Lethal anemia caused by interferon-b produced in mouse ... - Nature

© 2005 Nature Publishing Group http://www.nature.com/natureimmunology

ARTICLES

Lethal anemia caused by interferon-b produced in mouse embryos carrying undigested DNA Hideyuki Yoshida1,4, Yasutaka Okabe1,4, Kohki Kawane1,2, Hidehiro Fukuyama2,5 & Shigekazu Nagata1,2,3 The livers of DNase II–deficient mouse embryos contain many macrophages carrying undigested DNA, and the embryos die in utero. Here we report that erythroid precursor cells underwent apoptosis in the livers of DNase II–deficient embryos and that in the liver, interferon-b mRNA was expressed by the resident macrophages. When the DNase II–deficient mice were crossed with mice deficient in type I interferon receptor, the resultant ‘double-mutant’ mice were born healthy. The double-mutant embryos expressed interferon-b mRNA, but the expression of a subset of the interferon-responsive genes dysregulated in DNase II–deficient embryos was restored to normal. These results indicate that the inability to degrade DNA derived from erythroid precursors results in interferon-b production that induces expression of a specific set of interferon-responsive genes associated with embryonic lethality in DNase II–deficient mice.

Two types of erythropoiesis, primitive and definitive, take place in the fetus1,2. Primitive erythropoiesis occurs from embryonic day 7.5 (E7.5) in the yolk sac; this switches at E9.5 to definitive erythropoiesis, which occurs in the liver. Erythrocytes produced by primitive erythropoiesis have nuclei; those produced by definitive erythropoiesis lack them because the nuclei are expelled from erythroid precursor cells late in their differentiation. Red blood cells in the adult also do not have nuclei and are produced by definitive erythropoiesis in the bone marrow. The definitive erythropoiesis in fetal liver and bone marrow seems to take place in anatomic units called ‘blood islands’3,4, which consist of a macrophage surrounded by developing erythroid cells of different stages. The macrophage in each blood island is thought to support erythropoiesis by producing cytokines and/or providing a scaffold for erythroid precursor cells4. DNase II, encoded by Dnase2a, is an endonuclease located in lysosomes5,6. It is expressed in various tissues and cells7, including macrophages8,9. DNase II–deficient embryos suffer from severe anemia and die in utero9,10. The macrophages in the blood islands in the livers of DNase II–deficient embryos contain a large amount of undigested DNA, suggesting that the nuclei expelled from erythroid precursor cells are engulfed by the macrophage in the center of each blood island and that DNase II in the macrophages degrades the DNA. Here we report that many erythroid precursor cells in Dnase2a–/– fetal liver underwent apoptotic cell death. Analyses of gene expression with a gene array showed that interferon-inducible genes were strongly activated in the livers of Dnase2a–/– embryos. Macrophages positive for the macrophage-specific marker F4/80 constitutively expressed interferon-b (IFN-b) mRNA in Dnase2a–/– fetal liver. When

Dnase2a–/– mice were crossed with mice deficient in interferon type I receptor (IFN-IR), which mediates the IFN-b signal, the embryos deficient in both DNase II and IFN-IR (DNase II–IFN-IR doubledeficient) were born at the normal mendelian ratio and seemed healthy. The DNase II–IFN-IR double-deficient embryos were not anemic, despite the presence of undigested DNA in the macrophages in the blood islands. Gene array analysis showed that the expression of a subset of interferon-inducible genes that were dysregulated in Dnase2a–/– embryos were restored to normal in DNase II–IFN-IR double-deficient embryos. These results suggest that if the DNA of nuclei expelled from erythroid precursor cells is not properly digested, IFN-b produced in the fetal liver activates a set of interferon-inducible genes and kills the embryos. RESULTS Apoptosis in Dnase2a–/– fetal liver DNase II–deficient mouse embryos have very few mature erythrocytes and suffer from severe anemia9. There are two possible explanations for the anemia in Dnase2a–/– fetal liver. One is that the Dnase2a–/– fetal liver has lost the ability to synthesize growth factors that support erythropoiesis. The other is that cytokines that have cytotoxic activity are produced in the liver. To determine the cause of the anemia, we stained the liver sections of wild-type and DNase II–deficient embryos by TUNEL (terminal deoxinucleotidyltransferase-mediated dUTPbiotin nick end-labeling) to detect apoptotic dying cells. The DNase II–deficient but not wild-type fetal livers contained many large TUNEL-positive foci (Fig. 1a), which were probably F4/80-positive macrophages carrying DNA expelled from erythroid precursor cells

1Department of Genetics, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. 2Laboratory of Genetics, Integrated Biology Laboratories, Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan. 3Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, Osaka 565-0871, Japan. 4These authors contributed equally to this work. 5Present address: Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New York, New York 10021, USA. Correspondence should be addressed to S.N. ([email protected]).

Published online 28 November 2004; doi:10.1038/ni1146

NATURE IMMUNOLOGY

VOLUME 6

NUMBER 1

JANUARY 2005

49

ARTICLES

a

WT

b

DNase II KO

WT

DNase II KO

0.5%


102 103 TUNEL

104

4.6%

105

102

103

104

105

Figure 1 Apoptotic cell death of fetal liver cells in Dnase2a–/– embryos. (a) Histochemical analysis of fetal liver. Liver sections from E14.5 Dnase2a+/+ (left; WT) and Dnase2a / (right; DNase II KO) embryos were stained using TUNEL. Scale bar, 100 mm. (b) Flow cytometry of fetal liver cells for apoptotic cells. Liver cells from E14.5 Dnase2a+/+ (left) and Dnase2a–/– (right) embryos were stained with Ter-119 monoclonal antibody as well as by the TUNEL procedure. Histograms show the TUNEL staining profile of the Ter-119-positive population. Numbers in histograms indicate the percentage of TUNELpositive cells. The experiments were done with two independent fetal livers and average numbers are presented.

