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Regulation of Myeloid Growth and Differentiation by the Insulin-Like Growth Factor I Receptor¹

Laboratories of Immunophysiology (Y.M.L., D.H.S., Q.L., S.A., N.R., K.W.K.) and Muscle Biology (R.H.M.), Department of Animal Sciences, University of Illinois, Urbana-Champaign, Illinois 61801; The Picower Institute for Medical Research (Y.M.L), Manhasset, New York 11030; the Department of Biology, Illinois State University (S.A, N.R.), Normal, Illinois 61790; and the Laboratory of Integrative Neurobiology, INRA-INSERM, U-934 (R.D.), Bordeaux, France

Address all correspondence and requests for reprints to: Dr. Keith W. Kelley, Laboratory of Immunophysiology, University of Illinois, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, Illinois 61801.

	Abstract

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Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (>79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (<1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1–25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 ± 8%) or IGF-II (59 ± 8%), but not with des-IGF-I (3 ± 1%). In contrast, the anti-IGF-IR monoclonal antibody (mAb; IR-3) inhibited the DNA synthesis caused by 10 ng/ml exogenous IGF-I (67 ± 6%), IGF-II (72 ± 8%), and des-IGF-1 (82 ± 9%). Proliferation of KG-1 myeloblasts, however, was neither stimulated by the IGFs nor inhibited by the anti-IGF-IR mAb. In the absence of exogenous IGF-I, the mAb directed against the IGF-IR significantly suppressed basal DNA synthesis of HL-60 promyeloid (72 ± 5%) and U-937 monocytic (39 ± 7%) cells, but did not affect DNA synthesis of KG-1 myeloblasts (8 ± 1%) compared to an isotype-matched control mAb. Similarly, the IR-3 mAb abrogated vitamin D₃-induced differentiation of the HL-60 cells into macrophages in serum-free medium, as assessed by expression of the leucam surface protein, CD11b. As the IR-3 mAb inhibits DNA synthesis in the presence and absence of exogenous IGF-I on receptor-bearing cells, but not IGF-IR-negative cells, these data demonstrate that both endocrine and autocrine IGF-I are potent growth factors in human myeloid cells where expression of the surface receptor, rather than the ligand, is the critical control element. More importantly, these data support the hypothesis that autocrine IGF-I may play a significant role in the differentiation of promyeloid cells into macrophages.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A NUMBER OF hormones and growth factors are now known to be synthesized by cells of the immune system, including insulin-like growth factor I (IGF-I) and IGF-II (1). Thymic, alveolar, and bone marrow-derived macrophages express messenger RNA (mRNA) for IGF-I and synthesize this peptide, and exogenous IGF-I stimulates a number of activities of leukocytes (2). IGF-I is a progression factor in the cell cycle, and we have established that it acts on myeloid cells to promote their growth (3) and inhibit their apoptotic cell death (4). Two types of IGF receptors, designated IGF-IR and IGF-IIR, are expressed in a wide variety of tissues (5). The IGF-IR consists of two peptide-binding -subunits and two membrane-spanning ß-subunits that have intrinsic tyrosine kinase activity, whereas the IGF-IIR consists of a single chain polypeptide. Binding of radiolabeled ligands has been used to identify binding sites for both IGF-I and IGF-II on human T lymphocytes, B lymphocytes, macrophages, and neutrophils (6). The existence of IGF-binding sites on leukocytes that also synthesize this ligand, such as macrophages, suggests the possibility of autocrine regulation of cell growth and differentiation.

Although macrophages are known to synthesize IGF-I, the potential autocrine function of this peptide in the growth and differentiation of myeloid cells is unknown. This is a particularly important concept in view of recent data which indicate that induction of autocrine IGFs may be critical for cell transformation (7). For example, simian virus 40 T antigen induces IGF-I synthesis, and a functional IGF-IR is required for this viral oncogene to transform fibroblasts (8, 9, 10). These findings have been significantly extended by the demonstration that growth of rat IGF-secreting glioblastoma (11) and human rabdomyosarcoma (12) cells is inhibited in vivo by transfection with an antisense IGF-IR plasmid or treatment with an antibody against the IGF-IR, respectively. A similar role has been proposed for endogenous IGF-II, acting via the surface IGF-IR, in promoting the growth of mesenchymal tumors (13). Both human and murine T and B lymphocytes synthesize very little IGF-I (14), suggesting that IGF-I may be an autocrine growth factor for myeloid, but not lymphoid cells; the latter appear to use an endocrine or paracrine source of IGF-I (15, 16).

