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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2815-2822
RP105 Is Associated With MD-1 and Transmits an Activation Signal
in Human B Cells
By
Yoshihiro Miura,
Rintaro Shimazu,
Kensuke Miyake,
Sachiko Akashi,
Hirotaka Ogata,
Yoshio Yamashita,
Yutaka Narisawa, and
Masao Kimoto
From the Department of Immunology and the Division of Dermatology in
the Department of Internal Medicine, Saga Medical School, Saga, Japan.
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ABSTRACT |
RP105 was originally discovered as a mouse B-cell surface molecule
that transmits an activation signal. The signal leads to resistance
against irradiation-induced apoptosis and massive B-cell proliferation.
Recently, we found that mouse RP105 is associated with another
molecule, MD-1. We have isolated here the human MD-1 cDNA. We show that
human MD-1 is also associated with human RP105 and has an important
role in cell surface expression of RP105. We also describe a monoclonal
antibody (MoAb) that recognizes human RP105. Expression of RP105 is
restricted to CD19+ B cells. Histological studies showed
that RP105 is expressed mainly on mature B cells in mantle zones.
Germinal center cells are either dull or negative. RP105 is thus a
novel human B-cell marker that is preferentially expressed on mature B
cells. Moreover, the anti-RP105 MoAb activates B cells, leading to
increases in cell size, expression of a costimulatory molecule CD80,
and DNA synthesis. The B-cell activation pathway using RP105 is
conserved in humans.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE LEUCINE-RICH REPEAT (LRR) is a
protein motif that has been implicated in protein-protein interaction.
It is present in a number of proteins with diverse functions and
cellular locations.1 Some members have a role in defense
against pathogens. Tomato disease resistance (R) genes cf-2 and cf-9
are transmembrane proteins that possess LRRs in extracellular
domains.2,3 They recognize specific pathogen molecules with
LRRs and activate plant's defense responses.4 Similarly,
the Toll receptor, another LRR molecule of Drosophila, elicits defense
responses against the fungal pathogens.5 A human homologue
of Toll was recently cloned.6 Human Toll is expressed on
lymphocytes and monocytes and transmits an activation signal. Thus, LRR
molecules seem to have a basic role in the immune system among diverse
species.
RP105 is another LRR molecule that is expressed on mouse B lymphocytes.
It was first identified by establishing the RadioProtective (RP)
monoclonal antibody (MoAb) that protected spleen B cells from
irradiation-induced apoptosis.7,8 The RP105 molecule, when
cross-linked by the MoAb, transmits the activation signal that leads to
massive B-cell proliferation as well as resistance against apoptosis.
Interestingly, these activated and proliferating B cells arrest their
growth and undergo apoptosis upon a signal from the antigen
receptor.9 RP105 may be involved in regulation of B-cell
growth and an antigen-induced death. We have recently identified MD-1
as a molecule that is physically associated with RP105.10 MD-1 was originally reported as a
v-myb-regulated gene.11 It is a secretory molecule
but tethered to the cell surface by RP105. MD-1 seems to regulate cell
surface expression of RP105.10
A human homologue of RP105 was previously identified.12 The
amino acid sequence shows 75% identity to that of mouse RP105, and the
deduced molecular structure, including LRRs, is well conserved. Despite
such structural similarities, cell surface expression, transduction of
an activation signal, and association with human MD-1 are not clear
yet. To address these issues, we conducted molecular cloning of human
MD-1 and established an MoAb against human RP105.
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MATERIALS AND METHODS |
Cells and antibodies.
A mouse B-cell lymphoma M12 was transfected with an expression vector
pEFBOS (a gift from Dr Shigekazu Nagata, Osaka University Medical
School, Osaka, Japan) that codes for human RP105 with the flag epitope
at the carboxy-terminus. Expression of human RP105 was confirmed with
immunoblotting with an anti-flag MoAb M2 (Eastman Kodak Co, New Haven,
CT) and with cell surface biotinylation and subsequent
immunoprecipitation by the anti-flag MoAb. The cell line was referred
to as M12HRP. The BaHRP cell line was prepared by transfecting the same
vector as above into an mouse interleukin-3 (IL-3)-dependent line
Ba/F3. Ba/F3 cells do not express either MD-1 or RP105. Expression of
human RP105 was detected with immunoprecipitation and immunoblotting
with the anti-flag MoAb. The BaHRPMD cell line was established by
further transfection of BaHRP cells with an expression vector encoding
human MD-1 of which the carboxy-terminus was tagged with the flag
epitope. Cells bearing MD-1 on a cell surface were sought by staining
with the anti-flag MoAb. Although already transfected human RP105 also
bears the flag epitope, it is located inside the cell. Therefore, the
flag epitope on human RP105 is not detectable with cell surface
staining.