and/or apoptotic cells9. In addition, Dnase2a–/– livers contained relatively small TUNEL-positive cells that most likely represented erythroid precursor cells undergoing apoptosis. We stained cells from fetal liver with Ter-119 monoclonal antibody, which is specific for erythroid cells11, and analyzed the cells by flow cytometry. About 80–90% of the fetal liver cells in wild-type and DNase II–deficient embryos were Ter-119 positive (data not shown). Among the Ter-119positive cells, about 4.6% stained by TUNEL in DNase II–deficient fetal liver (Fig. 1b), whereas wild-type liver contained few TUNEL-positive cells. This result suggests that erythroid cells undergo apoptosis in Dnase2a–/– fetal liver. Interferon-regulated genes expressed in Dnase2a–/– liver We examined the gene expression profile of wild-type and DNase II– deficient fetal liver with oligonucleotide arrays consisting of more than 20,000 mouse genes and determined the changes in gene expressions involved in erythropoiesis (Table 1). The expression of a- and b-globin mRNA, the main mRNA in erythrocytes, was slightly reduced (to 80–90% of normal) in Dnase2a–/– fetal livers. The expression of cytokines (erythropoietin, stem cell factor and interleukin 7) and their receptors and of GATA-1 transcription factor was comparable in wild-type and DNase II–deficient fetal livers or was upregulated or downregulated by a maximum of 50% in Dnase2a–/– fetal liver. These results suggest that the capacity to support erythropoiesis was not compromised substantially in Dnase2a–/– fetal liver.

In contrast, gene array analysis indicated that the expression of approximately 60 genes was increased 20- to 200-fold in Dnase2a–/– fetal liver compared with wild-type liver (Table 2) and the expression of six genes was suppressed to less than 10% of wild-type by deficiency in the gene encoding DNase II (Table 3). RNA hybridization confirmed that the genes shown to be upregulated by gene array analysis were strongly expressed in Dnase2a–/– fetal liver but not in Dnase2a+/+ or Dnase2a+/– fetal liver (Fig. 2a). Interferon genes expressed in Dnase2a–/– liver The 60 genes upregulated in DNase II–deficient fetal liver (Table 2) are known to be induced by interferon (IFN-a, IFN-b or IFN-g). This suggests that interferons were produced in the Dnase2a–/– fetal liver. Because the signals for IFN-a, IFN-b and IFN-g mRNA were too low to be detected by the gene array analysis, we did RNA hybridization using a sensitive digoxigenin (DIG)–labeled probe12 and poly(A) RNA from fetal liver (Fig. 2b). Neither wild-type nor Dnase2a+/– fetal liver expressed IFN-a, IFN-b or IFN-g mRNA. However, we detected strong bands for IFN-b and IFN-g mRNA in the DNase II–deficient fetal liver. IFN-a is encoded by a family of genes with 80–90% homology at the nucleotide level13,14. No signal was detected for IFNa-1 even in low-stringency hybridization conditions (data not shown), suggesting that most members of the IFN-a gene family were not expressed in Dnase2a–/– fetal liver.

Table 1 Expression of genes involved in erythropoiesis Dnase2a–/–

Wild-type Product (symbol) Hemoglobin, a, adult chain 1 (Hba-a1)

Signal

Signal

Dnase2a–/–Ifnar1–/– ‘Fold’

Signal

‘Fold’

631.70

481.20

0.76

546.80

0.87

1031.80 203.10

894.30 302.50

0.87 1.49

993.50 237.10

0.96 1.17

Kit ligand (Kitl) Kit oncogene (Kit)

16.20 9.10

7.66 13.00

0.47 1.43

10.81 8.85

1.41 0.97

Erythropoietin (Epo) Erythropoietin receptor (Epor)

0.34 10.90

0.87 4.70

2.56 0.43

0.53 6.80

1.56 0.62

0.25 0.46

0.44 0.61

1.76 1.32

0.36 0.40

1.44 0.87

16.68

7.63

0.46

12.40

0.74

Hemoglobin, b, adult major chain (Hbb-b1) Liver transferrin (Trf)

Interleukin 7 (Il7) Interleukin 7 receptor (Il7r) GATA binding protein 1 (Gata1)

The Codelink Bioarray System was used for gene array analysis. Signals were obtained by division of the raw hybridization data with the median value. The median values were 59.89, 69.74 and 64.89 for wild-type, Dnase2a–/– and Dnase2a–/–Ifnar1–/– fetal liver RNA, respectively. The ‘fold increase’ in the gene expression was determined by division of the signal obtained with Dnase2a / or Dnase2a–/–Ifnar1–/– fetal liver RNA by the signal obtained with wild-type RNA. Representative genes involved in erythropoiesis are listed.

50

VOLUME 6

NUMBER 1

JANUARY 2005

NATURE IMMUNOLOGY

ARTICLES Table 2 Genes upregulated in the Dnase2a–/– fetal liver Dnase2a–/–


Wild-type

Dnase2a–/–Ifnar1–/–

Product (symbol)

Signal

Signal

‘Fold’

Signal

‘Fold’

Heat-shock protein 68 (Hsp68) VHSV-induced gene 1 (Vig1)

0.51 0.18

93.28 24.79

182.3 137.7

2.28 0.32

4.5 1.8

Apolipoprotein L–related (2310016F22Rik) T cell–specific GTPase (Tgtp)

0.16 0.73

21.08 96.00

131.8 131.5

0.29 24.86

1.8 34.1

Interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) Vomeronasal 1 receptor, B9 (V1rb9)

0.37 0.12

45.70 13.31

123.5 110.9

1.84 0.21

5.0 1.7

2¢-5¢oligoadenylate synthetase like-1 (Oasl1)