IGF-I enhances cellular differentiation in both skeletal and neuronal systems (17), but data supporting a role for IGF-I in the differentiation of hematopoietic cells is only beginning to accumulate. Most of these efforts have been directed at the developmental events in human B cells (16, 18, 19, 20), but nothing is known about the potential autocrine role of IGF-I in macrophage differentiation. Here we show that the DNA synthesis in U-937 human monocytic cells, which express IGF-I mRNA and secrete the peptide (21, 22), is significantly inhibited by a monoclonal antibody (mAb) against the IGF-IR (IR-3). The proliferation of human KG-1 myeloblasts, which express very little IGF-IR protein, is not affected by the IR-3 mAb. More importantly, vitamin D₃ is unable to differentiate human promyeloid HL-60 cells into macrophages in the presence of the IR3 antibody, whereas it induces a 5-fold increase in the expression of a surface integrin subunit, CD11b, on HL-60 cells incubated with an isotype-matched antibody. Collectively, these results establish that IGF-I can serve as an autocrine regulator of both myeloid cell growth and differentiation.

	Materials and Methods

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Tissue culture and reagents
Tissue culture medium was prepared from powdered RPMI 1640 (MediaTech, Herndon, VA) supplemented with 2 g/liter sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma Chemical Co., St. Louis, MO). FBS (Sigma Chemical Co.) as well as other reagents and plasticware were determined to be free of endotoxin (<25 pg/ml; Limulus amoebocyte lysate assay, Associates of Cape Cod, Woods Hole, MA). The FBS was heat inactivated at 56 C for 30 min. Recombinant human IGF-I and IGF-II were purchased from Intergen (Purchase, NY). Nonglycosylated and glycosylated (CHO) recombinant human IGF-binding protein-3 (IGFBP-3) were kindly provided by Celtrix (Santa Clara, CA). Des-(1, 3)-IGF-I (des-IGF-I), which has the first three amino-terminal amino acids deleted and results in poor binding affinity to IGFBP-3 compared to untruncated IGF-I (23, 24), was a gift from Genentech (South San Francisco, CA). Mouse antihuman IGF-IR mAb (IR3, IgG₁, free of sodium azide) was purchased from Oncogene Science (Uniondale, NY), the rat anti-human CD11b mAb (IgG_2b) was obtained from BioSource International (Camarillo, CA), and the irrelevant azide-free isotype-matched murine IgG₁ as well as the control rat IgG_2b mAb were purchased from Sigma Chemical Co. The F(ab')₂ fragment of fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (absorbed against rat and human Ig) and FITC-conjugated goat antirat antibody were obtained from Cappel (Durham, NC). The human myeloid cell lines, HL-60 (promyeloid), U-937 (monocytic), and KG-1 (myeloblast), were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 medium supplemented with 10% FBS, whereas all proliferation and differentiation assays were performed on washed cells cultured for 24 h in serum-free RPMI 1640 medium.

Flow cytometry to detect IGF-I receptors
Flow cytometry was carried out as described previously for identification of cell surface markers (25). Briefly, 3 x 10⁵ cells were washed with cold PBS (1.5 M NaCl, 19 mM Na₂HPO₄·H₂O, and 8.4 mM Na₂HPO₄) supplemented with 1% FBS. The viability of the cells before staining was at least 95%. Cells were pelleted in microfuge tubes by centrifugation at 800 x g at 4 C for 3 min, and then 50 µl anti-IGF-IR mAb (100 ng) diluted with PBS were added. As controls, 50 µl irrelevant mouse IgG₁ mAb in PBS at the same concentration as the primary antibody were added. Cells were incubated for 30 min at 4 C and then washed three times with PBS and 0.1% FBS. After the final wash, 50 µl of a 1:100 dilution of FITC-conjugated goat antimouse IgG antibodies [F(ab')₂ fragments] were added to each tube. The cells were incubated for an additional 30 min at 4 C and then washed three times. Finally, cells were resuspended in 200 µl PBS and maintained on ice for immediate analysis by flow cytometry, or they were analyzed less than 24 h later after fixation with PBS and 2% paraformaldehyde.

Stained cells were examined on an EPICS V flow cytometer (Coulter Instruments, Hialeah, FL). Bitmaps were established to include a uniform population of cells with the small proportion of dead cells (<10% in all cases) excluded from the analysis. Green fluorescence was monitored by single parameter histogram analysis, and within a bitmap, at least 10,000 cells were counted for each sample. The mean fluorescence intensity of the control sample, which was stained with a murine isotype-matched irrelevant antibody and FITC-conjugated secondary antibody, was considered the background. Histograms of anti-IGF-IR and control antibody staining were overlayed and analyzed by standard flow cytometry methods (Coulter Instruments).

