Comparison of biological and immunological activities of human monocyte-derived interleukin 1 beta and human recombinant interleukin 1 beta.
Recombinant human interleukin 1 beta (rhIL-1 beta) and supernatants of Escherichia coli lipopolysaccharides-stimulated human monocyte (Mo) cultures, containing native human IL-1 beta (nhIL-1 beta), demonstrate significant differences when tested in the mouse co-stimulatory thymocyte (lymphocyte activating factor [LAF]) assay. The aims of the present study were to investigate this characteristic difference between rhIL-1 beta and Mo culture supernatants (Mo supernatants), and to compare the biological and the immunological activity of preparations of rhIL-1 beta and nhIL-1 beta during each step of an identical purification procedure. The biological activity of rhIL-1 beta/nhIL-1 beta preparations was characterized by the use of the LAF assay and the rat islet insulin release assay. An IL-1 beta enzyme-linked immunosorbent assay (ELISA) was established in order to compare the biological and immunological responses of the IL-1 beta preparations. We report that the significant difference between rhIL-1 beta and supernatants of Mo cultures, which was only demonstrable in the LAF assay, is due to the presence of interleukin 6 (IL-6) in the Mo supernatants. We describe a simple cation exchange chromatography separating nhIL-1 beta and IL-6 of Mo supernatants. The highly purified rhIL-1 beta possessing the correct amino-terminal sequence and nhIL-1 beta have identical biological and immunological activities demonstrating a specific biological activity (SBA) of 3 x 10(2) U/ng IL-1 beta. Thus, we have no indications of secondary or tertiary structural differences between rhIL-1 beta and purified nhIL-1 beta. In contrast, both in the LAF assay and in the rat islet insulin release assay the SBA of an amino-extended rhIL-1 beta form, Met-Glu-Ala-Glu-rhIL-1 beta, was only 1-2% of the SBA of rhIL-1 beta, suggesting that structural changes were introduced into the molecule by the amino-terminal extension. In the present study we have demonstrated that systematic combined testing of IL-1 beta preparations in two different biological assays and an immunological assay is useful for the characterization and comparison of the activity of recombinant and native IL-1 beta preparations purified by the use of exactly the same procedures.
Mølvig
J
J
Steno Memorial Hospital, Gentofte, Denmark.
Sehested Hansen
B
B
Worsaae
H
H
Hejnaes
K R
KR
Helle
M
M
Dalbøge
H
H
Nerup
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007375:Interleukin-1 / Q000276:immunology* / Q000302:isolation & purification; D007376:Interleukin-2 / Q000276:immunology; D015850:Interleukin-6 / Q000276:immunology; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects; D014407:Tumor Cells, Cultured
D007375:Interleukin-1; D007376:Interleukin-2; D015850:Interleukin-6; D011994:Recombinant Proteins