Growth Hormone–Induced Stimulation of Multilineage Human Hematopoiesis
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《干细胞学杂志》
a Gladstone Institute of Virology and Immunology, San Francisco, California, USA;
b Department of Medicine, University of California, San Francisco, California, USA;
c Department of Microbiology and Immunobiology, University of California, San Francisco, California, USA
Key Words. Hematopoiesis ? Growth factor ? Hematopoietic progenitor ? In vitro ? Marrow stromal cells ? Bone marrow cells
Correspondence: Joseph M. McCune, M.D., Ph.D., Gladstone Institute of Virology and Immunology, 1650 Owens Street, San Francisco, California 94158-2261, USA. Telephone: 415-734-5060; Fax: 415-826-8449; e-mail: mmccune@gladstone.ucsf.edu
ABSTRACT
Growth hormone (GH) and its proximal mediator, insulin-like growth factor I (IGF-I), have been shown to play an important role in T lymphopoiesis . Administration of GH or IGF-I reverses thymic involution and enhances T lymphopoiesis in older rodents and accelerates immune reconstitution in immunodeficient animals . Recently, we extended these observations to humans, demonstrating that GH treatment is associated with increases in thymic mass and circulating na?ve CD4+ T cells in adults infected with human immunodeficiency virus, type 1 (HIV-1) . These data suggest that de novo T-cell production may be inducible in immunodeficient humans.
T lymphopoiesis is dependent upon the migration of prethymic bone marrow (BM) progenitor cells to the thymus , and age-related declines in T lymphopoiesis have been attributed to both decreased hematopoietic capacity of BM progenitors and involution of the thymus (reviewed in Miller and Globerson ). Thus, GH may enhance human T lymphopoiesis by acting on cellular targets in the BM and/or in the thymus. BM appears to be an important target of GH action, as demonstrated by rodent studies showing a significant effect of GH on multilineage hematopoiesis. In studies of mice and aging rats, administration of GH and IGF-I facilitates early stages of T-cell development by increasing the number of multilineage BM progenitors and by enhancing the migration and engraftment of progenitor cells into the thymus . Similarly, in vitro analyses showed that GH stimulates erythroid and myeloid colony formation from murine and human BM progenitors. These effects appear to be mediated, at least in part, by IGF-I .
In the current study, we sought to more specifically identify cellular targets of GH and IGF-I action within the human fetal BM (FBM). We hypothesized that GH may stimulate hematopoiesis either through direct effects upon multilineage or lineage-restricted hematopoietic progenitor cells or, indirectly, by inducing FBM stromal cells to produce cytokines that facilitate the survival or maturation of FBM progenitors. We found that GH and IGF-I have direct effects on the proliferation and survival of both multilineage and lineage-committed progenitor cells, and that these hormones also enhance cytokine production by FBM stroma. These findings further delineate the effects of GH and IGF-I within human BM and provide insight into mechanisms that may contribute to GH– and IGF-I–mediated enhancement of hemato-lymphopoiesis.
MATERIALS AND METHODS
GH Stimulates Proliferation of Primitive Multilineage Hematopoietic Progenitor Cells
To identify potential GH targets within the FBM, the expression of cell-surface GHR was assessed using a biotinylated antiGHR antibody on freshly acquired FBM mononuclear cells. Various subpopulations of multilineage and lineage-restricted hematopoietic progenitor cells were discriminated on the basis of their phenotypic markers, as described in Figure 1. The frequency of each of these subpopulations in the FBM was determined for six donors (Table 1). As shown in Figure 2A (left panel), GHR was expressed on a sizeable fraction of total FBM mononuclear cells (mean 19.4%, range 15%–28.8% in seven independent experiments). Using antibodies against the lineage markers shown in Figure 1, we found that these progenitor subpopulations expressed varying levels of GHR (Fig. 2A). Incubation of total FBM mononuclear cells or CD34+ cells with GH for at least 4 days resulted in a higher yield of total cells relative to untreated controls (p < .05) (Fig. 2B, left panel). Amongst the subpopulations included within FBM, statistically significant increases were observed in the primitive CD34+CD38– multilineage progenitors as well as the CD34+CD38+CD10+ lymphoid progenitors (Fig. 2B). There was also a strong positive correlation between the fraction of cells expressing GHR within a given subpopulation and the subsequent recovery of that subpopulation after the addition of GH (Fig. 2C).