Cellular DNA synthesis
HL-60, U-937, and KG-1 cells were washed three times and incubated in serum-free RPMI 1640 medium for 24 h before each assay. Cell proliferation assays were performed as previously described (26). Briefly, cells were washed again twice with RPMI 1640 medium and adjusted to the appropriate cell concentration in RPMI 1640 medium. All assays were carried out in serum-free RPMI 1640 medium supplemented with 12.5 µg/ml iron-saturated human transferrin and 30 nM sodium selenite (Sigma Chemical Co.). Under basal conditions in the absence of exogenous IGFs, HL-60, U-937, and KG-1 cells were incubated with the anti-IGF-IR mAb, IR3 (4 µg/ml), or a similar amount of IgG₁ isotype-matched control antibody. Cells were also stimulated with various concentrations of IGF-I, IGF-II, or des-IGF-I in the absence or presence of the isotype-matched control antibody (IgG1), IR3 (2 µg/ml), IGFBP-3 (500 ng/ml), or IGFBP-3CHO (500 ng/ml). Cells were incubated for 30 h at 37 C in 7% CO₂ and 95% relative humidity in flat-bottomed 96-well microtiter plates (Becton Dickinson, Lincoln Park, NJ). The cells were then pulsed with 1 µCi/well tritiated thymidine (6.7 Ci/mmol; ICN, Irvine, CA) in RPMI 1640 medium for 6 h and harvested onto glass microfiber filters (Whatman, Clifton, NJ) with a 24-channel PHD cell harvester (Cambridge Technology, Cambridge, MA). Filter discs were then dried, and [³H]thymidine incorporation was measured by the addition of 3 ml Omnifluor scintillation cocktail (DuPont, Boston, MA) and counting in a Beckman LS 6000IC liquid scintillation counter (Beckman Instruments, Fullerton, CA).

Differentiation of HL-60 cells with vitamin D₃
HL-60 cells undergo differentiation to a monocyte/macrophage phenotype after the addition of 1,25-dihydroxyvitamin D₃ (27). We used the serum-free system described above along with the IR3 monoclonal antibody to determine the requirement for autocrine IGF-I in monocytic differentiation. Washed cells maintained in serum-free RPMI 1640 for 24 h were washed twice more, resuspended (1 x 10⁶ cells/ml) in RPMI 1640 medium containing 12.5 µg/ml human transferrin and 30 nM sodium selenite, and then preincubated with IR3 (5 µg/ml) or an isotype-matched control antibody (5 µg/ml) at 37 C for 1 h. Subsequently, 1,25-dihydroxyvitamin D₃ (1 µM; Hoffman LaRoche, Nutley, NJ) was added, and the cells were then cultured for an additional 48 h. Monocytic differentiation was determined using flow cytometry by assessing the increase in the percentage of cells binding a mAb specific for the myeloid leucam surface marker CD11b. Vitamin D₃-treated cells (1 x 10⁶) were washed once in PBS containing 0.5% FBS and 0.25% BSA. Cells were then incubated with a rat antihuman CD11b mAb (2 µg/ml) or the appropriate isotype-matched control (rat IgG_2b) for 30 min at 4 C. After two washes with the same buffer, cells were then incubated with a secondary FITC-conjugated goat anti-rat F(ab')₂ fragment for 30 min at 4 C. The cells were washed twice and fixed in PBS containing 1% formaldehyde and subsequently analyzed by flow cytometry (EPICS V, Coulter). For each sample, the immunofluorescence intensity of cells stained with the isotype-matched control was used to establish a bitmap of at least 5000 cells of uniform size.

Statistical analysis
Data were analyzed using the Statistical Analysis System (28), with Student’s t test used to detect differences between treatments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
HL-60 and U-937, but not KG-1, cells express detectable binding to the IGF-IR antibody
Flow cytometric analysis was used to directly identify the IGF-IR protein on the surface of three types of hematopoietic cells at various stages of differentiation along the monocyte/macrophage lineage. The application of indirect fluorescence using flow cytometry in conjunction with the IR3 mAb has previously been shown to yield nearly identical results as those obtained with radioligand binding, being able to easily detect 250 IGF-I-binding sites/human thymocyte with a K_d of 0.1 nM (29). Here we used this sensitive approach of indirect immunofluorescence by staining cells with the mouse antihuman IGF-IR mAb (IR3) or with a murine IgG₁ isotype-matched irrelevant mAb followed by staining with a secondary goat antimouse FITC-conjugated F(ab')₂ antibody fragment. The fluorescence histogram of HL-60 and U-937 cells stained with anti-IGF-IR mAb showed a clear increase in fluorescence above that in cells treated with the isotype-matched control antibody, indicating that these cells express abundant IGF-IR protein (Fig. 1). The results of six independent experiments showed that background immunofluorescence with the isotype-matched control antibody for HL-60, U-937, and KG-1 cells was 5 ± 0.3%, 5 ± 0.3%, and 6 ± 0.5%, respectively, whereas these values after staining with the IR3 mAb were 84 ± 2% (P < 0.01), 93 ± 2% (P < 0.01), and 7 ± 1% (P > 0.10). The small increase of 1% above the isotype-matched control mAb in IGF-IR-positive KG-1 cells that were stained with the IR3 mAb was not significant (P > 0.10).

