von Willebrand factor clearance - biological mechanisms and clinical significance.

The mechanisms involved in regulating von Willebrand factor (VWF) clearance remain poorly understood. However recent studies have shown that macrophages play a critical role in regulating the half-life of VWF, and have identified specific lectin (including asialoglycoprotein, macrophage galactose-type lectin, Sigec-5 and C-type lectin domain family 4 member M) and scavenger receptors (including low-density lipoprotein receptor-related protein-1, scavenger receptor A1 and stabilin-2) that are involved in VWF clearance. Further studies will be required to determine the relative importance of these individual receptors with respect to physiological and pathological VWF clearance. Nevertheless, recent clinical data have highlighted the importance of enhanced VWF clearance in the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, increased clearance also contributes to reduced VWF levels in many patients with type 2 and type 3 VWD. Improved understanding regarding VWF clearance is not only of direct biological relevance, but may also have important implications for the development of novel therapeutic agents with extended plasma half-lives for the treatment of both VWD and haemophilia A.

MICL controls inflammation in rheumatoid arthritis.

BACKGROUND: Myeloid inhibitory C-type lectin-like receptor (MICL, Clec12A) is a C-type lectin receptor (CLR) expressed predominantly by myeloid cells. Previous studies have suggested that MICL is involved in controlling inflammation. OBJECTIVE: To determine the role of this CLR in inflammatory pathology using Clec12A(-/-) mice. METHODS: Clec12A(-/-) mice were generated commercially and primarily characterised using the collagen antibody-induced arthritis (CAIA) model. Mechanisms and progress of disease were characterised by clinical scoring, histology, flow cytometry, irradiation bone-marrow chimera generation, administration of blocking antibodies and in vivo imaging. Characterisation of MICL in patients with rheumatoid arthritis (RA) was determined by immunohistochemistry and single nucleotide polymorphism analysis. Anti-MICL antibodies were detected in patient serum by ELISA and dot-blot analysis. RESULTS: MICL-deficient animals did not present with pan-immune dysfunction, but exhibited markedly exacerbated inflammation during CAIA, owing to the inappropriate activation of myeloid cells. Polymorphisms of MICL were not associated with disease in patients with RA, but this CLR was the target of autoantibodies in a subset of patients with RA. In wild-type mice the administration of such antibodies recapitulated the Clec12A(-/-) phenotype. CONCLUSIONS: MICL plays an essential role in regulating inflammation during arthritis and is an autoantigen in a subset of patients with RA. These data suggest an entirely new mechanism underlying RA pathogenesis, whereby the threshold of myeloid cell activation can be modulated by autoantibodies that bind to cell membrane-expressed inhibitory receptors.

Discoidin domain receptor 1 is a novel transcriptional target of ZEB1 in breast epithelial cells undergoing H-Ras-induced epithelial to mesenchymal transition.

The epithelial-to-mesenchymal transition (EMT) process allows carcinoma cells to dissociate from the primary tumor thereby facilitating tumor cell invasion and metastasis. Ras-dependent hyperactive signaling is commonly associated with tumorigenesis, invasion, EMT, and metastasis. However, the downstream effectors by which Ras regulates EMT remain ill defined. In this study, we show that the H-Ras pathway leads to mesenchymal-like phenotypic changes in human breast epithelial cells by controlling the ZEB1/microRNA-200c axis. Moreover, H-Ras suppresses the expression of the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, via ZEB1, thus identifying ZEB1 as a novel transcriptional repressor of DDR1. Mutation studies on the putative promoter of the DDR1 gene revealed that bipartite Z- and E-box elements play a key role in transcriptional repression of DDR1 in Hs578T and MDA-MB-231 breast carcinoma cell lines by ZEB1. Furthermore, we found an inverse correlation between ZEB1 and DDR1 expression in various cancer cell lines and in human breast carcinoma tissues. Consistently, overexpression of DDR1 reduced the invasive phenotype of mesenchymal-like triple-negative breast cancer cells in 3D cultures and in vivo. Thus, ZEB1's role in maintenance of EMT in breast carcinoma cells is mediated in part by its ability to suppress DDR1 expression and consequently contribute to the activation of the invasive phenotype. Taken together, our results unveil a novel H-Ras/ZEB1/DDR1 network that contributes to breast cancer progression in triple-negative breast cancers.