Figure 1. CD34+ progenitor cell subpopulations in FBM. (A): Flow diagram representing multilineage human hematopoiesis. Cell surface markers designate those used to identify the different progenitor cell populations . (B): Flow cytometry plots show gating strategy used for phenotypic data collection. The first panel represents gating for CD34/CD38 populations, after gating on mononuclear cells gated by forward and side scatter (not shown). The large dashed box shows the total CD34+ gate. This CD34+ population was subdivided using CD38 (small boxes, labeled 34+38– and 34+38+). The CD34+CD38+ population was first subdivided with CD10 (next panel) into CD34+CD38+CD10+ and CD34+CD38+CD10– subpopulations. The remaining panels show gating used to discriminate CD34+CD19+, CD34+CD14+, and BrdU+ cell subpopulations, respectively, after first gating on mononuclear cells (not shown). Abbreviations: BFU-E, burst-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte; CFU-GM, colony-forming unit-granulocyte-monocyte; DC, dendritic cell; FBM, fetal bone marrow; NK, natural killer.
Table 1. Frequency of FBM progenitor subpopulations
Figure 2. Effects of GH on FBM progenitor cells. (A): Expression of GHR (shaded histograms) relative to isotype control staining (open histograms) after first gating on a mononuclear cell gate. These results are representative of seven independent experiments carried out on FBM progenitor cells from seven different donors. The numbers above the bars represent the percentage of GHR+ cells, that is, the percentage of cells in given subpopulation with levels of GHR staining above those found with the isotype control. (B): Effect of GH on the yield of cells within different FBM subpopulations as a mean percentage of untreated control cultures (unfilled bars). An increase in the absolute number of many subpopulations is seen after the addition of GH (100–250 ng/ml for 4–8 days) (filled bars) to either total FBM mononuclear cells (BM) or purified CD34+ cells (*p < .05). No difference was noted between using 100 ng/ml of GH versus 250 ng/ml; these two concentrations have accordingly been grouped for the analyses shown here. These results are pooled from seven independent experiments carried out on FBM cells from seven different donors. (C): Relationship between the percentage of GHR+ cells in different hematopoietic subpopulations (as shown in A) and the mean cell yield (as shown in B) after incubation of CD34+ cells with GH (r2 = 0.767, p < .0001). (D): BrdU incorporation of CD34+38– cells after stimulation of total FBM or CD34+ cells with GH (100–250 ng/ml for 4–7 days) (filled bars) compared with untreated controls (unfilled bars). Data are pooled from six independent experiments using six different donors and are represented as a percentage of control. The increase is significant after stimulation of CD34+ cells (*p < .05). Abbreviations: BM, bone marrow; FBM, fetal bone marrow; GH, growth hormone; GHR, growth hormone receptor.
GH-associated increases in the total number of hematopoietic progenitor cells could be due to enhanced proliferation or decreased levels of apoptosis. To discriminate between these possibilities, total FBM mononuclear cells or CD34+ cells were treated with GH and analyzed by flow cytometry to detect proliferation (BrdU incorporation) and apoptosis (annexin-V binding). Levels of proliferation were significantly increased in the CD34+CD38– subpopulation after GH stimulation of CD34+ cells (Fig. 2D), whereas none of the FBM subpopulations showed significant changes in apoptosis (data not shown).
To confirm that GH stimulation resulted in an increase in functionally competent human hematopoietic progenitor cells, total FBM mononuclear cells were cultured for 6 days in the presence or absence of GH and then plated into methylcellulose cultures. As shown in Table 2, GH treatment resulted in a statistically significant (p < .05) increase in the number of CFU-GM and CFU-GEMM, but no change in the number of BFU-E.
