Damage •Virus/infection •Toxin/ethanol •Autoimmunity · Acute hepatitis Chronic hepatitis...

Acute hepatitis

Resolution

Damage•Virus/infection•Toxin/ethanol•Autoimmunity

Acute hepatitis

Chronic hepatitis

Resolution


Acute hepatitis

Chronic hepatitis

Resolution

Cirrhosis


Acute hepatitis

Chronic hepatitis

Resolution

Cirrhosis

Hepatocellularcarcinoma


Interface hepatitis with periportal fibrosis

Established cirrhosis

Adams and Eksteen Nature Reviews Immunology 6, 244–251 (March 2006) | doi:10.1038/nri1784

The hepatic sinusoids are a unique, low-flow vascular bed

Endothelium

Lymphocyte

CaptureLittle role for SelectinsWong et al 1997; Adams et al 1996VAP-1McNab et al 1996, Lalor et al 2002

ActivationChemokinesCXCR3

ArrestICAM-1+VCAM-1

Chemokines

TransmigrationVAP-1ChemokinesICAM-1

Chemokines

TransmigrationChemokines

ActivationChemokines

Hepatic stellate cells(myofibroblasts)

Liver Infiltrating Lymphocytes

Non-enzymaticdigestion

Collagenase digestionImmunomagnetic

separationEnzymatic perfusion

technique

Human liver tissue from Birmingham transplantation

programme

Cultured in HGF+VEGF+Human sera

Sinusoidal endothelial cells

CD4 CD8 NK CELLS

NKT CELLS

CCR2 (+)

CCR5 + + + +

CCR6 + +

CCR7 (+)

CCR9 (+) (+)

CCR10 (+)

CXCR1 (+)

CXCR2 (+)

CXCR3 + + + +

CXCR4 + + +

CXCR6 + + + +

CX3CR1 + + + +

Chemokine receptor expression on human liver-infiltrating lymphocytes

Boisvert et al 2003; Shields et al 1999; Heydtmann et al 2006

A

B

C

Normal PBL Normal LIL PSC PBL PSC LIL

CXCR3

CD

3

CXCR3 is increased on liver-infiltrating T cells

CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (ITAC)Upregulated on sinusoids at sites of interface hepatitisShields et al 1999Critical in progression of inflammatory damage and fibrosisHelbig Hepatol 2004; Harvey J Leuk Biol 2003, Narumi J Immunol 1997

CXCL9

CXCR3 ligands are interferon-inducible chemokines that are increased at interface

hepatitis and which predict outcome in HCV

Video Recorder

T.V.TV

Syringe Pump

37°C Chamber

Lymphocytes

valve

Phase contrastmicroscope

Endothelial cellsin microslide

0.05-0.1 Pa

Lymphocytes from inflamed human liver

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0.8 1.3 1.8 2.3 2.8 3.30.0

5.0

10.0

15.0

20.0

25.0

30.0

0 . 8 1. 3 1. 8 2 . 3 2 . 8 3 . 3Control Control Control Controlα-CXCR3 PTX α-ICAM-1 CXCR3L

CXCR3 promotes transendothelial migration through cytokine-activated HSEC

Tran

send

othe

lial m

igra

tion

Curbishley 2005 Am J Path

Liver LobulePortal Tract

CXCR3 Receptor

CXCL9,CXCL10

Induction of CXCR3 ligands with inflammation• Upregulated on sinusoidal vessels at sites of interface hepatitis• Secreted by endothelium, hepatocytes and stellate cells• Paracrine presentation of CXCR3 ligand critical in progression of

inflammatory damage and fibrosis

IFN-γ + TNF-α

Portal Tract

Bile ducts

IFNγ+TNFα CXCR3

CXCL9 CXCL10

Can CXCR3 ligands secreted “upstream”by cholangiocytes be captures and

presented on sinusoidal endothelium?

Curbishley et al Am J Pathol 2005

Do paracrine signals from activated myofibroblasts regulate lymphocyte recruitment via endothelium?

