Advances in Bioscience and Biotechnology, 2013, 4, 908-917 ABB Published Online September 2013 (
Cooperative apoptosis of coelomocytes of the holothurian
Eupentacta fraudatrix and its modulation by
Olga A. Zaika, Lyudmila S. Dolmatova
Il’ichev Pacific Oceanological Institute, Far Eastern Branch, Russian Academy of Sciences, Vladivostok, Russia
Received 27 June 2013; revised 27 July 2013; accepted 20 August 2013
Copyright © 2013 Olga A. Zaika, Lyudmila S. Dolmatova. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
The capacities of phagocytes of subpopulation P1 (F)
and morula cells (MC) of holothurian Eupentacta frau-
datrix to modulate apoptosis of each other as well as
cytokine-dependent mechanisms and hormonal regu-
lation of these cells’s inte raction were studied. Th e 18-h
treatment of F with supernatants, obtained after cen-
trifugation of MC preincubated for 3 h with phos-
phate buffered saline (PSB) at the temperature of
22˚C (SMC3) resulted in a significant growth of apop-
tosis level. A 30-min incubation of F with supernat-
ants of MC, preincubated for 24 h (SMC24), on the
contrary, reduced the apoptosis level and increased the
level of interleukine-1α (IL-1α)-like substances, and
24-h incubation did not influence apoptosis and re-
duced level of IL-1α-like substances. Thus, proapop-
totic effects of MC’s supernatants in F inversely de-
pended on time of their preincubation with PSB and
directly on time of incubation with F. Additionally,
this effect was opposite to variations in the level of
IL-1α-like substances. The level of apoptosis declined
after 30 min of incubation but elevated after 24 h at
the inverse treatment of MC with supernatant, ob-
tained after preincubation of F during 24 h (SF24).
The level of IL-1α-like substances dropped after 30
min and insignificantly decreased after 24 h. Hence,
SF24 proapoptotic effect di rectly depended on time of
incubation with MC and did not correspond to varia-
tions in the level of IL-1α-like substances. 100 μM dexa-
methasone stimulated apoptosis in F and MC in an
inverse time-dependent manner during 24-h preincu-
bation, and supernatants of cell suspensions obtained
after such preincubations, stimulated apoptosis and
reduced the IL-1α-like substances level in target cells
at both types of interaction. IL-1α-like substances are
supposed to be mediators for MC’s effects in F, but
not for F’s action on MC. In holothurians, steroid
hormones apparently may participate in the regula-
tion of the immune response and cell cooperation.
Keywords: Phagocytes; Morula Cells; Cell Cooperation;
Dexamethasone; Apoptosis; Cytokines
Over the last two decades, immunity of marine inverte-
brates has been intensively studied all over the world.
This knowledge is especially urgent to understand evolu-
tion of the immune system, and to has a notion about the
use of immune cells in modeling the immune response in
lower animals, and it can also be applied for the inven-
tion of medicines and in environmental monitoring.
Echinoderms, being lower-organized animals, took a
particular position among invertebrates—right at the roo t
of Deuterostomia evolutional tree. Therefore, a number
of factors of the innate immunity, typical for higher ani-
mals, manifest themselves completely in echinoderms
too. Thus, in response to stimulation, immune cells of
echinoderms produce reactive oxygen species (ROS), ni-
trogen oxide (NO), etc. [1]. Additionally, cytokine-like
substances such as interleukin IL-1-, IL-2-, IL-6-, as well
as tumor necrosis factor-like molecules, were found in
echinoderms also [2,3]. Several types of circulatory cells
named coelomocytes play the key role in immune re-
sponse of echinoderms, of which phagocytes and morula-
like cells are the most abundant [1,4]. Echinoderm pha-
gocytes are functionally similar to macrophages of verte-
brates, as they perform phagocytosis and encapsulation of
foreign material [4,5]. Morula cells synthesize a number
of humoral defense factors, including cytotoxic ones, and
participate in encapsulation of foreign microorganisms,
O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917 909
in wound healing and regeneration [4,6]. As is known,
the immune response in vertebrates proceeds through the
interaction of several types of immune cells, both as di-
rect cell-to-cell contacts and via humoral factors such as
the system of cytokines, excreted by cells into the envi-
ronment [7,8]. As for echinoderms, data on similar in-
teractions can be found only in few works [6] mostly due
to the lack of information abo ut the functional activ ity of
certain types of phagocytes. Furthermore, there are no
available data on probable regulatory mechanisms of the
immune response.
