Simple Method Using Gelatin-Coated Film for Comprehensively Assaying Gelatinase Activity in Synovial Fluid

Abstract

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to the irreversible destruction of cartilage and bone. An important step in the destruction of tissue is the degradation of extracellular matrix (ECM), whose major components are type II collagen and aggrecan in cartilage, and type I collagen in bone. The synovial fluid of RA patients often contains high concentrations of proteinases, such as those of the matrix metalloproteinase (MMP) and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS) families, which induce ECM degradation either directly or indirectly and participate in joint destruction. MMP-1, MMP-8, and MMP-13, which function as collagenases, have proteolytic activity towards type I and type II collagen (Chakraborti et al., 2003; Murphy & Nagase, 2008). MMP-2 and MMP-9, termed gelatinases, degrade denatured collagen, while MMP-3 degrades several ECM proteins (Chakraborti et al., 2003). All of them have the gelatinase activity. ADAMTS-4 and ADAMTS-5 are major enzymes involved in the degradation of aggrecan (Arner, 2002) and have low levels of gelatinase activity (Gendron et al., 2007; Lauer-Fields et al., 2007). The concentration and activity of MMP and ADAMTS enzymes in synovial fluid and serum are measurable using ELISA or enzyme-specific substrates. In particular, MMP-3 is often the target of laboratory tests as a biomarker for disease progression. However, for the elucidation of actual RA progression, it is important to measure the comprehensive activity of all enzymes. In situ zymography is a method for assessing endogenous protease activity in tissue section (Yan & Blomme, 2003). Film in situ zymography (FIZ) using gelatin-coated film is a useful method for assessing the enzymes involved in arthritis (Yoshida et al., 2009). In this method, a frozen tissue section is adhered onto a gelatin-coated film and gelatin degradation loci on the film are then analyzed after a suitable incubation period. A locus of gelatin degradation represents an area where comprehensive enzymatic activity is high. This chapter describes a method for assaying the comprehensive gelatinase activity in synovial fluid using gelatincoated films for FIZ.