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PubMed1979

Suppressive effect of ferritin on in vitro lymphocyte function.

This study describes the effect of ferritin on lymphocyte function in vitro. Peripheral blood lymphocytes isolated from normal donors were incubated with purified human splenic ferritin, and the mitogenic effect of phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and mixed lymphocyte reaction (MLR) were assessed by the uptake of 3H-thymidine (3H-TdR). Ferritin (0.25--5.0 micrograms/ml culture) caused a marked suppression of PHA nad Con A blastogenesis but had no suppressive effect on PWM-induced transformation. Maximal suppression was obtained at a ferritin concentration of 1 microgram/ml and this was not enhanced by increasing ferritin concentrations. Ferritin also reduced the Con A capping phenomenon in normal lymphocytes from 22% to 6%, suppressed the MLR reaction but had no effect on the ability of normal lymphocytes to form E, EA and EAC rosettes or on in vitro lymphocyte cytoxicity against the K-562 cell line. Visual proof of the suppressive effect of ferritin on mitogen induced blastogenesis was provided by scanning electron microscopy, and direct evidence for the ability of lymphocytes to bind ferritin was obtained from studies with radioiodine labelled ferritin. The above findings indicate that ferritin suppresses certain parameters of T-lymphocyte function in vitro. The relation of the present findings to recognized abnormalities of T-cell function encountered in certain neoplastic disorders associated with high serum ferritin levels is at present unknown.

Cytotoxicity, Immunologic↗
PubMed2005

Targeting antigen to CD19 on B cells efficiently activates T cells.

CD19 is a B cell-surface molecule that participates as an important regulatory signaling complex for antigen bound at the surface by Ig. Triggering of CD19 through its linkage with CD21 amplifies signals transduced through the Src family kinases and modulates B cell differentiation in response to antigen. This study examines the kinetics of antigen uptake and processing of antigen directly targeted to the CD19 protein on purified B cells. We have demonstrated that the antigen internalized within minutes through CD19 forms a cap at the B cell surface and can be found within lysosomes in the cytoplasm in 90 min. B cells acquiring antigen via CD19 express elevated levels of B7-1 and B7-2 co-stimulatory molecules. Moreover, antigen-anti-CD19 complexes administered intravenously bind B cells in vivo and activate antigen-specific T cells more efficiently than non-specific uptake and in a manner similar to antigen taken up through surface IgM on B cells. This work illustrates an important and previously unrecognized mechanism for targeting proteins to B lymphocytes for antigen presentation and activation of CD4 T cells.

Animals↗
PubMed1979

Cross-reactivity of rat, mouse, and human IgD.

Highly purified sheep anti-rat lymphocyte membrane IgD (mIgD) was used to detect cross-reactivity with the putative murine-delta chain on mouse lymphocytes. Cross-reactivity is demonstrated by indirect immunofluorescent staining and by immunoprecipitation of 125I-labeled lymphocyte membrane extracts followed by electrophoresis on 10% polyacrylamide gels. In addition, cross-reactivity of anti-rat-delta with human IgD is shown by gel diffusion analysis. The anti-rat-delta reagent stained both Ig5a+ and Ig5b+ lymphocytes. Preincubation of Ig5b+ (but not Ig5a+) cells with monoclonal allotype-specific antibodies (anti-Ig5b) under capping conditions caused inhibition of staining by the sheep anti-rat-delta reagent, indicating that it is the delta-chain that is recognized on mouse lymphocytes and that the anti-rat-delta reagents does not distinguish between mouse-delta allotypes. Furthermore, absorption of the sheep anti-rat-delta serum with purified human IgD reduced subsequent staining of mouse lymphocytes by approximately 50%; staining was not affected by absorption with human IgM. This xenogeneic anti-delta antiserum appears to detect determinants on the delta-heavy chain, which are shared by at least three species of mammals, suggesting that these determinants represent important molecular features conserved during evolution.

Animals↗
PubMed1989

Role of Ca2+ in the regulation of hormone receptor exposure during lymphocyte activation.

