IL5

IL-5 Gretchen T. F. Schwenger*, Viatcheslav A. Mordvinov, ReÂgis Fournier Peter Czabotar, Susanne Peroni and Colin J. Sanderson School of Biomedical Sciences, CURTIN University of Technology, Perth, Western Australia * corresponding author tel: 08 9224 0357, fax: 08 9224 0360, e-mail: [email protected] DOI: 10.1006/rwcy.2000.09002.

SUMMARY Interleukin 5 (IL-5) is a cytokine primarily involved in the pathogenesis of atopic diseases. It specifically controls the production, activation, and localization of eosinophils, the major cause of tissue damage in atopic diseases. IL-5 belongs to a gene family shared by IL-3, IL-4, and GM-CSF and is predominantly regulated at the transcriptional level. The homodimeric IL-5 protein is well conserved between species and primarily produced by T cells but also in low levels by mast cells, B cells, and eosinophils. A variety of stimuli and modulators have been identified to regulate production of IL-5 both in vivo and in vitro, indicating a highly complex series of control mechanisms. However, a better understanding of the biology of IL-5 and the regulation of its expression is critical for development of new therapeutic agents for allergic disease. This chapter covers the major biological, molecular, and structural aspects of IL-5 research.

BACKGROUND

Discovery Two lines of research converged when it was demonstrated that two very different biological activities were properties of IL-5. Mouse eosinophil differentiation factor (EDF) was identified in 1985 (Sanderson et al., 1985), using an assay based on the production of eosinophils in vitro. This murine molecule was shown to stimulate the production of eosinophils from human bone marrow, and is one of the few T cell-derived cytokines to show this cross-species activity. Human IL-5 was identified by molecular

techniques based on the mouse sequence, and shown to stimulate the production of human eosinophils and thus is an eosinophil colony-stimulating factor (Eo-CSF) and a human EDF.

Alternative names Shortly after its discovery, mouse EDF was shown to be identical to the murine B cell growth factor (BCGFII) (Sanderson et al., 1986). Similarly, BCGFII was shown to be identical with mouse T cell-replacing factor (TRF) (Harada et al., 1985). There are two intriguing aspects of these dual biological activities of mouse IL-5 on eosinophils and B cells. Firstly, although there is a well-known association between eosinophilia and IgE levels, IL-5 does not appear to be involved in the IgE response, where IL-4 is the major controlling cytokine. Secondly, although the in vitro activity on murine B cells in vitro is well characterized, there is little evidence for a biological role of IL-5 on the B cell system in vivo. Furthermore, human IL-5 is not active in assays on human B cells analogous to those used in the mouse system, and despite some claims for an activity in vitro there is little evidence that IL-5 is a player in the development of the antibody response in either the mouse or humans.

Structure IL-5 is a glycoprotein with an Mr of 40,000±45,000 and is unusual among the T cell-produced cytokines in being a disulfide-linked homodimer. It is the most highly conserved member of a group of evolutionally related cytokines, including IL-3, IL-4, IL-13, and GM-CSF, which are closely linked on human chromosome 5.

862 Gretchen T. F. Schwenger et al.

Main activities and pathophysiological roles IL-5 plays a unique and specific role in the control of eosinophil production and differentiation. It has activating activity on the closely related basophil, but is probably not the major factor controlling basophil production. The effect on eosinophil production is relatively direct. When IL-5 is expressed eosinophils are produced, and when IL-5 expression is inhibited by drugs or gene knockout eosinophil production and survival virtually cease. The role of IL-5 in eosinophilia, coupled with a better understanding of the part played by eosinophils in the development of tissue damage in chronic allergy, has made IL-5 a major target for a new generation of antiallergy drugs.

GENE AND GENE REGULATION

Accession numbers Human: AC004042, J02971 Mouse: D14461, S77891, X06270

Chromosome location IL-5 may be regarded as belonging to a gene family shared by IL-4, IL-3, and GM-CSF. They are part of the cytokine gene cluster located on chromosome 5 in humans (Campbell et al., 1987; Van Leeuwen et al., 1989) and chromosome 11 in the mouse (Van Leeuwen et al., 1989). Although there is no overall sequence homology at either the nucleotide or amino acid level between any of these four cytokines, the localization and gene structural similarities suggest a common evolutionary origin (Sanderson, 1992). Moreover, IL-5, IL-3, and GM-CSF have been shown to be involved in the regulation of hematopoietic progenitor cells, suggesting common biologic activities as well. Despite this, IL-5 alone has been shown to exert a unique control in eosinophilia.

