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The Journal of Immunology, 2002, 168: 1746-1752.
Copyright © 2002 by The American Association of Immunologists

Impaired NK Cell Development in an IFN-{gamma} Transgenic Mouse: Aberrantly Expressed IFN-{gamma} Enhances Hematopoietic Stem Cell Apoptosis and Affects NK Cell Differentiation1

Osamu Shimozato*, John R. Ortaldo*, Kristin L. Komschlies{dagger} and Howard A. Young2,*

* Laboratory of Experimental Immunology, Center for Cancer Research, and {dagger} Intramural Research Support Program, Science Applications International Corp.-Frederick, National Cancer Institute, Frederick, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aberrant expression of IFN-{gamma} has been demonstrated to cause a wide variety of alterations in cell function and development. Previously we reported that constitutive expression of IFN-{gamma} in bone marrow (BM) and thymus results in a total absence of B cells and a substantial decrease in the number of hematopoietic progenitor cells. In this study, we demonstrate a severe deficiency of NK1.1+CD3- cells in this transgenic mouse model. Compared with normal control littermates, we found a pronounced reduction of NK cells in IFN-{gamma} transgenic mouse spleen and liver despite maintenance of normal function. In addition, we observed a reduced number of BM cells in the IFN-{gamma} transgenic mouse despite normal expression of hematopoietic growth factors in the BM. Interestingly, these cells were less responsive to stem cell factor (SCF) despite c-kit expression on hematopoietic stem cells (HSCs). We observed that addition of exogenous IFN-{gamma} inhibited proliferation of HSCs and differentiation of NK precursors from HSCs in normal mice in response to SCF, IL-7, fms-like tyrosine kinase 3 ligand, and IL-15. Furthermore, we found that HSCs express the IFN-{gamma}R{alpha} subunit and undergo apoptosis in response to exogenous IFN-{gamma}. Thus, we have demonstrated the occurrence of a severe deficiency of NK cells and lower numbers of BM cells in an IFN-{gamma} transgenic mouse model. Furthermore, because exogenous IFN-{gamma} affects the responsiveness to hematopoietic growth factors such as SCF in vitro, our results indicate that chronic expression of IFN-{gamma} in vivo leads to widespread immune system defects, including alterations in NK cell differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Natural killer cells are a critical component of the innate immune response to viral and obligate intracellular parasitic infection. Although NK cells and T cells appear to differentiate from a common T/NK progenitor, NK cell cytolytic activity, unlike cytotoxic T cell-mediated lysis, does not require presensitization, is not MHC restricted, and is not dependent on a rearranged Ag-specific receptor. NK cell differentiation is a multistep process that involves the commitment of hematopoietic stem cells (HSCs)3 to the NK cell lineage followed by further differentiation to yield functional mature NK cells. This process occurs primarily within the bone marrow (BM) microenvironment and requires cell-to-cell interactions and soluble factors derived from BM stromal cells (1, 2, 3). Based on a series of studies, it has been determined that a specific population of HSCs differentiates into cells that express NK cell markers and are capable of mediating natural cytotoxic activity in vitro (4, 5). Early-acting cytokines, such as stem cell factor (SCF) (c-kit ligand or steel factor) and fetal liver kinase ligand/fms-like tyrosine kinase 3 ligand (Flt3L), act on HSCs to induce the {beta} subunit of IL-2R and drive these precursor cells into the NK lineage (6, 7, 8, 9).

IFN-{gamma} is an immunoregulatory lymphokine that is primarily produced by T cells and NK cells (10, 11). IFN-{gamma} has multiple effects on the immune system, including the following: inducing macrophage cytotoxicity, enhancing Th1 cell growth, inhibiting Th2 cell growth, enhancing cytotoxic activity of CD8+ T cells and NK cells, up-regulating the expression of MHC molecules, and stimulating B cell IgG2a production (12, 13, 14, 15, 16). Changes in IFN-{gamma} expression are associated with numerous clinical conditions. For example, aberrant IFN-{gamma} expression in transgenic mice can result in diabetes, hepatitis, and retinal degeneration (17, 18, 19, 20).

Previously, we have generated an IFN-{gamma} transgenic mouse by inserting an extra copy of the murine IFN-{gamma} genomic DNA containing an Ig {lambda}-chain enhancer in the first intron of the transgene into the mouse germline. This IFN-{gamma} transgenic mouse aberrantly expresses IFN-{gamma} mRNA and protein in BM and thymus, and the IFN-{gamma} serum level is increased. Unlike other IFN-{gamma} transgenic mice, our IFN-{gamma} transgenic mouse shows a pronounced reduction of B lineage cells in BM, spleen, and lymph nodes, an increased number of CD4+CD8- or CD4-CD8+ thymocytes, and a decreased frequency and reduced number of myeloid progenitor cells in the BM (21).

