involvement of cd44, “a molecule with thousand faces”, in autoimmune diseases nathalie...
TRANSCRIPT
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Involvement of CD44, a molecule with thousand faces, in autoimmune diseasesNathalie Assayag-Asherie, Itshak Golan, Lola Weiss, Lora Eshkar Sebban, Shimon Slavin, Itamar Raz and David Naor
Supported by: D CureDiabetes care in Israel andThe Israel Academy of SciencesIsrael Science Foundation
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The inflammatory process
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GENOMIC STRUCTURE OF THE CD44 MOLECULE
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CD44 LIGANDSECM 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
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In collaboration with Professor Yehiel Zick The Weizmann Institute
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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
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The inflammatory process
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Anti-CD44 mAbs reduce pathological activities in the following experimental diseases:
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Dr Lola Weiss
Department of Bone Marrow Transplantation and Cancer Research Laboratory, Hadassah University Hospital, Jerusalem, Israel
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Nathalie Assayag Asherie
The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem, Israel
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Anti-CD44 mAb reduces insulitis in NOD micehistopathological analysisPNAS,97:285,2000
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Shlomo Nedvetzki
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Day 0
Day 21
Type II
Collagen
Injection
Type II
Collagen
Injection
2
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10 days
Onset of
disease
Antibody injection
every second day
Generation of
Collagen
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Induced Arthritis (CIA) Model
Experimental Protocol
DBA
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1 Mice
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Joint Inflammation in Collagen-Induced Arthritis (CIA) Model
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In-Vivo Efficacy in CIA model
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The CD44-dependency of CIA
J.Autoimmunity,13:39,1999
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Multiple sclerosis
Diabetes
Ulcerative Colitis
DISEASE-SPECIFIC CD44
Atherosclerosis
Normal
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Dr Itshak Golan
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CD44vRA: exon v4 TTTCAACCACACCACGGGCTTTTGACCACACAAAACAGAACCAGGACTGGACCCAGTGG S T T P R A F D H T K Q N Q D W T Q WCD44v3-10: exon v4 TTTCAACCACACCACGGGCTTTTGACCACACAAAACAGAACCAGGACTGGACCCAGTGG S T T P R A F D H T K Q N Q D W T Q WCD44vRA: AACCCAAGCCATTCAAATCCGGAAGTGCTACTTCAGACAACCACAAGGATGACTGCA N P S H S N P E V L L Q T T T R M T A CD44v3-10: AACCCAAGCCATTCAAATCCGGAAGTGCTACTTCAGACAACCACAAGGATGACTGAT N P S H S N P E V L L Q T T T R M T DCD44vRA: exon v5 GATGTAGACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACAC D V D R N G T T A Y E G N W N P E A HCD44v3-10: exon v5 GTAGACAGAAATGGCACCACTGCTTATGAAGGAAACTGGAACCCAGAAGCACAC V D R N G T T A Y E G N W N P E A HCD44vRA: CCTCCCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCACATTCTACAAGCACAA P P L I H H E H H E E E E T P H S T S T CD44v3-10: CCTCCCCTCATTCACCATGAGCATCATGAGGAAGAAGAGACCCCACATTCTACAAGCACAA P P L I H H E H H E E E E T P H S T S T
Fig. 1
C.
B.
1 2 3 4 5 6 1 2 3 4 5 6 M N K C1 C2
1714
571
1111
Primershs5 / hs3
Primerspv3I / hs3
A.
Exon 1
Exon 20
hs 5
hs 3
pv 3I
C1 C2 C3 C4 C5 V2 V3 V4 V5 V6 V7 V8 V9 V10 C16 C17 C18 C20
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GENOMIC STRUCTURE OF THE CD44 MOLECULE
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Sequence of V4-V5 junction in CD44v3-v10 of Keratinocytes in normal donors VARIANT EXON 4AT GTA GACV DVARIANT EXON 5ATG ACT GM TDJ. Clin. Invest. 111:1211-1220 (2003)
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Sequence of V4-V5 junction in CD44vRA of synovial fluid cells in Rheumatoid Arthritis patients
VARIANT EXON 4
AT GTA GAC V D
VARIANT EXON 5
ATG ACT G M T A
CA GD
33/41 patients
alanine
J. Clin. Invest. 111:1211-1220 (2003)
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A.
B.
anti-pan-CD44
anti-CD44v6
anti-CD44v9
SYNOVIAL FLUID CELLS FROM PATIENT 117RA
HUMAN KERATINOCYTE DONOR M
CELL COUNTS
Fig. 4
MEAN FLUORESCENCE INTENSITY
HUMAN KERATINOCYTES (M)
HUMAN KERATINOCYTES (L)
SYNOVIALFLUID CELLS
II Ab
F8:334mg/ml
Selective binding of F8:33 anti-CD44 mAb to synovial fluid cells from RA patients
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SYNOVIAL FLUID CELLS FROM PATIENT 143 (RA)
MEAN FLUORESCENCE INTENSITY
CELL COUNTS
SYNOVIAL FLUID CELLS FROM PATIENT 139 (OA)
CELL COUNTS
F8:33100mg/ml50mg/ml20mg/ml
F8:332mg/ml200ng/ml20ng/ml
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Question
Is the binding of anti-CD44 mAb to cell surface CD44 delivers intracellular signaling leading to a functional outcome, such as apoptosis ?
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Anti-CD44vRA mAb +Anti-CD44vRA mAb +
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Anti-CD44vRA mAb Induces Selective Apoptosisin Synovial Fluid Cells of RA Patient in-vitro
0 1mg/ml 5mg/ml 10mg/ml 50mg/ml
SFCF8:33
PBLF8:33
SFCAnti-Pan-hCD44
PBLAnti-Pan-hCD44
97%
97%
95%
8%
90%
6%
92%
13%
86%
14%
77%
Propidium Iodide
Annexin V
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0
5
10
15
20
25
30
35
40
45
50
1
2
3
4
5
6
7
8
Antibody Concentration (
m
g/ml)
% of Apoptosis
SFC / anti-hCD44vRA (F8:33)
SFC / anti-pan-hCD44
PBL / anti-hCD44vRA (F8:33)
PBL / anti-pan-hCD44
0 0.1 0.5 1 5
10 50 100
Selective Induction of Apoptosis with Anti-CD44vRA mAb in Synovial Fluid Cells from RA patients
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Multiple sclerosis
Diabetes
Ulcerative Colitis
DISEASE-SPECIFIC CD44
Atherosclerosis
Normal