Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

pigmentary glaucoma. Pigmentary glaucoma is believed to be caused by the release of melanin granules from the iris pigment epithelium into the aqueous humor and leads to obstruction of the aqueous outflow pathway. The DBA/2J mouse strain is described as an animal model of progressive pigmentary glaucoma. This mouse strain presents with several hallmarks of the human pigmentary glaucoma including iris atrophy, pigment dispersion, and peripheral anterior synechiae that are associated with development of elevated intraocular pressure (IOP), retinal ganglion cell loss, and optic nerve head excavation. Despite these similarities, it is shortsighted to claim that pigmentary glaucoma within DBA/2J mice is exactly the same as human pigmentary glaucoma. Differences that determine glaucoma susceptibility most certainly exist between humans and animal model systems. Actually, the DBA/2J mouse develops a more severe form of pigmentary glaucoma than humans. Although there are important species differences between humans and mice, the study of disease mechanism(s) using this in vivo murine system will help to elucidate the molecular mechanisms involved in the pathogenesis of human pigmentary glaucoma. This chapter describes the genetic mutations and immune dysfunction found within the DBA/2J strain, and presents evidence to support the possible involvement of pro-inflammatory or autoimmune mediator interleukin-18 (IL-18) in the development of murine pigmentary glaucoma.

PDS AND PIGMENTARY GLAUCOMA

PDS is medically important as it is very common in both glaucoma patients and general population (2.45% in Caucasian population). Approximately 50% of individuals affected with pigment dispersion develop elevated IOP and pigmentary glaucoma. The molecular mechanisms that cause PDS and/or its progression to pigmentary glaucoma are not known. In some families, PDS appears to be inherited as an autosomal dominant trait and is likely to be the result of an alteration in a single gene (1), but in other families it is more complex and appears sporadic (2). The gene responsible for PDS was mapped to the telomeric end of the long arm of chromosome 7 (7q35-q36) (3).

Since its initial description over 50 years ago, pigmentary glaucoma has become one of the most commonly reported forms of secondary glaucoma (1,2,4). Hallmarks of this disease include mid-peripheral iris transillumination defects and heavily pigmented trabecular meshwork. The primary causes of these abnormalities are unknown, and their etiologies are poorly understood. Some studies suggest that abnormally liberated iris pigment and cell debris enter the ocular drainage structures, and accumulate within the trabecular meshwork, causing denudation, collapse, or sclerosis of the trabecular beams, which leads to increased IOP (3,5). Other studies suggest that pigmentary glaucoma is the result of a generalized mesodermal dysgenesis of the eye that leads to abnormal development of the anterior segment, including the trabecular meshwork and Schlemm’s canal (6).

The inheritance of glaucoma in patients with PDS is not strictly Mendelian, suggesting that multiple genes may participate in the more severe pigmentary glaucoma phenotype seen in some patients. Pigmentary glaucoma has a hereditary basis and is probably an autosomal dominant disorder with multifactorial inheritance (7). It was suggested that mutations in tyrosinase-related protein 1 (Tyrp1) as a secondary genetic factor may result in a qualitative or a quantitative change in iris pigment production

that may directly or indirectly lead to an increase in IOP (8,9). However, study on DNA sequence variants in the Tyrp1 gene did not show association with human pigmentary glaucoma (10). Thus, little is known about the molecular and biochemical mechanism(s) underlying this disorder in humans.

THE DBA/2J MOUSE IS A MODEL OF PROGRESSIVE PIGMENTARY GLAUCOMA

The DBA/2J strain is a valuable, inbred strain that is widely used in a number of research areas including, cardiovascular biology (11), neurobiology (12), and sensorineural research (13). Its characteristics are often contrasted with those of the C57BL/6J inbred strain. DBA/2J mice show a low susceptibility to developing atherosclerotic aortic lesions. They also exhibit high-frequency hearing loss between 3 and 4 weeks of age that becomes severe by 2–3 months of age. This strain possesses three recessive alleles that cause progressive cochlear pathology initially affecting the organ of Corti. Decreasing anteroventral cochlear nucleus volume and neuronal cell loss parallels the progression of peripheral hearing loss (13). Young DBA/2J inbred mice are susceptible to audiogenic seizures (14). DBA/2J mice also show an extreme intolerance to alcohol and morphine (15). Currently, the DBA/2J mouse is the only strain that has many clinical features in common with pigmentary glaucoma in humans.

