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Correspondence to: Natasa Kovacic, Croatian Inst. for Brain Research, Zagreb University School of Medicine, Salata 12, 10000 Zagreb, Croatia. E-mail: natasa@mef.hr
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Summary |
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Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb4Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg4Cer, and Gb4Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment. (J Histochem Cytochem 48:16771689, 2000)
Key Words: flow cytometry, gangliosides, glycosphingolipids, immunohistology, in vivo, lymph node, mouse, spleen, thymus
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Introduction |
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GLYCOSPHINGOLIPIDS (GSLs) are amphiphilic constituents of the outer leaflet of the plasma membrane, with their carbohydrate structures directed towards the extracellular space, but they also have subcellular localization ( and GD1c have been reported as markers for murine TH2-lymphocytes (Fig 1;
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The GSL expression pattern changes with the cells' functional status and can be influenced by a variety of microenvironmental factors, including cytokines. Interferon- alters the expression of endothelial cell surface GSLs (
Most of the above studies were performed by biochemical and structural analysis of GSL extracts from tissues or cultured cells, which may not reflect their expression in vivo. The GSL expression pattern is different in cultured cells vs those in vivo (
In this study we used a combination of immunohistochemistry, flow cytometry, and Western blotting analysis to obtain information about GSL expression in lymphoid cells and tissues.
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Materials and Methods |
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Antibodies
Chicken polyclonal antibodies were used to detect Gg3Cer, Gb3Cer, Gb4Cer, GM3, and GalNAc-GM1b, and a rabbit polyclonal antibody to detect Gg4Cer (Table 1). Chicken antibodies were of the IgY isotype, the equivalent of IgG in mammals. All antibodies were produced and characterized by the laboratory of Dr. J. Müthing and were used for thin-layer chromatography (TLC) immunostaining of separated gangliosides and neutral GSLs (see references in Table 1). Dr. Müthing can be contacted for all information on antibody availability. Polyclonal chicken anti-Gg3Cer antibody was produced with HPLC-purified Gg3Cer according to the method of
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Secondary alkaline phosphatase-conjugated affinity chromatography-purified rabbit anti-chicken and goat anti-rabbit antibodies were used for immunohistochemistry, and dichlorotriazinyl-amino fluorescein (DTAF)-conjugated antibodies (Dianova; Hamburg, Germany) for flow cytometry.
A monoclonal anti-mouse CD19 antibody, specific for the B-cell surface marker expressed by all mature B-cells, and a monoclonal anti-mouse CD3 antibody, specific for the T-cell receptor-associated molecule and expressed on all mature T-cells, both conjugated with phycoerythrin (PE), were used for double flow cytometric staining (Pharmingen; San Diego, CA).
Animals
Female C57BL/6 mice, 68 weeks of age, were used in all experiments. For immunohistochemistry, animals were perfused with 20 ml of cold (4C) 0.1 M PBS, pH 7.4, immediately after sacrifice by CO2 anesthesia, and then with 20 ml of 4% formaldehyde in 0.1 M PBS, pH 7.4. After perfusion, whole organs were removed and postfixed for 2 hr in cold (4C) 2% formaldehyde in 0.1 M PBS, pH 7.4. Tissue was then rinsed in PBS, chilled in isopentane (-80C) (Merck; Darmstadt, Germany), and stored at -80C. For flow cytometry, whole organs were excised in ice-cold 0.1 M PBS with 0.1% NaN3 and were homogenized to obtain a single-cell suspension. Erythrocytes, especially abundant in spleen homogenates, were removed using a lysis buffer (150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.4).
Immunohistochemistry
Tissue sections 5 µm thick were cut on a cryomicrotome (Leica; Nussloch, Germany) at -20C, mounted on gelatin-precoated glass slides, and air-dried for 2 hr. All subsequent steps were performed at room temperature. Nonspecific antibody binding was blocked by incubating the sections with 1% bovine serum albumin (BSA) in 0.1 M PBS, pH 7.4, for 1 hr. Blocking solution was poured off and each section was incubated for 2 hr with 30 µl of polyclonal primary antibody (Table 1), diluted with 0.1 M PBS supplemented with 0.5% BSA and 0.02% NaN3. Preimmune serum, derived from the same animal species in which antibody was raised, was used as a negative control at the same dilution as the primary antibody. Sections were then rinsed three times in 0.1 M PBS and incubated for the next 2 hr with alkaline phosphatase-conjugated secondary antibody diluted 1:500 using the same buffer as for the primary antibody dilution, followed by three washes in 0.1 M PBS. Visualization of antibody binding was achieved with naphthol-AS-MX phosphate and Fast Red substrate (Sigma; St Louis, MO), followed by hematoxylin counterstaining. Sections were mounted with Mowiol (Hoechst; Frankfurt, Germany) as previously described (
Antibody staining was evaluated under a standard light microscope and staining intensity was graded as - for no staining, + for weakly positive staining, ++ for moderate staining, and +++ for very intensive staining.
