The human eye, like the brain and other organs throughout the body, contains immune-privileged sites that are protected from entry by circulating cells by complex ocular barriers. These barriers are conformed by tight junctions between specific epithelial cells and between vascular endothelial cells. Barrier dysfunction may be linked to alterations in molecular composition, function, and dynamics of the tight junction proteins. Furthermore, it may contribute to inflammation by vascular leakage of blood-borne molecules into the eye.
Two types of barriers have been identified within the eye, each characterized by its distinct tissue localization, immunologic properties, and physiological functions. The blood-aqueous barrier (BAB) localized in the anterior segment, and the blood-retina barrier (BRB) in the posterior segment.
The BAB is established by a tight junction complex of proteins and adapters, between the nonpigmented epithelial (NPE) cells of the ciliary epithelium. Tight junctions between vascular endothelial cells in ciliary processes, in the iris vasculature, and in the inner wall endothelium of the Schlemm’s canal also contribute to the BAB. However, the BAB established by the NPE layer of the ciliary epithelium is the most extensively studied.
Two BRB can be distinguished in the posterior segment of the eye. On one hand, the outer BRB, formed by tight junctions between the retinal pigment epithelium and fenestrated choriocapillaries. On the other hand, the inner BRB located within the inner layers of the neuroretina (ie, ganglion nerve cell layer) formed by tight junctions between nonfenestrated capillary endothelia covered by astrocytes and Müller cell foot projections.
Tight junctions are viewed as gatekeepers of the paracellular transport limiting the selective diffusion of ions and small solutes through the space between neighboring cells. Tight junctions [ie, claudins, junctional adhesion molecules (JAMs), occludins, zonula occludens (ZOs), cingulin] are part of the apical junctional complex that also includes the adherens junctions (ie, cadherin-catenin and nectin-afadin complexes) and the gap junctions (ie, connexins). These junctional complexes are dynamically regulated and they respond rapidly to pharmacologic agents and physiological changes.
Tight junctions between the NPE cells of the ciliary epithelium limit the diffusion through the paracellular space and simultaneously block the backflow from the aqueous to the stroma. The tight junctions among NPE cells also determine their cell polarity, separating their apical and basolateral membrane domains. Thus, the basolateral membrane of NPE cells face the aqueous humor fluid, whereas the apical membrane apposes the apical membrane of PE cell layer of the ciliary epithelium (Fig. 1). This apical-to-apical configuration of NPE-PE cells is unique and it is a challenge from the physiological point of view to understand the regulated mechanisms of active secretion of aqueous humor by the ciliary epithelium. An accepted model of aqueous humor secretion by the ciliary epithelium, proposes that aqueous humor formation occurs preferentially through transcellular pathways via numerous ion transporters in both cell layers, where gap junctions between NPE and PE cells play a key role.1 Ions are taken up from the vascularized stroma by transporters located in the basolateral membrane of PE cells, diffuse through gap junctions connecting PE and NPE cells, and then they are released to the posterior chamber of the eye. Transport of these ions generates an osmotic gradient that draws water across the ciliary epithelium to produce the aqueous humor.
In contrast to strategies targeting transepithelial transporters, and pathways of secretion of aqueous humor secretion to reduce its production, modulators of tight junctions could enhance paracellular permeability of BAB for a number and variety of drugs, including hydrophilic compounds to lower intraocular pressure (IOP) in glaucoma. Cytokines, for example, has been shown to alter the permeability of tight junctions by increasing the paracellular permeation for ions and solutes.
The paracellular ion permeability and ion selectivity through tight junctions are determined by claudins, a family of tight junctional proteins.2 On the basis of the gene-expression profile of tight junctions in human ocular tissues, it revealed that claudin 2 is predominantly expressed in the ciliary epithelium (Fig. 2), claudin 5 in the trabecular meshwork, and claudin 19 in the iris (M.C.-P., unpublished results, 2013).
The expression of claudin 2, for example, is characteristic of leaky epithelial cells, and this is consisting with the low transepithelial electrical resistance (an indicator of permeability and resistance of tight junctions) across the ciliary epithelium. This finding supports the view that aqueous humor diffusion may occur through the paracellular space between PE-PE and NPE-NPE cells (Fig. 1). The lack of claudin 19, for example, is associated with visual impairment (ie, colobomata, horizontal nystagmus, and myopia).
JAMs, is another family of integral membrane proteins within the tight junction complex. They are involved in intercellular adhesion between the cells of barriers, as well as in adhesion between barriers and blood cells. The JAM2 gene is preferentially expressed in the trabecular meshwork, iris, and ciliary body.
Tight junction proteins ZO-1 or ZO-2 are predominantly detected in the trabecular meshwork and they are important for clustering of claudins and occludin, resulting in the formation of tight junctional strands. The ZOs and cingulin can provide a direct link to the actin cytoskeleton.
Adherens junctions are positioned immediately below tight junctions and are characterized by apposing membranes which are separated by approximately 20 nM, that run parallel over a distance of 0.5 m. The complex cadherin/catenin and nectin/afadin mediate, separately, cell-cell adhesion and tight junction formation. They are also found in both epithelial and endothelial barriers.
