Concise Review International Journal of Cell Cloning 10:12-17(1992)
Regulation of B Cell Differentiation By Bone Marrow Stromal Cells Kenneth Dorshkind," Kennerh S . Landrethh aDivision of Biomedical Sciences, University of California, Riverside, California. USA; bDepartmentof Microbiology and lmmunology and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia, USA Key Words. B Lymphocytes Cytokines Hemopoiesis Immunoglobulin Long-term bone marrow culture Stromal cells
Abstract. B lymphocyte development occurs in the intersinusoidal spaces of bone marrow in association with a sessile population of stromal cells. Development of long-term bone marrow culture systems that support B lymphopoiesis has allowed the isolation of stromal cells that support that process and permitted analysis of their role in controlling the growth and differentiation of B lineage cells in culture. In addition to direct interactions with developing lymphocytes, stromal cells secrete a variety of cytokines that affect lymphocyte growth and differentiation, and many of these molecules have been expressed in recombinant form. These relatively recent advances have made it possible to formulate a working model of the cells and molecules involved in regulating primary B cell production.
Introduction Mammalian B lymphocyte development is an antigen-independent process in which immature hemopoietic precursors become committed to the lymphoid developmental pathway and mature into immunoglobulin (Ig) expressing B lymphocytes competent to mediate a humoral immune response [I]. Multiple regulatory controls are operative during B lymphopoiesis and their failure may contribute to both neoplasias and immunodeficiencies that involve cells at various stages in the B cell developmental pathway. Consequently, obtaining a better understanding of how B cell differentiation is controlled has tremendous practical relevance. In addition, studies of B cell differentiation provide a model for analysis of Correspondence: Dr. Kenneth Dorshkind, Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, USA. Received August 29, 1991; accepted for publication August 29, 1991. 07 37-1454/92/$2.00/0OAlphaMed Press
how such parameters as cell-cell interactions, soluble mediators, and cell-matrix interactions control growth and differentiation during mammalian development.
B Cell Differentiation in the Bone Marrow During fetal life the yolk sac [2], placenta [3], and liver [4, 51 contain B lineage cells, but in normal adult mammals, primary B cell production takes place exclusively in the bone marrow in parallel with erythropoiesis and myelopoiesis [ 1,6]. The daily rate of B cell production is extremely high, and studies that have measured the proliferative status, renewal rate, and intercompartmental transit of murine B lineage cells have estimated that up to 5 X lo7 B lymphocytes are generated per day [7,8]. B lymphocytes develop from immature hemopoietic precursors [8] which could include the p h i potent hemopoietic stem cell. This cell is capable of generating all blood cells, including B cells [9, lo], and continual input from that compartment may be necessary to maintain steady state B lymphopoiesis. Another possibility is that a bipotential lymphoid precursor capable of generating T and B cells exists [ 111, but definitive identification of these cells has not been made. Despite these uncertainties, it has been possible to resolve more mature, committed stages in the B cell developmental pathway through the use of antibodies that recognize cell surface and cytoplasmic determinants [ I ] (Fig. 1). Committed B cell progenitors in murine bone marrow can be recognized based on their expression of the 220,000 MW form of the Ly5,,,, antigen [ 12. 131. Subsequently, immunoglobulin (Ig) p heavy chain appears in the cytoplasm of pre-B cells and this indicates that productive heavy chain rearrangements have occurred. There is evidence that membrane p can be
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Fig. 1. Summary of primary B cell differentiation. Ig gene rearrangement events are placed at the cellular stage at which they first occur. The ability of different stromal cells to support distinct stages of B lymphopoiesis is indicated using the S17 and S10 lines as examples. Placement of stromal cell line S17 in this diagram is consistent with its role as an early acting stromal cell that allows B220- precursors to mature to at least the pre-B cell stage, whereas placement of the S10 line indicates its role as a late acting stromal cell type which stimulates expression of surface Ig. Dashed lines indicate that the placement of particular developmental events and regulatory signals is an approximation. For example, while the placement of KL in the figure is based on observations that the factor synergizes with IL7 to stimulate proliferation of progenitor/pre-B cells, the dashed line to the left of the kit-ligand box is meant to indicate that an effect(s) of KL on more immature cells in the B lineage is not excluded. Similarly, the dashed line to the right of IL-7 indicates a role for the factor in the pre-B to B cell transition has been suggested.
expressed on the cell surface of pre-B cells in association with nonconventional light chain molecules [14-171. The final stage of primary B lymphopoiesis is the expression of kappa or lambda light chain protein [ 181. Kappa genes are expressed predominantly in the mouse, while in humans the kappa:lambda ratio is approximately 7:3 [19]. In addition to the phenotypic determinants described above, there are additional cell surface molecules that have been used to characterize B lineage cells. Another cell surface antigen, referred to as BP-1 or 6C3 [20-211, also appears on murine B lineage cells, and a recent report indicates it is expressed subsequent to the appearance of the B220 molecule and before surface IgM [ 2 2 ] . The status of Ig gene rearrangements in B lineage cells provides a molecular means of defining stages of differentiation. The Ig heavy chain is as-
sembled from distinct genetic loci that include a variable (V) region that encodes the antigen binding site as well as diversity (D), joining (J) and constant (C) regions while V, J, and C loci encode light chain protein [reviewed in 231. These loci are separated from one another in the germ line by intervening sequences and are joined together during B cell differentiation by an enzymatic machinery that excises the noncoding regions and juxtaposes the coding elements [24]. Initial rearrangement events occur in the heavy chain genes and involve joining of a D to a J, locus. Subsequently, one of many V, region genes is juxtaposed to the rearranged DJ, segment. The rearranged heavy chain gene is then transcribed, and posttranscriptional processing removes noncoding sequences present between the rearranged V,-(D)-J, segment and the Cplocus, resulting in formation of a VH-(D)-J,-Cv transcript that is translated into p heavy chain protein. Heavy chain protein is believed to signal the initiation of light chain gene rearrangements [25]. The latter process involves joining a V, segment to one of five light chain gene J, segments. As with the heavy chain, the rearranged kappa gene is transcribed, and post-transcriptional processing results in the formation of a V,-J,-C, transcript that is then translated. Expression of the assembled Ig molecule on the cell surface marks the transition from a pre-B cell to a newly formed B lymphocyte. Bone Marrow Structure and Its Analysis The cellular and molecular events described above occur in association with sessile supporting elements, collectively referred to as stromal cells, that form a three dimensional supporting framework in the intersinusoidal spaces of the medullary cavity [reviewed in 26, 271. It is now evident that stromal cells and soluble mediators produced by them affect the proliferation and differentiation of hemopoietic cells, and there has been considerable effort aimed at identifying these secreted products. Such efforts have been possible, in part, due to the development of in vitro methods for growing stromal cells and hemopoietic cells of various lineages. The different longterm bone marrow culture systems (LTBMC) have been particularly important in this regard. The common feature of LTBMC is the formation of an adherent stromal cell layer on which the sustained growth of blood cells is dependent. There are two main types of cultures, which differ in the conditions under which they are maintained, and these are optimal for B lymphopoiesis or myelopoiesis [re-
