Glycobiology of leukocyte trafficking in inflammation.

Glycobiology Advance Access published September 25, 2014

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Glycobiology of Leukocyte Trafficking in Inflammation

Rachael D. Wright1 and Dianne Cooper1,2 1

William Harvey Research Institute, Barts and the London School of Medicine

and Dentistry, London, UK 2

To whom correspondence should be addressed: Dianne Cooper, William

Dentistry, London, EC1M 6BQ, United Kingdom. Tel: +44 207 882 5644; Fax: +44 207 882 6076; e-mail: [email protected]

© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e‐mail: [email protected]

Downloaded from http://glycob.oxfordjournals.org/ at Yale University on September 30, 2014

Harvey Research Institute, Barts and the London School of Medicine and

2

Abstract

To fulfill their potential, leukocytes must be able to exit the vasculature and reach the site of inflammation within the tissue. This process of leukocyte extravasation is a tightly regulated sequence of events that is governed by a host of cell adhesion molecules, cytokines, chemokines and lipid mediators.

and lipids involved is the post-translational modification of these moieties by glycosylation. The glycosylation process is co-ordinated by multiple enzymes that add and remove saccharides to/from glycan structures on proteins and lipids resulting in a unique molecular signature that affords specificity to the molecules involved in leukocyte recruitment. This review will discuss how glycosylation impacts the function of these key molecules involved in the recruitment of leukocytes during inflammation and the function of specific lectins (carbohydrate binding proteins) that have a role in leukocyte trafficking.

Key Words: Galectin, Glycosylation, Leukocyte, Trafficking, Inflammation


Of major importance to this process and the function of many of the proteins

3 Introduction Inflammation is a protective response of the host to tissue injury. Leukocyte trafficking from the bloodstream to the inflammatory foci within the tissue is fundamental to mounting a successful inflammatory response, evidenced by the recurrent infections and poor survival rate of patients suffering from leukocyte adhesion deficiencies, a class of conditions in which neutrophil trafficking is compromised (Hanna, S. and Etzioni, A. 2012).

Leukocyte trafficking is initiated by the capture of free flowing cells in the blood by the activated endothelium, a process that is mediated by the selectin family of adhesion molecules and their counter ligands. Once captured a proportion of these cells will roll along the vessel wall, again a process that is mediated predominantly by selectins. This rolling allows leukocytes to sample the local endothelial microenvironment and come into contact with surface bound chemokines. Inside-out signaling triggered by ligation of chemokine receptors combined with the downstream signals from selectin binding results in a conformational change in leukocyte integrins such that they are able to bind members of the Ig superfamily of adhesion molecules (ICAM-1, VCAM-1) expressed by the endothelium. Interactions between integrins and their respective ligands result in firm adhesion of the leukocytes on the vessel wall. Although termed firm adhesion, the leukocytes are still dynamic at this stage of the process and are able to crawl along the endothelium in search of a permissive site to exit the blood vessel and enter the tissue. For an excellent


Leukocyte Trafficking in Inflammation

4 review detailing the molecular determinants of leukocyte recruitment, readers are directed to (Ley, K., Laudanna, C., et al. 2007).

The soluble mediators and adhesion molecules that drive the leukocyte recruitment process have been investigated thoroughly over the past two decades and much knowledge has been gained on their function (Kunkel, E.J., Dunne, J.L., et al. 2000, Ley, K., Laudanna, C., et al. 2007, Petri, B.,

A.J., et al. 2013, Woodfin, A., Voisin, M.B., et al. 2010). An exciting, more recent, aspect of this research is focused on understanding how the posttranslational glycosylation of many of the molecules involved in this process adds an additional level of control to this system. Glycosylation is an enzymatic process that results in the addition of saccharides to proteins, lipids and other saccharides resulting in an abundant range of glycans. These glycans have many important functions throughout biology. In the following pages the impact of glycosylation on the process of leukocyte recruitment during inflammation will be addressed with a particular focus on myeloid cell recruitment during acute inflammation. The role of glycan-binding proteins, such as galectin-1, in this response will also be addressed.

Leukocyte Capture and Rolling Leukocyte recruitment begins with the capture and subsequent rolling of leukocytes on the endothelium, a process that relies predominantly on the selectin family of C-type (calcium-dependent) lectins (Jung, U. and Ley, K. 1999). Three selectins have been identified in mammals and are expressed


Phillipson, M., et al. 2008, Sanz, M.J. and Kubes, P. 2012, White, G.E., Iqbal,

5 on endothelial cells (E-selectin; CD62E), platelets and endothelial cells (Pselectin; CD62P) and leukocytes (L-selectin; CD62L) (McEver, R.P. 2002). The endothelial selectins are not constitutively expressed; P-selectin is rapidly mobilized from Weibel-Palade bodies upon stimulation whilst E-selectin is regulated at the transcriptional level by inflammatory cytokines such as TNF- and IL-1 (Bevilacqua, M.P., Stengelin, S., et al. 1989, Eppihimer, M.J., Wolitzky, B., et al. 1996, Geng, J.G., Bevilacqua, M.P., et al. 1990, Hattori, R.,

