IL-1b

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IL-1 Charles A. Dinarello* Department of Infectious Diseases, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, B168, Denver, CO 80262, USA * corresponding author tel: 303-315-3589, fax: 303-315-8054, e-mail: [email protected] DOI: 10.1006/rwcy.2000.04004.

BACKGROUND

Discovery The `discovery' of IL-1 is found in the literature on the pathogenesis of fever. Although initially concerned with the pyrogenic properties of gram-negative bacterial endotoxins, pyrogenic protein of endogenous origin and free of endotoxins was described in 1948. The first studies were carried out by Paul Beeson in 1948 on the fever-producing properties of rabbit peritoneal exudate cells (Beeson, 1948). The historical events were reviewed by Atkins in 1960 (Atkins, 1960) and again more recently (Dinarello, 1999). Many people think that IL-1 was first described by Waksman and Gery (1972), but that important discovery was related to the ability of IL-1 to act as a co-activator of lymphocyte proliferation, the least durable property of IL-1 compared to its role in inflammation. The fact remains that the most potent endogenous pyrogen and inducer of the so-called `acute phase response' in rabbits and humans is, in fact, IL-1 and this molecule was first described for its role in fever and the acute phase response (Merriman et al., 1977).

Alternative names IL-1 was originally studied as an endogenous pyrogen (EP, fever production) (Murphy et al., 1974; Dinarello et al., 1977), leukocyte endogenous mediator (LEM, an inducer of hepatic acute phase reactants) (Merriman et al., 1977), mononuclear cell factor (MCF, an inducer of collagenases and PGE2) (Dayer et al., 1977), lymphocyte-activating factor (LAF, a costimulator of T and B lymphocyte proliferation) (Gery and Waksman, 1972) and several

other biological properties. These are reviewed in detail in Dinarello (1989). Before the molecular cloning of IL-1 , this molecule was characterized by its isoelectric point of 7.0 to distinquish it from IL-1 which had an isoelectric point of 5.2 (Dinarello et al., 1974; Hanson and Murphy, 1984). After the cloning of human, mouse, rabbit, and other species of IL-1 and IL-1, the two members of the IL-1 family of agonists were identified by their amino acid sequences which, indeed, reveal that IL-1 and IL-1 have isoelectric points of 7 and 5, respectively.

Structure IL-1 is initially synthesized as a precursor molecule (31,000) without a signal peptide. Mature IL-1 is a 17,500 Da molecule. Although the IL-1 -converting enzyme (ICE) is primarily responsible for cleavage of the precursor intracellularly (Black et al., 1988), other proteases can process the IL-1 precursor into an active cytokine (reviewed in Fantuzzi et al., 1997a). Proteinase 3 processes the IL-1 precursor extracellularly to an active molecule (Coeshott et al., 1999) (Figure 1).

Main activities and pathophysiological roles The biological activities of IL-1 are similar to those of IL-1 and the distinction should be made that IL1 is a secreted molecule (via ICE) whereas IL-1 is primarily a cell-associated molecule. However, there are no biological activities of IL-1 that are not observed with IL-1 and vice versa. Any reports of differences between these two molecules in terms of biological activities are due to species differences.

352 Charles A. Dinarello Figure 1 Production and processing of IL-1 .

It is best to characterize the biological activities of IL-1 as being proinflammatory. Moreover, the biological importance of IL-1 in pathophysiological roles is best revealed by examining the effect of IL-1 blockade in animal models of disease. Since the IL-1 receptor antagonist (IL-1Ra) blocks the IL-1R, any reduction in disease severity associated with IL-1Ra is due to the role of IL-1 in that disease. These are discussed in detail under Role in experiments of nature and disease states.

GENE AND GENE REGULATION

Sequence The sequence of the human IL-1 gene can be found in Clark et al. (1986).

Chromosome location See Webb et al. (1986).

Regulatory sites and corresponding transcription factors Unlike the promoter of IL-1, the promoter region for IL-1 contains a clear TATA box, a typical motif of inducible genes. The half-life of IL-1 mRNA depends upon the cell type and the conditions of stimulation. The most studied cells are freshly

obtained human blood monocytes and macrophage cell lines derived from myelomonocytic leukemias. The initial studies established that endotoxin triggers transient transcription and steady state levels of IL-1 mRNA which accumulate for 4 hours followed by a rapid fall due to synthesis of a transcriptional repressor (Fenton et al., 1987, 1988). Unlike most cytokine promoters, IL-1 regulatory regions can be found distributed over several thousand base pairs upstream and a few base pairs downstream from the transcriptional start site. The topic of IL-1 gene regulation has recently been reviewed in detail, concluding that IL-1 gene expression is regulated at different levels (Auron and Webb, 1994). Studies have revealed sequences in the IL-1 promoter required for transcription using a reporter gene transfected into human and mouse macrophage cell lines. There are two independent enhancer regions (ÿ2782 to ÿ2729) (Tsukada et al., 1994) and (ÿ2896 to ÿ2846) (Shirakawa et al., 1993) which appear to act cooperatively. The latter contains a cAMP response element, whereas the former is a composite cAMP response element/NF-IL6 which is responsive to LPS. The 80 bp fragment (ÿ2782 to ÿ2729) is required for transcription and contains, in addition to a cAMP response element, an NFB-like site. Activating protein 1 (AP-1) sites also participate in endotoxin-induced IL-1 gene expression. Proximal promoter elements between ÿ131 and ‡14 have also been identified (Shirakawa et al., 1993). Sequences in this region contain the binding sites for the novel nuclear factor NFA (Buras et al., 1994) which appears to be similar to nuclear factors termed NF 1 and NF 2 (Shirakawa et al., 1993). This proximal promoter is required for maximal IL-1 gene expression. Recently, the nucleotide-binding sequences of NF A were found to be identical to those of the transcription factor Spi-1/PU.1 (Fenton et al., 1994; Kominato et al., 1995), a well-established NF in cells of myeloid and monocyte lineage. The requirement for Spi-1/PU.1 for IL-1 gene expression imparts tissue specificity, since not all cells constitutively express this NF. Human blood monocytes, which constitutively express Spi-1/PU.1, are exquisitely sensitive to gene expression of IL-1 by 1±10 pg/ mL of LPS. Interestingly, the IL-1Ra promoter contains the proximal Spi-1/PU.1 site (Auron and Webb, 1994), which is also highly sensitive to LPS.

Cells and tissues that express the gene Although the blood monocyte and tissue macrophages are the primary sources of IL-1 , in health,

IL-1 these cells do not constitutively express IL-1 (Mileno et al., 1995; Shapiro and Dinarello, 1997; Puren et al., 1999). Reports of constitutive expression of IL-1 in health are due to the activation of the IL-1 transcriptional process by surface contact (Schindler et al., 1990a). However, there seems to be constitutive expression of IL-1 in the human hypothalamus (Breder et al., 1988). Many malignant tumors, such as acute myelogenous leukemia (Cozzolino et al., 1989; Rambaldi et al., 1991; Estrov et al., 1993; Wetzler et al., 1994) and juvenile myelogenous leukemia (SchiroÁ et al., 1993), express IL-1 as part of their neoplastic nature.

PROTEIN

Accession numbers P01584

Sequence The primary sequence of the human IL-1 precursor can be found in Auron et al. (1984).

Description of protein IL-1 is primarily translated as a precursor (31,000) lacking a signal peptide (Auron et al., 1984). The IL1 precursor requires cleavage by the IL-1 -converting enzyme (ICE) (Black et al., 1988), also known as caspase 1 (Alnemri et al., 1996). Mature IL-1 , molecular weight 17,500 Da, has a sequence Nterminus at alanine 117 and is biologically active. The propiece of IL-1 is thought to have biological activities (Higgins et al., 1993).

