Christine A. Towle

Expression of IL-1 genes in human and bovine chondrocytes: a mechanism for autocrine control of cartilage matrix degradation.

In this report we describe the presence of interleukin-1 activity in medium conditioned by bovine articular cartilage. Preparations partially purified by Sephacryl S200 chromatography (Mr 18000-25000) stimulate murine thymocyte proliferation in the lymphocyte activation factor assay. Furthermore, the factor(s) activate cartilage tissue to secrete a protease which is essential for the activity of purified synovial collagenase. We also demonstrate the presence of mRNA coding for IL-1 alpha and beta in human articular chondrocytes and conclude that the human monocytic and chondrocytic mRNAs are identical. Our results demonstrating cartilage expression of IL-1 genes suggest the possibility of an autocrine mechanism whereby chondrocyte production of matrix degrading proteases is initiated by chondrocyte derived IL-1.

Detection of interleukin-1 in the cartilage of patients with osteoarthritis: a possible autocrine/paracrine role in pathogenesis

The interleukin-1 (IL-1) cytokines stimulate the synthesis of degradative enzymes in joint tissues and may play a role in the pathological joint destruction in osteoarthritis (OA). In this study, we have used immunohistochemistry and Western blot analysis to identify IL-1 in human OA cartilage. IL-1 alpha and IL-1 beta were evident in chondrocytes at the articular surface, as well as distributed throughout the cartilage. In many specimens, IL-1 beta but not IL-1 alpha was detected as a diffuse staining of the extracellular matrix especially surrounding superficial zone chondrocytes. Although chondrocyte-associated IL-1 alpha and IL-1 beta were detected in most specimens, cartilages exhibiting early osteoarthritic changes had the highest intensity of staining and the highest frequency of positive cells. Western blot analysis revealed intense immunoreactive bands corresponding to the 35 kDa precursor form of IL-1 alpha in all four chondrocyte lysates tested. The processed 18 kDa IL-1 beta species was present in only one of four chondrocyte lysates, and there was no clear evidence of precursor form within these cells. The results of this study indicate increased IL-1 alpha in cartilage showing early degenerative changes, suggesting an autocrine/paracrine role for this cytokine in OA pathogenesis.

A role for the interleukin-1 receptor in the pathway linking static mechanical compression to decreased proteoglycan synthesis in surface articular cartilage.

Loading of articular cartilage during weight bearing is essential for the maintenance of cartilage function. Although certain cyclic loading protocols stimulate extracellular matrix synthesis, constant or static compression decreases proteoglycan and collagen synthesis in cartilage explants. The goal of this study was to determine whether the compression-induced decrease in proteoglycan synthesis involves an interleukin-1 (IL-1) signaling pathway. Cartilage explants were compressed 50% in the presence of IL-1 receptor antagonist (IL-1ra), and the incorporation of [35S]sulfate into macromolecules was measured. IL-1ra increased sulfate incorporation in compressed cartilage but not in cartilage maintained at the in situ thickness (0% compression). IL-1alpha and IL-1beta mRNAs were detected in cartilage compressed 50% for at least 3h, while nitric oxide synthase II mRNA was only detected in cartilage compressed 50% for 6h. The data support a role for the IL-1 receptor in the pathway linking static compression to reduced proteoglycan synthesis.

Purification and characterization of collagenase activator protein synthesized by articular cartilage

We have isolated an activator of collagenase from medium conditioned with articular cartilage. The activity is contained in an acidic protein appearing as a doublet band of Mr 57,000 and 56,000 on sodium dodecyl sulfate polyacrylamide gels. Both components of the doublet have identical isoelectric points as demonstrated by gel electrophoresis. Purified synovial collagenase has a high dependence on the presence of this factor for activity. Other known activators of latent proteolytic enzymes such as trypsin and mercurials will stimulate collagenase but only if activator protein is present. The activator protein is itself a latent metalloprotease because in the presence of p-aminophenylmercuric acetate and calcium it will digest casein. The caseinase activity and collagenase activation activity have identical heat inactivation profiles, both being stable to a temperature of 60 degrees C and partially inactivated at 80 degrees C. The synthesis of the activator is localized in the superficial zone of articular cartilage.

