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Draper-mediated and Phosphatidylserine-independent Phagocytosis
of Apoptotic Cells by Drosophila Hemocytes/Macrophages*
Received for publication, July 29, 2004, and in revised form, August 18, 2004
Published, JBC Papers in Press, September 1, 2004, DOI 10.1074/jbc.M408597200
Junko Manaka‡, Takayuki Kuraishi§, Akiko Shiratsuchi‡§, Yuji Nakai§, Haruhiro Higashida‡,
Peter Henson¶, and Yoshinobu Nakanishi‡§储
From the ‡Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan, the
§Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan, and
the ¶Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206
The mechanism of phagocytic elimination of dying
cells in Drosophila is poorly understood. This study was
undertaken to examine the recognition and engulfment
of apoptotic cells by Drosophila hemocytes/macro-
phages in vitro and in vivo.Inthein vitro analysis,
l(2)mbn cells (a cell line established from larval hemo-
cytes of a tumorous Drosophila mutant) were used as
phagocytes. When l(2)mbn cells were treated with the
molting hormone 20-hydroxyecdysone, the cells ac-
quired the ability to phagocytose apoptotic S2 cells, an-
other Drosophila cell line. S2 cells undergoing cyclohex-
imide-induced apoptosis exposed phosphatidylserine on
their surface, but their engulfment by l(2)mbn cells did
not seem to be mediated by phosphatidylserine. The
level of Croquemort, a candidate phagocytosis receptor
of Drosophila hemocytes/macrophages, increased in
l(2)mbn cells after treatment with 20-hydroxyecdysone,
whereas that of Draper, another candidate phagocytosis
receptor, remained unchanged. However, apoptotic cell
phagocytosis was reduced when the expression of
Draper, but not of Croquemort, was inhibited by RNA
interference in hormone-treated l(2)mbn cells. We next
examined whether Draper is responsible for the phago-
cytosis of apoptotic cells in vivo using an assay for en-
gulfment based on assessing DNA degradation of apo-
ptotic cells in dICAD mutant embryos (which only
occurred after ingestion by the phagocytes). RNA inter-
ference-mediated decrease in the level of Draper in em-
bryos of mutant flies was accompanied by a decrease in
the number of cells containing fragmented DNA. Fur-
thermore, histochemical analyses of dispersed embry-
onic cells revealed that the level of phagocytosis of
apoptotic cells by hemocytes/macrophages was reduced
when Draper expression was inhibited. These results
indicate that Drosophila hemocytes/macrophages
execute Draper-mediated phagocytosis to eliminate
apoptotic cells.
Cells undergoing apoptosis are selectively and rapidly
eliminated from the organism by phagocytosis (1, 2). This
process contributes not only to the clearance of unnecessary
or spent cells from the body but also to maintaining tissue
homeostasis (3– 8). The mechanism underlying phagocytosis
of apoptotic cells consists of various distinct events such as
the migration of phagocytes toward the site of apoptosis, the
recognition and engulfment of apoptotic cells by phagocytes,
processing of engulfed apoptotic cells in phagocytes, and al-
teration of gene expression in engulfing phagocytes. Al-
though investigation of these phenomena has recently be-
come intensive, the molecular basis of each event largely
remains unclear. The phagocytic elimination of apoptotic
cells is considered to be an innate immune response, because
the underlying mechanism does not require the action of
protein products of rearranged genes (9 –11). Thus, clarifica-
tion of the mechanisms and consequences of this phenomenon
should lead to a better understanding of the cellular response
in innate immunity.
Apoptotic cells bind to phagocytes through recognition of
marker molecules that are expressed at their surfaces (4, 12),
the best characterized of which is the membrane phospholipid
phosphatidylserine (PS)
1
(13–16). PS is normally restricted to
the inner leaflet of the membrane bilayer but translocates to
the outer leaflet and is exposed on the cell surface during
apoptosis (13, 15, 17, 18). Externalized PS serves as a “phago-
cytosis marker” (i.e. phagocytes recognize apoptotic cells by
using either membrane-bound PS receptors or PS-binding sol-
uble “bridge” molecules) (15, 16). The importance of PS-medi-
ated phagocytosis of apoptotic cells for the development of
various organs and tissues has recently been shown in the
mouse (19, 20) by disrupting a gene coding for the macrophage
PS receptor (21). The existence of two seemingly independent
signaling pathways for the induction of engulfment has been
genetically identified in Caenorhabditis elegans (22). However,
the nature of receptors that exist farthest upstream of these
pathways has been unknown until recently. In C. elegans,
CED-1 has been suggested to serve as a receptor for one path-
way that involves CED-6, CED-7, and CED-10, although the
cognate ligand remains to be identified (14, 23). Surface recep-
tors responsible for the other pathway (which involves CED-2,
CED-5, CED-10, and CED-12) may include PSR-1, which is
encoded by psr-1, the C. elegans homologue of the gene coding
for the macrophage PS receptor (25). All of the above-men-
tioned genes possess mammalian counterparts, suggesting that
mammalian phagocytes use the same two pathways to engulf
apoptotic cells (26).
* This work was supported in part by a grant-in-aid for Scientific
Research from the Japan Society for the Promotion of Science. The costs
of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this
fact.
储To whom correspondence should be addressed: Graduate School of
Medical Science, Kanazawa University, Shizenken, Kakuma-machi,
Kanazawa, Ishikawa 920-1192, Japan. Tel.: 81-76-234-4481; Fax: 81-
76-234-4480; E-mail: nakanaka@kenroku.kanazawa-u.ac.jp.
1
The abbreviations used are: PS, phosphatidylserine; BSA, bovine
serum albumin; CAD, caspase-activated DNase; FBS, fetal bovine se-
rum; FITC, fluorescein isothiocyanate; ICAD, the inhibitor of CAD;
ISNT, in situ nick translation; PBS, phosphate-buffered saline; PFA,
paraformaldehyde; PC, phosphatidylcholine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 46, Issue of November 12, pp. 48466–48476, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org48466
by guest on August 18, 2018http://www.jbc.org/Downloaded from
On the other hand, much less is known about the interac-
tions between dying cells and phagocytes in Drosophila. Cro-
quemort, a member of the CD36 family of proteins (27), is
specifically expressed in hemocytes/macrophages (27) and has
been shown genetically to be involved in the clearance of apo-
ptotic cells in Drosophila embryos (28). Croquemort appears to
be structurally unrelated to either CED-1 or PSR-1, and how it
transduces signal(s) for phagocytosis, including the identity of
its ligand, remains unknown. On the other hand, Draper,
which is encoded by draper,isaDrosophila homologue of ced-1
and is expressed in two types of phagocytes, namely glia and
hemocytes/macrophages (29). Draper is therefore another can-
didate phagocytosis receptor in this organism. In fact, deletion
of the draper locus in embryos showed an increased number of
apoptotic neurons in the central nervous system, suggesting
the involvement of this molecule in glial phagocytosis of apo-
ptotic neurons (29). It is thus likely that at least one of the two
signaling pathways found in C. elegans also exists in Drosoph-
ila. Although the existence of the Drosophila homologue of the
gene coding for the macrophage PS receptor has been noted
(21), its involvement in phagocytosis in Drosophila is unknown.
A large number of cells undergo apoptosis during development
of Drosophila and are eliminated through phagocytosis by mac-
rophage-like hemocytes or glia (30, 31). In the present study,
we examined the mode of phagocytosis of apoptotic cells in
Drosophila both in vitro and in vivo, focusing on the involve-
ment of PS on target cells and Croquemort and Draper on
phagocytes.
