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

Glossary

Akt1 – Also known as Akt or protein kinase B, it is an important molecule in mammalian cellular signaling and inhibits apoptotic processes.

Eicosanoids – Signaling molecules made by oxygenation of 20-carbon essential fatty acids. There are four families of eicosanoids: the prostaglandins, prostacyclins, thromboxanes, and the leukotrienes.

IRF1 (interferon regulatory factor 1) – A member of the interferon regulatory transcription factor family. IRF1 serves as an activator of interferons alpha and beta transcription, and in mouse it has been shown to be required for double-stranded RNA induction of these genes. IRF1 also functions as a transcription activator of genes induced by interferons alpha, beta, and gamma. Further, IRF1 has been shown to play roles in regulating apoptosis and tumor-suppression. Keratitis – A condition in which the cornea becomes inflamed. The condition is often marked by moderate to intense pain and usually involves impaired eyesight.

LPS (lipopolysaccharide, endotoxin) – Major outer membrane component of Gram-negative bacteria comprising a lipid A core (endotoxin) and polysaccharide of varying length and composition. Toll-like receptor 4 (TLR4) and MD2 (myeloid differentiation 2) bind to the lipid A moiety.

MAPK (mitogen-activated protein kinase) –

Serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/ apoptosis.

Matrix metalloproteinase-9 (MMP-9) – Gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase is a biological enzyme. Proteins of the MMP family are involved in the breakdown of extracellular matrix in normal physiological processes, as well as in disease processes. Most matrix metalloproteinases (MMPs) are secreted as inactive pro-proteins which are activated when cleaved by extracellular proteinases. The enzyme encoded by this gene degrades type IV and V collagens.

NF-kB (nuclear factor-kappa B) – A protein complex that is 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.

Peroxynitrite – An anion, with the formula ONOO , it is an unstable valence isomer of nitrate, NO3 , which has the same formula but a different structure. Although peroxynitrous acid is highly reactive, its conjugate base, peroxynitrite is stable in basic solution.

SIGIRR (single immunoglobulin interleukin 1 receptor (IL-1R)-related protein) – An inhibitory member of this receptor superfamily. SIGIRR seems to temper cellular activation by IL-1, LPS, and probably other activators of receptors in the TLR–IL- 1R superfamily, such that the biological outcome will be the result of a balance between activation by a receptor and dampening by SIGIRR. SIGIRR therefore acts as a brake on the TLR system, which may be essential for regulating the detrimental effects of innate immunity, as occurs in sepsis and chronic inflammation.

siRNA (short interfering RNA or silencing RNA) –

A class of 20–25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference pathway, where it interferes with the expression of a specific gene.

T helper (Th) 1 (e.g., predominant IL-12 and interferon gamma production) and Th2 (predominant IL-4, 5, 10, 13) response –

Response involving effector T cells that play an important role in establishing and maximizing the capabilities of the immune system.

Introduction

In the United States, microbial keratitis is frequently associated with complications resulting from use of extended wear contact lenses with an incidence of about 30 000 cases annually. The cost of treatment is estimated at between $15 and $30 million, representing a considerable medical and economic impact. Of the bacterial organisms able to induce keratitis, Pseudomonas aeruginosa remains an important Gram-negative pathogen. The bacteria is often referred to as opportunistic, as it is capable of inducing keratitis not only in extended wear contact lens users, but

also in more tropical climates, and in patients that are either debilitated or hospitalized. Most of the complications of bacterial keratitis are structural alterations of the cornea, but other sight-threatening problems include development of secondary glaucoma and cataract. These consequences are largely caused by the host’s inflammatory response, but the influence of bacterial toxins, exoproducts, and toxicity from antibiotic treatment cannot be overlooked. There is also a recent increase in the incidence of reported antibiotic resistant strains and failure of an initially promising treatment using antimicrobial peptides to manage keratitis, suggesting that better understanding of the pathogenic mechanisms of disease induction by this pathogen will be critical to development of improved therapeutic strategies.

P. aeruginosa, like most other microorganisms, typically requires surface injury to permit corneal invasion. Because it has few nutritional requirements, it can adapt to a variety of ecological conditions and niches, such as preserved ophthalmic solutions and the hospital environment. Pseudomonal and other Gram-negative bacterial infections often present as a rapidly progressing, suppurative stromal infiltrate with a marked mucopurulent exudate. Yellowish coagulative necrosis surrounded by inflammatory epithelial edema is distinctive and stromal ulceration can lead to stromal tissue destruction and vision loss. A ring infiltrate may appear in the surrounding paracentral cornea and hypopyon (a dense inflammatory coagulum) is usually present; in addition, descemetocele (ulcer penetrated through cornea) formation or corneal perforation are not uncommon. Animal models of bacterial keratitis continue to be of value in the study of this disease and are produced by topical bacterial application after abrading the epithelium, by intrastromal inoculation, or by placing a contaminated suture or contact lens on the cornea. These approaches and models have led to increased understanding of the mechanisms of corneal inflammation and innate immunity that are operative in bacterial keratitis.

