Macrophage involvement for successful degeneration of apoptotic organs in the colonial urochordate Botryllus schlosseri
1 Department of Anatomy and Cell Biology, The Bruce Rappaport Faculty of
Medicine, Technion Israel Institute of Technology, Haifa,
Israel
2 National Institute of Oceanography, Oceanographic and Limnological
Research, Tel-Shikmona, PO Box 8030, Haifa 31080, Israel
Author for correspondence (e-mail:
buki{at}ocean.org.il)
Accepted 21 April 2004
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Summary |
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Key words: apoptosis, phagocytosis, macrophage, BHT, antioxidant, tunicate, B. schlosseri
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Introduction |
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Botryllus schlosseri is a colonial marine organism; each colony is
composed of numerous genetically identical modules (zooids) and is derived
from a single, sexually produced tadpole larva, which immediately settles upon
release. The colony grows through a highly synchronized and cyclical
developmental phenomenon called blastogenesis
(Berrill, 1950). Blastogenesis
is divided into four major stages, AD
(Mukai and Watanabe, 1976
),
and lasts about 1 week (at 1820°C). It is a highly tuned cycle in
which: (1) a new set of zooids is established through the development and
maturation of 14 primary buds per zooid, and (2) the parent set of
zooids deteriorates and is morphologically eliminated. During stages
AC, bud tissues are differentiated and internal organs are formed,
while four of the zooids remain active. At stage D, lasting 2436 h, all
zooidal tissues in the colony die, mainly by an apoptotic process, and are
phagocyted by specialized blood cells, the macrophages (Lauzon et al.,
1992
,
1993
), which, at this stage,
increase infrequency among circulating blood hemocytes
(Ballarin et al., 1998
). The
developing primary buds then, simultaneously, mature into the new generation
of functional zooids, replacing the old generation of zooids. Zooid apoptosis
proceeds in B. schlosseri colonies in a wave-like fashion, beginning
at the anterior end of each zooid and gradually advancing towards the
posterior end (Lauzon et al.,
1992
). This unique developmental phenomenon, in which every week
all functional soma go through an apoptotic process, remains an empirical and
theoretical challenge. To date, only a limited number of methodologies have
been found to successfully alter the blastogenic rhythm, including changes of
water temperature regimen (Boyd et al.,
1986
; Rinkevich et al.,
1998
), use of allogeneic fusions
(Rinkevich and Weissman,
1987a
), or zooid/bud removals (Sabbadin,
1956a
,b
;
Lauzon et al., 2002
) or by
employing ionization radiation (Rinkevich
and Weissman, 1990
). All the above protocols, however, had not
revealed much of the nature of this unique phenomenon.
Recently, we (Voskoboynik et al.,
2002) showed that blastogenesis was arrested and colonies
deteriorated to a morphologically chaotic state in clonal replicates of B.
schlosseri that had been treated with high doses of the antioxidant
butylated hydroxytoluene (BHT). Rescued colonies resorbed BHT-treated zooids,
regenerated entirely new sets of zooids, and then revealed enhanced growth
rates and also, in many cases, significant extension of post-treatment life
expectancies. Here, we further analyze blastogenesis in Botryllus
schlosseri colonies. The results reveal that macrophages, in addition to
their role in corpse removal, play an important role as mediators in this
whole body apoptotic event.
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Materials and methods |
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Histology
Specimens were fixed in Bouin's fixative
(Gretchen, 1967) for
4060 min at room temperature, dehydrated in a graded ethanol series
(70100%) and butanol, and embedded in Paraplast (Sigma, Israel). Cross
sections (5 µm) were cut by hand-operated microtome (Leica, Nussloch,
Germany) and were stained with Azan Heidenhains
(Gretchen, 1967
) for general
morphology, or were used in the Klenow fragEL assay (see below). Outcomes were
observed under an Olympus (Tokyo, Japan) BX50 microscope.
Klenow fragEL assay
Apoptotic nuclei were stained using a Klenow fragEL DNA fragmentation
detection kit (QIA21 Calbiochem, Darmstadt, Germany), alkaline phosphatase
conjugate (Zymed, San Francisco, CA, USA) and BCIP/NBT substrate kit (Zymed),
according to the manufacturer's protocols. Endogenous alkaline phosphatase was
inhibited by Levamisole solution (Zymed). Each slide contained six sections,
three for DNA fragmentation detection and three for negative control. Negative
controls were generated by substituting the Klenow in the reaction mixture
with dH2O.
