P35-sensitive caspases, MAP kinases and Rho modulate ß-adrenergic induction of apoptosis in mollusc immune cells

Arnaud Lacoste*, Anne Cueff and Serge A. Poulet

Station Biologique de Roscoff, CNRS, Université Paris VI, INSU Place Georges Teissier, B.P. 74, F-29682 Roscoff cedex, France

* Author for correspondence (e-mail: lacoste{at}itsa.ucsf.edu )

Accepted 1 November 2001


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 Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is an important mechanism for the preservation of a healthy and balanced immune system in vertebrates. Little is known, however, about how apoptotic processes regulate invertebrate immune defenses. In the present study, we show that noradrenaline, a catecholamine produced by the neuroendocrine system and by immune cells in molluscs, is able to induce apoptosis of oyster Crassostrea gigas hemocytes. The apoptosis-inducing effect of noradrenaline was mimicked by isoproterenol and blocked by propranolol, which indicates that noradrenaline triggers apoptosis via a ß-adrenergic signaling pathway. Exposure to the pan-caspase inhibitor Z-VAD-FMK or expression of the caspase inhibitor P35 under the transcriptional control of a mollusc hsp70 gene promoter reduced the number of apoptotic cells among noradrenaline-treated hemocytes. These results suggest that P35-sensitive caspases are involved in the apoptotic process triggered by ß-adrenergic signaling. Complementary experiments suggest that mitogen-activated protein kinases and Rho, a member of the Ras GTPase family, may be involved in antiapoptotic mechanisms that modulate the apoptotic effect of noradrenaline. Taken together, these results provide a first insight into apoptotic processes in mollusc immune cells.

Key words: Mollusc, Immune cell, Noradrenaline, Apoptosis, ß-adrenergic signaling


    Introduction
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 Introduction
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Advances in the understanding of apoptotic processes in invertebrates are mainly due to genetic analyses using the nematode Caenorhabditis elegans (Hengartner, 1999Go). Recently, genome analysis and a number of molecular and biochemical studies have demonstrated the existence of cell death effectors such as caspases in the arthropod Drosophila melanogaster, which provided new insights into the role of apoptosis in important biological processes such as development or the removal of unnecessary cells in mature organisms (Kumar and Doumanis, 2000Go). However, although apoptosis is recognized as essential for the maintenance of homeostasis in the mammalian immune system (Osborne, 1996Go), few studies have explored its role in invertebrate immunity.

Recently, the need for improved knowledge of molluscan immunity has increased for at least three reasons. First, several molluscs constitute important sources of protein in several regions of the planet and thus have a great ecological and socio-economical value that is permanently threatened by disease outbreaks. Second, certain cultured marine molluscs that are susceptible to infection by invertebrate pathogens are suspected to promote interspecies transmission of these infectious organisms to other marine molluscs (Arzul et al., 2001Go), which may have important ecological consequences. Third, bivalves and several other molluscs are vectors of human pathogens such as viruses (e.g. the hepatitis A virus, the Norwalk-like virus and a number of enteric viruses in bivalve molluscs), bacteria (e.g. pathogenic Vibrios in bivalve molluscs) or eukaryotic parasites (e.g. schistosomes in gastropods). Some of these pathogens interfere with immune functions or take advantage of transient immunodepression episodes to replicate and accumulate in mollusc tissues (De Jong-Brink, 1995Go; Yoshino and Vasta, 1996Go; Genthner et al., 1999Go; Lacoste et al., 2001aGo). Consequently, the elucidation of molluscan vector immunity is expected to help identify factors that influence the vectorial capacity of molluscs and to provide new avenues to understand parasite transmission by these organisms.

The molluscan immune system involves both humoral responses and various immune cell reactions, including reactive oxygen species production (Pipe, 1992Go), antimicrobial peptide secretion (Mitta et al., 2000Go), encapsulation and phagocytosis (Sminia and Van Der Knaap, 1987Go). Previous studies have shown that bivalve hemocytes synthesize and secrete catecholamines (CA) including noradrenaline and dopamine (Ottaviani and Franceschi, 1996Go). Moreover, CA are known to circulate in the hemolymph of both gastropods and bivalves (Santhanagopalan and Yoshino, 2000Go; Pani and Croll, 2000Go; Lacoste et al., 2001bGo; Lacoste et al., 2001cGo), suggesting that these hormones are present in the microenvironment of hemocytes. In molluscs, CA play essential roles in several physiological processes, including feeding (Teyke et al., 1993Go), locomotion (Sakharov and Salànski, 1982Go), respiration (Syed and Winlow, 1991Go), reproduction (Martínez and Rivera, 1994Go) and development (Pires et al., 1997Go). Moreover, bivalve and gastropod immune cells express specific receptors for CA such as dopamine (Salzet et al., 1998Go) and noradrenaline (Lacoste et al., 2001dGo; Lacoste et al., 2001dGo; Lacoste et al., 2001fGo); however, little is known about how these hormones modulate mollusc immune functions.

