Basic immunology

The immune system is the body’s natural defence in combating organisms. Immunology has developed rapidly over the past decade owing to the refinements madein the molecular tests employed in this area of research. Therefore, the keen reader isencouraged to peruse the ophthalmic and immunological literature in order to keepabreast of the latest developments in this field.

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Owing to the complex nature of this subject, it isfar beyond the scope of this article to cover allaspects of immunology. Rather, the aims of thearticle are twofold: first to acquaint the busypractitioner with the basic concepts of theimmune system; and second, to introduce thereader to the more specific topic of ocularimmunology – the study of the ocular immunesystem.

Finally, since it is envisaged that optometristswill one day prescribe therapeutic agents, thediscussion is limited to the anterior segment andanterior uvea.

Innate & adaptive immune systemsThe immune system can be thought of as havingtwo “lines of defence”: the first, representing anon-specific (no memory) response to aannttiiggeenn(substance to which the body regards as foreignor potentially harmful) known as the iinnnnaatteeiimmmmuunnee ssyysstteemm; and the second, tthhee aaddaappttiivveeiimmmmuunnee ssyysstteemm, which displays a high degree ofmemory and specificity. The innate systemrepresents the first line of defence to anintruding pathogen. The response evolved istherefore rapid, and is unable to “memorise” thesame said pathogen should the body be exposedto it in the future. Although the cells andmolecules of the adaptive system possess slowertemporal dynamics, they possess a high degreeof specificity and evoke a more potent responseon secondary exposure to the pathogen.

The adaptive immune system frequentlyincorporates cells and molecules of the innatesystem in its fight against harmful pathogens.For example, ccoommpplleemmeenntt (molecules of theinnate system - see later) may be activated byaannttiibbooddiieess (molecules of the adaptive system)thus providing a useful addition to the adaptivesystem’s armamentaria.

A comparison of the two systems can be seenin TTaabbllee 11..

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Gregory Heath BSc (Hons), MCOptom, Dip. Clin. Optom

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Cells of the innateimmune system

PhagocytesAlthough sub-divided into two main types,namely neutrophils and macrophages, theyboth share the same function - to engulfmicrobes (phago - I eat, Latin).

NeutrophilsMicroscopically, these cells possess acharacteristic, salient feature - amultilobular nucleus ((FFiigguurree 22)). As such,these cells have been referred to aspolymorphonuclear leukocytes (PMNs) andhave a pivotal role to play in thedevelopment of acute inflammation. Inaddition to being phagocytic, neutrophilscontain granules and can also be classed asone of the granulocytes. The granulescontain acidic and alkaline phosphatases,defensins and peroxidase - all of which

represent the requisite molecules required forsuccessful elimination of the unwantedmicrobe(s).

MacrophagesMacrophages (termed monocytes when in theblood stream) have a horseshoe-shaped nucleusand are large cells. Properties of macrophagesinclude phagocytosis and antigen presentationto T cells (see later). Unlike neutrophils (whichare short-lived cells), they are seen in chronicinflammation as they are long-lived cells.

Mononuclear phagocytic systemThe cells comprising the monocyte phagocyticsystem are tissue bound and, as a result, arefurther sub-divided depending on their location.A list of the cells together with theircorresponding location can be found in TTaabbllee 22.

Table 1: Cells and molecules of the innate and adaptive immune systems

Immunity Cells Molecules

Innate Natural killer (NK) cells CytokinesMast cells ComplementDendritic cells Acute phase proteinsPhagocytes

Adaptive T and B cells CytokinesAntibodies

Components of the immune system can be seen in Figure 1.

Figure 1 The principle components of the immune system are listed, indicating which cellsproduce which soluble mediators. Complement is made primarily by the liver, with somesynthesised by mononuclear phagocytes. Note that each cell only produces a particular set ofcytokines, mediators etc

Figure 2 Morphology of the neutrophil. Thisshows a neutrophil with its characteristicmultilobed nucleus and neutrophilic granulesin the cytoplasm. Giemsa stain, x 1500

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Table 2: Examples of cells of themononuclear phagocytic system and theirrespective locations

Cells Location

Monocytes Blood stream

Alveolar macrophages Lungs

Sinus macrophages Lymph nodes and spleen

Kupffer cells Liver

Dendritic cellsDendritic cells consist of Langerhans’ andinterdigitating cells and form an importantbridge between innate and adaptive immunity,as the cells present the antigenic peptide to theT helper cell (adaptive immunity). Such cells aretherefore known as professional antigenpresenting cells ((AAPPCCss)). TTaabbllee 33 illustrates thevarious types of dendritic cells together with anexample of their location.

EosinophilsEosinophils (so called because their granulesstain with eosin – FFiigguurree 44) are granulocytesthat possess phagocytic properties. Despite thefact that they represent only 2-5 % of the totalleukocyte population, they are instrumental inthe fight against parasites that are too big to bephagocytosed.

Phagocytosis - the processPhagocytosis is the process by which cells engulfmicroorganisms and particles ((FFiigguurree 33)). Firstly,the phagocyte must move towards the microbeunder the influence of chemotactic signals, e.g.complement (see later). For the process tocontinue, the phagocyte must attach to themicrobe either by recognition of the microbialsugar residues (e.g. mannose) on its surface orcomplement/antibody, which is bound to thepathogen. Following attachment, thephagocyte’s cell surface invaginates and themicrobe becomes internalised into aphagosome. The resultant pphhaaggoossoommee fuses withmultiple vesicles containing O2 free radicals andother toxic proteins known as lysosomes to forma phagolysosome. The microbe is subsequentlydestroyed.

Opsonisation (“to make tasty” - Greek) OOppssoonniinnss are molecules, which enhance theefficiency of the phagocytic process by coatingthe microbe and effectively marking them fortheir destruction. Important opsonins are thecomplement component C3b and antibodies.

Natural killer (NK) cellsNNKK cells are also known as “large granularlymphocytes” (LGLs) and are mainly found in thecirculation. They comprise between 5-11% of thetotal lymphocyte fraction. In addition topossessing receptors for immunoglobulin type G(IgG), they contain two unique cell surfacereceptors known as killer activation receptor andkiller inhibition receptor. Activation of theformer initiates cytokine (“communication”)molecules from the cell whilst activation of thelatter inhibits the aforesaid action.

