Macrophage Lectin Research Articles

Mannan decelerates the clearance of human red blood cells in SCID mouse

Mannans and its related compounds decelerated human (Hu) red blood cell (RBC)-clearance in severe combined immunodeficiency (SCID) mice by inhibiting erythro-phagocytosis of macrophages. Chimeric SCID mice for Hu-RBC which are generated by repeated transfusions with mature Hu-RBCs are described recently as a model for Plasmodium falciparum infection, though the Hu-RBC clearance in the mice at present is very rapid and the parasitemia in the mice is only erratic. Here, we aimed to study the method to decelerate Hu-RBC clearance in SCID mice, to establish a suitable mouse model for malaria parasites. Yeast and Candida mannans as well as lactoferrin, a glycoprotein containing both oligomannoside- and N-acetyllactosamine-type glycans, decelerated Hu-RBC clearance, but instead other saccharides such as carboxymethyl chitin, N-acetylglucosamine, and d-glucose did not. Yeast mannan and lactoferrin interfered significantly with in vitro Hu-RBC-phagocytosis which was also inhibited by mannopentaose and mannotoriose. d-mannose exhibited a moderate inhibitory activity. N-acetyl-d-glucosamine, however, showed only a slight inhibitory activity, but d-glucose had no inhibitory activity on Hu-RBC phagocytosis. These results may postulate that Hu-RBC clearance in SCID mouse might be mediated by receptor–ligand binding by a macrophage lectin like receptor with mannose specificity.

Tumor site-selective localization of an adoptively transferred T cell line expressing a macrophage lectin.

CTLL-2 cells were transfected with an expression vector that contained cDNA of a mouse macrophage galactose/N-acetylgalactosamine-specific calcium-type lectin (MMGL) and a stable transfectant (CTL-ML) was established. These cells and mock transfectant cells (CTL-CEP) were labeled with a long-term fluorescent cell tracer, DiI. The labeled cells were intravenously injected into mice that contained established lung metastases produced by OV2944-HM-1 ovarian tumor cells. Analyses with fluorescence microscopy of a series of frozen lung sections from the recipient mice revealed that CTL-ML preferentially accumulated in the lung metastatic nodules, whereas CTL-CEP did not. Time course studies showed that the preferential accumulation was evident 3 days after adoptive transfer. We also found that OV2944-HM-1 cells bound peanut agglutinin and Vicia villosa agglutinin B4, whose sugar specificity overlaps with the specificity of MMGL. These results suggested that MMGL molecules expressed on CTLL-2 cells contributed to their selective trafficking or retention in tumor foci possibly through recognition of tumor-associated cell surface carbohydrate antigens. These results also suggested that MMGL could be used for the selective targeting of effector cells in adoptive immunotherapy.

Unique tissue distribution of a mouse macrophage C-type lectin.

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specific C-type lectin using a rat monoclonal antibody (mAb) specific for this lectin (mAb LOM-14). The binding of mAb LOM-14 was detected in detergent extracts from tissue by means of immunoblotting analysis. It was shown that this mAb did not cross-react with mouse hepatic lectins, a structural homologue. The macrophage lectin was widely distributed among various mouse tissues as judged by the affinity isolation followed by the immunochemical detection. The exceptions were brain, liver, kidney, small intestine, and peripheral blood. Extracts from these organs exhibited, at best, very weak signals upon mAb LOM-14 binding, despite the presence of cells expressing macrophage markers. The most intense signal was observed in the extract from skin, suggesting that cells expressing this lectin are abundant in skin. The tissues shown to contain this lectin were further investigated by immunohistochemical staining of the sections. Cells were distributed in the connective tissue and in the interstice, particularly the dermis and subcutaneous layer of skin. Cells localized in the epithelium of skin (epidermis) or other epithelia that we examined were not stained. Perivascular localization of cells stained with mAb LOM-14 was also demonstrated in cardiac and skeletal muscle tissues. Immunoelectron microscopy revealed the presence of this lectin along the rough endoplasmic reticulum. In conclusion, the distribution of C-type lectin specific for galactose/N-acetylgalactosamine in mice was unique. The connective tissue-specific distribution should provide important information on the biological role of this lectin.

A molecular model of the carbohydrate recognition domain of a rat macrophage lectin and analysis of its binding site

A three-dimensional model of the carbohydrate recognition domain of a rat macrophage C-type lectin has been constructed by comparative modeling and assessed by inverse folding analysis. Comparative modeling in the presence of low sequence similarity was based on information provided by comparison of X-ray structures and sequence-structure alignments. The sequence-structure compatibility of the model was sound. Its binding site was analyzed in comparison to the X-ray structure of a galactose-specific mutant of the mannose-binding protein. The specificity of the macrophage lectin was discussed in light of mutagenesis data on asialoglycoprotein receptors.

