Selectins as potential targets of therapeutic intervention in inflammatory diseases

BB

Bioch~ic~a

et Biophysica A~ta

ELSEVIER

Biochimica et Biophysica Acta 1197 (1994) 215-226

Selectins as potential targets of therapeutic intervention in inflammatory diseases Joseph K. Welply *, Jeffery L. Keene, Jon J. Schmuke, Susan C. Howard Monsanto Corporation, Division of Glycosciences, Department of Immunology, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA (Received 22 September 1993; revised manuscript received 16 February 1994)

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

215

2. Selectin: structural motif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

216

3. Unique biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

217

4. Carbohydrate ligands and counter-receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Oligosaccharide ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Counter-receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Critical amino acids for binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

218 218 219 220

5. Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Immunochemical observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Effect of antagonists in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Disease targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221 221 222 223

6. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

223

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

223

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

223

I. Introduction * Corresponding author. Fax: + 1 (314) 6948949. Abbreviations: sLex, sialyl-Lewis x, S A a 2 - 3 G a l f l l - 4 ( F u c a l 3)GIcNAc; CLA, cutaneous lymphocyte antigen; PMN, polymorphonuclear leukocyte; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; GlyCAM-1, glycosylation dependent cell adhesion molecule-l; LAD, leukocyte adhesion deficiency; MBP, mannose binding protein; CR, complement regulatory domain; EGF, epidermal growth factor; fuc, fucose; NeuAc, SA, N-acetylneuraminic acid; gal, galactose; glcNAc, N-acetyl glucosamine; OLR1, potential O-linked region 1; OLR2, potential O-linked region 2; RA, rheumatoid arthritis; ICAM-1, intercellular adhesion molecule-l; TGF, transforming growth factor; HUVEC, human umbilical vein endothelial cell; PAF, platelet activating factor; LTB4, leukotriene B4; sLea, sialyl Le a, SAa2-3Gal/31-3(Fucal-4)GlcNAc; INFy, interferon y; PMA, phorbol 12-myristate 13-acetate; NF, nuclear factor; NOD, non-obese diabetic. 0304-4157/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

SSDI 0 3 0 4 - 4 1 5 7 ( 9 4 ) 0 0 0 0 5 - X

Cell surface lectins frequently recognize carbohydrates on other cells, thereby facilitating cell to cell adhesion [1]. A set of lectins which enables adhesion between endothelial cells and leukocytes was discovered four years ago [2-4]. The three proteins that comprise this class are referred to as the selectins, E-, P- and L-selectin. Selectins are cell surface proteins. Eand P-selectin are expressed on the surface of endothelial cells; L-selectin is on the leukocyte surface. Pselectin is also found on the surface of activated platelets. The selectins are highly related to each other by primary structure. They are multi-domain proteins,

21b

.LK. Welplv et al. /Biochimica et Biophysica Acta 1197 (1994) 215-22t~

having a carbohydrate recognition portion, or lectin domain, at the amino-terminus. The lectin domain is the primary, biologically functional part of the selectin protein. Selectins bind to mucin-like counter-receptors on the surface of leukocytes or endothelial ceils. Under non-inflammatory conditions E-selectin is not expressed, and P-selectin is not involved in cell adhesion, in that it is sequestered in intracellullar pools rather than on the cell surface. In inflammation, E- and P-selectin appear in the endothelial membrane within the tissue at the site of the insult. There, E-and Pselectin recognize mucin-like counter-receptors on the surface of the leukocyte. The binding to mucin-like receptors causes an association between the cells which results in the rolling of leukocytes on the endothelium as the leukocytes are pushed along by vascular flow [5-8]. The rolling interaction initiates subsequent events in the process of leukocyte extravasation [8], an event which is crucial for host defense, permitting lymphocytes to circulate into the lymph system through peripheral lymph nodes and leukocytes to extravasate into injured tissue. L-selectin, the third member of the family, is also involved in the rolling of leukocytes on endothelium. L-selectin is constitutively expressed on leukocyte cell surfaces, including those of lymphocytes, neutrophils, and monocytes. A class of circulating lymphocytes attaches to specialized endothelial cells in venules in lymph nodes due to the binding of L-selectin to mucin-like counter-receptor(s) in the endothelial cell membrane of the nodes. This interaction enables the entry of peripheral lymphocytes into the lymphatic system for immune surveillance [9]. In other inflamed tissues, L-selectin on neutrophils and monocytes binds to inducible mucin-like counter-receptors that are expressed on the surface of endothelial cells at the site of insult. In this manner, L-selectin participates in leukocyte rolling within inflamed tissue. Thus, the selectin family enables binding between endothelial cells and leukocytes leading to leukocyte extravasation. In addition to defense of the host, the selectins also may participate in various pathophysiological states by facilitating leukocyte extravasation that is deleterious to the host. The establishment of the role of the selectins in the trafficking of ieukocytes during inflammation and inflammatory disease has caused intense interest in these proteins. The selectins represent molecular targets for generation of drugs to treat inflammation and inflammatory diseases, e.g., rheumatoid arthritis, psoriasis, inflammatory bowel disease and asthma. They have received widespread attention from those who desire to generate antagonists, including neutralizing antibodies and small molecule inhibitors. The way in which selectins recognize carbohydrate is being examined in detail. The mucin-like counter-receptors are being identified. The structure of the receptors may reveal

clues to aid in the discovery of antagonists. Studies in animal models are revealing potential inflammatory diseases in which anti-selectin therapy may be useful. This review will concentrate on recent progress in these areas. The reader is referred to a series of excellent reviews for additional information [l I)-13].

