Interleukin 5 and its receptor

Progressin GrowthFactorResearch,Vol. 3, pp. 87-102, 1991 Printedin Great Britain.All rightsreserved.

0955-2235/91 50.00 + .50 9 1991PergamonPresspie

I N T E R L E U K I N 5 A N D ITS R E C E P T O R Kiyoshi Takatsu and Akira Tominaga Department of Biology Institute for Medical Immunology Kumamoto University Medical School 2o2-I Honjo, Kumamoto 860, Japan

IL-5 is a cytokhze mahdy pro&wed by T lymphocytes, especially when the)' are sensitized with microorganisms, which hrduce eosflwphils and [,)'-1 positive B lineage cells, both of which are probably engaged hi the prhnary protection agahrst microorganisms. These possibilities are discussed by analyzhzg IL-5 transgenic mice. IVe also discuss the possibility of ushlg these mice as anhnal models for the diseases which may be caused by hwreased levels of eoshwphils. AIthough 1L-5 is trot produced by bone marrow stromal cells, it is hwolved ht the early development of eoshwphils and Ly-1 positive B-lineage cells that can differentiate hzto macrophages. The chte to the role of IL-5 may exist hi the constitution of 1L-5 receptor. Tire IL-5 receptor consists of ct and fl chains. The ~ chahl is a 60 kDa glycosylated protehl which bhrds IL-5, by itself, with low affinity. On the other hand, the 130 kDa fl chahr does trot bhrd IL-5 by itself, but forms high affinity IL-5 receptors together with the ct chaht. Surprishrgly, this fl chahr is probably shared with the GM-CSF receptor and is very homologous to the IL-3 receptor. It seems that the fl chah~ is expressed h~ the very earl)" stage o f hematopoiesis. The ot chahl may be directly related to the cell lineage commitment. Keyvvords: IL-5, IL-5 receptor, G M - C S F receptor, Ly-I positive B cells, eosinophils. INTRODUCTION T h e B cell response to an a n t i g e n has been s h o w n to be r e g u l a t e d b y a helper T cell r e s p o n d i n g to, a n d specific for, the s a m e antigen molecule. H e l p e r T cells recognize antigenic f r a g m e n t s in r e l a t i o n to the class II m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x ( M H C ) molecules on B cells a n d r e n d e r B cells susceptible to the s u b s e q u e n t a c t i o n o r B cell s t i m u l a t o r y factors ( B S F s ) which can induce B cell g r o w t h a n d differentiation. Once B cells are a c t i v a t e d a n d p r o l i f e r a t e d , their differentiation into i m m u n o g l o b u l i n (lg)-secreting cells requires soluble T cell-derived p r o d u c t s .

Acknowledgenwnts--We express our sincere thanks to all the collaborators for their outstanding contributions. This work was supported in part Grant-in-Aids for Scientific Research and for Special Project Research, Cancer Bioscience, from the Ministry of Education, Science, and Culture; by Special Coordination Funds for Promoting Science and Technology of the Science and Technology Agency, Japan; and by a Research Grant from Tokyo Biochemical Research Foundation. The author is grateful to Drs Hideo Hayashi and Kaoru Onoue for helpful suggestions and encouragement during the course of this study. Ms Misako Nakao is acknowledged for her secretarial assistance. 87

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The study of IL-5 originated in the search for one of the B cell growth and differentiation factors, named T cell-replacing factor (TRF), that induces antigenstimulated B cells to differentiate into plasma cells [1,2] or the growth of BCL, B cell tumors (BCGFII) [3]. On the other hand, eosinophil differentiation factor (EDF) [4] is known as a diffusible factor from the thoracic duct lymphocytes of parasite-infected rats [5]. cDNA cloning [6,7,8] and the monoclonal antibody (mAb) against IL-5 [9] enabled us to identify this molecule as a cytokine that has pleiotropic activity on various target cells, besides B cells and eosinophils, [10]. IL-5 is an inducible hormone produced by T cells after antigen stimulation such as Mycobacterhtm tuberculosis or Toxocara canis [11,12], and by mast cells upon stimulation with allergen/lgE complex or calcium ionophore [13]. A key question regarding the action of IL-5 in responsive cells has been the molecular mechanism of signal transduction after IL-5 binding to the functional IL5R. The pleiotropic activity of IL-5 on target cells is directly dependent on initial binding to specific cell surface receptors. Like other cytokines, IL-5 interacts with target cells with biphasic equilibrium binding kinetics, reflecting two classes ofbinding sites with high and low affinity [I 4]. We carried out the cloning of the cDNA for the IL5R [15] by utilizing a high efficiency COS7 cell expression system with a CDM8 vector and anti-IL-5R mAbs. This cDNA clone encodes 60 kDa protein which binds IL-5 with low affinity by itself. We will describe the biological functions of IL-5 and molecular identification of the second component of the high affinity IL-5 receptor system. STRUCTURESOFI~5 The cDNAs encoding both murine and human IL-5 have been cloned [6,7,8]. Murine IL-5 consists of 133 amino acids, including a hydrophobic signal sequence of 20 amino acids and the secreted core polypeptide with a molecular mass of 12.3 kDa. Three potential N-glycosylation sites, as well as three cysteine residues are present in the polypeptide sequence. The deduced amino acid sequence ofmurine IL-5 and sequences of known proteins, including all the cytokines so far identified, fail to show any significant homology except for short sequences ofmurine IL-3, murine granulocytemacrophage colony-stimulating factor (GM-CSF) and murine IFN-T/[6]. Using the mouse IL-5 cDNA as a probe, we isolated the human IL-5 cDNA [7]. Human IL-5 consists of 134 amino acids with a typical signal sequence of 19 residues (Table 1). Comparison of the cDNA sequence of murine ]L-5 with that of human shows a sequence homology of 77% at the DNA level and 70% at the protein level [7]. The chromosomal genes for murine and human IL-5 were isolated using IL-5 cDNA as probes [16,17]. Both murine and human IL-5 genomic genes consist of 4 exons and 3 introns. The exon-intron organization and the location of the cysteine codons of the IL-5 genes resemble those of the GM-CSF, IL-2, IL-4 genes, but are quite distinct from GM-CSF. By hi situ hybridization, the human IL-5 gene was mapped to chromosome 5q23.3-31.1 on which IL-4, GM-CSF, and IL-3 genes are also mapped. IL-5 genes are clustered with the GM-CSF and IL-3 genes on human chromosome 5 and murine chromosome 11 and that these genes might be derived by duplications of a common ancestral gene. The mature IL-5 molecule is heavily glycosylated and consists of a homodimer of

