OCULAR IMMUNOLOGY Christine Watté, D.V.M., Dipl.ECVO Vetsuisse-University of Bern, Switzerland The immune system is capable of producing robust responses to protect against infections and injury, but has also evolved mechanisms that harness immune responses to preserve delicate tissues from immune damage. A tailored immune protection is particularly important for the tissues that compose the visual axis, as these tissues typically have limited regenerative capacity. Excessive injury inflicted by the immune system may lead to functional loss of the eye. This course will start by giving a brief overview of general immune principles. Thereafter, the distinct regulatory mechanisms found at the ocular surface, within the cornea and in the inner parts of the eye will be presented in separate sections. Some selected ocular diseases with an immunological etiology will then be discussed at the end of each section. I. GENERAL PRINCIPLES OF IMMUNOLOGY Immunology was originally defined as the study of the body’s (aka host) response against infectious microbes. Immune responses can however be triggered against a much broader panel of molecules and chemicals that are considered not to belong to oneself (non-self or foreign), regardless of the intrinsic pathogenicity of these substances. In autoimmune disease for example, self-molecules become the target of the immune system. Today’s definition of immunology therefore includes all cellular and molecular responses to molecules and cells that are –rightly or wrongly– identified as foreign by the immune system. The immune system has been divided into two broad types of reactions: reactions of innate immunity and reactions of adaptive immunity. Innate and adaptive immunity can be thought of as two equally important arms of the immune system. They differ with respect to the effector cell types that they utilize, specificity for different classes of microbes, speed of intervention and duration of response to pathogens. Generally speaking, immunity can be decomposed into the early reactions of innate immunity and the later more prolonged response of adaptive immunity. I.1 INNATE IMMUNITY The distinctive characteristics of the innate immune response are that: (1) defense mechanisms are largely in place before infection, (2) the response is directed against broad patterns that are common to a large class of molecules rather than to a specific microbe, (3) the response remains largely unchanged in the event of reinfection with the same pathogen. Innate immunity includes several layers of complementing barrier systems. The epithelial cell layers and their adnexa (hairs, secretory glands) constitute a primary physical and chemical barrier on body surfaces and orifices. Examples of such barriers include tight cellular junctions between cells that serve to prevent microbial invasion, mucus that trap particles and microbes, antimicrobial peptides that have direct bacteriostatic or bactericidal activities. The next barrier of innate immunity consists of specialized cells called macrophages and Dendritic Cells (DCs) that are residing in tissues and positioned at critical places to detect invading microbes in epithelial and mucosal surfaces, such as the skin’s Langerhans cells or alveolar macrophages. Such sentinel cells are also found in internal organs such as the liver’s Kupffer cells or splenic macrophages and contribute to detection of blood born microbes. An important function of these cells is to engulf microbes and particles (phagocytosis) and degrade them through the actions of various enzymes targeting proteins, lipids and carbohydrates. A second important function of these phagocytes is to produce proteins called cytokines and lipid mediators (leukotrienes) that contribute to amplify and coordinate the inflammatory response. The cardinal signs of inflammation (redness, swelling, heat and pain) described by the Ancient Greeks reflect a broad array of molecular and cellular innate responses that contribute to diluting, neutralizing and eliminating the source of toxic signals. Among these responses, the recruitment of other innate immune cells such as neutrophils, eosinophils and monocytes is an essential arm of natural immunity. Innate lymphoid cells such as Natural Killer (NK) cells contribute to the elimination of virus-infected cells and tumors. The innate immune system is finally composed of many secreted proteins found in blood such as the complement proteins, pentraxins and lectins that contribute to recognize, neutralize and destroy pathogens through their direct effects or in helping innate immune cell functions. The innate immune system detects microbes through pattern recognition receptors (PRRs). These are proteins expressed by cells of the innate immune system which serve to identify two classes of molecules: pathogen-
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OCULAR IMMUNOLOGY
Christine Watté, D.V.M., Dipl.ECVO Vetsuisse-University of Bern, Switzerland
The immune system is capable of producing robust responses to protect against infections and injury, but has also evolved mechanisms that harness immune responses to preserve delicate tissues from immune damage. A tailored immune protection is particularly important for the tissues that compose the visual axis, as these tissues typically have limited regenerative capacity. Excessive injury inflicted by the immune system may lead to functional loss of the eye. This course will start by giving a brief overview of general immune principles. Thereafter, the distinct regulatory mechanisms found at the ocular surface, within the cornea and in the inner parts of the eye will be presented in separate sections. Some selected ocular diseases with an immunological etiology will then be discussed at the end of each section. I. GENERAL PRINCIPLES OF IMMUNOLOGY Immunology was originally defined as the study of the body’s (aka host) response against infectious microbes. Immune responses can however be triggered against a much broader panel of molecules and chemicals that are considered not to belong to oneself (non-self or foreign), regardless of the intrinsic pathogenicity of these substances. In autoimmune disease for example, self-molecules become the target of the immune system. Today’s definition of immunology therefore includes all cellular and molecular responses to molecules and cells that are –rightly or wrongly– identified as foreign by the immune system. The immune system has been divided into two broad types of reactions: reactions of innate immunity and reactions of adaptive immunity. Innate and adaptive immunity can be thought of as two equally important arms of the immune system. They differ with respect to the effector cell types that they utilize, specificity for different classes of microbes, speed of intervention and duration of response to pathogens. Generally speaking, immunity can be decomposed into the early reactions of innate immunity and the later more prolonged response of adaptive immunity. I.1 INNATE IMMUNITY The distinctive characteristics of the innate immune response are that: (1) defense mechanisms are largely in place before infection, (2) the response is directed against broad patterns that are common to a large class of molecules rather than to a specific microbe, (3) the response remains largely unchanged in the event of reinfection with the same pathogen. Innate immunity includes several layers of complementing barrier systems. The epithelial cell layers and their adnexa (hairs, secretory glands) constitute a primary physical and chemical barrier on body surfaces and orifices. Examples of such barriers include tight cellular junctions between cells that serve to prevent microbial invasion, mucus that trap particles and microbes, antimicrobial peptides that have direct bacteriostatic or bactericidal activities. The next barrier of innate immunity consists of specialized cells called macrophages and Dendritic Cells (DCs) that are residing in tissues and positioned at critical places to detect invading microbes in epithelial and mucosal surfaces, such as the skin’s Langerhans cells or alveolar macrophages. Such sentinel cells are also found in internal organs such as the liver’s Kupffer cells or splenic macrophages and contribute to detection of blood born microbes. An important function of these cells is to engulf microbes and particles (phagocytosis) and degrade them through the actions of various enzymes targeting proteins, lipids and carbohydrates. A second important function of these phagocytes is to produce proteins called cytokines and lipid mediators (leukotrienes) that contribute to amplify and coordinate the inflammatory response. The cardinal signs of inflammation (redness, swelling, heat and pain) described by the Ancient Greeks reflect a broad array of molecular and cellular innate responses that contribute to diluting, neutralizing and eliminating the source of toxic signals. Among these responses, the recruitment of other innate immune cells such as neutrophils, eosinophils and monocytes is an essential arm of natural immunity. Innate lymphoid cells such as Natural Killer (NK) cells contribute to the elimination of virus-infected cells and tumors. The innate immune system is finally composed of many secreted proteins found in blood such as the complement proteins, pentraxins and lectins that contribute to recognize, neutralize and destroy pathogens through their direct effects or in helping innate immune cell functions. The innate immune system detects microbes through pattern recognition receptors (PRRs). These are proteins expressed by cells of the innate immune system which serve to identify two classes of molecules: pathogen-
associated molecular patterns (PAMPs) that are expressed on microbial pathogens, and damage-associated molecular patterns (DAMPs) that are associated with cell components released during cell damage or cell death and strongly contribute to enhance the immune response. Microbe-specific molecules (or PAMPs) include bacterial carbohydrates of Gram-negative bacteria (such as lipopolysaccharide), cell wall components of Gram-positive bacteria (such as lipoteichoic acid), bacterial peptides (such as flagellin), fungal glucans and chitin, microbial nucleic acids (e.g. double stranded RNA of viruses, unmethylated CpG motifs of bacterial DNA…). Although the ligands of PRRs are referred to as pathogen-associated molecular patterns (PAMPs) these molecules are highly conserved in the microbial world and shared by pathogenic as well as non-pathogenic microbes. They may perhaps be more correctly called Microbial Associated Molecular Patterns (MAMPs). On the basis of function, PRRs may be divided into endocytic PRRs or signaling PRRs. Endocytic PRRs promote the attachment, phagocytosis and destruction of microorganisms by phagocytes (such as neutrophils, monocytes, macrophages, mast cells, and dendritic cells). Signaling PRRs include the large families of membrane-bound Toll-like receptors and cytoplasmic NOD-like receptors. Their activation results in the production of co-stimulatory molecules, pro-inflammatory cytokines and chemokines (chemotactic or chemoattractant cytokines) that signal to other cells of the immune system and provide a link between innate and adaptive immunity. Importantly, each type of PAMP activates only a subset of PRRs resulting in an immune response corresponding to the type of pathogen with production of specific cytokines and chemokines, which in turn will help recruit and enhance the activity of innate and adaptive responses. I.2 ADAPTIVE IMMUNITY While the innate immune system is fast at controlling initial microbial invasion, pathogens may develop ways to evade innate immune mechanisms and cause infection. The adaptive immune system is called into action to fight pathogens that are not quickly cleared. Phagocytes ingest microbes that pass through the epithelial barrier. Macrophages in particular secrete cytokines that produce inflammation and attract lymphocytes. Following phagocytosis of microbes macrophages and dendritic cells act as Antigen Presenting Cells (APCs), which activate cells of the adaptive immune system. Similarly, adaptive responses strongly enhance antimicrobial activities of innate cells. Thus, innate and adaptive immunity may be considered as a cooperative, integrated system. The characteristics of adaptive immune responses are: (1) specificity and diversity: each response is exquisitely specific for a distinct molecule and the repertoire of receptors is extremely large (107-109), (2) the ability to learn: mechanisms are stimulated following exposure to infectious agents, (3) memory: each repeated exposure to a particular microbe leads to a defense response of a higher magnitude. Lymphocyte effector functions and their products (cytokines and antibodies) are the principal components of adaptive immunity. T lymphocytes and antibodies produced by B-lymphocytes recognize and respond to foreign molecules that are referred to as antigens. In fact, the T cell receptor and the antibody recognize only small parts of antigens, called epitopes. Adaptive immune responses can be broadly divided into two types of responses - namely humoral immunity and cell-mediated immunity - with each targeting different types of microbes. Humoral immunity is mediated by antibodies produced by B cells and principally targets pathogens in the extracellular milieu. Antibodies neutralize toxins or directly bind to pathogens, thereby inducing activation of the complement system, phagocytosis and killing of the microbe. Cell-mediated immunity involves T cells and is particularly efficient against intracellular pathogens (viruses and some bacteria), which evade humoral immunity by residing within cellular compartments. Subsets of T lymphocytes expressing the CD8 molecule are called cytotoxic T cells and directly kill virus-infected and tumor cells. Other subsets expressing the CD4 molecule are called helper T cells and enhance the killing activity of innate cells that have ingested microbes or help B cells produce antibodies. As will be described in a following section, CD4 helper T cells are the orchestrators of the immune response through their ability to differentiate into effector cells that promote an immune response tailored to a particular type of pathogen. The repertoire of antigenic epitopes is extremely vast and because the initial numbers of lymphocytes that are specific for a particular epitope are low (tens to a few hundred in the whole body), T and B cells need to circulate through lymphoid organs to enhance their chances of meeting their cognitive antigen. In steady state conditions, naïve lymphocytes (naïve meaning that they have not yet encountered their cognate antigen) enter lymph nodes via the blood circulation and scan many APCs. In the absence of their cognate antigen, lymphocytes exit the lymph node, enter the efferent lymphatic vessels leading to the blood circulation and the
cycle starts over. This continuous patrolling of secondary lymphoid organs is called lymphocyte recirculation. When an infection occurs, APCs take up antigens in infected tissues, change their phenotype to become mature APCs and migrate through afferent lymphatics towards the regional lymph node. This maturation process induces the expression of chemokine receptors, which serve to direct migration to the lymph nodes, and increases the expression of Major Histocompatibility Complex (MHC) molecules, on which peptide antigens are presented to T cells. Alternatively, or in parallel, antigens and sometimes even whole microbes directly take the lymphatic route to lymph nodes to be engulfed by specialized macrophages and dendritic cells (DC). CD4 T cells recognize antigens presented on MHC II molecules at the surface of APCs. CD8 T cells recognize antigens presented on MHC I molecules that are expressed on the surface of all nucleated cells of the body. For a strong adaptive response to take place, T cells need to receive 3 forms of messages from the APCs: (1) recognition of the cognate antigen presented on the MHC, (2) accessory signals which promote adhesion (integrins) and costimulation from activating molecules (such as CD80 and CD86) concurrently with a lack of inhibitory signals (such as CTLA4 and PD-1) on the surface of the APC, (3) secretion of stimulatory cytokines that will induce proliferation and differentiation of T cells into effector and memory T cells. Stimulated lymphocytes will proliferate to increase the number of cells that recognize the specific antigen. This clonal expansion enables the adaptive immune response to catch up and control highly proliferating pathogens. Following control of the pathogen, a phase of contraction occurs where the vast majority of the effector lymphocytes undergo apoptosis. A certain number of antigen-specific cells will persist and upon secondary exposure to the same antigen, these memory cells will produce a faster and stronger (both quantitatively and qualitatively) adaptive immune response. It is worth noting that the type and intensity of stimulation of PRRs by PAMPs and DAMPs strongly determines the phenotype of the APC, which in turn will orient the response of antigen-specific lymphocytes. B cells can produce different kinds of antibodies, and CD4+ helper T cells can specialize into diverse differentiation phenotypes that are adapted to specific types of pathogens. For example, Th1 cells produce interferon-gamma, which enhances the killing activity of macrophages through increased expression of inducible nitric oxide synthase. Other types of differentiation programs of CD4 T cells include Th2 cells, which stimulate IgE and eosinophil reactions; and Th17 cells, which contribute to strong neutrophilic responses. Finally, the ability of the immune system to discriminate foreign from self-antigens is one of the most striking properties of the mammalian immune system. Unresponsiveness or tolerance to self-antigens is regulated at multiple levels. These mechanisms include elimination of lymphocytes that express receptors specific for some self-antigens, inactivation of self-reactive lymphocytes, or suppression of these cells by the actions of regulatory cells. Failure in these regulatory mechanisms at any level may lead to autoimmunity. II DEFENSE MECHANISMS OF TEARS AND OCULAR SURFACE The eye contacts the external world at the ocular surface where it is exposed to a diverse microbial flora. Hence, an important function of the ocular surface is to form an effective barrier that prevents invasion by microorganisms. The ocular surface possesses multiple interconnected mechanisms of protection that concur to limit microbial invasion. These include anatomic and physical barriers (such as the eyelids and eyelashes, the tear film and the surface epithelium), chemical factors within the tears, and innate and adaptive immune cells that reside within these tissues. By the concerted action of these different mechanisms, immune protection at the ocular surface aims to limit the growth of microorganisms and promote tolerance against non-pathogenic microbes. But when strong measures are called for it is also capable of mounting a robust immune response in response to pathogenic invasion. II.1 INNATE IMMUNE DEFENSE MECHANISMS OF THE OCULAR SURFACE Innate immune defenses comprise a range of non-specific mechanisms, many of which are shared across mucosal surfaces. II.1.a Anatomic and physical barriers. Eyelashes limit the access of particulate matter and dust to the ocular surface. The continuous production of aqueous tears washes the ocular surface, and the physical action of the eyelids wipes it clean. This concerted action of washing and wiping helps to remove cellular debris, foreign particles and microorganisms through dilution and drainage, thereby diminishing the antigenic load on the ocular surface.
II.1.b Mucins. The tear film is composed of an outer lipid layer, middle aqueous layer and inner mucin layer. Although these constituents have traditionally been subdivided in 3 demarcated layers, more recent research shows that these layers are tightly interconnected. Mucins are a diverse family of high molecular weight, heavily glycosylated proteins that have the capacity to retain large amounts of water to lubricate the ocular surface. Soluble mucins are produced mainly by the lacrimal glands, gel-forming mucins are secreted by goblet cells, and membrane bound mucins constitute the glycocalyx of the corneal and conjunctival epithelium. Mucins of the glycocalix interlace with the secreted mucins to create a gradient of mucin in the tear film. This flexible layer coats the ocular surface, stabilizes the he tear film and allows a gradual transition between the hydrophobic epithelium and aqueous tears. The mechanical properties of mucins serve to trap and immobilize microorganisms and debris to facilitate their evacuation. During blinking, secreted mucins with entrapped microbes move easily over the glycocalyx and are shunted towards the nasolacrimal apparatus. Mucins also bind antimicrobial peptides and proteins can bind to mucins, which helps to improve their retention and distribution within tears. Some bacteria even have specific affinity for certain carbohydrate moieties on mucins. This enhances their entrapment and facilitates the activity of antimicrobial proteins. Species-specific variations in mucin glycosylation may influence not only the composition of the normal microbial flora, but also species-specific susceptibility to certain pathogens 1-3. Membrane-bound mucins of the glycocalix form a dense layer at the epithelial-tear film interface. This serves to distance the epithelium from the microbial flora and furthermore provides binding sites for neutrophils that constantly surveil the tear film. When pathogens invade the glycocalix mucins are cleaved at their base near the epithelial membrane to allow elimination of the invading microbe. Simultaneous intracellular signaling will render the epithelial cell more resistant to infection3,4. During inflammation pro-inflammatory mediators, such as IL-6, interferons and tumor necrosis factor-alpha, increase mucus secretion and alter glycosylation. Taken together, increased production, glycosylation and cleavage of the glycocalyx unfavorably changes the environment for pathogens and promotes their elimination. In chronic inflammatory conditions (such as in canine keratoconjunctivitis sicca), mucin stores in goblet cells become depleted 5,6. Topical therapy with cyclosporine A, among its multiple biological actions, helps restore mucin production in conjunctival goblet cells 7. II.1.c Antimicrobial proteins and peptides are primarily secreted by the ocular surface epithelia and lacrimal glands, with minor contributions from serum exudates and infiltrating immune cells. The tear film contains a plethora of such substances among which the most famous include lysozyme, lactoferrin, lipocalin, secretory phospholipase A2, cathelicidin and several types of defensins. Together, these molecules constitute a chemical barrier. Definitive proof of antimicrobial activity for certain substances is sometimes lacking since some peptides are present in too low quantity to be effective, but there are examples of synergistic activity which may facilitate their individual actions even at low concentrations 8. Lysozyme was the first tear protein to be identified and accounts for approximately 4% of the total tear protein concentration in normal dogs, but increases with inflammation 9. This enzyme disrupts bacterial and fungal cell walls, which are then no longer capable of maintaining a stable osmotic environment and undergo cell lysis. Lactoferrin’s main function is to bind and deplete iron from the tear film, which is required for microbial metabolism and growth. Lipocalin A inhibits iron uptake by microorganisms by blocking the microbial iron transport mechanisms. Each animal species seems to have it’s own panel of anti-microbial substances that are constitutively expressed in tears 10,11. There is furthermore a dynamic regulation of these substances whose presence and concentration can be modulated according to the need for anti-microbial protection. For example, the composition of tears during sleep (closed eye tears) is different than basal tears or reflex tears. And during infection or inflammation, secretion of certain substances, such as defensins and lysozyme for example, is increased in the tear film 9-12. Although the main activity of these substances is to limit the growth of microorganisms, they also serve several other functions. Several substances participate in the immune response by recruiting and activating immune cells, regulating different aspects of the inflammatory response, and promoting adaptive immunity. Lysozyme for example, enhances bactericidal activity of neutrophils and attenuates excessive inflammatory responses 13. Defensins promote immune cell chemotaxis and modulate cytokine production. Anti-microbial substances also increase epithelial defense mechanisms during infection and assist in wound healing by stimulating cell proliferation and angiogenesis 14. Lactoferrin for example can inhibit bacterial invasion or viral adhesion to epithelial cells, and increases apoptotic signals in infected cells 13. II.1.d Complement. Low levels of functionally active complement factors are constitutively expressed in tears. Abundant C3 and factor B suggests that activation via the alternative pathway (i.e. spontaneous hydrolysis of
