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Cutting Edge: Identification of a Hybrid Cytokine Consisting of IL-7 and the β-Chain of the Hepatocyte Growth Factor/Scatter Factor

This information is current as Laijun Lai and Irving Goldschneider of September 23, 2021. J Immunol 2001; 167:3550-3554; ; doi: 10.4049/jimmunol.167.7.3550 http://www.jimmunol.org/content/167/7/3550 Downloaded from References This article cites 30 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/167/7/3550.full#ref-list-1

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Cutting Edge: Identiﬁcation of a Hybrid Cytokine Consisting of IL-7 and the ␤-Chain of the Hepatocyte Growth Factor/Scatter Factor1

Laijun Lai and Irving Goldschneider2

initiate or up-regulate the expression of terminal deoxynucleotidyl Pre-pro-B cell growth-stimulating factor (PPBSF) is a het- transferase, cytoplasmic Ig␮ heavy chain, and IL-7R␣-chain (Ref. erodimer of IL-7 and a 30-kDa cofactor. Unlike monomeric 5, and C. Wei, L. Lai, and I. Goldschneider, manuscript in prepara-

IL-7, PPBSF selectively induces proliferation and differentiation). The latter events appear to enable newly formed pro-B cells Downloaded from tion of pre-pro-B cells and up-regulates IL-7R␣-chain expres- to proliferate and differentiate in response to monomeric IL-7 (3). sion. Here we clone the PPBSF cofactor from bone marrow PPBSF-coF is produced constitutively by IL-7Ϫ/Ϫ BM stromal stromal cells and identify it as a variant ␤-chain of hepatocyte cells in our culture system (3, 6), and antisera raised to native growth factor (HGF), a pleiotropic cytokine homologous to PPBSF contain separable reactivities for IL-7 and PPBSF-coF (4). plasminogen that regulates cell growth, motility, and morpho- By itself, PPBSF-coF maintains the viability of pre-pro-B cells,

genesis. We further demonstrate that, in the presence of low whereas in the presence of IL-7 it forms functional complexes of http://www.jimmunol.org/ m.w. heparin sulfate-derived oligosaccharides, rHGF␤ com- PPBSF. However, despite interacting with IL-7, the PPBSF-coF bines with rIL-7 to form a biologically active heterodimer hav- differs functionally and serologically from stem cell factor (SCF), ing the properties of PPBSF. The results indicate that PPBSF insulin-like growth factor-1, thymic stromal lymphopoietin, is a novel form of cytokine (hybrid cytokine) consisting of the flk2/flt3 ligand, and pre-B cell growth-stimulating factor/stromal bioactive components of two unrelated cytokines. Based on its derived factor-1␤. heparin-binding and mitogenic properties, we postulate that In the present study we determined by amino acid sequencing, the HGF␤-chain in PPBSF enables IL-7 to participate in cog- RT-PCR analysis, and cDNA cloning that the PPBSF-coF is the ␤ nate interactions at the stromal cell surface and to transduce free mitogenic -chain of hepatocyte growth factor (HGF)/scatter factor, a heparin-binding, stromal cell-derived, multifunctional cy- by guest on September 23, 2021 signals effectively at low levels of IL-7R. The Journal of Im- tokine closely homologous to plasminogen (reviewed in Ref. 7). munology, 2001, 167: 3550Ð3554. This finding was wholly unexpected, as HGF is a pleiotropic factor that promotes parenchymal cell growth, motility, and morphogen- onsiderable progress has been made in identifying stro- esis in a broad spectrum of tissues; and as the HGF ␤-chain had not mal cell-derived growth factors that regulate pro-B cell previously been found to be produced independently of the HGF C and pre-B cell development. However, little is known ␣-chain (receptor-binding domain for c-MET), with which it is about the factors that regulate