Table 2. GH and IGF-I effects on CFU-Cs
GH Stimulates Proliferation of FBM Stromal Cells
The above data indicate that GH may interact directly with and induce the proliferation of primitive multilineage human hematopoietic progenitor cells as well as of lineage-restricted progenitor cells. Given the high expression levels of GHR on CD34+CD14+ myeloid progenitor cells, some of which might mature into FBM stromal macrophages , we hypothesized that GH might also indirectly regulate hematopoiesis through the FBM stroma. As a first test of this hypothesis, expression levels of GHR were assessed on recently plated (
Figure 3. Effects of GH on FBM stromal cells. (A): Representative surface staining of GHR on FBM stromal cells (n = 9, a mean 3.4% of total, range 0.91%–11.5%, were positive for GHR) (second panel). Gates were set using the isotype control (left panel). Many of the GHR+ cells were also CD45+ (n = 9, mean 65.3%, range 25%–97%). GHR+CD45+ FBM stromal cells are IGFR+ (n = 3, range 76%–99%) (solid line) whereas GHR+CD45– FBM stromal cells are IGFR low or negative (dashed line) (third panel). CD45+GHR+ FBM stromal cells are also CD14+ (n = 3, range 68%–93%) (solid line, right panel). (B): Incubation of FBM stromal cells with GH (100 ng/ml) results in an increase in the total number of total FBM stromal cells, stromal cells that are GHR+IGFR+CD45+, and stromal cells that are CD14+IGFR+CD45+. These panels represent pooled data from four separate experiments. Cell yields in GH-treated cultures are expressed as a mean percentage of untreated control cultures. (C): BrdU incorporation into the FBM stromal cell populations after 1 day in culture with GH addition (100 ng/ml). *p < .05 in (B, C). Abbreviations: FBM, fetal bone marrow; GH, growth hormone; GHR, growth hormone receptor; IGFR, antitype I insulin-like growth factor receptor.
Incubation of FBM stromal cultures with exogenous GH for 1 day resulted in an increased yield of stromal cells relative to untreated control cultures (p < .05) (Fig. 3B). Additionally, there was an increase of GHR+IGFR+CD45+ and CD14+IGFR+CD45+ stromal cells compared with untreated controls (Fig. 3B). Labeling studies with BrdU or annexin-V showed that these increases in cell yield were associated with increased levels of proliferation (p < .05) (Fig. 3C) without changes in apoptosis (data not shown). Thus, GH appears to induce the proliferation of myeloid-like FBM stromal cells.
IGF-I Effects on Human Multilineage Hematopoiesis
GH effects are often mediated through IGF-I . Indeed, in three FBM stromal cultures treated with exogenous GH (100 ng/ml), a significant increase (p < .05) in IGF-I production was observed relative to untreated controls (999 pg/ml versus 1,421 pg/ml, respectively; data not shown). These findings suggest that the effects of GH might be partly attributable to IGF-I.
To determine potential cellular targets of IGF-I, IGFR expression was assessed on FBM mononuclear and stromal cells. Surface expression of IGFR was detected on CD45+CD14+ myeloid stromal cells (see above, Fig. 3A) and on a large fraction of FBM progenitor cells (Fig. 4A). Additionally, mature CD10+CD45– fibroblast-like stromal cells expressed a low level of IGFR (data not shown).
Figure 4. Effects of IGF-I on FBM progenitor cells. (A): Expression of IGFR (shaded histograms) on FBM mononuclear subpopulations relative to isotype control staining (open histograms). These results are representative of three independent experiments carried out on FBM cells from three different donors. The numbers above the bars represent the percentage of IGFR+ cells, that is, the percentage of cells in given subpopulation with levels of IGFR staining above those found with the isotype control. (B): Effect of IGF-I on the yield of cells within different FBM mononuclear subpopulations as a percentage of the mean of the untreated control cultures (unfilled bars). An increase in the absolute number of many subpopulations is seen after the addition of IGF-I (100 ng/ml) (filled bars) to either total FBM mononuclear cells or CD34+ cells. These results represent pooled data from eight independent experiments carried out on FBM progenitor cells from eight different donors. (C): Effect of IGF-I on the percentage of apoptotic (annexin-V staining) cells within FBM mononuclear subpopulations as a percentage of the mean of the untreated control cultures (unfilled bars). These results represent pooled data from eight independent experiments carried out on FBM progenitor cells from eight different donors. In (B, C), *p < .05. Abbreviations: FBM, fetal bone marrow; IGF-I, insulin-like growth factor I; IGFR, antitype I insulin-like growth factor receptor.
