Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis

Leukocyte infiltration into the liver is paramount to the development of liver injury in hepatitis. Hepatitis occurring after the administration of Con A in mice is felt to be a T lymphocyte-mediated disease. In this study, we report that neutrophils are the key initiators of lymphocyte recruitment and liver injury caused by Con A. The objectives of this study were to investigate the involvement of neutrophils in Con A-induced hepatitis in vivo via intravital microscopy. After Con A administration, we observed a significant increase in leukocyte rolling flux, a decrease in rolling velocity, and an increase in leukocyte adhesion to the hepatic microvasculature. Fluorescence microscopy identified that within 4 h of Con A administration only a minority of the recruited leukocytes were T lymphocytes. Furthermore, immunohistochemistry showed a significant increase in neutrophils recruited to the liver post-Con A treatment in association with liver cell damage, as reflected by elevated serum alanine aminotransferase levels. Using flow cytometry, we observed that Con A could bind directly to neutrophils, which resulted in a shedding of L-selectin, an increase in beta(2)-integrin expression, and the production of reactive oxidants. Following neutrophil depletion, a significant inhibition of Con A-induced CD4+ T lymphocyte recruitment to the liver resulted and complete reduction in hepatic injury, as assessed by serum alanine aminotransferase levels. In summary, the present data support the concept that neutrophils play an important and previously unrecognized role in governing Con A-induced CD4+ T cell recruitment to the liver and the subsequent development of hepatitis.     

Effects of concanavalin A on the net formation of zoospores in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

In the green alga Hydrodictyon reticulatum (L.) Lager‐heim (Chlorococcales, Chlorophyceae), zoospores are arranged in a regular fashion to form an intricate hexagonal network during the asexual reproductive cycle. Polypeptides that bind concanavalin A (Con A) in zoospores increased in amount during net formation and decreased after the completion of the adhesion between zoospores. Fluorescein isothiocyanate‐Con A‐binding sites corresponded to the contact sites of zoospores immediately after cessation of the movement. Treatment with 25 μg mL^?1 Con A inhibited adhesion of isolated immotile zoospores obtained from parental cells, and Con A‐treated zoospores could not form hexagonal nets. Moreover, when isolated immotile zoospores were treated with Con A, the cessation of the zoospore movement was retarded in dose‐dependent manner. These results suggest that the Con A‐binding sites may participate in the adhesion of zoospores during hexagonal net formation.     

Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors

The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.     

Response of superoxide anion production by guinea pig eosinophils to various soluble stimuli: comparison to neutrophils

The effect of various soluble stimuli on the superoxide production by guinea pig eosinophils was studied in comparison to neutrophils. Phorbol myristate acetate, A23187, digitonin, NaF, concanavalin A (Con A), and cytochalasin E stimulated eosinophils and neutrophils to release O2-. The O2- production by these active agents, excluding Con A and cytochalasin E, was much greater in eosinophils than in neutrophils. Formyl-Met-Leu-Phe stimulated the O2- production in neutrophils but not in eosinophils. Neither histamine nor Val/Ala-Gly-Ser-Glu stimulated the O2- production in both types of leukocytes. A23187- or Con A-stimulated O2- production was greatly enhanced by cytochalasin B pretreatment in neutrophils but not in eosinophils. Lineweaver-Burk analysis of NADPH oxidase in particulate fractions showed that eosinophils possessed the same Km values as neutrophils and greater Vmax values than neutrophils, suggesting that eosinophils have a similar, but more active, O2- -generating enzyme system than neutrophils.     

Concanavalin A-induced impairment of fibroblast spreading on laminin but not on fibronectin

The effect of concanavalin A (Con A) on the adhesion of 8-day-old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM alpha-methylmannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM-Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM-Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.     

A novel accessory role of neutrophils in concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis has been investigated as a model of T cell-mediated liver injury, in which IFN-gamma plays an essential role by inducing apoptosis of liver cells. Since a large number of neutrophils infiltrate into the liver in the model, the role of neutrophils was investigated in this study. Con A hardly caused liver injury in neutrophil-depleted mice, as assessed as to the plasma alanine aminotransferase level as well as histochemistry. Neutrophil-depleted mice also failed to produce IFN-gamma. Intracellular IFN-gamma staining revealed that, among liver leukocytes, T and NK cells but not neutrophils are the main producers of IFN-gamma. Nylon wool-purified "T cells", however, failed to produce IFN-gamma in response to Con A in vitro, while the production was restored by the addition of neutrophils. Overall, this study suggests that neutrophils play a novel accessory role in IFN-gamma production in Con A-induced hepatitis.     

Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.     

Concanavalin A stimulation of O2 consumption in electropermeabilized neutrophils via a pertussis toxin-insensitive G protein

Electropermeabilized human neutrophils were used to investigate the possible role of G-proteins in the respiratory burst elicited by concanavalin A (Con A). The Con A-induced activation of the NADPH oxidase was not inhibited by either pertussis toxin or cholera toxin. However, the burst was inhibited by GDP and GDP beta S providing evidence for the involvement of a G-protein(s). O2 consumption in Con A-stimulated cells was dependent on both ATP and Mg2+. ATP could be substituted by ATP gamma S but not by the non-hydrolyzable analog AMP-PNP, suggesting involvement of phosphotransferase reactions. It is concluded that at least two distinct types of G-proteins are capable of inducing the respiratory burst in neutrophils and that accumulation of phosphorylated intermediates may be essential for activation of the respiratory burst by the lectin.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Cell spreading on laminin substrate involves Con A-binding proteins

Concanavalin A (Con A), a tetravalent lectin, was shown to impair 8 chick embryo fibroblast (8 d CEF) spreading on a laminin (LM) substrate but not on a fibronectin substrate (FN), suggesting that cell surface Con A binding proteins could be involved in 8 d CEF spreading on a LM substrate. The interaction of Con A-binding proteins with Con A is dependent upon the carbohydrate moieties of the isolated glycoproteins; since they interact strongly with Con A-Sepharose and are eluted with 0.3 Mol/l alpha-methylmannopyranoside, the isolated Con A binding-proteins inhibit 8 d CEF adhesion to a Con A substrate to the same extent as alpha-methylmannopyranoside. Furthermore, the isolated Con A binding proteins specifically inhibit in a dose-dependent manner 8 d CEF spreading on LM but not on FN.     

Effects of endothelial basement membrane on neutrophil adhesion and migration

The influence of the sub-endothelial basement membrane (BM) on the adhesion and migration of leukocytes is not well-defined. We therefore investigated the behaviour of human neutrophils on purified BM proteins and on BM deposited by short- or long-term cultures of endothelial cells (EC). The adhesion, but not migration velocities, of neutrophils activated with interleukin-8 was dependent on the coating concentrations of purified collagen, laminin or fibronectin. In contrast, adhesion was similar on matrices deposited by 3-day or 20-day cultures of EC, but neutrophils migrated more slowly on the distinct BM that formed over 20 days. In addition, while adhesion on all surfaces was greatly reduced when neutrophils were treated with antibody against β₂-integrins, antibody against β₁-integrins only inhibited adhesion to the 20-day BM. Thus, the native BM has distinct effects on integrin usage and migration by neutrophils, which are not reproduced by purified proteins or matrix deposited early during endothelial culture.     

Adhesion of sea-urchin embryonic cells to substrata coated with cell adhesion molecules

Summary— A cell-to-substratum adhesion assay is developed to study the adhesion of sea-urchin embryonic cells to coated substrata. The involvement in this process of both carbohydrate and protein molecules is reported. Concanavalin A (Con A) increases the attachment of cells to the substratum in a dose-dependent manner and this effect is completely abolished when the incubation is carried out in the presence of the specific monocarbohydrate Con A-inhibitor, α-methyl-d -mannoside. A Con A-mediated enhancement of cell-to-substratum adhesion was also detected on cells deprived of toposome, a glycoprotein complex responsible for cell-to-cell adhesion. The involvement of other molecules as well as toposome in the process of cell-to-substratum adhesion is also investigated. Results of these in vitro experiments indicate that all the molecules tested contribute to the process of cell-to-substratum adhesion.     

