IL18

IL-18 Haruki Okamura*, Hiroko Tsutsui, Shin-ichiro Kashiwamura, Tomohiro Yoshimoto and Kenji Nakanishi Hyogo College of Medicine, Nishinomiya, Japan * corresponding author tel: 81-0798-45-6744, fax: 81-0798-45-6746, e-mail: [email protected] DOI: 10.1006/rwcy.2000.04003.

SUMMARY IL-18 is a strategically important proinflammatory cytokine. It is detectable in the clinical samples of various diseases and its pathophysiological roles are currently under intensive investigation.

BACKGROUND

Discovery Since the cloning of IL-18 in 1995 (Okamura et al., 1995), many investigators have studied the molecular mechanism of its processing, identification of its receptor molecules, its signal transduction pathways, and some of its central bioactivities. This early information was recently reviewed (Dinarello, 1998; Dinarello et al., 1998; Gillespie and Horwood, 1998; Okamura et al., 1998b). Note added in proof: Since this article was prepared, a number of papers have been published. Unexpectedly, it has been found that IL-18 exerts its actions not only in innate immunity and TH1 responses but also in TH2 responses.

Main activities and pathophysiological roles To date, the roles of IL-18 in immune responses, both innate and acquired, have been analyzed and considerable knowledge has been obtained. However, studies on its physiological and pathological roles based on these fundamental activities have only just started. IL-18 appears to play important roles in the

host defenses against infections and tumors, but it also seems to be involved in the pathogenesis of various diseases. The roles of IL-18 produced in tissues other than immune organs, such as intestine, secretory gland, and skin, remain unclear. IL-18 may also play other physiological roles which are still to be determined. Like IL-1, IL-18 may connect the immune system to the endocrine, nervous, and other systems.

GENE AND GENE REGULATION

Accession numbers Human IL-18: D49950 Mouse IL-18: D49949

Chromosome location The genes for IL-18 and its receptor map to different chromosomes. Human IL-18 gene is located on chromosome 11q22.2-22.3, closely linked to the dopamine receptor D2 (DRD2) locus, while the IL-18R gene maps to chromosome 2q13-21, on which human IL-1 family members IL-1, IL-1 , IL-1Ra, and IL-1R type I are located (Parnet et al., 1996; Nolan et al., 1998). The relative gene order of IL-18 is: ATM-IL-18-DRD2-THY1, which is the same as that of murine IL-18 (Rothe et al., 1997a). Preliminary experiments on nonobese diabetic (NOD) mice revealed that murine IL-18 gene localizes within the Idd2 interval, which suggests that IL-18 may be an NOD-susceptible gene on chromosome 9 (Rothe et al., 1997a).

338 Haruki Okamura et al. The murine IL-18 gene is composed of seven exons that distribute over 26 kb, and exons 1 and 2 are noncoding (Tone et al., 1997).

PROTEIN

Accession numbers D49949

Description of protein cDNAs for murine and human IL-18 are shown to encode 192 and 193 amino acids, respectively (Okamura et al., 1995; Ushio et al., 1996). They contain unusual leader sequences composed of 25 amino acids, like IL-1 (Okamura et al., 1995). In addition, homology in the secondary structure of IL-18 to IL-1 was pointed out by the fold recognition method showing the conserved amino acid sequence in the position of sheets (Bazan et al., 1996). It was also predicted that IL-18 is processed by a protease-like IL-1 -converting enzyme (ICE), and this was later proven (Ghayur et al., 1997; Gu et al., 1997). Moreover, the receptor for IL-18 was shown to be a member of the IL-1 receptor family (Parnet et al., 1996; Torigoe et al., 1997). Thus, IL-18 is similar to IL-1 in many respects.

Posttranslational modifications As predicted from the homology in the tertiary structures of IL-18 and IL-1, IL-18 was shown to require cleavage by ICE (caspase 1) to become bioactive (Ghayur et al., 1997; Gu et al., 1997). This was proven by cotransfection into COS cells with proIL-18 cDNA and cDNAs encoding various caspases (Gu et al., 1997), and by direct cleavage action on pro-IL-18 by recombinant caspase 1 (Ghayur et al., 1997). Caspase 1-deficient mice are unable to process IL-18, and these mice are defective in IFN production induced by LPS (Gu et al., 1997). Caspase 4 has cleaving activity on pro-IL-18, but with much lower affinity than caspase 1 (Gu et al., 1997). Caspase 3 also acts on both precursor and mature IL-18 but yields smaller sizes of fragments (Akita et al., 1997; Gu et al., 1997). These small fragments do not induce IFN production. Pro-IL-1 and pro-IL18 are both substrates for caspase 1, but pro-IL-18 has a higher substrate affinity for caspase 1 than does pro-IL-1 .

