Sera of Patients With Celiac Disease and Neurologic Disorders Evoke a Mitochondrial-Dependent Apoptosis In Vitro
2007; Elsevier BV; Volume: 133; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.04.070
ISSN1528-0012
AutoresElisabetta Cervio, Umberto Volta, Manuela Verri, Federica Boschi, O. Pastoris, Alessandro Granito, Giovanni Barbara, Claudia Parisi, Cristina Felicani, Marcello Tonini, Roberto De Giorgio,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoBackground & Aims: The mechanisms underlying neurologic impairment in celiac disease remain unknown. We tested whether antineuronal antibody–positive sera of patients with celiac disease evoke neurodegeneration via apoptosis in vitro. Methods: SH-Sy5Y cells were exposed to crude sera, isolated immunoglobulin (Ig) G and IgG-depleted sera of patients with and without celiac disease with and without neurologic disorders, and antineuronal antibodies. Adsorption studies with gliadin and tissue transglutaminase (tTG) were performed in celiac disease sera. Apoptosis activated caspase-3, apaf-1, Bax, cytochrome c, cleaved caspase-8 and caspase-9 and mitochondrial respiratory chain complexes were evaluated with different methods. Results: SH-Sy5Y cells exposed to antineuronal antibody–positive sera and isolated IgG from the same sera exhibited a greater percentage of TUNEL-positive nuclei than that of antineuronal antibody–negative sera. Neuroblasts exposed to antineuronal antibody–negative celiac disease sera also showed greater TUNEL positivity and apaf-1 immunolabeled cells than controls. Antigliadin- and anti-tTG–depleted celiac disease sera had an apoptotic effect similar to controls. Anti–caspase-3 immunostained cells were greater than controls when exposed to positive sera. The mitochondrial respiratory chain complex was reduced by positive sera. Western blot demonstrated only caspase-9 cleavage in positive sera. Cytochrome c and Bax showed reciprocal translocation (from mitochondria to cytoplasm and vice versa) after treatment with positive sera. Conclusions: Antineuronal antibodies and, to a lower extent, combined antigliadin and anti-tTG antibodies in celiac disease sera contribute to neurologic impairment via apoptosis. Apaf-1 activation with Bax and cytochrome c translocation suggest a mitochondrial-dependent apoptosis. Background & Aims: The mechanisms underlying neurologic impairment in celiac disease remain unknown. We tested whether antineuronal antibody–positive sera of patients with celiac disease evoke neurodegeneration via apoptosis in vitro. Methods: SH-Sy5Y cells were exposed to crude sera, isolated immunoglobulin (Ig) G and IgG-depleted sera of patients with and without celiac disease with and without neurologic disorders, and antineuronal antibodies. Adsorption studies with gliadin and tissue transglutaminase (tTG) were performed in celiac disease sera. Apoptosis activated caspase-3, apaf-1, Bax, cytochrome c, cleaved caspase-8 and caspase-9 and mitochondrial respiratory chain complexes were evaluated with different methods. Results: SH-Sy5Y cells exposed to antineuronal antibody–positive sera and isolated IgG from the same sera exhibited a greater percentage of TUNEL-positive nuclei than that of antineuronal antibody–negative sera. Neuroblasts exposed to antineuronal antibody–negative celiac disease sera also showed greater TUNEL positivity and apaf-1 immunolabeled cells than controls. Antigliadin- and anti-tTG–depleted celiac disease sera had an apoptotic effect similar to controls. Anti–caspase-3 immunostained cells were greater than controls when exposed to positive sera. The mitochondrial respiratory chain complex was reduced by positive sera. Western blot demonstrated only caspase-9 cleavage in positive sera. Cytochrome c and Bax showed reciprocal translocation (from mitochondria to cytoplasm and vice versa) after treatment with positive sera. Conclusions: Antineuronal antibodies and, to a lower extent, combined antigliadin and anti-tTG antibodies in celiac disease sera contribute to neurologic impairment via apoptosis. Apaf-1 activation with Bax and cytochrome c translocation suggest a mitochondrial-dependent apoptosis. Celiac disease is an autoimmune chronic inflammatory intestinal disease resulting from sensitivity to ingested gluten-containing foods.1Green P.H.R. Jabri B. Coeliac disease.Lancet. 