Ae2-Deficient Mice Develop Antimitochondrial Antibodies and Other Features Resembling Primary Biliary Cirrhosis
2008; Elsevier BV; Volume: 134; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2008.02.020
ISSN1528-0012
AutoresJanuary T. Salas, Jesús M. Bañales, Sarai Sarvide, Sergio Recalde, Alejandro Ferrer, Iker Uriarte, Ronald P.J. Oude Elferink, Jesús Prìeto, Juan Francisco Medina Gallardo,
Tópico(s)Liver Disease and Transplantation
ResumoBackground & Aims: Cl−/HCO3− anion exchanger 2 (AE2) is involved in intracellular pH (pHi) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2a.b-deficient mice. Methods: In this murine model, splenocyte pHi and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Results: Ae2a,b−/− mice exhibit splenomegaly, elevated pHi in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8+ T-cell population, and under represented CD4+FoxP3+/regulatory T cells. Most Ae2a,b−/− mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2a,b−/− mice had extensive portal inflammation with CD8+ and CD4+ T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2a,b−/− mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Conclusions: Ae2 deficiency alters pHi homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC. Background & Aims: Cl−/HCO3− anion exchanger 2 (AE2) is involved in intracellular pH (pHi) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2a.b-deficient mice. Methods: In this murine model, splenocyte pHi and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Results: Ae2a,b−/− mice exhibit splenomegaly, elevated pHi in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8+ T-cell population, and under represented CD4+FoxP3+/regulatory T cells. Most Ae2a,b−/− mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2a,b−/− mice had extensive portal inflammation with CD8+ and CD4+ T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2a,b−/− mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Conclusions: Ae2 deficiency alters pHi homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC. Cl−/HCO3− anion exchanger 2 (AE2; also known as SLC4A2) mediates electroneutral Na+-independent Cl−/HCO3− exchange across the plasma membrane.1Romero M.F. Fulton C.M. Boron W.F. The SLC4 family of HCO3− transporters.Pflugers Arch. 2004; 447: 495-509Google Scholar, 2Alper S.L. Molecular physiology of SLC4 anion exchangers.Exp Physiol. 2006; 91: 153-161Google Scholar This widely expressed exchanger is a relevant acid loader involved in the regulation of intracellular pH (pHi).1Romero M.F. Fulton C.M. Boron W.F. The SLC4 family of HCO3− transporters.Pflugers Arch. 2004; 447: 495-509Google Scholar, 2Alper S.L. Molecular physiology of SLC4 anion exchangers.Exp Physiol. 2006; 91: 153-161Google Scholar Moreover, AE2 is involved in transepithelial acid-base transport, including proton gastric secretion3Gawenis L.R. Ledoussal C. Judd L.M. et al.Mice with a targeted disruption of the AE2 Cl−/HCO3− exchanger are achlorhydric.J Biol Chem. 2004; 279: 30531-30539Google Scholar, 4Recalde S. Muruzabal F. Looije N. et al.Inefficient chronic activation of parietal cells in Ae2a,b−/− mice.Am J Pathol. 2006; 169: 165-176Google Scholar and secretin-stimulated biliary HCO3− excretion.5Banales J.M. Arenas F. Rodriguez-Ortigosa C.M. et al.Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.Hepatology. 2006; 43: 266-275Google Scholar Alkalinization of pHi in lymphocytes has been reported to affect their proliferation, differentiation, and activation status.6Lardner A. The effects of extracellular pH on immune function.J Leukoc Biol. 2001; 69: 522-530Google Scholar Because AE2 activity might influence acid-base equilibrium and cell function, not only in lymphocytes but also in biliary epithelial cells, alterations of this transporter were postulated to play an etiopathogenic role in primary biliary cirrhosis (PBC).7Prieto J. Qian C. Garcia N. et al.Abnormal expression of anion exchanger genes in primary biliary cirrhosis.Gastroenterology. 