There is a possibility that IL-18 is processed by other enzymes apart from caspase 1, because ICE gene targeting does not completely impair IL-1 release, and some of the metalloproteinases (MMP-2, -3, -4) cleave pro-IL-1 into a biologically active form (Fantuzzi et al., 1997; SchoÈnbeck et al., 1998). Whether IL-18 is also processed by these metalloproteinases needs to be examined. On the other hand, IL-1 and IL-18 may not be the only substrates for caspase 1: several protein kinase C isoforms are also cleaved by this protease (Bras et al., 1997). The regulation of caspase 1 activation is important for the understanding of IL-18 secretion, since caspase 1 by itself is produced in a biologically inactive form. However, how caspase 1 is activated is not well understood. Caspase 1 was considered to be activated by autocatalytic processing. Recent investigations indicate that caspase 11, which is activated by LPS, cleaves caspase 1 into the active tetramer (Wang et al., 1998). Mutant mice deficient in caspase 11 are resistant to endotoxic shock, and production of IL-1 is also blocked in these mice. Thus, LPS can activate caspase 1, dependent or independent on the activation of caspase 11, in a human macrophage cell line or freshly isolated human monocytes (Schumann et al., 1998). Recently, IL-12 has been shown to be involved in caspase-dependent processing of IL-18 (Fantuzzi et al., 1999). The administration of IL-12 increased serum levels of IL-18 in wild-type mice but not totally in caspase 1-deficient mice. It is of interest that nitric oxide (NO), a biologically active gas essential for clearance of intracellular bacteria, including Listeria monocytogenes, directly inhibits caspase 1, resulting in prevention of IL-1 and IL-18 secretion (Kim et al., 1998). Downregulation of caspase 1 may be important for protection of tissues, because the prolonged production of these cytokines may be harmful. There are two opposite views on the roles of caspase 1 in cell apoptosis. One insists that caspase 1 activation is not connected to apoptosis because apoptosis is inducible in macrophages even when caspase 1 is inhibited (Fiordalisi et al., 1995). The other insists that caspase 1 is involved in apoptosis in many cases, such as mammary epithelial cell apoptosis, when deprived of extracellular matrix (Boudreau et al., 1995). The probability that caspase 1 is involved in apoptosis is also proven by the induction of apoptosis in COS cells coexpressing caspase 1 and IL-1 (Friedlander et al., 1996). This was also demonstrated when neuronal apoptosis caused by ischemic brain injury was not observed in caspase 1-deficient mice (Friedlander et al., 1997; Schelke et al., 1998). However, it remains to be clarified whether activation

IL-18 339 of caspase 1 and processing of IL-18 or IL-1 are coupled with apoptosis of macrophages. Recent experiments have revealed that IL-1 is processed by a caspase other than caspase 1 (Miwa et al., 1998). Peritoneal exudate neutrophils stimulated with FasL release mature IL-1 in association with an apoptotic response in a caspase 1-independent manner. Mature IL-18 is also secreted from Fas-expressing macrophages on stimulation with FasL (Tsutsui et al., 1999). Propionibacterium acneselicited macrophages, Kupffer cells, and splenic macrophages release biologically active IL-18 when stimulated with FasL. This is also the case for P. acnes-elicited macrophages from caspase 1-deficient mice, whereas the same macrophages do not secrete IL-18 after stimulation with LPS, as expected. Macrophages from nontreated mice, either wild-type or caspase 1-deficient, do not secrete IL-18 on FasL stimulation. This is because macrophages do not express Fas under normal conditions, but gain Fas expression after treatment with P. acnes. Fas expression is mediated by the endogenous accumulation of IFN during P. acnes-priming, because P. acneselicited macrophages from IFN -deficient mice do not express Fas even after treatment with P. acnes. IL-18 secretion from P. acnes-elicited macrophages on FasL stimulation is inhibited by simultaneous incubation with general caspase inhibitors. Therefore, IL-18 secretion from FasL-stimulated macrophages is mediated by a caspase other than caspase 1. Proteinase 3, a serine protease stored in the granules of neutrophils and macrophages, is also shown to participate in the processing of IL-18 (Coeshott et al., 1999; Fantuzzi and Dinarello, 1999). Recombinant proteinase 3 has the capacity to cleave pro-IL-18, but at a different site from caspase 1. Proteinase 3 is usually released from cells according to their degranulation after they have been appropriately stimulated. To date, the in vivo role of proteinase 3-dependent processing of IL-18 has not yet been elucidated.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Activated macrophages were first shown to express high levels of IL-18. Subsequent investigations revealed that a wide range of cell types, including mononuclear cells (Puren et al., 1999), keratinocytes (Stoll et al., 1997), osteoblastic cells (Udagawa et al., 1997; Saha et al., 1999), intestine epithelial cells

