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28 Interleukin- 1 receptor antagonist [IL-1F3] W i l l i a m P. A r e n d I a n d C h r i s t o p h e r H. E u a n s e 1University of Colorado Health Sciences Center, Denver, CO, USA 2Harvard Medical School, Boston, MA, USA It is better to be h a t e d for w h a t y o u are t h a n to be loved for s o m e t h i n g you are not. Andr6 Gide

INTRODUCTION The pleiotropic and ubiquitous inflammatory activities of interleukin- 1 (IL-1) suggested to many investigators that natural inhibitory mechanisms or molecules must exist. IL-1 inhibitors theoretically could work at the levels of synthesis, secretion, receptor, or post-receptor intracellular pathways in cells. Early studies had described IL-1 inhibitory bioactivities in human urine and in a variety of cell supernatants (reviewed in Mend, 1993). However, many of these activities turned out to be artifacts of bioassay systems or remained uncharacterized. At a symposium in Ann Arbor, MI, in June 1985, two different groups first reported bioactivities now known to be due to interleukin-1 receptor antagonist (IL-1Ra). Dayer and colleagues described that the dialyzed and concentrated urine of a patient with acute monocytic leukemia and fever inhibited the effects of IL-1 on induction of PGE2 and collagenase production by cultured human dermal fibroblasts. Arend and col-

The Cytokine Handbook, 4th Edition, edited by Angus W. Thomson & Michael T Lotze ISBN 0-12-689663-1, London

leagues reported that the supernatants of human monocytes cultured on adherent immune complexes exhibited an activity inhibitory towards IL-1 stimulation of proliferation of murine thyrnocytes. During the next 5 years both research groups further characterized these IL-1 inhibitory bioactivities. The semi-purified inhibitor from the urine of patients with monocytic leukemia also inhibited IL-1-induced PGE2 and collagenase production in human synovial cells, as well as proliferation of thymocytes. In addition, both serum and urine from febrile patients with systemic juvenile chronic arthritis contained an IL-1 inhibitor that was absent during afebrile periods. Further purification of the urine material indicated a size of 18-25 kDa with inhibitory activities towards both IL-la and IL-113 induction of PGE2 production by fibroblasts. Most importantly, Dayer and colleagues first demonstrated that the urine-derived inhibitor blocked the binding of IL-1 to receptors on EL4-6.1 murine thymoma cells. The material in the supernatants of human monocytes stimulated by adherent

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i m m u n e complexes also was -22 kDa in size and inhibited IL-1, but not IL-2, effects on both thymocytes and chondrocytes. Furthermore, similar to the IL-1 inhibitor in urine, the semi-purified monocytederived material also functioned as a specific IL-1 receptor antagonist. This period culminated with the description in 1990 of the purification, sequencing, cDNA cloning, and expression of the IL- iRa molecule (reviewed in Mend, 1993). IL- IRa is the first described naturally occurring specific receptor antagonist of any cytokine or hormonelike molecule. Thus, it occupies a unique position in biology. The pattern of production of IL- IRa, immediately following that of IL-1 and as an acute phase protein by the liver, indicates that IL-1Ra may serve an important role in regulation of the potentially injurious effects of IL-1. Furthermore, the spontaneous development of vasculitis (Nicklin et al., 2000) or arthritis (Horai et al., 2000) in different inbred strains of mice rendered genetically deficient in IL-1Ra production suggests that maintenance of a balance between local levels of IL-1Ra and IL-1 is important in prevention of disease. Knowledge about the biochemistry and biology of the IL-1Ra family of molecules rapidly expanded during the 1990s (Mend, 1990, 1991, 1993; M e n d et al., 1990, 1998; Dinarello, 1991, 1993; Dinarello and Thompson, 1991; Lennard et al., 1992; Bresnihan and Cunnane, 1998). At least four isoforms of IL- iRa are now known: secreted IL- iRa, or sIL- iRa and types 1, 2 and 3 intracellular IL-1Ra, icIL-1Ral, iclL-1Ra2 and icIL-1Ra3. Furthermore, during the 1990s the mechanism of action of IL-1Ra was established, the anti-inflammatory role of endogenous IL-1Ra was determined, and the therapeutic use of recombinant IL-iRa in h u m a n diseases was explored. Sections in this chapter will review characteristics of the IL-1Ra gene and protein isoforms, biological role in normal physiology and in disease, potential therapeutic uses of exogenous administration, and areas of current inquiry and research.

STRUCTURE OF THE IL-1Ra GENE AND REGULATION OF TRANSCRIPTION The structure of the 16.5 kb IL-1Ra gene (IL1RN) is depicted in Figure 28.1 (Lennard et al., 1992; Mend,

1993). Four exons encode sIL-1Ra, with 6.4 kb comprising the region for sIL-1Ra DNA including a short 3' UTR. An additional exon encoding icIL-1Ral is located 9.6 kb upstream, with the intervening region constituting a large first intron for this structural variant. Human IL1RN contains three Alu repeats, two located in the intron 3' from the first exon for icIL-1Ral and the third located in intron 3 for sIL-iRa. A potentially important allelic polymorphism is present in the sIL-1Ra second intron caused by variable numbers of an 86-bp tandem repeat (Tarlow et al., 1997). Allele 2 of this polymorphism is associated with a variety of h u m a n diseases, largely of epithelial cell origin, as discussed below. This intronic allele appears to influence production of different isoforms of IL-1Ra in a cell-specific manner. It is not known whether the allele affects transcription, translation, or both. IL1RN was mapped to the long arm of h u m a n chromosome 2 at band 2q14.2 (reviewed in Mend, 1993). The IL-la and IL-1 [3loci are also located in this region of chromosome 2 and the genes for IL-1Ra, IL-la and IL-I~) all contain similar exon-intron structures. An analysis of sequence comparisons and mutation rates for these three genes suggest an origin by gene duplication from a primordial precursor. A physical map of the region on chromosome 2 described the genes for the three IL-1 family members to be present on a 430-kb restriction fragment flanked by two CpG islands (reviewed in M e n d et al., 1998). The three genes were mapped relative to one terminal CpG island with the following intervals: ILIA between +0 and +35 kb, IL1B between +70 and +110 kb, and IL1RN between +330 and +430 kb. It is of interest that the genes for h u m a n IL-1 receptors, both types I and II, are also located on the long arm of chromosome 2, although not close to the genes for the three IL- 1 ligands. The separate 5' regulatory regions for both sIL-iRa and icIL-1Ral have been mapped, and some of the cis-acting DNA regions involved in regulation of transcription have been characterized (reviewed in Mend et al., 1998). To study the slL-iRa promoter, 1680 bp of 5' flanking DNA was isolated, sequenced and cloned into a luciferase expression vector for use in gene transfer experiments. The sIL-1Ra promoter possessed a TATAAbox at -26, with consensus sequences for possible NF-KB-, NFIL-I[3A-, AP-1- and CRE-

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STRUCTURE OF THE IL-1Ra GENE AND REGULATION OF TRANSCRIPTION Exons

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FIGURE 28.1 IL-1Ra gene structure, three mRNAs and four proteins. The extended IL-1Ra gene contains four originally described exons encoding sIL-iRa. Two additional upstream exons were subsequently identified, producing mRNAs for icIL- 1Ral and icIL- 1Ra2 by alternative transcriptional splice mechanisms. The four IL- iRa protein isoforms described to date result from translation of these three mRNAs, with icIL- 1Ra3 formed by alternative translational initiation primarily from the sIL-IRa mRNA. The three ic (intracellular) isoforms of IL-iRa contain no leader peptides, are formed in the cytoplasm and generally remain within cells. (Reproduced by permission from Gabay, C. (2000). IL-1 inhibitors. Novel agents in the treatment of rheumatoid arthritis Exp. Opin. Invest. Drugs 9, 113-127). binding sites located further upstream. Cloning of this sIL-IRa promoter construct into a variety of cell lines indicated a pattern of activity identical to that of the endogenous IL-IRa gene, i.e. primarily in monocytes and macrophages. A series of 5'-truncated mutants with a common 3' end at +27 were generated to further characterize active regions in the sIL-1Ra promoter. Removal of sequences between - 2 9 4 and - 1 4 8 led to a >90% decrease in both basal and LPSinduced promoter activity. Further deletion to - 8 5 led to an almost complete loss of promoter activity. These early results indicated that sites in the proximal region of the sIL-lra promoter potently regulated both basal and induced activity. Further studies indicated the presence of one inhibitory and three positive-acting LPS response elements within the proximal 294 bp of the sIL-iRa promoter. An element between - 2 9 4 and -250 masked the LPS response, representing an inhibitory region. Three positive LPSresponse elements were located between - 2 5 0 and - 200, -200 and - 148, and - 148 and - 31, with co-

operativity demonstrated between these sites for maximal promoter activity. The most proximal LPSresponse element contained a NF-~:B binding site between - 9 3 and -84. Subsequent studies indicated the presence of a PU.1 binding site between - 9 0 and - 8 0 of the sIL-1Ra promoter, overlapping the NF-KB site (Smith et al., 1998). Moreover, this region demonstrated interactions with GA-binding protein, as well as with NF-KB and PU.1. Mutation studies indicated that cooperativity between all three transcription factors acting on this proximal region was involved in sIL-IRa promoter activity. A second PU.1 binding site was centered at - 2 3 0 with mutation of both PU.1 sites leading to an almost complete loss of promoter activity. Thus, the two PU.1 sites in the proximal sIL-1Ra promoter represent the major response elements for LPS-induced sIL-iRa gene expression. To study transcriptional regulation of the icIL-1Ral gene, 4.5 kb of the 5' flanking region was isolated, sequenced and cloned into a luciferase expression vector. The promoter for icIL-1Ral lacked a traditional

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TATAA or CAAT motif so must use an alternative mechanism of transcriptional initiation. This promoter construct demonstrated a pattern of activity identical to that of the endogenous icIL-1Ral gene, with constitutive expression in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937 or the lymphocyte cell lines Raji and Jurkat. However, inducible expression was demonstrated in response to LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. Studies with deletional mutants indicated that constitutive expression of the icIL- 1Ral promoter in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, - 2 8 8 to -156, and - 1 5 6 to -49. In contrast, basal expression of the icIL-1Ral promoter in RAW 264.7 cells was regulated by an inhibitory element between -4525 to -1438 and a strong positive element between - 156 and -49. Lastly, LPS induction of the icIL-1Ral promoter in RAW 264.7 cells was regulated by strong positive DNA regions between bases -1438 to -909 and - 1 5 6 to -49. Thus, the proximal region of the icIL-1Ral promoter, between bases - 1 5 6 to -49, contained positive cis-acting elements necessary for expression in both epithelial and macrophage cell lines. However, the ability of this proximal region in the icIL-1Ral promoter to regulate transcription was strongly influenced in both positive and negative manners by other upstream elements in a cell type-specific pattern. Recent studies have examined transcriptional regulation of the icIL-1Ral gene in IL-la-stimulated primary mouse keratinocytes (La and Fischer, 2001). Regulatory elements for both C/EBP and NF-~cB b e t w e e n - 5 9 8 a n d - 2 8 8 in the icIL-1Ral promoter were found to be involved in mediating the response to IL- la. Post-transcriptional regulation of IL-1Ra production may be influenced by the 3'-untranslated (3'-UTR) region of the IL-1Ra mRNA (Yamshchikov et al., 2002). The h u m a n 3'-UTR led to a 5.7-fold and 3.9-fold, respectively, decrease in expression of a luciferase reporter gene in the murine macrophage cell line RAW264.7 or in the h u m a n macrophage cell line U937. This reporter gene construct was under the control of the CMV promoter with the IL-1Ra 3'-UTR placed downstream of the luciferase gene. The IL-iRa mRNA levels were not changed significantly, suggesting that the 3'-UTR influenced translation.

IL- 1Ra PROTEIN ISOFORMS The purification to homogeneity of IL-1Ra from the supernatants of monocytes cultured on adherent IgG indicated a protein of 17 kDa and 152 amino acids (Mend, 1993). This isoform of IL-1Ra is secreted as variably glycosylated species of 22-25 kDa and is now known as secretory IL-iRa (sIL-iRa). Another laboratory subsequently purified the same molecule from the supernatants of the h u m a n myelomonocytic leukemia cell line U937 after differentiation with phorbol myristate acetate (PMA) and stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF). Both laboratories showed that this molecule functioned as a specific inhibitor of receptor binding of IL-1 and exhibited an absence of agonist properties, sIL-1Ra is produced in large amounts by monocytes, macrophages and neutrophils, but can be synthesized by virtually any cell that produces IL-1 with the possible exception of epithelial and endothelial cells. Sequencing the primary structure of this IL-1 inhibitor led to cloning using degenerate oligonucleotide probes. The IL-1Ra cDNA was cloned from a ~.gtl0 library prepared from monocytes cultured on adherent IgG or from U937 cells (Mend, 1993; Carter et al., 1990). The cDNA contained an open reading frame encoding a protein of 177 amino acids, a short 5' UTR of 14 nucleotides, and a long 3' UTR of 1133 nucleotides. The N-terminal 25 residues possessed an internal hydrophobic stretch representing a signal peptide. Recombinant IL-1Ra was obtained after cloning and expression in E. coli. The purified recombinant nonglycosylated protein showed identical structure as the purified native molecule. IL-IRa exhibited 26% amino acid sequence homology to IL-1~ and 19% homology to IL-la, similar to the degree of homology between the two forms of IL-1. Subsequent structural studies on IL-1Ra from other species indicated that it is a highly conserved molecule, again similar to IL-1. A single Asn-linked glycosylation site is located at 333-339, and the native sIL-1Ra is variably glycosylated accounting for the size range of this secreted molecule. However, glycosylation is not thought to influence receptor binding of this molecule as both native purified sIL-1Ra and the recombinant molecule appeared to have identical biological properties in early studies.

