Journal of Neuroscience Research 29:319-325 (1991)
Human Monoclonal Antineurofilament Antibody Cross-Reacts With a Neuronal Surface Protein S.A. Sadiq, L.H. van den Berg, F.P. Thomas, K. Kilidireas, A.P. Hays, and N. Latov Departments of Neurology (S.A.S., L.H.v.d.B., F.P.T., K.K., N.L.) and Pathology, Division of Neuropathology (A.P.H.), Columbia Presbyterian Medical Center, College of Physicians and Surgeons, Columbia University, New York, New York Increased titres of anti-neurofilament antibodies have been reported in neurodegenerative disorders, and it has been suggested that such antibodies might be pathogenic. We investigated the specificity of an IgA monoclonal antibody (MAb) from a patient with amyotrophic lateral sclerosis which reacted with neurofilaments and bound to the surface of neuroblastoma cells. In Western blots, the immunoaffinity-purified IgA bound to the 220-kD, high-molecular-weightneurofilament protein (NFH) and cross-reacted with several closely migrating protein bands with apparent mobility of 62-68 kD in neuroblastoma cells and extracts of normal human spinal cord. Following crosslinking to the surface of radiolabeled neuroblastoma cells, the IgA MAb immunoprecipitated a 65kD protein, indicating that the protein was present on the cell surface and available to the antibodies for binding. Several other MAbs to NFH did not immunostain the surface of neuroblastoma cells or bind to the 65-kD protein, indicating that the protein was not a fragment of NFH. Thus, antibody binding to the 65-kD protein, possibly by cross-reacting with NFH, may have contributed to the neuronal degeneration. Key words: amyotrophic lateral sclerosis, immunoglobulin A, autoantibody, neuronal surface antigen INTRODUCTION Antineurofilament antibodies are common constituents of the human immune repertoire, but increased titres have been reported in some neurodegenerative diseases, and it has been suggested that the antibodies might be pathogenic (Gajdusek, 1985). We investigated the specificity of a monoclonal antibody (MAb) that reacted with the high-molecular-weight neurofilament protein (NFH) and immunostained the surface of intact cultured neuroblastoma cells. The MAb was obtained from a patient with amyotrophic lateral sclerosis (ALS) and patho0 1991 Wiley-Liss, Inc.
logical studies at postmortem revealed accumulations of the antibody in the cell bodies and processes of surviving spinal motor neurons (Hays et al., 1990). We now report that the antigen on the surface of neuroblastoma cells is a protein with an apparent molecular weight of -65 kD that shares antigenicity with, but is distinct from, NFH.
METHODS Serum IgA Monoclonal Antineurofilament Antibody Serum was obtained from a patient V.C. who had ALS and an IgA monoclonal gammopathy. The clinical and pathological studies of this patient were described elsewhere (Hays et al., 1990). Immunocytochemical studies at postmortem revealed accumulations of the IgA MAb in surviving motoneurons and their processes. The MAb immunostained neurons and their axons in the central and peripheral nervous systems and cross-reacted with the surface of the human neuroblastoma cell line, LAN-5. Neuroblastoma Cells Neuroblastoma cells were grown in tissue culture flasks containing DME with 15% FCS supplemented with 2 mM glutamine, 1 mM pyruvate, and 1% penicillin/streptomycin (Gibco, Grand Island, NY) at 37°C in 5% CO,. The cells were harvested by gentle shaking, washed twice in PBS (0.2 M NaCl, 0.05M NaH,PO, pH 7.4) at 4"C, and stored at -20°C until use. Neural Tissues Normal human spinal cord and peripheral nerve samples were obtained at autopsy within I2 h after death Received October 15, 1990; accepted December 3 , 1990. Address reprint requests to Dr. Saud A. Sadiq, Department of Neurology, Columbia University, Black Building Room 3-323, 630 W 168th St., New York. NY. 10032.
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from patients who died with non-neurological disease. Myelin and nonmyelin-containing neuron and axon membrane preparations were obtained by sucrose-density gradient centrifugation from spinal cord (Norton, 1974) and from peripheral nerve (Micko and Schlaepfer, 1978). The preparations were lyophilized and kept desskated at -70°C until use.
Neurofilament Preparation Neurofilaments were prepared from bovine spinal cord as previously described (Tokutake et al., 1983). The isolated neurofilaments contained the neurofilament triplet proteins, as well as actin, tubulin, and glial fibrillary acidic protein (GFAP). Preparations were stored at -70°C in 0.3 M phosphate buffer (pH 7.0) in 8 M urea until use. Dephosphorylation of Neurofilaments The neurofilament preparation was dialyzed overnight against Tris buffer containing 1 mM ZnSO,, 100 mM NaCl, and 50 mM Tris-HC1 (pH 8.0) at 4°C (Braxton et al., 1989). The filaments were dephosphorylated with 10 units of alkaline phosphatase (Sigma, St. Louis, MO) per mg of purified neurofilament protein for 18 h at 37°C (Carden et a1., 1985). After removal of the enzyme by centrifugation, the pellet was washed twice with 400 mM Na phosphate, 50 mM EDTA, and 100 mM NaCl pH 7.0, resuspended in 0.5M Tris-HC1 pH 6.8 and stored at -70°C until use. Expression of NFH as a Fusion Protein in Eschenchia coli A 2.1-kb cDNA clone containing a 1.5-kb sequence encoding 500 amino acids at the C-terminus region of rat NFH was expressed as a fusion protein with the bacterial protein trpE in E. coli HBlOl as previously described (Braxton et al., 1989). Anti-NFH Antibodies We used four MAbs to NFH: a mouse monoclonal IgM designated NF2 (Tokutake et al., 1983), a mouse IgG MAb designated NE-14 (Sigma), a mouse IgG1 MAb (Chemicon), and a rabbit monoclonal IgG NF-200 (Sigma). Each was used at dilutions of 1:500.