pre-pro-B cell development. To ap- secreted as an inactive single chain precursor (pro-HGF). We also proach this problem, we developed a long-term bone marrow demonstrated that, in the presence of low m.w. heparin sulfate (BM)3 lymphoid culture system that selectively supports the self- (HS)-derived oligosaccharides, rHGF␤ complexes with rIL-7 to replication of pre-pro-B cells and their differentiation to pro-B form a heterodimer having the functional, physical, and serological cells (1, 2). Using this system, we have purified a pre-pro-B cell properties of native PPBSF. Hence, PPBSF appears to represent a growth-stimulating factor (PPBSF) and demonstrated that it is a functionally unique and heretofore undescribed form of cytokine heterodimer of IL-7 and a 30-kDa cofactor (PPBSF-coF) (3, 4). (hybrid cytokine) consisting of the complexed bioactive portions Unlike monomeric IL-7, the IL-7/PPBSF-coF complex stimulates of two independently generated cytokines. The possible dual func- proliferation and differentiation of pre-pro-B cells and helps to tion of the IL-7/HGF␤ hybrid cytokine in the induction of B lineage commitment and the regulation of early B cell development is discussed. Department of Pathology, School of Medicine, University of Connecticut Health Cen- ter, Farmington, CT 06030 Materials and Methods Received for publication May 21, 2001. Accepted for publication August 14, 2001. Animals The costs of publication of this article were defrayed in part by the payment of page ϫ Ϫ/Ϫ ϩ/ϩ charges. This article must therefore be hereby marked advertisement in accordance (129 B6)F2 IL-7 and IL-7 mice (generously provided by Dr. R. with 18 U.S.C. Section 1734 solely to indicate this fact. Murray, DNAX Research Institute of Cellular and Molecular Biology, Palo 1 This study was supported in part by National Institutes of Health Grant AI 32752. Alto, CA) were used as donors of primary BM-adherent cell feeder layers Ͼ 2 and of enriched ( 98%) populations of stromal cells generated by serial Address correspondence and reprint requests to Dr. Irving Goldschneider, Depart- passage in vitro (3). Male 4- to 6-wk-old Lewis strain rats were used as ment of Pathology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030. E-mail address: [email protected] donors of BM lymphoid precursor cells. 3 Abbreviations used in this paper: BM, bone marrow; CM, conditioned medium; coF, Cytokines and Abs cofactor; HGF, hepatocyte growth factor; HS, heparin sulfate; PPBSF, pre-pro-B cell growth-stimulating factor; SCF, stem cell factor; EPO, erythropoietin; CHO, Chinese Recombinant murine IL-3, IL-7, GM-CSF, HGF, and erythropoietin (EPO) hamster ovary. were purchased from R&D Systems (Minneapolis, MN), as were anti-HGF

● The Journal of Immunology 3551 and anti-IL-7 Abs. Affinity-purified goat polycolonal Ab reactive with human and mouse HGF␤, and HRP-linked anti-goat IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Amino acid sequence analysis of PPBSF PPBSF was purified from IL-7ϩ/ϩ BM stromal cell conditioned medium (CM) on an anti-IL-7 immunoaffinity column as previously described (3, 4). The bound Ag was eluted, dialyzed for 16 h in PBS (pH 7.2) at 4¡C, and loaded on a 12% SDS-PAGE gel under reducing conditions. After elec- trophoresis, the proteins were transferred onto ProBlott membrane (Ap- plied Biosystems, Foster City, CA) using a trans-Blot SD Semidry Transfer FIGURE 1. Partial NH2-terminal amino acid sequence identity of puri- Cell (model 200/2.0; Bio-Rad, Hercules, CA), then stained with Coomassie fied mouse PPBSF-coF from enriched IL-7ϩ/ϩ BM stromal cells. Com- brilliant blue R-250 (Bio-Rad). The 30-kDa band was excised and sent to parison with the predicted amino acid sequences for the HGF ␤-chain in the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale mouse, rat, and human as derived from the published nucleotide sequences. University for direct NH -terminal sequencing. 2 Positions of reported nucleotide substitutions are indicated in boxes. Molecular cloning and sequencing of HGF␤

