INNATE IMMUNITY IN LOPHOTROCHOZOANS: THE ANNELIDS
Michel Salzet1,
Aurélie Tasiemski1, Edwin Cooper2
In the course of evolution
annelids have developed different immunodefense strategies against microbes
living in water or soil that are ingested during feeding or introduced into the
body after injury [1]. For understanding immune
processes, it is necessary to briefly review annelid anatomy. Earthworms (Oligochaeta,
oligochaetes) and
leeches (Hirudinea, achaetes) are characterized by the presence of two
compartments containing free cells: (a) the blood system with haemocytes: this
compartment does not seem to be actively implied in immunity and (b) the coelom
that includes several coelomocytes populations playing a role in the immune
defense Fig. (1). We focus in the present review on Oligochaeta and.
Hirudinea in which most of the work on immunodefense has been performed. Both
are important for certain biomedical applications [2-4].
I- Cellular immune
responses in annelids
A Phagocytosis by LC and NK-like
lysis by SC
In
oligochaetes, spontaneous allogeneic
cytotoxicity of coelomocytes from Eisenia
fetida and Lumbricus terrestris
has been investigated in vitro using
different assays (trypan blue, lactate dehydrogenase and 51Cr
release; FACS analyses) [5, 6]. Suzuki and Cooper
investigated xenogenic reactions against human tumor cells [6]. TEM and SEM studies showed that close contact of target
cells with Lumbricus effector cells (coelomocytes with different mitochondrial
properties) [7] is followed by lysis. The
results confirm that effector cell/target cell contact is essential for
cytotoxicity to occur. These cytological events support analogies with
cytotoxic activities that are affected when
vertebrate natural killer cells are co-cultured with appropriate targets.
Cytotoxicity supports the postulate of Cooper [8] and later Franceschi[9] that a primitive natural killer cell-like (analogue vertebrate NK
cells) evolved early in phylogenesis [8-11] and the following results
support this view. As in the NK system, the target specificity is exceedingly
broad since xenogenic, allogenic, and even syngenic red blood targets are
killed under appropriate conditions. The specificity of these cytotoxic cells
may be directed at cell-surface glycoproteins on target cell surfaces since
several defined mono- and disaccharides can block killing. Suzuki and Cooper
examined whether modifying cell surface antigens can affect the level of
cell-mediated cytotoxicity[12]. When co-cultured with small
coelomocytes (SC) with NK-sensitive cells lines (K592), they become agitated,
extending numerous pseudopodia that bind to and kill targets cells [13]Figs. (2A, 2B). Large coelomocytes (LC)
aggregate around the lysed targets and encapsulate them before forming
granulomas [13]. These results suggest that
phagocytosis and cytotoxicity are independently mediated by two different cell
types; earthworm leukocytes as a group are polyfunctional and not exclusively
phagocytic [4]. This supports the view of
early divergence of phagocytosis and lysis.
The use of monoclonal
antibodies directed against several human CD markers enabled Cossarizza et al to focus on these two coelomocytes
in E. fetida [14]: (1) the SC also called
killer cells which are positive for CD11a+, CD45RA+,
CD45RO+, CDw49b+, CD54+, b2-m+ and Thy-1+ (CD90) and (2)
the phagocytic LC that are negative for these markers [15]; [16]. Both cell types were
negative for numerous other CD and MHC
class I and class II markers. [17]. SC are active during
recognition, rapidly binding to targets and LC are phagocytic. One cell group
the SC effects recognition, binding and killing, whereas cleaning up the
remaining debris rests with the other cell type the LC.
In
achaetes,
differential CD staining at different time points following grafting
experiments (four months between two transplants and specimen examination after
1 month), assume the presence of two different cell types, i.e. NK-like cells and CD8+ cells in the medicinal leech
(generally classified as cytotoxic lymphocytes in vertebrates) [18, 19]. These coelomocytes are
selectively stimulated by different antigens [19]. De Eguileor and colleagues
using morphological and immunocytochemical approaches have analyzed the
migratory behavior of cells involved in inflammatory responses of Glossiphonia complanata [18] as well as angiogenesis
processes [20, 21]. Like other annelids, leech
leukocytes phagocytose and encapsulate foreign material selectively depending
upon size [22]. Encapsulation is optimally
visible when a parasite has been completely coated and isolated by a thick
melanotic capsule [22]. Wounding in leeches is
accompanied by “leucopoiesis”, as it is generally accepted in vertebrates. In
fact, in transplanted/wounded leeches, massive proliferation of lymphocyte-like
cells stemming from a single cluster has been observed [22].