EXPERIMENTAL PROCEDURES
Cell Culture, Fly Maintenance, and Apoptosis Induction—l(2)mbn
cells (provided by S. Natori) established from larval hemocytes of a
tumorous Drosophila melanogaster mutant (32) were maintained at
25 °C in Schneider’s Drosophila medium (Invitrogen) containing 12%
(v/v) fetal bovine serum (FBS), 100 units/ml penicillin, and 100
g/ml
streptomycin. l(2)mbn cells at about 80% confluence were incubated
with 20-hydroxyecdysone (Sigma) (1
M) for 48 h. S2 cells (provided by
S. Natori) were maintained at 25 °C in Schneider’s medium containing
10% heat-inactivated FBS, 50 units/ml penicillin, and 50
g/ml strep-
tomycin and induced to undergo apoptosis by incubation with cyclohex-
imide (Sigma) (1.5
g/ml) for 24 h. Macrophages were prepared from
peritoneal lavage after thioglycollate administration to mice (C57BL/6,
8-week-old females) as described previously (33). Jurkat cells, a human
leukemic T-cell line, were cultured in RPMI 1640 medium containing
10% FBS at 37 °C with 5% (v/v) CO
2
in air and induced to undergo
apoptosis by incubation with doxorubicin (Sigma) (1.5
g/ml) for 30 – 40
h. The occurrence of apoptosis was monitored by examining the follow-
ing changes: caspase activation detected by immunohistochemistry (see
below), chromatin condensation detected by staining cells with the
DNA-binding fluorescent dye Hoechst 33342, DNA breakage detected
by in situ nick translation (ISNT) (see below), and PS externalization
detected by determining the binding of fluorescence-labeled annexin
V (34) or the fluorochrome PSS-380 (35) (provided by B. Smith). For
the inhibition of apoptosis, S2 cells were incubated with the pan-
caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (R&D
Systems Inc.) at the concentrations indicated in the figures for 1 h
prior to, and during, treatment with cycloheximide. Wild-type yw
strain and dICAD-null flies (provided by S. Nagata), the latter of
which is a P-element insertion mutant with no expression of either
the inhibitor of caspase-activated DNase (dICAD) or caspase-
activated DNase (dCAD) (36), were maintained with standard fly food
and a photoperiod of 12 h dark/12 h light (lights off, 10 a.m.–10 p.m.)
at 24 °C. Eggs were collected on agar plates containing grape juice
and allowed to develop at 16 °C. For induction of apoptosis, embryos
that had developed for4hat24°Cafter egg laying were exposed to
x-rays (4,000 rads) and incubated for1hat24°Cbefore analysis as
described previously (30).
Antibody—Anti-Croquemort antiserum was generated by immuniz-
ing rats (Donryu, 2– 4-month-old males) with a 420-amino acid region at
the carboxyl end of Croquemort, which was expressed in Escherichia
coli as a fusion protein with glutathione S-transferase, solubilized with
urea, and affinity-purified. Anti-Draper rabbit antiserum, which was
raised against an intracellular region of Draper (29), and rabbit anti-
serum to activated drICE, which was raised against a synthetic peptide
corresponding to a region specific for activated drICE (37), were pro-
vided by M. Freeman and B. Hay, respectively. Anti-Repo mouse mono-
clonal antibody, clone 8D12, was obtained from the Developmental
Studies Hybridoma Bank.
Preparation of Liposomes and Liposome Incorporation Assay—Lipo-
somes were formed using either phosphatidylcholine (PC) only (PC
liposomes) or a combination of PC and PS at a molar ratio of 7:3 (PS
liposomes) as described previously (34). Fluorescence-labeled liposomes
were prepared by including N-(lissamine rhodamine B sulfonyl)-L-
␣
-
phosphatidylethanolamine (Avanti Polar Lipids, Alabaster, AL) in lipo-
somes at 1% (in molar ratio) of total phospholipids. To examine the
incorporation of liposomes, l(2)mbn cells maintained with serum-free
Schneider’s medium were incubated with rhodamine-labeled liposomes
(0.2 mM)for2hat25°Candwashed with phosphate-buffered saline
(PBS) containing 0.1% (w/v) bovine serum albumin (BSA), and the
extent of incorporation was examined either by fluorescence microscopy
or flow cytometry.
Western Blotting—To obtain protein samples for Western blotting,
l(2)mbn cells were suspended in lysis buffer consisting of 10 mMTris-
HCl (pH 7.4), 1% (v/v) Nonidet P-40, 0.1% (w/v) sodium deoxycholate,
0.1% (w/v) SDS, 0.15 MNaCl, and 1 mMEDTA, sonicated, and incubated
at 4 °C for 30 min with continuous agitation. The resulting whole-cell
lysates were centrifuged at 10,000 ⫻gfor 10 min, and the supernatants
were collected. Proteins in this fraction were solubilized in SDS sample
buffer (0.0625 MTris-HCl (pH 6.8), 2.5% SDS, and 2.5% (v/v) 2-mercap-
toethanol), separated by SDS-7.5% (w/v) polyacrylamide gel electro-
phoresis, and electrophoretically transferred onto a polyvinylidene flu-
oride membrane. The membrane was blocked with 5% (w/v) dry skim
milk, incubated with primary antibody, and washed. It was then re-
acted with alkaline phosphatase-conjugated secondary antibody, and
the signals were visualized using the Immun-Star system (Bio-Rad).
For the analysis of embryonic proteins, embryos were treated with 2.5%
(v/v) sodium hypochlorite for dechorionation, suspended in SDS sample
buffer, sonicated, and centrifuged at 10,000 ⫻gfor 10 min, and proteins
in the supernatants were analyzed as described above.
Immunohistochemistry—For immunohistochemical detection of acti-
vated drICE in S2 cells, cells smeared on silane-coated glass slides were
fixed with PBS containing 2% (w/v) paraformaldehyde (PFA) and 0.1%
(w/v) glutaraldehyde and subsequently permeabilized by treatment
with 0.1% (v/v) Triton X-100. The cells were then incubated with PBS
containing 3% BSA, washed with PBS, and reacted with anti-activated
drICE antibody. They were washed, incubated with fluorescein isothio-
cyanate (FITC)-conjugated anti-rabbit IgG antibody, enclosed with 50%
(v/v) glycerol, and examined by fluorescence/phase-contrast microscopy.
