Mice lacking asparaginyl endopeptidase developdisorders resembling hemophagocytic syndromeChi-Bun Chana,1, Michiyo Abeb,1, Noriyoshi Hashimotob, Chunhai Haoa, Ifor R. Williamsa, Xia Liua, Shinji Nakaoc,Akitsugu Yamamotod, Chengyun Zhenge,f, Jan-Inge Hentere, Marie Meethse,f, Magnus Nordenskjoldf, Shi-Yong Lia,Ikuko Hara-Nishimurag, Masahide Asanob,2, and Keqiang Yea,2

aDepartment of Pathology and Laboratory Medicine, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322; bDivision of TransgenicAnimal Science, Advanced Science Research Center, and cDepartment of Cellular Transplantation Biology, Graduate School of Medical Science, KanazawaUniversity, 13-1 Takara-machi, Kanazawa 920-8640, Japan; dDepartment of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266Tamura-machi, Nagahama 526-0829, Japan; gDepartment of Botany, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku,Kyoto 606-8502, Japan; eDepartment of Woman and Child Health, Childhood Cancer Research Unit, and fDepartment of Molecular Medicineand Surgery, Clinical Genetics Unit, Karolinska Institute, Karolinska University Hospital, SE-141 86 Stockholm, Sweden

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved November 10, 2008 (received for reviewOctober 1, 2008)

Asparaginyl endopeptidase (AEP or legumain) is a lysosomal cys-teine protease that cleaves protein substrates on the C-terminalside of asparagine. AEP plays a pivotal role in the endosome/lysosomal degradation system and is implicated in antigen pro-cessing. The processing of the lysosomal proteases cathepsins inkidney is completely defective in AEP-deficient mice with accumu-lation of macromolecules in the lysosomes, which is typically seenin lysosomal disorders. Here we show that mutant mice lacking AEPdevelop fever, cytopenia, hepatosplenomegaly, and hemophago-cytosis, which are primary pathological manifestations of hemoph-agocytic syndrome/hemophagocytic lymphohistiocytosis (HLH).Moreover, AEP deficiency provokes extramedullary hematopoiesisin the spleen and abnormally enlarged histiocytes with ingestedred blood cells (RBCs) in bone marrow. Interestingly, RBCs fromAEP-null mice are defective in plasma membrane components.Further, AEP-null mice display lower natural killer cell activity, butnone of the major cytokines is substantially abnormal. Theseresults indicate that AEP might be a previously unrecognizedcomponent in HLH pathophysiology.

hematopoiesis � macrophage � legumain � lysosomal disorder

Hemophagocytic syndrome (hemophagocytic lymphohistio-cytosis, HLH) is a life-threatening condition caused by

hyperinflammatory response. It can be classified into familialand acquired forms (1). The primary symptoms of HLH includeprolonged fever, cytopenia, hepatosplenomegaly, and hemoph-agocytosis by activated macrophages (2). Whereas acquiredHLH is induced by a variety of infectious organisms, such asviruses, bacteria, protozoa, or fungi (1), genetic HLH is adisorder inherited in an autosomal recessive or X-linked manner.Several loci have been implicated in the pathophysiology ofgenetic HLH, including PRF1 (3), UNC13D (4), RAB27A (5),LYST (6), and SH2D1A (7, 8). Most of these known defectivegenes associated with HLH are involved in the cytotoxic-granuleexocytosis pathway of T lymphocytes, suggesting the critical roleof the lysosomal granule in maintaining normal cellular immu-nity. For example, mutation of UNC13D in human impairs therelease of granzyme and perforin from cytolytic granules at theimmunological synapse (4). Nevertheless, up to 70% of familialHLH is caused by unknown genetic defects. The identification ofnew genes that are involved in the development of HLH wouldthus be beneficial to our understanding of the disease patho-physiology and our current therapeutic regimen to treat HLH.

Asparagyl endopeptidase (AEP or legumain) is a lysosomalcysteine protease that cleaves protein substrates on the C-terminal side of asparagine (7, 8). AEP is highly expressed inkidney and localizes in the late endosomes and lysosomes of thekidney-proximal tubule cells. Disruption of AEP leads to lateendosomes and lysosomes augmentation and dislocation from

the apical region of the kidney-proximal tubule cells and theabnormal lysosomes contained in electron-dense and/or mem-branous materials (9, 10). AEP activation is autocatalytic andrequires sequential removal of C- and N-terminal propeptides atdifferent pH thresholds. Recently, we have reported that neu-ronal AEP is involved in neuronal apoptosis by degrading theDNase inhibitor SET during excitoneurotoxicity (11). AEP alsoparticipates in antigen presentation, the activity of which isinversely proportional to myelin basic protein (MBP) epitopepresentation (9, 10).

In this report, we show that AEP-knockout mice developclassical syndromes resembling HLH with extramedullary he-matopoiesis in spleen and abnormally enlarged histiocytes withingested RBCs in bone marrow. Moreover, higher body temper-ature and reduced natural killer cell activity are observed inAEP-null mice. These results indicate that AEP might be apreviously unrecognized component in HLH pathology.

