Department of Cell Biology DNAX Research Institute of Molecular and Cellular Biology 901 California Avenue Palo Alto, CA 94304 USA

Introduction

Hematopoiesis is a complexed process of cell proliferation and differentiation that are regulated by a number of cytokines (Arai et al., 1990). Among such cytokines, interleukin-3 (IL-3) stimulates multipotential hematopoietic progenitors as well as lineage-committed cells including granulocytes, macrophages, megakaryocytes, erythroid cells, and mast cells, resulting in formation of the colonies of multiple lineages. Thus, IL-3 is also known as a multi-colony stimulating factor (multi-CSF). Granulocyte macrophage colony stimulating factor (GM-CSF) stimulates not only granulocytes and macrophages, but also various hematopoietic cells and exhibits activities similar to IL-3. In contrast, IL-5 has a narrow target cell specificity and mainly stimulates eosinophils and CD5-positive B cells. Interestingly, in factor-dependent cultured cells, these three cytokines are strong mitogens which induce almost identical biochemical responses such as tyrosine phosphorylatin of intracellular proteins and activation of signaling pathways (Miyajima et al., 1993). Although IL-3, GM-CSF, and IL-5 show an overlapping biological activity on a certain type of cells (e.g. eosinophils), both GM-CSF and IL-5 do not possess multi-CSF activity on the primitive progenitor cells and mast cell stimulation activity. Biological activities of IL-3, GM-CSF and IL-5 are mediated by their specific high affinity receptors which consist of alfa and ß subunits, members of the class I cytokine receptor family (Miyajima et al., 1993). The alfa subunits are glycoproteins of 60- 70 kilodalton (kD) and bind their specific ligand with low affinity. Reconstitution experiments have shown that the high affinity receptors for IL-3, GM-CSF, and IL-5 share the ß subunit (ßc) that is a glycoprotein of 120130 kD. The ßc does not bind any cytokine by itself, but forms high affinity receptors with anyone of the three alfa subunits: i. e. IL-3Ralfa , GM-CSFR alfa , and IL-5R alfa . Thus, the alfa subunits determine the cytokine specificity of the high affinity receptors. When cytokine and the alfa and ß subunits form a complex on the cell surface, various intracellular signaling cascades are activated. Although intracellular domains of both alfa and ß subunits are required for transmitting a proliferation signal (Takaki et al., 1994 ), the ß subunit plays a major role in signaling including Ras/Raf activation, myc induction, and JAK2 kinase activation (Sato et al., 1993; Quelle et al., 1994). Thus the common biological responses are elicitted by IL-3, GM-CSF, and IL-5 through the common signal transducer, ßc. It should be noted that in mice there are two homologous ß subunits, ß c and IL-3 specific ß, ßIL-3, which binds IL-3 with low affinity and form a high affinity receptor with only IL-3R alfa . The two high affinity IL-3Rs formed with either ßc or ßIL-3 show any functinal differences (Takaki et al., 1991; Hara and Miyajima, 1992; Park et al., 1992). Expression of alfa and ß subunits of IL-3/GM-CSF/IL-5 receptors is mainly restricted in hematopoietic cells. The ß subunits are expressed in myeloid progenitor cells, macrophages, mast cells, and CD5-positive B cells, but not in T cells and fibroblasts (Gorman et al., 1990). In contrast, expression of the alfa subunits is restricted to each cytokineresponsive cells (Hara and Miyajima, 1992). To better understand the function of each subunit in hematopoietic cell development, we have studied mice that show altered expression of IL-3R alfa and IL-5R alfa. In this article, we describe recent findings on the role of the IL-3R alfa subunit and the IL 3/IL-3R system in hematopoiesis.

Multi-CSF activity is mediated by the ectopically expresed functional IL-5R:
IL-3R alfa and IL-5R alfa are functionally equivalent.

In the bone marrow, primitive hematopoietic progenitor cells are self-renewing and they are capable of differentiating into various lineage of mature hematopoietic cells upon stimulation with IL-3. Such a multipotent progenitor population can be isolated by using antibodies against several cell surface antigens (Heimfeld et al., 1991). As we expected, both alfa and ß subunits (ßc and ßIL-3) of IL-3R are expressed in the multipotential progenitor cells (Fig. 1 ).

Fig. 1 Expression of the IL-3R subunits in sorted hematopoietic stem cells. Bone marrow cells were collected from mice and the stem cell population (Lin-, Sca-1+, and Thy –1 high 10) was isolated according to the published procedure (Heimfeld et al., 1991). RNA expression of the a. and two ß subunits of mouse IL-3R was examined by reverse transcriptase-PCR by using specific primers. The PCR products were detected by hybridization using 32p-labelled internal oligonucleotides as probes.