Regulatory sites and corresponding transcription factors The coding sequence of the IL-5 gene forms four exons. The introns show areas of similarity between the mouse and human sequences, although the mouse has a considerable amount of sequence (including repeat sequences) which is not present in the human

gene. The mouse includes a 738 bp fragment in the 30 untranslated region (also known as the Alu-like repeat) which is not present in the human gene and thus the mouse mRNA is 1.6 kb while the human is 0.9 kb (Campbell et al., 1988). Each of the exons contains the codons for an exact number of amino acids. Expression of IL-5 appears to be predominantly regulated at the transcriptional level (Naora and Young, 1994). A study of the 50 and 30 untranslated regions of murine IL-5 (mIL-5) found no evidence to suggest that they play a role in mRNA stability or translation efficiency (Thomas et al., 1999a). The 50 flanking region of the IL-5 RNA initiation site contains the TATA box and additional motifs involved in transcription of the gene. There is a short sequence called CLE0 located immediately upstream of the TATA box in the IL-5 promoter region. CLE0 is highly conserved among the regulatory regions of several lymphokine genes such as IL-3, IL-4, and GM-CSF (Masuda et al., 1993). Although most studies using deletion and mutation analysis have shown CLE0 to be critical for IL-5 expression (Naora and Young, 1994; Bourke et al., 1995; Lee et al., 1995, 1998; Siegel et al., 1995; Mori et al., 1997), a few have indicated that it may not be essential (Stranick et al., 1995, 1997). The CLE0 element contains sequences similiar to the binding sequences for AP-1 and NFAT, but only the AP-1 moiety has been demonstrated to be important for inducible complex formation in EL4 cells (Siegel et al., 1995; Karlen et al., 1996). Fos and Jun proteins have been found to bind to this sequence in phorbol myristate acetate (PMA)/ cAMP-stimulated EL4 cells (Siegel et al., 1995; Karlen et al., 1996). Surprisingly, in another study, protein complexes binding to CLE0 in the mouse T cell clone D10.G4.1, were found to be constitutive, and a consensus oligonucleotide for AP-1 was unable to inhibit complex formation (Stranick et al., 1995). It has been suggested that CLE0-binding protein 1 (CLEBP-1) and high mobility group 1/2 (HMG1/2) proteins may play a role in facilitating expression of IL-5 CLE0 (Marrugo et al., 1996). The AP-1 members binding to hIL-5 CLE0 in an inducible fashion have recently been identified in the human leukemic T cell line PER-117 as JunD and Fra-2. The same report also identified constitutive binding of Oct1 and inducible binding of Oct2 to hIL-5 CLE0. These octamer factors were shown to be involved in positive regulation of the hIL-5 gene (Thomas et al., 1999a). The CLE0 element is thought to work in concert with other activation elements in the IL-5 promoter. The binding of Oct (Gruart-Gouilleux et al., 1995) and GATA (Siegel et al., 1995; Yamagata et al., 1995;

IL-5 863 Zhang et al., 1997; Lee et al., 1998) proteins at two sites immediately upstream of the CLE0 element has been reported. Binding of proteins to the Oct element was found to be dependent on activation, whereas binding of GATA was constitutive. Mutations in the GATA element have been shown to abolish IL-5 expression (Zhang et al., 1997; Lee et al., 1998), whereas the functional role of the octamer motif in this region is not clear. Recent results have found that GATA-3 is involved in the TH2-specific expression of the IL-5 gene (Lee et al., 1998). An additional Oct element located at positions ÿ244 to ÿ237 has been identified (Gruart-Gouilleux et al., 1995). This element forms complexes with factors antigenically related to members of the Oct group, and is involved in positively regulating the IL-5 promoter. Two palindromic regulatory elements, mPRE1IL-5 (ÿ79 to ÿ90) and mPRE2-IL-5 (ÿ459 to ÿ470) have been identified in the murine IL-5 promoter (Schwenger et al., 1998). Although these elements appear to bind proteins constitutively, mutations of specific motifs within these elements, which act to abolish protein binding, also significantly reduce IL-5 promoter activity. These results suggest that these elements are essential for enhancing mIL-5 gene expression. Stranick et al. also report the presence of a protected region which corresponds to mPRE1-IL-5 around position ÿ76 to ÿ90 which is likely to be involved in positive regulation of the mIL-5 gene (Stranick et al., 1998). Recently, similar palindromic regulatory elements have been reported in the hIL-5 gene. Mordvinov et al. have identified a negative regulatory element between positions ÿ79 to ÿ90 within a protected region (binding region 3) in the hIL-5 gene. This negative regulatory element overlaps with the hPRE1-IL5 element which is similar to mPRE1-IL-5 and binds Oct-1, octamer-like and YY1 nuclear factors (Mordvinov et al., 1999). A second palindromic element in the distal hIL-5 gene was identified by Schwenger et al. as hPRE2-IL-5. This element is in a similar position to mPRE2-IL-5, has sequence homology with hPRE1-IL-5 and contains overlapping binding sites for YY1 and NFAT (ÿ447 to ÿ459). The hPRE2-IL-5 element also acts to repress expression of the hIL-5 gene, displaying a novel function for NFAT (Schwenger et al., 1999). It is interesting to note the difference in the function of the PRE-IL-5 elements between the human and murine IL-5 genes when considering the conservation of sequence and position of these elements and the conservation of function of the IL-5 gene between species. NFAT, in conjunction with AP-1 family members, has been found to bind to the mIL-5P sequence located at positions ÿ117 to ÿ92 in EL4 cells