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approximate molecular mass 20-25 kDa [11,18-20]. Deglycosylated IL-5 exerts IL-5 activity on B cells [18], indicating that the carbohydrate moiety is not essential for its binding to IL-5 receptor. Since the monomeric IL-5 failed to exert its activity [20], the dimer formation is essential for expressing biological activity. This may suggest that the formation of 4-c~helical bundles is required for IL-5 to be active as suggested for other cytokines by Bazan [21]. While mouse and human IL-5 are equally active in human ceil assays, human IL-5 is 1000-fold less active than murine IL-5 in mouse cell assays [19]. McKenzie et aL [22] recently analyzed structure-function of IL-5 utilizing mouse/human chimeric molecules and found that the changing ofonly eight residues in the C-terminus region of human IL-5, to those of mouse IL-5, resulted in the hybrid exerting biological activity comparable to mouse IL-5 and competing with the IL-5 binding to target cells, showing that the C-terminus region probably interacts with the receptor. IL-5 AND GROWTH AND DIFFERENTIATION OF B CELLS It has been reported that B cells are generated from pluripotent hematopoietic stem cells. The earliest stage of B cell development represented by pro-B cells contains the germ-line pattern ofimmunoglobulin heavy chain (IgH) genes and expresses B-lineage cell characteristic surface phenotypes [23,24]. Pro-B cells can differentiate into pre-B cells accompanying the rearrangement of IgH genes and expression of cytoplasmic p chains [24]. Pre-B cells proliferate, rearrange light (L) chain genes and express surface IgM (slgM) giving rise to mature B cells [25]. Palacios et aL [26] first established Ly-I + pro-B cell lines (Lyd9, LyH7, and Lyb9) by culturing bone marrow cells depleted of granulocytes, Ia + and Thy I + cells in the presence oflL-3. They can grow in response to IL-3, or to a much lesser extent IL-4 or IL-5, strongly suggesting that the IL-5R is expressed as well as the IL-3R and IL-4R on the celt from an early phase of B cell development. By utilizing an IL-5 and a stromal cell line, ST2, we established eighteen different clones, eight of which showed a germ-line configuration of the IgH genes [27,28]. We also observed 2 clones which had rearranged IgH genes in both chromosomes and 8 clones with the rearranged configuration of one IgH gene. Their surface phenotype was CD45R § Ly-I +, Lyb-2 § clgM-, slgM-, Ia-, Mac-1-, Thy1-, and IL-2R(Tac) § When we treated two clones (J8 and Jl0), which had germ-line type IgH, genes with 5-azacytidine for 24h followed by co-culture with ST2 and IL-5, they became Ly-I § slgM +, B cells and Ly- 1§ Mac- l § macrophage-like cells, respectively. The generation of slgM§ cells from IL-3 responsive Ly-1 + pro-B cells in response to 5-azacytidine followed by culturing on adherent cells with LPS was clearly demonstrated by Palacios's group and by us [26,28,29]. When we maintained other cell lines (J 1 and J 13) by co-culture with ST2 and IL-5 for more than a year, they showed a rearranged IgH configuration. These cell lines expressed Cp-mRNA and A5-mRNA [28] consistent with normal pre-B cells. ST2 dependent and IL-5 independent MJ88-1 cells [30] established from the same bone marrow culture expressed marginal levels of Ly-1 antigen and showed Cp and 25 mRNA expression. Intriguingly, J 1 and J 13 expressed a higher level of c-fins mRNA than that of M J88-1. When they were co-cultured with ST2 and GM-CSF in place of ST2 and IL-5, we found growth of Ly-I + cells with macrophage morphology that had lost their p-chains and acquired Mac-I expression.

