Blocking both E-Selectin and P-Selectin Inhibits ... - Science Direct

CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY

Vol. 83, No. 1, April, pp. 45–52, 1997 Article No. II964324

Blocking both E-Selectin and P-Selectin Inhibits Endotoxin-Induced Leukocyte Infiltration into the Eye SCOTT M. WHITCUP, ALEXANDER T. KOZHICH, MARK LOBANOFF, BARRY A. WOLITZKY,*

AND

CHI-CHAO CHAN

National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892; and *Roche Research Center, Hoffmann-La Roche, Incorporated, Nutley, New Jersey 07110

vessel wall involves an initial rolling of inflammatory cells along the vascular endothelium and appears to be mediated by selectins. This primary but transient contact is followed by a more firm adherence between leukocytes and the vascular endothelium and diapedesis involving the interaction of integrins and members of the immunoglobulin superfamily (3–5). The selectins are a family of molecules that share a highly conserved structure consisting of an N-terminal lectin domain, an epidermal growth factor-type domain, a variable number of complement regulatory protein-like domains, a single transmembrane domain, and short cytoplasmic domains. The selectins all recognize ligands that contain specific carbohydrate moieties such as sialyl-Lewis X (6). There are three members of the selectins: E-selectin (CD62E), P-selectin (CD62P), and L-selectin (CD62L). E-selectin, previously known as ELAM-1, is expressed on activated vascular endothelium, and was first identified as a molecule that is up-regulated within several hours after activation of vascular endothelial cells by inflammatory cytokines (7, 8). P-selectin, also known as PADGEM, is expressed on both vascular endothelium and platelets. Unlike Eselectin and L-selectin, P-selectin is prestored in the Weibel–Palade bodies of endothelial cells and is rapidly mobilized for cell-surface expression (9). L-selectin, previously known as gp90MEL, is expressed on most lymphocytes, neutrophils, monocytes, and eosinophils and was initially identified as a homing receptor for lymph nodes (10). In this study, we investigate the role of E-selectin and P-selectin on endotoxin-induced uveitis, an animal model for acute intraocular inflammation in humans. Endotoxin-induced uveitis is characterized by the acute infiltration of neutrophils and monocytic cells into the anterior chamber of the eye and into the vitreous in the area of the optic nerve head in susceptible animals (11–13). Recently, a murine model of endotoxin-induced uveitis has been described in C3H/HeN mice (14, 15). Our studies on endotoxin-induced uveitis in C3H/ HeN mice showed that neutrophils begin entering the eye within 6 hr after endotoxin administration and peak about 24 hr following endotoxin injection (16). We

The initial contact between leukocytes and the vascular endothelium at sites of inflammation is mediated by selectins. The purpose of this study was to investigate the role of the two selectins expressed on the vascular endothelium, E-selectin and P-selectin, in the pathogenesis of endotoxin-induced uveitis. Endotoxininduced uveitis was produced in female C3H/HeN mice using Salmonella typhimurium endotoxin injected into one hind footpad. At the time of endotoxin injection mice were treated with an intraperitoneal injection of a monoclonal antibody against E-selectin or Pselectin, a combination of both anti-selectin antibodies, or isotype-matched control antibodies. In a second set of experiments, antibody treatment was administered 6 hr after endotoxin injection, when inflammatory cells are already entering the eye. Ocular inflammation was graded histologically by a masked observer. When administered at the time of endotoxin injection, anti-P-selectin antibody decreased ocular inflammation by 37% compared to control animals (P Å 0.05). There was no statistical decrease in ocular inflammation in animals treated with anti-E-selectin antibody. The combination of anti-P-selectin and anti-Eselectin antibodies decreased infiltrating inflammatory cells by 61% (P õ 0.01). When treatment was delayed until 6 hr after endotoxin injection, the combination of anti-P-selectin and anti-E-selectin antibodies again decreased ocular inflammation by 60% (P õ 0.01). Immunohistochemical staining showed decreased ICAM-1 expression in the eyes of animals treated with the combination of anti-P- and anti-E-selectin antibodies. Blocking both P-selectin and E-selectin resulted in a significant decrease in endotoxin-induced intraocular inflammation. q 1997 Academic Press

INTRODUCTION

The migration of leukocytes to areas of inflammation is dependent on the expression of cell adhesion molecules. The process of leukocyte adherence to the vascular endothelium and the subsequent extravasation of cells into inflamed tissues involves several steps (1, 2). The first interaction between leukocytes and the blood 45

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0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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endotoxin (Difco Laboratories, Detroit, MI) in 0.1 ml of phosphate-buffered saline into one hind footpad. Monoclonal Antibodies

