Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Pertubation of membrane and

loss of intracellular contents = microbial death

+

+

Figure 2 Mechanism of action of antimicrobial peptides. Positively charged peptides (shown in yellow) such as defensins and cathelicidin (LL-37) interact with negatively charged microbial membranes leading to disruption of the membrane, and possibly transient or stable pore formation, which results in leakage of intracellular contents, disturbance of metabolism, and death of the organism.

nutrient for growth. Furthermore, a highly basic sequence at the N-terminus (referred to as lactoferricin) allows lactoferrin to act as a cationic detergent and disrupt the cell membrane of some organisms. Tear lipocalin represents approximately 25% of the reflex tear proteins and was recently shown to be capable of binding siderophores produced by a range of bacteria and fungi. Siderophores are chelating compounds that transport iron into microorganisms. Thus, like lactoferrin, lipocalin exerts a bacteriostatic effect by interfering with the ability of pathogens to take up iron.

The tear film also contains members of the collectin family of C-type lectins, surfactant proteins (SP)-A and -D. Collectins bind to carbohydrates on the surface of various microorganisms and to receptors on phagocytic cells, which in turn promotes their phagocytic activity. SP-A and SP-D are produced by lacrimal gland cells and also corneal and conjunctival epithelial cells. SP-D is known to exert growth inhibitory effects on some Gram-negative bacteria, to promote pathogen phagocytosis by mononuclear cells and through an interaction with lipopolysaccharide (LPS), which is present on the outer membrane of Gram-negative bacteria, can inhibit bacterial adhesion to target cells. SP-D inhibits corneal epithelial cell invasion by P. aeruginosa (a Gram-negative pathogen that commonly causes ulcers in contact lens wearers), possibly via an LPS-dependent mechanism.

Secretory IgA (sIgA) is the predominant immunoglobulin in the tear film. This antibody is of great importance as it facilitates removal of pathogens right at the point of entry at the ocular surface. sIgA is not a particularly efficient activator of complement (important for preventing unwanted inflammation) or a good opsonin (although

neutrophils do have receptors for sIgA which when engaged could trigger phagocytosis). The major effector mechanism of sIgA is neutralization, which prevents attachment to host cells. sIgA can also bind to lectin-like adhesin molecules on pathogens causing them to aggregate and trapping them within the tear film. sIgA is produced by plasma cells (terminally differentiated B lymphocytes) residing in the lacrimal gland and in specialized areas of the conjunctiva referred to as con- junctival-associated lymphoid tissue. sIgA binds to specific receptors on lacrimal gland acinar cells, conjunctival epithelial cells, and is taken up by endocytosis, and then traverses the cell by transcytosis. This antibody is then released into the tears attached to a protein called secretory component, a fragment of the receptor to which the antibody was bound during its passage through the cells. Secretory component stabilizes the antibody and masks proteolytic sites so conferring resistance to host and pathogen proteases.

Low levels of functionally active complement and complement regulatory proteins have also been detected in tears. An overview of the complement pathway is presented in Figure 3. The relative amounts of different components, namely abundant C3 and factor B, but less C1q, suggest that activation via the alternative pathway (i.e., spontaneous hydrolysis of C3) is the predominant mechanism. Possible sources of the various complement components are leakage of plasma through the conjunctival vessels during sleep, infiltrating neutrophils, and local synthesis by corneal and conjunctival epithelial cells. Activation of the complement pathway generates fragments involved in acute inflammatory responses, fragments that act as opsonins which facilitate target recognition by neutrophils and results in the formation of membrane attack complexes that can lyse pathogens (and host cells). The complement pathway is believed to be most active when the eyes are closed (see comments below on closed-eye tears). To prevent unnecessary activation and hence tissue damage, the complement pathway is regulated by a number of factors. This pathway is inhibited by molecules such as lactoferrin and vitronectin both of which are present in the tears and CD55 (decay accelerating factor) as well as CD59 which are membranebound molecules expressed by corneal and conjunctival epithelial cells.

In immediate apposition to the superficial epithelial cells is a blanket of mucus, composed primarily of the gelforming mucin MUC5AC which is secreted by goblet cells in the conjunctiva in response to parasympathetic stimulation. This blanket interacts with the glycocalyx coating the superficial cells. Membrane spanning mucins MUC 1, 4, and 16 produced by the epithelial cells are important components of the glycocalyx and can be cleaved from the cell surface and released into the tear film. Mucins are known to help prevent bacteria from

reaching epithelial surfaces. This function has been attributed to a number of mechanisms, for example, mucin can bind and trap the bacteria, which are then effectively removed from the ocular surface by blinking. It should be noted, however, that the ability to interact with mucins varies widely among different organisms. There is also evidence that sIgA and positively charged proteins such as lysozyme and SLPI accumulate in the mucous blanket, thus providing a reservoir of antimicrobial agents. Therefore, mucins may trap microbes, which are then killed by accumulated antimicrobials or aggregated by sIgA and then cleared by blinking.

