Download Regulators and signalling in insect haemocyte immunity

Cellular Signalling 21 (2009) 186–195
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Cellular Signalling
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c e l l s i g
Review
Regulators and signalling in insect haemocyte immunity
Vassilis J. Marmaras ⁎, Maria Lampropoulou
Department of Biology, University of Patras, 26500 Patras, Greece
a r t i c l e
i n f o
Article history:
Received 6 August 2008
Accepted 24 August 2008
Available online 28 August 2008
Keywords:
Haemocytes
Signalling
Phagocytosis
Nodulation
Encapsulation
Melanization
a b s t r a c t
The innate immune system of insects relies on both humoral and cellular immune responses that are
mediated via activation of several signalling pathways. Haemocytes are the primary mediators of cellmediated immunity in insects, including phagocytosis, nodulation, encapsulation and melanization. The last
years, research has focused on the mechanisms of microbial recognition and activation of haemocyte
intracellular signalling molecules in response to invaders. The powerful tool, RNA interference gene silencing,
helped several regulators involved in immune responses, to be identified. In this review, we summarize
recent advances in understanding the role(s) of receptors and intracellular signalling molecules involved in
immune responses.
© 2008 Elsevier Inc. All rights reserved.
Contents
1.
2.
Introduction . . . . . . . . . . . . . . . . . . . . .
Haemocyte defence responses . . . . . . . . . . . . .
2.1.
Phagocytosis . . . . . . . . . . . . . . . . . .
2.2.
Nodulation . . . . . . . . . . . . . . . . . .
2.3.
Encapsulation . . . . . . . . . . . . . . . . .
2.4.
Melanization . . . . . . . . . . . . . . . . .
3.
Regulators of haemocyte defense responses . . . . . .
3.1.
Humoral receptors . . . . . . . . . . . . . . .
3.2.
Haemocyte-surface receptors . . . . . . . . . .
3.3.
Intracellular signalling molecules . . . . . . . .
3.3.1.
Rho . . . . . . . . . . . . . . . . . .
3.3.2.
FAK (Focal Adhesion Kinase) . . . . . .
3.3.3.
Src . . . . . . . . . . . . . . . . . .
3.3.4.
Syk . . . . . . . . . . . . . . . . . .
3.3.5.
Protein kinase C . . . . . . . . . . . .
3.3.6.
MAP kinases . . . . . . . . . . . . .
3.3.7.
PI-3K . . . . . . . . . . . . . . . . .
3.3.8.
Elk-1-like protein, protein containing the
3.3.9.
CED intracellular components . . . . .
3.3.10.
JAK/STAT. . . . . . . . . . . . . . .
3.3.11.
Eicosanoids. . . . . . . . . . . . . .
3.3.12.
ProPO activation system . . . . . . .
3.3.13.
Ddc activity . . . . . . . . . . . . .
4.
Intracellular signalling pathways. . . . . . . . . . . .
5.
Summary . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: +30 2610 969251; fax: +30 2610 994797.
E-mail address: [email protected] (V.J. Marmaras).
0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.cellsig.2008.08.014
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V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
1. Introduction
Multicellular animals as well as humans are surrounded by a
plethora of pathogens, both prokaryotic and eukaryotic. To defend
themselves against pathogens, vertebrates have developed two
interconnected powerful defence mechanisms, known as innate and
acquired immunity. The acquired immune system is mediated by B
and T lymphocytes.
Insects lack an acquired immune system and hence B and T
lymphocytes, but they have a well-developed innate immune system
that allows a general and rapid response to infectious agents. The
innate immune system of insects relies on both humoral and cellular
responses [1,2]. Humoral immune responses include several antimicrobial peptides, enzymic cascades that regulate coagulation and
melanization of haemolymph, and the production of reactive oxygen
species (ROS) and reactive nitrogen species (RNS). Cellular responses
include phagocytosis, nodulation and encapsulation [3].
The insect body cavity (haemocoel) contains haemolymph, which
serves a function analogous to blood in mammals in that it transports
nutrients, waste products and several micro-and macromolecules. In
addition, several types of haemocytes circulate in insects' haemolymph, originated from mesodermally derived stem cells that
differentiate into specific lineages. The most common types of
haemocytes are prohaemocytes, granulocytes, plasmatocytes, spherulocytes and oenocytoids [3]. However, all these haemocyte types do
not exist in all insect species [4–6]. The circulating haemocytes are
essential for the insect immunity, while their number decreases
drastically during an infection and new haemocytes are produced
from haematopoietic tissues, to balance the lost haemocytes. Lymph
glands are the larval haematopoietic organs where haematopoiesis
occurs during the embryonic and larval stages. Lymph gland-derived
haemocytes are secreted into haemolymph during late third instar
larvae, just before the onset of pupation.
2. Haemocyte defence responses
Haemocytes are responsible for a number of defense responses in
insects, among which phagocytosis, nodulation, encapsulation and
melanization have been documented. These processes appear to be
discrete immune responses in terms of gene expression and outcome.
However, these certain immune responses share a number of common
elements that function in concert to clear pathogens from the
haemolymph. Below we have outlined the current data on these
defense responses and their relationships.
2.1. Phagocytosis
Phagocytosis refers to the recognition, engulfment and intracellular destruction of invading pathogens and apoptotic cells by
individual haemocytes. Phagocytosis in mammals is mainly achieved
by mononuclear phagocytes (monocytes and macrophages) and
polymorphonuclear cells (neutrophils). Monocytes and neutrophils
circulate in the blood, whereas macrophages are tissue residing. In
insects, phagocytosis is achieved mainly by the circulating plasmatocytes or granulocytes, in the haemolymph. [5–9]. Other haemocytes
such as oenocytoids may also take up pathogens [10]. Recently it has
been reported that phagocytes of Manduca sexta have distinct
functions like vertebrate phagocytes. Plasmatocytes are involved in
phagocytosis of non-self microsphere beads, whereas granulocytes
are involved in phagocytosis of self dead cells [11].
Phagocytosis of a microbe by a phagocytic cell is an extremely
complex and diverse process which requires multiple successive
interactions between the phagocyte and the pathogen as well as
sequential signal transduction events. Phagocytosis is induced when
phagocyte surface receptors, are activated by target cells. Since
phagocytosis is a widely conserved cellular process that occurs in
187
many protozoa and all metazoans, it could be hypothesized that insect
phagocytosis is also similar with mammalian phagocytosis. Phagocytosis is also important during insect development, as it participates in
the clearance of apoptotic haemocytes. However, the mechanisms of
phagocytosis of apoptotic haemocytes in insects have not been
intensely investigated [12].
It must be emphasized that the haemocyte response to various
bacteria differs. For example, in A. aegypti the response of haemocytes
to E. coli is phagocytosis, whereas the response of haemocytes to Micrococcus luteus is melanization [13–15], with some melanized M.
luteus being subsequently phagocytosed by granulocytes. Furthermore, it has also been demonstrated that differences exist in the
efficiency and speed of phagocytosis among different bacteria. It has
been shown that E. coli is more readily phagocytosed than S. aureus, in
A. gambiae, and Drosophila cell lines as well as in isolated medfly
haemocytes [5,16,17]. These results strongly suggest that several
distinct molecular mechanisms regulate phagocytosis in insects. It is
also clear that phagocytes are required to regulate the majority of
bacteria infections.
2.2. Nodulation
Nodulation is a predominant cellular defense mechanism in insects
and refers to multicellular haemocytic aggregates that entrap a large
number of bacteria. Melanized or non-melanized nodules are formed
in response to a number of invaders. Nodule formation is lectinmediated process but has not yet been fully characterized. The only
available information so far is that eicosanoids mediate nodulation in
many insect species [18], and prophenoloxidase (PO) and dopa
decarboxylase (Ddc) are involved in the nodulation of medfly
haemocytes [19].
2.3. Encapsulation
Encapsulation refers to the binding of haemocytes to larger targets,
such as parasites, protozoa, and nematodes. Encapsulation can be
observed when parasitoid wasps lay their eggs in the haemocoel of
Drosophila larvae. Haemocytes after binding to their target they
form a multilayer capsule around the invader, which is ultimately
accompanied by melanization. Within the capsule the invader is
killed, by the local production of cytotoxic free radicals ROS and RNS,
or by asphyxiation [20,21].
2.4. Melanization
Melanization refers to the pathway leading to melanin formation.
Melanization has a central role in defense against a wide range of
pathogens and participates in wound healing as well as in nodule and
capsule formation in some lepidopteran and dipteran insects, such as
Pseudolusia and Drosophila [1,2]. Melanization depends on tyrosine
metabolism. Briefly, tyrosine is converted to dopa, an important
branch point substrate, by activated PO (Fig. 1). Dopa may be either
decarboxylated by Ddc to dopamine or oxidised by PO to dopaquinone. Dopamine is also an important branch point substrate, because
dopamine-derived metabolites either via PO or through other
enzymes are used in several metabolic pathways, participating in
neurotransmission, cuticular sclerotization, cross-linking of cuticular
components via quinone intermediates, phagocytosis, wound healing
and melanization in immune reactive insects [11,22,23].
