IL-1a

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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/rcwy.2000.04001.

SUMMARY IL-1 is distinct from the other agonist member of the IL-1 family, IL-1 . Although IL-1 triggers the same IL-1 receptor and although many of the biological effects of IL-1 are similar to those of IL-1 , in humans IL-1 is predominantly an intracellular molecule. In fact, there is evidence that IL-1 has both intracellular functions as a precursor molecule due to a nuclear localization sequence. IL1 as an unprocessed precursor is biologically as active as the processed form. IL-1 is also found consitutively in epithelial cells, whereas constitutive expression of IL-1 is rare. In many ways, IL-1 appears to be closer to the fibroblast growth factor family than the secreted IL-1 form. Therapeutic stratiegies for blocking IL-1 predominate over those for blocking IL-1. Many humans have circulating neutralizing antibodies to IL-1 but not IL-1 .

BACKGROUND

of IL-1 was carried out with supernatants from this cell line.

Alternative names See the chapter on IL-1 for alternative names. Hematopoietin 1 was originally described as IL-1 because the cell line primarily produced IL-1 rather than IL-1 . The purification and cloning of mouse IL-1 was from a cell line that also primarily produced IL-1 as opposed to IL-1 (Lomedico et al., 1984).

Structure IL-1 is initially synthesized as a (31 kDa) precursor molecule without a signal peptide (Lomedico et al., 1984), mature IL-1 being a 17.5 kDa molecule. Calpain, a calcium-activated cysteine protease, is primarily responsible for the cleavage of the IL-1 precursor (Kobayashi et al., 1990; Miller et al., 1994; Watanabe and Kobayashi, 1994).

Discovery Please see section on Discovery in the IL-1 chapter. In 1974, IL-1 was reported to be an endogenous pyrogen with a pI of 5 (Dinarello et al., 1974). Purified rabbit endogenous pyrogen with an isoelectric focusing point at 5.0 was shown to possess activity identical to that for lymphocyte-activating factor (LAF) (Hanson and Murphy, 1984). The pI of 5.0 indicates that this was probably IL-1. Mizel and Oppenheim reported extensively on murine LAF with a pI of 5, which was thus identified as IL-1 (Lomedico et al., 1984). The murine cell P388 produces IL-1 and not IL-1 ; hence, the purification

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 IL-1 is not a secreted molecule but a primarily cellassociated molecule. It is best to characterize the biological activities of IL-1 as proinflammatory. In addition, because keratinocytes and other epithelial cells produce IL-1 constitutively, IL-1 appears to have a greater role in cell proliferation than does IL-1 , which is not expressed constitutively.

308 Charles A. Dinarello

GENE AND GENE REGULATION

Sequence

Accession numbers

The primary sequence of the human IL-1 precursor is described in March et al. (1985).

See Lomedico et al. (1984) for the mouse and March et al. (1985) for the human gene accession numbers. M28983

Regulatory sites and corresponding transcription factors The IL-1 promoter does not contain a clear TATA box, a typical motif of inducible genes. Inducible gene expression for IL-1 involves both a distinct promoter that is 4.2 kb upstream from the start site as well as a proximal promoter region of 200 bp (Mora et al., 1990). The upstream sequences are sufficient for induction by phorbol esters, and the downstream sequences can be deleted without affecting gene expression (Mora et al., 1990; Auron and Webb, 1994). A construct containing sequences ÿ1437 to ‡19 does not allow for the stimulation of specific expression, but an additional 731 bp spanning exon I, intron I, and a segment of exon II control a 20-fold increase in stimulation over background level in murine macrophage cells. Interestingly, using the same contruct in human leukemic cells, only a 2-fold increase was observed. These additional 731 base pairs contain nuclear factor (NF) IL-6 and NFB within intron I.