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Intracellular isoforms of IL-IRa have been described which possess variations in the N-terminal amino acid sequence (Table 28.1) (reviewed in Arend et al., 1998). The first intracellular isoform of IL-1Ra, now termed type 1 or icIL- 1Ral, was identified from a cDNA isolated from a h u m a n blood cell library. The cDNA was identical to that for sIL-iRa except at the 5' end where 85 bp were replaced by a different sequence of 130 bp derived from an upstream exon. This segment was spliced into an internal splice acceptor site within the exon encoding the leader sequence (bp 87 and 88) of the sIL-1Ra cDNA. The resultant protein lacked the N-terminal 21 amino acids of the 25-residue signal sequence of sIL-1Ra, which was replaced by three different amino acids. icIL-1Ral is a non-glycosylated intracellular protein of 159 amino acids and 18 kDa which exhibits binding to IL-1 receptors with the same characteristics as sIL-1Ra (Malyak et al., 1998a). This intracellular isoform ofIL-iRa is produced primarily by keratinocytes and other epithelial cells, but is a delayed transcriptional product of monocytes and macrophages (Malyak et al., 1998b). Additional species of intracellular IL-1Ra have subsequently been described. The distance between the 5' UTR of sIL-1Ra and the alternative exon for

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icIL-1Ral is 9.6 kb. A cDNA identical to that for icIL-1Ral except for an additional in-frame 63-bp sequence located three codons downstream of the translation start site of icIL-1Ral was cloned from h u m a n polymorphonuclear cells. This additional sequence is inserted between the first and second exons of icIL-1Ral and is coded by an extra exon located 2 kb downstream from the first exon for icIL-1Ral. This second isoform of intracellular of IL-1Ra was termed type 2 or icIL-1Ra2 with the predicted protein of 25 kDa and 180 amino acids possessing an extra sequence of 21 amino acids in the N-terminal region. However, icIL- 1Ra2 has never been shown to exist as a naturally occurring protein in vivo and may not be translated to any degree in living cells. A third intracellular isoform of IL- iRa, now termed type 3 or icIL-1Ra3, was originally described as a 143 amino acid and 16 kDa protein in a variety of cells (Malyak et al., 1998a). This protein is derived by alternative translational initiation from the mRNAs for both sIL-iRa and icIL-1Ral, but particularly from the former. Thus, icIL-1Ra3 lacks a signal peptide and is found only in the cytoplasm of cells. In direct binding studies using surface immobilized receptors, this low molecular weight isoform of IL-1Ra bound to IL-1 receptor type I (IL-1RI) four- to five-fold less avidly

TABLE 28.1 IL-1Ra isoforms Secretory IL- 1Ra (slL- 1Ra) Gene contains four exons Synthesized as a protein of 177 amino acids with a signal peptide of 25 residues Processed to a 17 kDa molecule containing 152 amino acids Secreted as variably glycosylated species of 22-25 kDa 26% sequence homology to IL-I[3 and 19% homology to IL-I~ Produced by monocytes, macrophages, neutrophils, and other cells Intracellular IL-1Ra (iclL-1Ra) Type 1 icIL-1Ra (icIL-1Ral) An additional upstream exon is spliced into an acceptor site within the exon encoding the leader sequence for sIL- IRa cDNA Sythesized in the cytoplasm as an 18 kDa molecule containing 159 amino acids Binds to IL-1 receptors equally as well as sIL-1Ra A major product of keratinocytes and other epithelial cells, endothelial cells, and fibroblasts Type 2 icIL-iRa (icIL-1Ra2) cDNA found in h u m a n cells encoded by an additional upstream first exon Predicted protein of 25 kDa and 180 amino acids may not be translated in vivo Type 3 icIL- 1Ra (icIL- 1Ra3) Produced by alternative translation initiation or by an alternative transcriptional splice Synthesized in the cytoplasm as a non-glycosylated 16 kDa molecule of 143 amino acids Binds poorly to type I IL-1R, but binds well to type II IL-1R A major protein in hepatocytes and neutrophils with smaller amounts found in macrophages

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than did sIL-1Ra or icIL-1Ral. However, all three isoforms of IL-iRa bound equally well to immobilized IL-1RII. IL-1RI is the functional form of the IL-1 receptor as IL-1RII exhibits no induction of intracellular responses after ligand binding, icIL-1Ra3 is found in monocytes, macrophages and epithelial cells, but is also present in large amounts in hepatocytes and neutrophils (Malyak et al., 1998b; Gabay et al., 1999). A recently described variant IL-1Ra cDNA, cloned from human articular cartilage and subsequently identified in keratinocytes, theoretically could give rise to an intracellular IL-1Ra species of 143 amino acids and 16 kDa by alternative transcriptional splicing and translation from an internal methionine (Weissbach et al., 1998). Thus, icIL-1Ra3 may arise by both alternative transcriptional and translational mechanisms. Additional structural variants of the IL-1 family have been described that have most similarity to IL-1Ra, although none of these proteins to date have been shown to have IL-1 receptor antagonist biologic activity. The results of initial studies on the effects of IL-IRa in IL-l-stimulation of target cells indicated that a great excess of IL-1Ra was necessary to inhibit IL-1. Up to 100-fold or greater amounts of IL-1Ra over IL-1 were required to give 50% inhibition of IL-1augmented proliferation of thymocytes or IL-1induced production of PGE2 and collagenase by cultured human synovial cells (Mend, 1990; Mend et al., 1990). This requirement is because cells are exquisitely sensitive to very small amounts of IL-1, exhibiting full biological responses with occupancy of only a few IL-1 receptors per cell. Since cells have an excess of IL-1 receptors, and IL-1Ra binds with near the same affinity as IL-1,100-fold or greater amounts of IL-IRa need to be present to effectively inhibit the binding of only a few molecules of IL-1.

PRODUCTION AND TISSUE LOCALIZATION The characteristics of production of the major IL-IRa isoforms, as well as cell and tissue localization, have been determined, sIL-1Ra is primarily a product of monocytes and tissue macrophages, but also can be secreted by neutrophils, hepatocytes, dendritic cells and other cells, icIL-1Ral is a major constitutive pro-

tein in keratinocytes and epithelial cells lining the entire gastrointestinal tract, but also can be produced by fibroblasts and endothelial cells, icIL-1Ra2 appears not to be produced in detectable level,~ by any cell in vivo. icIL-1Ra3 is created by alternative translational initiation primarily from the sIL-1Ra mRNA, and in much lower amounts from the icIL-1Ral mRNA. Thus, icIL-1Ra3 can theoretically be found in any cell secreting sIL-1Ra, but is present in particularly large levels in neutrophils and hepatocytes. The stimuli for sIL-1Ra production by monocytes and macrophages have been summarized in previous reviews (Dinarello, 1991; Arend, 1993; Arend et al., 1998). The major inducing factors are adherent IgG, GM-CSF and IL-1 itself. Early studies showed that IL-1 and IL-iRa were both produced by the same cell. In fact, IL-4 stimulation of human monocytes led to reciprocal regulation of IL-1 and IL-1Ra production, inhibited IL-1 while enhancing IL-IRa. High levels of icIL-1Ral are present constitutively in keratinocytes with enhanced production during cell differentiation. Both TNF-a and IL-la also increase icIL-1Ral production in keratinocytes (Kutsch et al., 1993; La and Fischer, 2001). iclL-1Ral is produced constitutively in small amounts by fibroblasts, but is up-regulated by PMA, LPS, TNF-R and IL-1 (Krzesicki et al., 1993; Martel-Pelletier et al., 1993; La and Fischer, 2001). Studies on the tissue localization of IL-1Ra isoforms in the mouse indicated that sIL-1Ra mRNA was not present in any tissue in control mice, but was upregulated primarily in the lung, spleen and liver after LPS injection (Gabay et al., 1997a). In contrast, icIL- 1Ral mRNA was present constitutively in the skin and epithelial cells lining the gastrointestinal tract. A unique characteristic of sIL-iRa is production by the liver as an acute phase protein. Both HepG2 hepatoma cells and fleshly isolated human hepatocytes produced sIL-1Ra mRNA and protein in response to stimulation with IL-1[3 and IL-6. Both NF-KB and C/EBP binding sites on the sIL-1Ra promoter mediated transcription of the sIL-1Ra gene. Furthermore, IL-4 enhanced the stimulatory effects of IL-113 on sIL-1Ra production by HepG2 cells and hepatocytes (Gabay et al., 1999). This IL-4 effect on transcription was mediated through a STAT6 binding element within the sIL-iRa promoter./n vivo studies showed that after either LPS injection or induction of local inflammation with turpentine injection, hepatic

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production of sIL-1Ra was up-regulated and paralleled the rise in serum levels (Gabay et al., 2001a, 2001b). The total amount of sIL-1Ra present in the liver after LPS injection was six- and ten-fold higher than in the lung and spleen, respectively. By in situ hybridization, icIL- 1Ral mRNA, but not sIL- 1Ra mRNA, was present in hepatocytes, but not Kupfer cells, after LPS injection. These studies proved conclusively that sIL-IRa was produced by the liver as an acute phase protein, explaining the high levels of this protein found in the circulation of patients with inflammatory, infectious, or neoplastic diseases (see below).

CRYSTAL STRUCTURE, RECEPTOR BINDING AND MECHANISM OF ACTION The results of detailed studies on the crystal structure ofthe three IL-1 ligands (IL-la, IL-lfi, and IL-1Ra) and the two IL-1 receptors has clarified how the ligands bind to the receptors and the mechanism, whereby IL- 1Ra fails to stimulate cells (reviewed in Arend et al., 1998). Examination of the crystal structure of sIL-1Ra at 2.0-A resolution revealed the presence of the same [~-trefoil structure as IL-1R and IL-lfi as well as a similar hydrophobic core. However, structural differences were observed between sIL-1Ra and the two IL-1 agonists, particularly at the open end of the [~-barrel. Structural studies by NMR spectroscopy yielded similar results, suggesting that differences in side chains may contribute to the difference in biological properties between IL-1Ra and IL-1. Additional studies on the structure of sIL-1Ra by NMR spectroscopy indicated conservation of residues between sIL-1Ra and IL-I~ thought to be important in receptor binding, but lack of conservation of residues critical for receptor activation. Analysis of crystals of sIL-1Ra bound to soluble type I IL-1 receptors revealed a 1:1 receptor-ligand complex, suggesting that sIL-1Ra bound a single receptor and did not induce receptor aggregation. The binding of sIL-1Ra to type I IL-1 receptors on murine EL4 thymoma cells indicated an affinity equal to that of IL- lcz and IL- 113 (Kd = 150 pM). Surfacebound IL-1Ra failed to activate the protein kinase activity responsible for down-modulation of the

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EGF receptor on the murine 3T3 fibroblast cell line and did not undergo receptor-mediated internalization. Extensive site-directed mutagenesis experiments described candidate regions in IL-I[~ and IL-1Ra that might be involved in receptor binding. Conversion of IL-1Ra into a partial agonist was seen following mutation of Lys-145(K) into Asp(D), suggesting the possible importance of the region surrounding Asp-145 in IL-I[~ for triggering biological responses after receptor binding. Additional studies indicated that the insertion of the [~-bulge of IL-1 [~ at the corresponding region of IL-1Ra K145D resulted in a three to four-fold increase in agonist activity, with a further enhancement in activity following the coexpression of a second substitution of His-54 to Pro. Most importantly, these investigators also described that the bioactivity of the triple mutant IL-1Ra K145D/H45P/fi-bulge was dependent upon an interaction with the IL-1 accessory protein (IL-1R AcP). Cloning and characterization of the IL-1R AcP demonstrated that this molecule was present in the plasma membrane of cells responsive to IL-1 and formed a complex with the single chain type I IL-1R and either IL-I~ or IL-lfi, but not IL-1Ra (Greenfeder et al., 1995). IL-1R AcP was shown to be indispensable in IL-1 induction of signal transduction pathways (Wesche et al., 1996; Korherr et al., 1997; Cullinan et al., 1998). An early event in IL-1 signaling is the recruitment of the Ser/Thr kinase IRAK to the intracellular domain of the type I IL-1 receptor; IRAK was recruited to the IL-1 receptor complex through its association with IL-1R AcP (Huang et al., 1997). Over-expression of an IL-1R AcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-l-induced NF-~:B activation. Thus, the mechanism whereby IL-1Ra inhibits cell responses to IL-1 is by competitively binding to type I IL-1 receptors and preventing the interaction of the IL-R AcP molecule with the single chain receptor. The crystal structure at 2.7A resolution of the soluble extracellular portion of type I IL-1 receptor complexed to sIL-1Ra indicated that the receptor possessed three Ig-like domains. Residues of all three domains contacted the sIL-1Ra molecule, including five critical residues previously identified by sitedirected mutagenesis as important in receptor binding. However, a region in IL-I[~ thought to be

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important in biological function, the receptor trigger site, was not in direct association with the receptor in the IL-1Ra complex. The possibility exists that a small structural change in the IL-1Ra molecule conformationally separates the receptor trigger site from association with the type I IL-1 receptor in a manner that blocks interaction of the IL-1R AcP with the complex. Type II IL-1 receptors possess a short cytoplasmic domain and fail to trigger cells after ligand binding (Colotta et al., 1994; Sims et al., 1994). Thus, type II IL-1 receptors on the cell surface act as further inhibitors of IL-1 effects by competing for binding of the IL-1 agonists. The extracellular portions of both types of IL-1 receptors are enzymatically cleaved from the plasma membrane of activated cells and are present in the microenvironment of cells and in body fluids, particularly soluble type II IL-1 receptors. The results of binding studies demonstrated that IL-I~ bound more avidly to soluble IL-1RII than IL-la or IL-1Ra, primarily because of a slow dissociation rate (Mend et al., 1994; Burger et al., 1995). In contrast, IL-1Ra bound more avidly to soluble IL-1RI than either IL-1 agonist, again because of a very slow dissociation rate. Further experiments indicated that some IL-IRa and IL-1 may bind soluble IL-1R in body fluids in vivo, obscuring measurement of these cytokines by ELISA. A further implication is that naturally occurring soluble IL-1RI may bind to endogenous or exogenously administered IL-1Ra, reducing its IL-l-inhibitory effects. Lastly, IL-1R AcP interacts with both IL- 1RI and IL- 1RII in the plasma membrane of cells (Lang et al., 1998; Malinowsky et al., 1998). Upon IL- 1 binding, IL- 1RII may recruit IL- 1R AcP into a nonfunctional complex, removing the IL-1R AcP from possible interaction with IL-1RI. Thus, IL-1RII may function as a natural IL- 1 inhibitor through three different mechanisms that all involve reduction in binding of IL-la or IL- 113to membrane-bound IL- 1RI: competing on the cell surface for ligand binding, competing for ligand binding as a soluble receptor in the cell microenvironment, and competing on the cell surface for interaction with IL- 1R AcE

EXPRESSION IN NORMAL ANIMALS AND HUMANS Tissue distribution Examination of tissues recovered from normal mice suggests the constitutive expression of icIL-1Ral mRNA and protein in the skin (Gabay et al., 1997a). This is consistent with data from analysis of human keratinocytes and epithelial cells, which also spontaneously synthesize icIL-1Ral. Analysis of normal human skin by immunohistochemistry has also identified IL-1Ra in the sebaceous glands, eccrine sweat glands, dermal dendritic cells and upper dermal blood vessels. The intestine is an additional site of constitutive icIL-1Ral production in mice and rabbits (Cominelli et al., 1994). The secreted isoform of IL-1Ra, in contrast, does not appear to be synthesized spontaneously in mice. However, IL-IRa has been detected in normal, human synovial fluid (Cameron et al., 1994), tears (Solomon et al., 2001), blood and cerebrospinal fluid (Tarkowski et al., 2001), using ELISA methods that do not distinguish between the different isoforms of IL-1Ra. Moreover, IL-1Ra has been identified by immunohistochemistry in certain neurons in the neocortex and hippocampus of the normal brain (Yasuhara et al., 1997). Low levels of icIL-1Ral, but not sIL-1Ra, are synthesized ex vivo by orbital fibroblasts recovered from the eyes of normal donors (Muhlberg et al., 1997). In addition, the epithelial cells of the normal human cornea synthesize both sIL-1Ra and icIL-1Ral, while the stromal cells produce only the intracellular variant (Kennedy et al., 1995). Human mast cells recovered from normal bronchoalveolar lavage fluid constitutively synthesize IL-1Ra (Hagaman et al., 2001). As described below, IL-1Ra is produced by the normal ovary in a cyclical manner around the time of ovulation, and during gestation and child birth. Human milk also contains high concentrations of IL-1Ra that may contribute to the improved development and immune function of breast-fed infants.