NK cells serve an important role in attackingvirally-infected cells in addition to certaintumour cells. Destruction of infected cells isachieved through the release of perforins andgranyzymes from its granules, which induceapoptosis (programmed cell death). NK cells arealso able to secrete interferon-γ (IFN-γ). Thisinterferon serves two purposes: first, to preventhealthy host cells from becoming infected by avirus; and second, to augment the T cellresponse to other virally infected cells (see later).

Mast cells and basophilsMorphologically, mast cells and basophils arevery similar in that both contain electron densegranules in the cytoplasm. Basophils are so-called owing to the fact that their granules

stain with a basic dye. Unlike mast cells, whichare present in close proximity to blood vessels inconnective tissue, basophils reside in thecirculation.

Both cell types are instrumental in initiatingthe acute inflammatory response. Degranulationis achieved either by binding to components ofthe complement system or by cross-linking ofthe IgE antibody which results in the release ofpro-inflammatory mediators including histamineand various cytokines. The former inducesvasodilation and augments vascular permeabilitywhilst the latter are important in attractingboth neutrophils and eosinophils.

Table 3: Dendric cells and location

Cells Location

Langerhans cell Limbus, skin

Interdigitating cell T cell areas in lymph nodes

Figure 4 Morphology of the eosinophil. Themultilobed nucleus is stained blue and thecytoplasmic granules are stained red.Leishman stain, x 1800

Figure 3Phagocytes arrive at a site of inflammationby chemotaxis. They may then attach tomicroorganisms via their non-specific cellsurface receptors. Alternatively, if theorganism is opsonised with a fragment ofthe third complement component (C3b),attachment will be through the phagocyte’sreceptors for C3b. If the phagocytemembrane now becomes activated by theinfectious agent, it is taken into aphagosome by pseudopodia extendingaround it. Once inside, lysosomes fuse withthe phagosome to form a phagolysosomeand the infectious agent is killed. Undigestedmicrobial products may be released to theoutside

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for the successful eradication of an invadingvirus by the innate immune system.

Type II IFN, IFN-γ, is produced by T Helpercells and NK cells and is able to augment boththe antigen presenting properties together withthe phagocytic properties of the APCs (e.g.macrophages and dentritic cells).

Adaptive immunityAs mentioned previously, there is a great deal ofsynergy between the adaptive immune systemand its innate counterpart. The adaptive immunesystem comprises two main types of leukocyteknown as B and T lymphocytes. Before describingthese important cell types, it is necessary toacquaint the reader with both the primary andsecondary lymphoid organs and tissues in thebody. These are summarised in TTaabbllee 44.The bone marrow represents the dominant sitefor haemopoiesis (production of blood cells andplatelets). Although most of the haemopoieticcells maturate in this region, T lymphocytes doso in the thymus. In the thymus, premature Tcells undergo a process of positive and negativeselection whereby the former are allowed toprogress to maturity whilst the latter are markedfor termination via apoptosis (see centraltolerance).

LymphocytesMorphologically, there are three types oflymphocytes: T, B and NK cells. However, only Tand B lymphocytes exhibit memory andspecificity and, as such, are responsible for theunique quality of the adaptive immune system.

Resting B lymphocytes are able to react withfree antigen directly when it binds to their cellsurface immunoglobins which act as receptors. Tlymphocytes do not react with free antigen andinstead make use of APCs to phagocytose theantigen and then to express its component

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Molecules of the innateimmune systemThere are many molecules, which work in concertwith the cells of the innate immune system andwhich also foster close functional links with theiradaptive counterpart. The three major moleculesare:• Complement• Acute phase proteins (APP)• Interferons (IFNs)

ComplementThe complement system represents a large groupof independent proteins (denoted by the letter Cand followed by a number), secreted by bothhepatocytes (liver cells) and monocytes.Although these proteins maybe activated by boththe adaptive immune system (ccllaassssiiccaall ppaatthhwwaayy)or innate immune system (aalltteerrnnaattiivvee ppaatthhwwaayy),the nomenclature is derived from the fact thatthe proteins help (“complement”) the antibodyresponse.

Activation of complement via the microbeitself is known as the alternative pathway. Theclassical pathway requires the interaction ofantibody with specific antigen. The C3component is the pivotal serum protein of thecomplement system. Binding of the antigen toC3 results in two possible sequelae. In eithercase, C3 component becomes enzymaticallyconverted to C3b. The bacterial cell wall caneither remain bound to C3b and becomeopsonised (since phagocytes have receptors forC3b) or act as a focus for other complementproteins (namely C5, 6, 7, 8 and 9). The latterform the membrane attack complex (MAC), whichinduces cellular lysis.

The functions of the complement system maybe summarised as follows:• Opsonisation• Lysis (destruction of cells through damage/

rupture of plasma membrane)• Chemotaxis (directed migration of immune

cells)• Initiation of active inflammation via direct

activation of mast cells

It is important that complement is regulated toprotect host cells from damage and/or their totaldestruction. This is achieved by a series ofregulatory proteins, which are expressed on thehost cells themselves.

Acute phase proteinsThese serum proteins are synthesised byhepatocytes and are produced in high numbersin response to cytokines released frommacrophages.

Interferons (IFNs)IFNs are a group of molecules, which limit thespread of viral infections ((FFiigguurree 55)). There aretwo categories of IFNs, namely type I and type II.Type I IFNs maybe sub-divided further into IFN-αand β. IFN-γ is the sole type II interferon. Type IIFNs are induced by viruses, pro-inflammatorycytokines and endotoxins from gram negativebacterial cell walls. Their presence remains vital

proteins on the cell surface adjacent to specialhost proteins called major histocompatibilitycomplex (MHC) class II molecules. As discussed,antigen presenting cells which express MHC classII molecules include dendritic cells andmacrophages. This “afferent” phase must occurin order for the T cell to recognise the antigen.The “efferent” phase occurs when activatedlymphocytes enter the tissue and meet antigenagain. This results in multiplication and secretionof cytokines or immunoglobins in order todestroy the antigen.