(Lee et al., 1995), and to a similiar position of the human IL-5 (hIL-5) promoter in the human T cell clone SP-B21 (Stranick et al., 1997). The role of this site in IL-5 gene expression remains controversial, as mutation analysis in some studies has suggested that this site does play a critical role (Lee et al., 1995, 1998; Stranick et al., 1997), while others have shown little or no effect (Siegel et al., 1995; Zhang et al., 1997). The NFAT site has been found to cooperate with the downstream GATA consensus site to regulate the hIL-5 promoter in a mouse mast cell line (Prieschl et al., 1995). This site has recently been shown to be involved in positive regulation of hIL-5 in both PER117 cells and peripheral blood lymphocytes (De Boer et al., 1999). Upstream of the NFAT site another GATA consensus site has been identified. Electrophoretic mobility shift assays (EMSA) with an oligonucleotide encompassing the hIL-5 promoter from positions ÿ177 to ÿ80 has suggested that GATA does bind here in IgE/antigen-stimulated mouse mast cells (Prieschl et al., 1995). No functional significance of binding was determined in this study. Whether GATA may bind to this region in T cells remains to be determined. Through deletion and mutation analysis, several distal promoter elements that may play a positive role in IL-5 gene expression have been identified. Mutations in the IL-5A (ÿ948 to  ÿ933) element were found to decrease IL-5 promoter activity in EL4 cells by 60% upon PMA/cAMP stimulation (Lee et al., 1995). Using stable transfectants of EL4 cells, deletion analysis revealed a positive element located between positions ÿ1016 to ÿ929 upon PMA stimulation (Bourke et al., 1995). Mutation of a CTF/ NF1 consensus site within this region converted the stable transfectants to constitutive expression, suggesting that this site may be important for inducible expression. Negative regulatory elements have also been identified in the IL-5 promoter. In the mIL-5 promoter, two negative regulatory elements, NREI and NREII, were mapped to the regions between positions ÿ431 and ÿ392 and ÿ300 to ÿ261 respectively (Stranick et al., 1995). In addition, investigations of the hIL-5 promoter in mouse T cells has demonstrated that two negative regulatory elements lie between positions ÿ404 and ÿ312 (Gruart-Gouilleux et al., 1995) and ÿ172 to ÿ127 (Stranick et al., 1997). The activity of these elements is dependent on activation of the cells since deletion of the regions containing these elements results in a marked increase in inducible promoter activity. Nuclear proteins that may interact with these negative regulatory elements in the IL-5 promoter have not yet been characterized.

864 Gretchen T. F. Schwenger et al.

Cells and tissues that express the gene IL-5 is produced by activated CD4+ T cells (Okudaira et al., 1995; Till et al., 1997), natural killer cells (Warren et al., 1995; Walker et al., 1998), mast cells (Barata et al., 1998; Csonga et al., 1998), B cells (Paul et al., 1990), eosinophils (Bao et al., 1996; Barata et al., 1998), and bone marrow microvascular endothelial cells (Mohle et al., 1997). Support that IL-5 may be produced by a non-T cell in vivo was shown by Castro et al. (1995), in which SCID mice deficient in T cells were found to contain IL-5 mRNA in spleen cells.

Within each IL-5 monomer reside four  helices laid end to end. In addition, a short degree of antiparallel pleated sheet formation occurs between opposing monomers (Milburn et al., 1993). Extensive mutagenesis of the IL-5 molecule has been performed to identify the residues involved in receptor interaction. Such work identified residues His38, Lys39, and His41 in the second helix, Glu89 and Arg91 in the strand and Thr109, Glu110, Trp111, and Iso112 in the fourth helix region as important in interaction with the hIL-5R chain (Graber et al., 1995; Tavernier et al., 1995). In addition these studies identified Glu13 as a contact point for the chain of the IL-5 receptor.

Discussion of crystal structure

PROTEIN

Accession numbers SwissProt: Human IL-5: P05113 PDB: 1HUL Murine IL-5: P04401

Sequence See Figure 1.

Description of protein IL-5 is a homodimeric molecule of between 45 and 60 kDa (Tominaga et al., 1990). Each monomer is 115 residues in length (113 in mouse), giving a predicted molecular weight of 24 kDa for the homodimer. The extra mass is provided by posttranslational modification in the form of glycosylation. It is unlikely, however, that this glycosylation is required for biological activity as both deglycosylated expressed IL-5 and recombinant Escherichia coli-expressed IL-5 show full receptor binding and cell proliferative activity (Tominaga et al., 1990, Graber et al., 1993).

Solving the crystal structure for IL-5 revealed the structural characteristics which allow dimerization to occur (Figure 2) (Milburn et al., 1993). The dimeric form of the molecule is maintained by cysteine bonds between opposing monomers at residues 44 and 86 (Minamitake et al., 1990) and is further stabilized by antiparallel sheet formation between opposing monomers at residues 23±35 and 89±92. This dimeric nature provides IL-5 with two sets of four-helix bundles. Each bundle consists of the first three helices from one monomer combining with the fourth from the opposing monomer. The tight packaging provided by this structure forms a molecule consisting of two main faces composed of either the A and D helices or the B and C helices. Within the AD face of the molecule reside the residues of IL-5, which are identified as playing a role in receptor interaction (Graber et al., 1995; Tavernier et al., 1995).

Important homologies The IL-5 protein has both sequence and structural homologies with both the IL-3 and GM-CSF proteins. Each of these cytokines consists of four  helices arranged in an up/down/up/down configuration. In addition they share similarities in sequence.