View larger version (21K): [in this window] [in a new window]   Figure 1. Flow cytometric analysis of myeloid cells to characterize the expression of IGF-IR. Cells were first stained with either an anti-IGF-IR mAb or isotype-matched (IgG1) control mAb and then stained with fluorescence-labeled secondary F(ab')2 antibody fragments. At least 10,000 cells of each sample were analyzed by flow cytometry within a bitmap. Flow cytometry data using the anti-IGF-IR mAb (IR3) were plotted as cell number (ordinate) vs. relative units of fluorescence intensity (abscissa). The control IgG1 mAb histogram was considered background fluorescence. An increase in fluorescence intensity was clearly observed in HL-60 and U-937, but not in KG-1, cells.

IGF-I, IGF-II, and des-IGF-I increase DNA synthesis in HL-60 and U-937, but not KG-1, cells
To determine whether stimulation of IGF-I receptors affected the activity of these myeloid cells, a serum-free culture system was developed and then used to test the effects of four different treatments (Table 2) on three concentrations (1, 10, and 25 ng/ml; Tables 1 and 2) of three ligands (IGF-I, IGF-II, and des-IGF-I). As shown in Table 1, increasing amounts of IGF-I or des-IGF-I caused a dose-dependent increase in DNA synthesis in HL-60 cells by a minimum of nearly 2-fold at 1 ng/ml (P < 0.01; basal proliferation was 40,504 ± 3,521 cpm without IGFs). Similarly, 10 and 25 ng/ml, but not 1 ng/ml, IGF-I or IGF-II augmented DNA synthesis (P < 0.01) of U-937 cells (Table 1; basal, 85,607 ± 19,588 cpm). In contrast, IGF-I (25 ng/ml) did not increase DNA synthesis of KG-1 cells (6 ± 4%; n = 3; P > 0.10), and indeed, the enhancement of DNA synthesis in these cells by IGF-I, IGF-II, or des-IGF-I at any concentration tested never resulted in an increase that was statistically significant (P < 0.10; Table 1; basal, 85,358 ± 9,659 cpm). This is consistent with the finding that KG-1 cells lack significant amounts of IGF-IR protein on their extracellular surface, as determined by flow cytometric analysis.

IGF-IR mediates IGF-I, IGF-II, and des-IGF-I-induced proliferation of HL-60 cells
To determine the degree to which the IGF-IR is involved in IGF-induced cell proliferation in HL-60 cells, we examined the effect of the IR3 anti-IGF-IR mAb on IGF-I-, IGF-II-, and des-IGF-I-induced DNA synthesis (Table 2). In these experiments, the IR3 mAb was added to serum-free cultures of HL-60 cells. An isotype-matched IgG₁ antibody was used as a control, which had no effect on DNA synthesis in HL-60 cells (Table 2; P > 0.10). However, the anti-IGF-IR mAb significantly suppressed DNA synthesis in HL-60 cells incubated with 10 ng IGF-I, IGF-II, or des-IGF-I (67 ± 6%, 72 ± 8%, and 82 ± 9%, respectively; P < 0.01; Table 2). These results clearly demonstrate that the effects of exogenous IGF-I and IGF-II on the proliferation of HL-60 cells are primarily mediated by the IGF-IR.

IGFBP-3 inhibits IGF-I-induced DNA synthesis in HL-60 cells
Although IGFBPs can inhibit or potentiate (17) IGF-induced cell proliferation in different types of cells, the influence of IGFBPs on either lymphoid or myeloid cell proliferation is only beginning to be elucidated (3). We, therefore, examined the effects of both nonglycosylated (IGFBP-3) and glycosylated (IGFBP-3CHO) IGFBP-3 on DNA synthesis in HL-60 cells cultured with IGF-I, IGF-II, and des-IGF-I. As shown in Table 2, DNA synthesis induced by 10 ng IGF-I was significantly inhibited by 500 ng IGFBP-3 (53 ± 8%; P < 0.01) or IGFBP-3CHO (57 ± 10%; P < 0.01), whereas proliferation of HL-60 cells induced by 10 ng IGF-II was also inhibited by the same dose of IGFBP-3 (59 ± 8%; P < 0.01) and IGFBP-3CHO (53 ± 6%; P < 0.01). In contrast, neither form of IGFBP-3 inhibited DNA synthesis of HL-60 cells cultured with any dose of des-IGF-I (Table 2). This is probably due to the poor binding affinity of des-IGF-I to IGFBP-3 (23, 24). These results show that IGFBP-3 inhibits IGF-I- and IGF-II-induced DNA synthesis in HL-60 cells and that glycosylation of IGFBP-3 is not required for this inhibition.