(Takeuchi et al., 1997), dendritic cells (Stoll et al., 1998; Gardella et al., 1999), chondrocytes (Olee et al., 1999), and neuroblastomas (Hunter et al., 1999) express IL-18 on the basis of the particular structure of the IL-18 gene. Other types of cells may also produce IL-18. It is probable that IL-18 is produced by macrophages or dendritic cells and it is probably involved in immune responses or in host defenses against infection and tumor through IFN production. Although the physiological meaning is not known, keratinocytes express high levels of IL-18 without expressing caspase 1 (Stoll et al., 1997). This expression is upregulated by the stimulation of contact sensitizers. Augmented expression of IL-18 can also be observed in a mouse model for contact hypersensitivity (Xu et al., 1998a). IL-18 may thus be implicated in the pathogenesis of murine skin diseases. IL-18 produced by osteoblastic cells was shown to suppress osteoclast formation via the induction of GM-CSF in lymphocytes (Udagawa et al., 1997; Horwood et al., 1998). Therefore, IL-18 may affect the balance of bone metabolism. IL-18 is clearly detectable in intestinal epithelial cells on top of the villi (Takeuchi et al., 1997). Interestingly, it has been reported that the active lesions of patients with Crohn's disease produce mature IL-18 in association with the presence of active caspase 1, suggesting possible involvement of locally released mature IL-18 in this disease and probably other inflammatory bowel diseases (Monteleone et al., 1999; Pizarro et al., 1999). IL-18 mRNA is induced by IL-1 in chondrocytes, and mature IL-18 is secreted by stimulation with IL-1 (Olee et al., 1999). Both active caspase 1 and IL-18 are also expressed in osteoarthritic cartilage (Saha et al., 1999). IL-18 may regulate chondrocyte functions and contribute to cartilage degradation. It is notable that a variety of endocrine and exocrine glandules, such as pituitary gland, adrenal cortex, and pancreas, express this cytokine (Conti et al., 1996; Okamura et al., 1998b). In particular, IL-18 mRNA is augmented by cold stress in the zona reticularis and fasciculata of the adrenal gland where glucocorticoid is produced. The relationship between IL-18 production and hormone secretion needs to be investigated. Although, on the face of it, these IL-18producing cells have little in common, they do tend to belong to tissues where the cells turn over rapidly. This suggests that production of IL-18 in these cells may be connected to apoptosis. Another common feature of these cells is that they are mostly the secretory cells, and so activation of IL-18 may be linked to secretion of hormones or enzymes. Thus, this cytokine may connect the immune system to

340 Haruki Okamura et al. other systems, including the endocrine system, as do IL-1 and TNF.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators IL-18 is expressed in a variety of cells, including both immune and nonimmune tissues. Analysis of the genomic structure to reveal the mechanism for transcriptional control revealed that two distinct promoters were identified (Tone et al., 1997; Kim et al., 1999). The IL-18 gene comprises seven exons distributed over 26 kb, and promoter activity was detected upstream of noncoding exons, exon l and exon 2, respectively. The former seems to be upregulated in response to LPS, and the latter seems to be constitutively active. This may support the constitutive expression of IL-18 mRNA in macrophages such as Kupffer cells, peritoneal exudate cells, and microglia (Okamura et al., 1995; Conti et al., 1999; Prinz and Hanishc, 1999). Both of these promotors are TATA-less and G+C poor, suggesting IL-18 expression in a wide spectrum of cell types. Furthermore, the IL-18 gene contains only one or no RNAdestabilizing element (Tone et al., 1997). Usually, cytokine genes contain so many copies of RNAdestabilizing elements that the cytokine mRNA has a short half-life, limiting the cytokine production to a restricted period. NFB has been suggested to be involved in upregulation of IL-18 gene expression, because an NFB recognition sequence is located in the promoter region. Moreover, there are also two potential Oct1 recognition sequences, interacting with the viral transcriptional activator protein VP16, which may also upregulate the expression. Taken together, IL-18, presumably pro-IL-18, may be produced in the cytoplasm of macrophages under normal conditions, which may characterize macrophages as cells in the front line to release active IL-18 in prompt response to an appropriate stimulation, such as viral infection and LPS. IL-18 requires regulation at both transcriptional and processing levels.