Since IL-5 does not stimulate these multipotential progenitor cells, we attempted to express IL-5R alfa in these cells to examine whether IL-5 exhibits multi-CSF activity when IL-5R alfa is expressed. For this purpose, we established the transgenic mouse expressing IL-5R alfa cDNA which is driven by the PGK-1 promoter (Takagi et al., 1995). As shown in Fig. 2, bone marrow cells derived from the IL-5R alfa transgenic mice gave rise to a various lineage of hematopoietic colonies in response to IL-5, which was the same as the colonies stimulated by IL-3. In the presence of erythropoietin, erythroid colonies also appeared in response to IL-5. Furthermore, when bone marrow cells from the transgenic mice were cultured in liquid medium containing IL-5 for a long time (> one month), IL-5-dependent mast cells started to grow (M. Takagi and A. M., unpublished data). Therefore we concluded that IL-5R has the same potential as IL-3R when the alfa subunit is ectopically expressed. In other words, identical signals which lead to cell differentiation can be delivered from both IL-3R and IL-5R. This result indicates that specific biological activities of IL-3 are due to the regulated expression of the IL-3Ralfa in certain populations of hematopoietic cells. Intriguingly, Longmore et al. has shown that erythropoietin acts as a multi-CSF on bone marrow cells infected with retrovirus carrying erythropoietin receptor (Longmore et al., 1994 ). Although further studies on other cytokine receptor systems are needed, hematopoietic cell differentiation signals could be transmitted by multiple signal transducing cytokine receptors when they are expressed in the immature precursor cells.

Fig. 2 Colony formation assays. Bone marrow cells (2 x 10 high 4 ) from IL-5Ralfa transgenic mice or normal littermates were cultured in the presence of FCS and various cytokines as indicated. Total number of various kinds of hematopoietic colonies was scored. Abbreviations of colony types are as follows: OM, granulocyte/macrophage; Mf, macrophage; Eo, eosinophil; GMM, granulocyte/macrophage/megakaryocyte; GEM, granulocyte/erythrocyte/macrophage; GEMM, granulocyte/erythrocyte/macrophage/megakaryocyte; EM, erythrocyte/megakaryocyte; Meg, megakaryocyte; BFU-E, erythroid bursts; and Mast, mast cells.

Characterization of the IL-3 nonresponsive mice

So far as we know, IL-3 is the only cytokine which strongly stimulates multipotential hematopoietic progenitor cells. Hence it is of interest to know whether hematopoiesis is affected in mice lacking the functional IL-3R. Interestingly, IL-3 nonresponsive mice have been reported (Morris et al., 1990) and recently we found that expression of IL-3R in these mice are impaired (Ichihara et al., 1994; T. H., M. Ichihara, M. Takagi, and A. M., submitted). Bone marrow cells isolated from these mouse strains (A type) are nonresponsive to IL-3 in colony assays, however, they respond to GM-CSF normally, indicating that they lack an ILR specific component or signaling molecule. Detailed characterization has revealed that they are indeed deficient in expression of IL-3R alfa (Fig. 3A). The IL-3-nonresponsive A type mouse strains possess a common mutation in intron 7 of the IL-3R alfa gene. This mutaion, a 5 base-pairs deletion, disrupts the branchpoint consensus sequence for RNA splicing (Fig. 3B ). Consequently, the alternatively spliced IL-3R alfa transcript is produced in A type mice. Since the IL-3R alfa protein produced from an alternatively spliced RNA lacks exon 8 encoding10 amino acid residues in the extracellular domain, it is discriminated from the normal product and is localized intracellularly but not translocated to the cell surface. The IL-3-nonresponsive mouse strains thus do not express a normal level of IL-3R alfa nor high affinity IL-3Rs. Remarkably, we have found 10 IL-3-nonresponsive A type mouse strains out of 27 inbred mouse strains (Table 1). The A type mouse strains do not apparently have a defect in constitutive hematopoiesis nor disadvantage In forming mouse colonies. Thus the A type IL-3R alfa gene appears to be an allele which is not eliminated from the mice population in the laboratory settings. Interestingly, however, only 1 out of 21 wild-derived mouse strains we examined carry the A type IL-3R alfa gene (T. H., M. Ichihara, M. Takagi, and A. M., submitted). This result may indicate some selective advantage of mice that possess the IL-3/IL-3R system in wild populations.

Table 1. Genotype of IL-3Ralfa gene in inbred mouse strains

Fig. 3 Expression of the IL-3R alfa protein and sequence comparison of the IL-3R alfa gene in A and B type mouse strains. A. Bone marrow cells from A type mouse strain (A/J) or B type strain (C57BL/6J) were analyzed by flow cytometry using monoclonal antibodies against ßIL-3 or IL-3R alfa. Blank areas show staining profiles with an isotype control antibody. B. Nucleotide sequence of intron 7 including a 5 base-pairs deletion (boxed) in A type mouse strains is shown. The consensus sequence for a branch point is also shown.

Many cytokines are coodinatedly produced and acting on a variety of cell types in the bone marrow where constitutive hematopoiesis is taking place. In the A type mouse strains, IL-3 alone does not stimulate the colony formation, however , IL-3 in combination with stem cell factor (SCF) stimulate multipotential primitive cells at a level similar to that of normal IL-3-responsive mice. This synergy is not caused by upregulation of the number of IL-3R by SCF. It is more likely that a small number of functional IL-3R, which is still present in the A type mice, delivers some signals that are not sufficient for triggerring cell division but can be complemented by other cytokine signals. The A type mice and cell lines derived from them would be useful tools to dissect signals and to find critical molecules for IL-3mediated cell proliferation and differentiation. We have described the role of IL-3R, particularly the a subunit, in hematopoiesis: 1) a functional similarity between the a subunits of IL-3, GM-CSF, and IL-5 receptors, and 2) normal hematopoiesis in IL-3R alfa-deficient mice. To further understand the role of IL-3R system, characterization of ß knockout mice is underway in this laboratory.

Acknowledgements

The authors would like to thank Drs. S. Hudak, D. Rennick, M. Takagi, and M. Ichihara for their help
and contributions to these studies. DNAX Research Institute of Molecular and Cellular Biology
is supported by Schering-Plough Corporation.

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