2003; 362: 383-391Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar, 2Lock R.J. Pitcher M.C. Unsworth D.J. IgA anti-tissue transglutaminase as a diagnostic marker of gluten sensitive enteropathy.J Clin Pathol. 1999; 52: 274-277Crossref PubMed Scopus (85) Google Scholar, 3Maki M. Collin P. Coeliac disease.Lancet. 1997; 349: 1755-1759Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 4Dieterich W. Ehnis T. Bauer M. Donner P. Volta U. Riecken E.O. Schuppan D. Identification of tissue transglutaminase as the autoantigen of celiac disease.Nat Med. 1997; 3: 797-801Crossref PubMed Scopus (1839) Google Scholar Although the disease primarily affects the gastrointestinal tract, celiac disease is a classic example of a systemic disorder involving many organs, such as skin, thyroid, pancreas, liver, and heart, as well as joints, muscles, bones, the central nervous system (CNS), and the peripheral nervous system.5Alaedini A. Green P.H. Narrative review: celiac disease: understanding a complex autoimmune disorder.Ann Intern Med. 2005; 142: 289-299Crossref PubMed Scopus (269) Google Scholar Approximately 10% of patients with celiac disease display neurologic symptoms related to a wide array of disorders, such as epilepsy, myoclonus, cerebellar ataxia, multifocal leukoencephalopathy, dementia, chorea, migraine, multiple sclerosis, memory/attention impairment, and peripheral axonal and demyelinating neuropathies.3Maki M. Collin P. Coeliac disease.Lancet. 1997; 349: 1755-1759Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 6Luorostainen L. Pirttila T. Collin P. Coeliac disease presenting with neurological disorders.Eur Neurol. 1999; 42: 132-135Crossref PubMed Scopus (121) Google Scholar, 7Wills A.J. The neurology and neuropathology of coeliac disease.Neuropathol Appl Neurobiol. 2000; 26: 493-496Crossref PubMed Scopus (46) Google Scholar, 8Ciclitira P.J. AGA technical review on celiac sprue.Gastroenterology. 2001; 120: 1526-1540Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar The nature of this association is unclear, and whether a specific neurologic complication occurs in celiac disease remains unsettled. Malabsorption may lead to vitamin and trace element deficiencies. Therefore, patients who develop neurologic dysfunction should be carefully screened for these alterations. However, malabsorption alone does not explain the pathophysiology and clinical course of many of the associated neurologic disorders. Other mechanisms proposed include gluten toxicity,9Pengiran Tengah D.S. Wills A.J. Holmes G.K. Neurological complications of coeliac disease.Postgrad Med J. 2002; 78: 393-398Crossref PubMed Scopus (68) Google Scholar genetic factors,10Schuppan D. Current concepts of celiac disease pathogenesis.Gastroenterology. 2000; 119: 234-242Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar and autoimmunity.11Volta U. De Giorgio R. Petrolini N. Stanghellini V. Barbara G. Granito A. De Ponti F. Corinaldesi R. Bianchi F.B. Clinical findings and anti-neuronal antibodies in coeliac disease with neurological disorders.Scand J Gastroenterol. 2002; 37: 1276-1281Crossref PubMed Scopus (64) Google Scholar The concept that autoimmunity can act as a mechanism triggering neurologic dysfunction is strengthened by the identification of lymphocytic infiltration in the central and peripheral nervous systems12Hadjivassiliou M. Grünewald R.A. Chattopadhyay A.K. Davies-Jones G.A. Gibson A. Jarratt J.A. Kandler R.H. Lobo A. Powell T. Smith C.M. Clinical, radiological, neurophysiological, and neuropathological characteristics of gluten ataxia.Lancet. 1998; 352: 1582-1585Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 13Hadjivassiliou M. Grünewald R.A. Kandler R.H. Chattopadhyay A.K. Jarratt J.A. Sanders D.S. Sharrack B. Wharton S.B. Davies-Jones G.A. Neuropathy associated with gluten sensitivity.J Neurol Neurosurg Psychiatry. 2006; 77: 1262-1266Crossref PubMed Scopus (94) Google Scholar as well as circulating antineuronal antibodies.14Wills A.J. Turner B. Lock R.J. Johnston S.L. Unsworth D.J. Fry L. Dermatitis herpetiformis and neurological dysfunction.J Neurol Neurosurg Psychiatry. 2002; 72: 259-261Crossref PubMed Scopus (23) Google Scholar, 15Hadjivassiliou M. Grunewald R.A. Davies-Jones G.A. Gluten sensitivity as a neurological illness.J Neurol Neurosurg Psychiatry. 