In this study, we have investigated the possibility that aberrantly expressed IFN-{gamma} may alter NK cell development. Here we report the cellularity and phenotype of NK cells in the IFN-{gamma} transgenic mouse and examine whether exogenous IFN-{gamma} affects the differentiation of HSCs to NK precursor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 mice and IFN-{gamma} transgenic mice were supplied by the Animal Production Area (National Cancer Institute, Frederick, MD). Mice were kept in a specific pathogen-free facility and maintained in isolator cages on water and mouse chow ad libitum.

Reagents

Recombinant human (rh)IL-2 was obtained from Hoffmann-LaRoche (Nutley, NJ). Recombinant mouse (rm)IL-7 and rhFlt3L were purchased from R&D Systems (Minneapolis, MN). rmIL-12, rmSCF, and rhIL-15 were obtained from PeproTech (Rocky Hill, NJ). PE- and biotin-conjugated anti-mouse B220 (RA3-6B2), mouse CD11b (M1/70), mouse CD11c (HL3), Gr-1, TER-119, mouse CD4 (RM4-5), mouse CD8{alpha} (53-6.7), and NK1.1 (PK136); PE-conjugated anti-mouse fms-like tyrosine kinase 3 (Flt3) (A2F10.1); FITC-conjugated anti-mouse CD3 molecular complex (17A2) and anti-mouse DX5 (DX5); PE-conjugated anti-mouse CD27 (LG.3A10), anti-mouse CD44 (IM7), anti-mouse IL-2R{gamma} (TUGm2), and anti-mouse c-kit (2B8); biotin-conjugated anti-mouse CD2 (RM2-5), anti-mouse IL-2R{beta} (TM-{beta}1), anti-mouse CD244 (2B4), and anti-mouse CD16/32 (2.4G2); FITC-, PE-, and biotin-conjugated isotype-matched control Ig; and annexin V-FITC apoptosis determining kit I were purchased from BD PharMingen (San Jose, CA). Cell death detection ELISAplus was purchased from Roche Molecular Biochemicals (Indianapolis, IN). PMA and ionomycin were purchased from Calbiochem (San Diego, CA).

In vivo treatment of mice with IL-2, NK cell isolation, and 51Cr-release assay

Splenic NK cells were isolated from spleens of IFN-{gamma} transgenic and normal littermate control mice as previously described (22). Briefly, nylon wool nonadherent cells were depleted of T/NKT cells by Ab and complement. In vivo IL-2 treatment was performed as previously described (23). IFN-{gamma} transgenic mice and control littermates were injected with 6 x 105 IU of rhIL-2 twice a day for 3 days (36 x 105 IU/total/head). On day 4, livers and spleens were harvested. Cytolytic activity against YAC-1 cells was measured by a standard 6-h 51Cr-release assay.

Surface Ag analysis by flow cytometry

Spleen and liver cells were isolated as previously described (22). BM cells were isolated from tibia and femur of IFN-{gamma} transgenic and normal littermate control mice. Cells were directly stained using PE- and FITC-labeled primary Abs or indirectly stained using a biotinylated primary Ab followed by streptavidin-PerCP (BD PharMingen). Anti-mouse CD16/CD32 mAb (2.4G2; BD PharMingen) was used to block the nonspecific binding.

Cytokine assay

Purified liver NK cells were treated with different stimuli for 24 h as indicated in Results. The cell-free supernatants were collected and assayed for cytokine production by ELISA. The specific ELISA kits for mouse (m)IFN-{gamma}, mIL-13, and mTNF-{alpha} were purchased from R&D Systems.

HSC isolation and quantification of viability of HSC in response to SCF

HSCs were prepared as described (8). In brief, HSCs were sorted from BM cells as c-kit+lineage markers (lin)- (B220, CD11b, CD11c, Gr-1, TER-119, NK1.1, CD4 and CD8) using the MoFlo sorter (Cytomation, Fort Collins, CO), and then cultured with mSCF (50 ng/ml) and mIFN-{gamma} (200 U/ml). To evaluate the responsiveness to SCF, the numbers of viable cells were determined by trypan blue staining and dead cells were determined by flow cytometry analysis (FCA) following propidium iodide (PI) staining. Apoptotic cell death was determined by annexin V staining (BD PharMingen) and cell death detection ELISAplus (Roche Molecular Biochemicals) according to the manufacturer’s instructions.

