Immunology of the Eye

I.INTRODUCTION

The immune system provides protection against potentially harmful microorganisms or the development of tumor cells. It can also cause injury and disease under certain conditions (autoimmune disease, allergy or transplant rejection.)

Immunology is the study of the body's reaction to foreign substances. It involves: 1. Cellular and molecular events 2. Recognition of self vs. non-self 3. Effector mechanisms ("taking action")

II.CLASSIFICATION OF THE IMMUNE SYSTEM A. Innate (natural) immunity B. Adaptive (specific) immunity

III.INNATE (NATURAL, NONSPECIFIC) IMMUNITY

A. Characteristics 1. Nonspecific, first-line defense system 2. Effective at birth 3. Present prior to exposure to foreign substance 4. Does not discriminate among foreign substances 5. Not enhanced by previous exposure 6. Present in all multicellular organisms 7. Provides a rapid, incomplete host defense until slower, acquired immune response develops B. Components 1. Biochemical: antibacterial enzyme lysozyme (tears), pH.

2. Physical defense: intact epithelial surface (cornea), mechanical effect of blinking, washing effect of tears, mucous membranes, cough reflex.

3. Complement System: a group of proteins that are present in the blood as inactive precursors. Microorganisms or antibodies can activate these proteins in a cascade reaction. The activated proteins can lyse the membranes of some micro-organisms, attract phagocytes, opsonize (coat) microbes and facilitate their phagocytosis.

4. Interferon: a group of proteins that are produced by virally infected cells and leukocytes. Interferons protect neighboring cells, affecting microoganisms and cancer cells.

5. Phagocytes: Monocytes – blood Macrophages - tissue Neutrophils Natural killer cells (a type of lymphocyte)

IV.ADAPTIVE (SPECIFIC) IMMUNITY

A. Characteristics 1. Present at birth but not fully functional 2. Induced or stimulated by exposure to a specific foreign substance (immunogen, antigen) 3. Defense components will retain memory for a specific antigen (years, life) 4. Enhanced with each exposure (increased magnitude) 5. Initial contact with an immunogen triggers a chain of events a. lymphocytes are activated b. antibody and cytokines are produced c. memory cells develop

B. Components 1. Immunogen is any agent capable of inducing an immune response. 2. Antigen is usually a foreign substance of high molecular weight (protein, polysaccharides) with a specific chemical site (antigenic determinant or epitope). It can bind specifically to components of the immune response, such as lymphocytes and antibodies. 3. Hapten is a foreign substance (drug, dye). It is of small molecular weight and cannot cause an immune response by itself. However, it can attach itself to a larger molecule in the body, such as a body protein. This changes the structure of the protein, which now becomes "foreign" or an recognizable antigen; triggering an immune response. A hapten is an antigen but not an immunogen. 4. Antibodies are special molecules that are produced by B lymphocytes. They are immunoglobulins (glycoproteins) and are divided into 5 classes: IgG (most prevalent), IgA (tears), IgM, IgD and IgE. Each antibody consists of four chains--two light and two heavy chains.

Each antibody has 2 antigen binding sites (FAB) and one portion (FC) that binds to cells (phagocytes) and activates the complement system.

Reactions between antibody and antigen occur on the surfaces of cells and they mediate recognition events that are transmitted to special lymphocytes.

CLASSES OF IMMUNOGLOBIN Present in majority of B cells; neutralizes viruses and toxins, IgG enhances destruction of microorganism or tumor cell by the host’s natural killer cells. Activates complement, binds to macrophages and is the only Ig capable of crossing placenta. Predominant Ig in body secretions; (tears, saliva, respiratory IgA secretions, breast milk); protects mucous membranes. Protects against local infection, prevents certain bacteria and viruses from attaching to host cell.

IgM Prominent in early immune response; activates complement

IgD May be involved in maturation of B cell lymphocytes

IgE Binds to mast cell and basophils; involved in allergic reactions, and parasitic infections.

4. Lymphocytes known as B cells produce antibodies. Special lymphocytes known as B cells produce antibodies. Special lymphocytes known as T cells are involved in virus recognition. T cells are divided into different classes (suppressor, helper, cytotoxic). C. Immunity: protection against foreign agents

Humoral immunity can be transferred to non-immunized individuals via antibodies from the plasma or serum (no cells) of an immunized person. Cell mediated immunity can be transferred to non-immunized individuals with cells from an immunized person (lymphocytes responsible for specific antigen recognition).

