Brief Communication 781
Interactions between the cellular and humoral immune
responses in Drosophila
Monicia Elrod-Erickson, Smita Mishra and David Schneider
Drosophila has highly efficient defenses against
infection. These include both cellular immune
responses, such as the phagocytosis of invading
microorganisms, and humoral immune responses, such
as the secretion of antimicrobial peptides into the
hemolymph [1,2]. These defense systems are thought
to interact, but the nature and extent of these
interactions is not known. Here we describe a method
for inhibiting phagocytosis in Drosophila blood cells
(hemocytes) by injecting polystyrene beads into the
body cavity. This treatment does not in itself make a fly
susceptible to Escherichia coli infection. However,
when performed on flies carrying the mutation immune
deficiency (imd), which affects the humoral immune
response [3], the treatment results in a striking
decrease in resistance to infection. We therefore
carried out a sensitized genetic screen to identify
immunocompromised mutants by co-injecting beads
and E. coli. From this screen, we identified a new gene
we have named red shirt and identified the caspase
Dredd as a regulator of the Drosophila immune
response. The observation that mutants with defects in
the humoral immune response are further
immunocompromised by blocking phagocytosis, and
thus inhibiting the cellular immune response, shows
that the Drosophila cellular and humoral immune
responses act in concert to fight infection.
Address: Whitehead Institute, 9 Cambridge Center, Cambridge,
Massachusetts 02142, USA.
Correspondence: David Schneider
E-mail: dschneider@wi.mit.edu
Received: 25 April 2000
Revised: 18 May 2000
Accepted: 22 May 2000
Published: 16 June 2000
Current Biology 2000, 10:781–784
0960-9822/00/$ – see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Results and discussion
When fluorescent sub-micrometer particles are injected
into an adult fly they are quickly removed from the circu-
lation by hemocytes, providing a convenient means of visu-
alizing these cells. Many hemocytes were found to gather
at the anterior end of the dorsal vessel in the fly abdomen
(Figure 1a). Hemocytes are also scattered throughout the
body and are attached to a variety of tissues. Most of these
cells appear to be sessile, as we were unable to find many
hemocytes in the hemolymph of adult flies or after perfu-
sion. We injected a variety of particles and found that these
cells were able to collect living and dead Gram-negative
bacteria, dead Gram-positive bacteria, yeast and poly-
styrene beads. Confocal microscopic examination of hemo-
cytes following injection of particles suggested that the
particles were being phagocytosed (Figure 1b,c).
To show that particles were being phagocytosed, we
developed an in vivo assay for phagocytosis. E. coli labeled
with fluorescein isothiocyanate (FITC) were injected into
the hemocoel, followed at various time points by injection
of trypan blue. This dye quenches the fluorescence of all
FITC-labeled particles it contacts but cannot quench par-
ticles that have been phagocytosed. Immediately after
injection, E. coli gather on the dorsal vessel (Figure 2a) but
few of these bacteria have been phagocytosed, as their flu-
orescence can be quenched by trypan blue (Figure 2b). If
flies are incubated for 30 minutes between the E. coli and
trypan blue injections, many bacteria are seen to be
phagocytosed (Figure 2c).
We found that phagocytosis can be inhibited by injecting
polystyrene beads into the hemocoel. These beads are
phagocytosed by hemocytes, but bacteria injected after
the beads are not (Figures 1b,2d). Bacteria injected
30 minutes after bead injection are bound by hemocytes
but not internalized, whereas bacteria injected 1 day after
bead injection are not bound. Bead injection thus provides
a simple method of inhibiting a potentially important
aspect of the fly’s cellular immune response. This proce-
dure is particularly useful as it can be used in any genetic
background and, unlike mutations that decrease hemocyte
numbers but are lethal [4,5], it can be used to study adults.
To determine whether non-phagocytosing flies are
immunocompromised, we injected a concentration of E. coli
that we had found kills imd/imd flies. Injection of either
bacteria or beads, or co-injection of bacteria and beads, did
not affect the survival of wild-type flies (Figure 3a). In con-
trast, bead and bacteria co-injection sensitized imd/imd flies
to E. coli infections (Figure 3b). Under our experimental
conditions, 60% of imd/imd flies injected with E. coli died
after 3 days incubation at 29°C. Flies that survived this
incubation appeared to recover from the infection. In
imd/imd flies co-injected with beads and bacteria, the death
curve was accelerated by 2 days: about half the flies died
after 24 hours instead of after 72 hours, and only 5% of flies
survived past 3 days. We visualized bacterial growth in flies
by injecting E. coli expressing green fluorescent protein
782 Current Biology Vol 10 No 13
Figure 1
Particles injected into the Drosophila
(a)
hemocoel are collected by sessile hemocytes.
