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Expression and function of c-kit in hemopoietic progenitor cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

T he protooncogene c-kit encodes a receptor tyrosine kinase belonging to the platelet derived growth factor (PDGF)t/CSF-1 receptor subfamily (1) . Recent studies have shown that c-kit is allelic with the dominant spotting locus (W) of mice (2, 3). Since mice homozygous with the W allele which encodes a nonfunctional c-kit gene product die perinatally of severe anemia (4), it is clear that c-kit and its ligand play an essential role in intramarrow hemopoiesis in the adult mouse. More recently, a gene encoding the ligand for c-kit was cloned by several groups (5)(6)(7)(8)(9) . This ligand was demonstrated to have colony stimulating activity ofmultipotent progenitors (9)(10)(11). Thus far, this newest hemopoietic growth factor and CSF-1 are the only two factors whose actual roles in vivo are well understood, owing to the availability of the mice which are defective in these genes (12)(13)(14).
Despite the considerable understanding of the essential role of c-kit and its ligand on the regulation of constitutive hemopoiesis in adult bone marrow (15, 16), several questions remain to be resolved. First, it is not clear which types of intramarrow hemopoietic cells express c-kit, although expression of c-kit on the surface ofmast cells has been clearly shown (17). To closely examine the expression ofc-kit in bone marrow cells, it is necessary to produce mAbs against the extracellular domain ofc-kit. Secondly, recent studies have suggested that multipotent stem cells, erythroid progenitors and some B lineage progenitors can be stimulated to form colonies in vitro by the recombinant c-kit ligand (5,(9)(10)(11). However, this does not necessarily mean that the maintenance of these progenitor cells in the bone marrow is actually dependent on c-kit . If a mAb which can antagonize the function ofc-kit is obtained, an in vivo blocking experiment may resolve this question. This kind of experiment is important for such a molecule as c-kit whose expression does not necessarily indicate that it is functioning . For example, although c-kit has been shown to be expressed in the brain (3, 18), development ofthe central nervous system appears to be normal even in the W/ W mouse, which does not express functional c-kit at all .
Recently, we developed mAbs against the extracellular domain of c-kit, some of which can block the function of c-kit both in vitro and in vivo (46). An antagonistic anti-c-kit mAb injected into pregnant mice could be transported to the embryo through the placenta, and was able to block the colonization of melanocyte precursors into the epidermal layer of the skin, thereby producing unpigmented offspring from a pigmented mouse. In the present study, we used these antic-kit mAbs and attempted to determine the expression and function of c-kit in hemopoietic cell progenitors . Our results demonstrate that c-kit is expressed on almost all hemopoietic cell progenitors clonable in vitro by various soluble hemopoietic growth factors and on most spleen colony-forming cells (CFCs) . We also show that this receptor molecule actually functions in these cells in vivo.
Antibody Injection. B6 male mice were injected with 100 ug or 1 mg purified antibodies intravenously every other day. Mice were then anesthetized and sacrificed by cervical dislocation, and bone marrow cells were analyzed for lineage markers and colony forming ability.
Colonrforming Unit-S (CFU-S) Assay. Female B6 mice were X-irradiated (9 Gy) . After 24 h, the mice were injected intravenously with 5 x 10^or 2.5 x 105 cells suspended in Eagle's MEM. Mice were anesthetized and sacrificed by cervical dislocation at 8 or 13 d after injection (31,32). The spleens were removed and fixed in formalin/acetic acid/alcohol and colonies were counted. Controls injected with cell-free suspending medium had 2 colonies per 13 spleens .
Depletion ofc-kit* Cells from Bone Marrow Preparation . Ten million bone marrow cells harvested from normal B6 mice were incubated with ACK4 or RA3-6B2, and resuspended in 1 ml ofEagle's MEM supplemented with 5% FCS. The paramagnetic microspheres coated with sheep anti-rat IgG (Dynabeads M-450; Dynal, Oslo, Norway) (33) were washed and suspended with same medium at the concentration of4 x 108 beads/ml. One ml of the beads suspension was mixed with the cell suspension prepared above and incubated on ice for 30 min with gentle shaking . The mixture was then diluted 1:5 by adding medium. The beads were removed twice by a magnetic concentrator (MPC1 ; Dynal) together with the cells bound to the beads. The cell recoveries were always around 30% . An aliquot of the remaining negative cells was stained again with the corresponding antibody and checked by flow cytometry. The remainder was tested in the colony assay described above.

