Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

Abstract

Background: Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.

Methods: The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system.

Results: We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals.

Conclusion: Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.

Fig 1. Expression of recombinant LRP4.

(A) Flow cytofluorimetric analysis of parental untransfected HEK293T cells labeled with the rabbit anti-LRP4 antiserum (shaded area), compared to LRP4fl-HEK293T transfected cells (full line). (B) Precipitated supernatants from EGFP-HEK293T LRP4ecto-HEK293T cells were analyzed by anti-c-Myc immunoblotting; the band corresponding to the ecto-LRP4-myc tag fusion protein is indicated by arrow.

Fig 2. Anti-LRP4 detection by FACS analysis.

Each of 101 MG s and 85 control sera was tested on both parental untransfected (shaded area) and on LRP4fl-HEK293T transfected cells (full line). None of the 45 NHS but only 14 MG sera showed a clear cut shift of mean fluorescence value with a ratio transfected/untransfected > 1.5. We show the immunoreactivity of one NHS, one dSN-MG (sample#8), one AChR-MG (sample#9) and one MuSK-MG serum (sample#14) (A,B,C,D respectively) as representative plots.

Fig 3. Anti-LRP4 detection by immunoprecipitation of pools of sera.

Supernatants from LRP4ecto HEK293T cells were immune-precipitated with the indicated pools (p) of sera that scored positive at FACS analysis: pool#1 and #2 from dSN-MG, Musk-MG pool and AChR-MG pool. Immuno-complexes were subdued to western blotting and probed with anti-c-Myc. Aliquots of total supernatants from EGFP-HEK293T and from LRP4ecto HEK293T cells were blotted alongside with immune-precipitates as negative and positive control, respectively. The specific, uppermost band is pointed by the arrow.