Inhibition of discoidin domain receptor 2-mediated lung cancer cells progression by gold nanoparticle-aptamer-assisted delivery of peptides containing transmembrane-juxtamembrane 1/2 domain.

The delivery of biologically functional peptides into mammalian cells can be a direct and effective method for cancer therapy and treatment of other diseases. Discoidin domain receptor 2 (DDR2) is a collagen-induced receptor tyrosine kinase recently identified as a novel therapeutic target in lung cancer. In this study, we report that peptides containing the functional domain of DDR2 can be efficiently delivered into lung malignant cancer cells via a gold nanoparticle-DNA aptamer conjugate (AuNP-Apt)-based system. Peptide delivery resulted in the abrogation of DDR2 activation triggered by collagen. Moreover, the peptide delivered by the AuNP-Apt system inhibited cancer cell proliferation and invasion mediated by DDR2 activation. Thus, these results suggest that peptide loaded onto AuNP-Apt conjugates can be used for the development of peptide-based biomedical applications for the treatment of DDR2-positive cancer.

ECM: chemoattraction but not adhesion.

The mechanisms that regulate 3-dimensional (3D) neutrophil chemotaxis are poorly understood. In this issue of Blood, Afonso et al demonstrate that the collagen receptor Discoidin domain receptor 2 (DDR2) promotes neutrophil chemotaxis in 3D by triggering matrix metalloproteinase (MMP) activity and the generation of chemotactic collagen peptides.

Discoidin domain receptor 2 regulates neutrophil chemotaxis in 3D collagen matrices.

Neutrophils express a variety of collagen receptors at their surface, yet their functional significance remains unclear. Although integrins are essential for neutrophil adhesion and migration on 2-dimensional (2D) surfaces, neutrophils can compensate for the absence of integrins in 3-dimensional (3D) lattices. In contrast, we demonstrate that the inhibition of the tyrosine-kinase collagen receptor discoidin domain receptor 2 (DDR2) has no impact on human primary neutrophil migration on 2D surfaces but is an important regulator of neutrophil chemotaxis in 3D collagen matrices. In this context, we show that DDR2 activation specifically regulates the directional migration of neutrophils in chemoattractant gradients. We further demonstrate that DDR2 regulates directionality through its ability to increase secretion of metalloproteinases and local generation of collagen-derived chemotactic peptide gradients. Our findings highlight the importance of collagen-derived extracellular signaling during neutrophil chemotaxis in 3D matrices.

Discoidin domain receptors: a proteomic portrait.

The discoidin domain receptors (DDRs) are collagen-binding receptor tyrosine kinases that have been implicated in a number of fundamental biological processes ranging from growth and development to immunoregulation. In this review, we examine how recent proteomic technologies have enriched our understanding of DDR signaling mechanisms. We provide an overview on the use of large-scale proteomic profiling and chemical proteomics to reveal novel insights into DDR therapeutics, signaling networks, and receptor crosstalk. A perspective of how proteomics may be harnessed to answer outstanding fundamental questions including the dynamic regulation of receptor activation kinetics is presented. Collectively, these studies present an emerging molecular portrait of these unique receptors and their functional role in health and disease.

Carbohydrate clearance receptors in transfusion medicine.

BACKGROUND: Complex carbohydrates play important functions for circulation of proteins and cells. They provide protective shields and refraction from non-specific interactions with negative charges from sialic acids to enhance circulatory half-life. For recombinant protein therapeutics carbohydrates are especially important to enhance size and reduce glomerular filtration loss. Carbohydrates are, however, also ligands for a large number of carbohydrate-binding lectins exposed to the circulatory system that serve as scavenger receptors for the innate immune system, or have more specific roles in targeting of glycoproteins and cells. SCOPE OF REVIEW: Here we provide an overview of the common lectin receptors that play roles for circulating glycoproteins and cells, and present a discussion of ways to engineer glycosylation of recombinant biologics and cells to improve therapeutic effects. MAJOR CONCLUSIONS: While the pharmaceutical industry has learned how to exploit carbohydrates to improve pharmacokinetic properties of recombinant therapeutics, our understanding of how to improve cell-based therapies by manipulation of complex carbohydrates is still at its infancy. Progress with the latter has recently been achieved with cold-stored platelets, where exposure of uncapped glycans lead to rapid clearance from circulation by several lectin-mediated pathways. GENERAL SIGNIFICANCE: Understanding lectin-mediated clearance pathways is essential for progress in development of biological pharmaceuticals.