EDTA, 0.23 mM PMSF, and 3 mM benzamidine (Sigma) (Hoffman et al., 1987). The insoluble precipitates were then removed by centrifuging at 14,000 rpm for 15 min at room temperature and the protein concentrations determined (Lowry et al., 1951). The proteins were then separated by 10%or 12% SDS-PAGE (Laemmli, 1970). In experiments of antibody binding to neurofilament proteins, 5 pg of the neurofilament preparation was applied in each lane. In experiments of antibody binding to neuroblastoma cells, we applied 50 pg of the protein per lane. Nerve and spinal cord proteins were examined at 100 pg protein per lane. Following separation, the proteins were transferred electrophoretically onto nitrocellulose sheets (Towbin et al., 1979) and unreactive binding sites were saturated for 1 h at room temperature in a solution containing 8% BSA, 0.15 M NaCl, and 0.01 M Trizma base, pH 7.4, and nitrocellulose strips washed in washing solution containing 1% BSA, 0.05% Nonidet P-40 (NP 40), 0.15 M NaCl, and 0.01 M Trizma base, pH 7.4. For investigations of human antibody binding, the blots were incubated with patient serum, diluted 1:1,000 in washing solution overnight at 4 ° C and control strips were incubated with normal serum or with washing solution only. After washing, the strips were incubated for 2 h at 4°C with biotinylated, affinity purified F(ab) fragments of antibodies to human IgA, TgG, IgM, or K and A light chains (Sigma) diluted 1: 1,000 in washing solution. The strips were washed and incubated with avidin-biotin-peroxidase complexes (ABC method, Vector, Burlinghame, CA) for 1 h at room temperature. Reaction products were then developed in 0.025% hydrogen peroxide, 0.5% diaaminobenzidine, 0.015 M imidazole, 0.15 M NaCl, and 0.01 M Trizma base, pH 7.4. Blots were also immunostained with mouse or rabbit NFH antibodies diluted 1:500 in washing solution and counterstained with biotinylated antibodies to mouse or rabbit Ig (Sigma), diluted 1: 1,000, and developed in the ABC system.
Immunoaffinity Purification of Monoclonal IgA by Elution From NFH The monoclonal IgA was affinity purified by elution from NFH on nitrocellulose blots (Olmsted et al., 1981) as follows. Nitrocellulose strips containing the separated neurofilament proteins were incubated with the patient's serum at a dilution of 1:200, and parallel Western Blot Analysis strips were counterstained with anti-NFH antibodies to Neuroblastoma cells and the central and peripheral visualize the NFH bands. The anti-NFH monoclonal IgA nervous system myelin and nonmyelin fractions were was then eluted from the coresponding regions on the delipidated with acetone and the proteins solubilized by strips by incubation in 0.2 M glycine-HC1, pH 2.8 for 15 heating for 10 min at 64°C in 2% sodium dodecyl sulfate min and neutralized with NaOH. The eluted antibody (SDS) containing the protease inhibitors 0.7 pM pepsta- was then tested for binding to immunoblots containing tin, 1.1 p M leupeptin, 50 pg/ml trypsin inhibitor, 2 mM the neurofilament preparation, dephosphorylated neuro-
Human MAb Cross-Reacts With NFH
filaments, the NFH fusion protein, and the neuroblastoma proteins.
NFH Immunoabsorption of Patient Serum With Neurofilament Protein To absorb anti-NFH antibodies, the patient’s serum was incubated with the neurofilament preparation as follows: 1 ml of the purified bovine neurofilament preparation was dialysed against PBS overnight at 4°C; 100 p1 of patient serum diluted 1: 100 was then incubated with 100 pl of the dialysed neurofilament for 2 h at 4”C, and the neurofilaments were then removed by centrifuging at 10,000 rpm for 10 min. The procedure was repeated two more times. The absorbed serum was tested for binding to neuroblastoma proteins and to NFH by western blot analysis. Immunoprecipitation of Surface Antigen From Neuroblastoma Cells The target antigen was immunoprecipitated from the surface of radiolabeled LAN-5 neuroblastoma cells after crosslinking to the monoclonal IgA (Brenner et al., 1985; Hamada and Tsuruo, 1987) as follows. Neuroblastoma cells in tissue culture flasks were incubated in methioninedeficent, serum-free medium for 2.5 h, and 100 pCi/ml of L-[”S]methionine (1,169 Ciimmol, New England Nuclear, Wilmington, DE) was added for an additional 5 h (Salton et al., 1983). The cells were rinsed with PBS three times, and patient serum, diluted 1 5 0 0 in PBS, was added for 2 h at 4°C. After washing with PBS, the cells were suspended in crosslinking buffer (PBS containing 1 mM MgCl, and 0.2% sodium azide, pH 8.3), and the crosslinking reagent, dithiobissuccinimidylpropionate (DSP) (Pierce Chemical, Rockford, IL) 50 mM in DMSO (Sigma) was added at a final concentration of DSP of 1 mM. The cells were kept at room temperature for 1 h, washed once in quenching buffer (100 mM Tris-HC1 pH 8 .0 and 140 mM NaC1) , and then solubilized in buffer A (20mMTris-HClpH 8.0, 140mMNaC1,O.l mMPMSF) containing 1% NP-40 for 30 min at 4°C. After centrifugation at 10,000 rpm for 20 min, to remove the insoluble matter, the supernatant was added to 200 p l ( 5 mg/ml) of immunobead rabbit antihuman IgA (BioRad, CA) and incubated with constant mixing at 4°C for 30 min. The immunoprecipitates were washed four times with 1% BSA containing 0.2% NP-40 in PBS and twice with PBS containing 0.2% NP-40, and the bound antigen released from the immunobead-antibody-antigen complexes by suspension in 100 pl of Laemmli sample buffer (Laemmli, 1970) containing 5% 2-mercaptoethanol and boiling for 5 min. The radiolabeled and immunoprecipitated surface antigen was separated by SDS-PAGE, transferred to nitrocellulose, and its mobility determined by exposure to Kodak X-OMAT film overnight at -70°C.
