Earthworm Leukocyte Populations

Earthworm leukocyte populations specifically harbor lysosomal

enzymes that may respond to bacterial challenge

May 11, 2004

By P. Engelmann . L. Moln?r . L. P?link?s . E. L. Cooper .

P. N?meth

Abstract Earthworm leukocytes (coelomocytes) are responsible

for innate cellular immune functions such as

phagocytosis and encapsulation against parasites and

pathogens. Microbial killing results from the combined

action of the phagocytic process with humoral immune

factors such as agglutinins (e.g., lectins), lysosomal

enzymes (e.g., acid phosphatase, lysozyme), and various

cytotoxic and antimicrobial molecules. There is also

evidence of weak adaptive immune responses against

foreign transplants. This study focused on aspects of the

innate immune response. First, anti-human acid phosphatase

(anti-AcP) polyclonal antibody characterized different

acid hydrolase patterns in coelomocytes. Second, flow

cytometry identified a strongly immunoreactive coelomocyte

population. Third, ultrastructural and cytochemical

analyses revealed acid phosphatase in discrete granules

(lysosomes) of effector hyaline and granular coelomocytes

but not in mature chloragocytes. Coelomocytes were

exposed to bacteria to assess how phagocytosis influences:

(a) the production of acid phosphatase using Western blot,

and (b) release of acid phosphatase using ELISA from

cell-free coelomic fluid. Fourth, after phagocytosis, acid

phosphatase levels differed between controls and experimentals.

Fifth, we found a 39-kDa molecule that reacted

intensely with anti-AcP. Our results suggest that effector

earthworm coelomocytes may not eliminate pathogens

only by phagocytosis but also by extracellular lysis.

Keywords Innate immunity . Coelomocyte . Acid

phosphatase . Cytochemistry . Immunocytochemistry .

Flow cytometry . Western blot . Earthworm, Eisenia

foetida (Annelida)

Introduction

Invertebrates exhibit different immune mechanisms

against environmental pathogens. In earthworms, the

cellular functions of innate and adaptive immunity are

effected by different coelomocytes (leukocytes) located in

the coelomic cavity whose discrete characteristics, like

those of other functional cell types, depend largely upon

available techniques and assays (Cooper et al. 2002). First,

for example, based on their ultrastructural and cytochemical

properties, in this investigation, three main populations

of earthworm leukocytes can be defined and

confirmed: hyaline, granular amoebocytes and chloragocytes

(Cooper 1996). Second, these populations have been

previously divided into more subpopulations by several

authors (Stein et al. 1977; Jamieson 1981a). Third,

physical parameters as measured by flow cytometry can

distinguish two categories (small and large coelomocytes),

which have different functional characteristics (Cooper et

al. 1995; Cossarizza et al. 1996; Quaglino et al. 1996;

Cooper et al. 1999). Fourth, our own flow cytometric

measurements identified three different populations of

coelomocytes (R1, R2, R3), which correspond to these

previously identified major populations of coelomocytes

(Engelmann et al. 2002a). Among the different species

from other phyla there is essentially no information

concerning homology of immune cells. However, with

respect to function there are many instances of the same

conserved mechanisms (Cooper et al. 2002).

Hyaline and granular amoebocytes of earthworms are

capable of phagocytosis and encapsulation; however,

granular amoebocytes engulf less foreign particles than

other coelomocyte types. Chloragocytes effect no phagocytic

activities but they exert several functions including

nutrition, excretion, as well as production of cytotoxic and

antibacterial molecules (Valembois et al. 1985; Dales and

Kalaç 1992). Granular amoebocytes frequently degranulate

their contents into the extracellular space, where they

are then located in the coelomic fluid, the equivalent of

vertebrate serum (Cooper and Stein 1981). These effector

cells (all coelomocytes) have been investigated for their

roles in the innate immune response and their functions in

analyses involving environmental toxicants (Cooper 1969;

Eyambe et al. 1991; Cancio et al. 1995b; Ville et al. 1995,

1997; Roch et al. 1996).

Several papers discuss chloragocytes, a characteristic

immunodefense population of earthworm leukocytes. The

cytoplasm of chloragocytes is filled with granules called

chloragosomes. These organelles range from 1 to 3 µm

and are positive for several enzymatic activities (acid

phosphatase, ß-glucuronidase, peroxidase, a-naphthyl

acetate esterase enzyme activity). These same enzymes

are related to the vertebrate lysosomal characteristics

(Prentø 1986; Hønsi and Stenersen 2000). Therefore many

authors have suggested that chloragocyte chloragosomes

have lysosomal origins (Varute and More 1972; Cancio et

al. 1995a). The chloragosomes show histochemical staining

for phospholipids and acid phosphatase (Varute and

More 1973; Prentø 1986; Peeters-Joris 2000). Other

earthworm immunocytes have not been investigated so

extensively for enzyme characteristics; however, these

cells also contain acid phosphatase (Stein and Cooper

1978).