Ca2+ is known to be required for mitogen-mediated lymphocyte activation. In order to further define the regulatory role of Ca2+, we have examined the activation events which occur following treatment with ionomycin (a Ca2+ ionophore), as compared to those occurring following concanavalin A (Con A) treatment of mouse splenic T-lymphocytes. Our results indicate that ionomycin and Con A induce the exposure of both interleukin-2 (IL-2) and insulin receptors on the surface of the lymphocytes within the first 5 min of treatment. The exposed insulin and IL-2 receptors have the following properties: (1) they consist of both high- and low-affinity receptors; and (2) they appear on the cell surface in small clusters (i.e., patches) or, occasionally, a large aggregate (i.e., cap). c-myc gene expression and DNA synthesis occur in both the ionomycin and Con A-treated lymphocytes when either IL-2 or insulin is present in the culture medium. Furthermore, the exposure of both hormone receptors can be inhibited by either EGTA (a Ca2+ chelator), bepridil (a Ca2+ channel blocker), W-7 (a calmodulin antagonist) or cytochalasin D (a microfilament inhibitor). Treatment with these inhibitors also blocks the expression of c-myc gene and DNA synthesis which occur at later times during IL-2 and insulin-induced activation of ionomycin- and Con A-treated lymphocytes. These findings suggest that a Ca2+ and calmodulin-mediated contractile system is involved in the exposure of certain hormone receptors which appear to be required for complete lymphocyte activation.

Animals↗
PubMed1990

Effects of pentoxifylline administration on blood viscosity and leukocyte cytoskeletal function in patients with intermittent claudication.

We have previously shown that pentoxifylline, a drug used in intermittent claudication, causes depolymerization of actin in leukocytes in vitro. In this study we evaluated several parameters in peripheral blood obtained from 17 patients receiving pentoxifylline, before therapy and at 1 and 2 months after initiation of drug therapy. Total blood viscosity decreased at 1 month and was further reduced at 2 months. The plasma viscosity remained unchanged during the course of the therapy (1.770 +/- 0.147, 1.776 +/- 0.162, and 1.772 +/- 0.164 centipoise at 0, 1, and 2 months, respectively; mean +/- SD, n = 14 to 17). No changes were observed in stimulus-induced actin polymerization in granulocytes, concanavalin A-induced capping in granulocytes and lymphocytes, and anti-IgG-induced caps in lymphocytes, before and after therapy. Similarly, there was no difference in the magnitude of depolymerization caused by pentoxifylline when added in vitro. Thus none of the parameters altered by pentoxifylline treatment in vitro have been observed ex vivo in patients receiving this drug. However, the decrease in total blood viscosity along with unaltered plasma viscosity suggests that the rheology of the cellular elements is being affected by the administered drug. In addition to the direct effects on the cell membrane and the cytoskeleton, pentoxifylline may exert indirect effects through its inhibitory action on cytokine production. Subtle changes in a number of parameters in leukocytes, which taken alone fail to show demonstrable changes, might ultimately be responsible for the therapeutic benefit noted with pentoxifylline.

Actins↗
PubMed1979

Immunosuppressive properties of a virion polypeptide, a 15,000-dalton protein, from feline leukemia virus.

The 15,000-molecular-weight polypeptide (p15) of feline leukemia virus (FeLV) was shown to impair normal lymphocyte function in vitro and to abrogate immunity to feline oncornavirus disease in vivo. FeLVp15 suppressed concanavalin A-induced blast transformation of normal feline lymphocytes by 68%, while other virion proteins had no effect. p15 suppression was not due to toxicity, nor was p15 a competitive inhibitor of concanavalin A binding. Capping of receptors for concanavalin A on normal feline lymphocytes also was inhibited by either inactivated FeLV or FeLV p15. Groups of cats were immunized with either killed feline oncornavirus-associated cell membrane antigen bearing tumor cells or tumor cells plus FeLV p15. After challenge with feline sarcoma virus, three of four p15-treated cats developed progressive fatal fibrosarcoma as compared to one of five non-p15-treated cats. The cats receiving p15 also had lower cytotoxic antibody titers against feline oncornavirus-associated cell membrane antigen (mean peak titer, 1:6) than did the non-p15 group (1:74). These data support the hypothesis that the immunosuppression in cats infected with FeLV is mediated by FeLV p15.

Animals↗
PubMed1981

Antigenic modulation of lymphocytic surface immunoglobulin yielding resistance to complement-mediated lysis. II. Relationship to redistribution of the antigen.