-
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- 9 2.5 -69 -46
-30
-21.5
- 14.3 A
B
Fig. 1. Immunoglobulin isotype staining of bovine neurofilaments. Blots were incubated with serum from patient V.C. and the bound immunoglobulin detected with affinity-purified biotinylated antibodies to (A) IgA and (B) A light chains. There is strong staining of IgA A antibodies to the 200-kD highmolecular-weight neurofilament protein (arrow). No staining was observed with IgG, IgM, or K light chains (not shown).
RESULTS IgA Binding to NFH When tested for binding to neurofilaments on Western blots, the serum IgA bound to the 200-kD, highmolecular-weight neurofilament protein (NFH) . Binding was specific for IgA and for h light chains, the same heavy and light chain type as the serum paraprotein (Fig. 1). No binding to NFH was seen with the patient’s IgM, IgG, or K light chains. IgA Binding to Neurobiastoma Proteins In Western blots, the serum IgA bound to several proteins in neuroblastoma cells. To detennine whether the observed binding was due to the monoclonal IgA, the IgA was immunoaffinity-purified by elution from NFH on nitrocellulose blots and tested for binding to the neuroblastoma proteins. The eluted IgA bound strongly to several closely migrating neuroblastoma protein bands with apparent mobilities of 62-68 kD; the most prominently staining band was found at 65 kD. The antibody also bound to dephosphorylated NFH and to a fragment of NFH expressed as a fusion protein in E . coli, suggest-
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kD
200 92.5
+
69-
65kD+
30 -+
21.5
--t
a
b
C
d
Fig. 2. IgA binding to neuroblastoma and neurofilament proteins. Affinity-purified IgA eluted from the NFH band, bound several proteins in neuroblastoma cells with the most prominent migrating 62-68 kD (a). The IgA also bound to the 200kD NFH (b), to the 165-kD dephosphorylated NFH (c), and to the 100-kD NFH fragment expressed as a fusion protein in E . coli (d).
ing that the IgA recognized an unmodified peptide epitope (Fig. 2). Preabsorption of serum with the neurofilament preparation greatly diminished binding to the neuroblastoma proteins, confirming that the same antibody bound to both antigens (Fig. 3 ) . Four mouse and rabbit monoclonal antibodies to NFH did not cross-react with the 65kD neuroblastoma protein, indicating that it was not a breakdown fragment of NFH (Fig. 4).
Immunoprecipitation of the 65-kD Protein From the Surface of Neuroblastoma Cells by Monoclonal IgA Following crosslinking to the surface of radiolabeled neuroblastoma cells, the antigen-antibody complex was solubilized and immunoprecipitated, and the radiolabeled antigen identified by autoradiography after separation by SDS-PAGE. The immunoprecipitated antigen migrated with an apparent molecular weight of -65 kD (Fig. 5 ) and was immunostained by the patient’s serum IgA, indicating that the 65-kD protein (previously identified by Western blot) was present on the cell sur-
a
b
Fig. 3. Binding of serum IgA to neuroblastoma proteins on Western blots prior to and following absorption with neurofilaments. Binding of IgA to neuroblastoma proteins (a) was greatly diminished by preabsorbing the antibody with the neurofilament protein preparation (b). face and available to the IgA autoantibody for binding. Control sera did not immunoprecipitate the 65-kD Ag .
Presence of the 65-kD Protein in Human Spinal Cord By Western blot analysis, the serum IgA bound to protein bands of -65 kD in the nonmyelin (axonal and neuronal) membrane fractions of human spinal cord that comigrated with the neuroblastoma antigen (Fig. 6). The 65-kD band was not present in the myelin fractions of human peripheral nerve or spinal cord or in the peripheral nerve nonmyelin preparation (Fig. 6). Binding was specific for IgA and A light chains, the same as the monoclonal IgA light chain type. DISCUSSION These studies indicate that the monoclonal IgA in this patient with ALS recognized a protein on the surface
Human MAb Cross-Reacts With NFH
I
2
323
3
Fig. 4. Binding of an anti-NFH monoclonal antibody to neuroblastoma proteins. Serum IgA from patient V.C. bound most prominently to a 65-kD protein in neuroblastoma cells (la) and to NFH in the neurofilament preparation (lb). A mouse monoclonal IgM antibody to NFH (NF2) bound to several proteins in neuroblastoma cells (2a) and the neurofilament preparation (2b) but not to the 65-kD neuroblastoma protein. As control, biotinylated antimouse IgM antibody did not bind to the 65-kD protein (3a) or to NFH (3b). Three other commercially available monoclonal anti-NFH antibodies similarly did not bind the 65-kD protein.
