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Blood, 1 May 2004, Vol. 103, No. 9, pp. 3362-3364. Prepublished online as a Blood First Edition Paper on January 8, 2004; DOI 10.1182/blood-2003-09-3245. This Article Abstract Full Text (PDF) All Versions of this Article: 2003-09-3245v1 103/9/3362    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kunisaki, Y. Articles by Fukui, Y. Search for Related Content PubMed PubMed Citation Articles by Kunisaki, Y. Articles by Fukui, Y. Related Collections Hematopoiesis and Stem Cells Immunobiology Phagocytes Red Cells Apoptosis Brief Reports Related Article in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMATOPOIESIS Brief report Defective fetal liver erythropoiesis and T lymphopoiesis in mice lacking the phosphatidylserine receptor Yuya Kunisaki, Sadahiko Masuko, Mayuko Noda, Ayumi Inayoshi, Terukazu Sanui, Mine Harada, Takehiko Sasazuki, and Yoshinori Fukui From the Division of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; the Department of Anatomy and Physiology, Saga Medical School, Saga, Japan; Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; the International Medical Center of Japan, Tokyo, Japan; and PRESTO, Japan Science and Technology Agency, Saitama, Japan.    Abstract Top Abstract Introduction Study design Results and discussion References   Clearance of apoptotic cells by macrophages is considered important for prevention of inflammatory responses leading to tissue damage. The phosphatidylserine receptor (PSR), which specifically binds to phosphatidylserine (PS) exposed on the surface of apoptotic cells, mediates uptake of apoptotic cells in vitro, yet the physiologic relevance of PSR remains unknown. This issue was addressed by generating PSR-deficient (PSR-/-) mice. PSR-/- mice exhibited severe anemia and died during the perinatal period. In the PSR-/- fetal livers, erythroid differentiation was blocked at an early erythroblast stage. In addition, PSR-/- embryos exhibited thymus atrophy owing to a developmental defect of T-lymphoid cells. Clearance of apoptotic cells by macrophages was impaired in both liver and thymus of PSR-/- embryos. However, this did not induce up-regulation of inflammatory cytokines. These results indicate that during embryonic development, PSR-mediated apoptotic cell uptake is required for definitive erythropoiesis and T lymphopoiesis, independently of the prevention of inflammatory responses. (Blood. 2004;103:3362-3364)    Introduction Top Abstract Introduction Study design Results and discussion References   The phosphatidylserine receptor (PSR) has been identified as a molecule expressed on macrophages, fibroblasts, and epithelial cells that specifically binds to phosphatidylserine (PS) exposed on apoptotic cells.1 However, several molecules have been also implicated in the recognition and ingestion of apoptotic cells by macrophages. These include cell-surface molecules such as CD14, class A scavenger receptor, adenosine triphosphate binding cassette transporter 1, receptor tyrosine kinase Axl/Mer/Tyro3, and V3 integrin, which, in association with CD36, binds to thrombospondin recognized by an undefined ligand on apoptotic cells.2-7 In addition, 2 soluble molecules, growth arrest-specific gene 6 product and milk fat globule-endothelial growth factor-factor 8, have been reported to bind to PS.8,9 Therefore, although in vitro experiments clearly indicate that PSR is involved in anti-inflammatory clearance of cells undergoing apoptosis,1,10,11 the physiologic relevance of PSR remains unclear. To address this issue, we generated PSR-deficient mice by homologous recombination in embryonic stem (ES) cells.    Study design Top Abstract Introduction Study design Results and discussion References   Targeted disruption of the PSR gene The targeting vector was constructed to replace a 2.8-kilobase fragment from the EcoRI site located upstream of exon 1 to the EcoRI site within exon 3 with a neomycin resistance cassette. Correctly targeted ES clones were microinjected into C57BL/6 blastocysts, and chimeras obtained were crossed with C57BL/6 mice. Mice heterozygous for the mutant allele were crossed to obtain PSR-/- embryos. Northern blot analysis Poly(A) mRNA was prepared from mouse pups and hybridized with a cDNA probe. Blood analysis Hemoglobin (Hb) concentration and hematocrit (Ht) were determined with an automatic cell analyzer (Sysmex, Kobe, Japan). Blood smear samples were stained with Giemsa solution (Merck, Darmstadt, Germany). Flow cytometric analysis Cells were stained with the relevant monoclonal antibodies (PharMingen, San Diego, CA) and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Tissue staining Fresh frozen sections were stained with terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate-fluorescein nick end labeling (TUNEL) and then incubated with phycoerythrin-conjugated monoclonal antibody to F4/80 (Caltag Laboratories, Burlingame, CA). Reverse transcriptase (RT)-polymerase chain reaction (PCR) Total RNA samples treated with RNase-free DNase I (Invitrogen, Carlsbad, CA) were reverse transcribed using oligo(dT) and subjected to PCR with specific primers.    