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,¶,
,§
*
Cancer Center and Departments of
Microbiology and Immunology,
Pediatrics, and
§
Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642; and
¶
Eastman Dental Center, Rochester, NY 14620
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) cells simultaneously display macrophage characteristics, such
as phagocytosis and F4/80 expression, while retaining B cell features,
including expression of surface Ig, CD5, B220, and rearranged Ig genes.
The present study investigated the fibroblast-secreted factor that
promotes this phenotypic change from B cell to B/M cell. RT-PCR
analysis demonstrated that mRNA for M-CSF is produced by splenic
fibroblasts. Recombinant M-CSF (CSF-1) could replace
fibroblast-conditioned medium to elicit the development and survival of
B/M cells from splenic B lymphocytes. In addition, neutralization of
fibroblast-secreted M-CSF with specific mAbs abrogated the ability of
conditioned supernatants to promote outgrowth of B/M cells. The
transition from B lymphocyte to B/M cell was marked by the kinetic
appearance of mRNA for the M-CSF receptor, c-fms, at day
3 following culture initiation. These results demonstrate that M-CSF is
important in the development and physiology of mouse B/M cells and
potentially in the growth of human biphenotypic hematological
malignancies. Interestingly, the presence of IFN-
in splenic B
lymphocyte cultures abrogated the effect of fibroblast-conditioned
medium or M-CSF on outgrowth of B/M cells. Furthermore, these
findings suggest that a Th1 microenvironment favored by typical
macrophages is detrimental to the outgrowth of B/M
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 cells
(5). These biphenotypic cells represent the normal
counterpart of phagocytic endpoints of malignant
CD5+ B lymphocyte differentiation (reviewed in
Ref. 6). B/M cells simultaneously expressed the B cell
markers B220, functional surface IgM, and surface IgD along with the
macrophage markers F4/80 and Mac-1 and phagocytized opsonized sheep RBC
(5). Additionally, they retained the genetic fingerprint
of the B cell lineage, rearranged Ig genes. The identity of the
fibroblast-secreted factor, which promoted this striking phenotypic
change of normal B lymphocytes to B/M cells, has not been
discovered.
At least three CSFs may participate in macrophage differentiation. Of
these, GM-CSF and IL-3 not only give rise to macrophages, but also
promote differentiation of granulocytes (reviewed in Ref.
7). M-CSF, however, is thought to be specific for the
macrophage lineage. The diversity of CSFs is reflected in the
heterogeneity of the macrophage population. For example, M-CSF-derived
macrophages are larger, more mature, contain more differentiated
organelles, and display numerous cellular projections compared with
GM-CSF-derived macrophages (8). IL-3-derived macrophages
are of an intermediate phenotype (8). In addition,
macrophages elicited with M-CSF ingest more latex beads than their
GM-CSF-derived counterparts, but GM-CSF induces a macrophage population
that expresses higher levels of Ia Ags and more effectively kill
intracellular bacteria (7). Besides the diversity
generated by the action of the three CSFs, heterogeneity of macrophage
precursors could also be responsible for anatomically and functionally
unique macrophage subsets (9). The nonmonocytic origin of
certain macrophages is suggested by the fact that destruction of the
bone marrow monocyte precursor pool does not eliminate tissue
macrophages (10). In another study, depletion of
radiosensitive bone marrow cells with 89Sr
selectively abolished blood monocytes, but not resident peritoneal
macrophages (11). The results demonstrating that
CD5+ B lymphocytes can generate macrophage-like
descendants further support a nonmonocytic origin for some
"macrophages." For development of normal B/M cells from B
lymphocytes in vitro, the requirement for a fibroblast cytokine(s) is
absolute, since B/M cells cannot be elicited in medium
alone or with fixed fibroblasts (5). As such, a critical
regulatory mechanism must exist such that B cells do not typically
