MICR 304 Immunology & Serology Lecture 13 Apoptosis; Failures of the Immune System;...

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