Elsevier

Bone

Volume 51, Issue 4, October 2012, Pages 771-780
Bone

Original Full Length Article
IL-4 inhibits TNF-α-mediated osteoclast formation by inhibition of RANKL expression in TNF-α-activated stromal cells and direct inhibition of TNF-α-activated osteoclast precursors via a T-cell-independent mechanism in vivo

https://doi.org/10.1016/j.bone.2012.06.024Get rights and content

Abstract

It has been reported that osteoclastogenesis is induced by tumor necrosis factor (TNF)-α. Interleukin (IL)-4 is the most important cytokine involved in humoral immunity. However, no studies have investigated the effect of IL-4 on TNF-α-mediated osteoclast formation in vivo. In this study, we investigated the effect of IL-4 on TNF-α-mediated osteoclast formation in vivo. TNF-α was administered with and without IL-4 into the supracalvariae of mice. The number of osteoclasts and the levels of mRNA for cathepsin K and tartrate-resistant acid phosphate, both osteoclast markers, in mice administered TNF-α and IL-4 were lower than those in mice administered TNF-α alone. The level of tartrate-resistant acid phosphatase form 5b (TRACP5b) as a marker of bone resorption in mice administered both TNF-α and IL-4 was also lower. We showed that IL-4 inhibited TNF-α-mediated osteoclast formation in osteoclast precursors in vitro. Expression of receptor activator of NF-κB ligand (RANKL) in TNF-α-activated stromal cells was also inhibited. Furthermore, we investigated whether IL-4 had effects on both stromal cells and osteoclast precursors in TNF-α-mediated osteoclast formation in vivo. Using mice whose stromal cells and osteoclast precursors were chimeric for the presence of TNF receptors, IL-4 inhibited TNF-α-mediated osteoclast formation in the presence of TNF-α-responsive stromal cells, and TNF-α-responsive osteoclast precursors in vivo. IL-4 also inhibited TNF-α-induced RANKL expression in the presence of TNF-α-responsive stromal cells in vivo. This event is dependent on p38 inhibition in vitro. Additionally, IL-4 inhibited TNF-α-mediated osteoclast formation in T cell-depleted mice. In summary, we conclude that IL-4 inhibited TNF-α-mediated osteoclast formation by inhibiting expression of RANKL in TNF-α-activated stromal cells, and directly inhibited TNF-α-activated osteoclast precursors in vivo via a T cell-independent mechanism.

Highlights

► IL-4 significantly inhibited TNF-α induced osteoclastogenesis in vivo. ► IL-4 inhibited TNF-α-induced RANKL mRNA expression in vitro and in vivo. ► IL-4 affects both stromal cells and osteoclast precursors in TNF-α-mediated oeteoclastogenesis in vivo. ► IL-4 also inhibited TNF-α-induced osteoclastogenesis in mice blocked T cell.

Introduction

Osteoclasts derived from bone marrow cells regulate bone resorption and remodeling [1]. Osteoclast formation and activation require two factors: receptor activator of NF-κB ligand (RANKL), and macrophage colony stimulating factor (M-CSF) [2]. It has been reported that tumor necrosis factor (TNF)-α mediates osteoclast formation in vitro [3], [4], [5] and in vivo [6], [7]. TNF-α is known to play a major role in host defense and it exerts proinflammatory activities in various cells, such as mononuclear phagocytes, in which it is responsible for the activation of bactericidal and cytocidal systems [8]. TNF-α is produced mainly by macrophages but also by a variety of other cell types in response to bacterial toxins, inflammatory products and other invasive stimuli [9]. TNF-α-mediated osteoclast recruitment is probably central to the pathogenesis of disorders with inflammatory osteolysis such as periprosthetic bone loss, rheumatoid arthritis and periodontal disease [10], [11], [12]. TNF-α is a known cause of postmenopausal osteoporosis [13]. The inhibition of TNF-α has been found to be an effective therapy for treating rheumatic diseases [14].

