Biology of Blood and Marrow Transplantation
Volume 10, Issue 1 , Pages 32-39, January 2004

Effects of organ-specific loss of insulin-like growth factor-I production on murine hematopoiesis1

  • Lisbeth A Welniak

      Affiliations

    • Department of Microbiology & Immunology, University of Nevada School of Medicine, Reno, Nevada, USA
    • ,
  • Michael Karas

      Affiliations

    • National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland, USA
    • ,
  • Shoshana Yakar

      Affiliations

    • National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland, USA
    • ,
  • Miriam R Anver

      Affiliations

    • Pathology/Histotechnology Laboratory, SAIC-Frederick; National Cancer Institute-Frederick, Frederick, Maryland, USA
    • ,
  • William J Murphy

      Affiliations

    • Corresponding Author InformationCorrespondence and reprint requests: William J. Murphy, PhD, Department of Microbiology & Immunology, University of Nevada School of Medicine, Applied Research Bldg., MS 199, Reno, NV 89557, USA
    • Department of Microbiology & Immunology, University of Nevada School of Medicine, Reno, Nevada, USA
    • ,
  • Derek LeRoith

      Affiliations

    • National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, Maryland, USA

Received 7 August 2003; accepted 8 September 2003.

Article Outline

Abstract 

To determine whether circulating insulin-like growth factor (IGF)-I has a role in hematopoiesis, we examined hematologic parameters in mice with markedly reduced serum levels resulting from a liver-specific inactivation of the IGF-I gene. These mice have normal postnatal growth and development, suggesting that local production of IGF-I can maintain anabolic effects. Liver-specific IGF-I-deficient (LID) mice were compared with control littermates with regard to hematopoietic parameters. Spleen cellularity was decreased in the LID mice compared with control mice. Spleen myeloid progenitors, as determined by colony-forming units-granulocyte/monocyte (CFU-GM) and colony-forming units-high proliferative potential (CFU-HPP), were significantly decreased in the LID mice. Immune parameters, as indicated by the absolute number of B and T cells, did not significantly differ between the knockout and control mice. In contrast to the decreased cellularity and myelopoiesis in the spleen, bone marrow cellularity was not different between the 2 groups, but the total femoral content of CFU-GM and CFU-HPP was significantly increased in the LID mice. The decrease in splenic myelopoiesis was not due to the inability of progenitors to exit the bone marrow, because CFU-GM and burst-forming units-erythroid were significantly increased in the blood of LID mice compared with normal littermates. Administration of exogenous IGF-I to the LID mice for 4 days partially restored myelopoietic parameters in the spleen. Liver production of IGF-I and, therefore, normal serum levels of this hormone, although not necessary for general organ growth and development, seems necessary for survival or transition of myeloid progenitors into the spleen.

Keywords:  Myelopoiesis, Growth hormone, Mobilization, Progenitors

 

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Introduction 

The insulin-like growth factors (IGFs) are members of the insulin-related peptide family, which includes IGF-I, IGF-II, and insulin [1]. IGF-I and IGF-II, acting through the IGF-I receptor, are essential for normal growth and development 2, 3, 4.

The original somatomedin hypothesis suggested that growth hormone (GH), produced in the anterior pituitary, stimulated the production and secretion of IGF-I by the liver, which then mediated the effects of GH in various tissues. However, it has been recently shown that liver-derived IGF-I, the major source of this hormone in the circulation, is not needed for normal postnatal growth [5]. Local production of IGF-I active either in paracrine/autocrine or endocrine fashion seems to be sufficient for GH-induced somatic growth 5, 6.

IGF-I has been shown to promote hematopoietic cell growth in vitro [7], but the role of endogenous IGF-I in hematopoiesis is unclear. Examination of the role of IGF-I in hematopoiesis through the use of null mutation experiments has been hampered by the severity of the growth retardation, which complicates evaluation of bone and hematopoiesis in IGF-I-deficient animals.

Administration of either GH or IGF-I has been shown to improve hematopoietic function in the bone marrow and spleens in aged rats [8] after myelosuppression 9, 10 or after bone marrow transplantation [11]. Although these agents can improve recovery of stressed or injured marrow, previous studies have also shown that administration or overexpression of GH or IGF-I results in increased splenic myelopoiesis, with increases in DNA synthesis and hematopoietic progenitors but does not change the cellular content of the bone marrow 9, 10, 12. In this liver-specific IGF-I-deficient (LID) model, which exhibits low serum IGF-I and has a 4.5-fold increase in serum GH [5], we were able to examine the effect of increased serum GH with concomitant low serum IGF-I levels on hematopoiesis to dissect the putative overlapping roles of GH and IGF-I on hematopoiesis.

