Complement factors and tubulitis Steven Sacks King’s College London.

Complement factors and tubulitis

Steven Sacks

King’s College London

C3b

Graft

Wbc

iC3bC3d

FI

INFLAMMATIONINFLAMMATION

MEMBRANEINJURY

MEMBRANEINJURY

IMMUNEACTIVATION

IMMUNEACTIVATION

C3aCR1CR3CR2

C3aR/C5aR

C3Activating factors

C5b C6 C7 C8

C9 C9

C5a

•Macrophages

•Dendritic cells

•NK cells

•Neutrophils

•T and B cells

CD59DAFCR1MCP

C3biC3bC3d

CR3CR2 CR1

Graft

Wbc

FI

C5b C6 C7 C8

C9 C9

C3a C5a

C3aR/C5aR

Complement Control Proteins

Vulnerability of tubules to complement

Make C3, C4, C2, B Low regulators Activating factors

Ischaemia reperfusion injuryRenal C3 mRNA expression

Ischaemia reperfusion injuryRenal C3 mRNA expression

0 35 35 40 400.0

0.2

0.4

0.6

0.8

Rat

io o

f C

3/ -

acti

n

0.0

0.5

1.0

0 6 24 48

Minutes Hours

C3Actin

Ratio of C3/-actin

Ischaemia Reperfusion

C3 protein Neutrophils

Ischemia reperfusion injuryIschemia reperfusion injury

0

200

400

600

800

1000

0 1 2 3 4 5 6

0

20

40

60

80

100

0min 60min 4hr 24hr

P-selectinC3

PMNL

30min

Mean area/section (m2)

Mouse nativeKidney model

Mouse nativeKidney model

Syngeneic C3+/+ donor kidney transplanted into a C3-/- recipientSyngeneic C3+/+ donor kidney transplanted into a C3-/- recipient

Tubular deposition of C3

Specific blockade of renal IR injurySpecific blockade of renal IR injury

C3

C3aC5

C5a

C5b-9

Classical Lectin Alternative

C1C4C2

MBLMASPC4C2

C3bBD

C6C7C8C9

Non-inhibitory

Inhibitory

Zhou 2000

Thurman 2003

De Vries 2003

***

*

****

***

***

Blocking effect: *

Late blockade is as effective as

early blockade

Summary 1Summary 1

Complement-mediated ischaemia reperfusion injury Primarily a tubular injury Dependant on membrane attack Alternative pathway driven Intensifies over 24-48 hours

Renal allograft rejectionRenal allograft rejection

Donor C3 TUBUAR

Total C3

+GLOMERULAR

+INFILTRATE

Mouse kidney allograftMouse kidney allograftC3 mRNAPAS

C3-/- donor

C3+/+ donor

t t

tt

t

C3 protein

Deficient local synthesis of C3 prolongs allograft survivalDeficient local synthesis of C3 prolongs allograft survival

0 70

50

100

8 10 12 14 20 40 60 80 100

Days after transplantation

Recipient survival (%)

C3-/- donor (n=10)

C3+/+ donor (n=10)

C57BL/6, H-2b donor kidney B10Br, H-2k recipient

C57BL/6, H-2b donor kidney B10Br, H-2k recipient

Recipient anti-donor T cell responseRecipient anti-donor T cell response

C3-/- donor

C3+/+ donorNo donor

Antidonor T cell proliferationCells / well (x105)

0 1 2 3 4 5 6 7 80.0

2.5

5.0

7.5

Day of culture

P < 0.001

The honey pot and the flyThe honey pot and the fly

C3 mRNA

tu

T

C3 protein

tu tu

T

Complementreceptor 1/2

CR/CD4

CD4

CR

Predicted model

C3-binding receptor

Covalently bound C3

T cell

PTEC

thioesterC3

Diapedesis

•Increased T cell stimulation•Increased T cell stimulation

•Increased T cell transmigration•Increased T cell transmigration

T cell cytokine response on stimulation by C3-coated PTEC T cell cytokine response on stimulation by C3-coated PTEC

24h 48h 72h 96h0

5

10

15

20

25

30NS

HIS

IFN

-

(ng/

ml)

C5-deficient serum - as effective as NS

C3-deficient serum - equivalent to HIS

T cell migration across epithelial monolayerT cell migration across epithelial monolayer

C3+/+ C3-/-0

100000

200000

300000

Primed B10.Br T cellsNaive B10.Br T cells

B6 tubular epithelial cells

Mig

rate

d ce

ll nu

mbe

r < O.05

Summary 2Summary 2

In acute renal allograft rejection, absent local synthesis of C3 Reduced tubulitis Reduced antidonor T cell activation/migration Prolonged graft survival

Ascending pyelonephritisAscending pyelonephritis

C3-/-

C3+/+

2/22

14/22

Renal Infection:

Internalisation of E. coli by PTEC is complement dependentInternalisation of E. coli by PTEC is complement dependent

Non infected Infected cells

+ E. coli

+ C3Log-differenceLog-difference

BACTERIALINTERNALISATION

? Replication? Dormancy? Invasion

Cell signallingCytoskeleton

URINARYSPACE

TUBULAREPITHELIUM

C3

Adhesin

E. coli

Adhesin C3 Receptor

1

5

2

3

4

Summary 3Summary 3

In ascending pyelonephritis Human E. coli exploit local complement to

invade the upper urinary tract Epithelial secretion of C3 drives bacterial

uptake leading to tubulitis

ConclusionConclusion

Complement activation in the extravascular compartment is a potent cause of tubulitis

Intragraft production of C3 causes local inflammation and stimulates antidonor T cell reaction and trans-epithelial migration

