Immunohistochemical localization of citrullinated proteins in adult rat brain.

Abstract

By using hybridoma technology, an IgM monoclonal antibody (F95) against multiple citrullinated synthetic and natural peptides was recently developed and used to stain immunohistochemically subsets of astrocytes and myelin basic protein (MBP) from selected regions of human brain (Nicholas and Whitaker [2002] Glia 37:328-336). With this antibody, the present study provides a more detailed localization of citrullinated epitopes in the central nervous system (CNS) by examining immunohistochemical staining patterns for F95 in the normal adult rat brain. Thus, immunohistochemical labeling for citrullinated epitopes was seen in white matter areas consistent with myelin staining; however, in general, it was more prominent and uniform in the caudal CNS (spinal cord, medulla oblongata, pons, and cerebellum) than in more rostral areas. F95 staining was also seen in cells and fibers often intimately associated with blood vessels and/or ventricular surfaces. By using dual-color immunofluorescence, the vast majority of this latter staining was colocalized within a subset of astrocytes also immunoreactive for glial fibrillary acidic protein (GFAP). By using Western blot analysis of rat brain proteins, multiple GFAP- and MBP-immunoreactive proteins and peptide fragments were seen, and many of them were also reactive with the F95 antibody. Thus, the present study not only demonstrates that citrullinated epitopes in normal rat brain are most concentrated in subsets of myelin and astrocytes but also provides evidence that GFAP, like MBP, may be present as multiple citrullinated isoforms.