Download Macrophage- and dendritic cell–dependent

IMMUNOBIOLOGY
Macrophage- and dendritic cell–dependent regulation of human B-cell
proliferation requires the TNF family ligand BAFF
Andrew Craxton, Dario Magaletti, Elizabeth J. Ryan, and Edward A. Clark
Macrophages and dendritic cells play an
important role in regulating B-cell responses, including proliferation to antigens such as trinitrophenyl (TNP)–Ficoll
and TNP-Brucella abortus. However, the
mechanisms and molecule(s) that regulate these processes are relatively undefined. In this report, we show that human
macrophages generated in vitro strongly
costimulate proliferation of dense human
tonsillar B cells ligated via their B-cell
antigen receptor (BCR) but not proliferation via CD40. Similarly, dendritic cells
also markedly enhance BCR-activated Bcell proliferation. Soluble molecule(s) are
required for human macrophages to costimulate proliferation of B cells triggered
via their BCR. Importantly, a TACI (transmembrane activator and CAML interactor)–Fc fusion protein inhibits both macrophage- and dendritic cell (DC)–dependent
BCR-activated B-cell proliferation, indicating a requirement for at least one of the
known TACI ligands, BAFF and/or APRIL.
Consistent with a major role for BAFF,
macrophages release BAFF at levels sufficient to potently costimulate BCR-induced B-cell proliferation. In addition,
BAFF is more than 100-fold more potent
than APRIL in enhancing BCR-mediated
human B-cell proliferation. Furthermore,
immunodepletion of APRIL under conditions that prevent APRIL-mediated B-cell
costimulation does not block macro-
phage enhancement of B-cell proliferation. Finally, there is no correlation between the high levels of a proliferationinducing ligand (APRIL) expressed by
macrophages compared with DCs and
the similar abilities of macrophages and
DCs to enhance BCR-stimulated B-cell
proliferation. In summary, our results suggest that macrophage- and DC-derived
B-cell–activating factor belonging to the
TNF family (BAFF) represents a key molecule by which macrophages and DCs
directly regulate human B-cell proliferative responses to T-cell–independent
stimuli. (Blood. 2003;101:4464-4471)
© 2003 by The American Society of Hematology
Introduction
Antibody responses to antigens can be broadly divided into 2 types;
T-cell dependent (TD) and T-cell independent (TI).1 T-cell–
dependent antibody responses require the tumor necrosis factor
receptor (TNFR) homolog, CD40, a costimulatory molecule expressed on B cells and dendritic cells (DCs).2,3 Engagement of
CD40 is mediated by CD154 (CD40 ligand), principally expressed
on activated T cells.
Less is known about TI responses initiated by bacterial cell wall
lipopolysaccharides (type 1 antigens) and repetitive polysaccharides (type 2 antigens). The splenic marginal zone (MZ) provides a
primary line of defense against blood-borne pathogens, including
encapsulated bacteria in mice.4-6 Anatomically, the MZ lies between the white and red pulp of the spleen and is composed of B
cells, macrophages, and DCs.7 The MZ B cells are adjacent to the
marginal sinuses and are among the first cell population encountered by blood-borne antigens. TI type 2 immune responses require
MZ B cells. Thus, mice deficient in the tyrosine kinase Pyk-2 that
lack splenic MZ B cells have defective antibody responses to the TI
type 2 antigen, trinitrophenyl (TNP)–Ficoll.8 DCs localized within
the MZ express phenotypic markers typically associated with
macrophages and have been referred to as myeloid DCs.9 In
vitro–derived myeloid DCs initiate humoral immune responses in
mice to TI antigens, expressed by the intact gram-positive extracellular bacteria Streptococcus pneumoniae.10 Recently, a specific
population of murine DCs that can promote the generation of
antigen-specific plasmablasts from naive MZ B cells has been
identified. These DCs are blood-derived, CD11clo DCs, which
capture antigen and then migrate from the blood to the spleen.11
However, a supporting or accessory role for other cell types in
regulating B cells such as macrophages is also possible. Indeed,
bacteria-primed splenic murine macrophages also enhance MZ
B-cell survival in vitro.11
Earlier in vitro studies suggested that the TI B-cell response in
mice to TNP-haptenated antigens such as TNP-Ficoll, TNPlipoprotein (LP), and TNP–Brucella abortus directly required
macrophages.12-15 Interestingly, interleukin 1 (IL-1) appeared to
substitute for the requirement of macrophages in B-cell responses
to TI antigens. In addition, IL-1–secreting cells reconstitute the
response of macrophage-depleted splenic cells to TI antigens.15
A newly characterized tumor necrosis factor (TNF) ligandreceptor system has recently been implicated in the regulation of
both TI and TD immune responses. B-cell–activating factor
belonging to the TNF family (BAFF; also known as TALL-1,
THANK, BlyS, and zTNF4) is a novel TNF ligand superfamily
member that is a potent regulator of B-cell development and
function.16-20 BAFF protein is expressed by myeloid lineage cells
and exists as both cell surface–associated and soluble, released
From the Department of Microbiology, University of Washington, Seattle;
Department of Immunology, University of Washington, Seattle; and the
Regional Primate Research Center, University of Washington, Seattle.
Reprints: Andrew Craxton, Department of Microbiology, University of
Washington, Seattle, WA 98195; e-mail: [email protected].
Submitted October 16, 2002; accepted January 8, 2003. Prepublished online as
Blood First Edition Paper, January 16, 2003; DOI 10.1182/blood-2002-10-3123.
Supported by grants GM37905, DE13061, and RR00166 from the National
Institutes of Health and a grant from Zymogenetics.
4464
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2003 by The American Society of Hematology
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
MACROPHAGE-INDUCED B-CELL PROLIFERATION REQUIRES BAFF
forms.19,21 BAFF binds to 3 distinct TNF receptors: TACI (transmembrane activator and CAML interactor), BCMA (B-cell maturation antigen), and BAFF-R/BR3 (BLyS receptor 3).20,22-26 Two of
these receptors, TACI and BCMA, also bind another TNF ligand
that shares about 50% sequence homology in its extracellular
region with BAFF, known as a proliferation-inducing ligand
(APRIL), TRDL-1, or TALL-2.17,27-30 Like BAFF, APRIL is also
released as a biologically active protein from cells.31
Overexpression or injection of BAFF increases MZ B-cell
numbers and serum immunoglobulin levels in mice.32 Furthermore,
TACI-deficient mice have defective responses to TI-2 antigens.33,34
In addition to its role in T-cell–independent immune responses,
BAFF also appears to modestly enhance T-cell–dependent immune
responses.35
In this study, we investigated a potential role for human
macrophages in directly enhancing proliferation of human tonsillar
B cells. We show that human macrophages directly enhance
proliferation of human B cells triggered via their B-cell antigen
receptor (BCR) but not CD40, a process that requires soluble
molecule(s). Moreover, we show that macrophage- and mono-DC–
regulated BCR-induced human B-cell proliferation is inhibited by
TACI-Ig (immunoglobulin), demonstrating a requirement for one
or more of the TACI ligands, BAFF, and/or APRIL. Consistent with
a major role for BAFF, macrophages released BAFF at doses
sufficient to maximally costimulate BCR-induced B-cell proliferation. Moreover, immunodepletion of APRIL did not block macrophage enhancement of BCR-induced B-cell proliferation.