FIG. 1. Bar graph showing the number of inflammatory cells infiltrating the eye 24 hr after endotoxin injection. At the time of endotoxin injection animals were treated with a single injection of anti-E-selectin mAb (n Å 10), anti-P selectin mAb (n Å 10), anti-Eplus anti-P-selectin mAb (n Å 10), or isotype-matched control IgG1 plus IgG2b mAb (n Å 10). Treatment with anti-P-selectin mAb or the combination of anti-E- and anti-P-selectin mAb resulted in a significant reduction in ocular inflammation compared to controls.

also previously showed that E-selectin expression in the eye is up-regulated before endotoxin-induced inflammatory cell migration into the eye (22). Since Eselectin and P-selectin are both expressed on vascular endothelium and are important for the initial contact of neutrophils along the blood vessel wall, we predicted that these selectins would play an integral role in the pathogenesis of endotoxin-induced uveitis. MATERIALS AND METHODS

Animals Female C3H/HeN mice, 6 to 8 weeks of age, were obtained from NIH stock (Frederick, MD) and kept under standard pathogen-free conditions. This research adhered to the ARVO ‘‘Statement for the Use of Animals in Ophthalmic and Vision Research.’’ Endotoxin-Induced Uveitis Endotoxin-induced uveitis was produced in C3H/HeN mice by injecting 0.2 mg of Salmonella typhimurium

Monoclonal antibody (mAb) against murine P-selectin (5H1) was generated by methods previously detailed (17). This rat anti-mouse P-selectin antibody (IgG1) recognizes native P-selectin induced by tumor necrosis factor on murine eEnd.2 endothelial cells, blocks the adhesion of HL60 cells and bone marrowderived neutrophils to recombinant murine P-selectin, and does not cross-react with either murine E-selectin or murine L-selectin. Monoclonal antibody against murine E-selectin (10E6) was also generated using methods previously described (18). This rat anti-mouse E-selectin antibody (IgG2b) also does not cross-react to murine E-selectin or L-selectin. Rat IgG1 (mAb 8B9) and IgG2b (mAb 2-4A1) were used as isotype-matched control antibodies. Monoclonal antibody against ICAM-1 (CD54) was produced from the YN/1.7.4 hybridoma obtained from the ATCC. This rat anti-mouse ICAM-1 antibody (IgG2b) was isolated from the supernatant of the YN1/ 1.7.4 hybridoma grown in a Cellulosic MPS bioreactor (Cellco, Germantown, MD) and purified by affinity chromatography on a protein G column, and has been previously described in detail (19). Effect of Anti-E-selectin and Anti-P-selectin Antibodies on Endotoxin-Induced Uveitis Endotoxin-induced uveitis was produced in C3H/ HeN mice as described above. At the time of endotoxin injection mice received an intraperitoneal injection of 0.25 mg of anti-E-selectin antibody (n Å 10), 0.25 mg of anti-P-selectin antibody (n Å 10), a combination of anti-E-selectin and anti-P-selectin antibody (n Å 10), or a combination of control antibodies (n Å 10). Mice were euthanized 24 hr after endotoxin injection and both eyes were enucleated. The right eyes were fixed in 4% glutaraldehyde for 30 min and transferred to 10% formalin for at least 24 hr. Eyes were embedded in methacrylate, and 4-mm anterior–posterior vertical sections containing the cornea, the pupil, and the optic nerve were stained with hematoxylin and eosin. Infiltrating inflammatory cells per histologic section (anterior chamber and vitreous) were then counted by a masked observer. Left eyes were immediately snap frozen in a dry ice and methylbutane slurry and embedded

FIG. 2. Histologic sections from animals treated with a single injection of anti-E- plus anti-P-selectin mAb or isotype-matched control IgG1 plus IgG2b mAb. Numerous inflammatory cells (arrows) and eosinophilic material can be seen in the anterior chamber (A) and the vitreous (B) of the IgG-treated control animals. Most of these infiltrating inflammatory cells are clearly identified as neutrophils (A, inset). In contrast, few inflammatory cells are found in the anterior or posterior chamber (C) or vitreous (D) of the animals treated with anti-Eand anti-P-selectin mAbs. Hematoxylin and eosin, original magnification 1200; inset 1400.