differentiate into B/M cells unless exposed to a cytokine in an
appropriate microenvironment requiring macrophage-like effector
function. This report demonstrates that M-CSF is a critical factor in
the development of biphenotypic B/M cells from their B lymphocyte
precursors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purification of B lymphocytes was performed as previously described (5). Briefly, a single cell suspension was prepared from spleens of 6- to 10-wk-old male B6D2(F1) mice (The Jackson Laboratory, Bar Harbor, ME) by mechanical disruption. Erythrocytes were lysed by brief incubation in buffered ammonium chloride. The erythrocyte-depleted splenocyte suspension was incubated for 2 h at 37°C in a humidified atmosphere with 7% CO2 in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 5% FBS (HyClone, Logan, UT), 5 x 10-5 M 2-ME (Eastman Kodak, Rochester, NY), 10 mM HEPES (United States Biochemical, Cleveland, OH), 2 mM L-glutamine (Life Technologies), and 50 µg/ml gentamicin (Life Technologies), which resulted in adherence of macrophages. Nonadherent cells were washed from the plates, pelleted, and resuspended in a mixture of the following hybridoma supernatants: 30H12 (anti-Thy 1.2), GK 1.5 (anti-CD4), and 3.155 (anti-CD8). This suspension was incubated for 30 min on ice. T lymphocytes were depleted by the addition of low toxicity rabbit complement (Accurate Chemicals/Cedarlane, Westbury, NY) and incubation at 37°C for 45 min. These cells are <1% Thy 1.2- and <1% F4/80-positive as measured by indirect immunofluorescence and flow cytometric analysis of 10,000 cells. Furthermore, they are nonresponsive to Con A, nonphagocytic, and nonspecific esterase-negative.
Cell lines
The isolation of normal mouse splenic fibroblasts and their passage in vitro was described previously (12). Line 1, a mouse small cell lung carcinoma was kindly provided by Dr. Edith M. Lord (University of Rochester, Rochester, NY). LADMAC, a bone marrow monocytic cell line that is transfected with a form of myc and secretes M-CSF (13), was generously provided by Dr. William S. Walker (St. Jude Children’s Research Hospital, Memphis, TN). Conditioned medium from these cell lines was collected from confluent cultures and passed through 0.2-µm syringe filters (Gelman Sciences, Ann Arbor, MI) before addition to purified B lymphocytes.
Culture of B lymphocytes to elicit B/M cells
A total of 3 x 106 purified B
lymphocytes was incubated in 50% splenic fibroblast-conditioned medium
(SFCM), which was harvested from confluent cultures of normal mouse
spleen fibroblasts as described above. As indicated, recombinant human
(rh)M-CSF (Genzyme, Cambridge, MA) or recombinant mouse (rm)M-CSF (R&D
Systems, Minneapolis, MN) was added as a substitute for 50%
fibroblast-conditioned medium. Two units (10 ng/ml) of rhM-CSF was
comparable to 2 ng/ml rmM-CSF in its ability to elicit B/M cell
outgrowth (shown in Fig. 3
D). In some experiments, rmIFN-
or rmIL-4 (both from Genzyme) was added to the B cell cultures. Cells
were incubated at 37°C for at least 6 days before analysis of B/M
cell outgrowth. At the time points indicated in the figure legends
(ranging from 6–21 days), nonadherent dead conventional B lymphocytes
were vigorously washed from the adherent B/M cell monolayer with two
changes of medium. PBS containing 0.1% EDTA was added, and plates were
incubated 10 min at room temperature to promote detachment of B/M
cells from the surface. After gentle tapping of the culture plates,
cell scrapers were used to detach firmly adherent cells and did not
result in decreased viability. B/M cells harvested in this manner
are morphologically and functionally identical to those previously
described (5), coexpressing surface B220 and F4/80, and
are phagocytic.
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Total RNA was isolated from cells using Tri-Reagent (Molecular
Research Center, Cleveland, OH) according to the manufacturer’s
instructions. RNA was reverse transcribed using a modification of the
method of Dallman et al. (14). Briefly, oligo(dT) (2 µg;
Pharmacia, Piscataway, NJ) annealing to RNA was accomplished by a
60°C incubation for 5 min followed by a 3-min incubation on ice.