II- Humoral immune responses in annelids
A. Is there evidence of
complement like activity?
Both, cellular and humoral immune
reactions in annelids are increased in response to an inflammatory or
intracoelomic injection of foreign material. Certain annelid immune mechanisms,
like NK-like activity and pore formation resemble functionally perforin and/or
complement activity of vertebrates. The protective mechanisms of invertebrates consist, beyond cell immunity,
of lysis and agglutination enhanced by
components from the coelomic fluid like antigen binding protein, cytokines,
antimicrobial substances. Results in the field of research are numerous,
especially in oligochaetes. In several experiments designed to test the
possible existence of complement like activity, Cooper [23]co-cultured earthworm
leukocytes with various NK dependent and independent targets [23]. Since washed coelomocytes
were always used in cytotoxic assays, the experiments were done to test the
capacity of cell-free supernatants. Adding supernatant from coelomocytes
cultured for 10 min at 22oC caused significant cytotoxicity against
K562 and other human cell lines. Cytotoxicity was not evident when supernatants
from the same (autogenic) or different (allogenic) earthworms were added
immediately after washing, nor did cytotoxicity occur when supernatants were
heated at 56oC for 30 min before addition to K562 cultures. This suggests that a humoral factor-mediated
component that may be complement like protein is involved in a significant
percentage of target cell killings. Cytotoxicity cannot be interpreted as
effected primarily by earthworm leukocytes.
B- Hemolytic substances
1.
A recognition component
A coelomic cytolytic factor (CCF)
has been isolated from coelomic fluid of the earthworm E.
fetida [24].CCF-1
shared functional analogy with vertebrate TNF based on N,N'-diacetylchitobiose
lectin like domain/activity, despite a gene or protein homology [25]. This suggests that CCF and TNF lectin domain have been functionally
conserved in the course of evolution as a recognition mechanism during innate
immune responses [26]. CCF-1
is capable of lysing different mammalian tumor cell lines but by contrast with
TNF, this cytolytic activity is mediated by lysis and is not due to
proteolysis. Interestingly, CCF-1
recognizes lysozyme-predigested Gram-positive bacteria or the peptidoglycan
constituent muramyl dipeptide as well as muramic acid (Fig.3) [27]. The broad specificity of CCF for pathogen-associated molecular patterns
results from the presence of two distinct pattern recognition domains. One
domain, which shows homology with the polysaccharide and glucanase motifs of
beta-1, 3-glucanases and invertebrate defense molecules located in the central
part of the CCF polypeptide chain,
interacts with LPS and beta-1,3-glucans. The C-terminal tryptophan-rich domain
mediates interactions of CCF with
N,N'-diacetylchitobiose and muramic acid Fig.(3). These data provide
evidence for the presence of spatially distinct carbohydrate recognition
domains within this invertebrate immunodefense molecule [28] [27].
2.
Pore forming molecules
Besides CCF-1,
pore-forming proteins have been characterized in the earthworm coelom: i.e. eiseniapore [29, 30], lysenin [31] and fetidin[32] (table 1) These
molecules bind to and disturb the lipid bilayer only when particular sphingolipids,
(e.g. sphingomyelin), are present which are lacking in annelids[33, 34] . As for lysenin, cholesterol enhances
eiseniapore lytic activity toward sphingomyelin-containing vesicles. Leakage of
vesicles was most efficient when the lipid composition resembled that of the
outer leaflet of human erythrocytes [35] [33]. Electron microscopy of erythrocyte membranes confirmed
ring-shaped structures (pores). The channel complex consists of six monomers [29, 30] suggesting a plausible explanation for the mechanism by
which components of the earthworm's immune system destroy non-self components Fig.