These lysosomal enzymes play a role in earthworm

immune mechanisms including microbicidal action

(Marks et al. 1981) as well as in wound healing (Cooper

and Roch 1992; Ville et al. 1995; Cikutovic et al. 1999)

and graft rejection processes (see early reviews: Cooper

1975a, 1975b). Other invertebrate cells exhibit different

acid hydrolase activity in their lysosomes that correlates

with the anti-pathogenic responses (Canesi et al. 2002). In

infected mollusks elevated levels of serum acid phosphatase

were responsible for destroying the parasite Schistosoma mansoni sporocysts (Granath and Yoshino 1983;

Cheng and Dougherty 1989). Hemocytes of the clam

Tapes phillipinarium possess hydrolytic and oxidative

enzymes following stimulation with yeast cells (Cima et

al. 2000). Vertebrate immune cells (macrophages) increase

their acid phosphatase levels during phagocytosis, and

acid phosphatase is co-localized with the phagocytosed

Staphylococcus bacteria in the phagolysosomes (Raisanen

et al. 2001). Another possible role of acid phosphatase

concerns its association in toxicological analyses using

heavy metals. Heavy metals and other xenobiotics act

upon lysosomal membranes, where they cause structural

and physiological changes such as lysosomal fragility and

release of acid hydrolases. These alterations are components

of the inflammatory process that are followed by cell

death (Cancio et al. 1995b). The question of cell death is

still open with respect to earthworm cytotoxic processes,

i.e., necrosis or apoptosis (Nasi et al. 2002). In our current

experiments we tested acid phosphatase positivity in

earthworm coelomocytes by cytochemical and various

immunological methods (immunocytochemistry, flow cytometry

and immunoblot). By these procedures that are

different from earlier approaches, our recent work

confirms evidence of acid phosphatase in different

coelomocytes and proposes new undescribed features

attributed to their functional differences.

Materials and methods

Earthworms

Mature earthworms [Eisenia foetida (Lumbricidae, Oligochaeta)]

were obtained from the Department of General Zoology and

Neurobiology, University of P?cs. Earthworms were maintained in

small, dark plastic boxes containing moist wood pulp at around

20°C. Two days prior to the experimental procedure, earthworms

were placed on wet cotton wool, allowing defecation, to avoid

contamination during the harvesting of coelomocytes.

Coelomocyte isolation and harvesting

Earthworms were placed into Petri dishes containing cold extrusion

buffer as published previously (Eyambe et al. 1991; Diogen? et al.

1997). The modified extrusion buffer contains 71.2 mM NaCl, 5%

v/v ethanol, 50.4 mM guaicol-glyceryl-ether, 5 mM EGTA, pH 7.3.

Earthworms rapidly extruded coelomocytes through dorsal pores.

The coelomocytes were then pipetted into tubes filled with LBSS

(Lumbricus balanced salt solution; 71.5 mM NaCl, 4.8 mM KCl,

1.1 mM MgSO4 ×7H2O, 0.4 mM KH2PO4, 0.3 mM NaH2PO4,

4.2 mM NaHO3, pH 7.3). The coelomocytes were washed twice in

cold LBSS and counted by trypan-blue dye exclusion.

Reagents

Anti-human acid phosphatase antibody (Sigma), horseradish peroxidase

(HRP) conjugated goat anti-rabbit antibody (Dakopatts), biotin

conjugated anti-rabbit antibody (Amersham), HRP conjugated

streptavidin (Amersham), fluorescein isothiocyanate (FITC) conjugated

streptavidin, and R-phycoerythrin conjugated streptavidin

were used (Dakopatts).

Enzyme assay

Coelomocytes were spread on glass slides using cytospin (Shandon,

USA), washed with 0.1 M sodium acetate buffer (pH 5.2) for 10 min

and incubated for 3 h with reaction mixture. The reaction mixture

contained 10 mg naphthol AS-BI phosphate (Sigma) in 400 µl

dimethylformamide (DMF) and 400 µl of a 4% aqueous solution of

NaNO2. After the incubation coelomocytes were washed in sodium

acetate buffer and counterstained with Mayer’s hematoxylin

(Reanal).

