IL-1β inhibits human osteoblast migration

Abstract

Bone has a high capacity for self-renewal and repair. Prolonged local secretion of interleukin 1β (IL-1β), however, is known to be associated with severe bone loss and delayed fracture healing. Since induction of bone resorption by IL-1β may not sufficiently explain these pathologic processes, we investigated, in vitro, if and how IL-1β affects migration of multipotent mesenchymal stromal cells (MSC) or osteoblasts. We found that homogenous exposure to IL-1β significantly diminished both nondirectional migration and site-directed migration toward the chemotactic factors platelet-derived growth factor (PDGF)-BB and insulin like growth factor 1 (IGF-1) in osteoblasts. Exposure to a concentration gradient of IL-1β induced an even stronger inhibition of migration and completely abolished the migratory response of osteoblasts toward PDGF-BB, IGF-1, vascular endothelial growth factor A (VEGF-A) and the complement factor C5a. IL-1β induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK) activation and inhibition of these signaling pathways suggested an involvement in the IL-1β effects on osteoblast migration. In contrast, basal migration of MSC and their migratory activity toward PDGF-BB was found to be unaffected by IL-1β. These results indicate that the presence of IL-1β leads to impaired recruitment of osteoblasts which might influence early stages of fracture healing and could have pathological relevance for bone remodeling in inflammatory bone disease.

Figure 1

Influence of IL-1β on osteoblast migration under basal conditions (A) and toward C5a (B). In vitro migration was analyzed in a modified Boyden chamber using 8 μm poly-carbonate filters. IL-1β was diluted in serum-free DMEM at 10 (light gray bars), 100 (dark gray bars) or 1000 (black bars) pg/mL and added either to both upper and lower or only to lower wells of the Boyden chamber as indicated. Serum-free DMEM was used as control (white bars). C5a was diluted in serum-free DMEM at 100 ng/mL. Columns represent mean number of totally migrated cells, bars represent SEM from three independent donors. Significance was calculated with two-way ANOVA followed by a Bonferroni post hoc test; *P < 0.05. (A) Migration of osteoblasts with IL-1β in upper and lower (left) or only in lower wells (right). (B) Migration of osteoblasts toward a C5a gradient with IL-1β in upper and lower (left) or only in lower wells (right).

Figure 2

Influence of IL-1β on osteoblast migration toward several growth factors with IL-1β present in a uniform concentration (A) or as a gradient (B). In vitro migration was analyzed in a modified Boyden chamber using 8 μm polycarbonate filters. IL-1β was diluted in serum-free DMEM 100 pg/mL (black bars) and added either to both upper and lower (A) or only to lower (B) wells of the Boyden chamber as indicated. Serum-free DMEM was used as control (white bars). Growth factors were diluted in serum-free medium at the following concentrations: PDGF-BB (10 ng/mL), IGF-1 (100 ng/mL) and VEGF-A (100 ng/mL). Columns represent mean number of totally migrated cells, bars represent SEM from three independent donors. Significance was calculated with two-way ANOVA followed by a Bonferroni post hoc test; *P < 0.05. (A) Migration of osteoblasts toward growth factors with IL-1β in upper and lower wells. (B) Migration of osteoblasts toward growth factors with IL-1β only in lower wells.

Figure 3

Influence of IL-1β on osteoblast adhesion. In vitro adhesion assay was performed in a modified Boyden chamber using 8 μm polycarbonate filters. IL-1β was diluted in serum-free DMEM at 100 pg/mL and added either to both upper and lower (gray bars) or only to lower wells (black bars) of the Boyden chamber as indicated. Serum-free DMEM was used as negative control (white bars). Columns represent mean number of adhered cells from one donor.

Figure 4

Influence of an IL-1β gradient on MSC migration toward PDGF-BB. In vitro migration was analyzed in a modified Boyden chamber using 8 μm polycarbonate filters. IL-1β was diluted in serum-free DMEM at 100 pg/mL and added to the lower wells of the Boyden chamber as indicated (black bars). Serum-free DMEM was used as control (white bars). PDGF-BB was diluted in serum-free DMEM at 10 ng/mL. Columns represent mean number of totally migrated cells, bars represent SEM from three independent donors.

Figure 5

Expression of IL-1R1 in MSC and osteoblasts. Immunocytochemistry with an anti-IL-1R1 antibody was performed on MSC and osteoblasts cultured on glass slides as well as on migrated MSC and osteoblasts. For in vitro basal migration and migration toward PDGF-BB, a modified Boyden chamber with 8-μm polycarbonate filters was used. Positive IL-1R1 staining in cultured MSC (A) and osteoblasts (B). Negative control for cultured MSC (C) and osteoblasts (D). Positive IL-1R1 staining in MSC (E) and osteoblasts (F) that migrated toward PDGF-BB. Positive IL-1R1 staining in MSC (G) and osteoblasts (H) that migrated under basal conditions. Scale bars, 100 μm.

Figure 6

Human Phospho-MAPK Array of control and IL-1β-stimulated osteoblasts. Adherent osteoblasts were left untreated (upper filter, white bars) or stimulated with 100 pg/mL IL-1β (lower filter, black bars) in serum-free DMEM and lysed for analysis with the Human Phospho-MAPK Array.

Figure 7

Restoration of IL-1β-induced inhibition of osteoblast basal migration (A) and osteoblast migration toward PDGF-BB (B)—both through inhibition of ERK 1/2 and JNK signaling in osteoblasts. In vitro migration was analyzed in a modified Boyden chamber using 8-μm polycarbonate filters. IL-1β was diluted in serum-free DMEM at 100 pg/mL and added to the lower wells of the Boyden chamber as indicated (black bars). Serum-free DMEM was used as control (white bars). PDGF-BB was diluted in serum-free DMEM at 10 ng/mL. For inhibition of chemotaxis with MAPK inhibitors, 10 μmol/L PD 98059 (ERK1/2 inhibition) or JNK inhibitor II was added to upper and lower wells. Columns represent mean number of totally migrated cells, bars represent SEM from three independent donors. Significance was calculated with two-way ANOVA followed by a Bonferroni post hoc test; **P < 0.01. Inhibition of ERK 1/2 and JNK signaling in osteoblasts in the presence of IL-1β (A) or both IL-1β and PDGFBB (B).