Adhesion Molecule Strategies

Pulmonary Pharmacology & Therapeutics (1999) 12, 137–141 Article No. pupt.1999.0189, available online at http://www.idealibrary.com on

PULMONARY PHARMACOLOGY & THERAPEUTICS

Adhesion Molecule Strategies P. G. Hellewell Section of Medicine – Vascular Biology, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK INTRODUCTION

SELECTINS

In order to accumulate in airway tissue in diseases such as asthma, the leukocyte must adhere to the endothelium lining the blood vessels of the bronchial microcirculation, penetrate the vessel wall and migrate to the airway lumen. Cell adhesion molecules are involved in all stages of this process. In addition, adhesion molecules present on other cells in the lung including mast cells, dendritic cells, macrophages and nerves also contribute to the inflammatory response in this and other organs.

Selectins are restricted to the vascular system. They are differentially expressed on endothelium (E-selectin, P-selectin), platelets (P-selectin) and leukocytes (Lselectin).1 The three members of the selectin family share an N-terminal lectin domain that is directly responsible for ligand binding. L-selectin is expressed constitutively on surface microvilli of all leukocyte classes, although only a subpopulation of memory T cells and NK cells are positive. Exposure of eosinophils to inflammatory mediators causes L-selectin to be rapidly shed from the cell surface via the action of a protease. This shedding is thought to facilitate the development of firm adhesion. Some leukocytes (including eosinophils) can reexpress their surface Lselectin following transmigration, suggesting that this molecule may be also be important for leukocyte behaviour in tissue. P-selectin is synthesized by endothelial cells and stored in Weibel-Palade bodies. Following exposure of endothelial cells to inflammatory mediators, P-selectin is rapidly released to the cell surface. P-selectin is also up-regulated transcriptionally by inflammatory cytokines, including IL4, which may be important for allergic inflammation. E-selectin is expressed exclusively on endothelial cells where it is transcriptionally regulated by cytokines and LPS. Expression peaks around 4 h after exposure to cytokines and persists for 12 h. Numerous studies have noted functional overlap between the selectins, demonstrating for instance that following mild stimulation, P-selectin alone is sufficient to support substantial leukocyte rolling and inflammatory cell accumulation.2 On the other hand, more severe stimuli (e.g. TNF-a stimulation) produce rolling that is dependent on more than one of the selectins.3

CELL ADHESION MOLECULES AND THE ADHESION CASCADE Adhesion molecules involved in leukocyte trafficking are grouped into three families based on structural features: the selectins, the integrins and the immunoglobulin (Ig) gene superfamily. Studies of adhesion mechanisms in vitro and observation of the living microcirculation using intravital microscopy have delineated the coordinated sequence of events responsible for accumulation of leukocytes. In the absence of inflammation, circulating leukocytes rarely interact with vessel wall. However, with the development of an inflammatory response, leukocytes are tethered by a transient adhesive interaction that results in leukocyte rolling along the endothelium of post-capillary venules. The selectins and their glycoprotein ligands largely mediate this process. Subsequent activation by chemoattractants (e.g. chemokines, anaphylatoxins, formylated peptides, lipid mediators) causes the rolling leukocyte to arrest, firmly adhere and flatten (reducing exposure to shear forces and increasing surface area in contact with endothelium). Integrins and immunoglobulin superfamily members mediate these steps. Finally, the leukocytes migrate between endothelial cells (diapedesis) into the interstitium and move towards the source of the stimulus (chemotaxis). 1094–5539/99/020137+05 $30.00/0

SELECTIN LIGANDS Physiological selectin ligands are a diverse group of glycosylated proteins and glycolipids that recognize 137

