IL9

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IL-9 Jean-Christophe Renauld* Ludwig Institute for Cancer Research and Experimental Medicine Unit, Universite Catholique de Louvain, 74 Avenue Hippocrate, Brussels, B-1200, Belgium * corresponding author tel: ‡32 2 764 74 64, fax: ‡32 2 762 94 05, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03004.

SUMMARY Interleukin 9 (IL-9) is a multifunctional cytokine produced by activated TH2 clones in vitro and during TH2-like T cell responses in vivo. Although IL-9 was initially described as a T cell growth factor, its role in T cell responses is still unclear. While freshly isolated normal T cells do not respond to IL-9, this cytokine induces the proliferation of murine T cell lymphomas in vitro, and in vivo overexpression of IL-9 results in the development of thymic lymphomas. In the human, the existence of an IL-9-mediated autocrine loop has been suggested for some malignancies such as Hodgkin's disease. Various observations indicate that IL-9 is actively involved in mast cell responses by inducing the proliferation and differentiation of these cells. In addition, both genetic and experimental evidence points to the implication of IL-9 in the pathogenesis of asthma. Other potential biological targets for IL-9 include B lymphocytes, eosinophils, and hematopoietic progenitors, for which higher responses were observed with fetal or transformed cells as compared with normal adult progenitors. The IL-9 receptor is a member of the hematopoietin receptor superfamily and interacts with the chain of the IL-2 receptor for signaling.

cells and antigen, provided that supernates from activated TH2 cells were added to the culture. The growth factor present in such supernates was further purified, designated P40, and its corresponding cDNA was cloned (Van Snick et al., 1989). Independently, HuÈltner and coworkers observed that the proliferation of mast cell lines, such as L138, in response to IL-3 or IL-4 could be enhanced by a factor, designated MEA for mast cell growth enhancing activity (HuÈltner et al., 1989). Incidentally, some similarities were noticed between MEA and a T cell growth factor activity provisionally called TCGFIII (Moeller et al., 1990). The molecular cloning of a murine P40 cDNA and the availability of recombinant protein led to the demonstration that the same factor, now termed IL-9, was responsible for all these biological activities (HuÈltner et al., 1990). The human IL-9 cDNA was identified by expression cloning of a factor stimulating the proliferation of a human megakaryoblastic leukemia, Mo7E (Yang et al., 1989) and by cross-hybridization with a mouse probe (Renauld et al., 1990a).

Alternative names P40 (Uyttenhove et al., 1988); TCGF-III (Moeller et al., 1990); MEA (HuÈltner et al., 1989).

BACKGROUND

Discovery

Main activities and pathophysiological roles

The study of murine IL-9 originated in the search for a growth-promoting activity that allowed for the antigen-independent proliferation of T helper cell clones (Uyttenhove et al., 1988). Stable T cell lines, such as TS1, could be derived in the absence of feeder

Although IL-9 was initially described as a T cell growth factor, its role in T cell responses is still unclear. While freshly isolated normal T cells do not respond to IL-9, this cytokine induces the proliferation of murine T cell lymphomas in vitro, and in vivo

156 Jean-Christophe Renauld overexpression of IL-9 results in the development of thymic lymphomas. In the human, the existence of an IL-9-mediated autocrine loop has been suggested for some malignancies such as Hodgkin's disease. Various observations indicate that IL-9 is actively involved in mast cell responses by inducing the proliferation and differentiation of these cells. In addition, both genetic and experimental evidence points to the implication of IL-9 in the pathogenesis of asthma. Other potential biological targets for IL-9 include B lymphocytes, eosinophils, and hematopoietic progenitors, for which higher responses were observed with fetal or transformed cells as compared with normal adult progenitors.

GENE AND GENE REGULATION

Accession numbers Human: M30135, M86593, M55519 Mouse: M30136

Sequence A similar structure is shared by the human and murine IL-9 genes with five exons and four introns stretching over about 4 kb (Renauld et al., 1990). The five exons are identical in size for both species and show homology levels ranging from 56 to 74%. In contrast, no significant sequence homology was found in the introns, although their size is roughly conserved. However, 30 and 50 flanking regions show a high level of identity, supporting a possible involvement of these sequences in the transcriptional or posttranscriptional regulation of IL-9 expression. In particular, numerous ATTTA motifs, frequently present in cytokine mRNAs and supposedly involved in the modulation of the mRNA stability, were found in the 30 untranslated region of both genes (Renauld et al., 1990b).

Chromosome location The human IL-9 gene is a single copy gene and was mapped on chromosome 5, in the 5q31!q35 region (Modi et al., 1991), which contains various growth factor and growth factor receptor genes such as IL-3, IL-4, IL-5, GM-CSF, and CSF1R. In the mouse, the IL-9 gene does not seem to be linked to the same gene cluster, as the IL-3, IL-4, IL-5, and GM-CSF genes are located on chromosome 11, while the IL-9

gene has been localized on mouse chromosome 13 (Mock et al., 1990).

Relevant linkages In the human, the 5q31!q35 region has been shown to be deleted in some hematological disorders (Sokal et al., 1975), and is involved in asthma, atopy, and bronchial hyperresponsiveness (Doull et al., 1996). The mouse IL-9 locus is also involved in bronchial hyperresponsiveness (Nicolaides et al., 1997). Interestingly, a similar linkage has been reported for the human IL-9R locus (Holroyd et al., 1998).

Regulatory sites and corresponding transcription factors The promoter of the IL-9 gene contains a TATA box sequence and potential recognition sites for several transcription factors such as AP-1, IRF-1 (interferon regulatory factor 1), which were identified in both promoters but whose physiological relevance remains elusive (Renauld et al., 1990). Other consensus sequences present in the 50 flanking region of the human gene include SP-1, NFB, Octamer, AP-3, AP-5, glucocorticoid-responsive element, and a cAMP response element (Kelleher et al., 1991). Functional analysis of the IL-9 promoter by phorbol esters and PHA demonstrated the role of the AP-1 and NFB sites in IL-9 induction in a T cell leukemia (Zhu et al., 1996).