A tale of two collagen receptors, integrin ß1 and discoidin domain receptor 1, in epithelial cell differentiation.

As increase in collagen deposition is no longer taken as simply a consequence but, rather, an inducer of disease progression; therefore, the understanding of collagen signal transduction is fundamentally important. Cells contain at least two types of collagen receptors: integrins and discoidin domain receptors (DDRs). The integrin heterodimers α(1)ß(1), α(2)ß(1), α(10)ß(1), and α(11)ß(1) are recognized as the non-tyrosine kinase collagen receptors. DDR1 and 2, the tyrosine kinase receptors of collagen, are specifically expressed in epithelium and mesenchyme, respectively. While integrin ß(1) and DDR1 are both required for cell adhesion on collagen, their roles in epithelial cell differentiation during development and disease progression seem to counteract each other, with integrin ß(1) favoring epithelium mesenchyme transition (EMT) and DDR1 inducing epithelial cell differentiation. The in vitro evidence shows that the integrin ß(1) and DDR1 exert opposing actions in regulation of membrane stability of E-cadherin, which itself is a critical regulator of epithelial cell differentiation. Here, we review the functional roles of integrin ß(1) and DDR1 in regulation of epithelial cell differentiation during development and disease progression, and explore the underlining mechanisms regarding to the regulation of membrane stability of E-cadherin.

Discoidin domain receptor 2 (DDR2) regulates proliferation of endochondral cells in mice.

Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase that is activated by fibrillar collagens. DDR2 regulates cell proliferation, cell adhesion, migration, and extracellular matrix remodeling. The decrement of endogenous DDR2 represses osteoblastic marker gene expression and osteogenic differentiation in murine preosteoblastic cells, but the functions of DDR2 in chondrogenic cellular proliferation remain unclear. To better understand the role of DDR2 signaling in cellular proliferation in endochondral ossification, we inhibited Ddr2 expression via the inhibitory effect of miRNA on Ddr2 mRNA (miDdr2) and analyzed the cellular proliferation and differentiation in the prechondrocyte ATDC5 cell lines. To investigate DDR2's molecular role in endochondral cellular proliferation in vivo, we also produced transgenic mice in which the expression of truncated, kinase dead (KD) DDR2 protein is induced, and evaluated the DDR2 function in cellular proliferation in chondrocytes. Although the miDdr2-transfected ATDC5 cell lines retained normal differentiation ability, DDR2 reduction finally promoted cellular proliferation in proportion to the decreasing ratio of Ddr2 expression, and it also promoted earlier differentiation to cartilage cells by insulin induction. The layer of hypertrophic chondrocytes in KD Ddr2 transgenic mice was not significantly thicker than that of normal littermates, but the layer of proliferative chondrocytes in KD-Ddr2 transgenic mice was significantly thicker than that of normal littermates. Taken together, our data demonstrated that DDR2 might play a local and essential role in the proliferation of chondrocytes.

DDR2 plays a role in fibroblast migration independent of adhesion ligand and collagen activated DDR2 tyrosine kinase.

Discoidin domain receptor-2 (DDR2) is a cell surface tyrosine kinase receptor that can be activated by soluble collagen and has been implicated in diverse physiological functions including organism growth and wound repair. In the current studies, we used fibronectin and collagen-coated 2D surfaces and collagen matrices in combination with siRNA technology to investigate the role of DDR2 in a range of fibroblast motile activities. Silencing DDR2 with siRNA inhibited cell spreading and migration, and similar inhibition occurred regardless whether cells were interacting with fibronectin or collagen surfaces. Under the assay conditions used, DDR2 tyrosine kinase activation was not observed unless soluble collagen was added to the incubation medium. Finally silencing DDR2 also inhibited human fibroblast migration in 3D collagen matrices but had no effect on 3D collagen matrix remodeling and contraction. Taken together, our findings suggest that DDR2 is required for normal fibroblast spreading and migration independent of adhesion ligand and collagen activation of DDR2 tyrosine kinase.

