Abstract
The biochemical nature of endogenous interleukin-6 (IL-6) as it exists in human serum or plasma was investigated. Serum from a patient following bone marrow (BM) transplantation and fresh plasma samples from patients with epidermolysis bullosa or psoriasis, as well as from normal volunteers, were fractionated through G-200 columns and each of the eluted fractions assayed for IL (interleukin)-6 content using enzyme-linked immunosorbent assays (ELISAs) based on the monoclonal antibody (mAb) pairs IG61/5IL6 or 4IL6/5IL6 and in the B9 hybridoma growth factor bioassay. The IG61/5IL6 ELISA and the B9 assay detected IL-6 in BM serum almost exclusively of molecular mass approximately 20 kDa. In contrast, the 4IL6/5IL6 ELISA detected strong IL-6 immunoreactivity in complexes of size 100-150 and 400-500 kDa. IL-6 present in the 100-150- and 400-500-kDa complexes was purified by immunoaffinity chromatography through a 5IL6 mAb column. The 5IL6 mAb immunoaffinity column eluate of the respective pools from BM serum contained IL-6 at concentrations approaching 1 microgram/ml as characterized by Western blotting. Sufficient IL-6 and associated proteins were purified by 5IL6 mAb immunoaffinity column chromatography of the 100-150-kDa complex from 0.8 ml of BM serum to allow (i) verification of three of the polypeptides as IL-6 by amino-terminal sequencing (estimate of IL-6 in original serum sample: 5-10 micrograms/ml), (ii) identification by amino acid sequencing of the "associated" proteins as complement factor C3b (carboxyl-terminal of the alpha-chain), complement factor C4b (gamma-chain), C-reactive protein, and albumin, and (iii) detection of an "associated" polypeptide consistent with the soluble IL-6 receptor. Taken together, these data establish that IL-6 is present at unexpectedly high concentrations in human blood in novel biochemical complexes that include other plasma proteins, which in turn, can camouflage IL-6 immunoreactivity and bioactivity as measured in conventional assays.
Highlights
The biochemical nature of endogenous interleukin-6 Thecytokineinterleukin-6(IL-6)’has beenincreasingly (IL-6) as it exists in human serum or plasma was in- studied as a protein with diverse and profound effects on a vestigated
Fig. 1shows that all of the B9-active IL-6 was found in a low molecular mass (13-20 kDa) peak that coincided with the bulk of the IG61/ 5IL6-detectable IL-6 antigen. (Monomeric rIL-6 of 21-kDa mass by SDS-PAGEelutes from a Sephadex gel filtration column with a Stokes radius consistent with that of a 13.5kDa protein; see Ref. 40)
ELISA; this result is consistent with the observation that the The data in this article raise novel questionsabout the biochemical nature of rIL-6 and fibroblast-derived IL-6 is transport andbioavailability of human IL-6 inthe peripheral circulation
Summary
Sephadex G-200Gel Filtration of Bioactive and Immunoreactive IL-6 inSerum and Plasma-Previously published descriptions of the apparentsize of serum-derived IL-6 eluted off a gel filtration column [38, 39] were confirmed using the B9 growth factor assay or the IG61/5IL6 ELISA. G-200 column fractionation of fresh plasma (with or without the addition of heparin or of EDTA) from patients with active cutaneous disease (epidermolysis bullosa or psoriasis) or even from disease-free volunteers revealed that virtually all of the IL-6 antigen reactive in the 4IL6/51L6 ELISA was reproducibly present in the two 100-150- and 400-500-kDa molecular mass peaks at concentrations in the ng/ml range (Fig. 2). This high molecular mass IL-6 antigen, or any IL-6 antigen for that matterw, as minimally detectable in the IG61/ 5IL6 ELISA (Fig. 2). It is noteworthy that high levels of IL-6
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