HPLC thiobarbituric acid test
Fatty acid oxidation in B. schlosseri homogenates from different
blastogenic stages, BHT-treated ramets, and their controls, were determined by
using high performance liquid chromatography based on thiobarbituric acid
tests (Chirico, 1994). The
thiobarbituric acid (TBA) test measures malondialdehyde (MDA), which is formed
in peroxidizing lipid systems (Chirico,
1994
). B. schlosseri homogenates were prepared as
described. Protein concentration was estimated using a Bio Rad Protein Assay
Dye Reagent (Bio Rad, Hercules, CA, USA).
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Results |
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Stage D zooid apoptotic resorption was significantly delayed at all tested BHT doses (0.0315 mg l-1). While treatment with alcohol did not affect the duration of this stage (regular colonies, 1.0±0.2 days, N=23 ramets; ethanol-treated colonies, 1.1±0.3 days, N=15; P>0.05), under BHT treatment, stage D was significantly prolonged to 2.7±0.7 days (P<0.05; N=9) at low BHT doses (0.031.2 mg l-1), and up to 8.4±3.3 days at higher BHT doses (2.515.0 mg l-1, N=21; one-way ANOVA, P<<0.001; Duncan's test, P<0.05). Exposing B. schlosseri ramets to low BHT doses (0.031.2 mg l-1) resulted in the lengthening of stage D, although it did not affect the successful outcome of the resorption process. At higher BHT doses, however, morphological resorption of zooids was completed only after the colonies were removed from the BHT environment.
Morphological changes in colonies arrested in stage D
Exposure of the colonies to doses >2.5 mg BHT l-1 seawater
dramatically arrested blastogenesis, followed by the development of a chaotic
morphology (Fig. 1) and zooid
deterioration that eventually led to their death. Arrested blastogenesis was
further accomplished by an accumulation of pigment cells in the zooids, buds
and blood vessels with peripheral ampullae shrinkage, sluggish blood flow
rates throughout the colony, dispersion of zooids within the tunic matrix and
abnormal development of primary and secondary buds. Zooids at arrested stage D
remained inactive (closed siphons) but alive (beating hearts) within the tunic
matrix. The primary buds continued their development and grew into the stage
where they are supposed to replace the old generation of zooids (with opened
siphons). Then, instead of creating new systems, they were dispersed in the
tunic as abnormal functional zooids together with the old generation zooids
(Fig. 1D,F). After several
days, the old generation of zooids became very condensed and partly resorbed,
while the newly formed functional zooids and their buds developed additional,
abnormal morphologies such as round zooids instead of the regular pearl-like
shapes. At the same time, the typical synchronization between successive
generations in the colony was lost, and zooids and buds of different sizes and
generations survived together within the tunic. The tunic became softer and
opaque (Fig. 1F). Ethanol
control colonies developed none of these morphological changes, with the
exception of light pigmentation of zooids and ampullae and an opaque tunic in
a few ramets.
|
Histological characteristics of colonies arrested at stage D
The morphological changes in ramets arrested in stage D were studied at
various stages following the onset of treatment. A histological comparison of
a stage D control colony vs. a 3-day arrested stage D (6 mg BHT
l-1, Day 10 exposure) is shown in
Fig. 2. In normal blastogenic
stage D zooids (mid takeover stage; Fig.
2A,C,E) the internal organs are already at various degrees of
deterioration (Fig. 2A).
However, the tissues of BHT arrested stage D ramets seemed to be functional,
even after 3 days (Fig. 2B).
That was further confirmed by examining, at higher magnification, organs such
as the branchial sac stigmata, endostyle and siphons, which still existed
after 3 days arrested at stage D (Fig.
2D,F). In the control (Fig.