Bacterial eradication by neutrophils (PMN) involves phagocytosis, lysosomal degranulation and bacterial killing within the acidic lysosomal compartment of the cell. Phagocytosis and intracellular degranulation by PMN also involve oxidative attack, production of toxic oxygen metabolites, triggering of the respiratory burst, biosynthesis of superoxide anions, and other oxidizing agents such as hydrogen peroxide and formation of peroxynitrite. Phagocytic secretion and lysis result in release of extracellular lysosomal enzymes, including, but not limited to, elastase, collagenase and myeloperoxidase (MPO). These enzymes and the oxygen-derived free radicals cause stromal destruction by breaking down collagen, digesting glycosaminoglycans, and disrupting stromal keratocytes. Nitric oxide (NO) also mediates vasodilation and can be important in bacterial killing as well as in bystander tissue

damage. These and other substances released from activated PMN and other inflammatory cells (e.g., macrophages, Mf) contribute to stromal necrosis and corneal edema during bacterial disease. Bacterial endotoxin and exotoxins also stimulate Mf to release biologically active substances including, but not limited to, interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a, cytokines that synergize to elicit inflammatory events. Selected cytokines, certain eicosanoids, and other molecular mediators are also involved in ulceration and angiogenesis during bacterial keratitis.

Maintenance of leukocyte recruitment during inflammation requires intercellular communication between infiltrating leukocytes, the epithelium, neuropeptides such as substance P (SP) and vasoactive intestinal peptide (VIP), vascular endothelium, and resident stromal cells. These events are mediated by generation of early response cytokines (e.g., IL-1), the expression of adhesion molecules such as intercellular adhesion molecule-1, and the production of chemotactic cytokines and chemokines.

PMN, Cytokines, and Chemokines

If leukocytes such as PMN persist within the cornea, destructive pathology ensues, including stromal scarring and perforation, potentially requiring corneal transplantation. PMN infiltration into inflamed tissue is controlled largely by local production of inflammatory mediators. In the mouse, two members of the CXC family of chemokines, MIP-2 (functional homolog of human IL-8) and KC, are potent chemoattractants and activators of PMN. In corneal infections, MIP-2 has been shown to be the major chemokine that attracts PMN into the P. aeruginosa infected cornea and persistence of PMN in the cornea of susceptible (cornea perforates) C57BL/6 versus resistant BALB/c (no corneal perforation) mice (Table 1) was found to correlate with higher MIP-2 chemokine levels (both mRNA and protein). IL-1, produced by Mf, monocytes, and resident corneal cells also influences PMN infiltration into tissues. When tested, IL-1a and b (mRNA and protein) were elevated in the infected cornea of C57BL/6 over BALB/c mice. Furthermore, after infection, MMP-9 was shown to upregulate chemotactic cytokines/chemokines (IL-1b and MIP-2), contribute to degradation of collagen IV, and overall, enhanced P. aeruginosa keratitis. In contrast, neutralization of IL-1b in infected C57BL/6 mice reduced

Table 1 Response to P. aeruginosa corneal infection in mice

disease severity, evidenced by reduction of PMN in cornea (MPO assay), decreased bacterial load, and decreased levels of MIP-2 at both the mRNA and protein levels. The use of caspase 1 (enzyme that processes IL-1b to generate the mature cytokine) inhibitor treatment in C57BL/6 mice confirmed these data, even when inhibitor treatment was initiated 18 h after disease onset. In addition, improvement was augmented when the caspase 1 inhibitor was given after infection together with the antibiotic ciprofloxacin. A live attenuated P. aeruginosa vaccine also has been tested and found to elicit outer membrane protein-specific active and passive protection against corneal infection.

T Cells and IL-12

The role of other cells, such as T cells in P. aeruginosa corneal infection, was first studied in inbred C57BL/6 wild type and cytotoxic, CD8(+) T deficient, b2 microglobulin knockout mice (on the C57BL/6 background). Corneas of both groups of mice perforated by 7 days post-infection (p.i.) and histopathology was similar, with infiltration of PMN within 24 h p.i. In contrast, corneas of wild-type mice antibody depleted of helper, CD4(+) T cells and infected with P. aeruginosa did not perforate at 7 days p.i., versus mice depleted of CD8(+) T cells or treated with an irrelevant antibody. Antibody neutralization of IFN-g before infecting C57BL/6 mice also prevented corneal perforation and was associated with a lower delayed type hypersensitivity response when compared with C57BL/6 mice similarly treated with an irrelevant antibody. These data support that a CD4(+) T cell T helper type 1 (Th1) dominant response following P. aeruginosa infection is associated with genetic susceptibility and corneal perforation in C57BL/6 mice and provided the first evidence that CD4(+) T cells are important in development of severe keratitis and eventual corneal perforation. In addition, use of gene array studies confirmed a Th1 versus Th2 bias of C57BL/6 versus BALB/c mice to infection with Pseudomonas. Other studies investigated whether IL-12 (IL-12 p40) was associated with IFN-g production and the susceptibility response of C57BL/6 mice after P. aeruginosa challenge. IL-12 p40 knockout mice (C57BL/6 background) versus wild-type mice were tested to examine disease progression in endogenous absence of the cytokine. When tested, both groups of mice were susceptible to corneal challenge with P. aeruginosa and corneal perforation was observed at 5–7 days p.i. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analyses confirmed that IL-12 p40 message and protein levels were elevated after infection in the wild type over the expected absence of IL-12 p40 in the knockout mouse cornea. Immunostaining for IL-12 in wildtype C57BL/6 mice revealed that stromal PMN were at least one source of the cytokine.