3. Regulators of haemocyte defense responses
The first step of insect haemocyte-mediated immune responses
includes the recognition of pathogens and other entities from self. The
insect components responsible for the recognition of non-self bind
conserved pathogen-associated molecular patterns (PAMPs), are
188
V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
Fig. 1. Tyrosine metabolism via PO in insects haemocytes.
synthesized by bacteria and fungi [22]. PAMPs are essential and
unique components of virtually all microorganisms, but absent in
higher organisms [24]. Most of identified PAMPs are microbial cellwall components like lipopolysaccharides (LPS) of Gram-negative
bacteria, lipoteichoic acid and peptidoglycans of Gram-positive
bacteria and β-1,3 glucans of fungi.
A number of different haemocyte receptors involved in the
recognition of invaders, leading to both humoral and cellular
responses, have been reported in insect haemocytes. Important
insights into characterization of receptors have been gleaned mainly
from studies in Drosophila. Some of the haemocyte receptors appear
to be unique to Drosophila, whereas others have direct homologues to
mammals and other insect species (Table 1). Very probably, the
number of Drosophila homologues to other insect species will increase
as the research in this field expanded (Table 1). The activation of
receptors in response to invaders leads to the activation of certain
intracellular signalling molecules that are required for the completion
of immune responses (Table 2). The functional analysis of several
regulators in insect haemocytes has become possible with the
development of RNA interference technology. This technique is used
to silence specific gene expression by introducing double-stranded
RNA into the haemocytes that matches the nucleotide sequence of the
targeted mRNA. However, further identification and characterization
of regulators is required for a better understanding of the roles of
haemocytes in immune responses. The regulators of haemocyte
defense responses can be tentatively classified into three major
classes; a) secreted molecules (humoral receptors), b) putative
transmembrane receptors (surface receptors), and c) intracellular
Table 1
Haemocyte-surface homolog receptors in insects and mammals involved in immune
responses
Organisms receptors
Drosophila
Mosquitoes
Lepidoptera
Medfly
Mammals
Integrins
Dscam
Nimrod
PGRP-CL
dSR-Cl
Croquemort
Eater
Draper
Peste
LRP
Noduler
+
+
+
+
+
+
+
+
+
−
−
+
+
+
+
−
−
−
−
−
+
−
+
−
−
−
−
−
−
−
−
−
+
+
−
−
−
−
−
−
−
−
−
−
+
+
+
+
−
+
+
+
+
+
−
signalling molecules, including scaffold and adaptor proteins. These
three classes of candidate regulators are outlined below.
3.1. Humoral receptors
Humoral pattern recognition receptors circulate in the haemolymph of insects. Two humoral immune responses have been wellcharacterized; the induction and secretion of a battery of antimicrobial peptides by the fat body, an organ functionally equivalent to the
liver in humans [25] and melanization [26,27]. Some candidate
humoral regulators participating in cell-mediated immune responses
are listed below.
1) Immulectins are members of the C-type (calcium-dependent)
lectins, containing a dual carbohydrate receptor domain (CRD).
They have been identified in several insect species including M.
sexta [28–31], B. mori, Hyphantria cunea, Drosophila and A. gambiae,
[32,33]. Immulectins bind to LPS, lipoteichoic acid and fungal β1,3-glucan [29–31,34], and are implicated both in humoral and
cellular immune responses, such as proPO activation, phagocytosis
[35], nodule formation [36], encapsulation and melanization
[35,37].
2) Thioester-containing proteins (TEPs), are a major component of
the innate immune response of insects to invading microbes.
TEPs form a discrete group of proteins that includes the α2macroglobulins and complement factors in vertebrates. The
Table 2
Signalling molecules involved in haemocyte immune responses
Mammalian
signalling
molecules
Insect homologues
Drosophila
Mosquitoes
Medfly
Lepidoptera
ERK
JNK
P38
PI-3K
CEDs
FAK
Ets
STAT
Syk
TEPs
JAK
Rho
Rac1, Rac2, cdc42
Src
Eicosanoids
+
+
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
+
+
+
−
+
+
+
−
−
−
−
−
+
+
+
+
−
+
+
−
−
−
−
+
−
+
−
+
+
−
+
−
−
−
−
+
−
−
−
−
−
+
V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
3)
4)
5)
6)
7)
8)
Drosophila genome encodes for six TEPs, three of which are
upregulated after an immune challenge, while in A. gambiae
nineteen TEPs have been identified [38]. RNAi strategy confirmed
the role of the TEPs in phagocytosis in Drosophila [39] and in A.
gambiae [40].
LPS-binding protein, a C-type lectin, has been isolated and
characterized in B. mori [32]. It is a pattern recognition protein
that recognizes the lipid A of LPS and is implicated in several
cellular immune responses.
Peptidoglycan recognition proteins (PGRPs) [41–43]. PGRPs are
present in insects, molluscs, echinoderms, and vertebrates, but not
in nematodes or plants. Insects have up to 19 PGRPs, classified into
two categories; the short (S) and long (L) forms. The short forms
are mainly present in the haemolymph, cuticle, and fat body, and
the long forms are mainly expressed in haemocytes. Insect PGRPs
activate Toll or immune deficiency (Imd) signalling pathways or
induce proteolytic cascades that generate antimicrobial products,
phagocytosis, hydrolyze peptidoglycan, and protect insects against
infections [44]. In Drosophila, the three expressed PGRPs, one
soluble and two transmembranes, have been implicated in both
humoral and cellular immune responses [45].
The Gram-negative bacteria-binding protein (GNBP) was first
isolated from the silk worm B. mori as a protein that could bind
to the cell wall of Gram-negative bacteria [46]. Drosophila has three
different GNBPs (DmGNBP-1, -2, -3); DmGNBP-1 has been shown
to exist in both soluble and membrane-bound forms [47]. The
DmGNBPs contain a β-1,3 glucanase-like domain homologous to
that of Bacillus circulans β-1,3 glucanase, but lacking the two active
glutamic acid residues [46]. The Toll pathway in D. melanogaster is
activated by pattern recognition receptors, such as the Gramnegative bacteria-binding protein (GNBP) [47,48].
Beta 1,3-glucan recognition protein (βGRP), a component of the
surface of fungi and bacteria, was initially identified in the
silkworm B. mori and subsequently in several other lepidopterans.
By interacting with beta 1,3-glucan, βGRP initiates activation of
cell-free haemolymph propheloloxidase, a key enzyme in the
signalling pathway leading to melanotic encapsulation in insects
[49].
Haemolin, is a member of the immunoglobulin superfamily
[50,51]. Haemolin has been demonstrated in cell-free haemolymph
of several lepidopteran species [42]. However, no homologues have
been identified in Drosophila or A. gambiae genomes. The synthesis
of lepidopteran haemolin mRNA and protein are strongly induced
by microbial challenge [49,50]. Haemolin binds to both haemocytes and bacteria and therefore it appears to play a central role in
lepidopteran haemocyte immune responses in M. sexta [52].
B. mori multibinding protein (BmMBP). Two C-type lectins, BmLBP
and Bm MBP, were purified from B. mori plasma, identified as PRPs
and are shown to play a role in nodulation [53].
2)
3)
4)
5)
6)
7)
8)
3.2. Haemocyte-surface receptors
Several haemocyte-surface proteins are implicated in the immune
responses against invading microbes, by insect haemocytes. Their role,
if any, in the immune processes in response to bacteria has not been
documented, yet [3]. Two notable exceptions are the Drosophila
scavenger receptor dSR-Cl [54] and peptidoglycan recognition protein
[45]. As far as we know some of the haemocyte-surface receptors have
direct mammalian homologues, whereas others appear to be unique
in insects (Table 1). The most well known candidate cellular receptors
involved in recognition of pathogens in several insect species are
listed below:
1) The scavenger receptor dSR-CI in Drosophila is expressed in
haemocytes during embryonic development. It recognizes Grampositive and Gram-negative bacteria, but not yeast [54,55]. SR-CI
9)
189
expression is upregulated in larvae after exposure to bacteria [56].
It is conserved from insect to humans and may represent one of
the most primitive forms of bacterial recognition.
Peste is another class B scavenger receptor that can bind M.
fortuitum and has been identified in S2 Drosophila cells. This
finding suggest that mammalian B scavenger Receptors may also
be involved in the recognition of mycobacteria [57].