To confirm the lipid nature of antibody binding structures, sections were pretreated with methanol and then with chloroform/methanol (1:1, v/v), each for 10 min, before immunostaining (
Flow Cytometry
Single-cell suspensions of spleens, thymi, and lymph nodes were prepared in ice-cold 0.1 M PBS with 0.1 % sodium azide. After centrifugation, 106 cells were incubated with primary anti-GSL antibodies (Table 1) and/or 1 µg of PE-conjugated antibodies reactive to mouse CD19 or CD3 (Pharmingen) for 30 min on ice. Antibodies were diluted in 0.1 M PBS with 0.1% NaN3. After two washes in 0.1 M PBS with 0.1% sodium azide, 0.5 µg of secondary DTAF-conjugated, affinity chromatography-purified rabbit anti-chicken IgY and goat anti-rabbit IgG antibodies (Dianova) was added and incubated on ice for the next 30 min. Finally, cells were resuspended in 1 ml of 0.1 M PBS with 0.1% sodium azide.
Two-color fluorescence was measured at the excitation wavelength of 496 nm, using a FACSCalibur (BectonDickinson; San Jose, CA). Fluorescence was further quantified on the population of lymphocytes gated according to FSC (forward scatter, proportional to cell size) vs SSC (side scatter, proportional to cell complexity) dot-plots. A total of 104 cells was analyzed. Negative controls for anti-CD19 and anti-CD3 antibodies were non-immune species-matched, PE labeled immunoglobulins. Negative controls for anti-GSL antibodies were preimmune sera derived from the same species in which antibody was raised. They were used in the same dilution as the primary antibody, followed by the incubation with DTAF-labeled secondary antibody. Nonspecific binding of secondary antibody was excluded by incubating the cells only with the DTAF-labeled secondary antibody.
Western Blotting Analysis
Thymi, spleens, and lymph nodes were dissected out from their fibrous capsules and homogenized in ice-cold 20 mM imidazole, 250 mM sucrose, pH 7.38, with the addition of protease inhibitor (No. 1873580; Boehringer, Mannheim, Germany). The homogenate was centrifuged at 1500 rpm and 4C for 15 min. Supernatant was removed into a clean tube and centrifuged for another 10 min at 14,000 rpm at 4C. Protein concentration was determined using a commercial kit (No. 500-0001; Bio Rad, Vienna, Austria). Samples were then mixed with sample buffer (Bio Rad) containing 2% SDS, and heated at 100C for 10 min. Equal amounts of proteins (20 µg) were separated on a 12% polyacrylamide gel and electrophoretically transferred onto a nitrocellulose membrane. After blocking with 3% BSA in Tris-buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 7.4), membranes were incubated with anti-GSL primary antibody diluted in TBS 1:500. After four washes in TBS with 0.02% Tween-20, the membranes were incubated with affinity chromatography-purified alkaline phosphatase-conjugated rabbit anti-chicken or goat anti-rabbit secondary antibody, diluted 1:100 in TBS. Membranes were then washed three times in 0.02% Tween-20 in TBS and once in TBS. Finally, proteins were visualized using a 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP/NBT) developing system (Kirkegaard & Perry Laboratories; Gaithersburg, MD) according to the manufacturer's instructions. A low molecular weight electrophoresis calibration kit was obtained from Amersham Pharmacia (New York, NY).
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Results |
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Biochemical analysis of lymphoid tissues from C57BL/6 mice showed that GSL fractions isolated and purified from thymus, spleen, and lymph nodes contain GM1b, GalNAc-GM1b, and GM3 gangliosides, as well as Gb3Cer, Gb4Cer, Gg3Cer, and Gg4Cer neutral GSLs (
Table 2 Table 3 Table 4 summarize the data on GSL expression in the thymus, spleen, and lymph nodes obtained by immunohistochemistry and flow cytometry. The data are from a representative experiment from a series of three experiments with similar results.
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Thymus
Histologically, the thymus consists of a cortical part harboring immature thymocytes, closely associated with specialized cortical epithelium (; deeper cortical cells are immature thymocytes going through T-cell receptor gene rearrangement, resulting in a low expression of CD3
. Mature T-cells that have completely rearranged their T-cell receptor gene are located in the medulla and express high levels of the CD3
molecule.