Figure 1 Amino acid sequence for human and mouse IL-5. The leader sequence is underlined. Human IL-5 MRMLLHLSLLALGAAYVYAIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNH QLCTEEIFQGIGTLESQTVQGGTVERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQ FLGVMSTEWAMEG EFLGVMNTEWIIES Mouse IL-5 MRRMLLHLSVLTLSCVWATAMEIPMSTVVKETLTQLSAHRALLTSNETMRLPVPTHKNHQ LCIGEIFQGLDILKNQTVRGGTVEMLFQNLSLIKKYIDRQKEKCGEERRRTRQFLDYLQE

IL-5 865 Figure 2

Crystal structure for IL-5.

In particular a glutamic acid residue in the first  helix of these molecules is conserved. This residue is involved in interaction with the common chain shared by these receptors.

Posttranslational modifications Disulfide bridges are formed between opposing monomers of IL-5 at Cys44 and Cys86 to form a dimeric molecule (Minamitake et al., 1990). Other posttranslational modification occurs in the form of glycosylation which, unlike disulfide bridge formation, is not essential for biological activity. Glycosylation of the human IL-5 molecule occurs in the form of N-linked glycosylation at Asn28 and O-linked glycosylation at Thr3 (Minamitake et al., 1990). Glycosylation also occurs at the equivalent mIL-5 residues in addition to an extra N-linked glycosylation site at Asn55 (Kodama et al., 1992).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce All the original reports on characterization, cloning, and purification of mIL-5 utilized T cell lines or lymphomas as a source of the protein, suggesting that

T cells are an important source of the cytokine. Native mIL-5 has been isolated from T cell supernatants (Sanderson et al., 1985), while human IL-5 has not been purified from natural sources. The fact that eosinophilia is a T cell-dependent phenomenon illustrates the central role of the T cell in producing IL-5. However, IL-5 mRNA production has also been demonstrated in mast cell lines (Burd et al., 1989; Plaut et al., 1989) and human Epstein±Barr virustransformed B cells (Paul et al., 1990). Furthermore, eosinophils themselves have been demonstrated to produce IL-5 (Broide et al., 1992), although they do not produce enough to maintain their own survival. In a careful study of cells producing IL-5 in bronchial biopsies from subjects with asthma it was concluded that T cells are the major cellular source of IL-5. The apparent dominance of mast cells in some studies was attributed to the fact that mast cells store IL-5 in their granules, whereas T cells secrete IL-5 rapidly as it is synthesized. Thus, immunohistological staining for IL-5 underrepresents the number of T cells compared with in situ hybridization (Ying et al., 1997).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Eosinophilia without increases in numbers of any other leukocytes is evidence of IL-5 expression in vivo.

866 Gretchen T. F. Schwenger et al. As such, when investigating IL-5-eliciting stimuli it is necessary to identify stimuli that elicit eosinophilia. Eosinophilia is characteristic of a limited number of disease states, most notably parasitic infections and allergy but also including several tumors, as described above. Toxocara canis infection of mice caused the development of eosinophilia accompanied by the appearance of IL-5 mRNA in the spleen (Yamaguchi et al., 1990). Elevations in IL-5 precede eosinophilia in onchocerciasis (Hagan et al., 1996) and significant IL-5 levels were discovered in the serum of patients with idiopathic hypereosinophilia (Owen et al., 1989). Infection of volunteers with hookworms (Necator americanus) resulted in eosinophilia as the only significant change (Maxwell et al., 1987). In these cases, the eliciting stimuli are antigens derived from the parasites. In the case of IL-5 production stimulated by allergic diseases such as allergic rhinitis and atopic asthma, the IL-5-eliciting stimuli can be a variety of innocuous antigens to which the specific individual is allergic. Typical examples include house dust mite, Dermatophagoides pteronyssinus, pollens such as grass pollen, and domestic pet dander. IL-5 production has also been shown to be both upregulated and downregulated in vitro by prostaglandin, depending on whether stimulation is via the T cell receptor (TCR) or rhIL-2 (Snijdewint et al., 1993; Kaminuma et al., 1997). Inhibitory stimuli for IL-5 production may include other cytokines such as IFN and IL-10 (Schandene et al., 1994) which have been shown to have inhibitory effect in vitro but little work has been carried out in this area in vivo. Corticosteroids, specifically glucocorticoids, have been demonstrated to be exogenous modulators of IL-5 activity, downregulating IL-5 production. Corticosteroid treatment in patients with moderate asthma revealed a decrease in bronchial responsiveness, bronchial eosinophilia, and the number of bronchoalveolar lavage (BAL) cells expressing mRNA for IL-5 (Robinson et al., 1993; Leung et al., 1995). In another study, corticosteroid treatment was found to significantly decrease peripheral blood eosinophil counts and serum IL-5 (Corrigan et al., 1993). These studies indicate the effectiveness of glucocorticoids in downmodulating the expression of IL-5 in asthmatic patients. Other drugs that have been used in the treatment of asthma include cyclosporin A (CsA), FK506, and rapamycin, all of which have been shown to inhibit IL-5 production (Mori et al., 1994; Valentine and Sewell, 1997). Modulation of IL-5 expression in vivo could also be considered to be affected by factors that influence the balance of helper T cell subsets. An increase in

prostaglandin E2 has been shown to influence the balance of helper T cell subsets in favor of the TH2 subset in vitro (Betz and Fox, 1991; Snijdewint et al., 1993) and antigen-presenting cell-derived IL-12 skews towards the TH1 subset (Kapsenberg et al., 1996). Therefore, prostaglandin E2 and IL-12 are indirectly modulating IL-5 production.

