Ординатура / Офтальмология / Английские материалы / The Retina and its Disorders_Besharse, Bok_2011

Glossary

Endocytosis – The process by which cells absorb molecules, such as proteins, from outside the cell by engulfing them with their cell membrane to form an endosome.

Endophthalmitis – An infection of the posterior of the eye.

Opsinization – The process by which a pathogen or infected cell is marked for destruction by a phagocyte.

Phagocytosis – The engulfment of solid particles, such as bacteria, by the cell membrane to form an internal phagosome.

Introduction

Innate immunity comprises a large number of molecules and cells that recognize and respond rapidly to pathogens, providing immediate defense against infection. However, innate immunity also carries with it the potential of highly destructive inflammation that presents an important dilemma for the eye. Inflammation is necessary for successfully eradicating pathogens. An ideal response would eliminate the microorganisms before they are able to directly damage any ocular tissues. The innate immunity would be limited and produce little or no damage to the surrounding normal tissues. However, some types of ocular infections trigger inflammation that is either (1) insufficient to clear the microorganisms, resulting in direct destruction of ocular tissue by the pathogen, or (2) excessive inflammation that clears the microorganisms, but destroys a significant amount of normal tissue. Either of these two scenarios is undesirable and can lead to significant loss of vision. Therefore, a delicate balance must be achieved between the amount of inflammation required for pathogen clearance and the amount of nonspecific tissue damage.

The innate immune system of the eye is similar to other mucosal surfaces. The first tier is passive consisting of several anatomic, physical, and chemical barriers that work together to prevent infection without inducing inflammation. The second tier is active consisting of cellular and secretory components that together cause acute inflammation aimed at eradicating the pathogen. The delicate

tissues of the eye that make up the visual axis (cornea, lens, and retina) have a very low tolerance for inflammation, as a very small amount of damage can produce a significant loss of vision. The two-tiered system helps to prevent unnecessary inflammation and the active mechanisms of innate immunity are only turned on once the passive barriers have been breached. Both the passive and active arms of ocular innate immunity are the focus of this article.

Passive Innate Defense System

Anatomic and Physical Barriers

Several anatomic and physical barriers protect the anterior and posterior of the eye from invading pathogens (Figure 1). The active arm of innate immunity is only triggered when pathogens breach these barriers. The cornea is exposed to the external environment, making the anterior segment highly vulnerable to potential pathogen invasion. Therefore, the anterior segment possesses a multilayer barrier system that includes: eyelids and eyelashes, tear film, and the corneal epithelium. By contrast, the posterior segment is not exposed to the external environment, and is therefore less vulnerable to infection. The critical barriers of the posterior segment include

(1)the retinal pigment epithelium (RPE), which lies between the blood-rich choroid and the neural retina, and

(2)the posterior lens capsule that forms the barrier between the anterior and posterior segments. Each component of the passive innate defense system is described briefly below.

Eyelids and eyelashes

The outermost barrier of the ocular surface consists of the eyelids and eyelashes. The eyelashes protect the ocular surface from dust and foreign debris. The regular blinking action of the eyelids moves the tears across the ocular surface, washing away potentially colonizing or infecting organisms.

Tear film

Tears form the second barrier, lubricating and protecting the ocular surface. Tears also posses a potent defense system that limits the growth, colonization, and survival of microorganisms. The tear film consists of three layers: the outermost lipid layer, an aqueous layer, and the inner mucus layer (Figure 2). The lipid layer lubricates the eyelid and slows evaporation of the aqueous tear film layer. The aqueous layer forms the major component of the tear film and contains numerous antimicrobial

Bowman’s

membrane Stroma

Descemet’s membrane

Endothelium

Eye lid and eye lashes

Corneal epithelium

Posterior lens capsule

Figure 1 Anatomic and physical barriers of the eye. The eyelid, eye lashes, tear film, and corneal epithelium serve as barriers of the anterior segment of the eye. The posterior lens capsule and RPE serve as barriers of the posterior segment of the eye.

