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Antigen Receptor Engagement Selectively
Induces Macrophage Inflammatory Protein-1
α (MIP-1α) and MIP-1β Chemokine
Production in Human B Cells
Roman Krzysiek, Eric A. Lefèvre, Weiping Zou, Arnaud
Foussat, Jérôme Bernard, Alain Portier, Pierre Galanaud and
Yolande Richard
J Immunol 1999; 162:4455-4463; ;
http://www.jimmunol.org/content/162/8/4455
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Copyright © 1999 by The American Association of
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References
Antigen Receptor Engagement Selectively Induces Macrophage
Inflammatory Protein-1a (MIP-1a) and MIP-1b Chemokine
Production in Human B Cells1
Roman Krzysiek, Eric A. Lefèvre, Weiping Zou, Arnaud Foussat, Jérôme Bernard,
Alain Portier, Pierre Galanaud, and Yolande Richard2
C
hemokines ensure the continuous recirculation of immune cells among the various anatomical microenvironments, which is essential for maintaining immunological
homeostasis in vivo. They also control the selective recruitment of
specific subsets of leukocytes at sites of immune responses and
inflammation (1–3). Chemokines rapidly stimulate integrin-dependent lymphocyte adhesion and are important regulators of other
biological functions, including lymphocyte activation/differentiation, and cytotoxicity (4, 5). They are immobilized by components
of the extracellular matrix or are sequestered on the cell surface by
membrane-bound glycosaminoglycans and create the local gradient of attractant required for the selective compartmentalization of
immunocompetent cells and for direct cell-to-cell interactions (6,
7). Much is known about T cell trafficking in lymphoid tissue, but
few reports have focused on the production of chemokines by B
cells or the regulation of B cell migration.
One recently identified chemokine, B cell-attracting chemokine
1, is involved in the B cell positioning in the primary follicles of
the spleen and Peyer’s patches (8, 9). Another CXC chemokine,
stromal cell-derived factor-1a (SDF-1a),3 expressed around ger-
Institut National de la Santé et de la Recherche Médicale, Unit 131, Institut Paris-Sud
sur les Cytokines, Clamart, France
Received for publication October 7, 1998. Accepted for publication January 21, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants and a fellowship (to E.A.L.) from the Agence
Nationale de Recherche sur le SIDA, Institut National de la Santé et de la Recherche
Médicale, and the Association Claude Bernard, the Université Paris-Sud.
2
Address correspondence and reprint requests to Dr. Yolande Richard, Institut National de la Santé et de la Recherche Médicale, Unit 131, 32 rue des Carnets, 92 140
Clamart, France. E-mail address: [email protected]
3
Abbreviations used in this paper: SDF, stromal cell-derived factor; GC, germinal
center; BCR, B cell Ag receptor; FDC, follicular dendritic cells; MIP, macrophage
inflammatory protein; PE, phycoerythrin; CCR, CC chemokine receptor; CM, complete medium; SAC, Staphylococcus aureus Cowan I; MCP, monocyte chemotactic
Copyright © 1999 by The American Association of Immunologists
minal centers (GC), may attract lymphocytes to this site (10). Two
functionally related CC chemokines, C6kine/Exodus-2/secondary
lymphoid tissue chemokine and MIP-3b/Exodus-3/CKb11/EBVinduced molecule 1 ligand, are probably major mediators of lymphocyte trafficking into and through the secondary lymphoid organs (11, 12). Both chemokines preferentially attract naive T cells,
so they are probably not involved in cognate T/B cell interactions.
Ag receptor engagement profoundly affects the migration of mature B cells, blocking their homing to primary follicles (13, 14).
Indeed, B cells responding to Ag undergo arrest in the outer T cell
zone and proliferate in response to ligation of a critical number of
B cell Ag receptors (BCR) (15). Thereafter, maturation of the B
cell response depends on the availability of primed T cells and the
ability of Ag-specific B cells to selectively recruit them. Ag-binding B cells are programmed to die unless they are rescued by
signals delivered by primed T cells (15, 16). Ag-specific B cells
must develop efficient mechanisms enabling them to establish cognate T/B interactions to stop the BCR-induced cell death program.
This first step of cognate T/B interactions takes place rapidly after
Ag stimulation in vivo and is followed by the migration of selected
lymphocytes into the follicles (14, 15, 17). The subsequent differentiation of B cells within the follicles requires further interactions
not only with Ag-specific T cells but also with follicular dendritic
cells (FDC). These interactions generate a secondary repertoire of
high affinity Abs and memory B cells (18). Data obtained in vivo
suggest the existence of highly sophisticated control mechanisms
ensuring the rapid colocalization of Ag-specific T and B cells at
precise anatomical sites during an ongoing immune response, but
the factors regulating this process are unknown.