IL-18 and IFN-g

The role of IL-18 and IFN-g production in the resistance response of the predominantly Th2 responding BALB/c mouse was also tested. Semi-quantitative RT-PCR detected IFN-g mRNA expression levels in the cornea of infected mice at 1–7 days p.i. Cytokine levels were significantly upregulated when compared with control, uninfected normal mouse corneas. Using RT-PCR, IL-18 mRNA expression was detected constitutively in the normal, uninfected cornea, but levels were significantly elevated 1–7 days p.i. To test whether IL-18 regulated IFN-g production, BALB/c mice were injected with an anti-IL-18 monoclonal antibody. Treatment decreased corneal IFN-g mRNA levels and both bacterial load and disease severity increased when compared to immunoglobulin G injected control mice. Data provided evidence that IL-18 is critical to the resistance response of BALB/c mice by induction of IFN-g and that IFN-g is required for bacterial killing/ stasis in the cornea. Another separate study showed that the killing effect of IFN-g was indirect, through regulation of NO levels in the infected cornea.

IFN-g and SP

Further study of the resistance response in BALB/c mice examined the role of the pro-inflammatory neuropeptide, SP in IFN-g production. This study provided evidence that natural killer (NK) cells were required to produce IFN-g; that the cells expressed the neurokinin-1 receptor (NK1R, the major SP receptor); and that they directly and tightly regulated IFN-g production through SP interaction with this receptor, suggesting a unique link between the nervous system and development of innate immunity in the cornea. On the other hand, a disparate distribution of SP in the infected cornea of susceptible C57BL/6 (higher levels) versus resistant BALB/c (lower levels) mice also has been documented and blocking the interaction of SP with its major receptor (NK1R) in C57BL/6 mice improved disease outcome, supporting a role for SP in development of the susceptible phenotype after P. aeruginosa corneal infection. Thus, the amount of SP and its interaction with available NK1R sites after infection contributes either to resistance or susceptibility, and appears an important component of keratitis outcome in these murine models.

Neuropeptides

SP

In this regard, SP involvement in the pathophysiology of inflammatory disease generally has been evidenced by aberrant levels of SP and SP containing nerve fibers,

as well as NK1R, in diseased tissues. SP has been shown to elicit cytokine secretion (IL-2, IL-4, IL-10, and IFN-g) from mouse T cells. In addition, it was demonstrated that human bronchial epithelial cells produce IL-6, IL-8, and TNF-a after SP treatment. SP-induced cytokine production and secretion by leukocytes, including T cells, Mf, and dendritic cells leads to the release of a number of inflammatory mediators such as additional cytokines, oxygen radicals, arachidonic acid derivatives, and histamine, all of which further amplify the inflammatory response.

VIP

Recent studies have also provided ample evidence for another neuropeptide, VIP, functioning as a potent endogenous anti-inflammatory molecule affecting the immune response antithetically when compared to SP. VIP regulates inflammatory mediators via several transduction pathways and transcription factors essential for gene activation, such as nuclear factor-kappa B, interferon regulatory factor-1 (IRF-1), mitogen-activated protein kinase (MAPK), and cAMP response element. VIP downregulates the production of several pro-inflammatory cytokines, including: TNF-a, IL-1, IL-6, IL-12, and IFN-g, while stimulating production of anti-inflammatory cytokines IL-10, IL-1 receptor (R) antagonist, and TGF-b. Investigation of the effect of VIP in a murine endotoxin challenge model showed that after treatment with VIP, levels of TNF-a and IL-6 in serum and peritoneal fluid were reduced by almost 50%. Regarding the eye, VIP treatment converted the susceptible phenotype (corneal perforation) to resistant (no perforation) in a mouse model of P. aeruginosa-induced infection via downregulation of pro-inflammatory mediators, upregulation of anti-inflammatory molecules, and modulation of host inflammatory cell activation. Thus VIP, a 28-amino-acid peptide, delivered by several types of neurons to immune organs and lymphoid tissues in the heart, gastrointestinal tract, lungs, kidney, cornea and skin, is anti-inflammatory. In fact, evidence indicated a differential response to VIP between infected BALB/c (more) and C57BL/6 (less) mice, due to disparate VIPR1 expression by Mf (which can be induced in a dose-dependent manner by VIP itself). Mf are known to play a key role in regulating/ balancing proand anti-inflammatory activity in the resistant (BALB/c) and susceptible, C57BL/6 murine models; therefore, evidence that VIP influences the functional behavior of these cells further supports a more salient role for this neuropeptide in regulating the inflammatory response.

