Patterns of antibody binding to aquaporin-4 isoforms in neuromyelitis optica

Abstract

Background: Neuromyelitis optica (NMO), a severe demyelinating disease, represents itself with optic neuritis and longitudinally extensive transverse myelitis. Serum NMO-IgG autoantibodies (Abs), a specific finding in NMO patients, target the water channel protein aquaporin-4 (AQP4), which is expressed as a long (M-1) or a short (M-23) isoform.

Methodology/principal findings: The aim of this study was to analyze serum samples from patients with NMO and controls for the presence and epitope specificity of IgG and IgM anti-AQP4 Abs using an immunofluorescence assay with HEK293 cells expressing M-1 or M-23 human AQP4. We included 56 patients with definite NMO (n = 30) and high risk NMO (n = 26), 101 patients with multiple sclerosis, 27 patients with clinically isolated syndromes (CIS), 30 patients with systemic lupus erythematosus (SLE) or Sjögren's syndrome, 29 patients with other neurological diseases and 47 healthy controls. Serum anti-AQP4 M-23 IgG Abs were specifically detected in 29 NMO patients, 17 patients with high risk NMO and two patients with myelitis due to demyelination (CIS) and SLE. In contrast, IgM anti-AQP4 Abs were not only found in some NMO and high risk patients, but also in controls. The sensitivity of the M-23 AQP4 IgG assay was 97% for NMO and 65% for high risk NMO, with a specificity of 100% compared to the controls. Sensitivity with M-1 AQP4 transfected cells was lower for NMO (70%) and high risk NMO (39%). The conformational epitopes of M-23 AQP4 are the primary targets of NMO-IgG Abs, whereas M-1 AQP4 Abs are developed with increasing disease duration and number of relapses.

Conclusions: Our results confirm M-23 AQP4-IgG Abs as reliable biomarkers in patients with NMO and high risk syndromes. M-1 and M-23 AQP4-IgG Abs are significantly associated with a higher number of relapses and longer disease duration.

Figure 1. Different staining patterns of NMO-IgG in M-1 and M-23 AQP4 transfected cells.

Anti-AQP4 IgG (red) in NMO patient's serum targets AQP4 (green), which is expressed by transiently transfected HEK cells. Performing the assay for M-23 AQP4 (A, green) versus M-1 AQP4 (B, green), results in different staining patterns of NMO-IgG (red). Weaker binding was observed to M-1 AQP4, which contrary to M-23 AQP4 forms only few orthogonal arrays of particles.

Figure 2. NMO-IgG staining patterns in AQP4-EmGFP versus AQP4 expressing cells.

Fusion of EmGFP to AQP4 molecules has no effect on the formation of the different staining patterns of NMO-IgG in M-1 and M-23 AQP4 transfected cells. NMO-IgG has the same laminar staining pattern when binding M-23 AQP4 with and without EmGFP fusion (A). In contrast, cells transfected with M-1 AQP4-EmGFP and M-1 AQP4 have a more point shaped staining pattern (B).

Figure 3. M-1 and M-23 AQP4-IgG titer values in follow-up samples.

Higher titer values of NMO-IgG in two patients with recurrent ON (1^st sample) after conversion into NMO (2^nd sample) after 2.6 and 8.7 years. With increasing M-23 AQP4-IgG titers, patient one developed Abs against full length AQP4, whereas patient two remained M-1 AQP4-IgG negative.