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First published online April 26, 2007
Stem Cells Vol. 25 No. 7 July 2007, pp. 1814 -1822
doi:10.1634/stemcells.2007-0002; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

A Novel Function of Interleukin-10 Promoting Self-Renewal of Hematopoietic Stem Cells

Young-Ju Kanga, Seung-Jip Yanga, Gyeongsin Parka, Bin Chob, Chang-Ki Mina, Tae-Yoon Kimc, Joon-Sung Leeb, Il-Hoan Oha

aCatholic High-Performance Cell Therapy Center and Department of Cellular Medicine;
bDepartment of Pediatrics;
cDepartment of Dermatology, The Catholic University of Korea, Seoul, Republic of Korea

Key Words. Stem cells • Hematopoiesis • Transplantation • Self-renewal

Correspondence: Il-Hoan Oh, M.D., Ph.D., Catholic High-Performance Cell Therapy Center, The Catholic University of Korea, 505, Banpo-Dong, Seocho-Ku, Seoul, Korea 137-701. Telephone: 82-2-590-2515; Fax: 82-2-591-3994; e-mail: iho{at}catholic.ac.kr

Received on January 2, 2007; accepted for publication on April 5, 2007.

First published online in STEM CELLS EXPRESS  April 26, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Self-renewal of hematopoietic stem cells (HSCs) is key to their reconstituting ability, but the factors regulating the process remain poorly understood. Here, we show that Interleukin-10 (IL-10), a pleiotropic immune modulating cytokine, can also play a role in regulating HSC self-renewal. First, a quantitative decrease of primitive hematopoietic cell populations, but not more matured cells, was observed in the bone marrows of IL-10 disrupted mice as determined by long-term in vitro cultures or in vivo competitive repopulation assays. In contrast, normal HSCs from 5-fluorouracil treated marrows cultured on the IL-10 secreting stroma displayed an enhanced repopulating activity compared with cells grown on control stroma, with ninefold higher numbers of donor-derived HSCs in the reconstituted recipient marrows. Moreover, limiting dilution transplantation assay demonstrated that exogenous addition of IL-10 in the stroma-free cultures of purified Lin–Sca-1+c-kit+ cells caused three- to fourfold higher frequencies of HSCs in the 5-day short-term culture without indirect inhibitory effect of IL-10 on tumor necrosis factor-{alpha} or interferon-{gamma} secretion. Interestingly, primitive hematopoietic cells, including Lin–Sca-1+c-kit+ or side population cells, expressed the surface receptor for IL-10, and microenvironmental production of IL-10 was sharply increased in the osteoblasts lining the trabecular regions of the radiation-stressed marrow but not in the steady-state marrows. These results show that IL-10 may be a ligand that can stimulate self-renewal of HSCs to promote their regeneration in addition to being a ligand for immune regulation.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hematopoietic stem cells (HSCs) constitute a rare subpopulation in hematopoietic tissues with the ability to give rise to all types of mature blood cells in response to physiological demand [1]. These HSCs exhibit long-term repopulating activities when transplanted into myeloablated hosts through their unique ability to execute self-renewal during regeneration [2, 3]. The repopulating capacity of individual HSCs following transplantation into recipients constitutes a functional endpoint for the specific quantification of HSCs in limiting dilution transplantation assay [4, 5]. The cells thus defined are referred to as competitive repopulating units (CRUs), and quantitative increments in these CRU numbers have been the key evidence for self-renewal of HSCs [6, 7].

Thus, the key issue for HSCs has been identifying extracellular and intracellular signals that can govern their fates during their asymmetric division [8] to promote self-renewal. Although extensive studies have identified numerous intrinsic transcription factors [9–15] and extrinsic factors [16–23], including microenvironment, that can influence their self-renewal [24–26], physiological control of HSC self-renewal remains yet unclear, warranting further identification of factors involved in the control of HSC behavior.

Interleukin-10 (IL-10) is a pleiotropic immune-regulatory cytokine that inhibits cytokine secretion and effector functions of T cells, monocytes, and macrophages [27]. Targeted disruption of IL-10 (IL-10 knockout [KO]) leads to an uncontrolled immune response, causing chronic enterocolitis [28, 29] and a greater susceptibility to allergic encephalitis and autoimmune diseases such as rheumatoid arthritis [30]. In addition to its immune-modulating function, a possible role of IL-10 for hematopoiesis has been suggested by several studies showing variable influence of IL-10 on hematopoietic progenitor cells from both normal or aplastic anemia bone marrow cells [31–35]. However, many of the indirect effects of IL-10 on hematopoietic cells have been suggested [35–38], and it is not clear yet whether IL-10 could function as a direct ligand for hematopoietic cells, including the primitive hematopoietic cell populations.