1993; 105: 572-578Crossref Scopus (121) Google Scholar, 8Medina J.F. Martinez A. Vazquez J.J. et al.Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis.Hepatology. 1997; 25: 12-17Google Scholar, 9Melero S. Spirli C. Zsembery A. et al.Defective regulation of cholangiocyte Cl−/HCO3− and Na+/H+ exchanger activities in primary biliary cirrhosis.Hepatology. 2002; 35: 1513-1521Google Scholar PBC is an autoimmune disease of unknown pathogenesis that results in destruction of small- and medium-sized bile ducts.10Talwalkar J.A. Lindor K.D. Primary biliary cirrhosis.Lancet. 2003; 362: 53-61Google Scholar, 11Kaplan M.M. Gershwin M.E. Primary biliary cirrhosis.N Engl J Med. 2005; 353: 1261-1273Google Scholar The disease occurs mainly in middle-aged women and is characterized by portal inflammation surrounding interlobular bile ducts and the presence of antimitochondrial antibodies (AMA). Indeed, up to 90%–95% of patients develop AMA against some components of the 2-oxo dehydrogenase complexes, mainly the inner lipoyl domain in the E2 component of the pyruvate dehydrogenase complex (PDC-E2).12Surh C.D. Coppel R. Gershwin M.E. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis Implication for a conformational autoepitope.J Immunol. 1990; 144: 3367-3374Google Scholar Although AMA might not be involved in the pathogenesis of the disease, T-cell immune responses against PDC-E2 determinants seem to be implicated in organ damage.11Kaplan M.M. Gershwin M.E. Primary biliary cirrhosis.N Engl J Med. 2005; 353: 1261-1273Google Scholar, 13He X.S. Ansari A.A. Ridgway W.M. et al.New insights to the immunopathology and autoimmune responses in primary biliary cirrhosis.Cell Immunol. 2006; 239: 1-13Google Scholar Despite the strong association with autoimmune phenomena, PBC responds poorly to classic immunosuppressants. The fact that ursodeoxycholic acid, a bile acid that induces bicarbonate-rich choleresis, may improve the clinical course of PBC14Corpechot C. Carrat F. Bahr A. et al.The effect of ursodeoxycholic acid therapy on the natural course of primary biliary cirrhosis.Gastroenterology. 2005; 128: 297-303Abstract Full Text Full Text PDF Scopus (313) Google Scholar is compatible with the concept that defective Cl−/HCO3− exchange may play a role in the pathogenesis of the disease.9Melero S. Spirli C. Zsembery A. et al.Defective regulation of cholangiocyte Cl−/HCO3− and Na+/H+ exchanger activities in primary biliary cirrhosis.Hepatology. 2002; 35: 1513-1521Google Scholar, 15Prieto J. Garcia N. Marti-Climent J.M. et al.Assessment of biliary bicarbonate secretion in humans by positron emission tomography.Gastroenterology. 1999; 117: 167-172Google Scholar Consistent with this view, AE2 gene expression has been reported to be reduced in liver biopsy specimens and blood mononuclear cells from patients with PBC.7Prieto J. Qian C. Garcia N. et al.Abnormal expression of anion exchanger genes in primary biliary cirrhosis.Gastroenterology. 1993; 105: 572-578Crossref Scopus (121) Google Scholar, 8Medina J.F. Martinez A. Vazquez J.J. et al.Decreased anion exchanger 2 immunoreactivity in the liver of patients with primary biliary cirrhosis.Hepatology. 1997; 25: 12-17Google Scholar Although regulation of pHi appears to be important to modulate lymphocyte function, there is no information on the consequences of altered Cl−/HCO3− exchange activity on the homeostasis of the immune system. Similarly, it is not known whether defective AE2 function may be damaging for bile duct cells. Previously we had generated Ae2a,b−/− mice with a widespread disrupted expression of the 3 major Ae2 variant isoforms Ae2a, Ae2b1, and Ae2b2.16Medina J.F. Recalde S. Prieto J. et al.Anion exchanger 2 is essential for spermiogenesis in mice.Proc Natl Acad Sci U S A. 2003; 100: 15847-15852Google Scholar Early in life, these mice show increased perinatal mortality and male sterility due to azoospermia,16Medina J.F. Recalde S. Prieto J. et al.Anion exchanger 2 is essential for spermiogenesis in mice.Proc Natl Acad Sci U S A. 2003; 100: 15847-15852Google Scholar as well as impaired stimulated gastric acid secretion,4Recalde S. Muruzabal F. Looije N. et al.Inefficient chronic activation of parietal cells in Ae2a,b−/− mice.Am J Pathol. 