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J. Biol. Chem., Vol. 279, Issue 39, 40368-40375, September 24, 2004
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Is Generated by Cleavage That Involves the Tumor Necrosis Factor--converting Enzyme (TACE/ADAM17)*
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From the
Center for Cancer Drug Discovery and Development, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, the Department of Immunology and Cell Biology, Research Center Borstel, D-23845 Borstel, Germany, and the **Institute of Biochemistry, Christian-Albrechts-University, D-24118 Kiel, Germany
Received for publication, April 14, 2004 , and in revised form, June 21, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15R (sIL-15R) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15R is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor--converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15R, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15R. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15R shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15R, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15R generation in mice. The existence of natural sIL-15R offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Both membrane-bound and soluble proteases can mediate ectodomain shedding (5, 6). Members of the protease superfamily, including the matrix metalloproteinases (MPs)1, membrane-tethered matrix MPs, and zinc-dependent ADAM (a disintegrin and metalloproteinase) family MPs have been shown to be responsible for the cleavage of the majority of shed proteins (6). Among these, members of the ADAM family are particularly important (6–8). ADAM17 or TNF-converting enzyme (TACE) has surfaced recently as a central mammalian ectodomain sheddase (7, 8). TACE plays a critical role in the ectodomain shedding of many soluble proteins, including TNF (9), TNFRI and II (10), L-selectin (7), IL-6R (11), CD30 (12), CD40 (13), growth hormone receptor (14), erythropoietin receptor (15), transforming growth factor 
(16), and others. Of more than 30 other members of the ADAM family known up to date, ADAM10/Kuzbanian shares significant sequence homology with TACE. Both TACE and ADAM10 have been implicated in the shedding of the amyloid precursor protein (17), cellular prion protein (18), IL-6 receptor after cellular cholesterol depletion (19), and TNF (20). In addition, ADAM10/Kuzbanian is involved in cleavage of Notch (20), Notch ligand Delta (21), and fractalkine (22).
The IL-15 receptor (IL-15R) chain is a specific high affinity receptor that constitutes together with the IL-2 receptor (IL-2R) and the IL-2 receptor 
(IL-2R/c) subunits a trimeric receptor for IL-15 (23, 24). IL-15R is structurally related to the IL-2R chain and alone is capable of high affinity binding of IL-15 (Kd 
10–11 M) (23). Both IL-15R and IL-15 are expressed by a variety of tissues and cell types, including monocytes/macrophages, keratinocytes, fibroblasts, nerve, muscle, and epithelial cells (25–29). Several different isoforms of human and murine IL-15R as a result of alternative splicing of the IL-15R gene were recently described (23, 30, 31). Recombinant soluble IL-15R prevents collagen-mediated arthritis (32), inhibits short-term carrageenan-induced inflammation (33), and enhances cardiac allograft survival (34). Although recombinant soluble IL-15R has been used for several in vivo studies (32–34), no data concerning the existence of natural sIL-15R were reported thus far. In this study, we demonstrated the presence of natural soluble IL-15R (sIL-15R) in mouse serum. Furthermore, murine fibroblasts constitutively release sIL-15R into the culture medium, and this process is further stimulated by PMA. We provide several lines of evidence that constitutive and PMA-inducible IL-15R cleavage involves the activity of TACE by using specific MP inhibitors, by overexpression of the enzyme, and by using TACE-deficient fibroblasts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were purchased from TEBU (London, UK). Lipopolysaccharide (LPS), cycloheximide, phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). Antibodies against IL-15R (N-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against ADAM17 and ADAM10 were from Chemicon (Hofheim, Germany), and biotinylated goat-anti-mouse IL-15R was from R&D Systems (Wiesbaden, Germany). Rabbit anti-goat horseradish peroxidase conjugates (Amersham Biosciences) were used as secondary antibodies. IL-15-IgG2b and IL-2-IgG2b fusion proteins (FPs) were produced as described previously (35). Recombinant sIL-15R was produced as described previously (32). In brief, the His6-tagged recombinant protein was expressed in Escherichia coli (strain BL21), extracted from bacteria under denaturing conditions and purified using a nickel agarose purification system (Qiagen, Dorking, UK) according to the manufacturer's recommendations. The purity of recombinant sIL-15R was analyzed by SDS-PAGE and Western blotting using specific anti-IL-15R antibodies. Purified recombinant sIL-15R inhibited IL-15-but not IL-2-mediated proliferation of CTLL cells (data not shown).
