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Division of Cancer and Hematology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Key Words. Eosinophil formation • Culture • Mouse • Stem cell factor
Donald Metcalf, M.D., Division of Cancer and Hematology, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050 Australia. Telephone: 61-3-9345-2555; Fax: 61-3-9347-0852; e-mail: metcalf{at}wehi.edu.au
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on these considerations, mice with inactivation of the gene encoding the ß common receptor chain (ßc-/- mice) have no known agent able to stimulate eosinophil proliferation and should, therefore, lack eosinophils. ßc-/- mice do exhibit significantly fewer eosinophils in the blood than control mice, but there is not a complete absence of such cells [5]. Furthermore, immature and mature eosinophils are present in the ßc-/- bone marrow, albeit at reduced levels, and mature eosinophils can be found in those tissues, such as the uterus, that usually contain eosinophils. These data have been supported by observations on continuing low-level production of eosinophils in mice with inactivation both of the ßc gene and the gene for IL-3 [9]. The conclusion is that at least one additional eosinophil-stimulating factor must exist and be responsible for stimulating the low level of the eosinophil formation occurring in ßc-/- and ßc-/- IL-3-/- mice.
Because IL-3-responsive eosinophil progenitor cells are present in ßc-/- marrow populations [5], IL-3 can be used as a positive stimulus to verify their presence in each ßc-/- marrow cell suspension used. These cells can then be used to search for novel eosinophil-active factors, particularly in organ-conditioned media, because such media lack IL-3 [8]. The cells can also be used to test the activity of known cytokines with the confidence that any observed eosinophil stimulation cannot be indirectly mediated by the induced production either of GM-CSF or IL-5.
To date, only a single type of lineage-committed eosinophil progenitor cell has been described that is able to generate colonies of maturing eosinophils (Eo colonies [10]). However, as the present studies progressed, it became obvious that an additional type of committed progenitor cell exists in the mouse that is able to generate colonies containing exclusively neutrophilic granulocytes and eosinophils (GEo colonies). From these studies, it has emerged that stem cell factor (SCF) is an additional regulator with an ability to stimulate the formation of eosinophils by both types of eosinophil progenitor cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Marrow Cultures
Agar cultures of bone marrow cells were performed containing from 25,000-500,000 cells per ml in Dulbecco’s modified Eagle’s medium containing a final concentration of 20% (volume by volume) newborn calf serum and 0.3% agar [11]. The 1-ml agar cultures were prepared in 35-mm plastic Petri dishes, and stimuli for colony formation were added in 0.1 ml volumes before the addition of the agar medium containing the target bone marrow cells. After gelling, cultures were incubated for 7 days at 37°C in a fully humidified atmosphere of 10% CO2 in air. After incubation, colony counts were performed at 35x magnification using an Olympus dissection microscope (Olympus; Tokyo, Japan; http://www.olympus.com) then all cultures were fixed by the addition of 1 ml of 2.5% glutaraldehyde. After fixation for 4 hours, intact gels were floated onto glass slides and, after drying, were stained with acetylcholinesterase then Luxol Fast Blue (to identify eosinophils) and counterstained with hematoxylin. Coverslips were mounted using mounting medium, then all colonies present in each culture were enumerated and their cellular composition determined at 100x or 200x magnification. Previous studies have documented that Luxol Fast Blue positivity (green stain) is specific for colony eosinophils [10], and the segmented ring shape of the nucleus in hematoxylin-stained preparations is equally specific for mature neutrophilic granulocytes (granuloctyes).
Stimuli
Media were harvested from cultures of continuous cell lines at confluence or from cultures of minced organs incubated for up to 4 days in serum-free medium as described previously [8,12]. After millipore filtration, 0.1-ml volumes were added to duplicate marrow cultures at 1:1 and 1:2 dilutions.
The final concentration in the cultures of the purified recombinant growth factors tested were: murine GM-CSF, human G-CSF, murine macrophage (M-CSF), murine IL-3 (10 ng/ml), murine IL-6 (100 ng/ml), murine SCF (100 ng/ml), murine flk-ligand (500 ng/ml), human IL-1 (10 U/ml), murine IL-4 (10 ng/ml), murine IL-5 (10 ng/ml), murine thrombopoietin (TPO) (50 ng/ml), human IL-11 (10 ng/ml), murine leukemia inhibitory factor (10 ng/ml), human erythropoietin (Epo) (2 U/ml), murine eotaxin (200 ng/ml), murine interferon 
(2 x 103 U/ml), human oncostatin M (100 ng/ml), and human IL-2 (100 U/ml). All recombinant materials were either produced in this laboratory or purchased from Pepro Tech (Rocky Hill, NJ; http://www.peprotech.com).
