Painful trigeminal neuropathy associated with anti-Plexin D1 antibody

Abstract

Objective: To determine whether anti-Plexin D1 antibody (Plexin D1-immunoglobulin G [IgG]), which is associated with limb and trunk neuropathic pain (NP) and binds to pain-conducting small unmyelinated dorsal root ganglion (DRG) neurons, exists in patients with idiopathic painful trigeminal neuropathy (IPTN) and whether Plexin D1-IgG binds to trigeminal ganglion (TG) neurons.

Methods: We enrolled 21 consecutive patients with IPTN and 35 age- and sex-matched controls without NP (25 healthy persons and 10 with neurodegenerative diseases). We measured serum Plexin D1-IgG using a mouse DRG tissue-based indirect immunofluorescence assay (IFA) and by Western blotting (WB) using a recombinant human Plexin D1 (rhPlexin D1) accompanied by immunoadsorption tests with rhPlexin D1. The reactivity of Plexin D1-IgG toward mouse TG, brain, heart, and kidney was assessed by tissue-based IFAs.

Results: Serum Plexin D1-IgG was detected more frequently in IPTN than in controls by both IFA and WB (14.3% vs 0%, p = 0.048). Three Plexin D1-IgG-positive patients also had limb or trunk NP and commonly showed tongue pain. In tissue-based IFAs, IgG from 2 Plexin D1-IgG-positive patients immunostained small TG neurons, which was prevented by preincubation with rhPlexin D1. Moreover, Plexin D1-IgG immunostaining mostly colocalized with isolectin B4-positive pain-conducting unmyelinated TG neurons. IFAs of other tissues with the same IgG revealed weak immunoreactivity only in endothelial cells, which was prevented by preincubation with rhPlexin D1.

Conclusions: Plexin D1-IgG, which binds to pain-conducting small TG neurons in addition to DRG neurons, can be present in IPTN as well as limb and trunk NP.

Figure 1. Detection of Plexin D1-IgG by IFA using mouse DRG and WB with rhPlexin D1

(A) IFA with mouse DRG for case 1. IgG from case 1 (green) bound to small DRG neurons (upper images). The immunostaining (green) of small DRG neurons by IgG from case 1 was prevented by preincubation with rhPlexin D1 (2 µg/mL) (lower images). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) IFA with mouse DRG for case 2. IgG from case 2 (green) bound to small DRG neurons (upper images). The immunostaining (green) of small DRG neurons by IgG from case 2 was prevented by preincubation with rhPlexin D1 (2 µg/mL) (lower images). Nuclei are counterstained with DAPI (blue). (C) WB analysis using rhPlexin D1 and IgG from cases 1, 2, and 3, 3 patients with IPTN without Plexin D1-IgG, 3 healthy controls, 3 patients with neurodegenerative diseases, and a commercial anti-human Plexin D1 antibody. WB of rhPlexin D1 with IgG samples from cases 1, 2, and 3 showed a common immunoreactive band between 150 and 250 kDa, similar to the commercial anti-human Plexin D1 antibody (R&D Systems, Minneapolis). All other samples were nonreactive. Scale bars: (A and B) = 50 µm. DRG = dorsal root ganglion; IFA = immunofluorescence assay; IPTN = idiopathic painful trigeminal neuropathy; ND = neurodegenerative diseases; Plexin D1-IgG = anti-Plexin D1 antibody; rhPlexin D1 = recombinant human Plexin D1; WB = Western blotting.

Figure 2. IFA using mouse tissues and IgG from patients with IPTN with Plexin D1-IgG

(A) IFA with mouse TG for case 1. IgG from case 1 (green) bound to small TG neurons (left image). The immunostaining (green) of small TG neurons by IgG from case 1 was prevented by preincubation with rhPlexin D1 (2 µg/mL) (right image). Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (B) IFA with mouse TG for case 2. IgG from case 2 (green) bound to small TG neurons (left image). The immunostaining (green) of small TG neurons by IgG from case 2 was prevented by preincubation with rhPlexin D1 (2 µg/mL) (right image). Nuclei are counterstained with DAPI (blue). (C) Immunostaining for IB4 (red), a marker of unmyelinated C-fiber type TG neurons, mostly colocalized with IgG (green) in a representative patient with IPTN (case 1). (D) Immunostaining for NFH (red), a marker of myelinated Aβ and Aδ fiber type TG neurons, did not colocalize with IgG binding (green) in case 1. (E) IFAs with the mouse brain, heart, and kidney using IgG from a representative patient with IPTN (case 1). IgG from case 1 (green) restrictedly bound to endothelial cells (white arrows) in the cerebral cortex (upper image), heart (middle image), and kidney (lower image). The weak immunostaining (green) of endothelial cells by IgG from case 1 was prevented by preincubation with rhPlexin D1 (2 µg/mL). Nuclei are counterstained with DAPI (blue). Scale bars: (A–E) = 50 µm. IB4 = isolectin B4; IFA = immunofluorescence assay; IPTN = idiopathic painful trigeminal neuropathy; NFH = neurofilament heavy chain; Plexin D1-IgG = anti-Plexin D1 antibody; rhPlexin D1 = recombinant human Plexin D1; TG = trigeminal ganglion.