Phagocytosis

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Phagocytosis

  1. 1. A four-year-old boy comes with the H/O of several "boils" on his arm. The informant being his mother tells us that the boy has had similar lesions on several previous occasions that were treated successfully with antibiotics. She denies any history of eczema or typical childhood illnesses such as measles or chicken pox. The child has had all of his immunizations.
  2. 2. Laboratory examination CBC: N Ig levels : N B & T cell counts : N Complement levels: N Serum Ca+2 & PTH levels : N The NBT test : - ve CBC= Complete blood count , N=normal , PTH=parathyroid hormone , NBT : nitroblue tetrazolium.
  3. 3. The child had a defect of the phagocytosis called the Chronic granulomatous disease
  4. 4. What is Phagocytosis? It is defined as ingestion of particles of >0.5µm by cells.
  5. 5. Who discovered phagocytosis? Ellie Ilya Metchnikoff discovered it in 1882. Received Nobel prize for the same in 1906 Carl Fredrich Claus coined the term Phagocytosis
  6. 6. WHAT IS THE MAIN PURPOSE OF PHAGOCYTOSIS ?
  7. 7. infection Innate immunity x disease no disea se in re- fe n tio c adaptive immunity c re o ry ve x An Extensive cross talk is seen between the adaptive and innate immunity no disease
  8. 8. What are the cells involved in phagocytosis ? Professional phagocytes 1. Neutrophils 2. Monocytes 3. Macrophages 4. Dendritic cells 5. Mast cells 6. Eosinophils
  9. 9. Cells Function Other features Neutrophils Phagocytose antigens coated with antibodies and complement, intracellular killing, inflammation and tissue damage Short life span , have primary and secondary granules in cytoplasm,and segmented nucleus, main station is blood,CD66 membrane marker. Monocytes Phagocytosis and acts as APC, Precursors of macrophages and dendritic cells Longer life span , cytoplasm has granules , main station is blood Macrophages Phagocytosis intra & extracellular killing, acts as APC, tissue repair ,stores iron in bone marrow , play a potent role in tumor cell destruction, Longer life span , no granules in cytoplasm, different phenotypes based on station eg Kuffer cells in liver, CD14 membrane marker Dendritic cells Major role in phagocytosis and AP in viral infections Typical outgrowths , main station is skin and lining epithelium of RT or GIT , Derived from monocytes or ?? Lymphocyte precursor cells Mast cells Allergic responses , Phagocytose and act as APC against gram negative bacteria like salmonella Derived from basophils, main station is connective tissues Eosinophils Immunity against parasites , weakly phagocytose and kill the parasite Contain eosinophilic granules in the cytoplasm , main station is the blood, levels raised in parasitic infestation and atopy
  10. 10. Non Professionals Phagocytosis is not their main function , do not have specific receptors. Lymphocytes NK and LGL cells (Large Granular lymphocytes) Epithelial cells Endothelial cells Fibroblasts Paoletti R., Notario A. and Ricevuti G., (editors), Phagocytes: Biology, Physiology, Pathology, and Pharmacotherapeutics, The New York
  11. 11. How does it take place ?
  12. 12. Entry of the pathogen Recognition & phagocytosis by tissue phagocytes SOS signals Recruitment of phagocytes & inflammation
  13. 13. How does a phagocyte recognize the bugs ? By using PRRs Toll like receptors FC receptors Surface Complement receptors Scavenger receptors NOD receptors RIG1 MDA5 Intracellular OAS
  14. 14. Toll like receptors
  15. 15. Fcreceptors FcαR and Fc γ recognize microbes that are coated with antibodies. Fc receptors Fc γ R I Location Monocytes , Macrophages & Inf γ stimulated Neutrophils IIA Phagocytes & NK cells IIB T & B lymphocytes IIIA Monocytes & Macrophages IIIB Neutrophils FcαR Neutrophils, Monocytes & Macrophages
  16. 16. Complement receptors Bind complement components deposited on the microbes Complement receptor Special feature CR1 Found on RBC`s & WBC`s except eosino & basophils. Cannot trigger phagocytosis unless the phagocyte is preactivated CR3 Member of Integrin family of receptors, expressed abundantly on macrophages and poorly on neutrophils , activation increases its levels. Also recognizes ICAM , Factor X and yeast polysaccharide cell wall CR4 Member of Integrin family of receptors, expressed poorly on neutrophils and macrophages , monocyte – macrophage incresases its levels , mediates phagocytosis of iC3b opsonised particles and appears to be important for nonopsonic , sugar ligand dependent phagocytosis
  17. 17. NOD protein structure (Nucleotide oligomerization domain) CARD NBD LRRs N-term C-term NOD-1 Binds γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) CARD CARD NBD N-term LRRs C-term Binds muramyl dipeptide (MDP) CARD (Caspase-activating and recruitment domain) NBD (Nucleotide binding Domain) LRRs (Leucine-Rich Repeats) NOD-2
  18. 18. Other IC Rceptors RIG 1(Retinoic acid inducible gene) and MDA 5 (Melanoma differentiaton associated gene) : RNA helicase with tandem caspase activation and recruitment domains (CARDs) that binds viral dsRNA and ultimately stimulates the genes induced by interferon regulator factor 3 (IRF-3) and nuclear factor B (NF-B).  OAS: 2,5-oligoadenylate synthetase : recognises non self dsRNA and breaks them down.