A broad-spectrum hydroxamic acid-based MP inhibitor batimastate (BB94) was purchased from GlaxoSmithKline (Harlow, UK). GW280264X (a potent inhibitor of TACE and ADAM10 metalloproteinases) and GI254023X (an inhibitor of ADAM10) were described previously (22). Murine IL-15R was cloned into pcDNA3.1 expression vector (Invitrogen) as described previously (27). Murine TACE and human ADAM10 cDNA were cloned into pDC304 vector.
Mice—Female mice of the inbred strains Balb/c, CH3, and C57BL/6 were obtained from Charles River Laboratories (Sulzfeld, Germany). IL-15R–/–mice were bred in Research Center Borstel under specific pathogen-free conditions. Mice were bled from the tail vein, and sera were analyzed by ELISA and Western blotting.
Cell Culture, Stimulation, and Transfection Conditions—Ras-Myc-retrovirus-immortalized TACE–/–and simian virus large T-antigenimmortalized ADAM10–/–mouse embryonic fibroblasts (MEFs) and respective wild type cells were generated and characterized as described elsewhere (7, 36). Murine fibrosarcoma L929 (ECACC), its TNF-resistant derivative L929R, and primate fibroblast COS-7 (American Type Culture Collection) cell lines were maintained in RPMI 1640, and MEFs were cultured in Dulbecco's modified Eagle's medium. Culture medium was supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were stimulated with PMA (200 ng/ml) in fetal calf serum-free medium.
For transient transfection, cells were seeded at 5 x 105/well in 6-well plates. COS-7 cells were transfected using the GenePORTER 2 transfection kit (Gene Therapy Systems, San Diego, CA), and MEFs were transfected using LipofectAMINE 2000 (Invitrogen). Transfection efficiency was confirmed by Western blotting using specific antibodies. Conditioned medium was collected 48 h after transfection, concentrated 10-fold using 10-kDa cutoff filtration units (Vivaspin; Vivascience, Hannover, Germany), and analyzed by immunoprecipitation and Western blotting for the presence of sIL-15R.
ELISA for sIL-15R—A 96-well plate (Greiner, Hamburg, Germany) was coated overnight at 4 °C with 1 µg/ml of IL-15-IgG2b FP, which served as a capture protein for sIL-15R. Wells were blocked with 3% BSA in PBS for 2 h. Samples (50 µl/well) were added to the plate and incubated overnight. Serial dilutions of murine recombinant sIL-15R were used for standardization. Bound sIL-15R was detected using biotinylated anti-mouse IL-15R antibodies followed by incubation with streptavidin-peroxidase. Chromogenic substrate (R&D Systems) was used for visualization, and reaction was stopped after 20 min of incubation by addition of1NH2SO4. Optical density was determined at 450 nm using ELISA reader (Dynatech, Denkendorf, Germany). The detection limit of ELISA was 20 pg/ml of recombinant sIL-15R. The specificity of newly developed sIL-15R ELISA was convincingly demonstrated by the complete absence of detectable sIL-15R using IL-2-IgG2b FP as a coating reagent.
