Neuronal uptake of anti-Hu antibody, but not anti-Ri antibody, leads to cell death in brain slice cultures

Journal of Neuroinflammation

Noel G Carlson²  Troy D Jaskowski⁴  Ikuo Tsunoda¹  Stacey L Clardy⁵  Blair Wood⁵  Kenneth E Hill⁵  Susan A Clawson⁵  John E Greenlee³ 

[1] Department of Microbiology and Immunology, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport 71130, LA, USA;Center on Aging, University of Utah, 10 South 2000 East, Salt Lake City 84112-5880, UT, USA;Brain Institute, University of Utah, 383 Colorow Drive, Salt Lake City 84108, UT, USA;Institute for Clinical and Experimental Pathology, ARUP, 500 Chipeta Way, Salt Lake City 84108, UT, USA;Department of Neurology, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City 84132, UT, USA

关键词: ANNA-2;    Hu antigens ANNA-1;    Apoptosis;    Cell death;    Organotypic brain cultures;    Brain slice cultures;    Neurons;    Anti-Ri antibody;    Anti-Hu antibody;    Paraneoplastic syndromes;   

Others  :  1228267
DOI  :  10.1186/s12974-014-0160-0

 received in 2014-07-01, accepted in 2014-08-23,  发布年份 2014

【 摘 要 】

Background

Anti-Hu and anti-Ri antibodies are paraneoplastic immunoglobulin (Ig)G autoantibodies which recognize cytoplasmic and nuclear antigens present in all neurons. Although both antibodies produce similar immunohistological labeling, they recognize different neuronal proteins. Both antibodies are associated with syndromes of central nervous system dysfunction. However, the neurological deficits associated with anti-Hu antibody are associated with neuronal death and are usually irreversible, whereas neurological deficits in patients with anti-Ri antibody may diminish following tumor removal or immunosuppression.

Methods

To study the effect of anti-Hu and anti-Ri antibodies on neurons, we incubated rat hippocampal and cerebellar slice cultures with anti-Hu or anti-Ri sera from multiple patients. Cultures were evaluated in real time for neuronal antibody uptake and during prolonged incubation for neuronal death. To test the specificity of anti-Hu antibody cytotoxic effect, anti-Hu serum IgG was incubated with rat brain slice cultures prior to and after adsorption with its target Hu antigen, HuD.

Results

We demonstrated that: 1) both anti-Hu and anti-Ri antibodies were rapidly taken up by neurons throughout both cerebellum and hippocampus; 2) antibody uptake occurred in living neurons and was not an artifact of antibody diffusion into dead cells; 3) intracellular binding of anti-Hu antibody produced neuronal cell death, whereas uptake of anti-Ri antibody did not affect cell viability during the period of study; and 4) adsorption of anti-Hu antisera against HuD greatly reduced intraneuronal IgG accumulation and abolished cytotoxicity, confirming specificity of antibody-mediated neuronal death.

Conclusions

Both anti-Hu and anti-Ri antibodies were readily taken up by viable neurons in slice cultures, but the two antibodies differed markedly in terms of their effects on neuronal viability. The ability of anti-Hu antibodies to cause neuronal death could account for the irreversible nature of paraneoplastic neurological deficits in patients with this antibody response. Our results raise questions as to whether anti-Ri antibody might initially induce reversible neuronal dysfunction, rather than causing cell death. The ability of IgG antibodies to access and react with intracellular neuronal proteins could have implications for other autoimmune diseases involving the central nervous system.

【 授权许可】

   
2014 Greenlee et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 8.

Figure 9.