Total RNA was isolated with TRIzol Reagent (Life Technologies, Gaith- PPBSF-coF as the HGF ␤-chain was confirmed by Western blot ersburg, MD) from populations of enriched IL-7Ϫ/Ϫ BM stromal cells. Random-primed first-strand cDNA was generated using Moloney murine analyses, in which PPBSF-coF reacted with Ab against full-length HGF and the HGF ␤-chain (Fig. 2), but not the HGF ␣-chain (data leukemia virus reverse transcriptase (RETRO Script; Ambion, Austin, TX). Downloaded from PCRs were performed using Taq polymerase (Life Technologies) and not shown). In addition, both anti-HGF and anti-HGF␤ Abs neu- primers designed to amplify the entire coding sequence of mouse HGF: tralized the PPBSF activity in IL-7ϩ/ϩ CM (data not shown, but Ј Ј Ј Ј 5 -CAGTCTGCTCGAACTGCA-3 (in 5 flanking region), and 5 -TGGC see Fig. 5). CTCTTCTATGGCTA-3Ј (in 3Ј flanking region). PCR was performed as follows: 95¡C for 5 min, 30 cycles of 94¡C for 1 min, 60¡C for 1 min, and 72¡C for 1 min followed by a final extension at 72¡C for 5 min. The Identification and cloning of a variant of HGF mRNA amplified fragments were separated on 1% agarose gel and visualized by Two products were obtained when mRNA transcripts from IL- http://www.jimmunol.org/ ethidium bromide. The small amplified fragment was cloned into plasmid 7Ϫ/Ϫ mouse BM stromal cells were analyzed by RT-PCR (Fig. 3). vectors according to the instructions of the TA Cloning kit (Invitrogen, San Diego, CA). The plasmid DNA was purified and sequenced. One of these products corresponded to the full-length HGF cDNA (2230 bp), and the other to the coding sequence of HGF␤ (840 bp). ␤ Production of rHGF proteins The cDNA of the shorter product was cloned, and the nucleotide The small PCR-amplified fragment was subcloned into the mammalian sequence was found to concur precisely with the published mouse expression vector pcDNA3.1(ϩ) (Invitrogen) with a BamHI-XhoI site. The HGF␤ cDNA sequence. In addition, the signal sequence was iden- plasmid was transfected into Chinese hamster ovary (CHO) cells (LIPO- tical with that in full-length HGF cDNA. FECTAMINE Plus Reagent; Life Technologies). The serum-free superna- ␤ tant from the transfected CHO cells was evaluated for HGF␤ protein by The HGF -chain cDNA was then transfected into CHO cells or

ELISA. The HGF␤ gene was also subcloned into the prokaryotic fusion transformed into E. coli BL21 (DE3). HGF␤ protein was detected by guest on September 23, 2021 protein expression vector pCAL-n (Stratagene, La Jolla, CA) with a by ELISA in the supernatant of HGF␤ (but not empty vector)- BamHI-EcoRI site and transformed into Escherichia coli BL21(DE3). The transfected cells, and by SDS-PAGE and Western blotting after ␤ HGF and calmodulin-binding-peptide fusion protein was purified by cal- release from the prokaryotic fusion protein (data not shown). modulin affinity resin, and the peptide was released by thrombin. The purified proteins were electrophoresed in 12% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). The membranes were Formation and biological activity of a heterodimer of rIL-7 and incubated with anti-HGF Ab or anti-HGF␤ Ab and HRP-labeled anti-goat rHGF␤ IgG. Bound Ab was detected using the ECL system (Amersham Pharmacia As both IL-7 and HGF are heparin-binding molecules (9, 10), we Biotech, Piscataway, NJ). tested the ability of rIL-7 