Macrophage Inflammatory protein (MIP)-1
Osteolytic and Tumor-Promoting Effects in
Myeloma Bone Disease
Babatunde O . Oyajobi, M.D., Ph.D.
The University of Texas Health Science Center at San Antonio
San Antonio, TX
10th International Myeloma Workshop, Sydney 2005
Factors Implicated in Osteolysis
in Multiple Myeloma
· RANKL (RANK/OPG)
· DKK1
· MIP-1
· VEGF
· Lymphotoxin/TNF-
· HGF
· IL-1, IL-6, IL-3
· PTHrP
· M-CSF
· IL-17, IL-15
MIP-1 and Myeloma
· C-C chemokine - preferentially attract cells
of monocyte/macrophage lineage
· Stimulates formation of TRAP+ osteoclast-like
cells in vitro (Kukita et al. 1997 Lab Invest)
· MIP-1 overexpressed in marrow plasma of
myeloma patients (Choi et al. 2000 Blood)
· Produced by multiple myeloma cells
· Does MIP-1 stimulate osteoclast formation
and bone resorption in vivo?
· Does this require RANKL signaling via RANK?
· Experimental metastasis model
Stable Chinese hamster ovarian (CHO) cell
clones expressing human MIP-1
Intra-cardiac inoculation of cell transfectants
in athymic mice
· Direct injection of MIP-1
Subcutaneous tissue over calvariae
(normal Swiss ICR mice)
Stable Chinese Hamster Ovarian (CHO) Cell
Clone Expressing Human MIP-1
Human MIP-1
(pg/105 cells)
12500
(9993 ± 563)
10000
7500
5000
2500
0
CHOneo
CHO/MIP-1
Increased Osteolysis in CHO/MIP-1
Tumor-Bearing Mice
CHOneo
CHO/MIP-1
Hypercalcemia and Increased Osteolytic
Lesions Associated with CHO/MIP-1 Tumors
Whole blood
ionized calcium (mM)
Number of lytic lesions
Mice/group
1.4
*
10
*
9
8
6
1.3
4
6
4
1.2
2
1.1
0
PBS
neo
MIP-1
PBS
neo
MIP-1
CHO cells
CHO cells
Increased Osteoclast Activity Associated
with CHO/MIP-1 Tumors
TRAP staining
CHO/MIP-1
CHOneo
MIP-1 Stimulates Osteoclast Formation and
Bone Resorption In Vivo
Effect of MIP-1 on Osteoclastogenesis
In Vitro Requires RANKL
300
RAW 264.7 cells
*
*
250
*
# p <0.05 vs veh
200
TRAP+ Ocl
*p <0.05 vs
( 3 nuclei )
RANKL + M-CSF
150
#
100
50
0
RANKL (50ng/ml)
-
+
--
-
++
+
-
+
--
-
++
+
M-CSF (10ng/ml)
--
0.1
1
10
0.1
1
10
MIP-1 (ng/ml)
Effect of MIP-1 on Bone Resorption In Vivo is
Dependent on Intact RANK Signaling
Radl Model of Human Multiple Myeloma
5TGM1 myeloma cells
(MIP-1-secreting)
C57BL/KaLwRij mice
· Serum `M' protein (IgG2b) spike
· Osteolysis in recipient mice
· Splenomegaly ( 0.1g)
(Garrett et al. 1997 Bone; Dallas et al. 1999 Blood; Oyajobi et al. 2003 Blood)
Experimental Protocol
5TGM1 cells
sacrifice
Monoclonal rat anti-mouse MIP-1
Isotype rat IgG2a
(twice weekly 100µg i.p. injection)
Day 0
14
28
X-ray
IgG2b
X-ray
IgG2b
IgG2b
Histology
Anti-MIP-1 Antibodies Inhibit Development of
Osteolytic Lesions in Myeloma-bearing Mice
Number of osteolytic lesions
3.5
3
2.5
2
1.5
1
0.5
*
0
PBS + Ctrl IgG
anti-MIP-1
Decreased Osteoclast Activity in Myeloma-Bearing
Mice Treated with Anti-MIP-1 Antibodies
TRAP staining
* Tumor
*
Tumor
Tumor
marrow
Tumor
Ctrl IgG
anti-MIP-1
Neutralizing Anti-MIP-1 Antibodies Reduce
Tumor Burden in Myeloma-Bearing Mice
20
Line, median
Serum IgG2b 15
Box, 25-75%
(mg/ml)
*
Bar, 95% confidence interval
10
5
0 ctrl IgG + PBS anti-MIP-1
Anti-MIP-1 Antibodies Reduce Incidence of
Splenomegaly in Myeloma-Bearing Mice
20
r = 0.83
anti-MIP-1
15
Isotype IgG2a
(mg/ml)
+
PBS
10
IgG2b
Serum
5
0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Spleen wet weight (g)
RANK.Fc Inhibits Development of Osteolytic
Lesions in Myeloma-Bearing Mice
Lesion Number
Mean surface Area (mm2)
3.5
0.25
n 8
3
0.2
2.5
0.15
2
1.5
*
0.1
1
0.05
0.5
*
*
0
0
0
10
100
0
10
100
RANK.Fc (µg/day)
RANK.Fc Decreases Tumor Load in Bones of
Myeloma-Bearing Mice
Vehicle
RANK.Fc
tumor
tumor
tumor
Osteolysis in Myeloma Bone Disease
(vicious cycle)
MIP-1
(secreted)
RANK.Fc X
X Anti-MIP-1
mAb
CCR-1/5
RANKL
antagonists
(membrane-
bound)
(Abe et al. 2003 Blood; Seshasayee et al. 2004 J Biol Chem)
Acknowledgements
UTHSC at San Antonio
Picower Institute
· Paul Williams
· Barbara Sherry
· Donna Pulkrabek
· Giovanni Franchin
· Anjana Gupta
· Rami Käkönen
Amgen Washington
· Steve Munoz
· Dirk Anderson
· Greg Mundy
· Bill Dougall
International Myeloma Foundation
Multiple Myeloma Research Foundation
NIH/NCI