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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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

E. coli phagocytosis by medfly hemocytes, in contrast to mammalian macrophages, associates with E. coli-challenged hemocyte secretion by mitogen activating protein (MAP) kinases. In the present work, we examined whether this system links with the proteolytic activation of prophenoloxidase (proPO). ProPO and prophenoloxidase-activating proteinases (PAPs) were initially identified within freshly isolated medfly hemocytes. Moreover, flow cytometry and immunocytochemical analysis revealed the constitutive expression of proPO and its stable association with hemocyte surface. The expression level of hemocyte surface proPO is not affected by E. coli infection. In addition, flow cytometry analysis in freshly isolated hemocytes showed that E. coli phagocytosis is markedly blocked by antibodies against proPO or PAPs, as well as by several serine protease inhibitors, strongly supporting the involvement of proPO cascade in the phagocytosis process. Similarly, it was shown that melanization process depends on proPO activation. MAP kinases appeared to control both phagocytosis and melanization, since they regulate PAPs secretion, a prerequisite for the conversion of proPO to active PO. From this and previous studies, hemocytes appear to be central to immune response in medfly.

In response to LPS/E. coli treatment, extracellular signal-regulated kinase (ERK) is activated in medfly hemocytes. To explore the molecular mechanisms underlying LPS/E. coli/latex beads endo- and phagocytosis, we studied the signalling pathways leading to p38 and c-jun N-terminal kinase (JNK) activation. JNK and p38-like proteins were initially identified within medfly hemocytes. Flow cytometry analysis revealed that mitogen-activated protein kinases (MAPK) are required for phagocytosis. Inhibition of specific MAPK signalling pathways, with manumycin A, toxin A, cytochalasin D and latrunculin A, revealed activation of p38 via Ras/Rho/actin remodelling pathway and activation of JNK that was independent of actin cytoskeleton reorganization. ERK and p38 pathways, but not JNK, appeared to be involved in LPS-dependent hemocyte secretion, whereas all MAPK subfamilies seemed to participate in E. coli-dependent secretion. In addition, flow cytometry experiments in hemocytes showed that the LPS/E. coli-induced release was a prerequisite for LPS/E. coli uptake, whereas latex bead phagocytosis did not depend on hemocyte secretion. This is a novel aspect, as in mammalian monocytes/macrophages LPS/E. coli-triggered release has not been yet correlated with phagocytosis. It is of interest that these data suggest distinct mechanisms for the phagocytosis of E. coli and latex beads in medfly hemocytes.

A doublet of medfly hemocyte proteins with a molecular mass of about 55 and 50 kDa were precipitated with LPS. Antibodies raised against human CD14 recognize the same doublet of proteins. These results support that mammalian CD14 and the doublet of protein bands in medfly hemocytes share common epitopes. This doublet of protein bands is released from hemocytes upon LPS triggering. A portion of the released protein is clustered on the surface of a distinct hemocyte type and the other remains soluble. The membrane-bound LPS-binding protein is involved in LPS internalization and Escherichia coli phagocytosis but not in LPS signaling., (Copyright 2003 Wiley-Liss, Inc.)