IL-1 mediates TNF-induced osteoclastogenesis

Abstract

TNF-induced receptor activator NF-kappaB ligand (RANKL) synthesis by bone marrow stromal cells is a fundamental component of inflammatory osteolysis. We found that this process was abolished by IL-1 receptor antagonist (IL-1Ra) or in stromal cells derived from type I IL-1 receptor-deficient (IL-1RI-deficient) mice. Reflecting sequential signaling of the cytokines TNF and IL-1, TNF induces stromal cell expression of IL-1 and IL-1RI. These data suggest that TNF regulates RANKL expression via IL-1, and, therefore, IL-1 plays a role in TNF-induced periarticular osteolysis. Consistent with this posture, TNF-stimulated osteoclastogenesis in cultures consisting of WT marrow macrophages and stromal cells exposed to IL-1Ra or in cocultures established with IL-1RI-deficient stromal cells was reduced approximately 50%. The same magnitude of osteoclast inhibition occurred in IL-1RI-deficient mice following TNF administration in vivo. Like TNF, IL-1 directly targeted osteoclast precursors and promoted the osteoclast phenotype in a TNF-independent manner in the presence of permissive levels of RANKL. IL-1 is able to induce RANKL expression by stromal cells and directly stimulate osteoclast precursor differentiation under the aegis of p38 MAPK. Thus, IL-1 mediates the osteoclastogenic effect of TNF by enhancing stromal cell expression of RANKL and directly stimulating differentiation of osteoclast precursors.

Figure 1

TNF induces RANKL gene expression by murine and human marrow stromal cells. (A) VCAM-1–positive and –negative murine marrow cells were exposed to PBS (Control), 1,25-dihydroxyvitamin D (Vit. D; 10^–8 M), or TNF (10 ng/ml) for 24 hours. (B) hMSCs were cultured for 1 week and then exposed to 1 or 10 ng/ml TNF for 24 hours. In both circumstances, total RNA was extracted and analyzed by RT-PCR. GAPDH mRNA served as loading control. Results are expressed as AU relative to control (arbitrarily set as 1).

Figure 2

TNF-induced RANKL gene expression is mediated by IL-1. (A and C) VCAM-1–positive WT or IL-1RI–deficient (IL-1RI KO) murine marrow cells or (B) hMSCs were exposed to designated cytokines or 1,25-dihydroxyvitamin D (10^–8 M) for 24 hours. In some circumstances TNF was added conjointly with IL-1Ra. RNA was extracted and subjected to RT-PCR. Numbers in parentheses represent concentrations in ng/ml.

Figure 3

TNF induces IL-1 and IL-1RI in stromal cells. (A) VCAM-1–positive murine marrow cells and (B) hMSCs were exposed to vehicle (Control) or indicated cytokines for 24 hours. Numbers in parentheses represent concentrations in ng/ml. Total RNA was extracted and analyzed by RT-PCR.

Figure 4

IL-1 mediates TNF-induced osteoclastogenesis in vitro. WT or IL-1RI–deficient, VCAM-1–positive murine marrow stromal cells were cocultured with bone marrow macrophages derived from p55/p75 TNF receptor–deficient mice in the presence of only the indicated cytokines. IL-1Ra (100 ng/ml) was added to half the cultures containing WT stromal cells. Medium was changed every 3 days. (A) After 7 days, the cells were stained for TRAP activity and (B) the number of multinucleated TRAP-positive cells determined. Values are mean ± SD for 6 determinations per group (**P < 0.01 vs. WT). OC, osteoclasts.

Figure 5

IL-1 mediates TNF-induced osteoclastogenesis in vivo. WT and IL-1RI–deficient mice were subcutaneously injected daily for 5 days at the base of the skull with vehicle or 0.75 or 1.5 μg TNF. The animals were sacrificed on day 6 and TRAP-stained histological sections of calvariae histomorphometrically analyzed for osteoclast number. (A) Representative histological sections. (B) Histomorphometric analysis of osteoclast number (*P < 0.05, **P < 0.01 vs. WT).

