Identification of a sulfoglycolipid epitope shared by cells of neuroectodermal and hematopoietic origin

Journal of Neuroimmunology, 23 (1989) 233-240 Elsevier

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JNI 00800

Identification of a sulfoglycolipid epitope shared by cells of neuroectodermal and hematopoietic origin Folke Schriever, Gert Riethmi~ller and Judith P. Johnson Institute of Immunology, Unioersityof Munich, D-8000 Munich, F.R.G. (Received 11 July 1988) (Revised, received 23 January 1989) (Accepted 23 January 1989)

Key words: Monoclonal antibody; Melanoma glycolipid; Enzyme-linked immunosorbent assay; Immunohistochemistry

Summary Monoclonal antibody (mAb) SNH.1 detects an epitope which is restricted to cells of neuroectodermal and hematopoietic origin. The mAb was obtained by immunization of a mouse with liposomes containing a crude extract of human melanoma acidic glycolipids. The SNH.1 antigen isolated from melanoma was identified as a sulfated glycolipid, closely related or identical to sulfogalactosyl-ceramide. When tested with different lipids, mAb SNH.1 reacted as well with other sulfoglycolipids. The staining of mAb SNH.1 is restricted to the cytoplasm and often localized to the perinuclear region. Therefore, the SNH.1 mAb epitope may be detectable only during the biosynthesis of sulfoglycolipids.

Introduction

Since the original observation that Thy-1, the classical marker of mature murine T cells (Reif and Allen, 1964) is also expressed by neurons (Pruss, 1979), several differentiation antigens have been found to be shared among cells of the neuroectodermal and hematopoietic lineages. The ganglioside Go3 , colnmon on neuroectodermal tissues, has been found to characterize a T cell subpopulation (Welte et al., 1987) and the CD4 molecule, a T cell differentiation antigen and the HIV-1 receptor, recently has been detected on neurons (Funke et al., 1987). The HNK-1 epitope, Address for correspondence: Judith P. Johnson, Institute of Immunology, D-8000 Munich 2, F.R.G. Requests for reprints should be addressed to Folke Schriever at the Dana-Farber Cancer Institute, Division of Tumor Immunology, 44 Birmey Street, Boston, MA 02115, U.S.A.

a sulfated glucuronic acid containing carbohydrate structure (Chou et al., 1986) which marks human natural killer cells (Abo and Balch, 1981), has also been found on the neural adhesion molecules (NCAM) and myelin-associated glycoprotein (MAG) (Kruse et al., 1984). This study describes a new antigen common to neuroectodermal and hematopoietic cells which has been identified by the monoclonal antibody (mAB) SNH.1. The epitope recognized by mAb SNH.1 is located on a sulfated glycolipid.

Materials and methods Materials Tissues, cells and monoclonal antibodies.

Tissues were snap frozen in liquid nitrogen immediately after surgical removal and stored at - 7 0 ° C . All cells were grown in RPMI 1640

0165-5728/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

234 supplemented with 10% fetal calf serum, 1 mM sodium pyruvate and antibiotics. Cell lines were obtained through exchange or established in our laboratory. The mAbs S1 (anti-human leukocyte antigen (HLA)-DQ, IgM), R1 (anti-HLA-DQ, IgM) and R3 (anti-HLA-DR, IgM) have been produced in our laboratory (Johnson and Wank, 1984). The mAb 141.11 (anti-H2-Kk, -Kb, -Dk, -IgM) was provided by G. H~immerling, German Cancer Center, Heidelberg, F.R.G. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) unless otherwise noted. Glycolipids. The sulfoglycolipids SB1 (GgOse4Cer-II3,IV3-bis-sulfate), SB2 (GgOse 3 Cer-II 3, III3-bis-sulfate), SM1 (GgOseaCer-II3-sulfate), SM2 (GgOse3Cer-IIa-sulfate), SM4g (seminolipid, Gal(fll-3)alkylacylGro-I3-sulfate) and SM4s (GalCer-I3-sulfate) were a generous gift of Ineo Ishizuka, University School of Medicine, Tokyo, Japan. SM4 (GalCer-I3-sulfate), galactosyl-ceramide, cholesterol-3-sulfate, L-et-phosphatidylcholine (dimyristoyl), L-a-phosphatidyl-D-L-glycerol, L-et-phosphatidylethanolamine and L-a-phosphatidylinositol were obtained from Sigma. The gangliosides GDla, GDlb, GTlb, GD3, GMa and GM2 (abbreviations according to IUPAC-IUB Commission on Biochemical Nomenclature, 1977 and Svennerholm, 1963) were purchased from Fidia Research (Abano Terme, Italy). G M3 was obtained from Dr. Pallmann (Munich, F.R.G.).

