Ophthalmic Res 1991:23:73-78
Histochemical Analysis of Experimental Granulomatous Uveitis1 Michaela Kristevaa, Jyotirmay Biswasa, Geeta Pararajasegarama, Alex Sevanianb, Narsing A. Raoa aDohcny Eye Institute and Departments of Ophthalmology and Pathology, and bInstitute for Toxicology, University of Southern California School of Medicine. Los Angeles, Calif. USA
Key Words. Enzyme histochemistry • Lens-induced uveitis • Arachidonic acid • Lysosomal enzymes
Granulomatous uveitis is one of the most common forms of uveitis that can lead to visual impairment and blindness [1], The precise mechanisms of the granuloma forma 1 This work was supported in part by a research grant (No. F.Y 06953) from the National Eye Insti tute. National Institutes of Health. USA.
tion are not known, but it has been estab lished that macrophages are primarily in volved in this process. Granulomas in var ious tissues have been shown to develop in sequential stages from mononuclear phago cytes to mature epithelioid cell granulomas [2-5]. and lysosomal enzymes such as acid phosphatase, P-glucuronidase, succinate de
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Abstract. We have previously demonstrated the effects of various inhibitors of arachi donic acid metabolism on experimental lens-induced granulomatous uveitis. In the present study, we investigated the effect of these same inhibitors on the expression of lysosomal enzymes at different stages of choroidal inflammation in experimental lens-induced granu lomatous uveitis and compared this to the inflammation observed at each stage examined. Lysosomal enzymes such as acid phosphatase, P-glucuronidase and succinate dehydrogenase are known to be liberated during the maturation of mononuclear phagocytes to epithelioid cell granulomas. Although animals treated with nordihydroguaiaretic acid showed less severe inflammation than did indomethacin-treated or control animals, none of these agents appeared to affect the expression of acid phosphatase and P-glucuronidase, as determined histochemically. Succinate dehydrogenase could not be detected in any of the eyes examined, even though sections of liver and kidney from these same animals were positive for this enzyme.
74
hydrogenase and cytochrome oxidase are known to be liberated during this maturation process [6]. Experimental granulomatous uveitis is one of the standard animal models for hu man granulomatous uveitis and has greatly added to our understanding of some of the mechanisms of this disease [7, 8], In this ani mal model, uveitis is elicited by disruption of the lens capsule, following multiple im munizations with lens protein. The disease is best manifested in rats, but can be produced also in guinea pigs and mice [9], In rats, the pigmented Brown Norway strain develops a severe reaction around the lens, along with marked thickening of the choroid with mononuclear cells and multiple epithelioid cell granulomas, whereas albino strains pro duce an Arthus reaction around the lens [9], In an earlier study, we observed that various arachidonic acid metabolite inhibitors have modulating effects on the expression of gran ulomatous uveitis in the Brown Norway rat [10], The lipoxygenase inhibitors nafazatrom and nordihydroguaiaretic acid (NDGA) markedly suppressed the granulomatous in flammation, whereas the cyclo-oxygenase in hibitor indomethacin enhanced inflamma tion [10], In the study described herein, we investigated the effect of these inhibitors on the expression of lysosomal enyzmes in the choroidal inflammation that is characteristic of experimental lens-induced granulomatous uveitis.
Materials and Methods Seventy-two adult Brown Norway rats, were sensi tized to lens protein by subcutaneous injection of 10 mg of bovine lens protein in Freund’s complete adjuvant (Difco Laboratories. Detroit. Mich.. USA).