Thus, tears are equipped with a plethora of chemical entities capable of neutralizing invading pathogens. While many tear components have independent antimicrobial effects, several are thought to cooperate in a synergistic fashion to yield maximal effect. For example, sequestering of cations by lactoferrin destabilizes the cell wall of Gramnegative bacteria making the peptidoglycan layer more accessible to cleavage by lysozyme. Also, it should be brought to the reader’s attention that the composition of the tears changes during sleep. Open-eye and reflex tears have primarily lysozyme, lactoferrin, lipocalin, and sIgA, whereas closed-eye tears have increased amounts of sIgA (up to 80% of total tear protein), complement components, and of serum-derived proteins. There is also a large influx of neutrophils within 2–3 h of eye closure, which

provides additional defense factors in the guise of AMPs and reactive oxygen species, for example. Overall, these changes appear to represent a shift to a subclinical state of inflammation, which is believed to be necessary to protect the ocular surface from invasion by entrapped pathogens while the lids are closed. There is also an increase in proteins such as SLPI and elafin, which have potent antiprotease activity, and vitronectin, which inhibits complement, which serve to protect the ocular surface cells in this proinflammatory environment.

Defense Mechanisms of the Ocular

Surface Epithelia

Mechanical/Physical Defenses

The outermost superficial epithelial cells are bound by tight junctions, which effectively seal two cells together forming a barrier against free diffusion of fluids, electrolytes, and macromolecules as well as microorganisms and their secreted products. Tight junctions are also important in establishing and maintaining cell polarity. Polarized cells are characterized by differences in the composition and distribution of proteins and other surface molecules between apical and basolateral surfaces. This arrangement is maintained by the aforementioned tight junctions that segregate the domains and targeted delivery

that sends the molecules to their correct location. Disruption of polarity, such as occurs with corneal epithelial cells migrating to recover an injured area, has been shown to increase susceptibility to infection. This may be the result of a number of factors, for example, host cell receptors with which pathogens interact may be more abundant on basolateral surfaces. Also, loss of polarity disrupts the apical mucin-containing glycocalyx, which normally helps restrict bacterial attachment.

Another important feature contributing to defense is the constant turnover of the ocular surface epithelial cells. Both corneal and conjunctival epithelia have a population of strategically located stem cells that provide new cells to replace those being shed into the tear film. In the absence of a penetrating injury, infection begins in the most superficial epithelial layers and so through the constant renewal of cells, outer infected cells may be sloughed off before there is time for the infection to spread to the lower epithelial layers.

Pathogen Recognition

While the tear film acts to prevent pathogens from reaching the ocular surface epithelial cells in the first place, it is very important that the cells have a system to recognize the presence of invading organisms if they do happen to conquer the outer defenses. Host cell proteins that mediate pathogen recognition are often referred to as pathogen recognition receptors (PRRs) and they recognize pathogen-associated molecular patterns present on bacteria, viruses, and fungi. The primary PRRs on epithelial cells are toll-like receptors (TLRs). These are type I transmembrane glycoproteins, which have an extracellular leucine-rich domain and a cytoplasmic domain homologous to the signaling domain of the interleukin (IL)-1 receptor.

Of the 10 functional human TLRs that have been identified all have been reported to be expressed by corneal and conjunctival epithelial cells. Figure 4 shows a simplified diagram of the distribution of TLRs, their ligands, and signaling pathways. TLR1, 2, 4, 5, 6, and 10 are typically located at the cell surface. TLR2 forms heterodimers with TLR1 and with TLR6 and so can recognize a large variety of microbial products. For example, TLR2/6 heterodimers recognize lipoteichoic acid from Gram-positive bacteria and TLR2/1 heterodimers recognize triacyl lipoprotein/peptides of bacterial cell walls. TLR4 forms a complex with MD2 (also known as lymphocyte antigen 96) and cluster of differentiation 14 (CD14) protein and recognizes LPS from Gram-negative bacteria, while TLR5 recognizes flagellin, a component of bacterial flagella. TLR10, the ligand for which is unknown, is able to dimerize with TLR1 and TLR2. TLR3, 7, 8, and 9 are (typically) all located intracellularly, on endosomal membranes and recognize nucleic acids. TLR3 recognizes double-stranded RNA, a by-product of the replication of some viruses, whereas TLR7 and 8

recognize viral single-stranded RNA. TLR9 responds to unmethylated cytosine–phosphate–guanosine dinucleotide motifs found in both bacterial and viral DNA. Thus, by interacting with specific pathogen-derived molecules TLRs can detect the presence of a wide range of organisms, including those that replicate intracellularly.

