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MICR 304 Immunology &
Serology
MICR 304 Immunology &
Serology
Lecture 13Apoptosis; Failures of the Immune
System; SuperantigensChapter 5.15, 6.25, 6.26, 8.26-8.30,
Lecture 13Apoptosis; Failures of the Immune
System; SuperantigensChapter 5.15, 6.25, 6.26, 8.26-8.30,
Overview of Today’s Lecture
• Apoptosis– Definition– Triggers– Intracellular events– Assays to detect apoptosis
• Introduction to failures of immune system
• Superantigens
Apoptosis
Key Players in Immunology
Innate Adaptive
Cells Phagocytes(PMN, MP, DC)Epithelial Cells
NK Cells
Lymphocytes(B-Ly, T-Ly)
Defense Proteins ComplementAntimicrobial (Poly)Peptides
Antibodies
Apoptosis
• Programmed cell death• Cell suicide• Requires active participation of cell
– Caspase and DNAse activation
• Characterized by nuclear DNA fragmentation and condensation
• Contrasts necrosis (death from “without”, e.g. anoxia, poisoning)
Apoptosis is Important In Various Situations
• In general– Regulation of cell mass– Morphogenesis, organ development
• In host defense– Removal of infected host cells or tumor
cells– Epithelial cell shedding (“Falling of Leaves
from Trees”)• Skin• Intestine
– Tolerance (lymphocytes)– After antigen has been eliminatedNote: Some pathogens induce apoptosis; e.g., Salmonella typhimurium
Apoptosis in T Cell-Mediated Cytotoxicity
• Elimination of infected cells without destruction of healthy cells– Death induced within minutes– Mainly via cytotoxic granules
• Control of immune cells– Mainly via Fas and Fas-ligand interaction– Mutations in Fas lead to lymphoproliferative
disease associated with severe autoimmunity
Apoptosis Specific Cell Changes
Early LateAPOPTOSIS
Induction
CaspaseActivation
DNAFragmentation
Relocation ofPhosphatidylserine
Phagocytosis by
Macrophages
Induction of Apoptosis
• Perforin/granzyme• Fas/Fas-ligand• TNF-• Mitochondrial cytochrome C release
– Counteracted by Bcl-2
• Recognition of pathogens via TLRs– To remove infected cells?– To the benefit of the pathogen?
Induction of Apoptosis in Target Cells by CTLs
Healthy cell Condensed chromatinCell membrane intact
Membrane vesicle shedding
necrotic
apoptotic
Very condensed nucleus
Loss of cytoplasma
apoptotic
MHC I
CTLs Release Cytotoxic Effector Molecules in a Polarized Fashion
Target
CTL Cytotoxic granules
Fragmented nucleus
Complexed in granules with a proteoglycan
Fas:Fas-Ligand Mediated Initiation of Apoptosis
• Fas (CD95, Apo-1)– on many cells, especially lymphocytes– monomer
• Fas ligand (FasL)– on T-cells, stromal cells (bone marrow, thymus)– Trimer
• Fas receptor trimerization upon Fas-Ligand binding
• Cytoplasmic Fas-death domains activated• Adaptor proteins activated, cleave a
procaspase • TNF/ TNF receptors can enter this pathway
Fas:FasL Initiated Apotosis
Release of active caspase 8
TNF- mediated Apoptosis
Membrane bound TNF
DD: Death Domain
DD: Death Effector Domain
Apoptosis initiated by recruitment of signaling molecules to DED
Cell activation initiated by recruitment of signaling molecules to DD and not DED
Caspase Activation during Apoptosis
• Caspases are activated early– Cysteine proteases cleaving after
aspartic acid residues
• Caspases activate a DNAse (Caspase activated DNAse or CAD, late)
• DNAse translocates to nucleus and fragments DNA (200bp)
Phosphatidylserine Translocation during
Apoptosis• In normal cells located at the inner
membrane leaflet• In early apoptosis, after caspase
activation, translocation to the outer membrane leaflet
• Macrophages have receptor for phosphatitdylserine
Fate of Apoptotic Cells
• Condensed• Rapidly
phagocytosed by specialized macrophages– Recognize
phosphatidylserine• Residual apoptotic
bodies
Thymic Cortex
Red: apoptotic cellsBlue: macrophages
Detection of Apoptosis Specific Cell Changes
• Caspase activation (early)– Colorigenic or fluorigenic substrates
• Annexin V (early after caspases)– Relocation of phosphatidylserine (PS) to outer
membrane– Can be bound by annexin V**, a protein with
high affinity for PS
• DNA fragmentation (late)– Fragments: DNA gel electrophoresis, 200 bp
multimers– Strand breaks: TUNEL assay
Failures of the Immune System
Key Players in Immunology
Innate Adaptive
Cells Phagocytes(PMN, MP, DC)Epithelial Cells
NK Cells
Lymphocytes(B-Ly, T-Ly)
Effector Molecules
ComplementAntimicrobial (Poly)PeptidesAntimicrobial
Lipids?
