Current Eye Research
Voiiirnc I I numbci 10 1992, 955-961 ~
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~~~
Ultrastructural localization of hydrogen peroxide in experimental autoimmune uveitis
Guey-Shuang Wu. David C.Gritz, Lily R.Atalla, David A.Stanforth, Alex Sevanian’ and Narsing A.Rao Doheny l”,).eIn4tute and ‘Institute for Toxicology, University of Southern California School of Medicine, Los Angeles, CA. USA
ABSTRACT One of the most prominent features of S-anti en induced iiveitis is the massive infiltration of polymorp onuclear leukocytes (PMNs) and mononuclear cells in the ocular tissues and fluids. These inflammatory cells generate reactive oxygen metabolites as microbicidal agents and release these oxidants into the surrounding tissues. Using the cerium perhydroxide method, we have localized subcellular hydrogen peroxide in various inflamed ocular tissues. Most notably, the positive electron-dense granules were seen in the plasma membranes of PMNs that were infiltrating in the retina and uvea. These deposits were noted also i n PMNs located within the extravascular spaces. For the intravascular PMNs, the positive reaction products were seen in much lower concentrations. A direct demonstration of substantial concentrations of hydrogen peroxide in experimental autoimmune uveitis, therefore, suggests the possibility that this reactive metabolite is an inflammatory mediator in thk condition.
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
E
Although the rnflammarory response differs throughout [ l i t eke, the bdiic event\ include an arrival of infl:immatory sellel i n the retina. There are inflammatory cells located \ v i t h i n the ve\sc.l (mall arrows), as well as one adjacent to the vc.\iel (large arrow). The endothelial cell is marked a 3 I; ‘I he eutravascular inflammatory cell has rn‘trked depovtioii of electron-dense granules along its
situated close to the vessel wall. Polymorphonuclear leukocytes in the extravascular tissues, however, were stained extensively along their plasma membranes (Fig. 2 ) . Surrounding some of the extravascular inflammatory cells was a diffuse collection of electron-dense granules (Fig. 1). Perivascular cells in the choroid demonstrated electron-dense deposits interstitially along their plasma membranes (Fig. 3). The deposits were more abundant near the vessel wall and seemed to decrease in concentration in the interstitial spaces more distal to the choroidal vessels. Apical portions of the retinal pigment epithelium cells also showed positive electron-dense deposits. At the peak of inflammation (14 days postimmunization), there appeared to be a massive infiltration of PMNs in the inflamed retina. That these PMNs were activated was indicated by the existence of electron-dense granules in the plasma membranes of these cells (Fig. 4).
plasma membrane. The intravascular inflammatory cells, in contrast, do not demonstrate such abundant ranules. The intravascular inflammatory cell closest to t e vesssel wall (*), however, possesses more granules than do the other two intravascular cells. Bar = 10 1.1.
f
957
Current Eye
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Research
Figure 3. Transmission electron micrograph showing perivascular cells in the choroid. Electron-dense
granules are present in the interstitial spaces of these perivascular cells. Bar = 0.1 I.(.
Fi w e 4. Transmission electron micrograph showing the in iltration of activated PMNs in the retina at the peak of inflammation (14 days postimmunization). Cell types are
marked as follows: blood vessel lumen, BVL; blood vessel wall, BVW; retinal cell, RC and lymphocyte, LY. Bar = l o p .
F
958
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
All 12 inflamed eyes examined showed a positive reaction and the same general pattern of localization. The extensiveness of the granular deposits appears to vary someNhat according ito severity of the disease. Uterine endometrium revealed electron-dense deposits in the plasma membranes of the microvilli. None of the tissues processed in the absence of exogenous NADH or NADPH showed any positive reaction products.