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Regulation of B Cell Differentiation By Bone Marrow Strornal Cells
viewed in 281. The lymphoid cultures described by Whirlock and Wirte [29] selectively maintain the growth of B lymphocytes and their precursors, and in established cultures, mature myeloid cells and their progenitors are not present. Lymphoid cells in these cultures express the full range of phenotypic markers described above. A second type of myeloid long-term culture was developed by Dexter and colleagues [30]. The mature cells in these cultures are neutrophils and macrophages, and most myeloid and erythroid progenitors are also present. Cells that express Ig or the B220 antigen are not maintained, but lymphoid precursors are present. This can be demonstrated in vitro; upon transfemng an established Dexter culture to the Whitlock-Witte culture conditions, myelopoiesis ceases and B cell development initiates [31-321. Because blood cell production in the cultures is dependent upon stromal cells that contribute to the adherent layer, the long-term systems have provided a means to study how environmental components regulate B lymphopoiesis. Much of this work has relied upon the analysis of cloned populations of stromal cells isolated from the adherent layer of primary cultures [reviewed in 26,271. Several laboratories have generated stromal cell lines [33-381, and there are indications that they exhibit a heterogeneity based on the stages of B cell development supported (Fig. I). For example, one group of stromal cells, such as S17 [33,39,40], AC6 [35], and ST2 [34, 411 support maturation of B22@ precursors to at least the Cpexpressing pre-B cell stage. Distinct signals are apparently required for the maturation of precursors to B220 expression, since other lines, such as PA6, support self-renewal but not the maturation of B220- cells in culture [34]. These early acting stromal cell lines do not appear to be identical, however. Surface Ig positive B cells do not develop in response to S 17 [40] or AC6 [35] signals, although preliminary data indicate that kappa light chain genes are rearranged in the S17-supported preB cells. The ST2 line differs from S17 and AC6 in that cells mature to surface Ig expression [34, 411. These differences imply the existence of additional, relatively late-acting (Fig. 1) regulatory signals that allow the pre-B to B cell transition to occur, and results with other stromal cells support this premise [38,40]. For example, the S10 line can not potentiate the development of pre-B cells from B220- precursors [40], but conditioned medium from the line potentiates the formation of lymphoid colonies in which sIg+cells develop from slg- progenitors [33,40]. It is unclear whether these observations indicate that there are distinct stromal cell populations in the bone mar-
row, but the generation of stromal cell lines with differential support capabilities implies the existence of multiple signals that act at defined points in the B cell developmental hierarchy.
Regulation of B Cell Development by Stromal Cells The primary LTBMC and the stromal cell lines have been extremely valuable for studies of how environmental components regulate primary B lymphopoiesis. One means by which such effects are mediated is via direct cell-cell interactions with developing blood cells. This interaction is obvious from examination of the cultures in which lymphoid cells are intimately associated with the stroma. There is evidence to suggest that such direct cell-cell interactions may be required for long-term growth/survival of B220- precursors [22, 421. Two groups have provided evidence that developing B lymphocytes express the VLA-4 integrin that interacts with a ligand on stromal cells that appears to be VCAM-I [43,44]. Hyaluronate present on stromal cells has also been implicated in mediating stromal cell-lymphocyte interactions [45]. Another means by which stromal cells affect proliferation and differentiation of B lineage cells is via the secretion of soluble mediators. Considerable progress in identification of stromal cell factors has been made, and major efforts are underway to define the stages of B cell differentiation on which they act. The genes encoding two of these factors, interleukin 7 (IL-7) [46] and kir-ligand [47-501, have recently been cloned and the effects of the recombinant molecules on B cell production studied. The primary action of IL-7 is on stimulation of cell proliferation, although there are reports that IL-7 may act to potentiate the maturation of pre-B cells into sIg-expressing lymphocytes [4 11. Since little IL7 proliferation by B220- cells is observed, cells appear to become IL-7 responsive following expression of the B220 antigen, and once surface Ig is expressed, responsiveness is lost [5I]. Mutations at the steel locus of the mouse are known to result in a variety of hemopoietic abnormalities [52], and mice with such defects have been considered to have a stromal cell or microenvironmental defect. The gene product of the steel locus, variously referred to as kit-ligand, mast cell growth factor, or stem cell factor, was recently cloned and demonstrated to encode a cytokine which has a wide range of effects on the growth of cells from multiple
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hemopoietic lineages [47-501. With regards to B lymphopoiesis, the most dramatic effect of the factor, referred to herein as kit-ligand (KL), is its marked synergy with IL-7 in stimulating proliferation of B cell progenitors [53]. Most stromal cell lines secrete KL, and this activity appears to explain the observation that supematants from S 17 stromal cells synergize with IL-7 to stimulate pre-B cell growth (Blood, in press). While IL-7 and KL are able to synergistically stimulate proliferation of B lineage cells, neither cytokine appears to provide the early acting signal (Fig. 1) in which B220- cells differentiate into pre-B cells. When the factors are used individually or mixed together at various concentrations with bone marrow cells depleted of B220' cells, no B220' or Ig-expressing cells develop (Blood, in press). Since conditioned medium from stromal cells mediates this effect [39], this implies that there are additional stromal cell factors that potentiate the maturation of B220- cells into B220' ones. Such factors have not been cloned, but initial biochemical characterization suggests the involvement of molecules of approximately 6,00010,000 MW [39].
A Working Model of B Cell Development Much of our understanding of how B lymphopoiesis is regulated has resulted from development of techniques for establishing LTBMC that support lymphoid development. The availability of these systems has made it possible to isolate stromal cell lines, to test their B cell support capabilities, and to identify and clone stromal cell-derived factors that regulate primary B cell production. As a result of these efforts, it is possible to suggest a working model of B cell development mediated by stromal cells and their products. Studies from several laboratories have established that B cell differentiation can be divided into several stages based upon the particular stromal cell and soluble mediator(s) involved. Among the earliest events in B lymphopoiesis is the differentiation of B220- precursors into B220' progenitors, and this event can be supported by stromal cells [33-351. Although direct stromal cell contact may be needed for long-term survival of B220- cells [22, 34,421, maturation of these precursors into Cpexpressing pre-B cells can be potentiated by soluble mediators [39]. The factors that mediate this maturational step have not been cloned, and previously defined cytokines including IL-7 and/or KL do not appear to play a role (Blood, in press).