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They were nonspecific esterase-positive, and could ingest latex beads [28]. A converted myeloid line had the JH rearrangement pattern as its lymphoid parent and adhered to culture flasks. Attempts to regenerate B-lineage cells from macrophages by IL-5 were unsuccessful. M J88-1 did not convert to macrophage by the addition o f G M - C S F , suggesting that B-lineage cells maintained on ST2 in the presence of IL-5 may have distinct potential from those maintained in the absence o f IL-5. Several pre-B cell lines have been recognized to differentiate either to Ly-1 § B cell or to monocytes/ macrophages [31]. These results provide evidence for a close relationship between the myeloid and Ly-I § B-cell pathways of differentiation, and indicate that IL-5dependent clones are multipotential intermediates in differentiation from pro-B to B cells or to macrophages. By culturing one o f the Ly-I § early B cell line J-87, whose growth depends on IL-5 and ST2, in high density, we also obtained IL-5 dependent and ST2-independent cell lines (T88-M). N o change was observed in terms o f cell surface marker after becoming ST2 independent. Although cell lines thus established expressed increased levels of high affinity IL-5 binding sites, they could not differentiate into Ig-secreting cells in response to IL-5. TABLE 1. Structure, polypeptide,and biological activities.

(I) Apparent MW Total (2) Number of Mature amino acids } (3) N-Glycosylationsites (4) Chromosome localization (5) Genomic structure (6) Producer cells

Mouse

Human

40-50kDa 133 113 3 11 4 exons T cells Mast cells

30-40kDa 134 i15 2 5 4 exons T cells Reed Sternberg cells* EBV-transformed B cellst

(7) Biologicalactivity (1) Induction of differentiation (a) B cell differentiation (b) Induction of IL-2 receptor expression in B and T cells (c) Cofactor ofcytotoxic T cell differentiation (d) Differentiation ofeosinophils (e) Enhancement ofhistamine releaseby basophils (2) Stimulation of cell growth (a) Promotion of Ly-I § early B cell growth (b) Ly-I § cell proliferation (c) Clonal expansion of resting B cell (d) Eosinophil precursor cell growth * Samoszuk M, Nansen L. Blood; 1990; 75: 13-16. "]" Paul CC, Keller JR, Armpriester JM, Bauman, MA. Blood; 1990; 75: 1400-1403. Functional studies using recombinant murine IL-5 confirmed that activities o f B cells [6], although it has pleiotropic activities on various target cells (Table I) [10]. IL-5 acts on naturally activated B cells hi vivo, hi vitro LPS-stimulated B cells, and resting B cells to induce maturation and propagate proliferation [32]. IL-5 induced the increase in levels of secreted forms o f l t - m R N A in BCL~, or resting, as well as activated B cells [33,34]. Utilizing a single hapten-specific murine B cell assay system, it was found that

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murine IL-5 caused an increase in both the frequency of B cell proliferation and subsequent secretion of Ig [35]. Anti-IL-5 mAbs were found to inhibit the antigenspecific primary IgM response induced by cloned helper T cells in a MHC-restricted manner resulting in polyclonal Ig-production [36]. Interestingly, IL-5 can induce antigen-specific and polyclonal IgA production in antigen-primed B cells and in LPSstimulated B cells, respectively [37-39]. IL-5 acts on surface IgA positive B cells, but not on surface IgA-negative B cells, for tlhe induction of IgA production [39,40], suggesting that IL-5 acts as a maturation factor for B cells committed to become IgAsecreting cells, rather than as a class switching factor. On the other hand, TGF-fl seemed to work on surface IgA negative B cells to induce class-switch [40]. This hypothesis was supported by circular DNA analysis [41]. IL-5 AND GROWTH AND DIFFERENTIATION OF EOSINOPHILS A novel activity ofeosinophil differentiation factor (EDF) which also has BCGFII activity was initially reported by Sanderson et al. [4]. The cloning of cDNA encoding the murine EDF [17] revealed that the nucleotide sequence of the cDNA is completely identical to IL-5/TRF/BCGFII cDNA [6,7,8]. EDF and eosinophil colony-stimulating factor activities of murine IL-5 have been confirmed by other investigators, including us [8,42]. Murine IL-5 was also shown to maintain the viability of mature eosinophils, induce the production of superoxide anion in mature eosinophils and possess chemotactic activity for eosinophils [43]. Essentially similar activities of human IL-5 on human eosinophils and their precursors have been also described [44]. The synergistic effect of IL-5 and colony-stimulating factors on the expansion of eosinophils is supposed to contribute to the rapid mobilization of eosinophils at the time of helminthic infections and allergic responses [45]. However, it has been demonstrated that un-regulated IL-5 production may be involved in eosinophilia in patients with idiopathic hypereosinophilic syndrome (HES) [46,47]. In humans, only eosinophils express IL-5 receptors at a level that could be detected with radiolabeled ligand [48,49]. The effects of IL-5 on human B cells are still controversial [7,44]. PATHOPHYSIOLOGY IN THE IL-5 TRANSGENIC MICE To investigate the possible involvement oflL-5 in the development of Ly-1 +B cells hi vivo, transgenic mice carrying the mouse IL-5 gene ligated with a metallothionein