Immunoprecipitation and Western Blotting—Nonidet P-40 (0.5% final concentration) and mixture of protease inhibitors were added to supernatants and immunoprecipitation with anti-IL-15R antibodies was performed for 2 h at 4 °C. Immunocomplexes were captured on protein G-agarose. To analyze glycosylation, after immunoprecipitation, the samples were treated with 250 mU of N-glycosidase F (Roche) for 3 h at 37 °C according to the manufacturer's instructions. Samples were resuspended in SDS-PAGE sample buffer (62.5 mM Tris-HCL, pH 8.0, 1% glycerol, 2% SDS, 5% -mercaptoethanol, and 0.01% bromphenol blue), boiled for 5 min, and analyzed on 10% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose (Bio-Rad). Blots were blocked for 1 h in phosphate-buffered saline containing 0.05% Tween 20 (phosphate-buffered saline-T) and 3% BSA (Sigma). After incubations with primary and secondary antibodies and washing with phosphate-buffered saline/Tween 20, visualization of specific proteins was carried out by an enhanced chemiluminescence (ECL) method using ECL Western blotting detection reagents (Amersham Biosciences) according to the manufacturer's instructions. Cellular extracts were prepared and analyzed by Western blotting as described elsewhere (31).
Reverse Transcription-PCR—RNA was extracted from cells using TRIzol reagent (Invitrogen). cDNA was synthesized from 5 µg of total RNA using random oligonucleotides as primers and SuperScriptII kit (Invitrogen). cDNA was amplified by standard PCR procedure as described previously (31). The following primers were used: murine TACE: sense, 5'-GCGGCGTCTCCTCATCCT-3'; antisense, 5'-TTATATTCTGCCCCATCTGTGTTG-3'; and -actin: sense, 5'-GTGGGGCGCCCCAGGCACCA-3'; antisense, 5'-CTCCTTAATGTCACGCACGATTTC-3'. All primers were purchased from Metabion (Planegg-Martinsried, Germany). Amplification of -actin message was used to normalize the amount of cDNA. A mock PCR (without cDNA) was included to exclude contamination in all experiments.
CTLL Assay—Serial 2-fold dilutions of the supernatant were added to microtiter plates containing 1 x 104 CTLL-16 cells in a final volume of 200 µl. The cells were incubated for 20 h at 37 °C and pulse-labeled for another 4 h with 0.5 µCi of [3H]thymidine (5 Ci/mmol). Thymidine incorporation was quantified by liquid scintillation counting (PerkinElmer Life and Analytical Sciences). Standards of IL-15, IL-2, and sIL-15R were included in each experiment.
Flow Cytometric Analysis—Adherent cells were harvested from culture plates using acutase (PAA Laboratories, Coelbe, Germany). IL-15R expression was evaluated by incubation of cells with IL-15-IgG2b FP as described previously (31), and analyzed by flow cytometry using FACScalibur (BD Biosciences) and CELLQuest software. Negative controls consisted of isotype-matched, nonspecific antibodies (BD Phar-Mingen). The fluorescence signal of the labeled cells was calculated as median fluorescence intensity of the cell population.
Data Analysis—All experiments were performed in at least three independent assays that yielded highly comparable results. Data are summarized as mean ± S.D. Statistical analysis of the results was performed by Student's t test for unpaired samples. A p value of < 0.05 was considered as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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—Murine L929 fibrosarcoma cells abundantly express IL-15R mRNA and protein (27). Membrane-bound IL-15R could be detected on the cell surface of these fibroblasts by flow cytometry and confocal microscopy (data not shown). To investigate whether these cells release sIL-15R into the culture medium, conditioned media were collected from L929 fibroblasts after 3–5 days of culture and analyzed by CTLL assay for the ability to block specifically IL-15-mediated proliferation of CTLL cells. In parallel, the stimulation with IL-2 was assessed, which served here as a control. In fact, IL-15-but not IL-2-induced proliferation of CTLL cells was strongly inhibited by the addition of the conditioned medium from L929 fibroblasts (Fig. 1A).
