MAP kinases mediate phagocytosis and melanization via prophenoloxidase activation in medfly hemocytes

Abstract

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.

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

“…Immunofluoresence labeling also detects proPO protein only in oenocytoids (Jiang et al, 1997). But in the medfly, proPO is detected in the cytoplasm of the hemocytes, as well as on the surface of all hemocytes (Mavrouli et al, 2005). ProPO does not contain a signal secretion sequence, and it is released from hemocytes into the hemolymph by rupture (Jiang et al, 1997;Ashida and Brey, 1998).…”

“…PO is a key enzyme in cellular immune responses including melanotic encapsulation and phagocytosis (Ashida and Brey, 1998;Cerenius and Soderhall, 2004;Kanost et al, 2004;Mavrouli et al, 2005). The phagocytic function of insect hemocytes effectively destroys and removes small invading pathogens such as bacteria, as well as self dead cells and tissue debris (Lavine and Strand, 2002;Ling et al, 2003;Zhou et al, 2004;Ling and Yu, 2005a).…”

Regulation and function of the melanization reaction in Drosophila

“…66 Moreover, melanization has been reported to be associated with phagocytosis in mosquitoes and the medfly. [67][68][69][70] Similarly, increased PO activity enhances phagocytosis by hemocytes and resistance to infection in the fresh water crayfish P. leniusculus. 54 In Drosophila, the cellular immune response was reduced in a DDC mutant background lacking a melanin precursor, dopamine.…”

Development and evolution of insect pigmentation: Genetic mechanisms and the potential consequences of pleiotropy

“…Mutations in pale (encoding Tyrosine Hydroxylase) and Dopa-decarboxylase affect pigmentation and the melanic immune response [59,62,70,71]; and genes encoding phenoloxidases polymerize dopa and dopamine into melanins during pigmentation development as well as wound healing and encapsulation [69,[72][73][74][75]. Despite the production of similar pigments, melaniza-tion that occurs as part of the immune response and melanization that is used for body pigmentation, does not require all of the same genes.…”