T cells T cells can be broadly divided into both T helper(TH) and cytotoxic T cells (Tc). Furthermore, TH

cells may be sub-divided into TH1 and TH2. Theformer are pro-inflammatory T cells andstimulate macrophages whilst the latterorchestrate B cell differentiation and maturationand hence are involved in the production ofhumoral immunity (antibody mediated). T cellsexpress cell surface proteins, described by clusterdetermination (CD) numbers. TH cells express CD4molecules on their cell surface, which enable thelymphocyte to bind to a MHC class II molecule.The T cell receptor is unique in that it is onlyable to identify antigen when it is associatedwith a MHC molecule on the surface of the cell.

Cytotoxic T cells are primarily involved in thedestruction of infected cells, notably viruses.Unlike TH cells, cytotoxic cells possess CD8 cellsurface markers, which bind to antigenicpeptides expressed on MHC class I molecules.

B cells and antibodies(immunoglobulins - Ig)B cells are lymphocytes that produce antibodies(immunoglobulins) and can recognise freeantigen directly. They are produced in the bonemarrow and migrate to secondary lymphoidorgans. B cells are responsible for the

Figure 5When host cells become infectedby virus, they may produceinterferon. Different cell typesproduce interferon-α (IFN-α ) orinterferon-β (IFN-β); interferon-γ(IFN-γ) is produced by some typesof lymphocyte (TH) after activationby antigen. Interferons act on otherhost cells to induce a state ofresistance to viral infection. IFN-γhas many other effects as well

Table 4:Primary and secondary lymphoid organs

Primary lymphoid organs Secondary lymphoid organs

Bone marrow Lymph nodesMALT - mucosa associated lymphoid tissue (includes bronchus, gut, nasal and conjunctival associated mucosal tissues)

Spleen

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development of antibody mediated immunityknown as hhuummoorraall mmeeddiiaatteedd iimmmmuunniittyy.

When activated by foreign antigen, B cellsundergo proliferation and mature into antibodysecreting ppllaassmmaa cceellllss. The latter are rich inorganelles such as rough endoplasmic reticulumand mitochondria, which confer their ability tosecrete soluble proteins (antibodies). Not allproliferating B cells develop into plasma cells.Indeed, a significant proportion remain asmemory B cells through a process known asclonal selection. This process is vital ineliminating the antigen should the body becomere-exposed to it in the future. T cells are alsoclonally selected and this confers to theproduction of T memory cells.

Although T and B cells behave differently,both are able to recirculate around the bodymigrating from blood to tissue and vice versa.The ability to recirculate obviously increases theefficiency with which cells of the immune systemcan home onto the invading antigen.

AntibodiesAntibodies have two roles to play - the first is tobind antigen and the second is to interact withhost tissues and effector systems in order toensure removal of the antigen((FFiigguurree 66)).

There are five different types (known asiissoottyyppeess) of antibody in the human immunesystem - namely IgM, IgG, IgA, IgE and IgD. Inaddition, there are four sub classes of IgG (IgG1-4). The basic antibody unit consists of aglycosylated protein consisting of two heavy andtwo light, polypeptide chains. The region whichbinds to the antigen is known as the FFaabb region,while the constant region, FFcc, not onlydetermines the isotype but is the regionresponsible for evoking effector systems, e.g.mast cell activation. The term immune complexrefers to the combination of antigen andantibody and will be discussed later in the article(see type III hypersensitivity).

The antibody isotypes together with theircorresponding function are illustrated in TTaabbllee 55..

MHCMajor histocompatability complex (MHC) are cellsurface proteins classified as class I (also termedhuman leucocytic antigen [HLA] A, B and C),found on all nucleated cells and class II (termedHLA, DP, DQ and DR), found on all antigenpresenting cells (APCs). MHC molecules are thesine qua non of T cell induced immunity.

Clinically, there is a strong associationbetween HLA and certain systemic and oculardiseases (see later).

CytokinesCytokines (also termed interleukins [IL] meaning“between white blood cells”) are small moleculesthat act as a signal between cells and have avariety of roles including chemotaxis, cellulargrowth and cytotoxicity. Owing to their ability tocontrol immune activity, they have beendescribed as the “hormones” of the immunesystem.

CharacteristicsCrosses placenta thus providing newborn with useful humoral immunityHigh affinityPredominant antibody in blood and tissue fluid

Large pentameric structure in circulationPresent in monometric form on B cell surfaceSecreted form is predominant antibody in early immune response against antigenReaches 75% of adult levels at 12 months of age

Exists in both a monometric and dimeric formSecretory IgA (dimeric form) represents 1st line of defence againstmicrobes invading the mucosal surface, e.g. tears

Low levels in circulationIncreased levels in worm infectionsFc region has high affinity for mast cell thus involved in allergy

Antigen receptor on B cellsAbsent from memory cells

Table 5:Antibody isotypes and corresponding functions

Antibody IgG

IgM

IgA

IgE

IgD

Table 6:Various cytokines, their sources and functions

Cytokine Source FunctionIL-1 Macrophages 1. T, B cell activation

2. Mobilisation of PMNs3. Induction of acute phase proteins

IL-2 T cells Proliferation of T and NK cells

Il-4 Th2 cells B cell activationMast cells IgE response

IL-8 Macrophages Chemotaxis of PMNsT cellsFibroblastsKeratinocytes

IL-10 TH2 cells B cell activationMacrophages Suppress macrophages

IL-12 B cells Stimulate TH1

Inhibit TH2

TGF β (transforming growth factor) T cells Inhibits other cytokines

TNF α (tumour necrosis factor) Macrophages Inflammation

Figure 6When a microorganism lacks theinherent ability to activatecomplement or bind tophagocytes, the body providesantibodies as flexible adaptormolecules. The body can makeseveral million different antibodiesable to recognise a wide varietyof infectious agents. Thus theantibody illustrated binds microbe1, but not microbe 2, by its‘antigen-binding protein’ (Fab).The ‘Fc portion’ may activatecomplement or bind to Fcreceptors on host cells,particularly phagocytes.

