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Osaka University 100 Papers : 10 Selected Papers

ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2005-2006

Biology

Phosphatidylserine-Dependent Engulfment by Macrophages of Nuclei from Erythroid Precursor CellsPaper in journals : This is the first page of a paper published in Nature.[Nature] 437, 754-758 (2005)

Reprinted with permission from Nature (437, 754-758, 2005). Copyright: Nature Publishing Group

40 ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2005-2006

Phosphatidylserine-Dependent Engulfment by Macrophages of Nuclei from Erythroid Precursor CellsYOSHIDA Hideyuki and NAGATA Shigekazu(Graduate School of Frontier Biosciences)

Abstract

Definitive erythropoiesis occurs in the bone marrow or fetal liver, where erythroblasts are associated with a cen-

tral macrophage in units called “erythroblastic islands” (1). Late in erythropoiesis, nuclei are expelled from the eryth-roid precursors and engulfed by macrophages in the islands (2). To investigate the mechanism behind the enucleation, we isolated erythroblasts from the spleens of phlebotomized mice. When these erythroblasts were cultured, nuclei spontane-ously protruded from erythroblasts. A weak physical force could disconnect the nuclei from reticulocytes. The released nuclei contained an undetectable level of ATP, and quickly exposed phosphatidylserine (PS) on their surface. Fetal liver macrophages efficiently engulfed the nuclei; masking PS on the nuclei prevented this engulfment. These results indicated that the nuclei are engulfed by macrophages only after they are disconnected from reticulocytes, and PS, which is often used as an “eat me” signal for apoptotic cells, is also used for the engulfment of nuclei expelled from erythroblasts.

Enucleation in erythroid precursor cellsPhlebotomy promotes the re-development of erythroid cells

in mouse spleen. This system was used to obtain a large number of erythroblasts at a synchronized developmental stage. When erythroblasts prepared from spleens 4 days after phlebotomy were cultured at 37°C, some cells started blebbing, and nucleus moved to one side of the cell. After 5 h, about 12% of the erythroblasts possessed an eccentric nucleus that could be dis-tinguished from irregularly shaped reticulocytes. Nucleus was surrounded by an intact plasma membrane, and was connected to the reticulocytes via a thin pseudopodium-like membrane extension (Fig. 1). This could be disrupted by weak physical stress. The production of reticulocytes from the erythroblasts was inhibited by cytochalasin B, indicating that the actin polymerization is involved in this process. Erythropoietin is indispensable for definitive erythropoiesis. But, it had no effect on the enucleation process.

Exposure of phosphatidylserine on nuclei from erythroid precursor

Apoptotic cells expose PS on their surface as a signal for macrophages to engulf them (3). As shown in Fig. 2, nucleated erythroblasts and reticulocytes were not recognized by Annexin V that binds to PS. Nuclei were Annexin V−. However, when the cultures were subjected to weak physical stress and then incubated further, the nuclei became Annexin V+. The plasma membrane surrounding the nuclei remained intact after the physical stress. PS is normally confined to the inner leaflet of plasma membrane (4). This asymmetry is maintained by a com-bination of an ATP-dependent aminophospholipid translocase

Fig. 1. Enucleation of erythroblasts. Erythroblasts from phlebotomized mice were incubated for 3 h, and observed by Electron microscopy. Boxed areas in left panels are enlarged in right panels. Bar, 1 µm.

Fig. 2. Exposure of phosphatidylserine on nuclei released from erthroblasts. a, Erythroblasts from phlebotomized spleens were cultured for 3 h, pipetted in and out 5 times, and further incubated for the indicated periods. The Annexin V-staining profiles for nuclei, reticulocytes, erythroblasts, and dead cells are shown.

The following is a comment on the published paper shown on the preceding page.

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Osaka University 100 Papers : 10 Selected Papers

ANNUAL REPORT OF OSAKA UNIVERSITY—Academic Achievement—2005-2006

that transports PS from the outer to inner leaflet of the plasma membrane, and a Ca2+-dependent but energy-independent scamblase that randomizes lipids across the bilayer. As shown in Fig. 3, the ATP level was reduced to the undetectable level in the nuclei expelled from reticulocytes, while the Ca2+ level was much higher in the nuclei than in the reticulocytes.

