The sea urchin complement homologue, SpC3, functions as an opsonin
1 Graduate Program in Genetics, Institute of Biomedical Sciences, George
Washington University, Washington DC, USA
2 Department of Biological Sciences, George Washington University,
Washington DC, USA
3 Department of Biological Sciences, Macquarie University, Sydney,
Australia
4 Department of Biochemistry, Medical University of South Carolina,
Charleston SC, USA
Author for correspondence (e-mail:
csmith{at}gwu.edu)
Accepted 29 March 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: phagocyte, opsonin, sea urchin, Strongylocentrotus purpuratus, echinoderm, complement, innate immunity, evolution
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A number of workers have suggested that the diversification of complement
gene families like TEPf was driven by whole genome duplication events that are
thought to have occurred early in vertebrate phylogeny (Lachman, 1979;
Dodds and Day, 1993;
Campbell et al., 1988
). In
particular, genome duplications seem to have provided the genetic diversity
necessary for the evolution of discrete lectin-mediated and classical
complement activation pathways. Similarities are also evident between the
classical and alternative pathways. This interpretation of complement
evolution implies that the ancestral genes predate the appearance of the
vertebrates. The ancient origin of complement components was first suggested
by studies of the green sea urchin Strongylocentrotus droebachienses
(Kaplan and Bertheussen, 1977; Bertheussen, 1981, 1982; Bertheussen and
Seljelid, 1982). Sea urchins are members of the phylum Echinodermata, which
belongs to the same deuterostome lineage as the chordates, which includes the
urochordates, cephalochordates and the vertebrates. Bertheussen and his
coworkers found that phagocytosis by S. droebachienses coelomocytes
could be significantly enhanced when target cells (yeast or red blood cells)
were opsonized with mammalian C3. This suggested that coelomocytes had cell
surface receptors for C3-like proteins and, by corollary, that sea urchins
expressed C3 homologues, which could function as ligands for those
receptors.
Molecular evidence of C3 homologues in echinoderms was first identified as
an expressed sequence tag (EST) from lipopolysaccharide (LPS)-activated
coelomocytes of the purple sea urchin, S. purpuratus
(Smith et al., 1996). Two
full-length cDNA sequences were identified as complement components; a C3
homologue (SpC3; Al-Sharif et al.,
1998
) and a factor B (Bf) homologue (SpBf;
Smith et al., 1998
).
Complement components have since been identified in both the urochordates
(tunicates) and cephalochordates (amphioxus), which are deuterostome
invertebrates related to echinoderms. The complement homologues in tunicates
include C3-like molecules, a Bf-like gene and members of the lectin-mediated
complement pathway (Ji et al.,
1997
; Nonaka et al.,
1999
; Nair et al.,
2000
; Marino et al.,
2002
; Raftos et al.,
2002
), in addition to a complement receptor similar to type 3 or
type 4 (Miyazawa et al.,
2001
). Furthermore, a number of additional putative complement
genes have been identified through data mining of the Ciona
intestinalis genome (Azumi et al.,
2003
). Complement components have also been identified in
Branchiostoma belcheri or amphioxus
(Suzuki et al., 2002
), in a
gorgonian Swiftia exerta (GenBank accession no. AAN86548), and in a
squid (M. McFall-Ngai, personal communication). Furthermore, two mosaic
proteins composed of domains found in complement regulatory proteins in higher
vertebrates have been characterized in the purple sea urchin (GenBank
accession nos. AY494840, AY494841:
Multerer and Smith, 2004
).
Overall, various forms of the complement system may be present throughout the
animal kingdom and be important for host defense.
The carboxy-terminal region of the SpC3 chain incorporates a number
of structural characteristics that are crucial to the opsonic activities of
the vertebrate counterparts. These include a single histidine, and two
prolines surrounding the motif, GCGEQ, in the
chain, which in mammals
act as the basis for forming covalent thioester bonds with either hydroxyl or
amino groups on target cell surfaces
(Isaac and Isenman, 1992
;
Dodds and Day, 1993
;
Al Sharif et al., 1998
). A
hydrophobic pocket that protects the thioester site from deactivation by the
aqueous environment has been characterized from the crystal structure of human
C3d and eleven functionally important hydrophobic amino acids have been
identified that create the hydrophobic pocket, which are located both near the
thioester site and throughout the C3d fragment
(Nagar et al., 1998
).
Comparisons between the C3d alignment in Nagar et al.