RECEPTOR UTILIZATION IL-5R: specific for IL-5 c: shared with the receptor complexes for IL-3 and GM-CSF.

IN VITRO ACTIVITIES

In vitro findings See Table 1.

Regulatory molecules: inhibitors and enhancers IL-5 can be induced by a variety of stimulants and modulators: the most efficient production requires activation of both the TCR and a second signaling pathway. IL-1, PMA, forskolin, and monoclonal antibodies (mAb) to CD2 and CD45 are all able to induce IL-5 expression (Naora and Young, 1994; Lee et al., 1998). The lectin concanavalin A (ConA) and the TCR idiotype-specific mAb 3D3 are also able to induce IL-5 synthesis and histamine has been shown to increase the production of IL-5 in activated T cells (Schmidt et al., 1994). Several combinations of regulatory molecules have been shown to induce IL-5 synthesis best in vitro. It has been shown that anti-CD28 mAb in combination with PMA are necessary for optimal induction of IL-5 synthesis (Kuiper et al., 1994; Schandene et al., 1994). On the other hand, induction of IL-5 synthesis by PMA was shown to be markedly increased by the addition of cAMP (Lee et al., 1993; Gruart-Gouilleux et al., 1995). PMA and calcium ionophore together have also been shown to induce efficient production of IL-5 (Naora and Young, 1994). It has been shown that different inhibitory molecules have a differential effect on IL-5 production depending on stimulation. For example, cAMP has been shown to downregulate IL-5 production as a

IL-5 867 Table 1 In vitro findings IL-5 species

In vitro findings

References

Murine

Induction of eosinophil production in liquid bone marrow cultures

Sanderson et al., 1985

Induction of B cell differentiation

O'Garra et al., 1986; Alderson et al., 1987

Induction of IgA production from B cells

Harriman et al., 1988

Augmentation of IL-2 receptor expression on B cells

Poudrier and Owens, 1994; Loughnan et al., 1987

Induction of eosinophil production in liquid bone marrow cultures

Lopez et al., 1986; Clutterbuck and Sanderson, 1988

Induction of basophil production in cord blood cultures

Dvorak et al., 1989

Priming human basophils for histamine and leukotriene production

Bischoff et al., 1990; Hirai et al., 1990

Enhancement of IgM and IgA production from unstimulated and Staphylococcus aureus Cowan-stimulated peripheral blood lymphocytes

Yokota et al., 1987

Enhancement of IL-2-dependent differentiation of T cells

Ramos, 1989; Nagasawa et al., 1991

Heparan sulfate augments the proliferative activity of IL-5 on the IL-5-dependent cell line Baf-IL-5

Lipscombe et al., 1998

Human

Controversial activities of hIL-5 Induction of IgM secretion from Staphylococcus aureus Cowan-activated human B cells

Azuma et al., 1986; Bertolini et al., 1993

Expression of IL-5R mRNA in human B cells

Huston et al., 1996

Enhanced Ig production by purified B cells stimulated with Moraxella catarrhalis

Huston et al., 1996

secondary effect to suppression of IL-2 production (Kaminuma et al., 1997). IL-5 production induced by ConA, 3D3 mAb and a combination of PMA and cAMP is reduced by the addition of CsA whereas PMA and CD28 mAB antibody induction of IL-5 is CsA-insensitive (Kuiper et al., 1994; Schandene et al., 1994; Karlen et al., 1996). Prostaglandin E2 has been demonstrated to affect IL-5 production in vitro; however, the effects are differential depending on the costimulatory signal used to induce IL-5 expression (Borger et al., 1998). IL-10 has been shown to inhibit B7/CD28-dependent IL-5 production but was unable to affect IL-5 secretion in response to PMA and calcium ionophore (Schandene et al., 1994). Glucocorticoids, specifically the synthetic glucocorticoid dexamethasone, have also been shown to inhibit IL-5 production in vitro depending on stimulation. Rolfe et al. (1992) reported that dexamethasone could inhibit IL-5 production

from cells stimulated with phytohemagglutinin (PHA), PMA, and rhIL-2 but not ionomycin. Mori et al. (1994) however report inhibition of PMA and ionomycin-induced IL-5 by dexamethasone. Other IL-5 inhibitors include FK506 which strongly inhibits IL-5 mRNA in cells stimulated with PHA, PMA, and/or ionomycin (Andersson et al., 1992; Rolfe et al., 1997) and a new agent, OM-01, which has been shown to suppress IL-5 protein production, mRNA expression, and transcriptional activity in peripheral blood mononuclear cells (PBMCs) with no effect on IL-2 or IL-4 (Okudaira et al., 1997).

Bioassays used Standard ELISA: protocols and products are commercially available.