Cyclooxygenase-2 Expression in Chondrosarcoma

Objective: In recent years it has become evident that tissue cyclooxygenase-2 (COX-2) may play a role in carcinogenesis and tumor malignancy. There is now a mounting body of information that strongly implies that COX-2 inhibitors may be of some value in the management of patients with carcinomas, and most recently several similar reports have appeared relating to sarcomas. Methods: The authors studied 32 samples of cartilage tumors from our tumor tissue bank for the presence of COX-2 by a Western blot technique. There were 29 patients from whom the samples were obtained, including 8 with enchondromas and 21 with chondrosarcomas. Results: Thirteen of the 24 chondrosarcoma samples and none of the 8 enchondromas were positive for COX-2. An attempt was made to correlate these results with clinical data including age, gender, staging according to the Musculoskeletal Tumor Society, anatomical site, ploidic pattern, presence of metastases and death rate but no statistically valid correlation could be found. Conclusion: It is evident that COX-2 may play some role in chondrosarcoma but not in the benign enchondroma and that further studies with COX-2 inhibitors are warranted.

Insulin promoted decrease in the phosphorylation of protein synthesis initiation factor eIF-2

Insulin stimulates cellular protein synthesis in calf chondrocytes in suspension culture. This enhanced synthetic activity is seen in association with a decrease in phosphorylation of the alpha subunit of protein synthesis initiation factor eIF-2. [32P] associated with the alpha subunit is reduced approximately 50% by insulin treatment of chondrocytes incubated in [32P] containing media. Identical or closely located amino acids in the eIF-2 alpha subunit are phosphorylated by the chondrocyte kinase(s) and the rabbit reticulocyte hemin regulated kinase as indicated by comparative peptide fragment analysis of [32P] labeled alpha subunits.

Stimulation of the synthesis of collagenase activator protein in cartilage by a factor present in synovial-conditioned medium.

We have purified a low molecular weight protein from medium conditioned by calf synovium with physical and biological properties similar to the leukocyte cytokine interleukin 1 (IL-1). The factor is active in stimulating the synthesis (three- to fivefold) of collagenase activator protein (CAP) by the surface (1-2 mm) of articular cartilage while CAP synthesis in the deeper zones of articular cartilage is not affected. Recombinant mouse IL-1 and commercially available purified human IL-1 are also capable of stimulating cartilage to synthesize and secrete CAP. The synthesis of other proteins, including collagenase, appeared to be unaffected by either the synovial factors or the human and mouse IL-1.

Alternatively spliced annexin XI transcripts encode proteins that differ near the amino-terminus☆

Annexins are a family of structurally related calcium-dependent phospholipid binding proteins. We recently described a new member of this family, bovine annexin XI [1]. Two kinds of cDNAs were identified corresponding to annexin XI mRNA variants A and B, which are generated by alternative splicing of identical primary transcripts. Annexin XI isoforms are predicted to differ at the amino-terminus, suggesting that they may have distinct biological roles.

Purification of the ‘link proteins’ from bovine articular cartilage and comparison with ‘link proteins’ from nasal septum

Summary Proteoglycan links are glycoproteins present in cartilage ground substance which are reportedly involved in stabilizing the interaction between two other components of cartilage, proteoglycan subunit and hyaluronic acid. These proteins have been isolated and purified in the past by equilibrium density centrifugation. Presented here, for the first time, is evidence for the purification of the links from bovine articular cartilage using a new method which does not involve equilibrium density centrifugation. The results indicate that the articular cartilage links have properties similar to those of the nasal septum links.

Photodynamic treatment has chondroprotective effects on articular cartilage

Osteoarthritis (OA) is a disabling joint disease for which there is currently no cure. It is characterized by the destruction of articular cartilage. One strategy that is being explored for protecting the cartilage in OA is the administration of transforming growth factor‐β, which in vitro antagonizes cartilage degradation initiated by catabolic stimulants such as interleukin‐1 (IL‐1). The problems associated with selective delivery of the growth factor to chondrocytes, undesirable side‐effects on joint tissues, and short biological half‐life have led us to explore modalities aimed at activating transforming growth factor‐β that is stored in the cartilage as latent complexes. Photodynamic therapy is a two‐step protocol of tissue sensitization with a light‐activatable chemical called a photosensitizer followed at some interval by irradiation with the appropriate wavelength visible light. Biological effects are typically elicited through oxygen‐dependent photochemistry without heat generation. Transforming growth factor‐β1 can be activated by oxidative mechanism(s), prompting us to explore whether photodynamic technology can be harnessed to modulate cartilage metabolism. Disks of bovine articular cartilage were photosensitized by incubation with a chlorine6‐succinylated polylysine conjugate and irradiated with 1–2 J/cm2 red light (λmax = 671 nm). This two‐step regimen dramatically inhibited IL‐1‐stimulated proteoglycan degradation and concomitantly increased latent and active transforming growth factor‐β1 in culture medium. This research may lead to the development of minimally invasive photodynamic therapy in which light is delivered to locally activate a chondroprotective program in photosensitized cartilage in the context of OA.