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Cell proliferation is an obligate event in B lymphocyte development, but the stromal cell cytokines that potentiate the development of B220' cells from theirprecursors donot stimulate significant cell growth [39]. Therefore, molecules such as IL-7 may have evolved in order to expand the number of B cell progenitors and/or pre-B cells. Several lines of evidence would indicate that cells do not become IL-7 responsive until they express the B220 antigen, and data from two recent studies suggest that the cells most sensitive to the IL-7 proliferative stimulus are those in which D-J, rearrangements have occurred [22, 541. Interestingly, there is evidence that contact of developing lymphocytes with stromal cells may stimulate IL7 production by the latter population [55]. The synergistic interactions between IL-7 and KL provide a means to further amplify pre-B cells and imply that target cells for the latter factor exist at relatively late stages in the B cell developmental pathway. It is important to note two caveats that apply to this model. First, a recent report has raised the possibility that those progenitors/pre-B cells that proliferate in respone to KL and IL-7 may be a subset of B lineage cells [56]. Another point is that the effects of KL on B cell progenitors does not exclude the possibility that more immature B cell precursors that have not yet expressed B220 are also sensitive to its effects. The terminal event in primary B cell production is the transition of pre-B cells into surface Ig-expressing B lymphocytes. This also appears to be a regulated process in which stromal cells and their secreted products are involved. For example, medium conditioned by the SCL 160 and S10 stromal cell lines allows the development of sIg-expressing B cells from pre-B cells [38, 401. The SCL 160 activity is due to an IL-4-like molecule, while the S10 factor(s) has not been identified. As noted, it has been reported that IL-7 may also potentiate the pre-B to B cell transition [41]. The present studies have focused on positive regulation, but there is emerging evidence that B cell development may be sensitive to negative regulatory influences in culture and in vivo [57]. For example, recent observations from one of our laboratories suggest that T lymphocytes interact with stromal cells and play a role in suppressing B cell generation (unpublished observation). Various cytokines, including transforming growth factor-p[51], IL-1 [58,59) granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF, respectively) [59], may also inhibit primary B lymphopoiesis. Treatment of mice with GM-CSF systemically resulted in reduced numbers of pre-B cells in the marrow and
Regulation of B Cell Differentiation By Bone Marrow Stromal Cells
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recovery occurred only after cessation of adrninistration [60].Other studies have shown that treatment of stromal cells with G- or GM-CSF abrogates their ability to support pre-B cell differentiation and suggested that this negative effect of CSF may b e mediated by alterations of stromal cell function (unpublished observation). Further efforts aimed at characterizing these effects and in determining how the interaction of stirnulatory and inhibitory signals results in regulated, steady state B lymphocyte production should contribute substantially to our understanding of primary events in the development of imrnunocompetence.
References 1
2
3 4
5
6
7
8
9
10
11
12
1.7
Kincade PW. Experimental models for understanding B lymphocyte formation. Adv Immunol 1987;41:181267. Paige CJ, Kincade PW, Moore MAS, Lee G. The fate of fetal and adult B-cell progenitors grafted into immunodeficient CBA/N mice J Exp Med 1979;150:548563. Melchers F. Murine embryonic B lymphocyte development in the placenta. Nature 1979;277:219-221. Owen JJT, Cooper MD, Raff MC. In vitro generation of B lymphocytes in mouse fetal liver, a mammalian “bursa equivalent” Nature 1974;249:361-363. Phillips RA, Melchers F. Appearance of functional lymphocytes in fetal liver. J Immunol 1976;117:10991 103. Rosse C. Small lymphocyte and transitional cell populations of the bone marrow; their role in the mediation of immune and hemopoietic progenitor cell functions. Int Rev Cytol 1976;45:155-290. Landreth KS, Rosse C, Clagett J. Myelogenous production and maturation of B lymphocytes in the mouse. J Immunol 1981 ; 127:2027-2034. Opsteltin D, Osmond DG. Pre-B cells in the bone marrow: immunofluorescence stathmokinetic studies of the proliferation of cytoplasmic pchain bearing cells in normal mice. J Immunol 1983; 1312635-2640. Abramson S, Miller RG, Phillips RA. The identification in adult bone marrow of pluirpotent and restricted stem cells of the myeloid and lymphoid systems. J Exp Med 1977;145:1567-1579. Keller G, Snodgrass R. Life span of multipotential hematopoietic stem cells i n vivo. J Exp Med I990 171:1407-141 8. Fulop GM, Phillips RA. Use of scid mice to identify and quantitate lymphoid-restricted stem cells in longterm bone marrow cultures. Blood 1989;74:1537-1544. Kincade PW, Lee G, Watanabe T, Sun L, Scheid MP. Antigens displayed on murine B lymphocyte precursors in vitro. J Immunol 1981;127:22626-2268. Coffman RL. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B
cell development. Immunol Rev 1982;69:5-23. 14 Sakaguchi N, Melchers F. Lambda 5, a new lightchain related locus selectively expressed in pre-B lymphocytes. Nature 1986;324:579-582. 15 Pillai S , Baltimore D. Formation of disulphide-linked mu 2 omega 2 tetramers in pre-B cells by the 18K omega-immunoglobulin light chain. Nature 1987;329: 172-174. 16 Tsubata T, Reth M. The products of pre-B cell-specific genes (lamdba 5 and V preB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface. J Exp Med 1990;172:973-976. 17 Kudo A, Melchers F. A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be seletively expressed in pre-B lymphocytes. EMBO J 1987;6:2267-2272. 18 Reth MG, Ammirati P, Jackson S, Alt FW.Regulated progression of a cultured pre-B cell line to the B cell stage. Nature 1985;317:353-355. 19 Mcguire KL, Vitetta ES. Kappdambda shifts do not occur during maturation of murine B cells. J Immunol I98 1; 127:1670-1673. 