Generation of NK cells from HSC in vitro

In vitro NK cell development from HSC was performed as previously described (8). In brief, isolated HSCs were cultured with mSCF (50 ng/ml), hFlt3L (50 ng/ml), and hIL-7 (5 ng/ml) in the presence or absence of mIFN-{gamma} (200 U/ml). After 6 days, growth factors were removed and the viable cells (1 x 106 cells) were cultured in the presence of rhIL-15 (200 ng/ml) for an additional 6 days. HSC-derived NK cells were analyzed for the surface expression of NK1.1 as described above.

Statistical analysis

Student’s paired t test was used to determine the significance of differences between means, and a value of p < 0.05 was taken as indicating statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduced numbers of NK cells in the IFN-{gamma} transgenic mouse

It has been reported that a subpopulation of B220+CD19- cells in BM contains NK cell progenitors (24). Because our IFN-{gamma} transgenic mouse lacks mature and immature B cells in BM and peripheral lymphoid organs (21), we hypothesized that IFN-{gamma} impaired NK cell development as well as B cell development in this transgenic mouse. To examine this possibility, freshly isolated splenocytes from the IFN-{gamma} transgenic mouse were stained with mAbs to CD3 and a NK marker, NK1.1, and compared with splenocytes from a normal control mouse. We observed a significantly reduced percentage of the NK cell population in the spleens of the IFN-{gamma} transgenic mouse when compared with normal control mouse (3.3 vs 0.5%; Fig. 1Go). Furthermore, the cell number was reduced 15-fold in the NK cell population in the spleen and liver of the IFN-{gamma} transgenic mouse (Table IGo). Next, we administered IL-2 i.v. into both IFN-{gamma} transgenic mouse and normal control mouse to obtain cells generated by IL-2-induced leukocyte rebound in the spleen and liver (23). We observed a lower but substantial rebound of NK1.1+CD3- cells obtained following IL-2 treatment in the transgenic mouse when compared with the normal control mouse (19.2 vs 2.2%; Fig. 1Go and Table IGo). In addition to the results obtained with the splenic cell populations, we observed an NK cell deficiency in the liver of the IFN-{gamma} transgenic mouse even after IL-2 treatment (Table IGo). In contrast to NK cells, we observed the percentage of T and NKT cell populations were increased in the IFN-{gamma} transgenic mouse when compared with normal control littermates (83.1 vs 34.0% and 6.3 vs 2.0%, respectively) while the numbers of T and NKT cells were equivalent to the control mouse, despite the reduced numbers of total lymphocytes in the IFN-{gamma} transgenic mouse (Table IGo). We also observed a reduced percentage and number of NK1.1-CD3- cells in spleen and liver lymphocytes (Fig. 1Go and Table IGo). However, this phenomenon was consistent with our previous report that the IFN-{gamma} transgenic mouse lacks the B cell compartment in the spleen and BM (Ref. 21 and data not shown).



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  		FIGURE 1. Representative pattern of spleen cells in the IFN-{gamma} transgenic mouse. Splenocytes were isolated from HBSS or in vivo IL-2-treated animals as described in Materials and Methods. Cells were stained with FITC-conjugated anti-mCD3 molecular complex and PE-conjugated anti-NK1.1 mAb.

 

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  		Table I. Cellularity of spleen and liver MNC in IFN-{gamma} transgenic mouse1

 
Functional characterization of NK cells in the IFN-{gamma} transgenic mouse

Based on our findings of the numbers and reduced response to IL-2 in NK cells from the IFN-{gamma} transgenic mouse, we hypothesized that the alteration of the phenotype of NK cells could result from the aberrant expression of IFN-{gamma} in vivo. To examine this possibility, we first performed three-color FCA on splenocytes from the IFN-{gamma} transgenic mouse. We observed that the expression of DX5, CD244 (2B4), CD2, CD27, CD44, CD16/32 (Fc{gamma}RII), IL-2R{beta}, and IL-2R{gamma} on NK1.1+CD3- cells in the IFN-{gamma} transgenic mouse were the same as on NK cells from the normal littermate control mouse (Fig. 2Go). We also found that a lymphokine-activated killer cell marker, B220 (25), was induced on NK cells from the IL-2-treated IFN-{gamma} transgenic mouse to the same level as seen on NK cells from the normal control mouse (Fig. 3GoA).



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  		FIGURE 2. Surface markers on NK cells isolated from the IFN-{gamma} transgenic mouse. Representative patterns indicate the expression of DX5, 2B4, CD2, CD44, Fc{gamma}RII, CD27, IL-2R{beta}, and IL-2R{gamma} on NK1.1+CD3- gated cells.

