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)