In the present study, we show that noradrenaline has the capacity to induce the apoptosis of oyster hemocytes. Moreover, we provide evidence that P35-sensitive caspases, mitogen-activated protein (MAP) kinases and Rho modulate noradrenaline (NA)-induced apoptotic processes in mollusc immune cells.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 References
 
Drugs
NA, the {alpha}-adrenoceptor agonist phenylephrine, ß-adrenoceptor agonist isoproterenol (ISO), the {alpha}-adrenoceptor antagonist prazosin, the ß-adrenoceptor antagonist propranolol, the protein kinase C inhibitors staurosporine and calphostin C, the protein kinase A inhibitor H-89 and the MAP kinase kinase inhibitor PD059098 were all obtained from Sigma. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine was from Calbiochem and the cell permeable pancaspase inhibitor N-benzyloxycarbonyl-VAD-fluoromethylketone (Z-VAD-FMK) was from Promega.

Oyster hemocytes
Oysters Crassostrea gigas (65-70 g wet weight) were maintained in polyethylene tanks containing 110 1 of aerated and continuously flowing (50 l/hour) natural seawater at 14-15°C. Hemolymph (0.5-1 ml/oyster) was collected from the pericardial cavity using 2 ml syringes and 26 Gx1/2" needles and pooled to obtain 10-15 ml samples. Hemocyte concentration (ranging usually between 1-2.5x106 cell/ml) was then adjusted to 106 cell/ml by the addition of modified Hank's balanced salt solution (MHBSS) consisting of HBSS adjusted to ambient sea water salinity (31 ppm) and pH 7.4 and containing 2 g/l of D-glucose, 110 mg/l of sodium pyruvate (Gibco) and 55 mg/l of ascorbic acid (Sigma). 1 ml of cell suspension (106 cells) was dispensed into 35 mm Petri dishes together with 1 ml of MHBSS. Cells were allowed to attach for 20 minues and carefully rinsed with MHBSS. Hemocytes were then maintained at 15°C in IMDM (Gibco), adjusted to ambient salinity (31 ppm), containing 5% heat inactivated horse serum, 5% heat inactivated fetal bovine serum, penicillin G (50 units/ml) and streptomycin (50 µg/ml). NA, phenylephrine or ISO were added to the culture medium at concentrations indicated in the text. In some experiments, antagonists or inhibitors were added 30 minutes (or 90 minutes in the case of Z-VAD-FMK) prior to the addition of NA or ISO. At the end of the incubation period, the culture medium was removed and cells were detached from the dishes by incubation for 5 minutes at room temperature in 300 µl of 0.2% trypsin (Sigma) in MHBSS. Samples were then processed for further analyses.

Scanning electron microscopy
Cells were resuspended in MHBSS, left to attach on glass coverslips in 35 mm Petri dishes and maintained for 24 hours in modified IMDM containing NA at the concentrations mentioned in the text. At the end of the incubation period, cells were fixed for 10 minutes in 2.5% glutaraldehyde in cacodylate buffer. Samples were then dehydrated by graded ethanol solutions, critical-point dried and gold coated. Cells were then examined with a Joel JSM-5200 scanning electron microscope.

FITC-Annexin V and propidium iodide (PI) staining
Detached cells were pelleted by centrifugation (200 g for 15 minutes) and resuspended in 200 µl annexin-binding buffer (10 mM Hepes, 140 mM NaCl and 3.3 mM CaCl2, pH 7.4), then adjusted to ambient sea water salinity (31 ppm). FITC-Annexin V (Sigma) was added to a final concentration of 2.5 µg/ml and PI (Sigma) to a final concentration of 1 µg/ml. Cells were incubated for 30 minutes in the dark at 15°C and then processed for confocal microscopy or flow cytometry analyses.

Confocal microscopy
Cells stained with FITC-Annexin-V were fixed with 3.7% formaldehyde for 15 minutes and viewed under a IX Fluoview Olympus confocal microscope equipped with argon-krypton lasers.

Flow cytometry
Analyses were performed with a Becton Dickinson FACSort cytometer equipped with an air-cooled laser providing 15 mW at 488 nm and using the standard filter setup. For each experimental condition, 20,000 events were counted. FITC and PI-fluorescence were collected on the logarithmic scale for all experiments. Data were computed with the custom designed software CYTOWIN (Vaulot, 1989Go). Graphs were drawn with the WinMDI freeware (Joseph Trotter). The percentage of apoptotic cells (FITC positive/PI negative cells) was calculated and data were expressed as means and standard errors of at least three experiments. For comparison of two means, paired or unpaired t-tests were used where appropriate.

Construction of expression vectors
The baculovirus p35 gene, the mollusc Aplysia californica wild-type rho gene or a valine-14 mutant of the Aplysia rho gene (Perona et al., 1993Go) were cloned just downstream of the mollusc Bomphalaria glabrata heat shock protein 70 gene (hsp 70) promoter. A HindIII-XhoI fragment of the multicloning site of the pcDNA 3.1+ vector (Invitrogen) was ligated into the HindIII-XhoI locus of the pG construct (Yoshino et al., 1998Go) just downstream of the 1068 bp B. glabrata hsp70 gene promoter fragment to generate the construct pGMCS. The baculovirus p35 gene was excised from the pNN1 vector (Sah et al., 1999Go) by KpnI and BamHI and cloned in pGMCS. The wild-type (rhoWT) and mutant (rhoV14) rho genes were excised respectively from the pZIP-neo-rhoWT and pZIP-neo-rhoV14 vectors (Perona et al., 1993Go) by BamHI and cloned into pGMCS.

