1. 1. Dr. M. Krishna Vasudev Immunoglobulins: Structure and Function
2. 2. Immunoglobulin Immunoglobulin is a glycoprotein that is made in response to an antigen and can recognize and bind to the antigen that caused its production.
3. 3. Are gamma globulins Synthesized by plasma cells Constitute 25-30 % of total serum proteins Antibodies are present in serum, tissue fluids and mucosal surfaces. All antibodies are immunoglobulins, but all immunoglobulins may not be antibodies
4. 4. Basic structure Composed of 4 polypeptide chains. 2 identical light and 2 identical heavy chains Linked by disulphide bonds Light chains similar in all immunoglobulins Light chains occur in 2 varieties kappa and lambda Light and Heavy chains are subdivided into variable and constant region. Each heavy and light chain contains amino terminal in variable region carboxy terminal in constant region
5. 5. Heavy chains are structurally and antigenically distinct for each class Each immunoglobulin peptide chain has intra chain disulphide bonds- form loops Each loop is compactly folded to form a globular structure- domain Light chain contains a single variable domain (VL) and a single constant domain (CL). Heavy chain contains one variable domain (VH) and 3 constant domains (CH1, CH2, CH3) Hinge region is the segment in heavy chain - between CH1, CH2
6. 6. Digestion with proteolytic enzymes Papain enzyme Peptide bonds in the hinge region are broken Produces 3 fragments 2 identical fragments called Fab fragments antigen binding activity. Other fragment called Fc fragment (Fraction crystallizable)
7. 7. Pepsin digestion Produce a single fragment composed of two Fab like subunits F(ab)2 binds antigen Fc fragment is not recovered- digested to small numerous peptides.
8. 8. Classification Based on structure and antigenic nature of H chain the immunoglobulins are classified into 5 classes. Ig G- (gamma) Ig A- (alpha) Ig M- (mu) Ig D- (delta) Ig E - (epsilon)
9. 9. Immunoglobulin G (Ig G) Most abundant class in serum Constitutes 80% total immunoglobulin Present in blood, plasma and tissue fluids Contains less carbohydrate than other immunoglobulins It has a half life of 23 days: the longest of all of the immunoglobulin isotypes
10. 10. Crosses placenta and provide natural immunity to foetus and neonate at birth Acts against bacteria and viruses by opsonizing Neutralize toxin Activate complement by classical pathway Catabolism of IgG is unique in that it varies with its serum concentration
11. 11. Sub classes of Ig G Ig G1, Ig G2, Ig G3, Ig G4.
12. 12. Biological function of subclasses IgG1, IgG3, IgG4 cross placenta and protect foetus IgG3 activates complement IgG1 and IgG3 binds to Fc receptor on phagocytic cells, monocytes and macrophages and mediate opsinization.
13. 13. Immunoglobulin A (Ig A) Constitutes 10-15 % of total immunoglobulins Present in milk, saliva, tears, mucous of respiratory tract, digestive tract and genitourinary tract. In serum exist as monomer In external secretions exist as dimer called secretory Immunoglobulin. Has J chain and secretory piece. Half life: 6-8 days
14. 14. Formation of secretory Ig A Dimeric Ig A binds to the receptor on the surface of the epithelial cells - endocytosed and transported across the cell to the luminal surface After reaching the surface, the poly-Ig receptor is cleaved The portion of the receptor that remains attached to the Ig A dimer secretory component Secretory piece protects Ig A from digestive enzymes and denaturation by
15. 15. Functions Provides local immunity. Secretory Ig A binds to surface antigens of microorganism and prevent its attachment and invasion of the mucosal surfaces of respiratory and digestive tract- immune elimination. Secretory IgA provides important line of defense against salmonella, Vibrio cholerae, N. gonorrhoeae, influenza virus and poliovirus. Secretory IgA present in breast milk protects newborn during first months of life. Activates complement by the alternative pathway Promotes phagocytosis and intracellular killing of microorganisms
16. 16. Immunoglobulin M (Ig M) Accounts for 5-10% of total serum proteins Polymer of five monomeric units (pentamer) Held together by disulfide bonds and J chain Mol. Wt. of 900,000- 10,00,000 (millionaire molecule) Half life: 5 days
17. 17. Most of IgM (80%) present intravascularly Present in low concentration in intercellular tissue fluids Cannot cross placenta Presence of IgM antibody in serum of newborn indicate congenital infection. Earliest immunoglobulin to be synthesized by foetus (20 weeks) First immunoglobulin to be produced in primary response to antigen Relatively short-lived hence its demonstration in the serum indicates recent infection Monomeric IgM appears on the surface of unstimulated B lymphocytes and act as receptors