Cells and tissues that express the gene So far, IL-9 expression seems to be mainly restricted to activated T cells. In the human IL-9 was shown to be mainly produced by CD4‡CD45RO‡ T cells (Renauld et al., 1990; Houssiau et al., 1995), HTLV-Iinfected T cell leukemias (Yang et al., 1989; Kelleher et al., 1991) and Hodgkin cell lines (Merz et al., 1991).

PROTEIN

Accession numbers SwissProt: Human: P15248 Mouse: P15247

IL-9 157

Figure 1 Amino acid sequences for mouse and human IL-9. Signal sequence is underlined. Mouse IL-9: MLVTYILASV LLFSSVLGQR CSTTWGIRDT NYLIENLKDD PPSKCSCSGN VTSCLCLSVP TDDCTTPCYR EGLLQLTNAT QKSRLLPVFH RVKRIVEVLK NITCPSFSCE KPCNQTMAGN TLSFLKSLLG TFQKTEMQRQ KSRP Human IL-9: MLLAMVLTSA LLLCSVAGQG CPTLAGILDI NFLINKMQED PASKCHCSAN VTSCLCLGIP SDNCTRPCFS ERLSQMTNTT MQTRYPLIFS RVKKSVEVLK NNKCPYFSCE QPCNQTTAGN ALTFLKSLLE IFQKEKMRGM RGKI

Sequence See Figure 1.

Description of protein Both human and murine protein sequences contain 144 residues with a signal peptide of 18 amino acids. The overall identity reached 69% at the nucleotide level and 55% at the protein level. In accordance with the heavy glycosylation observed with the natural murine protein, four potential N-linked glycosylation sites were noticed in both sequences. The sequence is also characterized by the presence of 10 cysteines, which are perfectly matched in both mature proteins and a strong predominance of cationic residues, which explains the elevated pI (  10) measured with purified natural IL-9.

Posttranslational modifications Cleavage of the N-terminal signal peptide. Heavy glycosylation (sugars represent > 50% of the Mr of natural IL-9).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce So far, IL-9 expression seems to be mainly restricted to activated T cells. In the mouse, IL-9 is preferentially produced by TH2 clones in vitro (Schmitt et al., 1989; Gessner et al., 1993). In the human, a link between dysregulated IL-9 production and lymphoid malignancies has been suggested by the observation that lymph nodes from patients with Hodgkin's lymphoma and large cell anaplastic lymphomas constitutively produce IL-9 (Merz et al., 1991). Constitutive IL-9 expression was

also detected in HTLV-I transformed T cells (Kelleher et al., 1991) and in Hodgkin cell lines (Merz et al., 1991; Gruss et al., 1992).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators In the mouse, in vivo, IL-9 production is also associated with TH2-like T cell responses such as anti-IgD-mediated polyclonal activation (Svetic et al., 1991), helminth infections (Svetic et al., 1993a), and Leishmania major infection of susceptible but not resistant mice (Gessner et al., 1993). In the human, the regulation of IL-9 expression has been studied in vitro using freshly isolated peripheral blood mononuclear cells (PBMC). While no IL-9 message could be detected in these cells in the absence of any stimulation, T cell mitogens such as phytohemagglutinin (PHA) or anti-CD3 mAb induced a substantial IL-9 expression by T cell-enriched lymphocyte populations, and more specifically, by CD4‡CD45RO‡ T cells (Renauld et al., 1990a; Houssiau et al., 1995). When human peripheral T cells are activated by lectins or other mitogens, IL-9 mRNA expression appears in the late stages of T cell activation, with a peak at 28 hours. IL-2 was identified as a major mediator of IL-9 expression, since anti-IL-2 receptor antibodies completely block this process (Houssiau et al., 1992). Moreover, in patients with a primary immunodeficiency disease affecting IL-2 gene expression, reduced levels of IL-9 were found in response to antigen-specific T cell stimulation and this defect was corrected by addition of exogenous IL-2 (Hauber et al., 1995). Further analysis of IL-9 expression by peripheral T cells unraveled a cascade of cytokines with IL-2 being required for IL-4 production, a combination of IL-2 and IL-4 for IL-10 production, and a combination of IL-4 and IL-10 for IL-9 production (Houssiau et al., 1995). As mentioned

158 Jean-Christophe Renauld above, IL-9 is produced by CD45RO‡ T cells but not by CD45RA‡ T cells. However, the latter T cell subset became able to produce IL-9, provided that both exogenous IL-4 and IL-10 were added to the cultures. The central role played by IL-2 and IL-10 for IL-9 expression by human T cells was also confirmed in mouse models, as IL-9 production is significantly reduced in IL-2-deficient T cells (Schmitt et al., 1994), and in vivo in IL-10 targeted mice (Monteyne et al., 1997). As regards IL-4, contradictory results obtained in in vitro and in vivo experimental models suggest that IL-9 can be expressed by both IL-4-dependent and independent pathways (Schmitt et al., 1994; Monteyne et al., 1997). In addition, IL-1 plus TGF was also shown to be a potent inducer of IL-9 production by murine peripheral T cells (Schmitt et al., 1991, 1994), whereas IL-12, IFN , and IFN/ inhibit IL-9 expression (Svetic et al., 1993b; Lauwerys et al., 1998). Another characteristic of IL-9 expression is its association with HTLV-I, a retrovirus involved in adult T cell leukemia. Indeed, it was observed that HTLV-I-transformed T cells produce IL-9 constitutively (Yang et al., 1989; Kelleher et al., 1991). Although it is not yet clear which protein of HTLV-I is responsible for this induction, Kelleher and colleagues suggested an implication of the Tax transactivator through an NFB consensus site in the IL-9 promoter (Kelleher et al., 1991). Interestingly, in another system of T cell transformation by murine polytropic retroviruses, viral infection also resulted in IL-9 expression (Flubacher et al., 1994). However, in vivo, the TH1-promoting effect of most viral infections is likely to downregulate any putative direct IL-9-inducing activity of viruses.