Strong binding of GalNAc-GM1b antibody was localized in the medullar region of the thymus (Fig 2A, upper panels) and was mostly of lipid nature, with trace positivity still visible in the perivascular spaces after pretreatment with organic solvents (Fig 2A, lower panels). Weak binding that was not affected by lipid extraction also persisted in the thymic cortex.
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Because a TLC immuno-overlay detected rather small amounts of GalNAc-GM1b in the thymus of C57BL/6 mice (
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Flow cytometry showed that the anti-GalNAc-GM1b binding structure was expressed on the surface of almost all thymocytes (Fig 4A), including mature CD3+high thymocytes (Fig 4B).
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Anti-Gg4Cer antibody stained both the thymic cortex and medulla (Fig 2B, upper panels). Lipid extraction confirmed a completely lipid nature of the bound antigen in the thymic cortex, whereas weak binding remained in the thymic medulla (Fig 2B, lower panels). Flow cytometry revealed that about a third of thymocytes expressed Gg4Cer on their surfaces. These cells belonged to both mature CD3+high and immature CD3+low thymocytes. The majority of CD3- immature thymocytes did not express Gg4Cer on the cell surface (Fig 4B; Table 2).
Anti-Gb3Cer antibody strongly stained the cortex (Fig 2C, upper panels), and this staining was not altered by chloroform/methanol extraction. In contrast, intensely stained positive patches in the medulla were almost completely removed by lipid extraction, leaving only weak and diffuse perivascular staining (Fig 2C, lower panels). In flow cytometry, more than 80% of thymocytes bound anti-Gb3Cer (Fig 4A). Cells that did not bind anti-Gb3Cer were mature CD3+high thymocytes (Fig 4B; Table 2).
The histological distribution of anti-Gg3Cer antibody binding was similar to that of anti-Gg4Cer antibody, although more intense. Lipid extraction removed most of the medullar staining, except for the perivascular spaces, but did not affect staining of the cortex (Table 2). In flow cytometry, more than 80% thymocytes bound anti-Gg3Cer antibody (Fig 4A). Double staining revealed that those cells were immature CD3- or CD3+low thymocytes (Fig 4B). Mature CD3+high thymocytes did not bind anti-Gg3Cer antibody (Table 2).
Anti-Gb4Cer antibody immunohistochemically labeled mostly perivascular spaces in the thymus, with the most prominent staining around blood vessels in the corticomedullar junction. The staining was readily removed with chloroform/methanol pretreatment (Table 2). In flow cytometry, 5.1% of thymocytes bound anti-Gb4Cer antibody (Fig 4A), those mostly belonging to the mature CD3+high population (Fig 4B).
Immunohistochemistry with anti-GM3 antibody yielded a similar staining pattern as the anti-Gb3Cer antibody, although somewhat more intense (Table 2). Flow cytometry revealed that half of the thymocytes bound anti-GM3 antibody (Table 2). These were mostly immature CD3- or CD3+low thymocytes (Fig 4A), because mature CD3+high thymocytes did not show anti-GM3 binding structures (Fig 4B).
Spleen
Spleen tissue consists of the white pulp, which harbors lymphocytes, and the red pulp, which is the site of erythrocyte destruction (
Anti-GalNAc-GM1b antibody bound to the white pulp of the spleen (Fig 2A, upper panels), without specificity for T- or B-cell-dependent zones, and to the marginal zone. It also stained the red pulp, but less intensely than other anti-GSL antibodies. Staining of the T-cell-dependent periarteriolar sheaths was completely abolished by lipid extraction, but some staining still remained in B-cell-dependent peripheral regions (Fig 2A, lower panels). Western blotting analysis revealed a band just above the 43-kD protein marker (Fig 3). In accordance with intensive immunohistochemical staining of the white pulp, antibody bound to almost 90% of splenocytes in flow cytometry (Fig 5A). A small proportion of cells that did not react with GalNAc-GM1b antibody were exclusively CD3+ T cells (Fig 5B). The entire CD19+ B-cell population was stained by GalNAc-GM1b antibody (Fig 5C).
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Gg4Cer antibody staining was most intense in central periarteriolar sheaths of the white pulp, a T-cell-dependent zone (Fig 2B, upper panels). The periphery of the white pulp showed some positive staining and the red pulp was intensely stained. Lipid extraction completely abolished staining with Gg4Cer antibody (Fig 2B, lower panels). Anti-Gg4Cer antibody bound to more than 90% of all splenocytes (Fig 5A), and with even distribution between T- and B-lymphocyte populations of the spleen (Fig 5B and Fig 5C, respectively; Table 3).
