IL-1Ra

IL-1Ra Danielle Burger* and Jean-Michel Dayer Division of Immunology and Allergy, Department of Internal Medicine, University Hospital, CH-1211, Geneva 14, Switzerland * corresponding author tel: 41-22-372-9376, fax: 41-22-372-9369, e-mail: [email protected] DOI: 10.1006/rwcy.2000.04002.

SUMMARY Inflammation is an important homeostatic mechanism that limits the effects of infectious agents. However, inflammation might be self-damaging and therefore has to be tightly controlled or even abolished by the organism. Interleukin 1 (IL-1) is a critical mediator of the inflammatory response, playing an important part in the development of pathologic conditions leading to chronic inflammation. Although IL-1 production may be downmodulated or its effect limited by so-called anti-inflammatory cytokines, IL-1 inflammatory effects are inhibited and can be abolished (in vitro) by one particularly powerful inhibitor, IL-1 receptor antagonist (IL-1Ra). To date, three forms of IL-1Ra have been identified, one soluble form and two intracellular forms, referred to as sIL-1Ra, icIL-1RaI, and icIL-1RaII, respectively. Although sIL-1Ra functions obviously as an IL-1 inhibitor by competitively binding to IL-1 receptor without inducing signal transduction, the functions of icIL-1RaI and icIL-1RaII remain to be determined. The three forms of IL-1Ra are transcribed from the same gene, two different promoters regulating the transcription of sIL-1Ra and icIL1RaI. In mice, sIL-1Ra is predominantly found in peripheral blood cells, the lung, spleen, and liver, while icIL-1RaI is mainly found in the skin. IL-1Ra knockout mice display growth retardation after weaning and an increased susceptibility to endotoxininduced injury and collagen-induced arthritis, suggesting that IL-1Ra plays an important part in both health and pathologic conditions. IL-1Ra is produced by hepatic cells as an acute phase protein. The circulating blood levels of sIL-1Ra are low in normal condition and elevated in individuals with allele 2 polymorphism and in several human infectious, autoimmune, and chronic inflammatory diseases, as well as

in numerous experimental models of diseases in animals. Because of its beneficial effects in many animal disease models, IL-1Ra has been used as therapeutic agent in human patients. IL-1Ra has failed to show beneficial effects in septic shock but seems to improve the condition of rheumatoid arthritis patients.

BACKGROUND Interleukin 1 receptor antagonist (IL-1Ra) is a member of the IL-1 family. Three forms of IL-1Ra have been described, two of them being expressed as intracellular proteins (icIL-1RaI and icIL-1RaII) and one being secreted (sIL-1Ra). The three forms of IL1Ra result from the same gene. The function(s) of the intracellular forms of IL-1Ra are still elusive whereas sIL-1Ra binds competitively to IL-1 receptor I without inducing signal transduction and thus inhibits IL-1 and IL-1 actions.

Discovery An IL-1 inhibitory activity was first observed in the urine of febrile patients (Balavoine et al., 1986). Subsequently this activity was further attributed to sIL-1Ra, which blocks IL-1 binding to its receptor (Seckinger et al., 1987). sIL-1Ra cDNA was cloned from a human monocyte library (Eisenberg et al., 1990). Thereafter, sIL-1Ra was found to be produced by many other cells including neutrophils, fibroblasts, corneal epithelial cells, and microglial cells. Indeed, sIL-1Ra production is inducible in most cell types. Natural sIL-1Ra is a 22±26 kDa glycoprotein (Seckinger et al., 1987; Hannum et al., 1990; Mazzei et al., 1990), whose recombinant 17 kDa form retains full IL-1-inhibitory capacity (Carter et al., 1990;

320 Danielle Burger and Jean-Michel Dayer Eisenberg et al., 1990). Since it potently inhibits the various effects of IL-1, sIL-1Ra is considered an important regulator of the inflammatory and overall immune response mediated by IL-1. sIL-1Ra was soon regarded as a potential marker of disease severity (Prieur et al., 1987) and a therapeutic tool (Seckinger et al., 1990). icIL-1RaI was also cloned from a human monocyte library (Haskill et al., 1991). Natural and recombinant icIL-1RaI binds to the IL-1 receptor I with a similar affinity to sIL-1Ra in vitro (Gruaz-Chatellard et al., 1991; Haskill et al., 1991). icIL-1RaI is constitutively expressed in keratinocytes and epithelial cells (Arend, 1993; Arend et al., 1998). Another putative intracellular form of IL-1Ra was identified by polymerase chain reaction on mRNA from human polymorphonuclear cells (Muzio et al., 1995). However, the protein product of this high molecular weight icIL-1Ra mRNA form has never been detected. More recently, a 16 kDa icIL-1Ra was detected by western blot analysis of LPS-stimulated human neutrophils, monocytes, and the human hepatoma cell line HepG2 (Gabay et al., 1997b; Malyak et al., 1998). This low molecular weight form of icIL1Ra is now referred to as icIL-1RaII. Furthermore, a variant cDNA species containing an additional 171 nucleotides within the icIL-1RaI cDNA, that interrupts the coding region, was recently described (Weissbach et al., 1998). This mRNA variant was found to be expressed in keratinocytes and human articular cartilage. Translation is likely to be initiated at an alternate Met codon other than that utilized for icIL-1RaI, suggesting that this mRNA variant encodes a novel polypeptide. The function(s) of icIL-1Ra isoforms remain(s) unclear.

Alternative names IL-1Ra was also known as IL-1 receptor antagonist protein (IRAP), but it is now currently referred to as IL-1Ra or sIL-1Ra (secreted IL-1Ra) and its intracellular forms as icIL-1RaI and icIL-1RaII.