Therefore, in the current study, we investigated the possibility by analyzing various stages of hematopoiesis in IL-10 KO mice as well as by examining the effect of exogenous IL-10 on normal HSC during ex vivo culture. From this study, we demonstrate that IL-10 is a unique cytokine that, in addition to being able to modulate immune function, can influence self-renewal of HSCs, with their secretion being induced in a stressed bone marrow microenvironment.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Animals and Cell Purification
For congenic murine transplantation model, 8–12-week-old C57BL/6J-Ly 5.2 (CD45.2) (BL6) mice or C57BL/6J-Pep3b-Ly5.1 (CD45.1) (Pep3b) mice were used as recipients or donors. The cells from these mice can be distinguished with their surface phenotype, Ly5.1 or Ly5.2. IL-10 KO mice (Il10tm1Cgn; C57BL6) [28] were obtained from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). Homozygotes of IL-10 KO mice were screened with genomic polymerase chain reaction (PCR) and Southern blot analysis of genomic DNA after digestion with EcoRl (Fig. 1A). Some of these animals developed spontaneous enterocolitis with aging, as observed previously [28]. All animals were bred and maintained under sterile conditions in microisolator cages located in an air-filtered, positively pressured room in the animal facility of Catholic University of Korea. Experiments were undertaken with approval from the Animal Experiment Board of the Catholic University of Korea.


Figure 1
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  		Figure 1. In vitro assay for effect of IL-10 disruption on hematopoietic cells. (A): Genotype screening for IL-10 KO mice. Schematic structure of the targeting vector for the IL-10 gene and a representative Southern blot for wild-type and homozygote of IL-10 disrupted mice (IL-10 KO) are shown. (B): Comparison of bone marrow hematopoietic cell lineages. The percentage of myeloid (Mac-1/Gr-1), lymphoid (B220 or TB104) and erythroid (Ter119) cells in WT and IL-10 KO mice marrow is shown with error bars representing the SEM (n = 4 for WT, n = 5 for IL-10 KO, * p < .05). (C): Comparison of the in vitro colony-forming cells from WT and IL-10 KO mice bone marrow cells. Equivalent numbers (5 x 104) of bone marrow mononuclear cells from WT or IL-10 KO mice were plated in the semisolid medium for erythroid, myeloid, and mixed colonies. The mean number of each type of colony is shown, and the error bars represent the SEM (n = 4 for WT, n = 5 for IL-10 KO). (D): Comparison of long-term culture-initiating activity of WT and IL-10 KO bone marrow cells. Equal numbers (5 x 105) of BMCs of WT and IL-10 KO mice were subjected to long-term culture with weekly half-medium changes for 4 weeks then shifted into semisolid medium to measure total numbers of colonies generated. The mean number of total colonies obtained from three experiments of long-term cultures is shown (n = 4 for WT, and n = 5 for IL-10 KO; p = .02). Abbreviations: BFU-E, burst-forming unit-erythroid; BMCs, bone marrow mononuclear cells; CFU-E, colony-forming unit-erythroid; CFU-GEMM, colony-forming unit-mixed; CFU-GM, colony-forming unit-myeloid; IL-10, Interleukin-10; kb, kilobases; KO, knockout; LTC, long-term culture; No., number; WT, wild-type.

 
Cells
Murine bone marrow cells enriched with hematopoietic progenitors were obtained by 4-days-prior i.v. injection of mice with 150 mg/kg body weight of 5-fluorouracil (5-FU; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). For primitive hematopoietic populations, bone marrow cells were first depleted of lineage (CD5, CD45R, CD11b, TER119, Gr-1, 7–4)-positive cells using an immunomagnetic column (StemSep; StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) (Lin–) then sorted for Lin–Sca-1+c-kit+ (LSK) cells. Side population (SP) cells were purified as previously described [39]. Briefly, bone marrow mononuclear cells (BMCs) were incubated in buffer containing 5 µg/ml Hoechst 33342 (Sigma), 2% fetal bovine serum (FBS; StemCell Technologies), and 1 mM Hepes at 37°C for 90 minutes in the presence or absence of 50 µM verapamil (Sigma). SP cells were sorted on FACSVantage with excitation at 350 nm and emission at 450 nm (blue) and 675 nm (red).