ResultsAEP-null mice are fertile and viable with no overt behavioralabnormality, although their body weights are reduced comparedwith wild-type littermates (9, 10). We observed a significantenhanced body temperature in AEP-null mice compared withtheir control littermates (Fig. 1A). To clarify the cause of fever,we determined various inflammatory cytokine levels in theserum. TNF-� levels were increased in some of AEP�/� mice,suggesting that the fever is in part caused by increased TNF-�levels. However, most of AEP�/� mice showed normal inflam-matory cytokine levels (Fig. S1). A complete blood countrevealed that AEP-null mice, but not the control littermates, hadmarked low concentration of RBCs and hemoglobin, suggestiveof severe anemia (Table 1). We also compared the peripheralblood parameters from different ages of AEP �/� and �/� miceand found that the hematocrit values from AEP-null mice weregradually decreased in an age-dependent manner (Fig. 1B). Onthe other hand, the reticulocyte percentage progressively in-

Author contributions: C.-B.C., M. Abe, N.H., C.H., I.R.W., C.Z., J.-I.H., M. Asano, and K.Y.designed research; C.-B.C., M. Abe, N.H., C.H., I.R.W., X.L., S.N., A.Y., and M.M. performedresearch; I.H.-N. contributed new reagents/analytic tools; C.-B.C., M. Abe, N.H., C.H., I.R.W.,C.Z., M.M., M.N., S.-Y.L., M. Asano, and K.Y. analyzed data; and C.-B.C., M. Asano, and K.Y.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1C.-B.C. and M. Abe contributed equally to this work.

2To whom correspondence may be addressed. E-mail: asano@kiea.m.kanazawa-u.ac.jp orkye@emory.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809824105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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creased in AEP�/� mice compared with control mice, fittingwith the observation of anemia in AEP-deficient mice (Fig. 1C).In contrast, the total number of white blood cells (WBCs)increases as the AEP-knockout mice get older, which is causedpredominantly by leukocytosis (Table 2). Erythropoietin (Epo),which is produced by the kidney in the adult and by the liver inthe fetus, increases RBCs through supporting the survival oferythroid progenitor cells and stimulating their differentiationand proliferation via binding to Epo receptors (EpoRs) presenton the surface of immature erythroid cells (12). Defects in Epoproduction lead to severe anemia caused by the absence ofcirculating RBCs. To investigate whether anemia in AEP-

deficient mice is caused by a lack of Epo production, wedetermined the concentrations of Epo in the blood. Surprisingly,Epo production was markedly increased in an age-dependentmanner in both types of mice, but the circulating Epo wassignificantly higher in AEP-lacking mice than in wild-type mice(Fig. 1D). Concomitantly, EpoR was dramatically increased inbone marrow from AEP�/� mice compared with wild-typemice, fitting with the Epo increase in blood (Fig. 1E). Presum-ably, the elevated Epo and EpoR might compensate for the RBCloss in the AEP-lacking mice.

Next, we monitored the weight of different organs from theage-matched wild-type and knockout mice and found that thespleen was substantially larger in AEP�/� mice than in controlmice. The liver was also significantly enlarged in AEP�/� miceas compared with the normal counterparts, whereas otherorgans were comparable in size and weight. On average, thespleens from AEP-null mice were 5–10 times larger than thoseof wild-type controls (Fig. 2A). The difference in organ size wasage dependent. As AEP�/� mice grew older, the spleen grewmuch bigger than that of the control mice (Fig. 2B). Noticeably,the color of the spleen and liver from AEP-null mice was muchdarker than the wild-type control (Fig. S2), suggestive of anincrease in the RBCs in the organs. To test this possibility, weexamined the histology of the spleen and found a significantincrease in extramedullary hematopoiesis in the AEP�/� spleenas evident in an increase of morphologically normal megakaryo-cytes (Fig. 2C). Moreover, a substantial number of erythrocytes(Fig. 2D) and immature myeloid lineage cells (Fig. 2E) wereobserved in the AEP�/� spleen, further supporting that ex-tramedullary hematopoiesis was in progress. These findingsindicate the extramedullary hematopoiesis might be the cause ofsplenomegaly. However, the liver histology appeared normal(data not shown).

To confirm the hematopoiesis, the spleen cells from AEP-nullmice were subjected to hypotonic lysis of mature erythrocytes

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Fig. 1. Higher body temperature and anemia in AEP-deficient mice. (A) Bodytemperature of AEP-knockout (filled circles) and control heterozygous (opencircles) mice at 12 months of age. ���, P � 0.005, Student’s t test, �/�, n � 4,�/�, n � 7. Body core temperature was continuously recorded on a potenti-ometer by a copper–constantan thermocouple covered with polyethylenetubing (outside diameter 1 mm). (B) Age-dependent decrease of circulatinghematocrit was seen in AEP-null (filled circles) animals but not in the het-erozygous control mice (open circles). �, P � 0.05, Student’s t test, 3-month-old: �/�, n � 6, �/�, n � 6; 6-month-old: �/�, n � 7, �/�, n � 6; 15-month-old:�/�, n � 2, �/�, n � 4. (C) Age-dependent increase of circulating reticulocyteswas seen in AEP-null (filled circles) animals but not in the heterozygous controlmice (open circles) �, P � 0.05; ��, P � 0.01, Student’s t test). Data wereobtained from the same mice examined in B. (D) Circulating Epo from wild-type (filled bars) and AEP-knockout (open bars) were determined by ELISA. Aprogressive increase of serum Epo was recorded in AEP-null animals withincreased age. �, P � 0.05, Student’s t test, n � 6. (E) Bone marrow sectionsfrom femur of wild-type (�/�) and AEP-knockout (�/�) animals were stainedwith EpoR-specific antibody. IHC, immunohistochemistry. Enhanced EpoRexpression was detected in AEP-knockout mice. (Scale bar, 20 �m.)