Electron-microscopic examinations

After anesthesia in 10% ethanol, two to three postclitellar segments

of the body were excised from the worms. The dissected body parts

were fixed by immersion in a modified Karnovsky’s solution (final

concentration of glutaraldehyde was 2.5%) made with 0.2 M

cacodylate buffer (Karnovsky 1965) at pH 7.2 for 2 h. After

prefixation single segment samples were sliced with a razor blade

that was washed in cacodylate buffer containing 7.5% sucrose, then

postfixed in 2% buffered OsO4 for 2 h. These procedures were

carried out on ice. Following complete dehydration with graded

ethanol series the tissues were embedded in Durcupan ACM (Geyer

1973). Semithin sections were cut with a glass knife on an LKB

ultramicrotome and stained with Giemsa solution. Fine sections

were made with the same ultramicrotome, contrasted with uranyl

acetate and lead citrate and examined in a JEOL TEMSCAN-100C

transmission electron microscope (TEM).

Acid phosphatase (AcP) cytochemistry

The technique for the cytochemical localization of AcP (EC 3.1.3.2)

in chloragocytes was similar to that of Ericsson and Trump (cited in

Geyer 1973). Small pieces of segments obtained from earthworms

were fixed for 4 h at 4°C in 6.25% glutaraldehyde in 0.067 M

cacodylate buffer, pH 7.4. After aldehyde fixation, 30-µm-thick

cryostat sections were cut, and washed in 0.1 M cacodylate buffer

containing 7.5% sucrose at 4°C for 4 h. The buffer was changed four

times. To demonstrate AcP activity the slices were incubated in a

Gomori-type medium (freshly prepared mixture of Pb (NO3)2 and

glycerophosphate in 0.1 M acetate buffer, pH 5.2) for 30 min at

37°C. Controls consisted of sections incubated in Gomori-type

medium without the glycerophosphate substrate; no specific staining

developed in these sections. After incubation, samples were rinsed

in 0.1 M acetate buffer at pH 5.2 for 1 min and in 2% acetic acid for

30 s. Tissues were then processed as described for conventional

transmission electron microscopy without OsO4 postfixation

because granular structures of both chloragocytes and free

coelomocytes are extremely electron-dense structures.

Immunocytochemistry

Coelomocytes were spread on glass slides with cytospin (Shandon,

USA). After a 20-min fixation in 4% ice-cold paraformaldehyde

(PFA) the coelomocytes were immersed in 0.1% Triton X-100 for

20 min. Endogen peroxidase was inhibited by phenylhydrazine

hydrochloride (Sigma, 1 mg/ml in phosphate-buffered saline, PBS).

Non-specific binding was blocked with 5% bovine serum albumin

(BSA) saturation for 20 min. Anti-human prostate acid phosphatase

antibody (1:50, Sigma) was used as first antibody biotin labeled

anti-rabbit antibody (1:100, Dakopatts) or HRP conjugated anti-

rabbit antibody (1:100, Dakopatts) was used as the second reagent.

Following the biotin labeled antibody we incubated with streptavidin

peroxidase conjugate (1:500, Amersham Bioscience) at RT for

1 h. 3-amino-9-ethylcarbazole (Sigma) was used as chromogen in

0.1 M sodium acetate buffer (pH 5.2), and Mayer’s hematoxylin was

used for counterstaining. Controls were incubated with non-immune

rabbit serum instead of anti-AcP antibody.

Images of coelomocytes

Coelomocytes were analyzed with an Olympus BX61 microscope

and AnalySIS software.

Phagocytosis assay

Isolated coelomocytes were used for in vitro phagocytosis of

Escherichia coli and Staphylococcus aureus for 5 h with end over

end rotation (Drevets and Campbell 1991). A quantity of 2.5×106

coelomocytes and 2.5×107 bacteria were mixed to a 1-ml final

volume in 12×75-mm tubes (Falcon, BD Labware). After incubation,

coelomocytes were washed in LBSS and sonicated with

Ultrasonic (Cole-Palmer Inst. Co., USA, 600 W) for 2 min at 15%

efficiency. The protein content of the samples was measured by

micro-Bradford assay (Bradford 1976).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) and Western blots

The proteins of coelomocyte lysates were separated by SDS-PAGE

on 10% polyacrylamide gels, topped by a 4% stacking gel according

to Laemmli (1970) using the Mini-Protean 3 apparatus (Bio-Rad).