 1999 Academic Press

138

P. G. Hellewell

one or more of the selectins.1 However, the minimal carbohydrate epitope recognized by all three selectins is the sialylated, fucosylated tetrasaccharide sLex and its stereoisomer sLea. Of all the glycoproteins proposed as selectin ligands, P-selectin glycoprotein ligand-1 (PSGL-1) is the best characterized and most widely accepted.4 PSGL-1, a homodimer of two 120 kDa subunits, is localized to microvilli on all leukocytes and is therefore in a prime position to bind P-selectin. In spite of its name, PSGL-1 is also recognized by E- and L-selectin. Human PSGL-1 is sulphated on tyrosine residues and removal of sulphate eliminates binding to P-selectin and L-selectin but does not prevent binding to E-selectin. E-selectin ligand-1 (ESL-1) was identified in mouse as a high affinity Eselectin ligand expressing sLex.5 However, since it has strong homology with a putative chicken fibroblast growth factor receptor and identity with a marker of the Golgi, it’s importance as a physiological E-selectin ligand remains to be established. The ligand for Lselectin on post-capillary venules is not known. Lselectin ligands on high endothelial venules in mucosal and peripheral lymph nodes have been identified as MadCAM-1 and GlyCAM-1, respectively.1 CD34 is another proposed ligand for L-selectin that is expressed on the lumenal facia of many blood vessels. As it is concentrated on endothelial microvilli, it is ideally placed to mediate L-selectin binding. However, in view of the fact that CD34 knockout mice show no deficiencies in leukocyte rolling in venules the role of CD34 as an endothelial cell ligand for L-selectin is not confirmed.6 The enzymes involved in the synthesis of sLex and its isomer sLea have been identified as a2, 3 sialyltransferases and a1, 3 fucosyl-transferases. Mice genetically deficient in one of these enzymes, (a1, 3 fucosyl-transferase VII), show impaired leukocyte rolling.7 However, leukocyte expression of PSGL-1 and ESL-1 in these mice is normal illustrating the essential role of post-translational glycosylation with fucosylated oligosaccharides for selectin adhesion. INTEGRINS Integrins are heterodimeric proteins consisting of noncovalently linked a- and b-chains with cytoplasmic tails which associate with the cytoskeleton and provide a connection to the extracellular matrix. There are currently 17 a-chains and 8 b-chains that have been cloned and sequenced. Leukocytes express 13 different integrins that mediate binding to endothelial cells and matrix proteins; the most important for adhesion to endothelial cells are the b1, b2 and b7 integrins. The b2 integrin subfamily is abundantly expressed on leukocytes and consists of a common b subunit (b2/CD18) linked to one of four a subunits, CD11a

(aL), CD11b (aM), CD11c (aX) or CD11d (aD). Lymphocytes express primarily CD11a/CD18 while neutrophils, eosinophils and monocytes express all four b2 integrins. CD11d is more closely related to CD11b and CD11c than to CD11a and is expressed on all blood leukocytes, particularly monocytes. Exposure of leukocytes to chemoattractants results in increased adhesion to endothelial cells. CD11a and CD11b binding to ICAM-1 and ICAM-2 on the endothelial surface mediate this. On granulocytes, surface expression of CD11b is rapidly increased after exposure to chemoattractants due to mobilization from intracellular granule stores. By constantly being newly expressed on the cell surface, this intracellular pool appears to support sustained neutrophil movement on surfaces where adhesion is CD11b dependent. In contrast, CD11a is not stored and so its level on the cell surface is not increased; rather, there is a change in the conformation of this integrin that regulates affinity for ICAM-1. The b1 integrin VLA-4 (a4b1) is expressed on all blood leukocytes except neutrophils although these can be induced to express this molecule following transendothelial migration.8 VLA-4 is constitutively active and does not require a conformational change or increased expression to bind to its ligands VCAM1 or fibronectin although some stimuli can increase adhesion via VLA-4 up-regulation. The other a4 integrin, a4b7, is expressed on eosinophils and a subset of gut homing lymphocytes. On eosinophils a4b7 mediates binding to VCAM-1, fibronectin and MadCAM-1 and, unlike VLA-4, requires activation. a4 integrins can also support leukocyte rolling in vivo, although this cannot occur in the absence of selectins. Other b1 integrins expressed by leukocytes include a5b1 and a6b1 which mediate adhesion to matrix proteins. IMMUNOGLOBULIN SUPERFAMILY The immunoglobulin (Ig) superfamily includes ICAM-1, ICAM-2, ICAM-3, VCAM-1 and PECAM1. ICAM-1 is constitutively expressed at low levels on endothelial cells but is transcriptionally up-regulated by various cytokines and LPS. ICAM-2 is constitutively expressed on endothelial cells at higher levels than ICAM-1 and it is not up-regulated by inflammatory cytokines. In fact, TNF-a and IL-1 both reduce its expression on cultured endothelial cells, although the significance of this is not clear. ICAM3 is not normally found on endothelial cells but is present at high levels on leukocytes and is important in mediating interactions between leukocytes. VCAM1 is another member of the Ig superfamily that is expressed on endothelial cells. Basal expression of VCAM-1 on endothelial cells is very low but is increased by cytokines including IL-4. PECAM-1, which

