Species‐specific differences in heparin‐induced modulation of IL‐12 family cytokines

Abstract

IL‐12 family cytokines play a central role in the immune system by connecting the activities of innate and adaptive immunity. IL‐12 family members, IL‐12 and IL‐23, share a common p40 subunit that contains a prominent heparin binding domain. We have previously exploited this heparin binding segment for heparin affinity‐based purification of recombinant IL‐12. We also demonstrated that exogenous heparin can enhance IL‐12‐mediated interferon‐gamma (IFN‐g) production. However, the interaction between heparin and IL‐12/IL‐23 at the molecular level and the mechanisms by which heparin modulates the biological functions of these cytokines have not been explored. In addition, little is known about the species dependence of IL‐12 and IL‐23 in the context of sulfated glycosaminoglycans. Using isothermal titration calorimetry (ITC), we found that heparin can bind at micromolar concentrations to both human IL‐12 (hIL‐12) (Kd=9.8±0.2 mM) and murine IL‐12 (mIL‐12) (Kd=45.2±1.1 mM). ITC studies also showed that only heparin molecules longer that 8 saccharide units are able to bind to IL‐12. We confirmed the interaction of heparan sulfate (HS)‐derived compounds and hIL‐12/mIL‐12 via a novel, high throughput glycan microarray. Results from the microarray demonstrated that only HS‐derived molecules with 3 sulfate groups per disaccharide can bind to hIL‐12 and mIL‐12. In bioactivity studies, heparin was found to enhance hIL‐12 and human IL‐23 (hIL‐23) in human cells. On the other hand, though heparin did not increase the activity of mIL‐2 in 2D6 murine lymphocytes or C57BL/6 splenocytes, heparin enhanced the activity of mouse IL‐23 (mIL‐23) in C57BL/6 splenocytes. In cross‐species studies, heparin enhanced mIL‐12 activity in human NK‐9MI cells (1.55‐fold) whereas hIL‐12 was inactive in murine 2D6 cells and remained inactive in the presence of heparin. Interestingly, heparin was found to enhance mIL‐23 in human HEK Blue™ IL‐23 cells and hIL‐23 bioactivities in C57BL/6 splenocytes. Based on these and other data, we developed a working model in which heparin serves as a co‐receptor by stabilizing the hIL‐12/hIL‐12 receptor complex. We hypothesize that the mIL‐12/mIL‐12 receptor complex is sufficiently stable so that it does not benefit from heparin involvement. On the other hand, the interactions of hIL‐12/hIL‐12R, hIL‐23/hIL‐23R, and mIL‐23/mIL‐23R may be less stable so as to benefit from the addition of heparin. Ongoing ITC studies will evaluate the thermodynamics of the hIL‐12/hIL‐12R, mIL‐12/mIL‐12R, hIL‐23/hIL‐23R, and mIL‐23/mIL‐23R complexes in the presence and absence of heparin. Differences in binding affinity may help explain the species‐specific nature of heparin‐enhanced activity of the IL‐12 family cytokines.Support or Funding InformationThis work was supported by funding from the National Institutes of Health, National Cancer Institute (R01CA172631), the Arkansas Biosciences Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.