Haematologica 2003 - Supplements

155
Evidence for a Role for Bone Marrow Endothelial Cells
in the Development of Myeloma Bone Disease
Evy De Leenheer (1,2)Karin Vanderkerken (2)Gabrielle
Mueller (1)Claire Shipman (1)Marleen Bakkus (3)Ben Van
Camp (2)Peter Croucher (1)
(1) Nuffield Department of Orthopaedic Surgery, University of
Oxford, Oxford, United Kingdom(2) Hematology and Immunology,
Free University Brussels, Brussels, Belgium(3) Molecular
Haematology Laboratory, Academic Hospital of the Free University
Brussels, Brussels, Belgium
The growth of myeloma cells in the local bone marrow
environment is associated with the development of osteolytic
bone disease; however, the mechanisms involved in the
pathogenesis of this bone disease are poorly understood.
Recently, the ligand for receptor activator of NF-kB
(RANKL)/osteoprotegerin (OPG) system has been reported to
play an important role in the development of normal osteoclasts
and this system may be abnormally regulated in patients with
myeloma. Different cell types are present in the bone marrow
microenvironment and may express components of the
RANKL/OPG system. Endothelial cells are one such cell type
and these cells can be found in close association with bone,
making them ideally placed to influence bone turnover.
Furthermore, myeloma cells promote angiogenesis, suggesting
that local proliferation of endothelial cells may play an important
role in the development of multiple myeloma. Therefore, the aim
of the present study was to determine whether murine bone
marrow endothelial cells express RANKL and OPG and whether
myeloma cells can regulate expression of these molecules.
RT-PCR and flow cytometric analysis demonstrated the presence
of OPG mRNA in the murine bone marrow endothelial cells
STR10 and STR12. ELISA confirmed that these endothelial cells
release OPG. In contrast, OPG could not be detected in the
supernatant of cultures of LE1SVO lung endothelial cells. The
5T33MMvt cells, an in vitro growing, but clonally identical
variant of 5T33MMvivo cells were shown to express OPG
mRNA but OPG protein was not detected in the culture
supernatant. RANKL was also shown to be expressed by STR10
and STR12 cells.
Media conditioned by STR10 and STR12 cells and containing
OPG was able to inhibit the ability of tartrate resistant acid
phosphatase positive osteoclasts to resorb a mineralised substrate
in vitro.
The addition of 5T33MMvt cells to either STR10 or STR12 cells
resulted in a cell number-dependent decrease in OPG production,
as determined by ELISA. Quantitative RT-PCR confirmed that
expression of the mRNA for OPG was decreased in STR10 and
STR12 cells. When myeloma cells and endothelial cells were
physically separated a decrease in OPG was still observed. To
establish whether a soluble factor was responsible for the
decrease in OPG, medium conditioned by myeloma cells was
added to the endothelial cells. Surprisingly, no decrease in OPG
production was seen, suggesting that close contact but not
necessarily physical contact between myeloma cells and
endothelial cells is required to down-regulate OPG production.
In conclusion, these data demonstrate that bone marrow
endothelial cells express RANKL and produce OPG and that
production of OPG by these cells may be able to decrease bone
resorption in vitro. Myeloma cells decrease OPG production in
endothelial cells raising the possibility that this could contribute
to the development of myeloma bone disease.
156
Osteoprotegerin and sRANKL serum levels in multiple
myeloma patients
Maria Kraj, Piotr Centkowski, Barbara Kruk.
Institute of Haematology and Blood Transfusion, Warsaw, Poland
Receptor activator of NF-κB (RANK) is a TNF receptor
superfamily member expressed on the surface of osteoclasts and
their precursors that mediates their differentiation, survival, and
activation upon interaction with its ligand, RANKL, expressed by
osteoblasts, and stromal cells. RANKL is produced as a
membrane bound protein and cleaved into a soluble form by a
metalloprotease. The primary secreted form is produced by
activated T-lymphocytes. Osteoprotegerin (OPG) is a secreted
TNFR, it acts as a decoy receptor for RANKL and inhibitor of
RANK-RANKL interaction. Recent studies suggest that MM
triggers osteoclastogenessis by disrupting the balance between
RANKL and its natural inhibitor, OPG. Therefore in this study
we analyzed the concentrations of serum OPG and sRANKL in
MM patients at diagnosis. Determinations of serum OPG and
sRANKL concentrations were performed in 25 healthy subjects
and 94 MM patients (15 at stage I, 12-II, 55-IIIA, 12-IIIB acc. to.
D.S; 67 had lytic lesions at skeletal X-ray survey and 10 had
hypercalcemia; monoclonal protein IgG was in 61 patients, IgA-
22, IgD-1, Bence Jones-8, non secretory-2) by means of ELISA
method using Osteoprotegerin ELISA and sRANKL ELISA kits
(Biomedica GmbH, Wien, Austria). In the whole group of MM
patients, OPG concentrations in particular patients ranged from
40 to 371 pg/ml with a mean concentration of 111±62, median 94
pg/ml while in healthy persons OPG levels ranged from 49 to 130
pg/ml, mean 77±22, median 74 pg/ml (p=0,002). In MM patients
with renal failure mean level of OPG was 172±87, median 145
pg/ml while in MM patients with normal renal function they were
101±51 and 85 pg/ml respectively (p=0,0002). In MM patients
with hypercalcemia mean level of OPG was 169±98, median 143
pg/ml while in MM patients with normal serum calcium level
they were 104±54 and 88 pg/ml respectively (p=0,01). The
differences in OPG concentrations depending on occurrence of
osteolysis were not statistically significant: in patients with
osteolysis 116±64 median 99 pg/ml and in those without it
99±57, median 84 pg/ml (p=0,1). In MM patients serum
concentrations of OPG increased significantly with age (r=0,42;
p=0,00002). There was a positive correlation with OPG and β2M
serum concentrations (r=0,32; p=0,002). In MM patients
sRANKL concentrations in particular patients ranged from 0 to
63 pg/ml with a mean concentration of 2,77±7,7 pg/ml, while in
healthy persons sRANKL levels ranged from 0 to 43 pg/ml,
mean 5,0±10,1 pg/ml.
Conclusions: In MM patients at diagnosis serum OPG levels are
not reduced; in contrast in 20% of patients are elevated and it
may be a compensative reaction in relation to increased bone
destruction. Significantly increased OPG concentrations in MM
patients with renal failure may be related to its decreased
elimination. In the majority of MM patients serum sRANKL is
undetectable.
S157