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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Summary |
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Key words: Mollusc, Immune cell, Noradrenaline, Apoptosis, ß-adrenergic signaling
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Introduction |
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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., 2001),
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, 1995
;
Yoshino and Vasta, 1996
;
Genthner et al., 1999
;
Lacoste et al., 2001a
).
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, 1992), antimicrobial
peptide secretion (Mitta et al.,
2000
), encapsulation and phagocytosis
(Sminia and Van Der Knaap,
1987
). Previous studies have shown that bivalve hemocytes
synthesize and secrete catecholamines (CA) including noradrenaline and
dopamine (Ottaviani and Franceschi,
1996
). Moreover, CA are known to circulate in the hemolymph of
both gastropods and bivalves
(Santhanagopalan and Yoshino,
2000
; Pani and Croll,
2000
; Lacoste et al.,
2001b
; Lacoste et al.,
2001c
), 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., 1993
),
locomotion (Sakharov and
Salànski, 1982
), respiration
(Syed and Winlow, 1991
),
reproduction
(Martínez
and Rivera, 1994
) and development
(Pires et al., 1997
).
Moreover, bivalve and gastropod immune cells express specific receptors for CA
such as dopamine (Salzet et al.,
1998
) and noradrenaline
(Lacoste et al., 2001d
;
Lacoste et al., 2001d
;
Lacoste et al., 2001f
);
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.
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Materials and Methods |
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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, 1989). 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., 1993)
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.,
1998
) 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., 1999
) 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., 1993
)
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., 1998;
Cadoret et al., 1999
;
Lacoste et al., 2001f
). 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.,
1999
; Lacoste et al.,
2001f
) 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., 1999
). Cells were
then exposed to 1 µM ISO for 12 hours and monitored for apoptosis.
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Results |
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|
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The -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
-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.
|
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.
|
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., 1999
;
Zoog et al., 1999
;
LaCount et al., 2000
), 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.
|
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.
|
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., 1993), 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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Discussion |
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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-receptorcAMP-dependent signaling pathway is present
in mollusc foot muscle sarcolemma
(Pertseva et al., 1992), 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,
1982
).
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., 1993), and even
though caspase genes have been cloned in mammals and Drosophila
(Fraser and Evan, 1997
;
Salvesen and Dixit, 1997
),
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., 1996
;
Bose et al., 1998
). 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.,
1999
; Zoog et al.,
1999
; LaCount et al.,
2000
), 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.,
1994
; Sugimoto et al.,
1994
; Beidler et al.,
1995
; Xue and Horvitz,
1995
). 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., 2000). MAP
kinases are thought to exist in all eukaryotic cells from yeast to human
(Martin-Blanco, 2000
), 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., 2000
). 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, 1997
). Although
rho genes were first identified in Aplysia californica
(Madaule and Axel, 1985
), 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, 2000
), it is
plausible that growth factors and cytokines, which are known to exist in
invertebrates (Ottaviani and Franceschi,
1996
; Beck et al.,
2000
), antagonize the apoptosis-inducing effect of NA on oyster
hemocytes.
Interestingly, mollusc immune cells are known to secrete NA
(Ottaviani and Franceschi,
1996). 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., 1997
;
Su et al., 1998
), 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, 1995
; Wei et al.,
1996
; Watson et al.,
1998
).
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, 1996).
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Acknowledgments |
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