and rHGF␤ to form a heterodimer when Binding assays Bovine kidney HS (Sigma, St. Louis, MO) was digested with heparitinase Iat37¡ for 1 h, and the products were ultrafiltrated by Centriprep-3 (m.w. cut-off 3000; Amicon, Beverly, MA) (7). rIL-7 and rHGF␤ were mixed in the presence or absence of the low m.w. HS-derived oligosaccharides for 1 h. The mixtures were subjected to SDS-PAGE under nonreducing conditions and to Western blotting using anti-HGF␤ Ab and anti-IL-7 mAb, as described above. Pre-pro-B cell growth-stimulating activity Freshly harvested rat BM cells were added to 2 ml of RPMI 1640 containing 20% lot-selected, defined FBS in 35-mm-diameter culture plate ϫ 6 wells (2 10 cells/well) and incubated at 37¡Cin5%CO2 in the presence of 5 ng/ml rIL-7 and/or rHGF␤. Anti-HGF or anti-HGF␤ Ab was added to some cultures at the same time. Ten days later, the nonadherent cells were harvested for phenotypic analysis. Results Identification of the PPBSF-coF as the ␤-chain of HGF

Partial NH2-terminal amino acid sequence analysis of purified ϩ/ϩ PPBSF-coF from IL-7 mice (Fig. 1) showed that, when allow- FIGURE 2. Confirmation of the identity of the PPBSF-coF as HGF␤ by ances are made for known single nucleotide substitutions at posi- Western blotting analyses. Affinity purified PPBSF was electrophoresed tions 8, 10, and 11, the first 17 amino acid residues were identical under reducing conditions and developed with anti-HGF or anti-HGF␤ with those predicted by published cDNA nucleotide sequences for Abs. The 30-kDa PPBSF-coF, but not the 25-kDa IL-7 component, reacted mouse, rat, and human HGF ␤-chain (8). The identity of the with Abs against HGF␤. 3552 CUTTING EDGE: IDENTIFICATION OF AN IL-7/HGF␤ HYBRID CYTOKINE

able to stimulate their proliferation or differentiation to pro-B cells. However, when added concurrently, these reagents significantly enhanced the generation of pre-pro-B cells and pro-B cells (Fig. 5A), presumably by forming PPBSF complexes comparable to those present in stromal cell CM (3, 4). In the second approach, the purified rIL-7/rHGF␤ heterodimer preformed in the presence of HS-derived oligosaccharides was added directly to the culture medium (Fig. 5B). In both instances the bioactivity was neutralized by anti-HGF␤ Ab. To test the specificity of the interaction of IL-7 with HGF␤, several other heparin-binding factors involved in lymphohemopoi- esis (GM-CSF, IL-3, EPO) (11) were mixed with rIL-7 or rHGF␤ in the presence of HS-derived oligosaccharides. No detectable complexes were formed (data not shown). FIGURE 3. RT-PCR analysis of the HGF mRNA transcripts from Ϫ Ϫ mouse BM stromal cells. First-stand cDNA from cultured IL-7 / mouse Discussion BM stromal cells were subjected to PCR with primers designed to amplify Inasmuch as pro-HGF is converted by targeted protease digestion the entire coding sequence of mouse HGF. Both the 2230-bp PCR product to a disulfide-linked heterodimer comprised of a 60-kDa ␣-chain Downloaded from corresponding to full-length HGF and a novel 840-bp product were present. and a 30-kDa ␤-chain (7), it could be argued that the free HGF␤- chain (PPBSF-coF) observed in culture supernatants (3) is an en- equal concentrations (250 ng/ml) were mixed in serum-free me- zymatic cleavage fragment that normally would be degraded in dium in the presence or absence of low m.w HS-derived oligosac- vivo. However, both RT-PCR analysis of mRNA transcripts and charides. The results in Fig. 4 show that rHGF␤ and rIL-7 mi- cDNA cloning of the shorter product proved otherwise. Rather, the ␣ grated at 30 and 14.5 kDa, respectively, in the absence of the PPBSF-coF is a variant HGF that lacks the -chain domain. While