Results
Establishment of Anti-c-kit Monoclonal Antibody. IL-3-dependent mast cells derived from normal mouse bone marrow highly express the c-kit gene and are able to proliferate in the presence of the ligand of c-kit without the addition of IL3 (5, 6, 10, 16). On the other hand, mast cells derived from W/ W mice produce a c-kit molecule from which the transmembrane domain is deleted (4,34) . Consequently, the cells are expected to fail to express c-kit on the cell surfaces. This fact prompted us to make anti-c-kit mAbs by immunizing a rat with 11,3-dependent normal mast cells followed by selection on the basis of the ability to bind with the mast cells derived from a normal littermate mouse but not from a WI W mouse. By these criteria we obtained four independent mAbs against c-kit . Fig . 1 shows the surface staining of mast cells from both a normal and W/W mouse by two of the obtained mAbs, designated ACK2 and ACK4. These two mAbs recognized normal mast cells while they could not bind to mast cells derived from the W/W mouse. ACK2 recognized surface molecules whose mol wt were 120 and 160 kD as determined by Western blotting analysis of normal mast cells (data not shown) . These sizes are in good accordance with those ofc-kit (18). Furthermore, ACK2 bound to the COS7 cells transfected with the c-kit cDNA which was cloned into an expression vector while the parent cells were ACK2 -(data not shown) . These data indicate that the ACK mAbs specifically bind to the extracellular domain of c-kit . Expression ofc-kit in Bone Marrow. By using the anti-c-kit mAb, ACK2, the expression of c-kit and other lineage markers in fresh bone marrow cells from adult C57BL/6 mouse was determined . As shown in Fig. 2, c-kit bright and dull populations coexist in bone marrow. The c-kit bright population Figure 2 . Expression of c-kit and lineage markers on B6 bone marrow cells. Cells were incubated with normal rat serum, then APC-conjugated anti-c-kit (ACK2) and FITC-conjugated anti-13220, Gr-1, Mac-1, or TER-119 antibody were added . A mixture of FITC-conjugated anti-13220, Gr-1, Mac-1, anti-CD4, and anti-CD8 antibodies were used to stain lineage markers (Lin .) positive cells . As a control, APC-conjugated mAbs against irrelevant antigens were used. Cells were washed, stained with propidium iodide (PI), and analyzed by flowcytometry. The dead cells stained with PI were gated out.
does not coexpress other lineage markers including 13220, CD4, CD8, TER-119, Gr=1, nor Mac-1. This population accounts for about 3.3 ± 0.3% (mean ± SD) of the total bone marrow cells . Most of the c-kit dull population coexpresses some of these lineages markers, although those which coexpress 13220 or TER 119 are extremely low in number. The dull c-kit staining of Gr-1 and Mac-1 positive cells seems to be specific, since the addition of isotype matched unrelated mAb or normal rat serum does not affect this staining (data not shown) . Thus, total c-kit positive cells account for 7 .8% ± 0 .6% of total bone marrow cells if the c-kit dull positive cells are included .
