Immune responses of mussel hemocyte subpopulations are

differentially regulated by enzymes of the PI 3-K, PKC, and

ERK kinase families.

Erick García-García1, Maria Prado-Álvarez2, Beatriz Novoa2, Antonio Figueras2,

and Carlos Rosales1*

1Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad

Nacional Autónoma de México, Mexico City, MEXICO, and 2Instituto de

Investigaciones Marinas, CSIC, Spanish National Reference Laboratory for Mollusc

Diseases, 36208 Vigo, SPAIN.

* Corresponding author: Carlos Rosales, Department of Immunology, Instituto de

Investigaciones Biomédicas - UNAM, Apdo. Postal 70228, Cd. Universitaria, México

D.F. - 04510, MEXICO. Phone: 52 - 55 - 5622 – 8951 FAX: 52 - 55 - 5622 – 9212.

Email: carosal@servidor.unam.mx

* Manuscript

2

Immune functions of mussel hemocyte subpopulations are differentially

regulated by enzymes of the PI 3-K, PKC and ERK kinases families

Abstract.

Various hemocyte cell types have been described in invertebrates, but for most

species a functional characterization of different hemocyte cell types is still lacking. In

order to characterize some immunological properties of mussel (Mytilus

galloprovincalis) hemocytes, cells were separated by flow cytometry and their capacity

for phagocytosis, production of reactive oxygen species (ROS), and production of nitric

oxide (NO), was examined. PI 3-K, PKC and ERK inhibitors were also used to

biochemically characterize these cell responses. Four morphologically distinct

subpopulations, designated R1 to R4, were detected. R1, R2, and R3 cells presented

different levels of phagocytosis towards zymosan, latex beads, and two bacteria species.

Similarly, R1 to R3, but not R4 cells produced ROS, while all subpopulations produced

NO, in response to zymosan. Internalisation of all phagocytic targets was blocked by PI

3-K inhibition. In addition, internalisation of latex particles, but not of bacteria, was

partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan

was impaired by PKC, or ERK inhibitors only in R2 cells. Zymosan-induced ROS

production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In

addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1

and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by

PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the

existence of profound functional and biochemical differences in mussel hemocytes, and

indicate that M. galloprovinciallis hemocytes are specialized cells fulfilling specific

tasks in the context of host defence.

3

Key words: mollusc, signal transduction, innate immunity, phagocytosis,

respiratory burst, wortmannin, staurosporine, PD98059, professional phagocytes.

1. Introduction.

Despite the lack of an adaptive immune system, invertebrates are able to survive

among potentially pathogenic viruses, bacteria, and fungi [1, 2], and respond to

infection by activating various defence mechanisms [2, 3]. This suggests a key role for

their apparently limited innate immune system, in protecting these invertebrates from

infections. Among the defence mechanisms described in invertebrates are phagocytosis,

activation of the phenoloxydase system, encapsulation, the respiratory burst, nitric oxide

production, and the production and release of various microbicidal molecules [3]. It is

however unclear which of the different cells integrating the invertebrate innate immune

system, usually named hemocytes, are responsible for these cell responses.

Different types of hemocytes have been described in various invertebrate

species, but an accurate universal classification of invertebrate hemocytes is still

lacking. A consensus hemocyte classification is not available yet, due in part to intrinsic

differences in the immune systems of even closely related invertebrate species [4-6],

and also to differences in the experimental methods used to study them [7-11].

Depending on the species, and the experimental methodology, hemocytes have been

classified in as few as two, or up to eleven categories [7, 11, 12]. In molluscs, hemocyte

classification has largely depended on cell granularity, determined either by microscopy

or by flow cytometry [6, 13-16]. However, as in most invertebrate phyla, no definitive

mollusc hemocyte classification exists.

Extensive studies have shown that blood cells appear to have conserved roles in

vertebrate organisms ranging from fish to mammals [17-19]. In contrast, few attempts

4

have been made to functionally characterize the different hemocyte subtypes of most

invertebrate organisms. For example, while little doubt exists regarding the phagocytic

and microbicidal abilities of macrophages [19], even in lower vertebrates such as fish

[18], the information regarding the specific defence mechanisms displayed by different

hemocyte subtypes is scarce, and has largely focused only on phagocytic capacity [8-10,

20-22].

In mammals, phagocytosis and the respiratory burst are fundamental defence

mechanisms of professional phagocytes, that often take place simultaneously [23-26].

Phagocytosis is the physical internalisation of microbes or cellular debris [25, 26]. The

respiratory burst on the other hand, is a series of biochemical reactions that produce

highly microbicidal reactive oxygen species (ROS), such as superoxide (O2-), hydrogen

peroxide (H2O2), and hydroxyl radical (OH.) [23, 24]. Phagocytosis and activation of

the respiratory burst have been observed in hemocytes of various mollusc species [8,

27-33]. However, the specific hemocyte subtypes that present these cell responses are

still unknown.

In the mammalian immune system, the production of nitric oxide (NO) is part of

the defence mechanisms displayed by professional phagocytes such as macrophages

[34, 35]. NO is a highly cytotoxic and microbicidal molecule [34, 35], also capable of

activating other leukocytes [36]. The expression of a nitric oxide synthase, and the

production of NO by hemocytes has been reported in various mollusc species [37-43].

Yet, no information is available on the capacity of specific hemocyte subtypes to

produce NO.