http://www.jimmunol.org/ HS-derived oligosaccharides, and at 45 kDa in their presence. At unique, this finding is consistent with the identification of several higher concentrations of IL-7 and HGF␤ (Ն1000 ng/ml), larger other variant HGFs and transcripts produced by alternative splic- complexes were also detected, but the heterodimeric form contin- ing of the HGF gene (12Ð15). Among these, an 85-kDa native and ␣ ued to predominate (data not shown). Comparable results were a 28-KDa variant HGF -chain is produced by human BM stromal obtained when FBS (5Ð20%) was substituted for HS-derived cells (16). oligosaccharides. Although a great deal is known about the actions of HGF in Two approaches were used to confirm that the IL-7/HGF␤ het- nonhemopoietic tissues, its role in the regulation of hemopoiesis, erodimer had biological activity. In the first, rat BM cells were and particularly lymphopoiesis, is fragmentary (7). HGF has been by guest on September 23, 2021 incubated in culture medium (plus 20% FBS) containing rIL-7 proposed to regulate hemopoiesis in mouse fetal liver and adult and/or rHGF␤. As anticipated (3), both rIL-7 and rHGF␤ were BM, where it apparently can substitute for the SCF and c-kit sys- able to maintain the viability of pre-pro-B cells, but neither was tem (17). HGF is produced by BM stromal cells and synergizes

FIGURE 4. Recombinant IL-7 forms a heterodimer with rHGF␤ in the presence of low m.w. HS-derived oligosacchrides. Recombinant IL-7 (250 ng) was added to serum-free culture medium containing rHGF␤ (250 ng) FIGURE 5. PPBSF activity is reconstituted by rIL-7/rHGF␤ complexes in the presence or absence of low m.w. HS-derived oligosaccharides. One formed in culture by adding 5 ng of rIL-7 and 5 ng of rHGF␤ to medium hour later, duplicate aliquots of each mixture were electrophoresed on a containing 20% FBS (A), or preformed in the presence of HS-derived oli- single gel under nonreducing conditions. The proteins were then trans- gosaccharides before being added to the cultures (B). Freshly harvested rat ferred to an Immobilon-P membrane, one half of which was developed BM cells (5 ϫ 105 cells/ml) were cultured in each well in the presence or with anti-HGF␤ Ab (A) and the other half with anti-IL-7 mAb (B). In each absence of anti-HGF␤ Ab. Nonadherent lymphoid cells were phenotyped instance, a 45-kDa heterodimer was observed in the presence (lane 2), but on day 10. Means of triplicate wells. Ab controls (normal goat serum) were not the absence (lane 1), of the HS-derived oligosaccharides. negative (data not shown). The Journal of Immunology 3553 with IL-3 or GM-CSF to support the growth of hemopoietic pro- tor that helps to regulate the earliest stages of B lymphocyte de- genitor cells and myeloid tumor cell lines, all of which express the velopment, but also because it may presage the existence of other HGF receptor, c-MET (18). In addition, HGF has been found to hybrid cytokines. Therefore, it will be important to determine how promote adhesion of hemopoietic progenitor cells to fibronectin signaling by PPBSF differs from that of monomeric IL-7 (and (18). In the presence of EPO, HGF induces the formation of col- HGF), and whether the HGF␤ moiety binds to the IL-7R complex onies along the erythroid lineage, whereas in the presence of EPO itself or to another receptor on the pre-pro-B cell surface. It will and SCF, HGF supports the growth of multipotent colonies (19). also be of interest to investigate the intriguing possibility that Furthermore, IL-11, which is thought to up-regulate c-MET, syn- PPBSF may help to induce the commitment of hemopoietic stem ergizes with HGF to support in vitro colony formation by hemo- cells to development along the B (and possibly T) lymphoid poietic stem cells (20). Thus, HGF appears to be an important pathways. mediator of paracrine interactions between stromal and hemopoietic cells that preferentially acts in the window of differentiation References between multipotent stem cells and committed progenitors. How- 1. Hayashi, J., E. S. Medlock, and I. Goldschneider. 