Chronic cardiac rejection in the LEW to F344 rat model. Blockade of CD28-B7 costimulation by CTLA4Ig modulates T cell and macrophage activation and attenuates arteriosclerosis.

CTLA4Ig, a fusion protein that blocks CD28-B7 costimulation, was studied in a LEW to F344 rat model of chronic cardiac rejection. In rats treated with a single dose of CTLA4Ig (0.5 mg intraperitoneally) 2 d after transplantation, allografts survived significantly longer ( > 70 d in 64%) than in untreated controls or rats treated with control Ig (all rejected within 25 d). Only 25% of grafts from rats treated with a single, high dose of cyclosporine A (25 mg/kg, 2 d after transplantation) survived longer than 70 d. Reverse transcriptase PCR and immunostaining analyses of tissue from 75-d, CTLA4Ig-treated allografts showed reduced expression of the T cell factor IFN-gamma and macrophage activation factors monocyte chemoattractant protein-1, inducible nitric oxide synthase, and galactose/N-acetylgalactosamine macrophage lectin, as well as TGF-beta. Grafts from longterm survivors ( > 120 d) treated with CTLA4Ig showed significant reductions in the frequency and severity of arteriosclerosis in comparison with cyclosporine A-treated rats. Thus, T cell activation is a proximal event in the cascade that culminates in the arteriosclerosis of chronic rejection. Strategies for blocking T cell costimulation may help prevent chronic rejection in clinical transplantation.

Molecular cloning and expression of cDNA encoding human macrophage C-type lectin. Its unique carbohydrate binding specificity for Tn antigen.

A human macrophage calcium-dependent (C-type) lectin cDNA clone was obtained from a library derived from IL-2-treated peripheral blood monocytes. The cDNA cloning was based on the structural homology to hepatic asialoglycoprotein receptors. The nucleotide sequence of this cDNA clone was homologous to those of the galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of rodents. In the putative carbohydrate recognition domain, deduced amino acid sequence revealed 60 and 63% homology to galactose- and N-acetylgalactosamine-specific C-type macrophage lectins of mice and rats, respectively. The cDNA clone was ligated into a mammalian expression vector and transfected into COS-1 cells. In the lysates of these cells, an MR 38,000 component, which bound to galactose-Sepharose, was identified after electrophoretic separation by its interaction with polyclonal antisera against synthetic polypeptides representing a portion of the carbohydrate recognition domain. The carbohydrate-binding specificity of the recombinant macrophage lectin was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures from immobilized macrophage lectins. When N-terminal octapeptides from human glycophorin A that bore NeuAc alpha 2-3 Gal beta 1-3 (NeuAc alpha 2-6) GalNAc and its sequentially deglycosylated derivatives were compared, glycopeptides carrying three constitutive GalNAc-Ser/Thr (Tn Ag) strongly bound to the recombinant human macrophage lectin. This is the first study to demonstrate that human macrophage cell surface lectin recognizes Tn Ag, a well-known human carcinoma-associated epitope.

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

Identification and upregulation of galactose/N-acetylgalactosamine macrophage lectin in rat cardiac allografts with arteriosclerosis.

Using differential mRNA display to uncover potential mediators associated with chronic rejection, we identified a cDNA fragment induced in Lewis to F344 rat cardiac allografts with arteriosclerosis but not Lewis syngrafts. The full-length cDNA (1.4 kb) isolated from a rat cardiac allograft cDNA library was 99% identical to galactose/N-acetylgalactosamine (Gal/GalNAc) macrophage lectin, a cell-surface receptor. This cDNA hybridized in Northern analysis with total RNA from eight cardiac allografts but not with host hearts, syngrafts, or other organs. There was a significant allograft-specific increase in transcript levels measured by reverse transcriptase PCR at days 7, 14, 28, and 75 in comparison with paired F344 host hearts (subject to same circulation but histologically normal), day-0 hearts, and syngrafts (P < 0.008, n = 4 at each time). Transcript levels in cardiac allografts were higher than those in paired host spleens (a major source of inflammatory cells) (P < 0.0001), indicating the localized nature of Gal/GalNAc lectin induction. By in situ hybridization and immunostaining, Gal/GalNAc lectin expression localized to a subset of inflammatory cells in cardiac allografts. These findings link Gal/GalNAc macrophage lectin to the chronic rejection process, as a possible mediator of macrophage infiltration.

