Increased cytotoxicity of normal rabbit serum for lectin-resistant mutants of animal cells.

Plant lectins are cytotoxic and can be used to select for mutants of animal cells that exhibit structural changes in cell surface carbohydrates reflecting glycosylation defects. We isolated eight lectin mutants of Chinese hamster ovary (CHO) cells that appear to represent three different phenotype classes. These lectin mutants were much more sensitive to the cytotoxic action of normal rabbit serum (NRS) than were the parental cells. This increased cytotoxicity was heat sensitive, specifically absorbed, and inhibited by simple and complex carbohydrates. No killing was observed under conditions in which only the alternate complement pathway was active. An NRS-resistant subclone that was isolated from one lectin mutant was shown to have also regained wild type behavior when tested with the lectins. The possibility that naturally occurring antibodies in rabbit serum are reacting with incomplete carbohydrate chains on the surface of the lectin mutants is discussed.

Migrating endothelial cells are distinctly hyperglycosylated and express specific migration-associated cell surface glycoproteins.

Migration of endothelial cells is one of the first cellular responses in the cascade of events that leads to re-endothelialization of an injured vessel and neovascularization of growing tissues and tumors. To examine the hypothesis that endothelial cells express a specific migration-associated phenotype, we analyzed the cell surface glycoprotein expression of migrating bovine aortic endothelial cell (BAECs). Light microscopic analysis revealed an upregulation of binding sites for the lectins Concanavalin A (Con A), wheat germ agglutinin (WGA), and peanut agglutinin after neuraminidase treatment (N-PNA) on migrating endothelial cells relative to contact-inhibited cells. These findings were confirmed and quantitated with an enzyme-linked lectin assay (ELLA) of circularly scraped BAEC monolayers. The expression of migration-associated cell surface glycoproteins was also analyzed by SDS-PAGE. The overall expression of cell surface glycoproteins was upregulated on migrating BAECs. Migrating BAECs expressed Con A- and WGA-binding glycoproteins with apparent molecular masses of 25 and 48 kD that were not expressed by contact-inhibited BAEC monolayers and, accordingly, disappeared as circularly scraped monolayers reached confluence. Subconfluent BAEC monolayers expressed the same cell surface glycoconjugate pattern as migrating endothelial cells. FACS analysis of circularly scraped BAEC monolayers showed that the phenotypic changes of cell surface glycoprotein expression after release from growth arrest occurred before the recruitment of the cells into the cell cycle (3 vs. 12 h). Suramin, which inhibits endothelial cell migration, abrogated the expression of the migration-associated phenotype and induced the expression of a prominent 28-kD Con A- and WGA-binding cell surface glycoprotein. These results indicate that endothelial cells express a specific migration-associated phenotype, which is characterized by the upregulation of distinct cellular glycoconjugates and the expression of specific migration-associated cell surface glycoproteins.

Wheat germ agglutinin blocks the acrosome reaction in Strongylocentrotus purpuratus sperm by binding a 210,000-mol-wt membrane protein.

Wheat germ agglutinin (WGA) binds to the entire surface of Strongylocentrotus purpuratus sperm, and inhibits the egg jelly-induced acrosome reaction. The binding was found to be species dependent and was completely inhibited by 5 mM N-acetyl-D-glucosamine. Blockage of the acrosome reaction by WGA was bypassed by a combination of the ionophores A23187 and monensin, although neither ionophore was effective individually. These experiments suggest that WGA blocks both Ca2+ uptake and Na+/H+ exchange in these sperm, which was confirmed by direct measurements of 45Ca2+ uptake and H+ efflux. The target of WGA in S. purpuratus sperm appears to be a membrane glycoprotein of Mr = 210,000. Treatment of this protein with neuraminidase or endo-beta-N-acetylglucosaminidase F abolished WGA binding.

Interaction of lectins with membrane receptors on erythrocyte surfaces.

The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.

Discoidin domain receptor 1 mediates collagen-induced inflammatory activation of microglia in culture.

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor tyrosine kinase with an extracellular domain homologous to discoidin 1 of a soil-living amoeba Dictyostelium discoideum. We have previously demonstrated that DDR1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Because collagen is one of the main components of extracellular matrix in the central nervous system, we hypothesized that collagen also induces inflammatory activation of brain microglia, and DDR1 may mediate collagen-induced microglial activation. Using BV-2 mouse microglial cells and mouse primary microglial cultures, we have demonstrated that (1) collagen induces inflammatory activation of microglia as evidenced by production of nitric oxide, expression of inducible nitric oxide synthase, COX-2, CD40, and matrix metalloproteinase-9; (2) DDR1 is expressed in microglia and is phosphorylated by collagen treatment; and (3) collagen-induced microglial activation is abrogated by DDR1 blockade but not by integrin neutralization. We have further shown that p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-kappa B are involved in the collagen-DDR1-induced microglial activation. Our results suggest that collagen can induce inflammatory activation of brain microglia and that DDR1 mediates this effect of collagen in an integrin-independent manner.