Experiments were carried out on guinea-pig L2C leukaemic lymphocytes to investigate the mechanism of antigenic modulation of their surface immunoglobulin (Ig) defined as the conferring by anti-Ig of resistance to lysis by anti-Ig plus complement. The phenomenon reflects, and is probably a consequence of, redistribution of the Ig molecules by bivalent antibody. Fab fragments of the antibody were completely ineffective. Parallel studies by indirect immunofluorescence of the movement of th surface antigen-antibody complexes revealed that modulation for syngeneic complement was apparent when the complexes were minimally aggregated: capping and extensive endocytosis were not necessary. Modulation for xenogeneic (rabbit) complement required more extensive movement but was still appreciable while complexes persisted on the surface. Sodium azide at 10 mM, which inhibits antibody-induced redistribution of surface molecules, diminished modulation. In experiments omitting pre-incubation with antibody alone, the presence of azide during incubations with anti-Ig plus syngeneic complement increased lysis from a low and variable to a consistently high level; there was no effect on the already high level of lysis occurring with the non-modulating anti-Ia plus syngeneic complement. This effect of azide provides further evidence that antigenic modulation can be a major factor determining a cell's survival when it is confronted simultaneously by antibody and complement.

Animals↗
PubMed1982

Activation of rat B lymphocytes. I. Characterization of anti-immunoglobulin responses and isotype density of rat B cells.

Spleen cells of two rat strains, Lewis and Brown Norway (BN), have been activated by lectins and by antibodies specific for immunoglobulin isotypes embedded in their cell membranes. Optimal concentrations of antibodies specific for mu, gamma, or delta-chains of rat augments in vitro incorporation of 3H-TdR 5 to 18-fold in Lewis B lymphocytes and 1.5 to 4-fold in BN B lymphocytes. In addition, F(ab')2 fragments of anti-Ig reagents induced Lewis splenic B cells but not BN B cells to incorporate 3H-TdR. Responses to LPS and dextran sulfate, B lymphocyte mitogens, measured by radioactive uptake, were five to 10 times greater in Lewis B cell populations than in BN B cell populations. Density of surface Ig isotypes and capping kinetics were similar in the two rat strains, although the percentage of T cells, T cell subsets, B cells, and Ia+ B cells differed in the spleens of these strains of rats. Both T lymphocytes and macrophages were needed in culture to effect an optimal response. IL-2 restored the response in B cell cultures depleted of T cells and macrophages, and enhanced 3H-TdR uptake in whole spleen cells of Lewis but not BN rats. The strain-dependent responsiveness of B cells to specific anti-Ig reagents or B cell mitogens appears to be associated with inherent (genetic) defects in T cells and B cells or defects in T cell to B cell cooperation in BN rats.

Animals↗
PubMed1990

Activation of mechanical responses in leukocytes.

Different kinds of leukocytes undergo cytoskeleton-dependent mechanical responses associated with their specific physiological functions. We have investigated cellular stiffening of several types of leukocytes using a method which measures the force resisting cellular indentation. We have found that lymphocytes stiffen in response to crosslinking cell surface antigens in a process associated with the much studied capping and patching processes. Further studies of myosin-deficient mutants of the ameba Dictyostelium discoideum suggest that this stiffening process results from a myosin dependent contractile process. Rat basophilic leukemia cells and pancreatic islet cells stiffen when triggered to secrete. The function of these cytoskeleton dependent processes is now unknown, but, at least in the islet cells, may be related to a regulation of the rate of secretion. Primary neutrophils stiffen in response to the chemotactic agent, fMet-Leu-Phe. This stiffening may be responsible for retention of these cells in the pulmonary microcirculation during response to inflammation. These observations pose the challenge of determining the structural basis, mechanism, and physiological function of each of these cellular responses.

Animals↗
PubMed1983

Lateral mobility and capping of rat lymphocyte membrane proteins.