Table 1. Complete blood count of AEP-deficient mice at the ageof 6 months

Parameter �/� (n � 8) �/� (n � 8)

Body weight, g 30.9 � 3.1 28.2 � 3.7Spleen weight, mg 153 � 45 330 � 142†

RBCs, 104/�L 971 � 50 798 � 120‡

Hemoglobin, g/dL 13.8 � 0.6 10.9 � 1.6‡

Hematocrit, % 47.2 � 1.8 38.2 � 5.1‡

Mean corpuscular volume, fL 48.6 � 1.2 48.0 � 1.0Mean corpuscular hemoglobin, pg 14.2 � 0.4 13.7 � 0.3*Mean corpuscular hemoglobin conc., g/dL 29.2 � 0.4 28.7 � 0.5*Total WBCs, 102/�L 36.5 � 13.8 36.1 � 9.7

Lymphocytes, 102/�L 23.6 � 13.2 21.3 � 5.6Monocytes, 102/�L 6.0 � 2.2 9.1 � 5.3Neutrophils, 102/�L 6.6 � 5.3 5.5 � 1.8

Platelets, 104/�L 79.8 � 16.5 58.2 � 33.4

Data are expressed as mean � SD. *, P � 0.05; †, P � 0.01; ‡, P � 0.005;Student’s t test, n � 8.

Table 2. WBC contents of AEP-deficient mice at the ageof 12 months

Parameter �/� (n � 5) �/� (n � 5)

Total WBCs, 102/�L 40.0 � 11.4 115 � 36*Lymphocytes, 102/�L 20.6 � 8.3 69.4 � 26.4*Monocytes, 102/�L 4.4 � 2.9 20.3 � 20.8Neutrophils, 102/�L 15.1 � 6.9 25.0 � 19.5

Data are expressed as mean � SD. *, P � 0.05, Student’s t test, n � 5.

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and analyzed by flow cytometric immunophenotyping for nu-cleated hematopoiesis precursor cells. As the weight of thespleen from AEP-null mice increased, the ratio of CD3 and B220double-negative cells was markedly elevated (Fig. 3A), indicatingthat non-T cell and non-B cell populations were augmented assplenomegaly was getting worse. To identify the non-T cell andnon-B cell populations, FACS analysis was further performed.The percentage of Ter-119�/CD45� erythroid lineage cells inthe spleen was dramatically increased in the AEP-null mice (e.g.,18.2% in a representative AEP-null mouse compared with just0.4% in a wild-type control) (Fig. 3B). Myeloid cells identifiedby Gr-1 and Mac-1 staining were also increased in the AEP-nullmice (e.g., 10.2% in AEP-null mice versus 1.2% in wild-type)(Fig. 3C). The presence of a large number of erythroid lineageTer-119� cells in the spleens of AEP-null mice confirms thatextramedullary hematopoiesis was taking place.

Compared with the wild-type mice, the bone marrow in AEP-nullmice was infiltrated with many enlarged histiocytes that displayeda small and eccentrically placed nucleus and a cytoplasm withcharacteristic crinkles or striations, resembling Gaucher cells inGaucher disease, a congenital storage disease due to lysosomalenzyme defect. We made the same observation in the spleen ofAEP-null mice (Fig. 4A). Numerous histiocytes revealed ingestedRBCs on one side and striated cytoplasm on the other side,suggesting accumulation of lipids from the digested RBCs. He-mophagocytes were observed in the bone marrow of AEP�/� mice

at 3 months of age and they increased remarkably with age (Fig.S3A). These Gaucher-like cells showed strong autofluorescence aswell as engulfed erythrocytes (Fig. S3D). Periodic acid/Schiffreagent (PAS) staining further supported that these were Gaucher-like cells (Fig. S3E). Hemophagocytes observed in the bone marrowof AEP�/� mice were further examined by Cytospin. Thesehemophagocytes were both F4/80 and CD68-positive histiocytes(Fig. S3F), confirming their identities as macrophages. Electronmicroscopic examination of bone marrow showed that the aberranthistiocytes engorged numerous RBCs, many of which were appar-ently intact, but some of which were partially or completelydegraded (Fig. 4B).

