Dendrobaena veneta - Journal of Cell and Molecular Biology

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Dendrobaena veneta - Journal of Cell and Molecular Biology

The role of opsonin in phagocytosis by coelomocytes of the earthworm

Dendrobaena veneta

Yeflim Kalaç*, Ayten Kimiran, Gülruh Ulako¤lu and Ayfl›n Çotuk

University of ‹stanbul, Faculty of Science, Department of Biology, 34459 Vezneciler, ‹stanbul, Turkey

(* author for correspondence)

Received 9 July 2001; Accepted 25 October 2001

Abstract

The phagocytes which reside in the coelomic cavity of the earthworms have roles in cellular immunity. The

phagocytic activity of coelomic cells of the earthworm Dendrobaena veneta against the pathogenic and

non-pathogenic bacteria was investigated in vitro. The coelomic cells of D. veneta having phagocytic activity was

separated by density gradient centrifugation method. It was observed that these cells have neutrophil and basophil

structure. Among the pathogenic bacteria, B. megaterium and A. hydrophila, and non-pathogenic bacteria

P. maltophilia and B. subtilis, to various earthworms, B. megaterium was significantly ingested by phagocytes

(p


8 Yeflim Kalaç et al.

(Cameron, 1932; Millar and Ratcliffe, 1994; Rittig et

al., 1996). The free coelomocytes of the coelomic

cavity of annelids play a key role in defence

mechanisms (Dales, 1978; Dales and Kalaç, 1992).

The coelomocytes of annelids vary widely in

morphology, not only between classes but also

between species of the same family (Stein and

Cooper, 1983). The coelomocytes are generally

divided in two main groups by light microscopy:

ameobocytes (phagocytes) and eleocytes

(chloragogen cells), which are not phagocytic

(Ratcliffe and Rowley, 1981). Eleocytes, which can

be found in almost all annelids, are generally big cells

and have granules (chloragosomes) that contain lipid,

lipid-like and protein substances. Although such cells

have no phagocytic features, they are responsible for

synthesizing humoral factors such as bacteriostatic

substances, hemolysins and hemagglutinins in

Oligochaeta (Ratcliffe et al., 1985; Tripp, 1992;

Valembois et al., 1982; Valembois et al., 1986).

Ameobocytic coelomocytes are extremely efficient

removing foreign particles, such as bacteria, fungi

and nematodes from the coelomic cavity, by either

phagocytosis, nodule formation or encapsulation

(Millar and Ratcliffe, 1994) and can reach all tissues

and all parts of the earthworm body (Bilej et al.,

1990a; Cameron, 1932; Cooper et al., 1974; Cooper,

1986; Dales and Kalaç, 1992).

Earthworms Lumbricus terrestris and Eisenia

foetida phagocytes generally have four cell types such

as basophils, neutrophils, acidophils and granulocytes

according to their morphological and cytochemical

properties (Anderson, 1975; Dales, 1978; Millar and

Ratcliffe, 1994; Ratcliffe and Rowley, 1981; Ratcliffe

et al., 1985; Stein and Cooper, 1978).

Earthworm coelomic cavity very often can contain

bacteria, fungi, protozoa and nematodes in spite of

interaction between outer environment and coelomic

cavity via dorsal pores and nephridiopores (Cameron,

1932).

As we know that annelids have both cellular and

humoral response to those infective agents, although

earthworms have both cellular and humoral defence

mechanisms, some researchers have demonstrated

that bacteria such as Aeromonas hydrophila, Bacillus

megaterium, Serratia marcescens (Çotuk and Dales,

1984a), Bacillus thuringiensis (Heimpel, 1966;

Smirnoff and Heimpel, 1961) and Yersinia ruckeri

(Çotuk and Kalaç, 1990) are pathogenic to some

species of earthworms. The primary cellular response

against invading microorganisms starts with

recognition step and is followed by ingestion and

killing. Following contact of the foreign particles with

the phagocytic cell, the next step is attachment.

Recognition and attachment may be mediated, in part,

in a non-specific way by physiochemical properties of

the foreign material such as surface change and

hydrophobicity (Anderson, 1975; Bilej et al., 1990b;

Millar and Ratcliffe, 1994). On the other hand

humoral factors may facilitate recognition and

subsequent ingestion of foreign material by

phagocytic cells. These factor, which facilitate or

enhance phagocytosis, are generally referred to as

opsonins (Cooper et al., 1974; Kelly et al., 1993;

Laulan et al., 1988).