The engagement of TLRs with their specific microbial ligand results in activation of intracellular signaling pathways, leading to a variety of functional changes in the ocular surface epithelial cells. The latter include production of inflammatory cytokines such as IL-6 and chemokines such as IL-8 that will attract neutrophils for phagocytosis and AMPs such as human b-defensin-2 that can directly kill invading pathogens (see the section entitled ‘Antimicrobial peptides’).

It is important that members of the normal ocular flora do not trigger TLR activation and hence cause unwanted inflammatory reactions at the ocular surface. To this end, it has been observed that flagellin from pathogenic, but not from nonpathogenic bacteria, can activate TLR5 in corneal epithelial cells. Expression of TLR5 (and possibly TLR4) appears to be restricted to basal and wing cells suggesting that TLR5 will only be activated when there is a breach in the corneal epithelium. Also, rather than being surface bound, TLR4 may be expressed intracellularly and so would not be available.

Evidence for the expression of another class of PRRs, the cytoplasmic nucleotide-binding and oligomerization domain (NOD) proteins, has yet to be investigated for the human ocular surface. However, mouse anterior eye tissue expressed both NOD1 (which recognizes meso-DAP, a component of peptidoglycan in Gram-negative organisms) and NOD2 (which recognizes muramyl dipeptide found in both Gram-positive and -negative bacteria).

Antimicrobial Peptides

AMPs are small peptides, most less than 50 amino acids, that are amphipathic and typically carry an overall positive charge (+2 or greater) due to a relative excess of amino acids such as arginine and lysine. These peptides show a broad spectrum of antimicrobial activity and many have additional effects on mammalian cell behavior.

The two major categories of mammalian AMPs are the defensins and cathelicidins. Human defensins are characterized by the presence of six cysteine residues that interact to form three disulfide bonds (the specific pattern of connectivity gives rise to two classes referred to as a and b). Both corneal and conjunctival epithelial cells express at least three b-defensins (hBDs). hBD-1 and hBD-3 are constitutively expressed, whereas the expression of hBD-2 is variable, being expressed by normal tissue only occasionally. Ocular surface hBD-2 expression is known to be inducible by exposure to both Gram-negative and -positive bacteria and

MD2

TLRTLR

4 4

CD14

bacterial products such as LPS, peptidoglycan, and lipoproteins. This upregulation is chiefly mediated via the activation of TLRs such as TLR2. In a recent study, expression of a novel beta defensin gene DEFB 109 was detected in the ocular surface epithelia, and interestingly its expression was decreased in inflammation and infection. As noted earlier, a-defensins HNP-1 through -3 are produced by neutrophils and are present in the normal tear film. They can also be detected in the cornea and conjunctiva when neutrophils infiltrate in response to a specific stimulus.

The cathelicidins have a highly conserved N-terminal cathelin domain and a variable antimicrobial domain. Only one, LL-37, is expressed in humans. LL-37 is expressed by both corneal and conjunctival epithelial cells and its expression is increased in response to corneal epithelial injury and bacterial challenge with P. aeruginosa and S. aureus. LL-37 is also a major component of neutrophil granules;

thus, its ocular surface levels are expected to rise in situations leading to infiltration of these and other inflammatory cells.

While defensins and LL-37 represent the main AMPs present at the ocular surface, others have been reported including liver expressed AMP-1 and -2, statherin, CCL28 and CXCL-1 (two of many antimicrobial cytokines), MIP3a, and thymosin b-4. However, as most of these molecules have other recognized functions, it is unlikely that antimicrobial effects are the major facet of their action at the ocular surface.

The primary site of AMP action is the microbial cell membrane, electrostatic disruption of which leads to permeabilization, loss of essential intracellular components, and death (see Figure 2). However, intracellular targets may also be utilized leading to inhibition of protein, peptidoglycan, and nucleic acid synthesis and interference with the activity of bacterial heat-shock proteins. Epithelial b-defensins and LL-37 are effective against

common ocular surface bacterial pathogens in vitro with hBD-3 and LL-37 showing the broadest spectrum and most potent activity. Animal studies have revealed that experimental infection with P. aeruginosa in genetically modified mice unable to express cathelicidin causes much more severe disease and corneal damage than in normal wild-type mice, thus showing that AMPs are important for ocular surface defense in vivo.