upon the surface of most leukocytes and is generally shed upon leukocyte activation (Griffin, J.D., Spertini, O., et al. 1990, Jutila, M.A., Rott, L., et al. 1989). The counter-receptors for selectins are typically heavily glycosylated molecules, many of which bear terminal sialyl Lewis x (SLex) motifs (NeuAc2,3-Gal-1,4-(Fuc-1,3)-GlcNAc), production of which requires the concerted action of several glycosyltransferases, sialyltransferases and fucosyltransferases. The ligands for E- and P-selectin are expressed on circulating leukocytes whereas L-selectin binds to ligands on both leukocytes and the endothelium. As the focus of this review is myeloid cell trafficking during inflammation the role of the inflammatory selectins (E- and P-selectin) and the glycosylation of their respective receptors will be considered within. Lselectin, which has a fundamental role in lymphocyte homing will not be covered in detail here; for a recent review on the role of L-selectin in leukocyte recruitment the reader is guided to (Wedepohl, S., Beceren-Braun, F., et al. 2012).


Hamilton, K.K., et al. 1989). In contrast, L-selectin is constitutively expressed

6 Three physiological selectin ligands have been identified on murine neutrophils with P-selectin glycoprotein ligand-1 (PSGL-1) being the major ligand bound by all three selectins (it is the dominant ligand for P-selectin and it co-operates with other ligands for rolling on E-selectin) (Norman, K.E., Katopodis, A.G., et al. 2000, Norman, K.E., Moore, K.L., et al. 1995, Sreeramkumar, V., Leiva, M., et al. 2013). Core 2 O-linked glycans on the Nterminus of PSGL-1 are important for high affinity binding of all three selectins

optimal binding to P-selectin (Westmuckett, A.D., Thacker, K.M., et al. 2011). O-glycan synthesis is initiated by the enzyme polypeptide Nacetylgalactosamine (GalNAc) transferase (ppGalNAcT), which covalently links GalNAc to threonine or serine residues in the protein backbone. ppGalNAcT subsequently plays a role in leukocyte trafficking both homeostatically and during inflammation by providing a scaffold for generation of selectin ligands. Mice lacking this enzyme exhibit impaired P- and Eselectin dependent rolling and increased rolling velocity, which translates into reduced levels of adhesion and transmigration due to reduced expression of appropriately glycosylated selectin ligands by circulating neutrophils (Block, H., Ley, K., et al. 2012, Tenno, M., Ohtsubo, K., et al. 2007).

O-glycan elongation progresses through the action of core 1 and core 2 transferases; core 1 -1,3-galactosyltransferase (T-synthase) forms the core 1 backbone, which is then elongated to form extended core 1 structures or branched to form core 2 structures. Core 2 O-glycans required for selectin binding to PSGL-1 are generated by core 2 -1,6-N-


whilst sulfation of tyrosine residues near the N-terminus is also required for

7 acetylglucosaminyltransferase-1 (C2GnT-1); mice lacking this enzyme exhibit a mild neutrophilia, significant deficiencies in binding of P- and L-selectin and correspondingly impaired neutrophil trafficking (Ellies, L.G., Tsuboi, S., et al. 1998). Specifically, P-selectin dependent leukocyte rolling is severely diminished in C2GnT-1 null mice whereas E-selectin-dependent rolling is only partially reliant on C2GnT-1 indicating a role for other E-selectin ligands (Sperandio, M., Thatte, A., et al. 2001).

galactosyltransferases (4GalT) resulting in the synthesis of poly-Nacetyllactosamines and the generation of SLex in collaboration with -2,3sialyltransferase and -1,3-fucosyltransferase. Seven isoforms of 4GalT have been isolated but studies in 4GalT-I-deficient mice have identified that it is this isoform that is chiefly responsible for selectin ligand biosynthesis with reduced binding of soluble P-selectin to neutrophils and monocytes of these animals (Asano, M., Nakae, S., et al. 2003). These mice have neutrophilia and leukocytosis and reduced neutrophil recruitment in a model of zymosaninduced dermatitis indicating a defect in neutrophil trafficking in these animals (Asano, M., Nakae, S., et al. 2003). The importance of this enzyme in myeloid cell recruitment was further corroborated in a skin wound healing model with reduced neutrophil and macrophage recruitment in 4GalT-I-deficient mice (Mori, R., Kondo, T., et al. 2004).

Addition of sialic acid in an -2,3 linkage is a critical component of selectin ligands. Six sialyltransferases in the mammalian genome can generate the -


Core 2 O-glycans are extended through the action of -1,4-

8 2,3 linkage required for selectin binding and three of these (ST3Gal-III, -IV and -VI) sialylate type II oligosaccharides that can be processed to form SLex. Investigations in mice lacking sialyltransferases have identified a role for ST3Gal-IV in generating ligands for P- and E-selectin, however treatment of neutrophils from ST3Gal-IV null mice with sialidase further reduces selectin binding suggesting that other sialyltransferases may also generate functional selectin ligands (Ellies, L.G., Sperandio, M., et al. 2002). There is evidence to

human neutrophils on E-selectin with inhibition of glycolipid synthesis impairing rolling on E-selectin in vitro (Nimrichter, L., Burdick, M.M., et al. 2008), although their contribution to rolling in vivo is currently unknown.