Results and discussion Top Abstract Introduction Study design Results and discussion References   Although PSR+/- mice were healthy and fertile, no PSR-/- mice were found among the progeny of the PSR+/- intercrosses when analyzed at 4 weeks after birth. At various embryonic stages, homozygous mutant mice were present at a frequency roughly consistent with Mendelian inheritance. Close examination at parturition revealed that some PSR-/- pups were born alive, but died within 24 hours. Lack of PSR expression in mutant mice was confirmed by Northern blot analysis using whole neonates (Figure 1A). View larger version (29K): [in this window] [in a new window]   Figure 1.. Developmental defects of erythroid and T-lymphoid cells in PSR-/- embryos. (A) PolyAmRNA(2 µg) prepared from PSR+/- or PSR-/- neonates was hybridized with PSR or control (TGF-3) cDNA. (B) Phenotypic comparison of PSR+/- (left) and PSR-/- (right) littermates at E15.5. Original magnification x 8. (C) Hb concentration and Ht of PSR+/+ (, n = 6), PSR+/- (, n = 12), and PSR-/- (, n = 3) embryos are compared at E16.5. (D) Blood smears of PSR+/- (left) and PSR-/- (right) littermates are compared at E16.5. Original magnification x 1000. (E) The number of E13.5 fetal liver cells (left) or E18.5 thymocytes (right) is compared among PSR+/+ (, n = 9 for fetal liver cells and n = 7 for thymocytes), PSR+/- (, n = 21 for fetal liver cells and n = 9 for thymocytes), and PSR-/- (, n = 6 for fetal liver cells and n = 3 for thymocytes) embryos. (F) The E13.5 fetal liver cells of PSR+/- and PSR-/- littermates were stained for Ter119 and c-kit. Numbers in each quadrant indicate the percentage of each subset of the cells. (G) The number of Ter119+c-kit- (left) or Ter11+c-kit+ (right) fetal liver cells is compared among PSR+/+ (, n = 4), PSR+/- (, n = 9), and PSR-/- (, n = 4) embryos at E13.5. (H) Thymus sections of E19.0 PSR+/- and PSR-/- littermates stained with hematoxylin and eosin. Original magnification x 250. (I) Thymocytes of E18.5 PSR+/- and PSR-/- littermates were stained for CD4 and CD8. Numbers in each quadrant indicate the percentage of each subset of the cells.   Although PSR-/- embryos were apparently normal until embryonic day 12.5 (E12.5), the mutant embryos at E15.5 were pale and small, compared with PSR+/- littermates (Figure 1B). Therefore, we first analyzed Hb concentration and Ht of peripheral blood at E16.5. Although no difference was found between PSR+/+ and PSR+/- embryos, both Hb concentration and Ht were remarkably reduced in PSR-/- embryos (Figure 1C). In peripheral blood obtained from PSR+/- embryos, enucleated erythrocytes characteristic of definitive erythropoiesis were the preponderant cell types found (Figure 1D). In contrast, peripheral blood from PSR-/- embryos contained predominantly larger, nucleated erythrocytes derived from the yolk sac (Figure 1D). Definitive erythropoiesis in the liver starts around E11.5 to E12.5. At E13.5, the total number of fetal liver cells in PSR-/- embryos decreased to less than 20% of the wild-type levels (Figure 1E). While the proportion of Ter119+c-kit- cells significantly decreased in PSR-/- embryos, PSR-/- fetal livers showed a 4-fold increase in the proportion of Ter119+c-kit+ cells, compared with that of PSR+/- embryos (Figure 1F). Hence, although the number of Ter119+c-kit- erythroid cells was remarkably reduced in PSR-/- fetal livers, the number of Ter119+c-kit+ immature erythroid cells was almost comparable between PSR-/- and PSR+/+ or PSR+/- embryos (Figure 1G). All erythroid cells after the colony-forming unit-erythroid (CFU-E) stage become Ter119-positive,12 whereas c-kit is expressed on the cell surface up to the basophilic erythroblast stage.13 Therefore, it is suggested that in PSR-/- fetal livers, erythroid differentiation is blocked at an early erythroblast stage, most likely the proerythroblast or basophilic erythroblast stage. Another feature of PSR-/- embryos was impaired thymic development. Histologic analysis revealed that both the cortex and the medulla were poorly developed in the PSR-/- thymus (Figure 1H). At E18.5, the total number of thymocytes in PSR-/- embryos was reduced to less than 20% of the wild-type levels (Figure 1E). While more than 60% of the PSR+/- thymocytes were CD4+CD8+ thymocytes, this population was reduced to less than 15% in the PSR-/- thymus (Figure 1I), indicating that thymocyte differentiation is blocked at the CD4-CD8- stage in the PSR-/- thymus. We then examined whether clearance of apoptotic cells was impaired in PSR-/- embryos. While most TUNEL-positive cells colocalized with F4/80+ macrophages in E13.5 fetal liver sections of PSR+/- embryos (Figure 2A), PSR-/- fetal livers exhibited a 3-fold increase in the frequency of unphagocytosed TUNEL-positive cells (Figure 2A-B). Similar results were obtained when E16.5 thymus sections were analyzed (Figure 2E-F). The number of F4/80+ macrophages was reduced in both liver and thymus of PSR-/- embryos (Figure 2C,G). However, it is unlikely that this caused the increased frequency of remnant apoptotic cells, because the number of TUNEL-positive cells was also reduced in the PSR-/- tissues (Figure 2D,H). Therefore, it is suggested that clearance of apoptotic cells by macrophages is impaired