Oxidative Stress and Cataract

donors (reductants) such as hydrogen sulfide or hydroxylamine. The first ‘‘energy crisis’’ arose when these reduced compounds were exhausted (oxidized) in their aqueous environments. The solution of the problem for the ancestors of cyanobacteria was ‘‘inventing’’ photosystem II, i.e. a second photosystem containing a chlorophyll modification with an E0o as high as þ830 mV thus allowing them to utilize water as an inexhaustible electron source for free. This novel photosystem produced oxygen, protons and electrons in a light-dependent reaction involving manganese as catalytic redox converter as an electron-trap with water as electron donor. This strategy was so efficient that it allowed to assemble high densities of these organisms, accumulating as pure carbon, geologically designated as graphite (coal is approximately 2.5–3 billion years younger!).

All this actually happened in Bavaria: In the community of Hauzenberg, approximately 30 km northeast of the city of Passau, the only graphite mine in Middle Europe is still being exploited and worth wile visiting (‘‘graphite museum’’).

THE BENEFITS AND THE PROBLEM

From this time other unicellular organisms, devoid of chlorophyll (heterotrophes), took advantage of these novel ‘‘energy-unlimited’’ cells, using them as food source or even incorporating them as cellular organelles in a sense of photovoltaic elements: coevolution started and multicellular, higher organisms could develop.

The trade–in of water-splitting by photosystem II was ‘‘oxygen toxicity’’, however. The worst case is, when light and oxygen are operative at the same place. Thus, it is not astonishing, that photosystem II, where a light–dependent oxygen liberation from water is achieved, is perfectly protected against oxygen toxicity by a wealth of cooperative systems involving ‘‘electron idling’’ as well as antioxidants such as tocopherol, carotenoids as well as a set of enzyme systems.1 Actually our eyes have to envisage similar problems as the photosystems in plants: they only operate in the light exposed to high oxygen tensions; therefore it is also not astonishing, that the solutions to the problem of ROS toxicity might ask for similar solutions.

In the first couple of hundred million of years the problem for oxygen evolving cells was not that dramatic since most oxygen was bound and sedimented by the process of iron IIþ oxidation. An intermediate period might have allowed to re-reduce oxidized nitrogen (‘‘nitrate-respiration’’) thus supporting the original ‘‘one-photosystem’’ organisms as well as primitive heterotrophes living without oxygen. Finally, when land-plants developed and oxygen accumulated in the atmosphere, nitrate respiration was substituted by the more efficient oxygen respiration utilizing the ‘‘counterpart’’ of water-splitting, namely water formation via oxygen reduction by cytochrome a/a3. This system involved both iron and copper as redox converters in analogy to the manganeous system in photosystem I, now functioning as oxygen trap.

Water splitting and water formation, i.e. oxygen formation from water and oxygen reduction to water are four-electron steps. Thus, another problem

arose: four electrons could not be transferred simultaneously, but step by step thus involving superoxide, hydrogen peroxide and OH-radical as intermediates on the way of oxygen to water, according to:

O2 þ 4e þ 4Hþ ! 2H2O

Since these intermediates are of great chemical reactivity, the mechanism of their production had to be ‘‘cryptic’’, i.e. with an excellent isolation towards its surroundings, similar to an atomic power plant. Other redox systems in internal metabolism were also thermodynamically able to reduce oxygen potentially producing the above toxic species. All aerobic organisms therefore had to develop antioxidative strategies and synthesize antioxidants in order to survive. In the following, both phototrophic (algae, higher plants) and heterotrophic organism (bacteria, fungi, animals) developed cooperative and adaptive strategies for detoxification: Again, the heterotrophes took advantage of the much better synthesizing capacities of the plants: they just ‘‘forgot’’ to build bioenergetically ‘‘expensive’’ (ATP-consuming) molecules such as aromats— with some exceptions. The ROS–detoxifying systems were developed synergistically and allowed both plants and animals to utilize oxygen activation as defence systems (‘‘respiratory burst’’) exhibiting homologous external and internal battle fields such as the apoplasts of the plant and the phagosome of the animals. Traditional and modern medicine use microbial and higher plant’s products i.e. their antioxidants as drugs, preventive therapies and food additives.2–4

CATARACT

What Is Cataract Chemically?

Cataract, the turbidity of the eye lens, is due to protein cross linking via sulfhydryl oxidation and protein glycation, dependent on the individual patterns of pathometabolism.5 In most cataractogenic reactions oxygen seems to be involved. This seems to be clearly supported by the recent finding, that ‘‘vitrectomy surgery increases oxygen exposure to the lens’’ with the risk of nuclear cataract formation.6 Fundamentally, extremely different influences may govern cataractogenic processes, measurable at different sites in the lens as outlined in the following Table 1 and Figure 1.

Principally, there are initiating processes triggered by radiation of different qualities, and others initiated by certain metabolites operating also in the dark via reductive oxygen activation, as outlined in ref.5 and below.

Principle Effects of Light

As demonstrated below there are several compounds, such as riboflavine or tryptophan derivatives, which are absorbing light quanta, transiently forming an activated state (P*) which can transfer this activity onto molecular oxygen thus yielding highly reactive ‘‘Singlet oxygen’’, 1O2. This reaction is called ‘‘Type II’’

Table 3 Photodynamic Drugs

In cataract formation the ‘‘oxidation-sensitive’’ amino acid tryptophan has been shown to act as one predominant precursor for two compounds operating as photodynamic enhancers: 3-hydroxy kynurenine-glucoside and xanthurenic acid 8-0-b-D-glucoside (Figure 2). Both substances stem from oxidative splitting of tryptophan via N-formylkynurenine and subsequent deformylation

Figure 2 Formation of xanthurenic acid.

and glycosylation. Xanthurenic acid undergoes some more transformations, including N-heterocyclic ring formation and additional hydroxylation. Xanthurenic acid accumulates as non-diabetic brunescent colour in cataractic eyes and acts as endogenous chromophore/fluorophore and UV region sensitizer with an excitation at 338nm and an emission at 440nm, efficiently generating singlet oxygen.7 Singlet oxygen again produces long living peroxides thus promoting and extending the initial damage8

Reductive Events

Since the redox potential of the pair O2/O2 is 330mV, many electronegative compounds may represent potential candidates as initiators of monovalent oxygen reduction. Some of them were designed for this purpose, i.e. anti-cancer drugs such as adriamycin.

Generally, benzo-, naphthoand anthraquinones are well known as redox cyclers in biochemistry, as shown for a naphthoquinone in Figure 3.

