Blockade of recombinant human IL-6 by tocilizumab suppresses matrix metalloproteinase-9 production in the C28/I2 immortalized human chondrocyte cell line.

Evan C Meszaros,Wissam Dahoud,Sam Mesiano,Charles J Malemud

doi:10.15761/imm.1000158

Abstract

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6 or rh-TNF-α increased the production of matrix metalloproteinase-9 (MMP-9). The effect of rhIL-6 on neutrophil gelatinase-associated lipocalin (NGAL) was also assessed. Although C28/I2 chondrocytes incubated with rhIL-6 (50 ng/ml) increased MMP-9 production which could not be mimicked by the T/C28a2 chondrocyte line, the effect of rhTNF-α on MMP-9 was more robust than with rhIL-6. The combinations of rhIL-6 and soluble IL-6 receptor-α (sIL-6Rα) or rhIL-6 and tocilizumab (TCZ), a fully-humanized recombinant monoclonal antibody that neutralizes the interaction between IL-6 and IL-6R significantly reduced MMP-9 production by C28/I2 chondrocytes. However, TCZ had no effect on rhTNF-α-induced MMP-9 production. By contrast, rhIL-6 did not increase the production of NGAL by C28/I2 chondrocytes although the number of NGAL-positive cells was significantly reduced by sIL-6R compared to its control group, but not by the combination of rhIL-6 plus TCZ compared to rhIL-6. In summary, these results showed that rhIL-6 stimulated the production of MMP-9, but not NGAL, in the C28/I2 chondrocyte line. TCZ or sIL-6Rα suppressed rhIL-6-induced MMP-9 production.

Highlights

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 [1]
The contribution of IL-6 to MMP-9 production by cultured human chondrocytes remains to be fully elucidated. 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) [5] inhibits recombinant human-IL6-mediated MMP-9 production was determined in the immortalized human juvenile T/C28a2 and C28/I2 chondrocyte lines
The ICC of anti-MMP-9 antibody-positive C28/I2 chondrocytes treated with rhIL-6 (50 ng/ml) for 24 hrs is shown in Figure 5 and compared to C28/I2 chondrocytes in the “no additions” control or 10% fetal bovine serum (FBS) control group

Summary

Introduction

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 [1]. The contribution of IL-6 to MMP-9 production by cultured human chondrocytes remains to be fully elucidated 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) [5] inhibits recombinant human (rh)-IL6-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 [6,7]. T/C28a2 and C28/I2 chondrocytes expressed several other molecules characteristic of authentic human chondrocytes, most notably the molecular signature SOX9 gene, considered the “master” transcriptional regulator of several cartilagespecific genes as the type II collagen (COL2A1) gene and the aggrecan (AGRN) gene [6,7,8]

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