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The Sushi Domain of Soluble IL-15 Receptor Is Essential for Binding IL-15 and Inhibiting Inflammatory and Allogenic Responses In Vitro and In Vivo
Xiao-qing Wei, Michael Orchardson, J. Alastair Gracie, Bernard P. Leung, Bao-mei Gao, Hui Guan, Wanda Niedbala, Gavin K. Paterson, Iain B. McInnes and Foo Y. Liew J Immunol 2001; 167:277-282; ; http://www.jimmunol.org/content/167/1/277
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

The Sushi Domain of Soluble IL-15 Receptor Is Essential for Binding IL-15 and Inhibiting Inammatory and Allogenic Responses In Vitro and In Vivo1
Xiao-qing Wei,2* Michael Orchardson,* J. Alastair Gracie, Bernard P. Leung,* Bao-mei Gao,* Hui Guan,* Wanda Niedbala,* Gavin K. Paterson,* Iain B. McInnes, and Foo Y. Liew2*
IL-15 is a pleiotropic cytokine that plays important roles in both innate and adaptive immunity. It is associated with a range of immunopathology, including rheumatoid arthritis and allograft rejection. IL-15 functions through the trimeric IL-15R complex, which consists of a high afnity binding -chain and the common IL-2R - and -chains. Characterization of IL-15/IL-15R interactions may facilitate the development of improved IL-15 antagonists for therapeutic interventions. We previously constructed soluble murine IL-15R (sIL-15R ) by deleting the cytoplasmic and transmembrane domains. To localize the functional domain of IL-15R , we have now constructed various truncated versions of sIL-15R . The shortest region retaining IL-15 binding activity is a 65-aa sequence spanning the Sushi domain of IL-15R . Sushi domains, common motifs in protein-protein interactions, contain four cysteines forming two disulde bonds in a 1-3 and 2-4 pattern. Amino acid substitution of the rst or fourth cysteine in sIL-15R completely abolished its IL-15 binding activity. This also abrogated the ability of sIL-15R to neutralize IL-15induced proinammatory cytokine production and anti-apoptotic response in vitro. Furthermore, the mutant sIL-15R lost its ability to inhibit carrageenan-induced local inammation and allogenic cell-induced T cell proliferation and cytokine production in vivo. Thus, the Sushi domain is critical for the functional activity of sIL-15R . The Journal of Immunology, 2001, 167: 277282. nterleukin-15, a cytokine produced by multiple cell types, stimulates T cell proliferation (1) and NK cell development (2). It is also chemotactic for T cells (3), acts on neutrophils to induce morphological cell shape changes (4), and stimulates IL-8 production (5). IL-15 and IL-15R knockout mice lack NK cells and activated CD8 T cells (6, 7). IL-15 also provides survival signals to support mature lymphocytes (8). Moreover, IL-15 is expressed by activated endothelial cells and regulates the capacity of lymphocytes and neutrophils to migrate across endothelial barriers into inamed tissues (9). We have demonstrated that IL-15 is present in the synovium of patients with rheumatoid arthritis and may recruit and activate synovial T cells in the relative absence of IL-2 (10, 11). Following IL-15-mediated activation, synovial T cells both secrete TNF- directly and induce TNFsynthesis by macrophages through cognate interactions (12, 13), indicating an important role for IL-15 in the inammatory cascade within the synovium. Furthermore, IL-15 expression has been detected in several diseases, including inammatory bowel diseases, sarcoidosis, and chronic active hepatitis (14 16), suggesting that such proinammatory pathways may be of general importance. IL-15 functions through a trimeric receptor complex, which consists of a unique high afnity -chain, the IL-2R -chain and the