20 Cooper MD, Mulvaney D, Coutinho A. Cazenae P. A novel cell surface molecule on early B-lineage cells. Nature 1986;321:616-618. 21 Wu Q, Tidrnarsh GF, Welch PA, Pierce JH, Weissman IL, Cooper MD. The early B lineage antigen BP- I and transformation-associated antigen 6C3 are on the same molecule. J Immunol 1989;143:3303-3308. 22 Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K. Resolution and characterization of proB and pre-pro B cell stages in normal mouse bone marrow. J Exp Med 1991;173:1213-1225. 23 Yancopoulos GD, Alt FW. Regulation of the assembly of variable-region gene expression. Ann Rev Immunol 1986;4:339-368. 24 Oettinger MA, Schatz DG, Gorka C. Baltimore D. RAG-I and RAG-2, adjacent genes that synergistically activate V(D)J recombination, Science l988;248: 15 17-1523. 25 Reth M, Petrac E, Wiese P, Lobel L, Alt FW.Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J 1987;6:3299-3305. 26 Kincade PW, Lee G, Pietrangeli CE, Hayashi SI, Gimble JM. Cells and molecules that regulate B lymphopoiesis in bone marrow. Annu Rev Immunol 1989;7:111-143. 27 Dorshkind K. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol 19908:111-137. 28 Dorshkind K, Witte ON. Long-term murine hemopoietic cultures as model systems for analysis of B lymphocyte differentiation. Curr Top Microbiol Immunol 1987; 135~24-41. 29 Whitlock CA, Witte ON. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc Natl Acad Sci USA 1982;79:3608-3612. 30 Dexter TM, Allen TD, Lajtha LG. Conditions control-
DorshkindLandreth
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
ling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977;91:334-344. Dorshkind K. In vitro differentiation of B lymphocytes from immature hemopoietic precursors present in long-term bone marrow cultures. J Immunol 1986;1361422-429. Denis KA, Witte ON. In vitro development of B lymphocytes from long-term cultured precursor cells. Proc Natl Acad Sci USA 1986;83:441-445. Collins LS, Dorshkind K. A suomal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J Immunol 1987;138:1082-1087. Nishikawa SI, Ogawa M, Nishikawa S, Kunisada T, Kodama H. B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation. Eur J Immunol 1988;18:1767-1771. Whitlock CA, Tidmarsh GF, Muller-Sieburg C, Weissman IL. Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule. Cell 1987;48:10091021. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON. A single bone marrow-derived stromal cell type suppons the in vitro growth of early lymphoid and myeloid cells. Cell 1987; 48:997-1007. Pietrangeli CE, Hayashi SI, Kincade PW. Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors. Eur J Immunol 1988;18:863-872. King AG, Wierda D, Landreth KS. Bone marrow stroma1 cell regulation of B-lymphopoiesis I. The role of macrophages, IL- I , and IL-4 in pre-B cell maturation. J Immunol 1988;141:2016-2026. Landreth KS, Dorshkind K. Pre-B cell generation potentiated by soluble factors from a bone marrow stroma1 cell line. J Immunol 1988;140:845-852. Henderson AJ, Johnson A. Dorshkind K. Functional characterization of two stromal cell lines that support B lymphopoiesis. J Immunol I990 145:423-428. Gunji Y, Sudo T, Suda J, Yamaguchi Y, Nakauchi H, Nishikawa SI, Yanai N, Obinata M, Yanagisawa M, Miura Y, Suda T. Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7. Blood 1991;77:2612-2617. Kiemey PC, Dorshkind K. B lymphocyte precursors and myeloid progenitors survive in diffusion chamber culture but B cell differentiation requires close association with stromal cells. Blood 1987;70:1418-1424. Miyake K, Weissman IL, Greenberger JS, Kincade PW. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med I99 1; 173:599-607. Kina T, Majumdar AS, Heimfeld S, Kaneshima H, Holzmann B, Katsura Y, Weissman IL. Identification of a 107-kD glycoprotein than mediated adhesion between stromal cells and hematolymphoid cells. J Exp Med 1991;173:373-381. Miyake K, Underhill CB, Lesley J, Kincade PW. Hyaluronate can function as a cell adhesion molecule
17
46
47
48
49
50
51
52 53
54
55
56
57
58
59
60
and CD44 participates in hyaluronate recognition. J Exp Med 1990;172:69-75. Namen AJ3, Lupton S, Hjemld K, Wagnall J, Mochizuki DY, Schmierer A, Mosley B , March C, Urdal D, Gillis S, Cosman D, Goodwin RG. Stimulation of Bcell progenitors by cloned murine interleukin-7. Nature 1988;333:571-573. Williams DE, Eisenman J, Baird A, et al. Identification of a ligand for the c-kit proto-oncogene. Cell 1990;63:167-174. Flanagan JG, Leder P. The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell 1990;63:185-194. Zsebo KM, Wypych J, McNiece IK, et al. Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liverconditioned medium. Cell 1990;63:195-201. Huang E, Nocka K, Beier DR, et al. The hematopoietic growth factor KL is encoded at the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 199063:225-233. Lee G. Namen AE,Gillis S , Ellingsworth LR, Kincade PW. Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-,!?. J Immunol 1989;142:3875-3883. Russel ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genetics 1979;20:357-459. McNeice IK, Langley KE, Zsebo KM. The role of recombinant stem cell factor in early B cell development. J Immunol 1991; 146:3785-3790. Rolink A, Kudo A, Karasuyama H, Kikuchi Y,Melchers F. Long-term proliferating early pre-B cell lines and clones with the potential to develop to surface Igpositive, mitogen reactive B cells in vitro and in vivo. EMBO J 1991;10:327-336. Suda T, Ito M, Ogawa Y,et al. Interleukin 7 production and function in stromal cell-dependent B cell development. J Exp Med 1989;170:333-338. Ogawa M, Matsuzaki Y,Nishikawa S, et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63-72. Billips LG, Petitte D, Landreth KS. Bone marrow stroma1 cell regulation of B lymphopoiesis: interleukin- 1 (IL-I) and IL-4 regulate stromal cell support of pre-B cell production in vitro. Blood 1990;75:611-619. Suda T, Okada S, Suda J, et al. A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B-cell colony formation and an inhibitory effect of IL-la. Blood 1989; 74:1936-1941. Dorshkind K. IL-1 inhibits B cell differentiation in long term bone marrow cultures. J Immunol 1988;141:53 1-538. Dorshkind K. In vivo administration of recombinant granulocyte-macrophage colony-stimulating factor results in a reversible inhibition of primary B lymphopoiesis. J Immunol 1991; 146:4202-4208.
