Heparan sulfate and inflammation Christopher R Parish Entry of leukocytes into tissues is a key feature of inflammation. New data suggest the polysaccharide heparan sulfate is required for several stages of this entry process.
H
eparan sulfate is a ubiquitously expressed polysaccharide that appears on cell surfaces and in extracellular matrices as a proteoglycan. It shows great structural diversity and has been implicated in numerous biological processes1,2. In the case of inflammation, heparan sulfate is a potential ligand for P- and L-selectin, two key molecules involved in the adhesion of leukocytes to inflamed endothelium3. Similarly, heparan sulfate can bind proinflammatory chemokines, transport them across the endothelium and present them to leukocytes3,4. Until now, however, the precise role of heparan sulfate during in vivo inflammatory responses has been unclear. In this issue of Nature Immunology, Wang et al.5 show that endothelial cells expressing partially sulfated heparan sulfate are unable to recruit leukocytes into inflammatory sites. This effect is due to heparan sulfate being an essential adhesion ligand for L-selectin and being required for the transport of chemokines across the endothelial barrier for presentation to leukocytes. In contrast, leukocytes expressing partially sulfated heparan sulfate were unimpaired in their response to inflammatory stimuli. Overall these findings unequivocally demonstrate that endothelial cell expression of heparan sulfate is essential for multiple stages of an inflammatory response. It has been known for many years that the entry of leukocytes into sites of inflammation is a highly ordered process involving a range of adhesion receptors and ligands. Initial attachment and rolling of leukocytes on the
Christopher Parish is at the John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory 2601, Australia. e-mail:
[email protected]
inflamed vascular endothelium can involve up to three calcium-dependent (C-type) lectins, termed selectins6,7. L-selectin is constitutively expressed by leukocytes, whereas P- and E-selectin are displayed on the surface of endothelial cells that have been activated by various proinflammatory stimuli. After a reduction in rolling velocity mediated by selectins, leukocytes are able to interact with endothelially bound chemokines8, a process
Neutrophil
Rolling Heparan sulfate proteoglycan
that activates leukocyte integrins and results in more stable leukocyte–endothelial cell adhesion. Finally, the stably adherent leukocytes follow chemokine gradients within the blood vessel wall and enter the target tissue, in a process called extravasation (Fig. 1). Numerous complex carbohydrate ligands have been identified that interact with the different selectins. P- and E-selectin recognize sialylated, fucosylated mucins expressed
Chemokine activation of integrins
Extravasation
Lumen L-selectin PSGL-1
Heparan sulfate
IntegrinICAM
P-selectin
Endothelial cells
TNF receptor Chemokines Cytokines
Bacteria
Macrophage
Tissues
Figure 1 Role of heparan sulfate in leukocyte entry into sites of inflammation. In response to inflammatory stimuli, such as a bacterial infection, resident macrophages in tissues produce both chemokines (such as interleukin-8) that attract leukocytes into tissues and cytokines (such as tumor necrosis factor, TNF) that trigger the display of preformed P-selectin on the luminal surface of endothelial cells. Cytokines can also induce the expression of E-selectin by endothelial cells (not shown). Endothelial heparan sulfate is depicted as playing an important role in L-selectin binding, in chemokine presentation to chemokine receptors on neutrophils, and in the transportation of chemokines, produced by tissue macrophages (and neutrophils), across the endothelial cell barrier. ICAM, intercellular adhesion molecule; PSGL, P-selectin glycoprotein ligand-1.
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NEWS AND VIEWS
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
NEWS AND VIEWS on the surface of leukocytes with PSGL-1, which consists of O-linked sialyl Lewis X and three adjacent sulfated tyrosine residues, being usually the dominant ligand6,7. L-selectin has an additional function in that it not only is involved in the rolling of leukocytes on inflamed endothelium but also mediates the selective binding of lymphocytes to high endothelial venules9. In fact, the latter process is crucial in the entry of lymphocytes into lymph nodes. Much is known about the L-selectin ligands on high endothelial venules, which are predominantly sulfated, sialylated and fucosylated mucins9, but the identity of the ligands for L-selectin on inflamed endothelium is unclear. To add to the confusion, there is abundant evidence that both L- and P-selectin, as well as interacting with mucinous ligands, also bind heparan sulfate and heparin3 (a highly sulfated version of heparan sulfate that is restricted to mast cell granules and is widely used as an anticoagulant). Thus, before the study by Wang et al.5, a key unanswered question had been whether endothelial heparan sulfate is involved in leukocyte adhesion or, alternatively, whether mucinous ligands dominate. Wang et al.5 resolve this issue by using elegant molecular genetic techniques to clearly demonstrate that heparan sulfate can act as the dominant L-selectin ligand on inflamed vascular endothelium (Fig. 1). Wang et al.5 also noted that, in contrast to endothelial cells, leukocytes expressing undersulfated heparan sulfate functioned normally in inflammatory responses. The simplest interpretation of this finding is that heparan sulfate is not an important Pselectin ligand on leukocytes. Certainly earlier reports showing that P-selectin has a lower binding affinity for heparan sulfate than Lselectin support this view10. Thus, on the basis of earlier studies, PSGL-1 seems to be the most likely leukocyte ligand for P-selectin (Fig. 1). Yet heparan sulfate cannot be totally discounted as a possible P-selectin ligand. Heparan sulfate is structurally extraordinarily diverse, mainly as a result of differences in the position of O-linked and N-linked sulfate groups along the polysaccharide chain. In the study by Wang et al.5, an enzyme involved in N-sulfation of heparan sulfate was selectively inactivated in either leukocytes or endothelial cells, and the inactivation also resulted in a partial reduction in O-sulfation of the heparan sulfate molecules. It is conceivable, however, that P-selectin may bind to less sulfated heparan sulfate molecules.