2002; 72: 560-563Crossref PubMed Scopus (158) Google Scholar These antibodies bind to CNS and enteric nervous system (ENS) neurons. Using appropriate tissue sections (eg, cerebellum, brain cortex, and intestine), sera of patients with antineuronal antibodies provide labeling of either CNS or ENS neurons at indirect immunofluorescence. Two different patterns have been so far recognized: one characterized by a predominant neuronal nuclear labeling (ie, "Hu-like pattern") and the other with a cytoplasmic staining of CNS neurons such as Purkinje cells ("Yo-like pattern").11Volta U. De Giorgio R. Petrolini N. Stanghellini V. Barbara G. Granito A. De Ponti F. Corinaldesi R. Bianchi F.B. Clinical findings and anti-neuronal antibodies in coeliac disease with neurological disorders.Scand J Gastroenterol. 2002; 37: 1276-1281Crossref PubMed Scopus (64) Google Scholar, 14Wills A.J. Turner B. Lock R.J. Johnston S.L. Unsworth D.J. Fry L. Dermatitis herpetiformis and neurological dysfunction.J Neurol Neurosurg Psychiatry. 2002; 72: 259-261Crossref PubMed Scopus (23) Google Scholar, 15Hadjivassiliou M. Grunewald R.A. Davies-Jones G.A. Gluten sensitivity as a neurological illness.J Neurol Neurosurg Psychiatry. 2002; 72: 560-563Crossref PubMed Scopus (158) Google Scholar A high prevalence of these antibodies has been shown to correlate with cerebellar ataxia, epilepsy, and peripheral neuropathy related to celiac disease.11Volta U. De Giorgio R. Petrolini N. Stanghellini V. Barbara G. Granito A. De Ponti F. Corinaldesi R. Bianchi F.B. Clinical findings and anti-neuronal antibodies in coeliac disease with neurological disorders.Scand J Gastroenterol. 2002; 37: 1276-1281Crossref PubMed Scopus (64) Google Scholar However, whether antineuronal antibodies elicit neurologic impairment still remains unclear. The present study was designed to gain insights into the pathogenetic mechanisms of patients with neurologic celiac disease by testing the hypothesis that sera containing antineuronal antibodies present in a subset of such patients may evoke neuronal damage. For this purpose, we elected to use a neuroblastoma cell line of human origin (ie, SH-Sy5Y) because these cells provide a reliable model for analyzing molecular pathways involved in different neurodegenerative disorders. In this report, we specifically focused on neuronal apoptosis and related pathways as indicators of neuronal damage/degeneration resulting from sera of patients with celiac disease with and without neurologic manifestations. This study included 9 selected patients (6 women and 3 men; age range, 21–61 years) with celiac disease and concomitant CNS or peripheral nervous system dysfunction associated with the presence of antineuronal antibodies (Table 1). The study included also 6 patients with celiac disease (5 women and 1 man; age range, 30–46 years) without neurologic abnormalities and without antineuronal antibodies. In 2 patients, one from the group with celiac disease and antineuronal antibodies and the second from the group with celiac disease without neurologic abnormalities and without antineuronal antibodies, sera were collected and immunoglobulin (Ig) G purified as indicated in the following text. Patients with celiac disease were diagnosed at the Departments of Internal Medicine, Cardioangiology, Hepatology, and Internal Medicine and Gastroenterology of the University of Bologna. Celiac disease–related antibodies (antigliadin, antiendomysial, and anti–tissue transglutaminase [anti-tTG]) tested positive in all patients.11Volta U. De Giorgio R. Petrolini N. Stanghellini V. Barbara G. Granito A. De Ponti F. Corinaldesi R. Bianchi F.B. Clinical findings and anti-neuronal antibodies in coeliac disease with neurological disorders.Scand J Gastroenterol. 2002; 37: 1276-1281Crossref PubMed Scopus (64) Google Scholar, 16Volta U. Molinaro N. De Franceschi L. Fratangelo D. Bianchi F.B. IgA anti-endomysial antibodies on human umbilical cord tissue for celiac disease screening: save both money and monkeys.Dig Dis Sci. 1995; 40: 1902-1905Crossref PubMed Scopus (113) Google Scholar Diagnosis of celiac disease was confirmed by endoscopic duodenal biopsy. Histologic findings were graded according to Marsh's revised criteria.17Marsh M.N. Gluten, major histocompatibility complex, and the small intestine A molecular and immunobiologic approach to the spectrum of gluten sensitivity.Gastroenterology. 