Gb3Cer antibody strongly bound to the spleen red pulp (Fig 2C, upper panels) and less intensely stained B-cell-dependent peripheral white pulp regions. T-cell-dependent zones of the white pulp were completely unstained except for a narrow acellular region around the central arteriole. Chloroform/methanol extraction completely abolished the positive staining of the red pulp but not of the B-cell-dependent periphery of the white pulp (Fig 2C, lower panels). Flow cytometry revealed that more than half of all splenocytes bound anti-Gb3Cer antibody (Fig 5A). These were mostly CD19+ B-cells (Fig 5C), whereas only a minor proportion of CD3+ T-cells bound anti-Gb3Cer (Fig 5B; Table 3).
Anti-Gg3Cer antibody intensely stained the B-cell-dependent peripheral parts of the white pulp, whereas the central periarteriolar sheath and the marginal sinus were not stained (Table 3). Staining of the white pulp was not altered by lipid extraction. The red pulp was also intensely stained with anti-Gg3Cer antibody, but this positivity was completely removed by lipid extraction. In flow cytometry, about half of all splenocytes were anti-Gg3Cer-positive (Fig 5A). These were mostly CD19+ B-cells (Fig 5C), whereas less than 5% Gg3Cer-positive cells were CD3+ T-cells (Fig 5B; Table 3).
Anti-Gb4Cer antibody reacted exclusively with the spleen red pulp and the narrow periarteriolar area, whereas the white pulp remained unstained (Table 3). Anti-Gb4Cer binding was abolished by chloroform/methanol pretreatment of the sections. Although the white pulp was completely unstained on spleen sections, the antibody bound to about 40% of the splenocytes in flow cytometry (Fig 5A), mostly to CD19+ B-cells (Fig 5C).
Anti-GM3 antibody intensely stained peripheral B- cell-dependent areas of the white pulp and the marginal sinus, but not the central periarteriolar T-cell-dependent zones (Table 3). White pulp positivity was resistant to lipid extraction and the red pulp positivity could be only partially abolished by this treatment. In accordance with the immunohistochemical findings, flow cytometry showed that anti-GM3 antibody bound almost exclusively to B-cells (Fig 5C) but only to a small percentage of T-cells (Fig 5B).
Lymph Node
Histologically, the lymph node consists of an outer cortex, which contains lymphoid follicles composed mostly of B-cells (
GalNAc-GM1b antibody weakly stained B-cell follicles in the cortical region and, somewhat more intensely, the T-cell-dependent paracortical area (Fig 2A, upper panels). Staining was not affected by lipid extraction (Fig 2A, lower panels). Flow cytometry was in accordance with such a diffuse staining pattern. Anti-GalNAc-GM1b antibody bound to more than 90% of lymph node lymphocytes (Fig 6A). The majority of T-cells and all lymph node B-cells expressed GalNAc-GM1b antibody binding epitope (Fig 6B and Fig 6C).
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By immunohistochemistry, anti-Gg4Cer antibody stained almost exclusively the T-cell-dependent paracortical region, with less intense staining in the medullar region (Fig 2B, upper panels). Lipid extraction completely abolished the staining (Fig 2B, lower panels). Flow cytometry showed positive binding to 80% of the lymph node lymphocytes (Fig 6A). Not only the CD3+ T-cells but also CD19+ B-cells were stained (Fig 6B and Fig 6C; Table 4), although the B-cell-dependent cortical region was unreactive in immunohistochemistry.
The most intense staining with anti-Gb3Cer antibody was observed in the medullar area and cortical vessels (Fig 2C, upper panels). Trace positivity was observed in the cortical region. Chloroform/methanol pretreatment partially diminished anti-Gb3Cer antibody binding (Fig 2C, lower panels). Flow cytometry revealed that about 20% of all lymph node lymphocytes bound the antibody (Fig 6A). They were mostly CD19+ B-cells (Fig 6C), whereas the majority of CD3+ T-cells were unreactive (Fig 6B; Table 4).
The staining with anti-Gg3Cer antibody was diffuse and weak (Table 4). The staining was more intense in B-cell follicles of the cortical region, whereas a trace positivity was present in the paracortical and medullar areas. Lipid extraction decreased but did not abolish the staining (Table 4). In flow cytometry, 25% of all lymph node lymphocytes bound the anti-Gg3Cer antibody (Fig 6A). Most of them were CD19+ B-cells (Fig 6C and Fig 6B; Table 4).
Staining with anti-Gb4Cer antibody was present only in the medullar area and cortical blood and/or lymph vessels, and chloroform/methanol pretreatment abolished the staining (Table 4). Flow cytometry revealed that about 30% of all lymph node lymphocytes bound anti-Gb4Cer antibody (Fig 6A). All CD19+ B-cells and a small fraction of CD3+ T-cells bound the antibody (Fig 6B and Fig 6C; Table 4).