Chronic cardiac rejection: identification of five upregulated genes in transplanted hearts by differential mRNA display.

Transplant arteriosclerosis, the major manifestation of chronic rejection, develops after allogeneic (Lewis to F344) but not syngeneic (Lewis to Lewis) rat cardiac transplantation. To identify transcriptionally regulated mediators associated with chronic cardiac rejection, we adapted the differential mRNA display technique for in vivo transplant specimens. Gene transcript patterns in four allogeneic hearts showing early signs of chronic rejection were compared with those in two syngeneic hearts exposed to the same surgical procedure but histologically normal. Twelve differentially expressed cDNA bands were identified. We improved the probability of isolating one or more allograft-specific cDNAs from a single display band by first using recovered and reamplified PCR products as probes in RNA blot analysis. cDNA fragments cloned from individual bands were then used in a second RNA blot analysis, which allowed for the correlation of specific mRNA transcripts with cDNA clones. Five cDNA clones produced time-dependent, allograft-specific hybridization. Sequence analysis demonstrated that two of these cDNAs corresponded to unknown genes, whereas the other three represented known genes not previously associated with chronic rejection. The latter group included the macrophage lectin specific for galactose/N-acetylgalactosamine (a cell-surface receptor), the nuclear P1 gene (a homologue of a yeast replication protein), and a ubiquitin-like gene. Our application of the differential display technique allowed the direct identification of potential mediators under in vivo conditions that preserve the environment of the disease process--including infiltrating cell populations critical to the inflammatory response.

Interaction of immobilized recombinant mouse C-type macrophage lectin with glycopeptides and oligosaccharides.

Inflammatory and tumoricidal macrophages express galactose- and N-acetylgalactosamine-specific Ca(2+)-dependent lectins on their surfaces. This lectin is a family member of membrane-bound C-type animal lectins and consists of 304 amino acid residues (molecular weight 34,595). In the present study, expression vectors containing a nucleotide sequence corresponding to the carbohydrate-binding domain of mouse macrophage lectin cDNA have been prepared. The carbohydrate-binding specificity of the recombinant macrophage lectin expressed in Escherichia coli was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures on immobilized lectins. When elution profiles of high mannose-type and complex-type Asn-linked carbohydrate chains were compared, the degree of retardation from immobilized macrophage lectin column was in the order tetraantennary complex-type with terminal galactosyl residues > triantennary complex-type with terminal galactosyl residues > biantennary complex-type with terminal galactosyl residues > high mannose-type glycopeptides. N-Terminal octapeptides from human glycophorin A that bore three NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc serine/threonine-linked tetrasaccharide chains and their sequentially deglycosylated derivatives were also applied to this column. Glycopeptides carrying three constitutive GalNAc-Ser/Thr(Tn-antigen) had the strongest affinity, whereas those with fully sialylated carbohydrate tetrasaccharide chains showed weak interaction. The association kinetics of Asn-linked glycopeptides from bovine asialofetuin to recombinant macrophage lectin was determined by surface plasmon resonance spectroscopy. The results indicate k(assoc) value of 1.63 x 10(4) M-1 s-1. The calculated value for Ka was 6.20 x 10(7) M.

Dual function of macrophage galactose/N-acetylgalactosamine-specific lectins: glycoprotein uptake and tumoricidal cellular recognition.

We investigated whether the interaction of peritoneal macrophages with extracellular ligands is mediated by C‐type lectins specific for galactose and N‐acetylgalactosamine. The carbohydrate‐binding domain of mouse galactose/N‐acetylgalactosamine‐specific lectin was prepared in a recombinant form. The purified recombinant lectins were tested for competitive inhibition against glycoprotein uptake and against tumoricidal effect. Thioglycolate‐elicited macrophages internalized galactosylated bovine serum albumin in vitro. The internalization was blocked by recombinant macrophage lectins. Activated macrophages obtained after intraperitoneal injection of a nonspecific immune potentiator, OK432, did not internalize galactosylated bovine serum albumin. These cells elicited a cytotoxic effect against P815 murine mastocytoma cells, and the effect was blocked by recombinant macrophage lectins. These results indicated that galactose/N‐acetylgalactosamine‐specific C‐type lectins expressed on the surface of inflammatory macrophages and on activated tumoricidal macrophages mediate two distinct functions, i.e. glycoprotein uptake and tumoricidal effector mechanisms.

