Transcript slides - Smith Lab

Immunology of
Age Related Macular
Degeneration
Kyle C. McKenna, Ph. D.
Associate Professor of Biology, Franciscan University of Steubenville
Associate Professor of Ophthalmology, University of Pittsburgh
[email protected]
412-802-8437
Age Related Macular Degeneration
Leading cause of blindness in individuals over
the age of 60
Due to atophy of the macula area of the retina
where central
vision is focused.
Retinal Architecture
Bruch’s
membrane
Photoreceptors
RPE
Choroid
Sclera
Macular Degeneration Types
• Dry Form
– Loss of RPE and overlying retina resulting in
“geographic atrophy”
• Wet Form
– Neovascularization of macula, inflammation,
retinal scarring, associated with severe vision loss
AMD Pathogenesis
Normal Aging causes
Thickening of Bruch’s
Membrane
Toxic products of
Phototransdution
Accumulate in RPE
RPE cell death
Dry AMD
AMD Pathogenesis
Thickening of Bruch’s
Membrane
Toxic products of
Phototransdution
Accumulate in RPE
Choroidal neovascularization,
Retinal edema
Scar formation
RPE cell death
Wet AMD
AMD Risk Factors
•Age. In the United States, macular degeneration is the leading cause
of blindness in people age 60 and older.
•Cigarette smoking. Exposure to cigarette smoke doubles the risk of
macular degeneration.
•Low levels of nutrients. This includes low blood levels of minerals,
such as zinc, and of antioxidant vitamins, such as A, C and E.
Antioxidants may protect cells from oxygen damage
(oxidation), which may partially be responsible for the effects of
aging and for the development of certain diseases such as
macular degeneration.
•Family History of AMD.
SNP associations in AMD
• SNP Y402H in Complement Factor H associated with
• increased risk
• SNP LOC 387715 which localizes to a mitochondrial
protein is associated with increased risk
Het. At either Y402H or Loc 387715: 2.8 fold risk
Het At both Y402H or Loc 387715: 5.8 fold risk
Hom At either locus:
Y402H: 7.1 risk, Loc 387: 8.1
Hom at both loci: 57-fold risk
What is the Complement?
• Collection of heat-labile soluble proteins constitutively
produced primarily by the liver that are found in blood,
lymph and extracellular fluids
–
–
–
–
C1 (C1q:C1r2:C1s2), C4(C4b, C4a), C2(C2a, C2b), C3 (C3a,C3b)
Collectins (MBL) Ficolins
Factor D, Factor B, Properdin
C5 (C5a, C5b), C6, C7, C8, C9)
• Many complement proteins are proteases that are
synthesized as inactive pro-enzymes (zymogens)
– C1(C1q:C1r2:C1s2)
– Collectins or Ficolins (MASP1, MASP2)
What is the function of Complement?
• Opsonization to promote phagocytosis
• Stimulation of inflammation
• Clearance of Immune Complexes, apoptotic cells,
cellular debris
• Lysis of bacteria, viruses, and cells that are
damaged or infected.
