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Stem Cells, Vol. 17, No. 6, 357-365, November 1999
© 1999 AlphaMed Press

Age-Dependent Abnormalities of Hematopoietic Stem Cells in (NZW x BXSB)F1 Mice

Akira Sugiharaa,b, Yasushi Adachia, Muneo Inabaa, Hiroko Hishaa, Kikuya Sugiuraa, Shigeo Miyashimac, Yasuo Amohd, Shigeru Taketanie, Haruki Oyaizua,f, Kazuya Ikebukuroa,g, Masayo Kawamuraf, Hisae Genbah, Takeshi Horiob, Susumu Ikeharaa

a First Department of Pathology;
b Department of Dermatology;
c Department of Orthopedic Surgery;
d Third Department of Internal Medicine;
e Department of Hygiene;
f First Department of Internal Medicine;
g Second Department of Surgery;
h Second Department of Physiology, Kansai Medical Unversity, Moriguchi City, Osaka, Japan

Key Words. W/BF1 mice • BMT • Leukocytosis • Cytokines • Stem cells • Autoimmune diseases

Dr. Susumu Ikehara, First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8606, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The (NZW x BXSB)F1 (W/BF1) mouse is known as an autoimmune-prone strain which develops lupus nephritis, thrombocytopenia due to platelet-specific autoantibodies, leukocytosis, and myocardial infarction. In this experiment, we investigated the age-dependent abnormalities of the hematopoietic stem cells (HSCs) and hematopoiesis in this mouse. White blood cell counts (especially Mac-1- or Gr-1-positive cells) in the peripheral blood of 12-week-old W/BF1 mice increased in comparison with those of four-week-old W/BF1 or normal mice. To investigate whether the abnormal hematopoiesis can be attributed to the HSCs of W/BF1 mice, colony-forming unit in spleen (CFU-S) and colony-forming unit in culture (CFU-C) assays were performed. Day 12 CFU-S counts of 12-week-old W/BF1 mice significantly increased in comparison with those of four-week-old W/BF1 mice or normal mice. In the CFU-C assay, CFU-GEMM and CFU-GM counts in 12-week-old W/BF1 mice increased in comparison with those of four-week-old W/BF1 or control mice. The bone marrow cells (BMCs) from 12-week-old W/BF1 mice showed a high level of G-CSF and a low level of GM-CSF in mRNA expression. To examine the effect of HSCs from 12-week-old W/BF1 mice on the onset of autoimmune diseases and the abnormal hematopoiesis, T- and B-cell-depleted BMCs of four-week-old or 12-week-old W/BF1 mice were transplanted to C3H mice. Recipient C3H mice that had received the BMCs from 12-week-old W/BF1 mice showed an earlier onset of autoimmune diseases and a shorter survival rate than those that had received the BMCs from four-week-old W/BF1 mice. These data suggest that the HSCs from 12-week-old W/BF1 mice showing the symptoms of autoimmune diseases have the capacity to induce autoimmune diseases earlier than the HSCs from four-week-old W/BF1 mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
We have previously demonstrated that allogeneic bone marrow transplantation (BMT) can be used to prevent and treat both systemic and organ-specific autoimmune diseases in (NZB x NZW)F1 (B/WF1), BXSB [1, 2], and MRL/MP-lpr/lpr (MRL/lpr) mice [3]. We have also reported that BMT has a preventive effect on insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice [4], and that the combination of BMT plus pancreas grafts can be used as a treatment [5]. This was the case when we used a non-insulin-dependent (type II) diabetic model mouse, the KK-Ay mouse; BMT completely ameliorated the symptoms, such as urine sugar, hyperinsulinemia, hyperlipidemia, and diabetic nephropathy [6].