2006; 169: 165-176Google Scholar growth retardation, bone abnormalities, and deafness (see Recalde et al4Recalde S. Muruzabal F. Looije N. et al.Inefficient chronic activation of parietal cells in Ae2a,b−/− mice.Am J Pathol. 2006; 169: 165-176Google Scholar). We now describe that mature/elderly Ae2a,b−/− mice show immunologic and hepatobiliary features similar to those found in PBC. Our present data therefore indicate that the universal deficiency of Ae2 function in Ae2a,b−/− mice may eventually disturb the immune system and the biliary epithelium besides other tissues, predisposing the animals to develop a PBC-like syndrome in addition to the previously documented PBC-unrelated phenotypic alterations. Ae2a,b-targeted mice were generated as described.16Medina J.F. Recalde S. Prieto J. et al.Anion exchanger 2 is essential for spermiogenesis in mice.Proc Natl Acad Sci U S A. 2003; 100: 15847-15852Google Scholar Most experimental animals were bred in heterozygous couples against an FVB/N background. Confirmatory experiments were also performed in mice bred in heterozygous couples against different backgrounds (129/Sv, Balb/c, and SJL). Breeding was performed at the CIMA animal facility (University of Navarra). Each Ae2a,b−/− mouse was housed together with respective control littermates of the same sex in a separated wire mesh cage. All animal procedures were approved by the Institutional Animal Care and Use Committee. Blood was extracted from anesthetized mice at 6, 12, and 15 months of age, and serum aliquots were stored at −20°C for simultaneous analysis. Serum levels of alanine aminotransferase and total alkaline phosphatase were determined in a Hitachi 911 analyzer (Roche/Hitachi, Indianapolis, IN), including a normalizing external control. Total alkaline phosphatase values were corrected for the hepatic fraction of alkaline phosphatase (hALP) by determining in each mouse the relative levels of hALP after its separation from the bone-derived alkaline phosphatase by automated gel electrophoresis in Hydragel ISO-PAL agarose gel in the Hydrasys system (Sebia, Norcross, GA). For visualization and densitometric analysis of separated bands, we used a Hyrys-2 densitometer (Sebia). Total immunoglobulin (Ig) M and IgG levels were measured with mouse enzyme-linked immunosorbent assay kits (Assay-Designs, Ann Arbor, MI, and Bethyl Laboratories, Montgomery, TX, respectively). After preparation of PDC extracts from mouse hearts as described,17Jones D.E. Palmer J.M. Yeaman S.J. et al.Breakdown of tolerance to pyruvate dehydrogenase complex in experimental autoimmune cholangitis: a mouse model of primary biliary cirrhosis.Hepatology. 1999; 30: 65-70Google Scholar extract proteins were electrophoresed (10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis; 15 μg/lane) and electrotransferred to nitrocellulose membranes. Blocked membrane strips were incubated with 1:3000 diluted mouse sera (room temperature, 1 hour) and washed and incubated with 1:10,000 diluted peroxidase-labeled goat anti-mouse IgG plus IgM antibodies (Pierce, Rockford, IL). Once Western Lightning Chemiluminescence Reagent (Perkin Elmer, Boston, MA) was added, chemiluminescent bands were detected with an ImageQuant ECL system (GE Healthcare, Buckinghamshire, England). For quadrupole/time-of-flight mass spectrometry (Q-TOF/MS) of the immunoreactive PDC band, extract aliquots were loaded in parallel gels (10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis): one for band visualization with SimplyBlue Safestain (Invitrogen, Carlsbad, CA) and