Native IL-1 Beta Research Articles

Ligation of CD40 Activates Interleukin 1β-converting Enzyme (Caspase-1) Activity in Vascular Smooth Muscle and Endothelial Cells and Promotes Elaboration of Active Interleukin 1β

Inflammation contributes to a variety of arterial diseases including atherosclerosis. Interleukin 1beta (IL-1beta) in its activated mature 17-kDa form may mediate aspects of vascular inflammation. As shown previously, human vascular wall cells, such as smooth muscle cells (SMC), express the IL-1beta precursor upon stimulation and the IL-1beta-converting enzyme (ICE) constitutively but do not produce mature IL-1beta or express ICE activity. How SMC, the most numerous cell type in arteries, may release active IL-1beta has therefore remained a perplexing problem. We report here that stimulation of human vascular SMC and endothelial cells (EC) through CD40 ligand, a mediator recently localized in human atheroma, induced elaboration of the IL-1beta precursor as well as activation of cell-associated ICE. In addition to the constitutively expressed 45- and 30-kDa immunoreactive ICE proteins, vascular cells incubated with recombinant human CD40 ligand (rCD40L) (but not IL-1 or TNF) showed an increase of a 20-kDa immunoreactive ICE protein by Western blot analysis. Furthermore, SMC and EC stimulated through rCD40L processed recombinant human IL-1beta precursor (pIL-1beta), generating a cleavage product of approximately 17 kDa. Appearance of both the 20-kDa immunoreactive ICE protein and pIL-1beta processing activity required at least 6 h of stimulation with 0.3 or 1.0 microg/ml rCD40L, respectively, and was inhibited by pre-incubation of the ligand with an anti-CD40L antibody. Stimulation of vascular SMC and EC through rCD40L resulted in the release of biologically active IL-1beta, indicating processing of the native IL-1beta precursor induced by the ligand. These findings establish a novel mechanism of IL-1beta activation in human vascular cells and, moreover, indicate a new pathway of ICE-activation, which could participate in inflammatory aspects of atherogenesis and other disease states.

Structure of an interleukin-1.beta. mutant with reduced bioactivity shows multiple subtle changes in conformation that affect protein-protein recognition

Site-specific mutagenesis was used to obtain the human interleukin-1 beta mutant protein with glycine substituted for threonine at position 9 (IL-1 beta Thr9Gly). The mutant maintains receptor binding but exhibits significantly reduced biological activity. The crystal structure of IL-1 beta Thr9Gly has been determined at 2.4-A resolution by molecular replacement techniques and refined to a crystallographic R-factor of 19.0%. IL-1 beta Thr9Gly crystallizes in a different space group (P6(5)22) than does native IL-1 beta (P4(3)); thus the molecules pack differently. Their overall structure is similar, nevertheless, with both composed of 153 amino acids which form 12 antiparallel beta-strands. However, significant conformational differences both close to and far from the site of the mutation may explain the mutant's altered properties.

Chemical modification of interleukin-1 beta: biochemical characterization of a carbodiimide-catalyzed intramolecular cross-linked protein.

We have modified recombinant human Interleukin-1 beta using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide at pH 6.5, resulting in the formation of an internally cross-linked protein. The major product (30% yield) of the reaction (17 kD; pI = 6.2) was purified and fully characterized by peptide mapping using Endoproteinase Lys C. When digests were conducted under nondenaturing conditions, we found that the modified protein is different from the native protein. The native protein yielded 14 peptides after digestion, whereas only two large peptides and a tetrapeptide, Asn-Tyr-Pro-Lys, were released from the cross-linked protein (i.e., cleavage occurs only at residues Lys88 and Lys92). Using gel filtration, the two peptides were found to co-elute as a single species (15 kD), which represent a noncovalent complex of the amino terminal and C-terminal portions of the molecule. Further analysis of the modified protein by peptide mapping under denaturing conditions and by FAB MS analysis showed that Glu111 and Lys138 were internally cross-linked. The cross-linked protein had bioactivity (T-cell proliferation), fluorescence, and circular dichroism spectra similar to native IL-1 beta. In contrast, while having similar secondary structure, the digested cross-linked protein had less than 1% of T-cell proliferative activity of the undigested protein. These data show that the structural integrity surrounding and perhaps including the Asn-Tyr-Pro-Lys region may be crucial for the biological activity of rIL-1 beta and may be important for the binding of IL-1 to its receptor.

High-level secretion of correctly processed recombinant human interleukin-1β in Kluyveromyces lactis

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.

Effects of IL-1 muteins on cartilage degradation and as inducers of acute inflammation

IL-1 peptides with N-terminal amino acid mutations were cloned and expressed to help characterize structural requirements for activity. Addition of Thr-Asn to the N-terminus (DP516) or substitution of the first two residues (Ala-Pro) of mature, native IL-1 beta with Thr-Met (DuP118) had no effect on the potency of the muteins in stimulating proteoglycan breakdown and inhibiting proteoglycan synthesis in vitro, or inducing mouse paw swelling in vivo. When Arg in position 4 of DuP118 was replaced by Glu (Glu-4), proteoglycan-synthesis-inhibitory activity was reduced to 20% and proteoglycan-degrading activity to 2% of that induced by native IL-1 beta. Glu-4 was much less active in inducing mouse paw swelling and gave maximal swelling about 40% that of native IL-1 beta. The data suggest that the presence of the positively charged side chain of Arg in position 4 may be important for the activity of IL-1 and may be useful in designing specific IL-1 receptor agonists/antagonists.

Comparison of Biological and Immunological Activities of Human Monocyte‐Derived Interleukin 1β and Human Recombinant Interleukin 1β

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.

Synthetic C-terminal peptide of IL-1 functions as a binding domain as well as an antagonist for the IL-1 receptor

Fragments of IL-1 beta were chemically synthesized and tested for biological activity as well as binding of radiolabelled peptides to the IL-1 receptor. A peptide from the extreme C-terminal region of IL-1 beta was found to antagonize intact, native IL-1 beta in the thymocyte bioassay. In addition, this C-terminal region peptide, when radiolabelled, can function as a ligand for the IL-1 receptor on murine cell lines and effectively compete with intact radiolabelled recombinant IL-1 beta.