The Croquemort, a homologue of the mammalian CD36 family of
B scavenger receptors, is present in several insect species [58]. It is
expressed exclusively in plasmatocytes during embryogenesis,
and is mainly localized at the membrane surface of subcellular
vesicles that contain apoptotic cell corpses. It participates in
apoptotic cell removal in Drosophila embryos and in phagocytosis
of S. aureus [59]. However, it is not known whether this receptor
can trigger internalization by itself without assistance from coreceptors, since mammalian CD36 functions in concert with
vitronectin and phosphatidylserine receptors to engulf apoptotic
cell corpses [60]. It must be underlined that in Drosophila, a
phosphatidylserine homologue, expressed in phagocytic cells has
already been described, but no functional studies have yet shown
a role for this receptor for apoptotic cell removal [61].
The Eater, a transmembrane protein with epidermal growth factor
(EGF)-like repeats in its extracellular domain, mediates phagocytosis, as has been demonstrated with knockdown experiments, in
a broad range of bacteria in Drosophila [62]. It is the first EGF-like
repeat receptor shown to be involved in microbial recognition.
Eater is expressed in plasmatocytes and appears to recognize a
broad range of pathogens.
Nimrod, a putative phagocytic receptor in Drosophila plasmatocytes, is involved in the phagocytosis of bacteria. Nimrod-like
genes are also conserved in other insects such as Anopheles and
Apis melliphica. The Nimrod proteins are transmembrane proteins
with 10 EGF-like repeats that are similar to those found in Eater
and Draper from Drosophila and CED1 from C. elegans [63].
The Noduler, a protein identified in Antheraea myllita participates
in the clearance of bacteria by forming nodules of haemocytes
with bacteria. RNA interference experiments reduce significantly
the number of nodules. Noduler specifically binds LPS, lipoteichoic acid, and beta-1,3 glucan components of microbial cell walls
[64].
The Draper, a single-path membrane protein with EGF repeats
[65,66], an homologue of C. elegance CED-1 [67], is essential for
the clearance of apoptotic cells. It appears to be also a candidate
receptor for phagocytosis of apoptotic cells by embryonic
haemocytes in Drosophila. In addition, a recent report, provided
evidence that calreticulin could be the “eat me” signal in the
Draper pathway in Drosophila.
The Down syndrome cell-adhesion molecule (Dscam) is an Ig
superfamily receptor that binds to bacteria and is required for the
efficient phagocytosis by Drosophila plasmatocytes [54]. Dscam
can produce about 38,000 isoforms, via alternative splicing, with
distinct extracellular portions. Therefore, it can be concluded that
Dscam is sufficient to provide a wide range of microbial
recognition receptors in Drosophila. Similarly, it has been recently
demonstrated that in A. gambiae, depletion of Dscam, by RNAi in
cultured cells, decrease phagocytosis of E. coli and S. aureus. The
Dscam gene of A. gambiae, can also produce over 31,000 potential
alternative splice forms [68]. Dscam soluble forms have been
demonstrated in cells soluble phase and in the haemolymph.
Integrins or proteins with RGD-binding motifs have been demonstrated in insect haemocytes. RGD sequence is the primary
adhesive motif in many extracellular matrix molecules that
contains the amino-acid triplet Arg-Gly-Asp. In Drosophila
haemocytes they are required for the proper encapsulation of
wasp eggs [69]; in P. includens plasmatocytes and granulocytes,
they take part in recognition of both microbial and abiotic targets
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[70]; in S. damnosum, they kill microfilarial parasites [71]; in
medfly C. capitata, they are involved in bacteria (Gram-positive
and Gram-negative) phagocytosis by plasmatocytes, but not for
LPS or abiotic targets [5,72]; in A. gambiae, a new b integrin,
BINT2, is involved in E. coli engulfment [40] and in M. sexta, they
play a key role in stimulating haemocytes adhesion leading to
encapsulation [73,74].
10) The peptidoglycan recognition protein LC (PGRP-LC), participates
in phagocytosis of Gram-negative but not Gram-positive bacteria,
by S2 Drosophila cells [45]. Similarly, the mosquito PGRP-LC was
found to be involved in the phagocytosis of bacteria [40]. This
receptor is also required for activation of the Imd pathway by
Gram-negative bacteria [75].
11) Drosophila Toll is the first identified Toll family member and
consists of an extracellular leucine-rich repeat [76]. The ligand for
Toll receptor is Spatzle which is a secreted serine protease [77,78],
leading to a signalling pathway controlling the potent resistances
to fungi and Gram-positive bacteria. Toll receptor of the tobacco
hornworm, Manduca sexta, MsToll, is also a typical single-pass
transmembrane protein containing characteristic domain architecture of Toll and Toll-like receptors [79]. MsToll is expressed
in haemocytes, fat body, malpighian tubule, midgut and epidermis. Real-time PCR analysis showed that MsToll mRNA in
haemocytes is significantly induced by E. coli, as well as by yeast
(Saccharomyces cerevisiae) and Micrococcus lysodeikticus. It is suggested that MsToll may play a role in innate signalling in response
to E. coli infections.
The above data clearly shows that certain of the recorded regulators
are involved in several humoral (secretion of antibacterial peptides,
melanization) or cellular (phagocytosis, nodulation, encapsulation) or
both defense processes, supporting that these receptors are not
characterized by specificity and their classification in humoral or
cellular receptors is rather arbitrary. It must be also underlined that the
function attributed to the majority of these regulators for haemocytemediated immune responses, is not convincingly demonstrated
because their role in these processes in vivo hasn't been shown, yet.
It must be also pointed out that insect haemocytes surface
receptors, besides bacteria and fungi, have the ability to recognize,
many other living targets (nematodes, protozoan, insect parasites etc),
as well as abiotic targets (nylon, latex beads etc) that are made of
synthetic materials, normally not encountered in nature, during
evolutionary time. Usually different surface receptors are responsible
for different invaders, but in some cases, the same receptor recognizes
different ligands. For example, the RGD-binding receptors in P.
includens recognize abiotic targets, whereas RGD-binding receptors
in medfly haemocytes do not recognize abiotic targets [5,70].
3.3. Intracellular signalling molecules
Among the intracellular signalling molecules linking cell-surface
receptors with bacteria and other invaders, central role(s) have several
scaffold and adaptor proteins. Scaffold proteins are proteins that bind
other proteins that usually function in sequence. Adaptor proteins are
proteins that augment cellular responses by recruiting other proteins
to a complex. They usually contain several protein–protein interaction
domains. In other words, in addition to the direct interactions between protein kinases and their substrates, these molecules function
as organizing platforms that recruit both the kinase and the substrate
in the same complex [80].
In insects, innate immune responses are triggered by the immune
challenge and therefore involve signalling processes. Most of the data
concerning innate immune responsive signalling pathways stems
from the synthesis of antibacterial peptides by the fat body (Fig. 2a).
Although several humoral and haemocyte-surface proteins have been
proposed as candidate receptors in various insect species, participat-
ing in cell-mediated immune responses, the intracellular signalling
pathways, downstream of these receptors activated in response to
several invaders are not yet known and therefore, the available
information is limited. Bioinformatics approach strongly supports a
high homology of insect haemocyte signalling molecules participating
in immune responses to mammalian counterparts. It must be
underlined that the most data for intracellular signalling molecules
involved in immune responses is data for Drosophila (Table 2).
Evidently, the number of Drosophila homologues to other insect
species and vice versa will increase as the data in this field are
accumulated. Below we will try to delineate several homologues of the
vertebrate signalling molecules that appear to function in haemocytemediated immune responses.
3.3.1. Rho
Rho is a family of monomeric G proteins, comprising Rho, Rac and
Cdc42 that are homologous to Ras. These molecules are the main
regulators of actin cytoskeleton [81,82]. In the nematode Caenorhabditis elegens, Rac controls phagocytosis of apoptotic cells [59,83]. The
effect of Rho proteins during phagocytosis and engulfment in insect
model systems is also well documented. The medfly homologues of
Ras and Rho proteins have been shown to affect phagocytosis in
response to LPS and E. coli via Ras/Rho/MAPs pathway [84]. The Drosophila homologue Rac2 is required for bacterial engulfment [57,85],
for resistance to Pseudomonas aeruginosa and to a large set of Gramnegative and Gram-positive bacteria [86] and for the control
phagocytosis of apoptotic cells [59,83]. Recently, it has been also
reported that in Drosophila the signalling homologue molecules Rac1
and Rac2 are necessary for the proper encapsulation of eggs from the
parasitoid wasp Leptopilina boulardi [87,88].
3.3.2. FAK (Focal Adhesion Kinase)
Focal adhesions are highly specialized cell-adhesion structures
that connect actin filaments to the extracellular matrix via integrins,
and FAK is considered to be a component of central importance in
integrin signalling cascade [89]. FAK functions as a phosphorylationregulated scaffold to recruit Src. Upon integrin binding to pathogens,
FAK is autophosphorylated at Y397, which is a docking site for the SH2
domain of Src family kinases as well as PI-3K. As a consequence of Src
binding, other tyrosine residues of FAK, including Y576 and Y925 are
also phosphorylated, resulting to increased kinase activity of FAK and
additional docking of other (cytoskeleton-associated) adapters and
signalling molecules. This coordinated activation of FAK is critical in
diverse cellular processes such as focal adhesion formation/turnover,
cell spreading and migration, cell proliferation and apoptosis [89,90].
