Immunohistochemical Detection and Immunochemical Analysis of Type 11 Collagen Degradation in Human Normal , Rheumatoid , and Osteoarthritic

Articular cartilage destruction and loss of function in arthritic diseases involves proteolytic degradation of the connective tissue matrix. We have investigated the degradation of cartilage collagen by developing immunochemical methods that permit the identification and analysis of type II collagen degradation in situ. Previously, a technique to specifically identify type II collagen degradation in situ in articular cartilage did not exist. These methods utilize a polyclonal antiserum (R181) that specifically reacts with unwound a-chains and CNBr-derived peptides, al(II)CB11 and al(II)CB8, of human and bovine type II collagens. The experimental approach is based on the fact that when fibrillar collagens are cleaved the helical collagen molecule unwinds, exposing hidden epitopes. Here we demonstrate the use of R181 in studying type II collagen degradation in bovine articular cartilage that has been cultured with or without IL-1 and in human normal, rheumatoid, and osteoarthritic articular cartilages. Compared to cartilages either freshly isolated or cultured without IL-1, bovine cartilage cultured with IL-1 for 3-5 d showed an increase in both pericellular and intercellular immunohistochemical staining. Extracts of these cartilages contained type II collagen a chains that were increased in amount after culture with IL-1 for 11 d. In addition, culture with IL-1 resulted in the appearance of a chain fragments of lower molecular weight. All human arthritic tissues examined showed areas of pronounced pericellular and territorial staining for collagen degradation as compared with nondiseased tissues, indicating that chondrocytes are responsible in part for this degradation as compared with nondiseased tissues. In most cases rheumatoid cartilage was stained most intensely at the articular surface and in the deep and midzones, whereas osteoarthritic cartilage usually stained more in the superficial and mid-zones, but less intensely. Distinct patterns of sites of collagen degradation reflect differences in collagen destruction in these diseases, suggesting possible different sources of chondrocyte activation. These experiments demonstrate the application of immunological methods to detect collagen degradation and demonstrate an increase of collagen degradation in human arthritides and in IL-1-treated viable bovine cartilage. Address reprint requests to Dr. Poole, Joint Diseases Laboratory, Shriners Hospital for Crippled Children, Department of Surgery, McGill University, 1529 Cedar Avenue, Montreal, Quebec H3G 1A6, Canada. The present address of Dr. Dodge is Department of Pathology and Cell Biology, Thomas Jefferson University, Jefferson Alumni Hall, Room 249, 1020 Locust Street, Philadelphia, PA 19107. Receivedfor publication 18 May 1988 and in revisedform 3 August 1988. Introduction Most arthritic diseases result in the destruction of articular cartilage with a loss of function. The destruction is due, in part, to the degradation of the extracellular matrix, which is composed primarily of fibrillar type II collagen and aggregating proteoglycans. In articular cartilage, type II collagen fibrils are responsible for the tensile strength whereas the proteoglycans provide the compressive stiffness necessary for normal articulation and function (1). The precise mechanisms by which these connective tissue components are degraded are not fully understood. In mammals, one mechanism involves collagenase, an enzyme capable of a site-specific cleavage of helical (native) collagen (2). Incapable of maintaining a helical structure at physiological temperatures, collagenase-cleaved collagens unwind and become susceptible to further degradation by other proteinases in the extracellular space. In this regard, collagenase can be considered the rate limiting enzyme involved in collagen degradation. A variety of studies have provided indirect evidence that proteolytic enzymes are implicated in the destruction of articular cartilage, although there has been no direct evidence other than morphological (3, 4) that fibrillar collagen is being degraded at the tissue level, such as in cartilage. Limited proteolytic degradation of collagen in cartilage has been demonstrated to result in a loss of the tensile strength of cartilage with negligible release of collagen from the cartilage matrix, measured biochemically (5). Since the maintenance of tissue integrity is essential to normal function, factors that influence the synthesis, secretion, and activity of proteinases, such as collagenase, are important to examine. The cytokine, IL1, has been shown to stimulate collagenase synthesis and secretion from a variety of cells including synovial cells, fibroblasts, and chondrocytes (6-8). Even though IL1 can be produced by different connective tissue cells and cells of the inflammatory response, its relationship to the pathogenesis of connective tissue diseases, such as rheumatoid and osteoarthritis, remains speculative. In vitro, stromelysin or neutral metalloproteinase has been shown to activate collagenase (9, 10): it is also coordinately released with collagenase after stimulation with agents such as IL-1 or urate crystals (10). One major limitation on our ability to detect and study collagen degradation in health and disease has been the lack of a technique for identifying collagen breakdown in situ. We describe here both immunohistochemical and immunochemical methods that permit one to detect type II collagen degradation. These methods have been used to analyze collagen degradation in human and bovine articular cartilages. They employ an antibody (R181) that specifically reacts with fragmented and denatured human and bovine type II collagen a-chains and does not react with native helical collagens. Also, R181 does not react with bovine type V, VI, IX, and XI collagens, all of which are collagens found in cartilage matrix, nor with any Articular Cartilage Degradation 647 J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/89/02/0647/15 $2.00 Volume 83, February 1989, 647-661 other molecules in bovine and human cartilage extracts. The studies are based on the premise that when fibrillar collagens are cleaved by collagenase the subsequent unwinding and fragmentation results in the exposure of hidden epitopes that are not exposed in the native molecule. Earlier work (1 1-14) identified antibody reactive epitopes on unwound collagen a chains. These determinants are usually sequestered in the triple helix and are not detectable until this unwinds. Experimentally, an antibody that only recognizes epitopes on unwound or fragmented type II collagen, provides a tool to study cartilage collagen degradation as a normal function ofcartilage remodeling and in pathology. We report here experiments in which type II collagen degradation was demonstrated in situ and analyzed in explants ofbovine articular cartilage, cultured with IL-1, and in human articular cartilages from normal, rheumatoid, and osteoarthritic patients.