Adhesion Molecule Strategies

has 6 Ig domains, is expressed constitutively on endothelial cells, leukocytes and platelets. On endothelial cells, stimulation with the same inflammatory cytokines that induce up-regulation of ICAM-1 results in a redistribution of PECAM-1 to the cell periphery without affecting the total amount expressed by each cell. This process may facilitate leukocyte migration between adjacent endothelial cells.

ADHESION MOLECULE SIGNALLING In general, integrins are not constitutively active and require activation to bind to a ligand. Within minutes of exposure to a stimulus integrins on the cell surface acquire the ability to attach to other cell surface and extracellular ligands. This is thought to occur by conformational changes in their ligand binding sites. Recent studies have suggested that the cellular machinery controlling this change in integrin affinity for ligands include CD98, an early marker of T cell activation.9 Clustering of CD98 by unknown ligands is thought to induce an intracellular signalling cascade that leads to the conformation change. Other molecules including CD47 (integrin associated protein) and members of the transmembrane 4 family of proteins (CD53, CD63, CD81, CD82) have also been shown to be involved in integrin signalling.10 This type of signalling is known as ‘inside-out’ signalling and, in cells stimulated with chemoattractants, involves G-proteins including Rho and Rac, although kinases including PI-3K and PKC are also involved. The other type of signalling, known as ‘outside-in’ signalling, is the result of an integrin transmitting a signal to the cell machinery leading to functions such as chemotaxis, secretion of specific cytokines, killing of invasive pathogens, gene expression and apoptosis. For example, ligation of the eosinophil b2 integrin subunit triggers cell activation and degranulation; ligation of L-selectin has similar signalling effects on leukocytes.

ADHESION MOLECULES ON OTHER CELLS In asthma, altered expression of adhesion molecules is not limited to endothelial cells and leukocytes. Bronchial epithelial cells express ICAM-1 although, unlike lung microvascular endothelial cells they do not express VCAM-1, PECAM-1 or selectins.11 Epithelial cell ICAM-1 mediates adhesion of eosinophils and may be an important mechanism by which these two cells are brought into close proximity leading to epithelial cell injury.12 Other adhesion molecule expressing cells in the lung include mast cells which express b1 integrins that bind to matrix proteins.

139

Effective mast cell degranulation requires a4-mediated adhesion to fibronectin.13 Macrophage activation is enhanced by adhesion to other cells and surfaces. Adhesion molecules expressed by lung macrophages include ICAM-1, CD11b, VLA4, PSGL-1 and L-selectin. Similarly, VLA-4 and PSGL-1 expression on lung dendritic cells may facilitate activation via adhesion to fibronectin and Lselectin, respectively. The airway localization of these adhesion molecules may favour local treatment to the lung with agents that modulate their function. For example, preventing adhesion to matrix proteins may deny an eosinophil important signals for cell survival leading rapidly to apoptotic cell death.14 Airway nerves express a ligand for VLA-4, possibly VCAM-1, which mediates eosinophil adhesion and subsequent nerve damage, in a manner analogous to eosinophil-mediated epithelial cell damage.15

MODULATORS OF ADHESION MOLECULES AS THERAPEUTICS A number of approaches to modulate adhesion molecule expression and function are possible (Table 1). Blocking the cytokines that induce adhesion molecule expression is one such possibility although will be a rather non-specific effect. Antisense is highly specific and an oligonucleotide to ICAM-1 reduces inflammation in vivo and has been shown in clinical studies to be effective in Crohn’s disease.16 Blocking adhesion molecule function with mAbs has been extensively tested in vitro and in vivo. For example, antiICAM-1 mAb (BIRRI) reduces eosinophil trafficking and airway hyperresponsivness in a monkey model,17 although this mAb has not been tested in asthma in humans. A humanized anti-E-selectin mAb (CDP850) is currently in clinical trial in psoriasis, the rationale being that E-selectin-cutaneous lymphocyte antigen (CLA, a form of PSGI-1) is a lymphocyte homing pair in skin. Soluble adhesion molecule-IgG constructs (immunoadhesins) are effective as inhibitors of leukocyte trafficking in animals models of lung inflammation but have not been tested in allergic diseases. While blocking antibodies directed against integrins have been used successfully in animal models, these are unlikely to be used clinically in allergic inflammation because of the problems associated with repeated antibody administration. Integrin inhibitors such as neutrophil inhibitory factor (NIF), a 41 kDa protein that is secreted by canine hookworm, may be more useful. NIF binds to the cation binding site in the A domain of CD11b to inhibit neutrophil adhesion to endothelial cells in vitro. Overexpression of NIF in endothelial cells in vivo prevents LPS-induced lung

