Basic immunology

Introduction
History
Types of immunity
Tissues of immunity
Cells of immunity
Basic aspects of immunology
Major histocompatibility complex
Cytokines
Disorders of immune system
Immune responses in periodontal pathogenesis
Periodontal vaccine
Host modulation
Conclusion
References

1. BASIC IMMUNOLOGY Hema Duddukuri 1 st year PG Dept. of Periodontics Panineeya institute of dental sciences

2. Introduction History Types of immunity Tissues of immunity Cells of immunity Basic aspects of immunology Major histocompatibility complex Cytokines Disorders of immune system Immune responses in periodontal pathogenesis Periodontal vaccine Host modulation Conclusion References CONTENTS

3. INTRODUCTION Immunity: resistance to disease, specifically infectious disease. Immune system: The collection of cells, tissues, and molecules that mediate resistance to infections. Immune response: the coordinated reaction of these cells and molecules to infectious microbes . Immunology: the study of the immune system, including its responses to microbial pathogens and damaged tissues and its role in disease.

4. Traditional concept: Immunity refers to protection against infectious diseases . Modern concept: Immunity is a function of which an individual recognizes and excludes antigenic foreign substances. It is normally beneficial, but sometimes, it is injurious.

5. 1796 – First demonstration of  smallpox vaccination  ( Edward Jenner ) 1862 –  Phagocytosis  ( Ernst Haeckel ) 1877 –  Mast cells  ( Paul Ehrlich ) 1883 – 1905 – Cellular theory of immunity via phagocytosis by macrophages and microphages ( polymorphonuclear leukocytes) ( Elie Metchnikoff ) 1890 – Demonstration of antibody activity against   diphtheria and tetanus toxins . Beginning of humoral theory of immunity - Emil von Behring and Kitasato Shibasaburō 1900 – Antibody formation theory ( Paul Ehrlich ) HISTORY

6. 1965 – Discovery of " immune interferon" (gamma interferon) (E.F. Wheelock) 1971 – Peter Perlmann and Eva Engvall at Stockholm University invented ELISA 1973 – Dendritic Cells first described by Ralph M. Steinman 1975 - Discovery of Natural Killer cells ( Rolf Kiessling , Eva Klein, Hans Wigzell ) 1980-1983 – Discovery and characterization of interleukins (IL-1 IL-2 ) ( Robert Gallo, Kendall A. Smith, Tadatsugu Taniguchi ) 1985 – Identification of Toll-like receptors (Christiane Nusslein Volhard ) 2000 - Characterization of M1 and M2 macrophage subsets by  Charles Mills

7. Importance of the immune system in health and disease :

8. TYPES OF IMMUNITY

9. Resistance to infections which, an individual possesses by virtue of his genetic and constitutional make-up. Not affected by prior contact with microorganisms or immunization. Nonspecific - when it indicates a degree of resistance to infections in general. Specific - where resistance to a particular pathogen is concerned. Innate or native immunity:

10. Species immunity: refers to the total or relative refractoriness, to a pathogen, shown by all members of a species. Racial immunity: Within a species, different races may show differences in susceptibility to infections. Such racial differences are known to be genetic in origin, Individual immunity: The differences in innate immunity exhibited by different individual in a race.

11. Factors influencing the level of innate immunity:

12. Mechanisms of innate immunity: Epithelial surfaces: Antibacterial substances in blood and tissues : Complement system Beta-lysine Basic polypeptides- leukins , plakins Acidic substances like lactic acid Enzymes - lactoperoxidase , lysozymes Interferons

13. Microbial antagonisms: Cellular factors: Phagocytic cells , discovered by Metchinikoff Microphages : PMN Macrophages: Monocytes Natural killer cells Innate Lymphoid Cells

14. Inflammation: Fever: A rise of temperature following infection is a natural defense mechanism. Accelerates the physiological processes and destroy the infecting pathogens. Stimulates the production of interferon. and aids recovery from viral infections.

15. Acute phase proteins: Infection or injury leads to a sudden increase in plasma concentrations of certain proteins. These include: C reactive protein (CRP), Mannose binding protein, Alpha-1-acid glycoprotein, Serum amyloid P component etc., A ctivate the alternative pathway of complement. Enhance host resistance, prevent tissue injury and promote repair of inflammatory lesions.