Thereafter, a RT reaction mix was added that contained 1x reaction
buffer (Life Technologies), 200 µM dNTPs (Pharmacia), 40 U RNasin
(Promega, Madison, WI), and 200 U Moloney murine leukemia virus-reverse
transcriptase (Life Technologies) in a total volume of 40 µl. For
each RNA isolate, a control not containing reverse transcriptase was
included to insure that bands obtained in the PCR were not a result of
DNA contamination. RT was conducted in a Perkin-Elmer (Norwalk, CT)
Thermal Cycler for 60 min at 37°C followed by denaturation of the
enzyme at 95°C for 10 min. The sample was diluted by adding an equal
volume of water. A total of 10 µl cDNA was used in a 100-µl PCR
reaction containing 200 µM dNTPs, 1.5 mM MgCl2,
50 mM KCl, 10 mM Tris-HCl, 1 µM each primer, and 2.5 U Taq
polymerase (Boehringer-Mannheim, Indianapolis, IN). Primer sequences
for M-CSF analysis were: 5'-CCTGCAGCAGTTGATCGACAG and
3'-CAGGCTTGGTCACCACATCTC. The expected PCR product size for M-CSF cDNA
using these oligonucleotides is 413 bp. M-CSF receptor
(c-fms) cDNA was amplified using primers:
5'-ACTCTCCAACCTGCATCGGCT and 3'-GTCCACAGCGTTGAGACTGAG. These
primers are expected to yield a 756-bp product. Primers used to detect
IFN- cDNA were: 5'-TGTTACTGCCACGGCACAGTC and
3'-TCCGCTTCCTGAGGCTGGATTC, which amplify a 395-bp product. All primers
were synthesized by Genosys Biotechnologies (The Woodlands, TX). PCR
was conducted using an initial denaturation at 94°C for 2 min
followed by 40 cycles of denaturation at 94°C for 1 min, annealing at
60°C for 1 min, and polymerization at 72°C for 3 min. A total of 18
µl of each PCR reaction was diluted in loading buffer and
electrophoresed in a 2% agarose gel in a chamber containing 1x Tris
borate EDTA (TBE) with ethidium bromide. Gels were photographed under
UV illumination.
Flow cytometric analysis
B/M cells were resuspended in PBS containing 0.1% azide and
1% BSA (PAB) for staining. To block FcR, samples were incubated in a
1:20 dilution of normal rat serum (Jackson ImmunoResearch, West Grove,
PA) for 20 min on ice. Primary Abs or isotype-matched controls were
then added for 30 min at a concentration of 1 µg per 1 x
106 cells. FITC-conjugated rat anti-mouse
CD45RA (B220, clone RA36B2) and biotin-conjugated rat anti-mouse
F4/80 (Caltag Laboratories, Burlingame, CA) or irrelevant FITC-rat
IgG2a and biotin-rat IgG2b were employed for two-color flow cytometry
(PharMingen, San Diego, CA). After three washes with PAB, 0.4 µg
streptavidin-Tricolor (Caltag) was added to each sample and incubated
for 30 min on ice. Samples were washed and analyzed for simultaneous
expression of B220 and F4/80 using an Epics Elite-ESP flow cytometer
(Coulter Electronics, Hialeah, FL). Ten thousand events were examined
based on gates set according to the negative controls.
mAb preparation
The 5A1 hybridoma, which produces a rat IgG1-neutralizing Ab reactive with mouse M-CSF, was generously provided by Dr. Hsiu-San Lin (Washington University, St. Louis, MO) (15). The hybridoma was grown in complete RPMI medium described above until confluence. The culture supernatant was first concentrated using the Millipore (Marlboro, MA) Minitan System. Affinity purification was done by passing the concentrated supernatant over an anti-rat IgG agarose column (Sigma, St. Louis, MO) followed by elution with 100 mM glycine (pH 3.0). The purified Ab was concentrated and dialyzed using Centricon-10 Microconcentrators (Amicon, Beverly, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cells
Highly purified splenic B lymphocytes, when cultured in RPMI
medium alone, remain nonadherent and die rapidly in the absence of
stimulation. However, when viable but not fixed splenic fibroblasts are
present, adherent B/M cells are found in the B cell cultures
(5). Similar B/M cells are elicited when B lymphocytes
are cultivated in the presence of 50% SFCM, as described in the
Materials and Methods. To determine whether the development
of B/M cells was stimulated by fibroblast-specific cytokines, or a
cytokine potentially produced by many cell types, the ability of other
cell culture supernatants to support B/M cell outgrowth was tested.