DISCUSSION In EAU, the occurrence of disease is signified by a massive infiltration of PMNs in the inflammatory sites (Fig. 41, IJsing chemiluminescence measurements, we previoiisly detected increased free radical activity in the retina and choroid of animals with EAU. The chemilurninescence count!; exhibited by the inflamed tissues were more than 20.-foldgreater than those of uninflamed control tissues (12). Among the most important primary oxygen metabolites found in this pool were superoxide anions (12). In inflammation, superoxide anions are formed presumably by the action of' NAUPH oxidase located in the plasma membranes of phagocytes. (1. 13). These primary radicals are then rapidly converted to hydrogen peroxide at the site of griier;~rionby two known pathways, a spontaneous dismutation a n d a scavenging reaction by superoxide dismutase. 'These sequence of reactions are depicted in the Following schematic form:
I n prior localizations of hydrogen peroxide in activated phagocytes, the electron-dense granules appeared primarily on the cell s~irfdce,within plasma membranes. Localization was observed less frequently in the iniernalized plasma membranes, such as phagocytic vacuole inein tiranes and intracellular vesicles (3,5,7). In o u r localization of hydrogen peroxide in inflamed ocular tissues, including cornea, ciliary body, iris, retina and thoroid. we consistently observed positive reaction product\ only within the plasma membranes of PMNs in these tissues. Although there appeared to be some weak cytoplasmic staining, further studies are needed to confii-niihia finding. Several reasons could account for
the staining being localized primarily in the plasma membranes as observed in this study. In our localization experiments, 3-amino-1,2,4-triazole was used to inhibit the activity of catalase. Although this agent appears to totally abolish the activity of catalase in isolated PMNs, it might not have been sufficient to exert this effect in the tissue preparations used in the present study. When dealing with the whole retina, choroid or ciliary body, penetration of 3-amino-1,2,4-triazole beyond the plasma membranes might require more stringent incubation conditions. The staining of plasma membranes only (4) or of plasma membranes plus some occasional intracellular vesicles (7) have been previously reported. Another possible cause for the limited localization may be that the cerium chloride, which had been dissolved in buffer, might not diffuse sufficiently into the intracellular compartments to produce concentrations high enough for the reaction to take place under the present incubation conditions. Although lipid hydroperoxides will also react with cerous chloride to form electron-dense granules Seen in the present study, the formation of such hydroperoxides in the plasma membranes of PMNs is unlikely due to the following two reasons: 1) The morphological changes of PMNs during phagocytosis has been well documented. These changes on the plasma membranes include formation of bleb-like or cylindrical structures on the plasma membranes followed by breaking-off of these formations (14,15). A possible change in the fluidity of plasma membranes, which increases susceptibility for peroxidation, was never indicated ( 1 4 ~ 5 ) .2) Rat peritoneal PMNs, when stimulated with 200 ng of phorbol myristate acetate for up to one hour at 37'C produced no detectable quantity of conjugated dienes (16). The detection of conjugated dienes is one of the most sensitive method for measuring peroxidation of membrane polyunsaturated fatty acids (16). Therefore, it appears that the membrane lipids of phagocytic leukocytes are well protected from bactericidal oxygen metabolites by the abundant quantities of superoxide dismutase, catalase and reduced glutathione in the membranes. Only when these enzymes, especially superoxide dismutase, are depleted by any of the genetic defects, does the oxidative damage to phagocytic leukocytes become apparent (17,18).
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
-
