Characterization of the heparin-binding properties of IL-6.
We establish, using an ELISA approach, that recombinant human and murine IL-6 bind to an immobilized heparin-BSA complex. In the case of human IL-6, this binding is displaceable by soluble heparin, IC(50) approximately 2 microg/ml, corresponding to approximately 200 nM. This binding is specific because chondroitin sulfates B and C fail to compete, whereas chondroitin sulfate A and several heparan sulfates are weak inhibitors. Of a range of chemically modified heparins examined, the strongest competitor was the 2-O:-desulfated product, but even this showed a considerably reduced IC(50) ( approximately 30 microg/ml). The epitopes of five IL-6-specific mAbs were still accessible in heparin-bound IL-6, and the dimer formed from the association of rIL-6 with its truncated soluble receptor polypeptide, srIL-6alpha, still bound to heparin. Further analysis showed that heparin competed partially and weakly with the binding of srIL-6 to IL-6; however, it competed strongly for the binding of the rIL-6/srIL-6Ralpha dimer, to soluble glycoprotein 130. In studies of the proliferation of IL-6-sensitive Ba/F3 cells expressing glycoprotein 130, we were unable to detect any effect of either the removal of cell surface heparan sulfate, or addition of soluble heparin. By contrast, heparin was able to protect IL-6 from digestion by the bacterial endoproteinase Lys-C. Overall, our findings show that IL-6 is a heparin-binding cytokine. This interaction will tend to retain IL-6 close to its sites of secretion in the tissues by binding to heparin-like glycosaminoglycans, thus favoring a paracrine mode of activity. Moreover, this binding may serve to protect the IL-6 from proteolytic degradation.
Mummery
R S
RS
Division of Biochemistry, Royal Holloway University of London, Egham, Surrey, United Kingdom.
Rider
C C
CC
D000818:Animals; D001667:Binding, Competitive / Q000276:immunology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects / Q000276:immunology; D002460:Cell Line / Q000187:drug effects / Q000276:immunology; D002704:Chlorates / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006131:Growth Inhibitors / Q000494:pharmacology; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D019947:Receptors, Interleukin-6 / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism
D002704:Chlorates; D006131:Growth Inhibitors; D006493:Heparin; D006497:Heparan Sulfate; D015850:Interleukin-6; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; C032706:sodium chlorate