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NEOPLASIA
From the Division of Endocrinology and Metabolism,
Department of Medicine, University of Texas Health Science Center at
San Antonio, San Antonio, TX; Department of Biochemistry, Osaka
University Faculty of Dentistry, Suita, Osaka, Japan
Myeloma is a unique hematologic malignancy that exclusively homes
in the bone marrow and induces massive osteoclastic bone destruction
presumably by producing cytokines that promote the differentiation of
the hematopoietic progenitors to osteoclasts (osteoclastogenesis). It
is recognized that neighboring bone marrow stromal cells influence the
expression of the malignant phenotype in myeloma cells. This study
examined the role of the interactions between myeloma cells and
neighboring stromal cells in the production of osteoclastogenic factors
to elucidate the mechanism underlying extensive osteoclastic bone
destruction. A murine myeloma cell line 5TGM1, which causes severe
osteolysis, expresses Multiple myeloma is unique among hematologic
malignancies in its capacity to cause massive osteoclastic bone
destruction, leading to serious skeletal complications such as
intractable bone pain, pathologic bone fracture, and
hypercalcemia.1,2 Histologic observations of osteolytic
lesions in patients with myeloma demonstrate the occurrence of
osteoclastic bone destruction adjacent to nests of myeloma
cells,3 suggesting that myeloma cells influence the
differentiation of hematopoietic progenitors to osteoclasts
(osteoclastogenesis) and subsequent activation of these osteoclasts to
destroy bone. However, the mechanism by which myeloma cells regulate
osteoclastogenesis and activate osteoclasts remains unclear.
A large body of evidence suggests that the support of bone marrow
stromal cells is indispensable for the differentiation, growth, and
homing of cells of the B-cell lineage in the bone marrow.4-7 Moreover, recent studies also suggest that the
vascular cell adhesion molecule-1 (VCAM-1) that is constitutively
expressed in bone marrow stromal cells8 plays an important
role in regulating the behavior of neighboring myeloma
cells,9-14 which constitutively express
In the present study, we investigated this hypothesis using a murine
myeloma cell line, 5TGM1, that causes severe osteolysis in syngeneic
mice.17 5TGM1 is a subclonal cell line we established from
5T33 multiple myeloma that was originally described by Radl and
colleagues18 and expresses
Cells
Antibodies
Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was prepared from 5TGM1, primary culture of bone marrow stromal cells, and ST2 marrow stromal cell line by the single-step RNA isolation method using TRIzol reagent (GIBCO). RNA (3 µg) was incubated with 50 ng of random hexamer at 70°C for 10 minutes and chilled on ice, then converted to first-strand complementary DNA (cDNA) using reverse transcriptase (Perkin-Elmer, Branchburg, NJ) according to the manufacturer's instruction. The primers used for PCR were as follows: murine VCAM-1 5'-primer, 5'-OH-GCTGCGCGTCACCATTGTTCTC-3'-OH; murine VCAM-1 3'-primer, 5'-OH-ACCACCCTCTTGAAGCCTTGTG-3'-OH; murine integrin 4 5'-primer, 5'-OH-CCCCTCAACACGAACAGATAGG-3'-OH;
murine integrin 4 3'-primer,
5'-OH-GCCTT- GTCCTTAGCAACACTGC-3'-OH; murine GAPDH 5'-primer,
5'-OH-TTGAAGGGTGGAGCCAAACG-3'-OH; murine GAPDH 3'-primer,
5'-OH-ACACATTGGGGGTAGGAACACG-3'-OH. PCR was performed for 30 cycles
consisting of 1 minute at 94°C, 1 minute at 55°C, and 2 minutes at
72°C. The PCR reaction mixture (total 50 µL) contained 10 µL
first-strand cDNA, 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 8.3), 2 mmol/L
MgCl2, deoxy-NTP mix (0.2 mmol/L each), the pair of primers
(0.15 µmol/L each), and 2 U Taq DNA polymerase (Perkin-Elmer). The
PCR products were separated on 2.5% agarose gels containing ethidium
bromide and visualized under UV light. The size of the fragments was
confirmed by reference to molecular weight markers. GAPDH served as
a control.
Flow cytometric analysis of VCAM-1 and
4-integrin antibody (1:1
dilution). Expression of the VCAM-1 or 4-integrin on
cell surface was determined using a flowcytometer (FACStar plus; Becton Dickinson, San Jose, CA).