Quantitative measurement of carbohydrate binding activity of mouse macrophage lectin

A simple ELISA assay measuring lectin activity of a mouse macrophage galactose/ N-acetylgalactosamine-specific C-type lectin (MMGL) was developed. The binding of galactosylated poly-lysine (termed a ligand) to the immobilized soluble form of MMGL (rML) was measured quantitatively. Consistent with the characteristics of MMGL, the binding was calcium dependent and inhibited by galactose and N-acetylgalactosamine. An antiserum against rML inhibited the ligand binding, demonstrating the usefulness of this method for the screening of blocking antibodies. Using this assay, we found a significant interaction between MMGL and carrageenans, a group of sulfated polygalactans. The inhibitory effect of carrageenans was not attributable to a nonspecific interaction because other types of sulfated polysaccharides, such as glycosaminoglycans and fucoidin, did not interfere with the ligand binding. The relevance of the present finding to the biological activities of carrageenans is discussed.

Genetic exchange of determinants for capsular polysaccharide biosynthesis between Klebsiella pneumoniae strains expressing serotypes K2 and K21a

The production of a capsular polysaccharide (CPS; K antigen) is characteristic of Klebsiella pneumoniae, but CPS structure varies among strains, and many different serotypes are now known. In this study, cps gene clusters encoding the elements of capsular polysaccharide biosynthesis were exchanged by homologous recombination between strains expressing different serotypes. The wild-type K. pneumoniae strains used for genetic exchange were KPA1 (cpsK2), expressing K2 CPS, and KPB1 (cpsK21a), expressing K21a CPS. Plasmid R68.45 was used to mobilize fragments of chromosomal DNA from auxotrophic derivatives of donor strains. Auxotrophic his alleles introduced into recipient strains provided selectable markers to coinherit the adjacent cps gene clusters from donors expressing a heterologous CPS. Each of the capsule-switched recombinants, KPA5 (cpsK21a) and KPB20 (cpsK2), was shown to have a CPS that was immunologically identical to the serotype of the respective donor. The recombinants retained their respective recipient strain background, as evidenced by a genetic marker and demonstration of a distinctive restriction fragment length polymorphism in genomic DNA. KPB1 CPS contained a sequence (mannose-alpha-2-mannose) that binds to a macrophage lectin and may be responsible for their higher susceptibility to macrophage binding and phagocytosis compared with KPA1, whose CPS lacked such sequences. The recombinant strains expressing heterologous cps genes inherited the macrophage-binding phenotype of the donor, thus confirming that relative susceptibility to phagocytosis was determined by the capsule type expressed. KPA1 was highly virulent in a mouse lethality assay, which is a feature typical of K2 strains, whereas KPB1 was not virulent in mice. Recombinant KPA5 retained relatively high virulence in mice, even though it produced the heterologous K21a CPS, which suggests that a virulence factor other than capsule biosynthesis is encoded by the KPA genomic strain background. In contrast, KPB20 gained marginal virulence in the mouse lethality assay through the inheritance and expression of the K2 CPS from the virulent strain. Thus, pathogenesis in K. pneumoniae may be multifactorial. Specific antibody was used to stabilize the CPS on the surface of K. pneumoniae, and the structural organization of the homologous and heterologous capsules was examined by electron microscopy. Recombinant KPB20, expressing heterologous K2 CPS, had a uniform layer of capsule surrounding the organism that was similar to that seen on the surfaces of the parental strains. However, KPA5, expressing the heterologous K21a CPS, was unusual in that the uniform capsular layer was physically separated from the cell wall by approximately 50 nm.(ABSTRACT TRUNCATED AT 400 WORDS)

動物レクチン：マクロファージアシアロ糖タンパク質結合タンパク質の構造と機能

動物レクチン：マクロファージアシアロ糖タンパク質結合タンパク質の構造と機能

Expression of a functional asialoglycoprotein receptor through transfection of a cloned cDNA that encodes a macrophage lectin.

