Con A-treated Cells Research Articles

Protective effect of quercetin against macrophage-mediated hepatocyte injury via anti-inflammation, anti-apoptosis and inhibition of ferroptosis

Yinchenhao Decoction (YCHD) is a classic prescription in traditional Chinese medicine (TCM). It appears to play an important role in anti-inflammation and autoimmunity protection. As one of the key active ingredients in YCHD, quercetin is a novel anti-inflammatory metabolite that exerts protective effects in many autoimmune diseases. However, its role in autoimmune hepatitis (AIH)-related hepatic injury has not been studied. The aim of this study was to reveal the hepatocyte protective mechanism of quercetin. In this study, we used Concanavalin A (Con A) to establish an in vitro hepatocyte injury-associated AIH model. Brl3a hepatocyte injury was induced by the supernatant of J774A.1 cells treated with Con A. We found that quercetin mitigated Con A-induced via macrophage-mediated Brl3a hepatocyte injury. Quercetin administration reduced the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the supernatant of Con A-treated Brl3a cells and attenuated the infiltration of J774A.1 macrophages induced by Con A. Moreover, quercetin effectively inhibited the expression of proinflammatory cytokines including interleukin-1β (IL-1β) by Con A. Furthermore, quercetin decreased hepatocyte apoptosis and ferroptosis levels in the macrophage-induced hepatocyte injury model. In conclusion, our study indicates that quercetin alleviates macrophage-induced hepatocyte damage by reducing the inflammatory response, apoptosis and ferroptosis. Our work suggests that quercetin might be a potential therapeutic strategy for AIH.

Concanavalin A Disrupts the Release of Fibrous Material Necessary for Zygote Formation of a Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

The Closterium peracerosum–strigosum–littorale (C. psl.) complex is the best characterized charophycean alga with respect to the processes of sexual reproduction. We examined the effect of concanavalin A (Con A) on physiological and ultrastructural changes during the conjugation of the C. psl. complex. Two heterothallic gametangial cells formed a sexual pair as usual; however, the release of gametes was completely blocked by the addition of Con A. Fluorescein isothiocyanate-labeled Con A bound to the outermost layer of the conjugation papillae of paired cells. In the absence of Con A, the disruption of outer cell walls on the conjugation papillae and the secretion of fibrous materials from the conjugation papillae were observed using a transmission electron microscope, but Con A-treated cells did not show these changes. Instead, a highly electron-dense layer was observed in the outermost papillae, and the excess fibrous materials remained at the inside of the layer. These results suggest that an unknown molecule(s) recognized by Con A is essential for the diffusion of fibrous materials at the conjugation papillae, which is an indispensable step for gamete release during conjugation of the C. psl. complex.

Rapid regulation of tonic GABA currents in cultured rat hippocampal neurons

Subacute and chronic changes in tonic GABAergic inhibition occur in human and experimental epilepsy. Less is known about how tonic inhibition is modulated over shorter time frames (seconds). We measured endogenous tonic GABA currents from cultured rat hippocampal neurons to evaluate how they are affected by 1) transient increases in extracellular GABA concentration ([GABA]), 2) transient postsynaptic depolarization, and 3) depolarization of presynaptic cells. Transient increases in [GABA] (1 μM) reduced tonic currents; this reduction resulted from GABA-induced shifts in the reversal potential for GABA currents (E(GABA)). Transient depolarization of postsynaptic neurons reversed the effects of exogenous GABA and potentiated tonic currents. The voltage-dependent potentiation of tonic GABA currents was independent of E(GABA) shifts and represented postdepolarization potentiation (PDP), an intrinsic GABA(A) receptor property (Ransom CB, Wu Y, Richerson GB. J Neurosci 30: 7672-7684, 2010). Inhibition of vesicular GABA release with concanamycin A (ConA) did not affect tonic currents. In ConA-treated cells, transient application of 12 mM K(+) to depolarize presynaptic neurons and glia produced a persistent increase in tonic current amplitude. The K(+)-induced increase in tonic current was reversibly inhibited by SKF89976a (40 μM), indicating that this was caused by nonvesicular GABA release from GABA transporter type 1 (GAT1). Nonvesicular GABA release due to GAT1 reversal also occurred in acute hippocampal brain slices. Our results indicate that tonic GABA currents are rapidly regulated by GABA-induced changes in intracellular Cl(-) concentration, PDP of extrasynaptic GABA(A) receptors, and nonvesicular GABA release. These mechanisms may influence tonic inhibition during seizures when neurons are robustly depolarized and extracellular GABA and K(+) concentrations are elevated.