Loss of discoidin domain receptor 2 promotes hepatic fibrosis after chronic carbon tetrachloride through altered paracrine interactions between hepatic stellate cells and liver-associated macrophages.

Hepatic stellate cells (HSCs) interact with fibrillar collagen through the discoidin domain receptor 2 (DDR2) in acute hepatic injury, generating increased fibrosis. However, the contribution of DDR2 signaling to chronic liver fibrosis in vivo is unclear, despite its relevance to chronic human liver disease. We administered carbon tetrachloride (CCl(4)) to DDR2(+/+) and DDR2(-/-) mice twice weekly, and liver tissues and isolated HSCs were analyzed. In contrast to changes seen in acute injury, after chronic CCl(4) administration, DDR2(-/-) livers had increased collagen deposition, gelatinolytic activity, and HSC density. Increased basal gene expression of osteopontin, transforming growth factor-ß1, monocyte chemoattractant protein-1, and IL-10 and reduced basal gene expression of matrix metalloproteinase-2, matrix metalloproteinase-13, and collagen type I in quiescent DDR2(-/-) HSCs were amplified further after chronic CCl(4). In concordance, DDR2(-/-) HSCs isolated from chronically injured livers had enhanced in vitro migration and proliferation, but less extracellular matrix degradative activity. Macrophages from chronic CCl(4)-treated DDR2(-/-) livers showed stronger chemoattractive activity toward DDR2(-/-) HSCs than DDR2(+/+) macrophages, increased extracellular matrix degradation, and higher cytokine mRNA expression. In conclusion, loss of DDR2 promotes chronic liver fibrosis after CCl(4) injury. The fibrogenic sinusoidal milieu generated in chronic DDR2(-/-) livers recruits more HSCs to injured regions, which enhances fibrosis. Together, these findings suggest that DDR2 normally orchestrates gene programs and paracrine interactions between HSCs and macrophages that together attenuate chronic hepatic fibrosis.

CLEC-1 Acts as a Negative Regulator of Dectin-1 Induced Host Inflammatory Response Signature in Aspergillus fumigatus Keratitis.

Purpose: C-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro. Methods: Quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages. Results: The expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1ß production. Conclusions: These findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1ß production in response to A. fumigatus keratitis.

Interactions between receptors for interleukin 2 and interleukin 4 on lines of helper T cells (HT-2) and B lymphoma cells (BCL1).

IL-2 and IL-4 induce a synergistic proliferative response in HT-2 cells, suggesting that IL-2Rs and IL-4Rs may interact. The purpose of this study was to examine the effect of IL-4 on the expression and function of IL-2Rs. Preincubation of HT-2 and BCL1-3B3 cells with IL-4 for 60 min at 4 degrees C or 37 degrees C resulted in a partial decrease in the number, but not the affinity of high affinity IL-2Rs as evidenced by Scatchard analysis of binding data. The decrease in the number of high affinity receptors correlated with decreased internalization of IL-2. After preincubation with IL-4, crosslinking of 125I-IL-2 to high affinity IL-2Rs also demonstrated a approximately 50% reduction in the number of high affinity IL-2Rs. Another lymphokine, IL-1, which acts on HT-2 cells, had no measurable effect on the affinity or number of IL-2Rs. Taken together, these results indicate that IL-4 downregulates the expression of high affinity IL-2Rs on some cells. It is not known whether this occurs by a direct ligand-mediated receptor interaction, by the sharing of a common receptor subunit, or by interaction of the two receptors with another membrane molecule or cytoskeletal component.

Heterogeneity of helper/inducer T lymphocytes. I. Lymphokine production and lymphokine responsiveness.