Hepatic stellate cells are myofibroblasts thata) drive liver fibrosis in response to injury b) are intimately associated with the overlying sinusoidal endotheliumc) act as PERICYTES

Acute inflammation Chronic inflammation

Hepatic stellate cells secrete chemokines

5000

500

pg/m

l

Control

αSMA

CD90

Vimentin

Desmin

CK-19

Electronic valve

sample

Fluid reservoir

MicroscopeSyringe pump

37 C enclosureo

Parallel plateflow chamber

Cell

Flow Based Adhesion Assay Incorporating the Coculture Parallel Platein collaboration with Ed Rainger and Gerard Nash

Endothelial cells

Myofibroblasts

Cells on opposing sides of porousmembrane in secretory and physicalcontact

HSEC Alone(TNF 1ng/ml-1)

HSEC Co-cultured withaLMF (PBC P3)

(TNF 1ng/ml-1)

HSEC

AdherentLymphocytes

Insert

0.4µm pore membrane

Adhesion ++Rolling +

aLMFHSEC

Adhesion ++++Rolling ++

In collaboration with Ed Rainger, Gerard Nash and Chris Buckley

HSEC co-cultured with aLMF support increased lymphocyte adhesion from flow.

HSEC alone HSECSkin FB

HSECaLMF

PTX

0

50

100

150A

dher

ent c

ells

/mm

2/m

illio

n in

fuse

d

CD106 CD45

Stellate cells are pericytes is close association with sinusoidal endothelium

Can they direct migration beyond the endothelium into the

parenchyma?

LeucoLeuco--stromal interactions are dynamic stromal interactions are dynamic pseudoemperipolesis

Bradfield et al Arthritis Rheum. (2003)

Adh

eren

t l c

ells

/HPF

0

20

40

60

80

100

120

TOTAL ADHESION PHASE BRIGHT PHASE DARK

* ** Liver

Skin

TOTAL ADHESION

PHASE DARK

PHASE BRIGHT

Activated liver myofibroblasts support ICAM-1 and VCAM-1 dependent pseudoemperipolesis

0

20

40

60

80

100

120

IgG aENA-78 aCCL2 aCXCR3 PTx aVEGF aIL-6 aHGF

%M

igra

tion

with

isot

ype

cont

rol

***p<0.01 *p<0.05

n=9

*

***

***

******

Chemokine-dependent mechanisms support pseudoemperipolesis on activated liver myofibroblasts

+++ SURVIVAL SIGNALSRETENTION

CXCR3 ligands

ACTIVATED HSCVCAM/VLA-4

ICAM/LFA-1 ICAM-1

NEO-MATRIX

Lymphocyte

APH 2006

VAP-1 VCAM-1

PROLIFERATION

What retains T cells in the liver?

Red = HEA 125

Green = CD45

Lymphocyte

Integrins

CCR5

CXCR3

CXCR6

CXCR6 is expressed on tissue infiltrating effector T cellsJJ Campbell et al J Immunol 2001; Kim et al JCI 2001

α4β1αLβ2

CXCR6

Liver-infiltrating T cells

Blood T cells

Heydtmann et al J Immunol 2005

Boisvert J Hepatol 2003

CXCL16 is expressed on inflamed epithelial cells in the liver

And on human biliary epithelial cells in vitro

Hepatocytes

Bile ducts

VCAM-1 ICAM-1

α4β1αLβ2

CXCR3

PSC Liver endothelium

Biliary epithelial cell or hepatocyte

VAP-1

VAP-1R

CXCL10

VCAM-1

CXCR3

CXCL16

α4β1

VAP-1R

CXCR6

CXCR3

PROMOTES RECRUITMENT

CXCR6 Target cells to epithelium

CXCR6

EFFECTOR CELLS

Heydtmann et al J Immunol 2005 and 2006

CCL28 is expressed on inflamed bile ducts in chronic inflammatory liver disease

Eksteen et al J Immunol 2006

What is the nature of the CCR10+ T cells in the liver?

CCR10+ T cells from the liver are functional Tregs

CCR10+ T cells from the liver have a tissue-infiltrating phenotype

Liver-derived CCR10+CD4+CD25+

Blood CD4+CD25+

CCR10 CXCR6

Proliferation/Local differentiation

Apoptosis

RecruitmentCXCR3

Emigration

CCR10 CXCR6

Chemokines involved in chronic liver inflammation

Effector cellsRegulatory cells

Distribution of Liver infiltrating Treg in autoimmune hepatitis; HCV and primary biliary cirrhosis

DAPI Grey

FOXP3 Green

CD3 Blue

CD4 Red

Distribution of Treg in portal lymph nodes

DAPI Grey

FOXP3 Green

CD3 Blue

CD4 Red

R18 R19

R20 R21

100 101 102 103 104Normal liver CD4 Violet 1 Log Comp

100

101

102

103

104

Nor

mal

liver

FO

XP3

PE L

og C

omp

Gated on FOXP3high and CD127low Liver-derived lymphocytes

Normal Liver

CD

25

CD4

R11 R12

R13 R14

100 101 102 103 104LDL CD4 Violet 1 Log Comp

100

101

102

103

104

LDL

CD

25 P

E-C

y5 L

og C

omp

2.4%0.3%

Diseased liver contains a higher proportion of Tregs compared with normal human liver