Inhibition of collagen-induced discoidin domain receptor 1 and 2 activation by imatinib, nilotinib and dasatinib.

Imatinib, nilotinib and dasatinib are protein kinase inhibitors which target the tyrosine kinase activity of the Breakpoint Cluster Region-Abelson kinase (BCR-ABL) and are used to treat chronic myelogenous leukemia. Recently, using a chemical proteomics approach another tyrosine kinase, the collagen receptor Discoidin Domain Receptor1 (DDR1) has also been identified as a potential target of these compounds. To further investigate the interaction of imatinib, nilotinib and dasatinib with DDR1 kinase we cloned and expressed human DDR1 and developed biochemical and cellular functional assays to assess their activity against DDR1 and the related receptor tyrosine kinase Discoidin Domain Receptor2 (DDR2). Our studies demonstrate that all 3 compounds are potent inhibitors of the kinase activity of both DDR1 and DDR2. In order to investigate the question of selectivity among DDR1, DDR2 and other tyrosine kinases we have aligned DDR1 and DDR2 protein sequences to other closely related members of the receptor tyrosine kinase family such as Muscle Specific Kinase (MUSK), insulin receptor (INSR), Abelson kinase (c-ABL), and the stem cell factor receptor (c-KIT) and have built homology models for the DDR1 and DDR2 kinase domains. In spite of high similarity among these kinases we show that there are differences within the ATP-phosphate binding loop (P-loop), which could be exploited to obtain kinase selective compounds. Furthermore, the potent DDR1 and DDR2 inhibitory activity of imatinib, nilotinib and dasatinib may have therapeutic implications in a number of inflammatory, fibrotic and neoplastic diseases.

Expression of high affinity interleukin-4 receptors on human renal cell carcinoma cells and inhibition of tumor cell growth in vitro by interleukin-4.

Previously, Puri et al. (Puri, R. K., M. Ogata, P. Leland, G. M. Feldman, D. Fitzgerald, and I. Pastan. 1991. Cancer Res. 51:3011-3017) have demonstrated that murine sarcoma and colon adenocarcinoma cells express high affinity interleukin-4 receptors (IL-4R) which are internalized after binding to a chimeric ligand consisting of IL-4 and Pseudomonas exotoxin. In the present study, we have tested primary cultures of human renal cell carcinoma (RCC) cells, generated from tumor specimens obtained after nephrectomy, for the expression of IL-4R and their modulation by IL-4. By using iodinated IL-4 in a receptor binding assay, we observed that renal cell carcinoma cells expressed a single class of high affinity IL-4R ranging from 1,425 +/- 207 (mean +/- SEM) to 3,831 +/- 299 (mean +/- SEM) IL-4R molecules/cell with a Kd ranging from 112 +/- 11 pM to 283 +/- 71 pM. Northern blot analysis for IL-4R gene expression, performed with a cDNA probe to IL-4R, revealed that all RCC cells exhibited a single mRNA species of 4 kb. IL-4 downregulated the surface expression of IL-4R on one RCC tumor cell line. The function of IL-4R expression on RCC tumor cells was further determined by investigating the effect of IL-4 on tumor cell growth in vitro and comparing it with IL-4 effect on growth of normal fibroblast and endothelial cell lines. Tumor cell growth, as measured by [3H]thymidine incorporation, was inhibited by IL-4 from 20 to 68% in a dose-dependent manner. A neutralizing antibody to human IL-4 was able to reverse the growth inhibitory effect of IL-4. Normal human fibroblast and endothelial cell lines also expressed high affinity IL-4R, however, IL-4 did not inhibit their growth in vitro. In fact, IL-4 caused modest stimulation of their growth. Taken together, our findings can help develop strategies for the treatment of RCC in which IL-4R may be used as a target for IL-4 itself, for IL-4 toxin therapy or, alternatively, in gene therapy.

Immunosuppressive therapy.