Fig. 5. Immunoprecipitation of neuroblastoma cell surface proteins. Autoradiograph showing the mobility of the cell surface protein following immunoprecipitation from the radiolabeled neuroblastoma cells by the monoclonal IgA. The radiolabeled antigen is visualized at approximately 65 kD (b, arrow). The protein was not immunoprecipitated when control serum was substituted for the IgA (a).
of human neuroblastoma cells and in normal human spinal cord. The protein migrated with an apparent mobility of -65 kD and was available to the autoantibodies for binding. The antibody also cross-reacted with the 200kD high-molecular-weight neurofilament protein, and it probably recognized an unmodified peptide epitope in both NFH and the 65-kD protein because it bound to dephosphorylated NFH and to NFH expressed as a fusion protein in E. coli. Whether the 65-kD protein is present on the surface of neurons in vivo or is specific to motor neurons is not known; this could not be determined by immunocytochemical studies because binding of the IgA to the 65-kD protein could not be distinguished from binding to NFH. Availability of antibodies specific to the 65-kD protein would make these studies feasible. The monoclonal IgA that accumulated in motor neurons could have contributed to the pathogenesis of
ALS in this patient. Although serum antibodies may be taken up passively, antibodies to neuronal components such as the monoclonal IgA may also be taken up preferentially, possibly by receptor-mediated endocytosis (Fabian, 1988). Once internalized, they could bind to NFH and disrupt cellular functions. Similarly, antibodies to dopamine (3-hydroxylase have been shown to be taken up by sympathetic neurons and to cause neuronal degeneration (Fillenz et al., 1976; Zeigler et al., 1976; Furness et al., 1977), and uptake of myeloma proteins has been implicated in a case of neuropathy and multiple myeloma (Burges and Busis, 1985). In addition, antibody binding to the neuronal cell surface might interfere with intercellular interactions or receptor functions. In preliminary studies, the monoclonal IgA inhibited the proliferation of cultured neuroblastoma cells as measured by uptake of radiolabeled thymidine (Apostolski et al., 1990), indi-
a
b
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kD
* 200 +
92.5
that were similar to those found in this patient (Engelhardt et al., 1989). Further characterization and investigation of the 65-kD protein may reveal whether it has a role in neuronal degeneration.
4-69 65 k D +
ACKNOWLEDGMENTS
4-46
+
30
+21.5
This work was supported by a grant from the Aaron Diamond Foundation and by center grants from the Muscular Dystrophy Association and NINCDS (NS 11766) to Columbia University. Dr. Sadiq and Dr. Thomas are research fellows of the Muscular Dystrophy Association and the Dana Foundation and Dr. Kilidireas is supported from the State Scholarship Foundation of Greece. We would like to express our appreciation to Dr. Lewis P. Rowland for his support and for critically reviewing this manuscript.
* 14.3
a b c d e 1
a b c d e 2
Fig. 6. Western blot analysis of binding o f patient serum IgA to spinal cord proteins. Serum IgA was tested for binding to protein extracts from (la) spinal cord nonmyelin membrane fraction, (lb) spinal cord myelin, (lc) peripheral nerve nonmyelin niembrane fraction, (Id) peripheral nerve myelin, and (le) neuroblastoma. The 65-kD protein was immunostained in l a and l e . In panel 2, as control, biotinylated antibody to the IgA alone did not stain the two bands.
cating that the antibody could have had a physiological effect. Anti-NFH antibodies are present in serum from normal patients (Braxton et al., 1989; Stefannson et a]., 1985) but seem to be found more frequently and in higher concentrations in patients with neurodegenerative diseases and in aging (Bahmanyar et al., 1983; Kurki et al., 1986; Toh et al., 1985; Elizan et al., 1983) and MAbs to intermediate filaments have previously been reported in patients with neuropathy (Dellagi et al., 1982). Most antineurofilament antibodies are probably not pathogenic, but if a subset of the antibodies crossreact with surface antigens or are taken up by neurons, they might cause clinical disease. Similar cross-reactivity with surface antigens has been implicated in the pathogenesis of anti-DNA antibodies in systemic lupus erythematosus (Raz et al., 1989). Immunoglobulin deposits have been reported in motor neurons of patients with ALS (Engelhardt and Appel, 1990) and immunization with a motor neuron preparation caused an experimental motor neuron disease with intraneuronal accumulations of immunoglobulins
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port of neurofilament as a common pathogenetic mechanism in certain diseases of the central nervous system. N Engl J Med 3 12:714-7 19. 2. Hays AP, Roxas A, Sadiq S , Vallejos H, D’Agati V, Thomas F, Torres R , Sherman W, Braxton D, Hays A, Rowland L, Latov N ( 1 990): A monoclonal IgA lambda in motor neuron disease reacts with neurofilaments and the surface of neuroblastoma cells. J Neuropathol Exp Neurol 49:383-398. 3. Norton WT (1974): Isolation of myelin from nerve tissue. Methods Enzymol 32:435-444. 4. Micko S, Schlaepfer WW (1978): Protein composition of axons and myelin from rat and human peripheral nerve. J Neurochem 30: 1041-1049. 5 . Tokutake S , Hutchison SB, Pachter JS, Leim RK (1983): A batchwise purification procedure of neurofilament proteins. Anal Biochem 135:102-1 05. 6. Braxton DM, Williams M, Kamali D, Chin S , Liem R, Latov N (1989): Specificity of human anti-neurofilament autoantibodies. J Neuroimmunol 2 1: I93 -203. 7. Carden MJ, Schlaepfer WW, Lee VMY (1985): The structure, biochemical properties and imrnunogenieity of neurofilament peripheral regions are determined by phosphorylation state. J Biol Chem 260:9803-9817. 8. Hoffman EP, Brown RH, Kunkel LM (1987): Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell 5 11919-928. 9. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951): Protein measurement with the Fohn phenol reagent. J Biol Chem 193: 265 -275. 10. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. 11. Towbin H, Stachelin T, Gordon J (1979): Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:43504354. 12. Olmsted JB (1981): Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Bio. Chem 256: 11955-1 1957. 13. Brenner MB, Trowbridge IS, Strominger JL (1985): Crosslinking
