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Department of Pharmacology, University of Bern, Bern, Switzerland
1Correspondence: Department of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. E-mail: hus{at}pki.unibe.ch
ABSTRACT
The regulation of cell death is a key element in building up and maintaining both innate and adaptive immunity. A critical role in this process plays the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family of death receptors. Recent work suggests that sialic acid binding immunoglobulin (Ig) -like lectins (Siglecs) are also empowered to transmit death signals, at least into myeloid cells. Strikingly, death induction by Siglecs is enhanced when cells are exposed to proinflammatory survival cytokines. Based on these recent insights, we hypothesize that at least some members of the Siglec family regulate immune responses via the activation of caspase-dependent and caspase-independent cell death pathways.—von Gunten, S., Simon, H.-U. Sialic acid binding immunoglobulin-like lectins (Siglecs) may regulate innate immune responses by modulating the life span of granulocytes.
Key Words: apoptosis • caspases • eosinophils • inflammation • neutrophils • non-apoptotic cell death • Siglecs
THE IMMUNE SYSTEM is tightly held in check to avoid autoreactivity and bystander tissue damage caused by overwhelming immune reactions. Elimination of cells by apoptosis is one important mechanism in this regulatory process (1)
. Apoptosis is mediated via two main routes, often referred to as the intrinsic and extrinsic pathways (2)
. The intrinsic pathway is triggered by cellular stress, for instance by survival factor withdrawal. The extrinsic pathway is induced by ligand-mediated activation of death receptors of the tumor necrosis factor (TNF)/nerve growth factor (NGF) family containing a functional death domain (DD) within their cytoplasmic region.
Both pathways converge into common mechanisms involving activation of cysteine-containing aspartate-specific proteases (caspases) (3)
. Caspases have been shown to cut multiple cellular substrates by limited proteolysis that results in either activation or inactivation of proteins involved in RNA splicing, DNA repair, maintenance of cell structure, and others. Although caspases are not the only proteases involved, their action in apoptosis is critical.
Besides death receptors, another family of receptors, called sialic acid binding immunoglobulin (Ig) -like lectins (Siglecs) (4)
, has recently been shown to be associated with cell death regulation of myeloid cells (5
6
7)
. The cytokine microenvironment appears to greatly influence the efficacy of death induction by Siglecs, suggesting they play a particular role in cell death regulation under inflammatory conditions. In this article, we would like to draw the attention of the reader to these new findings and hypothesize that Siglecs contribute to the limitation of innate immune responses by regulating the life span of neutrophils and eosinophils. Therefore, additional knowledge about the role of Siglecs under inflammatory conditions may provide important new insights for the pathogenesis and treatment of infectious, autoimmune, and allergic diseases.
SIGLECS: A BRIEF FAMILY PORTRAIT AND SOME EVOLUTIONARY ASPECTS
Siglecs are an evolutionary relatively young family and characterized by a homologous N-terminal V-set Ig-like domain and between one (CD33) and 16 (sialoadhesin) C2-set Ig-like domains (4
, 8
, 9)
. In humans, 11 members of the Siglec family have been identified: sialoadhesin (Siglec-1, CD169), CD22 (Siglec-2), the myelin-associated glycoprotein (Siglec-4, MAG), and additional members, which constitute a subgroup including CD33 (Siglec-3) and CD33-related Siglecs (Siglec-5 to -11) (8
, 10)
. Siglecs are found on the surface of cells of the hematopoietic system, with the exception of Siglec-4, which is also expressed by oligodendrocytes and Schwann cells in the nervous system (11)
. Whereas some family members are rather broadly expressed (e.g., Siglec-9) (12)
, others are restricted to one cell type (e.g., CD22 is selectively expressed by B cells) (13
, 14)
.
CD22 is the best-characterized member of the Siglec family and known to negatively regulate certain B cell responses (15)
. Like CD22, members of CD33-relatedsubgroup have also been shown to act as inhibitory receptors in various hematopoietic cells (4
, 8
, 9)
. Their inhibitory function is ascribed to the presence of conserved immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic regions. ITIMs have been implicated in the regulation of apoptosis (16
, 17)
. Therefore, it should not be too surprising that Siglecs, at least under certain conditions (see below), also control cell death.
It is assumed that Siglecs appeared once the sialic acid biosynthesis pathways had already been established (9)
. The Siglecs seem to have tailored their binding properties to the evolutionary changes that occurred in their oligosaccharide ligands. Such adaptations are present even in closely related species like humans and great apes. For instance, humans lack the sialic acid Neu5Gc due to a mutation in the CMP-Neu5Ac hydroxylase, which occurred after evolutionary divergence from great apes (18
, 19)
. As a consequence, whereas chimpanzee Siglec-11 shows robust binding to Neu5Ac, human Siglec-11 lost most of the binding capacity to this oligosaccharide ligand (20)
.
