Unbiased determination of cytokine localization in bone: Colocalization ofinterleukin-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 localiza tion of interleukin-6 (IL-6) relative to ostcoblasts (alkaline phosphate [A LP]-positive cells) and osteoclasts (tartrate-resistant acid phosphate [TRA P]-positive cells) in midsagittal, paraffin-embedded serial sections of tho racic 13 (T-13) vertebrae from 49 female cynomolgus monkeys. Serial section s 1 and 4 were immunostained for IL-6, section 2 was histochemically staine d for TRAP, and section 3 was immunostained for ALP, Sixteen centrally loca ted fields were measured in the cancellous compartment and grid alignment a mong sections was verified using image analysis. Using a Merz grid, IL-6 lo calized to 6% of the bone surface on sections 1 and 4, whereas TRAP localiz ed to 8.5% and ALP to 12% of the bone surface, Colocalization was defined a s positive staining within an 80 x 80 mu m block in the first serial sectio n that "overlapped" staining in either the corresponding block or its eight surrounding blocks within the second serial section. For each section, 160 0 blocks were analyzed. Using Monte Carlo simulations, random colocalizatio n was calculated to determine the statistical significance of experimental colocalizations. Colocalization of approximately 90% between the two IL-6 s ections verified staining reproducibility and proper grid alignment among s ections. Colocalization of TRAP and ALP was not statistically different fro m random (p > 0.3), As identified using ALP- or TRAP-positive surfaces, the re was significant IL-6 colocalization with osteoblasts (p < 0.003), but no t with osteoclasts (P > 0.3), These in vivo colocalization data support the hypothesis that osteoblasts produce and respond to IL-6, (C) 2000 by Elsev ier Science Inc. All rights reserved.