 


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  		FIGURE 3. B220 induction and cytolytic activity of NK cells in in vivo IL-2-treated IFN-{gamma} transgenic mice. A, B220 induction on cytokine-activated NK cells. Splenocytes were isolated from in vivo IL-2-treated animals. Cells were stained with FITC-conjugated anti-B220, PE-conjugated anti-NK1.1, and biotin-labeled anti-mCD3 mAb followed by streptavidin-PerCP. Representative patterns demonstrate the expression of B220 on NK1.1+CD3- splenocytes. B, NK activity. NK cells were isolated from spleen of in vivo IL-2-treated IFN-{gamma} transgenic mice and normal littermates as described in Materials and Methods. Cytolytic activity against YAC-1 cell was measured by a 6-h 51Cr release assay at the indicated E:T cell ratios. Data represent mean ± SD of triplicate wells.

 
It has been reported that NK cells produced various cytokines, such as IFN-{gamma}, IL-13, and TNF-{alpha}, in response to IL-2, IL-12, and IL-18 (22); therefore, we investigated the cytokine production in response to IL-2, IL-12, and IL-18 (Table IIGo). We observed a strong IFN-{gamma} production in NK cells from the IFN-{gamma} transgenic mouse. We also detected hyperproduction of TNF-{alpha} in NK cells from the IFN-{gamma} transgenic mouse even when the cells were not stimulated. In contrast, we observed the same level of IL-13 production in NK cells from the IFN-{gamma} transgenic mouse in response to IL-2 plus IL-18 stimulation when compared with NK cells from the normal control mouse. However, IL-2 alone induced much higher IL-13 production in cells from the transgenic mouse.


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  		Table II. Cytokine production by NK cells in the IFN-{gamma} transgenic mouse1

 
Furthermore, to evaluate the cytolytic activity of NK cells in the IFN-{gamma} transgenic mouse treated with IL-2 in vivo, we purified splenic NK cells from the IFN-{gamma} transgenic mouse (purity of NK1.1+CD3- cell was >80% with no contaminating any CD3+ T or NKT cells; data not shown) and compared the cytolytic activity of purified NK cells with normal control littermates. As shown in Fig. 3GoB, we did not detect any difference in YAC-1 cell-targeted cytolytic activity of NK cells between the IFN-{gamma} transgenic mouse and control littermates. These data indicated that the expression of cell surface molecules and the cytolytic activity of NK cells in the IFN-{gamma} transgenic mouse is normal while the absolute numbers and cytokine production was altered in the cells obtained from the transgenic mouse.

Reduced numbers, but normal phenotype, of BM progenitor cells in the IFN-{gamma} transgenic mouse

The microenvironment of the BM plays an important role in stem cell maturation and differentiation. BM stromal cells produce various growth factors for HSCs, including SCF, IL-3, IL-6, and IL-7, which induce stem cell proliferation and differentiation of lineage-committed hematopoietic progenitor cells (1, 2, 3). Previously, we had reported that the absolute numbers and frequency of BM progenitor cells in the IFN-{gamma} transgenic mouse were reduced (21). As reduced numbers of BM cells in the IFN-{gamma} transgenic mouse could result from impaired expression of these early-acting cytokines, we prepared total RNA from whole BM cells from both IFN-{gamma} transgenic and the normal control mouse and performed ribonuclease protection assay analysis to determine the gene expression of these growth factors in BM. We observed that the mRNA expression of these growth factors in the BM of the IFN-{gamma} transgenic mouse was the same as that observed in BM from normal control littermates (data not shown). It has been reported that SCF is the most primitive growth factor for HSC proliferation and also acts to enhance the commitment of HSCs into NK cells using in vitro culture systems and c-kit gene-mutated mouse, W/Wv (26, 27, 28, 29, 30). To test the hypothesis that aberrantly expressed IFN-{gamma} affects the expression of c-kit, we investigated the expression of c-kit on BM stem cells in the IFN-{gamma} transgenic mouse; however, we detected normal expression of c-kit on BM stem cells in theIFN-{gamma} transgenic mouse when compared with BM from the normal control mouse (Fig. 4GoA). Next, to determine whether there is an altered response of HSC from the IFN-{gamma} transgenic mouse to these hematopoietic growth factors, both whole BM cells and sorted HSCs were cultured in the presence of SCF for 6 days in vitro. As shown in Fig. 4GoB, whole BM cells in the IFN-{gamma} transgenic mouse demonstrated significantly lower responsiveness to SCF in this in vitro culture system (p value is 0.0093). However, we observed the recovery of the lower responsiveness to SCF of HSCs in the IFN-{gamma} transgenic mouse by eliminating the lin+ cells in BM, although the responsiveness of HSCs from the IFN-{gamma} transgenic mouse is substantially reduced (p value is 0.0091). These data indicated that aberrantly expressed IFN-{gamma} in BM lin+ cells affects the HSC proliferative response to SCF although it does not affect the expression of SCF, IL-3, IL-6, IL-7 and its receptors on BM cells in the IFN-{gamma} transgenic mouse.