To determine whether these cells were functionally responsive to IGF-I, total FBM mononuclear cells or purified CD34+ mononuclear cells were incubated with IGF-I. Treatment with IGF-I for 4–8 days resulted in an increase in the yield of FBM CD34+ progenitor cells relative to untreated control cultures (p < .05) (Fig. 4B, second panel from left). As in the case of GH stimulation, this increase in cell number was reflected by an increased yield of multiple hematopoietic subpopulations, including primitive, multilineage CD34+CD38– progenitor cells (Fig. 4B, remaining panels). In contradistinction to the effects of GH, the increased cell numbers observed after IGF-I treatment appeared to be primarily due to increased cell survival, with substantially lower levels of apoptosis (annexin-V staining) observed in multiple subpopulations after 4–8 days of culture (Fig. 4C). No consistent changes in the proliferation of these subpopulations (as assessed by BrdU incorporation) were observed (data not shown). IGF-I treatment of FBM stroma resulted in an increase in proliferation (as assessed by BrdU incorporation) of CD45+CD14+GHR+IGFR+ stromal cells (data not shown). This cell subpopulation also revealed an IGF-I–associated decrease in apoptosis (as assessed by annexin-V staining, data not shown).
In contrast to the effects of GH, treatment of FBM cells with IGF-I resulted not only in an increase in CFU-GM and CFU-GEMM but also in an increase in BFU-E (p < .005 in each case) (Table 2).
GH and IGF-I Induce Cytokine Secretion by FBM Stromal Cells
We hypothesized that GH and IGF-I might facilitate multilineage hematopoiesis by inducing the secretion of key hematopoietic cytokines from FBM stromal cells. To investigate this possibility, FBM stroma cultures were plated in replicate, stimulated with exogenous GH or IGF-I for 1 day, treated with brefeldin A to block secretion of induced cytokines, and then assessed by flow cytometry for the presence of intracellular IL-3. Relative to untreated cultures (Fig. 5, upper panels), IL-3 production was consistently stimulated by IGF-I (Fig. 5, middle panels) and, to a more variable and lesser degree, by GH (Fig. 5, lower panels). When lineage markers were included in the analysis, most IL-3–producing cells were found to be nonhematopoietic (CD45–CD10+) mature fibroblast-like FBM stromal cells (data not shown). Although increases in IL-3 production were observed in four of five stromal cultures incubated with GH (average = 2.2-fold) and in five of five cultures incubated with IGF-I (average = 5.9-fold), these changes were variable, even within a given experiment (and as evidenced in the replicates of Fig. 5). In addition to IL-3, we observed sporadic increases in the expression of other hematopoietic cytokines (e.g., IL-6, IL-7, and SCF; data not shown). In aggregate, these results suggest that GH may act, directly or indirectly, to induce both IGF-I and IL-3 from FBM stroma, but that the stromal cell(s) making these secondary mediators are rare.
Figure 5. Induction of IL-3 production by GH and IGF-I. FBM stromal cells were incubated in triplicate with medium alone (upper panels), 100 ng/ml IGF-I (middle panels), or 100 ng/ml GH (lower panels) for 24 hours, and then assessed by flow cytometry for the presence of intracellular IL-3. Triplicate results from a single donor are displayed. This experiment is representative of five independent experiments carried out with cells from five different donors. Abbreviations: FBM, fetal bone marrow; GH, growth hormone; IGF-I, insulin-like growth factor I; IL, interleukin.
DISCUSSION
This study was supported by National Institutes of Health (NIH) grants R37 AI40312 and R01 AI43864 (to J.M.M.) and K08 AI01597 (to L.A.N). J.M.M. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist and a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and of the NIH Director’s Pioneer Award.