868 Gretchen T. F. Schwenger et al. The measurement of cell viability is the basis for a number of cell-based assays. For example, the proliferation of growth factor-dependent cell lines in vitro is used to detect cytokines and their agonists and antagonists. These assays are extremely sensitive to cytokine concentrations and to any perturbations of the biological activity that may be caused by an agonist or antagonist. A number of techniques are currently available for the measurement of cell proliferation. The expressed luciferase viability assay (ELVA) (Coombe et al., 1998) involves cotransfection of a plasmid containing luciferase and a plasmid containing the human IL-5R  chain into the factordependent cell line Ba/F3, and selecting in IL-5. This produces an IL-5-responsive cell line expressing luciferase in a single step without the need for antibiotic selection. The eosinophil differentiation activity assay (EDAA) (Warren and Sanderson, 1985) assay determines IL-5 concentration through its differentiation activity on eosinophil progenitors in bone marrow cultures. Eosinophil peroxidase assay or eosinophil numbers on stained cytocentrifuge preparations can be used to determine activity.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles In mice, as with humans, no normal physiological role has been identified for IL-5. IL-5 is produced as part of the immune response.

Species differences Highly conserved in mammals, although there are differences in specific activity when tested across species.

Knockout mouse phenotypes The generation of mice with an inactive IL-5 gene (IL-5 knockout mice) has confirmed the key role of IL-5 in the control of eosinophilia (Foster et al., 1996; Kopf et al., 1996). No eosinophils were produced in response to either a parasite infection or aeroallergen sensitization with ovalbumin. In fact, the low background level of eosinophils seen in normal control mice was substantially reduced in nonsensitized knockout mice, to leave a very small number of

IL-5-independent eosinophils. The lack of effect on other cell types or on antibody production confirmed the unique specificity of IL-5 for the eosinophil lineage. The knockout mice have provided an important animal model to test the biological role of IL-5 and eosinophils. Following the induction of eosinophils in the lung by the challenge of sensitized mice with an antigen aerosol, a high degree of lung inflammation develops. However, in knockout mice, lung eosinophilia is not observed, and there is very little development of inflammation and lung damage (Foster et al., 1996). There is also no effect on antibody production in these animals.

Transgenic overexpression As IL-5 is normally a T cell product and the gene is transcribed for only a relatively short period of time after antigen stimulation, transgenic mice in which IL-5 is constitutively expressed by all T cells have been produced (Dent et al., 1990). These mice have detectable levels of IL-5 in the serum. They show a profound and lifelong eosinophilia, with large numbers of eosinophils in the blood, spleen, and bone marrow. This indicates that the expression of IL-5 is sufficient to induce the full pathway of eosinophil differentiation. It therefore seems likely that, because eosinophilia can occur without a concomitant neutrophilia or monocytosis, a mechanism must exist by which IL-5 is specifically and independently induced by the T cell system in eosinophilia. Another important aspect of these transgenic animals is that, despite their massive, long-lasting eosinophilia, the mice remained normal. This illustrates that an increased number of eosinophils is not of itself harmful, and that the tissue damage seen in allergic reactions and other diseases must be due to agents which trigger the eosinophils to degranulate.

Pharmacological effects Key effect is the development of allergic reactions.

Interactions with cytokine network There appears to be an interaction between IL-5 and eotaxin. Interactions with IL-3 and GM-CSF which have been demonstrated in vitro have not been validated in vivo. Clinical administration of IL-2 results in the production of IL-5 and the development of eosinophilia.

IL-5 869

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects IL-5 is a cytokine that is not encountered at high levels in healthy individuals. The control of IL-5 protein production takes place at the level of transcription. The transcription of the gene is inducible, and the protein is predominantly produced by activated CD4+ T lymphocytes. The production of IL-5 is thus part of the immune response that leads to a cell-mediated immunity. Although the major disorder involving an elevated level of IL-5 is asthma, a series of disorders exist which show a high level of IL-5.

Role in experiments of nature and disease states Allergic Disease Asthma is a respiratory disease involving bronchial inflammation and its incidence is becoming more and more important. It is a disease in which cell-mediated immunity orchestrates the accumulation of neutrophils and, more importantly, eosinophils. There is much evidence that the release of eosinophil major basic protein and eosinophil-derived neurotoxin, which indicates local degranulation at the sites of injury, are responsible for the bronchial epithelial damage within the lungs of asthmatics. Many studies of the inflammatory infiltrate of patients with asthma show that, amongst all present cytokines, IL-5 was highly represented and that the majority of the cells were eosinophils (Sanderson, 1992). Indeed, antigen challenge of asthmatic patients results in the infiltration and accumulation of eosinophils in the lung. IL-5 has been implicated in the pathogenesis of asthma and it has been shown that it has a central role in the disease because of its role in eosinophil differentiation, proliferation, survival, and recruitment. In patients suffering from acute asthma, IL-5 gene expression is more significantly increased than in patients with a stable disease. The presence of eosinophil cationic protein (ECP), a marker of eosinophil activation, is also increased in patients with acute asthma. Patients presenting with allergic rhinitis display higher levels of IL-5 and IL-4 than control subjects during natural exposure to antigen.