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from murine fibroblasts. To this end, L929 cells were treated with PMA for different time intervals, or left untreated. Interestingly, these experiments revealed that PMA had the ability to up-regulate the amount of membrane IL-15R within the first hour of stimulation as demonstrated by IL-15-IgG2b FP staining of the membrane-bound IL-15R and flow cytometry analysis (Fig. 1B). However, the increase in membrane-linked IL-15R was transitory and dropped substantially within the second hour of PMA treatment. After 2 h, L929 fibroblasts clearly had less membrane-bound IL-15R, resulting in 52% reduction of median fluorescence intensity compared with the untreated control (Fig. 1B). Given that down-modulation of IL-15R from the cell surface may result from its release into the culture medium, cell-free supernatants from PMA-stimulated and untreated cells were assayed for the presence of the soluble chain by ELISA. Indeed, PMA treatment induced the release of the soluble form to the culture medium from L929 cells, increasing the amount of sIL-15R 3-fold (Fig. 1C). These results were further corroborated by Western blotting analysis of the culture medium. Conditioned media from these cells were harvested after 2 h of culture, and sIL-15R was immunoprecipitated using specific antibodies. As illustrated in Fig. 1D, immunoprecipitation and Western blotting experiments confirmed that sIL-15R is present in the cell supernatants as a single band protein with molecular mass of about 30 kDa. Re-appearance of the membrane-bound IL-15R was detected after 24 h of PMA removal (data not shown).
It has been reported that certain ligands could induce release of respective receptors to the culture medium. Such liganddriven release of soluble form of the cognate receptor has been demonstrated for IL-4 and TNF (10, 39). Thus, the ability of IL-15 to induce sIL-15R release was tested. Parallel with IL-15, we also tested for the ability to induce release of sIL-15R several other agents, including TNF and LPS. In L929 cells, TNF can reportedly induce apoptosis, necrosis, or even both at once (27, 40), whereas LPS treatment triggers expression of chemokines by these cells.2 L929 cells were treated with IL-15, TNF, or LPS for different time intervals, and shedding of the IL-15R chain was evaluated by FACS and ELISA. It is interesting that only TNF was able to induce shedding of IL-15R in L929 cells to the levels comparable with PMA treatment, leading to the 2.5-fold increase of sIL-15R concentration in the culture medium (Fig. 1C), whereas IL-15 and LPS had no effect. Furthermore, TNF treatment mimicked PMA action, also leading to the increase of the membrane-bound IL-15R within 1 h of stimulation followed by down-regulation of the protein within next hour, as assessed by flow cytometry. The time-course changes in the expression of membrane-linked IL-15R are shown in Fig. 1E. Taken together, these experiments demonstrate that murine L929 fibroblasts release sIL-15R into the culture medium under natural conditions, whereas stimulation with PMA or TNF enhances this process.
The Soluble IL-15R Is N-glycosylated—The molecular mass of the natural sIL-15R is about 30 kDa, which is approximately 5 kDa higher than the reported molecular mass of the recombinant sIL-15R produced in bacteria (32). The difference in the molecular mass might result from the presence of one potential N-glycosylation site in the extracellular part of IL-15R (24). To analyze a glycosylation pattern of sIL-15R, IL-15R was precipitated from L929-conditioned medium using specific antibodies. The immunoprecipitates were treated with N-glycosidase, and the resulting products were analyzed by Western blotting. As shown in Fig. 1F, treatment of sIL-15R with N-glycosidase leads to the shift of the specific band to lower molecular mass position (about 26 kDa), which corresponds to non-glycosylated recombinant sIL-15R (32).
Metalloproteinase Inhibitors That Discriminate between TACE and ADAM10 Differentially Block Constitutive and Inducible Shedding of IL-15Ra—Disintegrin-like metalloproteinases (ADAMs) have been implicated in the constitutive as well as the PMA-inducible ectodomain shedding of a number of cell surface-expressed molecules (5, 38). Both types of shedding are blocked by broad-spectrum metalloproteinase inhibitors, such as batimastat (22). To test whether the shedding of IL-15R is mediated by metalloproteinases, cells were first incubated with this inhibitor. These experiments demonstrated the ability of batimastat to suppress constitutive and PMA-induced IL-15R shedding, as assessed by ELISA (Fig. 2A) and Western blotting (Fig. 2B).