Anti-IGF-I IR mAb suppresses basal DNA synthesis in HL-60 and U-937 cells
These data established that HL-60 and U-937 cells express specific IGF-IRs and that stimulation of these receptors with exogenous IGF-I, IGF-II, or des-IGF-I increases DNA synthesis after binding to these IGF-IRs. These findings are consistent with an earlier report showing that the growth of some T and B acute lymphoblastic leukemia cells can be inhibited by the IR3 antibody when grown in 5% FBS as a source of IGF-I (31). Therefore, we next determined the effect of this antibody on DNA synthesis of these cells grown in serum-free medium and in the absence of recombinant IGF-I. Table 3 presents a summary of several experiments which demonstrate that the anti-IGF-IR IR3 mAb significantly inhibited DNA synthesis in both HL-60 and U-937 cell lines by 72 ± 5% and 39 ± 7% (P < 0.01), respectively. The murine isotype-matched control antibody did not affect (P > 0.10) proliferation in any of the three types of cells. Consistent with the finding that KG-1 cells do not express the IGF-IR, the anti-IGF-IR mAb did not alter the proliferation of KG-1 cells, nor did the isotype-matched control mAb (P > 0.10).

An antibody to the IGF-IR prevents monocyte/macrophage differentiation of HL-60 cells
To test the hypothesis that myeloid cell differentiation is also regulated by IGF-I, we used the well established system of treating HL-60 cells with vitamin D₃ in serum-free medium to induce their differentiation into monocytes/macrophages. Very few HL-60 cells expressed the surface integrin -subunit CD11b (4 ± 1%; Fig. 2). However, the addition of vitamin D₃ caused these cells to differentiate along the monocyte/macrophage pathway, as shown by a 5-fold increase in the expression of CD11b (22 ± 3%). Coincubation of vitamin D₃-treated HL-60 cells with the anti-IGF-IR mAb (IR-3) effectively reduced the level of CD11b expression to that of control cells (7 ± 2%; Fig. 2). Importantly, incubation of HL-60 cells with an isotype-matched IgG₁ control antibody had no effect (21 ± 3%) on the ability of vitamin D₃ to induce differentiation in these cells.

View larger version (21K): [in this window] [in a new window]   Figure 2. Inhibition of HL-60 differentiation with an antibody to the IGF-IR. Cells were preincubated with the antiinterleukin-IR mAb (IR-3) or an isotype-matched control mouse IgG1 (IgG) antibody in serum-free medium for 1 h, followed by the addition of 1 µM vitamin D3 (D3). After 2 days of incubation, the proportion of cells expressing the CD11b surface antigen of mature myeloid cells was determined by flow cytometry. Data were expressed as the mean ± SEM of six experiments; means with different superscripts differ significantly (P < 0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recent findings showing that expression of the IGF-IR is required for simian virus 40-mediated fibroblast transformation in vitro and that disruption of the IGF-IR on tumor cells limits their growth in vivo suggest that this receptor is critical for the development of the transformed phenotype and subsequent proliferation of transformed cells (7). Similarly, activation of the tumor-suppressor gene p53 has recently been shown to induce IGFBP-3 expression, which suppresses IGF-I-driven cellular proliferation in colon carcinoma cells (34). The present experiments support these recent findings by demonstrating a regulatory role for IGFBPs, because IGFBP-3 inhibited the IGF-I-induced enhancement of DNA synthesis in HL-60 cells. These data suggest that IGF-I, IGF-IR, and at least one of the IGFBPs, collectively known as the IGF-I triad, are involved in regulating the survival and proliferation of transformed cells. Interestingly, IGFBP-3 did not inhibit the basal proliferation of HL-60 and U-937 cells (data not shown) or the Saos-2 cells used in the studies of Buckbinder et al. (34). Instead, our findings suggest that endogenous IGF-I acts via its surface receptor to promote both the growth and differentiation of myeloid cells. This conclusion is supported by the finding that an antibody to the IGF-IR inhibited basal DNA synthesis in both U-937 and HL-60 cells and prevented the developmental expression of CD11b during myelopoiesis. Furthermore, KG-1 cells, which do not express significant amounts of the IGF-IR, failed to respond to exogenous IGF-I, and their proliferation was not inhibited by the anti-IGF-IR antibody.

Three types of myeloid cells that represent different stages of cell development (27) were used to examine the expression of IGF-IR protein on these cells. Binding sites for IGF-I have been shown to be expressed on some hematopoietic cells by binding assays employing ¹²⁵I-conjugated IGF-I (6). Although this method detects binding sites for IGF-I, this technique does not directly and readily define which type of IGF receptor is responsible for the binding of [¹²⁵I]IGF-I, because this ligand can bind to both the IGF-IR and IGF-IIR, albeit with different affinities (17). Furthermore, the IR-3 mAb will bind to an occupied IGF-IR, whereas radiolabeled IGF-I cannot, and IGF-II binds nearly as well as IGF-I to the IGF-IR (35). Although IGF-I and IGF-II usually bind to the insulin receptor with a low affinity, they bind to atypical insulin receptors on the human lymphoid cell line, IM-9, with a moderately high affinity (36).