(1998
) and the alignment of
several members of the thioester protein family, including SpC3 in Al-Sharif
et al. (1998
), indicates that
seven of the hydrophobic amino acids are identical between the human C3 and
SpC3, and three are conserved (W/F or Y/F). One of the hydrophobic amino acid
positions in SpC3, which is located near the functional histidine, is
consistent with mammalian C5 (Q/P) rather than C3. Although there is no
crystal structure data available for SpC3, alignments with other C3d sequences
plus predictions of the secondary structure based on the primary sequence of
the SpC3d region
(http://www.compbio.dundee.ac.uk/
www-jpred)
indicates that the locations of the 12
helices in human C3d that form
an
barrel and defines its three-dimensional structure,
are conserved in SpC3. The conservation of the putative
helices plus
the positions of the relevant hydrophobic amino acids in the SpC3d region,
suggests that there may be a hydrophobic pocket in the sea urchin protein that
protects the thioester site in a fashion similar to that in human C3. The
appearance of these functionally critical structural elements suggests that
SpC3, like its vertebrate homologues, acts as a humoral opsonin. Thioester
dependent opsonic activity has been characterized previously for tunicate C3
homologues (Nonaka et al.,
1999
; Raftos et al.,
2001
) and SpC3 exhibits functional characteristics that are
typical of thioester-mediated opsonic activity, such as methylamine binding
and autolysis (Smith, 2002
).
In this study, we expand on preliminary data
(Smith, 2001
) to confirm that
SpC3 acts as a humoral opsonin to augment the phagocytosis of target cells by
phagocytic coelomocytes.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sea urchins and lipopolysaccharide injections
SpC3 secretion into the coelomic fluid (CF) was stimulated by injecting LPS
into sea urchins Strongylocentrotus purpuratus Stimpson prior to
opsonization experiments. The wet masses of sea urchins were used to calculate
the amount of LPS (from Vibrio cholerae; Sigma Chemicals) to inject
according to the formula described by Smith et al.
(1992). Sea urchins were
injected with sufficient LPS to yield an estimated final concentration of 2
µg ml1 of CF. Control, sham-injected animals received
sterile artificial seawater (ASW; Instant Ocean, Mentor, OH, USA) equivalent
to 2 µl ASW ml1 of CF. Injections of both LPS and ASW
were performed three times per animal with injections given at 2-day
intervals. CF was collected for analysis 24 h after each of the three LPS
injections or after the ASW injections. Sea urchins were housed at 14°C in
a 400 l aquarium containing recirculating aerated ASW and equipped with
several types of filters and UV sterilization
(Shah et al., 2003
).
Coelomic fluid collection
To collect coelomocytes for phagocytosis assays or immunocyotchemistry,
whole CF (wCF) was withdrawn from the coelomic cavity into calcium- and
magnesium-free sea water containing 30 mmol l1 EDTA and 50
mmol l1 imidazole (CMFSW-EI) as described previously
(Clow et al., 2000; Gross et
al., 1999
,
2000
). To prepare cell-free
CF, wCF was collected in the absence of CMFSW-EI, centrifuged for 5 min at 10
000 g (4°C) and the CF (supernatant) was decanted for use
in various assays.
Quantitation of SpC3 in coelomic fluid
The concentration of SpC3 in CF was determined by western blotting and
densitometry using anti-SpC3-6H according to the method of Clow et al.
(2000). The relative
intensities of different bands were determined from digital images of blots
using Scion Image software (US National Institutes of Health, Bethesda MA,
USA) according to the method of Green et al.
(2003
). This technique allowed
SpC3 titers to be quantified with accuracy equivalent to that of enzyme-linked
immunosorbant assay (ELISA; Clow et al.,
2000
).
Target cell preparation
Baker's yeast Saccharomyces cerevisiae (type II; Sigma Chemicals)
were used as target cells for phagocytosis. Yeast (100 mg) were suspended in
0.5 ml of phosphate-buffered saline (PBS; 150 mmol l1 NaCl,
10 mmol l1 phosphates, pH 7.0), killed in a boiling water
bath for 30 min and washed six times in PBS. The suspension was diluted
90-fold in PBS and incubated at 37°C for 30 min in the dark with either
fluorescein isothiocyanate (FITC; Sigma Chemicals) or rhodamine isothiocyanate
(RITC; Sigma Chemicals) at a concentration of 1.5 µg FITC or
RITC/108 yeast. Stained yeast were washed five times in PBS and
then three times in ASW before being resuspended in ASW at
1x108 yeast ml1. Stained yeast were stored
at 4°C in the dark. Before use, yeast were diluted 1:10 in ASW and counted
to ensure that 1x107 yeast were employed in opsonization
experiments.
Opsonization assays
Yeast stained with FITC (FITC-yeast; 1x107cells
ml1 ASW) were mixed with an equal volume of CF or ASW on a
shaker for 40 min at room temperature. Non-opsonized controls were prepared by
incubating FITC-yeast with an equal volume of ASW for 40 min. In some cases,
CF (diluted 1:1 in ASW) was pre-incubated for 2 h with anti-SpC3-6H (1:20) or
anti-profilin (1:20) before being used to opsonize yeast. Alternatively,
CF-opsonized yeast were acid washed by incubation in 1 mol
l1 glycine (pH 2.0) for 10 min before being washed twice in
50 mmol l1 Tris (pH 7.0) and resuspended in ASW.