Attachment of 5TGM1 myeloma cells onto ST2 mouse bone marrow stromal cells The ST2 cells were cultured in-MEM supplemented with 10%
FBS until confluency in 48-well culture plates (Coster, Cambridge, MA).
Growing 5TGM1 cells were labeled with 10 µCi
[methyl-3H] thymidine (New England Nuclear, Boston,
MA) for 24 hours. 3H-labeled 5TGM1 cells (20 000
cells, 8654 ± 244 cpm) were then incubated on the ST2 cell monolayer
in the absence or presence of antibodies to VCAM-1 or
4 1-integrin for 1 hour. Nonadherent cells
were removed by washing with 5% trichloroacetic acid twice and
phosphate-buffered saline (PBS) twice, and adherent cells were
solubilized in 300 µL of 0.25 mmol/L NaOH and neutralized with the
same volume of 0.25 mmol/L HCI; the radioactivity was determined in a
liquid scintillation counter.
Double-staining of the co-cultures for tartrate-resistant acid phosphatase (TRAP) and VCAM-1 expression Co-cultures were fixed with 3.7% formaldehyde and stained first for TRAP as described below. TRAP-stained cultures were next treated with 0.6% hydrogen peroxide for 15 minutes and then with 0.8% rabbit serum for 1 hour. Subsequently, the cultures were incubated with anti-VCMA-1 antibody (M/K-2.7, 1:10 dilution) at room temperature for 45 minutes, washed with PBS containing 1% rabbit serum (× 4), incubated with secondary antibody (rabbit antirat IgG, Vector Laboratories, Burlingame, CA) at room temperature for 45 minutes, washed with PBS containing 1% rabbit serum (× 4), and visualized using a commercial kit (Vectastatin Elite ABC kit, Vector Laboratories).Formation of TRAP-positive multinucleated osteoclasts and resorption pits in co-culture of 5TGM1 myeloma cells and primary mouse bone marrow cells Mouse bone marrow cells were obtained from 5-week-old male C57BL mice as described previously.23,24 Femurs and tibiae were dissected aseptically, cut off both ends. Bone marrow cells were flushed out, collected, and incubated in-MEM supplemented with 10%
FBS (Hyclone, Logan, UT) in 100-mm culture dishes (Becton Dickinson
Labware, Bedford, MA) for 2 hours and nonadherent cells containing
hemopoietic osteoclast precursors and stromal cells were harvested.
Bone marrow cells (1 × 106/well) and 5TGM1 cells (1000 cells/well) in 300 µL of the culture medium were plated onto 48-well
culture plates (day 1). On day 2, 300 µL of fresh culture medium was
gently added to each well, and on day 4, 300 µL of spent medium was
replaced with the same volume of fresh medium. On day 6, the cultures
were fixed and stained for TRAP using commercial kits (Sigma).
TRAP-positive multinucleated cells with more than 3 nuclei were defined
as osteoclasts and manually counted under the microscope. As a positive
control, a potent osteoclastogenic agent, 1,25-dihydroxyvitamin
D3 (1,25-D3, Biomol, Plymouth Meeting, PA), was
added to bone marrow cultures. The co-cultures were conducted in the
absence of 1,25-D3 unless indicated.
To confirm that these TRAP-positive multinucleated osteoclasts have the capacity to resorb bone, 5TGM1 cells and marrow cells were co-cultured on 5 × 5-mm whale dentine slices in the same condition, and resorption pits formed on these dentine slices were examined by scanning electron microscopy as described.25 In some experiments, co-cultures of 5TGM1 myeloma cells and marrow cells were performed using transwell inserts (24-well, Becton Dickinson Labware) to prevent the direct contact between these 2 types of cells. Conditioned medium The ST2 marrow cells (5 × 105/dish) and 5TGM1 myeloma cells (5 × 106/dish) were plated together onto 60-mm culture dishes (Becton Dickinson) in IMDM supplemented with 10% FBS and cultured overnight, washed with serum-free IMDM twice, and incubated in 5 mL of serum-free IMDM with 0.1% bovine serum albumin (BSA). After 48 hours, conditioned media were harvested, centrifuged to remove cell debris, and stored at 20°C until use. Conditioned
medium was also harvested from the co-cultures in which 5TGM1 myeloma
cells were cultured with monolayer of ST2 stromal cells that had been
fixed with 2.5% paraformaldehyde.