Discussion of crystal structure Crystal structural analysis of the mature form of IL-1 reveals that the molecule is comprised of all sheets (Priestly et al., 1988). IL-1 has two sites of binding to IL-1 receptor type I (IL-1RI). There is a primary binding site located at the open top of its barrel shape (Gruetter et al., 1994), which is similar but not identical to that of IL-1 (LambriolaTomkins et al., 1993). There is a second site on the back side of the IL-1 molecule (Gruetter et al., 1994). IL-1Ra also has two binding sites similar to those of IL-1 (Evans et al., 1994; Vigers et al., 1994). However, the back side site in IL-1Ra is more homologous to that of IL-1 than the primary

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binding site (Evans et al., 1994). The formation of the heterodimer consisting of the IL-1RI and IL-1R accessory protein (IL-1R AcP) (Greenfeder et al., 1995) probably explains the failure of IL-1Ra to trigger a signal. From the structural differences described above between IL-1 and IL-1Ra, one can propose that the second binding site missing from the IL-1Ra is, in fact, the site which binds the accessory protein. The crosslinked complex of radiolabeled IL-1Ra to the type I receptor was not precipitated by a specific antibody to the accessory protein (Greenfeder et al., 1995). Mutational analyses reveal two distinct areas for binding to IL-1RI: site A and site B. For IL-1 , site A is on the back side of the molecule (Gruetter et al., 1994), whereas site B is at the open end of the barrel. Site A on IL-1 is shared with Tyr16, Glu20, Tyr34, Glu36 and Tyr147 of IL-1Ra (Evans et al., 1994). Site B, which in IL-1 comprises Arg4, Lys92/Lys94, Gln48/Glu51, Lys103, and Glu105 and has been proposed to form the receptor `trigger site'. This site is missing from IL1Ra. It is likely, however, that signal transduction for the two IL-1 agonists results in conformational changes in the IL-1RI when both A and B sites bind. Therefore, the possession of a single binding area on IL-1Ra may account for its binding and receptor occupancy. Most importantly, without the second binding site, the critical amino acids for receptor triggering may not become engaged and receptor conformations remain unchanged. This would explain the failure of IL-1Ra to transduce a signal. A nonapeptide within the mature IL-1 structure which possesses immunostimulatory properties is absent from IL-1Ra (Antoni et al., 1986, 1989). A single point mutation in the carboxyl end of the IL-1Ra molecule (lysine to aspartic acid) converts IL-1Ra from an antagonist to a partial agonist (Ju et al., 1991) but this may be due to malfolding of the molecule. In general, it appears that no single amino acid substitution accounts for the differences between the binding and signal transduction of IL-1 compared to IL-1Ra.

Important homologies The relevant homology of IL-1 are to the acidic fibroblast growth factor (Murzin et al., 1992) and to IL-18 (Okamura et al., 1995). Also, IL-1 shares homology to soybean trypsin inhibitor. The -barrel structure of IL-1 is closely related to that of fibroblast growth factor (Murzin et al., 1992) which possesses some IL-1-like activities. The primary amino acid homology of mature human IL-1 to mature IL-1 is 22%. Importantly, IL-1 is more

354 Charles A. Dinarello closely related to IL-1Ra than to IL-1; the homology of IL-1 to IL-1Ra is 26%. Since each member of the IL-1 family binds to the same IL-1 receptors, it is not surprising that IL-1, IL-1 , and IL-1Ra share structural topology. For example, Arg4 and Arg12 of mature IL-1 and IL-1, respectively, are required for binding and biological activity and both arginines occupy the same relative location in their respective crystallographic structures (Nanduri et al., 1991). IL-1 can act as a receptor antagonist of biological responses to IL-1 (Boraschi et al., 1990). Changing the aspartic acid at tyrosine 151 in the mature IL-1 results in loss of PGE2 induction and fibroblast growth but the T cell responses are unaffected (Yamayoshi et al., 1990).

Posttranslational modifications The primary translational product of the IL-1 gene is a precursor (31,000) lacking a signal peptide (Auron et al., 1984). The IL-1 precursor requires intracellular cleavage by the cysteine protease called the IL-1 -converting enzyme (ICE) (Black et al., 1988), also known as caspase 1 (Alnemri et al., 1966). Mature IL-1 , molecular weight 17,500 Da, has a sequence N-terminus at Ala117 and is biologically active. Following synthesis, the IL-1 precursor remains primarily cytosolic until it is cleaved and transported out of the cell. The IL-1 propiece (amino acids 1± 116) is myristoylated on lysine residues (Stevenson et al., 1993). Some IL-1 is found in lysosomes (Bakouche et al., 1987) or associated with microtubules (Rubartelli et al., 1990; Stevenson et al., 1992) and either localization may play a role in the secretion of IL-1 . In mononuclear phagocytes, a small amount of proIL-1 is secreted from intact cells (Auron et al., 1987; Beuscher et al., 1990) but the pathway for this secretion remains unknown. The release of mature IL-1 appears to be linked to processing by ICE (Black et al., 1988). Although well-controlled in the setting of laboratory cell culture, death and rupture of inflammatory cells is not an unusual occurrence in vivo. There are several sites in the N-terminal 16 kDa part of proIL1 which are vulnerable to cleavage by enzymes in the vicinity of Ala117. These are trypsin, elastase, chymotrypsin, a mast cell chymase, and a variety of proteases (Hazuda et al., 1990, 1991), which are commonly found in inflammatory fluids. Proteinase 3 also cuts the IL-1 precursor into an active molecule (Coeshott et al., 1999). The extent of the role that these proteases play in the in vivo conversion of proIL-1 to mature forms is uncertain, but in each case a biologically active IL-1 species is produced. In the

discussion on the soluble IL-1 receptor type II, it is pointed out that the affinity of proIL-1 for this constitutively produced soluble receptor is high and may prevent haphazard cleavage of the precursor by these enzymes in inflammatory fluids. The cDNA encoding ICE has been reported (Cerretti et al., 1992). The 45 kDa precursor of ICE requires two internal cleavages before becoming the enzymatically active heterodimer composed of a 10 and a 20 kDa chain. The active site cysteine is located on the 20 kDa chain. ICE itself contributes to autoprocessing of the ICE precursor by undergoing oligomerization with itself or homologs of ICE (Wilson et al., 1994; Gu et al., 1995). In the presence of a tetrapeptide competitive substrate inhibitor of ICE, the generation and secretion of mature IL-1 is reduced and precursor IL-1 accumulates mostly inside but also outside the cell (Thornberry et al., 1992). This latter finding supports the concept that proIL-1 can be released from a cell independent of processing by ICE. Methods for measuring IL-1 detect primarily the mature form of IL-1 (Herzyk et al., 1992). Hence, agents and conditions reported to reduce the synthesis of IL-1 may, in fact, only inhibit secretion of mature IL-1 . Futhermore, prevention of the cleavage of the ICE precursor may also account for an apparent inhibition of IL-1 synthesis. Due to alternate RNA splicing, there are five isoforms of human ICE (Alnemri et al., 1995): ICE, , , , and ". ICE cleaves the ICE precursor and the IL-1 precursor; it is presumed that ICE and ICE also process precursor ICE. ICE" is a truncated form of ICE which may inhibit ICE activity by binding to the p20 chain to form an inactive ICE complex. Enhancement of processing and secretion can be also be regulated; adding ATP to LPS-stimulated cells increases IL-1 secretion whereas blocking anion transport reduces the secretion of mature IL-1 (Laliberte et al., 1994), an effect which may be due to inactivation of ICE. A reduction in cellular potassium is associated with increased processing of the IL-1 precursor (Walev et al., 1995).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce The primary sources of IL-1 are monocytes, macrophages, and dendritic cells. B lymphocytes and NK cells are also sources. Keratinocytes will produce IL-1 when stimulated, although these

IL-1 cells constitutively express IL-1. Fibroblasts and epithelial cells generally do not produce IL-1 . In health, circulating human blood monocytes or bone marrow aspirate do not constitutively express IL-1 (Mileno et al., 1995; Shapiro and Dinarello, 1997; Puren et al., 1999). Reports of constitutive expression of IL-1 in health are due to the activation of the IL-1 transcriptional process by surface contact (Schindler et al., 1990a). However, there seems to be constitutive expression of IL-1 in the human hypothalamus (Breder et al., 1988). Many malignant tumors express IL-1 as part of their neoplastic nature, including acute myelogenous leukemia (Cozzolino et al., 1989; Rambaldi et al., 1991; Estrov et al., 1993; Wetzler et al., 1994) and juvenile myelogenous leukemia (SchiroÁ et al., 1993).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Nearly all microbes and microbial products induce the production of IL-1 . Depending on the stimulant, IL-1 mRNA levels rise rapidly within 15 minutes but start to fall after 4 hours. This decrease is thought to be due to the synthesis of a transcriptional repressor and/or a decrease in mRNA half-life (Fenton et al., 1988; Jarrous and Kaempfer, 1994). Using IL-1 itself as a stimulant of its own gene expression, IL-1 mRNA levels were sustained for over 24 hours (Schindler et al., 1990b; Serkkola and Hurme, 1993). Raising cAMP levels in these same cells with histamine enhances IL-1-induced IL-1 gene expression and protein synthesis (Vannier and Dinarello, 1993). In human peripheral blood mononuclear cells (PBMCs), retinoic acid induces IL-1 gene expression but the primary precursor transcripts fail to yield mature mRNA (Jarrous and Kaempfer, 1994). Inhibition of translation by cycloheximide results in enhanced splicing of exons, excision of introns, and increased levels of mature mRNA (superinduction) by two orders of magnitude. Thus, synthesis of mature IL-1 mRNA requires an activation step to overcome an apparently intrinsic inhibition to process precursor mRNA. Stimulants such as the complement component C5a (Schindler et al., 1990c), hypoxia (Ghezzi et al., 1991), adherence to surfaces (Schindler et al., 1990a) or clotting of blood (Mileno et al., 1995) induce the synthesis of large amounts of IL-1 mRNA in monocytic cells without significant translation into the IL-1 protein. This dissociation between transcription and translation is characteristic of IL-1 but