promoter were generated [50]. Transgenic mice carrying the IL-5 gene exhibited elevated levels of IL-5 in the serum (2-20 ng/ml) and an increase in the levels of serum IgM and IgA. We stained peritoneal cells, spleen cells, and bone marrow ceils from IL-5 transgenic mice with anti-Ly-1 mAb together with anti-IL-5 receptor (IL-5R) mAb. FACS analysis revealed that the proportions of IL-5R § and Ly-I § cells in peritoneal cells from the transgenic mice were unchanged compared with those from a littermate control. However, a distinctive Ly-l-positive and B220-positive population became apparent. This population has higher density of IgM on its surface and also expresses IL-5R (H7 antigen). This distinctive Ly-I § and B220 § population was also seen in the bone marrow cells, though it was not as clear as in the spleen. These results suggest that in IL-5 transgenic mice, IL-5R-positive cells are selectively expanded and some of them

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also coexpress Ly-1 antigens simultaneously. The increase in the total number of Ly-I + and IL-5R§ cells in the peritoneal cavity and spleen in the IL-5 transgenic mice prompted us to examine the production of autoantibodies in the transgenic mice, because Ly-1 § cells are supposed to produce autoantibodies. In the IL-5 transgenie mice, we could detect IgM autoantibodies against ssDNA, dsDNA, cardiolipin, poly(ADP-ribose) and TNP, and all the antibody activity was absorbed with DNAcoupled Sepharose beads. Another striking feature of the transgenie mice was a marked increase in the number of peripheral blood white cells (PBL), of spleen cells and of peritoneal cavity cellularity. The increase in the numbers of eosinophils in PBL was 75-fold compared to those of age-matched control normal mice [50]. The cell cycle analysis of purified eosinophils revealed that most of the eosinophils ( ~ 91%) in the peritoneal cavity were in the G0/Gt phase of the cell cycle, but less than 1% were in G2M phase [51], strongly suggesting that proliferation of eosinophils in response to IL-5 is rarely seen. Infiltration of eosinophils into various tissue was also obvious, particularly after cadmium injection. The spleen was enlarged (about 5 times in cell number) and had white patches consisting of a mass of eosinophils. Large parts of bone marrow were occupied by mature and immature eosinophils. In general, it seems that eosinophils have a tendency to lodge in places rich in connective tissue. There may either be a homing receptor for eosinophils in these connective tissues or perhaps the growth ofeosinophils depends on connective tissue. Figure la shows a picture of a Glisson's capsule of IL-5 transgenic mice. Eosinophils have invaded around the bile duct. The capsule ofthe spleen has also been infiltrated with eosinophils and thickened (Fig. lb). Around the muscle there are membrane-like structures as indicated by arrows, these structures consist of eosinophils (Fig. lc). In Fig. ld, eosinophils are also observed in muscle itself. There is a typical example ofthe disappearance of striation of the muscle and the derangement of sarcolemma, which may be a sign ofmyositis. Although it is not as clear as in femoral muscle, these signs can also be observed in heart. Eosinophils are also present in lymphnodes, particularly those along the trachea. However, those eosinophils seem to be resting: exposure oflL-5 transgenic mice to antigen or hard exercise may drive them to be activated and to release granules. So, we think these IL-5 transgenic mice will give us feasible animal models of certain diseases such as myositis, fascitis, or asthma with eosinophils, besides idiopathic hypereosinophilic syndrome and allow us to test antagonists oflL-5. Dent et al. [52] also generated IL-5 transgenic mice which indicated that induction of IL-5 is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. It is suggested, therefore, that aberrant expression of the IL-5 gene may induce accumulation of Ly-1 § cells and eosinophils. STRUCTURE OF IL-5 RECEPTOR The High Affinity 1L-5-Bhlding Sites Transduce I1-5 Signals

Cloning o f c D N A for murine IL-5 and production of anti-IL-5 mAb enabled us to yield large quantities of highly purified recombinant IL-5 and has made it possible to examine binding sites for IL-5. By using either 35S-labeled IL-5 or radioiodinated IL-5, IL-5 binding assay was initiated by using IL-5-responding BCL~-B20 (in vitro line) and

Interleukh~ 5 and its Receptor

93

t..

9

.

~

.II1~.

.~, . ~ , , . , - ~lp~ "~r

~.-o

57

~.~ ~1

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.i

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F I G U R E 1. Preferential lodging of eosinophiis in 11.-5 transgenic mice. Liver, spleen, and femoral muscle were fixed in 10% formalin and sections of each tissue ~ere stained with hematoxylin and eosin, a, liver; b, spleen; c and d, femoral muscle and surrounding tissue. Photographs were taken at • 300 for a, • 1000 for b, x 75 for c, and x 600 for d.

a subline (T88-M) of IL-5-dependent early B cell line, T-88 [53,54]. Like other cytokines, IL-5 specifically interacts with responsive cells with biphasic equilibrium