【 参考文献 】

• [1]Dalmau J, Graus F, Rosenblum MK, Posner JB: Anti-Hu associated paraneoplastic encephalomyelitis/sensory neuronopathy: a clinical study of 71 patients. Medicine 1992, 71:59-72.
• [2]Moskovitz DN, Robb KV: Small cell lung cancer with positive anti-Hu antibodies presenting as gastroparesis. Can J Gastroenterol 2002, 16:171-174.
• [3]Brieva-Ruiz L, Diaz-Hurtado M, Matias-Guiu X, Marquez-Medina D, Tarragona J, Graus F: Anti-Ri-associated paraneoplastic cerebellar degeneration and breast cancer: an autopsy case study. Clin Neurol Neurosurg 2008, 110:1044-1046.
• [4]Hormigo A, Dalmau J, Rosenblum MK, River ME, Posner JB: Immunological and pathological study of anti-Ri-associated encephalopathy. Ann Neurol 1994, 36:896-902.
• [5]Luque FA, Furneaux HM, Ferziger R, Rosenblum MK, Wray SH, Schold SC Jr, Glantz MJ, Jaeckle KA, Biran H, Lesser M, et al.: Anti-Ri: an antibody associated with paraneoplastic opsoclonus and breast cancer. Ann Neurol 1991, 29:241-251.
• [6]Shams'ili S, Grefkens J, de Leeuw B, van den Bent M, Hooijkaas H, van der Holt B, Vecht C, Sillevis Smitt P: Paraneoplastic cerebellar degeneration associated with antineuronal antibodies: analysis of 50 patients. Brain 2003, 126:1409-1418.
• [7]Sutton IJ, Barnett MH, Watson JD, Ell JJ, Dalmau J: Paraneoplastic brainstem encephalitis and anti-Ri antibodies. J Neurol 2002, 249:1597-1598.
• [8]Weizman DA, Leong WL: Anti-Ri antibody opsoclonus-myoclonus syndrome and breast cancer: a case report and a review of the literature. J Surg Oncol 2004, 87:143-145.
• [9]Brashear HR, Li L, Herr JC: Antineuronal antibody in two patients with paraneoplastic opsoclonus-ataxia. Neurology 1992, 42(Suppl 3):457.
• [10]White D, Beringer T: Paraneoplastic limbic encephalitis in an elderly patient with small cell lung carcinoma. Ulster Med J 2010, 79:22-24.
• [11]Harloff A, Hummel S, Kleinschmidt M, Rauer S: Anti-Ri antibodies and limbic encephalitis in a patient with carcinoid tumour of the lung. J Neurol 2005, 252:1404-1405.
• [12]Ohmer R, Golnik KC, Richards AI, Kosmorsky GS: Ophthalmoplegia associated with the anti-Ri antibody. J Neuroophthalmol 1999, 19:246-248.
• [13]Greenlee JE, Brashear HR, Herndon RM: Immunoperoxidase labelling of rat brain sections with sera from patients with paraneoplastic cerebellar degeneration and systemic neoplasia. J Neuropathol Exp Neurol 1988, 47:561-571.
• [14]Liu J, Dalmau J, Szabo A, Rosenfeld M, Huber J, Furneaux H: Paraneoplastic encephalomyelitis antigens bind to the AU-rich elements of mRNA. Neurology 1995, 45:544-550.
• [15]Szabo A, Dalmau J, Manley G, Rosenfeld M, Wong E, Henson J, Posner JB, Furneaux HM: HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to Elav and Sex-Lethal. Cell 1991, 67:325-333.
• [16]Lewis HA, Chen H, Edo C, Buckanovich RJ, Yang YY, Musunuru K, Zhong R, Darnell RB, Burley SK: Crystal structures of Nova-1 and Nova-2 K-homology RNA-binding domains. Structure 1999, 7:191-203.
• [17]Brashear HR, Caccamo DV, Keeney PM: Localization of antibody in the central nervous system of a patient with paraneoplastic encephalomyeloneuritis. Neurology 1991, 41:1583-1587.
• [18]Greenlee JE: Treatment of paraneoplastic neurologic disorders. Curr Treat Options Neurol 2010, 12:212-230.
• [19]Uchuya M, Graus F, Vega F, Rene R, Delattre JY: Intravenous immunoglobulin treatment in paraneoplastic neurological syndromes with antineuronal autoantibodies. J Neurol Neurosurg Psychiatry 1996, 60:388-392.