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Schlaepfer WW, Helgason CM (1985): Circulatory autoantibodies to the 200,000 Dalton protein of neurofilaments in the serum of healthy individuals. Science 228:1117-1 119. Bahmanyar S , Moreau-Dubois MC, Brown P, Cathala F, Gajdusek DC (1983): Serum antibodies to neurofilament antigens in patients with neurological and other diseases and in healthy controls. J Neuroimmunol 5:165-172. Kurki P, Helve T, Dahl D, Virtanew I (1986): Neurofilament antibodies in systemic lupus erythematosis. J Rheumatol 13:6973. Toh BH, Gibbs CJ, Gajdusek DC, Goudsmit J , Dahl D (1985): The 200- and 150-dK neurofilament proteins react with IgG autoantibodies from patients with Kuru, Crentzfeldt-Jakob disease, and other neurologic diseases. Proc Natl Acad Sci USA 82:34853489. Elizan TS, Casals J, Yahr MD (1983): Auto-neurofilarnent antibodies in postencephalitic and idiopathic Parkinson’s disease. J Neurol Sci 59:341-347. Dellagi K, Brovet JC, Perreau J, Paulin D (1982): Human monoclonal IgM with autoantibody activity against intermediate filaments. Proc Natl Acad Sci USA 79:446-450. Raz E, Braegis M, Rosenmann E, Eilat D (1989): Anti-DNA antibodies bind directly to renal antigens and induce kidney dysfunction in the isolated perfused rat kidney. J Immunol 142: 3076-3082. Engelhardt JI, Appel SH (1990): IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis. Arch Neurol 47: 12 1 0- I 2 16. Engelhardt JI, Appel SH, Killian JM (1989): Experimental autoimmune motoneuron disease. Ann Neurol 26:368-376.
Human Monoclonal Antineurofilament Antibody Cross-Reacts With a Neuronal Surface Protein S.A. Sadiq, L.H. van den Berg, F.P. Thomas, K. Kilidireas, A.P. Hays, and N. Latov Departments of Neurology (S.A.S., L.H.v.d.B., F.P.T., K.K., N.L.) and Pathology, Division of Neuropathology (A.P.H.), Columbia Presbyterian Medical Center, College of Physicians and Surgeons, Columbia University, New York, New York Increased titres of anti-neurofilament antibodies have been reported in neurodegenerative disorders, and it has been suggested that such antibodies might be pathogenic. We investigated the specificity of an IgA monoclonal antibody (MAb) from a patient with amyotrophic lateral sclerosis which reacted with neurofilaments and bound to the surface of neuroblastoma cells. In Western blots, the immunoaffinity-purified IgA bound to the 220-kD, high-molecular-weightneurofilament protein (NFH) and cross-reacted with several closely migrating protein bands with apparent mobility of 62-68 kD in neuroblastoma cells and extracts of normal human spinal cord. Following crosslinking to the surface of radiolabeled neuroblastoma cells, the IgA MAb immunoprecipitated a 65kD protein, indicating that the protein was present on the cell surface and available to the antibodies for binding. Several other MAbs to NFH did not immunostain the surface of neuroblastoma cells or bind to the 65-kD protein, indicating that the protein was not a fragment of NFH. Thus, antibody binding to the 65-kD protein, possibly by cross-reacting with NFH, may have contributed to the neuronal degeneration. Key words: amyotrophic lateral sclerosis, immunoglobulin A, autoantibody, neuronal surface antigen INTRODUCTION Antineurofilament antibodies are common constituents of the human immune repertoire, but increased titres have been reported in some neurodegenerative diseases, and it has been suggested that the antibodies might be pathogenic (Gajdusek, 1985). We investigated the specificity of a monoclonal antibody (MAb) that reacted with the high-molecular-weight neurofilament protein (NFH) and immunostained the surface of intact cultured neuroblastoma cells. The MAb was obtained from a patient with amyotrophic lateral sclerosis (ALS) and patho0 1991 Wiley-Liss, Inc.
logical studies at postmortem revealed accumulations of the antibody in the cell bodies and processes of surviving spinal motor neurons (Hays et al., 1990). We now report that the antigen on the surface of neuroblastoma cells is a protein with an apparent molecular weight of -65 kD that shares antigenicity with, but is distinct from, NFH.
METHODS Serum IgA Monoclonal Antineurofilament Antibody Serum was obtained from a patient V.C. who had ALS and an IgA monoclonal gammopathy. The clinical and pathological studies of this patient were described elsewhere (Hays et al., 1990). Immunocytochemical studies at postmortem revealed accumulations of the IgA MAb in surviving motoneurons and their processes. The MAb immunostained neurons and their axons in the central and peripheral nervous systems and cross-reacted with the surface of the human neuroblastoma cell line, LAN-5. Neuroblastoma Cells Neuroblastoma cells were grown in tissue culture flasks containing DME with 15% FCS supplemented with 2 mM glutamine, 1 mM pyruvate, and 1% penicillin/streptomycin (Gibco, Grand Island, NY) at 37°C in 5% CO,. The cells were harvested by gentle shaking, washed twice in PBS (0.2 M NaCl, 0.05M NaH,PO, pH 7.4) at 4"C, and stored at -20°C until use. Neural Tissues Normal human spinal cord and peripheral nerve samples were obtained at autopsy within I2 h after death Received October 15, 1990; accepted December 3 , 1990. Address reprint requests to Dr. Saud A. Sadiq, Department of Neurology, Columbia University, Black Building Room 3-323, 630 W 168th St., New York. NY. 10032.
