IL4

IL-4 Achsah D. Keegan* Immunology Department, Holland Laboratory of the American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855, USA * corresponding author tel: 301-517-0326, fax: 301-517-0344, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03002.

SUMMARY IL-4 is a type I cytokine that is produced by activated T cells, mast cells, and basophils. It elicits many biological responses, two of which stand out as being of great importance: the regulation of helper T cell differentiation to the TH2 type and the regulation of IgE and IgG1 production by B cells. Over the last 15 years, numerous studies using gene targeting and transgenic approaches have examined the structure of IL-4 and its in vivo role in infection and disease models. As a result of these studies, novel approaches either to inhibit IL-4 or to enhance its effects are being tested for their effects on disease models in animals and in human patient populations. Strategies to block IL-4 have shown promise in preventing or ameliorating allergic diseases. Strategies to target IL-4 to various tissues are being tested for effects on tumor growth and on skewing pathologic TH1 responses to nonpathologic TH2 responses. These strategies could ultimately lead to the ability to harness or eliminate the powerful effects of IL-4 for the improvement of human health.

BACKGROUND

Discovery Interleukin 4 (IL-4) was initially discovered in 1982 as a T cell product distinct from IL-2, which could stimulate the proliferation of B cells that had been treated with anti-IgM antibodies (for review see Paul, 1991). It was also independently characterized as a T cell factor capable of inducing the differentiation of mouse B cells into IgG1-secreting plasma cells. The development of a monoclonal antibody capable of inhibiting the B cell costimulation assay (termed

11B11) greatly facilitated the characterization of IL-4 functions. This antibody was used to purify a protein from the supernatants of activated T cells that possessed the ability to costimulate B cell proliferation. The same purified protein was also capable of acting on resting B cells to increase expression of MHC class II molecules and inducing IgG1 production in the presence of LPS. Subsequent studies revealed that IL-4 can stimulate many different biological responses in lymphocytes, as well as in other cell types (Table 1). There are currently over 12,000 publications on IL-4, making it difficult thoroughly to discuss the whole literature in one review. The reader is directed to a number of extensive reviews on IL-4 as listed in the reference section.

Alternative names IL-4 was initially termed B cell growth factor (BCGF) due to its ability to stimulate the proliferation of B cells treated with anti-IgM antibodies. It was also termed B cell differentiation factor (BCDF ). Once it became apparent that BCGF and BCDF were the same protein, and that a number of biological responses could be elicited by this protein in resting B cells, the term B cell stimulatory factor 1 (BSF-1) or B cell stimulatory factor provisional 1 (BSF-p1) was proposed. Subsequent to the cloning of the cDNA encoding this protein in 1986 and the realization that cell types other than B cells were affected, the name IL-4 was assigned.

Structure IL-4 is an  20 kDa secreted glycoprotein. It exists in a globular structure comprising four short  helices (Powers et al., 1992; Wlodawer et al., 1992).

128 Achsah D. Keegan Table 1 IL-4-mediated responses. IL-4 elicits a huge number of different biological responses in many diverse cell types Immune system Acts as a costimulant for growth in B cells, T cells, and mast cells Increases the survival of cultured T cells, B cells, and other hematopoietic cell types Directs immunoglobulin class switching to the IgG1 and IgE isotypes in mice and IgG4 and IgE in humans Directs T helper cell differentiation to the TH2 type Diminishes the inflammatory functions of monocytes and macrophages while enhancing their antigen-presenting functions Other systems Inhibits the growth of transplanted tumors in vivo and diminishes the growth of cancer cells in vitro Induces growth and chemotaxis in human fibroblasts and the production of IL-6, extracellular matrix proteins, and ICAM-1 Regulates expression of VCAM-1 on human endothelial cells and VLA-4 on T cells and eosinophils Reduces Cl± secretion by intestinal epithelial cells

