Abstract ISJ 7: 146-148, 2010 ISSN 1824-307X VISIONS AND PERSPECTIVES Cytokine network in invertebrates: the very next phase of comparative immunology D Malagoli Department of Biology, University of Modena and Reggio Emilia, Modena, Italy Accepted May 20, 2010 Abstract Information on invertebrate cytokines has been growing impressively in the past five years. However, molecular characterization of newly discovered cytokines has not proportionally improved our understanding of the main reason underpinning their conservation among metazoans. One possible explanation is that cytokines have been conserved for the fundamental processes they control, but in mammals a single cytokine can hardly be considered as controller of complex reactions. In mammals, cytokines constitute a network of communication, and only this network can be considered the real controller of the effects that cytokines exert on immune or developmental functions. In all, the capability of constituting a network could represent the principal reason for cytokine evolution and conservation through diversification of metazoans. Key Words: cytokines; innate immunity; invertebrates; evolution Introduction In mammals, cytokines are described as molecules responsible for the regulation of the maturation, proliferation, differentiation and survival of lymphocytes, macrophages and dendritic cells, and are further classified as lymphokines, monokines, interleukins and chemokines based on their origin and function (Corbellini, 2010). Among invertebrates (paraphiletic term but useful for the purposes of the manuscript), cytokines are present but much less characterized. No more than 6 years ago, the knowledge on invertebrate cytokines was limited and based on indirect evidence (Ottaviani et al., 2004), and the very existence of possible counterparts of vertebrate cytokines was disputed (Beschin et al., 2004). Recent experiments have introduced dramatic changes in this scenario, so that it could be said now we are moving into the “second age” of invertebrate cytokines. The “second age” of invertebrate cytokines: state of art and future perspectives In the last three years, several reports on invertebrate immunity have used the terms “putative cytokine”, “cytokine-like” etc. (Malagoli et al., 2007; Parrinello et al., 2008; Roberts et al., 2008; Zhang et al., 2008; De Zoysa et al., 2009, 2010; Schikorski et ___________________________________________________________________________ Corresponding author: Davide Malagoli Department of Biology, University of Modena and Reggio Emilia, Via Campi 213/D, 41125 Modena, Italy E-mail: davide.malagoli@unimore.it al., 2009), but the factors that are commonly accepted as full-title invertebrate cytokines at present are Spätzle (Ferrandon et al., 2004) and Unpaired (Upd)-3 (Agaisse et al., 2003) in Drosophila melanogaster, Insect chemotactic peptide (ICP) in the moth Pseudaletia separata (Nakatogawa et al., 2009) and Astakine-1 in Pacifastacus leniusculus (Söderhall et al., 2005). These cytokines do not present traits of similarity with vertebrate cytokines, but seem to be widespread in different invertebrate models (An et al., 2010; Hsiao et al., 2010), and their receptor and the signalling pathway they elicit appear to be conserved among metazoans (Malagoli et al., 2010). Interestingly, while the research for cytokine in invertebrates have been prompted mainly by evolutionary-aimed studies (Beschin et al., 2004; Ottaviani et al., 2004), very few considerations on the evolution of cytokines have been made after the discovery of full-title cytokines. It may appear a paradox, but more speculations on the possible history of cytokines can be realized starting from molecules that at present appear to be candidate cytokines. Indeed, some of those molecules, namely Drosophila Helical Factor (DHF) (Malagoli et al., 2007), Ciona intestinalis Tumor necrosis Factor (CiTNF) (Parrinello et al., 2008) and Hirudo medicinalis endothelial monocyte-activating polypeptide II (HmEMAP-II) (Schikorski et al., 2009) promise to give a significant contribution for the understanding of the evolution of cytokines. DHF, or more simply HF as indicated in FlyBase (www.flybase.org), is a molecule with a predicted structure typical of the vertebrate helical 146 cytokines (Huising et al., 2006). The discovery of HF (Malagoli et al., 2007) represented a significant contribution supporting the hypothesis that the molecular structure is a component of equal importance to gene and protein sequences in evolutionary studies (Malagoli and Ottaviani, 2007). CiTNF is another molecule in which sequence conservation is limited to key region of the molecule (Parrinello et al., 2008), and the same is true for HmEMAP-II (Schikorski et al., 2009). In these respects, it has to be remarked that differently from HF, there is still no information about the activity of CiTNF and HmEMAP-II in Ciona and Hirudo, respectively, because their discovery has not been followed by a detailed functional analysis, yet. Functional assays, based for instance on the utilization of native and recombinant molecules, are however necessary in order to unravel if the structure/sequence conservation corresponds to similar function. However, even among the speculations on the evolution of cytokines, there is a fundamental aspect raised by the discovery of cytokines in invertebrates that has been neglected by comparative immunologists. In their efforts to isolate and characterize cytokines in invertebrates, researchers have been keeping their focus on the specific molecule under study. This led to very detailed and complete characterizations (Nakatogawa et al., 2009), but some studies seemed almost concluded with the discovery and characterization themselves. The risk for the next future is to have a long list of new cytokines well-characterized in terms of molecular aspects, but very limited information on their implications for evolutionary biology. The presence of cytokines in organisms with quite different evolutionary histories make it difficult to understand the basis of their conservation. The most obvious explanation is that these molecules are ancient signals that, regulating reactions fundamental for survival and homeostasis maintenance, have been conserved in structure and function. But if we step back to the principle reference of comparative biologists, i.e., mammals and, above all, humans, we can observe that the term "cytokine" indicates a soluble factor that necessarily acts together with many other cytokines in order to determine an effect (Corbellini, 2010). In mammals, the importance of cytokines relies principally on their capability of constituting molecular networks, and the functional meaning of a single cytokine is almost absent if we do not consider that molecule acting within a specific molecular context with many other players. Accordingly, human immunologists have articulated numerous and complex proposals to describe the interactions between different systems and mediators, e.g., the bow-ties (Ottaviani et al., 2008). Molecules identified as cytokines have been conserved in diverging metazoan taxa for hundreds million years, and it should be asked if this is due to their capacity to interact and constitute molecular networks, or to function as single signal molecules. The object of natural selection have been cytokines or cytokine networks? Admittedly, only in D. melanogaster more than a cytokine has been identified at present, but if we consider the amount of data collected in the past five years and the possibilities offered today by bioinformatic approaches and the high throughput technologies, the discovery of several other invertebrate cytokines appears just a matter of time. Now the complete molecular characterization of single factors is a reality in several invertebrate models, the very next phase of comparative immunology must be the identification of networks of cytokines as occurs in vertebrate species. 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