Cells and tissues that express the gene The blood monocytes and tissue macrophages are the primary sources of IL-1. However, unlike IL-1 , IL-1 is found constitutively expressed in keratinocytes and other epithelial cells. In fibroblasts derived from pathological tissues, for example, in renal failure, IL-1 is expressed constitutively; in contrast, there is no consitutively expressed IL-1 in dermal fibroblasts from healthy subjects (Lonnemann et al., 1995a,b).

PROTEIN

Accession numbers IL-1: P01584

Description of protein IL-1 is primarily translated as a (31 kDa) precursor lacking a signal peptide (Lomedico et al., 1984; March et al., 1985). Cleavage of the precursor is via the cysteine protease calpain (Kobayashi et al., 1990; Watanabe and Kobayashi, 1994).

Discussion of crystal structure On analysis of the crystal structure, the mature form of IL-1 is found to be similar to that of IL-1 , the analysis revealing that the molecule is comprised entirely of sheets. IL-1 has two sites of binding to IL-1RI, the primary binding site located at the open top of its barrel shape being similar but not identical to that of IL-1 (Lambriola-Tomkins et al., 1993).

Important homologies The relevant homology of IL-1 is to the acidic fibroblast growth factor (Murzin et al., 1992). Changing the aspartic acid at tyrosine 151 in mature IL-1 results in a loss of PGE2 induction and fibroblast growth, but T cell responses are unaffected (Yamayoshi et al., 1990).

Posttranslational modifications Unlike IL-1 , most of the 31 kDa precursor of IL-1 that is synthesized in monocytes and other cells remains in the precursor state. Processing by calpain takes place and is followed by secretion, but to a far greater extent than takes place following the processing of IL-1 by ICE (Schindler et al., 1990a). Also unlike IL-1 , the IL-1 precursor is biologically active (Mosley et al., 1987) via specific cell binding. Membrane IL-1 Precursor IL-1 can be found on the surface of several cells, particularly on monocytes and B lymphocytes after stimulation in vitro. Approximately 10±15% of the IL-1 is myristoylated (Stevenson et al., 1993), this form being thought to be transported to the cell surface, where it is called `membrane' IL-1 (Kurt-Jones et al., 1985). The myristoylation of

IL-1 specific lysine residues facilitates the passage to the cell membrane (Stevenson et al., 1993). This `membrane' IL-1 is biologically active, its biological activities are neutralized by anti-IL-1 rather than anti-IL-1 antibodies, and it appears to be anchored via a lectin interaction involving mannose residues (Brody and Durum, 1989). Using a high concentration of IL-1Ra to prevent IL-1 binding to the cell surface IL-1R during fixation, the biological activity of membrane IL-1 is unaffected. In contrast, a mannose-like receptor appears to bind membrane IL-1 (Kaplanski et al., 1994). Although IL-1 has glycosylation sites, recombinant forms of mature IL-1 are biologically active when expressed in Eschericia coli that lack the ability to glycosylate proteins. Since membrane IL-1 is probably a glycosylated or myristoylated form of the cytokine, it accounts for no more than 5% of the total pro-IL-1 synthesized by the cell. There has been some dispute over whether membrane IL-1 represents a `leak' of intracellular IL-1 (Minnich-Carruth et al., 1989), but with prolonged fixation, leakage does not account for the activity of membrane IL-1 (Brody and Durum, 1989; Bailly et al., 1990).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Nearly all the studies on the biological activities of IL-1 have been performed using the recombinant form of IL-1, which is the mature, 17 kDa C-terminal peptide. Even under conditions of cell stimulation, human blood monocytes neither process nor readily secrete mature IL-1 (Endres et al., 1989; Lonnemann et al., 1989; Schindler et al., 1990a). The 31 kDa IL-1 precursor is, unlike most proteins translated in the endoplasmic reticulum, synthesized in association with cytoskeletal structures (microtubules) (Stevenson et al., 1992). When the cells die, precursor IL-1 is released and can be cleaved by extracellular proteases (Kobayashi et al., 1991). Precursor IL-1 can also be cleaved by activation of the calcium-dependent, membrane-associated calpains (Kobayashi et al., 1990; Miller et al., 1994). In transformed cell lines constitutively synthesizing precursor IL-1, the addition of a calcium ionophore stimulates the calpain, which cleaves the precursor. Hence, the release of the 17 kDa IL-1 can take place in the absence of cell death (Watanabe and Kobayashi, 1994). For reasons that are unclear, mice deficient in ICE have a reduced ability to secrete IL-1 (Li et al., 1995).