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K. Takatsu and A. Tombtaga

binding kinetics, reflecting two classes oflL-5 binding sites. Approximately 10-15% of the receptors display high affinity binding (dissociation constant, Kd of -,, 150 pM) and the remainder show low affinity binding (Kd of approx 20 nM). The number of high affinity binding sites for IL-5 on BCL,-B20 cells can be up-regulated by stimulation with LPS [54], whereas it decreased to one-third of the control level by culturing them with either IL-5 or IL-2, both ofwhich can stimulate BCL~-B20 for their differentiation into IgM-secreting cells. IL-5-dependent early B cell lines expressed large numbers of high affinity IL-5R. The order of IL-5 responsiveness and numbers ofhigh affinity IL-5R was in good agreement. LPS- or IL-5 stimulated normal B cells expressed detectable numbers of IL-5 binding sites [54,55], whereas normal resting B cells, bone marrow cells, and Con A-stimulated T cell blasts expressed few, ifany. The concentration of IL5 necessary to elicit a response and the detection of high affinity IL-5 binding to IL-5 indicate that biological responsiveness to IL-5 depends on interactions with the high affinity form of the receptor. Chemical crosslinking of IL-5 binding proteins on T88-M by using disuccinimidyl tartarate (DST) revealed that two crosslinked bands of approximately 160 kDa and 100 kDa specific for IL-5 were observed under nonreducing conditions [54]. The 160 kDa complex was weakened with the use of DSS and was barely visible if EGS was used. If we assume the MW of IL-5 is 40 kDa, it is likely that two polypeptide chains of approximately 60 kDa and 120-130kDa are involved in the formation of the high affinity IL-5R. The intensity of the crosslinked band of 160 kDa correlates with the number of the high affinity IL-5R. We, then, prepared two mAbs, designated H7 and T21 against murine IL-5R [56,57]. Both mAbs could inhibit the binding of IL-5 to, and the proliferation of, IL-5-dependent Ly-I + early B cell lines (T88-M and Y16), whereas they did not inhibit other cytokine activities. The 60kDa protein (p60) was immunoprecipitated by H7 mAb from the membrane of IL-5R bearing cells and the immunoprecipitation was inhibited by unlabeled IL-5 [56], clearly indicating that H7 and T21 mAbs can recognize p60 that can bind IL-5. More than 70% of B cells and all of Ly-1 § cells in peritoneal cavity expressed H7 protein (IL-5R) on their surface [57]. Less than 10% of splenic B cells expressed IL-5R on their surface and most of them were Ly-l-negative. Approximately one out of three IL-5R+B cells in the peritoneum or one out of two hundred in spleen responded well to IL-5 for IgM production [57]. These results indicate that IL-5R detected by mAbs on the normal B cell population is functional and quite similar to that on Ly-I + early B cell lines. Passive administration of H7 mAb into the IL-5 transgenic mice caused the decrease in the number of Ly-I + and IL-5R § B cells by 50% (unpublished observation). Molecular Clonhzg and Expression of the Low Affinity IL-5 Receptor Complementary DNAs encoding the murine IL-5 binding polypeptide have been isolated by the expression cloning procedures with anti-IL-5R mAbs (H7 and T21) [15]. A cDNA library was constructed from an I L-5-dependent early B cell line, Y 16 in the mammalian expression vector, CDM8. A cDNA library was expressed in COS7 cells and the H7-positive cells were screened by panning procedures using H7 and T21 mAbs and anti-rat IgG or F(ab')2 fragments of anti-rat IgG-coated dishes. We obtained four different clones (plL-5R.8, plL-5R.13, plL-5R.2, and plL-5R.39). The sequence of the cDNAs (plL-5R.8 and plL-5R.13) encoding the IL-5R indicates that the IL-5R is a transmembrane protein with glycosylation of 415 amino acids (MW

lnterleukht 5 attd its Receptor

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45,284), including an N-terminal signal peptide (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids), and a cytoplasmic tail (54 amino acids). The nucleotide sequences ofpIL-5R.2 and pIL-5R.39 were identical to that ofpIL-5R.8 except that they lacked short regions of sequence [15]. These deletions will cause altered translational reading frames, resulting in absence of the transmembrane domain. The sequence analysis of the IL-5R reveals significant homology in the extracellular domain with those ofseveral receptors for cytokines, growth hormone and prolactin. The extracellular region of IL-5R contains two pairs of cysteine residues and the 'WSXWS' motif located close to the transmembrane domain. The IL-5R may, therefore, belong to a cytokine receptor gene family [21], and may have evolved from a common ancestor of other cytokine receptors. The cytoplasmic domain of the IL-5R is unique and does not exhibit consensus sequences for either a tyrosine kinase domain or a catalytic domain of any protein kinase. Northern blot analysis revealed the presence of two mRNAs (,-~ 5.0 kb and 5.8 kb) whose expression was restricted to cell lines that have been identified as bearing IL-5R. The level of mRNA expression correlated well with the number of low affinity IL-5R per cell. Analysis of the gene amplification of IL-5R eDNA by PCR techniques revealed the existence of variant transcripts corresponding to both the membranebound and soluble forms of IL-5R eDNA in cell lines bearing IL-5R [15]. The transcripts for the membrane-bound form appeared to be expressed most abundantly among transcripts for the IL-5R. After transfection of COS7 cells with a cDNA encoding p60, cell surface murine IL5R were expressed at a density of 10~-106per cell as a single binding class of low affinity (Kd of 6-9 nM). Despite careful analysis, no high affinity IL-5 binding was detected [15]. It is of particular interest that FDC-SR cells, established from IL-3-dependent FDCPI cells (IL-5R-negative) by transfection with murine IL-5R eDNA, expressed ~ 500 binding sites per cell for IL-5 with an apparent Kd of 30pM (high affinity) and 8,000 binding sites per cell with Kd of 6 n~l (low affinity). These are close to the values determined for the IL-5 binding on IL-5 responsive cells. FDC-PI cells became responsive to IL-5 for DNA synthesis, whereas neither parental FDC-PI nor FDC-PI transfected pSV2Neo alone responded to IL-5 [15]. These observations suggest that the low affinity IL-5R participates in the formation of the high affinity receptor complex, but that other cell-specific molecules are required for the generation of the high affinity receptor for transmembrane signaling.