In mammalian leukocytes, the signalling pathways regulating immune

responses, such as phagocytosis, differ depending on the type of phagocyte involved

[44], the specific nature of the receptor involved [45], and the differentiation and

5

activation state of the phagocytic cells [44, 46, 47]. Kinases of the phosphatidylinositol

3-kinase (PI 3-K), and protein kinase C (PKC) families are key regulators of

phagocytosis mediated through various membrane receptors [25, 26]. In addition, the

extracellular signal-regulated kinase (ERK) is an enzyme involved in phagocytosis

regulation, depending on the phagocytic receptor involved [25], and also on the

phagocytic cell type [44, 48, 49]. The role of these signalling molecules in the

regulation of hemocyte phagocytosis is unclear. Similarly, the participation of these

signalling enzymes in the regulation of the respiratory burst or NO production in

invertebrate organisms is poorly defined.

In order to characterize some immunological properties of the mussel M.

galloprovincialis hemocyte subtypes, cells were separated by flow cytometry and their

capacity for phagocytosis, production of reactive oxygen species (ROS), and production

of nitric oxide (NO), was examined. PI 3-K, PKC and ERK inhibitors were used to

biochemically characterize these cell functions. Four morphologically distinct

subpopulations, designated Large granular (R1), Large semigranular (R2), Small

semigranular (R3), and Small hyaline (R4) hemocytes, were detected in mussel

hemolymph. While Large granular (R1), Large semigranular (R2), and Small

semigranular (R3) cells were phagocytic, and capable of activating the respiratory burst,

Small hyaline (R4) cells lacked these two capabilities. Nevertheless, all hemocyte

subpopulations were capable of NO production. Biochemical characterization of

hemocyte functions showed a clear role for PI 3-K in phagocytosis and the respiratory

burst, and also for NO production in some cell types. PKC or ERK participated in

phagocytosis regulation, depending on the hemocyte subtype and the phagocytic target,

but had no role in the regulation of zymosan-induced respiratory burst. While zymosan-

induced NO production was independent of PKC, this cell response required ERK, but

6

only in some cell types. These data revealed the existence of critical functional and

biochemical differences in mussel hemocytes, and indicate that M. galloprovinciallis

hemocytes are specialized cells fulfilling specific tasks in the context of host defence.

2. Materials and Methods.

2.1 Reagents

Sea water was obtained from the Ría de Vigo (NW Spain). It was filtered

sequentially through 0.45 μm and 0.22 μm porous membranes before use. Flourescein-

labelled 1μm latex beads (catalogue number F8823), flourescein-labelled zymosan

bioparticles (catalogue number Z-284), flourescein-labelled E. coli bacteria (catalogue

number E-2861), the reactive oxygen species (ROS)-specific intracellular indicator

chloromethyl-H2DCFDA (catalogue number C6827), and the nitric oxide (NO)-specific

intracellular indicator DAF-FM/ DAF-FM diacetate (catalogue number D-23844), were

all from Molecular Probes (Eugene, OR). Fluorescein-labelled Vibrio alginolyticus

bacteria were prepared as described previously [50]. PD98059 (catalogue number

V1191) was from Promega, (Madison, WI). Wortmannin (catalogue number 19545-26-

7), phorbol myristate acetate (PMA) (catalogue number P8139), staurosporine

(catalogue number 55921), and all other chemicals were from Sigma Chemical

Company (St. Louis, MO).

2.2 Mussel hemocytes

Mussels (Mytilus galloprovincialis) were obtained from the Ría de Vigo (NW

Spain). They were maintained in filtered sea water tanks at 15 ºC with aeration, and fed

daily with the alga Tetraselmis svecica. Mussel shells were notched, and one to three ml

of hemolymph per individual were withdrawn from the adductor muscle. All

7

experiments were performed with hemolymph pools from a minimum of three

individuals.

2.3 Flow cytometry analysis of hemocyte subpopulations

Freshly isolated hemocytes diluted two fold with cold filtered sea water, or fixed

for 20 minutes with cold 2 % paraformaldehyde in sea water, were analysed in a

FACScalibur flow cytometer (Becton Dickinson; Franklin Lakes, NJ) equipped with

cell sorting and cell concentrator modules. Cell size (Forward light Scatter, FSC) and

cellular complexity (Side light Scatter, SSC) parameters were used to distinguish

different hemocyte subpopulations in dot-plots. Instrument settings were as follows:

FSC set in log scale at E-1, SSC set in log scale at 287, the primary parameter for

threshold was FSC set at 217. Ten thousand events were recorded for each hemolymph

sample. No differences were observed in dot-plots between fresh or 2 %

paraformaldehyde-fixed hemolymph samples (data not shown).

2.4 Morphological analysis of hemocyte subpopulations.

Cell sorting of paraformaldehyde-fixed cells was performed using sea water as

sheath fluid. Cells were recovered from the concentrator module, centrifuged at 300 x g

in Eppendorf tubes for 10 minutes, resuspended in 3.5 μl 10 % bovine serum albumin in

PBS, and placed on glass slides. Cells were then either placed under glass cover-slips,

and observed directly with a Nikon Optiphot phase-contrast microscope (Nikon

Instruments; Melville, NY), or air-dried and stained with hemacolor (catalogue

numbers: Red 1.11956.2500, Blue 1.11957.2500; Merk KGaA, Darmstadt, Germany)

for observation in a Nikon Optiphot light transmission microscope (Nikon Instruments;

Melville, NY).