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Most experimental models of disease employ the mouse or rat. Unlike human cells, mouse cells readily produce and release IL-1. The relative contribution of IL-1 and IL-1 in these disease models can be resolved by comparing the responses of the IL-1 deficient with those of the IL-1RI knockout mouse, assuming that the differences arise from the role of IL-1. Specific neutralizing antibodies to either murine IL-1 or murine IL-1 have shown that some disease models are IL-1 dependent (Geiger et al., 1993; van den Berg et al., 1994). Anti-IL-1 but not anti-IL-1 reduces collagen-induced rheumatoid arthritis in mice (van den Berg et al., 1994). Intracellular IL-1 Because of the lack of a leader peptide, the precursor IL-1 remains in the cytosol after translation, and there is no appreciable accumulation of IL-1 in any specific organelle. Immunohistochemical studies of IL-1 in endotoxin-stimulated human blood monocytes reveal a diffuse staining pattern, but, in comparison in the same cell, the IL-1Ra is localized to the Golgi apparatus (Andersson et al., 1992). In experimental inflammatory bowel disease, there is a better correlation of disease severity with the colonic tissue level of IL-1 than with that of IL-1 (Cominelli et al., 1990) as a result of the cellassociated nature of IL-1. IL-1 is not commonly found in the circulation or in body fluids except during severe disease (Wakabayashi et al., 1991).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Nearly all microbes and microbial products induce the production of IL-1 (see the section on IL-1 ). Raising the cAMP level in human PBMCs with histamine enhances IL-1-induced IL-1 gene expression and protein synthesis (Vannier and Dinarello, 1993). This is thought to be the mechanism by which PGE2 enhances IL-1-induced IL-6 (Vannier and Dinarello, 1994). Also see the relevant section in the IL-1 chapter for the ability of `cytokine-suppressing anti-inflammatory drugs', or CSAIDs, to inhibit the synthesis of IL-1 (Young et al., 1994).

RECEPTOR UTILIZATION The high-affinity binding of IL-1 for the IL-1RI varies from 100 to 450 pM. The concentrations of

310 Charles A. Dinarello IL-1 that can elicit a biological response lie in the range 10±100 fM. There are two affinities; in cells expressing large amounts of IL-1R-AcP, the highaffinity binding of the IL-1R/IL-1R-AcP complex may explain which two classes of binding have been observed. Human recombinant 17 kDa IL-1 binds more avidly to IL-1RI than to the nonsignal transducing type II (IL-1RII). IL-1 binding to the soluble form of the IL-1RI is higher compared than that to the cell-bound receptor. The most dramatic differences between IL-1 and IL-1 binding to cells can, however, 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). In comparison, IL-1 and IL-1Ra bind with 50- and 100-fold or lower affinities (Arend et al., 1994; Dower et al., 1994; Symons et al., 1994). Moreover, pro-IL1 also preferentially binds to IL-1 soluble RII (Symons et al., 1991, 1993).

