Differentiation of Early Plasma Cells on Bone Marrow Stromal Cells
Requires Interleukin-6 for Escaping From Apoptosis
By Michio M. Kawano, Keiichiro Mihara, Naihui Huang, Takako Tsujimoto, and Atsushi Kuramoto
The bone marrow (BM) is well known to be the major site
of Ig production in secondary immune responses; thus, the
microenvironment of BM is considered to be essential for
final differentiation of plasma cells. We identified in the pe-
ripheral blood (PB) early plasma cells (CD38++CD19'VLA-5-)
committed to entering the BM. The sorted early plasma cells
rapidly entered apoptosis in vitro, but these cells could sur-
vive and further differentiate into mature plasma cells
(CD38+++CD19+) just as BM plasma cells in the presence of
a BM-derived stromal cell line (KM-102). Culture superna-
tants of KM-l02 cell lines could also support survival of these
LASMA CELLS ARE finally differentiated cells of B-
cell lineage. More than half amounts of serum IgG are
reported to be produced by plasma cells in the bone marrow
(BM)."3 Thus, most plasma cells are considered to finally
differentiate in the BM; microenvironments of BM support
differentiation of plasma cells. However, the exact pathway
and regulation of differentiation of plasma cells have not
been clarified in detail. We recently found that phenotypes
of plasma cells in the BM could be clearly analyzed by two-
color staining with fluorescein isothiocyanate (F1TC)-anti-
CD38 an t ib~dy .~ The cells located at CD38-highest positive
(CD38+++) fraction were all plasma cells, and there were no
plasma cells at CD38- or CD38+ fractions. By using this
two-color analysis with anti-CD38 antibody, we first clari-
fied heterogeneity of expression of adhesion molecules on
human myeloma cells.536 BM myeloma cells (malignant
plasma cells) consisted of CD3tFTLA-5- immature and
CD38+++VLA-5+ mature myeloma cells. Immature my-
eloma cells proliferated markedly in vitro and responded
to interleukin-6 (IL-6), a growth factor for myeloma cells:
whereas mature myeloma cells showed very low prolifera-
tive activities and no responses to IL-6, but attached to BM
stromal cells more tightly than immature myeloma cells at
least through the VLA-5 molecule.536 These findings are also
the case for normal plasma cells in the BM; immature plasma
cells (CD38+++VLA-5-) and mature plasma cells
(CD38+++VLA-5+) are both detected, although most of the
BM plasma cells are mature cells in healthy donors and
patients with polyclonal gamm~pathy.~ According to these
findings, we postulated that the BM plasma cells might be
derived from the early stage compartments, so-called early
plasma cells, outside the BM, because the secondary immune
responses could not occur in the BM. Hence, we detected
early plasma cells in the peripheral blood (PB) and examined
whether these early plasma cells could differentiate into ma-
ture plasma cells just like BM plasma cells in vitro. In addi-
tion, we discussed the significant role of BM stromal cells
on the final differentiation of plasma cells in the BM.
P
MATERIALS AND METHODS
Cell separation. PB or BM mononuclear cells were isolated by
Ficoll-Hypaque centrifugation from healthy donors or patients with
polyclonal gammopathy such as septicemia and collagen disease.
Informed consent was obtained before BM aspiration procedure in all
Blood, Vol 85, No 2 (January 15). 1995: pp 487-494
cells, and antibody to interleukin-6 (IL-6) completely blocked
the effect of these supernatants. Furthermore, recombinant
11-6, but not 11-1 or IL-3, could support their survival and their
differentiation into mature plasma cells (CD38+++CD19+VLA-
5+) with expression of V U - 5 mRNA. Therefore, here is direct
evidence that early plasma cells found in the PB differenti-
ated into mature plasma cells with stromal cell-derived IL-
6 in vitro; thus, BM stromal cells control the final checkpoint
of plasma cell differentiation with secretion of IL-6 in the
BM.
0 1995 by The American Society of Hematology.
cases. The cells were washed and suspended in RPMI-1640 medium
(Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum
(FCS; M.A. Bioproducts, Walkersville, MD).
Two-color j-70~ cytometry. The mononuclear cells (PB or BM;
5 X lo5 cells) were stained at 4°C for 30 minutes with monoclonal
anti-CD19 (J4.119) or anti-VLA-5 (SAM1) antibody (Immunotech
S.A., Marseille, France). After washing with phosphate-buffered sa-
line (PBS; pH 7.2) and bovine serum albumin (BSA; 200 pg/mL), the
cells were incubated with phycoerythrin (PE)-labeled goat antimouse
IgG (Immunotech S.A. or Tago, Inc, Burlingame, CA) at 4°C for
30 minutes. The cells were then washed and subsequently incubated
with 50 pg of normal mouse IgG to block nonspecific binding at
4°C for 20 minutes. The cells were then incubated with FITC-labeled
anti-CD38(T16) antibody (Immunotech S.A.) at 4°C for 30 minutes.
Immunofluorescein of the membrane was measured by a cell sorter
(Epics Elite: Coulter, Hialeah, FL). Two-color cytograms using flu-
orescence contour plots of the expression of CD38 antigen (x axis,
log scale) and CD19 or VLA-5 (y axis, log scale) are presented.
Cell sorting. BM or PB mononuclear cells were stained in the
sterile condition with FITC-anti-CD38 and PE-anti-CD19 (Immuno-
tech S A . ) as described above. The cells (l X 10') were applied to
a cell sorter (Epics Elite).
In vitro culture of sorted early plasma cells. The sorted cells (1
X IO4), as described above, were cultured in 6-well microtiter plates
(no. 25810; Coming, Coming, NY) with RPMI-1640 medium sup-
plemented with 10% FCS and 1 X m o m 2-mercaptoethanol
(2-ME) for 1 to 3 days at 37°C in a 5% CO2 humidified atmosphere.
One or 2 days before the experiments were performed, a BM-
derived stromal cell line, KM-l02 (kindly provided from Dr K.
Harigaya, Chiba University, Chiba, Japan),' was plated on 6-well
microtiter plates at a density of approximately 1 X IO5 cells per
From the Department of Hematology and Oncology, Research
Institute for Radiation Biology and Medicine, Hiroshima University,
Hiroshima, Japan.
Submitted April 22, 1994; accepted September 26, 1994.
Supported in part by grants from the Ministry of Education Sci-
ence and Culture, and the Ministry of Health and Welfare, Japan.
Address reprint requests to Michio M. Kawano, MD, Myeloma
Study Group, Department of Hematology and Oncology, Research
Institute for Radiation Biology and Medicine, Hiroshima Universiq,
1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8502-0022$3.00/0
487
only.
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