Figure 4 represents compounds (besides the mentioned naphthoquinones like juglone, and the anthraquinone rein), pyrroloquinolin quinones (PQQ), quat salts such as paraquat and nitroaromats such as nitrofuran which may act as redox cyclers, being ‘‘unspecifically’’ reduced by flavoprotein (FP)-oxidoreductases (diaphorases) inducing ROS-production and thus oxidative destruction.

Figure 3 Redox cycle of 2-methyl naphthoquinone producing superoxide.

Figure 4 Redox substrates of NAD(P)H oxidoreductases (diaphorases).

Protein Glycosylation: Diabetic Events

Aldehyde groups of sugars can react with amino groups forming Schiff bases (aldimines). These Schiff bases undergo so-called Amadori rearrangements finally forming enediols. Enediols are compounds that may form complexes with transition metals such as copper or iron (mostly as chelates) which easily

Figure 5 Model reaction for protein glycation and transition metal catalyzed oxidations.

autooxidize forming superoxide and the well known follow-up ROS, hydrogen peroxide and OH-radical thus again starting the well known initiation of destructive events. This cascade is represented in Figure 5.

In the experiment in vitro, Amadori products i.e. the ‘‘pure’’ mechanism of ene-diol oxidation, can be substituted by dihydroxyfumaric acid, HOOC-C(OH) =C(OH)-COOH (DHF): in the presence of iron-ADP-complexes, DHF transfers ‘‘two times one’’ electron onto oxygen yielding diketosuccinate (DKS) and again superoxide, H2O2 and OH-radical (Table 4).

Protein glycosylation can also be simulated in vitro by incubation of lens proteins with ascorbic acid, which also contains such an enediol configuration acting as prooxidant in this situation. Thus situations may occur where ‘‘ascorbylations’’ represent glycation models producing superoxide and so on, as outlined

Table 4 Autoxidation of Dihydroxyfumaric Acid (DHF)

Table 5 Experimental Cataract Induction

ØNaphtalin, Selenite, UV, Smoking, Transition metals, Redox cyclers (PMS)

ØDiabetes: sugars via aldose reductase and/or Amadori reaction

ØVitrectomy: increase of oxygen tension in the eye

Øgenetic and/or ethnic

by Linetsky et al.9 These models are very important to learn about basic reaction mechanisms; whether these situations are of clinical significance, however, is a matter of ongoing debate around the problem: when are antioxidants prooxidative?

Altogether, there are certain possibilities to explore mechanisms of cataractogenesis by a wealth of models, mimicking physiological aspects thus allowing to test for possible amendments of procataractic events. Although we have little influence on the genetic basis yet, genetically favoured cataract formation (see below and),10 for example in the (Emory)-mouse model also greatly contributed to the field.

Experimentally by naphthalin induced cataract seems to be mediated via its hydroxylation to 1,2-hydroquinone and following superoxide formation, as shown by the protection by SOD.11 Other examples are selenite (via interaction with sulfhydryl groups) or the redox cycler phenazonium methosulfate (PMS, c.f. ref. 12) representing valuable tools in cataract research. Some well known conditions for cataract induction, including experimental models, are summarized in Table 5.

First Biochemical Signs of Cataract Formation

Before cataract becomes evident as measurable or visible lens opafication, certain biochemical processes can be measured in advance (Table 6), some of which were already mentioned above. Three points shell be especially addressed here:

ØFormation of protein-bound dihydroxyphenylalanin (DOPA) by high

energy radiation as one more ene-diol mediated, transition metal-catalysed ROS generator.13

Table 6 Early Events in Cataract Formation

ØIncrease of methionine sulfoxide and cystin (electron donors for riboflavin-type I photooxidation);

ØIncrease of oxidation products of tryptophan as singlet oxygen generators and thus amplificators (3-hydroxy-kynurenine and its cyclic derivates xanthurenic acid and xanthurenic-8-glycoside);

ØIncrease of peroxides (lipid-OOH, Tyr-OOH) and hydroxynonenal;

ØInduction of DT-Diaphorases;

ØDecrease of GSH and ATP-ases;

ØProtein binding of DOPA as amplificator

ØInduction of the formation of so-called DT-diaphorases which reduce p- quinones to the corresponding hydroquinones. By subsequent catalysis of SOD, semiquinones and superoxide, produced by the above mentioned autoxidation, are detoxified in a ‘‘hetero-dismutation’’.

ØPeroxides14 and aldehydes such as hydroxynonenal and other aldehydes15 have to be under strict metabolic (enzymatic) control.

Intrinsic Light Reactions

As demonstrated in Figure 6, lens homogenates from calf eyes drive timeand light-dependent ethene formation from a-keto-S-methyl-butyric acid (KMB), a sensitive indicator for ROS.16 Thus, an intrinsic photodynamic activity in these preparations is indicated.

Prevention of Cataract in Model Reactions In Vitro and Ex Vivo

One of the dominating late processes in cataract formation is protein agglomeration by S-S bridge formation and other condensations producing high molecular weight (HMW)-aggregates. This process can be followed by means of protein electrophoresis or FPLC chromatography.17 In the experiment, lens homogenates are illuminated in the presence of mM concentrations of riboflavin and FPLC chromatograms are developed after illumination in the presence or absence of iodide (KJ) as ‘‘quenching’’ electron donor (Figure 7).

Figure 6 Ethene formation from KMB by lens homogenates after illumination as indicator for intrinsic photodynamic activities and ROS formation.

Figure 7 FPLC chromatogramme of lens homogenate (LH) illuminated for 120 min with 50mM riboflavin (Rib) in the absence or presence of 10mM iodide (KJ).

As shown in Figure 7 high molecular weight (HMW)-protein with a retention time around 15 min increases after illumination in the presence of the photodynamic activator, riboflavin. This process is partially reversed in the presence of KJ as electron donor. It should be mentioned here that KJ was in use as topical anticataractic in the 1970s–1980s.

With the same method it could be shown that photodynamic formation of

several HMW-aggregates can be prevented by antioxidants such as (dihydro)- thioctic acid,6,18 which is also in use as drug for the prevention of certain

neurological disorders (Figure 8).

Models for Investigating Topical Penetration Rates

Enucleated rabbit eye bulbs were used in a ‘‘droplet apparatus’’ (Figure 9) to investigate on potential penetration rates of drugs in the interior of the eye, i.e., vitreous humor and lens tissue.19 In this apparatus, tear flow and eye lid movements are simulated by a paper strip on top of a copper net, thus connecting the bulb surface with an electrolyte reservoir. After the corresponding droplet applications, the increase of concentration of drug can be analyzed timedependently in the individual compartments of the eye (compared to the electrolyte reservoir), by means of inhibition of light-dependent, riboflavin-driven ethene formation from KMB, as demonstrated in Figure 6.