RECEPTOR UTILIZATION The IL-9 receptor interacts with the chain of the IL2 receptor, which is required for signal transduction but not for IL-9 binding (Kimura et al., 1995), and is shared by the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors (Demoulin and Renauld, 1998).

IN VITRO ACTIVITIES

In vitro findings T Lymphocytes Initial observations in a murine system suggested that the activity of IL-9 was apparently restricted to some T helper cell clones (Uyttenhove et al., 1988). Noticeably, freshly isolated T lymphocytes never

responded to this cytokine, the only exception being murine fetal thymocytes (Suda et al., 1990). However, it appeared that the sensitivity to the growthpromoting activity of IL-9 is not a characteristic of a particular T cell subpopulation, but can be acquired gradually by long-term in vitro culture. In human systems, significant proliferation could be induced by IL-9 when PBMCs were preactivated for only 10 days with PHA, IL-2, and irradiated allogeneic feeder cells, thereby indicating that responses to IL-9 require previous activation. When human T cell clones were derived from established PHA-stimulated T cell lines, most of these clones proliferated in response to IL-9, irrespective of their CD4 or CD8 phenotype (Houssiau et al., 1993). IL-9 was found to significantly stimulate the in vitro proliferation of primary lymphomas induced either by chemical mutagenesis in DBA/2 mice or by X-ray radiation in B6 mice (Vink et al., 1993). Moreover, IL-9 was found to protect such tumor cells against dexamethasone-induced apoptosis, even for cell lines whose in vitro proliferation is completely independent of any cytokine. With some cell lines, IL-9 turned out to be more potent in protecting cells against apoptosis rather than in inducing proliferation. By contrast, IL-2 seems to be more efficient as a proliferation inducer (Renauld et al., 1995a). This activity could therefore be particularly relevant for the oncogenic potential of IL-9 in vivo. B Lymphocytes For mouse B lymphocytes, although IL-9 alone fails to stimulate Ig production, it acts synergistically with suboptimal doses of IL-4 for the production of IgE and IgG1 by LPS-activated semipurified B cells (Petit-FreÁre et al., 1993). The influence of IL-9 on the IL-4-induced IgG1 production correlated with an increase in the number of IgG1-secreting cells. By contrast, IL-9 did not affect the IL-4-induced CD23 expression by LPS-activated B cells, indicating that its activity is not a simple upregulation of the IL-4 responsiveness by the B cells. In the human, similar observations have been reported with semipurified peripheral B cells (Dugas et al., 1993). In this experimental system, IL-9 cooperated with IL-4 in the production of IgE and IgG but not of IgM. Moreover, IL-9 also potentiated the IL-4-induced IgE production by sorted CD20‡ human B cells upon costimulation by irradiated EL4 murine T cells, thereby suggesting a direct activity on B cells (Dugas et al., 1993). In another experimental model, anti-IL-9 antibodies were found to inhibit IgE production by human PBMCs stimulated by IL-4 and IL-7 (Jeannin et al., 1998).

IL-9 159

Mast Cells IL-9 induces the proliferation of most permanent mast cell lines such as L138, MC-6, H7 and MC-9 (Williams et al., 1990). The effect of IL-9 on bone marrow-derived mast cells (BMMCs) varies according to the length of culture in vitro. Early after derivation of primary BMMCs from bone marrow progenitors, IL-9 alone is not sufficient to sustain mast cell growth, but increases the survival of the cells and synergistically enhances the proliferation induced by IL-3 or steel factor. After further time in vitro, when stable mast cell lines are obtained, IL-9 alone becomes capable of inducing their proliferation, without any need for additional factors. Besides this growth-promoting activity, IL-9 may play a key role in mast cell differentiation by regulating the expression of mast cell proteases. Indeed, stimulation of BMMCs by IL-9 induces the expression of transcripts encoding mMCP-1, mMCP-2, and mMCP-4 proteases. By contrast, IL-4 and IL-3 seem to suppress the differentiation of BMMCs, since they inhibit the IL-9-induced expression of mast cell proteases (Eklund et al., 1993). Other protease genes belonging to the granzyme family, such as granzyme B, are also produced by mast cells in response to IL-9 (Louahed et al., 1995). Moreover, IL-9 similarly upregulates the mRNA expression of the  chain of the high-affinity IgE receptor (Louahed et al., 1995) and induces IL-6 secretion by mast cell lines (HuÈltner and Moeller, 1990; HuÈltner et al., 1990). Taken together, these observations indicate that, beyond its activity on the proliferation, this factor could be an important mediator of mast cell differentiation.

to be dependent on the presence of T cells (Williams et al., 1990). By contrast, granulocyte or macrophage colony formation (CFU-GM, CFU-G or CFU-M) was usually not affected by IL-9. However, an activity on early multipotential progenitors was observed by a two-step liquid culture assay with CD34‡ CD33ÿ DRÿ cells (Lemoli et al., 1994). In this assay, the majority of the colonies observed with IL-9 or IL-9 and SCF corresponded to CFU-GM. Noticeably, Schaafsma and colleagues observed that IL-9 also promoted some granulocytic as well as monocytic colony (CFU-GM) growth from CD34‡CD2ÿ progenitors from some bone marrow donors (Schaafsma et al., 1993). Experiments comparing the effects of IL-9 on fetal and adult progenitors have shown that addition of IL-9 to cultures of fetal progenitors induced maturation of CFU mix and CFU-GM while IL-9 is also more effective on fetal cells of the erythroid lineage (Holbrook et al., 1991). In addition, IL-9 was found to increase the in vitro proliferation of human myeloid leukemic cells in a clonogenic assay in methylcellulose, suggesting a preferential activity on transformed myeloid cells as compared with their normal progenitors (Lemoli et al., 1996). Neurons A role for hematopoietic cytokines in the differentiation of neuronal cells has been suggested by studies on immortalized murine embryonic hippocampal progenitor cell lines, with little evidence of morphological maturation. In combination with bFGF and TGF, IL-9 enhanced neurite outgrowth as well as other morphological modifications, and conferred electrical excitability to these cells (Mehler et al., 1993).