• [20]Keime-Guibert F, Graus F, Fleury A, Rene R, Honnorat J, Broet P, Delattre JY: Treatment of paraneoplastic neurological syndromes with antineuronal antibodies (Anti-Hu, anti-Yo) with a combination of immunoglobulins, cyclophosphamide, and methylprednisolone. J Neurol Neurosurg Psychiatr 2000, 68:479-482.
• [21]Bien CG, Vincent A, Barnett MH, Becker AJ, Blumcke I, Graus F, Jellinger KA, Reuss DE, Ribalta T, Schlegel J, Sutton I, Lassmann H, Bauer J: Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain 2012, 135:1622-1638.
• [22]Gazic B, Pisem J, Dolenc-Groselj L, Popovic M: Paraneoplastic encephalomyelitis/sensory motor peripheral neuropathy - an autopsy case study. Folia Neuropathol 2005, 43:113-117.
• [23]Prestigiacomo CJ, Balmaceda C, Dalmau J: Anti-Ri-associated paraneoplastic opsoclonus-ataxia syndrome in a man with transitional cell carcinoma. Cancer 2001, 91:1423-1428.
• [24]Hunter SF, Parisi JE, Mastovich SL, Power W, Delahoussaye B, Duncan PR, Lennon VA: Chronic progressive paraneoplastic syndrome with prominent brainstem and spinal cord involvement associated with Type-2 anti-neuronal antibodies (ANNA-2) and breast carcinoma. J Neuropathol Exp Neurol 1995, 54:464.
• [25]Bataller L, Graus F, Saiz A, Vilchez JJ: Clinical outcome in adult onset idiopathic or paraneoplastic opsoclonus-myoclonus. Brain 2001, 124:437-443.
• [26]Digre KB: Opsoclonus in adults: report of three cases and review of the literature. Arch Neurol 1986, 43:1165-1175.
• [27]Dropcho EJ, Kline LB, Riser J: Antineuronal (anti-Ri) antibodies in a patient with steroid-responsive opsoclonus-myoclonus. Neurology 1993, 43:207-211.
• [28]Pittock SJ, Lucchinetti CF, Lennon VA: Anti-neuronal nuclear autoantibody type 2: paraneoplastic accompaniments. Ann Neurol 2003, 53:580-587.
• [29]Graus F, Saiz A, Dalmau J: Antibodies and neuronal autoimmune disorders of the CNS. J Neurol 2009, 257:509-517.
• [30]Dalmau J, Rosenfeld MR: Paraneoplastic syndromes of the CNS. Lancet Neurol 2008, 7:327-340.
• [31]Greenlee JE, Parks TN, Jaeckle KA: Type IIa (¿anti-Hu¿) antineuronal antibodies produce destruction of rat cerebellar granule neurons in vitro. Neurology 1993, 43:2049-2054.
• [32]Schafer KH, Klotz M, Mergner D, Mestres P, Schimrigk K, Blaes F: IgG-mediated cytotoxicity to myenteric plexus cultures in patients with paraneoplastic neurological syndromes. J Autoimmun 2000, 15:479-484.
• [33]Sillevis Smitt PA, Manley GT, Posner JB: Immunization with the paraneoplastic encephalomyelitis antigen HuD does not cause neurologic disease in mice. Neurology 1995, 45:1873-1878.
• [34]Greenlee JE, Clawson SA, Hill KE, Wood BL, Tsunoda I, Carlson NG: Purkinje cell death after uptake of anti-Yo antibodies in cerebellar slice cultures. J Neuropathol Exp Neurol 2010, 69:997-1007.
• [35]Hill KE, Clawson SA, Rose JW, Carlson NG, Greenlee JE: Cerebellar Purkinje cells incorporate immunoglobulins and immunotoxins in vitro: implications for human neurological disease and immunotherapeutics. J Neuroinflammation 2009, 6:31. BioMed Central Full Text
• [36]Moll JW, Antoine JC, Brashear HR, Delattre J, Drlicek M, Dropcho EJ, Giometto B, Graus F, Greenlee J, Honnorat J, Jaeckle KA, Tanaka K, Vecht CJ: Guidelines on the detection of paraneoplastic anti-neuronal-specific antibodies: report from the Workshop to the Fourth Meeting of the International Society of Neuro-Immunology on paraneoplastic neurological disease, held October 22¿23, 1994, in Rotterdam, The Netherlands. Neurology 1995, 45:1937-1941.
