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First published online October 25, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 534 -542
doi:10.1634/stemcells.2007-0741; www.StemCells.com
© 2008 AlphaMed Press

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THE STEM CELL NICHE

Adenoviral Vector-Mediated Transfer of the Indian Hedgehog Gene Modulates Lymphomyelopoiesis In Vivo

Masayoshi Kobunea,b, Junji Katoa, Yutaka Kawanoa, Katsunori Sasakic, Hiroaki Uchidad, Kohichi Takadaa, Sho Takahashia, Rishu Takimotoa, Yoshiro Niitsua

aFourth Department of Internal Medicine and
bDepartment of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan;
cCore Facility for Therapeutic Vectors and
dDepartment of Surgery and Bioengineering, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Key Words. Hedgehog • Adenovirus • Lymphocyte • Hematopoiesis • Spleen • Thymus

Correspondence: Correspondence: Masayoshi Kobune, M.D., Ph.D., Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Chuo-ku, South-1, West-16 Sapporo, Hokkaido 060-8556, Japan. Telephone: 81-11-611-2111, ext. 3254; Fax: 81-11-612-2136; e-mail: mkobune{at}sapmed.ac.jp

Received on September 4, 2007; accepted for publication on October 19, 2007.

First published online in STEM CELLS EXPRESS  October 25, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Indian hedgehog (Ihh) plays an essential role in angiogenesis, hematogenesis, and epiphysis formation during embryogenesis. In the present study, we injected an adenoviral vector (Adv) carrying the mock-control (Adv-control) or Ihh (Adv-Ihh) gene into severe combined immunodeficiency (SCID) or BALB/c mice to evaluate the effects of lhh on the regulation of postnatal hematopoiesis in vivo. After the i.v. injection of Adv-Ihh, the expression of vector-derived Ihh mRNA was detected in the liver. Four weeks after administration of Adv-Ihh to SCID mice, we observed an increase in the number of c-Kit+ cells and clonogenic cells per 105 mononuclear cells in the bone marrow compared with Adv-control-administered mice. Moreover, after administration of Adv-Ihh to BALB/c mice, the number of splenic B220+IgMlowCD23intCD21int B lymphocytes and CD4+ T lymphocytes was strongly increased. Furthermore, the number of thymic double-negative (DN)2, DN3, CD8+ immature single-positive, and CD4+/CD8– cells was significantly elevated relative to the number in mice that received the control Adv vector. Our results suggest that enhanced signaling by Ihh can modulate the proliferation and differentiation of splenic B lymphocytes and thymic T lymphocytes during bone marrow hematopoiesis in vivo. Thus, modulation of the hedgehog signaling pathway may provide a therapeutic strategy to stimulate lymphomyelopoiesis in vivo.

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
 
The hedgehog (Hh) protein family is a group of secreted intercellular signaling molecules. Members of this protein family are subject to two types of lipid modification, cholesterol coupling and palmitoylation [1]. Secreted Hh protein binds to a receptor, Patched (Ptc), expressed on the plasma membrane of target cells. This interaction triggers the dissociation of Ptc from Smoothened (Smo), which is a constitutively active signal transducer [2]. Hh signals are eventually transmitted to the transcription factors, Gli1, Gli2, and Gli3, whose downstream signaling leads to entry of the cells into the cell cycle [3], inhibition of apoptosis [4], maintenance of self-renewal of stem cells in various tissues [5, 6], regulation of stem cell differentiation [7], and modulation of tissue polarity [8].

Three types of Hh protein exist in mammalian cells: Indian hedgehog (Ihh), Sonic hedgehog (Shh), and Desert hedgehog (Dhh). Hh family proteins have been shown to function at various stages, regions, and cell types during embryogenesis, and the tissue distribution of individual members is generally mutually exclusive or at least only partially physically overlapping [9, 10]. In particular, Ihh is thought to be a key inducer of primitive hematopoiesis and angiogenesis in the developing mouse yolk sac [11–13] and of definitive hematopoiesis in zebrafish embryos [14] and has also been shown to be essential for bone formation [15–17]. Moreover, we have recently shown that Ihh mediates an interaction between hematopoietic stem/progenitor cells and bone marrow (BM) stromal cells that leads to augmentation of the proliferation of primitive hematopoietic cells in vitro, suggesting that Ihh might play a significant role in postnatal hematopoiesis [18].

More recently, it has been demonstrated that Hh signaling plays an essential role in T-lymphocyte differentiation in the mouse thymus [19, 20]. Both Shh and Ihh are expressed in the mouse thymus. Shh is produced by the thymic epithelium and thymus medulla [21, 22], and Ihh expression is mainly associated with blood vessels in the thymic medulla [23].