Hematopoiesis The first evidence for an involvement of IL-9 in the hematopoietic system was provided by the identification and cloning of the human protein as a growth factor for the megakaryoblastic leukemia Mo7E, a cell line displaying early markers of differentiation, such as CD33 and CD34, and markers for bipotent erythromegakaryoblastic hematopoietic precursors (Yang et al., 1989). In fact, IL-9 did not seem to be active on normal megakaryoblastic precursors, but supported the clonogenic maturation of erythroid progenitors in the presence of erythropoietin (Donahue et al., 1990). This activity was confirmed by several groups and reproducibly observed with highly purified progenitors after sorting for CD34‡ cells and T cell depletion (Lu et al., 1992; Sonoda et al., 1992; Schaafsma et al., 1993), particularly in synergy with SCF (Lemoli et al., 1994). In the mouse, a similar erythroid burstpromoting activity has been described but appeared

HIV Infection Two puzzling observations suggest that IL-9 plays a role in HIV infection of T lymphocytes. On the one hand, IL-9 was shown to significantly increase HIV replication in CD4‡ T cells (Mackewicz et al., 1994). On the other hand, IL-9 receptor expression was reported to be associated with the anti-HIV activity of CD8‡ T cells (Hossain et al., 1998). Further experiments are definitely needed to understand the mechanisms and physiological significance of these observations.

Regulatory molecules: Inhibitors and enhancers Many activities of IL-9 have been demonstrated in synergy with other cytokines: IL-3 for mast cell

160 Jean-Christophe Renauld proliferation (HuÈltner et al., 1990); IL-2 for the proliferation of fetal thymocytes (Suda et al., 1990); SCF for proliferation of mast cells or hematopoietic precursors (Miyazawa et al., 1992); EPO for proliferation and differentiation of erythroid progenitors (Donahue et al., 1990); IL-4 for IgE and IgG1 production by B_cells (Dugas et al., 1993).

Bioassays used The original bioassay for murine IL-9 is based on the proliferation of a factor-dependent T cell line called TS1, the half-maximal proliferation being obtained with 15 pg/mL of purified IL-9 (Uyttenhove et al., 1988). These cells are not responsive to IL-2 but proliferate in response to IL-4. Most of the IL-9dependent lines have lost the  chain of the IL-2 receptor as well as the capacity to proliferate in the presence of IL-2, with one exception, the ST2.K9 line, responsive to IL-2 and IL-9 but not to IL-4 (Schmitt et al., 1989). Leukemia-inhibiting factor (LIF) was also found to stimulate the proliferation of some IL9-responsive T cell lines (Van Damme et al., 1992). Murine mast cell lines such as MC-9 also respond to IL-9 in addition to IL-3, IL-4, and IL-10. Human IL-9 was identified as promoting growth activity for the Mo7E cell line, isolated from a child with acute megakaryoblastic leukemia (Yang et al., 1989). This cell line is also responsive to steel factor, GM-CSF, and IL-3 which are more potent stimulators of these cells (Hendrie et al., 1991) ± a clear disadvantage when IL-9 must be measured in complex cytokine mixtures. While human and mouse IL-9 are equally active in an Mo7E proliferation assay, human IL-9 is not active on murine cells. An easier and more specific bioassay for human IL-9 was obtained when murine TS1 cells were transfected with the human IL-9 receptor cDNA (Renauld et al., 1992). Among the human factors described so far, only LIF/HILDA and insulin were shown to promote the proliferation of this transfected murine cell line.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Preliminary results suggest that IL-9 might play an important role in the immune response against intestinal parasites such as helminths, probably by contributing to the development of mucosal mast cell

hyperplasia induced by worm infections. Resistance to Trichuris muris was found to correlate with the production of IL-5 and IL-9 in mesenteric lymph nodes (Else et al., 1992).

Transgenic overexpression IL-9 transgenic mice that overexpress this cytokine in most tissues have been generated; 5±10% of these mice spontaneously developed lymphoblastic lymphomas (Renauld et al., 1994). Interestingly, no preneoplastic T cell hyperplasia has ever been observed in these mice, thereby confirming the lack of activity of IL-9 on normal resting T cells in vitro. This contrasts with other transgenic models such as IL-7 transgenic mice, in which a proliferation of normal T cells in the skin precedes the onset of lymphomas (Rich et al., 1993). Moreover, the IL-9 transgenic mice were highly susceptible to chemical mutagenesis, as all transgenic animals developed T cell lymphomas after injection of doses of a mutagen (N-methyl-N-nitrosourea) that were totally innocuous in control mice. Similarly, these transgenic mice exhibit a high sensitivity to the tumorigenic effect of gamma irradiation (Renauld et al., 1995b). In all transgenic animals, increased mast cell infiltration was found inside the gastric and intestinal epithelium as well as in the upper airways epithelium and kidneys, but not in other organs, notably the skin. Although IL-9 by itself did not induce mast cell development in vitro, it was strongly synergistic with steel factor or stem cell factor (SCF) to promote the growth and differentiation of mast cells from bone marrow progenitors. In vivo, antibodies directed against c-kit, the SCF receptor, blocked the IL-9 transgenic mastocytosis. Since a similar constitutive SCF expression was observed in both IL-9 transgenic and control mice, observations indicate that neither SCF nor IL-9 are sufficient to induce mastocytosis, but that the synergistic activity of these cytokines is responsible for the in vivo amplification of this cell population in IL-9 transgenic mice (Godfraind et al., 1998). Unexpectedly, intestinal IL-9 transgenic mast cells showed a phenotype related to connective type mast cells, usually found in the skin and in the peritoneal cavity. The cells were stained by safranin and strong expression of mMCP-4, and mMCP-5 proteases was found in this organ. However, they also expressed proteases related to the mucosal mast cells such as mMCP-1 and mMCP-2. Interestingly, mast cells derived in vitro in the presence of IL-9 and SCF expressed the same protease pattern as that observed in IL-9 transgenic mice. These observations suggest that the synergistic activity of IL-9 and SCF induces