Transfection assays
Protocols used for oyster hemocyte transfection were inspired by previous studies showing that cationic lipids allow foreign gene transfer into mollusc cells (Yoshino et al., 1998Go; Cadoret et al., 1999Go; Lacoste et al., 2001fGo). Oyster hemocytes resuspended in MHBSS were left to attach in 35 mm Petri dishes (2.106 cells/dish) for 20 minutes, rinsed with MHBSS and incubated for 1 hour at 17°C in MHBSS containing 20% DMEM (Gibco) adjusted to ambient salinity (31 ppm). Cells were then rinsed twice in MHBSS containing 20% DMEM and exposed for 2 hours at 17°C to a 1:5 mixture of 10 µg DNA precomplexed to Plus reagent (Gibco) and lipofectamine (Gibco) in 1 ml MHBSS containing 20% DMEM. To increase transfection efficiency a multiple transfection protocol (Yamamoto et al., 1999Go; Lacoste et al., 2001fGo) was used (transfection was repeated for a total of four times over an 8 hour period). The volume of medium was then increased to 3 ml by the addition of modified IMDM (as described above), and the cells were maintained for 2 hours at 17°C. Heat shock treatment was then given at 37°C for 1 hour followed by incubation for 5 hours at 17°C for the expression of p35 or rho genes (Sah et al., 1999Go). Cells were then exposed to 1 µM ISO for 12 hours and monitored for apoptosis.


    Results
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 Materials and Methods
 Results
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 References
 
Scanning electron micrographs of oyster hemocytes (Fig. 1A,B) show that a 24 hour exposure to 1 µM NA caused marked plasma membrane changes and the formation of blebs characteristic of cells undergoing programmed cell death. In addition, FITC-Annexin V binds to the membrane of hemocytes incubated in the presence of NA (Fig. 1C,D), which demonstrates that externalization of phosphatidylserine, one of the earliest events in apoptosis, is occuring. Further analyses using flow cytometry (Fig. 2) show that, in the presence of 1 µM NA, the percentage of apoptotic hemocytes increased over time and reached 16.1% after 24 hours. The apoptosis-inducing effect of NA was dose dependent and significant (P<0.01) at concentrations ranging between 1 and 10 µM NA (Fig. 3).



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Fig. 1. Exposure of oyster hemocytes to 1 µM NA for 24 hours induces membranal changes that are indicative of cells undergoing apoptosis. (A) A scanning electron micrograph of a control hemocyte maintained in vitro for 24 hours in the absence of NA. The cell tends to spread on the substrate (arrowhead) and deploys several pseudopods (arrows), whereas (B) an oyster hemocyte incubated in the presence of 1 µM NA exhibits visible membrane blebbing (arrow). (C) Confocal microscopy analysis after FITC-AnnexinV-PI staining shows that phosphatidylserine is absent from the external leaflet of the plasma membrane, whereas (D) FITC-Annexin V binds to the membrane of hemocytes incubated in the presence of NA, demonstrating that in addition to membrane blebbing (arrow), externalization of phosphatidylserine, one of the earliest events in apoptosis, is occuring. Cells in (C) and (D) were fixed after FITC-Annexin V staining to allow localization of nuclear DNA by PI. Scale bars: (A,B) 1 µm, (C,D) 2 µm.

 


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Fig. 2. Flow cytometric analysis of FITC-Annexin V-PI stained hemocytes incubated in the presence of 1 µM NA for 0, 6, 12 and 24 hours. Region R1 includes viable cells, which excude PI and are negative for FITC-Annexin V binding. Region R2 includes apoptotic cells, which are FITC-Annexin V positive but impermeable to PI, and region R3 includes non-viable, necrotic or late apoptotic cells, which are positive for FITC-Annexin V staining and for PI uptake. The number of apoptotic cells increases over time and reaches about 16% of the total number of cells after 24 hours of exposure to 1 µM NA. One representative experiment out of three is shown.

 


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Fig. 3. The apoptosis-inducing effect of NA is dose dependent and significant (P<0.01) at concentrations >=1 µM. Cells were incubated for 24 hours in the presence of NA. Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the absence of NA.

 

The {alpha}-adrenoceptor agonist phenylephrine had no significant effect on the percentage of apoptotic hemocytes at concentrations ranging between 0.1 and 10 µM (Fig. 4A), whereas in the presence of the ß-adrenoceptor agonist ISO (0.1-10 µM) a dose-dependent increase in the percentage of apoptotic cells was observed (Fig. 4B). Moreover, the {alpha}-adrenoceptor antagonist prazosin had no significant effect on oyster hemocyte NA-induced apoptosis (Fig. 4C), whereas the ß-adrenoceptor antagonist propranolol significantly (P<0.01) reduced the apoptosis-inducing effect of NA at concentrations >=10 µM (Fig. 4D). These data suggest that NA acts through ß-adrenoceptors to induce oyster hemocyte apoptosis.