TLR

Mouse eye infection models have also been used to study the role of Toll receptors in disease. The Toll family of

receptors, conserved throughout evolution from flies to humans, is central in initiating innate immune responses. This family of receptors, composed of trans-membrane molecules, links the extracellular compartment where contact and recognition of microbial pathogens occurs and the intracellular compartment, where signaling cascades leading to cellular responses are initiated. Gene array data showed that the expression of TLRs and related molecules including CD14, soluble IL-1 receptor antagonist, TLR-6, and IL-18R-accessory-protein were significantly elevated in susceptible (C57BL/6) versus resistant (BALB/c) mice following challenge with P. aeruginosa. In another model system involving induction of sterile keratitis, when C3H/HeJ (Toll-like receptor 4, TLR4, point mutation) versus control mice were treated with lipopolysaccharide (LPS) from P. aeruginosa, a significant increase in stromal thickness and haze was seen in the cornea of TLR4 sufficient control, but not in TLR4 deficient mutant mice. The severity of disease coincided with PMN stromal infiltration, indicating that TLR4 signaling enhances corneal disease. In contrast, in a bacterial keratitis model, TLR4 mRNA expression was markedly increased in the cornea of resistant BALB/c mice after bacterial infection. These data led to testing corneas from TLR4-deficient and wild-type control (BALB/c) mice after challenge with live P. aeruginosa to determine the role of TLR4 in bacterial keratitis. Given that TLR4 deficiency was suggested to be protective in sterile keratitis, we might predict that corneas of TLR4-deficient mice would be less susceptible and exhibit a decreased inflammatory response to bacterial infection. In marked contrast, TLR4-deficient versus control mice exhibited significantly increased inflammation and corneal perforation instead of healing after infection. Furthermore, data from clinical score, slit lamp, and histopathology confirmed that TLR4-deficient versus wild-type control mice exhibited significantly increased corneal disease with more opacity and more severe stromal swelling and destruction. In addition, bacterial load (more than 10-fold higher) and PMN recruitment (MPO activity) were markedly upregulated in the infected cornea of TLR4-deficient versus TLR4-sufficient control mice. The data provide strong evidence that TLR4 is required for the resistance response of BALB/c mice to P. aeruginosa challenge and, unlike the sterile keratitis model, TLR4 is required for disease resolution in bacterial keratitis.

Overall, it appears conflicting that TLR4 is critical in the pathology of corneal disease in sterile keratitis, while it is protective in bacterial keratitis and required for host resistance. In fact, these data illustrate the characteristic double-edge sword activity of TLR4 activation. On the one hand, in the keratitis model, TLR4 recognized LPS, a component of P. aeruginosa, and initiated an innate immune response that was important for bacterial clearance. TLR4 deficiency impaired bacterial clearance, led

to overgrowth of bacteria, an overwhelming PMN infiltrate and excessive pro-inflammatory cytokine production. This in turn contributed to corneal destruction and perforation. In the sterile keratitis model, activation of LPS-TLR4 signaling in TLR4 sufficient mice, lead to pro-inflammatory cytokine production and PMN infiltration, increased stromal thickness and contributed to haze production which increased, albeit transiently, corneal perturbation when compared with TLR4 mutant mice.

Negative regulators of TLR are also of importance and recent evidence showed that one of them, single immunoglobulin IL-1R related molecule is differentially expressed in BALB/c (resistant) versus C57BL/6 (susceptible) mice. This Toll receptor is critical in resistance to P. aeruginosa infection in BALB/c mice, functioning to downregulate type 1 immunity and negatively regulating sustained IL-1 and TLR4 signaling. siRNA treatment to knockdown TLR9 was also found to influence the outcome of bacterial keratitis, and lead to reduced inflammation, but with the unwanted effect of decreased bacterial killing.

Apoptosis

Delay in apoptosis in bacterial keratitis, as evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, also contributes to corneal perforation in C57BL/6 mice. Consistent with this finding, Bcl-2, an anti-apoptotic gene, was significantly upregulated in C57BL/6 mouse cornea at 18 h p.i., suggesting that the delayed onset of apoptosis in C57BL/6 mouse cornea may be, in part, due to upregulation and signaling of this gene. These data are also consistent with previous studies showing that over-expression of Bcl-2 reduces lymphocyte apoptosis in P. aeruginosa-induced pneumonia.

In the process of apoptosis, execution of cells largely depends on proteolytic cleavage and activation of caspase 3. In this regard, BALB/c versus C57BL/6 mouse cornea expressed more intense staining for activated caspase 3 at 1 day p.i. compared to the delayed peak intensity in C57BL/6 mice. The two mouse groups were also disparate for expression of caspase 9, with significantly more mRNA expression in BALB/c over C57BL/6 mice. Although only hypothetical, these data suggest that different pathways of apoptosis may be operative in the infected cornea of the two groups of mice. Our studies, using a combination of TUNEL and immunostaining, as well as PMN depletion, also provided evidence that the corneal apoptotic cells identified in both groups of mice were predominantly PMN and confirmed that apoptosis of these cells is delayed in C57BL/6 mice. We hypothesize that earlier apoptosis of PMN in resistant BALB/c mice is consistent with effective elimination of invading bacteria, while inducing minimal tissue damage due to unresolved and persistent inflammation.

Neuropeptides

The balance between apoptosis and cell survival, as well as the tissue milieu and timing of apoptosis, is critical in immune defense. In this regard, the neuropeptide SP, mentioned above, is a potent anti-apoptotic regulator and can exacerbate inflammation.