TTaabbllee 66 summarises some of the cytokinespertinent to ocular immunology, their functionsand their progenitors. Since interferons havebeen discussed earlier in the article, they havebeen omitted from the table.

Central and peripheraltolerance: nature’s way ofcontaining the immuneresponseSince various cells of the immune system arecapable of reacting with self-antigens, it istherefore essential that the human body hasmechanisms to suppress/eliminate autoreactivecells. Failure to do so, can, in some cases, leadto the development of autoimmune diseases(see later).

Central toleranceCentral tolerance refers to the process wherebyboth immature B and T cell lymphocytes, whichreact against normal, healthy cells (self-antigens), are eliminated via apoptosis.

Peripheral toleranceThis involves the removal of mature lymphocytes,which are not tolerant to healthy cells.

Ocular immune privilegeThere are numerous sites in the body wherebytissue may be grafted with minimal risk ofrejection. Such regions include, inter alia, thetestis, thyroid lens, anterior chamber, cornea,iris and ciliary body1,2.

It is important that immune privilege is notsimple interpreted as the host’s inability toinitiate an immune response to a transplantedtissue. Rather, it is an area of the body in whichthere exists a paucity of various elements of thehuman immune system in response to anantigen.

FactorsThe factors purported by investigators thatcontribute to the phenomenon of ocular immuneprivilege include:• Isolation from a vascular supply• Isolation from a lymphatic supply• Presence of a vascular barrier• Ability to suppress the immune response• Anterior chamber associated immune

deviation (ACAID)

Vascular supplyThe healthy cornea is a good example of anocular site devoid of a vascular network. Theevidence to support the role a vascular networkhas to play in the mechanism of graft rejectionis unequivocal since the risk of failure correlatespositively with the degree of hostvascularisation3.

Vascular barrierThere is a plethora of evidence in theophthalmic literature to support the existence ofa blood-ocular barrier. Furthermore, the samesaid barrier encompasses different elementsincluding tight junctions between retinal

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endothelial cells and the presence of junctionalcomplexes linking retinal pigment epithelialcells4.

Lymphatic roleThe fact that skin allografts were not rejectedfollowing lymph node removal5 led investigatorsto hypothesise that immune privilege was solelydue to the absence of the same said system at aparticular anatomical site. However, althoughcertain immune privileged sites do indeed lacklymphatic drainage, others such as the testes6

and eye7 do possess such a system. It appearsthat a proportion of the aqueous humour drainsvia the uveoscleral pathway into the lymphaticvessels in the head and neck.

The eye, APCs & MHCsAs mentioned previously, APCs, through theirability to express MHC class II molecules, arepotent progenitors of the immune response.Moreover, such cells are capable of activating Tcells within the tissue itself. It is therefore notunreasonable to assume that a paucity of APCsmay play an important role in immuneprivilege. In addition, failure to express MHCclass I molecule would make a tissue immuneagainst the lytic action of the cytotoxic T cells.

Although the aforementioned mechanismsare theoretically plausible, cells expressing bothMHC class I and II molecules have beendetected in the eye. TTaabbllee 77 illustrates therelationship between histocompatability classand ocular cell type.

It is noteworthy that the epithelial cells ofthe crystalline lens are devoid of class Iexpression14 and that the Langerhans’ cells(class II expression) are absent from the centralcornea15.

It is interesting that not all cells, whichexpress MHC class II act as professional APCs inthe eye. Indeed, it has been shown that suchcells reside in the iris and ciliary body and notonly fail to present alloantigens to T cells, buthave the ability to suppress mixed lymphocytereactions16.

The failure to incite the inflammatory

response has attracted a great deal of interestamongst ophthalmologists and immunologistsalike. It appears such suppression is achieved byvarious factors present in the aqueous humour(e.g. transforming growth factor - β).

Anterior chamber associated immunedeviation (ACAID)As a result of experiments with rats,investigators discovered that antigens placed inthe anterior chamber resulted in systemicinhibition of delayed type hypersensitivity (DTHor type IV hypersensitivity) reactions to thesame said antigens17. This phenomenon has beencoined anterior chamber associated immunedeviation (ACAID). The anterior chamber is thusable to suppress delayed type hypersensitivityreactions and inhibit the production ofcomplement fixing antibodies18,19. However, ithas no inhibitory effect on cytotoxic T cellactivity and has a minimal influence on theproduction of non-complement fixingantibodies.

With respect to the endothelium, twoadaptations prevent it from immunologicalinjury: first, avoidance of cytotoxic T lymphocyte(CTL)-mediated lysis; and second, inhibition ofDTH responses in the anterior chamber22. Itachieves the former through the cells inabilityto express MHC class I molecules21. The corollaryof this, however, is that virally infected cellsmay persist in this region. The râison d’etre ofACAID is to protect the eye from the DTHresponse to pernicious antigens. As a result ofits location in relation to the anterior chamber,the corneal endothelium seems well placed toreap the benefits of ACAID.

ACAID is beneficial in reducing the incidenceof stromal keratitis in herpes simplex virusinfection. It therefore seems reasonable toassume that such an unwanted corneal side-effect occurs as a result of a DTH responserather than the toxic effect of the virus per se22.

Corneal graft rejection may be due, in part,to failure to invoke ACAID. Streilein at al23 notonly discovered that the immunosuppressiveeffects of the cornea were abolished in corneas

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Table 7:MHC and ocular cell type8-13

Ocular cell type MHC class I MHC class IICorneal epithelium •

Corneal stroma •

Corneal endothelium •

Trabecular meshwork •

Pigmented and non pigmented •cells of ciliary body

Anterior iris •

RPE cells •

Corneal limbus •

Iris and ciliary body •

Uveoscleral pathway •

Ora serrata •

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that had ACAID removed via cauterisation orkeratoplasty but also abolished in corneas thathad been denervated.