2.5 Phagocytosis assays

8

Five hundred μl of hemolymph were placed into individual wells of tissue-

culture 24-well plates. Hemocyte number in each sample was estimated by counting

cells in a hemocytometer. Hemocytes were allowed to adhere to the well bottom by

incubating them for 30 minutes at 15 ºC in the dark. Hemolymph supernatant was then

removed by blotting onto paper towels. Five hundred μl of filtered sea water were then

added to each well. Flourescein-labelled 1μm latex beads, flourescein-labelled zymosan

bioparticles, flourescein-labelled E. coli bacteria, or fluorescein-labelled Vibrio

alginolyticus bacteria were added at a 10:1 target:hemocyte ratio. After a two hour

incubation at 15 ºC in the dark, uninternalised particles were removed by washing wells

twice with 500 μl PBS. Cells were finally resuspended in 500 μl PBS by gently

detaching them from the well bottom, using a rubber cell scraper. Fifty μl of 0.8 %

trypan blue in PBS were added to each sample to quench external fluorescence. Ten

thousand cells were then analysed by flow cytometry. Cell fluorescence was analysed

through the FL-1 channel set at log scale. FL-1 was adjusted for each phagocytic target,

so that positive cells (cells that internalised al least one particle) fell within the same

fluorescence range, independently of the phagocytic target used. Phagocytosis was

expressed as the percentage of cells that internalised at least one fluorescent particle

(positive cells). In selected experiments PI 3-K, PKC, or ERK inhibitors, or only the

DMSO solvent, were added to the cells and incubated at 15 ºC in the dark for 30

minutes before mixing them with the fluorescent particles.

2.5 Analysis of the respiratory burst and nitric oxide production

Two hundred and fifty μl of hemolymph pools were placed into individual wells

of tissue-culture U-bottom 96-well plates. After 30 minutes at 15 ºC in the dark, the

hemolymph supernatant was removed, and 200 μl filtered sea water, containing 0.4 %

DMSO and 5 μg/ml of chloromethyl-H2DCFDA, or DAF-FM/ DAF-FM diacetate were

9

added per well. In some experiments PI 3-K, PKC, or ERK inhibitors were added

together with the ROS or NO indicators. After 30 minutes at 15 ºC in the dark, the

supernatant was removed, and wells washed twice with filtered sea water. Zymosan (1

mg/ml), or PMA (1-1000 ng/ml) were then added in 200 μl sea water. Cells were

incubated for 1 hour at 15 ºC in the dark, washed twice with PBS, and finally

resuspended in 200 μl PBS. Cell fluorescence was analysed by flow cytometry through

the FL-1 channel, set at log scale. The mean fluorescence intensity (MFI) values of the

different hemocyte subpopulations in the resting state, was considered to be 100%. The

change in MFI value, induced by zymosan or PMA stimulation, was then plotted as a

percentage of the resting state value for each hemocyte subpopulation..

2.6 Statistical analysis

Data were analysed by paired-samples T-tests; or by one-way ANOVA with

post-hoc multiple-comparisons tests (Dunnett), using the Statistical Package for the

Social Sciences v8.0 software (SPSS Inc, Chicago, IL). A significance level of P < 0.05

was used. The maximum p values for the statistically different points in each data series

are given in the figure legends.

3. Results

3.1 Characterization of hemocyte subpopulations.

M. galloprovincialis hemolymph, withdrawn from the adductor muscle,

presented a mixed population of adherent cells differing in size and morphology (Fig.

1A). Flow cytometry analysis was used to characterize the cell types present in mussel

hemolymph. Based on measurements of cell size (Forward light Scatter, FSC) and

complexity (Side light Scatter, SSC), four morphologically distinct hemocyte

subpopulations could be separated. These hemocyte subpopulations were designated R1

10

to R4 (Fig. 1B). R1 are large granular cells with high SSC values, sizing between 15

and 20 μm in diameter (Fig. 1B and 1C), and possessing a relatively small nucleus

located eccentrically (Fig. 1C). R2 are large semigranular cells with intermediate SSC

values, sizing between 15 and 20 μm in diameter, and presenting a larger nucleus (Fig.

1B and 1D). R3 cells are smaller semigranular cells with intermediate SSC values (Fig.

1B and 1E), presenting a small nucleus and several membrane extensions (Fig. 1E). R4

cells are small agranular (hyaline) cells, with the lowest SSC values (Fig. 1B and 1F),

and a prominent nucleus that covers most of the cytoplasm. Small hyaline cells were

around 10 to 15 μm in diameter (Fig. 1F).

3.2 Large granular, Large semigranular, and Small semigranular hemocyte

subpopulations are phagocytic.

To assess the phagocytosis potential of the various mussel hemocyte subtypes,

cells were mixed with several fluorescein-labelled phagocytic targets, allowed to ingest

these targets, and then analysed by flow cytometry. Hemocytes in regions R1 (Large

granular hemocytes), R2 (Large semigranular hemocytes), and R3 (Small semigranular

hemocytes) were all capable of phagocytosis, although at different levels (Fig. 2).

Zymosan (Fig. 2B), latex beads (Fig. 2C), and the bacteria V. alginolyticus (Fig. 2D)

were ingested efficiently by all Large granular (R1), Large semigranular (R2), and

Small semigranular (R3) hemocytes. In contrast, the bacteria E. coli, which appears to

be non-pathogenic for molluscs [50, 51], was poorly phagocytosed (Fig. 2E). Small

hyaline (R4) hemocytes were not phagocytic at all (Fig. 2). Large granular (R1)

hemocytes were the most efficient phagocytic cells, followed by Large semigranular

(R2) hemocytes, and then Small semigranular (R3) hemocytes (Fig. 3). Zymosan was

ingested by about 40 % of the total of Large granular (R1) cells, and by about 25 % of

the total of Large semigranular (R2) or Small semigranular (R3) cells (Fig. 3A). Latex

11

beads were also internalised by about 40 % of Large granular (r1) cells, and by about 30

% of Large semigranular (R2) cells, but only by about 10 % of Small semigranular (R3)

cells (Fig. 4B). In addition, the bacteria V. alginolyticus, was efficiently ingested by

about 60 % of the total of Large granular (R1) cells, by about 45 % of Large

semigranular (R2) cells, and by almost 30 % of Small semigranular (R3) cells (Fig. 3C).

