Review Article Purinergic Receptors in Ocular Inflammationeprints.ucm.es/42239/1/320906purinergic receptors 2014.pdf · Purinergic Receptors in Ocular Inflammation ... A 1 and A 3

Review ArticlePurinergic Receptors in Ocular Inflammation

Ana Guzman-Aranguez12 Xavier Gasull234 Yolanda Diebold56 and Jesuacutes Pintor12

1 Department of Biochemistry and Molecular Biology IV Faculty of Optics and Optometry Universidad Complutense de MadridCArcos de Jalon 118 28037 Madrid Spain

2 Spanish Cooperative Thematic Research Network in Ocular Prevalent and Chronic Pathology (RETIC)Instituto de Salud Carlos III Madrid Spain

3 Neurophysiology Lab Department of Physiological Sciences I Medical School Universitat de Barcelona Barcelona Spain4 Biomedical Research Institute August Pi i Sunyer (IDIBAPS) Barcelona Spain5 Ocular Surface Group Institute for Applied Ophthalmobiology (IOBA) University of Valladolid Valladolid Spain6 Biomedical Research Networking Center on Bioengineering Biomaterials and Nanomedicine (CIBER-BBN) Spain

Correspondence should be addressed to Jesus Pintor jpintorucmes

Received 25 April 2014 Accepted 17 June 2014 Published 14 July 2014

Academic Editor Mireia Martın-Satue

Copyright copy 2014 Ana Guzman-Aranguez et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Inflammation is a complex process that implies the interaction between cells and molecular mediators which when not properlyldquotunedrdquo can lead to disease When inflammation affects the eye it can produce severe disorders affecting the superficial andinternal parts of the visual organ The nucleoside adenosine and nucleotides including adenine mononucleotides like ADP andATP and dinucleotides such as P1P4-diadenosine tetraphosphate (Ap

4A) and P1P5-diadenosine pentaphosphate (Ap

5A) are

present in different ocular locations and therefore they may contributemodulate inflammatory processes Adenosine receptorsin particular A

2A adenosine receptors present anti-inflammatory action in acute and chronic retinal inflammation Regarding theA3receptor selective agonists likeN6-(3-iodobenzyl)-51015840-N-methylcarboxamidoadenosine (CF101) have been used for the treatment

of inflammatory ophthalmic diseases such as dry eye and uveoretinitis Sideways diverse stimuli (sensory stimulation largeintraocular pressure increases) can produce a release of ATP from ocular sensory innervation or after injury to ocular tissuesThenATP will activate purinergic P2 receptors present in sensory nerve endings the iris the ciliary body or other tissues surroundingthe anterior chamber of the eye to produce uveitisendophthalmitis In summary adenosine and nucleotides can activate receptorsin ocular structures susceptible to suffer from inflammatory processes This involvement suggests the possible use of purinergicagonists and antagonists as therapeutic targets for ocular inflammation

1 Introduction

Inflammation is the biological process triggered when avascular tissue needs to be repaired from an injury or facesa microbial challenge This process is normally self-limitedhowever in the absence of a proper return to homeostasis itcan turn into a damaging condition for tissues [1 2] It worksthroughout complex and specific interactions between cellsand mediator molecules in the damaged tissue that requirea fine and well-tuned molecular regulation In the absenceof a prompt resolution of the acute inflammatory responseaffected tissue may progress to a chronic inflammatory statusthat leads to disease [3]

As many organs human eye suffers important andsometimes devastating inflammatory diseases In such cases

inflammation can be either cause or consequence of disordersaffecting different structures in the anterior part of the eyethe intraocular compartment or both Examples of such con-ditions include lacrimal keratoconjunctivitis severe allergicdiseases severe cicatrizing conjunctivitis uveitis (intraocularinflammation of the uvea retina vitreous body andoroptic nerve head) age-relatedmacular degeneration diabeticretinopathy and its major complication and proliferativevitreoretinopathy Also it is important to note that ocularinfections traumas and surgery also involve inflammatoryprocesses that can lead to a vision-threatening situation ifnot properly controlled Many efforts have been invested inthe development of therapeutic strategies to confront suchdiseases (for recent reviews see [4ndash6]) even using the aid of

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 320906 11 pageshttpdxdoiorg1011552014320906

2 Mediators of Inflammation

novel technology-related strategies such as drug delivery sys-tems or gene therapy (for recent reviews see [7 8]) Howevera much deeper knowledge of molecular pathophysiologymechanisms andmediators underlying ocular inflammation-related disorders is necessary

The eye has its own mechanism to protect itself frominflammation which is the so called immune privilege [9]The biological significance of such ldquoprivilegerdquo is the activetolerance to foreign antigens exerted by the ocular immunesystem In addition the tightly sealed blood-ocular barriersprevent the passage of inflammatory cells andmolecules fromthe blood into the eye Altogether the collateral inflamma-tion that is associated with the normal immune responseis avoided However the immune privilege can fail andinflammation can eventually develop with the involvement ofdifferent mediators

There are several families of molecules that have beendescribed as active participants in ocular inflammatory dis-easesThese include immunemediators such as cytokines andchemokines and enzymes such as matrix metalloproteinaseslipids growth factors and their receptors and neurotransmit-ters and their receptors These last ones are especially inter-esting because they are involved in maintaining the immuneprivilege [10] among other physiological activities Differentneuropeptides and their receptors have been identified inocular structures including the cornea sclera iris ciliarybody ciliary process and the retina [11] In addition there areexamples of the involvement of not only neuropeptides butalso biogenic amines amino acids acetylcholine or purinesin physiological processes of the eye that can be affected byinflammationThus the knowledge of the clinical impact thatneuropeptides and their receptors may have is growing inparallel with their envisioned therapeutical applications inocular inflammatory diseases

The role of parasympathetic and sympathetic nerves inconjunctival goblet cell functioning has been reported (forreview see [12])The parasympathetic nerves contain the neu-rotransmitters acetylcholine and vasoactive intestinal peptide(VIP) and the sympathetic nerves contain norepinephrineand neuropeptide Y Also purinergic receptor P2Y

2agonists

such as UTP and ATP are capable of stimulating both gobletcell mucin secretion and stratified squamous cell fluid (waterand electrolytes) secretion to tear film [12] Sensory nervesof cornea and conjunctiva contain neurotransmitters suchas substance P calcitonin gene-related peptide and galaninwhich can activate a neural reflex to stimulate conjunctivalgoblet cell secretion [13] The reduction in corneal sensationthat takes place in chronic inflammatory disease of theocular surface can impair the secretion of mucin and watercomponents of the tear film

Histamine produced by conjunctival sensitized mast cellsis a well-known mediator of the allergic pathology affectingthe eye [14] It is secreted to conjunctival tissues and the tearfilm along with other mast cell-derived irritant mediatorsafter the allergen challenge and triggers different allergicinflammation-related processes [15] It not only increasesvasodilation and vascular permeability to immune cellsbut also directly acts upon specific receptors present inconjunctival epithelial cells to stimulate goblet cell mucin

secretion [16] Besides histamine exerts a chemotactic effecton various immune cell types through a complex cytokinenetwork thus amplifying its biological activities

The eye has a small piece of brain in it that is the retinaIt is long known the importance of neurotransmitters andtheir receptors for the processing of visual informationwithinthe retinal tissue [17] Catecholamine neurotransmittersmainly dopamine are key for retinal neuron functioningin the vertebrate retina Also the role exerted by excitatoryand inhibitory amino acids as well as acetylcholine in thevisual process is well established [18] Glutamate aspartategamma-aminobutyric acid taurine and glycine are normalneurotransmitter or neuromodulatory agents for photore-ceptors and other retinal neurons such as horizontal andamacrine cells Muller cells the main glial cell of the retinaalso secrete neurotransmitters and express a wide varietyof neurotransmitter receptors reflecting their participationin the physiological signaling between neurons and glialcells [19] However glutamate-mediated loss of retinal gan-glion cells occurs in glaucoma and retinal vessels occlusion(central and branch retinal artery and retinal vein) [20]Cooperation between inflammatory cytokines and glutamatereceptors has been proposed as one of the mechanismsresponsible for a toxic damage on retinal cells related toglutathione depletion [21] Muller cells protect neurons fromglutamate toxicity It is now widely accepted that almostany retinal degenerative disease is associated with Mullercell gliosis which is a complex series of functional changesthat occurs as a consequence of retinal inflammation accom-panying the degenerative process Gliosis impedes Mullercell protective role against glutamate toxicity and impairstheir neurotransmitter recycling activity in the glioneuronalinteractions [22]