Concanavalin A/IFN-Gamma Triggers Autophagy-Related Necrotic Hepatocyte Death through IRGM1-Mediated Lysosomal Membrane Disruption

Interferon-gamma (IFN-γ), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ−/− mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

Paradigmatic identification of MMP‐2 and MT1‐MMP activation systems in cardiac fibroblasts cultured as a monolayer

Activations of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin-dependent MMP-2 and MT1-MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP-2 and MT1-MMP activations by potential non-matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype-producing agent. We showed that treatment of cells with ConA activated pro-MMP-2, and that this activation concurred with elevated levels of cellular MT1-MMP and TIMP-2. The presence of active MT1-MMP and 43 and 36 kDa processed forms of MT1-MMP in a fraction of intracellular proteins prepared from ConA-treated cells suggests the possible internalization of differential forms of MT1-MMP. The appearance of 36 kDa processed form of MT1-MMP in conditioned media prepared from ConA-treated cells indicates the possible extracellular release of the further processed MT1-MMP fragment. Inhibition of furin in ConA-treated cells attenuated pro-MT1-MMP processing and the cellular TIMP-2 level, plus it reduced cell-released active MMP-2 in a time-dependent manner. These results suggest the involvement of furin in the ConA-induced activations of MT1-MMP and MMP-2. Furthermore, the existence of furin inhibitor-insensitive pro- and active MMP-2 species associated with ConA-treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP-2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif.

The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large ‘vacuole-like structure’ or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.

Role of Mitochondrial Membrane Potential in Concanavalin A-Induced Apoptosis in Human Fibroblasts

Lectins induce apoptosis in a wide variety of cell types but the mechanisms of apoptotic induction are unknown. We examined the role of mitochondrial membrane potential (Ψm) in concanavalin A-induced apoptosis in human diploid fibroblasts. Cells were treated with Con A for 0.5, 1, 3, 5, and 24 h. Con A induced a time-dependent increase of the proportion of TUNEL+ve cells over 24 h. Ψmwas examined by staining cells with the mitochondria-specific fluorescent cationic dye JC-1. Comparison of JC-1 fluorescence within mitochondria by flow cytometry showed that after 3 h, Con A reduced Ψmin a subpopulation of apoptotic cells with smaller cell volume and with apoptotic nuclear morphology. In contrast, Ψmwas unchanged in a separate population of viable cells with normal volume and normal nuclear morphology. Cyclosporin A protected cells against reduction of Ψmand also against nuclear condensation and morphological apoptosis. Measurement of intracellular calcium ion concentration ([Ca2+]i) by ratio fluorimetry of fura 2-loaded cells showed that Con A did not affect [Ca2+]iin viable cells but induced a progressive depletion of [Ca2+]iwith generation of calcium oscillations in apoptotic cells. Assessment of Bcl-2 in Con A-treated cells demonstrated an initially strong increase in Bcl-2 protein and mRNA but the appearance of degraded Bcl-2 protein at 3 and 5 h after treatment, indicating an inadequate protective response to the Con A stimulation. Collectively, these data indicate that lectin-induced apoptosis in fibroblasts is associated with breakdown of Ψm, loss of [Ca2+]ihomeostasis, and induced Bcl-2 expression.

Calmodulin antagonists increase the expression of membrane-type-1 matrix metalloproteinase in human uterine cervical fibroblasts.