Human cell lines, including Daudi, Ramos, Nalm-6, U937, K562, JY,
RPMI8866, CEM, and Molt4, were obtained from Japan Cancer Resources
Banks (JCRB, Osaka, Japan). Peripheral blood leukocytes were isolated from heparinized blood by Ficoll-Hypaque density gradient
centrifugation (Pharmacia, Uppsala, Sweden). Normal tonsil tissues were
obtained from tonsillectomies at the Saga Medical School Hospital
(Saga, Japan). In the cell proliferation assay (see Table
2), tonsillar B cells were enriched by negative selection using
anti-CD2 MoAb-coupled Dynabeads (Dynal Inc, Oslo, Norway).
Anti-CD19 MoAb SG/16 was established in this laboratory. Another
anti-CD19 MoAb conjugated with fluorescein isothiocyanate (FITC) was obtained from Becton Dickinson Immunocytometry
Systems (San Jose, CA). The biotinylated anti-CD80 MoAb was purchased from PharMingen (San Diego, CA). The MHR73 MoAb (IgG1/ ) was
established from BALB/c mice immunized with the M12HRP cell line (see
Results). The MoAb was purified from ascitic fluid with the ABx column
chromatography (J.T. Baker Inc, Phillipsburg, NJ).
An Est cDNA clone and sequencing.
An Est cDNA clone encoding human MD-1 (Genbank accession no. T84854)
was obtained from Genome Systems Inc (St Louis, MO). Sequencing was
conducted with ALFexpress DNA sequencer and Thermo Sequenase cycle
sequencing kit (Pharmacia Biotech Japan, Tokyo, Japan).
Cell surface biotinylation, immunoprecipitation, and Western
blotting.
Cell surface biotinylation and immunoprecipitation was conducted as
described previously.13 Briefly, cells were washed in Hanks' balanced salt solution (HBSS) and adjusted to 5 × 107/mL in saline containing 100 mmol/L HEPES (pH
8.0). Sulfosuccinimidobiotin (Pierce, Rockford, IL) was added to cell
suspension at 0.5 mg/mL. After 30 minutes of incubation at room
temperature with occasional shaking, cells were washed in HBSS and
lysed in a buffer consisting of 50 mmol/L Tris/HCl (pH 7.5), 150 mmol/L
NaCl, 1% Triton X-100, 50 mmol/L iodoacetamide, 1 mmol/L phenylmethyl
sulfonyl fluoride (PMSF), 10 µg/mL soybean trypsin
inhibitor, 5 mmol/L EDTA, and 0.1% sodium azide. After 30 minutes of
incubation on ice, lysate was centrifuged and supernatant was
recovered. Anti-RP105-coupled Hi-Trap beads (Pharmacia Biotech Japan)
were added to cell lysate and rotated for 2 hours at 4°C. Beads
were washed in the lysis buffer and bound proteins were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Western blotting. Biotinylated proteins
were detected with streptavidin-peroxidase (Amersham Japan, Tokyo,
Japan) and Supersignal chemiluminescent substrate (Pierce). The flag
epitope was demonstrated with the anti-flag MoAb followed by goat
antimouse IgG-horseradish peroxidase (BioSource International,
Camarillo, CA).
Northern hybridization.
Total RNA was extracted with Isogen (Nippon Gene, Toyama, Japan) and
subjected to agarose electrophoresis (10 µg/lane). After transfer to
a nylon membrane (Hybond N+; Amersham Japan, Tokyo, Japan), RNA was
hybridized to a probe that had been labeled by random priming of the
cDNA clone encoding human MD-1. Hybridization buffer consisted of 10%
dextran sulfate (Pharmacia), 1 mol/L NaCl, 1% SDS, 50 mmol/L Tris/HCl
(pH 7.5). Hybridization was conducted at 65°C for 20 hours. Washing
was performed in 2× SSC with 0.1% SDS at up to 65°C.
