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Blood, 15 June 2002, Vol. 99, No. 12, pp. 4646-4647
CORRESPONDENCE
To the editor:
Immunocytochemistry reveals RANKL expression of myeloma cells
Two highly interesting papers related to osteoclastic activation
in multiple myeloma were published in the December 15 issue of
Blood.1,2 These studies were related to the
imbalance of 2 molecules involved in the regulation of bone resorption: receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin. Croucher and colleagues2 found that mouse myeloma
cells expressed RANKL mRNA and that RANKL protein could be demonstrated
on the cell membrane. Contrary to these findings, Giuliani and
colleagues1 could detect neither RANKL mRNA in
human myeloma cells nor RANKL protein using immunohistochemistry of
formalin-fixed, decalcified, and paraffin-embedded bone marrow
specimens in patients with multiple myeloma. Although these
discrepancies might let the reader assume that there are crucial
differences between the murine model used2 and human
myeloma disease, we wish to make the readers aware of our results,
which show a strong RANKL expression of plasma cells in patients with
multiple myeloma and osteolytic lesions. Pearse et
al3 demonstrated that myeloma stimulates
osteoclastogenesis by triggering an increase in RANKL and a decrease in
osteoprotegerin expression in stromal cells, but RANKL expression could
not be demonstrated in myeloma cells.3 On the other hand,
human myeloma cell lines have been shown to express RANKL by other
investigators.4 The immunohistochemical evaluation of
formalin-fixed, decalcified, and paraffin-embedded tissue sections may
be associated with some limitations, since it is known that
decalcification of formalin-fixed tissue samples reduces its
antigenicity.5 To avoid the potential problems associated
with this technique, we studied the RANKL expression in multiple
myeloma patients with osteolytic lesions by immunocytochemistry using
bone marrow smears and cytospins. Intracellular staining of bone marrow
cells was performed using the same monoclonal mouse antibody against
human RANKL as Giuliani and colleagues (clone 70525.11, R&D Systems,
Minneapolis, MN). The antibody was applied for 30 minutes in a 1:10 dilution
(antibody-diluent with background reducing components, Dako,
Carpinteria, CA) for detection of RANKL-expressing cells. Rabbit
antimouse (secondary antibody, Dako, Glostrup, Denmark) was applied for 30 minutes in a 1:50 dilution. Visualization was performed by the
alkaline phosphatase/anti-alkaline phosphatase double bridge technique
(APAAP; Dako). For intensification, the secondary antibody was used
again for another 10 minutes of incubation, followed by another APAAP
application for 10 minutes (Fuchsin + Substrate-Chromogen-System, Dako). Counterstaining was done with hematoxylin. Control stainings were performed the same way but without using the primary antibody or
using an irrelevant antibody (pancytokeratin). SaOs-2 cells, a human
osteosarcoma cell line, which is known to express RANKL, served as a
positive control. We could detect a strong cytoplasmatic expression of RANKL in bone
marrow plasma cells in all 6 multiple myeloma patients investigated
(Figure 1). Negative controls showed no
staining reaction, and SaOs-2 cells were strongly positive for RANKL.
Complementary to these results of cytoplasmatic RANKL expression, we
performed flow cytometry for the analysis of RANKL expression on the
cell surface using the same bone marrow aspirates. Plasma cells were identified as CD38 strongly positive (++) and CD138 (B-B4) positive (+)
cells as described previously.6 Immunofluorescence
staining of RANKL on the cell surface was performed using the same
monoclonal mouse antibody against human RANKL we used for
immunocytochemistry and its isotype control (mouse IgG2b), and
fluorochrome conjugated F(ab')2 fragment goat anti-mouse
IgG. Using flow cytometry, we could detect a strong expression of RANKL
on bone marrow plasma cells in all multiple myeloma patients
investigated (Figure 2). Furthermore, we
performed Western blot analysis in 6 human myeloma cell lines (IM-9,
NCI-H929, LP-1, OPM-2, RPMI 8226, U266) and found RANKL protein
expression in all of them.
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| Figure 1.
Immunocytochemistry of bone marrow myeloma cells and
controls.
(A) RANKL immunostaining shows strong positivity in myeloma cells
obtained by bone marrow aspirate from a patient with multiple myeloma
and osteolytic lesions. (B) Negative control staining using an
irrelevant antibody (pancytokeratin). (C) SaOs-2 cells, a human
osteosarcoma cell line, which is known to express RANKL, served as a
positive control.
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| Figure 2.
Flow cytometry of bone marrow myeloma cells.
In the same patient as in Figure 1, flow cytometry reveals RANKL
expression on the surface of bone marrow plasma cells. (A)
Identification of plasma cells according to their CD38++/ B-B4
expression. (B) RANKL expression (solid line) of the gated plasma cells
compared with isotype control (dotted line).