IN VITRO ACTIVITIES

In vitro findings Induction of IFN in T Cells IL-18 was originally discovered as an IFN -inducing factor. However, IL-18 by itself induces only a trace

amount of IFN production in purified spleen T cells. This is also the case for IL-12. Combined stimulation of IL-18 and IL-12 induces remarkably enhanced production of IFN in these cells (Okamura et al., 1995, 1998a). The underlying mechanism for this strong synergism was analyzed and it was found that IL-12 induces the IL-18 receptor on CD4+ T cells (Ahn et al., 1997; Yoshimoto et al., 1998). The promoter region of the IFN gene contains the consensus sequence for STAT4, AP-1, NF-AT, and NFB (Sica et al., 1997; Barbulescu et al., 1998). Since IL-12 is known to activate STAT4, IL-12 may be involved in the expression of both IL-18 receptor and IFN . IL-18, as well as IL-12, is also shown to activate AP-1 (Barbulescu et al., 1998). So, IL-18 together with IL-12 may synergistically induce transcription of the IFN gene through activation of STAT4 and AP-1. On the other hand, IL-18 has been shown to utilize IRAK and TRAF-6 and activates NFB (Matsumoto et al., 1997; Muzio et al., 1997; Robinson et al., 1997; Adachi et al., 1998; Tsuji-Takayama et al., 1999). The role of NFB in IFN induction has not been investigated, although it is known that the NFB consensus sequence is in the IFN promoter. IFN induction in CD8 Cells CD8+ T cells are an important source of IFN , and these cells produce high levels of IFN in response to IL-18 (Tomura et al., 1998a). Resting CD8+ cells stimulated with anti-CD3 antibody plus anti-CD28 antibody express IL-12R but not IL-18R on them; subsequent addition of IL-12 induces IL-18R, and then IL-18 stimulates IFN production. So, the same synergistic mechanism for IFN induction seems to work on CD8+ T cells, although IL 18 directly activates the cytotoxicity of CD8+ T cells (Kohyama et al., 1998). Effect of IL-18 on TH1 Cells When IL-18 was first being researched, it was considered to be another factor that could differentiate TH1 cells, like IL-12. Recent investigations have revealed that IL-18 itself has no ability to drive the differentiation of TH1 cells but strongly induces them to express their functions and induce synthesis of IFN or Fas ligand (Dao et al., 1996; Kohno et al., 1997; Robinson et al., 1997; Yang et al., 1999). However, IL-18 does not activate TH2 cells (Kohno et al., 1997). IL-12 receptor on TH1 cells is induced by the stimulation of anti-CD3 and anti-CD28 mAbs. As described above, IL-12 was also shown to induce IL-18 receptor on T cells (Ahn et al., 1997;

IL-18 341 Yoshimoto et al., 1998; Okamura et al., 1998a). This was further confirmed by demonstrating that functional IL-18 receptor is selectively expressed on TH1 cells but not on TH2 cells (Xu et al., 1998b). Thus, IL-18 receptor can be regarded as a differentiation marker of TH1 cells. However, surprisingly, it was recently demonstrated that IL-18 enhances production of the TH2 cytokine IL-13 in T and NK cells (Hoshino et al., 1999). The induction of IL-13 was also suggested to be regulated by IFN . These results seem important, because IL-18 may be involved in the amplification of TH1 responses as well as the regulation of TH1 responses through production of TH2 cytokines. IFN Induction in NK Cells IL-18, as well as IL-12, was shown to augment NK cell activity (Okamura et al., 1995; Tomura et al., 1998b). IL-18 also acts on NK cells to produce IFN (Hunter et al., 1997; Tomura et al., 1998b). Like its actions on T cells, IL-18 alone, as well as IL-12 alone, induces NK cells to produce only a little IFN , and a striking synergy between IL-18 and IL-12 has been observed in the induction of IFN in NK cells (Hunter et al., 1997; Fehniger et al., 1999). NK cells were proved constitutively to express IL-18 receptor and augment their cytolytic activity in spleen cells of mice lacking IL-12, IFN , or IL-2 in response to IL-18 (Hyodo et al., 1999). Thus, IFN -inducing activity of IL-18 on NK cells is dependent on IL-12, while its stimulatory activity on cytolysis of NK cells is independent of IL-12. This may indicate that the activation of a signal-transducing system needed for IFN production is not the same as that for perforin/ granzyme. It has been shown that a combination of anti-CD28 antibody and IL-12 exhibits a synergistic effect on IFN induction in NK cells. However, IL-18 and anti-CD28 antibody employ different signal-transducing mechanisms (Walker et al., 1999). Unexpectedly, it was shown that IL-10 and IL-18 synergistically upregulated IFN production as well as cytotoxicity in NK cells (Cai et al., 1999; Micallef et al., 1999). The mechanism for these phenomena must be clarified.

