Immunology of Age Related Macular Degeneration Kyle C. McKenna, Ph. D. Associate Professor of Biology, Franciscan University of Steubenville Associate.

Immunology ofAge Related Macular

Degeneration

Kyle C. McKenna, Ph. D.

Associate Professor of Biology, Franciscan University of SteubenvilleAssociate Professor of Ophthalmology, University of Pittsburgh

mckennakc@upmc.edu412-802-8437

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 centralvision is focused.

Retinal Architecture

PhotoreceptorsRPE

Bruch’smembrane

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 causesThickening of Bruch’sMembrane

Toxic products ofPhototransdutionAccumulate in RPE

RPE cell death

Dry AMD

AMD PathogenesisThickening of Bruch’sMembrane

Toxic products ofPhototransdutionAccumulate 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 : C5C3aR : 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

Menu FB

Inflammation

• Certain complement proteins generated during the complement cascade are inflammatory (anaphylotoxins)– C3a– C4a– C5a

C5a>C3a>C4a

Potency

InflammationIncrease vascular permeabilityPromote Chemotaxis of Immune Cells (neutrophils)Promote Mast Cell DegranulationIncreased 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

Functions of the Complement System ©

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

Menu FB

(d)

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

MAC Pores

Summary of Complement Functions

In addition to providing protection from pathogens, complement plays anImportant role in normal physiology by removing apoptotic and damaged cellsand 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)

AMPLIFICATION

INITIATION

EFFECTORS

ACTIVATION

INITIATION: Classical Pathway

• C1 complex can bind:– 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

C1 complex

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

The exception to the “b” rule

FORMATION OF C3 CONVERTASE

C3b deposition on cell surface

C4b2a is a C3 convertase

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

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

Ficolins have a fibrinogen-like domainThat binds acetylated sugars but not Mannose

n-linked glycoproteins are terminatedwith sialic acid residues.

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

-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)

FORMATION OF C3 CONVERTASE

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)

C3 converta

se feeds

into alternative

pathway

First step in MAC assembly

EFFECTOR (Assembly of Membrane Attack Complex (MAC))

Classical/Lectin Alternative

Formation of C5 convertase Cleavage of C5 convertase

Subsequent steps in MAC assemblyC5b is labile and will be inactivated w/i two minutes unless stabilized by C6

EFFECTOR (Assembly of Membrane Attack Complex (MAC))

Hydrophobic Domain

All complement pathways converge to this process

EFFECTOR What happens to the complement fragments

not deposited on the surface?

-C3a, C4a and C5a are anaphylatoxins that induce local inflammatory responses

ANAPHYLATOXINS

Summary

Complement Regulation

(Not Janeway)

Also, C4

-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

-Factor I cleaves C4b and C3b but requires cofactors to yield inactive proteins:-C4 binding protein (C4BP) aids Factor I in cleaving C4b

C4BP displaces C2a from the C4b2a complex. C4b bound by C4BP is cleaved by a soluble Factor I to inactive forms C4d and C4c

C4c

C4d

Complement Regulation

(Not Janeway)

Complement Regulatory Proteins Found in Plasma

-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

-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

(Not Janeway)

Complement Regulation

Complement Regulatory Proteins Embedded in Cell Membranes

-CD59 (protectin) prevents complete assembly of MAC

Unregulated Complement in AMDB-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 byOxidative 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 unregulatedComplement activation on the cell surface leading to retinal inflammation

InflammasomeInflammasome activation is associated with geographic atrophy in AMD patients

Tarallo V et al. 2012 Cell 149:847-859

Apoptic Cell

Photoreceptors

RPE

BM

Choroid

BMDrusen

C1q

C4

C4b

C4a

C2 C4b2a

C2b

C3

C3a

C3b C5 C5b

C5a

C6 – C9B D

C3bBb

Ba

C4b2aC3bC3b

C3b2Ba

Mac MacMac VEGF

OxidizedPhospholipids

Photoreceptors

RPE

BM

Choroid

BMDrusen

C1q

C4

C4b

C4a

C2 C4b2a

C2b

C3

C3a

C3b C5 C5b

C5a

C6 – C9B D

C3bBb

Ba

C4b2aC3bC3b

C3b2Ba

Mac Mac Mac

FACTOR H

InhibitsC3 convertase formation Masks oxidized

phospholipids

Binds to CRP

Retinal Damage

• Induces anti-retinal antibodies along with antibodies to carboxypyrole adducted phospholipids