• [37]Rothstein JD, Jin L, Dykes-Hoberg M, Kuncl RW: Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci U S A 1993, 90:6591-6595.
• [38]King MA: Detection of dead cells and measurement of cell killing by flow cytometry. J Immunol Methods 2000, 243:155-166.
• [39]Bedner E, Smolewski P, Amstad P, Darzynkiewicz Z: Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res 2000, 259:308-313.
• [40]Darzynkiewicz Z, Bedner E, Smolewski P, Lee BW, Johnson GL: Detection of caspases activation in situ by fluorochrome-labeled inhibitors of caspases (FLICA). Methods Mol Biol 2002, 203:289-299.
• [41]Mori S, Sternberger NH, Herman MM, Sternberger LA: Leakage and neuronal uptake of serum protein in aged and Alzheimer brains. A postmortem phenomenon with antemortem etiology. Lab Invest 1991, 64:345-351.
• [42]Sillevis SPA, Manley GT, Posner JB: High titer antibodies but no disease in mice immunized with the paraneoplastic antigen HuD. Neurology 1994, 44(Suppl 2):A378.
• [43]Congdon EE, Gu J, Sait HB, Sigurdsson EM: Antibody uptake into neurons occurs primarily via clathrin-dependent Fcgamma receptor endocytosis and is a prerequisite for acute tau protein clearance. J Biol Chem 2013, 288:35452-35465.
• [44]Gu J, Congdon EE, Sigurdsson EM: Two novel Tau antibodies targeting the 396/404 region are primarily taken up by neurons and reduce Tau protein pathology. J Biol Chem 2013, 288:33081-33095.
• [45]Greenlee JE, Sun M: Immunofluorescent labelling of nonhuman cerebellar tissue with sera from patients with systemic cancer and paraneoplastic cerebellar degeneration. Acta Neuropathol Berl 1985, 67:226-229.
• [46]Furneaux HM, Wong E: Incubation of tumor cells with anti-Hu monoclonal results in apoptosis. Neurology 1994, 44(Suppl 2):A377.
• [47]De Giorgio R, Bovara M, Barbara G, Canossa M, Sarnelli G, De Ponti F, Stanghellini V, Tonini M, Cappello S, Pagnotta E, Nobile-Orazio E, Corinaldesi R: Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility. Gastroenterology 2003, 125:70-79.
• [48]Hinman MN, Lou H: Diverse molecular functions of Hu proteins. Cell Mol Life Sci 2008, 65:3168-3181.
• [49]Ince-Dunn G, Okano HJ, Jensen KB, Park WY, Zhong R, Ule J, Mele A, Fak JJ, Yang C, Zhang C, Yoo J, Herre M, Okano H, Noebels JL, Darnell RB: Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability. Neuron 2012, 75:1067-1080.
• [50]King PH, Redden D, Palmgren JS, Nabors LB, Lennon VA: Hu antigen specificities of ANNA-I autoantibodies in paraneoplastic neurological disease. J Autoimmun 1999, 13:435-43.
• [51]Greenlee JE, Parks TN, Jaeger F, Brashear HR: Interaction of Type IIA (¿Anti-Hu¿) and Type IIB (¿Anti-Ri¿) antibodies with primary cultures of rat cerebellar granule cells: a possible model for paraneoplastic neuronal injury in man. Brain Pathol 1992, 2:256.
• [52]Buckanovich RJ, Posner JB, Darnell RB: Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system. Neuron 1993, 11:657-672.
• [53]Ratti A, Fallini C, Colombrita C, Pascale A, Laforenza U, Quattrone A, Silani V: Post-transcriptional regulation of neuro-oncological ventral antigen 1 by the neuronal RNA-binding proteins ELAV. J Biol Chem 2008, 283:7531-7541.
• [54]Buckanovich RJ, Yang YY, Darnell RB: The onconeural antigen Nova-1 is a neuron-specific RNA-binding protein, the activity of which is inhibited by paraneoplastic antibodies. J Neurosci 1996, 16:1114-1122.