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Materials and methods 

Mice 

Mice with a liver-specific deletion of the igf1 gene were generated by using the Cre/loxP system as previously described [5]. Sex-matched littermates with the igf1 gene flanked by loxP sites but lacking the Cre-transgene and thus expressing liver-specific IGF-I served as controls [5]. Mice were bred and maintained under specific pathogen-free conditions in the National Institutes of Health Bethesda facility. Male mice at 4 weeks of age were used for immune and hematopoiesis studies. These studies were approved by the Animal Care and Utilization Committee of the National Institute of Diabetes and Digestive and Kidney Disease. All animals used in this research project were cared for and used humanely according to the following policies: the US Public Health Service Policy on Humane Care and Use of Animals (1996); the Guide for the Care and Use of Laboratory Animals (1996); and the US Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (1985). All National Cancer Institute-Frederick animal facilities and the animal program are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

Cell preparation 

Bone marrow cells were extracted by flushing bone marrow plugs from femurs with an RPMI medium-filled syringe fitted with a 22-gauge hypodermic needle, and single-cell suspensions were prepared by repeated aspirations of the plugs. Single-cell suspensions of spleens were prepared by gently crushing the organs in RPMI-1640 supplemented with 10% fetal bovine serum, l-glutamine, and antibiotics (100 U/mL penicillin and streptomycin). Spleen cell preparations and heparinized peripheral blood cells were treated with ACK lysis buffer (Quality Biologicals, Gaithersburg, MD) to remove red blood cells. Nucleated spleen, bone marrow, and peripheral blood cell counts were performed with a Z1 Coulter counter (Coulter Electronics, Hialeah, FL).

Colony assays 

For colony-forming units-granulocyte/monocyte (CFU-GM) and burst-forming units-erythroid (BFU-E), bone marrow cells, spleen cells, or peripheral blood cells were plated in 35-mm petri dishes at a concentration of 5 × 104, 5 × 105, or 3 × 105 nucleated cells per plate, respectively. Colony formation was stimulated with 10 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor, 10 ng/mL recombinant murine interleukin (IL)-3 (Peprotech Inc., Rocky Hill, NJ), and 5 U/mL erythropoietin (Stem Cell Technologies, Vancouver, British Columbia, Canada). Plates were incubated at 37°C for 7 days in 5% CO2 and 100% humidity. Colonies were defined as aggregates of more than 50 cells. Colonies that contained only red blood cells were scored as BFU-E. Colonies that consisted of only white blood cells were scored as CFU-GM. For colony-forming unit-high proliferative potential (CFU-HPP), bone marrow cells or spleen cells were plated in 60-mm petri dishes at a concentration of 2.5 × 104 or 5 × 105 nucleated cells per plate, respectively. Colony formation was stimulated with 20 ng/mL recombinant human IL-6, 20 ng/mL recombinant murine IL-3, and 100 ng/mL stem cell factor. Plates were incubated at 37°C for 10 days in 5% CO2 and 100% humidity. CFU-HPP were defined as colonies with a diameter of >2 mm and an estimated cell number of >10 000.

Flow cytometry 

Lymphocyte subsets in the spleen were evaluated by 2-color flow cytometric analysis on a FACScan affixed with a 488-nm laser (Becton Dickinson, Mountain View, CA). Spleen cells (106 cells) were labeled with primary antibody for 20 minutes at 4°C, washed, and fixed with 1% paraformaldehyde. Data were collected on viable lymphocytes on the basis of forward and side scatter, and 104 cells were analyzed for expression of surface markers. Analysis was performed by using CellQuest software (Becton Dickinson). Monoclonal antibodies (BD Pharmingen, San Diego, CA) included fluorescein isothiocyanate-conjugated anti-CD8 monoclonal antibodies (clone 53–6.7), anti-immunoglobulin M (clone R6–60.2), and the phycoerythrin-conjugated antibodies anti-CD4 (RM4–5) and CD45R (clone RA3–6B2). Isotype-matched controls were used to quantify nonspecific background binding, which was subtracted from all results.

Histology 

Mice were killed by CO2 asphyxiation. Tissues were removed and fixed in 10% buffered neutral formalin. Femurs were decalcified. Tissues were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. Tissue sections were reviewed by a veterinary pathologist.