In vertebrates opsonins are mainly

immunoglobulins and the third component of

complement and specific recognition may also take

place through carbohydrate-binding proteins called

lectins. In invertebrates the body fluids and some

tissues may have lectins and may act as recognition

molecules. It has been suggested that earthworm

coelomic fluid acts as an opsonin against some

microbial agents (Bilej et al., 1990a; Laulan et al.,

1988; Stein et al., 1977). In vitro investigations by

Stein and Cooper (1981) underlined the importance of

time and temperature in the phagocytosis of yeast by

L. terrestris phagocytes.

In this study cellular response to pathogenic and

non-pathogenic bacteria in earthworm Dendrobaena

veneta was investigated in vitro. We worked on

recognizing the cell type of this species of

earthworms by light microscopy. The phagocytosis

experiments were run for different incubation periods

and temperatures. The opsonin effect of the coelomic

fluid on phagocytosis was studied using pathogenic

and saprophytic bacteria to understand probable

specific recognition.

Materials and Methods

Earthworms

Dendrobaena veneta was derived from a single

source in Istanbul University garden, and has been


maintained in the laboratory for a number of years.

Bacteria

The bacteria Aeromonas hydrophila 9926 and

Bacillus megaterium that are pathogenic to D. veneta

and Pseudomonas maltophilia KUEN 1297 and

Bacillus subtilis ATCC 6633 that are saprophytic

were obtained from different reference laboratories.

Aeromonas hydrophila 9926 was grown at 22°C, the

other bacteria were grown at 37°C for 24 h then

centrifuged and the pellet was washed three times

with saline solution. Bacteria were adjusted to 3x10 8

cells/ml by Nephelometry method (Mc Farland,

1907), before the experiment.

The preparetion of coelemic cells

Coelomic cells were liberated via the dorsal pores by

5 V stimulation (Roch, 1979). Cell free fluid was

obtained by centrifugation at 20000 g for 15 min.

Coelomic cells were prevented from clumping by

liberation into ice-cold Ca 2+ /Mg 2+ free Holtfreter

saline, containing 5 mM ethylen glycol-bis (ßaminoethyl

ether) N,N,N',N'- tetraacetic acid (EGTA)

(Sigma) adjusted to pH 7.02 and 200 mOsm.

Coelomic fluid was filtered through a 0.2 µm pore

size membrane filter and then used.

Separation of phagocytes and short term cell cultures

Phagocytes were separated from eleocytes by

density gradient centrifugation at 2500 g. Following

the centrifugation, eleocytes were carefully removed

from the interface, and the lower phase containing the

phagocytes and non-adherent cells were diluted two

times with Ca 2+ /Mg 2+ free Holtfreter containing 5 mM

EGTA, recentrifuged and resuspended according to

Dales and Kalaç (1992) methods. Cell suspensions

were handled in silicone-coated glass tubes (2%

dimethyldichlorosilane in 1,1,1-trichloroethane)

(Sigma) to prevent probable clumping then they were

counted by haemocytometer before using.

Phagocytes (2x10 6 cells/ml) were allowed to

attach to sterile glass coverslips for 30 min. and then

immersed in 5 cm Petri dishes containing 5 ml sterile

medium. Medium was based on Eagles minimum

essential medium with Earl's salts (Sigma) adjusted

by addition of 0.42 g CaCl2·2H2O, 0.09 g KCl, 0.88 g

MgSO4·7H2O, 1.27 g NaCl, 5.0 mg adenosine, 0.5 mg

sodium pyruvate, 1.0 mg peptone per liter and

adjusted to 200 mOsm and pH 7.2. An antibiotic

cocktail was added to keep the culture for long-term

(Kalaç, 1997). After the separation, attached cells

were stained with fluorescein isothiocyanate (FITC)

(Sigma) (Bellinati-Peres, 1989) and acridine orange

(Sigma) (Oda and Maeda, 1986) for 45-60 seconds

and modified Wright's stain (Sigma) for 2 min (Stein

and Cooper, 1981) to distinguish microscopically

different types of D. veneta coelomocytes.

Phagocytosis

For the measurement of phagocytosis rate the

suspension of bacteria were added into the cell culture

in such a way that cells and the bacteria ratio was

1:50. Opsonin effect was investigated by using

coelomic fluid collected by electrical stimulation

(Roch, 1979) and sterilized by filtration as described

above. 500 µl steril coelomic fluid was added into the

cell cultures contained 5 ml medium. The cells and

bacteria were incubated for 30 min and 60 min both at

22°C and 37°C. Bacteria were not added to controls.

