Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Discovery and development of antiandrogens wikipedia , lookup
Psychopharmacology wikipedia , lookup
Discovery and development of angiotensin receptor blockers wikipedia , lookup
Cannabinoid receptor antagonist wikipedia , lookup
Theralizumab wikipedia , lookup
NK1 receptor antagonist wikipedia , lookup
Investigative Ophthalmology & Visual Science, Vol. 32, No. 8, July 1991 Copyright © Association for Research in Vision and Ophthalmology Lipid Mediators of Tumor Necrosis Facfor-a-lnduced Uveitis Lloyd N. Fleisher, Jenny B. Ferrell, Mariberh G. Smith, and M. Christine McGahan The authors tested the hypothesis that platelet-activating factor (PAF) and cyclooxygenase metabolites of arachidonic acid mediate the ocular inflammatory response to intravitreally injected tumor necrosis factor-a (TNFa). Rabbits were treated with the PAF receptor antagonist SRI 63-441, the cyclooxygenase inhibitors indomethacin and naproxen, or SRI 63-441 and indomethacin. At 24 hr after intravitreal injection of TNF (20,000 U), the severity of inflammation was assessed based on iridal hypermia, aqueous humor leukocyte number and aqueous humor protein, immunoreactive-prostaglandin E (I-PGE), and leukotriene B4 (LTB4) concentrations. Although all of the treatments significantly reduced the severity of anterior uveitis, SRI 63-441 plus indomethacin was the most effective, indomethacin and naproxen were intermediately effective, and SRI 63-441 was the least effective. The results of this study are consistent with an important role for cyclooxygenase metabolites of arachidonic acid in the inflammatory response to TNFa, particularly with respect to dilation of iridal blood vessels. Although naproxen was nearly as effective as indomethacin in reducing aqueous humor I-PGE levels, indomethacin conferred significantly more protection to the blood- aqueous barrier as shown by lower protein levels in the aqueous humor. Thus, although cyclooxygenase inhibition may partially explain the protection afforded the blood-aqueous barrier by these nonsteroidal anti-inflammatory agents, the data suggest that indomethacin may also exert anti-inflammatory effects that are independent of cyclooxygenase inhibition. Furthermore, the data are consistent with TNFa releasing PAF in the eye and PAF acting both directly, by increasing vascular permeability, and indirectly, by promoting release of cyclooxygenase and lipoxygenase metabolites of arachidonic acid. Invest Ophthalmol Vis Sci 32:2393-2399,1991 Tumor necrosis factor-a (TNFa) is a cytokine that is released by monocytes/macrophages in response to bacterial lipopolysaccharide and other stimuli.12 In nonocular tissues, TNFa is a pluripotent inflammatory mediator because of its ability to enhance neutrophil and monocyte chemotaxis,3'4 neutrophil-endothelial cell adherence,5'6 endothelial cell permeability,7 endothelial cell procoagulant properties,8 and neutrophil and monocyte/macrophage degranulation, respiratory burst, and cytotoxicity.9"12 In the eye, effects of TNFa are just being denned. TNFa produces uveitis when it is injected into the vitreal chamber of the rabbit eye, as characterized by hyper- From the Department of Anatomy, Physiological Sciences, and Radiology, North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina. Supported by grant #EY-08688 from the National Institutes of Health and a grant from the State of North Carolina. Presented in part at the meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, 1990. Submitted for publication: September 10, 1990; accepted March 10, 1991. Reprint requests: Lloyd N. Fleisher, PhD, Department of Anatomy, Physiological Sciences, and Radiology, North Carolina State University, College of Veterinary Medicine, Raleigh, NC 27606. emia of iridal blood vessels, disruption of blood-ocular barriers, infiltration of leukocytes, and elevation of prostaglandin (PG) levels in aqueous humor.1314 TNFa stimulates the production of platelet-activating factor (PAF)1516 and cyclooxygenase1718 and lipoxygenase19'20 metabolites of arachidonic acid (eicosanoids) in nonocular tissues. We hypothesized that the ocular inflammatory effects of intravitreally injected TNFa might be due to the release of PAF and eicosanoids. To test this hypothesis, uveitis was induced in rabbit eyes by intravitreal injection of TNFa. The effects of the cyclooxygenase inhibitors, indomethacin and naproxen, and the PAF receptor antagonist, SRI 63-441, on the severity of inflammation were assessed by biomicroscopic, cellular, and biochemical criteria. Materials and Methods Induction of Inflammation and Drug Treatments All experiments were conducted in accordance with the ARVO Resolution on the Use of Animals in Research. Male 2-kg New Zealand white rabbits were anesthetized with intramuscular injections of xylazine (12 mg/kg) and ketamine (60 mg/kg). Human recombinant TNFa (0.1 ml; 20,000 U; 1 Mg protein; 2393 Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 2394 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1991 Genzyme Corp., Boston, MA) was injected into the right eye; the left eye received an equal volume of sterile vehicle that consisted of phosphate-buffered saline that contained 0.1 % low-endotoxin bovine serum albumin (Fraction V; Sigma Chemical Co., St. Louis, MO). Injections were made through a 30-g needle that was attached to a syringe, approximately 3 mm posterior to the limbus. Animals were divided into five treatment groups: group I animals received injections of drug vehicles. The schedule and route of administration of vehicle injections corresponded to those given to the four groups of drug-treated animals (groups II-V). Group II animals received intraperitoneal injections of the cyclooxygenase inhibitor indomethacin (10 mg/kg) at 0.5 hr before TNFa and 8 hr after TNFa injection. Indomethacin was delivered as the trihydrate after reaction with equimolar amounts of sodium carbonate. Group III animals received intraperitoneal injections of the cyclooxygenase inhibitor naproxen sodium (50 mg/kg) 0.5 hr before TNFa and 7, 14, and 23 hr after TNFa injection. Group IV animals received intravenous injections of the PAF receptor antagonist SRI 63-441 (10 and 20 mg/kg) 0.5 hr before and 2, 4, and 6 hr after TNFa injection. Group V animals were treated with indomethacin and SRI 63-441 as described for groups II and IV. SRI 63-441 was initially given at a dose of 20 mg/kg.21"23 Because this dose produced hemolysis in three of six animals, it was subsequently reduced to 10 mg/kg. SRI 63-441-induced hemolysis has also been reported in equine erythrocytes.2425 The results reported are the combined means of four animals that received the lower dose and three (in which hemolysis did not occur) that received the higher dose of SRI 63-441. The same dose of indomethacin was used as that reported to reduce PGE2 levels in aqueous humor after intravitreal injection of bacterial lipopolysaccharide.26 However, we found that two injections over 24 hr reduced the PGE2 levels in aqueous humor after intravitreal injection of lipopolysaccharide (aqueous humor PGE2 levels: indomethacin treatment, 0.18 ± 0.31 ng/ml, n = 3; indomethacin vehicle treatment, mean: 20.17, range = 12.10 - 28.23, n = 2) as effectively as the three doses given in the previous study. To determine the proper dose regimen for naproxen, a dose-response experiment was performed (Table 1). Assessment of Inflammation and Removal of Ocular Fluids Twenty-four hours after TNFa injection, animals were examined with a slit lamp for intensity of iridal hyperemia and were rated on a scale of 0-3, with 3 being the most intense response.27 Animals were then Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 Vol. 32 Table 1. Effect of dosage regimen of naproxen sodium on I-PGE levels in aqueous 24 hr after intravitreal injection of E. coli lipopolysaccharide. Dose (mg/kg) Injections/24 hr 0 2-4 20 2 40 2 50 2 50 4 Aqueous humor I-PGE (ng/ml) 6.55 19.21 9.98 7.89 1.79 9.70 5.69 13.62 0.99 0.90 Data represent individual values for each animal (n = 2). killed by exposure to an atmosphere of 100% CO2, their eyes were removed, and the aqueous and vitreous humors were aspirated into heparin-treated syringes. Aliquots of aqueous humor (5-10 n\) were removed for determination of protein and total leukocyte cell number. Aqueous humor (100 fi\) was then centrifuged in a Micro-centrifuge (Model 235c; Allied Fisher