The rapid evolution of the Siglecs, which is thought to be due to extensive gene duplication and exon shuffling (8)
, has caused major differences in the genetic sequence of the various family members as well as among Siglecs in different species (9)
. Furthermore, the tissue expression of Siglecs varies between species. For instance, Siglec-11 expression in microglia cells is absent in chimpanzee, but gained prominent expression in humans (20)
. In addition, despite the homology of some human and mouse Siglecs, such as human Siglec-5 with mouse Siglec-F (49% sequence identity) or the human Siglecs-7, -8, and -9 all with mouse Siglec-E (53%, 54%, and 53% identity; ref 9
), one has to consider the large differences. Therefore, investigations in vivo using genetically modified mice will require careful and sophisticated strategies.
SIGLECS: A NEW FAMILY OF DEATH RECEPTORS?
Several groups recently reported the induction of apoptosis upon ligation of certain Siglecs. For instance, CD33 ligation resulted in apoptosis in leukemic cells of patients with acute myeloid leukemia (AML) (5)
. Moreover, ligation of Siglec-8 induced apoptosis in eosinophils (6)
, and activation of Siglec-9 triggered apoptosis in neutrophils (7)
. Caspase activation via Siglec-9 appeared to be somehow delayed compared with Fas-triggered apoptosis (7)
, perhaps because Siglecs do not contain a DD, which is able to immediately activate caspase-8. The absence of a DD in the cytoplasmic domain of Siglecs might also explain the delayed (at least compared with TNF/NGF family members) realization of their deadly potential.
If Siglecs do not directly activate caspases, then what is the mechanism of caspase activation? It was recently shown that Siglec-8 and -9-mediated apoptosis is largely dependent on reactive oxygen species (ROS) (7
, 21)
, which represent an evolutionary conserved machinery involved in cell death effector mechanisms. For instance, ROS involvement in programmed cell death has also been demonstrated in plants (22)
. Similarly, ROS are known to play a role in proapoptotic pathways in mammalian cells (23
, 24)
.
After ligation of Siglec-9, increased and sustained levels of ROS can be measured (Fig. 1
) (7)
. ROS involvement in Siglec signaling was demonstrated by experiments using ROS scavengers and inhibitors of the NADPH oxidase (7
, 21)
. Furthermore, neutrophils from chronic granulomatous disease (CGD) patients, cells that lack a functional NADPH oxidase (an enzyme that generates high levels of ROS in neutrophils), failed to undergo Siglec-9-induced apoptosis (7)
. The action of ROS in Siglec signaling was localized upstream of mitochondria and is thought to disrupt mitochondrial membrane integrity, causing the release of proapoptotic proteins such as cytochrome c (21)
. It is remarkable that the phylogenetically young Siglecs utilize this ancient ROS system for mediating the cell death function. The question, by which mechanism Siglec-9 (and probably other Siglecs) increases ROS production, however, remains to be addressed.
|
SIGLEC-8 AND -9: TURNING SURVIVAL FACTORS INTO KILLERS
It has been demonstrated that ligation of death receptors abolished the anti-apoptotic activity of several cytokines in neutrophils (16
, 19)
. Similar experiments were performed in which Siglec receptors were ligated in the presence of survival factors in both eosinophils and neutrophils. Strikingly, the survival factors IL-5 and GM-CSF considerably enhanced Siglec-8-mediated eosinophil apoptosis (6)
. Similarly, GM-CSF, which blocks proapoptotic pathways both proximal (25
26
27
28)
and distal (29)
to mitochondria (Fig. 1)
, enhanced cell death induced by Siglec-9 in neutrophils (7)
. Based on these data, at least Siglec-8 and -9-transduced signals appear to be especially cytotoxic in a scenario in which certain survival factors are present. Their raison d’être is most likely in disease, when cells are exposed to a proinflammatory microenvironment. Under these conditions, uncontrolled accumulation of inflammatory cells should be avoided by the activation of cytotoxic mechanisms in spite of concurrent survival factor signaling. This hypothesis is underscored by the finding that inflammatory neutrophils obtained from blood of septic patients or from joint fluids of rheumatoid arthritis patients are highly sensitive to Siglec-9-mediated cell death (7)
. Clearly, these studies expanded our knowledge on the cross-talk between survival and death signals that likely occurs in vivo (30)
.
The kind of Siglec-9-mediated neutrophil death occurring under proinflammatory conditions was largely caspase-independent (Fig. 1)
(7)
. Morphological investigations by transmission electron microscopy demonstrated the cytosolic presence of multiple vacuolar structures with dense inclusions, reminiscent of autophagic vacuoles (31)
. Apoptosis and autophagy may be interconnected, depending on the cellular setting, and inhibition of apoptosis may convert cell death morphology to autophagic, and vice versa (32)
. Other studies are required to gain a better understanding of the in vivo importance of Siglecs in switching these types of programmed cell death.
SIGLECS: MOLECULAR TOOLS CONTRIBUTING TO IMMUNE PRIVILEGE AND/OR TOLERANCE?