Fluorescent microsphere phagocytic assay with the peritonealmacrophages demonstrated that AEP depletion evidently en-hanced the phagocytic activity, suggesting that phagocytic activityof macrophages (histiocytes) in AEP�/� mice was elevated (Fig.4C). To further explore whether the hemophagocytic activity inAEP�/� mice results from augmented RBC engulfing activity bymacrophages, we isolated RBCs and peritoneal macrophages fromAEP�/� and �/� mice. At 2 months of age, the macrophages fromboth mice engulfed opsonized RBCs with comparable activities(data not shown). By contrast, at 12 months of age, the macro-phages from AEP�/� mice engulfed more opsonized RBCs, butnot unopsonized RBCs, than those from AEP�/� mice, no matterwhether the RBCs were isolated from AEP�/� or �/� mice,confirming that phagocytic activity of macrophages from AEP�/�mice was elevated (Fig. 4D). To examine whether unopsonizedRBCs are engulfed by macrophages, macrophages and RBCs wereincubated for 24 h instead of 1 h. Phagocytic activity of macro-phages from AEP�/� mice against unopsonized RBCs was ele-vated compared with macrophages from AEP�/� mice, regardlessof the genotype of RBCs, although the phagocytic index was verylow (Fig. S4A). In contrast to the enhanced macrophage activity,the natural killer cell activity in the AEP-null animal is significantlyreduced (Fig. 4E).

The histological observation that the histiocytes specificallyengulfed mature RBCs suggests the possible defect in the RBCs.To explore whether the plasma membrane in RBCs fromAEP�/� mice contains any defect, we monitored the resolvingpattern of RBC membrane proteins on SDS/PAGE and foundthat the some of the erythrocyte proteins in AEP-null micedisplayed an age-dependent reduction compared with thosefrom wild-type mice (Fig. S4B). Proteomic analysis of theproteins from AEP-null circulating erythrocytes demonstratedthat valosin-containing protein, MPP1, and �-subunit of ATPsynthase were substantially altered in AEP-null mice. We furtherperformed immunoblotting analysis to confirm the proteomicfindings. As a control, the RBC membrane-associated protein4.1R remained stable in both wild-type and AEP-knockout mice.In contrast, EpoR was substantially decreased in AEP-null miceand valosin-containing protein was completely lost in AEP-depleted mice, whereas they remained stable in the wild-typecontrol (Fig. S4C). The deficiency of EpoR in RBCs fromAEP-null mice at least in part explains why AEP-lacking micedevelop anemia. The loss of EpoR and valosin-containingprotein in AEP-null erythrocyte membrane might result in thedeformity of the RBCs and phagocytosis by histiocytes once theyare produced in bone marrow.

DiscussionOur results show that AEP-knockout mice develop major char-acteristic features of HLH. Clinically, AEP-null animals haveprolonged body temperature elevation and progressive hepato-splenomegaly. Histologically, hemophagocytosis in both thebone marrow and spleen were seen in AEP-knockout mice.Moreover, AEP-knockout mice display cytopenia-reduced NKcell activity. These hallmarks for HLH suggest that AEP mightplay an unexplored role in development of HLH.

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Fig. 2. Hepatosplenomegaly inAEP-knockoutmice. (A)Theweightsofboththeliver and spleen from AEP-null animals (open bar) were significantly higher thanthoseofage-matchedwild-typecontrols (filledbar).�,P�0.05;���,P�0.001,n�8. (B) Age-dependent increase of spleen weight in AEP-knockout mice. AEP-knockout mice (filled circles) and heterozygous control (open circles) of variousages as indicated were killed and the spleens were weighed (3-month-old: �/�,n � 6, �/�, n � 6; 6-month-old: �/�, n � 6, �/�, n � 6; 9-month-old: �/�, n �3, �/�, n � 10; 12-month-old: �/�, n � 4, �/�, n � 3; 15-month-old: �/�, n � 3,�/�,n�10;18-month-old:�/�,n�6,�/�,n�8.). (C)H&Estainingof thespleenfrom both AEP-heterozygous (�/�) and homozygous mice (�/�). (Scale bar, 500�m.) (D) H&E staining of AEP-null spleen showing numerous hemophagocytes.(Scale bar, 500 �m.) (E) Giemsa staining of splenocytes from AEP�/� mice show-ing immature myeloid lineage cells.

470 � www.pnas.org�cgi�doi�10.1073�pnas.0809824105 Chan et al.

Although the phenotypes displayed in AEP-null mice highlyresemble the primary clinical symptoms associated with HLH,no significant elevation of circulating cytokine is detected inAEP-knockout mice as shown in HLH patients (13, 14). Apossible explanation to this discrepancy lies in the timing of thedisease onset. HLH can be a rapid fatal condition, but thesymptoms may present with less severe signs. Since AEP-knockout mice show no aberrant death in early age (data notshown), it is thus suggested that the HLH development inAEP-null mice progresses slowly. The serum cytokine levels arenot statistically changed in AEP-null mice up to 18 months ofage. Nevertheless, some of the AEP mice have drastically highercytokine levels than the age-matched controls, suggesting aprogression of the disease. It has been reported that IFN-� is thekey mediator in HLH development in mice (15), but we could notdetect the same elevation in AEP-knockout mice. However, thereport by Jordan et al. (15) that IFN-� is only essential for HLHdevelopment in a lymphocytic choriomeningitic virus (LCMV)-induced fashion indicates that the IFN-� level might not neces-sarily be elevated in physiological condition in mice.