The treatment of human uterine cervical fibroblasts with concanavalin A (ConA), or a specific calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or trifluoperazine resulted in accumulation of an active form of matrix metalloproteinase 2 (MMP-2, gelatinase A). In contrast, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weaker antagonist of calmodulin, did not modulate the activation of proMMP-2. The activation of proMMP-2 was confirmed by the enhanced activity on gelatin and the conversion of proMMP-2 to a 62-kDa form by zymography and western blotting. The plasma membrane, but not the conditioned medium, of the W-7- or trifluoperazine-treated cells activated proMMP-2; this activation was blocked by membrane-type-1 MMP (MT1-MMP) antibody and EDTA. The plasma membrane from trifluoperazine- or ConA-treated cells contained MT1-MMP and tissue inhibitor of metalloproteinases 2. Both trifluoperazine treatment and ConA treatment increased the steady-state levels of MT1-MMP mRNA and proMMP-2 mRNA. These results, together with our previous observations on the production of proMMP-1 (interstitial procollagenase) and proMMP-3 (prostromelysin 1) [Ito, A., Sato, T., Ojima, Y., Chen, L.-C., Nagase, H. & Mori, Y. (1991) J. Biol. Chem. 266, 13598-13601], suggest that calmodulin negatively regulates the matrix turnover by suppressing the production of a number of proMMPs including proMMP-1, proMMP-3 and MT1-MMP, and the activation of proMMP-2 in human uterine cervical fibroblasts.

Contribution of phosphoinositides and phosphatidylcholines to the production of phosphatidic acid upon concanavalin A stimulation of rat thymocytes

Stimulation of rat thymocytes by concanavalin A (Con A) results in a very early increase of the cellular level of phosphatidic acid (PA), while that of diacylglycerol (DAG) was not affected. As the biological activity of PA is very likely to be determined by its molecular species composition, the present study aims to investigate the pathways leading to the production of PA in Con A-stimulated rat thymocytes. Prelabeling the cells with [3H]arachidonic acid, [3H]myristic acid, [3H]choline, or [14C]lysophosphatidylcholine allowed us to determine that PA is formed by both phosphoinositide (PIs) and phosphatidylcholine (PC) hydrolysis. We then investigated whether PA derived from PC was formed by phospholipase C (PLC) or phospholipase D (PLD) hydrolysis. In the presence of 1-butanol, the production of phosphatidylbutanol was only observed in tetradecanoyl phorbol acetate (TPA)-stimulated cells. The use of a specific PC phospholipase C inhibitor resulted in a decrease of Con A-stimulated PA production in cells labeled with [3H]myristate. When cells were labeled with [3H]choline, only TPA stimulation induced a release of labeled choline. All together, these experiments suggest that PA is originated from two phospholipid sources, predominantly PI via PLC hydrolysis and to a lesser extent PC, by PLC hydrolysis also. Molecular species analyses by reverse phase HPLC are in agreement with this hypothesis, as diacyl-GP molecular species composition is similar to that of diacyl-GPC and DAG in resting cells, but resembles that of diacyl-GPI in Con A-treated cells. Thus, in stimulated cells, the amount of 18:0/20:4 species doubled while those of saturated and monounsaturated species decreased.

Activation of phosphatidylinositol 3-kinase by concanavalin A through dual signaling pathways, G-protein-coupled and phosphotyrosine-related, and an essential role of the G-protein-coupled signals for the lectin-induced respiratory burst in human monocytic THP-1 cells.