Apoptosis (prog rammed cell d eath) is known to be one
of important elements of the immune response in verte-
brates. This is a physiological p rocess, aimed to maintain
homeostasis of the organisms, and it may intensify under
some stress conditions [9]. As an interesting fact, a high
level of background apoptosis, different for various types
of immunocytes, is observed in freshly isolated coelo-
mocytes of holothurians [10]. Therefore, the issue of a
probable exchange with apoptotic signals between cer-
tain cells and the regulation of this exchange during the
immune response in holothurians is of great importance.
The numerous studies of regulatory mechanisms of
immune response in vertebrates revealed the important
role of glucocorticoid hormones (GH), as the physio-
logical inductors of apoptosis of immune cells in par-
ticular [11,12]. Hormones assumed probably the leading
role in homeostasis regulation in lower animals—they
are found in plants and animals at various levels of de-
velopment, whereas in vertebrates, the system of hormo-
nal regulation reaches the highest complexity and diver-
sity [13].
Steroid molecules are observed also in all the phyla of
invertebrates [14]. Among echinoderms, the steroid com-
position was studied most thoroughly in starfish es, which
were found to have corticocosterods in their tissu es [15].
As is stated in some of reports, GH may regulate func-
tions in aquatic invertebrates. In particular, chronically
given synthetic GH dexamethasone (Dex) inhibited the
population growth of freshwater crustacean C. dubia [16].
Our previous data on concentration-dependent effect of
dexamethasone on the apoptosis level in normal coelo-
mocyte fractions of the holothurian E. fraudatrix [10]
demonstrated that hormonal regulation of immune re-
sponse, apparently, exists also in echinoderms, holothu-
rians in particular.
Until recently, no adequate attention has been paid to
issues of the immune response control in holothurians,
neither the probability of cooperation of their immuno-
cytes. Meanwhile many of holothurians are a highly de-
manded resource for the pharmaceutical industry [17]
that entails the necessity of their cultivation. For this
reason, the study of a probable exchange with apoptosis-
modulating signals between immune cells in holothu-
rians and a control of this exchange is not only of theo-
retical, but also practical significance.
Holothurian Eupentacta fraudatrix inhabits coastal wa-
ters of the Sea of Japan. Earlier, two fractions of phago-
cytes P1 and P2, and a fraction, enriched with morula
cells, have been isolated in them [18].
The aim of the study is to investigate the mechanisms
of apoptosis-modulating interaction between phagocytes
and morula cells as well as clarify the probability of their
hormonal regulation in E. fraudatrix.
2.1. Animals
E. fraudatrix individuals (4 - 6 cm in length) were col-
lected in Peter the Great Bay in autumn and winter
2003-2004 and 2007-2008. Prior to experiments, they
were kept in an aquarium with aerated seawater for 2
2.2. Coelomocyte Separation
Coelomic fluid of each animal dissected was added into
10-ml glass flasks with the following anticoagu lant solu-
tion (1:1, v/v), 30 mM ethylenediaminetetraacetic acid
(EDTA), 31 g/l NaCl, and 50 mM Tris-HCl, pH 7.6 [4].
Combined fluid samples from 15 - 25 animals were ap-
plied to a ficoll-verographine discontinous gradient with
the following proportions between ficoll-verographine
and anticoagulant solution (v/v): Step 1, 1:0.4; Step 2,
1:1; and Step 3, 1:2 as described earlier [17]. The cen-
trifugation was carried out at 300 × g for 15 min at 5˚C.