(Yoshimoto et al., 1997). Similarly, these cytokines synergistically induce IFN in B cells from chronic graft-versus-host disease mice and inhibit Ig production (Lauwerys et al., 1998). In human PBMCs, CD19+ B cells constitutively express IL-18R and IL-18 was suggested to modulate B cells directly (Kunikata et al., 1998). Effect of IL-18 on NK Activity IL-18 shares its biological functions on NK cells with IL-12, including IFN induction, enhancement of NK cytotoxicity, and proliferation. NK cells, unlike T cells, constitutively express IL-18 receptor and IL-18 stimulates their cytotoxicity independently of IL-12, IL-2, and IFN (Hyodo et al., 1999). This cytotoxicity is exerted through activation of perforin and granzyme. Unlike the case of IFN induction in NK cells, there seems to be no synergistic effect of IL-12 and IL-18 on augmentation of NK cytotoxicity. IL-18 also has a critical role in the proliferation of NK cells (Tomura et al., 1998b). In vitro experiments revealed that NK1.1 CD3ÿ cells proliferated in response to IL-12 and IL-18, even after incubation of NK1.1+CD3ÿ cells. These NK1.1ÿCD3ÿ cells became NK1.1+CD3ÿ cells after incubation with IL-2. Both types of cells produce large amounts of IFN and exhibit strong NK cell cytolytic activity in response to IL-12 plus IL-18. Moreover, functional development of NK cells is defective in IL 18-deficient mice (Takeda et al., 1998). Thus, IL-18 seems to be involved in both the development and activation of NK cells. Effects of IL-18 on NK T Cells The liver contains various types of cytotoxic lymphocytes, such as NK1.1+CD4ÿ cells, NK1.1+ CD4+ cells, IL-2R +CD3+ cells, and IL-2R + CD3- cells. IL-18 has been shown to enhance the killing activity of NK1.1+CD4+ T cells (NK T cells) against liver lymphocytes (Dao et al., 1998). It exerts its activity independently of a Fas-mediated mechanism, but in a perforin-dependent manner. These cells may be involved in the clearance of activated lymphocytes which are reactive with autoantigens.

IFN Induction in B Cells

Effect of IL-18 on Fas Ligand Expression

The combination of IL-12 and IL-18 was said to induce IFN production in B cells (Yoshimoto et al., 1997; Kunikata et al.,1998; Lauwerys et al., 1998). B cells activated with anti-CD40 anitbody produce high levels of IFN in response to IL-18 and IL-12, and may regulate IgE/IgG1 and IgG2a responses through production of IFN , both in vivo and in vitro

IL-18 is also involved in another mechanism for cell death. It strongly induces Fas ligand expression in established liver NK cells (Tsutsui et al., 1996) or in TH1 cells (Dao et al., 1996). However, the physiological meaning of this function remains unknown. It may be concerned with activation-induced cell death (AICD) of lymphocytes, but IL-18-deficient

342 Haruki Okamura et al. mice do not appear to manifest symptoms of autoimmune diseases. It is of interest that IL-1 precursor serves as a negative regulator of Fas-mediated cell death (Tatsuta et al., 1996). Precursor IL-18 may also have such a role. However, mature IL-18 induces Fas ligand, and so, the roles of IL-18 in apoptosis, relevant to caspases, remain to be elucidated. Intestinal epithelial cells are said to express Fas antigen, both in normal and in disease-bearing mice (French and Tschopp, 1996; Inagaki-Ohara et al., 1997; Lin et al., 1998), and these cells also express IL-18 (Takeuchi et al., 1997; Pages et al., 1999). It will be of interest to examine the possibility that IL-18 is involved in the turnover of intestinal cells. Other Actions; Induction of Chemokines or Cytokines by IL-18 IL-18 has the ability to induce cytokines other than IFN . It stimulates production of GM-CSF in T cells (Ushio et al., 1996), IL-8, and TNF (Puren et al., 1998a,b). IL-18 was also shown to act on chondrocytes to express genes such as IL-6, inducible NO synthase, inducible cyclooxygenase, and stromelysin. It also augmented the release of glycosaminoglycans from the articular cartilage (Olee et al., 1999). Detailed analyses of the characteristics of the responding cells or of the synergism with IL-12 are not reported for these cytokines or enzymes.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Knockout mouse phenotypes IL-18-deficient mice have normally developed phenotypes of lymphoid organs. Flow cytometric analysis revealed normal cell numbers of B cells, T cells, and NK cells in their spleen, and normal development of T cells in the thymus. Although T cell responses to anti-CD3 mAb in IL-18 mutant mice are equivalent to those in wild-type littermates, NK activity in knockout mice is slightly reduced. IL-12-deficient mice also showed decreased NK activity of spleen cell (Takeda et al., 1998). However, the decreased NK activities in either mutant mice are improved by exogenous stimulation with IL-18 and/or IL-12, indicating that IL-18 and IL-12 independently exert their action on the functional development of NK cells and on their activation.

