Elsevier

Cytokine

Volume 54, Issue 1, April 2011, Pages 92-99
Cytokine

IL-6 plays an essential role in neutrophilia under inflammation

https://doi.org/10.1016/j.cyto.2011.01.007Get rights and content

Abstract

In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3 h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte–macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation.

Introduction

In rheumatoid arthritis (RA) patients, many neutrophils migrate into the inflamed joints and the number of neutrophils in the circulating blood is substantially increased. Neutrophils play an essential role in the course of inflammation, and removal of neutrophils by column or depletion of neutrophils ameliorates joint inflammation in RA patients and several animal arthritis models [1], [2], [3]. Migrated neutrophils are deeply involved in the onset and maintenance of inflammation through their robust production of inflammatory mediators such as prostaglandins, reactive oxygen species, complements, proteases and cytokines. The mechanisms underlying the trans-migration of neutrophils into inflamed sites have been actively investigated, and it is clear that chemokines and adhesion molecules are involved in neutrophil trans-migration [4]. Neutrophilia also facilitates the trans-migration of neutrophils into affected tissues; however, the precise mechanisms by which neutrophilia induces this trans-migration are not fully understood in RA patients.

The mechanism of neutrophilia is thought to be the mobilization of neutrophils from the marginal pool to the circulation [5]. In the marginal pool, leukocyte rolling and adhesion is mediated by the sequential interaction of adhesion molecules on the leukocyte surface with counter-ligands on the vascular endothelium and extra-vascular structures [6]. Constitutive expression of P-selectin and ICAM-1 on vascular endothelial cells is sufficient to support neutrophil rolling and adhesion, respectively [7], [8]. Once neutrophils are activated, the expression levels of receptor molecules (CD162/PSGL-1, CD62L/L-selectin, and CD11b) can change depending on their exposure to stimulants. Following stimulation, neutrophils translocate from the marginal pool to the circulating blood. For example, G-CSF increases the neutrophil count in circulating blood by reducing CD62L expression, thus decreasing the rolling of neutrophils on the endothelium [3].

IL-6 is a pro-inflammatory cytokine and is produced by several kinds of cells in affected tissues. IL-6 participates in neutrophil migration by inducing production of chemokines such as IL-8 and MCP-1 and by inducing expression of adhesion molecules such as ICAM-1 on endothelial cells [9], [10]. IL-6 blockade reduces infiltration of neutrophils into arthritic joints in a monkey model of arthritis [11]. Moreover, interestingly, IL-6 injection rapidly causes neutrophilia [12], [13]. However, there are few reports indicating how IL-6 increases neutrophilia. Suwa et al. reported that IL-6 decreased CD62L expression on neutrophils and promoted their trafficking in rabbits [14]. However, in their study, they did not address the discrepancy between the effect of IL-6 on CD62L expression in vivo and that in vitro, nor did they verify the involvement of other adhesion molecules in neutrophilia induced by IL-6. In the present study, we studied the involvement of IL-6 in neutrophilia induction under inflammatory conditions.

Section snippets

Reagents

Recombinant humanized anti-human IL-6 receptor (IL-6R) antibody (tocilizumab) and human IL-6 were prepared in our laboratories as previously described [15], [16]. Cynomolgus monkey IL-6 cDNA was cloned from cynomolgus monkey thymus by PCR and transfected into Chinese hamster ovary cells, and the recombinant cynomolgus monkey IL-6 was purified by gel filtration. Recombinant human IL-8 and GM-CSF were purchased from Wako Pure Chemical Industries, Osaka, Japan.

Animals

Cynomolgus monkeys (Macaca

Anti-IL-6 receptor antibody decreased neutrophil counts in arthritic monkeys

The neutrophil counts increased rapidly in the first week after the 1st immunization, and then remained in a steady state until week 5. The elevated neutrophil counts were rapidly decreased by tocilizumab treatment (Fig. 1). After the initial rapid decrease, neutrophil counts gradually began to increase.

In this model, arthritis was first observed at around day 28 and swelling of joints reached the maximum at day 35. We previously reported that serum IL-6 levels were clearly elevated after the

Discussion

The present report describes one mechanism of neutrophilia in arthritis. We showed that (1) IL-6 blockade rapidly improved neutrophilia in arthritic monkeys; (2) IL-6 injection increased neutrophil counts and remarkably reduced CD162 expression on circulating neutrophils in monkeys; (3) IL-6 injection induced GM-CSF and IL-8 production in monkeys; (4) GM-CSF and IL-8 reduced CD162 expression in human isolated neutrophils, but IL-6 itself did not; and (5) GM-CSF and IL-8 promoted shedding of CD162 in

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