the other for excision of the 65-kilodalton band. After proteolytic digestion and reverse-phase separation of tryptic digests, peptides were analyzed by mass spectrometry followed by data processing with MassLynx 4.0 and ProteinLynx Global Server 2.0 (Waters, Manchester, UK) connected to the Swiss-Prot database.18Santamaria E. Munoz J. Fernandez-Irigoyen J. et al.Molecular profiling of hepatocellular carcinoma in mice with a chronic deficiency of hepatic s-adenosylmethionine: relevance in human liver diseases.J Proteome Res. 2006; 5: 944-953Google Scholar We expressed in Escherichia coli the mouse PDC-E2 inner lipoyl domain peptide 170–313 (National Center for Biotechnology Information protein database NP_663589), fused to an N-terminal His-Patch thioredoxin sequence and a C-terminal 6xHis tag (total mol wt, 31,340). It contains the 24-aa sequence GDLLAEIETDKATIGFEVQEEGYL, which encompasses the major PBC-specific autoantigens (previously referred to as peptides 167–186, 163–176, and 165–174).11Kaplan M.M. Gershwin M.E. Primary biliary cirrhosis.N Engl J Med. 2005; 353: 1261-1273Google Scholar, 13He X.S. Ansari A.A. Ridgway W.M. et al.New insights to the immunopathology and autoimmune responses in primary biliary cirrhosis.Cell Immunol. 2006; 239: 1-13Google Scholar PDC-E2 complementary DNA fragment (432 base pairs long) produced by polymerase chain reaction (PCR) on mouse liver complementary DNA (see Table 1 for primers used) was subcloned into pBAD/Thio TOPO vector (ThioFusion Expression Kit; Invitrogen) for l-arabinose–induced expression in E coli. After fast protein liquid chromatography purification, recombinant peptide was electrophoresed (1 μg/lane) and electrotransferred. Immunoblots with 1:3000 diluted mouse sera and chemiluminescence detection were performed as described previously with mouse PDC extract (see detailed supplementary methodology; supplementary material available online at www.gastrojournal.org). Type determination of AMA was performed by using peroxidase-labeled secondary antibodies against either mouse IgM or IgG (Pierce).Table 1Specific Mouse Primers Designed for PCR AmplificationaAmplified PDC-E2 complementary DNA sequence was used to express the recombinant peptide. and Real-Time PCRProductPrimersCatalase (Cat)5′-GTTCGATTCTCCACAGTCAC-3′ (sense)5′-GAAACCTGATGGAGAGACTC-3′ (antisense)Glutathione peroxidase 2 (Gpx2)5′-AGTTCGGACATCAGGAGAAC-3′ (sense)5′-TCAAAGTTCCAGGACACGTC-3′ (antisense)Glutathione peroxidase 3 (Gpx3)5′-GAAGTCTAAGACAGACTGCC-3′ (sense)5′-AGGTATTGGTCTGTCAGACC-3′ (antisense)Heme oxygenase 1 (Hmox1)5′-GCACTATGTAAAGCGTCTCC-3′ (sense)5′-CAGAGTGTTCATTCGAGCAC-3′ (antisense)NADPH oxidase organizer 1 (Noxo1)5′-TCCAGACATTTGCCTTCTCC-3′ (sense)5′-AAACGTACCAGTCCTCGACC-3′ (antisense)Superoxide dismutase 3 (Sod3)5′-CCTGACAGGTGCAGAGAACC-3′ (sense)5′-CCTATCTTCTCAACCAGGTC-3′ (antisense)Thioredoxin interacting protein (Txnip)5′-GCTATGAAGACACACTTCTCC-3′ (sense)5′-GATCGAGAAAAGCCTTCACC-3′ (antisense)Thioredoxin reductase 1 (Txnrd1)5′-CAGGATTTCTGGCTGGTATC-3′ (sense)5′-GTTCCTGCTTCGATCTGTTC-3′ (antisense)Thioredoxin reductase 3 (Txnrd3)5′-CTGTATTACGAGTGACGACC-3′ (sense)5′-TAGGATCCCACTTTCTCTGC-3′ (antisense)Histocompatibility 2, D region, locus 1 (H2-D1)5′-GTGTGGACTTGGTGACAGAC-3′ (sense)5′-GTTGGCTGTGGAAGGGAAC-3′ (antisense)Ubiquitin-specific peptidase 2 (Usp2)5′-TTTCCTTCTGGATGGTCTCC-3′ (sense)5′-TCAGGGTAACCTCTCTTTGC-3′ (antisense)Glyceraldehyde-3-phosphate dehydrogenase (Gapdh)5′-CCAAGGTCATCCATGACAAC-3′ (sense)5′-TGTCATACCAGGAAATGAGC-3′ (antisense)PDC-E2 peptideaAmplified PDC-E2 complementary DNA sequence was used to express the recombinant peptide.5′-CAGGATATTGAGGCCTTTAAAAATTATACATTGGAT-3′ (sense)5′-TAAGCTGGTCACTTCTGTTGGC-3′ (antisense)NADPH, reduced nicotinamide adenine dinucleotide phosphate.a Amplified PDC-E2 complementary DNA sequence was used to express the recombinant peptide. Open table in a new tab NADPH, reduced nicotinamide adenine