Anti-GM3 immunohistochemistry was similar to that of anti-Gg3Cer (Table 4), with dispersed, moderately intense staining in all lymph node regions, and was unaltered by lipid extraction. Flow cytometry revealed that the anti-GM3 antibody predominantly bound to CD19+ B-cells (Fig 6A6C; Table 4).
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Discussion |
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This study provides a comprehensive analysis of tissue and cellular expression of GSLs in lymphoid organs in vivo, using well-characterized anti-GSL polyclonal antibodies that recognize specific carbohydrate epitopes in lipid extracts from different mouse tissues (
According to our data, mature thymocytes expressed a more restricted repertoire of GSLs compared to immature thymocytes. Immature T-lymphocytes bound all tested anti-GSL antibodies, except Gb4Cer antibody. Mature CD3+high thymocytes retained the expression of Gg4Cer and GalNAc-GM1b epitope and acquired the expression of Gb4Cer epitope.
During prenatal development, thymocytes change their ganglioside profile, shifting from a high expression of GM1a-type gangliosides towards GM1b-type gangliosides (
The anti-Gb4Cer antibody was the only one that specifically bound to mature thymocytes, in accordance with biochemical studies that characterized globoside as a marker for small subpopulation of mature T-cells (Fig 1;
T-lymphocytes from the secondary lymphoid organs bound only anti-GalNAc-GM1b and anti-Gg4Cer antibodies, whereas other GSL structures were detected only on a minor proportion of cells, confirming that T-lymphocytes maintain their GSL phenotype after maturation and release from the thymus (Fig 1). However, there was a difference in the distribution and level of expression of GalNAc-GM1b epitope between the spleen and the lymph nodes. Anti-GalNAc-GM1b antibody strongly stained T-cell-dependent periarteriolar sheets in the spleen, and homogeneously stained the entire CD3+ population in flow cytometry. In contrast, staining of the lymph node with this antibody was less intense and diffuse. Flow cytometry detected two populations of GalNAc-GM1b-positive cells: a smaller one with high fluorescence intensity staining (indicating high density of the epitope on the cell surface), comprising 510% of lymph node lymphocytes, and a larger one with low cellular fluorescence. It is difficult to explain these differences, especially in view of the finding that anti-GalNAc-GM1b antibody also recognized a specific glycoprotein in a Western blot. It is possible that the functional differences between the cellular microenvironments of these two lymphoid organs contribute to the observed differences. The spleen and the lymph nodes differ in the way of presenting an antigen, entering the lymph node via the lymph and the spleen from the blood (
The entire B-lymphocyte population from secondary lymphoid organs was recognized by all anti-GSL antibodies tested. This indicates that mature, unstimulated murine B-lymphocytes do not have a specific GSL profile as do T-lymphocytes. The possibility that such a broad positivity could be due to the polyclonal character of the antibodies, i.e., nonspecific recognition of surface immunoglobulins, has been ruled out by complete absence of binding of control preimmune sera in the dilutions used for immunohistology or flow cytometry. Moreover, the polyclonal anti-Gb3Cer antibody used in this study has the same specificity as anti-Gb3Cer monoclonal antibodies on human tonsil lymphocytes (
Anti-GSL antibodies stained B-cell-dependent zones of the secondary lymphoid organs even after lipid extraction with chloroform/methanol, suggesting that non-lipid epitopes were recognized by these antibodies. However, Western blotting analysis revealed a specific acceptor glycoprotein only for the GalNAc-GM1b sequence. Binding of anti-GalNAc-GM1b antibody to a specific protein is a novel finding and requires further analysis concerning the biological significance and biochemical character of the detected protein. This protein may be responsible for immunoreactivity of immature thymocytes and B-lymphocytes which, according to previous biochemical studies, were not expected to react with GalNAc-GM1b antibody. GalNAc-GM1b antibody did not detect the GalNAc-GM1b sequence in a biochemical analysis of ganglioside fractions from cultured B-cells (3Galß4Glc-R is structurally almost homologous with the oligosaccharide Neu5Ac
3Galß4GlcNAc-R, so that anti-GM3 antibody crossreacts with glycoproteins (
14Gal disaccharide, responsible for the recognition of the CD77 molecule (Gb3Cer), could be simultaneously recognized as a glycoprotein-bound determinant (
Despite the fact that there was no specific staining of protein extracts by other anti-GSL antibodies in the Western blotting analysis, most antibodies stained tissue cryosections even after lipid extraction with chloroform and methanol, which should remove all lipid-specific binding. These discrepancies can be explained by differences in tissue preparation for Western and immunohistochemical analysis. GSLs may be insoluble in detergents or organic solvents because of their membrane compartmentalization (
For some antibodies, there was a discordance in immunohistochemical and flow cytometric data. For example, GalNAc-GM1b was detected on almost all thymocytes by flow cytometry, whereas in immunohistochemistry it was almost undetectable in the thymic cortex, which harbors maturing T-lymphocytes. In addition, anti-Gg4Cer and anti-Gb4Cer antibodies did not react with B-cell-dependent zones in the secondary lymphoid organs, whereas they bound to B-cells in flow cytometry. A possible explanations for such discrepancy is a change in the surface density and composition of membrane GSLs and gangliosides during preparation of single-cell suspensions, which may affect recognition by anti-GSL antibodies (
Despite these methodological issues, which require experimental clarification, our study clearly showed that murine T- and B-lymphocytes differ in their expression of glycolipids and that immunohistochemistry and flow cytometry using biochemically well-characterized GSL antibodies may provide important information complementing standard biochemical analyses of glycolipids.