Ocular immunologyAnterior segment immunology may be sub-divided into the following aspects:• Tear film• Corneal immunology• Conjunctival immunology• Scleral immunology• Uveal immunology

Tear filma) Mucous layerThis layer is produced both by the conjunctivalgoblet and epithelial cells. The glycocalyxsynthesised by the corneal epithelial cells servesto attach the mucous layer and in doing so bindsto immunoglobulin in the aqueous24. It has beensuggested that the latter immunological sign mayhave antiviral effects. The evidence to supportthis is that certain intestinal mucosa, which havesimilar binding properties to those seen in theeye, are able to exert an inhibitory action againstviral replication25.

b) Aqueous layerThe aqueous layer contains the followingantimicrobial factors:1. Lactoferrin2. Lysozyme3. IgA4. Miscellaneous proteins

1. LactoferrinLactoferrin is produced via the acinar cells of thelacrimal gland. Its main function is to bind iron,which is required for bacterial growth. Ittherefore possesses both bacteriostatic andbacteriocidal properties. Furthermore, it is ableto enhance the effects of certainimmunoglobulins.

2. LyszomeProduced by type A cells in the lacrimal gland,lysozome constitutes up to 25% of the total tearprotein. It is surprising somewhat that despitebeing effective at lysing gram positive bacterialcell walls, staphylococcus aureus appearsrecalcitrant to its action26. However, it isinstrumental in enhancing IgA against gramnegative bacteria.

3. IgAThis is the predominant isotype found in thehuman tears in the non-inflamed eye. Very littleis present in serum with the majority secretedthrough epithelial cells of structures such as thelacrimal gland and the lactating breast. The IgApresent in the tears is produced by plasma cellssituated underneath the secretory cells lining theacini in the lacrimal gland.

IgA is very effective at binding microbes and,as such, prevents the microbe from adhering tothe mucosal surface. The antibody achieves thiseither by interfering with the binding sitedirectly or by agglutination. Successful binding of

the microorganism enables the tears to washthem away. IgA possesses other functionsincluding opsonisation and inactivation ofvarious bacterial enzymes and toxins. Inaddition, it may be involved in antibodydependent cell-mediated cytotoxicity. It must beemphasised, however, that IgA is unable to bindto complement and, as such, is not involved inthe classical pathway27.

4. Miscellaneous proteinsβ-Lysin, a bacteriocidal cationic protein, ispresent both in the tears and aqueous humour28.Its bacteriocidal properties are conferredthrough their ability to disrupt the micro-organisms’ walls. Various components ofcomplement are present in varying degreesdepending on whether the eye is closed or not.

Closed eye vs open eye

Open eyeIn addition to the components of complementmentioned above, the tears are rich in lysozyme,lactoferrin and lipocalin (tear pre albumin)together with low levels of IgA29,30.

Closed eyeIn the closed eye environment, levels of IgA andcomplement components are increased. Inaddition, neutrophils are also recruited. The eyecan be interpreted as being in a state ofsubclinical inflammation. However, the eye isprotected from damage induced by eithercomplement or neutrophils by DAF (DecayedAccelerating Factor [an inhibitory component ofthe complement system])31 and α1 –antitrypsin33.

Corneal immunologyParadoxically, the cornea, an immune privilegedsite, is capable of evoking an immunologicalresponse evidenced by the presence ofsubepithelial infiltrates, keratic precipitates,epithelial and stromal keratitis under certainclinical conditions.

Cytokine releaseNumerous cytokines are synthesised in thecornea including IL-1, IL-6, IL-8, TNF-α, IFN-γ,C5a and prostaglandins33. Phagocytosis of certainantigens via corneal epithelial cells initiate therelease of IL-1 which, in turn, gives rise to therecruitment of Langerhans’ cells from the limbusto the central cornea. It is worthy of note thatthe same said APC may be recruited centrally inresponse to chemical or localised damage34,35.Investigators have discovered that stromalfibroblasts synthesise IL-8 in response to infection by the herpessimplex virus36. Furthermore, it seems plausiblethat the latter cytokine release may beresponsible for the neutrophilic infiltrationobserved in cases of herpetic keratitis.

ImmunoglobulinsThe three isotypes detected in the cornea areIgM, G and A. IgG predominates in the centralcornea37. By contrast, IgM predominates in the

limbal region. The latter antibody is restrictedfrom the central cornea owing to its large size.Antibodies in the cornea are found in thestroma since they are cationically charged. Dueto their positive ionic charge, they bind toproteoglycans and the anionicglycosaminoglycans38.

Antigen/antibody complexes may sometimesbe observed in the corneal stroma in a varietyof pathological conditions (e.g. herpes simplexkeratitis), and because of their ringed shapedappearance are known as “Wessley rings”.

ComplementThe elements of complement found in thecornea include C1-739 in addition to theregulatory proteins H and I and C1 inhibitor40.Interestingly, C1 is present in highconcentrations in the limbus and is restrictedfrom the central cornea. This finding issignificant since the limbal region is susceptibleto ulceration following complement activationand immune complex deposition. It has beensuggested that C1 is produced by cornealfibroblasts whereas the remaining componentsdetected in the cornea appear to be derivedfrom the plasma via linked vessels.

Conjunctival immunologyThe conjunctival associated lymphoid tissue(CALT) is part of the more general mucosaassociated lymphoid tissue (MALT). Langerhans’cells and lymphocytes exist within theconjunctival epithelial layer. In the substantiapropria, neutrophils, lymphocytes, IgA and IgG,dendritic cells and mast cells all reside. It isnoteworthy that eosinophils and basophils arenot present in the healthy conjunctiva.

Langerhans’ cells and dendritic cells presentthe antigenic peptide to the conjunctival Thelper cells. Following antigenic presentation,the T cells secrete the cytokine IFN-γ whichserves to promote antigen elimination bymacrophages. This is the delayed-typehypersensitivity (DTH) response (see later) andis characteristic of conjunctival pathology suchas phlyctenulosis.

Scleral immunologyThere exists only a small number of immunecells in the sclera compared to the conjunctivasince it is relatively avascular. In its restingstate, IgG appears to be present in largeamounts41. However, a sclera under stress, maybecome immunologically active as a result ofmigrating cells from the overlying episcleral andunderlying choroidal vasculature42,43.