Among the plethora of transmitters present in the ocularstructures nucleosides and nucleotides emerge as remarkablemolecules with the ability to regulate many biochemical andphysiopathological processes Their actions are mediated bymembrane receptors termed purinergic receptors that canbe divided into adenosine P1 or A receptors and nucleotidereceptors named as P2 Adenosine receptors can be dividedinto A

1 A2A A2B and A

3and these receptors are only

sensitive to the nucleoside adenosine On the contrary P2receptors are divided into two main groups ionotropic P2Xandmetabotropic P2Y receptors and are sensitive to adenineto guanine nucleotides and also to dinucleotides such asdinucleoside polyphosphates

The diversity of receptors in the eye structures reflectsthe importance of these molecules in processes such as tearsecretion intraocular pressure homeostasis lens accommo-dation or retinal functioning Moreover use of nucleosidesand nucleotides naturally occurring and synthetic has beensuggested to rescue the eye from some pathological con-ditions Indeed there is a chance for the development ofpatents based on nucleotides as a therapeutic approach sinceit has been possible to relate nucleosidenucleotide levels withpathological conditions such as dry eye or glaucoma forexample The review of the patent literature did not bringany document related to ocular inflammatory processes Inthis sense it might be of interest the development of new

Mediators of Inflammation 3

Cornea

Sclera

Corneal epitheliumendothelium

Anterior chamber

Iris

LensCiliaryprocesses

Ciliarymuscle

Pupil

Sensory innervationParasympathetic innervation

Sympathetic innervation

Iridocorneal angle

TG

P2Y1 P2Y2 P2Y4 P2Y6 P2Y11

P2X7A2B

ConjunctivaP2Y2

P2X4 P2X7

P2Y1 P2Y2 P2Y4 P2Y11

A1 A2 A3

IrisP2Y1 P2Y2 P2Y4

Ciliary bodyP2Y1 P2Y2 P2Y4 P2Y11

P2X2

A1 A2A A2B A3

Trigeminal ganglionsensory neuronsP2Y1 P2Y4

P2X1 P2X2 P2X3 P2X4 P2X5 P2X6

A2B

Iridocorneal angletrabecular meshwork

Figure 1 Purinergic receptors identified in the ocular anterior segment Purinergic receptors localized in the different ocular partsstructuresof the ocular anterior segment are shown

inventions for the treatment of ocular inflammation based onpurinergic agonists and antagonists

The scientific literature that studies the relation of purinesand the eye have provided a disperse number of papersdescribing the involvement of these molecules in ocularinflammatory processes In this sense the present workreviews and groups the existing works in the field by struc-turing them in two main groups on the one hand actionsmediated by means of adenosine receptors and on the otherhand those occurring by nucleotide receptors

2 Adenosine Receptors

Adenosine is elevated at sites of tissue damage resulting frominflammation or hypoxia [23 24] Adenosine can be formedintracellularly and diffuse into the extracellular space viaequilibrative nucleoside transporter or extracellularly fromreleased ATP by ectonucleotidases CD39 and CD73 Understress and ischemic conditions the local tissue concentrationof extracellular adenosine is increased due to its synthesisfrom the released ATP Adenosine has been proposed tomodulate a variety of physiological responses includinginflammation and immunity by stimulating specific adeno-sine receptors (AR) [25 26] To date four adenosine receptorsubtypes A

1 A2A A2B and A

3have been identified that

belong to the family of seven transmembrane G protein-coupled receptors [27] The A

1and A

3adenosine receptors

preferentially couple to Gi protein to inhibit adenylate cyclaseand consequently the production of cyclic AMP (cAMP)and the A

2A and A2B subtypes stimulate the production of

cAMP by coupling to Gs Expression of adenosine receptorshas been described in different eye locations (Figure 1)

The presence of A2B adenosine receptors [28] has been

detected on bovine corneal endothelium In the ciliaryepithelium A

1 A2A and A

2B adenosine receptor mRNAswere found in the ciliary processes of rat using in situhybridization [29] Later A

3adenosine receptor mRNA

expression was also detected in cultured human ciliaryepithelial cells and rabbit ciliary processes by RT-PCR [30]In the retina A

2A adenosine receptor mRNA expression wasmainly found in the inner nuclear layer and ganglion cell layerand to a lesser extent in the outer nuclear layer LikewiseA1and A

3adenosine receptor mRNAs were identified in the

ganglion cell layer of the retina [29 31] In addition A2A and

A2B adenosine receptors are also present in retinal pigment

epithelial cells [29 32] as well as in Muller cells [33]

21 A1Adenosine Receptors Conflicting conclusions about

the effect of A1adenosine receptors on inflammation have

been reported Thus A1adenosine receptor has been impli-

cated as a potent anti-inflammatory mediator in variousinflammatory models of several organ systems including thekidney [34] heart [35] liver [36] and brain [37] On thecontrary in the lung pharmacologic blocking of A

1adeno-

sine receptors attenuated lipopolysaccharide (LPS)-inducedlung injury in cats [38] Likewise in an allergic mousemodel of asthma A

1adenosine receptors have been shown

responsible for altered vascular reactivity increased airwayhyperresponsiveness and systemic inflammation [39]

In the eye there is no data about the role of this receptorin ocular inflammation To date it has been only showedthat A

1adenosine receptor mediates IL-6 trophic effect on

retinal ganglion cells [40] IL-6 is a pleiotropic cytokineclassically denominated proinflammatory but additionally


it has been demonstrated that this cytokine is able to increasethe survival of retinal ganglion cells [41] It remains unknownwhether A

1adenosine receptor could also take part in some

proinflammatory actions induced by this cytokine in theretina apart from the IL-6 trophic effect on retinal ganglioncells

22 A2119860

Adenosine Receptors Substantial lines of evidencehave suggested that the anti-inflammatory effects of extracel-lular adenosine aremainlymediated by A

2A adenosine recep-tors [25 42] The anti-inflammatory action of A

2A adenosinereceptors in acute and chronic retinal inflammation has beendemonstrated [43 44] Using cultured retinal microglia cellsactivated by LPS as an in vitro model of acute neuroinflam-mation Liou et al [44] showed that A

2A adenosine receptoractivation in the stressed retinal microglial cells efficientlyinhibited LPS-induced TNF-120572 release The protective roleof A2A adenosine receptor in chronic retinal inflammation

associated to diabetic retinopathy has also been examined[43 45] Diabetic retinopathy has been categorized as avascular-neuroinflammatory disease Among the early signsof diabetic retinopathy are retinal inflammatory reactionsbreakdown of the blood-retinal barrier function and lossof retinal neurons [46ndash48] As the disease progresses theretina may be damaged by oxidative stress induced by hyper-glycemia or advanced glycation end products [49 50] Thisstress damages vascular and neuronal tissues of the retinaand activatesmicroglial cells [51] Activatedmicroglia furtherexacerbate the damage by releasing cytotoxic molecules(glutamate reactive oxygen species) and proinflammatorymediators such as TNF-120572 [52 53] Thus local inflamma-tion has a relevant contribution in the pathogenesis ofdiabetic retinopathy To elucidate the role of A

2A adenosinereceptor in diabetic retinopathy the effect of A

2A adeno-sine receptor ablation on diabetic mice was analyzed [43]Knockout A

2A adenosine receptor mice had significantlymore retinal terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL)-positive cells TNF-120572 releaseand intercellular adhesion molecule 1 (ICAM-1) expressioncompared with diabetic wild type [43] Interestingly togetherwith these changes an altered microglia phenotype wasobserved in the knockout A

2A adenosine receptor miceIn this sense in a diabetic milieu microglia transformedfrom their ramified resting state into an amoeboid shapethe activated and cytokine-releasing state and this pheno-typic configuration was more evident in the knockout A

2Aadenosine receptor diabetic mice than in diabetic wild-type[43] Moreover treatment of diabetic mice with the A

2Aadenosine receptor agonist CGS21680 (3-[4-[2-[[6-amino-9-[(2R 3R 4S 5S)-5-(ethylcarbamoyl)-34-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid) attenu-ated the morphological transformation of ramified microgliainto an activated ameboid microglia and resulted in markeddecreases in diabetes-induced retinal cell death and TNF-120572 release [43] Inhibition of reactive microglial phenotypeacquisition is not the only mechanism by which A

2A adeno-sine receptor regulates inflammation in diabetic retinopathyAdditional studies using microglial retinal cells treated withamadori-glycated albumin (AGA) (a risk factor in diabetic

disorders) showed that activation of A2A adenosine receptor

attenuated AGA-induced TNF-120572 release by repressing theinflammatory cascade C-Rafextracellular signal-regulatedkinase (ERK) in activated microglia (Figure 2) [43 45]

