Published online before print May 3, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
,
,1
* Departments of Hematopoiesis and
Immunology, American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland; and Departments of
Anatomy and Cell Biology and
Immunology, The George Washington University, Washington, D.C.
1Correspondence: Hematopoiesis Department, American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail: quc{at}usa.redcross.org or buntingk{at}usa.redcross.org
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and STAT6 have also recently been found to be enriched for expression in primitive hematopoietic stem cells (HSCs) in gene expression-profiling studies. Therefore, we have investigated whether defects in hematopoietic function in mice lacking STAT6 expression extended into the primitive hematopoietic compartments of the bone marrow. Here, we report that STAT6 deficiency increased bone marrow-committed myeloid progenitors but did not alter the number of cells enriched for HSC/multipotent progenitors, primitive cobblestone area-forming cells assayed in vitro, or bone marrow short-term or long-term repopulating cells assayed in vivo. Therefore, the requirement for STAT6 activation during hematopoiesis is limited, and primitive hematopoietic cell types are insulated against possible effects of cytokine stimulation by Th1 cells.
Key Words: interleukin-4 • knockout mouse • hematopoietic stem cell • hematopoiesis
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The roles that STATs play in hematopoiesis depend in large part on the cytokine receptor expression level in particular stages of hematopoietic development. In the case of STAT6, the interleukin-4 receptor (IL-4R) chain associates with the common 
chain of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 to promote lymphocyte development [6
, 10
, 11
]. These same lymphocytes are known to release growth factors that can alter hematopoiesis. The exact role of IL-4 in hematopoiesis is complicated by studies showing positive or negative effects on hematopoiesis. For example, the release of granulocyte macrophage-colony stimulating factor (GM-CSF) or G-CSF from T lymphocytes can be enhanced by IL-4 stimulation [12
], which would presumably favor myelopoiesis. IL-4 also appears to be capable of modulating the interaction of hematopoietic progenitors and stromal cells [13
]. However, there is also conflicting evidence supporting a negative regulatory role for IL-4 in modulating hematopoiesis via inhibition of GM-CSF production [14
]. Other studies have found that costimulation of IL-4 and stem cell factor (SCF) enhances stromal cell-dependent hematopoiesis [15
], and IL-4 has been reported to have direct effects on stimulation of G-CSF but inhibition of formation of macrophage colonies [16
, 17
]. These complicated, dual regulatory roles for IL-4 have made it difficult to assess the cell-intrinsic function in colony-forming activity of primary bone marrow cells. Therefore, IL-4 actions may be indirect and result from alteration of the microenvironment.
The role of STAT6 signaling in hematopoiesis has not been characterized extensively. In the absence of STAT6 in mice, T helper cell type 1 (Th1) cells accumulate because of a block in differentiation to the Th2 stage [6 , 7 ]. In IL-4-induced T lymphocytes from STAT6–/– mice, proliferative responses are reduced because of defective p27(Kip1) expression and dysregulation of cell-cycle progression [18 , 19 ]. These Th1 cells then modulate the hematopoietic microenvironment in tissues, such as the spleen and bone marrow, to increase the number of myeloid progenitors [20 , 21 ]. It has been demonstrated that Th1 cells can regulate hematopoietic progenitor numbers by secretion of oncostatin M (OsM) [21 ], which is an early-acting, hematopoietic cytokine that is activated downstream of JAK/STAT signaling. OsM is a target gene for STAT5, and it is believed to be important for myelopoiesis. Overexpression of constitutively activated STAT5 or OsM in mice results in leukemia [22 ], and OsM–/– mice have mild defects in erythroid and megakaryocytic differentiation [23 ].
Gene expression-profiling studies have identified the IL-4R and STAT6 as genes that are differentially expressed at high levels in primitive hematopoietic cells [24
]. This suggested that IL-4 or STAT6 signaling might play a functional role in hematopoietic stem cells (HSCs). Based on the results of prior studies showing alteration of myeloid progenitor homeostasis in STAT6–/– mice, we initiated experiments to determine whether these effects were also seen in populations of bone marrow cells enriched for primitive, multipotent progenitors or bona fide, long-term, repopulating HSCs. The results indicate that STAT6 is not essential for bone marrow HSC homeostasis and that the effects of dysregulated cytokine production in these mice are uniquely limited to the granulocyte/erythroid/monocyte/macrophage (GEMM) progenitor compartment.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
32P]-Deoxy-cytidine 5'-triphosphate was obtained from Amersham (Arlington Heights, IL). NcoI restriction enzyme was obtained from New England Biolabs (Beverly, MA).