The separated proteins were blotted onto nitrocellulose membranes

in blotting buffer overnight. Then nitrocellulose sheets were blocked

with 2% non-fat milk powder (Sigma) in PBS for 1 h. Following

incubation with anti-AcP antibody (1:500), biotinylated anti-rabbit

antibody (1:1,000) was used as second reagent. Alkaline phosphatase-

conjugated avidin or HRP-conjugated streptavidin was used in

the third step for 1 h on RT. For the color reactions nitroblue

tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP)

mixture in alkaline phosphatase substrate buffer (100 mM TRIS-

HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.5) and enhanced

chemiluminescent (ECL) reagent (Amersham) were used.

PBS/0.05% Tween 20 (Sigma) was used for washing the nitrocellulose

sheets between the reaction steps. Pharmacia low molecular

weight proteins (a-lactalbumin 14 kDa, soybean trypsin inhibitor

20 kDa, carbonic anhydrase 30 kDa, ovalbumin 45 kDa, BSA

67 kDa, phosphorylase 94 kDa) were used as standards. The blots

were analyzed for densitometry by ScionImage software for

Windows.

Immunoserology (simple binding indirect enzyme linked

immunosorbent assay, ELISA)

Microtiter plate (Nunc, Microelisa) wells were coated with 50 µlof

the cell free coelomic fluid (in LBSS) after phagocytosis overnight

at 4°C. Non-specific binding was blocked with 0.5% gelatin for half

an hour at 37°C and the wells incubated with anti-AcP antibody

(1:500) for 1 h at 37°C. Following three washings, the plates were

incubated with 50 µl of anti-rabbit Ig (goat) horseradish peroxidase

conjugated antibody (Dakopatts, Denmark) for 1 h at 37°C. Color

reaction was developed with orthophenylene diamine (OPD) and

stopped with 4 M H2SO4. After each step the plates were washed

with PBS containing 0.05% Tween 20. A Dynatech ELISA reader at

490 nm wavelengths measured the color reaction.

Flow cytometry

Isolated coelomocyte populations were washed in RPMI 1640

medium with 10% fetal calf serum (FCS, Sigma) twice by mild

centrifugation (500 rpm, 5 min), and 106 cells/sample were stained.

The samples were fixed in 4% PFA/LBSS for 20 min. The washing

steps, one with LBSS containing 0.1% NaN3, were followed by a

permeabilizing buffer (LBSS was performed with 0.1% saponin and

0.1% NaN3). The antibodies (anti-acid phosphatase 1:50, biotin

labeled anti-rabbit antibody 1:100, streptavidin-FITC 1:100) were

added to the samples in the same buffer and incubated with the cells

for 30 min at 4°C. After incubation we washed the samples twice in

saponin buffer and once in LBSS/BSA/NaN3. Finally the samples

were fixed in LBSS containing 0.1% formalin and measured by a

Becton Dickinson FACS Calibur cytometer and analyzed with the

FCS Express software.

Results

Acid phosphatase enzyme activity in earthworm

effector coelomocytes

Coelomocytes stained positively for acid phosphatase

using naphthol AS-BI phosphate substrate. This staining

shows various patterns in different coelomocyte populations.

The enzyme was active mainly in the highly

adherent cells known as hyaline amoebocytes (Fig. 1a),

but other coelomocyte types (granular amoebocytes,

chloragocytes) showed weaker reactions. Figure 1 shows

several reactions: a large negative cell or unstained

chloragocyte (a), positive small coelomocytes (a and b)

Fig. 1a–e Acid phosphatase

enzyme was detected by cytochemistry

in different coelomocyte

subpopulations. Earthworm

coelomocytes stained for acid

phosphatase enzyme activity

showed intense reaction in the

discrete granules (lysosomes) of

the leukocytes. Arrows indicate

the positive cytoplasmic granules,

while asterisks denote cells

negative for AcP enzyme activity.

Bar 10 µm

as well as evidence of high levels of enzyme activity (c

and d). The intense granular reaction is rather spectacular.

Acid phosphatase reactivity is localized in discrete granules

that are occasionally numerous (as in Fig. 1c, granular

amoebocyte). The enzymatically negative region of the

cell is around the nucleus, while the positive reaction fills

the cytoplasm.

Ultrastructure and acid phosphatase content of certain

coelomocytes

Various types of free coelomocytes are located in the

coelomic cavity of earthworms: eleocytes (detached

chloragocytes called eleocytes by several authors; Jamie-

son 1981b), and granular and hyaline amoebocytes with

the detailed ultrastructure. The most prominent features of

agranular (hyaline) coelomocytes are the numerous

Fig. 2a–d Ultrastructure and AcP content of some coelomocytes.