C3) is the predominant mechanism. Their presence is thought to enable rapid activation of the complement cascade upon pathogen recognition. Activated complement helps clear infection through a multitude of different ways, such as (1) the generation of inflammatory factors and vasodilation, (2) chemotaxis of phagocytes (neutrophils and monocytes), (3) opsonisation and phagocytosis of antibody coated cells, (4) assisting antibodies to clear pathogens, (5) lysing bacteria and virus-infected cells 15. It is most active during sleep as their concentration is increased in closed-eye tears. To prevent unnecessary activation complement proteins are kept under tight control by a number of regulatory proteins. In addition, both lysozyme and lactoferrin have also been found to limit complement activation 8. II.1.e Immunoglobulins. The tear film possesses antigen-specific antimicrobial substances in the form of secretory IgA, IgG and IgM, with secretory IgA (sIgA) being the most abundant immunoglobulin in tears. Total immunoglobulin concentrations within dog tears vary with age, time of day, and show large day-to-day variations 16. Closed eye tears contain an increased amount of IgAs which may provide increased protection to the ocular surface when bacterial clearance through lid movement is reduced 17. IgA is produced by locally residing plasma cells that are located in the lamina propria of the conjunctiva and stromal space of lacrimal glands. IgAs are taken up by lacrimal gland acinar cells and conjunctival epithelial cells and traverse the cells by receptor-mediated transcytosis. Secretory IgA is released in tears by cleavage of the extracellular portion of the receptor on the apical side of cells. This portion of the receptor remains attached to the antibody to confer increased stability to the secreted antibody and also serves as a non-specific pathogen-binding site. Once secreted within the tear film sIgA can remain soluble or bind to mucins for an increased retention time and better distribution on the ocular surface. Secretory-IgAs do not kill microbes directly but exert their protective effect by neutralizing, aggregating and immobilizing pathogens, so as to facilitate their removal from the tear film during blinking. Other functions in immune homeostasis include neutralization of bacterial virulence factors, influencing the composition of the microbial flora, transport of antigens to antigen presenting cells (APC) in the subepithelial space and down-regulation of inflammatory responses 18,19. Secretory IgA that bind to the epithelial glycocalix immobilize commensal bacteria that form a biofilm occupying a strategic niche close to the epithelium. This hinders access of pathogenic bacteria, depletes the environment of essential nutrients and produces toxins that kill bacteria of non-related species. IgG is normally present in low concentrations, but increases during inflammation. This immunoglobulin is first produced during re-exposure to the same pathogen. In contrast to IgA producing plasma cells, plasma cells that produce IgG reside mostly in secondary lymphoid organs and the bone marrow. IgG travels through the circulation and its relative small molecular weight allows it to diffuse into tears alongside other serum proteins, particularly when there is inflammation. IgGs are most efficient in killing microbes through activation of classical complement mediated cellular lysis and facilitates phagocytosis by macrophages. IgM antibodies are the first antibodies to be produced after primary infection. They appear early in the course of infection. IgM antibodies are pentamers. Their higher molecular weight restricts them mainly to the circulation and limits their diffusion in normal tears. II.1.f Epithelial tight junctions. The outermost superficial epithelial cells of the conjunctiva and cornea possess tight junctions that seal cells together forming a barrier against microbial invasion. Their rapid turnover rate with regular shedding of the most superficial cells reduces the likelihood of corneal infection, as potentially infected cells will be sloughed off before there is time for the infection to spread to the deeper epithelial layers. II.1.g Pattern recognition receptors. Epithelial cells are equipped with a predetermined set of receptors that can immediately recognize the presence of microbes that contact them. Pattern recognition receptors (PRR) on epithelial cells and immune cells recognize conserved molecular patterns (microbe or pathogen-associated molecular patterns) that are shared by many microbes. The main class of PRRs are Toll-like receptors (TLR) and are expressed on cell surfaces and in intracellular endosomes. A total of 13 TLRs have been identified across all mammalian cells and appear to be highly conserved among different species. They are capable of recognizing different classes of pathogens, such as bacteria, viruses and fungi, with each receptor recognizing one or a limited set of Pathogen Associated Molecular Patterns. In humans, a total of 10 TLRs have been identified and all of them have been identified the ocular surface. Multiple TLRs were recently identified in the equine cornea and conjunctiva 20.
Stimulation of TLRs signals the earliest events in inflammation through release of inflammatory mediators, chemotaxis of immune cells and expression of adhesion molecules that enable the migration of immune cells in infected tissues. In doing so, TLRs trigger an immediate innate immune response and activate adaptive immunity 21-23. TLR expression is spatially regulated within the epithelium. Expression is generally restricted to basal cells and wing cells or intracellular compartments. For example TLR5, which recognizes bacterial flagellin, is present mostly in the basal epithelial layers. This restricts activation to situations where epithelial damage reaches the deeper layers. Further regulation of their activation is achieved by limiting the expression of certain receptors to the intracellular compartments. For example TLR2 and 4, that recognize lipopeptide and lipopolysaccharide respectively, are mostly present in the endosomal compartments of epithelial cells, which limits their activation to situations where bacteria have been phagocytized 22,24,25. Further regulatory mechanisms modulate the epithelial cell’s capacity to signal inflammation. For example only TLR5 stimulation by flagellin from pathogenic bacteria, such as Pasteurella multocida, induces the release of strong inflammatory mediators, whereas non-pathogenic bacteria do not transmit inflammatory signals. This mechanism enables ocular surface epithelial cells to produce measured responses upon TLR5 stimulation by harmless commensal bacteria versus pathogenic bacteria 26. Another class of PRRs, the cytoplasmic nucleotide-binding and oligomerization domain-like receptors (NOD-like receptors) are located within the cellular cytoplasm. They can recognize intracellular microbes and endogenous danger signals such as uric acid 27,28. II.2 ADAPTIVE IMMUNE DEFENSE MECHANISMS OF THE OCULAR SURFACE Eye associated lymphoid tissue (EALT) is composed of organized and diffuse lymphoid cells within the lacrimal glands (LGALT), conjunctiva (CALT), and drainage apparatus (LDALT). Organized lymphoid tissue is particularly noticeable on the bulbar surface of the third eyelid in dogs, and becomes hyperplastic upon stimulation. Different mucosal tissues throughout the body are linked functionally, via the traffic of immune cells, to comprise a physiological system called the mucosal immune system. The mucosal immune system has the challenge not only to defend the ocular surface from pathogenic invasion, but also to form useful partnerships with commensal microbes that are of benefit to the host. When the mucosal barrier fails and there is pathogenic invasion of the ocular surface tissues, conventional innate immune responses, relayed by the adaptive immune response can mount robust inflammatory response to clear the pathogen, but this is typically associated with more tissue damage and may bear permanent consequences for the ocular tissues. Much of our understanding of mucosal immunology is derived from studies on the gastrointestinal tract. Mucosal immune responses can be subdivided into sites of immune induction, where specific recognition of pathogens takes place, and immune effector sites, where immune effector cells protect the ocular tissues. II.2.a Mucosal inductive sites. The conjunctival epithelium that overlies lymphoid follicles in dogs and cats has typical features of mucosal inductive sites in that it has a reduced thickness, goblet cells are absent, and it contains specialized cells that morphologically and functionally resemble Membranous cells (M-cells) 29,30. M-cells phagocytize microbes and molecules at the surface to relay them to Antigen Presenting Cells (APCs) located at their basolateral surface. The antigenic material is engulfed and processed for presentation to lymphocytes. Signals received early in the encounter with the antigenic material are decisive in determining the functional activation of the APC. It influences their subsequent interactions with other cell types, and ultimately determines the outcome of the adaptive immune response. These signals are received not only by the nature of the presented microbe, but also by the local signaling milieu in which the APC resides. The normal conjunctiva contains a multitude of neurotransmitters and soluble factors, such as TGFβ, IL-10 and IL1R-antagosist that naturally bias the adaptive immune responses towards anti-inflammatory immune responses and favor the production of IgA antibodies. II.2.b Mucosal effector sites. After lymphocytes become activated, they will relocalize to the lacrimal glands, conjunctiva and nasolacrimal apparatus. B cells preferentially relocate to the lacrimal gland. Signals received in the mucosal effector sites induce IgA+ B cells to mature into long-lived IgA secreting plasma cells 31. T cells relocate in greater numbers to the conjunctival epithelium. CD8+T regulatory cells are found mostly in the basal layer of the conjunctival epithelium, whereas CD4+T helper cells are more prominent in the lamina propria. Regulatory T cells within the conjunctiva of normal subjects likely contribute to both tolerance to commensal
