Antibodies against the flotillin-1/2 complex in patients with multiple sclerosis

Abstract

Multiple sclerosis is a tissue-specific autoimmune disease of the central nervous system in which the antigen(s) remains elusive. Antibodies targeting the flotillin-1/2 complex have been described in 1-2% of the patients in a recent study. Other candidate antigens as anoctamin-2 or neurofascin-155 have been previously described in multiple sclerosis patients, although their clinical relevance remains uncertain. Our study aims to analyse the frequency and clinical relevance of antibodies against neurofascin-155, anoctamin-2 and flotillin-1/2 complex in multiple sclerosis. Serum (n = 252) and CSF (n = 50) samples from 282 multiple sclerosis patients were included in the study. The control group was composed of 260 serum samples (71 healthy donors and 189 with other neuroinflammatory disorders). Anti-flotillin-1/2, anti-anoctamin-2 and anti-neurofascin-155 antibodies were tested by cell-based assays using transfected cells. We identified six multiple sclerosis patients with antibodies against the flotillin-1/2 complex (2.1%) and one multiple sclerosis patient with antibodies against anoctamin-2 (0.35%). All multiple sclerosis patients were negative for anti-neurofascin-155 antibodies. Three of the anti-flotillin-1/2 positive patients showed anti-flotillin-1/2 positivity in other serum samples extracted at different moments of their disease. Immunoglobulin G subclasses of anti-flotillin-1/2 antibodies were predominantly one and three. We confirm that antibodies targeting the flotillin-1/2 complex are present in a subgroup of patients with multiple sclerosis. Further studies are needed to understand the clinical and pathological relevance of anti-flotillin-1/2 autoantibodies in multiple sclerosis.

Keywords: antigens; autoantibodies; multiple sclerosis.

Graphical Abstract

Figure 1

Immunocytochemistry of anti-ANO2 antibodies. HEK293 cells transfected with mammalian-expression vectors encoding human ANO2 using Lipofectamine 2000 (A–F) or HEK293 cells non-transfected (G–I); double-stained with serum (B, E, H) and with commercial antibody against ANO2 (A, D, G). MS patient’s IgG bind to transfected cells (B) and colocalize with ANO2 ab (C); in contrast with the healthy control (E) that does not show any reactivity against ANO2 antibodies.

Figure 2

Immunocytochemistry of anti-FLOT1/2 antibodies (commercial slides). Commercial biochips (Euroimmun) containing FLOT-1/2 transfected HEK293 cells (A, C) or non-transfected HEK293 cells (B, D); incubated with serum from MS Patient 1 (A, B) and from a healthy donor (C, D). Patient 1 showed strong IgG reactivity against co-transfected cells (A) in comparison with non-transfected cells (B) and with the negative control (C).

Figure 3

Immunocytochemistry of anti-FLOT1/2 antibodies (in house transfection). HEK293 cells co-transfected with mammalian-expression vectors encoding human FLOT1 and FLOT2 using Lipofectamine 2000; double-stained with serum (B, E) and with commercial antibody against FLOT1 (A, D). MS patient’s IgG bind to co-transfected cells (B) and colocalize with FLOT1 ab (C); in contrast with the healthy control (E) that doesn’t show any reactivity against FLOT-1/2 antibodies (F).

Figure 4

Immunoadsorption of anti-FLOT1/2 antibodies. HEK293 cells co-transfected with mammalian-expression vectors encoding human FLOT1 and FLOT2 double-stained with serum immunoadsorbed with non-transfected cells (A), FLOT1-transfected cells (D), FLOT2-transfected cells (G) or FLOT1 and FLOT2 co-transfected cells (J); and with commercial antibody against FLOT1 (B, E, K) or FLOT2 (H). Reactivity against the FLOT-1/2 complex was lost after serum pre-adsorption with HEK cells co-expressing FLOT1 and FLOT2 (L), but not after pre-adsorption with cells transfected with FLOT1 (F) or FLOT2 (I) alone or with non-transfected HEK cells (C).