Shh signaling plays an important role in the regulation of thymic cellularity and the development of CD4–CD8– DN and CD4+ CD8+ double-positive (DP) thymocytes [21, 23, 24]. Analysis of Shh–/– and Gli3–/– thymi has shown that Hh signaling is a positive regulator of the early stages of thymocyte development, controlling homeostasis of DN progenitors and differentiation from DN1 to DN2 [22, 25, 26]. In addition, the effector function of peripheral CD4+ T cells is modulated by Shh [27, 28]. Nevertheless, conditional deletion of Smo from T-lineage cells failed to show any influence of loss of Hh signaling after the DN2 stage, and no defect in T-cell activation was detected in Smo-deficient lymphocytes [22]. Therefore, the efficacy of using the Hh gene to modulate T-cell development still remained unclear. Furthermore, the effect of Hh on differentiation of B cells is poorly understood [29, 30]. One obstacle to a extensive analysis of the effects of the Hh protein in the postnatal period in vivo is the difficulty of obtaining or producing sufficient quantities of active Hh proteins with lipid modification.

The use of adenoviral vectors (Advs) is one of approaches to confer the high-level ectopic expression of certain soluble factors, because they allow for regional expression of a given factor for a sufficient duration to exert a physiological effect in mice. The i.v. injection of Adv into mice results primarily in the localization of the Adv to extramedullary organs, in particular the liver [31]. Hence, administration of Adv expressing Ihh in mice is an easily established strategy to mimic sustained plasma release of Ihh, as previously shown in analyzing the effect of vascular endothelial growth factor (VEGF) and angiopoietin-1 in vivo via adenovirus-mediated gene transfer [32]. Moreover, this strategy is useful to gain insight into the efficacy of Ihh delivery for clinical application and to screen for possible side effects of Hh gene therapy [33, 34]. In the present study, we investigated the comprehensive effects on the hematopoietic and immune systems following adenoviral delivery of Ihh gene in vivo.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Recombinant Adenoviral Vectors
Replication-deficient Adv with RGD-mutated fibers was used as a mock control (Adv-control) [35, 36]. The pGEM-T easy-Ihh-N plasmid containing human Ihh cDNA corresponding to the biologically active N terminus of the Ihh protein fused to a secretion signal peptide (Ihh-N) [18] was digested with EcoRI (New England Biolabs, Beverly, MA, http://www.neb.com) and ligated into the EcoRI site in the chicken β-actin promoter with cytomegalovirus enhancer (CA promoter)-based plasmid vector pCAcc vector, and the resulting plasmid was designated pCAhIhh-N. The plasmid pCAhIhh-N was digested with ClaI, and the fragment containing the human Ihh-N cDNA expression unit was inserted into pWEAx-F/RGD [36], resulting in pWEAxCAhIhh-N-F/RGD. pWEAxCAhIhh-N-F/RGD was digested with PacI and transduced into human embryonic kidney (HEK) 293 cells using Lipofectamine 2000 (Invitrogen, Tokyo, http://www.invitrogen.com). The adenoviral human Ihh-N expression vector, AxCAhIhh-N-F/RGD (henceforth referred to as Adv-Ihh), was isolated, screened, propagated, and purified by CsCl equilibrium centrifugation. We did not detect contamination of the preparation with the E1 region and/or wild-type fiber by polymerase chain reaction (PCR), as described previously [37]. Viral titers were determined by an end point titer assay (tissue culture infective dose of 50%) using HEK293 cells or by estimation of adenoviral particle number, calculated from the optical density at 260 nm (OD260) [38]. The titers of each virus are shown in Table 1.


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  		Table 1. Infectious titers of Adv preparation

 
Animals and Administration of Adv Vectors
Immunodeficient age- (8 weeks), weight- (20 g), and sex-matched severe combined immunodeficiency (SCID) mice (on BALB/c background) and BALB/c were purchased from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org) and maintained in filtered-air Thorensen units. The study was approved by the institutional review board at our university. All mice received Adv-Ihh or the Adv-control in a volume of 100 µl, by single i.v. administration on day 0.

Expression and Activity of Ihh-N Protein by Adv-mediated Ihh-N cDNA Transfer
Adenovirus-mediated gene transduction into NIH3T3 cells was performed and expression of Ihh-N protein was confirmed by immunoblot analysis as described previously [18]. Briefly, cells were transduced by exposure to Adv-control or Adv-Ihh at various numbers of particles (particles per cell) for 1 hour in quadruplicate cultures. The culture medium was harvested 72 hours after transduction. To confirm expression of Ihh-N protein, 20 µl of culture medium was subjected to electrophoresis through a 4%–20% SDS-polyacrylamide gradient gel and transferred to a polyvinylidene difluoride membrane using a semidry transfer apparatus (Bio-Rad, Tokyo, http://www.bio-rad.com). Anti-human Hh antibody (Ab) (MAB1705; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) was used as the primary Ab. Proteins were visualized using the enhanced chemiluminescence method (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com).

The lhh reporter plasmid (TK-6GBS-Luc) expressed luciferase under control of the thymidine kinase (TK) promoter and was a gift of Dr. Jun Aruga (Developmental Neurobiology Laboratory, Brain Science Institute, RIKEN, Japan) [39]. The plasmid contained six tandem copies of the GL1-target site, 5'-TGGGTGGTC-3', inserted upstream of the TK promoter. The parental luciferase reporter plasmid was used as a control (TK-0GBS-Luc). For analysis of Ihh-N activity in transduced cells, NIH3T3 cells were exposed to the supernatant from cells transduced with Adv-Ihh for 24 hours. Subsequently, cells exposed to the supernatant were transfected with 200 ng of the luciferase reporters TK-0GBS-Luc or TK-6GBS-Luc, together with 2.5 ng of phRL-TK as an internal standard (Renilla luciferase) using Lipofectamine 2000. Luciferase activities were measured according to the manufacturer's recommendation (Promega, Madison, WI, http://www.promega.com) using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany, http://www.bertholdtech.com). Firefly luciferase activity was normalized to the Renilla luciferase activity. The relative fold activation is presented as the ratio of the normalized value to the activity observed in the cells transduced with the empty vector.