High Affinity IL-5 Bhlding Requires Coexpression of the Low Affinity IL-5 Receptor and the IL-3R Homolog, AIC2B As described above, the high affinity IL-5R is composed of at least two membrane polypeptide chains; one is IL-5 binding (p60) and the other chain is 120-130 kDa protein (p130) detectable by chemical crosslinking studies with IL-5. R52.120, a mAb reactive with the mouse IL-5R that has been described by Rolink et aL [58], partially suppressed the mouse IL-5 induced proliferation of BI3 cells (a mouse pre-B cell line that also responds to IL-3), T88-M and YI6 [59]. Addition of R52.120 mAb together with H7 or T21 mAb caused more striking inhibition of the IL-5-dependent proliferation of T88-M than that caused by either one of them alone. R52.120 mAb down-regulated the number and Kd ofhigh affinity IL-5 binding sites on T88-M without

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affecting the levels of those with low affinity [59]. Intriguingly, R52.120 mAb immunoprecipitates p130/140 on B13 [58] and FDC-P1 as well as T88-M [59,60], whereas it did not react with recombinant IL-5R (p60). Furthermore, R52.120 mAb had inhibitory effects on IL-3-driven proliferation of FDC-PI. Moreover, IL-3 and IL5 could induce phosphorylation of a similar set of proteins in an IL-5-dependent cell line, T88-M [61]. As described above, an IL-3-dependent cell line, FDC-PI, became responsive to IL-5 when the low affiniity IL-5R (P60) cDNA was expressed [15]. These results strongly suggest that the p130/p140 protein, recognized by R52.120 mAb is indispensable, together with the low affinity IL-5R (p60), for the formation of high affinity IL-5R and appears to be involved in the IL-3R system. The above possibility was further supported by the fact that anti-IL-3R (anti-Aic-2) mAb reacted with all five IL-5 responsive cell lines (YI6, T88-M, BCL~, MOPCI04E, and T-88). Since anti-Aic-2 mAb has been shown to recognize the low affinity IL-3R (AIC2A) and its homolog (AIC2B), the identity of proteins recognized by either R52.120 or anti-Aic~ mAb was verified using stable L cell transfectants. R52.120 mAb indeed reacted with L cell transfectants expressing AIC2A (L-2A) as well as AIC2B (L2B) [60]. As expected, L cell transfectants expressing p60 IL-5R alone (L-5R) expressed the low affinity IL-5R. L-5R derived clones transfected with AIC2A cDNA (L-5R-2A) and AIC2B cDNA (L-5R-2B) expressed almost equal amounts of the respective cDNA products when assessed by flow cytofluorometry. Only L-5R-2B reconstituted both high (Kd of 15 pM) and low affinity (Kd 0f2.2 nM) IL-5R, whereas L-5R-2A showed low affinity IL-5R like L-5R [60]. L-2A or L-2B did not show any specific binding for IL-5 at concentrations up to 4 nM. When L-5R-2B was crosslinked with radiolabeled IL-5, the p170 band was detected in addition to the pl00 band. The crosslinking patterns were identical among IL-5-responsive cells bearing high affinity IL-5R. These results clearly indicate that AIC2B hardly binds IL-5 by itself, but it contributes to the formation of the high affinity IL-5R by interacting with p60 IL-5R/IL-5 complex. The dissociation of IL-5 from L-5R-2B was much slower than from L-5R, whereas association kinetics of IL-5 to both L-5R-2B and L-5R were almost similar. AIC2B may therefore stabilize the binding of IL-5 to p60 IL-5R. The deduced amino acid sequence of the AIC2B cDNA revealed that AIC2B also belongs to a cytokine receptor family. Evidence that AIC2B also appears to be a subunit of GM-CSFR and is essential for signal transduction was recently presented [62]. Ifthis is correct, IL-5R and GM-CSFR share AIC2B protein as a flchain, although the role of AIC2B in signal transduction is not clear. DISCUSSION It has been reported that various cytokines induce hi vitro growth of Ly-I +B-cell tumor cells or Ly-1 + early B cell lines [3,26,32]. In particular, IL-4, IL-5, and GM-CSF can induce the proliferation of BCL~ cells and IL-3, IL-5, and IL-7 can induce the proliferation of Ly-I + early B cell lines. In contrast, TGF-fl and IFN-?' are shown to inhibit the proliferation induced by the above cytokines. Among these cytokines, IL-5 appears to be well characterized for its activity on Ly-1 +B-lineage cells. The mechanism ofthe preferential increase of a distinctive Ly-la~H+B cell population in the spleen of IL-5 transgenic mice is not fully understood, but we assume the direct involvement of IL-5 is highly possible. Early B cell lines established by our bone