Opsonization & Phagocytosis
• Complement activation deposits C3b on the
surface of bacteria, viruses, or cells that are
infected or damaged
• C3b receptors are present on phagocytes
– CR1 (CD35): C3b macrophages, neutrophils, RBC
– CR2 (CD21): C3dg macrophages and B cells
– CR3 (CD11b): iC3b macrophages and neutrophils
– CR4 (CD11c) : iC3b dendritic cells
CR1 Binds C3b and Augments
Phagocytosis
(Not Janeway)
Phagocytosis may require additional
activation via
C5aR : C5
C3aR : C3
Opsonization & Phagocytosis
• Phagocytosis is enhanced by antibodies
binding to Fc receptors on the surface of
phagocytes
• Hence, the antibody response (adaptive) is
complemented by the innate response
(complement)
Opsonization
Inflammation
• Certain complement proteins generated
during the complement cascade are
inflammatory (anaphylotoxins)
– C3a
– C4a
– C5a
Potency
C5a>C3a>C4a
B
Menu
F
Inflammation
Increase vascular permeability
Promote Chemotaxis of Immune Cells (neutrophils)
Promote Mast Cell Degranulation
Increased phagocytosis
Removal of Immune Complexes
• Complement also helps clear immune complexes
(Antigen/Antibody conjugates)
• Immune complexes can cause many problems, esp. in
autoimmune diseases
• Immune complexes bind C3b
• Recognized by CR1, esp. on RBC’s
• Phagocytosed in spleen and liver
Removal of
Immune
Complexes via
C3b and the
CR1 receptor
Removal of Apoptotic Cells
• Phosphocholine, present in bacterial
phospholipids is recognized by C-reactive
protein
• C-reactive protein activates complement
• Apoptotic cells express phosphocholine on the
cell surface which activates complement for
subsequent removal
MAC lysis
• The MAC pore size is 70-100Å
• Allows ions and small molecules to diffuse out
• Disrupts osmotic stability and lyses bacteria,
virus, or cells that are infected or damaged
(d)
B
Menu
F
MAC Pores
Summary of Complement Functions
In addition to providing protection from pathogens, complement plays an
Important role in normal physiology by removing apoptotic and damaged cells
and cell debris
How is Complement Cascade Initiated?
• By Direct Binding of Pathogen Surfaces
(Mannose/Lectin Pathway)
• By Indirectly Binding of Pathogen Surfaces
(Classical Pathway) via engagement of
antibodies or C-reactive protein
• Spontaneously (Alternative Pathway)
INITIATION
ACTIVATION
AMPLIFICATION
EFFECTORS
INITIATION: Classical Pathway
• C1 complex can bind:
C1
complex
– Antibodies complexed with
antigens
• Natural
• Adaptive
– C-reactive protein
• Acute phase protein that
binds phophocholine in
bacterial polysaccharides and
apoptotic cells
– Microbe surfaces
• Lipoteichoic acid (Gm+)
• Some bacterial proteins
INITIATION: Classical Pathway
Not Janeway
-The initial steps in classical pathway initiation are very similar when C1 binds C-reactive protein
(or a pathogen surface).
INITIATION: Classical Pathway
-NOTE: Cleavage of complement proteins yields “a” and “b” products.
-The “b” product is ALMOST ALWAYS the larger (big) one and binds to the surface
INITIATION: Classical Pathway
FORMATION OF C3 CONVERTASE
The exception to the “b” rule
C4b2a is a C3 convertase
C3b deposition on cell surface
INITIATION: Lectin Pathway
- Present in low concentrations in plasma; production by liver
increased during acute phase response
- Two- to six-headed that forms complex with two protease zymogens
- MASP-2 closely related to C1r and C1s
INITIATION: Lectin Pathway
Ficolins have a fibrinogen-like domain
That binds acetylated sugars but not
Mannose
n-linked glycoproteins are terminated
with sialic acid residues.
Complement activation highly similar to classical pathway
-Upon binding a pathogen surface, a conformational change in MASP-2
occurs resulting in cleavage of C4 and C2, formation of C3 convertase
(C4bC2a)
-C3b is deposited on the cell surface
INITIATION: Alternative Pathway
-In plasma, circulating C3 spontaneously undergoes hydrolysis to form
C3(H2O) which binds factor B
-Factor B on C3(H2O) is cleaved by Factor D to form C3(H2O)Bb, a C3
convertase
-C3b is deposited on the cell surface
INITIATION: Alternative Pathway
FORMATION OF C3 CONVERTASE
-Factor B binds C3b on cell surface which is subsequently cleaved by
Factor D to form C3bBb, a C3 convertase
-C3 convertase is stabilized by Factor P (properdin)
(-properdin: t1/2 = 5 min; + properdin; t1/2 = 30 min)
Properdin (Factor P) is made by neutrophils
AMPLIFICATION
Classical & Lectin
C3 convertase
Alternative C3 convertase
-One molecule of C3 convertase can cleave up to 1000 molecules of C3 into C3b
-Many C3b molecules deposited on cell surface (2 x 106 C3b molecules
deposited in <5 min)
EFFECTOR
(Assembly of Membrane Attack Complex (MAC))
Formation of C5 convertase
Cleavage of C5 convertase
Classical/Lectin Alternative
First step in MAC assembly
EFFECTOR
(Assembly of Membrane Attack Complex (MAC))
All complement pathways converge to this process
Hydrophobic Domain
Subsequent steps in MAC assembly
C5b is labile and will be inactivated w/i two minutes unless stabilized by C6
EFFECTOR
What happens to the complement fragments
not deposited on the surface?
ANAPHYLATOXINS
-C3a, C4a and C5a are anaphylatoxins that induce local inflammatory responses
Summary
Complement Regulation
Also, C4
(Not Janeway)
-C3b, C4b rapidly inactivated by water unless
allowed to immediately bind to protein or
carbohydrate on cell surface
Complement Regulation
• C1 inhibitor (C1INH).
– Dissociates C1r and C1s from C1 thereby removing
the enzymatic activity necessary to cleave C4 and
continue the complement cascade
Complement Regulation
Complement Regulatory Proteins Found in Plasma
C4BP displaces C2a from the
C4b2a complex. C4b bound by
C4BP is cleaved by a soluble
Factor I to inactive forms C4d
and C4c
C4d
C4c
-Factor I cleaves C4b and C3b but requires cofactors to yield inactive proteins:
-C4 binding protein (C4BP) aids Factor I in cleaving C4b
Complement Regulation
Complement Regulatory Proteins Found in Plasma
(Not Janeway)
-Factor H also acts as a cofactor to Factor I to cleave C3b to yield inactive protein
Complement Regulation
Complement Regulatory Proteins Embedded in Cell Membranes
(Not Janeway)
-Decay-accelerating factor (DAF) and Membrane Cofactor Protein (MCP) prevent formation of
convertases by displacing C3b
-MCP further acts as a cofactor to Factor I to yield inactive C3b
-Other: CR1 also prevents formation of C3 convertase by displacing C2a and/or Bb and acts as
cofactor to Factor I to cleave C4b and/or C3b
Complement Regulation
Complement Regulatory Proteins Embedded in Cell Membranes
-CD59 (protectin) prevents complete assembly of MAC
Unregulated Complement in AMD
B-amyloid in Drusen inhibits Factor I cleavage of C3b
Wang J. et al. 2008. J. Immunol. 181:712
mRNA expression of Factor H by RPE is reduces by
Oxidative stress (smoking).
Wu Z. et al. 2007. J. Biol. Chem. 282:22414
Phagocytosis of oxidized photoreceptor cells by RPE
Inhibits Factor H production by RPE
Chen M. et al. 2007. Exp. Eye Res. 84:635
Y402H Polymorphism Effects
• Y402H Polymorphism in Factor H decreases
the affinity of Factor H for CRP
– Prossner B. E. et al. 2007. J. Exp. Med. 204:2277
• Y402H Polymorphism in Factor H decreases
the affinity of Factor H for the oxidized
lipoprotein malondialdehyde (MDA)
– Weismann D et al. 2011 Nature 478:76-81
CFH mutations prevent binding of CFH to cell surface thereby causing unregulated
Complement activation on the cell surface leading to retinal inflammation
Inflammasome
Inflammasome activation is associated with geographic atrophy in AMD patients
Tarallo V et al. 2012 Cell 149:847-859
Photoreceptors
Apoptic
Cell
RPE
C4b
C4
C4b2a
C2
C3
C3b C3bBb C3b
B
C4a
C2b
C4b2aC3b
D
C5 C5b
C6 – C9
C5a
C3a
C3b2Ba
Ba
Oxidized
Phospholipids
Drusen
BM
Choroid
Mac
Mac
Mac
VEGF
Photoreceptors
Binds to CRP
RPE
C4b2a
C2
C4b
C3
C3b C3bBb C3b
B
C4
C4a
C2b
C4b2aC3b
D
C5 C5b
C6 – C9
C5a
C3a
C3b2Ba
Ba
Inhibits
C3 convertase formation
Masks oxidized
phospholipids
FACTOR H
Drusen
BM
Choroid
Mac
Mac
Mac
Retinal Damage
• Induces anti-retinal antibodies along with
antibodies to carboxypyrole adducted
phospholipids