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PubMed1978

Microtubules and cyclic AMP in human leukocytes: on the order of things.

We have shown previously that the beta-adrenergic agonist isoproterenol (2muM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.

Adult↗
PubMed1980

Carrier detection in Duchenne muscular dystrophy.

Serum creatine kinase, myoglobin, and percentage lymphocyte capping was determined in ten patients with Duchenne muscular dystrophy, 12 carriers (nine definite and three probable), 16 other female relatives, and eight normal controls. There was no detectable difference in lymphocyte capping ability between any of these clinical groups. Significant myoglobinaemia was present in all the affected males, but the difference in levels between carriers and controls suggested that this test has no advantage over creatine kinase estimations in carrier detection.

Creatine Kinase↗
PubMed1981

Development of capping ability during differentiation of HL-60 human promyelocytic leukemia cells.

Developmental changes in cell surface and cytoskeletal elements have been studied in human promyelocytic leukemia cels (line HL-60) which differentiate into functionally mature myeloid cells when grown in dimethyl sulfoxide (DMSO)-supplemented medium. Both differentiated and undifferentiated HL-60 cells bind fluorescent concanavalin A (F-Con A) in a diffuse pattern over the entire cell surface. As with normal neutrophils, pretreatment of the differentiated HL-60 cells with colchicine before incubation with Con A causes the formation of large cytoplasmic protrusions over which the lectin associates into a cap. On the other hand, similarly treated undifferentiated HL-60 cells do not form the cytoplasmic protuberances and are unable to cap the Con A. Transmission electron microscopy reveals that the number and distribution of microtubules and microfilaments change during differentiation. Thus, developing myeloid cells undergo important alterations in the structure and function of the cytoskeleton as they differentiate into mature phagocytes.

Cell Differentiation↗
PubMed1996

Association of murine splenocyte CD3 complex to the cytoskeleton: absence of modulation by exogenous fatty acids.

The cytoplasmic regions of the CD3 complex are presumably involved in signal transduction following ligand-receptor binding. We investigated the effects of incubating either stearic or oleic acid on the association of murine lymphocyte CD3 complex with the cytoskeleton. Both cytochalasin D, an inhibitor of microfilament formation, and W7, an inhibitor of calmodulin, inhibited capping of CD3. The association of CD3 with the cytoskeleton was confirmed by confocal laser scanning microscopy studies, which showed co-localization of the cross-linked CD3 receptors and the membrane attachment proteins ankyrin and fodrin. Although exogenous oleic acid increased plasma membrane fluidity, neither expression nor capping of CD3 receptors was increased. Nonetheless, oleic acid did increase uptake of tritiated thymidine after binding of anti-CD3 antibodies. Lymphoproliferation was progressively inhibited by both cytochalasin D and W7, confirming the importance of intact cytoskeleton for cellular activation.

Animals↗
PubMed1985

Studies of the effects of spherulin from Coccidioides immitis on human polymorphonuclear leukocytes.

The effects of spherule lysate (spherulin) on human polymorphonuclear leukocyte (PMN) function was examined. PMN adherence to glass and capping was increased by spherulin, findings which may account for spherulin's interference with complement-mediated migration through cellulose ester filters. In contrast, PMN attachment to yeast, killing of bacteria, and effects on spherules were virtually unaffected by spherulin, suggesting that it does not directly inhibit PMN antimicrobial function.

Antigens, Fungal↗
PubMedDate not supplied

Capping and internalization of a monoclonal antibody-surface antigen complex: a possible mode of interaction of monoclonal antibodies and tumor cells.

Our results demonstrate that upon incubation of 125I-3G5 (a monoclonal IgM against a membrane ganglioside antigen on RINm5F cells) with rat insulinoma RINm5F cell monolayers at 37 degrees C, the IgM is rapidly internalized. Cell-bound radioactivity, detectable within 10 to 15 minutes, reaches a peak at 4 hours. By 24 hours the intracellular radioactivity has decreased to about 37.5% of the 4-hour value, accompanied by an increase in free 125I in the incubation medium. The incubation of 125I-3G5 with RINm5F cell monolayers at 4 degrees C shows that this series of events is inhibited by low temperature. Microautoradiography confirms these events indicating the presence of radiolabeled antibody on the plasma membrane as well as distinct capping processes and diffuse radioactive deposits within the cells as early as 5 to 10 minutes after initiating incubation at 37 degrees C. Electron microscopy autoradiography provides a detailed demonstration of the capping phenomenon and of endocytic vacuoles, followed at later times by the distribution of radioactive deposits throughout the cell. This model constituted by the capping of the 125I-3G5-ganglioside complex on rat insulinoma RINm5F cells may be useful in elucidating a possible mode of interaction of monoclonal antibodies and tumor cells.

