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Definition of an extracellular matrix protein in rostral portions of the human central nervous system
Brain Research, 438 (1988) 315-322
315
Elsevier BRE 22668
Definition of an extracellular matrix protein in rostral portions of the human central nervous system Wolfgang J. Rettig I Pilar Garin Chesa 2, H. Richard Beresford l Myron R. Melamed 2 and Lloyd J. Old 1 •I
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1Laboratory of Human Cancer hnmunology and :Department of Pathology. Memorial Sloat~-Kettering Cancer Center, New York, N Y 10021 ( U. S. A. )
(Accepted 22 September 1987) Key words: Extraceilular matrix: Central nervous system: lmmunohistochemistrv; Brain segment: Monoclonal antibody
We have identified and characterized an extracellular matrix (ECM) glycoprotein of cultured astrocytomas, NEC1. that is expressed in normal human brain p,~renchyma. Detailed immunohistochemical analysis reveals a region-specific NEC1 pat~,e~lalong the rostrocaudal axis of the cen.tral nervous system (CNS). with strong expression throughout the white matter of telencephalon and diencephalon, scant expression in some areas of mesencephalon, and no expression in pons, cerebellum, medulla, spinal cord, and peripheral nervous system. NEC 1 is not co-distributed with any known neural cell type, suggesting that expression of specific ECM proteins in the CNS is segmentally controlled. The development and maintenance of complex patterns of cellular connectivity in the nervous system are intriguing but poorly understood phenomena. Several controlling mechanisms have been proposed for morphogenesis in neural and non-neural tissues, including chemotaxis 2~. differential expres. sion of cell surface recognition and cell adhesion molecules ~1"!6, and cellular interactions with extracellular matrix (ECM) components- ~0. Fibronectin (FN), one of the well-characterized ECM proteins, is thought to guide the migration of neural crest-derived peripheral nervous system (PNS) precursors along preformed ECM pathways to their final tissue locations 3°, but attempts to detect FN or other ECM proteins in the parenchyma of the neural tube-derived central nervous system (CNS) have generally failed 4"7"19"2°. In the present study, we have identified an ECM protein which shows a unique pattern of expression in white matter of the most rostral, telencephalic and diencephalic portions of the human CNS, but is absent from more caudal CNS portions and the PNS. °
Monoclonal antibody (MAb) NEC1 (IgG1 subclass) was derived from a mouse immunized with SKMG-10 human astrocytoma cells 6 using standard hybridization and cloning procedures ~'-~6.The MAb was selected for reactivity with the cell-free ECM of astrocytomas (Fig. 1) and lack of reactivity with ECM produced by other cell types. Indirect immunofluorescence staining 17 and analysis with the highly sensitive immune hemadsorption assay (I-HAA) 8"-'~'25 showed that NEC1 is present in the substrate attachment matrix 1°-23-31 (Figs. 1 and 2A, C) but not on the surface of intact astrocytoma cells (Fig. 2A). NEC1 was equally found in ECM prepared by removal of astrocytoma ceils either with E D T A (0.02% in phosphate buffered saline (PBS)) or non-ionic detergent (0.5% Nonidet P40 (NP40), 150 mM NaCi, 2 mM MgCI, 10 mM Tris, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 20 U/ml kallikrein inhibitor) or by shaking off confluent monolayers of intact cells, lmmunofluorescence staining for NECI or I-HAA reactivity were not reduced when these matrices were further extracted with NP40 buffer (0.5% in
Correspondence: W.J. Rettig. Section 6801. Box 148. Memorial Sloan-Kettering Cancer Center. 1275York Avenue, New York, NY
10021, U.S.A. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
316 Tris buffer, pH 7.4; 30 min at room temperature) or treated with neuraminidase 27. However, reactivity was abolished by heating the E C M (5 min at 100 °C) or treatment with trypsin or pronase -'7.
Fig. 1. Immunofluorescence staining of ceil-free ECM prepared from SK-MG-10 astrocytoma cultures and tested with MAb NEC1. Insert (lower right corner): control experiment with unrelated MAb. Note intense staining of substrate attachment material (each round "footprint' corresponds to a single cell attachment site) and weak staining of culture surfaces between attachment sites. Cell-free ECM was prepared by removing cells of subconfluent SK-MG-10 cultures with EDTA (0.02% in PBS, 15 min at room temperature) and subsequent extraction of substrate attachment rmteria123 with non-ionic detergent (0.5% NP40, 150 mM NaCI, 2 mM MgCI, 10 mM Tris, pH 7.4; 10 min at RT), followed by 3 washes with PBS. Indirect immunofluorescence staining was carried out as described tT. x68.