proteins including: lysozyme, lactoferrin, defensins, secretory IgA (sIgA), and complement. Many of these antimicrobial proteins are constitutively expressed and provide early, broad-spectrum protection against invading pathogens and also prevent the overgrowth of commensal bacteria. The innermost mucus layer of the tear film is made up of secreted and membrane-bound mucins that protect the epithelium from debris, pathogens, and desiccation. Mucins are high-molecular weight glycoproteins characterized by extensive O-glycosylation. Membrane-bound mucins expressed by the ocular surface epithelia include MUC1, MUC4, and MUC16. Secreted mucins are also found in the mucus layer and include MUC2, MUC5AC, and MUC19. The membrane-bound mucins anchor the ocular tear film to the corneal epithelium and are thought to act as a physical barrier against pathogen penetrance. Secreted mucins bind to pathogens in the tear film, facilitating their clearance from the ocular surface. Under normal conditions, mucin production and secretion by goblet cells and corneal epithelial cells are constitutive. However, mucin production can also be induced via Tolllike receptors (TLRs) expressed on the surface of corneal epithelial cells. Moreover, inflammatory cytokines, such as IL-1b, IL-6, and TNFa, have also been shown to induce mucin production and secretion. Together, these data reveal that constitutively expressed mucins make up

a critical component of the passive defense system, while at the same time, upregulation of mucin production and secretion can also be a product of the active arm of innate immunity in the eye.

Corneal epithelium

The final barrier of the ocular surface consists of nonkeratinized stratified epithelial cells bound together by tight junctions. The corneal epithelium acts as a physical barrier to invasion of microorganisms due to the presence of epithelial intercellular tight junctions and the rapid renewal of epithelial cells with frequent shedding of the superficial layers of potentially infected epithelium. As mentioned in the previous section, the epithelium also expresses membrane-bound mucins that inhibit bacterial binding to the epithelial surface and produce several of the antimicrobial factors that are present in the ocular tear film.

Posterior lens capsule

The posterior lens capsule forms a physical barrier between the anterior and posterior segments of the eye after extracapsular cataract surgery and prevents the spread of microorganisms from the anterior chamber into the posterior chamber in the postsurgical eye. The best example of this is the fact that an intact posterior lens capsule is critical in preventing endophthalmitis following cataract surgery. Contamination of the aqueous humor can occur during cataract surgery. However, the pathogens are quickly cleared and endophthalmitis does not develop. By contrast, when the posterior capsule is breached, the rate of endophthalmitis increases significantly. This supports the finding that the anterior segment is much more efficient at clearing bacteria as compared to the posterior segment. Studies suggest the difference in ability to clear pathogens in the anterior versus posterior of the eye may be linked to expression of antimicrobial peptides. One major difference is that the AH is continuously secreted and drained, whereas the vitreous humor is not. The vitreous also offers greater opportunity for microbes to bind its fibrils. However, the exact molecular mechanisms involved remain unclear.

Retinal pigment epithelium

The RPE consists of a single layer of cells joined by tight junctions that lie between the photoreceptors of the neural retina and the blood-rich choroid. The RPE serves multiple functions aimed at protecting and maintaining the health of the neural retina. RPE cells (1) phagocytose shed disks from the photoreceptor outer segments and recycle their components; (2) transport nutrients from the choroid to the retina; (3) absorb light; (4) provide adhesive properties for the retina; and (5) serve as a rich source of cytokines, chemokines, and growth factors. More recently, RPE have also been linked to immunity and have been

Lipid layer

Aqueous

layer

Mucin layer

Epithelium

Peter Mallen

shown to behave as antigen-presenting cells that phagocytose pathogens, produce cytokines, and present pathogenderived peptides to sensitized T cells. Therefore, while RPE acts as a physical barrier to the posterior segment, it is also important in shaping and regulating the adaptive immune response once this barrier has been breached.

Chemical Barriers

In addition to the anatomic and physical barriers designed to block invasive pathogens, there are also a number of soluble factors that inhibit bacterial growth, adherence, and survival. Several of these factors are constitutively expressed at low levels, providing a baseline of protection from foreign pathogens. However, many of these factors can also be upregulated in response to pathogens and inflammation; thus, these become an important product of the active arm of innate immunity. Some of the more important factors are discussed in more detail below.

Lysozyme

Lysozyme is bacteriacidal and makes up 20–40% of the total tear protein. Lysozyme kills bacteria by (1) binding to and creating pores in the bacterial cell wall, or (2) dissolving bacterial membranes by enzymatic digestion.

Secretory phospholipase A2

Secretory phospholipase A2 exhibits potent antibacterial activity against Gram-positive bacteria. Similar to lysozyme, secretory phospholipase A2 dissolves bacterial membranes by hydrolyzing the principal phospholipid, phosphatidylglycerol.

