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• Stimulatory autoantibodies against PDGF receptor in scleroderma
  Armando Gabrielli
  Published on: 13 April 2016
• Autoantibodies to PDGF receptor and systemic sclerosis
  Jean-Baptiste Demoulin
  Published on: 13 April 2016

• Published on: 13 April 2016

  Stimulatory autoantibodies against PDGF receptor in scleroderma

  • Armando Gabrielli
  • Other Contributors:
    • Silvia Svegliati, Enrico V. Avvedimento

  Dear Editor,

  We read with interest the work by Balada et al. publishedin Ann Rheum Dis 2008 Feb 13; [Epub ahead of print].

  Although we noticed the authors pointed out in the title and along the text that anti-PDGFR antibodies in their study were detected by non-functional assays, we thought necessary a comment on their findings, for a better understanding of the subject, mainly for those readers who may n...

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  Dear Editor,

  We read with interest the work by Balada et al. publishedin Ann Rheum Dis 2008 Feb 13; [Epub ahead of print].

  Although we noticed the authors pointed out in the title and along the text that anti-PDGFR antibodies in their study were detected by non-functional assays, we thought necessary a comment on their findings, for a better understanding of the subject, mainly for those readers who may not be familiar with the immunological methods described in the manuscript.We have noticed several methodological problems, that might limit or question the authors’ conclusions.

  1. The authors did not provide information on the specificity of binding assays employed in their study, since no control peptide (e.g. EGFR extracellular domain) was included in either assays. This is a mandatory and important control, considering that the immunoassays, chosen by the authors to detect anti-PDGFR antibodies, were performed under denaturing conditions. In fact, it is likely that once the 550 aa PDGFR polypeptide is used to coat plastic wells (ELISA assay) or is denatured prior to electrophoresis (western blot) several linear epitopes are unmasked which non specifically bind highly concentrated IgG contained in 1:5 diluted human serum. Immunoprecipitation and immunoblot will solve this problem by using these assays, we did not find anti PDGFR binding antibodies in normal subject (2).

  2. Carrying out the experiments using a serial dilution of sera would better discriminate between positive and negative samples: the authors used a 1:5 serum dilution in all the experiments! Considering 1:5 dilution, the serum IgG concentration is approximately 2 mg/ml, that is at least ten fold higher than the one we used in our report (2) to discriminate IgG purified from serum of SSc patients from IgG purified from serum of controls.

  3. We would like to point out that in supplemental data (2) we also used a highly sensitive ECL detection system to reveal the immunoblots. The main difference between our and their immunoassay in detecting denatured PDGFR, is in the primary reagent: we used immunopurified IgG at a concentration of 200 micrograms/ml, whereas Balada et al. used sera at 1:5 dilution (1). This may account for the different results.

  In conclusion, we wish to call attention to the conditions that may be used by the authors to reproduce our experimental data on the detectionof specific anti PDGFR antibodies. In the absence of adequate controls, the presence of specific of anti-PDGFR autoantibodies in healthy humans is questionable.

  References

  1. Balada E, Simeón-Aznar CP, Ordi-Ros J, Rosa-Leyva M, Selva-O'Callaghan A, Pardos-Gea J, Fonollosa-Pla V, Vilardell-Tarrés M.Anti-PDGFR-{alpha} antibodies measured by non-bioactivity assays are not specific of systemic sclerosis. Ann Rheum Dis 2008 Feb 13 [Epub ahead of print].

  2. Svegliati Baroni S, Santillo MR Bevilacqua F, Luchetti M, Spadoni T, Mancini M , Fraticelli P, Sambo P, Funaro A , Kazlauskas A, Avvedimento EV, Gabrielli A Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis (scleroderma). N Engl J Med 2006;354;2667-2676.

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  Conflict of Interest:
  None declared.

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• Published on: 13 April 2016

  Autoantibodies to PDGF receptor and systemic sclerosis

  • Jean-Baptiste Demoulin
  • Other Contributors:
    • Fredrik Rorsman, Bernard Lauwerys and Johan Lennartsson

  Dear Editor,

  While the debate on the existence of autoantibodies against the PDGF receptor is amplified by two publications (1, 2) that contradict the data published by Gabrielli and colleagues (3), we wish to point to one important detail in the paper of Balada et al (4).

  The protein that is sold by Upstate biologicals (now Millipore) and was used in the assay developed by Balada and colleagues corresponds to t...

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  Dear Editor,

  While the debate on the existence of autoantibodies against the PDGF receptor is amplified by two publications (1, 2) that contradict the data published by Gabrielli and colleagues (3), we wish to point to one important detail in the paper of Balada et al (4).

  The protein that is sold by Upstate biologicals (now Millipore) and was used in the assay developed by Balada and colleagues corresponds to the intracellular domain of the PDGF receptor alpha (from amino-acid 550 to the end), fused to a N-terminal polyhistidine tag. Thus the antibodies detected in this report are, by definition, unable to bind to intact cells, by contrast to those described by Gabrielli and colleagues, which were shown to bind to the extracellular domain of the PDGF receptors. In other words, the two reports studied antibodies that recognize completely different epitopes. This has been overlooked in Balada’s conclusions.

  In addition, the report does not include any control ruling out the possibility that the patient antibodies recognized the histidine tag instead of the PDGF receptor.

  We believe that this point is a key to the understanding of Balada’s data, which may be misleading to scientists who are not familiar with receptor biochemistry.

  References

  1. Classen JF, Henrohn D, Rorsman F, et al. Lack of evidence of stimulatory autoantibodies to platelet-derived growth factor receptor in patients with systemic sclerosis. Arthritis Rheum 2009;60:1137-44.

  2. Loizos N, Lariccia L, Weiner J, et al. Lack of detection of agonist activity by antibodies to platelet-derived growth factor receptor alpha in a subset of normal and systemic sclerosis patient sera. Arthritis Rheum 2009;60:1145-51.

  3. Baroni SS, Santillo M, Bevilacqua F, et al. Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 2006;354:2667-76.

  4. Balada E, Simeón-Aznar CP, Ordi-Ros J et al. Anti-PDGFRa antibodies measured by non-bioactivity assays are not specific for systemic sclerosis. Ann Rheum Dis 2008;67:1027-1029.

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  Conflict of Interest:
  None declared.

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