Structure Mature human sIL-1Ra is a 152 amino acid residue protein which is generated by the cleavage of a 25 amino acid hydrophobic signal sequence from a cytoplasmic precursor of 177 amino acid residues. Although IL-1Ra, IL-1, and IL-1 genes seem to have arisen through duplication and divergence of a common ancestral gene, sIL-1Ra displays no more than 19% and 26% amino acid sequence homology with IL-1 and IL-1 , respectively. However,

sIL-1Ra sequence was conserved during evolution, displaying > 75% homology in human, rat, rabbit, mouse, bovine, and horse sIL-1Ra. sIL-1Ra interacts with the same receptors as IL-1 and IL-1 suggesting structural rather than sequence similarities between these cytokines. It has been proposed that sIL-1Ra binds to the IL-1 receptor I through one interacting domain as compared with two such domains on IL-1 and IL-1 . The lack of a receptor-interacting domain might be the cause of the lack of signal transduction upon binding of sIL1Ra to IL-1 receptor I (for review see Lennard, 1995).

Main activities and pathophysiological roles sIL-1Ra competitively binds the IL-1 receptor I without inducing signal transduction. It is therefore likely that sIL-1Ra regulates (inhibits) cellular functions affected by IL-1 and/or IL-1 . The function(s) of intracellular forms of IL-1Ra are less obvious. Some hypotheses were recently reviewed (Arend et al., 1998) claiming that icIL-1Ra might counteract intracellular IL-1 activities and destabilize and/or degrade mRNAs induced by IL-1 . However the latter functions remain to be demonstrated. More recently, it has been shown that resting and cytokine-stimulated human pulmonary epithelial cells release icIL-1RaI in the extracellular space, where it can antagonize cell surface IL-1R (Levine et al., 1997). Therefore, icIL-1Ra might be an epithelial store of IL-1Ra liable to immediate release upon stimulation. Furthermore, the expression of icIL1RaI in Caco-2 cells decreased IL-1 -induced IL-8 secretion (Bocker et al., 1998). Whether icIL-1RaI is released by the latter cells remains to be determined.

GENE AND GENE REGULATION

Accession numbers The IL-1Ra gene is referred to as IL-1RN. The sequence of human IL-1RN, bases 1±33,414, is accessible in GenBank at number U65590. cDNA sequences are accessible at number M63099 (human sIL-1Ra) and X84348 (human icIL-1RaI) and X52015, X64532, X53296, and M55646. Mouse IL1RN is accessible at number MGI:92197.

Chromosome location Human IL-1RN has been mapped in the long arm of chromosome 2 at bands q14-21 (Steinkasserer et al.,

IL-1Ra

one minor and two major sites, 14, 15, and 16 nucleotides, respectively, upstream to the cDNA sequence. icIL-1RaII is derived from both icIL-1RaI and sIL-1Ra mRNA by alternative translation initiation from the second 50 ATG (Malyak et al., 1998). A variable number of tandem repeats polymorphism has been described in intron 2 of the IL-1Ra gene (Tarlow et al., 1993). Allele 2 of this polymorphism is associated with the severity of many chronic inflammatory diseases (Table 1), although the mechanism of this association remains elusive. Furthermore, carriage of at least one copy of the A2 allele was found to be associated with reduced bone loss at the spine in early postmenopausal patients (Keen et al., 1998). In normal human subjects, IL-1Ra allele 2 has a clear influence on IL-1Ra circulating levels, i.e. its carrier individuals had 10-fold higher levels (745 ng/mL) than the noncarrier individuals (627 pg/mL). This also required the presence of the IL-1 -511 allele 2 or absence of the IL-1 +3953 allele 2, indicating that the IL-1 gene participates in the regulation of IL1Ra production in vivo (Hurme and Santtila, 1998). Reciprocally, the presence of the IL-1Ra allele 2 is associated with enhanced IL-1 production in mononuclear cells in vitro (Santtila et al., 1998). The activity of sIL-1Ra promoter is highly controlled by tissue restricted factor(s), being inactivated in T cells (Jurkat), B cells (Raji), and epithelial cells (HeLa, HT29) (Smith et al., 1992; Butcher et al., 1994). Furthermore, the sIL-1Ra gene is not constitutively expressed in resting or unstimulated monocytes, and established myeloid cell lines such as THP1, in contrast to macrophages. Promoter elements controlling the induction of human sIL-1Ra

1993). Mouse IL-1RN has also been mapped at chromosome 2 (10.00 cM) (Zahedi et al., 1991).

Relevant linkages Human IL-1 and IL-1 genes (IL-1A and IL-1B) are also located in the long arm of chromosome 2. There are only 75 kb between IL-1A and IL-1B and 200 kb between IL-1B and IL-1RN. The genes of the mouse IL-1 family are mapped in chromosome 2 but IL-1RN is far from IL-1A and IL-1B which are closely linked. In the human system the IL-1 receptor genes map in chromosome 2 close to the IL-1 gene cluster, whereas this is not the case in the mouse.

Regulatory sites and corresponding transcription factors sIL-1Ra, icIL-1RaI, and icIL-1RaII are translates originating from the same gene (Figure 1). sIL-1Ra and icIL-1RaI are produced by the alternative splicing of two different exons 1: 1s and 1ic. Exon 1ic is located upstream of exon 1s. Exon 1ic splices into exon 1s excluding a large proportion of the leader peptide sequence (Butcher et al., 1994). The transcription of icIL-1Ra and sIL-1Ra is controlled by two different promoters, Pic and Ps, respectively. Pic is located upstream of exon 1ic and Ps is located upstream of exon 1s, in the 9.4 kb intron which separates exon 1ic from 1s. Three transcription start sites have been identified for human sIL-1Ra mRNA,

Figure 1 IL-1Ra isoforms are generated from the same gene. icIL-1Ra is produced by splicing of exon 1ic into exon 1s excluding a large proportion of the leader peptide sequence (dashed zone). The production of sIL-1Ra is controlled by promoter Ps. sIL-1Ra pro-peptide is processed and glycosylated prior to secretion. Pic

1ic

Ps

1s

2

3

4

Gene 5'UTR

5'UTR

3'UTR

Primary RNA 5'UTR

3'UTR

mRNA 5'UTR

3'UTR IcIL-1Ra

5'UTR

3'UTR

pro-sIL-1Ra

Intracellular protein sIL-1RA Secreted protein

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322 Danielle Burger and Jean-Michel Dayer Table 1

Association of two allele repeat (IL1RN*2) with inflammatory diseases References

Associated diseases Alopecia aerata

Cork et al. (1995), Tarlow et al. (1994)

Systemic lupus erythematosus

Suzuki et al. (1997), Blakemore et al. (1994)

