Fluorogenic Probe for Detecting Active Matrix Metalloproteinase-3 (MMP-3) in Plasma and Peripheral Blood Neutrophils to Indicate the Severity of Rheumatoid Arthritis

Diagnosis of patients with rheumatoid arthritis (RA) is essential for early and accurate drug treatment to protect the patient from joint bone erosion and relieve symptoms of the disease. In some cases, the RA patient's X-ray images and other clinical diagnostic methods are often difficult to distinguish from different diseases, such as gout, osteoarthritis, and other inflammatory conditions. Thus, methods for diagnosis of disease activity and real-time monitoring of therapeutic effect and accurate differentiation from other bone diseases are needed. In this article, we suggest a matrix metalloproteinase-3 (MMP-3)-specific protease-activated probe immobilized in vitro kit and cell staining for flow cytometry analysis as methods to support clinical diagnosis. To overcome interindividual differences, we used phorbol 12-myristate 13-acetate (PMA)-activated plasma from 269 RA patients, 49 osteoarthritis patients, and 30 healthy volunteers. The in vitro kit developed for PMA-activated plasma showed potential for identifying disease severity and distinguishing RA from other bone diseases. In particular, expression of active MMP-3 increased until the moderate disease activity and then sharply decreased at severe disease. We suggest an analysis of intracellular MMP-3-specific protease-activated probe staining by flow cytometry. Compared with anti-MMP-3 antibody staining, the results for active MMP-3 in neutrophils using the probe exactly matched the results obtained with the in vitro kit. We also confirmed that expression of active MMP-3 was mainly from neutrophils. Together, these results suggest that the MMP-3-specific protease-activated probe might be a promising noninvasive tool for accurate diagnosis of disease severity and differentiation from similar bone diseases as well as for monitoring therapeutic efficacy.