Figure 5.

Heparin-induced enhancement of IL-12 depends on sulfation level. hIL-12 binding to heparin oligosaccharides was quantified via a novel microarray. The binding of Alexa Fluor 488-labeled hIL-12 (A) or mIL-12 (B) to the different heparin oligosaccharides was visualized and quantified via fluorescence microscopy. Six dodecasaccharides, including the high binding compounds 18 and 19, were selected for bioactivity studies (C and D). hIL-12 activity as measured by IFN-γ production by NK-92MI cells (C) or secreted alkaline phosphatase by HEK-Blue^TM IL-12 cells (D) was measured after co-culture with heparin compounds 18, 19, 21, 23–25 or LMWH (10 μg/ml). Heparin compounds 18, 19, and LMWH significantly increased the bioactivity of hIL-12 in NK-92MI cells and HEK-Blue^TM IL-12 cells (p < 0.0001 versus hIL-12 alone via Tukey's post-test). Heparin compounds 21, 24, and 25 did not enhance the bioactivity of IL-12 in NK-92MI cells and HEK-Blue^TM IL-12 cells (p > 0.05 versus hIL-12 alone via Tukey's post-test). Heparin compound 23 did not enhance hIL-12 bioactivity in NK-92MI cells (***, p < 0.0001 versus IL-12 alone via Tukey's post-test) and modestly enhanced IL-12 bioactivity in HEK-Blue^TM IL-12 cells (p < 0.0001 versus IL-12 alone and p < 0.0001 versus IL-12 plus LMWH via Tukey's post-test). Data bars in the bioactivity studies represent mean ± S.D. of triplicate measurements. Experiments were performed in triplicate and repeated three times with similar results.