Although Cyclosporin A has improved transplant outcome, its use has serious limitations due to its narrow therapeutic window. New approaches to broaden this window exploit alternative drug formulations, pharmacokinetic profiling and new immunosuppressive agents, such as Rapamycin and Brequinar, which act in a synergistic fashion. There is no evidence to suggest that the pharmacological alternative to Cyclosporin A, FK-506, displays a broader therapeutic window, although it may be tenfold more potent. Similarly, despite the specificity of the IgG2a mouse anti-human CD3 monoclonal antibody, it displays a significant range of clinical side effects, delayed therapeutic action and frequently stimulates generation of human anti-mouse monoclonal antibodies. Recent advances in monoclonal antibody technology seek not only to produce antibodies against determinants involved in alloactivation, but also to 'humanize' the antibodies for reduced side effects. The availability of this array of potential agents highlights the need to develop guidelines for clinical trial methodologies to address the unique needs and demands of organ transplantation.

Internalization of ricin in Chinese hamster ovary cells.

Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells.

Binding of peanut lectin to breast epithelium, human carcinomas, and a cultured rat mammary stem cell: use of the lectin as a marker of mammary differentiation.

We investigated the binding of fluorescence-labeled peanut agglutinin (PNA) to breast epithelium. Specific binding of PNA to the mammary glands of female Sprague-Dawley rats increased as the gland matured. Sexually immature rats showed relatively little fluorescence, but this increased in mature and pregnant animals. A maximum was reached in lactating rats in which significant labeling of material within the lumen was observed. PNA was bound exclusively to the epithelial and not the myoepithelial or mesenchymal cells. In tissue culture, a rat mammary epithelial stem cell line, which can be stimulated to differentiate to alveolus-like secretory or myoepithelial cells, showed evidence of PNA binding only on the secretory cells and not on unstimulated or myoepithelial cells. Fibroblast cultures also failed to show significant binding of PNA. Receptor sites on the secretory cells were masked mainly by sialic acid. Human breast sections, like those of the rat, showed fluorescent labeling at the apical region of the epithelial cells; this labeling increased if the tissue had prior treatment with neuraminidase. Breast carcinomas that were morphologically differentiated showed more labeling with PNA than did undifferentiated tumors, which often had weak or sometimes negative labeling. When significant fluorescence was observed, it was localized mainly in the cytoplasm. By contrast, labeling was restricted to the cell periphery in differentiated carcinomas. The use of PNA as a marker for breast epithelial cell differentiation is therefore proposed.

Participation of common surface receptor(s) in the activation of murine macrophages by Sarcophaga lectin and wheat germ agglutinin.

Mouse peritoneal macrophage surface proteins which bind Sarcophaga lectin were studied. Two major binding proteins with molecular masses of 170 and 110 kDa were identified. Sarcophaga lectin and wheat germ agglutinin were found to share common binding proteins for activating macrophages, although their hapten sugars are different. Antibody raised against the Sarcophaga lectin-binding proteins inhibited both the production of tumor-specific cytotoxic protein by the macrophage-like cell line J774.1 cells and the lectin-dependent macrophage-mediated cytotoxic reaction induced by Sarcophaga lectin or wheat germ agglutinin. Thus the 170-kDa and/or 110-kDa protein is important in activation of macrophages.

Modular Total Syntheses of the Alkaloids Discoipyrroles A and B, Potent Inhibitors of the DDR2 Signaling Pathway.

The title natural product 1 has been synthesized by treating the 1,2,3,5-tetrasubstituted pyrrole 23 with oxoperoxymolybdenum(pyridine) (hexamethylphosphoric triamide) (MoOPH). Compound 23 was itself prepared in seven steps from parent pyrrole using Ullmann-Goldberg and Suzuki-Miyaura cross-coupling, Vilsmeier-Haack formylation, electrophilic bromination, and Wittig olefination reactions as key steps. Related chemistry has been used to prepare discoipyrrole B (2).

Identification and characterization of the pulmonary alveolar type II cell Maclura pomifera agglutinin-binding membrane glycoprotein.

The lectin Maclura pomifera agglutinin (MPA) binds to the apical surface of pulmonary alveolar type II but not type I cells. We show that MPA binds to a single membrane glycoprotein in type II cells with a molecular mass of 230 kDa in the rabbit and 200 kDa in the rat. The glycoprotein has an abundance of terminal N-acetylgalactosamine residues. It is a hydrophilic integral membrane protein suggesting that it has an extensive extramembrane domain or is an ion channel. The glycoprotein is similar in rat and rabbit, with the exception that the rat glycoprotein is partially sialylated and is trypsin sensitive. The MPA-binding glycoprotein represents a new integral membrane marker of the apical domain of the pulmonary alveolar type II cell.