In summary, our studies using human cells suggest that
macrophages directly regulate B-cell proliferation via the TNF
ligand, BAFF, and support the idea that macrophages may play an
important accessory role during TI immune responses.
Materials and methods
Materials
Purified soluble recombinant FLAG-tagged human BAFF (residues 141285) and a recombinant TACI-Ig fusion protein consisting of the extracellular domain (amino acids 1-154) of human TACI fused to the Fc portion of
the human IgG␥1 heavy chain were generous gifts from Dr Jane Gross
(Zymogenetics, Seattle, WA). Purified recombinant human TACI-Ig was
also purchased from Alexis (San Diego, CA). Equivalent results were
observed with TACI-Ig from both sources. Recombinant soluble FLAGtagged human APRIL (residues 105-250) and soluble human CD40 ligand
(CD40L) were obtained from Alexis. A preservative-free rabbit anti-APRIL
polyclonal serum (catalog no. 2415) was purchased from ProSci (Poway,
CA). Rabbit anti-BAFF polyclonal serum (catalog no. 07-167) was
obtained from Upstate Biotechnology (Lake Placid, NY). Mouse antihuman BAFF monoclonal antibodies (mAbs) were purchased from either
Research Diagnostics (Flanders, NJ) or ID Labs (London, ON). Rabbit
anti-p38 mitogen-activated protein kinase (MAPK) polyclonal serum
(sc-535) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Goat anti-human IgM F(ab⬘)2 fragments (anti-IgM) were purchased from
Jackson ImmunoResearch (West Grove, PA). Macrophage colonystimulating factor (M-CSF) and IL-4 were obtained from Research
Diagnostics. Human granulocyte-macrophage colony-stimulating factor
(GM-CSF) (Leukine/Sargramostim; Immunex, Seattle, WA) for research
purposes was purchased from the University of Washington School of
Medicine Pharmacy (Seattle).
Generation of CD14ⴙ monocyte-derived human macrophages
and dendritic cells
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral
human blood of anonymous individual donors by centrifugation over
4465
Ficoll-Hypaque (Robbins Scientific, Sunnyvale, CA). T cells were depleted
by rosetting with sheep erythrocytes. CD14⫹ cells were subsequently
isolated by positive selection with magnetic anti-CD14 microbeads according to the manufacturer’s instructions (Miltenyi Biotech, Auburn, CA). For
human monocyte–derived macrophages (subsequently referred to as “macrophages”), CD14⫹ cells (1 ⫻ 106/mL) were cultured in complete RPMI
1640 medium containing 100 ng/mL M-CSF at 37°C in a humidified
incubator for 6 to 8 days. Macrophage preparations were more than 95%
CD14⫹ and were also mannose receptor (MR)⫹, CD71⫹, and CD3⫺, and
many of the cells morphologically appeared spindle-like and adherent.
Human monocyte–derived DCs (subsequently referred to as “mono-DCs”)
were generated in vitro from CD14⫹ cells cultured with GM-CSF (100
ng/mL), IL-4 (30 ng/mL), and 2-mercaptoethanol (50 ␮M) for up to 7 days
and were more than 95% CD14⫺ and strongly CD1a⫹.36
Isolation of dense human tonsillar B cells
Dense B lymphocytes were isolated from human tonsils derived from
different individual donors by sheep erythrocyte rosetting to deplete T cells
and Percoll density gradient centrifugation as described.37 These cells were
more than 95% CD20⫹ and more than 70% naive IgD⫹CD38⫺ B cells and
were the source of all B cells used in this study.
B-cell proliferation assays
Macrophages, mono-DCs, or monocytes (0.3-5 ⫻ 104) or macrophageconditioned culture medium (MCCM) were cultured for 72 hours with
dense tonsillar B cells (2 ⫻ 105) in the presence of anti-IgM (10 ␮g/mL),
CD40 (G28-5) mAb (250 ng/mL), or soluble human CD40L (100 ng/mL) in
the presence of enhancer (1 ␮g/mL) as described by the manufacturer in
96-well flat-bottomed polystyrene culture plates (Becton Dickinson, Franklin Lakes, NJ). Cells were incubated for the final 16 to 24 hours with 0.0185
MBq (0.5 ␮Ci) [3H]-thymidine. Incubations were performed in triplicate,
and results are presented as the mean ⫾ SD. In some experiments,
macrophages, MCCM, mono-DCs, or soluble BAFF or APRIL were
preincubated for at least 30 minutes at 37°C with either 10 ␮g/mL TACI-Ig
or control-Ig fusion protein prior to coculture with B cells.
In some experiments, B cells and either macrophages or mono-DCs
were cultured in separate compartments using Transwells (6.5 mm diameter, 0.4-␮m tissue culture–treated polycarbonate membrane; Costar, Corning, NY). Macrophages or mono-DCs (7.5 ⫻ 104) in a volume of 0.1 mL in
the upper compartment were cultured with B cells (2 ⫻ 105) in a volume of
0.6 mL in the lower chamber for 72 hours. Cells were pulsed for the final 16
to 24 hours with 0.0555 MBq (1.5 ␮Ci) [3H]-thymidine. After 72 hours, B
cells were divided into 3 wells of a flat-bottomed 96-well plate, and
[3H]-thymidine incorporation was quantified by liquid scintillation counting. Results are presented as the mean ⫾ SD of each triplicate.
Immunodepletion of macrophage-conditioned culture medium
Recombinant APRIL or MCCM was incubated for 3 hours at 4°C with
constant mixing with either 0 to 10 ␮g/mL rabbit anti-human APRIL
polyclonal IgG or normal rabbit IgG. Protein A–Sepharose beads (30 ␮L
packed beads) were added for the final 90 minutes to bind immune
complexes. After centrifugation at 14 000g for 5 minutes at 4°C, immunodepleted supernatants were sterile filtered (0.2 ␮m) and cocultured with B
cells in the presence of anti-IgM (10 ␮g/mL) as described above.