After the incubation, the coverslips were gently

washed in Ca 2+ /Mg 2+ free Holtfreter and coverslips

were stained with a modified Wright's stain (Sigma)

for 2 min. All experimental groups also were tested

for viability by using 0,1 % nigrosin (w/v) (Sigma)

(Dales and Kalaç, 1992). Then air dried coverslips

were stuck on clean slides by entellan (Sigma). The

cells were counted under the light microscope and

both phagocytic index (PI) and percentage of

phagocytosis (%P) were calculated for every slide

using equations below respectively (Çotuk et al.,

1990).

PI=

Statistical Analysis

Role of opsonin in phagocytosis 9

Number of cell phagocytosed bacteria

Number of total cells

Number of cell phagocytosed bacteria

% P= X 100

Number of total cells

Data were evaluated by Student's t test and variant

analysis. All values are expressed as the mean ±


10 Yeflim Kalaç et al.

standard error of the mean. Number of replicates is

given as N.

Results

Coelomocytes of D. veneta

The coelomocytes of D. veneta have been

distinguished, based on morphological properties as

seen by light and U.V. microscope stained by

flouroscent dyes. According to our observations

D. veneta has two groups of cells, phagocytes and

eleocytes (chloragocytes). Eleocytes were easily

separated by gradient centrifuge method and

distinguished by their yellow color. Eleocytes are not

responsible for phagocytosis. Also they clump to each

other more than other cells. Following Wright's stain,

our observations show that phagocytic cells can be

grouped in two different types. The first one is likely

to be basophils, as L. terrestris cells, with

distinguishable nucleus. The other group is likely to

be neutrophils, has a nucleus and a large cytoplasm

containing vacuoles with bacteria (Figures 1, 2, 3, 4).

Furthermore while we counted the cells we saw that

the number of basophils were more than neutrophils.

In vitro phagocytosis experiments

Our results have shown that D. veneta phagocytes had

a phagocytosis activity for all kinds of bacteria used

in the experiments (Table 1 and Figure 5). Percentage

of phagocytosis rate at 22°C after 30 and 60 min.

incubation without coelomic fluid (opsonin) was

6-9% for B. megaterium, 7-16% for A. hydrophila,

2-3% for P. maltophilia and B. subtilis (Figure 6).

With respect to our evaluation the phagocytosis rates

were found to be insignificant at 22°C after 30 and 60

min. incubation both with and without opsonin.

Among the bacteria we tested at 37°C, percentage of

phagocytosis rates for A. hydrophila and B. subtilis

showed that the increase of phagocytosis rates were

significant (p0.05) was observed

between percentages of phagocytosis rates at 22°C for

A

B

Figure 1: Basophilic (A) and neutrophilic (B) cells stained

by Wright dye.

Figure 2: Eleocyte (el) and phagocytic (ph) cells stained by

fluorecein isothiocyanate dye (FITC).

all bacteria with and without opsonin. As it is seen

from Figure 5, B. megaterium was the most ingested

one among the tested bacteria without opsonin, for 30


Figure 3: Phagocytic cells stained by acridine orange dye.

and 60 min. incubation (p


12 Yeflim Kalaç et al.

Table 1: Phagocytic index and percentage of phagocytosis of D. veneta phagocytes against certain bacteria at different

temperatures and times.

Bacteria °C

B.megaterium 22

37

A.hydrophila 22

9926

37

P.maltophilia 22

KUEN 1297

37

B. subtilis 22

ATCC 6633

37

30’

* Values expressed as means ± S.E.

** N = Number of replicates.

A

B

Phagocytic Index* Percentage of Phagocytosis*

Without Opsonin With Opsonin Without Opsonin With Opsonin

0.099±0.07

(N=7)**

0.096±0.04

(N=7)

0.028±0.01

(N=5)

0.04±0.007

(N=9)

0.064±0.03

(N=7)

0.075±0.04

(N=7)

0.02±0.007

(N=8)

0.04±0.001

(N=6)

60’

0.137±0.04

(N=7)

0.154±0.04

(N=6)

0.61±0.273

(N=9)

0.162±0.04

(N=8)

0.025±0.01

(N=7)

0.044±0.02

(N=7)

0.02±0.007

(N=7)

0.04±0.009

(N=7)

30’

0.028±0.011

(N=8)

0.01±0.005

(N=7)

0.037±0.006

(N=6)

0.023±0.007

(N=7)

0.015±0.004

(N=7)

0.016±0.006

(N=7)

0.018±0.005

(N=8)

0.003±0.001

(N=6)

Figure 5: Percentage of phagocytosis of D. veneta

phagocytes against different bacteria in 30 and 60 minutes

with and without opsonin at 22˚C (A) and 37˚C (B).

