Griffonia Simplicifolia IB4 Research Articles

Immunohistochemical study of cell proliferation and differentiation in epidermis of mice after administration of cholera toxin

Cholera toxin causes reversible epidermal hyperplasia. We observed maximal thickness of the epidermis on the fourth day after treatment and a return to pretreatment values by day 7. The increase in thickness occurred in the basal and intermediate layers, with these layers becoming two to three times thicker than those of normal epidermis. The time sequence of epidermal proliferation was studied using bromodeoxyuridine (BrdU) labelling. We observed a maximum number of labelled basal cells within the first 24 h. Only a few cells were labelled 7 days after toxin injection. Griffonia simplicifolia-IB4 (GSA-IB4), Ulex europaeus-I (UEA-I) and Griffonia simplicifolia-II (GSA-II) lectins were used for the analysis of epidermal cell differentiation in the tissue sections. To study keratinocyte differentiation, further immunological staining was performed using two anticytokeratin antibodies, PKK2 and PKK3 mouse monoclonal antibodies. From the immunocytochemical results, we conclude that synchronous differentiation of the epidermis occurs after cholera toxin administration.

Lectin binding sites on head structures of the spermatid and spermatozoon of the mosquito Culex quinquefasciatus (Diptera, Culicidae).

The presence of intranuclear and acrosomal lectin binding sites in spermatids and spermatozoa of the mosquito Culex quinquefasciatus was analysed. Direct and indirect lectin-gold techniques were used on LR White-embedded cells. The nuclear compartment was the structure most intensely labelled. Early spermatid nucleus showed moderate labelling for peanut agglutinin (PNA), Griffonia simplicifolia IB4 (GS-IB4) and Ricinus communis agglutinin (RCA), and light labelling for the other lectins tested. The sperm nucleus was intensely labelled by all lectins. The acrosome, an enzyme-containing structure, was labelled by some lectins. The anterior acrosomal region was labelled by PNA, while the proximal acrosomal region was labelled by PNA and G. simplicifolia II (GS II) lectins, and showed the presence of fucose residues with the use of Ulex europaeus I (UEA-I) lectin. The spermatozoa stored in the spermatheca showed the same pattern of labelling as that observed in spermatozoa localized in testis and seminal vesicles for all lectins tested. Carbohydrate residues in the nuclear compartment may be involved with the process of chromatin condensation. In the acrosomal region these residues may play a role in the process of sperm-oocyte interaction.

Macrophage membrane glycoprotein binding of Griffonia simplicifolia I-B4 induces TNF-alpha production and a tumoricidal response.

Thioglycollate-elicited macrophages (m phi), upon binding the lectin Griffonia simplicifolia IB4 (GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor-alpha (TNF-alpha), belongs to the group of elicited proteins. This specific m phi response is directly influenced by the dose of GSIB4 used and the time in contact with the cells. At 40 micrograms/ml GSIB4, the maximum dose of lectin used, the m phi activity was equal to that achieved when the cells were incubated with an interferon-gamma/lipopolysaccharide (IFN/LPS) stimulus alone. Moreover, the data showed that TNF-mediated tumoricidal activity was significantly influenced by GSIB4 binding to the m phi membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand-receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4 with m phi significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor-ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3 levels were significantly enhanced upon m phi-GSIB4 binding. Collectively, the data show that GSIB4 binding to specific glycoproteins in the m phi membrane induces TNF-alpha production and facilitates TNF-alpha dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that m phi activity is influenced by the sequence in which GSIB4 is presented to the m phi relative to the IFN/LPS treatment.

Specific ligation of surface α-D-galactosyl epitopes markedly affects the quantity of four major proteins secreted by macrophages

Activated macrophages (M phi s) have terminal alpha-D-galactosyl (alpha D-Gal) residues on their membranes that are not apparent on resting cells. Ligation of these epitopes with Griffonia simplicifolia I-B4 (GSI-B4), a lectin that has specificity for alpha-D-Gal residues, alters selected M phi functions. To explore the mechanism(s) that may be responsible for some of the functional changes, alterations in the secretory pattern of [35S]methionine-labeled proteins were assessed when cells were cultured with or without this ligand. The proteins were identified by Western blots and quantitated. Interestingly, alpha-D-Gal ligation proved to decrease the secretion of some proteins while increasing the secretion of others. Some of the most significant changes were observed in four proteins: fibronectin and transglutaminase were down-regulated by 55 and 66% respectively, while plasminogen activator inhibitor type 2 was increased by 259% and collagenase was increased 1000-fold. These observations show that the emergence of new oligosaccharide epitopes, such as alpha-D-Gal, concomitant with M phi activation may serve to mediate the transduction of signals that cause quantitative changes in the elaboration of diverse M phi products. The biologic significance of the four identified proteins has been well established. Fluctuations in their levels are likely to play a role at sites of chronic inflammation.