dinucleotide phosphate. Following the killing of the animals, spleens were removed and weighed. Isolated splenocytes (100-μL aliquots with 1.5 × 106 cells in Dulbecco phosphate-buffered saline) were incubated with CD3 rat anti-mouse PE-Cy5 conjugated and either CD4 or CD8 rat anti-mouse fluorescein isothiocyanate labeled (each from BD Pharmingen, San Diego, CA). Similarly, splenocytes were triple labeled for CD4, CD25, and FoxP3 with Mouse Regulatory T-Cell Staining Kit (eBioscience, San Diego, CA). Populations were analyzed in a FACSCalibur flow cytometer with CellQuest Pro software (BD Biosciences, San Jose, CA). FoxP3 immunostaining was performed on sections of formalin-fixed spleen pieces embedded in paraffin. Following dewaxing, hydration, microwave antigen retrieval (in TE buffer, 750 W and 375 W, 10 minutes each), and treatment with peroxidase-blocking reagent (Dako, Glostrup, Denmark), slides were incubated overnight with 1:100 diluted affinity-purified anti-mouse/rat FoxP3 (eBioscience). Washes were followed by incubations with 1:200 diluted polyclonal rabbit anti-rat immunoglobulins, adding EnVision horseradish peroxidase against rabbit and DAB+ liquid chromogen (all from Dako) and counterstaining with Mayer's hematoxylin solution (Merck-KGaA, Darmstadt, Germany). FoxP3+ cell ratio (area of stained splenocytes/area of total splenocytes) was calculated using image analysis software for random acquisition with a motorized Axioplan 2ie microscope (Zeiss, Oberkochen, Germany), operated through MetaMorph imaging software (Molecular Devices, Downingtown, PA), and further image segmentation, kindly designed by Dr Ortiz-de-Solórzano (CIMA, University of Navarra). A total of 20 × 106 splenocytes loaded with 2.5 μmol/L 2′,7′-bis(carboxyethyl)-5,6-carboxyfluoroscein (Molecular Probes, Eugene, OR) in supplemented Tris/3-(N-morpholino)propanesulfonic acid, pH 7.4, were measured in the FACSCalibur flow cytometer for the ratio of emission wavelengths 530/650 nm upon excitation at 488 nm, essentially as described.19Musgrove E. Rugg C. Hedley D. Flow cytometric measurement of cytoplasmic pH: a critical evaluation of available fluorochromes.Cytometry. 1986; 7: 347-355Google Scholar We estimated pHi values from calibration curves performed for each sample using the nigericin clamp technique,20Thomas J.A. Buchsbaum R.N. Zimniak A. et al.Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.Biochemistry. 1979; 18: 2210-2218Google Scholar in which 2′,7′-bis(carboxyethyl)-5,6-carboxyfluoroscein–loaded splenocytes were incubated in different aliquots of nigericin-containing media at pH 7, 7.2, 7.4, 7.6, 7.8, and 8. Ae2a,b−/− and wild-type splenocytes (2 × 105 in 200 μL of RPMI complete medium) in 96-well U-bottom plates were incubated with either hamster anti-mouse CD3ε antibody (BD Pharmingen; clone 145-2C11; 0.1 μg/well) or simply medium as controls (n = 6 each). After 72-hour incubation (37°C), pooled supernatants (150 μL from each well) were analyzed for 22 cytokines using RayBio Mouse Cytokine Antibody Array I membranes (RayBiotech, Norcross, GA). Chemiluminescence signals (duplicates) were quantified with the ImageQuant ECL system and normalized with the kit membrane-positive controls. Sections of formalin-fixed liver specimens embedded in paraffin were used for H&E and Masson's trichrome stainings. For immunohistochemistry, we also used OCT liver sections fixed with acetone and blocked with 0.3% H2O2 in methanol and then with SuperBlock buffer (Pierce). Incubations with 1:100 diluted antibodies (rat anti-mouse CD4, CD8, CD11b, and CD19; BD Pharmingen) were followed by incubations with rabbit anti-rat Igs and further staining procedures as described previously for FoxP3 staining of spleen sections, visualizing cell populations with the motorized Axioplan 2ie microscope. For FACScan analysis of T-cell populations, fresh liver pieces were incubated for 15 minutes (37°C) in 10 mL