The reaction, H202 + CeC13 Ce(OH)200H, used in the localization procedure appears to be specific for detecting hydrogen peroxide only (33). The other oxidative processes of NADPH such as the reaction of dihydrolipoamide dehydrogenase (NADPH diaphorase) oxidizes NADPH but produces no hydrogen peroxide, are apparently not detected by this procedure (19). It is of interest to note that the positive deposits were seen in the plasma membranes of PMNs located predominantly in the interstitial tissues, and not in PMNs within intravascular spaces (Fig. 2). This could be due to the concentration of hydrogen peroxide generated in the plasma membranes of intravascular PMNs being diminished by the presence of abundant quantities of intravascular catalase. This rationale has been proposed by Guy and collaborators (20) for a similar phenomenon observed in experimental optic neuritis. Another explanation could be that the phagocytes situated intravascularly are not activated, and thus do not produce hydrogen peroxide in an appreciable quantity. The perivascular distribution of hydrogen peroxide that we noted in the preparations of choroid was very similar to the findings reported by Guy and co-workers (20). In their study, hydrogen peroxide was observed in perivascular distribution temporal to sites of severe inflammation of the optic nerve, and was seen at a time point similar to that in our model. This perhaps reflects the diffusion of hydrogen peroxide into the interstitial spaces from adjacent PMNs. These results, however, were not always consistent in the sections examined. In conclusion, we have demonstrated the presence of subcellular hydrogen peroxide in ocular tissues from animals with EAU. Although hydrogen peroxide is generally considered to be a weak oxidant, in the presence of myeloperoxidase it can oxidize halides to produce the hypohalous acids. These potent acids and their salts react with a wide variety of biological molecules ( 1 ~ 3 ) .Hydrogen peroxide can also produce hydroxyl radicals via the Haber-Weiss reaction. Hydroxyl radicals are extremely powerful oxidant capable of reacting with a variety of biological molecules at diffusion controlled rates (1,13). In a previous experiment we demonstrated oxidative damage of the retinas induced by reactive oxygen species in inflamed retina (21). In EAU, the lymphocytes and their immunological effects are
960
probably the most profound factors governing the overall consequence of the disease. However, PMNs appear at early stages of the disease and release superoxide and hydrogen peroxide to the surrounding tissues. Considering the fact the photoreceptors contain abundant quantities of polyunsaturated fatty acids (22), which are extremely susceptible to peroxidation, the existence of hydrogen peroxide at the inflammatory sites is definitely also an important factor in amplication and oxidative destruction of the involved tissues. ACKNOWLEDGEMENTS Supported in part by grant EY 05662 from the National Institutes of Health, and a grant from Research to Prevent Blindness, Inc. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 1989, and in the thesis (N.A.R.) required for membership in the American Ophthalmological Society. CORRESPONDING AUTHOR Narsing A. Rao, MD, Doheny Eye Institute, 1355 San Pablo Street, Los Angeles, CA 90033. REFERENCES 1. Weiss, S.J. and LoBuglio, A.F. (1982) Phagocyteenerated oxy en metabolites and cellular injury. b. Invest. Q, .$ 5-18. 2. Rao, N.A., Sevanian, A., Fernandez, M.A.S., Romero, J.L., Faure, J.P., de Kozak, Y., Till, G.O. and Marak, G.E., Jr. (1987) Role of oxy en radicals in ex erirnental allergic uveitis. Invest. phthalmol. Vis. [ci. 28, 886-892. 3. Briggs, R.T., Drath, D.B., Karnovsky, M.L. and Karnovsky, M.J. (1975) Localization of NADH oxidase on the surface of human polymo honuclear leukocytes by a new cytochemical meth0T.l. Cell Biol. 62,566-586. 4. Atalla, L.R., Sevanian, A. and Rao, N.A. (1988) Hydrogen peroxide localization in ocular tissue: an electron microscopic study. Curr. Eye . cvtochemical . Res., I , 931-936. 5. Karnovsky, M.J., Robinson, J.M., Briggs, R.T. and Karnovsky, M.L. (1981) Oxidative cytochemistry in hagocytosis: the interface betweefi structure and Function. Histochem. J. U, 1-22. 6 . Ohno, Y.I., Hirai, K.I., Kanoh, T., Uchino, H. and Ogawa, K. (1982) Subcellular localization of H 0 2 production in human neutrophils stimulated w& particles and an effect of cytochalasin-B on the cells. 253-260. Blood, 7. Ohno, Y.I., Hirai, K.I., Kanoh, T., Uchino, H. and Ogawa, K. (1982) Subcellular localization of hydrogen peroxide production in human
%
%
a,
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
olymorphonuclear leukocytes stimulated with r'electron ectins, phorbol myristate acetate, and di itonin: an microscopic study using CeC13. blood, a,