Uveal immunologyThe uveal tract is an important site from animmunological perspective for two reasons:first, it is highly vascular in nature; and second,the majority of vessels are highly fenestratedwhich facilitates the recruitment of leukocytesduring the inflammatory cascade. The uveacontains numerous cellular components of theimmune system including macrophages, mastcells, lymphocytes and plasma cells in addition

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to an appreciable number of immune factors.Although IgG and IgM have been detected in

both the choroid and iris, they exist in greaternumbers in the former structure. The reason forsuch disparate levels is the dearth of anionicantibody binding sites on the iris surface44. Bycontrast, the ciliary body harbours tremendousamounts of IgG by virtue of its anionic tissuesites.

Immunopathology and the eyeImmunopathology encompasses both pathologyas a result of an over-active immune system(hhyyppeerrsseennssiittiivviittyy and aauuttooiimmmmuunniittyy) togetherwith that acquired through an individualsinability to fight off infection – namelyimmunodeficiency. Unfortunately, it is farbeyond the scope of this article to describe thelatter and its ophthalmic correlates.

In this section, the classification systempertaining to hypersensitivity reactions will bedescribed and each subtype with an anteriorsegment manifestation will be discussed. Theassociation between HLA and systemic andophthalmic disease will be illustrated togetherwith a brief overview of the concepts pertainingto autoimmunity.

HypersensitivityThe term hypersensitivity refers to the processwhereby the adaptive immune response over-reacts to a variety of infectious and inertantigens resulting in damage to the host tissue.Five types of hypersensitivity reactions exist –all of which vary in their timing followingcontact with the antigen ((TTaabbllee 88)).

• Type I hypersensitivity: allergyAllergies may affect approximately 17% of thepopulation45. The term atopic is used to describethose individuals who possess a geneticpredisposition to allergy. Allergies may occur tootherwise innocuous antigens (known asaalllleerrggeennss) and infectious agents, e.g. worms.Type I hypersensitivity exists in two phases, thesensitisation and effector phases.

Firstly, a harmless allergen causes productionof IgE antibody on first exposure. This IgE

February 8, 2002 OT

diffuses throughout the body until it comes intocontact with mast cells and basophils. Boththese cell types have receptors for IgE antibody.Although the patient experiences no symptomsafter the initial binding, reintroduction of theantigen/allergen induces the production ofmore IgE and, furthermore, increase thelikelihood of cross-linking with existingantibodies on the mast cell surface. Such cross-linking induces the mast cell to degranulate andrelease a host of inflammatory mediators suchas histamine, prostaglandins and bradykinin.

Histamine characteristically causes the itchysymptoms experienced by patients and as aresult of binding to H1 receptors in the eye,induces vasodilation and enhances mucoussecretion by the goblet cells. Bradykininaugments vascular permeability, decreasesblood pressure and contracts smooth muscle.Prostaglandins are also powerful inflammatorymediators.

Ocular correlates:The following anterior segment conditions aredue, if not only in part, to type Ihypersensitivity:• Seasonal allergic conjunctivitis (type I)• Giant papillary conjunctivitis (types I and IV)• Vernal keratoconjunctivitis (VKC)

(type I and IV)• Atopic keratoconjunctivitis (AKC)

(types I and IV) ((FFiigguurree 77))

Seasonal allergic conjunctivitis can be easilyrecognised by chemosis and hyperaemia of theconjunctiva, lid swelling and excessivelacrimation. The cornea is not affected.

GPC is well known to optometrists as anallergic response to contact lenses, prostheticlenses, protruding corneal sutures and scleralbuckles. Histological examination of eyessuffering from GPC have revealed degranulatedmast cells (type I hypersensitivity)46 and CD4+ Tcells (type IV)47, thus corroborating the theorythat such a condition is mediated by both typesof hypersensitivity.

Vernal conjunctivitis can present either in apalpebral form characteristically exhibiting

giant cobblestone papillae, or in a limbal formwith gelatinous deposits known as Trantas’-dots,which represent degenerating epithelial cells andeosinophils. The first corneal change is apunctate epithelial keratitis, which if leftunchecked develops into a macroerosion ((FFiigguurree 88)) and finally a corneal plaque develops((FFiigguurree 99)). The conjunctiva itself ischaracteristically oedematous and contains anarray of immunological cells includinglymphocytes and mast cells. Evidence of type Iinvolvement includes the histological detectionof, inter alia, degranulated mast cells,eosinophils and increased levels of IgE inaffected eyes49. The detection of CD4+ T cells andmacrophages is indicative of a delayedinflammatory component49.

Table 8:Hypersensitivity reactions

Hypersensitivity Appearance Mediators Mechanismtype time

I. Immediate 2-30 mins IgE Mast cell response(enhance acute inflammation)

II. Cytotoxic 5-8 hours IgM and IgG Antibody and complement

III. Immune 2-8 hours IgM and IgG Antibody/antigen complex immune complexes complexes

IV. Delayed type 24-72 hours CD4 and CD8 T cells, T cell mediatedAPCs Macrophages activated

Include granulomatous reactions

V. Stimulatory Autoantibodies Autoantibodies against hormone

Figure 8:Epithelial macroerosion in VKC

Figure 9:Corneal plaque in VKC

Figure 7:Corneal thinning and vascularisation in AKC,excess mucous is present

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Type II hypersensitivity: cytotoxicThis classification of hypersensitivity involveseither IgG or IgM antibodies, which may inducecellular lysis due to the involvement of theclassical complement pathway (as seen in bloodtransfusion reactions) or recruit and activateinflammatory cells via complement. Thecomponents of complement include the C5a,which serves to attract inflammatory cells to thesite of interest. The hypersensitivity reaction is aresult of the excessive amount of extracellularmediators released by the inflammatory cells toantigens that are too big to be completelyphagocytosed.

Antibodies to self-antigens, such as theacetylcholine receptor in myasthenia gravis, isnot only another example of type IIhypersensitivity but also an example ofautoimmune disease.

