IL13

IL-13 Andrew N.J. McKenzie* and David J. Matthews Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK * corresponding author tel: 01223-402377, fax: 01223-412178, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03007.

SUMMARY Interleukin 13 (IL-13) is produced predominantly by T helper 2 cells, but also by mast cells and NK cells. IL-13 is related to IL-4 and maps to a gene cluster that includes IL-4, IL-5, IL-3, and GM-CSF. The biological functions of IL-13 include the regulation of IgE secretion by B cells, the modulation of TH2 cell development, and the suppression of inflammatory responses due to regulation of macrophage function. Significantly, recent studies have indicated that IL-13 plays a central part in TH2 responses, mediating key roles in the expulsion of gastrointestinal parasites and in the development of asthma and allergy.

Main activities and pathophysiological roles IL-13 has been shown to have anti-inflammatory functions on monocytes and macrophages and to induce B cells to proliferate and isotype-switch to the production of IgE (McKenzie and Heath, 1996). Blocking IL-13 activity has been shown profoundly to inhibit the pathophysiology of asthma (Grunig et al., 1998; Wills-Karp et al., 1998). Studies using IL-13deficient mice have identified impaired TH2 cell development in the absence of IL-13 (McKenzie et al., 1998b) and indicated an important role for IL-13 in the expulsion of parasitic gastrointestinal helminths (McKenzie et al., 1998a).

BACKGROUND

Discovery

GENE AND GENE REGULATION

In 1989 Brown et al. identified several novel cDNA sequences that were induced upon activation of mouse lymphocytes in vitro. One of these sequences was termed P600 and it represents the first cloning of an IL-13 cDNA (Brown et al., 1989); however, at that time no biological activity was assigned to the protein product of P600. It was not until the human homolog was cloned and its activities on cells of the immune system identified that it was given the designation IL-13 (McKenzie et al., 1993a; Minty et al., 1993).

Accession numbers

Alternative names Mouse IL-13 (mIL-13) protein and cDNA have also been described as P600. Human IL-13 (hIL-13) cDNA has also been referred to as NC30.

GenBank: Bovine gene: AJ132441 Mouse cDNA: M23504 Mouse gene: L13028 Rat cDNA: L26913 Human cDNA: L06801 Human gene: L13029

Sequence Table 1 shows the nucleotide identities between human, rat, mouse, and bovine sequences.

204 Andrew N.J. McKenzie and David J. Matthews Table 1 Interspecies IL-13 sequence identity Nucleotide identity Human

Rat

Mouse

Cattle

±

74%

66%

81%

Amino acid identity Human Rat

63%

±

87%

71%

Mouse

58%

79%

±

71%

Bovine

71%

57%

58%

±

Chromosome location Whilst the human IL-13 gene is located on the long arm of human chromosome 5 (5q31), the mouse IL-13 gene is located on the syntenic region of mouse chromosome 11 (McKenzie et al., 1993b; Smirnov et al., 1995; Frazer et al., 1997), and bovine IL-13 gene is located on chromosome 4q23-31 (Buitkamp et al., 1999). All these genes have four exons and three introns and span approximately 4.5 kb (McKenzie et al., 1993b; Buitkamp et al., 1999).

Relevant linkages The IL-13 gene is closely linked to the genes encoding IL-4, IL-5, IL-3, and GM-CSF. Indeed, the IL-13 gene maps approximately 12 kb upstream of the IL-4 gene in the mouse and the human (Smirnov et al., 1995; McKenzie and Heath, 1996). The sequence of the entire 1 Mb region on human chromosome 5 containing these cytokine genes is available via the internet at http://www-hgc.lbl.gov/sequence-archive.html (Frazer et al., 1997).

Regulatory sites and corresponding transcription factors Little is known about IL-13 transcriptional regulation. The 50 flanking regions of the mouse and human genes contain a number of conserved sites for transcriptional regulators, including potential sites for AP-1, AP-2, and AP-3, interferon responsive sites, an NF-IL6 site, a GATA-3 site and a TATA-like sequence (McKenzie et al., 1993b; Smirnov et al., 1995; Dolganov et al., 1996). It is noteworthy that in IL-13 transgenic mice an IL-13 transgene containing 3 kb of 50 flanking genomic sequence retained its T cell inducible expression (Emson et al., 1998).