Here we show that Ag receptor engagement, but not stimulation by
CD40 mAb and/or IL-4, induced the coordinated production of two T
cell chemoattractants, MIP-1a and MIP-1b, by B cells. In chemotaxis
protein; sIg, surface immunoglobulin; DC, dendritic cells; sn B SAC, conditioned
medium from SAC-activated B cells.
0022-1767/99/$02.00
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We show herein that B cell Ag receptor (BCR) triggering, but not stimulation by CD40 mAb and/or IL-4, rapidly induced the
coordinated expression of two closely related T cell chemoattractants, macrophage inflammatory protein-1b (MIP-1b) and MIP1a, by human B cells. Naive, memory, and germinal center B cells all produced MIP-1a/b in response to BCR triggering. In
contrast to MIP-1a/b, IL-8, which is spontaneously produced by germinal center B cells but not by naive and memory B cells, was
not regulated by BCR triggering. Culturing follicular dendritic cell-like HK cells with activated B cells did not regulate MIP-1a/b
production, but it did induce production of IL-8 by HK cells. Microchemotaxis assays showed that CD41CD45RO1 T cells of the
effector/helper phenotype actively migrated along a chemotactic gradient formed by BCR-stimulated B cells. This effect was
partially blocked by anti-MIP-1b and anti-CC chemokine receptor 5 Ab, but not by anti-MIP-1a Ab suggesting that MIP-1b plays
a major role in this chemoattraction. Since maturation of the B cell response to a peptide Ag is mostly dependent on the availability
of T cell help, the ability of Ag-stimulated B cells to recruit T cells via MIP-1a/b, may represent one possible mechanism enabling
cognate interactions between rare in vivo Ag-specific T and B cells. The Journal of Immunology, 1999, 162: 4455– 4463.
4456
BCR TRIGGERING INDUCES MIP-1a/b PRODUCTION BY HUMAN B CELLS
assays conditioned medium from BCR-stimulated B cells had chemotactic activity and selectively recruited T cells of the helper/effector
phenotype. Thus, BCR-activated B cells may create chemoattractive
gradients favoring cognate interactions with T cells.
Materials and Methods
Flow cytometric analysis
Cell surface Ags were detected using the following mAbs: anti-CD19-PE,
anti-CD14-FITC, anti-CD62L-FITC, and anti-CD44-FITC (all from Diaclone, Besançon, France); anti-IgM-FITC (Southern Biotechnology Associates, Birmingham, AL); anti-IgD-PE (PharMingen, San Diego, CA); antiCD38-PE and anti-CD95-PE (from Becton Dickinson, Mountain View,
CA); and anti-CD20-FITC (Immunotech, Marseille, France). The CD77
Ag was visualized by indirect immunofluorescence with uncoupled rat antiCD77 mAb, provided by J. Wiels (19), and FITC-conjugated goat anti-rat
IgM Ab (SBA). Anti-CD4, -CD3, and -CD8 mAbs (Becton Dickinson);
anti-CD45RO and -CD45RA mAbs (Diaclone); and anti-CCR5 (2D7)
mAb (National Institute of Biological Standards and Controls, Potters Bar,
U.K.) and then by dichlorotriazinylaminofluorescein (DTAF)-conjugated
goat anti-mouse IgG (H1L) F(ab9)2 (Immunotech) were used for indirect
immunofluorescence. For intracellular labeling, cells were permeabilized
with saponin before staining with uncoupled anti-Ki67 (Dako, Glostrup,
Denmark), and anti-bcl2 (Becton Dickinson) mAbs and then by DTAFconjugated goat anti-mouse IgG (H1L) F(ab9)2 (Immunotech) as previously described (20). Mouse isotype-matched FITC- and PE-conjugated
control Igs were purchased from Diaclone and Becton Dickinson, respectively. Uncoupled control mouse Igs were purchased from ICN (Costa
Mesa, CA). A FACScan flow cytometer (Becton Dickinson) with a logarithmic scale was used for immunofluorescence analysis. After gating on
viable cells, 5000 cells/sample were analyzed.