Mesenchymal stromal cells (MSCs) were obtained by culturing murine bone marrow cells in Dulbecco's modified Eagle's medium containing 10% FBS and 1% Penicillin-Streptomycin (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) as previously described [40]. Established MSCs were subcultured until all cells become negative for hematopoietic marker CD45.

Ex Vivo Culture and Retroviral Transduction
Stroma-free culture of LSK cells was performed in serum-free medium containing Iscove's modified Dulbecco's medium and BIT (StemCell Technologies) supplemented with 10–4 M 2-mercaptoenthanol plus 40 µg/ml low density lipoprotein (Sigma) and 100 ng/ml murine steel factor (mSF; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 100 ng/ml human Flt-3 ligand (R&D Systems), and 50 ng/ml human thrombopoietin (TPO) (CytoLab, Rehovot, Israel, http://www.cytolab.cz/Peprotech, Rocky Hill, NJ, http://www.peprotech.com) in the presence or absence of 100 ng/ml murine IL-10 (mIL-10; CytoLab/Peprotech) for 5 days. For preparation of engineered MSCs secreting IL-10, murine IL-10 cDNA was first cloned into MSCV-IRES-GFP (MIG) retroviral vector [41], and retroviral particles were produced by cotransfection of 293 T cells with each retroviral vector (MIG or MIG-IL-10) plus plasmids containing gag-pol, vesicular stomatitis virus-glycoprotein, and gibbon ape leukemia virus envelope as previously described [42]. Supernatants were then collected, concentrated by ultracentrifugation, and used to infect MSCs. After transduction, transduced MSCs were purified by sorting for green fluorescent protein (GFP)+ cells. Concentration of IL-10 in the supernatants of each feeder was measured by enzyme-linked immunosorbent assay (ELISA) at the end of the 96-hour culture. Coculture of 5-FU treated bone marrow cells on each stroma was performed in long-term culture medium (MyeloCult; StemCell Technologies) supplemented with the same cytokines for 5 days.

In Vitro Assay for Colony-Forming Cells and Long-Term Culture-Initiating Cells
For in vitro assay for colony-forming cells, cells were plated in methylcellulose medium (MethoCult; StemCell Technologies) containing 3 U/ml erythropoietin (StemCell Technologies), 50 ng/ml mSF, 10 ng/ml human IL-6 (R&D Systems), and 10 ng/ml mIL-3 (R&D Systems) and scored for types and numbers. For long-term culture assay, stromal feeder cells were first established from murine bone marrow cells by culturing in myeloid long-term culture medium (MyeloCult) containing 10–6 M hydrocortisone sodium hemisuccinate (Sigma) followed by irradiation (1,500 cGy). BMCs were cultured on this feeder in the same medium for 4 weeks with weekly half-medium changes followed by transfer of the cells to semisolid methylcellulose medium to count the number of colonies formed [43].

In Vivo Repopulation and CRU Assay
Donor cells were intravenously injected into irradiated (900 rad) recipient mice along with 1 x 105 helper cells derived from recipient origin. Repopulation of transplanted cells in the recipients was assessed by flow cytometry to measure the proportion of leukocytes expressing donor-origin (Ly5.1 or Ly5.2) surface antigen in their blood or bone marrows. Lineages of repopulated hematopoietic cells were analyzed by staining with anti-Mac-1 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml), anti-Gr-1 antibody (BD Pharmingen) for myeloid cells and with anti-TB104 (BD Pharmingen), anti-B220 antibodies (BD Pharmingen) for lymphoid engraftment as described previously [41].

To quantitatively measure number of HSCs with in vivo repopulating abilities, CRU assays were performed using principles of limiting dilution analysis as previously described [4]. Briefly, cells in serially diluted dose were transplanted into lethally irradiated (900 cGy) mice along with 1 x 105 helper cells, and the levels of lymphoid and myeloid engraftment in the recipient mice blood were determined after 16 weeks. Recipient mice whose white blood cells contained 1% or more of the donor-derived lymphoid and myeloid cells were scored as positive. The cell dose calculated to result in 37% of the mice tested being negative was defined as 1 CRU [4]. CRU frequencies and 95% confidence intervals (CI) were calculated by applying Poisson statistics to the proportion of negative mice in groups of recipients given different numbers of cells using L-Calc software (StemCell Technologies).