In familial HLH, genetic studies mapped the HLH-associatedgenes in a variety of chromosome locations, including 9q21.3–22(unknown gene), 10q21–22 (PRF1), 17q25 (UNC13D), 6q24(STX11), 15q21(RAB27A), 1q42.1–42.2 (LYST), and Xq25(SH2D1A) (1). However, our screening of DNA sequence from10 familial HLH patients revealed no mutation in the codingregion of the AEP gene (data not shown). Depending on theracial and ethnic backgrounds of patients, the frequency offamilial HLH caused by unknown genetic defect ranges from17% to 70% (16). Therefore, more sequences from a larger poolof patients is necessary before we can exclude the possibility thata defect in AEP is implicated in HLH.

Analysis of RBC membrane proteins on SDS/PAGE indicatesthat AEP plays an essential role for maintaining RBC membraneintegrity. During the maturation of reticulocytes into erythrocytes,the membrane structures are remodeled such that a portion of thecell membrane is eliminated by fusion with a lysosome (17).Presumably, protease activities in AEP erythrocytes are impaired,leading to abnormal membrane protein destruction during theerythrocyte development. Indeed, activity of dipeptidyl peptidaseII, a protease that can be found in erythrocytes in which activitydepends on the maturation status (18), is dramatically elevated in

AEP-null tissues (19). The defective RBCs were actively engulfedby the histiocytes in the spleen and bone marrow, which mightexplain why the RBC count was evidently decreased in AEP-nullmice. Nevertheless, EpoR was substantially elevated in the bonemarrow from the AEP-null mice compared with wild-type mice.Concomitantly, Epo in blood was also markedly enhanced inAEP-deficient mice, which might be a reconciling mechanism forgenerating more mature RBCs in the anemic mice. Epo is primarilyproduced in the peritubular fibroblasts of the kidney in response toanemia, hypoxia, and, to a lesser extent, vasoconstriction (20). Epomay be nonphysiologically increased in association with kidneyabnormalities (21). Previous study shows that AEP depletion incursaberrant lysosomal storage in kidney-proximal tubule cells. Pre-sumably, AEP-deficiency-provoked renal dysfunction provides an-other molecular mechanism for the overproduction of Epo inAEP-null mice.

We observed that phagocytic activity of AEP-deficient macro-phages against fluorescent microspheres and RBCs was slightly butsignificantly elevated, fitting the observation that numerous he-mophagocytes were observed in AEP�/� mice. However, the onlyslight enhancement of phagocytic activity of AEP-deficient mac-rophages does not account for the remarkable hemophagocytosis inAEP�/� mice. One possibility is that phagocytic activity of Gau-cher-like cells in the bone marrow and spleen, but not peritonealmacrophages, in AEP�/� mice might be severely enhanced. An-other possibility is that impaired digestive activity of macrophagesagainst RBCs as well as enhanced phagocytic activity might causethe remarkable hemophagocytosis. As described, erythrocytemembrane integrity of AEP�/� mice was impaired in an age-dependent manner, suggesting that the defect in RBCs fromAEP�/� mice might cause hemophagocytosis in the bone marrow.However, circulating RBCs from AEP�/� mice and �/� micewere equally engulfed by macrophages in the phagocytic assay.Engulfed RBCs in the bone marrow, but not circulating RBCs,might be morphologically impaired with membrane deformity.Conceivably, these morphologically aberrant RBCs might be en-gulfed in the bone marrow, so that they are not circulating in theperipheral tissues. Obviously, further characterization of Gaucher-like cells and RBCs in AEP�/� mice is necessary to clarify thediscrepancy.

It has been proposed that the pathology of HLH largelydepends on the failure of cytotoxic granule discharge in T cells.

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Fig. 3. Extramedullary hematopoiesis of myeloid lineage in AEP-knockout mice. (A) Non-T and non-B cells increased in splenocytes with age and spleen weightin AEP-knockout mice. Spleen weight is shown above each graph. (B and C) A substantial increase of erythroid cells (Ter-119/CD45) (B) and small increase ofmyeloid cells (Gr-1/Mac-1) (C) occurred in spleen from AEP-null mice compared with wild-type mice.

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This dependence prevents the ultimate execution of the antigen-presenting cells, and thus preventing the termination of theantigen presentation and T cell activation, leading to the exces-sive cytokine production and systemic inflammation (15). Al-though the role of AEP in cytotoxic processing remains un-known, it has been reported that AEP is essential for microbialtetanus toxin antigen presentation (9). Presumably, defectiveantigen presentation by AEP deficiency might be one of thecauses for the hyperimmunological response in HLH.

Materials and MethodsMice. AEP �/�, �/�, and �/� mice on a mixed 129/Ola and C57BL/6 back-ground were generated as reported (19) and kept under specific pathogen-free conditions in an environmentally controlled clean room at the WhiteheadBiomedical Research Building and Institute for Experimental Animals at Ka-nazawa University. Mice were housed at 22 °C on a 12-h/12-h light/dark cycle.Food and water were provided ad lib. The experiments were conductedaccording to the institutional ethical guidelines for animal experiments andapproved by the Institutional Animal Care and Use Committee (IACUC) atEmory University and at Kanazawa University.

