Stem Cells, Vol. 14, No. 6, 605-618,
November 1996
© 1996 AlphaMed Press
Function and Signal Transduction Mediated by the Interleukin 3 Receptor System in Hematopoiesis
Takahiko Hara,
Atsushi Miyajima
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan
Key Words. IL-3 • Cytokine receptor • Common ß subunit • JAK-STAT pathway • Ras • Colony-stimulating factor
Dr. Takahiko Hara, Institute of Molecular and Cellular Biosciences (IMCB), The University of Tokyo, 1-1-1 Yayoi Bunkyo-ku, Tokyo 113, Japan.
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Abstract
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Interleukin 3 (IL-3) promotes development of hematopoietic cells through activation of the IL-3 receptor (IL-3R) complex consisting of 
and ß subunits. The subunit binds IL-3 with low affinity and forms a high-affinity receptor with the common ß subunit (ßC). The ßC subunit does not bind any cytokine by itself but is involved in the formation of high-affinity functional receptors for IL-5 and GM-CSF. As the subunits provide the specificity to cytokines and ßC plays a major role in signal transduction, IL-3, GM-CSF and IL-5 exhibit similar functions when they act on the same cells. Surprisingly, no apparent hematological defect other than a reduced number of eosinophils was found in knock-out mice lacking an entire function of IL-3, GM-CSF and IL-5; this indicates a remarkable functional overlap with other cytokine systems for hematopoiesis. Binding of the cytokines to the receptor induces activation of the JAK2 tyrosine kinase that associates with ßC and triggers the signaling events. The membrane proximal region of ßC is responsible for activation of JAK2 and STAT5, as well as for induction of c-myc. The signals induced by this region are required for cell-cycle progression and DNA synthesis. Activation of the Ras pathway requires the distal region of ßC and is involved in the suppression of apoptosis. Proliferation of hematopoietic cells requires signals for both DNA synthesis and anti-apoptosis. In this review, we describe the recent findings of the function and signal transduction mediated by the IL-3R system.
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Introduction
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A small number of hematopoietic stem cells, mainly present in the adult bone marrow, proliferate and differentiate to lineage-committed progenitors which eventually generate all kinds of mature, terminally differentiated blood cells [1, 2]. These processes are regulated by a number of cytokines produced from activated T cells, macrophages and stromal cells [3]. Among such cytokines, interleukin 3 (IL-3) and GM-CSF stimulate multilineages of hematopoietic cells, while IL-5, G-CSF, macrophage colony-stimulating factor, erythropoietin (EPO) and thrombopoietin (TPO) stimulate more restricted lineages of hematopoietic cells. As IL-3 and GM-CSF act on broad target cells, they exhibit a number of different biological activities depending upon the target cells. In contrast, IL-5 chiefly stimulates colony formation of eosinophils and proliferation/differentiation of some murine B cells [4]. Interestingly, IL-3, GM-CSF and IL-5 exhibit almost identical biologic activities on their common target cells, such as eosinophilic progenitors (for IL-3, GM-CSF and IL-5) and pre-B cells (IL-3 and IL-5) [5]. The pleiotropic function associated with each cytokine and the functional overlap among different cytokines are two fundamental characteristics of cytokines [6].
Molecular cloning of the receptor components for IL-3, GM-CSF and IL-5, and reconstitution of the functional receptors have revealed that the receptors for the three cytokines share the same signaling subunit, ßC [7], which can explain the functional overlap that they exhibited. On the other hand, molecular cloning of the receptors indicates that there is only one specific receptor for each cytokine except for murine IL-3. Therefore, the pleiotropic function of a cytokine must be explained by the different signaling machinery depending upon the target cells. Thus, studying the mechanism of signal transduction initiated from receptor activation by cytokines in various hematopoietic cells is primarily important for understanding their function. In this review, we describe recent findings of the signal transduction by IL-3 and GM-CSF.
IL-3, GM-CSF and IL-5 are major hematopoietic growth factors produced by activated T cells; however, their expression in bone marrow stroma cells is minimal, if any. Therefore, it has been hypothesized that the major role of these cytokines is to stimulate hematopoiesis associated with the inflammatory reaction. Recent results using mutant mice deficient in cytokines or their receptors have revealed that the function of these three cytokines is dispensable [8]. It implies a remarkable functional overlap among various cytokines. We describe studies using such receptor-deficient mice and discuss the in vivo role of the IL-3, GM-CSF and IL-5 receptor systems.