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b Department of Medicine, University of California, San Francisco, California, USA;
c Department of Microbiology and Immunobiology, University of California, San Francisco, California, USA
Key Words. Hematopoiesis ? Growth factor ? Hematopoietic progenitor ? In vitro ? Marrow stromal cells ? Bone marrow cells
Correspondence: Joseph M. McCune, M.D., Ph.D., Gladstone Institute of Virology and Immunology, 1650 Owens Street, San Francisco, California 94158-2261, USA. Telephone: 415-734-5060; Fax: 415-826-8449; e-mail: mmccune@gladstone.ucsf.edu
ABSTRACT
Growth hormone (GH) and its proximal mediator, insulin-like growth factor I (IGF-I), have been shown to play an important role in T lymphopoiesis . Administration of GH or IGF-I reverses thymic involution and enhances T lymphopoiesis in older rodents and accelerates immune reconstitution in immunodeficient animals . Recently, we extended these observations to humans, demonstrating that GH treatment is associated with increases in thymic mass and circulating na?ve CD4+ T cells in adults infected with human immunodeficiency virus, type 1 (HIV-1) . These data suggest that de novo T-cell production may be inducible in immunodeficient humans.
T lymphopoiesis is dependent upon the migration of prethymic bone marrow (BM) progenitor cells to the thymus , and age-related declines in T lymphopoiesis have been attributed to both decreased hematopoietic capacity of BM progenitors and involution of the thymus (reviewed in Miller and Globerson ). Thus, GH may enhance human T lymphopoiesis by acting on cellular targets in the BM and/or in the thymus. BM appears to be an important target of GH action, as demonstrated by rodent studies showing a significant effect of GH on multilineage hematopoiesis. In studies of mice and aging rats, administration of GH and IGF-I facilitates early stages of T-cell development by increasing the number of multilineage BM progenitors and by enhancing the migration and engraftment of progenitor cells into the thymus . Similarly, in vitro analyses showed that GH stimulates erythroid and myeloid colony formation from murine and human BM progenitors. These effects appear to be mediated, at least in part, by IGF-I .
In the current study, we sought to more specifically identify cellular targets of GH and IGF-I action within the human fetal BM (FBM). We hypothesized that GH may stimulate hematopoiesis either through direct effects upon multilineage or lineage-restricted hematopoietic progenitor cells or, indirectly, by inducing FBM stromal cells to produce cytokines that facilitate the survival or maturation of FBM progenitors. We found that GH and IGF-I have direct effects on the proliferation and survival of both multilineage and lineage-committed progenitor cells, and that these hormones also enhance cytokine production by FBM stroma. These findings further delineate the effects of GH and IGF-I within human BM and provide insight into mechanisms that may contribute to GH– and IGF-I–mediated enhancement of hemato-lymphopoiesis.
MATERIALS AND METHODS
GH Stimulates Proliferation of Primitive Multilineage Hematopoietic Progenitor Cells
To identify potential GH targets within the FBM, the expression of cell-surface GHR was assessed using a biotinylated antiGHR antibody on freshly acquired FBM mononuclear cells. Various subpopulations of multilineage and lineage-restricted hematopoietic progenitor cells were discriminated on the basis of their phenotypic markers, as described in Figure 1. The frequency of each of these subpopulations in the FBM was determined for six donors (Table 1). As shown in Figure 2A (left panel), GHR was expressed on a sizeable fraction of total FBM mononuclear cells (mean 19.4%, range 15%–28.8% in seven independent experiments). Using antibodies against the lineage markers shown in Figure 1, we found that these progenitor subpopulations expressed varying levels of GHR (Fig. 2A). Incubation of total FBM mononuclear cells or CD34+ cells with GH for at least 4 days resulted in a higher yield of total cells relative to untreated controls (p < .05) (Fig. 2B, left panel). Amongst the subpopulations included within FBM, statistically significant increases were observed in the primitive CD34+CD38– multilineage progenitors as well as the CD34+CD38+CD10+ lymphoid progenitors (Fig. 2B). There was also a strong positive correlation between the fraction of cells expressing GHR within a given subpopulation and the subsequent recovery of that subpopulation after the addition of GH (Fig. 2C).
