Introduction

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A characteristic common to all inflammatory reactions is the local accumulation of specific subsets of leukocytes. For example, during ischemia-reperfusion injury or bacterial pneumonia, large numbers of circulating neutrophils collect at the site, whereas in parasitic or allergic inflammatory responses, the localized cellular infiltrate is notable for eosinophil accumulation. Some of these responses are considered useful to host immunity (e.g., in pneumonia and in parasitic disease), whereas in others the response is felt to be detrimental (e.g., in reperfusion injury and in asthma). As a result, when cell accumulation is deleterious, treatment of the latter types of conditions with anti-inflammatory drugs that prevent cell accumulation would be desirable (1, 2).

Among the earliest events that occur during inflammatory responses is the interaction of circulating leukocytes with vascular endothelium adjacent to the inflammatory site. Microscopically, these events can be seen as cell margination, whereby leukocytes tether and roll along the lumen of the blood vessel, typically at the level of the postcapillary venule. It is now well recognized that these cell-cell interactions are dependent on the function of a family of adhesion molecules, the selectins, that include E-selectin, L-selectin, and P-selectin (3). Each consists of an N-terminal domain of 117-120 amino acids possessing calcium- dependent (C-type) lectin activity; this region is the most critical portion of the molecule for adhesion (4).

A variety of carbohydrate-containing mucin-like ligands for selectins have been identified (5). Although the tetrasaccharide sialyl Lewis X (sLe^x), which contains 2,3-linked terminal sialic acid residues and 1,3-linked fucose, can bind to all three selectins (6), a number of relatively selectin-specific ligands have been identified, such as P-selectin glycoprotein ligand-1 (PSGL-1) for P-selectin; E-selectin ligand-1 (9) and myeloglycans (10) for E-selectin; and CD34 (11), glycosylated cell adhesion molecule-1 (12), mucosal addressin cell adhesion molecule-1 (13), and an as-yet- unidentified cytokine-inducible endothelial structure (14, 15) for L-selectin.

The importance of selectins and their carbohydrate-containing counterligands are clearly demonstrated by studies of protein-based antagonists or of mice with deficiencies in these molecules (reviewed by Lowe and Ward [16]). It is therefore not surprising that these adhesion molecules have become targets for the development of novel anti- inflammatory compounds. One such approach has focused on the complex carbohydrates themselves (17) because they are concise informational packages (18) that represent a great potential for biologic specificity and leads for the generation of glycomimetic therapeutics (19, 20). As part of ongoing efforts to generate anti-inflammatory selectin antagonists for possible use in inflammatory diseases in which neutrophil or eosinophil influx is considered pathogenic, we tested new glycomimetic compounds mimicking sLe^x and a glycomimetic derived from the natural substance glycyrrhizin (17, 21) for their ability to interfere with selectin-dependent neutrophil or eosinophil adhesion in a variety of in vitro assays. In these compounds, different sugars were substituted for the glucuronic acids and were found to inhibit E-selectin-, L-selectin-, and P-selectin-mediated adhesion of both leukocyte types.

	Materials and Methods

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Antibodies

All antibodies used had functional adhesion-blocking activity and were used at saturating concentrations. For selectin-based adhesion assays, F(ab')₂ preparations of murine monoclonal antibodies (mAbs) recognizing human E-selectin (mAb 7A9, immunoglobulin G₁ [IgG₁] from Dr. Walter Newman, Otsuka American Pharmaceutical, Inc., Rockville, MD [22]) and human P-selectin (mAb G1, IgG₁ from Dr. Rodger McEver, University of Oklahoma, Oklahoma City, OK [23]) were generously provided. The L-selectin antibody LAM 1-3 (IgG₁) was generously provided by Dr. Thomas Tedder, Duke University, Durham, NC (15).

For integrin-based adhesion assays, F(ab')₂ preparations of murine mAb recognizing human intercellular adhesion molecule-1 (ICAM-1) were obtained from Caltag (South San Francisco, CA). Also used were murine mAbs recognizing human CD11a (MHM24, IgG₁) and CD11b (H5A4, IgG₁), both generously provided by Dr. James Hildreth, Johns Hopkins University, Baltimore, MD (24).