Figure 8 Prevention of photodynamic HWM-aggregate formation by reduced thioctic acid (Lip(SH)2) LH: lens homogenate, FPLC-separated crystalline proteins with a molecular weight (MG) of more than 300 kD and of ca. 45 kD respectively; riboflavin 2,5 mM; illumination: 15 min with 30 klux.

As shown in Figure 10, potassium iodide (KI) as potential anticataractic is present in all the compartments under investigation. It also became clear that in a realistic time of 2 min. no KI was found in the anterior lens cortex. Further incubation for 20 min. increased this rate considerably, especially in the aqueous humor.

Figure 11 Corneal penetration of KI as compared to lateral transport.

PROTECTION BY ANTIOXIDATIVE STRATEGIES

Protection from Oxygen Stress

Ocular diseases in respect to ROS have been addressed in recent reviews: ‘‘Oxygen free radicals in ocular diseases’’ have dealt with by Schempp and Elstner20 and by Varma et al.21

ROS have to be continuously under strict control of integral detoxification processes, detoxificating enzymes and organic antioxidants. One principle way to deal with oxygen toxicity is ‘‘avoidance’’, i.e. circumventing one or two electron donating processes towards oxygen. This can be achieved by ‘‘tight’’ coupling of electron transport chains operating at the electronegative region of oxygen activation or by stoichiometric coupling of oxygen activating processes with utilization of activated oxygen. Another possibility is the inhibition or inactivation of oxygen activating processes or enzymes. This has been shown for xanthine oxidase, lipoxygenases, prostaglandine cyclase, NAD(P)H oxidases and other enzymes by a wealth of compounds used in medicine. The so-called NSAIDs (non-steroidal antiinflammatory drugs) and several flavonoids are good examples for this principle.

Detoxifying Enzymes

Detoxification by enzymatic processes is only possible, if the reactivity of the respective oxygen species is reasonably low under physiological conditions so that the enzymatic reactions allow k-values of at least 2–3 orders of magnitude between the reaction under enzyme catalysis and the non-catalyzed, spontaneous reaction between the oxygen species and any reaction partner in its ‘‘molecular’’

neighbourhood. Therefore, the reactions of OH ,1O2, RO , ROO and HOO are not under enzymatic control; their reaction constants with potential reaction partners in their typical ‘‘environments’’ is too fast (generally k>>108) for enzyme catalysis. Thus, the reactions of biomolecules with these oxygen species have to be ‘‘amended’’ after damage. In order not to ‘‘flood’’ these repair processes the above mentioned antioxidative molecules serve as scavengers and quenchers of activated states.

Enzyme-catalyzed detoxifications thus mainly concern superoxide, peroxides, semiquinones and epoxides (produced by cytochrome-P450-activities) as more or less ‘‘stable’’ reduced oxygen species.

In most aerobic cells catalase (CAT), superoxide dismutases (SODs), monoor dehydro-ascorbate reductase, glutathione peroxidase (GSH-POD), glutathione reductases, DT-diaphorases and different peroxidases (PODs) either individually or cooperatively remove stable reactive oxygen species. Different individual physiological parameters or ‘‘stresses’’ may induce different enzymatic patterns.

Microperoxidase, a ferriheme undecapeptide, derived from cytochromes, has been shown to degrade peroxides (similar to a-keto-acids; see below) was suggested as protective against oxidative stress in the lens.22

One early event in cataract induction is the appearance of organic peroxides (c.f. Table 5) partially of lipophilic character, which experimentally is reflected by tert-butyl-hydroperoxide (TBOOH). Exposure towards TBOOH induces resistance towards hydrogen peroxide in immortal murine lens epithelial cells probably via a whole set of defence enzymes: out of more than 12.000 gene expressions tested, 16 genes were found to account for protection including glutathione-S-transferases, SOD, zeta-crystallin, NADPH-quinone reductase, toxic lipoprotein degradation, control of iron metabolism and aldehyde detoxification.10 Thus it seem evident that the fate of cataract development is clearly under genetic control. If this control fails, some other potentials seem to be available: Nutritional low molecular weight supplements promise to support possibly failing intrinsic defence lines, sometimes with extremely doubtful prerequisites, however.

External Helpers: Phenolic Derivatives Protect from Oxidative Stress

Phenolic compounds play an important role in this context acting as antioxidants, inducers of enzymes, transition metal chelators thus avoiding Haber-Weiss- (Fenton)-chemistry and cofactors of regulation of enzymatic activities.

Detoxification in a wider sense thus also concerns the replacement of damaged molecules such as DNA, proteins and membrane lipids by a complex ‘‘crew’’ of integrated repair enzymes and replacement processes. A continuous involvement of these repair processes, however, would render them inactive since they also continuously function as targets of these reactive oxidants. Therefore, another batch of first aid molecules such as phenolics is biologically more than logic.

The only ‘‘help’’ for the final repair teams are small molecules with ‘‘Kamikazee-type’’ properties, representing antioxidants with or without chance to be metabolically repaired themselves.

Phenolic redox reactions are fundamentally involved in stress metabolism both in plants and in animals comprising redox processes and antioxidative functions including the formation of phenoxyl radicals, semiquinone radicals and o- or p-quinones undergoing electron donating reactions towards reactive radicals. Dependent on the neighbourhood the formed phenolic radical may be rather stable awaiting reduction by available electron donors such as ascorbate and a-tocopherol. In a ‘‘pecking order’’23 of these two important antioxidants, radical states in biomembranes are quenched where ascorbate or thiols such as reduced glutathione or lipoic acid (thioctic acid) regenerate the reduced state of phenolics such as tocopherol or ubiquinol in the interphase between lipophilic and hydrophilic plasmatic phases.24,25

With certain initiator radicals phenolics may be converted into alkoxyl radicals (RO ) or semiquinones thus acting as prooxidants depending on the substituents in the neighbourhood of the phenoxyl radical group; tocopherols acting as prooxidants are good examples for this process. In the presence of ubiquinol, however, the prooxidative activity of vitamin E is converted into an antioxidative function as shown for LDL-oxidation.26 Thus cooperative effects of diverse phenolics are indicated where the over-all antioxidative effect is due to ‘‘total phenolics’’ and not a single substance where additive, synergistic and supplementory effects are observed. In the case of transition metal catalysis (Fentonor Haber-Weiss-chemistry), phenolics may act as chelators for ironor copper-ions. In this respect they both may stimulate or inhibit oxidative reactions, strongly dependent on the model reaction or the type of damage looked at. Phenolics may simply act as radical scavengers or radical-chain breakers thus extinguishing strongly oxidative free radicals such as OH.; they also may react with non-radical species such as hypochlorous acid or peroxinitrite yielding products with much lower oxidative capacities as compared to the parent compounds.27–29

Some molecules such as quercetin seem to have more than just one function: a strong antioxidative (scavenger) function as well as iron chelating and enzyme-inhibitor properties. Very recently, Fiorani et al30 reported on the prevention of dehydroascorbate (DHA)-dependent GSH depletion in red blood cells due to the presence of quercetin. The mechanism was not simply a chemical interaction of quercetin with DHA or GSSG, but an activation of enzymatic GSSG-reduction downstream to this primary redox events.

