title: Breakthrough of immune self-tolerance to calreticulin induced by CpG-oligodeoxynucleotides as adjuvant.
abstract: Reportedly, bacterial DNA containing unmethylated cytosine-guanosine dinucleotide motif-containing oligodeoxynucleotides (CpG-ODNs) can induce Th1-type adjuvant effects. We produced autoantibodies and induced hepatitis in mice using extracted proteins from human hepatocytes with CpG-ODNs as adjuvant. Western blot analysis was performed of sera from immunized mice and two patients with autoimmune hepatitis (AIH). When a common band was detected, N-terminal amino acid sequencing was performed to determine its site. For detection of antibodies against the identified protein (calreticulin), ELISA was performed of sera of 50 patients with AIH: 45 with primary biliary cirrhosis (PBC), 24 with chronic hepatitis C (CH), and 24 healthy controls. Mice were immunized with calreticulin protein with CpG-ODNs as adjuvant. Several reacted bands were detected in their sera; in addition, a common band to the sera of patients with AIH was detected at 60 kDa. Subsequent N-terminal amino acid sequencing revealed that the protein was human calreticulin. ELISA showed that, of patients with AIH, PBC, and CH, 30.0% (15/50), 17.8% (8/45), and 12.5% (3/24), respectively, were positive for anti calreticulin antibodies. Splenocytes from immunized mice produced IFN-gamma after they were pulsed with calreticulin protein. Histological analyses of liver specimens taken from mice immunized with calreticulin protein together with CpG-ODNs showed spotty and focal necrosis. Immunofluorescence analysis showed increased expression of calreticulin in the liver treated with CpG-ODNs. These results suggest that a breakthrough of immune self tolerance to calreticulin is induced with CpG-ODNs as adjuvant and that calreticulin protein might be a target antigen in this model.
authors.affiliation: Department of Internal Medicine II, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan.
authors.forename: Kazumichi
authors.initials: K
authors.lastname: Abe
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Ohira
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Hironobu
authors.initials: H
authors.lastname: Saito
authors.forename: Atsushi
authors.initials: A
authors.lastname: Takahashi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Rai
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kanno
authors.forename: Kyoko
authors.initials: K
authors.lastname: Monoe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Atsushi
authors.initials: A
authors.lastname: Irisawa
authors.forename: Yukio
authors.initials: Y
authors.lastname: Sato
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D037282:Calreticulin / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019693:Hepatitis, Autoimmune / Q000276:immunology*; D006801:Humans; D007108:Immune Tolerance; D007114:Immunization; D007371:Interferon-gamma / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*
chemical_list: D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D037282:Calreticulin; D009838:Oligodeoxyribonucleotides; D015847:Interleukin-4; D007371:Interferon-gamma