in the absence of PSR. View larger version (41K): [in this window] [in a new window]   Figure 2.. Defective apoptotic cell uptake by PSR-deficient macrophages without induction of inflammatory cytokines. (A,E) The E13.5 liver sections (A) and E16.5 thymus sections (E) of PSR+/- and PSR-/- littermates were stained with TUNEL (green) and anti-F4/80 antibody (red). Typical examples of phagocytosed or unphagocytosed apoptotic cells are shown by the arrows or arrowheads, respectively. The white bar indicates 100 µm. (B,F) The percentage of unphagocy tosed apoptotic cells in E13.5 liver sections (B) or E16.5 thymus sections (F) is compared between PSR+/- (, n = 640 for liver sections and n = 106 for thymus sections) and PSR-/- (, n = 176 for liver sections and n = 44 for thymus sections) littermates. (C,G) The number of F4/80+ macro phages per 100 000 µm2 of E13.5 liver sections (C) or E16.5 thymus sections (G) is compared between PSR+/- () and PSR-/- () littermates. F4/80+ cells with more than 60 µm2 of surface area were counted. (D,H) The total number of TUNEL-positive cells per 100 000 µm2 of E13.5 liver sections (D) or E16.5 thymus sections (H) is compared between PSR+/- () and PSR-/- () littermates. (I) The E18.5 thymuses of PSR+/- and PSR-/- littermates were analyzed for the expression of TNF-, IFN-, IFN-, and TGF- by RT-PCR. The primers used to amplify IFN- were described elsewhere.14 For amplification of TNF-, IFN-, and TGF-, sets of specific primers were obtained from Toyobo (Osaka, Japan). The gene encoding hypoxanthine phosphoribosyl transferase (HPRT) used as a control was amplified with the following primers: 5'-CACAGGACTAGAACACCTGC-3' and 5'-GCTGGTGAAAAGGACCTCT-3'. Amplification increases by 3 cycles, from the left to the right, starting at 22 cycles for TGF- and HPRT, or at 25 cycles for TNF-, IFN-,and IFN-. Error bars (B-D, F-H) indicate standard deviation.   Semiquantitative RT-PCR analysis revealed that inflammatory cytokines, tumor necrosis factor (TNF-), interferon (IFN-), and IFN-, as well as transforming growth factor (TGF-) were comparably expressed in PSR+/- and PSR-/- thymuses (Figure 2I). In the fetal liver, the expression of TNF- and TGF- was comparable between PSR+/- and PSR-/- embryos, but no expression was detected for IFN- and IFN- in both embryos (data not shown). It is thus clear that the developmental defects of erythroid and T-lymphoid cells in PSR-/- embryos do not result from inflammatory responses. The lack of inflammatory responses in the PSR-/- fetal liver and thymus might reflect the fact that the absolute number of remnant apoptotic cells in these tissues was unchanged between PSR+/- and PSR-/- embryos (Figure 2B,D,F,H). We have shown that PSR deficiency causes severe developmental defects of erythroid and T-lymphoid cells in mouse embryos. These phenotypes are similar to those of mice lacking DNase II, a lysosomal DNase in macrophages,14-16 suggesting that PSR-mediated uptake of apoptotic cells is a major pathway leading to DNA degradation by DNase II in macrophages. At this stage, we do not know the precise mechanism by which PSR regulates erythroid and T-lymphoid differentiation. However, we found that PSR deficiency causes repression of apoptosis in several tissues including fetal liver and thymus, but not in intestine whose epithelial cells are removed independently of phagocytosis (data not shown). This raises the possibility that PSR-mediated uptake of apoptotic cells regulates the cell-death machinery in developing cells through a feedback mechanism between phagocytes and developing cells, as described in Caenorhabditis elegans.17,18 This feedback mechanism may play an important role in terminal differentiation of erythroid and T-lymphoid cells, in which apoptosis-related signaling is required.19-23 Note added in proof. After submission of this manuscript, Li et al24 reported that PSR is required for normal development of lung and brain.    Acknowledgements   We thank Dr Lurie Erickson for comments on this manuscript.    Footnotes   Submitted September 24, 2003; accepted December 31, 2003. Prepublished online as Blood First Edition Paper, January 8, 2004; DOI 10.1182/blood-2003-09-3245. Supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Japan Science and Technology Agency. An Inside Blood analysis of this article appears in the front of this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Yoshinori Fukui, Division of Immunogenetics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; e-mail: fukui{at}bioreg.kyushu-u.ac.jp.    References Top Abstract Introduction Study design Results and discussion References   Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RAB, Henson PM. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: 2003-09-3245v1 103/9/3362    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kunisaki, Y. Articles by Fukui, Y. Search for Related Content PubMed PubMed Citation Articles by Kunisaki, Y. Articles by Fukui, Y. Related Collections Hematopoiesis and Stem Cells Immunobiology Phagocytes Red Cells Apoptosis Brief Reports Related Article in Blood Online Social Bookmarking                   What's this?

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