Considering these findings about the protective roleof A2A adenosine receptor activation in diabetes-induced

retinal inflammation abnormality in adenosine metabolismcould have influence on retinal complications in diabeticretinopathy In this context an increased expression andactivity of catabolic enzyme adenosine deaminase-2 (ADA2)which represent a critical checkpoint in the regulation ofextracellular adenosine levels and consequently in the con-trol of receptor stimulation and function have been identifiedin human andporcine retinaswith diabetes aswell as inAGA-treated porcine and human microglia cells [54] MoreoverTNF-120572 release was induced in AGA-treated microglia cellsand that TNF-120572 release was blocked by ADA2-neutralizingantibody or ADA2 siRNA [54] These results confirm thatabnormality in adenosine metabolism can contribute toretinal inflammation in diabetic retinopathy and suggest thatthe anti-inflammatory activity of A

2A adenosine receptorsignaling can be impaired in diabetic retinopathy due toincreased ADA2 activity

Taking into advance the ability of A2A adenosine receptor

to offer protection against retinal inflammation in diabeticretinopathy the use of the A

2A adenosine receptor agonistCGS21680 in other ocular retinal pathologies in which proin-flammatory mediators are released has also been examined[55] The A

2A adenosine receptor agonist administration sig-nificantly attenuated the expression of inflammatory (TNF-120572 IL-6 and ICAM-1) and cell death markers in a mousemodel of traumatic optic neuropathy (a disease characterizedby retinal ganglion cell death which is closely related to thelocal production of reactive oxygen species and inflammatorymediators from activated microglial cells) [55] A

2A adeno-sine receptor agonist anti-inflammatory action was mediatedby blocking ERK activation and subsequent cytokine releasein traumatic optic neuropathy activated microglia cells(Figure 2)

On the other hand it has been described the contributionof adenosinergic pathway throughA

2A adenosine receptor onprotective regulatory immunity in a mouse model of humanautoimmune uveitis [56] Thus A

2A adenosine receptoractivation on T cells was associated with antigen-presentingcells (APC) induction and activation of Tregs (regulatory Tcells) which mediate a postexperimental autoimmune uveo-retinitis regulatory immune response to ocular autoantigensprotecting from recurrence of uveitis [56]

23 A2119861

Adenosine Receptors Discrepancy between anti-inflammatory and proinflammatory effects has beenobserved in several tissues for A

2B adenosine receptors [57]This apparent contradiction might be related to differencesbetween the acute and chronic models of inflammationstudied playing the receptor different roles at different pointsduring the progression of inflammation Furthermore A

2Badenosine receptors may play different roles even in similartypes of inflammation but occurring in different tissues[57ndash59]


G protein

ERKAMPc

Adenosine

A cyclase

Retinal inflammation neuronalvascular

abnormalities

C-Raf

TNF-120572

A2A adenosine receptor

Activation of retinal

microglial cells

(mdash) (mdash)

(mdash)

Figure 2 Regulation of retinal inflammation by A2A adenosine receptor Pathways proposed to be involved in anti-inflammatory effect of A

2Aadenosine receptor in the retinal microglial cells during pathologies such as diabetes or traumatic optic neuropathy A

2A adenosine receptoractivation reduces TNF-120572 release by repressing the inflammatory cascade C-RafERK in activated retinal microglia

Little is known about the role of A2B adenosine receptor

in the eye A gradual increase in A2B adenosine receptor

has been reported after alkali burn-induced corneal inflam-mation and neovascularization As A

2B adenosine receptorwas not expressed by normal cornea it suggests that theA2B adenosine receptor detected after alkali burns was pro-

duced in the cornea by infiltrated inflammatory cells [60]In agreement with this finding it has been detected thatA2B adenosine receptor seems to be mainly expressed in

inflammatory cells [61]

24 A3Adenosine Receptors The A

3adenosine receptor is

highly expressed in inflammatory cells whereas low or almostno expression is found in normal cells [62] rendering theA3adenosine receptor as a potential therapeutic target A

3

adenosine receptor upregulation can be attributed to severalfactors including elevated adenosine and cytokines whichare characteristic of the microenvironment of inflammatory

cells [63] Under these conditions the binding of adenosineto their cell surface receptors might induce through anautocrine pathway the expression of its own receptors [6465] Additionally it has been proposed that the proin-flammatory cytokine TNF-120572 can induce an increase of thephosphatidylinositol 3-kinase (PI3K) and protein kinase B(PKB)Akt expression levels resulting in upregulation ofcAMP response element-binding (CREB) and nuclear factor-kappaB (NF-120581B) which translocate to the nucleus to act as A

3

adenosine receptor transcription factors [62]Selective A

3adenosine receptor agonists are being

developed for the treatment of inflammatory diseasessuch as rheumatoid arthritis osteoarthritis psoriasis andinflammatory bowel diseases [66] One of these ago-nists is the compound CF101 (N6-(3-iodobenzyl)-51015840-N-methylcarboxamidoadenosine) which exerts a robust anti-inflammatory effect in experimental animal models ofinflammatory diseases [67ndash70] The mechanism of action


mediating the anti-inflammatory effect of CF101 includesdownregulation of NF-120581B signaling pathway leading toinhibition of proinflammatory cytokines (TNF-120572 IL-6 andIL-12) macrophage inflammatory proteins (MIPs-1aMIP-2)and receptor activator ofNF-120581B ligand (RANKL) resulting inapoptosis of inflammatory cells [68 71] In addition a directantiproliferative effect of CF101 towards autoreactive T cellshas been observed [72]

The anti-inflammatory effects of CF101 via A3adenosine

receptor has prompted to explore its use for the treatment ofinflammatory ophthalmic diseases such as dry eye and uve-oretinitis Dry eye syndrome is an inflammatory conditionof the eye characterized by a massive production of proin-flammatory cytokines [73ndash75]Desiccating stress induces tearhyperosmolarity activating intracellular signaling pathwaysthat initiate the production of proinflammatory cytokinesThese inflammatory mediators promote the activation (mat-uration) of immature APCs and induce their migrationto draining lymphoid tissues The APCs are responsiblefor priming naive T cells in the lymphoid compartmentleading to the expansion of autoreactive CD4+ helper T cell(TH) subtype 1 and TH17 cell subsets T cells subsequentlyinfiltrate the ocular surface where they secrete additionalproinflammatory cytokines [76]

A phase II clinical study (randomized multicenterdouble-masked placebo-controlled and parallel group)exploring the effect of CF101 on patients with moderateto severe dry eye syndrome has been performed CF101administrated orally (1mgday for 12 weeks) induced astatistically significant improvement in the corneal stainingand an improvement in the tear break-up time and tearmeniscus height in patients with dry eye syndrome [77] Ingood agreement with previous trials [78] the drug was verywell tolerated and no severe adverse effects were detectedIt has been suggested that the improvement in the cornealstaining and tear break-up time in the study group mightbe due to reduced inflammation on the ocular surfacefollowing direct interaction between CF101 and its receptorson inflammatory cells [79] However additional proofs ofreduction of inflammation are required to fully confirm thisnotion

An experimental mice model of uveitis has been usedto test the anti-inflammatory action of CF101 Oral treat-ment with CF101 (10 120583gkg twice daily) initiated upondisease onset improved uveitis clinical score measured byfundoscopy and ameliorated the pathological manifestationsof the disease [72] A decrease in PI3K and STAT (signaltransducer and activator of transcription) protein levels in thelymph nodes of experimental autoimmune uveitis mice wasdetected upon CF101 treatment Both proteins are known tobe involved in the production of proinflammatory cytokines[80 81] and indeed inhibition of interleukin-2 TNF-120572 andinterferon-120574 (IFN-120574) production was also found in CF101-treated animals [72] Furthermore CF101 suppressed theantigen-specific proliferation of autoreactive T cells Overallthese results indicate the marked anti-inflammatory effectof CF101 and support further investigation of this drug foruveitis treatment

3 Ocular Sensory Innervation andPurinergic Receptors P2 Involved inOcular Inflammation

The trigeminal ganglion through the ophthalmic nerve pro-vides nonvisual sensory innervation of the eye Sensoryneurons innervating the eye detect noxious or potentiallynoxious stimuli in order to protect the eyeball elaborateresponses to minimize damage and promote tissue repairThese sensory neurons transduce mechanical thermal andchemical stimuli in the noxious range or close to it Mostof the sensory nerve endings innervate the front of the eyein particular the cornea and conjunctiva but importantinnervation is present in the uvea where it has a critical roleon ocular inflammation [82]