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  		FIGURE 4. Normal c-kit expression but lower responsiveness to SCF of BM cells in the IFN-{gamma} transgenic mouse. A, Representative pattern of lineage markers on BM cells. BM cells were stained with FITC-conjugated anti-c-kit, PE-conjugated anti-lineage markers as described in Materials and Methods. B, Reduced responsiveness to SCF of BM stem cells isolated from the IFN-{gamma} transgenic mouse. BM cells (left) and sorted HSC (right) from the IFN-{gamma} transgenic mouse (filled bar) and normal control littermates (open bar) were cultured with mSCF (50 ng/ml) for 6 days. Data represent mean ± SD of the number of viable cells in triplicate wells. *, p < 0.01 vs control littermates.

 
Decreased responsiveness to Flt3L of HSCs in the IFN-{gamma} transgenic mouse

Based on the analysis of IL-15 and IL-2R{beta} gene-targeted mice, IL-15 can play an important role in NK cell development (1). Recently, it has been reported that Flt3/Flt3L can play a role in the differentiation of IL-2R{beta}- HSCs to IL-15-reactive IL-2R{beta}+ HSCs, a representative NK progenitor cell in the BM (8). To examine whether aberrantly expressed IFN-{gamma} affects the development of HSCs to NK cells, we analyzed the differentiation of HSCs to NK progenitor cells in the response to SCF, IL-7, and Flt3L, followed by IL-15. After 6 days of secondary growth with IL-15, we performed FCA of the expression of NK1.1 on HSCs to evaluate the differentiation of HSCs from the IFN-{gamma} transgenic mouse and normal littermates. As shown in Fig. 5GoA, although NK1.1 expression was not observed on HSCs from both transgenic and normal animals stimulated with SCF, IL-7, and Flt3L (<1%, data not shown), the IL-15-induced NK1.1 expression on HSCs from the IFN-{gamma} transgenic mouse was significantly reduced as compared with HSCs from normal littermate controls (19.44 and 50.94%, respectively). Decreased responsiveness to IL-15 could result from the lower responsiveness of Flt3L in HSCs from the IFN-{gamma} transgenic mouse. To examine this hypothesis, we performed FCA to determine whether aberrant expression of the IFN-{gamma} in transgenic mouse had effects on the expression of Flt3 on lin-c-kit+ BM cells. As shown in Fig. 5GoB, reduced expression of Flt3 on lin-c-kit+ BM cells in the IFN-{gamma} transgenic mouse was observed as compared with normal control mice (11.4 and 20.58%, respectively). Collectively, these data indicated that aberrantly expressed IFN-{gamma} could affect the expression of Flt3 on lin-c-kit+ BM cells, which has been reported to represent one of the NK progenitor cells, in the IFN-{gamma} transgenic mouse, and lower expression of Flt3 could cause reduced differentiation of HSC to IL-15-reactive NK progenitors in response to Flt3L.



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  		FIGURE 5. Reduced numbers of NK progenitors in the BM of the IFN-{gamma} transgenic mouse. A, Representative pattern of NK1.1 expression on HSC-derived NK cells. HSCs were prepared from BM cells of the IFN-{gamma} transgenic mouse or normal control littermates and cultured with hematopoietic growth factors for 12 days as described in Materials and Methods. The cells were stained with PE-conjugated anti-NK1.1 mAb. B, Representative pattern of Flt3 on HSC. BM cells were stained with FITC-conjugated anti-c-kit, PE-conjugated anti-mFlt3, and biotin-conjugated lineage marker mAbs followed by PerCP-conjugated streptavidin. The expression of Flt3 represents the cells electrically gated on lin-c-kit+ cells.