Isolation of Human Leukocytes

Human neutrophils were purified from peripheral blood using density gradient centrifugation, and red blood cells were removed by hypotonic lysis (25). Human eosinophils from allergic donors were purified by negative selection using an immunomagnetic bead technique (25). Purity and viability for eosinophils and neutrophils always exceeded 95%.

Leukocyte Adhesion Assays

E-selectin-dependent adhesion (37°C, 10 min) was performed with interleukin (IL)-1-treated (5 ng/ml, 4-6 h; Upstate Biotechnology, Lake Placid, NY) human umbilical vein endothelial cell (HUVEC) monolayers (25). L-selectin dependent adhesion was performed as for E-selectin adhesion except that the assay was performed under rotating conditions (80 revolutions/min) and at 4°C (15). P-selectin-dependent adhesion (45 min, room temperature) was done on 96-well plates coated with 1% glutaraldehyde-fixed immobilized human peripheral blood platelets stimulated with phorbol myristate acetate (10⁷ M, 37°C, 10 min [26]). For E-selectin assays, HUVECs were preincubated with compounds for 30 min at 37°C prior to addition of ⁵¹Cr-labeled leukocytes. For L-selectin assays, cells were preincubated with compounds or mAbs for 30 min at 37°C prior to adhesion. In the case of the L-selectin assay, microscopic cell counts were used instead of percent adherence (15).

To determine whether 1-mM concentrations of these selectin antagonists had any activity in integrin-based adhesion assays, adhesion of unstimulated or platelet-activating factor (Sigma Chemical Co., St. Louis, MO)-treated (1 µM, 5 min) neutrophils to an optimal concentration of immobilized soluble intercellular adhesion molecule-1 (ICAM-1) (500 µg/ml; R&D Systems, Minneapolis, MN) was also tested in the presence or absence of compounds.

Leukocyte and Endothelial Cell Viability

Viability of neutrophils, eosinophils, or cytokine-activated HUVEC monolayers was determined by light microscopic analysis of erythrosin B dye exclusion as previously described (27).

Statistical Analysis

Paired Student's t test was used to analyze differences among treatments. Values for P < 0.05 were considered statistically significant.

	Results

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Several carbohydrate-based compounds mimicking sLe^x were synthesized (Figure 1) and tested in adhesion assays for their ability to inhibit selectin-mediated adhesion of eosinophils and neutrophils in vitro. These included novel compounds that mimic sLe^x, derived from glycyrrhetinic acid. Using 1-mM concentrations, results of statistically significant antagonistic effects in E-selectin-, L-selectin-, or P-selectin-dependent adhesion assays are displayed in Figure 2. Stimulus-induced adhesion was always at least 4-fold higher than without stimulus (see figures), and selectin dependence was confirmed with blocking mAbs. As shown in Figure 2A, compounds GM1292 and GM2296 caused significant inhibition of both eosinophil and neutrophil adhesion to IL-1-stimulated HUVEC under static conditions (a predominantly E-selectin-dependent assay). For neutrophils, sLe^x with a lipid tail (sLe^x-Lipid) also inhibited adhesion. Compound GM2296 was the most effective ( 60-75% inhibition) and its inhibitory effect was similar to that seen with an E-selectin-blocking mAb. In contrast, compounds GM1925 and GM1380 had no significant effect on eosinophils or neutrophils (n  4, data not shown). In the predominantly L-selectin-dependent rotational assay using IL-1-treated HUVECs, two of the same compounds, GM1292 and GM2296, caused significant inhibition of both eosinophil and neutrophil adhesion, with compound GM2296 being the most effective ( 70-95% inhibition, Figure 2B). Inhibitory effects seen with compound GM2296 were at least as great as those seen with the L-selectin-blocking mAb. As in the previous assay, compounds GM1925 and GM1380 had no significant effect on eosinophils or neutrophils; however, here sLe^x-Lipid also failed to inhibit adhesion (n  4, data not shown). In Figure 2C, significant effects on leukocyte adhesion to immobilized phorbol myristate acetate-activated platelets (a predominantly P-selectin-dependent assay) were again seen with compounds GM1292 and GM2296, with compound GM2296 being the most effective ( 50-70% inhibition) and at least as effective as the P-selectin-blocking mAb. Again, compounds GM1925, GM1380, and sLe^x-Lipid had no significant effects on eosinophils or neutrophils (n  4, data not shown). To determine whether the active antagonists might have inhibitory effects in non-selectin-based adhesion assays, 1-mM concentrations of active compounds were tested in ICAM-1-dependent adhesion assays (using neutrophils, with or without prior activation with 1 µM platelet-activating factor). As expected, no inhibition of adhesion was seen (n  2, data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1.   Structures of five sulpho-Lex, sLex, and triterpene-based sLex glycomimetics tested for their ability to antagonize selectin-dependent adhesion of human eosinophils and neutrophils in vitro.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Effect of active oligosaccharide compounds on neutrophil (filled bars) and eosinophil (open bars) adhesion. Inhibitions seen with indicated mAbs are shown on the right side of each panel. Values represent means ± SEM; *P < 0.05. (A) Static adhesion to IL-1-treated HUVECs. Adherence to unstimulated and stimulated HUVECs was 5 ± 2% and 24 ± 4% for neutrophils and 5 ± 2% and 21 ± 5% for eosinophils, respectively (n = 4 -9). (B) Rotational adhesion to IL-1-treated HUVECs. Adherence of neutrophils and eosinophils to unstimulated and IL-1-stimulated HUVECs was 119 ± 48 and 950 ± 116 cells per four high-power fields for neutrophils, and 95 ± 50 and 578 ± 64 cells per four high-power fields for eosinophils, respectively (n = 3-6). (C) Adhesion to immobilized activated platelets. Adherences to unstimulated and stimulated platelets were 5 ± 2% and 58 ± 6% for neutrophils, and 7 ± 3% and 49 ± 5% for eosinophils, respectively (n = 3-7).