To monitor expression of vector-specific Ihh-N mRNA in injected mice, we performed reverse transcription (RT)-PCR using a forward primer specific for human Ihh and a reverse primer that binds the rabbit β-globin terminator in the Adv-Ihh vector. One microgram of total RNA from murine liver was reverse-transcribed by SuperScript II (Invitrogen) and amplified using the Advantage GC 2 Polymerase Mix (Clontech, Tokyo, http://www.clontech.com). The forward primer was 5'-TGCGGGCCGGGTCGGGTGGTG-3', and the reverse primer was 5'-AGATGCTCAAGGGGCTTCATGATG-3'. A murine/rat primer pair specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from R&D Systems.

Hematological Analysis
We analyzed total peripheral blood mononuclear cells from lateral tail veins with a Semi-Automated Microcell Coulter Model F820 (Sysmex, Kobe, Japan, http://www.sysmex.com) using a size distribution curve of 30–300 fl for leukocyte determinations. The peripheral blood was further analyzed by multiplying the total leukocyte count from an animal by the percentage of each cell type in the corresponding differential analysis performed by flow cytometry as previously reported [40]. Total leukocytes and lymphocytes were also evaluated using a Neubauer hematocytometer (Fisher Scientific, Pittsburgh, PA, http://www.fishersci.com) and stained by May-Giemsa staining, and differential leukocyte counts were obtained by examining the blood smears from each mouse (200 cells counted per smear).

Flow Cytometric Analysis
Flow cytometric analysis of peripheral blood mononuclear cells was performed every 3 weeks using phycoerythrin (PE)-conjugated Gr-1, CD3, or B220 Abs, including the monoclonal immunoglobulin isotype control (all from BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) as previously described [40]. Lymphocytes harvested from spleen of Adv-control or Adv-Ihh-injected mice were analyzed phenotypically by flow cytometry. Single-cell suspensions of BM, spleen, or thymus were prepared and filtered through a 40-µm nylon mesh (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). The cells were blocked in phosphate-buffered saline (PBS) (Life Technologies, Grand Island, NY, http://www.lifetech.com) with 10% normal mouse serum (BD Pharmingen) for 10 minutes at 4°C. One million cells were incubated with fluorescein isothiocyanate-conjugated CD3, B220, Mac-1, CD8, TCR{gamma}{delta}, or isotype control and CD3-PE, CD4-PE, CD43-PE, CD21-PE, CD23-PE, CD25-PE, CD19-APC, CD44-APC, or IgM-PE-Cy7 and the appropriate isotype control (BD Pharmingen) for 30 minutes at 4°C and washed twice with PBS containing 0.1% bovine serum albumin (BSA). The cells were analyzed by flow cytometric analysis using a FACSCalibur instrument (Becton Dickinson).

Determination of Cell Cycle and Apoptosis
Cell cycle analysis was performed by staining with equal volumes of 2 mg/ml RNase A in PBS and 0.6% Nonidet P40 containing 0.1 mg/ml propidium iodide (Calbiochem, La Jolla, CA, http://www.emdbiosciences.com) in PBS at 4°C for 30 minutes. Thereafter, cell cycle distribution was analyzed by flow cytometry as previously described [41].

Analysis of Clonogenic Cells
A clonogenic assay was performed as previously described [40]. Briefly, bone marrow cells were cultured in triplicate in Iscove's modified Dulbecco's medium containing 30% fetal calf serum, 1% BSA, 2% penicillin/streptomycin, 2 mmol/l L-glutamine (Life Technologies), 1 x 10–4 mol/l β-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 4 U/ml human erythropoietin (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 100 ng/ml recombinant rat stem cell factor, 100 U/ml murine interleukin-3 (10 ng/ml; Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and 0.8% methylcellulose (MethoCult M3134; StemCell Technologies). Progenitor colonies were counted after 14 days of culture in a humidified environment at 37°C and 5% CO2.

Histological Examination of Spleen, Thymus, BM, and Other Tissues
The spleen, thymus, BM, and other nonhematological tissues were collected for histological analysis. The spleen, thymus, and other organs were fixed with formalin and embedded in paraffin, and 10-µm sections were cut. These tissues were stained by hematoxylin and eosin, and BM tissue was stained after decalcification.

