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Innate Immunity - Methods

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					    Innate Immunity


               Debra Laskin
           Professor and Chair
Department of Pharmacology and Toxicology
    Ernest Mario School of Pharmacy

        laskin@eohsi.rutgers.edu
       What is the Innate Immune
              Response?
   Universal and evolutionarily conserved mechanism of
    host defense against infection; first line of defense;
   Predates adaptive immune response
     – Found in all multicellular organisms
         (adaptive only in vertebrates)
     – Uses receptors and effectors that are ancient in their
       lineage
     – Provides protection against a wide variety of
       pathogens
   Distinguishes self from non-self perfectly
   Defects in innate immunity are very rare and almost
    always lethal
Elie Metchnikoff: “Father of Macrophages”
1908 Nobel prize
Watched the reaction to a splinter inserted into a starfish.
Hemocytes (amoeba-like cells) arrived and tried to ingest the
foreign body; if they could not, they walled it off. Mammals do
exactly the same thing to foreign bodies.




Phagocytosis- to devour
    Oyster hemocyte                Mouse macrophage


Similarity in appearance invertebrate and mammalian
phagocyte striking; also use many of the same cytotoxic
mechanisms (e.g., production ROS)
   Innate Immunity: Functions


 Provides   a barrier to prevent the spread of
 infection
  – Physical
     » Skin (epithelial cells); Wounds, burns, insect bites
     » Mucosal surfaces (respiratory, GI, Reproductive)
  – Mechanical (tight junctions, movement)
  – Chemical (fatty acids, enzymes, pH,
     antimicrobial peptides)
  – Microbiological (normal flora)
      Innate Immunity: Functions


 Identifies   and eliminates pathogens
   – Non-adaptive recognition systems
   – Activates molecules that target the microbe
     and aid in it’s identification
      » These factors may be surface expressed (TLR),
        released from immune cells (antibodies) or present
        within circulatory system (complement)
   Innate Immunity: Functions

 Initiates   an inflammatory response
   – Reaction to injury or infection
      » Trauma to tissues or cells
      » Presence of foreign material (splinter)
      » Infectious agents (viruses, bacteria, fungi)
   – Delivers immune cells and effector molecules to
     the site of injury/infection
   – Components
      » Granulocytes, MP, inflammatory mediators
      » Blood vessels (endothelium)
      » Plasma proteins
 Innate Immunity: Functions


 Provides signals to alert the adaptive
 immune system to activate an effective
 specific immune response
  – Antigen processing and presentation
    (activation of T helper cells)
  – Upregulation of co-stimulatory molecules
     » MHC class II, CD80/86
  – Induction of cytokine/chemokine response
     » IL-4, IL-12
      Innate Immunity:
    Cellular Components

 Granulocytes
  – Polymorphonuclear leukocytes
      (PMN, neutrophils)
  – Eosinophils
  – Basophils (blood)
  – Mast Cells (tissues)
 Mononuclear      Phagocytes (RES)
  – Monocytes (blood)
  – Macrophages (tissue)
      Origin and Development of
    Macrophages and Granulocytes
Bone Marrow                  Blood         Tissue
  monoblast    promonocyte   monocyte

                                           macrophage

              basophilic       basophil
Phagocytic    promyelocyte
precursors


              eosinophilic                    mast
                              eosinophil
              promyelocyte                    cell
 myeloblast
              neutrophilic    Neutrophil
              promyelocyte    (PMN)
Neutrophils
(PMN)


  Present   in blood (60-70% of WBC)
  Not normally present in tissues
  Short lifespan - 12 hours
  Functions:
   – First cell to respond to infection or injury
     (inflammatory site)
   – Ingest and kill pathogens
   – Release cytotoxic/proinflammatory mediators
     (ROS, proteolytic enzymes, bioactive lipids,
     chemokines)
Mononuclear
Phagocytes