In addition to exerting direct antimicrobial effects, AMPs have other properties that help protect against pathogens and their destructive actions. For example, LL37 is known to bind and neutralize LPS. The latter is a product from Gram-negative bacteria and through activating TLR4 induces an inflammatory response and likely mediates much of the ocular surface damage that results from infections with pathogens such as P. aeruginosa. Thus, LL-37 may have a role in dampening LPS-mediated ocular surface inflammation and damage. Also, both defensins and LL-37 have been shown to be chemotactic for a variety of immune and inflammatory cells, including lymphocytes, monocytes, and immature dendritic cells thus may help draw these cells to a site of infection. AMPs also stimulate inflammatory and immune cell cytokine production, which in turn can modulate cellular functions. Additionally, hBD-3 has been shown to activate dendritic cells, raising the possibility that corneal/conjunctival epithelial hBD-3 may be able to activate epithelial Langerhans cells and stromal dendritic cells which in turn may initiate an adaptive immune response.

Other Contributions to Ocular Surface Epithelial Defense

As noted earlier, the outermost superficial cells of both the cornea and conjunctiva are coated in a matrix of carbohydrate referred to as the glycocalyx. By projecting from the epithelial cell surface, membrane spanning mucins MUC 1, 4, and 16 of the glycocalyx physically prevent pathogens from reaching the cell membrane. Also, some organisms are repulsed by negatively charged glycosaminoglycans present on the mucin.

The ocular surface epithelial cells also produce a variety of cytokines and chemokines that are important in protection from microbial invasion. IL-1 is an important cytokine released in response to trauma and injury and among a plethora of activities serves to regulate production of other molecules such as IL-6, growth regulated oncogene (GRO)-a, -b, -g, TNF-a, and IL-8, which in turn modulate inflammatory and immune cell infiltration and activation.

Dispersed between the epithelial cells of the cornea and conjunctiva are bone-marrow-derived dendritic cells called Langerhans cells. These cells are highly potent antigen-presenting cells which capture antigen and when mature present it to T lymphocytes in nearby secondary lymphoid tissues so activating adaptive immunity.

The Langerhans cells are typically found between basal epithelial cells and in the cornea their density is lowest in the central region and gradually increases toward the periphery. Also, most cells in central cornea appear to be small immature cells. In the periphery, larger mature major histocompatibility complex class II (MHC II) expressing cells with prominent dendritic processes are observed. Overall Langerhans cells perform a surveillance function, screening their environment for pathogens that have breached the defenses of the tear film and the epithelial barrier. If successful in their search they then activate adaptive immunity to help eliminate the invader.

Sensory nerves, which are particularly abundant in the cornea, also provide an important contribution to ocular surface defense. When triggered nerves induce the production of reflex tears, which, by virtue of their increased volume, help wash pathogens from the ocular surface and dilute out their toxic products. Release of neuropeptides such as substance P from the nerve termini may also affect epithelial cell cytokine production that, as noted above, may modulate other aspects of host defense.

The presence of a complement of nonpathogenic organisms also assists in preventing infection. Such commensals deplete the tears of nutrients, occupy attachment sites so preventing binding of pathogens, and produce bacteriocins that kill members of nonrelated species.

Concluding Remarks

In summary, the ocular surface and tears possess a wide range of chemicals and physical attributes that help prevent infection. Such redundancy is commonplace in biological systems, but is particularly important at the ocular surface where the inability to control and eliminate an infection can have dire consequences for visual function. Having multiple protective mechanisms is also necessary as pathogens are very adept at developing strategies to circumvent host defenses. While countering one specific mechanism is relatively easily achieved, developing multiple strategies is rather more challenging.

Acknowledgments

The author acknowledges grant support from NIH, NSF, and the State of Texas for her work on ocular surface antimicrobial peptides and thanks Kimberly Thompson of the University of Houston College of Optometry audiovisual department for drawing the figures.

See also: Antigen-Presenting Cells in the Eye and Ocular Surface; Conjunctiva Immune Surveillance; Corneal Epithelium: Cell Biology and Basic Science; Corneal Nerves: Anatomy; Corneal Nerves: Function; Immunopathogenesis of HSV Keratitis; Immunopathogenesis of

Pseudomonas Keratitis; Overview of Electrolyte and Fluid

Transport Across the Conjunctiva; Pathogenesis of

Fungal Keratitis.

Further Reading

Chen, G., Shaw, M. H., Kim, Y. G., and Nunez, G. (2009). Nod-like receptors: Role in innate immunity and inflammatory disease. Annual Review of Pathology 4: 365–398.

Evans, D. J., McNamara, N. A., and Fleiszig, S. M. J. (2007). Life at the front: Dissecting bacterial–host interactions at the ocular surface.