of RPMI (Gibco-Invitrogen, Paisley, UK) plus GlutaMAX (Gibco-Invitrogen) with collagenase (400 Mandl-U/mL) and deoxyribonuclease I (50 μg/mL). Digested tissues were passed through a 70-μm nylon mesh, and mononuclear cells were isolated in 35% Percoll gradient (room temperature). Fluorescence-activated cell sorter (FACS) analysis of liver lymphocytes was performed as described for splenocytes. Intrahepatic bile duct units were isolated from male wild-type and Ae2a,b−/− mice (each 5 months old) as described.21Mennone A. Alvaro D. Cho W. et al.Isolation of small polarized bile duct units.Proc Natl Acad Sci U S A. 1995; 92: 6527-6531Google Scholar Intrahepatic bile duct units were resuspended in fully supplemented Dulbecco's modified Eagle medium/F-12 medium (Gibco-Invitrogen)22Salter K.D. Roman R.M. LaRusso N.R. et al.Modified culture conditions enhance expression of differentiated phenotypic properties of normal rat cholangiocytes.Lab Invest. 2000; 80: 1775-1778Google Scholar and seeded on 250-mL Cellstar flasks (Grenier, Frickenhausen, Germany) coated with rat tail collagen (∼2-mm-thick monolayer).23Vroman B. LaRusso N.F. Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.Lab Invest. 1996; 74: 303-313Google Scholar Cholangiocytes, grown as clusters derived from intrahepatic bile duct units in a monolayer fashion, were passaged at ∼90% confluence, being rendered free of fibroblasts by treatment with dispase at the time of passages (1.66 mg/mL of Dulbecco's modified Eagle medium/F-12, 1 hour at 37°C) and also between passages (1 mg/mL, for 20 minutes) if contaminating fibroblasts were observed when the medium was changed every 48 hours. Pure differentiated cholangiocytes (positive staining for cytokeratin 7 and negative for vimentin and desmin) were obtained by passages 3–4, the differentiated phenotype being maintained for over 15 passages. Basal pHi was determined exactly as previously described for rat cholangiocytes by Banales et al.5Banales J.M. Arenas F. Rodriguez-Ortigosa C.M. et al.Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.Hepatology. 2006; 43: 266-275Google Scholar Mouse cholangiocytes with 9 passages were restored for cell polarity (see detailed methodology in supplementary material online at www.gastrojournal.org), and TRIzol (Gibco-Invitrogen) was added for total RNA extraction. A preliminary pathway-focused gene expression test was performed in one sample of each genotype by using the RT2 Profiler PCR Array for Mouse Oxidative Stress and Antioxidant Defense (catalog #PAMM-065A), with the RT2 Real-Time PCR Master Mix SYBR Green (both from Superarray Bioscience Corp, Frederick, MD), in an iQ5 Apparatus (Bio-Rad, Hercules, CA). Further validation/analysis of expression changes was performed separately (n = 5 for each genotype) by real-time PCR (duplicates) in the iQ5 Apparatus with iQ SYBR Green Supermix and specific primers (Table 1), using Gapdh as normalizing control. Expression changes for genes involved in antigen presentation (Table 1) were similarly analyzed. Statistical analysis was performed with SPSS version 12.0 (SPSS Inc, Chicago, IL). Results are expressed as mean ± SD. Analysis of variance residuals for each variable had a normal distribution (Shapiro–Wilk test), and data were analyzed using analysis of variance (with genotype as fixed factor and littermate as random factor), followed by the Tukey HSD test. Two-tailed P values <.05 were considered statistically significant. At 15 months of age, Ae2a,b−/−, Ae2a,b+/−, and Ae2a,b+/+ mice were killed and spleens and livers were removed. Ae2a,b−/− mice exhibited gross splenomegaly, the average size of their spleens being nearly twice as large as that of Ae2a,b+/+ mice (Figure 1A). In fact, the spleen/body weight ratio was significantly higher in Ae2a,b−/− mice than in wild-type littermates (Figure 1B). Measurement of pHi in splenocytes from Ae2a,b−/− mice showed increased values compared