It has been reported that interleukin (IL)-12, IL-18 and interferon (IFN)-γ, which are T-helper 1 (Th1)-type cytokines, inhibit TNF-α-mediated osteoclast formation in vitro [15], [16], [17] and in vivo [17], [18], [19]. However, Th2 cell-derived cytokines, such as IL-4 and IL-10, inhibit RANKL-induced osteoclast formation in vitro [20]. IL-4, a Th2 cytokine, is pleiotropic and produced by antigen-activated T cells and plays important roles in inflammatory and immune responses. IL-4 also regulates growth, activity and survival of the lymphoid lineage [21]. IL-4 has been reported to suppress IL-17, RANKL and bone resorption for mouse collagen arthritis in vivo [22]. Furthermore, IL-4 inhibited RANKL-induced osteoclast differentiation through direct action on osteoclast precursors that was independent of stromal cells in vitro [23], [24], [25]. Additionally, IL-4 inhibited TNF-α-mediated osteoclast formation in vitro [26]. However, no studies have investigated the effect of IL-4 on TNF-α-mediated osteoclast formation in vivo.

IL-4 is known to influence both osteoclasts and osteoblasts. Ovariectomy or parathyroid hormone-related protein-stimulated bone resorption is inhibited by IL-4 in vivo [27], [28]. Mice overexpressing IL-4 develop systemic bone loss akin to type 2 osteoporosis. It is characterized by attenuated remodeling activity in which osteoblast and osteoclast recruitment is suppressed [29]. TNF-α exerts an osteoclastogenesis effect by stimulating stromal cells to produce RANKL and also directly activates the osteoclast precursors, as shown in an experiment on chimeric mice using wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFR KO) mice in vivo [7]. Previous reports have shown that IL-4 inhibits TNF-α-induced RANKL expression in murine stromal cells and human mesenchymal stem cells (hMCSs) in vitro [30]. There is a possibility that IL-4 affects TNF-α-activated stromal cells and TNF-α-activated osteoclast precursors during TNF-α-mediated osteoclast formation in vivo.

In this study, we determine how IL-4 affects TNF-α-mediated osteoclast formation, whether IL-4 directly inhibits differentiation from osteoclast precursors to osteoclasts and IL-4 indirectly inhibits TNF-α-mediated osteoclast formation by inhibition of TNF-α-mediated RANKL expression in stromal cells. In addition, we focus on the issue of whether the effect of IL-4 on osteoclast formation requires the presence of a T cell population in vivo.

Section snippets

Mice and reagents

Nine- to ten-week-old male C57BL6/J mice were purchased from CLEA Japan (Tokyo, Japan) and TNFR KO mice (Tnfrsf1atm1lmxTnfrsf1btm1lmx) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All animal procedures were in accordance with Tohoku University regulations. Recombinant mouse IL-4 was purchased from R&D Systems (Minneapolis, MN, USA). TNF-α was a recombinant mouse TNF-α [6], and M-CSF was a recombinant mouse M-CSF developed from an M-CSF expressing cell line (CMG14-12) [31].

IL-4 inhibits TNF-α-mediated osteoclast formation in vivo

In the TNF-α-administered group, many osteoclasts were observed; however, the number of osteoclasts was significantly reduced in the group administered both TNF-α and IL-4 (Figs. 1A and B). The expression levels of TRAP and cathepsin K mRNAs were higher in the TNF-α-administered group than in the PBS group. The expression levels of both TRAP and cathepsin K were significantly lower in the group administered both TNF-α and IL-4 than those in the TNF-α-administered group (Figs. 1C and D). The

Discussion

In the present study, we examined the effects of IL-4 on TNF-α-mediated osteoclast differentiation in vivo. We demonstrated for the first time that IL-4 could inhibit TNF-α-induced osteolysis by inhibition of osteoclast formation in vivo. It has been reported that osteoclast precursors are direct targets of TNF-α in vitro [32]. We previously reported that both stromal cells and osteoclast precursors demonstrate TNF-α-induced osteoclastogenic properties. In that study, the results showed that

Acknowledgments

This work was supported by a Grant for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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