Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0903440 on May 3, 2004

Published online before print May 3, 2004
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(Journal of Leukocyte Biology. 2004;76:484-490.)
© 2004 by Society for Leukocyte Biology

Increased numbers of committed myeloid progenitors but not primitive hematopoietic stem/progenitors in mice lacking STAT6 expression

Kevin D. Bunting*,{dagger},1, Wen-Mei Yu*, Heath L. Bradley*, Eleonora Haviernikova*, Ann E. Kelly-Welch{ddagger}, Achsah D. Keegan{ddagger},§ and Cheng-Kui Qu*,{dagger},1

* Departments of Hematopoiesis and
{ddagger} Immunology, American Red Cross, Jerome H. Holland Laboratory for the Biomedical Sciences, Rockville, Maryland; and Departments of
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal transducer and activator of transcription-6 (STAT6) plays important roles in cytokine signaling via interleukin-4 and -13 receptors (IL-4R and IL-13R). Mice in which STAT6 has been disrupted by homologous recombination show defects in T helper cell type 2 (Th2) lymphocyte production, resulting in an accumulation of Th1 cells. In addition to defects in differentiation and proliferation of T lymphocytes, STAT6-deficient mice show increased cell-cycle activation and frequency of myeloid progenitors. Although this has been shown to be mediated through Oncostatin M production by T cells, IL-4R{alpha} 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal transducer and activator of transcription (STAT) molecules are activated downstream of hematopoietic cytokines and play important roles in immune function. STAT1 [1 , 2 ] and STAT2 [3 ] are required for interferon (IFN) signaling, whereas STAT4 [4 , 5 ] and STAT6 [6 , 7 ] are required for T cell proliferation and differentiation. The roles of STAT3 and STAT5 are more pleiotropic, as a large number of cytokines and hormones use these signaling molecules [8 ]. STATs are activated by Janus kinase (JAK) family members, inducing dimerization and translocation to the nucleus, where target genes are transactivated. The JAK/STAT signaling axis is evolutionarily conserved and appears to be required for a wide range of cellular functions, including cell proliferation, differentiation, apoptosis, and movement [9 ].

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 {alpha} (IL-4R{alpha}) chain associates with the common {gamma} 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{alpha} 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Long-term culture medium for primitive murine hematopoietic cells was purchased from StemCell Technologies (Vancouver, BC, Canada). [{alpha}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 [{alpha}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To investigate the role of STAT6 during hematopoietic development, we obtained STAT6–/– mice, which were bred onto the BALB/c genetic background. WT BALB/c mice were used for comparison. The first analyses performed were on peripheral blood of mice, which contains the most differentiated cell types. The results are shown in Table 1 . The total cellularity of WBCs, RBCs, or PLTs was not changed in mice lacking STAT6 expression. The hematocrit levels were also unchanged, which is consistent with the normal, total RBC count. The differential counts were also not statistically different. However, there were significant variation and increases in the absolute neutrophil count (ANC) for some STAT6–/– mice (Fig. 1 ). Differential counts were also confirmed in some mice by scoring Wright-Giemsa-stained peripheral blood smears. We found that 31% of STAT6–/– mice had an ANC above 1800/µl, and no WT BALB/c mice had an ANC above this level. Furthermore, flow cytometry for Gr-1+ leukocytes showed four of 11 STAT6–/– mice with a percentage above 30%, whereas only one of five BALB/c WT mice had Gr-1+ cells above this level (data not shown). Although not statistically significant, these results suggest that STAT6 deficiency predisposes mice to an increased number of circulating neutrophils.


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  		Table 1. Comparison of Blood Cell Counts between WT and STAT6–/– Mice

 


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  		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.

 
We were next interested to determine whether changes in myeloid differentiation could be observed in the bone marrow. As bone marrow is the primary site of hematopoiesis and contains the majority of hematopoietic lineage-committed progenitors and differentiating progeny, we wanted to assess the total bone marrow cellularity. The total number of nucleated cells per femur was not changed between STAT6–/– and BALB/c WT mice (Fig. 2A ). However, the frequency of myeloid progenitors in the bone marrow measured following plating in methylcellulose medium was altered. STAT6–/– mice showed significant increases in the frequency of BFU-E, CFU-GM, and CFU-GEMM (Fig. 2B) . As the total bone marrow cellularity was not significantly decreased, the increased frequency of STAT6–/– myeloid progenitors also corresponded with an increased absolute number of myeloid progenitors. The increases were observed in multiple myeloid cell types, indicating that the effects were at a GEMM level-committed progenitor stage. Therefore, the bone marrow compartment of STAT6–/– mice had increased steady-state myelopoiesis. These results are consistent with previously reported observations in STAT6–/– bone marrow and spleen [21 ].