Surface immunoglobulin (SIg), RT1 (rat histocompatibility proteins) and thy-1 present on rat lymphocytes cap with similar kinetics and to the same extent, the only difference being the antibody requirement. SIg requires one cross-linking antibody to induce capping, while RT1 and thy-1 require two antibodies. Fluorescence recovery after photobleaching experiments show that there is heterogeneity in the diffusion characteristics of lymphocyte membrane proteins. SIg is relatively immobile when labeled by Fab' and is immobilized by F(ab')2. RT1 diffuses 2.7 times as fast as SIg and the mobile fraction of RT1 is greater. When RT1 is cross-linked by relatively low concentrations of F(ab')2 its mobility is about the same as that of SIg. When the concentration of F(ab')2 is increased, RT1 is immobilized. Thy-1 diffuses faster than SIg and has a higher mobile fraction. When thy-1 is labeled with F(ab')2 it remains mobile and is immobilized when a second antibody layer is added. The relative order of mobility is thy-1 much greater than RT1 greater than SIg.

Animals↗
PubMed1988

Actin depolymerization and inhibition of capping induced by pentoxifylline in human lymphocytes and neutrophils.

Pentoxifylline is used clinically for the treatment of intermittent claudication. It is believed to exert its effect by altering the rheologic properties of blood. The cytoskeleton plays an important role in the maintenance of cell structure and function. In particular, alterations in the state of actin seem to play an important role in cell motility. Therefore, we examined the effect of pentoxifylline on the actin state in human polymorphonuclear leukocytes (PMN) and mononuclear cells. Pentoxifylline (10 mM final concentration) decreased F-actin content in both PMN and mononuclear cells. Pentoxifylline also inhibited concanavalin A-induced capping in PMN and mononuclear cells. Similarly, surface immunoglobulin capping in B lymphocytes was also inhibited. Pretreatment of cells with pertussis toxin did not inhibit pentoxifylline-induced decrease in F-actin, suggesting pentoxifylline does not act through pertussis toxin-sensitive G-proteins. Dibutyryl cyclic AMP failed to show any significant effect on the F-actin content in PMN. Therefore, the effect of pentoxifylline cannot be attributed to changes in cyclic AMP levels. Chemotactic peptide-induced actin polymerization was unaffected in PMN when expressed as percent changes in F-actin. The observations reported here suggest that the rheological effects of pentoxifylline might be due to its effects on the actin state in the cellular elements of the blood. Further studies on the mechanism of action of pentoxifylline on actin state in leukocytes will prove useful in delineating the physiological mechanisms regulating actin state in leukocytes.

Actins↗
PubMed1978

The capacity of microsomally-activated cyclophosphamide to induce immunosuppression in vitro.

Cyclophosphamide (CY) was activated in vitro with washed rat liver microsomes and cofactors. Pretreatment of mouse spleen cells in vitro with the activated drug abolished their capacity to give a primary antibody response to SRBC and levan on transfer to irradiated syngeneic recipients. However, responsiveness returned if challenge was delayed for 7 or more days after transfer. Part of this was shown to be of donor origin by an allotype marker. The treatment of normal spleen cells with activated CY in vitro also prevented B cells from regenerating their immunoglobulin receptors after capping with anti-immunoglobulin serum. The induction of suppression required contact between lymphocytes and activated CY for at least 30 min at 37 degrees and did not appear following incubation for 1 h at 0 degrees. Since the antibody response of drug-treated spleen cells to SRBC could not be restored with purified normal B or T cells, it is probable that B and T lymphocytes are both susceptible to suppression by activated CY in vitro. Similar pretreatment abrogated the graft-versus-host (GVH) reactivity of spleen cells as measured by survival and in a popliteal lymph node assay. B cell chimerism in F1 recipients of drug-treated parental spleen cells was demonstrated by the presence of congenic allotype markers. This suggests a possible approach for the attenuation of GVH disease which is associated with bone marrow transplantation in man.

Animals↗
PubMed1988

Normal human peritoneal macrophages are unable to cap and internalize class II antigens.

Normal human peritoneal macrophages show a restricted capacity to differentiate into inflammatory macrophages in vivo. We now report that these cells are unable to cap and internalize HLA-DR, as compared to endometriosis, and other macrophages. Immunoelectron microscopy indicated that lack of modulation was not due to the presence of preclustered antigenic sites. Northern blot analysis demonstrated transcripts for HLA.DR, c-fms, and c-fos, indicating that the surface defects were not likely to be associated with a general depression of transcriptional activity. There was no correlation between the mobility of class II molecules and the ability to present antigen as determined by autologous lymphocyte responses to tetanus toxoid. The inability of normal peritoneal macrophages to modulate class II antigens may represent a normal and more general environmental alteration required to permit peritoneal cells a scavenging function without developing the deleterious effects leading to a peritoneal inflammatory response.