Cooperative Effects of Antioxidants

LDL Oxidation

LDL oxidation is supposed to be one initiating factor in atherogenesis and seem to be a good model reaction to study lipid peroxidation in vitro. There have been

Figure 12 Cooperative detoxification of alkoxyl (LO ) and peroxyl (LOO ) radicals. DHLA: dihydrolipoic acid; LA: lipoic acid; Qred: reduced quinone; Qox: oxidized quinone

numerous publications in the past 10–15 years reporting on prevention of LDL oxidation by a ‘‘pecking order’’-principle23,26 by food supplements such as genistein,31 cocoa,32 grape seed powder33 and many others. Besides the well

known cooperative ‘‘repair teams’’, tocopherol-ascorbate and tocopherol- biquinole-ihydrolipoic acid.26 and refs therein In this case, the oxidized quinones are

re-reduced by a-keto acids via the diaphorase-thiocitic acid (dihydrolipoic (DHLA) and lipoic acid (LA)) pathway as shown schematically in Figure 12.

a-keto acids such as ketoglutarate, KMB (see also above) or pyruvate34 react chemically with peroxides in an ionic process thus acting antioxidatively per se, in addition to act as potential electron donors in the above electron transport system.

It has been mentioned that antioxidants such as vitamin C may act prooxidatively. This can be shown in the case of LDL oxidation using the copper model where 1mM ascorbate accelerates the lag phase of dienconjugation of intrinsic linolenic acid. In the presence of the flavonoid rutin, however, this prooxidative effect is reversed and ascorbate and rutin work cooperatively in protection35 (Figure 13).

Jet other ‘‘lipid protecting teams’’ in LDL may be operating, involving intrinsic carotenoids (b-carotene, lycopene, lutein): carotenoid oxidation was strongly delayed by the lemon oil terpene, g-terpinene,36,37 in a similar manner as tocopherol by ubiquinole as shown above.

Herbal Extracts

Neuronal disorders such as Parkinson’s disease or Alzheimer’s disease38 and eye diseases such as AMD (age related macular degeneration) and cataract gain increasing importance due to increasing age of our population.

Herbal extracts are in use against mental and generally neuronal disorders since the old times and envisage dramatic revitalization in our days. Prominent examples are Ginkgo biloba extracts,39–41 Ayurvedic medications42 and extracts from St. John’s wort, Hypericum perforatum.43,44 Since the onset of atherosclerosis

Figure 13 Cooperative effects of rutin and ascorbate in LDL protection.

has some basic similarities to cataract induction, i.e. glycations and formation of organic peroxides, medication of both may be indicated. Formation of glycation end products may be prevented or ameliorated by drugs called ‘‘amadorins’’ such as aminoguanidine and pyridoxamine (‘‘Pyridorin’’).45,46

Furthermore, additional uptake of both vitamins C and E together with moderate physical exercise have been shown to strengthen the antioxidative defense system in an animal model.47

Neuronal hypoxia as quite ‘‘normal’’ age-related anatomic change, varying from mild deficits to massive neuropathological events, implies pharmacological benefits for GBE by means of its antioxidant flavonoids. GBE have been proven in many studies to be advantageous for the amelioration of the blood vessel system thus protecting cells from oxidative damage in connection with inflammatory processes. Since neurological (cerebral) disorders are based on inflammatory processes and limitations in blood circulation (ischemic situations), an attenuation by antioxidants is indicated especially if accompanied by other activities.

Animal experiments with rats after occlusion of the carotid arteries showed that pre-ischemic administration of GBE (150 mg/kg p.o.) protected against postischemic injury measured as malondialdehyde (MDA), glutathion (GSH)-status,

phospholipid levels as well as superoxide dismutase (SOD) and lactate dehydrogenase (LDH) activities48 and other metabolic activities, as recently outlined.49 Oyama et al50 studied the metabolism of brain neurons in resting and calcium-loaded cells and the effects of myricetin and quercetin as GBEconstituents on oxidative events by means of increased fluorescence after 20,70- dichlorofluorescein oxidation: 3 nM myricetin and 10 nM quercetin reduced oxidation significantly indicating that these ingredients of GBE may be partly responsible for the observed beneficial effects in the cells after ischemic events. Lipid peroxidation during experimental spinal cord injury (‘‘paraplegic animals’’) was measured (MDA-test) by Koc et al51 either in the absence or presence of GBE, methylprednisolon (MP) or thyrotropin-releasing hormone (TRH). Both MP and GBE exhibit protective effects due to their antioxidative properties.

Subarachnoid hemorrhage, where NO-levels in serum are decreased but increased in the brain, are followed by cerebral vasospasms and neuronal damage. GBE antagonizes these effects thus reversing pathological NO-alteration and relieving cerebral vasospasms.52

Glutamate-induced cytotoxicity in neuronal (HAT-4) cells is associated with glutathion depletion and thus oxidative stress. GBE and also maritime pine bark extracts (‘‘Pycnogenol’’) were able to protect against glutamate-induced damage.39

On the other hand AMPAand NMDA-receptors are antagonized by 6-hydroxykynurenic acid (6-HKA) and kynurenic acid, which can be extracted from Ginkgo leaves. Therefore 6-HKA is suggested as a useful tool for the analysis of glutamate-mediated synaptic responses.53

Staurosporine (ST)-induced neuronal apoptosis was inhibited by GBE and some of its components: After treatment with ST (200nM) for 24 h, 74% apoptosis in chick neurons was observed. This was reduced to 24%, 62% and 31% by GBE (100mg/l), ginkgolide J (100mM) and ginkgolide B (10mM), respectively.54

Age-related problems in terms of nutritive aspects are addressed by Riedel et al.55 They recommend that supplementations with antioxidants and cofactors like folate, b-carotene and tocopherole, caffeine (in low doses) and GBE are beneficial for enhancing cognitive functions in elderly people.

GBE, due to its ability to improve peripheral blood flow to the eye and general neuroprotection, may thus be also advantageous for the treatment of glaucoma.56 Glaucoma, an eye disease with increased intraocular pressure, is normally treated with b-blockers and calcium channel inhibitors.