IL-9 161 the proliferation and differentiation of a new mast cell subset expressing an extended pattern of proteases characteristic of both connective tissue type and mucosal mast cells. In line with these observations, IL-9 transgenic mice exhibit a mast cell-dependent resistance to intestinal parasites such as Trichinella spirelis and Trichuris muris (Faulkner et al., 1997, 1998). Besides intestinal mastocytosis, IL-9 transgenic mice also show an increase in pulmonary mast cells (Godfraind et al., 1998; Temann et al., 1998), thereby supporting the hypothesis of the implication of IL-9 in asthma originally raised by genetic linkage observations (Doull et al., 1996; Nicolaides et al., 1997; Holroyd et al., 1998). In this respect, IL-9 transgenic mice exhibit a markedly increased airway hyperresponsiveness (McLane et al., 1998; Temann et al., 1998). Like mast cells, eosinophils are supposed to play an important role in the pathogenesis of asthma. Interestingly, accumulation of eosinophils has also been observed in bronchoalveolar lavages and in the peritoneal cavity of IL-9 transgenic mice (McLane et al., 1998; Temann et al., 1998; Dong et al., 1999; Renauld et al., unpublished data). Although preliminary data indicate that IL-5 is required for the IL-9-induced eosinophilia in vivo, further experiments are needed to determine if IL-9 has a direct or indirect effect on these cells. The possibility remains that IL-9 can both directly promote the proliferation of eosinophil progenitors, and indirectly induce their migration into the lungs, as suggested by the observation that IL-9 upregulates the expression of eotaxin and other chemokines by lung epithelial cells (Dong et al., 1999). Contrasting with the rather weak activity of IL-9 on in vitro immunoglobulin production, preliminary observations made in IL-9 transgenic mice strongly support its implication in humoral responses in vivo. Both basal titers of all immunoglobulin classes and antigen-specific antibody responses are increased in the serum of these animals. In addition, these mice are characterized by a dramatic increase in the number of peritoneal B1b cells, but not in conventional B cells, suggesting that IL-9 may be specifically active in B cell responses involving this particular subset, such as autoimmune processes (Vink et al., 1999).

IN THERAPY

Preclinical ± How does it affect disease models in animals? The potential activity of IL-9 in autoimmunity models is illustrated by preliminary data obtained in nonobese diabetic (NOD) mice. NOD mice are

considered as a model of cell-mediated autoimmunity since within a few months they spontaneously develop a T cell-mediated insulitis leading to diabetes. This autoimmune process can be inhibited by TH2 cytokines such as IL-4 and IL-10, or accelerated by a TH1-promoting cytokine such as IL-12. In addition, NOD mice are also susceptible to autoimmune processes such as iodide-induced thyroiditis. When a high iodide dose is administered to goitrous NOD mice the iodide-induced thyroid cell necrosis is followed by diffuse infiltration by macrophages and CD4 and CD8 T cells, leading to follicular destruction similar to Hashimoto's thyroiditis in the human. A short course of IL-9 treatment completely abrogated T lymphocyte and macrophage infiltration. In addition, IL-9 induced an increase in germinal center formation in draining lymph nodes, indicating that the inhibition of the cell-mediated response is accompanied by B cell activation. However, no significant change in TH1 or TH2 cytokine expression was detected in the thyroid or lymph nodes of IL-9-treated NOD mice. A similar inhibition of cellular infiltrate was observed in the pancreatic islets of NOD mice, whereas treatment with IL-9 for 4±6 days significantly suppressed the insulitis (Many et al., unpublished data). Taken together, these data indicate that IL-9 favors humoral autoimmunity but inhibits cellmediated autoimmune processes.

References Demoulin, J.-B., and Renauld, J.-C. (1998). Signalling by cytokines interacting with the interleukin-2 receptor chain. Cytokines Mol. Ther. 4, 243±256. Donahue, R., Yang, Y., and Clark, S. (1990). Human P40 T-cell growth factor (interleukin-9) supports erythroid colony formation. Blood 75, 2271±2275. Dong, Q., Louahed, J., Vink, A., Sullivan, C. D., Messler, C. J., Zhou, Y., Haczku, A., Huaux, F., Arras, M., Holroyd, K. J., Renauld, J. C., Levitt, R. C., and Nicolaides, N. C. (1999). Interleukin-9 induces chemokine expression in lung epithelial cells and baseline airway eosinophilia in transgenic mice. Eur. J. Immunol. 29, 2130±2139. Doull, I. J., Lawrence, S., Watson, M., Begishvili, T., Beasley, R. W., Lampe, F., Holgate, T., and Morton, N. E. (1996). Allelic association of gene markers on chromosomes 5q and 11q with atopy and bronchial hyperresponsiveness. Am. J. Respir. Crit. Care Med. 153, 1280±1284. Dugas, B., Renauld, J.-C., PeÁne, J., Bonnefoy, J. Y., PetitFreÁre, C., Braquet, P., Bousquet, J., Van Snick, J., and Mencia-Huerta, J.-M. (1993). Interleukin-9 potentiates the interleukin-4-induced immunoglobulin (IgG, IgM and IgE) production by normal human B lymphocytes. Eur. J. Immunol. 23, 1687±1692. Eklund, K., Ghildyal, N., Austen, F., and Stevens, R. (1993). Induction by IL-9 and suppression by IL-3 and IL-4 of the levels of chromosome 14-derived transcripts that encode lateexpressed mouse mast cell proteases. J. Immunol. 151, 4266±4273.