The animals were injected twice in the dorsal nuchal region at an interval of 2 weeks. One week after the second injection, the lens in 1 eye of each animal was surgically disrupted with a bent 30-gauge needle. To prevent infection, broadspectrum antibiotic ointment was applied to the lensdisrupted eyes immediately following the surgical procedure and once daily for the course of each exper iment. The rats were divided into two major groups of 36 animals, and these two groups were each further divided into three subgroups of 12 rats each. Each of these subgroups received intraperitoneal injections beginning on the days of lens disruption, as follows: subgroup 1 - control animals treated daily with intra peritoneal injections of 0.5 ml of diluent (0.01 M so dium hydroxide in normal saline solution); subgroup 2 - treated daily with intraperitoneal injections of 0.5 ml NDGA (Sigma, St. Louis, Mo., USA), 10 mg/ kg of body weight, and subgroup 3 - treated daily with intraperitoneal injections of 0.5 ml indomethacin (Sigma) 5 mg/kg of body weight. In both groups, 6 animals of each subgroup were killed on the 3rd day of treatment and the remaining animals on day 7. All eyes from the first group (36 rats) were fixed in formal calcium at 4 °C for 18 h, washed in tap water and impregnated with gum su crose at 4 °C for 18 h, then slowly frozen in embed ding medium for frozen tissue specimens (OCT; Miles, Indiana, USA) and stored at -7 0 ° C . These eyes were processed to detect the presence of the lyso somal enzymes, P-glucuronidase and acid phospha tase. For the detection of P-glucuronidase, the method of Hayashi et al. [11] was employed. In brief, cryostat sections were incubated in a medium containing naphthol AS-BI glucuronide and hexazonium pararosanilin at 37 °C for 30 min. The sections were washed in distilled water and countcrstaincd with 2% methyl green. P-Glucuronidase activity is manifested by the development of a red coloration. To detect the presence of acid phosphatase, the azo dye coupling method was used, as described pre viously [12]. Sections were incubated in sodium naphthyl phosphate in 0.1 M acetate buffer for 30 min, washed in distilled water and counterstained with methyl green. Acid-phosphatase-positive cells appear red when examined microscopically. From the second group (36 rats), all eyes were fresh frozen in OCT with liquid nitrogen. These eyes
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Kristeva/Biswas/Pararajasegaram/Sevanian/Rao
Granulomatous Uveitis
were processed for the detection of succinate dehy drogenase, as described previously [12], Unfixed cryostat sections were incubated at 37 °C for 45 min in a solution containing tetrazolium salt. Succinate dehydrogenase activity was detected by the develop ment of a purple deposit in positive cells. Eight-micrometer-thick sections were used for all histochemical stains. A piece of liver and of kidney were excised from each animal and used as positive controls during histochemical analysis of these en zymes. One section from each eye was stained with hematoxylin and eosin to study the histologic changes as welll as for comparison with the histo chemical staining. The sections were coded to ensure that the pathologist remained unaware of the treat ment. Enzyme activity was evaluated by grading the intensity of staining: trace or minimal activity was graded as 1+, moderate staining as 2+ and intense reaction as 3+.
75
Table 1. Histochemical analysis of lysosomal en zymes in experimental lens-induced granulomatous uveitis Days after lens disruption
Enzymes Grading of staining reaction assayed ------------------------------------diluent NDGA indo methacin
3
AP ß-G SD
1.3 1.8 ND
1.2 1.1 ND
1.6 1.2 ND
7
AP ß-G SD
2.3 2.8 ND
3.0 2.9 ND
3.0 3.0 ND
AP = Acid phosphatase; p-G = ß-glucuronidase; SD = succinate dehydrogenase; ND = not detectable.
Histopathologic examination of the eyes of all animals showed disruption of the ante rior lens capsule with fragmentation of the cortical lens material. Control animals killed on the 3rd day fol lowing lens disruption showed uniform thickening of the uveal tract due to infiltra tion of polymorphonuclear leukocytes, mononuclear cells and occasional epithelioid cells. There was no distinct granuloma for mation. Control animals killed on the 7th day after lens disruption showed an increase in thickening of the choroid, with marked infiltration of mononuclear, epithelioid cells and a few giant cells. The animals treated with NDGA showed less severe inflammation than did the con trol group on both the 3rd and 7th days fol lowing lens disruption, while animals treated with indomethacin showed histologic fea tures similar to those of the control group on both the 3rd and 7th days.
Histochemically, the enzymes acid phos phatase and (i-glucuronidase were found in mild to moderate (1+ to 2+) amounts in con trol animals killed on the 3rd day after lens disruption. Animals treated with NDGA or indomethacin showed no variation in these two enzymes when compared with the con trol animals (table 1). While an increase (3+) in the amount of acid phosphatase (fig. 1A) and P-glucuronidase (fig. ID) was observed in the control animals on the 7th day, compared with that observed on the 3rd day, there was no differ ence in the staining reaction of enzymes be tween the control groups and the groups treated with indomethacin (fig. IB. E) or NDGA (fig. 1C. F) at this time interval (ta ble 1). The lysosomal enzyme succinate dehy drogenase was not detected in any of the globes removed on the 3rd or 7th day after lens disruption. Sections of the liver and kid-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Results
76
Krisieva/Biswas/Pararajasegaram/Sevanian/Rao
ney from these same animals, however, did show a positive reaction for succinate dehy drogenase at both time points examined.
Discussion In our study, histologic examination of the control animals (treated with diluent) on day 3 revealed the presence of predomi nantly mononuclear phagocytes and occa sional lymphocytes. On day 7. these control animals showed a marked increase in the number of macrophages/epithelioid cells, and mature granulomas were also observed. In the bacille Calmette-Guerin (BCG) in duced skin granuloma, Dannenberget al. [6]
observed polymorphonuclear leukocytes and mononuclear cells on the 5th day following immunization, while on the 12th day, poly morphonuclear leukocytes were less promi nent. and macrophages assumed the charac teristics of immature epithelioid cells. By the 15th day, mature epithelioid cells were ob served. In our model, earliest indications of granuloma formation were seen on the 3rd day after lens disruption, and mature granu lomas were seen on the 7th day. The re sponse to antigenic stimuli in the lens-in duced granulomatous inflammation thus ap pears to be more marked than that observed in the skin granulomas. Enzyme histochemical studies revealed the presence of two lysosomal enzymes, acid
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Fig. 1. The choroidal inflammation exhibiting acid phosphatase reaction product in an animal treated with diluent (A), indomethacin (B) and NDGA (C). Note that there is no difference in the distribution of acid phosphatase. Note the presence of P-glucuronidase in the uveal inflammation of an animal treated with diluent (D). with indomethacin (F.) or with NDGA (F). There is a reduction in choroidal thickness in animals treated with NDGA. P-Glucuronidase is present in similar amounts in all three groups of animals. X 240.
phosphatase and (3-glucuronidase. The activ ity of these enzymes was found to be mark edly increased from the 3rd to the 7th day after lens disruption, which corresponds to the time course of granuloma formation ob served histologically. This observation was similar to that of Dannenberg et al. [6], who observed a marked increase in the lysosomal enzymes acid phosphatase, (3-glucuronidase, succinic dehydrogenase and cytochrome oxi dase at different stages of the maturation process in BCG-induced granulomas. We could not detect the enzyme succinate dehydrogenase in our model of lens-induced granulomatous uveitis. In a separate experi ment, skin granulomas produced by subcuta neous injection of lens protein revealed min imal activity of this enzyme on day 7 and moderate amounts on day 14; the enzyme was not detectable in skin granulomas exam ined on the 3rd day following immunization [unpubl. observations]. Our inability to de tect succinate dehydrogenase in the choroid may be due either to the absence of this enzyme in this region, or to the enzyme being present at levels below the level of detection of our histochemical staining tech nique. The concentration of NDGA and of indomethacin used in this study was based on previously reported investigations [10, 13]. Both drugs are known to be nonspecific in hibitors of arachidonic acid metabolism when used at high concentrations [ 13]. How ever. at the doses employed in the current study, NDGA and indomethacin have been shown to selectively inhibit the lipoxygenase and cyclo-oxygenase pathways of the arachi donic acid metabolism, respectively [ 10, 13]. Histologically, there was suppression of in flammation in the choroid in the NDGAtreated groups compared with that in the
77
control or the indomethacin-treated groups of rats. This was similar to our earlier obser vation [10]. Although there was a marked reduction in the number of granulomas as well as in the choroidal thickness in the NDGA-treated group compared with the indomethacintreated group or with the control animals, there was no apparent difference in the amounts of acid phosphatase or (3-glucuronidase detected in the three groups. This is probably due to the fact that although lip oxygenase inhibitors can suppress inflamma tion. the maturation process of granulomas is not regulated by these agents. Hence, the amounts of lysosomal enzymes generated would remain unaltered. Another possibility is that the intensity of histochemical staining of these enzymes does not correspond to the amount present; the histochemical detection of these enzymes may be an all or nothing reaction. In conclusion, our study demonstrates an increase in lysosomal enzymes concurrent with maturation of choroidal granuloma at different stages of lens-induced granuloma tous uveitis. There is no substantial differ ence in the lysosomal enzyme activity be tween control animals and those treated with cyclo-oxygenase or lipoxygenase inhibitors. The granulomatous inflammation, however, was reduced with lipoxygenase inhibitors in comparison with the other treatments, which correlates with our previous findings [10], These findings suggest that although arachidonic acid metabolites regulate the in flammatory response in lens-induced granu lomatous uveitis, they do not play a major role in the expression of lysosomal enzymes during development of the granulomatous inflammation observed in our model of uveitis.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Granulomatous Uveitis
Kristeva/Biswas/Pararajasegaram/Sevanian/Rao
References 1 Woods AC, Abrahams IW: Uveitis survey. Am J Ophthalmol 1961;51:761-780. 2 Hogan MJ, Zimmerman LE: Ophthalmic Patholo gy: An Atlas and Textbook, ed 2. Philadelphia, Saunders, 1962, pp 373-402. 3 Boros DL: Granulomatous Inflammations. Prog Allergy 1978:24:183-267. 4 Adams DO: The granulomatous inflammatory re sponse: A reveiw. Am J Pathol 1976;84:164— 191. 5 Adams DO: The biology of the granuloma; in Joa chim HI. (cd): Pathology of Granulomas. New York. Raven Press. 1983. pp 1-20. 6 Danncnberg AM Jr, Meyer OT, Esterly JR, Kambara T: The local nature of immunity in tubercu losis, illustrated histochemically in dermal BCG lesions. J Immunol 1968:100:931-941. 7 Marak GE Jr: Abrogation of tolerance to lens pro tein; in Sears ML (ed): New Directions in Oph thalmic Research. New Haven. Yale University Press, 1981. pp 47-61. 8 Rao NA, Marak GE Jr: Experimental granuloma tous uveitis: An electron-microscopic study of pig ment containing giant cells. Invest Ophthalmol VisSci 1985:26:1303-1305. 9 Marak GE Jr. Front RL. Rao NA: Strain differ ences in autoimmunity to lens protein. Ophthal mic Res 1981;13:320-329.
10 Rao NA, Patchett R, Fernandez MA, Sevanian A, Kunkel SL, Marak GE Jr: Treatment of experi mental granulomatous uveitis by lipoxygenase and cyclo-oxygenase inhibitors. Arch Ophthalmol 1987;105:413-415. 11 Hayashi M, Nakajima Y, Fishman WH: The cyto logic demonstration of p-glucuronidase employing naphthol AS-BI glucuronide and hexazonium pararosanilin: A preliminary report. J Histochem Cytochem 1964:12:293-297., 12 Bancroft JD: Enzyme histochemistry; in Bancroft JD, Stevens A (eds): Theory and Practice of His tological Techniques. Edinburgh, Churchill Liv ingstone. 1982, pp 379-405. 13 Kunkel SL, Chensue SW, Plewa M. Higashi GI: Macrophage function in the Schistosoma mansoni egg-induced pulmonary granuloma. Role of arachidonic acid metabolites in macrophage la anti gen expression. Am J Pathol 1984; 114:240-249.
Received: May 17, 1990 Accepted: August 21,1990 Narsing A. Rao, MD 1355 San Pablo Street Los Angeles, CA 90033 (USA)
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78
Histochemical Analysis of Experimental Granulomatous Uveitis1 Michaela Kristevaa, Jyotirmay Biswasa, Geeta Pararajasegarama, Alex Sevanianb, Narsing A. Raoa aDohcny Eye Institute and Departments of Ophthalmology and Pathology, and bInstitute for Toxicology, University of Southern California School of Medicine. Los Angeles, Calif. USA
Key Words. Enzyme histochemistry • Lens-induced uveitis • Arachidonic acid • Lysosomal enzymes
Granulomatous uveitis is one of the most common forms of uveitis that can lead to visual impairment and blindness [1], The precise mechanisms of the granuloma forma 1 This work was supported in part by a research grant (No. F.Y 06953) from the National Eye Insti tute. National Institutes of Health. USA.
tion are not known, but it has been estab lished that macrophages are primarily in volved in this process. Granulomas in var ious tissues have been shown to develop in sequential stages from mononuclear phago cytes to mature epithelioid cell granulomas [2-5]. and lysosomal enzymes such as acid phosphatase, P-glucuronidase, succinate de
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Abstract. We have previously demonstrated the effects of various inhibitors of arachi donic acid metabolism on experimental lens-induced granulomatous uveitis. In the present study, we investigated the effect of these same inhibitors on the expression of lysosomal enzymes at different stages of choroidal inflammation in experimental lens-induced granu lomatous uveitis and compared this to the inflammation observed at each stage examined. Lysosomal enzymes such as acid phosphatase, P-glucuronidase and succinate dehydrogenase are known to be liberated during the maturation of mononuclear phagocytes to epithelioid cell granulomas. Although animals treated with nordihydroguaiaretic acid showed less severe inflammation than did indomethacin-treated or control animals, none of these agents appeared to affect the expression of acid phosphatase and P-glucuronidase, as determined histochemically. Succinate dehydrogenase could not be detected in any of the eyes examined, even though sections of liver and kidney from these same animals were positive for this enzyme.
74
hydrogenase and cytochrome oxidase are known to be liberated during this maturation process [6]. Experimental granulomatous uveitis is one of the standard animal models for hu man granulomatous uveitis and has greatly added to our understanding of some of the mechanisms of this disease [7, 8], In this ani mal model, uveitis is elicited by disruption of the lens capsule, following multiple im munizations with lens protein. The disease is best manifested in rats, but can be produced also in guinea pigs and mice [9], In rats, the pigmented Brown Norway strain develops a severe reaction around the lens, along with marked thickening of the choroid with mononuclear cells and multiple epithelioid cell granulomas, whereas albino strains pro duce an Arthus reaction around the lens [9], In an earlier study, we observed that various arachidonic acid metabolite inhibitors have modulating effects on the expression of gran ulomatous uveitis in the Brown Norway rat [10], The lipoxygenase inhibitors nafazatrom and nordihydroguaiaretic acid (NDGA) markedly suppressed the granulomatous in flammation, whereas the cyclo-oxygenase in hibitor indomethacin enhanced inflamma tion [10], In the study described herein, we investigated the effect of these inhibitors on the expression of lysosomal enyzmes in the choroidal inflammation that is characteristic of experimental lens-induced granulomatous uveitis.
Materials and Methods Seventy-two adult Brown Norway rats, were sensi tized to lens protein by subcutaneous injection of 10 mg of bovine lens protein in Freund’s complete adjuvant (Difco Laboratories. Detroit. Mich.. USA).
The animals were injected twice in the dorsal nuchal region at an interval of 2 weeks. One week after the second injection, the lens in 1 eye of each animal was surgically disrupted with a bent 30-gauge needle. To prevent infection, broadspectrum antibiotic ointment was applied to the lensdisrupted eyes immediately following the surgical procedure and once daily for the course of each exper iment. The rats were divided into two major groups of 36 animals, and these two groups were each further divided into three subgroups of 12 rats each. Each of these subgroups received intraperitoneal injections beginning on the days of lens disruption, as follows: subgroup 1 - control animals treated daily with intra peritoneal injections of 0.5 ml of diluent (0.01 M so dium hydroxide in normal saline solution); subgroup 2 - treated daily with intraperitoneal injections of 0.5 ml NDGA (Sigma, St. Louis, Mo., USA), 10 mg/ kg of body weight, and subgroup 3 - treated daily with intraperitoneal injections of 0.5 ml indomethacin (Sigma) 5 mg/kg of body weight. In both groups, 6 animals of each subgroup were killed on the 3rd day of treatment and the remaining animals on day 7. All eyes from the first group (36 rats) were fixed in formal calcium at 4 °C for 18 h, washed in tap water and impregnated with gum su crose at 4 °C for 18 h, then slowly frozen in embed ding medium for frozen tissue specimens (OCT; Miles, Indiana, USA) and stored at -7 0 ° C . These eyes were processed to detect the presence of the lyso somal enzymes, P-glucuronidase and acid phospha tase. For the detection of P-glucuronidase, the method of Hayashi et al. [11] was employed. In brief, cryostat sections were incubated in a medium containing naphthol AS-BI glucuronide and hexazonium pararosanilin at 37 °C for 30 min. The sections were washed in distilled water and countcrstaincd with 2% methyl green. P-Glucuronidase activity is manifested by the development of a red coloration. To detect the presence of acid phosphatase, the azo dye coupling method was used, as described pre viously [12]. Sections were incubated in sodium naphthyl phosphate in 0.1 M acetate buffer for 30 min, washed in distilled water and counterstained with methyl green. Acid-phosphatase-positive cells appear red when examined microscopically. From the second group (36 rats), all eyes were fresh frozen in OCT with liquid nitrogen. These eyes
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Kristeva/Biswas/Pararajasegaram/Sevanian/Rao
Granulomatous Uveitis
were processed for the detection of succinate dehy drogenase, as described previously [12], Unfixed cryostat sections were incubated at 37 °C for 45 min in a solution containing tetrazolium salt. Succinate dehydrogenase activity was detected by the develop ment of a purple deposit in positive cells. Eight-micrometer-thick sections were used for all histochemical stains. A piece of liver and of kidney were excised from each animal and used as positive controls during histochemical analysis of these en zymes. One section from each eye was stained with hematoxylin and eosin to study the histologic changes as welll as for comparison with the histo chemical staining. The sections were coded to ensure that the pathologist remained unaware of the treat ment. Enzyme activity was evaluated by grading the intensity of staining: trace or minimal activity was graded as 1+, moderate staining as 2+ and intense reaction as 3+.
75
Table 1. Histochemical analysis of lysosomal en zymes in experimental lens-induced granulomatous uveitis Days after lens disruption
Enzymes Grading of staining reaction assayed ------------------------------------diluent NDGA indo methacin
3
AP ß-G SD
1.3 1.8 ND
1.2 1.1 ND
1.6 1.2 ND
7
AP ß-G SD
2.3 2.8 ND
3.0 2.9 ND
3.0 3.0 ND
AP = Acid phosphatase; p-G = ß-glucuronidase; SD = succinate dehydrogenase; ND = not detectable.
Histopathologic examination of the eyes of all animals showed disruption of the ante rior lens capsule with fragmentation of the cortical lens material. Control animals killed on the 3rd day fol lowing lens disruption showed uniform thickening of the uveal tract due to infiltra tion of polymorphonuclear leukocytes, mononuclear cells and occasional epithelioid cells. There was no distinct granuloma for mation. Control animals killed on the 7th day after lens disruption showed an increase in thickening of the choroid, with marked infiltration of mononuclear, epithelioid cells and a few giant cells. The animals treated with NDGA showed less severe inflammation than did the con trol group on both the 3rd and 7th days fol lowing lens disruption, while animals treated with indomethacin showed histologic fea tures similar to those of the control group on both the 3rd and 7th days.
Histochemically, the enzymes acid phos phatase and (i-glucuronidase were found in mild to moderate (1+ to 2+) amounts in con trol animals killed on the 3rd day after lens disruption. Animals treated with NDGA or indomethacin showed no variation in these two enzymes when compared with the con trol animals (table 1). While an increase (3+) in the amount of acid phosphatase (fig. 1A) and P-glucuronidase (fig. ID) was observed in the control animals on the 7th day, compared with that observed on the 3rd day, there was no differ ence in the staining reaction of enzymes be tween the control groups and the groups treated with indomethacin (fig. IB. E) or NDGA (fig. 1C. F) at this time interval (ta ble 1). The lysosomal enzyme succinate dehy drogenase was not detected in any of the globes removed on the 3rd or 7th day after lens disruption. Sections of the liver and kid-
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Results
76
Krisieva/Biswas/Pararajasegaram/Sevanian/Rao
ney from these same animals, however, did show a positive reaction for succinate dehy drogenase at both time points examined.
Discussion In our study, histologic examination of the control animals (treated with diluent) on day 3 revealed the presence of predomi nantly mononuclear phagocytes and occa sional lymphocytes. On day 7. these control animals showed a marked increase in the number of macrophages/epithelioid cells, and mature granulomas were also observed. In the bacille Calmette-Guerin (BCG) in duced skin granuloma, Dannenberget al. [6]
observed polymorphonuclear leukocytes and mononuclear cells on the 5th day following immunization, while on the 12th day, poly morphonuclear leukocytes were less promi nent. and macrophages assumed the charac teristics of immature epithelioid cells. By the 15th day, mature epithelioid cells were ob served. In our model, earliest indications of granuloma formation were seen on the 3rd day after lens disruption, and mature granu lomas were seen on the 7th day. The re sponse to antigenic stimuli in the lens-in duced granulomatous inflammation thus ap pears to be more marked than that observed in the skin granulomas. Enzyme histochemical studies revealed the presence of two lysosomal enzymes, acid
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Fig. 1. The choroidal inflammation exhibiting acid phosphatase reaction product in an animal treated with diluent (A), indomethacin (B) and NDGA (C). Note that there is no difference in the distribution of acid phosphatase. Note the presence of P-glucuronidase in the uveal inflammation of an animal treated with diluent (D). with indomethacin (F.) or with NDGA (F). There is a reduction in choroidal thickness in animals treated with NDGA. P-Glucuronidase is present in similar amounts in all three groups of animals. X 240.
phosphatase and (3-glucuronidase. The activ ity of these enzymes was found to be mark edly increased from the 3rd to the 7th day after lens disruption, which corresponds to the time course of granuloma formation ob served histologically. This observation was similar to that of Dannenberg et al. [6], who observed a marked increase in the lysosomal enzymes acid phosphatase, (3-glucuronidase, succinic dehydrogenase and cytochrome oxi dase at different stages of the maturation process in BCG-induced granulomas. We could not detect the enzyme succinate dehydrogenase in our model of lens-induced granulomatous uveitis. In a separate experi ment, skin granulomas produced by subcuta neous injection of lens protein revealed min imal activity of this enzyme on day 7 and moderate amounts on day 14; the enzyme was not detectable in skin granulomas exam ined on the 3rd day following immunization [unpubl. observations]. Our inability to de tect succinate dehydrogenase in the choroid may be due either to the absence of this enzyme in this region, or to the enzyme being present at levels below the level of detection of our histochemical staining tech nique. The concentration of NDGA and of indomethacin used in this study was based on previously reported investigations [10, 13]. Both drugs are known to be nonspecific in hibitors of arachidonic acid metabolism when used at high concentrations [ 13]. How ever. at the doses employed in the current study, NDGA and indomethacin have been shown to selectively inhibit the lipoxygenase and cyclo-oxygenase pathways of the arachi donic acid metabolism, respectively [ 10, 13]. Histologically, there was suppression of in flammation in the choroid in the NDGAtreated groups compared with that in the
