Interleukin-1 agonists

Both IL-1α and IL-1β bind to the same receptors, and there are no significant differences in the spectrum of activities of recombinant IL-1α or IL-1β when studied in vitro or in vivo in diverse experimental systems. IL-1 is a highly potent pro-inflammatory cytokine. Intravenous injection of only few hundred nanograms of IL-1β into humans, induces fever, leukocytosis, thrombocytosis,

hypotension, and the release and production of cytokines such as IL-6 which, in turn, trigger the synthesis of the hepatic acute-phase proteins serum amyloid A and C-reactive protein(12;89).

IL-1α and IL-1β are synthesized as 31–33 kD precursors lacking signal peptides, and proteolytic processing, by the membrane-associated cysteine proteases, calpains(90) and IL-1-converting enzyme (ICE)/caspase-1(91;92), respectively, generates the 17.5 kD carboxyl-terminal fragments. Whereas IL-1β precursor is inactive, IL-1β is active in its secreted form. Mononuclear cells display the strongest secretory capacity of IL-1β, whereas diverse nonphagocytic cells generally secrete low levels of IL-1β. Although circulating levels of IL-1β are measurable, these levels are usually in the low pg/ml range, even in sepsis. In humans, IL-1α is mainly active intracellularly or in its membrane-associated form (23 kD) when it engages the IL-1 surface receptor by a mechanism of activation commonly termed “juxtacrine”(93;94), but is only marginally active as a secreted 17.5 kD molecule. IL-1α may be secreted by activated macrophages. It is not commonly detected in blood or in body fluids, except during severe diseases, in which case the cytokine may be released from dying cells. Processing of the IL-1α precursor form (pIL-1α) to the mature form appear to be deficient in a variety of cells(89), leaving the intracellular precursor form in abundance. The IL-1α amino-terminal propiece (ppIL-1α) includes a latent nuclear localization sequence that is functional after cleavage of the precursor(95). In fact, ppIL-1α translocates to the nucleus in a variety of cells in response to cytokines and Toll-like receptor (TLR) ligands, and this appears to be necessary for specific downstream events(96). More specifically, overexpression of intracellular IL-1α is often seen in chronic diseases. In such context, it has been proposed that ppIL-1α might play a pivotal role in the pathogenesis and maintenance of chronicity via the activation of the transcriptional machinery together with the synthesis of pro-inflammatory cytokines(97).

IL-1α and IL-1β differ from most other cytokines by lacking a signal sequence, thus they do not traffick through the endoplasmic reticulum-Golgi pathway. To date the mechanisms of IL-1 secretion is still an ill-defined process. Following the synthesis by TLR ligands of the IL-1β precursors, a part moves into specialized secretory lysosomes to co-localize with procaspase-1, while most reside in the cytosol(98). It is generally believed that in resting cells, procaspase-1 is bound to a large inhibitor molecule, which prevents its activation, and that through initiation of IL-1β production, there is activation of caspase-1 by a complex of proteins termed the "IL-1β inflammasome", which then processes the IL-1β precursor form into a mature form ready for secretion(12;99) (Figure 2). Many studies have shown the requirement of P2X7 receptors for ATP-induced caspase-1 activation and subsequent IL-1β release(100;101). P2X7 receptor activation is supposed to mimic a hypotonic stress situation. Hence, to date, at least two systems are able to cause

activation of caspase-1, one as a result of bacterial agents and the other following alterations in the intracellular ionic environment.

Because IL-1β production is critical for the control of infections and that excessive cytokine production is harmful to the host, it is therefore not surprising that IL-1β activity is controlled at several levels. These include the regulation gene transcription(102-109), mRNA turnover (presence of AREs)(110;111) and translation(112;113). As stated above, IL-1β activity is furthermore regulated by the post-translational processing of the protein precursor by caspase-1(92), secretion, and receptor association (reviewed in (114)).

Despite numerous reports on IL-1 and the obvious importance of IL-1 in the cytokine network, very little is known regarding the molecular details of IL-1 gene regulation. The structure of the IL-1α and IL-1β gene promoters differ. The IL-1α promoter, unlike that of IL-1β, does not have a typical TATA box(102;115), suggesting that both genes have distinct physiological modes of regulation. Both human promoters contain NF-κB regulatory elements, binding sites for NF-IL6, activator protein (AP)-1 proteins and cyclic adenosine 3’,5’-cyclic monophosphate (cAMP) response element binding protein (CREB). IL-1β mRNA is absent from cells until stimulated by extracellular signals. Constitutive activity of murine IL-1β promoter is controlled by a transcriptional

Figure 2. Steps in the processing and secretion of IL-1β. A TLR ligands such as endotoxins trigger synthesis of the IL-1β precursor, which remains in the cytosol. In the same cell, inactive procaspase-1 is bound to components of the IL-1β inflammasome. The IL-1β inflammasome is kept in an inactive state by binding to a putative inhibitor. B After TLR signals, there is a transient uncoupling of the inhibitor from the procaspase-1, which then colocalizes with the IL-1β in secretory lysosomes. C Autocrine activation of the P2X7 receptor by ATP initiates the efflux of potassium from the cell via a potassium channel. The efflux of potassium activates the autocatalytic processing of procaspase-1. Active caspase-1 cleaves the IL-1β precursor in an active cytokine. D The efflux of potassium ions results in the influx of calcium ions, which in turn activate phospholipases.