18. 18. Functions It agglutinates bacteria Activates complement by classical pathway Causes opsonization and immune heamolysis Believed to be responsible for protection against blood invasion by microorganisms
19. 19. Immunoglobulin E (Ig E) Structure is similar to Ig G Has 4 constant region domains. Mol. Wt. 1,90,000 Half life: 2 days Heat labile (inactivated at 560C in 1 hour) Normal serum concentration 0.3 ug/ml Mostly present extra cellularly Does not cross placenta
20. 20. Produced in the lining of respiratory and intestinal tract Known as reagin antibody Does not activate complement nor agglutinate antigens Binds to the Fc receptors on the membranes of blood basophils and tissue mast cells Mediates immediate hypersensitivity reaction and P.K. reaction Responsible for symptoms of anaphylactic shock, hay fever and asthma. Play a role in immunity against helminthic parasites
21. 21. IgE binds to Fc receptors on the membrane of blood basophils and tissue mast cells. When two IgE molecules on the surface of these cells are cross linked by binding of the same antigen- cells degranulates. Release histamine and pharmacological mediators of anaphylaxis from cell. The physiological role of IgE appears to be protection against pathogens by mast cell degranulation and release of inflammatory mediators
22. 22. Mediates P.K. reaction (PRAUSNITZ & KUSTNER) The presence of a serum component responsible for allergic reaction was first demonstrated by Prausnitz and Kustner in 1921. Kustner was suffering from atopic hypersensitivity to certain species of fish Kustners serum was injected intracutaneously in Prausnitz After 24 hrs small quantity of cooked fish antigen was injected at the same site A wheal and flare reaction occurred within minutes.
23. 23. Immunoglobulin D (Ig D) Structure is similar to IgG Serum concentration 30 micrograms per ml Constitutes 0.2% of total immunoglobulins Half life: 3 days IgD together with IgM is major membrane bound immunoglobulin on unstimulated B lymphocytes-acts as recognition receptors for antigens
24. 24. Properties and biological activities of Immunoglobulins Ig G Ig A Ig M Ig D Ig E 1. Structure Monomer Monomer in serum Dimer in secretion Pentamer Monomer Monomer 2. Heavy chain CH domain Gamma Three Alfa Three Mu Four Delta Three Epsilon Four 3. Mol. Wt. 1,50,000 1,60,000 9,00,000 1,80,000 1,90,000 4. Serum concentration (mg/ml) 12 2 1.2 0.03 0.00004 5. Present on membrane of mature B cell _ _ + + _ 5. Intravascular Distribution (%) 45 42 80 75 50 6. Crosses placenta + - - - - 7. Present in milk + + - - - 8. Selective secretion by seromucous glands - + - - - 9. Activation of complement Classical Alternate + - - + + - - - - - 10 Binds to FC receptor of phagocytes + - - - - 11 Induces mast cell degranulation - - - - +
25. 25. Complement System
26. 26. Consists of approx. 20 proteins that are present in normal human serum synthesized mainly by liver Heat-labile inactivated by heating serum at 560C for 30 minutes Able to augment the effects of other components of the immune system Important component of innate host defenses
27. 27. Three main effects: 1. Lysis of cells (bacteria, allografts, tumor cells) 2. Generation of mediators of inflammation 3. Opsonization enhancement of phagocytosis
28. 28. Sequential activation of complement components occurs via one of three pathways: 1. Classic pathway 2. Lectin pathway 3. Alternative or Properdin pathway Lectin and alternative pathways are more important the first time we are infected by microorganisms because antibody required to activate the classic pathway is not yet present
29. 29. Part of acquired or adaptive immunity Activated by Ag-Ab complexes Immunoglobulins involved: IgM and IgG (except IgG4) Involves activation of C1 Composed of C1q, C1r, and C1s binds to Fc portion of IgG and IgM Requires calcium for activation Classic Pathway
30. 30. Other activators include: 1. Viruses Murine and Retroviruses 2. Bacteria Mycoplasma 3. Polyanions, especially bound to cations a. PO4 3- - DNA, lipid A, cardiolipin b. SO4 2- - dextran, heparin, chondroitin 4. Arrays of terminal mannan groups Classic Pathway
31. 31. Classic Pathway Components of the Classical Pathway Native component Active component(s) Function(s) C1(q,r,s) C1q Binds to antibody that has bound antigen, activates C1r. C1r Cleaves C1s to activate protease function. C1s Cleaves C2 and C4. C2 C2a Unknown. C2b Active enzyme of classical pathway; cleaves C3 and C5. C3 C3a Mediates inflammation; anaphylatoxin. C3b Binds C5 for cleavage by C2b. Binds cell surfaces for opsonization and activation of alternate pathway. C4 C4a Mediates inflammation. C4b Binds C2 for cleavage by C1s. Binds cell surfaces for opsonization.