1992; 102: 330-354Abstract PubMed Google Scholar, 18Oberhuber G. Granditsch G. Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists.Eur J Gastroenterol Hepathol. 1999; 11: 1185-1194Crossref PubMed Scopus (1455) Google Scholar A formal neurologic assessment was routinely performed in all patients with celiac disease on presentation, including analysis of the time of onset of any neurologic symptoms with respect to diagnosis of celiac disease. In addition, we included 4 non–celiac disease patients with neurologic disorders (2 patients with cerebellar ataxia and 2 with epilepsy; 1 male and 3 females; age range, 5–37 years) without (n = 2) and with antineuronal antibodies (n = 2, titer 1:200). Control sera, including 10 sex- and age-matched blood donors, tested negative to all of the aforementioned antibodies as expected. All patients gave their informed consent to participate in the present study, which was approved by the St Orsola-Malpighi University Hospital Ethics Committee (reference no. 1804/2006).Table 1List of Patients With Celiac Disease With Concomitant Neurologic Disorders and Antineuronal AntibodiesPatients with celiac diseaseAge (y)SexAntigliadin antibodies IgA (titer)Anti-tTG IgA (AU)Antineuronal antibodies IgG (titer)Associated neurologic disorder137F1:4015CNS (1:200)Multiple sclerosis224F1:160>20CNS (1:100)Cerebellar ataxia361F1:108CNS (1:200)Cerebellar ataxia428M1:8018CNS (1:50)Epilepsy542F1:20>20ENS (1:1600)Cognitive disorders645F1:80>20ENS (1:200)Memory/attention impairment761F1:4011CNS (1:200)Cerebellar ataxia859M1:2012ENS (1:100)Epilepsy921M1:8014ENS (1:100)Moyamoya diseaseNOTE. Antigliadin antibodies of the IgA class were detected by immunofluorescence with a cutoff value ≥1:10; anti-tTG of the IgA class were detected by enzyme-linked immunosorbent assay with a cutoff value of >7 AU. Open table in a new tab NOTE. Antigliadin antibodies of the IgA class were detected by immunofluorescence with a cutoff value ≥1:10; anti-tTG of the IgA class were detected by enzyme-linked immunosorbent assay with a cutoff value of >7 AU. The presence of antineuronal antibodies to the CNS and ENS was detected by indirect immunofluorescence on 5-μm cryostat sections of monkey and rat cerebellum as well as rat ileum and colon (Medic, Turin, Italy). Sera were tested at the initial dilution of 1:10 (in phosphate-buffered saline [PBS]) and, when positive, were titrated up to the end point. Rabbit anti-human IgG and IgA (Dako, Copenhagen, Denmark) were used as secondary antibody at the appropriate working dilution (1:60 and 1:100 on rat and monkey tissue, respectively).11Volta U. De Giorgio R. Petrolini N. Stanghellini V. Barbara G. Granito A. De Ponti F. Corinaldesi R. Bianchi F.B. Clinical findings and anti-neuronal antibodies in coeliac disease with neurological disorders.Scand J Gastroenterol. 2002; 37: 1276-1281Crossref PubMed Scopus (64) Google Scholar Sera of 2 patients with celiac disease with neurologic disorders and with or without antineuronal antibodies, respectively, were used. IgG were purified from sera using a protein G agarose column according to the manufacturer's instructions (KPL, Gaithersburg, MD). An automatic immunoturbidimetric assay (Modular, Roche Diagnostics, Basel, Switzerland) showed that the IgG aliquots obtained at the end of the procedure contained more than 95% of total IgGs and no IgA or IgM Igs, whereas the IgG-depleted samples contained only IgA and IgM Igs. Commercially available human pure IgG and IgA used in this study were purchased by Bethyl Laboratories Inc (Montgomery, TX). Six sera obtained from previously examined patients with celiac disease without neurologic disorders and antineuronal antibodies, positive for anti-tTG and antigliadin antibodies, were incubated overnight at 4°C, under continuous shaking, with guinea pig liver transglutaminase (Sigma Chemical Co, St Louis, MO) or crude gliadin (Sigma Chemical Co) at a concentration of 10 mg of protein/mL of undiluted serum. Antibody-antigen complexes were then separated from sera by ultracentrifugation at 100,000g for 30 minutes.19Granito A. Muratori P. Cassani F. Pappas G. Muratori L. Agostinelli D. Veronesi L. Bortolotti R. Petrolini N. Bianchi F.B. Volta U. Anti-actin IgA antibodies in severe coeliac disease.Clin Exp Immunol. 