In the 1940s Sir Peter Medawar coined the term "immune privilege" based on the observation that skin and other types of grafts placed in special anatomical sites (anterior chamber of the eye and the brain) exhibited prolonged survival (33
, 34)
. Since that time, immune privilege was considered to be a phenomenon restricted to adaptive immunity. Recently, however, it was suggested that innate immune privilege may also exist (35)
. Experimental evidence on Siglecs exists that appears to support this notion. KSGal6ST is a sialotransferase required for the biosynthesis of 6'-sulfo-sLex, the putative ligand of Siglec-8 (see below). Based on the fact that this enzyme is expressed in the central nervous system, Bochner suggested that expression of the Siglec-8 ligand might participate in the elimination of inflammatory cells that have infiltrated the central nervous system (36)
. This is in analogy with the hypothesis that Fas ligand (FasL) expression at immunoprivileged sites protects from invading Fas-positive immunocompetent cells (37)
. Clearly, such FasL-Fas receptor interactions are certainly not the only way to mediate immune privilege (38
, 39)
, and Siglec-mediated death might be an additional mechanism by which these tissues are protected, at least from invading granulocytes (Fig. 2
).
|
Species-specific glycosylation patterns are a major obstacle to xenotransplantation. Hyperacute rejection of xenogenic porcine tissues is largely mediated by human natural antibodies directed against galactose-
-1,3-galactose (40)
. In view of a potential regulatory role of the Siglecs, several questions remain to be answered. For instance, it is unclear whether insufficient expression of cognate Siglec ligands leads to hypersensitive immune responses in xenogenic tissue by breaking human-specific innate tolerance. Another important question is whether subversion of Siglec function by microorganisms could constitute an immune evasion mechanism (4
, 8)
. This suggestion has been based on the observation that some bacterial pathogens have acquired sophisticated strategies to synthesize sialic acids and to present them at their surface. Such a mechanism might override the initial defense by the innate immune system leading to infestation of the host and expansion of the pathogens.
THE SEARCH FOR FUNCTIONAL SIGLEC LIGANDS
Sialic acid is a generic term for a large family of 9-carbon sugars that all derivate from neuraminic acid (Neu) or keto-deoxynonulosonic acid (KDN). The two major sialic acids in mammalians are N-acetylneuraminic acid (Neu5Ac) and N-glycolyl-neuraminic acid (Neu5Gc), which are abundantly expressed on secreted and cell-bound glycoproteins and glycolipids. Siglecs bind the terminal sialic acids on such glycoconjugates, each member recognizing different oligosaccharide structures as preferred ligands. Recognition is influenced by the nature of sialic acid, its linkage to substituted sugars, and the underlying neutral oligosaccharides (4
, 8)
.
At present, one experimental limitation is caused by the lack of the physiological functional ligands for CD33-related Siglecs. Therefore, functional studies in human primary cells are currently restricted to cross-linking approaches using monoclonal antibodies. However, recent studies in this active field of research provide some clues regarding potential Siglec ligands. Members of the CD33-related Siglec subgroup have been shown to bind with variable selectivity to the gangliosides GT1b, GQ1b, GD3, GM2, GM3, and GD1a (41)
. In another approach, 10 Siglec-Ig chimeras were screened for binding to 28 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids (42)
. Such binding studies are now being facilitated by The Consortium for Functional Glycomics (www.functionalglycomics.org) funded by the National Institutes of General Medical Sciences to support research commitment on glycan binding proteins.
In cooperation with this Consortium, 6'-sulfo-sLex has been identified as a ligand for Siglec-8 (36)
, a structure closely related to 6-sulfo-sLex, which is a candidate ligand for Siglec-9 (www.functionalglycomics.org). It is hoped that more complete information on the nature and the expression patterns of the ligands will provide decisive insights into how Siglecs regulate the immune system.
CONCLUSIONS
Previous work suggested that Siglecs control immunity by acting as inhibitory receptors. Recent data demonstrated that at least some members of the Siglec family are also able to transduce death signals in myeloid cells. Death induction via these Siglecs occurs in spite of concurrent survival factor signaling. The death induced under these conditions is highly efficient but exhibits caspase independence in both eosinophils and neutrophils. Moreover, in neutrophils the type of death is at least related to autophagic cell death and appears to be relevant in inflammatory diseases, such as sepsis and rheumatoid arthritis. Still in its infancy, Siglec research shares the dilemma common to all fields: the more that is known, the more questions arise. A substantial number of remaining questions need to be addressed by orchestrated engagement in various fields such as immunology, glycobiology, and cell death research. However, the goal is worth such an effort. Based on an understanding of how Siglecs contribute to the regulation of the immune system, novel therapeutic strategies could be developed in the future.
The laboratory of the authors is supported by the Swiss National Science Foundation (grant no. 310000-107526), the Bonizzi Theler Foundation (Zurich), the OPO Foundation (Zurich), and the Foundation for Supporting Research at the University of Bern (Bern).
Received for publication November 1, 2005. Accepted for publication December 8, 2005.
REFERENCES
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