B and T cell preparations
Human mononuclear cells were obtained from palatine tonsils removed from
children with chronic tonsillitis by gentle dissociation with forceps. B cellenriched populations were obtained by one cycle of rosette formation and
depletion of residual T cells with CD2 magnetic beads (Dynabeads M-450,
Dynal, Oslo, Norway). The resulting B cell populations consistently contained
$95% CD191, #1% CD141, and #1% CD31 and DRC11 cells. For some
experiments tonsillar B cells were separated into IgD1 and IgD2 populations
by incubating them for 30 min with anti-IgD mAb (IADB6, SBA) and removing IgD1 cells with goat anti-mouse IgG magnetic beads (Dynal). IgD2
B cells were further separated into CD441 and CD442 B cells using a similar
protocol with CD44 mAb (BF24, Diaclone). All purification procedures were
conducted at 4°C to prevent apoptosis. The B cell phenotypes of the various
subsets are illustrated in Fig. 1.
T cells were recovered from the first rosetting cell fraction and were
depleted of residual B cells using CD19 magnetic beads (Dynal). T cells
were further depleted of CD81 and CD45RA1 cells by incubation for 30
min with CD8 magnetic beads (Dynal) and CD45RA (ALB11, Immunotech)-coated goat anti-mouse IgG magnetic beads (Dynal). The resulting
populations contained $95% CD41CD45RO1 T cells.
B and T cell stimulation
All cells were cultured in RPMI 1640 medium (Life Technologies, Paisley,
Scotland) containing 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate, and 10% heatinactivated FCS (complete medium (CM)). B cells (1 3 106 cells/ml) were
activated by incubation in CM for 2 days, unless otherwise indicated, with
polyclonal anti-IgM Ab coupled to beads (Irvine Scientific, Santa Anna,
CA; 5 mg/ml), CD40 mAb (G28.5; 1 mg/ml), IL-4 (Schering Plough, Kenilworth, NJ; 20 ng/ml), or a combination of these. In some experiments B
cells were also stimulated in CM by Staphylococcus aureus Cowan I (SAC;
1/104, v/v). The concentration of endotoxin in the culture medium and the
concentrations of the reagents used were consistently ,1 ng/ml.
Purified CD41CD45RO1 tonsillar T cells (1 3 106 cells/ml) were cultured in 12-well plates in CM for 12 days with 400 IU/ml IL-2 (Chiron,
Amsterdam, The Netherlands). These IL-2-conditioned T cells were .95%
CD45RO1, .80% CD41, ,20% CD8low, .75% CCR51, ,2% CD141,
and ,1% CD201 as assessed by flow cytometry.
HK cell line and cocultures with B cells
The HK cell line was obtained from Y. S. Choi (Alton Ochsner Medical
Foundation, New Orleans, LA). It has been shown to preferentially adhere
to and cooperate with GC B cells (21). The HK cell line was treated with
13 trypsin/EDTA (Life Technologies) and was cultured at a density of 105
cells/ml in CM for 3 days. On the third day, the supernatant was discarded,
and HK cells were cultured in fresh CM for 4 more days. For the coculture
assay, HK cells were gamma irradiated at 30 grays (137Cs source) 1 day
before coculture and were seeded at 2 3 103 cells/well in 96-well microtiter plates (Costar, Cambridge, MA). B cells (105/well) were added and
cultured with HK cells either alone or with various stimuli in 200 ml of
CM, for 2 (chemokine production) or 3 (proliferation assay) days. In some
experiments HK cells were seeded at 2 3 103 cells/well in 96-well microtiter plates for 4 h, treated by incubation with 1% paraformaldehyde in 13
PBS for 15 min, and extensively washed before coculture with B cells.
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FIGURE 1. Cell surface phenotype of human tonsillar B cell subsets. A, Tonsillar B cell subsets were isolated according to the expression of surface
IgD and CD44 antigen as described in Materials and Methods. B, Phenotypic analysis by flow cytometry showed that the total B cell population was 93 6
4% CD191, 56 6 5% IgM1, 59 6 6% IgDhigh, 81 6 16% CD441, 21 6 6% CD38high, 15 6 7% CD77high, 42 6 12% CD951, 21 6 6% Ki671, 71 6
12% Bcl21, and 42 6 6% CD62L1; naive B cells were 96 6 4% CD191, 88 6 13% IgM1, 83 6 4 IgDhigh, 97 6 2 CD441, 7 6 3% CD38high, 3 6 3%
CD77high, 7 6 3% CD951, 2 6 2% Ki671, 90 6 8% Bcl21, and 74 6 10% CD62L1; memory B cells were 92 6 6% CD191, 33 6 12% IgM1, 20 6
6 IgDhigh, 73 6 17% CD441, 10 6 10% CD38high, 3 6 3% CD77high, 54 6 8% CD951, 4 6 2% Ki671, 86 6 4% Bcl21, and 73 6 13% CD62L1; and
GC B cells were 94 6 3% CD191, 13 6 2% IgM1, 11 6 4% IgDhigh, 11 6 10% CD441, 90 6 2% CD38high, 44 6 12% CD77high, 84 6 4% CD951,
75 6 2% Ki671, 7 6 4% Bcl21, and 13 6 5% CD62L1. Data are representative of five independent experiments.
