New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy

New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.     

Apical and basal Forssman antigen in MDCK II cells: a morphological and quantitative study.

We have studied the surface distribution of a glycosphingolipid (the Forssman antigen) in MDCK II and CCL39 cells. The Forssman antigen is mobile on the surface of both these cell lines. Its surface distribution is homogenous on non-polarized cells. Under conditions where MDCK II cells are well polarized, the Forssman antigen is present in equal amounts on the apical membrane and on the basal membrane and its processes. Very little Forssman antigen can be detected on the lateral membrane. The nature of the mechanism excluding the Forssman antigen from the lateral domain remains to be determined. This surface distribution is established within hours after plating and was observed with cells grown on different types of filters. The surface density of the Forssman antigen on the apical and on the basal domain has been estimated. No involvement of the basal Forssman antigen in cell attachment could be demonstrated. However, the apical Forssman antigen appears to be essential to the establishment of the cells in culture.     

Forssman antigen and phase specific fetal antigens: an evaluation of their role in SV40 tumor immunity.

Forssman heterophile antigen was detected on hamster fetal cells which had been previously shown to be capable of eliciting transplantation resistance to syngeneic hamster SV40 tumors. The expression of Forssman antigen continued throughout fetal development and could be detected postpartum in the tissues of neonate hamsters. In contrast, fetal antigen(s) evoking immunity to SV40 tumors was also present on early fetal cells but, unlike Forssman antigen, was not expressed after the tenth day of gestation. Immunization of hamsters with guinea pig kidney cells or sheep erythrocytes which carry Forssman antigen failed to demonstrate significant protection against SV40 tumor development. Again by contrast, immunization with fetal cells was effective in evoking tumor immunity. Evaluation of serological responses to the FORSSMAN A ANTIGEN IN HAMSTERS INDICATED THAT THE HEMOLYTIC REACTIVITY PRODUCED BY IMMUNIZATION WITH GUINEA PIG KIDNEY CELLS OR SHEEP ERYTHROCYTES WAS ELICITED AGAINST ISOANTIGENS AND NOT THE Forssman antigen. A response to the Forssman determinant could only be detected in the serum from animals receiving exhaustive hyperimmunization with fetal cells or SV40 tumor cells. These data would eliminate a possible role of the Forssman heterophile antigen in the tumor protection evoked by immunization with fetal cells bearing embryonic antigens.     

Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution

Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freeze-substitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK I cells, even though they were connected by tight junctions. The labeling density on the apical plasma membrane was 1.4-1.6 times higher than basolateral. Roughly two-thirds of the gold particles were found intracellularly. The Golgi complex was labeled for Forssman as were endosomes, identified by endocytosed albumin-gold, and lysosomes, defined by double labeling for cathepsin D. In most cases, the nuclear envelope was Forssman positive, but the labeling density was 10-fold less than on the plasma membrane. Mitochondria and peroxisomes, the latter identified by catalase, remained free of label, consistent with the notion that they do not receive transport vesicles carrying glycosphingolipids. The present method of lipid immunolabeling holds great potential for the localization of other antigenic lipids.     

CELL MITOTIC CYCLE SYNTHESIS OF NIL HAMSTER GLYCOLIPIDS INCLUDING THE FORSSMAN ANTIGEN

The synthesis of phospholipids and glycolipids during the cell mitotic cycle of an established hamster line, NIL, has been studied. Cells were synchronized with excess thymidine and mitotically harvested by shaking. Cells were radioactively labeled for 4 h with palmitate, glucosamine, or galactose. Lipids were analyzed by thin-layer chromatography. As cells progressed through the mitotic cycle, incorporation into phospholipids increased but the fraction represented by each remained constant. Similarly, ceramide monohexoside, dihexoside, and hematoside were labeled equally in all phases. Ceramide trihexoside and tetrahexoside were labeled only during G₁ and S. Ceramide pentahexoside (the Forssman antigen) shows density-dependent synthesis, accumulation, and reactivity. Ceramide pentahexoside was labeled during all phases of the mitotic cycle but the rate of incorporation decreased in S and G₂. The total amount of lipid assayed immunologically in cell extracts gradually increased. Exposure of the Forssman antigen in untreated or trypsin-treated cells was studied using binding of chemically labeled antiForssman antiserum. The amount of antigen detected in trypsinized cells increased during G₁ and early S but then remained constant. Mitotic cells exposed all detectable antigen. As cells progressed through the mitotic cycle, a large fraction of the Forssman antigen became cryptic.     