As shown in Table I, LADMAC, a monocytic
line secreting high amounts of M-CSF (13), could replace
50% SFCM to promote B/M cell outgrowth at numbers comparable to
fibroblast supernatant. Supernatant from line 1, a mouse small cell
lung carcinoma line, also possessed this property; however, B/M cell
yield was reduced 
3-fold compared with fibroblast-conditioned medium
(Table I). However, 50% supernatant from Con A-stimulated splenocytes
did not result in B/M cell development, rather, the cultured B cells
remained nonadherent.
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growth synthesize M-CSF mRNA
To further determine whether permissive supernatants express a
common cytokine pattern, cytokine mRNA profiles were obtained by RT-PCR
analysis of the cells that supported or did not support B/M
outgrowth (Fig. 1). Candidate cytokines
important for expansion of B/M cells in vitro would likely be
members of the colony-stimulating family, known to participate in the
differentiation of typical M. Splenic fibroblasts did not express
IL-3 by RT-PCR; therefore, that cytokine was not pursued in this study
(data not shown). The housekeeping gene, ß-actin, was used as a
positive control for each PCR sample. Bands were not observed in
negative controls lacking reverse transcriptase during the cDNA
synthesis (data not shown). While splenic fibroblasts and other
permissive cell types produced mRNA for M-CSF (Fig. 1) and GM-CSF (data
not shown), the nonpermissive, Con A-treated spleen cells contained
M-CSF plus IFN- mRNA (Fig. 1). Line 1 carcinoma cells also displayed
a faint band amplified by IFN- -specific primers. These data, along
with results obtained in Table I
, suggest that the relative
concentration of IFN- in culture supernatants either will not
support outgrowth of B/M cells, or will support outgrowth of these
cells at a lower cell number (as occurs using supernatant from line 1
cells). The question of whether or not IFN- was an inhibitor of
B/M outgrowth was assessed next. The addition of 10–100 U/ml
rmIFN- to cultures of purified B lymphocytes in 50% SFCM inhibited
the development of B/M cells (Fig. 2),
and doses of 150 U/ml completely abrogated this process (data not
shown). 50% line 1-conditioned medium produced fewer B/M cells than
SFCM, perhaps due to the production of very low levels of IFN-
protein, which are undetectable in an ELISA with a sensitivity of 0.5
ng/ml (Dr. E. M. Lord, unpublished observations). Addition of
exogenous IFN- to line 1 supernatant also prevented the expansion of
B/M cells from splenic B lymphocytes (Fig. 2). Thus, conditioned
media lacking IFN- support the optimal outgrowth of B/M cells
from their normal splenic B lymphocyte precursors.
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cells from B lymphocytes
The mRNA data obtained in Fig. 1 suggest that a cytokine common to
permissive supernatants is M-CSF. To further characterize the
B/M-promoting effects of the CSFs, rhM-CSF (2.0 U/ml) was added to
purified B lymphocyte cultures, and, at day 13, the resultant spindly
adherent cells were compared with those control B/M cells elicited
by 50% SFCM. Since B/M cells have a striking morphology that
differs from typical macrophages with reniform nuclei, cytocentrifuge
preparations of B/M cells differentiated with 50% SFCM (Fig. 3A) or rM-CSF (Fig. 3B) were stained with Wright-Giemsa solution. In both
samples, enlarged vacuolar granular cells with eccentric round nuclei
were present that were morphologically identical to B/M cells
(5). Furthermore, this morphologic analysis showed that
the B/M cells retrieved after culture in SFCM or rM-CSF were
homogeneous and did not contain any typical B lymphocytes or
conventional macrophages. Next, adherent cells were harvested from the
rM-CSF cultures and stained for expression of B220 and F4/80, a
hallmark of B/M cells growing in vitro. Fig. 3C
demonstrates that M-CSF was the only exogenous factor required to
promote B/M development in purified B lymphocyte cultures, as
adherent B/M cells elicited by rhM-CSF expressed B220 as well as
F4/80. While GM-CSF did promote proliferation of B/M cells already
differentiated from B lymphocytes with 50% SFCM, it did not promote
their differentiation when added at the onset of B cell culture (data
not shown). Thus, M-CSF is sufficient to promote B/M differentiation
from splenic B lymphocytes in vitro. The effect of rM-CSF was
antagonized by rmIFN- (Fig. 3D), as was shown for
unpurified conditioned cell supernatants (Fig. 2). Even at high doses
of rmM-CSF (40 ng/ml), inhibition of B/M cell outgrowth was 94% in
the presence of 100 U/ml rmIFN- (Fig. 3D).