SP has been shown to stimulate phosphorylation of the anti-apoptotic molecule Akt in colonic mucosa both in vivo and in vitro, preventing apoptosis in humans with colitis. In another in vitro study, SP induced p53, Bcl-2, and NO expression in peritoneal Mf, blocking apoptotic signals. SP delays spontaneous apoptosis of PMN in a dose-dependent fashion by its interaction with the NK1R in vitro, and this effect could be inhibited by application of the NK1R antagonist GR82334.

In this regard, C57BL/6 mice treated with another NK1R antagonist, Spantide I, showed significantly improved disease outcome and an earlier onset of apoptosis, similar to the pattern observed in naturally resistant BALB/c mice. Consistent with earlier onset of apoptosis, mRNA expression of caspase 3 was also significantly upregulated earlier in the cornea of Spantide I, versus control treated animals. The data suggest that the protective mechanism of Spantide I treatment in C57BL/6 mice involves induction of earlier PMN apoptosis in the infected cornea, with less bystander tissue damage. Whether in C57BL/6 mice, the effects of Spantide I are directly mediated via the PMN or indirectly through Mf regulation of PMN also required resolution. Clodronate depletion of Mf with or without Spantide I treatment revealed that in the absence of the Mf, apoptosis was reduced/absent in the cornea. To further explore the role of this cell in susceptibility and resistance, Mf from both groups of mice (C57BL/6 and BALB/c) were incubated in the presence of SP together with LPS or with LPS alone. Significantly fewer apoptotic cells were detected in cells from C57BL/6 mice in the presence of SP and LPS versus LPS alone, while the same combined treatment (SP and LPS) did not decrease the number of LPS induced apoptotic Mf from BALB/c mice. To determine the mechanism for the disparate response to SP treatment between Mf from the two mouse groups, expression levels of the NK1R were comparatively tested. Although cells from both groups expressed the receptor, with a slightly weaker signal in BALB/c cells, after LPS stimulation, mRNA expression for the NK1R was only detected in cells from C57BL/6 mice. These data suggest that the possible mechanism for the absence of the anti-apoptotic effects observed in BALB/c Mf after SP treatment may involve a low level of NK1R expression on the cells and possible rapid depletion of the receptor upon LPS stimulation. In this regard, VIPR1, the major receptor for the neuropeptide VIP, was also reported to be expressed disparately in Mf from C57BL/6 (less) versus BALB/c (more) mice.

Figure 1 P. aeruginosa infection in Marmoset monkey model.

Mf also may provide distinct activation signals for Th1/Th2 differentiation. In this regard, others have reported that Leishmania major infected Mf enhanced the proliferation and IL-4 secretion of Th2 T cells, but inhibited the response of Th1 Tcells. When testing for this possibility, we detected that Mf from C57BL/6 mice expressed significantly more IL-12, while BALB/c Mf expressed more IL-10 after LPS stimulation. IL-10 appears protective in the BALB/c infected cornea, as after subconjunctival injection of clodronate-containing liposomes to deplete these cells, higher levels of IFN-g and lower levels of IL-10 were detected and resistant mice were converted to the susceptible phenotype. It was also reported that VIP treated C57BL/6 mice showed improved disease outcome and increased IL-10 expression after P. aeruginosa corneal infection. Furthermore, the data suggest that SP, which acts in an anti-apoptotic manner toward activated C57BL/6 mouse Mf, may also enhance an IL-12 driven, Th1 type immune response and thus further contribute to the susceptibility of this mouse strain to P. aeruginosa infection.

New Animal Model

Without doubt, experimental animal models of bacterial infection with P. aeruginosa have provided us with important insights into mechanisms underlying ocular infection and inflammation and our understanding of the effector and regulatory mechanisms involved in disease continues to grow. However, our understanding and knowledge of the precise mechanisms operative in human cases of keratitis (sterile and infectious) remains much more limited. In this regard, development of a new primate model of keratitis would be a timely and important translational approach to facilitate eventual human application of what has been learned in rodent and other species. The Marmoset monkey, with 98% homology to the human genome, provides such a model. Initial studies have been undertaken in the monkey and show that with scarification of the cornea (similar to that done in the mouse), infection can be

initiated and progresses similar to the human pattern. A photograph of the new animal model is provided in Figure 1. This model will allow us to explore treatment paradigms that have been efficacious in the mouse and other rodent species and may more quickly result in clinical trial of successful candidates.

See also: Corneal Epithelium: Response to Infection.

Further Reading

Delgado, M., Pozo, D., and Ganea, D. (2004). The significance of vasoactive intestinal peptide in immunomodulation. Pharmacological Reviews 56(2): 249–290.

DeVane, L. The fibromyalgia community, substance P: A new era, a new role. http://fmscommunity.org/subp.htm (accessed June 2009).

Dinarello, C. A. (2005). Blocking IL-1 in systemic inflammation. Journal of Experimental Medicine 201(9): 1355–1359.

Ferguson, T. A. and Griffith, T. S. (2007). The role of Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) in the ocular immune response. Chemical Immunology and Allergy 92: 140–154.