Figure 1. CD34+ progenitor cell subpopulations in FBM. (A): Flow diagram representing multilineage human hematopoiesis. Cell surface markers designate those used to identify the different progenitor cell populations . (B): Flow cytometry plots show gating strategy used for phenotypic data collection. The first panel represents gating for CD34/CD38 populations, after gating on mononuclear cells gated by forward and side scatter (not shown). The large dashed box shows the total CD34+ gate. This CD34+ population was subdivided using CD38 (small boxes, labeled 34+38– and 34+38+). The CD34+CD38+ population was first subdivided with CD10 (next panel) into CD34+CD38+CD10+ and CD34+CD38+CD10– subpopulations. The remaining panels show gating used to discriminate CD34+CD19+, CD34+CD14+, and BrdU+ cell subpopulations, respectively, after first gating on mononuclear cells (not shown). Abbreviations: BFU-E, burst-forming unit-erythroid; CFU-GEMM, colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte; CFU-GM, colony-forming unit-granulocyte-monocyte; DC, dendritic cell; FBM, fetal bone marrow; NK, natural killer.
Table 1. Frequency of FBM progenitor subpopulations
Figure 2. Effects of GH on FBM progenitor cells. (A): Expression of GHR (shaded histograms) relative to isotype control staining (open histograms) after first gating on a mononuclear cell gate. These results are representative of seven independent experiments carried out on FBM progenitor cells from seven different donors. The numbers above the bars represent the percentage of GHR+ cells, that is, the percentage of cells in given subpopulation with levels of GHR staining above those found with the isotype control. (B): Effect of GH on the yield of cells within different FBM subpopulations as a mean percentage of untreated control cultures (unfilled bars). An increase in the absolute number of many subpopulations is seen after the addition of GH (100–250 ng/ml for 4–8 days) (filled bars) to either total FBM mononuclear cells (BM) or purified CD34+ cells (*p < .05). No difference was noted between using 100 ng/ml of GH versus 250 ng/ml; these two concentrations have accordingly been grouped for the analyses shown here. These results are pooled from seven independent experiments carried out on FBM cells from seven different donors. (C): Relationship between the percentage of GHR+ cells in different hematopoietic subpopulations (as shown in A) and the mean cell yield (as shown in B) after incubation of CD34+ cells with GH (r2 = 0.767, p < .0001). (D): BrdU incorporation of CD34+38– cells after stimulation of total FBM or CD34+ cells with GH (100–250 ng/ml for 4–7 days) (filled bars) compared with untreated controls (unfilled bars). Data are pooled from six independent experiments using six different donors and are represented as a percentage of control. The increase is significant after stimulation of CD34+ cells (*p < .05). Abbreviations: BM, bone marrow; FBM, fetal bone marrow; GH, growth hormone; GHR, growth hormone receptor.
GH-associated increases in the total number of hematopoietic progenitor cells could be due to enhanced proliferation or decreased levels of apoptosis. To discriminate between these possibilities, total FBM mononuclear cells or CD34+ cells were treated with GH and analyzed by flow cytometry to detect proliferation (BrdU incorporation) and apoptosis (annexin-V binding). Levels of proliferation were significantly increased in the CD34+CD38– subpopulation after GH stimulation of CD34+ cells (Fig. 2D), whereas none of the FBM subpopulations showed significant changes in apoptosis (data not shown).
To confirm that GH stimulation resulted in an increase in functionally competent human hematopoietic progenitor cells, total FBM mononuclear cells were cultured for 6 days in the presence or absence of GH and then plated into methylcellulose cultures. As shown in Table 2, GH treatment resulted in a statistically significant (p < .05) increase in the number of CFU-GM and CFU-GEMM, but no change in the number of BFU-E.