Statistical Analysis
Results are expressed as the mean ± SD. Each data set was first evaluated for normality of distribution by the Kolmogorov-Smirnov test to decide whether a nonparametric rank-based analysis or a parametric analysis should be used. Two groups were compared by either the Student's t test or the Mann-Whitney U test. p values of less than 0.05 were considered to be statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
The Expression of Functional Ihh Protein in Ihh-N Gene-Transduced NIH3T3 Cells and the Duration of Ihh-N Expression in Mice
We first evaluated the expression and activity of the Ihh-N protein following transduction of NIH3T3 cells with an Adv encoding Ihh-N (Adv-Ihh). To do so, we harvested the supernatants of Adv-Ihh-transduced NIH3T3 cells 48 hours after transduction of the cells at various numbers of particles and subjected them to immunoblot analysis for lhh (Fig. 1A). We detected expression of the Ihh protein in cells transduced with as little as 5,000 particles (pt) per cell in a dose-dependent manner (Table 1). We next evaluated the activity of the expressed lhh with a luciferase reporter assay. We used a reporter plasmid comprising the luciferase gene under control of a TK promoter containing six tandem repeats of a GL1-binding sequence, which is activated in response to hedgehog signaling. Serum-starved NIH3T3 cells were exposed for 24 hours to the supernatant from NIH3T3 cells that had been transduced with Adv-Ihh at various numbers of particles. The cells were then transfected with the luciferase reporter plasmid to assess lhh activity. Luciferase activity was normalized to the activity of an internal transfection control (Renilla luciferase). As shown in Figure 1B, Hh activity detected by this assay was greater in cells exposed to supernatant from cells transduced with higher number of particles of Adv-lhh, indicating that Adv-Ihh transduced cells produced functional Ihh-N protein.


Figure 1
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  		Figure 1. Expression and activity of Ihh-N protein following adenovirus-mediated gene transfer. (A): Expression of human Ihh-N in NIH3T3 cells transduced with Adv-Ihh encoding Ihh-N. Ihh expression was measured 48 hours after transduction of cells with Adv-Ihh at various numbers of pts per cell. The supernatants of the transduced NIH3T3 cells were analyzed by immunoblot analysis. (B): Analysis of the activity of the Ihh-N protein. The Ihh-N activity in the supernatants of cells transduced with Adv-Ihh was evaluated using a luciferase reporter assay. Serum-starved NIH3T3 cells were incubated for 24 hours to supernatant from cells transduced with Adv-Ihh. Subsequently, the incubated cells were transfected with Ihh-responsive luciferase reporter plasmids. Luciferase activity was normalized to the activity of an internal control (Renilla luciferase). The data are presented as the mean ± SD from triplicate assays. (C, D): RT-polymerase chain reaction (PCR) analysis of the expression of vector-derived Ihh-N mRNA in the livers of severe combined immunodeficiency (C) or BALB/c mice (D). Adv-Ihh was injected as a single i.v. injection of 1 x 1010 viral pts on day 0, mice were sacrificed weekly, and the expression of vector-derived Ihh-N was analyzed by RT-PCR, along with GAPDH as an internal standard. RT reaction was used as a negative control. Abbreviations: Ihh, Indian hedgehog; mGAPDH, mouse-glyceraldehyde-3-phosphate dehydrogenase; Pre, pre-injection; pt, particle; RT, reverse transcription.

 
We next determined the tolerability of delivery of Adv-lhh delivery to SCID mice by lateral tail vein injection. We injected 1 x 109, 5 x 109, or 1 x 1010 pt of Adv-Ihh or Adv-control into SCID mice. All mice that received 1 x 1010 pt or fewer of Adv-control or Adv-Ihh survived for more than 28 days. We therefore determined that a single administration of 5 x 109 and/or 1 x 1010 pt of Adv was sufficient to evaluate the efficacy and the toxicity of Adv-Ihh on hematopoietic tissues and nonhematopoietic tissues in injected animals. We next examined the duration of Ihh-N expression following administration of Adv-Ihh into SCID and BALB/c mice. We previously showed that most of the Adv resulted in liver transduction following tail vein injection [31]. We assessed the expression of vector-derived exogenous lhh-N mRNA in the liver by RT-PCR analysis, as we are unable to distinguish human and mouse lhh by immunoblot analysis due to the identical sequence of the active N-terminal Ihh protein (human Ihh-N, 28–202 amino acids [aa], NP_002172.2 [GenBank] ; mouse Ihh-N, 66–240 aa, NP_034674 [GenBank] ). Vector-derived Ihh-N mRNA was detected in the livers of injected SCID mice up to 28 days following Adv-Ihh administration (Fig. 1C). In contrast, we were able to detect expression of lhh-N mRNA for 14 days following administration of the vector to BALB/c mice (Fig. 1D). However, the significant decline in expression of vector-derived Ihh-N mRNA by day 21 in BALB/c was observed (Fig. 1D).

The Effect of Ihh Expression on Peripheral Blood, Bone Marrow Progenitor Cells, and the Structure of the Bone Marrow Cavity
Hh is reported to stimulate the production of angiopoietin-1 and VEGF, which mobilize endothelial progenitor cells and hematopoietic stem/progenitor cells [32]. Therefore, we examined counts and phenotypes of peripheral blood (PB) cells in mice injected with the Adv-control and Adv-lhh. Unexpectedly, we did not observe a significant difference between the number of PB mononuclear cells (MNCs) in animals injected with the Adv-control and Adv-Ihh. In addition, neither the hemoglobin concentration nor platelet counts differed in animals that received either the control or lhh vector (data not shown). We next assessed the percentage of c-Kit+ or Flk-1+ cells in the PB by flow cytometric analysis. We did not observe an increase of either cell population in PB MNCs, indicating that Ihh did not mobilize either the hematopoietic stem/progenitor or endothelial progenitor cell populations (Fig. 2A–2C).