IGF-I administration 

Mice received subcutaneous injections of recombinant human IGF-I (Genentech, San Francisco, CA; 1 mg/kg) daily for 4 days.

Statistical analysis 

Statistical analysis was performed by using the Student t test. Values shown are means ± SEM. Differences of P < .05 were considered significant.

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Results 

Initial examination of LID mice revealed normal body and organ development except for a decrease in spleen weight [5]. Serum IGF-I levels were reduced by >50% in LID mice compared with control mice containing both loxP-flanked igf1 alleles [5]. Because IGF-I affects immune cell number, function, and hematopoiesis (see reviews 13, 14), we investigated the reduced size of the spleen by examining both the lymphoid and hematopoietic compartments of this tissue. The 22% reduction in nucleated spleen cells (Figure 1A) was consistent with the original observation of decreased spleen weight in the LID mice relative to controls [5]. Normal splenic architecture was maintained in the LID mice (data not shown). In addition to normal architecture, the frequencies of T and B cells were not significantly different between LID mice and controls (Table 1). These findings were not unexpected because previous reports found no defect in B- or T-cell development in either lit/lit mice, which do not secrete GH from the anterior pituitary, or IGF-I-deficient mice 13, 15.

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  • Figure 1. 

    Decreased splenic myelopoiesis in LID mice. A, Total number of nucleated cells per spleen in LID and control mice (P < .05). B, Frequency of committed myeloid (CFU-GM; P < .0005) and erythroid (BFU-E; not significant) progenitors and primitive progenitors (CFU-HPP; P < .001) in the spleens of LID mice and control littermates. C, Total number of CFU-GM (P < .0005) and BFU-E progenitors and CFU-HPP (P < .001) per spleen in LID mice compared with control littermates. Results of 4 independent experiments with a combined total of 7 mice per group are shown.

Table 1. Distribution of Spleen Lymphocytes in LID Mice and Littermates
Cell PhenotypeControl Mice (%)LID Mice (%)
B220+, immunoglobulin M+35.7 ± 2.135.5 ± 1.1∗
CD4+23.6 ± 1.929.3 ± 2.5∗
CD8+4.7 ± 0.44.9 ± 0.8∗

Spleen cell lymphoid subsets were unaltered in LID mice. Splenocytes from naive animals were labeled with directly conjugated antibodies and analyzed by flow cytometry. No differences were observed in the frequency of B cells (B220+ and immunoglobulin M+) or T cells (CD4+ or CD8+ subsets). Results of 4 independent experiments with a combined total of 7 mice per group are shown.

∗ No significant difference from control (Student t test).

In contrast to the lymphoid compartment of the spleen, a significant reduction in the frequency (Figure 1B) and absolute number of myeloid progenitors was observed (Figure 1C) in LID mice compared with controls. Both the relatively mature population of progenitors, measured by CFU-GM, and the more immature population, measured by CFU-HPP, were affected (P < .005 and P < .001, respectively). No significant changes in erythroid progenitors were observed, although a mild relative decrease in the myeloid to erythroid cell ratio was noted in 2 of 3 histologic specimens compared with littermate controls. These results indicate that the contribution of liver-derived IGF-I plays an important role in the splenic myeloid cell compartment.

Investigation of femoral bone marrow hematopoiesis revealed normocellular bone marrow in both LID and control mice, although increased numbers of megakaryocytes were present in 2 of 3 LID specimens (data not shown). In agreement with the histologic examination of the femurs, there was no significant difference in the total number of nucleated cells per femur in either control or LID mice (Figure 2A). Although no gross abnormality was observed in the bone marrow, a significant increase in both committed and primitive progenitors was observed in LID mice relative to control littermates (Figure 2B and 2C). Peripheral blood cell counts mirrored the bone marrow findings. Erythrocytes, granulocytes, and monocytes were within normal parameters (data not shown), whereas the platelet counts were increased (689 ± 75 million platelets per milliliter of peripheral blood) in the LID animals compared with control mice (265 ± 11 million platelets per milliliter of peripheral blood), which is consistent with the increase in bone marrow megakaryocytes described previously. Taken together, these findings suggest that the marked decrease in serum IGF-I in LID mice has a limited effect on murine bone marrow hematopoiesis through increased myeloid progenitor cell content and thrombocytosis.

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  • Figure 2. 