Stimulation of monocytic THP-1 cells by a lectin, concanavalin A (Con A), resulted in protein-tyrosine phosphorylation and association of some of the thus phosphorylated proteins with the 85 kDa regulatory subunit of PtdIns 3-kinase. Both actions of Con A were not inhibited by wortmannin, a PtdIns 3-kinase inhibitor, or by prior exposure of cells to pertussis toxin which uncouples certain G-proteins from receptors. The binding of PtdIns 3-kinase to the tyrosine-phosphorylated proteins increased upon Con A stimulation; there was a marked increase in the enzymic activity in the anti-phosphotyrosine immuno-precipitates from Con A-treated cells. The increase was abolished by wortmannin but not affected by pertussis toxin. The incorporation of 32P into PtdInsP3 also increased during incubation of [32P]P(i)-prelabelled cells with Con A, reflecting activation of whole-cell PtdIns 3-kinase which could not be accounted for solely by the increase in the phosphotyrosine-bound enzyme activity from the following aspects: (1) different concentration dependencies for Con A; and (2) almost total susceptibility of the incorporation to pertussis toxin. This notion appears to be supported by different time courses between increases in PtdInsP3 production and the phosphotyrosine-bound activity. The susceptibility to the toxin may reflect involvement of the toxin-sensitive G-proteins. In contrast, insulin-induced increases in PtdInsP3 production, as well as increases in phosphotyrosine-bound PtdIns 3-kinase activity, were blocked by wortmannin, but never affected by prior exposure of cells to pertussis toxin, excluding a possible involvement of G-proteins in the insulin-induced activation. Con-A-induced O2- production was almost inhibited by either pertussis toxin or wortmannin. These results suggest that oligomerization of cell-surface glycoproteins with Con A gives rise to activation of G-protein(s) and certain tyrosine kinase(s), both of which were responsible for PtdIns 3-kinase activation; the G-protein-mediated activation led to the respiratory burst.

ConcanavalinA receptors and the chemosensory behaviour of Tetrahymena thermophila

The relationship between concanavalin A (ConA) receptors and the chemosensory behaviour of the ciliated protozoan Tetrahymena thermophila was studied using the peptide chemoattractants proteose peptone and fibroblast growth factor. Studies on the chemosensory behaviour in semisolid methylcellulose showed that 50 μg/ml ConA selectively inhibited the persistent element of swimming behaviour by reducing time runs of cells responding to proteose peptone from 12.2±4.5 min to 0.8±0.3 min. Methyl-alpha-D-mannoside, but not methyl-alpha-D-galactoside, abolished the inhibitory effect of ConA, suggesting that mannoside-containing ConA receptors are involved in maintaining a persistent swimming behaviour. Control experiments, carried out in liquids where persistent swimming is less important for cellular behaviour, showed that ConA did not affect proteose-peptone-induced chemoattraction under these conditions as measured by a two-phase assay for chemoattraction. Also, no inhibitory effect of ConA could be found on swimming rates when individual velocities of ConA-treated cells were determined. When tested in liquid chemoattraction assays, ConA was found to be a weak but significant chemoattractant. Studies of the cellular location of ConA receptors on the plasma membrane of starved cells showed an unequal distribution. A preferential clustering of receptors at the anterior end of the cell was observed when determined at high concentrations (100 μg/ml) of fluorescent ConA. Methyl-alpha-D-mannoside but not methyl-alpha-D-galactoside abolished the fluorescent ConA labelling, indicating a preferential clustering of these mannoside-containing receptors at the anterior part of the plasma membrane and cilia. At lower concentrations (25 μg/ml), FITC-ConA produced more general labelling of the entire cell membrane. The results suggest that ConA receptors are necessary for the persistent element of swimming and that binding of ConA to its receptors interferes with processes related to signal transduction rather than by limiting the free movement of cilia required for locomotion. The gradient of receptors seen at high FITC-ConA concentrations may be important for a putative spatial chemosensory mechanism, i.e. chemotaxis.

GD2 ganglioside-specific monoclonal antibody reacts with murine cytotoxic T lymphocytes reactive with FBL-3N erythroleukaemia.

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4+CD8-, CD4-CD8+, and CD4-CD8- phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.