The final step in synthesis of selectin ligands is fucosylation, which in leukocytes is performed by either -1,3-fucosyltransferase-IV (FucT-IV) or FucT-VII. Mice deficient in FucT-VII, the rate-limiting enzyme for sLex synthesis, show a loss of all selectin ligands and subsequent defects in neutrophil trafficking in a model of thioglycollate-induced peritonitis (Malý, P., Thall, A., et al. 1996). Some high velocity rolling is still apparent in FucT-VII null mice but this is abolished in FucT-IV null and FucT-IV/VII double knockout mice (Homeister, J.W., Thall, A.D., et al. 2001, Weninger, W., Ulfman, L.H., et al. 2000). Antibody blocking studies confirmed that FucT-VII generated ligands control tethering efficiency through interaction with P-selectin whilst both enzymes influence rolling velocity predominantly through fucosylation of E-selectin ligands (Weninger, W., Ulfman, L.H., et al. 2000). The importance of fucosylation in leukocyte trafficking is underscored in patients suffering from


suggest that sialylated glycosphingolipids may also play a role in rolling of

9 leukocyte adhesion deficiency II (LADII). These patients have a deletion in the GDP-fucose transporter gene resulting in a lack of selectin ligands which manifests as recurrent infection, persistent leukocytosis and mental and growth retardation (Etzioni, A., Frydman, M., et al. 1992, Frydman, M., Etzioni, A., et al. 1992).

Whilst PSGL-1 has been identified as the major receptor for P-selectin, E-

neutrophils (Katayama, Y., Hidalgo, A., et al. 2005, Levinovitz, A., Mühlhoff, J., et al. 1993, Norman, K.E., Katopodis, A.G., et al. 2000, Yang, J., Hirata, T., et al. 1999). Neutrophils lacking either ESL-1, CD44 or PSGL-1 only show partial defects in E-selectin binding underscoring co-operativity between ligands (Hidalgo, A., Peired, A.J., et al. 2007). A recent study (Yago, T., Fu, J., et al. 2010) has uncovered an essential role for core 1 O-glycans in Eselectin binding using mice lacking the enzyme -1,3-galactosyltransferase (T-synthase), which forms the core 1 backbone Gal1,3GalNAc1-Ser/Thr, specifically in cells of the hematopoietic system. As was seen for mice lacking core 2 O-glycans, these mice have neutrophilia and defects in neutrophil trafficking. Specifically, T-synthase null neutrophils failed to bind to E-selectin to a greater degree than was observed for C2GnT-1 null cells indicating binding to O-glycans other than core 2. Neutrophil tethering and rolling on Eselectin in vitro was also severely impaired as it was in TNF- inflamed venules in vivo. Ultimately T-synthase null neutrophils failed to transmigrate into the peritoneum following challenge with thioglycollate with a comparable response to that observed in mice lacking all three selectins indicating its


selectin has been shown to bind PSGL-1, ESL-1 and CD44 on murine

10 absolute requirement for synthesis of selectin ligands (Yago, T., Fu, J., et al. 2010).

In addition to binding core 1 and 2 O-glycans on its ligands, E-selectin binds fucosylated N-glycans on both CD44 and ESL-1 as demonstrated by retention of rolling on E-selectin following removal of all surface O-glycans, which again underscores how glycosylation is used to achieve discrete differences in

recruitment in different vascular beds (Katayama, Y., Hidalgo, A., et al. 2005, Lenter, M., Levinovitz, A., et al. 1994, Levinovitz, A., Mühlhoff, J., et al. 1993, Steegmaier, M., Levinovitz, A., et al. 1995). Figure 1 indicates the different selectin ligands and their glycosylation patterns on neutrophils.

The above studies indicate a role for glycobiology in guiding the inflammatory response, with glycosylation of selectin receptors imperative for the initial capture and rolling of neutrophils during inflammation. However, much of the data accrued is derived from acute models of inflammation in knockout mice, with little information gained using human cells or tissues or in complex animal models that more closely mimic inflammatory pathologies. It would be of interest to determine how glycosylation patterns are altered in chronically inflamed tissues and whether this is as a result of changes in enzyme expression levels or activity. Lessons can be learnt from the field of cancer biology where it is known that selectin ligand expression correlates with cancer progression, with elevated levels of glycosyltransferases detected in cancer patients (Häuselmann, I. and Borsig, L. 2014).


ligand binding that translate into subtle differences in leukocyte subtype

11

Firm Adhesion Slow rolling on E-selectin allows leukocytes to be exposed to surface bound chemokines that, through interaction with specific GPCRs on the leukocyte surface results in integrin activation and increased affinity for binding to cognate receptors on the endothelium. There is evidence to suggest that glycosylation is also important at this stage of leukocyte recruitment, both in

(Diamond, M.S., Staunton, D.E., et al. 1991, Döring, Y., Noels, H., et al. 2014, Frommhold, D., Ludwig, A., et al. 2008, Ludwig, A., Ehlert, J.E., et al. 2000, Sriramarao, P., Berger, E., et al. 1993).