(3).
In addition, Kauschke et al. have demonstrated the existence of a perforin-like protein [36] [37]. Perforin and other lytic
molecules seemed to be linked. One explanation is that perforin will generates
the holes in target cell membranes facilitating the entry the lytic molecules
like lysenins, lysins, fetidins and the hemolysins (H1, H2,
H3) 33. [38] [39] [32] (Table 1). Each of these molecules has been demonstrated to be
active towards certain human diseases (Nieman-Pick; blood clots).
C.
Cytokines and mitogenic factors
Most of the data obtained at the
present time on cytokines in invertebrates are by antibody recognition and not
by sequence similarity. In annelids, the most data concerns the oligochaetes [40] and also leeches [41, 42]. With respect to mitogenic
factors, one was initially discovered in Lumbricus that exerts activity
on T cells [43]. Later, a 60-kDa component
with mitogenic activity on murine splenocytes was identified in the coelomic fluid of E. fetida and named CMF (Coelomic
Mitogenic Factor) [44]. CMF was found to bind ConA
that could account for its ability to inhibit ConA-induced spleen cell
proliferation. CMF is a trimer of a 20-kDa protein. N-terminal amino acid
sequence of monomeric CMF reveals partial sequence homology with phospholipase
A2 (PLA2). Moreover, CMF-enriched
coelomic fluid exerts phospholipase activity comparable with that of bovine
pancreatic PLA2. These results
suggest that coelomic fluid of E. foetida
contains an ubiquitous PLA2-like
enzyme which might be involved in immune reactions in earthworms such as
anti-bacterial mechanisms [44].
D. Antigen-binding proteins (ABP)
Laulan et al. reported
synthesis by L. terrestris of
specific substances in response to an immunization with a synthetic hapten [45]. Tuckova et al identified a 56 kDa molecule, antigen-binding protein consisting
of two disulfide-linked polypeptide chains (31 and 33 kDa) both of which
participate in the formation of the antigen-binding site [46]. The kinetics of ABP formation revealed that the response reached a
maximum level between the 4th and the 8th day after the first dose and
approximately 4 days after the second challenge. The degree of specificity of
the ABP after the secondary in vivo challenge increased, but even so
it was considerably lower than that of vertebrate immunoglobulin [47].
E. Antimicrobial proteins
The most studied
antimicrobial protein in annelids is the lysozyme [48] Table (1). This is an
enzyme that cleaves the β-1-4 bonds between N acetylglucosamine and N
acetylmuramic acid of Gram positive bacterial cell walls. Lysozyme is often
detected using its action against
Microccocus lysodeikticus.
In oligochaetes, background activity was 20-fold enhanced by one injection of either
Gram positive or Gram negative bacteria and sheep red blood cells. Maximum
induced activity occurred 4-5 hr after injection. Transcription and translation
regulation of lysozyme have been investigated by Hirigoyenberry et al [49]. Besides, lysozyme activity,
the coelomic fluid of the earthworm E.fetida
andrei exhibits antibacterial, hemolytic and hemagglutinating activities
(See the cytotoxicity section). These activities are mainly mediated by two
proteins, named fetidins, of apparent molecular mass 40 kDa and 45 kDa,
respectively [32]. The recombinant protein was
showed to inhibit Bacillus megaterium
growth. Near its hemolysis activity, the protein also exhibits a peroxydase
activity and a peroxydase signature was identified in residues 52-62 [32] Table (1).
In
achaetes, an intrinsic
lysozyme-like activity was demonstrated for destabilase [50, 51]. Several isoforms of
destabilase constitute a protein family with at least two members characterized
by lysozyme activity [50, 51]. The corresponding gene
family implies an ancient evolutionary history of the genes although the
function(s) of various lysozymes in the leech remains unclear. Differences in
primary structures of the destabilase family members and members of known
lysozyme families allow one to assign the former to a new family of lysozymes.