Main activities and pathophysiological roles As highlighted in Table 1, IL-4 can elicit many diverse biological responses. These responses include the regulation of gene expression, costimulation of B cell and T cell proliferation, the protection of cells from spontaneous and induced apoptosis, and the regulation of chloride ion transport by intestinal epithelial cells. However, two responses stand out as being the dominant functions of IL-4. The first is the ability of IL-4 to regulate the differentiation of naive CD4+ T cells (Mosmann and Coffman, 1989; Paul, 1991; Swain, 1994; O'Garra et al., 1994; Seder and Paul, 1994), driving them into a helper phenotype called TH2 cells (Figure 1). The TH2 cells secrete a set of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13 that tend to favor a humoral immune response while suppressing a cell-mediated immune response controlled by TH1 cells. The second dominant function is the ability of IL-4 to drive immunoglobulin class switching to the IgG1 and IgE isotypes (Rothman, 1993). The ability of IL-4 to regulate the T helper cell phenotype has direct relevance to the outcome of disease (Table 2). Excessive IL-4 production by TH2 cells has been associated with elevated IgE production and allergy (Song et al., 1996; Hopkin, 1997; Rosenwasser, 1999). In addition, a strong tendency towards TH2 cell differentiation can result in a failure to efficiently cure certain infections by intracellular pathogens such as Leishmania major and lead to immunopathology (Scott et al., 1989; Reiner and Locksley, 1995).

Figure 1 Model of T helper cell differentiation. Naive T cells can differentiate into two general types of helper cells termed TH1 cells and TH2 cells. This differentiation is guided by the cytokines found in the local environment during an antigenic challenge. Differentiation to the TH1 type is guided by IL-12 and IL-18. Mature TH1 cells are characterized by the production of IL-2, IFN and lymphotoxin. Differentiation to the TH2 type is guided by IL-4. Mature TH2 cells are characterized by the production of IL-4, IL-13, IL-5, IL-6, and IL-10.

GENE AND GENE REGULATION

Accession numbers The cDNA encoding mouse and human IL-4 were both cloned in 1986 (GenBank accession numbers: mouse IL-4, M25892; human IL-4, M13982). Use of the recombinant IL-4 definitively demonstrated that IL-4 could elicit a number of responses in a variety of different mouse and human cell types (Paul, 1991). To date, cDNA encoding IL-4 have been cloned from

IL-4 129 Table 2 Generalized paradigm for the roles of TH1 and TH2 cells in disease. In general, TH1 cells drive cell-mediated immune responses such as the classic delayed-type hypersensitivity response, while TH2 cells drive an IgG1/IgE antibody response and an eosinophilic inflammatory response. The products of one class of helper T cells tends to inhibit the differentiation/function of the other class. This paradigm has direct relevance to the outcome of several disease models summarized here Disease

TH1 cells

TH2 cells

Experimental leishmaniasis

Cure

Progression

Experimental autoimmune encephalitis (multiple sclerosis)

Progression

Prevention

Atopy

Prevention

Progression

Type I diabetes (NOD)

Progression

Prevention

a large number of species, including hamster, dog, and sheep and are listed in the GenBank database (www.ncbi.nih.gov).

Figure 2 IL-4 locus. The IL-4 locus for both human and mouse has been sequenced. The organization of the IL-4 family of cytokine genes and other closely linked genes is shown.

Chromosome location The IL-4 gene resides on mouse chromosome 11 and on the long arm of human chromosome 5 at position 5q23-31 within a complex that contains genes for other cytokines. This complex has been sequenced (Frazer et al., 1997) and both the mouse and human complexes exhibit similar organization (Figure 2).

Relevant linkages The IL-4 gene is closely linked to the IL-13 gene, which lies immediately downstream of the IL-4 locus. The gene for another TH2 cytokine, IL-5, is also closely linked. Interferon regulatory factor 1 (IRF-1) lies 200 kb telomeric to IL-4. The IL-3 and GM-CSF genes are located in this chromosomal region as well. The close proximity of the cytokines IL-4, IL-13, and IL-5 suggest that there may be some type of locus control to allow for coordinate expressions of these cytokines in TH2 cells.

Regulatory sites and corresponding transcription factors The IL-4 gene consists of four exons and three introns. The second intron is long, between 4 and 5 kb. In IL-4-expressing mouse mast cells, but not T cells, there is a unique DNase I hypersensitivity site in this intron, suggesting there may be some cell typespecific regulation of IL-4 production (Brown and Hural, 1997).