IL-10 Receptor Expression, Cytokine Assay, and Immunohistochemistry of Bone Marrows
Cells were incubated with antibody against the {alpha} subunit of murine IL-10 receptor (BD Pharmingen) for flow cytometry. For detection at the transcription level, total RNA was subjected to reverse transcription (RT)-PCR using the primer sets 5'-GAG TCT CCA GGG CTA CAG GC-3' and 5'-CAC AGC TAA CCA CAC CCA GG-3'. To measure the concentration of tumor necrosis factor (TNF)-{alpha} or interferon (IFN)-{gamma} in culture, supernatants were taken after 2 or 5 days of ex vivo culture of LSK cells and subjected to ELISA according to the manufacturer's instruction (R&D Systems). The values of individual samples were analyzed by interpolation from standard curves assayed on individual plates. To detect IL-10 production in normal and irradiated bone marrows, immunohistochemical analysis was performed as previously described [44]. Briefly, deparaffinized femur tissues were antigen retrieved in sodium citrate solution (121°C, 3 minutes) and incubated in serum blocking solution (ChemMate Detection Kit; Dako, Glostrup, Denmark, http://www.dako.com) containing anti-mouse IL-10 antibody (BioLegend, San Diego, http://www.biolegend.com) (10 µg/ml) for 30 minutes. After washing for 15 minutes, slides were stained with secondary goat anti-rat antibody (BioLegend), treated with 0.3% H2O2 to block endogenous peroxidase, then visualized by using streptavidin-horseradish peroxidase (Dako) and H&E staining. The production of IL-10 in the fresh bone marrows was also analyzed by RT-PCR using the primers 5'-CAG TAC AGC CGG GAA GAC AA-3' and 5'-CAG CTT CTC ACC CAG GGA AT-3'.

Statistical Analysis
The significance of the differences between groups was analyzed using Student's t test (p < .05). CRU frequencies and 95% CI were calculated by applying Poisson statistics to represent ± 2 SEM.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Primitive Hematopoietic Cell Populations Are Affected in IL-10 KO Mice
To investigate the potential influence of IL-10 on the hematopoietic system, we first examined the bone marrow cells in the IL-10 KO mice and compared these with the cells from wild-type (WT; C57BL6) mice. IL-10 KO mice exhibited slightly fewer cells than WT mice in their marrows (14.8 ± 3 vs. 21.2 ± 2 x 107 cells, respectively; n = 4–5 in each group). The lineages of mature hematopoietic cells were similar in the two groups except for a modest decrease of erythroid (TER119) cells in the IL-10 KO marrows (Fig. 1B). Similarly, the number and types of colonies generated in semisolid medium from the equivalent numbers of BMCs did not differ between groups (Fig. 1C), suggesting lack of significant effects for more mature cells as previous noted [28].

In contrast, significant difference was seen in the ability of more primitive hematopoietic cells to sustain long-term culture (i.e., equivalent number of BMCs from IL-10 KO marrow produced 24 ± 4 colonies, whereas WT marrow cells produced 67 ± 17 colonies after 4-week long-term culture) (Fig. 1D) (three experiments; p < .05; n = 4 for WT, and n = 5 for IL-10 KO). These results suggested that primitive, rather than more matured, hematopoietic cells could have been selectively affected in the IL-10 KO mice. To further examine this possibility, we next examined for long-term in vivo repopulating cells by comparing the reconstitution levels achieved after transplantation of each group of BMCs into lethally irradiated recipients. As shown (Fig. 2A, 2B), BMCs from IL-10 KO mice showed lower engraftment levels of donor-derived cells than BMCs from WT mice being evident from 3 weeks after transplantation (45% vs. 70%), and the difference was sustained throughout the observation period of 12 weeks (46% vs. 88% after 12 weeks, p = .02) (Fig. 2B). However, the different engraftment levels in the recipients were not accompanied by changes in lymphoid-myeloid distribution of the donor-derived cells (Fig. 2C), suggesting that the difference might be derived from the HSC level rather than from progenitors of a particular lineage. To examine the possibility, quantitative assessment of HSCs in the bone marrows of each group was performed by employing competitive repopulation analysis. The result of this assay showed that IL-10 KO mice had fewer CRUs in their marrows than WT mice (1,580 CRUs vs. 4,340 CRUs, respectively) (Table 1). Interestingly, older (>20 weeks) IL-10 KO mice that had spontaneously developed enterocolitis had further lowered numbers of CRUs (<376 CRUs), indicating the progression of HSC loss with aging in the affected mice. Taken together, these results show a quantitative decrease of primitive hematopoietic populations in the marrow of IL-10-disrupted mice.