Flow Cytometric Analysis. Single-cell suspensions of spleen were prepared bysqueezing the spleen through a nylon mesh. Mature erythrocytes were re-moved by lysis in pH 7.4 Tris-buffered hypotonic ammonium chloride solution(144 mM). Directly conjugated monoclonal antibodies to Ter119, CD45, Gr-1,Mac-1, CD3, and B220 (all from BD Biosciences PharMingen) were used to stainthe cells. Nonviable cells were excluded from analysis by staining with 7-ami-noactinomycin D (BD Biosciences PharMingen) or propidium iodide (Sigma). A

four-color FACSCalibur cytometer (BD Biosciences) was used to acquire aminimum of 10,000 events. Data were analyzed with FloJo software (Treestar).

Serum Epo Determination. Mouse serum Epo levels were determined by usingthe Erythropoietin Quantikine ELISA Kit (R&D Systems).

Immunohistochemistry Staining and Histological Examination. Immunohisto-chemical assays were performed on formalin-fixed paraffin-embedded sections.Three- to 5-�m thick sections from femur bone marrow and the spleen were cut,deparaffinized in xylene, rehydrated in graded alcohols, and subjected to hema-toxylin and eosin (H&E) or periodic acid–Schiff (PAS) staining using standardmethods. The autofluorescence of bone marrow cells was analyzed by confocalmicroscopy (LCM5, Carl Zeiss). For cytospins, spleen and bone marrow cell sus-pensions were prepared, spun onto slides, and stained with Giemsa stain. Forimmunohistochemical analysis, the slides were treated with 0.3% hydrogenperoxide and SuperBlock blocking buffer (Pierce) and incubated with mAbagainst F4/80 (Abcam) and CD68 (Serotec). Next, they were treated with biotin-conjugated Ab against rat IgG (Vector Laboratories), and then with an ABC kit(Vector Laboratories). Finally, horseradish peroxidase activity was detected byDAB substrate and the cells were counterstained with hematoxylin. For Eporeceptor staining, endogenous peroxidase activity was blocked by 3% hydrogenperoxide for 5 min and all slides were boiled in 10 mM sodium citrate buffer (pH6.0) for 10 min. EpoRs on the tissues were stained by using a Zymed Histo-SP AECkit (Invitrogen) and specific antibody against EpoR (sc-697, 1:100 dilution, SantaCruz Biotechnology). Slides were then counterstained with hematoxylin. Theslides were examined under an Olympus BX41 microscope.

RBC Membrane Protein Analysis. RBC membrane was isolated as previouslydescribed (22). Purified RBC ghosts were then solubilized, resolved by SDS/

A B E

C D

Fig. 4. Enhanced phagocytic activity against RBCs in AEP-null macrophages. (A) H&E staining of the bone marrow and spleen from wild-type and AEP-lackingmice. AEP-null mice display ‘‘Gaucher-like cells’’ in bone marrow with engulfed RBCs. The bone marrow and spleen from AEP-null mice contained numerousGaucher-like cells. The histiocytes in AEP-lacking mice contained numerous engulfed RBCs (arrows). (Scale bar, 10 �m.) (B) Electron microscopic analysis ofhistiocytes in bone marrow from AEP-null animals. R, RBC. (Scale bar, 2 �m.) (C) Enhanced phagocytic activity of peritoneal macrophages from AEP-knockoutmice. Peritoneal macrophages from 2-month-old AEP-null mice (�/�) and control heterozygous mice (�/�) were used for the phagocytosis assay with fluorescentmicrospheres. (Left) Representative results. (Right) Results are shown as a histogram; data are expressed as mean � SD. �, P � 0.05, Student’s t test, n � 6. (D)Peritoneal macrophages from AEP-knockout mice (�/�) and control heterozygous mice (�/�) were used for the phagocytosis assay against RBCs fromAEP-knockout mice and control heterozygous mice (�/�). RBCs were untreated (�) or opsonized (�) with anti-mouse RBC antibody before the assay.Macrophages (M�) and RBCs were taken from 12-month-old mice and incubated for 1 h. Data are expressed as mean � SD. ���, P � 0.001 against untreated group;��, P � 0.001 against opsonized group in different genotype using the same RBCs, Student’s t test, n � 6. (E) Reduced natural killer cells activity in AEP-knockoutmice. Percent cytotoxicity of NK cells against P815 mastocytoma from aged-matched wild-type (filled bar) and AEP-knockout (open bar) were measured by flowcytometry. Results are expressed as mean � SEM. �, P � 0.05, Student’s t test, n � 3.

472 � www.pnas.org�cgi�doi�10.1073�pnas.0809824105 Chan et al.

10% PAGE, and subjected to silver staining. Protein bands of interest wereexcised from the gel and analyzed by proteomic analysis.

Electron Microscopy. Mice were fixed by injecting 2.5% glutaraldehyde intothe heart, and the femurs were decalcified by 10% EDTA; the bone marrowswere subjected to standard electron microscopic techniques as describedpreviously (19, 23). Ultrathin sections were observed under a Hitachi H7600electron microscope.

