Drosophila that are deprived of haemocytes are clearly sensitized to infection. This was shown in larvae carrying a mutation called domino which results in extremely reduced haemocyte counts. When this mutation is combined with mutations affecting humoral immunity (imd) or melanization, resistance of larvae to infection is strongly decreased compared to single mutants (Braun et al., 1998). Similarly, inactivation of adult haemocytes by massive injection of polystyrene beads reduced the ability of imd flies to fight bacterial infection (Elrod-Erickson et al., 2000). Haemocytes are responsible for a number of immune functions in Drosophila, among which phagocytosis, encapsulation and melanization have been documented. We have outlined the current data on these immune reactions below.
In vertebrates, the elimination of microorganisms and the removal of apoptotic cells are achieved by the same phagocytes. However, whereas phagocytosis of non-self particles induces inflammation, that of apoptotic cells either downregulates or suppresses inflammation (Vandivier et al., 2002). Phagocytosis occurs in several steps. The first consists in the attachment of the phagocyte to the targeted particle followed by alteration of the cytoskeleton and internalization. The engulfed target is then destroyed within phagosomes by lysosomal enzymes, reactive oxygen species and nitric oxide (Jones et al., 1999). The latter phases are likely conserved between invertebrates and vertebrates, involving namely actin-remodelling and vesicule trafficking (Rämet et al., 2002a).
It has been proposed that phagocytosis of both apoptotic cells and microorganisms requires two processes: tethering, followed by actin-dependent engulfment (reviews in Aderem and Underhill, 1999; Fadok and Chimini, 2001; Hoffmann et al., 2001; Greenberg and Grinstein, 2002). Tethering of the target to the phagocyte is achieved by receptors that will either directly recognize determinants on microorganisms or apoptotic cells, or bind through opsonizing molecules. Most of these receptors have a broad recognition spectrum for both endogenous and exogenous molecules, and may participate in the phagocytosis of both types of targets. Mammalian examples are the Scavenger Receptor family (qualified as ‘molecular fly papers’ by Krieger et al., 1993). Tethering favours a tight association between the target and the phagocyte, and clustering of the binding proteins then mediates phagocytosis. The release of the signal triggering the appropriate on/off switch of inflammation seems to be dependent on the engulfment receptor that is simultaneously recruited. A few receptors are able to initiate engulfment, and they also participate in the recognition of the target. For ingestion of microorganisms, well studied receptors are the Mannose Receptor, that binds sugar moieties on microorganisms, two proteins of the integrin family, CR3 (αMβ2) and CR4 (αXβ2) that are receptors for the opsonizing complement factor C3bi, and the Fcγ receptors that recognize antibodies. Phagocytosis of apoptotic cells is dependent on the recognition of phosphatidylserine by its receptor (PS-R), but can also be mediated by two integrins αVβ3 and αVβ5, the tyrosine kinase MER and CD91 (Henson et al., 2001; Huynh et al., 2002).
In Drosophila, plasmatocytes are responsible for the disposal of both microorganisms (Fig. 1) and apoptotic cells. The mechanisms by which they recognize and engulf their targets are poorly understood. Phagocytosis of apoptotic cells at the embryonic stage requires wild-type function of the croquemort gene, which encodes a CD36 homologue (Franc et al., 1996, 1999). In mammals CD36 is a class B Scavenger Receptor which acts in concert with the vitronectin receptor (αVβ3) and PS-R to engulf apoptotic corpses (Fadok et al., 1998). A homologue of PS-R has also been described in Drosophila but no functional studies have yet demonstrated a role for this receptor in apoptotic cell removal (Henson et al., 2001). A scavenger receptor, dSR-CI with similar broad ligand recognition as mammalian class A Scavenger Receptor was identified by Pearson et al. (1995). dSR-CI can mediate binding to both Gram-negative and Gram-positive bacteria when transfected into CHO cells and participates to some extent to the binding of these microorganisms to S2 cells, a primary Drosophila blood cell culture (Rämet et al., 2001). PGRP-LC, a Peptidoglycan Recognition Protein, mediates phagocytosis of Gram-negative, but not Gram-positive bacteria by S2 cells (Rämet et al., 2002a). Interestingly, PGRP-LC belongs to a family of 12 members in Drosophila (Werner et al., 2000). This putative membrane bound protein is involved in the induction of the fat body Imd transduction pathway, leading to the production of antibacterial peptides after an infection with Gram-negative bacteria (Gottar et al., 2002). Similarly, PGRP-SA which is a soluble PGRP, is required for the proper induction of the Toll pathway after infection with Gram-positive bacteria (Michel et al., 2001). Gottar et al. (2002) have suggested that PGRPs are sensors which define the type of microbial infection and drive the proper signalling pathway. An additional family of recognition proteins found in insects are the Gram-negative bacteria-binding proteins or GNBPs which all contain gluconase-like domains. Three cDNAs encoding GNBPs have been cloned from a Drosophila blood cell line library. Although their role in phagocytosis has not been demonstrated, it was shown that one GNBP (DGNBP-1) can bind LPS and β-1,3-glucan (Kim et al., 2000). Although there is good evidence that Croquemort, dSC-RI or PGRP-LC can mediate phagocytosis of target particles, there is no demonstration that they can by themselves trigger internalization without assistance from co-receptors.