Nuclear Localization of IL-1 Mizel and coworkers (1987) initially reported that radiolabeled 17 kDa recombinant IL-1 bound to the cell surface receptors was rapidly internalized and, after 2±3 hours, was found to be associated with the nucleus. It was unclear whether the nuclear binding comprised the IL-1/IL-1R complex or just the ligand. Curtis et al. (1990) reported that internalized IL-1 was still bound to its receptor and that internalized IL-1R correlated with increased signal transduction. It was later shown that the IL-1/IL-1R complex but not the 17 kDa IL-1 bound to immobilized DNA could be eluted under the same salt conditions as that of the estrogen receptor (Weizmann and Savage, 1992). The cytoplasmic domain of IL-1RI is highly conserved (see below for a discussion of Toll protein), and contains a consensus sequence (residues 517±529) similar to those which transport viral proteins (Heguy et al., 1991). If precursor IL-1 plays an essential role in keratinocyte cellular differentiation (Hauser et al., 1986; Hammerberg et al., 1992), this is probably not in conjunction with the type I IL-1 receptor since mice deficient in this receptor appear to have a normal phenotype, including that seen on gross examination of the skin and fur. The response of the IL-1RIdeficient mouse to IL-1 signaling is absent, and it is anticipated that the response to external challenges will be attenuated in a similar manner to that seen in mice treated with neutralizing antibodies to the type I receptor. Since precursor IL-1, whether recombinant (Mosley et al., 1987) or naturally membrane bound (Beuscher and Colten, 1988; Kaplanski et al., 1994),

binds to the extracellular IL-1RI indistinguishably from 17 kDa IL-1, precursor IL-1 could also be involved in nuclear localization. Using antibodies directed specifically at precursor IL-1 (Stevenson et al., 1992) and transfection with plasmids containing the first 115 amino acids of precursor IL-1 (also called the IL-1 propiece), it appears that the propiece rather than the C-terminal, mature segment of IL-1 localizes to the nucleus (Maier et al., 1994). This concept is supported by the observation that a specific peptide in the propiece of IL-1 binds to DNA (Wessendorf et al., 1993). Phosphorylation (Beuscher et al., 1988) and myristoylation (Stevenson et al., 1993) of the IL-1 propiece may facilitate nuclear localization. Myristoylation takes place on lysine residues 82 and 83 of the IL-1 propiece, which is found in the nuclear localization sequence KVLKKRR (Wessendorf et al., 1993). Transfecting endothelial cells with a plasmid containing this sequence has revealed nuclear localization (Maier et al., 1990). Transfecting cells with the propiece of IL-1 results in a slower rate of proliferation (Maier et al., 1990, 1994) consistent with a role for IL-1 in early endothelial senescence (Maier et al., 1990). The transfection of an intracellular IL-1-producing plasmid increases IL-2 production in thymoma cells, a biological effect that is prevented by antisense IL-1 (Falk and Hofmeister, 1994), suggesting that IL-1 without its receptor is functional as an intracellular molecule. The N-terminal IL-1 propiece, when transfected into glomerular mesangial cells, acts as a oncoprotein and results in malignant transformation (Stevenson et al., 1997). The inhibition of IL-1 production by FK506 results in the suppression of anthralin-induced tumors (Yamamoto et al., 1994).

IN VITRO ACTIVITIES

In vitro findings IL-1 stimulates cells in vitro in the picomolar to femtomolar range. The list of IL-1 activities on cells in vitro is best understood at the level of gene expression (See Table 1). Most studies shown in this table include the IL-1 stimulation of gene expression as well as the IL-1 suppression of gene expression. Differences Between IL-1 and IL-1 Blocking IL-1 in disease with IL-1Ra (see the chapter on IL-1 ) is a sensible strategy, but the next question is how much of the effectiveness of IL-1Ra in animal models as well as in human disease results from

