«Niederkorn JY, Kaplan HJ (eds): Immune Response and the Eye. Chem Immunol Allergy. Basel, Karger, 2007, vol 92, pp 36–49 Anatomy and Immunology of ...»
Niederkorn JY, Kaplan HJ (eds): Immune Response and the Eye.
Chem Immunol Allergy. Basel, Karger, 2007, vol 92, pp 36–49
Anatomy and Immunology of the
Erich Knopa, Nadja Knopb
Research Laboratory of the Eye Clinic CVK, Charité-University School of Medicine,
Berlin, bDepartment for Cell Biology in Anatomy, Hannover Medical School,
The ocular surface, in a strict sense, consists of the cornea and its major support tissue, the conjunctiva. In a wider anatomical, embryological, and also functional sense, the ocular mucosal adnexa (i.e. the lacrimal gland and the lacrimal drainage system) also belong to the ocular surface. This definition includes the source and the eventual drainage of the tears that are of utmost importance to ocular surface integrity. The ocular surface is directly exposed to the external environment, and therefore is endangered by a multitude of antigens and pathogenic microorganisms. As a mucosa, it is protected by the mucosal immune system that uses innate and adaptive effector mechanisms present in the tissue and tear film. Immune protection has two partly opposing tasks: the destruction of invading pathogens is counterbalanced by the limitation of inflammatory events that could be deleterious to the subtle structure of the eye. The immune system of the ocular surface forms an eye-associated lymphoid tissue (EALT) that is recognized as a new component of the mucosal immune system. The latter consists of the mucosa-associated lymphoid tissues in different organs of the body. Mucosa- and hence eye-associated lymphoid tissues have certain characteristics that discriminate them from the central immune system. The mechanisms applied are immunological ignorance, tolerance, or an immunosuppressive local microenvironment, all of which prefer non-reactivity and anti-inflammatory immunological responses. The interaction of these mechanisms results in immune privilege of the ocular surface. During eye closure, the ocular surface appears to have different requirements that make an innate pro-inflammatory environment more attractive for immune defense. The structural and functional components that contribute to this special immune regulation will be the focus of this chapter.
Copyright © 2007 S. Karger AG, Basel Anatomy of the Immune System at the Ocular Surface and Adnexa Cornea The cornea consists of a transparent connective tissue (stroma) covered by epithelia on both sides. The endothelium that lines the anterior chamber is a monolayer and the outer border of the cornea is a stratified non-keratinized squamous epithelium that is 5–7 cells thick . It seals the stroma from the external environment by luminal junctions and forms a physical barrier against external antigens. This is supplemented by a physicochemical barrier of the epithelial-derived mucin layer that protects against the adhesion and entrance of antigens and by mechanical washing effects of the tear fluid and lid wiping combined with the action of protective proteins .
In the normal cornea, very few cells can assist in immune defense.
Lymphoid cells do not occur under physiological conditions. The central cornea is avascular because blood and lymph vessels end in the limbal zone  and hence prevent an access of the vast majority of immunologically relevant cells.
Major histocompatibility complex (MHC) class II-positive dendritic antigenpresenting Langerhans cells are present in the epithelium of the peripheral cornea and their absence from the central cornea was assumed to be a major reason for corneal immune privilege. Other dendritic cells (DCs) that are negative for markers of cell activation were recently observed in the central cornea of mice . Further bone marrow-derived DC precursors or macrophage-like cells were reported in the anterior stroma and in the posterior stroma.
Morphology The conjunctiva consists of an epithelium and an underlying loose connective tissue, known as the lamina propria; both are separated by the epithelial basement membrane. The epithelial histology is stratified non-squamous and consists of two-to-three cell layers having cuboidal morphology in most parts. The lamina propria is rich in bone marrow-derived cells that form a mucosal immune system known as the conjunctiva-associated lymphoid tissue (CALT) and of blood vessels of different kinds. Apart from capillaries and lymph vessels, specialized high endothelial venules  for the regulated migration of lymphoid cells are present in the conjunctiva . They are a normal component of ocular lymphoid tissue, have a characteristic ultrastructure as in other lymphoid tissues, and express cell adhesion molecules.