0.41

40.01

97.6

2.38

5.8

2¢-5¢oligoadenylate synthetase like-2 (Oasl2) Nuclear antigen SP100 (Sp100)

0.75 0.25

58.36 18.78

77.8 75.1

1.32 0.32

1.8 1.3

Z-DNA-binding protein 1 (Zbp1) Glycoprotein nmb (Gpnmb)

0.19 0.03

12.67 2.24

66.7 65.8

0.57 1.67

3.0 49.1

Interferon-inducible GTPase (Iigp) Chemokine (C-C motif) ligand 5 (Ccl5)

0.16 0.21

9.62 11.06

60.1 52.7

1.58 7.38

9.9 35.1

Chemokine (C-X-C motif) ligand 1 (Cxcl1) Interferon-regulatory factor 7 (Irf7)

0.09 3.75

4.89 192.60

52.6 51.4

3.07 2.68

33.0 0.7

Membrane-spanning 4 domains subfamily A (Ms4a4c) Myxovirus-resistant 1 (Mx1)

0.73 0.09

35.68 4.31

48.9 48.9

0.89 0.10

1.2 1.1

28k IFN-a-responsive protein (5830458K16Rik) Lymphocyte antigen 6 complex, locus A (Ly6a)

1.05 1.52

43.10 54.47

41.0 35.8

0.74 1.08

0.7 0.7

Interferon-induced protein with tetratricopeptide repeats 3 (Ifit3) Transporter 1, ATP-binding cassette (Tap1)

0.57 0.14

20.28 4.80

35.6 34.3

0.41 1.12

0.7 8.0

Chemokine (C-X-C motif) ligand 2 (Cxcl2) Chemokine (C-C motif) ligand 7 (Ccl7)

0.10 0.12

3.00 3.53

30.0 29.4

0.95 2.70

9.5 22.5

Ubiquitin-specific protease 18 (Usp18) Guanylate-binding nucleotide protein 2 (Gbp2)

1.98 0.19

55.32 5.25

27.9 27.6

2.09 1.99

1.1 10.5

Cholesterol 25-hydroxylase (Ch25h) Guanylate nucleotide-binding protein 4 (Gbp4)

0.15 0.43

3.97 11.33

26.6 26.4

0.18 1.27

1.2 3.0

Fibrinogen-like protein 1 (Fgl1) Chemokine (C-C motif) ligand 10 (Cxcl10)

0.11 2.80

2.93 70.16

26.4 25.1

0.84 33.48

7.6 12.0

Hepcidin antimicrobial peptide (Hamp)

5.43

111.65

21.7

13.74

2.5

Signals and ‘fold increases’ in gene expression were determined as described in Table 1. Genes that were upregulated more than 20-fold in the Dnase2a–/– fetal liver are listed in descending order. Genes whose expression in Dnase2a–/–Ifnar1–/– fetal liver returned to less than twofold of wild-type are underlined.

The fetal livers of Dnase2a–/– embryos contain many F4/80-positive macrophages carrying undigested DNA9. To identify which cells expressed interferon in fetal livers, we made single-cell suspensions from livers of E14.5 wild-type or DNase II–deficient embryos and sorted the cells by magnetic-activated cell sorting (MACS) using antibody to F4/80 (anti-F4/80). We collected both the cells that were trapped on the column (F4/80 positive) and those that passed through the column (F4/80 negative). F4/80-positive macrophages were

enriched by approximately sixfold in the positive fraction, whereas macrophages were completely removed from the negative fraction (Fig. 3a). Real-time PCR analysis indicated that IFN-b mRNA was expressed exclusively in the F4/80-positive fraction from Dnase2a–/– but not wild-type fetal liver. We detected no IFN-b mRNA in the F4/80-negative fractions. In contrast, IFN-g mRNA, which was strongly upregulated in Dnase2a–/– fetal liver cells, was expressed by F4/80-positive macrophages and by F4/80-negative cells. In situ

Table 3 Genes downregulated in the Dnase2a–/– fetal liver Dnase2a –/–

Wild-type

Dnase2a–/–Ifnar1–/–

Product (symbol)

Signal

Signal

Percent

Signal

Percent

Riken cDNA (4930413022Rik)

20.01

0.37

1.84

0.42

2.10

Carboxylesterase 3 (Ces3) Carbonic anhydrase 3 (Car3)

1.22 16.29

0.05 0.87

3.93 5.34

0.27 2.52

21.96 15.47

Cholinergic receptor, nicotinic, a 1 (Chrna1) Serine proteinase inhibitor (Serpina1a)

1.11 55.98

0.07 5.11

6.72 9.17

0.13 32.98

11.90 58.91

Macrophage receptor (Marco)

41.11

3.98

9.68

13.45

32.71

Signals and gene expression (percent of wild-type expression) were determined as described in Table 1. Genes that were downregulated more than tenfold in Dnase2a–/– fetal liver are listed in descending order.