Phosphatidylcholine-specific phospholipase C (PC-PLA-2) facilitates lysosomal exocytosis and secretion of IL-1β. (From (12)).

repressor(107). Actually, proIL-1β gene is under the control of both activators and repressors of transcription(116).

Two receptors have been characterized. IL-1RI is an 80 kD transmembrane molecule with a signal transducing cytoplasmic domain, through which all IL-1-mediated responses are relayed (Figure 3)(117). Therefore, IL-1RI-deficient mice fail to respond to IL-1 and display reduced inflammatory responses(118). IL-1RI contains three immunoglobulin domains, which domains 1 and 2 bind IL-1 agonists with low affinity but bind IL-1Ra (sIL-1Ra and icIL-1Ra1) with high affinity(119;120). Besides, the conformational changes induced in the ligand-receptor complex by tight binding of the IL-1 agonists to domain 3 of IL-1RI, that did not occur with IL-1Ra, may allow a secondary interaction of this complex with the IL-1 receptor accessory protein (IL-1RAcP) (Figure 3). Thus, domain 3 of IL-1RI is necessary to achieve high-affinity binding with the two IL-1 agonists, in addition to generation of agonist activity. Although the IL-1RAcP molecule itself does not bind the IL-1 agonists, association of IL-1RAcP with the ligand-receptor complex drives a fivefold increase in the affinity of binding of 1 agonists to the receptor(121). An excess of IL-1Ra, commonly the soluble form, is necessary to block the biological effects of IL-1 agonists because the binding of only few molecules of IL-1 per cell suffices to stimulate a full biological response. In addition to IL-1RI, which mediates the effect of IL-1 binding, the type II receptor (IL-1RII) is a smaller molecule (68 kD) with a truncated cytoplasmic domain, which functions as a nonsignaling decoy target for IL-1 receptor ligands (Figure 3)(45;122). IL-1 receptors play an additional role in the control of IL-1 activities through the proteolytic cleavage of their extracellular

Figure 3. Natural mechanisms for reducing IL-1 activities. A IL-1 receptor type I (IL-1RI) binds IL-1β, which then recruits the IL-1 receptor accessory protein (IL-1RAcP) to transmit signalling. When secreted IL-1 receptor antagonist (sIL-1Ra) occupies the IL-1RI, the IL-1RAcP is not recruited, and there is no signal. The affinity of sIL-1Ra for the IL-1RI is greater than that for IL-1β. IL-1RII is termed “decoy” receptor since it has a greater binding affinity to IL-1β than the type I receptor, and lacks a significant intracellular segment and hence does not signal. IL-1β bound to the IL-1RII can also form a high-affinity complex with the IL-1RAcP. Soluble IL-1RAcP may form a complex with IL-1RI and IL-1β but without initiating a signal. B The extracellular (soluble) domains of IL-1RI (1RI) bind IL-1Ra with a greater affinity than that for IL-1α or IL-1β. sIL-1RI may act as a sink for sIL-1Ra. The sIL-sIL-1RII binds IL-1β and neutralizes its activities. The sIL-1RAcP does not bind IL-1β but rather forms a high-affinity complex with the sIL-1RII and neutralizes IL-1β activities. (Adapted from ref. (11)).

domains, although soluble-type IL-1RII receptor (sIL-1RII) is the predominant shed form in vivo(43). Soluble IL-1RI (sIL-1RI) is considered as a pro-inflammatory moiety, since it retains the ability of membrane-bound IL-1RI to bind sIL-1Ra and IL-1α with greater affinity than IL-1β, and therefore further enhanced the inflammatory effects of IL-1 on target cells (Figure 3)(119;123-125).

These findings might explain the lack of efficacy of recombinant human IL-1RI in clinical trials in RA patients(126). In contrast, sIL-1RII binds to sIL-1Ra and IL-1α with much lower affinity than IL-1β and may thus increase the anti-inflammatory effects of 1Ra(119;123;127-129). Thus, sIL-1RII contributes to IL-1 antagonism through the preferential neutralization of IL-1β activity(123;128;130). The administration of sIL-1RII in experimental models resulted in a manifest inhibition of joint damage and joint swelling(131;132). Recently, an alternative splice transcript of the membrane IL-1RAcP, encoding a smaller and soluble protein (sIL-1RAcP) has been described(133;134). This sIL-1RAcP is mainly produced by the liver and circulates systemically.

Overexpression of sIL-1RAcP ameliorates joint and systemic manifestations of collagen-induced arthritis (CIA) in mice(135). A possible explanation is that sIL-1RAcP can interact with sIL-1RII, thus forming a high-affinity IL-1 scavenger (Figure 3)(136).