1195-1202. Armstrong, D., Sohal.,R. and Cutler, R. (1984) Free radicals in Molecular biology. Aging Dis. 22,307353. Kao, K.A., Fenandez, M.A., Cid, L.L., Romero, J.L. and Sevanian, A. (1987) Retinal lipid peroxidation in experimental uveitis. Arch. Ophthalmol. u)5,17121716. Dorey, C., Cozette, J . and Faure, J.P. (1982) A simple and rapid method for isolation of retinal S antigen. Ophthalmic IRes. l4,249-255. Ishikawa, Y,,Hirai, K..I. and Ogawa, K. (1984) Cytocheniical localization of hydrogen peroxide production in rat uterus. J. Histochem. Cytochem. .32, - h74-676. Wu, C.S., Goto, H., Sevanian, A. and Rao, N. A. (100 1) Generation of chemiluminescence in experimental autoimmune uveitis. Curr. Eye Res. U, 009-9 17. Habior, B.M., Curnutte, J.T. and Okamura, N. (1988) The respiratury burst oxidase of the human neutrophil. I n "Oxygen Radicals and Tissue Injury", (Ed. Halliwell, B.). Pp. 43-48. Federation of American Societies for Experimental Biology, Uerhesda, MD. Radwey, J.A., Curnutte, J.T., Robinson, J.M., Berde, .D.,Karnovsky, M.J. and Karnovsky, M.L. (1984) ffect of free Fatty acids on release of superoxide id oil change of sha ie b human neutrophils. J. iol. Chern. 32,7876-78J7. Graham, K.C., Jr., Kamovsky, M.J., Shafer, A. W., Glass, E.A. and Karnlovsky, M.L. (1967) Metabolic and morphological observations on the effect of burface-active agents on leukocytes. J. Cell Biol. 2, 629-647. Ward,P.A., 'Till, G.O..,Hatherill, J.A., Annesley, 'T'.M. and Kunkel, R.G. (1985) Systemic complement activation, lung injury and products of lipid peroxidation. J. Clin. Invest. 517-527. Roos, D., Weening, R..S. and Voetman, A. A. (1979) Host-defense by and self-defense of phagocytic leukocytes. In "The Molecular Basis of Immune Cell Function", (Ed. Kaplan, J.G.). Pp. 259-272. E,lsevier/North I-Iolland Biomedical Press, Anis t e rd a m . Roo>, D., Weening, H . S . and Voetman, A.A. (1980) Protection o f human neutrophils against oxidative cl:!magz. "4gents Actions, 528-535. 'ey, V. (1966) Lipoyl dehydrogenase from pig heart. Methods Enzymol. 9, 272-278. tiuy, J., Ellis, E.A. and Rao, N.A. (1990) Hydrogen peroxicfe localization in experimental optic neuritis. Arch. Oph t ha1mol . 108,1614-1621. Wu, G. S., Goto, H., Atalla, L.R., Chen, F., Sevanian, A. and Rao, N.A. (1989) Detection of chemi luminescence and lipid peroxidation products in experimental uveitis. Invest. Ophthalmol. Vis. Sci. 30 i,Suppl.j,2x7. Anderson, R.E. and Andrews, L.D. (1982) Biitchemiatry of retinal photoreceptor membranes in vertebrates and invertebrates. In "Visual Cells in Evolution", (Ed. Westfall, J.A.). Pp. 1-22. Raven Pres,, Nev; Y o r L
x,
u,
96 1
Voiiirnc I I numbci 10 1992, 955-961 ~
~
~~
~~
~~~
Ultrastructural localization of hydrogen peroxide in experimental autoimmune uveitis
Guey-Shuang Wu. David C.Gritz, Lily R.Atalla, David A.Stanforth, Alex Sevanian’ and Narsing A.Rao Doheny l”,).eIn4tute and ‘Institute for Toxicology, University of Southern California School of Medicine, Los Angeles, CA. USA
ABSTRACT One of the most prominent features of S-anti en induced iiveitis is the massive infiltration of polymorp onuclear leukocytes (PMNs) and mononuclear cells in the ocular tissues and fluids. These inflammatory cells generate reactive oxygen metabolites as microbicidal agents and release these oxidants into the surrounding tissues. Using the cerium perhydroxide method, we have localized subcellular hydrogen peroxide in various inflamed ocular tissues. Most notably, the positive electron-dense granules were seen in the plasma membranes of PMNs that were infiltrating in the retina and uvea. These deposits were noted also i n PMNs located within the extravascular spaces. For the intravascular PMNs, the positive reaction products were seen in much lower concentrations. A direct demonstration of substantial concentrations of hydrogen peroxide in experimental autoimmune uveitis, therefore, suggests the possibility that this reactive metabolite is an inflammatory mediator in thk condition.
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
E
Although the rnflammarory response differs throughout [ l i t eke, the bdiic event\ include an arrival of infl:immatory sellel i n the retina. There are inflammatory cells located \ v i t h i n the ve\sc.l (mall arrows), as well as one adjacent to the vc.\iel (large arrow). The endothelial cell is marked a 3 I; ‘I he eutravascular inflammatory cell has rn‘trked depovtioii of electron-dense granules along its
situated close to the vessel wall. Polymorphonuclear leukocytes in the extravascular tissues, however, were stained extensively along their plasma membranes (Fig. 2 ) . Surrounding some of the extravascular inflammatory cells was a diffuse collection of electron-dense granules (Fig. 1). Perivascular cells in the choroid demonstrated electron-dense deposits interstitially along their plasma membranes (Fig. 3). The deposits were more abundant near the vessel wall and seemed to decrease in concentration in the interstitial spaces more distal to the choroidal vessels. Apical portions of the retinal pigment epithelium cells also showed positive electron-dense deposits. At the peak of inflammation (14 days postimmunization), there appeared to be a massive infiltration of PMNs in the inflamed retina. That these PMNs were activated was indicated by the existence of electron-dense granules in the plasma membranes of these cells (Fig. 4).
plasma membrane. The intravascular inflammatory cells, in contrast, do not demonstrate such abundant ranules. The intravascular inflammatory cell closest to t e vesssel wall (*), however, possesses more granules than do the other two intravascular cells. Bar = 10 1.1.
f
957
Current Eye
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Research
Figure 3. Transmission electron micrograph showing perivascular cells in the choroid. Electron-dense
granules are present in the interstitial spaces of these perivascular cells. Bar = 0.1 I.(.
Fi w e 4. Transmission electron micrograph showing the in iltration of activated PMNs in the retina at the peak of inflammation (14 days postimmunization). Cell types are
marked as follows: blood vessel lumen, BVL; blood vessel wall, BVW; retinal cell, RC and lymphocyte, LY. Bar = l o p .
F
958
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
All 12 inflamed eyes examined showed a positive reaction and the same general pattern of localization. The extensiveness of the granular deposits appears to vary someNhat according ito severity of the disease. Uterine endometrium revealed electron-dense deposits in the plasma membranes of the microvilli. None of the tissues processed in the absence of exogenous NADH or NADPH showed any positive reaction products.
DISCUSSION In EAU, the occurrence of disease is signified by a massive infiltration of PMNs in the inflammatory sites (Fig. 41, IJsing chemiluminescence measurements, we previoiisly detected increased free radical activity in the retina and choroid of animals with EAU. The chemilurninescence count!; exhibited by the inflamed tissues were more than 20.-foldgreater than those of uninflamed control tissues (12). Among the most important primary oxygen metabolites found in this pool were superoxide anions (12). In inflammation, superoxide anions are formed presumably by the action of' NAUPH oxidase located in the plasma membranes of phagocytes. (1. 13). These primary radicals are then rapidly converted to hydrogen peroxide at the site of griier;~rionby two known pathways, a spontaneous dismutation a n d a scavenging reaction by superoxide dismutase. 'These sequence of reactions are depicted in the Following schematic form:
I n prior localizations of hydrogen peroxide in activated phagocytes, the electron-dense granules appeared primarily on the cell s~irfdce,within plasma membranes. Localization was observed less frequently in the iniernalized plasma membranes, such as phagocytic vacuole inein tiranes and intracellular vesicles (3,5,7). In o u r localization of hydrogen peroxide in inflamed ocular tissues, including cornea, ciliary body, iris, retina and thoroid. we consistently observed positive reaction product\ only within the plasma membranes of PMNs in these tissues. Although there appeared to be some weak cytoplasmic staining, further studies are needed to confii-niihia finding. Several reasons could account for
the staining being localized primarily in the plasma membranes as observed in this study. In our localization experiments, 3-amino-1,2,4-triazole was used to inhibit the activity of catalase. Although this agent appears to totally abolish the activity of catalase in isolated PMNs, it might not have been sufficient to exert this effect in the tissue preparations used in the present study. When dealing with the whole retina, choroid or ciliary body, penetration of 3-amino-1,2,4-triazole beyond the plasma membranes might require more stringent incubation conditions. The staining of plasma membranes only (4) or of plasma membranes plus some occasional intracellular vesicles (7) have been previously reported. Another possible cause for the limited localization may be that the cerium chloride, which had been dissolved in buffer, might not diffuse sufficiently into the intracellular compartments to produce concentrations high enough for the reaction to take place under the present incubation conditions. Although lipid hydroperoxides will also react with cerous chloride to form electron-dense granules Seen in the present study, the formation of such hydroperoxides in the plasma membranes of PMNs is unlikely due to the following two reasons: 1) The morphological changes of PMNs during phagocytosis has been well documented. These changes on the plasma membranes include formation of bleb-like or cylindrical structures on the plasma membranes followed by breaking-off of these formations (14,15). A possible change in the fluidity of plasma membranes, which increases susceptibility for peroxidation, was never indicated ( 1 4 ~ 5 ) .2) Rat peritoneal PMNs, when stimulated with 200 ng of phorbol myristate acetate for up to one hour at 37'C produced no detectable quantity of conjugated dienes (16). The detection of conjugated dienes is one of the most sensitive method for measuring peroxidation of membrane polyunsaturated fatty acids (16). Therefore, it appears that the membrane lipids of phagocytic leukocytes are well protected from bactericidal oxygen metabolites by the abundant quantities of superoxide dismutase, catalase and reduced glutathione in the membranes. Only when these enzymes, especially superoxide dismutase, are depleted by any of the genetic defects, does the oxidative damage to phagocytic leukocytes become apparent (17,18).