In general, the dynamic changes that undergo tight junctions including disintegration and remodeling, or reversible changes in paracellular permeability are regulated by signaling pathways such as protein kinases A, C and G, Rho kinases, myosin light chain kinase, and the mitogen-activated protein kinase system.3
Connexins are a large family of proteins involved in formation of gap junctions, which mediate the movement of ions and low–molecular-weight metabolites between the cytoplasm of adjacent cells. Connexins form hexameric hemichannels (termed “connexons”) in the endoplasmic reticulum, which are then translocated into the plasma membrane. The connexon then “docks” with a connexon of an adjacent cell to form a functional channel termed a “gap junction.” Among connexins expressed in the ciliary epithelium, Cx43 plays a key role in gap junction formation between PE and NPE cells, it is the primary ion-conducting pathway between these cells and it is required for aqueous humor secretion. Inactivation of this connexin in the NPE cells of the mouse eye results in a significant reduction in IOP.4 Mutations in the connexin gene GJA1 (Cx43) cause oculodendrodigital dysplasia, and mutations in lens connexins GJA3 (Cx46) and GJA8 (Cx50) cause cataract.5
Evidence that the BAB surrounds an immune-privileged site (ie, anterior chamber) has been well documented. There is evidence that leukocytes can gain access to the aqueous humor through the ciliary epithelium, supporting the view that BAB serve as active and selective immune-skewing gate under steady state conditions. In an excellent review, Shechter et al6 suggested that blood barriers constituted by endothelial cells are absolute immunologic barriers associated to destructive inflammation, whereas blood barriers formed by epithelial cells serve as immunoregulatory and selective gates. It is known that the loss of barrier control can lead to pathologic conditions including inflammation. The epithelial and endothelial cells of the BAB express many immunoregulatory factors that block cytotoxic T lymphocyte, natural killer cell functions, and promote immune deviation.6
Breakdown of the BAB, independently of the etiology, is manifested by an increase in the aqueous humor protein concentration. However, recent studies have shown that an increase in protein not always is associated with the disruption of the BAB.7 Mice lacking the lysyl oxidase-like 1 (LOXL1) gene, a major genetic risk factor for exfoliation syndrome and exfoliation glaucoma, exhibit increased diffusion of fluorescein at the BAB, suggesting disruption of the ciliary epithelial barrier.8 Interestingly, the observed increase in permeability through this cellular barrier was not accompanied by an increase deposition of aqueous humor proteins, nor to an increased IOP, suggesting other mechanisms are likely involved. What may then cause the disruption of the BAB in Loxl1 −/− (null) mice? As tight junctions are targets of proteinases including matrix metalloproteinases (MMPs), further investigation is warranted. For example, to explore whether claudins/occludins in NPE cells might be prone to proteolytic degradation more rapidly in Loxl1 −/− mice than in normal mice by MMPs (ie, MMP-1, MMP-3, MMP-9) leading to an increased cellular permeability at the BAB.
- BAB and BRB barriers separate immune-privileged tissues from the circulation.
- Cell entry to immune-privileged sites through barriers composed of tight junction-interconnected endothelium is associated with destructive inflammation.
- Border structures comprised of fenestrated vasculature enveloped by tightly regulated epithelium (ie, BAB) serve as active and selective immune-skewing gates in the steady state.
- The BAB is not completely sealed but rather serves as a selective gate for cell trafficking.
- The BAB is patrolled by leukocytes and is suggested as a primary site for leukocyte infiltration preceding retinal inflammation.
- Retinal injury induces bone marrow-derived macrophage migration into the eye through the ciliary body, without damaging the BRB.
1. Coca-Prados M, Escribano J. New perspectives in aqueous humor secretion and in glaucoma: the ciliary body as a multifunctional neuroendocrine gland. Prog Retin Eye Res. 2007; 26:239–262.
2. Rizzolo LJ. Development and role of tight junctions in the retinal pigment epithelium. Int Rev Cytol. 2007; 258:195–234.
3. Niessen CM. Tight junctions/adherens junctions: basic structure and function. J Invest Dermatol. 2007; 127:2525–2532.
4. Calera MR, Wang Z, Sanchez-Olea R, et al.. Depression of intraocular pressure following inactivation of connexin43 in the nonpigmented epithelium of the ciliary body. Invest Ophthalmol Vis Sci. 2009; 50:2185–2193.
5. Pfenniger A, Wohlwend A, Kwak BR. Mutations in connexin genes and disease. Eur J Clin Invest. 2011; 41:103–116.
6. Shechter R, London A, Schwartz M. Orchestrated leukocyte recruitment to immune-privileged sites: absolute barriers versus educational gates. Nat Rev Immunol. 2013; 13:206–218.
7. Freddo TF, Neville N, Gong H. Pilocarpine-induced flare is physiological rather than pathological. Exp Eye Res. 2013; 107:37–43.
8. Wiggs JL, Pawlyk B, Connolly E, et al.. Disruption of the blood-aqueous barrier
and lens abnormalities in mice lacking lysyl oxidase-like 1 (LOXL1). Invest Ophthalmol Vis Sci. 2014; 55:856–864.