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also of TNF (Schindler et al., 1990a). It appears that the above stimuli are not sufficient to provide a signal for translation despite a vigorous signal for transcription. Without translation, most of the IL-1 mRNA is degraded. Although the IL-1 mRNA assembles into large polyribosomes, there is little significant elongation of the peptide (Kaspar and Gehrke, 1994). However, adding bacterial endotoxin or IL-1 itself to cells with high levels of steady-state IL-1 mRNA results in augmented translation (Schindler et al., 1990a, 1990c) in somewhat the same manner as the removal of cycloheximide following superinduction. One explanation is that stabilization of the AU-rich 30 untranslated region takes place in cells stimulated with LPS. These AU-rich sequences are known to suppress normal hemoglobin synthesis. The stabilization of mRNA by microbial products may explain why low concentrations of LPS or a few bacteria or Borrelia organisms per cell induce the translation of large amounts of IL-1 (Miller et al., 1992). Another explanation is that IL-1 stabilizes its own mRNA (Schindler et al., 1990b) by preventing deadenylation as it does for the chemokine GRO (Stoeckle and Guan, 1993). Removal of IL-1 from cells after 2 hours increases the shortening of poly(A) and IL-1 apparently is an important regulator of gro synthesis because it prevents deadenylation. In fact, of the several cytokines induced by IL-1, large amounts of the chemokine family are produced in response to low concentrations of IL-1. For example, 1 pM of IL-1 stimulates fibroblasts to synthesize 10 nM of IL-8 (Shapiro et al., 1994). Studies have taken advantage of pyridinyl-imidazol compounds which block the synthesis of IL-1 without affecting transcription or steady-state levels of mRNA. mRNA levels for either IL-1 or TNF in PBMCs stimulated with LPS in the presence of these compounds are indistinguishable from those in PBMCs stimulated with C5a, hypoxia, or adherence. In other words, there is ample cytokine mRNA but no cytokine protein. These are interesting compounds which have entered clinical medicine. Some are cyclooxygenase/lipoxygenase inhibitors because, by classification, they block these enzymes. Hence they are often called `dual inhibitors'. However, their mechanism of action in suppressing IL-1 and TNF have never been linked to their ability to suppress either cyclooxygenase or lipoxygenase (Sirko et al., 1991). These and other imidazol-like drugs have recently been called `cytokine-suppressing anti-inflammatory drugs' or CSAIDs (Young et al., 1994). These CSAID compounds reduce IL-1 and TNF translation because they bind and inactivate a mitogen-activating protein (MAP) kinase (Lee et al., 1994). Like most MAP kinases, phosphorylation of serine/threonine residues

356 Charles A. Dinarello on various proteins is observed; however, these CSAID-associated MAP kinases apparently phosphorylate proteins which are required for translation of cytokine mRNAs into their respective proteins (Lee et al., 1994). They share homology with that of the yeast hyperosmolarity glycerol 1 gene (HOG-1) and in the human are identical to the 38 kDa MAP kinase which is phosphorylated in cells stimulated with LPS or hyperosmolar concentrations of NaCl (Galcheva-Gargova et al., 1994; Han et al., 1994, 1995). These HOG-1-related MAP kinases are also the same as those which are phosphorylated during IL-1 signal transduction (Freshney et al., 1994; Kracht et al., 1994). Thus, these observations are consistent with the ability of IL-1 or LPS to augment the translation of cytokine mRNA (Schindler et al., 1990c). Of relevance is that initiation factor eIF-4E requires a MAP-like phosphorylation step in order to dissociate from a translational regulatory molecule.

large amounts of IL-1R AcP, the high-affinity binding of the IL-1R/IL-1R AcP complex may explain why two classes of binding have been observed. Human, recombinant 17 kDa IL-1 binds more advidly to the nonsignal transducing type II receptor (IL-1RII) at 100 pM. IL-1 binding to the soluble form of the IL-1RI is lower than to the cell-bound receptor. However, the most dramatic differences in IL-1 binding can be seen at the level of the soluble form of the type II receptor. Of the three ligands, the most avid binding is that of mature IL-1 (500 pM). By comparison, IL-1 and IL-1Ra bind with 50-fold or lower affinities. In addition to the highest affinity, IL-1 binding to IL-1sRII is nearly irreversible due to a long dissociation rate (2 hours) (Arend et al., 1994; Dower et al., 1994; Symons et al., 1994). Moreover, pro-IL-1 also preferentially binds to IL-1 soluble RII (Symons et al., 1991, 1993). Interactions of IL-1 with the receptors is summarized in Figure 2.

RECEPTOR UTILIZATION

IN VITRO ACTIVITIES

The high-affinity binding of IL-1 for the IL-1RI ranges from 100 to 450 pM. The concentrations of IL1 which can elicit a biological response are 10± 100 fM. There are two affinities: in cells expressing

In vitro findings IL-1 stimulates cells in vitro in the picomolar to femtomolar range. The list of IL-1 activities on cells

Figure 2 Crosslinking of IL-1R type I and IL-1R accessory protein by IL-1 or IL-1 induces IL-1R signaling. IL-1Ra binds to IL-1R but does not activate. Type II receptor, either on membrane or in solution, acts as a decoy. Ra

IL-1 in vitro is best understood at the level of gene expression. As summarized in Table 1, upon exposure to IL1 , gene expression is either increased or decreased.

Regulatory molecules: Inhibitors and enhancers The most important regulatory molecule for IL-1 is the IL-1Ra which is produced usually in 10- to 100fold molar excess (Granowitz et al., 1991; Fischer

et al., 1992) and reviewed in (Arend et al., 1998). In addition, the soluble form of the IL-1R type II has a high affinity for IL-1 and the IL-1 precursor and is produced in 5±10 molar excess. Enhancers of IL-1 production are not common, whereas natural inhibitors include IFN and IFN. For example, IFN inhibited IL-1-induced PGE2 in human blood monocytes (Browning and Ribolini, 1987). IFN also inhibited IL-1-induced IL-1 (Schindler et al., 1990b; Ghezzi and Dinarello, 1988). IFN also inhibits IL-1-induced IL-10 (Schindler

Table 1 Summary of the effect of exposure to IL-1 on gene expression Genes that increase following exposure to IL-1 Cytokines

IL-1, IL-1Ra, TNF, IL-2, IL-3, IL-6, GM-CSF, TGF 3, G-CSF, M-CSF, steel factor, LIF, IFN, IL-8 and chemokine family, MIP-1

Cytokine receptors

IL-2 (p55), IL-2, IL-3, IL-5, GM-CSF ( c receptor chain), c-kit

Proinflammatory mediators

Cyclooxygenase type 2 Cytosolic and secretory phospholipase A2, type 2 Nitric oxide synthase Endothelin 1 Gammaglutamyltransferase

Hepatic acute phase reactants

Mn superoxide dismutase C-reactive protein (PTX3), serum amyloid A Complement C2, C3, factor B Metallothioneins, ceruloplasmin, lysozyme Xanthine dehydrogenase, xanthine oxidase

Growth factors

PDGF A chain, FGF, KGF Hepatocyte growth factor Nerve growth factor MGSA (gro-, , ) Insulin-like growth factor 1 Activin A

Clotting factors

Fibrinogen Urokinase plasminogen activator Type 1 and 2 plasminogen activator inhibitor Protease nexin 1