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Subunit Structure of the IL-3R System
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Figure 1
schematically shows the structure of the functional receptors for IL-3, GM-CSF and IL-5, which have been proven by reconstitution with cloned receptor subunit cDNAs [7]. The high-affinity receptors for IL-3, GM-CSF and IL-5 consist of and ß subunits. Both are members of the class I cytokine receptor superfamily, type I transmembrane receptors with an extracellular domain at the N-terminus, a transmembrane domain and a cytoplasmic domain without any intrinsic enzymatic activity. There are one or two conserved modules, cytokine receptor modules (CRMs), in the extracellular domains of these receptors. A CRM consists of two repeats of a fibronectin type III domain, one with four spaced cysteine residues and the other with the WSXWS motif (Fig. 1). The subunits are glycoproteins of 60-70 kDa and bind their specific ligand with low affinity. Reconstitution experiments using molecularly cloned receptor subunits have established that the high-affinity receptors for IL-3, GM-CSF and IL-5 share the common ß subunit (ßC), a glycoprotein of 120-130 kDa cDNAs [7]. As the ßC subunit is required for and shared by the high-affinity binding of the three cytokines, they compete with each other for binding to the high-affinity receptors when they are expressed on the same cells [9]. The ß subunit has two repeats of CRMs in the extracellular domain and a large cytoplasmic domain with two motifs known as "box-1" and "box-2," which are conserved in other signaling subunits of cytokine receptors, such as gp130, G-CSF receptor (G-CSFR), IL-2Rß and erythropoietin receptor (EPOR) [10]. The ßC does not bind any cytokine by itself, but forms high-affinity receptors with any one of the three subunits, IL-3R, GM-CSFR and IL-5R. Thus, the subunits determine the cytokine specificity of the high-affinity receptors. As described below, the ß subunit is responsible for transmitting various intracellular signals upon binding of the cytokines to the /ß subunits on the cell surface.
Curiously, in mice, there are two homologous ß subunits, ßC and the IL-3-specific ß, ßIL-3, which binds IL-3 with low affinity and forms a high-affinity receptor with only IL-3R [11]. No functional differences of IL-3 have been found between the two high-affinity IL-3Rs formed with either ßC or ßIL-3. As described below, the targeting of the ßIL-3 gene has no apparent effect at all [12, 13], while targeting of the ßC gene abrogates the response to GM-CSF and IL-5 without affecting the IL-3 response [12, 14]. Similarly, although the physiological significance of the presence of two IL-3R systems in mice still remains unknown, the subunit structure of the three cytokine receptors has provided the molecular basis for the common biological activities, as well as the common signaling pathways by these three cytokines in the same cells [15]. Shared receptor subunits have subsequently been found in other cytokine receptors, e.g., IL-6 and its family members, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 share the signal tranducing subunit known as gp130. (In addition, the LIF receptor ß subunit is also shared by LIF, OSM, CNTF) [16, 17]. The IL-2 family of cytokines (IL-2, IL-4, IL-7, IL-9 and IL-15) share the common 
subunit originally identified as the IL-2R subunit [18, 19].
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Receptor Activation
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The intracellular region of human ßC consists of 432 amino acid residues, while those of the subunits are about 50 amino acid residues. Deletion of the cytoplasmic domains of either or ß subunits abrogates signal transduction, although such mutant /ß receptors lacking the cytoplasmic domain still bind their ligand with high affinity, indicating that both cytoplasmic domains are necessary for signaling [20-23]. However, an essential role of the ß subunits in signaling has been shown by constructing several chimeric receptors. Since the EPOR forms a homodimer upon EPO binding, a chimeric receptor consisting of the extracellular domain of the EPOR and the intracellular domain of ßC or ßIL-3 was constructed and its function examined. EPO induced proliferation signals in Ba/F3 cells through the chimeric receptors, indicating that the homodimerization of the ß subunit is necessary and sufficient for receptor activation [24]. Likewise, the mutant GM-CSFR in which the cytoplasmic domain is replaced with that of the ßC forms a high-affinity receptor with the normal ßC and transduced proliferation signals, again indicating the importance of the dimerization of the ß cytoplasmic domain [25]. In contrast, the ßC mutant in which the cytoplasmic domain is substituted with that of GM-CSFR, forms a high-affinity GM-CSFR with the GM-CSFR, but is unable to induce the proliferation signal, indicating that the dimerization of the GM-CSFR cytoplasmic domain is not sufficient for signaling [25]. These results using chimeric receptors have established the importance of the dimerization of the ß subunit.
