Ocular Biochem: Ocular Immunochemistry

Immunology

a science that deals with the manner in which organisms defend themselves against foreign invasion

What can initiate an immune reaction?

-any foreign substance

-individuals may reject some of their own tissue by an autoimmune reaction

-immune reactions are normally limited to the region of anterior segment

Immunoglobulins

-large proteins with at least 4 polypeptide chains in each protein

-bind to foreign substance, identify and initiate an immunological response

-aka antibodies

-have an overall conformation similar to the letters Y or T

How many domains do heavier immunoglobulin chains have?

4 domains each connected by random coil sequences

How many domains do lighter immunoglobulin chains have?

2 domains

What is each domain in immunoglobulins held together by?

intrachain disulfide bond - both heavy and light chains are associated by interchain disulfide bonds

What is the amino acid sequence in each C domain like?

always the same for the particular immunoglobulin that it constitutes

What is the amino acid sequence in each V domain determined by?

the antigen with which the antibody must bind

Antigen Binding Domain

the space between two variable domains where binding takes place

What is the binding region for simple immunoglobulins?

-hinge regions: represent sequences of amino acids between C1 and C2 of the heavy regions that can twist or flex

-two binding regions per molecule

-in this way, the immunoglobulin molecules can twist back and forth from a Y to a T in order to bind to one or two antigenic molecules

What is the role of the V-domain?

binding

What is the role of the C1 and C2 domains?

bind to the complement

What is the role of the C3 domain?

bind to receptor proteins on macrophages and monocytes (WBCs)

What is the role of the light chain C domain?

primarily structural in function

What is the role of oligosaccharides attached to immunoglobulins?

-increase water solubility

-protect against enzymatic degradation

-facilitate in secretion from cells producing them

IgA

-tends to form a two molecule unit (dimer) when secreted by cells that make it

-secretory IgA is joined stem-to-stem by a polypeptide called a J chain (15,000 Da) where J strands are for joining

-S chain (secretory; 60,000 Da) is wrapped around the two immunoglobulin tetramer units

What is the role of the S chain in IgA?

-prevents proteolytic degradation of the immunoglobulin

-this is necessary as the precorneal tear film and other secretions are rich in proteolytic enzymes

IgD

-plays a significant role in connective tissue defense and is present on the membranes of many B cells

-may play a role in lymphocyte differentiation and is not normally found in the anterior ocular fluids

IgE

-occurs in very small quantities in the precorneal tear film

-causes release of histamine from mast cells after binding to their surface

-associated with hypersensitivity in asthma and hay fever

IgG

-forms about 80% of all immunoglobulins

-found in blood serum, significant quantities in both precorneal tear film and aqueous humor

-smallest of the immunoglobulins; found in interstitial fluids in highest quantities

-able to diffuse through the cornea by itself

-made in high quantities in response to infection

-forms second line of defense while IgA is the first line of defense to a specific antigen

-can bind to surface receptor proteins of B lymphocytes in order to stimulate those cells

-second most important antibody molecule in ocular tissues

IgM

-very large molecule

-found in precorneal tear film

-composed of five tetrapeptide units (a pentamer as well as J polypeptide

-one unit has 10 antigen binding sites

-first immunoglobulin to be synthesized after the immuno recognization involving binding of IgA to the antigen

-tends to aggultinate to form large molecule complexes around foreign bacteria

Where does the antigen-antibody reaction take place?

between the ends of the variable domains of the heavy and light chains

Antigen-Antibody Reaction

-complex and involves noncovalent forces

-requires correct conformational requirements between an enzyme and a substrate

-each antibody is specific only for a few antigens

-immunoglobulins binds to only a portion of the antigen for most immune antigen-antibody reactions

Antigen

-a substance that will produce an antibody to which it will bind

-it binds to antibodies that are part of the surface of B cells and T cells of the immune system

Epitope (Antigenic Determinant)

-part of an antigen that is recognized by the immune system specifically by antibodies, B cells, or T cells

-could be a short peptide, oligosaccharide, polysaccharide, lipid molecule, or even a nucleic acid fragment

-polypeptides and large polysaccharides may be antigenic

Hapten

-substances that becomes antigenic when combined with a large carrier molecule

-e.g. organic diazonium compounds

Hydrogen Bond

sharing of a hydrogen atom due to partial charges between two atoms

Electrostatic Bond

full positive and/or negative charge(s) on antigen and/or antibody, respectively

van der Waals Reactions

induced partial charge between two atoms (not involving hydrogen)

What are the different factors that contribute to antigen-antibody reactions at the antigen-binding region of an immunoglobulin?