Anatomy and Immunology of the Ocular Surface 37 Diffuse Leukocyte Subpopulations Over the last decades, evidence has accumulated that leukocytes, including lymphoid cells, are normal, non-inflammatory components of the ocular surface [for review see ref. 7].
Lymphocytes and plasma cells are the main populations of leukocytes  and form a diffuse lymphoid tissue throughout all conjunctival zones, with predominant expression in the tarso-orbital conjunctiva . Lymphocytes occur in the basal layer of the epithelium as intraepithelial lymphocytes (IEL) and more frequently in the lamina propria [8, 9] as lamina propria lymphocytes. Several lines of evidence indicate that the ocular surface has a mucosal immune system with common characteristics: CD8 suppressor/cytotoxic T cells dominate over CD4 T helper (Th) cells in IEL and a reverse distribution occurs in lamina propria lymphocytes [9, 10]. It is assumed that most of the CD8 cells act in the suppressor mode and hence provide an immunosuppressive environment . Conjunctival lymphocytes are activated cells (CD45Ro and CD25 ) and express human mucosal lymphocyte antigen-1 [10, 11]. Local plasma cells regularly occur in the lamina propria [6, 11–13]. They mainly produce IgA and the joining molecule (J chain) that forms the dimeric type of IgA. Its transepithelial transporter molecule secretory component (SC) is found in the epithelium, as verified by immunohistochemistry  and molecular biology (RT-PCR) .
The conjunctiva hence produces secretory SIgA on its surface and constitutes a secretory immune system . Interspersed B lymphocytes are rarely found as they are restricted to organized lymphoid follicles [6, 9, 11].
Other bone marrow-derived accessory leukocyte subpopulations exist in the conjunctiva and mainly act for the innate immune system. Macrophages enable the engulfment and destruction of pathogens and remnants of dead cells, and their potential antigen presentation to lymphocytes. They are frequent in the lamina propria but difficult to detect in conventional histological specimens. An immunohistological study reported CD68 macrophages as the second most frequent leukocyte population in the conjunctiva . Dendritic Langerhans cells, which aid in the uptake and professional presentation of antigens to lymphocytes, are regularly found . They express activation markers such as MHC class II or ATPase. Depending on their maturation and migratory behavior, they are critical regulators of immunity and link innate and adaptive immune effector mechanisms . Mast cells are resident accessory leukocytes in the lamina propria . They produce several factors, including cytokines, which recruit other leukocytes and orchestrate inflammatory reactions for the destruction of pathogens. Although their role in physiological host defense is poorly understood, they are potentially useful cells. They are mainly known, however, for their deleterious inflammatory activity during IgE-mediated allergic disease . Granulocytes of different subtypes (neutrophils, basophils, and
Follicles Lymphoid follicles involved in the production of lymphoid effector cells are regularly observed on normal human whole-mount conjunctivas [6, 12, 13], and in several other species , being mostly secondary follicles . Their frequency is age dependent ; increased levels are noted before onset of puberty which decrease with age. About 60% of individuals in their mid-70s still have follicles in the conjunctiva, with an average number of 10 follicles per conjunctival sac .
Follicles show typical mucosal characteristics: they consist of B cells with parafollicular T cells and associated high endothelial venules and have an apical follicleassociated epithelium. It is thin, highly permeated by lymphocytes, and includes M cells for antigen uptake in several species , but lacks the IgA transporter SC.
The human lacrimal gland is anatomically continuous with the conjunctiva via 10–12 lacrimal excretory ducts. It is a tubulo-acinar gland with shortbranched tubules that end in secretory acini . Between the secretory acini is a loose connective tissue resembling that of the conjunctiva and, in fact, continuous with it along the excretory ducts. Plasma cells are more frequent than lymphocytes, IEL are fewer, and CD8 suppressor/cytotoxic T lymphocytes are generally more frequent than CD4 Th cells in the gland in contrast to the conjunctiva . Plasma cells are mainly positive for IgA, and the acinar epithelium expresses the IgA transporter SC . Therefore, the lacrimal gland is an established component of the secretory immune system and was until recently considered as the only source of IgA proteins present in the tear film [9, 24]. T cells are reported to form groups around intralobular ducts  but ordinary lymphoid follicles are very rarely observed and may not be physiologically relevant.
