1. MICR 304 Immunology & Serology
Lecture 13
Apoptosis; Failures of the Immune System; Superantigens
Chapter 5.15, 6.25, 6.26, ,

2. Overview of Today’s Lecture
Apoptosis
Definition
Triggers
Intracellular events
Assays to detect apoptosis
Introduction to failures of immune system
Superantigens

3. Apoptosis

4. Key Players in Immunology
Innate
Adaptive
Cells
Phagocytes
(PMN, MP, DC)
Epithelial Cells NK Cells
Lymphocytes
(B-Ly, T-Ly)
Defense Proteins
Complement
Antimicrobial (Poly)Peptides
Antibodies

5. 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)

6. 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 eliminated
Note: Some pathogens induce apoptosis; e.g., Salmonella typhimurium

7. 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

8. Apoptosis Specific Cell Changes
Phagocytosis by
Macrophages
Induction
APOPTOSIS
Early
Late
Relocation of
Phosphatidylserine
Caspase
Activation
DNA
Fragmentation

9. Induction of Apoptosis
Perforin/granzyme
Fas/Fas-ligand
TNF-a
Mitochondrial cytochrome C release
Counteracted by Bcl-2
Recognition of pathogens via TLRs
To remove infected cells?
To the benefit of the pathogen?

10. Induction of Apoptosis in Target Cells by CTLs
MHC I
necrotic
apoptotic
apoptotic
Healthy cell
Condensed chromatin
Cell membrane intact Membrane vesicle shedding
Very condensed nucleus
Loss of cytoplasma

11. CTLs Release Cytotoxic Effector Molecules in a Polarized Fashion
Target
CTL
Cytotoxic granules
Fragmented nucleus
Complexed in granules with a proteoglycan

12. 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
TNFa/ TNF receptors can enter this pathway

13. Fas:FasL Initiated Apotosis
Release of active caspase 8

14. TNF-a 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

15. 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)

16. 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

17. Fate of Apoptotic Cells
Thymic Cortex
Red: apoptotic cells
Blue: macrophages
Condensed
Rapidly phagocytosed by specialized macrophages
Recognize phosphatidylserine
Residual apoptotic bodies

18. 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

19. Failures of the Immune System

20. Key Players in Immunology
Innate
Adaptive
Cells
Phagocytes
(PMN, MP, DC)
Epithelial Cells NK Cells
Lymphocytes
(B-Ly, T-Ly)
Effector Molecules
Complement
Antimicrobial (Poly)Peptides
Antimicrobial Lipids?
Antibodies

21. 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

22. Microbial Evasion Microbes involved are pathogenic
Otherwise healthy adults are affected
Specific disease with typical symptoms
Not recurrent

23. Superantigens

24. Key Players in Immunology
Innate
Adaptive
Cells
Phagocytes
(PMN, MP, DC)
Epithelial Cells NK Cells
Lymphocytes
(B-Ly, T-Ly)
Defense Proteins
Complement
Antimicrobial (Poly)Peptides
Antibodies

25. 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 Vb gene segments
Massive T cell activation

26. 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

27. Superantigens Cross-Link TCR and MHC II molecules

28. Superantigens Bind to a Subset of TCRs
Superantigens are specific for certain Vb domains
Can bind to one or a few different Vb chains
20 – 50 different Vb gene segments known
Massive stimulation of selected CD4 + cells
Between 2 – 20% of all T cells can be simultaneously

29. T-Helper Cell Cytokines
TH1 Cytokines
IFN-g (Mph, NK )
LT-b (formerly TNF-b, Phagocytes, lymphotoxic)
IL2 (T Cell proliferation)
IL3/ GM-CSF (Hematopoiesis)
TNF-a
MCP-1 (chemotactic Monocytes/Mph)
TGF-b ()
TH2 Cytokines
IL4 (IgE production)
IL5 (Eosinophil )
TNF-a
TH3 Cytokines
IL10 ()
TGF-b ()
Shock!!!

30. Consequences of T-Helper Cell Activation
Uncontrolled hyperactivation of the immune system
Proliferation of activated T-cells
Systemic toxicity
Shock (TNF-a, IFN-g-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)

31. 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

32. 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)

33. Additional Resources http://www.aafp.org/afp/20000815/804_f6.jpg
Accessed 5/14/2008