The extra-intestinal manifestations of IBD

Dr Allister Grant Consultant Hepatologist University Hospitals Leicester

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

Margination of Lymphocytes Blood Blood Flow 1. Rolling on Selectins Endothelium Tissue

Endothelial Cell E-selectin P-selectin PNAd mucin L-selectin CLA mucin PSGL-1 mucin LYMPHOCYTE

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

a

-sub family C

b

-sub family C CXC CC C IL-8 ENA-78 GRO C MCP-1 MIP-1

a

MIP-1

b

RANTES IP-10 HuMIG ITAC

Proteoglycans in the endothelial glycocalyx retain and present chemokines to lymphocytes Lymphocyte Integrin activation Cytoskeletal reorganisation Migration Endothelium

3. Firm Adhesion

Endothelial Cell ICAM-1 VCAM-1

a

Inactive integrin

b

LYMPHOCYTE Active LFA-1 Active

a

4ß1

Integrins Addressins Activation signal

Arrest of Lymphocytes on Integrins

Ctyoskeletal Rearrangement

(Chemokine Mediated) 4. Transmigration

Adhesion cascade Blood Flow Lymphocyte Endothelium

Tethering Rolling

Selectins Integrins Carbohydrate ligands Addressins

Arrest & Activation

Integrins & Chemokine receptors Addressins & Chemokines

Transmigration

Chemokine receptors Chemokines

Thymocytes TISSUE

DC

THYMUS Naïve T cells BLOOD LYMPH NODE

DC

BLOOD Memory lymphocytes Effector T cells

TISSUE SPECIFIC HOMING OF MEMORY EFFECTOR T LYMPHOCYTES

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

BLOOD Lymph Node BLOOD TISSUE Memory T cell Skin Naïve T cell Memory T cell Gut

Tissue specific T cell recruitment Naïve T cell

CCR7 + L-selectin

Memory/effector T cell

CCR4 + CLA CCR9 + a 4 b 7

PNaD CCL21(SLC) LYMPH NODE E-selectin CCL17(TARC) SKIN MAdCAM-1 CCL25(TECK) GUT

Blood Flow Endothelium

Tethering & Rolling Activation

Lymphocyte

Arrest

CCR5 VAP-1 receptor

?

CCR5 ligands glycocalyx VAP-1 Liver VAP-1 receptor

?

VAP-1

EXPRESSION OF VAP-1 IN HUMAN ENDOTHELIUM

Salmi et al. J. Ex. Med.1993; McNab et al Gastro 1996

LIVER vascular sinusoidal LYMPH NODE GUT KIDNEY SKIN SYNOVIUM BRAIN HEART NORMAL ++ +++ +++ _ + _ _ _ + INFLAMED + +++ +++ +++ + ++ ++ _ +

MAdCAM-1 Expression in PSC Liver

Grant et al Hepatology 2001, Hillan et al Liver 1999

Positive Staining of Portal Vascular Endothelium

ACT-1(a4b7) STAINING OF PSC LIVER

Immunofluorescent Staining of a Portal Vessel in PSC MAdCAM-1

(red) a4b7 (green)

Adhesion Assay Under Conditions of Shear Stress

Media reservoir 37  C Incubator Cell sample Microslide Syringe pump Microscope Video/computer analysis system 2-way electronic valve www.expertreviews.org

Lymphocyte rolling

Lymphocyte Transmigration

Adhesion Assays Under Conditions of Shear Stress

100 90 80 70 60 50 40 30 20 10 0 Control MAdCAM-1 a4b7 Target of Blocking Monoclonal Antibody CD62L

Tissue specific T cell recruitment

CXCR3 + VAP-1r CCR9 + a 4 b 7

VAP-1 IP-10 NORMAL LIVER VAP-1 MAdCAM-1 IP-10 ?

INFLAMED PSC LIVER MAdCAM-1 CCL25(TECK) GUT

A C B D

P 1 P 2 P 3 P 4 P 5 P 6 NL 1 NL 2 NL 3 PB 1 PB 2 ALD 1 ALD 2 P 7

CCL25

b

-actin Western Immunoblotting for CCL25.

P = PSC NL = Normal PB = PBC ALD = Alcoholic Liver Disease

Tissue specific T cell recruitment

CXCR3 + VAP-1r CCR9 + a 4 b 7

VAP-1 IP-10 NORMAL LIVER VAP-1 MAdCAM-1 IP-10 CCL25 INFLAMED PSC LIVER MAdCAM-1 CCL25(TECK) GUT

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

CD3+ Flow

a4b7 a4b1

Gut Endothelium MAdCAM-1 VCAM-1 IL-2 IL-12 Th1 Response IL-18 TNF-

a

Pro-inflammatory CD3 + CD4/8 IL-11 Regulatory cytokines IL-10 +

CD3+

a L b 2

Flow Leukocyte Gut Endothelium ICAM-1 ICAM-2 IL-2 IL-12 Th1 Response IL-18 TNF-

a

Pro-inflammatory CD3 + CD4/8 IL-11 Regulatory cytokines IL-10 +

“Abstracts from America”

PSC and Crohn’s disease 2 patients with colonic CD Rx with infliximab GGT 486 ALP 979 ALT 188 AST 221 232 669 52 92 GGT 446 ALP 1377 ALT 106 AST 154 193 408 49 51

CD3+ Flow

a4b7 a4b1

Gut Endothelium MAdCAM-1 VCAM-1 IL-2 IL-12 Th1 Response IL-18 TNF-

a

Pro-inflammatory CD3 + CD4/8 IL-11 Regulatory cytokines IL-10 + Natulizamab LDP-02 MLN-02

CD3+ Flow

a4b7 a4b1

Gut Endothelium Daclizumab Basiliximab IL-2 MAdCAM-1 VCAM-1 J695 IL-12 Th1 Response IL-18 TNF-

a

Pro-inflammatory CD3 + CD4/8 + IL-11 Numega Regulatory cytokines IL-10 IL-10

Lactobacillus Lactis

CD3+ Flow

a4b7 a4b1

Gut Endothelium MAdCAM-1 VCAM-1 IL-2 IL-12 Th1 Response IL-18 TNF-

a

Pro-inflammatory CD3 + CD4/8 + Visilizumab IL-11 Regulatory cytokines IL-10

“ 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)