(a) Red fluorescent beads were injected into
this fly 60 min before the photograph was
taken. The beads were visualized by merging
a photograph taken under white light with one
taken using TRITC epifluorescence. Red
beads concentrated on the dorsal vessel are
indicated by the arrow. (b) Red polystyrene
beads were injected into the hemocoel of a
fly, followed 30 min later by FITC-labeled phagocytosed, whereas the E. coli decorate
E. coli (green). The beads have been the surface of the cell. (c) Differential
(GFP) (Figure 4). By 24 hours after co-injection, bacteria
were abundant throughout the hemocoel of the imd/imd
flies, but were not visible in wild-type flies.
We used these observations to develop a genetic screen
for immunocompromised flies. As our assay, we co-
injected beads and bacteria and monitored the flies for
death at 2 days. We also monitored the growth of the
GFP-expressing bacteria by fluorescence microscopy. We
reasoned that this assay might reveal mutations that were
not immunocompromised in isolation and therefore could
only be observed in combination with a cellular immune
response defect. By this method, we could also identify
genes that work outside the signal transduction cascades
studied using antimicrobial peptide reporter genes [6].
Using this assay, we screened 1095 viable lines that had
been mutagenized by ethylmethane sulfonate (EMS) on
chromosome 2 and identified one mutant with a strong
phenotype. We also used this assay as a secondary screen
for EP lines [7] that we had previously identified as being
slightly more susceptible than wild type to E. coli infec-
tion using another assay (data not shown). One EP line
was found to have a moderately immunocompromised
phenotype following bacteria and bead co-injection.
Figure 2
(a) (b)
(c) (d)
(b) (c)
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interference contrast image of the hemocyte
in (b). Scale bar represents 10 mm.
We called the recessive EMS mutation red shirt (rsh) after
the minor characters who were always the first to die in
Star Trek episodes. Rsh/rsh flies are more sensitive to bac-
terial infections than are imd homozygotes, showing a 90%
death rate at 48 hours. Bead treatment shifts this death
rate forward 24 hours (Figure 3c). Meiotic mapping placed
rsh on the right arm of chromosome 2 near the marker Pin.
We localized red shirt further by crossing our allele to defi-
ciencies in this region. One deficiency, Df(2R)Dll-MP [8]
which maps to 60E1,2-60E6, failed to complement rsh.
The P-element line EP(X)1412 [7] was originally selected
in our laboratory as giving E. coli a slight in vivo growth
advantage, and its immune defect was confirmed using
the bead and bacteria co-injection assay. Whereas bacterial
injection into EP(X)1412 homozygotes has a minor affect
on viability, co-injection of bacteria and beads leads to
pronounced bacterial growth and death of the flies
(Figures 3d,4d). The insertion site of EP(X)1412 was
sequenced by the Berkeley Drosophila Genome Project.
This P element is inserted 10 base pairs upstream of the
ATG in the 5¢-untranslated region of the gene dredd,
which encodes a caspase that has been implicated in trans-
ducing cell-death signals in the Drosophila embryo [9].
Bacteria are phagocytosed by Drosophila
hemocytes, and this process is blocked by
bead injection. Flies injected with
FITC-labeled E. coli were (a) incubated for
3 min with no trypan blue injection;
(b) incubated for 3 min before trypan blue
injection; or (c) incubated for 30 min before
trypan blue injection. (d) Flies injected with
polystyrene beads 24 hours before E. coli
injection were then incubated for 30 min
before trypan blue injection.
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Figure 3
(a) 100 (b) 100
80 80
Survival (%)
Survival (%)
60 60
40 40
20 20
0 0
0 2 4 6 8 10 0 2 4 6
Day post-infection Day post-infection
(c) 100 (d) 100
80 80
Survival (%)
Survival (%)
60 60
40 40
20 20
0 0
0 2 4 6 8 10 0 2 4 6
Day post-infection Day post-infection
Current Biology
Bead injection enhances the lethality of live E. coli injection. Survival
curves were prepared using at least 100 flies of each genotype in
groups of 20. The genotypes were (a) Oregon R (wild type),
(b) imd/imd, (c) rsh/rsh, and (d) dredd/dredd. Filled squares indicate
bead injection alone; filled circles with dotted line, injection of bacteria
alone; filled triangles, bead and bacteria co-injection.