Supporting the hypothesis that the cytoplasmic domain of ßC is essential for delivering signals for cell proliferation, several mutations and genetic changes in ßC cause autonomous growth of IL-3/GM-CSF-dependent cell lines. They include: A) replacement of the extracellular domain with an intron-encoded segment by retroviral insertion [26]; B) point mutation in the transmembrane or extracellular region [27]; C) duplication of a short segment including the WSXWS motif [28], and D) deletion of most of the extracellular domain above the WSXWS motif [29]. By analogy to the oncogenic v-mpl [30], the constitutive active mutations of EPOR [31], the c-neu proto-oncogene [32], and the structural changes of ßC caused by those genetic alterations may facilitate ligand-independent homodimerization of ßC, possibly through an extracellular cysteine bridge or the WSXWS motif [33]. However, it should be noticed that the intracellular domains of the subunits are also important for signaling through the natural receptors [22, 23]. Since a dimer of the ß subunit can be detected without ligand binding [34], a cytokine may induce conformational change of the ß subunit to induce signaling through the intracellular domain of the subunit.
Homo- or heterodimerizaion of the cytokine receptor members is a general mechanism of activation of the cytokine receptors. Members of IL-6 and its related cytokines induce either homo-dimerization of gp130 or heterodimerization of gp130 with the LIF receptor ß [35, 36]. IL-2 family cytokines induce heterodimerization of a unique receptor subunit for each cytokine with the common chain [18]. EPO, TPO, G-CSF, growth hormone (GH) and prolactin (PRL) are known to induce homodimerization of the cognate receptors [37-41].
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Signal Transduction through the ß Subunits
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Despite the lack of an intrinsic tyrosine kinase in the receptors, IL-3, GM-CSF and IL-5 induce rapid tyrosine phosphorylation of various cellular proteins, including the ß subunits [20, 42, 43], phosphoinositide 3 (PI-3) kinase [44, 45], Vav [46] and Shc [47-49]. They also trigger the activation of Ras, Raf, microtubule-associated protein kinase (MAPK), and PI-3 kinase as well as gene induction of c-fos, c-jun, pim-1 and c-myc [44]. Various mutant receptors were used to localize the cytoplasmic region of ßC responsible for generating these signals, as shown in Figure 2. The membrane proximal region is necessary for the induction of c-myc as well as pim-1, while the more distal portion is required for the activation of Ras, Raf, MAPK, and PI-3 kinases as well as the induction of c-fos and c-jun mRNAs [44]. The C-terminal portion appears to be involved in negative regulation, since the deletion of this region enhances tyrosine phosphorylation of ßC and Shc [20, 44]. Presumably, a tyrosine phosphatase may bind to this region and negatively regulate the signaling. The best candidate for this negative regulator is hematopoietic cell phosphatase (HCP), because it has been shown to bind to ßC [50] and its negative regulatory role in EPO signaling has been known [51].
Many attempts had been made to identify a tyrosine kinase that associates with the ß subunits or that is immediately activated upon cytokine stimulation. Lyn, Fps/Fes and Tec tyrosine kinases [52-54] were shown to be involved in IL-3/GM-CSF signaling in certain cell types. However, since some cells lacking these tyrosine kinases are able to respond to IL-3/GM-CSF, the role of these kinases in the IL-3/GM-CSF signaling pathway remains unknown. Btk tyrosine kinase has been implicated in IL-5 signaling in B cells [55]. However, it is not clear whether a similar type of tyrosine kinase is involved in IL-3/GM-CSF signaling in myeloid cells.