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. Given the important role of TACE and ADAM10 in ectodomain shedding and soluble receptor generation, we investigated the involvement of these two proteases. A panel of hydroxamate-based metalloproteinase inhibitors was recently used to analyze the involvement of the ADAM family members in fractalkine shedding (22). Among these inhibitors, two compounds were found to differ in their ability to abrogate TACE and ADAM10 action. GW280264X inhibitor blocked both enzymes, whereas GI254023X compound had potent ADAM10 inhibitory activity with minimal influence on TACE. Thus, the effect of these compounds on constitutive and inducible shedding of IL-15R in L929 cells was analyzed. To this end, cells were treated with 10 µM of either inhibitor for 1 h followed by PMA stimulation for another 2 h, and concentration of sIL-15R in the cell supernatants was assessed by ELISA (Fig. 2C). The presence of sIL-15R was also evaluated by Western blotting analysis after immunoprecipitation from the cell supernatants (Fig. 2D). These experiments showed that GW280264X compound clearly inhibited constitutive and PMA-induced IL-15R shedding, whereas GI254023X had no effect. These chemical agents also had similar effect upon TNF-induced release of sIL-15R (data not shown). It is noteworthy that the inhibition of the IL-15R shedding by the respective metalloproteinase inhibitors (the broad-spectrum inhibitor batimastat and the inhibitor of TACE and ADAM10 GW280264X) was associated with an increase of IL-15R on the cell surface or in the cell lysates, whereas all used inhibitors did not affect the total protein content in the cell lysates and cell viability within 48 h, compared with cycloheximide (data not shown). These experiments provide evidence that those chemical compounds do not act through nonspecific inhibition of protein synthesis. The selective action of the inhibitors upon respective metalloproteinase(s) is in agreement with previous studies (22, 41). Taken together, these results show that TACE plays a major role in the constitutive and inducible IL-15R cleavage, whereas ADAM10 seems to be dispensable for this process.
Overexpression of TACE Enhances Constitutive and Inducible IL-15R cleavage—To assess the role of TACE and ADAM10 in the IL-15R shedding in more detail, we overexpressed IL-15R alone or together with TACE or ADAM10 in COS-7 cells. To this end, COS-7 cells were transiently transfected with a vector coding for IL-15R in combination with TACE or ADAM10. Co-transfection of IL-15R with an empty vector was used as a control. The shedding of IL-15R was analyzed 48 h after transfection by ELISA and Western blotting. Overexpression of IL-15R alone resulted in an increased release of sIL-15R into the culture medium, which was further up-regulated by PMA (Fig. 3A). These experiments indicate that endogenous MP activity is present in COS-7 cells. Co-transfection of TACE and IL-15R significantly enhanced both constitutive and PMA-inducible IL-15R cleavage (2.1- and 1.7-fold increase, respectively, versus overexpression of IL-15R alone). These results were further confirmed by the detection of sIL-15R in the culture medium by Western blotting after immunoprecipitation with specific antibodies (Fig. 3B). The equal expression of transfected proteins was evaluated by probing the membrane with respective antibodies (Fig. 3C). ADAM10 is detectable as a dual band with 90- and 65-kDa molecular mass corresponding to the precursor and mature protein. Mature TACE is detectable as a band with molecular mass of 80 kDa, whereas IL-15R is a 65-kDa protein. Thus, the constitutive and PMA-induced IL-15R cleavage upon overexpression in COS-7 cells is mediated by TACE.