The Journal of Immunology
4457
Table I. QC-RT-PCR amplification primersa
Sequence of Primers for PCR
MIP-1a
MIP-1b
b-actin
Sequence of Biotin-Labeled Primers
Sense
Antisense
Recognizing the cDNA
Recognizing the competitor
59-GTCATCTTCC
TAACCAAGCG-39
59-AGGAAGCTTC
CTCGCAACTT-39
59-GGGTCAGAAG
GATTCCTATG-39
59-TGTGGCTGTT
TGGCAACAAC-39
59-AGTCCTGAGT
ATGGAGGAGA-39
59-GGTCTCAAAC
ATGATCTGGG-39
59-biotin GCCGGCAGG
TCTGTGCTGACCCC-39
59-biotin CCAGCAGCC
TCTGCTCCCAGCC-39
59-biotin CGACGAGGC
CCAGAGCAAGAGA-39
59-biotin GGGATCCTT
TGCATCGAACT-39
59-biotin AGACCCCA
GCAGAGAATGGAA-39
59-biotin GTGAGGGAC
ATGCTCACGCAGC-39
a
The software program BISANCE (Université Paris V, CITI 2) was used for designing the primer pairs and selecting annealing temperatures using
sequences obtained directly form GenBank, with the exception of the b-actin sequences.
Proliferation assays
Chemokine production
Cell-free supernatants of 2 3 105 B cells/well were collected on day 2
unless otherwise indicated and were stored at 220°C until tested. Chemokine production was measured with specific ELISA kits purchased from
R&D Systems (MIP-1a and MIP-1b) and Diaclone (IL-8) according to the
manufacturer’s recommendations. Results are expressed as the mean concentration (picograms per milliliter; 6SD) of triplicate determinations.
Competitive RT-PCR
RNA was extracted from 10 3 106 B cells using RNAzol (Bioprobe, Systems, Montreuil, France) and was treated with 10 U of RNase-free DNase
(Boehringer Mannheim, Meylan, France) for 30 min at 37°C. The mixture
was subjected to phenol/chloroform extraction, and RNA was precipitated
in ethanol, recovered by centrifugation, and suspended in 10 ml of water.
The cDNA was prepared by reverse transcription using Superscript (Life
Technologies). Samples were treated at 42°C for 60 min, and the cDNA
concentration was measured by spectrophotometry.
Competitive PCR for b-actin was performed for each sample as previously described (22). In brief, 50 ng of each cDNA was denatured for 5 min
at 94°C, and b-actin was amplified in the presence of graded concentrations
of pQB2 plasmid as competitor. Competitive PCR for MIP-1a was performed in the same conditions as those used for b-actin except that the
amount of cDNA input was equivalent to 106 molecules of b-actin cDNA.
Competitive PCR for MIP-1b was performed with the same amount of
cDNA as that used for MIP-1a, but 35 cycles of PCR were performed, each
cycle consisting of 1 min at 94°C, 1 min at 55°C, and 1.5 min at 72°C. The
MIP-1a and MIP-1b competitor was pQB2 plasmid as previously described (23). The primers used for b-actin, MIP-1a, and MIP-1b are shown
in Table I. A PTC-100TM Programmable Thermal Controler (MJ Research, Watertown, MA) was used for all PCR reactions.
Quantification of PCR products by colorimetry
Aliquots of the amplified products of competitive PCR were subjected to
an additional elongation cycle in the presence of biotinylated oligonucleotides recognizing either the cDNA or the competitor (Table I) and digoxigenin-labeled dUTP. Labeled products were quantified by ELISA in
streptavidin-coated microtiter plates using peroxidase-conjugated Fab fragments of sheep anti-digoxigenin Ab. All reagents were obtained from
Boehringer Mannheim. Results are expressed as the number of chemokine
mRNA copies per 106 copies of b-actin mRNA in the sample.
Chemotaxis assay
IL-2-stimulated CD41CD45RO1 T cells were extensively washed in
RPMI for the chemotaxis assay. Cell migration was assessed in a 48-well
microchemotaxis chamber (NeuroProbe, Gaithersburg, MD) as previously
described (24). The lower wells of the chamber were filled with 27.5 ml of
conditioned medium from SAC-activated B cells or control chemokines
Statistical analysis
Student’s t test was used to detect significant differences. A p
value of ,0.05 was regarded as significant.