2. Selectin: structural motif

The selectins are multi-domain proteins whose overall structure is unique (Fig. 1). At the amino-terminal there is a lectin domain homologous to those found in several mammalian proteins which bind carbohydrate in a Ca 2+ dependent manner [14]. The iectin domain is followed by an epidermal growth factor (EGF)-like domain, which is followed by a variable number of repeated domains homologous to the short consensus repeats found in several of the complement-binding proteins (CR) [15]. The remainder of the selectin polypeptide consists of a transmembrane portion and a short cytoplasmic tail. In L-, E-, and P-selectins from humans, there are 2, 6, and 9 CR domains, respectively. In other mammalian species the number of CR domains vary. A comparison of the primary structure of the human selectins reveals that the sequences are highly homologous (65%) throughout the lectin and EGF-like domains. The CR domains are less conserved (40%). Proteins containing deletions within the lectin domain of the selectins do not bind to cells or to carbohydrate ligands [16] and the vast majority of the neutralizing antibodies against the selectins recognize the lectin domain [17,18], implying that the lectin domain is essential for the function of the protein. Constructs containing the E G F and lectin domain, but not the lectin domain alone, bind to carbohydrate, suggesting that the the E G F domain may be important for stabilizing the structure of the protein and influencing

Selectin motif

L=2 E=(4-6) P=(8-9)

• Transmembrane [] Cytoplasmictail Fig. 1. (left) S, signal sequence; lectin, C-type lectin domain; EGF, epidermal growth factor like domain; CR, complement regulatory domains; transmembrane domain and cytoplasmic tail. Human derived L-, E- and P-selectin contain 2, 6, and 9 CR domains, respectively. The number of CR domains vary in E-selectin from different species.

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the ability to bind carbohydrate [19]. CR domains do not appear to be essential for binding to carbohydrate because variants missing CR domains still bind to cells [16]. Nevertheless, the CR domains of murine L-selectin have been reported to facilitate lectin activity, perhaps by contributing to conformational stabilization or to the orientation of the selectin in the membrane [20]. One of the neutralizing antibodies against the selectins recognizes a common epitope within the CR domains of E- and L-selectin [21]. That the cytoplasmic domain is important in regulating the affinity of L-selectin can also be inferred [22]. The cytoplasmic domain of L-selectin is highly conserved across species [23]. Deleting eleven amino acids from the carboxyl-terminus of L-selectin has been found to decrease binding to endothelial cells and to decrease rolling of lymphocytes on mesenteric venules. Interestingly, binding to carbohydrate ligands is unaffected by the deletions. These observations suggest that the cytoplasmic domain of L-selectin may modulate cell-cell adhesion. The cytoplasmic tail of P-selectin has been shown to contain a sorting signal, as yet undefined, which directs the protein to storage granules [24]. Phosphorylation and selective dephosphorylation of P-selectin following platelet activation may be important for function and signal transduction. Likewise, E-selectin may be phosphorylated on one or more serine residues, and this modification could be involved in internalization of the protein in TNF-activated HUVECs [25,26]. P-selectin is also acylated at Cys-766 through a thioester linkage [27]. These modifications may regulate intracellular trafficking or other functions of the selectins. Tetramers of P-selectin bind to myeloid cells with greater avidity than do monomers [28]. The mechanism for formation of the tetramer is unclear.

3. Unique biochemistry Table 1 contains information concerning biochemical properties exhibited by the selectins. E-, L- and P-selectin are cell surface proteins that bind to receptors on other cell types, i.e., leukocytes and endothelial cells. Binding of the selectins to the counter-receptors initiates a binding interaction between blood-borne leukocytes and endothelium. Due to the shear forces of vascular flow, the leukocytes actually roll on the endothelium. The rolling behavior occurs on endothelium within tissue that is expressing P- or E-selectin on the surface of the endothelium [5-7]. In a similar manner, L-selectin promotes roiling by binding to its counterreceptor(s) when they are expressed on endothelium. Injection of L-selectin protein, or neutralizing antibodies to L- or P-selectin prevent leukocyte rolling in inflamed mesentery vessels [30,31]. Locally released chemoattractants, and integrins on the leuckocyte provide additional steps necessary for extravasation [8,32]. The process is outlined in Fig. 2. Binding of chemoattractants to receptors on the leukocyte causes release of L-selectin [32] and activation of cell-surface integrins which bind tightly to receptors on the endothelium. Subsequently, the leukocytes migrate through the endothelial layer to the site of the insult. Why selectins are suited to promote leukocyte roiling is not understood. The positioning or distribution of the selectins on the cell surface could be important. Alternatively, fast on and off rates, typical of the carbohydrate-lectin interaction, may facilitate the rolling process. The selectins appear to work in concert. Since Pselectin is pre-made and stored in granules and rapidly mobilized (min) to the cell surface upon exposure to inflammatory mediators, e.g., histamine or thrombin, it