Psoriasis

Tarlow et al. (1997)

Diabetic nephropathy

Blakemore et al. (1996)

Relapsing/remitting multiple sclerosis

de la Concha et al. (1997)

Henoch±SchoÈnlein

Liu et al. (1997)

SjoÈgren's syndrome

Perrier et al. (1998)

Nonassociated diseases Graves' disease

Muhlberg et al. (1998), Cuddihy and Bahn, (1996)

Ulcerative colitis

Hacker et al. (1997), Bioque et al. (1996)

Rheumatoid arthritis

Tarlow et al. (1994)

Crohn's disease

Mansfield et al. (1994)

were identified in mouse (RAW 264.7) and human (THP1) myeloid cell lines (Smith et al., 1992; Butcher et al., 1994). sIL-1Ra promoter is active in transfected cell lines, suggesting a dysregulation in such systems. Nucleotide deletion downstream of -294 had no significant effect on promoter activity, although this region might contain elements required for the inducible activation of sIL-1Ra transcription in myeloid cells. Indeed, most proximal 294 bp of the human sIL-1Ra promoter are sufficient for full basal activity and LPS responsiveness. Four LPS-responsive elements (LRE) have been identified in the sIL1Ra promoter. One LRE masking the response to LPS is located between -294 and -250 (INH). Three other positive-acting sites were identified between 250 and -200 (LRE3), -200 and -148 (LRE2), and -93 and -84 (LRE1) (Smith et al., 1994). LRE1, LRE2, and LRE3 exhibited cooperativity in mediating the responses to LPS. LRE1 was identified as an NFBbinding site. The transcription factors binding to LRE2 and LRE3 remain to be identified. Recently, two PU.1-binding sites were identified (Smith et al., 1998), one of which centered at -230 whose mutation resulted in a 50% decrease in LPS-responsive promoter activity. The other PU.1-binding site, which partially overlapped the NFB site, was revealed to be a novel composite NFB/PU.1/GAbinding protein-binding site. Both PU.1-binding sites are major responsive elements for LPS-induced sIL1Ra gene expression. Two STAT-binding elements (SBEs) were identified in a region between -250 and 200. Upon IL-4 activation of monocyte-macrophages, STAT6 binds to the more distal SBE, inducing

transcriptional activation of sIL-1Ra gene (Ohmori et al., 1996). In transfection studies, a promoter/luciferase fusion construct containing 4.5 kb of the 50 flanking sequence of the human icIL-1Ra gene exhibited a pattern of expression in epithelial cell lines and macrophage cell lines similar to that of the endogenous icIL-1Ra gene (Jenkins et al., 1997). Mutants of the latter promoter construct indicated that the constitutive expression in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, -288 to -56, and -156 to -49. In contrast, basal activity of the icIL1RaI promoter in transfected but unstimulated RAW 264.7 cells was under the control of a weak inhibitory region located between -4525 and -1438 bp and a strong positive element between -156 and -49. Induction of icIL-1Ra transcription by LPS in RAW 264.7 cells was regulated by strong positively acting DNA regions between bases -1438 to -909 and -156 to -49. In summary, the proximal region of the icIL-1Ra promoter, between bases -156 and -49, contains positive cis-acting elements that are required for expression in both epithelial cell and monocytic cell lines.

Cells and tissues that express the gene sIL-1Ra is an inducible gene in most cells with maybe the exception of astrocytes (Liu et al., 1998), whereas

IL-1Ra icIL-1Ra is expressed constitutively in keratinocytes and intestinal epithelial cells (for review see Arend et al., 1998). Furthermore, icIL-1RaI mRNA is constitutively expressed in the epithelial cell lines A431 and HT29, but not in the macrophage cell lines RAW 264.7 and U937, or in the lymphocyte cell lines Raji and Jurkat. However, icIL-1Ra mRNA expression was induced in response to stimulation with LPS in RAW 264.7 cells and to PMA and LPS in U937 cells (Jenkins et al., 1997). In normal mice and those injected with LPS, icIL1Ra mRNA was found only in the skin, whereas sIL1Ra mRNA was not detected in any tissues of normal mice but was upregulated in spleen, lung, and liver of LPS-treated mice (Gabay et al., 1997a). In normal rabbits, IL-1Ra was constitutively produced in all tissues examined (Apostolopoulos et al., 1996; Matsukawa et al., 1997). All tissues produced sIL1Ra whereas thymus, cecum, skin, and kidney produced both sIL-1Ra and icIL-1RaI.

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Figure 2 The binding of IL-1Ra to IL-1R type I does not trigger the formation of a trimolecular complex, thus blocking signal transduction. Residue 145 confers the agonistic (aspartic acid, D) or the antagonistic (lysine, K) activity to the ligand.

D

IL-1β

K

IL-1Ra

IL-1RI

IL-1R AcP K

D

Low affinity bimolecular complex

High affinity trimolecular complex

PROTEIN

Accession numbers SwissProt: Human: p18510 Rabbit: p26890 Mouse: p25085 Rat: p25086 cDNA sequences for bovine and horse IL-1Ra have been described recently (Kato et al., 1997; Kirisawa et al., 1998; Howard et al., 1998) but are not yet accessible in data banks.

Sequence Human sIL-1Ra precursor is a 177 amino acid glycoprotein comprising a signal sequence (amino acids 1±25), a disulfide bound between Cys69 and Cys116 (Schreuder et al., 1995), a potential Nglycosylation site at Gln109 and a cis-Pro at position 53. Human 18 kDa icIL-1Ra displays a sequence similar to pro-sIL-1Ra with the N-terminal sequence MAL instead of MEICRGLRSHLITLLLFLFHS, giving rise to a 159 amino acid unglycosylated protein (Haskill et al., 1991). Rabbit, rat, mouse, bovine, and horse sIL-1Ra predicted amino acid sequences display around 75% sequence homology with human sIL1Ra, the rat and mouse sIL-1Ra precursor exhibiting a signal sequence of 26 residues, being one amino acid longer than that of human and rabbit sIL-1Ra. The three forms of IL-1Ra found in humans, i.e. sIL-1Ra,

icIL-1RaI, and icIL-1RaII, were also identified in mouse and rabbit (Goto et al., 1992; Cominelli et al., 1994; Zahedi et al., 1994; Gabay et al., 1997a; Matsukawa et al., 1997).