Results
MIP-1a and MIP-1b release is selectively up-regulated upon
cross-linking of the BCR
Human tonsillar B cells cultured for 2 days in CM spontaneously
released 352 6 243 pg/ml MIP-1a, 361 6 401 pg/ml MIP-1b, and
1867 6 1152 pg/ml IL-8 into culture supernatants (n 5 8). In the
same culture supernatants, the concentrations of the other two CC
chemokines tested, MCP-1 and RANTES, were much lower: 64 6
24 and 93 6 24 pg/ml, respectively. Activation of tonsillar B lymphocytes by anti-IgM Ab significantly increased MIP-1a and
MIP-1b production (Fig. 2A; 3.6 6 2.3 times more for MIP-1a and
7.2 6 3.2 times more for MIP-1b; n 5 5), whereas the addition of
CD40 mAb with or without IL-4 had no effect on their secretion.
In contrast, the addition of IL-4 and CD40 mAb increased the
anti-IgM Ab-induced secretion of MIP-1a and MIP-1b by 1.5- to
2.3-fold. In these five separate experiments using five different B
cell donors, the increased production of MIP-1a/b observed after
stimulation with anti-IgM Ab alone ( p , 0.05) or with anti-IgM
Ab and CD40 or IL-4 ( p , 0.005) was significant.
MIP-1b production involved rapid protein release. After 12 h,
significantly more immunoreactive MIP-1b was detected in culture supernatants from anti-IgM Ab-stimulated B cells than in
those from medium- or CD40 mAb-treated cells. After BCR triggering, the concentration of MIP-1b increased until 48 h and then
plateaued. The plateau value for MIP-1b at 48 h was 30 times
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Proliferation was measured by supplying the cultures with a pulse of 0.5
mCi/well of [methyl-3H]thymidine (Amersham, Les Ulis, France) for the
last 12 h of the third day of incubation. Cells were collected by filtration
through a glass-fiber filter, and [3H]thymidine incorporation was measured
in a beta scintillation counter (Betaplate 1205, EGG Wallac, Turku, Finland). Results are expressed as counts per minute (mean of triplicates 6
SD). In some experiments mouse anti-human IL-8, goat anti-human MIP1a, and anti-human MIP-1b neutralizing Ab (all from R&D Systems,
Abingdon U.K.) were added to a final concentration of 5 mg/ml.
diluted in HEPES-buffered RPMI 1640 medium, pH 7.4, containing 1%
heat-inactivated human plasma (assay buffer). The upper wells were filled
with 50 ml of cells (2 3 106 cells/ml) in assay buffer. The lower and upper
wells were separated by polyvinylpyrolidone-free polycarbonate membranes with 3-mm pores (Osmonics, Livermore, CA). The surface facing
the lower wells was coated with 5 mg/ml collagen type IV (Sigma, St.
Louis, MO) by incubation for 1 h at 37°C before the assay. Assays were
conducted at 37°C for 3 h in a humidified atmosphere of 5% CO2 in air.
Filters were collected, and the surface facing the upper well was washed
carefully with 13 PBS, fixed, and stained with Diff-Quick dye. The number of cells that had migrated to the underside of the membrane was
counted in five randomly selected high power fields (3400 magnification).
All assays were performed in triplicate. Results are expressed as the
mean 6 SD of the number of cells in five high power fields for each well.
Human recombinant SDF-1a (Diaclone), MIP-1a, (Diaclone), and
MIP-1b (R&D) were used at a concentration of 100 ng/ml, and IL-8 (72
amino acid form) (Diaclone) was used at a concentration of 50 ng/ml.
Neutralizing polyclonal anti-MIP-1a Ab, anti-MIP-1b Ab, and monoclonal
anti-IL-8 Ab (R&D) were used at a concentration of 5 mg/ml. The antiCCR5 (2D7) mAb (National Institute of Biological Standards and Controls) was used at a concentration of 10 mg/ml for blocking experiments. In
some experiments T cells were preincubated at 37°C for 2 h with 200 ng/ml
of Bordetella pertussis toxin (Calbiochem, La Jolla, CA) before chemotaxis assay.
4458
BCR TRIGGERING INDUCES MIP-1a/b PRODUCTION BY HUMAN B CELLS
FIGURE 2. MIP-1a and MIP-1b production by tonsillar B cells. A,
Tonsillar B cells (2 3 105/well, $95% purity) were cultured in medium
alone or with the activators indicated. Cell-free culture supernatants were
harvested on day 2, and MIP-1a and MIP-1b production was assessed by
ELISA. Results (picograms per milliliter) are the means of triplicate determinations from five donors. The SD was ,10% of the mean. B, B cells
were cultured in medium alone or with the activators indicated for various
times. MIP-1b production was assessed by ELISA in cell-free culture supernatants harvested at the time points indicated. Results (picograms per
milliliter) are the means of triplicate determinations. The SD was ,10% of
the mean. Representative data from four independent experiments are
shown. Student’s t test was used to compare the effect of various stimuli on
chemokine production (p values).
higher after activation with SAC than after activation with antiIgM Ab (Fig. 2B). Similar results were observed for MIP-1a production (data not shown). The addition of 10 mg/ml cycloheximide
during BCR triggering totally abolished the increase in MIP-1a/b
production, suggesting that it was dependent on de novo protein
synthesis (data not shown).
In contrast, the production of IL-8 was not significantly affected
by any of the stimuli used, and that of RANTES and MCP-1 remained ,120 pg/ml. The addition of the cytokines, IL-2, IL-12,
IL-13, or IFN-g, did not significantly affect spontaneous or BCRinduced MIP-1a, MIP-1b, and IL-8 production (data not shown).
Thus, B cells selectively released two potent T cell chemoattractants, MIP-1b and MIP-1a, after BCR triggering, whereas IL-8
production was insensitive to in vitro B cell activation.
MIP-1a and MIP-1b mRNA levels after BCR or CD40
triggering
MIP-1b and MIP-1a transcript levels were determined by competitive RT-PCR before and after B cell activation. Unstimulated
B cells contained low levels of both mRNA species before activation (46 3 104 copies of MIP-1a mRNA and 35 3 104 copies
of MIP-1b mRNA/106 copies of b-actin mRNA; Fig. 3A). The
amounts of both mRNA species rapidly increased after BCR crosslinking, peaking at 8 h and decreasing thereafter. At 8 h, the number of MIP-1a mRNA copies reached a maximum of 735 3 104/
106 copies of b-actin mRNA, much higher than the 58 3 104
copies in medium-treated cells (12.7 times higher). The activation
of B cells by anti-IgM Ab resulted in 9.3 times more copies of
MIP-1b mRNA (1490 3 104 copies/106 copies of b-actin in BCRstimulated cells vs 159 3 104 in medium-treated cells; Fig. 3B).
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FIGURE 3. Kinetics of MIP-1a and MIP-1b mRNA expression. Tonsillar B lymphocytes ($95% purity) were cultured for the times indicated
in medium alone or with anti-IgM Ab or CD40 mAb. A, RT-PCR analysis
using a primer pair designed to amplify the mRNA species encoded by the
human MIP-1a, MIP-1b, and b-actin genes was performed as described in
Materials and Methods. B, Stimulation indexes for MIP-1a and MIP-1b
expression in B cells after stimulation with anti-IgM or CD40 mAb vs
medium, 8 h postactivation. Representative data from three independent
experiments are shown.
The Journal of Immunology
4459
BCR triggering induces MIP-1a and MIP-1b production in all
subsets of tonsillar B cells
FIGURE 4. MIP-1a/b production and proliferation of tonsillar B cells.
Tonsillar B cells (2 3 105 cells/well, $95% purity) were cultured alone or
in the presence of the activators indicated. B cell proliferation was measured on day 3 by [3H]TdR incorporation. Cell-free culture supernatants
were harvested on day 2, and MIP-1a/b production was quantified by
ELISA. Results are expressed as counts per minute (6SD) and means
(nanograms per milliliter; 6SD) for triplicate cultures. Representative data
from four independent experiments are shown.
There were time-dependent changes in MIP-1a/b mRNA levels
after BCR cross-linking, in contrast to the steady state levels of
both species in medium- and CD40-treated B cells.
Secretion of MIP-1a and MIP-1b and proliferative response are
differently regulated in tonsillar B cells
BCR triggering induces both B cell proliferation and MIP-1a/b
production, so we tested whether these two processes were regulated by similar signals. BCR triggering was itself sufficient to
markedly up-regulate MIP-1b secretion and the proliferation of
unfractionated tonsillar B cells (sevenfold increase in both cases;
Fig. 4). The proliferation induced by anti-IgM Ab B cell stimulation was increased by the addition of IL-4 or CD40 mAb (10 and
4 times higher, respectively), but BCR-induced MIP-1b production was only 1.5–2 times higher after these additions. MIP-1b
production was also unaffected by the potent mitogenic stimulation
of the combination of CD40 mAb and IL-4. A similar pattern was
observed for MIP-1a production. Therefore, the production of
MIP-1a and MIP-1b does not depend on the proliferative state of
B cells. This conclusion is further supported by the observation
that anti-MIP-1a/b neutralizing Ab did not inhibit B cell proliferation (data not shown). Thus, B cell proliferation and MIP-1a/b
production seem to be controlled by independent mechanisms.