NATURE IMMUNOLOGY

VOLUME 6

NUMBER 1

JANUARY 2005

51

ARTICLES


a

b

Figure 2 Induction of interferon genes in the fetal liver of Dnase2a –/– mice. (a) Induction of interferon-inducible genes. Total RNA (9 mg) from E14.5 Dnase2a+/+ (WT), Dnase2a+/– (HET) and Dnase2a–/– (KO) livers was analyzed by RNA hybridization. Probes (cDNA) were isolated by RT-PCR from Dnase2a –/– fetal liver and were labeled with 32P: Tgtp, T cell–specific GTPase38; Ifit3, IFN-induced protein with tetratricopeptide repeats 3 (ref. 39); Oas1g, 2¢-5¢ oligoadenylate synthetase 1g40; Irf7, interferon-regulatory factor 7 (ref. 41); Cxcl10, chemokine ligand 10 (ref. 42); and G1p2, IFN-a-inducible protein43. Bottom right, agarose gel stained with ethidium bromide for ribosomal RNA (rRNA) to confirm equivalent amounts of RNA were analyzed. (b) Induction of IFN genes. Poly(A) RNA (1 mg) from the livers of E14.5 Dnase2a+/+, Dnase2a+/– and Dnase2a / embryos was analyzed by RNA hybridization with mouse cRNA for IFN-b (Ifnb1), IFN-g (Ifng) and IFN-a1 (Ifna). To confirm the same amount of RNA was loaded in each lane, filters were hybridized with a b-actin (Actb) or glyceraldehyde phosphodehydrogenase (Gapd) cDNA probe. As a positive control for IFN-a mRNA, dendritic cells were prepared by culture of mouse bone marrow cells for 7 d in the presence of mouse granulocyte-monocyte colony-stimulating factor, as described44,45 and were treated for 4 h at 37 1C with 10 mg/ml of poly(I:C) and 10 mg/ml of lipofectamine. Poly(A) RNA (0.2 mg) from the poly(I:C)-treated bone marrow–derived dendritic cells (poly(I:C) DC) was separated by electrophoresis. End hybridized with Ifna CDNA. The experiments were done at least twice.

hybridization with the antisense but not the sense probe for IFN-b produced specific signals on tissue sections from Dnase2a–/– fetal liver (Fig. 3b). In contrast, we detected no specific signal on tissue sections from wild-type fetal liver (data not shown). The IFN-b mRNA expression profile of Dnase2a–/– fetal liver was consistent with the staining profile for F4/80-positive macrophages, as described before9. These results indicated that IFN-b mRNA was upregulated in macrophages carrying undigested DNA. Interferon-dependent death of Dnase2a–/– embryos Type I interferons (IFN-a and IFN-b) have strong cytotoxic effects in newborn mice15,16. The lethal effect of IFN-g has also been noted in mice deficient in suppressor of cytokine signaling 1 (refs. 17,18). To determine whether IFN-b or IFN-g induced erythroid cell death in DNase II–deficient fetal liver, we cultured fetal liver cells in the presence of IFN-b or IFN-g. IFN-b inhibited the growth of erythroid cells in a dose-dependent way, whereas IFN-g had

a

IFN-β

F4/80

HET

KO

HET

b

IFN-γ

KO

HET

little effect (Fig. 4a). To confirm that IFN-b was responsible for the lethality of DNase II deficiency, we crossed Dnase2a+/– mice with mice deficient in Ifnar1, which encodes the common receptor for IFN-a and IFN-b19. Because Ifnar1 / mice develop normally20, we generated and intercrossed Dnase2a+/–Ifnar1–/– mice. Genotype analysis of the littermates born from these intercrosses indicated that IFN-IR deficiency ‘rescued’ the lethality of DNase II deficiency (Fig. 4b). That is, Dnase2a–/–Ifnar1–/– mice were born at the normal mendelian ratio and were apparently healthy, at least until 8 weeks of age. Dnase2a–/– embryos suffered from severe anemia (hematocrit, 10.8 7 6.0) and had a substantial number of nucleated cells in the peripheral blood (Fig. 4c). In contrast, the hematocrit of Dnase2a–/–Ifnar1–/– embryos was similar to that of wild-type embryos (hematocrit, 44.4 7 1.7) and no nucleated erythroid cells were present in the periphery. These results indicated that signaling through the interferon type I receptor was responsible for the anemia of Dnase2a–/– embryos.

KO

Figure 3 Identification of interferon-producing cells in Dnase2a –/– fetal liver. (a) Cells from Dnase2a+/– or Dnase2a –/– E14.5 fetal livers were sorted by MACS with anti-F4/80. Cells that attached to (filled bars) or passed through (gray bars) the column were collected and RNA was prepared from these. RNA was also prepared from an aliquot of the cells before sorting (open bars). F4/80 antigen46, IFN-b33 and IFN-g34 mRNA was quantified by realtime PCR and results are expressed as copy number/ng total RNA. Each experiment was done three times, and average numbers are plotted. (b) In situ hybridization with the probe for IFN-b. Sections from E14.5 Dnase2a –/– fetal liver were hybridized to DIG-labeled antisense or sense RNA. Bottom right, sections were also stained with methyl green and pyronine yellow, which label DNA and RNA, respectively. Scale bars, 100 mm. Experiments were done three times.

52

VOLUME 6

NUMBER 1

JANUARY 2005

NATURE IMMUNOLOGY

ARTICLES


a

c

Figure 4 ‘Rescue’ of the embryonic lethality of DNase II deficiency by deficiency in IFN-b signaling. (a) Cytotoxic effect of IFN-b on fetal liver cells. Cells (1.0 106 cells/ml) from the wild-type C57BL/6 fetal liver at E14.5 were incubated at 37 1C for 48 h in the presence of various amounts (horizontal axes) of mouse IFN-b (specific activity, 1.2 107 U/mg protein) or IFN-g (specific activity, 4 106 U/mg protein). The assay was done three times and average numbers of viable cells are plotted with s.d. (bars). (b) Normal development of Dnase2a –/– Ifnar1–/– embryos. Dnase2a+/–Ifnar1+/+ or Dnase2a+/–Ifnar1–/– mice were intercrossed and the genotypes of the progeny were determined by PCR. Below each lane, number of mice born with that genotype. Right margin, molecular sizes. (c) Lack of anemia in Dnase2a –/–Ifnar1–/– embryos. Peripheral blood of E17.5 embryos was smeared and stained with Wright-Giemsa. The hematocrit (Hct) was determined for at least nine blood samples for each group and average values are presented with s.d. Mice that were wild-type or heterozygous for Dnase2a or Ifnar1 gave indistinguishable results and these data were therefore combined. IFN-IR KO, Ifnar1–/–; DNase II KO, Dnase2a –/–; DKO, Dnase2a –/–Ifnar1 –/–.