In contrast with these tyrosine kinases, JAK kinases are now known to play the most critical role in cytokine signaling [56]. The JAK kinase family, which includes JAK1, JAK2, JAK3 and Tyk2, are cytoplasmic proteins of approximately 130 kDa, and which possess a characteristic structure of conserved regions at the N-termini followed by a kinase-like domain and a tyrosine kinase domain at the C-terminus. The importance of JAK kinases in cytokine-mediated signal transduction was originally discovered in the study of interferon (IFN) signaling leading to the IFN-specific gene induction [57]. IFN stimulation results in rapid activation of the receptor-associated JAK kinases; i.e., JAK1 and Tyk2 are activated by IFN-/ß, while JAK1 and JAK2 are activated by IFN-. The activated JAK kinases phosphorylate tyrosine residues of the latent cytoplasmic transcription factors known as STAT (signal transducer and activator of transcription), which are DNA-binding proteins of 80-120 kDa in size with an SH2 domain and tyrosine phosphorylation sites at the C-terminus. The tyrosine-phosphorylated STATs then form a homo- or a heterodimer and translocate to the nucleus, where they bind to a DNA motif sequence known as a gamma-IFN-activated site (GAS), a positive promoter element for the IFN-inducible genes [57]. Thus, this signaling pathway links the receptors to the genes directly.
The JAK/STAT pathways are subsequently found to be important for many other cytokine signaling systems, including the IL-3, GM-CSF and IL-5 receptor systems [56, 58]. The membrane proximal regions of various cytokine receptors known as "box-1" and "box-2" are responsible for the binding and activation of JAKs [59]. The ß subunit binds JAK2 through the box-1 region [59]. As JAKs associate with cytokine receptors in the absence of stimulation [59] and cytokines induce dimerization of their receptors, it is now generally accepted that despite the lack of an intrinsic tyrosine kinase, cytokine receptors activate the receptor-associated JAKs by receptor aggregation induced by cognate cytokines. Thus, JAKs appear to be the most critical tyrosine kinases activated by cytokines. Supporting this idea, the expression of a dominant negative form of JAK2 abrogates GM-CSF response entirely in Ba/F3 cells expressing the human GM-CSFR [60].
Currently, seven members of the STAT family have been reported, and each cytokine activates a relatively specific set of JAKs and STATs (Table 1). IL-3, GM-CSF and IL-5 mainly utilize STAT5a and STAT5b [61, 62] which are also activated by many other cytokines such as IL-2, EPO, TPO, GH and PRL [63-65]. STAT5 was originally isolated as a PRL-responsive positive transcription factor for ß-casein expression in the lactating sheep mammary gland [66]. Subsequently, two homologous STAT5s, STAT5a and STAT5b, were isolated in mouse and human and have been shown to be involved in many cytokine signaling systems. The two STAT5s are highly homologous and their functions are indistinguishable [61]. Interestingly, there is no specific association between JAKs and STATs as shown in Table 1, e.g., IL-2 activates STAT5 through JAK1/JAK3 [63], whereas IL-4 activates STAT6 through JAK1/JAK3 [67, 68]. IL-6/LIF utilizes STAT3 and STAT1 through JAK1/JAK2/TYK2 [69, 70]. As STATs possess an SH2 domain and can be recruited to the tyrosine-phosphorylated receptors, the specificity of STATs activated by a given cytokine is determined by the receptor. However, activation of STAT5 by IL-3/GM-CSF does not necessarily require the phosphorylated tyrosine residues in ßC, as the truncated mutant ßC lacking all of the tyrosine phosphorylation sites is still capable of activating STAT5 [61]; this suggests that JAK2 may directly activate STAT5. Alternatively, an additional factor may mediate the interaction between JAK2 and STAT5.