Mice and peripheral hematology analysis
Wild-type (WT) control BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). STAT6–/– mice on the BALB/c genetic background were obtained from Dr. William E. Paul [Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Bethesda, MD] and were bred in-house. Mice were housed at the American Red Cross Holland Laboratory Vivarium (Rockville, MD). All animal procedures complied with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Four- to 8-week-old mice were bled from the retro-orbital venous sinus following puncture with a heparinized microcapillary tube. Whole blood was then analyzed using a Cell Dyn 3700 hematology analyzer (Abbott Laboratories, Abbott Park, IL). Hematocrit readings were performed manually in centrifuged microhematocrit tubes. Differential counts were independently confirmed by manually scoring Wright-Giemsa-stained peripheral blood smears.
Flow cytometry
Peripheral blood leukocytes obtained from mice were analyzed on a BD Biosciences (San Jose, CA) LSR flow cytometer following staining for Ly-6G (Gr-1). For c-kit-lin-Sca-1 (KLS) and c-kit-lin (KL) quantitation, whole bone marrow cells were isolated from hindlimbs of WT and STAT6–/– mice by flushing into phosphate-buffered saline (PBS)/2% fetal bovine serum (FBS), followed by staining with a cocktail of phycoerythrin-conjugated antibodies to lineage markers that included Ly-6G (Gr-1), CD11b (Mac-1), CD45R/B220, CD4 (L3T4), CD8 (Ly-2), Ter119/Ly-76, CD90.2 (Thy1.2), and NK1.1 (NKR-P1B and NKR-P1C). The cells were also stained with antibodies to fluorescein isothiocyanate-conjugated Ly-6A/E (Sca-1) and biotin-conjugated CD117 (c-Kit). All antibodies for these studies were obtained from BD PharMingen (San Diego, CA).
Cobblestone area-forming cell (CAFC) assay
Bone marrow cells were harvested from hindlimbs of WT and STAT6–/– mice, and long-term cultures with limiting dilution to quantitate the primitive progenitor cells were performed as reported [25
]. To prepare stromal layers, mouse bone marrow-nucleated cells were cultured at 33°C in long-term culture medium [-minimum essential medium with 12.5% horse serum, 12.5% FBS, 0.2 mM I-inositol, 16 µM folic acid, 10–4 M 2-mercaptoethanol (2-ME), 2 mM L-glutamine, and 10–6 M hydrocortisone]. After 2 weeks, confluent stromal layers were trypsinized, irradiated (1500 rads), and subcultured in 96-well flat-bottomed plates at a density of 2.5 x 104 cells/well. Cultures were then seeded with serially diluted single-cell suspensions of bone marrow cells in the same medium. Bone marrow-nucleated cells pooled from two to three animals of each genotype were seeded at twofold dilutions (105–1562 cells/well). Culture medium was gently half-changed with fresh medium weekly, and the CAFCs were scored after 5 weeks [26
]. The frequency of CAFC was calculated using the L-CALC software (StemCell Technologies) for limiting dilution analysis.
Hematopoietic progenitor differentiation assay
Bone marrow cells were harvested from hindlimbs of WT and STAT6–/– mice, and cells (2x104 cells/ml) were assayed for colony-forming units (CFUs) in 0.9% methylcellulose Iscove’s modified Dulbecco’s medium containing 30% FBS, glutamine (10–4 M), 2-ME (3.3x10–5 M), and a combination of hematopoietic growth factors {5% pokeweed mitogen-stimulated spleen cell-conditioned medium, 20 ng/ml IL-3, 50 ng/ml SCF, 2 units/ml erythropoietin (EPO), and 0.1 mM hemin [27
, 28
]}. After 7 days of culture at 37°C in a 5% CO2 incubator, hematopoietic cell colonies were counted under an inverted microscope.
Competitive repopulation assays
Bone marrow cells were harvested from hindlimbs of WT and STAT6–/– mice by flushing into PBS/2% FBS. Male WT or STAT6–/– bone marrow cells were mixed at an equal donor equivalent ratio with female WT competitor bone marrow. The mixed bone marrow was then injected into 850 rads irradiated female BALB/c recipients. Following bone marrow transplant, mice were killed at 12 weeks (Experiment #1) and 29 weeks (Experiment #2), and the tissues were collected for Southern blot analysis of engraftment with Y-chromosome-positive cells. Standard curves were generated by mixing male and female DNA, and standard curves were run with each set of Southern blot gels.
