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Discrepant effects of mammalian factors on molluscan cell motility, chemotaxis and phagocytosis: divergent evolution or finely tuned contingency?
Davide Malagoli and Enzo Ottaviani1
Department of Biology, University of Modena and Reggio Emilia, Modena, Italy
Cell motility, cell migration and phagocytosis are distinct, though frequently sequential, processes. They are fundamental for the maintenance of homoeostasis in single cells as well as in pluricellular organisms. Like vertebrates, invertebrate immune functions are strictly dependent on cell motility, chemotaxis and phagocytosis. Several comparative immunobiology experiments have tested the effects of mammalian factors on cell migration and phagocytic activity in invertebrate immune-competent cells. The discrepancies that were found suggest various hypotheses, e.g. species-specific reactions to heterologous factors. Here, we reconsider data concerning the effects of POMC (proopiomelanocortin)-derived peptides, cytokines and growth factors on molluscan immunocytes in the light of recent findings that also encompass the effects of experimental conditions.
Key words: cell migration, innate immunity, invertebrate, phagocytosis
Abbreviations: ACTH, adrenocorticotropic hormone, CRH, corticotrophin-releasing hormone, IL, interleukin, PDGF-AB, platelet-derived growth factor, POMC, proopiomelanocortin, TGF-β1, transforming growth factor-β, TNF-α, tumour necrosis factor-α, YTX, yessotoxin
1To whom correspondence should be addressed (email email@example.com).
Part of a series marking the 70th birthday of the Cell Biology International Editor-in-Chief Denys Wheatley
According to Manske and Bade (1994), cell shape changes are an expression of cell motility, while chemotaxis is connected with cell migration.
Chemotaxis, defined as non-random locomotion, allows the cell to move towards a chemoattractant, which is recognized. Moreover, if the chemoattractant is particulate material such as bacteria, chemotaxis may be followed by engulfment and phagocytosis. These processes may be used by animal organisms to discriminate ‘self’ from ‘non-self’, and they may be divided conceptually into recognition and attachment of a particle to the surface of the phagocyte and internalization of the bound particle (Rabinovitch, 1970; Silverstein et al., 1977; Klebanoff and Clark, 1978).
The assumption that chemotaxis is followed by phagocytosis is not always true. For instance, chemotaxis and phagocytosis experiments performed in molluscs using different mammalian molecules as chemoattractants for the circulating immunocytes, e.g. POMC (proopiomelanocortin)-derived peptides such as ACTH (adrenocorticotropic hormone) and β-endorphin, cytokines and growth factors, displayed results that changed significantly as a function of the stimulant. However, from the collected results, it is possible to describe the probable sequence of events that follows the stimulation of invertebrate immunocytes. The first event that is always observed is a change of cell shape, which may be followed by chemotaxis and/or phagocytosis.
2. Cell shape changes
In the late 1990s, morphological changes of immunocytes were analysed in the mollusc, Mytilus galloprovincialis, using computer-assisted microscopy image analysis testing for ACTH (1–24) (Sassi et al., 1998), PDGF-AB (platelet-derived growth factor), CRH (corticotrophin-releasing hormone) (Malagoli et al., 2000), TGF-β1 (transforming growth factor-β) (Ottaviani et al., 1997b, 1998) and IL (interleukin)-8 (Ottaviani et al., 2000).
These molecules trigger cell shape modifications via different signal transduction pathways. For instance, ACTH (1–24) exerts its effects via an adenylate cyclase/cAMP/protein kinase A pathway, as well as through the activation of protein kinase C (Sassi et al., 1998). The expression of receptors for CRH has been demonstrated in M. galloprovincialis, and image analysis experiments have shown that human CRH promotes shape changes in molluscan cells through a synergistic activity of both cAMP- and phosphoinositide-activated pathways (Malagoli et al., 2000).
Mussel immunocytes present PDGF receptors (-α and -β) and TGF-β-receptor (type II)-like molecules. The growth factors PDGF-AB and TGF-β1 transduce their signals along the phosphoinositide signalling pathway. It is noteworthy that the molluscan immunocyte responses are dose-correlated for PDGF-AB and dose-dependent for TGF-β1. Furthermore, the response to PDGF-AB is independent of the entrance of extracellular calcium, while the effects of TGF-β1 are calcium-dependent. Malagoli et al. (2010) indicate that invertebrate cytokines are a relatively recent discovery, and several aspects of their signalling remains obscure (Royet, 2004; Silverman et al., 2009; Arnot et al., 2010). Human recombinant IL-8 causes cell shape changes in molluscan immunocytes via protein kinase A and C pathways, and these changes are related to a redistribution of actin microfilaments. Indeed, immunocytochemical experiments have shown that after incubating the cells with the cytokine, F-actin is not uniformly distributed beneath the plasma membrane as observed in controls, but is concentrated in those areas where the cell is in contact with the substrate (Ottaviani et al., 2000).