In contrast, the bacteria E. coli, was poorly phagocytosed by all hemocyte subtypes

(Fig. 3 D). These results indicated that Large granular (R1) and Large semigranular

(R2) mussel hemocytes are efficient phagocytes, with different phagocytic abilities

depending on the target they are presented.

3.3 Phagocytosis regulation by PI 3-K, PKC, or ERK.

To investigate the participation of PI 3-K, PKC, and ERK enzymes in

phagocytosis, mussel hemocytes were treated with specific inhibitors for these enzymes

before allowing them to phagocytose fluorescein-labelled particles. Phagocytosis of

zymosan was inhibited by wortmannin (the PI 3-K inhibitor) in the three phagocytic

hemocyte subpopulations (Fig. 4 A). Phagocytosis by Large granular cells was the most

sensitive to PI 3-K inhibition. Inhibition of PKC, and ERK (by staurosporine, and

PD98059, respectively) did not affect zymosan phagocytosis in either Large granular or

Small semigranular hemocytes (Fig. 4B and 4C). In contrast, zymosan phagocytosis by

Large semigranular hemocytes was significantly reduced after treatment with

staurosporine or PD98059 (Fig. 4B and 4C). None of the inhibitors affected cell

viability at the highest concentrations used: 85.2 ± 5.5 % hemocytes were viable after

DMSO treatment, 86 ± 2.6 % after wortmannin treatment, 87.6 ± 1.9 % after

staurosporine treatment, and 83.6 ± 4.2 % after PD98059 treatment. These data showed

that all types of hemocytes require PI 3-K activity for phagocytosis of zymosan, and

that only Large semigranular hemocytes also needed PKC and ERK for zymosan

12

internalisation. Phagocytosis of latex beads was also inhibited by wortmannin in the

three phagocytic hemocyte subpopulations (Fig. 5A). All cells seemed to be similarly

sensitive to PI 3-K inhibition (Fig. 5A). In contrast, inhibition of PKC and ERK caused

only a minute decrease in the level of phagocytosis by all phagocytic hemocytes (Fig.

5B and 5C).

Because the phagocytic activity of mussel hemocytes seemed to vary depending

on the type of bacteria being ingested (Fig. 4), the biochemical requirements for these

forms of phagocytosis were evaluated using the inhibitors for PI 3-K, PKC, and ERK.

Phagocytosis of the bacteria V. alginolyticus was inhibited, about 50 %, by wortmannin

in the three phagocytic hemocyte subpopulations (Fig. 6A). In contrast, inhibition of

PKC and ERK did not affect V. alginolyticus phagocytosis by any type of hemocytes

(Fig. 6B and 6C). Interestingly, even though phagocytosis of the bacteria E. coli was

very poor in all types of hemocytes (Fig. 4), it was also inhibited about 50 % by

wortmannin in the three phagocytic hemocyte subpopulations (Fig. 7A). Similarly to V.

alginolyticus, inhibition of PKC and ERK did not affect E. coli phagocytosis by any

type of hemocytes (Fig. 7B and 7C). These data suggested that all types of hemocytes

require PI 3-K activity for phagocytosis of bacteria, and that this type of phagocytosis is

independent of PKC and ERK activities. Data also suggested that efficient phagocytosis

of different bacterial species probably involves other mechanisms besides the use of PI

3-K.

3.4 Large granular, Large semigranular, and Small semigranular hemocyte

subpopulations produce reactive oxygen species.

Zymosan was used to determine which of the different hemocyte subpopulations

presented was capable of producing reactive oxygen species (ROS). Large granular

(R1), Large semigranular (R2), and Small semigranular (R3) hemocytes were all

13

capable of ROS production at comparable levels (Fig. 8). Small hyaline (R4) hemocytes

showed a modest increase in ROS, but this increment was not statistically different from

control (unstimulated) cells (Fig.8).

3.5 Regulation of reactive oxygen species production by PI 3-K or PKC.

Similarly to phagocytosis, the participation of certain enzymes, such as PI 3-K,

PKC, and ERK, in ROS production was investigated with the use of pharmacological

inhibitors. Zymosan-induced ROS production was inhibited by wortmannin in the three

phagocytic hemocyte subpopulations (Fig. 9A). Large semigranular cells were the most

sensitive to PI 3-K inhibition. Inhibition of PKC, and ERK did not affect ROS

production by any of the hemocyte subpopulations (Fig. 9B and 9C). Although, PKC

inhibition by staurosporine did not affect zymosan-induced ROS production, it has been

reported in other cell types that PKC is in fact a good activator of the respiratory burst

[52, 53]. Treatment of mussel hemocytes with the phorbol ester PMA, a well known

activator of PKC, indeed resulted in strong ROS production independently of any other

stimulus. This increase in ROS production was three to four-fold in the phagocytic

hemocyte subpopulations (Fig. 9D). In Small hyaline (R4) cells, PMA-induced

activation of PKC caused only a small increment in ROS production (not shown). These

data showed that Large granular (R1), Large semigranular (R2), and Small semigranular

(R3) hemocyte subtypes require PI 3-K activity for zymosan-induced ROS production,

and that neither PKC, nor ERK, participate in this cell response. However, PKC seems

to participate in other pathways that lead to an even stronger activation of the

respiratory burst.

3.6 All hemocyte subpopulations produce nitric oxide.

All hemocyte subpopulations were capable of producing nitric oxide (NO) in

response to zymosan (Fig. 10). Large granular (R1), and Large semigranular (R2)

14

hemocytes presented NO production that doubled the basal NO levels of unstimulated

cells (Fig. 10A and 10B). Small semigranular (R3) and Small hyaline (R4) hemocytes

showed a smaller increase in NO production, but this increment was statistically

different from control (unstimulated) cells (Fig. 10C and 10D).