Radioactive signals were visualized with an image-analyzer BAS2000
(Fuji Film Co Ltd, Tokyo, Japan). The same membrane was reprobed for
Glyceraldehyde-3-phosphate dehydrogenase.14
Flow cytometry and cell permeabilization.
Cells were incubated with the MHR73 MoAb or the anti-flag MoAb for 20 minutes on ice. After washes with a staining buffer (phosphate-buffered
saline [PBS] containing 2% fetal calf serum [FCS] and 0.1% azide), goat antimouse IgG-FITC was
added. Propidium iodide was included in the second incubation to
exclude dead cells. For dual-staining, the biotinylated MHR73 followed
by avidin-phycoerythrin and FITC-labeled anti-CD19 MoAb were used.
Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA).
For cell permeabilization, 0.1% saponin detergent was added to the
staining buffer as described.15
Cell proliferation assay.
B cells enriched from a normal human tonsil were cultured in a 96-well
plate for 4 days with an indicated MoAb. Cells were pulsed with 1 µCi
of [3H] TdR (ICN Radiochemicals, Irvine, CA) for the last
6 hours. They were then harvested onto glass fiber filters and the
incorporated radioactivity was determined on a Beta plate flat-bed
liquid scintillation counter (Pharmacia-Wallac, Gaithersburg, MD). The
results are presented as a mean ± SD of triplicate wells.
Immunohistochemistry.
Normal tonsil tissues were embedded in Tissue-Tek II OCT compound
(Miles Inc, Elkhart, IN), frozen in liquid nitrogen, and stored at
 80°C. Acetone-fixed cryostat sections of 4 µm were incubated with the anti-RP105 MoAb MHR73. After washes, ENVISION+/HRP (DAKO Japan, Inc, Tokyo, Japan) was added. RP105 was finally visualized with 3,3 -diaminobenzidine.
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RESULTS |
Molecular cloning of human MD-1.
A cDNA encoding human MD-1 was isolated. We used the expressed-sequence
tag (EST) database at the National Center for Biotechnology Information
(NCBI, Bethesda, MD) to obtain the human MD-1 cDNA. A
search with the mouse MD-1 nucleotide sequence identified a cDNA clone
from human fetal liver and spleen (Genbank accession no. T84854). A
whole sequence of the clone was determined. It contained the full
coding region as well as a poly(A) signal (residue 847-852, underlined
in Fig 1A). The cDNA clone encodes 162 amino acids. There is a hydrophobic stretch from the 1st to the 19th
amino acid residue. This portion is likely to be a signal peptide
(underlined in Fig 1A). The mature peptide consists of 143 amino acids,
and does not have any additional stretches of hydrophobic amino acids.
Therefore, human MD-1 seems to be a secretory molecule. Two canonical
N-glycosylation sites are contained (underlined in Fig 1A). The amino
acid sequence of human MD-1 has 66% and 38% identity to mouse and
chicken MD-1, respectively (Fig 1B). Six of seven cysteine residues in
the mature human MD-1 peptide are conserved in all three species.
Northern hybridization showed that the MD-1 transcript is about 1 kb
and demonstrable in all four B-cell lines (Nalm-6, Ramos, Daudi, and
RPMI8866) and a monocyte line U937, but not in two T-cell lines
(CEM and Molt-4) or an erythroleukemia line K562
(Fig 2).
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| Fig 1.
A nucleotide sequence of human MD-1 and alignment of its
amino acid sequence with mouse and chicken MD-1. (A) The nucleotide
sequence of human MD-1 is shown. A poly(A) signal is underlined. An
encoded amino acid sequence is also shown. A signal sequence and two
canonical N-glycosylation sites are underlined. (B) Amino acid
alignment of human, mouse, and chicken MD-1. Identical residues in
mouse and chicken MD-1 are denoted by (). Five gaps are introduced
in chicken to optimize alignment and denoted by blanks.
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| Fig 2.
Northern hybridization of human MD-1. Total RNA
from human cell lines were electrophoresed, blotted, and hybridized
with the human MD-1 probe. An approximately 1-kb signal is apparent and
indicated as huMD-1. The same blot was reprobed for GAPDH and is shown
below.