Ocular manifestations• Mooren’s ulcer• Cicatrical pemphigoid

Mooren’s ulcer is a rare peripheral ulcerativekeratitis that exists either as a unilateral, non-progressive form which has a predilectionfor elderly patients or as a more severe,progressive form affecting both eyes of relativelyyoung individuals. The signs range from a smallpatch of grey infiltrate near the margin to frankulceration involving the entire cornealcircumference and, in some cases, the centralregion as well. The healing process results in athin, vascularised, opaque cornea. Investigatorshave identified a significant number oflymphocytes, neutrophils and plasma cells50 inthe cornea, thus providing unequivocal evidenceto support the theory that this condition has animmunopathological aetiology.

Cicatrical pemphigoid is a chronic, blisteringdisease, which has a predilection for both theocular and oral mucous membranes. Unlike itsself-limiting counterpart, pemphigus vulgaris,cicatrical pemphigoid rarely affects the skin.Ocular cicatrical pemphigoid is a serious,bilateral condition that represents effectiveshrinkage of the conjunctiva. Although theinitial presentation may be subacute and non-specific, it frequently progresses tosymblepharon, entropion with secondarytrichiasis, dry eye, ankyloblepharon andconjunctival fornix shortening. There is evidenceto support the presence of IgG antibodiesdirected against self-antigen in the basementmembrane of both the skin and eye51. Binding ofthe aforementioned antibodies may activatecomplement with subsequent recruitment ofinflammatory cells into the area. The process ofcicatrisation is achieved through the secretion ofcollagen via fibroblasts as a result of stimulationvia cytokines released from the invadinginflammatory cells.

Type III hypersensitivity: immune complexLarge pathogens with multiple antigenic siteshave several antibodies bound to them forming

immune complexes. Normally, these complexesare removed by the mononuclear phagocytes inthe liver and spleen with no adverse sequelae.However, persistence of immune complexes doesoccur in certain individuals leading to theirdeposition in tissue. As a consequence of thelatter action, complement may be activated thuspaving the way for inflammatory cells to enterthe deposition site. Since blood vessels (whichfilter plasma at high pressure and exhibit a greatdeal of tortuosity) are more susceptible forimmune complex deposition, the ciliary body isparticularly vulnerable to this type ofhypersensitivity reaction.

Ocular manifestations• Uveitis (Crohn’s disease)• Peripheral corneal lesions associated with

rheumatoid arthritis• Stevens Johnson syndrome• Sjögren’s syndrome

The signs and symptoms of the above will bedescribed in future articles in the series.

Type IV hypersensitivity: delayed-typehypersensitivity (DTH)The term DTH has been used to describe such areaction owing to its prolonged time-scalerelative to the other hypersensitivity types.Although DTH can be transferred by T cells thathave been previously sensitised by an antigen, itcannot be transferred in serum.

The sequence of events leading to DTH beginswith initial presentation of the antigen peptideto T cells by APCs (e.g. Langerhans’ cells). Theprimed T cell migrates to the site of antigenicentry whereby it releases pro-inflammatorymediators such as TNF. The release of thesecytokines facilitates blood flow and extravasationof plasma contents to the area. The activation ofCD4 T helper and CD8 cells results in the releaseof IFN-γ and, as a consequence, enhancesmacrophage activity in that area. Resolution ofDTH is dependent on the efficacy with whichsuch phagocytes can remove the offendingantigen.

Recalcitrant infectious agents result in achronic DTH that causes the chronically activatedmacrophages to fuse together and formmultinucleated giant cells. In an attempt tocontain the infectious agent, macrophages mayundergo further inter connections to resemble anepithelial layer. Owing to the similarity to thislayer they are referred to as epitheloid cells.Both epitheloid and multinucleated giant cellssecrete factors that induce fibrosis resulting ingranuloma formation. Thus granulomata are thehallmark of chronic inflammation. Damage andloss of function of the neighbouring tissuesfrequently ensues until the agent is removedeither chemically or surgically.

Ocular manifestations • Ocular allergies (VKC, AKC GPC)• Idiopathic uveitis• Sympathetic ophthalmia• Phlyctenulosis

Type V hypersensitivityThis relatively new category encompasses theconcept of autoantibodies binding to hormonereceptors that mimic the hormone itself. Thisresults in stimulation of the target cells.Examples include thyrotoxicosis.

AutoimmunityThe ability to react against self-antigens isknown as autoimmunity. However, a significantnumber of people exist who harbour auto-antibodies and yet remain asymptomatic. Thecorollary of this is that the presence ofautoreactive cells per se is not sufficient totrigger autoimmune disease. In fact,autoimmune disease is a result of breakdown ofone of the immunoregulatory mechanisms.Furthermore, autoimmune disease may beclassified as either being organ specific (e.g.insulin dependent diabetes mellitus, Grave’sdisease) or non-organ specific (e.g. Sjögren’ssyndrome, ankylosing spondylitis).

It is important to realise that the causes ofautoimmune diseases are multifactorial. Themain predisposing factors are age, gender,infection and genetics. However, one of themost important factors of interest toimmunologists and clinicians alike is theassociation between HLA and autoimmunedisease. When determining the likelihood ofcontracting a disease both epidemiologists andclinicians refer to the relative risk. In the case ofHLA antigen, the relative risk compares thechance of a person who has a particular HLAantigen acquiring a disease to those individualswho do not have such an antigen. Theassociation is exemplified by the relative risk ofsuffering from ankylosing spondylitis (AS) inindividuals who possess HLA-B27. In patientssuffering from AS, the prevalence of HLA-B27 is90% and this figure rises to 95% in patientswho suffer both with the disease and acuteiritis. Indeed, in the UK approximately 45% ofpatients who present with acute iritis willharbour HLA-B2752.

It is therefore important that patients whopresent with anterior uveitis are screened forHLA-B27 because although a positive result maynot necessarily be diagnostic, its presence willcertainly improve the sensitivity of furtherradiological tests.

TTaabbllee 99 compares the HLA associations withboth ophthalmic disorders together with thosesystemic disorders relevant to the ophthalmicpractitioner.

