Peripherin-IgG association with neurologic and endocrine autoimmunity

Abstract

Peripherin-IgG has been reported a pertinent autoantibody in non-obese type 1 diabetic (NOD) mice. However, it has not previously been recognized in any human disease. In blinded evaluation of serum for markers of neurological autoimmunity in a high-volume diagnostic laboratory, we incidentally identified 26 patients (61% female) with an IgG that bound selectively to neural elements in enteric ganglia, sympathetic nerve trunks and discrete nerve tracts in mid-brain and hind-brain. The target antigen was identified as peripherin, a 55kDa - type III intermediate filament protein. Review of clinical histories revealed that 54% of seropositive patients had dysautonomia (predominantly gastrointestinal dysmotility), 30% had neuropathies with varied sensory symptoms and 35% had clinical or serological evidence of endocrinopathy (type 1 diabetes, thyroiditis or premature ovarian failure). Collectively, 73% had autonomic dysfunction or endocrinopathy. None of 173 healthy subjects was seropositive. Subsequent western blot evaluation of archival sera from patients with small fiber/autonomic neuropathies (with or without endocrinopathy) revealed a 33% seropositivity rate for peripherin-IgG. Our further demonstration that peripherin-immunoreactive autonomic fibers in pancreas, thyroid and ovary are juxtaposed to endocrine epithelium, complement our clinical observations in suggesting that neuronal elements may be a pertinent initial target for immune attack in multiple forms of endocrine autoimmunity (intermolecular epitope spreading). It remains to be determined whether or not peripherin-IgG is predictive for development of small fiber neuropathy (autonomic or somatic).

Fig. 1

Novel IgG autoantibody binds to neural elements in sections of mouse stomach and kidney and to discrete fiber tracts in mid-hind brain. Bound IgG was visualized using fluorescein-conjugated anti-human IgG. The characteristic staining pattern of this autoantibody is prominent in myenteric ganglia, nerve fibers and nerve trunks in the enteric nervous system (upper panel), sympathetic nerve trunks and fibers adjacent to arterioles in the gastric submucosa and kidney (center panel) and discrete nerve bundles in the mid-hind brain (lower panel). G: ganglion; NT: nerve trunk; NF: nerve fibers; SN: sympathetic nerve

Fig. 2

The autoantigen is restricted to neurons. Differentiated cell lines used as substrates: pheochromocytoma (PC12; upper panels), astrocytes (CG4; middle panels) and skeletal muscle (L6; bottom panels). Informative IgG probes included: Neurofilament (neuronal, column 1), GFAP (glial, column 2) and striational (skeletal muscle sarcomere, column 3), and patient serum (column 4). Only the neuronal cell line was immunoreactive with patient serum.

Fig. 3

Novel IgG binds to a protein Mr-55-60kDa that is confined to the neuronal cytoskeleton. (a) Using PC12 lysate as a source of antigen, proteins were separated, immunoblotted, and probed with patient or normal human sera. A common band (∼55kDa by reference to molecular weight standards) was revealed by IgG in patients' sera (lanes 1-4), but not by IgG in control human serum (lanes 5-7). To verify specificity, patient IgG was affinity purified on the putative antigenic band and a control band. Eluates from the putative antigenic band (b) and control band (c) were reapplied to the composite mouse tissue substrate slide, and compared to the original immunostaining pattern of whole patient serum IgG on enteric nerve fibers (d). An identical staining pattern was observed in eluates from putative antigenic band. To determine the subcellular distribution of the antigen, PC12 lysates were fractionated by differential detergent extraction. Proteins in each fraction were resolved electrophoretically, transblotted, and probed with patient sera (e). IgG bound exclusively to a protein in the cytoskeletal fraction.

Fig. 4

Identification of peripherin as the autoantigen. Duplicate preparations of the cytoskeletal fraction of PC12 cells were separated further by sequential isoelectric focusing and gel electrophoresis. One gel was silver stained (a), and the replica was transblotted and probed with patient serum (b). Unique peptides yielded by in-gel digestion from the silver stained gel of the common spots (numbered 1-4) identified the antigen to be peripherin.

Fig. 5

Patient IgG colocalizes with peripherin immunoreactivity in brain and endocrine organs. Tissues (brain [a-c], thyroid [d-f], pancreas [g-i], ovary [j-l] and liver [m-o]) were harvested from a 6-8 week old female mouse, cryosections (8 μm) were cut and stained with rabbit anti-peripherin-IgG (left columns), and patient IgG (center columns). All merged images (right column) show nuclear DAPI staining (blue) except for pancreas, where endocrine islet cells (i) were identified by IgG specific for the β-cell transcription factor PDX-1 (pseudo-colored purple).