Microwell Assays
The Ba/F3 cell line used for the specific bioassay of IL-3 responds by proliferation in microwell cultures to concentrations of IL-3 as low as 1.5 pg/ml [12,13]. For the specific assay of G-CSF or M-CSF, stable Ba/F3 sublines expressing inserted murine G-CSF or M-CSF receptors (Ba/F3GR and Ba/F3MR cell lines) were used, as previously described [13]. Because no organ-conditioned medium in fact contained detectable IL-3, these cell lines were able to provide specific assays for their respective ligands and were able to detect concentrations as low as 100 pg/ml for G-CSF and M-CSF.
The FDC-P1 cell line proliferates in response to both GM-CSF and IL-3 [13]. Because of the absence of IL-3 from organ-conditioned media, the FDC-P1 cell line was able to be used as a specific bioassay for GM-CSF (lower detection limit, 100 pg/ml) [12].
Cell lines for use in microwell assays were washed three times by centrifugation in 10 ml of medium to remove the CSF used in the maintenance cultures. Microwell assays were performed as described previously [12]. All assays included a titration of 1 ng/ml IL-3 to certify the viability and responsiveness of the Ba/F3, Ba/F3GR, and Ba/F3MR cell lines and also a titration of the appropriate purified recombinant regulator starting with an initial concentration of 100 ng/ml for G-CSF and M-CSF. For FDCP-1 cells, the standard titrated was 1 ng/ml GM-CSF.
Recloning Experiments
Cultures of 25,000 C57BL bone marrow cells were stimulated by 100 ng SCF or 100 ng SCF plus 10 ng IL-3. After 7 days of incubation, sequential individual multicentric blast cell colonies were removed using a fine pipette and resuspended in 8 ml of agar medium. One-ml volumes of this suspension of colony cells were recultured using 0.1 ml of pokeweed mitogen-stimulated BALB/c spleen-conditioned medium [11] as a source of GM-CSF and IL-3. After 7 days of incubation, the secondary cultures were fixed, stained, and scored for colony formation as above.
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RESULTS |
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, the young adult ßc-/- marrow contains both eosinophilic myelocytes and mature eosinophils, although in lower combined numbers than ßc+/+ marrow. Unlike the situation in op/op mice, lacking M-CSF, where the macrophage deficiency is corrected with increasing age [14], an analysis of 8-month-old ßc-/- mice showed no change from the situation shown in Table 1 for younger ßc-/- mice (data not shown). The presence of some eosinophils in ßc-/- mice indicates that there must be one or more unrecognized agents in the mice that have some ability to stimulate eosinophil proliferation.
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A similar negative outcome was obtained when organ-conditioned media prepared from C57BL, ßc-/-, or ßc+/+ thymus, heart, lung, salivary gland, spleen, kidney, pancreas, muscle, bladder, bone shaft, or bone marrow tissues were assayed on ßc-/- marrow cells. Many of these organ-conditioned media stimulated granulocyte and/or macrophage colony formation, correlating with their content of G-CSF and M-CSF, but no eosinophil colonies were observed. Parallel assays of the ßc-/- and ßc+/+ organ-conditioned media on Ba/F3 cells failed to detect IL-3 (Table 2), eliminating the possibility that tissues in ßc-/- mice had acquired an aberrant capacity to produce IL-3. The content of GM-CSF, G-CSF, and M-CSF in media conditioned by ßc-/- organs did not differ substantially from that of media conditioned by ßc+/+ organs (Table 2).
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, cell crowding of both ßc-/- and ßc+/+ cells did result in some endogenously stimulated granulocyte and macrophage colony formation but no eosinophil colonies were observed in cultures of ßc-/- cells.
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, tumor necrosis factor, epidermal growth factor and, importantly, the eosinophil chemotactic agent, eotaxin. None of these had eosinophil stimulating activity at the concentrations tested. However, one agent was found to have such activity—SCF.
SCF-Stimulated Eosinophil Colonies
SCF stimulates the formation of two dominant colony types in cultures of mouse bone marrow cells—blast cell colonies and neutrophilic granulocytic colonies [15]. The compact nature of these colonies made it easier to recognize the additional presence in SCF-stimulated cultures of far less frequent eosinophil-containing colonies. Only approximately 1 in 100 colonies developing in such cultures contained eosinophils. These eosinophil-containing colonies were of two types: compact colonies composed entirely of eosinophils (Eo colonies) and colonies with a dominant population of neutrophilic granulocytes but also containing a minority population of mature eosinophils (GEo colonies)—the eosinophils being distinguishable from neutrophilic granulocytes by their green cytoplasm and nonsegmented nuclei (Fig. 1).