Description of protein IL-1Ra displays a hydrophobic core and the typical -trefoil structure with 12 strands which is found in the IL-1 family of proteins (Stockman et al., 1992, 1994; Vigers et al., 1994). Residues Trp16, Gln20, Tyr34, Gln36, and Tyr147 are crucial for the binding of sIL-1Ra to the receptor (Evans et al., 1995). Residue 145 (Lys in sIL-1Ra, Asp in IL-1 ) is crucial in determining agonist or antagonist activity (Ju et al., 1991). However, this region does not interact directly with the receptor, either in IL-1Ra or in IL-1 (Schreuder et al., 1997), suggesting that this residue might be important for the interaction with the IL-1 receptor accessory protein (IL-1R AcP) (Arend et al., 1990; Schreuder et al., 1997) (Figure 2).

Discussion of crystal structure The 3D structure of IL-1Ra has been determined by X-ray crystallography and in solution by NMR spectroscopy (Stockman et al., 1992, 1994; Vigers et al., 1994; Schreuder et al., 1995, 1997). IL-1Ra has 12 strands connected by loops. Six of the strands form a barrel structure that is closed at one end by the

324 Danielle Burger and Jean-Michel Dayer remaining sheet. The overall structure of IL-1Ra is similar to that of IL-1 and IL-1. Differences in the amino acid sequence of the two types of protein account for the interactions with the receptor, triggering or not signal transduction through the formation of the trimolecular complex IL-1/IL-1RI/ IL-1R AcP.

Important homologies Recombinant human sIL-1Ra binds to mouse, bovine, and rabbit IL-1 receptor with similar avidity. This suggests that IL-1Ra has been well conserved through evolution. Indeed, the amino acid sequences of all the mammal IL-1Ra described to date display > 75% homology, e.g. the deduced amino acid sequence of bovine IL-1Ra demonstrated 80%, 78%, 78%, 77%, and 76% homology with human, mouse, rat, rabbit, and horse sequences, respectively (Kirisawa et al., 1998). There is poor sequence homology between IL-1Ra and IL-1 or IL-1 , although all three proteins are derived from a unique primordial precursor gene which gave rise to a common IL-1/IL-1 gene and the IL-1Ra gene after a gene duplication event which occurred some 350 million years ago (Eisenberg et al., 1991).

Posttranslational modifications The only posttranslational event which occurs in sIL1Ra is glycosylation. Indeed, a potential N-glycosylation site is present in the IL-1Ra sequence of all species described to date. However, the presence of the oligosaccharide moiety does not seem to affect the inhibitory activity of sIL-1Ra since both natural, glycosylated sIL-1Ra and recombinant, unglycosylated sIL-1Ra display similar avidity to bind to the IL-1 receptor. Therefore, glycosylation might protect sIL-1Ra from proteolytic degradation and thus prolong its lifespan in the extracellular space. Since the intracellular forms of IL-1Ra are not translated in the endoplasmic reticulum and are consequently not processed through the Golgi, icIL-1RaI and icIL1RaII are not glycosylated.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce It is very likely that all cell types able to produce IL-1 and/or IL-1 will also express sIL-1Ra or

icIL-1Ra or both forms. However, by far the majority of the studies have focused on the expression of sIL1Ra in monocyte-macrophages, neutrophils, and fibroblasts and of icIL-1Ra in epithelial cells. The induction of IL-1Ra production in various cells has been extensively reviewed (Lennard, 1995; Dinarello, 1996; Arend et al., 1998). The differential expression of IL-1 and IL-1Ra in different cell types of the central nervous system has recently been described. IL-1Ra (protein and mRNA) is constitutively expressed in neurons of the paraventricular nucleus and supraoptic nucleus. After administration of endotoxin in rats, no additional cell expressed immunoreactive IL-1Ra whereas IL-1 and IL-1 were induced in the same cell types, i.e. macrophages in meninges and choroid plexus and microglial cells in various brain regions. This suggests that in the brain IL-1Ra is constitutively expressed by cell types other than those expressing IL-1 (van Dam et al., 1998).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The production of IL-1Ra, and particularly that of sIL-1Ra, has been extensively studied. Indeed, since sIL-1Ra is usually produced by the same cell as IL-1, the identification of factors able to modulate differentially these productions has remained a challenge. LPS induces both IL-1 and IL-1Ra in monocytes. However, the expression kinetics of the two proteins are different, the production of IL-1 and sIL-1Ra reaching its peak at 2 hours and 4 hours, respectively (Vannier et al., 1992). Similarly, differential production of IL-1 and sIL-1Ra was observed in vivo. LPS administered to human volunteers induced the production of IL-1 and sIL-1Ra which peaked 1 hours and 2 hours after injection, respectively. Furthermore, peak plasma concentrations of IL-1Ra were about 100-fold higher than those of IL-1 (Granowitz et al., 1991). In vitro, the most potent inducer of IL-1Ra in monocytes is adherent IgG which weakly induces IL-1 production. In monocytes activated by adherent IgG, high concentrations of LPS reduce the expression of sIL1Ra but not that of IL-1 . Together, these studies indicate that different signaling pathways are involved in the induction of IL-1 and IL-1Ra production. Another important inducer of sIL-1Ra in monocytes is direct cellular contact with stimulated T cells (Burger and Dayer, 1998) which also concomitantly induces the production of IL-1 and sIL-1Ra. In the latter system, IL-1 and sIL-1Ra production is