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IL- 1Ra in b l o o d IL-1Ra cannot be detected in the blood of normal mice (Gabay etal., 1997a), despite the fact that sIL- 1Ra mRNA is constitutively present in whole blood RNA as a result of its expression in neutrophils. Plasma levels of IL-1Ra increase dramatically in mice within 4 h of LPS injection. The major sites of sIL-1Ra expression following the administration of LPS to mice are the lung, spleen and liver. The major blood cell contributing to circulating sIL-1Ra in response to LPS appears to be the neutrophil (Gabay et al., 1997a). Equivalent results have been obtained using rats. Unlike the case with mice, sIL-1Ra is detectable in h u m a n serum recovered from normal individuals, usually at concentrations of I ng/ml or less. Administration of LPS to healthy volunteers leads to a rapid increase in circulating IL-1Ra levels in approximately the same time-frame as seen with mice (Granowitz et al., 1991). TNF-~, IL-1 and IL-6 have also been injected intravenously into normal volunteers, with a rapid and marked increase in plasma IL-1Ra concentrations; a slower elevation occurs following injection of IFN-7. The rapid induction of IL-1Ra by LPS, IL-1 and IL-6 is explained by the demonstration (Gabay et al., 1997b) that IL-1Ra is an acute phase protein. In cell culture, both normal h u m a n hepatocytes and the hepatoma cell line HepG2 produce sIL-1Ra in response to IL-1 and IL-6. As with other acute phase proteins, synthesis of sIL-1Ra is increased by dexamethasone. Infection and the onset of inflammatory conditions lead to rapid induction of sIL-1Ra synthesis by the liver, resulting in serum concentrations in excess of 50 ng/ml in certain patients with sepsis (Rogy et al., 1994). Concentrations of sIL-1Ra are also strongly elevated in the peripheral blood of patients with conditions such as juvenile rheumatoid arthritis, polymyositis-dermatomyositis, systemic lupus erythematosis, rheumatoid arthritis (RA), active multiple sclerosis, peripheral artery disease, heart failure and Gaucher disease, as well as following trauma, burns, hemodialysis, stroke, vigorous exercise and acute mental stress. The effects of trauma, stress and exercise may be secondary to the influence of adrenaline, the infusion of which elevates plasma IL-1Ra concentrations. Plasma levels of IL-1Ra have been reported to increase markedly during pregnancy.

The role in biology of sIL-1Ra appears to be to regulate the effects of IL-1 in the cell microenvironment. IL-1Ra is synthesized and released from cells immediately following IL-1, and the ratio of IL-1Ra found locally to IL-1 influences the relative stinulatory potential of the IL-1 present. This ratio may be imbalanced either because of sustained stimulation of production of IL-1 or inadequate local production of sIL-1Ra. An imbalance in the IL-1Ra/IL-1 ratio may lead both to abnormalities in normal cyclical organ function, or to actual disease where the imbalance is prolonged. The role in biology of the intracellular isoforms of IL-lra is less clear. These molecules may play unique roles inside cells that do not involve receptor binding. Precursor IL-la possesses a nuclear localization sequence in the propiece, which mediates movement to and entry into the nucleus of cells (Wessendorf et al., 1993). Pre-IL-la acts as a negative regulator of the growth of endothelial cells (McMahon et al., 1997) and as a transforming nuclear oncoprotein (Stevenson et al., 1997). However, it is not known if icIL-1Ra isoforms influence these intracellular functions of pre-IL-la. The presence of icIL-1Ral in ovarian carcinoma cells was associated with a decrease in mRNA stability for the chemokines IL-8 and GRO (Watson et al., 1995). No more recent reports have investigated this observation further, and its relevance must remain unclear. In addition, scleroderma fibroblasts overexpress both pre-IL-l~ and icIL-1Ral, with the pre-IL-la actually inducing expression of icIL-1Ral (Higgins et al., 1999). The relationship of this finding to the abnormal phenotype of scleroderma fibroblasts is not known. Under certain conditions icIL-1Ral may be secreted from keratinocytes and other epithelial cells and function as a competitive inhibitor of IL-1 receptor binding. Larger, more differentiated keratinocytes secrete iclL-1Ral in culture, suggesting that these cells in the upper layers of the skin may also carry out this function (Corradi et al., 1995). Human airway epithelial cells also release icIL-1Ral in response to stimulation with IL-4, IL-13 or IFN-7, all cytokines that also induce production of sIL-1Ra in monocytes and macrophages (Levine et al., 1997). Lastly,

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icIL-1Ral is released from respiratory epithelial cells in vivo and in vitro after infection with rhinovirus, and may help resolve the symptoms of rhinovirus infections of the upper respiratory tract (u et al., 1999). In all of these studies, the release of icIL-1Ral by the epithelial cells was not due to cell death, as cytoplasmic enzymes were not found in the cell supernatants. Although these results indicate that one function of icIL-1Ral may be to block extracellular IL-1, other as yet poorly characterized intracellular effects of icIL-1Ra may exist.

Reproductive system The concept that ovulation resembles a cyclical, inflammatory process driven by IL-1 has become popular in recent years. Among the evidence for this hypothesis is the ability of IL-1 to induce ovulation and to synergize with luteinising hormone (LH) in this regard (Brannstrom et al., 1993), the ability of IL-1Ra to inhibit ovulation (Simon et al., 1994b), and the expression of IL-1 in the ovary around the time of ovulation (Hurwitz et al., 1991). This being so, IL-1Ra may serve as an endogenous modulator of ovulation. IL-iRa is not expressed in the immature rat ovary, but is rapidly induced after treatment with chorionic gonadotrophin. Transcripts encoding sIL-1Ra and icIL-1Ra are localized to the mural, antral and cumulus granulosa cells, as well as the oocytes. It is likely that IL-IRa synthesis is stimulated by prior induction of IL- 1 in the ovary. In agreement with the studies of rat ovaries, analysis of cervical mucus in humans revealed that IL-iRa was constitutively expressed during the entire menstrual cycle, but concentrations were higher in the ovulatory than in the follicular phase (Kanai et al., 1997). Immunohistochemistry identified epithelial cells of the endometrium as a source of IL-1Ra (Fukuda et al., 1995). Macrophages within the endometrial stroma also stained positively for IL-ira. RT-PCR analysis confirmed that stromal and glandular cells of the human endometrium expressed icIL-1Ra (Simon et al., 1995). IL-1 is also intimately involved with embryonic implantation. At the two-cell stage, murine embryos do not express IL-iRa transcripts. However, starting at the eight-cell stage, embyos start to express IL-1Ra at

increasing frequencies, such that 74% of blastocytes give a positive RT-PCR signal for IL-1Ra (Kruessel et al., 1997). This timing is likely to be of great significance, as IL-1Ra inhibits embryonic implantation (Simon et al., 1994a), possibly by inhibiting the expression of adhesion molecules on the endometrial epithelium. IL- iRa is further likely to play a key role in gestation and delivery. Maternal serum levels ofIL-iRa increase with increasing gestational age and during labor activity (Ammala et al., 1997). IL-1Ra is synthesized by the placenta at the time of birth; cells within the amnion, chorion and decidua are sources of IL-1Ra. Amniotic fluid also contains high concentrations of IL-iRa, but much of this is thought to originate from the fetal urine. According to one study, urinary IL- i r a concentrations are higher in female newborns than male newborns, even though newborn serum concentrations of IL-1Ra are not dependent upon gender (Bry et al., 1994).

Host defense and inflammation IL-1 is a central orchestrator of the body's response to infection, being involved in both innate and acquired immunity at various levels (see Chapter 27). As well as activating macrophages, IL-1 serves as a costimulatory molecule for lymphocyte proliferation, is involved in antigen presentation, and increases T celldependent antibody production. By modulating the activities of IL- 1, the IL- 1Ra molecule plays a key role in regulating these processes. Its importance in the normal homeostasis of immune and inflammatory reactions is indicated by the phenotypes of mice with disrupted IL-1Ra genes. Such animals spontaneously develop inflammatory joint diseases (Horai et al., 2000), arterial inflammation (Nicklin et al., 2000), and are more sensitive to the lethal effects of endotoxin.

Central nervous system There is growing evidence that IL-1 is involved in normal sleep regulation, and that IL-iRa is a physiological regulator of this process. Administration of IL-IRa into the lateral cerebral ventricle of normal rabbits transiently reduced sleep, induced non-rapid eye movements, and blocked fever (Opp et al., 1992).

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IL-1Ra IN DISEASE Infection IL-1 is a key component of host defense responses (see Chapter 27). There are numerous examples confirming the marked increase in circulating IL-1Ra levels that occurs during viral and bacterial infections. Intravenous administration of endotoxin or TNF into normal human volunteers provokes a rapid rise in serum IL-1Ra concentrations, peaking 3-6 h after injection (Granowitz et al., 1991). As noted above, IL-1Ra is an acute phase protein synthesized rapidly in the liver in response to infection and endotoxin (Gabay et al., 1997b). Concentrations of IL-1Ra may also be elevated by local synthesis within various organs during infection. Much of the IL- 1Ra produced under these conditions is likely to be by infiltrating leukocytes, particularly macrophages, or by the cells that are normally resident within the tissue. Plasma levels of IL-1Ra can be particularly high during sepsis. The protective role of IL-1Ra has been well established in experimental animals where disruption of the IL-1Ra gene, or administration of neutralizing antibodies to IL-1Ra, worsens disease and increases mortality. Conversely, administration of recombinant IL-1Ra has the opposite effect (Wakabayashi et al., 1991) and has led to clinical trials, described below, of its use in human sepsis.

Arthritis The role of IL-1 in both the inflammatory and erosive components of RA is well established (reviewed in van den Berg, 2001). IL-1 synthesized within the synovium by both resident synoviocytes and infltrating leukocytes contributes to the recruitment of inflammatory cells, the formation of pannus, and the invasion of the adjacent bone and cartilage. Moreover, IL-1 produced by the articular chondrocytes appears to be the major stimulus for endogenous breakdown of the cartilagenous matrix ('chondrocytic chondrolysis'). It also inhibits synthesis of the catilagenous matrix, and thus serves as a particularly powerful agent of cartilage damage. IL-iRa is present in the synovial fluid of normal joints, and its concentration rises considerably in rheumatoid arthritis and other inflammatory joint conditions. Macrophages and neutrophils are proba-

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bly the major sources of the IL-1Ra found in the synovial fluid; the amounts produced by chondrocytes are quite low. Although synovial fibroblasts synthesize considerable quantities of IL-1Ra, it is predominantly the intracellular isoform(s) that are produced (Firestein et al., 1994). The fact that RA persists in many patients despite the intraarticular presence of considerable quantities of IL-1Ra probably reflects the high molar ratio of IL-1Ra:IL-1 that is needed to suppress the biological activities of the latter. This problem is highlighted by a study of Firestein et al., who measured the ex vivo synthesis of IL-1 and IL-1Ra by explants of articular tissues recovered from the joints of patients with RA (Firestein et al., 1994). IL-1Ra:IL-1 ratios of 1.2-3.6 were noted, values far below those needed to provide strong protection from the intraarticular activities of IL- 1. An improvement in the ratio of IL- 1Ra:IL- 1 synthesized by peripheral blood monocytes from patients whose rheumatoid arthritis responded to treatment by methotrexate and gold drugs, has been noted (Seitz et al., 1995). The importance of IL-1Ra has also been demonstrated in a study of acute knee arthritis in patients with Lyme disease, where those who recovered more rapidly had higher synovial fluid ratios of IL-1Ra:IL-1 (Miller etal., 1993). IL-1 may be particularly important in osteoarthritis (OA), especially in view of its unique potency as a mediator of cartilage breakdown, the major pathology in this condition. There are no convincing studies in which IL-1Ra:IL-1 ratios have been measured in OA, but administration of recombinant IL-1Ra (Caron et al., 1996), or delivery of a cDNA encoding IL-1Ra (Pelletier et al., 1997; Frisbie et al., 2002), reduces disease activity in animal models. In rotator cuff diseases of the shoulder, there is expression of icIL- 1Ral by the synovial lining cells and of sIL- iRa by cells of the sublining; expression of IL-1Ra correlates with shoulder pain (Gotoh et al., 2001). IL-1Ra is also elevated in patients with temporomandibular joint disease. Blood levels of IL-1Ra are elevated in RA (Chikanza et al., 1995), suggesting an involvement in modulating the extraarticular and systemic components of the disease. High serum levels of IL-1Ra are also associated with systemic lupus erythematosus (Suzuki et al., 1995), where IL-1Ra concentrations correlate with disease severity. Response to immunoglobulin therapy in patients with the multisystem autoimmune

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disease cicatricial pemphigoid is also associated with increased levels of circulating IL-1Ra (Kumari et al., 2001). Elevated concentrations of circulating IL- 1Ra have been reported for patients with juvenile rheumatoid arthritis (Prieur et al., 1987) and polymyositis (Gabay et al., 1994). Interestingly, synovial fluid concentrations of IL-1Ra have been reported to fall following rupture of the anterior cruciate ligament, and to remain chronically reduced in symptomatic patients (Cameron et al., 1994). Whether this is related to the secondary development of OA in such patients is a matter for speculation.