-chemical bond: hydrogen, electrostatic, van der Waals

-nonpolar bond: hydrophobic

-conformation: lock and key

Hydrophobic Bond

association of molecules away from a water (polar) environment

Lock and Key Conformation

complementary shape of antigen and hypervariable part of V domain

Radioimmunoassay

-sensitive assays

-antigen-antibody reactions are coupled to radioactive compounds to give extremely sensitive assays

-used to detect hormones or other compounds present in low concentration in tissues

What occurs in a radioimmunoassay?

-in the assay, a radioactive antigen competes with a non-radioactive antigen for the possession of a binding site on an antibody

-the assay ultimately measures the radioactivity on the bound antibody

-greater the binding of a nonradioactive antigen on an antibody, the lower the level of radioactivity measured

How does the control of the radioimmunoassay construct a standard curve?

-control gives the reference radioactivity for 100% binding of radioactive antigen

-standard known amounts of nonradioactive antigens are reacted in the upper row and compared with the control

-from this, a standard curve is constructed and one or more unknown samples are run

-percent of radioactivity in the antibodies are measured and intersected with the standard curve to obtain the concentration of antigen

-useful for determining very low concentrations of hormones present in blood

B Cells or B Lymphocytes

-site of immunoglobulin synthesis

-formed in the bone marrow

-transported to lymphoid tissues prior to full development as B plasma cells

-can only produce nonspecific IgM and IgD when they leave the bone marrow

-nonspecific IgM and IgD bind to the cell surface membrane at the stem side of the molecule

Why are B cells induced in lymphoid tissues?

to make specific immunoglobulins

Where does the synthesis of immunoglobulins by developing B cells begin?

bone marrow

How are B cells involved in the synthesis of immunoglobulins?

-B cells become committed to the synthesis of light chains initially and then heavy chains to produce IgM and then IgD

-these immunoglobulins have hydrophobic tails that bind to the membranes of B cells

-when antigens encounter IgM and IgD bound to the nascent B cells, they bind to these antibodies and transmit a signal to being specific antibody synthesis

-such short-lived, high output B lymphocytes are called plasma cells

Where are uncommited B cells present?

bone marrow or fetal liver

What do DNA of B cells code for?

-300 separate sequences of the variable domain (V genes)

-4 separate peptides that will connect or join the V and C domains (J genes)

What do J genes code for?

a joining peptide between V and C domains

What is the code for the constant domain contained in?

C gene

What occurs when the stem cell becomes committed to B cell development in the antibody diversity hypothesis?

-the DNA containing all these codes recombines by excision of part of the DNA

-all of the V genes 30 through 300 have been excised and V gene 29 has been directly joined to J gene 2

-J gene 1 is also excised

-even though V genes 1 through 28 are still present, they are not directly connected in sequence to the J2 gene and are therefore inactive

-this is also true for J3 and J4 genes

-this is the form of DNA that exists in a B cell that is ready to begin IgG synthesis

-in the transcription state of forming hnRNA, only V gene 29, J genes 2, 3, 4, and the C gene are transcribed

What occurs when hnRNA is processed to mRNA in the antibody diversity hypothesis?

-all the introns, as well as the inactive J3 and J4 exons, are spliced out of the gene sequence

-this leaves the V29, J2, and C codes for the specific light chain of IgG to be translated and assembled into an IgG, which will bind to a specific antigen

-the processing of the heavy chains is similar, but more complex

What are the three antibodies that occur in significant amount in ocular tissues?

-IgA

-IgG

-IgM

IgE is present, but tightly bound to mast cells, DOES NOT occur in free form in normal cornea

IgA in Precorneal Tear Film

-primarily the secretory type

-assembled from dimeric IgA in the lacrimal gland epithelial cells

-these epithelial cells synthesize the S chain polypeptide that surrounds the IgA molecules and protects them from degradation

What ocular tissue is IgM involved with?

due to its large size, it is limited in penetration of the cornea

What parts of the eye are the occurrence of immunoglobulins limited to?

-precorneal tear film

-aqueous

-ocular blood vessels

-cornea

-sclera

not present in deeper tissues such as lens, vitreous, and non-vascular parts of retina since no antibodies are normally found

What happens to the content of immunoglobulins when tear production is more?

the content of immunoglobulins and other protein contents are less due to reflex dilution

Where is IgA mainly located in the eye?

any part that involves secretions

Where is IgD and IgM mainly located in the eye?

blood vessels

Where is IgE mainly located in the eye?

mast cells

Why is the manner of collection of tears for assay important?

-tears collected with small sponges tend to be contaminated with conjunctival secretions

-tears obtained with filter paper (Schirmer strips) may cause reflex stimulation

-tears drawn into glass capillary tubes do not have these drawbacks if collection is done carefully

What is the first antibody to respond to the herpes virus from precorneal tear film?

