infant and maternal cells was increased only 2.35- and 1.65-fold. Corresponding values for diamide were 11.94-, 2.27, and 2.49-fold for control, infant, and maternal neutrophils, respectively. Many cellular processes are involved in capping, and the mechanisms responsible for aberrant capping of infant neutrophils remain undefined. However, this is another property that distinguishes infant neutrophils from those of older individuals.

Adult↗
PubMed1988

Association of cytoskeletal re-organization with capping of the complement decay-accelerating factor on T lymphocytes.

Recent studies have identified cell-associated proteins that are membrane anchored by glycosyl-inositol-phospholipid structures but the biologic implications of this mode of membrane attachment are incompletely understood. Among proteins anchored in this way is the decay-accelerating factor (DAF), a complement (C) regulatory factor that functions on blood cell surfaces to prevent autologous C attack. As one approach to investigate the functional consequences of glycosyl-inositol-phospholipid-anchoring of DAF in T lymphocytes, the effects of crosslinking surface DAF molecules were compared to those of crosslinking conventionally by anchored cluster of differentiation (CD) proteins. Upon incubation with anti-DAF mAb and anti-murine IgG, DAF re-distributed to a pole of the cell with a t1/2 at 37 degrees C of 4.4 min as compared to t1/2 of 3.5 to 7 min for CD3, CD4, and CD8. Re-distribution of DAF occurred independently of CD2, CD3, CD4, or CD8. Anti-DAF immunoprecipitates of membrane extracts of cells chemically cross-linked with dithiobis(succinimidylpropionate) contained only monomeric DAF. Immunofluorescent staining demonstrated clustered actin, tubulin, and vimentin beneath the capped DAF protein. Pre-treatment of cells with colchicine or 8-azidoadenosine 3',5'-cyclic phosphate, but not lumicolchicine, resulted in reduction of the t1/2 for DAF to 1 to 2.6 min. Conversely, treatment of cells with cytochalasins B or D completely blocked DAF capping. The results indicate that, upon cross-linking, glycosyl-inositol-phospholipid-anchored DAF molecules undergo capping similar to conventionally anchored CD molecules and that DAF capping is associated with cytoskeletal reorganization.

Adult↗
PubMed1989

Polymorphonuclear leukocytes cap a derivative of wheat germ agglutinin upon stimulation with formyl peptide and C5a but not leukotriene B4.

Previously, we reported that a derivative of wheat germ agglutinin (termed WGA-D) specifically inhibits human polymorphonuclear leukocyte (PMN) chemotaxis to FMLP by blocking reexpression (or recycling) of formyl peptide receptors. WGA-D (? formyl peptide receptor probe) binds to a protein on the PMN membrane that exhibits the same m.w. as the formyl peptide receptor. Since clustering (i.e., capping) of ligand-receptor complexes most likely precedes their internalization, we examined the ability of normal and stimulated PMN to cap fluoresceinated WGA-D. We found that, in contrast to capping of fluoresceinated Con A, PMN cap WGA-D in a chemotactic factor-specific fashion. Fluoresceinated WGA-D (5.0 to 20 micrograms/ml) alone did not induce either PMN shape changes (i.e., activation) or capping. Both FMLP (1 to 1000 nM) and human C5a (0.1 to 1.0 nM) induced PMN to polarize and to cap bound WGA-D, in a concentration-dependent fashion. Interestingly, leukotriene B4 (LTB4) (5.0 nM), while inducing the same degree of PMN polarization as FMLP (100 nM) and C5a (0.5 nM), failed to induce PMN to cap bound WGA-D. In contrast, FMLP (100 nM), C5a (0.5 nM), and LTB4 (5.0 nM) induced PMN to cap bound fluoresceinated Con A (10 micrograms/ml) to the same extent. The effect of suboptimal concentrations of FMLP and C5a on capping of WGA-D by PMN was additive. LTB4 did not enhance either FMLP or C5a-induced capping of WGA-D by PMN. Also, FMLP and C5a (but not LTB4) were capable of inducing both desensitization and cross-desensitization of WGA-D capping by PMN. Studies using rhodamine-labeled WGA-D and a fluoresceinated analog of FMLP revealed that both capped to the same place on the PMN membrane. Thus, the data suggest that WGA-D binds to a site on the PMN membrane that is either the FMLP receptor or very closely associated with it.

Adult↗