Tissue remodeling

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Stromelysin, gelatinases, elastase, collagenases Tissue inhibitor of metalloproteinases 1, transin

Neuropeptides

Pro-opiomelanocorticotropin, corticotropin-releasing factor

Lipid synthesis

Triglyceride-increasing Apo CIII

Oncogenes

c-jun, c-abl, c-fms, c-myc, c-fos

Adhesion molecules

ICAM-1, ELAM, VCAM-1

Receptors

Low-density lipoprotein

Apolipoprotein J

358 Charles A. Dinarello

Table 1 (Continued) FGF IL-1R type II Extracellular matrix

Aortic smooth muscle cell decorin Collagen type IV Beta amyloid precursor Basement membrane protein 40 Laminin B1 and B2

Others

Constitutive heat shock protein p70 Ornithine decarboxylase Heme-oxygenase G protein  subunit Aromatic 1-amino decarboxylase

Genes which decrease in steady-state levels following exposure to IL-1 Housekeeping genes

Albumin, alkaline phosphatase

Receptors

IL-1R type I

Cytochrome p450c17, p450 IIBI, IID PDGFR Cytokines Extracellular matrix proteins

TGF 1, insulin-like growth factor I (in Leydig cells) Proteoglycans (chondroitin sulfate) Type II collagen, fibronectin and thrombospondin

Others

CD34 on endothelium Thyroid peroxidase Thyroglobulin Glutaminase in fibroblasts

et al., 1990b). In cultured chondrocytes, IFN reduced IL-1-induced expression of collagenase (Andrews et al., 1989, 1990). Spontaneous production of IL-1 by synovial fluid macrophages from patients with rheumatoid arthritis was inhibited by IFN (Ruschen et al., 1989). Although IL-10 inhibits IL-1 synthesis, it does not inhibit IL-1Ra production. IL-10 injected into humans inhibits the synthesis of IL-1 ex vivo (Chernoff et al., 1995). There are a number of reports that focus on the ability of IL-10 to suppress gene expression and synthesis of inflammatory cytokines (reviewed by Moore et al., 1993). Cell signaling following the engagement of IL-10 to its receptor includes phosphorylations of JAK1 and TYK2, very similar to that of IFN. Some studies have shown that IL-10 inhibits the translocation of NFB. Most studies on the anti-inflammatory properties of IL-10 have focused on suppression of macrophage cytokines. IL-10 suppresses IL-1, IL-1 , TNF, IL-6, IL-8, IL-12,

GM-CSF, G-CSF, M-CSF, MIP-1, RANTES, LIF, and IL-10 itself. Explants of human rheumatoid synovium release IL-1 and TNF as well as IL-10. However, specific neutralization of IL-10 resulted in an increase in the production of IL-1 and TNF, suggesting that the IL-10 constitutively produced in rheumatoid synovium acts as a natural suppressor of IL-1 and TNF production (Katsikis et al., 1994). Neutralization of IL-10 also resulted in detectable IFN . IL-13 and TGF inhibit IL-1 production (Vannier et al., 1996). Although IL-6 is found in a variety of inflammatory, hematological, and infectious diseases, IL-6 suppresses IL-1-inducible cyclooxygenase (Hauptmann et al., 1991); in addition, IL-6 suppresses gene expression and synthesis of inflammatory cytokines (Aderka et al., 1989; Schindler et al., 1990d). Similarly to IL-6, CNTF binds to its specific soluble receptor, and when incubated with human blood PBMCs, the CNTF/

IL-1 soluble receptor complex suppresses IL-1-induced IL-8 and PGE2 synthesis (Shapiro et al., 1994).

Bioassays used There are several bioassays for IL-1 in vitro. Although the early studies focused on IL-1 activation of murine thymocytes (Dinarello et al., 1986a) and the TH2 cell line D10 (Orencole and Dinarello, 1989), currently the best in vitro bioassay for IL-1 is the induction of IL-8 or IL-6 from fibroblasts (Shapiro et al., 1994; Kaplanski et al., 1994). In addition, IL-1 induction of PGE2 is also a reliable bioassay for IL-1 .

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Because a null mutation in the IL-1 gene results in a phenotypically normal mouse, there is probably no normal physiological role for IL-1 in health. The IL-1 -deficient mouse is now nearly 4 years in producing offspring and there are no signs of increased susceptibility to disease or rapid aging.

Species differences In general, nearly all species tested respond to IL-1 . Human IL-1 induces a variety of in vivo responses in mice, rabbits, rats, etc. that are also observed with mouse and rabbit IL-1 (Cannon et al., 1989a).

Knockout mouse phenotypes As stated above, the IL-1 -deficient mouse is without abnormal findings after 4 years of continuous breeding. However, upon challenge, IL-1 -deficient mice exhibit specific differences from their wild-type controls. The most dramatic is the response to local inflammation followed by a subcutaneous injection of turpentine (50±10 mL). Within the first 24 hours, IL-1 -deficient mice injected with turpentine do not manifest an acute phase response, do not develop anorexia, have no circulating IL-6 and have no fever (Zheng et al., 1995; Fantuzzi et al., 1997a). These

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findings are consistent with those reported in the same model using anti-IL-1R type I antibodies in wild-type mice (Gershenwald et al., 1990). IL-1 deficient mice also have reduced inflammation following zymosan-induced peritonitis (Fantuzzi et al., 1997b). Additional studies have also found that IL-1 -deficient mice have elevated febrile responses to IL-1 and IL-1 (Alheim et al., 1997). In addition, there appears to be some reduction in body temperature responses and behavioral changes in IL-1 mice. In contrast, IL-1 -deficient mice have nearly the same responses to LPS as do wild-type mice (Fantuzzi et al., 1996) with one notable exception. IL-1 deficient mice injected with LPS have little or no expression of leptin mRNA or protein (Faggioni et al., 1998). In pregnant IL-1 -deficient mice there is a normal response to LPS-induced premature delivery; however, in these mice there are decreased uterine cytokine levels following LPS (Reznikov et al., 1999). The reduction in LPS-induced cytokines is not found in nonpregnant IL-1 -deficient mice, suggesting that the combination of the hormonal changes in pregnancy and the state of IL-1 deficiency act together to reduce the responsiveness to LPS. The mechanism for the reduced cytokine production in pregnant IL-1 deficient mice appears to be a reduction in the constitutive level of the p65 component of NFB (Reznikov et al., 2000). No differences were noted in plasma elevations of glucocorticoid steroids between IL-1 -deficient and wild-type mice following injection of LPS, indicating that IL-1 is not required for activation of the HPA axis during endotoxemia (Kozak et al., 1998). The data demonstrate that in the mouse, IL-1 is critical for the induction of fever during local inflammation. Another characterized IL-1 actvity was studied by feeding live influenza virus in IL-1 -deficient mice. Body temperature and activity were lower in IL-1 deficient mice (Kozak et al., 1995). The anorexic effects of influenza infection were similar in both groups of mice. The mice deficient in IL-1 exhibited a higher mortality to influenza infection than the wild-type mice.

Transgenic overexpression There are no reports of mice overexpressing IL-1 .

Pharmacological effects Unlike TNF, even large doses of IL-1 do not result in death. However, adrenectomized mice have

360 Charles A. Dinarello increased susceptibility to the lethal effects of IL-1 (Bertini et al., 1988). Injection of IL-1 in primates induces neutrophilia and acute phase proteins. Injection of modest doses IL-1 into mice (1±10 mg/kg) induces fever, anorexia, and circulating IL-6. Injection of IL-1 intravenously into rabbits induces a shock-like state (Okusawa et al., 1988). The most dramatic responses to the pharmacological effects of IL-1 are observed in humans.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects

Although there are many interactions of IL-1 with other cytokines, the most consistent and the most clinically relevant is the synergism of IL-1 and TNF. In rabbits, this synergism is manifested with a severe shock-like state and acute respiratory distress and death (Okusawa et al., 1988). In fact, there are few examples in which the synergism between IL-1 and TNF has not been demonstrated. These include radioprotection, Shwartzman reaction, PGE2 synthesis, sickness behavior, nitric oxide production, nerve growth factor synthesis, insulin resistance, loss of mean body mass, and IL-8 and chemokine synthesis. An illustration of the molecular and cellular interactions initiated by IL-1 is shown in Figure 3.