Figure 2
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  		Figure 2. Comparison for in vivo repopulating activity of bone marrow mononuclear cells (BMCs) from WT and IL-10 KO mice. Equal numbers (2 x 105) of BMCs from WT or IL-10 KO (Ly5.2) mice were transplanted into lethally irradiated Pep3b mice (Ly5.1) along with 1 x 105 normal Pep3b BMCs per recipient. Representative flow cytometry plots for (A) and mean % ± SEM of donor-derived (Ly5.2) cells in the recipient blood at indicated times are shown (B) (three experiments, n = 17 for each group, *; p < .05). (C): Lineage distribution of engrafted cells, as defined by myeloid (Mac-1/Gr-1) and lymphoid (B220) cells, was examined in the recipient blood. Percent of each lineage in the donor-derived (Ly5.2) cells are shown, and error bar represents SEM. Abbreviations: APC, allophycocyanin; FSC, forward scatter; IL-10, Interleukin-10; KO, knockout; wk(s), week(s); WT, wild-type.

 


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  		Table 1. Quantitative comparisons of CRU number in the WT and IL-10 KO mice marrows

 
IL-10 Secreting Stroma Can Enhance Total Regenerative Activity of Normal HSCs
Since a quantitative loss of HSCs was seen IL-10 KO mice, we next questioned if addition of IL-10 could, in turn, exert an opposite effect on the quantity of HSCs. Although we found a quantitative loss of HSCs in IL-10 KO mice, it could not be excluded that systemic in vivo effects such as alteration of cytokine milieu in IL-10 KO mice [45] could have been involved. Therefore, to see if IL-10 can influence the quantity of HSCs in the absence of such systemic effects, HSCs from normal mice were cultured ex vivo under the conditions that can mimic the stromal microenvironment with or without IL-10 supply (Fig. 3A). Thus, bone marrow derived mesenchymal stromal cells were engineered to secrete IL-10 by transduction with a retroviral vector encoding mIL-10 (IL-10/stroma) or a control vector (MIG/stroma) then sorted for transduced (GFP+) cells. IL-10 concentration in the supernatants of the feeder cells reached 60–70 ng/ml after 4 days for IL-10-secreting stroma, whereas the IL-10 concentration was only 20–30 pg/ml for control stroma. The stromal cells thus established exhibited a typical surface phenotype of MSCs with no hematopoietic component (Fig. 3B), and transplantation of stromal cells alone did not cause any hematopoietic reconstitution of the recipients for either type of feeder cell (data not shown). When 5-FU-treated BMCs were cocultured on each feeder cell and transplanted into lethally irradiated mice, a significantly higher level of reconstitution was seen in the mice given cells from IL-10-secreting stroma than from cells from the control stroma throughout the period of observation (37% ± 9% vs. 13% ± 7% at 16 weeks, p < .05) (Fig. 3C). Of note, the higher levels of repopulation in the IL-10 stroma group were associated with higher numbers of donor-derived (Ly5.1+) HSCs regenerated in the recipients, as determined by limiting-dilution transplantation of the reconstituted primary marrows into secondary recipients (550 CRUs vs. 65 CRUs, respectively), which corresponded to expansion of 34-fold (550/16) and 4-fold (65/16) from initial input cells (16 CRUs in 1 x 105 5-FU bone marrow cells [46]) (Fig. 3D). These results showed that effect of IL-10 to increase regeneration of HSCs could be reproduced independent of systemic in vivo effects.