Lacrimal Drainage System
The lacrimal drainage system is continuous with the conjunctiva via the lacrimal puncta and canaliculi into the lacrimal sac and through the nasolacrimal duct into the nose. Like the conjunctiva, it represents a moist mucous membrane.
The epithelium is a stratified squamous non-keratinized layer inside the canaliculi Anatomy and Immunology of the Ocular Surface 39 and transforms into a pseudostratified epithelium with columnar ciliated cells in the lacrimal sac and nasolacrimal duct . The mucosa contains diffuse lymphoid tissue  that contributes to the secretory immune system, and also follicles similar to the conjunctiva [25, 27]. Its mucosa-associated lymphoid tissue was accordingly integrated as a lacrimal drainage-associated lymphoid tissue (LDALT) into the mucosal immune system . The reported frequency of organized lymphoid follicles with typical morphology varies from 41  to 56% in old age human populations .
Tear Film and Integrated Proteins
The tear film is an important functional component of immune defense in the ocular mucosal surface. Apart from a cleansing effect induced by lid wiping, it contains specific IgA antibodies that are secreted by the lacrimal gland and by the ocular mucosal surfaces. In addition, there is an ever-increasing number of reported peptides and proteins of the immune system . Some of them have a direct antimicrobial effect whereas others (e.g. chemokines and cytokines) recruit and activate leukocytes, including lymphoid cells.
Historically, and due to their relative concentration, three secreted antimicrobial proteins are most important. Lysozyme destroys the bacterial cell wall, lactoferrin binds iron, and tear-specific prealbumin (lipocalin) acts as a scavenger of bacterial products; complement occurs as a transudate from the serum.
Angiogenin is a newly described tear protein found at high concentrations in virtually all tear samples . It appears to have primarily an antimicrobial effect within the tear film. Other multifunctional antimicrobial molecules are predominant in the closed eye during sleep, e.g. specific leukocyte protease inhibitor, elafin, and neutrophil gelatinase-associated lipocalin. CXC and CC chemokines, such as interleukin (IL)-8, epithelial neutrophil-activating peptide 78, interferon- -inducible protein-10, growth-regulated oncogene or macrophage chemoattractant protein-1 and macrophage inhibitory protein-1 are able to recruit leukocytes into the tear film. Inflammatory cytokines such as IL-6 and macrophage colony-stimulating factor appear to occur in every normal tear film . Most of these tear proteins show an inverse correlation with the amount of aqueous tear secretion and their concentration strongly increases in the closed-eye tear film, when lacrimal secretion has almost ceased.
Mucosal Immune Defense Mechanisms at the Ocular Surface The anatomy and leukocyte cell types clearly show that a mucosal immune system is maintained at the normal human ocular surface and mucosal adnexa.
Fig. 1. The eye-associated lymphoid tissue (EALT) is the mucosa-associated lymphoid tissue for immune protection of the ocular surface and its mucosal adnexa. It is anatomically continuous from the lacrimal gland throughout the conjunctiva- and lacrimal drainage-associated lymphoid tissue (i.e. CALT and LDALT, respectively). It consists of a diffuse lymphoid tissue of T lymphocytes and IgA-secreting plasma cells, including accessory leukocyte populations in all organs and of lymphoid follicles in conjunctiva- and lacrimal drainage-associated lymphoid tissue (in the drawing, large blue cells represent plasma cells, small blue cells represent B cells and small black cells represent T cells). Protective as well as aggressive factors inside the tear film, which connects the different parts of the ocular surface and protects it from the external environment, are a major component of ocular surface immunity. The organs are also connected by lymphocyte recirculation via specialized vessels with each other and with the rest of the immune system.