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Glycosulfopeptides modeled on P-selectin glycoprotein ligand 1 inhibit P-selectin-dependent leukocyte rolling in vivo ¹

ANNE E. R. HICKS^*,2, ANNE LEPPÄNEN^,2, RICHARD D. CUMMINGS, RODGER P. MCEVER^¶, PAUL G. HELLEWELL^* and KEITH E. NORMAN^*3

^* Cardiovascular Research Group, Division of Clinical Sciences (North), University of Sheffield, Sheffield, UK; and
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, and
^¶ Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA

³Correspondence: Cardiovascular Research Group, Clinical Sciences Centre, Northern General Hospital, Sheffield S5 7AU, UK. E-mail: k.norman{at}shef.ac.uk

SPECIFIC AIMS

The aim was to investigate whether rationally designed selectin antagonists (glycosulfopeptides) inhibit P-selectin-dependent leukocyte rolling in living blood vessels.

PRINCIPAL FINDINGS

1. Glycosulfopeptides (2-GSP-6, 4-GSP-6) modeled on 18 amino acids from the NH₂ terminus of mature human P-selectin glycoprotein ligand 1 (PSGL-1) enzymatically modified in vitro to express sialyl Lewis^x (sLe^x) and sulfated tyrosine at appropriate locations inhibited preexisting P-selectin-dependent leukocyte rolling in vivo (Fig. 1 ). Control glycosulfopeptides (2-GSP-1, 4-GSP-1) carrying only N-acetylgalactosamine in place of sLe^x did not inhibit P-selectin-dependent leukocyte rolling.

View larger version (23K): [in this window] [in a new window]   Figure 1. a) Effects of different GSP on proportion of leukocytes rolling through observed venules. Baseline leukocyte rolling was determined from a 1 min observation recorded 30 min after surgical preparation of the cremaster for intravital microscopy. Glycosulfopeptides were injected at 31 min and effects determined between 32 and 33 min. P-selectin (RB40.34) or PSGL-1 (2PH1) blocking antibodies were injected at 45 min as a positive control. Numbers in parentheses indicate the dose of GSP in µmol/kg. Asterisks indicate significant difference from baseline rolling (*P<0.05, **P<0.01, ***P<0.001). b) Time course of effect of GSP on leukocyte rolling. Individual vessels were tracked for up to 10 min after injection of GSP. Open diamonds indicate rolling after 4.3 µmol/kg 2-GSP6. Filled circles, squares, and triangles indicate rolling after 1.43, 4.3, and 12.9 µmol kg 4-GSP-6, respectively. Data in panels a, b are presented as mean ± SE of n = 9–12 venules from 3–4 mice per treatment.

2. A low dose (1.43 µmol/kg) of active glycosulfopeptide increased leukocyte rolling velocity (Fig. 2 ). Higher doses (4.3 and 12.9 µmol/kg) of glycosulfopeptides reduced the proportion of leukocytes rolling and increased the velocity of cells continuing to roll (Figs. 1 and 2) . The extent of inhibition was similar to that seen with PSGL-1 blocking antibody, but did not match that given by P-selectin blocking antibody.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of different doses of 4-GSP-6 on leukocyte rolling velocity. Average rolling velocities were determined every minute for 10 min after injection of 4-GSP-6. Data are from 9–12 venules from 3–4 mice per treatment. Asterisks indicate significant difference from baseline rolling (*P<0.05, **P<0.01).

3. The proportion and velocity of leukocytes rolling were altered for only 1–2 min after injection of lower doses (4.3 µmol/kg) of glycosulfopeptides. Renal clearance of intact material to urine was confirmed using ¹²⁵I-labeled 4-GSP-6.

4. 4-GSP-6 injected at 12.9 µmol/kg gave a sustained (>10 min) elevation of leukocyte rolling velocity (Fig. 2) , suggesting that redistribution of some material to total body water may have occurred, prolonging its activity.

CONCLUSIONS AND SIGNIFICANCE

The belief that inhibiting selectins will provide benefit in diverse inflammatory diseases has been widespread since it was demonstrated that this family of C-type lectins supports the first detectable event (leukocyte rolling) in the recruitment of leukocytes from the systemic circulation to sites of inflammation. Leukocyte rolling is a dynamic event requiring constant formation of new bonds at the front of a cell coupled with detachment at the rear. The constant need for new bond formation in this system suggests that it may be susceptible to drug intervention (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Proposed mechanism of action of selectin antagonists. a) Leukocyte rolling is critically dependent on constant bond formation and rupture. b) After a slight delay ( 20 s from jugular to cremaster), injected inhibitor reaches observed venules. Soluble selectin antagonists compete with cell-bound selectin ligands and increase leukocyte rolling velocity (double arrow). c) If sufficient material is applied and remains in the circulation, all endothelial selectins may be blocked. Rolling cells will detach and new attachments will be prevented. Although many putative P-selectin antagonists have been described, glycosulfopeptides are the first rationally designed, structurally defined molecules to demonstrate this mechanism of action against P-selectin in vivo. There is no in vivo evidence that previously described small molecule P-selectin inhibitors act in this manner.

Early identification of carbohydrate structures (sLe^x and variants thereof) integral to all selectin ligands provided lead structures for rational development of competitive selectin inhibitors. A vast collection of sLe^x mimetics has been generated, many examples of which inhibit selectin-dependent events in vitro. High throughput screening of random compound libraries has also identified diverse structures that inhibit selectin-dependent events in vitro. None of these molecules have been found to convincingly inhibit P-selectin-dependent leukocyte rolling in vivo in the manner depicted in Fig. 3 .

We and others have studied the relationship between structure and function of the selectin ligand PSGL-1 to enhance our understanding of its contribution to leukocyte recruitment in vivo and provide clues as to how this response might be modified for clinical therapy. Glycosulfopeptides modeled on the NH₂ terminus of PSGL-1 were generated and found to bind with high affinity to P-selectin in vitro and to inhibit binding of human neutrophils to immobilized P-selectin in a static adhesion assay.

In the current investigation, we tested the effects of glycosulfopeptides on established P-selectin-dependent leukocyte rolling in vivo. The velocity of rolling leukocytes was increased by low doses of glycosulfopeptides, and the proportion of rolling cells was reduced by higher doses. These observations fit the model depicted in Fig. 3 wherein soluble P-selectin ligand mimetics compete with cell surface bound PSGL-1 to either weaken or reduce P-selectin-dependent leukocyte rolling. Although rapid clearance of GSP limits their utility, derivatives of these prototype compounds may provide therapeutic benefit in conditions where P-selectin-dependent leukocyte recruitment contributes to pathology.

Ours is the first demonstration that a precisely defined molecule, rationally based on a natural selectin ligand, can competitively inhibit P-selectin-dependent leukocyte rolling in vivo.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0075fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.02-0075fje

² A.E.R.H. and A.L. contributed equally to this work.

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