To determine whether the P-element insertion in the dredd
locus is responsible for the immunocompromised pheno-
type of EP(X)1412 homozygotes, EP(X)1412 was crossed to
Df(1)R194, which deletes dredd [9]. Transheterozygous
females were immunocompromised, as assayed by both the
growth of GFP-expressing E. coli and death of the flies.
Transheterozygotes for EP(X)1412 and a chromosome car-
rying both Df(1)R194 and a transgene containing the entire
dredd locus [9] were, however, indistinguishable from wild
type. Reduction in Dredd activity is thus responsible for the
phenotype observed in the EP(X)1412 line. We note that
the P-element insertion does not result in a null allele of
dredd, as quantitative RT-PCR analysis reveals that tran-
scription of dredd is reduced but not abolished in EP(X)1412
homozygotes (B.R. Levin and D.S., unpublished results).
Northern analysis of infected rsh and dredd homozygous
mutants reveals that both mutations affect the infection-
induced expression of antimicrobial peptide genes
(Figure 5). Rsh eliminates or greatly reduces the E. coli-
induced expression of all the antimicrobial peptide genes
tested (attacin, diptericin and drosomycin), suggesting
that the rsh gene product, like Relish [10], is required for
defense against a broad range of microorganisms. In con-
trast, mutation of dredd decreases the expression of the
antibacterial peptides attacin and diptericin but has little
or no effect on induction of the antifungal peptide dro-
somycin. In this respect, the dredd phenotype resembles
that of imd [3,11,12]. A null allele of dredd might, however,
have a greater effect on induction of these genes.
We have described a simple method for inhibiting phago-
cytosis in adult Drosophila by injecting beads into the
Brief Communication 783
Figure 4
(a) (b)
8 10
(c) (d)
8 10
Current Biology
Bacterial growth can be visualized in immunocompromised mutants.
GFP-expressing E. coli and red polystyrene beads were co-injected into
the hemocoel of the following genotypes: (a) Oregon R (wild type);
(b) imd/imd; (c) rsh/rsh; (d) dredd/dredd. (a–c) were photographed
20 h post-infection; (d) was photographed 25 h post-infection as
bacterial growth is slower in this mutant. Massive bacterial proliferation
is visible in (b–d) as indicated by the green color. Only a faint red
fluorescence due to the injected beads is visible in wild-type flies (a).
hemocoel. We observed that impairing the cellular
immune response in this way enhances the humoral
immunity defect of imd homozygotes, and we used this
observation as the basis for a genetic screen. By monitor-
ing fly survival and bacterial growth after co-injection of
beads and E. coli, we identified two mutations that impair
immune function: an EMS allele of a gene we have named
red shirt and a P-element insertion allele of dredd.
Rsh homozygotes fail to induce expression of several
antimicrobial peptide genes and are very susceptible to
infection by E. coli (in both the presence and absence of
beads). We suggest that the rsh gene product may function
upstream of the transcription factor Relish, as Relish
mutants also fail to induce a broad range of antimicrobial
peptide genes upon infection [10]. Further analysis will,
however, be required to define the role of rsh in the
immune response.
Dredd homozygotes show impaired inducibility of the
antibacterial peptide genes tested, but are not particularly
susceptible to infection by E. coli in the absence of bead
co-injection. (We note, however, that our P-element inser-
tion allele of dredd does not appear to be a null allele.) The
involvement of the caspase Dredd in the insect immune
system is unexpected and intriguing. We propose three
models for its possible role. First, bacterial toxins can
induce apoptosis of adjacent cells [13], and Dredd might
be involved in regulating apoptosis in response to the bac-
terial infection. If, in the absence of Dredd function,
784 Current Biology Vol 10 No 13

Figure 5 G. Walker for the GFP-expressing E. coli. Microscopy was carried out in the
Keck microscope facility at the Whitehead Institute. This work was sup-
ported by the Whitehead Institute Fellows program. M.E. is an Irvington
Institute for Immunological Research Postdoctoral Fellow.

dredd/dredd
Wild type
Wild type
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We have demonstrated that in three cases (imd, rsh and
dredd), mutants with defects in the humoral immune
response are further immunocompromised by blocking
phagocytosis and impairing the cellular immune response.
This indicates that the cellular and humoral immune
responses can, and do, cooperate to combat bacterial infec-
tions in the fly.

Supplementary material
Supplementary material including methodological details is available at
http://current-biology.com/supmat/supmatin.htm.

Acknowledgements
We thank B. Lemaitre, A. Rodriguez, J. Abrams, the Berkeley Drosophila
Genome Project, and the Bloomington Stock Center for fly stocks and