162 Jean-Christophe Renauld Else, K. J., HuÈltner, L., and Grencis, R. (1992). Cellular immune responses to the murine nematode parasite Trichuris muris. Immunology 75, 232±237. Faulkner, H., Humphreys, N., Renauld, J.-C., Van Snick, J., and Grencis, R. (1997). Interleukin-9 is involved in host protective immunity to intestinal nematode infection. Eur. J. Immunol. 27, 2536±2540. Faulkner, H., Renauld, J.-C., Van Snick, J., and Grencis, R. (1998). Interleukin-9 enhances resistance to the intestinal nematode Trichuris muris. Infect. Immun. 66, 3832±3840. Flubacher, M., Bear, S., and Tsichlis, P. (1994). Replacement of interleukin-2-generated mitogenic signals by a mink cell focusforming (MCF) or xenotropic virus-induced IL-9-dependent autocrine loop: implications for MCF virus-induced leukemogenesis. J. Virol. 68, 7709±7716. Gessner, A., Blum, H., and RoÈllinghoff, M. (1993). Differential regulation of IL-9 expression after infection with Leishmania major in susceptible and resistant mice. Immunobiology 189, 419±435. Godfraind, C., Louahed, J., Faulkner, H., Vink, A., Warnier, G., Grencis, R., and Renauld, J.-C. (1998). Intraepithelial infiltration by mast cells with both connective tissue-type and mucosal-type characteristics in gut, trachea, and kidneys of IL-9 transgenic mice. J. Immunol. 160, 3989±3996. Gruss, H.-J., Brach, M., Drexler, H.-G., Bross, K., and Herrmann, F. (1992). Interleukin 9 is expressed by primary and cultured Hodgkin and Reed-Sternberg cells. Cancer Res. 52, 1026±1031. Hauber, I., Fisher, M. B., Maris, M., and Eibl, M. M. (1995). Reduced IL-2 expression upon antigen stimulation is accompanied by deficient IL-9 gene expression in T cells of patients with CVID. Scand. J. Immunol. 41, 215±219. Hendrie, P., Miyazawa, K., Yang, Y.-C., Langefeld, C., and Broxmeyer, H. (1991). Mast cell growth factor (c-kit ligand) enhances cytokine stimulation of proliferation of the human factor-dependent cell line, MO7e. Exp. Hematol. 19, 1031±1037. Holbrook, S., Ohls, R., Schibler, K., Yang, Y.-C., and Christensen, R. (1991). Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors. Blood 77, 2129±2134. Holroyd, K. J., Martinati, L. C., Trabetti, E., Scherpbier, T., Eleff, S. M., Boner, A. L., Pignatti, P. F., Kiser, M. B., Dragwa, C. R., Hubbard, F., Sullivan, C. D., Grasso, L., Messler, C. J., Huang, M., Hu, Y., Nicolaides, N. C., Buetow, K. H., and Levitt, R. C. (1998). Asthma and bronchial hyperresponsiveness linked to the XY long arm pseudoautosomal region. Genomics 52, 233±235. Hossain, M. M., Tsuchie, H., Deterio, M. A., Shirono, H., Hara, C., Nishimoto, A., Saji, A., Koga, J., Takata, N., Maniar, J. K., Saple, D. G., Taniguchi, K., Kageyama, S., Ichimura, H., and Kurimura, T. (1998). Interleukin-9 receptor alpha chain mRNA formation in CD8‡ T cells producing antihuman immunodeficiency virus type 1 substances. Acta Virol. 42, 47±48. Houssiau, F., Renauld, J.-C., Fibbe, W., and Van Snick, J. (1992). IL-2 dependence of IL-9 expression in human T lymphocytes. J. Immunol. 148, 3147±3151. Houssiau, F., Renauld, J.-C., Stevens, M., Lehmann, F., LetheÂ, B., Coulie, P. G., and Van Snick, J. (1993). Human T cell lines and clones respond to IL-9. J. Immunol. 150, 2634±2640. Houssiau, F., SchandeneÂ, L., Stevens, M., Cambiaso, C., Goldman, M., Van Snick, J., and Renauld, J.-C. (1995). A cascade of cytokines is responsible for IL-9 expression in human T cells: involvement of IL-2, IL-4 and IL-10. J. Immunol. 154, 2624±2630.