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Acknowledgments |
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Supported by a research grant from the Croatian Ministry of Science and Technology ("Inflammation in the nervous system, the role of cytokines and chemokines," no. 1080110, A. Marui
), a grant from the Deutsche Forschungsgemeinschaft (DFG, SFB 549 "Macromolecular Processing and Signaling in the Extracellular Matrix," project B07, J. Müthing), and was performed under the framework of a bilateral scientific cooperation between Germany and Croatia (BMBF project KRO-002-99).
We also thank Dr D. Batini (Zagreb University School of Medicine) for critical help during this study and Dr R. Antolovi
(Pliva Research Institute) for critical help with the Western blotting analysis, Ms
.
avar for excellent technical assistance with flow cytometry, and Ms Baranski and Dr M. Krohn (International Bureau of the BMBF) for administrative help.
Received for publication March 7, 2000; accepted July 20, 2000.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bethke U, Kniep B, Mühlradt PF (1987) Forssman glycolipid, an antigenic marker for a major subpopulation of macrophages from murine spleen and peripheral lymph nodes. J Immunol 138:4329-4335
Bethke U, Müthing J, Schauder P, Conradt P, Mühlradt PF (1986) An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms. J Immunol Methods 89:111-116[Medline]
a
i
M, Müthing J, Kra
un I, Neumann U, WeberSchürholz S (1994) Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining. Glycoconjugate J 11:477-485[Medline]
a
i
M,
o
tari
K, WeberSchürholz S, Müthing J (1995) Immunohistological analysis of neutral glycosphingolipids and gangliosides in normal mouse skeletal muscle and in mice with neuromuscular diseases. Glycoconjugate J 12:721-728[Medline]
Chammas R, Sonnenburg JL, Watson NE, Tai T, Farquhar MG, Varki NM, Varki A (1999) De-N-acetyl-gangliosides in humans: unusual subcellular distribution of a novel tumor antigen. Cancer Res 59:1337-1346
Ebel F, Schmitt E, PeterKatalini J, Kniep B, Mühlradt PF (1992) Gangliosides: differentiation markers for murine T helper lymphocyte subpopulations TH1 and TH2. Biochemistry 31:12190-12197[Medline]
Esko JD (1993) Special considerations for proteoglycans and glycosaminoglycans and their purification. In Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. Vol 2. Preparation and Analysis of Glycoconjugates. New York, Wiley, 17.2.117.2.9
Gallagher SR, Smith JA (1993) One dimensional gel electrophoresis of proteins. In Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K, eds. Current Protocols in Molecular Biology. Vol 2. Analysis of Proteins. New York, Wiley, 10.2.110.2.21
Gillard BK, Jones MA, Turner AA, Lewis DE, Marcus DM (1990) Interferon- alters expression of endothelial cell-surface glycosphingolipids. Arch Biochem Biophys 279:122-129[Medline]
Gillard BK, Thurmon L, Marcus DM (1992) Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells. Cell Motil Cytoskel 21:255-271[Medline]
Gillard BK, Thurmon LT, Marcus DM (1993) Variable subcellular localization of glycosphingolipids. Glycobiology 3:57-67[Abstract]
Habu S, Kasai M, Nagai Y, Tamaoki N, Herzenberg LA, Okumura K, Tada T (1980) The glycolipid asialo-GM1 as a new differentiation antigen of fetal thymocytes. J Immunol 125:2284-2288
Hakomori S, Handa K, Iwabuchi K, Yamamura S, Prinetti A (1998) New insights in glycosphingolipid function: "glycosignaling domain" a cell surface assembly of glycosphingolipids with signal transducer molecules involved in cell adhesion coupled with cell signaling. Glycobiology 8:xixix
Hakomori S, Igarashi Y (1995) Functional role of glycosphingolipids in cell recognition and signaling. J Biochem 118:1091-1103[Abstract]
HefferLauc M, a
i
M, Juda