Tonsillar B cells consist of several phenotypically and functionally
different subsets, so we investigated whether particular B cell subsets were involved in BCR-induced chemokine production. MIP1a/b production was measured in culture supernatants of IgDhigh
naive, IgD2CD441 memory, and IgD2CD442 GC B cells. AntiIgM Ab-treated naive B cells produced 11276 pg/ml MIP-1b,
whereas anti-IgM Ab-treated memory B cells and GC B cells produced 357 and 266 pg/ml, respectively (Fig. 5). As surface IgM1
(sIgM1) cells make up a small proportion of memory B cells, we
compared the production of MIP-1a and MIP-1b in memory and
naive B cells after stimulation by SAC, a potent sIg cross-linker
acting on the BCR of various isotypes. SAC-activated naive and
memory B cells produced large amounts of MIP-1b (51,101 and
62,026 pg/ml, respectively) and MIP-1a (26,046 and 66,932 pg/
ml, respectively). Stimulation by anti-IgM Ab did not increase
MIP-1a/b production by GC B cells, but 22 times more MIP-1b
(3,086 vs 137 pg/ml) and 10 times more MIP-1a (1,776 vs 175
pg/ml) were produced in the presence of SAC. This suggests that
production of these two closely related CC chemokines is a constitutive phenomenon accompanying BCR triggering in all subsets
of mature B cells.
Regulation of MIP-1a/b secretion and B cell proliferation by
FDC-like HK cells
FDC retain Ag in an unprocessed form in vivo and strongly increase B cell survival and proliferation within GC. Therefore, we
assessed whether they regulated MIP-1a and MIP-1b production
in various B cell subsets. We used the human FDC-like HK cell
line to mimic the effect of FDC. Coculture with irradiated HK cells
did not significantly affect spontaneous overall B cell proliferation,
whereas it strongly increased B cell proliferation in the presence of
CD40 mAb with or without IL-4 or anti-IgM Ab (Fig. 6A). In
striking contrast, the production of MIP-1a/b by B cells was not
affected by coculture with HK cells alone. However, in the presence of anti-IgM and CD40 Ab or IL4, coculture with HK cells led
to a decreased production of both chemokines (Fig. 6B). Moreover,
coculture with HK cells did not affect the production of MIP-1a/b
by naive, memory, and GC B cells, whereas it increased their
proliferation in the presence of various stimuli (data not shown).
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FIGURE 5. MIP-1a/b production by various tonsillar B cell subsets in
response to BCR triggering. GC (IgD2CD442), memory (IgD2CD441),
and naive (IgDhigh) B cells (2 3 105/well) were cultured in medium alone
or in the presence of anti-IgM Ab or SAC. Cell-free culture supernatants
were harvested on day 2, and the production of MIP-1a (M) and MIP-1b
(f) was assessed by ELISA. Results (nanograms per milliliter) are the
means of triplicate determinations (6SD). Representative data from four
independent experiments are shown.
4460
BCR TRIGGERING INDUCES MIP-1a/b PRODUCTION BY HUMAN B CELLS
B cell-derived chemokines induce T cell chemotaxis
FIGURE 6. Effect of HK cells on B cell proliferation and MIP-1b release in tonsillar B cells. A, Tonsillar B cells (2 3 105 cells/well) were
cultured alone (M) or with irradiated FDC-like HK cell monolayers (2 3
103 cells/well; f) in the presence of the activators indicated. B cell proliferation was measured on day 3 by determining [3H]TdR incorporation.
Results for proliferation are expressed as counts per minute (mean of triplicate determinations 6 SD) for each culture condition. Radioactivity
(270 6 80 cpm) was incorporated into irradiated HK cells only. B, Cellfree culture supernatants were harvested on day 2, and MIP-1a/b production was quantified by ELISA. MIP-1a/b production is expressed as picograms per milliliter (mean of triplicate determinations 6 SD) for each
culture condition. Representative data from four independent experiments
are shown.
MIP-1a/b selectively attract activated T lymphocytes, so we assessed the extent to which conditioned medium from activated B
cells (sn B SAC) induced the migration of IL-2-activated
CD41CD45RO1 T cells. sn B SAC produced significantly higher
levels of T cell migration (2.2 times higher than medium alone).