The Drosophila genome harbours six genes encoding thiolester-containing proteins or TEPs (Lagueux et al., 2000). These proteins are related to the α2macroglobulin/complement factor C3 family as they share the 12 signature domains characteristic of these proteins, which comprise the well conserved thiolester motif region. However, TEPs clearly form a new group in this family due to a distinct cystein arrangement in their C-terminal region. All putative TEPs possess a signal peptide indicating that they are secreted proteins. The transcription of three out of six tep genes (tep1, 2 and 4) is upregulated after an immune challenge and tep1 and tep4 are mainly expressed in haemocytes (Lagueux et al., 2000; and M. Lagueux, unpubl. obs.). We have proposed that TEPs function during an immune response either as opsonins to promote phagocytosis, in a C3-like manner, or as protease inhibitors, in an α2-macroglobulin manner. The best evidence so far, in favour of the opsonin hypothesis, comes from studies in an other insect, Anopheles gambiae. Levashina et al. (2001) found similar TEP proteins in this model insect and could show that phagocytosis of Gram-negative bacteria in an Anopheles blood cell line is strongly reduced when transcription of the atep1 gene is impaired.
It is likely that plasmatocytes also send signals to other immunocompetent tissues. When larvae are infected per os with the phytopathogenic Erwinia carotovora, they respond by the activation in the fat body of the Imd pathway with subsequent production of antibacterial peptides (Basset et al., 2000). In mutant larvae with a reduced number of haemocytes, such as domino larvae for instance, the Imd pathway is not activated in response to infection. This indicates that haemocytes act as messengers that somehow convey information to the fat body in the case of bacterial infection. However, the nature of the signal is still unknown.
Some 50 hymenopteran species are reported parasites of Drosophila (Carton et al., 1986). The wasp females lay their eggs in the haemocoel of young larvae. This foreign body is detected by plasmatocytes which are the first line immune supervisors in circulation. They readily attach to the chorion of the egg (Russo et al., 1996) and a few hours later a strong cellular reaction is observed in the haematopoietic organ with enhanced proliferation, increase in crystal cell numbers (Sorrentino et al., 2002), and massive differentiation of lamellocytes (Lanot et al., 2001). Lamellocytes then form a multilayered capsule around the invader, which is ultimately accompanied by blackening due to melanization (Fig. 1). Within the capsule, the parasite is eventually killed, by asphyxiation or by the local production of cytotoxic free radicals, quinones or semiquinones (Nappi et al., 1995; 2000). The whole process of parasite encapsulation raises several intriguing questions. The first relates to the signal that is produced by plasmatocytes once they have recognized an invader which they cannot phagocyte. This signal is perceived by prohaemocytes in the lymph glands which then differentiate into an adapted haemocyte lineage. The identity of the signal has yet to be determined. Thus lamellocytes, to some extent, exhibit adaptive characteristics as they only differentiate in response to a specific immune challenge. They form several layers of cells around the parasite, to which they are attracted either by the previously attached plasmatocytes, or by wasp determinants. The nature of this mechanism is also unknown.
Humoral melanization in arthropods produces black pigment as a result of the activation of a biochemical pathway that converts tyrosine to melanin (reviews in Ashida and Brey, 1997; Söderhäll and Cerenius, 1998). Melanization is controlled by a cascade of serine proteases that ultimately cleave the zymogen prophenoloxidase to its active form. Phenoloxidase then catalyses the oxidation of phenols to quinones, which polymerize non-enzymatically to form melanin. It was shown in lepidopterans that the cascade is activated by initial recognition of non-self molecular patterns such as β-1,3-glucans, peptidoglycan or LPS. The different elements of the cascade are not yet elucidated in Drosophila, but a key control serpin has been recently identified that restricts phenoloxidase activity to the site of injury or infection (De Gregorio et al., 2002; Ligoxygakis et al., 2002). Serpin-27 A regulates the melanization cascade through the specific inhibition of prophenoloxidase processing by the terminal serine protease. Three genes encoding prophenoloxidases [Fujimoto et al., 1995; Berkeley Drosophila Genome Project (BDGP)] are present in the Drosophila genome, and in larvae they are specifically expressed in crystal cells (M. Meister, unpubl. obs.), which, once activated, readily disrupt and deliver their content into the haemolymph where the enzymes can function. A number of intermediate compounds formed during melanin synthesis are cytotoxic (see above, killing of the parasite), it is thus important to strictly control the localization of the reaction. This is achieved not only by the regulatory serpins, but also by the fact that activated phenoloxidase shows a tendency to aggregate (Ashida and Brey, 1997).
Melanization was proposed to participate in the sealing of a wound before the more elaborate epithelial wound healing process (Lai-Fook, 1966) that takes several days. This is based namely on the observation that Drosophila melanization-deficient mutants show defects in wound healing with excessive bleeding and reduced survival (Rämet et al., 2002b). These observations could indicate a role for melanization in the coagulation process, or that the proteolytic cascades leading to melanization and to coagulation share components. Nothing is known to date on molecular mechanisms responsible for coagulation in Drosophila. It will thus be challenging to determine how melanization and coagulation are activated in this model, and how closely they are related.
Extracellular matrix production
Drosophila haemocytes are known to produce abundant extracellular matrix material. Indeed many proteins have been purified from the supernatant of Kc cell cultures which are of haematopoietic origin (review in Fessler et al., 1994; Nelsson et al., 1994; Goto et al., 2001). Among these, laminins, collagen IV, tenascinm, glutactin, peroxidasin and haemolectin were subsequently found to be strongly expressed in embryonic and larval haemocytes. However, the almost complete absence of haemocytes in domino larvae does not result in apparent structural tissue defects and so the significance of this synthesis is not yet clear. Some of these molecules exhibit peculiar features, namely peroxidasin which contains a putative peroxidase domain associated with several leucine-rich repeats similar to those found in the extracellular domain of Toll family members (Nelsson et al., 1994). Haemolectin displays similarity with the von Willebrand factor which in vertebrates plays a role in haemostasis (Goto et al., 2001). However, as no mutants for both these genes are available, their function has not been assessed to date.