W/BF1 mice are also known to be autoimmune-prone and to develop lupus nephritis [7], thrombocytopenia [8-10] leukocytosis [8], hypertension [11], hypergammaglobulinemia [12], thymic atrophy [13] and myocardial infarction [7, 11]. W/BF1 mice also show various autoantibodies, including anti-DNA antibodies (Abs) [12], anti-platelet Abs [8], and anti-cardiolipin Abs [14]. This autoimmunity is more severe in male than female W/BF1 mice, due to the Yaa gene, which is localized on the Y chromosome [15]. We have previously demonstrated that allogenic BMT also has curative effects on autoimmune diseases in W/BF1 mice [8, 9, 16]. These findings suggest that autoimmune diseases are due to disorders in the hematopoietic stem cells (HSCs) themselves or their developmental pathways, followed by autoreactive lymphocyte accumulation. Actually, we have demonstrated that the transplantation of bone marrow cells (BMCs) or an HSC-enriched population of autoimmune-prone W/BF1 into normal C3H mice induces autoimmune diseases such as lupus nephritis, myocardial infarction, and immune thrombocytopenic purpura (ITP) in the C3H recipients [17].

In this report, we investigate the age-dependent abnormalities of the HSCs of W/BF1 mice and clarify the mechanisms underlying leukocytosis and "stem cell disorders" using W/BF1 mice, since W/BF1 mice show autoimmune diseases from the age of two months (early onset), whereas B/WF1 and BXSB mice show autoimmune diseases from the age of six months (late onset).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
(NZW x BXSB)F1 (W/BF1)(H-2z/b) mice were obtained from Kiwa Laboratory Animals (Wakayama, Japan), and C3H/ He (H-2k) mice were obtained from SLC (Shizuoka, Japan).

Antibodies
The following Abs were used for cell surface analyses: fluorescein isothiocyanate (FITC)-labeled anti-CD45 Ab, phycoerythrin (PE)-labeled anti-B220 Ab, PE-labeled anti-Mac-1 Ab, PE-labeled anti-CD3 Ab, and PE-labeled anti-Gr-1 Ab. FITC or PE-labeled Abs were purchased from Pharmingen (San Diego, CA). Anti-GM-CSF Ab and anti-M-CSF Ab were from Santa Cruz Biotechnology (Santa Cruz, CA).

Bone Marrow Transplantation (BMT)
C3H/He mice were exposed to 9.5 Gy from a 60Co source and then reconstituted with iv. injection of 1 x 107 W/BF1 BMCs that had been depleted of T and B cells using monoclonal Ab (mAb) against Thy1.2 (ATCC; Rockville, MD) and anti-B220 mAb (ATCC) plus rabbit complement.

White Blood Cell (WBC) and Platelet Counts
Blood was collected from the orbital vein, and the WBCs and platelets were counted using microscopy after lysing red blood cells using Unopette (Becton Dickinson; Franklin Lakes, NJ). For flow cytometric analyses, blood was collected from the tail vein using heparinized microlube and separated into plasma and blood cells by centrifugation. The blood cells were stained with appropriate FITC- or PE-labeled mAbs and analyzed using a FACScan® (Becton Dickinson; Mountain View, CA).

Colony-Forming Unit-Spleen (CFU-S) Assay
The C3H/He mice were irradiated and reconstituted with i.v. injection of 1 x 105 T- and B-cell-depleted BMCs from W/BF1 mice. Twelve days later, the mice were killed, the CFU-S counted, and the spleen weighed.

Colony-Forming Unit-Culture (CFU-C) Assay
Fourteen-day CFU-C assays were performed using "Metocart" (Stemcell Technologies Inc.; Vancouver; BC, Canada), following the manufacturer's instructions. In brief, 3 x 103 BMCs were mixed with 1.0 ml Metocart and were placed in 24-well culture plates.

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for Detection of mRNA Expression
RNA preparation, cDNA synthesis, and PCR were carried out. Total cellular RNA was prepared using a nucleic acid extractor (TRIZOL Reagent, Life Technologies, Inc.; Grand Island, NY) followed by chloroform extraction and isopropanol precipitation. cDNA was synthesized using reverse transcriptase (M-MLV RTase in RT-PCR high [RT-PCR Kit], TOYOBO; Tokyo, Japan) and Oligo(dT)20-P7 primers ( RT-PCR high). PCR was performed on the cDNA using the following primers for GM-CSF (Maxim Biotech, Inc.; San Francisco, CA), G-CSF [18], M-CSF [19] and G3PDH (RT-PCR high) with thermal cycling amplification using a Takara PCR Thermal Cycler MP (Takara; Otsu, Japan). PCR products were separated on a 1.2% agarose gel (GIBCO BRL; Rockville, MD) and visualized by ethidium bromide (Nakarai; Kyoto, Japan) staining.