Cathelicidin (LL-37)

LL-37 is a small cationic peptide with potent antimicrobial activity against Gram-positive and Gram-negative bacteria as well some viruses. The precise mechanism of action is incompletely understood, but it is widely believed that the antimicrobial activity is due to disruption of the microbial membrane or viral envelope.

Defensins

Beta defensins are expressed in epithelial cells that line mucosal surfaces such as the cornea. Similar to LL-37, defensins have a broad spectrum of antimicrobial activity and are effective against: Gram-positive and Gramnegative bacteria, fungi, and enveloped viruses. In addition to their antimicrobial activities, defensins modulate a variety of cellular activities including immune cell chemotaxis, epithelial proliferation, cytokine secretion, and stimulation of histamine release from mast cells. Human

corneal epithelial cells constitutively express human beta defensin-1 (hBD-1) and hBD-3. By contrast, hBD-2 is induced in response to corneal injury, infection, or inflammation, and is approximately 10-fold more potent than hBD-1 with an even wider antibacterial spectrum. Therefore, while hBD1 and hBD-3 provide a baseline defense to protect the cornea from infection, upon injury or microbial invasion, hBD-2 is upregulated and displays increased antimicrobial activity.

Lactoferrin

Lactoferrin is bacteriostatic and binds to and depletes iron from the tear film, which is required for microbial metabolism and growth. Lactoferrin is also bactericidal and permeabilizes membranes of Gram-positive and Gramnegative bacteria. Additional functions of lactoferrin have also been described: (1) inhibits biofilm development,

(2) inhibits bacterial adhesion to host cells, (3) inhibits intracellular invasion, (4) amplifies apoptotic signals in infected cells, and (5) enhances bactericidal activity of neutrophils.

Lipocalin-A

Lipocalin A prevents bacteria from obtaining iron, an essential nutrient for microorganism survival. However, unlike lactoferrin, lipocalin-A does not bind iron directly. Lipocalin-A inhibits the iron acquisition system of microbes by binding to and blocking microbial sidephores used to transport iron into bacteria.

Secretory IgA

sIgA protects the ocular surface against colonization and possible invasion by pathogenic microorganisms by binding to bacteria and facilitating clearance. In addition, sIgA can opsonize bacteria for phagocytosis.

Complement

Complement components (such as C3 and C4) are constitutively expressed in the tear film and are involved in phagocytic chemotaxis, opsonization, and lysis of bacteria. The eye is unusual in that there is a constitutive low level of activated complement that is present even in uninfected normal eyes. It is believed that this low level of activated complement provides innate immune surveillance of microbes and allows for a rapid activation of the full complement cascade upon pathogen invasion.

Active Innate Defense System

Pattern Recognition Receptors

Innate immunity develops rapidly and is described historically as nonspecific, while adaptive immunity develops slowly and is antigen-specific. However, the discovery of pattern recognition receptors (PRRs) that detect unique

pathogen-associated molecular patterns revealed a level of specificity in innate immunity that not only allows discrimination between self and non-self, but also allows the development of innate immunity tailored to specific pathogens, such as bacteria, viruses, and fungi.

There are two classes of PRRs: (1) TLRs and (2) nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) (Table 1). TLRs are expressed on the cell surface and detect pathogens at the cell membrane. TLRs are also expressed within endosomes and detect pathogens that have been endocytosed. By contrast, NLRs are expressed within in the cytoplasm and detect the presence of microbial molecules inside the host cell. These two classes of PRRs are discussed in detail below.

Toll-like receptors

TLRs are type 1 transmembrane proteins with an extracellular domain for ligand binding composed of leucine rich repeats and a cytoplasmic domain for intracellular signaling which is known as the Toll/IL-1 receptor (TIR) domain. TLRs recognize bacteria, viruses, fungi, protozoa, and endogenous ligands, such as heat shock proteins and fibrinogen. Triggering of the TLR leads to activation of the transcription factor, nuclear factor-kappa B (NF-kB), and the expression of pro-inflammatory molecules, such as TNF-a, IL-1, and IL-2. To date, 10 human TLRs have been identified and each TLR has a unique ligand specificity (Table 1). TLRs were first identified on innate immune cells: neutrophils, macrophages, monocytes, and dendritic cells. More recently, TLRs were also identified on epithelial cells that lie at the host/environment interface: skin, gastrointestinal tract, respiratory tract, and urogenital tract. Furthermore, several TLRs have been identified on ocular tissue in both the anterior and posterior segment of the eye, including cornea, iris, ciliary body, choroid, and RPE.