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
-
The reaction, H202 + CeC13 Ce(OH)200H, used in the localization procedure appears to be specific for detecting hydrogen peroxide only (33). The other oxidative processes of NADPH such as the reaction of dihydrolipoamide dehydrogenase (NADPH diaphorase) oxidizes NADPH but produces no hydrogen peroxide, are apparently not detected by this procedure (19). It is of interest to note that the positive deposits were seen in the plasma membranes of PMNs located predominantly in the interstitial tissues, and not in PMNs within intravascular spaces (Fig. 2). This could be due to the concentration of hydrogen peroxide generated in the plasma membranes of intravascular PMNs being diminished by the presence of abundant quantities of intravascular catalase. This rationale has been proposed by Guy and collaborators (20) for a similar phenomenon observed in experimental optic neuritis. Another explanation could be that the phagocytes situated intravascularly are not activated, and thus do not produce hydrogen peroxide in an appreciable quantity. The perivascular distribution of hydrogen peroxide that we noted in the preparations of choroid was very similar to the findings reported by Guy and co-workers (20). In their study, hydrogen peroxide was observed in perivascular distribution temporal to sites of severe inflammation of the optic nerve, and was seen at a time point similar to that in our model. This perhaps reflects the diffusion of hydrogen peroxide into the interstitial spaces from adjacent PMNs. These results, however, were not always consistent in the sections examined. In conclusion, we have demonstrated the presence of subcellular hydrogen peroxide in ocular tissues from animals with EAU. Although hydrogen peroxide is generally considered to be a weak oxidant, in the presence of myeloperoxidase it can oxidize halides to produce the hypohalous acids. These potent acids and their salts react with a wide variety of biological molecules ( 1 ~ 3 ) .Hydrogen peroxide can also produce hydroxyl radicals via the Haber-Weiss reaction. Hydroxyl radicals are extremely powerful oxidant capable of reacting with a variety of biological molecules at diffusion controlled rates (1,13). In a previous experiment we demonstrated oxidative damage of the retinas induced by reactive oxygen species in inflamed retina (21). In EAU, the lymphocytes and their immunological effects are
960
probably the most profound factors governing the overall consequence of the disease. However, PMNs appear at early stages of the disease and release superoxide and hydrogen peroxide to the surrounding tissues. Considering the fact the photoreceptors contain abundant quantities of polyunsaturated fatty acids (22), which are extremely susceptible to peroxidation, the existence of hydrogen peroxide at the inflammatory sites is definitely also an important factor in amplication and oxidative destruction of the involved tissues. ACKNOWLEDGEMENTS Supported in part by grant EY 05662 from the National Institutes of Health, and a grant from Research to Prevent Blindness, Inc. Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 1989, and in the thesis (N.A.R.) required for membership in the American Ophthalmological Society. CORRESPONDING AUTHOR Narsing A. Rao, MD, Doheny Eye Institute, 1355 San Pablo Street, Los Angeles, CA 90033. REFERENCES 1. Weiss, S.J. and LoBuglio, A.F. (1982) Phagocyteenerated oxy en metabolites and cellular injury. b. Invest. Q, .$ 5-18. 2. Rao, N.A., Sevanian, A., Fernandez, M.A.S., Romero, J.L., Faure, J.P., de Kozak, Y., Till, G.O. and Marak, G.E., Jr. (1987) Role of oxy en radicals in ex erirnental allergic uveitis. Invest. phthalmol. Vis. [ci. 28, 886-892. 