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Fig. 4. (A) A 24 hour exposure to the {alpha}-adrenoceptor agonist phenylephrine has no significant effect on the percentage of apoptotic hemocytes, whereas (B) a 24 hour exposure to the ß-adrenoceptor agonist isoproterenol mimicks the apoptosis-inducing effect of NA. (C) Addition of the {alpha}-adrenoceptor antagonist prazosin 30 minutes prior to the addition of 1 µM NA has no significant effect on the percentage of apoptotic cells whereas (D) addition of the ß-adrenoceptor antagonist propranolol blocks the apoptosis-inducing effect of NA. Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the absence of adrenoceptor agonist (A,B) or in the presence of 1 µM NA alone (C,D). Bas (basal level) indicates the percentage of apoptotic cells in samples incubated in the absence of drugs.

 

At concentrations >=10 µM, the adenylate cyclase inhibitor 2',5'-dideoxyadenosine caused a significant reduction in the percentage of apoptotic hemocytes (Fig. 5A). The protein kinase C inhibitors staurosporine (25 µM) and calphostin C (80 nM) had no significant (P<0.01) effect on the percentage of apoptotic hemocytes, whereas, in the presence of the protein kinase A inhibitor H-89 (25 µM), the apoptosis-inducing effect of ISO (1 µM) was significantly (P<0.01) reduced (Fig. 5B). Taken together, these results suggest that adenylate cyclase and a cAMP-dependent protein kinase are involved in the apoptosis-inducing effects of ISO.



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Fig. 5. (A) The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) reduces the percentage of apoptotic cells among ISO-treated hemocytes. (B) The protein kinase A inhibitor H-89 but not the protein kinase C inhibitors staurosporine (Stau) and calphostin C (CalC) reduce the percentage of apoptotic cells among ISO-treated hemocytes. These results indicate that adenylate cyclase and protein kinase A are required for the ß-adrenergic induction of apoptosis. Cells were maintained for 24 hours in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the presence of ISO alone.

 

To determine whether caspases are involved in oyster hemocyte NA-induced apoptosis, the effects of the cell permeable pan-caspase inhibitor Z-VAD-FMK were examined. At concentrations >=10 µM, Z-VAD-FMK significantly (P<0.01) reduced the apoptosis-inducing effect of ISO (Fig. 6A). The effects of the baculovirus P35 protein, a caspase inhibitor (Fisher et al., 1999Go; Zoog et al., 1999Go; LaCount et al., 2000Go), were also tested. Transfection of oyster hemocytes with a recombinant plasmid containing the baculovirus p35 gene under the transcriptional control of the gastropod hsp70 gene promoter was able to significantly (P<0.01) reduce apoptosis among ISO-treated hemocytes (Fig. 6B). Control constructs containing the hsp70 gene promoter alone or the p35 gene alone had no significant effect on hemocyte ISO-induced apoptosis.



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Fig. 6. Caspase inhibitors reduce the apoptosis-inducing effect of ISO on oyster hemocytes. (A) The pan-caspase inhibitor Z-VAD-FMK reduces the percentage of apoptotic cells among ISO-treated hemocytes, and (B) transfection of oyster hemocytes with a recombinant plasmid containing the baculovirus p35 gene under the transcriptional control of the gastropod hsp70 gene promoter (hsp p35) was able to reduce apoptosis among ISO-treated hemocytes. Control constructs containing the hsp70 gene promoter alone (hsp only) or the p35 gene alone (p35 only) had no significant effect on hemocyte ISO-induced apoptosis. Cells were maintained for 24 hours in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the presence of ISO alone.

 

The MAP kinase kinase inhibitor PD098059 was used to examine a possible role for the MAP kinase cascade in the apoptotic process induced by ISO. PD098059 alone at concentrations ranging between 10 and 100 µM had no significant effect on the percentage of apoptotic hemocytes (Fig. 7). When hemocytes were exposed to ISO, the MAP kinase kinase inhibitor (50 and 100 µM) caused a significant (P<0.01) increase in the percentage of apoptotic cells, suggesting that MAP kinase signaling is involved in mechanisms that protect mollusc immunocytes from ISO-induced apoptosis.



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Fig. 7. The MAP kinase kinase inhibitor PD098059 (PD) increases the percentage of apoptotic cells among ISO-treated hemocytes, indicating that MAP kinase signaling is involved in mechanisms that protect hemocytes from apoptosis induced by ß-adrenergic signaling. PD098059 alone had no significant effect on the percentage of apoptotic hemocytes. Cells were maintained for 24h in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (P<0.01) differences from samples incubated in the presence of ISO alone.

 

Finally, the possible involvement of Rho in ISO-induced hemocyte apoptosis was examined. Transfection of oyster hemocytes with a recombinant plasmid containing the wild-type Aplysia californica rho gene under the transcriptional control of the gastropod hsp70 gene promoter was able to significantly (P<0.05) reduce apoptosis among ISO-treated hemocytes (Fig. 8). Constructs containing the rhoV14 gene, a mutant with higher activity than the wild-type rho gene (Perona et al., 1993Go), also induced a significant (P<0.01) reduction of the percentage of apoptotic cells among ISO-treated hemocytes. Control constructs containing the hsp70 gene promoter alone or the rhoWT or rhoV14 gene alone had no significant effect on ISO-induced hemocyte apoptosis. These results suggest that Rho is also involved in mechanisms that protect hemocytes from ISO-induced apoptosis.



