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Toxicology Course Vienna MODULE 12 Immunotoxicology, Allergy July 2, 2008 Immunity of the Mucosa Innate immunity at mucosa Prof. Erika Jensen-Jarolim, MD Dept. of Pathophysiology Medical University Vienna Gastrointestinaltrakt: Mukosale Pathophysiologie und Immunologie Jensen-Jarolim, Schöll, Szalai. Springer 2006 Wörterbuch Allergologie und Immunologie, Ferencik, Jensen-Jarolim et al, Springer 2004 Immunologie (Charles A. Janeway and P. Travers) Absorption of Nutrients - Barrier for Pathogens Mucus production: Goblet cells • Basic secretion • Lumen: Secretory fluid defense and Flora • Sessile: static defense IEL Squamous epithelia • Mobile and recruitable LC • Irritant - stimulated secretion: exocytosis of granula • Asthma: IL-4 and IL-13 bind to IL-4 Rα chain, but only IL-13 induces Goblet cell hyperplasia, fibrosis (Kondo et al. American J. of Respiratory Cell and Molecular Biology 2002) Epithelium: Langerhans cell (LC), intraepithelial Lymphocyte (IEL) Connective Tissue: DD MC plasma cell Cylindric epithelia Mast cells (MC), Dermal Dendrocyte (DD) • PMNs, Monocytes, Lymphocytes Fluid phase: Mucous Mucin glycoproteins (MGs): 30 – 90% carbohydrate with serin- or threonine-galactosaminyl glycopeptide linkage Fluid phase: Mucous Mucin glycoproteins (MGs): 30 – 90% carbohydrate with serin- or threonine-galactosaminyl glycopeptide linkage • Lubricating fluid • Lubricating biofilm • Protection from physical and chemical harm • Protection from physical and chemical harm Mucin-2: • Prevents colonization and viral infection (HIV, HSV) by fluid phase carbohydrates 1 – 2 μm layer Mucin-1 (MG1): bigger 1 – 2 μm layer • Barrier against toxins, enzymes, acids, carcinogens • AB0 and Lewis Antigens • sIgA • Complexes with Amylase, Prolin-rich-Proteins (PRPs), Statherin, Histatins 1 Endo- und exocrine cells: participate in mucosal defense Fluid phase: Serous Nonspecific immune factors Paneth cells: Enterochromaffine cells: • Lysozyme • Lactoferrin „Yellow cells“: Belong to APUD PRP: Baxter et al Biochemistry 1997 • Peroxidase . • Histatins: Histidin-rich α-helical peptides, bacteriostatic activity, In saliva most important anti-fungal factor (Candida a.), decrease – HIV infections • PRPs: Proline-rich proteins (25 – 40%), bind plantpolyphenols, - supersaturate saliva with Ca-phosphate • Cystatins: from Monocytes/Macrophages, bind bacterial pili – anti-adhesive effect Specific factor: sIgA They produce Serotonin, Gastrin, Secretin, Somatostatin, VIP, Enteroglucagon, Cholecysteokinin. Lysozyme, Peptidase α- and β- DEFENSINs : antimicrobial polypeptides Contain IgG, IgA Lysozyme (Muramidase) • Cationic protein (e.g Parotis: 1-10mg/L) Defensins • Cleaves β-1,4-linkage between N-acatylglucosamine & N-acetylmuramic acid • In vertebrates, invertebrates, plants • Lyse bacterial cell wall NAM NAG From UCLA-DOE Inst. for Genomics and proteomics α-Defensins β-Defensins β- pleated sheet β- pleated sheet, different disulfides Murein Cationic antibiotic peptides Cationic antibiotic peptides Fungicidal and bactericidal activity against Gram positive bacteria Origin: neutrophils Polymerisation Origin: Paneth cells – mice cryptidins Lysozyme (Muramidase) • Binds NAG-NAM structures • Activity against Gram-positive bacteria • kills also organisms without peptidoglycan (Fungi) due to cationic nature Gram positive Bacillaceae Lactoferrin: from neutrophils and epithelial cells • Transferrin family • Iron-binding glycoprotein (e.g Parotis: 1-10mg/L) • 10-20 mg/L in saliva, 1g/L (!) in milk Micrococcaceae Mycobacteriaceae Peptococcaceae Gram negative Acetobacteriaceae Gram positive cell wall • eliminates Fe++, cofactor for bacterial growth • destabilization of bacterial outer membrane allows attack by microbicidal peptides Alcaligenaceae Bacteroidaceae Chromatiaceae Enterobacteriaceae Legionellaceae Neisseriaceae Nitrobacteriaceae Pseudomonadaceae Rhizobiaceae Rickettsiaceae Gram negative cell wall Spirochaetaceae Vibrionaceae 2 Facts on gut flora • • We are born sterile Factors for successful colonization in neonates Natural sequential colonization through ingestion – development of imm.system • E. c oli Bacteroides, anaerobic, 30% Bifidobacteria, Eubacteriae, anaerobic E. Coli, facultative anaerobic • Breast versus formula feeding • Breast milk: undigestable carbohydrates – prebiotics support bifidabacteria and lactobacilli (probiotics) • Adult human body 1 x 10^14 cells - 10% actually belong to us!! • Delivery: Maternal flora (colon, vagina) constitutes principal child´s flora • Colon: > 400 species density of 1 * 10^11 organism / mL • Th2 dominance – counterbalance by bacteria stimulating TGFβ and IL-10 • Compete pathogenic populations by competing for substrates, unfavorable pH and Colicins - antibacterial substances. • Accelerated IgA response and natural defense Epithelial cells modulate immune responses Slow bacterial infection: Helicobacter pylori bacteria activated enterozyt / keratinocyte: 1.) H. pylori: damage - danger – Ion secretion, permeability – Antigen presentation (MHC I and II) 2.) Insufficient response – akt. Suppressor T-Zellen (oral Tolerance) 3.) PMNs: NO, Radicals – poly-IgR and secretory component – ICAM Expression and No elimination, Langerhans migration IFNγ, TNFα – produces IL1, IL-5, IL-6, IL-8, TNF-α Defense damages mucosa Gastritis chronical, with limited activity Mucosal-associated Lymphoid Tissue – MALT Gut: - GALT, Bronchi: - BALT, Vascular - VALT Inducer site Antigen from Lumen Villi intest. Adaptive immunity at mucosa 67 % of antibody prod. M-cells Goblet 0-niveau MALT 85 % of lymphoid tissue Lamina propria Glandulae intestinales (Lieberkühn crypts) Peyers patch Musc. mucosae 3 Mucosal Defense at the effector sites Epithelia decide where to handle which antigen small things Small things Effector Big things Endocytosis Phagosomes Lysosomes Antibodies, T-lymphocytes Villi intest. B Goblet M T Epithelial cells: MHC II Presentation, but no costimulation B-cells,macrophages: HLABII presentation and costimulation T TOLERANCE via Anergy IMMUNITY or TOLERANCE 0-niveau Lamina propria Peyers patch Glandulae intestinales (Lieberkühn crypts) Musc. mucosae Intraepithelial Lymphocytes: IEL CD8+ • Thymus-independent and dependent T-cells Pathways for IgA • 80% CD8+ • 50% bear TCR αα or αβ (TCR-2), 50% γδ TCR (TCR-1) • γδ-cells differentiate in small intestines TCR-1 γδ TCR−2 Vγ Vδ αβ αα Vα Vβ Vα Vα Inducer site •Peyers patch Cγ Cδ Cα Cβ Cα Cα Mesenteric lymph node: B-cell maturation and proliferation 60% B-cells 40% T-cells • The αα−TCR-2 scans non-classical MHC I on