Figure 2
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  		Figure 2. Analysis of peripheral blood and bone marrow (BM) cells after administration of adenoviral vector (Adv)-control or Adv-Ihh. A single dose of 5–10 x 109 particles (pt) of Adv-control or Adv-Ihh was administered intravenously into severe combined immunodeficiency mice. (A): Total number of PBMCs at 7, 14, 21, and 28 days. (B): Total number of c-Kit+ cells in mononuclear cells (MNCs). (C): Total number of Flk1+ endothelial progenitor cells in MNCs. The hatched line indicates control mice injected with the Adv-control (1 x 1010 pt [n = 6]), and the solid line indicates mice injected with Adv-Ihh (1 x 1010 pt [n = 6]). Four weeks after vector administration, BM cells were analyzed by flow cytometry and a clonogenic assay. (D): The total number of c-Kit+ cells in Adv-control (open columns) and Adv-Ihh treated (filled columns) mice in BM cells. (E): The total number of Flk-1+ cells in Adv-control (open columns) and Adv-Ihh treated (filled columns) mice in BM cells. The results are expressed as means ± SD. *, p < .01, control group versus Ihh-N group. Abbreviations: Ihh, Indian hedgehog; PBMC, peripheral blood mononuclear cells.

 
To examine the BM cells, SCID mice were sacrificed and BM cells were harvested from femurs 28 days after administration of either Adv-control or Adv-Ihh. As shown in Figure 2D and 2E, c-Kit+ hematopoietic cells and Flk-1+ cells were increased in BM MNCs. Concomitantly, BM progenitor cells were also increased in the BM cavity of Adv-Ihh-injected SCID mice, although no significant change of BM cells was observed in Adv-control-injected SCID mice (Fig. 3A–3D). These results are consistent with previous reports showing that Shh and Ihh signaling stimulates the proliferation of hematopoietic stem/progenitor cells [18, 42]. We next analyzed BM histology, because BM cellularity in Adv-Ihh-injected SCID mice decreased slightly (Fig. 3A). Unpredictably, the BM sinusoidal space appeared to be dilated, and there was evidence for proliferation of capillaries in all Adv-Ihh-treated SCID mice, whereas there were no remarkable changes in Adv-control-treated mice (Fig. 3E). This increase in endothelium could potentially account for the slight decrease of BM cells. These results suggest that in addition to hematopoietic cells, BM endothelial cell populations respond to Ihh. In addition, we did not observe an effect of lhh overexpression on any other nonhematopoietic tissue that we evaluated (supplemental online Fig. 1).


Figure 3
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  		Figure 3. The analysis of BM clonogenic cells and histological findings after administration of Adv-control or Adv-Ihh. (A–D): The number of total BM cells (A), CFU-C (B), BFU-E (C), and CFU-Mix (D) in Adv-control (open columns) and Adv-Ihh treated (filled columns) mice. The results are expressed as means ± SD. *, p < .01, control group versus Ihh-N group. (E): Histological analysis of BM in severe combined immunodeficiency (SCID) mice 28 days after Adv administration. Tissues were stained by hematoxylin and eosin after decalcification. (Ei): 1 x 1010 particles (pt) of Adv-control-injected SCID mouse. (Eii): 1 x 1010 pt of Adv-Ihh-injected SCID mouse. Original magnifications, x100 (Ei, Eii). Images are representative of four independent histological analyses. Abbreviations: BFU-E, blast forming unit-erythroid; BM, bone marrow; CFU, colony-forming unit; Ihh, Indian hedgehog.

 
The Effect of Overexpression of Ihh-N on Splenocytes in BALB/c Mouse
To evaluate lymphatic tissue, BALB/c mice were injected intravenously with 1 x 1010 pt of Adv-control or Adv-Ihh, and the injected mice were sacrificed 2 weeks later. The size of the spleen was increased in all Adv-Ihh-treated mice compared with the Adv-control-treated mice (Fig. 4A) without any histological changes in liver tissue (supplemental online Fig. 2). We observed increased cellularity and follicular hyperplasia in the spleens of Adv-Ihh-treated mice (Fig. 4B, 4C). We next evaluated the immunophenotype of splenocytes by flow cytometric analysis using anti-Mac-1 antibody (a granulocyte/monocyte marker), anti-B220 antibody (a pan-B lymphocyte marker), and anti-CD3 antibody (a pan-T lymphocyte marker). We observed similar numbers of Mac-1+ splenocytes in mice injected with Adv-control or Adv-lhh. In contrast, the number of B220+ and CD3+ lymphocytes in Adv-Ihh-treated mice was greater than the number in Adv-control-treated mice (Fig. 4D).