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FIG. 2—Continued 48

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in optimum cutting temperature compound (Miles Laboratory, Naperville, IL). Eight-micrometer frozen sections were placed on gelatinized slides for immunohistochemical staining using an avidin–biotin–peroxidase complex technique (20). Monoclonal antibodies against CD54 (ICAM-1) and IgG1 and IgG2b (controls) were used as the primary antibodies. Biotin-goat antirat IgG (Vector Laboratories, Burlingame, CA) was used as the secondary antibody. After incubation with the avidin–biotin–peroxidase complex (Vector Laboratories), the slides were developed in 3,3-diaminobenzidine (Mallinckrodt, Paris, KY) and dehydrated and counterstained with 1% methyl green in methanol. Immunohistochemically stained sections were then graded by a masked observer. Previous studies showed that leukocyte infiltration into the eye occurs by 6 hr after endotoxin injection (16). To study the effect of antibodies against E-selectin and P-selectin on preexisting ocular inflammation, endotoxin-induced uveitis was produced in C3H/HeN mice. Six hours after endotoxin injection, animals were then treated with intraperitoneal injection of 0.25 mg of anti-E-selectin antibody (n Å 4), 0.25 mg of antiP-selectin antibody (n Å 6), a combination of anti-Eselectin and anti-P-selectin antibody (n Å 6), or a combination of control antibodies (n Å 8). Mice were euthanized 24 hr after endotoxin injection and the eyes were enucleated and processed as described above. RESULTS

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FIG. 3. Bar graph showing the number of inflammatory cells infiltrating the eye 24 hr after endotoxin injection. Six hours after endotoxin injection, animals received a single injection of anti-Eselectin mAb (n Å 10), anti-P-selectin mAb (n Å 10), anti-E- plus anti-P-selectin mAb (n Å 10), or isotype-matched control IgG1 plus IgG2b mAb (n Å 10). Only treatment with the combination of antiE-selectin and anti-P-selectin mAbs resulted in a significant reduction in ocular inflammation compared to control animals.

Immunohistochemical staining (Fig. 4) showed decreased expression of ICAM-1 on the endothelium and ciliary body epithelium in eyes treated with the combination of antibodies against E- and P-selectin when compared to control animals. There was no difference in ICAM-1 expression in eyes from animals treated with anti-E-selectin or anti-P-selectin antibody alone compared to control animals.

The effect of antibodies against E-selectin or P-selectin or a combination of both antibodies on preventing endotoxin-induced uveitis is shown in Fig. 1. There was no difference in ocular inflammation in mice treated with anti-E-selectin antibody when compared to conDISCUSSION trols. Anti-P-selectin resulted in a 37% decrease in the number of infiltrating inflammatory cells (P Å 0.05). The combination of anti-P-selectin antibody and antiOur data show that a combination of mAb against E-selectin antibodies resulted in a 61% decrease in in- P-selectin and E-selectin inhibits endotoxin-induced filtrating inflammatory cells (P õ 0.01). Histopathology migration of inflammatory cells into the eye. Blocking (Fig. 2) showed that the combination of anti-P-selectin E-selectin or P-selectin alone had small inhibitory efand anti-E-selectin antibodies inhibited infiltration of fects that were not statistically or clinically significant. both neutrophils and monocytic cells into both the ante- E-selectin and P-selectin are not expressed on neutrorior chamber and the vitreous cavity of the eye. phils and previous studies show that these anti-selectin Figure 3 shows the effect of the anti-selectin anti- antibodies do not deplete neutrophils from the periphbodies administered 6 hr after endotoxin injection eral circulation (17). Rather, their anti-inflammatory when infiltrating inflammatory cells were already effects can be attributed to inhibition of homing and present in the eye. Anti-E-selectin antibody had no migration. Endotoxin-induced uveitis is known to be effect on ocular inflammation, and anti-P-selectin re- dependent on neutrophil migration, and this cell trafsulted in a small (15%), but not statistically signifi- ficking is mediated, in part, by cell adhesion molecules. cant, decrease in infiltrating inflammatory cells (P Å We previously showed that the interaction of integrin 0.47). Again, the combination of anti-P-selectin and molecules and immunoglobulin superfamily members anti-E-selectin antibodies resulted in a larger (60%) is involved in the development of endotoxin-induced and statistically significant decrease in ocular in- ocular inflammation. Monoclonal antibodies against flammatory disease (P õ 0.01). Mac-1 (16), and against LFA-1 and ICAM-1 (21), sig-