epithelial cells upon activation – secretory (Leishman et al. Science 2002) 0,4% myeloid cells • Genito-urinary tract • Gut • Salivary and lacrimal glands • Breasts • Respiratory tract • TCR-1: true IEL, dendritic epidermal T-cell (DETC), IL-6 prod., participation in wound repair (Jameson et al. Science 2002) • Participation in attack of viral infections and tolerance ? IgA: secretory immunoglobulin Effector sites • IgA: most prominent isotype in mucosa • Most produced Ig-class, 2/3 is sIgA epithelial cell • Main source bone marrow poly-IgR Plasma cell IgA-dimer with J-chain Production (mg/day) IgA IgG Circulation 2100 2100 Saliva 200 2 Tears 5 ? Bile 400 160 Small intestines 5200 600 Large intestines 1200 140 Nasopharynx 45 15 • Antigen transport Urine 3 3 • Endogeneous reinfection, e.g. EBV Total 9200 3000 IgA-Dimer + secretory component Phosphorylation Endo- and Transcytosis • IgA secretion - neutralization with 5 Ig-like domains Mestecky 1986; Clin Immunol Immunopathol. 4 Fluid phase: Specific Defense Secretory IgA: Dimer - (2α2L)2,J Serum IgA: Monomeric – (2α2L) Bact. IgAspecific proteases IgA1 Bact. IgAspecific proteases Disulfide bridges Disulfide bridges J-chain: plasma cells IgA1 J-chain: plasma cells sc: polyIgR Streptococcus sanguis, S.pneumoniae, Hämophilus influenzae, Neisseria gon,, N. mening. sc: polyIgR IgA1: common in serum mIgA Extended, highly glycosylated hinge sIgA Isotypic and allotypic variation of IgA IgA Deficiency – defect of immune elimination IgA2: 50% of IgA in mucosa Allotypes: A2m(1) and A2m(2) (98% in caucasians) Bact. IgAspecific proteases IgA1 1.) B-cell defects: Bruton – total Ig 2.) selective IgA-deficiency: 1: 800 Functional Adaption: IgA2 lacks hinge region (aa 217-241 deleted) (2α)2,J 2L • Shortened, inflexible hinge, • Soluble 2L IgA2 Mucosal immunity in neonates Colostrom: 2 days Milk: ~ 5 months 10 – 20 mg/ml sIgA 0,5 mg/ml sIgA (IgG: 0,3 – 0,5 mg/ml in milk) Passive oral immunotherapy chronic diarrhoea immune complex diseases more food allergies recurrent infections, in respiratory tract and gut: salmonella, campylobacter, giardia associated with celiac disease, Crohn´s disease, Colitis ulcerosa Vaccination 2 Compartments: peripheral and mucosal immunity • Antigen handling by lymphnodes – peripheral immunity Protective IgM, IgG response and cell mediated Immunity Th1 IL-12 IL-2, IFNγ IgG • by respiratory or gastrointestinal tract - mucosal immunity Protective IgA antibodies and IEL, but usually tolerance ! Th2 – Th3 TGFβ IL-4, IL-5, IL-6, IL-10 IgA 5 Oral vaccination Aim: systemic & mucosal immunity Toxicology Course Vienna • Adjuvants: Particles – big things • Mucosal targeting – functionalization MODULE 12 Immunotoxicology, Allergy July 2, 2008 CTB www.ualberta.ca LT www.csb.yale.edu Immunity of the Mucosa Prof. Erika Jensen-Jarolim, MD • Vehicles Conjugation of antigen to Cholera Toxin (immunog. any route) Heat-Labile Enterotoxin of E. coli. (LT) Expression of antigen in/by Bacterial vectors, Plants - Edible vaccines Dept. of Pathophysiology Medical University Vienna Gastrointestinaltrakt: Mukosale Pathophysiologie und Immunologie Jensen-Jarolim, Schöll, Szalai. Springer 2006 Wörterbuch Allergologie und Immunologie, Ferencik, Jensen-Jarolim et al, Springer 2004 Immunologie (Charles A. Janeway and P. Travers) 6