3. Briggs, R.T., Drath, D.B., Karnovsky, M.L. and Karnovsky, M.J. (1975) Localization of NADH oxidase on the surface of human polymo honuclear leukocytes by a new cytochemical meth0T.l. Cell Biol. 62,566-586. 4. Atalla, L.R., Sevanian, A. and Rao, N.A. (1988) Hydrogen peroxide localization in ocular tissue: an electron microscopic study. Curr. Eye . cvtochemical . Res., I , 931-936. 5. Karnovsky, M.J., Robinson, J.M., Briggs, R.T. and Karnovsky, M.L. (1981) Oxidative cytochemistry in hagocytosis: the interface betweefi structure and Function. Histochem. J. U, 1-22. 6 . Ohno, Y.I., Hirai, K.I., Kanoh, T., Uchino, H. and Ogawa, K. (1982) Subcellular localization of H 0 2 production in human neutrophils stimulated w& particles and an effect of cytochalasin-B on the cells. 253-260. Blood, 7. Ohno, Y.I., Hirai, K.I., Kanoh, T., Uchino, H. and Ogawa, K. (1982) Subcellular localization of hydrogen peroxide production in human
%
%
a,
Current Eye Research
Curr Eye Res Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
olymorphonuclear leukocytes stimulated with r'electron ectins, phorbol myristate acetate, and di itonin: an microscopic study using CeC13. blood, a,
1195-1202. Armstrong, D., Sohal.,R. and Cutler, R. (1984) Free radicals in Molecular biology. Aging Dis. 22,307353. Kao, K.A., Fenandez, M.A., Cid, L.L., Romero, J.L. and Sevanian, A. (1987) Retinal lipid peroxidation in experimental uveitis. Arch. Ophthalmol. u)5,17121716. Dorey, C., Cozette, J . and Faure, J.P. (1982) A simple and rapid method for isolation of retinal S antigen. Ophthalmic IRes. l4,249-255. Ishikawa, Y,,Hirai, K..I. and Ogawa, K. (1984) Cytocheniical localization of hydrogen peroxide production in rat uterus. J. Histochem. Cytochem. .32, - h74-676. Wu, C.S., Goto, H., Sevanian, A. and Rao, N. A. (100 1) Generation of chemiluminescence in experimental autoimmune uveitis. Curr. Eye Res. U, 009-9 17. Habior, B.M., Curnutte, J.T. and Okamura, N. (1988) The respiratury burst oxidase of the human neutrophil. I n "Oxygen Radicals and Tissue Injury", (Ed. Halliwell, B.). Pp. 43-48. Federation of American Societies for Experimental Biology, Uerhesda, MD. Radwey, J.A., Curnutte, J.T., Robinson, J.M., Berde, .D.,Karnovsky, M.J. and Karnovsky, M.L. (1984) ffect of free Fatty acids on release of superoxide id oil change of sha ie b human neutrophils. J. iol. Chern. 32,7876-78J7. Graham, K.C., Jr., Kamovsky, M.J., Shafer, A. W., Glass, E.A. and Karnlovsky, M.L. (1967) Metabolic and morphological observations on the effect of burface-active agents on leukocytes. J. Cell Biol. 2, 629-647. Ward,P.A., 'Till, G.O..,Hatherill, J.A., Annesley, 'T'.M. and Kunkel, R.G. (1985) Systemic complement activation, lung injury and products of lipid peroxidation. J. Clin. Invest. 517-527. Roos, D., Weening, R..S. and Voetman, A. A. (1979) Host-defense by and self-defense of phagocytic leukocytes. In "The Molecular Basis of Immune Cell Function", (Ed. Kaplan, J.G.). Pp. 259-272. E,lsevier/North I-Iolland Biomedical Press, Anis t e rd a m . Roo>, D., Weening, H . S . and Voetman, A.A. (1980) Protection o f human neutrophils against oxidative cl:!magz. "4gents Actions, 528-535. 'ey, V. (1966) Lipoyl dehydrogenase from pig heart. Methods Enzymol. 9, 272-278. tiuy, J., Ellis, E.A. and Rao, N.A. (1990) Hydrogen peroxicfe localization in experimental optic neuritis. Arch. Oph t ha1mol . 108,1614-1621. Wu, G. S., Goto, H., Atalla, L.R., Chen, F., Sevanian, A. and Rao, N.A. (1989) Detection of chemi luminescence and lipid peroxidation products in experimental uveitis. Invest. Ophthalmol. Vis. Sci. 30 i,Suppl.j,2x7. Anderson, R.E. and Andrews, L.D. (1982) Biitchemiatry of retinal photoreceptor membranes in vertebrates and invertebrates. In "Visual Cells in Evolution", (Ed. Westfall, J.A.). Pp. 1-22. Raven Pres,, Nev; Y o r L
x,
u,
96 1