The proliferative response of promyeloid HL-60 and U-937 monocytic cells to both endogenous and exogenous IGF-I depends on the expression of IGF-IR on their cell surface. In contrast, KG-1 myeloblasts, which lack readily detectable IGF-IR on their cell surface, do not respond to IGF-I, IGF-II, des-IGF-I, or the anti-IGF-IR antibody. This is consistent with the original observation that the level of [¹²⁵I]IGF-I binding in hematopoietic cells is dependent upon their differentiation status (30) and with the well recognized heterogeneity of myeloid cell populations leading from a stem cell to mature macrophages (37). Our data showing that the effect of both IGF-I and IGF-II on DNA synthesis of HL-60 cells is mediated through the IGF-IR is consistent with a recent report showing that both IGF-I and IGF-II bind to the IGF-IR on human basophils and enhance histamine release (38) and with the conclusion that the IGF-IR mediates in vivo growth induced by both IGF-I and IGF-II, as assessed in mice with targeted disruption of the IGF-IR (39). Our data also support the previous conclusion (40) that expression of the IGF-IR is critical for the proliferation and differentiation of hematopoietic cells to both endogenous and exogenous IGFs.

IGFBPs are important regulators of the biological actions of IGF-I (17). Cytokines, such as transforming growth factor-ß and tumor necrosis factor-, modulate the sensitivity of human fibroblasts to stimulation with IGF-I by altering IGFBP production (41). Neely et al. (42) reported that 6 of 12 human lymphoblast T and B cell lines expressed IGFBP-4 or IGFBP-2, as identified by Western ligand blotting. We have recently developed a sensitive affinity cross-linking technique with [¹²⁵I]IGF-I to show that murine macrophages synthesize and secrete IGFBP-4 (22). However, although new results show that IGFBPs are clearly involved in pathways that can inhibit the growth of both colon carcinoma (33) and breast cancer (43) cells, the potential role of IGFBPs in regulating myeloid cell development and transformation is only beginning to be explored. The present data show that IGFBP-3 suppresses IGF-I-induced proliferation of both HL-60 and U-937 cells. Des-IGF-I, which is bound with 5- to 100-fold lower affinity to IGFBP-3 (23, 24), appears to be more effective at lower concentrations than IGF-I and IGF-II in stimulating the proliferation of HL-60 and U-937 cells. Reduced binding of des-IGF-I to IGFBP-3 probably explains why neither form of IGFBP-3 inhibited the proliferation of HL-60 or U-937 cells cultured with des-IGF-I.

Substantial evidence supports the contention that IGF-I is directly involved in the growth and differentiation of myeloid, lymphoid, and erythroid cells (2, 6). This autocrine regulation of IGF-I could have both physiological and pathological significance. Wound macrophages, activated human alveolar macrophages, and monocytes secrete IGF-I, and colony-stimulating factor-1-induced synthesis of IGF-I by bone marrow-derived macrophages (3, 14) is potently inhibited by interferon- (44). Activated macrophages in ischemic cerebral cortex express mRNAs for IGF-I, IGF-II, and the IGF-IIR (45). Furthermore, IGF-I and IGF-II have been shown to promote the growth of nonmyelogenous tumors (13). It is, therefore, possible that IGF-I acts in an autocrine as well as an endocrine fashion to promote progression through late G1 in some leukemias (2, 6), such as we found in both HL-60 and U-937 cells. However, in the absence of the IGF-IR, cells do not respond to either IGF-I or IGF-II, as demonstrated by our results with KG-1 myeloblasts. The finding that IGFBP-3 inhibits IGF-I- and IGF-II-induced DNA synthesis in HL-60 as well as in primary murine CSF-1-differentiating bone marrow cells (3) further indicates that IGFBPs might be an important regulatory factor that affects both the growth and differentiating activities of IGF-I on myeloid cells.

In summary, these data establish that myeloid cells are heterogeneous in the expression of IGF-IR, a characteristic that is most likely related to the stage of development of these cells. Expression of IGF-IRs on cells of the myeloid lineage enhances DNA synthesis in response to either endogenous or exogenous IGF-I and promotes the ability of these cells to differentiate into a more mature phenotype. Both endogenous and exogenous IGF-I and IGF-II as well as IGFBP-3 regulate the growth and differentiation of myeloid lineages bearing the IGF-IR.

	Footnotes

 
¹ This work was supported by a grant (to K.W.K.) from the NIH (AG-06246).