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Signals Generated from the Membrane Proximal Region of the ß Subunit
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The membrane proximal region (amino acid residues 455-517), including the box-1 region, is indispensible not only for JAK2/STAT5 activation but also for the induction of DNA synthesis [44, 71]. GM-CSF is able to promote the cell cycle from G1 to S phase and to induce DNA synthesis through the mutant ß subunits truncated at either 517 or 544, indicating that the signals induced by the membrane proximal region are necessary and sufficient for these functions [71]. However, the mechanism to induce DNA replication by cytokine stimulation is entirely unresolved. In addition, not only in the IL-3/GM-CSF signaling but also in the case of other cytokines, target genes of STAT5 had been largely unknown despite their wide usage by many cytokines (Table 1
). While STAT5 was originally identified as a transcription factor for the ß-casein gene induced by PRL, it is not generally induced by IL-3 or other cytokines, suggesting the requirement for additional cell-type-specific cofactors for the gene induction by STAT5 in hematopoietic cells.
To identify the target genes of STAT5, we have isolated cytokine-inducible early-response genes by using the truncated receptor mutant that activates JAK2/STAT5, but not the Ras/Raf/MAPK cascade. The involvement of STAT5 for the induction of these early genes was further verified by expression of a dominant negative form of STAT5. By this procedure, we identified several immediate early-response genes induced by IL-3/GM-CSF through STAT5 in IL-3-dependent cell lines. These genes include cis, osm, Id-1, Pim-1, and c-fos [72-74].
The promoter of the mouse cis gene contains STAT5 binding sites, and cis mRNA is rapidly induced by various cytokines that activate STAT5. The cis gene is a novel gene encoding an SH2 domain without any other known motif, and it binds to the tyrosine-phosphorylated ß subunits of IL-3R. As overexpression of CIS protein suppresses IL-3-dependent cell proliferation, CIS may be an important negative feedback regulatory molecule in the cytokine receptor-mediated signaling machinery [72].
The promoter region of the mouse osm gene also possesses the GAS sequences which are binding sites for the activated STAT5 [73]. OSM protein is a member of the IL-6/LIF cytokine subfamily that utilizes gp130 as a signal transducer. Although OSM is produced by hematopoietic cells upon cytokine stimulation, it has no significant effect by itself on proliferation and differentiation of hematopoietic cells. Thus, OSM may act upon other cell types, such as stroma and bone-related cells, which interact with hematopoietic cells in bone marrow.
In contrast to these genes, promoter regions of the mouse pim-1 and id-1 genes do not have a typical STAT5 binding site [75, 76], but their expression was clearly suppressed by the dominant-negative form of STAT5 [74]. Id-1 is a helix-loop-helix (HLH) protein lacking a DNA-binding region. Hence Id-1 antagonizes the function of E-box HLH transcription factors. It is known to block differentiation of certain cell types, such as pro-B cells and erythroid cells [77]. Pim-1 is a serine/threonine kinase that was originally found to collaborate with v-myc to form B-cell lymphoma [78]. However, its role remains unclear. As c-fos expression has been shown to be controlled by Ras, suppression of c-fos expression by the dominant-negative STAT5 was rather surprising [74]. Perhaps optimal induction of c-fos requires multiple signals.
Although the membrane proximal domain of the ß subunit is also responsible for cytokine-induced c-myc expression, c-myc expression is not affected at all by the expression of the dominant-negative STAT5 [74], indicating that c-myc expression is independent of STAT5. Multiple signaling pathways are therefore activated through the membrane proximal region of ßC. Induction of c-myc has been implicated in the cell-cycle progression. Since the net result of the expression of dominant-negative STAT5 was suppression of DNA synthesis, some of the genes controlled by STAT5 must also contribute to the cell cycle progression.
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Role of the Ras Pathway in the IL-3/GM-CSF/IL-5 Function
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The membrane distal region of ßC (544-763) is required for activation of the Ras-Raf-MAPK pathway [44, 79]. Analyses using the mutant ß subunits with C-terminal truncation or substitution of tyrosine residues to phenylalanine within the ßC cytoplasmic domain suggest that multiple molecules mediate the Ras activation [44, 79]: Shc is an adaptor molecule that binds to the tyrosine-phosphorylated ßC and recruits SOS, the Ras-guanine nucleotide exchange factor, to the cell membranes where Ras activation (conversion of GDP form to GTP form) occurs. Phosphorylation of Shc by GM-CSF requires the tyrosine residue at 577 of ßC [79]. A protein tyrosine phosphatase, PTP-1D, is also known to serve as an adapter to recruit SOS. Tyrosine phosphorylation of PTP-1D is also mediated through tyrosine 577 as well as through other tyrosine residues in ßC [79]. Thus, activation of Ras appears to be mediated by multiple pathways.