16. Acquired Immunity : The resistance that an individual acquires during life is known as acquired Immunity. also known as adaptive immunity as it represents an adaptive response of t he host to a specific pathogen or other antigen. Types:

17. Comparison of active and passive immunity: Active immunity: Passive immunity: Produced actively by host's immune system. Received passively. No active host participation. Induced by infection or by immunogens . Readymade antibody treatment. Durable effective protection. Transient, less effective. Immunity effective only after lag period. Immediate immunity. Immunological memory present. No memory. Booster effect on subsequent dose. Subsequent dose less effective. Negative phase may occur. No negative phase. Not applicable in the immunodeficient . Applicable in immunodeficient .

18. Types of acquired immunity:

19. Properties of acquired immunity:

20. Phases of adaptive immune response:

21. TISSUES OF IMMUNE SYSTEM The tissues of the lymphoid organs can be classified into: Central / primary lymphoid organs Peripheral / secondary lymphoid organs Central lymphoid organs: Lymphoepithelial structures in which the precursor lymphocytes proliferate, develop and acquire immunological capability. Thymus Bone marrow Peripheral lymphoid organs: After acquiring immunocompetence , the lymphocytes migrate along blood and lymph streams, accumulate in the peripheral lymphoid organs and following antigenic stimulus, effect the appropriate immune response. Lymph nodes Spleen Mucosa associated lymphoid tissue (MALT)

22. Thymus:

23. Lymph nodes: Encapsulated nodular aggregates of lymphoid tissues. Lymph: tissues  lymphatic vessels  lymph node  blood circulation. APCs :- pick up antigens of microbes from epithelia and other tissues and transport these antigens to the lymph nodes. On antigenic stimulation: primary lymphoid follicles  germinal centers T cell zone: inbetween cortex and medulla which has t lymphocytes

24. Lymph nodes of head & neck region

25. Spleen:

26. Mucosa associated lymphoid tissue (MALT):

27. Waldeyers ring

28. CELLS OF IMMUNE SYSTEM

29. Cell Life span T cells Weeks to months Memory cells - years B cells 5-6 weeks Macrophages 6-16 days Monocytes 2-5 days Neutrophils 2-5 days Dendritic cells Few months

30. Lymphocytes: C irculate through lymphoid organs and nonlymphoid tissues. R ecognize foreign antigens and initiate adaptive immune responses. O.5 – 1 x 10 12 Lymphocytes. Arise from stem cells in bone marrow. Types: B-lymphocytes: B1 B2 T-lymphocytes: CD4+ T cells – Helper T cells Special subset of CD4+ T cells – Regulatory T lymphocytes. CD8+ T cells – Cytotoxic T lymphocytes. Null cells/large granular lymphocytes

31. MATURATION OF LYMPHOCYTES

32. CLASSES OF LYMPHOCYTES

33. STAGES IN THE LIFE HISTORY OF LYMPHOCYTES

34. CLONAL SELECTION

35. Natural killer cells: Natural killer (NK) cells recognize infected and stressed cells and respond by killing these cells by secreting the macrophage-activating cytokine IFN- γ

36. Dendritic cells: Leucocytes with cytoplasmic processes. Dermal dendrocytes are also known as histiocytes . Langerhans cells peripheral dendritic cells Possess receptors for C3a Express MHC class II, ICAM Molecules. B ridge between innate and adaptive immune response. Can express MMP in response to bacterial challenge and can contribute to periodontal destruction.

37. Mast cells: Important in immediate inflammation Has receptors for complement components (C3a, C5a) and Fc portion of IgE and IgG ( Fcxr and Fcyr). Express Toll like receptors Stimulation of receptors causes activation of vasoactive substances leading to inflammatory process. Contain lysozymes, histamine, neutrophil chemotactic factor, heparin. They can synthesize slow reactive substances like SRS-A, TNFa and IL6.

38. Neutrophils: First cells to arrive at the site of inflammation Possess receptors for c1,c3,c4 & c5 P rimary granules: Beta glucoronidases , betagalactosidase , Elastase , Myeloperoxidase, Lysozyme Secondary granules: Lactoferrin , Lysozyme, Antimicrobial peptides Beta defensins Cathelicidins cystatins

39. Macrophages: Monocytes enter tissue and differentiate into macrophages Antigen presenting cells Ingest antigens and coat with MHC molecules and present to T cell receptor Secrete variety of cytokines like IL1, IL6, TNFA, IFN, Complements and growth factors and can phagocytose antigens.

40. Phagocytosis:

41. Transendothelial migration

42. BASIC ASPECTS OF IMMUNOLOGY Antigens Antibodies Antigen-antibody reactions Complement system

43. ANTIGENS Antigen has been defined as any substance which when introduced parenterally into the body, stimulates the production of an antibody with which it reacts specifically and in an observable manner. Attributes of antigenicity: Immunogenicity – induction of an immune response Immunological reactivity – specific reaction with antibodies

44. Based on the ability of antigens to carry out these two functions, they are classified as : Complete antigen – able to induce antibody formation and produce a specific and observable reaction with the produced antibody. Haptens : substances incapable of inducing antibody formation on their own. Complex haptens – can precipitate with specific antibodies Simple haptens - nonprecipitating . Biological classes of antigens: T cell dependent antigens (TD) T cell independent antigens (TI)

45. Paratope The combinig area on the antibody molecule, corresponding to the epitope Epitope Antigen

46. Synthesized by plasma cells & to some extent by lymphocytes . Immunoglobulins are glycoproteins, each molecule consisting of two pairs of polypeptide chains, the light (L) & heavy (H) chains . ANTIBODIES

47. ABNORMAL IMMUNOGLOBULINS: Bence jones proteins in multiple myeloma. IgM in waldenstrom’s macroglobulinemia . Cryoglobulinemia A different disorder is found in ‘ heavy chain disease’, a lymphoid neoplasia characterized by over production of Fc parts of heavy chains.

48. ANTIGEN – ANTIBODY REACTIONS They serve several functions. In the body, they form the basis of humoral immunity. In the laboratory, they help in the diagnosis of infections, in epidemiological surveys, in the identification of infectious agents. The reaction occur in 3 stages 1. Primary reactions 2. Secondary reactions 3. Tertiary reactions

49. When an antigen is bound by an antibody, any of four actions may occur: Antigen may no longer function properly (inactivation by antibody ) Complement may be activated antigen and/or attached microorganism may clump (agglutination) or even precipitate out of solution Antibody-dependent cell-mediated cytotoxicity may occur .

50. GENERAL FEATURES: Reaction is specific. Entire molecule react & not fragments. No denaturation during the reaction. Combination occurs at the surface. Combination is firm but reversible. AFFINITY– intensity of attraction AVIDITY– strength of the bond

51. SEROLOGICAL REACTIONS: PRECIPITATION REACTION AGGLUTINATION REACTION

52. RADIO IMMUNOASSAY (RIA) ENZYME IMMUNOASSAY A technique for determining antibody levels by introducing an antigen labelled with a radioisotope and measuring the subsequent radioactivity of the antibody component.

53. IMMUNOCHROMATOGRAPHIC TESTS

54. THE COMPLEMENT SYSTEM coined by Ehrlich . Refers to a system of factors which occur in normal serum & are activated by Ag- Ab reaction & subsequently mediate a number of biologically significant consequences . Acts as mediator & amplifier of many immune responses & inflammation. The complement system belong to a group of biologic effector mechanisms called Triggered enzyme mechanisms . Only IgM & IgG1,2,3 fixes the complement . Complement as a whole is heat labile.

55. COMPONENTS: A complex 9 different fractions called C1- C9. C1 is present in serum as a calcium dependant ion which chelates with EDTA. COMPLEMENT ACTIVATION: C cascade is a series of reactions in which the preceding components act as enzymes on the succeeding components, cleaving them into dissimilar components. Larger fragment join the cascade, smaller fragments often possess the biologic effects which contribute to defense mechanisms by various mechanisms.

56. C cascade can be activated by 1. Classical pathway 2. Alternative pathway 3. lectin pathway

57. Ag- ab,DNA,trypsin like enzymes,retro viruses Properidin , factor D, Zymogen, bacterial endotoxins & IgA MBL binds to mannose

58. REGULATION OF C- ACTIVATION: Unchecked C-activity can cause not only exhaustion of the C-system but also serious damage to tissues. Two different type of regulators are A) Inhibitors B) Inactivators Various inhibitors include inhibitor of C1a esterase. This does not prevent normal C-cascade but checks it’s autocatalytic prolongation . The S protein binds to C567a & modulates the cytolytic action of the MAC.

59. Various inactivators are 1. Factor I 2. Factor H 3. Anaphylatoxin inactivator 4. C4 binding protein

60. MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) MHC is a locus in the short arm of Chromosome 6 that encodes for MHC classI , II & III molecules which are involved with antigen uptake and presentation. Class I – present in all cells in association with self derived antigens (intracellular) presented to CD8 & NK cells Class II – Antigens from Extracellular sources are presented by APCS (peripheral DCs, monocyte derivitives & B cells) in association with Class II molecules – HLA- DR, DP, DQ Class III – complement factors B, C2and C4

61. Genes Of MHC Organized In 3 Classes Class I MHC genes Glycoproteins expressed on all nucleated cells Major function to present processed Ags to T C Class II MHC genes Glycoproteins expressed on B-cells, dendritic cells. Major function to present processed Ags to T H Class III MHC genes Products that include secreted proteins that have immune functions. Ex. Complement system, inflammatory molecules.

62. DEFENSINS Endogenous , small, cysteine-rich antimicrobial peptides that are produced by leukocytes and epithelial cells . M ultifunctional and, by interacting with host cell receptor(s), participate in both immune responses. C ontain cysteine residues that form characteristic disulfide bridges. Human defensins are classified into two subgroups: Six alpha- defensins (hND-1 to hND-6 )  PMNs Four beta- defensins (hBD-1 to hBD-4 )  Mucosal epitheial cells

63. CYTOKINES Low molecular wt. proteins secreted by many cells (previously known as Lymphokines ) Assist in the regulation and development of Immune effector cells Cell to Cell communication Direct effector function Autocrine , Paracrine or endocrine Pleiotrophic

64. CELLULAR RECEPTORS FOR MICROBES AND DAMAGED CELLS Receptors used to detect microbes and damaged cells are expressed on phagocytes, dendritic cells and many other cell types. Based on their location, they are of 3 types:

65. These receptors for PAMP and DAMP belong to several protein families. Toll-like receptors NOD-like receptors and the inflammasome Others : the RIG-like receptor Cytosloic DNA sensors lectin receptors

66. Signaling functions of TLRs

67. The Inflammasome

68. IMMUNODEFICIENCY DISEASES Conditions where the defense mechanisms, of the body are impaired, leading to repeated microbial infections of varying severity and sometimes enhanced susceptibility to malignancies . Deficiencies of defence mechanisms may involve: S pecific immune functions - humoral immunity, cell mediated immunity or both N onspecific mechanisms - such as phagocytosis and complement. Immunodeficiencies may be classified as: P rimary or S econdary. Primary immunodeficiencies : from abnormalities in the development of the immune mechanisms. Secondary immunodeficiencies : are consequences of disease, drugs, nutritional inadequacies and other processes that interfere with the proper functioning of the mature Immune system.

69. Classification of primary Immunodeficiency syndromes:

70. AUTOIMMUNITY Condition in which structural or functional damage is produced by the action of immunologically competent cells or antibodies against the normal components of the body. Pathogenesis : Autoantibody formation may be a result of tissue injury They may cause damage by the cytotoxic (type II) or by toxic complex (type III) reactions Sensitized T-lymphocytes may also result in autoimmune diseases through type IV reactions. Humoral and cellular responses may act synergistically in the production of some autoimmune diseases.

71. Classification of autoimmune diseases:

72. HYPERSENSITIVITY The term hypersensitivity refers to the injurious consequences in the sensitized host, following contact with specific antigens. Traditionally they are classified into: Immediate hypersensitivity: Anaphylaxis Atopy Antibody mediated cell damage Serum sickness Delayed hypersensitivity: Infection (tuberculin) type Contact dermatitis

73. Coombs and Gell (1963) classification: Type I reactions ( IgE dependent): cytotropic IgE antibodies fixed on the surface of tissue cells (mast and basophils) in sensitized individuals binds to Ag. Occurs in two forms: Anaphylaxis – acute, fatal and systemic Atopy – chronic, recurrent, nonfatal, localized form Tissues involved - target tissues or shock organs.