The concurrent expression of Griffonia simplicifolia-IB4 binding and tumor necrosis factor-alpha differs between alveolar and peritoneal macrophages.

As a corollary to their anatomic location, alveolar macrophages (AM) have a lower threshold for generating some physiologic functions than peritoneal macrophages (PM). In this study, we examined both of these populations for their ability to bind the lectin Griffonia simplicifolia-IB4 (GSIB4) and to produce tumor necrosis factor (TNF)-alpha. The results showed that these two responses were concurrently expressed in activated macrophages, although they differed in magnitude when AM and PM were compared. Following in vitro incubation, AM from lipopolysaccharide-treated rats demonstrated a higher percentage of GSIB4 positivity and TNF production when compared with their respective PM. Since prostaglandin E2 can regulate the expression of some macrophage activities, experiments were conducted to determine whether this could also affect the ability of macrophages to bind the GSIB4 lectin. Neither the administration of indomethacin nor exogenous prostaglandin E2 altered the expression of this marker. Conversely, these treatments produced significant changes in TNF-alpha production in both alveolar and peritoneal macrophages. When the concurrent expression of GSIB4 lectin binding and TNF-alpha production was analyzed, AM from lipopolysaccharide-treated rats demonstrated both superior GSIB4 positivity and TNF-alpha production compared with all other macrophages examined. The results of this work show that AM and PM differ in their expression of GSIB4 binding and TNF-alpha production. These differential responses may be important in determining the level of activity of macrophages that are participating in an immune response.

Fc-γ Receptor-Ligand Interactions Enhance Macrophage GSIB4-Binding Activity

Activated macrophages have an increased ability to bind the lectin Griffonia simplicifolia-IB4 (GSIB4). Since macrophages readily use the Fc-receptor (FcR) during several immunologic and inflammatory processes, it is important to determine whether interactions with this moiety affect GSIB4-binding ability. Peritoneal macrophages cultured in vitro with Fc fragments of immunoglobulin G (IgG), whole IgG, or heat-aggregated IgG demonstrate an increase in this function. Conversely, treatment of macrophages with (Fab')2 fractions alone has no direct effect on this activity. Although the GSIB4-binding response is minimally expressed by normal macrophages, it is more markedly apparent on macrophages from LPS-treated animals. In both cases, however, pretrypsinization of the cells renders them refractory to IgG-mediated induction of the GSIB4-binding response. Moreover, macrophages cultured independently with IgG subclasses 1, 2a, or 3 demonstrate that the magnitude of their response to this signal is directly associated with the type of subclass used. Although each subclass enhanced the response, in this study interactions with IgG2a produced the best results. Overall, however, the greatest GSIB4-binding activity is generated when FcRs are crosslinked by aggregated IgG rather than simply bound by independent monomeric interactions at the FcRs. This suggests that the event of appropriately interacting with the FcRs amplifies the GSIB4-binding function. Such a mechanism could play a key role in coordinating the humoral, cell-mediated, and innate responses of the immune system.

Differential Induction of Macrophage GSIB4-Binding Activity

Activated macrophages display a terminal galactopyranosyl group on their membrane surface that binds the lectin Griffonia simplicifolia-IB4 (GSIB4). Using FITC-conjugated GSIB4, we examined the induction and subsequent expression of this corresponding marker on peritoneal macrophages from normal (NMO) and LPS-treated (LPS MO) mice. Although the percentage of fresh LPS MO explants that bound GSIB4 was always higher when compared to the NMO counterparts, marker expression on the latter was readily enhanced by culturing the cells in vitro either alone or with stimuli. Moreover, we found that an increase in this activity was promoted by either nonspecific phagocytosis of latex beads, or gamma-interferon (gamma-IFN) treatment. Further investigation showed that a prerequisite sequence of signal delivery to the macrophages was associated with maximal expression of the GSIB4 binding. When gamma-IFN treatment preceded latex bead ingestion, maximum GSIB4 binding occurred. Data obtained from using short-term (1 hr) and long-term (24 hr) exposure to latex beads showed that metabolic processing of induction signals was required to enhance the response over time. This yielded better GSIB4-binding activity when responses to these pulses were analyzed in freshly explanted macrophages. The overall results of this study demonstrated that macrophage binding of GSIB4 was differentially associated with stimuli induction. Moreover, select signals in the form of soluble mediators, or the mechanical events characteristic of internalization were capable of eliciting an increase in the percentage of macrophages that were positive for binding GSIB4. Thus, the enhanced affinity for binding this lectin may serve as a useful marker to determine the magnitude of macrophage responsiveness when these cells are examined following their exposure to different stimuli.