The cells of phagocyte fraction P1 (F) were collected
from the coelomic fluid sample/Step 3 interface, and c ells
of fraction enriched with morula cells (MC)-from Step
2/Step 1 interface. The cells were washed with phos-
phate-buffered saline (PBS) (36 g/l NaCl, pH 7.4) and
resuspended in medium 199 supplemented with 16.41 g/l
NaCl, 0.264 g/l KCl, 0.87 g/l CaCl2, 4.98 g/l MgCl2 ×
6H2O, 3.87 g/l MgSO4 × 7H2O, 22.74 g/l glycine, 0.1 g/l
glucose, 2.5 g/l bovine serum albumin, and 50 mg/l so-
dium oxacillin as modification of method described by
Odintsova [19]. Cells were counted in a Goryaev cham-
2.3. Experimental Design
2.3.1. Dexamethasone Effects on Cell Apoptosis
The 1 ml cell suspensions of each fraction were incu-
bated in round-botto m plates (5 × 105 - 1 × 106 cells/well)
at 22˚C with PBS (control) or 100 μM Dex (KRKA) in
PBS. All incubations were performed in two parallel
series. The samples were taken after 0, 30 min, 18 h and
24 h of incubation.
A portion of cell suspensions was centrifuged at 1000
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O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917
Copyright © 2013 SciRes.
× g for 5 min at 5˚C and the pellet was fr ozen and s tored
until DNA isolation .
2.3.2. Cell Co operation
Scheme of the experiment is shown at Figure 1. The
following vari ants of studies were pe rf ormed:
1) The influence of morula cells on phagocytes. The
cell suspension of MC (106 cells/ml) was incubated at
22˚C for 3 h or 24 h (M C prei ncu bat i o n).
Thereafter, the cells were separated from incubation
medium by centrifugation, and supernatants obtained in
3 h or 24 h (SMC3 or SMC24, respectively) were added
(1:1, v/v) to freshly isolated F (106 cells/ml). Further
incubation of F was performed at 22˚C for 30 min or 24 h.
Figure 1. Design of experiments. F: cells of phagocyte 1 fraction; MC: cells of fraction enriched with morula cells; SF24:
supernatants obtained after centrifugation of phagocytes preincubated with PSB for 24 h; S(F + Dex)24: supernatants
obtained after centrifugation of phagocytes preincubated with dexamethasone for 24 h; SMC3 and SMC24: supernatants
obtained after centrifugation of the suspension of the fraction enriched with morula cells preincubated with PSB for 3 h or
24 h, respectively; S(MC + Dex)24: supernatants obtained after centrifugation of the suspension of the fraction enriched
with morula cells preincubated with Dex for 24 h.
O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917 911
2) The influence of phagocytes on morula cells. The
suspension of F (106 cells/ml) was incubated at 22˚C for
24 h (F preincubation). After centrifugation, supernatant
obtained (SF24) was added (1:1, v/v) to freshly isolated
MC (106 cells/ml). Further incubation of MC was per-
formed at 22˚C for 30 min or 24 h.
3) The effect of Dex on cell cooperation. In some cases,
100 μM Dex was added to cell suspensions of F or MC
during their preincubation for 24 h. Supernatants obtain ed
(S(F + Dex)24 or S(MC + Dex)24, respectively) were
added to corresponding target cells MC or F followed by
24 h incubation.
After all experiments were finished, a portion of each
cell suspension was frozen in liquid nitrogen and stored
at 18˚C until enzyme activity and IL-1 α-like substances
assays. Another portion of cell suspensions was centri-
fuged at 1000 × g for 5 min at 5˚C and the pellet was
frozen and stored until DNA isolation or fixed with 4%
formalin until Hoechst 33342 stain ing.
2.4. DNA Isolation
DNA isolation was performed by the method described
by Dolmatov et al. [18] with using 4 M guanidine hy-
drochlori de (Sigma-Aldri ch ) f or de pr ot ei ni zation [20].
2.5. Measurement of Apoptosis Level
Apoptosis was estimated with using two methods: DNA
agarose gel electrophoresis and Hoechst 33342 staining.
2.5.1. DNA Electrophoresis
DNA fragmentation was detected by agarose gel elec-
trophoresis in Tris-borate buffer (pH 8.3) [21]. After el ec-
trophoresis, the gel was stained with ethidium bromide
(Bio-Rad laboratories) and DNA fragments were visual-
ized under UV light (240 nm).