Ras sequentially activates a cascade of kinases from Raf to MAPK. The PI-3 kinase may also be activated by Ras, although this has not formally been proven, and controversial results have been reported. MAPK is believed to be a positive regulator for the gene induction of the AP-1 transcription factor family including c-fos and c-jun. Since the activation of the Ras-Raf-MAPK pathway was abolished when the box-1 region was deleted from ßC, the box-1 cooperatively works with the distal part of ßC to maintain the function of this pathway. This is also supported by the finding that expression of a dominant-negative JAK2 abrogates GM-CSF-induced c-fos expression [60]. While activation of Ras alone can induce c-fos expression, the level of expression is much lower than that produced by IL-3/GM-CSF stimulation [74]. As described above, the expression of the dominant-negative STAT5 also suppresses c-fos induction [74]; the optimal induction of c-fos appears to be achieved by a combination of the two pathways generated from two regions of ßC.
BaF3 cells expressing the human GM-CSFR with truncation at 544 of ßC, which lacks the ability to activate Ras, proceed in the cell cycle from G1 to S and synthesize DNA in response to GM-CSF. However, these cells do not proliferate, and they undergo apoptosis even in the presence of GM-CSF [71]. Expression of a constitutively active form of Ras blocks apoptosis and confers upon the cells expressing the truncated ßC the ability to proliferate in response to GM-CSF [71]. Thus, by utilizing the mutant receptor, signals for DNA synthesis and the suppression of apoptosis are completely segregated. The mechanism by which Ras suppresses apoptosis is still unknown. Bcl-2 and Bcl-x are known to be anti-apoptotic proteins in various types of apoptosis and are induced by activated Ras. However, the degree of suppression of apoptosis by overexpression of Bcl-2 is significantly weaker than that of activated Ras, indicating that additional molecules are involved in the suppression of apoptosis induced by Ras [80]. It is of note that deficiency of the NF-1 gene, which is implicated in juvenile chronic myeloid leukemia, leads to GM-CSF hypersensitivity of hematopoietic cells [81]. Thus, activation of Ras can be the first event of leukemogenesis.
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Role of the Receptor Subunits in Hematopoiesis
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Expression of and ß subunits of the IL-3/GM-CSF/IL-5 receptors is mainly restricted in hematopoietic cells [11, 82], while their expression is also found in nonhematopoietic tissues such as testis, placenta and brain [83-85]. The ßC and ßIL-3 are expressed in various myeloid progenitor cells, macrophages, mast cells, CD5-positive B cells, and some endothelial cells, but not in erythroblast cells, mature T cells or fibroblasts. The expression of the subunits is more restricted to each cytokine-responsive cell; e.g., IL-3R, but not IL-5R, is expressed in mast cells and multipotential progenitor cells which form colony-forming unit-mixture [86] and (Takahiko Hara, Atsushi Miyajima, unpublished data). In contrast, IL-5R is predominantly expressed in eosinophils and B cells which produce IgM or IgD upon IL-5 stimulation [87]. IL-5 is a major cytokine for eosinophils, but not for other hematopoietic cells. Bone marrow cells of transgenic mice expressing IL-5R ubiquitously form colonies of multiple lineages in response to IL-5 in a manner similar to that in IL-3 [88]. This result indicates that the limited activity of IL-5 is due to the restricted expression of IL-5R, and that IL-5R is functionally equivalent to IL-3R. The results further suggest that cells have their own differentiation program that is not affected by the subunits.