Antibodies, Monoclonal↗
PubMed1994

The alpha/beta sheath and its cytoplasmic tyrosines are required for signaling by the B-cell antigen receptor but not for capping or for serine/threonine-kinase recruitment.

The B-cell antigen receptor is composed of membrane immunoglobulin sheathed by an alpha/beta heterodimer. The complex is noncovalently associated with protein kinase activity, and crosslinking of the receptor leads to capping and transmembrane signaling. Here we show that the sheath is not necessary either for this capping or for the association of membrane immunoglobulin with the detergent-insoluble cytoskeletal fraction that occurs following crosslinking. It is also not required for association of membrane immunoglobulin with a casein-kinase-like serine/threonine kinase. The sheath is essential, however, for transmembrane signaling. Provision of just the cytoplasmic domain of the beta sheath polypeptide to a mutant, unsheathed IgM molecule was sufficient to restore full signaling capability as judged by the phosphorylation of a variety of cellular proteins, including the B-cell-specific transmembrane protein CD22. This signaling was destroyed by mutating one of the tyrosines in the beta cytoplasmic domain. These results not only suggest that receptor signaling is mediated through phosphorylation of the tyrosines in the sheath's cytoplasmic domains but, together with previous work, indicate that different motifs within the sheath mediate presentation and signaling.

Amino Acid Sequence↗
PubMed1982

The status of concanavalin A receptors on the lymphocytes of leukemic AKR mice: inhibition of redistribution by high concentrations of the lectin.

Cell-electrophoretic and fluorescence microscopic investigations were carried out on the interaction of concanavalin A with the splenic lymphocytes of normal and spontaneously leukemic AKR mice. The normal splenic lymphocytes (NSL) showed a biphasic profile of electrophoretic mobility (EPM) as a function of the concentration of Con A, under capping conditions. The mean EPM of NSL increased at low concentrations of the lectin and was reduced below that of untreated cells at higher (greater than or equal to 15 micrograms/ml) concentrations of the lectin. The leukemic cells (LSL) also showed enhancement in EPM at low concentrations of Con A. At high concentrations of the same, however, the mean EPM of LSL was the same as that of untreated cells. In the case of NSL the reduction in EPM at high concentration of Con A is known to be brought about by post-redistributional binding of excess lectin to new receptor sites which emerge after capping of the first type of receptors. Similar investigations of the electrokinetic characteristics of Con A-receptor interaction on leukemic cells revealed that only a single type of receptor was present on LSL. These receptors were inducible to redistribution at low concentrations of Con A. High concentrations of the lectin, however, inhibited the redistribution of the receptors to Con A on LSL. This was confirmed when LSL treated with high concentrations of Con A were relieved from this inhibition by moderate concentrations of alpha-methyl glucoside, which dissociates cell bound Con A. Very low or very high concentrations of alpha-MG were ineffective. The receptors to Con A on LSL were thus behaviorally distinguishable from those on NSL. These data clearly demonstrate that the malignant transformation in AKR mice is also associated with alterations in the properties of receptors to a multivalent ligand Con A.

Animals↗
PubMed1993

Human CD38 is associated to distinct molecules which mediate transmembrane signaling in different lineages.

The CD38 antigen displays restricted functional associations with surface molecules involved in immune system and complement. Capping of the CD38 molecule in normal or neoplastic T cells is followed by rapid and specific co-modulation of the CD3-T cell receptor (TcR) complex. In normal and tumor cells of B lineage, CD38 was found to be also associated with surface Ig (sIg) and with the complement receptor 2 (CR2)/CD19 complex. The CD38 molecule expressed by purified natural killer cells displayed an association with the low affinity IgG Fc receptor (Fc gamma RIII) CD16. These observations suggest that specialized areas in the plasma membrane contain co-modulating structures, including different receptors involved in the transduction of extracellular signals. We propose a model whereby TcR, CR2 and CD16 are ligand binding structures in their respective lineages, while CD38 is a molecule involved in the intracellular transduction of the signals.

ADP-ribosyl Cyclase↗
PubMed1987

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells.

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.

Animals↗
PubMed1981

Intramembrane particles and the organization of lymphocyte membrane proteins.

An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- beta-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.

Animals↗