2.5.2. Hoechst 33342 Staining
Cell pellets fixed with formalin were smeared and sta ined
with Hoechst 33342 (Sigma-Aldrich) [22]. Apoptosis
was calculated as percent content of bright blue fluores-
cent cells with using fluorescent microscope technique.
2.6. IL-1α-Like Substances Assay
The level of IL-1α-like sub stances was determined using
kit for immune-enzyme analysis of human IL-1α (Cyto-
kin) according to manufacturer’ instruction. Protein was
quantified using Coomassie G-250 (Sigma-Aldrich) dye
2.7. Statistical Analysis
To compare the groups, data (means ± SE) were ana-
lyzed using unpaired t test. Means were considered sig-
nificantly different when p < 0. 05.
3.1. Apoptosis Level in F and MC during
Incubation of F at 22˚C during 30 min induced increase
in apoptosis level b y 42%, as it was established with both
DNA agarose gel electrophoresis (Figure 2(a)) and
Hoechst 33342 staining (Figure 2(b)) methods. Further
incubation for 18 - 24 h revealed a tendency to apoptosis
level lowering compared to that in 30 min. The addition
of Dex, however, induced an increase in apoptosis level
after 24 h (Figure 2(a)).
In MC (Figures 2(c) and (d)) the level of apoptosis
also reached maximal value at 30 min of incubation, and
then there was a tendency to its decline by 24 h. Treat-
ment of MC with Dex for 24 h induced the increase in
apoptosis level compared to that in untreated cells (Fig-
ure 2(c)).
Figure 2. Apoptosis in cell fractions enriched with phagocytes
type 1 ((a), (b)) and morula cells ((c), (d)) during preincubation
at 22˚C measured by methods using agarose gel electrophoresis
((a), (c)) and staining with Hoechst 33342 ((b), (d)). Experi-
mental treatments: 1-PSB, preincubation for 30 min; 2-PSB,
preincubation for 18 h; 3-PSB, preincubation for 24 h; 4-Dex
(100 μM), preincubation for 24 h. *p < 0.05 compared to the
control (PSB).
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3.2. The Influence of SMC and S(MC + Dex)24
on Apoptosis in F
To study the possible effects of MC on F, we treated F
with supernatants obtained after centrifugation of MC
being preincubated with PSB.
During 30 min-incubation of F with SMC24, DNA deg-
radation was almost not observed (Figure 3(a)). The
anti-apoptotic effect of supernatants of MC on F was
confirmed with staining—the apoptosis level in F under
the effect of SM C 24 was 1.3 times as low (Fi gu re 3(b)).
Meanwhile, a 24 h incubation of F with SMC24 did
not result in significant variations in the apoptosis level
in comparison with its level in con trol F incuba ted for 24
h. Thus, longer time of incubation of F and SMC24 c au s ed
a reduction of the anti-apoptotic effect of the latter. How -
ever, 24 h incubation of F and S(MC + Dex)24 led to the
former’s apoptosis increased by half over control (Fig-
ures 3(a) and (b)).
Figure 3. The influence of supernatants obtained after cen-
trifugation of preincubated MC on the apoptosis level in F,
measurements were ma de by methods using agar ose gel el ect ro-
phoresis (a) and staining with Hoechst 33342 (b). Experimental
treatments: 1, 3, 6-PSB (control); 2, 4-SMC24; 5-S(MC +
Dex)24; 7-SMC3. Abscissa axis: time of incubation. *p < 0.05
compared to the control (PSB), **p < 0.05 compared to that
under treatment with SMC24.
The influence of duration of MC’s pre-incubation on
the effect of SMC was also studied. An addition of SMC3
to F followed b y 18 h incubation resulted in the 1.4-time
growth of the apoptosis level in F. Thus, at a short time
of pre-incubation of MC, its supernatant had a pro-apop-
totic effect on F (Figures 3(a) and (b)).