Received July 17, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Weigent DA, Blalock JE 1995 Associations between the neuroendocrine and immune systems. J Leukocyte Biol 58:137–150[Abstract]
2. Minshall C, Liu Q, Arkins S, Kelley KW 1996 Growth hormone and immunology. In: Torosian MH (ed) Growth Hormone in Critical Illness–Research and Clinical Studies. Landes, Austin, pp 161–186
3. Arkins S, Rebeiz N, Brunke-Reese DL, Minshall C, Kelley KW 1995 The colony-stimulating factors induce expression of insulin-like growth factor-I messenger ribonucleic acid during hematopoiesis. Endocrinology 136:1153–1160[Abstract]
4. Minshall C, Arkins S, Freund GG, Kelley KW 1996 Requirement for phosphotidylinositol 3'-kinase to protect hematopoietic progenitors against apoptosis depends upon the extracellular survival factor. J Immunol 156:939–947[Abstract]
5. LeRoith D, Werner H, Beitner-Johnson D, Roberts Jr CT 1995 Molecular and cellular aspects of the insulin-like growth factor-I receptor. Endocr Rev 16:143–163[CrossRef][Medline]
6. Kooijman R, Hooghe-Peters EL, Hooghe R 1996 Prolactin, growth hormone and insulin-like growth factor-I in the immune system. Adv Immunol 63:000–000
7. Baserga R 1994 Oncogenes and the strategy of growth factors. Cell 79:927–930[CrossRef][Medline]
8. Porcu P, Ferber A, Pietrzkowski Z, Roberts CT, Adamo M, LeRoith D, Baserga R 1992 The growth stimulatory effect of simian virus 40 T antigen requires the interaction of insulin-like growth factor-I with its receptor. Mol Cell Biol 12:5069–5077[Abstract/Free Full Text]
9. Porcu P, Graña X, Li S, Swantek J, De Luca A, Giordano A, Baserga R 1994 An E2F binding sequence negatively regulates the response of the insulin-like growth factor I (IGF-I) promoter to simian virus 40T antigen and to serum. Oncogene 9:2125–2134[Medline]
10. Sell C, Rubini M, Rubin R, Liu JP, Efstratiadis A, Baserga R 1993 Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type-I insulin-like growth factor receptor. Proc Natl Acad Sci USA 90:11217–11221[Abstract/Free Full Text]
11. Resnicoff M, Sell C, Rubini M, Coppola D, Ambrose D, Baserga R, Rubin R 1994 Rat glioblastoma cells expressing an antisense RNA to the insulin-like growth factor-I (IGF-I) receptor are nontumorigenic and induce regression of wild-type tumors. Cancer Res 54:2218–2222[Abstract/Free Full Text]
12. Kalebic T, Tsokos M, Helman LJ 1994 In vivo treatment with antibody against IGF-I receptor suppresses growth of human rhabdomyosarcoma and down-regulates p34^cdc2. Cancer Res 54:5531–5534[Abstract/Free Full Text]
13. Daughaday WH 1990 The possible autocrine/paracrine and endocrine roles of insulin-like growth factors of human tumors. Endocrinology 127:1–4 (Editorial)[Medline]
14. Arkins S, Rebeiz N, Biragyn A, Reese DL, Kelley KW 1993 Murine macrophages express abundant IGF-I class I, Ea and Eb transcripts. Endocrinology 133:2334–2343[Abstract]
15. Gjerset RA, Yeargin J, Volkman SK, Vila V, Arya J, Haas M 1990 Insulin-like growth factor-I supports proliferation of autocrine thymic lymphoma cells with a pre-T cell phenotype. J Immunol 145:3497–3501[Abstract]
16. Landreth KS, Narayanan R, Dorshkind K 1992 Insulin-like growth factor-I regulates pro-B cell differentiation. Blood 80:1207–1212[Abstract/Free Full Text]
17. Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:3–34[CrossRef][Medline]
18. Gibson LF, Piktel D, Landreth KS 1993 Insulin-like growth factor-I potentiates expansion of interleukin-7-dependent pro-B cells. Blood 82:3005–3011[Abstract/Free Full Text]
19. Kimata H, Fujimoto M 1994 Growth hormone and insulin-like growth factor I induce immunoglobulin (Ig)E and IgG4 production by human B cells. J Exp Med 180:727–732[Abstract/Free Full Text]
20. Robbins K, McCabe S, Scheiner T, Strasser J, Clark R, Jardieu P 1994 Immunological effects of insulin-like growth factor-I-enhancement of immunoglobulin synthesis. Clin Exp Immunol 95:337–342[Medline]
21. Nagaoka I, Trapnell BC, Crystal RG 1990 Regulation of insulin-like growth factor-I gene expression in the human macrophage-like cell line U-937. J Clin Invest 85:448–455
22. Li YM, Arkins S, McCusker RH Jr, Donovan SM, Liu Q, Jayaraman S, Dantzer R, Kelley KW 1996 Macrophages synthesize and secrete a 25-kilodalton protein that binds insulin-like growth factor-I. J Immunol 156:64–72[Abstract]
