Fukushima J. Med. Sci.,
Vol. 53, No. 2, 2007

[Original Article]

BREAKTHROUGH OF IMMUNE SELF-TOLERANCE TO CALRETICULIN
INDUCED BY CpG-OLIGODEOXYNUCLEOTIDES AS ADJUVANT

KAZUMICHI ABE¹⁾, HIROMASA OHIRA¹⁾, HIROKO KOBAYASHI¹⁾,
HIRONOBU SAITO¹⁾, ATSUSHI TAKAHASHI¹⁾, TSUYOSHI RAI¹⁾,
YUKIKO KANNO¹⁾, KYOKO MONOE¹⁾, HIROSHI WATANABE¹⁾,
ATSUSHI IRISAWA¹⁾ and YUKIO SATO²⁾

Departments of ¹⁾Internal Medicine II and ²⁾Health Center, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan

(Received June 2, 2006, accepted October 9, 2007)

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-γ 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.

Key words: autoimmune hepatitis, primary biliary cirrhosis, autoantibody, hepatocyte, Western blot

阿部和道，大平弘正，小林浩子，斉藤広信，高橋敦史，雷　　毅，菅野有紀子，物江恭子，渡辺浩志，入澤篤志，佐藤由紀夫

Corresponding author: Hiromasa Ohira
E-mail: h-ohira@fmu.ac.jp