The expression of an insect FAK homologue, DFak56, has been
reported in the central nervous system, embryonic brain, epidermis,
nerve cord and visceral mesoderm of D. melanogaster [91]. DFAK56
shows a strong homology to mammalian FAK in domains critical for
the FAK function. Recently, knockdown experiments indicated that
dsRNA corresponding to FAK largely decreased the uptake of bacteria
and latex beads by medfly haemocytes. On the other hand, dsRNA for
FAK had no effect on the level of LPS uptake [90]. In addition, another
report in mosquitoes also shows that FAK facilitates the endocytosis of
West Nile virus (WNV), into C6/36 cell line [92], whereas DFAK56
controls morphogenesis of the Drosophila optic stalk [93]. In
mammalian systems the results concerning the involvement of FAK
in phagocytosis are controversial. It has been reported that FAK
promotes phagocytosis of integrin-bound photoreceptors [94] and is
involved in the uptake of Yersinia [95,96], whereas extracellular
pressure stimulates macrophage phagocytosis by inhibiting a pathway
involving FAK [97].
3.3.3. Src
Src belongs to the Src family of tyrosine kinases, the largest of the
non-receptor-tyrosine-kinase families. Src pathway controls a variety
V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
Fig. 2. Signalling pathways involved in haemocytes immune responses.
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V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
of biological processes, from cell proliferation and differentiation to
cytoskeletal rearrangements. Drosophila c-terminal Src kinase appears
to involve in maintaining epithelial integrity via c-terminal Src kinase
regulation [98]. Recently, it has been shown that certain Src signalling
levels have distinct outcomes in Drosophila. In particular, apoptotic
cell death was triggered specifically at high Src signalling levels; lower
levels directed antiapoptotic signals while promoting proliferation
[99]. Src is also involved in fusome development and karyosome
formation during Drosophila oogenesis [100]. The involvement of Src
in phagocytosis has been shown in Drosophila [101] and in medfly
haemocytes, as demonstrated with dsRNA for FAK [5,84,102].
3.3.4. Syk
Syk is a non-receptor tyrosine kinase, with a well established and
essential role in mammalian FcR signalling, which triggers several
pathways, leading, among others, to gene expression, actin cytoskeleton rearrangement and phagocytosis [22,103]. Although there is no
direct evidence, yet, involving Syk in insect haemocyte phagocytosis,
proteins with sequence similarity to human enzyme, containing the
conserved motifs, are encoded in the genomes of both Drosophila and
A. gambiae [104]. Shark, the homologue of Syk in Drosophila is
essential for Draper-dependent glial phagocytic activity [101].
3.3.5. Protein kinase C
Protein kinase c (PKC) appears to regulate the uptake of WNV virus
by mosquito C6/36 cells. This assumption is based on inhibition
experiments. Indeed, specific inhibitor for PKC is highly effective in
blocking WNV uptake by C6/36 cells [92].
3.3.6. MAP kinases
The MAP kinase family is a well-characterized intracellular,
evolutionary conserved, phosphorylation protein cascade, which is
implicated in the regulation of differentiation, cell proliferation,
development, inflammatory response, apoptosis and phagocytosis
[9,90,105–107].Three major MAP kinase subfamilies have been
characterized; the extracellular signal-regulated kinases (ERKs), the
c-jun N-terminal kinases (JNKs), also known as stress-activated
protein kinases (SAPKs), and the p38 mitogen activated protein. In
Drosophila, the following genes that encode MAPK have been
characterized a) rolled, the homologue of mammalian ERK, b) basket,
the homologue of mammalian JNK and c) dMAPKp38a and dMAPKp38b
the homologue of mammalian p38 [108].
Many studies in mammalian macrophages have shown that several
external stimuli, including LPS, E. coli and latex beads activate MAP
kinases [109].The same external stimuli have been also shown to
activate MAP kinases in insects. In particular, in Aedes albopictus cell
line C6/36 the pharmacological inhibitor SP600125 inhibits phagocytosis suggesting that the JNK-like protein regulates phagocytosis [110].
In the same system it was also found that JNK-like protein regulates
endocytosis of LPS and synthetic beads. In M. sexta haemocytes,
inhibition of ERK-like protein also decreased phagocytosis [104]. In
medfly haemocytes pharmacological inhibitors for ERK, JNK and p38
like proteins, largely decrease phagocytosis [5,9,84]. Similar results
were obtained in other invertebrate systems such as in freshwater snail
Lymnaea stagnalis [111], and in marine mussels Mytilus haemocyte.
In addition, in medfly haemocytes, FAK expression silencing in
response to FAK dsRNA treatment, in bacteria challenged haemocytes,
reduced the phosphorylation of MEK, ERK, JNK and p38 MAP kinases,
convincingly demonstrating that MAP kinases function downstream
of FAK, and their activation is vital for the efficient uptake by medfly
haemocytes [5]. Furthermore, it is known that alternative signalling
pathway(s) may also activate MAP kinases in response to the same
ligands, as well as MAP kinases are implicated in the regulation of
several other cellular processes. This data raises another, yet unanswered question of how cells discriminate the intracellular
signalling pathways promoted by external stimuli.
3.3.7. PI-3K
PI-3K is a heterodimeric enzyme consisting from a catalytic
110 kDa subunit and a regulatory 85 kDa (p85) subunit. PI-3K binds
to FAK via Y397 in vivo and in vitro [112]. Pharmacological inhibitors,
such as wortmannin, appear to decrease phagocytosis of bacteria in M.
sexta [104] and endocytosis of LPS in medfly haemocytes [84]. PI-3K
also appeared to be involved in apoptosis and autophagy in Drosophila
[113,114], as well as in medfly apoptosis [90]. PI-3K is also involved
in bacterial phagocytosis by haemocytes of the mollusc Lymnaea
stagnalis [115] and in macrophages [116].
3.3.8. Elk-1-like protein, protein containing the Ets DNA-binding site
In Drosophila eight Ets-related genes have been identified all
showing homology to genes in vertebrate species [117]. In Drosophila,
the Ets transcription factor, pointed, known to be downstream of the
Ras/MAPK pathway, appears to regulate cellular proliferation and
differentiation [117]. Ets proteins have also been identified in the fat
body of lepidoptera [118–120] and in the epidermis of M. sexta [121].
Bioinformatics and biochemical analysis strongly support that a
homologue protein with sequence similarity to human Elk-1 containing the conserved motif Ets, is encoded in the genome of medfly [12].
This protein, called Elk-1-like protein, as well as FAK, were demonstrated in both cytoplasm and nucleus of medfly haemocytes.
Interestingly, these proteins were found to be co-localized only in
the nucleus of haemocytes in response to pathogens [5,12].These
results strongly support that Elk-1-like protein is a novel proteinbinding partner for FAK, a finding that significantly broadens the
potential functioning of FAK and Elk-1 generally.
In addition, knockdown experiments using dsRNA for FAK and
osmotic loading experiments using Elk-1 antibodies [12], resulted the
blockage of Elk-1-like protein phosphorylation, indicating that an Elk1-like protein may be a candidate for the regulation of phagocytosis of
bacteria. Data from HK-2 human cells, based on knockdown experiments, indicate that Elk-1 may act as a potential physiological substrate
and regulator of FAK and MAP kinases expression [122].Evidently, the
HK-2 cells data must be confirmed in medfly haemocytes.
3.3.9. CED intracellular components
In A. gambiae knockdown experiments demonstrated that a
number of genes homologous to CED of C. elegans significantly affect
phagocytosis of bacteria [123]. In particular, in A. gambiae, silencing of
either CED5 or CED6 homologues resulted in 50% diminution of
phagocytosis of Gram-negative and Gram-positive bacteria. In addition, simultaneous inactivation of both CED5 and CED6 resulted in an
80% decrease of phagocytosis suggesting that at least two distinct
signalling pathways regulate the process of phagocytosis in A. gambiae
[40].
3.3.10. JAK/STAT
JAK/STAT signalling pathway takes place in the fat body and the
lymph gland-derived haemocytes (Fig. 2a,b). The main components of
this pathway are the ligand, Unpaired (Upd3), the receptor Dome, the
Hopscotch (JAK/Hop), and the STAT. Upd3 is a secreted protein, but
homologues have not been yet identified in vertebrates [124]. JAK/
STAT signalling pathway is implicated in the haematopoietic process
as well as in humoral and cellular responses [125]. JAKs are signalling
proteins that function downstream of many tyrosine kinase receptors
and activate the transcription factor STAT, which translocate to the
nucleus.