Animals↗
PubMed1979

Effects of hexachlorocyclohexane isomers on concanavalin A 'capping' in bovine lymphocytes.

The effects of alpha, beta, gamma, and delta isomers of hexachlorocyclohexane on concanavalin A 'capping' in bovine lymphocytes were evaluated, gamma and delta hexochlorocyclohexane inhibited 'capping' whereas the alpha and beta isomers were without effect. In addition, gamma-hexachlorocyclohexane has been shown to antagonize the maintenance of preformed 'caps' and cause the rapid dispersal of the concanavalin A-receptor complexes over the surface of cells by a temperature-dependent mechanism. The possible role of a gamma-hexachlorocyclohexane-sensitive process in the organization of microflow patterns in the lectin-activated lymphocyte membrane is discussed.

Animals↗
PubMed1987

Organizational fate of vimentin during redistribution of surface immunoglobulin in mouse splenic lymphocytes.

We have used immunofluorescence to examine the organizational fate of vimentin and its spatial relationship to the microtubule system during antibody-induced redistribution of surface immunoglobulin (sIg) in control and drug-treated mouse splenic lymphocytes. In control cells, vimentin is relocalized as a diffuse accumulation underneath the site of the cap during sIg redistribution. Observations on cells that were treated with colcemid or taxol prior to induction of sIg redistribution have further shown that vimentin accumulation corresponds to a dynamic rearrangement of this filamentous system which is related to, but is not required for, the energy-dependent translocation of sIg.

Actin Cytoskeleton↗
PubMed1984

Electrophoretic characterization of specific antibody effects on lymphocytes.

The electrophoretic mobility of lymphocytes can be influenced by incubation with the antibodies. The effects of specific antibodies on the electrophoretic mobility of B and T lymphocytes were determined. Comparison of results obtained under the capping and non-capping conditions suggests strongly that changes in the surface charge of lymphocytes after the interaction with the antibodies we used are not caused by antibody-induced redistribution of surface antigens. Most of the investigated anti-T and anti-B sera react specifically with human and mouse T and B lymphocytes, respectively. This was shown by the reduced electrophoretic mobilities of the relevant populations in the range of 0.06 to 0.22 electrophoretic units. Non-specific interactions of the antisera used with other cells can be excluded. The results of electrophoresis were found to correlate with the results of immunological tests used for the characterization of antibodies, e.g. cytotoxicity, E- and EAC-rosette inhibition, effects on antibody forming cells. Cell electrophoresis can therefore be used to determine antibody specificity. Results with an anti-T suppressor serum show that it is basically possible to detect interactions between highly specific antibodies and T lymphocyte subpopulations by electrophoresis.

Animals↗
PubMed1978

Improvement of polymorphonuclear leucocyte oxidative and bactericidal functions in chronic granulomatous disease with 4-amino-4'-hydroxylaminodiphenyl sulphone.

In an effort to restore oxidant-dependent capabilities to chronic granulomatous disease (CGD) polymorphonuclear leucocytes (PMN), we studied a dapsone derivative, 4-amino-4'-hydroxylaminodiphenyl sulphone (DDS-NOH), known to generate H2O2. After incubation of CGD PMN with 0.2 and 1.0 mM DDS-NOH for 30 min, the rate of glucose-1-14C oxidation via hexose monophosphate (HMP) shunt increased 2--4-fold and that of iodination of ingested zymosan particles 1.5--2.7-fold. Both effects could be further enhanced by superoxide dismutase (SOD) but inhibited by catalase. In three patients, 0.2 mM DDS-NOH improved in vitro killing of Staph. aureus. DDS-NOH 0.02 mM induced capping of Concanavalin A (Con A) receptor complexes suggesting interference by the drug with microtubule-associated function. Thus, optimal concentrations of DDS-NOH may be employed as an oxidant to improve metabolic and bactericidal functions of PMN from patients with CGD.