Nervous system There is evidence to suggest that IL-1 is involved in hypoxia-reperfusion injury and the sequelae of blunt trauma to the brain (Sanderson et al., 1999). IL-iRa is expressed constitutively in neocortex and hippocampus, and is induced by IL-1 and ischemia (Loddick et al., 1997) in additional areas of the brain, including the cerebellum. Administration of IL-1Ra cDNA reduces the severity of the response to blunt trauma in the rat brain (DeKosky et al., 1996). There is growing appreciation of the role of inflammatory processes in neurodegenerative conditions. Of interest in this regard is the recent finding that the cerebrospinal fluids of patients with Alzheimer's disease contain lower concentrations of IL-1Ra than those of unaffected control individuals (Tarkowski et al., 2001). IL-113was not detectable in fluids recovered from patients or unaffected controls. Nevertheless, in a separate study of the immunohistochemical localization of IL-1Ra in brain tissue, Alzheimer's disease was associated with increased numbers of positively staining neurons, as well as increased levels of staining (Yasuhara et al., 1997). IL-1Ra was additionally expressed in globular deposits in senile plaques and some extracellular neurofibrillary tangles., Increased expression of IL-1Ra was also noted in Pick's disease, another neurodegenerative condition (Yasuhara et al., 1997). IL-1Ra has been implicated in Guillain-Barr6 syndrome, an autoimmune disease affecting the peripheral nervous system. During the development of a rodent model of this condition, the Schwann cells surrounding the sciatic nerves start to express both IL-la and IL-I[3 during the pre-clinical stage of the

disease (Skundric et al., 2001). IL- 1Ra is not detectable at this time, but its expression occurs as the disease becomes clinically manifest. Of possible significance is the observation that IL-1Ra immunoreactivity co-localizes with myelin-associated glycoprotein, a marker for paranodal regions essential for proper impulse transmission (Skundric et al., 2001). Multiple sclerosis, an autoimmune condition affecting the central nervous system, is treated by the injection of IFN-[3 .Treatment with weekly, intramuscular injections of 30 mg IFN-13 increased serum levels of IL-1Ra, suggesting a role for IL-1Ra in the clinical improvement noted in these patients (Nicoletti et al., 1996). Experiments in various animal models suggest that IL-1 is an important contributor to neuropathic pain. This leads to the suggestion that IL-iRa may serve an analgesic function under the appropriate conditions. Using a rodent L5 spinal nerve transection model, it was shown that, although administration of IL-1Ra alone failed to reduce pain, it improved the ability of soluble TNF receptors to do so. This was associated with reduced expression of IL-6, but not IL-1, in the spinal cord (Sweitzer et al., 2001). Patients with fibromyalgia, a condition associated with chronic pain, have higher levels of circulating IL-iRa. Expression of IL-1Ra correlates with the degree of shoulder pain experienced by individuals with rotator cuff disease (Gotoh et al., 2001). IL-1Ra may also be involved in other aspects of neurological function. For instance, associations with autism and attention deficit hyperactivity have been suggested, indicating that the IL-1/IL-1Ra axis may play subtle roles in pyschobiological development and function.

Skin Expression ofIL-iRa appears to be disturbed in psoriasis. The expression of IL-iRa increases in the lesions of psoriatic skin (Debets et al., 1997). Of interest is the finding that although levels of IL-1Ra protein appeared to increase, as judged by immunohistochemistry, levels of IL-1Ra message, as judged by in situ hybridization, did not (Debets et al., 1997). Serum levels of IL-1Ra are higher in patients with psoriatic arthritis. The expression of IL-1Ra during keratinocyte cell division and differentiation has been studied with normal and psoriatic epidermis (Hammerberg et al.,

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1998). IL-1Ra expression increases as keratinocytes differentiate from basal stem cells into transient amplifying cells. Production of IL-1Ra by the latter cells normally increases throughout the cell cycle, but in cells derived from psoriatic skin, IL-1Ra production falls slightly (Hammerberg et al., 1998). Dermal fibroblasts recovered from patients with systemic sclerosis express higher levels of icIL-1Ral and intracellular pre-IL-la than controls (Higgins et al., 1999). These events may be coupled, as transduction of normal skin fibroblasts with a cDNA encoding pre-IL-la increases expression of icIL-1Ral (Higgins et aI., 1999). Serum levels ofIL-1Ra increase during flares of contact allergy, and other conditions involving dermatitis, such as exposure to sunlight, and atopic dermatitis (Pastore et al., 1998). The expression of IL-1Ra increases in murine hair follicular cells as they cycle from early anagen to telogen (Tokura et al., 1997). At the latter stage, contact photosensitivity is much lower, possibly due to the elevated levels of IL-1Ra that are present. In agreement with this, administration of IL- 1Ra inhibits contact hypersensitivity in mice (Kondo et al., 1995).

Lung diseases In asthma, levels of IL-1Ra increase both in the serum (Yoshida et al., 1996) and locally in the bronchial epithelium (Sousa et al., 1996). Moreover, serum concentrations of IL-1Ra are higher in patients during asthma attacks than under stable conditions (Yoshida et al., 1996). In vitro studies suggest that mast cells within the lung increase synthesis of IL-1Ra when stimulated with IgE (Hagaman et al., 2001), suggesting one way in which asthmatic attacks may result in increased IL-1Ra levels. Corticosteroids induce the synthesis of icIL-iRa by human bronchial epithelium (Levine et al., 1996), and this may contribute to their efficacy in treating asthma. Alterations in the pulmonary production of IL-1Ra have also been associated with a number of inflammatory and fibrotic diseases of the lung, including tuberculosis, smoking, malignancy, interstitial lung disease, pneumonia, acute respiratory distress syndrome, cystic fibrosis, high altitude edema, chest trauma, panbronchiolitis, and sarcoidosis. In sarcoidosis, elevated IL-1Ra production is localized to

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the sarcoid granulomas. In patients with idiopathic pulmonary fibrosis, IL-1Ra production has been localized by immunohistochemistry and in situ hybridization to hyperplastic type II pneumocytes, macrophages and local stromal cells (Smith et al., 1995). Human bronchogenic carcinoma cells also produce elevated quantities of IL-1Ra (Smith et al., 1993). Intraperitoneal administration of IL-1Ra protects mice from the pulmonary fibrotic response to bleomycin or silica (Piguet et al., 1993). Moreover, low serum concentrations of IL-1Ra and IL-10 are associated with a poor prognosis in patients with acute respiratory distress syndrome (Parsons et al., 1997).

Cardiovascular diseases There is currently much interest in the relationship between inflammation and atherosclerosis. Inflammatory events within atherosclerotic plaques are considered to be major determinants of disease progression and clinical outcome. Baseline plasma levels of IL-1Ra are increased in patients with atherosclerosis (Fiotti et al., 1999). According to Fiotti et al. (1999), these levels correlate with the occurrence and stage of the disease so strongly as to be predictive. Expression of icIL-1Ral is elevated in the endothelial lining of human, atherosclerotic cardiomyopathic arteries. When patients were subjected to a treadmill test, IL-1Ra levels increased in both control subjects and those with atherosclerosis (Fiotti et al., 1999). Administration of IL-1Ra is able to inhibit the formation of fatty streaks on the intimal surfaces of blood vessels in apolipoprotein E-deficient mice (Elhage et al., 1998). Moreover, mice lacking a functional IL-1Ra gene spontaneously develop arterial inflammation (Nicklin et al., 2000). Circulating levels of IL-1Ra also increase after severe, acute myocardial infarction and rise to higher levels than those found in patients with uncomplicated acute myocardial infarctions (Shibata et al., 1997). Moreover, plasma concentrations of IL-1Ra correlate closely with the severity of hemodynamic changes, and are significantly higher in non-survivors than survivors (Shibata et al., 1997). Serum IL- 1Ra levels are also elevated in patients with severe chronic congestive heart failure and correlate with their symptom-limited oxygen consumption. The possible correlation between high plasma levels of IL-1Ra and

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a poor prognosis for heart conditions of this nature is supported by a study of patients hospitalized for angina. Those patients with an uneventful course had lower levels of IL-1Ra at entry, and these levels further declined during the hospital stay. Those patients with in-hospital complications had higher levels of IL-iRa at entry, and these levels increased during the hospital stay (Biasucci et al., 1999).

Kidney diseases Renal clearance represents the major excretion pathway of IL-1Ra, and elevated plasma levels occur in patients with renal failure. Serum levels of IL-1Ra increase in patients with pyelonephritis, chronic renal failure, end-stage renal disease, and other conditions that severely impair kidney function. Conversely, urinary levels are lower than controls as a result of impaired IL-1Ra renal clearance. IL-1Ra is synthesized by the glomerular cells of the kidney during experimental glomerulonephritis in rats (Karkar et al., 1995), indicative of a local contribution to the increase in circulating I L - i r a that occurs under these conditions. Low circulating levels of IL-1Ra, in contrast, are correlated with kidney involvement in patients with lupus (Sturfelt et al., 1997). Plasma concentrations of IL-1Ra increase during hemodialysis, and it has been suggested that the level of IL-IRa synthesis by peripheral blood mononuclear cells of patients receiving dialysis may have value as a predictor of morbidity (Balakrishnan et al., 2000).

Reproductive system As noted earlier in this chapter, IL-1 and IL-1Ra play important roles in ovulation, implantation, gestation and childbirth. Disruption of their interplay is associated with various disorders of reproduction. Plasma levels of IL-1Ra are elevated in women with preeclampsia (Greer et al., 1994). These levels do not correlate with disease severity, but have 58% positive predictivity, 100% negative predictivity, 50% specificity and 100% sensitivity for pre-eclampsia during gestational weeks 20-25 (Kimya et al., 1997). IL-1Ra expression in the placenta is increased during chorioamnionitis, preterm labor, and intrauterine infection (Romero et al., 1994). However, IL-1Ra levels in cord blood are not affected by labor pain or fetal

distress (Hata et al., 1996). Reduced concentrations of IL-iRa in the plasma of pregnant women have been associated with maternal depression and higher rates of maternal complications after childbirth (Schmeelk et al., 1999).

Osteoporosis IL-1, along with TNF and IL-6, is a powerful inducer of osteoclastic activity. There is good evidence that these mediators are responsible for the loss of bone that occurs following human menopause and ovariectomy in both humans and experimental animals (Pacifici, 1996). The importance of IL-1 to the process has been demonstrated by administration of IL-1Ra, as a recombinant protein or by gene transfer, to ovariectomized rats and mice (Kimble et al., 1994b; Kitazawa etal., 1994; Baltzer etal., 2001). Bone loss in these animals is strongly inhibited. Mice lacking IL- 113or type I IL-1 receptors are completely protected from the loss of bone that accompanies ovariectomy (Lorenzo et al., 1998). Clinical studies are largely, but not unanimously, in support of the relevance of these animal studies to osteoporosis in humans. IL-1Ra plasma levels tend to be lower in osteoporotic women compared with age-matched non-osteoporotic controls, although the differences are quite small and not apparent in all studies. In a recent analysis, cytokine mRNA levels were measured in bone biopsies recovered from early post-menopausal women and related to measurements of bone mineral density. Slower bone loss was associated with higher IL-1Ra:IL-I~ mRNA levels (Abrahamsen et al., 2000b). Of interest is the suggestion that the actions of estrogen on bone may be associated with an influence on IL-I:IL-1Ra ratios. Thus, the cells in whole blood recovered from women receiving hormone replacement therapy produce IL-1 and IL-1Ra at a lower ratio than controls (Abrahamsen et al., 2000a). Addition of 17D-estradiol to blood recovered from post-menopausal women decreases the ratio of IL-I:IL-1Ra that is spontaneously produced in vitro (Rogers and Eastell, 2001). In the bone biopsy study discussed in the previous paragraph, hormone replacement therapy in post-menopausal women normalized IL-I[3 mRNA levels without affecting the abundance of IL-1Ra message (Abrahamsen et al., 2000b).

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an attempt by the body to limit the disease (Sandberg et al., 1994).

Plasma levels of IL-1Ra are higher than normal in patients with active ulcerative colitis, Crohn's disease and other inflammatory bowel diseases. Levels are lower in patients with inactive disease, but remain above normal (Kuboyama, 1998). In a study of biopsied colonic material, IL- 1Ra secretion was elevated in moderately to severely involved tissue samples, compared with non-inflamed tissue (Dionne et al., 1998). IL-1Ra:IL-113 ratios were lower in involved inflammatory bowel disease tissue (Dionne et al., 1998). Similar imbalances have been found in Crohn's disease, ulcerative colitis, diverticulitis and infectious colitis. Immunohistochemical studies have identified cells within the lamina propria, especially macrophages, as the major source ofIL- 1Ra in inflammatory bowel disease tissue (Nishiyama et al., 1994). Administration of antibodies to IL-1Ra exacerbates and prolongs disease in a rabbit model of colitis (Ferretti et al., 1994). Mice lacking a functional IL-10 gene develop spontaneous inflammatory changes in the gastrointestinal tract, and this may be related to IL-1Ra (Kuhn et al., 1993). It is interesting to note that IL-10 down-regulates IL-1 and up-regulates IL-1Ra in mononuclear phagocytes and lamina propria present in biopsies of colon taken from patients with inflammatory bowel disease (Schreiber et al., 1995). A similar response was noted following administration of an IL-10 enema (Schreiber et al., 1995). According to one study, serum levels of IL-1Ra are reduced in patients with colorectal cancer (Iwagaki et al., 1997). However, the results from a similar type of investigation suggested increased IL-1Ra in patients with this disease (Ito and Miki, 1999). The incidence of sIL- i r a mRNA increases in tumorous tissue recovered from patients with gastric cancer, and correlates with lymph node and liver metastasis (Iizuka et al., 1999).

Insulin-dependent diabetes mellitus Circulating concentrations of IL-1Ra are elevated in patients with longstanding insulin-dependent diabetes mellitus (Netea et al., 1997). Data from experiments in mice suggest that this response may indicate

Cancer There is considerable evidence that expression of IL-1Ra is dysregulated in various cancers (Kurzrock, 2001). In esophageal squamous cell carcinoma, for instance, expression of IL-1Ra is reduced two-fold (Hu et al., 2001). This also occurs in chronic myelogenous leukemia, where high leukocyte levels of IL-l[3 and low levels of IL-1Ra are seen in advanced disease and correlate with reduced survival (Kurzrock, 2001). In view of this, it is interesting that transfection of a murine skin carcinoma cell line with icIL-1Ral reduced its growth rate in vitro and slowed the development of tumors in vivo (La et al., 2001). Treatment of children with hematological malignancies using the chemotherapeutic agent cytarabine increases IL-1Ra blood levels (Ek et al., 2001). It is possible that IL-1 serves as a growth factor for certain types of tumors, with IL-1Ra acting to restrain tumor growth. Nevertheless, high levels of IL-lRa are found in the sera of patients with Hodgkin's lymphoma (Shin et al., 1995), and IL-1Ra is produced by bronchogenic carcinoma cells (Smith et al., 1993). It is also expressed by 20% of human hepatocellular carcinomas, and in pituitary tumor cells (Sauer et al., 1994). In these cases, IL-1Ra may help the tumor evade the immune system. Serum levels of IL-1Ra are altered in patients with soft tissue sarcomas (Ruka et al., 2001).