Scientific, Atlanta, GA) for 1 min, and the supernatant was aspirated and transferred to polypropylene centrifuge tubes. Aqueous humor supernatants were then subjected to organic extraction as described below. An inflammatory index was designed to facilitate the comparison of the effects of each drug treatment on the severity of the inflammatory response. For each inflammatory parameter, the drug treatment that produced the greatest effect was assigned a rating of 4, and the treatment that produced the least effect was assigned a rating of 1. Drug effects on vitreous protein concentration were not included in the computation of the inflammatory index because this concentration was not affected by intravitreal injection of TNFa (see Results section). The inflammatory index was designed to provide a simple way to visualize the effects of several drug treatments on multiple inflammatory parameters. It is meant to complement the actual results that are summarized in Table 3, not substitute for them. Protein Concentration and Leukocyte Number in Aqueous Humor Protein concentration in aqueous humor was determined with the use of the Lowry method28 that was modified for reduced volumes. Total leukocyte number in aqueous humor was determined with the use of a hemacytometer. TUMOR NECROSIS FACTOIVa-INDUCED UVEITIS / Fleisher er ol No. 8 2395 Effects of Cyclooxygenase Inhibitors and a PAF Receptor Antagonist on TNFa-Induced Uveitis Extraction of Aqueous Humor and Radioimmunoassay (RIA) of Eicosanoids Aqueous humor supernatant was acidified to pH 3.5 with 5 jul H3PO4 (0.49 M), extracted into six volumes of ethyl acetate (twice), dried under vacuum in a Speed-Vac Concentrator (Savant, Hicksville, NY), and reconstituted in 500 /x\ methanol. The efficiency of extraction, based on recovery of [3H]-PGE2 and [3H]-LTB4, was 63.8% and 74.2%, respectively. Aliquots of the methanol reconstitute were dried under vacuum, reconstituted in 300 n\ of a modified Krebs —Henseleit buffer (pH = 7.4), and PGE and LTB4 were measured by RIA. Details of the PGE RIA have been described.14 The antiserum used in this assay does not distinguish between PGE, and PGE2. Therefore, PG levels are referred to as immunoreactive PGE (I-PGE). LTB4 levels were measured with the use of a highly specific commercially available RIA kit (Amersham, Inc., Arlington, IL). Data Analysis Effects of drug treatment on TNFa-induced uveitis were evaluated using one-way ANOVA. Multiple means were compared with the use of Tukey's Omega test. Differences between TNFa-treated and vehicletreated eyes were determined with a paired student t-test. Means were considered significantly different at P < 0.05. Data are expressed as mean ± SEM. Results Effects of TNFa in Animals That Received Drug Vehicles No inflammation was seen in any eyes injected with TNFa vehicle. In group I animals, comparison of TNFa-injected eyes with the paired vehicle-injected eyes showed significant uveitis in the anterior segment based on hyperemia of iridal blood vessels and increases in aqueous humor leukocyte number and I-PGE and LTB4 concentrations (Table 2). However, based on vitreous humor protein concentration, the posterior segment blood-ocular barrier was not affected at 24 hr post-TNFa injection. These results are consistent with those of a previous report.14 Twenty-four hours after intravitreal injection of TNFa, hyperemia of iridal blood vessels, disruption of the blood-aqueous barrier, leukocyte infiltration into aqueous humor and accumulation of I-PGE and LTB4 in aqueous humor, were significantly reduced by treatment with the cyclooxygenase inhibitors, indomethacin and naproxen, or with the PAF receptor antagonist, SRI 63-441 (Table 3). However, differences were seen in the effectiveness of the drug treatments on each of the five inflammatory indices that were measured. With the possible exception of LTB4 concentration in aqueous humor (see below), TNFainduced uveitis was more effectively diminished by cyclooxygenase inhibition than by PAF receptor antagonism. Furthermore, aqueous humor I-PGE concentration and leukocyte number were more effectively reduced by PAF receptor antagonism plus cyclooxygenase inhibition than by cyclooxygenase inhibition or PAF receptor antagonism alone. Hyperemia of iridal blood vessels was especially sensitive to either of the cyclooxygenase inhibitors, whereas integrity of the blood-aqueous barrier, as indicated by protein concentration in aqueous humor, was equally protected by indomethacin or indomethacin plus SRI 63-441, but less protected by naproxen or SRI 63-441 alone. With respect to the 5-lipoxygenase metabolite, LTB4, differences in the effects of the four drug treatments were less pronounced. Indomethacin or SRI 63-441 reduced aqueous humor LTB4 concentrations to levels lower than those seen after treatment with naproxen. However, the effect of treatment with SRI 63-441 plus indomethacin was not significantly different from that of indomethacin, SRI 63-441, or naproxen. In Table 4, the relative effectiveness of drug treatments is expressed in terms of the inflammatory index (see Materials and Methods for details). Based on this index, indomethacin or indomethacin plus SRI 63441 most effectively reduced the inflammatory response to intravitreally injected TNFa. Treatment with the PAF receptor antagonist or with naproxen afforded the least protection. Table 2. Effects of intravitreal injections of TNFa and TNFa vehicle in animals that receive systemic treatment with drug vehicles Intravitreal injection Iridal hyperemia Aqueous cells (\d) Aqueous protein (vg/nO TNFa TNFa vehicle 2.1* 0.0 1882 ± 160* 0.0 11.1 ± 1.4* 0.9 ±0.1 1 Significantly different from TNFa vehicle (P < 0.05). Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 Vitreous protein (ng/nl) 0.8 ±0.1 0.6 ±0.1 Mean ± SEM (n = 10-12). Aqueous I-PGE (ng/ml) Aqueous LTB4 (ng/ml) 3.78 ± 0.62* 0.66 ±0.12 0.154 ±0.030* 0.050 ±0.012 2396 Vol. 32 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1991 Table 3. Effects of cyclo-oxygenase inhibition and PAF receptor antagonism on TNFa-induced uveitis Treatment TNF a TNF a Indomethacin TNF a Naproxen TNF a SRI 63-441 TNF a Indomethacin SRI 63-441 Aqueous cells (id) Aqueous protein (vg/id) Aqueous I-PGE (ng/ml) Aqueous LTB4 (ng/ml) 1882 ± 160 (11) 715±315*§" (11) 477 ± 139*§" (6) 1235±317*tt" (7) 251 ± 85*f*§ (9) 11.1 ± 1.4 (12) 2.1 ±0.2*t§ 3.78 ± 0.62 (12) 0.61 ± 0.15*§N (11) 0.95 ± 0.16*§N (6) 1.63 ± 0.3 l*tt" (7) 0.23 ± 0.1 O*tt§ (9) 0.154 ±0.030 (10) 0.043 ±0.011** (11) 0.077 ± 0.012*t§ (6) 0.042 ± 0.010** (7) 0.068 ± 0.012* (9) Iridal hyperemia 2.1 (12) 0.1 *§ (11) 0.3*§ (6) 1.2*tt" (7) 0.1 *§ (9) (ID 6.7 ± 0.8*t" (6) 7.9 ± 1.7*t" (7) 2.1 ±0.5*J§ (9) § Significantly different from treatment 4 (P < 0.05). Significantly different from treatment 5 (P < 0.05). Data represent mean ± SEM for the numbers of animals in parentheses. * Significantly different from treatment 1 (P < 0.05). t Significantly different from treatment 2 (P < 0.05). i Significantly different from treatment 3 (P < 0.05). 11 Discussion The results of this study support key roles for eicosanoids and PAF in the anterior uveitis seen 24 hr after intravitreal injection of human recombinant TNFa. Dilation of iridal blood vessels, leakage of plasma proteins into aqueous humor, and infiltration of leukocytes into aqueous humor were all reduced by the cyclooxygenase inhibitors, indomethacin and naproxen, or the PAF receptor antagonist, SRI 63-441. Uveitis that was induced by intravitreal injection of interleukin-1, a cytokine with many properties similar to TNFa, was also dependent on eicosanoid and PAF production.29 SRI 63-441 (tetrahydrofuranylmethoxyphosphinyloxy-ethylquinolinium) is a selective PAF receptor antagonist that prevents or reduces many of the hemodynamic and biochemical changes that are induced by infusion of PAF or endotoxin in many species.21"2330 Based on the results shown in Table 3 and in the inflammatory index (Table 4), SRI 63-441 plus indomethacin most effectively reduced TNFa-induced uveitis. This finding highlighted the ability of this drug combination to reduce aqueous cell number and I-PGE levels. Indomethacin was more effective than naproxen as an inhibitor of TNFa-induced uveitis (Tables 3 and 4). For example, indomethacin caused a greater reduction in aqueous humor protein and LTB4 levels than naproxen. Although the reason for the superior efficacy of indomethacin is not known, it may not be due to differen- tial cyclooxygenase inhibition because these agents did not differ significantly in this respect. However, aqueous humor I-PGE levels that follow indomethacin, although not statistically different from those that follow naproxen, were approximately 36% lower. This decrease may contribute to the superior efficacy of indomethacin over naproxen. Although inflammation was assessed 24 hr after intravitreal injection of TNFa, the PAF receptor antagonist was only administered for the first 6 hr after TNFa injection. Because of the relatively short duration of action of SRI 6 3-441,30 it is unlikely that there was significant PAF receptor antagonist activity 24 hr after induction of uveitis. This suggests that PAF release is a relatively early event in the uveitic response to TNFa. Furthermore, it raises the possibility that a PAF receptor antagonist with a longer duration of action might more dramatically reduce TNFa-induced uveitis. The vascular changes and disruption of the bloodaqueous barrier that occur during uveitis are believed to be caused by the combined effects of iridal vasodilation, increased vascular permeability, and alterations in the nonpigmented epithelium of the ciliary processes.3132 Inhibition of cyclooxygenase activity would reduce production of vasodilatory PGs, such as PGE2, and cause less dilation of iridal blood vessels. As shown in Table 3, iridal hyperemia was most effectively reduced by indomethacin or naproxen. Iridal hyperemia was also reduced by PAF receptor antago- Table 4. Relative effectiveness of drug treatments on the inflammatory response to intravitreally injected TNFa Inflammatory index Drug vehicle Indomethacin Naproxen SRI 63-441 Indomethacin SRI 63-441 0.0 3.2*f 2.4* 2.0* 3.6*f * Significantly different from drug vehicle treatment (P < 0.05). t Significantly different from naproxen and SRI 63-441 treatments (P < 0.05). Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 Data represent the mean rank for five inflammatory parameters. No. 8 TUMOR NECROSIS FACTOR-a-INDUCED UVEITIS / Fleisher er ol nism, but to a lesser extent than with indomethacin or naproxen. Thisfindinghas several implications. First, it is consistent with TNFa-induced release of PAF in the eye, a process that has been shown in nonocujari5,i6,2o a n ( j o c u i a r tissues.33"37 Second, because PAF is generally not a vasodilator,38*39 it is not likely to directly induce iridal hyperemia. However, PAF could act indirectly by releasing vasodilatory PGs. Indeed, PAF-induced release of cyclooxygenase metabolites has been shown in nonocular24-4041 tissues and in rabbit iris.42 Third, because SRI 63-441 only partially reduced aqueous humor I-PGE levels, compared with indomethacin and naproxen, PAF-induced PG release can account for only part of the total PG production that is induced by TNFa. The remainder of PG release is presumably caused by a direct action of TNFa on ocular tissues and/or on leukocytes. This dual mode of action for TNFa-induced stimulation of cyclooxygenase metabolite production is consistent with the effects of drug treatments on I-PGE concentration in aqueous humor (Table 3). Compared with group I, the blockade of PAF receptors with SRI 63441 reduced aqueous humor I-PGE levels by 57%, whereas inhibition of cyclooxygenase activity with indomethacin or naproxen reduced these levels by 84% and 75%, respectively. However, PAF receptor blockade plus cyclooxygenase inhibition (indomethacin plus SRI 63-441) reduced I-PGE levels by 94%. PAF potently alters vascular permeability as shown by its ability to increase cutaneous43'44 and conjunctival3545 vascular permeability. Furthermore, PAF has been implicated in the increased uveal permeability that accompanies laser irradiation of rabbit iris37 and the increased vascular permeability that accompanies endotoxin-induced uveitis and paracentesis in rabbits.36 The decrease in aqueous humor protein concentration after treatment with SRI 63-441 may be due to antagonism of the effects of PAF on vascular permeability. However, antagonism of PAF receptors with SRI 63-441 did not reduce aqueous humor protein concentration as effectively as indomethacin. Indomethacin-induced decreases in iridal