Harrison, S. and Geppetti, P. (2001). Substance P. International Journal of Biochemistry and Cell Biology 33(6): 555–576.

Hazlett, L. D. (2004). Corneal response to Pseudomonas aeruginosa infection. Progress in Retinal and Eye Research 23(1): 1–30.

Janeway, C. A., Jr. and Medzhitov, R. (2002). Innate immune recognition. Annual Review of Immunology 20: 197–216.

Kernacki, K. A., Barrett, R. P., Hobden, J. A., and Hazlett, L. D. (2000). Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection.

Journal of Immunology 164(2): 1037–1045.

Lighvani, S., Huang, X., Trivedi, P. P., Swanborg, R. H., and Hazlett, L. D. (2005). Substance P regulates natural killer cell interferon-gamma production and resistance to Pseudomonas aeruginosa infection.

European Journal of Immunology 35(5): 1567–1575.

McClellan, S. A., Huang, X., Barrett, R. P., van Rooijen, N., and Hazlett, L. D. (2003). Macrophages restrict Pseudomonas aeruginosa growth, regulate polymorphonuclear neutrophil influx, and balance proand anti-inflammatory cytokines in BALB/c mice. Journal of Immunology 170(10): 5219–5227.

Meek, B., Speijer, D., de Jong, P. T., de Smet, M. D., and Peek, R. (2003). The ocular humoral immune response in health and disease.

Progress in Retinal and Eye Research 22(3): 391–415.

Rudner, X. L., Kernacki, K. A., Barrett, R. P., and Hazlett, L. D. (2000). Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation.

Journal of Immunology 164(12): 6576–6582.

Strand, F. L. (1999). Neuropeptides: Regulators of Physiological Processes. Cambridge, MA: MIT Press.

Substance P: A modulator of inflammation. (1998). http://www. woongbee.com/Cytokine/Cytokine%20bulletin/Spring%201998/ spring1998-3.htm (accessed June 2009).

Szliter, E. A., Lighvani, S., Barrett, R. P., and Hazlett, L. D. (2007). Vasoactive intestinal peptide balances proand anti-inflammatory cytokines in the Pseudomonas aeruginosa-infected cornea and protects against corneal perforation. Journal of Immunology 178(2): 1105–1114.

Taylor, P. R., Martinez-Pomares, L., Stacey, M., et al. (2005). Macrophage receptors and immune recognition. Annual Review of Immunology 23: 901–944.

Todar, K. (2008). Todar’s Online Textbook of Bacteriology: Immune Defense against Bacterial Pathogens: Innate Immunity. http:// textbookofbacteriology.net/innate.html (accessed June 2009).

Winkler, J. D. (ed.) (1999). Apoptosis and Inflammation. Basel: Birkhauser Verlag.

Relevant Websites

http://www.diseasesdatabase.com – Diseases Database Ver 1.8; Medical lists and links Diseases Database, Vasoactive Intestinal Peptide.

http://www.macrophages.com – Macrophages.com. http://users.rcn.com – RCN Corporation: Apoptosis. http://users.rcn.com – RCN Corporation: Innate Immunity. http://www.ResearchApoptosis.com – Research Apoptosis.

Glossary

Clodronate – Drug that preferentially kills macrophages, but has no known deleterious effects on other cells of the innate or adaptive immune responses.

Interferon-g (IFN-g) – Cytokine produced by T cells that activates macrophages and enhances their capacity to kill Acanthamoeba trophozoites.

Mannose-binding protein (MBP) – Lectin receptor that is expressed on Acanthamoeba trophozoites and facilitates their adherence to mannosylated proteins that are expressed on corneal epithelial cells.

Major histocompatibility complex (MHC) class-I antigens – Antigens that allow cytotoxic

T lymphocytes to recognize and kill cells infected with viruses and some protozoal parasites.

Mannose-induced protease 133 (MIP-133) – The 133-kDa protease that is induced when Acanthamoeba trophozoites engage mannose in the cell walls of bacteria or upon mannosylated proteins on corneal epithelial cells. This protease facilitates invasion of Acanthamoeba trophozoites by degrading basement membranes and it also induces apoptosis of corneal cells.

Mucosal immunity – Immune responses that are primarily in the form of secretory immunoglobulin

A (IgA) antibodies, which preferentially accumulate in the milk, tears, and in mucosal secretions. This form of T-cell-dependent immunity acts primarily to prevent pathogens from entering the body via mucosal surfaces and rarely directly kills microorganisms.

Trophozoite – Amoebic phase of Acanthamoeba spp. Acanthamoeba spp. can exist as either dormant cysts or as the active trophozoite (amoebic) phase. Unlike cysts, trophozoites are invasive, produce pathogenic proteases, and directly kill host cells by apoptosis and direct cytolysis.