2C,E), a few hours after the onset of stage D, the general
structure of epithelia was in advanced deterioration phase and macrophages
carrying debris were seen all over. It is evident that the typical apoptotic
wave of stage D colonies (Lauzon et al.,
1992) was either suspended or failed to start under the impact of
BHT treatment.
|
Significant morphological changes were observed in the primary and secondary buds of the BHT-treated colonies (Fig. 3AH). Whereas only a few cells were found circulating in the blood vessels and lacunas around organs of stage D control buds (Fig. 3A), large numbers of blood cells appeared in those blood vessels during BHT treatment (3 days arrested at stage D colonies; Fig. 3B). Three examples are depicted here: (1) the bud endostyle of the treated colonies surrounded by a mass of blood cells, as compared to the relatively empty space around the control bud endostyle (Fig. 3C,D); (2) the digestive system area of the treated buds packed with blood cells, as compared to the control section (Fig. 3E,F; a high number of blood cells found in the peripheral-treated bud blood vessels, as compared to the control (bv in Fig. 3E,F). (3) The internal morphology of the secondary bud in the BHT-treated colony (Fig. 3H) was adversely affected with dilated blood lacunas filled with many cells. In the control, the secondary bud had large empty spheres lined by the epithelium of thin blood vessels (Fig. 3G).
|
Changes in the vascular system in colonies arrested at stage D
Various modifications resulting from BHT treatment were observed within the
vascular system. The thickness of the peripheral blood vessels epithelium at
stage D, which in control zooids averaged 0.8±0.5 µm
(N=25), increased to 5.6±3.6 µm (N=23) in zooids
on the third day of arrested stage D (P<<0.001; t test).
The thickness of the bud's peripheral blood vessel epithelium increased from
0.9±0.6 µm (N=28) in stage D control colonies, to
4.9±2.8 µm (N=30) in third-day arrested buds
(P<<0.001; t test). Dilated peripheral blood vessels in
the 3-day stage D arrested zooids and buds were 9.5- to 10-fold wider than in
the controls. The number of blood cells within the lumen of peripheral blood
vessels also increased from an average of 11±6 cells (N=6) per
100 µm2 section area of control stage D peripheral blood vessels
lumen to 192±125 in stage D arrested ramets (N=12;
P=0.002, t test).
We further examined the appearance and number of macrophages in histological sections. The following numbers reflect counts per 3500 µm2 section area. Empty macrophage-like cells (cells without phagocyte inclusions) were observed only in stage D arrested ramets [11.7±5.8 (N=5) and 8.2±5.3 (N=7) zooid and bud sections, respectively; Fig. 4] and could not be recorded in either the zooid or bud section of stage D control ramets. Conversely, the number of macrophages with phagocytic inclusions reached 6.6±1.5 (N=4) and 4.2±2.8 (N=4) per section area, in stage D zooid and bud control ramets, respectively, as compared to 1.3±1.9 (N=5; P=0.002, t test) and 0.35±0.6 (N=7; P=0.005, t test) at arrested stage D colonies, zooid and bud, respectively.
|
Effect of BHT on apoptotic processes
We followed the apoptotic status of five different colonies arrested at
stage D (6 mg BHT l-1), and their corresponding stage D control
ramets using Klenow FragEL, a DNA fragmentation detection staining kit.
Typical sections representing a control stage D zooid and a 3-day arrested
stage D zooid are shown in Fig.
5. Surprisingly, a similarly intensive staining was documented in
both types of treatments (Fig.
5A,C).
|
Effect of BHT on lipid oxidation
Only slight and insignificant (one way ANOVA, P>0.05) changes
in the lipids oxidative MDA levels were recorded in 40 tissue samples taken
from regular colonies at different blastogenic stages
(Fig. 6A). The minimal level
was recorded on the second day of blastogenic stage C (5.2±4.4 ng 10
µg-1 protein), whereas the maximum level was found on the third
day of blastogenic stage C (9.5±3.8 ng 10 µg-1 protein).
After BHT treatment (6 mg l-1, 13 days at arrested stage D)
MDA levels decreased significantly to a level of 4.8±2.2 ng MDA 10
µg-1 protein (7 ramets; one-way ANOVA, P<0.05), as
compared to the average MDA level of additional seven control ramets at
blastogenic stage C, day 3, (10.1±3.4 ng MDA 10 µg-1
protein) and 11.4±6.7 ng MDA 10 µg-1 protein in six
ethanol controls (Fig. 6B).
|
Recovery from the BHT treatment
In ramets rescued from BHT treatment, zooid resorption was immediately
initiated and completed within 17 days through a massive phagocytosis
process. In most cases, old generations of zooids and buds were cleared at
once, even before the first new takeover stage. Blood vessels were cleared of
their unusually deep pigmentation after 48 days; the excess zooidal
pigmentation cleared within 13 weeks and regular sausage-like ampullae
developed after 37 days. Few remaining active zooids created the new
colonies. Those post-BHT-treated ramets revealed higher growth rates, fast
blood flow, pale pigmentation and were characterized by active and long
peripheral ampullae, all resembling young colonies
(Voskoboynik et al.,
2002).