Opsonized yeast and non-opsonized controls were mixed 1:1 with coelomocytes (1x106 coelomocytes ml1) for periods of up to 45 min at room temperature to allow phagocytosis to occur. Following phagocytosis, an equal volume of Trypan Blue (0.06 mg ml1 in ASW) was added to portions of the yeast/coelomocyte suspensions to quench the fluorescence of non-phagocytosed yeast. After quenching, the number of coelomocytes and fluorescent (phagocytosed) yeast were counted in ten fields of view (400x magnification) using an Axioscope fluorescence microscope (Zeiss, Germany). Data were calculated either as the number of yeast phagocytosed per 100 coelomocytes or as the phagocytic stimulation index (PSI). PSI represents the mean percentage of coelomocytes that had taken up opsonized yeast divided by the mean percentage of coelomocytes that had phagocytosed non-opsonized yeast (i.e. yeast incubated in ASW only).
Immunocytology and confocal microscopy
RITC-yeast (1x105 cells ml1 in ASW) were
incubated with an equal volume of CF for 40 min and then mixed 1:1 with
coelomocytes (1x106 cells ml1) for 45 min
to allow phagocytosis to occur. The yeast/coelomocyte mixtures (30 µl) were
centrifuged at 1000 g for 7 min onto poly-L-lysine
coated slides using a cytospin rotor at 4°C (Eppendorf, Engelsdorf,
Germany). The cells were fixed in 4% paraformaldehyde in CMFSW-EI for 5 min,
washed in CMFSW-EI, and blocked for 1 h with sea urchin cytology blocking
buffer (CBB; 10% v/v normal goat serum and 10% v/v bovine serum albumin in
CMFSW-EI). After blocking, slides were incubated at room temperature for 1 h
with anti-SpC3'pep (1:50 in CBB) followed by washing in ASW and
further incubation in G
RIg-A (1:5000 in CBB) for 1 h.Fluorescence was
prolonged by using Slow-Fade (Pierce, Rockford, IL, USA) as the mounting
medium. Cells were observed using an Olympus IMT2-RFC (Olympus, Melville, NY,
USA) inverted microscope and images were captured with an MRC 1024 Confocal
Laser Scanning System (BioRad, Hercules, CA, USA).
Detection of SpC3 on yeast cell surfaces
To measure the amount of SpC3 that had bound to yeast surfaces, CF was
diluted 1:1 with ASW and mixed with heat-killed yeast (1x107
cells ml1; not FITC stained) for 40 min at room temperature.
Unless stated otherwise, subsequent incubations were performed on ice while
shaking. After mixing with CF, yeast were washed three times by centrifugation
through PBS before being incubated with PBS containing 5% (w/v) bovine serum
albumin (PBS-BSA) for 1 h. Yeast were then incubated with
anti-SpC3'pep (1:1,000 v/v in PBS-BSA) for 1 h followed by
G
RIg-AP (1:20 000 v/v in PBS-BSA) for a further 1 h. Yeast were washed
three times by centrifugation through PBS after each of these incubations.
Finally, yeast were resuspended to 1x107 cells
ml1 in PBS. Three x 100 µl from each sample were
transferred to separate wells of 96-well microtiter plates (Costar, Cambridge
MA, USA) and incubated at room temperature with alkaline phosphatase substrate
(100 µl per well; 4-nitrophenol phosphate tablets; Sigma Chemicals). After
1 h, absorbance at 415 nm was read on a SpectraMax 340 microplate
spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Data were adjusted
for the absorbance in wells containing substrate only.
Statistical analysis
Statistical analyses were performed with the SPSS software package
(Chicago, IL, USA). The statistical significance of differences between mean
values was determined using Student's two-tailed t-tests.
Correlations between the opsonic activity and SpC3 titer of CF were tested for
significance using the Pearson product moment correlation coefficient,
r. Differences between mean values and correlations were considered
to be significant if P<0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The opsonic activity of CF from LPS-stimulated sea urchins was strictly dose dependent. Significantly enhanced phagocytic activity, relative to non-opsonized controls, could only be detected when yeast were opsonized with CF concentrations greater than 10% v/v (Fig. 2, opsonized vs. non-opsonized controls, P<0.05). Phagocytic activity reached a plateau when yeast were opsonized with CF concentrations of greater than 25% v/v. To assess whether SpC3 was responsible for the opsonic activity of LPS-activated CF, the amount of SpC3 present in the CF used for opsonization assays was investigated. Results indicated that the enhanced opsonic activity after LPS injection was mirrored by increasing levels of SpC3 in CF (Fig. 3). The concentration of SpC3 in CF on day 5 after the initial LPS injection was 5.3 times greater than prior to injection (Fig. 3, day 5 vs. day 1, P<0.05) and 1.7 times higher than on day 1 after the first LPS injection. This close association between opsonic activity and SpC3 concentration was also evident from plotting the SpC3 titers of CF from individual sea urchins against their opsonic activities before and after LPS injection (Fig. 4, r=0.914, P<0.05). These data demonstrated that a strict correlation existed between opsonic activity and the concentration of SpC3 in CF.