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*Department of Immunology and Bacteriology and Center of Rheumatic Disease, University of Glasgow, Glasgow, United Kingdom Received for publication January 19, 2001. Accepted for publication April 23, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by the Wellcome Trust, the Medical Research Council, U.K., the Arthritic Research Campaign, and the Chief Scientists Ofce, Scotland. 2 Address correspondence and reprint requests to Dr. Xiao-quing Wei or Dr. Foo Y. Liew, Department of Immunology and Bacteriology, University of Glasgow, Glasgow, U.K. G11 6NT. E-mail address: F.Y.Liew@clinmed.gla.ac.uk or xqw1r@clinmed.gla.ac.uk

common -chain for signal transduction (1719). As part of our investigation into the functional role of IL-15 in rheumatoid arthritis, we have cloned and expressed a soluble fragment of IL15R and found that this protein profoundly suppressed the development of collagen-induced arthritis in DBA/1 mice (20). More recently, we demonstrated that soluble murine IL-15R (sIL15R )3 administered for a short period at the beginning of transplantation markedly prolonged the survival of allogenic heart grafts (21). These ndings suggest that sIL-15R or its analogs may have considerable therapeutic potential in a wide range of diseases. We therefore conducted further investigation into the nature and functional motif of sIL-15R to facilitate the potential designing of therapeutic agents. The extracellular region of IL15R contains a Sushi domain, which is a common motif in protein-protein interaction. Sushi domains are also known as short consensus repeats or type 1 glycoprotein motifs. They have been identied on a number of protein-binding molecules, including complement components C1r, C1s, factor H, and C2m as well as the nonimmunologic molecules factor XIII and 2-glycoprotein (22, 23). A typical Sushi domain has approximately 60 aa residues and contains four cysteines (24). The rst cysteine forms a disulde bond with the third cysteine, and the second cysteine forms a disulde bridge with the fourth cysteine. The two disulde bonds are essential to maintain the tertiary structure of the protein. We report here that the Sushi domain of IL-15R is critical for the binding and function of this protein. Furthermore, substitution of any of the four cysteine residues completely abrogated the ability of sIL-15R to inhibit acute inammation and T cell response to allogenic Ags in vivo. These results not only established the critical role of the Sushi domain for IL-15R activity, but revealed the therapeutic potential of truncated sIL-15R in a range of diseases in vivo.

Abbreviation used in this paper: sIL-15R , soluble murine IL-15R . 0022-1767/01/$02.00

Copyright 2001 by The American Association of Immunologists

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followed by addition of HRP-ExtrAvidin (Sigma; 1/1000 dilution) and was developed with 100 l/well tetramethylbenzidine substrate (Kirkegaard & Perry, Gaithersburg, MD). The OD was read at 630 nm.

Materials and Methods

Cloning, expression, and purication of serially truncated sIL15R
Using PCR and restriction sites PstI and RcaI, four truncated sIL-15R proteins were cloned from previously synthesized cDNA of sIL-15R (T1). T1 consists of 182 aa spanning the whole IL-15R extracellular domain, including the Sushi domain, linker, and Pro/Thr-rich region (17) (see Fig. 1). The other truncated proteins, designated T2, T3, T4, and T5, are also shown in Fig. 1. T2 retains 85 residues of the N-terminal end of T1. T3, T4, and T5 have sequential 20 residues truncated off from the Cterminal end of T2. The PCR primers for the constructs are as follows: T-P1, 5 -TTT CCT GCA GAA TTC ATT AAA GAG GAG CCT GCA GAA TTC ATT AAA GAG GAG A-3 ; T2P, 5 -TCA CTG TGG TTT CCA CTG GAA GTA CTG TCT-3 ; T3P, 5 -GGA GTT CAC GTA GTC TCT GGA GTA CTA TCG-3 ; T4P, 5 -GTG GGA CTA ACT CAC ACA CTA GTA CTT CTT-3 ; and T5P, 5 -ACA CTT GAG GTC CCT CTC CAA GTA CTC ATT-3 . T2, T3, T4, and T5 were amplied with T-P1/ T2P, T-P1/T3P, T-P1/T4P, and T-P1/T5P primer pairs, respectively. PCR products were digested with PstI and RcaI, then inserted into pEGFP-1 (Clontech, Basingstoke, U.K.). The recombinant six-histidine-tagged proteins were expressed in Escherichia coli (XL-1 Blue, Stratagene, La Jolla, CA) following isopropyl -D-thiogalactoside (Stratagene) induction and puried by a nickel-agarose purication system (Qiagen, Crawley, U.K.), according to the manufacturers recommendations. Puried proteins were analyzed by SDS-PAGE. The purity was 97% for all recombinant proteins. Two additional truncated proteins, T6 and T7 (see Fig. 1), were cloned using T2 DNA as template for PCR. PCR products were digested with NcoI and BglII before insertion into pQE60 (Qiagen) to produce Cterminal six-histidine-tagged proteins. The PCR primers for T6 and T7 clones were: T6P, 5 -ATC ACC ATG GCC TCC CTA GCT CAC TAC AGT CC-3 ; T7P, 5 -GAG TCC ATG GTC AAC AAG AAC ACA AAT GTT GC-3 ; and T-P2: CGC TAG ATC TGT ACA GCT CGT CCA TGC CGA GA-3 . The primer pairs used for T6 and T7 were T6P/T-P2 and T6P/T-P2, respectively. T6 and T7 proteins were expressed and puried as described above. LPS was not detected by the Limulus amebocyte test ( 0.01 ng/ g; E-Toxate; Sigma, St. Louis, MO).