IL-1Ra controlled by different intracellular mechanisms involving the differential effect of serine/threonine phosphatases (Vey et al., 1997), the latter enzymes having a positive effect on the induction of sIL-1Ra and a negative one on the induction of IL-1 . This has been confirmed in a study demonstrating that Raf-1 kinase, a serine/threonine kinase, is involved in one of the pathways leading to sIL-1Ra production. Interestingly, the Raf-1 and LPS pathways are mutually antagonistic (Guthridge et al., 1997). The ability of stimulated T lymphocytes to induce IL-1 and IL-1Ra is differentially modulated by drugs such as leflunomide (DeÂage et al., 1998). IL-3 induces sIL1Ra production in monocytes to levels equivalent to those induced by GM-CSF and LPS, whereas IL-1 and IL-4 are weak inducers (Jenkins and Arend, 1993). Macrophages differentiated either in vivo (lung alveolar macrophages, synoviocytes) or in vitro (GMCSF-treated monocytes) spontaneously produce large amounts of sIL-1Ra (Roux-Lombard, 1998). This production is increased by GM-CSF and IL-4 whereas LPS remains ineffective. Furthermore, C-reactive protein (CRP) triggers the production of IL-1 and sIL-1Ra in peripheral blood monocytes but inhibits it in alveolar macrophages (Tilg et al., 1993; Pue et al., 1996). In polymorphonuclear neutrophils (PMNs), sIL1Ra, but not icIL-1Ra, is mainly induced by LPS, GM-CSF, and TNF (Malyak et al., 1994). Interestingly, TNF is a poor inducer of sIL-1Ra in monocytes. The induction of sIL-1Ra in PMNs is synergistically enhanced by IL-4 and IL-10, but not by IL-13 and TGF . IFN increases the release of sIL-1Ra in LPS- and fMLP-stimulated PMNs (McDonald et al., 1998). In human microglia, IFN induces IL-1Ra production and enhances LPS- and IL-4-induced IL-1Ra production, whereas it suppresses LPS- and IL-1-induced IL-1 production. In contrast, IFN inhibits both IL-1 - and IL-1Ra-induced production (Liu et al., 1998). sIL-1Ra and IL-1 production by PMNs is also differentially induced by microcrystals, favoring that of IL-1 (Roberge et al., 1994). The potent induction of sIL-1Ra by either LPS or GM-CSF might be modulated by other cytokines such as IL-4, IL-10, and IL-13 which potentiate sIL1Ra production and simultaneously inhibit IL-1 production (Jenkins and Arend, 1993; Jenkins et al., 1994). Interestingly, although exogenous IL-1 is a poor sIL-1Ra inducer in monocytes, the induction of endogenous IL-1 by TGF in monocytes is required for the triggering of sIL-1Ra production (Wahl et al., 1993). Activin A, a member of the TGF family, has been shown to regulate the production of IL-1Ra and

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IL-1 in monocytic cells. Activin A inhibits the production of IL-1 and enhances that of IL-1Ra in activated THP1 and U937 human monocytic cells. It is noteworthy that activin A regulates cytokine production at a posttranscriptional level (Ohguchi et al., 1998). IFN also differentially modulates IL-1Ra and IL-1 production in PHA-stimulated peripheral blood mononuclear cells (PBMCs) by inhibiting the production of IL-1 and enhancing that of IL-1Ra (Coclet-Ninin et al., 1997). Retinoic acid differentially regulates IL-1 and IL-1Ra production by alveolar macrophages (Hashimoto et al., 1998).

RECEPTOR UTILIZATION IL-1Ra secreted form interacts with both IL-1 receptors. Natural and recombinant (unglycosylated) sIL-1Ra display similar affinity for the mouse IL-1 receptor I to that of human IL-1 and IL-1 (dissociation constant  200 nM) (Seckinger et al., 1987; Hannum et al., 1990; Eisenberg et al., 1990). sIL-1Ra binding to IL-1 receptor I does not induce signaling because it does not engage IL-1R AcP (Cullinan et al., 1998). However, in addition to sIL1Ra, the biological activity of IL-1 is counterbalanced by IL-1 soluble receptors (IL-1sR) which bind IL-1 and diminish the free concentration of soluble cytokine, thus hampering its binding to the cell surface receptor. Since IL-1sR can also bind IL-1Ra, the two types of inhibitors might abolish their respective inhibitory activity. Interestingly, the inhibitory effect of human sIL-1Ra is enhanced by type II IL-1sR and hindered by type I IL-1sR (Burger et al., 1995). Recombinant human sIL-1Ra, icIL-1RaI, and icIL-1RaII display similar affinity constants for type II IL-1sR (2.9±3.8  107 Mÿ1 ), whereas icIL-1RaII binds type I IL-1sR with a 4- to 5-fold lower affinity constant (3.0  109 Mÿ1 ) than sIL-1Ra and icIL-1RaI (1.2 and 1.4  1010 Mÿ1 ) (Malyak et al., 1998). In spite of very similar affinity constants for IL-1 receptor type I, large molar excess (10- to 100-fold) of sIL-1Ra is required to efficiently inhibit IL-1 activity in vitro (Arend et al., 1990). Similarly, preinjection of 100to 1000-fold molar excess of sIL-1Ra is required to block systemic responses to IL-1 in animals (Fischer et al., 1991).

IN VITRO ACTIVITIES In vitro, sIL-1Ra has mainly been used as a specific inhibitor of IL-1 and IL-1 activity, since it is a cytokine whose only known action is the competitive inhibition of the binding of IL-1 to its receptor.

326 Danielle Burger and Jean-Michel Dayer

In vitro findings IL-1Ra has been shown to inhibit all described IL-1 activities due to the binding of the agonist to its receptor, although the concentration required to abolish IL-1 effects is usually a 10- to 100-fold molar excess of IL-1Ra over IL-1. More studies are required to define the functions of intracellular forms of IL-1Ra.

Regulatory molecules: Inhibitors and enhancers Since it does not induce signal transduction, the `activity' of sIL-1Ra is regulated only by its levels of production which are controlled by the above mentioned inducers, inhibitors, and enhancers.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles In addition to the constitutive expression of icIL-1Ra in epithelial cells and keratinocytes, sIL-1Ra seems to be expressed at low levels in normal human subjects and animals, despite some species-dependent differences. However, the function of IL-1Ra in normal, i.e. `noninflammatory' conditions, remains elusive. It has been postulated that tissue IL-1Ra is involved in health maintenance by masking coexisting IL-1 activity in tissues (Matsukawa et al., 1997). In healthy rabbits, sIL-1Ra is produced constitutively in most tissues (lung, liver, spleen, thymus, cecum, skin, kidney, heart, and brain), while thymus, cecum, skin, and kidney produced both sIL-1Ra and icIL1Ra. In contrast, IL-1 activity has not been detected in any tissues with the exception of skin and heart, but became detectable after preincubation of the samples with neutralizing antibodies to IL-1Ra, demonstrating that constitutive but low expression of sIL-1Ra inhibits IL-1 activity in physiological conditions. On the other hand, studies on chick embryo fibroblasts have shown that the balance between the secreted cytokine network and IL-1Ra levels may represent a homeostatic mechanism aimed at controlling normal embryogenesis (Bodo et al., 1998). The physiological roles of endogenously produced IL-1Ra have been investigated in mice that either lack

IL-1Ra or overproduce it under the control of the endogenous promoter.