vasodilation that are secondary to the inhibition of vasodilatory PG production would contribute to reductions in aqueous humor protein concentration. However, the findings suggest that processes that are independent of cyclooxygenase inhibition may also be involved in indomethacin-induced preservation of the bloodaqueous barrier. Despite similar reductions in aqueous humor I-PGE concentrations, naproxen was less effective than indomethacin in reducing the aqueous humor protein concentration. Furthermore, we have reported that although another cyclooxygenase inhibitor, flunixin meglumine, was as effective as indomethacin in reducing aqueous humor PGE2 lev- Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 2397 els during endotoxin-induced uveitis, only indomethacin significantly reduced iridal hyperemia and aqueous humor protein concentration.26 The reduction in aqueous humor LTB4 levels after PAF receptor antagonism is consistent with the blockade of PAF-induced release of 5-lipoxygenase metabolites of arachidonic acid.4041-46 PAF has also been implicated in the stimulation of 5- and 12-hydroxyeicosatetraenoic acid release in a rabbit model of corneal inflammation.34 Because LTB4 levels in aqueous humor from TNFa-vehicle injected eyes (Table 2) were not different from those in SRI 63-441-treated animals (group IV from Table 3; student t-test, P > 0.3), TNFa-induced stimulation of LTB4 release may be secondary to the release of PAF. The decrease in aqueous humor LTB4 levels that is induced by the cyclooxygenase inhibitors is not likely due to a direct effect on the lipoxygenase pathway, but may be a reflection of the reduction in ocular inflammation. Also, nonsteroidal anti-inflammatory drugs, of which indomethacin and naproxen are representatives, inhibited neutrophil functions through effects on membrane function that is independent of PG formation.47 Leukocyte number in aqueous humor was most effectively reduced by a combination of cyclooxygenase inhibition plus PAF receptor antagonism; cyclooxygenase inhibition was intermediately effective; PAF receptor antagonism was the least effective (see Table 3). We have shown that the leukocyte response 24 hr after TNFa injection was largely monocytic (67%); neutrophils represented approximately 24% of the leukocytes that were present at this time.14 Although LTB4 is chemotactic for neutrophils,4849 the chemotactic factors for monocytes during uveitis are poorly understood. Therefore, it was not surprising that the blockade of PAF receptors, which was accompanied by maximal inhibition of LTB4 production, did not reduce leukocyte infiltration more dramatically. Because cyclooxygenase metabolites are not believed to possess significant chemotactic properties,5051 the association between cyclooxygenase inhibition and reduced leukocyte number in aqueous humor is more difficult to explain. This could be an indirect effect that is secondary to the anti-inflammatory action of these agents. Further investigation is required to elucidate the mechanisms by which eicosanoids and PAF mediate TNFa-induced uveitis. Based on the results shown, it is clear that these potent autacoids are important mediators of the ocular inflammatory response to intravitreally injected TNFa. Key words: tumor necrosis factor, platelet-activating factor, eicosanoids, SRI 63-441, uveitis 2098 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1991 Acknowledgments The authors thank Dean A. Handley, PhD, Platelet Department, Sandoz Research Institute, East Hanover, New Jersey, for the SRI 63-441. References 1. Nedwin GE, Svedersky LP, Bringman TS, Palladino MA, Jr, and Goeddel DV: Effect of interleukin-2, interferon-gamma, and mitogens on the production of tumor necrosis factors-alpha and beta. J Immunol 135:2492, 1985. 2. Beutler BA and Cerami A: Cachectin: More than a tumor necrosis factor. N Engl J Med 316:379, 1987. 3. Ming WJ, Bersani L, and Mantovani A: Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes. J Immunol 138:1469, 1987. 4. Rampart M, de Smet W, Fiers W, and Herman AG: Inflammatory properties of recombinant tumor necrosis factor in rabbit skin in vivo. J Exp Med 169:2227, 1989. 5. Gamble JR, Harlan JM, Klebanoff SJ, and Vadas MA: Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Sci U S A 82:8667, 1985. 