Immunoblotting
Whole-cell lysates were prepared from approximately 5 ⫻ 106 cells as
described.38 Lysates (50 ␮g protein) were resolved by 12.5% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Proteins
were transferred to nitrocellulose in non-SDS–containing transfer buffer
(25 mM Tris (tris(hydroxymethyl)aminomethane), 0.2 M glycine, 20%
methanol, pH 8.5). For APRIL immunoblotting, nitrocellulose membranes
were blocked overnight at 4°C with blocking buffer (TBST [Tris-HCl,
NaCl, Tween 20] plus 5% [wt/vol] nonfat dry milk) and subsequently
incubated overnight at 4°C with 1 ␮g/mL rabbit anti-APRIL serum diluted
4466
CRAXTON et al
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
in blocking buffer. As a control for protein loading, nitrocellulose membranes were reprobed with 0.25 ␮g/mL rabbit anti-p38 MAPK serum
diluted in TBST containing 5% (wt/vol) bovine serum albumin (BSA).
BAFF ELISA
High-capacity 96-well enzyme-linked immunosorbent assay (ELISA) plates
(MaxiSorp, Nunc, VWR International, Brisbane, CA) were coated overnight at 4°C with anti-human BAFF mAb (5 ␮g/mL) diluted in phosphatebuffered saline (PBS) as a capture antibody. Following blocking with PBS
containing 1% (wt/vol) BSA for at least 2 hours at room temperature,
culture supernatants or soluble BAFF as a standard were incubated
overnight at 4°C with the anti-BAFF–coated 96-well plates. Samples were
then incubated for 2 hours at room temperature with 2 ␮g/mL rabbit
anti-human BAFF serum and finally with goat anti-rabbit horseradish
peroxidase (HRP) serum (1:2500; Santa Cruz Biotechnology, Santa Cruz,
CA) for 1 hour at room temperature. HRP enzyme activity was quantified
using 3, 3⬘, 5, 5⬘-tetramethylbenzidine (TMB; Sigma, St Louis, MO) as a
substrate at 450 nm. All values are the mean ⫾ SD of triplicate samples.
BAFF and APRIL reverse transcriptase polymerase chain
reaction (RT-PCR)
Total RNA was isolated from cells using a guanidine isothiocyanate-based
method (Isoquick kit; Orca Biosciences, Bothell, WA), followed by a
30-minute incubation at 37°C with 5 to 10 U RNase-free DNase (Promega,
Madison, WI) to digest residual genomic DNA. Total RNA was further
purified using a RNA clean-up protocol supplied with a RNeasy kit
(Qiagen, Valencia, CA). First-strand cDNA was synthesized from total
RNA (1 ␮g) using oligo(dT) primers (0.5 ␮g) and Superscript II reverse
transcriptase (200 U) exactly as described by the manufacturer (Invitrogen,
Carlsbad, CA). APRIL, BAFF, and glyceraldehyde phosphate dehydrogenase (GAPDH) were amplified by PCR using the following gene-specific
primers: APRIL (129 base pair [bp]), 5⬘-CCGATGCCCTGGAAGCCTGG-3⬘ and 5⬘-AGTCATCCTTGGAGGTGGCG-3⬘; BAFF (368 bp),
5⬘-TCACGCCTTACTTCTTGC-3⬘ and 5⬘-GACCCTGAACGGCACGCTTATT-3⬘; GAPDH (452 bp), 5⬘-ACCACAGTCCATGCCATC AC-3⬘ and
5⬘-TCCACCACCCTGTTGCTGTA. PCR reactions were performed using
1 ⫻ thermophilic buffer, 0.5 mM each deoxynucleoside triphosphate
(dNTP), and 0.5 ␮M sense and antisense primers with 1 U Taq polymerase
(Promega) under the following conditions: APRIL, 2 mM MgCl2, 52°C
annealing temperature, 32 cycles; BAFF, 1 mM MgCl2, 50°C annealing
temperature, 35 cycles; GAPDH, 1.5 mM MgCl2, 50°C annealing temperature, 35 cycles. PCR products were resolved on 2.5% or 3% (wt/vol)
NuSieve 3:1 agarose (FMC BioProducts, Rockland, ME) in 1 ⫻ TBE (Tris
Borate EDTA (ethylenediaminetetraacetic acid)) buffer.
Results
Myeloid lineage cells enhance BCR-stimulated human B-cell
proliferation
Earlier studies have shown that monocytes and different populations of in vitro-generated human DCs directly enhance the
proliferation of CD40-activated B cells.39-41 Because macrophages
represent another discrete myeloid cell lineage that interacts with B
cells particularly in the splenic MZ,7 we were interested in testing
whether monocyte-derived macrophages (“macrophages”) may
also directly increase human B-cell proliferation in vitro. Dense
human tonsillar B cells (“B cells”) were stimulated with either
anti-IgM, which crosslinks the BCR, or alternatively treated with
soluble CD40 ligand (CD40L) or CD40 mAb. These serve as a
surrogate for CD40L expressed on activated T cells and also
stimulate both human B cells and macrophages.
In the absence of a costimulus, macrophages induced little
increase in B-cell proliferation (Figure 1A). However, in the
Figure 1. Macrophages and mono-DCs strongly enhance BCR- but not CD40stimulated B-cell proliferation. (A) Macrophages strongly increase BCR-activated
B-cell proliferation. Macrophages were cultured with dense tonsillar B cells in the
absence (䡺) or presence of goat anti-human IgM F(ab⬘)2 fragments (f) for 72 hours.
Cells were incubated for the final 16 to 24 hours with [3H]-thymidine. Incubations were
performed in triplicate, and representative results are presented as the mean ⫾ SD
from 1 of 6 similar experiments. The mean fold maximal increase in BCR-induced
B-cell proliferation by macrophages was 3.9 ⫾ 1.5 in 6 independent experiments. (B)
Effect of macrophages on CD40-induced B-cell proliferation. Macrophages were
incubated with B cells in the absence (‚) or presence of soluble CD40L (Œ), CD40
mAb (F), or an isotype control mAb (MOPC21, E) for 72 hours. Cells were pulsed for
the final 16 to 24 hours with [3H]-thymidine. Incubations were performed in triplicate,
and results are presented as the mean ⫾ SD from 1 of 3 similar experiments. (C)
Dendritic cells strongly enhance BCR-activated B-cell proliferation. Mono-DCs were
cultured with B cells in the absence (䡺) or presence of goat anti-human IgM F(ab⬘)2
fragments (f) for 3 days. Thymidine incorporation was quantified as described in
panel A. Incubations were performed in triplicate, and data are from 1 of 3 similar
experiments. The mean fold maximal enhancement of BCR-induced B-cell proliferation by mono-DCs was 4.0 ⫾ 1.2 in 3 separate experiments. (D) Monocytes weakly
increase BCR-stimulated B-cell proliferation. CD14⫹ monocytes were cultured with B
cells in the absence (䡺) or presence of goat anti-human IgM F(ab⬘)2 fragments (f) for
72 hours. Thymidine incorporation was quantified as described in panel A. Incubations were performed in triplicate, and results are presented as the mean ⫾ SD from 1
of 3 similar experiments. The mean fold maximal increase in BCR-induced B-cell
proliferation by monocytes was 2.2 ⫾ 1.0 in 3 independent experiments. (E) Effect of
dendritic cells on CD40-induced B-cell proliferation. Mono-DCs were incubated with
B cells in the presence of medium (䡺), CD40 mAb (F), or an isotype control MOPC21
mAb (E) for 72 hours. Cells were incubated for the final 16 to 24 hours with
[3H]-thymidine. Incubations were performed in triplicate, and results are presented as
the mean ⫾ SD from 1 of 3 independent experiments.
presence of anti-IgM, macrophages strongly enhanced BCRinduced B-cell proliferation up to 10-fold in a cell number–
dependent manner. The enhanced proliferation of BCR-activated B
cells by macrophages also correlated with increased numbers of
trypan blue–negative B cells, suggesting that macrophages also
increased B-cell survival (data not shown). In contrast, macrophages weakly augmented either CD40L- or anti-CD40–stimulated
B-cell proliferation at low numbers of macrophages relative to
B cells (Figure 1B). Surprisingly, the same high numbers of
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
MACROPHAGE-INDUCED B-CELL PROLIFERATION REQUIRES BAFF
macrophages that strongly enhanced BCR-induced proliferation,
significantly reduced both CD40L- and anti-CD40–induced thymidine incorporation into B cells (Figure 1B). Furthermore, MCCM
also significantly inhibited anti-CD40–stimulated proliferation of
B cells in a dose-dependent manner (data not shown).
Similar to macrophages, mono-DCs also potentiated BCRinduced B-cell proliferation (Figure 1C). Using CD14⫹ cells
isolated from the same donor, in vitro–derived macrophages and
mono-DCs enhanced anti-IgM–induced B-cell proliferation to
similar degrees with a 4- to 6-fold enhancement in thymidine
incorporation. In contrast, CD14⫹ monocytes weakly enhanced
BCR-activated B-cell proliferation only 2-fold (Figure 1D).
Similar to our results with monocyte-derived macrophages
(Figure 1B), mono-DCs also weakly enhanced CD40-stimulated
proliferation of B cells about 2-fold at lower ratios of mono-DCs to
B cells (Figure 1E). In contrast to macrophages, highest numbers of
mono-DCs did not markedly inhibit anti-CD40–induced B-cell
proliferation (Figure 1E).
Macrophages and mono-DCs costimulate B-cell proliferation
via soluble factor(s)
We then tested whether the effect of macrophages on BCRstimulated B-cell proliferation could be mediated by a soluble
factor(s), by incubating MCCM with B cells in the presence or
absence of anti-IgM. In the absence of BCR ligation, MCCM, like
macrophages (Figure 1A), did not significantly increase B-cell
proliferation (Figure 2A). However, in the presence of anti-IgM,
MCCM strongly increased B-cell proliferation in a dose-dependent
manner up to 5-fold (Figure 2A). This effect was independent of
M-CSF in the culture media, which did not itself enhance
BCR-induced B-cell proliferation (Figure 2A).
In addition, we compared the stimulatory effects of macrophages separated from B cells by a permeable membrane in
Figure 2. Macrophages and BCR ligation costimulate B-cell proliferation via
soluble factor(s). (A) Macrophage-conditioned culture medium strongly enhances
BCR-stimulated B-cell proliferation. Serial 2-fold dilutions of 7-day culture supernatants from macrophages (f,䡺) or M-CSF–containing control supernatants (F,E)
were cultured with B cells in either the presence (f,F) or absence (䡺,E) of goat
anti-human IgM F(ab⬘)2 fragments for 72 hours. [3H]-thymidine incorporation was
quantified as described in Figure 1A. Incubations were performed in triplicate, and
results are presented as the mean ⫾ SD of each triplicate from a representative
experiment. The mean fold maximal increase in BCR-induced B-cell proliferation by
macrophage-conditioned culture medium was 3.8 ⫾ 1.4 from 4 independent experiments. (B) Enhancement of BCR-induced B-cell proliferation by macrophages does
not require direct cell-cell contact. Macrophages, BAFF, or APRIL in the upper
compartment were cultured with B cells in the presence of goat anti-human IgM
F(ab⬘)2 fragments in the lower chamber of a 24-well Transwell culture plate for 72
hours. For comparative purposes, macrophages were also cocultured in direct
contact with B cells in a standard 24-well tissue culture plate under identical
conditions. Cells were pulsed for the final 16 to 24 hours with [3H]-thymidine.
[3H]-thymidine incorporation was quantified as described in Figure 1A. Incubations
were performed in triplicate, and data are presented as the mean ⫾ SD from 1 of 2
similar experiments. Similar results were also observed using mono-DCs. No
statistical difference (P ⬍ .05) was observed between the control and Transwell
groups for each of the 3 stimulated conditions (anti-IgM ⫹ BAFF, anti-IgM ⫹ APRIL,
anti-IgM ⫹ macrophages).
4467
Transwells with macrophages cocultured in direct contact with B
cells in standard wells. Soluble forms of the TNF ligands BAFF
and APRIL, which have been shown to augment BCR-induced
B-cell proliferation,19,29 were added to the upper chamber of the
Transwell separate from human B cells in the lower well as controls
to demonstrate that the Transwell membrane was functional
(Figure 2B). Macrophages separated from B cells enhanced
BCR-dependent B-cell proliferation to the same level observed
with macrophages cultured in direct contact with B cells (Figure
2B). Similarly, mono-DCs cultured apart from B cells also
increased BCR-induced B-cell proliferation (data not shown).
These data suggest that direct cell contact is not essential for
macrophages and DCs to increase B-cell proliferation. Thus, the
ability of both macrophages and mono-DCs to enhance BCRdependent human B-cell proliferation is mediated by a
soluble factor(s).