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  		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.

 
To extend these findings toward more primitive hematopoietic progenitors, total bone marrow cells were used in long-term culture experiments and assayed for the CAFC activity after 5 weeks of culture and also analyzed by fluorescence-activated cell sorter (FACS) for primitive stem/progenitor fractions (Table 2 ). For three separate experiments directly comparing WT and STAT6–/– mice, there was no significant difference observed in the CAFC frequency or number. To determine whether there were changes in the immunophenotype of primitive HSC/multipotent progenitor fractions, the absolute number of c-Kit+lin–Sca-1+ cells was quantitated. To determine whether there were changes in the oligopotent progenitor fraction that was committed to the common myeloid or common lymphoid progenitor, the c-Kit+lin–Sca-1– cell absolute number was quantitated. Both primitive bone marrow fractions were present in normal numbers in STAT6-deficient mice as compared with age-matched WT BALB/c control mice. Therefore, the abnormal myeloid expansion observed in the absence of STAT6 was not the result of expansion of a primitive multipotent progenitor population found within the bone marrow.


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  		Table 2. Comparison of Primitive Bone Marrow Populations in WT and STAT6–/– Mice

 
Although the FACS and CAFC assays measured cells with primitive stem/progenitor phenotype or function, the gold standard test for hematopoietic repopulating activity is reconstitution of hematopoiesis in irradiated recipient mice. To test the short-term radioprotection ability of STAT6-deficient or WT BALB/c bone marrow, mice were irradiated with 500 rads or 800 rads, and survival was determined (Fig. 3 ). All mutant and WT mice died at a similar rate in response to 800 rads irradiation, and all mice survived 500 rads of radiation. The trend toward greater survival in STAT6–/– mice at 800 rads could reflect a mild increase in myeloerythroid progenitor (MEP) activity [29 ]. However, there were not large differences in survival following radiation exposure.



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  		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.

 
To directly compare STAT6–/– and WT BALB/c mouse HSC fractions, two independent, competitive repopulation experiments were performed by mixing bone marrow from STAT6–/– versus WT bone marrow. Control competitions were set up with WT versus WT bone marrow. In all experiments, male bone marrow cells were mixed with female bone marrow cells to distinguish between the two donor grafts. At 12 weeks (Experiment #1, Fig. 4 ) and 29 weeks (Experiment #2, Fig. 5 ) following transplant, the mice were killed, and bone marrow, spleen, and thymus were collected for Southern blot analysis of male engraftment. For the WT versus WT control, the input value was 50% of each graft, but it is expected that because of female nonhematopoietic contribution to bone marrow, spleen, and thymus, the output values would be reduced from this percentage. As total engraftment for both experiments, we observed 21 ± 7% male BALB/c WT engraftment (n=10) and 21 ± 15% male STAT6–/– engraftment (n=7) in the bone marrow. We observed 27 ± 10% male BALB/c WT engraftment (n=10) and 21 ± 10% male STAT6–/– engraftment (n=8) in the spleen. We observed 25 ± 9% male BALB/c WT engraftment (n=10) and 23 ± 6% male STAT6–/– engraftment (n=7) in the thymus. Therefore, the mixing gave comparable levels of engraftment with mutant or WT HSCs, and the relevant comparison was between the WT versus STAT6–/– engraftment, which was identical in all tissues from two independent experiments. This result indicates that the increased bone marrow myeloid progenitor number was not a result of expansion at the HSC or primitive progenitor level.



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  		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.

 


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  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD4+ T lymphocytes can be divided into two types of Th cells. Th1 cells secreting cytokines such as IL-2, IFN-{gamma}, 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 ).



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  		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.

 
Other than the increased numbers of committed myeloid progenitors, we found that mature neutrophils were increased in a subset of mice. Overall, these numbers were statistically unchanged as a result of a fairly large standard error, but we cannot rule out that during induced inflammatory stress, STAT6–/– bone marrow might be capable of producing greater numbers of neutrophils. The increased severity of inflammation reported by others in STAT6–/– mouse models for experimental autoimmune encephalomyelitis [30 ], arthritis [31 ], or septic peritonitis [32 ] might be explained twofold. First, increased production of the proinflammatory cytokines IL-12, TNF-{alpha}, and IFN-{gamma} 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{alpha} 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 {gamma} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by NIHR01HL068212 (C-K. Q.), NIHR01AI038985 (A. D. K), NIHR01AI045662 (A. D. K.), NIHR01DK059380 (K. D. B.), NIHR01HL073738 (K. D. B.), and NIHR21HL071171 (K. D. B.).

Received September 24, 2003; revised January 23, 2004; accepted February 2, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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