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• Lack of serum antibodies to membrane-bound carbonic anhydrase iv in primary biliary cirrhosis
  Pietro Invernizzi
  Published on: 18 April 2016

• Published on: 18 April 2016

  Lack of serum antibodies to membrane-bound carbonic anhydrase iv in primary biliary cirrhosis

  • Pietro Invernizzi, MD, PhD
  • Other Contributors:
    • Carlo, Selmi, Massimo Zuin, Mauro Podda

  Dear Editor,

  Nishimori and colleagues have recently reported the presence of autoantibodies against carbonic anhydrase IV (anti-CA IV) in patients with autoimmune pancreatitis.[1] Further, serum antibodies to CA II (anti-CA II) were observed in several autoimmune conditions. We have now investigated the presence of anti-CA IV and anti-CA II in a large series of sera from patients with primary biliary cirrhosis (P...

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  Dear Editor,

  Nishimori and colleagues have recently reported the presence of autoantibodies against carbonic anhydrase IV (anti-CA IV) in patients with autoimmune pancreatitis.[1] Further, serum antibodies to CA II (anti-CA II) were observed in several autoimmune conditions. We have now investigated the presence of anti-CA IV and anti-CA II in a large series of sera from patients with primary biliary cirrhosis (PBC) and controls.

  CA II is known to be expressed in the cytosol of various types of epithelial cells including those lining bile ducts, renal tubules, and salivary ducts. For this reason, CA II was suggested as a common antigen in conditions characterized by an autoimmune aggression against epithelia. In autoimmune pancreatitis serum anti-CA II are useful diagnostic tools while in PBC they were first detected in 5/6 sera from patients with antimitochondrial antibody (AMA) negative PBC [2]. Subsequent studies, however, demonstrated prevalence rates as high as 46% in PBC sera but failed to confirm their specificity for AMA-negative sera [3,4]. Interestingly, anti-CA II were also shown to inhibit enzyme activity [5].

  Besides the cytosolic CA II, the CA family also includes a highly active membrane-bound enzyme that was coined CA IV [6]. Both CA II and CA IV are abundantly expressed in human bile duct epithelial cells. Interestingly, mainly due to the sequence homology between CA II and CA IV and the CA IV localization on cell membranes, Nishimori and colleagues hypothesized that the exposed CA IV might be more immunogenic than the cytosolic CA II [1].

  Seventy sera from patients with PBC [60 AMA-positive; all anti-HCV negative; 63 women; mean age¡ÓSD, 60¡Ó10 years] who attended our tertiary referral centre were consecutively enrolled in the study. Control sera were obtained from 50 healthy subjects matched with patients for sex and age class („T50 years vs. >50 years). All sera were tested by immunoblotting for anti-CA IV and anti-CA II as previously described [3]. Briefly, proteins were denaturated and separated (10ƒÝg/lane) on a 1.5 mm SDS-PAGE 12%. Proteins were then transferred onto nitrocellulose (pore size 0.45 mm) using a Semi-Dry transfer system. The nitrocellulose membrane was cut into 4-mm strips, and, after blocking with 5% non-fat milk, all strips were incubated with serum samples diluted 1:100 and 1:200 for anti-CA IV and 1:100 for anti-CA II. A Rabbit HRP-conjugated antibodies against human immunoglobulins G, A, and M (Dako, Glostrup, Denmark) was diluted 1:1000 and used as secondary antibody. Peroxidase development was obtained with 0.05% 4-chloro-1-naphthol in TBS containing 20% methanol and 0.05% H2O2. A rabbit polyclonal anti-human CA IV antisera [7] was used as positive control throughout the study. CA IV antigen and anti-CA IV antisera were provided by Dr William S. Sly (St. Louis, MO, USA).

  Results demonstrated no reactivity against CA IV in any of the PBC or healthy control sera. On the other hand, similar to previous reports, anti -CA II were detected in 6/70 (9%) sera from patients with PBC but were absent in control sera.

  In summary, we submit that the hypothesis that antibodies against the membrane-bound CA IV might play a role in PBC should be rejected, based on our experimental data on a large series of sera. Our finding may be secondary to a different cellular expression of CA IV in the target organ (i.e., pancreatic and bile ducts), but only specific tissue studies will give an answer. At the present status of knowledge, therefore, anti-CA IV should be regarded as specific to autoimmune pancreatitis and research should then focus to better define their possible role in this condition.

  References

  1. Nishimori I, Miyaji E, Morimoto K, et al.: Serum antibodies to carbonic anhydrase IV in patients with autoimmune pancreatitis. Gut 2005,54:274-281.

  2. Gordon SC, Quattrociocchi-Longe TM, Khan BA, et al.: Antibodies to carbonic anhydrase in patients with immune cholangiopathies. Gastroenterology 1995,108:1802-1809.

  3. Invernizzi P, Battezzati PM, Crosignani A, et al.: Antibody to carbonic anhydrase II is present in primary biliary cirrhosis (PBC) irrespective of antimitochondrial antibody status. Clin Exp Immunol 1998,114:448-454.

  4. Nishimori I, Akisawa N, Miyaji E, et al.: Serum antibody to carbonic anhydrase II in patients with chronic viral hepatitis: a review of its prevalence in liver diseases. Hepatol Res 2004,30:210-213.

  5. Botre F, Botre C, Podesta E, et al.: Effect of anti-carbonic anhydrase antibodies on carbonic anhydrases I and II. Clin Chem 2003,49:1221-1223. 6. Sly WS, Hu PY: Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 1995,64:375-401.

  7. Zhu XL, Sly WS: Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney. J Biol Chem 1990,265:8795-8801.

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  Conflict of Interest:
  None declared.

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