b

Interferon-inducible genes in Dnase2a–/–Ifnar1–/– liver The fetal livers of Dnase2a–/–Ifnar1–/– mice contained macrophages with undigested DNA similar to that of fetal livers of Dnase2a–/– mice (Fig. 5a). IFN-b and IFN-g mRNA in Dnase2a–/–Ifnar1–/– fetal liver, as quantified by real-time PCR, was comparable to that of Dnase2a–/– fetal liver (Fig. 5b). Some of interferon-inducible genes upregulated in

a

Dnase2a–/– fetal liver are also induced by IFN-g21. To identify the genes that showed reduced expression due to a deficiency in type I interferon signaling and that were therefore involved in the lethal effect mediated by IFN-b production, we did gene array analysis using RNA from Dnase2a–/–Ifnar1–/– fetal liver. The expression of 13 interferon-induced genes that were increased more than 20-fold in DNase II–deficient

IFN-β mRNA (×10–4)

b WT DNase II KO IFN-IR KO DKO 0

2

4

6

80

120

IFN-γ mRNA (×10–4) WT DNase II KO IFN-IR KO DKO 0

40

Figure 5 Expression of interferon genes in Dnase2a –/–Ifnar1–/– fetal liver. (a) Accumulation of DNA in macrophages of Dnase2a –/–Ifnar1 –/– fetal liver. Livers from E14.5 wild-type, Ifnar1 –/–, Dnase2a–/– and Dnase2a –/–Ifnar1 –/– embryos were stained with hematoxylin and eosin. Scale bar, 100 mm. (b) Interferon mRNA in the fetal liver. RNA was prepared from the livers of wild-type, Dnase2a –/–, Ifnar1–/– and Dnase2a –/–Ifnar1–/– embryos. IFN-b and IFN-g mRNA was quantified by real-time PCR and results are expressed as the value relative to b-actin mRNA.

NATURE IMMUNOLOGY

VOLUME 6

NUMBER 1

JANUARY 2005

53

ARTICLES


liver returned to normal (or were at most twofold higher than wildtype) in Dnase2a–/–Ifnar1–/– fetal liver (Table 2). These genes included V1rb9, Oasl2, Sp100, Mx1 and Irf7. DISCUSSION DNase II is an endonuclease present in the lysosomes of macrophages5,6 that cleaves DNA after macrophages engulf apoptotic cells or the nuclei are expelled from erythroid precursor cells9,22. DNase II– deficient embryos accumulate many macrophages containing undigested DNA and die in utero late in embryogenesis9,10. Because the DNase II–deficient embryos suffer from severe anemia, we hypothesized that the anemia causes the animals’ death9. However, it has been suggested that the large DNA-containing bodies cause malformation of the diaphragm, leading to the death of the animals by asphyxiation at birth10. Here we have shown that the gene encoding IFN-b (Ifnb1) was activated in Dnase2a–/– fetal liver and the lack of signals from the interferon type I receptor ‘rescued’ the anemia and the lethality. The Dnase2a–/–Ifnar1–/– embryos still had many macrophages carrying undigested DNA, indicating that the undigested DNA may not be itself a direct cause of the animals’ death. Dnase2a–/– embryos produced not only IFN-b but also IFN-g. But, in contrast to IFN-b, IFN-g had little cytotoxic effect on erythroid cells. Preliminary analysis has shown that deficiency in IFN-g receptor has no apparent effect on the embryonic lethality of DNase II deficiency (data not shown), suggesting that IFN-b is mainly responsible for inhibiting erythropoiesis and killing the embryos. RNA hybridization analysis indicated that Ifnb1 was activated in Dnase2a–/– fetal liver, but expression was low and we did not detect interferon activity in the serum of Dnase2a–/– embryos (data not shown). In contrast, in situ hybridization detected IFN-b mRNA in the macrophages carrying undigested DNA in the blood islands of Dnase2a–/– fetal liver. We therefore believe that IFN-b produced in the fetal liver is responsible for inhibiting erythropoiesis that occurs in association with macrophages at the blood islands3,23. Many cytokines have more potent activity in a membrane-associated form than in a soluble form24,25. It is likely that IFN-b expression by macrophages in the blood islands, even in low concentrations, has a deleterious effect on erythropoiesis. Type I interferon has cytotoxic effect in some cell types. Understanding how it induces this cytotoxicity has remained elusive. IFN-b and IFN-g regulate a set of genes called interferon-stimulated genes19. Some interferon-stimulated genes respond to either type I interferon or IFN-g or both21. Both IFN-b and IFN-g were produced in Dnase2a–/– as well as in Dnase2a–/–Ifnar1–/– fetal liver, but IFN-b signals caused anemia only in Dnase2a–/– fetal liver. Thus, certain genes that respond to IFN-b in vivo and whose expression returned to normal in Dnase2a–/–Ifnar1–/– fetal liver are probably responsible for inhibiting erythropoiesis. The genes with these characteristics include Oasl2, Sp100 and Usp18. Further studies of the genes regulated by IFN-b in Dnase2a–/– embryos will be useful for elucidation of the cytotoxic activity of IFN-b and will aid in the more efficient use of interferon in treating human patients26,27. Ifnb1 is activated in Dnase2a–/– fetal thymus, where many thymocytes undergo programmed cell death22. Here we also found constitutive Ifnb1 expression in Dnase2a–/– fetal liver. In both the fetal thymus and liver, we noted undigested DNA in macrophages, and the macrophages expressed IFN-b mRNA. These results suggest that DNA directly stimulated the macrophages to express Ifnb1. Activation of Ifnb1 is one of the markers for the activation of innate immunity28. It is activated by bacterial DNA, double-stranded RNA and endotoxins, mediated by Toll-like receptors 9, 3 and 4, respectively29. Mammalian