Figure 4
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  		Figure 4. Analysis of splenocytes of BALB/c mice 14 days after administration of Adv-control (control) or Adv-Ihh (lhh). (A): Representative macroscopic analysis of the spleen. (B): The total number of splenic mononuclear cells after injection with Adv-control or Adv-Ihh. The data are presented as the mean ± SD. *, p < .01, Adv-control versus Adv-Ihh. (C): Histological analysis of spleen in Adv-control or Adv-Ihh treated mice. (D): Splenic mononuclear cells were immunolabeled by B220 (pan-B cell marker), CD3 (pan-T-cell marker), or Mac-1 (granulocyte/monocyte marker) and analyzed by flow cytometry. The y-axis indicates the number of B220, CD3 or Mac-1+ splenocytes. The results are expressed as mean ± SD. *, p < .01, Adv-control versus Adv-Ihh. Abbreviations: Ihh, Indian hedgehog; pt, particles.

 
We next analyzed the maturation of splenic B cells by analyzing the expression of IgM, which is expressed by mature, splenic B lymphocytes [29, 30]. The percentage of B220+sIgM+ cells was greatly reduced in the spleens of Adv-Ihh-treated mice compared with Adv-control-treated mice (27.7 ± 2.1 vs. 4.9 ± 1.1; p < .01) (Fig. 5A, 5B). Conversely, the percentage of B220+sIgM– cells in the spleens of Adv-Ihh-treated mice was significantly elevated compared with Adv-control-treated mice (24.4 ± 1.8 vs. 36.9 ± 4.2; p < .05) (Fig. 5A). Similarly, the absolute numbers of B220+IgM+ and B220+IgM– splenocytes were decreased and increased, respectively, in Adv-Ihh-treated mice compared with the numbers in Adv-control-treated mice (Fig. 5B). These results suggest that Adv-lhh treatment leads to an increase in the number of immature splenic B lymphocytes and a decrease in the number of IgM+ mature splenic B lymphocytes. We further analyzed the expression of B220, CD23, and CD21 in splenic B lymphocytes by flow cytometric analysis. The percentage of splenic B220+CD23int or B220+CD21int B lymphocytes in mice treated with Adv-Ihh was likely to increase compared with that in mice treated with Adv-control (Fig. 5A). These results suggest that overexpression of the Ihh gene induces an increase in follicular B cells (B220+IgMlowCD23intCD21int) in vivo. We further analyzed the expression of B220, CD43, and CD19 in BM by flow cytometric analysis as described previously [30]. The percentage of B220+CD19+CD43+ and B220+CD19+CD43– (pro-B cells) cells was similar in mice treated with either Adv-Ihh or Adv-control (supplemental online Fig. 3). These results suggest that administration of Adv-lhh did not affect the number of B cell precursor cells in BM.


Figure 5
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  		Figure 5. Flow cytometric analysis of splenocytes in BALB/c mice 14 days after administration of 1 x 1010 particles of Adv-control or Adv-Ihh. (A): Expression of IgM, CD23, or CD21 on B220+ splenic B lymphocytes in Adv-control or Adv-Ihh treated mice. Data are representative of three independent experiments of three showing similar results. (B): The absolute number of IgM+B220+ mature splenic B lymphocytes and IgM-B220+ immature splenic B lymphocytes in Adv-control (open columns) and Adv-Ihh-treated (filled columns) mice. The results are expressed as means ± SD. *, p < .01, Adv-control versus Adv-Ihh. (C): Absolute number of CD4+ or CD8– splenocytes was compared in mice treated with Adv-control (open columns) or Adv-Ihh (filled columns). Data are the average from three independent experiments, each done in triplicate. The data are presented as the mean ± SD. *, p < .01, Adv-control versus Adv-Ihh. (D): Cell cycle analysis of splenic CD4+ and B220+ cells by propidium iodide staining. Splenic CD4+ or B220+ cells were labeled by FITC-conjugated anti-CD4 or B220 antibody and separated using anti-FITC microbeads and a magnetized column. Abbreviations: FITC, fluorescein isothiocyanate; Ihh, Indian hedgehog; PE, phycoerythrin.

 
We next examined CD4 or CD8 expression in the T-cell subsets. The number of CD4+ but not CD8+ T lymphocytes was increased in spleens of Adv-Ihh-treated mice (Fig. 5C). This result is consistent with a previous report showing that Shh promoted cell cycle progression in peripheral CD4+ T lymphocytes [28]. We therefore performed cell cycle analysis after separation of splenic CD4+ cells, as well as B220+ cells. As expected, the cell cycle of splenic CD4+ cells of Adv-Ihh-treated mice was enhanced compared with that of Adv-control-treated mice (Fig. 5D). Similarly, cell cycle progression of B220+ splenocytes were enhanced in Adv-Ihh-treated mice compared with the numbers in Adv-control-treated mice (Fig. 5D). These results indicate that overexpression of Ihh stimulates the proliferation not only of splenic CD4+ cells but also of B220+ cells.

