Impaired thymic development in mouse embryos deficient in ... - Nature

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Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation Kohki Kawane1, Hidehiro Fukuyama1,*, Hideyuki Yoshida1, Hiroko Nagase1, Yoshiyuki Ohsawa2,Yasuo Uchiyama2, Kazuhisa Okada3,Tetsuya Iida3 and Shigekazu Nagata1,4,5 Published online 13 January 2003; doi:10.1038/ni881

Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD –/– thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II.The thymus of DNase II –/–CAD –/– embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-β gene was strongly up-regulated in the thymus of DNase II –/–CAD –/– embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development. Apoptosis is defined as a death process that is accompanied by the condensation and fragmentation of cells and nuclei1. This process removes harmful or surplus cells generated during the development of metazoans and helps maintain homeostasis by removing senescent, virally infected and tumor cells2,3. Apoptosis is regarded as a cell-autonomous process4 and can be induced by a variety of stimuli5–7. Most apoptotic stimuli activate a cascade of caspases (cysteine proteases) that kill the cells by cleaving cellular proteins8–10. Cell integrity is maintained at the initial stage of apoptosis, but cells undergo secondary necrosis late in the process and release their contents1. To prevent the release of cellular materials into the circulation, which may cause inflammation, apoptotic cells are swiftly phagocytosed by phagocytes such as macrophages or dendritic cells11. Apoptosis is often accompanied by the degradation of chromosomal DNA12,13. We and others have identified a DNase (CAD, caspase-activated DNase; also called DFFB, DNA fragmentation factor 40 kDa, β-polypeptide) that is activated during apoptosis14,15. CAD is complexed with ICAD (inhibitor of CAD; also called DFFA, DNA fragmentation factor 45 kDa, α-polypeptide) in proliferating cells. When apoptotic stimuli activate the cascade of caspases, caspase 3, which functions downstream in the cascade, cleaves ICAD at two positions16. The cleaved ICAD loses its affinity for CAD17, and CAD, thus released from ICAD, digests the chromosomal DNA. In addition to its inhibitory activity, ICAD is required to produce the properly folded CAD polypeptide14,18. Cells deficient in ICAD or cells that express a caspase-resistant form of ICAD do not undergo apoptotic DNA fragmentation19,20, supporting the idea that CAD is responsible for the apoptotic DNA fragmentation. However, the possibility that other DNases that are regulated by ICAD might be involved in this process has not been ruled out. In addition, endonuclease G, which is

released from mitochondria during the apoptotic process, has recently been proposed as a DNase that causes apoptotic DNA fragmentation21–23. ICAD-deficient cells die efficiently19,20, indicating that the degradation of DNA is not a prerequisite for cells to die. In addition, ICAD-deficient mice develop normally, are fertile and show no apparent abnormality, indicating that apoptotic cell death takes place normally during their development without CAD-mediated DNA fragmentation. When the apoptotic cells in these mutant mice were examined under the electron microscope, they were found in macrophages20. Furthermore, when macrophages were cultured with apoptotic cells, they engulfed the apoptotic cells and digested their DNA in lysosomes20,24. From these results, we proposed that there are two auxiliary systems that degrade the DNA of apoptotic cells: a cellautonomous program mediated by CAD-ICAD in the dying cells; and a non–cell-autonomous system mediated by a DNase in macrophages20. DNase II, an acid DNase localized to the lysosomes of macrophages25, was likely to be responsible for digesting the DNA of apoptotic cells in macrophages, and we and others have established mouse lines lacking the DNase II gene26,27. Here, we established CAD-null mouse lines by gene targeting and used them to investigate the relative importance of CAD and DNase II in apoptotic DNA degradation. CAD–/– thymocytes did not undergo apoptotic DNA fragmentation, but their DNA was digested by thymic macrophages that effectively phagocytosed the apoptotic thymocytes. In embryos lacking both the CAD and DNase II genes, thymic development in the fetus was severely blocked at the pro-T stage. Of the several cytokine genes examined, the interferon (IFN)-β gene was found to be up-regulated in the thymus of CAD–/–DNase II–/– embryos. These results indicate that the DNA of apoptotic cells is degraded by two complementary DNases, CAD and

Departments of 1Genetics and 2Cell Biology and Neuroscience, Osaka University Medical School, Osaka 565-0871, Japan 3Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan 4Laboratory of Genetics, Integrated Biology Laboratories, Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan 5Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka 565-0871, Japan *Present address: Laboratory of Molecular Genetics and Immunology,The Rockefeller University, New York, NY 10021, USA. Correspondence should be addressed to S.N. ([email protected]). 138

