MICR 304 Immunology & Serology

1. MICR 304 Immunology & Serology Lecture 13 Apoptosis; Failures of the Immune System; Superantigens Chapter 5.15, 6.25, 6.26, 8.26-8.30,

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

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. Cytotoxic granules CTL Target Fragmented nucleus CTLs Release Cytotoxic Effector Molecules in a Polarized Fashion 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. Thymic Cortex Red: apoptotic cells Blue: macrophages Fate of Apoptotic Cells • 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

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

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 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 Superantigens Bind to a Subset of TCRs

29. 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 () T-Helper Cell Cytokines 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. 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 Toxic Shock Syndrome (Alcamo, 6th edition, p 309)

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