A large number of investigators have studied the regulation of transcription of the IL-4 gene in T cells using heterologous reporter constructs (for review see Szabo et al., 1997). The 50 untranslated region of the IL-4 gene contains numerous cis-acting sequence elements that can interact with trans-acting factors that work together to regulate IL-4 transcription (Figure 3). The minimal IL-4 promoter ( 500±700 bp) contains five sites termed P elements (P0±P4) that exist in a consensus site for nuclear factor of activated T cells (NF-AT). The NF-AT family of transcription factors comprises at least four members: NF-ATp, NF-ATc, NF-AT3, and NF-AT4. All five NF-AT sites participate to some degree in the regulation of IL-4 promoter activity. Blocking the receptormediated activation of NF-AT with cyclosporin A blocks IL-4 transcription. Expression of an NF-ATbinding protein, NIP-25, greatly enhances IL-4 transcription. However, the precise role of the individual NF-AT family members in the regulation of IL-4 transcriptional activity is not clear. Recent results suggest that NF-ATp suppresses IL-4 transcription. Between the P1 and P2 sites is a Y-box capable of binding the transcription factor NF-Y. This interaction seems to regulate overall promoter activity.

130 Achsah D. Keegan Figure 3 IL-4 promoter. The regions of the IL-4 promoter shown to be important for the regulation of IL-4 transcription and shown to interact with trans-acting factors are presented in cartoon form.

Adjacent to the P1 and P4 sites in the IL-4 promoter are sites for activation protein 1 (AP-1). These sites can interact with AP-1 family members Fra-1, Fra-2, JunB, and JunD. Mutation of the AP-1 sites decreases IL-4 promoter activity by 50%. NF-AT and AP-1 family members are expressed in both TH1 cells and TH2 cells. While these factors participate in the receptor-mediated regulation of IL-4, they are probably not responsible for the restricted pattern of IL-4 production. The IL-4 promoter contains a site termed MARE that interacts with the c-maf transcription factor. c-maf is expressed in TH2 cell clones but not in TH1 cell clones, B cells, or monocytes. Expression of c-maf is induced in primary T cells during differentiation to TH2 cells. The c-maf transcription factor may be one of a group of factors that determine tissue-specific expression of IL-4. Multiple potential binding sites for the transcription factor GATA-3 are present throughout the whole IL-4 locus, including in the IL-4 promoter (Ranganath et al., 1998). Expression of GATA-3 is suppressed in TH1 cells and enhanced in TH2 cells. T cells derived from GATA-3 transgenic mice express not only TH1-type cytokines, but also the TH2 cytokines IL-4, IL-6, and IL-10. These results suggest that GATA-3 may act as a general regulator of the set of TH2 cytokines. Since IL-4 itself regulates the TH2 differentiation, IL-4-activated transcription factors are candidates for regulators of IL-4 transcription. The IL-4 promoter contains interaction sites for two such factors, STAT6 and HMGI(Y). The IL-4 promoter contains a STAT6-binding site that can impart IL-4 inducibility to a heterologous reporter plasmid. Its precise role in regulation of the endogenous IL-4 gene is still unclear; however, recent results indicate that expression of c-maf and GATA-3 in developing T helper cells is dependent on STAT6. The HMGI(Y) protein has been shown to bind the P1 site and block NF-AT binding. IL-4-induced phosphorylation of HMGI(Y) could free the P1 site for binding by the TCR-activated NF-AT, thus enhancing transcription. Recent studies from several groups indicate that IL-4 may also alter the chromatin structure of the

IL-4 locus, potentially one allele at a time, in T cells (Agarwal and Rao, 1998; Bix and Locksley, 1998; Riviere et al., 1998). Chromatin remodeling (as defined by DNase I hypersensitivity and loss of methylation sites) in T cells requires STAT6 but is independent of c-maf. A picture is beginning to emerge where IL-4-activated STAT6 enhances expression of GATA-3 and induces c-maf. GATA-3 would then participate in the chromatin remodeling of the IL-4 locus, while c-maf could directly regulate the IL-4 promoter.