ELISA Assay
Serum GM-CSF was measured using an ELISA kit for GM-CSF (R & D Systems; Minneapolis, MN) following the manufacturer's instructions.

Western Blotting
Serum and BMCs were harvested and lysed in lysis buffer. The samples were sonicated and boiled. Adjusted proteins were analyzed by SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. Immunoblotting was performed using polyclonal Ab (anti-GM-CSF or anti-G-CSF Ab).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increases in WBC and Mac-1- or Gr-1-Positive Cell Counts in Peripheral Blood of W/BF1 Mice
At four weeks of age, W/BF1 mice with normal levels of proteinuria showed no abnormalities in WBC or platelet counts, whereas at 12 weeks of age, the mice developed proteinuria due to lupus nephritis, leukocytosis, and thrombocytopenia due to anti-platelet Abs (Fig. 1AGo [9]). Therefore, we performed the following experiments using four-week-old or 12-week-old W/BF1 and normal mice. As shown in Figure 1AGo, the WBC counts were significantly higher in the peripheral blood of 12-week-old W/BF1 mice than in four-week-old W/BF1 and normal mice; normal mice and four-week-old W/BF1 mice showed 2,000-8,000/mm3, while 12-week-old W/BF1 mice with the symptoms of autoimmune diseases showed more than 20,000/mm3. To examine which populations increased in the peripheral white blood cells (PWBCs) of 12-week-old W/BF1 mice, we analyzed the PWBCs from W/BF1 and control normal mice (C3H and BALB/c) using flow cytometry. For flow cytometric analyses, we used anti-CD3, anti-B220, anti-Gr-1, and anti-Mac-1 Abs to detect T cells, B cells, myeloid cells, and monocytes, respectively. As shown in Figure 1BGo, the CD3-positive cell counts were higher in both the four-week-old and 12-week-old W/BF1 mice than in the C3H mice; they were, however, similar to those of BALB/c mice. The B220-positive cell counts were also higher in the 12-week-old W/BF1 mice than in the four-week-old W/BF1 mice or normal mice, possibly due to the increase in autoreactive B cell counts (Fig. 1CGo). As shown in Figures 1D and 1EGo, the absolute numbers of Gr-1- or Mac-1-positive cells were significantly higher in the PWBCs of the 12-week-old W/BF1 mice than in the four-week-old W/BF1 or age-matched C3H or BALB/c mice.



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  		Figure 1. WBC counts and populations in the peripheral blood of male W/BF1 mice. A: WBC counts were measured using Unopett. Peripheral WBCs were stained with FITC-anti-CD45 Ab and PE-anti-CD3 Ab (B), PE-anti-B220 Ab (C), PE-anti-Gr-1 Ab (D) or PE-anti-Mac-1 Ab (E). At first, CD45-positive cells were gated, and the percentages of CD3 (+), B220 (+), Gr-1 (+) or Mac-1 (+) cells in CD45 (+) cells were analyzed, and their absolute numbers (/mm3) were calculated. Each group consisted of three mice.

 
Increases in Day 12 CFU-S Counts in 12-Week-Old W/BF1 Mice
It is well known that day 12-14 CFU-S or CFU-C counts reflect the hematopoietic ability of HSCs and progenitors [20, 21]. To examine the hematopoietic activity of HSCs and progenitors in W/BF1 mice, we first performed CFU-S assays; the recipient mice used were eight weeks old, since mice younger than eight weeks old are radiosensitive. Abnormalities in the hematopoietic functions of four-week-old and 12-week-old W/BF1 mice BMCs were compared in CFU-S assays. As shown in Figure 2Go, irradiated C3H (H-2k) mice that had received the BMCs from 12-week-old W/BF1 (H-2z/b) mice had twofold higher CFU-S counts than those that had received the BMCs from four-week-old W/BF1 mice; these findings are compatible with our previous findings that a major histocompatibility complex restriction exists between HSCs and stromal cells but not between abnormal HSCs and stromal cells [22, 23]. This was the case when syngeneic eight-week-old W/BF1 mice were used as recipients, although the CFU-S counts were much higher because of the syngeneic donor-recipient combination. In contrast, the hematopoietic function of BMCs from four-week-old and 12-week-old C3H mice was almost the same, and there was no sex difference between donor/recipient combinations in CFU-S counts. CFU-S counts of four- or 12-week-old C3H mice into W/BF1 mice (eight weeks old) showed almost the same results as those of four-week-old W/BF1 mice into C3H mice (data not shown). Therefore, the increased hematopoietic activity was due to the BMCs (HSCs or progenitors) of the 12-week-old W/BF1 mice. These findings were also confirmed when the spleen was weighed (data not shown). These results suggest that the BMCs from 12-week-old W/BF1 mice contain more HSCs or progenitors than those from four-week-old W/BF1 or age-matched C3H mice.