Table 1 Biological properties of the selectins Name

Cell location

Expression kinetics

Cells bound

Function

L-selectin

leukocytes

HEV in PLN, endothelium

E-selectin

endothelium

constitutive; shed during activation temporary (2-6 h) up: LPS, IL-1, TNFa, IFNy, IL-3

lymphocyte recirculation PMN extravasation PMN extravasation lymphocyte extravasation

down: TGF, IL-4 P-selectin

platelets endothelium

transient (5-20 min) activated by thrombin histamine, superoxide

neutrophils CLA + lymphocytes monocytes eosinophils tumor cells monocytes neutrophils lymphocyte subsets

metastasis PMN extravasation hemostasis

PLN, periperal lymph node; HEV, high endothelial venules; LPS, lipopolysaccharide; IL, interleukin; TNFa, tumor necrosis factor; CLA, cutaneous lymphocyte antigen.

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J.K. Welply et al. / Biochimica et Biophysica Acta 1197 (1994) 215 2267

Leukocyte Localization to Sites of Inflammation Roiling

m,

Activati~m

~

Sticking

l~ukocy~e

Selectins

77 ( henmattraetants

Integrins

#

P

Fig. 2. Three step model of leukocytemigration to sites of inflammation.

tion [55]. Soluble P-selectin is anti-inllammatory in vitro, blunting integrin dependent responses of neutrophils [56]. Thus, non cell-bound forms of the selectins may be anti-inflammatory by limiting leukocyte interaction with endothelium through direct interference with counter-receptors or by activating integrins at a time which precedes the normal timing that is critical to the extravasation process [57-61].

4. Oiigosaccharide ligands and counter-receptors

4.1. Oligosaccharide ligands has been suggested that P-selectin functions early during inflammatory responses. This idea is supported by the finding that neutrophil migration is delayed and compromised in P-selectin deficient mice [33]. 3 to 4 h after the onset of inflammation and release of cytokines, E-selectin becomes maximally expressed on the endothelium [34,35], at the time when increasing numbers of neutrophils begin rolling and extravasate. Leukocyte rolling, mediated by L-selectin, is enhanced upon expression of inflammation-induced counter-receptor(s) in the inflamed endothelium (see below). The expression of E-selectin is a highly regulated process. Multiple cytokines including LPS, TNF, IL-1, IL-3, IFN and thrombin induce expression of E-selectin. Expression is de novo. Maximal levels on the surface of the cell are found 3 - 4 h post induction with LPS, TNF, IL-3 or IL-1 [2,36]. IFN appears to prolong the expression of E-selectin, not by stabilizing the message [37], but presumably by inhibiting degradation of the protein. TGF, IL-4 and dexamethasone inhibit expression of E-selectin [38-40]. T G F and dexamethasone decrease transcription of E-selectin message [39,41]. Inhibition upon treatment with dexamethasone was greater when induction was by exposure to LPS or IL-1 than when expression was induced by TNF. An NF-KB site in the promoter for the E-selectin gene has been shown to be necessary for expression [42,43]. Additional elements are required for maximal expression [44,45]. Thus, it appears that regulation of the expression of E-selectin may reflect a balance between pro- and anti-inflammatory agents within the inflamed tissue. De novo expression of P-selectin is also induced by T N F [46]. Secreted or shed selectins are present in serum and their levels vary with infection [47-52]. An m R N A that codes for a truncated P-selectin lacking transmembrane and cytoplasmic domains has been identified [49]. Lower molecular weight forms of the other selectins have also been found. Evidence suggests that the non-cell bound forms may have anti-inflammatory properties. Injection of soluble L and E-selectin have been shown to lessen neutrophil recruitment in rodent models of pulmonary [53,54] and peritoneal inflamma-

Extensive efforts have been made to define the carbohydrates which the selectins bind. In late 1990 and early 1991 several groups demonstrated that Eand P-selectin recognize carbohydrates containing the terminal tetrasaccharide structure, sialyl-Lewis x or sLex [ S A a 2 - 3 G a l / 3 1 - 4 ( F u c a l - 3 ) G I c N A c ] [62-68]. Shortly thereafter, sLea, [ S A a 2 - 3 G a l / 3 1 - 3 ( F u c a l - 4 ) G l c NAc], a steroisomer of sLex, was also shown to be a ligand [69,70]. Binding to sLex and sLea was shown to be Ca 2÷ dependent, consistent with the binding properties of the lectin domain of Ca 2+ dependent lectins which are homologous to the selectins. Data supporting the recognition of sLex and sLea by L-selectin came later [71,72]. Several ceils and tissues, including cancer cells [73] and lymphoid tissue [74], were shown to express sLea or sLex, or both, and to be bound by selectins, thus supporting the conclusion that the sLex family is the ligand for the selectins. Studies have showed that the selectins also recognize sulfated oligosaccharides such as 3' sulfo-Lex and 3' sulfo-Lea [75,76]. The selectins also have affinity for sulfated carbohydrates unrelated to sLex, such as sulfatides [77,78] and P- and L-selectin bind sulfated glucuronyl glycolipids, SGNL [79,80]. L-selectin also recognizes anionic carbohydrates such as fucoidan and phosphomannan in a Ca 2+ independent manner [81,82]. Binding of Eselectin to 3' sulfo-Le x is Ca 2+ dependent, while binding by P-and L-selectin is partially Ca 2+ independent [83]. L- and P-selectin may bind more tightly to sulfated species than to sLeL Differences in the affinity of binding could have interesting biological ramifications, i.e., tighter binding may result in permanent leukocyte to endothelium attachment rather than the rolling interaction. To date, there are no reports addressing the affinity of interaction between the selectins and any of the carbohydrate ligands. Millimolar concentrations of free sLex are required to inhibit binding of leukocytes to endothelium in in vitro assays. Ligands with higher affinity may yet be discovered. Following the discovery that sLex was a ligand for the selectins, several derivatives were synthesized and the potencies of the compounds, relative to sLe ~ (Fig.

219

J.l~ Welply et al. /Biochimica et Biophysica Acta 1197 (1994) 215-226

CHs'~t""~ t ~ OH

o~c,.~

e0H

a O

O~(CN~IHs 10 ~

o

HO~0H HO~ ~IIi5 CHs'-'~O

Sialyl Lewis x Fig. 3. Structure of sLex.

3), were established by comparing the ability of the derivatives to inhibit the binding of the selectins to immobilized sugars. The majority of the studies were done with E-selectin [67,83,84]. From these it can be concluded that the a 2 - 3 linkage of sialic acid to galactose, and the fucose residue, are critical determinants of the ligand for E-selectin. P-selectin has reduced affinity for sialic acid linked in an a 2 - 6 linkage [71]. Sialic acid can be replaced with sulfate, as in 3' sulfoLex. Conformational studies of solution sLea and sLex indicate that an intramolecular interaction may occur between sialic acid and fucose [85,86] and that this interaction may be important for recognition by Eselectin. Recently, a compound having only sialic acid and fucose was shown to be bound by E-selectin [87] supporting the importance of the interaction with these residues or similarly charged species replacing sialic acid. E-and L-selectin require hydroxyl groups at the 2, 3, and 4 positions of the the fucose residue while P-selectin requires only the 3-position hydroxyl group [83]. Removal of carbons 8 and 9 did not alter binding to E-selectin [67]. N-acetyl and N-glycolyl forms of sialic acid are bound equally well [67]. Substitution of hydroxyl for the N-acetyl group at carbon 5 of sialic acid gives mixed results [83,84]. Modifications of the GlcNAc result in derivatives with enhanced activity [83,84]. The most potent reported are the amino derivatives of GlcNAc, which are approximately 10-fold more potent than the parent compound [84].

immunochemistry, but P-selectin was not observed to bind to the cells with high affinity [88]. In addition, Pand E-selectin were clearly shown to bind to different receptors on myeloid cells. Exposure of myeloid cells to a variety of proteinases [89], including treatment with O-sialylation dependent proteinase, a proteinase that cleaves proteins having mucin-like chains [90], eliminated binding of P-selectin to the cells, while binding by E-selectin was unaffected. Lastly, several glycoproteins which bind tightly to the selectins have been isolated by chromatography on resins containing purified selectin proteins [91-99]. These glycoproteins have been isolated from cells or tissue that the selectins bind and represent putative counter-receptors for the selectins. The first putative counter-receptor to be purified, sequenced and subsequently cloned was GIyCAM-1 [91]. The protein was isolated from lymph nodes and binds to L-selectin. The protein contains both sialic acid and sulfate [100,101] and both residues appear to be crucial for binding by L-selectin. The primary structure of the GIyCAM-1 polypeptide indicates that the protein has many potential sites for O-glycosylation and could therefore be mucin-like. A mucin-like structure is consistent with the overall carbohydrate content of GlyCAM-1, which has been estimated at 70% carbohydrate by weight. Potential mucin-like sites are present in two regions of the polypeptide (Fig. 4). If mucin-like, the GlyCAM polypeptide should possess the ability to present extremely dense patches of sLex a n d / o r other sulfated oligosaccharide ligands for Lselectin. The extreme density of terminal sugar structures presented within the oligosaccharide-rich patches may provide the valency that is necessary for high affinity binding by the selectins. Secondly, since mucins form rod-like, extended structures, they should expose the oligosaccharide patch at the outermost boundary of the cell surface and thereby facilitate binding to the selectins. Following identification of GIyCAM-1, two additional putative counter-receptors for L-selectin have been identified and cloned [94,95]. One is CD34 and the other MAdCAM-1. CD 34 is a marker for pluripotent stem cells in bone marrow and it is also OLR1 N-Tenmmua