In chronic sinusitis, it has been noted that the pathomechanism of tissue eosinophilia in nasal polyposis is associated with an increased synthesis of IL-5. In the chronic allergic eye diseases vernal keratoconjunctivitis, atopic keratoconjunctivitis, and giant papillary conjunctivitis, allergic tissues express increased levels of messenger for IL-3, IL-4, and IL-5. Like asthma, chronic cough is associated with high levels of IL-5 and GM-CSF gene expression. These cytokines are likely responsible for the persistent inflammatory state of the airways. Acute and chronic atopic dermatitis lesions are associated with increased activation of IL-4 and IL-5 genes; initiation of acute skin inflammation in atopic dermatitis is associated with a predominance of IL-4 expression, whereas maintenance of chronic inflammation is predominantly associated with increased IL-5 expression and eosinophil infiltration. Dermatitis herpetiformis is a chronic subepidermal blistering disease in which a cellular infiltrate mainly composed of CD4+ T cells together with neutrophils and eosinophils has been shown to be important for blister formation. IL-5 is intensely expressed at the intracellular level by eosinophils and T cells. Parasitic Infection In many helminth infections, the number of eosinophils increases dramatically, often without any concurrent increase in the number of other leukocytes, so that eosinophils become the dominant cell type. The presence of microfilariae in the lungs is sometimes associated with severe asthmatic symptoms and airway hyperresponsiveness. Helminth-induced airway hyperresponsiveness is associated with eosinophilia, but can be dissociated from asthma by the positive effects of antihelminth treatment. In helminth-infected individuals, a circulating population of IL-4- and IL-5-secreting cells has been shown to be expanded by parasite-derived antigen from nematodes (Mahanty et al., 1993). In Schistosoma haematobium-infected patients, IL-5 expression is higher than in normal individuals. After reinfection, resistant patients have a higher number of schistosome adult worm antigen-specific T cells secreting IL-5 or IL-4 than susceptible patients. IL-5 serum levels were also higher in the resistant patients. The resistance to these parasites is associated to the presence of eosinophils. Peripheral blood mononuclear cells stimulated by antigen from Ecchinococcus multilocarius have an enhanced expression of TH2-like cytokines and especially IL-5. The induction of the expression of IL-5 indicates a critical role of this cytokine in the

870 Gretchen T. F. Schwenger et al. manifestation of the infestation of the human by the parasite. Tumors In cutaneous T cell lymphoma and SeÂzary syndrome, cells from skin biopsies tend to have a TH2 cytokine profile. In all stages of cutaneous T cell lymphoma, IL-4 and IL-5 are detectable. Hodgkin's disease is a peculiar type of human malignant lymphoma characterized by a very low frequency of tumor cells. Eosinophilia is a common histopathologic feature observed in patients with Hodgkin's disease. IL-5 was present in all patients with Hodgkin's disease, but at higher levels in those with eosinophilia. Others In subcutaneous angioblastic lymphoid hyperplasia with eosinophilia (Kimura's disease), IL-5 is produced and released from the site of a granuloma and lymph nodes after stimulation with Candida antigen. Peripheral blood eosinophils have a prolonged viability. These results strongly suggest that locally produced IL-5 induced by Candida antigen contributes to the eosinophilia in this disease. Serum levels of IL-5 are significantly higher in patients with bullous pemphigoid than in control subjects and the level of IL-5 is correlated with the severity of the disease. IL-5 may be one of the cytokines involved in the formation of the blisters in bullous pemphigoid. Analysis of cytokine patterns in skin biopsies of lupus erythematosus patients show that IL-5 messenger was associated with many lupus erythematosus specimens. The two autoimmune thyroiditis diseases Graves' disease and Hashimoto's thyroiditis are associated with an increased serum level of IL-5. Orbital T cells from patients suffering thyroidassociated ophthalmopathy express increased amount of IL-5. Eosinophilia±myalgia syndrome is associated with the ingestion of L-tryptophan. The patient presents an augmentation of the viability of the eosinophils, which was shown to be inhibited in vitro by addition of anti-IL-5 antibody. An association with the effects of pollution has been shown. After intranasal challenge of volunteers with diesel exhaust particle, the level of IL-5 messenger, among other cytokine messengers, was readily detectable in cells of the nasal mucosa. A rapidly progressive obstructive lung disease due to the ingestion of the vegetable Sauropus androgynus was described in people from Taiwan, in which an