54

genomic DNA that does not contain nonmethylated CpG nucleotide does not activate this innate immune system30. However, our results indicate that mammalian DNA can activate innate immunity in certain conditions. When we crossed our Dnase2a–/– mice to mice deficient in Toll-like receptor 9, we found that Ifnb1 was still activated in the fetal liver, suggesting that Toll-like receptor 9 may not be essential in the activation of the innate immunity by endogenous DNA (data not shown). The receptors and signaling molecules responsible for this process remain to be determined. METHODS Mice. Mice with a homozygous deletion in Ifnar1, which encodes the a-chain of the type I interferon receptor20, were obtained from M. Aguet (Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland) through T. Taniguchi (The University of Tokyo, Tokyo, Japan). Mice deficient in DNase II have been described9. Ifnar1+/ and Dnase2a+/– mice were backcrossed to the C57BL/6 strain for at least eight generations, and genotyping with primers for a mouse marker set31 indicated that the backcrossed mice had the C57BL/6 background at 98%. Dnase2a–/–Ifnar1–/– mice were generated by crossing of Dnase2a+/–Ifnar1–/– parents. All mice were housed in a specific pathogen–free facility at Osaka University Medical School and all animal experiments were carried out in accordance with protocols approved by the Osaka University Medical School Animal Care and Use Committee (Osaka, Japan). For determination of the Dnase2a and Ifnar1 genotypes, genomic DNA was prepared from embryonic tissues or tail snip tissue from adult mice32 and was analyzed by PCR. For the wild-type and mutant alleles of Dnase2a, a sense primer specific for the wild-type allele (5¢-GCCCATCTAGACTAACTTTC-3¢) or mutant allele (5¢-GATTCGCAGCGCATCGCCTT-3¢; a sequence in the neomycin-resistance gene) was used with a common antisense primer (5¢-GAGTCTTAGTCCTTTGCTCCG-3¢). The wild-type and mutant alleles of Ifnar1 were detected by a similar method with a wild-type-specific (5¢-AAGATGTGCTGTTCCCTTCCTCTGCTCTGA-3¢) or mutant-specific (5¢-CCTGCGTGCAATCCATCTTG-3¢) antisense primer and a common sense primer (5¢-ATTATTAAAAGAAAAGACGAGGCGAAGTGG-3¢). Gene array analysis. Total RNA was prepared from the fetal liver with an Isogen RNA extraction kit (Nippon Gene) and poly(A) RNA was selected with an mRNA Purification Kit (Amersham Biosciences). Gene array analysis was done as a ‘custom order’ by Kurabo with a Uniset Mouse 20K Codelink Bioarray (Amersham Biosciences), which displays probe DNAs for about 20,000 mouse genes and expressed sequence tags. Double-stranded cDNA was prepared using RNA from fetal livers of Dnase2a+/+, Dnase2a–/– and Dnase2a–/–Ifnar1–/– mouse embryos (E14.5) and was transcribed in vitro with biotin-labeled dUTP as a substrate. The complementary RNA was used as probe for hybridization of the array and the hybridized RNA was detected with indodicarbocyanine-conjugated streptavidin. The arrays were scanned with a Biochip Reader (Applied Precision) and the array image was analyzed with CodeLink System Software (Amersham Biosciences). RNA blot analysis. Poly(A) RNA was separated by electrophoresis through a 1.5% agarose gel containing 2% (weight/volume) formaldehyde and was transferred to a nylon membrane (Roche Diagnostics). For detection of IFN-a, IFN-b and IFN-g mRNA, cDNA for IFNa-1 (ref. 13), IFN-b33 and IFN-g34 was inserted into pGEM-T Easy and DIG-labeled RNA probes were prepared with a DIG RNA labeling kit (Roche Diagnostics). Hybridization was carried out at 68 1C with DIG Easy Hyb buffer according to the instructions provided by the supplier. After hybridization, membranes were washed at 68 1C in 0.1 SSC and 0.1% SDS and were treated at 25 1C for 10 min with 5 mg/ml of RNase A. The hybridized probes were detected with alkaline phosphataseconjugated anti-DIG and the signals were visualized with the CDP-Star system (Roche Diagnostics). RNA blot hybridization for the interferon-inducible genes used high-stringency conditions with 32P-labeled probe DNA as described35. Real-time PCR. A LightCycler (Roche Diagnostics) was used for real-time PCR. Primers were as follows: 5¢-CCACCACAGCCCTCTCCATCAACTAT-3¢ and 5¢-CAAGTGGAGAGCAGTTGAGGACATC-3¢ for IFN-b; and

