Anti-HuD-induced neuronal apoptosis underlying paraneoplastic gut dysmotility.
OBJECTIVE
The role of autoimmunity underlying paraneoplastic gut dysmotility remains unsettled. Because anti-Hu antibodies may impair enteric neuronal function, we tested whether anti-HuD-positive sera from patients with paraneoplastic gut dysmotility or commercial anti-HuD antibodies activated the apoptotic cascade in a neuroblastoma cell line and cultured myenteric neurons.


METHODS
Anti-HuD antibodies from patients with severe paraneoplastic gut dysmotility were characterized by immunofluorescence and immunoblot. SH-Sy5Y neuroblasts and cultured myenteric neurons were exposed to sera containing anti-HuD antibodies or 2 commercial anti-HuD antibodies. Cells were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique to evaluate apoptosis. Immunofluorescence was used to identify activated caspase-3 and apaf-1, along with microtubule-associated protein 2.


RESULTS
In SH-Sy5Y cells, the percentage of TUNEL-positive nuclei observed after exposure to anti-HuD-positive sera (32% +/- 7%) or anti-HuD antibodies (23% +/- 2%) was significantly greater than that of control sera or fetal calf serum (P < 0.001). The time-course analysis showed a significantly greater number of apoptotic neuroblastoma cells evoked by the 2 commercial anti-HuD antibodies at 24, 48, and 72 hours versus controls. The number of TUNEL-positive myenteric neurons exposed to anti-HuD antibodies (60% +/- 14%) was significantly greater than that of fetal calf serum (7% +/- 2%; P < 0.001). Apaf-1 and caspase-3 immunolabeling showed intense cytoplasmic staining in a significantly greater proportion of cells exposed to anti-HuD-positive sera or to commercial anti-HuD antibodies compared with controls.


CONCLUSIONS
Anti-HuD antibodies evoked neuronal apoptosis that may contribute to enteric nervous system impairment underlying paraneoplastic gut dysmotility. Apaf-1 activation suggests participation of a mitochondria-dependent apoptotic pathway.
De Giorgio
Roberto
R
Department of Internal Medicine, University of Bologna, Italy. deg@orsola-malpighi.unibo.it
Bovara
Monica
M
Barbara
Giovanni
G
Canossa
Marco
M
Sarnelli
Giovanni
G
De Ponti
Fabrizio
F
Stanghellini
Vincenzo
V
Tonini
Marcello
M
Cappello
Silvia
S
Pagnotta
Eleonora
E
Nobile-Orazio
Eduardo
E
Corinaldesi
Roberto
R
D017209:Apoptosis / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology / Q000494:pharmacology; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; D005260:Female; D005769:Gastrointestinal Motility; D006801:Humans; D020287:In Situ Nick-End Labeling; D007410:Intestinal Diseases / Q000276:immunology* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009197:Myenteric Plexus / Q000166:cytology / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000276:immunology*; D009447:Neuroblastoma; D009474:Neurons / Q000166:cytology* / Q000276:immunology; D020364:Paraneoplastic Polyneuropathy / Q000276:immunology* / Q000473:pathology; D016601:RNA-Binding Proteins / Q000276:immunology*; D014407:Tumor Cells, Cultured
D001323:Autoantibodies; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; C498010:ELAVL4 protein, human; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins