Current Eye Research
Volume I I supplcmcnt 1992, 135- 140
Suppression of experimental autoimmune uveoretinitis by prazosin, an a,-adrenergic receptor antagonist J.M.Ruiz-Moreno, M.Misiuk-Hojlo, B.Thillaye and Y.de Kozak
Curr Eye Res Downloaded from informahealthcare.com by Stanford University on 11/19/14 For personal use only.
Laboratoire d’Immunopathologie de I’Oeil, INSERM U 86, Paris, France
ABSTRACT S-antigen (S-Ag)-inducedexperimental autoimmune uveoretinitis (EAU) was suppressed in Lewis and PVG rats by treatment beginning 4 days post immunization with prazosin, a specific al-adrenergic receptor antagonist. A significant suppression of EAU was observed at clinical and histological levels in both treated groups compared to a severe EAU which developed in controls. Fluorescein angiography showed no leakage of dye from the optic disc of a treated PVG rat presenting no ocular inflammation by clinical examination. The treatment had no effect on the titer of anti-S-Ag antibodies. Perivascular infiltrates of T-lymphocytes and macrophages together with alterations of blood-retinal barrier permeability are early events in EAU. Prazosin, by acting on the vascular al-adrenoreceptors, inhibits vasospasm, preserves blood-retinal-barrier integrity and prevents vascular edema and early inflammatory cell infiltration observed in EAU . INTRODUCTION Experimental autoimmune uveoretinitis (EAU) is a mostly T-cell dependent inflammatory disease of the eye (1) that has been studied extensively as a model of human uveitis. After S-antigen immunization in the footpads, sensitized T-cells and macrophages react with antigen-presenting cells in the retina : retinal pigment epithelial cells (21, vascular endothelial cells ( 3 ) and Muller cells ( 4 ) . Migration of sensitized cells from the blood stream to the target in the retina involves specific lymphocyteendothelium adhesion receptors, alteration
of retinal vascular endothelium and changes of the blood-retinal barrier ( 5 , 6). The vasoactive amines released by sensitized T-cells and choroidal mast cells ( 7 , 8 ) induce vasospasm and breakdown on the blood-retinal barrier. Prazosin by acting on vascular al-adrenergic receptor could inhibit vasospasm, prevent opening of bloodretinal barrier and inflammatory cell infiltration observed in EAU. We present here the inhibitory effect of prazosin on clinical and histological EAU induced by S-antigen (S-Ag) in Lewis and PVG rats. MATERIALS AND METHODS Induction of EAU Ten female Lewis rats (8-12 weeks old, weighing approximately 2009) and fifteen female PVG rats (weighing approximately 1509) were immunized with a single footpad injection of 30 pg or 50 pg respectively of purified bovine S-Ag ( 9 ) in 0.1 ml of saline mixed with 0.1 ml of complete Freund’s adjuvant (CFA, Difco Laboratories, Detroit, MI, USA). At the same time, rats were intraperitoneally injected with 5 x 10 killed Hemophilus pertussis (Institut Merieux, Lyon, France). Treatment of EAU Prazosin (Sigma) was suspended by sonication in saline and injected intraperitoneally twice a day at the dose
Received on January 2. 1992; accepted on March 23. 1992
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Curr Eye Res Downloaded from informahealthcare.com by Stanford University on 11/19/14 For personal use only.
Current Eye Research of 2 mg/70 g weight per day from day 4 to day 14 after S-Ag immunization in Lewis and from day 4 to day 20 in PVG rats. The control groups were injected with saline. cal s c o r u The animals were observed clinically from day 10 after S-Ag immunization using a slit lamp. The severity of EAU was graded from 0 to 4 according to the dilation of the iris after instillation of a mydriatic drug, and the density of cellular infiltration in the lewis rat anterior chamber ( 1 0 ) or in the PVG rat vitreous body (11). * . Quantitation of vascular Dermeabilitv Animals were intraperitoneally anesthetized with Nembutal (Abbott Laboratories, Saint-Remy-sur Avre, France) ( 3 0 mg/kg) then 0 . 3 ml of 5% sodium fluorescein was given by intracardiac injection. The angiogram was recorded using a Kowa fundus camera with standard fluorescein filters and black-and-white film. Xbtolocrv Lewis and PVG rats were sacrificed 14 or 20 days respectively after S-Ag immunization, i.e. 3-4 days after disease onset in control rats. The eyes were enucleated, fixed in Bouin's solution and embedded in paraffin. Sections were stained with hematoxylin-eosin. Retinal destruction was graded from 0 to 7, according to the extent (limited or total) of alterations in the various layers of the retina: 1, 2 = rod and cone layer; 3, 4 = outer nuclear layer; 5. 6 = inner nuclear layer; 7 = ganglion cell layer. Titration of anti - S- Aa antibodies At the time of sacrifice, blood samples were taken. The titer of anti-S-Ag antibodies was determined by ELISA. Rat sera, diluted, were distributed in microtitration plates coated with S-Ag (1 pg/ml). After washing, the plates were
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incubated with biotinylated sheep anti-rat Ig antibody followed by 10-3 streptavidin-biotinylated horseradish peroxidase complex (Amersham,UK) . o-Phenylenediamine was used as peroxidase substrate. The reaction was stopped with 4N sulfuric acid and read using an ELISA reader. Experimental animals were treated according to the rules and regulation of che European Common Community Board concerning the use of experimental animals (86/609/CEE). RESULTS co-Datholoaical features of E m Compared to untreated controls, administration of prazosin in Lewis and PVG rats delayed the onset of EAU (p
Volume I I supplcmcnt 1992, 135- 140
Suppression of experimental autoimmune uveoretinitis by prazosin, an a,-adrenergic receptor antagonist J.M.Ruiz-Moreno, M.Misiuk-Hojlo, B.Thillaye and Y.de Kozak
Curr Eye Res Downloaded from informahealthcare.com by Stanford University on 11/19/14 For personal use only.
Laboratoire d’Immunopathologie de I’Oeil, INSERM U 86, Paris, France
ABSTRACT S-antigen (S-Ag)-inducedexperimental autoimmune uveoretinitis (EAU) was suppressed in Lewis and PVG rats by treatment beginning 4 days post immunization with prazosin, a specific al-adrenergic receptor antagonist. A significant suppression of EAU was observed at clinical and histological levels in both treated groups compared to a severe EAU which developed in controls. Fluorescein angiography showed no leakage of dye from the optic disc of a treated PVG rat presenting no ocular inflammation by clinical examination. The treatment had no effect on the titer of anti-S-Ag antibodies. Perivascular infiltrates of T-lymphocytes and macrophages together with alterations of blood-retinal barrier permeability are early events in EAU. Prazosin, by acting on the vascular al-adrenoreceptors, inhibits vasospasm, preserves blood-retinal-barrier integrity and prevents vascular edema and early inflammatory cell infiltration observed in EAU . INTRODUCTION Experimental autoimmune uveoretinitis (EAU) is a mostly T-cell dependent inflammatory disease of the eye (1) that has been studied extensively as a model of human uveitis. After S-antigen immunization in the footpads, sensitized T-cells and macrophages react with antigen-presenting cells in the retina : retinal pigment epithelial cells (21, vascular endothelial cells ( 3 ) and Muller cells ( 4 ) . Migration of sensitized cells from the blood stream to the target in the retina involves specific lymphocyteendothelium adhesion receptors, alteration
of retinal vascular endothelium and changes of the blood-retinal barrier ( 5 , 6). The vasoactive amines released by sensitized T-cells and choroidal mast cells ( 7 , 8 ) induce vasospasm and breakdown on the blood-retinal barrier. Prazosin by acting on vascular al-adrenergic receptor could inhibit vasospasm, prevent opening of bloodretinal barrier and inflammatory cell infiltration observed in EAU. We present here the inhibitory effect of prazosin on clinical and histological EAU induced by S-antigen (S-Ag) in Lewis and PVG rats. MATERIALS AND METHODS Induction of EAU Ten female Lewis rats (8-12 weeks old, weighing approximately 2009) and fifteen female PVG rats (weighing approximately 1509) were immunized with a single footpad injection of 30 pg or 50 pg respectively of purified bovine S-Ag ( 9 ) in 0.1 ml of saline mixed with 0.1 ml of complete Freund’s adjuvant (CFA, Difco Laboratories, Detroit, MI, USA). At the same time, rats were intraperitoneally injected with 5 x 10 killed Hemophilus pertussis (Institut Merieux, Lyon, France). Treatment of EAU Prazosin (Sigma) was suspended by sonication in saline and injected intraperitoneally twice a day at the dose
Received on January 2. 1992; accepted on March 23. 1992
135
Curr Eye Res Downloaded from informahealthcare.com by Stanford University on 11/19/14 For personal use only.
Current Eye Research of 2 mg/70 g weight per day from day 4 to day 14 after S-Ag immunization in Lewis and from day 4 to day 20 in PVG rats. The control groups were injected with saline. cal s c o r u The animals were observed clinically from day 10 after S-Ag immunization using a slit lamp. The severity of EAU was graded from 0 to 4 according to the dilation of the iris after instillation of a mydriatic drug, and the density of cellular infiltration in the lewis rat anterior chamber ( 1 0 ) or in the PVG rat vitreous body (11). * . Quantitation of vascular Dermeabilitv Animals were intraperitoneally anesthetized with Nembutal (Abbott Laboratories, Saint-Remy-sur Avre, France) ( 3 0 mg/kg) then 0 . 3 ml of 5% sodium fluorescein was given by intracardiac injection. The angiogram was recorded using a Kowa fundus camera with standard fluorescein filters and black-and-white film. Xbtolocrv Lewis and PVG rats were sacrificed 14 or 20 days respectively after S-Ag immunization, i.e. 3-4 days after disease onset in control rats. The eyes were enucleated, fixed in Bouin's solution and embedded in paraffin. Sections were stained with hematoxylin-eosin. Retinal destruction was graded from 0 to 7, according to the extent (limited or total) of alterations in the various layers of the retina: 1, 2 = rod and cone layer; 3, 4 = outer nuclear layer; 5. 6 = inner nuclear layer; 7 = ganglion cell layer. Titration of anti - S- Aa antibodies At the time of sacrifice, blood samples were taken. The titer of anti-S-Ag antibodies was determined by ELISA. Rat sera, diluted, were distributed in microtitration plates coated with S-Ag (1 pg/ml). After washing, the plates were
136
incubated with biotinylated sheep anti-rat Ig antibody followed by 10-3 streptavidin-biotinylated horseradish peroxidase complex (Amersham,UK) . o-Phenylenediamine was used as peroxidase substrate. The reaction was stopped with 4N sulfuric acid and read using an ELISA reader. Experimental animals were treated according to the rules and regulation of che European Common Community Board concerning the use of experimental animals (86/609/CEE). RESULTS co-Datholoaical features of E m Compared to untreated controls, administration of prazosin in Lewis and PVG rats delayed the onset of EAU (p
