Figure 2

Confirmation of increase of neutrophil gelatinase-associated lipocalin (NGAL) in neutrophils stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and in the synovial fluid of patients with rheumatoid arthritis (RA). (a) The intensity of the peptide with m/z 1,791.0, detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and identified as NGAL by de novo sequencing using MS/MS and protein database searching, was compared between the organelle/membrane fractions of GM-CSF-treated and untreated neutrophils. (b) The increase of NGAL indicated by the mass spectrometric detection was further confirmed by western blotting using neutrophil lysate. Neutrophils treated with GM-CSF for 18 h or untreated were lysed, and separated on 12.5% SDS-PAGE gels. Then NGAL was probed by antibodies to human NGAL. The bound antibodies were visualised by horseradish peroxidase (HRP)-labelled secondary antibody and 3,3'-diaminobenzidene (DAB). NC, negative control – no first antibody and only HRP-labelled secondary antibody was used.(c) NGAL mRNA expression measured by real-time polymerase chain reaction (PCR) analysis. Total RNA was isolated from neutrophils treated with or without GM-CSF for 4 and 18 h. The amount of NGAL mRNA was expressed as a relative value, compared to that of the constitutively expressed housekeeping gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are presented as mean ± standard deviation (SD) (n = 3). (d) Concentration of NGAL in synovial fluid was measured by ELISA. The horizontal bars indicate the mean values. Each open circle indicates a concentration of NGAL in synovial fluids from individual patients.