Forssman penta- and tetraglycosylceramide are xenoantigens of ostrich kidney and liver.

The heterophile antigens Galalpha1-->3Gal and N-glycolylneuraminic acid are the major obstacle to grafting mammal organs, especially from pig, to man. Lack of expression of these common xenoantigens by birds has raised interest in ostrich as a potential organ donor for xenotransplantation. Glycosphingolipids of ostrich liver and kidney were investigated for their carbohydrate determinants. Both organs were found similar in their glycolipid composition with three major species, mono-, di-, and pentaglycosylceramide. The pentaglycosylceramide was characterized as the Forssman antigen. In both organs, the ceramide portion was highly hydroxylated with prevalence of alpha-hydroxylated fatty acids, C18 phytosphingosine in kidney and C18 sphingosine in liver Forssman glycolipid. These data indicate that hydroxylation of kidney glycosphingolipids, which is found in mammals, has been maintained since the divergence of birds from other vertebrates. Characterization of a minor glycolipid as a Forssman tetraglycosylceramide built on the galabiosylceramide core indicates that the Forssman tetraglycosylceramide also exists in vivo. Its precursors, galactosyl- and galabiosylceramide, were characterized in kidney and liver. The Forssman antigen is the third heterophile antigen against which man raises natural antibodies. Its localization in the vascular endothelium and connective tissue makes ostrich an unpromising organ or cell donor for xenotransplantation to man.     

Two kinds of P-fimbrial variants of uropathogenic Escherichia coli recognizing forssman glycosphingolipid.

Various types of fimbriae on pathogenic Escherichia coli strains have been classified by their antigenicities and recognition specificities for receptors. However, the antigenicity of fimbrial proteins does not always correlate with the fimbrial recognition specificity. In this communication, the exact carbohydrate structures recognized by the fimbriae of two human uropathogenic E. coli strains, KS71 (O4) and IH11024 (O6), that have P-fimbrial antigen, were examined. Strain KS71 showed mannose-resistant (MR) hemagglutination (HA) of human blood group OP1 phenotype erythrocytes, and its HA was inhibited by blood group Pk antigen, Gal(alpha,1-4)Gal(beta,1-4)Glc-ceramide and P antigen, GalNAc(beta,1-3)Gal (alpha,1-4)Gal(beta,1-4)Glc-ceramide but not by Forssman antigen, GalNAc(alpha,1-3)GalNAc(beta,1-3)Gal(alpha,1-4)Gal (beta,1-4)Glc-ceramide, as previously described in many papers. The cells also showed MR HA of sheep erythrocytes, which was potently inhibited by Forssman, and weakly by P and Pk antigens. These phenomena could not be explained by the above P adhesin specificity. This adhesin was called Forssman-like adhesin. Strain IH11024 also caused MR HA of sheep erythrocytes but not of human erythrocytes. The HA was inhibited specifically by Forssman but neither by Pk nor P antigen. This adhesin was completely different from P adhesin and Forssman-like adhesin in recognition of the carbohydrate epitope. This adhesin, until now called a pseudotype of P fimbriae, was renamed Forssman adhesin.     