To detect Croquemort in l(2)mbn cells, smeared cells were successively
treated with 4% PFA-containing PBS, methanol (⫺20 °C), and 5% (v/v)
swine serum in PBS and washed with 0.1% BSA in PBS. The cells were
then reacted with anti-Croquemort antibody, washed with 0.1% BSA in
PBS, and incubated successively with biotin-labeled anti-rat IgG anti-
body and FITC-conjugated avidin. The cells were washed with 0.1%
BSA in PBS, enclosed with 50% glycerol, and examined under a fluo-
rescence/phase-contrast microscope. To detect Draper in l(2)mbn cells,
smeared cells were successively treated with a mixture of methanol and
acetone (1:1 in volume) (⫺20 °C) and 3% BSA in PBS. The cells were
then reacted with anti-Draper antibody, washed with 0.1% BSA in PBS,
and incubated with FITC-conjugated anti-rabbit IgG. The samples were
enclosed with 50% glycerol and examined by microscopy. For detection
of activated drICE in Drosophila embryos, dechorionated embryos were
incubated with a mixture (1:1) of 3.7% (v/v) formaldehyde-containing
PBS and heptane for 30 min for fixation and then vortexed successively
with methanol and heptane to remove vitellin membranes. They were
then washed with 0.1% (v/v) Tween 20-containing PBS, permeabilized
by treatment with PBS containing 0.3% Triton X-100 and 0.3% sodium
deoxycholate for 30 min, and incubated for 1 h with PBS containing 5%
BSA and 0.1% Tween 20 for blocking. The samples were then incubated
with PBS containing 5% BSA, 0.1% Tween 20, and anti-activated drICE
antibody overnight at 4 °C. The embryos were washed with 0.1% Tween
20-containing PBS, treated with FITC-conjugated anti-rabbit IgG an-
tibody, enclosed with 50% glycerol, and examined by fluorescence
microscopy.
In Situ Nick Translation—To analyze S2 cells for the presence of
nuclei containing fragmented DNA, the cells were smeared on silane-
coated glass slides, fixed with 4% PFA, permeabilized with methanol,
and washed with PBS containing 0.3% Triton X-100. They were then
subjected to in situ DNA synthesis in the presence of tetramethylrho-
damine-conjugated dUTP, DNA polymerase I (150 units/ml), and other
nucleotides and reagents for 40 –90 min at room temperature. The
Draper-mediated Phagocytosis by Drosophila Hemocytes 48467
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samples were washed with PBS containing 0.3% Triton X-100, enclosed
with 50% glycerol, and examined under a fluorescence microscope. For
the analysis of dispersed embryonic cells, embryos (200 –1,000) were
suspended with PBS and hand-crushed through a mesh using a rubber
pestle, and the resulting suspension was centrifuged. The sedimented
materials were resuspended with PBS, treated successively with 0.25%
(w/v) collagenase and 0.25% (w/v) trypsin and then with FBS to termi-
nate the action of proteases, and filtered through a 70-
m mesh. The
resulting dispersed cells were pelleted by centrifugation, suspended
with PBS, and used for further analyses. Dispersed embryonic cells
were analyzed by ISNT as for S2 cells, except that the samples were
incubated with Hoechst 33342 before enclosing, and fluorescence sig-
nals derived from tetramethylrhodamine and Hoechst 33342 were in-
dividually detected. For the analysis of whole embryos, dechorionated
embryos were incubated with a mixture (1:1) of 4% PFA-containing PBS
and heptane for 30 min for fixation and vortexed successively with
methanol and heptane to remove vitellin membranes. They were fur-
ther treated with heptane, washed with methanol, and incubated in
PBS containing 0.3% Triton X-100. The embryos were then subjected to
in situ DNA synthesis, washed, and microscopically examined as done
for S2 cells.
In Vitro Phagocytosis Assay—l(2)mbn cells (2.5 ⫻10
5
) seeded on
coverslips in 24-well culture plates were incubated with Shields and
Sang M3 medium (Sigma) containing 1% heat-inactivated FBS for 2 h
at 25 °C, washed three times with serum-free Shields and Sang M3
medium, and mixed with S2 cells or Jurkat cells (2.5 ⫻10
6
), which had
been surface-labeled with biotin (sulfo-N-hydroxysuccinimide-LC-Bio-
tin; Pierce), in serum-free Shields and Sang M3 medium. The mixture
was incubated at 25 °C for the periods indicated in the figures, washed
with PBS, and successively treated with PBS containing 2% PFA, 0.1%
glutaraldehyde, and 0.05% Triton X-100, methanol, and FITC-labeled
avidin. The samples were examined by fluorescence/phase-contrast mi-
croscopy, and the number of l(2)mbn cells that incorporated fluorescent
target cells was determined and expressed relative to the total number
of l(2)mbn cells (in percentage; the phagocytic index). To examine the
phagocytosis of latex beads or zymosan particles, l(2)mbn cells treated
or not treated with 20-hydroxyecdysone were incubated with the tar-
gets, FITC-labeled latex beads (Fluoresbrite Carboxylate Microspheres;
diameter ⫽1.58
m; Polysciences, Warrington, PA) (phagocyte/target
ratio 1:30) or 5-carboxyfluorescein-labeled zymosan (zymosan A; Sigma)
(phagocyte/target ratio 1:5), for the periods indicated in the figures,
washed with PBS, fixed as described above, and examined by fluores-
cence microscopy. The extent of phagocytosis was determined as done
for the assay with cultured cells as targets. Mouse peritoneal macro-
phages were incubated with unlabeled or biotinylated S2 cells (macro-
phages/targets ratio 1:10 –20), which had been induced to undergo
apoptosis by treatment with cycloheximide as described above, in RPMI
1640 medium containing 10% FBS for2hat37°Cinthepresence or
absence of liposomes. In reactions using unlabeled target cells, the
samples were washed, fixed, and stained with hematoxylin, and the
FIG.1.Induction of apoptosis in S2 cells by cycloheximide. A, S2 cells treated (⫹) or not treated (⫺) with cycloheximide were incubated
with Hoechst 33342 and examined by fluorescence microscopy. Phase-contrast and fluorescence views of the same microscopic fields are shown. The
arrows point to cells containing condensed chromatin. Bar,10
m. B, S2 cells treated (⫹) or not treated (⫺) with cycloheximide were subjected to
ISNT and examined by fluorescence microscopy. Phase-contrast and fluorescence views of the same microscopic fields are shown. The arrows point
to cells containing fragmented DNA. Bar,10
m. C, S2 cells treated (⫹) or not treated (⫺) with cycloheximide were subjected to immunofluores-
cence with anti-activated drICE antibody and examined by fluorescence microscopy. Phase-contrast and fluorescence views of the same microscopic
fields are shown. The arrows point to cells positive for activated drICE. Bar,10
m. D, S2 cells treated (⫹) or not treated (⫺) with cycloheximide
were incubated with 5-carboxyfluorescein-labeled annexin V or PSS-380 (50
M) in the presence of propidium iodide and analyzed by flow
cytometry or fluorescence microscopy. The left panel is the result from the flow cytometric analysis, in which cells less intensely stained with
propidium iodide (cells with intact plasma membranes) were gated and examined for the level of bound annexin V. The vertical line indicates peak
fluorescence obtained in an experiment with no annexin V added (the level of fluorescence was almost the same with cycloheximide-treated and
-untreated cells). In the microscopic analysis, phase-contrast and fluorescence views of the same microscopic fields are shown in each row.Bar,50
m. E, caspase dependence. S2 cells were preincubated with the pancaspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone (z-VAD-fmk)
at the indicated concentrations, treated with cycloheximide in the presence of benzyloxycarbonyl-VAD-fluoromethyl ketone, and analyzed for the
occurrence of chromatin condensation as in A. The ratio (in percentage) of cells containing condensed chromatin is shown.
Draper-mediated Phagocytosis by Drosophila Hemocytes48468
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extent of phagocytosis was determined by phase-contrast microscopy.
In reactions using biotinylated S2 cells as targets, the extent of phag-
ocytosis was determined as done for the reaction using l(2)mbn cells as
phagocytes.