Knockout mouse phenotypes Mice lacking IL-1Ra are more susceptible than controls to lethal endotoxemia but less susceptible to listeriosis (Hirsch et al., 1996). Furthermore, IL-1Ra knockout mice had a significantly earlier onset of collagen-induced arthritis, with increased severity (Ma et al., 1998). This confirms the important role of IL1Ra in the regulation of IL-1 activity during infection and inflammation. More intriguing is the fact that IL-1Ra knockout mice have decreased body mass compared with wild-type controls or IL-1 knockout mice due to growth retardation after weaning (Hirsch et al., 1996; Horai et al., 1998). This implies that IL-1Ra plays an important but still elusive part in normal physiology.

Transgenic overexpression The effect of IL-1Ra overexpression has been assessed in several animal models of disease in which IL-1 was claimed to be involved. Mice overexpressing the IL1Ra gene under the control of its endogenous promoter had a significant reduction in both incidence and severity of collagen-induced arthritis suggesting that the endogenous expression of IL-1Ra is a critical determinant of susceptibility to the disease (Ma et al., 1998). Interestingly, after immunization with type II collagen, IL-1Ra mRNA was overexpressed in the spleens, but not in the injection paws, of transgenic mice (Ma et al., 1998). On the other hand, IL-1Ra overproducers are protected from the lethal effects of endotoxin while being more susceptible to listeriosis. Following an endotoxin challenge serum levels of IL-1 are decreased in IL-1Ra-null mice and increased in IL-1Ra overproducers in comparison to controls (Hirsch et al., 1996). Transgenic mice with distal airway epithelial cell expression of human IL-1Ra were partially protected from IL-1-induced airway inflammation and injury (Wilmott et al., 1998).

Pharmacological effects Since IL-1Ra is able to counteract the proinflammatory effects of IL-1, recombinant sIL-1Ra has been used as therapeutic agent in many animal disease models. These models were recently reviewed (Arend et al., 1998) and covered mainly infectious and

IL-1Ra inflammatory diseases. Exogenous IL-1Ra, usually administered intravenously, led to at least partial prevention or treatment of disease. However, the necessity of systemic IL-1Ra injections in large amounts limits the application of this therapy in human patients. New approaches have recently been tested applying exogenous IL-1Ra directly at the inflammatory site. These gene therapy trials were mainly carried out in animal models of rheumatoid arthritis and osteoarthritis using either adenoviral gene transfer into the joint or ex vivo gene transfer in synoviocytes and autograft (Arend et al., 1998). Interestingly, endogenously produced IL-1Ra was mainly chondroprotective, displaying only weak antiinflammatory properties. Therefore, IL-1Ra gene therapy displays some efficacy in inflamed animal joints mainly by diminishing cartilage destruction. Few trials have been carried out in other systems, although the transfer of IL-1Ra gene into neuronal cells, hematopoietic stem cells and lungs has been successful (Davidson et al., 1993; Boggs et al., 1995; McCoy et al., 1995). IL-1Ra overexpression in mouse brain by adenoviral vector attenuates the activation of inflammatory cells during focal cerebral ischemia ( Yang et al., 1998). IL-1Ra may also be upregulated in rat brain by peripheral administration of kainic acid (Eriksson et al., 1998).

Interactions with cytokine network IL-1 being a potent inducer of many other cytokines, the inhibition of its effects by IL-1Ra clearly has an important impact on the cytokine network. Furthermore, since IL-1 may induce the production of its inhibitor, sIL-1Ra might regulate its own production in some circumstances.

Endogenous inhibitors and enhancers IL-1Ra is usually produced by the same cells which produce IL-1 and/or IL-1. Besides, similar stimuli induce the production of both agonist and antagonist cytokines. Therefore, many studies have been devoted to the study of factors affecting the balance between IL-1Ra and IL-1 production, i.e. IL-4, IL-13, and IL10 favoring the production of sIL-1Ra over that of IL-1 . Interestingly, both TH1 and TH2 cytokines, i.e. IFN and IL-4, antagonize the inhibition of sIL-1Ra production by LPS-stimulated monocytes in the presence of glucocorticoids (Parsons et al., 1997), although IFN but not IL-4 acts through an

327

IL-1-dependent mechanism. Therefore, coinfusion of glucocorticoids and cytokine should favor the production of IL-1Ra over that of IL-1 .

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY The role of IL-1Ra in normal physiology of healthy humans remains elusive. Only animal models have shed light on the possible role of IL-1Ra in normal biological processes. Indeed, in contrast with IL-1 knockout mice, IL-1Ra knockout mice display growth retardation, suggesting an important role for IL-1Ra in normal physiology where it could mask coexisting IL-1 activity in tissues. However, additional studies are required to clarify the roles of IL-1Ra isoforms in normal biologic processes particularly in humans. Because sIL-1Ra levels are elevated in the peripheral blood of patients with various diseases, it has recently been suggested that IL-1Ra is an acute phase protein (Gabay et al., 1997b). Furthermore, the use of neutralizing antibodies has demonstrated the importance of IL-1Ra as a natural anti-inflammatory protein in animal models of diseases. In analogy a similar role for IL-1Ra has been suggested in humans. Indeed, as recently reviewed (Arend et al., 1998), extensive evidence indicates that IL-1Ra is produced by many tissues in healthy humans and that its production is upregulated in the host response to infection and acute and chronic inflammation.