Figure 3
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  		Figure 3. Enhanced regenerative activity of normal hematopoietic stem cells cultured in the IL-10 secreting stroma. (A): Schematic illustration of the experimental scheme; 5-FU-treated bone marrow mononuclear cells (Pep3b, Ly5.1) were cultured either on the control stroma (MIG/stroma) or IL-10 secreting stroma (IL-10/stroma) supplemented with steel factor, Flt-3 ligand, and thrombopoietin. After 5 days of culture on each stroma, cells were transplanted into lethally irradiated recipients (Ly5.2, BL6) to compare repopulating activities. The frequencies of donor-derived CRUs in the reconstituted marrows were then determined by transplanting primary marrow cells into secondary recipients at limiting-dilution doses to calculate the total number of donor-derived (Ly5.1) CRUs in each primary recipient marrow. (B): Phenotypic characterization of primary mesenchymal stromal cells (MSCs) in established feeder cells. Shown are the representative fluorescence-activated cell sorting plots for IL-10 secreting stroma displaying typical surface phenotype of MSCs without hematopoietic components. Dotted lines represent isotype control and thick lines staining with indicated antibodies. (C): Engraftment levels achieved in the primary recipient blood after transplantation of cells (initially contained 1 x 105 cells) cultured in MIG/stroma (MIG) or IL-10/stroma (IL-10). Shown are the mean ± SEM of the percentage of donor-derived cells (Ly5.1) in the recipient blood white blood cells at each indicated time (two experiments, n = 11 for the control group, and n = 8 for the IL-10 group, *; p < .05). (D): CRU numbers in reconstituted primary mice marrows. The marrows of primary mice in (C) were harvested and pooled 24 weeks after transplantation for secondary transplantation. The CRU frequency of donor-derived cells was determined by Poisson statistics 16 weeks after secondary transplantation, and the total number of CRUs was calculated assuming that two femurs and tibias represent 25% of the total marrow [62]. Values are the mean for the total number of donor-derived (Ly5.1+) CRUs per mouse (n = 15 for each group of secondary mice). Error bars represent upper and lower limits of 95% confidence interval (CI) equivalent to ± 2 SEM. Mean CRUs for MIG group was 65 (95% CI;17–250) and 550 CRUs (240–1,260) for IL-10 group. Abbreviations: CRU, competitive repopulating unit; IL-10, Interleukin-10; MIG, MSCV-IRES-GFP; No., number.

 
IL-10 Is a Ligand for HSC Self-Renewal Being Induced Under Stressed Microenvironment
Although enhanced HSC regeneration was observed in the stromal cultures secreting IL-10, it was also shown that IL-10 could inhibit secretion of cytokines such as TNF-{alpha} and IFN-{gamma} from mononuclear cells [37, 38], and that these cytokines could suppress primitive hematopoietic cell populations [47, 48]. Therefore, to exclude the possibility of such indirect cytokine inhibitory effects in HSC expansion, we tested whether IL-10 can promote HSC self-renewal to cause a difference in the frequency of HSCs under the conditions devoid of accessory cells or stromal cells during a 5-day short-term culture period, which could minimize generation of mature accessory cells [49]. Thus, purified LSK cells were cultured in stroma-free culture conditions supplemented with growth factors (steel factor, Flt-3 ligand, and TPO) in the presence or absence of exogenously added IL-10 then subjected to limiting dilution transplantation into recipients (shown schematically in Fig. 4A). CRU assays thus performed showed that exogenous addition of IL-10 increased the CRU frequencies by three- to fourfold in these cultures compared with those without IL-10 addition (4.3-fold and 2.8-fold for each experiment, respectively) (Table 2). Phenotypic analysis for cultured cells also displayed comparable increase of total LSK cells after culture with IL-10 addition (8.3-fold and 2.9-fold, respectively) (Table 2) in consistence with the previous observation of correlation between LSK cells and repopulating stem cells after culture [50]. No difference was seen in the cell cycle distributions or survival of LSK cells between the two cultures as determined by propidium iodide staining (data not shown). Of note, the concentrations of TNF-{alpha} and IFN-{gamma} produced in the IL-10-containing medium were, at least, not lower than those in the control medium after 2 days or 5 days of culture (Fig. 4B), suggesting that the higher CRU frequencies observed with IL-10 addition were not caused by indirect inhibitory effects of IL-10 on cytokine secretion [37]. To further see if IL-10 can be a ligand for stimulation of HSCs, expression of the IL-10 receptor was examined in a primitive hematopoietic population. As shown in Figure 4C, IL-10 receptor was detected readily in immature hematopoietic cells including Lin– cells, LSK cells, and SP cells, as determined by an antibody against surface receptors as well as by expression of its transcript. IL-10 receptor expression was also seen in the subpopulation of SP cells (tip-SP cells) that had been characterized as long-term hematopoietic cells [51], indicating that primitive hematopoietic cells can be stimulated by IL-10 through their receptors (Fig. 4D).