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Figure 1. Targeted disruption of the CAD gene by homologous recombination. (a) Targeting strategy. Endogenous CAD locus (top bar), targeting construct (middle bar) and targeted CAD locus (bottom bar) are shown schematically.The seven exons (coding regions) of the CAD gene are represented by black boxes, and numbered. The recognition sites for the BamHI (B), EcoRI (E), XhoI (Xh), SmaI (S), and XbaI (X) restriction enzymes are shown.There are several more XbaI sites in the 5′ region. The neomycin-resistance gene (neo) and the gene encoding the diphtheria toxin A fragment (DTA), used for positive and negative selection, respectively, are indicated. The probe used for Southern hybridization is indicated by the filled box under the top bar. The XbaIdigested fragments detected by the probe are depicted. Scale bar, 1 kb. (b) Southern blot analysis of tail DNA isolated from pups generated by CAD+/– pairs. Using the probe shown in the targeting schematic, a neo gene inserted by homologous recombination into the CAD locus results in the shift of a single 15.6-kb band to a 4.5-kb band upon XbaI digestion.The sizes of the marker DNAs are shown in kb at left. (c) Northern hybridization analysis. Poly(A) RNA from the spleen and thymus of CAD+/+, CAD+/– and CAD–/– mice was analyzed by northern hybridization with CAD cDNA as the probe. In the bottom panel, the filter was stained with methylene blue for rRNAs. (d) DNase activity in CAD–/– thymocytes. Plasmid DNA (left panel) or mouse liver nuclei (right panel) was incubated with the cell extracts from the WT (lanes 2 and 3) or CAD–/– (lanes 4 and 5) thymocytes in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of human caspase 3. DNA was analyzed by electrophoresis on an agarose gel. In lane 1, plasmid DNA or nuclei was treated as above without the cell extracts. (e) Apoptotic DNA fragmentation.Thymocytes from the WT or CAD–/– mice were incubated for the indicated periods of time (h) with dexamethasone, and their chromosomal DNA was analyzed by electrophoresis on an agarose gel. (f) Cleavage of chromosomal DNA into high-molecular-weight DNA. Thymocytes from the WT or CAD–/– mice were treated for the indicated periods of time (h) with dexamethasone, and their DNA was analyzed by the pulse-field gel electrophoresis.


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DNase II. If the DNA of apoptotic cells is left undigested, it causes adverse effects on thymic development by activating innate immunity.

Results Generation of CAD–/– mice To assess the physiological role of CAD in the apoptotic process, we generated CAD–/– mice by gene targeting. The murine CAD gene is encoded by seven exons within 11.0 kb of genomic DNA, which is localized to chromosome 4 (Fig. 1a)28. We constructed a replacementtype targeting vector that contains the 5.4-kb upstream and 4.5-kb downstream flanking sequences of the CAD gene (Fig. 1a). Exons 1–6 and part of exon 7 were replaced with the neo gene. Mice derived from two clones had identical phenotypes, and those from one representative clone were characterized in detail. Heterozygous animals were intercrossed and the tail DNA of the resulting littermates was analyzed by Southern blot hybridization with a probe containing a sequence from outside the targeting vector (Fig. 1a). XbaI-digested DNA gave a single 15.6-kb band for CAD+/+, a 4.5-kb band for CAD–/– and both for CAD+/– mice (Fig. 1b). Southern blot hybridization of BglII-digested DNA with the neo gene probe gave a single band in CAD+/– or CAD–/– mice (data not shown). The bands detected with the CAD and neo gene probes were exactly as predicted for the homologous recombination of the CAD gene with the targeting vector (Fig. 1a). The spleen and thymus of wild-type (WT) mice expressed relatively high amounts of the 2.5-kb CAD mRNA (Fig. 1c) and CAD protein (data not shown). This expression was reduced in CAD+/– mice and absent from CAD–/– mice, confirming the null mutation of the CAD–/– allele. The three genotypes (CAD+/+, CAD+/– and CAD–/–) were represented generally according to normal mendelian inheritance. www.nature.com/natureimmunology

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CAD–/– mice showed no gross developmental abnormalities and were fertile with normal fecundity. Cell extracts from the WT and CAD–/– thymocytes were treated with caspase 3 to examine the presence of other ICAD-regulated DNases. As reported previously14, this treatment generated a strong DNase activity with extracts from the WT thymocytes (Fig. 1d). In contrast, cell extracts from CAD–/– thymocytes did not show DNase activity upon the same treatment, indicating that there is no other CAD-like DNase that can be activated by caspase 3. The requirement for CAD in apoptotic DNA fragmentation was then examined by treating the thymocytes with dexamethasone to induce apoptotic cell death. All chromosomal DNA was fragmented into nucleosomal units within 24 hours in CAD+/+ thymocytes, but not in CAD–/– thymocytes (Fig. 1e). CAD-dependent apoptotic DNA fragmentation was also obtained with thymocytes using other apoptotic stimuli (such as Fas ligand and staurosporine) and with other cell types (splenocytes, hepatocytes and embryonal fibroblasts; H.N., K.K., H.F. and S.N., unpublished results), confirming the absolute requirement for CAD in cell-autonomous apoptotic DNA fragmentation. The apoptotic fragmentation of DNA into nucleosomal units is preceded by large-scale chromatin fragmentation29. When WT thymocytes were treated with dexamethasone and their DNA was analyzed by pulse-field gel electrophoresis, broad smearing bands with a peak at about 50 kb were observed (Fig. 1f). In contrast, treatment of CAD–/– thymocytes with dexamethasone did not cause large-scale chromatin degradation, indicating that this type of DNA degradation is also mediated by CAD.