Site-directed mutagenesis of the IL-15R Sushi domain

Site-directed mutants were generated based on the T1 construct. Two pairs of primers were designed to replace the rst and fourth cysteines with arginine (R; protein M1) and aspartic acid (D; protein M4), respectively (see Fig. 3A). After amplication of the whole plasmid with pfu DNA polymerase (Stratagene), the DNA was phosphorylated with T4 kinase (Roche, Lewes, U.K.), followed by digestion with DpnI to remove template DNA plasmid. The PCR fragments were puried for ligation and transformation into XL-1 Blue. M1 and M4 cDNA were sequenced to conrm the mutations. The proteins were puried as before and analyzed by SDSPAGE and Western blot. LPS was not detectable by the amebocyte Limulus test.

Western blot
Different concentrations of puried T1 and M4 were run on 12% SDSPAGE gel and transferred to nitrocellulose membranes (Bio-Rad, Hemel Hempstead, U.K.). The membranes were blocked with 2% BSA in PBS and incubated with sheep anti-sIL-15R Ab (Diagnostics Scotland, Carluke, U.K.; 1/5000 dilution). After washing, this was incubated with 10 ng/ml rIL-15 (R&D Systems, Abingdon, U.K.). For the rst blot, HRPanti-sheep IgG was used; in the second blot, biotin-conjugated anti-IL-15 (R&D Systems) and HRP-ExtrAvidin (Sigma) were used. The bands were developed with the ECL system (Amersham Pharmacia, Freiburg, Germany).

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CTLL proliferation assay

The capacity of sIL-15R and the truncated proteins to neutralize IL-15driven proliferation was assessed using a CTLL cell line (American Type Culture Collection, Manassas, VA). Following several washes to remove IL-2 or IL-15 from the maintenance growth medium, cells (5 103/well) were plated in 96-well plates. Cells were cultured for 48 h in the presence of 0.1 ng/ml IL-15 (Immunex, Seattle, WA) or with IL-15 that had been previously incubated for 30 min at 37C with graded concentrations of the test proteins. Cells were pulsed during the last 6 h of culture with 1 Ci [3H]thymidine, and incorporated radioactivity was measured in a Packard Matrix 96 beta counter (Pangbourne, U.K.).

Competitive ELISA for in vitro binding assay

Ninety-six-well plates (Immulon 4, Dynatech, Chantilly, VA) were coated with 1 g/ml sIL-15R (T1) in 0.1 M NaH2CO3, pH 8.6, at 4C overnight. After blocking with 10% FCS (Life Technologies, Glasgow, U.K.) in PBS, graded concentrations of the truncated IL-15R proteins, previously incubated with 500 pg/ml rIL-15 (R&D Systems, Oxon, U.K.), were added and incubated at 37C for 1 h. IL-15 binding was detected by addition of biotin-conjugated anti-IL-15 Ab (1/500 dilution; R&D Systems). This was

Effect of sIL-15R on apoptosis

A variation of the standard apoptosis assay was performed using the IL2/IL-15-dependent human T cell line BDB2, previously called IDB (25). These cells rapidly undergo apoptosis/necrosis in the absence of IL-2 or

FIGURE 1. Diagrammatic representation of murine sIL-15R and its serially truncated derivatives. The full-length IL-15R consists of the Sushi domain, the linker, the Pro/Thr-rich region, the transmembrane (TM) sequence, and the cytoplasmic domain. The various truncated proteins are shown to consist of various amino acid residues.