V. GENERAL SCHEME OF CELLS AND ORGANS INVOLVED IN THE IMMUNE SYSTEM Lower animals have more primitive immunologic defense mechanisms. Proteins with low specificity can recognize and agglutinate a wide variety of antigens (low specificity). Phagocytes engulf and digest antigens.

Vertebrates also produce proteins and phagocytes which provide innate or nonspecific immunity. In addition, lymphocytes are present for specific immunity.

VI. CELLS AND ORGANS OF THE IMMUNE SYSTEM A. Cells of the immune system originate from the pluripotent stem cell in the bone marrow.

megakaryocyte Tcytotoxic Hematopoietic Stem cell eosinophil Thelper common myeloid common lymphoid precursor precursor basophil Tsuppressor

neutrophil natural killer cell B cell mast cell (non-T, B cell null cell) monocyte

macrophage plasma cell

1. Lymphocytes: 20-30% of circulating leukocytes 2 types: T cells (thymus) B cells (fetal liver, bone marrow) Circulating blood contains small lymphocytes that are either B or T cells. Each cell type is distinguished by different cell surface proteins or "markers". During development, B and T cells acquire receptors for a specific antigen and are committed to reacting only with that antigen (immunologic specificity).

Both B and T cells become: 1. Effector cells = destroy antigen 2. Memory cells = formed following initial exposure to antigen. Revert to inactive state but can rapidly increase immune response if encounter subsequent antigen. a. B cells  Plasma cells (antigen exposure)  Produce antibody Each plasma cell produces only one type of antibody. The antibody binds to the surface of the B cell. If an antigen binds, the cell is activated and proliferates into two cell lines:

1) effector cells: produce antibodies to this specific antigen. 2) memory cells: "memorizes" the antigen and can produce antibodies immediately at a later contact.

b. T cells  helper/inducer (CD4+)  suppressor/cytotoxic (CD8+)

T cells continually re-circulate between blood, lymph, lymph nodes, spleen and non-lymphoid tissues. These cells recognize antigen that is presented (processed or broken down) by another cell such as an Antigen Presenting Cell (APC).

The T cell becomes activated and proliferates into two cell lines: 1) effector cells: produce lymphokines which are chemical messengers for other cells. Different types affect T, NK, or B cells, macrophages and promote B cell differentiation into plasma cells. 2) memory T cells

c. Natural killer cells (NK): large lymphocytes No markers Kill foreign cells, nonspecific Kill tumor cells, virus-infected cells

2. Mononuclear phagocytes: 2 functions: 1) Phagocytosis 2) Antigen presenting cell Cell Types: Blood monocytes Tissue macrophages: produces cytokines

3. Polymorphonuclear granulocytes 1) Nonspecific phagocytosis 2) Important to acute inflammation

A. Cell types: Neutrophils - release lysozyme, collagenase, lipase Eosinophils - release major basic protein (MBP), peroxidase, synthesizes leukotrienes. Basophils and Mast cells - release histamine Platelets - activate Complement system and attract leukocytes.

B. Lymphoid Organs: cells in immune system are organized into tissues and organs.

1. Primary lymphoid organs = major sites of lymphocyte development. Thymus: T cell maturation Fetal liver and bone marrow: B cells

2. Secondary lymphoid organs = efficient in trapping and concentrating antigen; creates environment for lymphocytes to interact with each other and with antigen. Main site of antibody production and generation of antigen - specific T cells. Lymph nodes Spleen Mucosa - Associated Lymphoid Tissue (MALT)

1. consists of mucosal tissues of the gastrointestinal, respiratory, genitourinary tracts, mammary glands and the eye. Components include: a. intraepithelial lymphocytes (predominantly CD8+ T cells) b. aggregates of lymphocytes known as lymphoid follicles or nodules (predominantly B cells in central area). 1) Peyer’s patches - lymphoid follicles in intestine 2) Gut - associated lymphoid tissue (GALT) - diffusely distributed lymphocytes along digestive tract. 3) Bronchus - associated lymphoid tissue (BALT) 4) Conjunctival - associated lymphoid tissue (CALT) c. Interaction between different sites of mucosa - associated lymphoid tissue: Antigen - stimulated B cells from gut can travel to the lung or eyes and initiate immune response there.

VII. HISTOCOMPATIBILITY ANTIGENS (Human leukocyte antigens - HLA) HLA are antigens carried on the surfaces of nucleated cells of most tissues. The major function of the HLA molecules is to present antigens to T cells.