The Effect of Overexpression of Ihh-N on Thymocytes In Vivo
As described above, the number of CD4+T lymphocytes was elevated in Adv-Ihh-treated mice compared with that in Adv-control-treated mice. It has been reported that Hh signaling effects thymocyte differentiation and thymus cellularity [21–24, 43]. Therefore, we performed analysis of the thymocytes after Adv-control or Adv-Ihh administration. The cellularity of the thymus was increased in Adv-Ihh-treated mice compared with that in Adv-control-treated mice (Fig. 6A). We next evaluated the immunophenotype of thymocytes from the treated mice using flow cytometry to classify the CD4+ and CD8+ T cells. The number of CD4–CD8– DN thymocytes, CD4+CD8– thymocytes, and CD4–CD8+ thymocytes was significantly greater in Adv-lhh-treated mice than in Adv-control-treated mice (Fig. 6B). We further assessed thymic DN1–4 subsets by analysis for CD44 and CD25 expression. Notably, DN2 and DN3 in Adv-Ihh-treated mice were significantly higher than those in Adv-control-treated mice (Fig. 6C). The absolute number of CD4+CD8+ DP thymocytes in mice treated Ihh vector showed a tendency (but not significant [p = .06]) to be higher than that in mice treated with the control vector [20, 23]. We further assessed thymic immature single-positive CD8s (ISPs) by staining with anti-CD3 antibody. As a result, CD3-CD8+ ISPs were significantly increased in mice after administration of Adv-Ihh compared with control mice (Fig. 6D). This result suggested that increment of thymic immature single positive cells (CD8+ ISPs) contributes to increment of CD4–CD8+ thymocytes. Collectively, Adv-Ihh administration could alter thymic cellularity and the composition of immature thymic T-cell compartment.


Figure 6
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  		Figure 6. Analysis of thymocytes in BALB/c mice 14 days after administration of 1 x 1010 particles of Adv-control or Adv-Ihh. (A): The total number of thymocytes in mice treated with Adv-control and Adv-lhh. Data are the average from three independent experiments; bars, SD. (B): The absolute number of CD4/CD8-expressing thymocytes. Thymocytes were immunolabeled by anti-CD4 and anti-CD8 antibody and analyzed by flow cytometry. Absolute number of CD4–CD8–, CD4+CD8+, CD4+CD8–, and CD4–CD8+ thymocytes was compared in mice treated with Adv-control (open columns) or Adv-Ihh (filled columns). Each p value (Adv-control vs. Adv-Ihh) is indicated at the top of the bar. Data are the average from three independent experiments, each done in triplicate; bars, SD. (C): Analysis of CD4–CD8– DN thymocytes. Thymocytes were immunolabeled with anti-CD4-fluorescein isothiocyanate (FITC), CD8-FITC, TCR{gamma}{delta}-FITC, anti-CD25-phycoerythrin (PE), and anti-CD44-APC. FITC-positive cells were gated out, and DN thymocytes were classified by CD25 and CD44 expression. Data are the average from three independent experiments, each done in triplicate. The data are presented as the mean ± SD. *, p < .01, Adv-control versus Adv-Ihh. (D): Analysis of immature single-positive CD8s in thymocytes. Thymocytes CD8 were stained with anti-CD3-PE and anti-CD8-FITC to assess the immature single-positive cells (CD3–CD8+ thymocytes). Data are the average from three independent experiments, each done in triplicate; bars, SD. Abbreviations: DN, double-negative; Ihh, Indian hedgehog.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
We have shown here that overexpression of Ihh following adenovirus vector-mediated i.v. gene transfer to mice results in an elevation of splenic B-lymphocyte and thymic T-lymphocyte cellularity and clonogenic hematopoietic cells. In addition, we observed no apparent histological change in nonhematological tissues following Adv administration via the tail vein.

We previously reported that overexpression of lhh in human stromal cells led to the expansion of hematopoietic progenitors and in vivo myeloid- and lymphoid-repopulating hematopoietic cells [18]. Likewise, Shh and Dhh have been shown to stimulate proliferation of hematopoietic progenitor cells and long-term hematopoietic repopulating cells in ex vivo culture [42]. Asn-50 and Ser-156 have been shown to be essential for Shh activity, and these residues are located close to the Ptc-binding site [44]. We compared the sequences of Ihh and Dhh with that of Shh by a Human Protein Reference Database BLAST search (http://www.hprd.org) and found that Ihh and Dhh are highly homologous to Shh at the amino acid level (88% and 74%, respectively). Furthermore, the Ptc-binding site in Shh is completely preserved in Ihh and Dhh, although the N terminus of the three proteins was quite divergent. Therefore, each of the three Hh family members functions via Ptc-Smo signal transduction to stimulate proliferation of hematopoietic stem cells, at least in ex vivo culture. Recently, Trowbridge et al. have made use of Ptc-1+/– cells to show that Hh regulates postnatal hematopoietic homeostasis and regeneration [45]. Consistent with these reports, Adv-mediated transfer of the Ihh gene led to an increase in the number of hematopoietic progenitor cells in mouse BM (Fig. 3A–3D). However, we also observed an altered BM microenvironment characterized by vascular distension and proliferation of capillaries (Fig. 3E). Both Ihh and Shh have been reported to regulate the differentiation of mesenchymal stem cells into cells of the osteoblastic lineage [46]. In addition, Hh signaling induces the expression of angiogenic factors such as VEGF and angiopoietin-1, suggesting involvement in angiogenesis during tissue regeneration [32]. Furthermore, Ihh has recently been shown to regulate skeletal angiogenesis, perichondrial maturation, and the formation of bone and the hematopoietic stem cell-supporting osteoblastic niche that lines endosteal bone [47]. Taken together, the results suggest that an increase in the level or duration of Ihh expression might induce altered BM structure in Adv-Ihh-treated mice.