Note the cytoplasm of the hyaline coelomocyte (a) is granule free; it

contains a few cisternae of endoplasmic reticulum (arrow) and small

vacuolar structures (arrowheads). The cytoplasm of the granular

coelomocyte (b) is filled with large (arrow) and small (asterisks)

granules. In detached chloragocytes (c) a few dense granules

pseudopods sprouting off their surface and the nucleus

surrounded by a small cytoplasmic rim. In addition, they

have moderate numbers of GER cisternae, a small Golgi

apparatus and a few vacuolar structures. The number of

mitochondria in these cells is usually low (Fig. 2a).

A medium size (10–20 µm) cell that contains numerous

granules is a characteristic granular coelomocyte (Fig. 2b).

Most electron-dense granules are 1–3 µm in diameter and

have a compact, osmiophilic matrix. Some of the granules

contain a small amount of dense material or they may be

filled with membrane whorls, while in others a large

number of membrane vesicles and debris are present. At

higher magnification several invaginations of the plasma

membrane were observed. In addition, cell-free electron-

dense granular structures of various forms and sizes were

often observed in the coelomic cavity and most probably

represented extruded granules from chloragocytes. Numerous

highly granular detached/liberated chloragocytes

were also identified (Fig. 2c).

Figure 2d shows the results of the AcP cytochemical

localization in granular coelomocytes. Moderately to

highly intense reactions were found in large granular

structures, while high enzyme activity was always present

in medium and small granules. Morphological analysis of

(arrows) and a high number of electron-lucent vesicles (asterisks)

can be seen. In granular coelomocytes (d) a few lysosomes (arrows)

with high AcP activity and some residual bodies (Rb) with no or

moderate lysosomal enzyme activity can be detected (Nu nucleus).

Bar 1 µm

AcP positive structures showed that the small spherical or

ovoid structures could be primary lysosomes while the

larger structures were secondary lysosomes. In the latter

structures, high AcP activity was found not only at the

periphery of the granules but also in the deeper regions of

their matrices.

Acid phosphatase-positive cells in the coelomocyte

populations defined by immunocytochemistry

In addition to detecting the ability directly, by virtue of its

enzymatic activity, a polyclonal anti-human prostate acid

phosphatase antibody was also used to locate acid

phosphatase in intracellular structures of coelomocytes

(Fig. 3a–e). Immunoreactivity was detected for AcP in

discrete coelomocyte granules except in Fig. 3a and b,

where a cell surface/extracellular reaction was shown.

Chloragocytes did not reveal any AcP detectable by

immunostaining (Fig. 3c, b). The rest of the coelomocyte

subtypes such as the hyaline and granular amoebocytes

were positive for the enzyme using the polyclonal anti-

AcP antibody. Diffuse reaction marks intracellular components

and cell surface staining (Fig. 3e). In controls

Fig. 3a–f Immunocytochemical

localization of acid phosphatase

in earthworm coelomocytes.

Arrows indicate the positive

granules in the cytoplasm,

while the asterisks denote the

negative cells for anti-AcP immunoreactivity.

Bars 20 µm (a),

10 µm (b–f)

(using non-immunized normal rabbit serum) we found no Changes in acid phosphatase level of coelomocytes

reaction in coelomocytes (Fig. 3f). during phagocytosis

To assess any alteration upon phagocytosis we analyzed

the amount of acid phosphatase. Coelomocytes eliminate

foreign particles during in vitro experimental procedures

Table 1 Percentage of positive cells for acid phosphatase in

different coelomocyte subpopulations (R1, R2, R3). The values

represent means of three experiments. More than 106 cells were

counted in each experiment

R1 19.38±10.74%

R2 43.3±19.15%

R3 11.01±11.69%

as revealed by their capacity to engulf different bacterial

strains (Escherichia coli and Staphylococcus aureus).

Coelomocytes were homogenized after a 5-h-long phagocytosis

assay. The coelomocyte suspension was used for

SDS-PAGE and Western blot analysis. Using polyclonal

anti-AcP antibody we found a protein fraction at 39 kDa,

which reacted specifically and intensely in Western blot

(Fig. 4a). We identified differences between activated

samples (coelomocytes after phagocytosis) and controls.

Levels of acid hydrolase enzyme in the phagocytosed

samples were lower than in control samples (Fig. 4b)

measured by densitometry. When S. aureus bacteria were

engulfed by coelomocytes we detected lower levels of acid

phosphatase than in the case of coelomocytes that had

engulfed E. coli. To test antibody specificity, we used

bacterial lysate (from the same bacterial strains) and found

no reaction by Western blots (data not shown). We found

an elevated level of acid phosphatase in cell-free coelomic

fluid after phagocytosis using ELISA (Fig. 4c). The

samples after phagocytosis contained higher levels of

serum acid phosphatase than the controls.