Saccharide residues in human gingiva as revealed with fluorochrome‐coupled lectins

The histochemical binding of 16 fluorochrome-conjugated lectins to human marginal gingiva was investigated. Of a total of 14 galactose/N-acetylgalactosamine (Gal/GalNAc)-specific lectins, Dolichos biflorus (DBA), Helix pomatia (HPA), and Helix aspersa agglutinins (HAA) were blood group A-reactive whereas Griffonia simplicifolia I-B4 (GSA-I-B4) and Sophora japonica (SJA) agglutinins were blood group B-reactive. HPA, HAA and GSA-I-B4 bound to all suprabasal epithelial cells and to vascular endothelia in tissues with compatible blood groups and detected only upper epithelial cells in tissues lacking the respective blood group antigens. SJA, on the other hand, bound to suprabasal epithelial cells and to endothelial cells in specimens from blood group B, AB and A individuals. DBA gave a heterogeneous labeling of upper epithelial cells in blood group A, AB and B specimens but not in O specimens and did not react with endothelia in any of the tissue samples. DBA bound, instead, consistently to mast cells in gingival lamina propria. Of the other Gal/GalNAc-reactive lectins, 2 bound to suprabasal epithelial cells and 7 to all viable cell layers in gingival epithelium. The binding of these lectins was blood group-independent. Of the fucose-specific lectins, Ulex europaeus agglutinin I (UEA-I) gave an intense suprabasal cell membrane-type of epithelial fluorescence in blood group O specimens and a more diffuse staining in other specimens and recognized endothelial cells in a blood group-independent way. Anguilla anguilla agglutinin (AAA) gave a blood group-independent epithelial staining and bound heterogeneously to endothelial cells only in blood group O samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells.

The study of the expression of alpha-galactosyl epitopes on various mammalian cells is of particular interest, since as much as 1% of circulating IgG antibodies in humans interact with this carbohydrate residue. This natural antibody, designated "anti-Gal," was previously found to bind to terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R on biochemically defined glycolipids (Galili, U., Macher, B. A., Buehler, J., and Shohet, S. B. (1985) J. Exp. Med. 162, 573-582; Galili, U., Buehler, J., Shohet, S. B., and Macher, B. A. (1987) J. Exp. Med. 165, 693-704). The expression of anti-Gal binding epitopes on nucleated cells from various mammalian species was studied by immunostaining with this antibody. The binding of anti-Gal to various cells was correlated with the binding of the lectin Bandeiraea (Griffonia) simplicifolia IB4 (BS lectin). The BS lectin also interacts with alpha-galactosyl residues and particularly with high affinity with Gal alpha 1----3Gal beta 1----4GlcNAc residues. We observed a striking evolutionary pattern in the expression of these epitopes on mammalian nucleated cells. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and lymphoid cells of nonprimate mammals, prosimians, and New World monkeys readily bound both anti-Gal and BS lectin. However, no such binding was detectable on cells of Old World monkeys, apes, and humans. Measurment of the binding of radiolabeled BS lectin to the various nucleated cells suggests that cells binding anti-Gal express 10(6) to 3.5 x 10(7) alpha-galactosyl epitopes, most of which, based on the anti-Gal specificity, seem to have the structure of Gal alpha 1----3Gal beta 1----4GlcNAc-R. The absence of these epitopes from human cells results from diminished activity of the enzyme alpha 1----3 galactosyltransferase, which catalyzes the following reaction. Gal beta 1----4GlcNAc-R + UDP-Gal(alpha 1----3-galactosyltransferase)----Gal alpha 1----3Gal beta 1----4GlcNAc-R + UDP This enzyme, which participates in the glycosylation of cell membrane glycoconjugates in nonprimate mammals, prosimians, and New World monkeys, appears to have been suppressed in Old World primates as a result of evolutionary events which occurred 20-30 million years ago. It is argued that an anomalous activity of this enzyme in man may result in initiation of autoimmune diseases because of the de novo expression of Gal alpha 1----3Gal beta 1----4GlcNAc-R epitopes recognized by anti-Gal.