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nificantly inhibit the development of endotoxin-induced uveitis. We also demonstrated that endotoxin up-regulates the expression of E-selectin on the vascular endothelium in the eye prior to leukocyte infiltration (22) and predicted that the selectins should play an important role in the development of endotoxin-induced uveitis. The selectins appear to be uniquely able to mediate leukocyte-endothelial adhesion under conditions of flow. Both E- and P-selectin have been demonstrated to play integral roles in the development of inflammatory disease. P-selectin transcription and expression are induced by a number of cytokines as well as endotoxin (23). Antibody against P-selectin inhibits pulmonary inflammation induced by cobra venom factor or IgA immune complexes (24). P-selectin-deficient mice are viable but do have deficient neutrophil infiltration into the peritoneum early after thioglycollate injection; however, neutrophil accumulation normalized later, suggesting that other adhesion molecules could substitute for P-selectin (25). Similarly, E-selectin is involved in the inflammatory process in a number of animal models. Anti-E-selectin antibody inhibits neutrophil trafficking in animal models of lung inflammation (26), delayed-type hypersensitivity in the skin (27), and airway obstruction (28). In this study, we showed that neutrophil migration into the eye can only be effectively inhibited by blocking both endothelial selectins with a combination of antibodies against E-selectin and P-selectin. Antibody against E-selectin had little effect on endotoxin-induced uveitis in any experiment. Anti-P-selectin mAb did decrease ocular inflammation by 37% when administered at the time of endotoxin injection but had minimal effect when administered 6 hr after endotoxin injection. These data are in agreement with other investigators. Labow et al. showed that E-selectin-deficient mice exhibited no significant change in the trafficking of neutrophils in thioglycolate-induced peritonitis or altered delayed-type contract hypersensitivity (17). However, blocking both endothelial selectins by treating E-selectin-deficient mice with an anti-murine Pselectin antibody significantly inhibited neutrophil emigration. More recently, Frenette et al. showed that knockout mice deficient in both P- and E-selectins exhibited a greater than 5-fold reduction in neutrophil influx in chemically induced peritonitis compared to wild-type mice and a 2.5-fold reduction relative to Pselectin knockouts (29). These data suggest that E-selectin and P-selectin are functionally redundant, and

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that both selectins need to be blocked to effectively inhibit acute inflammatory cell migration across the vascular endothelium. The data also suggest that blocking both E- and P-selectin can effectively reduce inflammatory cell recruitment despite expression of Lselectin or other cell adhesion molecules. Unfortunately, reproducible endotoxin-induced uveitis has only been elicited in C3H/HeN mice, making studies with the currently available selectin-knockout mice less feasible. Small molecules that block all three selectins have now been developed, and one of these molecules has been shown to inhibit endotoxin-induced leukocyte migration into the aqueous humor of rats (30). Therefore, although drugs that block only a single selectin molecule may result in only minimal inhibition of inflammatory disease, agents that block both E-selectin and P-selectin or all three selectin molecules may be useful for treating acute ocular inflammatory disease in humans. REFERENCES 1. Butcher, E. C., Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 67, 1033–1036, 1991. 2. Springer, T. A., Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76, 301– 314, 1994. 3. Lawrence, M. B., and Springer, T. A., Leukocytes roll on a selectin at physiologic flow rates: Distinction from and prerequisites for adhesion through integrins. Cell 65, 859–873, 1991. 4. Lawrence, M. B., and Springer, T. A., Neutrophils roll on E-selectin. J. Immunol. 151, 6338–6346, 1993. 5. Von Andrian, U. H., Chambers, J. D., McEnvoy, L. M., Bargatze, R. F., Arfors, K. E., and Butcher, E. C., Two-step model of leukocyte-endothelial cell interaction in inflammation: Distinct roles for LECAM-1 and the leukocyte b2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88, 7538–7542, 1991. 6. Bochner, B. S., Sterbinshky, S. A., Bickel, C. A., Werfel, S., Wein, M., and Newman, W., Differences between human eosinophils and neutrophils in the function and expression of sialic acidcontaining counterlignads for E-selectin. J. Immunol. 152, 774– 782, 1994. 7. Bevilacqua, M. P., Pober, J. S., Mendrick, D. L., Cotran, R. S., and Gimbrone, M. A., Jr., Identification of an inducible endothelial-leukocyte adhesion molecule. Proc. Natl. Acad. Sci. USA 84, 9238–9242, 1987. 8. Bevilacqua, M. P., Stengelin, S., Gimbrone, M. A. J., and Seed, B., Endothelial leukocyte adhesion molecule-1: An inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243, 1160–1164, 1989. 9. McEver, R. P., Beckstead, J. H., Moore, K. L., Marshall-Carlson, L., and Bainton, D. F., GMP-140, a platelet alpha-granule mem-

FIG. 4. Immunohistochemically stained sections showing strong expression of ICAM-1 on the nonpigmented ciliary body epithelium (arrow) in control animals (A). In contrast, there is substantially less ICAM-1 expression on the nonpigmented ciliary body epithelium (arrow) in animals treated with the combination of anti-E-selectin and anti-P-selectin mAbs (B). The pigmented ciliary body epithelium appears dark black in both animals. Original magnification 1200.

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Received September 23, 1996; accepted with revision December 16, 1996

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