74. Type II reaction: cytolytic and cytotoxic: A ntibodies bind with the antigenic determinants on the surface of cells leading to cytotoxic or lytic effects Ex: Autoimmune anemias and hemolytic disease of new born Long acting throid stimulators (LATS) in graves disease. Myasthenia gravis

75. Type III reactions: Immune complex diseases: Arthus reaction: Local manifestation of generalized hypersensitivity. Seen as an intense local reaction with edema,induration and hemmorhagic necrosis. Serum sickness: Systemic form of type III hypersensitivity. The clinical syndrome consists of fever, lymphadenopathy, splenomegaly, arthritis, glomerulonephritis, endocarditis, vasculitis,urticarial rashes, abdominal pain, nausea and vomiting.

76. Type IV reactions: Delayed hypersensitivity: Induced by sensitized t cells ( Tdth , Th1,Th2,Tc), which on contact with specific antigens causes biological effects on leucocytes, macrophages and tissue cells Tuberculin type (infection): S mall dose of tuberculin when injected intradermally in an sensitized individual, an indurated inflammatory reaction develops at the site within 48-72 hours. Contact dermatitis type: DH develops from skin (esp. inflamed area)contact with chemicals. Contact with the allergen in sensitized individuals. Varies from macules, papules to vesicles.

77. DRUG ALLERGY TESTS

78. PERIODONTAL DISEASE IMMUNOLOGY

79. First line of defense. Includes: Saliva GCF Epithelial barrier Commensals Orchestrated by a broad range of cytokines, chemokines , cell surface receptors. Innate immunity in periodontal diseases:

80. The PRR’s The primary response to pathogens in the innate immune system is triggered by Pattern Recognition Receptors (PRRs) that bind Pathogen-Associated Molecular Patterns (PAMPs ), found in a broad type of organisms. These receptor types include: toll-like receptors (TLR), nucleotide-binding oligomerization domain (NOD) proteins, cluster of differentiation 14 (CD14), complement receptor-3, lectins and scavenger receptors.

81. TOLL LIKE RECEPTORS 10 TLRs are identified so far. Among them TLR-2 and TLR-4 are most defined members. TLR 2 responds to lipoteichoic acid and microbial lipoproteins TLR 4 responds to LPS TLR 5 responds to Flagellin TLR 9 responds to bacterial DNA Porphyromonas gingivalis LPS stimulates TLR-2 , and not TLR-4 Only Aggregatibacter actinomycetemcomitans and Veillonella parvula were capable of stimulating both TLR-2 and TLR-4 . Chronic stimulation of TLRs results in over-production of pro-inflammatory cytokines and may lead to tissue destruction.

82. Adaptive immunity in periodontal disease: The importance of adaptive immunity in periodontal diseases is endorsed by the histologic studies of established lesions, which revealed: B cells and plasma cells in progressive lesions. T cells especially T-helper subset (CD4) in stable lesions. Antigen presenting cells – Generate Ag specific response T cells – alterations in th1 and th2 balance leads to tissue destruction B cells – control extracellular antigens. Antibodies – mainly IgG , but also IgM and A. Complement system - Provide barrier against spread of bacterial infections.

83. CYTOKINES & PERIODONTAL DISEASE

84. The MMPs Family of proteolytic enzymes that degrade extracellular matrix molecules. Produced by variety of cells like PMN, macrophages, fibroblast, epithelial cells., Key inhibitors of mmps : glycoprotein, antitrypsin, macroglobulin.

85. PERIODONTAL DISEASE OSTEOIMMUNOLOGY tnfa , il1a, 1b, 6, 11,15, 17 stimulate bone resorption Il 4,5,10,13,18,tgfb inhibit bone resorption . Th2 cells: inhibit ocl by il 4,10 Th17 cells: promote ocl by il 23,17 and RANKL.

