Dr Allister GrantConsultant Hepatologist

University Hospitals Leicester

The extra-intestinal manifestations of IBD

Aims

• Ease you in

• Explain the adhesion cascade

• Explain how adhesion molecules introduce tissue specific homing properties to activated lymphocytes

• Experimental data/techniques

• Anti-adhesion/inflammation strategies in IBD

Inflammatory Bowel Disease

Erythema Nodosum

Pyoderma Gangrenosum

Episcleritis

Ankylosing spondylitis

Cirrhosis

Expanded Portal Tracts(Blue)

BACKGROUND

Inflammatory Bowel Disease and PSC Incidence of IBD = 3-9 per 100 000

Prevalence of IBD = 40-200 per 100 000

2-10% of patients with IBD will subsequently develop PSC

~ 70% of patients with PSC have evidence of IBD

20-40% of patients with end stage PSC develop cholangiocarcinoma

Lymphocytes on endothelium

The Adhesion Cascade

Tissue

Margination of Lymphocytes

Blood Flow

Blood

Endothelium

1. Rolling on Selectins

P-selectinE-selectin

Endothelial Cell

LYMPHOCYTE

L-selectin

PNAd mucin

CLA mucin

PSGL-1 mucin

2. Lymphocyte Activation

Chemokine family of cytokines

• Small 8 - 12 kD proteins

• Over 50 human chemokines

• Produced by many cell types

• Secreted in large quantities

• Rapid effect on target cells by activating specific G-protein linked receptors

• Ability to trigger adhesion in seconds

Chemokines

-sub family -sub family

C

CC

C

C

CXC

C

IL-8

ENA-78GRO

IP-10HuMIGITAC

MCP-1MIP-1MIP-1RANTES

Lymphocyte

Endothelium

Integrin activation

Cytoskeletal reorganisation

Migration

Proteoglycans in the endothelial glycocalyx retain and present chemokines to lymphocytes

3. Firm Adhesion

ICAM-1

Endothelial Cell

LYMPHOCYTE

Inactive integrin

VCAM-1

Active 4ß1

Active LFA-1

Addressins

Integrins

Activation signal

Arrest of Lymphocytes on Integrins

Ctyoskeletal Rearrangement

(Chemokine Mediated)4. Transmigration

Transmigration Tethering Rolling Arrest & Activation

Endothelium

Blood Flow

Lymphocyte

Selectins Integrins Integrins & Chemokine receptors Chemokine receptors

ChemokinesAddressins & ChemokinesCarbohydrate ligands Addressins

Adhesion cascade

DC

TISSUELYMPH NODE

BLOOD

DC

BLOOD

Naïve T cells

Effector T cells

THYMUS

Thymocytes

Memory lymphocytes

BLOOD TISSUE Lymph Node BLOOD

Naïve T cell

Memory T cell

Memory T cell

Skin

Gut

I Weissman, E Butcher, C Mackay, S Shaw and S Jalkanen

TISSUE SPECIFIC HOMING OF MEMORY EFFECTOR

T LYMPHOCYTES

Tissue specific T cell recruitment

Naïve T cell

CCR7 + L-selectin

Memory/effector T cell

PNaD

CCL21(SLC)

E-selectin

CCL17(TARC)

MAdCAM-1

CCL25(TECK)

LYMPH NODE SKIN GUT

CCR4 + CLA CCR9 + 47

Tethering & Rolling Activation Arrest

Endothelium

Blood Flow

Lymphocyte

CCR5

glycocalyx

CCR5ligands

VAP-1 VAP-1

VAP-1 receptor VAP-1 receptor

? ?

Liver

NORMAL INFLAMED

LIVERvascular ++ +sinusoidal +++ +++

LYMPH NODE +++ +++GUT _ +++KIDNEY + +SKIN _ ++SYNOVIUM _ ++BRAIN _ _

HEART + +

EXPRESSION OF VAP-1 IN HUMAN ENDOTHELIUMSalmi et al. J. Ex. Med.1993; McNab et al Gastro 1996

MAdCAM-1 Expression inPSC Liver

Grant et al Hepatology 2001, Hillan et al Liver 1999

Positive Staining of Portal Vascular Endothelium

ACT-1(a4b7) STAINING OF PSC LIVER

MAdCAM-1(red)

(green)

Immunofluorescent Staining of a Portal Vessel in PSC

37CIncubator

Media reservoir

2-way electronic valve

Cell sample

Microslide

Microscope

Video/computeranalysis system

Syringe pump

Adhesion Assay Under Conditions of Shear Stress

www.expertreviews.org

Lymphocyte rolling

Lymphocyte Transmigration

Control MAdCAM-1 a4b7 CD62L0

102030405060708090

100

Ad

he

sio

n o

f P

SC

PB

La

s %

of

Co

ntr

ol

Adhesion Assays Under Conditions of Shear Stress

Target of Blocking Monoclonal Antibody

Tissue specific T cell recruitment

CXCR3 + VAP-1r

VAP-1

IP-10

MAdCAM-1

CCL25(TECK)

NORMAL LIVER GUT

CCR9 + 47

VAP-1 MAdCAM-1

IP-10

INFLAMED PSC LIVER

?

A

C D

B

P1 P2 P3 P4 P5 P6 NL1 NL2 NL3 PB1 PB2 ALD1ALD2 P7

CCL25

-actin

Western Immunoblotting for CCL25.P = PSCNL = NormalPB = PBCALD = Alcoholic Liver Disease

Tissue specific T cell recruitment

CXCR3 + VAP-1r

VAP-1

IP-10

MAdCAM-1

CCL25(TECK)

NORMAL LIVER GUT

CCR9 + 47

VAP-1 MAdCAM-1

IP-10

INFLAMED PSC LIVER

CCL25

Summary

• Populations of memory lymphocytes arise as a consequence of bowel inflammation and these cells express homing receptors that direct their subsequent migration not only to the gut but also to the liver.

• Long-lived cells may re-circulate to the liver for many years and, in the absence of a local activating stimulus, will not cause damage.

Summary

• If lymphocytes are subsequently activated in the liver this leads to the development of inflammation and tissue damage which promotes the recruitment of more mucosal lymphocytes resulting in persistent inflammation and disease.

• Recent findings that MAdCAM-1 and CCL25, previously thought to be restricted to the gut, are up-regulated in the liver during inflammatory liver diseases that complicate IBD support the concept that common mechanisms control lymphocyte recruitment to the inflamed liver and gut

Gut Endothelium

MAdCAM-1 VCAM-1

Flow

CD3+

Th1Response

IL-2

IL-12

IL-18

TNF-

CD3+CD4/8+

Pro-inflammatory

IL-11 IL-10

Regulatory cytokines

Gut Endothelium

Flow

Leukocyte

CD3+

Th1Response

IL-2

IL-12

IL-18

TNF-TNF-

CD3+CD4/8+

Pro-inflammatory

IL-10IL-11

Regulatory cytokines

L2

ICAM-1 ICAM-2

“Abstracts from America”

PSC and Crohn’s disease

2 patients with colonic CD Rx with infliximab

GGT 486 232 GGT 446 193ALP 979 669 ALP 1377 408ALT 188 52 ALT 106 49AST 221 92 AST 154 51

Gut Endothelium

MAdCAM-1 VCAM-1

Flow

CD3+

Th1Response

IL-2

IL-12

IL-18

TNF-

CD3+CD4/8+

Pro-inflammatory

IL-11 IL-10

Regulatory cytokines

NatulizamabLDP-02MLN-02

Gut Endothelium

MAdCAM-1 VCAM-1

Flow

CD3+

Th1Response

IL-2

IL-12

IL-18

TNF-

CD3+CD4/8+

Pro-inflammatory

IL-11 IL-10

Regulatory cytokines

DaclizumabBasiliximab

J695

Numega IL-10Lactobacillus Lactis

Gut Endothelium

MAdCAM-1 VCAM-1

Flow

CD3+

Th1Response

IL-2

IL-12

IL-18

TNF-

CD3+CD4/8+

Pro-inflammatory

IL-11 IL-10

Regulatory cytokines

Visilizumab

“ This is not the end. This is not even the beginning of the end. But it is, perhaps, the end of the beginning”

Winston Churchill(1942)