IL-1

Table 1 A comparison of IL-1 and IL-1 IL-1

IL-1

Pro-IL-1 is active

Pro-IL-1 is inactive

Active membrane form is IL-1

Membrane IL-1 not observed

Mature IL-1 does not circulate

Mature IL-1 circulates

Nuclear localization

No nuclear localization

Intracellular role for pro-IL-1

No intracellular role for pro-IL-1

Neutralizing autoantibodies

Non-neutralizing autoantibodies

Calpain cleavage of pro-IL-1

ICE and PR-3 cleavage of pro-IL-1

No disease link with calpain expression

Disease link with ICE expression

No correlation

Correlation with bone resorption

IL-1 knockout mice normal

IL-1 knockout mice resistant to disease

Anti-IL-1 ineffective in CIA

Anti-IL-1 effective in CIA

IL-1 expression in AML absent

IL-1 expression in AML present

No data

ICE inhibition # brain ischemia

No data

ICE inhibition # AML proliferation

No data

ICE antisense # AML proliferation

blocking IL-1 compared with blocking IL-1. It has been thought that the biological role of IL-1 in disease is as a membrane-bound cytokine. Indeed, membrane-bound IL-1 activity has consistently been shown to be that of IL-1 rather than IL-1 . IL-1 is secreted whereas IL-1 remains biologically active as a cell-associated cytokine (Brody and Durum, 1989; Kaplanski et al., 1994). Unlike the situation with IL-1 , normal skin and epithelial cells constitutively express precursor IL-1. In addition, autoantibodies to IL-1 are common, many being neutralizing antibodies (Svenson et al., 1992). Differences between IL-1 and IL-1 are shown in Table 1. One of the most interesting aspects of the difference between the in vitro biological effects of IL-1 and IL-1 lies in the induction of PGE2. In general, IL-1 is a more potent inducer of PGE2 in cultured cells than is IL-1 (Chin et al., 1988; Kent et al., 1993).

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Precursor IL-1 as an Autocrine Growth Factor The concept that IL-1 can be an autocrine growth factor takes into account three distinct mechanisms: first, that precursor IL-1 is synthesized and remains inside the cell, where it exerts a direct effect by binding to the nucleus; second, that intracellular precursor IL-1 complexes to an intracellular pool of IL-1RI before exerting an effect as a ligand/receptor complex; and third, that either precursor IL-1 or mature IL-1 bound to surface IL-1RI is internalized, with subsequent translocation to the nucleus (similar to what happens to steroid receptors). Each mechanism has supporting experimental data. Some investigators have considered that intracellular precursor IL-1 regulates normal cellular differentiation, particularly in epithelial cells and ectodermal cells. In the case of keratinocytes, the constitutive production of a large amount of precursor IL-1 is found in healthy human skin (Hauser et al., 1986). In support of the concept that precursor IL-1 functions as an intracellular messenger in certain cells, an antisense oligonucleotide to IL1 reduces senescence in endothelial cells (Maier et al., 1990), a prostaglandin-dependent process. In fibroblasts, antisense IL-1 does not have this effect (Wessendorf et al., 1994), raising the possibility that an autocrine effect of precursor IL-1 is cell specific. In the murine TH2 cell line, IL-1 was proposed as an essential autocrine and paracrine growth factor using an antisense IL-1 oligonucleotide or anti-IL-1 antibodies (Zubiaga et al., 1991). Thymic epithelium produces IL-1, and a requirement for IL-1 has been demonstrated in the expression of CD25 (IL-2 receptor  chain) and the maturation of thymocytes (ZuÂnÄiga-PfluÈcker et al., 1995). This must, however, take into account the report that in mice deficient for IL-1, there are no demonstrable defects in growth and development, including of skin, fur, epithelium and gastrointestinal function (Horai et al., 1998). The large amount of precursor IL-1 in normal skin keratinocytes is thought to affect terminal differentiation. If there is a role for intracellular precursor IL-1 in normal cell function, this should be carefully regulated. The presence of a large amount of an intracellular form of the IL-1Ra (icIL-1Ra) (Haskill et al., 1991; Hammerberg et al., 1992) produced in the same cells expressing precursor IL-1 is thought to compete with the intracellular pool of precursor IL-1 for nuclear binding sites. The IL-1deficient mouse does not support this concept. On the other hand, there is no dearth of reports that cell lines derived from various cell types spontaneously express IL-1 mRNA (reviewed in Lonnemann et al., 1995a,b); the spontaneous gene

312 Charles A. Dinarello expression and synthesis of IL-1 can, however, result from contamination of the tissue culture medium with endotoxins or from stimulation by serum factors (Watanabe and Kobayashi, 1994). This latter consideration is important since nearly all cultured cells require either fetal calf serum, defined animal serum substitutes, or human serum ± each known to contain platelet-derived and other growth factors. An essential role for IL-1 (presumably IL-1) in the growth of kidney-derived fibroblasts can be demonstrated when the serum concentration is lowered from 5% to 1%, otherwise cell growth at higher serum concentrations is unaffected by IL-1 receptor blockade (Lonnemann et al., 1995a). Hence, the concept of pro-IL-1 as an autocrine growth factor during in vitro culture should be mindful of the possible stimulatory effects of serum-derived growth factors.

