Methods: Osteoclasts were isolated from long bones of neonatal rats and rabbits. Morphology and motility were monitored using time-lapse videomicroscopy. Filamentous actin and αvβ3 integrins were fluorescently labeled and visualized by confocal microscopy.
Results: Vitaxin, a humanized monoclonal antibody that blocks human and rabbit αvβ3 integrins, induced significant retraction of rabbit osteoclasts only when pretreated with M-CSF. In contrast, control antibody had no significant effect. Similarly, VNR-149, a monoclonal antibody that blocks rat αvβ3 integrins, induced transient retraction of M-CSF-treated rat osteoclasts (planar cell area was reduced by 27 ± 6% at 30 min). In contrast to its effects on retraction, VNR-149 did not inhibit M-CSF-induced chemotaxis of rat osteoclasts. Both M-CSF and VNR-149 decreased the proportion of rat osteoclasts exhibiting actin rings (specialized structures associated with resorption). In contrast, only VNR-149 induced a unique punctate pattern of actin staining that may reflect disruption of actin rings. M-CSF caused redistribution of αvβ3 integrins, possibly explaining the greater sensitivity of M-CSF-treated osteoclasts to the blocking antibodies.
Conclusions: Following treatment with M-CSF, αvβ3 integrin blockers caused osteoclast retraction consistent with decreased adhesion to the extracellular matrix. This was accompanied by disruption of the actin cytoskeleton, a change associated with decreased resorptive activity. These findings provide further evidence that the αvβ3 integrin is an appropriate target for development of anti-resorptive therapeutics for periodontitis and other inflammatory and metabolic diseases affecting bone.
Supported by the Canadian Institutes of Health Research, Canadian Arthritis Network and MedImmune Inc.