In some experiments, 5TGM1 mouse myeloma cells and IM-9, U266B1, and ARH-77 human myeloma cells (1 × 106/mL/24-well) were cultured for 24 hours in IMDM with 5% FBS in plates coated with or without 1µg/mL recombinant soluble VCAM-1 (rsVCAM-1) that lacks transmembrane and cytoplasmic domains (kindly provided by Dr Lobb, Biogen, Cambridge, MA). Conditioned media were then harvested and assessed for bone-resorbing activity in fetal rat long bone assay as described below and for the capacity to stimulate osteoclastogenesis in mouse marrow cultures. IMDM incubated without cells in the presence and absence of rsVCAM-1 served as controls. In other experiments, ST2 mouse bone marrow cells
(2 × 105/24 well) were cultured for 24 hours on a
monolayer of CHO cells that had been transfected with
Organ cultures of 45Ca-labeled fetal rat long bones Conditioned media harvested as described above were assayed for bone-resorbing activity by organ cultures of 45Ca-labeled fetal rat long bones as described previously.23 Pregnant rats were injected with 250 µCi of 45Ca (New England Nuclear) on the 18th day of gestation. Radius and ulna bone shafts were excised from 19-day-old fetuses under the dissecting microscope and precultured for 24 hours in Biggers-Gwatkin-Jackson medium (Sigma) supplemented with 0.1% BSA between air and liquid phase on stainless mesh grids. Bones were then cultured in the presence of conditioned media (50% v/v) or in control medium for 120 hours. The media were changed once at 48 hours of the culture. At the end of the culture, bones were harvested and treated in ice-cold 5% trichloroacetic acid for 2 hours; 45Ca radioactivity in bones and media was determined in a liquid scintillation counter. Bone resorption was expressed as the percentage of 45Ca released into the medium from bones as calculated by 45Ca count in medium/45Ca count in medium and bone × 100.Statistical analysis All data were presented as the mean ± SEM and analyzed by analysis of variance, followed by a paired t test.
Expression of VCAM-1 and 4-integrin and VCAM-1 in myeloma cells and bone marrow
stromal cells, respectively. The 5TGM1 myeloma cells expressed
4-integrin as reported previously,19 whereas ST2 stromal cells did not show 4-integrin
expression (Figure 1Ai). Both ST2 stromal
cells and primary bone marrow stromal cells expressed VCAM-1, whereas
5TGM1 did not (Figure 1Aiii).
Expression of VCAM-1 on ST2 cells (Figure 1Bi) and
In a separate experiment, we also examined the expression of
Attachment of 5TGM1 myeloma cells to ST2 cell monolayer in the
absence or presence of antibodies to VCAM-1 and
4-integrin play
a role in the attachment between ST2 stromal cells and 5TGM1 myeloma cells. The 5TGM1 cells grow in suspension. In contrast, almost 100%
5TGM1 cells adhered to ST2 cell monolayer (Figure
2). The anti-VCAM-1 antibody (10 µg/mL) significantly and
anti-41-integrin antibody (10 µg/mL)
more profoundly inhibited the attachment of 5TGM myeloma cells to ST2
monolayer (Figure 2), suggesting that the attachment of 5TGM1 myeloma
cells to ST2 stromal cells is mediated via 4-integrin
and VCAM-1. The results also demonstrate that VCAM-1 and
41-integrin expressed on these cells are
biologically functional and that these antibodies have neutralizing
activity. Increase in the concentration of these antibodies to 20 µg/mL did not show further inhibition of the attachment (data not
shown). Moreover, combined treatment with anti-VCAM-1 antibody (10 µg/mL) and anti-41-integrin antibody
(10 µg/mL) did not further inhibit the attachment of 5TGM1 myeloma
cells to ST2 monolayer (Figure 2).
TRAP-positive multinucleated osteoclast formation in the co-cultures of 5TGM1 myeloma cells and mouse bone marrow cells We next examined whether the cell-cell interaction between myeloma cells and stromal cells caused a generation of osteoclasts. To determine this, 5TGM1 myeloma cells were co-cultured with bone marrow cells that contain both stromal cells and hematopoietic osteoclast progenitor cells. In preliminary experiments, we found that the co-culture consisting of 1 million bone marrow cells and 1000 5TGM1 myeloma cells produced the greatest number of TRAP-positive multinucleated osteoclasts. Increase in number of 5TGM1 myeloma cells rather decreased osteoclast formation due to the overgrowth of 5TGM1 myeloma cells during 6-day culture.Bone marrow cells alone did not form TRAP-positive multinucleated
osteoclasts (Figure 3A). In contrast,
bone marrow cells cultured in the presence of 10 nmol/L
1,25-D3 formed numerous TRAP-positive multinucleated
osteoclasts after 6 days of culture, as previously
described.24,26 In the co-cultures of bone marrow cells
and 5TGM1 myeloma cells, many TRAP-positive multinucleated osteoclasts
formed (Figure 3A,Bi). These TRAP-positive multinucleated cells
exhibited resorption pit formation on dentine slices (Figure 3Bii),
demonstrating that these cells were capable of resorbing bone and
possessed the osteoclast phenotype. When these TRAP-stained co-cultures
were subsequently immunostained with anti-VCAM-1 antibody, we observed
fibroblast-like cells surrounding TRAP-positive multinucleated osteoclasts were positive for VCAM-1 (Figure 3Biii), suggesting that
these cells are stromal cells. Nonmyeloma RAW8.1 B-cell lymphoma cells
co-cultured with bone marrow cells did not cause TRAP-positive multinucleated osteoclasts formation (Figure 3A).