    Increased myeloid progenitors in the bone marrow of LID mice. A, Total number of nucleated cells per femur in LID and control mice. B, Frequency of committed myeloid (CFU-GM; P < .05) and erythroid (BFU-E) progenitors and primitive progenitors (CFU-HPP; P < .005) in the bone marrow of LID mice and control littermates. C, The total number of CFU-GM (P < .005), BFU-E, and CFU-HPP (P < .01) per femur in LID mice was compared with that in control littermates. Results of 4 independent experiments with a combined total of 7 mice per group are shown.

We hypothesized that the presence of increased numbers of primitive and myeloid progenitors in the bone marrow may be due to a decreased ability of these cells to migrate out of the bone marrow and into the circulation of animals with low serum IGF-I. This finding would also account for a decrease in primitive and myeloid progenitors in the spleen. To test this hypothesis, the number of hematopoietic progenitors in the peripheral blood of LID mice and their littermates was determined. Contrary to our expectations, we observed a 3-fold increase in CFU-GM and a 7-fold increase in BFU-E in the peripheral blood of LID mice with reduced serum IGF-I compared with control littermates (Figure 3). These findings suggest that hematopoietic progenitors of both myeloid and erythroid lineages can egress from the bone marrow into the peripheral blood of LID mice, but in the environment of reduced serum IGF-I, the progenitors cannot migrate into the spleen; this results in an accumulation of progenitors in the circulation.

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  • Figure 3. 

    Increased myeloid progenitors in peripheral blood of LID mice. The total number of CFU-GM and BFU-E per milliliter of peripheral blood was compared in 4-week-old male littermates. Results of 3 independent experiments with a combined total of 5 mice per group are shown.

To confirm these findings, we attempted to correct the IGF-I defect and associated depression in splenic hematopoiesis in LID mice through a short-course administration of exogenous recombinant human IGF-I (1 mg/kg/d for 4 days). The results are presented in Figure 4A. Administration of IGF-I for 4 days increased the number of splenic hematopoietic progenitors in LID mice compared with LID mice that received vehicle only (CFU-GM; P < .001). At the same time, no significant changes in bone marrow cellularity or hematopoietic progenitor cell content were observed (Figure 4B). These results demonstrate that correction of circulating IGF-I levels can restore the myeloid compartment in the spleen of LID mice.

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  • Figure 4. 

    Restoration of splenic hematopoietic progenitors in LID mice after IGF-I administration. The total number of CFU-GM per spleen and bone marrow in mice treated with IGF-I (1 mg/kg twice daily for 3 days, subcutaneously; 4 mice per treatment) or phosphate-buffered saline (PBS; twice daily for 3 days, subcutaneously; 2 mice per treatment) was determined the day after completion of cytokine administration. A, Significant increases in splenic CFU-GM (P < .001) were observed in IGF-I-treated LID mice compared with LID mice that received PBS as a vehicle control. B, No significant differences in CFU-GM content were observed in the femurs of IGF-I-treated LID mice compared with LID mice that received PBS as a vehicle control.

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Discussion 

In this study, we have shown that in the LID mice, which exhibit low circulating IGF-I and high circulating GH levels, myeloid progenitors had a significant decrease in the spleen but increased numbers in the bone marrow and peripheral blood. Administration of exogenous IGF-I restored the number of splenic hematopoietic progenitors comparable to wild-type levels. Given the normal growth and body weight in LID animals and the presence of IGF-I messenger RNA (mRNA) in the spleen [5], the data presented in this study suggest that circulating IGF-I may mediate the movement of myeloid progenitors into the spleen or allow for their maintenance in the spleen.

A recent study demonstrated the efficacy of recombinant human GH as a mobilizing agent for peripheral blood stem cell collection when added to recombinant human granulocyte colony-stimulating factor in patients who did not achieve adequate mobilization with conventional protocols [16]. Of note, although there were no significant increases in the peak white blood cell count compared with recombinant human granulocyte colony-stimulating factor alone, the combined treatment yielded significantly higher numbers of blood CD34+ cells and primitive and myeloid progenitors.