32. 32. Classic Pathway Components of the Membrane-Attack Complex Native component Active component(s) Function(s) C5 C5a Mediates inflammation; anaphylatoxin, chemotaxin. C5b Initiates assembly of the membrane-attack complex (MAC). C6 C6 Binds C5b, forms acceptor for C7. C7 C7 Binds C5b6, inserts into membrane, forms acceptor for C8. C8 C8 Binds C5b67, initiates C9 polymerization. C9 C9n Polymerizes around C5b678 to form channel that causes cell lysis.
33. 33. Classic Pathway
34. 34. Alternative Pathway Also known as the Properdin Pathway Part of innate immunity Bypasses C1, C4, and C2 Does not require an antigen-binding protein Does not wait for antibody to be formed for activation Acts synergistically with the classical pathway
35. 35. Alternative Pathway Usually activated by products of micro-organisms like endotoxin Other activators include: 1. Complexes containing IgA 2. Some virus-infected cells (e.g. EBV) 3. Many gram negative and gram positive organisms 4. Parasites Trypanosomes, Leishmania 5. Dextran SO4 6. Erythrocytes 7. Carbohydrates (agarose)
36. 36. Alternative Pathway Components of the Alternate Pathway Native component Active component(s) Function(s) C3 C3a Mediates inflammation; anaphylatoxin. C3b Binds cell surfaces for opsonization and activation of alternate pathway. Factor B B Binds membrane bound C3b. Cleaved by Factor D. Ba Unknown. Bb Cleaved form stabilized by P produces C3 convertase. Factor D D Cleaves Factor B when bound to C3b. Properdin P Binds and stabilizes membrane bound C3bBb.
37. 37. Alternative Pathway
38. 38. Lectin Pathway Also known as the MBL Pathway Activated by binding of mannose-binding lectin (or mannose-binding protein) to surface of microbes bearing mannan (polymer of the sugar mannose) in a calcium dependent manner Binding causes activation of MASP (MBP- associated serine proteases) cleave C2 and C4
39. 39. Lectin Pathway
40. 40. All three pathways lead to production of C3b central molecule of complement cascade Presence of C3b on surface of a microbe marks it as foreign and targets it for destruction C3b with two important functions: 1. Combines with other complement components to generate C5 convertase 2. Opsonizes bacteria
41. 41. Biologic Effects: 1. Opsonization C3b & C1q; enhance phagocytosis 2. Chemotaxis C5a and C5,6,7 complex attract neutrophils C5a enhance adhesiveness of neutrophils to the endothelium 3. Anaphylatoxin (C3a, C4a, C5a) Cause degranulation of mast cells Bind directly to smooth muscles of bronchioles bronchospasm
42. 42. Biologic Effects: 4. Cytolysis (MAC) Disrupt the membrane & the entry of water and electrolytes into the cell 5. Enhancement of antibody production Binding of C3b to its receptors on the surface of activated B cells enhanced antibody production
43. 43. Regulation of Complement System 1. C1 inhibitor (C1-INH) Important regulator of classic pathway A serine protease inhibitor (serpin) Irreversibly binds to and inactivates C1r and C1s, as well as MASP in lectin pathway 2. Factor H Regulate alternative pathway Reduce amount of C5 convertase available With both cofactor activity for the factor I- mediated C3b cleavage, and decay accelerating activity against C3bBb (C3 convertase)
44. 44. Regulation of Complement System 3. Properdin Protects C3b and stabilizes C3 convertase 4. Factor I Cleaves cell-bound or fluid phase C3b and C4b inactivates C3b and C4b 5. Decay accelerating factor (DAF) Glycoprotein on surface of human cells Prevents assembly of C3bBb or accelerates disassembly of preformed convertase no formation of MAC Acts on both classical and alternative
45. 45. Regulation of Complement System 6. C4b-binding protein (C4BP) Inhibits the action of C4b in classical pathway Splits C4 convertase and is a cofactor for factor I 7. Complement Receptor 1 (CR-1) Co-factor for factor I, together with CD46 8. Protectin (CD59) and Vitronectin (S protein) Inhibits formation of MAC by binding C5b678 Present on self cells to prevent complement from damaging them
46. 46. Clinical Aspects 1. Deficiency of C5-C8 & Mannan-binding lectin Predispose to severe Neisseria bacteremia 2. Deficiency of C3 Severe, recurrent pyogenic sinus & resp. tract infections 3. Deficiency of C1 esterase inhibitor Angioedema inc. capillary permeability and edema 4. Deficiency of DAF Increased complement-mediated hemolysis paroxysmal nocturnal hemoglobinuria
47. 47. Clinical Aspects 5. Transfusion mismatches Activation of complement generate large amounts of anaphylatoxins & MAC red cell hemolysis 6. Autoimmune diseases Immune complexes bind complement low complement levels + activate inflammation tissue damage 7. Severe liver disease Deficient complement proteins predispose to infection with pyogenic bacteria
48. 48. Clinical Aspects 8. Factor I deficiency Low levels of C3 in plasma due to unregulated activation of alternative pathway recurrent bacterial infections in children Mutations in factor I gene implicated in development of Hemolytic Uremic Syndrome
49. 49. Dr. M. Krishna Vasudev Dept of General Medicine Immunodeficiency disorders
50. 50. Types Primary-usually genetic, congenital Secondary-Acquired
51. 51. Mechanisms of Immunodeficiency Loss or reduction of: Cell type Cell numbers Cell function
52. 52. Loss of Cell Function Receptors Cell signaling Cytokine production Ig production Co stimulation impairment Intracellular killing Extravasation impairment
53. 53. Primary Immunodeficiency Myeloid lineage Congenital agranulocytosis Leukocyte-adhesion deficiency Lymphoid lineage Severe combined immunodeficiency (SCID) B cells Agammaglobulinemia Hypogammaglobulinemia Specific Ig Deficiencies T cells DiGeorge Syndrome Wiskott Aldrich Syndrome
54. 54. Secondary Immunodeficiency Drug related Disease related Cancer AIDS HIV T helper cell as target
55. 55. SCID Various genetic defects No TCR or defective TCR Defective cell signaling Defective IL 2 Recurrent infections Death at early age
56. 56. Wiskott Aldrich Syndrome X linked disorder Affects platelet numbers/function Affects T cell function Cytoskeleton of lymphocytes affected Lower amounts of IgM Increased susceptibility to certain bacterial infections
57. 57. Brutons Agammaglobulinemia Low levels of IgG B cell signal transduction affected Defective BCR Recurrent bacterial diseases starting at end of first year of life Short life span
58. 58. DiGeorge Syndrome Poorly developed or functioning thymus Associated with other developmental conditions Depression of T cell numbers Absence of T cell response Humoral response to T independent antigens only
59. 59. Secondary Immunodeficiency Drugs Irradiation Cancer AIDS
60. 60. AIDS HIV as agent T helper as target cell Attacked by own CD8 cells Susceptible to opportunistic infections Kaposis sarcoma CMV Candida Pneumocystis carinii