To confirm that a direct contact between 5TGM1 myeloma cells and bone marrow stromal cells is necessary for TRAP-positive multinucleated osteoclast formation, we carried out co-culture experiments using trans-well inserts in which 5TGM1 cells were separated from bone marrow cells by a membrane. In this co-culture condition, there were very few osteoclasts formed (data not shown). The result indicates that the direct contact is essential. Effect of antibodies to VCAM-1 and
4-integrin in osteoclast formation in the co-cultures
using the neutralizing antibody to VCAM-1 or
41-integrin. Both anti-VCAM-1 antibody (VCAM-1 Ab, 10 µg/mL) and
anti-41-integrin antibody
(41 Ab, 10 µg/mL) dramatically
inhibited TRAP-positive multinucleated osteoclast formation (Figure
4). On the other hand, antibody against ICAM-1 (ICAM-1 Ab, 10 µg/mL) and control IgG had no effect on the
osteoclast formation (Figure 4).
To determine whether this inhibition by VCAM-1 Ab and
Effect of conditioned medium harvested from the co-cultures of 5TGM1 myeloma cells and ST2 stromal cells on bone resorption The results obtained in these co-culture experiments suggested to us that the direct contact between 5TGM1 myeloma cells and bone marrow stromal cells mediated via41-integrin
and VCAM-1 produces a soluble factor that stimulates osteoclast
formation and function. Accordingly, we tested the effects of
conditioned medium of the co-cultures of 5TGM1 myeloma cells and ST2
stromal cells on osteoclastic bone resorption. The conditioned medium at 40% (v/v) from the co-cultures showed increased bone-resorbing activity in fetal rat long bone assays (Figure
5). The conditioned medium at 20% also
exhibited significant bone-resorbing activity (percent increase over
control = 34 ± 3) but there was no effect at 10%. The 5TGM1
myeloma cells co-cultured with ST2 stromal cells fixed with 2.5%
paraformaldehyde also produced bone-resorbing activity. Conditioned
medium from 5TGM1 myeloma cells alone showed marginal bone-resorbing
activity. Co-culture of ST2 cells and nonmyeloma RAW8.1 B-cell lymphoma
cells did not produce bone-resorbing activity. Conditioned medium from
ST2 cells alone showed no bone-resorbing activity.
Production of osteoclastogenic and bone-resorbing activity in 5TGM1 myeloma cells in contact with rsVCAM-1 From the results described above, it seemed likely that 5TGM-1 cells were the producer of the bone-resorbing activity. To clarify this, 5TGM-1 myeloma cells were cultured in rsVCAM-1-coated plates in the absence of stromal cells. Of note, 5TGM1 myeloma cells became tightly attached to rsVCAM-1-coated plates. Conditioned medium was harvested from these cultures and assayed for osteoclastogenic activity using mouse bone marrow cultures. The conditioned medium showed strong osteoclastogenic activity (Figure 6A) and bone-resorbing activity in fetal rat long bone assay (Figure 6B). On the other hand, conditioned medium of 5TGM1 cells cultured on noncoated plates exhibited no activity of osteoclast formation and bone resorption. The rsVCAM-1 itself had no effects on bone resorption (data not shown).
