Charles J Malemud
Abstract
In this study, we employed the immortalized human juvenile chondrocyte lines, T/C28a2 and C28/I2, to determine whether recombinant human (RH)-IL-6 caused STAT3 to be phosphorylated (p-STAT3). WHI-P131, a JAK3-selective small molecule inhibitor was used to validate the JAK/STAT response to rhIL-6 since WHI-P131 should decrease p-STAT3 without altering total STAT3 (STAT3). Tocilizumab (TCZ), a monoclonal antibody which neutralizes the interaction between IL-6 and its receptor(s) was also employed to determine if matrix metalloproteinase-9 (MMP-9) production was coupled to the predicted rhIL-6-mediated JAK/STAT response. Western blots revealed that the T/C28a2 and C28/I2 chondrocyte lines produced STAT3 protein. However, constitutive p-STAT3 was detected only in T/C28a2. C28/I2 chondrocytes incubated with rhIL-6 (50 ng/ml) for 30 min increased p-STAT3 which was inhibited by WHI-P131. Furthermore C28/I2 chondrocytes incubated with rhIL-6 increased MMP-9 synthesis. Importantly, the combination of rhIL-6 and TCZ (200 ng/ml) significantly decreased MMP-9 production after 60 min which was sustained after 4 hrs and rhIL-6 plus TCZ significantly reduced the number of MMP-9- positive C28/I2 chondrocytes. Of note, sIL-6R also significantly reduced the number of MMP-9-positive cells compared to rhIL-6 alone. In contrast the combination of rhIL-6 and sIL-6R significantly increased MMP-9 cell positivity. These results indicated that rhIL-6-mediated STAT3 phosphorylation was coupled to MMP-9 production in C28/I2 chondrocytes where MMP-9 production was significantly reduced by TCZ or sIL-6R. These findings also support the view that TCZ likely inhibits rhIL-6-mediated MMP-9 production in C28/I2 chondrocytes by neutralizing all 3 IL-6-mediated-signaling pathways. Matrix metalloproteinase-9 (MMP-9; gelatinase B; 92 kDa gelatinase; 92 kDa type IV collagenase) is a critical MMP in mediating the progression of various arthritic conditions . MMP-9 has the capacity to degrade several articular cartilage extracellular matrix (ECM) proteins, including aggrecan, link protein, and type II collagen, all of which help maintain normal cartilage biomechanical functions . Importantly, in various types of arthritis, chondrocyte MMP-9 gene expression is significantly up-regulated in response to the elevated levels of pro-inflammatory cytokines in the synovial fluid milieu, exemplified by intereukin-(IL-6), IL-1β, IL-17, and tumor necrosis factor-α (TNF-α) . To probe the contribution of each of those cytokines to MMP-9 gene expression by articular chondrocytes in vitro would generally require that specific inhibitors for each of them be individually tested. In that regard, the effect of IL-1β or TNF-α blockade on MMP synthesis was previously reported with the results showing that IL-1 receptor antagonist or TNF-α blocking monoclonal antibodies inhibited MMP production . However, the contribution of IL-6 to MMP-9 production by cultured human chondrocytes remains to be fully elucidated. Therefore, to achieve this objective, the extent to which tocilizumab (TCZ), a recombinant fully humanized IgG1(κ) monoclonal antibody that neutralizes the interaction between IL-6 and the IL-6 receptor-α (IL-6Rα) inhibits recombinant human (rh)-IL-6-mediated MMP-9 production was determined in the immortalized human juvenile T/C28a2 and C28/I2 chondrocyte lines. These human chondrocyte lines were employed for this analysis because they had been previously shown to express cartilage-specific extracellular matrix protein genes. T/C28a2 and C28/I2 chondrocytes also expressed several other molecules characteristic of authentic human chondrocytes, most notably the molecular signature SOX9 gene, considered the “master” transcriptional regulator of several cartilage-specific genes as the type II collagen (COL2A1) gene and the aggrecan (AGRN) gene . The effect of rhIL-6 on the synthesis of chondrocyte-derived neutrophil gelatinase-associated lipocalin (NGAL) was also evaluated. The rationale for analyzing NGAL production by C28/I2 chondrocytes stemmed from our previously reported finding that chondrocytes obtained from human osteoarthritis knee cartilage synthesized NGAL in response to IL-1β. In addition, we showed that NGAL in synovial fluids obtained from patients with end-stage osteoarthritis was found in a complex with MMP-9. Moreover, we proved that the MMP-9/NGAL complex preserved MMP-9 activity by demonstrating that this complex prevented MMP-9 from being degraded, thus preserving MMP-9 activity. Biography Charles J Malemud received PhD from George Washington University in 1973 and completed Post-doctoral studies at the State University of New York at Stony Brook in 1977. Since 1977, he has been a member of the faculty at Case Western Reserve University School of Medicine where he is presently Professor of Medicine and Anatomy in the division of rheumatic diseases and Senior Investigator in the Arthritis Research Laboratory. He has published over 200 papers, chapters and reviews primarily in the field of chondrocyte biology. He is an editorial board of several rheumatology, immunology and musculoskeletal journals and is Editor-in-Chief for the journals of clinical and cellular immunology. Note: This work is presened from 4th International Conference and Exhibition on Immunology on September 28-30, 2015 at TX, USA