Highly sensitive assays for detecting IL-1 in the circulation of healthy humans reveals levels less than 10 pg/mL. Following a challenge injection of LPS into healthy humans, IL-1 levels increase in the circulation from 3 to 6 hours and then return to the baseline levels (Cannon et al., 1990). Increases are also seen after exercise (Cannon et al., 1989b). However, unlike IL-6 or TNF or IL-8, levels of IL-1 in human disease are remarkably low. Even in patients with severe sepsis, the levels of circulating IL-1 are low (Casey et al., 1993). Nevertheless, these levels do correlate with severity of illness (Casey et al., 1993; Dinarello and Cannon, 1993). What is the explanation? IL-1 is a very potent molecule when present in the circulation as evidenced by the response in humans to intravenously injected IL-1 (see below). The levels of IL1 measured in the circulation during disease are in the femtomolar range, consistent with the levels that would be reached following intravenous injection.

Endogenous inhibitors and enhancers

Role in experiments of nature and disease states

The endogenous inhibitors of IL-1 activity are TGF , IL-10, IFN , IFN, IL-13, and members of the gp130 family (IL-6 and CNTF). These are described in the section on In vitro activities.

The role of IL-1 in human disease states is best revealed by two groups of experiments: (1) the response of humans to parenterally administered IL1 , and (2) the response of humans with pre-existing

Interactions with cytokine network

Figure 3 Interactions between IL-1 and the endothelium lead to capillary leak and leukocyte recruitment.

IL-1 disease to IL-1 blockade either using the IL-1Ra or soluble IL-1R type I.

Response of Humans to Parenterally Administered IL-1 Although the systemic effects of IL-1 are normally studied in animals, IL-1 has been injected in patients with various solid tumors or as part of a reconstitution strategy in bone marrow transplantation. Acute toxicities of either IL-1 were greater following intravenous compared to subcutaneous injection; subcutaneous injection was associated with significant local pain, erythema, and swelling (Kitamura and Takaku, 1989; Laughlin et al., 1993). Chills and fever are observed in nearly all patients, even in the 1 ng/kg dose group (Tewari et al., 1990). The febrile response increases in magnitude with increasing doses (Crown et al., 1991, 1993; Smith et al., 1992, 1993; Nemunaitis et al., 1994) and chills and fever were abated with indomethacin treatment (Iizumi et al., 1991). In patients receiving IL-1 (Crown et al., 1991; Nemuanaitis et al., 1994), nearly all subjects experienced significant hypotension at doses of 100 ng/kg or greater. Systolic blood pressure fell steadily and reached a nadir of 90 mmHg or less 3±5 hours after the infusion of IL-1. At doses of 300 ng/kg, most patients required intravenous pressors. By comparison, in a trial of 16 patients given IL-1 from 4 to 32 ng/kg subcutaneously, there was only one episode of hypotension at the highest dose level (Laughlin et al., 1993). At 30±100 ng/kg of IL-1 patients exhibited had a sharp increase in cortisol levels 2±3 hours after the injection. Similar increases were noted in patients given IL-1. In 13 of 17 patients given IL-1 , there was a fall in serum glucose within the first hour of administration and in 11 patients, glucose fell to 70 mg/100 mL or lower (Crown et al., 1991). No changes were observed in coagulation parameters such as prothrombin time, partial thromboplastin, or fibrinogen degradation products. This latter finding is to be contrasted to TNF infusion into healthy humans which results in a distinct coagulopathy syndrome (van der Poll et al., 1990). Not unexpectedly, IL-1 infusion into humans significantly increased circulating IL-6 levels in a dosedependent fashion. Humans injected with IL-1 (Bargetzi et al., 1993) exhibit a rapid increase in circulating IL-1Ra and TNF soluble receptors (p55 and p75) and were observed following a 30-minute intravenous infusion. Because of the acute toxicities associated with doses of IL-1 above 30 ng/kg, a study injected six humans with metastatic melanoma with 3 ng/kg intravenously over a 30-minute infusion

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(Ogilvie et al., 1996). One hour after the start of the infusion, there was evidence of hypotension. There was fever (3 hours) and tachycardia. Plasma levels of IL-6 reached a peak of 25 pg/mL and IL-8 reached a peak of 311 pg/mL after 2 hours. There was also a statistically significant increase in plasma nitrite/ nitrate. IL-1 induced neutrophilia (200% increase) after 5 hours.

Response of Humans with Pre-existing Disease to IL-1 Blockade either using the IL-1Ra or Soluble IL-1R Type I Numerous in vitro studies have implicated a role for IL-1 in the pathogenesis of rheumatoid arthritis. These are based on incubating tissue explants and cell lines with IL-1 and measuring the production of PGE2, metalloproteinases, collagenases, chemokines, and the synthesis of several other cytokines and chemokines. Various animal models of inflammatory joint disease also focus on IL-1 as a key player in the disease. For example, infusion of IL-1 into the joint space of healthy rabbits results in inflammation and cartilage and bone loss (Feige et al., 1989). Most importantly, however, are animal models such as collagen-induced arthritis in mice and adjuvant arthritis in rats. Other models include bacterial cell wallinduced inflammation. In genetically altered mice overexpressing TNF, spontaneous rheumatoid arthritis-like lesions develop in the joints with progressive inflammation, cellular proliferation, and bone destruction. Although these findings strongly suggest that TNF mediates the pathological tissue remodeling of the disease, when these mice are treated with an antibody to the IL-1R type I the disease is dramatically reduced. In fact, in mice overexpressing TNF but deficient in the IL-1R type I, there is little or no disease (Siegel et al., 1995). These findings are highly consistent with the well-established ability of TNF to induce IL-1 (Dinarello et al., 1986b). Using explants of synovium from patients with rheumatoid arthritis, antibodies to TNF reduce the spontaneous production of IL-1 (Brennan et al., 1989). The strongest case for a pivotal role for IL-1 in rheumatoid arthritis is that the administration of antiIL-1 (Gieger et al., 1993) or IL-1Ra to animals, both highly specific in blocking IL-1, has reduced the intensity and destructive nature of the disease. IL-1Ra treatment of rats with established collagen-induced arthritis resulted in a near-complete suppression of all parameters of the disease (Bendele et al., 1999). In general, blocking IL-1 with IL-1Ra in collageninduced arthritis after the establishment of disease results in decreased joint destruction compared to

362 Charles A. Dinarello blocking TNF (van den Berg et al., 1994; van den Berg, 1998). Since IL-1Ra has been administered to humans trials of septic shock or rheumatoid arthritis, one can make specific conclusions about the role of IL-1 in human disease. In experimental endotoxemia, because endotoxin signals its own receptor, the systemic endotoxin model may not be optimal for testing the clinical utility and appropriate dose of anti-cytokine therapy for human disease. Nevertheless, the results of these studies shed light on the actions of cytokine blockade in a model of systemic inflammatory response syndrome, commonly termed SIRS. Pretreatment of subjects with 10 mg/kg of IL-1Ra prior to intravenous endotoxin did not reduce fever or systemic symptoms, although there was a statistically significant but modest decrease (40%) in circulating neutrophils (Granowitz et al., 1993). In another study, volunteers were pretreated with soluble IL-1R type I or placebo and then challenged with endotoxin. Again, no effects on fever or systemic symptoms were noted. Although there was a decrease in the level of circulating IL-1 compared to placebo-treated volunteers, there was also a decrease in the level of circulating IL-1Ra ( p < 0.001) due to complexing of the soluble receptor to endogenous IL-1Ra (Preas et al., 1996). This was dose-dependent and resulted in a 43-fold decrease in endotoxin-induced IL-1Ra. High doses of soluble IL1R type I were also associated with higer levels of circulating TNF and IL-8 as well as cell-associated IL-1 (Preas et al., 1996). These results support the concept that soluble IL-1R type I binds endogenous IL-1Ra and reduces the biological effectiveness of this natural IL-1 receptor antagonist in inhibiting IL-1. As discussed below, patients with rheumatoid arthritis treated with soluble IL-1R type I do not exhibit improved clinical outcome and the mechanism is likely to be the binding of endogenous IL-1Ra with a reduction in its biological role. IL-1Ra has been in given to patients with septic shock, rheumatoid arthritis, and steroid-resistant graftversus-host disease. A phase II randomized, placebocontrolled, open-label trial in 99 patients with septic shock was carried out. Patients received either placebo, or a loading bolus of 100 mg followed by a 3-day infusion of 17, 67, or 133 mg/hour IL-1Ra (Fisher et al., 1993). A dose-dependent improvement in 28day mortality was observed; mortality was reduced from 44% in the placebo group to 16% in the group receiving the highest dose of IL-1Ra ( p ˆ 0.015). In that study, there was a dose-related fall in the circulating levels of IL-6 24 hours after the initiation of IL-1Ra infusion. This fall in IL-6 levels is consistent with the well-established control of circulating