The analysis of the temporal dependence of SMC ef-
fects showed that the apoptosis-inhibiting effect of SMC
in F dropped with the increase of incubation time—the
maximum inhibiting effect appeared after 30 min of in-
cubation of F with SMC24. It is notable that a gradual
decline of the apoptosis level also took place in the con-
trol F during 24 h of incubation.
In this study, 24 h incubation of F with S(MC + Dex)24
led to the former’s apoptosis increased by half over con-
trol. It confirms the assumption that pro-apoptotic cleav-
age products accumulate in MC due to the increased
apoptosis level in these cells, and S(MC + Dex)24, in
turn, caused F’s apoptosis to increase, compared with
effect of SMC24 (apoptosis level in MC24 themselves
was close to the control one).
3.3. The Influence of SF and S(F + Dex)24 on
Apoptosis in MC
When examining reverse effect of supernatants, obtain ed
after F preincubation for 24 h, on MC, decrease in DNA
fragmentation, as compared to the control, was detected
in as little as 30 min (Figure 4 (a)). The Hoechst 33342
staining indicated that apoptosis level had halved (Fig-
ure 4(b)).
Yet it was increasing, by comparison with control,
during 24 h incubation of MC with SF24. Preincubation
of F with Dex promoted further apoptosis increase in MC
that were incubated with S(F + Dex)24 for 24 h (Figures
4(a) and (b)). Under effect of SF24 and S(F + Dex)24,
apoptosis level increased by 1.9 and 2.8 times respec-
tively, as compared to 24-h control. Thus, antiapoptotic
effect of SF24 was followed by proapoptotic one, with
MC and SF incubation period elongating, while F and
Dex preincubation promoted the former’s proapoptotic
3.4. IL-1α-Like Substances in F and MC during
Their Humoral Interactio n
Both types of the cells proved to excrete IL-1α-like sub-
stances, and their level elevated in control cells insig-
nificantly after the 24-h incubation as compared to the
30-min one—1.3 times in MC and 1.7 times in F (Fig-
ures 5(a) and (b)). However, when SMC24 was added to
F, for the first 30 minutes of incubation, the level of
IL-1α-like substances in F increased 1.6 times, compared
to the control, and dropped 2.2 times after 24 h (Figure
5(a)). The pre-incubation of MC with Dex caused sub-
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O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917 913
Figure 4. The effects of supernatants obtained after centrifuga-
tion of preincubated F on the apoptosis level in MC, measure-
ments were made by methods using agarose gel electrophoresis
(a) and staining with Hoechst 33342 (b). Experimental treat-
ments: 1, 3-PSB (control); 2, 4-F24; 5-S(MC + Dex)24. Ab-
scissa axis: time of incubation. *p < 0.05 compared to the con-
trol (PSB), **p < 0.05 compared to that under treatment with
stances to return to the control level after 30 min and
decreased even more as compared to the effect of S MC24
after 24 h.
Incubation of MC with SF24 during 30 min resulted in
the 2.4-time decrease of the level of IL-1α-like sub-
stances as compared to the control (Figure 5(b)).
If MC were incubated duri ng 30 m in wit h S(F + Dex)24,
the level of IL-1α-like substances returned almost to the
control one. After 24 h of incubation of MC both with
SF24 and with S(F + Dex)24, the observed decline of
IL-1α-like substances’ concentration reached 10% and
54%, respectively, compared to the control.
Studying the probability of transmission of apoptosis-
modulating signals between F and MC and influences of
Dex on the cell responses in E. fraudatrix, at the first
stage, we examined the influences of preincubation con-
dition and Dex on the cells studied. Earlier, we had re-
Figure 5. The level of IL-1α-like substances in F (a) treated
with SMC24 and in MC (b) treated with SF24. Experimental
treatments for (a): 1-PSB, control; 2-MC24; 3-S(MC + Dex)24.