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  		Figure 2. Day 12 CFU-S counts in the recipients of BMCs from four-week-old or 12-week-old W/BF1 mice. Recipient mice were exposed to 8.0 Gy from a 60Co source and then reconstituted with i.v. injections of 1 x 105 T- and B-cell-depleted BMCs from four-week-old or 12-week-old W/BF1 or normal mice to avoid graft-versus-host-disease. Twelve days after BMT, CFU-S were counted. CFU-S counts are shown in mean ± SD.

 
Increases in CFU-GEMM and CFU-GM Counts in 12-Week-Old W/BF1 Mice
We next performed day 14 CFU-C assays. W/BF1 mice (12 weeks old) showed higher colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) and CFU-granulocyte/macrophage (CFU-GM) counts than age-matched normal and four-week-old W/BF1 mice (Fig. 3Go). W/BF1 mice (12 weeks old) showed approximately 10 CFU-GEMM counts/10,000 cells, while four-week-old W/BF1 and normal mice showed 4 CFU-GEMM counts/ 10,000 cells. Similar results were obtained in CFU-GM counts. However, the CFU-M counts of 12-week-old W/BF1 mice were similar to those of 4-week-old W/BF1 or age-matched control mice. These data suggest that BMCs from aged W/BF1 mice contain many HSCs and progenitors, especially granulocyte-monocyte progenitors. This reflects the increases in Mac-1-positive or Gr-1-positive cell counts in the peripheral blood of 12-week-old W/BF1 mice.



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  		Figure 3. Increased CFU-C counts in 12-week-old W/BF1 mice. The BMCs from four-week-old or 12-week-old male W/BF1 mice or age-matched control mice were cultured with "Metocart". After 14-day culture, CFU-GEMM, CFU-GM, and CFU-M were counted. Each group consisted of three mice.

 
Early Onset of Autoimmune Diseases and Leukocytosis in C3H Mice Transplanted with BMCs from 12-Week-Old W/BF1 Mice
As we have previously reported [24], BMT from W/BF1 to normal mice induces autoimmune diseases in the recipients. To compare the ability to induce autoimmune diseases and leukocytosis between BMCs from four-week-old W/BF1 mice and BMCs from 12-week-old W/BF1 mice, we carried out BMT and observed the symptoms and survival rates. Lethally irradiated male C3H (H-2k) mice (eight weeks old) were reconstituted with T- and B-cell-depleted BMCs from four-week-old or 12-week-old male W/BF1 (H-2Kz/b) mice. From eight weeks after BMT, WBCs and platelets were counted every four weeks. The WBC counts in the recipients of BMCs from 12-week-old W/BF1 mice increased earlier than those in the recipients of BMCs from four-week-old W/BF1 mice. The WBC counts in the mice that had received BMCs from 12-week-old W/BF1 mice reached the same level as in the untreated W/BF1 mice 12 weeks after BMT (Fig. 4AGo). Figure 4BGo shows the kinetics of platelet counts. The recipients of BMCs from 12-week-old W/BF1 mice showed an earlier decline in platelet counts than the recipients of BMCs from four-week-old W/BF1 mice. These findings clearly show an earlier onset and more marked symptoms of autoimmune diseases in the recipients of BMCs from 12-week-old W/BF1 mice.