Autonomic parasympathetic innervation of the eye issupplied by the Edinger-Westphal nucleus in the brainstemthrough the oculomotor nerve [83 84] Parasympatheticnerve fibers synapse in the ciliary ganglion and enter theocular globe through the short ciliary nerves to innervatethe iris the ciliary body and ciliary muscle and partsof the iridocorneal angle (uveal trabecular meshwork andscleral spur) Some parasympathetic fibers come from thepons through the geniculate ganglion (Petrosal) Later theysynapse in the pterygopalatine ganglion before entering theeye [85] In parallel sympathetic nerve fibers arise from thesuperior cervical ganglion and enter the eyeball though thelong and short ciliary nerves They innervate the ciliary body(central stroma and stroma of the ciliary processes) the irisandparts of the iridocorneal angleNonsignificant autonomicinnervation is present in the cornea which is innervatedexclusively by sensory fibers

Different studies have provided evidence for the presenceof purinergic receptors in sensory neurons from the trigem-inal ganglion (Figure 1) P2X

3receptor mRNA and protein

are found in the cell bodies of both small and large sensoryneurons which has the highest level of expression amongthese neurons and in particular in peptidergic neurons [86]In contrast only a small percentage of IB4-binding neuronsexpress this receptor in trigeminal ganglia Lower levels arefound for P2X

1 P2X2 P2X4 P2X5 and P2X

6[86ndash88] mRNA

and protein for P2Y1and P2Y

4receptors are also present

and in many neurons colocalized with P2X3receptors [89]

Despite the studies in trigeminal ganglion neurons thereis a lack of specific studies on purinergic receptors in thesensory nerve endings innervating the anterior part of theeye (cornea sclera and conjunctiva) or the uvea (iris andciliary body) Although no information is available for ocularnerves purinergic receptors P2Y

1 P2Y2 P2Y4 and P2Y

6are

present in the corneal epithelium and endothelium cells [90](Figure 1) In fact injury to corneal epithelial cells results innucleotide release and mobilization of a calcium wave fromthe epithelium to the neurons [91] It has been hypothesizedthat ATP is initially released from epithelial cells and thenfollowed by a release of ATP and glutamate from neuronalprocesses that activate purinergic and N-methyl-D-aspartate(NMDA) receptors contributing to the wound response [91]In humans P2X

7receptor mRNA is also found in the cornea


and upregulated in diabetic patients Evidence indicates thatcorneal epithelial cells express full-length and truncatedforms of P2X

7 allowing P2X

7to function as a multifaceted

receptor that can mediate cell proliferation and migration orcell death [92]

In parallel the conjunctiva thewetmucosalmembrane ofthe eye is highly exposed to the environment and at the sametime very sensitive to the damaging effects of inflammationThe ocular surface therefore requires a carefully balancedmechanism to initiate inflammation only when absolutelynecessary Here hybridization to P2Y

2receptor mRNA has

been observed in the palpebral and bulbar conjunctivalepithelium including goblet cells the corneal epitheliumand in meibomian gland sebaceous and ductal cells [93] Inaddition recent studies [94] have reported that the purinergicreceptors P2X

4and P2X

7and the bacterial Toll-like receptor

2 (TLR2) are present and functional in conjunctival gobletcells and are involved in the priming and activation of theNLRP3 inflammasome initiated by danger associatedmolec-ular patterns (DAMPs) such as ATP The P2X

7receptor-

NLRP3 inflammasome complex modulates the release of theinflammatory cytokines IL-1b and IL-18 and it seems to beinvolved in the primary Sjogrenrsquos syndrome pathology in thesalivary glands and likely in Sjogrenrsquos derived ocular dryness(xerophthalmia) [95]

In the anterior uvea purinergic receptors P2Y1 P2Y2 and

P2Y4have been found in the iris [90]The same receptors and

P2Y11

have also been observed in both layers of the ciliarybody epithelium (pigmented and nonpigmented) in therabbit and monkey eye (Figure 1) in addition to a variety ofstructures within the choroid [90 93] Functional evidence ofP2Y2receptor activity has also been reported in these tissues

[96 97] In turn ocular ciliary epithelial cells are known tostore and release ATP an endogenous P2Y

2receptor agonist

providing a potential source of extracellular nucleotides forautocrine regulation of intraocular pressure [98] In thissense ATP it is known to be released from antidromicallystimulated trigeminal sensory nerve endings in the ciliarybody and as a consequence a significant increase of ATP isfound in the aqueous humor [99]This provides evidence thatATP released by ocular sensory innervation or after injuryof ocular tissues can activate both sensory nerve endingsand purinergic receptors present in the iris ciliary bodyor other tissues surrounding the anterior chamber of theeye to produce uveitisendophthalmitis In addition to thecornea and sclera abundant sensory nerve terminals arepresent in the iris and anterior uvea which detectmechanicalthermal and chemical stimuli contributing to neurogenicinflammation (inflammation of neural origin) by releasingproinflammatory neuropeptides like substance P and CGRP[82 100] As stated before releasedATPmight stimulate thesesensory nerve endings to enhance neurogenic inflammationand to maintain an inflamed state in the eye after a noxiousinsult

Circulating ATP nucleotides and dinucleotides releasedinto the aqueous humor can also stimulate purinergic recep-tors present in the trabecular meshwork a tissue located atthe iridocorneal angle of the anterior chamber of the eyeand involved in the regulation of aqueous humor outflow

mRNA protein and functional evidence have been foundfor purinergic receptors P2Y

1 P2Y

2 P2Y

4 and P2Y

11in

the bovine trabecular meshwork (Figure 1) [101 102] andin the human HTM-3 cell line [103] Depending on thepurinergic receptor activated an increase or decrease inaqueous humor outflow is found In this sense selectiveagonists of P2Y

1receptor increase the facility of aqueous

humor outflow and have been proposed as possible drugsfor ocular hypertension [102] On the other hand ocularinflammationuveitis produces the opposite effect on outflowfacility (decrease) and it has been proposed that ATP andother inflammatorymediatorsmight be involved in this effect[101 104ndash106]

4 Conclusions

The eye has evolved to curb intraocular inflammationprotecting the delicate visual elements from damage thatwould be detrimental to visual acuity This ability of theeye to limit and control immune responses is known asocular immune privilege However the immune privilege canfail and inflammatory processes can occur The nucleosideadenosine and nucleotides such as ATP are emerging as novelmolecules related to ocular inflammatory diseases To datethe anti-inflammatory effects of adenosine and their agonistsCGS21680 and CF101 acting via A

2A and A3adenosine

receptors respectively have encouraged exploring their usefor the treatment of inflammatory ophthalmic conditionssuch as ocular retinal pathologies and dry eye and clinicaltrials are being developed In contrast to adenosine thenucleotide ATP exhibits proinflammatory actions mediatedby purinergic P2 receptors present in sensory nerve endingsor in other eye locations Altogether the effects of nucleotidesand dinucleotides suggest the development of some of thesecompounds as therapeutic agents mainly based on the useof P2 receptor antagonists Also indirectly the use of P2Y

2

agonists on the ocular surface to treat dry eye could reduceocular surface inflammation but it is necessary to be awarethat the anti-inflammatory effect is a consequence of therestorage of aqueous andmucin productionUnder these newnormal conditions friction of the lids with the ocular surfaceis diminished and therefore inflammation is reduced In anycase to our knowledge apart from the commented effects ondry eye there is a lack of patents claiming the use of agonistsor antagonists for the treatment of ocular inflammationalthough in the recent years our knowledge about the rela-tion of these molecules with ocular inflammatory processesis increasing However a better understanding of their exactcontribution in the different ocular inflammatory diseases(dry eye severe cicatrizing conjunctivitis uveitis and soforth) is an important step to reveal additional pathologicmechanisms and designing new therapies based on the useof purinergic agonists and antagonists

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

This work was supported by the Ministry of Economy(Project SAF 201016024 and SAF-2013-44416-R) and theInstitute Carlos III (RETICS RD1200340003) Funding toXG was provided by Instituto de Salud Carlos III Spain (FISPI1101601) and Generalitat de Catalunya (2009SGR869)Funding to YD was provided by the Spanish Ministry ofEconomy (FEDER-CICYT Grant MAT2010-20452-C03-01)

References

[1] V Chesnokova and S Melmed ldquoMinireview neuro-immuno-endocrine modulation of the hypothalamic-pituitary-adrenal(HPA) axis by gp130 signaling moleculesrdquo Endocrinology vol143 no 5 pp 1571ndash1574 2002

[2] C Nathan ldquoPoints of control in inflammationrdquoNature vol 420no 6917 pp 846ndash852 2002

[3] CN Serhan ldquoResolution phase of inflammation novel endoge-nous anti-inflammatory and proresolving lipid mediators andpathwaysrdquo Annual Review of Immunology vol 25 pp 101ndash1372007