 Blood - monocytes (1-6% WBC)
 Tissues - macrophages
  – mature form of monocytes
  – found in tissues (ex., gastrointestinal tract, lung, liver,
    brain, skin, spleen); reticuloendothelial system (RES)
 Functions:
  – Inflammation- respond to injury, infection, other foreign
    substances
  – Phagocytize and kill pathogens
  – Wound repair, angiogenesis
  – Antigen presentation (activate adaptive immunity)
  – Tumor surveillance and cytotoxicity
Inflammatory Responses
Injury or
First Step: Activation of Vascular
Endothelial Cells by Macrophages

Endothelium
   – Form blood vessels
   – Macrophages present release TNFa and IL-1: upregulated
     expression of adhesion molecules on endothelium

        » P-selectin
        » E-selectin
                          Immediate release        Initiate PMN
        » ICAM-1                                   rolling/adhesion
                               Released 2 hours

   –   Initiate PMN rolling, adherence (LFA-1)
   –   Release chemokines (e.g., IL-8) induce PMN extravagation
   –   PMN first to respond (within hours)
   –   Monocyte second wave (24-48 hr)
            Leukocyte Adhesion, Diapedesis
                   and Emigration




selectins
  Localization and Destruction of
Pathogens and Foreign Substances
Chemotaxis: migration to injured or infected site;
 mediated by chemotactic factors; complement, fMLP,
 C-X-C (PMN, IL-8) and C-C (mono, MCP-1) chemokines
Phagocytosis: ingestion of foreign substances;
 receptor mediated, active process, requires energy
Localization and Removal of
   Foreign Substances

Metabolic Destruction
 intracellular digestion, killing

  » Oxygen independent: defensins and granular
    cationic proteins, lactoferrin, lysozyme, acid
    hydrolases

  » Oxygen dependent: myeloperoxidase,
    hydrogen peroxide, superoxide anion, hydroxyl
    radicals, nitric oxide, peroxynitrite
Inflammation is More than a Local
       Tissue Responses
Secretory Functions of Macrophages

    Binding proteins (transferrin, fibronectin)
    Complement components
    Proteolytic enzymes (lysozyme)
    Enzyme inhibitors (a2-macroglobulin)
    Endogenous pyrogen (IL-1)
    ROS (superoxide, hydrogen peroxide, hydroxyl radical)
    RNS (nitric oxide, peroxynitrite)
    Bioactive lipids (PAF, PG, LT, TBX)
    Chemokines (C-C and C-X-C)
    Growth factors (FGF, EGF, CSF)
    Proinflammatory cytokines (IL-1, TNFa, IL-6)
    Angiogenic factors: VEGF
    Matrix remodeling proteins: TGFb, MMP
   Stopping Inflammation:
Wound Repair and Angiogenesis

   Inflammatory macrophages release
    mediators that
    – Down regulate inflammation (eg., IL-10)
    – Inhibit inflammatory cell recruitment
    – Block specific immune responses
    – Initiate wound repair; matrix remodeling
      (MMPs, TGFb)
    – Recruit fibroblasts (FGF)
    – Induce angiogenesis (VEGF)
What Happens When Inflammation
       Fails to Resolve?

    Frustrated Phagocytosis
      Foreign Body Response
       – Macrophages wall off injurious agent
    Chronic inflammation
    Graunulomas
    Tissue Injury
    Cancer
Foreign Body Reactions




                                                Silicone droplets
    Suture material
                                         Clear globules of silicone released breast implant
Suture material is not digestible by     cannot be ingested; MP accumulate to wall off the
macrophages, so it has been walled off   material; note MP that has become a multi-
by fibroblasts.                          nucleated giant cell showing the “asteroid bodies”
                                         characteristic of the foreign body reaction.
Macrophage Mediators Can Damage Host
              Tissues


  Bioactive                  ROI
   Lipids
                Activated
               Macrophage


  TNF-a                      Proteolytic
  IL-1                       Enzymes
  chemokines
                 RNI
Reactive Oxygen Species


              H2O2
              O2-
              OH-

    Lipid Peroxidation
 Membrane, Protein and DNA
         Damage
Reactive Nitrogen Intermediates