Experimental treatments for (b): 1-PSB, control; 2-SF24; 3-
S(F + Dex)24. Abscissa axis: time of incubation. *p < 0.05
compared to the control (PSB), **p < 0.05 compared to that
under treatment with supernatants obtained after centrifugation
of the non-treated with Dex cells.
vealed the existence of pronounced spontaneous apop-
tosis in freshly isolated cells of MC but not F [10]. How-
ever, in both cell type studied, incubation for 30 min
under stress temperature conditions (22˚C) caused sig-
nificant increase in apoptosis level with further its de-
cline by 24 h. Dex stimulated apoptosis in both F and
MC in 24 h compared to control. Such effect of Dex on
holothurian immune cells corresponds to the results of
numerous studies on vertebrates, which have shown that
glucocorticoids promote apoptosis in mainly immature
lymphocytes [12], neutrophiles [24], and macrophages
[25]. However, further studies showed that SMC24 de-
creased apoptosis in F in reversed time-dependent man-
ner. Similarly, MC supernatant’s influence depended on
the time of preincubation of MC, and SMC3 had even
pro-apoptotic effect on F. Taking into account that a less
pronounced apoptotic effect was found in MC preincu-
bated for 24 h than that at 30 min, we can assume that
MC cells excrete some substances during incubation, hav-
ing a modulating effect on F. Apoptosis-increasing effect
of S(MC + Dex)24 in F confirms the assumption that
pro-apoptotic cleavage products accumulate in MC due
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O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917
to the increased apoptosis level in these cells, and S(MC
+ Dex)24, in turn, causes F’s apoptosis to increase. The
obtained results evid ence that the humoral products from
MC are capable of modulating apopto sis in F in time-and
concentration-dependent manner. Since the anti-apop-
totic effect of SMC depended on the time of incubation
with F, decreasing in the course of incubation along with
the reduction of apoptosis in F, it can be supposed that
these humoral substances exert a pronounced anti-apop-
totic effect only at a significant level of apoptosis in the
target cells.
Similarly, antiapoptotic effect of SF on MC had a re-
versed dependence on time of incubation with target cell,
which changed even into proapoptotic one by 24 h. The
effect was also concentration-dependent, as apoptosis in
MC increased when MC were treated with S(F + Dex)24.
Given the fact that, after 24 h incubation of MC, their
apoptosis levels were decreasing, compared to 30 min
one, as mentioned above, it is possible to conclude that
apoptosis-modulating effect of SF24 on MC depended on
the apoptosis levels of the latter. Thus, F’ humoral prod-
ucts may apparently regulate apoptosis in MC, and their
effect depends on the level of apoptosis in target cell.
The mechanism of such dependencies of both SMC
and SF effects on apoptosis level in target cells remains
unclear, but it is probably complex and multifactorial
like the most of apoptosis-modulating ones [26]. One of
the key mechanisms of development of apoptosis under a
stress is related to stimulation of the transcription factor
p53 [27] through stimulation of ROS [28]. Besides, p53
can participate also in inhibition of the expression of
anti-apoptotic genes [26]. However, according to the data
in recent publications, p53 may exert also the anti-apop-
totic effect by the feedback mechanism [29]. It seems
probable that at the background of a high level of apop-
tosis in F in present work, humoral sub stances from MC,
containing the pro-apoptotic factors, “switch on” the
anti-apoptotic mechanism by the feedback scheme and
induce its pro-apoptotic effect at the values of apoptosis
close to initial ones.
Given the facts that maximum apoptosis activation in
F during incubation takes place within first hours, and
development of oxidative stress (“respiratory burst”) pre-
cedes apoptosis of immunocytes [30], it is possible to
suppose that MC3’ products stimulate functional activity
of the F within at least the first 18 h, but not in 24 h of
In its turn, during 24-h incub ation, when control MC’s
apoptosis level decreased, compared to that of 30 min,
SF24, by contrast, promoted apoptosis in MC. Since the
role of some types of cells in the immune response in
holothurians is studied insufficiently, physiological rea-
sons for the effect like this canno t be determined for sure.
However, as MC are considered to be analogues of mast
cells of vertebrates [4], and facts on stimulation of mast
cell activity (histamine release) with apoptosis-decreas-
ing agents are known [31], apoptosis increasing effect of
SF24 was, apparently, followed by inhibiting functional
activity of MC. Apparently, cells of both types have
time-dependent apoptosis-modulating effect on each ot her .