IL-6 levels by IL-1 and the correlation of disease severity and outcome with IL-6 levels. This phase II trial was followed by a large placebocontrolled, double-blind study. In this phase III trial, 893 patients were randomized to receive placebo or a loading bolus of 100 mg followed by a 3-day infusion of 1 or 2 mg/kg/hour IL-1Ra or placebo. There was no statistically significant reduction in all-cause mortality in the entire group (Fisher et al., 1994). However, a retrospective analysis of patients with a predicted risk of mortality of 24% or greater revealed a significant reduction in 28-day mortality (45% in the placebo group and 35% in patients receiving 2 mg/kg/hour for 72 hours, p=0.005) (Fisher et al., 1994). In addition, the post-study analysis of data revealed an increase in survival time with IL-1Ra in patients with one or more organ failures (n=563; p=0.009). Although there was no overall statistically significant benefit in 28-day all-cause mortality, the results revealed a trend in the group receiving IL-1Ra and warranted another trial with the primary endpoint targeted to patients with a high risk of mortality (Fisher et al., 1994). This first phase III study was analyzed by Knaus and coworkers (1993). Although the mortality rate in the placebo group was 41.8% and 41.5% in the IL-1Ra group, in patients who were bacteremic at study entry, the mortality was 37% in the placebo group and 28% in the IL-1Ra group ( p ˆ 0.071). In patients without bacteremia, a 36% mortality rate was observed in both groups. Also, in patients who had a documented pathogen identified at study entry had a lower mortality rate if they had received IL-1Ra (31.5%) compared to placebo (37.5%), p ˆ 0.12. Patients without a documented infection had a slightly higher mortality rate than the placebo group but this difference did not reach statistically significant levels ( p=0.21). A second phase III trial in 91 academic centers in North America and Europe was initiated intending to randomize 1300 patients to either placebo or IL-1Ra. The IL-1Ra was administered as an intravenous bolus injection of 100 mg followed by 3 days of constant infusion of 2.0 mg/kg/hour. The primary endpoint was survival time in patients with end-organ dysfunction and/or shock at the time of entry. There were 512 patients who met these entry criteria. However, there were 184 patients who had been enrolled and randomized but who did not meet the primary endpoints. The mortality was 24.2% in the placebo group and 18.3% in the IL-1Ra group ( p ˆ 0.33). A mid-trial analysis was undertaken after 696 patients had been enrolled. The study was terminated during an interim analysis because a reduction in overall 28-day mortality would probably not reach statistical significance. The patient groups were well matched in that 52.9% of the placebo patients

IL-1 and 50.9% of the IL-1Ra group were in shock at the time of study entry. There was no excess mortality in patients receiving IL-1Ra. The 28-day all-cause mortality was 33.1% in the IL-1Ra group (116/350) compared to 36.4% (126/346) in the placebo group ( p ˆ 0.36), a 9% reduction in mortality ( p=0.36). However, as in each of the IL-1Ra trials in patients with septic shock, subgroups appeared to have benefitted. For example, patients with gram-positive infection had a mortality rate of 35.4% compared to 28.9% in the IL-1Ra-treated group ( p=0.40). This was offset by increased mortality in the group receiving IL-1Ra for unknown organism(s) (41.8% versus 28.6%, p=0.21). Nevertheless, unless such a hypothesis is retested in another prospective trial, the subgroup analysis remains unproven. A survival analysis was made in those patients who had major organ dysfunction (target population) and, like the overall group, there was no difference in probability of survival. These analyses support the view that (1) IL-1Ra administration in patients with life-threatening sepsis is safe, (2) there is a consistent but small improvement in survival with IL-1Ra, (3) there are patients with particular presentations at trial entry that clearly benefit from IL-1Ra treatment, and (4) the overall patient group, although with improved survival, is too heterogeneous to show statistically significant differences. Are we asking too much from monotherapy in sepsis? Similar results have been reported for anti-TNF-based therapy in these patients (Abraham et al., 1995, 1997, 1998; Fisher et al., 1996). One concept is to combine IL-1Ra with anti-TNF strategies for treating septic shock. IL-1Ra was initially tested in a trial in 25 patients with rheumatoid arthritis. In the group receiving a single subcutaneous dose of 6 mg/kg, there was a fall in the mean number of tender joints ( p< 0.05) (Lebsack et al., 1991). In patients receiving 4 mg/kg per day for 7 days, there was a reduction in the number of tender joints from 24 to 10, the erythrocyte sedimentation rate fell from 48 to 31 and C-reactive protein decreased from 2.9 to 1.9 mg/mL. In this group the mean plasma concentration of IL-1Ra was 660  240 ng/mL. In an expanded double-blind trial, IL-1Ra was given to 175 patients (Campion et al., 1996). Patients enrolled into the study had active disease and were taking nonsteroidal anti-inflammatory drugs and/or up to 10 mg/day of prednisone. There was an initial phase of 3 weeks during which patients were given either 20, 70, or 200 mg IL-1Ra one, three, or seven times per week. Thereafter, patients received the same dose once a week for 4 weeks. Placebo was given to patients once a week for the entire 7-week study period. To maintain the blindness of the study,

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patients received daily injections of either IL-1Ra or placebo on the days IL-1Ra was not administered. Four measurements of efficacy were used: number of swollen joints, number of painful joints, and patient and physician assessment of disease severity. A reduction of 50% or greater in these scores from baseline was considered significant in the analysis. After 3 weeks, a statistically significant reduction in the total number of parameters was observed with the optimal improvement in patients receiving 70 mg per day. Daily dosing appeared more effective than weekly dosing when assessed by the number of swollen joints, the investigator and patient assessments of disease activity, pain score, and C-reactive protein levels. A large double-blind, placebo-controlled multicenter trial of IL-1Ra in 472 patients with rheumatoid arthritis has been reported (Bresnihan et al., 1998). The study group comprised patients who had discontinued use of disease-modifying agents such as gold and methotrexate 6 weeks prior to entry. Patients had active and severe RA (disease duration 8 years) and were recruited into a 24-week course of therapy. They were divided into a placebo group and three IL-1Ra groups. Patients had stable disease of two or more years duration which was controlled on nonsteroidal anti-inflammatory agents. Some were taking less than 10 mg of prednisone daily. Three doses of IL-1Ra administered subcutaneously were used: 30, 75, and 150 mg/day for 24 weeks. At entry, age, sex, disease duration, and percentage of patients with rheumatoid factor and joint bone erosions were similar in each of the groups. After 24 weeks, 43% of the patients receiving 150 mg/day of IL-1Ra met the American College of Rheumatology criteria for response (the primary efficacy measure), 44% met the Paulus criteria, and statistically significant ( p ˆ 0.048) 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 and Health Assessment score. In addition, there was a dose-dependent reduction in C-reactive protein level, and erythrocyte sedimentation rate. Importantly, the rate of radiologic progression in the patients receiving IL-1Ra was significantly less than in the placebo group at 24 weeks, as evidenced by the Larsen score and the erosive joint count. The reduction in new bone erosions was assessed by two radiologists who were blinded to the patient treatment as well as blinded to the chronology of the X-ray films. This finding suggests that IL-1Ra is blocking the osteoclast-activating factor property of IL-1, as has been reported in myeloma cell cultures (Torcia et al., 1996). This study confirmed both the

364 Charles A. Dinarello efficacy and the safety of IL-1Ra in a large cohort of patients with active and severe rheumatoid arthritis. The only side-effect was local skin rash which was observed primarily during the first 2 weeks of therapy. After 24 weeks, there were no statistically significant increases in infection in the IL-1Ra group compared to placebo. When the trial was extended for another 24 weeks, there was also no statistically significant increases in infection. Some patients have now been treated with IL-1Ra for over 3 years and there are no reports of increased infection or cancer. Another trial of IL-1Ra in patients with rheumatoid arthritis is being carried out in patients randomized to receive treatment with methotrexate or different doses of IL-1Ra plus methotrexate treatment. Soluble IL-1R type I was administered subcutaneously to 23 patients with active rheumatoid arthritis in a randomized, double-blind, two-center study. Patients received subcutaneous doses of the receptor at 25, 250, 500, or 1000 mg/m2/day or placebo for 28 consecutive days. Although 4 of 8 patients receiving 1000 mg/m2/day showed improvement in at least one measure of disease activity, only 1 of these 4 patients exhibited clinical improvement (Drevlow et al., 1996). Similar to the placebo-treated patients, lower doses of the receptor did not result in any improvement by acceptable criteria. Despite this lack of clinical or objective improvement in disease activity, cell surface monocyte IL-1 expression in all patients receiving the soluble IL-1R type I was significantly reduced. Other parameters of altered immune function that are common in patients with rheumatoid arthritis also showed reduction. One possible explanation for the lack of clinical response despite efficacy in suppressing immune responses could be the inhibition of endogenous IL-1Ra. This was observed in volunteers receiving soluble IL-1R type I before challenge by endotoxin (Preas et al., 1996).