Southern blotting
Cells were lysed overnight at 37°C in a solution containing 0.6 mg/ml proteinase K in 50 mM Tris, pH 8.0, 1% sodium dodecyl sulfate (SDS), 100 mM NaCl, and 10 mM EDTA, pH 8.0. Genomic DNA was then extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and precipitated with 2.5 vol ice-cold ethanol and 1/10 vol sodium acetate. DNA (15 µg) was digested overnight with NcoI restriction endonuclease and separated on a 0.8% agarose gel by electrophoresis. Gels were blotted onto Hybond N+ nylon membrane (Amersham), UV-cross-linked, and hybridized with a [32P]-labeled mouse Y-chromosome-specific DNA probe and a ß-actin DNA probe for normalization of loading. Blots were washed at a final stringency of 0.5x saline sodium citrate/0.5% SDS at 65°C and were exposed overnight, and autoradiographic images were obtained using a Molecular Dynamics (Sunnyvale, CA) StormTM phosphorimager.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
View this table: [in a new window] |
Table 1. Comparison of Blood Cell Counts between WT and STAT6–/– Mice
|
 View larger version (11K): [in a new window] |
Figure 1. ANC of peripheral blood leukocytes from WT and STAT6–/– mice. WT and STAT6–/– mice were bled from the retro-orbital venous plexus using heparinized microcapillary tubes. The total WBC count was determined by automated counting on a hematology analyzer. The differential counts were determined by the hematology analyzer and independently confirmed in some cases by manually scoring Wright-Giemsa-stained blood smears. The ANC was determined by multiplying the total WBC/µl by the percentage of neutrophils. The ANC represents the number of neutrophils per µl whole blood. ANC is plotted on the y-axis, and each individual circle on the graph represents a single mouse. The number of mice analyzed is shown above each vertical point scatter-plot.
|
 View larger version (13K): [in a new window] |
Figure 2. Total cellularity and myeloid progenitor content of WT and STAT6–/– mouse bone marrow. (A) WT and STAT6–/– mice were killed, and hindlimbs were removed. The bone marrow cells from the femur were flushed out using a syringe loaded with PBS/2% FBS. The total numbers of nucleated cells per femur were counted using a hemacytometer under a standard microscope. The results shown are the average of eight WT and eight STAT6–/– mice. (B) Bone marrow (BM) cells were plated into methylcellulose culture medium in the presence of 5% pokeweed mitogen-stimulated spleen cell-conditioned medium and added IL-3, SCF, and EPO. Hematopoietic colonies were scored 7 days later for colonies with the distinct erythroid burst-forming units (BFU-E), CFU-GM, or CFU-GEMM morphology. The results shown are the average of three WT and three STAT6–/– mice.
|
|
View this table: [in a new window] |
Table 2. Comparison of Primitive Bone Marrow Populations in WT and STAT6–/– Mice
|
 View larger version (15K): [in a new window] |
Figure 3. Short-term radioprotective capacity of endogenous WT and STAT6–/– bone marrow cells. Mice were irradiated with 800 rads or 500 rads from a Cs137 source, and survival of mice was determined by checking mice daily. The numbers of mice irradiated with each dose is shown in the key. The percentage of surviving mice is shown on the y-axis, and the number of days following irradiation is shown on the x-axis. Any mice that were found moribund were killed, according to protocols approved by the Holland Laboratory Institutional Animal Care and Use Committee.
|
 View larger version (40K): [in a new window] |
Figure 4. Competitive repopulation Experiment #1 to compare bone marrow from WT and STAT6–/– mice for HSC activity. Bone marrow cells were collected from donor male WT and STAT6–/– mice and also from female WT mice. The male WT and female WT bone marrow samples were mixed 1:1 as a control. Male STAT6–/– bone marrow was mixed 1:1 with female WT bone marrow cells to assess the relative ability of STAT6–/– bone marrow to engraft competitively. All cells were injected into 850 rad-irradiated female BALB/c recipients. The bone marrow (BM), spleen (SPL), and thymus (THY) were collected 12 weeks later, and the relative male engraftment was determined by Southern blotting. (A) A standard curve of mixed male/female DNA controls; (B) the engraftment levels in tissues from analyzed mice. The percent male engraftment below each lane was normalized based on the ß-actin signal. The results are from a total of five WT and three STAT6–/– competitions into recipient mice. Y-probe, Y-Chromosome-specific DNA probe.