Methods Production of MAb SNH.1.

A 3-month-old male C57BL/6 × BALB/c F1 mouse was injected ].p. with a suspension of liposomes containing approximately 200 #g of a crude acidic glycolipid extract from malignant melanoma. Spleen cells were fused 3 days later with P3X63Ag8.653 myeloma cells using standard procedures. Hybrids were selected for glycolipid reactivity using a solid-phase enzyme-linked immunosorbent assay (ELISA), tested for their immunohistochemical reaction with tissue sections of malignant melanoma and finally examined by a thin-layer chromatogram (TLC) immunostaining assay.

Preparation of acidic glycolipids from malignant melanoma. A lymph node melanoma metastasis (40 g wet weight) was extracted twice with chloro-

f o r m / m e t h a n o l / H 2 0 (1:2:0.5). A phase separation according to Folch et al. (1956) was performed by adding H20 and the upper phase was dialyzed against distilled H20 and dried in a rotary evaporator. This lipid extract was used for immunization and ELISA screening. The sulfoglycolipid recognized by mAb SNH.1 was extracted and purified as described by Tadano et al. (1982). Briefly, the melanoma lymph node metastasis which had served as source for the crude lipid extract was extracted in three steps with chloroform/methanol (2: 1, v/v), chloroform/methanol/0.88% KC1 in H20 (60 : 120 : 9, V/V/V) and chloroform/methanol/0.4 M sodium acetate in H20 (60: 35:8, v / v / v ) . The extract was dried, treated with 4 M KOH in methanol for 8 h at 50 ° C, neutralized with 4 M acetic acid, dialyzed against distilled HzO and dried. The extract was applied to a DEAE-Sephadex A25 column (acetate form, Pharmacia, Uppsala, Sweden) and acidic glycofipids were eluted using a stepwise gradient of chloroform/methanol/0.05-0.3 M ammonium acetate in H20 (5 : 10 : 1, V/V/V). Desulfation of sulfoglycolipids was performed according to Ilyas et al. (1986). Briefly, 10-30/~g lipid was incubated with 0.1 M acetylchloride in dry methanol overnight at room temperature and dried under a stream of nitrogen. Thin-layer chromatography. TLC was performed on glass-backed high-performance thinlayer chromatogram-silica gels (Merck, Darmstadt, F.R.G.). The solvent system used for developing plates was chloroform/methanol/0.2% CaC12 in H20 (60: 35:8, v / v / v ) . Lipids with neutral sugar residues were identified by orcin/ sulfuric acid spray reagent. Sialic acid-containing glycolipids were stained with resorcin/hydrochloric acid spray reagent and phospholipids with ammonium molybdate/sulfuric acid. Production of liposomes. The method of Brunner et al. (1976) was used with modification. Melanoma crude lipid extract (10 mg) was dissolved in 2.5 ml 10 mM triethanolamine hydrochloride buffer (TEA) (Merck, Darmstadt, F.R.G.), pH 7.2, containing 24 mg asolectin (L-aphosphatidylcholine III S; Sigma) and 45 mg noctyl glucoside (Sigma) and incubated for 5-10 min. To remove the detergent the suspension was applied to a Sephadex-G25-PD10 column (Phar-