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  		Figure 4. Changes in WBC counts (A) and platelet counts (B) after BMT. BMT was performed as described in Materials and Methods. A: WBC counts in C3H mice transplanted with BMCs from male four-week-old or 12-week-old W/BF1 mice. The open squares (-{square}-) represent the recipients of the BMCs from four-week-old W/BF1 mice (n = 6), and the open diamonds (-{diamond}-) represent the recipients of the BMCs from 12-week-old W/BF1 mice (n = 6). The open triangles ({triangleup}) represent 12-week-old W/BF1 mice (n = 4). The open circles ({circ}) represent 4-week-old C3H mice (n = 4). Vertical bars show standard errors. B: Platelet counts in C3H mice transplanted with the BMCs from four-week-old or 12-week-old W/BF1 mice. The open squares (-{square}-) represent the recipients of the BMCs from four-week-old W/BF1 mice (n = 6), and the open diamonds (-{diamond}-) represent the recipient of the BMCs from 12-week-old W/BF1 mice (n = 6). The open triangles ({triangleup}) represent 12-week-old W/BF1 mice (n = 4). The open circles ({circ}) represent 4-week-old C3H mice (n = 4). Vertical bars show standard errors.

 
Shorter Survival Rates in Mice Transplanted with BMCs from 12-Week-Old W/BF1 Mice
The earlier onset of autoimmune diseases in the recipients of BMCs from 12-week-old W/BF1 mice was also confirmed by their survival rates, as shown in Figure 5Go. The recipients of BMCs from four-week-old W/BF1 mice survived longer than those of BMCs from 12-week-old W/BF1 mice. The recipients transplanted with the BMCs from 12-week-old W/BF1 mice showed an earlier onset of the diseases and more marked symptoms than those of mice transplanted with the BMCs from four-week-old W/BF1 mice. For example, 16 weeks after BMT, the mice transplanted with the BMCs from 12-week-old W/BF1 mice showed proteinuria (++ to +++), increased WBC counts (approximately 40,000 cells/µl on average) and decreased platelet counts (approximately 50 x 104/µl, on average; mouse normal platelet counts: >100 x 104/µl), while, in the mice transplanted with the BMCs from four-week-old W/BF1 mice, WBC and platelet counts showed approximately, 23,000/µl and 100 x 104/µl on average, respectively (Figs. 4, 5GoGo). These data suggest that HSCs or progenitor cells from 12-week-old W/BF1 mice accelerate not only the onset of autoimmune disease but also autoimmune symptoms.



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  		Figure 5. Survival rates of C3H mice transplanted with BMCs from male W/BF1 mice at four weeks (•••) (n = 11) or 12 weeks (—) (n = 11) of age. C3H mice (eight weeks old) were transplanted with T- and B-cell-depleted BMCs from four-week-old or 12-week-old W/BF1 mice, and survival rates were examined.

 
Upregulation of G-CSF and Downregulation of GM-CSF in BMCs from 12-Week-Old W/BF1 Mice
It has been reported that many cytokines are associated with hematopoiesis. Especially, G-CSF and GM-CSF are crucial for the production of granulocytes, and M-CSF and GM-CSF are crucial for the level of monocytes [25]. Therefore, we first examined the protein production of GM-CSF and M-CSF in the sera and the lysates of BMCs from four-week-old W/BF1 or 12-week-old W/BF1 and 12-week-old C3H mice by ELISA and Western blot analyses. However, we could not detect these cytokines in the sera or the lysates of BMCs from any mice (data not shown). It is conceivable that serum levels of these cytokines were too low to be detected in the protein levels, because these cytokines should work in a very small area, the so-called microenvironment of the bone marrow. Therefore, we performed RT-PCR to detect the mRNA of GM-CSF, G-CSF and M-CSF in the BMCs from four-week-old W/BF1, 12-week-old W/BF1 and 12-week-old C3H mice. As shown in Figure 6Go, in four-week-old W/BF1 and 12-week-old C3H mice, the mRNA of GM-CSF was detected but not the mRNA of G-CSF. Surprisingly, the BMCs from 12-week-old W/BF1 mice showed very low levels of GM-CSF mRNA and high levels of G-CSF mRNA in comparison with normal or four-week-old W/BF1 mice. These results suggest that high G-CSF mRNA and low GM-CSF mRNA could be associated with the increases in monocyte and granulocyte counts of aged W/BF1 mice.