The Journal of Immunology

IL-15. The cells were cultured in the presence of IL-15 or with IL-15 previously incubated with sIL-15R or the truncated proteins. Following culture for 24 h apoptosis was assessed by the annexin V/propidium iodide system according to the manufacturers protocol (BD PharMingen, San Diego, CA). Briey, cells were incubated with 5 l (100 g/ml) FITCconjugated annexin V and 5 l (50 g/ml) propidium iodide for 15 min at 20C in the dark. Binding buffer (400 l, pH 7.4, buffered HEPES/25 mM CaCl2) was then added. Cells were analyzed within 30 min by ow cytometry (FACScan Becton Dickinson, Mountain View CA) to reveal the three expected populations: live cells (AN /PI ), apoptotic cells (AN / PI ), and necrotic cells (AN /PI ).

279

Carrageenin-induced footpad inammation

Three groups of BALB/c female mice (6 8 wk old, obtained from Harlan Olac, Bicester, Oxon, U.K.) were injected on the left footpad with 300 g -carrageenan (Sigma) mixed with 5 g T1 or M4 or with the diluent PBS alone. Footpad swelling was measured at 4, 8, 24, and 32 h after injection using a dial caliper (Kroeplin, Munich, Germany). The difference in thickness between the left footpad and the uninjected right footpad was calculated to determine the degree of inammation (26).

Allogenic cellular activation in vivo

BALB/c female mice were injected in the left footpad with 106 mitomycin C-treated spleen cells from DBA/1 mice (males and females, 6 10 wk old; Harlan Olac) mixed with either 5 g T1 or M4 or the diluent PBS alone. Recombinant proteins (40 g) were then injected i.p. daily for 7 days. Mice were then sacriced, and spleens were removed and weighted individually. Spleen cell populations were cocultured at (2 106) with mitomycin Ctreated DBA/1 spleen cells (106/ml) for 48 h in medium (RPMI (Life Technologies/BRL, Glasgow, U.K.) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin, 25 mM HEPES buffer, and 10% FCS (all from Life Technologies)) at 37C in 5% CO2. Proliferation assays were performed in triplicate as described previously (20). Supernatants from parallel triplicate cultures were collected up to 72 h and stored at 70C until estimation of cytokine content by ELISA.

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FIGURE 2. Analysis of the ability of sIL-15R and its derivatives to bind IL-15 by competitive ELISA. Ninety-six-well plates were coated with sIL-15R (T1) and incubated with rIL-15 in the presence of increasing concentrations of the test proteins (T1T7). Following washing, the bound IL-15 was detected with anti-IL-15 Ab. A decrease in bound IL-15 indicates effectiveness in competition and hence the IL-15 binding capacity of the protein. Data are the means SD of triplicate tests and are representative of three or four experiments.

Cytokine assays
Murine TNF- , IFN- , IL2, IL-4, IL-6, and IL-10 were assayed by ELISA using paired Abs (BD PharMingen) according to the manufacturers instructions. Lower limits of detection were as follows: IL-4, IL-6, and TNF- were all at 10 pg/ml; IL-10 was at 40 pg/ml; and IFN- was at 80 pg/ml.

Statistical analysis
Statistics was performed using Minitab software for Macintosh. The analyses were performed using Students t test.