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macia) and eluted with 3 ml TEA. The eluate contained spontaneously formed vesicles which were subsequently transformed to large unilamellar vesicles by a 3 min incubation with 0.5 M CaC12 in TEA and addition of 0.25 M EDTA in TEA (Papahadjopoulos et al., 1975). The liposomes were used immediately for immunization. Antibody binding assays. Binding of antibodies to the crude lipid extract was assessed using a solid-phase ELISA described by Thurin et al. (1985). Melanoma lipid extract was coated onto 96-well flexible polyvinyl chloride microtiter plates (Dynatech Lab., Alexandria, VA, U.SA.), and the plates were blocked with 2% bovine serum albumin in phosphate-buffered saline (PBS). Binding of antibodies was detected using a peroxidasecoupled rabbit anti-mouse Ig (Nr. P161, Dakopatts, Copenhagen, Denmark) and a substrate solution of o-phenylenediamine (2 mg/ml) in 0.1 M citrate buffer, pH 5.0, containing 0.006% H202. Absorption was measured with an ELISA reader at 477 nm (SLT Labinstruments, Austria). For inhibition studies soluble antigen in PBS at a final concentration of 0.25-320 ng/~tl were added to the wells immediately before incubation with the first antibody. Immunoperoxidase staining of frozen tissue sections and cytospin preparations was performed as described by Lehmann et al. (1987) using an affinity-purified peroxidase-conjugated rabbit anti-mouse Ig (Dianova, Hamburg, F.R.G.) and as substrate 3-amino-9-ethylcarbazole (0.25 mg/ml in 0.1 M acetate buffer, pH 4.9) with 0.003% H202. For inhibition experiments the first antibody was mixed 1:1 with varying amounts of soluble antigen in PBS (0.5-400 ng/#l final concentration). Immunofluorescence of cell suspensions was done on live cells using a fluoresceinconjugated rabbit anti-mouse Ig (Dakopatts, Glostrup, Denmark). Immunoreactivity on thin-layer chromatogram plates (immuno-TLC) was carried out according to a modification of the method described by Dippold et al. (1987). Developed TLC plates were blocked in PBS containing 1% bovine serum albumin and 0.05% Tween 20. Peroxidase-coupled rabbit anti-mouse Ig (1 : 100) was used as second antibody and 4-chloro-l-naphthol (0.4 mg/ml in PBS) with 0.03% H202 as substrate. The minimal

amount of antigen that could be detected was 50 ng.

Results

MAb SNH.I reacts with cells of neuroectodermal and hematopoietic origin MAb SNH.1 (IgM) was produced against a crude acidic glycolipid extract of fresh melanoma. TABLE 1 TISSUES AND CELLS REACTIVE WITH mAb SNH.1 Embryologic origin Neuroectodermal Normal neural tissues Cortex parietalis Cerebellum Peripheral nerve Basal ganglia Nervus opticus Neural tumors Neuroblastoma Glioblastoma Astrocytoma Neurofibromatosis Melanocytic lesions Nevocellular nevi Spitz nevi Dysplastic nevi b Malignant melanomas Primary tumors Lymph node metastases Skin metastases Neuroblastoma cell lines Melanoma cell lines Lymphoid Spleen Red and white pulp Tonsil and thymus Lymph follicles Kupffer cells of the liver B cell lines T cell lines Others Kidney Glomeruli and distal tubules Renal carcinoma Smooth muscle

Number (positive/tested)

2/2 a 1/1 1/1 1/1 1/1 1/1 3/3 4/4 1/1 38/38 2/2 4/4 19/20 23/24 1/1 3/3 7/7 2/2 2/2 4/4 5/5 1/1 5/5 1/1 54/54 b

a Dysplastic nevi were defined according to Elder et al. (1982) and Clark et al. (1985). b Sections containing epidermis and gastrointestinal tract.

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Fig. 1. Immunoperoxidase reactivity of mAb SNH.1 (a) and isotype control 141.11 (b) with a melanoma lymph node metastasis. Original magnification, x 125.

Fig. 2. Immunoperoxidase reactivity of mAb SNH.1 with a cytospin preparation of a melanoma cell line. (a) mAb SNH.1, long arrows: cytoplasmic region positive with mAb SNH.1, short arrows: nucleus; (b) isotype control with mAb 141.11 (IgM), short arrows: nucleus. Original magnification, x500.