Receptors for a cytotoxic lectin, abrin and their role in cell intoxication.

The nature of binding of abrin to Chinese hamster ovary cells was examined in relation to the ensuing intoxication of the treated cells. Approx. 20% of [125I] abrin bound to CHO cells at 37 degree C was found to be resistant to the addition or presence of 0.1 M lactose. The extent of lactose-resistant binding depended inversely upon the temperature of incubation. Among various proteins, lectins and sugars, only non-labeled abrin could strongly inhibit the lactose-resistant binding of [125I] abrin. Lactose-resistant binding could lead to an inhibition of cellular protein synthesis and to a loss of cell viability. Abrin molecules bound at the lactose-sensitive and lactose-resistant binding sites apparently have an equal probability of being internalized by CHO cells. Binding of approx. 3.10(3) abrin molecules per CHO cell was required to elicit 50% loss of cell viability regardless of whether the binding occurs in the presence or absence of lactose. The result of a cross-linking experiment suggested that a membrane protein with an Mr of about 45 000 may be responsible for the lactose-resistant binding of abrin.

The binding of the B-chain of ricin to Burkitt lymphoma cells. A new approach to ligand-receptor interaction studies.

It is shown that conformational changes of receptor proteins brought about by binding of a ligand induce changes in the lipid environment of the receptor that can be monitored by fluorescent lipid probes. On this basis a new approach to studies of ligand-receptor binding is proposed. Using the interaction of the ricin B-chain with Burkitt lymphoma cells as an example and fluorescent labelled sphingomyelin as a probe, the ligand-induced changes of fluorescence anisotropy were shown to be concentration-dependent and to permit determination of the binding constant and the number of receptor-binding sites. The method was found to be specific and highly sensitive, allowing detection of the action of one RB molecule per cell. Scatchard analysis of the binding of 125I-RB demonstrated the presence on the cell surface of two binding sites with Kd approximately 10(-10) and approximately 10(-8) M, respectively. Only the high-affinity sites were detected by the fluorescence technique. Saturation of these sites resulted in maximum inhibition of protein synthesis.

Hydrogen peroxide inhibits epidermal growth factor receptor internalization in human fibroblasts.

Several cellular signal transduction cascades are affected by oxidative stress. In this study, the effect of hydrogen peroxide (H2O2) on the endocytosis of the epidermal growth factor (EGF) receptor was investigated. Exposure of HER14 cells to H2O2 resulted in a concentration-dependent inhibition of EGF receptor internalization. Binding studies demonstrated that this H2O2-induced inhibition in internalization was not due to altered binding of EGF to its receptor. Addition of H2O2 at different time points during internalization showed that EGF receptor internalization was rapidly reduced, suggesting that one of the first steps in the internalization process is inhibited. In addition, H2O2 inhibited the internalization of a different receptor, the chicken hepatic lectin receptor, in a concentration-dependent manner as well. Treatment of cells with another inducer of oxidative stress, cumene hydroperoxide, also resulted in a decreased internalization. Finally, we showed that H2O2 inhibited EGF-induced mono-ubiquitination of the EGF receptor pathway substrate clone 15, a process that normally occurs during EGF receptor endocytosis. These results clearly show that oxidative stress interferes with EGF signaling.

Lectin-carbohydrate interactions and infectivity of human immunodeficiency virus type 1 (HIV-1).

The aim of this study was to determine whether mannosyl-specific lectins, especially Concanavalin A (ConA), may bridge HIV-1 env glycoproteins to cell membranes to increase virus binding to its targets, and to what extent this lectin-carbohydrate interaction can modify HIV-1 infectivity for monocytic compared with lymphoid cells. Monocytic U937 and lymphoid CEM cells, which both express surface mannose, were utilized. Whether first incubated with env glycoprotein or with the cells, lectins bound both to the cells and to radiolabeled recombinant gp160 (rgp160). Thus, they enhanced rgp160 adsorption to the cells in a methyl-alpha-mannose inhibitable manner. ConA did not appear to bind to the V1 domain of CD4 at the U937 cell surface since Leu3a binding was not blocked in the presence of ConA, nor was recombinant CD4 retained on a ConA-agarose affinity matrix. Moreover, enhanced rgp160 binding to the cells was CD4 independent, since it was not modified by preincubating the cells with Leu3a. Finally, ConA did not inhibit the binding of CD4-IgG3 chimeric molecules to virions immobilized on nitrocellulose membrane, which argues against the possibility that it interferes with the interaction of gp120 and CD4. However, both when incubated with the virus or with the cells and despite mediating enhanced binding of env glycoprotein, ConA neutralized HIV-1 infectivity for monocytic U937 as well as for lymphoid CEM cells. In this respect, ConA behaves like neutralizing antibodies which do not interfere with CD4 binding of gp120 but rather with some later event that leads to virus entry. These findings obtained with plant lectins may be of relevance in vivo, inasmuch as endogenous mannosyl-binding proteins, which are known to function as opsonins, have been reported to inhibit in vitro infection by HIV-1.