References

1. Bessis M, Mize C, Prenant M. Erythropoiesis: comparison of in vivo and in vitro amplification. Blood Cells, 4, 155-174 (1978).

2. Kawane K, Fukuyama H, Kondoh G, Takeda J, Ohsawa Y, Uchiyama Y, Nagata S. Requirement of DNase II for definitive erythropoiesis in the mouse fetal liver. Science, 292, 1546-1549 (2001).

3. Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM. The role of phosphatidylserine in recognition of apoptotic cells by phagocytes. Cell Death & Differ., 5, 551-562 (1998).

4. Balasubramanian K, Schroit AJ. Aminophospholipid asymmetry: A mat-ter of life and death. Annu. Rev. Physiol., 65, 701-734 (2003).

5. Kawane K, Fukuyama H, Yoshida H, Nagase H, Ohsawa Y, Uchiyama Y, Iida T, Okada K, Nagata S. Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation. Nat. Immunol., 4, 138-144 (2003).

6. Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S. Identification of a factor that links apoptotic cells to phagocytes. Nature, 417, 182-187 (2002).

7. Strehler EE, Treiman M. Calcium pumps of plasma membrane and cell interior. Curr Mol Med, 4, 323-335 (2004).

8. Hanayama R, Tanaka M, Miyasaka K, Aozasa K, Koike M, Uchiyama Y, Nagata S. Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice. Science, 304, 1147-1150 (2004).

Fig. 4. Phosphatidylserine-dependent engulfment of nuclei. a, Fetal liver mac-rophages were co-cultured with nuclei in the absence or presence of D89E. The cells were then stained with Wright-Giemsa. Bar, 50 µm. b, Engulfment of nuclei by fetal liver macrophages was carried out in the presence of the indicated concentration of D89E.

Phosphatidylserine-dependent engulfment of nuclei by macrophages

Nuclei released from erythroblasts were isolated by a cell sorter and used as prey for macrophages from DNase II−/− embryos (5). Macrophages efficiently engulfed nuclei, and the phagocytosis index (the number of engulfed nuclei per macro-phage) was 2.4 after a 2-h incubation (Fig. 4). MFG-E8 binds PS via its Factor VIII-homologous domains in the C-terminal half, and macrophages via its RGD motif at the N-terminus (6). Thus, a point mutant (D89E) of MFG-E8 in the RGD motif binds PS exposed on apoptotic cells, and inhibits their engulf-ment. Similarly, the D89E mutant of MFG-E8 dose-depend-ently inhibited the engulfment of nuclei by macrophages. These results indicated that PS exposed on the expelled nuclei was required for their engulfment by macrophages.

DiscussionWe showed that nucleus spontaneously protruded from eryth-

roblasts, remaining connected via a thin membrane extension. Exposure of such erythroblasts carrying protruded nuclei to shear stress at the blood island may be sufficient to physi-

cally release reticulocytes from nuclei and release them into the bloodstream. The extruded nuclei contained a low level of ATP, because of no ATP supply in the nuclei. In return, Ca2+ concentra-tion increased in the nuclei due to the inactivation of the plasma membrane Ca2+ ATPase (PMCA) that pumps Ca2+ out of the cells in an ATP-dependent manner (7). Thus, aminophospholipid

translocase is inactivated, while scramblase is activated, causing the exposure of PS to the surface of nuclei. Several molecules expressed in macrophages have been proposed to bind PS for engulfment of apoptotic cells (6). Whether these molecules are also involved in engulfment of the expelled nuclei remains to be studied. We recently reported that inefficient engulfment of apoptotic cells causes autoimmunity in mice, by inducing the production of anti-nuclear and anti-double stranded DNA antibodies (8). Adult human produces 2 × 1011 erythrocytes per day, suggesting that blockage of the engulfment of nuclei may also contribute to the development of autoimmunity.

Fig. 3. Loss of ATP in nuclei, and Ca influx. a. The ATP level for erythrob-lasts (EB), reticulocytes (RE) and Nuclei (N) was measured using an ATP Bioluminescence Assay. b, Influx of Ca into nuclei. Erythroblasts were loaded with Fura-2. The bar at right shows the reference image for F340/F380. High F340/F380 ratios reflect high intracellular Ca2+ concentrations. In left panel, cells were stained for nuclei (red).