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). In cultures stimulated by G-CSF, no eosinophil-containing colonies developed, regardless of colony size. More intriguingly, as shown in the pooled data in Table 4, in 10 independent experiments using C57BL bone marrow, the combination of G-CSF with IL-5 was not synergistic for colony number or size and did not significantly stimulate the formation of GEo colonies—either in the form of granulocytes in eosinophil colonies or eosinophils in granulocytic colonies. No colonies resembling those in Figure 1
were seen, and in most cases, the colonies scored as GEo were highly dispersed colonies of mature granulocytes in which one, or at most two, mature eosinophils were present. In most cultures, the frequency of these single eosinophils was similar in colony-free areas of the agar, and most of the cells appeared to have been IL-5-activated eosinophils surviving from the initial marrow cells cultured. SCF, therefore, was exceptional in that it was the only eosinophil-active factor stimulating the formation predominantly of GEo colonies.
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Enhancement of SCF Action by G-CSF
Figure 2 presents data from four separate experiments in which cultures of 100,000 ßc-/- marrow cells were compared with paired cultures of 100,000 ßc+/+ marrow cells. The figure shows the mean number of Eo and GEo colonies developing in duplicate cultures stimulated either by SCF, SCF plus G-CSF, or IL-3. The data make several points of interest. Of necessity, the content of eosinophil progenitors can only be assessed in ßc-/- marrow by the use of IL-3. With this as the standard stimulus, it was clear that ßc-/- marrow does contain eosinophil progenitor cells, although in slightly lower frequency than in the ßc+/+ marrow. For both marrow types, when stimulated by IL-3, Eo colonies outnumbered GEo colonies. SCF stimulated eosinophil colony formation by both ßc-/- and ßc+/+ marrow cells. The numbers of colonies developing were small and GEo tended to be the more common colony type. Addition of G-CSF to SCF not only greatly enhanced the size of blast and granulocytic colonies in the cultures, as previously described [16], but also reproducibly increased the number of eosinophil-containing colonies, although the magnitude of the change was less than that for blast or granulocytic colonies. It was a surprising, but consistent, observation that the combination of SCF with G-CSF enhanced Eo colony formation more than GEo, and numbers of the latter type actually fell in many experiments using the combined stimulus.
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The low frequency of SCF-responsive eosinophil progenitors made it impractical to attempt single-cell cultures or clone transfer experiments to verify a direct action of SCF on the clonogenic cells. However, in experiments varying the numbers of cultured C57BL marrow cells from 10,000 to 200,000 per culture, eosinophil colony formation was linear with cultured cell numbers (data not shown).
Enhancement of SCF-stimulated blast and granulocytic colony formation comparable with that elicited by G-CSF can also be elicited by the addition of IL-6 [17]. However, as shown by the representative experiment in Figure 3, combination of IL-6 with SCF did not enhance eosinophil colony formation. Similarly, although combination of Epo or TPO with SCF enhanced megakaryocyte colony formation, the combination did not modify SCF-stimulated eosinophil colony formation. Combination of SCF with all the remaining cytokines listed above, except GM-CSF or IL-3, also failed to enhance SCF-stimulated eosinophil colony formation. G-CSF, therefore, appeared to play a special role in being able to enhance SCF-stimulated eosinophil colony formation.
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None of the 113 blast colonies analyzed were composed solely of Eo or GEo progenitors. Blast colonies contained progenitors able to form granulocytic, granulocyte-macrophage, macrophage, or eosinophil-containing colonies, although not all colonies contained progenitors of all types. As noted in earlier studies, the absolute number of progenitor cells in individual colonies varied widely [16,18]. Furthermore, as in primary marrow cultures, no eosinophils were observed in secondary macrophage or granulocyte-macrophage colonies.
The calculated total content of granulocytic progenitor cells for 113 individual blast colonies comparing those containing eosinophil progenitors with those lacking these progenitors is shown in Figure 4. The content of granulocytic progenitors varied from 0 to 1,406 per colony, and progenitor numbers were higher where colony size had been enhanced by combination of IL-3 with SCF. Where blast colonies contained eosinophil progenitors, these were approximately tenfold less numerous than granulocytic progenitors in the colony, and Eo progenitors were present in only 24 of 113 colonies analyzed. As shown previously [16], eosinophil progenitors were more often present in blast colonies when IL-3 had been combined with SCF, and there was a weak association between the occurrence of Eo progenitors and overall total granulocytic progenitor cell numbers.
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, blast colonies containing GEo progenitors were even less frequent than those containing Eo progenitor cells, and only 10 of 113 colonies analyzed contained GEo progenitor cells. The occurrence of GEo progenitors was significantly associated with a high colony content of granulocytic progenitors (
2 = 8.8), and GEo progenitors were more common in blast colonies stimulated by SCF plus IL-3 than in colonies stimulated only by SCF. A comparative analysis of blast colony content of macrophage progenitors and either Eo or GEo progenitors showed less evident associations (data not shown), and in fact, 9 of the 24 blast colonies containing eosinophil progenitors contained no macrophage progenitor cells.