Elevation of IOP

Elevation of IOP appears to be an important risk factor for developing glaucomatous damage in DBA/2J mice. We used a TonoVet impact tonometer to non-invasively measure IOP. This is an impact (rebound) tonometer, which is clinically used by veterinary ophthalmologists as a non-invasive tool for measuring rat and mouse IOPs

(16–18).

Several groups have found a strong correlation between the true IOP and the IOP as measured by the impact tonometer (16–18). We have found the IOP alteration pattern in DBA/2J mice to be consistent with the observations reported by Dr. John’s group at the Jackson Laboratory (19,20). Using the impact tonometer, we measured 578 eyes from C57BL/6J mice and 398 eyes from DBA/2J mice at ages ranging from 1 month to 25 months (21). The measurements were performed in awake, non-sedated mice. Eyes were topically anesthetized with 0.5% tetracaine before IOP measurement. Topical administration of 0.5% tetracaine did not affect the measurement of the IOPs. The IOPs of C57BL/6J mice were stable with age, although there was a slight increase after 20 months. There was no significant difference in IOP levels between the C57BL/6J and the DBA/2J mice at 1 and 3 months of age, whereas a significant increase in IOP was seen in some DBA/2J mice by 4 months of age. This increase reached its peak between 6 and 9 months and then slightly declined by 12 months (see Fig. 1C) (21).

Iris Atrophy and Loss of Retinal Ganglion Cells

Aging DBA/2J mice develop progressive eye abnormalities that closely mimic human pigmentary glaucoma (20–22). These defects include iris atrophy, pigment dispersion, and peripheral anterior synechiae (see Fig. 1A and B) and are associated

Fig. 1. Clinical appearance of anterior segments and iris morphology in DBA/2J mice. (A and B) 9-month-old DBA/2J mouse with severe iris atrophy and pigment dispersion. (C) Measurement of intraocular pressure (IOP) of 398 eyes from 201 DBA/2J mice using an impact tonometer. (D and F) Significant loss of retinal ganglion cells (RGC) and neurofibers in a 9-month-old DBA/2J mouse. (E and G) Normal retinal ganglion cells and neurofibers in a wild-type C57BL/6J mouse.

with both the development of elevated IOP (see Fig. 1C) and the loss of retinal ganglion cells and neurofibers (8,9,20,21) (see Fig. 1D and F). The onset of disease symptoms in DBA/2J mice generally begins between 3 and 5 months of age. Initial signs include iris pigment epithelium loss and defects in transillumination of the peripheral iris. Between 6 and 7 months of age, all the DBA/2J mice demonstrate significant widespread transillumination defects and thickening of the iris border. By 9 months of age, dramatic iris atrophy is seen (see Fig. 1B). A significant loss of ganglion cells is observed in some DBA/2J mice as early as 5 months of age. By 9 months, a loss of more than 50% of retinal ganglion cells is seen (see Fig. 1D arrows). Flat whole-mounted retinas from DBA/2J mice (see Fig. 1F) and from age-matched wild-type C57BL/6J mice (see

Fig. 3. Correlation of intraocular pression (IOP), age, contrast sensitivity, and visual acuity in the eyes of DBA/2J mice. (A) The set-up of Virtual Optomotor System. (B) Increase in IOP level with age. (C) Decrease in visual acuity with increase in IOP level. (D) Decrease in visual acuity with age. (E) Increase in contrast with increase in IOP level. (F) Increase in contrast with age.

frequency that the mouse tracked was identified as the threshold. There was a clear positive correlation between elevation of the IOP and age (see Fig. 3B), and clear decreases in visual acuity occurred with increases in IOP and in age (see Fig. 3C and D) of DBA/2J mice. The contrast sensitivity was measured starting with low contrast (40%) at a spatial frequency of 0.4 cycle/deg. When sine wave grating contrast percentage rose, the contrast sensitivity fell. The contrast sensitivity decreased with increases in IOP and in age (see Fig. 3E and F). Thus, there was a strong correlation between elevated IOP and vision in DBA/2J mouse.