Cells and tissues that express the gene IL-13 mRNA is produced primarily by CD4+ T helper 2 cells (TH2) and clones (Brown et al., 1989; Zurawski and de Vries, 1994) and mast cells (Burd et al., 1995). However, TH1 cell clones, TH0 cell clones and CD8+ T cell clones have all been shown to produce IL-13 mRNA (de Waal Malefyt et al., 1995). IL-13 gene transcription has also been detected in a number of B cell malignancies (Fior et al., 1994). Recently, natural killer (NK) cells have also been shown to secrete IL-13 (Hoshino et al., 1999b).

PROTEIN

Accession numbers Cattle IL-13: CAB46636 Human IL-13: P35225 Mouse IL-13: P20109 Rat IL-13: P42203

Sequence See Figure 1.

Description of protein IL-13 is secreted as a monomeric peptide of  10 kDa (McKenzie et al., 1993a; Minty et al., 1993). This molecular mass increases with glycosylation, resulting in a range of protein species with Mr of 14,000±40,000 (McKenzie et al., 1993a). In addition to the three N-linked glycosylation sites conserved between mIL13 and hIL-13, hIL-13 also contains one extra site.

IL-13 205 Figure 1 Amino acid sequences for mouse, rat, bovine, and human IL-13. Bold characters indicate signal sequence; cysteine residues are underlined. Mouse IL-13: MALWVTAVLA LACLGGLAAP GPVPRSVSL PLTLKELIEE LSNITQDQTP LCNGSMVWSV DLAAGGFCVA LDSLTNISNC NAIYRTQRIL HGLCNRKAPT TVSSLPDTKI EVAHFITKLL SYTKQLFRHG PF Rat IL-13: MALWVTAVLA LACLGGLATP GPVRRSTSPP VALRELIEEL SNITQDQKTS LCNSSMVWSV DLTAGGFCAA LESLTNISSC NAIHRTQRIL NGLCNQKASD VASSPPDTKI EVAQFISKLL NYSKQLFRYG H Cattle IL-13: MALLLTAVIV LICFGGLTSP SPVPSATALK ELIEELVNIT QNQKVPLCNG SMVWSLNLTS SMYCAALDSL ISISNCSVIQ RTKKMLNALC PHKPSAKQVS SEYVRDTKIE VAQFLKDLLR HSRIVFRNER FN Human IL-13: MALLLTTVIA LTCLGGFASP GPVPPSTALR ELIEELVNIT QNQKAPLCNG SMVWSINLTA GMYCAALESL INVSGCSAIE KTQRMLSGFC HKVSAGQFSS LHVRDTKIEV AQFVKDLLLH LKKLFREGRF N

However, glycosylation is not necessary for biological activity (McKenzie et al., 1993a; Minty et al., 1993). The four cysteines in the mature protein are conserved between species. Due to variation in the splicing of exon three to exon four, an extra glutamine residue may be included in the hIL-13 protein at position 98 (McKenzie et al., 1993a). Interestingly, the amino acid sequences of IL-13 and IL-4 are approximately 30% homologous, although this is low it represents a significant level of similarity, which is not evident between any other interleukins (Zurawski and de Vries, 1994). Although there is no crystal structure reported for IL-13 it is likely that it will have a structure similar to IL-4, which is an anti-parallel four  helix bundle protein. This is supported by circular dichroism spectra which indicate a high level of  helical structure (Zurawski et al., 1993).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-13 is produced primarily by mouse and human TH2-like cells, but is also expressed by TH0 and TH1 clones and CD8‡ T cell clones (Brown et al., 1989; Zurawski and de Vries, 1994; de Waal Malefyt et al., 1995). NaÈ ve CD4‡ T cells have also been reported to secrete IL-13 upon stimulation.