1. Differences in HLA antigens of tissue from an unrelated donor results in transplantation rejection. 2. Similarities in specific HLA antigens are associated with certain diseases (HLA-B27 and ankylosing spondylitis.) 3. The major histocompatibility complex (MHC) consists of a region of genes on chromosome 6 that are responsible for the human leukocyte antigens. The MHC contains genes that code for molecules that can be classified into three categories: a. Class I genes and products = involve immunosurveillance and the transplant antigen region. Class I antigens are present on virtually all nucleated cells and platelets The genes are designated as HLA-A, HLA-B and HLA-C. Class I antigens are important for the presentation of viral antigens to CD8+ cytotoxic T cells.

b. Class II = involves the immune response and regulation of T cell, B cell and macrophage processing. Class II antigens are present mainly on antigen-presenting cells, B cells and some activated T cells. These antigens are coded for in a region known as HLA-D. Class II antigens are important for the presentation of antigens to CD4+ helper T cells. c. Class III = products of these genes comprise certain components of the complement system

VII. OCULAR "IMMUNE" SYSTEM The cornea and conjunctiva are exposed to the environment and under constant assault from foreign antigens. The ocular surface is protected by several nonspecific mechanisms:

1. Reduced temperature of the central cornea inhibits certain microbes. 2. Blinking mechanically removes antigens 3. Irrigation by tears (lysozyme, lactoferrin) 4. Intact epithelial surface 5. Normal bacterial flora produce acidic end products and prevent colonization by more virulent pathogens.

A. Conjunctiva

1. Conjunctival associated lymphoid tissue (CALT). Lymphocytes of mucosal follicles remain largely confined to mucosal tissues. Lymphocytes can be arranged into single or multiple lymphoid follicles at limbus or follicles in the fornix.

2. Other cells: Mast cells Macrophages/Monocytes Antigen presenting cells (APC) Dendritic cells - phagocytic cells located in connective tissue and epithelium (Langerhans) cells

Antigen presenting cells reside in the tissue, encounter antigen, internalize and process it. APC's migrate via the lymphatic vessels to the preauricular and submandibular lymph nodes and present antigen to B and T cells. This leads to activation of B and T cells, with proliferation of these cells in the draining lymph node. B cells become plasma cells and secrete IgA.

B. Lacrimal tissue The accessory and main lacrimal gland are a part of the mucosal-associated lymphoid system. Lymphoid aggregates associated with the intra-lobular ducts contain B cells and plasma cells. The plasma cells produce IgA, which is important in promoting phagocytosis of microorganisms, inhibiting their binding to ocular surfaces and enhancing antibody-dependent cell-mediated reactions. Tear antibodies are directed against many bacteria, viruses and chlamydia. In addition, activated B cells from the intestine (GALT), travel to the lacrimal gland and develop into IgA-secreting plasma cells.

C. Cornea The normal healthy cornea is avascular and lacks a lymphatic system. Limbal vessels and the tear film probably account for the presence of limited amounts of immunoglobulin (IgG and IgA) and certain complement components. Antigen presenting cells, or Langerhans cells, are present throughout the corneal epithelium, but decrease in numbers toward the central cornea.

The cornea is often described as an "immunologically privileged site" or one that tolerates the presence of foreign antigens, such as a corneal graft. This characteristic may be due to the absence of blood vessels and the relative lack of antigen presenting cells, accounting for poor recognition of antigen.

Pathologic conditions of the cornea can lead to vascularization, the development of lymphatic channels and an increase in Langerhans cells. These factors can increase the immunologic and inflammatory response of the cornea, causing the scarring seen in many corneal diseases.

VIII. CLINICAL CORRELATE: REVIEW OF AN OCULAR IMMUNE RESPONSE Adenovirus

A. Infect epithelial cells of pharynx, conjunctiva, cornea and small intestine 1. Virus attachment to epithelial cells 2. Penetration and uncoating 3. Replication of viral DNA in cells nucleus 4. Assembly of infectious particles in cell cytoplasm B. Local ocular response: follicular conjunctivitis (may also have papillary response), diffuse hyperemia, chemosis, lacrimation, SPK (caused by live virus). May develop pseudomembrane.

C. Preauricular lymphadenopathy: 1. Viral particles are carried via conjunctival lymphatic system to preauricular lymph nodes. 2. Antigen encounters B and T cells in the node. a. Proliferation of lymphocytes causes the node to swell. b. B cells proliferate and become: 1) plasma cells: produce IgG neutralizing antibody in the case of Adenovirus infection (impedes infectivity of viruses for host cell). 2) memory cells c. T cells proliferative, differentiate and produce lymphokines d. T cells, B cells and antibody enter circulation and return to tissue.

D. Subepithelial infiltrates are thought to represent a delayed hypersensitivity reaction, with T cells attracted to viral antigen in the cornea. 1. Infiltrates suppressed with topical steroids but until inciting antigen “washes out” over time, infiltrates will reappear when steroids discontinued.