|
 View larger version (54K): [in a new window] |
Figure 5. Competitive repopulation Experiment #2 to compare bone marrow from WT and STAT6–/– mice for HSC activity. Bone marrow cells were collected from donor male WT and STAT6–/– mice and also from female WT mice. The male WT and female WT bone marrow samples were mixed 1:1 as a control. Male STAT6–/– bone marrow was mixed 1:1 with female WT bone marrow cells to assess the relative ability of STAT6–/– bone marrow to engraft competitively. All cells were injected into 850 rad-irradiated female BALB/c recipients. The bone marrow (BM; A), spleen (SPL; B), and thymus (THY; C) were collected 29 weeks later, and the relative male engraftment was determined by Southern blotting. Each panel includes a standard curve of mixed male/female DNA controls. The percent male engraftment below each lane was normalized based on the ß-actin signal. Each lane on the gel represents analysis of male DNA contribution from an individual recipient mouse. Y-probe, Y-Chromosome-specific DNA probe.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, and tumor necrosis factor ß (TNF-ß) are involved in delayed hypersensitivity and have cytolytic activity. Th2 cells secreting cytokines such as IL-4, IL-5, IL-6, and IL-10 represent mature Th cells capable of promoting immunoglobulin class-switching and B lymphocyte development. STAT6 is known to play a key role in Th2 cell development. In the absence of STAT6, mice accumulate Th1 cells because of the Th2 block. An interesting, recent finding was that STAT6 deficiency can regulate myeloid progenitor cell homeostasis through an indirect mechanism, likely mediated by secretion of a growth factor [21
]. However, although the defects in STAT6–/– mice were limited to the relatively mature myeloid progenitor compartment, it remained unclear whether STAT6 was important in other primitive hematopoietic cell types. We have now found no effects in stem cells or primitive progenitors indicating that the ability of Th1 cells to regulate hematopoiesis is rather limited (Fig. 6
).
 View larger version (22K): [in a new window] |
Figure 6. Model for the role of Th1 T lymphocytes in regulating hematopoiesis. The STAT6-deficient mouse has a block in Th2 differentiation leading to increased Th1 levels. Secretion of bioactive cytokines by Th1 cells regulates hematopoiesis in a narrow GM or GEMM population of committed myeloid progenitors. Th1 cells may regulate mature neutrophil differentiation but have no effect on primitive HSC/progenitor populations. These effects of Th1 cells are unique from activated T cells, where more primitive levels of hematopoiesis can be affected. The reason for these differences is not yet known. MPP, Multipotent progenitor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte/monocyte progenitor.
|
, and IFN- has been described in STAT6–/– mice [32
] and STAT6-defective human B lymphoblast cell lines [33
]. Second, we propose that the expanded CFU pool could promote rapid production of neutrophils upon appropriate stimulation. The significant variation between individual STAT6–/– mice but not WT BALB/c mice in terms of neutrophil numbers could result from this predisposition. It is interesting to note that STAT6 deficiency increased the CFU numbers significantly but had at best only minimal effects on the MEP function as measured by in vivo survival following sublethal irradiation. Short-term survival is determined by the radioprotective capacity of the endogenous bone marrow cells and also the relative radiation sensitivity of the two types of mice being compared. As our studies in fibroblasts and thymocytes from STAT6–/– mice have not shown differences in radiation sensitivity (data not shown), we conclude that the radioprotective capacity was also unchanged or at best, only moderately increased. It is possible that compensating pathways provide the appropriate downstream survival and proliferation signals independent of STAT6. One possibility is that a protein tyrosine-binding (PTB) protein, such as the insulin receptor substrate (IRS1 or IRS2) or the Shc adaptor protein, may signal cross-talk with other compensating pathways. IRS2 is known to interact with the IL-4R PTB region to regulate activation of the STAT6 signaling pathway [34 ].