Introduction

Acanthamoeba spp. are the causative agents for Acanthamoeba keratitis (AK) and can be isolated from virtually any terrestrial, aquatic, and marine environment. Viable Acanthamoeba

spp. have even been isolated from eyewash stations, bottled water, and contact lens cases of asymptomatic contact lens wearers. Acanthamoeba can exist either as a dormant cyst or as the active amoebic stage called the trophozoite. Trophozoites are the active vegetative stage that normally exist as free-living amoebae and feed on bacteria and fungi. Trophozoites are approximately the size of a leukocyte (10–25 mm) and are readily identified by their spiny pseudopodia that give them a sea urchin-like appearance. Acanthamoeba cysts are the dormant stage and are approximately half the size of the trophozoite. The cyst wall is comprised primarily of protein and cellulose. Interestingly, the latter molecule is not normally found in animals, but is restricted to members of the plant kingdom including bacteria and fungi. The cyst is remarkably resistant to environmental agents and can remain viable even after 20 years of storage at room temperature or following treatment with over 250 000 rads of gamma irradiation or doses of ultraviolet B (UVB) irradiation that are known to kill every category of mammalian cells. Although AK is believed to be caused by corneal infections produced by trophozoites adhering to contact lenses, cysts can also adhere to contact lenses, and, under certain circumstances, can produce corneal infections in experimental animals. Cysts can persist in corneal tissue for up to 31 months following antiamoebic treatment and may be the underlying cause for recrudescence in patients, especially those who receive corneal transplants to restore the vision lost as a consequence of AK. Corticosteroids are often used to extinguish the inflammation that is provoked in AK. However, corticosteroids have been shown to induce cysts to excyst and transform into infectious trophozoites. Moreover, corticosteroids activate trophozoites and render them more pathogenic and invasive. Thus, corticosteroid treatment may unwittingly exacerbate AK and contribute to the recrudescence that has been reported in AK patients who are treated with topical corticosteroids to prevent immune rejection of their corneal transplants.

In spite of the ubiquitous distribution of Acanthamoeba spp. in the environment and widespread contact lens wear, AK is remarkably rare. Moreover, environmental exposure to Acanthamoeba spp. is commonplace; up to 100% of the normal individuals with no history of AK possess serum antibodies to Acanthamoeba antigens, suggesting previous environmental exposure to Acanthamoeba spp. Viable Acanthamoebae can be isolated from the contact lens cases of individuals with no symptoms or history of AK. Collectively, these findings suggest that a large portion of the population is exposed to Acanthamoeba spp. and

may express some degree of immunity against corneal Acanthamoeba infection. This proposition begs the obvious question as to which immune component provides protection and how does it do it.

Anatomical and Physiological Barriers to Corneal Infections with Acanthamoeba

Clinicians have long suspected that contact lenses served as vectors for transmitting Acanthamoeba trophozoites to the corneal surface, and that antecedent injury to the corneal surface created epithelial defects that permitted trophozoites to gain a foothold in the cornea. Less than one-third of the cases of AK involve both eyes, even though contact lens wearers typically store their lenses in the same contact lens case, use the same contact lens solutions, and use the same finger for inserting their contact lenses. If a preexisting corneal epithelial barrier defect were not necessary, one would expect that the overwhelming majority of AK cases would be bilateral. Studies in animal models of AK confirm the importance of preexisting corneal epithelial defects in the development of AK that is produced by applying Acanthamoeba-laden contact lenses to the abraded corneal surface. However, other animal studies have shown that breaching the intact corneal epithelium by direct injection of trophozoites into the corneal stroma produces AK. Thus, the intact corneal epithelium provides a barrier for preventing the establishment of ocular infections with Acanthamoeba trophozoites.

In addition to the physical barrier provided by an intact corneal epithelium, the ocular surface is bathed in tears, which contain multiple factors that inhibit trophozoite binding and cytopathic effects. Although never formally proven, it is suspected that the shear forces produced by the blinking eyelid interfere with trophozoite adherence to the corneal epithelium and reduce the likelihood of the trophozoites gaining a foothold in the cornea.

Innate Immune System and Resistance to

Acanthamoeba Infections

The immune system is divided into two functionally distinct components: (1) the innate immune apparatus – which is characterized by its nimble response, but lack of antigen specificity – and (2) the adaptive immune apparatus – which, although slower in its response, provides long-lasting immunity and memory. Elements of the innate immune apparatus are the first responders via their detection of pathogen-associated molecular patterns (PAMPS) that are widely and promiscuously expressed by many microorganisms. By utilizing PAMPS, macrophages and neutrophils are able to rapidly identify invading microorganisms and mount an initial response that restrains the

infectious agents, thereby providing much-needed time for the development of the adaptive immune response.

Role of Macrophages in the Resistance to

Acanthamoeba Infections

In vitro studies have demonstrated that macrophages are capable of killing Acanthamoeba trophozoites. Moreover, depletion of periocular macrophages by subconjunctival injection of liposomes containing the macrophagicidal drug clodronate results in a dramatic exacerbation of AK in experimental animals. Likewise, in vitro or in vivo exposure to liposomes containing interferon-g (IFN-g) activates macrophages, increases their capacity to kill Acanthamoeba trophozoites, and mitigates AK in experimental animals. In animal models, AK is a self-limiting disease that resolves in 4–5 weeks. However, depletion of conjunctival macrophages results in progressive AK that does not resolve and mimics the human counterpart. The extraordinarily low incidence of AK is not commensurate with the enormous number of contact lens wearers and the ubiquitous distribution of Acanthamoeba spp. This suggests that the presence of an additional risk factor is involved in the development of AK. It is tempting to speculate that patients who develop AK represent a small population of individuals who have underlying deficiencies in their conjunctival macrophage population or altered macrophage function.