In the next set of experiments, the compounds found to be active in Figure 2 in each of the respective adhesion assays were titrated. Thus, compounds GM1292 and GM2296 were tested at several concentrations in all three assays, whereas sLe^x-Lipid was also tested in the E-selectin-dependent adhesion assay. Results displayed in Figure 3A for the E-selectin-dependent adhesion assay show similar or slightly greater effects of these compounds on neutrophils compared with eosinophils, with rank order of potency GM2296 > GM1292  sLe^x-Lipid. For compound GM2296, the concentrations producing 50% inhibition of adhesion (IC₅₀s) with both cell types were approximately 0.5 to 0.8 mM. Although IC₅₀s for the other compounds could not be determined, significant inhibition was seen at 3 mM for compound GM1292 (neutrophils and eosinophils) and sLe^x-Lipid (neutrophils only, data not shown). In the P-selectin-dependent assay, compounds GM2296 and GM1292 were slightly less active, yielding IC₅₀s of 0.7 and 1.0 mM for neutrophils (Figure 3B). As shown in Figure 3C, when tested in the L-selectin-dependent adhesion assay, slightly greater effects were again seen on neutrophils compared with eosinophils, with rank order of potency GM2296 > GM1292, and IC₅₀s for GM2296 with both cell types slightly better, approximately 0.2 to 0.5 mM. In each assay, any observed inhibition was completely reversible upon removal of the antagonist, and none of the compounds had any effect on endothelial or leukocyte viability as detected by dye exclusion (n  3, data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Titration of effects of active compounds on neutrophil (filled symbols) and eosinophil (open symbols) adhesion. Values represent means ± SEM, *P < 0.05. (A) E-selectin-mediated adhesion was inhibited by GM1292 (circles), sLex-Lipid (diamonds), and GM2296 (squares). IC50s for GM2296 were 0.5 and 0.8 mM for neutrophils and eosinophils, respectively. (B) P-selectin-mediated adhesion was inhibited by GM1292 (circles) and GM2296 (squares). IC50s for GM2296 and GM1292 were 0.7 and 1 mM for neutrophils, respectively. IC50s for eosinophils could not be determined; however, significant inhibition of the adhesion of both neutrophils and eosinophils was seen at 3 mM. (C) L-selectin-dependent adhesion was inhibited by GM1292 (circles) and GM2296 (squares). IC50s for GM2296 were 0.2 and 0.45 mM for neutrophils and eosinophils, respectively. IC50s for GM1292 could not be determined; however, significant inhibition on both neutrophils and eosinophils was seen at 3 mM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present experiments tested the inhibitory effects of five novel glycomimetics on E-selectin-, L-selectin-, and P-selectin-mediated human eosinophil and neutrophil adhesion in vitro. Among these compounds, GM2296 was the most effective antagonist, significantly inhibiting all three selectin-mediated adhesive events for eosinophils and neutrophils in vitro with IC₅₀s ranging between 0.2 and 1 mM. Compound GM1292 also significantly inhibited selectin-mediated adhesion of both cell types, but was less active. The sLe^x tetrasaccharide, sLe^x-Lipid, significantly inhibited E-selectin-mediated neutrophil but not eosinophil adhesion, and failed to alter significantly adhesion mediated by the other selectins. The remaining two compounds did not have significant activity when tested at up to 1-mM concentrations. These estimates of IC₅₀s may be somewhat higher than in actuality, because calculations were based on 50% inhibition of adhesion even though each of the selectin-dependent adhesion assays had a small but consistent CD18-dependent component (data not shown). This was evidenced, for example, in Figure 2 by the inability of the selectin-blocking mAb to yield complete inhibition of adhesion. In contrast, these antagonists did not have any effect on integrin-mediated adhesion because they failed to affect neutrophil adhesion to ICAM-1.