In particular, MC can stimulate F’s functional activity
within the early period of incubation of cells (until 3 - 18
h). Later (in 24 h), effects of both types of cells aimed at
limitation of functional activity of each other. Such in-
teraction confirms the existence of cell cooperation at the
level of humoral products. Examples of a cooperation
like these in vertebrates are the direct influence of mast
cells on proliferation and differentiation of T-lympho-
cytes in vitro [32], as well as a variant of indirect coop-
eration when mast cells produce mediators that ensure
T-lymphocyte’s adhesion to fibroblasts [33]. Moreover,
T-cells also have an influence on mast cells [34]. In par-
ticular, lymphocytes can initiate the synthesis of cyto-
kines in mast cells [35].
According to the modern views, immune response of
vertebrates is not limited to quick and efficient activatio n,
but also includes adequate downregulation permitting to
reduce very high costs of overshootin g immune reactions
[36]. But, while the limiting mechanisms, including cell
interaction at the humoral level, particularly—immuno-
modulatory effect of anti-inflammatory cytokines [37],
have been described for vertebrates, there is no such data
on inveretebrates, with only description of morphological
isolation of the cytotoxic immune effectors in the body
cavity of Cimicidae [38]. Thus, our data are the first to
point out to the possibility of time-dependent “+” and
” interaction between immune cells of echinoderms,
particularly in holothurians.
When considering the reasonability of this interaction
in the pair F-MC, it seems probable that both types of
cells have ability to stimulate functional activity at early
phase of immune response but at the later phases they
either do not stimulate or even reduce the activity of
other cell type to ensure the expression of adequate im-
mune response without damage to surrounding cells and
tissues by ROS, production of which is directly related to
functional activity of phagocytes, or by cytotoxic sub-
stances, resulted from MC activation. Hence, in teractions
of this kind provide a high degree of stability o f immune
Additionally, data obtained points out to the fact that
apoptosis in MC and F of holothurians can be regulated
with glucocorticoids, similarly to immune cells of verte-
brates, and pro-apoptotic signals induced by Dex can be
transmitted both from MC to F, and from F to MC. No-
tably, that apoptosis-sti mulating effect in F, in con trast to
MC, is rather related to stimulation of the F’s activity
and increase in ROS generation. Kraaij et al. [39] have
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O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917 915
shown that Dex could increase the ROS producing ca-
pacity of rat macrophages leading to suppression of T
cell responses in a ROS-dependent manner. Similarly, in
present work the stimulation of F by dexamethasone ap-
parently could lead to suppression of MC. Taking into
consideration that lymphocyte-like cells were also found
in holothurians [40], we can not exclude ability of Dex to
induce general immunosuppressive effects in holothu-
rians via stimulation of ROS generation in F.
In vertebrates, cytokines play the important role in
humoral cooperation of immune cells, [7]. Further, we
studied the variation in levels of IL-1α-like substances
during cooperation of F and MC.
The level of IL-1α-like substances in certain types of
coelomocytes in echinoderms is not described in litera-
ture. Nevertheless it was shown that moru la cells of s ome
of marine organisms are the main producers of IL-1α-
like substances among coelomocytes [41]. Our data evi-
dences a lack of significant difference between control
cells studied during incubation. However, during 24-h
incubation, there was a tendency to elevation of the lev-
els of IL-1α-like substances in both types of cells. Such
variations can indicate that IL-1α-like substances are
excreted in response to stress conditions of preincubation
of the cells. Nevertheless, the level of substances was
significantly elevated after 30 min incubation of F with
SMC24, but decreased in MC treated with SF24. In 24 h
of incubation, SMC24, on the contrary, decreased the
level of these substances in F, and SF24 elevated their
level up to the control in MC.