:

4.2. Counter-receptors Several observations suggest that there may be specific counter-receptors for each of the selectins. First it was observed that expression of sLex on cells is not sufficient to enable high affinity binding by the selectins; HT-29 carcinoma and neo-Lewis x CHO ceils were found to express high levels of sLex detectable by

OLR2

--

•

!: Conserved amino acids Noncowerved amino acids

't P°tenti~d ! P°tential ! P°tential / O-linked Conserved O-llnked site 0-linked cluster cluster (Mouseonly)

Mo~

Fig. 4. Conserved potential O-glycosylation sites of rat and mouse GlyCAM-1. The amino-termini of the mature peptides are indicated by the arrow. OLR1, potential O-linked region 1; OLR2, potential O-linked region 2. A 'cluster' is defined as two adjacent potential O-glycosylation sites.

221}

J.K. [4~,lplv et al. / Biochimica et BiophyslCa Acta 1197 (1994) 215- 22~

expressed in endothelium, but its expresssion is not restricted to lymph nodes. MAdCAM-1 is found primarily on endothe[ium in mucosal tissue. The protein participates in the homing of lymphocytes to mucosal tissue. Both CD-34 and MAdCAM-1 contain potential mucin-like regions, consistent with the role that this region may play in the binding of L-selectin to GIyCAM. MAdCAM-I is a multi-domain protein and is also a ligand for the integrin c~4/37. A putative glycoprotein receptor for P-selectin has been isolated and cloned. This protein is also mucin-like [96]. P-selectin binds to denatured protein suggesting that the conformation of the polypeptide is not critical. Treatment with a 2 - 3 specific neuraminidase eliminated binding of P-selectin, implying that sialic acid is essential [98]. Expression of the glycoprotein in cells that make sLex generated a product with binding activity, suggesting that presentation of sLex on the polypeptide may lead to binding. Three putative counter-receptors for E-selectin have been isolated but not yet cloned. One is a neutrophilderived 150 kDa protein [99] that, when treated with neuraminidase, loses binding activity. This protein may not be mucin-like because treatment of neutrophils with the mucin-specific O-sialylglycoproteinase does not eliminate binding by E-selectin. Another putative counter-receptor for E-selectin is the cutaneous lymphocyte antigen, CLA, characteristic of skin-derived memory lymphocytes (102). Binding of E-selectin to CLA-positive cells is prevented by anti-CLA and by neuramindase treatment. CLA is immunologically distinct from sLex. Treatment of CLA-positive cells with neuraminidase reveals Lex, implying that the CLA antigen may contain a derivative of sialic acid not present in sLex, or that CLA may be a disialylated structure. The third putative counter-receptor was recently isolated from bovine T cells [103]. These cells carry determinants distinct from sLex and CLA. The cells were recruited to the skin of animals following the induction of E-selectin in that organ. Similar to Lselectin, E-selectin may contain several different counter-receptors which are expressed in an organspecific manner. Another critical aspect of the counter-receptors for the selectins involves oligosaccharide processing. The GIyCAM-1 polypeptide is expressed in mammary tissue, but does not carry sulfated sugars and is not recognized by L-selectin [104]. Thus, the biological activity of the counter-receptors may be controlled by organ specific expression of the oligosaccharide processing enzymes. The structure of the oligosaccharides on the counter-receptors may be influenced by the presence of inflammatory mediators or other factors. To determine the role in leukocyte trafficking of each of the counter-receptors, antagonists to each must be tested in animal models of inflammation. Recently,

heparin like structures on non-lymphoid endothelial cells were implicated as counter-receptors for L-selectin [105]. The heparin-like structures might be expressed on GIyCAM-I, CD-34 or MAdCAM-I polypeptides. 4.3. Critical a m i n o acids ~br binding