TACI-Ig inhibits macrophage- and mono-DC–induced
costimulation of B-cell proliferation
Two novel, related TNF superfamily members, BAFF and APRIL,
bind to TACI and can be blocked by soluble TACI-Ig.20 Myeloid
lineage cells express the highest levels of BAFF.19,21 Although
BAFF is constitutively expressed by macrophages as both membrane-associated and secreted forms, it is undetectable on the
surface of myeloid DCs.21 However, a soluble form of BAFF is
secreted by DCs.21 In contrast, little is known about APRIL protein
expression in primary cells.27
To test whether BAFF and/or APRIL plays a role in macrophageinduced B-cell costimulation, we preincubated TACI-Ig with either
macrophages or MCCM prior to coculture with B cells. Under
experimental conditions in which TACI-Ig blocked BAFF-induced
B-cell costimulation, TACI-Ig but not a control IgG1 fusion protein
also completely inhibited macrophage-mediated B-cell proliferation (Figure 3A). Similarly, B-cell costimulation via anti-IgM and
MCCM was also strongly blocked by a TACI-Ig but not a control
IgG1 fusion protein (Figure 3A). Anti-IgM did not increase
thymidine incorporation by macrophages themselves. This result
excludes the possibility that proliferation of the macrophages
accounts for the strong increase in BCR-induced B-cell proliferation by macrophages (Figure 3A). Furthermore, TACI-Ig, but not a
control IgG1 fusion protein, also markedly inhibited mono-DC–
enhanced BCR-induced B-cell proliferation (Figure 3B). These
results show that BAFF and/or APRIL are required for macrophage- and DC-induced B-cell costimulation.
Myeloid cells express BAFF and APRIL mRNA and protein;
regulation by cytokines and bacterial cell wall components
Because our results strongly suggest that a macrophage- and
mono-DC–derived TACI ligand mediates B-cell costimulation, we
next examined the expression of BAFF and APRIL in different
human myeloid cells. Initially, RT-PCR was used to quantify
relative mRNA levels. Both BAFF and APRIL were expressed at
similar relative mRNA levels in macrophages, mono-DCs, and
monocytes (Figure 4A, upper and middle panels). APRIL but not
BAFF mRNA was also expressed by dense tonsillar B cells (data
not shown). Reports indicate that BAFF can be processed into a
truncated soluble and biologically active polypeptide.16,19,21 Thus,
we quantified release of soluble BAFF using capture ELISA.
Supernatants from M-CSF–derived macrophages contained BAFF
in the range of 3 to 23 ng/mL during a 6- to 8-day incubation period
from approximately 20 different donors (Figure 4B). Treatment of
4468
CRAXTON et al
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
Figure 3. TACI-Ig inhibits macrophage- and mono-DC–induced costimulation of B-cell proliferation. (A) Enhancement of BCR-induced B-cell proliferation by
macrophages requires a TACI ligand. Macrophages, 7-day conditioned macrophage culture supernatant, or BAFF was preincubated for at least 1 hour at 37°C with PBS,
TACI-Ig, or a control-Ig fusion protein prior to coculture with B cells in the presence of goat anti-human IgM F(ab⬘)2 fragments. [3H]-thymidine incorporation assays were
performed as described in Figure 1. Incubations were performed in triplicate, and results are presented as the mean ⫾ SD from 1 of 3 independent experiments. (B) A TACI
ligand is required for mono-DC–mediated increases in BCR-induced B-cell proliferation. Dendritic cells or BAFF were pretreated for 1 hour at 37°C with PBS, TACI-Ig, or a
control immunoglobulin fusion protein prior to coculture with B cells in the presence of goat anti-human IgM F(ab⬘)2 fragments. [3H]-thymidine incorporation assays were
performed as described in Figure 1. Incubations were performed in triplicate, and results are presented as the mean ⫾ SD from 1 of 2 experiments. (C) TACI-Ig partially reduces
anti-IgM–induced B-cell proliferation. B cells in the presence or absence of BAFF were preincubated for 1 hour at 37°C with PBS, TACI-Ig, or control-Ig fusion protein prior to
culture in the presence of goat antihuman IgM F(ab⬘)2 fragments. [3H]-thymidine incorporation assays were performed as described in Figure 1. Incubations were performed in
triplicate, and results are presented as the mean ⫾ SD of triplicate incubations from 1 of 2 separate experiments.
Figure 4. Expression of BAFF and APRIL in myeloid lineage cells. (A) Myeloid
lineage cells express comparable levels of BAFF and APRIL mRNAs. First-strand
cDNA was synthesized from RNase-free DNase-digested total RNA using oligo(dT)
primers and Superscript II reverse transcriptase. BAFF, APRIL, and GAPDH were
amplified by PCR using gene-specific primers. PCR products were resolved on
NuSieve 3:1 agarose. DNA molecular size markers are indicated in the far left lanes.
(B-C) Macrophages constitutively release BAFF; cytokines and bacterial cell wall
components induce BAFF release from macrophages. Anti-human BAFF mAbcoated ELISA plates were incubated with PBS/BSA for 2 hours at room temperature.
After blocking, culture supernatants or soluble BAFF as a standard was incubated
overnight at 4°C with anti-BAFF–coated plates. Samples were incubated for 2 hours
at room temperature with rabbit anti-human BAFF polyclonal serum and subsequently with goat anti-rabbit HRP for 1 hour at room temperature. HRP enzyme
activity was quantified using TMB as a substrate at 450 nm. All values are the mean ⫾
SD of triplicate samples. (D-E) Macrophages but not mono-DCs or monocytes
constitutively express high relative levels of APRIL protein. APRIL is induced by LPS
and interferon-␥ in mono-DCs. Cell lysates (50 ␮g total protein) from B cells,
macrophages, mono-DCs, and monocytes were resolved by SDS-PAGE, transferred
to nitrocellulose membranes, and immunoblotted with APRIL antiserum (upper panel)
or reprobed with p38 MAPK antiserum (lower panel) as a control for protein loading.
macrophages with either IL-10 or interferon-␥ (IFN-␥) but not IL-4
enhanced BAFF levels approximately 2- to 6-fold compared with
untreated controls, consistent with another study21 (Figure 4C). In
addition, the gram-positive cell wall component, peptidoglycan,
also moderately increased BAFF release 2- to 3-fold (Figure 4C).
APRIL protein was detected by immunoblotting cell lysates
using an APRIL-specific polyclonal antiserum. APRIL was expressed as 2 distinct polypeptides of approximately 25 and 27 kDa
(Figure 4D). The existence of multiple forms of APRIL is
consistent with previous studies that identified 3 alternatively
spliced human APRIL cDNAs (TRDL-1␣, TRDL-1␤, and TRDL1␥) and that are predicted to encode proteins of 27.4, 25.7, and 27.1
kDa, respectively.28 Macrophages expressed high relative levels of
the smaller APRIL polypeptide (APRIL-short, Figure 4D). This
shorter APRIL species was not detected in unstimulated monocytes, mono-DCs, or B cells (Figure 4D). In contrast, the larger
APRIL species (APRIL-long) was expressed at low levels in B
cells, but not significantly expressed in monocytes, mono-DCs, or
macrophages (Figure 4D). Consistent with a minor role for
endogenously produced APRIL driving B-cell proliferation, a
TACI-Ig but not a control-Ig fusion protein partially inhibited
anti-IgM–stimulated B-cell proliferation (Figure 3C). Furthermore,
B-cell and myelocytic leukemia tumor-derived cell lines (Raji,
T5-1, HL-60, and THP-1) exclusively expressed high levels of the
larger APRIL species (data not shown), consistent with reported
mRNA expression data.27 Treatment of mono-DCs with either
lipopolysaccharide (LPS) or IFN-␥ markedly increased levels of
the smaller APRIL species but not to the same levels expressed in
macrophages (Figure 4E). Neither LPS nor IFN-␥ further enhanced
the constitutively very high levels of APRIL observed in
macrophages.
