Maria Lampropoulou

Regulators and signalling in insect haemocyte immunity.

The innate immune system of insects relies on both humoral and cellular immune responses that are mediated via activation of several signalling pathways. Haemocytes are the primary mediators of cell-mediated immunity in insects, including phagocytosis, nodulation, encapsulation and melanization. The last years, research has focused on the mechanisms of microbial recognition and activation of haemocyte intracellular signalling molecules in response to invaders. The powerful tool, RNA interference gene silencing, helped several regulators involved in immune responses, to be identified. In this review, we summarize recent advances in understanding the role(s) of receptors and intracellular signalling molecules involved in immune responses.

Innate immunity in insects: surface-associated dopa decarboxylase-dependent pathways regulate phagocytosis, nodulation and melanization in medfly haemocytes

Phagocytosis, melanization and nodulation in insects depend on phenoloxidase (PO) activity. In this report, we demonstrated that these three processes appear to be also dependent on dopa decarboxylase (Ddc) activity. Using flow cytometry, RNA interference, immunoprecipitation and immunofluorescence, we demonstrated the constitutive expression of Ddc and its strong association with the haemocyte surface, in the medfly Ceratitis capitata. In addition, we showed that Escherichia coli phagocytosis is markedly blocked by small interfering RNA (siRNA) for Ddc, antibodies against Ddc, as well as by inhibitors of Ddc activity, namely carbidopa and benzerazide, convincingly revealing the involvement of Ddc activity in phagocytosis. By contrast, latex beads and lipopolysaccharide (LPS) did not require Ddc activity for their uptake. It was also shown that nodulation and melanization processes depend on Ddc activation, because antibodies against Ddc and inhibitors of Ddc activity prevent haemocyte aggregation and melanization in the presence of excess E. coli. Therefore, phagocytosis, melanization and nodulation depend on haemocyte‐surface‐associated PO and Ddc. These three unrelated mechanisms are based on tyrosine metabolism and share a number of substrates and enzymes; however, they appear to be distinct. Phagocytosis and nodulation depend on dopamine‐derived metabolite(s), not including the eumelanin pathway, whereas melanization depends exclusively on the eumelanin pathway. It must also be underlined that melanization is not a prerequisite for phagocytosis or nodulation. To our knowledge, the involvement of Ddc, as well as dopa and its metabolites, are novel aspects in the phagocytosis of medfly haemocytes.

Distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes

In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme‐linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram‐negative and Gram‐positive) is dependent on RGD‐binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD‐binding‐receptor‐independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD‐binding‐receptor‐independent and FAK/Src‐independent but MAPK‐dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal‐regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.

A β integrin subunit regulates bacterial phagocytosis in medfly haemocytes.

We have recently reported that the activation of focal adhesion kinase (FAK) and its downstream targets upon pathogen challenge regulate phagocytosis in medfly haemocytes. The goal of this study was to further explore the signalling pathway underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate the haemocyte surface receptor, through which the extracellular signals in response to bacteria are transmitted intracellularly. The presented data demonstrate the expression of a beta integrin subunit in the surface of medfly haemocytes that transmits signals upon pathogen triggering to FAK and its downstream targets, Src, MAP kinases and Elk-1-like protein, for the engulfment of pathogen. Interestingly LPS is not internalized through integrins.

Elk‐1 associates with FAK, regulates the expression of FAK and MAP kinases as well as apoptosis in HK‐2 cells

Focal adhesion kinase (FAK), MAP kinases and the nuclear transcription factor Elk‐1 have been reported to be implicated in the same cellular processes, however, their direct or indirect interaction and potential function(s) has not been documented. Here, we explored the association of FAK with Elk‐1, the implication of Elk‐1 in the regulation of FAK and MAP kinases expression as well as apoptosis, in HK‐2 cells. Biochemical and immunofluorescence approaches strongly support the association of low molecular weight protein bands, recognized by FAK antibodies, with Elk‐1 or pser383Elk‐1. The FAK/Elk‐1 complex is found, mainly, in the cytoplasm, near the nuclear membrane periphery, raising the possibility that Elk‐1 may have alternative extranuclear function(s) in HK‐2 cells. Furthermore, we demonstrated that Elk‐1 siRNA‐mediated knockdown experiments, increased apoptosis. By contrast, Elk‐1 siRNA decreased significantly the expression of FAK and MAP kinases, supporting the hypothesis that Elk‐1 may act as a potential physiological substrate and regulator of FAK and MAP kinases expression. These results strongly support that Elk‐1 protein is a novel binding‐protein partner for FAK, a finding that significantly broadens the potential functioning of FAK and Elk‐1. J. Cell. Physiol. 216: 198–206, 2008.

Alkaline phosphatase in the integument of Ceratitis capitata: developmental profile and functional properties

Abstract Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine- O -phosphate. Tyrosine- O -phosphate is the preferred substrate for the integumental enzyme form with a K m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine- O -phosphate.

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling.

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double‐stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI‐3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre‐incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI‐3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin‐positive and annexin‐negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI‐3K p85a, Akt, and ERK appeared in annexin‐positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI‐3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution. J. Cell. Biochem. 101: 331–347, 2007.

Elk‐1 is a novel protein‐binding partner for FAK, regulating phagocytosis in medfly hemocytes

Focal adhesion kinase (FAK) and its downstream signaling targets, mitogen‐activated protein kinase (MAPKs), are implicated in the process of phagocytosis by insect hemocytes. The goal of this study was to explore further the signaling pathways underlining the process of phagocytosis. The combination of bioinformatics, biochemical, and immunofluorescence approaches strongly support the expression of Elk‐1‐like protein in medfly hemocytes. Elk‐1 is phosphorylated in E. coli or latex beads‐challenged hemocytes and osmotic loading experiments as well as flow cytometry analysis demonstrated that Elk‐1‐like protein regulates the uptake of bacteria. RNA interference (RNAi) and pharmacological inhibitors show that the signaling for Elk‐1 phosphorylation is transmitted via FAK/Src and MAPKs pathways. Furthermore, confocal analysis clearly shows that FAK and the phosphorylated FAK at Y397 are localized in the nucleus and cytoplasm, whereas, the phosphorylated Elk‐1‐like protein is exclusively localized in the nucleus. Finally, co‐immunoprecipitation and reciprocal co‐immunoprecipitation analysis demonstrated the association of low molecular weight protein bands recognized by FAK antibodies, with Elk‐1 or phospho‐Elk‐1 at ser 383 and confocal microscopy specifies that this association occurs only in the nucleus. These results are strongly supporting that Elk‐1‐like protein is a novel protein‐binding partner for FAK, a finding that significantly broadens the potential functioning of FAK and Elk‐1 generally. Evidently, the complex participates in the process of phagocytosis in medfly hemocytes. J. Cell. Biochem. 103: 1895–1911, 2008.

Hydrogen peroxide is produced by E. coli challenged haemocytes and regulates phagocytosis, in the medfly Ceratitis capitata. The active role of superoxide dismutase.

Hydrogen peroxide (H(2)O(2)) participates as a second messenger in cell signaling. In this paper, the role of H(2)O(2) was investigated, in Escherichia coli phagocytosis by the haemocytes of the medfly Ceratitis capitata. Block of H(2)O(2) synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (NEM) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and H(2)O(2). Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H(2)O(2) synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced H(2)O(2) in the differential phosphorylation of MAP kinases.