3.7 Regulation of NO production by PI 3-K, PKC, or ERK.

Similarly to ROS production, the participation of PI 3-K, PKC, and ERK, in NO

production was investigated with the use of pharmacological inhibitors. Zymosan-

induced NO production was inhibited by wortmannin in the Large granular and Large

semigranular hemocyte subpopulations (Fig. 11A). Large semigranular cells were also

the most sensitive to PI 3-K inhibition. The small zymosan-induced increase in NO

production by Small semigranular and Small hyaline hemocytes was not affected by

wortmannin (Fig. 11A). Inhibition of PKC, did not affect NO production in any of the

hemocyte subpopulations (Fig. 11B), suggesting that PKC is not used for zymosan-

induced NO production. Similarly to phagocytosis (Fig. 4C) NO production was only

affected by EKR inhibition in Large semigranular hemocytes (Fig. 11C). Interestingly,

in absence of any other stimulus, PKC activation by PMA resulted in diminished levels

of NO in phagocytic hemocytes (Fig. 11D). These data suggest that efficient NO

production requires PI 3-K (in Large granular and Large semigranular cells), and

possibly ERK (in Large semigranular cells) activities.

15

4. Discussion.

The information regarding cell functions in invertebrate hemolymph has

increased substantially over the last decade, but still hemocyte classification remains

controversial. This is partially due to dissimilar classification criteria adopted for each

species. Classifications have been based on morphology and cytochemical properties for

the most part, and to a lesser extent on cell functions. Using flow cytometry techniques,

we have identified four morphologically distinct hemocyte subpopulations in the mussel

M. galloprovincialis: Large granular cells (R1), Large semigranular cells (R2), Small

semigranular cells (R3), and Small Hyaline cells (R4). Through the use of flow

cytometry we were able to show, for the first time, the capacity for phagocytosis, ROS

production, and NO production, of clearly defined hemocyte subpopulations in a

mollusc species. From the four M. galloprovincialis hemocyte subpopulations, we

identified two professional phagocytes: Large granular cells (R1), and Large

semigranular cells (R2). As in mammals [19, 23, 24, 34, 35], the two M.

galloprovincialis professional phagocytes present high phagocytic capacity, and

produce high levels of ROS and NO upon activation. M. galloprovincialis Small

semigranular (R3) hemocytes appear to play a minor role in phagocytosis, and ROS and

NO production, and it is possible that they participate in other immune functions, such

as coagulation, encapsulation, or defence against viruses. M. galloprovincialis Small

hyaline hemocytes (R4) present no phagocytic capacity, and is very likely that these

cells are specialized for coagulation or encapsulation, as has been suggested for

hyalinocytes of other invertebrates [10]. It is thus clear that M. galloprovinciallis

hemocytes are specialized cells fulfilling specific tasks in the context of host defence.

In agreement with our finding that M. galloprovincialis Large granular and

Large semigranular hemocytes are highly phagocytic, others have reported in various

16

invertebrates that granulocytes are efficient phagocytes [9, 20, 21, 30, 32]. M.

galloprovincialis Small hyaline M. galloprovincialis hemocytes were, on the other

hand, non-phagocytic. Contradictory information exists regarding the phagocytic

capacity of hyaline hemocytes. While some reports indicate a lack of phagocytic

potential in these cells [9, 32], other reports indicate a low phagocytic potential in

hyaline hemocytes [21, 30]. Considering that hyaline hemocytes are reported to posses,

at the best, a low level of phagocytosis, we are inclined to believe that these cells

perform defence mechanisms other than phagocytosis.

Many marine molluscs posses great economical importance as a source of

nourishment for humans; and also as possible sources of microbicidal molecules that

could be used to treat human diseases [54]. Therefore, the interest in understanding the

basic mechanisms that govern invertebrate immune systems has risen in the last few

years. In mammalian leukocytes, enzymes of the PI 3-K, PKC, and ERK families

appear to be central in the regulation of host defence mechanism including

phagocytosis, the respiratory burst, and NO production [25, 44, 52, 53, 55-61]. The

participation of PI 3-K, PKC, and ERK families of kinases in invertebrate defence

mechanisms is just starting to be explored. The existence of enzymes of these families

in mollusc species is mostly inferred from experiments in which cell responses are

blocked by pharmacological inhibitors [33, 62-65]. In a few species, including M.

galloprovincialis, the existence of enzymes of these families has been demonstrated by

Western-blotting techniques, using antibodies directed against enzymes of mammalian

origin [64, 66-68]. Thus it appears that PI 3-K, PCK, and ERK enzymes have been

conserved through evolution.

Here we show for the first time that, as in vertebrates [25, 44], the regulation of

hemocyte phagocytosis by enzymes of the PI 3-K, PKC, and ERK families is subjected

17

to the nature of the cell subtype, and to the nature of the particle triggering the

phagocytic process. In M. galloprovincialis PI 3-K appears to have a conserved role in

phagocytosis regulation, independently of the hemocyte subtype, or the nature of the

phagocytic target. In mammals, PI 3-K also participates in the regulation of

phagocytosis mediated by various membrane receptors [25]. The role of PI 3-K in the

regulation of phagocytosis thus appears to be evolutionarily conserved. The role of ERK

and PKC in phagocytosis regulation, on the other hand, is less generalized, and is

possibly restricted to specific targets (e.g. yeast, or bacteria), or to specific hemocyte

subtypes (e.g. Large semigranular cells). It is possible that, as has been suggested in

mammals [25], these differences in the utilisation of PKC, or ERK for phagocytosis are

due to differential stimulation of phagocytic receptors. However, phagocytic receptors

in most invertebrates, including molluscs, are yet to be characterised.