Furthermore, IL-18-null mice have impaired TH1 development, although IL-18 is reported not to be a differentiation factor for TH1 by itself (Takeda et al., 1998). This was proved by the fact that TH1 development was much weaker in double knockout mice on IL-12 and IL-18 genes than in IL-12 single knockout mice. Thus, IL-18 is involved in the functional development of NK cells and also in TH1 development. IL-18-deficient mice are resistant to endotoxininduced liver injury but are highly susceptible to endotoxin shock (Sakao et al., 1999). As initially reported, IL-18 is a pivotal factor in acute liver injury induced by Propionibacterium acnes and LPS (Okamura et al., 1995). As expected, IL-18-null mice do not suffer from P. acnes and LPS-induced liver injury. Unexpectedly, however, the mutant mice die more rapidly and more frequently after LPS challenge in association with an extraordinarily higher serum level of TNF, when compared to wild-type mice. This was improved by exogenous administration of IL-18 before P. acnes treatment. Thus, IL-18 plays an important, accelerating role in endotoxininduced liver injury, whereas it simultaneously acts as a suppressive factor for the induction of lethal shock, suggesting that the mechanisms underlying endotoxin-induced liver injury and lethal shock might be distinct from each other.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Role in experiments of nature and disease states Role of IL-18 Against Intracellular Parasites Since IL-18 induces IFN production, it is easy to guess that this cytokine is involved in the defense mechanism against intracellular parasites. There are few reports on the suppression of intracellular microbes such as Mycobacteria, Listeria, and Trypanosoma cruzi, but on infection with Mycobacteria, higher expression of IL-18 as well as IFN is observed in resistant strains of mouse than in susceptible strains (Kobayashi et al., 1997, 1998). In resistant mice, IL-18, together with IL-12, may be engaged in the development of TH1 cells. When IL-18-deficient mice were infected with Mycobacteria, they developed marked granulomatous lesions in the

IL-18 343 lung and spleen (Sugawara et al., 1999). IL-18 gene expression, in correlation with IFN , is also induced by the infection of another intracellular parasite, T. cruzi (Meyer zum BuÈschenfelde et al., 1997), and Leishmania (Wei et al., 1999). IFN has been known to play a crucial role in the resistance to this pathogen, and IL-12 together with IL-18 is effectively expressed in resistant mice. Recently, IL-18 was shown to contribute to host defenses against intracellular microbe Salmonella typhimurium (Mastroeni et al., 1999). Treatment of mice with anti-IL-18 antibody impaired the resistance to infection with Salmonella, and increased growth of bacteria in the liver and spleen. In contrast, administration of IL-18 to mice infected with a lethal dose of a virulent strain of Salmonella decreased the bacterial numbers in the tissues, and protected them from death in an IFN -dependent manner. On the other hand, macrophages infected with Salmonella exhibited reduced expression of IL-18, suggesting the ability of this pathogen to suppress the production of IL-18 (Elhofy and Bost, 1999). Role of IL-18 Against Infection by Other Bacteria Endogenous IL-18 is also involved in the defense against infection with Yersinia enterocolitica, which is not an intracellular parasite (Bohn et al., 1998). Administration of anti-IL-18 antibody to mice infected with Y. enterocolitica caused a 100- to 1000-fold increase of bacterial count in the spleen. However, the mechanism of IL-18 involvement has not been studied. IL-18 exerts antifungal activity against Cryptococcus neoformans. Peritoneal exudate cells incubated with IL-18, together with IL-12, suppress the growth of C. neoformans (Zhang et al., 1997). The effect is dependent on both IFN production by NK cells and NO production by macrophages. This suggests that IL-18 plays a role in innate immunity. In fact, administration of IL-18 to mice strongly suppressed the growth of this parasite in the brain and the lung, resulting in the prolongation of survival (Kawakami et al., 1997). Anti-IFN antibody completely abrogated the suppressive effect of IL-18. Susceptibility of AIDS patients to C. neoformans was suggested to be associated with the reduced efficacy of response to IL-18 and IL-12 (Brummer, 1999). Effect of IL-18 on Viral Infection On infection with Epstein-Barr virus (Setsuda et al., 1999), influenza A virus, and Sendai virus (Pirhonen