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Discussion |
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By exposing B. schlosseri colonies to high doses of the
antioxidant BHT, we significantly affected this organopoptosis phase. This
stage was arrested while the supposedly resorbed zooids remained alive in the
tunic matrix without completing their programmed death. The apoptotic process
was completed only when the colonies were taken out of the BHT treatment.
Prolonged periods of exposure to BHT have always caused the death of treated
ramets. BHT was not the only antioxidant agent that had an impact on the
apoptotic stage. Administration of the antioxidant Trolox,
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, gave similar results
(Voskoboynik, 2001).
The analysis of arrested blastogenesis has revealed the possible important
role of the blood-borne phagocytic activity in blastogenesis. As in our
experiments, BHT treatment in mice also increased the number of phagocytes.
Following four weekly high doses of BHT injections to BALB mice, alveolar
macrophage numbers increased fivefold over basal levels
(Bauer et al., 2001). This
study did not examine the phagocyte state (empty or full). In our study,
however, the activity of phagocytosis was clearly reduced by BHT treatment, as
large numbers of `empty' macrophage-like cells were observed in blastogenesis
stage D arrested zooids. In these colonies, significantly, higher numbers of
empty phagocytic cells were found to circulate in wider blood vessels and
lacunae and zooid organs, such as the stomach, intestine and the branchial
sac, remained intact days after the expected date for their regression and
resorption. Phagocytic activity, however, was regained immediately following
BHT elimination and zooid resorption was completed within a few days.
In botryllid ascidians, two hemocyte types, the uni-or multivacuolated
macrophage-like cells and hyaline amoebocytes are involved in phagocytosis
(Ballarin et al., 1994; Cima et
al., 1996
,
2001
). Studies suggested that
both cell types represent two functional stages of a single cell type
(Ballarin et al., 1994
;
Cima et al., 1996
) and both
share common cytochemical properties, a common content of hydrolytic enzymes,
and are positive for specific lectins such as WGA and ConA
(Cima et al., 2001
). The
macrophage-like cells are usually large (1015 µm in diameter) round
cells containing one to a few vacuoles, ingested material of heterogeneous
appearance and, in some cases, such as during the takeover process of
blastogenesis, these phagosomes occupy most of the cell volume
(Lauzon et al., 1993
). The
macrophage-like cells in Botryllus are also classified by several
authors as macrophages (literature cited in
Cima et al., 2001
).
We propose four possible mechanisms as to how BHT may affect
Botryllus macrophage activity. The first possibility is that these
cells, in addition to their classical role in cellular elimination, have a
role in providing signals that promote apoptosis. Mutations in C.
elegans engulfment genes allow the survival and differentiation of cells
that are programmed to die (Hoeppner et
al., 2001; Reddien et al.,
2001
). Earlier, Diez-Roux and Lang
(1997
) had also shown that
elimination of macrophages from the anterior chamber of the rat eye resulted
in the survival of endothelial cells that were normally programmed to die.
Boyle et al. (2001
) also found
that human macrophages induced apoptosis in plaque derived vascular smooth
muscle cells. Similarly, we propose that in the Botryllus system,
macrophages are needed in vivo for the activation and the completion
of apoptosis. This can be developed through reactive oxygen species. It is
well known that macrophages produce high levels of the reactive oxygen species
NO., which can initiate apoptosis
(Chandra et al., 2000
;
Mates and Sanchez-Jimenez,
2000
). Activated macrophages were also found to direct apoptosis
of mesangial cell population in vitro via the release of
NO. (Duffield et al.,
2000
). The antioxidant BHT could interfere with this process in
the Botryllus system. It is also documented that various cellular
antioxidants, such as catalase and N-acetylcysteine, can block
apoptosis induced by diverse agents other than oxidants
(Mates and Sanchez-Jimenez,
2000
). The precise mechanism of action of ROS in the cascade
leading to cell death is largely unknown
(Feinendegen, 2002
;
Kagan et al., 2002
; Martin et
al., 2002). They represent attractive candidates for final common mediators of
apoptosis, yet a specific role for ROS in the execution or resolution of the
apoptotic program has not been established (Ragan et al., 2002). However, it
is interesting to note that high doses of BHT in plants (wheat seedlings) also
blocked the apoptotic process (Bakeeva et
al., 2001
).
