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To address the ongoing debate concerning the specificity of synovial citrullinated proteins for rheumatoid arthritis (RA) and to analyze their pathophysiologic relevance to the induction or perpetuation of the RA‐specific anti–citrullinated protein antibodies (ACPAs).Synovium of 19 RA patients and 19 non‐RA controls was immunostained for the presence of citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD‐2), and for the free citrulline–producing enzyme inducible nitric oxide synthase (iNOS). Extending the RA cohort to 61 patients, the findings of anticitrulline staining in synovium were related to serum and synovial fluid ACPA levels, as measured by enzyme‐linked immunosorbent assay.F95 staining indicated the presence of synovial intracellular citrullinated proteins in 53% of RA samples versus 5% of control samples, whereas extracellular staining was not RA specific. Immunoblotting and inhibition experiments confirmed that the antibody recognized citrullinated proteins but not free citrulline. Accordingly, iNOS was equally found in RA and control synovium and in intracellular citrullinated protein–positive and intracellular citrullinated protein–negative samples. In contrast, intracellular citrullinated proteins colocalized with PAD‐2, which was found in 59% of RA samples versus 17% of control samples. Independent of local disease activity, the presence of the RA‐specific synovial intracellular citrullinated proteins was associated with significantly higher systemic and local ACPA levels and with local ACPA production in the joint.These data confirm the presence of RA‐specific intracellular citrullinated proteins in synovium. The link with PAD‐2 and local and systemic ACPA levels emphasizes their pathophysiologic relevance for RA‐specific humoral autoimmunity. [ABSTRACT FROM AUTHOR]

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. J. Comp. Neurol. 459:251–266, 2003. © 2003 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]