Whether cytokines are actively involved in the commitment of differentiation has been a subject of dispute for many years. A typical example is erythroid differentiation. EPO is a major cytokine for erythroid differentiation, and several independent works show that EPO induces globin expression in an IL-3-dependent BaF3 cell when the EPOR is ectopically expressed [89-91], while IL-3 has no such activity. This indicates that EPOR delivers a specific signal for differentiation. However, in the transgenic mice expressing the human GM-CSFR, human GM-CSF induces formation of erythroid colonies in the absence of EPO [89]. The results clearly indicate that GM-CSF can substitute for the effect of EPO if the receptor is expressed. Similarly, the SKT6 erythroid cell line differentiated in response to EPO, and this differentiation can be induced by PRL when the PRL receptor is ectopically expressed in this cell line (H. Wakao, D. Chida, Atsushi Miyajima, submitted). Conversely, EPO stimulates multiple hematopoietic cell lineages when EPOR is expressed in hematopoietic progenitors by retrovirus [92]. While in the BaF3 cell some difference is found between EPO and IL-3, the current results rather suggest that cells differentiate by their own program and that no specific signals induced by a particular cytokine are necessary for differentiation. Expression of the cytokine receptor is crucial for the specific function of cytokines. Thus, it is interesting to understand the molecular basis for the cell-type-specific expression of each cytokine receptor. In the case of mouse IL-3R gene, a cell-type-specific promoter region contains potential binding sites for Myb, GATA and Ets transcription factors [93], although a single transcription factor alone does not seem to be responsible for expression of IL-3R.
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Lack of IL-3R Expression in Several Mouse Strains
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Gene targeting may be a standard way of investigating the role of the receptor system. Interestingly, however, natural mutant mice that lack the IL-3R expression exist. There are several mouse strains whose bone marrow cells show hyporesponsiveness to IL-3 [94]. Bone marrow cells of A/J mice form only a few colonies in response to IL-3, while GM-CSF stimulates normal colony formation [95]. This impaired response to IL-3 is due to very low-level expression of functional IL-3R, while the ß subunits' expression is normal. Detailed analyses of the IL-3R gene in A/J mice revealed the molecular basis of this defect, as shown in Figure 3. There is a five bp deletion in intron 7 of the IL-3R gene in A/J mice. Since the deleted five bases correspond to a potential branch point for RNA splicing, the deletion leads to an altered splicing by skipping exon 8. The A/J type IL-3R which lacks 10 amino acid residues in the extracellular domain (upstream of the WSXWS motif) encoded by exon 8 can be produced in the cytoplasm but not efficiently transported to the cell surface. Thus, cells carrying the A/J type deletion in both chromosomal IL-3R genes do not have high-affinity IL-3R, even in the presence of the ß subunits [95]. In addition, this defect in chromosome 14 is genetically cosegregated with the IL-3 nonresponsiveness in recombinant inbred mouse strains between A/J and control C57BL/6J [95, 96], confirming the molecular basis for the lack of IL-3 response in A/J mice. Surprisingly, the identical deletion in the IL-3R gene is found not only in A-related mouse strains but also in many other unrelated strains including NZB and C58/J [97] (Fig. 3). Ten out of 27 laboratory mouse strains examined possess the same deletion, and there are no common hematological abnormalities among these mouse strains; this suggests that the IL-3R system is not essential for hematopoiesis. However, as the same mutation was not found in the wild mice we examined [97], IL-3 might be required for some circumstance. In man, a similar defective IL-3R gene has not been identified.
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Mice Devoid of the IL-3, GM-CSF and IL-5 Functions
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The receptor model based on cloning and reconstitution of the receptors predicts that the ßC subunit is crucial for IL-5 and GM-CSF, whereas ßIL-3 may be dispensable, as there are two IL-3Rs. To test the role of each ß subunit, homologous recombination was used to create mice devoid of either ß subunit. As predicted, the mice deficient in ßIL-3 showed no apparent phenotype, and no defect of hematopoiesis was found [12, 13]. In contrast, bone marrow cells of ßC-deficient mice did not form colonies in the presence of either GM-CSF or IL-5, while IL-3 stimulated colony formation normally [12, 14], indicating the essential role of ßC in the GM-CSF and IL-5 responses. Significant reduction of the eosinophil number in the peripheral blood was noticed in the ßC-deficient mice, consistent with the idea that IL-5 is the major cytokine for eosinophil development [12, 14]. The ßC-deficient mice exhibited lung abnormalities, including accumulation of proteinous material in the alveolar spaces and peribronchovascular lymphocytic infiltration [12, 14]. These observations are probably attributable to the deficiency of GM-CSF function, as the same phenotype was found in the GM-CSF-deficient mice [98]. Alveolar macrophages may play a role in lung homeostasis by clearing surfactant and other debris from alveolar space, and these macrophages may have defects in the absence of GM-CSF function. Patients with alveolar proteinosis were found to have an alteration in the ßC gene (R. Nishinakamura, R. Murray, personal communication).