Primary mouse mast cells and mouse and human mast cell lines have also been shown to express IL-13 in response to activation (Burd et al., 1995). Primary mouse NK cells and the human NK cell line (NK3.3) also secrete IL-13 in response to activation (Hoshino et al., 1999b).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Antigen-specific activation via the T cell receptor, or polyclonal stimulation with calcium ionophore, phorbol ester, concanavalin A or CD3"-crosslinking have all been demonstrated to elicit IL-13 expression from T cells (Brown et al., 1989; McKenzie et al., 1993a; Minty et al., 1993; Zurawski and de Vries, 1994; de Waal Malefyt et al., 1995). Calcium ionophore, phorbol ester and IgE-crosslinking elicit IL-13 expression by mast cells (Burd et al., 1995). Calcium ionophore, phorbol ester or IL-2 with IL-18 elicit IL-13 secretion by human and mouse NK cells (Hoshino et al., 1999a,b).

RECEPTOR UTILIZATION IL-13R1, IL-13R2, and IL-4R.

206 Andrew N.J. McKenzie and David J. Matthews

IN VITRO ACTIVITIES

In vitro findings See Table 2.

Regulatory molecules: Inhibitors and enhancers Neutralizing monoclonal antibodies to both mouse IL-13 and human IL-13 are available from R&D Systems. A potent inhibitor of IL-13 activity has been produced by fusing the extracellular region of IL-13R2 inframe with the human IgG1 CH2/CH3 regions.

It has been reported that only 10 ng/mL of this fusion protein was required to neutralize 50% of the activity of 3 ng/mL of IL-13 in a B9 (mouse B cell plasmacytoma) proliferation assay (Donaldson et al., 1998). As IL-4R is part of both the IL-4R complex and the IL-13R complex, mutant IL-4 analogs that act as competitive antagonists of IL-4 also compete with IL-13 for interaction with the IL-4R. A mouse IL-4 mutant protein with amino acid substitutions of Q116D and Y119D forms unproductive complexes with IL-4R and is an in vitro antagonist of IL-4 and IL-13 (Grunwald et al., 1998). Similarly, a human IL4 homolog with a mutation of Y124D competes with both IL-4 and IL-13 and antagonizes B cell responses (Aversa et al., 1993). In addition, antibody to IL-4R inhibits the action of both IL-4 and IL-13 (Zurawski et al., 1995).

Table 2 In vitro effects of IL-13 Cell type

Upregulates

Downregulates

Monocytes/macrophages

(a) CD23, MHC class II, integrin family members (CD11b, CD11c, CD18, CD29, and CD49e) (de Waal Malefyt et al., 1993)

(a) IL-1, IL-1b, IL-6, IL-8, G-CSF, TNF, IL-12, nitric oxide (de Waal Malefyt et al., 1993; Doherty et al., 1993)

(b) IL-1Ra (de Waal Malefyt et al., 1993; Muzio et al., 1994), IL-1 `decoy' receptor (Colotta et al., 1994)

(b) CD16, CD32, CD64, and CD14

(c) Chemotaxis (Magazin et al., 1994)

(c) Acts downstream of TNF signal to suppress NFB, AP-1 and apoptosis (Manna and Aggarwal, 1998)

Human B cells

(a) Proliferation in presence of costimulation by anti-CD40 or anti-IgM (Punnonen et al., 1993) (b) B cell immunoglobulin switching to IgE (Punnonen et al., 1993) (c) CD23, sIgM, CD71, CD72, and MHC class II (Punnonen et al., 1993)

Human T cells

Chemotaxis of CD4‡ and CD8‡ T cells (Jinquan et al., 1995)

Bone marrow/ hematopoietic cells

Macrophages development from Lin±Sca-1+ bone marrow progenitors in presence of stem cell factor and G-CSF (Jacobsen et al., 1994)

Human lung fibroblasts

(a) VCAM and b1-integrin expression (b) IL-6 production (Doucet et al., 1998)

Human synovial fibroblasts

IL-1Ra expression (Jovanovic et al., 1998)

Polymorphonuclear neutrophils

Cyclooxygenase-2, Fc RIII, prostaglandin E2, complement receptor 1 (Yu et al., 1998), and IL-1 decoy receptor (Colotta et al., 1994)

Endothelial cells

IL-6 (Derocq et al., 1994)

IL-1b and TNF

IL-1 and TNF

IL-13 207

Bioassays used Mouse IL-13 and human IL-13 can be assayed by measuring the proliferation of the human myeloid cell line TF1 to these factors (McKenzie et al., 1993a). However, TF1 cells also proliferate to a broad range of other cytokines. The mouse B cell plasmacytoma B9 also proliferates in response to IL-13, although these cells are approximately 100-fold less sensitive to human IL-13 (Zurawski et al., 1993).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles IL-13 production is induced in response to immunological challenge.