Table 2. GH and IGF-I effects on CFU-Cs
GH Stimulates Proliferation of FBM Stromal Cells
The above data indicate that GH may interact directly with and induce the proliferation of primitive multilineage human hematopoietic progenitor cells as well as of lineage-restricted progenitor cells. Given the high expression levels of GHR on CD34+CD14+ myeloid progenitor cells, some of which might mature into FBM stromal macrophages , we hypothesized that GH might also indirectly regulate hematopoiesis through the FBM stroma. As a first test of this hypothesis, expression levels of GHR were assessed on recently plated (
Figure 3. Effects of GH on FBM stromal cells. (A): Representative surface staining of GHR on FBM stromal cells (n = 9, a mean 3.4% of total, range 0.91%–11.5%, were positive for GHR) (second panel). Gates were set using the isotype control (left panel). Many of the GHR+ cells were also CD45+ (n = 9, mean 65.3%, range 25%–97%). GHR+CD45+ FBM stromal cells are IGFR+ (n = 3, range 76%–99%) (solid line) whereas GHR+CD45– FBM stromal cells are IGFR low or negative (dashed line) (third panel). CD45+GHR+ FBM stromal cells are also CD14+ (n = 3, range 68%–93%) (solid line, right panel). (B): Incubation of FBM stromal cells with GH (100 ng/ml) results in an increase in the total number of total FBM stromal cells, stromal cells that are GHR+IGFR+CD45+, and stromal cells that are CD14+IGFR+CD45+. These panels represent pooled data from four separate experiments. Cell yields in GH-treated cultures are expressed as a mean percentage of untreated control cultures. (C): BrdU incorporation into the FBM stromal cell populations after 1 day in culture with GH addition (100 ng/ml). *p < .05 in (B, C). Abbreviations: FBM, fetal bone marrow; GH, growth hormone; GHR, growth hormone receptor; IGFR, antitype I insulin-like growth factor receptor.
Incubation of FBM stromal cultures with exogenous GH for 1 day resulted in an increased yield of stromal cells relative to untreated control cultures (p < .05) (Fig. 3B). Additionally, there was an increase of GHR+IGFR+CD45+ and CD14+IGFR+CD45+ stromal cells compared with untreated controls (Fig. 3B). Labeling studies with BrdU or annexin-V showed that these increases in cell yield were associated with increased levels of proliferation (p < .05) (Fig. 3C) without changes in apoptosis (data not shown). Thus, GH appears to induce the proliferation of myeloid-like FBM stromal cells.
IGF-I Effects on Human Multilineage Hematopoiesis
GH effects are often mediated through IGF-I . Indeed, in three FBM stromal cultures treated with exogenous GH (100 ng/ml), a significant increase (p < .05) in IGF-I production was observed relative to untreated controls (999 pg/ml versus 1,421 pg/ml, respectively; data not shown). These findings suggest that the effects of GH might be partly attributable to IGF-I.
To determine potential cellular targets of IGF-I, IGFR expression was assessed on FBM mononuclear and stromal cells. Surface expression of IGFR was detected on CD45+CD14+ myeloid stromal cells (see above, Fig. 3A) and on a large fraction of FBM progenitor cells (Fig. 4A). Additionally, mature CD10+CD45– fibroblast-like stromal cells expressed a low level of IGFR (data not shown).
Figure 4. Effects of IGF-I on FBM progenitor cells. (A): Expression of IGFR (shaded histograms) on FBM mononuclear subpopulations relative to isotype control staining (open histograms). These results are representative of three independent experiments carried out on FBM cells from three different donors. The numbers above the bars represent the percentage of IGFR+ cells, that is, the percentage of cells in given subpopulation with levels of IGFR staining above those found with the isotype control. (B): Effect of IGF-I on the yield of cells within different FBM mononuclear subpopulations as a percentage of the mean of the untreated control cultures (unfilled bars). An increase in the absolute number of many subpopulations is seen after the addition of IGF-I (100 ng/ml) (filled bars) to either total FBM mononuclear cells or CD34+ cells. These results represent pooled data from eight independent experiments carried out on FBM progenitor cells from eight different donors. (C): Effect of IGF-I on the percentage of apoptotic (annexin-V staining) cells within FBM mononuclear subpopulations as a percentage of the mean of the untreated control cultures (unfilled bars). These results represent pooled data from eight independent experiments carried out on FBM progenitor cells from eight different donors. In (B, C), *p < .05. Abbreviations: FBM, fetal bone marrow; IGF-I, insulin-like growth factor I; IGFR, antitype I insulin-like growth factor receptor.