HuÈltner, L., Moeller, J., Schmitt, E., JaÈger, G., Reisbach, G., Ring, J., and DoÈrmer, P. (1989). Thiol-sensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4. J. Immunol. 142, 3440±3346. HuÈltner, L., and Moeller, J. (1990). Mast cell growth-enhancing activity (MEA) stimulates interleukin 6 production in a mouse bone marrow-derived mast cell line and a malignant subline. Exp. Hematol. 18, 873±877. HuÈltner, L., Druez, C., Moeller, J., Uyttenhove, C., Schmitt, E., RuÈde, E., DoÈrmer, P., and Van Snick, J. (1990). Mast cell growth enhancing activity (MEA) is structurally related and functionally identical to the novel mouse T cell growth factor P40/TCGFIII (interleukin-9). Eur. J. Immunol. 20, 1413±1416. Jeannin, P., Delneste, Y., Lecoanet-Henchoz, S., Gretener, D., and Bonnefoy, J. Y. (1998). Interelukin-7 (IL-7) enhances class switching to IgE in the presence of T cells via IL-9 and sCD23. Blood 91, 1355±1361. Kelleher, K., Bean, K., Clark, S., Leung, W.-Y., Yang-Feng, T., Chen, J., Lin, P.-F., Luo, W., and Yang, Y.-C. (1991). Human interleukin-9: genomic sequence, chromosomal location, and sequences essential for its expression in human T-cell leukemia virus (HTLV)-I-transformed human T cells. Blood 77, 1436±1441. Kimura, Y., Takeshita, T., Kondo, M., Ishii, N., Nakamura, M., Van Snick, J., and Sugamura, K. (1995). Sharing of the IL-2 receptor chain with the functional IL-9 receptor complex. Int. Immunol. 7, 115±120. Lauwerys, B. R., Renauld, J. C., and Houssiau, F. (1998). Inhibition of in vitro immunoglobulin production by IL-12 in murine chronic graft-vs.-host disease: synergism with IL-18. Eur. J. Immunol. 28, 2017±2024. Lemoli, R. M., Fortiuna, A., Fogli, M., Motta, M. R., Rizzi, S., Benini, C., and Tura, S. (1994). Stem Cell Factor (c-kit ligand) enhances the Interleukin-9-dependent proliferation of human CD34‡ and CD34‡CD33ÿDRÿ cells. Exp. Hematol. 22, 919±924. Lemoli, R. M., Fortuna, A., Tafuri, A., Fogli, M., Amabile, M., Grande, A., Ricciardi, M. R., Petrucci, M. T., Bonsi, L., Bagnara, G., Visani, G., Martinelli, G., Ferrari, S., and Tura, S. (1996). Interleukin-9 stimulates the proliferation of human myeloid leukemic cells. Blood 87, 3852±3859. Louahed, J., Kermouni, A., Van Snick, J., and Renauld, J.-C. (1995). IL-9 induces expression of granzymes and high affinity IgE receptor in murine T helper clones. J. Immunol. 154, 5061± 5070. Lu, L., Leemhuis, T., Srour, E., and Yang, Y.-C. (1992). Human interleukin (IL)-9 specifically stimulates proliferation of CD34‡‡‡DR‡CD33± erythroid progenitors in normal human bone marrow in the absence of serum. Exp. Hematol. 20, 418±424. Mackewicz, C. E., Ortega, H., and Levy, J. A. (1994). Effect of cytokines on HIV replication in CD4‡ lymphocytes: lack of identity with the CD8‡ cell antiviral factor. Cell. Immunol. 153, 329±343. McLane, M. P., Haczku, A. H., van de Rijn, M., Weiss, C., Ferrante, V., MacDonald, D., Renauld, J. C., Nicolaides, N. C., and Levitt, R. C. (1998). Interleukin-9 promotes allergeninduced eosinophilic inflammation and airway hyperresponsiveness in transgenic mice. Am. J. Respir. Cell. Mol. 19, 713±720. Mehler, M. F., Rozental, R., Dougherthy, M., Spray, D. C., and Kessler, J. A. (1993). Cytokine regulation of neuronal differentiation of hippocampal progenitor cells. Nature 362, 62±65. Merz, H., Houssiau, F. A., Orscheschek, K., Renauld, J.-C., Fliedner, A., Herin, M., Noel, H., Kadin, M., MuellerHermelink, H. K., Van Snick, J., and Feller, A. C. (1991).

IL-9 163 IL-9 expression in human malignant lymphomas: unique association with Hodgkin's disease and large cell anaplastic lymphoma. Blood 78, 1311±1317. Miyazawa, K., Hendrie, P., Kim, Y.-J., Mantel, C., Yang, Y.-C., Se Kwon, B., and Broxmeyer, H. (1992). Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel factor to stimulate proliferation of the human factor-dependent cell line, MO7e. Blood 80, 1685±1692. Mock, B. A., Krall, M., Kozak, C. A., Nesbitt, N. M., McBride, O. W., Renauld, J.-C., and Van Snick, J. (1990). IL-9 maps to mouse chromosome 13 and human chromosome 5. Immunogenetics 1, 265±270. Modi, W. S., Pollock, D. D., Mock, B. A., Banner, C., Renauld, J.-C., and Van Snick, J. (1991). Regional localization of the human glutaminase (GLS) and interleukin-9 (IL9) genes by in situ hybridization. Cytogenet. Cell. Genet. 57, 114±116. Moeller, J., HuÈltner, L., Schmitt, E., Breuer, M., and DoÈrmer, P. (1990). Purification of MEA, a mast cell growth-enhancing activity to apparent homogeneity and its partial amino acid sequencing. J. Immunol. 144, 4231±4234. Monteyne, P., Renauld, J. C., Van Broeck, J., Dunne, D. W., Brombacher, F., and Coutelier, J. P. (1997). IL-4-independent regulation of in vivo IL-9 expression. J. Immunol. 159, 2616± 2623. Nicolaides, N., Holroyd, K. J., Ewart, S. L., Eleff, S. M., Kiser, M. B., Dragwa, C. R., Sullivan, C. D., Grasso, L., Zhang, L. Y., Messler, C. J., Zhou, T., Kleeberger, S. R., Buetow, K. H., and Levitt, R. C. (1997). Interleukin 9: A candidate gene for asthma. Proc. Natl Acad. Sci. USA 94, 13175±13180. Petit-FreÁre, C., Dugas, B., Braquet, P., and Mencia-Huerta, J.-M. (1993). Interleukin-9 potentiates the interleukin-4-induced IgE and IgG1 release from murine B lymphocytes. Immunology 79, 146±151. Renauld, J.-C., Goethals, A., Houssiau, F., Van Roost, E., and Van Snick, J. (1990a). Cloning and expression of a cDNA for the human homolog of mouse T-cell and mast cell growth factor P40. Cytokine 2, 9±12. Renauld, J.-C., Goethals, A., Houssiau, F., Merz, H., Van Roost, E., and Van Snick, J. (1990b). Human P40/IL-9 ± Expression in activated CD4‡ T cells, genomic organization, and comparison with the mouse gene. J. Immunol. 144, 4235±4241. Renauld, J.-C., Druez, C., Kermouni, A., Houssiau, F., Uyttenhove, C., Van Roost, E., and Van Snick, J. (1992). Expression cloning of the murine and human interleukin 9 receptor cDNAs. Proc. Natl Acad. Sci. USA 89, 5690±5694. Renauld, J.-C., van der Lugt, N., Vink, A., van Roon, M., Godfraind, C., Warnier, G., Merz, H., Feller, A., Berns, A., and Van Snick, J. (1994). Thymic lymphomas in interleukin 9 transgenic mice. Oncogene 9, 1327±1332. Renauld, J.-C., Vink, A., Louahed, J., and Van Snick, J. (1995a). IL-9 is a major anti-apoptotic factor for thymic lymphomas. Blood 85, 1300±1305. Renauld, J.-C., Kermouni, A., Vink, A., Louahed, J., and Van Snick, J. (1995b). Interleukin 9 and its receptor: involvement in mast cell differentiation and T cell oncogenesis. J. Leukoc. Biol. 57, 353±360. Rich, B., Campos-Torres, J., Tepper, R., Moreadith, R., and Leder, P. (1993). Cutaneous lymphoproliferation and lymphomas in interleukin 7 transgenic mice. J. Exp. Med. 177, 305±316. Schaafsma, M. R., Falkenburg, J. H., Duinkerken, N., Van Snick, J., Landergent, J. E., Willemze, R., and Fibbe, W. E. (1993). Interleukin-9 stimulates the proliferation of enriched human erythroid progenitor cells: additive effects with GMCSF. Ann. Hematol. 66, 45±49.