Characterization of acid phosphatase-positive

coelomocytes by flow cytometry

Extruded coelomocytes were analyzed by flow cytometry

according to their physical parameters and fluorescence

profiles. Three populations were found which were

different in size and granularity. One population (R3)

was of small size and contained highly autofluorescent

granules. The R3 population was a mixed cell population

but most of them were identified as chloragocytes or

eleocytes. The other two populations (R1 and R2) were

composed of the effector hyaline and granular coelomocytes

(Fig. 5a). These coelomocytes were stained positively

with anti-human acid phosphatase antibody, while

the controls gave no reaction (Fig. 5b, d). The R2

population (as hyaline amoebocytes) stained strongly with

anti-acid phosphatase antibody (40–50%) (Table 1), while

the other two populations, R1 (granular coelomocytes) and

R3, showed a weaker reaction (Fig. 5c).

Discussion

Non-self recognition is the main function of invertebrate

innate immunity (Engelmann et al. 2002b). Earthworm

coelomocytes possess several immunodefense related

biological functions. These effector cells participate

Fig. 4a–c Immunoblot analysis of earthworm coelomocyte lysate

and ELISA of coelomic fluid after phagocytosis. During the in vitro

phagocytosis assay both gram-positive (Staphylococcus aureus) and

gram-negative (Escherichia coli) bacteria were engulfed by the

coelomocytes. Anti-AcP antibody was used for monitoring the

intracellular level of the enzyme after phagocytosis by immunoblot

from lysate (a). The diagram shows differences between the samples

after phagocytosis and controls (b). The density of bands was

measured by ScionImage software. An indirect ELISA method was

used to detect the extracellular level (from coelomic fluid) of AcP

enzyme after phagocytosis. The acid phosphatase level is increased

in bacteria-contained samples compared to the controls (c). The

results in the figures are representative values from three experiments

mainly in cellular mechanisms, but chloragocytes and

maybe the granular population of coelomocytes produce

humoral factors which may mediate the cellular and

humoral responses as well.

Following engulfment foreign invaders are neutralized

by invertebrate immunocytes. Several hydrolytic enzymes

(i.e., acid phosphatase, peroxidase, non-specific esterase,

alkaline phosphatase, aryl sulfatase) can be found in

invertebrate leukocytes and in their vertebrate counterparts

(Cheng 1975; Pipe 1990; Hoeger 1994; Pipe et al. 1997;

Markova et al. 1998; Hillyer and Christensen 2002). These

enzymes are ubiquitous in invertebrates; however, their

exact functions are not completely clear.

Fig. 5a–d Coelomocytes have

different amounts of acid phosphatase

as shown by flow cytometry.

R2 populations of

earthworm coelomocytes express

the highest level of this

acid hydrolase

Acid phosphatase (AcP) is a specific lysosomal marker

in invertebrate and vertebrate immune cells, and has been

well conserved during evolution from bacteria through

plants to animals. This molecule has been analyzed in

Drosophila hemocytes in normal and tumor cell lines

(Dinan et al. 1983) as well as in hemocytes of different

molluscan species, again concentrating on the possible

immune function (Cheng 1978; Cheng and Butler 1979;

Granath and Yoshino 1983; Cheng and Dougherty 1989;

Carballal et al. 1997b).

Mussel hemocytes show functional differences and

different lysosomal characteristics, granulocytes are phagocytic

containing large amount of hydrolytical enzymes,

while hyalinocytes showed limited phagocytosis and

lower levels of acid phosphatase (Carballal et al. 1997a,

1997b). In oysters (Crassotrea virginica, Ostrea edulis)

different hemocyte populations, i.e., granulocytes, are

more active in phagocytosis, expressing higher levels of

lysosomal enzyme activity. C. virginica hemocytes have

been characterized into five subpopulations. One granulocyte

population shows the highest acid phosphatase

activity and a high serum level of acid phosphatase

especially after administering a high dose of bacterial

challenge (Cheng and Mohandas 1985; Cheng and Downs

1988). The snail Biomphalaria glabrata was challenged in

vivo with heat-killed Bacillus megaterium. This resulted in

significant elevations in hemocyte acid phosphatase

activity and in the serum at 2 and 4 h postinjection

(Cheng and Butler 1979). Parasite infected (S. manzoni)

snails have an elevated level of serum acid phosphatase

that may be involved in parasite destruction (Cheng and

Dougherty 1989).