New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve
molluscs suggesting that the new lysozyme family can be widely distributed
among invertebrates [50, 51]. Besides lysozyme, a 14 kDa
protein sharing a bacteriostatic activity and belonging to the hemerythrin
family has been found in fat cells [52]as well as glial cells of
leeches[53]. Hemerythrin is a non-hemic-iron
oxygen-transport protein acting as an iron scavenger towards bacteria in
polychaetes [54] Table (1).
F. Antimicrobial peptides
During
the last few years studies on the components of the innate immune system fully
demonstrated the contribution of the antimicrobial peptides to host defense.
Antibiotic peptides are small molecules (size comprised between 2kDa and 8kDa).
Their implication in natural resistance to infection is sustained by their strategic
location in phagocytes, in body fluids and at epithelial cell levels i.e. at interfaces between organisms and
its environment [55](Table (1).
In
oligochaetes, an antimicrobial
peptide named lumbricin I has been isolated and characterized from the
earthworm, Lumbricus rubellus.
Lumbricin I is a proline-rich antimicrobial peptide of 62 amino acids.
Lumbricin I showed antimicrobial activity in vitro against a broad spectrum
of microorganisms without hemolytic activity. A 29-amino acid peptide, named
lumbricin I (6-34), which was derived from residues 6-34 of lumbricin I, showed
marginally stronger antimicrobial activity than authentic lumbricin I. However
lumbricin I gene is not induced by bacterial infection, but was constitutively
expressed. Furthermore, the expression of lumbricin I gene was specific in
adult L. rubellus, no expression was
found in eggs and young L. rubellus [56].
In achaetes,
Tasiemski et al., have recently isolated four antimicrobial peptides from the coelomic
fluid of the leech Theromyzon tessulatum:
theromacin, theromyzin, peptide B [57-59] [60, 61] and lumbricin sharing high
sequence homology with the one isolated from earthworm (unpublished data). Theromacin is a
cationic 75 amino acids peptide with 10 cysteines residues arranged in a
disulfide array showing no obvious similarities with other known antimicrobial
peptides [59] [62]. Theromyzin and peptide B are
anionic peptides of 82 and 31 amino acid residues respectively [58, 59] [62] [63]. Theromacin and theromyzin
are processed from a larger precursor constituted by a signal peptide directly
followed by the active peptide. Interestingly, peptide B is processed from the
C terminal part of proenkephalin A (PEA), the major precursor of enkephalins.
Peptide B and theromyzin possess bacteriostatic activity by contrast with
theromacin which exhibit potent bactericidal activity towards Gram positive
bacteria. Theromyzin is a histidine rich peptide sharing a HXXE, divalent ion
active site like histatin in vertebrates [64]. Analysis of theromacin and theromyzin gene expression demonstrated both a developmental and a
septic injury induction in large fat cells (LFC), which constitute a specific
tissue that seems to be a functional equivalent of the insect fat body (Figure 2D, 2E). Both peptides
seem to exert their antimicrobial activity by a systemic action. All these data
suggest for the first time an innate immune response in a lophotrochozoan
comparable to the antibacterial response of an ecdysozoan i.e. D. melanogaster. Consequently, Theromyzon is an original invertebrate model which has developed
two modes of fighting infections by antimicrobial peptides: (i) storage of
antibacterial peptide derived from PEA, particularly in coelomocytes and/or in
nervous system, and release of the peptide into the coelomic fluid after immune
challenge (ii) induction after septic injury of gene coding for more classical
antimicrobial peptide, mainly in LFC, and rapid release into the body fluid of the
antibiotic peptides Fig.(4).
G. Cysteine and serine protease inhibitors
Serine proteases, such as trypsin,
thrombin, granzyme B, are important enzymes involved in regulating many
physiologic events including the extrinsic pathways of blood coagulation and
immune processes. In the immune system, killer lymphocytes combine the activity
of a membranolytic protein, perforin, and the serine protease granzyme B to
induce target cell apoptosis [65]. Similarly, cysteine
proteases such as capthepsins are implicated in processing functions of mammalian antigen
presenting cells (APCs) [66]. They allow Class
II-antigenic peptide binding by degradation of Ii chaperone in the initial
Class II-Ii complex [67].