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Establishment of an antihuman RP105 MoAb, MHR73.
An anti-RP105 MoAb is requisite to study expression/function of human
RP105 and its association with MD-1. We immunized BALB/c mice with a
mouse B-cell line M12HRP that expresses human RP105 complexed with
mouse MD-1 (Fig 3A and see below).
Immunized spleen cells were fused with the SP2/0 myeloma. Supernatant
from resultant hybridoma cells were used for staining of 293T cells
that had been transiently transfected with vectors encoding human RP105 and mouse MD-1. Positive Abs were further characterized by staining human B-cell lymphomas. One MoAb was finally chosen and referred to as
MHR73. The MHR73 MoAb reacted with M12HRP cells but not with the
original line M12. M12HRP was used for cell surface biotinylation and
immunoprecipitation. MHR73 precipitated three distinct antigens of 97, 25, and 22 kD (Fig 3A1). Introduced human RP105 had been tagged with
the flag epitope. Human RP105 corresponds to the 97-kD species, because
it bore the flag epitope (Fig 3A2). The smaller species are likely to
be mouse MD-1, because similar signals were coprecipitated with
endogenous mouse RP105 and identified as mouse MD-1.10 The
MoAb bound to two human B-cell lines, Ramos and Daudi. The size of the
target antigen on these cell lines was similar to ectopically expressed
human RP105 on M12 cells (Fig 3B1 and 3B2). Thus, MHR73 is an MoAb
against human RP105.
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| Fig 3.
The MHR73 MoAb recognizes RP105. (A) Cell surface
proteins on M12HRP cells were biotinylated. After extraction with a
lysis buffer, immunoprecipitation was conducted with the MHR73 MoAb.
Precipitated proteins were subjected to SDS-PAGE (12% acrylamide,
reduced condition) and blotted onto a nitrocellulose membrane. Blotted
proteins were detected with either avidin horseradish peroxides (for
cell surface molecules: lane 1) or an anti-flag MoAb followed by goat
antimouse IgG-horseradish peroxidase (for human RP105 that bears the
flag epitope at the carboxy-terminus: lane 2). Signals of about 50 and
28 kD are observed in lane 2. They correspond to the heavy chain and
the light chain of the MHR73 MoAb, because they were detected with goat
antimouse IgG-horseradish peroxidase alone (data not shown). (B)
Indicated cell lines were subject to cell surface biotinylation. Cell
surface molecules were then precipitated with either the MHR73 MoAb
(lanes 1, 2, and 4) or the anti-flag MoAb (lane 3). Precipitated
molecules were resolved on SDS-PAGE, blotted on an nitrocellulose
membrane, and detected with avidin-horseradish peroxidase. The MD-1
signal was very faint in Daudi or Ramos cells in this experiment.
However, we confirmed additional signals of 25 and 22 kD in other
experiments (data not shown).
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Human MD-1 positively regulates cell surface expression of human
RP105.
The BaHRP line was established by transfection of the mouse
IL-3-dependent line Ba/F3 with a vector encoding human RP105. Ba/F3
cells do not express either mouse RP105 or MD-1 (data not shown).
Introduced RP105 had been tagged with the flag epitope at the carboxyl
terminus. Expression of human RP105 in BaHRP cells was therefore
confirmed by immunoprecipitation and subsequent probing with the
anti-flag MoAb (data not shown). However, MHR73 MoAb did not react with
BaHRP cells (Fig 4A), and only a faint signal was obtained with cell surface biotinylation and
immunoprecipitation with the anti-flag MoAb (Fig 3B3). We assumed that
the majority of human RP105 proteins did not come out but stayed inside
the cell. To see intracellular proteins, BaHRP cells were permeabilized with saponin detergent and stained with MHR73 or the anti-flag MoAb.
Either antibody was able to demonstrate human RP105 inside the BaHRP
line (Fig 4C and D). Therefore, the majority of human RP105 is located
inside the cell. Another BaHRP line also had most RP105 molecules
inside the cell (data not shown). The human RP105 protein expressed
alone seemed to have difficulty in reaching the cell surface (see
Discussion).
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| Fig 4.