|
|
|
ASW injection does not alter the opsonic activity or SpC3 titer of coelomic fluid
In contrast to the increases in opsonic activity in the CF induced by LPS,
injection of ASW had little effect on the opsonic activity
(Fig. 1). Yeast opsonized with
CF collected from sea urchins on day 1 or day 5 after the initial ASW
injection increased phagocytosis by only 1.9- or 2.0-fold respectively,
relative to non-opsonized yeast. These modest opsonic activities were not
significantly different from that of CF from animals before ASW injection
(Fig. 1, day 1
vs. day 1 or day 5, P>0.05). The limited induction of
opsonic activity after ASW injection corresponded with a modest, statistically
insignificant increase in the titer of SpC3 in CF from ASW-injected sea
urchins (Fig. 3, day 1
vs. day 1 or day 5, P>0.05). The opsonic activities and
SpC3 titers in CF from sea urchins injected with ASW were also far lower than
those of LPS-injected animals (Figs
1,
3). 5 days after the initial
injection, CF from animals receiving LPS had 4.1 times the opsonic activity
and 4.5-fold more SpC3 than CF from ASW-injected sea urchins
(P<0.05). These results indicated that there was a significantly
more active response by the sea urchin complement system to challenge from
what may have been perceived as an invasion by gram-negative bacteria as
compared to responses induced by a simple injury.
LPS and ASW injections did not enhance the inherent phagocytic activities of coelomocytes
Increases detected in the phagocytosis of CF-opsonized yeast after LPS
injection could have been a result of increasing concentrations of opsonic
SpC3 in CF, as suggested from the data shown in
Fig. 4. Alternatively, the
results could have been due to an activation of the coelomocytes by contact
with LPS, which induced higher underlying phagocytic rates. Coelomocytes used
in the assays were taken from the same animals from which CF was extracted and
used for opsonization. Therefore, to differentiate between opsonization and
coelomocyte activation, the ability of LPS or ASW to increase the inherent
phagocytic activity of the coelomocytes was gauged by measuring the
phagocytosis of non-opsonized yeast. Results show that there were no
significant differences in the ability of coelomocytes collected from sea
urchins before or after the injection of either LPS or ASW to take up
non-opsonized yeast (Fig. 1,
LPS/not opsonized vs. ASW/not opsonized, P>0.05).
CF-opsonized yeast were phagocytosed by polygonal phagocytes
At least four morphological types of phagocytes have been identified in the
coelomocyte population in the purple sea urchin
(Johnson, 1969). Phagocyte
types can be distinguished based on a combination of both morphology and SpC3
expression (Gross et al.,
2000
). The two most common phagocytes have been defined as a large
discoidal type and a smaller polygonal form
(Edds, 1993
), and both of
these cell types have subsets that express SpC3
(Gross et al., 2000
). Confocal
images of cells that had phagocytosed opsonized yeast demonstrated that
polygonal phagocytes were exclusively responsible for phagocytosis
(Fig. 5). Immunocytochemistry
with anti-SpC3
'pep revealed that the subset of polygonal cells
that phagocytosed opsonized yeast also contained SpC3.
|
Anti-SpC3-6H inhibits the phagocytosis of CF-opsonized yeast
There are many types of opsonins that have been characterized in
invertebrates, including lectins and LPS binding proteins
(Arason, 1996) in addition to
complement homologues. Hence, to confirm the contribution of SpC3 to the
opsonic activity of S. purpuratus CF, we used anti-SpC3-6H to block
phagocytosis. When LPS-activated CF was incubated with anti-SpC3-6H before
being used to opsonize yeast, opsonic activity was decreased by 64% compared
to yeast opsonized with untreated CF (Fig.
6, yeast+CF+
-SpC3-6H vs. yeast+CF,
P<0.05). Pre-incubating CF with anti-profilin, which was used as
an irrelevant control antibody, did not significantly alter opsonic activity
(Fig. 6, yeast+CF+
-profilin vs. yeast+CF, P>0.05). These
results demonstrated that SpC3 appears to be a major contributor of the
opsonic activity in the CF.
|
The inhibitory effect of anti-SpC3-6H on phagocytosis could be abrogated
when acid washing was performed on yeast opsonized with LPS-activated CF that
had been pre-incubated with anti-SpC3-6H. The acid wash was used to remove the
antibody from SpC3 molecules that had been deposited on yeast cell surfaces,
while the bound SpC3 molecules would remain due to the covalent thioester
bonds formed with the target surfaces
(Smith, 2002). The opsonic
activity of yeast opsonized with CF pre-incubated with anti-SpC3-6H followed
by acid wash did not differ significantly from that of yeast opsonized with CF
alone (Fig. 6,
yeast+CF+
-SpC3-6H washed vs. yeast+CF, P>0.05).
