A defense response comprises three steps: a first is recognition of non-self which initiates the subsequent steps of the defense process; a second activates the defence mechanisms which in vertebrates can consist of the synthesis of various interleukins or the activation of the complement system by serine esterase cascades; a third neutralizes non-self material by various processes: agglutination, cytolysis. The main difference between invertebrates and vertebrates concerns the first step, i.e. recognition. Invertebrates and vertebrates can, by lectin-like receptors, recognize LPS, PHA and various molecules acting on lymphocytes as polyclonal activators. In addition to this polyclonal type of recognition, however, vertebrates are capable of monoclonal activation due to their possession of a new class of receptors and rearranging genes which control their presence (Cooper, E. et al).

In insects, immune systems were impressed by the strong and rapid antibacterial reactions induced in these animals after the injection of bacteria into the blood. Even notorious human pathogens, like the agents of pneumonia, bacterial dysentery, typhoid fever and cholera, were efficiently dealt with if the insects had first been immunized by attenuated bacteria . Unlike the B- and T- cell-based adaptive immunity in vertebrates, there is no evidence for clonal selection mechanisms, so that resistance to bacteria unrelated to the immunizing agent is also enhanced. Furthermore, the immune response usually shows no 'memory'. In many ways the insect immune system is reminiscent of the innate immune reactions in vertebrates, and it is possible that the two systems are related. One component of innate immunity is the acute phase response, whereby infection or trauma induces the liver to secrete a variety of proteins that play a protective role during the acute phase reaction (Ron et al). At least two dramatic reactions are immediately triggered in insects by wounds or foreign objects: the activation of phenoloxidase and the clotting of the hemolymph (Hultmark, D). The prophenoloxidase (proPo) activating system which acts as a major recognition and defence pathway in crustaceans and insects. This system comprises a complex cascade of serine proteases and other factors, located in invertebrate blood cells or haemocytes. This cascade apparently mediates recognition and directs blood cell activity in a manner similar to complement (Cooper E.L., et al). The fruit fly, Drosophila melanogaster, is an excellent model for a molecular and genetic approach to insect immunity, and some of the genes that are activated during the immune response have now been cloned from this species (Hultmark, D).

Drosophila responds to the inoculation of bacteria within a few hours by the de novo synthesis of several peptidic or polypeptidic molecules. Only a few of these peptides have been fully characterized to date. These antimicrobial peptides have been isolated from other species and tissues. For example, magainins have been purified from skin and gut of the African clawed frog Xenopus laevis and have been detected immunologically in human saliva; have also been isolated from pig intestine. Analogous antimicrobial peptides have been described in bovine neutrophils, bovine tracheal mucosa, the hemocytes of the horseshoe crab, and the lymph fluid of honeybees (Groisman, E.a., et al). The first information on the sequences of inducible antibacterial peptides of Drosophila was obtained from DNA cloning studies published in 1990 (Hoffmann, J.A. et al). These molecules generally have a wide spectrum of activity against Gram-positive and/or Gram-negative bacteria. To date, several distinct inducible antibacterial peptides or peptide families have been totally or partially characterized. They can be grouped as follows:

(1) The cecropins are 4-kDa cationic peptides active on both Gram-negative and Gram-positive cells (Steiner, H. et al)(Fig.1a). The cecropins are highly amphipathic molecules that form two amphipathic a -helice separated by a 3-residue hinge region and interact with lipid membranes, creating voltage-dependent ion channels of variable size. In bacteria, they cause the permeability barrier of the cell membrane to break down (Hultmark, D).

(2) A second major group of membrane-active peptides induced in immunized insects are the cysteine-containing insect defensins, or sapecins. The insect defensins are 4-kDa anti-Gram-positive peptides with six cysteins engaged in three intramolecular disulfide bridges (Hoffmann, J.A. et al). They consist of three distinct domains: an amino-terminal loop, an amphipathic a -helix, and a carboxyl-terminal antiparallel b -sheet (Cociancich, S. et al)(Fig.1a). The sequence homology among the four dipteran defecsins presented is very high. Insect defensins show structural similarity to potassium-channel-blocking scorpion toxins like charybdotoxin. In contrast to the cecropins, which cause lysis within a minute, killing of bacteria by insect defensins is relatively slow. It appears likely that the defensins act by a different mechanism from that of the cecropins (Hultmark, D). Cociancich, S. et al showed that defensin disrupts the permeability barrier of the cytoplasmic membrane of Micrococcus luteus, resulting in a loss of cytoplasmic potassium, a partial depolarization of the inner membrane, a decrease in cytoplasmic ATP, and an inhibition of respiration. It is also inhibited by divalent cations and by a decrease in the membrane potential below a threshold of 110 mV. They found that insect defensins form channels in giant liposomes. The synthesis of insect defensin is elicited by a variety of stimuli, including both live and heat-killed Gram-positive and Gram-negative bacteria, and complete Freund's adjuvant (Hoffmann, J.A. et al).

(3) Several polypeptides, ranging in size from 8 to 27 kDa, frequently rich in glycine residues, are active essentially on Gram-negative bacteria: the attacins, sarcotoxin II, diptericins, and coleoptericin (Fig.1b). The attacin-like proteins affect dividing cells of Escherichia coli and some other Gram-negative bacteria, causing them to grow in long chains (Ishikawa, M. et al). The mechanism of action has been investigated for attacin, which specifically blocks the synthesis of the major outer-membrane proteins in E. coli (Carlsson, A., et al), leading to the breakdown of the integrity of the outer membrane. The Drosophila diptericin gene codes for a 9 kDa antibacterial peptide and is rapidly and transiently expressed in larvae and adults after bacterial challenge. (4) Small sized, proline-rich peptides, active against Gram-negative cells, are found at the amino terminus of diptercin and sarcotoxin II, and a high proline content is also characteristic of a little -understood group of bactericidal peptides from honey-bees (apidaceins, abaecin) and Drosophila (Bulet, P et al). The mechanisms of these antibacterial peptides were summarized in Fig. 2.

Increasing evidence suggests that this "immune reaction" is analogous to innate immunity in vertebrate, which includes a spectrum of defense reactions that are not mediated by antibodies. Revealing common elements of pathway in invertebrates in important undertaking if the evolutionary origins of immune recognition and ultimate unraveling of immune responses can be achieved.

Tryselius, Y. et al cloned the cecropin locus from Drosophila meanogaster and mapped it to 99E on the third chromosome. From this locus, they characterized four cecropin genes that are induced by the injection of bacteria and the Andropin (Anp) gene coding for a male-specific antibacterial peptide (Fig.3). In vaccinated larvae and adults, most of the cecropin mRNA comes from two of the genes, CecA1 and CecA2, whereas the third gene, CecB is much less active. At these stages, the fat body was shown to be the major site of synthesis although some transcription could also be demonstrated in hemocytes (Samakovlis, C., et al). In the early pupa stage (metamorphosis) , a different pattern of cecropin gene expression was evident. Although total cecropin gene expression is lower, CecB and CecC gene are most active. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. All cecropin genes are coordinately induced after the injection of bacteria, and it was therefore natural to search the promoter regions for conserved sequence elements. When CecC is compared to the other cecropin genes, a region of sequences similarity becomes apparent upstream of all cecropin genes. The conserved upstream region consists of three separate motifs (Fig.4). The central AC-rich motif is common to all four genes, with ten or eleven base pairs matching the 12-nucleotide consensus sequence AAAAATCCCCGT. This sequence is centered approximately 40-50 bp upstream of the TATA box. 4 bp downstream of this motif is the perfectly conserved sequence GCCTTATC, present in the CecA and CecC genes, and 5-10 bp upstream of the AC sequence is a GT-rich region with the consensus GTGTACTTTT, which is absent in the CecC gene only.

The promoter of diptericin gene contains two 17 bp repeats located closely upstream of the TATA-box and harbouring a decameric k B-related sequence (Fig.5). Diptericin contain a single intronless gene which is expressed predominantly in the fat body shortly (1-2 h) after injection of low doses of bacteria. ( Ksppler, C.,et al). In promoter positions, there repeats function as cis-acting elements that confer inducible transcriptional activity. The ability of proteins from induced Drosophila to bind to the proximal promoter regions was first tested by a DNaseI footprinting approach. Five regions (Fig. 6) were protected with fat body extracts of challenged larvae. Region I extends over 30 bp and contains three motifs related to established binding elements of mammalian immune response genes, namely: an interferon consensus response element (ICRE) and sequences related to binding sites for NF-IL6 and NF-k B; these potential binding sites are partially overlapping. Region II contains a second, identical, k B-related binding site; region III an additional site related to the mammalian NF-IL6 binding site and region IV a second interferon consensus response element. In region V, a sequence is present which is frequently associated with liver specific expression in mammalian genes where it binds the activator HNF-5 (Hepatic Nuclear Factor 5)(Georgel, P., et al). The genes for several of the inducible immune proteins of insects have now been cloned and summarized in Fig.7. There include the Drosophila genes that encode the cecropins and diptercin genes discussed above. Many of the antibacterial proteins are encoded by families of closely related genes, some of which show different patterns of expression. The first clues to how transcription of immune genes is controlled came form the observation that several insect immune genes share an upstream motif that is reminiscent of the binding site for the mammalian transcription factor NF-k B, a well established regulator of immune and acute-phase responses in mammals (Hultmark, D.).

Insects look nothing like vertebrates, and their organ systems seem to be built on entirely different principles. Nevertheless, as we get a better understanding of how these systems operate at the molecular level, unexpected similarities are emerging (Hultmark 1994). A potential link between innate immunity in mammals and insect immunity is suggested by the demonstration that the insect fat body is the primary site of synthesis of bactericidal polypeptides (Hultmark, D. 1993). The fat body shares numerous common features with the mammalian liver, including the use of related regulatory factors to control Adh expression (Abel, T., et al; Falb, D., et al ). Moreover, the promoter regions of a number of insect immunity genes, including the cecropins and diptericin, contain k B-like binding sites, suggesting that they might be regulated by transcription factors related to NF-k B (Sun , S-C., et al 1992). This was the first indication of such a connection between insect and vertebrate immune responses. They noticed that many of insect antibacterial genes share a common upstream motif, similar to the binding site for NF-k B, a mammalian member of the Rel family of transcription factors (Fig.8). In vertebrate, there are two classes of acute phase proteins: class I proteins are induced by the interleukin-1 (IL-1) cytokine, while class II proteins are induced by IL-6( Baumann et al). Recent studies suggest that the class I (IL-1) pathway is mediated, at least in part, by NF-k B (Edbrooke et al), a regularity factor containing a Rel homology domain (Ruben et al). The biological function of mammalian NF-k B is to rapidly induce gene expression upon extracellular stimulations that signal distress and pathogen invasion (Baeuerle, P.A.). One of the established signals is LPS, and k B enhancers can serve as response elements for LPS. In mammals, NF-k B and other Rel proteins control the expression of acute phase proteins in the liver and participates in lymphoid cells in the inducible expression of cytokines and cell surface receptors. NF-k B is present in uninduced mammalian cells as an inactive complex with an inhibitor protein, Ik B, and the activation involves dissociation of this inhibitor from its cytoplasmic complex with NF-k B and nuclear translocation of the active transcription factor (Kappler C., et al). Ip, Y.T. et al presented evidence that these genes are indeed regulated by a Rel-containing protein, suggesting that innate immunity is an evolutionarily ancient process.

The maternal morphogen dorsal (dl) is also a Rel-containing gene and has been previously identified in Drosophila (Steward,R.). It is responsible for patterning the dorsoventral (DV) axis of the early embryo ( Roth et al). There are similarities in the regulation of gene expression along the DV axis of the Drosophila embryo and lymphoid-specific expression in the mammalian immune system. Reichhart et al found that the dorsal gene itself also may be involved in the immune response. This gene is expressed at a low level in fat body cells of larvae and adults, but levels of messenger RNA increase when the immune system is stimulated by lipopolysaccharide. Both depend on Rel-containing proteins (dl and NF-k B) that are regulated at the level of nuclear transport. Dl and NF-k B are retained in the cytoplasm by the inhibitory proteins cactus (Geisler, R. et al) and Ik B (Inoue, J.-I., et al), respectively, which are related and contain a series of ankyrin repeats. The sequence of cactus has some similarity to gene of Ik B family. The dl-cact and NF-k B-Ik B complexes might be dissociated by related intracellular signaling systems. Once dl and NF-k B are released to the nucleus they interact with target promoters that sometimes contain closely linked k B(or dl)-binding sites and E boxes ( binding sites for helix-loop-helix proteins). Cooperative interactions between dl and helix-loop-helix proteins have been shown to be important for initiating gene expression in the early embryo (Gonzalez-Crespo, S. et al). To test that dl forms a multimer complex with a genetically invisible Rel protein present in early embryos, Ip, Y.T. et al identified one novel gene called Dif (Dorsal-related immunity factor).

