IL-3. Alternative names. Structure. Discovery. Main activities and pathophysiological roles. John W. Schrader * SUMMARY BACKGROUND

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1 IL-3 John W. Schrader * The Biomedical Research Centre, University of British Columbia, 2222 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3 * corresponding author tel: , fax: , john@brc.ubc.ca DOI: /rwcy SUMMARY Interleukin 3 (IL-3) is a typical member of the family of four helix-bundle cytokines. It is synthesized and released by T lymphocytes when they are activated by specific antigens, and mast cells and eosinophils when multivalent antigens crosslink cytophilic antibodies bound to Fc receptors on their surface. IL-3 stimulates the growth, differentiation, survival, and function of a very broad range of cells derived from the pluripotential hematopoietic stem cell, including stem cells themselves, and progenitor cells and mature cells of many lineages, but is particularly important in stimulating the production of mast cells and basophils. BACKGROUND Discovery IL-3 was discovered and purified by a number of different groups working with different biological assay systems. One detected an activity that maintained the survival and growth of a population of mast cells that could be derived from bone marrow and other tissues in the presence of IL-3 (Schrader, 1981). Another, used for the initial purification of IL-3, was based upon detection of the induction of the enzyme 20-hydroxysteroid dehydrogenase in cultures of spleen cells from congenitally athymic mice(ihle et al., 1983), an assay which, in hindsight, was probably detecting effects on myeloid progenitor cells. IL-3 was also purified using assays which detected its ability to stimulate the growth from progenitor cells of colonies of mixtures of various lineages of cells of hematopoietic origin, or detected its ability to support the growth of immortalized lines of hematopoietic cells which depended on IL-3 for growth. Alternative names Reflecting the diversity of these assays, IL-3 was investigated in different laboratories under different names, including persisting (P) cell-stimulating factor, mast cell growth factor, burst-promoting activity, Thy-1-stimulating activity, histamine-producing cellstimulating activity, hematopoietic cell growth factor, multipotential colony-stimulating factor, multilineage hemopoietic growth factor, CFU-s-stimulating activity, stem cell-activating factor, hematopoietin 2, synergistic activity, CSF-2 and CSF-2. Structure The mature human IL-3 is a polypeptide of 133 amino acids and the mature murine IL-3 of 140 amino acids. IL-3 has the four helix-bundle fold characteristic of other members of the family of cytokines. It is heavily glycosylated, mainly through N-linked carbohydrate moieties. Main activities and pathophysiological roles IL-3 has the broadest target range of all of the hematopoietic growth factors. It stimulates the growth and differentiation, and supports the survival of pluripotential lymphohematopoietic stem cells able to give rise to all blood cells, including T and B lymphocytes, as well as multipotential hematopoietic progenitors, and progenitors committed to generating specific lineages including mast cells, eosinophils, neutrophils, macrophages, megakaryocytes, erythrocytes, dendritic cells, and basophils. IL-3 also maintains the survival of mature cells of certain

2 856 John W. Schrader lineages, e.g. mast cells, eosinophils, basophils, and megakaryocytes, and can augment or stimulate effector functions in mast cells, eosinophils, basophils, and macrophages. There is no evidence for the production of IL-3 in normal animals that are not undergoing an immune response. However, IL-3 is released from activated T cells during immunological responses to specific antigens, and from mast cells and eosinophils when Fc receptors are crosslinked by interaction of multivalent antigens with cell-bound IgE or other cytophilic classes of antibodies. GENE AND GENE REGULATION Accession numbers Mouse: K01850 Human: M14743 Chromosome location The human gene for IL-3 is on chromosome 5q23±31; the mouse gene on chromosome 11. Relevant linkages The IL-3 gene is clustered together with those for a number of four helix-bundle cytokines, in particular the closely related GM-CSF and IL-5, but also more distantly, IL-4, IL-13, and CSF-1 and its receptor c-fms. The IL-3 gene is close to GM-CSF gene, particularly in the human, where they are less than 1 kb apart and