IL-1α is known to reduce apoptosis in cells of verte-
brates [42]. It can be assumed that the increase of its
level in F treated with SMC24 for 30 min as well as its
decrease in 24 h are the reasons for inhibition or eleva-
tion of apoptosis level, respectively, in corresponding pe-
riods of time as it was described above. Additionally, a
significant reduction in IL-1α-like substances’ level in F
under the treatment of S(MC + Dex)24 was shown. That
corresponds to know n data on the Dex suppressive effect
on the IL-1α synthesis in macrophages of vertebrates that
is widely used in clinical practice [43]. The fact of si-
multaneously developing increase in apoptosis level and
reduction in IL-1α-like substances’ level in F under the
effect of S(MC + Dex)24 supports the idea on primary
role of cytokines in apoptosis-modulating effects of SMC
in F. In addition, Dex was shown to play also an anti-
inflammatory role in E. fraudatrix, the same as those in
vertebrates, for an excessive activation of IL-1α-like sub-
stances may result in damage to tissues of holothurian’s
On the contrary, the dynamics of variations in the l evel
of apoptosis and IL-1α-like substances in MC under the
effects of SF24 or S(F + Dex)24 indicates that in general
there was no correlation between them; thus, a decrease
in the IL-1α-like substan ces’ level was fo und bo th within
the first 30 minutes of incubation with SF24 (reduction
of apoptosis) and after 24 h-with S(F + Dex)24 (increase
in apoptosis). Apparently, cytokines do not play the main
role in transmission of apoptotic signal from F to MC.
The data obtained show that there are different mecha-
nisms of interaction between F and MC. This can be re-
lated to differences in functional activity of two types of
cells. Various ways of apoptosis regulation in diverse
types of cells are shown to exist also in vertebrates. e.g.,
neutrophils respond to signals, which are pro-apoptotic
for other leukocytes, in the oppo site way [30]. Moreover,
neutrophils and mononuclear lymphocytes differently
react to various stress stimuli that is observed particu-
larly as multidirectional variations in activity of antioxi-
dant enzymes [44]. This probably ensures their interac-
tion at the immune response.
In present work, the effect of MC on F is apparently
exerted just through the synthesis of IL-1α-like substan c es ,
SF, on the contrary, possibly may have an effect on MC
through any other molecules, e.g., ROS, generated by F.
Earlier, the level of ROS in MC was shown to be much
lower than that in F [45]. ROS are known to possess not
only cytotoxic properties—they also can serve as secon-
dary messengers, taking a part in regulation of the state
of intracellular redox systems, activ ity of protein kinases,
and regulation of cell reactions such as proliferation, dif-
ferentiation, and apoptosis [46]. Particularly Н2O2 acti-
vates a transcription al factor NF-kB that resu lts in ind uc-
tion of a number of pro-inflammator y cytokines in verte-
brates [47,48]. At the same time, there are numerous data
concerning H2O2 as a key signal in dexamethasone-in-
duced apoptosis [49]. Further studies on oxidant-antioxi-
dant balance of the cells during cooperation may clear
the question on ROS part in this process.
The present studies revealed an interaction of cells of
two fractions of coelomocytes of holothurians—F and
MC—at the humoral level for the first time. F and MC
had the apoptosis-stimulating or apoptosis-inhibiting ef-
fect on each other, depending on the level of functional
activity of both effector and target cells. Similarly, at
SMC’s effect on F, apoptosis developed along with the
decreasing level of IL-1α-like substances in F; at SF’s
effect on MC, vice versa, along with the growing level of
IL-1α-like substances. This indicates different mecha-
nisms of regulation of apoptosis level in two types of
cells. Thus, we may suppose that IL-1α-like substances
are the probable apoptosis-modulating humoral media-
tors of MC’s effects in F, but not F’s in MC. The re-
vealed interaction between cells apparently ensures the
expression of adequate immune response without dam-
Copyright © 2013 SciRes. OPEN ACCESS
O. A. Zaika, L. S. Dolmatova / Advances in Bioscience and Biotechnology 4 (2013) 908-917
age to surrounding cells and tissues by ROS and other
toxic substances.
Dex was shown to stimulate apoptosis in both type of
cells, and the products being formed in the cells, pre-
incubated with Dex, also stimulated apoptosis in target
cells at both kinds of interaction studied. This indicates
that Dex in holothurian’s organism can produce general
immunosuppressive effect, which is similar to that de-
scribed for vertebrates. This implies a probability that
steroids may participate in the regulation of the immune
response in holothurians.
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