flora and suppression of autoreactive lymphocytes 31,32. Interspersed though the conjunctival epithelium are also dendritic cells, which are highly efficient at sampling and presenting antigenic material to lymphocytes. Theses cells extend long cellular processes through the epithelium that reach the ocular surface and constantly sample the external milieu for antigenic material. II.3 CLEARANCE OF PATHOGENS In contrast to cellular interactions during immune homeostasis, which are mostly aimed at preventing infection and containing unwanted inflammatory reactions, pathogenic invasion unleashes inflammation and profoundly changes the local signaling milieu. It is accompanied by vasodilation, expression of adhesion molecules and chemoattractants (chemokines) that recruit immune cells to the site of infection. Innate immune cells are the first responders to infection. This includes neutrophils, macrophages and natural killer cells that phagocytize or kill microbes and infected cells. Macrophages also function as APCs that present antigens to T and B cells to mount an antigen-specific adaptive immune response. Alterations in the local signaling milieu provide strong stimulation of APC that adopt an inflammatory phenotype and influences their subsequent interactions with T and B cells to generate immune effector cells that are specifically targeted to the infecting pathogen. After clearance of infection the adaptive immune system also displays long term memory that enables it to respond more quickly upon subsequent encounter with the same pathogen. While inflammation and immune reactions are aimed at ridding the tissues of pathogens, they can potentially also cause significant tissue damage. In particular, the effector function of the innate immune system is less specific and causes more bystander cell death and tissue destruction. Inflammatory processes plausibly account for much of the cytopathology that is noted on the ocular surface and in the lacrimal glands, regardless of the inciting etiology. II.4 DRY EYE AND ASSOCIATED OCULAR SURFACE INFLAMMATION Tear film disorders in dogs have traditionally been classified into quantitative disorders (synonymous of aqueous deficiency) and qualitative disorders (lipid and mucin abnormalities). Keratoconjunctivitis sicca (KCS) in dogs can be caused by a variety of factors, including congenital tear deficiency, breed predisposition, drug toxicity, irradiation, neurogenic disease, iatrogenic tear deficiency, infectious adenitis (e.g. canine distemper), metabolic disease, trauma to the eye and orbit, chronic blepharoconjunctivitis, and immune-mediated lacrimal inflammation 33,34. Lymphoplasmacytic infiltration and consequently immune-mediated destruction of the lacrimal glands, probably represents the major cause of canine KCS 3,4,35-38. Although the early inciting events that initiate immune-destruction of the lacrimal glands remain elusive, they probably involve a combination of different factors such as genetic predisposition, environmental stress, lacrimal and/or immune dysregulation, and hormonal imbalances. A female predisposition has been identified in West Highland White Terriers and English Cockers, whereas male Shih Tzus and Cavalier King Charles Spaniels are predominantly affected 1,5,6, underscoring the importance of different hormonal factors in different breeds. KCS is typically associated with an increased bacterial burden at the ocular surface, which develops consequently to the altered composition of the remaining tears and an insufficient washing of the ocular surface 39. In the majority of dogs the only clinical signs are lacrimal gland hyposecretion and ocular surface inflammation. However, up to 40% of affected dogs may show concurrent immune dysregulation in the form of circulating rheumatoid factor antibodies, lymphocytic infiltrates in other glands (e.g. salivary glands) and hyper-gamma-globulinemia 36,40. Although the early inciting events that lead to immune destruction of the lacrimal gland in canine patients remain unknown, several animal models have unraveled early mechanisms that lead to Dry Eye Disease. The maintenance of a healthy pre-corneal tear film is regulated by an integrated system comprising the lacrimal glands, the ocular surface epithelia, the eyelids, and the nerve endings that connect them. Dysfunction of any component within this system can destabilize the tear film and lead to alterations in volume, composition, or distribution of tears over the ocular surface. Regardless of the inciting cause, tear film abnormalities are always accompanied by redness, irritation and inflammation, which in itself contributes to maintain a self-perpetuating cycle of inflammation and tissue destruction 41-43. In experimental models of Dry Eye Disease, corneal desiccation or increased tear osmolarity alone are sufficient to disrupt tight junctions between ocular surface epithelial cells, induce epithelial squamous metaplasia and cell death, reduce goblet cell numbers and mucin production, and initiate inflammation 44,45. Damaged epithelial cells and nerve endings produce inflammatory cytokines and lipid mediators that increase vascular permeability and upregulate vascular adhesion molecules to facilitate the invasion and activation of immune cells. Cellular components released by dying cells are
processed by APCs in an inflammatory context, which in turn activate autoreactive T cells. Autoreactive T cells either fuel the inflammatory cycle at the ocular surface or provide help to autoreactive B cells to support their differentiation into antibody producing plasma cells. Inflammatory cytokines may also impede neural transmission at the ocular surface. The neuronal reflex signaling to the lacrimal gland is interrupted and the unstimulated lacrimal gland atrophies and sheds cellular debris into tears. This provides further stimulation to autoreactive T cells that relocate to the lacrimal gland and exacerbate lacrimal gland destruction 42 46. As the pathogenesis of the dry eye disease progresses, loss of tear production that is initially attributable to an inflammatory process and/or sensory isolation of the lacrimal gland, ultimately culminates into permanent tissue damage III IMMUNE PRIVILEGE OF THE CORNEA Corneal transparency is essential for good vision. This state of corneal transparency relies on a number of anatomic, physiologic and immunologic properties that work in concert to keep the cornea clear under normal conditions. Although the conjunctiva and cornea are anatomically proximate and are bathed in the same tear fluid, immune homeostasis in these tissues is distinctly different. Within the cornea, there are several tissue specific immune regulatory mechanisms that concur to prevent or modulate inflammatory reactions and thereby prevent potentially destructive immune responses. These mechanisms have been broadly divided into corneal immune privilege and corneal angiogenic privilege, and although they are here explained separately, there is considerable overlap and interdependency between these two mechanisms. III.1 ANGIOGENIC PRIVILEGE OF THE CORNEA Although blood and lymphatic vessels are prevented from developing in the cornea during embryogenesis, the maintenance of an avascular cornea during life is more than a simple anatomic particularity. Corneal avascularity during normal conditions is maintained by an active process, through the balanced production of low levels of angiogenic factors that are tightly regulated by an excess of inhibitors of angiogenesis. Angiogenic molecules include vascular endothelial growth factors (VEGFs), basic fibroblast growth factors (bFGFs), and matrix metalloproteinases (MMPs). Inhibitors of angiogenesis in the cornea include angiostatin, endostatin, thrombospondins, pigment epithelial derived factor (PEDF), and soluble vascular endothelial growth factor receptors (sVEGFR1-3) 47-50. Although all these factors are involved in local regulation of corneal vascularization, the interplay between VEGFs (activators) and soluble VEGFRs (inhibitors) likely plays a predominant role in maintaining the cornea avascular 47,48,51. Pathological conditions such as epithelial loss, hypoxia or severe inflammation can modify this balance of pro and anti-angiogenic factors in favor of vascularization. Typically both blood and lymphatic vessels will sprout from the limbal vasculature to co-migrate into the cornea, although only blood vessels are visible by slit lamp biomicroscopy. The immune system actively participates in corneal vascularization as VEGF-A also serves to chemoattract macrophages to the cornea who, once in the cornea will, release several isoforms of VEGFs to fire up the stimulus for both vessel ingrowth and the recruitment of additional macrophages 51,52. Macrophages can also align to form tubular structures thereby actively participating in the formation of new lymphatic vessels 53. In experimental animal models depletion of macrophages prior to corneal grafting reduces corneal vascularization and improves the survival of corneal grafts 51,53. The role of VEGF is also being investigated in veterinary patients. The normal canine cornea constitutively expresses VEGF and VEGFR at the level of the epithelium and endothelium 54,55.Vascularized corneas show an increased level of VEGF expression thoughtout all corneal layers. 54.. Equine deep stromal abscesses that contain fungal hyphae show slow healing responses and delayed vascularization. This has been correlated with low VEGF-A expression, which is suggested to contribute to the delayed vascularization 56. Manatees are an exception in the animal kingdom, in the sense that they naturally have vascularized corneas, apparently in the absence of corneal disease or inflammation 57. This particularity has been attributed to a genetic deficiency in soluble VEGFR1 47. Corneal vascularization is detrimental not only to optical clarity and vision, but also alters the nature of the adaptive immune response that is induced in response to corneal antigens. In the normal cornea, locally residing APCs are kept in an immature state. Although they can adopt an activated (mature) phenotype during corneal inflammation, there is no pre-existing route allowing their egress out of the cornea. APCs may leave the cornea through migration via the anterior chamber, which is filled with immunomodulatory factors such as TGF-2, alpha-melanocyte stimulating hormone (MSH) and vasointestinal peptide (VIP) that alter the biologic
behavior of APCs that contact them and thereby modify the nature of the resulting immune response. Research has shown that the presence of corneal lymphatic vessels is associated with rejection of corneal grafts and conversely, selective and temporary inhibition of lymphatic vessel formation improves graft survival 52,58. With the ingrowth of lymphatic vessels, corneal APCs are provided with a route for migration out of the cornea to the regional lymph nodes where they are able to induce an immune response to corneal antigens. Corneal blood vessels, on the other hand, enhance the delivery of immune effector cells such as activated lymphocytes and additional APCs. Together corneal blood and lymphatic vessels facilitate the trafficking of immune cells in and out of the cornea thereby eroding corneal immune privilege. Given the importance of lymphatic vessels in the initiation of an adaptive immune response, novel therapeutic strategies that inhibit lymphatic vessels or induce their regression are actively being researched. III.2 CORNEAL IMMUNE PRIVILEGE The immune privileged status of the cornea relies on several mechanisms that cooperate to reduce the likelihood of inflammatory reactions within the cornea. These mechanisms can be summarized into (1) the overall low antigenicity of the corneal tissue; (2) the paucity and low reactiveness of locally residing APCs; (3) local immune suppression; and (4) systemic immune deviation. The cornea shows little antigenicity in comparison to other tissues in the body. Corneal cells express few Major Histocompatibility Class I (MHC I) molecules. The primary immunogenicity of corneal grafts resides within the epithelium. Split thickness corneal grafts (stroma plus endothelium) that are covered with host epithelium show increased graft survival 59. Furthermore, the corneal endothelium expresses several molecules that induce T cell apoptosis 60 and inhibit NK cell activity 61,62. The normal cornea contains a resident population of APCs that are normally kept in a quiescent state. Various subsets of APCs can be found within the basal epithelium and throughout the stroma. The density of APCs decreases from the periphery towards the center of the cornea. In the peripheral cornea, APCs with a more mature phenotype connect to one another via long dendrites, facilitating cell-to-cell communication in case of an inflammatory event 63. The central cornea however contains surprisingly low numbers of APCs with an immature phenotype. This decreases their reactiveness and facilitates local tolerance to antigens 64. The cornea expresses several molecules capable of modifying the activity of invading immune effector cells. FasL can induce apoptosis of activated T cells and neutrophils 65. Programmed death ligand (PDL-1) can inhibit T cell proliferation, secretion of pro-inflammatory cytokines and induce apoptosis 66. Non-classical MHC class Ib molecules and Macrophage Inhibitory Factor (MIF) inhibit NK cell activity 61,62,67. Complement regulatory proteins in the cornea and in the aqueous protect the cornea from complement-mediated cell lysis and excessive inflammation 68. The endothelium is furthermore in direct contact with a multitude of immunomodulatory factors that are present in the aqueous humor. These factors possess anti-inflammatory and immunosuppressive properties that are capable of modifying the biologic behavior of immune cells that enter the eye. The survival of corneal grafts relies at least in part on the development of a systemic immune deviation that is induced through the anterior chamber (Anterior Chamber Associated Immune Deviation or ACAID). Experimental interference with immune mechanisms that sustain ACAID reduces graft survival and conversely, induction of ACAID by injection of corneal alloantigens prior to corneal transplantation increases graft survival 69-71. III.3 CORNEAL TRANSPLANTATION Corneal transplantation in humans enjoys a high success rate and is one of the most commonly performed tissue transplants. Over 185 000 corneal transplants are performed every year throughout the world 72.. This surgery is performed routinely without prior HLA-matching (human equivalent of MHC) and with minimal or no immunosuppression. Successful corneal transplantation in a human patient was first performed in 1905, over 100 years ago, when there was scarcely any knowledge of the immune system’s recognition of self and non-self tissues. Successful transplantation of other organs that are subjected to normal immune responses would have to wait another 60 years until the development of potent immunosuppressive drugs. The high success rate of corneal transplantation in a normal corneal recipient bed is undoubtedly associated with corneal immune privilege. Interference with any single mechanism that contributes to the immune privileged status of the