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  		Figure 6. mRNA of GM-CSF, G-CSF, and M-CSF in BMCs of W/BF1 and C3H mice. mRNA was obtained from the BMCs of male 12-week-old C3H, male four-week-old W/BF1 and male 12-week-old W/BF1 mice, followed by RT-PCR, as described in Materials and Methods. Each group consisted of three mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, to elucidate the age-dependent abnormalities of HSCs of W/BF1 mice, we carried out allogeneic BMT, CFU-S, and CFU-C assays and examined the production of cytokines. We have found that W/BF1 mice clearly show hemopoietic abnormalities at the age of 12 weeks (but not four weeks), and that this is due to the abnormalities of HSCs. In the peripheral blood of 12-week-old W/BF1 mice, Gr-1-positive cells, Mac-1-positive cells, and B220-positive cells significantly increased (Fig. 1Go). In the day 12 CFU-S assay, the mice that had been transplanted with the BMCs of 12-week-old W/BF1 mice showed many colonies in the spleens (Fig. 2Go). In the day 14 CFU-C assay, CFU-GEMM and CFU-GM counts increased in BMCs from12-week-old W/BF1 mice (Fig. 3Go). The mice that had been transplanted with the BMCs of 12-week-old W/BF1 mice showed not only an earlier onset of autoimmune diseases but also a shorter survival rate than the mice transplanted with four-week-old W/BF1 BMCs (Figs. 4, 5GoGo). In 12-week-old W/BF1 BMCs, GM-CSF mRNA was downregulated, while G-CSF mRNA was upregulated (Fig. 6Go).

We have previously found that aged W/BF1 mice show not only lupus nephritis but also ITP and leukocytosis [8, 9]. In the hemogram, granulocyte and monocyte counts increased in the peripheral blood of 12-week-old W/BF1 mice, and leukocytosis was found not to be due to leukemia, because abnormal blasts did not increase in the bone marrow (data not shown). WBC counts in another lupus mouse, MRL-Mp-lpr/lpr (MRL/lpr), which is well known for its defective Fas, were also examined. However, the WBC counts of MRL/lpr did not increase even when these mice showed severe symptoms of autoimmune diseases (data not shown). These results suggest that autoimmune diseases are not directly associated with leukocytosis in aged W/BF1 mice. In humans, patients with SLE generally show leukopenia. This may be due to the presence of autoantibodies against leukocytes and also granulocyte progenitors [26]. Absolute numbers of Gr-1-positive cells, Mac-1-positive cells, and B220-positive cells (particularly Mac-1-positive cells) increased in the peripheral blood of 12-week-old W/BF1 mice. In the day 14 CFU-C assay, the CFU-GM and CFU-GEMM counts had increased in the 12-week-old W/BF1 mice. These results suggest that HSCs from 12-week-old W/BF1 mice have the potent ability to enhance hemopoiesis, especially Gr-1-positive or Mac-1-positive cell counts. Vieten et al. have described that male BXSB mice, a parental strain of the W/BF1 mice, show monocytosis, and that the day 7 CFU-C assay using BMCs from aged BXSB mice show high CFU-M and CFU-GM counts, whereas CFU-GEMM counts remain unchanged [27]. These differences between W/BF1 and BXSB mice are possibly due to the additional dormant abnormalities in NZW mice.

We next examined the possibility that cytokines are involved in the leukocytosis of W/BF1 mice. We thought that GM-CSF and/or both G-CSF and M-CSF levels should be high because of the increase in Gr-1-positive and Mac-1-positive cell numbers. Contrary to our expectation, the G-CSF mRNA expression increased, whereas the GM-CSF mRNA expression decreased. The downregulation of GM-CSF mRNA could be explained by negative feedback regulation. The increased mRNA expression of G-CSF may be due to an intrinsic abnormality of W/BF1 mice, which is not regulated by negative feedback mechanisms. The increased cell numbers of Gr-1- and Mac-1-positive cells as a result of high G-CSF production may lead to the low production of GM-CSF due to a negative feedback mechanism. It has been described that G-CSF increases not only granulocytes but also monocytes; the increase is much more marked in granulocytes than in monocytes [28, 29]. However, there was only one case report indicating that G-CSF induced leukemoid monocytosis in a patient with acute myeloid leukemia [30]. These results suggest that G-CSF plus other factors or intrinsic abnormalities of bone marrow HSCs induce this leukocytosis. In the day 12 CFU-S assay, the BMCs from 12-week-old W/BF1 mice showed significantly increased numbers of colonies in the spleen in comparison with those from four-week-old W/BF1 or normal mice. These findings are compatible with those that abnormal HSCs from autoimmune-prone mice are more resilient than HSCs from normal mice. There is no MHC restriction between abnormal HSCs and stromal cells either in vivo or in vitro [22-24]; we have found that abnormal HSCs can proliferate even in an allogeneic microenvironment [24], and that abnormal HSCs show a much higher proliferative response than normal HSCs in vitro [24].