The lectin domain of the selectins contains 13 of the 14 invariant residues [15] (Fig. 5) of the C-type lectin family as well as the 18 additional residues that are highly conserved within the family [106]. Thus, by primary sequence, the selectins closely resemble other mammalian lectins which bind sugar in a Ca 2+ dependent fashion. In addition, the lectin domains of the selectins have metal binding features in common with the mannose binding protein (MBP), the only C-type lectin family member whose X-ray structure has been determined. MBP has two binding sites for Ca 2+ [107,108]. Although site No. 1 is not conserved in the selectins, E-, P- and L-selectin all contain the amino acids which ligate Ca 2+ at site No. 2 of MBP and hydrogen bond with hydroxyl groups of mannose and fucose [108,109]. By comparison with MBP, it has been postulated that Glu-80 and Asn-82 of the selectins form hydrogen bonds with fucose 3-OH, and Glu-88 and Asp-106 hydrogen bond with the 2-OH of fucose within the sLex structure [109,110]. By analogy with MBP, the fifth member of Ca 2+ binding site No. 2 is

L se]ectin E selectin E selectin MBP-a Consensus

WTYHYSEKPM N W Q N A R N ~ WSYNTSTEAM TYDEASA WTYHYSTKAY SWNISRK KFFVTNHERM PFSKVKA .................

DNYTDLVA]Q Q~YTHLVAIQ N~TDLVAIQ ELRG}VAIPR

NN~iEYLEK~]I NK IEYLNS NK DYLNK NA~KAIQE N--

41 t selectin E-selectin P-selectin MBP-a Consensus

TLFFS W I I LSY W I VLFY N I VAKT...SAF ...... F

E-selectin P-selectin Consensus

RNI..GGIN IRKV..NSrVW iRKN..NKTW TDkg%EGQF

F

TWV; VWV~ TWV~ M9

NKSLT QKPLT EEAK KKALT NEAE R..I,T ..YS --

FF

NRQ ~NKRs ~ ---

PGE DNE KDE - -E

A V-I .......

TA. TA.

-.....

[] 14 'invariant'residuesofC-typelectins [] Essential for eitherP or g- selectinto bind sLex Inhibitory peptides Key ~mino acids for fucosebinding(speculative)

Fig. 5. Critical residues for selectin-carbohydrate interaction. A m i n o acid alignment of the C-type lectin domains from the h u m a n selectins and thc mouse mannose binding protein (MBP-a) based on conserved cystine residues. O p e n boxes, residues c o m m o n to all C-type lectins; shaded boxes, residues criticalfor binding of either Por E-selectin to sLcx; stippled boxes, peptide domains that inhibit Pand E-sclcctin binding; arrows, residues critical for M B P Ca z' coordination (site 2) and mannose binding and suspected to be part of the fucose binding pocket of the sclcctins. See text for discussion.

J.K. Welply et al. /Biochimica et Biophysica Acta 1197 (1994) 215-226

Asp-106 in the selectins. It is assumed that Asp-106 of the selectins also coordinates with Ca 2÷ at site No. 2. A variant of P-selectin that contains an Asn substitution for Asp-106 no longer binds myeloid cells, consistent with a functional role for this residue [110]. Studies with variants of P- and E-selectin indicate that Pro-46, Ser-47, Tyr-48, Arg-97, Lys-99, Lys-lll, and Lys-ll3 are also involved in binding to sLex [110,111]. Substitutions at positions 32, 67, 74, 84 and 86 do not effect binding. If it is assumed that positions 80, 82, 88, 105 and 106 are involved in binding to fucose and Ca 2+, then residues at positions 46, 47, 48, 97, 111 and 113 may form a charged pocket for binding sialic acid. Mouse and bovine P-selectin have a substitution of Glu at position 88 rather than Gin, a substitution that would not be expected to affect hydrogen bonding to fucose [112,113]. Equilibrium dialysis studies indicate that P-selectin binds two Ca 2÷, even though by sequence comparison, Ca 2÷ site No. 1 of MBP is not conserved [114]. The second site is not yet defined in the selectins. Binding of Ca 2÷ protects E-selectin from proteolysis (S.C. Howard, unpublished data) as has been observed for MBP. Peptides corresponding to residues 23-30 and 54-63 have been reported to bind to Ca ~+ and block both P- and E-selectin mediated cell adhesion [114,115]. Based on modeling studies, the sequences corresponding to these lie distant from those assumed to bind fucose and those shown by mutagenesis, 46, 47, 48, 97, 99, and 113, to be essential for binding sLex. It was recently reported that peptides from the $2 and $3 domains of pertussis toxin, corresponding to homologous regions of E-selectin (residues 23-38), inhibited neutrophil binding to immobilized E-selectin [116].