DNase II-dependent DNA degradation in macrophages Although CAD seems to be critical for the degradation of DNA in apoptotic cells, DNase II in macrophages may also degrade the DNA of •

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Figure 2. DNA degradation of apoptotic cells in macrophages. Primary macrophages from the thymus of WT (a–f) or DNase II–/– (g–l) mouse embryos were cultured in chamber slides.Thymocytes from WT (a–c,g–i) or CAD–/– (d–f,j–l) mice were treated with dexamethasone, added to the macrophage and allowed to be phagocytosed at 37 °C for 1 h (a,d,g,j) or 3 h (b,c,e,f,h,i,k,l). Floating cells were removed from the culture and adherent cells were stained with Feulgen.The genotypes used for thymocytes (T) and macrophages (M) are indicated at left. Original magnifications; ×150 for a,b,d,e,g,h,j,k; ×300 for c,f,i,l.

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apoptotic cells after phagocytosis by macrophages20. To examine the relative contribution of CAD and DNase II in degrading DNA, primary macrophages were prepared from the thymus of WT and DNase II–/– embryos26. When these macrophages were incubated with the apoptotic thymocytes, they efficiently phagocytosed WT or CAD–/– cells, as shown by the substantial number of apoptotic cells in each macrophage after 1 hour (Fig. 2). After 3 hours of coculture, WT macrophages digested the nuclei of WT or CAD–/– thymocytes, indicating that macrophages have the ability to degrade DNA of the apoptotic cells even when the apoptotic cells could not digest their own DNA. In contrast, DNase II–/– macrophages were not able to digest the engulfed nuclei from CAD–/– thymocytes after 3 hours. The accumulation of undigested DNA in DNase II–/– macrophages was less pronounced when WT thymocytes were used as targets. These results indicate that CAD in apoptotic cells and DNase II in macrophages cooperatively degrade the DNA of apoptotic cells.

Thymic development in CAD–/–DNase II–/– embryos The CAD–/– mice had no apparent abnormalities, but the DNase II–/– embryos suffered from severe anemia late in embryogenesis26. To examine the physiological role of apoptotic DNA degradation during embryogenesis, CAD–/–DNase II+/– or CAD+/–DNase II+/– mice were intercrossed to generate CAD+/–DNase II+/– or CAD+/–DNase II+/+ (WT), CAD–/–DNase II+/– or CAD–/–DNase II+/+ (CAD–/–), CAD+/+DNase II–/– or CAD+/–DNase II–/– (DNase II–/–), and CAD–/–DNase II–/– embryos. Similar to DNase II–/– mice, CAD–/–DNase II–/– embryos at embryonic day (E) 17.5 were anemic and their body size was slightly smaller than the WT or CAD–/– embryos (data not shown). Among the various tissues examined, the size of the brain and heart was normal in DNase II–/– and CAD–/–DNase II–/– embryos. In 140

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contrast, the thymus and kidney were severely affected by the mutation in DNase II and CAD. Thymus of DNase II–/– mice examined at E17.5 was smaller than the thymus of WT or CAD–/– embryos (Fig. 3a). This reduction in size was enhanced in CAD–/–DNase II–/– mice. The cellularity of the thymus of CAD–/– embryos was slightly smaller than that of the WT embryos (74 ± 55%). In contrast, the cellularity of the DNase II–/– and CAD–/–DNase II–/– thymus was reduced to 29 ± 13% and 12 ± 4%, respectively, of the WT thymus (Fig. 3b). Most of the cells (92–98%) prepared from E17.5 fetal thymus expressed Thy-1, and this percentage of Thy-1+ cells did not differ among WT, CAD–/–, DNase II–/– and CAD–/–DNase II–/– mice (data not shown). However, the percentage of CD4+CD8+ thymocytes in CAD–/–DNase II–/– embryos was reduced to about one-third of the percentage in WT or CAD–/– embryos, with a concomitant increase in the percentage of CD4–CD8– thymocytes (Fig. 3c). To determine more precisely how the progression of thymocytes was inhibited in CAD–/–DNase II–/– mice, we examined the expression of the CD44 and CD25 surface markers, which serve to discriminate the CD4–CD8– subpopulations of thymocyte progenitors30. The percentage of immature CD44+CD25– cells increased in DNase II–/– and CAD–/–DNase II–/– embryos, whereas there was a decreased percentage of the more mature CD44–CD25– cells (Fig. 3c). The absolute number of CD44+CD25– thymocytes per thymus was reduced in CAD–/–DNase II–/– mice, but this decrease was less obvious than the reduction in the total number of thymocytes (Fig. 3d). These results indicate that T cell development was inhibited at the early progenitor stage in CAD–/–DNase II–/– embryos.