Monoclonal antibodies detecting different epitopes on the Forssman glycolipid hapten

Two hybridomas, derived by fusing mouse myeloma cells with spleen cells from a rat immunized with mouse mammary tumors, have been shown to produce antibodies that recognize cell surface antigens on mesenchymal cells in a variety of tissues. Evidence presented in this report suggests that these antibodies detect overlapping epitopes on the Forssman glycolipid hapten (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). One antibody (33B12) reacts with the terminal sugar sequence GalNAc alpha 1-3GalNAc and is specific for Forssman. The other antibody (117C9) recognizes the internal sugar sequence GalNAc beta 1-3Gal. The terminal sugar sequence GalNAc beta 1-3Gal in globoside, as well as the internal sugar sequence GalNAc beta 1-4Gal in asialo-GM1, is not recognized as an antigenic determinant by 117C9. Nevertheless, the 117C9 antibody does not react exclusively with the Forssman antigen. In a lipid extract fractionated by Folch partition of mouse mammary tumors, the antibody also detects other glycolipids.     

Radioimmuno-thin-layer chromatographic detection of Forssman antigen in human carcinoma cell lines

Glycolipid antigen was examined by radioimmuno-thin-layer chromatography (RITLC), which is a combination of a thin-layer chromatography and radioimmunoassay. In this way Forssman antigen was studied in seven carcinoma cell lines. The usual Forssman antigen with a ceramide pentasaccharide structure was detected in cell lines of a gastric cancer and a breast cancer. In addition another glycolipid with slower mobility on thin-layer chromatography and with Forssman reactivity was found in cell lines of three gastric cancers and one lung cancer.     

Immunolatex localization by scanning electron microscopy of intestinal bacteria from cockroaches.

An immunolatex reagent was developed from antiserum to Fusobacterium varium, an obligate anaerobe isolated from the colon of the cockroach, Periplaneta americana L. A new technique that enabled the preparation of a highly efficient immunolatex conjugate was used to localize the bacterium with scanning electron microscopy, in situ, in the mixed gut contents.     

Polarity of the Forssman glycolipid in MDCK epithelial cells

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.     

Murine Cell Glycolipids Customization by Modular Expression of Glycosyltransferases

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.     

Comparative study of the glycosphingolipids of chicken bursa of Fabricius and of chicken, rat and human thymus.

1. The glycosphingolipid compositions of the thymus and bursa of Fabricius of young male chickens were compared. The two tissues were found to contain complex mixtures of both neutral glycosphingolipids and gangliosides. Both tissues contained mono-, di-, tri-, tetra- and penta-glycosylceramides; the pentaglycosylceramide displayed a reaction of identity with authentic Forssman antigen when tested against a specific anti-(Forssman antigen) serum. The ganglioside G(m3) containing N-acetylneuraminic acid was the principle ganglioside of both tissues. 2. The thymus contained appreciable amounts of the simple ganglioside N-acetylneuraminylgalactosylceramide, a compound not found in the bursa. The ganglioside G(d3) (disialohaematoside) was detected in both tissues. 3. Rat and human thymus, like sheep thymus (Narasimhan, Hay, Greaves & Murray (1976) Biochim, Biophys. Acta 431, 578-591), both contained a tetraglycosylceramide species as their most complex neutral glycosphingolipid and possessed little or no Forssman antigen. They also contained a complex mixture of gangliosides. 4. The possible significance of these results is briefly discussed.     

The relationship of common saprophytic and pathogenic fungi to the Forssman and other antigens of the sheep erythrocyte

Summary 1) Culture filtrates and mats from 20 different species of fungi cultured on Sabouraud dextrose broth and asparagine synthetic medium were examined for the presence of Forssman activity utilizing a hemolysis inhibition test.2) None of the uninoculated media contained any Forssman activity.3) Seventeen of the fungi exhibited no such activity.4) Single preparations of mats ofCandida tropicalis, Fusarium moniliforme andHistoplasma capsulatum appeared to contain Forssman activity. Further studies, however, employing different preparations of these fungi in the hemolysis inhibition test and the initial preparations in an hemagglutination inhibition test did not corroborate the original findings.5) Preparations of three of seven different fungi, none of which possessed Forssman activity, were found to contain blood group A activity.6) Preparations of six different fungi without Forssman activity, including the three with the blood group A properties, were found not to inhibit the anti-sheep erythrocyte agglutinins in the serum from a patient with infectious mononucleosis.7) The importance of the sheep erythrocyte as a heterogenetic antigen is discussed.This study was supported in part by USPHS GRANTS A1-01478-07, 5 T1 A1 52-05 and 5T1-AM-5265-05 and the Dermatologic Research Foundation of California, Inc.     