M, Müthing J (1996) Anti-GM3 (II3NeuAc-lactosyl ceramide) ganglioside antibody labels human fetal Purkinje neurons during the critical stages of cerebellar development. Neurosc Lett 213:91-94[Medline]
Hildebrandt H (1996) Antigen binding of antiganglioside antibodies in vitro is strongly influenced by the ganglioside composition of the sample. FEBS Lett 388:29-33[Medline]
Horikawa K, Yamasaki M, Iwamori M, Nakakuma H, Takatsuki K, Nagai Z (1991) Concanavalin Astimulated expression of gangliosides with GalNAcß1-4(NeuAc2-3)Galß structure in murine thymocytes. Glycoconjugate J 8:354-360[Medline]
Iber H, Zacharias C, Sandhoff K (1992) The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same glycosyltransferases that catalyze biosynthesis of asialo-, a-, and b- series gangliosides. Glycobiology 2:137-142[Abstract]
Janeway CA, Travers P (1996) Immunobiology. 2nd ed London, Current Biology
Kasai M, Iwamori M, Nagai Y, Okumura K, Tada T (1980) A glycolipid on the surface of mouse natural killer cells. Eur J Immunol 10:175-180[Medline]
Kirkeby S, Moe D, Cläesson MH (1998) Gal1-4Gal glycans are expressed on myofibrillar associated proteins. Cell Tissue Res 293:285-291[Medline]
Li R, Gage D, Ladisch S (1993) Biosynthesis and shedding of murine lymphoma gangliosides. Biochim Biophys Acta 1170:283-290[Medline]
Lloyd KO, Gordon CM, Thampoe IJ, DiBendetto C (1992) Cell surface accessibility of individual gangliosides in malignant melanoma cells to antibodies is influenced by the total ganglioside composition of the cells. Cancer Res 52:4948-4953[Abstract]
Madassery JV, Gillard B, Marcus DM, Nahm MH (1991) Subpopulations of B cells in germinal centers. J Immunol 147:823-829
Mangeney M, Richard Y, Couland D, Tursz T, Wiels J (1991) CD77: an antigen of germinal center B cells entering apoptosis. Eur J Immunol 21:1131-1140[Medline]
Markoti A, Lümen R, Marusi
A, Jonji
S, Müthing J (1999) Ganglioside expression in tissues of mice lacking the tumor necrosis factor receptor 1. Carbohydr Res 321:75-87[Medline]
Miyamoto D, Ueno T, Takashima S, Ohta K, Miyawaki T, Suzuki T, Suzuki Y (1997) Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's lymphoma and germinal centre B cells. Glycoconjugate J 14:379-388[Medline]
Mühlradt PF, Bethke U, Monner DA, Petzoldt K (1984) The glycosphingolipid globoside as a serological marker on cytolytic T lymphocyte precursors and alloantigen responsive proliferating T lymphocytes in murine spleen. Eur J Immunol 14:852-858[Medline]
Müthing J (1996) High-resolution thin-layer chromatography of gangliosides. J Chromatogr 720:3-25
Müthing J (1997) Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains. Glycoconjugate J 14:241-248[Medline]
Müthing J (in press) Mammalian glycosphingolipids. In FraserReid B, Tatsuta K, Thiem J, eds. Heidelberg, Springer-Verlag pp. Glycoscience: Chemistry and Chemical Biology. Vol 3. Glycolipids
Müthing J, a
i
M (1996) Comparison of gangliosides of human skeletal and heart muscles by immunostaining on thin-layer chromatograms. Croatian Med J 37:152-157
Müthing J, Egge H, Kniep B, Mühlradt PF (1987) Structural characterization of gangliosides from murine T lymphocytes. Eur J Biochem 163:407-416[Abstract]
Müthing J, Heitmann D, Duvar S, Hanisch FG, Neumann U, Lochnit G, Geyer R, PeterKatalini J (1999) Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells. Glycobiology 9:459-468
Müthing J, Maurer U, o
tari
K, Neumann U, Brandt H, Duvar S, PeterKatalini
J, WeberSchürholz S (1994a) Different distribution of glycosphingolipids in mouse and rabbit skeletal muscle demonstrated by biochemical and immunohistological analyses. J Biochem 115:248-256[Abstract]
Müthing J, PeterKatalini J, Hanisch FG, Unland F, Lehmann J (1994b) The ganglioside GD1
, IV3Neu5Ac, III6Neu5Ac-GgOse4Cer is a major disialoganglioside in the highly metastatic murine lymphoreticular tumor cell line MDAY-D2. Glycoconjugate J 11:153-162[Medline]