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marrow culture system using IL-5 were Ly-1 +, while those established in the absence o f IL-5 at the same time were mostly Ly-1 -. By culturing bone marrow cells from 8-week old IL-5 transgenic mice, but not from normal mice, we generated IL-5-responsive Ly1§ B-lineage cells even in the absence oflL-5. Our Ly- I § B-lineage cell lines are Mac- 1-. This feature is different from peritoneal Ly-I +, Mac-1 § cells whose progenitors are reported to be present in fetal liver omentum but are largely missing in adult bone marrow [63]. Progenitors of Ly-I § B-lineage cells detected in our culture system may have homing activity to spleen and might have different phenotypes from those in fetal liver. It is not clear whether polyreactive anti-DNA autoantibodies in the IL-5 transgenic mice are solely produced by a distinct subpopulation of Ly-I + and IL-5R + splenic B cells. We do not have evidence at this moment to answer this issue. Wetzel [64] reported that splenic Ly-I +B cells proliferate in response to IL-5, and IL-5R § and Ly-I -B cells from the peritoneum and spleen are also responsive to IL-5 and produce autoantibodies, though IL-5-induced proliferation of Ly-1 +B cells is greater than that of the corresponding Ly-1 -B cells. It was also reported that Ly-I -, IL-5R § splenic B cells do not differ (except in the expression of IL-5R) from the vast majority of the rest of splenic B cells regarding surface phenotype, VH family usage, and autoreactivity [65]. Experiments to see whether Ly-I § IL-5R § splenic B cells in IL-5 transgenic mice produce autoantibodies different from those produced by conventional B cells are currently ongoing. Since the IL-5 transgenic mice showed no symptoms of autoimmunity or tissue damages, we introduced our IL-5 transgene into mice having genetic backgrounds that facilitate the progression ofautoimmunity, such as MRL/Ipr or NZW, by mating. So far, no class switch of autoantibodies from IgM to IgG has been observed. We are currently trying to induce IL-5 transgenic mice to make a class switch from IgM to IgG. To explore the mechanisms of signal transduction induced by IL-5, it is necessary to determine the structure of its receptor. We proposed a two subunit model of the high affinity IL-5R that consists of two different polypeptide chains; p60 and p130 [54]. The isolation and expression of cDNAs encoding the IL-5R (p60) definitively demonstrated that p60 is the low affinity IL-5R and reacts with anti-IL-5R (H7) mAb [I 5]. The transfection of the p60 cDNA into IL-3-dependent FDC-PI induces the high affinity IL-5R, indicating that the low affinity IL-5R (p60) can convert to the high affinity IL-5R in association with another molecule. Further analysis using R52.120 mAb and antiIL-3R (anti-Aic-2) mAb and transfection experiments revealed that the associated molecule (p130) did not bind IL-5 by itself and was identified as AIC2B, a homolog of murine IL-3R [60]. We proposed to call p60 and p130 as the ~zand flchain of IL-5R, respectively [59]. The schematic model of the high affinity IL-5R is displayed in Fig. 2. It has been reported that functional high affinity IL-2R, GM-CSFR, and IL-6R consist of two different polypeptide chains; an ~ chain that binds the ligand with low affinity by itselfand a pchain which interacts with the c~chain resulting in the formation ofhigh affinity receptor. The/3chain does not bind to a particular ligand by itself. In the case of the IL-2R system, the ~chain binds IL-2 with low affinity and the//chain by itself also binds IL-2 with intermediate affinity in hematopoietic cells. Coexpression of both ~ and/3 chains leads to the formation of high affinity IL-2R. In the case of the IL-6R system, transfection of the IL-6R cDNA in COS 7 cells induces the expression of mainly low affinity IL-6R. IL-6 triggers the association oflL-6R and nonligand binding membrane glycoprotein, gp130 and generates the IL-6 signal. The complex between

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( cr chain (p60)

13chain (p130)

~

i

F I G U R E 2. Schematic representation of the constitution of I L-5 receptor. I L-5 is dimerized ~ith S - S bonds and presented as four 7 helical bundle structures according to the Bazan's hypothesis. The IL-5 receptor is illustrated in domain structures according to Bazan's hypothesis [21].