This suggested that even though anti-SpC3-6H could block the opsonic activity
of LPS-activated CF, it did not do so by blocking thioester activity and
preventing SpC3 from binding onto the yeast surface.
SpC3 binds to yeast cell surfaces
The ability of SpC3 to bind yeast surfaces was confirmed by using
anti-SpC3'pep in an immunosorbent assay of CF-opsonized yeast.
The results indicated that incubating yeast with CF resulted in substantial
deposition of SpC3 onto yeast surfaces
(Fig. 7). The
spectrophotometric absorbance of yeast that had been incubated with CF
followed by primary (anti-SpC3
'pep) and secondary
(G
RIg-AP) antibodies was four times greater than that of yeast that had
been processed at the same time, but without the CF opsonization step
(P<0.05). Consequently, non-specific binding of the secondary
antibody to the yeast was eliminated as a possible explanation for this
result. Similarly, no substantial binding activity could be detected when the
primary and secondary antibodies were omitted from the assay.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we have confirmed the contribution made by SpC3 in the sea
urchin immune response by demonstrating that SpC3 acts as an important
inducible opsonin. We found that opsonizing yeast with CF from LPS-activated
sea urchins significantly enhanced their phagocytosis by certain types of
phagocytic coelomocytes. Significantly lower rates of phagocytosis were
evident when yeast were opsonized with CF from immunoquiescent sea urchins
that were either not injected or were injected with ASW. This suggests that
the opsonic factor is not always present in the CF and that its expression can
be induced by pathogen associated molecular patterns such as LPS. The need for
induction is in agreement with our previous studies which have shown that
immunoquiescent sea urchins do not express substantial quantities of SpC3
(Gross et al., 1999) and that
SpC3 secretion can be induced by LPS (Clow
et al., 2000
). A link between SpC3 and the opsonic activity in the
CF was also supported by the correspondence between the SpC3 titers and the
opsonic activities of CF from individual sea urchins. Densitometry of SpC3
bands on western blots developed with anti-SpC3-6H showed that increasing SpC3
titers were strictly correlated with increasing opsonic activities of CF after
LPS injection. The opsonic potential of SpC3 was also inferred from the
results of an immunosorbent assay using anti-SpC3
'pep, which
showed that SpC3 binds onto the surface of target cells. The relationship
between SpC3 and the opsonic activity of CF was also confirmed using
anti-SpC3-6H antibodies to inhibit opsonization. Pre-incubating LPS-activated
CF with anti-SpC3-6H significantly decreased the opsonic activity relative to
yeast opsonized with CF in the absence of antibody, while the irrelevant
control antibody (anti-profilin) had no effect on opsonization. This clearly
demonstrates that SpC3 represents a key opsonic factor in the CF.
Although phagocytosis decreases when CF is pre-incubated with anti-SpC3-6H
prior to opsonization, this antibody does not appear to inhibit opsonization
by preventing SpC3 from binding onto yeast cell surfaces. When anti-SpC3-6H
was removed from the bound SpC3 by low pH, increased phagocytosis was
restored. This suggests that anti-SpC3-6H does not recognize epitopes on SpC3
that are critical for target cell binding. Instead, it might block epitopes
required for the interaction between SpC3 and putative C3 receptors on the
phagocytes that would be required for complement-mediated phagocytosis.
However, the data may also be explained by acid-induced exposure of a
previously cryptic ligand on the yeast particles to which a different opsonic
system might bind. The existence of SpC3 receptors in echinoderms was first
implied by Bertheussen and his coworkers, who showed that mammalian C3 could
act as an opsonin in the green sea urchin, S. droebachienses (Kaplan
and Bertheussen, 1977; Bertheussen, 1981, 1982; Bertheussen and Seljelid,
1982). In mammals, C3 interacts with its primary cellular receptor, complement
receptor type 1 (CR1 or CD35), via a series of acidic amino acids
clustered at the N terminus of the C3 ' chain
(Oran and Isenman, 1999
).
These residues are only exposed after C3 has been proteolytically activated.
SpC3 has a similar cluster of acidic amino acids in precisely the same region
of the predicted SpC3
' chain
(Al-Sharif et al., 1998
).
Anti-SpC3-6H was generated against a portion of the protein that included the
SpC3
' chain and so could have inhibited the phagocytosis of
CF-opsonized yeast by blocking the ability of these acidic residues to
interact with the putative cellular receptor on the phagocyte that recognizes
SpC3. This is indirect evidence that sea urchins, and perhaps other
deuterostome invertebrates, have type 1 complement receptors in addition to
the type 3 or type 4 receptor identified previously in a tunicate
(Miyazawa et al., 2001
)
The SpC3 receptors of S. purpuratus may be restricted to a
distinctive subset of coelomocytes. Confocal micrographs indicated that
CF-opsonized yeast were taken up exclusively by polygonal phagocytes, which
represent a subset of one of the four distinct coelomocyte types in S.
purpuratus (Edds, 1993).