Regulation of B Cell Differentiation By Bone Marrow Stromal Cells Kenneth Dorshkind," Kennerh S . Landrethh aDivision of Biomedical Sciences, University of California, Riverside, California. USA; bDepartmentof Microbiology and lmmunology and Mary Babb Randolph Cancer Center, West Virginia University School of Medicine, Morgantown, West Virginia, USA Key Words. B Lymphocytes Cytokines Hemopoiesis Immunoglobulin Long-term bone marrow culture Stromal cells
Abstract. B lymphocyte development occurs in the intersinusoidal spaces of bone marrow in association with a sessile population of stromal cells. Development of long-term bone marrow culture systems that support B lymphopoiesis has allowed the isolation of stromal cells that support that process and permitted analysis of their role in controlling the growth and differentiation of B lineage cells in culture. In addition to direct interactions with developing lymphocytes, stromal cells secrete a variety of cytokines that affect lymphocyte growth and differentiation, and many of these molecules have been expressed in recombinant form. These relatively recent advances have made it possible to formulate a working model of the cells and molecules involved in regulating primary B cell production.
Introduction Mammalian B lymphocyte development is an antigen-independent process in which immature hemopoietic precursors become committed to the lymphoid developmental pathway and mature into immunoglobulin (Ig) expressing B lymphocytes competent to mediate a humoral immune response [I]. Multiple regulatory controls are operative during B lymphopoiesis and their failure may contribute to both neoplasias and immunodeficiencies that involve cells at various stages in the B cell developmental pathway. Consequently, obtaining a better understanding of how B cell differentiation is controlled has tremendous practical relevance. In addition, studies of B cell differentiation provide a model for analysis of Correspondence: Dr. Kenneth Dorshkind, Division of Biomedical Sciences, University of California, Riverside, CA 92521-0121, USA. Received August 29, 1991; accepted for publication August 29, 1991. 07 37-1454/92/$2.00/0OAlphaMed Press
how such parameters as cell-cell interactions, soluble mediators, and cell-matrix interactions control growth and differentiation during mammalian development.
B Cell Differentiation in the Bone Marrow During fetal life the yolk sac [2], placenta [3], and liver [4, 51 contain B lineage cells, but in normal adult mammals, primary B cell production takes place exclusively in the bone marrow in parallel with erythropoiesis and myelopoiesis [ 1,6]. The daily rate of B cell production is extremely high, and studies that have measured the proliferative status, renewal rate, and intercompartmental transit of murine B lineage cells have estimated that up to 5 X lo7 B lymphocytes are generated per day [7,8]. B lymphocytes develop from immature hemopoietic precursors [8] which could include the p h i potent hemopoietic stem cell. This cell is capable of generating all blood cells, including B cells [9, lo], and continual input from that compartment may be necessary to maintain steady state B lymphopoiesis. Another possibility is that a bipotential lymphoid precursor capable of generating T and B cells exists [ 111, but definitive identification of these cells has not been made. Despite these uncertainties, it has been possible to resolve more mature, committed stages in the B cell developmental pathway through the use of antibodies that recognize cell surface and cytoplasmic determinants [ I ] (Fig. 1). Committed B cell progenitors in murine bone marrow can be recognized based on their expression of the 220,000 MW form of the Ly5,,,, antigen [ 12. 131. Subsequently, immunoglobulin (Ig) p heavy chain appears in the cytoplasm of pre-B cells and this indicates that productive heavy chain rearrangements have occurred. There is evidence that membrane p can be
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BCELL
Fig. 1. Summary of primary B cell differentiation. Ig gene rearrangement events are placed at the cellular stage at which they first occur. The ability of different stromal cells to support distinct stages of B lymphopoiesis is indicated using the S17 and S10 lines as examples. Placement of stromal cell line S17 in this diagram is consistent with its role as an early acting stromal cell that allows B220- precursors to mature to at least the pre-B cell stage, whereas placement of the S10 line indicates its role as a late acting stromal cell type which stimulates expression of surface Ig. Dashed lines indicate that the placement of particular developmental events and regulatory signals is an approximation. For example, while the placement of KL in the figure is based on observations that the factor synergizes with IL7 to stimulate proliferation of progenitor/pre-B cells, the dashed line to the left of the kit-ligand box is meant to indicate that an effect(s) of KL on more immature cells in the B lineage is not excluded. Similarly, the dashed line to the right of IL-7 indicates a role for the factor in the pre-B to B cell transition has been suggested.
expressed on the cell surface of pre-B cells in association with nonconventional light chain molecules [14-171. The final stage of primary B lymphopoiesis is the expression of kappa or lambda light chain protein [ 181. Kappa genes are expressed predominantly in the mouse, while in humans the kappa:lambda ratio is approximately 7:3 [19]. In addition to the phenotypic determinants described above, there are additional cell surface molecules that have been used to characterize B lineage cells. Another cell surface antigen, referred to as BP-1 or 6C3 [20-211, also appears on murine B lineage cells, and a recent report indicates it is expressed subsequent to the appearance of the B220 molecule and before surface IgM [ 2 2 ] . The status of Ig gene rearrangements in B lineage cells provides a molecular means of defining stages of differentiation. The Ig heavy chain is as-
sembled from distinct genetic loci that include a variable (V) region that encodes the antigen binding site as well as diversity (D), joining (J) and constant (C) regions while V, J, and C loci encode light chain protein [reviewed in 231. These loci are separated from one another in the germ line by intervening sequences and are joined together during B cell differentiation by an enzymatic machinery that excises the noncoding regions and juxtaposes the coding elements [24]. Initial rearrangement events occur in the heavy chain genes and involve joining of a D to a J, locus. Subsequently, one of many V, region genes is juxtaposed to the rearranged DJ, segment. The rearranged heavy chain gene is then transcribed, and posttranscriptional processing removes noncoding sequences present between the rearranged V,-(D)-J, segment and the Cplocus, resulting in formation of a VH-(D)-J,-Cv transcript that is translated into p heavy chain protein. Heavy chain protein is believed to signal the initiation of light chain gene rearrangements [25]. The latter process involves joining a V, segment to one of five light chain gene J, segments. As with the heavy chain, the rearranged kappa gene is transcribed, and post-transcriptional processing results in the formation of a V,-J,-C, transcript that is then translated. Expression of the assembled Ig molecule on the cell surface marks the transition from a pre-B cell to a newly formed B lymphocyte. Bone Marrow Structure and Its Analysis The cellular and molecular events described above occur in association with sessile supporting elements, collectively referred to as stromal cells, that form a three dimensional supporting framework in the intersinusoidal spaces of the medullary cavity [reviewed in 26, 271. It is now evident that stromal cells and soluble mediators produced by them affect the proliferation and differentiation of hemopoietic cells, and there has been considerable effort aimed at identifying these secreted products. Such efforts have been possible, in part, due to the development of in vitro methods for growing stromal cells and hemopoietic cells of various lineages. The different longterm bone marrow culture systems (LTBMC) have been particularly important in this regard. The common feature of LTBMC is the formation of an adherent stromal cell layer on which the sustained growth of blood cells is dependent. There are two main types of cultures, which differ in the conditions under which they are maintained, and these are optimal for B lymphopoiesis or myelopoiesis [re-
