Unbiased determination of cytokine localization in bone: colocalization of interleukin-6 with osteoblasts in serial sections from monkey vertebrae

Few data are available describing the in vivo localization of cytokines in bone. The objective of this study was to describe the histological localization of interleukin-6 (IL-6) relative to osteoblasts (alkaline phosphate [ALP]-positive cells) and osteoclasts (tartrate-resistant acid phosphate [TRAP]-positive cells) in midsagittal, paraffin-embedded serial sections of thoracic 13 (T-13) vertebrae from 49 female cynomolgus monkeys. Serial sections 1 and 4 were immunostained for IL-6, section 2 was histochemically stained for TRAP, and section 3 was immunostained for ALP. Sixteen centrally located fields were measured in the cancellous compartment and grid alignment among sections was verified using image analysis. Using a Merz grid, IL-6 localized to 6% of the bone surface on sections 1 and 4, whereas TRAP localized to 8.5% and ALP to 12% of the bone surface. Colocalization was defined as positive staining within an 80 × 80 μm block in the first serial section that “overlapped” staining in either the corresponding block or its eight surrounding blocks within the second serial section. For each section, 1600 blocks were analyzed. Using Monte Carlo simulations, random colocalization was calculated to determine the statistical significance of experimental colocalizations. Colocalization of approximately 90% between the two IL-6 sections verified staining reproducibility and proper grid alignment among sections. Colocalization of TRAP and ALP was not statistically different from random (p> 0.3). As identified using ALP- or TRAP-positive surfaces, there was significant IL-6 colocalization with osteoblasts (p< 0.003), but not with osteoclasts (p> 0.3). These in vivo colocalization data support the hypothesis that osteoblasts produce and respond to IL-6.