MD-1 has an important role in cell surface expression of
human RP105. Two IL-3-dependent lines, BaHRP (A through D) and BaHRPMD
(E through G), were stained with either the anti-RP105 MoAb MHR73 (the
left column) or the anti-flag MoAb M2 (the right column). Cell
permeabilization was conducted in (C), (D), and (G). RP105 and MD-1
were tagged with the flag epitope at carboxy-terminus. Carboxy-temini
of RP105 and MD-1 are located inside or outside cells, respectively.
Therefore, the anti-flag MoAb detects MD-1 but not RP105 in cell
surface staining of BaHRPMD cells (F).
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The BaHRP line is the mouse IL-3-dependent line that ectopically
expresses human RP105 (see Materials and Methods). The BaHRPMD line was
then established by further transfection of BaHRP cells with an
expression vector encoding the human MD-1 cDNA. The MD-1 cDNA was
tagged with the flag epitope. The anti-flag MoAb is therefore able to
detect MD-1. Human MD-1 does not have a transmembrane portion (Fig 1).
Nevertheless, it was expressed on a cell surface (Fig 4F). RP105 also
appeared on the cell surface (Fig 4E). Cell surface biotinylation and
subsequent immunoprecipitation with MHR73 showed a signal of about 30 kD as well as human RP105 (Fig 3B4). Immunoprobing with the anti-flag
MoAb showed that the 30-kD species bore the flag epitope (data not
shown). Human MD-1 is therefore associated with human RP105.
Ectopically expressed MD-1 was bigger than endogenous MD-1 that was
about 22 or 25 kD. A similar change in size between ectopic and
endogenous MD-1 also occurred to the mouse homologue.10
This may stem from a difference in posttranslational modification such
as glycosylation.
Introduction of MD-1 complemented cell surface expression of RP105 (Fig
4A and E) in the BaHRPMD line. Moreover, we conducted transient
transfection into a human kidney line 293T cells with vectors encoding
human RP105 or human MD-1. Cell surface staining with MHR73 showed that
human RP105 did not appear on a cell surface when human RP105 was
transfected alone, but did when human MD-1 was coexpressed (data not
shown). Thus, MD-1 has an important role in cell surface expression of
RP105.
Expression of human RP105.
Expression of RP105 was studied with flow cytometry staining. Results
are summarized in Table 1. Among cell lines
studied, only two B-cell lines (Ramos and Daudi) were positive.
Negative cell lines include two B-cell lines (Nalm-6 and RPMI8866), a
monocyte line (U937), an erythroleukemia line (K562), and two T-cell
lines (CEM and Molt-4). Results with Ramos and Daudi were
consistent with that of northern hybridization (Fig 2 and Table 1).
However, those with Nalm-6 and U937 were not. They express the
transcripts of RP105 and MD-1, but RP105 was not expressed on cell
surfaces. RPMI8866 had only MD-1 mRNA.
Normal leukocytes from peripheral blood and tonsils were also stained
with MHR73. RP105 was restricted to CD19+ B cells
(Fig 5). Interestingly, all B cells in
peripheral blood are positive, whereas some tonsillar B cells were
negative. Percentages of RP105 B cells in whole B
cells were variable from 10% to 30%. To see RP105
B cells in more detail, sections of a normal tonsil were stained with
MHR73 (Fig 6). RP105 was mainly expressed
on B cells in mantle zones. Cells in germinal centers were either dull
or negative. Considering that recirculating B cells reside in follicles
and mantle zones, the result of section staining is consistent with that of flow cytometry. B cells in follicles and mantle zones correspond to mature B cells. RP105 is thus a B-cell marker that is
preferentially expressed on mature B cells.
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| Fig 5.
RP105 expression is restricted to CD19+ B
cells. Leukocytes from peripheral blood or a tonsil were stained with
the FITC-labeled antibody against CD19 and biotinylated MHR73 followed
by avidin-phycoerythrin. Stained cells were analyzed on a FACScan.
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| Fig 6.
RP105 is mainly expressed on B cells in mantle zones.
Frozen sections from a human tonsil were stained with the MHR73 MoAb.
(A) RP105+ B cells are mainly located in a mantle zone that
surrounds a germinal center where expression of RP105 is dull or
negative (original magnification × 50). (B) Higher magnification
(original magnification × 200) of a boundary between the mantle zone
and the germinal center of (A). No significant signal was observed by
staining with the second reagent alone.