Species differences Initial studies performed in vitro demonstrated that IL-13 induced human B cells to undergo germline immunoglobulin gene rearrangement resulting in switching to the production of IgE (Punnonen et al., 1993). To date, attempts to induce mouse B cells to switch to IgE expression in vitro have proven unsuccessful; however, expression of an IL-13 transgene in vivo does induce IgE production even in the complete absence of IL-4, indicating that IL-13 can influence IgE expression by mouse B cells (Emson et al., 1998). However, it remains to be demonstrated whether IL-13 can stimulate mouse B cell switching directly.

Knockout mouse phenotypes Peripheral T cells from naÈ ve IL-13-deficient mice produce lower levels of TH2-like cytokines. Analysis of in vitro T helper cell differentiation assays indicated that this is due to an impairment in TH2 cell development (McKenzie et al., 1998b). However, immunization of IL-13-deficient mice with antigen or infection with nematode parasites can overcome these deficiencies (McKenzie et al., 1998a). IL-13-deficient mice also have lower basal levels of total serum IgE and develop slightly elevated antigen-specific IgG2b

responses following immunization with ovalbumin and alum (McKenzie et al., 1998b). The IL-13-deficient mice are significantly impaired in their ability to expel the parasitic gastrointestinal nematodes Nippostrongylus brasiliensis and Trichuris muris (Bancroft et al., 1998; McKenzie et al., 1998a). The inability to expel N. brasiliensis efficiently does not correlate with deficiencies in cytokine or antibody responses, but does appear to correlate with an inability to mount an effective response in the gut. Indeed, the goblet cell hyperplasia normally associated with mucus production in response to intestinal parasites was absent from infected IL-13deficient mice (Bancroft et al., 1998; McKenzie et al., 1998a). Animals carrying a combined deletion of the IL-4 and IL-13 genes have been reported recently (McKenzie et al., 1999). These animals have clearly demonstrated the complex interrelated roles of IL-4 and IL-13. Unlike IL-4-deficient and IL-13-deficient mice, the doubly IL-4/13-deficient animals failed to produce the eosinophil-rich lung granuloma, IL-5 and IgE normally induced by pulmonary insult with schistosome eggs, but instead produced a response characterized by the expression of IFN and IgG2a (McKenzie et al., 1999). However, gastrointestinal helminth infections of these animals have also demonstrated that even in the absence of both IL-4 and IL-13 alternative pathways exist for the production of IL-5, the development of eosinophilia and the expulsion of parasitic worms.

Transgenic overexpression Transgenic mice expressing the mouse IL-13 transgene, containing 3 kb of upstream sequence linked to the human CD2 locus control region, express IL-13 predominantly in lymphoid and myeloid tissue (Emson et al., 1998). The expression is regulated by activation signals and results in the expression of 10to 100-fold higher levels of IL-13 dependent on the number of transgene copies. Significantly, serum from naÈ ve transgenic mice contains high levels of IgE. When the IL-13 transgenic mice were crossed with IL-4-deficient mice (which fail to make IgE) the transgene expression resulted in the production of IgE even in the absence of IL-4, demonstrating for the first time that IL-13 can induce IL-4-independent expression of IgE in the mouse (Emson et al., 1998). It remains to be demonstrated whether this effect is due to a direct effect of IL-13 on mouse B cells. The transgenic mice also have highly perturbed thymocyte development from approximately 4 weeks

208 Andrew N.J. McKenzie and David J. Matthews of age. Although the thymus retains a normal size and gross morphology, the number of CD4+CD8+ thymocytes is reduced by 90%. Interestingly, this does not affect significantly the composition of the peripheral T cell populations (Emson et al., 1998). Expression of IL-13 under the direction of the lung Clara cell promoter has allowed the examination of the effects of IL-13 expression in tissue (Zhu et al., 1999). These animals developed airway epithelial cell hypertrophy, enhanced mucus secretion, subepithelial airway fibrosis, and enhanced expression of eotaxin in the lung.

