Breakthrough of immune self-tolerance to calreticulin induced by CpG-oligodeoxynucleotides as adjuvant

Fukushima J Med Sci. 2007 Dec;53(2):95-108. doi: 10.5387/fms.53.95.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adjuvants, Immunologic / pharmacology*
  • Amino Acid Sequence
  • Animals
  • Blotting, Western
  • Calreticulin / immunology*
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Hepatitis, Autoimmune / immunology*
  • Humans
  • Immune Tolerance*
  • Immunization
  • Interferon-gamma / biosynthesis
  • Interleukin-4 / biosynthesis
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Oligodeoxyribonucleotides / pharmacology*

Substances

  • Adjuvants, Immunologic
  • CPG-oligonucleotide
  • Calreticulin
  • Oligodeoxyribonucleotides
  • Interleukin-4
  • Interferon-gamma