BAFF strongly costimulates BCR- but not CD40-induced
human B-cell proliferation
Because macrophages and mono-DCs express both BAFF and
APRIL21 (Figure 4), we compared the effects of recombinant
BAFF and APRIL on the proliferation of B cells. In the absence of a
costimulus, soluble BAFF induced moderate increases in B-cell
proliferation (Figure 5A). However, in the presence of anti-IgM,
BAFF strongly costimulated B-cell proliferation more than 10-fold
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
MACROPHAGE-INDUCED B-CELL PROLIFERATION REQUIRES BAFF
4469
Figure 5. BAFF strongly enhances BCR- but not CD40-induced proliferation of B cells. (A) BAFF strongly augments BCR-activated B-cell proliferation. B cells were
incubated with the indicated doses of BAFF in the presence (f) or absence (䡺) of goat anti-human IgM F(ab⬘)2 fragments (10 ␮g/mL) for 72 hours. [3H]-thymidine incorporation
was determined as described in Figure 1. Incubations were performed in triplicate, and results are presented as the mean ⫾ SD from 1 of 4 experiments. (B) APRIL moderately
increases BCR-induced B-cell proliferation. B cells were treated with APRIL in the presence (f) or absence (䡺) of goat anti-human IgM F(ab⬘)2 fragments (10 ␮g/mL) for 72
hours. [3H]-thymidine incorporation was determined as described in Figure 1. Assays were performed in triplicate, and results are presented as the mean ⫾ SD from 1 of 2
similar experiments. (C) Effect of BAFF on CD40 ligand-stimulated B-cell proliferation. B cells were treated for 72 hours with increasing doses of BAFF in the absence (䡺) or
presence of soluble human CD40L at the following concentrations: 20 ng/mL (f), 50 ng/mL (E), or 100 ng/mL (F). [3H]-thymidine incorporation was determined as described in
Figure 1. Assays were performed in triplicate, and results are presented as the mean ⫾ SD from 1 of 3 similar experiments.
(Figure 5A). Importantly, BAFF levels (3 ng/mL) that maximally
costimulated BCR-induced B-cell proliferation were within the
range of BAFF levels constitutively released by macrophages
(Figure 4B). Similar to BAFF, soluble recombinant APRIL also
enhanced BCR-induced B-cell proliferation 2- to 3-fold, but the
concentrations of APRIL required to costimulate B-cell proliferation were more than 50-fold higher compared with BAFF (Figure
5B). We also tested the effect of soluble BAFF on CD40L-induced
B-cell proliferation. BAFF increased CD40L-induced B-cell proliferation less than 2-fold over a wide range of CD40L and BAFF
concentrations (Figure 5C). These data correlate with our earlier
results showing that macrophages and mono-DCs only weakly
augment CD40-induced proliferation of B cells (Figure 1B).
Macrophage-induced B-cell proliferation does not
require APRIL
To further distinguish between a possible role for BAFF or APRIL,
we immunodepleted APRIL using an APRIL antiserum. APRILdependent increases in BCR-induced B-cell proliferation were
completely blocked by preincubation of purified recombinant
APRIL with the APRIL antiserum but not control serum (Figure 6).
In contrast, MCCM-mediated enhancement of anti-IgM–induced
B-cell proliferation was unaffected following preincubation with
anti-APRIL (Figure 6). These results together with the observation
that APRIL is much less effective than BAFF in stimulating B cells
Figure 6. Immunodepletion of APRIL does not affect macrophage-induced
costimulation of B cells. Macrophage-conditioned culture supernatant or recombinant human APRIL were incubated for 3 hours at 4°C with constant mixing with PBS,
purified rabbit anti-human APRIL IgG, or control IgG (10 ␮g/mL). Protein A–Sepharose beads were added for the final 90 minutes to bind immune complexes. After
centrifugation at 4°C, immunodepleted supernatants were sterile filtered and cocultured with dense tonsillar B cells in the presence of anti-human IgM F(ab⬘)2 fragments
(10 ␮g/mL) as described in Figure 1. The asterisk indicates statistically significant
differences between samples within the bracketed groups (P ⬍ .05).
and also that APRIL protein is not expressed in unstimulated DCs
(Figure 4D-E, Figure 5), strongly suggest that BAFF is the major
macrophage- and mono-DC–derived factor costimulating BCRactivated human B cells.
Discussion
These studies show that both human monocyte–derived macrophages and DCs directly enhance proliferation of BCR-activated
human B cells via a soluble factor(s) (Figures 1-2). Moreover,
macrophage- and mono-DC–induced B-cell costimulation was
inhibited by a TACI-Ig fusion protein (Figure 3), which binds 2
known ligands, BAFF and APRIL. Although macrophages express
both BAFF and APRIL protein (Figure 4), immunodepletion of
APRIL did not block macrophage-induced B-cell costimulation
consistent with the finding that BAFF is the major factor required
for the enhancement of BCR-induced B-cell proliferation by
macrophages (Figure 6). Consistent with these results and as
previously reported,21 BAFF was secreted by macrophages at
levels sufficient to potently costimulate B-cell proliferation (Figures 4-5). Furthermore, BAFF was at least 50-fold more potent than
APRIL in augmenting anti-IgM–dependent proliferation of tonsillar B cells (Figure 5). Finally, there was no correlation between the
high levels of APRIL protein expressed in macrophages compared
with mono-DCs and the similar abilities of these 2 myeloid cell
types to costimulate B-cell proliferation (Figures 1 and 4D). This
finding is consistent with the model that APRIL does not play a
major role in augmenting BCR-mediated B-cell proliferation.
Indeed, a possible role for APRIL in regulating B-cell homeostasis
in vivo remains to be shown, as APRIL-deficient mice die in
utero.42 These results are also consistent with previous studies
using mice deficient in various BAFF receptors, which strongly
suggest a major positive regulatory role for BAFF-R, which binds
only BAFF, but not TACI or BCMA, in BCR-induced B-cell
costimulation.25,26,33,34,43
Our findings are consistent with a role in vivo for human
macrophage– and DC-derived BAFF in TI type 2 humoral immune
responses, in which macrophages and DCs located within the
splenic MZ produce and secrete BAFF. Soluble BAFF may
subsequently serve a supporting role in the generation of antigenspecific plasmablasts, as shown by a recent study in which
bacteria-primed splenic macrophages and DCs provide a survival
signal to MZ B cells.11
4470
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
CRAXTON et al
DCs play an established role in stimulating antigen-specific
naive T cells to proliferate, secrete cytokines, and express CD40L.