Interestingly, we observed splenomegaly in Adv-lhh-treated mice. It has recently been reported that germinal center B cells express both components of the Hh receptor, Ptc and Smo, and that Shh is produced by follicular dendritic cells mainly in the germinal center [29]. These results are consistent with our results showing that lymph follicles in the spleen of Adv-lhh-treated mice are enlarged (Fig. 4C) and that treated mice exhibit an increase in the number of B220+ B lymphocytes in the spleen (Fig. 4D). Intriguingly, the number of immature B220+IgMlowCD23intCD21in splenic B lymphocytes is increased in Adv-Ihh-treated BALB/c mice (Fig. 5A, 5B), and the percentage of B220+CD19+CD43– pro B cells in BM was similar in Adv-Ihh-treated and Adv-control-treated mice (supplemental online Fig. 3). One possible explanation for these results is that Hh might regulate the maturation of splenic B cells in addition to functioning as a survival factor for germinal center B cells [29]. Another possible explanation is that Ihh dominantly induces an increase in follicular B cells, because cell cycle analysis of B220+ cells indicated that progression of cell cycle is remarkably enhanced in Adv-Ihh-treated BALB/c mice (Fig. 5D) [29].

We found that there was an increase in the number of CD3+ T lymphocytes in the spleens of mice injected with Adv-lhh (Fig. 4D) and that CD4+ but not CD8+ subsets were specifically increased (Fig. 5C). Moreover, CD4+ lymphocytes in the spleens of mice injected with Adv-lhh, remarkably, entered into S-phase of the cell cycle (Fig. 5D). These results indicate that Adv-lhh treatment stimulated the proliferation of CD4+ lymphocytes in the spleen, which is consistent with a previous report that Hh signaling enhances the effector function and cell cycle progression of activated T cells in vitro [27, 28]. We also examined the production of CD4+ and CD8+ lymphocytes in the thymus. It has been reported that both Hh receptor molecules, Ptc and Smo, are expressed by CD4–CD8– DN thymocytes, and Shh protein has been shown to arrest thymocyte differentiation at the DN stage in vitro [23]. In the present study, both thymic cellularity and the absolute number of DN thymocytes were increased in Adv-Ihh-treated mice (Fig. 6A, 6B). Moreover, the absolute number of DN2 and DN3 subsets was elevated in Adv-Ihh-treated mice compared with Adv-control-treated mice (Fig. 6C). These findings are consistent with those of the previous study showing that Hh signaling promotes thymocyte proliferation and development from DN1 to DN2 and that high-dose Hh blocks differentiation of DN3 thymocytes in vitro [22, 23, 25]. Regarding CD8+ thymic lymphocytes, the number of CD8+ ISPs was elevated in Adv-Ihh-treated mice (Fig. 6D). Hence, increment of CD8+ ISPs largely accounts for elevation of CD4–CD8+ thymocytes in Adv-Ihh-treated mice. In this regard, it was revealed that Hh signaling induced differentiation from DN4 to CD8+ ISPs [25]. Thus, increment of CD8+ ISPs in Adv-Ihh-treated mice could be a response to the overexpression of the Ihh gene.

Our findings in this study and previous reports as mentioned above clearly revealed that Hh signaling affected the differentiation and proliferation of T-cell lineage [22, 23, 25, 26]. Nevertheless, conditional deletion of Smo, the only nonredundant regulator of the Hh pathway, from T-lineage cells did not show any influence of loss of Hh signaling after the DN2 stage, and no defect in T-cell activation was detected in Smo-deficient lymphocytes [22], suggesting that the Hh signaling to T lymphocytes could be transduced in more complicated system. In this regard, it has been recently shown that endogenous Hh suppressors, including Hh interacting protein (HIP) [48], growth arrest-specific 1 [49], potassium channel tetramerization domain containing 11 (RENKCTD11) [50], FK506-binding protein 8 (FKBP8) [51], and interference Hedgehog [52, 53], were involved in modulation of Hh signaling pathway. HIP expression is induced by Shh signaling in the pancreas [54] and endothelial cells [55], where it may function in a negative feedback loop. FKBP8 is highly expressed in the retina, liver, pancreas, and kidney but not other tissues [51]. Thus, contribution of these inhibitory components to Hh signaling may confer the delicate and different effect of Hh members in a variety of tissues [48, 54, 56].

In summary, administration of Adv-Ihh could modulate the proliferation and differentiation of splenic B lymphocytes and thymic T lymphocytes during BM hematopoiesis in vivo. Modulation of the Hh signaling pathway may provide a therapeutic strategy to regulate lymphomyelopoiesis in vivo.


   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
 
This article has been carefully reviewed by an experienced medical editor in Network Assistance International Inc. This work was supported in part by a grant from the Ministry of Health, Labor and Welfare of Japan (to M.K.).


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

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