3.3.11. Eicosanoids
Eicosanoids are oxygenated metabolites of certain fatty acids,
including arachidonic acid. Phospholipase A2 (PLA2) is the enzyme
responsible for releasing arachidonic acid from cellular phospholipids.
PLA2 is present in the fat body and haemocytes, and is increased in
response to bacteria challenge [126]. Many studies with several insect
V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
species have documented the involvement of eicosanoids in several
insect haemocyte immune responses to bacteria, including phagocytosis, nodulation, haemocyte spreading and microaggregation
[18,127–130]. In addition, eicosanoids mediate prophenoloxidase
cascade activation only at the post-transcriptional level by inducing
release of prophenoloxidase from oenocytoids in the best armyworm
Spodoptera exigua, via haemocyte rupture [130].
3.3.12. ProPO activation system
The proPO activation system constitutes an important part of the
immune system in several invertebrate groups, including insects,
molluscs and echinoderms [131,132]. ProPO activation system is
composed by several proteins, including prophenoloxidase (proPO),
serine proteases, and their zymogens, as well as proteinase inhibitors.
The proPO activation system in insects, is one of the few pathways
participating and at the same time linking several humoral and
cellular defence reactions such as melanization, wound healing,
cytotoxic reactions, phagocytosis, encapsulation, nodulation as well
as the hardening and darkening of insect cuticle through sclerotization process [19,131,133,134].
The proPO cascade is activated in response to bacteria-challenge.
During proPO cascade activation, cell-free and haemocyte-surface
inactive PO is converted to active PO (Fig. 1) after limited proteolysis
through serine proteases [133,135–137]. Activated cell-free and
haemocyte-surface proPO catalyze the formation of quinones that
are reactive intermediates for melanization, nodulation and phagocytosis in several insects [11,19,133,138].
3.3.13. Ddc activity
The activation of haemocyte-surface proPO, however, is not
enough for the bacteria uptake by medfly haemocytes [5]. Experiments using siRNA for medfly dopa decarboxylase (Ddc), flow
cytometry, ELISA and microscopy, strongly support the expression of
Ddc on medfly haemocyte surface and its involvement in phagocytosis, nodulation and melanization [19].
Ddc is also involved in wound healing, parasite defense, cuticle
hardening, melanization and in the behaviour of insects [139]. Ddc is
found in epidermal, neural and ovarian cells and in haemocytes. In
Pseudaletia separate haemocytes the expression of Ddc mRNA was
enhanced by injection of an insect cytokine, growth-blocking peptide
[140]. Furthermore, microarray analysis demonstrated that Ddc levels
in Drosophila increased 11-fold after 3 h of septic infection with a
mixture of E. coli and M. luteus [141].
4. Intracellular signalling pathways
In mammals, the intracellular signalling molecules are organized
as communication networks that process, encode and integrate
internal and external signals and their relay stations are formed by
multiprotein complexes. These pathways regulate many fundamental
cellular processes, through branch points. However, little is known on
how these branch points are coordinated. In addition, it has recently,
become apparent that distinct spatio-temporal activation profiles of
the same repertoire of signalling proteins result in different geneexpression patterns and diverge physiological responses [142–144].
In insects, in contrast to mammals, the data for haemocyte
signalling molecules involved in immune responses is limited (Tables
1, 2), and evidently very little is known concerning their organization
in communication networks. The highly conserved signalling pathways during evolution, however, suggest that the expression of certain
signalling molecules in haemocytes, probably signal analogous pathways to mammalian counterparts. However, the signalling molecules
expressed in haemocytes and affecting immune responses but are not
involved in a signalling pathway are not included. Only the signalling
pathways which have already been described in haemocytes of several
insects and appear to be involved in immune responses are presented
193
below. The insect haemocyte data have been gleaned mainly from
studies in Drosophila.
a) In Drosophila, several signalling pathways, regulating humoral
and cellular responses, have been well documented. As stated earlier,
the humoral responses are mainly induced in fat body and the cellular
responses are mainly induced in haemocytes. A hallmark of the
humoral response is the release of antimicrobial peptides by the fat
body via Toll and Imd pathways. The Toll receptor is activated by
Gram-positive bacteria and fungi, whereas the Imd receptor is
activated via Gram-negative bacteria as well as LPS and PGN [145]
(Fig. 2a). Toll activation is triggered via spatzle, the ligand for Toll. Its
binding to Toll activates the signalling pathway Myd88/Pelle/Tube.
This leads to the degradation of Cactus, and nuclear translocation of
Dorsal and Dif transcription factors [146–148]. Toll pathway is also
required for proper encapsulation (Fig. 2b) [149].
b) The Imd pathway is activated by PGRP receptor [150,151] which
activates the transcription factor Relish, via either FADD/DREDD, or
TAK1/IKKβ/γ pathways (Fig. 2a). In addition, Imd via TAK1 pathway,
activates JNK which participates in several processes, such as
morphological changes during development, cell cycle regulation,
apoptosis and phagocytosis [5,12,105,152].
c) A third pathway that takes place in the fat body and is activated
upon septic injury, is the JAK/STAT signalling pathway (Fig. 2a). This
pathway, however, depends on haemocyte activation. In particular,
septic injury activates the Upd3 expression in haemocytes. The Upd3
is presumably secreted in haemolymph and subsequently activates
JAK/STAT pathway in the fat body. This pathway in turn regulates the
expression of TEP1 and TOT peptides [153]. TEP1 expression promotes
the phagocytic activity of a haemocyte-like cell line in mosquitoes
(Fig. 2c) [16]. Consequently, the JAK/STAT pathway in the fat body
assists haemocytes to their immune functions.
d) The JAK/STAT pathway also functions in lymph glands (Fig. 2b).
In particular, upon wasp parasitization plasmatocytes spread on the
surface of the eggs and evidently send signals to the lymph glands.
These signals trigger massive JAK/STAT-dependent differentiation of
lamellocytes in the lymph glands that are released into haemolymph
and encapsulate the wasp eggs.
e) In Drosophila haemocytes, another important component
promoting phagocytosis of apoptotic cells is Draper, the homologue
of C. elegans engulfment receptor CED1 (Fig. 2b). Drosophila shark, a
non-receptor tyrosine kinase similar to mammalian Syk, can associate
physically with Draper via the Draper ITAM intracellular domain. In
addition, it has been demonstrated that phosphorylation of Draper by
Src is essential for Shark/Draper interaction. Consequently, Draperdependent phagocytosis is mediated by the Draper/Src/Syk signalling
pathway (Fig. 2b). This is the first report of this signalling pathway in
invertebrates.
f) In the mosquito A. gambiae, two functional groups of genes,
referred as CED5 and CED6 appear to regulate the efficiency of
phagocytosis in E. coli (Fig. 2c). Components of the CED5 pathway
include the secreted putative TEP4, the integrin, BINT, and the
intracellular component CED2. The components of the CED6 pathway
include the secreted proteins TEP1, TEP3 and LRIM1, and the receptor
LRP [123]. Since TEP1 expression in Drosophila is regulated by the JAK/
STAT pathway it can be suggested that this pathway promotes the
phagocytic activity of a haemocyte-like cell line in mosquitoes [16].
g) In medfly haemocyte, phagocytosis, nodulation and melanization have been started to uncover [5,19,133].
Generally, the bacteria E. coli and S. aureus stimulate the signalling
wave integrins/FAK/Src/MAP kinases that leads to the activation of an
Elk-1-like protein [12] and in the secretion of several bioactive
molecules including protein activated serine proteases as well (Fig. 2c)
[5]. These pathways have been demonstrated with siRNAs for integrin
and Ddc, dsRNAs for FAK, immunoprecipitation, confocal analysis and
specific pharmacological inhibitors. Other external stimuli, such as
latex beads or LPS, also stimulate MAP kinases, but through distinct
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V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
pathways. Latex beads activate MAPs via an as yet unknown receptor
(s) and FAK pathway, whereas LPS activates MAPs through several as
yet unknown signalling molecules (Fig. 2c). Therefore, in medfly
haemocytes in response to different stimuli, distinct signalling pathways activate MAP kinases [5]. Several lines of evidence indicate that
in mammalian macrophages, neutrophils, under certain conditions,
FAK is also a major regulator of MAP kinase activation following
integrin stimulation.
The activated Elk-1-like protein via MAP kinases in response to
bacteria challenge is exclusively localized in the nucleus and is
functionally and physically associated with FAK [12] and participates
via a currently unknown mechanism in phagocytosis. The functional
and physical association of FAK with Elk-1 was confirmed in human
HK-2 cells [122].