cornea, such as inducing corneal inflammation, stimulating corneal vascularization, or interference with mechanisms that sustain ACAID, results in decreased graft survival 51,58,69-71. Corneal transplantation techniques are constantly evolving to accommodate the growing demand for donor tissue and improve surgical outcome. Lamellar keratoplasties, whereby only the diseased tissue is replaced, are gaining popularity in human medicine. Descemet membrane endothelial keratoplasty (DMEK), whereby only Descemet’s membrane and the endothelium are transplanted, enjoy a 99% success rate 2 years after grafting, compared to a 82% success rate with penetrating keratoplasty 73. The extraordinary high acceptance rate of this technique can be attributed to multiple factors, including selection for patients that have no pre-existing corneal inflammation, reduced amount of transplanted tissue, refined surgical techniques with suture-less surgeries, inherent low antigenicity of the corneal endothelium, and direct contact of the transplanted tissue with immunosuppressive factors in the anterior chamber. Corneal graft acceptance in veterinary patients is generally far less successful. First, there are marked differences in acceptance rate of corneal grafts between species. Corneal transplants in cats are better tolerated and success rates seem to equal those in human subjects 74. Immune rejection is much more common in dogs and horses 75. Species differences can be attributed at least in part to differences in the genetic make-up of the immune system. Such differences have also been noted between animals of the same species. For example, in murine models of corneal transplantation donor corneas are harvested from C56Bl/6 mice and grafted onto Balb/c recipient mice, as the latter seem to better tolerate corneal transplants. C56Bl/6 mice are more prone to inflammation and reject all corneal allografts from Balb/c donor mice. Similar intra-species differences are also noted in veterinary patients. For example, superficial chronic corneal erosions in dogs often induce some amount of corneal vascularization. Boxers, however, are prone to develop excessive vascularization more frequently. Secondly, most corneal grafts in veterinary patients are performed for tectonic purposes on vascularized and inflamed corneas 75. Under these circumstances mechanisms of corneal immune privilege no longer prevail and immune rejection is the norm. The same drop in success rate is noted in human patients that have corneal inflammation, corneal vascularization, viral keratitis, or a history of previous corneal rejection 76. Even in the absence of pre-existing corneal conditions, the excellent success rate in people slowly drops over time to reach a 50% success rate 15 years after transplantation 77. Although the exact reasons for this gradual drop are not exactly known it is likely that small day-to-day insults or low-grade inflammatory or infectious conditions, accompanied by neovascularization, lie at the basis of graft failure. All these conditions erode corneal immune privilege and favor an inflammatory immune response, which eventually culminates in graft failure. Regardless of species differences, the same therapeutic strategies that have led to improved surgical outcomes in human patients could also be successfully applied to veterinary patients. These include appropriate case selection, early surgical intervention, refined surgical techniques, and probably combined suppression of the immune system and inhibition of corneal vascularization. Experimental studies have demonstrated that lymphangiogenesis (formation of lymphatic vessels) is more detrimental to graft survival than hemangiogenesis (formation of blood vessels)58. Promoting graft survival by inhibiting lymphangiogenesis is a new concept in human corneal transplantation. Several possibilities to suppress actively outgrowing corneal blood and lymphatic vessels are being pursued, including topical application of vascular endothelial growth factor (VEGF) inhibitors78-80, corticosteroids81, and antisense oligonucleotide eye drops against insulin receptor substrate-1
(Aganirsen eye drops - an intracellular signaling molecule that inhibits excess VEGF and Il-1β expression) 82,83, however these methods cannot regress mature corneal vessels. For preexisting corneal blood vessels, preclinical approaches like anti-VEGF-B antibody fragment have been used84, fine needle diathermy coupled with anti-VEGFs85, and corneal crosslinking, which reduces both mature. Blood and lymphatic vessles86. III.4 NON-ULCERATIVE IMMUNE MEDIATED KERATOPATHIES IN VETERINARY SPECIES There are numerous examples of presumed immune-mediated keratopathies that affect veterinary patients. These are conditions where corneal disease is either initiated or maintained by an abnormal activity of the immune system. The target antigen to which the immune response is directed at, as well as the molecular and cellular events that lead to the development of the aberrant immune response, remain however mostly unknown. Most of the time it is presumed that a self-antigen, or a modified self-antigen, is the target of the immune attack. Because self-antigens are an integral part of the body, the immune-attack often takes on a recurrent or chronic
nature. Typical examples such diseases are equine immune-mediated keratopathies (IMMK) or canine chronic superficial keratitis (CSK). Feline stromal keratitis is also a condition that is maintained by an exaggerated and inefficient immune response, but in contrast to the previous examples, the immune reaction is directed against viral antigens that are retained in the corneal stroma. CSK is a progressive bilateral fibrovascular infiltration and inflammation of the cornea. The main inflammatory cell type in affected corneas are CD4+ T lymphocytes. Macrophages, plasma cells and neutrophils are recruited to the site of inflammation probably as a consequence of local production of inflammatory cytokines. Dogs with early signs of CSK were also shown to have increased serum levels of VEGF87. Local secretion of the inflammatory cytokine IFNγ by infiltrating CD4+ T cells increases the expression of MHC II for the purpose of antigen presentation88,89. Multiple lines of evidence support an immunogenetic predisposition exacerbated by environmental factors. This is supported by the inability to identify an underlying infectious cause 90,91, a strong breed predisposition, a positive response to immunosuppressive therapy and an increased disease incidence at higher altitudes92-94. Although CSK can sporadically be encountered in any breed of dog, it has a particular high incidence in German shepherds and greyhounds. Leukocytes of affected dogs were shown to be hypersensitive to corneal antigens, and the condition can be controlled by topical immunosuppressants 95,96. Analysis of the German shepherd population in Northern Europe showed a strong association between a particular MHC II haplotype, DLA-DRB1*01501/DQA1*00601/DQB1*00301, and disease development. Dogs that are heterozygote for this MHC II haplotype are 2.7 times as likely to develop CSK whereas homozygosity is associated with an 8-fold increased risk for disease development 97. Single nucleotide polymorphism in the regulatory region that controls transcription of the MHC II genes has also been associated with increased disease incidence. Polymorphism in this region could affect the level or pattern of MHC expression 98. MHC II molecules play an important role not only in antigen presentation to T cells, but are also important in the selection of the T cell repertoire during T cell development. It is therefore conceivable that overexpression of an abnormal MHC class II molecule could predispose to the development of CSK. CSK is exacerbated in dogs residing at high altitude where there is increased ultraviolet radiation 92-94. UV radiation may trigger a chain of events that can damage DNA, RNA, and the extracellular matrix 99. Despite these multiple lines of research, we still fail to understand the exact molecular events that lead to development of this condition. Treatment of CSK typically aims at limiting all avenues implicated in the disease process. With the present state of knowledge, this means limiting UV exposure and suppressing the immune response. To date, the only study evaluating the effect of UV-blocking contact lenses could not demonstrate a beneficial therapeutic effect, but study results might have been affected by owner compliance and alterations in the UV-blocking capacity after contact lens cleaning 100. The other therapeutic avenue is to suppress the aberrant immune response. This is usually achieved by the initial combined use of topical cyclosporine A and corticosteroids at a tapering dose. Beta-radiation (strontium 90β plesiotherapy) and soft X-rays are beneficial in those patients that are difficult to control by medical means only 101,102. Irradiation may exert an anti-inflammatory effect through induction of apoptosis in inflammatory cells and inhibition of vascularization 103,104. Feline stromal keratitis is an immune reaction against viral antigens that are either retained or continuously expressed in the corneal stroma 105,106. The condition is characterized by a progressive inflammation, edema, and vascularization of the corneal stroma, with or without involvement of the overlying epithelium. While this disease has been associated with feline herpes virus-1 infection (FHV-1), most of the stromal pathology is induced by an exaggerated immune response, without obligate involvement of active viral replication. Stromal keratitis usually does not develop after primary FHV-1 infection, but may develop in some cats that have experienced multiple episodes of viral reactivation and shedding in the cornea. It is not known why some cats can harbor active virus in their corneas in the absence of clinical disease, while others will develop recurrent epithelial disease or stromal keratitis 107. It is possible that the development of stromal keratitis may be influenced by differences in viral strains, the genetic make-up of the infected animal, and environmental factors such as stress 108,109. Compared to the overall frequency of FHV-1 related ocular disease, stromal keratitis develops relatively rarely, but it may have a significant impact on corneal transparency, lead to gradual visual deterioration, cause recurrent or persistent ocular discomfort, and is often frustrating to manage. Some of the immunologic events that lead from herpetic infection to stromal keratitis have been elucidated in animal models. Stromal keratitis can reliably be induced in experimental cats if local immunosuppression, through subconjunctival steroid injection, is induced prior to ocular infection with FHV-1 110,111. Suppression of the immune response is associated with an increased viral burden, increased cellular damage, delayed viral clearance, and acquisition of viral antigens by the corneal stroma. When the immune system recovers it is
