Transcript "a molecule with thousand faces,"in autoimmune diseases.

Involvement of CD44,
“a molecule with thousand faces”,
in autoimmune diseases
Nathalie Assayag-Asherie, Itshak Golan, Lola Weiss, Lora Eshkar
Sebban, Shimon Slavin, Itamar Raz and David Naor
Supported by:
D Cure
Diabetes care in Israel
and
The Israel Academy of Sciences
Israel Science Foundation
The inflammatory process
GENOMIC STRUCTURE OF THE CD44 MOLECULE
CD44 LIGANDS
ECM
CELL SURFACE
* Hyaluronic acid
* Mucosal vascular addressin
* Collagen
* Serglycin/gp600
* Fibronectin
* Osteopontin (OPN)
* Fibrinogen
* Class II invariant chain (li)
* Laminin
* E-selectin
* Chondroitin Sulfate
* L-selectin
Galectin-8
In collaboration with Professor Yehiel Zick
The Weizmann Institute
CD44 FUNCTIONS
* Cell-cell and cell-matrix interactions
* Support of cell migration
* Signal delivery for cell growth and cell differentiation
* Transmission of apoptotic signals
* Signal delivery for cell survival
* Signal delivery for production of cytokines and chemokines
* Presentation of chemokines and growth factors to cells with
corresponding receptors
* Docking of proteolytic enzymes on the cell surface and their
presentation to relevant substrates
* Internalization and degradation of hyaluronic acid
The inflammatory process
Anti-CD44 mAbs reduce pathological activities in the
following experimental diseases:
Name of disease
Target CD44
epitope
Reference
Malignant lymphoma in BALB/c
mice
Constant
M.A. Zahalka, E. Okon, U. Gosslar, B.
Holzmann, and D. Naor. J. Immunol. 154:
5345, 1995.
Diabetes in NOD mice
Constant
L. Weiss, S. Slavin, S. Reich, P. Cohen, S.
Shuster, R. Stern, E. Kaganovsky, E. Oken,
A.M. Rubinstein, and D. Naor. PNAS 97:285,
2000.
Experimental allergic
encephalomyelitis in (PLxSJL)
F1 mice
Constant
S. Brocke, C. Piercy, L. Steinman, I.L.
Weissman, and T. Veromaa. PNAS 96:6896,
1999.
Experimental colitis in BALB/c
mice
v7
B. Witting, C. Schwärzler, N. Föhr, U.
Günthert, and M. Zöller. J. Immunol.
161:1069, 1998.
Collagen-induced arthritis in
DBA/1 mice
Constant
K. Mikecz, F.R. Brennan, J.H. Kim, and T.T.
Glant. Nature Med. 1:558, 1995; S. Nedvetzki,
M. Walmsley, E. Alpert, R.O. Williams, and D.
Naor. J. Autoimmunity 13:39, 1999.
Dr Lola Weiss
Department of Bone Marrow Transplantation and Cancer Research
Laboratory, Hadassah University Hospital, Jerusalem, Israel
Nathalie Assayag Asherie
The Lautenberg Center for General and Tumor Immunology,
Hebrew University-Hadassah Medical School, Jerusalem, Israel
Anti-CD44 mAb reduces insulitis in
NOD mice
histopathological analysis
PNAS,97:285,2000
Shlomo Nedvetzki
Generation of
Collagen-Induced Arthritis (CIA) Model
Experimental Protocol
DBA-1 Mice
Day 0
Day 21
Antibody injection
every second day
2-10 days
Type II
Collagen
Injection
Type II
Collagen
Injection
Onset of
disease
The CD44-dependency of CIA
J.Autoimmunity,13:39,1999
DISEASE-SPECIFIC
CD44
Ulcerative
Colitis
Rheumatoid
arthritis
Cancer
Normal
Atherosclerosis
Diabetes
Multiple
sclerosis
Dr Itshak Golan
GENOMIC STRUCTURE OF THE CD44 MOLECULE
Sequence of V4-V5 junction in
CD44v3-v10 of Keratinocytes
in normal donors
VARIANT EXON 4
ATG ACT G
M
T
D
VARIANT EXON 5
AT
GTA GAC
V
D
J. Clin. Invest. 111:1211-1220 (2003)
Sequence of V4-V5 junction in CD44vRA
of synovial fluid cells in Rheumatoid
Arthritis patients
VARIANT EXON 4
VARIANT EXON 5
ATG ACT G CA
G AT
GTA GAC
M
D
V
T
A
alanine
33/41 patients
D
Selective binding of F8:33 anti-CD44 mAb
to synovial fluid cells from RA patients
B.
A.
SYNOVIAL FLUID CELLS FROM PATIENT 117RA
anti-pan-CD44
anti-CD44v6
anti-CD44v9
HUMAN KERATINOCYTES (L)
II Ab
CELL COUNTS
HUMAN KERATINOCYTES (M)
HUMAN KERATINOCYTE DONOR M
MEAN FLUORESCENCE INTENSITY
Fig. 4
SYNOVIAL
FLUID CELLS
F8:33
4g/ml
Binding of F8:33 Anti-CD44vRA
mAb to Synovial Fluid Cells of an RA
Patient but not to his own PBLs
Question
Is the binding of anti-CD44 mAb to cell surface
CD44 delivers intracellular signaling
leading to a functional outcome, such as apoptosis ?
Anti-CD44vRA mAb Induces Selective Apoptosis
in Synovial Fluid Cells of RA Patient in-vitro
77%
86%
90%
92%
SFC
F8:33
Anti-CD44vRA
mAb +
F8:33
Anti-CD44vRA
mAb +
14%
100
101
102
Annexin V
103
104
100
101
13%
102
Annexin V
103
100
104
101
6%
102
Annexin V
0
104 10
103
8%
101
102
Annexin V
103
104100
101
102
Annexin V
103
104
Propidium Iodide
PBL
97%
95%
100
101
102
Annexin V
103
104
100
0
SFC
101
102
Annexin V
103
102
Annexin V
103
104
100
1g/ml
101
102
Annexin V
103
104
100
5g/ml
101
102
Annexin V
103
40
1010
10g/ml
101
102
Annexin V
103
104
50g/ml
97%
Anti-PanhCD44
100
101
102
Annexin V
103
104 100
101
104
100
101
102
Annexin V
103
4 0
1010
101
102
Annexin V
103
104
100
101
102
Annexin V
103
104
PBL
Anti-PanhCD44
100
101
102
Annexin V
103
104
100
101
102
Annexin V
103
104
100
101
102
Annexin V
103
Annexin V
104
100
101
102
Annexin V
103
104 100
101
102
Annexin V
103
104
Selective Induction of Apoptosis with Anti-CD44vRA
mAb in Synovial Fluid Cells from RA patients
SFC / anti-hCD44vRA (F8:33)
50
SFC / anti-pan-hCD44
45
PBL / anti-hCD44vRA (F8:33)
PBL / anti-pan-hCD44
% of Apoptosis
40
35
30
25
20
15
10
5
0
0 1
0.1
2
0.5
3
14
Antibody Concentration
55
( g/ml)
10
6
507
100
8
DISEASE-SPECIFIC
CD44
Ulcerative
Colitis
Rheumatoid
arthritis
Cancer
Normal
Atherosclerosis
Diabetes
Multiple
sclerosis