the two genes share regulatory elements. Regulatory sites and corresponding transcription factors The IL-3 promoter is complex, with both positive and negative regulatory sites being reported. Transcription factors binding to the promoter include Egr-1, AP-1, and Oct-1. Cells and tissues that express the gene The principal sources of IL-3 are activated T cells and NK cells. IL-3 is produced when the Fc receptors on mast cells and eosinophils are crosslinked by multivalent antigens. A number of murine myeloid leukemias exhibit rearrangements of the IL-3 gene that result in autocrine production of IL-3 that is responsible for maintenance of their survival and growth (Leslie and Schrader, 1989). The myelomonocytic cell line WEHI-3B is commonly used as a source of murine IL-3. PROTEIN Accession numbers Human: M14743 Murine: K01850 Sequence See Figure 1. Description of protein The three-dimensional structure of IL-3 has been determined using NMR (PDB Id: 1JLI). IL-3 is a globular protein which is heavily glycosylated, principally through the addition of N-linked sugars. There is no evidence for differences in the functional roles of the differentially glycosylated species. IL-3 exhibits the three-dimensional structure typical of cytokines of the four helix-bundle family Figure 1 Amino acid sequences for human and mouse IL-3. Human IL-3 MSRLPVLLLL QLLVRPGLQA PMTQTTSLKT SWVNCSNMID EIITHLKQPP LPLLDFNNLN GEDQDILMEN NLRRPNLEAF NRAVKSLQNA SAIESILKNL LPCLPLATAA PTRHPIHIKD GDWNEFRRKL TFYLKTLENA QAQQTTLSLA IF Murine IL-3 MVLASSTTSI HTMLLLLLML FHLGLQASIS GRDTHRLTRT LNCSSIVKEI IGKLPEPELK TDDEGPSLRN KSFRRVNLSK FVESQGEVDP EDRYVIKSNL QKLNCCLPTS ANDSALPGVF IRDLDDFRKK LRFYMVHLND LETVLTSRPP QPASGSVSPN RGTVEC

3 IL (Feng et al., 1996). The four helices are linked by loops and are arranged in the up-up, down-down configuration characteristic of this family of proteins. Residues in the N-terminal helix are critical for interactions with the chain of the IL-3 receptor. More C-terminal residues appear to be important for interacting with the chain of the receptor. Important homologies IL-5 and GM-CSF are closely related and share with IL-3 the capacity to bind to the common chain when they are complexed with their cognate receptor subunits. Posttranslational modifications IL-3 is a globular protein which is heavily glycosylated principally through the addition of N-linked sugars. There is no evidence for distinct functional roles for differentially glycosylated species. CELLULAR SOURCES AND TISSUE EXPRESSION Cellular sources that produce IL-3 is produced by T lymphocytes, NK cells, mast cells, and eosinophils. Eliciting and inhibitory stimuli, including exogenous and endogenous modulators The external stimuli eliciting the production of IL-3 are either multivalent antigens, which trigger mast cells or eosinophils by binding to antigen-specific cytophilic antibodies bound to their Fc receptors, or peptides generated by antigen-presenting cells from specific antigens and presented on MHC antigens, which activate antigen-specific T cells. RECEPTOR UTILIZATION IL-3 binds with low affinity to a specific receptor subunit named the IL-3 receptor subunit. This ligand±receptor complex then interacts with a receptor subunit to form a high affinity trimeric complex which activates intracellular signaling paths. This receptor subunit is termed common, reflecting the fact that it also performs an analogous role in the receptors for GM-CSF and IL-5, interacting with complexes of these ligands and their respective specific receptor subunits. In the mouse, the situation is made somewhat more complex by the existence of a second chain, encoded by a recent duplication of the common gene, which interacts exclusively with the IL-3 receptor subunit and itself has low affinity for IL-3. IN VITRO ACTIVITIES In vitro findings IL-3 stimulates the growth, differentiation, and survival of pluripotential hematopoietic stem cells and many of their progeny, including multipotential progenitors and progenitors committed to individual cell lineages and mature cells. These include stem cells capable of giving rise to all lymphohematopoietic cell lineages and multipotential progenitors capable of generating neutrophils, macrophages, megakaryocytes, and erythroid cells. More committed progenitors targeted by IL-3 include those giving rise to in vitro colonies of eosinophils, neutrophils, macrophages, megakaryocytes, mast cells, and erythroid cells. In combination with other molecules such as CD40 ligand, IL-3 