Transplantation The allograft response that leads to the rejection of transplanted organs is partly mediated by IL-1. Hepatic expression of IL-113 and IL-1Ra is increased during rejection following liver transplantation in humans. Moreover, low production of IL-1Ra is associated with steroid resistance of acute rejection (Conti et al., 2000). Acute graft-versus-host disease (GVHD) is a serious complication of marrow transplantation. Circulating levels of IL-1Ra are depressed 3-5 days after transplantation, but become elevated with the development of GVHD (Schwaighofer et al., 1997). The magnitude of the increase correlates with disease severity. Because IL-1 is thought to be an important

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component of the GVH response, this response has been interpreted as an attempt by the body to limit GVHD. In mice, administration of recombinant IL-iRa reduces the immunosuppression and mortality of GVHD without impairing the engraftment of hematopoietic stem cells (McCarthy et al., 1991). These results have led to a clinical trial of IL-1Ra in patients with GVHD (see below).

IL- 1Ra ALLELIC

POLYMORPHISMS AND DISEASE A polymorphism in intron 2 of the IL-1Ra gene, caused by two to six copies of an 86 bp tandem repeat, is associated with a variety of human diseases (Tarlow et al., 1993) (Table 28.2). Two single nucleotide polymorphisms exist within exon 2 of IL1RN, but polymorphisms at these sites are always linked with the allelic polymorphism in intron 2. Allele A2 containing two repeats is found in 21.4% ofthe normal Caucasian population and is present in increased frequencies in diseases largely of epithelial cell or tissue origin

TABLE 28.2 IL-1Ra allelic p o l y m o r p h i s m s a n d disease H u m a n diseases a s s o c i a t e d w i t h I L - 1 R a g e n e allele A 2 (IL1RN*2) Ulcerative colitis in certain population groups Severity of alopecia areata Lichen sclerosis Early-onset psoriasis Multiple sclerosis in certain population groups Systemic lupus erythematosus, particularly skin lesions Sj6gren's disease Juvenile chronic arthritis Hyochlorhydria and gastric cancer Diabetic nephropathy Susceptibility to sepsis Henoch-Sch6nlein purpura IgA nephropathy Early-onset periodontitis Bronchial asthma Fibrosing alveolitis Silicosis Severity of acute graft-versus-host disease in bone marrow transplant patients Idiopathic recurrent miscarriage

(Mend and Guthridge, 2000; Witkin et al., 2002). However, all of the associated diseases are likely polygenic and IL1RN allele A2 (IL1RN*2) represents only one of many genes that may predispose to a disease. Furthermore, ILIRN*2 may be more linked with the severity of the disease, and the association may not actually be with the IL-1Ra gene itself, but with another gene in close linkage disequilibrium. An association of IL1RN*2 with an increased prevalence of ulcerative colitis, but not always with regional enteritis or Crohn's disease, was described in some, but not all population groups (Mansfield et al., 1994; Andus et al., 1997; Roussomoustakaki et al., 1997; Bouma et al., 1999; Tountas et al., 1999; Craggs et al., 2001; Ishizuka et al., 2001). The association of IL1RN*2 with ulcerative colitis has been found in Americanbased Hispanic and Jewish populations, but the disease may be more heterogeneous in Northern European ethnic groups. However, in many of these reported studies, the number of subjects may have been too small to uncover a relatively weak association with IL1RN*2. A meta-analysis of eight European studies showed a significant association between carriage of allele 2 and UC with an odds ratio of 1.23 (Carter et al., 2001). IBD patients with IL-1Ra allele 2 demonstrated a decreased mucosal concentration of IL-1Ra protein (Andus et al., 1997; Carter et al., 1998). IL1RN*2 is also associated with some skin diseases, including the severity of alopecia areata (Tarlow et al., 1994), lichen sclerosis (Clay et al., 1994), and early onset psoriasis (Tarlow et al., 1997). IL1RN 2 was associated with multiple sclerosis in Dutch, Spanish and Italian patients (Crusius et al., 1995; de la Concha et al., 1997; Sciacca et al., 1999), particularly with a more aggressive disease (Schrijver et al., 1999). However, no association of IL1RN*2 with MS was demonstrated in French, American, Japanese or Finnish patients (Semana et al., 1997; Kantarci et al., 2000; Luomala et al., 2001; Niino et al., 2001). An association of IL1RN*2 was also described with HenochSch6nlein nephritis (Liu et al., 1997), idiopathic recurrent miscarriage (Unified et al., 2001), and a reduced risk for duodenal ulcer disease (Garcia-Gonzalez et al., 2001). No association with IL1RN*2 was found with endometriosis (Hsieh et al., 2001), sarcoidosis or idiopathic pulmonary fibrosis (Hutyrova et al., 2002), or with changes in serum levels of IL-1D or IL-1Ra after yellow fever vaccination (Hacker et al., 2001).

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IL-1Ra ALLELIC POLYMORPHISMS AND DISEASE The possible association of IL1RN*2 with rheumatic diseases has been explored in different population groups. In Caucasian patients in the UK, an increase in both frequency and carrier rate of IL1RN*2 was associated with systemic lupus erythematosus (SLE) (Blakemore et al., 1994). This association was strengthened with the presence of extensive disease and with discoid skin lesions. In a similar fashion, IL1RN*2 was associated with SLE in Japanese patients, particularly with photosensitive skin lesions (Suzuki et al., 1997). In a Swedish study, the presence of IL1RN*2 increased the susceptibility to SLE, and was associated with arthritis, although not with renal disease or overall severity (Tjernstrom et al., 1999). In all of these studies on IL-1Ra alleles and SLE, there was no relationship between the levels of IL-1Ra in serum and the presence or activity of disease. An association of definite Sj6gren's syndrome with IL- IRa allele A2 was described in a French study; in these patients the levels of IL-1Ra in serum were elevated, but the levels in salivary fluid were low (Perrier et al., 1998). Three studies failed to describe an association between any IL-1Ra allele and rheumatoid arthritis (Perrier et al., 1998; Cantagrel et al., 1999; Cvetkovic et al., 2002). However, IL-I~ allele 2 (+3954) was associated with more severe RA and was accompanied by decreased serum levels of IL-iRa (Buchs et al., 2000). IL1RN*2 was associated with juvenile chronic arthritis in Czech and Turkish patients, particular with patients exhibiting an extended oligoarticular course (Vencovsky et al., 2001). British patients with anl~losing spondylitis exhibited a significant increase in the carriage of IL1RN*2 compared with local controls (odds ratio 2.3) (McGarry et al., 2001). Lastly, IL1RN*I, but nor IL1RN*2, was found to be associated with juvenile idiopathic inflammatory myopathies in Caucasians, with an odds ratio of 2.5 (Rider et al., 2000). Additional studies on the association between IL-IRa, IL-1 and disease have emphasized the importance of the ratio between these opposing cytokines. Linkage disequilibrium was demonstrated between IL1RN*2 and an IL-I~-31T diallelic polymorphism that enhances IL-I~ production (Santtila et al., 1998; E1-Omar et al., 2000). This genetic combination was associated with hypochlorhydria and gastric cancer (E1-Omar et al., 2000). Furthermore, either IL1RN*2

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alone or in association with an IL-l[3 allele has been associated with diabetic nephropathy (Blakemore etal., 1996), susceptibility to sepsis (Fang et al., 1999), IgA nephropathy (Shu et al., 2000; Syrjanen et al., 2002), early-onset periodontitis (Parkhill et al., 2000), bronchial asthma (Mao et al., 2000), fibrosing alveolitis (Whyte et al., 2000), silicosis (Yucesoy et al., 2001), and severity of acute graft-versus-host disease in bone marrow transplant patients (Cullup et al., 2001). The mechanism of IL1RN*2 association with this variety of diseases, primarily of epithelial cell origin, remains unclear, but probably relates to a change in the ratio of IL-1Ra to IL-1[3. In early studies, increased secretion of sIL-iRa, but not levels of cellassociated icIL-1Ra isoforms, were observed in cytokine-stimulated monocytes from IL1RN*2 normal donors (Danis et al., 1995). However, the results of more recent studies have not substantiated these findings as total IL-IRa production, both secreted and cell-associated, was decreased in resting or stimulated monocytes from either normal donors or patients with ulcerative colitis each carrying the IL1RN*2 allele (Tountas et al., 1999). Studies in collagen-induced arthritis in mice, an animal model resembling rheumatoid arthritis, indicated that the disease activity subsided commensurate with a large increase in production of icIL-1Ral in the joint tissue and a marked decrease in IL-1 production (Gabay et al., 2001b). This finding supported the concept that disease suppression was possibly secondary to an increase in the local ratio of icIL- 1Ral to IL- 1. The possibility exists that an association between IL1RN*2 and disease is not related to changes in production of sIL-iRa by monocytes and other cells, but to decreased production of icIL-1Ral by epithelial and endothelial cells. This hypothesis is supported by the observations from many laboratories that icIL-1Ral levels are low in the intestinal lining of IL1RN*2 patients with ulcerative colitis. In addition, a genetic deletion in IL-1Ra production predisposes to the spontaneous development of arteritis (Nicklin et al., 2000) or arthritis (Horai et al., 2000), in different inbred strains of mice. These organs are rich in endothelial cells or fibroblasts where icIL-1Ral is the major isoform produced. The importance of decreased icIL-1Ral production in predisposition to disease is further supported by studies describing an association between IL1RN*2 and single-vessel

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coronary artery disease (Francis et al., 1999). Only iclL-1Ral mRNA, not sIL-1Ra mRNA, was demonstrated in different types of endothelial cells both in vivo and in vitro (Dewberry et al., 2000). Most importantly, cultured h u m a n umbilical vein endothelial cells from individuals carrying the IL1RN*2 allele produced less iclL-1Ral mRNA and protein in comparison with cells from donors possessing the other IL1RN alleles (Dewberry et al., 2000). However, further work is necessary to prove the hypothesis that the association of IL1RN*2 with the reported variety of h u m a n diseases is secondary to decreased production of icIL- 1Ral and to a reversal in the local ratio of total IL- i r a to IL- 1.

injected intravenously or into a body cavity such as the joint (Granowitz et al., 1992; Barrera et al., 2000). Subcutaneous injection provides a more sustained release, because the extracellular matrix of the dermis captures the IL-iRa and releases it progressively over the course of several hours (Campion et al., 1996); this is the route of administration of IL-1Ra in the treatment of RA. These issues are of particular concern to the therapeutic adminstration of IL-1Ra, because it needs to be present in a very large molar excess over IL-1 to suppress the latter's biological properties (Mend et al., 1998). As discussed below, transfer of the IL-iRa gene offers considerable advantages as a delivery strategy for achieving high, sustained concentrations of IL-1Ra within the body (Evans and Robbins, 1994).

POTENTIAL THERAPEUTIC USES OF IL- 1Ra TREATMENT OF ANIMAL MODELS OF DISEASE WITH IL-1Ra

Conceptual issues IL-1Ra has obvious therapeutic potential in conditions where dysregulated IL-1 activity is a key pathophysiological mechanism. A variety of inflammatory and degenerative diseases falls into this category, as do the sequelae of acute trauma. As discussed below, impressive data from a wide range of different animal models of disease support this conclusion. Nevertheless, application to h u m a n disease has been questioned on two main fronts. The first concern draws attention to the complexity of cytokine interactions in h u m a n diseases, where large numbers of interacting cytokines, with overlapping, pleiotrophic properties, appear to offer substantial redundancy and resistance to simple therapeutic manipulation. These circumstances led to the view that there was little therapeutic benefit to be gained by blocking the activities of a single cytokine, as other cytokines within the system would compensate. The recent, remarkable success of TNF-a antagonists in treating RA and Crohn's disease (Feldmann et al., 2001; Emery and Buch, 2002) has provided a powerful argument against this opinion, and has reinvigorated the development of cytokine antagonists as drugs. The second concern is delivery. Like other proteins, IL- i r a does not lend itself readily to convenient, longterm administration. It cannot be taken orally, and has a biological half-life of less than an hour when

Table 28.3 lists those animal models of disease that respond to recombinant IL-1Ra. A wide range of inflammatory, allergic and degenerative conditions, as well as responses to trauma, ischemia/reperfusion and injury, are represented. In most cases the mop TABLE 28.3 A n i m a l models o f diseases a n d disorders successfully treated with IL-1Ra Sepsis a Rheumatoid arthritis a Osteoarthritis Lupus (specific symptoms in certain models) Osteoporosis Traumatic, ischemic and excitotic brain injury Allergic reactions: Contact dermatitis Asthma Intestinal anaphylaxis Lung fibrosis Atherosclerosis Glomerulonephritis Colitis and other inflammatory bowel conditions Pancreatitis Diabetes Graft-versus-host disease a Allograft responses Pain a

Has been tested in human clinical trials.

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ecule has been administered by frequent injection, or by the insertion of a pumping device that ensures constant delivery.

Sepsis IL-1 is a major secondary mediator of the effects of endotoxins released during sepsis. Considerable research confirms the protective effect of IL-1Ra in experimental sepsis in a range of laboratory animals, including rats, rabbits, piglets and baboons. In septic rats, a variety of physiological disturbances are normalized by the intravenous infusion ofIL- 1Ra, including bradycardia, hypothermia, hypotension and arteriole vasoconstriction, leading to reduced mortality (Alexander et al., 1992). Various cellular and biochemical sequelae of sepsis are also responsive to IL-1Ra, including increased muscle protein breakdown, decreased muscle synthesis, altered IGF-1 concentrations in blood, liver, kidney and skeletal muscle, altered IGF-1 binding protein-1 in blood and liver, structural changes in the aortic endothelium, and elevated circulating TNF (Norman et al., 1995; Lang et al., 1996). However, the effects of IL-1Ra are not uniform in all cases. For instance, in rats treated with E. coli endotoxin, IL-1Ra attenuated synthesis of TNF-R, but not of nitric oxide (Norman et al., 1995). In a separate study, IL-1Ra was found to prevent the increase of IGF-1 binding protein-1 in blood and liver, but not muscle (Lang et al., 1996). Likewise, in rats with an abdominal abscess, IL-1Ra did not restore hepatic protein synthesis, despite doing so in the kidney and small intestine (Cooney et al., 1996). Moreover, in the latter it did so only in the seromuscular layer. In newborn rats infected with Klebsiella p n e u m o n i a , IL-1Ra reduced or enhanced mortality, depending on the dose and duration of IL-1Ra administration (Mancilla et al., 1993). In rabbits injected with Staphylococcus epidermidis, IL-1Ra inhibited the fall in mean arterial pressure and systemic vascular resistance, and the increase in circulating TNF and IL-1. However, leukopenia and thrombocytopenia were unaffected. Circulating levels of TNF and IL-1 were not responsive to IL-1Ra in rabbits infused with E. coli, but blood pressure was preserved and lethality reduced (Wakabayashi et al., 1991). In baboons treated with a LD 100 of live E. coli, infusion of IL-1Ra attenuated the decrease in mean

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arterial blood pressure and cardiac output, and improved survival. IL-1Ra also reduced circulating levels of IL-1 and IL-6, but not TNF (Fischer et al., 1992). A related study demonstrated that IL-1Ra also reduced levels of thrombin, plasminogen activator inhibitor, and neutrophil elastase in septic baboons (Jansen et al., 1995). As discussed below, IL-1Ra has been evaluated in the treatment of sepsis in humans.