Decreased DNA and increased IFN To determine the cause of the impaired thymic development in CAD–/–DNase II–/– embryos, we next performed histochemical analysis of february 2003

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Figure 3. Impaired thymic development in DNase-targeted mouse embryos. (a) Size of thymus from E17.5 WT, CAD–/–, DNase II–/– and CAD–/–DNase II–/– embryos. (b) Reduced number of thymocytes in DNase-targeted mouse embryos.The thymus was prepared from E17.5 mouse embryos of the indicated genotypes, and their cell numbers are plotted. Numbers of embryos examined are as follows:WT, 25; CAD–/–, 11; DNase II–/–, 12; CAD–/–DNase II–/–, 7. (c) Reduction of the CD4+CD8+ T cell population in the thymus of DNase-targeted embryos.Thymocytes from E17.5 mouse embryos of the indicated genotypes were stained with CD4 and CD8 antibodies, and the percentages of CD4+CD8+ double-positive cells were determined by FACS analysis. Numbers of embryos examined are as follows:WT, 25; CAD–/–, 11; DNase II–/–, 13; CAD–/–DNase II–/–, 7. Representative FACS profiles for each genotype are shown in the top panels. (d) Numbers of CD25–CD44+ immature thymocytes in DNase-targeted embryos.Thymocytes prepared from the E17.5 mouse embryos of the indicated genotypes were subjected to three-color FACS analysis using CD4, CD8, CD25 and CD44 antibodies.The percentage of CD25–CD44+ thymocytes in the CD4–CD8- population was determined.The absolute cell numbers for CD25–CD44+ per thymus were calculated from the total number of thymocytes, the percentage of CD4–CD8– thymocytes and the percentage of CD25–CD44+ thymocytes in the CD4–CD8– population (mean ± s.d.). Numbers of embryos examined are as follows:WT, 18; CAD–/–, 7; DNase II–/–, 7; CAD–/–DNase II–/–, 4. Representative FACS profiles for CD25 and CD44 in the CD4–CD8– population are shown in the top panels.

the fetal thymus. Wild-type and CAD–/– fetal thymuses showed normal architecture (Fig. 4a). Many abnormal foci were observed in thymus of DNase II–/– and CAD–/–DNase II–/– embryos, and these foci stained strongly with 4´,6-diamidino-2-phenylindole (DAPI). The number of abnormal foci and the average size of each focus in the CAD–/–DNase II–/– thymus were slightly larger than those observed in the DNase II–/– thymus. The foci were stained with Feulgen and found in F4/80-positive cells (data not shown), suggesting that they consisted of F4/80-positive macrophages carrying DNA. Sections of thymus from DNase II–/– and CAD–/–DNase II–/– embryos were then analyzed by transmission electron microscopy. Macrophage-like cells were present in the foci of the DNase II–/– and CAD–/–DNase II–/– fetal thymuses and they contained many nucleus-like structures (Fig. 4b). These were probably the nuclei of the engulfed apoptotic cells. There were some condensed structures that may www.nature.com/natureimmunology

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have represented the newly phagocytosed apoptotic nuclei, whereas other loosely packed structures may have been nuclei in which proteins were digested by lysosomal proteases. When the loosely packed structures were examined at higher magnification, there was a substantial difference in the appearance of these structures in the DNase II–/– and CAD–/–DNase II–/– mice. These structures were mainly composed of DNA-like strings. In DNase II–/– mice, the strings appeared fragmented; in contrast, they were rather long and intact in DNase II–/–CAD–/– embryos. We have shown that the thymic development of mouse embryos was severely inhibited when the endogenous DNA was not properly degraded during the apoptotic process. Type I IFNs (IFN-α and IFN-β), produced by activation of innate immunity during bacterial infection31, are known to cause thymic atrophy32,33. We therefore examined the expression of IFNs and tumor necrosis factor (TNF)-α in the DNase-null mice using real-time •

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Figure 4. Accumulation of DNA in thymic macrophages of DNase-targeted embryos. (a) Accumulation of DAPI-positive foci in the fetal thymus.Thymus from E17.5 WT, CAD–/–, DNase II–/– and CAD–/–DNase II–/– mice was stained with DAPI. Scale bar, 0.1 mm. (b) DNA-like strings in macrophages.Abnormal foci in thymus from E16.5 DNase II–/– or CAD–/–DNase II–/– embryos were analyzed by electron micrography.The left panels, at lower magnification, show the overall structure of an abnormal macrophage. N, nucleus-like structure; MN, macrophage nuclei. Scale bars, 2 µm.The right panels show the inclusion in lysosomes of an abnormal macrophage at higher magnification. Scale bars, 0.5 µm.