Cells and tissues that express the gene IL-4 gene expression is highly tissue specific. Its expression is induced in T cells and highly induced in differentiated TH2 cells and NK1.1+ T cells in response to stimulation through the cell surface T cell receptor (Paul, 1991). IL-4 message is also induced in mast cells, basophils, and eosinophils in response to stimulation through the cell surface receptor for IgE (Woerly et al., 1999). The need for receptor stimulation can be bypassed using the pharmacologic agents PMA plus a Ca2+ ionophore. The transcription of IL-4 in response to these stimuli can be blocked by the immunosuppressant cyclosporin A.

PROTEIN

Accession numbers The accession number for the amino acid sequence of human IL-4 is 1310839.

Sequence See Figure 4.

IL-4 131 Figure 4 Amino acid sequence for IL-4. 1 HKCDITLQEI IKTLLNSLTE QRTLCTELTV TDJFAASKNT TEKETFCRAA TVLRGFYSHH EKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI MREKYSKCSS 130

Description of protein IL-4 is an  20 kDa secreted globular glycoprotein. Its core polypeptide makes up 14 kDa with the remaining 6 kDa composed of three N-linked sugar moieties. IL-4 functions as a monomer.

Discussion of crystal structure The structure of IL-4 has been solved (Powers et al., 1992; Wlodawer et al., 1992). To view the structure go to www.ncbi.nlm.nih.gov/Structure/ and use PDB identifier 1HIK. IL-4 exists in a globular structure comprising four short  helical bundles (termed A, B, C, D) that are arranged in an up-up-down-down configuration (go to www.psynix.co.uk/cytweb/ cyt_strucs and select IL-4 to view a ribbon diagram). Because of this arrangement, there are relatively long overhand loops between helices A and B and helices C and D. Amino acid residues 6±19 make up helix A with residues 27±30 making up the linking strand and sheet structure. Amino acids 41±59 make up helix B. Residues 70±94 comprise helix C with residues 106±109 making up the looping strand and sheet. Residues 110±127 make up helix D. There are three pairs of intrachain disulfide bonds in human IL-4 formed between residues 3 and 127, 24 and 65, 46 and 99. IL-4 makes contact with its receptor the IL-4R  chain with residues in the A and C helices, while residues in the D helix (especially Tyr124 and Ser121) contact the associating receptor chains (Kruse et al., 1993) c or IL-13R. Mutations of these residues in the D helix generate IL-4 antagonists since they will still bind the IL-4R  chain, but are unable to cause heterodimerization to form an active receptor complex.

Important homologies IL-4 possesses structural homologies with the other members of the short  helical family of cytokines including IL-2 and IL-13. It is  30% homologous

to IL-13, a cytokine with similar biological functions and similar receptor utilization.

Posttranslational modifications IL-4 is synthesized as a 14 kDa precursor protein in the rough endoplasmic reticulum. Several (about three) N-linked oligosaccharides are added and processed in the Golgi, resulting in a final molecular weight of 20 kDa. The carbohydrate residues are probably not important in IL-4 function; recombinant IL-4 produced in a variety of systems, including E. coli, COS cells, yeast, and insect cells resulting in molecules lacking all N-linked oligosaccharides to molecules highly overglycoslyated, are all fully biologically active.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-4 is produced by T cells and highly produced by differentiated TH2 cells and NK1.1+ T cells. It is also produced by mast cells, basophils, and eosinophils.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators In T cell populations, IL-4 is produced in response to stimulation through the cell surface T cell receptor. In mast cells, basophils, and eosinophils, IL-4 is produced in response to stimulation through the cell surface receptor for IgE. The pharmacologic agents PMA plus a Ca2+ ionophore can also stimulate IL4 production in these cells. The production of IL-4 in response to these stimuli can be blocked by the immunosuppressant cyclosporin A.

RECEPTOR UTILIZATION IL-4 binds to two types of cell surface receptor complexes termed the type I and type II IL-4 receptors. One or the other of these receptor complexes is expressed on many different cell types in accordance with the ability of IL-4 to have effects on many different cell types.

