Gene transfer-mediated expression of physiological numbers of the type II decoy receptor in a myelomonocytic cellular context dampens the response to interleukin-1

ARTICLE

INTRODUCTION

IL-1 consists of two polypeptide mediators (IL-1alpha and IL-1ß) which are among the most potent and multifunctional cell activators. The spectrum of action of IL-1 includes cells of hematopoietic origin, from immature precursors to differentiated leukocytes, vessel wall elements and cells of mesenchymal, nervous and epithelial origin [1, 2]. The production and action of IL-1 are regulated by multiple control pathways, some of which are unique to this cytokine. This complexity and uniqueness is best represented by the term "IL-1 system" [3]. The IL-1 system consists of two agonists, IL-1alpha and IL-1ß, a specific activation system (IL-1 converting enzyme, ICE), a receptor antagonist (IL-1ra) produced in different isoforms [4, 5], and two high affinity surface-binding molecules [3].

Two receptors for IL-1, termed type I and type II (RI and RII, respectively), usually coexpressed in different cell types, have been identified and cloned [5-7]. Recent evidence indicates that AcP, also called RIII, plays an essential role in IL-1 signaling [8, 9]. An accessory protein, which increases the binding affinity of RI for IL-1ß has recently been identified [10]. IL-1 signaling activity appears to be mediated exclusively via RI [11-14], whereas IL-1RII has no signaling property and acts in myelomonocytic cells as a "decoy" for IL-1 (decoy RII), inhibiting its activity by preventing IL-1 from binding to the signaling RI [3, 15, 16]. Consistent with this model of decoy function of RII, molecules with anti-inflammatory activity, including glucocorticoid hormones, IL-4 and IL-13, induce increased expression and release of RII in human polymorphonuclear leukocytes (PMN) [17, 18]. Soluble decoy RII is found in biological fluids under a variety of pathophysiological conditions [19-22]. The decoy RII anti-IL-1 pathway does not interfere with the action of IL-1ra [16, 23]. Chemoattractants and TNF cause rapid shedding of RII, a phenomenon interpreted as a means to buffer IL-1 leaking from sites of inflammation [24-26].

The concept of a high affinity binding, nonsignaling molecule, whose only function is to prevent the ligand from interacting with the true receptor on the same cell, is without precedent. In an effort to test the "decoy" function model for RII directly, gene transfer experiments were performed [27, 28]. It was found that decoy RII gene transfer in a fibroblast and keratinocyte context inhibits responsiveness to IL-1. Inhibition of response by decoy RII gene transfer was specific for IL-1, mediated by surface expressed RII and was independent of the cytoplasmic portion of the molecule [27]. In these previous experiments, gene transfer was performed in cells (e.g. fibroblasts) usually not expressing abundant levels of RII [3]. Expression levels obtained were higher than those observed in cells which normally express RII. It could therefore be argued that the inhibition of IL-1 responsiveness observed after decoy RII gene transfer reflected an unphysiological cellular context and receptor number. It was therefore important to test the decoy function model for RII using a myeloid recipient and receptor numbers comparable to those found in normal neutrophils and monocytes.

MATERIALS AND METHODS

Constructs.

The full length cDNA for human IL-1 RII, SaII-SaII insert [6, 27] and a cytoplasmic deletion construct, SaII-BglII insert (see below) were ligated back into the polylinker of the pCEP4beta expression vector (Invitrogen, S. Diego, CA). pCEP4beta is an episomial, high copy number expression vector containing the hygromicin resistance gene and the insert cDNA under the control of the CMV promoter. A cytoplasmic deletion construct in which the coding region terminates after the first 3 aminoacids of the cytoplasmic portion (HisArgArg) was constructed, resulting in the insertion of a stop codon after the first 3 amino acids of the cytoplasmic portion [27].

Cells and transfection.

The human myeloid line U937 was grown in RPMI 1640 (Gibco, Glasgow, Scotland) with 10% heat-inactivated fetal calf serum (FCS, Hyclone, Logan, UT). Cells were transfected by electroporation. After 48 h, transfected cells were selected for hygromicin (Boehringer Mannheim, Mannheim, Germany) resistance (40 µg/ml) and the pool of resistant cells was further analyzed.

Northern and PCR analysis.

Total RNA was isolated by the guanidium isotiocyanate method. Ten micrograms of total RNA was analyzed by electrophoresis through 1% agarose/formaldehyde gels, followed by Northern blot transfer to Gene Screen Plus sheet (New England Nuclear, Boston, MA). Probes were: a EcoRI-HindIII 477-bp fragment from hIL-1RI cDNA, a EcoRI-SaII 750-bp fragment from hIL-1RII cDNA, a full length hIL-8 cDNA. Membranes were washed twice with 2X SSC/1% SDS at 60° C and exposed for 18 h at ­ 80° C. RNA transfer to membranes was checked by UV irradiation. For RT-PCR amplification of RI, fragments corresponding to nucleotide 123-694 and 3201-3682 of the Gen Bank sequence (No. M27492) were amplified using specific oligos.

IL-1 binding.