IN THERAPY

Preclinical ± How does it affect disease models in animals? There are three therapeutic uses for IL-1 : (1) pretreatment before a toxic or even lethal challenge, (2) as a bone marrow stimulator, and (3) as an adjuvant for vaccines. Each has been tested in animals in preclinical studies and IL-1 has been used in clinical trials to augment bone marrow transplantation. IL-1 has not been tested in clinical trials as a vaccine adjuvant to date.

(1) Pretreatment with IL-1 Before a Toxic Lethal Challenge in Animals In general, pretreatment of animals with a low dose of IL-1 , usually a dose which itself causes no apparent disease, affords protection against or reduces the severity of a subsequent challenge. The challenge event can be lethal infection, an inflammatory agent, or induction of an autoimmune process. For example, adminstration of 40 ng of IL-1 to neutropenic mice 24 hours prior to lethal Pseudomonas infection significantly prolongs survival even when the animals are treated with appropriate antibiotics (van der Meer et al., 1988). Most importantly, protection requires pretreatment, whereas administration of IL-1 at the time of challenge has no effect or worsens the disease. Animals used in most studies are healthy and not immunocompromised. However, in some experiments, the animals are first treated with bone marrowsuppressing drugs or radiation. In other models, mice susceptible to a particular disease due to genetic inheritance, an underdeveloped defense system, or acquired immunosuppression have also been used. Protection is also observed under these conditions. Almost without exception, similar protection can be accomplished using pretreatment with a low dose of endotoxin, TNF, or the combination of IL-1 and TNF (White and Ghezzi, 1989). In healthy animals, a single pretreatment dose of IL-1 has reduced hypotension, organ colony counts, or mortality to infection caused by Pseudomonas, Klebsiella pneumoniae, Escherichia coli, Listeria monocytogenes, Candida albicans, Plasmodium berghei, and Mycobacterium lepraemurium. Protection is often reported for lethal doses of LPS and has also been observed in mixed infections resulting from cecal ligation and puncture. In the nonobese diabetic mouse and the biobreeder rat, multiple IL-1 treatments before the onset of disease have completely prevented spontaneous autoimmune diabetes and thyroiditis as well as inhibited the infiltration of lymphocytes into the islets (Formby et al., 1992). In models of type II collagen, denatured albumin or bacterial cell wall-induced arthritis, IL-1 has reduced the severity of joint inflammation (Schwab et al., 1992; Drelon et al., 1993). Delayedtype skin hypersensitivity reactions are prevented by IL-1 pre-exposure (Wilson et al., 1990). Pretreatment with IL-1 protects against gastroduodenal ulcers and inflammatory bowel disease (Cominelli et al., 1990). In models of lung injury, pretreatment with IL-1 greatly prolongs survival after lethal exposure to hyperoxia (Tsan et al., 1991), reduces the edema and neutophilic infiltration after E. coli injection and the bronchoconstriction after intratracheal challenge in sensitized

IL-1 guinea pigs (Vannier et al., 1989). Myocardial injury following ischemia-reperfusion is prevented by pretreatment with IL-1 (Maulik et al., 1993). LPSinduced circulating TNF levels are reduced by a prior injection of IL-1 (LeContel et al., 1992; Vogels et al., 1994). Similar protection has been observed in immunocompromised animals with neutropenia, viral infection, exogenous stress and genetically immunosuppressed mice. In 24-hour-old rats, marked survival to live Klebsiella pneumoniae infection is observed following a single, low-dose pretreatment with IL-1 without antibiotic treatment (Mancilla et al., 1993). (2) IL-1 as a Bone Marrow Stimulator There is no evidence that IL-1 has a role in normal hematopoiesis. The IL-1 -deficient mouse is without evidence of hematological impairment. The best characterized role for IL-1 in hematopoiesis is its ability to increase the production of CSFs and stem cell factors, either by increasing their transcription or by stabilization of mRNA. A single, low dose of IL-1 can protect up to 90% of mice exposed to lethal radiation (Neta et al., 1987; Schwartz et al., 1987; Moreb et al., 1989). Several mechanisms may explain the ability of IL-1 to protect bone marrow cells; these include a protective effect on the pluripotent stem cell, the myeloid stem cell, and the early progenitor cells. In addition, arrest of cell cycling or increases in Mn superoxide dismutase and other antioxidants (Zucali et al., 1994) may be involved. IL-1 increases gene expression and synthesis of c-kit on bone marrow cells and this is thought to explain the synergy of IL-1 and stem cell factor in protecting against lethal radiation (Neta et al., 1994). Using IL-1-treated male marrow cells prior to irradiation and transplantation into female mice, administration of IL-1 protected both short- and long-term repopulating stem cells and accounted for the reconstitution of myeloid and lymphoid organs (Zucali et al., 1994). Animal models of myelosuppression often include IL-1 as part of the recovery protocol and studies in marrow transplantation have shown that a single, low-dose injection of IL-1 accelerates multilineage recovery (Tiberghien et al., 1993). (3) IL-1 as an Adjuvant for Vaccines IL-1 is a potent immunoadjuvant. It enhanced in vivo secondary antibody responses of mice to a protein antigen. The activity was found to be dose- and timedependent. The enhancing effect was obtained when IL-1 was injected 2 hours after the priming dose of antigen (Staruch and Wood, 1983). The studies of IL1 as an adjuvant for vaccines are also derived from

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experiments using the nonapeptide from the IL-1 sequence (163±171) (Antoni et al., 1986, 1989; Boraschi et al., 1992). The advantage of the nonapeptide over IL-1 itself was one of reduced toxicity. However, toxicity to IL-1 may be less when the cytokine is used for mucosal immunization. Also the synthetic nonapeptide VQGEESNDK showed in vivo immunomodulatory capacities qualitatively and quantitatively comparable to those of the mature human IL-1 protein but did not cause any of several inflammation-associated metabolic changes inducible by the whole IL-1 molecule in vivo (Boraschi et al., 1988). To avoid the toxicities of IL-1 , mucosal immunization has been reported. IL-1 was as effective as the most widely used and studied mucosal adjuvant, cholera toxin, for the induction of antigen-specific serum IgG, vaginal IgG and IgA (Staats and Ennis, 1999). IL-1 was administered intranasally with soluble protein antigen. These results indicate that IL-1 may be useful as an adjuvant for mucosal vaccines.

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. (1) Pretreatment with IL-1 Before a Toxic Challenge in Animals The spectrum of pathological models in which this protective phenomenon occurs does not suggest a common mechanism. Because blocking IL-1 activity converts a sublethal infection of Listeria monocytogenes to a lethal infection (Havell et al., 1992; Rogers et al., 1992), mechanisms related to IL-1 enhancement of bactericidal activity have been proposed. However, this is an unlikely mechanism in neutropenic mice. In cerebral malaria, nude mice have less of a protective response to IL-1, suggesting a role for T cells (Curfs et al., 1990). It is clear that a finite time period is needed between the administration of IL-1 and the challenge event, presumably for cellular changes to occur, synthesis of proteins or production of other molecules with protective properties. In general, pretreatment with IL-1 prior to a lethal or injurious challenge induces a state of cellular and organ resistance to death. IL-1-induced heat-shock proteins (Freshney et al., 1994), acute phase proteins (Vogels et al., 1993) or antioxidants (Tsan et al., 1991) could contribute to this state. Because IL-1 is a potent inducer of PLA2 and COX-2, endogenous PGE2 production may account for some of its effects. Indeed, protection in inflammatory bowel disease, skin hypersensitivity, and ulcerogenic conditions is mediated by IL-1-induced