2004; 137: 386-392Crossref PubMed Scopus (62) Google Scholar An enzyme-linked immunosorbent assay for the detection of anti-tTG and antigliadin antibodies was performed before and after adsorption of sera. The human neuroblastoma cell clone SH-Sy5Y was maintained in 100-cm2Lock R.J. Pitcher M.C. Unsworth D.J. IgA anti-tissue transglutaminase as a diagnostic marker of gluten sensitive enteropathy.J Clin Pathol. 1999; 52: 274-277Crossref PubMed Scopus (85) Google Scholar dishes in a 1:1 mixture of Dulbecco's modified Eagle medium (DMEM) and Ham's F-12 containing 15% fetal calf serum (FCS), penicillin (100 IU/mL), streptomycin (100 μg/mL), and nonessential amino acids (100 μg/mL) in an incubator at 37°C and gassed continuously with a mixture of 5% co2 and 95% o2. Cells were detached every 2 days with trypsin-EDTA (0.05% trypsin and 0.53 mmol/L EDTA), followed by centrifugation, resuspension, and finally seeding in new plates. All reagents were obtained from Gibco BRL Laboratories (Gaithersburg, MD). Passages never exceeded 30. For experimental analyses, cells were grown in either cell culture dishes or on poly-L-lysine–coated (Sigma Immunochemicals, St Louis, MO) (50 μg/mL) glass coverslips. SH-Sy5Y neuroblasts were exposed to sera of patients with celiac disease and neurologic disorders with or without antineuronal antibodies, sera of non–celiac disease patients with or without neurologic disorders, and control sera/FCS. Each of the above sera (5% or 1:20) was diluted in DMEM and Ham's F-12. Furthermore, neuroblasts were exposed to commercially available human pure IgG and IgA (both diluted in DMEM and Ham's F-12 to 1:1000 at 0.001 mg/mL concentration) and to isolated IgG from patients with celiac disease with neurologic disorders with or without antineuronal antibodies (both diluted 1:20 at 0.063 and 0.064 mg/mL concentrations, respectively) and related sera without IgG (diluted to 5%). Neuroblastoma cells were exposed to these sera for 12 hours (ie, activated apaf-1, caspase-3, cleaved caspase-8 and caspase-9) and 24 hours (ie, to identify apoptosis by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling [TUNEL] technique). Mitochondrial fraction from neuroblasts, cultured for 6 or 12 hours with antineuronal antibody–positive sera (from patients with celiac disease with neurologic impairment) and antineuronal antibody–negative sera (from patients with celiac disease without neurologic abnormalities and blood donors), was processed for respiratory chain complexes (including nicotinamide adenine dinucleotide [NADH]-ubiquinone oxidoreductase, succinate dehydrogenase, and cytochrome oxidase) and citrate synthase specific activities. Finally, an exposition time of 16 hours was used to evaluate Bax translocation and release of cytochrome c from mitochondria. This method was applied to prove the occurrence of nuclear condensation as a result of the apoptosis induced by sera of patients with celiac disease positive for antineuronal antibodies. Briefly, cells were cultured for 24 hours in DMEM containing 5% of antineuronal antibody–positive sera of patients with neurologic celiac disease, antineuronal antibody–negative sera of patients with celiac disease without neurologic abnormalities, or control sera, fixed in 3.7% buffered formaldehyde (10 minutes) and in methanol (20 minutes) at room temperature, washed in PBS, and stained with Hoechst 33258 dye (Sigma Immunochemicals) (10 μg/mL) for 15 minutes at room temperature, rinsed with PBS, and finally mounted upside down on glass slides in a drop of Mowiol (Calbiochem, Darmstadt, Germany). SH-Sy5Y cells were examined with a fluorescent microscope. Further control experiments were performed in parallel using cells cultured for 24 hours in DMEM containing 15% FCS. SH-Sy5Y neuronal cells were grown for 24 hours on poly-L-lysine–coated coverslips and then incubated for 24 hours in DMEM and Ham's F-12 containing 5% of celiac disease sera of patients with and without neurologic disorders and with and without antineuronal antibodies, sera of non–celiac disease patients with neurologic disorders with and without antineuronal antibodies, isolated IgG from patients with celiac disease with neurologic