can stimulate production of dendritic cells, and in vitro, supports the growth of early B cell progenitors. IL-3 stimulates the growth of mature murine macrophages as well as increases in spreading and phagocytosis. IL-3 supports the survival of mature mast cells, eosinophils, basophils, and megakaryocytes and also augments the effector functions of basophils, mast cells, and eosinophils. IL-3 also regulates other functions, for example, in mast cells, opposing the induction of class II MHC antigens by IFN. Regulatory molecules: Inhibitors and enhancers IL-3 synergizes strongly with other cytokines, the precise effects depending on its target. For example, in the case of primitive pluripotential hematopoietic stem cells, IL-3 synergizes with factors like IL-1, steel locus (stem cell) factor, or IL-6. In the case of mast cells, IL-3 synergizes with steel locus factor, IL-4, IL- 10, and IL-9. In the case of megakaryocytes, IL-3 synergizes with thrombopoietin and IL-11, and in the case of the erythroid lineage with erythropoietin.

4 858 John W. Schrader Bioassays used The most commonly used bioassays involve factordependent cell lines. For murine IL-3, cell lines such as Ba/F3 or FDCP-1 are useful. Ba/F3 is particularly convenient as it does not respond with continuous growth to any cytokine other than IL-3. However, bioassays should always be used in conjunction with a specific neutralizing antibody. IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS Normal physiological roles The normal physiological role of IL-3 is to act as a link between the immune system and the hematopoietic system. The synthesis of IL-3 is triggered by the presentation of antigenic peptides to activated T cells or the binding of multivalent antigens to cytophylic antibodies bound to mast cells or eosinophils. The effects of IL-3 depend on where it is released. The local release of IL-3 in mucosal surfaces or in lymph nodes results in the generation of mucosal-type mast cells from undifferentiated, committed mast cell progenitors present in these tissues. Systemic release of IL-3 results in increases in the number of hematopoietic progenitor cells and mast cell precursors in the spleen as well as increases in numbers of megakaryocytes, neutrophils, and mast cells. Species differences IL-3 stimulates a broad range of cells of hematopoietic origin in both mice and humans. Knockout mouse phenotypes Mice lacking functional IL-3 genes have no obvious phenotype. A minor defect in contact sensitization has been reported (Mach et al., 1998). However, when mice lacking functional IL-3 genes, or more dramatically both functional IL-3 and c-kit genes, are infected with the parasite Stronglyoides venezuelensis, they exhibit decreased responses in terms of generation of basophils and mast cells and delayed clearance of the parasite (Lantz et al., 1998). Thus IL-3 appears to have a role in the response to certain parasites, probably through its ability to increase the numbers of mast cells and basophils stimulated by the infection. Pharmacological effects Administration of recombinant or chemically synthesized IL-3 to mice results in mastocytosis in the gut and splenomegaly with increased numbers of neutrophils, megakaryocytes, mast cells, erythroid cells, and progenitors of mast cells and other lineages. PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY Normal levels and effects Levels of IL-3 in normal plasma are very low and are probably not biologically significant. Role in experiments of nature and disease states IL-3 can be detected in the serum in animals undergoing immunological activation, for example, during graft-versus-host (Crapper and Schrader, 1986) or following challenge with an allergenic antigen to which an animal has been sensitized. IL-3 is also present in the serum of mice bearing leukemias which are producing autocrine IL-3 as a result of gene rearrangements (Schrader and Crapper, 1983; Leslie and Schrader, 1989). IN THERAPY Effects of therapy: Cytokine, antibody to cytokine inhibitors, etc. Administration of recombinant human IL-3 by subcutaneous injection results in increases in the levels of neutrophils and eosinophils in the peripheral blood, as well as increases in platelet counts and lymphocyte numbers (Bukowski et al., 1996). Increases in number of CD34+ cells in the peripheral blood have been reported (Huhn et al., 1995). Pharmacokinetics Daily subcutaneous administration to humans results in reproducible effects on white cell counts in blood.