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Human RP105 transmits an activation signal.
We next studied whether the MHR73 MoAb was able to induce B-cell
activation. Peripheral blood mononuclear leukocytes were cultured in
the presence or absence of MHR73. CD19+ B cells were
examined about changes in cell size and expression of a costimulatory
molecule CD80. An increase in cell size, measured by forward scatter
with flow cytometry, was apparent as early as 24 hours after incubation
had started (Fig 7A). After 40 hours, the
change in cell size became more remarkable, and a costimulatory molecule CD80 was significantly induced (Fig 7). B cells were enriched
from tonsils and cultured with MHR73 for 3 to 4 days. An increase in
DNA synthesis was apparent (Table 2). With
peripheral blood leukocytes, a similar increase in DNA synthesis was
observed after 3 to 4 days of culture with MHR73 (data not shown).
Thus, B cells were activated in response to cross-linking of human
RP105.
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| Fig 7.
The anti-RP105 MoAb induces B cells to increase in cell
size and expression of a costimulatory molecule CD80. Peripheral blood
mononuclear cells were collected by Ficoll-Hypaque density gradient
centrifugation and cultured in a 24-well plate at 2 × 106
cells/well with or without the anti-MHR73 MoAb (20 µg/mL). After 24 hours (the left panel of A) or 40 hours (the right panel of A and B),
cells were harvested and stained with FITC-labeled anti-CD19 MoAb and
biotinylated anti-CD80 MoAb followed by avidin-phycoerythrin. Stained
cells were analyzed on a FACScan. Only CD19+ B cells were
examined, and profiles of forward scatter (A) or CD80 expression (B)
are shown. Solid lines and hatched lines depict histograms of cells
cultured with or without the MHR73 MoAb, respectively.
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DISCUSSION |
Human MD-1, a molecule interacting with RP105, was isolated in the
present study. Amino acid sequence showed 66% and 38% identity to
mouse and chicken MD-1, respectively. Mouse MD-1 binds to human RP105
ectopically expressed on mouse M12HRP cells. On the other hand, human
MD-1 was associated with mouse RP105 transiently expressed on 293 T
cells (Miyake et al, unpublished observation).
Interaction of RP105 and MD-1 is therefore well conserved between
humans and mice and works easily beyond the species barrier.
MD-1 plays an important role in cell surface expression of RP105.
Without MD-1, the majority of human RP105 was held inside the cell (Fig
4A and C). RP105 alone may have difficulty in intracellular maturation
or traffic to cell surfaces. In this regard, it has to be noted that
RP105 without MD-1 seemed to be smaller and less heterogeneous than
RP105 with MD-1. We compared RP105 molecules in BaHPRMD or BaHRP cells
by immunoprecipitation and subsequent probing with the anti-flag MoAb
(Miyake et al, unpublished observation). Two closely
located signals were obtained from BaHRPMD cells that expressed RP105
and MD-1. The bigger one appeared to be cell surface RP105, because it
was similar in size to the RP105 signal obtained by cell surface
biotinylation (Fig 3B4). The other, smaller and less heterogeneous
signal is likely to be intracellular precursor of RP105. Smaller size
and lesser heterogeneity of intracellular RP105 would stem from
immature glycosylation. Only the smaller signal was apparent from BaHRP
cells that expressed RP105 but not MD-1. In the absence of MD-1, the
RP105 protein seems to be held before or during maturation steps.
It remains to be clarified how MD-1 contributes to posttranslational
modification and subsequent cell surface expression of RP105. One
possibility is that RP105 and MD-1 is associated before maturation and
the association itself is important for maturation and subsequent cell
surface expression. Association of MD-1 might confer more stable
conformation on the RP105 molecule. RP105 alone could be unstable and
easily degraded. Further studies are under way to seeking for
differences between RP105 alone and RP105 associated with MD-1.
The MHR73 was able to recognize intracellular and immature RP105 in the
absence of MD-1 (Fig 4C). The epitope of MHR73 is therefore not
dependent on either glycosylation or association of MD-1, but on the
protein backbone.
It is important to know expression of MD-1 as well as RP105, because
cell surface expression of RP105 is dependent on MD-1 (Fig 4).