overwhelmed by viral antigens and dying cells. The response that is mounted under such conditions is accompanied by massive inflammation and non-specific tissue destruction, which ultimately results in scarring. Stromal keratitis has been extensively studied in rodent animal models, as the disease can conveniently be induced after primary infection with herpes simplex virus type 1 (human equivalent of FHV-1) 112,113. While there are bound to be differences between mice and cats, the murine models are nevertheless useful in unraveling some of the molecular and cellular events involved in disease development. In mice, primary infection is followed by superficial ulceration in an initial phase of the disease. Stromal keratitis only develops after most of the virus has been cleared from the ocular surface and the epithelium has re-epithelialized. Stromal keratitis is at its core an inflammatory reaction that is initiated by viral particles resulting in the release of a multitude of chemokines that attract immune cells to the cornea, and cytokines that activate these incoming cells. Natural killer cells, macrophages and neutrophils that are recruited to the cornea amplify the inflammatory process through release of additional cytokines and chemokines. Neutrophils are mostly inefficient at controlling viral replication, but are responsible for inflicting most of the corneal damage and scarring. CD4+ T helper cells play a major role in orchestrating the recruitment of other cell types and maintaining a chronic inflammatory state 112-114. Different subsets of CD4+ T cells have been implicated in stromal keratitis. Virus-specific CD4+ T cells with a Th1 or Th17 phenotype induce corneal disease. Bystander activated CD4+ T cells of irrelevant specificity (i.e. that do not recognize viral or corneal antigens) may become activated by the inflammatory response in the lymph nodes and then relocate to the cornea 115,116. These bystander T cells secrete cytokines and increase the severity of the pathology without contributing to viral control. Virus-specific CD4+ T cells that cross-react with corneal antigens could be induced with certain strains of HSV were identified in rodent models of stromal keratitis 117, but not in human patients 118. Neovascularization of the cornea is a prominent feature in the pathogenesis of herpes stromal keratitis. Infected corneas produce increased amounts of vascular endothelial growth factor (VEGF) and reduced levels of soluble VEGFR1, inducing a disequilibrium in the balance of angiogenic and anti-angiogenic factors and creating a permissive environment for the ingrowth of blood and lymphatic vessels into the cornea 119,120. The ingrowth of lymphatic vessels provides a conduit for the egress of corneal APCs to migrate to regional lymph nodes and induce lymphocyte activation. Corneal blood vessels provide enhanced access of activated CD4+ T cells to the cornea. Inhibition of neovascularization significantly improves stromal keratitis in experimental rodents and is currently investigated as a potential therapeutic avenue 121-123. Management of stromal keratitis is complicated by the fact that the immune cells that should eliminate the virus, are not only inefficient, but contribute or cause corneal disease. However, suppressing the immune response may potentially exacerbate viral replication. Treatment directed against the virus is helpful in some patients, but often insufficient to control the inflammatory damage to the cornea. The real difficulty in feline patients lies in the fact that they are often presented late in the course of disease, with established corneal lesions and permanent corneal vascularization. Ideally, therapy would have to target each individual avenue, namely FVH-1, the aberrant immune reaction and corneal vascularization, while at the same time presenting the least amount of undesirable side effects. IV OCULAR IMMUNE PRIVILEGE, ACAID, AND ITS THERAPEUTIC POTENTIAL The privileged relationship that the eye enjoys with the immune system was first discovered 2 centuries ago by the Dutch ophthalmologist van Dooremaal (1873). In his studies on the etiology of cataract, he placed skin grafts in the anterior chamber of dogs and observed that they experienced a prolonged survival time compared to other sites of transplantation. Seventy-five years later, Medawar (1948) confirmed this observation and further noted that skin grafts survived in the anterior chamber as long as new vessels did not grow into the transplanted tissue. He named this phenomenon “immune privilege” and originally thought it to be synonymous with “immunological ignorance”, namely the host’s immune system had not recognized the transplanted tissue, which was inaccessible because of the lack of direct lymphatic drainage in the eye and the presence of a blood aqueous barrier 124. It took another thirty years before Kaplan and Streilein showed that this lack of tissue rejection in the anterior chamber was not only due to passive immunological ignorance, but that a deviant systemic immune response was mounted to antigens placed in the ocular environment. These findings were extended to other sites in the eye, including the vitreous and the subretinal space where survival of transplanted cells was also prolonged 125. Current thinking holds that ocular immune privilege is an active mechanism providing the eye with immune protection, while avoiding the damaging effects of excessive inflammation. This particular relationship between the eye and the immune system may serve to preserve the delicate tissues of the eye that are essential for vision, and also have limited regenerative capability 126,127. While the concept of immune privilege seems simple, the mechanisms that sustain this privileged status are numerous and complex. At present it is thought that 3 major mechanisms contribute to the immune privileged status of the eye: (1) the
presence of anatomical, cellular, and molecular barriers that shield the eye; (2) the immune suppressive intraocular microenvironment; and (3) eye-derived immunological tolerance, or so-called Anterior Chamber-Associated Immune Deviation (ACAID). Together these mechanisms aim to restrict the access of pathogens and damaging immune cells to the eye and limit inflammation that may ensue following the induction of a conventional immune response. In the normal healthy eye, it is thought that the presence of efficient blood-ocular barriers and local immunosuppression are the two main mechanisms that keep the eye protected from inflammation. Systemic immune regulation is activated when the blood-ocular barrier is breached and additional measures are needed to protect the eye from damaging immune responses. The blood-ocular barriers provide the first line of protection to the eye. This is composed of tight junctions between the epithelial cells of the posterior iridal epithelium, non-pigmented ciliary body epithelium and retinal pigment epithelium, as well as in the vascular endothelium of the iridal and retinal vessels 128. The tight junctions between cells separate the immune privileged eye from the general circulation. This results in reduced access of inflammatory cells and solutes to the eye, but also regulates the passage of selected immune cells at certain checkpoints 129. Immune cells that exit the fenestrated vessels of the ciliary body and choroid can be deleted or granted selective passage through the ocular epithelia. After retinal damage for example, immune cells first enter the eye through the ciliary body region, suggesting that this area is not entirely impermeable to cell trafficking but serves as a selective gate for the trafficking of immune cells 130. The epithelial cells of the ciliary body express multiple immunoregulatory factors that can block innate immune functions; induce T cell anergy, deletion or suppression; or convert activated T cells into a regulatory phenotype and induce their expansion 126,131,132. The expression of FasL on ciliary body and RPE cells induces preferential apoptosis of Th1 cells, which express the highest levels of Fas, rather than inducing general T cell deletion. Inhibitory factors expressed on the RPE can also inhibit T cell activation, proliferation and cytokine production, or convert T cells into a regulatory phenotype. The inner blood-retinal barrier, located within the retinal vascular endothelium, is on the contrary a true barrier system that solely relies on tight junctions to seal the access of immune cells into the eye and does not possess immune-skewing properties. Breakdown of the inner blood retinal barrier, and passage of leukocytes through the vascular endothelium, is always associated with disease and can be clinically seen as retinal vasculitis or cuffing 129. A second line of protection to the eye is provided by soluble and cell bound immunosuppressive factors within the intraocular environment that are capable of suppressing activated immune cells and inflammatory molecules that could have gained access to the eye 126. The anterior chamber contains a multitude of factors that possess anti-inflammatory or immunosuppressive properties capable of influencing both innate and adaptive immune responses. Collectively, these factors act to suppress inflammatory responses within the eye, inhibit the activity of immune cells that can cause non-specific cellular damage, while still allowing for immune surveillance. Innate immune functions, such as nitric oxide production, NK cell and neutrophil activity are inhibited by
calcitonin gene related peptide (CGRP), macrophage migration inhibitory factor (MIF), α -melanocyte-
stimulating hormone (α-MSH) and FasL. In the eye, there is constant low-level activation of the complement cascade, which could potentially predispose the eye to inflammatory insults. However, complement activity is tightly regulated by soluble and membrane bound complement regulatory proteins that serve to prevent inadvertent inflammatory reactions 68,133,134. It is believed that locally residing macrophages maintain their capacity to phagocytize pathogens and cell debris within the eye, but are inefficient in recruiting other immune
cells that could cause inflammation 135. The activity of APCs in the eye is regulated by α-MSH, vasoactive
intestinal peptide (VIP), CGRP, TGF-β2, and trombospondin-1 (TSP-1). TGF-β2 and TSP-1 are both essential factors for the induction of ACAID, which will be discussed in a later section of this text. In response to a stimulus, APCs that have been in contact with aqueous humor produce less inflammatory cytokines, less
costimulatory molecules, and secrete the immunomodulatory cytokines TGF-β1 and IL-10. T cells that contact aqueous humor show reduced proliferation, cytotoxicity and production of inflammatory cytokines. They also
start producing the immune regulatory cytokine TGF-β and change their functionality to become regulatory T cells (Treg). Therefore, aqueous humor is capable not only of suppressing inflammatory T cells but also turn these cells around to themselves participate in immune regulation. It must be noted that although these soluble factors were first identified and described in the anterior chamber, several factors were subsequently identified in other parts of the eye, such as in the vitreous and sub-retinal space, indicating shared mechanisms leading to
