Activation of extracellular-signal regulated kinase is required for phagocytosis by Lymnaea stagnalis haemocytes

Abstract

Haemocytes are the primary defence cells of molluscs. In the present study, extracellular-signal regulated kinase (ERK) 1/2-like proteins were identified within Lymnaea stagnalis haemocytes, with apparent molecular weights of 44 and 43 kDa, respectively. Mitogen-activated protein kinase (MAPK) activity assays have confirmed that the L. stagnalis ERK possesses kinase activity towards Elk-1. Challenge of haemocytes with bacterial lipopolysaccharide (LPS) resulted in a transient activation of ERK, and immunocytochemistry revealed that phospho-ERK was present in both the perinuclear region and the nucleus following challenge. MAPK/ERK kinase (MEK) inhibitors blocked ERK activation confirming that MEK lies upstream of ERK in haemocytes. Moreover, phagocytosis assays, using various inhibitors, showed that ERK activity was vital for efficient phagocytosis and that ERK may be activated by both Ras-dependent and Ras-independent mechanisms. Overall, this study has furthered knowledge of ERK signalling in molluscan immunity and has shown that the ERK pathway regulates the phagocytic activity of molluscan haemocytes.

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the Defence Activity of Radix lagotis (Lymnaeidae) Haemocytes

“…Because signalling pathways are known to regulate haemocyte defence responses such as phagocytosis and H 2 O 2 output [7], [9]–[12], and because these defence responses were supressed in R. lagotis haemocytes as a result of T. regenti infection, we aimed to determine PKC and ERK activation in haemocyte monolayers derived from uninfected and infected R. lagotis . Western blotting of haemocyte proteins with anti-phosphospecific PKC and ERK antibodies, which detect only the active forms of these kinases in snails [7], [8], [27], followed by densitometric analysis of immunoreactive bands from several independent blots revealed that PKC and ERK phosphorylation were reduced by 57% and 55%, respectively, in haemocytes from infected snails when compared to those from uninfected snails (p<0.01; Figure 7A–B).…”

“…Western blotting of haemocyte proteins with anti-phosphospecific PKC and ERK antibodies, which detect only the active forms of these kinases in snails [7], [8], [27], followed by densitometric analysis of immunoreactive bands from several independent blots revealed that PKC and ERK phosphorylation were reduced by 57% and 55%, respectively, in haemocytes from infected snails when compared to those from uninfected snails (p<0.01; Figure 7A–B). We reasoned, therefore that ERK expression might also be suppressed.…”

“…To investigate the possible role of PKC and ERK in the regulation of phagocytosis by R. lagotis haemocytes, cells from uninfected snails were incubated with the PKC or MEK inhibitors GF109203X or U0126, respectively, compounds that have been shown to decrease PKC or ERK phosphorylation (activation) in L. stagnalis haemocytes [7], [27]. Phagocytosis was blocked in a dose-dependent manner, with 1 µM and 10 µM GF109203X significantly suppressing uptake of bioparticles by approximately 35% and 70%, respectively (p<0.01, p<0.001; Figure 8A).…”

“…Membranes were blocked with 5% non-fat dried milk (Bio-Rad) in 0.1% Tween/Tris-buffered saline (TTBS) at RT for 45 min, and incubated overnight at 4°C in either anti-phospho-PKC (pan) (βII Ser660) rabbit polyclonal antibodies or anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) rabbit monoclonal antibodies (CST) (1∶1000 in TTBS). These antibodies were previously validated for detection of exclusively phosphorylated (activated) forms of PKC and ERK in L. stagnalis haemocytes [7], [27], and were also used in other studies of molluscs [17]. Following further incubation at RT for 2 h, membranes were washed 3×5 min in TTBS and incubated for 2 h at RT in anti-rabbit IgG HRP-conjugated secondary antibodies (1∶4000 in TTBS) (CST).…”

“…Among the ROS, hydrogen peroxide (H 2 O 2 ) is an important metabolite known for killing sporocysts of the human parasite Schistosoma mansoni [4]. At the molecular level, snail haemocyte defence responses are regulated by complex networks of intracellular signalling pathways, including the evolutionarily conserved protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways [7]–[10]. Activation of PKC, p38 MAPK and/or extracellular signal-regulated kinase (ERK) is required for efficient phagocytosis and H 2 O 2 production by snail haemocytes; other kinases such as phosphatidylinositol 3-kinase also play a crucial role in these processes [7], [9]–[12].…”