Phagocytosis Assay. Peritoneal macrophages were harvested on day 4 afterintraperitoneal injection of 1.5 ml of 3% thioglycollate (Difco). Macrophages(2 � 105 cells) were plated on round 12-mm glass coverslips on a 24-well plate.Macrophages were incubated with FluoSpheres fluorescent microsphere (1�m, 2 � 107 beads, Molecular Probes) for 2 h, or RBCs (4 � 106 cells) for 1 h or24 h at 37 °C. After incubation, the cells were washed out to remove surface-bound beads. For erythrophagocytosis assay, unengulfed RBCs were removedby hypotonic lysis, fixed, and stained with Giemsa stain. Engulfed micro-spheres and RBCs were observed by fluorescence and light microscopy, re-spectively. Phagocytic index is expressed as the number of engulfed micro-spheres or RBCs per macrophage.

NK Cell Activity Assay. Spleen cell suspensions were prepared by squeezing theorgans through a nylon mesh (100 �m) in DMEM containing 5% FBS. The cells

were centrifuged and the RBCs were lysed in ACK buffer (150 mM NH4Cl, 1 mMKHCO3, and 0.1 mM EDTA, pH 7.2) at room temperature. The spleen suspen-sion was then incubated with P815 murine mastocytoma prelabeled withVybrant cell labeling solution (Invitrogen) at an effector/target ratio of 50:1.The mixture was incubated at 37 °C for 3 h. Fifteen microliters of propidiumiodide (50 �g/ml) was added to the effector/target cell mixture during incu-bation. Natural killer cell functions were analyzed by flow cytometry asreported (24).

Statistics. Data are presented as mean � SEM or S.D. as indicated in the figurelegends. Statistical evaluation was carried out by means of Student’s t test orWelch’s t test following Levene’s test for equality of variance between AEP-deficient and control mice. A two-sided level of P � 0.05 was accepted asstatistically significant. All statistical analysis was performed by the computerprogram Prism (GraphPad).

ACKNOWLEDGMENTS. We thank the late Dr. O. Miyaishi of Chubu RosaiHospital for his initial contribution on this work and Drs. A. Shiratsuchi and Y.Nakanishi at Kanazawa University for teaching us the phagocytic assay. Thiswork is supported by Grant R01 NS45627 from the National Institutes of Health(to K.Y.) and Grant 18500325 from the Ministry of Education, Culture, Sports,Science and Technology of Japan (to N.H.).

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CELL BIOLOGYCorrection for ‘‘Mice lacking asparaginyl endopeptidase developdisorders resembling hemophagocytic syndrome,’’ by Chi-BunChan, Michiyo Abe, Noriyoshi Hashimoto, Chunhai Hao, Ifor R.Williams, Xia Liu, Shinji Nakao, Akitsugu Yamamoto, Shi-YongLi, Ikuko Hara-Nishimura, Masahide Asano, and Keqiang Ye,which appeared in issue 2, January 13, 2009, of Proc Natl AcadSci USA (106:468–473; first published December 23, 2008;10.1073�pnas.0809824105).

The authors request that Chengyun Zheng, Jan-Inge Henter,Marie Meeths, and Magnus Nordenskjold be added to theauthors list between Akitsugu Yamamoto and Shi-Yong Li.Chengyun Zheng designed research and analyzed data, Jan-IngeHenter designed research, Marie Meeths performed researchand analyzed data, and Magnus Nordenskjold analyzed data.The online version has been corrected. The corrected author andaffiliation lines and related footnotes appear below.

Chi-Bun Chana,1, Michiyo Abeb,1, Noriyoshi Hashimotob,Chunhai Haoa, Ifor R. Williamsa, Xia Liua, Shinji Nakaoc,Akitsugu Yamamotod, Chengyun Zhenge,f, Jan-IngeHentere, Marie Meethse,f, Magnus Nordenskjoldf,Shi-Yong Lia, Ikuko Hara-Nishimurag, MasahideAsanob,2, and Keqiang Yea,2

aDepartment of Pathology and Laboratory Medicine, Emory UniversitySchool of Medicine, 615 Michael Street, Atlanta, GA 30322; bDivision ofTransgenic Animal Science, Advanced Science Research Center, andcDepartment of Cellular Transplantation Biology, Graduate School ofMedical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa920-8640, Japan; dDepartment of Bio-Science, Nagahama Institute ofBio-Science and Technology, 1266 Tamura-machi, Nagahama 526-0829,Japan; gDepartment of Botany, Graduate School of Science, KyotoUniversity, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; eDepartmentof Woman and Child Health, Childhood Cancer Research Unit, andfDepartment of Molecular Medicine and Surgery, Clinical Genetics Unit,Karolinska Institute, Karolinska University Hospital, SE-141 86 Stockholm,Sweden

Author contributions: C.-B.C., M. Abe, N.H., C.H., I.R.W., C.Z., J.-I.H., M. Asano, and K.Y.designed research; C.-B.C., M. Abe, N.H., C.H., I.R.W., X.L., S.N., A.Y., and M.M. performedresearch; I.H.-N. contributed new reagents/analytic tools; C.-B.C., M. Abe, N.H., C.H., I.R.W.,C.Z., M.M., M.N., S.-Y.L., M. Asano, and K.Y. analyzed data; and C.-B.C., M. Asano, and K.Y.wrote the paper.