Figure 4
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  		Figure 4. IL-10 can directly stimulate self-renewal of hematopoietic stem cells during ex vivo culture. (A): Schematic illustration of the experimental scheme. Purified LSK cells were cultured in the stroma-free cultures containing GF cocktails with or without exogenously added IL-10. Immediately after 5 days of culture, the cells were transplanted into irradiated recipient mice at serial dilutions to determine the CRU frequencies 16 weeks after transplantation (shown in Table 2). Cytokine concentrations in the supernatants of 2-day or 5-day cultures were measured to examine the inhibitory effect of IL-10 on cytokine secretion. (B): Concentrations of TNF-{alpha} and IFN-{gamma} during the stroma-free culture of LSK cells in the culture medium with or without IL-10 addition. Supernatants were taken at the end of the 5-day culture or after 2-day culture of LSK cells in separate experiments. The levels of indicated cytokines determined by enzyme-linked immunosorbent assay are shown in a mean value of each duplicate measurement. (C): Expression of the IL-10 receptor in immature hematopoietic cells. Lin–, LSK cells, or SP cells (representing 0.25% of mononuclear cells [MNCs]) were purified from normal murine bone marrow and examined for cell surface receptors and transcripts for the {alpha} subunit of the IL-10 receptor. The representative flow cytometry profiles of surface antibody staining (upper panel) and results of reverse transcription-polymerase chain reaction (RT-PCR) for the transcript (lower panel) are shown. RT-PCR for ß-actin control was performed with 1/10 dilution of cDNA mix. (D): Expression of IL-10 receptors in subpopulations of SP cells. Light-density MNC cells were stained with Hoechst 33342 and gates for proximal-SP (0.22% of total MNCs), tip-SP (0.02%), or MP (98.6%) were set in the presence of verapamil (left), and expressions of IL-10 receptor in each subpopulation were analyzed (right). Numbers in each gate represent the percentage of cells positively stained with surface antibody against IL-10 receptor under the gate set where 99% of isotype control was excluded. Abbreviations: CRU, competitive repopulating unit; GF, growth factor; IFN, interferon; IL-10, Interleukin-10; IL-10R, Interleukin-10 receptor; Lin–, lineage-negative population; LSK, Lin–Sca-1+c-kit+; MP, main population; NA, below detection limit; NC, negative control; SP, side population; TNF, tumor necrosis factor; W, whole bone marrow mononuclear cells.

 


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  		Table 2. Effect of exogenous IL-10 in the stroma-free culture of LSK cells

 
Next, to investigate the physiological significance of IL-10 for HSCs, we questioned if IL-10 is involved in the changes in microenvironment during the regenerative phase of reconstitution. For this end, we examined IL-10 production in the bone marrow microenvironment under the "steady-state" and "stress" conditions induced by radioablation. As shown, whereas production of IL-10 in nonirradiated, steady-state marrow is at basal level, its production is rapidly increased in the bone marrows of irradiated mice as determined by induction of transcript in the total bone marrow cells (Fig. 5A). Of note, IL-10 production was markedly increased in the osteoblasts lining the endosteal surface of trabecular region in the irradiated marrows. The induction of IL-10 production in the trabecular surface was maintained in the marrows up to 8 days after radiation, when most of hematopoietic cell components were ablated (Fig. 5B). Since trabecular bone is the primary site where HSCs reside and osteoblasts are a critical component of the stem cell niche for their self-renewal [52, 53], these results suggest that IL-10 could be an important ligand for HSC self-renewal during the stress-induced regeneration of HSCs in the normal bone marrow. Taken together, these results suggest that IL-10 may function as a physiological ligand that can promote preservation and/or self-renewal of HSCs during the regenerative phase of marrow.


Figure 5
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  		Figure 5. Induction of IL-10 in the microenvironment of stressed marrow. The mice were irradiated with lethal dose (900 rad), and bone marrows were harvested at each indicated day after radiation. (A): Transcripts of IL-10 in the total harvested bone marrows were examined by RT polymerase chain reaction. (B): Femurs from each mouse were examined for IL-10 production by immunohistochemistry using antibody against IL-10 and examined with light microscopy at indicated magnification. Lower panel is the magnification of boxed region, arrows indicate endosteal osteoblast, star-shaped mark indicates trabecula of the femur, and inlets show further magnification (x400) of dotted-line boxed region to show cytoplasmic staining of IL-10 in osteoblasts. Abbreviations: IL-10, Interleukin-10; RT, reverse transcription.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Inducing self-renewal of HSCs has been of major interest for hematopoietic reconstitution as well as for maintenance and/or expansion of primitive hematopoietic cells during ex vivo culture. Although an extending spectrum of extrinsic factors for HSCs is being identified [16–23], limited success has been reported for ex vivo expansion using multiple combinations of the growth factors [54], warranting further elucidation of factors in HSC regulation.