with those from heterozygous and wild-type littermates (Figure 1C). Upon stimulation of splenocytes with anti-CD3 antibody, cells from Ae2a,b−/− mice produced more interferon gamma and interleukin (IL)-12p70 than Ae2a,b+/+ splenocytes (Figure 1D). No significant differences were observed for other cytokines (such as IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, heterodimeric IL-12p40p70, and tumor necrosis factor α) included in the dot array used. FACS analysis of the spleen T-cell populations showed a striking increase in both the proportion and the absolute number of CD8+ lymphocytes in Ae2a,b−/− mice compared with heterozygous and wild-type littermates (Figure 2A and B). There were no differences in the total number of CD4+ lymphocytes between genotypes (not shown), but the proportion of these cells was dramatically reduced in Ae2a,b−/− mice as a consequence of CD8+ expansion (Figure 2A and B), with marked inversion of the CD4+/CD8+ ratio (Figure 2C). Natural regulatory T cells (Treg), a subset of CD4+ lymphocytes that express CD25 and the transcription factor FoxP3, are critical for peripheral immune tolerance to self-antigens. Recently, a deficiency of these cells was reported in PBC.24Lan R.Y. Cheng C. Lian Z.X. et al.Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis.Hepatology. 2006; 43: 729-737Google Scholar Using FACS analysis, we found that the proportion of CD4+ splenocytes expressing FoxP3 was lower in Ae2a,b−/− mice than in control littermates (Figure 2D). In confirmation of these results, immunohistochemical studies of paraffin-embedded spleen sections showed a significant decrease in FoxP3+ cells in Ae2a,b−/− mice compared with Ae2a,b+/+ littermates, with intermediate values in Ae2a,b+/− mice (Figure 2E and F). A hallmark of PBC is the development of specific AMA against components of the 2-oxo dehydrogenase complexes, mainly the inner lipoyl domain of the PDC-E2 subunit.12Surh C.D. Coppel R. Gershwin M.E. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis Implication for a conformational autoepitope.J Immunol. 1990; 144: 3367-3374Google Scholar Interestingly, we found that most Ae2a,b−/− mice spontaneously developed serum AMA that recognize a 65-kilodalton protein of mouse PDC extract (Figure 3A). Q-TOF/MS confirmed that the recognized 65-kilodalton band corresponds to Mus musculus PDC-E2 (Figure 3B). We further ascertained that AMA developed in Ae2a,b−/− mice are equivalent to AMA in patients with PBC by testing mouse sera against a recombinant mouse PDC-E2 peptide with the PBC autoepitope.12Surh C.D. Coppel R. Gershwin M.E. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis Implication for a conformational autoepitope.J Immunol. 1990; 144: 3367-3374Google Scholar Immunoblot analysis showed that 9 out of 11 Ae2a,b−/− mice had AMA against this epitope (Figure 4A). We also detected slight immunoreactivity in 6 out of 13 Ae2a,b+/− littermates, whereas the remaining heterozygotes, like all the wild-type animals, tested negatively (Figure 4A). Chronological analysis of AMA-positive Ae2a,b−/− mice and Ae2a,b+/− littermates showed a tendency to increase their immunoreactivity over time (Figure 4B and C). Typing of AMA developed in our targeted mice revealed that they are IgG autoantibodies. Elevated levels of total IgM and IgG in serum are also characteristic of PBC.11Kaplan M.M. Gershwin M.E. Primary biliary cirrhosis.N Engl J Med. 2005; 353: 1261-1273Google Scholar We found that Ae2a,b−/− mice have higher IgM levels (1.02 ± 0.62 mg/mL; n = 11) than wild-type littermates (0.49 ± 0.12 mg/mL; n = 6; P < .01). However, IgM values in heterozygous animals (0.70 ± 0.71 mg/mL; n = 10) showed no significant differences from those in Ae2a,b−/− (P = .074) or Ae2a,b+/+ littermates (P = .388). Similarly, IgG levels in Ae2a,b−/− mice (2.50 ± 0.16 mg/mL; n = 6) were higher than those in wild-t
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