 
Endogenous IFN-{gamma} triggers HSC cell death and inhibits differentiation to NK progenitors

We observed that high levels of IFN-{gamma} were secreted from lineage-committed cells in the BM of the IFN-{gamma} transgenic mouse (data not shown). Thus it is possible that aberrant IFN-{gamma} expression in the BM microenvironment could affect the responsiveness to hematopoietic growth factors. To examine whether aberrantly expressed IFN-{gamma} signaling is the basis for the altered cell responses described above, we investigated whether the receptor for IFN-{gamma} is expressed on HSCs. As shown in Fig. 6GoA, we detected the expression of IFN-{gamma}R{alpha} subunit on HSCs isolated from wild-type C57BL/6 mice. We investigated whether exogenous IFN-{gamma} could inhibit the proliferation of HSC from wild-type C57BL/6 mice in response to SCF. In fact, exogenous IFN-{gamma} inhibited the proliferation of HSC in response to SCF and this treatment resulted in an increase in the percentage of dead cells in the culture (Fig. 6GoB). To examine the possibility that IFN-{gamma} directly induces apoptosis in HSCs, we evaluated apoptosis in HSCs using annexin V staining. As shown in Fig. 7GoA, we detected the annexin V-positive cells in HSCs from C57BL/6 mice when the cells were cultured with exogenous IFN-{gamma}. Also, the percentage of the annexin V-positive cells increased in response to increasing levels of IFN-{gamma}. To exclude the possibility of necrotic cell death in HSCs, we analyzed the histone-associated DNA fragmentation in the HSCs, which is a typical feature of apoptosis (31). We observed that exogenous IFN-{gamma} increased DNA fragmentation in the cytoplasm in HSCs (Fig. 7GoB). These data demonstrated that IFN-{gamma} induces apoptotic cell death in HSC.



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  		FIGURE 6. Exogenous IFN-{gamma} abrogates the HSC proliferation and maturation of NK cells. A, Representative pattern of IFN-{gamma}R expression on HSCs. BM cells was stained with FITC-conjugated anti-c-kit, PE-conjugated anti-mIFN-{gamma}R{alpha} chain, and biotin-conjugated anti-lineage marker mAbs followed by PerCP-conjugated streptavidin. The data show the cells electronically gated on c-kit+lin- cells. B, Exogenous IFN-{gamma} triggers cell death in HSCs. HSCs were cultured with SCF in the presence or absence of IFN-{gamma} (filled bar or open bar, respectively) as described in Materials and Methods. Data represent mean ± SD of the number of viable cells and the percentage of dead cells in triplicate wells.

 


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  		FIGURE 7. IFN-{gamma} enhances apoptosis in HSC. A, Annexin V staining. HSCs were cultured with SCF in the presence of the indicated dose of IFN-{gamma} for 3 days. The cells were analyzed for the percentage of apoptosis by annexin V staining. The number represents the percentage of annexin V+PI+ cells and the number in parentheses represents the percentage of annexin V+PI- cells. B, DNA fragmentation. HSCs were prepared from BM of C57BL/6 mice and then cultured for 12 h in the presence or absence of IFN-{gamma}. DNA fragmentation was measured by the histone-associated DNA-specific ELISA. Data represent mean ± SEM.

 
Next, we examined the possibility that exogenous IFN-{gamma} could affect the differentiation of HSC to NK precursors during in vitro NK cell development. To focus on the effect of IFN-{gamma} on the Flt3L-dependent IL-2R{beta} induction, we investigated the capability of HSCs obtained from wild-type C57BL/6 mice to differentiate to NK precursors in the presence of exogenous IFN-{gamma} using in vitro culture systems with SCF, IL-7, and Flt3L, followed by IL-15. As shown in Fig. 8Go, in HSCs pretreated with IFN-{gamma} (Fig. 8Go, right panel) there was a reduced induction of NK1.1 expression on their surface in response to IL-15 when compared with HSCs pretreated without IFN-{gamma} (Fig. 8Go, left panel). These data indicated that exogenous IFN-{gamma} directly affected the induction of NK1.1 expression on NK precursors in response to IL-15.