Amelioration of ischemia-reperfusion injury with cyclic peptide blockade of ICAM-1

Neutrophils are pivotal in the pathogenesis of ischemia-reperfusion (I/R) injury leading to muscle damage. Firm adhesion of neutrophils to the endothelium is initiated by an interaction between intercellular adhesion molecular-1 (ICAM-1) on the endothelium and beta(2)-integrins on neutrophils. Inhibition of ICAM-1-dependent binding using monoclonal antibodies has been shown to be efficacious in ameliorating I/R injury by preventing the influx of neutrophils into the ischemic tissue. We recently described a cyclic peptide that is a potent and selective inhibitor of ICAM-1 (IP25) in vitro. In this study, we tested the hypothesis that IP25-mediated blockade of ICAM-1 would inhibit neutrophil influx during reperfusion of ischemic tissue and consequently attenuate muscle injury in a tourniquet hindlimb murine model of I/R injury. Varying amounts of peptide drug were injected at the beginning of the reperfusion period. The neutrophil influx and size of infarction at the end of 2 h of reperfusion were compared with those in untreated control mice and contralateral nonischemic limbs. Mice receiving IP25 immediately before reperfusion showed a 56% reduction in neutrophil infiltration in the ischemic muscle, accompanied by a 40% reduction in the infarct size. No effect on I/R injury was seen if IP25 administration was delayed for 60 min after reperfusion. We conclude that IP25 effectively inhibits ICAM-1-mediated adhesion of neutrophils to the endothelium in mice leading to a protective effect and suggests that synthetic peptide antagonists have a potential role as therapeutic tools.     

Double stimulation with FMLP and Con A restores the activation of the respiratory burst but not of the phosphoinositide turnover in Ca2+-depleted human neutrophils. A further example of dissociation between stimulation of the NADPH oxidase and phosphoinositide turnover

The results reported here show that the activation of the NADPH oxidase in neutrophils by formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A) may occur with a stimulus response coupling sequence that bypasses the activation of phosphoinositide hydrolysis, monitored as accumulation of inositol phosphates and glycerophosphoinositol, and the increase in [Ca2+]i. In fact: in Ca2+-depleted neutrophils FMLP and Con A do not induce the respiratory burst and the activation of phosphoinositide hydrolysis. The addition of Ca2+ restores both the respiratory and the phosphoinositide responses; the double treatment of Ca2+-depleted neutrophils with FMLP and Con A in sequence, before FMLP and then Con A and vice versa, or simultaneously, restores the capacity to respond to the second stimulus with the respiratory burst but not with the activation of phosphoinositide hydrolysis. These findings suggest that, for the activation of the NADPH oxidase by FMLP and by Con A: the transduction pathway including the stimulation of phosphoinositide turnover, the Ca2+ changes and the activity of the protein kinase C is not required, or is not the unique, and one stimulus may trigger more than one transduction pathway. Possible transduction pathways are discussed.     

Cross-linking of cell surface receptors enhances cooperativity of molecular adhesion

Cooperativity of molecular adhesion has been proposed as a mechanism for enhanced binding strength of adhesion molecules on the cell surface. Direct evidence for its mechanism, however, has been lacking until now. Atomic force microscopy (AFM) was used to measure the adhesive strength between concanavalin A (Con A) coupled to an AFM tip and Con A receptors on the surface of NIH3T3 fibroblast cells. Cross-linking of receptors with either glutaraldehyde or 3, 3'-dithio-bis(sulfosuccinimidylproprionate) (DTSSP) led to an increase in adhesion that could be attributed to enhanced cooperativity among adhesion complexes. An increase in loading rate due to greater stiffness of fixed cells also contributed to the twofold increase in binding strength. These results show that receptor cross-linking can greatly contribute to a total increase in cell adhesion by creating a shift toward cooperative binding of receptors.     