In contrast to leukocyte capture and rolling, where O-glycosylation plays a major role in leukocyte trafficking through modification of proteins on the leukocyte, N-glycan synthesis is thought to play a role in modulating firm adhesion through modification of endothelial expressed molecules. Human ICAM-1 contains eight N-glycosylation sites (Bloom, J.W., Madanat, M.S., et al. 1996) and the size and complexity of N-linked glycans on ICAM-1 regulates binding to its integrin ligands, Mac-1 and LFA-1. Mac-1 binds with higher avidity to molecules of ICAM-1 with smaller N-linked oligosaccharide side chains, produced following treatment with the -mannosidase inhibitor deoxymannojirimicin (DMJ). A functional effect of this was demonstrated by increased neutrophil adhesion to DMJ-treated HUVEC (Sriramarao, P., Berger, E., et al. 1993). However, unlike Mac-1, LFA-1 binds more favourably if ICAM-1 has a more complex carbohydrate side chain (Diamond, M.S.,


terms of adhesion molecule interactions and responsiveness to chemokines

12 Staunton, D.E., et al. 1991) again illustrating how discrete changes in glycosylation could lead to subtle changes in leukocyte recruitment. VCAM-1 potentially contains multiple N-glycan sites and is -2,6 sialylated in response to pro-inflammatory cytokine stimulation (Hanasaki, K., Varki, A., et al. 1994), although a later study determined that although endothelial sialylation inhibited VCAM-1-dependent leukocyte adhesion under flow, this was not due to sialylation of VCAM-1 itself (Abe, Y., Smith, C.W., et al. 1999).

leukocytes, a further activation step is required for exposure of the active epitope of integrins. This activation is mediated by chemokines presented on the endothelial cell surface (again by glycans such as heparan sulphate (Middleton, J., Patterson, A.M., et al. 2002)) that interact with specific GPCRs on the leukocyte leading to arrest and spreading of the cell. Glycosylation of chemokine receptors has been shown to protect the receptor from proteolytic attack, as is the case for CXCR2 on the surface of neutrophils (Ludwig, A., Ehlert, J.E., et al. 2000), as well as increasing the binding affinity of the chemokine for the receptor as shown for CXCR4 and CCR5 (Bannert, N., Craig, S., et al. 2001, Zhou, H. and Tai, H.H. 1999). Specifically, sialylation has been found to be key for binding of CCL3 and CCL4 to CCR5 leading to further studies investigating the sialyltransferase, ST3Gal-IV, responsible for this modification. Studies in ST3Gal-IV null mice, which have mild defects in selectin-dependent leukocyte rolling, have also revealed an important role for sialylation in chemokine-induced neutrophil arrest (Döring, Y., Noels, H., et al. 2014, Frommhold, D., Ludwig, A., et al. 2008). Intravital microscopy studies


In conjunction to the adhesion molecules required for firm adhesion of

13 demonstrated that CXCR2-mediated firm adhesion is significantly impaired in the venules of ST3Gal-IV mice both following surgery-induced injury and in response to systemic administration of the CXCR2 ligands CXCL1 and CXCL8. The downstream effect of this deficit was a severe impairment in neutrophil (and eosinophil and monocyte) extravasation in response to TNF- stimulation, indicating the importance of ST3Gal-IV for a functional CXCR2 response (Frommhold, D., Ludwig, A., et al. 2008). A recent study has

Y., Noels, H., et al. 2014) with reduced arrest and migration of classical monocytes and neutrophils from St3Gal-IV null mice in response to CCL5. The impact of this defect was indicated following crossing of the St3Gal-IV mice with the atherosclerosis susceptible Apoe-/- mouse; plaque development and leukocyte infiltration into aortic lesions were reduced in the double knockout (St3GalIV-/-Apoe-/-) mice. It was proposed that sialylation either induces favourable conformational changes in CCL5 receptors or enforces electrostatic interactions of basic chemokine residues with negatively charged sialic acids attached to chemokine receptors which results in efficient CCL5 binding (Döring, Y., Noels, H., et al. 2014). Figure 2 indicates the enzymes involved in the glycosylation of chemokine receptors and members of the Ig superfamily expressed on the endothelium, that are responsible for firm adhesion of neutrophils.

It is clear from the studies mentioned above that glycosylation not only plays a role in the mechanics of leukocyte recruitment but that it also adds an extra layer of complexity to the inflammatory response both in terms of determining


identified a similar response through sialylation of CCR5 and CCR1 (Döring,

14 recruitment of specific leukocyte subtypes as well as the temporal and spatial qualities of the inflammatory response. This is achieved through subtle changes in receptor avidity as seen for increased binding of LFA-1 over Mac1 to ICAM-1 as a result of increased glycosylation (Diamond, M.S., Staunton, D.E., et al. 1991), or the increased binding of chemokines through receptor retention at the cell surface and increased binding affinity (Bannert, N., Craig, S., et al. 2001, Zhou, H. and Tai, H.H. 1999). Levels of chemokines such as

with acute respiratory distress syndrome; is glycosylation of chemokine receptors another mechanism that controls the number of recruited cells in such disease states? Also, is there a role for glycosylation in the function of silent chemokine receptors such as Duffy Antigen Receptor for Chemokines that function to “mop up” excess chemokines? It is known that this receptor contains occupied N-glycosylation sites (Czerwinski, M., Kern, J., et al. 2007), but what this means in terms of functionality is unknown. It would be interesting to investigate how the glycosylation profile of chemokine receptors is altered in humans under inflammatory conditions. For example in chronic inflammatory pathologies is the glycosylation profile of immune cells altered in such a way that facilitates prolonged neutrophil recruitment or that leads to a failure of resolution in chronic inflammation?

