Anti-IL-6 receptor antibody increases blood IL-6 level via the blockade of IL-6 clearance, but not via the induction of IL-6 production

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Abstract

We explored the mechanism for the increase of blood IL-6 level after anti-IL-6 receptor (IL-6R) antibody injection. First, we examined whether anti-IL-6R antibody stimulates IL-6 production. Single injection of tocilizumab (anti-IL-6R antibody) in monkeys with collagen-induced arthritis (CIA) caused a marked increase in blood IL-6 and IL-6R levels, but did not increase IL-6 mRNA and IL-6R mRNA expression in liver, spleen, lymph nodes, synovium or whole blood 1, 3 and 7 days later. This suggests that tocilizumab did not induce IL-6 and IL-6R production. Second, we investigated whether anti-IL-6R antibody releases IL-6 from IL-6 complexes in the blood. When plasma from CIA monkeys was incubated with tocilizumab, the IL-6 concentration was not affected. Finally, we studied whether anti-IL-6R antibody affects the clearance of IL-6 from the blood. When MR16-1 (anti-mouse IL-6R antibody) was injected into IL-6-deficient mice continuously infused with human IL-6, blood human IL-6 levels significantly increased. These results suggest that the elevation of blood IL-6 after the administration of anti-IL-6R antibody is the result of inhibition of the clearance of IL-6 due to IL-6R blockade, and that it is not the result of induction of IL-6 production or release of IL-6 from complexes.

Introduction

IL-6 is a multifunctional cytokine that plays essential roles in host defense mechanisms through the immune system, hematopoiesis and central nervous system [1]. Two different molecules participate in IL-6 signaling. One is IL-6 receptor (IL-6R), an IL-6-specific 80 kDa chain; and the other is gp130, a non-ligand-binding signal transducer. Furthermore, there are two types of IL-6R, membrane bound IL-6R and soluble IL-6R (sIL-6R); both types can form a complex with IL-6 and mediate IL-6 signaling [2].

The humanized anti-human IL-6R antibody, tocilizumab, binds to both types of IL-6R, inhibiting the binding of IL-6 to IL-6R [3] and completely neutralizing IL-6 activities. Clinical trials have shown that tocilizumab can ameliorate the disease activity of chronic inflammatory diseases characterized by elevated IL-6 production, such as Castleman's disease, rheumatoid arthritis (RA), Crohn's disease (CD) and systemic-onset juvenile idiopathic arthritis [4], [5], [6], [7].

During clinical trials in patients with RA and CD, it was noted that tocilizumab increased blood sIL-6R [6], [8]. Wendling et al. have reported a similar phenomenon that anti-IL-6 antibody increased blood IL-6 in RA patients during the administration period [9].

Of interest, tocilizumab also increased blood IL-6 level. Since RA and CD patients have elevated blood IL-6 even without the administration of anti-IL-6R antibody [10], [11], we suspected that anti-IL-6R antibody may further augment IL-6 production to compensate for the rapid blockade of IL-6 signaling. Another possible source for the extra IL-6 induced by anti-IL-6R antibody is the large amount of IL-6 complex (including IL-6/sIL-6R, IL-6/sIL-6R/soluble gp130 (sgp130)) that can be assumed to be present in the blood of RA and CD patients, who have high levels of IL-6 in addition to the high levels of sIL-6R and sgp130 found even in healthy subjects [12], [13]. Since tocilizumab can dissociate the IL-6/sIL-6R complex [3], we considered it possible that dissociation of IL-6 complexes causes the increased IL-6 seen after the administration of anti-IL-6R antibody. A third possibility we considered is that the anti-IL-6R antibody may inhibit the clearance of IL-6 from the blood.

The present study was therefore conducted to look for evidence of these three possible mechanisms for the elevation of blood IL-6 by anti-IL-6R antibody. We used collagen-induced arthritis (CIA) monkeys because they have elevated blood IL-6 and because tocilizumab has been found to increase blood IL-6 even further [14], [15], reflecting the situation in RA and CD patients [6], [8].

Firstly, we examined the effects of tocilizumab administration on IL-6 mRNA expression in whole blood and various organs that may participate in the production of blood IL-6 in CIA monkeys. Secondly, we added tocilizumab to blood from CIA monkeys to see whether it releases IL-6 from IL-6 complexes. Thirdly, using IL-6-deficient mice continuously infused with human IL-6, we examined whether anti-mouse IL-6R antibody affects the clearance of IL-6 from the blood.

Section snippets

Reagent

Tocilizumab (humanized anti-human IL-6R monoclonal antibody, human IgG1) [16] and MR16-1 (rat anti-mouse IL-6R monoclonal antibody, rat IgG1) [17] and human recombinant IL-6 were prepared in our laboratories.

Animals

Female cynomolgus monkeys (Macaca Fascicularis, 3 to 6 years old), were purchased from the Guangxi Research Center of Primate Laboratory Animals (Guangxi, China). About 108 g of solid food (Teklad Global Certified 25% Protein Primate Diet, Harlan Sprague Dawley, Indianapolis, IN) was

Plasma IL-6 and sIL-6R levels

A single injection of tocilizumab increased blood IL-6 and sIL-6R concentrations, and the increases were still present on day 7 after injection (Table 1, Table 2). This confirmed out previous finding that tocilizumab increased blood IL-6 and sIL-6R levels in monkey CIA [14], [15].

CRP and joint swelling

To confirm IL-6 signal inhibition by tocilizumab, we measured CRP levels. Tocilizumab caused a marked decrease in plasma CRP level in all animals on days 3 and 7, whereas there was no such rapid and marked decrease in

Discussion

In the present study, we analyzed the mechanism for the elevation of blood IL-6 levels by anti-IL-6R antibody. The results were as follows: (1) Anti-IL-6R antibody did not augment the expression of IL-6 mRNA (or IL-6R mRNA) in various relevant organs or whole blood. (2) When added to plasma from arthritis monkey, anti-IL-6R antibody did not increase the concentration of IL-6 (or sIL-6R). (3) Anti-IL-6R antibody increased blood IL-6 levels in IL-6-deficient mice continuously infused with human

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