Bioassays used There are several bioassays for IL-1 in vitro. Although the early studies focused on the IL-1 activation of murine thymocytes (Dinarello et al., 1986) and the TH2 cell line D10 (Orencole and Dinarello, 1989), the best in vitro bioassay for IL-1 is currently the induction of IL-8 or IL-6 from fibroblasts (Kaplanski et al., 1994; Shapiro et al., 1994). In addition, the 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 Regulatory molecules: Inhibitors and enhancers The most important regulatory molecule for IL-1 activity is IL-1Ra, which is usually produced in a 10± 100-fold molar excess (Granowitz et al., 1991; Fischer et al., 1992; reviewed in Arend et al., 1998). In addition, the soluble form of the IL-1R type I has a high affinity for IL-1 and is produced in a 5±10 molar excess. IFN also inhibits the formation of IL-1-induced IL-1 (Ghezzi and Dinarello, 1988; Schindler et al., 1990b), and IFN also inhibits IL-1-induced IL-1 production (Schindler et al., 1990b). In cultured chondrocytes, IFN reduces the IL-1-induced expression of collagenase (Andrews et al., 1989, 1990). Although IL-10 inhibits IL-1 synthesis, it does not inhibit IL-1Ra production. A large number of reports focus on the ability of IL-10 to suppress gene expression and the synthesis of inflammatory cytokines (reviewed in Moore et al., 1993). Cell signaling following the engagement of IL-10 to its receptor includes the phosphorylation 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 the suppression of macrophage cytokines. IL-10 suppresses IL-1, IL1 , TNF, IL-6, IL-8, IL-12, GM-CSF, G-CSF, MCSF, MIP-1, RANTES, LIF, and IL-10 itself. CNTF binds to its specific soluble receptor and, when incubated with human blood PBMCs, the CNTF/ soluble receptor complex suppresses IL-1-induced IL-8 and PGE2 synthesis (Shapiro et al., 1994).

Because a null mutation in the IL-1 gene results in a phenotypically normal mouse (Horai et al., 1998), there is probably no normal physiological role for IL-1 in health. The IL-1-deficient mouse is now over 3 years into 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 human IL-1 but not IL-1. This is particularly the case when testing the adrenal hypothalamic axis response to IL-1, in which case rats are used. Rats respond to human IL-1 but not IL-1. In contrast, rabbits appear to respond equally to the fever-producing property of both human IL-1 and human IL-1 (Cannon et al., 1989).

Knockout mouse phenotypes As stated above, the IL-1-deficient mouse has demonstrated no abnormal findings after 3 years of continuous breeding. Upon challenge to local inflammation induced by a subcutaneous injection of turpentine (50±100 mL), IL-1 -deficient mice exhibit a marked reduction in fever, cortisone level and other indicators of the systemic response to inflammation (see the chapter on IL-1 ). In sharp contrast, these same responses are not diminished in IL-1-deficient mice (Horai et al., 1998). In IL-1-deficient mice,