Normal levels and effects Except for allele 2 carriers, low levels of circulating sIL-1Ra have been detected in healthy humans (400± 800 pg/mL) which were drastically increased in various pathological conditions (Table 2). The increase in IL-1Ra level in such diseases does not seem to counteract efficiently the inflammatory effects of IL-1. However, studies using neutralizing anti-IL1Ra antibodies in animal models of disease indicate an anti-inflammatory effect of endogenous IL-1Ra. On the other hand, the levels of IL-1Ra were found to be locally enhanced in acute and chronic disorders such as rheumatoid arthritis (Firestein et al., 1992; Malyak et al., 1993), osteoarthritis, and Lyme arthritis (Miller et al., 1993), and nonperforating Crohn's disease (Gilberts et al., 1994).

328 Danielle Burger and Jean-Michel Dayer Table 2 Diseases with increased circulating IL-1Ra level Disease

Reference

Chronic arthritis

Jouvenne et al. (1998)

Insulin-dependent diabetes mellitus

Netea et al. (1997)

Systemic lupus erythematosus

Sturfelt et al. (1997)

Gynecological cancers

Fujiwaki et al. (1997)

Pediatric sepsis syndrome

Samson et al. (1997)

Pre-eclampsia

Kimya et al. (1997)

Acute myocardial infarction

Shibata et al. (1997)

Graft-versus-host disease

Schwaighofer et al. (1997)

Hemophagocytic lymphohistiocytosis

Henter et al. (1996)

Patients with burns

Endo et al. (1996)

Bronchial asthma

Yoshida et al. (1996)

Chronic renal failure

Deschamps-Latscha et al. (1995)

Systemic chronic juvenile arthritis

De Benedetti et al. (1995)

Polymyositis/dermatomyositis

Gabay et al. (1994)

Septic shock

Rogy et al. (1994)

Role in experiments of nature and disease states The serum levels of sIL-1Ra are elevated in many pathologies. However, only few studies contemplate the determination of IL-1Ra as a diagnostic test. In inflammatory bowel disease the balance between IL-1 and sIL-1Ra might influence disease expression. It was demonstrated that patients with active Crohn's disease had significantly higher serum sIL-1Ra levels than patients with active ulcerative colitis. Therefore, levels of sIL-1Ra in the peripheral blood of patients with inflammatory bowel disease are of clinical relevance, representing a marker of disease activity and a possible differential diagnostic marker (Propst et al., 1995). More recently, it has been shown that serum levels of sIL-1Ra and IL-6 were enhanced 2 days before the clinical manifestation of neonatal sepsis (Kuster et al., 1998). While circulating sIL-1Ra levels are increased in patients at risk for acute respiratory distress syndrome (ARDS) who die, it does not predict the development of the syndrome (Parsons et al., 1997). Furthermore, it was recently suggested that in chronic polyarthritis elevated levels of IL-1Ra may reflect increased production and activity of IL-1, in contrast with the levels of endogenous type II IL-1sR which may constitute a natural anti-inflammatory factor. This should be taken into account when considering these molecules

as prognostic markers or for therapeutical usage (Jouvenne et al., 1998). In renal transplant recipients, patients with high IL-1Ra/IL-1 ratios in urine are less prone to acute allograft rejection than patients with low IL-1Ra/IL-1 ratios (Teppo et al., 1998). Rejection episodes in orthotopic heart transplantation are accompanied by a renewed and more pronounced elevation in IL-1Ra (serum levels beyond 4000 pg/mL) for at least 2 days, indicating that IL1Ra might have a predictive value in the detection of acute allograft rejection (Thiele et al., 1998).

IL-1RA IN THERAPY

Preclinical ± How does it affect disease models in animals? As stated above, the therapeutic use of sIL-1Ra has been tested in many animal models of diseases (Table 3). This has been extensively reviewed (Arend et al., 1998). Some studies published in 1998 have shown that IL-1Ra was also efficient in the treatment of experimental allergic encephalomyelitis (EAE ), the animal model for multiple sclerosis (Badovinac et al., 1998), although IL-1Ra was administered before disease onset. Indeed, dark Agouti rats which were treated during the induction phase of EAE (days 0±6) with IL-1Ra (350 g/rat/day) developed milder

IL-1Ra

329

Table 3 Animal models of diseases improved by IL-1Ra administration Model of disease