132 Achsah D. Keegan

IN VITRO ACTIVITIES

In vitro findings There have been a huge number of biological activities described in the literature for IL-4 on a diverse group of cell types in in vitro assays (Table 1). IL-4 has effects on B cells, T cells, macrophages, mast cells, fibroblasts, epithelial cells, and endothelial cells. It has effects on a number of different tumor cell lines including the inhibition of growth of renal cell carcinomas. IL-4 acts as a viability factor for B cells and T cells and as a costimulant for their proliferation. It drives the differentiation of T cells to the TH2 phenotype, and regulates lymphocyte gene expression. It also induces the expression of CD23 and B7 on B cells and increases expression of MHC class II. IL-4 induces VLA-4 expression on T cells. In the presence of a costimulant, IL-4 causes isotype switching to the IG1 and IgE isotypes. IL-4 has a number of effects on monocytes. It increases expression of MHC class II molecules and IL-1Ra while downregulating production of the proinflammatory cytokines IL-1, TNF, IL-6, and IL-8. IL-4 is a growth and survival factor for mast cells. It enhances VCAM-1 expression on endothelial cells and downregulates IL-8 production. IL-4 induces eotaxin production by lung epithelial cells and Clÿ ion secretion in intestinal epithelial cells. While this is not an exhaustive list of activities, it indicates that IL-4 can cause a number of responses in many different types of cells.

Regulatory molecules: Inhibitors and enhancers In mouse cells there is a naturally occurring soluble version of the IL-4R that inhibits the binding of IL-4 to the cell surface receptor complex in vitro. For many of the biological responses of B cells, IFN will antagonize the effects of IL-4. This inhibition may be due to the induction of a gene called SOCS-1 by IFN that blocks the IL-4-induced activation of STAT6. For many of the responses on endothelial cells, TNF enhances the effect of IL-4.

Bioassays used Costimulation of proliferation of purified B cells treated with anti-IgM was the first bioassay for IL-4. The ability to induce expression of CD23 on B cells has also been used as a very sensitive assay for

both mouse and human IL-4. Proliferation of the mouse indicator T cell line HT-2 or CTLLs in the presence of anti-IL-2 antibodies was used extensively for mouse IL-4. This assay was replaced by using a derivative of CTLLs lacking the IL-2R  chain, termed CT.4s. Since IL-4 action is species specific, the mouse CT.4s cells transfected with the cDNA encoding the human IL-4 receptor were used in bioassays for human IL-4. Due to a lack of complete specificity, other assays are now used routinely to measure IL-4 levels, including ELISA, ELISPOT, and intracellular IL-4 staining.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles While IL-4 has many demonstrable functions in vitro, its normal role in vivo appears to be to regulate B cell homeostasis, to regulate T helper cell differentiation, and to regulate Ig isotype switching.

Species differences In mice, IL-4 causes switching to IgG1 and IgE, while in humans the subclass of IgG is the IgG4.

Knockout mouse phenotypes IL-4 knockout mice are healthy and show normal lymphocyte development. However, in general terms, they are TH2 deficient (Muller et al., 1994; Kopf et al., 1995) (Figure 1). They are able to mount a primary and secondary antibody response. However, they lack the ability to mount a strong TH2 response to protein antigens and show greatly diminished IgG1 and IgE responses. On a Balb/c background, mice lacking IL-4 are resistant to infection with certain strains of Leishmania major (Reiner and Locksley, 1995) but not others. IL-4 knockout mice also show a deficiency in the expulsion of intestinal parasites such as Heligmosomoides polygirus (Finkelman et al., 1997). In general, these results are similar to the effects of treatment of normal mice with anti-IL-4 antibody. There are certain challenges in vivo that can overcome these defects. For example, infection of IL-4 knockout mice with a virus that causes murineacquired immunodeficiency syndrome (MAIDS)

IL-4 133 induces a strong in vivo IgE response (Morawetz et al., 1996).

