ABSTRACT: INTRODUCTION: In rheumatoid arthritis (RA), synovial fluid (SF) contains a large number of neutrophils which contribute to the inflammation and destruction of the joints. The SF also contains granulocyte/macrophage-colony stimulating factor (GM-CSF), which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate effects of GM-CSF on neutrophils. METHODS: Neutrophils stimulated by GM-CSF were divided into four subcellular fractions of cytosol, membrane/organelle, nuclei, and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF MS). RESULTS: We detected 33 peptide peaks whose expression was up-regulated by more than 2.5 fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database search. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed roles of the increased NGAL in RA. We analyzed proteome alternation of synoviocytes from patient with RA by the treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), intensities of 21 protein spots were increased to more than 1.5-folds and those of 10 protein spots were decreased to less than 1/1.5-folds by the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins, which included transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2) increased to 4.8, 1.5 and 1.6-fold, respectively. 2-Dimensional electrophoresis followed by western blot analysis confirmed the up-regulation of TERA by the NGAL treatment, and moreover the western blot analysis showed that the NGAL treatment changes the protein spots caused by post-translational modification of TERA. Furthermore, NGAL cancelled proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells. CONCLUSION: Our results indicate that GM-CSF contributes to the pathogenesis of RA through up-regulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the up-regulated enzymes may play an important role in RA.