IL-1 body temperature (fever) rose from days 1±5 after the turpentine injection, as in wild-type mice. These observations clearly indicate that IL-1 but not IL-1 mediates the systemic response to local inflammation. In wild-type mice, turpentine induces an elevated brain IL-1 mRNA level, but in IL-1 deficient mice, there is a 30-fold reduction in IL-1 level (Horai et al., 1998). In brain tissue, however, IL-1 is found at a level comparable to that of IL-1deficient mice. In IL-1Ra-deficient mice, the IL-1 level in brain tissue following turpentine injection is elevated 2.5-fold, whereas the level of IL-1 is not affected. COX-2 expression in brain tissue is markedly elevated after turpentine injection, but its level is unaffected in IL-1-deficient mice, although it is suppressed in IL-1 -deficient mice (Horai et al., 1998). Similar results were observed for cortisol level. In mice in which zymosan peritonitis has been induced, IL-1 deficiency results in decreased chemokine production; however, after cotreatment with IL-1Ra, there is no further reduction in inflammation, indicating that IL-1 does not play a major role in this model of inflammation (Fantuzzi et al., 1997). 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, but in these mice, there is a decreased uterine cytokine level following the administration of LPS (Reznikov et al., 1999). The reduction in LPS-induced cytokine level is not found in non-pregnant 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 difference was noted in the plasma elevation of glucocorticoid steroid level between IL-1 -deficient and wild-type mice following the injection of LPS, indicating that IL-1 is not required for the activation of the HPA axis during endotoxemia (Kozak et al., 1995). The data demonstrate that, in the mouse, IL-1 is critical for the induction of fever during local inflammation. Another study characterized body temperature, activity and feeding live influenza virus in IL-1 -deficient mice. Body temperature and activity were lower in IL-1 -deficient mice (Kozak et al., 1995), but the anorexic effect of

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the influenza infection was similar in both groups of mice. The mice deficient in IL-1 exhibited a higher mortality from influenza infection than did the wildtype mice.

Transgenic overexpression Proliferation is enhanced in human vascular smooth muscle cells overexpressing IL-1 (Beasley and Cooper, 1999). Both IL-1(1±271) and IL-1(113± 271) stable transfectants produced a moderate level of IL-1. Human vascular smooth muscle cells transfected with either IL-1 1±271- or IL-1 113± 271-expression plasmids proliferated rapidly compared with nontransfected or vector-transfected cells. These results demonstrate that IL-1 precursor is an autocrine growth factor for human vascular smooth muscle cells and further indicate that amino acids 113±271 play a crucial role in its actions.

Pharmacological effects The injection of IL-1 into primates induces neutrophilia and the production of acute phase proteins, and the injection of modest doses (1±10 mg/kg) of IL-1 into mice, fever, anorexia and the production of circulating IL-6. The most dramatic responses to the pharmacological effects of IL-1 are observed in humans.

Interactions with cytokine network Although there are many interactions of IL-1 with other cytokines, the most consistent and most clinically relevant is its synergism with TNF. There are, in fact, few examples in which the synergism between IL-1 and TNF has not been demonstrated. These include radioprotection, the 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.

Endogenous inhibitors and enhancers 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).

314 Charles A. Dinarello

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects Highly sensitive assays for detecting IL-1 in the circulation of healthy humans reveal levels of less than 10 pg/mL. Following a challenge injection of LPS into healthy humans, the level of IL-1 in the circulation does not increase, unlike that of IL-1 (Cannon et al., 1990).

Role in experiments of nature and disease states The role of IL-1 in human disease states is best revealed by the response of humans to parenterally administered IL-1. Chills and fever are observed in nearly all patients, even in the 1 ng/kg dose group (Smith et al., 1991, 1992, 1993). The febrile response increases in magnitude with increasing dose, and chills and fever can be abated with indomethacin treatment. Nearly all the subjects observed experienced significant hypotension at doses of 50 ng/kg or greater. The systolic blood pressure fell steadily, reaching a nadir of 90 mmHg or less 3±5 hours after the infusion of IL-1. At a dose of 300 ng/kg, most patients required intravenous pressors. When compared, these findings are very similar to those seen in patients given IL-1 . Autoantibodies to IL-1 Several studies have established that anti-IL-1 IgG antibodies are present in the sera of patients with inflammatory diseases and that these autoantibodies are neutralizing (Bendtzen et al., 1990; Svenson et al., 1990, 1992). These anti-IL-1 antibodies are also found in commercial preparations of intravenous IgG (Svenson et al., 1993). In general, the autoanti-IL-1 antibodies circulate, but IL-1 is not bound to these IgG anti-IL-1 antibodies. Role of Anti-IL-1 in Rheumatoid Arthritis Circulating cytokine autoantibodies in 318 patients with chronic rheumatoid arthritis were examined (Jouvenne et al., 1997). Anti-IL-1 but not anti-IL1 IgG antibodies were detected in 9% of blood donors and 18.9% of chronic arthritis patients, being present at a higher level in patients with