Figure 6

IL-4 inhibits TNF- or IL-1–induced RANKL expression. VCAM-1–positive murine marrow cells or hMSCs were pretreated with IL-4 (10 ng/ml) for indicated periods, followed by exposure to vehicle (–) or (A) TNF (10 ng/ml) or (B) IL-1 (10 ng/ml) for 24 hours. RNA was extracted and analyzed by RT-PCR.

Figure 7

IL-4 inhibits IL-1–mediated p38 MAPK activation. hMSCs were pretreated without or with IL-4 (10 ng/ml) for 1 or 24 hours. Cells were then exposed to IL-1 (10 ng/ml) for the indicated periods. Cells were lysed, fractionated by SDS-PAGE, and analyzed by immunoblot using antibodies recognizing total and phosphorylated signaling molecules. β-actin served as loading control. p-, phospho-.

Figure 8

p38 MAPK mediates TNF- and IL-1–induced RANKL and IL-1RI expression. hMSCs were pretreated with SB203580 at the indicated concentrations overnight before exposure to vehicle (–) or (A) TNF (10 ng/ml) or (B) IL-1 (10 ng/ml) for 15 minutes. Cells were lysed and fractionated by SDS-PAGE, and activation of signaling molecules determined by immunoblot. ATF-2, activating transcription factor–2. (C) hMSCs were pretreated with decreasing concentrations of SB203580 for 30 minutes before exposure to PBS, TNF (10 ng/ml), or IL-1 (10 ng/ml) for 24 hours. RNA was extracted and analyzed by RT-PCR.

Figure 9

TNF stimulates IL-1 gene expression by osteoclast precursors in a p38-dependent manner. WT murine marrow macrophages were pretreated with decreasing concentrations of SB203580 for 30 minutes before exposure to PBS (Control) or TNF (10 ng/ml) for 24 hours. Numbers in parentheses represent concentrations in ng/ml. RNA was extracted and analyzed by RT-PCR.

Figure 10

IL-1 directly induces osteoclast precursor differentiation. (A) WT or p55/p75 TNF receptor–deficient (TNFR KO) murine marrow macrophages were cultured in human recombinant M-CSF (50 ng/ml) without or with RANKL and/or IL-1 at the indicated concentrations (ng/ml) at the initiation of culture. TRAP stain was performed on day 5. (B) Number of osteoclasts in cultures illustrated in panel A (* P < 0.05, ** P < 0.01, ^#P < 0.001 vs. no addition).

Figure 11

IL-1 induction of osteoclast precursor differentiation occurs within a broad temporal window. (A) WT marrow macrophages were cultured in the presence of M-CSF (50 ng/ml) and RANKL (10 ng/ml). IL-1 (1.25 ng/ml) or vehicle was added only once to each well. IL-1 addition occurred on sequential days from day 0 through day 4. TRAP stain was performed on day 5. (B) Osteoclast number in each culture condition described in A (^#P < 0.0001 vs. no IL-1 addition). (C) WT marrow macrophages were cultured in the presence of M-CSF (50 ng/ml) plus RANKL (100 ng/ml). Total RNA was extracted with time and analyzed by RT-PCR for abundance of IL-1RI and IL-1RII mRNA.

Figure 12

TNF and IL-1 induce IL-1RI expression in committed preosteoclasts in a p38-dependent manner. (A) Freshly isolated bone marrow macrophages (BMMs) and the same cells exposed to M-CSF plus RANKL for 3 days (preosteoclasts [pre-OCs]) were washed with PBS and then treated with TNF-α, IL-1, or RANKL for 24 hours. Total RNA was extracted with time and analyzed by RT-PCR for IL-1RI. (B) Preosteoclasts were pretreated with decreasing concentrations of SB203580 for 30 minutes before exposure to PBS (Control) or TNF (10 ng/ml), IL-1 (10 ng/ml), or RANKL (100 ng/ml) for 24 hours. RNA was extracted and analyzed by RT-PCR. (C) Preosteoclasts were treated with TNF (10 ng/ml) with or without IL-1Ra (100ng/ml) for 24 hours. Numbers in parentheses represent concentrations in ng/ml. RNA was extracted and analyzed by RT-PCR.

Figure 13

Mechanisms of TNF-induced osteoclastogenesis. As detailed in the text, TNF-induced osteoclastogenesis involves targeting of both stromal cells and osteoclast precursors.