Similar to other barrier epithelium, several TLRs have been identified on the RPE and corneal epithelial cells and are vital for sensing microbes and triggering a rapid response to eliminate the pathogen. The study of TLRs in ocular immunity is still relatively new and debate remains over which TLRs are expressed in the eye and whether TLRs are expressed on the cell surface or within endosomes. However, it is clear that TLRs are required for initiation of innate immunity in the eye. This is supported by studies using TLR knockout mice that demonstrate a significant decrease in inflammation characterized by decreased neutrophil infiltration and increased susceptibility to infection in the absence of TLRs.

NOD-like receptors

The NLRs comprise a large family of cytoplasmic PRRs that recognize bacteria and endogenous danger signals such as uric acid (Table 1). All members of the NLR family share a conserved NOD. However, the NLR family

can be subdivided into three groups based upon their N-terminal domains: caspase recruitment domain (NODs), pyrin domain (NALPs), or baculovirus inhibitor repeat (neuronal apoptosis inhibitor proteins, NAIPs). At present, the human NLR family comprises of 23 proteins, while at least 34 NLR genes have been identified in mice. NALPs are unique in that upon binding to their ligand, NALPs form a complex termed the inflammasome resulting in caspase-1 activation and the release of active IL-1b. Recently, NALP1, 3, and 10 were identified in corneal epithelial cells, but their potential function in regulating ocular innate immunity has not been determined. NAIPs inhibit caspase effectors and are mainly expressed in neurons where their primary role is to protect against apoptosis. NAIPs have not yet been found in ocular tissues. NODs are the most extensively studied members of the NLR family and are expressed in ocular tissue.

Both NOD1 and NOD2 are constitutively expressed within the eye; NOD1 in the anterior and posterior segments of the eye and NOD2 only in the anterior segment. However, which specific tissues express NODs is unknown. NOD1 and NOD2 recognize specific subcomponents of bacterial peptidoglycan. NOD1 recognizes D-g-glutamyl-meso-diaminopimelic acid, while NOD2 recognizes muramyl dipeptide. Recently, a mutation in NOD2 was linked to the development of uveitis in patients with Blau syndrome, suggesting that cytoplasmic

NODs may be important in ocular inflammation. While the importance of NOD receptors in innate immunity and the pathogenesis of inflammatory disease are recognized outside the eye, the study of NODs within the eye is at an early stage. A complete understanding of ocular host defense will require a better understanding of where NODs and other NLRs are expressed and how they regulate innate immunity within the eye.

Complement

Similar to PRRs, the complement system acts as an innate immune surveillance system, detecting the first signs of pathogen invasion. The complement system was first identified as a biochemical cascade of serum proteins that help or complement antibodies to clear pathogens and mark them for opsinization by phagocytes. It is now known, however, that the complement system can also be activated directly by microbial products via the alternative pathway. Several studies demonstrate that complement is constitutively active at low levels in the eye. This is thought to be a primary defense mechanism of the eye against pathogenic infection. Upon pathogen invasion the complement system is further activated to clear the infection through (1) generation of inflammatory factors (C3 and C5a), (2) chemotaxis of phagocytes (C3 and C5a),

(3) opsonization of Ab-coated cells (C3b), and (4) lysis of bacteria and virus-infected cells (C8 and C9).

Cytokines, Chemokines, and Effector Cells

Once pathogens breach the passive barriers of the eye and trigger the PRRs or the complement system, the primary function of innate immunity is to eliminate the invading pathogen as quickly as possible. To achieve this innate immunity must: (1) trigger an immediate immune response,

(2) amplify the response, (3) clear the pathogens, and

(4) activate the adaptive system if the pathogen cannot be cleared quickly. Cytokines, chemokines, and adhesion molecules play critical roles in regulating each of these stages.