et al., 1999), IL-18 expression was augmented, suggesting the participation if IL-18 in the defenses against viral infection. In fact, a protective effect of IL-18 against infection with herpes simplex virus (HSV) (Fujioka et al., 1999), Vaccinia virus (TanakaKataoka et al., 1999), and encephalomyocarditis virus (Tovey et al., 1999) was shown in mouse models. Administration of IL-18 to mice before infection of HSV remarkably improved the survival of mice. The effect is also exerted in athymic nude mice and in SCID mice, suggesting that IL-18 augmented innate immunity. The effect is dependent on IFN , independent of NK cells and NO, but the source of IFN is obscure. On the other hand, IL-18 increased HIV type 1 production by 5±30 times in chronically infected U1 monocytic cells (Shapiro et al., 1998). Because IL-18 induced these cells to produce TNF and IL-6 and antibodies to TNF or IL-6 reduced IL-18-induced HIV production, and because IL-18 activates NFB to translocate, IL-18 seems to activate HIV via production of TNF, IL-6, and on activation of NFB. The involvement of T cells in HIV activation or their implication in the onset of AIDS has not yet been evaluated. Antitumor Actions of IL-18 Although IL-18 exhibits antitumor activity against several types of cells, the mechanism and mediating factors seem to be different for each type of cell. Tumor growth of Meth A sarcoma cells inoculated in mice is suppressed by IL-18 and the mice survive longer than control mice (Micallef et al., 1997a,b). Depletion of NK cells by antiasialo GM1 antibody abrogates the effect of IL-18, and NK cells are considered to play crucial roles in the suppression of tumor growth. However, the surviving mice are resistant to challenge with the same cells, indicating that CTLs are also involved. IL-18 also suppresses another type of tumor cell, CL8-1 melanoma cells in an NK cell-dependent manner (Osaki et al., 1998, 1999; Hashimoto et al., 1999). Since this protective effect can be observed in IFN -deficient and in IL-12-deficient mice, IL-18 probably acts on NK cells directly. Using engineered SCK mammary carcinoma cells, it was shown that IL-12 and IL-18 synergistically induced regression in these cells (Coughlin et al., 1998). The effect was suggested to be due to the inhibition of angiogenesis using the matrigel implant assay. The combination of local secretion of IL-12 and systemic administration of IL-18 exerts an enhanced antitumor effect on the bladder cancer cells, MBT2 (Yamanaka et al., 1999). These effects are dependent on IFN , probably through production of IP-10. Thus, IL-18 exerts

344 Haruki Okamura et al. antitumor activity in several ways, dependently and independently of IFN . Tumor cells transfected with IL-18 gene exhibit reduced tumorigenicity and induce immunoprotective effects against parental tumor cells in an IFN dependent manner (Fukumoto et al., 1997; Tan et al., 1998; Heuer et al., 1999). However, human carcinoma AsPC-1 cells transduced with IL-18 gene do not exhibit any antitumor effect in nude mice, while cells transfected with IL-12 gene exhibit retarded tumor growth (Yoshida et al., 1998). The effect of intermediating IFN or the involvement of NK cells have not been examined to evaluate the difference between IL-18- and IL-12-transfectants. Pathological Roles in the Pancreas TH1 cells are considered to be involved in the pathogenesis of destructive diseases, including some types of autoimmune diseases. Abnormal production of TH1-inducing costimulatory factors by macrophages are considered to be important. Since IL-18 is a strong costimulator of TH1 cell development, this cytokine may be a mediator of tissue injury in such diseases. In animal models for insulin-dependent diabetes mellitus (IDDM), IL-18 is expressed in the pancreas in association with the activity of the disease (Rothe et al., 1997a,b; Rothe and Kolb, 1998). It is well established that IFN contributes to the destruction of cells (Von Herrath and Oldstone, 1997). In NOD mice, disease onset is accelerated by cyclophosphamide, associated with increase of IFN positive cells in the pancreas. The increase of IL-18 mRNA in the pancreas correlates with that of IL-12 mRNA and precedes the increase of IFN -producing cells. Insulin-producing cells can be destroyed in several ways, by activation of CTLs, induction of NO, and enhancement of Fas/FasL-mediated apoptosis (Iwahashi et al., 1998; Yoon et al., 1998). IL-18 is concerned with all of these activities, and abnormally reactive macrophages infiltrating the islets may be the source of IL-18 and IL-12 (Rothe and Kolb, 1998). However, unexpectedly, when singly administered to NOD mice, IL-18 suppressed the onset of IDDM (Rothe et al., 1999). This suggests the important roles of IL-18 and needs more analysis. The murine IL-18 gene was mapped in the locus of chromosome 9, idd2, insulin-dependent diabetes susceptibility gene (Rothe et al., 1997a). This also suggests the pathological importance of IL-18 in IDDM. However, according to a recent analysis of the human IL-18 gene, it is mapped to 11q22.2-22.3 and is closely linked to the dopamine receptor D2 (DRD2) gene locus, but distinct from the IDDM locus (Nolan et al., 1998).