PCR. The IFN-α1 and IFN-β genes were not expressed in WT or CAD–/– E17.5 fetal thymus, but high amounts of IFN-γ and TNF-α mRNAs were found, as reported previously34. IFN-α1 mRNA was not up-regulated in DNase II–/– and CAD–/–DNase II–/– fetal thymus (Table 1). However, the IFN-β gene was up-regulated by 5- to 8-fold in DNase II–/– fetal thymus and 70- to 100-fold in CAD–/–DNase II–/– fetal thymus. The expression of IFN-γ mRNA was also 8–12 times higher in DNase II–/– and CAD–/–DNase II–/– fetal thymus than in WT or CAD–/– fetal thymus. In contrast, expression of the TNF-α gene was only moderately up-regulated (three- to fourfold) in the DNase II–/– and CAD–/–DNase II–/– fetal thymus.

Discussion DNA fragmentation is regarded as a hallmark of apoptosis35 and is an indicator of apoptotic cells in vitro and in vivo36,37. We have shown here that apoptotic DNA degradation proceeds in two steps involving cellautonomous and non–cell-autonomous processes. CAD-null cells did not show large-scale chromatin degradation or fragmentation into nucleosomal units when cultured in vitro, suggesting that the cell-autonomous DNA degradation is mediated by CAD. Thus, the contribution of other DNases

Table 1. Induction of interferon gene expression Genotype wild type CAD–/– DNase II–/– CAD–/–DNase II–/–

IFN-α1

IFN-β

IFN-γ

TNF-α

1.0 0.6 0.7 1.1

1.0 1.9 8.1 86.3

1.0 1.0 8.7 11.8

1.0 1.2 2.9 3.6

RNA was prepared from thymus of the E17.5 WT, CAD–/–, DNase II–/– and CAD–/–DNase II–/– embryos.The expression of IFN-α, IFN-β, IFN-γ and TNF-α mRNA was determined by real-time PCR.The amounts of cytokine mRNA are expressed as a value relative to those in WT thymus. Numbers of embryos examined are as follows: wild-type, 3; CAD–/–, 2; DNase II–/–, 4; CAD–/–DNase II–/–, 2.

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(such as endonuclease G21, DNase I38 and a putative apoptosis-inducing factor (AIF)-activated DNase39) to this process, if any, must be very small. Genetic analysis in Caenorhabditis elegans indicated that NUC-1, a DNase II homolog, degrades DNA in dying cells40. Although we cannot rule out this possibility, the fact that DNase II–/– macrophages cannot digest the DNA of engulfed apoptotic cells suggests that DNase II must function within macrophages and work in a non–cell-autonomous manner. The apoptotic DNA in Drosophila melanogaster is also degraded in two steps41, indicating that this mechanism is conserved between mammals and Drosophila. Null mutation in the CAD of apoptotic cells does not cause the accumulation of DNA. CAD–/– cells are efficiently engulfed by macrophages, and their DNA may then be digested by DNase II in macrophages. It seems that the efficiency of DNase II in digesting the DNA of apoptotic cells is not affected by whether their DNA is pre-cleaved by CAD or not. This may explain why there is no adverse phenotype in CAD–/– mice during development. We previously observed that when mice were irradiated with γ-rays, many thymocytes underwent apoptotic DNA degradation outside macrophages20. It will be interesting to examine the role of CAD-mediated DNA degradation under such pathological conditions, in which the acute massive load of apoptotic cells may exceed the capacity of macrophages. DNase II–/– macrophages did not accumulate DNA when they were cultured in vitro with CAD+/+ apoptotic cells. However, DNase II–/– embryos accumulated undigested DNA in various fetal organs such as the thymus, kidney, interdigital area and brain27. During development, apoptotic cells that contain condensed and fragmented nuclei are usually found in macrophages42, indicating that these cells are rapidly engulfed by macrophages in vivo. Once the cells have been phagocytosed by macrophages, CAD would be inactivated or digested. Thus, the apoptotic cells may not have enough time for CAD to cause the cell-autonomous DNA fragmentation under these situations. In contrast, cells treated for 5 hours in vitro to cause apoptosis may have sufficient time for CAD to cause the cell-autonomous DNA fragmentation before they were added to february 2003