  19. 19. PRR Recognizes Pathogen associated molecular pattern Produce Transcription factors Induce Effector molecules
  20. 20. Underhill and Ozinsky. Annu. Rev. Immunol. 2002
  21. 21. Phagocyte response to infection The SOS Signals ◦ N-formyl methionine ◦ Clotting system peptides ◦ Complement products Phagocyte response ◦ Vascular adherence ◦ Diapedesis ◦ Chemotaxis ◦ Activation ◦ Phagocytosis and killing
  22. 22. Clinical correlation: Leukocyte adhesion defects
  23. 23. How does a pseudopodia form ? Clinical correlation : Wiskott Aldrich syndrome,neutrophil actin dysfunction
  24. 24. What is the mechanism of phagocytosis ? Two theories proposed to explain are Zipper theory Trigger theory
  25. 25. Zipper theory According to the zipper mechanism, ingestion occurs by sequential engagement of a phagocyte's membrane against the particle surface, and pseudopod advance proceeds no further than receptor-ligand interactions permit.
  26. 26. Trigger theory In contrast, in the trigger mechanism particle binding initiates an all-or-none phagocytic response
  27. 27. What is phagosome maturation? Clinical correlation: Microbial evasion of phagocytosis Cellular Microbiology, 1999, 1(3):195-203
  28. 28. How is the microbe killed ? Killing occurs by o2 dependent & independent mechanisms & by both intracellular and extracellular mechanisms
  29. 29. Clinical correlation: Degranulation defects
  30. 30. What happens in the phagolysosome ?
  31. 31. Active Resting O2 p22phox O2- Heme gp91phox FAD NADPH Rac Defective microbial killing PKC PI3K P PKA p40phox P p47phox P P P67phox MAPK
  32. 32. How does extracellular killing take place? LPS binds TLR4 IL-1 binds IL1-R1 TNFα binds TNF R1 and activates the iNOS
  33. 33. Citruline Arginine
  34. 34. Reactions Between ROS and RNS NO + O2- NO2 + ONOO- NONOates S-nitrothiols Nitrite Nitrous Acid
  35. 35. Mechanisms of Microbicidal Activity Polyunsaturated Lipids DNA Formation of Oxylipins disrupts membrane Enzyme Function of nucleosides Deamination Abasic sites Oxidization and Nitrosylation of SH groups Strand breaks of Tyrosine residues Nitrosylation Inactivation of metal ions at active site Depletion of antioxidants
  36. 36. “the act of physically escaping from something (an opponent or a pursuer or an unpleasant situation) by some adroit manoeuvre” Nature 3:11, 2002 editorial •Passive •Active
  37. 37. Avoiding contact with Phagocytes Inhibiting chemotaxis  Streptococcal streptolysin also kills them.  Clostridium θ toxin.  Mycobacterium tuberculosis. • Covering self with substance that is recognised as self T. pallidum binds fibronectin to its surface  GAS has a hyluronic acid capsule 
  38. 38. Inhibition of engulfment  Polysaccharide capsules of S. pneumoniae, Haemophilus influenzae, and Klebsiella pneumoniae  M protein and fimbriae of Group A streptococci  Surface slime (polysaccharide) produced as a biofilm by Pseudomonas aeruginosa  Vi antigen of Salmonella typhi  Cell-bound or soluble Protein A produced by Staphylococcus aureus. Protein A attaches to the Fc region of IgG and blocks the cytophilic (cell-binding) domain of the Ab.