 Nitric oxide and peroxynitrite
 Nitric oxide- formed from l-arginine by the
  enzyme nitric oxide synthase (NOS)
 Macrophages: (NOSII) induced by
  inflammatory cytokines (IFNg, TNFa) and
  bacterially-derived products (LPS)
 Highly labile; oxidizes nucleic acids,
  membranes, proteins
 Nitric oxide reacts with superoxide anion
  forming peroxynitrite
Proinflammatory Cytokines


 Tumor   necrosis factor-a
 Interleukin-1
 Interleukin-6
 Interleukin-18
 Chemokines
 Interferon-g
Tumor Necrosis Factor-a


Proinflammatory
Primes phagocytes to produce
 ROI and RNI
Cytotoxic
Induces apoptosis and necrosis
     Macrophage Processing of
            Antigens

 Macrophages    function as accessory cells or
  antigen processing cells (APC)
 Macrophage associated antigen is 1000x more
  immunogenic
 Processing of antigens involves change so that
  it binds MHC II (Ia) proteins; may involve
  unfolding, partial degradation, selection for
  epitope with high affinity for MHC II
 Required for T-helper cell recognition of
  antigens
 Other APC: B cells, epithelial cells, dendritic
  cells
How is it Possible for Macrophages
to Perform all of these Functions?
Macrophages functionally polarized into subpopulations
  by inflammatory signals in microenvironment

  M1 macrophages
    Activators: LPS, IFNg, TNFa, TLR ligands
    Cytotoxic (pathogens, tumor cells)/proinflammatory activity
    Release ROS, RNS, IL-12, TNFa, M1 chemokines
    Promote Th1 responses
  M2 macrophages (alternatively activated)
    Activators: IL-4, IL-13, IL-10, immune complexes
    Antiinflammatory/wound repair activity
    Release IL-10, TGFb, PDGF, VEGF, MMP, EGF, FGF
    Immunosuppressive
    Promote Th2 responses
           M2 Macrophages

 M2a-activated   by IL-4, IL13

 M2b-activated by immune
 complexes and TLR agonists or IL-1

 M2c (tumor associated macrophages)-
 activated by IL-10, TGFb, glucocorticoids
  **active in wound repair, angiogenesis, chronic
    inflammation
Tumor Associated Macrophages

  Found  within tumor microenvironment
  M2 phenotype
  Respond to cytokines (CSF-1) and
   chemokines (MCP-1) released by tumor cells
  Hijacked by tumor cells to release mediators
   that contribute to tumor promotion,
   progression, angiogenesis and metastasis
  EGF, VEGF, MMP, IL-1, IL-6, chemokines
How do Macrophages Recognize
        Pathogens?
Phagocytosis of Bacteria by
      Macrophages
How do Macrophages Identify Microbes?

  Pattern Recognition Receptors (PPR )
        – Recognize pathogen associated molecular
          patterns (PAMP); conserved molecular
          patterns on microbes
        – Identify a class of microbes; ex., LPS, LTA,
          peptidoglycan, lipoarabinomannan, dsRNA, mannose,
          b-glycans
        – PAMP are often essential for microbe survival
     Action Time
        – Immediate activation of effectors
        – Delays need for adaptive immunity
Pattern Recognition Receptors (PRR)
Three broad classes based on expression profile,
  localization, function
 PRR    that signal an infection
  Toll Receptor Family
   – Expressed externally or internally
   – Binding activates “pro-inflammatory” signaling pathways
 Phagocytic (endocytic) PRR
  – Expressed on the surface of phagocytic cells
  – Mediate uptake of microbe into phagocytes
 Secreted PRR
  – Secreted by MP, epithelial cells, hepatocytes
  – Activate complement, opsonins, function as accessory
    proteins for PAMP recognition
Toll-like Receptor (TLR) Family


  First discovered in Drosophila
  Thirteen receptors identified in mice and
    humans