Previous studies have examined the effect of increased GH concentrations on murine hematopoiesis. In those studies, increased serum GH was maintained through constitutive expression in transgenic models [12] or by systemic administration of recombinant human GH 9, 11. Comparison of the transgenic mice with high levels of both GH and IGF-I to the LID mice with high GH and low serum IGF-I reveals marked differences that suggest a role for circulating IGF-I in splenic hematopoiesis. In the GH transgenic animals, splenic hematopoiesis was enhanced; increased numbers of erythroid cells, megakaryocytes, and day 10 colony-forming units-spleen were noted [5]. A high proportion of myeloid progenitors were in cycle compared with controls [5]. In the bone marrow, which is 100% cellular in healthy young mice, hematopoiesis was only minimally perturbed with only increased megakaryocyte progenitors and an increase in the in vivo proliferation index [5]. We observed that LID mice, with high serum GH levels and low serum IGF-I, exhibit decreased splenic myelopoiesis and normoplastic bone marrow with increased myeloid and immature (CFU-HPP) progenitors, as well as increased megakaryocytes. On the basis of the cumulative observations in GH transgenic [5] and LID mice, it seems that the increase in hematopoiesis is most likely a direct result of increased GH levels or increased localized production of IGF-I. Given that we observed no evidence of blocked passage of myeloid or erythroid progenitors from the bone marrow to the periphery in either LID mice or control mice, circulating IGF-I does not seem to affect egress of these cells from the marrow space. However, migration of myeloid progenitors into the spleen does seem to be dependent on the presence of adequate levels of circulating IGF-I. An alternate explanation that cannot be fully ruled out is that maintenance of myeloid progenitors in the spleen is dependent on circulating IGF-I, although this is less likely given the localized production of IGF-I mRNA in spleen [5].

Loss of CD44 expression has been shown to result in an impaired migration of hematopoietic progenitors from the bone marrow into the bloodstream and subsequent homing to the spleen in mice [17]. In contrast, activation of some isoforms of CD44 has been shown to occur on stimulation of cells with IGF-I [18]. To date, no published studies have examined the effect of IGF-I stimulation on CD44 or other adhesion molecules on hematopoietic stem or progenitor cells. However, previous studies have shown enhanced lymphocyte homing to lymphoid tissues after recombinant human GH administration [19]. However, in the LID mice, no defect in splenic lymphocyte number or splenic architecture was observed. Whether this inconsistency is due to differences or the relatively short-term time frame of hormone administration versus congenital abnormalities is undetermined. It should be noted that in CD44-deficient mice, lymphocyte homing and function are also apparently unaffected [17]. Other potential targets for IGF-I modulation of molecules that affect homing are members of the integrin family, stromal-derived factor-1, CXCR4, vascular endothelial growth factor, and metalloproteinases. Inhibition of Gαi-coupled receptors on bone marrow cells with pertussis toxin before transplantation can block homing to the spleen, but not bone marrow [20]. Although no evidence exists to suggest that IGF-I acts indirectly through chemokines in hematopoietic cells, IGF-I may act through 1 or more molecules to inhibit migration into the spleen. Both chemokine receptors and CD44 are possible targets on the basis of the studies discussed in this article. IGF-I has been shown to activate integrin family members on other cell types 21, 22, 23. The α4-containing integrins are important mediators of mobilization and homing of hematopoietic stem and progenitor cells from and to the bone marrow, although not the spleen [23]. Vascular endothelial growth factor can stimulate mobilization, and its expression is decreased in LID mice [24] and restored with exogenous IGF-I administration [25]. Similarly, IGF-I can modulate metalloproteinase activity, including matrix metalloproteinase-9 [26], another important regulator of mobilization.

Failure to support and maintain splenic myelopoiesis in mice with reduced circulating IGF-I is an alternate explanation of the findings presented in this study. Previous studies have demonstrated a role for IGF-I in the survival of myeloid progenitors 27, 28. The presence of IGF-I mRNA in the spleen of LID mice has been previously reported [5]; however, adequate levels of IGF-I for splenic, in contrast to bone marrow, hematopoiesis could be dependent on circulating, as well as paracrine, sources.

Myeloid and high proliferative potential progenitors are decreased in the spleens of LID compared with control mice. We hypothesize that this maybe due to downregulation or deactivation of adhesion molecules inhibiting the movement of cells out of the peripheral blood and into the spleen. Although we cannot rule out that the low number of splenic hematopoietic progenitors is due to insufficient levels of IGF-I protein in the spleen, IGF-I mRNA is produced in the spleens of LID mice at greater levels than in controls [5].

Therefore, these results suggest that circulating IGF-I is important in hematopoietic progenitor homing to the spleen in mice and may be of interest to assess its role with regard to mobilization and bone marrow engraftment studies.

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Acknowledgements 

This project was funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-12400. The authors gratefully acknowledge the expert technical assistance provided by Steve Stull and Tanya Chanturya. We thank Laura Knott for outstanding secretarial services.

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PII: S1083-8791(03)00340-9

doi:10.1016/j.bbmt.2003.09.008

Biology of Blood and Marrow Transplantation
Volume 10, Issue 1 , Pages 32-39, January 2004

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