IX. MECHANISMS OF IMMUNE-MEDIATED INJURY (Hypersensitivity Reactions)

The immune response is usually beneficial and provides protection against microorganisms or the development of tumor cells. However, the immune response system can cause injury if the response is excessive. These type of responses are called hypersensitivity reactions and there are four different mechanisms. The reactions occur throughout the body, but will be discussed in the context of ocular conditions.

Types of Hypersensitivity Reactions: 1. Type I Hypersensitivity: Anaphylaxis or Immediate Hypersensitivity

Antigen reacts with IgE bound to the surface of mast cells or basophils, causing the cells to release their cytoplasmic granules. The granules contain pre-formed inflammatory mediators (histamine, serotonin, eosinophil chemotactic factors and proteases). In addition, other mediators are generated during the release reaction. These mediators are generated from cell membrane components and include leukotrienes, prostaglandins and thromboxane. Both the preformed and the newly generated mediators cause the signs and symptoms of allergy. The IgE response is under the control of T helper cells and there is a genetic factor involved (ie. allergic parents usually have allergic children). Although up to 50% of the general population produces IgE to airborn antigens on mucosal surfaces, only 10% show clinical symptoms of allergy.

Allergic conjunctivitis: Airborn antigen such a ragweed comes into contact with ragweed-specific IgE bound to the mast cells of the conjunctiva. The mast cells degranulate causing chemical mediators to be released. These chemical mediators cause vasodilation, vessel permeability and fluid leakage. The resulting signs are conjunctival hyperemia, chemosis, a mild papillary response, and mucoid discharge other findings include lid edema and possibly an "allergic shiner," resulting from a transient increase in periorbital pigmentation (due to impaired venous return in subcutaneous tissue). Conjunctival scrapings often reveal eosinophils.

The hallmark of atopic disease is the rapid onset of symptoms. The patient usually notes itching, red and watery eyes. Itching can resolve fairly quickly as long as the patient does not rub the eye which leads to further mast cell degranulation. Rhinitis is often present. These patients may have other forms of immediate hypersensitivity such as asthma, atopic dermatitis or food and drug allergies.

Treatment centers around reducing or removing the source of antigen. Topical vasoconstrictors, with or without antihistamine, topical and oral antihistamines, mast cell stabilizers and topical non-steroidal anti-inflammatory drugs can be used. Topical corticosteroids are effective but are not for chronic use. 2. Type II: Cytotoxic hypersensitivity

Antibodies are formed against antigens that are on the surface of specific cell or tissue types. The antibodies are either IgG or IgM. Once bound, they can activate the Complement system, stimulate release of chemical mediators or activate killer cells.

Cicatricial (ocular) pemphigoid Autoantibodies form to the basement membrane of conjunctival epithelium. The antibodies activate the complement system, resulting in membrane breakdown, bullae of the conjunctiva, subepithelial fibrosis, loss of goblet cells and formation of symblepharons.

The patient is usually elderly and presents with symptoms of a dry eye and injection. Often the condition is initially (and incorrectly) diagnosed as chronic conjunctivitis of unknown etiology.

A cicatricial "pemphigoid-like" syndrome has been associated with certain topical medications: 1. pilocarpine 2. echothiophate iodide 3. phenylephrine 4. gentamicin

Clinical signs include conjunctival scarring, shrinkage and symblepharon formation. The drugs could have acted as haptens, attaching to the ocular tissue and initiating an immune response to the tissue-drug complex. However, there is no conclusive evidence to support this.

Treatment of cicatricial pemphigoid is difficult and consists of managing patient symptoms and the use of topical corticosteroids for immunosuppression and to decrease the production of inflammatory mediators.

3. Type III: Immune complex hypersensitivity

Circulating antigen-antibody complexes become deposited in tissues, activate complement, and stimulate mediator release and phagocytosis by macrophages and other leukocytes. Antigens triggering this type of reaction include micro-organisms, drugs or autologous ("self") components. The injured cells of tissues are "innocent bystanders"; the antigen-antibody complex attaches to the cells and causes immune injury.

The architecture of the limbal vasculature may contribute to the deposition of immune complexes in the corneal periphery. Peripheral corneal infiltrates may occur in autoimmune diseases such as rheumatoid arthritis, Wegener's granulomatosis, systemic lupus erythmatosus and periarteritis nodosa. Ulceration of the epithelium and peripheral corneal melting can result.

Episcleritis and scleritis are also associated with these systemic diseases and are due to an immune complex vasculitis of the conjunctival or scleral vessels.