Antibodies
When Does the Immune System Fail?
• Microbial Evasion: microbes circumvent the defense
• Superantigens: exogenous overstimulation of immune system
• Hypersensitivities: endogenous overreaction
• Autoimmune diseases: self attack• Immune deficiencies: inherent
failure
Microbial Evasion
• Microbes involved are pathogenic• Otherwise healthy adults are
affected• Specific disease with typical
symptoms• Not recurrent
Superantigens
Key Players in Immunology
Innate Adaptive
Cells Phagocytes(PMN, MP, DC)Epithelial Cells
NK Cells
Lymphocytes(B-Ly, T-Ly)
Defense Proteins ComplementAntimicrobial (Poly)Peptides
Antibodies
Superantigens
• Trigger T-cell mediated immune response• Cross-link TCR and MHC II from outside• Act in native conformation
– Not loaded into MHC groove– Processing destroys activity
• Soluble or membrane bound• Bind to specific V gene segments• Massive T cell activation
Two Types of Superantigens
• Exogenous (soluble)– bacterial exotoxin
• Staphylococcal Toxic Shock Syndrome Toxin
• Enterotoxins
• Endogenous (membrane bound)– viral coded new membrane protein on
MHC II positive cells• Mouse Mammary Tumor Virus
Superantigens Cross-Link TCR and MHC II
molecules
Superantigens Bind to a Subset of TCRs
• Superantigens are specific for certain V domains
• Can bind to one or a few different V chains
• 20 – 50 different V gene segments known
• Massive stimulation of selected CD4 + cells
• Between 2 – 20% of all T cells can be simultaneously
T-Helper Cell Cytokines
• TH1 Cytokines– IFN-Mph, NK– LT-formerly TNF-
Phagocytes, lymphotoxic)
– IL2 (T Cell proliferation)– IL3/ GM-CSF
(Hematopoiesis)– TNF-– MCP-1 (chemotactic
Monocytes/Mph)– TGF-
• TH2 Cytokines– IL4 (IgE
production)– IL5 (Eosinophil – TNF-
• TH3 Cytokines– IL10 – TGF-
Shock!!!
Consequences of T-Helper Cell Activation
• Uncontrolled hyperactivation of the immune system
• Proliferation of activated T-cells• Systemic toxicity• Shock (TNF-, IFN--mediated macrophage
activation)• Followed by clonal depletion of reactive cells
and suppression of adaptive responses• Recovery probably mediated by delayed build-
up of suppressive cytokines (IL-10)
Examples for Exogenous Superantigens
• Staphylococcal superantigens– S. aureus– Over 20 described– TSST, exfoliatins, enterotoxins
• Streptococcal– S. pyogenes (Group A beta-
hemolysing streptococci)– Exotoxin A and C, and others
Toxic Shock Syndrome
• First described in menstruating women using certain types of tampons
• High fever, rash, skin peeling in palms, shock, multiple organ failure
• Staphylococcus TSST production triggered in these tampons
• TSST resorption through vaginal mucosa
(Alcamo, 6th edition, p 309)
Additional Resources
Accessed 5/14/2008
http://www.aafp.org/afp/20000815/804_f6.jpg