Initiation and amplification

PRRs (TLRs and NLRs) recognize microbial products in the earliest phase of host defense and activate many immune and inflammatory genes, the products of which are important in initiating and amplifying antimicrobial immunity. Triggering TLRs on either the corneal epithelium or the RPE leads to activation of NF-kB via the (1) MyD88-dependent or (2) MyD88-independent pathway. The MyD88-dependent pathway is utilized via most TLRs (TLR1, 2, 4, 5, 6, 7, 8, and 9) and leads to the production of proinflammatory cytokines and chemokines (IL-6, IL-8, IL-18, MIP-1, and TNF-a). By contrast, only TLR3 and TLR4 utilize the MyD88-independent pathway and leads to the production of IFN-a and IFN-b. Recruitment of neutrophils into inflamed tissues is controlled predominantly by two chemokines (MIP-2 (= IL-8 in humans) and KC). In Pseudomonas aeruginosa-induced corneal keratitis, elevated MIP-2 and KC correspond with increased infiltration of neutrophils. In the posterior segment, elevated TNF-a, IL-1b, and CINC (rat homolog of IL-8) contribute to the breakdown of the blood–retinal barrier and the recruitment of neutrophils in response to Staphylococcus aureus. The adhesion molecules ICAM-1 and E-selectin are also upregulated early in iris, ciliary body, and retinal vessels, serving to enhance the infiltration of neutrophils to the site of infection.

Clearing the pathogen

In response to inflammatory cytokines and chemokines, neutrophils and macrophages are recruited to the site of infection. Bacterial clearance by neutrophils is accomplished by (1) phagocytosis, (2) generation of reactive oxygen species, and (3) the release of granule-associated enzymes: cathepsin G, myeloperoxidase, lactoferrin, and elastase. While the recruitment of neutrophils to the site of infection is essential for clearance of the pathogen, the persistence of neutrophils and the prolonged release of inflammatory mediators is also associated with nonspecific host tissue damage. Similar to neutrophils, macrophages also phagocytose and directly kill microbes as part of innate immunity. However, macrophages are also antigenpresenting cells, and as such participate in the development

of the adaptive immune response. The natural killer (NK) cell is another important innate effector cell in host defense against viral infections of the cornea such as herpes simplex virus type-1. NK cells respond in an antigen-independent manner and kill virus-infected host cells through the release of perforin and granzymes or through binding of the death receptors Fas and TRAIL-R on the target cell. If innate effector cells fail to clear the infection, adaptive immunity will take over and finish eradicating the pathogen. However, if the infection is successfully cleared, the final and most important step of innate immunity is preventing nonspecific host tissue damage. This is accomplished in the eye through several mechanisms that together make up innate immune privilege.

Innate Immune Privilege

The primary role of innate immunity is to rapidly eradicate invading pathogens through the induction of inflammation. As a general rule, the level of inflammation is proportional to the size and virulence of the infection. Small, nonvirulent infections are cleared by mild inflammation, while larger, more virulent infections induce intense inflammation. The potential danger of inflammation occurs when intense and/or prolonged inflammation threatens the surrounding normal tissue, resulting in nonspecific tissue damage and scarring. The potential danger of inflammation in the eye is magnified by the presence of irreplaceable and highly sensitive ocular tissues. Therefore, it is not surprising that immune privilege in the eye has multiple mechanisms to control innate immunity and limit nonspecific tissue damage. The aqueous humor contains multiple factors that directly inhibit innate immunity including: (1) TGF-b, soluble Fas ligand, and alpha-melanocyte stimulating hormone (inhibit neutrophil activation); (2) macrophage migration inhibitory factor (inhibits NK cell-dependent lysis of target cells);

(3) calcitonin gene-related peptide (inhibits nitric oxide release from activated macrophages); and (4) complement regulatory factors: CD46, CD55, CD59, and Crry (inhibit complement activation). These factors work together to limit the damaging consequences of inflammation and to preserve the visual axis. Unfortunately, while these mechanisms evolved to limit local tissue destruction and preserve the visual axis, they may leave the eye more vulnerable to organisms whose virulence often requires a robust inflammatory response for eradication. Therefore, a delicate balance must be made between the amount of inflammation needed for eradicating the pathogen and the amount of nonspecific tissue damage.

Link between Innate and Adaptive Immunity

Innate and adaptive immunity have long been discussed as separate arms of the immune system. However, it is

increasingly clear that they are indeed not separate but highly integrated. Several studies, both outside and inside the eye, identified the dendritic cell as a central link between innate and adaptive immunity. Immature dendritic cells reside in peripheral tissues and through triggering of TLRs, participate in the primary immune response against microbial infections. Immature dendritic cells encounter invading pathogens, capture bacterial antigens, and migrate to the draining lymph node. Once in the lymph node, only mature dendritic cells can efficiently prime naı¨ve T cells and initiate adaptive immunity. Studies using TLR and MYD88 deficient mice, reveal that TLR signaling is required for bacteria-induced maturation of dendritic cells and induction of adaptive immunity. Therefore, TLRs on dendritic cells are essential for (1) sensing the microbe and initiating the immediate innate immune response, as well as (2) inducing the development of an adaptive immune response. While dendritic cells have been identified in the cornea and retina, additional studies are needed to completely understand their function in linking innate and adaptive immunity within the eye.