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Fig. 8. Rho signaling is involved in mechanisms that protect hemocytes from ISO-induced apoptosis. Transfection of oyster hemocytes with a recombinant plasmid containing the wild-type (hsp rhoWT) or a more active valine-14 mutant (hsp rho V14) Aplysia californica rho gene under the transcriptional control of the gastropod hsp70 gene promoter was able to reduce apoptosis among ISO-treated hemocytes. Control constructs containing the hsp70 gene promoter alone (hsp only) or the rho genes alone (rhoWT only or rhoV14 only) had no significant effect on hemocyte ISO-induced apoptosis. Cells were maintained for 12 hours in the presence of 1 µM ISO or in the absence of drugs (Bas, basal level). Data are means and standard errors of three replicate experiments. Asterisks denote significant (* for P<0.05, ** for P<0.01) differences from samples incubated in the presence of ISO alone.

 


    Discussion
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Apoptosis is used in virtually all metazoans to dispose of unneeded, infected or deleterious cells. Although this process has been recognized as essential for normal development and homeostasis in a number of vertebrates and invertebrates (for reviews, see Raff, 1992Go; White, 1996Go), data are still lacking concerning regulation through apoptosis in invertebrate immune cells.

In the present study, we show that NA induces apoptosis in oyster hemocytes via a ß-adrenoceptor-mediated activation of adenylate cyclase and a cAMP-dependent protein kinase. Previous studies have shown that a ß-adrenergic-receptor—cAMP-dependent signaling pathway is present in mollusc foot muscle sarcolemma (Pertseva et al., 1992Go), but our results show for the first time that this signaling pathway can trigger apoptosis in molluscs. Moreover, the NA concentrations used in the present study are known to exert physiological effects in bivalves (Muneoka and Kamura, 1982Go).

Interestingly, the induction of oyster immune cell apoptosis by ß-adrenergic signaling involves caspases, a family of cysteine proteases formerly known as the ICE/Ced-3 family. Although the critical role of caspases in apoptosis was first demonstrated by the isolation of the ced-3 gene in the nematode Caenorhabditis elegans (Yuan et al., 1993Go), and even though caspase genes have been cloned in mammals and Drosophila (Fraser and Evan, 1997Go; Salvesen and Dixit, 1997Go), information is missing concerning caspases in many invertebrate phyla including molluscs. Z-VAD-FMK is a non-selective caspase inhibitor whose activity has been demonstrated in mammals and Drosophila (Pronk et al., 1996Go; Bose et al., 1998Go). We show here that this inhibitor can also block apoptosis in the mollusc Crassostrea gigas. The baculovirus protein P35, another substrate inhibitor of death caspases (Fisher et al., 1999Go; Zoog et al., 1999Go; LaCount et al., 2000Go), was also able to reduce the percentage of apoptotic cells among ISO-treated oyster hemocytes. This result is consistent with previous studies showing that permanently or transiently expressed P35 can inhibit programmed cell death in diverse organisms, including insects, C. elegans and mammals (Hay et al., 1994Go; Sugimoto et al., 1994Go; Beidler et al., 1995Go; Xue and Horvitz, 1995Go). Information is now needed concerning the precise structure and regulation of molluscan caspases; however, the present results bring further evidence that molecular components leading to apoptosis are conserved among animal species.

A possible role of the MAP kinase cascade in NA-induced oyster hemocyte apoptosis was also examined because members of this family of serine-threonine kinases are known to be involved in the activation or inactivation of key elements of apoptotic processes in animals (Cross et al., 2000Go). MAP kinases are thought to exist in all eukaryotic cells from yeast to human (Martin-Blanco, 2000Go), and the use of the MAP kinase cascade inhibitor PD098059 has recently allowed the demonstration that this signaling pathway is involved in key cellular processes in bivalves (Katsu et al., 2000; Canesi et al., 2000Go). In the present study, inhibition of MAP kinase signaling by PD098059 resulted in increased apoptosis among ISO-treated oyster hemocytes. This result suggests that the MAP kinase cascade is involved in cellular processes that protect oyster hemocytes against apoptosis induced by ß-adrenergic signaling. The MAP kinase cascade is activated by various molecules including protein kinase C and small GTPases of the Ras family. Among these small GTPases, proteins of the Rho subfamily are involved in the regulation of cell growth and apoptosis (Lacal, 1997Go). Although rho genes were first identified in Aplysia californica (Madaule and Axel, 1985Go), data are lacking concerning Rho signaling in molluscs. We show here that transfection of oyster hemocytes with a recombinant plasmid containing the Aplysia californica rho gene under the transcriptional control of the gastropod hsp70 gene promoter tends to reduce the apoptotic effect of ISO in oyster hemocytes. The question of whether Rho and MAP kinases act through separate signaling pathways to inhibit oyster hemocyte apoptosis or whether the antiapoptotic signals relayed by Rho require the activation of the MAP kinase cascade now arises. Incubation of Rho-transfected hemocytes in PD098059 prior to treatment with ISO (data not shown) produced highly variable results, possibly because of the antagonistic effects of Rho and PD098059 on oyster hemocyte apoptosis. As a consequence, the question could not be answered in the present study. Nevertheless, our results clearly show that oyster immune cells are under the dual control of apoptotic-inducing signals involving NA and antiapoptotic signaling involving Rho and MAP kinases. Considering that both Rho and the MAP kinase cascade are activated by several growth factors and cytokines in vertebrates (Martin-Blanco, 2000Go), it is plausible that growth factors and cytokines, which are known to exist in invertebrates (Ottaviani and Franceschi, 1996Go; Beck et al., 2000Go), antagonize the apoptosis-inducing effect of NA on oyster hemocytes.