VOLUME 6

NUMBER 1

JANUARY 2005

NATURE IMMUNOLOGY

ARTICLES


5¢-TAGCTCTGAGACAATGAACGCTAC-3¢ and 5¢-GTGATTCAATGACGCTTATGTTGT-3¢ for IFN-g. As a control, b-actin mRNA was detected with primers 5¢-TGTGATGGTGGGAATGGGTCAG-3¢ and 5¢-TTTGATGTCACGCACGATTTCC-3¢. The amount of specific mRNA was quantified at the point at which the LightCycler System detected the upstroke of the exponential phase of PCR accumulation and was normalized to b-actin mRNA for each individual sample. Flow cytometry for apoptotic cells. Cells from the fetal livers were suspended in flow cytometry staining buffer (PBS containing 2% FCS and 0.02% NaN3), were incubated on ice for 30 min with 4 mg/ml of phycoerythrin-conjugated rat anti-mouse Ter-119 (BD PharMingen), were fixed with 1% paraformaldehyde and were treated with 0.1% Triton X-100. The TUNEL reactions included 200 units/ml of terminal deoxynucleotidyl transferase (Takara Shuzo) and were incubated at 371C for 45 min in 50 ml of 100 mM cacodylate buffer, pH 7.2, containing 1 mM CoCl2, 0.01% BSA and 2.5 mM fluorescein isothiocyanate– labeled dUTP (Roche Diagnostics), and were analyzed by flow cytometry with a FACSAria (BD Biosciences). Cell viability assay. Fetal liver cells from E14.5 embryos were suspended in aMEM containing 10% FCS, 2.0 units/ml of human erythropoietin (Kirin Brewery), 10 ng/ml of human stem cell factor (Kirin Brewery) and 500 mg/ml of transfererin (Sigma), as described36. Cells were incubated at 37 1C for 2 d and were stained with indodicarbocyanine-labeled Annexin V (BioVision) and propidium iodide. After analysis by flow cytometry, the number of viable cells was calculated. Cell sorting. Macrophages in E14.5 fetal liver were enriched by sorting with a MACS column (MS+ Separations; Miltenyi Biotec). The antibody that reacts with macrophages, hamster anti-F4/80 (ref. 37; clone 6-16A; a gift from M. Tanaka, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan) was biotinylated. Cells (2.0 107) from the fetal liver were incubated at 4 1C for 20 min in 4 ml of MACS buffer (PBS containing 0.5% BSA and 2 mM EDTA) with 2.5 mg/ml of anti-F4/80, followed by incubation with 80 ml of anti-biotin microbeads (Miltenyi Biotec). Cells were loaded onto a column placed in magnetic field and F4/80-positive and F4/80-negative cells were separated according to the instructions provided by the manufacturer. Histochemical analysis and in situ hybridization. Livers of E14.5 embryos were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, containing 4% sucrose, were embedded in paraffin, were sectioned at 4 mm and were stained with hematoxylin and eosin. For TUNEL staining, the paraformaldehyde-fixed sections were embedded in optimum cutting temperature compound (Sakura Fine Technical), were sectioned at 6 mm and were stained with an ApopTag fluorescein in situ apoptosis detection kit from Chemicon International. The sections were mounted with FluorSave (Calbiochem) and were visualized by fluorescence microscopy (Olympus IX-70). For in situ hybridization, fetal livers were fixed with 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3, then were embedded in paraffin, sectioned at 6 mm and mounted onto Matsunami adhesive silane–coated slides (Matsunami Glass). The sections were dewaxed, dehydrated, treated at 37 1C for 15 min with 50 mg/ml of proteinase K in PBS and were ‘refixed’ with 4% paraformaldehyde. Hybridization used DIG-labeled RNA probes at a concentration of 1.0 mg/ml in hybridization buffer (5 SSC and 50% formamide). After hybridization overnight at 42 1C, the sections were washed at 45 1C in 2 SSC containing 50% formamide and were treated at 37 1C for 5 min with 1.0 mg/ml of RNase A. The hybridized probes were then detected with alkaline phosphatase–conjugated anti-DIG and the signals were visualized with NBT-BCIP (nitro-blue tetrazolium chloride–5-bromo-4chloro-3¢-indolylphosphate p-toluidine salt). Geo accession numbers. Raw data sets, GSE 1859. ACKNOWLEDGMENTS We thank K. Miwa for genotyping the mice; and M. Fujii and M. Harayama for secretarial assistance. Supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture in Japan.

NATURE IMMUNOLOGY

VOLUME 6

NUMBER 1

JANUARY 2005

COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 20 September; accepted 9 November 2004 Published online at http://www.nature.com/natureimmunology/