Neutralization of M-CSF in fibroblast-conditioned medium prevents
B/M development
A key point in establishing M-CSF as the required B/M promoting
cytokine in fibroblast-conditioned medium was to employ a neutralizing
Ab to block this activity. SFCM was incubated with a dilution series of
5A1 Ab (rat anti-mouse M-CSF) or isotype control Ab 11B11 (rat
anti-mouse IL-4) on ice for 45 min before its addition to purified
splenic B cells. Fig. 4A shows
that cells treated with SFCM only or SFCM and isotype control Ab are
tightly adherent and viable with a spindly B/M morphology. By
contrast, when the neutralizing 5A1 Ab (50 µg/ml) was present in
purified splenic B cell cultures in SFCM, B/M development was
blocked and only dead nonadherent B cells were visible. These results
are depicted numerically in Fig. 4B, where B/M cells were
removed from the cultures with trypsin and counted using a
hemacytometer, and there were no detectable B/M cells in wells
containing the 5A1 Ab compared with isotype and fibroblast-conditioned
medium-only controls.
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cells, but not their precursors, express c-fms mRNA
The cytokine M-CSF has been shown to interact with a specific
receptor believed to be restricted to cells of the monocyte/macrophage
lineage (16). The c-fms protooncogene encoding
this receptor gives rise to a transmembrane product that contains an
intracellular tyrosine kinase domain (reviewed in Ref.
17). To examine whether the established, normal B/M
cells expressed mRNA for c-fms, RT-PCR was employed as
described in Materials and Methods. Using a malignant
B/M-like cell line, P388D1, as a positive control, data shown in
Fig. 5 was obtained. Both normal and
malignant B/M lines expressed c-fms mRNA. To assess
whether precursor B lymphocytes expressed c-fms mRNA, total
purified splenic B lymphocyte RNA was used in the RT-PCR assay. As
shown in Fig. 6A, the starting
B lymphocyte population did not express c-fms message, and
this was confirmed by a second round of PCR for a total of 80 cycles
(data not shown). Fluorescence-activated cell sorting of the starting
population into CD5+ and
CD5- B lymphocytes followed by RT-PCR for
c-fms also failed to demonstrate expression. These results
also indicate that the starting B lymphocyte population was pure and
did not contain typical macrophages or their precursors that would
express c-fms.
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cells, the precursor
cells must, at some point after culture initiation, express the
receptor for this cytokine. A preliminary kinetic analysis of only the
nonadherent B cells cultured in SFCM showed no evidence of
c-fms mRNA expression at any time point (data not shown). To
include any adherent intermediates, a kinetic analysis of adherent and
nonadherent cells in each culture was performed, and the results are
shown in Fig. 6B. While c-fms mRNA is
undetectable at days 1 and 2 following input of purified B lymphocytes
in 50% SFCM, a faint band was detectable at day 3. Message for
c-fms was easily detectable in B cell cultures harvested on
days 4–7 following their initiation, which correlated with the
appearance of adherent cells with B/M morphology and coexpression of
B220 and F4/80. Therefore, other events before the expression of
c-fms must take place to render the precursor mature B
lymphocytes responsive to M-CSF in the progression from B cell to
B/M cell. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell
phenotype in normal B lymphocytes was elucidated in this study. While
splenic fibroblasts and their conditioned medium were initially used to
induce B/M cell development from B lymphocytes (5),
this activity is not fibroblast-specific, as LADMAC and line 1 tumor
cell supernatants had this capability (Fig. 2). Moreover, rM-CSF could
replace conditioned media to induce B/M cell differentiation (Fig. 3). This is in contrast to observations of Cumano et al.
(18), who found that M-CSF could not replace S17
fibroblasts and IL-7 to differentiate B cells and macrophages from
bipotential precursors isolated from fetal liver. Since these are
earlier precursors and have unrearranged Ig loci, unlike B/M cells
(5), perhaps the cytokine requirements to allow macrophage
differentiation at that stage are more complex than at the level of the
mature Ig+ B cell to B/M transition. M-CSF is
produced by many cell types, including fibroblasts and macrophages
(reviewed in Ref. 19). Thus, multiple cell types may
promote B/M growth in vivo by secreting M-CSF.
Importantly, although splenic fibroblasts do synthesize mRNA for
another CSF, GM-CSF (data not shown), neutralizing Abs specific for
M-CSF completely blocked B/M outgrowth (Fig. 4, A and
B). This suggests that GM-CSF does not act in a redundant
fashion to elicit normal splenic B/M cells. Further indication that
GM-CSF is not the B/M-inducing factor came from incubation of B
lymphocytes in 50% supernatant from line 1 tumor cells transfected
with a GM-CSF expression vector, which did not result in the outgrowth
of B/M cells (M. A.Borrello and R. P. Phipps, data not
shown). This is in contrast to the work of Katoh et al.
(2), where GM-CSF in conjunction with stromal cells was
required for macrophage differentiation from a pre-B cell line in
long-term bone marrow culture. However, rGM-CSF did act as a
proliferative stimulus for already established B/M cells (data not
shown). Perhaps the two CSFs cooperate, such that splenic
fibroblast-secreted M-CSF is an initiation factor for B/M cell
differentiation from B lymphocyte precursors and GM-CSF is a growth
factor, sustaining their continued proliferation in vitro.
The inflammatory cytokine IFN- had a strong inhibitory effect on the
outgrowth of B/M cells. Con A-stimulated spleen cell-conditioned
medium displayed IFN- mRNA (Fig. 2) and is known to contain high
levels of IFN- protein (20). Although M-CSF mRNA is
also detectable in splenocytes treated with Con A, it appears that
IFN- overrides any positive signaling to prevent B/M
differentiation. Likewise, the dominant negative regulatory effect of
IFN- was shown by its addition to otherwise permissive culture
supernatants (splenic fibroblast, line 1) that blocked B/M outgrowth
(Fig. 2). IFN- can also act at a later stage, following B/M
differentiation, to abrogate their
[3H]thymidine incorporation in response to SFCM
(data not shown). Although few to no adherent cells are present in
IFN--treated B cell cultures in permissive conditioned media (Fig. 2) or rM-CSF (Fig. 3D), it is possible that the conversion
from B lymphocyte to B/M cell has progressed to an intermediate
phase. Information regarding this intermediate could be obtained by
adding IFN- at later time points (days 1–4) following culture
initiation to determine whether this inhibitory cytokine must be
present at the onset of B/M differentiation to abrogate their
appearance, or if multiple phases of B/M cell development are
IFN--sensitive. An intriguing finding would be that IFN- prevents
c-fms expression on developing B/M cells, especially in
light of data showing that this cytokine has no effect on transcription
of c-fms in differentiation of bone marrow-derived
macrophages in response to M-CSF (21).
Cell-mediated and humoral immunity are promoted by differing and highly
controlled mechanisms that have been the recent subject of intense
investigation. The cytokines IFN- and IL-4 reciprocally regulate
whether a cell-mediated (Th1) or a humoral (Th2) response occurs after
infection. In the mouse, IFN- promotes a Th1 response to the
exclusion of Th2 effectors, and IL-4 precludes Th1 development
(reviewed in Ref. 22). Conventional macrophages are
stimulated by IFN- to express activation and costimulatory
molecules, act as APCs, and promote a Th1 response. IL-4, in contrast,
has proven to be inhibitory to many aspects of macrophage function,
especially intracellular killing and expression of activation Ags
(23). Initial evidence that B/M cells participate in
immune responses independent of IFN- was their constitutive
expression of class II MHC, CD40, and B7-2 (5). The fact
that IFN- is highly inhibitory to B/M proliferation and prevents
their differentiation from B lymphocytes further supports the
hypothesis that B/M cells may be a Th2-type APC involved in humoral
immunity. The notion that B/M cells can present Ag in the absence of
IFN- priming is underscored by Bretz et al.’s (24)
finding that ras-transformed B/M cells presented Ag
comparably with peritoneal exudate macrophages without prior
activation. Whether IL-4 or other Th2 cytokines have a stimulatory
effect on B/M cells, as they do for B cells, such as inducing class
II MHC hyperexpression, remains to be investigated.