Interestingly, mollusc immune cells are known to secrete NA (Ottaviani and Franceschi, 1996Go). Thus, in addition to being under the influence of NA produced by the neuroendocrine system, hemocytes could regulate the apoptosis of other hemocytes by paracrine mechanisms. Hemocyte apoptosis could also be triggered by an autocrine loop. In this case, progression to apoptosis may be the normal default state of mollusc immune cells, and their survival would be contingent upon antiapoptotic signals emanating from the environment. Such a regulation is known to exist in several human immune cells (Murray et al., 1997Go; Su et al., 1998Go), including neutrophils, where progression towards apoptosis is a constitutive process negatively regulated by granulocyte-macrophage stimulating factor through the activation of extracellular signal-related kinases (also termed ERKs), a subfamilly of MAP kinases (Waterman and Sha'afi, 1995Go; Wei et al., 1996Go; Watson et al., 1998Go).

Further work is needed, however, to clarify these points and to determine the precise function of NA-induced hemocyte apoptosis. Classic hypotheses would be that such a process participates in the maintenance of the immune system homeostasis by controlling hemocyte longevity and clearance, by eliminating abnormal or infected cells or by promoting the removal of hemocytes from sites of inflammation, as in mammals (Osborne, 1996Go).


    Acknowledgments
 
We thank T. P. Yoshino for the constructs bearing the Bomphalaria glabrata hsp70 gene promoter, S. E. Hasnain for the pNN1 construct and J. C. Lacal for the pZIP-neo-rhoWT and pZIP-neo-rhoV14 constructs. We are grateful to S. Jacquet and J. Blanchot for advice concerning flow cytometric analyses and to J.-F. Lennon for his help concerning confocal microscopy. This work was supported by grants from the Conseil Régional de Bretagne, Département du Finistère, Côtes d'Armor et Ille-et-Vilaine and the Section Régionale Conchylicole de Bretagne Nord.


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Arzul, I., Renault, T., Lipart, C. and Davison, A. J. (2001). Evidence for interspecies transmission of oyster herpes virus in marine bivalves. J. Gen. Virol. 82,865 -870.[Abstract/Free Full Text]

Beck, G., Ellis, T. W. and Truong, N. (2000). Characterization of an IL-1 receptor from Asterias forbesi coelomocytes. Cell. Immunol. 203, 66-73.[Medline]

Beidler, D. R., Tewari, M., Friesen, P. D., Poirier, G. and Dixit, V. M. (1995). The baculovirus p35 protein inhibits Fas- and tumor necrosis factor-induced apoptosis. J. Biol. Chem. 270,16526 -16528.[Abstract/Free Full Text]

Bose, R., Chen, P., Loconti, A., Grullich, C., Abrams, J. M. and Kolesnick, R. N. (1998). Ceramide generation by the Reaper protein is not blocked by the caspase inhibitor, p35. J. Biol. Chem. 273,28852 -28859.[Abstract/Free Full Text]

Cadoret, J. P., Debon, R., Cornudella, L., Lardans, V., Morvan, A., Roch, P. and Boulo, V. (1999). Transient expression assays with the proximal promoter of a newly characterized actin gene from the oyster Crassostrea gigas. FEBS Lett. 460, 81-85.[Medline]

Canesi, L., Ciacci, C., Betti, M. and Gallo, G. (2000). Growth factor-mediated signal transduction and redox balance in isolated digestive gland cells from Mytilus galloprovincialis Lam. Comp. Biochem. Physiol. C 125,355 -363.

Cross, T. G., Scheel-Toellner, D., Henriquez, N. V., Deacon, E., Salmon, M. and Lord, J. M. (2000). Serine/threonine protein kinases and apoptosis. Exp. Cell. Res. 256, 34-41.[Medline]

De Jong-Brink, M. (1995). How schistosomes profit from the stress responses they elicit in their hosts. Adv. Parasitol. 35,177 -256.[Medline]

Fisher, A. J., Cruz, W., Zoog, S. J., Schneider, C. L. and Friesen, P. D. (1999). Crystal structure of baculovirus P35: role of a novel reactive site loop in apoptotic caspase inhibition. EMBO J. 18,2031 -2039.[Abstract/Free Full Text]

Fraser, A. G. and Evan, G. I. (1997). Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE. EMBO J. 16,2805 -2813.[Abstract/Free Full Text]

Genthner, F. J., Volety, A. K., Oliver, L. M. and Fisher, W. S. (1999). Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica). Appl. Environ. Microbiol. 65,3015 -3020.[Abstract/Free Full Text]