14
Regulation of B Cell Differentiation By Bone Marrow Strornal Cells
viewed in 281. The lymphoid cultures described by Whirlock and Wirte [29] selectively maintain the growth of B lymphocytes and their precursors, and in established cultures, mature myeloid cells and their progenitors are not present. Lymphoid cells in these cultures express the full range of phenotypic markers described above. A second type of myeloid long-term culture was developed by Dexter and colleagues [30]. The mature cells in these cultures are neutrophils and macrophages, and most myeloid and erythroid progenitors are also present. Cells that express Ig or the B220 antigen are not maintained, but lymphoid precursors are present. This can be demonstrated in vitro; upon transfemng an established Dexter culture to the Whitlock-Witte culture conditions, myelopoiesis ceases and B cell development initiates [31-321. Because blood cell production in the cultures is dependent upon stromal cells that contribute to the adherent layer, the long-term systems have provided a means to study how environmental components regulate B lymphopoiesis. Much of this work has relied upon the analysis of cloned populations of stromal cells isolated from the adherent layer of primary cultures [reviewed in 26,271. Several laboratories have generated stromal cell lines [33-381, and there are indications that they exhibit a heterogeneity based on the stages of B cell development supported (Fig. I). For example, one group of stromal cells, such as S17 [33,39,40], AC6 [35], and ST2 [34, 411 support maturation of B22@ precursors to at least the Cpexpressing pre-B cell stage. Distinct signals are apparently required for the maturation of precursors to B220 expression, since other lines, such as PA6, support self-renewal but not the maturation of B220- cells in culture [34]. These early acting stromal cell lines do not appear to be identical, however. Surface Ig positive B cells do not develop in response to S 17 [40] or AC6 [35] signals, although preliminary data indicate that kappa light chain genes are rearranged in the S17-supported preB cells. The ST2 line differs from S17 and AC6 in that cells mature to surface Ig expression [34, 411. These differences imply the existence of additional, relatively late-acting (Fig. 1) regulatory signals that allow the pre-B to B cell transition to occur, and results with other stromal cells support this premise [38,40]. For example, the S10 line can not potentiate the development of pre-B cells from B220- precursors [40], but conditioned medium from the line potentiates the formation of lymphoid colonies in which sIg+cells develop from slg- progenitors [33,40]. It is unclear whether these observations indicate that there are distinct stromal cell populations in the bone mar-
row, but the generation of stromal cell lines with differential support capabilities implies the existence of multiple signals that act at defined points in the B cell developmental hierarchy.
Regulation of B Cell Development by Stromal Cells The primary LTBMC and the stromal cell lines have been extremely valuable for studies of how environmental components regulate primary B lymphopoiesis. One means by which such effects are mediated is via direct cell-cell interactions with developing blood cells. This interaction is obvious from examination of the cultures in which lymphoid cells are intimately associated with the stroma. There is evidence to suggest that such direct cell-cell interactions may be required for long-term growth/survival of B220- precursors [22, 421. Two groups have provided evidence that developing B lymphocytes express the VLA-4 integrin that interacts with a ligand on stromal cells that appears to be VCAM-I [43,44]. Hyaluronate present on stromal cells has also been implicated in mediating stromal cell-lymphocyte interactions [45]. Another means by which stromal cells affect proliferation and differentiation of B lineage cells is via the secretion of soluble mediators. Considerable progress in identification of stromal cell factors has been made, and major efforts are underway to define the stages of B cell differentiation on which they act. The genes encoding two of these factors, interleukin 7 (IL-7) [46] and kir-ligand [47-501, have recently been cloned and the effects of the recombinant molecules on B cell production studied. The primary action of IL-7 is on stimulation of cell proliferation, although there are reports that IL-7 may act to potentiate the maturation of pre-B cells into sIg-expressing lymphocytes [4 11. Since little IL7 proliferation by B220- cells is observed, cells appear to become IL-7 responsive following expression of the B220 antigen, and once surface Ig is expressed, responsiveness is lost [5I]. Mutations at the steel locus of the mouse are known to result in a variety of hemopoietic abnormalities [52], and mice with such defects have been considered to have a stromal cell or microenvironmental defect. The gene product of the steel locus, variously referred to as kit-ligand, mast cell growth factor, or stem cell factor, was recently cloned and demonstrated to encode a cytokine which has a wide range of effects on the growth of cells from multiple
DorshkindLandreth
hemopoietic lineages [47-501. With regards to B lymphopoiesis, the most dramatic effect of the factor, referred to herein as kit-ligand (KL), is its marked synergy with IL-7 in stimulating proliferation of B cell progenitors [53]. Most stromal cell lines secrete KL, and this activity appears to explain the observation that supematants from S 17 stromal cells synergize with IL-7 to stimulate pre-B cell growth (Blood, in press). While IL-7 and KL are able to synergistically stimulate proliferation of B lineage cells, neither cytokine appears to provide the early acting signal (Fig. 1) in which B220- cells differentiate into pre-B cells. When the factors are used individually or mixed together at various concentrations with bone marrow cells depleted of B220' cells, no B220' or Ig-expressing cells develop (Blood, in press). Since conditioned medium from stromal cells mediates this effect [39], this implies that there are additional stromal cell factors that potentiate the maturation of B220- cells into B220' ones. Such factors have not been cloned, but initial biochemical characterization suggests the involvement of molecules of approximately 6,00010,000 MW [39].
A Working Model of B Cell Development Much of our understanding of how B lymphopoiesis is regulated has resulted from development of techniques for establishing LTBMC that support lymphoid development. The availability of these systems has made it possible to isolate stromal cell lines, to test their B cell support capabilities, and to identify and clone stromal cell-derived factors that regulate primary B cell production. As a result of these efforts, it is possible to suggest a working model of B cell development mediated by stromal cells and their products. Studies from several laboratories have established that B cell differentiation can be divided into several stages based upon the particular stromal cell and soluble mediator(s) involved. Among the earliest events in B lymphopoiesis is the differentiation of B220- precursors into B220' progenitors, and this event can be supported by stromal cells [33-351. Although direct stromal cell contact may be needed for long-term survival of B220- cells [22, 34,421, maturation of these precursors into Cpexpressing pre-B cells can be potentiated by soluble mediators [39]. The factors that mediate this maturational step have not been cloned, and previously defined cytokines including IL-7 and/or KL do not appear to play a role (Blood, in press).