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between parental and derivative L929 cell lines (referred to as L929R), which is resistant to the cytotoxic action of TNF (data not shown). Interestingly, that the spontaneous and PMA-induced release of sIL-15R from L929R cells was about 1.7–2.2-fold higher compared with parental L929 fibroblasts (Fig. 3D). L929R cells constitutively express higher amounts of TACE at mRNA and protein levels (Fig. 3, E and F). Noteworthily, both cell lines express equivalent amounts of IL-15R protein (Fig. 3F). Finally, we wanted to verify that the observed ability of TNF to increase the production of sIL-15R is not caused by its direct cytotoxic effect upon target cells. Importantly, TNF was able to induce the increased release of sIL-15R both in TNF-sensitive and -resistant cell lines (L929 and L929R, respectively), whereas the concentration of sIL-15R was 2.5-fold higher in the culture medium from L929R cells (Fig. 3D). Thus, TNF preserves the ability to stimulate sIL-15R production in the fibroblasts that are not sensitive to its cytotoxic action. Taken together, these results provide further support to the idea that a stable increase in the expression of TACE is sufficient to shed more IL-15R from the cell surface.
Cleavage of IL-15R Is Reduced in Murine Fibroblasts Lacking TACE but Remains Intact in ADAM10-deficient Cells— More evidence for the role of TACE and ADAM10 in the cleavage of IL-15R was obtained by using mouse embryonic fibroblasts that were generated from mouse embryos with a targeted deletion of TACE or ADAM10 as described previously (7, 36). The absence of TACE or ADAM10 in respective cells was confirmed by Western blotting using specific anti-ADAM10 or anti-TACE antibodies (Fig. 4A, top and bottom). However, the endogenous expression level of IL-15R was rather low in ADAM10–/–, TACE–/–, and respective wild type MEFs as detected by reverse transcription-PCR and Western blotting (data not shown). Therefore, MEFs from these mice were transiently transfected with a vector coding for IL-15R and analyzed for the constitutive or PMA-induced shedding of IL-15R into the culture medium 48 h after transfection. Both constitutive and PMA-induced release of sIL-15R was dramatically lower in TACE–/–MEFs compared with their normal WT counterparts (Fig. 4B). However, a small amount of sIL-15R was still detectable in the supernatants from these cells. As expected, the reconstitution of TACE into TACE–/–MEF (TACE–/–Re) completely restored sIL-15R generation, in either the presence or the absence of PMA. No significant changes in sIL-15R production were observed in ADAM10–/–versus ADAM10+/+ MEFs. The expression levels of IL-15R are shown as controls (Fig. 4A, middle). These results further support an important role for TACE in the constitutive and inducible release of sIL-15R.
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in Mouse Serum—To identify sIL-15R in the body fluids of mice, sera from at least five animals per strain were first tested by ELISA. As shown in Fig. 4C, Balb/c, C57BL/6, and CH3 mice have different levels of circulating sIL-15R varying from 18 ng/ml in Balb/c to 40 ng/ml in C57BL/6 mice. The specificity of the newly developed sIL-15R-ELISA was convincingly demonstrated by the complete absence of the detectable protein in the sera of IL-15R–/–mice (Fig. 4C). These results were further corroborated by Western blotting analysis, which showed the presence of a band representing a protein with a molecular mass of 30 kDa corresponding to the N-glycosylated form of sIL-15R after immunoprecipitation from serum using specific antibodies (Fig. 4D). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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chain in the mouse serum and cell-conditioned medium. Furthermore, our data indicate that proteolytic cleavage represents a major mechanism for sIL-15R generation in mice by providing several lines of evidence that unambiguously demonstrate an important role for TACE in the IL-15R shedding. Murine fibrosarcoma cells constitutively release detectable amounts of sIL-15R into the culture medium, and the generation of sIL-15R is further enhanced by PMA stimulation. Both a broad-spectrum metalloproteinase inhibitor batimastat and an inhibitor of TACE and ADAM10 (GW280264X) inhibited the spontaneous and inducible shedding of IL-15R, whereas a selective inhibitor of ADAM10 (GI254023X) did not affect sIL-15R production. Moreover, the release of sIL-15R by fibroblasts derived from mice with a homozygous disruption of the gene encoding TACE was dramatically reduced compared with shedding by corresponding wild-type cells. The reconstitution of active TACE expression in TACE-deficient cells restored both spontaneous and PMA-inducible IL-15R shedding.