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  		FIGURE 8. Exogenous IFN-{gamma} abrogates the maturation of NK progenitors. HSCs pretreated with SCF, IL-7, and Flt3L in the presence (right panel) or absence (left panel) of IFN-{gamma} for 6 days were cultured with IL-15 for an additional 6 days. IL-15-stimulated HSCs were analyzed for the expression of NK1.1 by FCA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have demonstrated that NK cells are dramatically reduced in a transgenic mouse aberrantly expressing IFN-{gamma} in the BM. Examination of spleen cells reveals a significantly reduced number of NK cells in this IFN-{gamma} transgenic mouse (Fig. 1Go and Table IGo). To examine the possibility that the transgene itself affected the phenotype of NK cells in the IFN-{gamma} transgenic mouse, we investigated the phenotype of the NK cells in this mouse. First, we have examined the expression of surface molecules, such as CD2, CD44, and CD27 (25, 32, 33), and the induction of B220 in response to IL-2 (25). Indeed, we did not observe any difference in the expression of these surface markers when comparing transgenic and normal mice (Figs. 2Go and 3GoA). Second, we have examined the cytolytic activity of NK cells against YAC-1 cell and we observed that NK cells in the IFN-{gamma} transgenic mouse demonstrated the same cytolytic activity against YAC-1 cells as NK cells from control littermates (Fig. 3GoB). In contrast, NK cells from the IFN-{gamma} transgenic mouse exhibited spontaneous hyperproduction of TNF-{alpha} and IL-13 following IL-2 activation in vivo (Table IIGo). We had previously reported that NK cells obtained from IFN-{gamma} knockout mice demonstrated increased production of IL-13 (34). In that report, we hypothesized that this increased production of IL-13 in IFN-{gamma} knockout mice could result from the deficiency of IFN-{gamma}, thus allowing expansion of an NK population producing IL-13. Although the current results would appear to contradict this model, it is possible that the IL-13-expressing cells represent precursors to NKT cells that have not yet expressed the CD3 molecule on their cell surface. Alternatively, the lack of B cells in this transgenic model might contribute, through unknown mechanisms, to the increased IL-13 production by permitting expansion of a cell population producing both IFN-{gamma} and IL-13. However, due to the lack of a suitable intracellular staining reagent for murine IL-13, we are unable to determine whether the NK cells are producing both IFN-{gamma} and IL-13. In contrast to the IL-13 results, it has been reported that IFN-{gamma} overcomes steroid suppression of TNF-{alpha} production in murine macrophages (35); therefore, it could be possible that a large amount of IFN-{gamma} expressed in the transgenic mouse induced TNF-{alpha} production in the NK cells. Further experimentation will be required to test these hypotheses.

Examination of lymphocyte populations in spleen and liver also reveals that T cell and NKT cell populations are increasing in the IFN-{gamma} transgenic mouse when compared with normal control littermates, whereas total cell number is reduced (Fig. 1Go and Table IGo). In our preliminary experiments, we found that the ratio of CD4:CD8 T cells in the spleen of the IFN-{gamma} transgenic mouse was lower than the ratio in splenic T cells from normal littermates (data not shown). Curiously, we also found that the number of CD8+TCR{alpha}{beta}+ T cells was increased in the BM of the IFN-{gamma} transgenic mouse (data not shown), although we had previously reported that the CD4/CD8 ratio of thymocytes in the IFN-{gamma} transgenic mouse was not affected (21). Thus, these data suggest that aberrantly expressed IFN-{gamma} expands the peripheral CD8+ T cells. Recently, it has been reported that IFN-{gamma} expands the NKT cell population in vivo (36), and these data are consistent with our findings. Thus, although it has been reported that NK, T, and NKT cells are developed from a common NK/T progenitor cell in BM (1, 2), the aberrantly expressed IFN-{gamma} might not cause a shift in the differentiation of a common NK/T progenitor cell to T and NKT cells. This hypothesis requires further experimentation.

Similar observations of a significant reduction in lymphopoiesis have been reported in IL-7/IL-7R, IL-15/IL-15R, common {gamma}-chain, and Flt3/Flt3L gene-targeted mice and W/Wv mice that contain a spontaneously mutated c-kit gene (1, 2, 3, 37, 38). Relevant to data reported in this study, our IFN-{gamma} transgenic mouse resembles the phenotype of IL-7/IL-7R, IL-15/IL-15, and Flt3/Flt3L gene-targeted mice and W/Wv mouse. Upon analysis of primitive hematopoietic precursor cells, we observed normal expression of HSC growth factors and receptors (Fig. 4GoA and data not shown). Interestingly, in vitro proliferation assays using BM cells from the IFN-{gamma} transgenic mouse revealed hyporesponsiveness to SCF in BM cells despite normal expression of c-kit. This hyporesponsiveness to SCF was reversed by the depletion of matured lin+ cells in BM (Fig. 4GoB). We have previously reported that this IFN-{gamma} transgenic mouse aberrantly produced IFN-{gamma} in BM and thymus and a major source of IFN-{gamma} in the thymus is the stromal cells (21). Furthermore, the experiment using HSCs obtained from wild-type C57BL/6 mouse demonstrated that the inhibition of HSC proliferation in response to SCF results from exogenous IFN-{gamma} that triggers apoptotic cell death in HSC in a dose-dependent manner (Fig. 7Go, A and B). This hypothesis is consistent with our previous report of the phenomenon of the reduced numbers of BM cells in this IFN-{gamma} transgenic mouse (21).

