Tocilizumab inhibits signal transduction mediated by both mIL-6R and sIL-6R, but not by the receptors of other members of IL-6 cytokine family
Introduction
Tocilizumab is a humanized anti-human IL-6 receptor (IL-6R) monoclonal antibody that inhibits IL-6 functions as reported previously [1]. In the clinical trials, tocilizumab shows significant improvement of disease activity of Castleman's disease, rheumatoid arthritis, Crohn's disease and system-onset juvenile idiopathic arthritis [2], [3], [4], [5].
IL-6 is a multifunctional cytokine and plays essential roles in host defense mechanisms through the immune system, hematopoiesis and the central nervous system [6]. IL-6 exerts its biological activities through two membrane molecules, a ligand-binding 80 kD chain (IL-6R) and a non-ligand-binding signal transducer gp130. After binding of IL-6 to membrane bound IL-6R (mIL-6R), IL-6/IL-6R complex associates with gp130 and then the signal is transmitted into the cell. In addition, soluble IL-6R (sIL-6R), which lacks trans-membrane and cytoplasmic domains, can associate with gp130 in the presence of IL-6 and transduce the signal through gp130. Namely, both mIL-6R and sIL-6R play essential roles in IL-6 signaling [7].
IL-6 and related cytokines, such as leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and IL-11 exhibit multiple functions and redundancy in biological activities with each other. All of these cytokine receptors utilize gp130 in their signal transduction systems, and constitute a cytokine receptor super family [8].
In this paper, we indicate that tocilizumab has the binding activity to both sIL-6R and mIL-6R and the neutralizing activity to IL-6, and that it does not have the ability to inhibit other IL-6 family cytokines in which their receptors are highly homologous to IL-6R.
Section snippets
Antibodies
Tocilizumab, MT18 (non-neutralizing anti-human IL-6R monoclonal antibody) [9] and recombinant soluble human IL-6R (sIL-6R) [10] were prepared in our laboratory.
Binding activity to sIL-6R and inhibitory activity of IL-6 binding to sIL-6R
Binding activity to sIL-6R and inhibitory activity of IL-6 binding to sIL-6R were measured by ELISA as previously described [1].
Dissociation constant analysis
Each 10 μL of 125I-tocilizumab (11 nmol/L, prepared in our laboratory) and tocilizumab (11, 33, 77, 165, 341 and 11000 nmol/L) were added to the IL-6R-expressing human myeloma cell line, U266B1 cells (200 μL, 5.5
Binding activity to sIL-6R
The binding activity of tocilizumab to sIL-6R was examined using the sIL-6R-coated plates. The results showed that tocilizumab is able to bind to sIL-6R in a dose-dependent manner at the concentration between 0.001 and 0.1 μg/mL. In contrast, hIgG1 did not show any binding activity to sIL-6R (Fig. 1A).
Inhibitory effect on the binding of IL-6 and sIL-6R
The binding of IL-6 (10 ng/mL) to sIL-6R was suppressed by the addition of tocilizumab in a dose-dependent manner at the concentration between 0.002 and 4 μg/mL. Complete inhibition was achieved
Discussion
There are two ways in IL-6 signal transduction, one is mIL-6R-mediated and the other is sIL-6R-mediated. In the cells expressing IL-6R on their cell surface, IL-6 binds to mIL-6R, this formed complex associates with the second IL-6 receptor called gp130, and then a signal is transmitted into the cells. Similarly, IL-6 can form a complex with sIL-6R in the body fluids and transmit a signal through gp130 [7]. Therefore, it is essential to inhibit both mIL-6R- and sIL-6R-mediated signaling for the
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