IL-15R is thus a novel substrate for TACE, although our results do not exclude a possibility that TACE is not the only MP involved in the proteolytic cleavage of IL-15R. The fact that some sIL-15R is still detectable in the supernatants of TACE–/–cells is a very strong argument for the involvement of other proteases. Current studies in our laboratory are focused on the identification of putative additional members of the ADAM family, which may contribute to the constitutive and/or inducible shedding of this receptor. Notwithstanding, how TACE recognizes its diverse targets and features in ADAM substrates that allow their proteolysis remains obscure (4). It has been suggested that structural and kinetic characteristics rather than minimal consensus shedding sequences and identity of amino acids consisting them regulate access of the protease to the cleavage site (42, 43), although the Ala-Val preference for TACE has recently been reported (44). The most consistent feature indicative of cleavage site among ADAM substrates is that they usually reside in a stalk region between the membrane and an initial globular extracellular subdomain (45). The identification of amino acids comprising a potential cleavage site for TACE in the extracellular region of IL-15R will provide helpful insights to our understanding of this process.
The proteolytic cleavage and release of transmembrane cell surface proteins or ectodomain shedding has emerged as an important post-translational mechanism to regulate the function of cell surface proteins (2, 7). Many structurally and functionally diverse proteins, which are initially synthesized as membrane-anchored moieties, can be shed from the cell surface in a biologically active form, including adhesion molecules, cytokines, growth factors, and their receptors (1, 2). Ectodomain cleavage and shedding of N-glycosylated IL-15R might have a particular physiological and/or pathological relevance for the biology of IL-15, and supposedly affect the function of this pleiotropic cytokine in several aspects. IL-15 plays important roles in both innate and adaptive immunity, and is associated with a wide range of immunopathological reactions, including rheumatoid arthritis and allograft rejection (32, 34). The bioavailability of sIL-15R might contribute to complex regulatory mechanisms of IL-15 secretion, which is tightly controlled at multiple levels (46). Regulation of the availability of free IL-15 may be an important protective mechanism against excessive IL-15 activity. The presence of sIL-15R in biological fluids could affect function of IL-15 in the first place by competing for the cytokine with the cellular receptors and inhibiting its action. Given that IL-15 has many features of an attractive therapeutic target in inflammatory synovitis (47), a potential ability of sIL-15R to serve as a natural antagonist of IL-15 could have a clinical relevance. On the other hand, sIL-15R might also affect the bioavailability of IL-15 by prolonging the cytokine half-life and slowing the release of the ligand from a complex between the soluble receptor and IL-15 to provide physiological concentrations of the cytokine in tissues. Furthermore, sIL-15R may expand IL-15 action from autocrine or juxtacrine to paracrine or endocrine modes, resulting in local or systemic effects of the cytokine, and influence the nature and/or duration of the signaling event. High or low levels of sIL-15R may correlate with distinct pathological conditions and possibly serve as a prognostic marker for certain types of diseases, such as allergy and inflammation. Moreover, membrane-bound IL-15 present on monocytic THP-1 cell line and human monocytes participates in reverse signaling, inducing activation of extracellular signal-regulated kinase 1/2 and p38, and production of IL-8 (48). The ability of membrane-bound IL-15 to mediate reverse signaling is also supported by data from our laboratory (57). Thus, sIL-15R can serve as a natural agonist for membrane-bound IL-15 to mediate distinct cellular responses under certain physiological or pathological conditions. It remains to be elucidated whether sIL-15R could also potentiate IL-15 signaling under some circumstances, as was already shown for soluble IL-6 receptor (49). In addition, future experiments are required to test whether difficulties in the detection of free IL-15 may be caused, at least in part, by the ability of sIL-15R to form receptor-ligand complexes with IL-15.