[4] C Evereklioglu ldquoOcular Behcet disease current therapeuticapproachesrdquo Current Opinion in Ophthalmology vol 22 no 6pp 508ndash516 2011

[5] S CMaloney KDGodeiro ANOdashiro andMN BurnierJr ldquoCurrent and emerging concepts in the management ofneovascular age-related macular degenerationrdquo Cardiovascularand Hematological Agents in Medicinal Chemistry vol 5 no 2pp 147ndash154 2007

[6] I Offiah and V L Calder ldquoImmune mechanisms in allergic eyediseases what is newrdquo Current Opinion in Allergy and ClinicalImmunology vol 9 no 5 pp 477ndash481 2009

[7] C J Chu S E Barker A D Dick and R R Ali ldquoGenetherapy for noninfectious uveitisrdquo Ocular Immunology andInflammation vol 20 no 6 pp 394ndash405 2012

[8] Y Diebold L Contreras-Ruiz I Arranz-Valsero and L Garcıa-Posadas ldquoDrug delivery systems for ophthalmic administra-tion of antiinflammatory agentsrdquo Anti-Inflammatory and Anti-AllergyAgents inMedicinal Chemistry vol 10 no 3 pp 203ndash2142011

[9] J Y Niederkorn and J Stein-Streilein ldquoHistory and physiologyof immune privilegerdquo Ocular Immunology and Inflammationvol 18 no 1 pp 19ndash23 2010

[10] A W Taylor ldquoNeuroimmunomodulation and immune privi-lege the role of neuropeptides in ocular immunosuppressionrdquoNeuroImmunoModulation vol 10 no 4 pp 189ndash198 2002

[11] T Tervo K Tervo and L Eranko ldquoOcular neuropeptidesrdquoMedical Biology vol 60 no 2 pp 53ndash60 1982

[12] D A Dartt ldquoRegulation of mucin and fluid secretion by con-junctival epithelial cellsrdquo Progress in Retinal and Eye Researchvol 21 no 6 pp 555ndash576 2002

[13] T L Kessler H J Mercer J D Zieske D M McCarthy andD A Dartt ldquoStimulation of goblet cell mucous secretion byactivation of nerves in rat conjunctivardquo Current Eye Researchvol 14 no 11 pp 985ndash992 1995

[14] A S Bacon P Ahluwalia A Irani et al ldquoTear and conjunctivalchanges during the allergen-induced early- and late-phaseresponsesrdquo Journal of Allergy and Clinical Immunology vol 106no 5 pp 948ndash954 2000

[15] M Ohbayashi B Manzouri K Morohoshi K Fukuda and SJ Ono ldquoThe role of histamine in ocular allergyrdquo Advances inExperimental Medicine and Biology vol 709 pp 43ndash52 2010

[16] D Hayashi D Li C Hayashi M Shatos R R Hodges andD A Dartt ldquoRole of histamine and its receptor subtypes instimulation of conjunctival goblet cell secretionrdquo Investigativeophthalmology amp visual science vol 53 no 6 pp 2993ndash30032012

[17] R G Pourcho ldquoNeurotransmitters in the retinardquo Current EyeResearch vol 15 no 7 pp 797ndash803 1996

[18] C J Pycock ldquoRetinal neurotransmissionrdquo Survey of Ophthal-mology vol 29 no 5 pp 355ndash365 1985

[19] R A de Melo Reis A L M Ventura C S Schitine M CF de Mello and F G de Mello ldquoMuller glia as an activecompartment modulating nervous activity in the vertebrateretina neurotransmitters and trophic factorsrdquo NeurochemicalResearch vol 33 no 8 pp 1466ndash1474 2008

[20] N J Sucher S A Lipton and E B Dreyer ldquoMolecular basis ofglutamate toxicity in retinal ganglion cellsrdquoVision Research vol37 no 24 pp 3483ndash3493 1997

[21] T Harada C Harada K Nakamura et al ldquoThe potential role ofglutamate transporters in the pathogenesis of normal tensionglaucomardquo Journal of Clinical Investigation vol 117 no 7 pp1763ndash1770 2007

[22] A Bringmann T Pannicke J Grosche et al ldquoMuller cells inthe healthy and diseased retinardquo Progress in Retinal and EyeResearch vol 25 no 4 pp 397ndash424 2006

[23] C Martin M Leone X Viviand M Ayem and R GuieuldquoHigh adenosine plasma concentration as a prognostic index foroutcome in patients with septic shockrdquo Critical Care Medicinevol 28 no 9 pp 3198ndash3202 2000

[24] B SperlaghMDodaM Baranyi andGHasko ldquoIschemic-likecondition releases norepinephrine and purines from differentsources in superfused rat spleen stripsrdquo Journal of Neuroim-munology vol 111 no 1-2 pp 45ndash54 2000

[25] S Gessi S Merighi D Fazzi A Stefanelli K Varani and PA Borea ldquoAdenosine receptor targeting in health and diseaserdquoExpert Opinion on Investigational Drugs vol 20 no 12 pp 1591ndash1609 2011

[26] G Hasko J Linden B Cronstein and P Pacher ldquoAdenosinereceptors therapeutic aspects for inflammatory and immunediseasesrdquo Nature Reviews Drug Discovery vol 7 no 9 pp 759ndash770 2008

[27] B B Fredholm A P Ijzerman K A Jacobson K Klotzand J Linden ldquoInternational Union of Pharmacology XXVNomenclature and classification of adenosine receptorsrdquo Phar-macological Reviews vol 53 no 4 pp 527ndash552 2001

[28] R J Walkenbach and W-T Chao ldquoAdenosine regulation ofcyclic AMP in corneal endotheliumrdquo Journal of Ocular Phar-macology vol 1 no 4 pp 337ndash342 1985

[29] A Kvanta S Seregard S Sejersen B Kull and B B FredholmldquoLocalization of adenosine receptor messenger RNAs in the rateyerdquo Experimental Eye Research vol 65 no 5 pp 595ndash602 1997

[30] C H Mitchell K Peterson-Yantorno D A Carre et al ldquoA3adenosine receptors regulate Cl- channels of nonpigmentedciliary epithelial cellsrdquoTheAmerican Journal of PhysiologymdashCellPhysiology vol 276 no 3 part 1 pp C659ndashC666 1999

[31] M Zhang M T Budak W Lu et al ldquoIdentification of theA3 adenosine receptor in rat retinal ganglion cellsrdquo MolecularVision vol 12 pp 937ndash948 2006


[32] C Blazynski ldquoCharacterization of adenosine A2 receptors inbovine retinal pigment epithelial membranesrdquo ExperimentalEye Research vol 56 no 5 pp 595ndash599 1993

[33] E A Newman ldquoCalcium increases in retinal glial cells evokedby light-induced neuronal activityrdquo Journal of Neuroscience vol25 no 23 pp 5502ndash5510 2005

[34] H T Lee G Gallos S H Nasr and C W Emala ldquoA1adenosine receptor activation inhibits inflammation necrosisand apoptosis after renal ischemia-reperfusion injury in micerdquoJournal of the American Society of Nephrology vol 15 no 1 pp102ndash111 2004

[35] Y Liao S Takashima Y Asano et al ldquoActivation of adenosineA1 receptor attenuates cardiac hypertrophy and prevents heartfailure in murine left ventricular pressure-overload modelrdquoCirculation Research vol 93 no 8 pp 759ndash766 2003

[36] J Kim M Kim J H Song and H T Lee ldquoEndogenous A1

adenosine receptors protect against hepatic ischemia reperfu-sion injury in micerdquo Liver Transplantation vol 14 no 6 pp845ndash854 2008

[37] S Tsutsui J Schnermann F Noorbakhsh et al ldquoA1 adenosinereceptor upregulation and activation attenuates neuroinflam-mation and demyelination in a model of multiple sclerosisrdquoJournal of Neuroscience vol 24 no 6 pp 1521ndash1529 2004

[38] C F Neely J Jin and I M Keith ldquoA1-adenosine receptorantagonists block endotoxin-induced lung injuryrdquo The Ameri-can Journal of Physiology vol 272 no 2 pp L353ndashL361 1997

[39] D S Ponnoth A Nadeem S Tilley and S J Mustafa ldquoInvolve-ment of A1 adenosine receptors in altered vascular responsesand inflammation in an allergic mouse model of asthmardquoThe American Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 299 no 1 pp H81ndashH87 2010

[40] R Perıgolo-Vicente K Ritt M R Pereira P M M Torres RPaes-de-Carvalho and E Giestal-de-Araujo ldquoIL-6 treatmentincreases the survival of retinal ganglion cells in vitro The roleof adenosine A1 receptorrdquo Biochemical and Biophysical ResearchCommunications vol 430 no 2 pp 512ndash518 2013