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These results extend our recent findings using flow cytometry alone in
10 previous patients,6 underline that RANKL is directly expressed by plasma cells in the bone marrow of multiple myeloma patients with osteolytic bone lesions, and suggest a direct role of
this ligand in the osteoclast activation in multiple myeloma.
Orhan Sezer, Ulrike Heider, Christian Jakob, Ivana Zavrski, Jan Eucker, Kurt Possinger, Christine Sers, and Veit Krenn
Correspondence: Orhan Sezer, Universitätsklinikum
Charité, Hematology and Oncology, Berlin, Germany; e-mail:
sezer{at}charite.de
References
1.
Giuliani N, Bataille R, Mancini C, Lazzaretti M, Barille S.
Myeloma cells induce imbalance in the osteoprotegerin/osteoprotegerin ligand system in the human bone marrow environment.
Blood.
2001;98:3527-3533[Medline]
[Order article via Infotrieve].
2.
Croucher PI, Shipman CM, Lippitt J, et al.
Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma.
Blood.
2001;98:3534-3540[Medline]
[Order article via Infotrieve].
3.
Pearse RN, Sordillo EM, Yaccoby S, et al.
Multiple myeloma disrupts the TRANCE/osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression.
Proc Natl Acad Sci U S A.
2001;98:11581-11586[Abstract/Free Full Text].
4.
Altamirano CV, Ma HJ, Parker KM, et al.
RANKL is expressed in malignant multiple myeloma cell lines [abstract].
Blood.
2000;96:365a.
5.
Shi SR, Tandon AK, Haussmann RR, Kalra KL, Taylor CR.
Immunohistochemical study of intermediate filament proteins on routinely processes, celloidin-embedded human temporal bone sections by using a new technique for antigen retrieval.
Acta Otolarygol.
1993;113:48-54[Medline]
[Order article via Infotrieve].
6.
Sezer O, Heider U, Zavrski I, Possinger K.
Differentiation of monoclonal gammopathy of undetermined significance and multiple myeloma using flow cytometric characteristics of plasma cells.
Haematologica.
2001;86:837-843[Abstract/Free Full Text].
7.
Sezer O, Heider U, Jakob C, Eucker J, Possinger K.
Human bone marrow myeloma cells express RANKL.
J Clin Oncol.
2002;20:353-354[Free Full Text].
Response:
OPGL/RANKL expression is not detectable in human myeloma cells
The data reported by Sezer et al show the expression
of receptor activator of NF-kappaB ligand (RANKL) or osteoprotegerin ligand (OPGL) in human myeloma cells by immunocytochemistry and flow
cytometry analysis. In contrast, we previously found no expression of
RANKL on myeloma cells checked at the protein level by Western blot and
immunohistochemistry and also at the RNA level by reverse transcription-polymerase chain reaction.1 Our
data, including positive controls and resulting from the analysis of 10 human myeloma cell lines, 26 samples of primary myeloma cells, and 15 bone marrow specimen from patients with or without osteolysis, are
consistent with those reported by Pearse et al2 but
contrast with the mouse model described by Croucher et al in which
RANKL expression has been observed in the mouse myeloma cell line
5T2MM.3 Furthermore, we have demonstrated by in vitro
experiments and bone marrow biopsies analyses that human myeloma cells
induced the overexpression of RANKL in the human bone marrow
environment especially in stromal cells, as observed by Pearse et al.
In conclusion, most of the published data indicate that myeloma bone
disease is associated with the increase of RANKL expression in bone
environment and that, at least in the human disease, the overexpression
of RANKL results from the up-regulation of RANKL expression in bone environment rather than the expression of RANKL by myeloma cells themselves.
Nicola Giuliani, Regis Bataille, Cristina Mancini, Mirca Lazzaretti, and Sophie Barille
Correspondence: Sophie Barille, Institut de Biologie,
9, INSERM U463, Nantes 44095 cedex 01, France; e-mail:
sbarille{at}nantes.inserm.fr
References
1.
Giuliani N, Bataille R, Mancini C, Lazzaretti M, Barille S.
Myeloma cells induce imbalance in the osteoprotegerin/osteoprotegerin ligand system in the human bone marrow environment.
Blood.
2001;98:3527-3533[Medline]
[Order article via Infotrieve].
2.
Pearse RN, Sordillo EM, Yaccoby S, et al.
Multiple myeloma disrupts the TRANCE/osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression.
Proc Natl Acad Sci U S A.
2001;98:11581-11586[Abstract/Free Full Text].
3.
Croucher PI, Shipman CM, Lippitt J, et al.
Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma.
Blood.
2001;98:3534-3540[Medline]
[Order article via Infotrieve].
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