Conclusion

Innate immunity is a critical first line of defense against ocular infections. However, the regulation of innate immunity within the eye is just beginning to be unraveled. The identification of TLRs and NLRs has provided new insights into the mechanisms of host defense and the pathogenesis of inflammatory diseases. A better understanding of how microbial agents and endogenous host factors interact with TLRs and NLRs in the eye will be critical in advancing our knowledge of the pathogenesis of infectious and noninfectious eye diseases. Moreover, a better understanding of these mechanisms will lead to the identification of new therapeutic targets for treating and preventing sight-threatening infections.

See also: RPE Barrier.

Further Reading

Banchereau, J. and Steinman, R. M. (1998). Dendritic cells and the control of immunity. Nature 392: 245–252.

Creagh, E. M. and O’Neill, A. J. (2006). TLRs, NLRs and RLRs: A trinity of pathogen sensors that co-operate in innate immunity. Trends in Immunology 27: 352–357.

Gregory, M., Callegan, M. C., and Gilmore, M. S. (2007). Role of bacterial and host factors in infectious endophthalmitis. Chemical Immunology and Allergy 92: 266–275.

Haynes, R. J., McElveen, J. E., Dua, H. S., Tighe, P. J., and Liversidge, J. (2000). Expression of human beta-defensins in intraocular tissues.

Journal of Investigative Ophthalmology and Visual Science 41: 3026–3031.

Holtkamp, G. M., Kijlstra, A., Peek, R., and de Vos, A. F. (2001). Retinal pigment epithelium–immune system interactions: Cytokine production and cytokine-induced changes. Progress in Retinal and Eye Research 20: 29–48.

Kaplan, H. J. and Niederkorn, J. Y. (2007). Regional immunity and immune privilege. Chemical Immunology and Allergy 92: 11–26.

Kawai, T. and Akira, S. (2007). TLR signaling. Seminars in Immunology 19: 24–32.

Kolls, J. K., McCray, P. B., and Chan, Y. R. (2008). Cytokine-mediated regulation of antimicrobial proteins. Nature Reviews Immunology

8: 829–835.

Pearlman, E., Johnson, A., Adhikary, G., et al. (2008). Toll-like receptors at the ocular surface. Ocular Surface 6: 108–116.

Rodriguez-Martinez, S., Cancion-Diaz, M. E., Jimenez-Zamudio, L., et al. (2005). TLRs and NODs mRNA expression pattern in healthy mouse eye. British Journal of Ophthalmology 89: 904–910.

Sack, R. A., Nunes, I., Beaton, A., and Morris, C. (2001). Host-defense mechanism of the ocular surfaces. Bioscience Reports 21: 463–480.

Sohn, J. H., Bora, P. S., Jha, P., et al. (2007). Complement, innate immunity and ocular disease. Chemical Immunology and Allergy

92: 105–114.

Streilein, W. J. and Stein-Streilein, J. (2000). Does innate immune privilege exist? Journal of Leukocyte Biology 67: 479–487.

Tosi, M. F. (2005). Innate immune responses to infection. Journal Allergy and Clinical Immunology 116: 241–249.

Van Vilet, S. J., den Dunnen, J., Gringhuis, S. I., Geijtenbeek, T. B., and Van Kooyk, Y. (2007). Innate signaling and regulation of dendritic cell immunity. Current Opinion in Immunology 19: 435–440.

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

Apoptosis – The molecular and biochemical process by which a cell soma (cell body) is able to disassemble all of its organelles and break apart to be engulfed by neighboring cells. As the process is intrinsic, and tightly regulated by the cell itself, it has often been called a cell suicide program. Apoptosis is the mechanism of cell elimination during development (programmed cell death) and is often the end-stage result of cell loss in a variety of diseases, particularly chronic neurodegenerative disorders. Retinal ganglion cell soma apoptosis is the mechanism of cell loss in glaucoma.