Leukocyte Transmigration There are several adhesion molecules that mediate transmigration of leukocytes through the endothelial wall including PECAM-1, ICAM-2 and JAM-A (Woodfin, A., Voisin, M.B., et al. 2009) and whilst it is known that some


CXCL8 and CXCL5 correlate with neutrophil number in the lungs of patients

15 of these, for example PECAM-1 are glycosylated, it is unknown at current whether this influences leukocyte transmigration directly. A recent study suggested there may be an indirect effect as PECAM-1 levels were reduced on endothelial cells isolated from ST6Gal-I null mice, suggesting that sialylation might be required for surface retention of PECAM-1 on the endothelium (Kitazume, S., Imamaki, R., et al. 2010).

interactions that regulates endothelial barrier function (reviewed in (Dejana, E., Spagnuolo, R., et al. 2001)); redistribution of VE-cadherin is required in order for neutrophil transmigration to occur (Alcaide, P., Newton, G., et al. 2008). VE-cadherin has seven potential N-glycosylation sites, all of which are in the extracellular domain; these are predominantly sialylated and fucosylated biantennary complex N-glycans with some expression of sialylated hybrid-type N-glycans (Breviario, F., Caveda, L., et al. 1995, Geyer, H., Geyer, R., et al. 1999, Suzuki, S., Sano, K., et al. 1991). Studies using bacterially-produced VE-cadherin suggested the protein forms trimers at the cell surface however this was recently demonstrated to be an artefact caused by a lack of glycosylation and it is now believed that the high level of sialylation provides the protein with a negative charge thus preventing association with molecules on the same cell and promoting association with molecules on opposing cells to maintain the “zipper-like” structure (Bibert, S., Jaquinod, M., et al. 2002, Brasch, J., Harrison, O.J., et al. 2011, Legrand, P., Bibert, S., et al. 2001). Whether glycosylation of VE-cadherin is modified


VE-cadherin, expressed on endothelial cells forms zipper-like cell-cell

16 during inflammation to permit increased neutrophil transmigration is not known.

As transmigration is the ultimate step of the leukocyte recruitment cascade it is a difficult stage to study in isolation. Alterations in glycosylation that affect cell rolling or firm adhesion may mask a specific role in transmigration. As such, there is still much to be deciphered with respect to the role glycosylation

in Figure 3. Static model systems that can allow the transmigration process to be studied in isolation may help to identify a role for glycosylation of adhesion molecules such as PECAM-1 that play a role specifically in this stage of trafficking.

Glycosylation as a navigation tool for leukocytes during inflammation Glycosylation of the endothelium has been proposed to act as a “zip code” for directing leukocyte subtype specific recruitment in different vascular beds in response to specific stimuli (Renkonen, J., Tynninen, O., et al. 2002). The glycosylation profile of the endothelium is fluid and is modified in response to different inflammatory stimuli; pro-inflammatory stimuli such as TNF-α and disturbed flow increase the level of hypoglycosylated, high mannose and hybrid, N-glycans on both arterial and venous endothelial cells, resulting in increased monocyte adhesion under flow (Chacko, B.K., Scott, D.W., et al. 2011). These changes correspond with altered expression of several genes encoding various glycosyltransferases (ST6Gal-1, FucT-I, FucT-IV and C2GnT-2) by activated endothelial cells (García-Vallejo, J.J., Van Dijk, W., et


plays in neutrophil transmigration and this is depicted by the question marks

17 al. 2006). Changes in N-glycan composition and a decrease in mannosidase activity, may be a feature of inflammatory pathology with a significantly reduced activity also observed in the aortas of diabetic rats (Wolinsky, H., Goldfischer, S., et al. 1978). Interestingly, transcripts for GlcNAc6ST-2 and C2 GlcNAcT are induced in the high endothelial venules of salivary glands from non-obese diabetic mice, indicating a potential role for these enzymes in a chronic inflammatory state (Hiraoka, N., Kawashima, H.,

impacts the binding of glycan binding proteins such as galectins that may through these interactions influence leukocyte trafficking. Treatment of the endothelium with immunosuppressive cytokines (IL-10 and TGF-) results in an increased expression of tri- and tetra-antennary N-glycans and polyLacNAc and a concomitant decrease in 2,6-linked sialic acid as detected by reduced binding of the lectin SNA (Croci, D.O., Cerliani, J.P., et al. 2014). The outcome of these modifications was an increase in binding of galectin-1 (Gal1), a protein known to modulate many functions of immune cells including leukocyte trafficking (Cooper, D., Norling, L.V., et al. 2008b, He, J. and Baum, L.G. 2006, Norling, L.V., Sampaio, A.L., et al. 2008). The actions of this family of glycan-binding proteins on leukocyte recruitment will be discussed below.