1984. A selective culture sys- ever, by itself, HGF does not appear to stimulate proliferation of tem for generating terminal deoxynucleotidyl transferase-positive lymphoid pre- hemopoietic precursors. This suggests, as one possibility, that the cursor cells in vitro. I. Description of the culture system. J. Exp. Med. 160:1622. 2. McKenna, S. D., E. S. Medlock, D. L. Greiner, and I. Goldschneider. 1994. A primary role of HGF in hemopoiesis is to enhance signal trans- selective culture system for generating terminal deoxynucleotidyl transferase- duction by lineage-specific cytokines. positive lymphoid precursor cells in vitro. IV. Properties and developmental re- lationships of the lymphoid cells in the adherent and nonadherent compartments Among lymphoid cells, mRNA for c-MET has been identified in of the culture. Exp. Hematol. 22:1164. Downloaded from thymocytes, and HGF increases the generation of mature T cells in 3. McKenna, S. D., F. Chen, L. Lai, and I. Goldschneider. 1998. Identification of an fetal thymus organ cultures (21). c-MET is also expressed on early IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). I. Production of the non-IL-7 component by bone marrow stromal cells from IL-7 gene-deleted B lineage cells in BM (19), and may help to regulate integrin- mice. J. Immunol. 160:2272. mediated adhesion and migration of B cells in germinal centers 4. Lai, L., F. Chen, S. D. McKenna, and I. Goldschneider. 1998. Identification of an (22). Furthermore, upon activation, B cells bind large amounts of IL-7-associated pre-pro-B cell growth-stimulating factor (PPBSF). II. PPBSF is a covalently linked heterodimer of IL-7 and a Mr 30,000 cofactor. J. Immunol.

HGF via HS moieties, thereby promoting the phosphorylation of 160:2280. http://www.jimmunol.org/ c-MET and of several substrates (7). 5. Wei, C., R. Zeff, and I. Goldschneider. 1999. Murine pro-B cells require IL-7 to Therefore, the question arises as to why the IL-7/HGF␤ hybrid upregulate IL-7R␣, TdT and c ␮ expression in vivo. J. Immunol. 164:1961. 6. McKenna, S. D., and I. Goldschneider. 1993. A selective culture system for cytokine, in contrast to HGF, HGF␤, and monomeric IL-7, selec- generating terminal deoxynucleotidyl transferase-positive lymphoid cells in vitro. tively supports the proliferation and differentiation of pre-pro-B V. Detection of stage-specific pro-B-cell stimulating activity in medium condi- cells. Two possibilities are immediately apparent: 1) the need for tioned by mouse bone marrow stromal cells. Dev. Immunol. 3:181. 7. van der Voort, R., T. E. Taher, P. W. Derksen, M. Spaargaren, R. van der Neut, cognate interactions of pre-pro-B cells with BM stromal cells (23); and S. T. Pals. 2000. The hepatocyte growth factor/Met pathway in development, and 2) the expression of only low levels of the IL-7R␣-chain (5). tumorigenesis, and B-cell differentiation. Adv. Cancer Res. 79:39. ␤ 8. Liu, Y., G. K. Michalopoulos, and R. Zarnegar. 1993. Molecular cloning and Our working hypothesis is that the heparin-binding IL-7/HGF characterization of cDNA encoding mouse hepatocyte growth factor. Biochim. hybrid cytokine, like HGF (11), functions primarily as a cell sur- Biophys. Acta. 1216:299. by guest on September 23, 2021 face or extracellular matrix-bound molecule; and that it, unlike 9. Ashikari, S., H. Habuchi, and K. Kimata. 