Antigen-specific, Ia-restricted helper/inducer T lymphocytes consist of subsets that can be distinguished by lymphokine secretion. One, called Th1, secretes IL-2 and the other, termed Th2, produces BSF-1/IL-4 in response to stimulation by lectin or antigen receptor signals, and each uses the respective lymphokine as its autocrine growth factor. Cloned lines representing Th2 cells proliferate in response to both IL-2 and their autocrine lymphokine, BSF-1/IL-4, but this proliferation is dependent on the synergistic costimulator activity of the monokine, IL-1. In contrast, Th1 clones proliferate only in response to IL-2, are unresponsive to BSF-1/IL-4, and their growth is unaffected by IL-1. These response patterns are not attributable to variations in culture conditions but apparently reflect intrinsic properties of the two T cell subsets. Moreover, the unresponsiveness of Th1 cells to BSF-1/IL-4 may be related to lower levels of expression of surface receptors for this lymphokine. These results may explain the observed heterogeneity among bulk populations of T cells in terms of lymphokine responsiveness and requirement for accessory factors (costimulators). In addition, our findings suggest that IL-2, unlike BSF-1/IL-4, is a fully competent growth factor that is potentially involved in antigen-independent expansion of bystander T cells present at sites of immune stimulation.

An interleukin 4 (IL-4) mutant protein inhibits both IL-4 or IL-13-induced human immunoglobulin G4 (IgG4) and IgE synthesis and B cell proliferation: support for a common component shared by IL-4 and IL-13 receptors.

Interleukin 4 (IL-4) and IL-13 share many biological functions. Both cytokines promote growth of activated human B cells and induce naive human surface immunoglobulin D+ (sIgD+) B cells to produce IgG4 and IgE. Here we show that a mutant form of human IL-4, in which the tyrosine residue at position 124 is replaced by aspartic acid (hIL-4.Y124D), specifically blocks IL-4 and IL-13-induced proliferation of B cells costimulated by anti-CD40 mAbs in a dose-dependent fashion. A mouse mutant IL-4 protein (mIL-4.Y119D), which antagonizes the biological activity of mouse IL-4, was ineffective. In addition, hIL-4.Y124D, at concentrations of up to 40 nM, did not affect IL-2-induced B cell proliferation. hIL-4.Y124D did not have detectable agonistic activity in these B cell proliferation assays. Interestingly, hIL-4.Y124D also strongly inhibited both IL-4 or IL-13-induced IgG4 and IgE synthesis in cultures of peripheral blood mononuclear cells, or highly purified sIgD+ B cells cultured in the presence of anti-CD40 mAbs. IL-4 and IL-13-induced IgE responses were inhibited > 95% at a approximately 50- or approximately 20-fold excess of hIL-4.Y124D, respectively, despite the fact that the IL-4 mutant protein had a weak agonistic activity. This agonistic activity was 1.6 +/- 1.9% (n = 4) of the maximal IgE responses induced by saturating concentrations of IL-4. Taken together, these data indicate that there are commonalities between the IL-4 and IL-13 receptor. In addition, since hIL-4.Y124D inhibited both IL-4 and IL-13-induced IgE synthesis, it is likely that antagonistic mutant IL-4 proteins may have potential clinical use in the treatment of IgE-mediated allergic diseases.

Antipsychotic drug-induced increases in ventral tegmental area dopamine neuron population activity via activation of the nucleus accumbens-ventral pallidum pathway.

Acute administration of antipsychotic drugs increases dopamine (DA) neuron activity and DA release via D2 receptor blockade. However, it is unclear whether the DA neuron activation produced by antipsychotic drugs is due to feedback from post-synaptic blockade or is due to an action on DA neuron autoreceptors. This was evaluated using two drugs: the first-generation antipsychotic drug haloperidol that has potent D2 blocking properties, and the second-generation drug sertindole, which is unique in that it is reported to fail to reverse the apomorphine-induced decrease in firing rate typically associated with DA neuron autoreceptor stimulation. Using single-unit extracellular recordings from ventral tegmental area (VTA) DA neurons in anaesthetized rats, both drugs were found to significantly increase the number of spontaneously active DA neurons (population activity). Apomorphine administered within 10 min either before or after sertindole reversed the sertindole-induced increase in population activity, but had no effect when administered 1 h after sertindole. Moreover, both sertindole- and haloperidol-induced increase in population activity was prevented when nucleus accumbens feedback was interrupted by local infusion of the GABAA antagonist bicuculline into the ventral pallidum. Taken together, these data suggest that antipsychotics increase DA neuron population activity via a common action on the nucleus accumbens-ventral pallidum-VTA feedback pathway and thus provide further elucidation on the mechanism by which antipsychotic drugs affect DA neuron activity. This provides an important insight into the relationship between altered DA neuron activity and potential antipsychotic efficacy.

