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Published on: March 2, 2011
High levels of "complexed" interleukin-6 in human blood
L T May1, H Viguet, J S Kenney
1Department of Microbiology and Immunology, New York Medical College, Valhalla 10595.
Insights
Endogenous interleukin-6 (IL-6) in human serum exists in high-concentration complexes with plasma proteins. These complexes can mask IL-6
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Endogenous interleukin-6 (IL-6) is a key cytokine involved in immune responses and inflammation.
- Understanding the native biochemical state of IL-6 in circulation is crucial for accurate assessment of its activity.
- Previous studies often relied on assays that might not detect IL-6 in complexed forms.
Purpose of the Study:
- To investigate the biochemical nature and molecular mass of endogenous IL-6 in human serum and plasma.
- To characterize the protein complexes containing IL-6 and identify associated proteins.
- To evaluate how IL-6 complexation affects its detection in conventional immunoassays and bioassays.
Main Methods:
- Fractionation of serum and plasma samples using G-200 gel filtration chromatography.
- Quantification of IL-6 using two distinct ELISA kits (IG61/5IL6 and 4IL6/5IL6) and a B9 hybridoma bioassay.
- Purification of IL-6 complexes via immunoaffinity chromatography using a 5IL6 monoclonal antibody, followed by Western blotting, amino-terminal sequencing, and amino acid sequencing.
Main Results:
- A low molecular mass form (~20 kDa) of IL-6 was detected by one ELISA and the bioassay.
- Higher molecular mass complexes (100-150 kDa and 400-500 kDa) containing IL-6 immunoreactivity were identified by the other ELISA.
- Purified complexes revealed IL-6 associated with complement factors (C3b, C4b), C-reactive protein, albumin, and the soluble IL-6 receptor.
Conclusions:
- IL-6 circulates in human blood at unexpectedly high concentrations within novel biochemical complexes.
- These IL-6-protein complexes can obscure IL-6's immunoreactivity and bioactivity in standard assays.
- The findings necessitate re-evaluation of IL-6 measurement methods and its physiological role in complexed forms.
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.
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