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Link to original content: https://pubmed.ncbi.nlm.nih.gov/12381835
A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity - PubMed Skip to main page content
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. 2002 Oct 15;99(21):13723-8.
doi: 10.1073/pnas.212519099.

A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity

Philip Bufler  1 , Tania Azam, Fabia Gamboni-Robertson, Leonid L Reznikov, Sanjay Kumar, Charles A Dinarello, Soo-Hyun Kim
Affiliations

A complex of the IL-1 homologue IL-1F7b and IL-18-binding protein reduces IL-18 activity

Philip Bufler et al. Proc Natl Acad Sci U S A. .

Abstract

IL-1F7 was discovered in expressed sequence tag databases as a member of the increasing family of proteins sharing sequence homology to IL-1alpha/beta, IL-1Ra, and IL-18. In the present study using immunohistochemical staining, IL-1F7 was localized in human peripheral monocytic cells, suggesting its role in immune regulation. Recombinant human IL-1F7b was shown to bind to the IL-18Ralpha but without IL-18 agonistic or antagonistic function. Using chemical cross-linking, we observed that, unlike IL-18, IL-1F7b fails to recruit the IL-18Rbeta chain to form a functionally active, ternary complex with the IL-18Ralpha chain. IL-1F7b shares two conserved amino acids with IL-18 (Glu-35 and Lys-124), which participate in the interaction of IL-18 with the IL-18Ralpha chain as well as the IL-18-binding protein (IL-18BP), a secreted protein that neutralizes IL-18 activity. In testing whether IL-1F7b interacts with IL-18BP, we unexpectedly observed that IL-1F7b enhanced the ability of IL-18BP to inhibit IL-18-induced IFNgamma by 25-30% in a human natural killer cell line. This effect was observed primarily at limiting concentrations of IL-18BP (3.12-12.5 ng/ml) and at a 50- to 100-fold molar excess of IL-1F7b. Similar results were obtained by using isolated human peripheral blood mononuclear cells. To study the molecular basis of this effect we performed binding studies of IL-1F7b and IL-18BP. After cross-linking, a high molecular weight complex consisting of IL-1F7b and IL-18BP was observed on SDS/PAGE. We propose that after binding to IL-18BP, IL-1F7b forms a complex with IL-18Rbeta, depriving the beta-chain of forming a functional receptor complex with IL-18Ralpha and thus inhibiting IL-18 activity.

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Figures

Figure 1
Figure 1
IL-1F7b neither stimulates nor inhibits IFNγ production induced by IL-18. (A) Human NKO cells, cultures of whole human blood, PBMC [costimulated with IL-12 (1 ng/ml)], and KG-1 cells [costimulated with TNFα (10 ng/ml)] were treated with 100 ng/ml recombinant IL-1F7b (pro or mature form) or IL-18. After 18 h (48 h for KG-1) IFNγ was measured in the supernatant. (B) Induction of NK cells by IL-18 (20 ng/ml) in the presence of IL-12 (1 ng/ml) and increasing concentrations of pro or mature IL-1F7b. The data represent mean ± SEM of three independent experiments.
Figure 2
Figure 2
IL-1F7b does not alter IL-12-induced IFNγ production. NKO cells were induced by IL-12 with or without IL-1F7b at a constant concentration of 250 ng/ml. After 18 h IFNγ was measured in the supernatant. Results are shown as mean ± SEM of three independent experiments.
Figure 3
Figure 3
Cross-linking of IL-1F7b and IL-18Rα-ECD 3. (A) Reducing SDS/PAGE of IL-1F7b cross-linked to IL-18Rα:D3. After blotting on nitrocellulose the cross-linked proteins were visualized by a mAb against the IL-18Rα. BS3, bis(sulfosuccinimidyl) suberate. (B) Formation of a ternary complex of the IL-18Rα- and -β-ECD in the presence of IL-18 but not IL-1F7b after chemical cross-linking. After Western blotting the complexes were visualized by an anti-His6 tag mAb against the His6-tagged IL-18Rβ.
Figure 4
Figure 4
Sequence similarity of human IL-18 and IL-1F7b. Human IL-18 (GenBank accession no. D49950) and human IL-1F7b (accession no. AF200496) are shown. Alignment was generated by using Expert Protein Analysis System (ExPasy) with additional manual adjustment. The amino acid identity of IL-18 with IL-1F7b is 28% and the similarity 55%. The underlined amino acids represent the caspase-1-cleavage site in IL-18 and the predicted cleavage site in IL-1F7b.
Figure 5
Figure 5
IL-1F7b enhances the ability of IL-18BP to inhibit the IL-18-induced IFNγ release by NKO cells. Mature IL-1F7b at 250 ng/ml (A, n = 9) or pro IL-1F7b at 250 ng/ml (B, n = 8), IL-18 (25 ng/ml) and a dilution of IL-18BP in RPMI/10% FCS were incubated in 96-well microtiter plates for 1 h before the addition of NKO cells (0.5 × 106 per ml) and IL-12 (1 ng/ml). After 16 h the supernatant was collected and IFNγ was measured by ECL. Values are expressed as the percent change of IFNγ produced by NKO cells stimulated with IL-18 (25 ng/ml) plus IL-12 (1 ng/ml) in the absence of IL-1F7b or IL-18BP. Statistical analysis was performed by using Student's paired t test (***, P < 0.001).
Figure 6
Figure 6
Cross-linking of IL-1F7b and IL-18BP. (A) Detection of cross-linked proteins (1.5 μg each) on a Western blot by using a rabbit anti-IL-18BP serum. (B) Immunoprecipitation of cross-linked proteins (10 μg each) with a mAb against IL-18BP. Cross-linked IL-1F7b/IL-18BP and the control lanes (IL-18BP with or without BS3) were stained with a rabbit anti-IL-1F7b serum. IL-18/ IL-18BP complex was detected with a rabbit anti-IL-18 serum. BS3, bis(sulfosuccinimidyl) suberate.
Figure 7
Figure 7
Expression of IL-1F7b in transfected RAW264.7 cells and human PBMC. (A) After stable transfection lysates of individual clones (5 × 106 cells) were separated by SDS/PAGE and tested for IL-1F7b expression by using Western blot analysis. The rabbit anti-IL-1F7b serum (1:500 dilution) specifically stained IL-1F7b-positive clones. (B) Stable transfectants of RAW264.7 cells (Mock or IL-1F7b clone 23) were stained with affinity-purified rabbit anti- IL-1F7b IgG and visualized with confocal digital microscopy. (C) Freshly isolated human PBMC were stained against IL-1F7b by using affinity-purified polyclonal rabbit anti-IL-1F7b-IgG. Mø, monocyte; Ly, lymphocyte. Red dye, anti-IL-1F7b; green dye, membranes; blue dye, nuclear stain.
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