PI 3-K played an important role in role in the regulation of ROS production by

different M. galloprovincialis hemocyte subtypes. This enzyme has also been reported

to regulate ROS production in vertebrates [57, 58], which suggests that regulation of the

respiratory burst by PI 3-K is an evolutionarily-conserved mechanism. Zymosan-

induced ROS production by M. galloprovincialis hemocytes was, on the other hand,

independent of PKC, or ERK. In agreement with our data, other reports suggest that, in

mussels, ERK is not required for bacterial killing mechanisms possibly related to ROS

production [63, 67]. So, it seems that ERK is not used by any type of mussel hemocytes

for ROS production. These observations also suggest the existence of alternative ERK-

and PKC-independent signalling pathways regulating the respiratory burst in M.

galloprovincialis hemocytes. In contrast, it has been reported that in snails, ERK is

required for the respiratory burst [65], and for regulation of ROS production [69]. One

possible explanation for this is that differences in ERK and PKC utilisation for the

18

regulation of ROS production are species-specific. The analysis of ROS production

among closely-related species will probably help to clarify this issue.

Participation of PI 3-K in the regulation of NO production was restricted to M.

galloprovincialis hemocytes presenting the highest levels of NO production: the

professional phagocytes Large granular, and Large semigranular hemocytes. It is thus

very likely that, in M. galloprovincialis professional phagocytes, PI 3-K is central in the

regulation of pathogen killing, by coordinating efficient phagocytosis with efficient

ROS and NO production. ERK, on the other hand, regulated NO production only in

Large semigranular hemocytes. In snails ERK was also reported to be required for NO

production [43], but the methodology used did not allow for the identification of the

hemocyte subtypes producing NO. Our data support the idea that ERK utilisation for the

regulation of NO production is hemocyte subtype-specific, since only one type of

hemocytes showed ERK participation in this cell response. However, ERK utilization

for NO production might still exist for other types of stimuli. This is to say, that ERK

participation may be just the result of differential membrane receptor stimulation. We

only tested NO production in response to zymosan. Other stimuli need to be tested in

order to decide whether ERK is used for NO production by only one type of hemocyte

as we show here, or whether ERK is needed depending on the receptor engaged on the

hemocyte plasma membrane. Both possibilities seem at the moment equally possible.

PKC played no role in the regulation of zymosan-induced NO production in any

of the M. galloprovincialis hemocyte subtypes, suggesting the existence of PKC

independent mechanisms for NO production. By measuring NO metabolites, rather than

NO itself, others have suggested that PKC indeed participates in the regulation of NO

production [38, 41-43]. Interestingly, we observed that direct activation of PKC with

PMA actually reduces the basal levels of NO in the ROS-producing Large granular,

19

Large semigranular, and Small semigranular hemocytes. This effect of PMA was more

marked in the Large granular and Large semigranular hemocytes. the cell types that also

presented the strongest respiratory burst. The formation of peroxynitrite, from

respiratory burst-derived superoxide anions and NO, has been recorded in M.

galloprovincialis hemocytes after PMA stimulation [38]. It is thus possible that the

PMA-induced reduction in NO levels is due to peroxynitrite formation; which would

consume the available NO required for the oxidation of the NO-specific indicator. If this

is the case, hemocyte-pathogen interactions that resulted in PKC activation would be

predicted to promote the production of large amounts peroxynitrite, and rapid pathogen

killing.

Lack of a consensus criterion for mollusc hemocyte classification makes it

difficult to integrate the information on the basic mechanisms of invertebrate host

defence, obtained by different research groups. See for example: [6, 14, 70, 71].

Unifying criteria for hemocyte classification is thus urgent, and should involve a

standardised methodology for the separation of hemocyte subtypes, as well as for their

morphological and functional characterization. Due to its fast and powerful analytic

capacity, allowing for accurate cell separation, we propose that flow cytometry could be

used as standard methodology for the classification of invertebrate hemocytes. Given

the availability of a wide variety of fluorescent phagocytic particles, and a wide array of

cellular probes tailored for the measurement of various cellular functions, flow

cytometry easily allows for the functional characterization of hemocyte subtypes. The

strong potential of flow cytometry for hemocyte classification is therefore illustrated by

our results, as we were able to specifically record phagocytosis, ROS production, and

NO production, in clearly defined hemocyte subpopulations.

20

It is important to bear in mind that the gating and separation of complex cell

mixtures requires careful selection of FSC and SSC amplification, and threshold

settings [72]. Failure to accurately adjust these critical flow cytometry parameters may

preclude the visualization of discernible cell subpopulations in dot-plots. For example, a

previous attempt to study M. galloprovincialis hemocytes by flow cytometry reported

only one hemocyte population visualized in dot-plots [73]. This is clearly at odds with

the large differences in hemocyte size and shape observed in mussel hemolymph

preparations under the microscope. Contradictory results, when using flow cytometry,

have also been reported in the American oyster Crassostrea virginica [71, 74]. The

selection of FSC and SSC amplification, and threshold settings is thus critical for the

identification of cell subpopulations. Fortunately, most flow cytometers are now

equipped with software containing tools that aid in the identification and gating of

separate cell populations. It is therefore very likely that flow cytometry can easily be

applied to the morphological and functional characterization of hemocytes of many

invertebrate species.

