M-CSF mediates TNF-induced inflammatory osteolysis

Abstract

TNF-alpha is the dominant cytokine in inflammatory osteolysis. Using mice whose BM stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, we found that both cell types mediated the cytokine's osteoclastogenic properties. The greater contribution was made, however, by stromal cells that express the osteoclastogenic cytokine M-CSF. TNF-alpha stimulated M-CSF gene expression, in vivo, only in the presence of TNF-responsive stromal cells. M-CSF, in turn, induced the key osteoclastogenic cytokine receptor, receptor activator of NF-kappaB (RANK), in osteoclast precursors. In keeping with the proproliferative and survival properties of M-CSF, TNF-alpha enhanced osteoclast precursor number only in the presence of stromal cells bearing TNF receptors. To determine the clinical relevance of these observations, we induced inflammatory arthritis in wild-type mice and treated them with a mAb directed against the M-CSF receptor, c-Fms. Anti-c-Fms mAb selectively and completely arrested the profound pathological osteoclastogenesis attending this condition, the significance of which is reflected by similar blunting of the in vivo bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b). Confirming that inhibition of the M-CSF signaling pathway targets TNF-alpha, anti-c-Fms also completely arrested osteolysis in TNF-injected mice with nominal effect on macrophage number. M-CSF and its receptor, c-Fms, therefore present as candidate therapeutic targets in states of inflammatory bone erosion.

Figure 1

Macrophage and stromal cell responsivity to TNF-α are required for optimal TNF-α–induced osteoclastogenesis in vivo. (A) Histological sections of calvaria excised from WT to WT (WT→WT), WT to KO, KO to WT, and KO to KO mice after 5 daily supracalvarial injections of increasing doses of TNF-α were stained for TRAP activity (red reaction product). Magnification, ×40. (B) The percentage of BM interface covered by osteoclasts was histomorphometrically determined in specimens derived from WT to WT, KO to WT, WT to KO, and KO to KO mice. *P < 0.01 compared with KO to KO mice. n = 3 mice per group.

Figure 2

(A) TNF-α increases RANK mRNA expression in vivo. BM was obtained from WT to WT, WT to KO, KO to WT, and KO to KO mice after 5 daily injections of TNF-α (3 μg/day) or vehicle. RNA was isolated, and RANK and RANKL mRNA expression was measured by RT-PCR. GAPDH mRNA served as loading control. (B). TNF-α and M-CSF increase RANK mRNA expression in vitro. WT BMMs were cultured without M-CSF. After 24 hours, the cells were placed in medium containing increasing amounts of TNF-α or M-CSF. Six hours later, RNA was isolated and RANK mRNA expression was measured by RT-PCR. GAPDH mRNA served as loading control. (C) TNF-α increases M-CSF mRNA expression in vivo. BM was obtained from WT to WT, WT to KO, KO to WT, and KO to KO mice after 5 daily injections of TNF-α (3 μg/day) or vehicle. RNA was isolated, and M-CSF mRNA expression was measured by RT-PCR. GAPDH mRNA served as loading control.

Figure 3

TNF-α–mediated increase in osteoclast precursor number in vivo requires cytokine-responsive stromal cells. The percentage of osteoclast precursors in BM recovered from WT to WT, WT to KO, KO to WT, and KO to KO mice after 5 daily injections of TNF-α (3 μg/day) or vehicle was determined by FACS, using FITC-conjugated anti-CD11b mAb (solid line) and FITC-conjugated isotype mAb (dotted line).

Figure 4

TNF-α induces M-CSF mRNA expression by BM stromal cells independent of IL-1. BM stromal cells were isolated on a VCAM-1 affinity column from WT or IL-1RI^–/– mice. VCAM-1–positive cells were then exposed to carrier (control) or TNF-α, IL-1α, and IL-1Ra alone or in combination for 24 hours. M-CSF mRNA in equal amounts of total RNA was determined by RT-PCR. GAPDH mRNA served as loading control.

Figure 5

TNF-α–mediated increase in osteoclast precursor number in vivo is IL-1 independent. The percentage of osteoclast precursors in BM recovered from WT, IL-1RI^–/–, and IL-1α^–/– mice after 5 daily injections of TNF-α (3 μg/day) or vehicle was determined by FACS using FITC-conjugated anti-CD11b mAb (filled profiles) and FITC-conjugated isotype mAb (open profiles).