Enzyme-linked immunosorption assays ( E L I S A ) were used to determine the presence of secreted NEC1 in spent media of a n u m b e r of cultured cell types. As illustrated in Fig. 3, NEC1 is secreted by all 5 astrocytoma lines tested but not by other cells which share a common neuroectodermal origin with glial cells, including m e l a n o m a (SK-MEL-23, .-64, -186, -109), neuroblastoma (SK-N-SH, -MC, SMSMSN, -KAN), retinoblastoma (Y79), medulloblastoma (TE671), and normal leptomeningeal cells. Immunoprecipitation experiments with spent media of [35S]methionine- and [3H]glucosamine-labeled astrocytoma cultures showed that NEC1 is a high-molecular weight glycoprotein which barely enters a 7.5% sodium dodecyl sulphate (SDS) polyacrylamide gel under non-reducing conditions (Fig. 4, lane A). Under reducing conditions, the antigen is resolved into two smaller subunits (Fig. 4, lane C), indicating that native NEC1 is composed of disulfide-linked 250kDa and 180-kDa glycoproteins. Identical glycoproteins were immunoprecipitated from spent media of
Fig. 2. MAb-mediated immune hemadsorption assays (I-HAA) with intact SK-MG-3 astrocytoma cells. A: reactivity with MAb NEC1 showing presence of antigen (as indicated by adherence of rabbit antimouse lgG-coated human erythrocytes) adsorbed to plas. tic culture surface between SK-MG-3 target cells but no reactivity with cell surface membranes. B" reactivity with MAb B5 showing erythrocyte adherence to SK-MG-3 cell surface membranes but not to the culture surface between target cells. Note reciprocal patterns of antigen localization in panels A and B. C: reactivity of MAb NECI with cell-free substrate attachment material of SK-MG-3 cells, prepared as described in Fig. 1. For I-HAA, SK-MG-3 cells were seeded into MicroWell plates (Nunc, Roskilde, Denmark) and gro~n in a 95% air/5% CO_, atmosphere for 12-18 h at 37 °C. Viable target cells were tested for antigen expression as described s'-'7 Unrelated MAbs were used as negative controls and did not produce any erythrocyte adherence to target cells or to the culture suhstrate.
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Fig 3. Solid-phase ELISA for detection of NECI in spent media of cultured human cells and in human brain tissue extracts. A: spent media of cultured astrocytoma (O, SK-MG-3; A, SK-MG-15; O , SK-MG-16; [], SK-GS-I), neuroblastoma ( ~ , SMS-KAN), melanoma (r-q SK-MEL-64), and norreal leptomeningeai cells (.__6,FB2 short-term culture) were adsorbed to MicroWell plates and tested with serial dilutions of MAb NEC1. C), negative control MAb tested with spent media of SK-MG-3 cells. B: spent media of SK-MG-13 astrocytoma cells fractionated on a gelatin-Sepharose column tested with MAb NECI: O, unfractionated: ~ . flowthrough fraction; ~ , FN-containing fraction eluted with 8 M urea in citrate buffer~Z; ©, negative control experiment with unrelated MAb on unfractionated spent media. C: extracts of fresh human brain (starting concentration 2.5 !~g protein/well) were adsorbed to MicreWell plates and tested by ELISA with MAb NECI: O, frontal lobe white matter; •, temporal lobe white matter; A, occipital lobe white matter; C), frontal lobe gray matter; 1-1, temporal lobe gray matter; A, cerebellum, white matter; ~ , negative omtrol experiment with unrelated MAb on frontal lobe white matter extract. For ELISA testing of spent media, confluent cultures were grown for 5 days in DMEM, 7.5-15% fetal bovine serum (FBS), non-essential amino acids, L-glutamine, and antibiotics. Culture sypernatants were cleared by centrifugation at 900 g for 20 min, incubated in MicroWell plates for 18 h at 4 °C or 1 h at 37 °C, washed with PBS;5% FBS, and incubated with MAb for 1 h at room temperature followed by alkaline phosphatase-conjugated rabbit h~ ,I ~,,~1.~ ~,,~ antimouse-lgG and F-m" t rop..~ny.phuov ..... (PNP,Sigma, 1 mg/ml in 10% diethanolamine buffer, pH 9.7). Changes in optical density were deterr, ined at 405 nm. Brain tissues were collected at autopsy, homogenized in 8 volumes of extraction buffer (30 mM diethvlamine, 1 mM EI2TA, pH 11.5) '~, cleared by centrifugation (900 g, 20 min), adjusted to a protein concentration of 0.25 mg/ml, serially diluted with extraction buffer, and incubated in MicroWell plates for 18 h at 4 °C before ELISA analysis.
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Fig. 4. Fluorograph of immunoprecipitates obtained with MAbs NECI and B5 (anti-mel-CSPG) and anti-human FN antibody from astrocytoma cultures. A B: concanavalin Abound fraction of spent media from [-~SS]methionine-labeled SK-MG-3 cells tested with MAb NEC1 (A) or unrelated control MAb (B); SDS-PAGE under nonreducing conditions (extraction buffer containing 14 mg/ml iodoacetamide). C - E : spent media of [3H]glucosamine-le,beled SK-MG-3 cells tested with MAb NEC1 (C), unrelated control MAb (D), or anti ICN-'O (E); SDS-PAGE under reducing conditions (extraction buffer containing 12 mg;ml dithiothreiioi). F: NP40-extract of [3H]glucosamine-labe!ed SK-MG-3 cells tested with MAb B5 (antimei-CSPG proteoglycan)-'-~: SDS-PAGE under reducing conditions. Molecular mass ef immunoprecipitated components is indicated to the left and "origin" indicates top of 7.5% SDS-polyacrylamide gel. lmmunochemical procedures have been described previously~'.
