Siglecs (sialic acid-binding Ig-like lectins) are mainly expressed in the immune system. Sn (sialoadhesin) (siglec-1), CD22 (siglec-2) and siglec-15 are well conserved, whereas the CD33-related siglecs are undergoing rapid evolution, as reflected in large differences in repertoires among the different mammals studied so far. In the present paper, we review recent findings on the signalling properties of the CD33-related siglecs and discuss the emergence of both inhibitory and activating forms of this family. We also discuss how Sn may function as a positive regulator of adaptive immune responses and its emerging role as an induced macrophage pattern-recognition molecule for sialylated pathogens, especially enveloped viruses.
- innate immune system
- sialic acid
Siglecs (sialic acid-binding Ig-like lectins) are members of the Ig superfamily characterized by their specificity for sialic acids attached to the terminal regions of cell-surface glycoconjugates. These type 1 transmembrane proteins comprise a sialic acid-binding N-terminal V-set domain, variable numbers of C2-set Ig domains, a transmembrane region and a cytosolic tail. On the basis of their sequence similarities and evolutionary conservation, two primary subsets of siglecs have been identified: the first subset includes Sn (sialoadhesin) (siglec-1), CD22 (siglec-2), MAG (myelin-associated glycoprotein) (siglec-4) and siglec-15, all of which are well-conserved in mammals. The second, rapidly evolving, subset is designated the CD33-related siglecs. In humans, these include CD33 and siglecs-5, -6, -7, -8, -9, 10, -11, -14 and -16, whereas, in mice, they comprise murine CD33 and siglecs-E, -F, -G and -H. With the exception of MAG, which is expressed in the nervous system, siglecs are differentially expressed on various subsets of leucocytes where they play a role in the positive and negative regulation of immune and inflammatory responses as discussed in recent reviews [1,2]. The present mini-review focuses on ITIM (immunoreceptor tyrosine-based inhibitory motif)-bearing CD33-related siglecs as negative immunoregulators and endocytic receptors and on the more recently discovered CD33-related siglecs that lack ITIMs, but associate with the ITAM (immunoreceptor tyrosine-based activating motif)-containing adaptor, DAP-12 (12 kDa DNAX-activating protein) which is implicated in both positive and negative immunoregulation [3,4]. We also discuss the role of Sn (which lacks tyrosine-based motifs) as a positive regulator of the immune system and a target for sialylated enveloped viruses and other sialylated pathogens.
Phosphorylation, ubiquitination and internalization: CD33-related siglecs as negative immunomodulators and endocytic receptors
The CD33-related siglecs are thought to play important roles in modulating leucocyte function by inhibiting cellular activation and proliferation, inducing apoptosis, modulating cytokine secretion and mediating endocytosis. As negative immunoregulators, their cytoplasmic tails harbour ITIMs and ITIM-like motifs. These motifs undergo phosphorylation by Src family tyrosine kinases, creating high-affinity binding sites for several SH2 (Src homology region 2)-domain-containing signalling molecules. The best characterized of these are SHP (SH2-domain-containing protein tyrosine phosphatase)-1 and SHP-2, which have been known for many years to be recruited to multiple ITIM-bearing inhibitory receptors, leading to ITIM dephosphorylation and the dampening of ITAM-dependent signalling . Although tyrosine phosphorylation is very important for triggering these responses, earlier work from our group demonstrated that even in the absence of tyrosine phosphorylation, the CD33-related siglec-5 was able to recruit SHP-1 weakly and trigger inhibitory signalling functions . Recently, Orr et al. [7,8] and Walter et al.  have shown that tyrosine-phosphorylated CD33 can recruit two other SH2-domain-containing proteins, SOCS3 (suppressor of cytokine signalling 3) and Cbl, which may result in polyubiquitination or mono-ubiquitination respectively. SOCS3-dependent polyubiquitination leads to proteasomal degradation, whereas Cbl-mediated mono-ubiquitination is thought to increase the rate of endocytosis and post-internalization into multivesicular bodies, thus removing it from the cell surface. Furthermore, by acting as adaptor proteins, SHP-1/SHP-2 binding to CD33 regulates its phosphorylation, its ubiquitination and its internalization, and ultimately lowers the rate of CD33 endocytosis which is also contributed to by the inefficient recruitment of the endocytic machinery to its cytoplasmic tail (Figure 1). These various SH2-domain-containing molecules are likely to compete with each other for phosphotyrosine binding and it remains to be established what factors determine the relative amounts of each of these that bind and hence the eventual outcome. This could include the relative affinities of SH2 domains for the phosphorylated ITIM and ITIM-like motifs that could differ between individual CD33-related siglecs and the relative intracellular concentrations of each molecule. For example, strong up-regulation of SOCS3 by inflammatory signals could tip the balance in favour of its binding, as opposed to SHP-1 and/or SHP-2 which are constitutively expressed in haemopoietic cells. These issues are also highly relevant for current leukaemia treatment strategies based on CD33 expression, since this molecule is expressed on immature and mature myeloid cells, B-cell subsets, activated T-cells and NK (natural killer) cells. Its expression on malignant cells such as those in AML (acute myeloid leukaemia), together with its internalization, has led to its exploitation in the targeted delivery of the immunotoxin, Gemtuzumab . Other siglecs such as siglec-9 are also expressed on subsets of AML and may provide additional therapeutic targets in the future [11,12].