5. Pathophysiology 5.1. Immunohistochemical observations

E-selectin has been detected on endothelium in a variety of acute inflammatory conditions including appendicitis, tonsillitits, delayed hypersensitivity reactions, sepsis and cutaneous inflammation [117,118]. It is also expressed in endothelium during chronic inflammmation, such as rheumatoid arthritis, inflammatory bowel disease, and gingivitis [119-123]. Surprisingly, E-selectin has also been observed at sites that are distant from the site of injury. For instance, E-selectin was found on endothelium within the skin in models of peritonitis [124] and in rat lung in a model of ischemic reperfusion of the hind limb [125]. The expression of E-selectin correlates with the kinetics of the extravasation of neutrophils in a variety of animal models. Further, in animal models, E-selectin is induced with delayed kinetics, similar to that

221

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0

0.25

0.5

1

2

4

im

¢n

Time (hr) Post LPS

C Fig. 6. R N A dot blot of E-selectin message induction in various organs following bacterial endotoxin treatment. Rats were treated with 100/zg/kg bacterial endotoxin (lipopolysaccharide; LPS), vehicle (saline) by i.v. injection or were untreated. Organs were harvested at the indicated times, total RNA isolated and 5 /~g RNA was probed with 32p labeled E-selectin cDNA.

observed in tissue culture following treatment with LPS or cytokines [126,127]. Expression of E-selectin mRNA in various organs following intravenous injection of LPS into the rat is shown in Fig. 6. Extremely high levels are found in the lung and considerably lower or undetectable levels are observed in spleen, liver, kidney and bowel. Also, similar to what has been observed in vitro, expression of E-selectin diminishes 6 h after injection of LPS. Intravenous injection of LPS into the mouse and baboon results in high expression in the lung as well as the heart, liver and kidney [34,126]. In certain situations, expression of E-selectin may be prolonged in the skin. Intradermal injection of TNF results in the expression of E-selectin that is maintained for more than 24 h [127]. Expression for longer times may reflect the involvement of E-selectin in the recruitment of lymphocytes that bear CLA [128]. Expression of P-selectin on the cell surface is difficult to measure by immunochemistry because staining of internally stored material frequently occurs during the normal fixation procedures. In cell culture, Pselectin expression at the cell surface is transient (min) but may be sustained in the presence of oxygen radicals [129]. In both Graves disease and rheumatoid arthritis, P-selectin is expressed [130,131]. Monocytes have been shown to bind P-selectin in frozen sections of inflammed synovium [131]. MECA 79 antibody [132] has been used to detect putative ligands bound by L-selectin. This antibody stains pancreatic islet cells after the onset of diabetes in non-obese diabetic (NOD) mice [133], suggesting that L-selectin could be operative in this disease. Re-

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J.K. Welplv el al. / Biochimica et Biophy~tca A ¢ m 1197 ~1q94) 215 22o

combinant L-selectin stains myelinated regions of the central nervous system in mice with multiple sclerosis, suggesting that a counter-receptor may be present in this tissue [134,135]. Myelinated areas within rat cerebellum and cerebrum are bound by lymphocytes in an L-selectin sensitive manner, consistent with a potential role for L-selectin in lymphocyte recruitment in diseases involving demyelination [135]. Staining with recombinant L-selectin has detected potential counterreceptors in lymph nodes, white matter, neurons, Purkinje ceils, choriod plexus of the central nervous system and distal tubes and capillary blood vessels of the kidney [136].

5.2. Effect of antagonists in animal models Evidence that the selectins play a direct rote in pathophysiology has been approached using three types of blocking reagents: neutralizing antibodies, recombinant selectins and oligosaccharide ligands. The results of these studies suggest that inflammation of the lung involves all of the selectins. In a model of acute inflammation brought on by activation of complement in the rat, P-selectin was found to be expressed on the pulmonary endothelium [137]. Neutrophil sequestration within the capillary bed of the lung was diminished by administration of anti-P-selectin or sLex ligand [137, 138]. Both reagents inhibited sequestration by up to 30-40% and tissue hemorrhage and increases in vascular permeability were significantly reduced. In another model of rat pulmonary inflammation induced by immune complex deposition, E-selectin was expressed in pulmonary endothelium 3-4 h post insult, coincident with the recruitment of neutrophils into the bronchial lavage fluid. Administration of anti E-selectin [139] (Table 2) decreased the influx of neutrophils by up to

Table 2 Inhibition of immune-complex alveolitis and dermatitis by E-selectin antibodies Percent inhibition

Neutrophil influx M P O content Hemorrhage index Permeability index

lung

skin

47 67 94 53

72 84 40

Rats were treated intratracheally or intradermally with B S A / a n t i BSA and alveolar neutrophils (influx), total tissue neutrophils (myeloperoxidase activity or MPO), hemorrhage and permeability were measured at 4 h. T r e a t m e n t animals received neutralizing anti-E-selectin antibody CL-3 F(ab') 2 fragments. Control animals received either no antibody or non-neutralizing anti-E-selectin F(ab') 2 fragments. Data compiled from Figs. 2, 3, 4 and 5 of Ref. 138.

Leukocyte Accumulation Promoting F i b r i n Deposition is Mediated by P-Selectin t,.

100

~

80

~=

60.

~

40

~

20.