Hay, B. A., Wolff, T. and Rubin, G. M. (1994). Expression of baculovirus P35 prevents cell death in Drosophila.Development 120,2121 -2129.[Abstract/Free Full Text]

Hengartner, M. O. (1999). Programmed cell death in the nematode C. elegans. Recent Prog. Horm. Res. 54,213 -222.[Medline]

Katsu, Y., Minshall, N., Nagahama, Y. and Standart, N. (1999). Ca2+ is required for phosphorylation of clam p82/CPEB in vitro: implications for dual and independent roles of MAP and Cdc2 kinases. Dev. Biol. 209,186 -199.[Medline]

Kumar, S. and Doumanis, J. (2000). The fly caspases. Cell Death Differ. 7,1039 -1044.[Medline]

Lacal, J. C. (1997). Regulation of proliferation and apoptosis by Ras and Rho GTPases through specific phospholipid-dependent signaling. FEBS Lett. 410, 73-77.[Medline]

Lacoste, A., Malham, S. K., Cueff, A. and Poulet, S. A. (2001a). Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus. Appl. Environ. Microbiol. 67,2304 -2309.[Abstract/Free Full Text]

Lacoste, A., Malham, S. K., Cueff, A., Jalabert, F., Gélébart, F. and Poulet, S. A. (2001b). Evidence for a form of adrenergic response to stress in the oyster Crassostrea gigas. J. Exp. Biol. 204,1247 -1255.[Abstract/Free Full Text]

Lacoste, A., Malham, S. K., Cueff, A. and Poulet, S. A. (2001c). Stress-induced catecholamine changes in the hemolymph of the oyster, Crassostrea gigas. Gen. Comp. Endocrinol. 122,181 -188.[Medline]

Lacoste, A., Malham, S. K., Cueff, A. and Poulet, S. A. (2001d). Noradrenaline modulates hemocyte reactive oxygen species production via ß-adrenergic receptors in the oyster Crassostrea gigas. Dev. Comp. Immunol. 25,285 -289.[Medline]

Lacoste, A., Malham, S. K., Cueff, A. and Poulet, S. A. (2001e). Noradrenaline inhibits phagocytosis by hemocytes of the oyster Crassostrea gigas via a ß-adrenoceptor/cAMP signalling pathway. Gen. Comp. Endocrinol. 122,252 -259.[Medline]

Lacoste, A., De Cian, M.-C., Cueff, A. and Poulet, S. A. (2001f). Noradrenaline and {alpha}-adrenergic signaling induce the hsp70 gene promoter in mollusc immune cells. J. Cell Sci. 114,3557 -3564.[Abstract/Free Full Text]

LaCount, D. J., Hanson, S. F., Schneider, C. L. and Friesen, P. D. (2000). Caspase inhibitor P35 and inhibitor of apoptosis Op-IAP block in vivo proteolytic activation of an effector caspase at different steps. J. Biol. Chem. 275,15657 -15664.[Abstract/Free Full Text]

Madaule, P. and Axel, R. (1985). A novel ras-related gene family. Cell 41, 31-40.[Medline]

Martin-Blanco, E. (2000). p38 MAPK signalling cascades: ancient roles and new functions. BioEssays 22,637 -645.[Medline]

Martínez, G. and Rivera, A. (1994). Role of monoamines in the reproductive process of Argopecten purpuratus. Invertebr. Reprod. Dev. 25,167 -174.

Mitta, G., Vandenbulcke, F., Noël, T., Romestand, B., Beauvillain, J.-C., Salzet, P. and Roch, P. (2000). Differential distribution and defence involvement of antimicrobial peptides in mussel. J. Cell Sci. 113,2759 -2769.[Abstract/Free Full Text]

Muneoka, Y. and Kamura, M. (1982). The multiplicity of neurotansmitters and neurohormones controlling mytilus muscle. Comp. Biochem. Physiol. 73C,149 -156.

Murray, J., Barbara, J. A., Dunkley, S. A., Lopez, A. F., Van Ostade, X., Condliffe, A. M., Dransfield, I., Haslett, C. and Chilvers, E. R. (1997). Regulation of neutrophil apoptosis by tumor necrosis factor-{alpha}: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro. Blood 90,2772 -2783.[Abstract/Free Full Text]

Ottaviani, E. and Franceschi, C. (1996). The neuroendocrinology of stress from invertebrates to man. Prog. Neurobiol. 48,421 -440.[Medline]

Osborne, B. A. (1996). Apoptosis and the maintenance of homeostasis in the immune system. Curr. Opin. Immunol. 8,245 -254.[Medline]

Pani, A. K. and Croll, R. P. (2000). Catechol concentrations in the hemolymph of the scallop, Placopecten magellanicus.Gen. Comp. Endocrinol. 118,48 -56.[Medline]

Perona, R., Esteve, P., Jimenez, B., Ballestero, R. P., Ramon y Cajal, S. and Lacal, J. C. (1993). Tumorigenic activity of rho genes from Aplysia californica. Oncogene 8,1285 -1292.[Medline]

Pertseva, M. N., Kuznetzova, L. A., Pestneva, S. A. and Grishin, A. V. (1992). ß-agonist-induced inhibitory-guanine-nucleotide-binding regulatory protein coupling to adenylate cyclase in mollusc Anodonta cygnea foot muscle sarcolemma. Eur. J. Biochem; 210,279 -286.[Abstract]