Results
The Sushi domain of sIL-15R is essential for IL-15 binding Five sequential C-terminal truncated proteins of IL-15R were constructed as T1, T2, T3, T4, and T5, which contain 182, 85, 65, 45, and 25 aa residues, respectively (Fig. 1). They were puried and tested for their ability to bind IL-15 in vitro using a competitive binding assay. As expected, T1 bound IL-15 strongly in a dose-dependent manner. T2 bound IL-15 indistinguishably from T1. The binding of IL-15 by T3 was signicantly weaker than that by T1 and T2. In contrast, T4 and T5 failed to bind (Fig. 2). We then investigated whether the C-terminal end of IL-15R contributed to the binding of IL-15. Two N-terminal truncated proteins, T6 and T7, were constructed using the same approach as that for T2T5. T6 and T7 contain 25 and 45 residues with a deleted or disrupted Sushi domain, respectively (Fig. 1). T6 and T7 completely failed to bind IL-15 in a standard binding assay (Fig. 2). These results therefore suggest that the Sushi domain of IL-15R is essential for binding IL-15 with contributions from the Pro/Thrrich region. The disulde bonds of the Sushi domain of IL-15R are critical for binding IL-15 The Sushi domain has two overlapping disulde bonds that contribute to the maintenance of the unique tertiary structure of IL-

15R . The rst cysteine forms a disulde bond with the third cysteine, and the second forms a bond with the fourth. To conrm the functional importance of the disulde bonds for sIL-15R , site-directed mutation was conducted to replace the rst cysteine with arginine and the fourth cysteine with aspartic acid to form M1 and M4, respectively (Fig. 3A). DNA sequencing was conducted to conrm the single residue mutation. M1 and M4 were then expressed in XL-1 blue as for T1. Competitive binding ELISA showed that both M1 and M4 completely failed to bind IL-15 (Fig. 3B). Western blot analysis demonstrated that although both M4 and T1 were equally recognized by an anti-IL-15R Ab, only T1 could bind IL-15, which was then recognized by an anti-IL-15 Ab (Fig. 3C). These results therefore clearly demonstrate that the disulde bonds of the Sushi domain of sIL-15R are critical for the binding of IL-15. The Sushi domain of IL-15R is required for the neutralization of IL-15-mediated T cell proliferation and rescue of apoptosis and necrosis CTLL cells proliferate in response to IL-15. Using this system, the ability of truncated and mutated sIL-15R proteins to inhibit IL-15 activity was analyzed. T1 and T2 were efcient in blocking the ability of IL-15 to induce CTLL cells proliferation, T3 was only partially so. In contrast, T4, T5, and M4 completely failed to inhibit this proliferation (Fig. 4). IL-15 is known to rescue T cells from apoptosis and necrosis (8, 27). A human T cell line, BDB2 (IBD in Ref. 25) rapidly undergoes apoptosis and necrosis when cultured in medium alone. This was almost completely prevented by the presence of IL-15. This rescuing ability of IL-15 was totally reversed by the presence of T1 or T2, but not by T5 or M4 (Fig.

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FIGURE 3. Site-directed mutation of cysteines in the Sushi domain abolished IL-15 binding activity of T1. A, Cysteine residues were replaced by arginine (R in M1) and aspartic acid (D in M4). B, Competitive ELISA (see Fig. 2) shows that both M1 and M4 have completely lost the IL-15 binding activity. C, Western blot analysis demonstrates that M4 is recognized by anti-IL-15R Ab, but was not able to bind IL-15 and hence was not recognized by anti-IL-15 Ab following blotting with rIL-15 and then anti-IL-15 Ab. The results are representative of three (B) and two (C) experiments.

FIGURE 4. Inhibition of CTLL cell proliferation by sIL-15R and its derivatives. CTLL cells proliferated vigorously in the presence of IL-15. This was inhibited in a dose-dependent manner by coculturing with T1, T2, and partially by T3, but not by T4, T5 (A), or M4 (B). Data are the means SD of triplicate cultures and are representative of three experiments.