Expression of c-kit on the Surface of Hemopoietic Progenitor Cells. Next we investigated whether c-kit is expressed on the surface of hemopoietic progenitor cells including CFU-S. Normal bone marrow cells were incubated either with antic-kit mAb ACK4 or anti-13220 mAb 6B2, and antibody-bound cells were depleted by using anti-rat Ig antibody-coated magnetic beads . More than 90% of the positive cells were depleted by this treatment (data not shown) . The number of CFC reactive to 110, GM-CSF, M-CSF, or lIr7 and spleen CFUs was measured (Table 1). When c-kit+ cells were removed from bone marrow cells, CFU-IIr3, CFU-GM, CFU-M, and CFU-S were depleted almost completely, while CFU-11,7 remained . Consistent with previous reports (30,35), deple-' The c-kit* or B220+ cells were depleted from the bone marrow of B6 mouse by using antibody coated magnetic beads. For in vitro CFC assay, 2 x 104 cells were incubated in 1 ml of semisolid medium containing various cytokines for 7 d and aggregates consisting of 40 or more cells were scored as a colony. For the CFU-S assay, 5 x 10 4 cells from control or B220+-depleted sample, or 2 .5 x 10 5 cells from ACK4*-depleted sample were injected into an irradiated mouse . The spleen was removed and fixed 8 d after injection and number of colonies was counted. t Mean ± SD for triplicates . However, it remained to be elucidated whether c-kit is functionally required for these progenitor cells in vivo. To address this question, we took advantage of an antagonistic anti-c-kit mAb, ACK2, which was shown to block the function of c-kit but not to be cytotoxic to c-kit' cells (46). If c-kit on the surface of hemopoietic progenitor cells is functioning in vivo, administration of ACK2 would induce severe anemia. In fact, ACK2 inhibited myelopoiesis in a longterm bone marrow culture with a stromal cell clone (our unpublished observation) .
One milligram of purified ACK2 was injected into B6 mice intravenously every second day and the number of B220+, TER-119+, or Mac-1+ cells in bone marrow was measured on various days after the initiation of ACK2 injection . Purified Mac-1 antibody was injected as a class matched control. Fig .  3 shows the morphology of the bone marrow cells from normal, Mac-l-injected, or ACK2-injected mice 12 d after the initial injection . No significant difference was observed between the bone marrow cells from normal and Mac-1 treated mice. In contrast, no polymorphonuclear cells or erythroblasts were present in the bone marrow of an ACK2 injected mouse, and most of the cells remaining in this bone marrow showed lymphoid morphology. This observation was confirmed by flow cytometry analysis (Fig. 4), which demonstrated that no Mac-1 + cells nor TER 119 * cells were present in the bone marrow of ACK2-injected mouse and that 90% of the total cells were B220+ B lineage cells including 16% of surface IgM+ cells . It is important to note that even after such a complete depletion ofmyeloid and erythroid cell lineages from bone marrow, the number of bone marrow cells recovered from ACK2-injected mice was nearly normal . This suggests that B lineage cells continued to grow in the ACK2-

66
Function of c-kit in Intramarrow Hemopoiesis treated mouse to fill the space from which myeloid and erythroid progenitor cells were purged. As shown in Fig. 6, CFU-IL3, CFU-GM, and CFU-M disappeared from the bone marrow much earlier than mature myeloid cells did, and a single ACK2 injection eliminated these CFCs almost completely. On the other hand, the number of CFU-IL7 increased five-to six-fold, suggesting that active proliferation of B cell precursors was actually induced in ACK2-treated bone marrow. Because the addition ofACK2 did not affect the formation of colonies from normal bone marrow cells in semisolid medium containing various cytokines, the disappearance of CFC was not due to a cytostatic effect of ACK2 during the culture assay period (data not shown) .
Furthermore, it is important to note that CFU-IL3, CFU-GM, and CFU-M decreased simultaneously rather than in  order of the proposed differentiation hierarchy of these progenitor cells (36,37) . This suggests that c-kit is functionally required for the maintenance of all of these CFCs in vivo. To further investigate whether or not each CFC has a different sensitivity to ACK2 treatment, the dose of ACK2 was decreased to 100 Ag and the numbers of CFCs were measured . The sensitivity of each CFC to ACK2 as estimated from Table  2 seemed not to differ significantly. Although only incomplete reduction of each CFC was induced by 10014g ACK2 in the bone marrow, the magnitude of reduction did not differ 67 Ogawa et al . significantly. We have also previously observed that 100 Ag is the dose at which adult spermatogenesis is completely blocked in vivo (Yoshinaga, K., S . Nishikawa, M . Ogawa, S .I. Hayashi, T. Kunisada, T. Fujimoto, and S .I. Nishikawa, manuscript submitted for publication) . Thus, the sensitivities to ACK2 seem to vary among c-kit-dependent tissues. Elimination ofColonyforming Units-S (CFU-S) by ACK27njection . Finally, we investigated whether the earliest hemopoietic progenitor cells which can give rise to spleen colonies upon transfer into lethally irradiated recipients are dependent on c-kit in vivo (38) . Bone marrow cells were harvested from mice which had been given 6 i.v. shots of 1 mg mAb, and the number of CFU-S was determined (Table 3). Again, most of CFU-S were depleted from the bone marrow of ACK2injected mouse irrespective of the day of the assay for spleen colony (31,32) . Interestingly, small but a significant number of day 13 CFU-S (approximately 10% of control) always re- mained in the bone marrow, and these colonies were large mixed type colonies (Fig. 7) . Therefore, a small fraction of day 13 CFU-S is resistant to the antagonistic activity of the anti-c-kit antibody.