Similar results were obtained from vertebrate species. A

macrophage function has been studied in red deer using

Mycobacterium bovis. Stimulation of cells with LPS

results in enhanced intracellular production of acid

phosphatase; the extracellular enzyme level is increased

after phagocytosis of zymosan particles (Cross et al.

1996). In a tartrate resistant acid phosphatase (TRAP)

knockout mouse, the inflammatory response and microbial

clearance has been measured. Interestingly, TRAP knockouts

had a reduced population of macrophages with

normal phagocytic activity and killing recruitment, but

showed a delayed clearance of the microbial pathogen

Staphylococcus aureus (Bune et al. 2001).

In the various subpopulations of earthworm coelomocytes

several enzyme activities with different patterns have

been defined. In this study we have shown that bacterial

infection of earthworms stimulated an increased number of

lysosomes in coelomocytes. It is well documented that low

lysosomal enzyme activity is characteristic of chloragocytes

from Lumbricus terrestris collected from its natural

habitat (Prentø 1986). In contrast, high AcP activity is

found in the chloragogenous tissue of lead-exposed

(Cancio et al. 1995b) as well as in starved and dehydrated

earthworms (Varute and More 1972). However, these

suboptimal conditions induced a transient increase in the

activity of lysosomal enzymes. In the first 8 days of both

starvation and dehydration, lysosomal activity increased,

but later the enzyme activity declined significantly and

returned to the baseline levels (Varute and More 1972).

These results indicate the rapid adaptation of the lysosomal

system of chloragocytes to suboptimal conditions. A

similar metabolic pathway could be typical of coelomo

cytes as well; however, secretion of lysosomal enzyme

may not be ruled out. The increase in AcP activity in the

cell-free coelomic fluid has been observed in autografted

and xenografted earthworms (Marks et al. 1981).

From our cytochemical results we propose that electron-

dense granules of coelomocytes are identical to residual

bodies that lack lysosomal enzymes or they are characterized

by very low acid hydrolase activity (Holtzmann

1989). It is well documented that chloragocytes as one

type of coelomocytes are rich reservoirs of phosphate

compounds such as lombricine kinase (Suzuki et al. 1997),

phosphatidyl ethanolamine and phosphatidyl choline (for

review see Jamieson 1981b). The above compounds could

also become incorporated into lysosomes, and become

modified through interactions with various molecules to

ortho-and pyrophosphates. These latter compounds could

be responsible for the inhibition, perhaps the total block,

of lysosomal enzymes in coelomocytes that result from

accumulation of partially digested organic compounds in

their matrices. This hypothesis is in agreement with the

results of biochemical observations showing that orthophosphates

and pyrophosphates are strong inhibitors of the

lysosomal enzyme activity of earthworm chloragocytes

(Varute and More 1973). It has been found that both

inhibition of lysosomal hydrolases and oxidative stress

play a significant role in lipofuscinogenesis (Marzabadi et

al. 1991).

Cytochemical methods were used to characterize earthworm

coelomocytes including their enzymes. In our

cytochemical experiments (Fig. 1), granular and hyaline

amoebocytes but not chloragocytes release this acid

hydrolase activity, in accordance with recent reports

(Hamed et al. 2002). Enzyme reactivity was bound to

discrete granules (e.g., lysosomes) in the cytoplasms of

coelomocytes as observed by electron microscopy; however,

the staining pattern was different (Fig. 2d). This

phenomenon may indicate different cell activation stages.

Coelomocytes released the contents of the granules

(including enzymes) into the coelomic cavity to fight

pathogens. With Western blotting we found a 39-kDa

peptide that reacts with anti-AcP antibody. Subsequently

intracellular acid phosphatase decreases compared to the

controls in the samples which contain bacterial strains

(Fig. 4a, b). In contrast, ELISA results show that the

samples had an increased level of extracellular acid

phosphatase after phagocytosis (Fig. 4c). An acid phosphatase

has been isolated from another earthworm species,

Eisenia veneta, which has two isoenzymes of acid

phosphatase, one being 113 kDa composed of identical

peptide chains of 36 kDa (Stubberud et al. 2000).

As bacteria are bound to the leukocytes they are

removed from circulation and may be destroyed by the

release of degradative enzymes, reactive oxygen metabolites,

or any antimicrobial molecules secreted from the

blood cells of the mussel Mytilus edulis (Pipe et al. 1997).