[41] P M M Torres and E G De Araujo ldquoInterleukin-6 increasesthe survival of retinal ganglion cells in vitrordquo Journal ofNeuroimmunology vol 117 no 1-2 pp 43ndash50 2001

[42] G Hasko and P Pacher ldquoA2A receptors in inflammation andinjury lessons learned from transgenic animalsrdquo Journal ofLeukocyte Biology vol 83 no 3 pp 447ndash455 2008

[43] A S Ibrahim M M El-shishtawy W Zhang R B Caldwelland G I Liou ldquoA2A adenosine receptor (A2AAR) as a thera-peutic target in diabetic retinopathyrdquo The American Journal ofPathology vol 178 no 5 pp 2136ndash2145 2011

[44] G I Liou J A Auchampach C J Hillard et al ldquoMediationof cannabidiol anti-inflammation in the retina by equilibrativenucleoside transporter and A2A adenosine receptorrdquo Investiga-tive Ophthalmology and Visual Science vol 49 no 12 pp 5526ndash5531 2008

[45] G I Liou S Ahmad M Naime N Fatteh and A S IbrahimldquoRole of adenosine in diabetic retinopathyrdquo Journal of OcularBiology Diseases and Informatics vol 4 no 1-2 pp 19ndash24 2011

[46] A J Barber E Lieth S A Khin D A Antonetti A GBuchanan and T W Gardner ldquoNeural apoptosis in the retinaduring experimental and human diabetes early onset and effectof insulinrdquo Journal of Clinical Investigation vol 102 no 4 pp783ndash791 1998

[47] A B El-Remessy M Al-Shabrawey Y Khalifa N Tsai RB Caldwell and G I Liou ldquoNeuroprotective and blood-retinal barrier-preserving effects of cannabidiol in experimental

diabetesrdquoAmerican Journal of Pathology vol 168 no 1 pp 235ndash244 2006

[48] A M Joussen V Poulaki M L Le et al ldquoA central role forinflammation in the pathogenesis of diabetic retinopathyrdquo TheFASEB Journal vol 18 no 12 pp 1450ndash1452 2004

[49] M Al-Shabrawey M Rojas T Sanders et al ldquoRole of NADPHoxidase in retinal vascular inflammationrdquo Investigative Ophthal-mology and Visual Science vol 49 no 7 pp 3239ndash3244 2008

[50] A W Stitt T Bhaduri C B T McMullen T A Gardiner andD B Archer ldquoAdvanced glycation end products induce blood-retinal barrier dysfunction in normoglycemic ratsrdquo MolecularCell Biology Research Communications vol 3 no 6 pp 380ndash388 2000

[51] E Rungger-Brandle A A Dosso and P M Leuenberger ldquoGlialreactivity an early feature of diabetic retinopathyrdquo InvestigativeOphthalmology and Visual Science vol 41 no 7 pp 1971ndash19802000

[52] G W Kreutzberg ldquoMicroglia a sensor for pathological eventsin the CNSrdquo Trends in Neurosciences vol 19 no 8 pp 312ndash3181996

[53] M Sayyah M Javad-Pour and M Ghazi-Khansari ldquoThebacterial endotoxin lipopolysaccharide enhances seizure sus-ceptibility in mice involvement of proinflammatory factorsnitric oxide and prostaglandinsrdquo Neuroscience vol 122 no 4pp 1073ndash1080 2003

[54] N M Elsherbiny M Naime S Ahmad et al ldquoPotential rolesof adenosine deaminase-2 in diabetic retinopathyrdquo Biochemicaland Biophysical Research Communications vol 436 no 3 pp355ndash361 2013

[55] S Ahmad N Fatteh N M El-Sherbiny et al ldquoPotential role ofA2A adenosine receptor in traumatic optic neuropathyrdquo Journalof Neuroimmunology vol 264 no 1-2 pp 54ndash64 2013

[56] D J Lee and A W Taylor ldquoBoth MC5r and A2Ar are requiredfor protective regulatory immunity in the spleen of post-experimental autoimmune uveitis in micerdquo The Journal ofImmunology vol 191 no 8 pp 4103ndash4111 2013

[57] I Feoktistov and I Biaggioni ldquoRole of adenosine A2119861

receptorsin inflammationrdquo Advances in Pharmacology vol 61 pp 115ndash144 2011

[58] V L Kolachala M Vijay-Kumar G Dalmasso et al ldquoA2Badenosine receptor gene deletion attenuates murine colitisrdquoGastroenterology vol 135 no 3 pp 861ndash870 2008

[59] Y Zhou A Mohsenin E Morschl et al ldquoEnhanced air-way inflammation and remodeling in adenosine deaminase-deficient mice lacking the A

2119861adenosine receptorrdquo Journal of

Immunology vol 182 no 12 pp 8037ndash8046 2009[60] Y Han Y Shao Z Lin et al ldquoNetrin-1 simultaneously sup-

presses corneal inflammation and neovascularizationrdquo Inves-tigative Ophthalmology ampVisual Science vol 53 no 3 pp 1285ndash1295 2012

[61] P Rosenberger J M Schwab V Mirakaj et al ldquoHypoxia-inducible factor-dependent induction of netrin-1 dampensinflammation caused by hypoxiardquo Nature Immunology vol 10no 2 pp 195ndash202 2009

[62] A Ochaion S Bar-Yehuda S Cohen et al ldquoThe anti-inflam-matory target A3 adenosine receptor is over-expressed inrheumatoid arthritis psoriasis and Crohnrsquos diseaserdquo CellularImmunology vol 258 no 2 pp 115ndash122 2009

[63] L Madi S Cohen A Ochayin S Bar-Yehuda F Barer andP Fishman ldquoOverexpression of A3 adenosine receptor inperipheral blood mononuclear cells in rheumatoid arthritis


involvement of nuclear factor-120581B in mediating receptor levelrdquoJournal of Rheumatology vol 34 no 1 pp 20ndash26 2007

[64] A Ochaion S Bar-Yehuda S Cohn et al ldquoMethotrex-ate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expressionrdquo ArthritisResearch andTherapy vol 8 no 6 article R169 2006

[65] U Schlotzer-SchrehardtM Zenkel U Decking et al ldquoSelectiveupregulation of the A3 adenosine receptor in eyes with pseu-doexfoliation syndrome and glaucomardquo Investigative Ophthal-mology amp Visual Science vol 46 no 6 pp 2023ndash2034 2005

[66] P Fishman S Bar-Yehuda B T Liang and K A JacobsonldquoPharmacological and therapeutic effects of A3 adenosinereceptor agonistsrdquo Drug Discovery Today vol 17 no 7-8 pp359ndash366 2012

[67] S Bar-Yehuda L Rath-Wolfson L Del Valle et al ldquoInduction ofan antiinflammatory effect and prevention of cartilage damagein rat knee osteoarthritis by CF101 treatmentrdquo Arthritis andRheumatism vol 60 no 10 pp 3061ndash3071 2009

[68] P Fishman S Bar-Yehuda L Madi et al ldquoThe PI3K-NF-120581B signal transduction pathway is involved in mediating theanti-inflammatory effect of IB-MECA in adjuvant-inducedarthritisrdquo Arthritis Research and Therapy vol 8 no 1 articleR33 2006

[69] J Mabley F Soriano P Pacher et al ldquoThe adenosine A3 recep-tor agonist N6-(3-iodobenzyl)-adenosine-51015840-N-methylurona-mide is protective in two murine models of colitisrdquo EuropeanJournal of Pharmacology vol 466 no 3 pp 323ndash329 2003

[70] L Rath-Wolfson S Bar-Yehuda L Madi et al ldquoIB-MECAan A3 adenosine receptor agonist prevents bone resorption inrats with adjuvant induced arthritisrdquo Clinical and ExperimentalRheumatology vol 24 no 4 pp 400ndash406 2006

[71] C Szabo G S Scott L Virag et al ldquoSuppression ofmacrophageinflammatory protein (MIP)-1120572 production and collagen-induced arthritis by adenosine receptor agonistsrdquo British Jour-nal of Pharmacology vol 125 no 2 pp 379ndash387 1998

[72] S Bar-Yehuda D Luger A Ochaion et al ldquoInhibition of exper-imental auto-immune uveitis by the A3 adenosine receptoragonist CF101rdquo International Journal of Molecular Medicine vol28 no 5 pp 727ndash731 2011

[73] A Acera G Rocha E Vecino I Lema and J A DuranldquoInflammatory markers in the tears of patients with ocularsurface diseaserdquo Ophthalmic Research vol 40 no 6 pp 315ndash321 2008