To confirm the age-dependent abnormalities of HSCs in the bone marrow of W/BF1 mice, we transplanted the BMCs from four-week-old or 12-week-old W/BF1 mice into C3H mice. The recipients (C3H) of BMCs from 12-week-old W/BF1 mice showed an earlier onset of autoimmune diseases than recipients of the BMCs from four-week-old W/BF1 mice, indicating age-dependent abnormalities of HSCs in W/BF1 mice. This was confirmed in other autoimmune-prone mice such as MRL/lpr, nonobese diabetic, and B/WF1 mice; for example, the transplantation of T-cell-depleted BMCs from autoimmune-prone mice with autoimmune diseases to normal mice induces autoimmune diseases in the recipients sooner than when the BMCs from autoimmune-prone mice before the onset of autoimmune diseases were transplanted (data not shown). The development of HSCs into T-cell, B-cell and myeloid lineages in the recipients occurred within eight weeks of BMT, and almost all peripheral cells expressed donor-type H-2 in the recipients of both BMCs from four-week-old and 12-week-old donors (data not shown). Since the HSCs in autoimmune-prone mice after the onset of autoimmune diseases are activated and form significantly higher CFU-S than in autoimmune-prone mice before the onset of autoimmune diseases, abnormal immunocompetent cells such as abnormal T cells, B cells, macrophages, and dendritic cells should differentiate from abnormal HSCs, which would result in the development of autoimmune diseases. However, the abnormalities such as thrombocytopenia, proteinuria, and lupus nephritis appeared later. We therefore suspect that the accumulation of auto-reactive T cells and/or B cells, which are derived from abnormal HSCs, in the peripheral organs, should be attributed to the onset of autoimmune diseases.

It remains to be clarified why W/BF1 mice show abnormalities in the HSCs from 12 weeks of age, and why B/WF1 mice do so from six months of age, even though the abnormalities should exist at the gene level (before birth). It is conceivable that endogenous or exogenous retroviruses, or exogenous bacterial infection are involved in the induction of these abnormalities. We are now in the process of examining which agents are involved, and how.


   Acknowledgments
 
We thank Ms. Matsui-Hashimoto, Ms. Shinno, Ms. Murakami-Shinkawa, Ms. Tokuyama and Ms. Miura for their expert technical assistance, and also Ms. Ando for the preparation of this manuscript.

A.S. and Y.A. contributed equally to this work.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ikehara S, Yasumizu R, Inaba M et al. Long-term observations of autoimmune-prone mice treated for autoimmune disease by allogeneic bone marrow transplantation. Proc Natl Acad Sci USA 1989;86:3306-3310.[Abstract/Free Full Text]

  2. Ikehara S, Inaba M, Ishida S et al. Rationale for transplantation of both allogeneic bone marrow and stromal cells in the treatment of autoimmune diseases. In: Champlin RE, Gale RP, eds. New Strategies in Bone Marrow Transplantation. UCLA Symposia on Molecular and Cellular Biology, New Series. New York, Chichester, Brisbane, Toronto, Singapore: Wiley-Liss, 1991;251-257.

  3. Ishida T, Inaba M, Hisha H et al. Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. J Immunol 1994;152:3119-3127.[Abstract]

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accepted for publication October 22, 1999.



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Y. Adachi, S. Taketani, J. Toki, K. Ikebukuro, K. Sugiura, H. Oyaizu, R. Yasumizu, M. Tomita, H. Kaneda, Y. Amoh, et al.
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