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

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-ODN s 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.