318 all 5 astrocytoma lines tested and also from NP40 extracts of astrocytoma cells collected in E D T A buffer (0.02% in phosphate-buffered saline (PBS)). No antigen was precipitated from spent media or cell extracts of melanoma or neuroblastoma cell lines. These results show that NEC1 is synthesized by astrocytoma cells as a detergent-soluble, multimeric glycoprotein which is both secreted into the culture media and incorporated into the detergent-insoluble substrate attachment matrix. A proportion of human astrocytoma cell lines produce FN and proteoglycans ~9"25that can be incorporated into the detergent-insoluble substrate attachment matrix l°. Therefore, we have compared NEC1 with astrocytoma-derived FN and meI-CSPG, a specific chondroitin-sulfate proteoglycan of astrocytomas recognized by M A b B5 (ref. 25). Fig. 4 shows that NEC1 differs in molecular size from both melCSPG and FN, and Fig. 2 shows that NEC1 also differs from meI-CSPG in subcellular localization: melCSPG is detected on the surface of intact astrocytoma cells whereas NEC1 is not present on the cell surface; although meI-CSPG is also shed into the spent media 5, it is not detectably adsorbed to plastic culture surfaces (Fig. 2A, B). FN is readily adsorbed to plastic surfaces but differs from NEC1 in gelatin binding: FN is strongly bound to gelatin 12 whereas NEC1 shows no binding to a gelatin-Sepharose column (Fig. 3B). MAb NEC1 also fails to react by E L I S A with purified human plasma FN 12 (50/~g/ml in Dulbecco's PBS (DPBS), adsorbed to MicroWell plates for I h at 37 °C). Finally, our biochemical and immunohistochemical results (see below) also distinguish NEC1 from laminin (LN) and other glycoproteins secreted by astrocytoma cell lines 1"2,4.19-21. Cultured astrocytomas produce several E C M proteins that are not produced by normal glial cells in vivo 4'7"19"21,and we have used immunohistochemical methods to examine NEC1 expression in neural tissues. The results show that NEC1 is specifically expressed in white matter of the most rostral portions of the brain (Fig. 5, Table I). Strong NEC1 immunostaining was found throughout the white matter of frontal, parietal, temporal, and occipital lobes of the cerebral hemispheres (Fig. 5A, D), corpus callosum, internal capsule, basal ganglia (Fig. 5B), and diencephalon. In contrast, the more caudal portions of the brain, including most areas of mesencephaion
FABLE I Distribution of ECM glycoproteins recognized by MAb NECI in normal neural tissues determined by the avidin-biotin immunoperoxidase procedure in frozen section,~
Tissues from 5 adult individuals were obtained at autopsy, embedded in OCT compound (Tissue-TEKll, Miles. Naperville, I L ) . quick-frozen in isopentane precooled in liquid nitrogen, and stored at -70 °C until used. Multiple tissue samples (cut surface ranging from 8 x 8 mm to 10 x 15 mm) were tested for each CNS region indicated in the table and for each of the 5 individuals. Frozen 7-/~m sections were cut, air-dried, fixed in cold acetone for 10 min at 4 °C, and tested with MAb NEC1 (hybridoma culture supernatant) or unrelated MAb by the avidin-biotin immunoperoxidase method as described B. Results are indicated as follows: + + + , ++, ,, strong, moderate, and weak immunoreactivity; -, anti~z~t-negative; +/+ + +, + +/+. -/+, regional heterogeneity in NEC1 staining intensity. Nervous system subdivision
Region
NEC1 expression
Telencephalon
Cerebral hemispheres (frontal, parietal, temporal, occipital lobes) Gray matter +++ White matter +++ Corpus callosum ++/+ Olfactory bulb Basal ganglia +/+++* Globus pailidus +* Putamen
Diencephalon
Internal capsule Thalamus Hypothalamus MammiUary body
++/+ ++1+* + +/+* ++/+*
Mesencephalon
Substantia nigra Crus cerebri Medial lemniscus
_/+ *
Metencephalon
Pons Cerebellum White matter Molecular layer Purkinje cell layer Granular layer Cerebellar peduncles (superior, inferior, middle) -
Myelencephalon Medulla oblongata Fiber tracts Inferior olivary nuclear complex Spinal cord
Spinal cord
PNS
Peripheral nerves Autonomic ganglia Sensory ganglia
* Heterogeneity of NECl-immunostaining generally followed the regional distribution of white (strong NEC1 staining) and gray matter (weak or no NEC1 staining) within rostral portions of the brain.
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Fig. 5. lmmunoperoxidase staining of human brain tissues with MAb NEC1 ( A - E ) or the astrocyte-specific MAb K4 (F). Hematoxylin counterstaining. A: temporal lobe, strong NEC1 staining throughout the subcortical white matter (w) (for high-power view see panel D) and in a thin superficial cortical layer (*) but not in bulk of cortical gray matter (g) or leptomeninges. B: globus pailidus, strong NEC1 staining showing ECM pattern; ceil bodies not labeled. C: mesencephalon, scant NECI staining with loose, fibrillary pattero D: subcortical white matter of temporal lobe, NEC1 staining with ECM pattern; cell bodies not labeled. E: cerebellum, granular layer (top) and white matter (bottom) NECIneg,tive. F: mesencephalon (parallel section to panel C), labeling of glial cells with the astrocyte-specific MAb K4 (unpublished data) -'7. Unrelated MAbs were routinely included as negative controls and did not produce any staining. Hematoxylin counterstaining of nuclei (blue) ns easily distinguished from immunoperoxidase staining (brown). Original magnification x5 (A), x 100 (B,C,E), x250 (D,F).