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from patients who died with non-neurological disease. Myelin and nonmyelin-containing neuron and axon membrane preparations were obtained by sucrose-density gradient centrifugation from spinal cord (Norton, 1974) and from peripheral nerve (Micko and Schlaepfer, 1978). The preparations were lyophilized and kept desskated at -70°C until use.
Neurofilament Preparation Neurofilaments were prepared from bovine spinal cord as previously described (Tokutake et al., 1983). The isolated neurofilaments contained the neurofilament triplet proteins, as well as actin, tubulin, and glial fibrillary acidic protein (GFAP). Preparations were stored at -70°C in 0.3 M phosphate buffer (pH 7.0) in 8 M urea until use. Dephosphorylation of Neurofilaments The neurofilament preparation was dialyzed overnight against Tris buffer containing 1 mM ZnSO,, 100 mM NaCl, and 50 mM Tris-HC1 (pH 8.0) at 4°C (Braxton et al., 1989). The filaments were dephosphorylated with 10 units of alkaline phosphatase (Sigma, St. Louis, MO) per mg of purified neurofilament protein for 18 h at 37°C (Carden et a1., 1985). After removal of the enzyme by centrifugation, the pellet was washed twice with 400 mM Na phosphate, 50 mM EDTA, and 100 mM NaCl pH 7.0, resuspended in 0.5M Tris-HC1 pH 6.8 and stored at -70°C until use. Expression of NFH as a Fusion Protein in Eschenchia coli A 2.1-kb cDNA clone containing a 1.5-kb sequence encoding 500 amino acids at the C-terminus region of rat NFH was expressed as a fusion protein with the bacterial protein trpE in E. coli HBlOl as previously described (Braxton et al., 1989). Anti-NFH Antibodies We used four MAbs to NFH: a mouse monoclonal IgM designated NF2 (Tokutake et al., 1983), a mouse IgG MAb designated NE-14 (Sigma), a mouse IgG1 MAb (Chemicon), and a rabbit monoclonal IgG NF-200 (Sigma). Each was used at dilutions of 1:500.
EDTA, 0.23 mM PMSF, and 3 mM benzamidine (Sigma) (Hoffman et al., 1987). The insoluble precipitates were then removed by centrifuging at 14,000 rpm for 15 min at room temperature and the protein concentrations determined (Lowry et al., 1951). The proteins were then separated by 10%or 12% SDS-PAGE (Laemmli, 1970). In experiments of antibody binding to neurofilament proteins, 5 pg of the neurofilament preparation was applied in each lane. In experiments of antibody binding to neuroblastoma cells, we applied 50 pg of the protein per lane. Nerve and spinal cord proteins were examined at 100 pg protein per lane. Following separation, the proteins were transferred electrophoretically onto nitrocellulose sheets (Towbin et al., 1979) and unreactive binding sites were saturated for 1 h at room temperature in a solution containing 8% BSA, 0.15 M NaCl, and 0.01 M Trizma base, pH 7.4, and nitrocellulose strips washed in washing solution containing 1% BSA, 0.05% Nonidet P-40 (NP 40), 0.15 M NaCl, and 0.01 M Trizma base, pH 7.4. For investigations of human antibody binding, the blots were incubated with patient serum, diluted 1:1,000 in washing solution overnight at 4 ° C and control strips were incubated with normal serum or with washing solution only. After washing, the strips were incubated for 2 h at 4°C with biotinylated, affinity purified F(ab) fragments of antibodies to human IgA, TgG, IgM, or K and A light chains (Sigma) diluted 1: 1,000 in washing solution. The strips were washed and incubated with avidin-biotin-peroxidase complexes (ABC method, Vector, Burlinghame, CA) for 1 h at room temperature. Reaction products were then developed in 0.025% hydrogen peroxide, 0.5% diaaminobenzidine, 0.015 M imidazole, 0.15 M NaCl, and 0.01 M Trizma base, pH 7.4. Blots were also immunostained with mouse or rabbit NFH antibodies diluted 1:500 in washing solution and counterstained with biotinylated antibodies to mouse or rabbit Ig (Sigma), diluted 1: 1,000, and developed in the ABC system.
Immunoaffinity Purification of Monoclonal IgA by Elution From NFH The monoclonal IgA was affinity purified by elution from NFH on nitrocellulose blots (Olmsted et al., 1981) as follows. Nitrocellulose strips containing the separated neurofilament proteins were incubated with the patient's serum at a dilution of 1:200, and parallel Western Blot Analysis strips were counterstained with anti-NFH antibodies to Neuroblastoma cells and the central and peripheral visualize the NFH bands. The anti-NFH monoclonal IgA nervous system myelin and nonmyelin fractions were was then eluted from the coresponding regions on the delipidated with acetone and the proteins solubilized by strips by incubation in 0.2 M glycine-HC1, pH 2.8 for 15 heating for 10 min at 64°C in 2% sodium dodecyl sulfate min and neutralized with NaOH. The eluted antibody (SDS) containing the protease inhibitors 0.7 pM pepsta- was then tested for binding to immunoblots containing tin, 1.1 p M leupeptin, 50 pg/ml trypsin inhibitor, 2 mM the neurofilament preparation, dephosphorylated neuro-
Human MAb Cross-Reacts With NFH
filaments, the NFH fusion protein, and the neuroblastoma proteins.