altered pattern of expression of cytokines, including IL-5, was causing the infiltration of eosinophils and neutrophils in the lung. Chronic eosinophilic pneumonitis is associated with long-standing respiratory symptoms accompanied by a massive pulmonary eosinophil infiltration. Amongst other cytokines, IL-5 is present at a very high level in the BAL fluid of the involved lung segment, but completely absent in the uninvolved segment. Cryptogenic fibrosing alveolitis is an inflammatory condition of the lungs resulting in scarring, pulmonary failure, and death. A TH2 pattern of cytokine, with increased levels of IL-4 and IL-5, is predominant in cryptogenic fibrosing alveolitis. In active coeliac disease, the eosinophils infiltrating the intestine express IL-5 mRNA. These eosinophils have the ability to synthesize IL-5. This IL-5 could act in a paracrine manner to interact with B cells and T cells and in an autocrine manner contribute to the differentiation and local infiltration of eosinophils. In eosinophilic gastroenteritis, plasma from a patient obtained during eosinophilia formed in vitro eosinophilic colonies whose formation was inhibited by addition of anti-IL-5 antibodies. High plasma levels of IL-5 were noted. In Crohn's disease, patients present a higher eosinophil infiltration in diseased areas of the neoileum, than in normal areas. This infiltration is associated with a high level of IL-5 expression at the local level. IL-5 could so be implied in the generation of early mucosal damage in Crohn's disease. In pediatric Crohn's disease, results suggest that eosinophils produced IL-5 at the site of inflammation and could be involved in the immune response in an autocrine fashion, and also by interaction with T cells and B cells. T cells isolated from inflamed ulcerative colitis mucosa produced increased amounts of IL-5. The culture supernatants of gingival mononuclear cells isolated from patients with severe stage of adult periodontitis contain IL-5 and IL-6. Eosinophilic endomyocardial disease represents a major evolutive risk in chronic eosinophilia-associated disorders. Eosinophil granule proteins appear to be involved in cardiac injury. The synthesis of IL-5 by eosinophils was detected in myocardial sections and blood cells. Results suggest that IL-5 can be produced by eosinophils at the sites of myocardial tissue damage and might participate in local eosinophil activation. IL-5 levels from the peritoneal fluid of patients with pelvic endometriosis, postpelvic inflammatory disease, advanced cancer, adenomyosis and benign ovarian tumor tend to be higher than those from peritoneal fluid from normal individuals. Primary biliary cirrhosis is an autoimmune disease of the liver that is associated with an increased level of

IL-5 871 IL-5. Eosinophils were described to be present within the infiltrate of cells invading the liver. Eosinophil proliferation driven by IL-5 may contribute to tissue damage. Eosinophilic granuloma of the soft tissue is a rare disease showing infiltration of eosinophils in the granuloma tissue. IL-5 produced locally by T cells may enhance the infiltration of eosinophils in the granuloma.

IN THERAPY

Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. Administration of IL-5 to mice results in an increase in eosinophils (Hitoshi et al., 1991). Mice infected with Nippostrongylus brasiliensis develop eosinophilia and increased levels of IgE. However, when treated with an anti-IL-5 antibody, no eosinophils were observed and the number of eosinophil precursors in the bone marrow was also depressed (Coffman et al., 1989). Similar results were seen with treatment of mice infected with Schistosoma mansoni, and Strongyloides venezuelensis (Sher et al., 1990; Korenaga et al., 1991). As the antibody does not block the effect of IL-3 or GM-CSF, these experiments demonstrate the essential role of IL-5 in the control of eosinophilia. An alternative approach is to use antibody to the IL-5 receptor. A single injection of anti-IL-5 receptor antibody reduced peripheral blood eosinophils in eosinophilic transgenic mice to control levels (Hitoshi et al., 1991). It is unclear whether the antibody acts by blocking IL-5 binding to its receptor, or whether cells expressing the receptor are eliminated by immune mechanisms.

Pharmacokinetics IL-5 has a short half-life in the blood but may be sequestered by the extracellular matrix.

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LICENSED PRODUCTS See Table 2.

Table 2 Suppliers of human IL-5 and related products Supplier

Catalog no.

Source

Quantity

Sigma

I3891

Yeast expressed

3 mg

Amersham

ARM19002

Sf21 insect cell expressed

2 mg

Human IL-5

Amersham

ARM19010

Sf21 insect cell expressed

10 mg

ICN

152402

E. coli expressed

2 and 10 mg

Endogen

R-IL-5-2

E. coli expressed

2 mg

IL-5 875 Table 2 (Continued ) Supplier

Catalog no.

Source

Quantity

Endogen

R-IL-5-10

E. coli expressed

10 mg

Genzyme

80-3455-01

E. coli expressed

10 mg

Anti-human IL-5 antibodies Endogen

M-550

Monoclonal rat IgG2a; 39D10

500 mg

Endogen

M-551

Monoclonal rat IgG2a; 5A10

500 mg

Endogen

P-551

Polyclonal rabbit

500 mg

Genzyme

2374-01

Monoclonal mouse IgG

500 mg

Genzyme

1722-01

Polyclonal rabbit

500 mg

Cambridge

Monoclonal rat, TRFK5

Bioscience Human IL-5 ELISA kits Amersham

RPN 2761

96 wells

Endogen

EH-IL-5

96 wells

Endogen

EH-IL-5-10

10  96 wells

Murine IL-5 protein Sigma

I4642

COS cell expressed

5000 U

ICN

158349

COS cell expressed

5000 U

ICN

195759

Sf21 insect cell expressed

5 mg

Genzyme

MIL-5

COS cell expressed

5000 U

Genzyme

1968-01

Monoclonal rat IgG

500 mg

Endogen

MM550D

Monoclonal rat IgG2a, TRFK4

500 mg

Endogen

MM550C

Monoclonal rat IgG1, TRFK5

500 mg

Antimurine IL-5 antibodies

Murine IL-5 ELISA kits Endogen

EM-IL-5

96 wells

Endogen

EM-IL-5-5

5  96 wells