Axons – The single extensions of neurons that act as the conduit for an electrical impulse or signal originating from the soma of the same neuron. They typically terminate in a synapse that contacts one of many dendrites of other neurons. In reference to glaucoma, axons of the retinal ganglion cells pass out of the eye into the optic nerve.

Glia – The abundant non-neuronal cell types in the nervous system. There are many different classifications of glia, but principally they are made up of astrocytes, Mu¨ller cells (in the retina), oligodendrocytes or Schwann cells (which synthesize myelin in the central and peripheral nervous systems, respectively), and microglia. Astrocytes and Mu¨ller cells are thought to function as neuronal support cells under normal conditions. Microglia are thought to play an important role in the innate immune response of neural tissue.

Lamina cribrosa – In higher primates, it is the connective tissue and neuronal structure comprising the scleral canal at the site where the optic nerve exits the eye. The connective tissue is formed as plates of collagen and basement membrane and the surface of these plates are occupied by astrocytes and a secondary cell type called lamina cribrosa cells. Pore structures exist between the plates through which retinal ganglion cell axons are bundled as they exit the eye. Rodents do not have a lamina cribrosa, but instead have columns of astrocytes that surround the bundles of exiting axons. Because of the lack of connective tissue, this structure has been termed the cellular lamina in these animals.

Optic nerve – It is anatomically described as the second of the 12 paired cranial nerves and is an

extension of the central nervous system. The nerve begins at the lamina cribrosa and extends to the lateral geniculate nucleus in higher primates and the superior colliculus in rodents. Shortly after it exits the eye, the fibers of the nerve, which are axons of retinal ganglion cells, become myelinated.

Retinal ganglion cells – The projection neurons that reside in the innermost layer of the retina, and extend axons out of the eye and into the optic nerve. They receive electrical stimulation from photoreceptors by way of bipolar neurons and then convey that stimulus to optical centers in the brain. Soma – Also known as the cell body, it is anatomically and functionally distinct from the dendrites, axon, and synapse, even though these compartments are all part of the same cell. The soma contains the basic cellular organelles including the nucleus and the majority of endoplasmic reticulum and Golgi bodies. Retinal ganglion cell somas reside in the ganglion cell layer of the retina.

Introduction – Intraocular Pressure as a Risk Factor for Glaucoma

Glaucoma is one of the world’s leading causes of blindness, estimated to affect over 60 million people worldwide by the year 2010. It is typically a disease of the elderly and often goes undetected until later stages because progression is usually not associated with pain, or the devastating loss of central vision. Instead, glaucoma progresses slowly, creating a series of small peripheral defects in vision (called scotomas) that are compensated for by processing in the visual centers of an affected individual. The development of these pathological blind spots is principally the result of the regional degeneration of the retinal ganglion cells, involving the loss of both the axon in the optic nerve, and the cell body (soma) in the retina. Once a critical number of cells are lost in the retina, light information from that specific region is unable to be transmitted to the brain.

Because of the slow progressive nature of the disease, early detection is paramount to establish treatments that can attenuate further damage to the retina and optic nerve. A critical part of the early detection arsenal is the association between elevated intraocular pressure (IOP) and glaucoma. Nearly every person with glaucoma has

either elevated IOP, or benefits from having existing levels of IOP lowered. In addition, experimentally induced ocular hypertension is a staple of virtually all animal models of the disease, while lowering elevated IOP in these models is associated with slowing the progression of glaucoma, thus providing experimental evidence for the causal relationship between eye pressure and optic nerve disease.

Currently, there is no clear mechanism that associates elevated IOP with the activation of optic nerve and retinal damage; however, studies over the last several decades have pointed to a viable model linking the two. At present, this model serves as a framework for both current and future hypothesis-driven studies that may help resolve this important question. Understanding the model requires a brief tutorial on the relevant anatomical structures of the eye. In essence, the mammalian eye is a closed hydrostatic system. The optics, which are designed to focus incoming light from the anterior segment of the eye onto the sensory retina lining the posterior segment of the eye, require that the globe be properly inflated. To do this, aqueous humor is secreted by cells of the ciliary body posterior to the iris and drained through the trabecular meshwork and Schlemm’s canal, anterior to the iris. As light passes into the eye, and is focused onto the sensory retina, photons are captured by photoreceptors, where they are converted into chemical signals that are then processed by the neural network of the retina until finally reaching the retinal ganglion cells. Ganglion cells transmit these signals through axons that exit the globe, enter the optic nerve, and connect to neurons in visual centers in the brain. Anatomically, the axons from the ganglion cells typically form bundles and exit the globe through a posterior hole in the sclera called the lamina. In small mammals, such as rodents, the axon bundles in the laminar region are supported by columns of glial cells, principally astrocytes. This structure has been called the cellular lamina by some researchers to distinguish it from the lamina cribrosa (LC) found in the eyes of larger mammals such as primates. The LC accommodates larger numbers of ganglion cell axons (in the range of 1 million in higher primates versus 50 000–100 000 in rodents), which necessitates the presence of collagenous beams and plates that support the spaces through which the axon bundles pass (Figure 1). Similar to rodents, this region of the optic nerve is also populated with cells, including astrocytes, specialized cells referred to as LC cells, and microglia.