  39. 39. Survival inside cells      •    •   Inhibition of fusion of the phagocytic lysosomes with the phagosome. M.Tuberculosis S. Typhi L. Pneumophila Leishmania Survival inside the phagolysosome Mycobacteria Br. Abortus B. Anthracis Escape from phagosomes Listeria Shigella
  40. 40. Products of bacteria that kill phagocytes Killing before phagocytosis Streptolysin O of GAS  Leukocidins of Staphylococci  Exotoxin A of P. Aeruginosa  • Killing after ingestion Mycobacteria  Brucella  Listeria 
  41. 41. What are the other functions of phagocytic system? Involved in apoptosis Silent clearance is achieved by expression of phosphatidyl serine on surface of apoptotic bodies Involved in immunological tolerance Cells carrying self antigens are killed by phagocytosis • In antigen processing and presentation
  42. 42. WHAT ARE THE CONDITIONS ASSOCIATED WITH DEFECTIVE PHAGOCYTIC SYSTEM? Phagocytosis: Dr.Vinaykumar . Hallur
  43. 43. Defects of degranulation Disease Etiology Clinical consequence Chediak Higashi Syndrome AR, impaired coalescence of lysosomal granules Neutropenia , recurrent pyogenic infections, oculocutaneous albinism Specific granule deficiency AR, mutation of C/EBP which regulates specific granule formation Recurrent deep abscesses
  44. 44. Defects of adhesion Disease Etiology Clinical consequences Leukocyte adhesion defect AR, absence of CD11/18 surface adhesive glycoprotein Neutrophilia, recurrent bacterial infection without pus Neutrophil actin dysfunction Altered polymerization of neutrophil actin Neutrophilia, recurrent bacterial infection without pus
  45. 45. Defects of cell motility Disorder Etiology Clinical consequences AR, Defective protein pyrin Recurrent fever and serositis, arthritis and amyloidisis Ethanol , steroids Frequent infections Immune complexes Bind Fc receptors on neutrophils in SLE ,RA etc Recurrent pyogenic infections Jobs Syndrome AD, variable expression of inhibitor from mononuclear cells , high levels of antistaphylococcal IgE Recurrent skin and sinopulmonary infections Increased Familial Mediterranean fever Decreased Direct inhibition
  46. 46. Defects of microbial killing Disorder Etiology Clinical consequence Chronic granulomatous disease AR/XLR defective NADPH oxidase G6PD deficiency <5% activity of G6PD Recurret pyogenic infections with catalase positive organisms eg S.Aureus , B. Cepacia ,S. Marcescens.↑Fungal infections Myeloperoxidase deficiency AR, failure to process precursor protein due to missense mutation None!!! RAC2 deficiency AR, mutant protein neutrophilia , recurrent bacterial infections
  47. 47. HOW TO EVALUATE A PATIENT OF DEFECTIVE PHAGOCYTOSIS ? Phagocytosis: Dr.Vinaykumar . Hallur
  48. 48.  2
  49. 49. NOD 2 and Crohn`s disease  Abnormal NOD-2 expression correlates with defective epithelial defense ◦ NOD-2 expressed in Paneth cells of intestine ◦ Enteric bacteria induce α-defensin through NOD-2 to kill luminal microbes. ◦ Clinical evidence: CD patients have decreased α-defensin expression in Paneth cells
  50. 50. What happens when the phagocytic system goes haywire ? ALI Septic Shock Renal failure Emphysema
  51. 51. Toll like receptors a boon or curse ? Recent studies have shown mice deficient in certain TLRs to be resistant to some infections.
  52. 52. Can we exploit the phagocytic sytem for our benefits ?  TLR4 agonist Imiquimod has been approved for the treatment of viral warts.  A TLR4 receptor agonist has been used recently as an adjuvant in drugs.  TLR7 receptor agonists have been used to treat Basal cell ca, melanoma and other cancers.  Drugs enhancing autophagy : Currently, a phase I/II trial of hydroxychloroquine with radiation therapy and concomitant and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme is being conducted through the New Approaches to Brain Tumor Therapy consortium.
  53. 53. Summary  Phagocytosis is a component of innate and aquired immunity. It is the principal means of destroying pathogenic bacteria and fungi. Phagocytosis initiates the process of antigen presentation.  Many phagocytic receptors recognize a diverse array of microbial pathogens. Some pathogens (e.g., S. pneumoniae) require opsonization by antibodies and complement for their clearance. However, bugs fight back.
  54. 54. Summary contd..    Phagocytic leukocytes employ oxidative and nonoxidative means of killing. The NADPH oxidase generates reactive oxidants, such as superoxide anion and hypochlorous acid (bleach). Innate immunity ushers in acquired immunity: innate immune activation of APCs results in up-regulation of co-stimulatory molecules and enhances the effectiveness of antigen presentation. Phagocytosis is an essential component of development and tissue remodelling. Ingestion of apoptotic bodies is immunologically “silent” and is normally accompanied by a suppression of inflammation. Failure of this mechanism may result in autoimmunity.
  55. 55. Science is like looking through a keyhole: The closer you get to the keyhole, the more you see of the room on the other side. -George Wald 1967 Nobel Laureate in Medicine
  56. 56. Thank you Phagocytosis: Dr.Vinaykumar . Hallur

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