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Immunohistochemical investigation showed homogeneous reactivity of mAb SNH.1 with cells and tissues of neuroectodermal and hematopoietic origin. As can be seen from Table 1, virtually all cells and structures belonging to these lineages were stained, as were tumors and cell lines derived from them. Cells and tissues of other embryonic origins were unreactive (Table 2) with the exception of kidney, skin sweat glands and smooth muscle (Table 1), structures which are frequently stained by mAbs against diverse antigens. The staining by mAb SNH.1 appeared cytoplasmic (Fig. 1) and this was confirmed by immunofluorescence analyses on reactive cell lines. No surface reactivity was observed with melanoma cell line Mel Wei nor with four glioma cell lines (N18, N59, N63, N64) (data not shown). In cytospin preparations, staining of the perinuclear region was often observed (Fig. 2), whereas in situ, an exact intracellular localization of the reactivity could not always be determined. The staining of fibrillary endings of astrocytoma cells is shown in Fig. 3. M A b S N H . 1 detects an epitope on sulfoglycolipids

MAb SNH.1 was initially selected because it reacted in a binding assay with the immunizing melanoma lipid extract. To define more exactly the specificity of the antibody, the various components of the lipid extract were separated by TLC

TABLE 2 TISSUES AND CELLS NONREACTIVE WITH mAb SNH.1 Epidermis (44 a) Duodenum (2) Stomach (3) Colon b (5) Hepatocytes (4) Pancreas Gallbladder Thyroid gland (4) Lung Placenta Mammary gland (3) Vagina Ovary (2) a Number of tissues tested, if more than one sample was examined. b Brush border of epithelium weakly positive.

Fig. 3. Immunoperoxidase reactivity of mAb SNH.1 with an astrocytoma. Arrows: SNH.l-positive fibrillary endings. Original magnification, × 200.

and examined for reactivity with mAb SNH.1 using immuno-TLC. The antibody bound to a single component which appeared as a double band. This component was identified as a glycolipid because of its positive reactivity with iodine vapor and orcin. It was negative with resorcin/ hydrochloric acid and ammonium molybdate indicating that it did not contain neuraminic acid and amino groups. When SNH.1 was tested with a panel of lipids which included gangliosides, neutral glycolipids, phospholipids, cholesterol-3sulfate and SM4, it bound only to SM4 (Fig. 4), indicating that the epitope recognized by SNH.1 may be expressed by a sulfated glycolipid. The IgM Mabs S1, R1, R3 and 141.11, directed against unrelated antigens were included as controls and showed no reactivity in these assays. To examine the melanoma antigen more closely, sulfoglycolipids were extracted from the lymph node metastasis. A SNH.l-positive glycolipid was eluted from a DEAE-Sephadex column with 0.2-0.3 M ammonium acetate, following the elution of GM3, as is reported for SM4 (Tadano et al., 1982). As can be seen from Fig. 4, this glycolipid migrates in the range of SM4. Treatment of

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Fig. 4. Thin-layer chromatography. Plate a: Lane 1, standard gangliosides, stained with resorcin/HC1; lane 2, neutral glycolipids, stained with orcin/H2SO4; lane 3, sulfated and lane 4, desulfated melanoma glycolipid; lane 5, sulfated and lane 6, desulfated sulfogalactosyl-ceramide; samples in lanes 2-6 have been stained with orcin/H2SO4; lane 7, cholesterol-3-sulfate, identified with iodine vapor; lanes 8 and 9, phospholipids stained with ammonium m o l y b d a t e / H 2 S O 4. Plate b: Immunoreactivity of mAb SNH.1 with the same samples as on plate a, which ran under identical conditions. CMH, ceramide monohexoside; CDH, ceramide dihexoside; CTH, ceramide trihexoside; CTetH, ceramide tetrahexoside; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdIns, phosphatidylinositol; PtdCho, phosphatidylcholine.

the melanoma antigen and SM4 with 0.1 M acetylchloride, which leads to a selective loss of sulfate groups, resulted in an altered mobility pattern of both glycolipids (Fig. 4). The desulfated melanoma antigen ran at the same height as the desulfated SM4 (i.e. galactosyl-ceramide) and in the range of ceramide monohexoside, indicating that after loss of its sulfate group it contains only one hexose. The similar migration pattern of the melanoma antigen and SM4 before and after desulfation suggests that they have related chemical structures. No binding of mAb SNH.1 to either of the two desulfated glycolipids was observed, indicating that the sulfate moiety is important for the formation of the SNH.1 epitope. MAb SNH.1 was further tested in immuno-TLC for reactivity with a series of different sulfoglycolipids with related chemical structures: SB1, SB2, SM2, SM3, SM4s and SM4g (data not shown). mAb SNH.1 reacted with each of these, suggesting that the terminal or subterminal sulfated galactose participates in the formation of the epitope. The specificity of mAb SNH.1 for sulfated glycolipids was confirmed using an ELISA inhibition assay (Fig. 5). mAb SNH.1 binding to SM4 could be completely inhibited b y the addition of 32 n g / # l SM4 but not by galactosyl-ceramide. The IgM mAbs R1 and R3 are shown as isotype controls and identical results were obtained with