Polygonal phagocytes are among the two subpopulations of phagocytic cells in
S. purpuratus that are responsible for producing SpC3
(Gross et al., 2000
). It
should be noted, however, that even though the expression of SpC3 by these
phagocytes was enhanced by LPS injections, their inherent capacity for
phagocytosis did not change. In the absence of opsonization, the rate at which
yeast were taken up by phagocytes was not increased by the injection of either
LPS or ASW. This failure to enhance cellular activity in response to antigenic
challenge means that, in sea urchins, inducible anti-pathogen responses might
rely heavily on secreted humoral opsonins such as SpC3 and not on the
`activation' of coelomocytes.
The results presented here imply that the sea urchin complement system is of major importance in the humoral defenses of S. purpuratus. We have shown that, when the opsonic activity of SpC3 is either blocked, deactivated or the concentration is too low to be of functional relevance, the level of phagocytosis decreases significantly and other opsonin systems are apparently not designed to compensate. We cannot, however, rule out the existence of additional opsonic systems because anti-SpC3 antibodies did not abrogate completely the opsonic activity of the CF (Fig. 6).
Echinoderms do not have homologues of the rearranging immunoglobulin class
of genes in their genomes, and so the activation of SpC3 cannot include a
pathway analogous to the classical complement pathway of higher vertebrates.
However, SpC3 activation may be mediated by mechanisms analogous to the
alternative or lectin pathways (Smith et al.,
1999,
2001
). Lectin-mediated
complement activation has been characterized in the tunicates Styela
plicata and Halocynthia roretzia
(Green et al., 2003
;
Ji et al., 1997
;
Nair et al., 2000
;
Raftos et al., 2001
), and a
number of collectin family members have been identified in the genome of
another tunicate, Ciona intestinalis
(Azumi et al., 2003
). A similar
genome-level analysis for the sea urchin may also reveal a collectin gene
family putatively involved in complement activation. The feedback loop that
functions in the mammalian alternative pathway is an effective and highly
efficient mechanism that acts quickly to coat foreign cells with complement
proteins (Dodds and Day,
1993
). It is also generally agreed that the opsonization function
of the complement system of higher vertebrates (in addition to the lytic
activities of the terminal pathway) is of significant importance for host
defense. The dynamic activation of thioester proteins by the C3-convertases
bound to the surface of foreign cells results in the cleavage and activation
of additional C3 in close proximity to the target surface (reviewed by
Xu et al., 2001
). This
amplification mechanism results in faster and more effective opsonization than
can be provided by a simple opsonin that does not rely on a cascade of events
to magnify its activation. It has been postulated that the alternative pathway
of complement in the sea urchin also functions as a feedback loop (Smith et
al., 1999
,
2001
). The establishment of
such an active cascade-based opsonization system in a basal deuterostome
ancestor may have provided significant selective advantages to the host for
recognizing and eliminating pathogens. This efficiency may be a core reason
why the complement system has been retained and expanded upon to become the
central immune effector system in both the innate and adaptive immune systems
of present-day vertebrates.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: L. A. Clow, United States Patent and Trademark Office,
Alexandria, VA 22313, USA
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Al-Sharif, W. Z., Sunyer, J. O., Lambris, J. D. and Smith, L.
C. (1998). Sea urchin coelomocytes specifically express a
homologue of the complement component C3. J. Immunol.
160,2983
-2997.
Arason, G. J. (1996). Lectins as defence molecules in vertebrates and invertebrates. Fish Shellfish Immunol. 6,277 -289.[CrossRef]
Azumi, K., De Santis, R., De Tomaso, A., Rigoutsos, I., Yoshizaki, F., Pinto, M. R., Marino, R., Shida, K., Ikeda, M., Ikeda, M. et al. (2003). Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: `waiting for Godot'. Immunogenet. 55,570 -581.[CrossRef][Medline]
Bentley, D. R. (1988). Structural superfamilies of the complement system. Expl. Clin. Immunogenet. 5, 69-80.
