MICR 304 Immunology & Serology Lecture 13 Apoptosis; Failures of the Immune System; Superantigens Chapter 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 Complement Antimicrobial (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 eliminated Note: 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 Phagocytosis by Macrophages Induction APOPTOSIS Early Late Relocation of Phosphatidylserine Caspase Activation DNA Fragmentation

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?

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

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 TNFa/ TNF receptors can enter this pathway

Fas:FasL Initiated Apotosis Release of active caspase 8

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

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 Thymic Cortex Red: apoptotic cells Blue: macrophages Condensed Rapidly phagocytosed by specialized macrophages Recognize phosphatidylserine Residual apoptotic bodies

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 Complement Antimicrobial (Poly)Peptides Antimicrobial 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 Complement Antimicrobial (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 Vb 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 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

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!!!

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)

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