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320 (Fig. 5C), the pons. cerebellum (Fig. 5E), and medulla oblongata, as well as the spinal cord are NEC1negative (Table I). No immunostaining for NECI was detected in the peripheral nervous system, leptomeninges, and cerebral blood vessels, structures known to express FN and L N 4"19"2° At the microscopic level, immunostaining for NEC1 defines a distinctive, fine-fibrillary meshwork in CNS parenchyma rather than a cellular or fiber tract pattern (Fig. 5 A - D ) . In contrast to this characteristic ECM pattern for NEC1, parallel tissue sections tested with MAbs to cellular antigens 14"17'26"27 showed well-defined cytoplasmic or cell membrane staining as illustrated in Fig. 5F. Therefore, NEC1 can be localized to intercellular spaces in rostral portions of the CNS, consistent with the presence of NEC1 in the ECM but not on the surface of cultured glial cells (Fig. 2A). The results of our immunohistochemical analysis of neural tissues were confirmed by ELISA. Frontal, temporal, and occipital lobe white matter extracts prepared in alkaline buffer (30 mM diethylamine, 1 mM EDTA, pH 11.5) 9 contained readily detectable NEC1 antigen, whereas adjacent areas of gray matter were NECl-negative (Fig. 3C). Extracts prepared from either white or gray matter of the cerebellum were also NECl-ne~,ative. Consistent with the localization of NEC1 to the detergent-insoluble ECM in vitro, the antigen was not found in extracts of forebrain white matter prepared in NP40 buffer (0.5% NP40 in Tris buffer, pH 7.4). NEC1 defines a unique region-specific pattern of ECM protein expression in human CNS parenchyma. Chicken cytotactin, an antigen first identified in embryonic chicken brain ~8. shares ~ome characteristics with human NEC1. It is a glycoprotein composed of disulfide-linked 220-kD, 200-kDa, and 190-kDa subunits and is expressed with an ECM pattern in chicken brain tissue 18. However, Crossin et al. have described widespread distribution of cytotactin in embryonic chicken CNS, including forebrain, cerebellum, medulla, and spinal cord, and a comparable pattern of expression in the adult animal, including forebrain and cerebellum 9. NECI has a different distribution in the adult human CNS (Fig. 5A, E, Table I), being restricted to the most rostral portions of the brain (telencephalon, diencephalon, some areas of mesencephalon). Cerebellum, pons, medulla, spinal
cord, and PNS tissues do not show any NEC1 expression. Therefore. chicken cytotactin and human NEC1 appear to be non-homologous ECM proteins or they may be structural homologues whose patterns of tissue-specific expression have diverged during evolution, as previously described for the Thy-1 antigen ~7. Amino acid sequencing of NECI and cytotactin will help to resolve this question. The identity of cells secreting NEC1 in human brain tissue is unknown, although astrocytes are likely candidates. However, the distinctive topographical pattern of NEC1 expression in brain tissue does not coincide with the distribution of any known subset of glial or neuronal cells or fiber system. One possible explanation for this unioue NEC1 pattern is the existence of an as yet uniden~.;ied group of NECI-secreting cells (for instance, a subset of astrocytes) that are restricted to rostral portions of the CNS. Alternatively, cells capable of secreting NEC1 may be present throughout the CNS, but they become NEC1 producers only when induced by local differentiation factors that are restricted to rostral portions of the CNS. We suggest that NEC1 is one of a group of molecules showing segmentally restricted expression in mammalian CNS tissues. Additional members of this group are two mouse homeo box genes 3"j5 ar:d a MAb-defined antigen of rabbit brain, designated 271A6, which is restricted to gray matter of the telencephalon -'2. The putative protein products of the mouse homeo box genes and the biochemical nature and subcellular localization of the 271A6 rabbit antigen are still unknown, precluding a detailed comparison with the NEC1 glycoproteins. It has been hypothesized that development and maintenance of precise patterns of cellular connectivity in the CNS are determined by gradients of diffusible factors or by interactions between cell surface recognition molecules on neurons and glial cells 11"16"2~'3°. The rostrocaudal patterning of NEC1 expression in the human CNS may represent an alternative organizing principle in which site-restricted expression of ECM components and cell type-restricted expression of ECM-receptors play a central role. According to this hypothesis, NEC1, for which we propose the designation human neuronectin, interacts with receptors on cells of the developing and adult CNS to guide cell movement and determine cellular differentiation within molecularly unique do-
321 mains of the CNS. It is unlikely that neuronectin-negative segments of the human CNS are devoid of ECM proteins and we are now seeking evidence for additional regionspecific ECM proteins.
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We thank S. Walker and G. Lark for excellent technical help. Dr. K.O. Lloyd for anti-FN antibody. and Dr. J. Sorvillo for purified FN. Supported in part by NCI Grants CA-08748 and CA-25803 and bv the Oliver S. and Jennie R. Donaldson Charitable Trust. .