Galectins Galectins are a family of 15 evolutionary conserved carbohydrate-binding proteins (Laderach, D.J., Compagno, D., et al. 2010) which share close sequence homology in their carbohydrate recognition domain (CRD). Galectins can be sub-divided into three distinct groups: 1) proto-type galectins


et al. 2004). Changes in the glycosylation status of the endothelium also

18 which have one CRD and are capable of homodimerisation (Gal-1, -2, -5, -7, 10, -13, -14, and -15), 2) chimera-type, of which Gal-3 is the only member, which contains a single CRD with an extended N-terminus and 3) tandem repeat type which have two distinct CRDs joined by a short linker peptide (Gal-4, -6, -8, -9, and -12) (Cooper, D.N. and Barondes, S.H. 1999, Leffler, H., Carlsson, S., et al. 2004, Liu, F.T. 2000). Galectins can be bi- or multivalent in terms of their ligand-binding activity which accounts for their ability to cross-

In contrast to the selectins, galectin binding to carbohydrates is calcium independent (Hughes, R.C. 2001). The majority of galectins (Gal-10 is an exception as it can bind mannose-containing saccharides) bind to Nacetyllactosamine (Gal1,3GlcNAc or Gal1,4GlcNAc), a common disaccharide found on many N- or O-linked glycans(Elola, M.T., Chiesa, M.E., et al. 2005). Structural analysis of the CRD of various galectins, have shown slight variations in their carbohydrate-binding specificities (Brewer, C.F., Miceli, M.C., et al. 2002) which may contribute to the distinct set of responses evoked by individual members of the galectin family.

Regulation of leukocyte trafficking by galectins Galectins differentially affect neutrophil trafficking with Gal-3, -8 and -9 enhancing, and Gal-1 inhibiting, neutrophil recruitment at various points of the leukocyte recruitment cascade (for a recent review detailing the effects of galectins on leukocyte trafficking see (Cooper, D., Iqbal, A.J., et al. 2012)). Data from our laboratory have indicated inhibitory roles for Gal-1 specifically


link cell surface glycoproteins.

19 on neutrophil rolling, adhesion and emigration both in vitro and in vivo (Cooper, D., Norling, L.V., et al. 2008b, La, M., Cao, T.V., et al. 2003). This data is supported by several other studies indicating an inhibitory role for Gal1 on neutrophil trafficking (Gil, C.D., Gullo, C.E., et al. 2010, Iqbal, A.J., Sampaio, A.L., et al. 2011, Rabinovich, G.A., Sotomayor, C.E., et al. 2000). The studies in vitro suggest that the neutrophil rather than the endothelial cell is the target of Gal-1 although studies in Gal-1 null mice and using Gal-1

Gal-1 which then targets the neutrophil to inhibit cell recruitment (Cooper, D., Norling, L.V., et al. 2008b). Increased binding of Gal-1 by neutrophils has been reported upon activation or post-transmigration suggesting an alteration in receptor expression or modification of the neutrophil glycome (Almkvist, J., Dahlgren, C., et al. 2002, Dias-Baruffi, M., Zhu, H., et al. 2003a). The mechanism for these inhibitory effects of Gal-1 on neutrophil trafficking has still to be elucidated but may be partly due to a distinct modulation of adhesion molecule expression on the neutrophil (Cooper, D., Norling, L.V., et al. 2008a, Gil, C.D., Gullo, C.E., et al. 2010). Whilst Gal-1 has apparent antiinflammatory actions, in the absence of any inflammatory stimuli it has a chemotactic role for neutrophils that is mediated through the sialoglycoprotein CD43 (Auvynet, C., Moreno, S., et al. 2013).

As galectins recognize multiple galactose 1-4-N-acetyllactosamine sequences displayed on N- and O-glycans the expression of glycosyltransferases responsible for this modification may determine susceptibility to the actions of Gal-1. Gal-1 binding can be blocked by


deficient endothelial cells indicates that the endothelium may be a source of

20 sialylation of ligands through the action of 2-6 sialyltransferase (ST6Gal1) as has been shown to be the case in certain T cell subsets which are resistant to the actions of Gal-1 due to sialic acid capping of receptors (Amano, M., Galvan, M., et al. 2003, Toscano, M.A., Bianco, G.A., et al. 2007). Studies have indicated that the glycosylation profile of neutrophils is indeed fluid. Neutrophils display altered glycosylation patterns upon transmigration and activation; this is the case for the sLex antigen, as selectin binding is lost once

D.G. 1991). This desialylation is due to the action of sialidases on the neutrophil cell surface (Gadhoum, S.Z. and Sackstein, R. 2008). Unpublished work from our laboratory has shown that upon transmigration through an endothelial monolayer neutrophils exhibit significant alterations in their expression of PNA-reactive asialo-core 1 O-glycans and L-PHA reactive β1-6 N-acetylglucosaminyltransferase (Mgat5)-modified N-glycans. SNA reactivity, as described previously is significantly reduced indicating a reduction in 2,6sialic acid linked residues. In line with sialylation capping prospective Gal-1 ligands enzymatic desialylation of resting neutrophils increases binding of recombinant Gal-1 although this was not associated with increased downstream effects (in this case phosphatidylserine exposure) (Dias-Baruffi, M., Zhu, H., et al. 2003b). Such alterations suggest that upon neutrophil transmigration galectin binding to neutrophils is likely to be modulated. Clearly more investigations into modulation of the neutrophil glycome during inflammation, particularly in the context of galectin binding are required.