Ocular Surface 5: 213–227.

Flanagan, J. L. and Willcox, M. D. P. (2009). Role of lactoferrin in the tear film. Biochimie 91: 35–43.

Fleming, A. (1922). On a remarkable bacteriolytic element found in tissues and secretions. Proceedings of the Royal Society Series B

93: 306–317.

Gupta, G. and Surolia, A. (2007). Collectins: Sentinels of innate immunity. BioEssays 29: 452–464.

Hamrah, P. and Dana, M. R. (2007). Corneal antigen-presenting cells.

Chemical Immunology and Allergy 92: 58–70. Hazlett, L. D. (2007). Bacterial infections of the cornea

(Pseudomonas aeruginosa). Chemical Immunology and Allergy 92: 185–194.

McDermott, A. M. (2009). The role of antimicrobial peptides at the ocular surface. Ophthalmic Research 41: 60–75.

Paulsen, F. P. and Berry, M. S. (2006). Mucins and TFF peptides of the tear film and lacrimal apparatus. Progress in Histochemistry and Cytochemistry 41: 1–53.

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

Shafer, W. M. (ed.) (2006). Antimicrobial Peptides and Human Disease.

Berlin: Springer.

Yu, F.-S. X. and Hazlett, L. D. (2006). Toll-like receptors and the eye.

Investigative Ophthalmology and Visual Science 47: 1255–1263.

Glossary

Caspases – Humans express 11 different cysteineaspartic acid proteases classified as either initiator or effector caspases, which play essential roles in apoptosis, necrosis, and inflammation.

CD44 – A type I transmembrane glycoprotein that regulates conformational changes of integrin heterodimers and their ability to microcluster and anchor to the actin cytoskeleton.

Desmosomes – A junctional complex of adhesion molecules and linking proteins in the plasma membrane for cell-to-cell adhesion and contributes to the structural integrity through linkage of keratin cytoskeletal filaments of adjoining cells.

Epidermal growth factor (EGF) – Expressed by corneal epithelial and stromal cells, it promotes cell migration and the attachment of corneal epithelial cells to fibronectin.

Hemidesmosomes – Integral membrane protein complexes of integrin hetrodimers in the basal cell plasma membrane anchoring cells to the extracellular matrix.

Langerhans cells – Unique subset of dendritic cells located in mucosal stratified squamous epithelium and skin epidermis; as professional antigenpresenting cells, they express toll-like receptors (TLRs) as well as C-type lectin receptors. Melanocytes – Pigment-producing cells located within the uvea of the eye.

Nuclear factor-kappa B (NF-kB) – A protein complex that functions as a transcription factor; NF-kB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, and bacterial or viral antigens.

Pathogen-associated molecular patterns (PAMPs) – Small, highly conserved molecular motifs present in bacteria, yeast, or viruses but not in mammalian cells and induce inflammatory signaling via interaction with receptors families, such as TLRs.

Tritiated thymidine-labeling – Technique used to label cells actively undergoing DNA synthesis; it estimates the proportion of S-phase cells in a cell population.

Trophic – It refers to a nutrition-derived function. Zonula occludens – Also known as tight junctions, these structures are primarily composed of occludins and claudins to form a virtual impermeable barrier between adjacent cell membranes.

Introduction

Basic Structure and Function

The corneal epithelium is a remarkably proficient tissue that combines structure and function to serve as the major refractive component of the eye and maintain ocular surface integrity; yet provide a critical barrier protecting the visual axis from the external environment. The corneal epithelium is the outermost layer of the cornea and is comprised of stratified, nonkeratinized, nonsecretory, squamous epithelial cells, intermixed with Langerhans’ cells and occasional dendritic melanocytes. This tissue layer is a highly organized structure that is avascular and almost perfectly transparent in order to preserve the optical properties of the cornea. It is 5–7 cell layers thick and contains three cell types (posterior to anterior): basal cells, wing cells, and superficial cells. The deepest layer is comprised of cuboidal basal cells. This single cell layer of progenitor cells undergoes mitosis at a rate of 10–15% per day, followed by intermediate differentiation of daughter cells into one to three layers of wing cells as they migrate toward the surface. Superficial to wing cells is a threeto four-cell thick layer of terminally differentiated squamous cells. These cells constantly degenerate and desquamate from the corneal surface in a continuous cycle of shedding of superficial cells and proliferation of cells in the basal layer resulting in complete renewal of the epithelium every 7 days, as has been demonstrated by mitotic rate measurements in basal cells and by tracking the migration of tritiated thymidine-labeled cells to the ocular surface. This regenerative process is further maintained by a constant renewal of basal cells from the limbal epithelium via stem cells located in the limbus that differentiate into basal cells, followed by centripetal migration into the cornea. Although the corneal epithelium is constructed as a highly effective, semipermeable membrane on the ocular surface,

it is also well equipped to participate in the host response to invading pathogens and infection as described in greater detail below.