On the other hand, the secretion of serine proteases via MAP kinases
in response to bacteria challenge activate the haemocyte-surface proPO
(Fig. 2c) [133], thus converting the circulating in the serum tyrosine to
dopa. Dopa is then converted to dopamine via haemocyte-surface Ddc
activity [19] (Figs. 1,2). It is likely, that dopamine-derived metabolites
that produce free radicals somehow are involved in the process of
engulfment, nodulation and melanization. However, phagocytosis and
nodulation depend on dopamine-derived metabolite(s), not including
the eumelanin pathway, whereas melanization depends exclusively on
the eumelanin pathway [19]. A key concept to emerge from these results
is that, in medfly haemocytes, a number of substrates and enzymes are
shared among phagocytosis, nodulation and melanization. It is not
known yet whether encapsulation also shares same components with
phagocytosis and nodulation.
In conclusion, the described insect models efficiently use phagocytosis as well as other immune responses to combat bacterial
infections. The documented different signalling pathways required for
efficient immune responses in the various insect species strongly
suggest that either these molecular mechanisms have diverged or
much work is still needed to explore the signalling pathways implicated in insect immune responses.
5. Summary
In this review we have highlighted our current knowledge of the
regulators as well as the signalling pathways required for insect
haemocyte immune responses. New techniques have uncovered
essential components of the haemocyte-mediated immunity. However, despite progress in understanding the complexity of immune
responses, our knowledge remains incomplete. Much work is still
needed to understand how haemocytes recognize invaders via transmembrane receptors and how the signals produced, are transmitted to
their targets. In addition, are phagocytosis, nodulation, encapsulation
and melanization linked and facilitate each other? Are there any
common signalling pathways in the various insect models or have
they diverged? Is there any extensive overlapping of signalling pathways between humoral and cellular immune responses?
Acknowledgments
We would like thank Dr. Sotiris Tsakas and Dr. Irene Mamali for the
critical reading of the manuscript.
References
[1]
[2]
[3]
[4]
M.D. Lavine, M.R. Strand, J Insect Physiol 47 (9) (2001) 965.
M.D. Lavine, M.R. Strand, Insect Mol. Biol. 12 (5) (2003) 441.
M.D. Lavine, M.R. Strand, Insect Biochem. Mol. Biol. 32 (10) (2002) 1295.
N.D. Charalambidis, C.G. Zervas, M. Lambropoulou, P.G. Katsoris, V.J. Marmaras,
Eur. J. Cell. Biol. 67 (1) (1995) 32.
[5] I. Lamprou, I. Mamali, K. Dallas, V. Fertakis, M. Lampropoulou, V.J. Marmaras,
Immunology 121 (3) (2007) 314.
[6] M. Meister, M. Lagueux, Cell Microbiol. 5 (9) (2003) 573.
[7] J.P. Gillespie, M.R. Kanost, T. Trenczek, Annu. Rev. Entomol. 42 (1997) 611.
[8] L.M. Henderson, J.B. Chappel, Biochim. Biophys. Acta 1273 (2) (1996) 87.
[9] I. Lamprou, S. Tsakas, G.L. Theodorou, M. Karakantza, M. Lampropoulou, V.J.
Marmaras, Biochim. Biophys. Acta 1744 (1) (2005) 1.
[10] P.G. Giulianini, F. Bertolo, S. Battistella, G.A. Amirante, Tissue Cell 35 (4) (2003)
243.
[11] E. Ling, X.Q. Yu, Insect Biochem. Mol. Biol. 35 (12) (2005) 1356.
[12] I. Mamali, K. Kapodistria, M. Lampropoulou, V.J. Marmaras, J. Cell Biochem. 103 (6)
(2008) 1895.
[13] S. Hernandez-Martinez, H. Lanz, M.H. Rodriguez, L. Gonzalez-Ceron, V. Tsutsumi,
J. Med. Entomol. 39 (1) (2002) 61.
[14] J.F. Hillyer, S.L. Schmidt, B.M. Christensen, Cell Tissue Res. 313 (1) (2003) 117.
[15] J.F. Hillyer, S.L. Schmidt, B.M. Christensen, J. Parasitol. 89 (1) (2003) 62.
[16] E.A. Levashina, L.F. Moita, S. Blandin, G. Vriend, M. Lagueux, F.C. Kafatos, Cell 104
(5) (2001) 709.
[17] L.F. Moita, G. Vriend, V. Mahairaki, C. Louis, F.C. Kafatos, Insect Biochem. Mol. Biol.
36 (4) (2006) 282.
[18] J.S. Miller, R.W. Howard, R.L. Rana, H. Tunaz, D.W. Stanley, J. Insect. Physiol. 45 (1)
(1999) 75.
[19] M. Sideri, S. Tsakas, E. Markoutsa, M. Lampropoulou, V.J. Marmaras, Immunology
123 (4) (2008) 528.
[20] A.J. Nappi, E. Ottaviani, Bioessays 22 (5) (2000) 469.
[21] A.J. Nappi, E. Vass, F. Frey, Y. Carton, Eur. J. Cell Biol. 68 (4) (1995) 450.
[22] A. Aderem, D.M. Underhill, Annu. Rev. Immunol. 17 (1999) 593.
[23] D.T. Fearon, Nature 388 (6640) (1997) 323.
[24] C.A. Janeway Jr, Cold Spring Harb. Symp. Quant. Biol. 54 (Pt 1) (1989) 1.
[25] A.M. Fallon, D. Sun, Insect Biochem. Mol. Biol. 31 (3) (2001) 263.
[26] B.M. Christensen, J. Li, C.C. Chen, A.J. Nappi, Trends Parasitol. 21 (4) (2005) 192.
[27] A.J. Nappi, B.M. Christensen, Insect Biochem. Mol. Biol. 35 (5) (2005) 443.
[28] X.Q. Yu, H. Gan, M.R. Kanost, Insect Biochem. Mol. Biol. 29 (7) (1999) 585.
[29] X.Q. Yu, M.R. Kanost, J. Biol. Chem. 275 (48) (2000) 37373.
[30] X.Q. Yu, E. Ling, M.E. Tracy, Y. Zhu, Insect Mol. Biol. 15 (2) (2006) 119.
[31] X.Q. Yu, M.E. Tracy, E. Ling, F.R. Scholz, T. Trenczek, Insect Biochem. Mol. Biol. 35
(4) (2005) 285.
[32] N. Koizumi, Y. Imai, A. Morozumi, M. Imamura, T. Kadotani, K. Yaoi, H. Iwahana, R.
Sato, J. Insect Physiol. 45 (9) (1999) 853.
[33] S.W. Shin, S.S. Park, D.S. Park, M.G. Kim, S.C. Kim, P.T. Brey, H.Y. Park, Insect
Biochem. Mol. Biol. 28 (11) (1998) 827.
[34] S.W. Shin, D.S. Park, S.C. Kim, H.Y. Park, FEBS Lett. 467 (1) (2000) 70.
[35] E. Ling, X.Q. Yu, Dev. Comp. Immunol. 30 (3) (2006) 289.
[36] N. Koizumi, M. Imamura, T. Kadotani, K. Yaoi, H. Iwahana, R. Sato, FEBS Lett. 443
(2) (1999) 139.
[37] X.Q. Yu, M.R. Kanost, Dev. Comp. Immunol. 28 (9) (2004) 891.
[38] G.K. Christophides, E. Zdobnov, C. Barillas-Mury, E. Birney, S. Blandin, C. Blass, P.T.
Brey, F.H. Collins, A. Danielli, G. Dimopoulos, C. Hetru, N.T. Hoa, J.A. Hoffmann, S.M.
Kanzok, I. Letunic, E.A. Levashina, T.G. Loukeris, G. Lycett, S. Meister, K. Michel, L.F.
Moita, H.M. Muller, M.A. Osta, S.M. Paskewitz, J.M. Reichhart, A. Rzhetsky, L.
Troxler, K.D. Vernick, D. Vlachou, J. Volz, C. von Mering, J. Xu, L. Zheng, P. Bork, F.C.
Kafatos, Science 298 (5591) (2002) 159.
[39] S.L. Stroschein-Stevenson, E. Foley, P.H. O'Farrell, A.D. Johnson, PLoS Biol. 4 (1)
(2006) e4.
[40] L.F. Moita, R. Wang-Sattler, K. Michel, T. Zimmermann, S. Blandin, E.A. Levashina,
F.C. Kafatos, Immunity 23 (1) (2005) 65.
[41] P. Bulet, C. Hetru, J.L. Dimarcq, D. Hoffmann, Dev. Comp. Immunol. 23 (4–5) (1999)
329.
[42] M.R. Kanost, H. Jiang, X.Q. Yu, Immunol. Rev. 198 (2004) 97.
[43] O. Schmidt, U. Theopold, M. Strand, Bioessays 23 (4) (2001) 344.
[44] R. Dziarski, D. Gupta, Genome Biol. 7 (8) (2006) 232.