Berthuessen, K. (1981). Endocytosis by echinoid phagocytes in vitro. I. Recoginition of foreign matter. Dev. Comp. Immunol. 5,241 -250.[CrossRef][Medline]
Berthuessen, K. (1982). Receptors for complement on echinoid phagocytes. II. Purified human complement mediates echinoid phagocytosis. Dev. Comp. Immunol. 6, 635-642.[Medline]
Berthuessen, K. and Seljelid, R. (1982). Receptors for complement on echinoid phagocytes. I. The opsonic effect of vertebrate sera on echinoid phagocytosis. Dev. Comp. Immunol. 6,423 -431.[Medline]
Campbell, R. D., Law, S. K. A., Reid, K. B. M. and Sim, R. B. (1988). Structure, organization and regulation of the complement genes. Ann. Rev. Immunol. 6, 161-195.[CrossRef][Medline]
Clow, L. A., Gross, P. S., Shih, C.-S. and Smith, L. C. (2000). Expression of SpC3, the sea urchin complement component, in response to lipopolysaccharide. Immunogenet. 51,1021 -1033.[CrossRef][Medline]
Dodds, A. W. and Day, A. J. (1993). The phylogeny and evolution of the complement system. In Complement in Health and Disease (ed. K. Whaley, M. Loos and J. M. Weiler). Immunol. Med. 20,39 -88. New York: Marcel Dekker Inc.
Edds, K. T. (1993). Cell biology of echinoid coelomocytes. I. Diversity and characterization of cell types. J. Invert. Pathol. 61,173 -178.[CrossRef]
Fujita, T. (2002). Evolution of the lectin-complement pathway and its role in innate immunity. Nat. Rev. Immunol. 2,346 -353.[Medline]
Green, P. G., Nair, S. V. and Raftos, D. A. (2003). Secretion of a collectin-like protein in tunicates is enhanced during inflammatory responses. Dev. Comp. Immunol. 27,3 -9.[CrossRef][Medline]
Gross, P. S., Al-Sharif, W. Z., Clow, L. A. and Smith, L. C. (1999). Echinoderm immunity and the evolution of the complement system. Dev. Comp. Immunol. 23,439 -442.
Gross, P. S., Clow, L. A., Shih, C.-S. and Smith, L. C. (2000). SpC3, the complement homologue from the purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes. Immunogenet. 51,1034 -1044.[CrossRef][Medline]
Isaac, L. and Isenman, D. E. (1992). Structural
requirements for thioester bond formation in human complement component C3.
Reassessment of the role of thioester bond integrity on the conformation of
C3. J. Biol. Chem. 267,10062
-10069.
Ji, X., Azumi, K., Sasaki, M. and Nonaka, M.
(1997). Ancient origin of the complement lectin pathway revealed
by molecular cloning of mannan binding protein-associated serine protease from
a urochordate, the Japanese ascidian, Haolcynthia roretzi. Proc.
Natl. Acad. Sci. USA 94,6340
-6345.
Johnson, P. T. (1969). The coelomic elements of sea urchins (Strongylocentrotus). I. The normal coelomocytes; their morphology and dynamics in hanging drops. J. Invert. Pathol. 13,25 -41.[Medline]
Kaplan, G. and Berthuessen, K. (1977). The morphology of echinoid phagocytes and mouse peritoneal macrophages during phagocytosis in vitro. Scand. J. Immunol. 6,1289 -1296.[Medline]
Lachmann, P. J. (1979). An evolutionary view of the complement system. Behring Inst. Mitt. 63, 25-37.
Lagueux, M., Perrodou, E., Levashina, E. A., Capovilla, M.
and Hoffmann, J. A. (2000). Constitutive expression of
a complement-like protein in toll and JAK gain-of-function mutants of
Drosophila. Proc. Natl. Acad. Sci. USA
97,11427
-11432.
Levashina, E. A., Moita, L. F., Blandin, S., Vriend, G., Lagueux, M. and Kafatos, F. C. (2001). Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae.Cell 104,709 -718.[CrossRef][Medline]
Marino, R., Kimura, Y., De Santis, R., Lambris, J. D. and Pinto, M. R. (2002). Complement in urochordates: Cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis.Immunogenet. 53,1055 -1064.[CrossRef][Medline]
Miyazawa, S., Azumi, K. and Nonaka, M. (2001).
Cloning and characterization of integrin alpha subunits from the solitary
ascidian, Halocynthia roretzi. J. Immunol.
166,1710
-1715.
Multerer, K. A. and Smith, L. C. (2004). Two cDNAs from the purple sea urchin, Strongylocentrotus purpuratus, encoding mosaic proteins with domains found in factor H, factor I, and complement components C6 and C7. Immunogenet. in press.
Nair, S. V., Pearce, S., Green, P. L., Mahajan, D., Newton, R. A. and Raftos, D. A. (2000). A collectin-like protein from tunicates. Comp. Biochem. Physiol. B 125,279 -289.[CrossRef][Medline]
Nagar, B., Jones, R. G., Diefenbach, R. J., Isenman, D. E. and
Rini, F. M. (1998). X-ray crystal structure of C3d: a C3
fragment and ligand for complement receptor 2. Science
280,1277
-1281.