• •

0

FIBRIN

Control Anti-P.Selectin

LEUKOCYTES PLATELETS Adapted from Palabrica et al., 1992

Fig. 7. Inhibition of leukocyte accumulation and fibrin deposition by P-selectin antibodies. Dacron grafts were implanted in a shunt between the femoral artery and vein in baboons. After 90 rain of reflow, bound I~lln-labeled platelets and leukocytes were measured using a G a m m a camera. Fibrin deposition was measured by immunoassay. Anti-P-selectin antibody GA6 F(ab')~ fragments (2 m g / k g body mass) was administered IV. Black bars, no antibody; striped bars, GA6 antibody. A non-neutralizing anti-P-selectin antibody (ACI.2) had no effect on leukocyte retention. Data taken at the 90 min timepoint from Figs. 2 and 4 of Ref. 142.

47% and hemorrhage of the tissue was lessened by up to 94%. Subsequently, recombinant, soluble forms of E- and L-selectin were shown to have protective effects in the immune complex model [54]. Soluble forms of Land P-selectin were also protective in the model of complement activation [54]. Both P- and L-selectin antagonists protect heart tissue during reperfusion after acute myocardial infarction in the cat, a model in which tissue damage is believed to be due to neutrophils trapped in capillaries [140,141]. The number of trapped neutrophils was diminished by treatments with the antagonists. Antagonists of P-selectin may be beneficial for blocking platelet-leukocyte interaction as thrombi develop. In baboons, administration of anti P-selectin decreased fibrin deposition into Dacron graft implants [142] (Fig. 7) without diminishing platelet accumulation into the grafts. The results suggest that the trapping of leukocytes, via interaction with platelets, may contribute to the deposition of fibrin. Blocking P-selectin should prevent this interaction and therefore, an antagonist of P-selectin may have utility as an anti-thrombogenic agent. Antagonists of L-selectin have been shown to lessen peritoneal inflammation [55] and to inhibit insulinitis and diabetes in NOD mice [143] suggesting that Lselectin antagonists may have utillity in a variety of inflammatory conditions and diseases.

J.K. Welply et al. /Biochimica et Biophysica Acta 1197 (1994) 215-226 5.3. Disease targets

A large body of data suggests that adhesion proteins are molecular targets for therapeutic intervention in inflammation. One of the best described is reperfusion injury where agents against L- and P-selectins, ICAM-1 [144] and CD-18 [145] have been shown to be effective. Adult respiratory distress may be an acute indication for P-selectin, based upon studies done in the rat as described above. Use of selectin antagonists for chronic problems, such as thrombogenesis, may have some limitations, although it has been shown that leukocytemediated host defense to bacterial peritonitis and soft tissue infection in rabbits is normal after administration of anti-P-selectin [146]. Thus, chronic diseases like rheumatoid arthritis and Graves disease may be targets for therapy with a P-selectin antagonist. Long term use of L- and E-selectin antagonists may be more problematical. Long term therapy with an E-selectin antagonist may lead to the LAD-2 phenotype. LAD-2 patients do not produce sLex and they have recurrent bacterial infections of the lung, skin and gingival tissue [147,148]. As anticipated, neutrophils from these patients do not roll on E-selectin, nor do they extravasate at normal levels. An L-selectin antagonist is expected to have a negative effect upon lymphocyte movement into the lymph system through peripheral nodes. Because there are now at least three counter-receptors for L-selectin, it may be possible to target one that is not involved in lymphocyte recirculation but is essential for progression of a specific disease, i.e., diabetes. Both E- and P-selectin also recognize a variety of tumor ceils [149151]. Thus metastasis may also be a target of antiselectin therapy. Cells with low metastatic potential have been converted to high potential when transfected with c D N A encoding a glycoprotein bearing sLex [152].

6. Concluding remarks D a t a gathered thus far suggest that agents which antagonize the selectins will help protect the host tissue from damage occuring during inflammation. Administration of three different types of selectin antagonists, i.e., neutralizing antibodies, recombinant soluble selectins and competing oligosaccharide ligands, resulted in decreased estravasation of leukocytes in a variety of animal models. Up to 70% inhibition of leukocyte extravasation has been achieved using selectin antagonists. Work to this point indicates that the discovery and development of potent small-molecule antagonists of the selectins will be difficult. Derivatives of sLex are 10-30-fold more potent than the parent sugar. The recent identification of counter-receptors for each selectin opens up unexplored routes for phar-

223

maceutical manipulation. Individual counter-receptors may play unique physiological roles. For instance, GlyCAM-1 may be essential for lymphocyte recirculation while MAdCAM-1 is essential for lymphocyte migration into mucosal tissue and CD-34 may be involved in neutrophil or monocyte migration during acute inflammation. Will each counter-receptor contain oligosaccharides having structural features that are not available in the other counter-receptors? Will the biosynthetic enzymes for synthesis of these oligosaccharides become targets for inhibition? The selectins have made the medical community aware of the physiological relevance of cell-surface lectins and their oligosaccharide counter-receptors. A new frontier for drug discovery is now apparent.

Acknowledgements We thank Kurt Drickamer, Pam Manning, Phil Streeter and William Westlin for helpful discussions.

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