disorders with or without antineuronal antibodies (and related sera without IgG), commercially available human pure IgG and IgA (1:1000), and control sera. Additional control experiments were performed in parallel using cells cultured for 24 hours in DMEM containing 15% FCS. All experiments were performed in duplicate. Fixed neuroblastoma cells were processed with either an in situ apoptosis detection kit (Neurotacs II, Trevigen, Gaithersburg, MD) or the dead-end fluorometric TUNEL system (Promega, Madison, WI) according to the manufacturer's instructions.20Migheli A. Attanasio A. Lee W. Bayer S.A. Ghetti B. Detection of apoptosis in weaver cerebellum by electron microscopic in situ end-labeling of fragmented DNA.Neurosci Lett. 1995; 199: 53-56Crossref PubMed Scopus (51) Google Scholar, 21Li X. Traganos F. Melamed M.R. Darzynkiewicz Z. Single-step procedure for labeling DNA strand breaks with floures.Cytometry. 1995; 20: 172-180Crossref PubMed Scopus (156) Google Scholar With the Neurotacs II method, the apoptotic nuclei appeared dark brown and could be visualized with phase-contrast light transmission microscopy (Leica, Westlar, Germany); with the TUNEL system, which allowed identification of fluorescein isothiocyanate–labeled DNA fragments, apoptotic cells were visualized with a microscope equipped with confocal laser scanning microscopy (Leica TCS-SP). As a positive control for apoptosis, cells were incubated for 12 hours with DMEM and dopamine (100 mmol/L), a well-established proapoptotic substance for this cell line.22Junn E. Mouradian M.M. Apoptotic signaling in dopamine-induced cell death: the role of oxidative stress, p38 mitogen-activated protein kinase, cytochrome C and caspases.J Neurochem. 2001; 78: 374-383Crossref PubMed Scopus (196) Google Scholar SH-Sy5Y cells were grown for 24 hours on poly-L-lysine–coated coverslips, cultured for 12 hours in DMEM containing 15% of FCS or 5% of celiac disease sera of patients with and without neurologic disorders and with and without antineuronal antibodies, sera of non–celiac disease patients with neurologic disorders with and without antineuronal antibodies, isolated IgG from patients with celiac disease with neurologic disorders with or without antineuronal antibodies (and related sera without IgG), commercially available human pure IgG and IgA (1:1000), and control sera. Cells were fixed as previously described and then incubated for 30 minutes in buffer containing 3% bovine serum albumin in PBS, pH 7.4, with 0.1% Triton X-100 (BSA-PBST). Primary rabbit antibodies against the active fragment of caspase-3 (1:1000; Trevigen) and anti–apaf-1 (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) were used in experiments performed incubating the SH-Sy5Y cells with sera and IgG/IgA as indicated previously, whereas primary mouse anti–cytochrome c (1:7500; BD PharMingen, Franklin Lakes, NJ) and rabbit anti-human Bax (1:1000; BD PharMingen) were used in experiments with either sera of patients with celiac disease with neurologic disorders and with antineuronal antibodies or control sera (blood donors/FCS). Cells were incubated in a humid chamber at 4°C overnight, rinsed with PBS, and incubated for 60 minutes at room temperature with the respective secondary antibodies (Molecular Probes Inc, Eugene, OR) diluted 1:500 in 3% BSA-PBST. Coverslips were finally rinsed with PBS and mounted upside down on glass slides in a drop of Mowiol. For double-labeling studies with cytochrome c and Bax in mitochondria, SH-Sy5Y cells were grown for 24 hours on poly-L-lysine–coated coverslips and then cultured for 12 hours in DMEM containing 15% of FCS or 5% of either antineuronal antibody–containing sera or control sera. Then, cells were incubated with the mitochondrial marker MitoTracker Deep Red 633 probe (Molecular Probes Inc), 500 nmol/L in growth medium, at 37°C for 30 minutes. Cells were then fixed with growth medium containing 3.7% formaldehyde at 37°C for 15 minutes. After fixation, cells were permeabilized with PBS containing 0.2% Triton X-100 (Sigma Chemical Co) at room temperature for 5 minutes. Mouse anti–cytochrome c monoclonal antibody (1:7500; BD Biosciences, Franklin Lakes, NJ) or rabbit anti-human Bax (1:1000; BD PharMingen) was applied for 1 hour at room temperature. Cells were then washed with PBS and