Figure 5 provides an analysis of the numbers of Eo or GEo progenitors in blast colonies containing one or both cell types. Eo progenitors were, in general, somewhat more numerous in positive colonies than GEo progenitors. Two points emerged from this analysis of relevance to the possible ancestral relationship between GEo and Eo progenitors. First, in 19 of the 26 blast colonies, only one type of eosinophil progenitor cell was present. Second, 16 of the 26 blast colonies contained Eo but not GEo progenitor cells. Both observations argue against the possibility that GEo might be the ancestors of the Eo progenitors. There were, however, 7 of 26 colonies that did contain both types of eosinophil progenitors and, at least for these, a common ancestor (blast colony-forming cell) has therefore been documented. This still leaves open the possibility that many Eo and GEo may be derived independently from possibly differing preprogenitor (blast colony-forming) cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present studies showed that ßc-/- marrow contains near-normal levels of eosinophil progenitor cells, but use of ßc-/- marrow cells as target cells failed to detect novel eosinophil colony-stimulating activity in a wide range of media conditioned by cell lines or organs from adult mice, including organs from ßc-/- mice.
Testing of known growth factors on ßc-/- marrow cells showed that SCF had a previously unrecognized capacity to stimulate the formation of small numbers of eosinophilcontaining colonies. This action of SCF was not restricted to ßc-/- cells, and comparable activity was observed with marrow cells from a range of mouse strains and cells from fetal liver and neonatal bone marrow. The eosinophil-stimulating action of SCF was consistently enhanced by combination with G-CSF.
The eosinophil stimulating action in vitro of SCF was weak compared with GM-CSF or multi-CSF, both in terms of eosinophil colony numbers and eosinophils per colony. However, previous experience with agents such as GM-CSF and G-CSF has shown that relative activities in vitro do not necessarily correlate with strength of action in vivo [19]. Moreover, the secreted form of SCF (as used in the present studies) is known to be less efficient than the membrane-displayed form in stimulating hematopoiesis [20]. Thus, the action of SCF on eosinophil formation may be stronger in vivo than evident from the present in vitro studies.
It has yet to be established that all lineage-committed progenitor cells originate from the preprogenitor (blast colony-forming cell) progeny of stem cells [21]. Some may be generated directly from stem cells without passing through a preprogenitor cell stage. This uncertainty aside, the present experiments showed that SCF-responsive preprogenitor cells can form both Eo and GEo progenitor cells. Thus, SCF has the ability not only to stimulate the generation of eosinophil progenitor cells but also to stimulate at least some of these to form mature eosinophils. In these actions, IL-3 has a strong synergistic action on eosinophil progenitor cell formation, and G-CSF a comparable action on the proliferation of the eosinophil progenitor cells.
The present study showed that G-CSF and IL-5 could not act synergistically on committed eosinophil progenitors, extending an earlier study by Yamaguchi et al. [3], although this earlier study did detect an interaction when the two factors were acting on less mature cells.
The blast colony recloning data showed that some Eo and GEo progenitors have the same ancestral preprogenitor cells but did not eliminate the possibility that others might have a different and unrelated ancestral origin.
The SCF-responsive Eo and GEo progenitors may well also be responsive to proliferative stimulation by GM-CSF or IL-3, but the present data indicate that at least most GEo progenitors are unresponsive to IL-5.
It remains for future studies to determine whether there are any functional differences between eosinophils generated from Eo and GEo progenitors.
The present studies have added SCF, acting alone or in combination with G-CSF, to the known factors able to stimulate eosinophil proliferation.
In early studies on the marrow of anemic W/Wv mice that lack functional receptors for SCF, a depleted content of marrow eosinophils was recorded but not commented upon [22]. It will require cross-mating of ßc-/- IL-3-/- mice with either W/Wv or Sld mice that are unable to respond to or produce SCF to establish whether the combined loss of stimulation by GM-CSF, IL-5, IL-3, and SCF leads to an absolute failure of all eosinophil formations. If not, then yet another growth factor must exist with an eosinophil-stimulating action in vivo.
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ACKNOWLEDGMENT |
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The authors are indebted to the AMRAD Corporation, Melbourne, for making available samples of culture media from their battery of normal and neoplastic murine cell lines.
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REFERENCES |
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) or SCF plus IL-3 (
) grouped according to whether or not the colony also contained Eo or GEo progenitor cells. Note that most colonies containing Eo or GEo progenitors had been initiated by SCF plus IL-3.