15
Cell proliferation is an obligate event in B lymphocyte development, but the stromal cell cytokines that potentiate the development of B220' cells from theirprecursors donot stimulate significant cell growth [39]. Therefore, molecules such as IL-7 may have evolved in order to expand the number of B cell progenitors and/or pre-B cells. Several lines of evidence would indicate that cells do not become IL-7 responsive until they express the B220 antigen, and data from two recent studies suggest that the cells most sensitive to the IL-7 proliferative stimulus are those in which D-J, rearrangements have occurred [22, 541. Interestingly, there is evidence that contact of developing lymphocytes with stromal cells may stimulate IL7 production by the latter population [55]. The synergistic interactions between IL-7 and KL provide a means to further amplify pre-B cells and imply that target cells for the latter factor exist at relatively late stages in the B cell developmental pathway. It is important to note two caveats that apply to this model. First, a recent report has raised the possibility that those progenitors/pre-B cells that proliferate in respone to KL and IL-7 may be a subset of B lineage cells [56]. Another point is that the effects of KL on B cell progenitors does not exclude the possibility that more immature B cell precursors that have not yet expressed B220 are also sensitive to its effects. The terminal event in primary B cell production is the transition of pre-B cells into surface Ig-expressing B lymphocytes. This also appears to be a regulated process in which stromal cells and their secreted products are involved. For example, medium conditioned by the SCL 160 and S10 stromal cell lines allows the development of sIg-expressing B cells from pre-B cells [38, 401. The SCL 160 activity is due to an IL-4-like molecule, while the S10 factor(s) has not been identified. As noted, it has been reported that IL-7 may also potentiate the pre-B to B cell transition [41]. The present studies have focused on positive regulation, but there is emerging evidence that B cell development may be sensitive to negative regulatory influences in culture and in vivo [57]. For example, recent observations from one of our laboratories suggest that T lymphocytes interact with stromal cells and play a role in suppressing B cell generation (unpublished observation). Various cytokines, including transforming growth factor-p[51], IL-1 [58,59) granulocyte and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF, respectively) [59], may also inhibit primary B lymphopoiesis. Treatment of mice with GM-CSF systemically resulted in reduced numbers of pre-B cells in the marrow and
Regulation of B Cell Differentiation By Bone Marrow Stromal Cells
16
recovery occurred only after cessation of adrninistration [60].Other studies have shown that treatment of stromal cells with G- or GM-CSF abrogates their ability to support pre-B cell differentiation and suggested that this negative effect of CSF may b e mediated by alterations of stromal cell function (unpublished observation). Further efforts aimed at characterizing these effects and in determining how the interaction of stirnulatory and inhibitory signals results in regulated, steady state B lymphocyte production should contribute substantially to our understanding of primary events in the development of imrnunocompetence.
References 1
2
3 4
5
6
7
8
9
10
11
12
1.7
Kincade PW. Experimental models for understanding B lymphocyte formation. Adv Immunol 1987;41:181267. Paige CJ, Kincade PW, Moore MAS, Lee G. The fate of fetal and adult B-cell progenitors grafted into immunodeficient CBA/N mice J Exp Med 1979;150:548563. Melchers F. Murine embryonic B lymphocyte development in the placenta. Nature 1979;277:219-221. Owen JJT, Cooper MD, Raff MC. In vitro generation of B lymphocytes in mouse fetal liver, a mammalian “bursa equivalent” Nature 1974;249:361-363. Phillips RA, Melchers F. Appearance of functional lymphocytes in fetal liver. J Immunol 1976;117:10991 103. Rosse C. Small lymphocyte and transitional cell populations of the bone marrow; their role in the mediation of immune and hemopoietic progenitor cell functions. Int Rev Cytol 1976;45:155-290. Landreth KS, Rosse C, Clagett J. Myelogenous production and maturation of B lymphocytes in the mouse. J Immunol 1981 ; 127:2027-2034. Opsteltin D, Osmond DG. Pre-B cells in the bone marrow: immunofluorescence stathmokinetic studies of the proliferation of cytoplasmic pchain bearing cells in normal mice. J Immunol 1983; 1312635-2640. Abramson S, Miller RG, Phillips RA. The identification in adult bone marrow of pluirpotent and restricted stem cells of the myeloid and lymphoid systems. J Exp Med 1977;145:1567-1579. Keller G, Snodgrass R. Life span of multipotential hematopoietic stem cells i n vivo. J Exp Med I990 171:1407-141 8. Fulop GM, Phillips RA. Use of scid mice to identify and quantitate lymphoid-restricted stem cells in longterm bone marrow cultures. Blood 1989;74:1537-1544. Kincade PW, Lee G, Watanabe T, Sun L, Scheid MP. Antigens displayed on murine B lymphocyte precursors in vitro. J Immunol 1981;127:22626-2268. Coffman RL. Surface antigen expression and immunoglobulin gene rearrangement during mouse pre-B
cell development. Immunol Rev 1982;69:5-23. 14 Sakaguchi N, Melchers F. Lambda 5, a new lightchain related locus selectively expressed in pre-B lymphocytes. Nature 1986;324:579-582. 15 Pillai S , Baltimore D. Formation of disulphide-linked mu 2 omega 2 tetramers in pre-B cells by the 18K omega-immunoglobulin light chain. Nature 1987;329: 172-174. 16 Tsubata T, Reth M. The products of pre-B cell-specific genes (lamdba 5 and V preB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface. J Exp Med 1990;172:973-976. 17 Kudo A, Melchers F. A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be seletively expressed in pre-B lymphocytes. EMBO J 1987;6:2267-2272. 18 Reth MG, Ammirati P, Jackson S, Alt FW.Regulated progression of a cultured pre-B cell line to the B cell stage. Nature 1985;317:353-355. 19 Mcguire KL, Vitetta ES. Kappdambda shifts do not occur during maturation of murine B cells. J Immunol I98 1; 127:1670-1673. 20 Cooper MD, Mulvaney D, Coutinho A. Cazenae P. A novel cell surface molecule on early B-lineage cells. Nature 1986;321:616-618. 