In Vivo Phagocytosis Assay—Phagocytosis of apoptotic cells in whole
embryos was examined by analyzing embryos of dICAD mutant flies (at
about 24 h of development at 16 °C) by ISNT as described above. For
examination of the involvement of Draper in phagocytosis, embryos of
dICAD mutant flies were subjected to RNA interference using double-
stranded RNA containing the Draper mRNA sequence as described
below and allowed to develop for 23.5 h at 16 °C. The embryos together
with embryos injected with solvent alone were dechorionated as de-
scribed above and incubated with a mixture (1:1) of 37% formaldehyde-
containing PBS and heptane for 20 min for fixation. They were placed
on double-sticky tapes, and vitellin membranes were removed manually
using needles. The samples were washed with methanol and subjected
to ISNT as described above, except that a 10-min treatment with
proteinase K (10
g/ml) was included prior to DNA synthesis reactions.
ISNT signals present in a whole single embryo were detected and
enumerated under a fluorescence microscope by gradually changing
focal planes. Twenty embryos in a group of each experiment were
analyzed in this way, and means and S.D. values of the number of
ISNT-positive signals were obtained. Phagocytosis in embryos was also
examined by histochemical examination of dispersed embryonic cells
from wild-type flies. For this purpose, embryonic cells were simulta-
neously analyzed by ISNT and immunofluorescence using antibody
recognizing phagocyte markers, and the ratio of phagocytes that had
engulfed apoptotic cells was determined. Embryonic cells were dis-
persed as described above, washed with PBS containing 0.2% Tween 20
and 0.1% BSA, and incubated with PBS containing 0.2% Tween 20,
0.1% BSA, and 10% FBS for blocking. The cells were then incubated
with anti-Croquemort (a marker of hemocytes/macrophages) or anti-
Repo (a marker of glia) antibody in the same solution used for block-
ing. The samples were washed with PBS containing 0.2% Tween 20
and 0.1% BSA and incubated with Alexa 488-conjugated anti-rat IgG
(for detection of Croquemort) or FITC-conjugated anti-mouse IgG (for
detection of Repo). The cells were then subjected to in situ DNA
synthesis, washed, stained with Hoechst 33342, and microscopically
examined as described above. About 4,000 cells in three different
microscopic fields were examined for the positivity in immunofluo-
rescence and ISNT. The number of Croquemort- or Repo-expressing
cells that contained ISNT-positive nuclei was determined and
expressed relative (in percentage) to total Croquemort- or
Repo-expressing cells as the phagocytic index.
RNA Interference—DNA corresponding to the region between nucle-
otide positions 495 and 1,294 (with the transcription start site num-
bered 1) of croquemort or the region between 1,322 and 2,463 of draper
was generated by polymerase chain reaction using the Croquemort (27)
(provided by N. Franc) or Draper (clone GH03529; obtained from the
Berkeley Drosophila Genome Project) cDNA as a template. The ampli-
fied DNAs were then used as templates for preparation of RNA from
both strands using a commercial kit (T7 RiboMAX Express Large Scale
RNA Production System; Promega Corp., Madison, WI). The synthe-
sized RNA was purified by gel filtration and allowed to form double
strands, and the samples were used in further experiments after the
formation of double strands was confirmed by gel electrophoresis.
l(2)mbn cells that had been maintained as described above were washed
with serum- and antibiotic-free Schneider’s medium, suspended in the
same medium containing the double-stranded RNA (50
g/ml) and
incubated for 30 min. The cultures were then supplemented with 2
volumes of Schneider’s medium containing 12% FBS, 100 units/ml
penicillin, and 100
g/ml streptomycin and further incubated at 25 °C
for 3 days. These cells were analyzed for phagocytic activity as well as
the level of Croquemort and Draper by Western blotting as described
above. To inhibit the expression of Draper in Drosophila embryos,
embryos were collected ⬃30 min after fertilization, lined up on glass
slides, and injected with 50 pl of RNA solution (1 mg/ml). The embryos
were then allowed to develop for 23.5 h at 16 °C and analyzed for the
level of Draper as well as for the occurrence of phagocytosis as described
above.
Analysis of Cell Surface Proteins—l(2)mbn cells (1 ⫻10
7
cells/ml),
either treated or not treated with 20-hydroxyecdysone, were incubated
with sulfo-N-hydroxysuccinimide-LC-biotin for 30 min at room temper-
ature and washed with PBS. The cells were suspended with lysis buffer,
sonicated, and incubated for 30 min on ice. The lysates were centrifuged
at 1,000 ⫻gfor 10 min at 4 °C, and supernatants were collected. The
supernatants (1 ⫻10
7
cells equivalent) were incubated with streptavi-
din-conjugated magnetic beads (Dynabeads M-280; Dynal Biotech,
Brown Deer, WI) for 30 min at room temperature with agitation. The
beads were collected, washed five times with lysis buffer, suspended
with SDS sample buffer, and heated for 5 min at 100 °C. The superna-
tants were then subjected to SDS-7.5% polyacrylamide gel electrophore-
sis, and the separated proteins were analyzed for the presence of
Draper by Western blotting as described above.
Statistical Analysis—Data from quantitative analyses are expressed
as the mean ⫾S.D. (n⫽3–20). Statistical analyses were done by
Student’s ttest, and pvalues of less than 0.05 were considered
significant.
RESULTS
Phagocytosis of Apoptotic Cells by 20-Hydroxyecdysone-
treated l(2)mbn Cells—The Drosophila cell line l(2)mbn, estab-
lished from tumorous larval hemocytes (32), differentiates into
several subpopulations that acquire the ability to phagocytose
one another or yeast when incubated with the molting hormone
20-hydroxyecdysone (38). We examined l(2)mbn cells treated
with 20-hydroxyecdysone for induction of the ability to phago-
cytose apoptotic cells. S2 cells, another Drosophila cell line,
were chosen as the target cells. Among various procedures
tested for inducing apoptosis in S2 cells, we found that treat-
FIG.2. Induction of phagocytic activity in l(2)mbn cells by 20-hydroxyecdysone. A, microscopic views of l(2)mbn cells containing
engulfed apoptotic cells. l(2)mbn cells treated with 20-hydroxyecdysone were subjected to a phagocytosis assay with apoptotic S2 cells as targets.
Phase-contrast and fluorescence views of the same microscopic field are shown. The arrow and arrowhead indicate fluorescent S2 cells engulfed
and not engulfed by l(2)mbn cells, respectively. Bar,10
m. B, dependence of phagocytosis on the hormone treatment of phagocytes and the
apoptotic state of target cells. Phagocytosis reactions were carried out for 2 h under the indicated conditions. benzyloxycarbonyl-VAD-fluoromethyl
ketone was used at 20
M.*,p⬍0.001. Data presented are from one experiment of three with similar results. C, time course of phagocytosis by
l(2)mbn cells. l(2)mbn cells treated (open circles) or not treated (closed circles) with 20-hydroxyecdysone were incubated with apoptotic S2 cells or
latex beads for the indicated periods, and phagocytic indices were determined. Data presented are from one experiment of three with similar
results.
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ment with the protein synthesis inhibitor cycloheximide effec-
tively caused apoptosis. Cycloheximide-treated S2 cells showed
condensation of chromatin (Fig. 1A), fragmentation of DNA
(Fig. 1B), activation of drICE (Drosophila version of caspase-3)
(Fig. 1C), and externalization of PS (Fig. 1D). The ratios of cells
with condensed chromatin, fragmented DNA, activated drICE,
and externalized PS were about 50, 20, 50, and near 100%,
respectively. The occurrence of chromatin condensation by cy-
cloheximide treatment was almost completely inhibited in the
presence of a pancaspase inhibitor (Fig. 1E). These results
indicate that cycloheximide-treated S2 cells exhibit typical
caspase-dependent apoptosis.