Different ACTH fragments, namely ACTH (1–4), (1–13), (1–17), (1–24), (4–9) and (11–24), can stimulate molluscan immunocytes. ACTH (4–11) antagonizes chemotactic inducers, such as ACTH (4–9) and TNF-α (tumour necrosis factor-α) (Genedani et al., 1994a). The effects of ACTH fragments on cell migration are independent of those generating steroidogenic activity, and those with behavioural and melanotropic activities (De Wied and Wolterink, 1988). Fragments of the N-terminal part of ACTH molecule [(1–4), (4–9), (4–11)], which in mammals are responsible for behavioural activity (De Wied and Wolterink, 1988), and the C-terminal part (11–24), which is devoid of both steroidogenic and behavioural effects (De Wied and Wolterink, 1988), allow molluscan immunocyte migration.
The whole β-endorphin sequence, its N- and C-terminal fragments, the sequence (2–17) which lacks both N- and C-terminals and its N-acetylated derivative can also stimulate immunocyte migration (Genedani et al., 1994b). This effect can probably be ascribed to the presence of opioid receptors on the plasma membrane of immunocytes. Indeed, naloxone antagonizes, at least in part, the stimulatory effect on cell migration of endorphin and its fragments (Genedani et al., 1994b). However, the co-occurrence of other non-opioid receptors in molluscs cannot be excluded (Stefano et al., 1989a, 1989b; Liu, 2008).
Experiments with microchemotaxis chambers have shown that mammalian cytokines, such as IL-1α, TNF-α and IL-8, and growth factors, such as PDGF-AB and TGF-β1, affect molluscan immunocyte migration. However, the chemotactic action of these molecules is different because diverse effects are observed in function in the tested species and at different concentrations. More precisely, the effects of IL-1α and TNF-α were species-specific in some molluscs (Ottaviani et al., 1995). Embryonic cells of Biomphalaria glabrata responded dose-dependently to human recombinant IL-1 (Steelman and Connors, 2009), while IL-8, PDGF-AB and TGF-β1 effects were dose-correlated (Ottaviani et al., 1997a, 2000).
Phagocytosis is a phenomenon of pivotal importance for nutrition and defence that is enhanced by opsonic factors. A vast range of foreign materials, e.g. bacteria, latex particles, dead cells, etc., can be phagocytized both in vitro and in vivo by invertebrate immunocytes, which present several typical characteristics of cells of the macrophage lineage (Ottaviani, 1992; Ottaviani and Franceschi, 1997).
With the exception of the ACTH fragments (4–9) and (1–17), β-endorphin and its related fragments, the majority of molecules mentioned above displaying chemotactic effects also stimulate phagocytosis (Ottaviani et al., 1994). In particular, phagocytic activity increases after incubation of immunocytes with the following molecules: ACTH (1–4), (1–24), (4–10) (Ottaviani et al., 1994), IL-1α, IL-2, TNF-α (Ottaviani et al., 1995), IL-8 (Ottaviani et al., 2000) and PDGF-AB and TGF-β1 (Ottaviani et al., 1997a).
ACTH fragments have different effects depending on the concentration and the species. Specifically, chemotactic and phagocytic effects are not directly correlated because peptides which influence cell migration do not always affect phagocytosis. Furthermore, as with cell migration (Genedani et al., 1994a, 1994b), the effect of an individual peptide on phagocytosis could also be species-specific and dose-dependent.
The influence of the cytokines IL-1α, IL-2, IL-8 and TNF-α on phagocytosis is more uniform than that on cell migration (Ottaviani et al., 1995; Ottaviani et al., 2000). Indeed, phagocytic activity increases independently of concentration in all the molluscan species tested. Conversely, the phagocytic response to PDGF-AB and TGF-β1 is species-specific in molluscs (Ottaviani et al., 1997a). However, the action of these factors seems to be dependent on other unknown molecules present in the haemolymph. For instance, the effects of TNF-α on mussel phagocytosis varied significantly when the cells were exposed to the cytokine in the presence of filtered seawater or haemolymph components (Betti et al., 2006).