Impaired protein kinase C activation as one of the possible mechanisms of reduced lymphocyte proliferation in iron deficiency in mice

We investigated the effects of iron deficiency in mice on protein kinase C (PKC) activation, an enzyme required for optimal lymphocyte proliferation. C57BL/6 mice were fed either an iron-deficient diet (ID; 10 mg Fe/kg diet), a control diet (C; 50 mg/kg diet), or were pair fed (PF) to ID mice for 34 d. PKC activity was studied in spleen cells by histone phosphorylation. Iron deficiency significantly reduced cytosol activity in unstimulated cells and membrane-bound activity in cells stimulated by concanavalin A (Con A) or phorbol-12-myristate-13-acetate (PMA), and the ratio of membrane-bound over cytosol activity in mitogen-stimulated cells. In PF mice the ratio of membrane-bound activity to cytosol activity was greater than normal in Con A-treated cells and only slightly decreased in PMA-treated cells. PKC activity positively correlated with iron status. We conclude that reduced PKC activity and poor translocation results in aberrant signal transduction, which in turn might be responsible for the impaired lymphocyte proliferation associated with iron deficiency.

Lectins modulate the internalization of recombinant interferon-alpha A and the induction of 2‘,5‘-oligo(A) synthetase.

After binding to specific cell surface receptors, interferon-alpha (IFN-alpha) along with its receptor is internalized by the cells. However, the physiological significance of the internalization of IFN is not known. We have found that the lectin concanavalin A (ConA), which does not inhibit the binding of 125I-rIFN-alpha A, inhibits both the internalization of 125I-rIFN-alpha A and the rIFN-alpha A-induced increase in the levels of 2',5'-oligo(A) synthetase mRNA and enzymatic activity in the B lymphoblastoid cell line Daudi. The reduced level of IFN-induced 2',5'-oligo(A) synthetase in ConA-treated cells was due neither to direct inhibition of the enzymatic activity nor to generalized inhibition of protein or RNA synthesis. The dose-response curves were similar for the effect of ConA to inhibit 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction. The correlation between the ConA-mediated inhibition of both 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction suggests that internalization of rIFN-alpha A plays a role in the responses to rIFN-alpha A. However, since ConA inhibits protein mobility in the plasma membrane, it is possible that ConA is also preventing aggregation of IFN receptors or interactions between IFN receptors and signal transducing proteins in the plasma membrane that may be necessary for responses to IFN.

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.

Studies of the mechanism by which gangliosides inhibit the proliferative response of murine splenocytes to concanavalin A

Gangliosides are known to inhibit the proliferative response of murine and human lymphocytes to antigens and mitogens in vitro. In this study the response of murine spleen cells to concanavalin A (Con A) was used as a model system. Analysis of the cellular events by flow cytometry revealed that during the first 24 hr of culture the effect of gangliosides on Con A-treated cells was minimal. At 48 hr, however, more of the ganglioside-treated cells were in G 0 G 1 , the cells contained more RNA, and fewer cells were in S phase. These data indicate that gangliosides inhibit the transition of the cells from G 0 G 1 into the S phase of the cell cycle. Expression of the interleukin 2 (IL-2) receptor, as measured by the binding of a monoclonal antibody to the receptor, was not inhibited by the gangliosides. Binding of 125I-labeled recombinant IL-2 to cells cultured for 48 hr with Con A was inhibited by ganglioside G Dla but not by asialo G MI. Inhibition was much more effective if the gangliosides were preincubated with IL-2 before addition of cells, but no inhibition was observed if the cells were preincubated with gangliosides and the unbound gangliosides were washed out prior to addition of the IL-2. These data suggest that interference with the binding of IL-2 to the high-affinity IL-2 receptor of activated T lymphocytes plays an important role in the inhibition of Con A-induced proliferation.

Fluorescence and electron microscopic study of intracellular F-actin in concanavalin A-treated and cytochalasin B-treated Ehrlich ascites tumor cells.

To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones.