T1, but not M4, inhibited allogenic Ag-induced T cell proliferation in vivo We have previously shown that sIL-15R (T1), administrated daily for 10 days from the day of transplantation, markedly prolonged the survival of allogenic heart graft in the mouse. This nding strongly suggests that IL-15 may contribute to the rejection of the transplanted allograft. However, the mechanism by which T1 suppressed the graft rejection is unclear. Using the recombinant proteins T1 and M4, we investigated the ability of sIL-15R to inuence the proliferative response and cytokine production by the recipient spleen cells following administration of allogenic cells in vivo. BALB/c mice were injected in the footpads with mitomycin C-treated allogenic (DBA/1) spleen cells. The mice were treated with daily injections of 40 g T1, M4, or PBS alone for 7 days. Mice were sacriced on day 8, and the spleens were collected. Spleens from mice treated with T1 were signicantly smaller than those treated with M4 or PBS (Fig. 7A). Cells from T1-treated mice also proliferated markedly less vigorously when cultured with DBA/1 spleen cells in vitro compared with cells from M1 or PBS-treated mice (Fig. 7B). Furthermore, cells from the T1-treated mice produced signicantly less IL-2, IFN- , TNF- , and IL-6 when cultured with DBA/1 cells in vitro compared with spleen cells from the M4- or PBS-treated mice (Fig. 7, CF). However, there was no signicant difference in the percentages of CD3 , CD4 , and CD8 T cells; B cells; or NK cells among the mice injected with PBS alone and those treated with T1 or M4 (data not shown). These results therefore demonstrate that sIL-15R is a potent inhibitor of the allogenic Ag-induced T response in vivo,

5). Thus, there is a direct correlation between the ability of sIL15R and its various derivatives to bind IL-15 and their ability to neutralize the IL-15 functions in vitro. Soluble IL-15R (T1), but not the mutant (M4), reduced acute inammation in vivo Carrageenan is a potent inducer of acute local inammation in vivo. It leads to a rapid recruitment of neutrophils to the site of administration and has been used extensively to investigate the mechanism of local inammation (26). Since IL-15 has been shown to play a signicant chemotactic role in cellular migration and inltration (3, 9, 13), we investigated the relative abilities of T1 and M4 to inuence carrageenan-induced local inammation. Carrageenan (300 g) was mixed with T1, M4 (5 g each), or PBS alone and injected into the footpads of BALB/c mice. Mice injected with carrageenan with PBS developed the expected local footpad swelling, which started 4 h after injection and was sustained for up to 48 h. This reaction was markedly reduced by the coapplication of T1, but not by M4 (Fig. 6). These results therefore demonstrate that IL-15 is a key mediator of carrageenan-induced local inammation, which was inhibited by T1, and that M4, with a disrupted Sushi domain, was completely inactive.

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FIGURE 5. Apoptosis and necrosis (A; annexin and propidium iodide ) of BDB cells was rescued by IL-15 (B). This ability to rescue was reversed by T2 (C) and T1 (E), but not by T5 (D) and M4 (F). The percentages of viable cells (bottom left quadrant) are: A, 7.6%; B, 80.5%; C, 14.4%; D, 83.2%; E, 15.1%; and F, 81.5.

and that this activity is completely abolished by disruption of the integrity of the Sushi domain.

FIGURE 7. Effect of sIL-15R on allogenic Ag-induced cellular response in vivo. BALB/c mice were injected in the footpad with 106 mitomycin C-treated BDA/1 spleen cells mixed with 5 g T1, M4, or PBS alone. Thereafter, mice were injected i.p. daily for 7 days with 40 g of the corresponding protein. Mice were sacriced on day 8, and spleen weights were recorded (A), T cell proliferation was assayed by [3H]thymidine incorporation (B), and cytokine production was analyzed by ELISA of the culture supernatants (CF). Results are the mean SD (n 5). , p 0.05; , p 0.01 (compared with PBS controls).