incubated for 1 hour at room temperature with an Alexa 488–conjugated goat anti-mouse or goat anti-rabbit IgG antibody (Molecular Probes Inc) diluted 1:500 in 3% BSA-PBST. Samples were finally counterstained with Hoechst 33258. Controls for double immunolabeling technique were performed to determine that the primary antibodies do not cross-react when mixed together and that the secondary antibodies recognize the appropriate antigen-antibody complexes. To determine enzyme activities, neuroblasts cultured for 6 or 12 hours in DMEM containing 15% of FCS or 5% of antineuronal antibody–containing sera were weighed and subsequently homogenized in 0.25 mol/L sucrose in a precooled Potter-Braun S homogenizer. The homogenate was diluted with 0.25 mol/L sucrose (ie, 1 g of cells in 10 mL of sucrose solution). This homogenate was then centrifuged at 800g for 15 minutes in a refrigerated centrifuge (Beckman J2-21, rotor JA-20, Beckman Coulter, Fullerton, CA), and the supernatant was stored in ice. The sediment was rehomogenized in 0.25 mol/L sucrose and centrifuged at 800g for 15 minutes. The 2 supernatants obtained were centrifuged at 14,000g for 20 minutes. The mitochondrial sediment was gently resuspended in sucrose solution at a final dilution of 100 mg/mL. An aliquot of this solution (60 μL) was used to assess the protein content, whereas the remaining portion was used to evaluate enzyme activities. The maximum rates of the following enzyme activities were evaluated in the mitochondrial fraction: citrate synthase for the tricarboxylic acid cycle and NADH-ubiquinone oxidoreductase, succinate dehydrogenase, and cytochrome oxidase for the electron transfer chain. Enzyme activities were recorded graphically for at least 3 minutes with a double recorder spectrophotometer (Shimadzu 1601; Shimadzu Biotech, Milan, Italy), and each value was calculated from 2 blind determinations on the same sample. Enzyme specific activities were expressed as nanomolar of substrate transformed per minute per milligram of protein.23Pastoris O. Dossena M. Vercesi L. Bruseghini M. Pagnin A. Ceriana P. Biochemical changes induced in the myocardial cell during cardioplegic arrest supplemented with creatine phosphate.J Cardiothorac Vasc Anesth. 1991; 5: 475-480Abstract Full Text PDF PubMed Scopus (14) Google Scholar After treatment with sera, neuroblastoma cells were first rinsed twice with ice-cold PBS and then lysed for 10 minutes on ice in 200 μL of ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 140 mmol/L NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L sodium orthovanadate, 2 g/mL aprotinin, 2 g/mL pepstatin, 2 g/mL leupeptin, and 1 μmol/L microcystin-LR and Triton X-100 1%). The lysates were sonicated for 10 seconds and then centrifuged at 9660g for 5 minutes at 4°C, and aliquots were taken for protein quantitation using the Pierce BCA protein assay kit (Rockford, IL). For the evaluation of the presence of cytochrome c and Bax in the cytoplasm and mitochondria, treated cells were first rinsed twice with ice-cold PBS and then lysed for 10 minutes on ice in 200 μL of ice-cold lysis buffer without Triton X-100 (50 mmol/L Tris-HCl, pH 7.5, 140 mmol/L NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mmol/L sodium orthovanadate, 2 g/mL aprotinin, 2 g/mL pepstatin, 2 g/mL leupeptin, and 1 μmol/L microcystin-LR). The lysates were sonicated for 10 seconds and then ultracentrifuged at 15,000g for 15 minutes at 4°C. Supernatant containing cytosol proteins was used to determine cytochrome c release from mitochondria. Sediment, containing membrane proteins, was rehomogenized in lysis buffer with Triton X-100 1% and ultracentrifuged at 15,000g for 15 minutes at 4°C, and aliquots of all samples were taken for protein determination using the Pierce BCA protein assay kit. Samples containing equal protein amounts (55 μg) were mixed with Laemmli's loading buffer, boiled for 5 minutes, and electrophoresed in a 9% or 15% sodium dodecyl sulfate/polyacrylamide gel electrophoresis minigel at 100 V. Resolved proteins were then electrophoretically transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA) for 1 hour at 4°C under a constant current of 100 V. Membranes were saturated with 5% low-fat dry milk in Tris-buffered saline (
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