[74] N Boehm A I Riechardt M Wiegand N Pfeiffer and F HGrus ldquoProinflammatory cytokine profiling of tears from dryeye patients by means of antibody microarraysrdquo InvestigativeOphthalmology and Visual Science vol 52 no 10 pp 7725ndash7730 2011

[75] H Lam L Bleiden C S de Paiva W Farley M E Stern andS C Pflugfelder ldquoTear cytokine profiles in dysfunctional tearsyndromerdquo American Journal of Ophthalmology vol 147 no 2pp 198ndash205 2009

[76] W Stevenson S K Chauhan and R Dana ldquoDry eye diseasean immune-mediated ocular surface disorderrdquo Archives ofOphthalmology vol 130 no 1 pp 90ndash100 2012

[77] I Avni H J Garzozi I S Barequet et al ldquoTreatment of dry eyesyndrome with orally administered CF101 data from a phase 2clinical trialrdquoOphthalmology vol 117 no 7 pp 1287ndash1293 2010

[78] A-R van Troostenburg E V Clark W D H Carey et alldquoTolerability pharmacokinetics and concentration-dependenthemodynamic effects of oral CF101 an A3 adenosine receptor

agonist in healthy young menrdquo International Journal of ClinicalPharmacology and Therapeutics vol 42 no 10 pp 534ndash5422004

[79] J N Ashar A Mathur and V Sangwan ldquoCF101 for dry eyerdquoOphthalmology vol 118 no 5 pp 1011ndash1012 2011

[80] B Renga M Migliorati A Mencarelli and S Fiorucci ldquoRecip-rocal regulation of the bile acid-activated receptor FXR and theinterferon-120574-STAT-1 pathway in macrophagesrdquo Biochimica etBiophysica Acta vol 1792 no 6 pp 564ndash573 2009

[81] S G Ward and P Finan ldquoIsoform-specific phosphoinositide3-kinase inhibitors as therapeutic agentsrdquo Current Opinion inPharmacology vol 3 no 4 pp 426ndash434 2003

[82] C Belmonte J Garcia-Hirschfeld and J Gallar ldquoNeurobiologyof ocular painrdquo Progress in Retinal and Eye Research vol 16 no1 pp 117ndash156 1997

[83] A Reiner H J Karten P D R Gamlin and J T ErichsenldquoParasympathetic ocular control Functional subdivisions andcircuity of the avian nucleus of Edinger-Westphalrdquo Trends inNeurosciences vol 6 no 4 pp 140ndash145 1983

[84] M P M Ten Tusscher H J M Beckers G F J M Vrensen andJ Klooster ldquoPeripheral neural circuits regulating IOP A reviewof its anatomical backbonerdquo Documenta Ophthalmologica vol87 no 4 pp 291ndash313 1994

[85] G L Ruskell ldquoThe orbital branches of the pterygopalatineganglion and their relationship with internal carotid nervebranches in primatesrdquo Journal of Anatomy vol 106 no 2 pp323ndash339 1970

[86] V Staikopoulos B J Sessle J B Furness and E A JenningsldquoLocalization of P2X2 and P2X3 receptors in rat trigeminalganglion neuronsrdquo Neuroscience vol 144 no 1 pp 208ndash2162007

[87] P M Dunn Y Zhong and G Burnstock ldquoP2X receptors inperipheral neuronsrdquo Progress in Neurobiology vol 65 no 2 pp107ndash134 2001

[88] H Kuroda Y Shibukawa M Soya et al ldquoExpression ofP2X1 and P2X4 receptors in rat trigeminal ganglion neuronsrdquoNeuroReport vol 23 no 13 pp 752ndash756 2012

[89] H Z Ruan and G Burnstock ldquoLocalisation of P2Y1 and P2Y4receptors in dorsal root nodose and trigeminal ganglia of theratrdquoHistochemistry and Cell Biology vol 120 no 5 pp 415ndash4262003

[90] J Pintor J Sanchez-Nogueiro M Irazu A Mediero T Pelaezand A Peral ldquoImmunolocalisation of P2Y receptors in the rateyerdquo Purinergic Signalling vol 1 no 1 pp 83ndash90 2004

[91] D J Oswald A Lee M Trinidad et al ldquoCommunicationbetween corneal epithelial cells and trigeminal neurons isfacilitated by purinergic (P2) and glutamatergic receptorsrdquoPLoSONE vol 7 no 9 Article ID e44574 2012

[92] C Mankus C Rich M Minns and V Trinkaus-RandallldquoCorneal epithelium expresses a variant of P2X 7 receptor inhealth and diseaserdquo PLoS ONE vol 6 no 12 Article ID e285412011

[93] M S Cowlen V Z Zhang L Warnock C F Moyer W MPeterson and B R Yerxa ldquoLocalization of ocular P2Y2 receptorgene expression by in situ hybridizationrdquo Experimental EyeResearch vol 77 no 1 pp 77ndash84 2003

[94] V E McGilligan M S Gregory-Ksander D Li et al ldquoStaphy-lococcus aureus activates the NLRP3 inflammasome in humanand rat conjunctival goblet cellsrdquoPLoSONE vol 8 no 9 ArticleID e74010 2013


[95] C Baldini C Rossi F Ferro et al ldquoThe P2X7 receptor-inflammasome complex has a role in modulating the inflam-matory response in primary Sjogrenrsquos syndromerdquo Journal ofInternal Medicine vol 274 no 5 pp 480ndash489 2013

[96] N A Farahbakhsh and M C Cilluffo ldquoP2 purinergic receptor-coupled signaling in the rabbit ciliary body epitheliumrdquo Inves-tigative Ophthalmology and Visual Science vol 43 no 7 pp2317ndash2325 2002

[97] M Shahidullah andW S Wilson ldquoMobilisation of intracellularcalciumbyP2Y2 receptors in cultured non-transformed bovineciliary epithelial cellsrdquo Current Eye Research vol 16 no 10 pp1006ndash1016 1997

[98] C H Mitchell D A Carre A M Mcglinn R A Stone and MM Civan ldquoA releasemechanism for storedATP in ocular ciliaryepithelial cellsrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 95 no 12 pp 7174ndash71781998

[99] E Maul and M Sears ldquoATP is released into the rabbit eye byantidromic stimulation of the trigeminal nerverdquo InvestigativeOphthalmology and Visual Science vol 18 no 3 pp 256ndash2621979

[100] G M Mintenig M V Sanchez-Vives C Martin A Gual andC Belmonte ldquoSensory receptors in the anterior uvea of the catrsquoseye an in vitro studyrdquo Investigative Ophthalmology amp VisualScience vol 36 no 8 pp 1615ndash1624 1995

[101] D Soto N Comes E Ferrer et al ldquoModulation of aqueoushumor outflow by ionic mechanisms involved in trabecularmeshwork cell volume regulationrdquo Investigative Ophthalmologyand Visual Science vol 45 no 10 pp 3650ndash3661 2004

[102] D Soto J Pintor A Peral A Gual and X Gasull ldquoEffects ofdinucleoside polyphosphates on trabecular meshwork cells andaqueous humor outflow facilityrdquo Journal of Pharmacology andExperimental Therapeutics vol 314 no 3 pp 1042ndash1051 2005

[103] C E Crosson P W Yates A N Bhat Y V Mukhin andS Husain ldquoEvidence for multiple P2Y receptors in trabecularmeshwork cellsrdquoThe Journal of Pharmacology and ExperimentalTherapeutics vol 309 no 2 pp 484ndash489 2004

[104] P Conquet B Plazonnet and J C le Douarec ldquoArachidonicacid induced elevation of intraocular pressure and anti inflam-matory agentsrdquo Investigative Ophthalmology vol 14 no 10 pp772ndash775 1975

[105] J G Ladas F Yu R Loo et al ldquoRelationship between aqueoushumor protein level and outflow facility in patients with uveitisrdquoInvestigative Ophthalmology and Visual Science vol 42 no 11pp 2584ndash2588 2001

[106] R S Moorthy A Mermoud G Baerveldt D S Minckler P PLee and N A Rao ldquoGlaucoma associated with uveitisrdquo Surveyof Ophthalmology vol 41 no 5 pp 361ndash394 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


novel technology-related strategies such as drug delivery sys-tems or gene therapy (for recent reviews see [7 8]) Howevera much deeper knowledge of molecular pathophysiologymechanisms andmediators underlying ocular inflammation-related disorders is necessary