Discussion
The major aim of the present study was to characterize the c-kit+ cells in adult mouse bone marrow and also to elucidate the functional role of this receptor tyrosine kinase molecule in this cell population . For this purpose, we used two mAbs against the extracellular domain of c-kit, ACK2 and ACK4 . These two mAbs recognize different epitopes on the c-kit molecule, because binding of one antibody was not blocked by the other. Moreover, in vitro hemopoiesis on PA6 stromal cell clone (39,40) or in Dexter's culture (41) was blocked completely by ACK2 but not by ACK4, suggesting that the former is antagonistic to c-kit function (our unpublished observation) .
By using these mAbs, we demonstrated the presence of c-kit+ cells in adult bone marrow. Interestingly, c-kit+ cells were further subdivided into c-kitbdghl and c-kitd°il populations, and c-kitbri ght cells did not coexpress other lineage Table 3 . Effects of Antibody Administration on Colonyforming Unit-S (CFU-S) in the Bone Marrow of B6 Mice B6 mice were given 6 shots of 1 mg antibodies on every other day. 50,000 bone marrow cells from the control mouse or Mac-1 treated mouse, or 2.5 x 105 bone marrow cells of ACK2 treated mouse were injected into irradiated recipients . The spleens were removed and fixed 8 or 13 d after the injection and the number of colonies was counted. t Mean ± SD for quadruplicates . S Not done. ' B6 mice were given 2 shots of 0.1 or 1 mg ACK2 on alternate days . The number of CFCs in bone marrow was determined as described in the legend for Table 1. t Mean ± SD for triplicates.   Table 3. Note that five-fold larger number of cells was injected into the recipients in the case of the ACK2treated mouse. 20 .0 ± 6.0 NDS markers while most of the c-kits°u population expressed myeloid markers such as Gr-1 and Mac-1. Probably, a small fraction of c-kitd°11 cells coexpressed B220, but no TER-119+ cells were included in this population. This staining pattern indicates that c-kit is highly expressed in the lineage marker negative (lin -) immature cell population and that c-kit expression decreases upon maturation into lin+ cells, although low level expression continues for some time. Since Willer-Sieburg et al. have shown that all CFCs were present in the lin -population (42), c-kitd" 11, lin+ population might have lost the progenitor activity. To confirm this, we are currently trying to sort each population to estimate the stem cell activity. In fact, a recent preliminary result suggests that CFU-S was highly enriched in a c-kitb6 ght, Thy-1duu, lin -population (Okada, S., H. Nakauchi, K. Nagayoshi, S. Nishikawa, S.I. Nishikawa, Y Miura, and T. Suda, manuscript submitted for publication). Our present result clearly indicated that almost all hemopoietic progenitor cells including CFCs reactive to IL 3, GM-CSF, or M-CSF and also CFU-S expressed c-kit on the cell surface, but B lineage progenitors which form colonies in response to IL-7 were c-kit-. Thus, we expect that c-kit expression would be additional and powerful markers in order to purify hemopoietic stem cells in various differentiation stages .