Müthing J, Schwinzer B, PeterKatalini J, Egge H, Mühlradt PF (1989) Gangliosides of murine T lymphocyte subpopulations. Biochemistry 28:2923-2929[Medline]
Müthing J, Steuer H, PeterKatalini J, Marx U, Bethke U, Neumann U, Lehmann J (1994c) Expression of gangliosides GM3 (NeuAc) and GM3 (NeuGc) in myelomas and hybridomas of mouse, rat and human origin. J Biochem 116:64-73[Abstract]
Nakamura K, Hashimoto Y, Yamakawa T, Suzuki A (1988) Genetic polymorphism of ganglioside expression in mouse organs. J Biochem 103:201-208[Abstract]
Nakamura K, Suzuki H, Hirabayashi Z, Suzuki A (1995) IV3(NeuGc
2
8NeuGc)-Gg4Cer is restricted to CD4+ T cells producing interleukin-2 and a small population of mature thymocytes in mice. J Biol Chem 270:3876-3881
Nakamura K, Suzuki M, Inagaki F, Yamakawa T, Suzuki A (1987) A new ganglioside showing choleragenoid-binding activity in mouse spleen. J Biochem 101:825-835[Abstract]
Noguchi M, Suping Y, Taguchi J, Hirano T, Hashimoto H, Hirose S, Iwamori M, Okumura K (1994) Unique T cell differentiation markers: gangliosides with cholera toxin receptor activity on murine fetal thymocytes. Cell Immunol 156:402-413[Medline]
Nores GA, Dohi T, Taniguchi M, Hakomori S (1987) Density-dependent recognition of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as possible immunogen: requirements for tumor-associated antigen and immunogen. J Immunol 139:1371-1376
Pörtner A, PeterKatalini J, Brade H, Unland F, Büntemeyer H, Müthing J (1993) Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes. Biochemistry 32:12685-12693[Medline]
Prasadarao N, Stuart A, Tobet A, Jungalwala FB (1990) Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids. J Histochem Cytochem 38:1193-1200[Abstract]
Rösner H, Greis Ch, Rodemann HP (1990) Density-dependent expression of ganglioside GM3 by human skin fibroblasts in an all-or-none fashion, as a possible modulator of cell growth in vitro. Exp Cell Res 190:161-169[Medline]
Schlosshauer B, Blum AS, MendezOtero R, Barnstable CJ, Constantine-Paton M (1988) Developmental regulation of ganglioside antigens recognized by the JONES antibody. J Neurosci 8:580-592[Abstract]
Schwarz A, Futerman AH (1997) Determination of the localization of gangliosides using anti-ganglioside antibodies: comparison of fixation methods. J Histochem Cytochem 45:611-618
Simons K, Ikonen E (1997) Functional rafts in cell membrane. Nature 387:569-572[Medline]
Tatewaki K, Yamaki T, Maeda Y, Tobioka H, Piao H, Yu H, Ibayashi Y, Sawada N, Hashi K (1997) Cell density regulates crypticity of GM3 ganglioside on human glioma cells. Exp Cell Res 233:145-154[Medline]
Tsunoda A, Nakamura M, Kirito K, Hara K, Saito M (1995) Interleukin-3-associated expression of gangliosides in mouse myelogenous leukemia NSF60 cells introduced with interleukin 3 gene: expression of ganglioside GD1a and key involvement of CMP-NeuAc:Lactosylceramide 2-3 sialyltransferase in GD1a expression. Biochemistry 34:9356-9367[Medline]
van Echten G, Sandhoff K (1993) Ganglioside metabolism. Enzymology, topology, and regulation. J Biol Chem 268:5341-5344
Wiels J, Mangeney M, Tetaud C, Tursz T (1991) Sequential shifts in the three major glycosphingolipid series are associated with B-cell differentiation. Int Immunol 3:1289-1300[Abstract]
Yang Z, Bergstrom J, Karlsson KA (1994) Glycoproteins with Gal alpha 4Gal are absent from human erythrocyte membranes indicating that glycolipids are the sole carriers of blood group P activities. J Biol Chem 269:14620-14624
Yohe HC, Ye S, Reinhold BB, Reinhold VN (1997) Structural characterization of the disialogangliosides of murine peritoneal macrophages. Glycobiology 7:1215-1227[Abstract]
Zhang S, CordonCardo C, Zhang HS, Reuter VE, Adluri S, Hamilton WB, Lloyd KO, Livingston PO (1997) Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides. Int J Cancer 73:42-49[Medline]