C-Type Lectin-Like Receptors: Head or Tail in Cell Death Immunity.

C-type lectin-like receptors (CLRs) represent a family of transmembrane pattern recognition receptors, expressed primarily by myeloid cells. They recognize not only pathogen moieties for host defense, but also modified self-antigens such as damage-associated molecular patterns released from dead cells. Upon ligation, CLR signaling leads to the production of inflammatory mediators to shape amplitude, duration and outcome of the immune response. Thus, following excessive injury, dysregulation of these receptors leads to the development of inflammatory diseases. Herein, we will focus on four CLRs of the "Dectin family," shown to decode the immunogenicity of cell death. CLEC9A on dendritic cells links F-actin exposed by dying cells to favor cross-presentation of dead-cell associated antigens to CD8+ T cells. Nevertheless, CLEC9A exerts also feedback mechanisms to temper neutrophil recruitment and prevent additional tissue damage. MINCLE expressed by macrophages binds nuclear SAP130 released by necrotic cells to potentiate pro-inflammatory responses. However, the consequent inflammation can exacerbate pathogenesis of inflammatory diseases. Moreover, in a tumor microenvironment, MINCLE induces macrophage-induced immune suppression and cancer progression. Similarly, triggering of LOX-1 by oxidized LDL, amplifies pro-inflammatory response but promotes tumor immune escape and metastasis. Finally, CLEC12A that recognizes monosodium urate crystals formed during cell death, inhibits activating signals to prevent detrimental inflammation. Interestingly, CLEC12A also sustains type-I IFN response to finely tune immune responses in case of viral-induced collateral damage. Therefore, CLRs acting in concert as sensors of injury, could be used in a targeted way to treat numerous diseases such as allergies, obesity, tumors, and autoimmunity.

Adhesivity and rigidity of erythrocyte membrane in relation to wheat germ agglutinin binding.

Binding of the plant lectin wheat germ agglutinin (WGA) to erythrocyte membranes causes membrane rigidification. One of our objectives has been to directly measure the effects of WGA binding on membrane rigidity and to relate rigidification to the kinetics and levels of WGA binding. Our other objective has been to measure the strength of adhesion and mechanics of cell separation for erythrocytes bound together by WGA. The erythrocyte membrane rigidity was measured on single cells by micropipette aspiration. The slope of the suction pressure-length data for entry into the pipette provided the measure of the membrane extensional modulus. Data were collected for cells equilibrated with WGA solutions in the range of concentrations of 0.01-10 micrograms/ml. Erythrocyte-erythrocyte adherence properties were studied by micropipette separation of two-cell aggregates. First, a "test" cell was selected from a WGA solution by aspiration into a small micropipette, then transferred to a separate chamber that contained erythrocytes in WGA-free buffer. Here, a second cell was aspirated with another pipette and maneuvered into close proximity of the test cell surface, and adhesive contact was produced. The flaccid cell was separated from the test cell surface in steps at which the force of attachment was derived from the pipette suction pressure and cell geometry. In addition, we measured the time-dependent binding and release of fluorescently labeled WGA to single erythrocytes with a laser microfluorometry system. The results showed that the stiffening of the erythrocyte membrane and binding of fluorescently labeled WGA to the membrane surface followed the same concentration and time dependencies. The threshold concentration for membrane stiffening was at approximately 0.1 microgram/ml where the time course to reach equilibrium was close to 1 h. The maximal stiffening (almost 30-fold over the normal membrane elastic modulus) occurred in concentrations greater than 2 micrograms/ml where the time to reach equilibrium took less than 1 min. The WGA binding also altered the normal elastic membrane behavior into an inelastic, plastic-like response which indicated that mechanical extension of the membrane caused an increase in cross-linking within the surface plane. Similar to the stiffening effect, we observed that the membrane adhesivity of cells equilibrated with WGA solutions greatly increased with concentration greater than 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and purification of an endogenous receptor for the lectin pallidin from Polysphondylium pallidum.

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges.