Insect Immunology

book.

1

INSECT AND VERTEBRATE IMMUNITY: KEY SIMILARITIES VERSUS DIFFERENCES

OTTO SCHMIDT*, ULRICH THEOPOLD** and NANCY E. BECKAGE†,     *Insect Molecular Biology, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, SA 5064, Australia; **Department of Molecular Biology and Functional Genomics, Stockholm University, S10691 Stockholm, Sweden; †Departments of Entomology and Cell Biology and Neuroscience and Center for Disease Vector Research, University of California-Riverside, Riverside, CA 92521, USA

ABSTRACT

Historically, the mammalian adaptive immune system was the first to be analyzed in depth, providing strong paradigms on mechanisms of immune recognition and the distinction of self and non-self. However, the differentiation power of the innate immune system and the possible diversity of defense devices emerging in non-mammalian organisms may offer new perspectives on how multicellular organisms recognize potentially damaging objects or substances.

Chapter Outline

1.1. Introduction

1.2. Similarities

1.2.1. Sensing Mechanisms

1.2.2. Effector Mechanisms

1.3. Differences

1.3.1. Acquired (Adaptive) Immune System in Higher Vertebrates and Immunological Memory Involving Clonally Selected Antibody-Producing Cells

1.3.2. Inducible Tolerance and Memory in Invertebrates

Abbreviations:

LM =

leverage mediated

LPS =

lipopolysaccharide

MHC =

major histocompatibility complex

PAMP =

pathogen-associated molecular patterns

PAMS =

pathogen-associated molecular structures

RNAi =

RNA interference.

1.1 INTRODUCTION

Insects are a large and diverse group of animals that have adapted to extreme environments, including endoparasitic lifestyles, where one insect develops inside another insect. Compared to insects, with more than 32 orders, some containing hundreds of thousands of species, vertebrates are a relatively small group and rather homogeneous in morphology and physiology. Nevertheless, higher vertebrates have evolved a unique defense system in the form of an anticipatory immune response in addition to the innate immune system, which is common to all animals. The adaptive immune system is developed during early mammalian ontogeny, where gene rearrangements create thousands of gene variants encoding a repertoire of binding proteins that are clonally selected against self-recognition in immune-specific cells. After induction by foreign immunogens, a process which requires the involvement of the innate arm of the immune system, immune cells remain in readiness for future encounters with a potentially damaging object or dangerous substance.

While the adaptive immune system has impressive features involving the ontogenetic generation of antibody diversity and immunological memory, we have only started to analyze the differentiation power of the innate recognition system in vertebrates (Vivier and Malissen, 2005). Likewise, given the broad range of extreme and challenging environments in which some invertebrates live (Loker et al., 2004), we have not yet grasped the possible diversity of defense devices that may exist in non-mammalian organisms (Little et al., 2005). Comparisons of immune genes among related insect species (Drosophila and Anopheles) belonging to the same order of Diptera suggest very different defense strategies, using different protein families as recognition proteins and receptors (Christophides et al., 2004; Zdobnov et al., 2002). While immune strategies in most invertebrates remain to be analyzed, some already reveal highly specific and effective defense mechanisms that rival those of higher vertebrates.

Historically, the mammalian adaptive immune system was the first to be analyzed in depth using molecular biology tools. This has provided strong conceptual paradigms on how we perceive the mechanisms of immune recognition and the distinction of self and non-self. In fact, the notion that the specificity of immune recognition is determined exclusively by the nature of protein–epitope interactions has emerged from the formative power of antibody–antigen interactions that are highly specific and crucial for the immune response in specific immune cells. It is only recently that cell-free defense reactions uncovered multi-protein complexes upstream of cellular receptors (Schmidt and Theopold, 2004) that are relevant to recognition processes in insects. For example, cellular uptake reactions may be based on multi-protein complexes with enhanced detection capabilities due to combinatorial interactions between phagocytic receptors (Stuart and Ezekowitz, 2005) and upstream regulatory processes (Rahman et al., 2006). While these regulatory cascades were known to exist in many animal species (Krem and Cera, 2002), their relevance to insect immunity (Lemaitre et al., 1996) and development (Anderson, 2000) was uncovered first in Drosophila.

Another consequence of the conceptual preeminence of the adaptive immune system is the habit of some immunologists to use particular mammalian gene functions synonymous with general immune functions. Such is the strength of the mammalian paradigm that the presence of major histocompatibility complex (MHC) genes is sometimes correlated with the functionality of histocompatibility and the absence of MHC-like genes in other organisms is taken as evidence that these mechanisms do not exist in those organisms. But histocompatibility has been shown to be performed in many multi-cellular organisms, such as sponges (Fernandez-Busquets et al., 2002; Muller and Muller, 2003) or primitive chordates (De Tomaso et al., 2005) as part of a self-recognition mechanism, using different sets of proteins to achieve it (Litman, 2005).

In this chapter we use a comparison between insect and higher vertebrate immune reactions to highlight some of the progress that has been made in our understanding of how innate immune recognition has evolved to protect multicellular organisms against potentially damaging organisms. This also provides an opportunity to remind us of the large gaps that exist in our basic conceptual framework of how biological recognition processes work, particularly when it comes to the integration of immune functions with developmental and basic cellular functions, such as recognition of self.

1.2 SIMILARITIES

1.2.1 SENSING MECHANISMS

1.2.1.1 Recognition of Pathogen-Associated Molecular Structures

Biological recognition processes are generally perceived to be performed exclusively by the specific interactions of proteins with molecular structures that indicate non-self or altered-self. For example, the binding properties of antibodies, enabling the variable binding domains to attach to almost any molecular structure (antigens) and the ontogenic elimination process of self-binding antibodies by clonal selection, allow higher vertebrates with adaptive immune systems to signal non-self structures (Boehm, 2006; Mak and Saunders, 2006). Likewise, the evolution of specific binding proteins that interact with protein, lipid or sugar determinants (epitopes) that are unique to other organisms, identifies potentially damaging organisms in plants (Jones and Dangl, 2006) and animals (Janeway and Medzhitov, 2002). Since the innate immune system lacks clonal elimination mechanisms of self-binding proteins, the distinction between self and non-self relies on the selective accumulation of recognition protein repertoires that bind to target sites that are diagnostic for potentially damaging organisms (Janeway, 1989). These pathogen-associated molecular patterns (PAMPs) (Janeway, 1989) or pathogen-associated molecular structures (PAMSs) (Beutler, 2003) were selected in host organisms as target sites for potential recognition proteins. Thus, the distinction between self and non-self is based on the absence of microbe-specific structures in the host. A precondition for microbe-specific recognition by a host organism is that these structures are essential to the target organism and conserved enough to allow the host to evolve binding proteins before the pathogen has been able to eliminate or modify the target site. While the PAMS approach has been useful in providing a conceptual underpinning of innate self versus non-self recognition, it is not the complete picture, given that innate immune responses are more differentiated toward microbes than PAMSs predict, such as commensal microbes displaying PAMS without eliciting a response.

Another interaction with potentially damaging organisms is based on the so-called gene-for-gene interactions. Some pathogens require host-specific sites of interaction to gain access to host cells, which allows a host to become protected to pathogen attack by eliminating potential binding sites, or by adapting the interaction into a signal that identifies the presence of a damaging organism to the host. These gene-for-gene interactions have been identified in pathogenic or parasitic relationships with strong selection pressure, such as plant/pathogen (Jones and Dangl, 2006), insect parasitoid (Carton et al., 2005) or insect pathogen (Gottar et al., 2006) interactions.

One of the shortcomings of the PAMS-based concept of non-self recognition is that it lacks an explanation to the observed immune recognition of synthetic or altered-self structures. This highlights the question of what are the actual selection processes that produce specific recognition proteins. The evolutionary mechanisms associated with these processes have recently been brought into focus by the discovery of recognition molecules that produce diversity through alternative splicing (Dong et al., 2006; Watson, 2005). While this is a mechanism that can potentially produce enough diversity to match the genetic variability of short-lived microbes and parasites, the chances of self-recognition by one of more of the variant proteins are increased accordingly. At this stage we do not know whether alternative splicing is regulated by mechanisms that exclude self-binding proteins or whether the selection is at the level of individuals that produce novel splice products.

This raises the more general question of how organisms evolve specific binding proteins (Beutler and Hoffmann, 2004). How do organisms fight newly emerging pathogens and what happens when recognition proteins become obsolete because the pathogens have changed? Are hosts defenseless without specific recognition pathways?

1.2.1.2 Extracellular Sensor Particles

As indicated above, a major conundrum for microbe-specific recognition models is the documented recognition of objects or substances that have not been encountered by the organism or any of its ancestors. In fact, innate immune responses to artificial objects, such as plastic beads (Lavine and Strand, 2001) or some adjuvance substances (Matzinger, 1998), constitute a conceptual problem that is difficult to solve in the context of recognition systems that are based on the evolution of microbe-specific proteins.

Recent observations in insects suggest that lipid particles may serve a functional role not only as a lipid carrier (Canavoso et al., 2001; Rodenburg and Van der Horst, 2005), but also as sensor particles, being involved in the recognition and detoxification of lipopolysaccharide (LPS) (Kato et al., 1994) and other toxins (Vilcinskas et al., 1997) as well as in the production of reactive oxygen (Arakawa et al., 1996). Thus, lipid metabolism and lipid carrier proteins appear to play critical roles in the systemic immune response to parasites and pathogens (Cheon et al., 2006).

The role of the exchangeable apolipoprotein in insect lipid particles has long been implied in immune reactions, given that apolipophorin III was not only sensitive to particle–lipid composition (Sahoo et al., 2000; Soulages et al., 1996), but also to immune elicitors (Halwani et al., 2000; Niere et al., 1999) changing its structure (Niere et al., 2001) and affecting plasma-derived (Halwani and Dunphy, 1999; Wiesner et al., 1997) and cellular (Iimura et al., 1998; Whitten et al., 2004) immune activities.

Moreover, an association between lipid particles and typical immune proteins, such as phenoloxidase (Mullen and Goldsworthy, 2003), was further uncovered by the observation that lipid particles carry prophenoloxidase, its activating proteases and recognition proteins, such as LPS- and peptidoglycan-binding proteins (Rahman et al., 2006). It appears that in immune-induced insects lipid particles become associated with immune proteins and turn into adhesive particles with the potential to form cell-free aggregates (Ma et al., 2006; Rahman et al., 2006) or interact with cells (see below).

Genetic evidence for regulatory processes involved in immune recognition came from the observation that immune signals are often developed upstream of membrane-bound receptors and used in very different contexts. For example, the Drosophila Toll ligand Spaetzle is required for developmental and immune signaling (Lemaitre et al., 1996) and is activated to form a dimer as an outcome of regulatory processes that are reminiscent of proteolytic enzyme cascades activating pro-coagulants (Krem and Cera, 2002) and prophenoloxidase (Soderhall and Cerenius, 1998; see chapter 4 by Mike Kanost). In fact, the activated Spaetzle ligand resembles arthropod coagulogens at the structural level (Bergner et al., 1997; Delotto and Delotto, 1998).

What is the significance of these extracellular regulatory cascades to recognition processes? It appears that invertebrates with an open circulatory system are using coagulation reactions not only for wound healing, but also for the inactivation of pathogens (Theopold et al., 2004). While these extracellular defense reactions have long escaped our notice due to the difficulty of analyzing covalently linked coagulation products at the biochemical level, it has become apparent that the regulatory cascades controlling coagulation and melanization (Cerenius and Soderhall, 2004) are part of an ancestral defense reaction that has been adapted to multiple functions in different organisms (Krem and Cera, 2002). But while lipid-containing particles, such as lipophorin (Duvic and Brehelin, 1998; Li et al., 2002) and vitellogenin (Hall et al., 1999), are known to be the pro-coagulants in arthropods, we have only recently become aware that some plasma components, including immune proteins, are associated with lipid particles (Ma et al., 2006) changing their properties in the presence of elicitors (Schmidt et al., invited review). For example, lipophorin particles in insects interact with exchangeable lipoproteins and other plasma proteins, such as apolipoprotein III (Niere et al., 2001), prophenoloxidase and its activating proteases (Rahman et al., 2006), imaginal disk growth factors (Ma et al., 2006) and morphogens (Panakova et al., 2005). These modified particles may be involved in multiple processes, including lipid metabolism (Canavoso et al., 2001), immunity (Whitten et al., 2004), growth and development (Panakova et al., 2005).

Likewise, proteomic analysis of mammalian lipid particle composition revealed an association with immune proteins (Vaisar et al., 2007). Moreover, apolipoprotein E knock-out mice are highly susceptible to bacterial attack (de Bont et al., 1999) and protection against LPS by lipid particles (Kato et al., 1994; Vreugdenhil et al., 2003) may involve LPS transfer to and distribution among lipid particles (Kitchens et al., 2003; Levels et al., 2001). Finally, recent observations in mammals revealed upstream processes involving soluble CD14 and MD-2 proteins (Miyake, 2004; Visintin et al., 2001) that resemble some of the upstream processes observed in insects. Given the closed circulatory system with its threat of thrombosis, the most likely place to find aggregation-based defense reactions in vertebrates is probably outside the blood vessels, in the gut lumen, lung and other epithelia and in secretions, such as the milk.

The presence of all-pervading lipid particles involved in homeostatic functions inside and outside of cells is compatible with a sensor function. In fact, some exchangeable proteins, required for lipid uptake and transport, such as apolipoprotein III in insects, register lipid composition (Van der Horst, 2003) and the presence of LPS (Whitten et al., 2004). Together with the observation that purified lipid particles respond to LPS by aggregation (Ma et al., 2006), it is conceivable that the first steps in the recognition process involve the incorporation of LPS into the lipid moiety of the particle (Rahman et al., 2006). Likewise, these particles can respond to other changes, such as lipid modification under oxidizing conditions, with changes in their properties. For example, some of the associated proteins can become activated, either directly (Mellroth et al., 2005; Yu and Kanost, 2002) or indirectly through regulatory cascades (Krem and Cera, 2002) to switch from non-adhesive to adhesive forms (Fig. 1.1).

FIGURE 1.1 Cell-free defense reactions involving recognition and aggregation around elicitors or damaging objects. Lipid particles, which can act as circulating sensor particles, are schematically depicted as a disk of lipid bilayer surrounded by ring-shaped apolipoproteins. Associated proteins respond to elicitors (LPS) or environmental cues by becoming adhesive either directly ( Mellroth et al., 2005) or indirectly through regulatory cascades ( Krem and Cera, 2002). Adhesive lipid particles aggregate by cross-linking lipid particles around damaging objects or substances.

Lipid particles are ideal biological sensors, being ubiquitous in and around all cells, containing lipids and glycolipids that are easily modified and oxidized, providing the ability to respond readily to many changes in the environment. Moreover, being similar to cellular membranes they are able to mimic damaging effects by responding to environmental clues and by changing their functional properties into immune effectors by becoming adhesive particles that can aggregate around toxins or pathogens (Rahman et al., 2006). Cell-free defense capacities combining afferent and efferent immune functions (Beutler, 2004) have long been implicated in invertebrate hemolymph, vertebrate blood plasma and milk, but whether these reactions are all based on coagulation reactions remains to be elucidated. The difficulty with identifying lipid particles involved in cell-free defense using genetic and RNAi screens is that abrogation of lipid carrier functions are deleterious to the organism hiding any possible immune phenotype. Linking lipid particles to immune functions may prove even more difficult in vertebrates, given that the closed circulatory system of vertebrates may have evolved lytic and phagocytic defense functions to inactivate potential pathogens, rather than coagulation reactions (see below).

Taken together these observations are compatible with the existence of cell-free defense reactions, involving coagulation and melanization reactions upstream of cell-bound receptors. The importance of cell-free defense reactions is the notion that an immune signal is developed upstream of membrane-bound receptors by extracellular sensors, such as lipid-containing particles that change properties and become adhesive after exposure to damaging environments and elicitors. Adhesive lipid particles that self-assemble around damaging objects (Rahman et al., 2006) can potentially inactivate potential pathogens and toxins before they reach the cell surface. Conversely, the existence of particles with the capacity to inactivate damaging objects and substances raises the question of how adhesive particles are removed from circulation by cells.

1.2.1.3 Recognition of Self (Histocompatibility and Self-incompatibility)

Two cells from the same organism or cells from identical twins form shared flat membranes when they interact. As much as this constitutes the most visible manifestation of self-recognition that is common to all multi-cellular organisms (Burnet, 1971), we nevertheless have problems in explaining the process using instructive models of cell recognition (Fig. 1.2). Firstly, there is no apparent signaling involved in self-recognition, even though the exchange of information anticipated for the formation of a flat shared membrane between two cells could be formidable, involving the fine-tuning of mutual cell surface processes, such as adhesive forces and cell turgor. Secondly, while there are cytoplasmic scaffolds involved in receptor anchorage (Takeichi and Abe, 2005), the driving forces shaping self-based cell interactions appear to originate from processes on the cell surface following rules of thermodynamics and energy minimization as seen in air-filled soap bubbles (Janmey and Discher, 2004). Thirdly, self, altered-self and non-self recognition are performed by the same group of gene functions, such as those found in the vertebrate MHC locus or other similar gene functions in invertebrates. The fact that some of these genes are involved in self as well as in non-self processes is difficult to reconcile with unique receptor functions triggering instructive signaling pathways (Pradeu and Carosella, 2006). Finally, the detection of incompatibility between cells from closely related individuals of the same species is usually based on small allelic differences involving the absence of epitopes that are difficult to recognize even with the most sophisticated anticipatory receptor repertoire. The fact that incompatibility is seen in primitive multi-cellular organisms, such as sponges (Fernandez-Busquets et al., 2002; Muller and Muller, 2003) or primitive chordates (De Tomaso et al., 2005), suggests that self-recognition is not based on anticipatory recognition processes identifying dominant epitopes, but deeply embedded in the biology and functionality of cell–cell interaction that establish multi-cellular organisms or tissues in the first place.

FIGURE 1.2 Instructive model of innate immune recognition and cellular response using the receptor-mediated endocytosis as an example. Elicitors bind to receptors using lock and key interactions. Subsequent structural changes of the elicitor–receptor complex initiate signal pathways in the cytoplasm. In this model the signal is required to activate cytoplasmic machineries for the cell response.

This basic conundrum forces us to step back and ask some fundamental questions. For example, do we need specific immune recognition proteins to achieve self-recognition? Or are self-recognition processes part of a basic cellular functionality that are reflected in homeostatic sub-routines that only require signaling pathways when external or internal circumstances cause a departure from the sub-routine. For example, the shared flat membrane may not be the result of instructive signaling pathways, but the outcome of a dynamic interactive process on the cell surface involving adhesive and cellular uptake reactions that form a balance of receptor stabilization and receptor uptake reactions (Schmidt and Schreiber, 2006). This paradigm shift engenders the simple notion that some processes, such as self-recognition, are based on hard-wired cell surface-driven processes that only require signaling when imbalances indicate altered-self or non-self.

In this interactive model the involvement of multi-protein assemblies on the cell surface engaging adhesive receptors in both attachments (adhesion) as well as in uptake reactions that potentially lead to de-adhesion provides a novel approach to recognition, where cells actively engage with outside objects and other cells and in the process decode self–non-self properties. In line with this model, a stable arrangement between two cells sharing flat shared membranes is the outcome of a tug of war, where self is defined by the absence of instabilities, whereas changes in cell properties (altered-self) or inherent genetic differences in surface properties will indicate non-self, resulting in the formation of vesicles or internalization of cells or objects.

What are these multi-protein assemblies and how can one and the same receptor engage in adhesion and de-adhesion? The crucial assumption is that extracellular driving forces can shape the membrane and internalize objects and receptors (Schmidt and Theopold, 2004). Most interactions of cells with outside influences can be described as instructive processes, where signals are generated by receptors and transmitted to the cytoplasm or nucleus, causing activation of cytoplasmic machineries or differential gene expression (Fig. 1.2). Yet all available data are also compatible with mechanisms, where signal transmission can be the result of extracellular driving forces that mediate cellular uptake reactions and receptor internalization. A number of observations, where receptors without a signaling capacity, such as GPI-anchored receptors, or recombinant receptors with deleted cytoplasmic domains are still performing uptake reactions, suggest that signal transmission may be a consequence of uptake reactions not a prerequisite.

What are these extracellular driving forces? A conceptual clue comes from an almost forgotten mechanisms implying a zipper-mediated phagocytosis of objects (Swanson and Baer, 1995), where surface receptors are wrapped around the object forming a phagosome by a velcro-like mechanism (Fig. 1.3). While velcro-like mechanisms are clearly visible only in a few cases, such as heavily opsonized objects, it is obvious that surface properties and opsonins recruit receptors to the phagocytotic cup (Stuart et al., 2007) and given the type of opsonin, the uptake can be achieved by multi-functional receptors, including GPI-anchored receptors, such as scavenger receptors (Stuart and Ezekowitz, 2005). This suggests that any particle or object potentially interacts with receptors to sculpture the cell membrane, provided it has receptor binding sites or cell adhesion proteins attached to it.

FIGURE 1.3 Cellular uptake of non-self objects involving attachment and interactions leading to recognition of non-self. Adhesion and uptake of objects and microbes are driven by mechanisms that involve either velcro-mechanisms, where uptake is dependent on adhesive receptors wrapping the cell membrane around the object (black round object covered with lectins as opsonins), or cellular membrane invaginations dragging the object into the cell. Given the size differences of lipid particles and receptors adhesive lipid particles can be regarded as opsonized objects taken up by cells using velcro-like phagocytosis reactions. Since this involves a tilting of membrane-bound receptors around the particle, we called this an LM-uptake reaction ( Schmidt and Theopold, 2004). Clustering of lipid particles on the cell surface may drive the uptake of solid and liquid cargo by a cellular clearance reaction based on dynamic adhesion processes on the cell surface ( Schmidt and Schreiber, 2006).

If we accept that lipid particles are sensors that change properties after encountering environmental or immunological cues (Fig. 1.1), the most instructive change in properties is that lipid particles become adhesive (Ma et al., 2006), causing particles to engage with cellular receptors, depending on the adhesive properties of the particle (Rahman et al., 2006). In this context, adhesive lipid particles can be visualized as small opsonized objects, which interact with opsonin-specific receptors. Although lipid uptake has been described in the literature as a classical case of receptor-mediated endocytosis reactions (Goldstein et al., 1985), the size and sensor properties of lipid particles are also compatible with a possible uptake by a velcro-like mechanism, where receptors are wrapped around the object creating the inverse curvature of the membrane required for the uptake reaction (Fig. 1.3). In an analogy with phagocytosis reactions, the interaction of adhesive receptors with lipid particles causes receptors to tilt, thereby producing a membrane curvature (Fig. 1.3). Clustering of particle–receptor complexes on the cell surface will drive uptake reactions in a so-called leverage-mediated (LM) process (Schmidt and Theopold, 2004), leading to membrane curvatures, receptor internalization, endocytosis and phagocytosis. Thus adhesive receptors that bind to external binding sites as well as to adhesive lipid particles can potentially become involved in two opposite reactions, the cell attachment to substrate or the detachment of spread cells due to receptor internalization (Fig. 1.4). It is this dynamic balance of forces between two cells that only form a shared flat membrane when the two cells are identical (Fig. 1.5). The unique properties of this tug of war between two cells are that minute alterations in the composition of these multiprotein complexes may create an imbalance, causing vesicle formation or complete phagocytosis of altered-self or non-self cells and objects.

FIGURE 1.4 Dynamic interaction of adhesion (attachment of receptors to external binding sites) and lateral cross-linking with adhesive particles on the cell surface, causing receptor internalization ( Schmidt and Schreiber, 2006). Since lateral cross-linking of receptors (receptor movements in two-dimensional membrane) are thermodynamically favored over receptor binding to external binding sites (requiring movements in three dimensions), uptake by LM-mechanisms are favored over adhesion. To retain adhesive properties on the cell surface requires anchorage to cytoplasmic scaffolds, such as actin-cytoskeleton. Conversely, destabilization of cytoplasmic scaffolds may enhance macropinocytosis of existing clusters of LM-assemblies, but also prevent the formation of clusters.

FIGURE 1.5 Cell adhesion and cell interaction leading to self and non-self recognition. After initial adhesive contact by adhesive receptors (black arrows) disruption of membrane attachments to cytoplasmic scaffolds allows lateral movement of receptors. This leads to LM-membrane invaginations pulling membranes inward (white arrows) bringing the cells closer together and producing more adhesive contacts. Eventually the two cells form a shared flat surface of two adjacent membranes if the combined adhesive and LM-mechanisms are equally balanced (below left). The implications of the model are that cells from two different genotypes produce forces at the membranes that are not equally balanced and generate vesicle formation and possible phagocytosis (below right).

1.2.1.4 Recognition of Altered-Self (Apoptotic and Tumor Cells)

Histoincompatibility reactions to individuals from the same species are found in vertebrates and many invertebrates (Buss, 1987), including primitive chordates and sponges. This suggests that the mechanism involved in these processes is performed by the innate immune system, where self and altered-self recognition is paradoxically mediated by the same receptors (Stuart and Ezekowitz, 2005) that also act as effectors. For example, the detection of apoptotic cells and its uptake by macrophage-like immune cells or by neighboring epithelial cells is achieved by immune and scavenger receptors (Franc et al., 1996; Greenberg and Grinstein, 2002; Platt et al., 1999). This raises the question, what are the immunogens and how is phagocytosis of apoptotic cells accomplished? While a number of gene functions have been identified in Drosophila and C. elegans (Horvitz and Reddien, 2004; Mangahas and Zhou, 2005), the mechanism of recognition and apoptosis is still unclear (Seimon et al., 2006).

However, the unique properties of self-recognition as a tug of war between two cells, where minute alterations in the composition of multi-protein complexes can create a difference in the balance of forces, provide a basis for the engulfment of apoptotic cells without a need for signaling. For example, epithelial cells that change membrane properties in the form of displaying phosphatidylserine on the outer membrane leaflet may generate instabilities at the site of cell–cell interactions, resulting in the formation of vesicles (Fig. 1.5) that indicate altered-self causing the uptake of apoptotic cells, by neighboring or macrophage-like cells.

A similar conundrum is the recognition of tumor cells (Houghton, 1994). In higher vertebrates tumor cells are inactivated when antibodies recognize dominant tumor markers from chromosome rearrangements (Argani et al., 2001), protein phosphorylation (Zarling et al., 2006), change of glycodeterminants (Kumar et al., 2005; Osinaga et al., 1996; Santos-Silva et al., 2005) or virus infection (Kurosaki et al., 2005). Thus the conceptual basis for tumor surveillance in higher vertebrates is the presence of new antigens in cancerous cells (Rosenberg, 2001). However, genetic analysis of insect tumors revealed mostly recessive tumor suppressor functions (Gateff, 1978; Sparrow, 1979) that can be used to target human genes involved in cancer (Bier, 2005; Brumby and Richardson, 2005). Moreover, investigations in the direct immunogenicity of mutated self suggest that the accumulation of small changes in proteins, mostly truncations, is responsible for detection of altered-self rather than single big changes (Engelhorn et al., 2006). While the loss of protein structures and functions is not easily detected by anticipatory recognition systems that are geared toward recognizing dominant gain of new antigens’ (Pardoll, 2003), the problem seems to be impossible for PAMS-based innate recognition system. In this context it is conceivable that surveillance mechanisms based on tug of war-like interactions can detect even minor changes in multi-protein complexes that form shared flat membranes.

1.2.2 EFFECTOR MECHANISMS

1.2.2.1 Antimicrobial Peptide Response

Antimicrobial proteins are important defense molecules against microbes with unique structural properties that allow the permeation and disruption of target membranes. Antimicrobial peptides are believed to kill microorganisms via non-receptor-mediated mechanisms, although peptides, such as nisin Z, exist that bind to bacterial cell wall components. According to Shai (2002) monomeric peptides that have random structure gain amphipathic structures and form oligomers in hydrophilic solutions, such that the hydrophobic regions are buried in the lumen of the oligomer and the hydrophilic regions are exposed to the solvent. Upon reaching the membrane the organization is reversed, i.e. the hydrophobic regions are exposed to the lipid constituents of the membrane and the hydrophilic regions are segregated in the lumen of the oligomer (if the peptide monomers insert into the membrane and oligomerize via the barrel’ mechanism), or are exposed to the solution (if the peptide lies on the surface of the membrane and acts via the carpet mechanism).

The main question is, why are these peptides inserted in bacterial but not in eukaryotic membranes? One suggestion is that the positive net charge of antibacterial peptides enables binding and permeation of the negatively charged phospholipid membranes of bacteria but not of zwitterionic membranes, which are the major constituents of the outer membrane leaflet of most eukaryotic membranes (Shai, 2002). However, there are important exceptions, which suggest that charge per se is not critical, but other factors contribute to target specificity, such as the secondary and tertiary structure of the peptide (Ahmad et al., 2006). Apart from the observation that alterations in a peptide’s sequence are more likely to destroy its activity against eukaryotic than prokaryotic cells, there are no identifiable amino acid sequences that are responsible for the observed specificity (Hancock and Rozek, 2002). The fact that diastereomeric analogs (Papo et al., 2002) and hybrid antimicrobial peptides (Merrifield et al., 1995) are active suggests that target specificity is dependent on the sterical properties rather than specific protein binding activities. The main argument put forward to explain peptide activity against bacterial membranes is that cholesterol and ergosterol, which are found in eukaryotic membranes, protect against the insertion of antibacterial peptides (Boman, 2003). Although this has been confirmed in artificial membrane systems for most peptides, it raises the question why some antifungal peptides and antimicrobial peptides, such as mellitin, are toxic to some fungal species and many eukaryotic cells?

Some of the interactive mechanisms described above may be involved in peptide interactions with the cell membrane (Schmidt et al., 2005) and drive insertion into the membrane (Schmidt and Theopold, 2004). However, we are far from providing testable models for these processes.

1.2.2.2 Phagocytosis (Clearance of Damaging Objects)

Phagocytosis, the cellular uptake of particulate substrate, is a fundamental cellular process in all eukaryotic organisms and essential for the clearance of damaging objects in multi-cellular organisms (Stuart and Ezekowitz, 2005). While many organisms have specialized cells that engage in phagocytosis, such as the professional phagocytes in higher vertebrates and macrophage-like hemocytes in insects, it is important to remember that the capability to recognize and engulf objects and substances is inherent to all cells. This has been demonstrated in nematodes, where epithelial cells are able to recognize altered-self properties in neighboring cells undergoing programmed cell death and subsequently remove dead cells by phagocytosis (Mangahas and Zhou, 2005). Given the basic requirements of uptake reactions to cell function it is quite surprising how little is known about the molecular forces that drive this reaction. The perception is that phagocytosis, like endocytosis, is mediated by trigger mechanisms, where receptors interact with the object and subsequently instruct the cytoplasmic machinery to engulf the object (Scott et al.,