Selenite-induced cataract (see above) is prevented by propolis, diclofenac, vitamin C and quercetin by 70, 60, 58, and 40%, respectively, whereas GBE has surprisingly no effect in this study.57 Likewise, death of glioma cells was prevented if apoptosis was induced by hydrogen peroxide but not if it was induced by the lipid-lowering drug, simvastatin, indicating different signaling pathways of these different apoptosis inducers.58

Age-related shortcomings such as ultrastructural changes of mitochondria in Muller (retinal) glial cells, accompanied with an increase of intrinsic glutathione, can be attenuated by GBE-feeding of the experimental animals.59

Other beneficial effects of GBE reported very recently are the protection from radiation-induced cataract60 or combined carrageenan-gamma radiation and acute inflammation61 or LPS-induced inflammation both in vitro and in vivo.62 As already mentioned above, amelioration of glutamate-induced neurotoxicity by GBE can also be shown in cultured retinal neurons thus definitely extending its importance63 for ‘‘extensions of the brain.’’

An overview on natural therapies on ocular disorders was presented by Head64 where especially regulatory functions of GBE were emphasized and by Christen and Maixent.65 The conclusion was that increased circulation to the optic nerve and antioxidative functions help to prevent, and potentially also to cure, cataracts and glaucoma.

Ischemic organs such as hearts after reperfusion showed much better performance if the animals were fed with 50 or 100 mg GBE before the experiment: especially the contractile function after global ischemia was strongly improved.66,67 Another study on cardioprotective effects, where GBE was compared with ginkgolides A and B and also bilobalide used hemodynamic properties and EPR spectroscopy as analytical tools. Anti-ischemic effects were observed after repeated feeding of either GBE (15 d 60mg/kg orally) or ginkgolide A (15 d 4mg/kg orally) as compared to placebo.

CONCLUSION

The goal, as in atherosclerosis and heart diseases, is to combine ‘‘safe’’ drugs (herbal extracts) with supplemented nutrition (‘‘novel food’’, ‘‘nutriceuticals’’, ‘‘functional food’’) in order to yield preventive protection. Two books addressing and perfectly summarizing these subjects should be mentioned in this context: the book comprising aspects of parmacognosy by Bruneton68 and that on functional food by Wildman.69 Both treatises discuss their respective fields exhaustively, not avoiding critical aspects.

In a recent review70 another very important new field is addressed: ‘‘Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants’’ open new visions and promise interesting future research areas. Recent developments of this rapidly growing research area of utmost medical and commercial importance are critically discussed, with respect to environmental concerns. One of the authors principle issues is that ‘‘plant derived biopharmaceuticals are cheap to produce and store, easy to scale up for mass production, and safer than those derived from animals’’. May be in many, or most cases! There is almost nothing to add: this issue is close to our own concern and research field if it is carefully integrated into classical procedures and treatments.

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and nitric oxide driven tissue damage whilst alternatively activated macrophages mediate Th2 cell differentiation, tolerance induction, down regulation of inflammation and healing. These opposing functional effects are controlled by cytokine or other polarising factors. Driving the balance towards an alternatively activated, healing phenotype is crucial to re-establish tissue homeostasis and disease remission. We have used rodent models to explore the role of macrophages in experimental autoimmune uveoretinitis (EAU), and how their function might be manipulated to limit retinal damage. In the normal CNS and retina, tissue resident macrophages and myeloid cells appear to be polarised towards alternatively activated phenotype, and this polarisation appears irreversible, providing a regulatory mechanism within the tissue that is over-ridden during autoimmune inflammation. Infiltrating classically activated monocyte-macrophages are essential for full expression of disease and our histological and trafficking experiments indicate that they are amongst the first cells to infiltrate the retina and may be the key cells initiating blood retina barrier breakdown. Infiltrating macrophages that are reactivated locally by T cell derived cytokines are also primary effectors of photoreceptor damage through nitric oxide and super-oxide generation but show greater resistance to apoptosis during EAU than would otherwise expected under normal inflammatory conditions, due to expression of a caspase 8 inhibitory molecule, FLIP. Down regulation of these classically activated macrophages through altering the cytokine microenvironment is key to controlling inflammation.

PATHOLOGY OF NITRIC OXIDE IN EAU

Effects of Nitric Oxide on Immune Function

It is now understood that all known isoforms of NO synthase catalyse the same reaction and all operate within the immune system. Neuronal NO synthase (nNOS or NOS1) and endothelial NO synthase (eNOS or NOS3) are constitutively expressed and regulated by Ca2+ flux and need not necessarily be expressed only by neurons and endothelium within the retina, but it is inducible NO synthase (iNOS or NOS2) that is frequently implicated in the inflammatory immune response.1,2 A hypothesis emerged that the constitutive forms of NO synthase were critical to normal physiology and their inhibition caused damage whist, induction of inducible NO synthase could be harmful (Figure 1). The generation of NOS2 deficient mice was supposed to provide an insight into the role of NOS2 in normal physiology and inflammation, but conflicting or contradictory results in various models raised even more questions than answers.3–5 In addition to well-described toxic effects, NOS2 has subsequently been shown to have multiple biological effects, including normal healing, regulation of T cell proliferation and differentiation.6 Considering that many of the targets of NO are themselves regulatory molecules (for example, transcription factors and components of various signalling cascades) it is evident that NO frequently exerts diverse phenotypic effects.7 NO mediatedstress will alter gene expression patterns, and the number of genes known to be involved is increasing. In addition, NO can act as powerful inducer of apoptosis or necrosis in some cells, it may also provide equally powerful protection from cell

Figure 1 Biological Effects of NO molecules. (i) NO may bind directly to the iron of a protein heme group in a reversible manner and exert signalling function. (ii) At higher concentrations, NO will react with O2 forming reactive nitrogen oxide intermediates that may S-nitrosate thiols. Under conditions of simultaneous oxidative and NO-mediated stress, NO may react with O2 to yield the unstable strong oxidant peroxynitrite anion, which can nitrate tyrosine residues. The stable NO oxidation product nitrite in the presence of peroxidases and H2O2 also leads to tyrosine nitration (adapted from8).

death in other situations. These effects may be in part due to differences in a cells capacity to cope with the stress of NO exposure.8

Within the immune system, many cells are capable of generating NO. Relevant to the eye these include microglia, dendritic cells, monocyte macrophages, granulocytes including mast cells, neutrophils and eosinophils.9 The expression of NOS2 is also regulated by cytokines often immune system derived.