Although most CD33-related siglecs potentially inhibit immune response activation by dampening ITAM-dependent signals or inducing apoptosis, they may also do so by enhancing the production of anti-inflammatory cytokines and suppressing the production of pro-inflammatory cytokines. A recent publication showed that overexpression of siglec-9 in macrophage cell lines was seen to inhibit production of pro-inflammatory cytokines such as TNFα (tumour necrosis factor α) and enhance the production of the anti-inflammatory cytokine IL (interleukin)-10 in an ITIM-dependent manner in response to Toll-like receptor signalling  (Figure 1).
Pathogen-induced evolution of siglecs as activating immunomodulators?
Unlike most other siglecs, human siglecs-14, -15 and -16, as well as murine siglec-H, lack ITIMs and interact instead via a positively charged residue in their transmembrane domain with DAP-12 which is an ITAM-containing receptor that can trigger both activating and inhibitory signalling [14–16] (Figure 1). Siglecs-14 and -16 are highly related to siglecs-5 and -11 respectively and are thought to have arisen from the corresponding genes by gene conversion. Similarly to other ‘paired’ receptors , it is tempting to speculate that this putative switch from negative to positive immunomodulation may serve as an adaptive evolutionary strategy of the host to counteract the manipulation of inhibitory siglecs by sialylated pathogens such as Campylobacter jejuni, Neisseria gonorrhoea, Group B Streptococci and Neisseria meningitidis [18–20]. Thus expression of these DAP-12-coupled siglecs in the innate immune system may contribute to host resistance by triggering cellular activation in response to sialylated pathogens. However, the DAP-12-coupled siglec-H which is predominantly expressed in murine plasmacytoid dendritic cells is able to inhibit production of IFN (interferon)-α when cross-linked with antibodies. The degree to which siglecs-14, -15 and -16 mediate activating or inhibitory signalling in the human immune system remains to be determined.
Sn as an immunomodulator and target for pathogens
Sn is expressed at high levels on discrete subsets of tissue macrophages, particularly those found in secondary lymphoid tissues . High expression is also seen in chronic inflammatory diseases such as rheumatoid arthritis , atherosclerosis  and models of inherited demyelinating diseases of the nervous system . In contrast with the CD33-related siglecs, there is currently little evidence that Sn mediates signalling functions via its transmembrane tail or cytoplasmic region which lacks obvious signalling motifs and is poorly conserved between mammalian species (Figure 1). Although antibody-mediated Sn cross-linking of porcine alveolar macrophages was shown recently to be associated with subtle alterations in MAPK (mitogen-activated protein kinase) and Wnt signalling, intact antibody was used that could have triggered Fc receptor-dependent signalling . The extracellular region of Sn is well-conserved, consisting of 17 Ig-like domains. Via its distal N-terminal domain, Sn can bind promiscuously to many sialylated glycoconjugates, with a preference for O-linked oligosaccharides terminating in Neu5Acα2,3Galβ1,3GalNAc. Its affinity for sialosides is low (10−3 M range), and high-avidity binding requires receptor and ligand clustering. This low affinity, together with a highly extended extracellular domain are key features in permitting Sn to mediate cell–cell interactions, particularly in the plasma microenvironment where the poorly clustered glycans of plasma glycoproteins are unable to compete efficiently with the highly clustered cell-associated glycans involved in avid Sn-binding. Furthermore, cell–cell and cell–matrix interactions are accentuated further by the extension of the N-terminal V-set domain beyond the reach of shorter cis-interacting inhibitory siglecs closer to the plasma membrane (Figure 1). Although the expression and features of Sn could potentially influence many macrophage cellular interactions relating to homoeostasis and immunity , evidence based on studies of Sn-deficient mice suggests that these may be more important in the regulation of adaptive immune responses. As reviewed recently , Sn-deficient mice exhibit reduced CD4+ T-cell and inflammatory responses in a model of autoimmune uveoretinitis and reduced CD8+ T-cell and macrophage recruitment in models of inherited demyelinating neuropathy in both the central and peripheral nervous systems [26,27]. Subtle effects on T-cells and IgM antibody levels were also seen in Sn-deficient mice kept under specific pathogen-free conditions . The sialomucin CD43 on T-cells  and MUC-1 (mucin 1)  on breast cancer cells have both been identified as putative Sn counter-receptors (Figure 1).