These activated T cells subsequently stimulate antigen-specific
naive B lymphocytes to proliferate and differentiate into germinal
center (GC) B cells or IgM-secreting plasma cells.44 Additional
studies also showed that DCs directly enhance proliferation and
immunoglobulin production of CD40-activated naive and memory
B cells.39,41 Although IL-12 and soluble IL-6R-␣ chain (spg 80) are
required for the differentiation of naive B cells into IgM-secreting
plasma cells,40,45 the mechanism(s) by which DCs directly costimulate B-cell growth remain uncharacterized. The factor(s) described
by Dubois et al39 that enhanced B-cell proliferation appeared to be
both soluble and produced in a CD40-independent manner. Similarly, we found using Transwells that both DCs and macrophages
enhance BCR-induced B-cell proliferation through the generation
of a predominantly soluble factor(s) (Figure 2). Nevertheless, it is
unlikely that BAFF is the DC-derived factor that enhances
CD40-activated proliferation of human tonsillar B cells,39,41 because BAFF only weakly enhances CD40-induced proliferation of
dense human tonsillar B cells (Figure 5C). In related studies,
macrophages, which strongly enhanced BCR-induced proliferation
(Figure 1A) and released BAFF at levels sufficient to potently
costimulate B-cell proliferation (Figure 4B), inhibited CD40Lstimulated proliferation of B cells (Figure 1B, data not shown),
suggesting that macrophages may differentially regulate B-cell
responses to TI versus TD B-lymphocyte signals.
Although our results suggest that APRIL does not appear to play
a major role in macrophage-induced costimulation of B cells
(Figures 4-6), we show for the first time that APRIL protein is
differentially expressed in myeloid lineage cells, with high constitutive levels observed in macrophages but not mono-DCs or
monocytes (Figure 4D). Our results also indicate that APRIL
expression may be regulated at the posttranscriptional level,
because APRIL mRNA was present in all 3 myeloid cell lineages
examined, yet high levels of protein were only observed in
macrophages (Figure 4). In addition, APRIL may be regulated after
translation via cleavage and secretion. It is currently unknown
whether endogenous APRIL is processed and released by macrophages, although recent studies using APRIL-transfected HEK293
cells show that APRIL, like BAFF, can be released as a soluble and
biologically active ligand.31 We were unsuccessful in attempts to
establish an APRIL-specific ELISA using a panel of commercially
available APRIL antisera to examine its possible release by
macrophages and mono-DCs, as we and others have observed for
BAFF21 (Figure 4B-C).
Although our studies were performed using dense human
tonsillar human B cells, a subset of these B cells, known as
subepithelial (SE) B cells, have many of the morphologic, phenotypic, and functional characteristics of splenic MZ B cells.46,47
These small, CD5⫺ SE B cells represent about 5% to 10% of the
total tonsillar B cells. Interestingly, SE B cells, but neither germinal
center nor follicular mantle tonsillar B-cell subsets, specifically
responded to the TI-II antigen, TNP-Ficoll, consistent with their
identification as a tonsillar B-cell counterpart of splenic MZ B
cells. Further experiments are necessary to examine whether
distinct human tonsillar B-cell subsets (naive, germinal center,
memory, and SE) differentially respond to BAFF.
In conclusion, our results demonstrate that monocyte-derived
macrophages and DCs directly enhance BCR-mediated human
B-cell proliferation via a soluble factor(s), which is specifically
blocked by TACI-Ig.
Acknowledgments
We thank Dr Jane Gross and colleagues (Zymogenetics Inc, Seattle,
WA) for generously providing purified soluble recombinant FLAGtagged human BAFF and TACI-Ig fusion protein. We are grateful
to Drs Helen Floyd and Hiro Niiro for critical reading and
comments during the preparation of this manuscript.
References
1. Mond JJ, Lees A, Snapper CM. T cell-independent antigens type 2. Annu Rev Immunol. 1995;
13:655-692.
2. Kehry MR. CD40-mediated signaling in B cells.
Balancing cell survival, growth and death. J Immunol. 1996;156:2345-2348.
3. Craxton A, Otipoby KL, Jiang A, Clark EA. Signal
transduction pathways that regulate the fate of B
lymphocytes. Adv Immunol. 1999;73:79-152.
4. Snapper CM, Mond JJ. A model for the induction
of T cell-independent humoral immunity to polysaccharide antigens. J Immunol. 1996;157:22292233.
5. Spencer J, Perry ME, Dunn-Walters DK. Human
marginal-zone B cells. Immunol Today 1998;19:
421-426.
6. Vos Q, Lees A, Wu Z-Q, Snapper CM, Mond JJ.
B-cell activation by T-cell-independent type 2 antigens as an integral part of the humoral immune
response to pathogenic microorganisms. Immunol Rev. 2000;176:154-170.
10. Colino J, Shen Y, Snapper CM. Dendritic cells
pulsed with intact Streptococcus pneumoniae
elicit both protein- and polysaccharide-specific
immunoglobulin isotype responses in vivo
through distinct mechanisms. J Exp Med. 2002;
195:1-13.
11. Balázs M, Martin F, Zhou T, Kearney JF. Blood
dendritic cells interact with splenic marginal zone
B cells to initiate T-independent immune responses. Immunity. 2002;17:341-352.
12. Chused TM, Kassan SS, Mosier DE. Macrophage
requirement for the in vitro response to TNP ficoll:
a thymic independent antigen. J Immunol. 1976;
116:1579-1581.
13. Boswell HS, Sharrow SO, Singer A. Role of accessory cells in B cell activation. I. Macrophage
presentation of TNP-ficoll: evidence for macrophage-B cell interaction. J Immunol. 1980;124:
989-996.
7. Fagarasan S, Honjo T. T-independent immune
response: new aspects of B cell biology. Science.
2000;290:89-92.
14. Corbel C, Melchers F. Requirement for macrophage or for macrophage- or T cell-derived factors in the mitogenic stimulation of murine B lymphocytes by lipopolysaccharides. Eur J Immunol.
1983;13:528-533.
8. Guinamard R, Okigaki M, Schlessinger J,
Ravetch JV. Absence of marginal zone B cells in
Pyk-2-deficient mice defines their role in the humoral response. Nat Immunol. 2000;1:31-36.
15. Sinha AA, Guidos C, Lee K-C, Diener E. Functions of accessory cells in B cell responses to thymus-independent antigens. J Immunol. 1987;138:
4143-4149.
9. Crowley MT, Reilly CR, Lo D. Influence of lymphocytes on the presence and organization of
dendritic cell subsets in the spleen. J Immunol.