1995. Characterization of heparin sulfate oligosaccharides that bind to hepatocyte growth factor. J. Biol. Chem. 270: IL-7, effectively transduces signals for proliferation and differen- 29586. tiation in the presence of low levels of IL-7R␣. We further pos- 10. Clarke, D., O. Katoh, R. V. Gibbs, S. D. Griffiths, and M. Y. Gordon. 1995. tulate that PPBSF enables the resulting pro-B cells to respond Interaction of interleukin 7 (IL-7) with glycosaminoglycans and its biological ␣ relevance. Cytokine 7:325. to monomeric IL-7 by up-regulating the expression of IL-7R 11. Roberts, R., J. Gallagher, E. Spoonceer, T. D. Allen, F. Bloomfield, and (Ref. 5, and C. Wei, L. Lai, and I. Goldschneider, manuscript in T. M. Dexter. 1998. Heparin sulphate bound growth factors: a mechanism for preparation). Our hypothesis is consistent with the demonstration stromal cell mediated haemopoiesis. Nature 332:376. 12. Rubin, J. S., A. M. Chan, D. P. Bottaro, W. H. Burgess, W. G. Taylor, A. C. Cech, that: 1) extracellular matrix glycoproteins can selectively increase D. W. Hirschfield, J. Wong, T. Miki, P. W. Ginch, and S. A. Aaronson. 1991. A the IL-7-dependent proliferation of early B lineage cells (24); 2) broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte differentiation of pre-pro-B cells to pro-B cells requires signaling growth factor. Proc. Natl. Acad. Sci. USA 88:415. 13. Jakubczak, J. L., W. J. Larochelle, and G. Merlino. 1998. NK1, a natural splice through high affinity IL-7R complexes (5); and 3) pre-BCR sig- variant of hepatocyte growth factor/scatter factor, is a partial agonist in vivo. Mol. naling can modulate the IL-7 response (25). It may also help to Cell. Biol. 18:1275. 14. Kinosaki, M., K. Yamaguchi, A. Murakami, M. Ueda, T. Morinaga, and explain why excess IL-7 fails to increase pre-pro-B cell generation K. Higashio. 1998. Identification of heparin-binding stretches of a naturally oc- in vivo (26, 27), and why IL-7 does not stimulate proliferation of curring deleted variant of hepatocyte growth factor (dHGF). Biochim. Biophys. pro-B cells from IL-7 knockout mice in vitro unless these cells are Acta 1384:93. 15. Miau, L., Y. Jan, B. Shen, W. Tsai, H. Lee, and S. Lee. 1996. Identification of a first treated with PPBSF (Ref. 5, and C. Wei, L. Lai, and I. Gold- novel variant hepatocyte growth factor secreted by spleen-derived stromal cells. schneider, manuscript in preparation). Biochem. Biophys. Res. Commun. 223:487. To our knowledge, this is the first demonstration of a naturally 16. Takai, K., J. Hara, K. Matsumoto, G. Hosoi, Y. Osugi, A. Tawa, and S. Okada. 1997. Hepatocyte growth factor is constitutively produced by human bone mar- occurring hybrid cytokine (i.e., a functional complex of the bio- row stromal cells and indirectly promotes hematopoiesis. Blood 89:1560. active portions of two or more disparate cytokines or growth fac- 17. Yu, S. Z., H. Hisha, Y. Li, Z. Lian, T. Nishino, J. Toki, Y. Adachi, M. Inaba, tors). Although IL-12 and related “composite” cytokines (e.g., IL- T. X. Fan, T. Jin, et al. 1998. Stimulatory effects of hepatocyte growth factor on hemopoiesis of CSF/c-kit system-deficient mice. Stem Cells 16:66. 23, cytokine-like factor-cardiotrophin-like cytokine complex) also 18. Weimar, I. S., N. Miranda, E. J. Muller, A. Kekman, J. M. Kerst, G. C. de Gast, are heterodimers, they are structurally analogous to cytokine-re- and W. R. Gerritsen. 1998. Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, ad- ceptor complexes rather than hybrid cytokines (28Ð30). However, ϩ hesion and survival of human hematopoietic progenitor cells (CD34 ). Exp. He- many cytokines other than HGF and IL-7 are known to interact matol. 