IL-12 Is a Heparin-Binding Cytokine

Abstract

Using an ELISA approach, we demonstrate that recombinant human IL-12 (rhIL-12) binds strongly to an immobilized heparin-BSA complex. This binding is completely displaceable with soluble heparin, IC50∼ 0.1 μg/ml, corresponding to ∼ 10 nM. By interpolation with our previous findings, this indicates an affinity for heparin greater than that of antithrombin III and comparable with that of FGF-2, two high-affinity heparin-binding proteins. Recombinant murine IL-12 also binds strongly to heparin. The binding of rhIL-12 to heparin shows specificity because chondroitin sulfates A and C fail to compete, whereas chondroitin B inhibits weakly. A highly sulfated heparan sulfate is a strong competitor, whereas other heparan sulfates show weak or no activity. Small heparin fragments inhibit binding, although activity decreases with size. An octasaccharide pool derived by cleavage of heparin with nitrous acid is a significantly stronger inhibitor than its heparinase I-derived counterpart, further indicating structural specificity in the interaction between rhIL-12 and heparin. The binding of recombinant p40 to heparin appears indistinguishable from that of the IL-12 heterodimer, implying that the heparin binding site is largely if not solely located in this subunit. These results show for the first time that IL-12 is a heparin-binding cytokine, a property common to the other Th1-response-inducing cytokines, IFN-γ and IL-2. Our findings strongly suggest that IL-12 will tend to be retained close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine role for IL-12.

Chitosan‐Low Molecular Weight Heparin Sodium Nanoparticles Regulate Treg/Th17 Immune Balance and Inflammation at the Maternal‐Fetal Interface to Ameliorate Pre‐Eclampsia by HB‐EGF

“…The interaction between heparin and glycosaminoglycan in cytokines has received significant attention in recent years. [68,69] Heparin binds to the p40 subunit of IL-12, [70] which is a crucial protein in Th17 differentiation. [71] Additionally, INF-𝛾 has a heparin-binding site.…”

Amino acid residues involved in the heparin-binding activity of murine IL-12 in the context of an antibody-cytokine fusion protein

“…GAG binding can also protect the cytokine from proteolytic degradation, as has been shown for basic and acidic FGF [41], IL-6 [42], IL-7 [38], IFN-γ [43]; and/or can facilitate the local accumulation of the cytokine as has been described for IL-2 [44] and IL-6 [42]. The possibility that IL-12 has heparin-binding activity was first proposed by Wolf et al [3], and this activity was found by Hasan et al [45], reporting high affinity binding of IL-12 to heparin/heparan sulfate. This latter group and more recently, Jayanthi et al and Garnier et al suggested that the heparin-binding activity of human and mouse IL-12 involves a cluster of basic amino acids within the C-terminal domain of the p40 subunit of human and mouse IL-12 [45][46][47].…”

“…The possibility that IL-12 has heparin-binding activity was first proposed by Wolf et al [3], and this activity was found by Hasan et al [45], reporting high affinity binding of IL-12 to heparin/heparan sulfate. This latter group and more recently, Jayanthi et al and Garnier et al suggested that the heparin-binding activity of human and mouse IL-12 involves a cluster of basic amino acids within the C-terminal domain of the p40 subunit of human and mouse IL-12 [45][46][47].…”

Spatiotemporally programming cytokine immunotherapies through protein engineering

“…ECM‐based retention is a promising strategy for anchoring cytokines due to the overexpression of, for example, collagen in solid tumors 113,114 . Furthermore, this strategy is actually biomimetic, as numerous cytokines (e.g., IL‐2, 22 IFNɣ, 115 and IL‐12 116 ) exhibit strong ECM interactions that keep their endogenous effects localized. Momin et al reported on a collagen‐anchoring platform that leverages these intratumoral collagen reserves for enhanced cytokine retention 24 .…”