60’

0.047±0.034

(N=8)

0.006±0.003

(N=9)

0.048±0.02

(N=7)

0.073±0.03

(N=6)

0.011±0.004

(N=7)

0.023±0.01

(N=5)

0.011±0.002

(N=9)

0.025±0.008

(N=9)

30’

5.68±3.54

(N=7)

4.95±1.70

(N=6)

6.92±4.11

(N=5)

2.98±0.47

(N=9)

3.14±1.7

(N=7)

4.8±2.19

(N=7)

1.9±0.6

(N=8)

0.34±0.12

(N=6)

60’

8.79±2.71

(N=7)

10.0±3.4

(N=6)

16.27±6.52

(N=9)

6.10±0.75

(N=9)

1.9±0.7

(N=7)

2.09±0.3

(N=7)

1.95±0.75

(N=7)

2.96±0.71

(N=7)

had no significant effect on phagocytosis ability of L.

terrestris coelomocytes and also L. terrestris showed

that 60 min. of phagocytosis time was better than 30

min.

Various annelid species possess agglutinins and/or

lysins. They are structurally unrelated to vertebrate

immunoglobulins and vary from species to species in

their molecular structure and biochemical

composition (Cooper et al., 1974; Millar and

Ratcliffe, 1994; Ohta et al., 2000). These substances

might have recognition molecules. Many authors

have also shown that the body fluid of invertebrates

acts as an opsonin (Lackie, 1980; Olafsen, 1988;

Ratcliffe et al., 1985; Renwrantz, 1983; Yeaton,

1981). The aim of some of our experiments was to

investigate whether the coelomic fluid acted as an

opsonin or not. We have observed that phagocytosis

activity of the D. veneta coelomocytes in both

saprophytic (P. maltophilia, B. subtilis) and

pathogenic (B. megaterium, A. hydrophila) bacteria

was suppressed by coelomic fluid (opsonin). This

suppression was found to be statistically significant at

37°C (p


Figure 6: Percentage of D. veneta phagocytes in 60

minutes against different bacteria with and without

opsonin at 37°C.

differences on phagocytosis rate of Halocynthic

roretzi ameobocytes when they phagocytosed sheep

red blood cells and latex beads in vitro. Another in

vitro study has shown that opsonin (hemolymph) did

not increase phagocytosis activity of mollusc

Mercenaria mercenaria phagocytic cells for

erythrocytes, bacteria and yeast (Tripp, 1992). Kelly

et al. (1993) declared that they saw a stimulating

effect of body fluid at 30 min. incubation in

Urochordate, Styela deva, but at 60 min. incubation

they did not find any differences in conditions with or

without opsonin. Stein and Cooper (1981) reported

that opsonins in the coelomic fluid of L. terrestris

facilitated phagocytosis of yeast by neutrophils but

not by basophils or granular ameobocytes. According

to Stein et al. (1977), the overall phagocytic capacity

of L. terrestris is not greatly effected by coelomic

fluid factors, since neutrophils constitute less than

20% of the coelomocyte population. In the cells

population the number of basophiles is higher (64%),

so that they may not be effected by opsonin. With

respect to the above results, a similar condition may

also fit for D. veneta coelomocytes. Because our

observations shows that the number of basophiles is

higher than neutrophils which seems to be more

phagocytic.

Our results showed that no significant difference

was observed in the phagocytosis of bacteria treated

with and without opsonin, except B. megaterium.

Though this explains why phagocytosis in many

basophils is not effected by opsonin, the whole

concept will be better understood by knowing the

ratio between the coelomic cells in D. veneta. An in

vitro study with cockroachs, Anderson (1975) shows

that the effect of the opsonin on phagocytosis of

erythrocytes coated by opsonin was under the control

values, similar to the results obtained in our study. In

some studies, another phagocytosis called “coiling

phagocytosis” has been reported both in vertebrate

and invertebrate during which the bacteria attract the

phagocytic cells without opsonin (Akbafl, 1996;

Rittig, 1996). Opsonins in invertebrates do not have

the ability to facilitate the phagocytic activity of cells

against foreign particles everytime. It appears that

there is a different response to foreign particles

among the earthworms species.

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