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macrophages. Nevertheless, the DNA that accumulated in the thymus of DNase II–/– embryos seemed to be more fragmented than that found in the CAD–/–DNase II–/– embryos, indicating that CAD-mediated DNA fragmentation contributes to the degradation of DNA during apoptosis in vivo. We previously reported that DNase II–/– embryos show no apparent abnormality in organ development except for a defect in erythropoiesis26. A close examination, however, indicated the presence of many abnormal foci containing undigested DNA throughout the embryos27 and, as described here, a defect in the development of T cells. The cellularity of the thymus of DNase II–/– embryos was severely reduced because T cell development was blocked at the pro-T cell stage. We do not think that the defect in thymic development was due to a secondary effect of anemia, because a similar accumulation of undigested nuclei was observed when the thymus from E13.5 DNase II–/– or CAD–/–DNase II–/– embryos was subjected to organ culture (K.K., H.F. and S.N., unpublished results). These results indicate that the DNA of apoptotic cells has an adverse effect on thymic development, when it is left undigested. This adverse effect seems to be due to the type I IFN produced in CAD–/–DNase II–/– embryos. As type I IFN is known to inhibit the development of other cells such as B cells33, it is likely that the development of B cells in the fetal liver or spleen is also defective in CAD–/–DNase II–/– embryos. Many cytokines are known to positively and negatively regulate thymic development32,33. We found that the IFN-β and IFN-γ genes were up-regulated in the DNase II–/– and CAD–/–DNase II–/– fetal thymus. The induction of the IFN-β gene was particularly high in the thymus of the CAD–/–DNase II–/– embryos, and its expression correlated well with the amount of thymic atrophy. Type I interferons have cytopathic effects on lymphocytes, as shown by the block in T cell development at the pro-T stage when type I IFN was administered to newborn mice33. In addition, the transient pancytopenia caused by lymphocytic choriomeningitis virus (LCMV) infection can be explained by the effect of type I IFNs produced in response to the virus infection, but not by IFN-γ43. Although we cannot rule out the possibility of the involvement of other cytokines and molecules, these results suggest that IFN-β may have an important role in causing the defect in thymic development in DNase II–/– and CAD–/–DNase II–/– embryos. Type I IFNs are produced in a variety of cells during infection by viruses and bacteria, although the ratio of IFN-β to IFN-α transcript varies with cell type and with the inducer44. We found that the IFN-β gene, but not the IFN-α gene, was specifically activated in the thymus of DNase II–/– and CAD–/–DNase II–/– embryos. A similar specific induction of the IFN-β gene was previously observed in macrophages activated by killed bacteria. IFNβ can induce the expression of the IFN-γ gene in lymphocytes31, which may explain the high concentration of IFN-γ transcripts in the DNase II–/– and CAD–/–DNase II–/– fetal thymus. How is the IFN-β gene activated in DNase II–/– and CAD–/–DNase II–/–? Non-methylated, CpG-rich bacterial DNA is known to activate the innate immunity through Toll-like receptor 9 (TLR-9)45,46. The CpG sequence is less abundant in mammalian chromosomal DNA and is usually methylated. However, there is a locus called the “CpG island” in the mammalian genome47, and earlier reports suggested that a complex of endogenous mammalian DNA with immunoglobulin can activate B cells to develop autoimmunity48. It is therefore likely that the large amount of DNA that accumulated in the lysosomes of the DNase II–/– macrophages directly activated the innate immunity to produce IFN-β. Although apoptosis is accompanied by DNA fragmentation, cells can die without it. Thus, the physiological role of DNA degradation in the apoptotic process has been elusive. Our results indicate that if the DNA of apoptotic cells is not properly disposed of, it has an adverse effect on mammalian development that involves the activation of innate www.nature.com/natureimmunology

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immunity. Whether the defect in definitive erythropoiesis in DNase II–/– embryos is also due to the abnormal activation of innate immunity remains to be studied.