This release of digestive enzymes may play a role in

autophagocytosis. Several reports have concluded that

other coelomocyte types clear degraded chloragogen cells.

Examinations have revealed chloragosomes inside “brown

bodies” (encapsulated particles in earthworms). These

bodies contain lipofuscin and melanin, which render them

capable of killing parasites and microbes and of clearing

altered cells (Valembois et al. 1994). In this process

different coelomocytes may have specialized tasks. Granular

coelomocytes may play a role in encapsulation, while

hyaline amoebocytes effect primarily phagocytic activity.

The functional distribution of different leukocyte types has

been characterized in other annelid species (Porchet-

Henner? 1990). Coelomocytes recognize non-self tissues

that consequently trigger a rejection process (Cooper

1968, 1969; Linthicum et al. 1977). The released acid

phosphatase in the coelom may exert an effect during this

response (Marks et al. 1981).

Some reports indicate that chloragogen cells (which

constitute the chloragogue tissue) have high acid phosphatase

activities (Cancio et al. 1995a). In contrast free

chloragogen cells exhibited low enzymatic activities in our

experiments. This coelomocyte type is found in all

lumbricid worms, and chloragocytes change during the

expulsion of granules and biochemical alterations of

cytoplasmic inclusions (Valembois 1971; Valembois and

Roch 1977; Valembois et al. 1985). Chloragocytes in the

coelomic fluid have low acid phosphatase content as

measured by flow cytometry, while immunocytochemical,

enzyme cytochemical examinations could not detect any

acid phosphatase in this coelomocyte subpopulation. This

may refer to a developmental stage of chloragocytes with

the loss of these hydrolytic enzymes, thus giving these

cells a status of secondary importance in the immune

mechanisms.

The immune system of other invertebrates, especially of

insects, has been widely studied and a number of effector

cells have been identified (Lavine and Strand 2002).

A key question of our study and invertebrate immunology

should be whether immune cells of annelids and

insects, originating from the same ancestor, developed

independently during evolution or whether there are

homologous cell lineages in these animals (Cooper et al.

1992). Undoubtedly there are certain immune cells in both

annelids and insects characterized by the same physiological

and histochemical characteristics. In Nereis diversicolor (Polychaeta, Annelida) as well as in insects,

coelomocytes participate and cooperate differentially in

the encapsulation process (Porchet-Henner? 1990; Pech

and Strand 1996).

The known nucleotide sequence of humoral immune

factors of earthworms is accumulating. Here is revealed

the family of fetidins, the lysenins that are large polypeptides.

In addition, an earthworm pattern recognition

molecule has been identified and cloned as coelomic

cytolytic factor (CCF), from the coelomic fluid of

earthworms. CCF is localized in chloragocytes and large

coelomocytes. CCF shows homology to ß-1, 3-glucan

recognition proteins of arthropods and with glucan

sensitive factor G from the horseshoe crab Limulus

polyphemus. There is also one antimicrobial peptide,

lumbricin I, which is the same size as those of arthropods.

There is also Eiseniapore, which has not been sequenced

but functions as the first three, primarily as a lytic

molecule. These have been outlined in the recent major

review by Cooper et al. in BioEssays (Cooper et al. 2002).

These molecules could help us to find some possible

connection between invertebrate immune cells of the

different invertebrate taxa.

Based on our recent results we can only hypothesize a

functional homology between some coelomocytes of

earthworms and blood cells of insects. Identification of

possible homologous immune cell lineages in these

animals needs further investigation.

Acknowledgements We thank Kriszti?n Kvell, P?ter Balogh and

Zolt?n R?k?si for careful reading of the manuscript and useful

advice.

E.L. Cooper has been supported by The Alexander von Humboldt

Foundation, a GAAC grant from the Federal Republic of Germany

and two grants from NATO, a Cooperative Research grant (971128)

and an Advanced Research Workshop grant (976680)

P. Engelmann (*). L. P?link?s . P. N?meth

Department of Immunology and Biotechnology, Faculty of

Medicine, University of P?cs,

P?cs, Hungary

e-mail: This email address is being protected from spam bots, you need Javascript enabled to view it

L. Moln?r

Department of General Zoology and Neurobiology, Faculty of

Sciences, University of P?cs,

P?cs, Hungary

L. Moln?r

Adaptation Biological Research Group of the Hungarian

Academy of Sciences,

P?cs, Hungary

E. L. Cooper

Laboratory of Comparative Neuroimmunology, Department of

Neurobiology, David Geffen School of Medicine at UCLA,

University of California,

Los Angeles, CA 90095-1763, USA

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