IgM, followed by increases in IgG and secretory IgA

Where are herpes antigens present in the eye as the herpes virus infection progresses?

-cornea: infected cells may have some of the viral antigens incorporated into their plasma membranes, will trigger antigen-body reactions against the cells themselves

-conjunctiva

-tear film

-iris

-trigeminal ganglion

What is the role of IgA in treating the herpes virus?

secretory IgA will attack the viruses that are extracellular - this hinders the virus form attaching to uninfected cells

What is the role of IgG in treating the herpes virus?

-causes phagocytosis of the antigen virus

-IgG4 levels increase by seven fold in response to herpes infection

What are levels of IgG like in response to infections?

-in patients with acute hemorrhagic conjunctivitis, the levels of IgG can reach up to 1300 micrograms/mL whereas its normal levels are 4 micrograms/mL

-free IgE has been detected in tears of individuals susceptible to pollen-born allergens

Complement

-a collection of relative proteins like proteolytic enzymes or membrane binding proteins

-an immune biochemical mechanism that is capable of destroying gram negative bacteria, some parasites and neutralizing some viruses

What are the two main functions of a complement?

-destruction of foreign organisms by membrane lysis

-activation of phagocytosis (cell engulfment) following chemotaxis (cell movement induced by chemicals)

What are complements comprised of?

-11 protein types namely C1q, C1r, C2, C3, C4, C5, C6, C7, C8, and C9

-more than one protein of the same type is used in the activation of complement complex and its byproduct formation

-mechanism invokes cascade amplification

Complement Activation

-begins with the binding of the C1 complex (C1q, two C1s, and two C1r molecules held together by Ca2+ ions) to the stem portion of either IgG or IgM molecules

-IgG or IgM molecules are themselves bound to the membrane antigens

-upon binding to the Ig, a conformational shift of the C1 complex induces autocatalytic properties in C1s and C1r

-protein cause their own activation

-C1s molecules ultimately become a protelytic enzyme for the next substrate proteins in the sequence C4 and C2

What happens to C4 in the complement activation?

-C4 is broken into fragments C4a and C4b

-C4a binds to the membrane in the vicinity of the antigen-antibody complex while C4b does not participate in the sequence

What happens to C2 in the complement activation?

-C2 is broken down into a C2a and C2b by C1s

-C2a binds to the C4b molecule and becomes an enzyme (C3 covertase)

What happens to C3 convertase in the complement activation?

-C3 convertase splits into C3a and C3b

-C3b fragment associates with C4b and C2a to form C5 convertase

What happens to C5 convertase in the complement activation?

-split into C5a and C5b

-C5b fragment binds to a nearby region of the antigen membrane and initiates formation of a complex that contains bound C6, C7, C8, and several molecules of C9

C9 Proteins

-channel forming proteins

-must pierce the cytoplasmic membrane of bacteria in order to be effective

What is the role of C9 proteins in complement activation?

-this complex forms a channel in the antigen membrane through which Na and water molecules quickly pass

-formation of the channel and rapid movement of water and ions causes lysis of gram negative bacterial membrane

-lysis occurs due to the osmotic shock produced by the lesion

What is the role of a lysozyme when the channel for complement activation allows it inside?

-lysozyme attacks the thinner peptidoglycan wall between the outer lipoprotein cover and the plasma membrane of the bacteria

-viruses exposed to the complement are neutralized

-complement prevents a virus from attaching to host cells by covering the viral surface with complement proteins

What is the order of immunoglobulins for lightest to heaviest?

-IgG (lightest)

-IgA

-IgD

-IgE

-IgM (heaviest)

Antigenic

ability to trigger antibody production

What is the effective distribution of complement in the eye limited by?

high molecular weight of the C1q component

Complement in the Eye

-this component only partially penetrates the cornea

-none of the complement proteins are normally transported to the aqueous chamber and the interstitial fluids of the retina

What is complement activation limited by?

the presence of inhibitory proteins that may protest the cornea from unwarranted complement activation

What is the destruction of A auereus enhanced by?

complement fixation

What does the destruction of gram positive organisms require?

lysozyme to destroy peptidoglycan coverings prior to direct complement destruction

Where does H gonorrhea occur in the eye?

occurs in both conjunctiva and cornea, elicits complement fixation

-a gram negative bacteria associated with gonorrhea infections

How does the complement protect the eye?