366 Charles A. Dinarello prostaglandin synthesis. Coadministration of cyclooxygenase inhibitors with IL-1 pretreatment prevents protection against lethal oxygen toxicity (White and Ghezzi, 1989). Even when IL-1 itself is given in order to precipitate an injury, protection can be accomplished by administration of free or liposome-bound PGE2 (Leff et al., 1994). On the other hand, in models of infection (van der Meer et al., 1988) and radioprotection (Neta, 1990), prostaglandins are clearly not involved. In models of autoimmune and hypersensitivity reactions in rats, treating animals with low doses of IL-1 results in a suppression of the disease, whereas treating with high doses accelerates the same disease (Wilson et al., 1990). Thus, a low dose of IL-1 acts in a similar fashion to immunosuppressive drugs. In the case of skin hypersensitivity reactions, prostaglandins appear to mediate the immunosuppression. This is not an unexpected finding since IL-1 induces PGE2, which is a well-known suppressor of immune responses and IL-2 production. The effect of cyclooxygenase inhibitors on IL-1-induced suppression of spontaneous autoimmune diabetes in the nonobese mouse (Formby et al., 1992) and biobreeder rat (Wilson et al., 1990) remains unknown. In some models of bacterial infection, induction of corticosteroids appears to be required because the protective effect of IL-1 is abolished in adrenalectomized mice (Fantuzzi and Ghezzi, 1993). In a nonlethal model, LPS-induced circulating levels of TNF are reduced in mice pretreated with a single low dose of IL-1 (LeContel et al., 1992; Vogels et al., 1994); in this model, induction of corticosteroids appears to mediate the protective effect of IL-1 (LeContel et al., 1992). Considerable data support the role of IL-1induced endogenous corticosteroids as a mechanism for IL-1-induced protection (Fantuzzi and Ghezzi, 1993). The ability of IL-1 to induce several antioxidants has been implicated as part of the protective mechanism. In the rat, IL-1-induced protection against lethal oxygen toxicity is associated with increases in catalase, glutathione, and Mn, Cu and Zn superoxide dismutases (Tsan et al., 1991). Similar mechanisms have been proposed for protection of bone marrow stem cells (Moreb et al., 1989; Moreb and Zucali, 1992). In a model of cardiac ischemia-reperfusion, an increase in these antioxidants and heat-shock protein 27 was thought to account for the protection afforded by IL-1 (Maulik et al., 1993). In humans, a single injection of IL-1 induces circulating levels of IL-1Ra and soluble TNF receptors (Tilg et al., 1994). In mice, a single, low dose of IL-1 induces IL-1Ra mRNA in various organs and decreases steady-state mRNA for the p55 TNF receptor (Vogels et al., 1994). In addition, steady-state mRNA levels of

IL-1RI in these organs are reduced, which is in agreement with a decrease in gene and surface expression for this receptor after exposure to IL-1 in vitro (Ye et al., 1992). Taken together, downregulation of these receptors by IL-1 pretreatment desensitizes cells against subsequent damage by IL-1 and TNF. In addition, the production of IL-1Ra and the release of soluble TNF receptors further decrease the activity of IL-1 and TNF. (2) IL-1 as a Bone Marrow Stimulator IL-1 synergizes with a variety CSFs. In fact, `hemopoietin-1', a factor which synergized with CSF, was due to IL-1. The synergism is most apparent on the ex vivo culture enriched with CD34‡ cells. In ex vivo expansion of enriched peripheral blood CD34‡ cells, IL-1 is often added to the cultures together with IL-3 and other CSFs (Brugger et al., 1993). Treatment of bone marrow endothelial cells with IL-1 increases the adherence of CD34‡ progenitor cells which may play a role in regulating the trafficking of pluripotent stem cells (Rafii et al., 1994). Purified mouse stem cells require IL-3, IL-6, and IL-1 for proliferation in vitro (Heimfeld et al., 1991), which suggests that primitive stem cells require multiple signals for growth. (3) IL-1 as an Adjuvant The mechanisms of IL-1 acting as an adjuvant include (a) the induction of IL-6 as a B cell activator, (b) the induction of chemokines to attract immunocompetent cells to the site of the immunogen, (c) induction of other cytokines which participate in the immune response.

Pharmacokinetics (1) Pretreatment with IL-1 Before a Toxic Challenge in Animals Because the doses of IL-1 that are administered to animals to afford protection are low, there are no data on the blood levels; however, it can be assumed to be in the femtomolar range. (2) IL-1 as a Bone Marrow Stimulator There are no specific studies on the best effective dose of IL-1 in these studies. (3) IL-1 as an Adjuvant for Vaccines The amount of IL-1 administered is variable. In the mucosal immune response, 4 mg of IL-1 are instilled

IL-1 intranasally with the protein adjuvant. The blood levels of IL-1 in these mice is unknown.

Toxicity (1) Pretreatment with IL-1 Before a Toxic Challenge in Animals In animals, there is no toxicity observed to the low doses of IL-1 which provides protection. In fact, the basis for IL-1 -induced protection is derived from (a) a low dose and (b) a time interval between the IL-1 injection and the challenge. Usually, this is 12±24 hours. In contrast, the toxicity of IL-1 in clinical trials is considerable and discussed in the section on Clinical results. (2) IL-1 as a Bone Marrow Stimulator Chronic administration of IL-1 to mice induces anemia and this is associated with a decrease in peripheral reticulocytes and suppression of mature CFUE (Furmanski and Johnson, 1990). However, the effect of IL-1 appears to be due to IL-1-induced TNF. This is not surprising since IL-1 induces TNF in vivo and in vitro (Ikejima et al., 1990) and blockade of IL-1 receptors in vivo is associated with decreased levels of circulating TNF (Shito et al., 1993; Aiura et al., 1993). In rats, TNF, but not IL-1, induces anemia which is due to both decreased red cell numbers and half-life (Moldawer et al., 1989). Why is there a discrepancy between the effects of chronic IL-1 in mice and rats? Part of the conflicting issue of IL-1's role in anemia is that in addition to inducing suppressing factors such as TNF and IFN, IL-1 has a positive effect on the primitive progenitors cells. In fact, while suppressing mature CFU-E, IL-1 increases colony formation of more primitive erythroid progenitors (Means and Krantz, 1992). (3) IL-1 as an Adjuvant for Vaccines There is no toxicity to the nonapeptide nor the intranasally administered IL-1 . However, there is toxicity (anorexia) in mice given parenteral IL-1 .

Clinical results

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been carried out. In part, these trials are testing the `protection' of bone marrow stem cells from the chemotherapy- and radiation-induced bone marrow suppression in bone marrow transplantation. (2) IL-1 as a Bone Marrow Stimulator IL-1 has been administered to patients during receiving autologous bone marrow transplantation. Daily treatment with 50 ng/kg IL-1 from day 0 of autologous bone marrow or stem cells resulted in an earlier recovery of neutropenia compared to controls (Nemunaitis et al., 1994). These results were reported in patients with acute myelogenous leukemia (AML) receiving 50 ng/kg/day of IL-1 for 5 days starting at the time of transplantation with purged or nonpurged bone marrow (Nemunaitis et al., 1994). In that study, an IL-1 treatment-associated decrease in infection rate and improved survival was observed. Using subcutaneously administered IL-1 , there were greater numbers of CFU-GM at day 21 compared to historical controls (Laughlin et al., 1993). Injecting humans with low doses of IL-1 confirms the impressive pyrogenic and hypotension-inducing properties of the molecules. The human studies also confirm the effects of IL-1 on stimulating the hypothalamic-pituitary-adrenal axis and on increased cytokine production, particularly IL-6. In many ways, the signs and symptoms following IL-1 injection into humans are indistinguishable from those of low doses of endotoxin (Wolff, 1973). Similar to endotoxin, IL-1 induces a general enhancement of hematopoiesis, particularly in increased neutrophil, monocyte, and platelet counts. In patients given marrow-suppressing chemotherapy, cotreatment with IL-1 decreases the nadir and the duration of the marrow suppression. However, the benefits of IL-1 therapy in these patients are clouded by its formidable toxicity. Low doses of IL-1 may be useful in combination with other hematopoietic growth factors for reducing myelosuppression during chemotherapy or bone marrow transplantation. (3) IL-1 as an Adjuvant To date there are no clinical trials of IL-1 as an adjuvant in either classic immunization schedules or via mucosal immunizations.

(1) Pretreatment with IL-1 Before a Toxic or Even Lethal Challenge in Animals

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