Immunology

Biography

Biography: 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 (50ng/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 (200ng/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. (Supported by Genentech/Roche, NEI P30-11373 and NICHD R01-069819)