Methods

Cells and mice. Mouse R1 ES cells were cultured as described49. Fetal thymic macrophages were prepared as described50. In brief, thymus lobes from E17.5 WT or DNase II–/– embryos were treated at 37 °C for 40 min with 0.05% collagenase (Roche Molecular Biochemicals, Indianapolis, IN) and 20 µg/ml DNase I (Boehringer Mannheim, Germany) in RPMI1640 medium, and gently disrupted. Cells from one lobe were suspended in 1.5 ml of α-MEM containing 10% fetal calf serum (FCS, Invitrogen, Carlsbad, CA) supplemented with a one-tenth volume of culture supernatant from CMG14-12 cells, which produce mouse M-CSF51, and cultured overnight in a 3.5-cm suspension culture dish (Corning Inc., Corning, NY). The cells were vigorously washed with the medium to remove non-adherent cells, and detached from the plate by incubating them in PBS containing 0.02% EDTA for 10 min at 37 °C. After a 3-week culture period with passage every 7–10 days, the cells expressed Mac-1 and were used as thymic macrophages. C57BL/6 mice were purchased from Nippon SLC Inc (Shizuoka, Japan). The DNase II+/– mice were previously described26. 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. Targeted disruption of the CAD gene. The plasmid carrying the mouse CAD chromosomal gene was described previously28. The CAD targeting vector was constructed by replacing the 10-kb SmaI-BamHI DNA fragment containing exons 1–6 and a part of exon 7 with a 1.2-kb neo gene driven by the thymidine kinase gene promoter. The 1.2 kb-DNA fragment coding for the diphtheria toxin A fragment under MC1 promoter was inserted downstream of the CAD gene for negative selection. The CAD-deficient mice were produced as described26. In brief, 3.0 × 107 R1 ES cells were transfected with 75 µg of the targeting vector by electroporation. G-418–resistant clones were screened for homologous recombination by PCR. The ES clones carrying the CAD-deficient allele were introduced into the host embryos by the aggregation method as described49, and used to produce chimeric mice. Chimeric mice with a high ES cell contribution were crossed with C57BL/6 mice to produce CAD+/– mice. CAD–/– mice were generated by crossing the CAD+/– parents. CAD–/–DNase II–/–- embryos were generated by crossing the CAD–/–DNase II+/– or CAD+/–DNase II+/– parents. PCR, Southern and northern blot analyses. Genomic DNA was prepared from embryonic tissues or adult tail snips52. The genotype of the CAD and DNase II genes was determined by PCR with Taq polymerase (Amersham Biosciences, Uppsala, Sweden) using a PerkinElmer 9700 cycler (Roche Molecular Systems, Pleasanton, CA). For the WT and mutant alleles of the CAD gene, a sense primer specific for the WT (5′-AAAAGAACAGTCGGGACTGC-3′) or mutant allele (5′-GATTCGCAGCGCATCGCCTT-3′; a sequence in the neomycin-resistant gene) was used with a common antisense primer (5′-TTCACACCAGGAGACATCTG-3′). The WT and mutant alleles of the DNase II gene were detected by a similar method using the WT (5′-GCCCATCTAGACTAACTTTC-3′) or mutant (5′-GATTCGCAGCGCATCGCCTT-3′) specific sense primer and the common antisense primer (5′-GAGTCTTAGTCCTTTGCTCCG-3′). For Southern hybridization, genomic DNA was digested with XbaI, separated by electrophoresis on a 0.7% agarose gel and transferred to a Hybond N+ membrane (Amersham Biosciences). Hybridization was carried out with a 0.6-kb DNA fragment that is located 5.0-kb downstream of the CAD gene. For northern hybridization, total RNA was prepared by the acid phenol-guanidine thiocyanate method and poly(A) RNA was selected using the QuickPrep mRNA purification kit (Amersham Bioscience). RNA was separated on a 1.5% agarose gel containing 2.2 M formaldehyde and transferred to a Hybond N+ membrane. Hybridization was done under high-stringency conditions as described53 using mouse CAD cDNA14 as probe. Real-time PCR. Total RNA (2–10 µg) from the thymus of E17.5 embryos was treated with RNase-free DNase I (Amplification Grade, Invitrogen), and reverse-transcribed in a total volume of 20 µl using Superscript II reverse-transcriptase (Invitrogen) with oligo(dT) as a primer. Aliquots of the reverse transcriptase products were amplified in the reaction mixture containing LightCycler-FastStart DNA Master SYBER Green I, 0.5 µM each primer and 3 mM MgCl2, using LightCycler (Roche Molecular Biochemicals) as described by the manufacturer. The primers were as follows: 5′-GCCTTGACACTCCTGGTACAAATGAG-3′ and 5′-CAGCACATTGGCAGAGGAAGACAG-3′ for IFN-α, 5′-CCACCACAGCCCTCTCCATCAACTAT3′ and 5′-CAAGTGGAGAGCAGTTGAGGACATC-3′ for IFN-β; _5′-CTTTGCAGCTCTTCCTCATGGCTGTTTCTG-3′ and 5′-TGACGCTTATGTTGTTGCTGATGGCCTG-3′ for IFNγ; 5′-CACAGAAAGCATGATCCGCGACGT-3′ and 5′-CGGCAGAGAGAGGAGGTTGAC TTTCT-3′ for TNF-α. As a control, expression of the β-actin gene was detected by primers 5′-TGTGATGGTGGGAATGGGTCAG-3′ and 5′-TTTGATGTCACGCACGATTTCC-3′. The amount of specific mRNA was quantified at the point where the LightCycler System detected the upstroke of the exponential phase of PCR accumulation, and normalized to β-actin expression in each individual sample. Assay for CAD, apoptotic DNA fragmentation and pulse-field electrophoresis. Thymocytes were prepared from the thymic lobes of 4-week-old mice by pressing them between glass slides.