Schmitt, E., van Brandwijk, R., Van Snick, J., Siebold, B., and RuÈde, E. (1989). TCGF III/P40 is produced by naive murine CD4‡ T cells but is not a general T cell growth factor. Eur. J. Immunol. 19, 2167±2170. Schmitt, E., Beuscher, U., Huels, C., Monteyne, P., Van Brandwijk, R., Van Snick, J., and Ruede, E. (1991). IL-1 serves as a secondary signal for IL-9 expression. J. Immunol. 147, 3848±3854. Schmitt, E., Germann, T., Goedert, S., Hoehn, P., Huels, C., Koelsch, S., KuÈhn, R., MuÈller, W., Palm, N., and RuÈde, E. (1994). IL-9 production of naive CD4‡ T cells depends on IL-2, is synergistically enhanced by a combination of TGF- and IL-4, and is inhibited by IFN . J. Immunol. 153, 3989±3996. Sokal, G., Michaux, J. L., Van Den Berghe, H., Cordier, A., Rodhain, J., Ferrant, A., Moriau, M., De BruyeÁre, M., and Sonnet, J. (1975). A new hematologic syndrome with a distinct karyotype: the 5q- chromosome. Blood 46, 519±533. Sonoda, Y., Maekawa, T., Kuzuyama, Y., Clark, S., and Abe, T. (1992). Human interleukin-9 supports formation of a subpopulation of erythroid bursts that are responsive to interleukin-3. Am. J. Hematol. 41, 84±91. Suda, T., Murray, R., Fischer, M., Yokota, T., and Zlotnik, A. (1990). Tumor necrosis factor-alpha and P40 induce day 15 murine fetal thymocyte proliferation in combination with IL2. J. Immunol. 144, 1783±1787. Svetic, A., Finkelman, F., Jian, Y., Dieffenbach, C., Scott, D., Mccarthy, K., Steinberg, A., and Gause, W. (1991). Cytokine gene expression after in vivo primary immunization with goat antibody to mouse IgD antibody. J. Immunol. 147, 2391±2397. Svetic, A., Madden, K. B., Di Zhou, X., Lu, P., Katona, I. M., Finkelman, F. D., Urban, J. F., and Gause, W. C. (1993a). A primary intestinal helminthic infection rapidly induces a gutassociated elevation of Th2-associated cytokines and IL-3. J. Immunol. 150, 3434±3441. Svetic, A., Jian, Y. C., Lu, P., Finkelman, F. D., and Gause, W. C. (1993b). Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN- in CD4‡ cells. Int. Immunol. 5, 877±883. Temann, U. A., Geba, G. P., Rankin, J. A., and Flavell, R. A. (1998). Expression of Interleukin 9 in the lungs of transgenic mice causes airway inflammation, mast cell hyperplasia, and bronchial hyperresponsiveness. J. Exp. Med. 188, 1307±1320. Uyttenhove, C., Simpson, R., and Van Snick, J. (1988). Functional and structural characterization of P40, a mouse glycoprotein with T cell growth factor activity. Proc. Natl Acad. Sci. USA 85, 6934±6938. Van Damme, J., Uyttenhove, C., Houssiau, F., Put, W., Proost, P., and Van Snick, J. (1992). Human growth factor for murine interleukin (IL)-9 responsive T cell lines: coinduction with IL-6 in fibroblasts and identification as LIFHILDA. Eur. J. Immunol. 22, 2801±2808. Van Snick, J., Goethals, A., Renauld, J.-C., Van Roost, E., Uyttenhove, C., Rubira, M. R., Moritz, R. L., and Simpson, R. J. (1989). Cloning and characterization of a cDNA for a new mouse T cell growth factor (P40). J. Exp. Med. 169, 363±368. Vink, A., Renauld, J.-C., Warnier, G., and Van Snick, J. (1993). Interleukin-9 stimulates in vitro growth of mouse thymic lymphomas. Eur. J. Immunol. 23, 1134±1138. Vink, A., Warnier, G., Brombacher, F., and Renauld, J.-C. (1999). IL-9-induced in vivo expansion of the B-1 lymphocyte population. J. Exp. Med. 189, 1413±1423. Williams, D., Morrissey, P., Mochizuki, D., de Vries, P., Anderson, D., Cosman, D., Boswell, H., Cooper, S.,

164 Jean-Christophe Renauld Grabstein, K., and Broxmeyer, H. (1990). T-cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro. Blood 76, 906±911. Yang, Y., Ricciardi, S., Ciarletta, A., Calvetti, J., Kelleher, K., and Clark, S. C. (1989). Expression cloning of a cDNA encoding

a novel human hematopoietic growth factor: human homologue of mouse T-cell growth factor P40. Blood 74, 1880±1884. Zhu, Y. X., Kang, L. Y., Luo, W., Li, C. C. H., Yang, L., and Yang, Y. C. (1996). Multiple transcription factors are required for activation of human Interleukin 9 gene in T cells. J. Biol. Chem. 271, 15815±15822.