NFH Immunoabsorption of Patient Serum With Neurofilament Protein To absorb anti-NFH antibodies, the patient’s serum was incubated with the neurofilament preparation as follows: 1 ml of the purified bovine neurofilament preparation was dialysed against PBS overnight at 4°C; 100 p1 of patient serum diluted 1: 100 was then incubated with 100 pl of the dialysed neurofilament for 2 h at 4”C, and the neurofilaments were then removed by centrifuging at 10,000 rpm for 10 min. The procedure was repeated two more times. The absorbed serum was tested for binding to neuroblastoma proteins and to NFH by western blot analysis. Immunoprecipitation of Surface Antigen From Neuroblastoma Cells The target antigen was immunoprecipitated from the surface of radiolabeled LAN-5 neuroblastoma cells after crosslinking to the monoclonal IgA (Brenner et al., 1985; Hamada and Tsuruo, 1987) as follows. Neuroblastoma cells in tissue culture flasks were incubated in methioninedeficent, serum-free medium for 2.5 h, and 100 pCi/ml of L-[”S]methionine (1,169 Ciimmol, New England Nuclear, Wilmington, DE) was added for an additional 5 h (Salton et al., 1983). The cells were rinsed with PBS three times, and patient serum, diluted 1 5 0 0 in PBS, was added for 2 h at 4°C. After washing with PBS, the cells were suspended in crosslinking buffer (PBS containing 1 mM MgCl, and 0.2% sodium azide, pH 8.3), and the crosslinking reagent, dithiobissuccinimidylpropionate (DSP) (Pierce Chemical, Rockford, IL) 50 mM in DMSO (Sigma) was added at a final concentration of DSP of 1 mM. The cells were kept at room temperature for 1 h, washed once in quenching buffer (100 mM Tris-HC1 pH 8 .0 and 140 mM NaC1) , and then solubilized in buffer A (20mMTris-HClpH 8.0, 140mMNaC1,O.l mMPMSF) containing 1% NP-40 for 30 min at 4°C. After centrifugation at 10,000 rpm for 20 min, to remove the insoluble matter, the supernatant was added to 200 p l ( 5 mg/ml) of immunobead rabbit antihuman IgA (BioRad, CA) and incubated with constant mixing at 4°C for 30 min. The immunoprecipitates were washed four times with 1% BSA containing 0.2% NP-40 in PBS and twice with PBS containing 0.2% NP-40, and the bound antigen released from the immunobead-antibody-antigen complexes by suspension in 100 pl of Laemmli sample buffer (Laemmli, 1970) containing 5% 2-mercaptoethanol and boiling for 5 min. The radiolabeled and immunoprecipitated surface antigen was separated by SDS-PAGE, transferred to nitrocellulose, and its mobility determined by exposure to Kodak X-OMAT film overnight at -70°C.
-
321
kD
-200
- 9 2.5 -69 -46
-30
-21.5
- 14.3 A
B
Fig. 1. Immunoglobulin isotype staining of bovine neurofilaments. Blots were incubated with serum from patient V.C. and the bound immunoglobulin detected with affinity-purified biotinylated antibodies to (A) IgA and (B) A light chains. There is strong staining of IgA A antibodies to the 200-kD highmolecular-weight neurofilament protein (arrow). No staining was observed with IgG, IgM, or K light chains (not shown).
RESULTS IgA Binding to NFH When tested for binding to neurofilaments on Western blots, the serum IgA bound to the 200-kD, highmolecular-weight neurofilament protein (NFH) . Binding was specific for IgA and for h light chains, the same heavy and light chain type as the serum paraprotein (Fig. 1). No binding to NFH was seen with the patient’s IgM, IgG, or K light chains. IgA Binding to Neurobiastoma Proteins In Western blots, the serum IgA bound to several proteins in neuroblastoma cells. To detennine whether the observed binding was due to the monoclonal IgA, the IgA was immunoaffinity-purified by elution from NFH on nitrocellulose blots and tested for binding to the neuroblastoma proteins. The eluted IgA bound strongly to several closely migrating neuroblastoma protein bands with apparent mobilities of 62-68 kD; the most prominently staining band was found at 65 kD. The antibody also bound to dephosphorylated NFH and to a fragment of NFH expressed as a fusion protein in E . coli, suggest-
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kD
200 92.5
+
69-
65kD+
30 -+
21.5
--t
a
b
C
d
Fig. 2. IgA binding to neuroblastoma and neurofilament proteins. Affinity-purified IgA eluted from the NFH band, bound several proteins in neuroblastoma cells with the most prominent migrating 62-68 kD (a). The IgA also bound to the 200kD NFH (b), to the 165-kD dephosphorylated NFH (c), and to the 100-kD NFH fragment expressed as a fusion protein in E . coli (d).
ing that the IgA recognized an unmodified peptide epitope (Fig. 2). Preabsorption of serum with the neurofilament preparation greatly diminished binding to the neuroblastoma proteins, confirming that the same antibody bound to both antigens (Fig. 3 ) . Four mouse and rabbit monoclonal antibodies to NFH did not cross-react with the 65kD neuroblastoma protein, indicating that it was not a breakdown fragment of NFH (Fig. 4).
Immunoprecipitation of the 65-kD Protein From the Surface of Neuroblastoma Cells by Monoclonal IgA Following crosslinking to the surface of radiolabeled neuroblastoma cells, the antigen-antibody complex was solubilized and immunoprecipitated, and the radiolabeled antigen identified by autoradiography after separation by SDS-PAGE. The immunoprecipitated antigen migrated with an apparent molecular weight of -65 kD (Fig. 5 ) and was immunostained by the patient’s serum IgA, indicating that the 65-kD protein (previously identified by Western blot) was present on the cell sur-
a
b
Fig. 3. Binding of serum IgA to neuroblastoma proteins on Western blots prior to and following absorption with neurofilaments. Binding of IgA to neuroblastoma proteins (a) was greatly diminished by preabsorbing the antibody with the neurofilament protein preparation (b). face and available to the IgA autoantibody for binding. Control sera did not immunoprecipitate the 65-kD Ag .