Biomechanical Engineering Studies

In a consideration of how elevated IOP causes the death of retinal ganglion cells, the lamina plays a central role. Several seminal studies on the pathophysiology of glaucoma have pointed to the laminar region of the optic nerve head as the first site of damage in glaucoma. The

evidence for this comes from a variety of observations, notably classical studies showing the disruption of both retrograde and anterograde axoplasmic transport in response to elevated IOP (Figure 2), and more recent electron microscopic studies showing very early axonal disruption in the nerve head of a mouse model of glaucoma.

Intuitively, the lamina causes something of a dilemma when considering that the eye must be under some kind of pressure in order to maintain normal function, since it is essentially a hole in an otherwise closed, spherical, hydrostatic system. Thus, one might expect that forces generated by hydrostatic pressure within this system would be focused onto the weakest point, such as this small hole. Given the requirement for some level of pressure in the eye, it is likely that the evolutionary development of the lamina would be sufficiently strong enough to support the axons passing through it, so that they are not adversely affected by the forces concentrated on this area. However, in the face of above-normal levels of IOP, which would increase the forces directed at the laminar structure, axons could be damaged if the forces focused on this region exceeded the structural capacity it was designed to withstand.

This rather speculative model has been tested by biomechanical engineers using finite element modeling. In this complicated field of applied engineering, real-life measurements of the tensile strength of biological tissues are taken and used to design small elements that are pieced together to create a three-dimensional model of the biological system in question. Early finite element models of the eye were created based on mathematics of a simple sphere under pressure, and these models helped confirm the basic intuitive idea that forces created by pressure inside the globe create stress and strain on the structural integrity of the lamina (Figure 1). Important advances in the field finite element modeling in the study of glaucoma have come from detailed element modeling of both the sclera of the eye and the different components of the primate LC.

Modulation of Glia Behavior at the Optic

Nerve Head and Dysfunction of Ganglion

Cell Axons

When IOP becomes elevated, resulting in an increase in the stress placed in the laminar region, the cells in this region respond. The precise nature of this response is not fully understood, but detailed gene expression profiles of the optic nerve head region of rats with experimental glaucoma clearly indicate that the glial cells in this region upregulate the expression of genes involved in proliferation and remodeling of the extracellular matrix (ECM). Similar changes in gene expression have been noted in optic nerve astroctytes and LC cells in higher primates

with experimental glaucoma and in the human disease. Early interpretations of these changes in gene expression generally assumed that cells in this region were responding to the loss of ganglion cell nerve fibers by laying down a glial scar. This notion has recently been challenged by biomechanical studies of the connective tissue composition of the laminas isolated from monkeys at the very early stages of experimental glaucomatous damage. In detailed three-dimensional reconstructions of serially sectioned nerve heads, in which each section was individually stained for ECM components, this group showed that connective tissue content increased in the laminar region well in advance of the loss of neuronal tissue. Surprisingly, the ratio of connective tissue to neural tissue remained relatively constant, indicative of an increase in

the thickness of the laminar region caused by the addition of new ECM material in the posterior region of the lamina and an actual widening of the scleral canal. Progressively, the connective tissue lamina begins to deform posteriorly partly as a function of the loss of ECM material in the anterior portion of the lamina, and, at later stages of glaucomatous progression, partly as a function of the loss of ganglion cell axons entering the optic nerve head. Eventually, the posterior deformation becomes permanent, resulting in the classic cupping of the optic nerve head observed clinically.

The interpretation of these changes in the optic nerve head in early glaucoma is that the glial cells in this region are responding to the tremendous increase in stress and strain induced by the elevation in IOP, by laying down