Since most of hemopoietic progenitors expressed c-kit, the next issue to be resolved was whether or not c-kit on the surface of these hemopoietic progenitors is functioning in vivo. The fact that the W1W homozygous mouse has no constitutive hemopoiesis in postnatal life indicates that c-kit is functioning at least in some progenitor cells (15). Previously, it was reported that CFCs do exist in the W1 W°mouse while CFU-S is absent (43) . This may well suggest that c-kit is functionally required only for the most immature progenitors, although it is expressed in both CFC and CFU-S. To address this question, we took advantage of a mAb capable of antagonistically blocking the function of c-kit. Injection of this antibody resulted in a complete and simultaneous elimination of all hemopoietic progenitor cells including CFU-S and CFCs reactive to IL3, GM-CSF or even M-CSF which has been considered to be a growth factor for the precursor restricted to the macrophage lineage. It could be that ACK2 is cytotoxic antibody eliminating all c-kit+ cells, thereby depleting all types of hemopoietic progenitors that express c-kit. We think, however, that this possibility is unlikely for the following reasons. First, although peritoneal mast cells highly express c-kit, i.p. injection of ACK2 does not affect the number ofmast cells in the peritoneal cavity (our unpublished observation) . Second, although all melanocytes in the adult hair follicle express c-kit, only those in the activated hair follicles are affected by ACK2 injection, suggesting that only c-kit function is blocked by this treatment (46). Third, although both spermatogonia and oocytes express c-kit, ACK2 injection blocked only spermatogenesis . Fourth, although Mac-1 used as a class matched control antibody also binds to myeloid cells, hemopoiesis in Mac-l-treated mice was normal . Lastly, rat mAbs of IgG2b class against various cell surface molecules have been used as antagonistic blockers of molecular interaction on the cell surface, and all previous results 6 9 Ogawa et al .
unequivocally indicated that rat-IgG2b mAb was cytostatic rather than cytotoxic (44). It could also be that ACK2 enhances the differentiation of the progenitor cells, thereby resulting in a depletion of stem cells. We think this possibility is also unlikely, because overgrowth of lineage marker bearing mature cells was never detected at any point after the injection of ACK2. Furthermore, despite extensive trials, we failed to detect any ligand mimicking activity of ACK2 to induce in vitro differentiation or proliferation of hemopoietic progenitors. On the other hand, the recombinant ligand for c-kit has recently been reported to have obvious in vitro activity to induce the proliferation of various hemopoietic precursor cells (5,(9)(10)(11) .
Consequently, it is very likely that ACK2 acts as an antagonistic antibody to c-kit function, although it remains to be elucidated whether ACK2 directly prevents the binding of the ligand to c-kit. Given that this is the case, our result must be interpreted as indicating that maintenance of hemopoietic progenitors in adult bone marrow is dependent on c-kit and its ligand. Because recent studies demonstrated that a recombinant ligand for c-kit induced colonies consisting of multiple cell lineages (9)(10)(11), the abolition ofall CFCs from ACK2-treated bone marrow may be due to the blockade of recruitment of lineage restricted progenitors from multipotent stem cells. However, it is important to note that all CFCs from those reactive to multi-CSF to even M-CSF disappeared from bone marrow simultaneously. If c-kit is functionally required for the maintenance of multipotent hemopoietic progenitor cells giving rise to lineage restricted progenitor cells such as CFU-M, there must be a time lag between disappearance of progenitors reactive to multi-CSF and to M-CSR In this context, it is interesting that the ligand for c-kit is able to cooperate with any ofother hemopoietic growth factors and enhances the number and size ofcolonies induced in bone marrow cells (10,11) . These results in conjunction with our present result imply that the maintenance of both multipotent and lineage restricted hemopoietic progenitor cells is in fact dependent on c-kit, although some other factors may also be required .
In contrast to the c-kit dependency ofmyeloid and erythroid progenitors, B lymphopoiesis was not significantly affected by ACK2 injection. More interestingly, a subset ofB lineage cells did overgrow to fill the bone marrow space from which other cell lineages were purged. On the other hand, it was demonstrated that the ligand for c-kit also remarkably enhanced the colony formation of B cell precursors in response to IL7 (11), suggesting the role of c-kit in intramarrow B lymphopoiesis . This result, however, is not inconsistent with our present result, because the previous studies clearly demonstrated that there is a subpopulation of B lineage cells which proliferates in response to IL7 alone (28,30,35,45). Therefore, the population capable of proliferating in response to IL-7 alone could be the subset that overproliferated in the ACK2-treated mouse. Alternatively, it could be that there is another subset ofB lineage precursors which requires yet unidentified signals for proliferation. Since the ligand for c-kit, and ACK2 which specifically inhibits the c-kit function are now available, this issue will be resolved soon.