soluble IL-6R and IL-6 also interacts with cell surface gpl30. Hayashida et al. [66] reported that the low affinity human GM-CSFR together with the KH97 protein encoded by a human cDNA homologous to murine IL-3R cDNA, forms a high affinity receptor for human GM-CSF. The KH97 protein does not bind GM-CSF. In the IL5R system, AIC2B does not bind IL-5 by itself, but can be crosslinked with IL-5 in the presence of the low affinity IL-5R chain (p60). IL-5 and soluble IL-5R complex did not interact with AIC2B (data not shown). Recently, Kitamura et al. [62] reported that cotransfection ofcDNAs encoding the low affinity human GM-CSFR and AIC2B into a mouse IL-2-dependent CTLL can induce the expression of the functional GM-CSFR. Therefore, in mice, it is highly likely that IL-5R and GM-CSFR share the fl chain, AIC2B, although the AIC2B protein can bind neither IL-5 nor GM-CSF. AIC2B may play an important role in the signal transduction in the IL-5R and GM-CSFR systems. The AIC2B protein appears to be always coexpressed with the AIC2A protein (the low affinity IL-3R). As we mentioned, Ly-I § pro-B and pre-B cell lines respond to IL-5 and GM-CSF for their growth and differentiation. This may be interpreted as follows: immature Ly-I § multipotential progenitor ceils may constitutively express the AIC2B protein with the AIC2A protein from early in development and respond to IL-3 for their growth; then they may differentiate into cells expressing low affinity receptors for IL-5 or GM-CSF later in ontogeny resulting in the construction of the high affinity receptor for the relevant cytokine, and acquire responsiveness for growth. CONCLUDING REMARKS Considering the fact that IL-5 is not produced by bone marrow stromal cells and is

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mainly produced by peripheral T cells, IL-5 may not be necessary for the development of Ly-I +B cells and eosinophils in bone marrow in constitutive hematopoiesis. The proliferation and maturation of hematopoietic progenitor cells from bone marrow appears to be remote-controlled by IL-5. It seems they can be expanded in case of emergency, such as microorganism infection, because IL-5 production is induced after the animals are infected with parasites. From this stand point, we can think of both LyI+B cells and eosinophils in the same category. They are engaged in the primary protection against infection by microorganisms, because the polyreactive antibodies secreted by Ly-I +B cells seem to be reactive with bacterial surface antigen, such as cardiolipin. Another important issue of IL-5 is that it is probably involved in the early development of hematopoiesis o f both eosinophil precursors and Ly-I+B cell precursors that can be induced to macrophage-like cells in response to GM-CSF. Uniting these two lineages is the fact that they both express receptors for GM-CSF, IL-5, and IL-3. We recently cloned c D N A encoding ligand binding moiety for IL-5 (60kDa) and found that it was responsible for the constitution of high affinity receptor for IL-5, when it was introduced into an IL-3 dependent myeloid cell line, F D C P - I . We also found a second chain of the IL-5 receptor (130 kDa) that does not bind either IL-5 or G M - C S F by itself and is probably shared between the IL-5 receptor and the G M - C S F receptor. This fact may shed light on why the IL-5 receptor is expressed on eosinophils and on Ly- 1+B-lineage cells that can develop into macrophages. This molecule, named AIC2B, may work as a basic receptor component for a stem cell at a certain stage. In other words, expression ofligand binding moiety may directly relate to the commitment ofthe cell lineage. This fact may be a clue as to why the IL-5 receptor is expressed on eosinophils and on Ly-I +B-lineage cells. We are in the process of examining how differentially IL-5 and G M - C S F transduce signals through cytoplasm into nuclei. REFERENCES 1. SchimplA, Wecker E. Replacement ofT-cell function by a T-cell product. Nature (New Biol.) 1972; 237: 15-17. 2. Takatsu K, Tominaga A, Hamaoka T. Antigen-inducedT cell-replacing factor (TRF). I. Functional characterization ofa TRF-producing helper T cell subset and genetic studies on TRF production. J lmmunol. 1980; 124:2414-2422. 3. SwainSL. Role of BCGF, in the differentiation to antibody secretion of normal and tumor B cells. J Immunol. 1985; 134:3934-3943. 4. SandersonCJ, Campbell HD, Young IG. Molecular and cellular biologyofeosinophildifferentiation factor (interleukin-5) and its effectson human and mouse B cells. Immunol Rev.1988; 102:29-50. 5. BastenA, BeesonPB. Mechanismsofeosinophilia. II. Role of the lymphocyte.J Exp Med. 1970; 131: 1288-1305. 6. Kinashi T, Harada N, Severinson E, Tanabe T, Sideras P, Konishi M, Azuma C, Tominaga A, Bergstedt-Lindqvist S, Takahashi M, Matsuda F, Yaoita Y, Takatsu K, Honjo T. Cloning of complementary DNA encoding T-cell replacing factor and identity with B-cell growth factor II. Nature 1986;324: 70-73. 7. Azuma C, Tanabe T, Konishi M, Kinashi T, Noma T, Matsuda F, Yaoita Y, Takatsu K, Hammerstrom L, Smith CIE, Severinson E, and Honjo T. Cloning of cDNA for human T-cell replacing factor (interleukin-5) and comparisonwith the murine homologue.NucleicAcids Res. 1986; 14: 9149-9158. 8. YokotaT, Coffman RL, Hagiwara H, Rennick DM, Takebe Y, Yokota K, Gemmell L, Shrader B, Yang G, MeyersonP, Luh J, Hoy P, P~neJ, Briere F, Spits H, BanchereauJ, De VriesJ, Lee FD, Arai N, Arai K-I. Isolation and characterization of lymphokinecDNA clones encodingmouseand human lgA-enhancing factor and eosinophil colony-stimulatingfactor activities: Relationship to interleukin 5. Proc Natl Acad Sci USA. 1987;84: 7388-7392.

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