Nonaka, M. and Azumi, K. (1999). Opsonic complement system of the solitary ascidian, Halocynthia roretzi.Dev. Comp. Immunol. 23,421 -427.[CrossRef][Medline]
Nonaka, M., Azumi, K., Ji, X., Namikawa-Yamada, C., Sasaki, M.,
Saiga, H., Dodds, A. W., Sekine, H., Homma, M. K., Matsushita, M.,
Endo, Y. and Fujita, T. (1999). Opsonic complement component
C3 in the solitary ascidian, Halocynthia roetzi. J.
Immunol. 162,387
-391.
Oran, A. E. and Isenman, D. E. (1999).
Identification of residues within the 727-767 segment of human complement
component C3 important for its interaction with Factor H and with complement
receptor 1 (CR1, CD35). J. Biol. Chem.
274,5120
-5130.
Pancer, Z. (2000). Dynamic expression of
multiple scavenger receptor cycteine-rich genes in coelomocytes of the purple
sea urchin. Proc. Natl. Acad. Sci. USA
97,13156
-13161.
Pancer, Z., Rast, J. P. and Davidson, E. H. (1999). Orgin of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. Immunogenet. 49,773 -786.[CrossRef][Medline]
Raftos, D. A., Green, P., Mahajan, D., Newton, R. A., Pearce, S., Peters, R., Robbins, J. and Nair, S. V. (2001). Collagenous lectins in tunicates and the proteolytic activation of complement. Adv. Exp. Med. Biol. 484,229 -236.[Medline]
Raftos, D. A., Nair, S. V., Robbins, J., Newton, R. A. and Peters, R. (2002). A complement component C3-like protein from the tunicate, Styela plicata. Dev. Comp. Immunol. 26,307 -312.[CrossRef][Medline]
Shah, M., Brown, K. and Smith, L. C. (2003). The gene encoding the sea urchin complement protein, SpC3, is expressed in embryos and can be upregulated by bacteria. Dev. Comp. Immunol. 27,529 -538.[CrossRef][Medline]
Smith, L. C. (2001). The complement system in sea urchins. In Phylogenetic Perspectives on the Vertebrate Immune Systems (ed. G. Beck, M. Sugumaran and E. Cooper). Adv. Exp. Med. Biol. 484,363 -372. New York: Kluwer Academic/Plenum Publishing Co.[Medline]
Smith, L. C. (2002). Thioester function is conserved in SpC3, the sea urchin homologue of the complement component C3. Dev. Comp. Immunol. 26,603 -614.[CrossRef][Medline]
Smith, L. C., Azumi, K. and Nonaka, M. (1999). Complement systems in invertebrates. The ancient alternative and lectin pathways. Immunopharmacol. 42,107 -120.[CrossRef][Medline]
Smith, L. C., Britten, R. J. and Davidson, E. H. (1992). SpCoel1: A sea urchin profilin gene expressed specifically in coelomocytes in response to injury. Mol. Biol. Cell 3,403 -414.[Abstract]
Smith, L. C., Chang, L., Britten, R. J. and Davidson, E. H. (1996). Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. J. Immunol. 156,593 -602.[Abstract]
Smith, L. C., Clow, L. A. and Terwilliger, D. P. (2001). The ancestral complement system in sea urchins. Immunol. Rev. 180,16 -34.[CrossRef][Medline]
Smith, L. C., Shih, C.-S. and Dachenhausen, S.
(1998). Coelomocytes specifically express SpBf, a homologue of
factor B, the second component in the sea urchin complement system.
J. Immunol. 161,6784
-6793.
Sottrup-Jensen, L., Stepanik, T., Kristensen, T., Lonblad, P., Jones, C., Wierzbicki, D., Magnusson, S., Domdey, H., Wetsel, R. and Lundwall, A. (1985). Common evolutionary origin of alpha 2-macroglobulin and complement components C3 and C4. Proc. Natl. Acad. Sci. USA 82,9 -13.[Abstract]
Suzuki, M. M., Satoh, N. and Nonaka, M. (2002). C6-like and C3-like molecules from the cephalochordate, amphioxus, suggest a cytolytic complement system in invertebrates. J. Mol. Evol. 54,671 -679.[CrossRef][Medline]
Theil, S., Sotrup-Jensen, T., Stover, C., Schwaeble, W., Laursen, S., Poulsen, K., Willis, A., Eggleton, P., Hansen, S., Holmskov, U., Reid, K. and Jensenius, J. (1997). A second serine protease associated with mannan-binding lectin that activates complement. Nature 386,506 -510.[CrossRef][Medline]
Volanakis, J. E. (1998). Overview of the complement system. In The Human Complement System in Health and Disease (ed. J. E. Volanakis and M. M. Frand), Immunol. Med. 20,9 -32. New York: Marcel Dekker Inc.
Xu, Y., Narayana, S. B. L. and Volanakis, J. E. (2001). Structural biology of the alternative pathway convertase. Immunol. Rev. 180,123 -135.[CrossRef][Medline]