immune privilege in other ocular compartments 135. Epithelial cells in the eye also express several cell surface molecules with immunosuppressive properties 60,136. Programmed death ligand-1 (PDL-1) is constitutively expressed on endothelial cells of the cornea, some stromal cells, iris–ciliary body epithelium, neural retina, and can be induced in other cell types during inflammation. Multiple cells within the eye also express membrane bound FasL. Together PDL-1 and FasL induce apoptosis of effector T cells, thereby suppressing the effector phase of the immune response 137-139. Other cell surface molecules such as CD86 and CTLA-2α are capable of inducing T regulatory cells 140,141. Lastly, should there be damage to the ocular tissues or introduction of antigen into the eye, the eye is capable of orchestrating a systemic regulatory immune response, also called systemic immune deviation or systemic tolerance. This immune deviation, of which the classical example is ACAID, is characterized by the production of antigen-specific regulatory T cells (Treg) that down-regulate immune responses that inflict uncontrolled tissue injury, while preserving mechanisms that provide immune protection without causing bystander cell death 126,127. To summarize, the collective activity of multiple factors within the eye inactivate inflammatory cells and locally induce Treg cells that maintain the unique immunologic properties of the ocular environment. They also lay the foundation for the generation of systemic immune deviation to antigens that are presented in the eye. Immune privilege should not be misinterpreted as being a total suppression of all immune responses, but rather is a fine-tuning of the immune response to suit the needs of the eye. IV.1 INDUCTION OF SYSTEMIC IMMUNE DEVIATION, OR SO-CALLED ANTERIOR CHAMBER IMMUNE DEVIATION (ACAID) Studies in the field of ACAID have revealed that immune deviation to antigens presented in the eye results from complex cellular and molecular interactions that involve the eye, spleen, thymus, nervous system, and is furthermore dependent on stimulation from light 126,127,142. So far, all experimental animals that have been studied (mostly rodent models and experimental primates) were shown to develop ACAID, suggesting that mechanisms of ACAID may be an evolutionary adaptation that is common to multiple species. Although ACAID is probably one of the best studied aspects of ocular immune privilege, it must be noted that immune privilege is not limited to the anterior chamber but probably extends to multiple, if not all, ocular compartments. In the experimental setting, ACAID is induced after injection of antigen in the eye. Locally residing or infiltrating APCs that enter the eye come in contact with the immunosuppressive factors of the eye, which change their biological behavior, phenotype, and also change the nature of the resulting systemic immune response. In comparison to mature APCs that induce conventional immune responses, ACAID-inducing APCs show decreased expression of molecules required for generating inflammatory responses, in combination with increased expression of anti-inflammatory molecules, and a unique pattern of chemokine receptors, which will allow them to leave the eye and migrate to lymphoid organs 143. Antigen processing and migration out of the eye is a slow process that can take up to 3 days. Because the normal eye lacks lymphatic drainage, APCs leave through the trabecular meshwork to join the systemic circulation and relocate to the thymus and spleen. How many APCs migrate out of the eye after antigen injection is not known but in the experimental setting as few as 20 ACAID-inducing APCs suffice to mount systemic immune deviation 144. This is made possible through an amplification process of the immune response that involves the recruitment of additional APCs, and other immune cells, that will amplify the ACAID signal. After egress out of the eye one subset of ACAID-inducing APCs will migrate to the thymus to interact with immature T cells, to induce the generation of CD4-CD8- invariant NKT cells (iNKT). iNKT cells are a subset of innate immune cells that express markers of NK cells and possess a conserved T cell receptor. They are important in ACAID induction through their rapid production of large quantities of IL-10 in early cell interactions, thereby driving the immune response towards tolerance. The newly generated iNKTs migrate to the spleen to take part in further cellular interactions. Another subset of eye-derived ACAID-inducing APCs migrate directly from the eye to the white pulp areas of the spleen, where interactions with multiple other immune cells take place. In contrast to conventional immune responses, the eye-derived APCs do not migrate to T cell rich areas, but rather accumulate in the marginal zone
(periphery) of the white pulp. The particular pattern of migration is directed by the expression of a set of chemokine receptors that is distinct from those on conventional APCs. Interactions between eye-derived APCs, marginal zone B cells, iNKT cells and CD4+T cells in the marginal zone of the spleen eventually lead to the generation of several populations of Treg cells. These Tregs are the effector cells that will inhibit cell-mediated immunity specific for the antigen presented in the eye. ACAID induces at least 2 types of antigen-specific Tregs. Afferent CD4+ Tregs can prevent the induction of a new immune response, but have no effect on an already established immune response. And efferent CD8+ Tregs capable of suppressing the effector stage of an established immune response, even if the animal was already sensitized to the antigen. How exactly these Treg cells exert their regulatory action is not entirely understood, although it is known that CD8+Tregs are functionally dependent on the inflammatory cytokine IFNγ and can induce apoptosis of effector T cells through a mechanism involving TNF-related apoptosis-inducing ligand (TRAIL) 145. It is also possible that still other Treg subsets are induced during ACAID, as at least some of the CD4+ Tregs were shown to express CD25 and Foxp3, which are common markers associated with a regulatory T cell phenotype in other models of immune tolerance 146,147. ACAID-Treg cells can be generated in response to various types of inflammatory stimuli, including Th1, Th2 and Th17 immune responses 148,149. In closing, this means that ACAID is a form of systemic immune regulation that helps to reduce or prevent inflammation in the eye through the generation of systemic Treg cells that are capable of suppressing both the priming and expression of immune responses. Finally, the eye is richly innervated by the nervous system with sensory, parasympathetic and sympathetic nerve fires. Studies suggest that all 3 types of innervation play a role in the maintenance of ocular immune privilege, although the specific mechanisms by which the nervous system suppresses inflammation in the eye is not entirely elucidated. This may occur through the release of neuropeptides that contribute to the ocular immunosuppressive environment, or may occur at the level of the thymus and spleen, which are also densely innervated by sympathetic fibers 150-152. IV.2 IS OCULAR IMMUNE PRIVILEGE UNIQUE? ACAID is a form of immunological tolerance that is induced after antigen is taken up in the eye. There are, however, numerous other tissues capable of inducing peripheral tolerance. These include the testis, brain, placenta, hair follicle, hamster cheek pouch, gut, and even certain tumors. The eye and the brain are anatomically connected and derive from the same embryologic tissue. Both brain and eye have limited regenerative capability. Hence, immune privilege in these organs can be considered as an adaptation to protect the eye and the brain from injury inflicted by inflammation. On the other hand, the testis, and the placenta are sites where stem cells reside. Protecting stem cells from inflammation makes sense in evolutionary terms 127. The discovery that a multitude of other sites can skew the immune response, sometimes only transiently, has expanded the notion of immune privilege. Nowadays, immune privilege is no longer considered to occur exclusively in privileged organs that are fenced behind anatomical barriers and lack lymphatic drainage. But rather, it can be induced in any location where immune cells expressing appropriate cell signals congregate and are bathed in an immunosuppressive micromilieu. Production of TGF-β appears to be a key component in these different sites of tolerance-induction 153. Nevertheless, the uniqueness of the eye probably lies in the fact that immunosuppressive cytokines are constitutively expressed within the ocular compartments. When comparing different models of tolerance induction, it must be noted that although a tissue may possess or can acquire the capacity to deviate the immune response, the exact molecular mechanisms that are employed to achieve tolerance and the type of regulatory cells that are produced, are not necessarily identical to those involved in ACAID. For example ACAID shares similarities with brain induced tolerance and oral tolerance to a low dose of antigens, but is different from intravenous tolerance or high dose oral tolerance 143. IV.3 THERAPEUTIC POTENTIAL OF ACAID ACAID plays a key role in promoting corneal graft survival. Injection of corneal donor cells in the anterior chamber prior to transplantation improves the survival of corneal grafts, whereas manipulations to prevent the induction of ACAID decrease the survival rate of corneal grafts 69-71,154. ACAID was also shown to decrease the severity of ocular autoimmune disease. The discovery that ACAID can be induced through in-vitro manipulation of APCs has bypassed the need for antigen injection in the eye and opened the door to a variety of therapeutic applications for this eye induced phenomenon. Principles of ACAID have been applied to non-ocular diseases that are induced by a deregulated immune response 155. APCs that are conditioned with TGFβ in-vitro, and pulsed with antigen, acquire a
tolerance-inducing potential that is similar to in-vivo generated ACAID-APCs after antigen inoculation into the eye. These in-vitro generated ACAID-like APCs were shown to be equally capable of inducing systemic tolerance. In-vitro grown ACAID-like APCs have been used in pre-clinical trials to reduce the immunopathology of autoimmune uveitis, autoimmune encephalomyelitis, allergic asthma and immune pulmonary fibrosis. To take these experiments one step closer to clinical applications, a humanized mouse model of allergic asthma was created and successfully treated using principles of ACAID. It must be noted that the ACAID-like APCs is actually not the only type of APCs capable of deviating the immune response. In this area of research, various types of in-vitro generated APCs have shown immune-skewing properties. Current research is now focusing on translating experimental knowledge into real clinical applications. Phase I clinical trials using in-vitro generated tolAPC, although different from the ACAID-tolAPC, have recently started for autoimmune diseases such as type-1 diabetes and rheumatoid arthritis. These novel therapies may offer an attractive alternative approach to minimize the use of immunosuppressive drugs in autoimmune diseases and transplantation 156. IV.4 IS THERE A DOWNSIDE TO OCULAR IMMUNE PRIVILEGE? Although the various mechanisms that sustain ocular immune privilege are beneficial in minimizing inflammation in the eye, it is possible that immune privilege may have unintended side effects. In the non-inflamed eye the ocular barrier system restricts the normal traffic of immune cells, and although this may protect the eye from unwanted inflammation associated with minor day-to-day insults, it may also have for consequence that naturally occurring autoreactive T cells become insufficiently tolerized to ocular antigens, leaving the eye more vulnerable to autoimmune disease after break-down of immune privilege (explained later in more detail) 157. Another possible downside may occur with intraocular presentation of pathogens whose elimination depends on delayed hypersensitivity responses, such as Herpes Simplex Virus-1. Suppression of delayed type hypersensitivity will result in increased viral replication and dissemination of the virus 158. Tumors that arise in the eye may also be protected from immune rejection and allowed to growth, eventually leading to destruction of the eye 158. IV.5 IMMUNE MEDIATED DISEASES OF THE UVEA Ocular immune privilege, as explained in the previous section, is not a rigid or unshakable mechanism that confers global protection against all immunological challenges in the eye. On the contrary, uveitis is a common ocular disorder that is frequently diagnosed in veterinary patients. Infectious agents, trauma, necrotic cell death or highly immunogenic tumors can induce strong inflammatory responses that modify the local signaling milieu and lead to erosion or frank loss of immune privilege 158-160. It must be remembered that the inflammatory response is intended as a defense mechanism to an inciting insult. But in the eye the risk of inflammation and uncontrolled tissue damage in response to a minor insult may prove more detrimental than useful. Factors that may predispose an animal to