1C.-B.C. and M. Abe contributed equally to this work.

2To whom correspondence may be addressed. E-mail: asano@kiea.m.kanazawa-u.ac.jp orkye@emory.edu.

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BIOCHEMISTRYCorrection for ‘‘Crystal structure of a near-full-length archaealMCM: Functional insights for an AAA� hexameric helicase,’’ byAaron S. Brewster, Ganggang Wang, Xian Yu, William B.Greenleaf, Jose Marıa Carazo, Matthew Tjajadia, Michael G.Klein, and Xiaojiang S. Chen, which appeared in issue 51,December 23, 2008, of Proc Natl Acad Sci USA (105:20191–20196; first published December 10, 2008; 10.1073/pnas.0808037105).

The authors note that the author name Matthew Tjajadiashould have appeared as Matthew Tjajadi. The online versionhas been corrected. The corrected author line appears below.

Aaron S. Brewster, Ganggang Wang, Xian Yu, William B.Greenleaf, Jose Marıa Carazo, Matthew Tjajadi,Michael G. Klein, and Xiaojiang S. Chen

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NEUROSCIENCECorrection for ‘‘Caloric restriction improves memory in elderlyhumans,’’ by A. V. Witte, M. Fobker, R. Gellner, S. Knecht, andA. Floel, which appeared in issue 4, January 27, 2009, of ProcNatl Acad Sci USA (106:1255–1260; first published January 26,2009; 10.1073/pnas.0808587106).

The authors note that the following two sentences should havebeen included on page 1257, left column, first full paragraph,after the sentence ending in the fifth line: ‘‘Note that one subjecthad to be excluded from the correlation analysis for insulin andone subject had to be excluded for hs-CRP due to a measurementerror in insulin or hs-CRP, respectively. Therefore, analyses wereconducted with 9 of initially 10 subjects defined by weight lossof �2 kg.’’ This error does not affect the conclusions of thearticle.

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CELL BIOLOGYCorrection for ‘‘Mice lacking asparaginyl endopeptidase developdisorders resembling hemophagocytic syndrome,’’ by Chi-BunChan, Michiyo Abe, Noriyoshi Hashimoto, Chunhai Hao, Ifor R.Williams, Xia Liu, Shinji Nakao, Akitsugu Yamamoto, Shi-YongLi, Ikuko Hara-Nishimura, Masahide Asano, and Keqiang Ye,which appeared in issue 2, January 13, 2009, of Proc Natl AcadSci USA (106:468–473; first published December 23, 2008;10.1073�pnas.0809824105).

The authors request that Chengyun Zheng, Jan-Inge Henter,Marie Meeths, and Magnus Nordenskjold be added to theauthors list between Akitsugu Yamamoto and Shi-Yong Li.Chengyun Zheng designed research and analyzed data, Jan-IngeHenter designed research, Marie Meeths performed researchand analyzed data, and Magnus Nordenskjold analyzed data.The online version has been corrected. The corrected author andaffiliation lines and related footnotes appear below.

Chi-Bun Chana,1, Michiyo Abeb,1, Noriyoshi Hashimotob,Chunhai Haoa, Ifor R. Williamsa, Xia Liua, Shinji Nakaoc,Akitsugu Yamamotod, Chengyun Zhenge,f, Jan-IngeHentere, Marie Meethse,f, Magnus Nordenskjoldf,Shi-Yong Lia, Ikuko Hara-Nishimurag, MasahideAsanob,2, and Keqiang Yea,2

aDepartment of Pathology and Laboratory Medicine, Emory UniversitySchool of Medicine, 615 Michael Street, Atlanta, GA 30322; bDivision ofTransgenic Animal Science, Advanced Science Research Center, andcDepartment of Cellular Transplantation Biology, Graduate School ofMedical Science, Kanazawa University, 13-1 Takara-machi, Kanazawa920-8640, Japan; dDepartment of Bio-Science, Nagahama Institute ofBio-Science and Technology, 1266 Tamura-machi, Nagahama 526-0829,Japan; gDepartment of Botany, Graduate School of Science, KyotoUniversity, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan; eDepartmentof Woman and Child Health, Childhood Cancer Research Unit, andfDepartment of Molecular Medicine and Surgery, Clinical Genetics Unit,Karolinska Institute, Karolinska University Hospital, SE-141 86 Stockholm,Sweden

Author contributions: C.-B.C., M. Abe, N.H., C.H., I.R.W., C.Z., J.-I.H., M. Asano, and K.Y.designed research; C.-B.C., M. Abe, N.H., C.H., I.R.W., X.L., S.N., A.Y., and M.M. performedresearch; I.H.-N. contributed new reagents/analytic tools; C.-B.C., M. Abe, N.H., C.H., I.R.W.,C.Z., M.M., M.N., S.-Y.L., M. Asano, and K.Y. analyzed data; and C.-B.C., M. Asano, and K.Y.wrote the paper.

1C.-B.C. and M. Abe contributed equally to this work.

2To whom correspondence may be addressed. E-mail: asano@kiea.m.kanazawa-u.ac.jp orkye@emory.edu.

www.pnas.org�cgi�doi�10.1073�pnas.0901254106

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