1999;163:4894-4900.
16. Schneider P, MacKay F, Steiner V, et al. BAFF, a
novel ligand of the tumor necrosis factor family,
stimulates B cell growth. J Exp Med. 1999;189:
1747-1756.
17. Shu H-B, Hu W-H, Johnson H. TALL-1 is a novel
member of the TNF family that is down-regulated
by mitogens. J Leukoc Biol. 1999;65:680-683.
18. Mukhopadhyay A, Ni J, Zhai Y, Yu G-L, Aggarwal
BB. Identification of a novel cytokine, THANK, a
TNF homologue that activates apoptosis, nuclear
factor-kappaB, and c-Jun NH2 terminal kinase.
J Biol Chem. 1999;274:15978-15981.
19. Moore PA, Belvedere O, Orr A, et al. BLyS: member of the tumor necrosis factor family and B lymphocyte stimulator. Science. 1999;285:260-263.
20. Gross JA, Johnston J, Mudri S, et al. TACI and
BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease. Nature.
2000;404:995-999.
21. Nardelli B, Belvedere O, Roschke V, et al. Synthesis and release of B-lymphocyte stimulator
from myeloid cells. Blood. 2001;97:198-204.
22. Von Bulow G-U, Bram RJ. NF-AT activation induced by a CAML-interacting member of the tumor necrosis factor receptor superfamily. Science. 1997;278:138-141.
23. Laabi Y, Gras MP, Carbonnel F, et al. A new gene,
BCM, on chromosome 16 is fused to the interleukin 2 gene by a t(4;16)(q26;p13) translocation in a
malignant T cell lymphoma. EMBO J. 1992;11:
3897-3904.
24. Madry C, Laabi Y, Callebaut I, et al. The characterization of murine BCMA gene defines it as a
new member of the tumor necrosis factor receptor superfamily. Int Immunol. 1998;10:1693-1702.
25. Thompson JS, Bixler SA, Qian F, et al. BAFF-R, a
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
MACROPHAGE-INDUCED B-CELL PROLIFERATION REQUIRES BAFF
newly identified TNF receptor that specifically interacts with BAFF. Science. 2001;293:2108-2111.
26. Yan M, Brady JR, Chan B, et al. Identification of a
novel receptor for B lymphocyte stimulator that is
mutated in a mouse strain with severe B cell deficiency. Curr Biol. 2001;11:1547-1552.
27. Hahne M, Kataoka T, Schröter M, et al. APRIL, a
new ligand of the tumor necrosis factor family,
stimulates tumor cell growth. J Exp Med. 1998;
188:1185-1190.
28. Kelly K, Manos E, Jensen G, Nadauld L, Jones
DA. APRIL/TRDL-1, a tumor necrosis factor-like
ligand, stimulates cell death. Cancer Res. 2000;
60:1021-1027.
29. Marsters SA, Yan M, Pitti RM, Haas PE, Dixit VM,
Ashkenazi A. Interaction of the TNF homologues
BLyS and APRIL with the TNF receptor homologues BCMA and TACI. Curr Biol. 2000;10:785788.
30. Wu Y, Bressette D, Carrell JA, et al. Tumor necrosis factor (TNF) receptor superfamily member
TACI is a high affinity receptor for TNF family
members APRIL and BLyS. J Biol Chem. 2000;
275:35478-35485.
31. López-Fraga M, Fernández R, Albar JP, Hahne N.
Biologically active APRIL is secreted following
intracellular processing in the golgi apparatus by
furin convertase. EMBO Rep. 2001;2:945-951.
32. Mackay F, Woodcock SA, Lawton P, et al. Mice
transgenic for BAFF develop lymphocytic disor-
33.
34.
35.
36.
37.
38.
39.
ders along with autoimmune manifestations. J
Exp Med. 1999;190:1697-1710.
Von Bulow G-U, van Duersen JM, Bram RJ.
Regulation of the T-independent humoral response by TACI. Immunity. 2001;14:573-582.
Yan M, Wang H, Chan B, et al. Activation and accumulation of B cells in TACI-deficient mice. Nat
Immunol. 2001;2:638-643.
Do RKG, Hatada E, Lee H, Tourigny MR, Hilbert
D, Chen-Kiang S. Attenuation of apoptosis underlies B lymphocyte stimulator enhancement of humoral immune responses. J Exp Med. 2000;192:
953-964.
Sallusto F, Lanzavecchia A. Efficient presentation
of soluble antigen by cultured human dendritic
cells is maintained by granulocyte/macrophage
colony-stimulating factor plus interleukin 4 and
downregulated by tumor necrosis factor-␣. J Exp
Med. 1994;179:1109-1118.
Clark EA, Shu G, Ledbetter JA. Role of the Bp35
cell surface polypeptide in human B-cell activation. Proc Natl Acad Sci U S A. 1985;82:17661770.
Craxton A, Jiang A, Kurosaki T, Clark EA. Syk and
bruton’s tyrosine kinase are required for B cell
antigen receptor-mediated activation of the kinase Akt. J Biol Chem. 1999;274:30644-30650.
Dubois B, Vanbervliet B, Fayette J, et al. Dendritic cells enhance growth and differentiation of
CD40-activated B lymphocytes. J Exp Med. 1997;
185:941-951.
4471
40. Dubois B, Massacrier C, Vanbervliet B, et al. Critical role of IL-12 in dendritic cell-induced differentiation of naı̈ve B lymphocytes. J Immunol. 1998;
161:2223-2231.
41. Dubois B, Bridon J-M, Fayette J, et al. Dendritic
cells directly modulate B cell growth and differentiation. J Leukoc Biol. 1999;66:224-230.
42. Mackay F, Mackay CR. The role of BAFF in B-cell
maturation, T-cell activation and autoimmunity.
Trends Immunol. 2002;23:113-115.
43. Xu S, Lam K-P. B-cell maturation protein, which
binds the tumor necrosis factor family members
BAFF and APRIL, is dispensable for humoral immune responses. Mol Cell Biol. 2001:21;40674074.
44. Liu Y-J, Arpin C. Germinal center development.
Immunol Rev. 1997;156:111-126.
45. Dubois B, Barthélémy C, Durand I, Liu Y-J, Caux
C, Brière F. Toward a role of dendritic cells in the
germinal center reaction: triggering of B cell proliferation and isotype switching. J Immunol. 1999;
162:3428-3436.
46. Dono M, Burgio VL, Tacchetti C, et al. Subepithelial B cells in the human palatine tonsil. I Morphologic, cytochemical and phenotypic characterization. Eur J Immunol. 1996;26:2035-2042.
47. Dono M, Zupo S, Augliera A, et al. Subepithelial B
cells in the human palatine tonsil. II. Functional
characterization. Eur J Immunol. 1996;26:20432049.