nondestructive arthritis. A negative association was found between the presence of anti-IL-1 antibodies and that of HLA-DR4. In a follow-up study, neutralizing autoantibodies to IL-1 were detected in a subset of chronic polyarthritis patients with SjoÈgren's syndrome (Jouvenne et al., 1997). The evolution of anti-IL-1 antibody levels was followed over 3 years, the level being higher in patients with a benign form of the disease. In contrast, the incidence and level of anti-IL-1 were lower in patients with rheumatoid arthritis. The relative risk of developing rheumatoid arthritis was 12 in the absence of high anti-IL-1 antibody levels. These studies suggest that anti-IL-1 autoantibodies are beneficial in patients with rheumatoid arthritis.

IN THERAPY

Preclinical ± How does it affect disease models in animals? The therapeutic uses of IL-1 are discussed under clinical trials in the chapter on IL-1 . Preclinical Studies on the Effect of IL-1 on Bone Marrow Function There is no evidence that IL-1 has a role in normal hematopoiesis (Horai et al., 1998). The IL-1-deficient mouse shows no evidence of hematological impairment. IL-1 synergizes with a variety CSFs. In fact, `hematopoietin 1', a factor which synergized with CSF, was due to IL-1. Subsequently it was shown that IL-1 also exhibited the property of `hemopoietin 1'. The synergism of either form of IL-1 with CSF is most apparent on the ex vivo culture enriched with CD34‡ cells. In the 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). The 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 (Rafi et al., 1994). Purified mouse stem cells require IL-3, IL-6, and IL-1 for their proliferation in vitro (Heimfeld et al., 1991), which suggests that primitive stem cells require multiple signals for growth. Preclinical Studies on IL-1 as an Adjuvant for Tumor Vaccines IL-1 is a potent immunoadjuvant for several tumors (Apte et al., 1993a,b). The immunostimulatory and

IL-1 antitumor effectiveness of IL-1 in experimental fibrosarcoma tumors has been demonstrated (Voronov et al., 1999). The expression of IL-1 by malignant T lymphoma cells results in activation and thus stimulates antitumor immune responses in vivo. Upon the intravenous inoculation of fibrosarcoma cells, hind leg paralysis and death occur. The shortterm expression of IL-1 results in a reduced tumorigenicity of approximately 75%. IL-1Ra reversed the reduced tumorigenicity and led to progressive tumor growth and the death of the mice. In contrast, IL-1 appears clearly to enhance tumor metastasis in several models. This may be due to the cell-associated (local) nature IL-1 in stimulating an immune response, whereas IL-1 is secreted and increases the expression of endothelial adhesion molecules (Dejana et al., 1988; Giavazzi et al., 1990; Lauri et al., 1991; VidalVanaclocha et al., 1994, 1996).

Pharmacokinetics There are no specific studies on the best effective dose of IL-1 in animal studies of bone marrow function.

Toxicity See the chapter on IL-1 .

Clinical results Clinical trials on IL-1 have been carried out that are specifically designed to mimic the protective studies in animals. These are discussed in the chapter on IL-1 . IL-1 has been administered to patients during receiving autologous bone marrow transplantation (Smith et al., 1993). The treatment with 50 ng/kg IL1 from day zero of autologous bone marrow or stem cells transfer resulted in an earlier recovery of thrombocytopenia compared with historical controls. There are to date no clinical trials on IL-1 as an adjuvant for tumors.

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