Pipe, R. K. (1992). Generation of reactive oxygen metabolites by the hemocytes of the mussel Mytilus edulis.Dev. Comp. Immunol. 16,111 -122.[Medline]

Pires, A., Coon, S. L. and Hadfield, M. G. (1997). CA and dihydroyphenylalanine in metamorphosing larvae of the nudibranch Phestilla sibogae, Bergh (Gastropoda, Opistobranchia). J. Comp. Physiol. 181A,187 -194.[Medline]

Pronk, G. J., Ramer, K., Amiri, P. and Williams, L. T. (1996). Requirement of an ICE-like protease for induction of apoptosis and ceramide generation by REAPER. Science 271,808 -810.[Abstract]

Raff, M. C. (1992). Social controls on cell survival and cell death. Nature 356,397 -400.[Medline]

Sah, N. K., Taneja, T. K., Pathak, N., Begum, R., Athar, M. and Hasnain, S. E. (1999). The baculovirus antiapoptotic p35 gene also functions via an oxidant-dependent pathway. Proc. Natl. Acad. Sci. USA. 96,4838 -4843.[Abstract/Free Full Text]

Sakharov, D. A. and Salànski, J. (1982). Effects of dopamine antagonists on snail locomotion. Experientia 38,1090 -1091.

Salvesen, G. S. and Dixit, V. M. (1997). Caspases: intracellular signaling by proteolysis. Cell 91,443 -446.[Medline]

Salzet, B., Stefano, G. B., Verger-Bocquet, M. and Salzet, M. (1998). Putative leech dopamine 1-like receptor molecular characterization: sequence homologies between dopamine and serotonin leech CNS receptors explain pharmacological cross-reactivities. Mol. Brain Res. 58,47 -58.[Medline]

Santhanagopalan, V. and Yoshino, T. P. (2000). Monoamines and their metabolites in the freshwater snail Biomphalaria glabrata. Comp. Biochem. Physiol. A 125,469 -478.

Sminia, T. and Van Der Knaap, W. P. W. (1987). Cells and molecules in molluscan immunology. Dev. Comp. Immunol. 11,17 -28.[Medline]

Su, X., Cheng, J., Liu, W., Liu, C., Wang, Z., Yang, P., Zhou, T. and Mountz, J. D. (1998). Autocrine and paracrine apoptosis are mediated by differential regulation of Fas ligand activity in two distinct Jurkat T cell populations. J. Immunol. 160,5288 -5293.[Abstract/Free Full Text]

Sugimoto, A., Friesen, P. D. and Rothman, J. H. (1994). Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode Caenorhabditis elegans.EMBO J. 13,2023 -2028.[Abstract]

Syed, N. I. and Winlow, W. (1991). Respiratory behavior in the pond snail Lymnaea stagnalis. II. Neural elements of the central pattern generator. J. Comp. Physiol. 169A,557 -568.

Teyke, T., Rosen, S. C., Weiss, K. R. and Kupfermann, I. (1993). Dopaminergic neuron B20 generates rhythmic neuronal activity in the feeding motor circuitry of Aplysia. Brain Res. 630,226 -237.[Medline]

Vaulot, D. (1989). CYTOPC: Processing software for flow cytometric data. Signal and Noise 2, 8.

Waterman, W. H. and Sha'afi, R. I. (1995). Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-{alpha} on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils. Biochem. J. 307,39 -45.[Medline]

Watson, R. W., Rotstein, O. D., Parodo, J., Bitar, R., Marshall, J. C., William, R. and Watson, G. (1998). The IL-1ß-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1ß. J. Immunol. 161,957 -962.[Abstract/Free Full Text]

Wei, S., Liu, J. H., Epling-Burnette, P. K., Gamero, A. M., Ussery, D., Pearson, E. W., Elkabani, M. E., Diaz, J. I. and Djeu, J. Y. (1996). Critical role of Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor. J. Immunol. 157,5155 -5162.[Abstract]

White, E. (1996). Life, death, and the pursuit of apoptosis. Genes Dev. 10, 1-15.[Medline]

Xue, D. and Horvitz, H. R. (1995). Inhibition of the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377,248 -251.[Medline]

Yamamoto, M., Okumura, S., Schwencke, C., Sadoshima, J. and Ishikawa, Y. (1999). High efficiency gene transfer by multiple transfection protocol. Histochem J. 31,241 -243.[Medline]

Yoshino, T. P. and Vasta G. R. (1996). Parasite-invertebrate host interactions. Adv. Comp. Environ. Physiol. 24,125 -167.

Yoshino, T. P., Wu, X. J. and Liu, H. D. (1998). Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line. Am. J. Trop. Med. Hyg. 59,414 -420.[Abstract/Free Full Text]

Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. and Horvitz, H. R. (1993). The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1ß-converting enzyme. Cell 75,641 -652.[Medline]

Zoog, S. J., Bertin, J. and Friesen, P. D. (1999). Caspase inhibition by baculovirus P35 requires interaction between the reactive site loop and the beta-sheet core. J. Biol. Chem. 274,25995 -26002.[Abstract/Free Full Text]