High-mobility group chromosomal box protein 1 (HMGB1) expression at different time points after immunization. Representative micrographs illustrating immunohistochemical staining of HMGB1. (a) In the non-proliferative synovial membrane of an unimmunized animal, a nuclear HMGB1 deposition is evident. (b) In addition to the nuclear expression, cytoplasmic HMGB1 staining appears in a large portion of cells in the synovial membrane 10 days after immunization, a time point preceding the expected clinical disease onset by 5 days. (c) An arthritic lesion, 28 days after immunization, in which an additional extracellular presence of HMGB1 is indicated by a brownish extracellular immunoreactivity surrounding cells displaying cytoplasmic HMGB1 staining. Original magnification ×500.