Pharmacological effects Administration of recombinant IL-13 to naÈ ve mice using an osmotic pump resulted in increased spleen cellularity characterized by increased immature erythroblasts and megakaryocytes, and enhanced numbers of hematopoietic precursors (Lai et al., 1996). Furthermore, IL-13 treatment resulted in increased numbers of circulating monocytes (Lai et al., 1996). In addition, further studies have demonstrated that in vivo administration of recombinant IL-13 can downregulate TNF production, but enhance IL-6 expression (Di Santo et al., 1997). Significantly, administration of IL-13 into the lungs of mice resulted in the potent induction of eotaxin (Li et al., 1999).

Interactions with cytokine network The anti-inflammatory function of IL-13 is manifested in its ability to suppress the expression of cytokines such as IL-1 and IL-1b, TNF, IL-8, and IL-12. Significantly, by inhibiting IL-12 it may also act to skew the T helper populations away from a TH1 phenotype and towards a TH2 phenotype (Zurawski and de Vries, 1994; McKenzie and Heath, 1996).

Endogenous inhibitors and enhancers Although there is no direct evidence to demonstrate that IL-13R2 acts as an endogenous in vivo inhibitor of IL-13 activity, its structure, expression patterns, high affinity, and the existence of a soluble form may imply a role as an IL-13 regulatory molecule (Gauchat et al., 1997; Zhang et al., 1997).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects IL-13 is not normally detectable in naÈ ve human systems.

Role in experiments of nature and disease states IL-13 expression correlates strongly with the occurrence of allergic asthma and atopy and the associated expression of IgE (Ghaffar et al., 1997; Koning et al., 1997; Doucet et al., 1998; Van der Pouw Kraan et al., 1998). Recent studies using mouse models of experimental airway hypersensitivity have demonstrated that IL-13 plays a central role in these responses that is independent of IgE and eosinophilia (Grunig et al., 1998; Wills-Karp et al., 1998). In contrast, the anti-inflammatory roles of IL-13 may prove important in controlling inflammation such as that encountered in osteoarthritis (Jovanovic et al., 1998). Interestingly, IL-13 has recently been demonstrated to be secreted by, and stimulatory for the growth of, Reed±Sternberg cells (Kapp et al., 1999).

IN THERAPY

Preclinical ± How does it affect disease models in animals? Administration of recombinant IL-13 to IL-13deficient mice or SCID mice resulted in improved expulsion of N. brasiliensis from helminth-infected animals (Barner et al., 1998; McKenzie et al., 1998a). However, the mechanism of this clearance is currently unknown. Recombinant IL-13 has been used to assess a potential contribution of this cytokine on the progression of simian immunodeficiency virus (SIV) infection. SIV-infected macaques given multiple administrations of IL-13 underwent body weight loss which correlated with intestinal tract damage with complete atrophy of duodenal villi (Zou et al., 1998). Thus, it was suggested that IL-13 should be considered when analyzing digestive manifestations of

IL-13 209 HIV infection as well as other intestinal epithelial disorders (Zou et al., 1998). Due to its anti-inflammatory roles, IL-13 treatment has been used in models of collagen-induced arthritis and LPS-induced endotoxemia. In a model of collagen-induced arthritis treatment with IL-13 resulted in an attenuation of the associated inflammatory response (Bessis et al., 1996). Similarly, if given before or at the time of challenge with LPS, IL-13 treatment significantly inhibited lethal endotoxic shock (Muchamuel et al., 1997; Nicoletti et al., 1997). The main feature of these disparate experiments was that TNF levels were reduced significantly upon administration of IL-13. In a mouse model of diabetes, prolonged treatment of nonobese diabetic (NOD) mice with IL-13 reduced the incidence of spontaneous type 1 diabetes and corresponded with a decrease in the production of proinflammatory cytokines (Zaccone et al., 1999). IL-13 has also been found to be potentially useful in the treatment of uveitis. In a monkey model of uveitis induced by the immunization of animals with human retinal S-antigen, the administration of IL-13 was reported to reduce intraocular inflammation (Roberge et al., 1998).

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