S2 cells treated with cycloheximide were surface-labeled
with biotin and mixed with l(2)mbn cells that had been incu-
bated in the presence of 20-hydroxyecdysone. When the mix-
ture was examined by fluorescence microscopy after membrane
permeabilization and the addition of FITC-conjugated avidin,
fluorescent S2 cells were detectable either within l(2)mbn cells
or by themselves (Fig. 2A). S2 cells present in l(2)mbn cells
were considered to have been engulfed, and the ratio (in per-
centage) of l(2)mbn cells that had engulfed S2 cells was deter-
mined and expressed as the phagocytic index. Treatment of the
l(2)mbn cells with 20-hydroxyecdysone was found necessary to
induce the capacity to phagocytose S2 cells (Fig. 2B). The
phagocytosis by hormone-treated l(2)mbn cells also depended
on treatment of target S2 cells with cycloheximide (i.e. viable
cells were not ingested, and the inclusion of a pancaspase
inhibitor during cycloheximide treatment inhibited S2 cells
from becoming susceptible to phagocytosis) (Fig. 2B). The ex-
tent of phagocytosis by 20-hydroxyecdysone-treated l(2)mbn
cells continued to increase for at least 4 h (Fig. 2C). Treatment
with 20-hydroxyecdysone augmented the level of phagocytosis
of zymosan particles by l(2)mbn cells (data not shown) as pre-
viously reported (38) but did not increase the phagocytosis of
latex beads (Fig. 2C). These results collectively indicate that
treatment with 20-hydroxyecdysone induces the ability of
l(2)mbn cells to phagocytose apoptotic cells without affecting
general phagocytic functions.
PS-independent Phagocytosis of Apoptotic S2 Cells by Hor-
mone-treated l(2)mbn Cells—We next examined whether or not
phagocytosis of PS-exposing S2 cells by 20-hydroxyecdysone-
treated l(2)mbn cells is mediated by PS. The effect of PS-
containing liposomes on phagocytosis was first examined, and
the results clearly showed that the level of phagocytosis was
not altered by the addition of liposomes either containing or not
containing PS (Fig. 3A). Macrophages prepared from peritoneal
fluids of thioglycollate-administered mice effectively phagocy-
tosed apoptotic but not viable S2 cells, and the phagocytosis
FIG.3.PS-independent phagocytosis of apoptotic S2 cells by hormone-treated l(2)mbn cells. A, effect of liposomes on phagocytosis.
Phagocytosis reactions were conducted for 2 h using l(2)mbn cells treated with 20-hydroxyecdysone and apoptotic S2 cells as targets in the presence
of PS liposomes (open circles) or PC liposomes (closed circles) at the indicated concentrations. Data presented are from one experiment of three with
similar results. B, phagocytosis of apoptotic S2 cells by mouse macrophages. Unlabeled S2 cells treated (⫹) or not treated (⫺) with cycloheximide
were subjected to a phagocytosis assay (2-h incubation) with mouse peritoneal macrophages as phagocytes in the presence or absence of liposomes
(1 mM). PS, PS liposomes; PC, PC liposomes. *, p⬍0.001. Data presented are from one experiment of three with similar results. C, phagocytosis
of apoptotic Jurkat cells by l(2)mbn cells. l(2)mbn cells treated with 20-hydroxyecdysone were subjected to a phagocytosis assay (2-h incubation)
with apoptotic S2 cells or Jurkat cells as targets. *, p⬍0.001. Data presented are from one experiment of three with similar results. D,
incorporation of liposomes by l(2)mbn cells. l(2)mbn cells treated (⫹) or not treated (⫺) with 20-hydroxyecdysone were incubated with no liposomes
(⫺), fluorescence-labeled PS liposomes (PS), or fluorescence-labeled PC liposomes (PC) and examined by fluorescence microscopy (left) or flow
cytometry (right). In the microscopic analysis, phase-contrast and fluorescence views of the same microscopic fields are shown (bar,20
m). In the
flow cytometric analysis, the vertical lines indicate peak fluorescence in the corresponding control experiments with no added liposomes.
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was specifically inhibited by the addition of PS-containing li-
posomes (Fig. 3B). To the contrary, hormone-treated l(2)mbn
cells phagocytosed apoptotic Jurkat cells, another PS-exposing
target, at only a minimum level (Fig. 3C), despite their effec-
tive, PS-dependent ingestion by mouse peritoneal macrophages
(data not shown). Finally, uptake of fluorescence-labeled lipo-
somes by hormone-treated l(2)mbn cells was examined. The
results showed that PS-containing liposomes and those consist-
ing of phosphatidylcholine alone were almost equally captured
by l(2)mbn cells (Fig. 3D). These results indicate that phago-
cytosis of S2 cells by l(2)mbn cells is not mediated by PS,
probably because l(2)mbn cells treated with 20-hydroxyecdys-
one do not effectively recognize PS.
Involvement of Draper but Not of Croquemort in Phagocytosis
by l(2)mbn Cells—We next sought candidate phagocytosis re-
ceptors in l(2)mbn cells. Two membrane proteins have previ-
ously been identified as possible phagocytosis receptors in Dro-
sophila hemocytes/macrophages: Croquemort, a member of the
CD36 family of proteins, and Draper, a Drosophila homologue
of C. elegans CED-1, both of which are expressed in hemocytes/
macrophages. Treatment with 20-hydroxyecdysone induced
l(2)mbn cells to become more flattened and acquire macroph-
age-like morphology (Fig. 4A). Examination of the expression of
Croquemort in l(2)mbn cells by immunofluorescence (Fig. 4B,
left panels) revealed that the proportion of cells expressing the
receptor increased from 35 to 95% after treatment. The punc-
tate distribution of Croquemort in l(2)mbn cells was similar to
that observed in COS cells ectopically expressing Croquemort
(27). The results of an immunofluorescence analysis showed a
pattern of distribution of Draper somewhat similar to that of
Croquemort, and there was no change in the overall intensity
or distribution of signals in l(2)mbn cells treated and not
treated with 20-hydroxyecdysone (Fig. 4B,right panels). The
augmented expression of Croquemort was confirmed by a West-
ern blot analysis of whole-cell lysates of l(2)mbn cells, in which
a distinct band at about 68 kDa was detected (Fig. 4C,left
panel). We then determined the level of Draper by Western
blotting. Whole-cell lysates of l(2)mbn cells gave a band at
FIG.4.Expression of Croquemort and Draper in 20-hydroxyecdysone-treated l(2)mbn cells. A, morphology of l(2)mbn cells. l(2)mbn
cells treated (⫹) or not treated (⫺) with 20-hydroxyecdysone were examined by microscopy. Phase-contrast and differential interference contrast
(DIC) views of the same microscopic fields are shown. Bar,20
m. B, expression of Croquemort and Draper in l(2)mbn cells analyzed by
immunofluorescence. l(2)mbn cells treated (⫹) or not treated (⫺) with 20-hydroxyecdysone were analyzed for the expression of Croquemort (left)
or Draper (right) by immunofluorescence. Phase-contrast and fluorescence views of the same microscopic fields are shown. Bar,20
m. C,
expression of Croquemort and Draper in l(2)mbn cells analyzed by Western blotting. In the analysis of Croquemort, whole-cell lysates (50
gof
protein) of l(2)mbn cells treated or not treated with 20-hydroxyecdysone (ecd) were examined. The pattern of separated whole-cell proteins that
were visualized by staining with Coomassie Brilliant Blue (CBB) is also shown. In the left panel of the analysis of Draper, whole-cell lysates (100
g of protein) of hormone-untreated l(2)mbn cells and total lysates (200
g of protein) of Drosophila embryos (stage 16) were analyzed. The
antibody was used with or without a 30-min preincubation in the presence of a protein containing an amino acid sequence used for generation of
the antibody (GST-DRPR) or glutathione S-transferase alone (GST). In the middle panel, whole-cell lysates (100
g of protein) of l(2)mbn cells
treated or not treated with 20-hydroxyecdysone were analyzed. The pattern of separated whole-cell proteins that were visualized by staining with
Coomassie Brilliant Blue is also shown. In the right panel, whole-cell lysates (100
g of protein) and biotin-selected cell surface proteins (1 ⫻10
7
cells equivalent) of l(2)mbn cells treated or not treated with 20-hydroxyecdysone were examined. Whole-cell lysates of biotinylated cells or
mock-selected surface proteins without biotinylation were also analyzed. The arrowheads point to the position of Croquemort or Draper. The
positions of markers are indicated at the left of each panel with molecular masses in kilodaltons.