Locally secreted C3 contributes to the pathogenesis of ascending pyelonephritis

Implications for targeted therapy

Thanks

Wuding Zhou Tony Farrar Shamim Basheer Julian Pratt Miriam Jones Jun Dong Neil Sheerin Tabitha Springall

Mike Carroll Mike Holers Greg Stahl

Funding MRC Wellcome Trust

Targeted complement inhibitionTargeted complement inhibition

- - -- - -Membrane

SCR (1-3) of sCR1

Membrane bindingpolylysineMembrane

inserting Myrostyl

0

10

20

30 ControlTagged inhibitorNaive response

Time (days)

Cells / well (x105)

0 1 2 3 4 5

Antidonor T cell response

Binding in donor kidney

ControlTagged inhibitor

BUN mg/dl

3 4 7 10 14

0

25

50

Days after transplantation

Graft function

F377 LEW

BLOODSPACE

SUPPORTINGTISSUE

URINARYSPACE

T

Complement C3

Membrane attack complex

T

T

T T lymphocyte

Effect of local complement synthesis on the allograft rejection response

Dendritic cell

LPS

Triptolide suppresses renal epithelial synthesis of C3 Triptolide suppresses renal epithelial synthesis of C3

0.0

2.5

5.0

7.5

10.0

C3

(ng

/105 ce

lls/

24

h)

Basal TNF CsA1000ng

FK5061000ng

Trip8ng

C3 ELISA

C3

GAPDH

M Basal 8TNF 4

Triptolide ng/ml

600bp

C3 transcript

Tripterygium Tilfordii Hook F.

C5b-9

Complement activation

Arachidonic acidPGE2Reactive oxygenIL-6 and TNF-aCollagen ISkeletal rearrangement

PTEC

Biancone 1994; David 1997

Human proximal tubular epithelial cells spontaneously activate C3

Dilute serum

LPS-stimulated production of C3 by mouse tubular epithelial cells

0 10 100 10000

1

2

3

LPS dose ng/ml

Supernatant C3 level g/ml

In situ hybridisation

ELISA

Also C2, C4, Factors B

and H

IFN-stimulated tubular epithelium with C3 deposit

C3+/+ PTEC C3-/- PTEC

O

C OS

C3b

=

Models for intragraft C3 interacting with T cells

Antigen-bound C3 stimulates APC

Donor epithelium

C3b

APC

C3b receptor

Ag

T cell

Tissue-bound C3 stimulates T cells

C3b

Ag

Donor epithelium

T cell

C3b receptor TCR

CR1/CR2 receptor blockade of migratory cellsCR1/CR2 receptor blockade of migratory cells

C3+/+ C3-/- 0

100000

200000

Primed B10.Br splenocytes

Primed B10.Br splenocytes + anti H2k

Primed B10.Br splenocytes + anti CR1/2

n.s< O.05

B6 tubular epithelial cells

Mig

rate

d C

ell N

umbe

r

C3-positive graft to C3-negative recipient

Glom

Tubules

C3+/+ graft C3-/- native

Terminal pathway plays a major role

C3

C5C6

MAC

CP AP

C3 (n=8) C4 (n=10) C5 (n=4) C6 (n=16)0

20

40

60

80

100

120C-defC-suf

Reduction 55% 30% 44%-14%

P value 0.005 ns ns 0.015

Seru

m U

rea

(mm

ol/L

)

Journal of Clin. Invest. 2000

C3a

C5a

C4

C3b

CR1CR3CR2

Graft

Wbc

iC3bC3d

FI

C5b C6 C7 C8

C9 C9

INFLAMMATIONINFLAMMATION

MEMBRANEINJURY

MEMBRANEINJURY

IMMUNEACTIVATION

IMMUNEACTIVATION

C3a C5a

C3aR/C5aR

C3

C3b

C3a

C5a

C5b

C6

C7

C8

C9

C9

MAC

C3aR, C5aR

InflammationInflammation

Membraneinjury

Membraneinjury

Activating factors

Graft

Interstitium

Wbc

Immunestimulation

Immunestimulation

CR1, CR2

CR3, CR4

Macrophages, Dendritic cells

NK cells, Neutrophils

T and B lymphocytes

Local synthesis

C3

C3b

C3a

C5a

C5b

C6

C7

C8

C9

C9

MAC

C3aR, C5aR

InflammationInflammation

Membraneinjury

Membraneinjury

Activating factors

Graft

Interstitium

Wbc

Immunestimulation

Immunestimulation

CR1, CR2

CR3, CR4

Macrophages, Dendritic cells

NK cells, Neutrophils

T and B lymphocytes

Local synthesis

C3

C3b

C3a

C5a

C5b

C6

C7

C8

C9

C9

MAC

Activating factors

Graft

Interstitium

Wbc

CD46, CD55

CD59

Complement Control Proteins

Overview of complement and renal transplantation

Hyperacute rejection Complement inhibited rats Alloimmune response Ischaemia reperfusion damage Local synthesis of complement Independent regulation Local contribution 5-15% circulating C3

Renal allograft rejectionRenal allograft rejection

Glomerulus

Tubules

Human transplant biopsy stained for donor C3

Properties of tubular epithelial cellsProperties of tubular epithelial cells

Abundant source of C4, C2, C3, Factor B Regulated by LPS, IFN-, IL-2 etc Low expression of complement regulators Vulnerable to complement attack Proinflammatory, profibrotic reaction

Culture insert

1. B6 PTECmonolayer

4. Migrated spleen cells at 24h

2. IFN- Treatmentfor 3d

C3b

3. Day-14, CD3-enriched spleen cells

C57BL/6 (H-2b)

B10.BR (H-2k)

Transmigration assay