Pathological Roles in the Brain IL-18 may also be engaged in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). IL-18 expression in the brain of EAE increases during the acute stages, together with the induction of caspase 1 (Jander and Stoll, 1998; Furlan et al., 1999). It was also demonstrated that neutralizing antibodies to rat IL-18 could prevent EAE in rats (Wildbaum et al., 1998). Microglial cells may be involved in the pathogenesis, producing and responding to IL-18 (Conti et al., 1999; Prinz and Hanisch, 1999). Pathological Roles in the Liver Endotoxin (LPS) causes serious hepatitis in mice pretreated with Propionibacterium acnes. There is a cascade of expression of cytokines, such as IL-12, IFN , TNF, and FasL in the liver after challenge with LPS (Tsutsui et al., 1997). Simultaneous administration of anti-IL-18 antibody with LPS abrogates the expression of IFN , TNF, and FasL, and the onset of hepatitis. Anti-IFN antibody reduces the expression of TNF but not FasL, and partially prevents the liver injury. Anti-TNF antibody also partially suppresses liver injury, and therefore it is suggested that both TNF and FasL mediate the hepatotoxic pathways in LPS-induced hepatitis. Transgenic mice expressing IFN in the liver manifest chronic active hepatitis (Toyonaga et al., 1994). Thus, IL-18 is considered to be situated upstream of the cascade of cytokines concerned with tissue injury. Pathological Roles in the Skin The possibility that IL-18 participates in skin diseases has also been suggested. Epidermal keratinocytes as well as Langerhans cells are known to be sources of cytokines that direct T cell development. IL-18 is one such cytokine produced by these cells. Low levels of IL-18 expression are detectable in epidermal cells of untreated mice, and a rapid upregulation of IL-18 mRNA is observable following administration of contact allergen (Stoll et al., 1997). IL-18 protein is also detectable in the lesion of contact hypersensitivity where inflammatory cells accumulate (Xu et al., 1998a). It has also been shown that caspase 1 (IL-1 converting enzyme) is activated by contact allergens in keratinocytes to secrete mature IL-1 (Zepter et al., 1997), suggesting that IL-18 is processed by this protease in keratinocytes. Epidermal Langerhans cells are the source of IL-18 in the skin (Stoll et al., 1998). It has been shown that IL-18, together with IL-12, stimulates the expression of IFN in the dendritic epidermal  T cells (Sugaya et al., 1999). The role of IL-18 in the repair of cutaneous wounds has also been

IL-18 345 suggested (Kampfer et al., 1999). However, the roles of IL-18 in the skin diseases remain to be clarified. Pathological Roles in the Kidney It is of interest that IL-18 was suggested to be involved in the pathogenesis of renal injury following ischemia±reperfusion of the kidney (Daemen et al., 1999). This indicates that IL-18 can be induced without any infection, and suggests that IL-18 secretion is related to various types of oxidative stress. Pathological Roles in the Lungs The possibility that IL-18 plays a role in the inflammatory reactions of asthma has been suggested (Kumano et al., 1999). IL-18, unlike IL-12 or IFN , augmented eosinophil infiltration in the airway of mice exposed to antigens. IL-12 has been shown to cause severe pathological changes accompanying lung edema in mice (Car et al., 1995). In this case, endogenous IFN plays a protective role and IL-12 causes pulmonary edema and is lethal in IFN -deficient mice. On the other hand, administration of IL-18 to mice apparently causes no pathological changes, but combined administration of IL-12 and IL-18 causes serious diarrhea, atrophy of various organs, and mortality (Okamura et al., 1998b). In contrast to the IL-12-induced pathology, IL-18 together with IL-12 fails to manifest any pathological changes in IFN -deficient mice. IL-18 Detectable in Clinical Material IL-18 is detectable in clinical samples such as blood and tissues of various diseases. For example, it is observed in the oral mucosa of patients with primary SjoÈgren's syndrome (Kolkowski et al., 1999), in the blood of hemophagocytosis (Takada et al., 1999), in the cartilage of patients with osteoarthritis (Saha et al., 1999), in the cerebrospinal fluid of patients with meningoencephalitis (Fassbender et al., 1999), in the demyelinating lesions of multiple sclerosis patients (Balashov et al., 1999), in the lesions of leprosy patients (Garcia et al., 1999), and in the intestinal mucosa of patients with Crohn's disease (Monteleone et al., 1999; Pizarro et al., 1999).

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