ConclusionThis article has only broached the fascinatingsubject of ocular immunology. A basicunderstanding of immunology is required ifpractitioners are to therapeutically manage theirpatients. Further articles in the series will helpto reinforce the concepts of this challengingsubject.

AcknowledgementThe author would like to thank Professor RogerBuckley for his permission to use FFiigguurreess 77 ttoo 99.

ot

February 8, 2002 OT www.optometry.co.uk34

Figures 1 to 6 reprinted fromImmunology, Third Edition, Roitt,Brostoff and Male, 1993 bypermission of the publisherMosby.

About the authorGregory Heath is an optometristworking part-time in privatepractice. He was recently awardedthe diploma in clinical optometryat City University and is currentlyreading medicine at the RoyalFree and University College,London, Medical School.

ReferencesReferences are available uponrequest. Please fax 01252-816176or email info@optometry.co.uk.

Table 9:HLA and ophthalmic disease

Disease HLA Relative risk 54 Ocularassociation manifestation

Ankylosing spondylitis B27 90 Anterior uveitis

Reiter’s disease B27 33 Mucopurulent conjunctivitisAnterior uveitisKeratitis

Rheumatoid DR4 7 Keratoconjunctivitis siccaKeratitisScleritis

Primary Sjögrens’ syndrome DR3, DR5 10 Keratoconjunctivitis sicca

Sarcoidosis DR3 Not known Anterior uveitis (acute and chronic)DacryoadentisRetinal vasculitis,neovascularisation,Optic nerve granulomata

Cicatrical pemphigoid DQw7 Not known Shrinkage of conjunctiva

Behcet’s disease B5 3 Anterior uveitisRetinitis, periphlebitis,retinal oedema

Sympathetic DR4, A11, Not known Panuveitisophthalmia B40

Systemic lupus DR2, DR3 3 Punctate epithelial keratopathyerythematosus Keratopathy

Necrotising scleritisRetinal lesions (cotton wool spots)Autoimmune optic neuropathy

11.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg iiss nnoott ppaarrtt ooff tthheeiinnnnaattee iimmmmuunnee ssyysstteemm??

a. Mast cellsb. Complementc. Phagocytesd. T cells

22.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissccoorrrreecctt rreeggaarrddiinngg tthhee iinnnnaattee iimmmmuunnee ssyysstteemm??

a. It is specificb. It evokes a more potent response on

secondary exposurec. It represents the first line of defenced. It is able to memorise pathogens on

subsequent exposures

33.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissccoorrrreecctt rreeggaarrddiinngg tthhee cceellllss ooff tthhee iinnnnaatteessyysstteemm??

a. Basophils are important phagocytesb. During phagocytosis the pathogen becomes

initially internalised as a phago-lysosomec. Eosinophils play an important role in

combating virally-infected cells d. Langerhans’ cells form a bridge between

innate and adaptive immunity

44.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissccoorrrreecctt rreeggaarrddiinngg tthhee aaddaappttiivvee iimmmmuunneessyysstteemm??

a. It consists of all types of lymphocytesb. T cells produce antibodiesc. T cells maturate in the thymusd. B cells are produced in the spleen

55.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissccoorrrreecctt rreeggaarrddiinngg TT cceellllss??

a. T cells can be subdivided into TH1 and TH2

subtypes onlyb. T cells alone can identify any type of antigenc. T cells express cell surface proteins denoted by

cluster determinant (CD) numbers d. All T cells are involved in initiating the

inflammatory response

66.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissiinnccoorrrreecctt??

a. B cells have antibodies as their cell surfacereceptor

b. There are five types of antibodyc. IgE is an important antibody in allergiesd. All B cells differentiate into plasma cells

77.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissccoorrrreecctt rreeggaarrddiinngg ooccuullaarr iimmmmuunnee pprriivviilleeggee??

a. There is an absence of Langerhans’ cells in thecentral cornea

b. Aqueous humour has no rolec. Abundant vascular supply is vitald. All ocular cells express MHC class II

88.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttssccoonncceerrnniinngg ooccuullaarr iimmmmuunnoollooggyy iiss iinnccoorrrreecctt??

a. Levels of IgA and complementincrease when the eyes are closed

b. IgA is the predominant antibodyin blood and tissue fluid

c. IgM, IgG and IgA antibody isotypes have beenidentified in the cornea

d. The sclera contains a smaller number ofimmune cells than the conjunctiva

99.. IInn aa ppaattiieenntt ssuuffffeerriinngg ffrroomm vveerrnnaall ccoonnjjuunnccttiivviittiiss,,wwhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissccoorrrreecctt??

a. Trantas’-dots represent infiltrating T cellsb. Affected eyes have increased levels of IgDc. Histologically, mast cells, lymphocytes and

macrophages have been identifiedd. Is due to type III Hypersensitivity

1100.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iissiinnccoorrrreecctt??

a. HLA-B27 is a risk factor for both anterior uveitisand ankylosing spondylitis

b. Granulomas are present in type IVhypersensitivity reactions

c. Histamine is an important vasoconstrictord. IgE mediated hypersensitivity is of rapid onset

1111.. WWhhaatt pprrooppoorrttiioonn ooff ppaattiieennttss wwiitthh aaccuuttee iirriittiiss wwiillllhhaarrbboouurr HHLLAA--BB2277??

a. 15%b. 25%c. 45%d. 65%

1122.. WWhhiicchh oonnee ooff tthhee ffoolllloowwiinngg ssttaatteemmeennttss iiss ccoorrrreeccttrreeggaarrddiinngg aa ttyyppee II hhyyppeerrsseennssiittiivviittyy rreeaaccttiioonn??

a. It always occurs in isolationb. It is characterised by the presence of

macrophagesc. It is associated with myasthenia gravisd. It involves the degranulation of mast cells

following the cross-linking of IgE bound to itscell surface

Multiple choice questions - Basic immunology Please note there is only one correct answer

An answer return form is included in this issue. It should be completed and returned to: CPD Initiatives (c4082c),OT, Victoria House, 178–180 Fleet Road, Fleet, Hampshire, GU51 4DA by March 6, 2002.