Transgenic overexpression Mice transgenic for IL-4 were initially difficult to generate, probably due to the toxicity of high levels of serum IL-4. Different transgenic lines show varying phenotypes. Transgenic mice with IL-4 targeted to T cells have been shown to have increased airway hyperreactivity, allergic conjunctivitis of the eye, and mild B cell hyperplasia (Tepper, 1994). Transgenic mice using the lck-proximal promoter to drive transcription demonstrated osteoporosis (Lewis et al., 1993). Transgenic mice with IL-4 targeted to the lung show enhanced mucus production from goblet cells (Temann et al., 1997). These phenotypes can be correlated with in vitro effects on specific cell types. The determining factor for the in vivo phenotype may be the location and level of expression of the IL-4 transgene.

Endogenous inhibitors and enhancers In mouse cells there is a naturally occurring soluble version of the IL-4R. In mouse models, the soluble receptor extends the half-life of serum IL-4 by acting as a carrier protein (Ma et al., 1996). Pharmacokinetic studies demonstrated that IL-4 is rapidly cleared from the circulation and secreted by the kidneys in a degraded form. The clearance of IL-4 is dependent on IL-4 receptors and internalization of the receptor complex. The soluble receptor may extend the halflife of serum IL-4 by blocking the binding of IL-4 to its cell surface receptor. A soluble version of the human IL-4R has been identified in human serum (Song et al., 1999) and may function as a carrier to extend the half-life of circulating IL-4.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY Information on serum IL-4 levels in humans is limited. The ability to measure detectable levels of serum IL-4 has been quite variable and a direct cause±effect relationship between serum IL-4 levels

and human disease has not yet been established (Ng et al., 1999; Rigano et al., 1999). However, polymorphisms in the IL-4 gene have been linked to atopy and asthma in human patients (Marsh et al., 1994). Several studies have shown that polymorphisms in the IL-4 promoter (Song et al., 1996; Rosenwasser, 1999) are associated with allergy. This would suggest that elevated transcription of IL-4 may contribute to the allergic phenotype in humans.

IN THERAPY

Preclinical ± How does it affect disease models in animals? IL-4 has been used in vivo to treat a number of disease models in mice. It has been the most successful at ameliorating autoimmune diseases that are caused by activated TH1 cells (Table 2). IL-4 treatment has been shown to prevent development of diabetes in NOD mice (Rabinovitch et al., 1995), a model for juvenile diabetes. IL-4 has also been shown to prevent the development of experimental autoimmune encephalitis (EAE) or neuritis (Racke et al., 1994; Deretzi et al., 1999), a model for multiple sclerosis. Although initially characterized as a growth and survival factor, IL-4 has been shown to suppress the growth of some cancer cells in vivo (Tepper, 1994). This inhibition is seen in the absence of T cells and depends on an eosinophilic infiltrate and inhibition of tumor angiogenesis. Local delivery of IL-4 containing retrovirus in situ by transduced packaging cells (Saleh, 1999), has resulted in the complete elimination of established intracranial gliomas and provided protection from further challenge with glioma.

Toxicity IL-4 has been used systemically as an aid to cell-based therapy for cancer (Rubin and Lotze, 1992). Bolus injection of IL-4 has resulted in flu-like symptoms, gastrointestinal upset, asymptomatic liver damage, severe allergy-type symptoms in the nasal mucosa, and vascular leak syndrome (Rubin and Lotze, 1992; Emery et al., 1992; Prendiville et al., 1993). The maximum tolerated dose was found to be  400 mg/m2/day. IL-4 was cleared rapidly with a t1/2 of 19 minutes. IL-4 does not have apparent toxicity if administered locally. Due to these intolerable sideeffects of systemic administration, most recent uses for IL-4 are designed for a method of local delivery.

134 Achsah D. Keegan

Clinical results During initial clinical trials for antimelanoma effects, systemic IL-4 caused serious side-effects and its use was terminated. Phase I clinical trials for the local delivery of IL-4 in several models of cancer are ongoing. Currently, melanoma cells transfected with IL-4 cDNA are being tested as vaccines (Belli et al., 1998) for patients with metastatic melanoma.

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