Arthritis and autoimmune diseases Animal models of RA are the most comprehensively investigated in the present context, and have led to the use of IL-1Ra to treat human RA. The results from these studies reveal interesting variations in the responses of different pathophysiological processes occurring within a single disease entity. IL-1 is thought to play an important role in both inflammatory and erosive events in RA. In almost all animal models of RA yet tested (e.g. Kuiper, 1998; Bendele, 1999, 2000; Joosten, 1996), administration of IL-1Ra strongly protects the articular cartilage from destruction, and is more powerful than TNF~ antagonists in this regard. However, the effects of IL-1Ra on inflammation and bone loss depend upon the disease model. For example, a weak antiinflammatory effect has been noted in antigen-induced arthritis in mice and rabbits, and adjuvant-induced arthritis in rats, despite almost complete protection of the articular cartilage (van de Loo et al., 1995; Otani et al., 1996). However, IL-1Ra has marked antiinflammatory properties in collagen-induced arthritis and streptococcal wall-induced arthritis in rats and mice (Joosten et al., 1996; Makarov et al., 1996; Bendele et al., 1999). Of interest is the observation that IL-1Ra had powerful anti-fibrotic properties in the knee joints of rabbits with antigen-induced arthritis (Lewthwaite et al., 1995). This agrees with the strong anti-fibrotic effects noted for IL-1Ra in experimental lung fibrosis (Piguet et al., 1993) and indicates a pathology against which IL-1Ra might be particularly effective. Because of the short biological half-life of injected IL-IRa and the substantial amounts needed to inhibit strongly the biological actions of IL-1, the most dramatic anti-arthritic effects are obtained with continuous administration; implantable pumps are frequently used for this purpose (e.g. van de Loo, 1995;

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Bendele, 1999). Data from experiments using rats and mice suggest that sustained IL-iRa serum concentrations in excess of 1 ~g/ml are necessary for a maximal anti-arthritic effect (van de Loo et al., 1995; Bendele et al., 1999). It is interesting that in a model such as collagen-induced arthritis, where high doses of IL-1Ra block both inflammation and erosion, a suboptimal dose of IL-iRa retains its anti-erosive properties, while failing to control inflammation (Bendele et al., 1999). Such data reinforce the conclusion that IL- 1 is a very important mediator of cartilage destruction in arthritic joints, but contributes less critically to articular inflammation. These pre-clinical findings are important, because a single, subcutaneous injection of IL-1Ra in humans does not sustain a serum concentration of IL-iRa above 1 ~g/ml for a full 24 h (Bendele et al., 1999). Data from rat studies further suggest that sub-optimal doses of TNF-a soluble receptors and IL-1Ra act synergistically in controlling collagen-induced and adjuvant arthritis (Bendele et al., 2000). Repeated, intraarticular injection of IL-1Ra also inhibits the development of OA in the canine, anterior cruciate transection model (Pelletier et al., 1997). This effect is consistent with a body of data from the study of experimental animals and human biopsy material suggesting that IL- 1 is an important mediator of cartilage loss in OA (Pelletier et al., 2001). IL-IRa has also beneficial effects in murine models of systemic lupus erythematosus. In NZB/W F1 mice, that spontaneously develop lupus-like symptoms, multiple, intraperitoneal injections ofIL-iRa at 100 ~g per mouse prevented the development of nephritis, reducing kidney damage and proteinuria (Sun et al., 1997). Serum levels of IL-1 were also reduced. However, IL-1Ra had no therapeutic effect in established disease in MRL+ / + mice (Kiberd and Stadnyk, 1995). In experimental autoimmune encephalomyelitis, a model of multiple sclerosis, IL-1Ra delayed disease onset, reduced the severity of paralysis and weight loss, and shortened the duration of the disease (Martin and Near, 1995). The mechanism may involve an influence on the activation and proliferation of encephalitogenic cells (Badovinac et al., 1998). Sustained administration of IL-1Ra using an osmotic pump protected against hyperglycemia and insulitis in a mouse model of diabetes (Sandberg, 1994). In a different approach related to the treatment of dia-

betes, IL-1Ra prolongs mouse islet allograft survival (Sandberg et al., 1993).

Nervous system IL-iRa is a powerful neuroprotective agent in animal models of brain injury, whether damage is traumatic, ischemic or excitotic (Relton and Rothwell, 1992). In seizures, neuronal cell death results from persistent stimulation of N-methyl-D-aspartate receptors, a process that is enhanced by IL-1. Intrahippocampal application of IL-IRa in mice strongly inhibits behavioral and EEG seizures induced by bicuculline (Vezzani et al., 2000). Intravenous injection of IL-1Ra also increases the survival time of rats exposed to heat stroke (Lin et al., 1995). Furthermore, continuous intravenous infusion of IL-IRa after the onset of heat stroke dramatically enhances resistance to further development of heat stroke (Chiu et al., 1996). Death from heatstroke is associated with neuronal cell death secondary to cerebral ischemia. Several independent studies have confirmed that IL-1Ra protects against neuronal cell death during cerebral ischemia (e.g. Garcia, 1995). Intracranial administration of IL-1Ra also attenuates neuronal death after trauma, and maintains cognitive function (Sanderson et al., 1999). In certain animal models, IL- iRa inhibits the hyperalgesia that accompanies inflammation. Thus in rats, IL-1Ra inhibits hyperalgesic responses to LPS, carrageenin, bradykinin, TNF and IL-1, but not those to IL-8, PGE 2, and dopamine (Cunha et al., 2000). In mice, IL-1Ra inhibits the nociceptive response to intraperitoneal injection of acetic acid. Intrathecal delivery of IL-1Ra in combination with soluble TNF receptor inhibits pain in a rat model of neuropathic pain (Sweitzer et al., 2001). Administration of IL- 1Ra also influences other aspects of neuronal function. For example, injection of 25 ng of IL-1Ra into the hypothalamus of rats increases food intake in tumor bearing rats (Laviano et al., 2000). This is of relevance to cancer-related anorexia.

Skin Local injection of IL-1Ra into sensitized mice inhibits contact hypersensitivity, as measured by ear swelling. At the highest intradermal dose of 100 ~tg per ear, swelling was reduced by 43% and both edema and the

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degree of inflammatory cellular infiltration were reduced. The sensitization and elicitation phases of contact hypersensitivity were also inhibited, but there was no effect on the non-specific inflammation elicited by phenol (Kondo et al., 1995).

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in this regard. TNF-R soluble receptors, in contrast, inhibited fatty streak formation in female, but not male, mice (Elhage et al., 1998). Nevertheless, IL-1Ra was unable to prevent the development of aortic abdominal aneurysms in rat model, unlike TNF-~ soluble receptors (Hingorani et al., 1998)

Lung A 50 ~tg bolus of aerosolized IL- 1Ra protects sensitized guinea pigs from bronchial hyperreactivity and pulmonary eosinophilia following antigen challenge (Watson et al., 1993). This observation suggests the possible utility of IL-1Ra in the treatment of asthma. Intraperitoneal administration of IL-1Ra is also effective in a guinea pig pulmonary hyperreactivity model (Selig and Tocker, 1992). IL-1Ra also reduces allergic reactions in the gastrointestinal tract and in the eye (Theodorou et al., 1993). IL-1 is known to promote the synthesis of type I collagen by fibroblasts. Consistent with this property, IL-1Ra protects mice from the pulmonary fibrosis that accompanies intratracheal administration of bleomycin or silica (Piguet et al., 1993). Not only does the continuous intraperitoneal infusion of IL-1Ra inhibit the development of fibrosis, but surprisingly, it also reduces the pulmonary hydroxyproline content in established fibrosis. IL- 1Ra treatment has also been evaluated in three different rat models of chronic pulmonary hypertension induced by adminstration of monocrotaline, inflammation, or hypoxia equivalent to that experienced at an altitude of 16 000 feet. Hypertension provoked by monocrotaline or inflammation, but not hypoxia, is reduced by IL- 1Ra (Voelkel et al., 1994).

Cardiovascular disease IL- 1 has been implicated in the development of atherosclerosis. Mice deficient in the apolipoprotein E gene develop atherosclerotic lesions spontaneously. IL-1Ra was administered to these mice at a dose of 25 mg kg -1 day -1 using an osmotic pump implanted in a dermal subcutaneous pocket (Elhage et al., 1998). This produced serum levels of over 2 ~tg IL-1Ra/ml in females and over 1 ~g/ml in males. These concentrations of circulating IL-1Ra dramatically reduced the formation of fatty streaks in the aortic sinus. IL-1Ra was as effective as the positive control, estradiol-1713

Kidney diseases Several animal models of antibody-mediated glomerulonephritis are responsive to treatment with IL-1Ra. In a model of crescentic glomerulonephritis induced by the adminstration of anti-glomerular basement mambrane antibodies, rats with established disease responded to constant infusion of IL-1Ra. Glomerular cell proliferation, crescent formation, glomerular sclerosis, tubular atrophy and interstitial fibrosis were all suppressed, with an impressive recovery of normal renal function (e.g. Lan et al., 1995). Protection was also seen in a spontaneously occurring IgA nephropathy in mice (Chen et al., 1997), but only modest effects occurred in rat anti-Thy-1 nephritis (Tesch et al., 1997). Nephritis is a prominent feature in murine models of lupus. In NZB/W F1 mice, intermittent intraperitoneal injection of IL-1Ra reduces renal damage and proteinuria, as well as normalizing certain aspects of immune function. Serum levels of TNF and IL-6, however, are not affected (Sun et al., 1997). Established nephritis in the MRL+/+ mouse, however, does not respond to continuous, intraperitoneal infusion of IL- 1Ra (Kiberd and Stadnyk, 1995).

Osteoporosis There is increasing evidence that the loss of bone associated with menopause is driven by IL-1 and other cytokines. Intravenous infusion of IL-1Ra strongly reduces bone loss in ovariectomized rats and mice (Kimble et al., 1994b; Kitazawa et al., 1994). Of interest is the observation that the protective effect of IL-1Ra persisted for several weeks after discontinuation of the treatment (Kimble et al., 1994a). We have observed a similar effect following IL-1Ra gene therapy in ovariectomized animals (Baltzer et al., 2001) (see below). The reason for this is not known. IL-1Ra may also help to regulate strain-induced remodeling of bone. In one study using gerbils, IL- 1Ra

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inhibited resorption, while having no significant effects on rates of bone apposition. Inhibition of resorption appeared to be related to a decrease in osteoclast cell surface area in response to IL-1Ra (Chole et al., 1995). Bone destruction by a multiple myeloma cell line in mice, in contrast, was not affected by IL-IRa (Ferguson et al., 2002).

Gastrointestinal tract Inflammatory conditions in various compartments of the gastrointestinal tract are responsive to treatment with IL-IRa. In a dose-related manner, the severity of experimentally induced colitis in both rats and rabbits is markedly reduced by administration of IL-1Ra (Thomas et al., 1991; Cominelli et al., 1992; Ferretti et al., 1994). This molecule also attenuates experimental pancreatitis in mice and rats, and reduces mortality (Norman, 1995). IL-1Ra also prevents the colonic motor and secretory changes induced by intestinal anaphylaxis in guinea pigs, suggesting a possible therapeutic role in food hypersensitivity (Thomas et al., 1991). There is also evidence that IL-1Ra may be of use in the treatment of hepatic inflammation secondary to amyloidosis (Grehan et al., 1997). IL-1Ra reduces liver injury and mortalityfollowing hepatic ischemia/reperfusion injury in the rat.

Diabetes Continuous infusion of IL-1Ra prevents the development of hyperglycemia and insulitis in mice treated with streptozotocin (Sandberg et al., 1994). Once the IL-1Ra is withdrawn, the disease returns. IL-1Ra has also been shown to prolong the survival and function of pancreatic islets after transplantation in mice (Sandberg et al., 1993).

Transplantation The influence of exogenously supplied IL-1Ra in the context of transplantation has been most comprehensively studied in GVHD. Early experiments demonstrated the ability of IL-1Ra to reduce the immunosuppression and mortality of GVHD in mice, without impairing engraftment of hematopoietic stem cells (McCarthy et al., 1991). Later research,

however, found that although IL-1Ra reduced the severity of GVHD in mice where the recipient and donors had only minor histocompatibility mismatch, there was no effect on disease in the presence of a major histocompatibility mismatch (Vallera et al., 1995). As discussed below, clinical trials of IL-1Ra in patients with GVHD have been carried out. IL-IRa has also been evaluated experimentally as a way of improving the outcome of solid organ transplants. In a murine corneal transplant model, topical application of IL-iRa improves the survival of corneal allotransplants (Dana et al., 1997). This is associated with reduced corneal inflammation, and attenuated infiltration of the graft by Langerhans cells. In subsequent studies, IL-1Ra did not alter the accelerated rejection that occurs in presensitized animals (Dekaris et al., 1999), and did not induce tolerance (Yamada et al., 2000). Nevertheless, in a murine model it sustains ocular immune privilege through a suppressive effect on Langerhans cell function. Locally applied IL-iRa also inhibits the post-operative inflammation that accompanies intraocular lens implantation (Nishi et al., 1994). IL-IRa prolongs the survival of cardiac allografts in rats, and is particularly effective when used in conjunction with cyclosporin. Survival of the allografts is associated with strongly reduced leukocytic infiltration (Shiraishi et al., 1995). IL-1Ra also prolongs the survival of pancreatic islet allografts in mice and prevents recurrence of diabetes in these animals (Sandberg et al., 1993, 1997).