Early nuclear events in lymphocyte proliferation: The role of DNA strand break repair and ADP ribosylation

The previously reported extensive DNA strand breakage in resting murine splenic lymphocytes is not an artifact of the extraction or assay procedure. The benzamide inhibitors of poly(ADP ribose) synthetase (pADPRS), such as 5-methoxybenzamide (MBA), had been shown to block the strand break repair occurring within 2 h of activation of splenic lymphocytes by the mitogen concanavalin A (conA); the inhibitors also blocked early events in proliferation, such as blast formation, as well as entry into S phase. Inhibitors of pADPRS blocked lymphocyte proliferation by inhibiting the activity of this enzyme, rather than by non-specific effects. Aphidicolin, an inhibitor of α-polymerase, also prevented DNA strand break repair in conA-stimulated cells but, unlike MBA, did not prevent blast formation. DNA strand breaks accumulated in the presence of MBA at the same linear rate (300–400/h) in both resting and conA-treated cells. We and others had hypothesized that this accumulation was due to a continuous production of strand breaks in lymphocytes, leading to their accumulation in presence of repair inhibitors. However, incubation of the cells with aphidicolin at concentrations that inhibited repair did not result in any increase in strand breaks. The hypothesis of continuous cycling of breaks is incorrect; accumulation of breaks was due to some indirect effect of MBA, such as a possible disinhibition of an ADP-ribosylation-sensitive endonuclease described in other cell types. All of the early stages of lymphocyte proliferation, including blast transformation (but not DNA synthesis) require ADP ribosylation. Repair of DNA strand breaks is not a precondition for blast formation, though experiments involving the combined effects of MBA and aphidicolin showed that repair of the breaks is essential in order for the cells to replicate their DNA. Our data are consistent with a model suggesting that DNA strand breaks introduced into differentiated cells act as an additional safety-catch mechanism that restrains them from replicating their genetic material but not from undergoing the early stages of proliferation.

Stimulation of proliferation of murine lymphocytes by the calcium ionophore A 23187 in the absence of serum: The requirement for thiols

The proliferation of mouse spleen cells and T-lymphocytes, initiated by the calcium ionophore A 23187 was studied by a serum-free culture technique. In contrast to Con A, A 23187 was capable of stimulating cells only if 2-mercaptoethanol, cysteine and glutathione (reduced form), respectively, were present in the culture medium. In the absence of one of these compounds a stimulating activity of A 23187 was observed only with high concentrations of cells (i.e., 10 7/ml). With glutathione present, the cells could be stimulated only at concentrations of A 23187 which were found to be suboptimal in cultures with 2-mercaptoethanol. Human serum, fetal calf serum and bovine serum albumin shifted the active and optimally stimulating concentrations of A 23187 to higher values. A similar effect was observed with sera- and Con A-treated cells. The effect of sera and albumin was paralleled by a protecting effect of cells against high concentrations of A 23187.

Changes in mouse MHC expression during the cell cycle of Con A-treated spleen cells.

This study was directed at correlating the expression of class I MHC determinants with different segments of the cell cycle by using dual laser flow microfluorometry to measure levels of both DNA and cell surface H-2Kd or H-2Dd determinants for single cells. Con A-treated mouse spleen cells were identified as being in the G2/M or G0/G1 phases of the cell cycle on the basis of propidium iodide or Hoechst 33342 dye bound to DNA. Monoclonal anti-H-2 antibodies, indirectly fluoresceinated with goat anti-mouse IgG, were used to detect MHC determinants. The average level of both Kd and Dd determinants expressed by G2/M cells was about 1.6-fold higher than that expressed by G0/G1 cells. These observations indicate that the average-size G0/G1 and G2/M cells have the same apparent surface density of Kd and Dd determinants, insofar as we estimate that these cells differ in surface area by a factor of about 1.5. We also analyzed the expression of Kd and Dd determinants by measuring how they changed as a function of the intensity of forward light scatter from cells. For both G2/M and G0/G1 cells, changes in light scatter intensity were associated with parallel changes in levels of Dd and Kd determinants, indicating a common mechanism(s) that controls their cell surface expression.