5 IL Antibodies to recombinant IL-3 have been reported in patients treated by subcutaneous injections (Bukowski et al., 1996). Toxicity Side-effects reported with IL-3 include headaches, flu-like symptoms and fever and rashes. Clinical results In general, progress in the clinical application of IL-3 has been slow. It may find use in combination with other cytokines such as G-CSF in mobilization of stem cells. References Bukowski, R. M., Olencki, T., Gunn, H., McLain, D., Budd, G. T., Sandstrom, K., Tuason, L., Redovan, C., Rayman, P., Tubbs, R., Resta, D., Elson, P., and Finke, J. (1996). Phase I trial of subcutaneous interleukin 3 in patients with refractory malignancy: hematological, immunological, and pharmacodynamic findings. Clin. Cancer Res. 2, 347±357. Crapper, R. M., and Schrader, J. W. (1986). Evidence for the in vivo production and release into the serum of a T-cell lymphokine, persisting-cell stimulating factor (PSF), during graftversus-host reactions. Immunology 57, 553±558. Feng, Y., Klein, B. K., and McWherter, C. A. (1996). Threedimensional solution structure and backbone dynamics of a variant of human interleukin-3. J. Mol. Biol. 259, 524±541. Huhn, R. D., Yurkow, E. J., Kuhn, J. G., Clarke, L., Gunn, H., Resta, D., Shah, R., Myers, L. A., and Seibold, J. R. (1995). Pharmacodynamics of daily subcutaneous recombinant human interleukin-3 in normal volunteers. Clin. Pharmacol. Ther. 57, 32±41. Ihle, J. N., Keller, J., Oroszlan, S., Henderson, L. E., Copeland, T. D., Fitch, F., Prystowsky, M. B., Goldwasser, E., Schrader, J. W., Palaszynski, E., Dy, M., and Lebel, B. (1983). Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cellstimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity. J. Immunol. 131, 282±287. Lantz, C. S., Boesiger, J., Song, C. H., Mach, N., Kobayashi, T., Mulligan, R. C., Nawa, Y., Dranoff, G., and Galli, S. J. (1998). Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 392, 90±93. Leslie, K. B., and Schrader, J. W. (1989). Growth factor gene activation and clonal heterogeneity in an autostimulatory myeloid leukemia. Mol. Cell Biol. 9, 2414±2423. Mach, N., Lantz, C. S., Galli, S. J., Reznikoff, G., Mihm, M., Small, C., Granstein, R., Beissert, S., Sadelain, M., Mulligan, R. C., and Dranoff, G. (1998). Involvement of interleukin-3 in delayed-type hypersensitivity. Blood 91, 778±783. Schrader, J. W. (1981). In in vitro production and cloning of the P cell, a bone marrow-derived null cell that expresses H-2 and Ia-antigens, has mast cell-like granules, and is regulated by a factor released by activated T cells. J. Immunol. 126, 452±458. Schrader, J. W., and Crapper, R. M. (1983). Autogenous production of a hemopoietic growth factor, persisting-cell-stimulating factor, as a mechanism for transformation of bone marrowderived cells. Proc. Natl Acad. Sci. USA 80, 6892±6896.