Cytokines such as IL-1, IFN-g and TNF-a activate the NOS2 gene promoter via transcription factors such as NF-kB and AP-1,10,11 but equally, type 1 interferons

can inhibit NOS2 transcription.12 TGF-b post-transcriptionally regulates the production of NOS2 through enhanced degradation13 and IL-4 inhibits gene expression and NO production via a different pathway.14 Another factor that determines NOS activity is the availability of its substrate arginine, and that is regulated enzymatically by production of arginase. In macrophages and dendritic cells, Th2 cytokines and TGF–b strongly increase arginase activity thus limiting availability of arginine,14,15 and preventing the induction of NOS2 by subsequent exposure to IFN-g plus TNF-a. Regulation of NOS2 can also be mediated by cell-cell contact, and uptake of apoptotic (but not necrotic) lymphocytes by macrophages down regulates expression and at the same time shifts arginine metabolism towards the arginase pathway.16

Approximately 200 genes, including genes related to inflammation, infection and apoptosis are subject to regulation by NO,17 illustrating the complexity of NO induction and regulation. Protective and toxic effects frequently seen in parallel are reviewed by Bogdan,9 and are summarized in Table 1.

POTENTIAL CELL SOURCES OF NITRIC OXIDE IN EAU

Together with other inflammatory mediators, NO is known to be involved in the induction of ocular inflammation.18–20 In experimental autoimmune uveoretinitis (EAU), the inflammation is characterised by a breakdown of the blood retina

110 Liversidge et al.

Table 1 No Effects on Immune Function

lImmunopathology*apoptosis, extracellular matrix effects

~Inflammation

Necrosis or fibrosis of parenchyma l Anti-inflammatory/immunosuppressive

T and B lymphocyte proliferation *apoptosis Antibodies*disruption of signalling

!Leukocyte recruitment *adhesion, chemokines

lImmune regulation

~Cytokine, chemokine and growth factor modulation.

! (signalling cascades, transcription factors, mRNA stability)

lT helper cell deviation

~Regulation of Th1/Th2 immune responses.

!IL-12 regulation?

barrier, primarily at the post-capillary venules21 and at the retinal pigment epithelium (RPE).22,23 The disease is induced in animal models by immunisation with various retinal antigens.24,25 In acute disease an increase in vascular permeability together with fibrin exudation is associated with polymorphonuclear neutrophils and affects the anterior as well as the posterior chamber of the eye, in addition to

the monocyte macrophages and T lymphocytes that characterise the delayed type hypersensitivity response in the retina in more moderate disease.22,25,26 The

cytokine response associated with the inflammation represents mainly an elevation

of Th1 type cytokines, such as IFN-g, TNF-a and IL-2 and other generally proinflammatory cytokines such as IL-1b and IL-6.27,28 Although NOS2 is not

expressed within the normal retina and choroid, it is not surprising therefore that NOS2 is induced within the eye during inflammation.29–31

Potential cell sources of NO in EAU are tissue resident cells and inflammatory cells. Tissue resident cells include the photoreceptors and these have recently been reported to express NOS2 very early in disease and before inflammatory infiltrates are evident.32 The possible implications of this are discussed more fully in another Chapter of this volume. Expression of NOS2 in vivo by microglia and Mu¨ller glia (astroglia) is well described.33 In Mu¨ller cells

NOS2 is associated with neurotoxicity,34 whilst microglia expressed NOS2 may be regulatory.20,35,36 Vascular endothelium forms the inner blood retina barrier

and together with perivascular pericytes also expresses NOS early in disease37

but whether this plays any role in leukocyte recruitment to the CNS is less clear.38,39

In contrast, the RPE that forms the outer blood retina barrier does not appear to express NOS2 in vivo.30,31 In common with the cell types mentioned

above, cultured RPE cells do express NOS2 and produce high levels of nitrite when stimulated with cytokines,40–44 but there is no clear evidence that they can

Figure 2 A. The RPE layer in vivo (arrow head) is NOS2 negative, whilst infiltrate (arrowed) is NOS2 positive. B. RT-PCR for cytokine expression by RPE cultured in vitro with activated T lymphocytes.

produce NO in vivo. Indeed the evidence is that RPE may, in fact be protective as

co-culture with T cells or cross-linking of CD2 ligands on RPE cells induces PGE2 secretion and TGF-b expression (Figure 2).40,45,46

Inflammatory cells appear to be the major sources of NOS2 within the eye

in EAU. In hyperacute uveitis models such as endotoxin induced uveitis (EIU), neutrophils as well as monocytes express high levels of NOS2,47–49 but in the

posterior chamber in EAU, infiltrating monocytes are the principal inflammatory cell expressing NOS2 and the major cause of tissue damage.30,31,36,50 The role of

NOS2 expression by leukocytes in EAU is discussed more fully in the following section.

PATHOGENESIS OF NITRIC OXIDE INDUCTION IN EAU

Nitric oxide production is an important aspect of the innate response to microbial or parasitic infection,51,52 and deleterious effects of NOS2 expression in inflammatory settings involving endotoxin induced shock or haemorrhage and resuscitation are well recognised.4 However in macrophage driven inflammation, including autoimmunity, there is evidence for both beneficial, and deleterious effects. This is highlighted by the contradictory evidence from EAU models using

NOS2 KO mice or inhibitors of nitric oxide synthase. Protective effects could be demonstrated using NOS2 deficient mice53,54 or with a NOS2 inhibitor.55 On the

other hand, also using a NOS2 KO model, NO donors or inhibitors of NOS, a pathogenic role for NO could also be demonstrated by others.29,30,56–58

The molecular basis for these contradictory results has been extensively reviewed.1,8,9 The most relevant mechanisms for autoimmune inflammation such as EAU would appear to involve cytotoxic effects leading to apoptosis or necrosis of local tissue cells as NO is a key stimulus for DNA damage and p53 activation. This occurs particularly in the presence of superoxide that drives formation of the strong oxidant peroxynitrite anion that nitrates tyrosine residues. In addition, although stable, nitrite can also lead to tyrosine nitration in the presence of peroxidases and H2O2 (Figure 1). On the other hand, in smaller quantities, NO appears to be regulatory, particularly with respect to T cell growth and differentiation. The effect of NO can be profound, suppressing T cell

proliferation in response to mitogens as well as inhibiting antigen specific T cell expansion.40,59

Nitric oxide production is regulated by two enzymes. Nitric oxide synthase generates NO, and arginase, an enzyme that limits the availability of arginine, a substrate for NO production as well as T cell growth. NOS2 can also interfere with T cell growth through blocking the phosphorylation of signalling molecules required for IL-2 receptor signalling.60 Cytokines that induce NOS2 are produced predominantly by Th1 cells, therefore NO can also control Th1 cell responses by providing a negative feedback regulator of autoimmune Th1 driven autoimmune responses.5 Equally, Th2 cytokines up-regulate arginase, limiting the availability of arginine, the substrate for NO production, thus reducing NO production. Where NO levels are damaging, as in early EAU, then this will clearly be protective. When both enzymes are produced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T cell apoptosis providing an additional regulatory mechanism during inflammation.61–64 Further regulation of immune responses driven by NO release is through the immunosuppressive cytokine TGF-b. This is affected via three routes, decreased stability and translation of NOS2 mRNA, and increased degradation of NOS2 protein.65 Macrophage cytotoxic activity is also reduced by T helper 2 cytokines IL-10 and IL-4 that can synergise with TGF-b to limit tissue damage.66 Thus in Th1 driven organ specific autoimmune diseases such uveitis, NO production is part of a natural, negative feedback mechanism designed to limit inflammatory damage and promote healing. Such a complex role for this molecule explains much of the conflicting data found in models eliminating NOS activity, either by gene manipulation of specific inhibitors, or models providing NO donors.