HUMAN TRIALS WITH IL-1Ra Sepsis The first clinical studies were conducted in patients with sepsis. As described above, studies in mice, rats, rabbits and baboons with septic shock confirmed that administration of recombinant IL-1Ra dramatically reduced mortality. Because of the acute nature of the disease, it was possible to administer IL-1Ra to patients by continuous intravenous infusion. During the phase I component of this study, IL-1Ra was infused into both patients and healthy volunteers to establish safety and dosing. This conclusively established the remarkably acute safety of IL-1Ra. Over

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the course of a 3-day infusion, certain groups of healthy volunteers received tens of grams of recombinant IL-1Ra without any change in the various physiological parameters and blood chemistries that were monitored (Granowitz et al., 1992). Whether IL-1Ra will prove to be equally safe upon chronic administration will become clear from the results of its use in RA. In a subsequent, phase II, multicenter trial, recombinant IL-1Ra or placebo was infused intravenously for 3 days into 99 patients with sepsis syndrome or septic shock (Fisher et al., 1994b). Patients in the treatment arm were administered an intravenous loading dose of 100 mg IL-1Ra, and then one of three different doses: 17, 67 or 133 mg h -1. Patients were evaluated for 28-day mortality. There was 44% mortality in the placebo group. Mortality was reduced to 32% among patients receiving the lowest dose of IL-1Ra, 25% among the middle dose group, and 16% in patients receiving the highest dose. These differences were statistically significant (Fisher et al., 1994b). In a phase clinical III trial, a total of 893 individuals with sepsis syndrome took part in a randomized, double-blind, placebo-controlled, multicenter, multinational trial (Fisher et al., 1994a). As in the phase II trial, patients were loaded with 100 mg IL-1Ra or placebo. They then received 72-h infusion of 1 or 2 mg kg -~ IL-1Ra or placebo. The 28-day incidence of mortality was again used as the end-point. This study failed to demonstrate a statistically significant increase in survival time for the IL-1Ra-treated patients (Fisher et al., 1994a). Secondary and retrospective analyses of efficacy suggested that IL-Ra did increase survival in those patients who entered the study with the most severe disease (predicted risk of mortality of 24% or greater) (Knaus et al., 1996). In a second phase III study involving 696 patients with severe sepsis or septic shock, patients received standard supportive care and antimicrobial therapy in addition to IL-1Ra or placebo (Opal et al., 1997). IL-IRa was delivered as a 100 mg intravenous loading dose, followed by infusion of 2 mg kg -1 h -~ for 72 h. Although 28-day mortality was the primary outcome measure, the study was stopped after an interim analysis showed that it was unlikely that this outcome would be met (Opal et al., 1997). No further clinical development of IL-1Ra for treatment of sepsis has occurred.

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Graft-versus-host disease There have been two clinical trials to evaluate the safety and efficacy of IL-1Ra in graft-versus-host disease. In a small, phase I/II open label trial, 17 patients with steroid-resistant GVHD received a continuous infusion of 400-3200 mg day -1 IL-1Ra over 7 days (Antin et al., 1994). Stage-specific improvement occurred in the skin, gut and liver. Overall, acute GVHD improved by at least one grade in 63% of patients, associated with a decrease in TNF-a mRNA levels in blood mononuclear cells. However, of the 17 patients treated, five died (Antin et al., 1994). IL-1Ra treatment was further evaluated in a doubleblind, placebo-controlled, randomized trial of aUogeneic stem cell transplantation in 186 patients. Patients received either saline placebo or IL-1Ra 0.5 mg kg -1 h -1 by continuous intravenous infusion from 4 days prior to 10 days after the transplantation. The results indicated no effect of IL-1Ra on the incidence of GVHD or on mortality (Antin et al., 1999).

Rheumatoid arthritis In an initial dose-ranging study, Campion et al. enrolled 175 patients with RA into a randomized, double-blind trial of IL-iRa administered by subcutaneous injection (Campion et al., 1996). During an initial 3-week phase, patients were treated with 20, 70 or 200 mg IL-iRa by subcutaneous injection once, three times or seven times per week. This was followed by a 4-week maintenance phase during which patients received IL-1Ra or placebo on a daily basis. Background NSAIDs or corticosteroids were permitted. IL-iRa was well tolerated, with adverse injection site reactions being the most c o m m o n side-effect. These were severe enough to cause 5% of patients to withdraw from the study. Insofar as it was possible to tell, daily dosing appeared to provide greater efficacy than weekly dosing (Campion et al., 1996). Under the name Anakinra, IL-1Ra has been evaluated in 472 European patients with active and severe RA of between 6 months and 8 years duration, recruited into a 24-week, double-blind, randomized, placebo-controlled, multicenter study (Bresnihan et al., 1998). Patients were randomized into one offour groups receiving placebo, or 30, 75 or 150 mg IL-1Ra/ day self-administered by subcutaneous injection.

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DMARDs were prohibited during the study, but continuation of NSAIDs and corticosteroids was permitted. The primary outcome measure for this study was an ACR 20 response that was achieved by 27% of the placebo group and 43% of patients receiving the highest dose of IL-1Ra ( P - 0.014). Statistically significant improvement was recorded for several indices of disease, including number of swollen joints, number of tender joints, Health Assessment Questionnaire scores, erythrocyte sedimentation rate, and levels of C-reactive protein. Clinical responses occurred after 2 weeks of therapy. Radiologic damage assessed by Genant's modification of Sharp's grading system, revealed a 58% reduction in the rate of joint space narrowing, and a 38% reduction in the rate of erosions (]iang et al., 2000). Synovial biopsy, performed in a small number of participants, showed that Anakinra reduced the numbers of intimal layer macrophages and subintimal macrophages and lymphocytes (Cunnane et al., 2001). This may reflect the down-regulation of various adhesion molecules. Those patients who experienced arrest of radiologic disease progression were those with the largest decline in macrophage infiltration. The initial study was followed by a further, 24-week extension phase in which 76 participants who had formerly received placebo were randomized among the three treatment groups (Bresnihan, 2001). Patients who had previously received Anakinra continued treatment at their previous levels. A total of 309 patients completed the extension phase of the study. Of the patients who had previously received placebo, 71% ofthe group now receiving IL- 1Ra at 150 mg day -1 achieved anACR 20 response. Of the patients who were already receiving Anakinra, 49% maintained an ACR 20 response. Radiologic assessment of patients in the extension study confirmed that the reduction in the rates of joint space narrowing and erosion was sustained during the second 24-week period. In a separate recent study, 419 RA patients on maintenance doses of methotrexate for at least 6 months were randomized into six groups receiving 0, 0.04, 0.1, 0.4, 1.0 or 2.0 mg Anakinra kg -1 day -1 as a subcutaneous injection (Cohen et aL, 2002). After 12 weeks, 19% of the placebo group had achieved an ACR 20 response. Forty-six percent of the group receiving 1 mg k g -1 d a y -1 Anakinra achieved an ACR 20 response, and 38% of those receiving 2 mg k g -1 d a y -1

did so. These improvements occurred after 2-4 weeks. Moreover, 24% of patients in the highest dose groups achieved an ACR 50 response, compared with 4% of the control group; 10% of the high-dose patients achieved an ACR 70 response. Monitoring of subjects in these trials indicated that Anakinra is safe. Injection site reactions were the most common side-effects, and occurred in 81% of individuals receiving the highest dose. Such reactions were usually mild and transient, but resulted in approximately 5-10% of participants withdrawing from the study. The FDA recently approved Anakinra, under the trade name Kineret, for the treatment of moderate to severe RA in adults who have failed one or more DMARDs. Kineret may be used alone, or in combination with DMARDs other than the TNF-a antagonists. Collectively, these data suggest that IL-1Ra holds promise in the treatment of patients with RA. However, the response is modest and much weaker than that seen in experimental animals subjected to continuous infusion with IL-iRa. Of relevance is the likelihood that, despite daily self-injection of Anakinra at high concentrations, IL-1Ra levels did not remain high enough for long enough within the study subjects. Daily subcutaneous dosing at the highest level yet tested (150 mg), may fail to provide an anti-erosive dose of IL-1Ra for a substantial part of the day, and may barely achieve an anti-inflammatory dose at any time (Bendele et al., 1999). This could well explain the modest effects of Anakinra in clinical trials, and highlights the potential advantage of using gene transfer to achieve sustained, endogenous 24-h production of therapeutic quantities of IL-1Ra (Evans and Robbins, 1994).

GENE THERAPY WITH IL- 1Ra General principles Athough IL-1Ra has therapeutic potential in numerous diseases, its usefulness as a drug is limited by several biological factors. These include its oral unavailability, its short biological half-life, and the need to maintain a large molar excess of IL-1Ra over IL-1 at its sites of action. A gene transfer approach to the administration of IL-1Ra arose in response to these challenges (Evans and Robbins, 1994). The basic

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strategy is to transfer a cDNA encoding IL-1Ra to the appropriate organ, so that it becomes a sustained, endogenous source of therapeutic quantities of IL- iRa. Depending upon the vector system employed, and the physiology of the target cells, it is possible to achieve prolonged IL-1Ra gene expression, potentially in a regulated fashion. There is also the possibility that the native molecule synthesized from the transgene has superior properties to the recombinant protein produced in bacteria. The general principles of gene transfer have been described in Chapter 59 by Paul Robbins, Walter Storkus and Andrea Gambotto, and will not be repeated here. For present purposes, it is sufficient to note that various viral and non-viral vectors can be used to transfer genes by direct, in vivo or indirect, ex vivo administration (Ghivizzani et al., 2001). Recombinant adenoviruses are most commonly used in expriments requiring in vivo delivery, and recombinant retroviruses are most commonly used for ex vivo delivery. Non-viral vectors, such as plasmid DNA and DNA-liposome complexes, are typically used in an in vivo fashion. Generally speaking, viral transgene delivery is more efficient than non-viral delivery. This makes viral vectors particularly useful in experimental work with animals, but they raise greater safety issues when human application is envisaged. Present vector technology permits the efficient transfer and high in vivo expression of transgenes in many organs. However, it is not always possible to achieve long-term gene expression, which is a limitation for the treatment of chronic conditions. Technologies permitting close regulation of the level of transgene expression are under development.

IL-1Ra GENE TRANSFER IN ANIMAL MODELS OF DISEASE Arthritis IL-1Ra gene therapy experiments were first attempted in the context of RA (Evans et al., 1999). Two approaches have been evaluated. In so-called systemic delivery, the IL-1Ra that is synthesized as a result of gene transfer gains ready access to the systemic circulation. In so-called local delivery, the

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IL-1Ra gene is transferred to sites, typically the joints, where local accumulation of the transgene product occurs. For treating RA, the former strategy has the advantage of exposing multiple joints simultaneously to the transgene product and enabling the treatment of systemic and extraarticular manifestations of disease. It has the disadvantage of exposing non-target organs to high concentrations of the transgene product and thus facilitating unwanted side-effects. Moreover, non-genetic technologies can, or will within the foreseeable future, be able to deliver proteins systemically in an equivalent manner. With local delivery, in contrast, the highest concentrations of the transgene occur locally within the joint, with minimal exposure of extraarticular organs. This is something that no other delivery system can conveniently achieve, or is likely to be able to achieve soon. There is also the possibility that IL-1Ra synthesized locally at sites of pathology within the joint has a more powerful effect than IL-1Ra which diffuses into the joint from an extraarticular location. Systemic delivery of IL-1Ra has been achieved by retroviral transfer of the gene to the hematopoietic stem cells of mice (Boggs et al., 1995). This led to lifelong circulating levels of several hundred nanograms IL-IRa per ml plasma. Although these mice were not subjected to detailed pathological scrutiny, they showed no obvious signs of distress, gained weight normally, had normal peripheral blood leukocyte profiles, lived a normal lifespan, and did not seem more prone to disease or infection. This observation agrees with the earlier safety data obtained for the recombinant protein. Most progress towards an IL-1Ra gene therapy for arthritis, however, has been made with local gene delivery to the synovium (Bandara et al., 1992). In vivo IL-1Ra gene transfer to the joints of experimental animals has been accomplished with various vectors, including adenovirus, herpes simplex virus, retrovirus, adeno-associated virus and lentivirus. In each case, levels of IL-1Ra production were sufficient to suppress experimental models of RA in these animals. It is of interest that, in several instances, the chondroprotective effects of IL-1Ra were more powerful than the anti-inflammatory effects (Ghivizzani et al., 1998), a result consistent with much of the data obtained using recombinant protein in animal models of RA.

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Despite impressive progress, the vectors used for the in vivo delivery of IL-iRa to the joints of experimental animals are not yet suitable for application in h u m a n joints. However, ex vivo IL-1Ra gene delivery using a retrovirus has been implemented in a recent phase I clinical study (Evans et al., 1996, 2000). This protocol stemmed from a considerable body of work using the rabbit knee joint as the model system. A process was developed whereby a synovial biopsy was surgically recovered from the rabbit and used as a source of autologous synovial fibroblasts (Bandara et al., 1992). These were cultured in vitro, transduced with a retrovirus carrying the IL-1Ra cDNA, and returned to the knees of the individual donor animals by intraarticular injection. The injected cells engrafted in the recipient synovium and continued to produce elevated amounts of IL-1Ra, at a declining rate, for several weeks (Bandara et al., 1993). The amounts produced were sufficient to suppress antigen-induced arthritis in rabbits, with a particularly impressive protective effect on the articular cartilage (Hung et al., 1994). Similar protocols have been applied successfully to streptococcal cell wallinduced arthritis in rats and zymosan- and collageninduced arthritis in mice (Makarov et al., 1996; Bakker et al., 1997). As a demonstration of the advantages of gene therapy, Makarov et al. (1996) have calculated that local, ex vivo delivery of the IL-1Ra gene is 104-fold more potent than the recombinant protein in treating streptococcal cell wall-induced arthritis in rats. In agreement with this, we have noted a strong anti-arthritic effect of the IL-1Ra gene in rabbit antigen-induced arthritis (Otani et al., 1996; Ghivizzani et al., 1998), whereas Lewthwaite et al. found little effect of recombinant IL-iRa (Lewthwaite et al., 1994), even though comparable intraarticular concentrations of human IL-iRa accumulated in the knee joints. Extensive testing has confirmed that the ex vivo delivery of the IL-iRa gene to the synovium is safe, and there is no gene transfer to the germ-line (Evans et al., 1996). Moreover, studies in SCID mice have confirmed that delivery of the IL-1Ra gene to h u m a n synoviocytes inhibits chondrocytic chondrolysis in h u m a n cartilage (Muller-Ladner et al., 1997). Local, intraarticular transfer of the IL-1Ra gene to the synovium may also be of value in treating osteoarthritis (OA). Using ex vivo, retroviral gene delivery, Pelletier et al. retarded the early loss of carti-

lage that follows transection of the anterior cruciate ligament in dogs (Pelletier et al., 1997). It was not possible to determine the long-term effects of the gene therapy because of loss of gene expression. In a subsequent study, the same group reported similar results in a rabbit model of OA in which naked DNA encoding IL-1Ra was injected into the joint space (Fernandes et al., 1999). This is a remarkable result given the low level and transience of transgene expression that typically follow the intraarticular administration of plasmid vectors, as well as the inflammatory response of synovium to large amounts of naked DNA. A recent experimental study in horses has provided a convincing demonstration of the potential of IL-1Ra gene therapy in treating OA. Frisbie et al. (2002) cloned equine IL-1Ra and used an adenoviral vector to deliver it to the joints of horses with experimental OA. This experimental model mimics a condition seem commonly in race horses, where an osteochondral fragment has become dislodged and remains within the joint to provoke synovitis and loss of articular cartilage. Delivery of the equine IL-1Ra cDNA 10 weeks after induction of disease provided powerful protection of the articular cartilage. As noted in several other settings, IL-1Ra had a more modest antiinflammatory effect. Most importantly, gene therapy provided clinical improvement, evidenced by a reduction in the horses' lameness scores (Frisbie et al., 2002). In an alternative approach to therapy, the IL-1Ra gene has been transferred to articular chondrocytes (Baragi et al., 1995). Chondrocytes transduced with an adenovirus carrying the IL-1Ra cDNA were transferred on to cartilage fragments, and exposed to IL-1. Under these in vitro conditions, the underlying cartilage resisted the catabolic effects of IL-1. The IL-1Ra cDNA has also been transferred to chondrocytic cells from the end plates of the spine, as a strategy for influencing intervertebral disc disease and other conditions of the the spine (Wehling et al., 1997).