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about 150 kDa with an antibody raised against bacterially
expressed Draper protein, and preincubation of the antibody
with the Draper protein blocked the staining (Fig. 4C,second
panel from left). From the size, it is probable that the largest of
three Draper mRNAs produced by alternative splicing (29) is
the predominant species present in l(2)mbn cells. Lysates of
Drosophila embryos at stage 16 showed a similar pattern in
Western blotting, indicating that the same form of Draper is
expressed in both l(2)mbn cells and embryos, at least at the
developmental stage examined. The level of Draper expression
in l(2)mbn cells, analyzed by Western blotting with whole-cell
lysates (Fig. 4C,third panel from left) or biotin-selected surface
proteins (Fig. 4C,rightmost panel), remained almost the same
after hormone treatment, although Draper protein from hor-
mone-treated cells appeared to migrate in a gel somewhat
faster than that from control cells. These results indicate that
treatment with 20-hydroxyecdysone causes an increase in the
level of Croquemort but not of Draper in l(2)mbn cells. This
raised the possibility that Croquemort participates in the phag-
ocytosis of apoptotic cells by l(2)mbn cells.
To examine the respective roles for Croquemort or Draper,
phagocytosis reactions were conducted using l(2)mbn cells in
which the level of expression of each protein was reduced by
RNA interference. To do so, l(2)mbn cells that had been treated
with 20-hydroxyecdysone were incubated in the presence of
double-stranded RNA that contained a portion of the mRNA
sequence of each protein. When whole-cell lysates of those cells
were examined by Western blotting, we found that incubation
with the RNA severely decreased the level of the targeted
receptor but had no effect on the expression of the other (Fig.
5A). These l(2)mbn cells together with cells that were not
subjected to RNA interference were used in phagocytosis reac-
tions with apoptotic S2 cells as targets. In contrast to our
expectation, inhibition of Croquemort expression did not affect
phagocytosis, and a decrease in the level of Draper instead
caused more than a 50% reduction of the level of phagocytosis
(Fig. 5B,left panel). This effect was not due to inhibition of the
general phagocytic activity of l(2)mbn cells but rather seemed
to be specific for phagocytosis of apoptotic cells, because phag-
ocytosis of latex beads or zymosan particles (yeast) by l(2)mbn
cells was not affected (Fig. 5B,middle and right panels). These
results indicate that Draper, but not Croquemort, is responsi-
ble, at least in part, for phagocytosis of apoptotic S2 cells by
20-hydroxyecdysone-treated l(2)mbn cells.
Involvement of Draper in Phagocytosis of Apoptotic Cells by
Hemocytes/Macrophages in Drosophila Embryos—To deter-
mine whether Draper is involved in the phagocytosis of apo-
ptotic cells by hemocytes/macrophages in vivo, it was first
necessary to develop a system to detect the occurrence of
phagocytosed apoptotic cells in situ. For this purpose, we used
embryos of a Drosophila mutant in which a gene coding for
dICAD is disrupted (36). dICAD mutant flies do not produce
functional dCAD, because proper folding of dCAD requires the
presence of dICAD (39). As a result, there is no apoptotic DNA
fragmentation during embryogenesis in the mutant flies (36).
We thus anticipated that DNA fragmentation in embryos of the
mutant fly, if it occurs at all, should be the consequence of
lysosomal degradation of apoptotic cell DNA after engulfment
by phagocytes. To test this possibility, embryonic cells were
histochemically examined for the presence of nuclei containing
fragmented DNA. Whole embryos (at 24 h of development at
16 °C) of wild-type or dICAD mutant flies were first analyzed
by ISNT. Both types of embryos gave ISNT signals, and the
number of ISNT-positive nuclei in the mutant embryos was
always smaller than that in wild-type embryos (Fig. 6A). To
examine whether ISNT signals detected in embryos were from
nuclei of engulfed cells, cells present in embryos were dis-
persed, subjected to ISNT, and microscopically analyzed. Two
types of ISNT signals were found: one located within another
cell (Fig. 6B,top panels) and the other free from other cells (Fig.
6B,bottom panels). We considered the former signals to be
derived from apoptotic cells that had been phagocytosed and
the latter to be from cells left unengulfed in embryos. Wild-type
embryos contained both types of ISNT signals (free/engulfed
ratio 1:3), whereas most ISNT-positive nuclei found in dis-
persed cells of the mutant embryos were located within other
cells (Fig. 6C). We finally confirmed that apoptosis does occur
in embryos of the dICAD mutant flies. For this purpose, em-
bryos at4hofdevelopment at 24 °C, when hemocytes/macro-
phages or glia had not yet emerged (40), were exposed to x-rays
for induction of apoptosis and histochemically examined for the
presence of apoptotic cells. Exposure to x-rays resulted in the
appearance of many cells containing activated drICE in either
wild-type or dICAD mutant embryos, but ISNT signals became
detectable only in wild-type embryos (Fig. 6D). These results
indicate that embryonic cells of the dICAD mutant did undergo
apoptosis, although it was not accompanied by DNA fragmen-
tation, after exposure to x-rays. From all of the above-described
FIG.5.Involvement of Draper, but not Croquemort, in phagocytosis of apoptotic cells by hormone-treated l(2)mbn cells. A, level
of Croquemort and Draper in hormone-treated l(2)mbn cells before and after RNA interference. l(2)mbn cells treated with 20-hydroxyecdysone were
incubated with (⫹) or without (⫺) double-stranded RNA (dsRNA) containing the sequence of Croquemort (CRQ) or Draper (DRPR) mRNA, and
whole-cell lysates (50
g of protein for Croquemort and 100
g of protein for Draper) were analyzed by Western blotting. The pattern of separated
proteins that were visualized by staining with Coomassie Brilliant Blue (CBB) is also shown. The arrowheads with the terms DRPR and CRQ point
to the positions of Draper and Croquemort, respectively. The positions of markers are indicated at the left with molecular masses in kilodaltons.