In conclusion, the immune system of M. galloprovinciallis is composed of at

least four hemocyte subtypes, specialized to fulfil specific tasks in the context of host

defence. Two out of these four hemocyte subtypes, Large granular and Large

semigranular cells, are professional phagocytes, that share features with mammalian

professional phagocytes. PI 3-K appears to have an evolutionarily-conserved role in the

regulation of phagocytosis, and in ROS and NO production. Differential utilisation of

PKC, or ERK for the regulation of phagocytosis of diverse targets, and for the

regulation of ROS and NO production, reveals hemocyte subtype-specific variations in

signalling mechanisms, which could be due to the differential expression of membrane

21

receptors. The search for these invertebrate “immune receptors” shall be an exciting

quest in the near future.

22

5. Figure Legends.

Figure 1. Four hemocyte subpopulations are present in mussel hemolymph.

(A) Mussel hemolymph was observed directly by phase-contrast microscopy at 40 X

magnification. (B) Dot-plots of hemolymph from four individual mussels, analysed by

size (FSC) vs. cellular complexity (SSC), reveal four distinct cellular subpopulations,

desiganted R1 through R4. Particles detected in the lower left corner of the dot-plots

were cell debris and unidentified material (Deb). C-F) Mussel hemocyte subpopulations

R1 to R4 were physically separated by flow cytometry. (C) R1 cells, (D) R2 cells, (E)

R3 cells, (F) R4 cells. Cells in each region were observed directly by phase-contrast

microscopy (upper panels), or stained with Hemacolor and observed by light

transmission microscopy (lower panels).

Figure 2. Mussel Large granular (R1), Large semigranular (R2), and Small

semigranular (R3) hemocyte subpopulations present phagocytic capacity. Mussel

hemocytes were incubated at 15 ºC for two hours with nothing (A), or with different

fluorescein-labelled phagocytic targets: (B) zymosan, (C) latex beads, (D) V.

alginolyticus, (E) E. coli. Left panels show fluorescence histograms of hemolymph

samples after phagocytosis. Right panels show dot-plots of cells within the fluorescence

gate (R5) in the corresponding left-hand histogram.

Figure 3. Mussel hemocyte subpopulations have different phagocytic

efficiency. Mussel hemocytes were incubated at 15 ºC for two hours with different

fluorescein-labelled phagocytic targets: (A) zymosan, (B) latex beads, (C) V.

alginolyticus, (D) E. coli. Phagocytosis is expressed as % of positive cells (cells

internalising at least one fluorescent particle) from the total number of cells within each

hemocyte region (R1, R2, or R3). Data are mean + S.D. of over 10 determinations.

23

Figure 4. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of

zymosan. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various

concentrations of wortmannin (A), staurosporine (B), or PD98059 (C) before allowing

them to phagocytose fluorescein-labelled zymosan. Phagocytosis for the different

hemocyte subpopulations, Large granular (R1), Large semigranular (R2), and Small

semigranular (R3) is expressed as a percentage of control samples (inhibitor

concentration = 0), considered 100 %. Open symbols represent data points with no

statistical difference from controls. Filled symbols represent data points that are

statistically different from controls. Data are mean + s.e.m of 12-16 determinations.

Maximum p values, for statistically different data points are: A) p < 0.04, n=12. B) p <

0.03, n=16. C) p < 0.012, n=16.

Figure 5. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of latex

beads. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various

concentrations of wortmannin (A), staurosporine (B), or PD98059 (C) before allowing

them to phagocytose fluorescein-labelled latex beads. Phagocytosis for the different

hemocyte subpopulations Large granular (R1), Large semigranular (R2), and Small

semigranular (R3) is expressed as a percentage of control samples (inhibitor

concentration = 0), considered 100 %. Open symbols represent data points with no

statistical difference from controls. Filled symbols represent data points that are

statistically different from controls. Data are mean + s.e.m of 12-20 determinations.

Maximum p values, for statistically different data points are: A) p < 0.04, n=16. B) p <

0.03, n=20. C) p < 0.03, n=12.

Figure 6. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of the

bacteria Vibrio alginolyticus. Mussel hemocytes were incubated at 15 ºC for 30

minutes with various concentrations of wortmannin (A), staurosporine (B), or PD98059

24

(C) before allowing them to phagocytose fluorescein-labelled V. alginolyticus.

Phagocytosis for the different hemocyte subpopulations Large granular (R1), Large

semigranular (R2), and Small semigranular (R3) is expressed as a percentage of control

samples (inhibitor concentration = 0), considered 100 %. Open symbols represent data

points with no statistical difference from controls. Filled symbols represent data points

that are statistically different from controls. Data are mean + s.e.m of 10 determinations.

Maximum p values, for statistically different data points are: A) p < 0.016, n=10.

Figure 7. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of the

bacteria E. coli. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various

concentrations of wortmannin (A), staurosporine (B), or PD98059 (C) before allowing

them to phagocytose fluorescein-labelled E. coli. Phagocytosis for the different

hemocyte subpopulations Large granular (R1), Large semigranular (R2), and Small

semigranular (R3) is expressed as a percentage of control samples (inhibitor

concentration = 0), considered 100 %. Open symbols represent data points with no

statistical difference from controls. Filled symbols represent data points that are

statistically different from controls. Data are mean + s.e.m of 12-20 determinations.

Maximum p values, for statistically different data points are: A) p < 0.02, n=12.

Figure 8. Mussel hemocytes produce reactive oxide species (ROS) in

response to zymosan. Mussel hemocytes were incubated at 15 °C for 30 minutes with a

reactive oxygen species (ROS)-sensitive intracellular indicator, and then stimulated

with 1 mg/ml zymosan. After one hour, ROS production was quantified by flow

cytometry in each hemocyte subpopulation: Large granular (R1), Large semigranular

(R2), Small semigranular (R3), and Small hyaline (R4). Zymosan-stimulated ROS

production was plotted as a percentage of the resting level, considered 100 %. Data are

25

mean + S.D. of 28-60 determinations. Asterisks (*) indicate that differences from

controls were statistically significant, p ≤ 0.0001.