140

P. G. Hellewell

Table 1 Possible therapeutic approaches to target cell adhesion molecules.

CONCLUSION

Modulate cell surface expression Cytokine and cytokine receptor antagonists Antisense oligonucleotides (e.g. ISIS 2302) Fucosyltransfersase inhibitors Metalloproteinase inhibitors (to reduce L-selectin shedding)

Adhesion molecules are critical in regulating leukocyte trafficking which is a fundamental feature of allergic diseases. Some of the same molecules are important in cell signalling and may facilitate leukocyte migration as well as controlling function once leukocytes have migrated. In addition, expression of adhesion molecules on cells other than leukocytes and endothelial cells has highlighted additional pathophysiological processes in which these molecules participate. The latter observations have implications for blocking adhesion molecules as a therapeutic intervention in allergic diseases. For example, local delivery of an adhesion molecule blocker to the lung may have additional beneficial effects that would not be found with systemic administration. Moreover, this may minimize systemic side-effects, for example interference with host defence reactions such as seen in Leukocyte Adhesion Deficiency patients whose leukocytes are deficient in the b2 integrin. Despite the enormous progress made in the adhesion molecule field and the proven efficacy of blocking the function of these glycoproteins in disease models, the role of each of them in human disease has not been established and awaits the outcome of clinical studies.

Block function Monoclonal anatibodies (e.g. BIRRI, CDP850) Soluble adhesion molecules/immunoadhesins Oligosaccharides (e.g. CGP69669A) Glycomimetics (e.g. TBC1269) Small molecule inhibitors (e.g. BIO1211, CY-9652, TBC772) Integrin inhibitors (e.g. NIF, CD98 inhibitors)

injury in the mouse,18 although it has not been tested in allergic inflammation. Other small molecule integrin inhibitors prevent VLA-4 binding to the CS1 binding domain of fibronectin. This includes CY-9652 that shows antiinflammatory activity and ameliorates airway dysfunction when given by the inhaled route.19 Other VLA-4 inhibitors (e.g. BIO1211, TBC772) are also under study in preclinical and clinical investigations. The effect of CY-9652 may be independent of an action on circulating leukocytes, as found with an anti-VLA-4 mAb.20 In other words, expression of VLA-4 on intrapulmonary cells including mast cells, macrophages and dendritic cells is an important target. If leukocyte adhesion does involve CD98, then inhibition of this CD98-induced integrin upregulation may prevent inflammation. Metalloproteinase inhibitors may be useful by preventing leukocyte Lselectin shedding resulting in impaired endothelial transmigration. Oligosaccharides such as sLex and sLea prevent leukocyte rolling in vivo and attenuate accumulation at inflammatory sites. In post-capillary venules in vivo, sLex is an E-selectin antagonist,21 and is in clinical trial for ischaemia-reperfusion in children following cardiopulmonary bypass. sLex is not being tested in allergic diseases but a glycomimetic inhibitor of E-, P- and L-selectin (TBC1269),22 has been tested in phase IIa clinical studies for asthma. This compound attenuates airway dysfunction in an animal model when given by inhalation;23 that it is effective by this route suggests that it may be having a local action on intrapulmonary cells rather than reducing leukocyte trafficking. This local action could include blocking signalling via PSGL-1 or L-selectin. In view of the critical role of fucosyltrasferases in conferring function on selectin ligands such as PSGL-1, specific inhibitors of these enzymes, in particular FUCT-IV and FUCTVII are potential therapeutic agents.