The Gal/GalNAc-specific lectin on rat peritoneal macrophages (macrophage asialoglycoprotein binding protein, M-ASGP-BP) is structurally similar to rat hepatic asialoglycoprotein-binding protein (ASGP-BP) or rat hepatic lectin (RHL) and is highly homologous with the major component of RHL, RHL-1 (Ii, M, Kurata, H., Itoh, N., Yamashina, I., and Kawasaki, T. (1990) J. Biol. Chem. 265, 11295-11298). We found in this study that transfection with a cDNA clone that encodes a single polypeptide, M-ASGP-BP, was sufficient for the expression of an endocytic receptor for asialoorosomucoid (ASOR) on the COS-1 cell surface. The Kuptake value for ASOR for the transfected cells was 12.5 nM, which is similar to that for peritoneal macrophages (23 nM), and the number of ASOR bound on the cell surface was 1-8 x 10(5)/cell, this value being hundreds of times larger than that for peritoneal macrophages. 125I-ASOR bound on the surfaces of the transfected cells was rapidly internalized on incubation at 37 degrees C, and after 90 min of incubation, most of the radioactivity was recovered in acid-soluble degraded products from the medium. These results confirmed that the cDNA cloned in our previous study does in fact encode M-ASGP-BP and also that the single polypeptide chain can form a homooligomeric receptor (probably a hexamer or octamer) exhibiting high affinity for ASOR. The latter property was distinct from that of the hepatic ASGP-BP in that simultaneous transfection of two cloned cDNAs that encode RHL-1 and RHL-2/3 was required to produce an active ASOR receptor (McPhaul, M., and Berg, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8863-8867). This M-ASGP-BP expression system may serve as a simple model with which to investigate the molecular mechanisms underlying carbohydrate-mediated endocytosis.

The mannose receptor and other macrophage lectins

The mannose receptor and other macrophage lectins

The mannose receptor and other macrophage lectins

The macrophage expresses a variety of cell surface lectins with activities that support specific functional roles and correspond to various differentiation states characteristic of this cell type. Recently, research has been carried out to investigate the mannose receptor, the advanced glycosylation end products receptor, the mannose-6-phosphate-receptor, the β-glucan receptor, sialoadhesin and several galactose-specific binding proteins.

Influence of the galactosyl ligand structure on the interaction of galactosylated liposomes with mouse peritoneal macrophages

Liposomes bearing at their surface mono- and triantennary galactosyl ligands were prepared and their interaction with the galactose receptor of mouse peritoneal macrophages studied. Triantennary structures were synthesized by coupling derivatives of 1-thio-beta-D-galactose to the amino groups of lysyl-lysine dipeptide. Galactosylated liposomes were obtained either by synthesis of neo-galactolipids followed by their incorporation into the vesicles or by neo-galactosylation of performed liposomes by reaction between thiol-functionalized galactosyl ligands and vesicles bearing maleimido groups. The interaction of the galactosylated liposomes with the macrophage lectin was remarkably sensitive to the topology of the ligands, i.e., a spacer-arm length about 3 nm was necessary and, in contrast to results obtained with the galactose receptor of other cells, the triantennary structure did not provide additional binding. Related to the strategy of drug delivery with targeted liposomes, these results indicate that lectins from different cells might possibly be distinguished by using multiantennary ligands having optimal geometries.

Molecular cloning of a human macrophage lectin specific for galactose.

The murine Mac-2 protein is a galactose- and IgE-binding lectin secreted by inflammatory macrophages. We describe here the cloning and characterization of a cDNA representing the human homolog of Mac-2 (hMac-2). The amino acid sequence derived from the hMac-2 cDNA indicates that the protein is evolutionarily highly conserved, with 85% of its amino acid residues being similar to those in the murine homolog. This conservation is especially marked in the carboxyl-terminal lectin domain. The amino-terminal half of the protein is less conserved but still contains the repetitive proline-glycine-rich motif seen in the mouse protein. hMac-2 synthesized in vitro is recognized by the M3/38 monoclonal antibody to Mac-2 and binds to the desialylated glycoprotein asialofetuin and to laminin, a major component of basement membranes. These findings are discussed in the context of the potential functions of hMac-2.

Molecular cloning and sequence analysis of cDNA encoding the macrophage lectin specific for galactose and N-acetylgalactosamine.

The primary structure of the macrophage lectin specific for galactose and N-acetylgalactosamine (macrophage asialoglycoprotein-binding protein, M-ASGP-BP) has been deduced from its cDNA sequence. The M-ASGP-BP cDNA encoded a protein consisting of 306 amino acid residues with a molecular mass of 34,242 daltons. The sequence was highly homologous with that of the rat liver asialoglycoprotein receptor (rat hepatic lectin, RHL), particularly that of RHL-1 (the major form of RHL), throughout its whole length, and especially so in its putative membrane-spanning region and carbohydrate recognition domain. There were two N-glycosylation sites in M-ASGP-BP, the location of which were identical to those in RHL-1. However, M-ASGP-BP was characteristic in having a shorter cytoplasmic tail, and an inserted segment of 24 amino acids containing an Arg-Gly-Asp sequence between the membrane-spanning region and carbohydrate recognition domain.