Receptors for glucocorticoids in lung tissue

Gas chromatography-mass spectrometric identification of partially methylated aminosugars has been developed: (a) various kinds of O-methylated 2-deoxy-2-(N-methyl)-acetamidohexitols were prepared from partially O-(1-methoxy)-ethylated 2-deoxy-2-acetamidohexoses, and their gas chromatography-mass spectrometric patterns were determined; (b) permethylated glycolipids gave a satisfactory yield of 2-deoxy-2-N-methyl-amidohexoses by acetolysis with 0.5 n sulfuric acid in 95% acetic acid, followed by aqueous hydrolysis; (c) the resulting partially methylated aminosugars and neutral sugars were analyzed after borohydride reduction and acetylation according to the procedure of Lindberg and associates (Björndal, Lindberg and Svennson, 1967; Björndal, Hellerqvist, Lindberg and Svensson, 1970).This method was successfully applied to analysis of aminosugar linkages in blood group B-active ceramide pentasaccharide from rabbit erythrocytes and in Forssman antigen of equine spleen. The structure of blood group B-active glycolipid of rabbit erythrocyte was found to be Galα1 → 3Galβ1 → 4G1cNAcβ1 → 3Ga11 → 4Glc → Cer, and that of Forssman antigen to be GaNAcα1 → 3GalNAcβ1 → 3Galα1 → 4Ga11 → 4Glc → Cer.     

Purification of Forssman and human blood group A glycolipids by affinity chromatography on immobilized Helix pomatia lectin

A method for the affinity purification of intact glycolipids having nonreducing terminal alpha 1-3 linked N-acetylgalatosamine residues has been developed. This technique relies on the retention of the carbohydrate-binding specificity of immobilized Helix pomatia lectin in aqueous solutions of tetrahydrofuran. Both Forssman glycolipid and a mouse blood group A-active hexaosylceramide were bound by columns of the lectin equilibrated in a solvent containing 95% tetrahydrofuran and 5% water. After application of a step gradient of increasing water content up to 50%, the specifically bound glycolipids were eluted in solvent containing N-acetylgalactosamine. The Forssman and A-active glycolipids were similarly purified in a single chromatographic step from total lipid extracts of sheep and human type A erythrocyte stroma, respectively. Nonspecifically bound lipids and glycolipids were eluted from this column by simply increasing the water content of the eluting buffer. The extension of this method to other carbohydrate-binding proteins including lectins and monoclonal antibodies may provide a rapid purification of glycolipids based on their carbohydrate structures.     

Localization of globoside and Forssman glycolipids on erythrocyte membranes

Using the freeze-etch technique, the membrane localization of globoside, a principal glycolipid in human erythrocytes, and Forssman antigen, the chief glycolipid in sheep erythrocytes was evaluated using ferritin and colloidal gold as morphological markers for rabbit antibodies prepared against these glycolipids. Brief trypsinization of human red cell ghosts markedly aggregated intramembranous particles and permitted labeling of globoside, which appeared in a clustered arrangement. The aggregates of ferritin-anti-globoside differed from those of ferritin-wheat germ agglutinin, a label for glycophorin, which corresponded with the aggregates of intramembranous particles. Double-labeling of human trypsinized ghosts with anti-globoside/ Staphylococcal protein A-colloidal gold and ferritin-wheat germ agglutinin indicated that the patterns of labeling were different and that the aggregates of globoside did not bear a direct relationship to the intramembranous particles, which represent transmembrane proteins. Resealed sheep erythrocyte ghosts labeled with ferritin-conjugated rabbit anti-Forssman showed small clusters of Forssman glycolipid on the erythrocyte surface, which could be markedly aggregated with a second goat anti-rabbit antibody, indicating relative mobility of the small glycolipid domains. The distribution of ferritin-anti-Forssman label in sheep ghosts treated at pH 5.5 to aggregate intramembranous particles also did not show definite correspondence between intramembranous particles and the clusters of ferritin-anti-Forssman.     