Corneal Infection

The corneal epithelium, similar to other mucosal epithelial linings in the body, constitutes the eye’s first line of defense against microbial pathogens. The cornea has an immuneprivileged status, which includes features such as avascularity of the cornea and dearth of antigen-presenting cells (APCs) in the central region of the cornea in order to protect the visual axis. Although resident dendritic cells or Langerhans cells, known for their global antigen-presenting properties, are present in the corneal periphery, they can readily be recruited into the central cornea when necessary. In addition, the corneal epithelium plays an active role in host defense against invading pathogens. Cells of the corneal epithelium: (1) recognize pathogens and their byproducts and, as a result, (2) respond through expression and secretion of a network of proinflammatory cytokines and chemokines that recruit inflammatory cells into the cornea,

(3) secrete antimicrobial products, and (4) promote wound healing and restoration of tissue homeostasis.

Keratitis is a condition of corneal inflammation, which can be caused by a number of bacterial, viral, and fungal pathogens. Clinically, it is associated with symptoms such as redness, tearing, reduced visual clarity, corneal discharge, and severe pain. Bacterial keratitis is the leading cause of corneal infection. Staphylococci are the most commonly occurring organisms in bacterial conjunctivitis and keratitis, including Staphylococcus (S.) aureus, S. pneumoniae, S. intermedius, and a-hemolytic streptococcus. Pseudomonas aeruginosa (P. aeruginosa) is most frequently encountered in keratitis cases associated with extended contact lens wear and constitute 19–42% of bacterial keratitis cases. Other bacteria known to cause keratitis include Escherichia coli and Morganella morganii. Common pathogens associated with viral keratitis include, yet are not limited to, herpes simplex virus (HSV) and adenovirus. HSV infection is the most common cause of corneal blindness in the United States at present time. Approximately 400, 000 people in the United States have been infected with ocular herpes and 50, 000 initial and recurring cases of HSV keratitis are diagnosed annually. Fusarium, Aspergillus (both filamentary fungi), and Candida albicans (a yeast) constitute those fungi associated with the majority of fungal keratitis cases. Sterile keratitis also incites an immune response from the corneal epithelium; however, instead of bacteria adhering to and invading the

ocular surface (requisite for bacterial keratitis), the epithelium responds to the presence of bacterial endotoxin (lipopolysaccharide or LPS). Sterile keratitis can also occur when bacteria colonize contact lenses and subsequently, release endotoxin onto the corneal surface, a condition known as CLARE or contact lens-induced acute red eye.

The effects of both the invading pathogen on corneal tissue and the host immune response to the pathogen lead to many structural alterations that are otherwise essential to maintaining transparency of the cornea and, as a result, directly compromise visual acuity. As such, components of the corneal epithelium have evolved to respond to infection and these are described in greater detail below.

TLRs and TLR-Related Molecules

Toll-like receptors (TLRs) are evolutionarily conserved type I transmembrane protein receptors that are expressed by corneal epithelial cells (as well as inflammatory cells) and function to respond to pathogens as the corneal epithelium’s first line of defense. These receptors initiate innate immunity and are essential for host defense against infection. TLRs recognize a broad spectrum of pathogen-associated molecular patterns (PAMPs), ranging from LPS (predominately recognized by TLR4), flagellin (TLR5), peptidoglycan (TLR2), singleor double-stranded RNA (TLR3,-7,-8), and unmethylated CpG DNA (TLR9). TLRs signal through several adaptor molecules, including the common adaptor protein myeloid differentiation factor (MyD)88 and MD-2; however, the reader is referred to Further Reading section for more information.

It has been proposed that corneal epithelial cells play a central role in regulation of inflammatory responses by expression of functional TLRs and adaptor molecules. Epithelial cells are thought to intrinsically respond to the presence of pathogens through TLR recognition of PAMPs. Of the 13 TLRs identified to date, human corneal epithelial cells have been shown to express TLR-1,-2,-4,-5,-6, and -9 either intercellularly or at the cell surface (and has yet to be fully elucidated). TLRs primarily associate as homodimers, with exceptions for TLR-1,-2, and -6, which form heterodimers. Upon recognition of PAMPs expressed by invading pathogens on the ocular surface, TLRs produce downstream signaling events which induce translocation of nuclear factor kappa B (NF-kB), a major transcription factor of numerous genes important in both innate and acquired immune responses. These genes include proinflammatory cytokines and chemokines, leading to activation of adhesion molecules, and subsequently resulting in macrophage and polymorphonuclear neutrophilic leukocyte (PMN) recruitment into the

cornea. Antimicrobial peptide expression by corneal epithelial cells is induced by TLR activation, as well. Although there is a common pathway for TLR activation (the myeloid differentiation protein–IL-1 receptor-associated kinase–tumor necrosis factor receptor-associated kinase (MyD88– IRAK–TRAF) pathway), individual TLRs most likely activate different, alternative, signaling pathways as well, and these remain under investigation (IL, interleukin).