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315
Elsevier BRE 22668
Definition of an extracellular matrix protein in rostral portions of the human central nervous system Wolfgang J. Rettig I Pilar Garin Chesa 2, H. Richard Beresford l Myron R. Melamed 2 and Lloyd J. Old 1 •I
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1Laboratory of Human Cancer hnmunology and :Department of Pathology. Memorial Sloat~-Kettering Cancer Center, New York, N Y 10021 ( U. S. A. )
(Accepted 22 September 1987) Key words: Extraceilular matrix: Central nervous system: lmmunohistochemistrv; Brain segment: Monoclonal antibody
We have identified and characterized an extracellular matrix (ECM) glycoprotein of cultured astrocytomas, NEC1. that is expressed in normal human brain p,~renchyma. Detailed immunohistochemical analysis reveals a region-specific NEC1 pat~,e~lalong the rostrocaudal axis of the cen.tral nervous system (CNS). with strong expression throughout the white matter of telencephalon and diencephalon, scant expression in some areas of mesencephalon, and no expression in pons, cerebellum, medulla, spinal cord, and peripheral nervous system. NEC 1 is not co-distributed with any known neural cell type, suggesting that expression of specific ECM proteins in the CNS is segmentally controlled. The development and maintenance of complex patterns of cellular connectivity in the nervous system are intriguing but poorly understood phenomena. Several controlling mechanisms have been proposed for morphogenesis in neural and non-neural tissues, including chemotaxis 2~. differential expres. sion of cell surface recognition and cell adhesion molecules ~1"!6, and cellular interactions with extracellular matrix (ECM) components- ~0. Fibronectin (FN), one of the well-characterized ECM proteins, is thought to guide the migration of neural crest-derived peripheral nervous system (PNS) precursors along preformed ECM pathways to their final tissue locations 3°, but attempts to detect FN or other ECM proteins in the parenchyma of the neural tube-derived central nervous system (CNS) have generally failed 4"7"19"2°. In the present study, we have identified an ECM protein which shows a unique pattern of expression in white matter of the most rostral, telencephalic and diencephalic portions of the human CNS, but is absent from more caudal CNS portions and the PNS. °
Monoclonal antibody (MAb) NEC1 (IgG1 subclass) was derived from a mouse immunized with SKMG-10 human astrocytoma cells 6 using standard hybridization and cloning procedures ~'-~6.The MAb was selected for reactivity with the cell-free ECM of astrocytomas (Fig. 1) and lack of reactivity with ECM produced by other cell types. Indirect immunofluorescence staining 17 and analysis with the highly sensitive immune hemadsorption assay (I-HAA) 8"-'~'25 showed that NEC1 is present in the substrate attachment matrix 1°-23-31 (Figs. 1 and 2A, C) but not on the surface of intact astrocytoma cells (Fig. 2A). NEC1 was equally found in ECM prepared by removal of astrocytoma ceils either with E D T A (0.02% in phosphate buffered saline (PBS)) or non-ionic detergent (0.5% Nonidet P40 (NP40), 150 mM NaCi, 2 mM MgCI, 10 mM Tris, pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 20 U/ml kallikrein inhibitor) or by shaking off confluent monolayers of intact cells, lmmunofluorescence staining for NECI or I-HAA reactivity were not reduced when these matrices were further extracted with NP40 buffer (0.5% in
Correspondence: W.J. Rettig. Section 6801. Box 148. Memorial Sloan-Kettering Cancer Center. 1275York Avenue, New York, NY
10021, U.S.A. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
316 Tris buffer, pH 7.4; 30 min at room temperature) or treated with neuraminidase 27. However, reactivity was abolished by heating the E C M (5 min at 100 °C) or treatment with trypsin or pronase -'7.
Fig. 1. Immunofluorescence staining of ceil-free ECM prepared from SK-MG-10 astrocytoma cultures and tested with MAb NEC1. Insert (lower right corner): control experiment with unrelated MAb. Note intense staining of substrate attachment material (each round "footprint' corresponds to a single cell attachment site) and weak staining of culture surfaces between attachment sites. Cell-free ECM was prepared by removing cells of subconfluent SK-MG-10 cultures with EDTA (0.02% in PBS, 15 min at room temperature) and subsequent extraction of substrate attachment rmteria123 with non-ionic detergent (0.5% NP40, 150 mM NaCI, 2 mM MgCI, 10 mM Tris, pH 7.4; 10 min at RT), followed by 3 washes with PBS. Indirect immunofluorescence staining was carried out as described tT. x68.