the neutrophil has entered the sub-endothelial space (Cross, A.S. and Wright,

21 Gal-3 has been proposed to act as a soluble cell adhesion protein. Addition of the exogenous protein promotes neutrophil adherence to endothelial cell monolayers, laminin and fibronectin in vitro (Kuwabara, I. and Liu, F.T. 1996, Liu, F.T. and Rabinovich, G.A. 2010, Sato, S., Ouellet, N., et al. 2002b) and it clusters at neutrophil:endothelium tricellular corners, points at which neutrophils are known to preferentially transmigrate (Nieminen, J., Kuno, A., et al. 2007) which further underscores its role as an adhesion molecule.

has been reported; Gal-3 accumulation in the alveolar space of mice infected with S. pneumonia correlates with the recruitment of neutrophils to the area, with both alveolar macrophages and endothelial cells identified as potential cellular sources of Gal-3 (Sato, S., Ouellet, N., et al. 2002a). Conversely, neutrophil recruitment is reduced in Gal-3 null mice again in response to S. pneumonia but not E. Coli which is dependent on β2-integrin in this model (Nieminen, J., St-Pierre, C., et al. 2008). These effects of Gal-3 are not however restricted to the lungs with evidence for a role in supporting neutrophil trafficking to the chronically inflamed peritoneum as well as L. major infected foot pad and air pouch (Bhaumik, P., St-Pierre, G., et al. 2013, Colnot, C., Ripoche, M.A., et al. 1998). The response to L. major was specific to this micro-organism and it was proposed that in this scenario release of Gal-3 into the extracellular environment allows it to function as a damageassociated molecular pattern that facilitates neutrophil recruitment (Bhaumik, P., St-Pierre, G., et al. 2013).


A role for Gal-3 in β2-integrin-independent neutrophil migration to the lungs

22 As well as neutrophils Gal-3 can also directly support rolling and adhesion of eosinophils in an 4 integrin-dependent manner, with an effect comparable to that evoked by VCAM-1 (Rao, S.P., Wang, Z., et al. 2007). In vivo studies in Gal-3 knockout mice have found significantly lower numbers of eosinophils recruited to the lungs and dermis in models of allergic airway inflammation. Modification of N-glycans by the glycosyltransferase Mgat5 results in generation of branched glycans with N-acetyllactosamine groups which are

Correspondingly, eosinophils from Mgat5 null mice fail to roll on Gal-3 under flow and eosinophil recruitment to the airways of allergen challenged Mgat5 knockout mice is significantly attenuated. Interestingly, neutrophil recruitment was significantly increased in these mice in response to numerous inflammatory stimuli (Bahaie, N.S., Kang, B.N., et al. 2011).

There is limited evidence to suggest that tandem-repeat galectins promote neutrophil and eosinophil adhesion with both Gal-8 and Gal-9 promoting neutrophil adhesion to endothelial monolayers in static assays, an effect that may be integrin dependent in the case of Gal-8 (Imaizumi, T., Kumagai, M., et al. 2002, Nishi, N., Shoji, H., et al. 2003, Yamamoto, H., Nishi, N., et al. 2008). More studies are required however to determine whether these galectins have any involvement in leukocyte trafficking in vivo during inflammation.

Clearly, important immunomodulatory functions have been uncovered for galectins, however further studies are required to investigate the glycosylation profile of cells/tissues in the various models used to investigate galectin


suitable ligands for Gal-3 (Liu, F.T. and Rabinovich, G.A. 2010).

23 function. Whether galectin expression itself may somehow feedback and influence the glycosylation profile of galectin receptors is also of interest, this is suggested by a study in which genes encoding glycosyltransferases were differentially regulated in the corneas of Gal-3 null mice when compared to their wild-type counterparts in a model of wound healing (Saravanan, C., Cao, Z., et al. 2009). Whether this is the case for other galectin null mice in other inflammatory models remains to be determined.

With the understanding of the leukocyte recruitment cascade came the hope that the pathway could be manipulated for therapeutic gain in the treatment of inflammatory disorders. Attempts to target adhesion molecules have not however been as successful as initially hoped, mainly due to potentially fatal side effects due to infection (Bloomgren, G., Richman, S., et al. 2012). The studies detailed above indicate the importance of glycosylation, predominantly of proteins but also of lipids in the inflammatory process and specifically in the recruitment of leukocytes. Understanding how glycosylation is modified during inflammation, particularly chronic inflammatory pathologies in humans, and the function of glycan binding proteins such as galectins may therefore provide a more subtle mechanism for targeting the process of leukocyte recruitment during inflammation, without having the same negative impact on the systemic inflammatory process. As such it is vital that this avenue of research is maintained and where possible performed in human cells/tissues or in animal models that closely model human pathologies to corroborate the findings produced to date in mice.


Targeting glycosylation for therapeutic gain in inflammatory pathologies

24

Funding Funding to the Authors’ laboratory for the study of galectin biology in inflammation comes from Arthritis Research UK (Career Progression Fellowship 20387) and the Medical Research Council.