21 Wu Q, Tidrnarsh GF, Welch PA, Pierce JH, Weissman IL, Cooper MD. The early B lineage antigen BP- I and transformation-associated antigen 6C3 are on the same molecule. J Immunol 1989;143:3303-3308. 22 Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K. Resolution and characterization of proB and pre-pro B cell stages in normal mouse bone marrow. J Exp Med 1991;173:1213-1225. 23 Yancopoulos GD, Alt FW. Regulation of the assembly of variable-region gene expression. Ann Rev Immunol 1986;4:339-368. 24 Oettinger MA, Schatz DG, Gorka C. Baltimore D. RAG-I and RAG-2, adjacent genes that synergistically activate V(D)J recombination, Science l988;248: 15 17-1523. 25 Reth M, Petrac E, Wiese P, Lobel L, Alt FW.Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J 1987;6:3299-3305. 26 Kincade PW, Lee G, Pietrangeli CE, Hayashi SI, Gimble JM. Cells and molecules that regulate B lymphopoiesis in bone marrow. Annu Rev Immunol 1989;7:111-143. 27 Dorshkind K. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol 19908:111-137. 28 Dorshkind K, Witte ON. Long-term murine hemopoietic cultures as model systems for analysis of B lymphocyte differentiation. Curr Top Microbiol Immunol 1987; 135~24-41. 29 Whitlock CA, Witte ON. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc Natl Acad Sci USA 1982;79:3608-3612. 30 Dexter TM, Allen TD, Lajtha LG. Conditions control-
DorshkindLandreth
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
ling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol 1977;91:334-344. Dorshkind K. In vitro differentiation of B lymphocytes from immature hemopoietic precursors present in long-term bone marrow cultures. J Immunol 1986;1361422-429. Denis KA, Witte ON. In vitro development of B lymphocytes from long-term cultured precursor cells. Proc Natl Acad Sci USA 1986;83:441-445. Collins LS, Dorshkind K. A suomal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. J Immunol 1987;138:1082-1087. Nishikawa SI, Ogawa M, Nishikawa S, Kunisada T, Kodama H. B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation. Eur J Immunol 1988;18:1767-1771. Whitlock CA, Tidmarsh GF, Muller-Sieburg C, Weissman IL. Bone marrow stromal cell lines with lymphopoietic activity express high levels of a pre-B neoplasia-associated molecule. Cell 1987;48:10091021. Hunt P, Robertson D, Weiss D, Rennick D, Lee F, Witte ON. A single bone marrow-derived stromal cell type suppons the in vitro growth of early lymphoid and myeloid cells. Cell 1987; 48:997-1007. Pietrangeli CE, Hayashi SI, Kincade PW. Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors. Eur J Immunol 1988;18:863-872. King AG, Wierda D, Landreth KS. Bone marrow stroma1 cell regulation of B-lymphopoiesis I. The role of macrophages, IL- I , and IL-4 in pre-B cell maturation. J Immunol 1988;141:2016-2026. Landreth KS, Dorshkind K. Pre-B cell generation potentiated by soluble factors from a bone marrow stroma1 cell line. J Immunol 1988;140:845-852. Henderson AJ, Johnson A. Dorshkind K. Functional characterization of two stromal cell lines that support B lymphopoiesis. J Immunol I990 145:423-428. Gunji Y, Sudo T, Suda J, Yamaguchi Y, Nakauchi H, Nishikawa SI, Yanai N, Obinata M, Yanagisawa M, Miura Y, Suda T. Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7. Blood 1991;77:2612-2617. Kiemey PC, Dorshkind K. B lymphocyte precursors and myeloid progenitors survive in diffusion chamber culture but B cell differentiation requires close association with stromal cells. Blood 1987;70:1418-1424. Miyake K, Weissman IL, Greenberger JS, Kincade PW. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med I99 1; 173:599-607. Kina T, Majumdar AS, Heimfeld S, Kaneshima H, Holzmann B, Katsura Y, Weissman IL. Identification of a 107-kD glycoprotein than mediated adhesion between stromal cells and hematolymphoid cells. J Exp Med 1991;173:373-381. Miyake K, Underhill CB, Lesley J, Kincade PW. Hyaluronate can function as a cell adhesion molecule
17
46
47
48
49
50
51
52 53
54
55
56
57
58
59
60
and CD44 participates in hyaluronate recognition. J Exp Med 1990;172:69-75. Namen AJ3, Lupton S, Hjemld K, Wagnall J, Mochizuki DY, Schmierer A, Mosley B , March C, Urdal D, Gillis S, Cosman D, Goodwin RG. Stimulation of Bcell progenitors by cloned murine interleukin-7. Nature 1988;333:571-573. Williams DE, Eisenman J, Baird A, et al. Identification of a ligand for the c-kit proto-oncogene. Cell 1990;63:167-174. Flanagan JG, Leder P. The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell 1990;63:185-194. Zsebo KM, Wypych J, McNiece IK, et al. Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liverconditioned medium. Cell 1990;63:195-201. Huang E, Nocka K, Beier DR, et al. The hematopoietic growth factor KL is encoded at the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 199063:225-233. Lee G. Namen AE,Gillis S , Ellingsworth LR, Kincade PW. Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-,!?. J Immunol 1989;142:3875-3883. Russel ES. Hereditary anemias of the mouse: a review for geneticists. Adv Genetics 1979;20:357-459. McNeice IK, Langley KE, Zsebo KM. The role of recombinant stem cell factor in early B cell development. J Immunol 1991; 146:3785-3790. Rolink A, Kudo A, Karasuyama H, Kikuchi Y,Melchers F. Long-term proliferating early pre-B cell lines and clones with the potential to develop to surface Igpositive, mitogen reactive B cells in vitro and in vivo. EMBO J 1991;10:327-336. Suda T, Ito M, Ogawa Y,et al. Interleukin 7 production and function in stromal cell-dependent B cell development. J Exp Med 1989;170:333-338. Ogawa M, Matsuzaki Y,Nishikawa S, et al. Expression and function of c-kit in hemopoietic progenitor cells. J Exp Med 1991;174:63-72. Billips LG, Petitte D, Landreth KS. Bone marrow stroma1 cell regulation of B lymphopoiesis: interleukin- 1 (IL-I) and IL-4 regulate stromal cell support of pre-B cell production in vitro. Blood 1990;75:611-619. Suda T, Okada S, Suda J, et al. A stimulatory effect of recombinant murine interleukin-7 (IL-7) on B-cell colony formation and an inhibitory effect of IL-la. Blood 1989; 74:1936-1941. Dorshkind K. IL-1 inhibits B cell differentiation in long term bone marrow cultures. J Immunol 1988;141:53 1-538. Dorshkind K. In vivo administration of recombinant granulocyte-macrophage colony-stimulating factor results in a reversible inhibition of primary B lymphopoiesis. J Immunol 1991; 146:4202-4208.