The 5TGM1 myeloma cells did not attach to rsVCAM-1-coated plates in
the presence of anti-VCAM-1 Ab (10µg/mL) or
anti- Of note, several human myeloma cells also showed the production of osteoclastogenic activity when they were cultured in rsVCAM-1-coated plates (Figure 6A). Among these cells, ARH-77 cells have been shown to induce myeloma bone disease.20 Production of osteoclastogenic activity in ST2 stromal cells
in contact with 41-integrin expressed on 5TGM1 myeloma
cells in the co-cultures, ST2 cells were cultured on monolayer of fixed
CHO cells expressing 4-integrin. We observed that
attachment of ST2 cells to fixed CHO cells expressing
4-integrin was increased compared with fixed CHO cells
with empty vector or noncoated plates (data not shown). However, none
of conditioned media harvested from these cultures showed
osteoclastogenic activity in the bone marrow culture assay (Figure
7). These data strongly suggest that
5TGM1 myeloma cells are responsible for the production of
osteoclastogenic and bone-resorbing activity in the co-cultures.
Effect of neutralizing antibodies to known osteoclastogenic cytokines on bone-resorbing activity producer by 5TGM1 cells In an attempt to identify 5TGM1-derived cytokine responsible for osteoclastogenic activity and bone resorption, conditioned media from the co-cultures were treated with a saturating concentration of neutralizing antibodies to several known bone resorption-stimulating cytokines including IL-1, IL-1, IL-6, tumor necrosis factor- (TNF), tumor necrosis factor- (TNF), and PTH-rP.
Bone-resorbing activity of the antibody-treated conditioned media were
then determined in the fetal rat long bone assay. In a preliminary
experiment using the fetal rat long bone assay, 10 7 mol/L
IL-1 and IL-1, 500 ng/mL IL-6 together with 500 ng/mL soluble
IL-6 receptor, 107 mol/L TNF and TNF, and 25 ng/mL
PTH-rP markedly stimulated bone resorption. We confirmed that each of
the antibodies at a concentration used here blocked the bone-resorbing
activity of each corresponding cytokine in this assay (data not shown).
However, none of these antibodies blocked bone-resorbing activity
present in the conditioned media (Figure
8).
Progressive osteoclastic bone destruction is one of the most detrimental complications of multiple myeloma. Earlier studies have suggested that production of osteoclast-activating cytokines by myeloma cells is responsible for the aberrant increase in osteoclastic bone destruction in patients.2,3 Here, we studied the murine myeloma cell line, 5TGM1, which causes typical myeloma bone diseases with extensive osteoclastic osteolysis in syngeneic mice,17 for its capacity to produce bone-resorbing cytokines to understand the mechanism responsible for myeloma-induced bone disease. Like many other human and murine myeloma cells,2 conditioned medium from 5TGM1 cultures showed marginal bone-resorbing activity. In contrast, our data showed that 5TGM1 cells in direct contact with the ST2 bone marrow stromal cells or primary mouse bone marrow stromal cells in the co-cultures produced increased osteoclastogenic activity. Prevention of contact between 5TGM1 and stromal cells in trans-well cultures resulted in a marked decrease in the production of this activity. More importantly, the results that 5TGM1 cells cultured in rsVCAM-1-coated plates produced osteoclast-stimulating activity in the absence of bone marrow stromal cells indicate that 5TGM1 myeloma cells are responsible for producing this activity in the co-cultures. These data demonstrate that the cell-cell contact with bone marrow stromal cells is essential for 5TGM1 myeloma cells to cause osteoclast stimulation. It is, therefore, suggested that the absence of stromal cells, a critical cellular component of bone marrow, may explain the failure of myeloma cells that are freshly isolated from patients with extensive osteolytic skeletal lesions or currently available human and murine myeloma cell lines to consistently produce discernible osteoclast-activating cytokines in culture. Figure 6A in which several human myeloma cell lines show the production of osteoclastogenic activity only in the presence of rsVCAM-1 further supports this notion. We next examined which CAMs were involved in the direct cell-cell
interactions between 5TGM1 cells and marrow stromal cells that are
necessary for the production of osteoclastogenic cytokines. Our
experiments using neutralizing antibodies to VCAM-1 and
However, it should be noted that solid tumors may behave differently
from myeloma in this regard. For example, others have shown that stable
transfection of Myeloma cells express not only
Different osteoclast-activating cytokines such as IL-1,32
IL-6,33,34 TNF- In conclusion, our data suggest that the direct cell-cell contact
between myeloma cells and marrow stromal cells via
The authors are grateful to Dr David Roodman for valuable comments
and discussion, Dr Roy Lobb for helpful discussion and providing the
recombinant soluble VCAM-1, and Dr Kensaku Miyake for supplying the
antibodies to VCAM-1, ICAM-1, and
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4
1-integrin enhances
production of osteoclast-stimulating activity
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Abstract
Introduction
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