Although selectin antagonists, including antibodies, have been shown to have inhibitory effects in several animal models of eosinophil and/or neutrophil recruitment (1, 2, 28), our data are the first to demonstrate inhibitory effects with glycomimetics on human cells and at potentially achievable concentrations. Furthermore, in view of studies in knockout mice showing additive effects when the function of more than one selectin is eliminated (1, 31), the ability of GM2296 and GM1292 to function as pan- selectin inhibitors suggests that these compounds or their derivatives may be effective anti-inflammatory agents when tested in vivo. Indeed, in the rotational, L-selectin-dependent assay, the ability of GM2296 to provide somewhat greater inhibition than that seen with the L-selectin mAb was probably due to its ability to inhibit the small component of E-selectin-dependent adhesion typically seen in this assay (Picker and colleagues [32] and data not shown).

The L-selectin assays were performed under rotational conditions designed to mimic shear forces under which the molecule functions (15), and should therefore provide a reasonable estimate of how effective the antagonists might be under similar conditions in vivo. However, one potential shortcoming of our studies was that the other two selectin-dependent assays were performed under static conditions. On the basis of recently published studies comparing adhesion of human eosinophils and neutrophils, however, it appears that static and flow adhesion assays yield similar results with these cell types. For example, experiments examining adhesion of eosinophils and neutrophils under static conditions found comparable levels of sialidase- and protease-sensitive adhesion among these cell types (23). In a subsequent study, nearly equivalent levels of PSGL-1 expression and rolling adhesion of eosinophils and neutrophils were observed (33). Similarly, human neutrophils bound better than eosinophils to E-selectin under either static (22) or rolling conditions (34). Therefore, although it is ultimately desirable to test these and other selectin antagonists under more physiologic conditions of shear stress, their use in static adhesion assays should give an initial indication as to their activity as selectin inhibitors.

It is interesting to note that the glycomimetics of the complex oligosaccharide sLe^x were more potent than the natural epitope. For example, the natural tetrasaccharide sLe^x epitope attached to a lipid tail, sLe^x-Lipid, had only minimal activity, whereas GM1925 (a de-fucosylated form of sLe^x-Lipid) and GM1380 (a sulfo-sLe^x) showed only trace inhibition to the three selectins. In contrast, GM1292 is a natural product that contains the key structural mimics necessary for selectin antagonism, namely the carboxylic acid and a mimic of the calcium-binding ability of L-fucose. GM2296, fucose-carbon-glycyrrhetinic acid, was constructed to contain the necessary carboxylic acid and a carbon fucoside, and would be expected to have the ability to flex enough to fit all three selectins. Although in vivo data with these compounds are limited, both GM1380 and GM1292 have been shown in vivo to reduce myocardial infarct size in a rabbit ischemia model (35, 36).

In summary, our results suggest that compounds GM2296 and, to a lesser degree, GM1292 are inhibitors of E-selectin-, L-selectin-, and P-selectin-mediated adhesion of eosinophils and neutrophils in vitro. These compounds, along with others being developed (30, 37), should serve as potential tools to discover effective therapeutics. Additional studies are needed to determine whether these or more potent pan-selectin inhibitors, given via different routes of administration, will be useful for antagonizing selectin- mediated inflammatory events in allergic airways inflammation and other disorders in vivo.