REFERENCES 1. Kansas G S. Blood 1996; 88: 3259–3287. 2. Mayadas T N, Johnson R C, Rayburn H, Hynes R O, Wagner D D. Cell 1993; 74: 541–554. 3. Kunkel E J, Ley K. Circ Res 1996; 79: 1196–1204. 4. McEver R P, Cummings R D. J Clin Invest 1997; 100: 485–491. 5. Steegmaier M, Levinovitz A, Isenmann S, Borges E, Lenter M, Kocher H P, Kleuser B, Vestweber D. Nature 1995; 373: 615–620. 6. Suzuki A, Andrew D P, Gonzalo J A, Fukumoto M, Spellberg J, Hashiyama M, Takimoto H, Gerwin N, Webb I, Molineux G, Amakawa R, Tada Y, Wakeman A, Brown J, McNiece I, Ley K, Butcher E C, Suda T, Gutierrez-Ramos J C, Mak T W. Blood 1996; 87: 3550–3562. 7. Maly P, Thall A D, Petryniak B, Rogers G E, Smith P L, Marks R M, Kelly R J, Gersten K M, Cheng G Y, Saunders T L, Camper S A, Camphausen R T, Sullivan F X, Isogai Y, Hindsgaul O, von Andrian U H, Lowe J B. Cell 1996; 86: 643–653. 8. Kubes P, Niu X F, Smith C W, Kehrli M E, Reinhardt P H, Woodman R C. FASEBJ 1995; 9: 1103–1111. 9. Fenczik C A, Sethi T, Ramos J W, Hughes P E, Ginsberg M H. Nature 1997; 390: 81–85. 10. Aplin A E, Howe A, Alahari S K, Juliano R L, Pharmacol Rev 1998; 50: 197–263. 11. Bloemen P G, Henricks P A, Nijkamp F P, Clin Exp Allerg 1997; 27: 128–141. 12. Burke-Gaffney A C, Hellewell P G, Am J Resp Cell Mol Biol 1998; 19: 408–418. 13. Yasuda M, Hasunuma Y, Adachi H, Sekine C, Sakanishi T, Hashimoto H, Ra C, Yagita H, Okumura K. Int Immunol 1995; 7: 251–258. 14. Anwar A R, Moqbel R, Walsh G M, Kay A B, Wardlaw A J. J Exp Med 1993; 177: 839–843. 15. Fryer A D, Costello R W, Yost B L, Lobb R R, Tedder T F,

Adhesion Molecule Strategies

16. 17. 18. 19. 20.

Steeber D A, Bochner B S. J Clin Invest 1997; 99: 2036–2044. Yacyshyn B R, Bowen-Yacyshyn M B, Jewell L, Tami J A, Bennett C F, Kisner D L, Shanahan W R. Jr. Gastroenterology. 1998; 114: 1133–1142. Wegner C G, Gundel R H, Reilly P, Haynes N, Letts G, Rothlein R. Science 1990; 247: 456–458. Zhou M Y, Lo S K, Bergenfeldt M, Tiruppathi C, Jaffe A, Xu N, Malik A B, J Clin Invest 1998; 101: 2427–2437. Abraham W M, Ahmed A, Sielczak M W, Narita M, Arrhenius T, Elices M J, Am J Respir Crit Care Med 1997; 156: 696–703. Henderson W R, Jr. Chi E Y, Albert R K, Chu S J, Lamm

141

W J, Rochon Y, Jonas M, Christie P E, Harlan J M. J Clin Invest 1997; 100: 3083–3092. 21. Norman K E, Anderson G P, Kolbinger F, Ley K, Ernst B E. Blood 1998; 91: 475–483. 22. Palma-Vargas J M, Toledo-Pereyra L, Dean R E, Harkema J M, Dixon R A, Kogan T P. Am Coll Surg 1997; 185: 365–372. 23. Abraham W M, Ahmed A, Sabater J R, Lauredo I T, Botvinnikova Y, Bjercke R J, Hu X, Revelle B M, Kogan T P, Scott I L, Dixon R A F, Yeh E T H, Beck P J. Selectin blockade prevents antigen-induced late bronchial responses and airway hyperresponsiveness in allergic sheep. Am J Respir Crit Care Med 1999; 159: 1205–1214.