Cytokines/Chemokines

The immune response of the corneal epithelium utilizes a variety of different substances to mount an attack upon organisms invading the ocular surface. Cytokines and chemokines are produced and endogenously released by corneal epithelial cells to directly and indirectly recruit and activate immune cells of both innate and adaptive immunity. Gram-negative bacterial endotoxin such as LPS, for example, elicit production of endogenous proinflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1. Protein levels for both IL-1a and IL-1b have been shown to be constitutively expressed in the normal human cornea. It has further been demonstrated that upon rupture of the epithelial cell membrane by infectious agents or trauma, IL-1a is passively released, contributing to increased vascular permeability, macrophage and lymphocyte infiltration and activation, angiogenesis, as well as regulatory effects on corneal fibroblasts. If left unchecked, these events would destroy the cornea; however, the epithelium also secretes both soluble and membrane-bound forms of IL-1RII, a receptor and natural antagonist of IL-1 in order to modulate the effects of this potent proinflammatory cytokine and consequently preserve visual function.

In addition to IL-1, in vitro studies using primary human corneal epithelial cells (HCECs) and telomeraseimmortalized HCECs have revealed expression and secretion of IL-6, IL-8, and TNF-a following either P. aeruginosa or HSV-1 challenge. TNF-a is a major proinflammatory mediator that is known to promote apoptosis, inflammation, and regulate immune cells. IL-6 is both a proand anti-inflammatory cytokine that can potentiate the inflammatory response, yet also modulate inflammation through its inhibitory effects on TNF-a and IL-1, while activating IL-1 receptor antagonist and IL-10. IL-8 is a chemokine that is secreted by the epithelium under stress and functions as a strong chemoattractant for neutrophils (PMN); MIP-3a, also released by corneal epithelial cells after infection, promotes directed migration of leukocytes, such as immature dendritic cells and effector T cells. TGF-a,-b1, and -b2 are expressed also by corneal epithelial cells and contribute to re-epithelization, an essential step in resuming normal corneal function after infection.

TGF-b1, in particular, is a potent anti-inflammatory cytokine that modulates lymphocyte activation and promotes wound healing; regarding the cornea, it is further known to promote proliferation and lamellar differentiation of corneal epithelial cells via keratocyte-mediated stimulation.

Antimicrobial Molecules

The corneal epithelium also employs the action of small (100 amino acids or less), positively charged (arginineand lysine-rich) molecules known as antimicrobial peptides to further assist in combating invading pathogens. Over 500 naturally occurring antimicrobial peptides have been identified in mammals. Typically these molecules are cationic polypeptides that disrupt bacterial membranes through charge interactions and hydrophobic amino acids. Many antimicrobial peptides and their microbicidal effects are induced locally by inflammatory stimuli at the site of infection and act synergistically with other anti-inflammatory mechanisms (cytokines, inflammatory cells) in defending against microbial pathogenesis. Of the four distinct structural classes of antimicrobial molecules, recent studies have shown that corneal epithelial cells secrete peptides from the defensin and cathelicidin families, which are thought to help protect the eye through broad spectrum activity against microorganisms including Gram-positive and -negative bacteria, fungi, and certain enveloped viruses. These molecules and their roles in preventing microbial invasion and managing infection are discussed in greater detail below.

b-Defensins

The defensins include a- and b-defensin subfamilies, all of which are characterized by a b-sheet-rich fold and three disulfide bridges. A third class of y-defensins has been recently identified in rhesus macaque leukocytes. Although y-defensin mRNA is detectable in humans, these transcripts contain a premature stop codon preventing translation of functional protein. Leukocytes (PMN) and various types of epithelial cells have been shown to express both a- and b-defensins; however, the corneal epithelium has been demonstrated to produce and secrete only members of the b-defensin subfamily.