B, level of phagocytosis by l(2)mbn cells before and after RNA interference of Croquemort or Draper. Left panel, l(2)mbn cells used in Atogether
with l(2)mbn cells not treated with the hormone were subjected to a phagocytosis assay (2-h incubation) with apoptotic S2 cells as targets. *, p⬍
0.001. Middle and right panels, hormone-treated l(2)mbn cells were incubated with (open circles) or without (closed circles) double-stranded RNA
containing the sequence of Draper mRNA and then subjected to a phagocytosis assay with latex beads or zymosan particles as targets. Data
presented are from one experiment of four (apoptotic S2 cells as targets) and two (latex beads or zymosan as targets) with similar results.
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results, we concluded that apoptotic cells in the mutant em-
bryos become ISNT-positive only after engulfment by phago-
cytes, and thus the occurrence of phagocytosis of apoptotic cells
in Drosophila embryos can be examined in situ using dICAD
mutant flies based on the appearance of cells with fragmented
DNA.
We then examined the involvement of Draper in phagocyto-
sis of apoptotic cells in embryos of dICAD mutant flies by
altering the level of Draper expression by RNA interference.
Embryos at an immediate early stage of development, while
they remained multinucleate single cells, were injected with
double-stranded RNA and analyzed by ISNT after they were
allowed to develop to stage 16, when hemocytes/macrophages
had already emerged (note that these cells appear at stage 10).
We first tested whether the expression of Draper decreases
upon injection of the same double-stranded RNA that was used
to inhibit Draper expression in l(2)mbn cells. The level of
Draper was reduced, with no effect on the level of Croquemort
(Fig. 7A), indicating that the injected RNA specifically inhib-
ited the expression of Draper in wild-type embryos. Almost the
same results were obtained using embryos of the dICAD mu-
tant flies (data not shown). When the mutant embryos were
analyzed by ISNT for the level of phagocytosis of apoptotic
cells, the number of positive signals in embryos injected with
the RNA was 60 –70% of that observed with embryos that
received injection of solvent alone or were left untreated (Fig.
7B). These results indicate that Draper is responsible at least
partly for the phagocytic elimination of apoptotic cells in Dro-
sophila embryos. The involvement of Draper in the phagocyto-
sis of apoptotic neurons by glia has been suggested (29), and
embryos at the stage examined here contain glia in addition to
hemocytes/macrophages. We thus determined the cell type of
phagocytes, hemocytes/macrophages, or glia, whose engulf-
ment of apoptotic cells in embryos is mediated by Draper. To do
so, cells in stage 16 embryos of wild-type flies that had been
injected with RNA or solvent alone were dispersed and exam-
ined simultaneously by ISNT and immunofluorescence. We
used antibody that recognizes the hemocyte/macrophage
marker Croquemort or the glia marker Repo in the immuno-
fluorescence analysis. We found that cells expressing either
marker protein contained ISNT-positive nuclei, indicating that
both hemocytes/macrophages and glia are involved in the phag-
ocytosis of apoptotic cells in Drosophila embryos (Fig. 7C).
Phagocytic indices were then determined for hemocytes/macro-
phages and glia. Repeated experiments (more than three times)
using embryos injected with no materials showed a constant
level of phagocytosis by each type of phagocyte; phagocytic
indices of about 30 and 15 were reproducibly obtained with
hemocytes/macrophages and glia, respectively (data not
shown). These values remained the same when embryos were
injected with solvent alone (Fig. 7D). We found that the level
of phagocytosis by either type of phagocyte was reduced by
30 – 40% when Draper expression was inhibited by RNA in-
terference (Fig. 7D). The number of either Croquemort-
positive hemocytes/macrophages or Repo-positive glia pres-
ent in dispersed embryonic cells did not change after
injection of the Draper RNA (data not shown). These results
allowed us to conclude that Draper is responsible, at least in
FIG.6. Establishment of in vivo phagocytosis assay using embryos of dICAD mutant flies. A,in situ detection of cells containing
fragmented DNA in embryos. Embryos (at 24 h of development at 16 °C) from wild-type or dICAD mutant flies were subjected to ISNT and
examined by fluorescence microscopy. Phase-contrast and fluorescence views of the same microscopic fields are shown. Bar, 100
m. B,
identification of engulfed (top) and unengulfed (bottom) apoptotic cells in dispersed embryonic cells. Embryonic cells (at 24 h of development at
16 °C) of wild-type flies were dispersed, subjected to ISNT, stained with Hoechst 33342, and examined by fluorescence microscopy. Phase-contrast
and fluorescence views of the same microscopic fields are shown at each row. The arrows and arrowheads indicate the nuclei of ISNT-positive cells
and of phagocytes, respectively. Bar,10
m. C, number of engulfed and unengulfed ISNT-positive cells in embryos. Embryonic cells (at 24 h of
development at 16 °C) of wild-type and dICAD mutant flies were analyzed as in B, and the number of unengulfed (free) and engulfed ISNT-positive
cells was determined and expressed relative (in percentage) to total cells. Data presented are from one experiment of five with similar results. D,
occurrence of apoptosis in embryos of dICAD mutant flies. Embryos (at4hofdevelopment at 24 °C) of wild-type or dICAD mutant flies were
exposed to x-rays (⫹) or left untreated (⫺), incubated for1hat24°C,subjected to ISNT or immunofluorescence with anti-activated drICE antibody,
and examined by fluorescence microscopy. Bar, 100
m.
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part, for phagocytic elimination of apoptotic cells by hemo-
cytes/macrophages and glia in Drosophila embryos. The in-
volvement of Draper in the phagocytosis of apoptotic cells by
glia (29) was thus confirmed here in a more direct analysis of
phagocytosis reactions.
DISCUSSION
Role of Draper, the Drosophila Homologue of C. elegans
CED-1, in Phagocytosis of Apoptotic Cells—It has been sug-
gested that there are two signaling pathways that lead to the
induction of engulfment in phagocytes. These pathways are
most likely to be triggered by distinct ligands, to be mediated
by distinct receptors, and to involve partially overlapping sig-
nal mediators and downstream uptake mechanisms. The mem-
brane receptors called CED-1 and PSR-1 have been genetically
shown to act farthest upstream in the pathways in C. elegans.
In Drosophila, Croquemort, a member of the CD36 family of
proteins, is the only protein known to be involved in phagocy-
tosis of apoptotic cells by hemocytes/macrophages. However,
Croquemort appears to be structurally unrelated to either
CED-1 or PSR-1, and the identity of its ligand or how it trans-
duces signal(s) for phagocytosis remains unknown. A Drosoph-
ila homologue of C. elegans CED-1 was recently suggested to
play a role in phagocytosis by glia (i.e. loss of Draper expression
caused an increase in the number of apoptotic neurons in the
central nervous system of Drosophila embryos) (29). In the
present study, we confirmed the role of Draper in glia in a more
direct analysis and, more importantly, found that Draper is
also involved in the phagocytosis of apoptotic cells by Drosoph-
ila hemocytes/macrophages. This indicates that at least one of
the two signaling pathways for phagocytosis of apoptotic cells
in C. elegans exists in Drosophila. On the other hand, the
existence of a Drosophila homologue of the mammalian PS
receptor has been noted (21), but its role in the phagocytosis of
apoptotic cells in Drosophila remains to be determined. Our
finding that Draper was not solely responsible for the phago-
cytosis by larval hemocyte-derived cell line l(2)mbn, embryonic
hemocytes/macrophages, or embryonic glia suggests the pres-
ence of other phagocytosis receptors in those phagocytes. It is,
however, unlikely, at least for l(2)mbn cells, that a homologue
of the PS receptor is a putative additional phagocytosis recep-
tor, because phagocytosis by 20-hydroxyecdysone-treated
l(2)mbn cells was shown not to be mediated by PS present on
FIG.7.Involvement of Draper in phagocytosis of apoptotic cells by hemocytes/macrophages in embryos. A, level of expression of
Draper and Croquemort in embryos. Embryos (stage 16) of wild-type flies were injected with double-stranded RNA containing a sequence of Draper
mRNA (DRPR dsRNA)(⫹) or with solvent alone (⫺), and their lysates (175
g of protein for Draper and 105
g of protein for Croquemort)
were subjected to Western blotting using anti-Draper (DRPR) or anti-Croquemort (CRQ) antibody. The arrowheads with the terms DRPR and
CRQ point to the positions of Draper and Croquemort, respectively. The pattern of separated proteins (35
g) that were visualized by staining
with Coomassie Brilliant Blue (CBB) is also shown. The positions of markers are indicated at the left with molecular masses in kilodaltons.