Dif mediates an immune response in Drosophila. The and dl genes are physically linked and map within 100 kb of each other. Dif is not significantly expressed in early embryos, at the time when dl initiates DV patterning, but instead shows peak expression during larval, pupal, and adult stages. Dif can bind k B-like motifs in the CecA1 cecropin promoter. An induced nuclear factor from mbn-2 cells that binds this sequence upon lipopolysaccharide (LPS) stimulation is specifically recognized by anti-Dif antibodies. Immunolocalization studies reveal that the Dif protein is normally localized in the cytoplasm of fat bodies but rapidly accumulates in the nucleus when larvae are injected with Escherichia coli or damaged by manual puncture. These results suggest that mammalian and insect immunity share a common evolutionary origin ( Ip, Y.T. et al). Fig.9 showed that comparison of three pathways of gene activation mediated by Rel protein. In mammals, NF-k B and other Rel proteins have a central role in the transcriptional activation of immune-related factors such as immunoglobulins, interleukins and the proteins of the acute phase response. Next, taking advantage of Drosophila as an experimental system, two groups demonstrated the functional importance of the k B-like motif in the Drosophila cecropin and diptericin genes, by introducing various gene constructs in transgenic flies, and in a Drosophila blood-cell line. There are in fact several surprising similarities between the signal system used by the fly to instruct the embryo which side is up and which is down, and the one that mediates the effects of interleukin-1 (IL-1) in mammalian immune system. In both cases, inactive Rel proteins (NFk B or dorsal) are bound to Ik B-like inhibitors (Ik B and cactus) in the cytoplasm. After activation, the Rel proteins are translocated into the nucleus where they mediate the transcriptional activation of target genes. Furthermore, the ventral-specific signal to the embryo is mediated by a membrane receptor, encoded by the Toll gene, which is homologous in its cytoplasmic part to the IL-1 receptor (IL-1R). The homologue of dorsal was discovered that is inactive in the embryo that is inactive in the embryo but becomes expressed during later stages of development. This gene, Dif , is preferentially expressed in the fat body, the main site of synthesis of the antibacterial proteins, and it was therefore considered to be a good candidate for the immunoresponsive transcription factor. Dif protein binds specially to the k B-like element form the cecropin gene, whereas the dorsal protein does not. Like other Rel proteins, Dif is normally cytoplasmic, but becomes nuclear in the fat body after induction with bacteria. Some cross-talk may be possible with components of the dorsoventral pathways; a mutant in which Toll is constitutively activated induces nuclear localization of Dif, as well as increased expression of the Dif gene (Hultmark D. 1994).

The comparison of the evolution of the vast numbers of species that have emerged and vanished, and in comparisons of the species living today, which have survived only because of their high level of adaptability, of which immune mechanisms undoubtedly formed a significant part. It is necessary to focus research efforts on nontraditional models (the contemporary knowledge of immunology is derived virtually solely form the immunology of the mouse) because the information thus gained is more than likely to be of extreme importance and even of practical use.

Baeuerle, P.A. (1991) Biochim. Biophys. Acta. 1072:63-80.

Baumann, H., et al (1989) Stimulation of hepatic acute phase response by cytokines and glucocorticoids. Ann. NY Acad. Sci. 557:280-95.

Bulet, P. et al (1993) A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution. J. Biol. Chem. 268(20): 14893-897.

Carlsson, A., et al (1991) Attacin, an antibacterial protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in Escherichia coli by interfering with omp gene transcription. Infect. Immunol. 59: 3040-45.

Cociancich, S., et al (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 26:19239-45.

Cooper, E.L., et al (1992) Invertebrate Immunity: Another Viewpoint. Scand. J. Immunol. 35:247:266.

Edbrooke, M.R., et al (1991) Constitutive and NF-k B-like proteins in the regulation of the serum amyloid A gene by interleukin-1. Cytokines 3, 380-88.

Groisman, E.A., et al (1992) Resistance to host antimicrobial peptides is necessary for Salmonella virulence. Proc. Natl. Acad. Sci. USA. 89:11939-43.

Hoffmann, J.A. et al (1993) The humoral antibacterial response of Drosophila. FEBS 325 (1,2):63-66.

Hoffmann, J.A. et al (1992) Insect defensins: inducible antibacterial peptides. Immunol. Today. 13:411-415.

Hultmark, D. (1993) Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet. 9: 178-183.

Inoue, J.-I., et al (1992) Ik Bg , a 70 kd protein identical to the C-terminal half of p110 NF-k B: a new member of the Ik B family. Cell 68: 1109-1120.

Ip, Y.T. et al (1993) Dif, a dorsal-related gene that mediates an immune response in Drosophila. Cell 75: 753-63.

Ishikawa, M., et al (1992) Purification and characterization of a diptericin homology from Surcophaga peregrina (flessh fly) Biochem. J. 287: 573-78.

Geisler, R., et al (1992) cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the Ik B gene family of vertebrates. Cell. 71:613-21.

Gonzalez-Crespo, S., et al (1993) Interactions between dorsal and helix-loop-helix proteins initiate the differentiation of the embryonic mesoderm and neuroectoderm in Drosophila. Genes Dev. 7:1703-13.

Ron, D., et al (1990) Transcriptional regulation of hepatic angiotensinogen gene expression by the acute phase response. Mol. Cell. Endocrinol. 74:97-104.

Roth, S., et al (1989) A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell 59: 1189-1202.

Ruben, S.M., et al (1991) Isolation of a rel-related human cDNA that potentially encodes the 65-kd subunit of NF-k B. Science 251:1490-93.

Samakovlis, C., et al (1990) The immune response in Drosophila: pattern of cecropin expression and biological activity. EMBO J. 9:2969-76.

Steiner, H. et al (1981) Sequence and specificity of two antibacterial proteins involoved in insect immunity. Nature 292: 246-248.

Steward, R. (1987) Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel. Science 238: 692-94.

Tryselius Y., et al (1992) CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae. Eur. J. Biochem. 204: 395-99.