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Another issue examined by Wang et al.5 was the hypothesis that heparan sulfate binds and transports chemokines from the abluminal to luminal side of endothelial cells, a process called transcytosis. It is generally accepted in the field that this occurs. In fact, most chemokines possess a C-terminal stretch of positively charged amino acids that interacts with heparin and heparan sulfate and is distinct from the domain that is recognized by chemokine receptors. Yet until the present report that heparan sulfate is required for the transport of chemokines across endothelial cell barriers, there had been no formal demonstration that heparan sulfate–mediated transcytosis of chemokines occurs in vivo. This work is consistent with earlier studies indicating that heparan sulfate on the luminal surface of endothelial cells is required to both tether and present chemokines to leukocytes (Fig. 1). Based on the findings reported by Wang et al.5, a number of additional studies can be envisaged. First, it will be important to establish whether in most vascular beds heparan sulfate is the dominant L-selectin ligand on inflamed endothelium. Consistent with heparan sulfate being a universal L-selectin ligand is the observation that in three separate models of inflammation, namely acute peritonitis, contact dermatitis and cutaneous air-pouch inflammation, leukocyte entry was defective in mice lacking appropriately sulfated endothelial heparan sulfate. This simple interpretation is complicated, however, by the fact that the heparan sulfate deficiency also inhibited chemokine transcytosis, an essential feature of the inflammatory response. Further studies will be required to resolve this issue. Second, the temporal relationship between P- and L-selectin binding needs to be ascertained. The initial attachment and rolling of leukocyte on inflamed endothelium is probably mediated by P-selectin, with L-selectin having an essential role in reducing rolling velocity, but additional studies are needed to establish whether this is indeed the case. The relative contribution of E-selectin to this process also requires further examination. Third, it is well established that leukocyte subsets can be selectively recruited into inflammatory sites by specific chemokines. An intriguing question is whether heparan sulfate is involved in the transcytosis of all chemokines or only a subset. In addition, it is conceivable that variations in heparan sulfate structure under normal or inflammatory conditions can change the repertoire of
chemokines that can bind to heparan sulfate and be transported to the endothelial surface. Such a process could be important in determining which leukocyte subsets enter tissues. The demonstration that endothelial heparan sulfate participates in multiple steps of an inflammatory response has considerable clinical implications. Heparin has been known for years to possess anti-inflammatory activity. It seems highly likely that the anti-inflammatory activity of heparin results from it interfering with the binding of L- and P-selectin to their respective ligands11. Heparin, however, suffers from the disadvantage that it is diverse in structure and, consequently, mediates multiple in vivo effects. Nevertheless, it should be possible to synthesize low-molecular-weight mimetics of heparin and/or heparan sulfate that target inflammation with minimal side effects, as such molecules should be structurally much more homogeneous than heparin. These drugs could act by inhibiting the Lselectin (and possibly P-selectin)-mediated interaction of leukocytes with inflamed endothelium, by displacing heparan sulfate–bound chemokines from the vascular wall, or both. Indeed, recent studies indicate that small heparan sulfate mimetics can show remarkable selectivity in blocking various protein–heparan sulfate interactions12. In addition, the position of the negatively charged sulfate groups in the mimetics was found to be the critical factor that determines specificity. With the increasing need for new anti-inflammatory drugs with modes of action different from those of existing drugs, future research in this area will be followed with great interest. ACKNOWLEDGMENTS The author thanks the National Health and Medical Research Council of Australia for support. 1. Turnbull, J., Powell, A. & Guimond, S. Trends Cell Biol. 11, 75–82 (2001). 2. Esko, J.D. & Selleck, S.B. Annu. Rev. Biochem. 71, 435–471 (2002). 3. Gotte, M. FASEB J. 17, 575–591 (2003). 4. Middleton, J. et al. Cell 91, 385–395 (1997). 5. Wang, L., Fuster, M., Sriramarao, P. & Esko, J.D. Nat. Immunol. 6, 902–910 (2005). 6. Lowe, J.B. Immunol. Rev. 186, 19–36 (2002). 7. Kannagi, R. Curr. Opin. Struct. Biol. 12, 599–608 (2002). 8. Ley, K. Microcirculation 10, 289–295 (2003). 9. Rosen, S.D. Annu. Rev. Immunol. 22, 129–156 (2004). 10. Nelson, R.M. et al. Blood 82, 3253–3258 (1993). 11. Wang, L., Brown, J.R., Varki, A. & Esko, J.D. J. Clin. Invest. 110, 127–136 (2002). 12. Freeman, C. et al. J. Biol. Chem. 280, 8842–8849 (2005).
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