-the complement has a protective effect against P aeruginosa, a gram positive bacteria that infects the cornea

-in case of herpes simplex, complement fixation occurs only when the virus penetrates to the stromal level

-the amount of antibodies present must have a high enough concentration to elicit the complement reaction

Cicatricial Pemphigoid

-an autoimmune disease

-immune mechanisms including complement are directed against basal epithelial cells of the conjunctiva

Moonren's Ulcer

-may have autoimmune origin

-involves wasting away of corneal tissue from the exterior

-activation of mast cells by CSa and other complement fragments takes place

Inflammation

a physiological reaction to an injury or an invasion by foreign antigens

What are the three series of biochemical reactions that are involved in inflammation?

-an increased blood supply to the affected area

-an increased capillary permeability that allows WBC's and large molecules to enter interstitial spaces

-migration of leukocytes to the exact site of injury of foreign invasion (chemotaxis)

What is the inflammation mechanism?

-C3a, 4a, and 5a peptides diffuse away from the site of complement fixation until they encounter nearby blood vessels and mast cells

-granules hold inflammatory agents such as histamine, serotonin, prostaglandins, leukotrienes, and platelet activating factory

-complement peptides or anaphylatoxins binds to the mast cell surface at receptor proteins

-upon binding, complement peptides inhibit the activity of adenylate cyclase via a G-protein

-inhibition of adenylate cyclase activity

-decreased activity of adenylate cyclase lowers the levels of cAMP

-this inturn cause the mast cells granules to fuse with the plasma membrane and release their contents into the tissue environment

Mast Cells

small white blood cells that contain numerous granules

What does the inhibition of activity of adenylate cyclase in mast cells involve in the inflammation mechanism?

involves C5a peptide and its receptor C5aR

the complex of C5a and C5aR also involves Gi protein

Chemotaxis

-the released histamine as well as other C3-4-5a peptides diffuse toward local blood vessels where they induce vasodilation

-this increases local blood supply, allow fluid to leak from the vessels

-also causes WBCs to adhere to the blood vessel walls and to squeeze through the wall (diapedesis) into the surrounding interstitial fluid

What do selectins and B2 integrins (cell surface proteins) cause in chemotaxis?

-cause the WBCs to bind to the walls of the blood vessels prior to their passing through them

-once released, the leukocytes are guided to the site of the immune reaction (complement fixation) by the chemical gradient created by the diffusing C3-4-5a peptides

What is the direction in which leukocytes travel determined by in the chemotaxis mechanism?

-relative density of C3-4-5a peptides

-location of C3-4-5a peptides bound to receptor proteins on a given region of the leukocyte membrane

What is the chemotaxis mechanism?

-a chemoattractant such as C5a binds to a cell receptor protein

-cell receptor protein is attached inturn to a G-protein that activates or inhibits a phosphate transferring enzyme

-G-protein activates a Phosphoinositol-3 kinase γ that sets off a biochemical cascade

-this cascade leads to production of motion-setting actin fibers

-it is necessary for WBC to be able to recognize the antigen upon arrival at the site of complement fixation where the offending antigens are present

Opsonization

-the coating which antigens receive from the binding of antibodies (IgG) and complement protein components - recognition labelling

-antigen are marked as targets for the leukocytes

-leukocytes possess receptor proteins for these opsonization components and begin an interesting transformation process after binding to them

What occurs during opsonization?

-antigens become surrounded by the leukocyte membranes = phagosome

-phagosome is internalized and fused with lysosomes where the antigenic particles or bacteria or viruses are destroyed

-the fused bodies at this point are called phagolysosomes

Where does the destruction of antigenic organisms begin?

in the phagosome with the formation of highly reactive forms of oxygen

What is the destruction of antigenic organisms initiated by?

a respiratory burst

What occurs during the destruction of antigenic organisms?

-leukocytes take up large quantities of oxygen to form superoxide radicals

-radicals are rapidly converted to other compounds such as hydrogen peroxide and singlet oxygen (highly reactive with tissue proteins and membrane lipids)

-superoxide is dismuted or disproportioned (one mol is reduced while the other is oxidized) to hydrogen peroxide by the enzyme superoxide dismutase

-some of the hydrogen peroxide is also converted to hypochlorous acid, a very highly reactive and destructive chemical

Superoxide Radicals

have unpaired electrons (highly reactive and unstable)

What is the influence of ROS (reactive oxygen species) on membrane proteins and lipids?

-all these oxidative species attack the membranes of the organism within the phagosome

-these oxidative species spill out to the surrounding environment to cause collateral damage to nearby cells

Myeloperoxidase

-an ROS (reactive oxygen species) that catalyzes formation of hypochlorous acid and has a heme group at its active site that has a green color

-green color is due to the green hue associated with pus formation

Pus Formation

buildup of cellular debris from the oxidative destruction and enzymatic onslaught

How many different kinds of hydrolytic enzymes act to break down the viral or bacterial structures?

-40 different kinds of hydrolytic enzymes

-includes proteases, nucleases, glycosidases, lipases, etc.