High levels of "complexed" interleukin-6 in human blood.
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.
May
L T
LT
Department of Microbiology and Immunology, New York Medical College, Valhalla 10595.
Viguet
H
H
Kenney
J S
JS
Ida
N
N
Allison
A C
AC
Sehgal
P B
PB
D000595:Amino Acid Sequence; D001681:Biological Assay; D001798:Blood Proteins / Q000378:metabolism; D015153:Blotting, Western; D001853:Bone Marrow / Q000737:chemistry; D002850:Chromatography, Gel; D006801:Humans; D015850:Interleukin-6 / Q000097:blood*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis
D001798:Blood Proteins; D015850:Interleukin-6; D046911:Macromolecular Substances; D010446:Peptide Fragments