Enzyme-linked immunosorption assays ( E L I S A ) were used to determine the presence of secreted NEC1 in spent media of a n u m b e r of cultured cell types. As illustrated in Fig. 3, NEC1 is secreted by all 5 astrocytoma lines tested but not by other cells which share a common neuroectodermal origin with glial cells, including m e l a n o m a (SK-MEL-23, .-64, -186, -109), neuroblastoma (SK-N-SH, -MC, SMSMSN, -KAN), retinoblastoma (Y79), medulloblastoma (TE671), and normal leptomeningeal cells. Immunoprecipitation experiments with spent media of [35S]methionine- and [3H]glucosamine-labeled astrocytoma cultures showed that NEC1 is a high-molecular weight glycoprotein which barely enters a 7.5% sodium dodecyl sulphate (SDS) polyacrylamide gel under non-reducing conditions (Fig. 4, lane A). Under reducing conditions, the antigen is resolved into two smaller subunits (Fig. 4, lane C), indicating that native NEC1 is composed of disulfide-linked 250kDa and 180-kDa glycoproteins. Identical glycoproteins were immunoprecipitated from spent media of
Fig. 2. MAb-mediated immune hemadsorption assays (I-HAA) with intact SK-MG-3 astrocytoma cells. A: reactivity with MAb NEC1 showing presence of antigen (as indicated by adherence of rabbit antimouse lgG-coated human erythrocytes) adsorbed to plas. tic culture surface between SK-MG-3 target cells but no reactivity with cell surface membranes. B" reactivity with MAb B5 showing erythrocyte adherence to SK-MG-3 cell surface membranes but not to the culture surface between target cells. Note reciprocal patterns of antigen localization in panels A and B. C: reactivity of MAb NECI with cell-free substrate attachment material of SK-MG-3 cells, prepared as described in Fig. 1. For I-HAA, SK-MG-3 cells were seeded into MicroWell plates (Nunc, Roskilde, Denmark) and gro~n in a 95% air/5% CO_, atmosphere for 12-18 h at 37 °C. Viable target cells were tested for antigen expression as described s'-'7 Unrelated MAbs were used as negative controls and did not produce any erythrocyte adherence to target cells or to the culture suhstrate.
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Fig 3. Solid-phase ELISA for detection of NECI in spent media of cultured human cells and in human brain tissue extracts. A: spent media of cultured astrocytoma (O, SK-MG-3; A, SK-MG-15; O , SK-MG-16; [], SK-GS-I), neuroblastoma ( ~ , SMS-KAN), melanoma (r-q SK-MEL-64), and norreal leptomeningeai cells (.__6,FB2 short-term culture) were adsorbed to MicroWell plates and tested with serial dilutions of MAb NEC1. C), negative control MAb tested with spent media of SK-MG-3 cells. B: spent media of SK-MG-13 astrocytoma cells fractionated on a gelatin-Sepharose column tested with MAb NECI: O, unfractionated: ~ . flowthrough fraction; ~ , FN-containing fraction eluted with 8 M urea in citrate buffer~Z; ©, negative control experiment with unrelated MAb on unfractionated spent media. C: extracts of fresh human brain (starting concentration 2.5 !~g protein/well) were adsorbed to MicreWell plates and tested by ELISA with MAb NECI: O, frontal lobe white matter; •, temporal lobe white matter; A, occipital lobe white matter; C), frontal lobe gray matter; 1-1, temporal lobe gray matter; A, cerebellum, white matter; ~ , negative omtrol experiment with unrelated MAb on frontal lobe white matter extract. For ELISA testing of spent media, confluent cultures were grown for 5 days in DMEM, 7.5-15% fetal bovine serum (FBS), non-essential amino acids, L-glutamine, and antibiotics. Culture sypernatants were cleared by centrifugation at 900 g for 20 min, incubated in MicroWell plates for 18 h at 4 °C or 1 h at 37 °C, washed with PBS;5% FBS, and incubated with MAb for 1 h at room temperature followed by alkaline phosphatase-conjugated rabbit h~ ,I ~,,~1.~ ~,,~ antimouse-lgG and F-m" t rop..~ny.phuov ..... (PNP,Sigma, 1 mg/ml in 10% diethanolamine buffer, pH 9.7). Changes in optical density were deterr, ined at 405 nm. Brain tissues were collected at autopsy, homogenized in 8 volumes of extraction buffer (30 mM diethvlamine, 1 mM EI2TA, pH 11.5) '~, cleared by centrifugation (900 g, 20 min), adjusted to a protein concentration of 0.25 mg/ml, serially diluted with extraction buffer, and incubated in MicroWell plates for 18 h at 4 °C before ELISA analysis.
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Fig. 4. Fluorograph of immunoprecipitates obtained with MAbs NECI and B5 (anti-mel-CSPG) and anti-human FN antibody from astrocytoma cultures. A B: concanavalin Abound fraction of spent media from [-~SS]methionine-labeled SK-MG-3 cells tested with MAb NEC1 (A) or unrelated control MAb (B); SDS-PAGE under nonreducing conditions (extraction buffer containing 14 mg/ml iodoacetamide). C - E : spent media of [3H]glucosamine-le,beled SK-MG-3 cells tested with MAb NEC1 (C), unrelated control MAb (D), or anti ICN-'O (E); SDS-PAGE under reducing conditions (extraction buffer containing 12 mg;ml dithiothreiioi). F: NP40-extract of [3H]glucosamine-labe!ed SK-MG-3 cells tested with MAb B5 (antimei-CSPG proteoglycan)-'-~: SDS-PAGE under reducing conditions. Molecular mass ef immunoprecipitated components is indicated to the left and "origin" indicates top of 7.5% SDS-polyacrylamide gel. lmmunochemical procedures have been described previously~'.