Figure 9. Effect of PI 3-K, PCK, or ERK inhibitors on the production of

reactive oxygen (ROS) species in response to zymosan. Mussel hemocytes were

incubated at 15 °C for 30 minutes with a ROS indicator and various concentrations of

wortmannin (A), staurosporine (B), or PD98059 (C), and then stimulated with 1 mg/ml

zymosan (A-C). After one hour, ROS production was quantified by flow cytometry in

each hemocyte subpopulation: Large granular (R1), Large semigranular (R2), and Small

semigranular (R3). ROS production was plotted as a percentage of the control samples

(inhibitor concentration = 0), considered 100%. In (D) cells were stimulated with

various concentrations of PMA in the absence of zymosan, and ROS production was

plotted as a percentage of the resting level (PMA concentration = 0), considered 100 %.

Open symbols represent data points with no statistical difference from controls. Filled

symbols represent data points that are statistically different from controls. Data are

mean + s.e.m of 8-12 determinations. Maximum p values, for statistically different data

points are: A) p < 0.03, n=12. D) p < 0.038, n=8

Figure 10. Mussel hemocytes present nitric oxide production in response to

zymosan. Mussel hemocytes were incubated at 15 °C for 30 minutes with a nitric oxide

(NO)-sensitive intracellular indicator, and then stimulated with 1 mg/ml zymosan. After

one hour, NO production was quantified by flow cytometry in each hemocyte

subpopulation: Large granular (R1), Large semigranular (R2), Small semigranular (R3),

and Small hyaline (R4). Zymosan-stimulated NO production was plotted as a

percentage of the resting level, considered 100 %. Data are mean + S.D. of 36

determinations. Asterisks (*) indicate that differences from control were statistically

significant, p ≤ 0.04.

26

Figure 11. Effect of PI 3-K, PCK, or ERK inhibitors on nitric oxide (NO)

production by mussel hemocytes. Mussel hemocytes were incubated at 15 °C for 30

minutes with a NO indicator and various concentrations of wortmannin (A),

staurosporine (B), or PD98059 (C), and then stimulated with 1 mg/ml zymosan (A-C).

After one hour, NO production was quantified by flow cytometry in each hemocyte

subpopulation: Large granular (R1), Large semigranular (R2), Small semigranular (R3),

and Small hyaline (R4). NO production was plotted as a percentage of the control

samples (inhibitor concentration = 0), considered 100%. In (D) cells were stimulated

with various concentrations of PMA in the absence of zymosan, and NO production was

plotted as a percentage of the resting level (PMA concentration = 0), considered 100 %.

Open symbols represent data points with no statistical difference from controls. Filled

symbols represent data points that are statistically different from controls. Data are

mean + s.e.m. of 12 determinations. Maximum p values, for statistically different data

points are: A) p < 0.001, n=12. C) p < 0.03, n=12. D) p < 0.009, n=8.

27

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74. Fisher, W, Ford, S. Flow cytometry: a tool for cell research in bivalve pathology. American Fisheries Society Special Publication 1988; 18: 286-292.

A B

R1 R2

R3 R4

Figure 1

R5

R5

R5

R5

R5

R5

R5

R5

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A

B

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D

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FL-1

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FL-1Figure 2

Figu

re 3

30

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0 1 5 7.5 10 15 20 50PD98059 [uM]

Phag

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(% o

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0 0.5 1 1.5 2 2.5 3.5 5 10 20 30 40Staurosporine [nM]

Phag

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0 1 5 10 20 50 100 200Wortmannin [nM]

Phag

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R1R2R3

*

R1R2R3

*

R1R2R3

*

Figure 4

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0 0.5 1 1.5 2 2.5 3.5 5 10 20 30 40Staurosporine [nM]

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0 1 5 7.5 10 15 20 50PD98059 [uM]

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R1R2R3

*

R1R2R3

*

R1R2R3

*

Figure 5

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0 1 5 7,5 10 15 20 50PD98059 [uM]

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0 1 5 10 20 50 100 200Wortmannin [nM]

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*

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*

R1R2R3

*

Figure 6

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0 1 5 7.5 10 15 20 50PD98059 [uM]

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0 0.5 1 1.5 2 2.5 3.5 5 10 20 30 40Staurosporine [nM]

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0 1 5 10 20 50 100 200Wortmannin [nM]

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Figure 7

050100

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Cont

rol

Zym

osan

ROS Production (% of control)050100

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osan

ROS Production (% of control)

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*

050100

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ROS Production (% of control)050100

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ROS Production (% of control)

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* *

A C

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Figu

re 8

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01

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00

PMA

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ROS fluorescence (% of control)

30405060708090100

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9805

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M]

ROS fluorescence (% of control)

30405060708090100

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55

1020

40St

auro

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ine

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]

ROS Production (% of control)

30405060708090100

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01

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Wor

tman

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]

ROS Production (% of control)

A C

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* R4

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* R4

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Figu

re 9

R1

R2 R4

R3

A C

B D

Figu

re 1

0

30405060708090100

110

01

57.

510

1520

50PD

9805

9 [u

M]

NO Production (% of control)

30405060708090100

01

1010

010

00PM

A [n

g/m

l]

NO Production (% of control)

30405060708090100

110

120

01

22.

55

1020

40St

auro

spor

ine

[nM

]

NO Production (% of control)

30405060708090100

110

01

510

2050

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200

Wor

tman

nin

[nM

]

NO Production (% of control)

R1

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* R4

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* R4

R1

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* R4

R1

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* R4

A C

B D

Figu

re 1

1