develop uveitis include genetic susceptibility, trauma, exposure to infectious agents, systemic disease, a persistent inflammatory condition, or a combination of these factors. Most of the time however the inciting cause and reasons for perpetuation or recurrence of uveitis remain unknown. An underlying autoimmune disorder is often suspected based on the absence of identifiable infectious organisms, lack of response to antibiotics, and a positive response to immunosuppressive treatment 161. IV.5.a Autoimmunity and the eye When immune privilege breaks down, it seems the eye is more vulnerable to autoimmunity compared to other organs in the body. While it is normal to have some autoreactive lymphocytes that circulate throughout the body, it is unusual for these cells to cause autoimmune disease. Self-reactive T cells are mostly removed from the circulation during T cell maturation in the thymus. Some autoreactive T cells that manage to escape thymic deletion will become tolerant or anergic after encounter their corresponding self-antigen in normal uninflamed peripheral tissues. Because trafficking of lymphocytes in the eye is restricted by the blood ocular barriers, self-reactive T cells that have escaped thymic deletion remain insufficiently tolerized to their corresponding ocular antigen and may render the eye more vulnerable to autoimmune attack 157. Infectious agents may also predispose the eye to autoimmune attack without necessarily needing to infect the eye. Antigenic epitopes expressed on pathogens that bear resemblance to self-proteins can induce an immune response that can eventually turn towards a self-peptide as a result of cross-reactivity. T cells that have been activated in the periphery may then be able to bypass the threshold of ocular suppression to induce uveitis. Furthermore, injury to the ocular tissues, whether caused by trauma or inflammation, may alter self-proteins and
thereby present new immunogenic epitopes. Cell damage may also liberate intracellular proteins to which the immune system has not been previously exposed 162. IV.5.b Animal models of Experimental Autoimmune Uveitis (EAU) Autoimmune uveitis can be induced in rodents after immunization of the animal with peptides derived from interphotoreceptor binding protein (IRBP) and adjuvants. Simply injecting IRBP subcutaneously without adjuvant does not lead to EAU. Common experimental adjuvants are made of an oily solution of heath-killed Mycobacterium tuberculosis. The bacterial components are potent triggers of PRRs on innate immune cells, which will then activate IRBP-reactive T cells in peripheral lymph nodes to migrate to the eye and cause inflammation. These animals develop mostly posterior uveitis, which is self-limiting and resolves spontaneously163. The same type of immunization is used in an equine model of ERU164. Although these experimental conditions are not identical to uveitis that is encountered in the clinical setting, the models provide great insight into mechanisms that breakdown immune privilege, and how disease resolution comes about. IV.5.c Feline lymphoplasmacytic uveitis (LPU) Despite the common occurrence of this condition there is only limited insight into the disease mechanisms of LPU. Although several causes of uveitis in cats have been identified, determination of the underlying cause of the condition can be challenging. The cause of uveitis remains unidentified in up to 70% of affected cats despite the use of multiple testing modalities. Our limited progress in the understanding of this condition is related to the late presentation of patients and lack experimental animal model. Lymphoplasmacytic uveitis is essentially a histologic diagnosis. Infectious agents are conspicuously absent from enucleated globes, and any potential target antigen(s) against which the immune response is directed remain unknown. Inflammatory infiltrates are commonly accompanied by pre-iridal fibrovascular membranes (PIFM). A canine study showed that the angiogenic factor VEGF is commonly elevated in aqueous humor of diseased eyes and likely plays a key role in the development of PIFM165. PIFM formation may have a significant impact on the dynamics of immune responses within the eye as it bypasses the blood ocular barrier and facilitates trafficking of immune cells in and out of the eye. IV.5.d Canine Uveodermatologic Syndrome Uveodermatologic syndrome (UDS) in dogs is manifested by bilateral uveitis and cutaneous depigmentation, predominately affecting the head. The syndrome has a higher prevalence in the Akita breed, but has also been reported sporadically in many other breeds. It was first described in Japan and named “Vogt-Koyanagi-Harada-like” syndrome (VKH), after a human syndrome that presents similar ocular and cutaneous lesions. The syndrome in humans is commonly accompanied by neurological symptoms and auditory deficits, but these are not clinical features observed in dogs. The condition affects primarily young adult dogs that otherwise appear healthy or at least have no history of concurrent systemic illness. Frequently observed ocular signs include bilateral anterior and posterior uveitis, depigmentation of the iris, RPE and choroid, retinal detachment, and retinal and optic nerve degeneration. Although both eyes are usually affected, there may be asymmetrical disease progression. Unilateral involvement may occur in dogs with heterochromia iridis, whereby only the pigmented eye shows disease development. Skin and fur depigmentation (vitiligo and poliosis, respectively) develop concurrently with the ocular symptoms or may follow in later stages of the disease. The mucocutaneous junctions of the head are primarily affected (eyelids, nasal planum, lips), but poliosis may progress to affect larger parts of the body. In humans, severe inflammatory infiltrates and loss of ocular and dermal melanocytes suggest a cell mediated autoimmune reaction to melanocyte antigens, such as tyrosinase and tyrosinase-related proteins. Tyrosinase proteins are enzymes involved in melanin formation that are specifically expressed in melanocytes. The disease can be induced in experimental animals after immunization with human tyrosinase-related protein 1 and adjuvant, providing further support that the natural occurring disease is driven against melanocyte antigens. However, why one develops autoimmunity to melanocyte antigens is still subject to debate. A possible infectious trigger, although not necessarily involving the eye, is suggested as flu-like symptoms often precede disease development in people. Recently molecular mimicry between cytomegalovirus and tyrosinase peptides was identified 166. Furthermore, eye-derived CD4+T cells from human VKH patients reacted to CMV antigen, providing further evidence that molecular mimicry may trigger the condition. The disease can also be induced in dogs by immunization with human tyrosinase-related protein-1 and adjuvant. The strong prevalence of uveodermatologic syndrome in the Akita breed throughout the world suggests a permissive immunogenetic
background. A study analyzing dog leukocyte antigen class II alleles (MHC II) showed an increased incidence of a certain allele (DQA1*00201) in UDS-affected Akita dogs in the United States. MHC II molecules are necessary in both the selection and activation of CD4+ T cells. It is conceivable that an abnormal MHC molecule might predispose to autoimmunity by biasing the T cell repertoire or faulty antigen presentation. Unfortunately, the identified allele as a marker for disease development as most affected dog do not carry the DQA1*00201 allele. Immunohistochemical studies in humans indicate a T cell mediated immune response in both eyes and skin and anti-melanin autoantibodies are also present in peripheral blood. One study in dogs suggested that inflammatory cells in the eye where mostly B cells and macrophages, whereas skin contained mostly T cells and macrophages 167. V. LENS ASSOCIATED IMMUNE DISEASES Lens-induced uveitis is a group of inflammatory responses to liberated lens protein. It regroups at least two different clinical manifestations: phacoclastic uveitis is associated with lens proteins liberated through a ruptured lens capsule; and phacolytic uveitis is associated with the leakage of protein fragments through an intact lens capsule168. While it is generally accepted that lens pathologies are accompanied by varying degrees of uveitis, the immunogenicity of lens proteins is not that straightforward. Crystallins (α, β and γ) are the main structural proteins that contribute to the transparency and refractive properties of the lens. They were previously thought to be expressed exclusively in the lens, but most of them have subsequently been identified in other parts of the eye (cornea, anterior chamber, uvea and retina) and elsewhere in the body (including mitochondria, endoplasmic reticulum, smooth muscle proteins, cell nuclei…)169,170. Crystallins are also expressed in the thymus where they serve to tolerize developing T cells. Consequently, T cells appear to be relatively tolerant to lens proteins, even after immunization of animals with α-crystallin and complete Freud’s adjuvant. B cells on the other hand, seem to be less tolerant to crystallins and can be stimulated to produce antibodies against lens proteins without requiring T cell help 171. The presence of anti-lens antibodies does not necessarily lead to ocular disease. Only repeated immunization or lens puncture will cause inflammation172. A substantial amount of healthy dogs (approx. 60%) have circulating anti-crystalline antibodies in the serum in the absence of ocular problems. The development of cataract and lens induced uveitis are not associated with increased circulating anti-crystallin antibodies and no anti-lens antibodies could be found in the aqueous humor of dogs regardless of the presence of cataracts173. Furthermore, antibodies against βH-crystallin are negatively associated with cataract. Nevertheless, research suggests that immunogenicity may be directed towards protein fragments or derivatives of βA1-Crystallin174. Lens proteins undergo post-translational modifications with aging and cataract development, such as glycation, denaturation, unfolding and proteolysis 175. Modified proteins from cataractous lenses may constitute new epitopes and induce the production of antibodies with a distinct affinity from the normal proteins. In people, antibodies against glycated forms of crystallins are found at increased concentrations in patients with cataracts versus age-matched healthy individuals 176. Induction of diabetes in experimental rodents increases glycation of lens proteins and results in the production of autoantibodies177. The immunogenicity of modified lens fibers could plausibly also account for the increased amount of uveitis that is noted after phacoemulsification in dogs. The delayed onset of a more persistent type of uveitis, after the immediate effects of surgical trauma have resolved suggest an adaptive immune response. In contrast to the low-grade inflammation that is associated with phacolytic uveitis, phacoclastic uveitis is a severe fibrinopurulent or granulomatous reaction that occurs following capsular rupture with massive release of lens fibers and intracellular contents. Lens rupture occurs most frequently in young dogs as a result of perforating trauma such as caused by cat scratch injuries. Other causes of lens rupture include blunt trauma, intumescent cataract in diabetic dogs and lenticular Encephalitozoon cunniculi infection. While there might be differences in the type of uveitis depending on the cause and duration of inflammation, phacoclastic uveitis is generally more severe and difficult to control medically. A latency period may be observed between the inciting trauma and the development of severe ocular inflammation, which suggests an adaptive immune response is being mounted. Anti-lens antibodies can form immune-complexes, activate the complement cascade and promote phagocytic activity by macrophages. Phacoclastic uveitis in clinical patients is probably a complex immune reaction triggered by a combination of different factors, including trauma, simultaneous inoculation of foreign antigens, and an exposure of lens proteins. In extreme cases inoculation of microorganisms into the lens can lead to delayed septic endophthalmitis 178.
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