In the retina, nitric oxide clearly has physiological functions.67 Neuronal NOS1 may be responsible for producing NO in photoreceptors and bipolar cells and may be required for stimulus of guanylate cyclase in photoreceptor rod cells increasing calcium channel currents as inhibition of NOS is known to impair phototransduction. Endothelial NOS3 is required to maintain vascular tone and inducible NOS2 in RPE and Mu¨ller cells may be required for phagocytosis of ROS. In EAU, the NOS inhibitors aminoguanidine and L-NAME cause enhanced rolling of leukocytes on vascular endothelium, but decreases firm adhesion and inhibits overall leukocyte infiltration, indicating that during

inflammation, NOS may contribute to the pro-inflammatory response.39 We have also found that L-NAME is protective in EAU.30,68 L-arginine was found to

enhance IFN-g and exacerbate retinal inflammation, whereas L-NAME significantly reduced NOS2 expression and severity of tissue damage via an IFN-g dependent mechanism.30 The principle source of NOS2 expression was found to be infiltrating monocytes in the target organ, whilst tissue resident macrophages in the choroid, and RPE cells did not express the enzyme. It was also evident that monocyte NOS2 expression peaked early in the inflammatory process, subsiding after peak disease despite increasing infiltrations of monocytes in later stages of the inflammation (Figure 3).

Figure 3 Infiltration of NOS2+ monocytes coincides with peak disease, but NOS2 monocytes continue to accumulate within tissue in later stages. Serial sections of rat retina were immuno-stained and then percentage of retina positive for NOS2 or ED1 monocyte marker were determined using computer assisted densitometric scanning, n–6.

Further analysis of the mechanisms of NO tissue damage revealed that tissue damage correlated with peroxynitrite formation within monocytes in the outer retina, together with extensive photoreceptor apoptosis and apoptosis of Fas+ T cells within the retina. However the monocytes, despite showing evidence of lipid peroxydation remained resistant to apoptosis. The protective effect of L-NAME could be attributed to dramatically reduced photoreceptor damage, absence of nitrotyrosine formation and overall reduced NOS2 protein expression. However, as T cell apoptosis was also reduced, accumulations of these cells was increased despite continued expression of FAS and Fas ligand indicating that normal regulation of T cells within the inflammatory lesion via activation induced cell death was compromised.57

THERAPEUTIC STRATEGIES TO REDUCE NITRIC OXIDE INDUCED TISSUE DAMAGE

From the preceding sections it is clear that inhibiting NO production during retinal inflammation may not have purely beneficial effects. Our work, and that of others show that inhibition of NOS can inhibit or exacerbate inflammation depending upon the model. Even specific inhibitors of NOS2 were not protective.55 Other approaches to target monocyte cytotoxicity may perhaps be more effective. Nitric oxide is a product of classically activated monocytes. The cytokines IFN-g and TNF-a induce NOS2 activity in monocytes, but targeting

IFN-g, a stimulator of classical activation, has not proved effective in controlling EAU.54,69 It is now known that IFN-g (and IL-4) inhibit IL-23 dependent IL-17

production,70 and as IL-23 is the major cytokine driving neural inflammation in EAE71 this result is perhaps less surprising. Targeting TNF-a has been more

successful,20,50 and is now used therapeutically to control intractable uveitis in the clinic with some success.72,73

We have also looked at the mechanisms controlling monocyte/macrophage resistance to apoptosis within the inflamed retina. Monocyte/macrophages within the retina appear to exert a double role in inflammation, producing tissue damaging NO during early stages, but also having a role in resolution of inflammation and tissue healing.50,78 If monocyte apoptosis could be induced early in disease it may be possible to prevent early photoreceptor damage. Using flow cytometry we isolated monocytes from uveitic retina and found that up to 30% of monocytes expressed the FLICE-inhibitory protein (FLIP). This protein inhibits caspase 8 activation and prevents caspase 3 cleavage that leads to apoptosis.74 To test whether FLIP expression in monocytes could be targeted therapeutically, we challenged cultured monocytes with IFN-g and TNF-a and treated them with either L-NAME, PKC inhibitors or an anti-TNF receptor fusion protein.50 Whilst L-NAME had no effect on FLIP expression, both the PKC inhibitor and the TNF-fusion protein reduced FLIP expression by up to 50%. This approach has yet to be tested in vivo, but may provide a clue to the efficacy of anti-TNF therapies.

CONCLUSION

In EAU we may hypothesise that early infiltrates of monocytes are classically

activated by IFN-g and TNF-a produced by T cells being reactivated by retinal antigen by local or infiltrating APC.25,75 This induces NOS2 in infiltrating

monocytes as well as susceptible tissue resident cells and the production of reactive oxygen species leads to activation induced cell death of the T cells. Uptake of apoptotic T cells by APC is known to induce IL-10 production that will in turn have an immuno-regulatory effect on the immune response reducing inflammation and driving alternative activation of monocytes towards a healing phenotype.76–78 As IL-10 and IL-4 produced by alternatively activated macrophages synergise with TGF-b, known to be present within ocular tissues, NOS2 expression is rapidly down regulated as the inflammation progresses. Thus down regulation of these classically activated monocyte/macrophages through altering the cytokine balance, or through manipulation of specific receptor agonists or antagonists will be the key to controlling ocular inflammation.

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disease, producing pro-inflammatory cytokines. Following T cell infiltration into the eye, antigen specific recognition leads to a cytokine cascade including IFNg and IL-2, which activates resident microglia as well as infiltrating macrophages. Tissue damage is predominantly mediated via reactive oxygen species (ROS) and lipid peroxidation of cell membranes secondary to nitrotyrosine formation.3 More specifically, during autoimmune inflammation of the retina, the driving systemic CD4þ T cell response activates the production of TNFa from macrophages4 which in turn is required for complete classical macrophage activation and subsequent nitric oxide (NO) production.5 To elucidate mechanisms we will describe a series of experiments utilising the EAU model and describing pathways of macrophage activation and NO production.