23. Forbes B, Szabo L, Baxter RC, Ballard FJ, Wallace JC 1988 Classification of the insulin-like growth factor binding proteins into three distinct categories according to their binding specificities. Biochem Biophys Res Commun 157:196–202[CrossRef][Medline]
24. Clemmons DR, Dehoff ML, Busby WH, Bayne ML, Cascieri MA 1992 Competition for binding to insulin-like growth factor (IGF) binding protein-2, 3, 4, and 5 by the IGFs and IGF analogs. Endocrinology 131:890–895[Abstract]
25. Li YM, Brunke DL, Dantzer R, Kelley KW 1992 Pituitary epithelial cell implants reverse the accumulation of CD4^-CD8^- lymphocytes in thymus glands of aged rats. Endocrinology 130:2703–2709[Abstract]
26. Franklin RA, Li YM, Arkins S, Kelley KW 1990 Glutathione augments in vitro proliferative responses of lymphocytes to concanavalin A to a greater degree in old than in young rats. J Nutr 120:1710–1717
27. Harris P, Ralph P 1985 Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J Leukocyte Biol 37:407–422[Abstract]
28. SAS Institute Inc 1989 SAS/STAT User’s Guide, version 6, ed 4, vol 2. SAS Institute, Cary
29. Kooijman R, Scholtens LE, Rijkers GT, Zegers BJM 1995 Type I insulin-like growth factor receptor expression in different developmental stages of human thymocytes. J Endocrinol 147:203–209[Abstract]
30. Pepe MG, Ginzton NH, Lee PDK, Hintz RL, Greenberg PL 1987 Receptor binding and mitogenic effects of insulin and insulin-like growth factors I and II for human myeloid leukemia cells. J Cell Physiol 133:219–227[CrossRef][Medline]
31. Baier TG, Jenne EW, Blum W, Schönberg D, Hartmann KKP 1992 Influence of antibodies against IGF-I, insulin or their receptors on proliferation of human acute lymphoblastic leukemia cell lines. Leukemia Res 16:807–814[CrossRef][Medline]
32. Prager D, Li H-L, Asa S, Melmed S 1994 Dominant negative inhibition of tumorigenesis in vivo by human insulin-like growth factor I receptor mutant. Proc Natl Acad Sci USA 91:2181–2185[Abstract/Free Full Text]
33. Neuenschwander S, Roberts Jr CT, LeRoith D 1995 Growth inhibition of MCF-7 breast cancer cells by stable expression of an insulin-like growth factor-I receptor antisense ribonucleic acid. Endocrinology 136:4298–4303[Abstract]
34. Buckbinder L, Talbott R, Velasco-Miguel S, Takenaka I, Faha B, Selzinger BR, Kley N 1995 Induction of the growth inhibitor IGF binding protein-3 by p53. Nature 377:646–649[CrossRef][Medline]
35. Steele-Perkins G, Turner J, Edman JC, Hari J, Pierce SB, Stover C, Rutter WJ, Roth RA 1988 Expression and characterization of a functional human insulin-like growth factor-I receptor. J Biol Chem 263:11486–11492[Abstract/Free Full Text]
36. Jonas HA, Cox AJ 1990 Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9). Biochem J 266:737–742[Medline]
37. Rutherford MS, Witsell A, Schook LB 1993 Mechanisms generating functionally heterogenous macrophages: chaos revisited. J Leukocyte Biol 53:602–618[Abstract]
38. Hirai K, Miyamasu M, Yamaguchi M, Nakajima K, Ohtoshi T, Koshino T, Takaishi T, Morita Y, Ito K 1993 Modulation of human basophil histamine release by insulin-like growth factors. J Immunol 150:1503–1508[Abstract]
39. Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor I (igf-i) and type I IGF receptor (ifg1r). Cell 75:59–72[Medline]
40. Reiss K, Porcu P, Sell C, Pietrzkowski Z, Baserga R 1992 The insulin-like growth factor-I receptor is required for the proliferation of hemopoietic cells. Oncogene 7:2243–2248[Medline]
41. Yateman ME, Claffey DC, Cwyfan Hughes SCC, Frost VJ, Wass JAH, Holly JMP 1993 Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production. J Endocrinol 137:151–159[Abstract]
42. Neely EK, Smith SD, Rosenfeld RG 1991 Human leukemic T and B lymphoblasts produce insulin-like growth factor binding proteins 2 and 4. Acta Endocrinol (Copenh) 124:707–714[Medline]
43. Martin JL, Coverley JA, Pattison ST, Baxter RC 1995 Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol. Endocrinology 136:1219–1226[Abstract]
44. Arkins S, Rebeiz N, Brunke-Reese DL, Biragyn A, Kelley KW 1995 Interferon- inhibits macrophage insulin-like growth factor-I synthesis at the transciptional level. Mol Endocrinol 9:350–360[Abstract]
45. Lee W-H, Clemens JA, Bondy CA 1992 Insulin-like growth factors in the response to cerebral ischemia. Mol Cell Neurosci 3:36–43[CrossRef]

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