To determine whether these cells were functionally responsive to IGF-I, total FBM mononuclear cells or purified CD34+ mononuclear cells were incubated with IGF-I. Treatment with IGF-I for 4–8 days resulted in an increase in the yield of FBM CD34+ progenitor cells relative to untreated control cultures (p < .05) (Fig. 4B, second panel from left). As in the case of GH stimulation, this increase in cell number was reflected by an increased yield of multiple hematopoietic subpopulations, including primitive, multilineage CD34+CD38– progenitor cells (Fig. 4B, remaining panels). In contradistinction to the effects of GH, the increased cell numbers observed after IGF-I treatment appeared to be primarily due to increased cell survival, with substantially lower levels of apoptosis (annexin-V staining) observed in multiple subpopulations after 4–8 days of culture (Fig. 4C). No consistent changes in the proliferation of these subpopulations (as assessed by BrdU incorporation) were observed (data not shown). IGF-I treatment of FBM stroma resulted in an increase in proliferation (as assessed by BrdU incorporation) of CD45+CD14+GHR+IGFR+ stromal cells (data not shown). This cell subpopulation also revealed an IGF-I–associated decrease in apoptosis (as assessed by annexin-V staining, data not shown).
In contrast to the effects of GH, treatment of FBM cells with IGF-I resulted not only in an increase in CFU-GM and CFU-GEMM but also in an increase in BFU-E (p < .005 in each case) (Table 2).
GH and IGF-I Induce Cytokine Secretion by FBM Stromal Cells
We hypothesized that GH and IGF-I might facilitate multilineage hematopoiesis by inducing the secretion of key hematopoietic cytokines from FBM stromal cells. To investigate this possibility, FBM stroma cultures were plated in replicate, stimulated with exogenous GH or IGF-I for 1 day, treated with brefeldin A to block secretion of induced cytokines, and then assessed by flow cytometry for the presence of intracellular IL-3. Relative to untreated cultures (Fig. 5, upper panels), IL-3 production was consistently stimulated by IGF-I (Fig. 5, middle panels) and, to a more variable and lesser degree, by GH (Fig. 5, lower panels). When lineage markers were included in the analysis, most IL-3–producing cells were found to be nonhematopoietic (CD45–CD10+) mature fibroblast-like FBM stromal cells (data not shown). Although increases in IL-3 production were observed in four of five stromal cultures incubated with GH (average = 2.2-fold) and in five of five cultures incubated with IGF-I (average = 5.9-fold), these changes were variable, even within a given experiment (and as evidenced in the replicates of Fig. 5). In addition to IL-3, we observed sporadic increases in the expression of other hematopoietic cytokines (e.g., IL-6, IL-7, and SCF; data not shown). In aggregate, these results suggest that GH may act, directly or indirectly, to induce both IGF-I and IL-3 from FBM stroma, but that the stromal cell(s) making these secondary mediators are rare.
Figure 5. Induction of IL-3 production by GH and IGF-I. FBM stromal cells were incubated in triplicate with medium alone (upper panels), 100 ng/ml IGF-I (middle panels), or 100 ng/ml GH (lower panels) for 24 hours, and then assessed by flow cytometry for the presence of intracellular IL-3. Triplicate results from a single donor are displayed. This experiment is representative of five independent experiments carried out with cells from five different donors. Abbreviations: FBM, fetal bone marrow; GH, growth hormone; IGF-I, insulin-like growth factor I; IL, interleukin.
DISCUSSION
This study was supported by National Institutes of Health (NIH) grants R37 AI40312 and R01 AI43864 (to J.M.M.) and K08 AI01597 (to L.A.N). J.M.M. is an Elizabeth Glaser Pediatric AIDS Foundation Scientist and a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and of the NIH Director’s Pioneer Award.
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