GENETIC MUTATIONS IN THE DBA/2J MOUSE

DBA/2J mice harbor two mutant genes, the b allele of tyrosinase-related protein 1 (Tyrp1b/b) and a stop codon mutation in the glycoprotein (transmembrane) nmb gene (GpnmbR150x) (3,8–10,20,22,25). The GpnmbR150x mutation (GpnmbR150x/R150x) can be found by assaying for the presence or absence of a unique Pvu II site (CAGCTG), which

IMMUNE DYSFUNCTION IN THE DBA/2J MOUSE

Several lines of evidence suggest that pigment dispersion in the eyes of DBA/2J mice may involve immune dysfunction and that a chronic subclinical inflammatory response occurs in the eyes of these mice because of compromising ocular immune privilege (26). Dendritic cells are normally present in the iris and in the ocular outflow pathway (27,28). Gpnmb was found in these dendritic cells (29), suggesting the possibility that the Gpnmb mutation alters dendritic cell function(s) and promotes iris disease through abnormalities in the ocular immune system. The eyes of DBA/2J mice reveal deficiencies in some aspects of immune privilege before dispersed pigment is evident (26). Also, Tyrp1 and melanin were identified as antigens relevant to inflammatory eye disease (30,31), and melanin can also exhibit adjuvant-like properties (30,32). It was demonstrated that the aqueous humor from eyes of affected DBA/2J mice lacks the capacity to suppress T-cell activation, an abnormality that actually precedes the onset of clinical evidence of pigment dispersion. Evidence of inflammation is already present by 4 months of age in DBA/2J mice (26), implying a possible pathogenic role for inflammation and autoimmunity in this form of mouse pigmentary glaucoma through both abnormal melanosomes and a susceptible immune abnormality that propagates the iris disease. Therefore, iris damage in the eyes of DBA/2J mice may be initiated by the leakage of toxic molecules from melanosomes and by the development of melaninassociated antigens (19). Histological analysis showed the infiltration of leukocytes within the iris and the aqueous humor, and also the accumulation of leukocytes in the inferior angle of the anterior chamber. CD69 is rapidly induced on activated T and B cells, NK cells, and granulocytes and can be used as a marker for activated T cells to indicate an inflammatory response. Immunofluorescent staining using anti-CD69 antibody showed CD69-positive cells in the iris and the anterior chamber in DBA/2J mice between 9 and 12 months of age (see Fig. 5A and B) (21).

Recent histological studies have shown that pigment-containing macrophages are present in the eyes of patients with PDS (33–35). Increased numbers of immatureappearing and abnormal melanosomes have been observed in the iris pigment epithelium of patients with pigmentary glaucoma (33,36). In addition, dendritic cells, which are important in controlling the immune response, are present in both the human and the mouse iris (27,28). Furthermore, numerous studies have suggested a role for immune response in the pathogenesis of glaucoma (37,38). These studies show the presence of autoimmune antibodies directed against antigens from the eye in the sera of patients with glaucoma (39–43). By studying differential gene expression in human trabecular meshwork, tissues dissected from control donors and from primary open-angle glaucoma donors, significant up-regulation of several genes associated with inflammation was evidenced (44). It should be emphasized that the DBA/2J mouse develops a more severe form of pigmentary glaucoma than humans. Although there is no current evidence to support a role of inflammation in pigmentary glaucoma in humans, the possibility that inflammatory components are important risk factors for disease susceptibility has not been reported. Research on the involvement of inflammatory components in the development of pigmentary glaucoma in this mouse model will provide insights into cellular or molecular mechanisms responsible for

Fig. 5. (A and B) Detection of activated T cells in the iris and anterior chamber of DBA/2J mice using anti-CD69 antibody. (C) Expression of interleukin (IL)-18 in the anterior chamber side of the iris in a 6-month-old DBA/2J mouse. (D) ELISA showed elevation of IL-18 protein concentration in the aqueous humor in DBA/2J mice with age. (E) Western blot analysis showed increases in IL-18 protein expression in the iris/ciliary body of DBA/2J mice between the ages of 3 and 6 months.

the pathogenesis of pigmentary glaucoma, and will have an important impact on the management of human pigmentary glaucoma.