318 all 5 astrocytoma lines tested and also from NP40 extracts of astrocytoma cells collected in E D T A buffer (0.02% in phosphate-buffered saline (PBS)). No antigen was precipitated from spent media or cell extracts of melanoma or neuroblastoma cell lines. These results show that NEC1 is synthesized by astrocytoma cells as a detergent-soluble, multimeric glycoprotein which is both secreted into the culture media and incorporated into the detergent-insoluble substrate attachment matrix. A proportion of human astrocytoma cell lines produce FN and proteoglycans ~9"25that can be incorporated into the detergent-insoluble substrate attachment matrix l°. Therefore, we have compared NEC1 with astrocytoma-derived FN and meI-CSPG, a specific chondroitin-sulfate proteoglycan of astrocytomas recognized by M A b B5 (ref. 25). Fig. 4 shows that NEC1 differs in molecular size from both melCSPG and FN, and Fig. 2 shows that NEC1 also differs from meI-CSPG in subcellular localization: melCSPG is detected on the surface of intact astrocytoma cells whereas NEC1 is not present on the cell surface; although meI-CSPG is also shed into the spent media 5, it is not detectably adsorbed to plastic culture surfaces (Fig. 2A, B). FN is readily adsorbed to plastic surfaces but differs from NEC1 in gelatin binding: FN is strongly bound to gelatin 12 whereas NEC1 shows no binding to a gelatin-Sepharose column (Fig. 3B). MAb NEC1 also fails to react by E L I S A with purified human plasma FN 12 (50/~g/ml in Dulbecco's PBS (DPBS), adsorbed to MicroWell plates for I h at 37 °C). Finally, our biochemical and immunohistochemical results (see below) also distinguish NEC1 from laminin (LN) and other glycoproteins secreted by astrocytoma cell lines 1"2,4.19-21. Cultured astrocytomas produce several E C M proteins that are not produced by normal glial cells in vivo 4'7"19"21,and we have used immunohistochemical methods to examine NEC1 expression in neural tissues. The results show that NEC1 is specifically expressed in white matter of the most rostral portions of the brain (Fig. 5, Table I). Strong NEC1 immunostaining was found throughout the white matter of frontal, parietal, temporal, and occipital lobes of the cerebral hemispheres (Fig. 5A, D), corpus callosum, internal capsule, basal ganglia (Fig. 5B), and diencephalon. In contrast, the more caudal portions of the brain, including most areas of mesencephaion
FABLE I Distribution of ECM glycoproteins recognized by MAb NECI in normal neural tissues determined by the avidin-biotin immunoperoxidase procedure in frozen section,~
Tissues from 5 adult individuals were obtained at autopsy, embedded in OCT compound (Tissue-TEKll, Miles. Naperville, I L ) . quick-frozen in isopentane precooled in liquid nitrogen, and stored at -70 °C until used. Multiple tissue samples (cut surface ranging from 8 x 8 mm to 10 x 15 mm) were tested for each CNS region indicated in the table and for each of the 5 individuals. Frozen 7-/~m sections were cut, air-dried, fixed in cold acetone for 10 min at 4 °C, and tested with MAb NEC1 (hybridoma culture supernatant) or unrelated MAb by the avidin-biotin immunoperoxidase method as described B. Results are indicated as follows: + + + , ++, ,, strong, moderate, and weak immunoreactivity; -, anti~z~t-negative; +/+ + +, + +/+. -/+, regional heterogeneity in NEC1 staining intensity. Nervous system subdivision
Region
NEC1 expression
Telencephalon
Cerebral hemispheres (frontal, parietal, temporal, occipital lobes) Gray matter +++ White matter +++ Corpus callosum ++/+ Olfactory bulb Basal ganglia +/+++* Globus pailidus +* Putamen
Diencephalon
Internal capsule Thalamus Hypothalamus MammiUary body
++/+ ++1+* + +/+* ++/+*
Mesencephalon
Substantia nigra Crus cerebri Medial lemniscus
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Metencephalon
Pons Cerebellum White matter Molecular layer Purkinje cell layer Granular layer Cerebellar peduncles (superior, inferior, middle) -
Myelencephalon Medulla oblongata Fiber tracts Inferior olivary nuclear complex Spinal cord
Spinal cord
PNS
Peripheral nerves Autonomic ganglia Sensory ganglia
* Heterogeneity of NECl-immunostaining generally followed the regional distribution of white (strong NEC1 staining) and gray matter (weak or no NEC1 staining) within rostral portions of the brain.
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Fig. 5. lmmunoperoxidase staining of human brain tissues with MAb NEC1 ( A - E ) or the astrocyte-specific MAb K4 (F). Hematoxylin counterstaining. A: temporal lobe, strong NEC1 staining throughout the subcortical white matter (w) (for high-power view see panel D) and in a thin superficial cortical layer (*) but not in bulk of cortical gray matter (g) or leptomeninges. B: globus pailidus, strong NEC1 staining showing ECM pattern; ceil bodies not labeled. C: mesencephalon, scant NECI staining with loose, fibrillary pattero D: subcortical white matter of temporal lobe, NEC1 staining with ECM pattern; cell bodies not labeled. E: cerebellum, granular layer (top) and white matter (bottom) NECIneg,tive. F: mesencephalon (parallel section to panel C), labeling of glial cells with the astrocyte-specific MAb K4 (unpublished data) -'7. Unrelated MAbs were routinely included as negative controls and did not produce any staining. Hematoxylin counterstaining of nuclei (blue) ns easily distinguished from immunoperoxidase staining (brown). Original magnification x5 (A), x 100 (B,C,E), x250 (D,F).
