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J Biol Chem. 2019 Mar 22;294(12):4412-4424. doi: 10.1074/jbc.RA118.006193. Epub 2019 Jan 22.

Molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity.

Author information

1
From the Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
2
the Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695.
3
the Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701.
4
the Division of Chemical Biology and Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and.
5
the Human Retrovirus Pathogenesis Section, Vaccine Branch, NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702.
6
From the Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, dazaharo@ncsu.edu.

Abstract

Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.

KEYWORDS:

IL-12; cytokine; glycosaminoglycan; heparan sulfate; heparin; heparin-binding cytokine; immunology; interleukin

PMID:
30670588
PMCID:
PMC6433073
[Available on 2020-03-22]
DOI:
10.1074/jbc.RA118.006193

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