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To assay for CAD activity, plasmid DNA or mouse liver nuclei was incubated with the cell extracts prepared from the thymocytes in the presence of human caspase 3 as described14. For DNA fragmentation during apoptosis, thymocytes were treated at 37 °C with 10 µM dexamethasone in DMEM medium containing 10% FCS as described20. After incubation, cells were collected, and the chromosomal DNA was analyzed by electrophoresis on a 1.5% agarose gel as described14. For pulse-field electrophoresis, cells (1 × 106 cells) were suspended in 50 µl ice-cold buffer (10 mM Tris-HCl, pH 8.0, 100 mM EDTA and 20 mM NaCl) and mixed with an equal volume of 1.6% low-melting point agarose (Amersham Bioscience). Agarose blocks were incubated at 50 °C for 48 h with 1 mg/ml proteinase K and 1% sarkosyl, and the reaction was stopped by incubating at room temperature for 1 h in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA. The blocks were then inserted into wells of a 1% agarose gel in 0.5 × TBE, and the electrophoresis was carried out with the CHEF Mapper (Bio-Rad Laboratories, Hercules, CA) according to the instructions provided by the supplier. In vitro phagocytosis. The in vitro phagocytosis assay was carried out essentially as described24. In brief, fetal thymic macrophages (3 × 104 cells) were cultured in α-MEM containing 10% FCS supplemented with a one-tenth volume of CMG14-12 cell supernatant on 8-well Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) that had been coated with 0.1% gelatin. Thymocytes (1.5 × 106 cells) prepared from 4- to 8-week-old mice were treated at 37 °C for 5 h in DMEM containing 10% FCS with 10 µM dexamethasone to induce apoptotic cell death in 35–45% of the cells. The thymocytes were then added to macrophages on chamber slides and incubated for various periods of time. After unengulfed thymocytes were removed by vigorous washing, the macrophages were fixed overnight at 4 °C in 85% methanol, 4% formaldehyde and 5% acetic acid, treated for 30 min with 5N HCl at room temperature and stained at room temperature for 90 min in Schiff’s reagent (Merck, Darmstadt, Germany) for Feulgen staining. The cells were then observed by light microscopy. Histochemical and electron microscopic analysis of embryos. For histological analysis, thymus of E17.5 embryos was fixed with 4% paraformaldehyde in 0.1 M Na-phosphate buffer (pH 7.2) containing 4% sucrose, embedded in paraffin and sectioned at 4 µm. The sections were mounted with FluorSave mounting reagent (Calbiochem, San Diego, CA) containing 1 µg/ml of DAPI (Dojindo Laboratories, Kumamoto, Japan), and observed under a fluorescence microscope equipped with filter combination UV-2A (Nikon Optiphot, Nikon Co, Tokyo, Japan). For electron microscopy, thymus from E16.5 embryos was fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). After washing with the same buffer containing 7.5% sucrose, samples were post-fixed with 1% OsO4 at 4 °C for 2 h and embedded in Epon 812. Sections (80 nm) were prepared with an ultramicrotome (Reichert Ultracut N, Nissei, Tokyo, Japan), stained with lead citrate and uranyl acetate, and observed with a Hitachi H-7100 electron microscope (Hitachi High-Technologies Co.). FACS analysis. To prepare fetal thymocytes, thymus was removed from E17.5 embryos and disrupted by pressing the tissue through a nylon mesh. After incubation at 4 °C for 5 min with a rat Fcγ receptor II/III antibody, thymocytes were stained with various antibodies for 30 min at 4 °C in the staining solution (PBS containing 2% FCS and 0.05% NaN3), and analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using Cell Quest software (Quest Software Inc., Irvine, CA). The antibodies used for staining CD4 and CD8 were phycoerythrin (PE)-conjugated CD4 and fluorescein isothiocyanate (FITC)-conjugated CD8. For staining CD25 and CD44 in the CD4–CD8– population, thymocytes were first incubated with FITC-conjugated CD4 and CD8 antibodies, PE-conjugated CD25 antibody and biotin-conjugated CD44 antibody. After washing with the staining solution, cells were incubated at 4 °C for 30 min with Cy-Chrome-conjugated streptavidin. All antibodies and staining reagents were purchased from BD Pharmingen (San Diego, CA). Acknowledgments We thank M.Adachi for help in the initial stage of this study; K. Miwa for PCR;A. Kudo for CMG-12 cells; K. Ishihara for the advice on the fetal thymus organ culture; and S.Aoyama and M. Harayama for secretarial assistance.This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture in Japan. K.K. is supported by a research fellowship from the Japan Society for the Promotion of Science. Competing interests statement The authors declare that they have no competing financial interests. Received 8 October 2002; accepted 3 December 2002. 1. Kerr, J.F.,Wyllie,A.H. & Currie,A.R.Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 (1972). 2. Jacobson, M.D.,Weil, M. & Raff, M.C. Programmed cell death in animal development. Cell 88, 347–354 (1997). 3. Vaux, D.L. & Korsmeyer, S.J. Cell death in development. Cell 96, 245–254 (1999). 4. Raff, M. Cell suicide for beginners. Nature 396, 119–122 (1998). 5. Nagata, S.Apoptosis by death factor. 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