Distribution of MD-1 mRNA seems to be broader than that of RP105. The
RP105 transcript was previously observed in four lines: Nalm-6, Ramos,
Daudi, and U937.12 All of them express the MD-1 transcript
as well (Fig 2). A B-cell line RPMI8866 expresses MD-1 but not RP105
mRNA (Fig 2 and Table 1). Moreover, we found two additional EST clones
similar to MD-1 from human fetal brain or lung, where RP105 would not
be present. Also, in mouse MD-1, the transcript was observed in liver
and brain as well as in lymphoid organs.10 In the absence
of RP105, eg, in liver or brain, MD-1 can be secreted or might be
associated with another LRR molecule. MD-1 may have additional roles
outside the immune system. The anti-MD-1 MoAb is needed to see
localization of MD-1 and association with other LRR molecules.
Recirculating B cells have a bigger chance of encountering a pathogen
and contributing to an inductive phase of B-cell responses than sessile
B cells. It is reasonable that recirculating B cells express molecules
that receive a signal from the innate immune system, a system that has
an important role in the development of an antibody
response.16 For example, CD21 is preferentially expressed
on mature B cells. It is a receptor for the C3d fragment of the
complement system that belongs to innate immunity. In concert with
CD19, CD21 augments B-cell responses against an antigen to which the
C3d fragment attaches.17 Thus, CD21 assists communication of B cells with the innate immune system and helps B cells to mount
antibody production. In the present study, we showed that RP105 is
expressed on mature and recirculating B cells and that RP105 does
transmit an activation signal leading to increases in cell size and DNA
synthesis and to induction of CD80 expression. Induced CD80 would help
B cells to interact with T cells and accomplish further activation.
RP105 may also contribute to the inductive phase of B-cell activation
in response to innate immunity. This hypothesis is supported by other
LRR molecules in the immune system. CD14 is an lipopolysaccharide
(LPS) receptor and activates macrophages.18 Activated macrophages then activate lymphocytes by secreting cytokines or expressing costimulatory molecules. The Drosophila Toll receptor and
tomato disease resistance gene cf-2 and cf-9 are requisite for
eliciting defense responses against fungal pathogens.2-5 A
human homologue of the Toll receptor is expressed on macrophages or
dendritic cells and is expected to activate them in responses against
pathogens.6 These LRR molecules are thought to augment innate immunity and to mount adaptive immune responses. RP105 may
belong to these LRR molecules in terms of a role in the immune system
as well as of molecular structure. A ligand for RP105 might be derived
from a pathogen or a molecule produced in cells working in innate
immunity.
Another expectation of a role of RP105 comes from our previous study in
mice.9 B cells activated with anti-RP105 MoAb showed growth
arrest and apoptosis in response to a signal through the antigen
receptor. This result suggests that RP105 might be involved in
regulation of an antigen-induced B-cell death. The antigen-induced death of mature lymphocytes is thought to contribute to establishment or maintenance of peripheral B-cell tolerance and to termination of
B-cell responses by downsizing activated B-cell clones.19 Loss of function or malfunction of RP105 could lead to augmented B-cell
responses and autoimmune diseases. The MHR73 MoAb opens a human study
of RP105. It is interesting to study B cells from patients suffering
from recurrent infections or autoimmune diseases. Abnormalities of B
cells may be shown with staining or stimulation with MHR73. These
studies would complement results from mouse studies.
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FOOTNOTES |
Submitted April 9, 1998;
accepted June 11, 1998.
Y.M. and R.S. contributed equally to this study.
Supported by grants from the Ministry of Education, Science, and
Culture of Japan; the Osaka Cancer Research Foundation; the Ichiro
Kanehara Foundation; the Ryoichi Naito Foundation for
Medical Research; and Ciba-Geygy Foundation (Japan) for the
Promotion of Science.
Sequence data from this article have been deposited with the DDBJ,
EMBL, and GenBank Data Libraries under Accession No. AF05718.
Address reprint requests to Kensuke Miyake, MD, PhD, Department of
Immunology, Saga Medical School, Nabeshima, Saga 849-8501, Japan;
email: miyake{at}post.saga-med.ac.jp.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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Abstract
Introduction
Abstract