Aminobiphenyl Compounds↗
PubMed1996

Subcellular localization of CD80 receptors is dependent on an intact cytoplasmic tail and is required for CD28-dependent T cell costimulation.

CD28 provides a major costimulatory signal to T cells when it is cross-linked with mAb, immobilized recombinant ligand (CD80Ig or CD86Ig), or ligand-bearing cells but not when it is bound by specific Fab fragments or monomeric ligand. We wanted to determine how monomeric CD80 could cross-link CD28 since CD80 is expressed as a monomer on the surface of APC. We found that CD80 may interact with the actin-based cytoskeleton. To test whether the interaction of CD80 with the cytochalasin B-sensitive cytoskeleton was necessary for T cell costimulation through CD28, we constructed a tailless form of CD80 and generated stable transfectants of Chinese hamster ovary epithelial cells and Reh B cells expressing either the tailless or wild-type CD80 molecules. Unlike control cells expressing wild-type CD80, the tailless CD80 transfectants expressing equivalent levels of surface CD80 were not able to provide a costimulatory signal for anti-CD3-induced T cell proliferation, up-regulation of CD25 (IL-2Ralpha) expression, or the induction of IL-2 secretion. Thus, the cytoplasmic tail of CD80 apparently is required to signal T cells. Confocal microscopic studies revealed that wild-type CD80 and tailless CD80 have different patterns of subcellular distribution in both epithelial and lymphoid cells. Furthermore, T cell contact induces more patching and capping of CD80 in wild-type CD80-expressing cells than in tailless CD80-expressing cells. This suggests that the cytoplasmic region of CD80 functions to localize CD80 in complexes required for effective T cell costimulation.

Animals↗
PubMed1982

Cell surface properties of HLA antigens on Epstein-Barr virus-transformed cell lines.

A number of monoclonal antibodies have been used to investigate the distributions and rates of lateral motion of the HLA-A,B, and-DR antigens on several Epstein--Barr virus-transformed B-cell lines. The lateral diffusion coefficients (D) of fluorescein conjugates of the monoclonal antibodies bound to the cell surface were determined by fluorescence recovery after pattern photobleaching. Ds of HLA-A and-B were found to be comparable and of the order of 10(-9) to 10(-10) cm2/sec for each of the seven monoclonal antibodies and four cell lines examined. The HLA antigens appear to be monomeric on the cell surface based on experiments using mixtures of arsanilic acid-conjugated and fluorescein-conjugated antibodies. Four monoclonal antibodies against DR antigens were examined. Two of these, Genox 3.53 and L243, labeled the cell surface uniformly and gave Ds comparable to those obtained for the HLA-A and -B antigens. The other two, DA2 and 2.06, rapidly patched on the cell surface and were immobile. The DA2, L243, and Genox 3.53 antibodies bound outside of the caps formed with the arsanilic acid-conjugated 2.06 antibody and a second-step rhodamine-conjugated rabbit anti-arsanilate antibody. This is consistent with recent biochemical evidence that there are multiple distinct antigens coded for by the HLA-DR region.

Antibodies, Monoclonal↗
PubMed1987

Perfluorinated fatty acids alter merocyanine 540 dye binding to plasma membranes.

We have evaluated the effect of the perfluorinated fatty acids pentadecafluoro-n-octanoic acid (PFOA) and nonadecafluoro-n-decanoic acid (NDFDA) on the ability of a human B-lymphoblastoid cell line to bind the lipid-binding, membrane-impermeant, fluorescent dye merocyanine 540 (MC540). Subtoxic concentrations of perfluorinated fatty acids (0.9 mM PFOA; 0.5 mM NDFDA) greatly diminish binding of MC540 by normal plasma membranes, as determined by fluorescence flow cytometry. When perfluorinated fatty acids are added to cells at toxic or lethal concentrations (1.2 mM PFOA; 0.75 mM NDFDA), MC540 binding increases dramatically, with entrance of dye to internal membrane domains. Neither perfluorinated fatty acid molecule reduces the ability of surface immunoglobulin to migrate laterally and cap on cells. Our data suggest that perfluorinated fatty acids either interact directly with lipid binding sites for MC540, and thereby inhibit dye intercalation, or alter membrane lipid architecture and lipid packing to diminish MC540 binding. Both possibilities support a direct, physical, membrane-altering mechanism for perfluorinated fatty acid toxicity on mammalian cells.