The effects of a signal molecule depend on its binding to cellular receptors, thus promoting a cell response. This response can include cell motility, cell migration and phagocytosis. A few studies have focused on receptors for immune-related factors in molluscs, but high throughput techniques have started to improve the available information (Roberts et al., 2009). A specific receptor for opioids has been found in Mytilus edulis immunocytes (Stefano et al., 1993), as well as an mRNA encoding for an ACTH receptor-like in immunocytes of M. galloprovincialis (Ottaviani et al., 1998). Furthermore, there is immunocytochemical evidence that these cells present PDGF receptor-α- and β-like molecules, TGF-β-receptor (type II)-like molecules (Kletsas et al., 1998) and IL-2 receptor subunits on their membranes (Barcia et al., 1999).
In molluscs, the mechanisms by which the tested molecules, and particularly ACTH fragments (consisting of four or five amino acids), stimulate or inhibit immune functions remain obscure. In this context, the influence of small peptides on the mammalian immune system has been described both for bacterial (muramil peptides) and animal peptides, such as thymic hormones, tuftin and peptides obtained from colostrum or milk (Werner et al., 1986).
It should, however, be emphasized that it is difficult to understand, from the studies performed in molluscs, whether cytokines and growth factors have any conserved role in cell motility, chemotaxis and phagocytosis. Indeed, a molecule may be able to modulate one or more of the above-mentioned immune functions in one species, but not in another.
5. Toxins, cellular immunity and stress
Owing to the unavailability of molluscan immune factors and the difficult interpretation of data collected with mammalian stimulants, we decided to investigate the effects of marine toxins on the immune response (especially phagocytic activity) in M. galloprovincialis. These experiments provided several intriguing results that may help to explain some of the discrepancies described above. For instance, the phycotoxin, YTX (yessotoxin), is apparently unable to influence immunocyte motility, but significantly increases cell shape changes induced by the bacterial tripeptide fMLP (N-formyl-Met-Leu-Phe) (Malagoli and Ottaviani, 2004). This indicates that experimental conditions or neglected contaminants/haemolymph factors may influence the response towards a specific activator (Betti et al., 2006). For example, YTX, at concentrations that do not affect cell motility, increases membrane permeability to Ca2+. As a consequence, immunocytes can change their expected reaction to further stimulation (Malagoli et al., 2006). We have also elucidated the influence of stress and experimental conditions on the outcomes of phagocytosis experiments. Short air exposure (30 min) and salinity stress increase the number of circulating immunocytes in M. galloprovincialis, but not their phagocytic activity. Only long air exposure (120 min) and mechanical stress significantly raise both parameters (Malagoli et al., 2007). Finally, experiments using the algal toxins, okadaic acid and palytoxin, confirmed that experimental conditions have a significant impact on the outcomes of phagocytic tests. More importantly, collected data clearly indicated that the immunocytes can change their signalling accordingly to specific conditions, e.g. temperature. This capability may significantly interfere with and modify the effects of immune-modulating factors (Malagoli et al., 2008). Furthermore, these in vitro observations have recently been confirmed by experiments that also consider the life history of bivalves as a fundamental parameter that can influence their ability to escape pathogens (Paul-Pont et al., 2010).
6. Concluding remarks
Although functional experiments performed in molluscs using heterologous factors have proved of pivotal importance in driving research on invertebrate chemoattractants, cytokines and growth factors, it is undeniable they have also provided controversial evidence. Discrepancies have also been reported in mammals, where it has long been known that molecules that can exert a chemotactic effect on human neutrophils can also inhibit phagocytosis in the same cells (Musson and Becker, 1976). Thus, the conflicting information collected in molluscs should not be linked to species-specific reactions to a heterologous factor, but rather to the combination of contingency and presently unknown homologous factors that can significantly influence the response.
The ability of immunocytes to tune their signalling continuously and finely blurs the boundary between cell motility, cell migration and phagocytosis. All three are cell functions (or more correctly, phenomena) that operators perceive as distinct, but each ultimately depends on signalling and complex molecular ‘equilibria’ (e.g. microtubule and/or microfilament interactions) that are very difficult to disentangle. Factors that increase cell motility might only appear to have no effect on phagocytosis, since this is only what can be observed or measured. Inside the cells, however, molecular signalling is flexible and, therefore, is able to respond differently to the same stimulus on the basis of contingency.
Work in the authors' laboratory is supported by FAR 2009 grants of the University of Modena and Reggio Emilia (Italy) (to D.M. and E.O.)
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Received 12 July 2010; accepted 26 July 2010
Published online 24 September 2010, doi:10.1042/CBI20100514
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