Enhanced Expression of Fc Receptors on Neutrophils from Calves with Leukocyte Adhesion Deficiency

The expression of Fc receptors for immunoglobulin G(IgG) and concanavalin A (con A)-binding receptors, luminol-dependent chemiluminescent (LDCL) responses, and the effect of anti-bovine IgG on LDCL responses were evaluated in neutrophils from Holstein calves with leukocyte adhesion deficiency (BLAD). Neutrophils from affected calves showed a 2.1- to 2.5-fold increase in Fc receptor expression compared with those of control calves by flow cytometric analysis. Con A-binding activities of neutrophils from affected calves were similar to those of control calves. Neutrophils from a calf with BLAD, when stimulated with zymosan opsonized with bovine serum (OPZ), heat-aggregated bovine IgG (Agg-bovine IgG), sheep red blood cells (SRBC) sensitized with anti-SRBC antibody (SRBC-anti-SRBC Ab), or con A had LDCL responses of 36 (P<0.05), 77, 126 and 119% of peak LDCL values of controls, respectively. The NBT-reducing value of neutrophils from a calf with BLAD when stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG was 116.5% of the values of neutrophils from control calves, but the difference was not significant. The LDCL responses of neutrophils from a control calf and a calf with BLAD stimulated with OPZ were inhibited markedly by pre-incubation with anti-bovine IgG antiserum at concentrations ranging from 1.25 to 20 or 40 μg/ml. Although an increase in Fc receptor expression on neutrophils from calves with BLAD was observed, the LDCL responses stimulated with SRBC-anti-SRBC Ab and NBT-reducing activity stimulated with Agg-bovine IgG after pretreatment with anti-bovine IgG did not correlate significantly with the increased Fc receptor expression. These results support that neutrophil functions mediated by the Fc receptors are associated synergistically with the presence of the complement receptor type 3 (CR3)(CD11b/CD18).     

Adhesion of human neutrophils to fibronectin is inhibited by comaruman,pectin of marsh cinquefoil <Emphasis Type="Italic">Comarum palustre</Emphasis> L., and by its fragments

Earlier, we detected antiinflammatory action of comaruman, pectin of the marsh cinquefoil Comarum palustre L. This effect can be explained by new data concerning inhibition of adhesion of human neutrophils to fibronectin by comaruman and its fragments. The galacturonan backbone fragment of molecular mass >10 kD appears to be the active region of the comaruman macromolecule. Comaruman CP (50-200 microg/ml) was found to decrease adhesion of neutrophils stimulated by phorbol 12-myristate 13-acetate (PMA, 1.625 microM) and by dithiothreitol (DTT, 0.5 mM). The fragments of comaruman CP-H (100 kD), CP-H1 (10-50 kD), and CP-H2 (100 kD) obtained by acidic hydrolysis and representing regions of linear polygalacturonan are shown to inhibit neutrophil adhesion more than the crude pectin. A fragment CP-E (<10 kD) obtained using pectinolysis and representing a branched region of the comaruman macromolecule failed to influence cell adhesion. The parent comaruman CP as well as fragments of its polygalacturonan backbone diminish PMA-initiated generation of oxygen radicals in neutrophils.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Purification and Amino-terminal Sequencing of a 68 kD Glycopolypeptide of the Plasma Membrane from Maize Sperm Cells

Based on author's previous work on detection and immunolocalization of glycoproteins of the plasma membrane of maize ( Zea mays L. ) sperm cells, a 68 kD peripheral specific glycopolypeptide of the plasma membrane from maize sperm cells was purified by IEF-SDS two-dimensional electrophoresis. It presents specif- ically positive reaction in Con A-HRP (concanavalin A-horseradish peroxidase) staining, and its pi value is 5.5. The search in protein sequence database reveals that the amino-terminal sequence of this glycopolypeptide is identical with that of Con A. But its difference from Con A in molecular weight and pi value indicates that it could be related to a Con A receptor on the plasma membranes of maize sperm cells instead of being Con A itself. It is fascinating to study further the function of the above glycopolypeptide in gametic recognition, adhesion and fusion of the double fertilization in maize.     

Concanavalin A induces microtubule assembly and specific granule discharge in human polymorphonuclear leukocytes

Human neutrophils stimulated by concanavalin A (Con A, 100 microng/ml) contained markedly enhanced numbers of microtubules and discharged peroxidase-negative (specific) but not peroxidase-position (azurophile) granules. Release of lysozyme from specific granules was dose and time dependent, could be inhibitied by alpha-methyl-D-mannoside, and enhanced by cytochalasin B. Many microtubules were associated with internalized plasma membrane bearing Con A binding sites.