[45] M. Ramet, P. Manfruelli, A. Pearson, B. Mathey-Prevot, R.A. Ezekowitz, Nature 416
(6881) (2002) 644.
[46] W.J. Lee, J.D. Lee, V.V. Kravchenko, R.J. Ulevitch, P.T. Brey, Proc. Natl. Acad. Sci.
U. S. A. 93 (15) (1996) 7888.
[47] Y.S. Kim, J.H. Ryu, S.J. Han, K.H. Choi, K.B. Nam, I.H. Jang, B. Lemaitre, P.T. Brey, W.J.
Lee, J. Biol. Chem. 275 (42) (2000) 32721.
[48] S. Pili-Floury, F. Leulier, K. Takahashi, K. Saigo, E. Samain, R. Ueda, B. Lemaitre,
J. Biol. Chem. 279 (13) (2004) 12848.
[49] Y. Wang, E. Willott, M.R. Kanost, Insect Mol. Biol. 4 (2) (1995) 113.
[50] N.E. Ladendorff, M.R. Kanost, Arch. Insect Biochem. Physiol. 18 (4) (1991) 285.
[51] S.C. Sun, I. Lindstrom, H.G. Boman, I. Faye, O. Schmidt, Science 250 (4988) (1990)
1729.
[52] I. Eleftherianos, F. Gokcen, G. Felfoldi, P.J. Millichap, T.E. Trenczek, R.H. ffrenchConstant, S.E. Reynolds, Cell Microbiol. 9 (5) (2007) 1137.
[53] M. Ohta, A. Watanabe, T. Mikami, Y. Nakajima, M. Kitami, H. Tabunoki, K. Ueda, R.
Sato, Dev. Comp. Immunol. 30 (10) (2006) 867.
[54] M. Ramet, A. Pearson, P. Manfruelli, X. Li, H. Koziel, V. Gobel, E. Chung, M. Krieger,
R.A. Ezekowitz, Immunity 15 (6) (2001) 1027.
[55] A. Pearson, A. Lux, M. Krieger, Proc. Natl. Acad. Sci. U. S. A. 92 (9) (1995) 4056.
[56] P. Irving, L. Troxler, C. Hetru, C. R. Biol. 327 (6) (2004) 557.
[57] J.A. Philips, E.J. Rubin, N. Perrimon, Science 309 (5738) (2005) 1251.
[58] Z. Nichols, R.G. Vogt, Insect Biochem. Mol. Biol. 38 (4) (2008) 398.
[59] N.C. Franc, P. Heitzler, R.A. Ezekowitz, K. White, Science 284 (5422) (1999) 1991.
[60] V.A. Fadok, D.L. Bratton, D.M. Rose, A. Pearson, R.A. Ezekewitz, P.M. Henson,
Nature 405 (6782) (2000) 85.
[61] P.M. Henson, D.L. Bratton, V.A. Fadok, Curr. Biol. 11 (19) (2001) R795.
[62] C. Kocks, J.H. Cho, N. Nehme, J. Ulvila, A.M. Pearson, M. Meister, C. Strom, S.L.
Conto, C. Hetru, L.M. Stuart, T. Stehle, J.A. Hoffmann, J.M. Reichhart, D. Ferrandon,
M. Ramet, R.A. Ezekowitz, Cell 123 (2) (2005) 335.
V.J. Marmaras, M. Lampropoulou / Cellular Signalling 21 (2009) 186–195
[63] E. Kurucz, R. Markus, J. Zsamboki, K. Folkl-Medzihradszky, Z. Darula, P. Vilmos, A.
Udvardy, I. Krausz, T. Lukacsovich, E. Gateff, C.J. Zettervall, D. Hultmark, I. Ando,
Curr. Biol. 17 (7) (2007) 649.
[64] A.S. Gandhe, S.H. John, J. Nagaraju, J. Immunol. 179 (10) (2007) 6943.
[65] M.R. Freeman, J. Delrow, J. Kim, E. Johnson, C.Q. Doe, Neuron 38 (4) (2003) 567.
[66] J. Manaka, T. Kuraishi, A. Shiratsuchi, Y. Nakai, H. Higashida, P. Henson, Y.
Nakanishi, J. Biol. Chem. 279 (46) (2004) 48466.
[67] T.L. Gumienny, E. Brugnera, A.C. Tosello-Trampont, J.M. Kinchen, L.B. Haney, K.
Nishiwaki, S.F. Walk, M.E. Nemergut, I.G. Macara, R. Francis, T. Schedl, Y. Qin, L.
Van Aelst, M.O. Hengartner, K.S. Ravichandran, Cell 107 (1) (2001) 27.
[68] Y. Dong, H.E. Taylor, G. Dimopoulos, PLoS Biol. 4 (7) (2006) e229.
[69] P. Irving, J.M. Ubeda, D. Doucet, L. Troxler, M. Lagueux, D. Zachary, J.A. Hoffmann,
C. Hetru, M. Meister, Cell Microbiol. 7 (3) (2005) 335.
[70] M.R. Strand, L.L. Pech, J. Gen. Virol. 76 (Pt 2) (1995) 283.
[71] H.E. Hagen, S.L. Klager, Parasitology 122 (Pt 4) (2001) 433.
[72] L.C. Foukas, H.L. Katsoulas, N. Paraskevopoulou, A. Metheniti, M. Lambropoulou,
V.J. Marmaras, J. Biol. Chem. 273 (24) (1998) 14813.
[73] D.M. Levin, L.N. Breuer, S. Zhuang, S.A. Anderson, J.B. Nardi, M.R. Kanost, Insect
Biochem. Mol. Biol. 35 (5) (2005) 369.
[74] S. Zhuang, L. Kelo, J.B. Nardi, M.R. Kanost, Dev. Comp. Immunol. 32 (4) (2008) 365.
[75] T. Kim, Y.J. Kim, J. Biochem. Mol. Biol. 38 (2) (2005) 121.
[76] D.S. Schneider, K.L. Hudson, T.Y. Lin, K.V. Anderson, Genes Dev. 5 (5) (1991) 797.
[77] E.A. Levashina, E. Langley, C. Green, D. Gubb, M. Ashburner, J.A. Hoffmann, J.M.
Reichhart, Science 285 (5435) (1999) 1917.
[78] A.N. Weber, S. Tauszig-Delamasure, J.A. Hoffmann, E. Lelievre, H. Gascan, K.P. Ray,
M.A. Morse, J.L. Imler, N.J. Gay, Nat. Immunol. 4 (8) (2003) 794.
[79] J.Q. Ao, E. Ling, X.Q. Yu, Mol. Immunol. 45 (2) (2008) 543.
[80] R.P. Bhattacharyya, A. Remenyi, M.C. Good, C.J. Bashor, A.M. Falick, W.A. Lim,
Science 311 (5762) (2006) 822.
[81] G.M. Bokoch, Trends Cell Biol. 15 (3) (2005) 163.
[82] A. Hall, Science 279 (5350) (1998) 509.
[83] P.W. Reddien, H.R. Horvitz, Nat. Cell Biol. 2 (3) (2000) 131.
[84] A.N. Soldatos, A. Metheniti, I. Mamali, M. Lambropoulou, V.J. Marmaras, Insect
Biochem. Mol. Biol. 33 (11) (2003) 1075.
[85] L.M. Stuart, J. Deng, J.M. Silver, K. Takahashi, A.A. Tseng, E.J. Hennessy, R.A.
Ezekowitz, K.J. Moore, J. Cell. Biol. 170 (3) (2005) 477.
[86] A. Avet-Rochex, J. Perrin, E. Bergeret, M.O. Fauvarque, Genes Cells 12 (10) (2007)
1193.
[87] M.J. Williams, I. Ando, D. Hultmark, Genes Cells 10 (8) (2005) 813.
[88] M.J. Williams, M.L. Wiklund, S. Wikman, D. Hultmark, J. Cell Sci. 119 (Pt 10) (2006)
2015.
[89] D.D. Schlaepfer, C.R. Hauck, D.J. Sieg, Prog. Biophys. Mol. Biol. 71 (3–4) (1999) 435.
[90] I. Mamali, M.N. Tatari, I. Micheva, M. Lampropoulou, V.J. Marmaras, J. Cell
Biochem. 101 (2) (2007) 331.
[91] R.H. Palmer, L.I. Fessler, P.T. Edeen, S.J. Madigan, M. McKeown, T. Hunter, J. Biol.
Chem. 274 (50) (1999) 35621.
[92] J.J. Chu, P.W. Leong, M.L. Ng, Virology 349 (2) (2006) 463.
[93] S. Murakami, D. Umetsu, Y. Maeyama, M. Sato, S. Yoshida, T. Tabata, Development
134 (8) (2007) 1539.
[94] S.C. Finnemann, Embo. J. 22 (16) (2003) 4143.