The eye has its own mechanism to protect itself frominflammation which is the so called immune privilege [9]The biological significance of such ldquoprivilegerdquo is the activetolerance to foreign antigens exerted by the ocular immunesystem In addition the tightly sealed blood-ocular barriersprevent the passage of inflammatory cells andmolecules fromthe blood into the eye Altogether the collateral inflamma-tion that is associated with the normal immune responseis avoided However the immune privilege can fail andinflammation can eventually develop with the involvement ofdifferent mediators

There are several families of molecules that have beendescribed as active participants in ocular inflammatory dis-easesThese include immunemediators such as cytokines andchemokines and enzymes such as matrix metalloproteinaseslipids growth factors and their receptors and neurotransmit-ters and their receptors These last ones are especially inter-esting because they are involved in maintaining the immuneprivilege [10] among other physiological activities Differentneuropeptides and their receptors have been identified inocular structures including the cornea sclera iris ciliarybody ciliary process and the retina [11] In addition there areexamples of the involvement of not only neuropeptides butalso biogenic amines amino acids acetylcholine or purinesin physiological processes of the eye that can be affected byinflammationThus the knowledge of the clinical impact thatneuropeptides and their receptors may have is growing inparallel with their envisioned therapeutical applications inocular inflammatory diseases

The role of parasympathetic and sympathetic nerves inconjunctival goblet cell functioning has been reported (forreview see [12])The parasympathetic nerves contain the neu-rotransmitters acetylcholine and vasoactive intestinal peptide(VIP) and the sympathetic nerves contain norepinephrineand neuropeptide Y Also purinergic receptor P2Y

2agonists

such as UTP and ATP are capable of stimulating both gobletcell mucin secretion and stratified squamous cell fluid (waterand electrolytes) secretion to tear film [12] Sensory nervesof cornea and conjunctiva contain neurotransmitters suchas substance P calcitonin gene-related peptide and galaninwhich can activate a neural reflex to stimulate conjunctivalgoblet cell secretion [13] The reduction in corneal sensationthat takes place in chronic inflammatory disease of theocular surface can impair the secretion of mucin and watercomponents of the tear film

Histamine produced by conjunctival sensitized mast cellsis a well-known mediator of the allergic pathology affectingthe eye [14] It is secreted to conjunctival tissues and the tearfilm along with other mast cell-derived irritant mediatorsafter the allergen challenge and triggers different allergicinflammation-related processes [15] It not only increasesvasodilation and vascular permeability to immune cellsbut also directly acts upon specific receptors present inconjunctival epithelial cells to stimulate goblet cell mucin

secretion [16] Besides histamine exerts a chemotactic effecton various immune cell types through a complex cytokinenetwork thus amplifying its biological activities

The eye has a small piece of brain in it that is the retinaIt is long known the importance of neurotransmitters andtheir receptors for the processing of visual informationwithinthe retinal tissue [17] Catecholamine neurotransmittersmainly dopamine are key for retinal neuron functioningin the vertebrate retina Also the role exerted by excitatoryand inhibitory amino acids as well as acetylcholine in thevisual process is well established [18] Glutamate aspartategamma-aminobutyric acid taurine and glycine are normalneurotransmitter or neuromodulatory agents for photore-ceptors and other retinal neurons such as horizontal andamacrine cells Muller cells the main glial cell of the retinaalso secrete neurotransmitters and express a wide varietyof neurotransmitter receptors reflecting their participationin the physiological signaling between neurons and glialcells [19] However glutamate-mediated loss of retinal gan-glion cells occurs in glaucoma and retinal vessels occlusion(central and branch retinal artery and retinal vein) [20]Cooperation between inflammatory cytokines and glutamatereceptors has been proposed as one of the mechanismsresponsible for a toxic damage on retinal cells related toglutathione depletion [21] Muller cells protect neurons fromglutamate toxicity It is now widely accepted that almostany retinal degenerative disease is associated with Mullercell gliosis which is a complex series of functional changesthat occurs as a consequence of retinal inflammation accom-panying the degenerative process Gliosis impedes Mullercell protective role against glutamate toxicity and impairstheir neurotransmitter recycling activity in the glioneuronalinteractions [22]

Among the plethora of transmitters present in the ocularstructures nucleosides and nucleotides emerge as remarkablemolecules with the ability to regulate many biochemical andphysiopathological processes Their actions are mediated bymembrane receptors termed purinergic receptors that canbe divided into adenosine P1 or A receptors and nucleotidereceptors named as P2 Adenosine receptors can be dividedinto A

1 A2A A2B and A

3and these receptors are only

sensitive to the nucleoside adenosine On the contrary P2receptors are divided into two main groups ionotropic P2Xandmetabotropic P2Y receptors and are sensitive to adenineto guanine nucleotides and also to dinucleotides such asdinucleoside polyphosphates

The diversity of receptors in the eye structures reflectsthe importance of these molecules in processes such as tearsecretion intraocular pressure homeostasis lens accommo-dation or retinal functioning Moreover use of nucleosidesand nucleotides naturally occurring and synthetic has beensuggested to rescue the eye from some pathological con-ditions Indeed there is a chance for the development ofpatents based on nucleotides as a therapeutic approach sinceit has been possible to relate nucleosidenucleotide levels withpathological conditions such as dry eye or glaucoma forexample The review of the patent literature did not bringany document related to ocular inflammatory processes Inthis sense it might be of interest the development of new


Cornea

Sclera


Anterior chamber

Iris


Ciliarymuscle

Pupil



Iridocorneal angle

TG


P2X7A2B

ConjunctivaP2Y2

P2X4 P2X7


A1 A2 A3

IrisP2Y1 P2Y2 P2Y4


P2X2

A1 A2A A2B A3



A2B







1 A2A A2B and A



1and A







1 A2A and A













1adeno-











22 A2119860


























23 A2119861





G protein

ERKAMPc

Adenosine

A cyclase


abnormalities

C-Raf

TNF-120572



microglial cells

(mdash) (mdash)

(mdash)













3



3

















6[86ndash88] mRNA





1 P2Y2 P2Y4 and P2Y

6are





7 allowing P2X




2receptor mRNA has


4and P2X



7receptor-




P2Y11



2receptor agonist




1 P2Y

2 P2Y

4 and P2Y

11in




4 Conclusions


2A and A3adenosine


2





Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cornea

Sclera


Anterior chamber

Iris


Ciliarymuscle

Pupil



Iridocorneal angle

TG


P2X7A2B

ConjunctivaP2Y2

P2X4 P2X7


A1 A2 A3

IrisP2Y1 P2Y2 P2Y4


P2X2

A1 A2A A2B A3



A2B







1 A2A A2B and A



1and A







1 A2A and A













1adeno-











22 A2119860


























23 A2119861





G protein

ERKAMPc

Adenosine

A cyclase


abnormalities

C-Raf

TNF-120572



microglial cells

(mdash) (mdash)

(mdash)













3



3

















6[86ndash88] mRNA





1 P2Y2 P2Y4 and P2Y

6are





7 allowing P2X




2receptor mRNA has


4and P2X



7receptor-




P2Y11



2receptor agonist




1 P2Y

2 P2Y

4 and P2Y

11in




4 Conclusions


2A and A3adenosine


2





Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















22 A2119860


























23 A2119861





G protein

ERKAMPc

Adenosine

A cyclase


abnormalities

C-Raf

TNF-120572



microglial cells

(mdash) (mdash)

(mdash)













3



3

















6[86ndash88] mRNA





1 P2Y2 P2Y4 and P2Y

6are





7 allowing P2X




2receptor mRNA has


4and P2X



7receptor-




P2Y11



2receptor agonist




1 P2Y

2 P2Y

4 and P2Y

11in




4 Conclusions


2A and A3adenosine


2





Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















G protein

ERKAMPc

Adenosine

A cyclase


abnormalities

C-Raf

TNF-120572



microglial cells

(mdash) (mdash)

(mdash)













3



3

















6[86ndash88] mRNA





1 P2Y2 P2Y4 and P2Y

6are





7 allowing P2X




2receptor mRNA has


4and P2X



7receptor-




P2Y11



2receptor agonist




1 P2Y

2 P2Y

4 and P2Y

11in




4 Conclusions


2A and A3adenosine


2





Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























6[86ndash88] mRNA





1 P2Y2 P2Y4 and P2Y

6are





7 allowing P2X




2receptor mRNA has


4and P2X



7receptor-




P2Y11



2receptor agonist




1 P2Y

2 P2Y

4 and P2Y

11in




4 Conclusions


2A and A3adenosine


2





Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















7 allowing P2X




2receptor mRNA has


4and P2X



7receptor-




P2Y11



2receptor agonist




1 P2Y

2 P2Y

4 and P2Y

11in




4 Conclusions


2A and A3adenosine


2





Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgments


References


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of










































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

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