The apparently normal hematopoiesis other than eosinophils in ßC-deficient mice may be due to the presence of the functional IL-3R composed of ßIL-3. Therefore, it would be interesting to produce a double knock-out mouse line lacking both ß subunits. However, because two ß subunit genes are tightly linked on chromosome 15 [26, 99], it was difficult to generate a double knock-out mouse line by mating two mutant lines. As the IL-3 knock-out mouse was generated and was apparently normal (V.L.J. Tybulewicz, unpublished data), we crossed the IL-3 ligand knock-out mouse with the ßC knock-out mouse to generate the mouse line lacking the function of IL-3, GM-CSF and IL-5. To our surprise, the mice developed normally and were fertile [8]. Hematopoiesis in these mice was similar to that in ßC knock-out and showed a low level of eosinophils. Lung disease similar to that in the ßC-deficient mouse was developed, but severity was not changed compared with that of ßC-mutant mice. Thus, IL-3, GM-CSF and IL-5 are dispensable for hematopoiesis in normal life. This may not be too surprising since the major source of these cytokines is activated T cells, and basal levels of these cytokines are almost negligible in normal bone marrow. It has been hypothesized, therefore, that the role of these cytokines is to promote hematopoiesis in emergency situations such as inflammation. To address the question of whether these cytokines are essential for inflammatory hematopoiesis, the mutant mice were infected with parasites and bacteria. However, there was no significant difference between the normal and mutant mice [8], indicating that IL-3, GM-CSF and IL-5 are dispensable even in an emergency situation. The defects can be compensated for by other cytokine systems. These results indicate a remarkable functional redundancy of cytokines to maintain the hematopoiesis.
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Concluding Remarks
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The mouse IL-3 gene was cloned as a mast cell growth factor in 1983, and a number of different biological activities have been assigned to this molecule. IL-3, together with GM-CSF and IL-5, is abundantly produced by activated T cells and had been supposed to play an essential role in stimulation of hematopoiesis associated with inflammatory reaction. However, recent gene-targeting experiments have clearly shown that not only IL-3 but also IL-5 and GM-CSF are dispensable for normal as well as inflammatory hematopoiesis despite their strong hematopoietic growth-stimulatory activity [8]. This surprising finding suggests that hematopoiesis is ensured by several different mechanisms, and that lack of IL-3, GM-CSF and IL-5 can be complemented by other cytokine systems. It is not clear at present what substitutes for the function of these cytokines. This may be achieved by an unknown new cytokine or a combination of known cytokines. Alternatively, the hematopoietic microenvironment may be responsible for the compensation of the deficiency of the three cytokines.
Whatever mechanism may exist to substitute for the function of these cytokines, IL-3, GM-CSF and IL-5 are very potent hematopoietic growth factors. The IL-3/GM-CSF/IL-5 receptor systems provide an excellent model for studying the mechanism of hematopoietic cell growth and differentiation. Molecular analysis of signals delivered by the IL-3, GM-CSF and IL-5 receptors will lead to an understanding of hematopoietic cell growth and may provide a clue to developing a way of regulating the cellular processes in hematopoiesis.
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Acknowledgements
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We are grateful to Drs. H. Wakao and T. Kinoshita (IMCB University of Tokyo, Japan), Dr. M. Ichihara (Nagoya University, Japan), Dr. A. Yoshimura (Kurume University, Japan), Drs. R. Nishinakamura and M. Takagi (Institute of Medical Science, University of Tokyo, Japan), and Drs. A. Mui and R. Murry (DNAX, Palo Alto, CA) for their major contributions to the studies we have described. We thank C. Mawson for the critical reading of this manuscript. Our work was supported by DNAX Research Institute for Molecular and Cellular Biology, Palo Alto, CA, and in part by Grants-in-Aid for Scientific Research of the Ministry of Education, Science, and Culture in Japan.
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Kinoshita T, Yokota T
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Abstract
Introduction