The second way with which BHT may interrupt macrophage activity is through
the respiratory burst initiated at the presence of a phagocytic stimulus. In
this process, NADPH oxidase, a membrane-associated enzyme that is dormant in
resting phagocytes, is activated and then catalyzes a reaction that produces
superoxide anion (O2-;
Halliwell and Gutteridge,
1999; Forman and Torres,
2001
). Phagocytosis in B. schlosseri is also associated
with a respiratory burst, as evidenced by superoxide anion production
(Ballarin et al., 1994
). During
blastogenic stage D, phagocytic cell frequencies increased among circulating
B. schlosseri hemocytes (Ballarin
et al., 1998
). Simultaneously, a significant increase in the
levels of hydrogen peroxide, acid phosphatase, reactive oxygen metabolite
production, and nitrite ion release were recorded
(Cima et al., 1996
). In
vitro studies further revealed that phagocytosis by B.
schlosseri blood cells was significantly reduced in the presence of
superoxide dismutase, which decreased superoxide levels
(Ballarin et al., 1994
). It is
therefore possible that the antioxidant capacity of BHT interferes with the
generation of superoxide anion, or NO., thus inhibiting the
phagocytic activity.
The third way in which BHT can affect phagocyte activity is by inhibiting
lipid oxidation. Phagocytic cells such as macrophages recognize apoptotic
cells by specific changes in their cell surface markers through receptors like
the scavenger receptors (SRs). SRs are lipoprotein receptors, which have high
binding affinity for chemically modified, acetylated or oxidized lipoproteins,
and can recognize oxidized low-density lipoproteins
(Sambrano et al., 1994). Their
wide ligand binding activity makes SRs attractive candidate receptors for the
recognition and phagocytosis of apoptotic cells
(Platt et al., 1998
).
Moreover, recent studies (Chang et al.,
1999
; Tyurina et al.,
2002
) have demonstrated that apoptotic cells express
oxidation-specific epitopes, including oxidized phospholipids, on their cell
surfaces. These epitopes serve as ligands for recognition and phagocytosis by
elicited macrophages. We found here that lipid oxidation was significantly
decreased in BHT-treated B. schlosseri ramets. It is likely,
therefore, that BHT, a good antioxidant of the lipid phase, also blocks the
scavenger receptor. A decrease in phagocytosis by macrophages was seen in
guinea pigs after feeding them with high doses of vitamin E, which, like BHT,
is an efficient antioxidant of lipid peroxidation
(De la Fuente et al., 2000
).
Then again, in vitro treatment with antioxidants such as vitamin E,
ascorbic acid, glutathione, N-acetylcysteine and thioproline causes
an increase in various macrophage functions, such as ingestion and superoxide
anion production (Del Rio et al.,
1998
). This may be the result of reduced levels of antioxidant
doses used and the in vitro system, as opposed to the in
vivo system of Botryllus.
The observation of high levels of DNA fragmentation in stage D arrested
zooids as well as reduced Bcl-2 levels (anti human monoclonal; details in
Voskoboynik, 2001) suggests
tissue specific apoptotic signals. However, in the presence of BHT, when the
first sets of dying cells are not engulfed and removed by the phagocytes, the
apoptotic process is arrested. We therefore suggest, that the apoptotic
process (the takeover phase) can proceed only in the presence of active
phagocytes. These results indicate that proper functioning of blastogenesis
requires continuous removal of dying cells and tight coordination between
death signals and clearance signals. Recently, we have demonstrated a rapid
growth and significant extension of life expectancies in colonies rescued from
BHT (Voskoboynik et al.,
2002
).
Additionally, one need not necessarily invoke defects in macrophage recognition and/or engulfment of apoptotic cells. A fourth possibility is that BHT could be affecting the ability of cells from adult tissues and organs to commit suicide. It is well documented that apoptosis requires energy input from the cell. While the observation of TUNEL-positive cells implies some attempts at initiating the death program, BHT could be affecting other intrinsic aspects of the dying cell, independent of the phagocytic process. This could explain why organs appear morphologically intact in day 3-arrested BHT animals. At this stage, in the absence of other markers of apoptosis (such as annexin staining), it is rather difficult to explore the idea. It is also possible that BHT treatment has additional impacts on either stem cells or on the programmed life span characteristic to colonies of this species (Rinkevich et al., 1992). These aspects need to be further elucidated.
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Acknowledgments |
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Footnotes |
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