86. IMMUNOLOGIC CONSIDERATIONS OF: Gingivitis: Initial lesion - PMNs Early lesion – T Lymphocyte Established lesion - Plasma cells & B lymphocytes Advanced lesion - Plasma cells Chronic periodontitis: Involves mainly alternative pathway of complement P gingivalis produces an enzyme that can cleave C5 MMP-8 is elevated & TIMP-1 are not Chymotrypsin released by T denticola activates MMPs Most of the collagenase activity of C.P is due to the MMP-8 released by PMNs. Aggressive periodontitis: 75% of LAP patients have dysfunctional neutrophils. Elevated levels of MMP-1 & TIMP-1 are present. Demonstrate elevated IgG2 antibodies against Aa . GAP is often characterized by defects in either PMNs/monocytes.

87. Immunological differences between chronic and aggressive periodontitis C linically the gingival lesion is often absent in aggressive periodontitis suggesting that it may not follow the same sequence of initiation and progression as chronic periodontitis (from gingival T-cell lesion to progressive B-cell lesion ). Ford et al.  Examined the possibility of subtle immunological differences. There are differences between these two forms of periodontitis with respect to the synthesis of b- defensins by host cells. In components of the adaptive immune system

88. Differences in neutrophil function: Patients with localized aggressive periodontitis had an inherited trait characterized, by lower than normal chemotaxis phagocytosis and intracellular microbial killing responses. Ryder  believed that some of the differential functions of neutrophils in aggressive forms of periodontitis may be due to a combination of inherited and acquired characteristics . At present it is not possible to identify real differences in the immunopathology of the two diseases. This may be because there are no differences , or because the differences only reflect variations in the degree of severity or susceptibility rather than actual different immunopathologies .

89. Periodontal vaccine The complexity of periodontopathic bacteria might be a problem in developing a periodontal vaccine. The vaccine development program involves identifying the bacterial peptides and proteins that trigger the immune response, and using these as the basis of vaccines. The vaccines are being trialed in mouse models of periodontal disease and following a positive response , a vaccine will progress to clinical trials . Administering the vaccine, in early adolescent age before there is any sign of periodontitis, helps in prolonging the healthy life of the periodontium and risk of any inflammation is reduced.

90. Host Modulation Modulation of Arachidonic Acid metabolites (NSAIDS ) Modulation of MMPs – SDD ( Doxycline - 20 mg twice daily ) Modulation of bone remodeling ( Bisphosphanates ) Inhibit Inflammatory cytokines (IL1 & TNF α ) through recombinant human IL11 (rhIL-11 ) Inhibition of Nitric Oxides by MEG ( Mercaptoalkylguanidines )

91. CONCLUSION Immunity is a war between pathogens and host defense system which ends in the “ SURVIVAL OF FITTEST ”. The immune responses team in unique ways, depending upon host, microbial and environmental controls, to maintain or re-establish homeostasis . However , these inherent good guys if went rogue would result in collateral damage of the periodontium . Immune system, thus could be a DOUBLE EDGED SWORD

92. REFERENCES Text book of microbiology-R . AnanthanarayAan , 6 th edition. Carranza’s Clinical periodontology-11 th edition. Clinical periodontology and implant dentistry-Jan Lindhe . Mim’s medical microbiology-4 th edition . Basic Immunology - Functions and disorders of the immune system 5e - Abbas ( 2016) Textbook of periodontics – jaiganesh r, 1 st edition.

93. Ishikawa I, Nakashima K, Koseki T, Nagasawa T, Watanabe H, Arakawa S, et al. Induction of the immune response to periodontopathic bacteria and its role in the pathogenesis of periodontitis.   Periodontol 2000. 1997;14:79–111 . Ebersole , J. L. et al. Periodontal disease immunology: ‘double indemnity’ in protecting the host . Periodontol . 2000. 2013; 62 , 163–202. SILVA , Nora et al . Host response mechanisms in periodontal diseases.  J. Appl. Oral Sci.,  Bauru ,  v. 23, n. 3, p. 329-355,  June  2015 . Ford, P. J., Gamonal , J. and Seymour, G. J. (2010), Immunological differences and similarities between chronic periodontitis and aggressive periodontitis. Periodontology 2000, 53: 111–123.