Enhancement of phagocytosis by calcitonin gene-related peptide (CGRP) in cultured mouse peritoneal macrophages.

Calcitonin gene-related peptide (CGRP) is widely distributed in sensory neurons and nerve fibers. To clarify the function of CGRP on the immune system, the effect of CGRP on phagocytosis by peritoneal macrophages was examined by means of flow cytofluorometry. CGRP enhanced phagocytosis of latex beads in a dose-dependent manner. Because the phosphodiesterase inhibitor 3-isobutyl, 1-methylxanthine (IBMX) enhanced the CGRP-induced enhancement of phagocytosis, the enhancement might be mediated by cAMP. In the presence of mannan, the phagocytosis was suppressed and the CGRP-induced enhancement was also blocked, suggesting that mannose receptors on macrophages were involved in mediating the phagocytosis of latex beads, and CGRP enhanced the mannose receptor-mediated phagocytosis. The present results indicate that CGRP can modulate the function of macrophages in nerve terminals of sensory neurons during the development and maintenance of inflammation.

The role of IL-4 in human myeloid leukemia: stimulation of RNA synthesis and transduction of differentiation signals through an IL-4 receptor leads to functional and HLA positive HL-60 cells.

The ectopic expression of lineage markers on irrelevant cell types may be of importance in the differentiation pathway(s) of these cells. One example, that is the subject of this study, is the presence of the interleukin-4 (IL-4) receptor on the surface of the human HL-60 myeloid leukemia cell line. The presence of such a receptor, that at first seems to be a simple genetic misprogramming, has an unusual biological function: It serves as a bridge to link the B cell growth factor IL-4 in order to transduce a number of differentiation signals in this M2 acute myeloid leukemia (AML) population. Signal transduction is followed by stimulation of RNA synthesis and subsequent induction of differentiation. Daily administration of low IL-4 dose yields proliferative senescent cells that exhibit 66% of growth inhibition in a 5-day tritiated thymidine incorporation assay. These cells clearly exit from the standard M2 morphology and show more mature characteristics as assessed by the Giemsa-Wright staining technique, followed by a 2-fold increase of the monocyte-granulocyte-specific Mac-1 surface antigen. Cellular function is also affected positively since phagocytosis of latex beads increases considerably after IL-4 treatment. Finally, as reported for normal human and murine monocytes and macrophages, the receptor-ligand interaction augments the levels of the class I and class II antigenic determinants by approximately 60%. Our results suggest that ectopic expression of markers may be a "distinct" event required during a short period in the differentiation of certain hemopoietic cells leading to mature and normal phenotypes.

Electron microscopic cytochemistry on the distribution of wheat germ agglutinin receptor on the platelet plasma membrane after treatment with convulxin isolated from Crotalus durissus terrificus venom.

The effects of convulxin, isolated from the venom of Crotalus durissus terrificus, on the localization and distribution of wheat germ agglutinin (WGA) binding sites on platelet surfaces have been investigated at an ultrastructural level. A post-embedding cytochemical technique using WGA-gold complexes was used and the quantitative intensity of WGA-labeling on the surface membrane of platelets after convulxin stimulation was determined. In the presence of Ca2+, convulxin induced platelet release and aggregation, while in its absence, the platelets formed pseudopodia and showed release reaction, but without aggregation. After treatment with convulxin, WGA-labeling on the surface membrane decreased compared with intact washed (control) platelets. In the presence of Ca2+, clusters of gold label were often found on the surface membrane. However, the WGA-labeling intensity of the membrane of the open canalicular system increased significantly compared with that of platelets stimulated by convulxin in the absence of Ca2+. Direct morphological evidence demonstrates qualitative and quantitative alterations of WGA-labeling on the surface membrane of platelets, after convulxin stimulation. The possibility is considered that WGA-binding glycoproteins on the surface membrane are involved in the aggregation response after convulxin stimulation.