Abrogation of the proliferation of HSC by aberrantly expressed IFN-{gamma} may not be sufficient to account for the impaired NK cell development in the IFN-{gamma} transgenic mouse. To address this issue, we examined the frequency of NK precursors and the possibility that IFN-{gamma} could affect differentiation of HSC to NK lineage. We demonstrated the lower capability of HSCs in the IFN-{gamma} transgenic mouse to differentiate to NK lineage due to the lower frequency of lin-c-kit+Flt3+ cells in BM cells obtained from the IFN-{gamma} transgenic mouse. This phenotype has been reported to be one of the NK progenitor cells (Fig. 5Go, A and B). Furthermore, experiments using HSC obtained from wild-type C57BL/6 mice revealed that the exogenous IFN-{gamma} decreased the differentiation capability of HSC to IL-15-reactive NK precursors (Fig. 8Go). Thus, we speculate that IFN-{gamma} can affect either the expression of Flt3 on HSC in early stages of hematopoiesis or interfere with Flt3/Flt3L signaling to inhibit the expression of IL-2R{beta} on NK progenitors. Interestingly, a number of reports have shown that Flt3/Flt3L signaling has a role in the expansion of immature B cells that develop in the BM (39, 40, 41, 42). These reports could at least partially explain the phenomenon of B cell deficiency in the IFN-{gamma} transgenic mouse that may result from the reduced expression of Flt3 on HSCs in the IFN-{gamma} transgenic mouse. Therefore, we propose that the phenomenon of NK cell, but not T and NKT cell, deficiency in the IFN-{gamma} transgenic mouse could result from aberrantly expressed IFN-{gamma} inhibiting the differentiation capability of HSC to NK precursors.

In our previous studies, we administered IL-7 in an attempt to restore B cell development in this IFN-{gamma} transgenic mouse, because IL-7 is an indispensable growth factor for common lymphoid precursors in adult mouse lymphopoiesis. Curiously, examination of the spleen and BM of IL-7-treated IFN-{gamma} transgenic mice revealed no increase in the numbers of B220+IgM- cells in the BM and spleen when compared with wild-type mice, where large increases in B220+IgM- cells in the BM and spleen were observed (21). In addition, previous studies have been reported that IFN-{gamma} inhibits cytokine IL-7 signaling in in vitro experimental systems (43). These data suggested that aberrantly expressed IFN-{gamma} in BM might block IL-7 signaling. Recently, interesting data have been reported using a transgenic mouse that overexpressed suppressor of cytokine signaling 1 (SOCS1). This SOCS1 transgenic mouse reveals impaired T cell development in thymus due to an SOCS1 block of IL-7 signaling as observed in the IL-7/IL-7R gene-targeted mouse (44). Furthermore, it has been reported that SOCS1 binds to multiple receptor tyrosine kinases such as c-kit and Flt3 and suppresses their signaling (45). Thus, it could be possible that aberrantly expressed IFN-{gamma} in our mice induces SOCS1 in HSC and abrogates B and NK cell development due to the inhibition of SCF, Flt3, IL-7, and IL-15 signaling. This hypothesis remains to be tested.

In summary, the IFN-{gamma} transgenic mouse shows decreased numbers of peripheral mature NK cells as well as decreased responsiveness of HSC to growth factors. These experiments support the hypothesis that both the reduced number of HSC and the low frequency of Lin-c-kit+Flt3+ NK precursor cells in the HSC of the IFN-{gamma} transgenic mouse are a direct consequence of the aberrant IFN-{gamma} protein expression in the BM microenvironment.


   Acknowledgments
 
We thank John Wine for performing the animal experiments; Gordon Weigand for cell sorting; D. Reynolds and S. Krebs for technical assistance; Dr. T. Hoshino (Kurume University School of Medicine, Fukuoka, Japan) for helpful discussions; and S. Charbonneau and C. Champion for typing and editing this manuscript.


   Footnotes
 
1 The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. N01-CO-12400. O.S. was supported by the Japan Society for the Promotion of Science Fellowships for Japanese Biological and Behavioral Research at the National Institutes of Health. Animal care was provided in accordance with the procedures outlined in "A Guide for the Care and Use of Laboratory Animals" (National Institutes of Health Publication No. 86-23, 1985). Back

2 Address correspondence and reprint requests to Dr. Howard A. Young, Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute, Building 560, Room 31-93, Frederick, MD 21702-1201. E-mail address: youngh{at}mail.ncifcrf.gov Back

3 Abbreviations used in this paper: HSC, hematopoietic stem cell; SCF, stem cell factor; BM, bone marrow; Flt3, fms-like tyrosine kinase 3; Flt3L, Flt3 ligand; FCA, flow cytometry analysis; lin, lineage marker; rh, recombinant human; rm, recombinant mouse, m, mouse; PI, propidium iodide; SOCS1, suppressor of cytokine signaling 1. Back

Received for publication June 22, 2001. Accepted for publication December 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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