Recently, IFNα, a potent antiviral cytokine and immune modulator, was shown to induce Sn expression in monocytes which normally do not express the receptor and also to increase Sn expression in macrophages . IFNγ produced by activated T-cells and NK cells has also been shown to induce Sn expression on monocytes [22,32]. The induction of Sn expression in cells of the monocyte–macrophage lineage by IFNs may play a role in the potentiation of inflammatory diseases including rheumatoid arthritis (where Sn serves as a restricted inflammatory marker for tissue macrophages), systemic sclerosis , SLE (systemic lupus erythematosus)  and proliferative glomerulonephritis . Monocytes play an important role in the pathophysiology of SLE where their accelerated apoptosis and subsequent defective clearance leads to the generation of autoantibody responses and the presentation of auto-antigens to CD4+ T-cells in response to IFNα stimulation. This has been reinforced by recent data showing a correlation between the frequency of circulating Sn+ monocytes and the titres of anti-dsDNA (double-stranded DNA) auto-antibodies, indicating that this expression of Sn closely parallels disease activity . Furthermore, treatment of SLE patients with glucocorticoids resulted in a strong decrease in Sn+ monocytes, suggesting that Sn may be a useful biomarker for disease monitoring in response to therapeutic treatments.
In addition to its role in cell–cell interactions, Sn has also been shown to facilitate pathogen interactions. For example, Sn can promote macrophage uptake of sialylated strains of Neisseria meningitidis  and functions in endocytosis of the macrophage/monocyte-tropic PRRSV (porcine reproductive and respiratory syndrome virus) [35,36] (Figure 1). Viral entry occurs by receptor-mediated endocytosis via clathrin-coated pits and vesicles, and is mediated by an initial attachment to the glycosaminoglycan heparan sulfate which concentrates virions at the cell surface. Internalization is thought to be triggered via interactions between Sn and sialylated N-linked glycans present on the four structural viral glycoproteins GP2–GP5 of PRRSV [37,38]
Very recently, another pathogen shown to interact with Sn is HIV-1 . During the acute period of HIV-1 infection, IFNγ is produced by NK cells and T-cells, and IFNα is released by pDCs (plasmacytoid dendritic cells) as part of the antiviral response (Figure 1). This may lead to induction of Sn on monocytes, which in turn binds avidly to the virus in a sialic acid-dependent manner. This may permit the effective trans-infection of permissive cells and the delivery and distribution of HIV-1 to target cells in the periphery. Hence Sn may be involved in both capture of free virus and engagement of sialylated glyconjugates on target cells such as T-cells, overcoming the electrostatic repulsion between sialylated virions and sialylated target cell surfaces, and enhancing the binding of HIV-1 gp120 (glycoprotein 120) to its cognate receptors .
The recent discoveries of newly evolved members of the human CD33-related siglec group such as siglec-14 and -16 have revealed the existence of paired inhibitory and activating receptors within this gene family. Although the primordial functions of the CD33-related siglecs are likely to be in ITIM-dependent dampening of immune responses and endocytosis, the emergence of ITAM-coupled paired receptors points towards a counter-strategy of the host towards sialylated pathogens. Clearly there is an intricate interplay between pathogens and the multiple immune receptors that determines the outcome of the immune response, and siglecs are part of this complex network that also includes many other lectin-like receptors. In contrast with the CD33-related siglecs, Sn is a well-conserved siglec whose primary function is likely to be in positive regulation of adaptive immune responses via cell–cell interactions with target cells. Superimposed on this, evidence is now emerging that this extended and abundant macrophage receptor can provide a target for sialylated enveloped viruses that promote infection. More work is needed to see whether Sn can also provide host protective functions towards sialylated pathogens, including bacteria and enveloped viruses such as HIV. It will also be of interest to determine whether the IFNα-dependent induction of Sn has evolved primarily to promote macrophage–host cell interactions in adaptive immunity or whether this is a determining factor in host resistance and/or susceptibility to certain sialylated pathogens.
23rd International Lectin Meeting (Interlec-23): Independent meeting held at Universities of Edinburgh and Stirling, Scotland, U.K., 11–16 July 2008. Organized and Edited by Dave Kilpatrick (Scottish National Blood Transfusion Service, National Science Laboratory, Edinburgh, U.K.).
Abbreviations: AML, acute myeloid leukaemia; DAP-12, 12 kDa DNAX-activating protein; IFN, interferon; ITAM, immunoreceptor tyrosine-based activating motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; MAG, myelin-associated glycoprotein; NK, natural killer; pDC, plasmacytoid dendritic cell; PRRSV, porcine reproductive and respiratory syndrome virus; SH2, Src homology region 2; SHP, SH2-domain-containing protein tyrosine phosphatase; siglec, sialic acid-binding Ig-like lectin; SLE, systemic lupus erythematosus; Sn, sialoadhesin; SOCS3, suppressor of cytokine signalling 3
- © The Authors Journal compilation © 2008 Biochemical Society