Amphiregulin as monitoring biomarker for life-threatening acute graft-versushost disease: secondary analysis of two prospective clinical trials.

Not available.

Development and characterization of a human articular cartilage-derived chondrocyte cell line that retains chondrocyte phenotype.

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage-derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3-dimensional matrix within 48 h post-infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3-dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL-1 or BMP-7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra-cellular matrix molecules. In addition, one clone (AL-4-17) showed similar responses as primary chondrocytes when treated with IL-1 or BMP-7. In summary, this report provides a novel procedure to develop human articular cartilage-derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology.

Bacterial production of biologically active canine interleukin-1beta by seamless SUMO tagging and removal.

Interleukin 1beta (IL-1beta) is a potent stimulator of extracellular matrix degradation in models of osteoarthritis (OA). In contrast to bovine explant models which effectively respond to recombinant human IL-1beta, canine models are relatively refractory to human IL-1beta stimulation. Canine IL-1beta cDNA was cloned in order to produce a fully potent species matched preparation of IL-1beta for use specifically in canine models of OA. Established methods for the production of various orthologous IL-1beta proteins from different species are problematic due to the exquisite sensitivity of the mature IL-1beta product to N-terminal variations and the intrinsic technical challenges associated with producing an unmodified product. We have applied a seamless method of SUMO tagging and removal in order to produce a homogeneous unmodified preparation of canine IL-1beta from Escherichia coli which was found to be a potent inducer of aggrecanase activity in isolated canine articular chondrocytes. This method combines highly efficient aspects of seamless plasmid engineering, protein purification, and precise tag removal.

Mechanisms and kinetics of glycosaminoglycan release following in vitro cartilage injury.

OBJECTIVE: Acute joint injury leads to increased risk for osteoarthritis (OA). Although the mechanisms underlying this progression are unclear, early structural, metabolic, and compositional indicators of OA have been reproduced using in vitro models of cartilage injury. This study was undertaken to determine whether glycosaminoglycan (GAG) loss following in vitro cartilage injury is mediated by cellular biosynthesis, activation of enzymatic activity, or mechanical disruption of the cartilage extracellular matrix. METHODS: Immature bovine cartilage was cultured for up to 10 days. After 3 days, groups of samples were subjected to injurious mechanical compression (single uniaxial unconfined compression to 50% thickness, strain rate 100% per second). GAG release to the medium was measured, and levels were compared with those in location-matched, uninjured controls. The effects of medium supplementation with inhibitors of biosynthesis (cycloheximide), of matrix metalloproteinase (MMP) activity (CGS 27023A or GM 6001), and of aggrecanase activity (SB 703704) on GAG release after injury were assessed. RESULTS: GAG release from injured cartilage was highest during the first 4 hours after injury, but remained higher than that in controls during the first 24 hours postinjury, and was not affected by inhibitors of biosynthesis or degradative enzymes. GAG release during the period 24-72 hours postinjury was similar to that in uninjured controls, but the MMP inhibitor CGS 27023A reduced cumulative GAG loss from injured samples between 1 day and 7 days postinjury. Other inhibitors of enzymatic degradation or biosynthesis had no significant effect on GAG release. CONCLUSION: Injurious compression of articular cartilage induces an initially high rate of GAG release from the tissue, which could not be inhibited, consistent with mechanical damage. However, the finding that MMP inhibition reduced GAG loss in the days following injury suggests a potential therapeutic intervention.

Aggrecan protects cartilage collagen from proteolytic cleavage.

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.

A microplate assay specific for the enzyme aggrecanase.

We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.

Induction of aggrecanase 1 (ADAM-TS4) by interleukin-1 occurs through activation of constitutively produced protein.

OBJECTIVE: To study the production of aggrecanase 1 (ADAM-TS4) in monolayer chondrocytes, capsular fibroblasts, and cartilage. METHODS: Bovine nasal and articular cartilage, monolayer chondrocytes, and capsular fibroblasts were incubated in the absence and presence of interleukin-1 (IL-1). ADAM-TS4 production was evaluated by immunofluorescence or by Western blot analysis. Aggrecanase activity was measured in cells grown on an immobilized peptide substrate, and peptide cleavage was monitored by enzyme-linked immunosorbent assay. RESULTS: There was constitutive production of ADAM-TS4 in both cells and tissue. The protein was associated with the extracellular matrix based on the observation that the staining could be reduced following treatment of chondrocytes with heparin or exposure to chondroitinase ABC. Interestingly, there was no detectable change in the abundance of ADAM-TS4 in response to IL-1. Western blot analysis of cell lysates from IL-1-stimulated chondrocytes showed no evidence of increased ADAM-TS4 production, but resulted in activation of ADAM-TS4. The activation was associated with an increased generation in the aggrecanase neoepitope NITEGE in nasal cartilage in response to IL-1. These data suggest that induction of aggrecanase activity both in cells and in cartilage by IL-1 may involve the stimulation of an activator of ADAM-TS4. Consistent with this observation, culture of chondrocytes on a solid support containing a peptide substrate resulted in the generation of aggrecanase-mediated cleavage that could be blocked by selective inhibitors of ADAM-TS4. CONCLUSION: These data support the hypothesis that ADAM-TS4 is constitutively produced in these cells and tissue, and that stimulation by IL-1 results in aggrecanase activation. Thus, the activator could be a potential target by which to control aggrecanase-mediated degradation in arthritic diseases.