26:885. with HS (11), and some of these in theory could form functional 19. Galami, F., G. P. Bagnara, L. Bonsi, E. Cottone, A. Follenzi, A. Simeone, and P. M. Comogio. 1994. Hepatocyte growth factor induces proliferation and dif- homodimeric or heterodimeric complexes. For example, HS-de- ferentiation of multipotent and erythroid hemopoietic progenitors. J. Cell Biol. rived oligosaccharides induce fibroblast growth factor to form ho- 127:1743. modimers that facilitate fibroblast growth factor receptor dimer- 20. Goff, J. P., S. D. Shields, B. E. Petersen, V. F. Zazjac, G. K. Michalopoulos, and ␤ J. S. Greenberger. 1996. Synergistic effects of hepatocyte growth factor on human ization (31). Hence, the discovery of the IL-7/HGF complex is cord blood CD34ϩ progenitor cells are the results of c-met receptor expression. significant not only because it provides a functionally unique fac- Stem Cells 14:592. 3554 CUTTING EDGE: IDENTIFICATION OF AN IL-7/HGF␤ HYBRID CYTOKINE

21. Tamura, S., T. Sugawara, Y. Tokoro, H. Taniguchi, K. Fukao, H. Nakauchi, and 27. Morrissey, P. J., P. Conlon, K. Charrier, S. Braddy, A. Alpert, D. Williams, Y. Takahama. 1998. Expression and function of c-Met, a receptor for hepatocyte A. E. Namen, and D. Mochizuki. 1991. Administration of IL-7 to normal mice growth factor, during T-cell development. Scand. J. Immunol. 47:296. stimulates B-lymphopoiesis and peripheral lymphadenopathy. J. Immunol. 147: 22. van der Voort, R., T. E. Taher, R. M. Keehnen, L. Smit, M. Groenink, and 561. S. T. Pals. 1997. Paracrine regulation of germinal center B cell adhesion through 28. Gately, M. K., C. Y. Wu, and D. A. Faherty. 1997. Interleukin-12: a het- the c-met-hepatocyte growth factor/scatter factor pathway. J. Exp. Med. 185: erodimeric cytokine that promotes cell-mediated immunity. In Cytokines in 2121. Health and Disease, 2nd Ed. D. G. Remick and J. S. Friedland, eds. Marcel- 23. Medlock, E. S., S. D. McKenna, and I. Goldschneider. 1993. A selective culture Dekker, New York, p. 167. system for generating terminal deoxynucleotidyl transferase-positive lymphoid 29. Oppmaun, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, cells in vitro. III. Structure of the bone marrow microenvironment for early lym- N. Yu, J. Wang, K. Singh, et al. 2000. Novel p19 protein engages IL-12 p40 to phopoiesis. Lab. Invest. 69:616. form a cytokine, IL-23, with biological activities similar as well as distinct from 24. Oritani, K., and P. W. Kincade. 1996. Identiﬁcation of stromal cell products that IL-12. Immunity 13:715. interact with pre-B cells. J. Cell Biol. 134:771. 30. Elson, G. C., E. Lelievre, C. Guillet, S. Chevalier, H. Plun-Farreau, J. Froger, 25. Marshall, A. J., H. E. Fleming, G. E. Wu, and C. J. Paige. 1998. Modulation of I. Suard, A. Bonoit de Coignac, Y. Delneste, J. Y. Bonnefoy, et al. 2000. CLF the IL-7 dose-response threshold during pro-B cell differentiation is dependent on associates with CLC to form a functional heterodimeric ligand for the CNTF pre-B cell receptor expression. J. Immunol. 161:6038. receptor complex. Nat. Neurosci. 3:867. 26. Mertsching, E., U. Grawunder, V. Meyer, T. Rolink, and R. Ceredig. 1996. Phe- 31. Schlessinger, J., A. N. Plotnikov, O. A. Ibrahimi, A. V. Eliseenkova, B. K. Yek, notypic and functional analysis of B lymphopoiesis in interleukin-7 transgenic A. Yayon, R. J. Linhardt, and M. Mohammadi. 2000. Crystal structure of a mice: expansion of pro/pre-B cell number and persistence of B lymphocyte de- ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR velopment in lymph nodes and spleen. Eur. J. Immunol. 26:28. binding and dimerization. Mol. Cell 6:743. Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021