Surface carbohydrates of hamster fibroblasts. I. Chemical characterization of surface-labeled glycosphingolipids and aspecific ceramide tetrasaccharide for transformants.

1. Neutral glycosphingolipids of hamster fibroblast NIL cells have been characterized as follows: glucosylceramide, lactosylceramide (betaGall yields 4Glc yields Cer), a digalactosylceramide (alphaGall yields 4betaGal yields Cer), a trihexosylceramide (alphaGall yields 4betaGall yields 4Glc yields Cer), two kinds of ceramide tetrasaccharides (A: alphaGa1NAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 1Cer, a new type of Forssman active glycolipid; B: globoside, betaGalNAcl yields 3alphaGall yields 4betaGall yields 4betaGlc yields Cer), and a ceramide pentasaccharide having a classical structure for Forssman antigen (alphaGalNAcl yields 3betaGalNAcl yields 3alphaGall yields 4betaGall yields 4Glc yields Cer). 2. Neutral glycosphingolipids of polyoma virus-transformed NIL cells (NILpy) have been characterized as having an additional ceramide tetrasaccharide which was absent in normal NIL cells. The structure of this specific glycolipid was identified as lacto-N-neotetraosylceramide (betaGall yields 4betaGlc-NAcl yields 3betaGall yields 4Glc yields Cer). Chemical quantities of ceramide tetra- and pentasaccharides in NILpy cells were much lower than in NIL cells. 3. All of these glycolipids, except glucosylceramide and lactosylceramide, were labeled externally by galactose oxidase and tritiated borohydride according to the method previously described (GAHMBERG, C. G, and HAKOMORI, S. (1973) J. Biol. Chem. 248, 4311-4317). The specific activities of the label in glycolipid of NIHpy cells were much greater than that in NIL cells, i.e. reactivity of glycolipids with galactose oxidase in NIHpy cells was much higher than for NIL cells. The surface label in glycolipids was cell cycle-dependent in NIL cells, and a remarkable exposure of a galactosyl residue of a ceramide tetrasaccharide was demonstrated only on the surface of NILpy cells, due to the presence of lacto-N-neotetraosylceramide.     

Isolation, characterization, and biosynthesis of Forssman antigen in human lung and lung carcinoma

A glycolipid was isolated from normal lung and lung carcinoma tissues. This glycolipid was identified as the Forssman antigen of pentaglycosyl ceramide by means of chemical and immunological methods. The presence of this antigenic glycolipid was observed in all the tissues examined of adult and embryo lungs, and of lung tumors irrespective of histological type. The extracts of human lung and lung tumors were capable of catalyzing the synthesis of Forssman antigen from globoside.     

Physiological studies on the pneumococcal Forssman antigen: a choline-containing lipoteichoic acid.

The cell concentration and possible biological activities of the pneumococcal Forssman (F) antigen (membrane lipoteichoic acid) were examined in a number of physiological situations. In test tube cultures of pneumococci the concentration of the Forssman antigen per bacterium showed no significant fluctuations within a typical culture cycle. Purified F antigen had no effect on the activation of pneumococci to competence for genetic transformation, DNA mediated genetic transformation or adsorption of the pneumococcal phage Dp-1 to bacteria. Pneumococci grown in the presence of different amino alcohols (ethanolamine, N-monomethylethanolamine, or choline) exhibit differences with regard to both their ability to stimulate heterophile (haemolytic) antibody production in rabbits and in their ability to bind such antibodies. Choline-grown bacteria seem to cross-react with sheep red blood cells better than do the analogue-grown bacteria.