Central nervous system IL-1Ra gene transfer shows beneficial effects in animal models of brain injury resulting from trauma or ischemia and reperfusion. The inflammatory reaction to blunt trauma in the rat brain is dramatically

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reduced by the transfer of fibroblasts that have been retrovirally modified to express large amounts of IL-1Ra (DeKosky et al., 1996). In a rat model of stroke, the intraventricular, adenoviral delivery of IL-1Ra reduced cerebral infart volume by 64% (Betz et al., 1995). Equivalent results were reported with a mouse model of ischemia-reperfusion. In this case, delivery of the IL-IRa gene reduced, in addition to the infarct size, the disruption of the blood-brain barrier and expression of ICAM-1 (Yang et al., 1999).

Insulin-dependent diabetes mellitus Transfer of the IL-1Ra gene to pancreatic islets is of interest both as a means of suppressing the autoimmune destruction of these cells during type I diabetes, and as a means of improving the survival of allografts. Human islets, transduced in vitro with adenovirus or lentivirus carrying the IL-1Ra gene, were protected from IL-1 mediated nitric oxide formation, impaired glucose-stimulated insulin production, and Fas-triggered apoptosis (Giannoukakis et al., 1999). However, liposomally mediated transfer of IL-1Ra cDNA to syngeneically transplanted murine islets failed to confer resistance to islet destruction in recipient NOD mice (Saldeen et al., 2000). It is possible that the level and duration of transgene expression were too low to achieve a protective effect.

Cardiovascular system The ability of the IL-1Ra gene to influence ischemiareperfusion injury during cardiac allograft has been evaluated in a rat model. In this system, the hearts were transfected with the gene by intracoronory infusion and allografted to the abdomen of a recipient rat. While in the abdomen, the transplanted heart was subjected to 30 min of ischemia followed by 24 h of reperfusion. In the presence of the IL-1Ra gene, infarct size, neutrophil infiltration, and cardiomyocyte apoptosis were strongly inhibited (Suzuki et al., 2001). In another application, mice were inoculated intraperitoneally with encephalomyocarditis virus. Immediately afterwards, the IL-1Ra gene was electroporated into the tibialis anterior muscles of mice leading to peak circulating levels of 10.5 ng IL-1Ra

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m1-1. This procedure lowered expression of TNF-a and nitric oxide, and reduced mortality (Nakano et al., 2001).

Bone As noted above, administration of recombinant IL- 1Ra protein is able to reduce bone loss in ovariectomized rodents. Baltzer et al. have demonstrated a similar effect using the IL-1Ra gene (Baltzer et al., 2001). These investigators treated ovariectomized mice with adenovirus carrying IL-1Ra cDNA by intramedullary injection. This procedure transduced lining osteoblasts, osteocytes and marrow cells, as well as adjacent muscle and draining lymph nodes. Transgene expression persisted for approximately 12 days, but bone loss was attenuated during the entire 5-week experiment. Why the effect should persist in this manner is unknown, but a similar response had been noted with the recombinant protein (Kimble et al., 1994a). Moreover, the protective effect of the IL-1Ra gene was not limited to bones receiving the intermedullary injection, but occurred in all bones that were evaluated. Another potential use of IL-1Ra gene therapy in bone is to inhibit the aseptic loosening of prosthetic joints. This process is thought to be triggered by mediators released from periprosthetic cells in response to wear debris. Transduction of macrophage cultures with IL-1Ra cDNA inhibits the cellular responses to wear particles in vitro (our unpublished data), and also suppresses in vivo responses to wear debris in a murine air pouch model (Sud et al., 2001).

Kidneys Two methods have been employed to transfer the IL-1Ra gene to the renal glomerulus. In one ex vivo approach, cultured rat mesangial cells were stably transfected and delivered to the glomeruli of rats via the renal circulation. Responses of the recipient glomeruli to IL-1 were blunted (Yokoo and Kitamura, 1996). In a second ex vivo approach, marrow derived CDllb+CD18 § cells were transduced with a recombinant adenovirus carrying the IL-1Ra gene (Yamagishi et al., 2001; Yokoo et al., 2001). The genetically modified cells were injected intravenously into mice with

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an experimental ureteral obstruction. Expression of ICAM- 1, the counterligand for CD1 lb and CD18, increases in the renal interstitium during this disease, thus retaining the genetically modified cells and enhancing local synthesis oflL-iRa. Under these conditions, macrophage infiltration and other aspects of inflammation were reduced (Yamagishi et al., 2001; Yokoo et al., 2001). In a related application, marrow cells were transduced with a recombinant retrovirus carrying IL-1Ra and infused into lethally irradiated recipients. After successful marrow transplant, an experimental glomerulonephritis was induced in the recipient mice. In those mice receiving the IL-1Ra gene, renal function and histology were maintained and the survival rate improved (Yamagishi et al., 2001; Yokoo et al., 2001).

Lungs Recombinant retrovirus has been used to transfer the IL-1Ra gene to the lungs of sheep in utero (Pitt et al., 1995). Recombinant adenovirus has been used for the same purpose in the lungs of adult mice and pigs (McCoy et al., 1995; Morrison and Murtaugh, 2001). In mice, the IL-1Ra produced as a result of gene transfer fails to inhibit the inflammatory response to the adenovirus (McCoy et al., 1995). In pigs, however, no inflammatory response occurred (McCoy et al., 1995).

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retrovirally transferred to one half of the cells, while the remaining cells served as unmodified controls. In a double-blind fashion, one week before MCP joint replacement surgery two of these joints received the genetically modified cells and the other two received unmodified cells by intraarticular injection. Articular tissue recovered at the time of MCP joint arthroplasty was then examined for evidence of successful gene transfer and gene expression. All nine subjects completed the study without problems, and no adverse events related to the procedure were noted. Expression of the transgene was detected by RT-PCR in all joints that received it. Elevated IL-1Ra protein synthesis was also noted in tissues recovered from most of the transduced joints. In situ hybridization and immunohistochemistry confirmed the presence of engrafted, transduced cells on the recipient synovia. Certain patients reported symptomatic relief during the study, but these anecdotal observations could be attributed to a placebo effect. A similar phase I study, with an intraarticular dwell time of the IL-1Ra gene of 1 month, is under way in Germany (Evans et al., 2000). The preliminary data from the German trial are similar to those of the American trial. A phase II study to determine efficacy is being planned. Future development of a convenient and effective IL-iRa gene treatment for RA requires a vector that can be administered by direct, intraarticular injection and achieves prolonged transgene expression at therapeutic levels. Recent data suggest that novel lentiviral vectors hold promise in this regard (our unpublished data).

Arthritis IL-1Ra gene delivery to synovium ex vivo using autologous synovial fibroblasts in conjunction with a Moloney-based retroviral vector, was employed in a phase I clinical trial to determine safety and efficacy (Evans et al., 1996). Nine post-menopausal women with advanced RA were recruited to the study. Among the entry requirements was the need for surgical replacement of the 2nd-5th metacarpophalangeal (MCP) joints on one hand, and surgery on at least one other joint. The latter procedure provided the opportunity to harvest autologous synovium from which to grow synovial fibroblasts. Cultures of autologous cells were divided into two. The h u m a n IL-1Ra cDNA was

AREAS OF ACTIVE RESEARCH AND INQUIRY Role of IL-1Ra in normal physiology and in host defense The fact that IL-1Ra can be detected in normal individuals suggests a role for this molecule in normal physiology. As discussed above, there is evidence that the IL-1/IL-1Ra axis plays important roles in the normal CNS, in bone remodelling and in host defense, among others. Furthermore, the spontaneous development of arthritis or arteritis in IL-1Ra knockout

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mice, in each case on a specific genetic background, indicates that IL-1Ra in tissues plays a counterbalancing role to IL-1 in preventing disease. Possibly host responses to injury or infection, which normally would be controlled, lead to a clinical disease in the presence of a cytokine imbalance. Determining what are the other predisposing genes in these animal models of disease may clarify some of the genes and their interacting proteins in polygenic human diseases. Particularly intriguing are the roles of the intracellular isoforms of IL-ira, and how they may perform unique roles in host defense. It has been known for over a decade that, in addition to triggering signal transduction by binding to cell surface receptors, IL-1/receptor complexes can be internalized and translocated to the cell nucleus. Precursor IL-I~ also affects cell growth through movement to the nucleus without ever being externalized. Since intracellular isoforms of IL-1Ra predominate in certain cells, it is tempting to ascribe a role for icIL-1Ra in regulating the intracellular activities of IL-1. However, exactly what this role might be remains unknown.

Delivery of IL- 1Ra in therapy It is clear from the examples given above, that IL-1Ra has wide therapeutic potential in a large number of diseases. Delivery is a major impediment to developing IL-1Ra as a drug, especially in chronic conditions where therapeutic concentrations need to be sustained within the patient for an extended period. As noted, for the treatment of patients with RA, daily injections of very large amounts of recombinant protein are required. Various slow-release formulations and pumping devices are being investigated as improved delivery systems. These approaches to therapy with IL-1Ra may allow better pharmacokinetics with more sustained blood and tissue levels of the delivered agent. However, the kinetics of delivery from slow release vehicles tend to be biphasic with most of the dose liberated into the system relatively quickly, followed by a slow, non-uniform leaching of the residual protein. There is also the issue of the stability of the protein under physiological conditions, as it remains stored within the delivery device. Pumps solve the problems associated with non-uniform release, but not protein lability, and they are invasive

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and complex. Small organic molecules, which mimic the receptor blocking effects of IL-1Ra, have been sought unsuccessfully, probably because of the complex interactions between IL-1 or IL-iRa and the IL-1 receptor. As discussed above, gene therapy is also being investigated as a means of solving problems related to delivery. Moreover, as a result of gene transfer and expression, it is the native, authentically processed molecule that is produced. The biological properties of the native and recombinant forms of IL-ira have not been compared in a detailed fashion, and important differences may exist. In addition to facilitating the administration of sIL-1Ra in various diseases, gene transfer approaches hold particular promise in the delivery of the icIL-1Ral isoform, as this molecule will need to accumulate within the target cells. Although the principle of IL-ira gene transfer is now well-established, there remain the problems of tissue or cell specificity of delivery and of long-term gene expression. Considerable research into these problems is under way, with particular attention being paid to vector design, cell turnover and the immunological constraints to transgene expression. Inducible transgene expression in specific tissues has become a reality in animal systems and may become applicable to gene therapy of arthritis. This would allow prolonged and regulated expression of a transgene in arthritic joints only at times of disease activity.

Combination therapy with IL- 1Ra The biological activities of IL-1 are inhibited physiologically not only by IL-1Ra, but also by soluble forms of the IL-1 type I and type II receptors. Which of these holds the greatest therapeutic promise is a matter of importance in treating IL-1-driven diseases. Because the affinity of the soluble type I receptor for IL-1Ra exceeds that of its affinity for IL-1, it is a poor candidate, and experimental data confirm this. The soluble type II receptor, however, preferentially binds IL-1. Recent data from gene transfer experiments confirm that the type I soluble receptor is a far weaker inhibitor of the actions of IL-1 on chondrocytes than either the type II soluble receptor or sIL-1Ra. The latter are of approximately equal potency both in vitro and in a rabbit model of RA; whether they act synergistically is unknown.

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Most inflammatory and degenerative diseases are complex and involve multiple cytokines. There is consequently much interest in combining IL-1Ra with other biological agents to improve treatment. Data from many animal experiments suggest that the combination of IL- iRa with a TNF-cz antagonist is particularly powerful, but the potential side-effects of employing two such potent therapeutic agents will require thorough evaluation. Other combinations under consideration are IL-iRa plus a type-2 cytokine (e.g. IL-4, IL-10) or IL-iRa plus a traditional drug such as, in the case of RA, methotrexate. It is also possible that IL-iRa may be combined in the future treatment of RA with other new therapies with different mechanisms of action, such as enzyme inhibitors or chemokine blockers. Combinations may allow each therapeutic agent to be used at lower and potentially less toxic concentrations.

Clinical trims in additional diseases

0 FIGURE 28.2 IL- 1Ra gene transfer to human, rheumatoid, metacarpophalangeal joints. (1) Synovial tissue is removed from a non-MCP joint. Synovial cells are isolated and cultured. (2) Half the cells are transduced with a retrovirus carrying the h u m a n IL-1Ra cDNA; the other half of the cells remain as controls. (3) The majority of each population of cells is cryopreserved; aliquots are subjected to safety testing. (4) Safetytested cells are thawed, recultured and prepared for injection. Two MCP joints are injected with transduced cells and two are injected with controls, untransduced cells. (5) Seven days later, the injected joints are surgically replaced with prosthetic joints, as previously indicated for the m a n a g e m e n t of the disease. (6) Tissues retrieved at surgery are analyzed for evidence of successful IL-1Ra gene transfer and intraarticular IL-1Ra transgene expression. (Reproduced, with permission, from Evans et al. (1998). Blocking cytokines with genes. ]. L e u k o c y t e Biol. 64, 55-61.)

As described above, recombinant IL-1Ra has been subjected to clinical trims in sepsis, GVHD and RA. Only the last of these has progressed to the point of being approved by the FDA as a drug. Yet it is clear that many more conditions potentially stand to benefit from treatment with IL-1Ra, including OA and a range of inflammatory and degenerative diseases. Given the time and cost of conducting large-scale clinical studies, the challenge is to select carefully from the list of candidate diseases. The recent success of TNF-a antagonists in psoriatic arthritis, ankylosing spondylitis and juvenile chronic arthritis, suggests that IL-1 inhibition also may be efficacious in these conditions.

REFERENCES (Because of limitations of space, these references are illustrative, not comprehensive) Abrahamsen, B., Bonnevie-Nielsen, V., Ebbesen, E.N. et al. (2000a). Cytokines and bone loss in a 5-year longitudinal study- hormone replacement therapy suppresses serum soluble interleukin-6 receptor and increases interleukin1-receptor antagonist: the Danish Osteoporosis Prevention Study. ]. Bone Miner. Res. 15, 1545-1554. Abrahamsen, B., Shalhoub, V., Larson, E.K. et al. (2000b). Cytoldne RNA levels in transiliac bone biopsies from healthy early postmenopausal w o m e n . B o n e 26, 137-145.

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