4%C

D25

Autoimmune hepatitis

CD

25

Fulminant hepatitis

CD4 CD4

Cells+ (range n=4)CCR1 2-3%CCR2 5%CCR3 2-3%CCR4 65- 87%CCR5 10-15%CCR6 2-5%CCR7 15-25%CCR8 2-3%CCR9 10-15%CCR10 8-12%CXCR1 1-4%CXCR2 2-4%CXCR3 35-64% CXCR4 10-15%CXCR5 2-4%

CXCR6 10-15%0 10 20 30 40 50 60 70 80

%cells +ve

Chemokine receptor expression on liver-derived CD4+CD25+CD127lowFoxP3+ Tregs

CCR4 is expressed on liver-infiltrating Treg CCR4 expression in matched LITreg and PBTreg (CD4+CD25+CD127low)

LDTreg

control

PBTreg

**

%+v

e

P<0.01%+v

e

Liver Treg BloodTreg

CXCR3 expression in matched LITreg and PBTreg(CD4+CD25+CD127low)

Contr\ol

PBTreg

LDTreg

**

CXCR3 is expressed on liver-infiltrating Tre g

%+v

e

P<0.01

Liver Treg BloodTreg

Video Recorder

T.V.TV

Syringe Pump

37°C Chamber

Lymphocytes

valve

Phase contrastmicroscope

Endothelial cellsin microslide

0.05-0.1 Pa

Adhe

rent

cel

ls/ m

m2 /

106

cells

per

fuse

d at

0.0

5Pa

** **

* *

** **

% A

dher

ent c

ells

tr

ansm

igra

ting

at 0

.05P

a

n=4 p<0.01

Treg adhesion and transmigration on TNFα and IFNγ stimulated HSEC under flow

IgG1 α-CXCR3 Ptx α-VCAM-1 α-ICAM-1 αICAM-1 +VCAM-1

IgG1 α-CXCR3 Ptx α-VCAM-1 α-ICAM-1 αICAM-1 +VCAM-1

0

1000

2000

3000

4000

5000

6000

0

CXCL12CXCL12

+Ptx

CCL22

CCL22+

Ptx

CCL17CCL1

7+Ptx

Cel

l mig

ratin

g

Liver-derived Treg migrate to CCR4 ligands

CCR4 ligand TARC (CCL17) detected in diseased liver but not normal liver and is absent from resting and cytokine activated liver endothelium

HSEC (TNF&IFN)

Diseased liver tissue

Normal liver

1 2 3 4 5 6 7 8 9

-30-20

-12

-8

CCL22 stains dendritic cells in parenchymal and portal infiltrates that also contain FoxP3+ cells

CCL22

FoxP3 brown

CCL22 red

VCAM-1 ICAM-1

α4β1αLβ2

CXCR3


Biliary epithelial cell or hepatocyte

VAP-1

VAP-1R

CXCL10

VCAM-1

CXCR3

CXCL16

α4β1

VAP-1R

CXCR6

CXCR3


CXCR6 Target cells to epithelium

CXCR6

EFFECTOR CELLS

VCAM-1 ICAM-1

α4β1αLβ2

CXCR3


Epithelial cell

VAP-1

VAP-1R

CXCL10

VCAM-1

CXCR3

α4β1

VAP-1R

CCR10

CXCR3


CCR10 Target cells to epithelium

CCR10

Regulatory cells

CCL28

VCAM-1

α4β1 CXCR3


Dendritic cells

CXCL10

CXCR3

α4β1 CCR4

CXCR3


CCR4 Target cells to intrahepatic DCs

CCR4

Regulatory cells

CCL22

CREDITS

CollaboratorsRon Gladue (Pfizer Groton)Mike Briskin (Millenium)Jim Campbell (Harvard)

THE UNIVERSITY OF BIRMINGHAMBirmingham

MRC Centre for Immune Regulation• Liver Group

Trish LalorEmma Haughton and Andy HoltYe OoStuart CurbishleyBertus Eksteen

• Dept RheumatologyChris Buckley Mike SalmonAndrew Filer

• Vascular BiologyEd Rainger Gerard Nash

• ImmunologyLucy WalkerDave Withers

Dept Pathology Stefan Hubscher

Damage •Virus/infection •Toxin/ethanol •Autoimmunity · Acute hepatitis Chronic hepatitis...

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