77
control or the indomethacin-treated groups of rats. This was similar to our earlier obser vation [10]. Although there was a marked reduction in the number of granulomas as well as in the choroidal thickness in the NDGA-treated group compared with the indomethacintreated group or with the control animals, there was no apparent difference in the amounts of acid phosphatase or (3-glucuronidase detected in the three groups. This is probably due to the fact that although lip oxygenase inhibitors can suppress inflamma tion. the maturation process of granulomas is not regulated by these agents. Hence, the amounts of lysosomal enzymes generated would remain unaltered. Another possibility is that the intensity of histochemical staining of these enzymes does not correspond to the amount present; the histochemical detection of these enzymes may be an all or nothing reaction. In conclusion, our study demonstrates an increase in lysosomal enzymes concurrent with maturation of choroidal granuloma at different stages of lens-induced granuloma tous uveitis. There is no substantial differ ence in the lysosomal enzyme activity be tween control animals and those treated with cyclo-oxygenase or lipoxygenase inhibitors. The granulomatous inflammation, however, was reduced with lipoxygenase inhibitors in comparison with the other treatments, which correlates with our previous findings [10], These findings suggest that although arachidonic acid metabolites regulate the in flammatory response in lens-induced granu lomatous uveitis, they do not play a major role in the expression of lysosomal enzymes during development of the granulomatous inflammation observed in our model of uveitis.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 8:08:02 AM
Granulomatous Uveitis
Kristeva/Biswas/Pararajasegaram/Sevanian/Rao
References 1 Woods AC, Abrahams IW: Uveitis survey. Am J Ophthalmol 1961;51:761-780. 2 Hogan MJ, Zimmerman LE: Ophthalmic Patholo gy: An Atlas and Textbook, ed 2. Philadelphia, Saunders, 1962, pp 373-402. 3 Boros DL: Granulomatous Inflammations. Prog Allergy 1978:24:183-267. 4 Adams DO: The granulomatous inflammatory re sponse: A reveiw. Am J Pathol 1976;84:164— 191. 5 Adams DO: The biology of the granuloma; in Joa chim HI. (cd): Pathology of Granulomas. New York. Raven Press. 1983. pp 1-20. 6 Danncnberg AM Jr, Meyer OT, Esterly JR, Kambara T: The local nature of immunity in tubercu losis, illustrated histochemically in dermal BCG lesions. J Immunol 1968:100:931-941. 7 Marak GE Jr: Abrogation of tolerance to lens pro tein; in Sears ML (ed): New Directions in Oph thalmic Research. New Haven. Yale University Press, 1981. pp 47-61. 8 Rao NA, Marak GE Jr: Experimental granuloma tous uveitis: An electron-microscopic study of pig ment containing giant cells. Invest Ophthalmol VisSci 1985:26:1303-1305. 9 Marak GE Jr. Front RL. Rao NA: Strain differ ences in autoimmunity to lens protein. Ophthal mic Res 1981;13:320-329.
10 Rao NA, Patchett R, Fernandez MA, Sevanian A, Kunkel SL, Marak GE Jr: Treatment of experi mental granulomatous uveitis by lipoxygenase and cyclo-oxygenase inhibitors. Arch Ophthalmol 1987;105:413-415. 11 Hayashi M, Nakajima Y, Fishman WH: The cyto logic demonstration of p-glucuronidase employing naphthol AS-BI glucuronide and hexazonium pararosanilin: A preliminary report. J Histochem Cytochem 1964:12:293-297., 12 Bancroft JD: Enzyme histochemistry; in Bancroft JD, Stevens A (eds): Theory and Practice of His tological Techniques. Edinburgh, Churchill Liv ingstone. 1982, pp 379-405. 13 Kunkel SL, Chensue SW, Plewa M. Higashi GI: Macrophage function in the Schistosoma mansoni egg-induced pulmonary granuloma. Role of arachidonic acid metabolites in macrophage la anti gen expression. Am J Pathol 1984; 114:240-249.
Received: May 17, 1990 Accepted: August 21,1990 Narsing A. Rao, MD 1355 San Pablo Street Los Angeles, CA 90033 (USA)
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