Data presented are from one experiment of four with similar results. B, level of phagocytosis in Drosophila embryos before and after RNA
interference of Draper. Embryos (stage 16) of dICAD mutant flies that received double-stranded RNA containing a sequence of Draper mRNA
or solvent alone together with embryos left untreated were subjected to ISNT. The number of ISNT-positive signals present in an embryo is
shown. *, p⬍0.001. C, identification of hemocytes/macrophages and glia containing apoptotic cells. Dispersed cells from embryos (stage 16)
of wild-type flies were simultaneously analyzed by ISNT and immunofluorescence with anti-Croquemort or anti-Repo antibody. Examples of
Croquemort-positive hemocytes/macrophages or Repo-positive glia that had engulfed ISNT-positive cells are shown. Phase-contrast and
fluorescence views of the same microscopic fields are shown in each row. The arrows and arrowheads indicate signals of ISNT and
immunofluorescence, respectively. Bar,10
m. D, level of phagocytosis by hemocytes/macrophages and glia before and after RNA interference
of Draper. Embryos (stage 16) of wild-type flies were injected with double-stranded RNA containing a sequence of Draper mRNA (DRPR
dsRNA) (⫹) or with solvent alone (⫺), and dispersed embryonic cells were analyzed as in C. The ratio of Croquemort-positive or Repo-positive
(Repo) cells that contained ISNT-positive nuclei is shown as the phagocytic index. *, p⬍0.002. Data presented are from one experiment of two
with similar results.
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the surface of target apoptotic cells. At this point, it is not clear
whether or not PS serves as a phagocytosis marker at all in
Drosophila.
Possible Ligand for Draper/CED-1—It is most probable that
Draper induces engulfment of apoptotic cells in phagocytes by
recognizing a specific ligand (i.e. a “phagocytosis marker” mol-
ecule present on the surface of target cells). The ligand for
Draper/CED-1, however, remains to be identified. PS appears
not to be recognized by this receptor, because we showed in this
study that phagocytosis of PS-exposing apoptotic S2 cells by
20-hydroxyecdysone-treated l(2)mbn cells is mediated by
Draper with no dependence on PS. A mammalian membrane
protein called low density lipoprotein receptor-like protein
(LRP, also known as CD91) shares some structural similarity
with CED-1 and is considered to be its mammalian homologue
(41). Henson and co-workers (42) have suggested that CD91
acts as a phagocytosis receptor due to its association with
calreticulin, a molecular chaperone present in the endoplas-
mic reticulum. In addition, the involvement of calreticulin in
the phagocytosis of yeast by hemocytes of another insect,
Pieris rapae, was recently reported (43). A Dictyostelium
mutant lacking both calreticulin and calnexin, another mo-
lecular chaperone in the endoplasmic reticulum, shows a
reduced level of phagocytosis of yeast, although both of these
proteins appear to act not at the recognition step but at some
downstream events during phagocytosis by Dictyostelium
(44). Furthermore, like calreticulin, calnexin has been shown
to translocate to the surface of mammalian cells under cer-
tain circumstances (45). Calreticulin and calnexin, which
both exist in Drosophila, are thus good candidates for the
ligand of Draper/CED-1.
Mechanism of Activation of l(2)mbn Cells by 20-Hydroxyec-
dysone—Treatment with 20-hydroxyecdysone did not alter the
general phagocytic activity of l(2)mbn cells but augmented the
level of phagocytosis of apoptotic cells or zymosan particles.
This suggested that the hormone causes quantitative and/or
qualitative changes in l(2)mbn cells that are specific, if not
solely responsible, for phagocytosis of apoptotic cells. A simple
explanation would be an increase in the amount of Draper
present at the surface of the phagocyte, but we showed that
this was not the case. In contrast, the amount of Croquemort
in l(2)mbn cells increased upon treatment with the hormone,
but the results of an RNA interference experiment showed
that this receptor was not involved in the phagocytosis of
apoptotic cells by l(2)mbn cells. Another possibility is that
Draper is functionally activated in hormone-treated l(2)mbn
cells. This might be true, because we found that migration of
Draper protein in a polyacrylamide gel became a little bit
faster after treatment with 20-hydroxyecdysone. Although no
evidence of modification of CED-1 or Draper has been re-
ported so far, our results suggest that Draper is structurally
modified, through for example dephosphorylation or deglyco-
sylation, and activated by 20-hydroxyecdysone. We are now
examining this issue.
Cellular Innate Immunity by Drosophila Hemocytes—The
innate immune system, like adaptive immunity, consists of
humoral and cellular reactions (46). In Drosophila, the hu-
moral responses, most of which are conducted through the
action of the fat body (a functional equivalent of the mamma-
lian liver), include production of antimicrobial peptides and
melanization at wound sites or around microorganisms (47).
On the other hand, the cellular responses in Drosophila consist
of phagocytosis and encapsulation of microorganisms by hemo-
cytes, both of which events lead to killing of the invaders (47).
Although the mechanism of the humoral responses has been
well characterized (47, 48), how Drosophila hemocytes act
against invading microorganisms remains to be clarified.
Phagocytosis of apoptotic cells by hemocytes is considered
part of the cellular immune response of Drosophila. Our data
showed that Draper is involved in hemocyte phagocytosis of
apoptotic cells but not of zymosans. We thus anticipate that
distinct systems are utilized by Drosophila hemocytes for the
recognition of endogenous apoptotic cells and invading micro-
organisms. To clarify these recognition systems is necessary
for a better understanding of cellular innate immunity in
Drosophila.
Acknowledgments—We thank N. Franc, M. Freeman, B. Hay,
S. Nagata, S. Natori, B. Smith, the Developmental Studies Hybridoma
Bank, the Berkeley Drosophila Genome Project, and the National In-
stitute of Genetics for materials. We are also grateful to R. Krieser for
advice.
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Peter Henson and Yoshinobu Nakanishi
Junko Manaka, Takayuki Kuraishi, Akiko Shiratsuchi, Yuji Nakai, Haruhiro Higashida,
Hemocytes/MacrophagesDrosophilaCells by
Draper-mediated and Phosphatidylserine-independent Phagocytosis of Apoptotic
doi: 10.1074/jbc.M408597200 originally published online September 1, 2004
2004, 279:48466-48476.J. Biol. Chem.
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