1. Palis, J. & Segel, G.B. Developmental biology of erythropoiesis. Blood Rev. 12, 106– 114 (1998). 2. Keller, G., Lacaud, G. & Robertson, S. Development of the hematopoietic system in the mouse. Exp. Hematol. 27, 777–787 (1999). 3. Bernard, J. The erythroblastic island: past and future. Blood Cells 17, 5–10 (1991). 4. Sasaki, K. & Iwatsuki, H. Origin and fate of the central macrophages of erythroblastic islands in the fetal and neonatal mouse liver. Microsc. Res. Tech. 39, 398–405 (1997). 5. Bernardi, G. Spleen acid deoxyribonuclease. in The Enzymes vol. 3 (ed. Boyer, P.D.) 271–287 (Academic Press, New York and London, 1971). 6. Evans, C.J. & Aguilera, R.J. DNase II: genes, enzymes and function. Gene 322, 1–15 (2003). 7. Yasuda, T. et al. Molecular cloning of the cDNA encoding human deoxyribonuclease II. J. Biol. Chem. 273, 2610–2616 (1998). 8. Chou, S.F., Chen, H.L. & Lu, S.C. Up-regulation of human deoxyribonuclease II gene expression during myelomonocytic differentiation of HL-60 and THP-1 cells. Biochem. Biophys. Res. Commun. 296, 48–53 (2002). 9. Kawane, K. et al. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science 292, 1546–1549 (2001). 10. Krieser, R.J. et al. Deoxyribonuclease IIa is required during the phagocytic phase of apoptosis and its loss causes lethality. Cell Death Differ. 9, 956–962 (2002). 11. Kina, T. et al. The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage. Br. J. Haematol. 109, 280–287 (2000). 12. Shifman, M.I. & Stein, D.G. A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues. J. Neurosci. Methods 59, 205–208 (1995). 13. Shaw, G.D. et al. Structure and expression of cloned murine IFN-a genes. Nucleic Acids Res. 11, 555–573 (1983). 14. Nagata, S., Mantei, N. & Weissmann, C. The structure of one of the eight or more distinct chromosomal genes for human interferon-a. Nature 287, 401–408 (1980). 15. Gresser, I., Tovey, M.G., Maury, C. & Chouroulinkov, I. Lethality of interferon preparations for newborn mice. Nature 258, 76–78 (1975). 16. Gresser, I. et al. Electrophoretically pure mouse interferon inhibits growth, induces liver and kidney lesions, and kills suckling mice. Am. J. Pathol. 102, 396–402 (1981). 17. Alexander, W.S. et al. SOCS1 is a critical inhibitor of interferon-g signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell 98, 597–608 (1999). 18. Marine, J.C. et al. SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell 98, 609–616 (1999). 19. Stark, G.R., Kerr, I.M., Williams, B.R., Silverman, R.H. & Schreiber, R.D. How cells respond to interferons. Annu. Rev. Biochem. 67, 227–264 (1998). 20. Muller, U. et al. Functional role of type I and type II interferons in antiviral defense. Science 264, 1918–1921 (1994). 21. Der, S.D., Zhou, A., Williams, B.R. & Silverman, R.H. Identification of genes differentially regulated by interferon a, b, or g using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95, 15623–15628 (1998). 22. Kawane, K. et al. Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation. Nat. Immunol. 4, 138–144 (2003). 23. Sadahira, Y. & Mori, M. Role of the macrophage in erythropoiesis. Pathol. Int. 49, 841–848 (1999). 24. Tanaka, M., Itai, T., Adachi, M. & Nagata, S. Down-regulation of Fas ligand by shedding. Nat. Med. 4, 31–36 (1998). 25. Selleri, C., Maciejewski, J.P., Sato, T. & Young, N.S. Interferon-g constitutively expressed in the stromal microenvironment of human marrow cultures mediates potent hematopoietic inhibition. Blood 87, 4149–4157 (1996). 26. Pfeffer, L.M. et al. Biological properties of recombinant a-interferons: 40th anniversary of the discovery of interferons. Cancer Res. 58, 2489–2499 (1998). 27. Samuel, C.E. Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778–809 (2001). 28. Beutler, B. & Rietschel, E.T. Innate immune sensing and its roots: the story of endotoxin. Nat. Rev. Immunol. 3, 169–176 (2003). 29. Beutler, B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature 430, 257–263 (2004). 30. Krieg, A.M. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20, 709–760 (2002). 31. Wakeland, E., Morel, L., Achey, K., Yui, M. & Longmate, J. Speed congenics: a classic technique in the fast lane (relatively speaking). Immunol. Today 18, 472–477 (1997). 32. Laird, P.W. et al. Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19, 4293 (1991). 33. Higashi, Y. et al. Structure and expression of a cloned cDNA for mouse interferon-b. J. Biol. Chem. 258, 9522–9529 (1983). 34. Gray, P.W. & Goeddel, D.V. Cloning and expression of murine immune interferon cDNA. Proc. Natl. Acad. Sci. USA 80, 5842–5846 (1983).

55


ARTICLES 35. Sambrook, J. & Russell, D.W. Molecular Cloning: a Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001). 36. Zhang, D. et al. An optimized system for studies of EPO-dependent murine pro-erythroblast development. Exp. Hematol. 29, 1278–1288 (2001). 37. Austyn, J.M. & Gordon, S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage. Eur. J. Immunol. 11, 805–815 (1981). 38. Carlow, D.A., Teh, S.J. & Teh, H.S. Specific antiviral activity demonstrated by TGTP, a member of a new family of interferon-induced GTPases. J. Immunol. 161, 2348–2355 (1998). 39. Lee, C.G., Demarquoy, J., Jackson, M.J. & O’Brien, W.E. Molecular cloning and characterization of a murine LPS-inducible cDNA. J. Immunol. 152, 5758–5767 (1994). 40. Rutherford, M.N., Kumar, A., Nissim, A., Chebath, J. & Williams, B.R. The murine 2–5A synthetase locus: three distinct transcripts from two linked genes. Nucleic Acids Res. 19, 1917–1924 (1991). 41. Au, W.C., Moore, P.A., LaFleur, D.W., Tombal, B. & Pitha, P.M. Characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes. J. Biol. Chem. 273, 29210–29217 (1998).

56

42. Luster, A.D., Jhanwar, S.C., Chaganti, R.S., Kersey, J.H. & Ravetch, J.V. Interferoninducible gene maps to a chromosomal band associated with a (4;11) translocation in acute leukemia cells. Proc. Natl. Acad. Sci. USA 84, 2868–2871 (1987). 43. Meraro, D., Gleit-Kielmanowicz, M., Hauser, H. & Levi, B.Z. IFN-stimulated gene 15 is synergistically activated through interactions between the myelocyte/ lymphocyte-specific transcription factors, PU.1, IFN regulatory factor-8/IFN consensus sequence binding protein, and IFN regulatory factor-4: characterization of a new subtype of IFN-stimulated response element. J. Immunol. 168, 6224–6231 (2002). 44. Miyasaka, K., Hanayama, R., Tanaka, M. & Nagata, S. Expression of MFG-E8 in bone marrow-derived immature dendritic cells. Eur. J. Immunol. 34, 1414–1422 (2004). 45. Inaba, K. et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176, 1693–1702 (1992). 46. McKnight, A.J. et al. Molecular cloning of F4/80, a murine macrophage-restricted cell surface glycoprotein with homology to the G-protein-linked transmembrane 7 hormone receptor family. J. Biol. Chem. 271, 486–489 (1996).

VOLUME 6

NUMBER 1

JANUARY 2005

NATURE IMMUNOLOGY