Figure 6

Stromal cells or BMMs mediate optimal inflammatory osteolysis. Mice in each chimeric group were injected with arthrogenic serum derived from K/BxN mice. (A) Appearance of paws of representative mice at day 7 after injection with arthrogenic serum (+) or PBS (–). (B) Paw thickness measured daily for 10 days for each group of chimeric mice injected with arthrogenic serum (+) or PBS (–). (C) H&E-stained histological sections of ankles at day 7 showing severe inflammation (**) in arthrogenic serum-treated (+) WT to WT, WT to KO, and KO to WT mice compared with the relatively mild changes occurring in similarly treated KO to KO animals. Minus sign indicates those injected with PBS. (D) TRAP-stained histological sections of ankles of serum-injected or serum-noninjected WT to WT, WT to KO, KO to WT, and KO to KO mice at day 7. (E) Histomorphometric quantitation of the percentage of bone surface covered by osteoclasts (OC) in ankles of PBS-treated (–) or arthrogenic serum–treated (+) WT to WT, WT to KO, KO to WT, and KO to KO mice.*P < 0.001 arthrogenic serum– vs. PBS-treated mice. n = 4 mice per group. Magnification, ×40.

Figure 7

Stromal cells or BMMs mediate optimal inflammation-induced osteoclast precursor number. The percentage of osteoclast precursors in BM recovered from WT to WT, WT to KO, KO to WT, and KO to KO mice 7 days after injections of arthrogenic serum or vehicle was determined by FACS using FITC-conjugated anti-CD11b mAb (thick lines) and FITC-conjugated isotype mAb (thin lines).

Figure 8

Anti–c-Fms antibody arrests osteoclastogenesis in vitro. (A) BMMs from WT mice were cultured with increasing amounts of anti–c-Fms mAb in the presence of 100 ng/ml M-CSF. After 3 days, the number of viable cells was measured using the MTT assay. MTT absorbance was determined at an OD of 570 nm. (B) The same number of BMMs were cultured in the presence of M-CSF (100 ng/ml) and RANKL (50 ng/ml) with increasing amounts of anti–c-Fms mAb for 3 days. The cells were stained for TRAP activity to identify osteoclasts. Magnification, ×400. (C) Number of osteoclasts generated in wells containing various amounts of anti–c-Fms antibody. *P < 0.001 vs. 0 ng/ml.

Figure 9

Anti–c-Fms mAb blocks inflammatory arthritis–induced osteoclastogenesis and bone resorption in vivo. Mice were injected with arthrogenic serum with or without daily administration of anti–c-Fms mAb or PBS. (A) Appearance of paws of representative mice at 7 days after injection with arthrogenic serum with or without anti–c-Fms mAb. (B) Paw thickness, measured daily for 7 days. (C) H&E- or TRAP- stained histological sections of ankles at day 7. TRAP reaction product identifying osteoclasts is red. Bottom panels are detailed views of boxed-in areas in middle panels. Magnification: ×40 (top and middle panels), ×200 (bottom panels). (D) Histomorphometric quantitation of the percentage of bone surface covered by osteoclasts in ankles of each group of mice. (E) Circulating TRACP 5b levels. (F). Representative TRAP-stained histological sections of proximal tibia of each group of mice. TRAP reaction product identifying osteoclasts is red. n = 4 mice per group. Magnification, ×40. *P < 0.001 compared with serum (+); ^#P < 0.001 compared with serum (–).

Figure 10

Anti–c-Fms mAb blocks TNF-α–induced osteoclastogenesis and bone resorption in vivo. (A) Histological sections of calvariae excised from mice after 5 daily supracalvarial injections of TNF-α, with or without intraperitoneal injection of anti–c-Fms mAb, were stained for TRAP activity (red reaction product). Magnification, ×40. (B) The percentage of bone/BM interface covered by osteoclasts was histomorphometrically determined. (C) Circulating TRACP 5b levels were determined by ELISA. (D) Abundance of CD11b-positive osteoclast precursors in BM recovered from TNF-injected mice with (red line) or without (green line) anti–c-Fms mAb. The black line represents FITC-conjugated isotype mAb. The illustration is representative of 4 separate experiments. n = 4 in all groups. *P < 0.001 compared with TNF-α alone; ^#P < 0.001 compared with control.