Animal

Acetic acid-induced colitis

Rats

Allergen-induced late asthmatic reaction

Guinea pigs

Allosensitization in corneal transplantation

Mice

Antigen-induced airway hyperreactivity

Guinea pigs

Antigen-induced arthritis

Rabbits

Antiglomerular basement membrane antibody-induced glomerulonephritis

Rats

Apolipoprotein E-deficient mice

Mice

Bacterial meningitis

Rabbits

Bleomycin- and silica-induced pulmonary fibrosis

Mice

Cardiac allograft immunoreactivity

Rats

Carrageenan-induced pleurisy

Rats

Cerulein-induced experimental acute pancreatitis

Mice

Collagen-induced arthritis

Mice

Crescentic glomerulonephritis

Rats

Cyclophosphamide-induced myelotoxicity

Mice

Diabetes recurrence after syngeneic pancreatic islet transplantation

Mice

Dimethylnitrosamine-induced hepatic fibrosis

Rats

Dinitrofluorobenzene-induced contact hypersensitivity

Mice

Endotoxin-induced mastitis

Cows

Endotoxin-induced uveitis

Rats, rabbits

Experimental allergic encephalomyelitis

Rats

Experimental heatstroke

Rats

Experimental shigellosis

Rabbits

Graft-versus-host disease

Mice

Immune complex-induced arthritis

Mice

Immune complex-induced colitis

Rabbits

Immune complex-induced lung injury

Rats

Intestinal anaphylaxis

Guinea pigs

Ischemia/reperfusion lung injury

Rats

Ischemic brain injury

Mice, rats

Islet allograft rejection

Mice

KMnO4-induced granuloma

Mice

LPS-induced pleurisy

Rabbits

Monocrotaline-induced pulmonary hypertension

Rats

Oleic acid-induced lung injury

Mice

Osteoarthritis

Dogs

Osteoclast formation and bone resorption in ovarectomized animals

Mice, rats

Oxidant-induced arthritis

Mice

Postcardiac transplant coronary arteriopathy

Piglets

Preterm delivery induced by IL-1

Mice

Septic shock

Mice, rats, rabbits, baboons

330 Danielle Burger and Jean-Michel Dayer Table 3 (Continued) Model of disease

Animal

Silver nitrate-induced secondary amyloidosis

Mice

Spontaneously occurring IgA nephropathy

Mice

Staphylococcal-induced arthritis

Rabbits

Streptococcal cell wall-induced arthritis

Rats

Streptococcal cell wall-induced colitis

Rats

Streptozotocin-induced diabetes

Mice

symptoms than control animals immunized with encephalitogen. Furthermore, isolated lymph node cells from myelin basic protein-primed rats which were restimulated in vitro in the presence of IL-1Ra (10 mg/mL) and transferred to naive syngeneic animals display diminished encephalitogenic capacity. This correlates with a lower proliferative response to antigen and mitogen and decreased expression of IL-2 receptors in lymph node cells treated with IL-1Ra. The beneficial effect of IL-1Ra in EAE is improved by coinfusion of type I TNF soluble receptor in terms of clinical severity, frequency of disease, loss of body weight, and day of onset (Wiemann et al., 1998). In the latter study, the treatment began at day 8 after EAE induction by myelin basic protein, i.e. 3 days prior to disease onset in untreated animals. Besides, suppression of allosensitization by topical application of IL-1Ra enhances corneal transplant survival (Yamada et al., 1998).

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. The beneficial effects of IL-1Ra as a therapeutic agent have been demonstrated in many animal models of diseases, although infused doses of IL-1Ra were usually very high. Despite the fact that humans are the most sensitive species to IL-1, i.e. 1 ng/kg of intravenous IL-1 in healthy volunteers induced symptoms (Dinarello, 1996), and that coinfusion of IL-1Ra and endotoxin did not reduce endotoxin-induced symptoms, fever, and tachycardia (Granowitz et al., 1993), IL-1Ra was used in clinical trials in patients with sepsis. In contrast with animal models, IL-1Ra was inefficient in enhancing the survival of sepsis patients (Arend et al., 1998). On the other hand, IL-1Ra might mediate the effects of other cytokines used as therapeutic agents. Indeed, IL-1Ra is elevated in healthy volunteers who received a single dose of IFN-2b (Reznikov et al., 1998) confirming previous observations in

IFN-treated patients with chronic hepatitis C (Naveau et al., 1997).

Pharmacokinetics Intravenously administered IL-1Ra displays a short lifespan in human, exhibiting an initial half-life of 21 minutes and a terminal half-life of 108 minutes (Granowitz et al., 1992). This might explain the limited action of IL-1Ra in therapy (Arend et al., 1998). Although this was considered a positive feature in treating acute diseases, IL-1Ra was revealed to be inefficient in the treatment of sepsis. This short halflife IL-1Ra was also used in clinical trials for the treatment of chronic diseases such as rheumatoid arthritis and graft-versus-host disease (Campion et al., 1996; Antin et al., 1994; Bresnihan and Cunnane, 1998). A recent study has shown that administration of IL-1Ra in a slow-release vehicle such as hylan fluid improves pharmacokinetics and efficacy in rat type II collagen arthritis (Bendele et al., 1998). Indeed, in female Lewis rats with established type II collagen arthritis, subcutaneous injection of single daily doses of IL-1Ra (100 mg/kg) in hylan fluid results in a slower release of IL-1Ra in the bloodstream and maintains therapeutic blood levels of IL-1Ra (1 mg/mL) for a longer period than does IL-1Ra in citrate-buffered saline with EDTA and polysorbate. Animals treated with single daily dose of IL-1Ra in hylan fluid displayed 78% inhibition of paw swelling over time whereas similar doses of IL-1Ra in the other vehicle were unefficient.

Toxicity IL-1Ra does not display toxicity. Indeed, the intravenous infusion of 10 mg/kg of IL-1Ra in healthy human volunteers, i.e. a 10 million-fold molar excess, is without effect (Dinarello, 1996). Healthy volunteers who for 3 hours had received a continuous intravenous infusion of IL-1Ra doses

IL-1Ra ranging between 1 mg/kg and 10 mg/kg displayed no significant clinical difference as compared with salineinfused individuals in terms of symptoms, physical examinations, complete blood counts, mononuclear cell phenotypes, blood chemistry profiles, and serum iron and serum cortisol levels. The main difference was that PBMCs obtained after completion of the IL1Ra infusion synthesized less IL-6 ex vivo than PBMCs from saline-injected controls. This suggests that the transient blockade of IL-1 receptors is safe and does not significantly affect homeostasis (Granowitz et al., 1992).

Clinical results Although IL-1Ra was inefficient in sepsis treatment, clinical trials are currently undertaken in inflammatory diseases. The results of one of these trials enrolling 472 patients with rheumatoid arthritis were recently published (Bresnihan et al., 1998). They demonstrate that sIL-1Ra is the first biologic agent displaying a beneficial effect on the rate of joint erosion. Treatment with sIL-1Ra was evaluated in four groups of patients who received placebo or a single, self-administered subcutaneous injection of sIL-1Ra at a daily dose of 30 mg, 75 mg, or 150 mg. IL-1Ra was well tolerated and no serious adverse effects were observed, an injection-site reaction being the adverse event most frequently observed. At 24 weeks, the rate of radiologic progression in the patients receiving sIL-1Ra was significantly less than in the placebo group. Of the patients who received 150 mg/day of sIL-1Ra, 43% met the American College of Rheumatology criteria for response (the primary efficacy measure), 44% met the Paulus criteria, and statistically significant improvements were seen in the number of swollen joints, number of tender joints, investigator's assessment of disease activity, patient's assessment of disease activity, pain score on a visual analog scale, duration of morning stiffness, Health Assessment Questionnaire score, Creactive protein level, and erythrocyte sedimentation rate. This study confirmed both the efficacy and the safety of IL-1Ra in a large cohort of patients with active and severe rheumatoid arthritis.

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LICENSED PRODUCTS Recombinant sIL-1Ra is commercialized under the name of Antril (Synergen/Amgen).