Alternative splicing of specific transcripts that gives rise to a polypeptide lacking transmembrane and/or other regions represents another mechanism, which accounts for the appearance of secreted forms of diverse membrane-linked proteins. For example, sIL-5R and sIL-7R are generated by alternative splicing (50, 51), but TNF receptors are cleaved from the cell surface (10), whereas for sIL-6R, sIL-4R, and growth hormone receptor, both mechanisms were shown (3, 52, 53). In addition, a machinery of soluble receptor generation may undergo evolutionary divergence in different species. A classic example of such phenomenon is generation of growth hormone receptor by proteolytic cleavage in rabbit and human and by alternative splicing in mouse and rat (42, 53, 54). Thus, cellular mechanisms accounting for the release of sIL-15R in mouse and human may be different. Moreover, a release of full-length p55 TNFR in exosome-like particles has recently been demonstrated as a novel mechanism for soluble cytokine receptor generation (55). Although our results support the idea that sIL-15R in mouse is produced mainly by proteolysis that involves TACE, it is not clear yet whether an alternative splicing mechanism and/or a release in exosome-like particles may also contribute to the generation of sIL-15R, and additional studies are required to address this question.
Despite the fact that certain ligands have been shown to enhance the release of their soluble receptors (10, 37), IL-15 treatment clearly had no effect on sIL-15R production by murine fibroblasts. Instead, TNF was able to trigger the release of sIL-15R from these cells. Noteworthily, the proteolytic cleavage of soluble TNF receptors I and II in human polymorphonuclear leukocytes reportedly relies upon TACE and is enhanced by TNF treatment (10). Thus, an increase in the production of sIL-15R may represent a concomitant event that results from an enhanced proteolytic activity of TACE induced by TNF stimulation. The involvement of TACE is further supported by the ability of GW280264X to block TNF-induced sIL-15R release into the culture medium, whereas a selective ADAM10 inhibitor GI254023X was ineffective (data not shown). Notwithstanding the above explanation, the mechanism underlying the ability of TNF to enhance sIL-15R production is not yet clear and deserves a systemic exploration in follow-up experiments. Furthermore, PMA-induced up-regulation of membrane-anchored IL-15R within the first hour of the treatment is interesting and is not seen with IL-6R and fractalkine (11, 23). Given that IL-15R could be detected as a protein associated with the endoplasmic reticulum, the Golgi, and perinuclear space by confocal microscopy and Western blotting (31), and the ability of this receptor to recycle between endosomes and the cell surface (56), the observed increase in the amount of membrane-bound IL-15R within the first hour of treatment with PMA or TNF is probably a result of mobilization of the receptor from intracellular pools.
In summary, our data provide evidence that, like many proteins with a single transmembrane domain, the high affinity -chain of the murine IL-15 receptor exists not only in membrane-anchored but also in soluble form, and implicate TACE as a metalloproteinase responsible for the shedding of this receptor. Given that an excessive or insufficient production of cytokines is often the main cause of the pathology in many types of diseases and the emerging importance of soluble cytokine receptors as novel potential immunotherapeutic agents, a dissection of the role played by sIL-15R in a complex biology of IL-15 under physiological or pathological conditions will facilitate the development of new tools for therapeutic intervention.
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FOOTNOTES |
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¶ Both authors contributed equally to this work.
|| To whom correspondence should be addressed: Department of Immunology and Cell Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. Tel.: 49-4537-188564; Fax: 49-4537-188403; Email: ebulanova{at}fz-borstel.de.
1 The abbreviations used are: MP, matrix metalloproteinases; ADAM, a disintegrin and metalloproteinase; TACE, TNF-converting enzyme; TNF, tumor necrosis factor ; IL, interleukin; IL-15R, IL-15 receptor ; sIL-15R, soluble IL-15R; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; FP, fusion protein; ELISA, enzyme-linked immunosorbent assay; MEF, mouse embryonic fibroblast; LPS, lipopolysaccharide; CTLL, cytotoxic T cell line.
2 E. Bulanova and S. Bulfone-Paus, unpublished observations.
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