In oligochaetes, three serine proteases have been
isolated from E. fetida leukocytes and one is related to trypsin [68]. In the same species, a
serine protease inhibitor of 14 kDa contained in the coelomic fluid has been
characterized [69]. This enzyme inhibitor shares
high homologies with several plant serine protease inhibitors. These molecules
seem to be implicated in the cytotoxic cascade involving fetidins, CCF and the prophenoloxidase system Fig. (3) [33].
In achaetes, a gene tandem cystatin B (Tt-cysb)-cathepsin L (Tt-catl) was identified in the leech T. tessulatum [70]. Tt-cysb belongs to the cysteine protease inhibitor family. Tt-cysb gene possesses an open reading
frame of 306 nucleotides without signal peptide. The deduced sequence presents
54% sequence identity with human cystatin B. No homologs of Tt-cysb are present in D. melanogaster and C. elegans genomes. The active site sequence (QXVXG) is conserved
as well as the glycine at position 5 allowing its interaction with its
substrate, the cathepsin L. Tt-cysb constitutes
the first cystatin B protein isolated in invertebrates. Tt-catl belongs to the cysteine proteinase family and presents 68%
sequence identity with D. melanogaster
cathepsin L. Tt-cysb is only expressed
in one circulating coelomic cells population. Bacterial challenge leads to an
enhancement of the Tt-cysb transcript
exclusively in these cells. Double in
situ hybridization showed cathepsin L gene (Tt-catl) to be expressed in the same cells suggesting that both Tt-cyst and Tt-catl gene products might play a role in immune regulation
through these circulating coelomic cells Fig.( 2F). These data are in line with those
evidenced in mammals or in drosophila in which cathepsin L is present in small
granules of haemocytes and might play a role in phagocytic events [71] [70].
III.
Perspectives concerning annelid immunity
Arthropods,
especially insects, represent an important group of invertebrates because of
their sheer numbers, and Drosophila has provided ample evidence that
they possess effective innate immune responses. Yet it is unwise to
over-generalize about the immune system of all invertebrates based upon this
single species. If one looks at metazoan phylogeny based on small subunit rRNA,
certain interesting revelations emerge.
There are two major groups, the pre-bilaterians and the bilateria, the
latter being subdivided into two major groups, the protostomes and
deuterostomes. Of the protostomes, in turn, there are two subgroups, the Lophotrochozoa,
to which the annelids and mollusks belong, and the Ecdysozoa to which the
arthropods and nematodes belong. Although it will not be discussed here, the
dominant body of evidence with respect to innate immunity and its relation to
vertebrate (mammalian) and plants concerns findings in two ecdysozoan species, Drosophila
and C. elegans. In contrast, lophotrochozoan systems show some distinct
differences and may have solved problems of immunodefense in a special manner
as has been revealed here with respect to two groups of annelids [3]. Annelids are among the first
coelomates and are therefore of special phylogenetic interest. Polychaetes
which we did not focus on in this review, are restricted to the marine domain
and are considered as the most primitive annelids, based on morphology,
physiology and development. Oligochaetes and achaetes are considered as being
more evolved as reflected in their immune systems. Compared to other
invertebrate groups, data on annelid’s immunity reveal heavier emphasis on the
cellular than on the humoral response suggesting that immune defense of annelids
seems to be principally developed as cellular immunity37. However,
the Expressed Sequence Tag (EST) sequencing programs on Lumbricus terrestris (LumbriBASE; http://www.earthworms.org and the leech Hirudo medicinalis (http://genome.uiowa.edu/projects/leech/), combined with
microarrays techniques developed on these models and imaging mass spectrometry
will allow acquisition of more information about leech and earthworm humoral
immunity. Our preliminary transcriptomic data obtained in leeches Table (2)
dealing with immune responses suggest that this strategy is feasible and that
substantial new knowledge will be obtained by analysing lophotrochozoans. This
will only lead to further expansion of the two existing schemes that have been
proposed concerning earthworm Fig. (3) and leech Fig. (4) immune
responses. In this expanse, we predict more relevant biomedical/clinical
applications further than what already exists.
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