Human b-defensins (hBDs) include 28 members, of which three have been associated with the corneal epithelium. Human b-defensin-1 (hBD-1) is constitutively expressed by corneal epithelial cells; while studies have shown that hBD-2 is induced by bacterial infection and bacterial products such as lipoprotein and lipotechoic acid, and is mediated by TLR2. In addition, cytokines TNF-a and IL-1 upregulate hBD-2 expression by corneal epithelial cells. Expression of hBD-3 is more variable,

whereby some studies have indicated solely constitutive expression by corneal epithelial cells; and others have demonstrated inducible expression by TNF-a and interferon-gamma (IFN-g). Regardless, hBD-3 was shown by McDermott and colleagues to exert most potent antibacterial activity against S. epidermidis, S. aureus, and P. aeruginosa using in vitro antimicrobial assays, followed by hBD-2; while hBD-1 showed moderate activity against Pseudomonas, but no activity against Staphylococcus strains. In vivo, it was recently demonstrated that murine (m) BD2, but not mBD1, was protective in the P. aeruginosa-infected mouse cornea. The importance of these antimicrobial peptides has been demonstrated by knocking out the mouse b-defensin-1 gene, which led to less effective clearance of Haemophilus influenzae from the lung and increased colonization of Staphylococcus in the bladder.

Cathelicidins

Of the numerous members of the cathelicidin family, only LL-37 has been described in humans. Cathelicidins express a highly conserved cathelin domain and a less conserved, more variable antimicrobial region. LL-37, as its name suggests, is a 37-amino acid linear peptide expressed by inflammatory/immune cells and epithelial tissue. It is derived by cleavage of the precursor, human cationic antimicrobial protein 18 (hCAP18) and appears to function as both an antibacterial peptide and immunomodulator. Low expression levels of LL-37 are detected constitutively and subsequently upregulated following injury, infection, and exposure to IL-1b, as well as heatkilled P. aeruginosa. Regarding antimicrobial properties, LL-37 is thought to function in a similar manner to that described for defensins and work synergistically with corneal epithelial proteins such as defensins, lactoferrin, and lysozyme (latter two present in tear film). LL-37 is able to bind and neutralize LPS and lipotechoic acid, thus reducing the inflammatory response associated with these molecules. In addition to eradicating ocular pathogens, studies have demonstrated that LL-37 enhances the innate and adaptive immune response in the corneal epithelium through modulation of cytokine/chemokine expression. Using an in vitro stimulation assay, LL-37 was shown to induce production of IL-1b, IL-6, IL-8, and TNF-a by human corneal epithelial cells. Furthermore, this molecule promotes wound repair through enhanced cell migration, including fibronectin-induced migration by stimulating corneal epithelial cells.

In addition to microbicidal activities of b-defensins and LL-37, these molecules also wield effects over immune cells and influence wound healing. They have been demonstrated to recruit and activate immune cells through the induction of cytokine and chemokine production by epithelial cells, which further actuate the cellular components of the immune response to corneal infection. Regarding

postinfection, both hBD-2 and LL-37 have been shown to be upregulated in an in vitro organ culture model of corneal epithelial wound healing suggesting roles for corneal epithelial cell migration and proliferation.

Complement System

The complement system also contributes to the first line of innate immune defense against corneal infection. This critical system is composed of a series of effector and regulatory proteins that sequentially activate one another to generate biologically active molecules, such as opsonins and chemotaxins. The complement system is continuously activated at low levels in the eye under normal conditions, as supported by detection of soluble and membraneassociated complement regulatory proteins (e.g., rsCD59), which are also strongly expressed in the corneal epithelium for tight regulation of aberrant activation. Components of complement are also more heavily distributed in the peripheral versus the central cornea, potentially due to the diffusion of complement molecules from corneal limbal vessels. In response to infection activation of complement can occur via both the classical and alternate pathways.

Secretory IgA

Although this molecule is not essential in ocular defense, secretory IgA (sIgA) does play a major role in the prevention of some corneal infections, including Pseudomonas and Acanthamoeba. In fact, over 75% of the general population contain anti-P. aeruginosa-specific IgAs in their tear film. sIgA protects the corneal epithelium by accumulating in the ocular mucin layer and displays an antigen–antibody clearance function. Aggregated sIgA opsonizes bacteria for PMN phagocytosis and processing via recognition by sIgA receptors expressed on the immune cell surface. IL-8, which is secreted by corneal epithelial cells during infection, further enhances the ability of sIgA to induce release of reactive oxygen species by PMN.

Adhesion Molecules

Under normal conditions, integrity of the corneal epithelium depends upon a number of factors, including adhesion molecules. Corneal epithelial cells are interdigitated, particularly in the middle layers, and largely interconnected by desmosomes. Basal cells are firmly attached to the basement membrane, neighboring basal cells and overlying wing cells via hemidesmosomes. Tight junctional complexes, or zonula occludens, found only between superficial cells are