Therapy usually involves topical corticosteroids (intact corneal epithelium). Non- steroidal anti-inflammatory drugs (NSAIDs) or systemic corticosteroids may be used in certain cases.

4. Type IV: Cell-mediated hypersensitivity

Antigen bound to a macrophage activates an antigen-specific T cell. The T cell then produces lymphokines that are responsible for mediating the response. The antigen can be bacteria, viruses, fungi, tissue allografts or neoplasms. Lymphokines attract other immune cells and aid in cytotoxicity. The Type IV reactions is often called delayed hypersensitivity because it takes time for production of lymphokines and for the clinical effects to develop.

Phlyctenulosis: In the past, the most common cause of phlyctenulosis was hypersensitivity to tuberculoprotein, but now it is most commonly associated with Staphylococcal antigens. Phlyctenulosis occurs in the first two decades of life, and is self-limited, with the first attack occurring at the limbus. The lesion is initially round or oval, 1-3 mm, and elevated. It is hard, red and surrounded by hyperemia. It follows a course of elevation, infiltration, ulceration and resolution. Subsequent lesions occur in the conjunctiva and cornea.

A phlyctenule is a cell-mediated reaction to an antigen that has been presented to the conjunctival sac either exogenously or endogenously. It consists of plasma cells, lymphocytes and macrophages. Polymorphonuclear leukocytes (PMN – neutrophils) invade when the epithelium undergoes necrosis.

Treatment involves removing the causative agent. Staphylococcal blepharitis is treated with lid hygiene and topical antibiotics. Topical corticosteroids lessen corneal scarring.

MAIN STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF LEUKOCYTES

Cells Morphological Estimated Circulation Time Chief Features or Lifespan Functions

Lymphocytes Blue cytoplasm; spherical Mean circulation time 10 Produce the nucleus; diameter either 7m hours for majority of cells; effector cells of (small lymphocytes) or 12 m variable time in lymphoid immune (large lymphocytes) organs and tissues; majority of responses cells are long-lived Monocytes Pale blue cytoplasm; kidney- Circulate for 1-3 days; variable Phagocytose shaped nucleus; diameter 12-20 time in tissues unwanted m material; give rise to macrophages and osteoclasts Neutrophils Mauve specific granules; purple Circulation time 6-10 hours; 2- Phagocytos and azurophilic granules; segmented 3 days in tissues kill bacteria nucleus (2-5 lobes); diameter 13 m Eosinophils Large red specific granules; Circulation time 1-10 hours; Have bilobed or segmented nucleus; up to 10 days in tissues antiparasitic and diameter 11 m antiallergic capabilities Basophils Large blue specific granules; Circulation time estimated as Release bilobed or segmented nucleus; 1-10 hours; variable time in inflammatory diameter 11 m tissues mediators in the course of systemic allergic reactions Adapted from Cormack, DH. Clinically Integrated Histology. Lippincott-Raven, 1998.

T LYMPHOCYTE SUBSETS: CHIEF FUNCTIONAL ACTIVITIES

SUBJECT DESIGNATION CHARACTERISTICS

 + Helper T cells TH CD4  Recognize antigen presented in association with MHC Class II surface glycoprotein  When activated by antigen, produce lymphokines that: 1) stimulate proliferation of B cells and antibody production in their progeny 2) stimulate proliferation and maturation of T cells 3) regulate hematopoiesis, and 4) activate macrophages

+ Suppressor T cells TS  CD8

Cytolytic CTL or Tc  CD8+ (cytotoxic) T cells  Recognize antigen presented with MHC Class I surface glycoprotein  Kill allogeneic cells (eg. in transplantedd organs), virus-infected cells, and fungi.  On contacting the target cell, release granules containing cytolytic molecules, notably perforin, a polymerizing protein that produces pores through which degradative enzymes and lymphokines can enter cell. Target cell killing is the result of (1) osmotic lysis, (2) digesting target cell DNA. Memory T cells  Long-lived, and (together with memory B cells) collectively responsible for immunological memory.

Self Assessment – Immunology of the Eye Practice Questions

1. What type of hypersensitivity reaction may cause corneal infiltrates from immune complex deposition and be associated with an autoimmune disease like Rheumatoid Arthritis (RA)?

a. Type I b. Type II c. Type III d. Type IV

2. What is the major cell that functions to phagocytize antigens in the blood?

a. Eosinophils b. Mast cells c. Neutrophils d. Monocytes

3. Where are B cells produced?

a. Pancreas b. Bone Marrow c. Thymus d. Kidney 1. C 2. D 3. B