Blood Physiological Phenomena↗
PubMed1981

Further molecular complexities of H-2 K- and D-region antigens.

The K and D regions of the H-2 gene complex are highly polymorphic and control cell-surface structures involved in the H-2 restriction of cytotoxic T lymphocytes. Originally it was assumed that each of these regions controlled only one type of antigenic molecule, H-2K and H-2D, respectively. The finding that the D region controls H-2L and H-2M, as well as H-2D, molecules suggests that the system of H-2 antigens is more complicated. We demonstrate here previously unknown H-2 molecules detected by anti-K- and D-region alloantisera in co-capping experiments. Three different types of molecule, distinct from Ia and Qa antigens, are distinguished in the products of the Ddx region, and two in the products of the Dk and Kd regions. Analysis of the antigenic heterogeneity of K- and D-region products may further understanding of the function of these regions in cell-mediated immunity.

Animals↗
PubMed1983

Possible role for C-reactive protein in the human natural killer cell response.

Functional NK activity can be removed from human PBL and from phagocyte- and T cell-depleted LGL preparations by treatment with antisera specific for C-reactive protein (CRP) in the presence of complement (C). Pretreatment of NK effector cells with high concentrations of anti-CRP in the absence of C also depletes functional activity. These results indicate that CRP or an antigenically similar molecule is present on a population of NK effector cells. Fluorescent antibody studies in which biotin-avidin amplification was used confirm the presence of surface CRP (S-CRP) on a small percentage of nonphagocytic peripheral blood mononuclear cells. S-CRP readily caps off, which suggest that removal by capping obviates killing by this cell population. This indicates that S-CRP or a molecule that co-caps with S-CRP may be required for successful effector-target cell interaction. The addition of exogenous CRP or CRP-CPS complexes, however, does not alter NK responses. A subpopulation of lymphoid cells responsible for functional NK activity therefore appears to bear surface CRP.

C-Reactive Protein↗
PubMed1990

The effects of new cytochalasins from Phomopsis sp. and the derivatives on cellular structure and actin polymerization.

The effects of ten 10-phenyl-[11]cytochalasins produced by Phomopsis sp. including novel compounds having 5,7- or 6,7-glycol structures and their derivatives, on the cell morphology and actin distribution in C3H-2K cells, as well as on lymphocyte capping and actin polymerization, were examined. The structure-activity relationship reported in the previous papers has been confirmed. The novel glycol type compounds showed little or no activity, suggesting the importance of the perhydroisoindol-1-one nucleus for the manifestation of the cytochalasin actions.

Actins↗
PubMed1990

Lymphocyte concanavalin A capping in hereditary cerebral haemorrhage with amyloidosis--Dutch type.

Lymphocyte capping with concanavalin A was studied in 11 patients with hereditary cerebral haemorrhage with amyloidosis (Dutch type) and 10 controls. No difference in capping was found between patients and controls. Abnormal lymphocyte concanavalin A capping has been reported in patients with the Icelandic type of cerebral amyloidosis and in patients with Alzheimer's disease, a disease in which cerebral amyloid angiopathy can also be found. The results suggest a difference in pathogenesis between the Dutch type of cerebral amyloidosis and the other amyloid diseases.

Aged↗
PubMed1979

Macromolecular insoluble cold globulin (MICG): a novel protein from mouse lymphocytes. IV. Evidence for the plasma membrane distribution of MICG.

Macromolecular insoluble cold globulin is a glycoprotein synthesized predominantly by T lymphocytes in the mouse. The present report details experiments demonstrating the plasma membrane distribution of MICG on T lymphocytes. By utilizing immunofluorescent techniques it was shown that MICG was located in the external cell surface of 98% of thymic lymphocytes and 60% of splenic lymphocytes. Furthermore, in spleen cells, it was demonstrated that T cells and not B cells were surface MICG positive. Antibody to MICG was able to cap all of the immunofluorescent-positive (60%) spleen cells. In contrast, anti-MICG antibody did not induce cap formation on thymus cells. Only when dilute solutions of antibody were used did MICG cap on the thymus cells. Employing limited proteolysis of thymus and spleen cells MICG was shown to be regenerated on the surface of T cells with a half-life of 3.5 hr. The distribution and cell surface characteristics of MICG are discussed in terms of a "receptor-like" function for this protein.

Animals↗