Materials and Methods

Animals and Induction of EAU

EAU was induced in C57Bl/6 and TNFRp55 / mice or Lewis rats as previously described5,6 (respectively). Briefly, mice were immunised with Interphotoreceptor retinoid-binding protein (IRBP) peptide 1–20 ((GPTHLFQPSLVLDMAKVLLD) (500mg/mouse)) in CFA (v/v) with additional intraperitoneal injection of 1.5mg of Bordetella pertussis toxin (PTX).7 Rats were immunised with retinal extract (RE; 5mg/ml) in CFA (v/v) with additional i.p. injection of 1mg of PTX. Animals were maintained in accordance with Home Office Regulations for Animal Experimentation, UK.

Immunohistochemistry

Eyes were enucleated for histological grading8 at time points indicated. Tissues were snap frozen, and fixed in acetone for 5 to 10 minutes and air-dried. Mouse sections were single stained for F4/80 and CD45 and visualised using VectorTM DAB. Stained slides were counterstained with haematoxylin and mounted in Histomount. Rat sections were dual fluorescent stained for ED1-FITC and NOS2 visualised with Texas Red.

Generation of Bone Marrow-Derived Macrophages

(BM-MF) and Retinal Myeloid Cell Isolation

Bone marrow cells were cultured as previously described9 in hydrophobic TeflonTM bags in M-CSF supplemented media. Retinal myeloid cells were isolated as previously described5,6 using a graduated density gradient (PercollTM).

Cytokine Stimulation of Macrophage Cultures

Macrophages were seeded at 5 105/ml/well in 24 well plates and stimulated with cytokines, IFNg (20U/ml), TNF-a (20U/ml; Peprotech EC, UK), and TGF-b (10ng/ml; R&D systems, UK), alone or administered sequentially in combination,

with the administration of each cytokine separated by a 4-hour period. Macrophage function was assessed 24 hours after addition of the first cytokine. IFN-g, followed 4 hours later by TNF-a (IFNg/TNFa), was used as a positive control for NO production, confirming previous in vitro studies.10

Quantification of NO Synthesis and Cytokine Analysis

NO generation was measured after 24 hours by assaying culture supernatants for the stable reaction product of nitric oxide (NO2; nitrite) using the Greiss reagent (0.5% sulphanilamide, 0.05% N-(1-napthyl) ethylenediamine dihydrochloride in 2.5% phosphoric acid), the optical densities were measured at 540nm, with a reference filter of 630nm.

Cytokines, IL-2, IFNg, IL-10, IL-12p40 and TNFa production were assayed by capture ELISA.

Statistical Analysis

Statistical analysis was performed by two-tailed unpaired t tests (GraphPad Instant software) amongst the groups and p values equal or less than 0.05 were considered significant, unless otherwise stated. Results are expressed as mean SEM. Disease incidence was compared using Fisher’s exact test (StatsDirect).

RESULTS

Retinal Microenvironment Controls Resident and Infiltrating

Macrophage Function During EAU

During EAU, macrophages show behavioural characteristics of cytokine conditioning at various phases of EAU.6 In summary, macrophages isolated from normal rat retina (consisting of perivascular ED2þ macrophages and microglia) generated little NO spontaneously, and furthermore, they remained unresponsive to further cytokine stimulation as there was no increase in NO production following stimulation with IFNg (Figure 1). The apparently stable state of the microglia, which has been termed a tonically deactivated state, may be secondary to either TGFb, found abundantly in the eye11 or via the negative signal received by the macrophage from CD200 receptor upon ligation with neuronal CD200;12 both mechanisms would render the cells unresponsive to IFNg-induced classical activation. Macrophages isolated from inflamed uveitic eyes at peak disease (corresponding to maximal macrophage infiltration in the retina) spontaneously produced significant amounts of NO (Figure 1). At this stage the population consists predominantly of infiltrating monocytes and not resident microglia. Evidence of nitrite production was supported by the increase in NOS2 expression just prior to maximal disease and nitrotyrosine expression on immunohistochemistry

Figure 1 Quantification of NO production by infiltrating macrophages during EAU.

Figure 2 (See color insert.) Immunohistochemical analysis of macrophage NOS2 expression during EAU. Two-colour immunofluorescence, with ED1 (FITC; green; arrow) and NOS2 (Texas Red; arrow head). A: An increased number of ED1þ NOS2þ macrophages were found during prepeak phase EAU only. B: NOS2 expression was absent in ED1þ macrophages during peak phase EAU.

(Figure 2). Furthermore, during the post-peak (days 13–15) and resolution (days 15–17) phases, when infiltrating monocyte/macrophage numbers are comparable to peak disease, the macrophages produced little NO and remained unresponsive to IFNg and TNFa stimulation (Figure 1).

In summary these set of experiments showed that resident retinal myeloidderived cells (predominantly microglia) are conditioned or tonically deactivated and thus remain resistant to further cytokine stimulation, at least in vitro. This was similar to the response seen in the larger number of macrophages isolated during EAU recovery. It was evident though that during peak inflammation, infiltrating macrophages adapt to the Th1 T cell response (IFNg/TNFa), inducing classical activation of cells and generating NO. Extrapolating the in vitro data

Figure 6 TNFRp55 / mice have display reduced incidence and severity of EAU.

Figure 6 shows that TNFRp55 / animals had significantly reduced histological scores at peak disease (day 18) with concomitant suppression of splenocyte proliferation, IL-2 and IFNg production (data not shown). At day 10, however, before onset of disease, TNFRp55 / splenocyte proliferative and IL-2 responses were reduced but associated with a significant increase in IFNg production, indicating normal T cell priming in these mice (data not shown; see ref.5).

Although T cell priming is relatively unaffected, macrophages lacking the TNFp55 receptor fail to produce NO following IFNg activation, because of a requirement for autocrine TNFa signalling through the TNFp55 receptor.5

DISCUSSION

Selected aspects of IFNg (adaptive immune system) activation are controlled by autocrine secretion of TNFa (Figure 7). NO production and MHC-class II upregulation are both critically dependent on autocrine secretion of TNFa, but only NO secretion requires signals from the TNFp55 receptor.5 However, data from TNFRp55 / macrophages demonstrate that other signals, notably from the innate immune system, via pathogen-associated molecular pattern (PAMP) recognising receptors such as the Toll family of receptors (e.g. TLR4) can induce NO production independent of signals through the TNFp55 receptor.5 This raises a question in autoimmune disease, when pathogens need not necessarily be present (for example sympathetic ophthalmia), of whether endogenous ligands for PAMP receptors contribute to the inflammatory milieu that promulgates disease. If this is the case it might provide an alternative therapeutic target for intervention.

Figure 7 Selective autocrine signalling from p55 and p75 induced by IFN-g.

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