Recognized motifs (PAMP)
  -lipopolysaccharide (LPS) from Gram-negative
        cell walls
  -peptidoglycans from the cell walls of both
        Gram-negative and Gram-positive bacteria
  -viral double-stranded RNA
  -CpG-rich bacterial DNA
Examples of TLR and Ligands
     Receptor                        Ligand
  (Pattern Recognition        (Pathogen-Associated
       Receptors)              Molecular Patterns)
         TLR1            Heterodimerizes with TLR2
         TLR2            PGN, some LPS, some LTA,
                         lipoproteins, AraLAM
         TLR3            dsRNA
         TLR4            Gram(-) LPS, Taxol, some LTA,
                         HSP60
         TLR5            Flagellin
         TLR6            Heterodimerizes with TLR2
         TLR7            Imidazoquinoline
         TLR9            Bacterial DNA (CpG)
Ligands are
      PAMP
 (pathogen-
 associated
  molecular
   patterns)

 Receptors
         are
       PRR
   (pattern-
recognition
 receptors)
Single ligand-single response vs. multiple
        ligands-complex response
Accessory Proteins for TLR-4

Binding requires several accessory molecules
  » LBP/MD-2 (Macrophages)
  » RP105 / MD-1 (B cells)



        MD-2            MD-2
                                       MD-2
Toll-like Receptor Signaling
                           Resulting in
                           the
                           activation of
                           gene
                           transcription

       MD-2




IRAK




        MAPK
            Intracellular PPR

 Protein   kinase receptor (PKR)
   – Activated upon binding to dsRNA (viruses)
      » Blocks viral and cellular protein synthesis (eIF2a)
      » Activates NF-kB, MAP kinase STAT & IRF signaling pathways
      » Induces apoptosis of infected cells and IFNa/b production
 2’-5’   Oligoadenylate Synthase and RNaseL
   – Family of IFN-inducible enzymes
      » Activated by dsRNA
      » RNaseL degrades viral and host RNA
      » Induces apoptosis
         Intracellular PRR


   Nucleotide-binding oligomerization domain
    (NOD) proteins


      -cytoplasmic surveillance proteins
      -bind peptidoglycans
       Surface Expressed PRR
  that Bind Bacterial Carbohydrates


 Mannose-binding      receptor (C-type lectin)
  – Recognizes patterns of mannose residues in a
    certain spatial orientation unique to microbes
  – Only found on macrophages (not monocytes
    or PMN)
 Glucan   Receptor
  – Present on all phagocytes
   Surface Expressed PRR that
 Bind other Bacterial Components

 Scavenger     Receptors
  – Recognize charged ligands
    » Polyanionic ligands (ds-RNA, LPS, LTA)
    » Acetylated low-density lipoproteins (LDL)
  – Found on all phagocytes (CD36; CD68)
  – MARCO (macrophage-specific, binds bacterial
    cell walls and LPS)
  – Phagocytosis of apoptotic cells
    » MFG-E8 (released from activated macrophages
      and binds to apoptotic cells via phosphatidylserine)
Secreted Pattern Recognition Molecules

Important in complement activation
Opsonization of microbial cells
Primarily produced by the liver but can be produced by
lung (SP) or phagocytes

                            Acute Phase Proteins
Secreted Pattern Recognition Molecules


 Collectins
    – Recognize microbial carbohydrate (CRD) domain
         Mannan-binding lectin
         Surfactant proteins (SP-A / SP-D) (lung)
 Pentraxin
     – Recognize phosphorylcholines on microbes;
   Lipid Transferases
    – Recognizes peptidoglycans
 Peptidoglycan recognition               proteins (PGRS)
  – LPS binding protein (LBP
               INNATE IMMUNITY


PHYSICAL           CELLS               CHEMICAL
BARRIERS           granulocytes,       BARRIERS
                   monocytes,          pH, lipids, enzymes
Skin, mucous
                   macrophages
membrane



               ADAPTIVE IMMUNITY

  HUMORAL                          CELL MEDIATED
    B cells           MP               T cells
    antibodies                      lymphokines
Cellular Interactions in the Immune System


   MP                           Activated
                                   MP              (+)

                    PA                                     Sensitized    Antigen
                               (+)             Th1
                                                               Th
                                      Th                                destruction
               MP
 AG                                                  (+)
                                       (-)
                                                   Tcyt
                                                                             Y
                         Th2
        (+)
              (-)              Treg          (-)
      B
               Sensitized
                                                                    plasma
                    B                      Bmem

				
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