In the present study, we have shown that IL-10 should be a ligand that can regulate primitive hematopoietic cell populations in addition to being an immune modulating cytokine. Although several studies also showed various influences of IL-10 on hematopoietic cells, those were largely focused on the hematopoietic progenitor cells being identified by in vitro assays, that is, addition of IL-10 in cytokine mixtures could increase progenitors of megakaryocytic [32], myeloid [34, 55], erythroid [35], or B-lymphoid [31, 56] lineages during the culture. In addition, it was also pointed out that many of the IL-10 effects on hematopoietic progenitor cells from normal or aplastic anemia patients were due to their indirect cytokine inhibitory effect [35–38], leaving the possibility of IL-10 being a direct ligand for primitive hematopoietic repopulating cells unexplored.

Previously, it was shown that heterogeneous hematopoietic populations exhibit distinct responses to cytokine stimulations with respect to their differentiation or ontological stages [22, 57, 58]. These observations raised the possibility that response to IL-10 could be distinct between the primitive in vivo repopulating hematopoietic cells and intermediate stage progenitor cells being identified in vitro. In coherence to the possibility, phenotypes or colony-forming ability of bone marrow cells in the IL-10 KO mice were not significantly affected except for anemia of systemic effect [28, 59]. In contrast, primitive hematopoietic populations were selectively affected by the lack of IL-10, as demonstrated by lower repopulating activities as well as decrease of total CRU numbers in their marrows, albeit to a moderate extent because of possible compensation by cytokines other than IL-10. The fact that CRU assays were performed on the mice in steady-state as well as compensation by other cytokines could account for the moderate extent of difference. Although a greater loss of CRUs were observed in mice with spontaneously developed colitis, it is not clear yet whether it reflects an accumulated loss of HSCs with aging, as these mice develop colitis at later ages [28].

On the other hand, more direct support for the effect of IL-10 on HSCs was obtained from ex vivo culture of normal hematopoietic cells, where a higher number of HSCs in the reconstituted recipient marrows was observed when the cells were cultured in the IL-10 secreting stroma. It was further supported by higher frequencies of CRUs in the stroma-free culture, where potential indirect effects from accessory cells or mature cells that can be generated during the culture [49] were minimized by employing a 5-day short-term culture of purified LSK cells. These findings, together with readily detectable expression of IL-10 receptors in primitive hematopoietic cell populations, suggest that IL-10 may function as a ligand that can stimulate HSC self-renewal independent of the indirect inhibitory effect on negative regulatory cytokine. Of note, whereas previous studies showed that multiple cytokine stimulations were necessary for maintenance of primitive hematopoietic cells [16–18], addition of IL-10 could promote self-renewal of HSCs further beyond the levels achieved by those known cytokine mixtures. Therefore, it is a possibility that IL-10 constitutes another growth factor that can stimulate HSC self-renewal in a nonredundant manner, although effects of IL-10 on other growth factors remain yet possible.

Of interest, IL-10 production was sharply induced in the osteoblasts lining the endosteum of trabecular bone region in the irradiated marrow. Previous studies have shown that osteoblasts in the trabecular regions are an important stem cell niche where HSCs reside and stimulate for self-renewal [52, 53]. The finding that IL-10 production is sharply increased in the microenvironment during stressed condition, but not in the homeostatic, steady-state marrow, is coherent with the view that IL-10 functions as a ligand for HSC self-renewal and further raises the possibility that IL-10 is an important soluble factor in the stem cell niche during the reconstitution of bone marrow after HSC transplantation.

The physiological significance of the current finding that IL-10, in addition to being a ligand for immune modulation, can regulate HSC self-renewal remains unclear. It would be possible to speculate that IL-10 may participate in the functional communication between the immune and hematopoietic systems. In this light, it is interesting to note that hematopoietic functions are often defective with manifestations of cytopenia in autoimmune diseases such as rheumatoid arthritis [60, 61]. Further studies are needed to explore this possibility. In summary, based upon the current findings for IL-10 functions to regulate primitive hematopoietic cells and their previously identified functions to modulate immune responses, we suggest that IL-10 may be a unique ligand that can exert its regulatory effects to both immune and hematopoietic systems for physiological coordination.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Ji-Yeon An for support in bone marrow analysis and Hijong An for support in flow cytometric sorting. This research was supported by a grant from the high-performance cell therapy R&D project (0405-DB01-0104-0006) by the Ministry of Health and Welfare, Republic of Korea. Y.J.K. and S.J.Y. contributed equally to this work.


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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