6. Pober JS, Gimbrone MA, Jr, Lapierre LA, Mendrick DL, Fiers W, Rothlein R, and Springer TA: Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J Immunol 137:1893, 1986. 7. Horvath CJ, Ferro TJ, Jesmok G, and Malik AB: Recombinant tumor necrosis factor increases pulmonary vascular permeability independent of neutrophils. Proc Natl Acad Sci U S A 85:9219, 1988. 8. Bevilacqua MP, Pober JS, Majeau GR, Fiers W, Cotran RS, and Gimbrone MA, Jr: Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: Characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci U S A 83:4533, 1986. 9. Philip R and Epstein LB: Tumour necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, g-interferon and interleukin-1. Nature 323:86, 1986. 10. Larrick JW, Graham D, Toy K, Lin LS, Senyk G, and Fendly BM: Recombinant tumor necrosis factor causes activation of human granulocytes. Blood 69:640, 1987. 11. Nathan CF: Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes. J Clin Invest 80:1550, 1987. 12. Warren JS, Kunkel SL, Cunningham TW, Johnson KJ, and Ward PA: Macrophage-derived cytokines amplify immune complex-triggered superoxide anion responses by rat alveolar macrophages. Am J Pathol 130:489, 1988. 13. Rosenbaum JT, Howes EL, Jr, Rubin RM, and Samples JR: Ocular inflammatory effect of intravitreally-injected tumor necrosis factor. Am J Pathol 133:47, 1988. 14. Fleisher LN, Ferrell JB, and McGahan MC: Ocular inflammatory effects of intravitreally injected tumor necrosis factor-alpha and endotoxin. Inflammation 14:325, 1990. 15. Camussi G, Bussolino F, Salvidio G, and Baglioni C: Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor. J Exp Med 166:1390, 1987. 16. Valone FH and Epstein LB: Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-l-b, tumor necrosis factor, or IFN-g1. J Immunol 141:3945, 1988. 17. Dayer JM, Beutler B, and Cerami A: Cachectin/tumor necrosis Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Vol. 32 factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts. J Exp Med 162:2163, 1985. Kawakami M, Ishibashi S, Ogawa H, Murase T, Takaku F, and Shibata S: Cachectin/TNF as well as interleukin-1 induces prostacyclin synthesis in cultured vascular endothelial cells. Biochem Biophys Res Commun 141:482, 1986. Huber M, Beutler B, and Keppler,D: Tumor necrosis factor a stimulates leukotriene production in vivo. Eur J Immunol 18:2085, 1988. Camussi G, Tetta C, Bussolino F, and Baglioni C: Tumor necrosis factor stimulates human neutrophils to release leukotriene B4 and platelet-activating factor. Induction of phospholipase A2 and acetyl-CoA:l-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase activity and inhibition by antiproteinase. Eur J Biochem 182:661, 1989. Chang S-W, Feddersen CO, Henson PM, and Voelkel NF: Platelet-activating factor mediates hemodynamic changes and lung injury in endotoxin-treated rats. J Clin Invest 79:1498, 1987. Handley DA, Tomesch JC, and Saunders RN: Inhibition of PAF-induced systemic responses in the rat, guinea pig, dog and primate by the receptor antagonist SRI 63-441. Thromb Haemost 56:40, 1986. Sessler CN, Glauser FL, Davis D, and Fowler AA, III: Effects of platelet-activating factor antagonist SRI 63-441 on endotoxemia in sheep. J Appl Physiol 65:2624, 1988. Morris DD and Moore JN: Equine peritoneal macrophage production of thromboxane and prostacyclin in response to platelet activating factor and its receptor antagonist SRI 63-441. Circ Shock 28:149, 1989. Carrick JB, Moore JN, and Morris DD: Inhibition of PAF-induced aggregation of equine platelets by a PAF-receptor antagonist (SDZ 63-441). In Platelet Activating Factor in Endotoxin and Immune Disorders, Handley DA, Saunders RN, Houlihan WJ, and Tomesch JC, editors. New York, Marcel Dekker, 1990, pp. 77-89. Fleisher LN: Effects of inhibitors of arachidonic acid metabolism on endotoxin-induced ocular inflammation. Curr Eye Res 7:321, 1988. Hogan MJ, Kimura SJ, and Thygeson P: Signs and symptoms of uveitis. I. Anterior uveitis. Am J Ophthalmol 47:155, 1959. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem, 193:265, 1951. Rubin MR and Rosenbaum JT: A platelet-activating factor antagonist inhibits interleukin-1-induced inflammation. Biochem Biophys Res Commun 154:429, 1988. Handley DA, Van Valen RG, Tomesch JC, Melden MK, Jaffey JM, Ballard FH, and Saunders RN: Biological properties of the antagonist SRI 63-441 in the PAF and endotoxin models of hypotension in the rat and dog. Immunopharmacol 13:125, 1987. Cole DF: The site of breakdown of the blood-aqueous barrier under the influence of vaso-dilator drugs. Exp Eye Res 19:591, 1974. Unger WG: Changes in the anterior uveal vessels and epithelium in the inflamed rabbit eye. Bibl Anat 18:278, 1978. Bussolino F, Gremo F, Tetta C, Pescarmona GP, and Camussi G: Production of platelet-activating factor by chick retina. J Biol Chem 261:16502, 1986. Bazan HEP, Reddy STK, Woodland JM, and Bazan NG: The accumulation of platelet activating factor in the injured cornea may be interrelated with the synthesis of lipoxygenase products. Biochem Biophys Res Commun 149:915, 1987. Gautheron PD, Coulbault L, and Sugrue MF: A study of PAF- •No. 8 36. 37. 38. 39. 40. 41. 42. 43. TUMOR NECROSIS FACTOR-a-INDUCED UVEITIS / Fleisher er ol induced ocular inflammation in the rat and inhibition by the PAF antagonist, L-652,731. J Pharm Pharmacol 39:857, 1987. Rubin RM, Samples JR, and Rosenbaum JT: Prostaglandinindependent inhibition of ocular vascular permeability by a platelet-activating factor antagonist. Arch Ophthalmol 106:1116, 1988. Verbey NL, Van Delft JL, Van Haeringen NJ, and Braquet P: Platelet-activating factor and laser trauma of the iris. Invest Ophthalmol Vis Sci 30:1101, 1989. Braquet P, Touqui L, Shen TY, and Vargaftig BB: Perspectives in platelet-activating factor research. Pharmacol Rev 39:97, 1987. Piper PP and Stewart AG: Antagonism of vasoconstriction induced by platelet-activating factor in guinea-pig perfused hearts by selective platelet-activating factor receptor antagonists. BrJ Pharmacol 90:771, 1987. Tanizawa H and Tai HH: Synergism between chemotactic peptide and platelet-activating factor in stimulating thromboxane B2 and leukotriene B4 biosynthesis in human neutrophils. Biochem Pharmacol 38:2559, 1989. Stewart AG, Dubbin PN, Harris T, and Dusting GJ: Plateletactivating factor may act as a second messenger in the release of icosanoids and superoxide anions from leukocytes and endothelial cells. Proc Natl Acad Sci U S A 87:3215, 1990. Yousufzai SYK and Abdel-Latif AA: Effects of platelet-activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle. Biochem J 228:697, 1985. Wedmore CV and Williams TJ: Platelet-activating factor (PAF), a secretory product of polymorphonuclear leucocytes, increases vascular permeability in rabbit skin. Br J Pharmacol 74:916P, 1981. Downloaded From: http://iovs.arvojournals.org/ on 06/12/2017 2399 44. Humphrey DM, McManus LM, Satouchi K, Hanahan DJ, and Pinckard RN: Vasoactive properties of acetyl glyceryl ether phosphorylcholine and analogues. Lab Invest 46:422, 1982. 45. Stock EL, Roth SI, Kim ED, Walsh MK, and Thamman R: The effect of platelet-activating factor (PAF), histamine, and ethanol on vascular permeability of the guinea pig conjunctiva. Invest Ophthalmol Vis Sci 31:987, 1990. 46. Lin AH, Morton DR, and Gorman RR: Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis in human polymorphonuclear leukocytes. J Clin Invest 70:1058, 1982. 47. Abramson SB, Cherksey B, Gude D, Leszczynska-Piziak J, Philips MR, Blau L, and Weissmann G: Nonsteroidal antiinflammatory drugs exert differential effects on neutrophil function and plasma membrane viscosity. Studies in human neutrophils and liposomes. Inflammation 14:11, 1990. 48. Ford-Hutchinson AW, Bray MA, Doig MV, Shipley ME, and Smith MJH: Leukotriene B: a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 286:264, 1980. 49. Bhattacherjee P, Hammond B, Salmon JA, Stepney R, and Eakins KE: Chemotactic response to some arachidonic acid lipoxygenase products in the rabbit eye. Eur J Pharmacol 73:21, 1981. 50. Bito LZ, Nichols RR, and Baroody RA: A comparison of the miotic and inflammatory effects of biologically active polypeptides and prostaglandin E2 on the rabbit eye. Exp Eye Res 34:325, 1982. 51. Kulkarni PS and Srinivasan BD: The effect of intravitreal and topical prostaglandins on intraocular inflammation. Invest Ophthalmol Vis Sci 23:383, 1982.