Role of Neutrophils in the Resistance to

Acanthamoeba Infections

The neutrophil is another constituent of the innate immune system that plays an important role in the resistance and resolution of AK. Neutrophils are consistently found in AK lesions – both in patients and in experimental animals. Neutrophils are highly effective in detecting the presence of both trophozoites and cysts. They kill both cysts and trophozoites in a myeloperoxidase-dependent manner. Blocking chemotactic responses of conjunctival neutrophils or depleting neutrophils with anti-neutrophilic antiserum results in progressive AK. Likewise, intracorneal injection of macrophage inflammatory protein-2 (MIP-2) – a potent chemoattractant for neutrophils – results in a swift infiltration of neutrophils into the central cornea and in an accelerated resolution of AK in experimental animals.

Ocular Acanthamoeba infections rarely progress beyond the cornea and are not known to produce endophthalmitis. Only three reports in the literature suggest that Acanthamoeba infections of the cornea progress to the posterior segment of the eye and involve the choroid or retina. Moreover, only one publication provides histopathological documentation of Acanthamoeba cysts in the posterior segment of the eye in a single AK patient. Moreover, the patient in this study had received four

separate corneal transplants in the affected eye. Likewise, more than 16 years of experience with both the pig and Chinese hamster models of AK has failed to produce any evidence of Acanthamoeba infections progressing posterior from the cornea or involving the uveal tract or retina. In vitro studies have shown that trophozoites can penetrate Descemet’s membrane and are, theoretically, capable of entering the anterior chamber. Further studies showed that intraocular injection of 1 million trophozoites into the anterior chamber of the eye in Chinese hamsters did not produce intraocular infection. A swift neutrophilic infiltrate eliminated the injected trophozoites without inflicting collateral damage to the intraocular tissues.

Humoral Factors of the Innate Immune System that Affect Resistance to Acanthamoeba Infections

Tears contain a potpourri of antimicrobial factors that protect the ocular surface from pathogenic insults. Among these are lysozyme, lactoferrin, and complement components. Lysozyme is active against Gram-negative bacteria and some fungi, but is ineffective against Gram-positive bacteria. By contrast, lactoferrin and transferrin are effective in controlling Gram-positive bacteria. Complement is present in tears and can be activated by the alternate pathway via bacterial products or by the classical pathway by antibodies. Thus, the complement system straddles the innate and adaptive immune systems. Complement appears to have little or no effect in controlling AK, as pathogenic Acanthamoeba spp. express complement-regulatory proteins – including decay-accelerating factor – which disable the complement system. Tears and milk also contain a factor that inhibits trophozoite adherence and cytolysis of corneal cells. Interestingly, the factor in both milk and tears is not an Ig. The milk-borne factor is >100 kDa and is inactivated by proteinase K, indicating that it is a protein. The two major proteins in milk – a1-antitrypsin and a1- antichemytrypsin – are not the milk-borne factors, as neither of these proteins blocks trophozoite adherence or cytolytic activity.

Thus, both humoral and cellular elements of the innate immune system can contribute to the resistance to AK (Table 1).

Adaptive Immune System and Resistance to Acanthamoeba Infections

Elements of the innate immune system serve as the first responders to pathogens and are characterized by capacity to act immediately in response to microbial infections. However, the innate immune system acting alone cannot clear all microbial infections. Unlike the innate immune system, the adaptive immune apparatus needs a jump start to generate its effector elements, but – once engaged – is crucial for the recovery from microbial infections and the establishment of immunity to future infections. The innate and adaptive immune systems do not function in isolation, but communicate with each other in a coordinated immune response. Macrophages and dendritic cells present antigens to T and B cells, which process is facilitated by another innate immune system component – complement. T and B cells have the capacity to generate an endless array of antigen receptors that, when confronted with antigens expressed on antigen-presenting cells, culminates in the generation of antibodies and T cells that possess exquisite specificity and are used by antibodies to identify and kill bacteria and neutralize viruses. T cells utilize their antigen receptors and CD8 surface molecules to identify and kill virus-infected cells. The adaptive immune system is characterized by its exquisite specificity and memory. The efficacy of preventive immunization relies entirely on the capacity of the vaccine to activate crucial elements of the adaptive immune system. One has to look no further than the biology of acquired immune deficiency syndrome (AIDS) to recognize the importance of the adaptive immune system. With few exceptions, recovery and survival from microbial infections is contingent upon the effective activation of the adaptive immune system. However, AK is a notable exception to this rule.

There is no evidence to date that patients with AIDS have an increased incidence of AK, suggesting that a disabled adaptive immune system does not increase the susceptibility to corneal infections with Acanthamoeba even though Acanthamoeba spp. express antigens that are capable of activating the adaptive immune system. Fifty to one hundred percent of the individuals with no past history of AK possess serum and tear antibodies specific for Acanthamoeba antigens – indicating that Acanthamoeba antigens can