The number and affinity of surface IL-1 receptors were determined as described [15]. Cells were washed with binding buffer (RPMI 1640-0.2% BSA, pH 7.4) and 2 x 10⁶ or 2 x 10⁵ cells were incubated with different concentrations of ¹²⁵I-IL-1ß (spec. activity 150 µCi/µg; NEN, Boston, MA) in the presence or absence of a 200 fold molar excess of non-radioactive cytokine in 50 µl binding buffer at 4° C for 4 h in 96 round bottomed well polystyrene microplates (Falcon, Lincoln Park, NJ) on a shaking platform. To separate bound from free ¹²⁵I-IL-1ß, cells were layered on the top of a 200 µl cushion of 20% sucrose (Merck)-1% BSA in 400 µl polypropylene tubes (Beckman Instruments, Palo Alto, CA) and centrifuged at 10,000 rpm for 30 seconds. Radioactivity in the cellular pellets was counted in a gamma-counter. Next, Scatchard analysis was performed by the LIGAND program to determine the kD and numbers of receptors for IL-1ß.

Affinity cross-linking.

Cross-linking of surface receptors was performed as described [15]. Briefly, for surface affinity cross-linking, 1 x 10⁷ cells were incubated in binding buffer with 1nM ¹²⁵I-IL-1ß for 4 h at 4° C. After addition of 1 mM disuccinimidyl suberate (DSS) (Pierce, Rockford, IL) at 4° C for 4 h, the cell pellet was lysed in 100 µl lysis buffer (0.5% Triton X-100, 25 mM hepes, 1mM PMSF). The debris-free supernatant was analyzed by 10% SDS-PAGE under reducing conditions and dried gels were exposed to autoradiography.

Cytokine assays and reagents.

3 X 10⁴ cells were plated in 96 flat-bottomed plates in 200 µl RPMI + 10% heat-inactivated FCS (culture medium). After 1 day, the medium was changed for culture medium containing stimuli (recombinant IL-1ß, from Dompè, L'Aquila, Italy, specific activity 10⁷ U/mg); and incubation was continued for a further 24 h. The supernatants were then harvested and kept at ­ 20° C. IL-8 was measured using a specific ELISA with mAb and a polyclonal serum (gifts from Dr M. Ceska, Sandoz, Vienna, Austria). Cytokine assays were conducted with 4 replicates and results are mean ± SE [27].

RESULTS AND DISCUSSION

Characterization of transfected cells.

U937 cells used in the present study had very low levels of RI mRNA transcripts, which required RT-PCR for detection (Figure 1A). Decoy RII mRNA was undetectable by Northern blot and surface expression of IL-1 receptor(s) was extremely low, with < 100 specific sites per cell (not shown). Yet, U937 cells responded to IL-1 with increased expression and production of IL-8 (see below). U937 cells were therefore selected for transferring the gene encoding the decoy RII or a cytoplasmic deletion mutant (delta 372-398) missing the cytoplasmic portion of the molecule. As shown in Figure 1, gene transfer resulted in expression of RII mRNA (Figure 1B) and protein as assessed by surface cross linking of a protein of the expected size (Figure 2). Decoy RII gene transfer did not affect expression of RI, as indicated by RT-PCR analysis (Figure 1A) and lack of a crosslinked species corresponding to the RI 80kD protein (Figure 2B). Control and transfected cells showed low, comparable amounts of AcP mRNA (not shown). After gene transfer, the number of specific binding sites was 988 and 1869 for RII and RII delta 372-398 respectively with comparable affinity (3.35 and 8.17 x 10^{­ 10}) (Figure 2A). These values are similar or lower than those found in normal neutrophils and monocytes [15, 24, 25].

Responsiveness to IL-1 of RII-transfected cells.

Vector-transfected U937 responds to IL-1 with mRNA expression and release of IL-8 (Figure 3). As shown in Figure 3, transfection of the full length decoy RII or with a cytoplasmic deletion mutant resulted in a substantial decrease of IL-1-induced expression and production of IL-8. For instance, induction of IL-8 mRNA was clearly visible with 0.1 ng/ml IL-1 in control cells, whereas it required >= 1 ng/ml in RII-transfected cells (Figure 3A). Levels of protein production were also reduced in RII-transfected cells (Figure 3B).

CONCLUSION

The decoy function model for RII was formulated on the basis of the following lines of evidence: 1) RII is not essential for responding to IL-1; 2) antibody-blocking experiments are consistent with a decoy function model; 3) certain anti-inflammatory molecules (e.g. glucocorticoid hormones) increase expression and release of this molecule [3, 15]. The decoy hypothesis was subsequently tested using a gene transfer approach [27, 28]. However, for these studies high, non-physiological numbers of receptors were forced in keratinocytes and fibroblasts. It could be argued that lineage-specific components are missing in these cell types, one of which, fibroblasts, does not normally express RII. Moreover, the forced expression of high numbers of RII might be regarded as non-physiological.

In the present study, we reconstructed a cellular system resembling normal myelomonocytic cells in terms of lineage and receptor numbers. U937 cells were used as recipients for gene transfer. These cells express extremely low levels of RI, as normal PMN and monocytes do, and have undetectable RII. After gene transfer, the levels of surface receptor expressed in U937 cells were comparable to those found in circulating myelomonocytic cells. RII gene transfer did not increase responsiveness of U937 cells to IL-1, it actually considerably dampened it. Consistent with a decoy explanation of this inhibition, truncated RII, lacking most of the cytoplasmic tail, was as effective as intact RII to block responsiveness to IL-1. One cannot formally exclude the possibility that RII may not only trap IL-1 (decoy) but also sequester components of the transduction pathway of AcP/RIII [8, 9]. Thus, the present study is consistent with the view that in a myelomonocytic cellular context, RII does not contribute to signaling and acts as a molecular sink (decoy) for IL-1. Consistent with this view, transgenic mice overexpressing RII in the skin show defective local inflammation [29].

Acknowledgements.

This work was supported by Istituto Superiore di Sanità, Project AIDS, and by the Italian Association for Cancer Research.

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