Discussion
The data presented here clearly demonstrate that the Sushi domain of IL-15R is critical for the binding of IL-15 and the biological activity of sIL-15R . The Pro/Thr-rich region, immediately outside the transmembrane domain, also contributes to this activity. The minimum length required to retain the biological functions of sIL-15R appears to be a sequence of 65 aa residues covering the entire Sushi domain, of which the integrity of the two disulde bridges is absolutely essential. The Sushi domains may have a broad general role in the protein-protein interaction essential for a range of ligand-receptor interactions and biological functions. IL2R has two such Sushi domains, which are also required for the binding to IL-2 (28). However, IL15R binds IL-15 with an afnity 1000-fold higher than that of IL-2 to IL-2R (17). Therefore, the Sushi domain of IL-15R is likely to be more important than that of IL-2 for the biological activities of their respective ligands. The unique and high afnity of the IL-15R -chain for binding IL-15 strongly suggests that it, particularly its Sushi domain, would be an important potential target for therapeutic interventions in diseases where overexpression of IL-15 is clearly implicated. Using the truncated sIL-15 (T1) and the single cysteine-substituted mutant (M4), we have explored further the therapeutic potential of T1 in vitro and in vivo. The results reported here extend our earlier ndings on the anti-inammatory role of sIL15 in collagen-induced arthritis (20) and the prolongation of allograft survival (21) in the murine models by providing insight into the mechanisms involved. It was previously shown that IL-15 is a growth factor capable of rescuing cells from apoptosis and necrosis, which occur through specic activation or neglect (8, 27). This was conrmed and extended in experiments reported here using a dened human T cell line (BDB2) that rapidly undergoes apoptosis/necrosis in the absence of IL-15 or IL-2. T1, but not M4, was able to reverse the rescuing effect of IL-15 of BDB2 cells. These results are the rst demonstration of the role of sIL-15R in cellular survival and strongly suggest the potential of T1 as an antiproliferative reagent in vivo.

FIGURE 6. Effect of sIL-15R (T1) and the inactive mutant (M4) on carrageenan-induced local inammation. BALB/c mice were injected in the footpad with 300 g carrageenan mixed with 5 g T1 or M4 or with PBS alone. Footpad swelling was measured at the times indicated. Results are the mean SEM (n 10, pooled from two experiments). , p 0.05 compared with T1 group.

282 We have investigated the effect of T1 in carrageenan-induced acute local inammation in the mouse. T1, but not M4, markedly inhibited the footpad swelling that resulted from rapid inltration of neutrophils and later the arrival of mononuclear cells (26). The effect of T1 was evident as early as 4 h after carrageenan administration. This probably reects the inhibition of the chemotactic activity of locally induced IL-15, which is a known to attract the migration of neutrophils across endothelial membrane into inamed tissues (9). IL-15 is also a chemoattractant for activated T cells (3), and this could account for the sustained inhibition of chronic inammation by T1. The nding that a short course of sIL-15R treatment can markedly prolong the survival of allogenic heart graft opens up the possibility that T1 could have a potential therapeutic value in preventing transplantation rejections (21). This nding also suggests that IL-15 is associated with the initiation of chronic allograft rejection process, including the amplication of acquired immunity. This is consistent with our observation that hosts accepting the heart graft, as the results of T1 treatment, were able to mount a normal rejection of a third-party skin graft (21). IL-15 is therefore required as an accessory signal for the full expansion of activated T cells, or alternatively, it acts as a paracrine growth factor for activated T cells. The results reported here demonstrate clearly that T1, but not M4, profoundly inhibited the specic proliferative response of spleen cells activated with allogenic cells in vivo. Furthermore, this is associated with the inhibition of the production of a number of proinammatory cytokines, including IL-2, IFN- , TNF- , and IL-6. These results thus provide a rational explanation to the strong suppressive effect of sIL-15R in allograft rejection. In conclusion, we report here the potential therapeutic role of sIL-15R in a number of experimental diseases. We have also dened the critical role of the Sushi domain in the functional activity of sIL-15Ra. The importance of the structural integrity of the Sushi domain through the maintenance of the disulde bridges implies that it may be of interest to examine polymorphisms within the Sushi domain and their possible association with disease. Additionally, any protein competitor for IL-15 should contain an intact Sushi domain. However, the general application of this principle to other receptor-ligand interactions and the mechanism(s) by which the Sushi domain of a receptor interacts with its ligand remain unclear, but is now amenable to further experimentation.

SUSHI DOMAIN OF sIL-15R

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