INVOLVEMENT OF IL-18 IN THE PATHOGENESIS OF GLAUCOMA IN THE DBA/2J MOUSE

Role of IL-18 in the Eye

IL-18, previously known as interferon- (IFN- )-inducing factor, has recently been described as a member of the IL-1 cytokine superfamily. IL-18 is now recognized as an important regulator of innate and acquired immune responses. Functions of IL-18 include the promotion of cytokine release, particularly tumor necrosis factor (TNF)- , granulocyte monocyte colony stimulating factor (GM-CSF), and IFN- , and cytotoxicity mediated by Fas and FasL. In addition, IL-18 induces NO release, activates NF- B, possesses prodegradative effects (45–47), up-regulates the expression of inducible NO synthase, stromelysin, cycloxygenase-2 (COX-2), IL-6, IL-8, IL-13, and matrix metalloproteinase (MMP) in many cells and tissues (48–50).

Although the role of IL-18 in the murine eye is not clear, concurrent administration of IL-12 and IL-18 to mice induced epithelial apoptosis and caused atrophy in the lacrimal glands and markedly elevated serum levels of IFN- (51). In the cornea, IL-18 is expressed in epithelial cells. Increased bioactive corneal IL-18 production is induced by a number of pro-inflammatory agents and may play an important role in initiating IFN- -mediated inflammatory responses (52). IL-18 is also expressed in the epithelial

family members, and leading to the phosphorylation of NF- B-inducing kinase and I B degradation, which allows NF- B nuclear translocation. A role for mitogenactivated protein kinases (MAPK) in IL-18 signaling was recently suggested (48,49). We demonstrated an increase in NF- B nuclear translocation and an increase in the phosphorylation of MAPK in the iris/ciliary body of DBA/2J mice, suggesting that both signaling pathways may be involved in the initial IL-18 cascade (21). IL-18- binding protein (IL-18BP), a constitutively secreted protein with high affinity to IL-18, is a natural antagonist to IL-18. It was shown that IL-18BP protects against contact hypersensitivity (55), and inhibits IL-18-induced IFNand IL-8 production and also inhibits NF- B activation (47,56,57).

Expression of IL-18 and activated MAPK and NF- B in the iris/ciliary body of DBA/2J mice increased before the development of elevated IOP and retinal ganglion cell loss, indicating a possible involvement of increased IL-18 expression in the regulation of specific pro-inflammatory cytokine expression in the iris tissue, in the development of a subclinical inflammation through activation of MAPK/NF- B- signaling pathways, and leading to iris disease or degeneration and pigment dispersion to cause glaucomatous damage.

OTHER BIOCHEMICAL ALTERATIONS IN THE IRIS

OF THE DBA/2J MOUSE

Increased MMP-2 Expression in the Iris of the DBA/2J Mouse

MMPs are a family of zinc endo-proteinases that can be either secreted or membrane bound. They play important roles in many biological processes including angiogenesis, inflammation, and cancer metastasis (58–60). Many cells can secrete the gelatinases MMP-2 and MMP-9 to degrade the extracellular matrix in response to cytokines and inflammatory mediators (61,62). MMPs are now being implicated in the pathogenesis of eye diseases (63). Elevated amounts of MMP-2 and MMP-9 are found in necrotizing scleritis (64), and pterygial tissue (65), in the aqueous humor and in infiltrating cells (macrophages, T lymphocytes, and neutrophils) of both patients and animal models with uveal inflammation (66). The expressions of MMPs have been localized in the iris and the ciliary body with staining mainly in the cytoplasm of both the non-pigmented and the pigmented epithelial cells (67). It was reported that in patients with primary open-angle glaucoma, the total MMP-2 protein concentration was double and MMP- 2 activity increased by 3.9 times compared with patients with cataracts, suggesting that the development of primary open-angle glaucoma may be associated with the abnormal expression of MMP-2 in the aqueous humor (68). MMP-9 has also been suggested to play a role in retinal ganglion cell death and degradation of laminin (62,69,70). We demonstrated that MMP-2 protein expression in the iris/ciliary body of DBA mice was clearly higher than the MMP-2 protein expression in age-matched C57BL/6J mice. Our gelatin zymograph analysis also demonstrated that the MMP-2 activity dramatically increased in the aqueous humor of DBA/2J mice at the age of 6 months (21). Furthermore, the gene and the protein expression of tissue inhibitor of MMP-1 (TIMP-1) were decreased with age in DBA/2J mice (21). This suggests that a degradation process regulated by MMPs and TIMPs may be involved in the pigment