Expression of the M-CSF receptor, c-fms, is considered to be
restricted to the macrophage lineage in mice. Borzillo et al.
(4) showed that forced expression of human
c-fms in mouse pre-B lymphoma cells was sufficient to induce
macrophage differentiation. Furthermore, Tanaka et al.
(25) used c-fms to characterize intermediates
in the spontaneous differentiation of the pre-B lymphoma 70Z/3 to
macrophage-like cells. Their development progressed from nonadherent
c-fms mRNA negative pre-B cells to an adherent
c-fms- intermediate, to adherent
macrophage-like, c-fms+ cells. In the
present study, c-fms mRNA was undetectable by RT-PCR in
purified B lymphocytes (Fig. 6A), but is detected on
transition cells by day 3 after culture initiation (Fig. 6B). Interestingly, M-CSF receptor expression has been
detected on the surface of normal and malignant human B lymphocytes
(26) and hairy cells of hairy cell leukemia patients,
which display B cell and monocytoid features (27). In
these studies, the level of c-fms gene product on human B
lymphocytes was lower than that of normal monocytes, suggesting that
the lack of detection of c-fms in mouse B lymphocytes may be
due to sensitivity issues, such as expression by a very small subset of
the total B cell population, or to species-specific differences. The
identification of a B lymphocyte subset expressing c-fms
would suggest their ability to differentiate into B/M cells in the
presence of M-CSF and would thus shed light on the B/M precursor.
Further studies of the constitutive expression of c-fms in
normal mouse B lymphocytes and the impact of induced expression on
development of macrophage characteristics are necessary to address
these issues.
There are at least two reports of human B cell lines capable of
differentiating to macrophage-like cells in vitro (28) or
displaying phosphoprotein patterns resembling cells able to
differentiate into macrophages (29). Additionally, there
are a number of malignant and nonmalignant diseases where biphenotypic
cells have been observed in bone marrow and peripheral sites (30, 31, 32). Patients bearing neoplasms with simultaneous B cell and
monocytoid characteristics display a low remission rate and poor
prognosis (30). Future work will examine the existence of
B/M cells in humans to identify a normal counterpart to these
neoplastic biphenotypic cells. The knowledge of the B/M growth
factor (M-CSF) and inhibitory factor (IFN-) may facilitate the
identification of these cells in vitro and, ultimately, in
understanding and controlling multiple human biphenotypic
malignancies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard P. Phipps, University of Rochester Cancer Center, 601 Elmwood Avenue, Box 704, Rochester, NY 14642. E-mail address:
3 Abbreviations used in this paper: B/M, B/macrophage cell; rh, recombinant human; rm, recombinant mouse; SFCM, splenic fibroblast-conditioned medium.
Received for publication October 27, 1998. Accepted for publication July 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by murine spleen cells and T cell lines: lack of correlation with Lyt 1,2 phenotype. Immunobiology 166:219.[Medline]
differentially regulate the transcriptional levels of proto-oncogenes and cytokine genes during the differentiation of colony-stimulating factor type-1-derived macrophages. Immunology 85:318.[Medline]
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 S. R. Almeida, L. S. Aroeira, E. Frymuller, M. A. A. Dias, C. S. B. Bogsan, J. D. Lopes, and M. Mariano Mouse B-1 cell-derived mononuclear phagocyte, a novel cellular component of acute non-specific inflammatory exudate Int. Immunol., September 1, 2001; 13(9): 1193 - 1201. [Abstract] [Full Text] [PDF]
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 D. J. Driver, L. J. McHeyzer-Williams, M. Cool, D. B. Stetson, and M. G. McHeyzer-Williams Development and Maintenance of a B220- Memory B Cell Compartment J. Immunol., August 1, 2001; 167(3): 1393 - 1405. [Abstract] [Full Text] [PDF]
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 L. J. McHeyzer-Williams, M. Cool, and M. G. McHeyzer-Williams Antigen-specific B Cell Memory: Expression and Replenishment of a Novel B220- Memory B Cell Compartment J. Exp. Med., April 3, 2000; 191(7): 1149 - 1166. [Abstract] [Full Text] [PDF]
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