Presence of the 65-kD Protein in Human Spinal Cord By Western blot analysis, the serum IgA bound to protein bands of -65 kD in the nonmyelin (axonal and neuronal) membrane fractions of human spinal cord that comigrated with the neuroblastoma antigen (Fig. 6). The 65-kD band was not present in the myelin fractions of human peripheral nerve or spinal cord or in the peripheral nerve nonmyelin preparation (Fig. 6). Binding was specific for IgA and A light chains, the same as the monoclonal IgA light chain type. DISCUSSION These studies indicate that the monoclonal IgA in this patient with ALS recognized a protein on the surface
Human MAb Cross-Reacts With NFH
I
2
323
3
Fig. 4. Binding of an anti-NFH monoclonal antibody to neuroblastoma proteins. Serum IgA from patient V.C. bound most prominently to a 65-kD protein in neuroblastoma cells (la) and to NFH in the neurofilament preparation (lb). A mouse monoclonal IgM antibody to NFH (NF2) bound to several proteins in neuroblastoma cells (2a) and the neurofilament preparation (2b) but not to the 65-kD neuroblastoma protein. As control, biotinylated antimouse IgM antibody did not bind to the 65-kD protein (3a) or to NFH (3b). Three other commercially available monoclonal anti-NFH antibodies similarly did not bind the 65-kD protein.
Fig. 5. Immunoprecipitation of neuroblastoma cell surface proteins. Autoradiograph showing the mobility of the cell surface protein following immunoprecipitation from the radiolabeled neuroblastoma cells by the monoclonal IgA. The radiolabeled antigen is visualized at approximately 65 kD (b, arrow). The protein was not immunoprecipitated when control serum was substituted for the IgA (a).
of human neuroblastoma cells and in normal human spinal cord. The protein migrated with an apparent mobility of -65 kD and was available to the autoantibodies for binding. The antibody also cross-reacted with the 200kD high-molecular-weight neurofilament protein, and it probably recognized an unmodified peptide epitope in both NFH and the 65-kD protein because it bound to dephosphorylated NFH and to NFH expressed as a fusion protein in E. coli. Whether the 65-kD protein is present on the surface of neurons in vivo or is specific to motor neurons is not known; this could not be determined by immunocytochemical studies because binding of the IgA to the 65-kD protein could not be distinguished from binding to NFH. Availability of antibodies specific to the 65-kD protein would make these studies feasible. The monoclonal IgA that accumulated in motor neurons could have contributed to the pathogenesis of
ALS in this patient. Although serum antibodies may be taken up passively, antibodies to neuronal components such as the monoclonal IgA may also be taken up preferentially, possibly by receptor-mediated endocytosis (Fabian, 1988). Once internalized, they could bind to NFH and disrupt cellular functions. Similarly, antibodies to dopamine (3-hydroxylase have been shown to be taken up by sympathetic neurons and to cause neuronal degeneration (Fillenz et al., 1976; Zeigler et al., 1976; Furness et al., 1977), and uptake of myeloma proteins has been implicated in a case of neuropathy and multiple myeloma (Burges and Busis, 1985). In addition, antibody binding to the neuronal cell surface might interfere with intercellular interactions or receptor functions. In preliminary studies, the monoclonal IgA inhibited the proliferation of cultured neuroblastoma cells as measured by uptake of radiolabeled thymidine (Apostolski et al., 1990), indi-
a
b
324
Sadiq et al.
kD
* 200 +
92.5
that were similar to those found in this patient (Engelhardt et al., 1989). Further characterization and investigation of the 65-kD protein may reveal whether it has a role in neuronal degeneration.
4-69 65 k D +
ACKNOWLEDGMENTS
4-46
+
30
+21.5
This work was supported by a grant from the Aaron Diamond Foundation and by center grants from the Muscular Dystrophy Association and NINCDS (NS 11766) to Columbia University. Dr. Sadiq and Dr. Thomas are research fellows of the Muscular Dystrophy Association and the Dana Foundation and Dr. Kilidireas is supported from the State Scholarship Foundation of Greece. We would like to express our appreciation to Dr. Lewis P. Rowland for his support and for critically reviewing this manuscript.
* 14.3
a b c d e 1
a b c d e 2
Fig. 6. Western blot analysis of binding o f patient serum IgA to spinal cord proteins. Serum IgA was tested for binding to protein extracts from (la) spinal cord nonmyelin membrane fraction, (lb) spinal cord myelin, (lc) peripheral nerve nonmyelin niembrane fraction, (Id) peripheral nerve myelin, and (le) neuroblastoma. The 65-kD protein was immunostained in l a and l e . In panel 2, as control, biotinylated antibody to the IgA alone did not stain the two bands.
cating that the antibody could have had a physiological effect. Anti-NFH antibodies are present in serum from normal patients (Braxton et al., 1989; Stefannson et a]., 1985) but seem to be found more frequently and in higher concentrations in patients with neurodegenerative diseases and in aging (Bahmanyar et al., 1983; Kurki et al., 1986; Toh et al., 1985; Elizan et al., 1983) and MAbs to intermediate filaments have previously been reported in patients with neuropathy (Dellagi et al., 1982). Most antineurofilament antibodies are probably not pathogenic, but if a subset of the antibodies crossreact with surface antigens or are taken up by neurons, they might cause clinical disease. Similar cross-reactivity with surface antigens has been implicated in the pathogenesis of anti-DNA antibodies in systemic lupus erythematosus (Raz et al., 1989). Immunoglobulin deposits have been reported in motor neurons of patients with ALS (Engelhardt and Appel, 1990) and immunization with a motor neuron preparation caused an experimental motor neuron disease with intraneuronal accumulations of immunoglobulins
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