[95] P.J. Bruce-Staskal, C.L. Weidow, J.J. Gibson, A.H. Bouton, J. Cell Sci. 115 (Pt 13)
(2002) 2689.
[96] K.A. Owen, K.S. Thomas, A.H. Bouton, Cell Microbiol. 9 (3) (2007) 596.
[97] H. Shiratsuchi, M.D. Basson, Am. J. Physiol. Cell Physiol. 286 (6) (2004) C1358.
[98] P.F. Langton, J. Colombani, B.L. Aerne, N. Tapon, Dev. Cell 13 (6) (2007) 773.
[99] M. Vidal, S. Warner, R. Read, R.L. Cagan, Cancer Res. 67 (21) (2007) 10278.
[100] I. Djagaeva, S. Doronkin, S.K. Beckendorf, Dev. Biol. 284 (1) (2005) 143.
[101] J.S. Ziegenfuss, R. Biswas, M.A. Avery, K. Hong, A.E. Sheehan, Y.G. Yeung, E.R.
Stanley, M.R. Freeman, Nature 453 (7197) (2008) 935.
[102] A. Metheniti, N. Paraskevopoulou, M. Lambropoulou, V.J. Marmaras, FEBS Lett.
496 (1) (2001) 55.
[103] M.T. Crowley, P.S. Costello, C.J. Fitzer-Attas, M. Turner, F. Meng, C. Lowell, V.L.
Tybulewicz, A.L. DeFranco, J. Exp. Med. 186 (7) (1997) 1027.
[104] P. de Winter, R.C. Rayne, G.M. Coast, J. Insect Physiol. 53 (10) (2007) 975.
[105] D.C. Goberdhan, C. Wilson, Bioessays 20 (12) (1998) 1009.
[106] G.L. Johnson, R. Lapadat, Science 298 ((5600)`) (2002) 1911.
[107] S.L. Pelech, Curr. Biol. 6 (5) (1996) 551.
195
[108] S.J. Han, K.Y. Choi, P.T. Brey, W.J. Lee, J. Biol. Chem. 273 (1) (1998) 369.
[109] M.J. Sweet, D.A. Hume, J. Leukoc. Biol. 60 (1) (1996) 8.
[110] T. Mizutani, M. Kobayashi, Y. Eshita, K. Shirato, T. Kimura, Y. Ako, H. Miyoshi, T.
Takasaki, I. Kurane, H. Kariwa, T. Umemura, I. Takashima, Insect Mol. Biol. 12 (5)
(2003) 491.
[111] L.D. Plows, R.T. Cook, A.J. Davies, A.J. Walker, Biochim. Biophys. Acta 1692 (1)
(2004) 25.
[112] H.C. Chen, P.A. Appeddu, H. Isoda, J.L. Guan, J. Biol. Chem. 271 (42) (1996) 26329.
[113] D.L. Berry, E.H. Baehrecke, Cell 131 (6) (2007) 1137.
[114] T.E. Rusten, K. Lindmo, G. Juhasz, M. Sass, P.O. Seglen, A. Brech, H. Stenmark, Dev.
Cell 7 (2) (2004) 179.
[115] L.D. Plows, R.T. Cook, A.J. Davies, A.J. Walker, J. Invertebr. Pathol. 91 (1) (2006) 74.
[116] E. Garcia-Garcia, R. Rosales, C. Rosales, J. Leukoc. Biol. 72 (1) (2002) 107.
[117] T. Hsu, R.A. Schulz, Oncogene 19 (55) (2000) 6409.
[118] G. Sun, J. Zhu, L. Chen, A.S. Raikhel, Proc. Natl. Acad. Sci. U.S.A 102 (43) (2005)
15506.
[119] G. Sun, J. Zhu, C. Li, Z. Tu, A.S. Raikhel, Mol. Cell Endocrinol. 190 (1–2) (2002) 147.
[120] G. Sun, J. Zhu, A.S. Raikhel, Mol. Cell Endocrinol. 218 (1–2) (2004) 95.
[121] G.E. Stilwell, C.A. Nelson, J. Weller, H. Cui, K. Hiruma, J.W. Truman, L.M. Riddiford,
Dev. Biol. 258 (1) (2003) 76.
[122] I. Mamali, P. Kotsantis, M. Lampropoulou, V.J. Marmaras, J. Cell Physiol. 216 (1)
(2008) 198.
[123] S.A. Blandin, E.A. Levashina, Immunol. Rev. 219 (2007) 8.
[124] D.A. Harrison, P.E. McCoon, R. Binari, M. Gilman, N. Perrimon, Genes Dev. 12 (20)
(1998) 3252.
[125] H. Agaisse, N. Perrimon, Immunol. Rev. 198 (2004) 72.
[126] H. Tunaz, Y. Park, K. Buyukguzel, J.C. Bedick, A.R. Nor Aliza, D.W. Stanley, Arch.
Insect Biochem. Physiol. 52 (1) (2003) 1.
[127] R.G. Downer, S.J. Moore, W LD-J, C.A. Mandato, J. Insect Physiol. 43 (1) (1997) 1.
[128] M.B. Figueiredo, E.S. Garcia, P. Azambuja, J. Insect Physiol. 54 (2) (2008) 344.
[129] J.S. Miller, Arch. Insect Biochem. Physiol. 59 (1) (2005) 42.
[130] S. Shrestha, Y. Kim, Insect Biochem. Mol. Biol. 38 (1) (2008) 99.
[131] P. Jiravanichpaisal, B.L. Lee, K. Soderhall, Immunobiology 211 (4) (2006) 213.
[132] K. Soderhall, L. Cerenius, M.W. Johansson, Ann. N. Y. Acad. Sci. 712 (1994) 155.
[133] M.D. Mavrouli, S. Tsakas, G.L. Theodorou, M. Lampropoulou, V.J. Marmaras,
Biochim. Biophys. Acta 1744 (2) (2005) 145.
[134] M. Sugumaran, S. Tan, H.L. Sun, Arch. Biochem. Biophys. 329 (2) (1996) 175.
[135] M. Ashida, P.T. Brey, Proc. Natl. Acad. Sci. U. S. A. 92 (23) (1995) 10698.
[136] H. Jiang, Y. Wang, M.R. Kanost, Adv. Exp. Med. Biol. 484 (2001) 313.
[137] M.R. Kanost, H. Jiang, Y. Wang, X.Q. Yu, C. Ma, Y. Zhu, Adv. Exp. Med. Biol. 484
(2001) 319.
[138] N.D. Charalambidis, L.C. Foukas, V.J. Marmaras, Eur. J. Biochem. 236 (1) (1996)
200.
[139] R.B. Hodgetts, S.L. O'Keefe, Annu. Rev. Entomol. 51 (2006) 259.
[140] H. Noguchi, S. Tsuzuki, K. Tanaka, H. Matsumoto, K. Hiruma, Y. Hayakawa, Insect
Biochem. Mol. Biol. 33 (2) (2003) 209.
[141] E. De Gregorio, S.J. Han, W.J. Lee, M.J. Baek, T. Osaki, S. Kawabata, B.L. Lee, S.
Iwanaga, B. Lemaitre, P.T. Brey, Dev. Cell 3 (4) (2002) 581.
[142] B.N. Kholodenko, Nat. Rev. Mol. Cell Biol. 7 (3) (2006) 165.
[143] C.J. Marshall, Cell 80 (2) (1995) 179.
[144] L.O. Murphy, S. Smith, R.H. Chen, D.C. Fingar, J. Blenis, Nat. Cell Biol. 4 (8) (2002)
556.
[145] J.A. Hoffmann, Nature 426 (6962) (2003) 33.
[146] J.L. Imler, J.A. Hoffmann, Curr. Top Microbiol. Immunol. 270 (2002) 63.
[147] Y.T. Ip, M. Reach, Y. Engstrom, L. Kadalayil, H. Cai, S. Gonzalez-Crespo, K. Tatei, M.
Levine, Cell 75 (4) (1993) 753.
[148] N. Nicolas, C.L. Gallien, C. Chanoine, Dev. Dyn. 213 (3) (1998) 309.
[149] R.P. Sorrentino, J.P. Melk, S. Govind, Genetics 166 (3) (2004) 1343.
[150] K.M. Choe, T. Werner, S. Stoven, D. Hultmark, K.V. Anderson, Science 296 (5566)
(2002) 359.
[151] M. Gottar, V. Gobert, T. Michel, M. Belvin, G. Duyk, J.A. Hoffmann, D. Ferrandon, J.
Royet, Nature 416 (6881) (2002) 640.
[152] H.K. Sluss, Z. Han, T. Barrett, D.C. Goberdhan, C. Wilson, R.J. Davis, Y.T. Ip, Genes
Dev. 10 (21) (1996) 2745.
[153] H. Agaisse, U.M. Petersen, M. Boutros, B. Mathey-Prevot, N. Perrimon, Dev. Cell 5
(3) (2003) 441.