mAbs $1 and 141.11. None of these mAbs bound to SM4 under the conditions used. Inhibition studies also showed that mAb SNH.1 reacts with a similar epitope on tissue sections, mAb SNH.1 staining both of neuroectodermal derived and lymphoid tissues could be completely inhibited by 400 n g / / d SM4 but was not influenced by

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galactosyl-ceramide suggesting that the antibody recognizes the same epitope in both cell lineages. No evidence was obtained for reactivity of mAb SNH.1 with a melanoma glycoprotein as Western blot analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-separated immunoprecipitates revealed no specific bands (data not shown).

Discussion The study presented here describes an mAb, SNH.1, which detects a sulfoglycolipid epitope expressed by neuroectodermal and hematopoietic cells. Although antibodies of the IgM isotype have been reported to bind non-specifically to sulfoglycolipids (Hofstetter et al., 1985; Roberts, 1986) mAb SNH.1 binding appears to reflect a specific reactivity, mAb SNH.1 did not bind to a variety of different neutral and acidic lipids, including gangliosides, phospholipids, and cholesterol-3sulfate. Binding to sulfoglycolipids was abrogated upon desulfation, and binding of mAb SNH.1 in solid-phase ELISA and in tissue sections could be completely inhibited by soluble SM4. Under the conditions used, none of the four control IgM mAbs bound to the sulfoglycolipids either in the ELISA or in the immuno-TLC. The observations that mAb SNH.1 did not react with the melanoma antigen nor with SM4 after desulfation and that it did not distinguish between different sulfoglycolipids indicate that a terminal or subterminal galactose group is essential for the formation of the epitope. The structure of the lipophilic part of the sulfoglycolipid does not seem to play an important role since mAb SNH.1 recognized sulfoglycolipids with ceramide (SM4s) as well as with alkylglycerol (SM4g) residues. Sulfoglycolipids are broadly distributed throughout the body and have been extracted from neural tissues (Ishizuka, 1978), kidney, spleen, red blood cells, testes, stomach and intestine (reviewed by Roberts, 1986). The universal occurrence of sulfated glycolipids contrasts with the limited pattern of reactivity of mAb SNH.1 and with the fact that only a single reactive com-

ponent was isolated from melanoma. While the latter may be explained by a predominance of SM4 in melanoma, the reactivity of mAb SNH.1 suggests that it recognizes an epitope which is not expressed in most tissues. Sulfoglycolipids at the cell surface participate in the formation of cellular receptors and have been shown to contribute to morphin (Cho et al., 1976), thrombospondin and laminin binding (Roberts et al., 1985). In contrast to the cell surface reactivity of other anti-SM4 mAbs (Hofstetter et al., 1984), mAb SNH.1 reactivity is restricted to the cytoplasm at least in melanoma and glioma cells, and is often localized to a perinuclear region where the Golgi apparatus is also localized. The Golgi apparatus is the site of the enzymatic processing of glycoproteins and glycolipids which occurs prior to their integration into the plasma membrane and this suggests that the SNH.1 epitope may be exposed or created only during a particular step of sulfoglycolipid biosynthesis. Although further studies are needed to determine the relationship between the SNH.1 epitope and those identified by other anti-SM4 mAbs, mAb SNH.1 may be a useful tool for the study of the biochemical mechanisms involved in sulfoglycolipid metabolism in neuroectodermal and hematopoietic cells.

Acknowledgements This work was supported in part by a grant from the Deutsche Krebshilfe, Mildred Scheel Stiftung Bonn, F.R.G. We gratefully thank Dr. I. Ishizuka for providing a panel of sulfoglycolipids, Dr. I. Funke and Dr. J.M. Gokel for providing various tissue samples and Dr. C. Hergersberg for helpful advice.

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