Neutrophil tethering and rolling is mediated by interactions between endothelial P-selectin, upregulated in response to inflammatory stimulation, and sialyl Lewis x (SLex) motifs (NeuAc-2,3-Gal-1,4-(Fuc-1,3)-GlcNAc) expressed on tyrosine sulphated Core 2 O-glycans displayed by PSGL-1 which is constitutively expressed on the neutrophil. The expression of the core 2 O-glycans is dependent on the actions of ppGalNAcT-1, T-synthase, C2GnT-1, ST3Gal-IV and FucT-VII. Neutrophil slow rolling is mediated by interactions between E-selectin expressed on the endothelium in response to inflammatory stimulation and N-glycans expressed on CD44/ESL-1, Core 1 Oglycans expressed on as yet unknown glycoproteins and Core 2 O-glycans expressed on glycolipids and PSGL-1. Slow rolling is dependent on the actions of T-synthase, ST3Gal-IV, FucT-VII and FucT-IV. Loss of the enzymes involved at either stage lead to a partial or complete loss of neutrophil trafficking.


Figure 1: The role of glycosylation enzymes in neutrophil rolling.

25 Figure 2: The role of glycosylation enzymes in neutrophil firm adhesion. Neutrophil firm adhesion is mediated by interactions between integrins expressed by leukocytes and their ligands on the activated endothelium. Slow rolling on E-selectin promotes the intermediate activation of the integrin molecules and these are fully activated by interactions between chemokines, which are immobilised on endothelial glycosaminoglycans, and their GPCR receptors. GPCR receptors require modification by ST6GalT-IV to bind

are N-glycosylated. Loss of enzymes at either stage leads to a partial loss of neutrophil trafficking.

Figure 3: The role of glycosylation enzymes in neutrophil transmigration. Neutrophil transmigration is mediated by interactions between cell adhesion molecules expressed on the leukocyte and the endothelium including PECAM-1, ICAM-2 and JAM-A. Of these PECAM-1 requires modification by ST6Gal-1 to be retained at the cell surface. VEcadherin is required to disassemble in order for trafficking to take place and is known to be glycosylated however it is unclear how this affects trafficking. Further studies are required to uncover the role of glycosylation in neutrophil transmigration


chemokines. Once fully active the integrins bind to ICAM-1 and VCAM-1 that

26

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complexities in neutrophil transmigration. Curr Opin Hematol, 17:9‐17.

PSGL-1

CD44/ESL-1

PSGL-1

? SO4 - Y

Glycolipid

P-selectin

ppGalNAcT-1 T-synthase C2GnT-1 β4GalT-1 ST3Gal-IV FucT-VII

GlcNAc GalNAc Glc Man Gal NeuAc Fuc

E-selectin

Vascular Endothelial Cell

Tethering and Rolling

ownloaded from http://glycob.oxfordjournals.org/ at Yale University on September 30, 2014

Neutrophil

Slow Rolling T-synthase ST3Gal-IV FucT-VII and FucT-IV

LFA-1

Mac-1 Integrin

Chemokine Receptor

Heparan Sulphate


Firm Adhesion ST3Gal-IV Mannosidase I

ICAM-1


Neutrophil

VCAM-1

GalNAc Man Gal NeuAc Fuc Chemokine

?

GalNAc Man Gal NeuAc

Integrin

Fuc

? PECAM-1


ICAM-2

? VE-cadherin

Transmigration ST6Gal-1

?

JAM-A


Neutrophil

Circadian rhythms in leukocyte trafficking.

Highly specific blockade of CCR5 inhibits leukocyte trafficking and reduces mucosal inflammation in murine colitis.

Circulating fibrocytes are involved in inflammation and leukocyte trafficking in neonates with necrotizing enterocolitis.

Imaging of Leukocyte Trafficking in Alzheimer's Disease.

CYR61: a new player in inflammation and leukocyte trafficking.

reperfusion injury.

Leukocyte Trafficking in Cardiovascular Disease: Insights from Experimental Models.

Interactions between CD44 and Hyaluronan in Leukocyte Trafficking.

Selectin-mediated leukocyte trafficking during the development of autoimmune disease.

Targeting leukocyte trafficking for the treatment of inflammatory bowel disease.

Leukocyte adhesion to endothelium in inflammation.

Glycobiology of ocular angiogenesis.

Leukocyte traffic to sites of inflammation.

Leukocyte Trafficking to the Small Intestine and Colon.

Glycobiology of Eosinophilic Inflammation: Contributions of Siglecs, Glycans, and Other Glycan-Binding Proteins.

Platelets in inflammation: regulation of leukocyte activities and vascular repair.

Parasite Glycobiology: A Bittersweet Symphony.

Glycobiology: drifting toward polymer perfection.

The coming of age of glycobiology.

Journeys in science: glycobiology and other paths.

Glycobiology of platelet-endothelial cell interactions.

Assessment of leukocyte trafficking in humans using the cantharidin blister model.

Latest advances in glycobiology highlighted and old challenges revisited at the 2013 Annual Conference of the Society for Glycobiology.

E6130, a Novel CX3C Chemokine Receptor 1 (CX3CR1) Modulator, Attenuates Mucosal Inflammation and Reduces CX3CR1+ Leukocyte Trafficking in Mice with Colitis.