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320 (Fig. 5C), the pons. cerebellum (Fig. 5E), and medulla oblongata, as well as the spinal cord are NEC1negative (Table I). No immunostaining for NECI was detected in the peripheral nervous system, leptomeninges, and cerebral blood vessels, structures known to express FN and L N 4"19"2° At the microscopic level, immunostaining for NEC1 defines a distinctive, fine-fibrillary meshwork in CNS parenchyma rather than a cellular or fiber tract pattern (Fig. 5 A - D ) . In contrast to this characteristic ECM pattern for NEC1, parallel tissue sections tested with MAbs to cellular antigens 14"17'26"27 showed well-defined cytoplasmic or cell membrane staining as illustrated in Fig. 5F. Therefore, NEC1 can be localized to intercellular spaces in rostral portions of the CNS, consistent with the presence of NEC1 in the ECM but not on the surface of cultured glial cells (Fig. 2A). The results of our immunohistochemical analysis of neural tissues were confirmed by ELISA. Frontal, temporal, and occipital lobe white matter extracts prepared in alkaline buffer (30 mM diethylamine, 1 mM EDTA, pH 11.5) 9 contained readily detectable NEC1 antigen, whereas adjacent areas of gray matter were NECl-negative (Fig. 3C). Extracts prepared from either white or gray matter of the cerebellum were also NECl-ne~,ative. Consistent with the localization of NEC1 to the detergent-insoluble ECM in vitro, the antigen was not found in extracts of forebrain white matter prepared in NP40 buffer (0.5% NP40 in Tris buffer, pH 7.4). NEC1 defines a unique region-specific pattern of ECM protein expression in human CNS parenchyma. Chicken cytotactin, an antigen first identified in embryonic chicken brain ~8. shares ~ome characteristics with human NEC1. It is a glycoprotein composed of disulfide-linked 220-kD, 200-kDa, and 190-kDa subunits and is expressed with an ECM pattern in chicken brain tissue 18. However, Crossin et al. have described widespread distribution of cytotactin in embryonic chicken CNS, including forebrain, cerebellum, medulla, and spinal cord, and a comparable pattern of expression in the adult animal, including forebrain and cerebellum 9. NECI has a different distribution in the adult human CNS (Fig. 5A, E, Table I), being restricted to the most rostral portions of the brain (telencephalon, diencephalon, some areas of mesencephalon). Cerebellum, pons, medulla, spinal
cord, and PNS tissues do not show any NEC1 expression. Therefore. chicken cytotactin and human NEC1 appear to be non-homologous ECM proteins or they may be structural homologues whose patterns of tissue-specific expression have diverged during evolution, as previously described for the Thy-1 antigen ~7. Amino acid sequencing of NECI and cytotactin will help to resolve this question. The identity of cells secreting NEC1 in human brain tissue is unknown, although astrocytes are likely candidates. However, the distinctive topographical pattern of NEC1 expression in brain tissue does not coincide with the distribution of any known subset of glial or neuronal cells or fiber system. One possible explanation for this unioue NEC1 pattern is the existence of an as yet uniden~.;ied group of NECI-secreting cells (for instance, a subset of astrocytes) that are restricted to rostral portions of the CNS. Alternatively, cells capable of secreting NEC1 may be present throughout the CNS, but they become NEC1 producers only when induced by local differentiation factors that are restricted to rostral portions of the CNS. We suggest that NEC1 is one of a group of molecules showing segmentally restricted expression in mammalian CNS tissues. Additional members of this group are two mouse homeo box genes 3"j5 ar:d a MAb-defined antigen of rabbit brain, designated 271A6, which is restricted to gray matter of the telencephalon -'2. The putative protein products of the mouse homeo box genes and the biochemical nature and subcellular localization of the 271A6 rabbit antigen are still unknown, precluding a detailed comparison with the NEC1 glycoproteins. It has been hypothesized that development and maintenance of precise patterns of cellular connectivity in the CNS are determined by gradients of diffusible factors or by interactions between cell surface recognition molecules on neurons and glial cells 11"16"2~'3°. The rostrocaudal patterning of NEC1 expression in the human CNS may represent an alternative organizing principle in which site-restricted expression of ECM components and cell type-restricted expression of ECM-receptors play a central role. According to this hypothesis, NEC1, for which we propose the designation human neuronectin, interacts with receptors on cells of the developing and adult CNS to guide cell movement and determine cellular differentiation within molecularly unique do-
321 mains of the CNS. It is unlikely that neuronectin-negative segments of the human CNS are devoid of ECM proteins and we are now seeking evidence for additional regionspecific ECM proteins.
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We thank S. Walker and G. Lark for excellent technical help. Dr. K.O. Lloyd for anti-FN antibody. and Dr. J. Sorvillo for purified FN. Supported in part by NCI Grants CA-08748 and CA-25803 and bv the Oliver S. and Jennie R. Donaldson Charitable Trust. .
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