It was known 16 years ago that the IL-4R is ubiquitously expressed in the hematopoietic lineage. IL-4R can be found on mature T and B lymphocytes, macrophages, and blast-like hematopoietic cell lines [35
]. In addition to the classical B lymphocyte stimulatory activity, at the progenitor cell level, IL-4 costimulation synergizes with G-CSF to promote granulocyte-colony formation, with EPO effects on BFU-E and megakaryocytopoiesis by IL-3 [36
, 37
]. Furthermore, the combination of IL-4 and IL-11 has been reported to stimulate more primitive, multipotential blast-cell colonies [38
]. The role of IL-4/IL-4R signaling in primitive hematopoiesis, however, is not well defined. Although IL-4R and STAT6 are expressed highly in the Sca-1+c-Kit+lin– HSC/multipotent progenitor-enriched fraction [24
], our results indicate that STAT6-mediated signaling does not play an intrinsic role in HSC survival or proliferation. The long-term, competitive repopulating assays presented here demonstrate unequivocally that there are no intrinsic alterations in HSC number or function in the absence of STAT6.
Studies of the role for T cells in engraftment of HSCs have primarily been focused on activated T cells. IL-2 along with other costimulatory molecules results in T cell activation and proliferation in vitro. It is interesting that the absence of IL-2 in knockout mice shows an accumulation of T cells with the activated or memory phenotype [39
], and some mice develop symptoms of an autoimmune disorder similar to ulcerative colitis [40
]. Defects in IL-2 production can cause aberrant myelopoiesis and can alter hematopoietic engraftment [41
]. Bone marrow HSCs from IL-2 knockout mice also have a deficiency in long-term repopulating activity [42
]. Therefore, defects in cytokine production by activated T cells may play some role in a type of autoimmune destruction of bone marrow cells. Mice lacking JAK3 also demonstrate an activated T cell phenotype that increases myelopoiesis as a result of alterations in cytokine signaling through the common chain [43
]. Therefore, in general, activated T cells secrete cytokines that promote myeloid development and inhibit HSC function. The STAT6-deficient phenotype appears to be distinct from these phenotypes perhaps as a result of the different type of T cell defect and type of cytokines released. The results of our study suggest that the consequences of cytokine production by Th1 cells differ from that of activated T cells, as HSCs and primitive progenitors are spared from the effects regulated by Th1 cells. The mechanism for the narrow sensitivity within the myeloid progenitor compartment will require further study. Perhaps a better understanding of the pathologic consequences of T cell defects on HSC-repopulating activity could lead to improved engraftment and use of HSCs for therapy of blood diseases.
Received September 24, 2003; revised January 23, 2004; accepted February 2, 2004.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and interleukin-4 reciprocally regulate the production of monocytes/macrophages and neutrophils through a direct effect on committed monopotential bone marrow progenitor cells Eur. J. Immunol. 23,1072-1077[Medline] and IFN Int. J. Oncol. 24,447-453[Medline]-chain in regulating activation of the insulin receptor substrate 2 and signal transducer and activator of transcription 6 pathways. Analysis by mutagenesis J. Biol. Chem. 273,9898-9905/ß T cells effectively eliminate abnormally activated T cells lacking the interleukin 2 receptor ß in vivo J. Exp. Med. 190,1561-1572This article has been cited by other articles:
 |
|
 |
|
  Z. Wang, G. Li, W. Tse, and K. D. Bunting Conditional deletion of STAT5 in adult mouse hematopoietic stem cells causes loss of quiescence and permits efficient nonablative stem cell replacement Blood, May 14, 2009; 113(20): 4856 - 4865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Thawani, M. Tam, and M. M. Stevenson STAT6-mediated suppression of erythropoiesis in an experimental model of malarial anemia Haematologica, February 1, 2009; 94(2): 195 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. G.P. Bumm, C. Elsea, A. S. Corbin, M. Loriaux, D. Sherbenou, L. Wood, J. Deininger, R. T. Silver, B. J. Druker, and M. W.N. Deininger Characterization of Murine JAK2V617F-Positive Myeloproliferative Disease Cancer Res., December 1, 2006; 66(23): 11156 - 11165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Giron-Michel, M. Giuliani, M. Fogli, D. Brouty-Boye, S. Ferrini, F. Baychelier, P. Eid, C. Lebousse-Kerdiles, D. Durali, R. Biassoni, et al. Membrane-bound and soluble IL-15/IL-15R{alpha} complexes display differential signaling and functions on human hematopoietic progenitors Blood, October 1, 2005; 106(7): 2302 - 2310. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