Significant T cell migration with six different sn B SAC were
obtained in eight independent experiments (2.0 6 0.4 times higher
migration than control medium; p , 0.005) using T cells from
different donors. Dilutions (1/1 to 1/10) of the sn B SAC were
tested, and the undiluted supernatant and a 1/1 (50%) dilution of sn
B SAC yielded comparable and maximal responses (data not
shown). The chemoattractant activity of sn B SAC was stronger
than that of 50 ng/ml IL-8, comparable to that of 100 ng/ml
MIP-1a and 1.6 and 3 times lower than that of 100 ng/ml MIP-1b
and SDF-1a, respectively. SDF-1a, a highly efficient chemoattractant for T cells, was used as a positive control (Fig. 7A). Pretreatment of CD41CD45RO1 T cells with 200 ng/ml of Bordetella
pertussis toxin inhibited migration toward sn B SAC to background levels, confirming the involvement of Gia protein-coupled
receptor(s) (Fig. 7B). A checkerboard-type analysis showed that
the migration of T cells toward sn B SAC was mostly chemotactic
and not chemokinetic because the cells did not migrate above the
background level if incubated in the absence of a chemotactic concentration gradient (Fig. 7C). The addition of neutralizing antiMIP-1b Ab reduced T cell migration by only 27 6 7%, but this
effect was significant ( p , 0.005; n 5 7). Interestingly, the addition of neutralizing anti-MIP-1a Ab with neutralizing anti-MIP-1b
Ab did not increase the blocking effect over that observed with
anti-MIP-1b Ab alone. The addition of anti-CCR5 blocking mAb
inhibited the chemoattractant activity of sn B SAC by 41 6 5%
( p , 0.05; n 5 4). In contrast, neutralizing anti-IL-8 mAb and
isotype-matched control Igs did not cause any significant inhibition (Fig. 7D). Thus, chemokines or chemotactic agents other than
MIP-1a/b, produced by BCR-stimulated B cells, also attract T
cells of the helper/effector phenotype.
Discussion
GC and in vitro activated B cells induce IL-8 secretion by
HK cells
In striking contrast to naive and memory B cells, GC B cells spontaneously secreted large amounts of IL-8 (,10, 68, and 1547 pg/
Maturation of the B cell response to a peptide Ag depends mostly
on the limiting amount of T cell help (25). This process depends on
the availability of Ag-specific T cells and on the ability of Agbinding B cells to efficiently recruit them. As B and T cells specific
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ml, respectively; Table II). None of the B cell activators tested
induced significant levels of IL-8 production by naive and memory
B cells or regulated its production by GC B cells. The IL-8 concentration in the supernatants of GC, but not of naive or memory
B cells, was strongly increased in the presence of irradiated HK
cells. If HK cells were treated with paraformaldehyde rather than
being irradiated before coculture, no increase in IL-8 production
was observed, suggesting that HK cells were indeed the main
source of IL-8 during HK-B cell coculture (data not shown). IL-8
production during GC B cell/HK cell cocultures was not further
increased by B cell activation (Table II). In contrast, stimulation of
naive B cells by anti-IgM Ab with or without IL-4 and/or CD40
mAb up-regulated IL-8 production by HK cells. Stimulation by
CD40 mAb and IL-4 of naive B cells also increased IL-8 production by HK cells. A similar pattern of IL-8 production was observed for memory B cells (data not shown). These results suggest
that strong induction of IL-8 production by HK cells during HK-B
cell coculture depends on B cell activation. GC B cells activated in
vivo spontaneously induce IL-8 production, whereas naive and
memory B cells require further activation in vitro.
The Journal of Immunology
4461
Table II. In vivo and in vitro activated B cells induce IL-8 production by FDC-like HK cellsa
IgDhigh B cells
Activators
None
Anti-IgM Ab
CD40 mAb
IL-4
CD40 mAb 1 IL-4
Anti-IgM Ab 1 CD40 mAb
Anti-IgM Ab 1 IL-4
Anti-IgM Ab 1 CD40 mAb 1 IL-4
2 HK cells
c
0
0
40
0
0
77
0
52
IgD2CD442 B Cells
1 HK cellsb
2 HK cells
1 HK cells
431
4,319
471
442
4,948
6,330
17,115
25,907
1,547
1,496
1,876
1,500
1,202
1,354
1,485
1,299
36,499
39,909
35,390
35,287
49,255
47,143
47,414
47,765
a
A total of 2 3 105 IgDhigh naive and IgD2CD442 GC B cells (.90% purity) were cultured alone or with 2 3 103 HK cells
in the presence of the activators indicated. IL-8 was measured on day 2 in cell-free culture supernatants by ELISA. Values
(pg/ml) are the means of triplicate determinations. SD were consistently ,15% of the mean. Representative data of four
independent experiments are shown.
b
The levels of IL-8 in culture supernatants of HK cells cultured alone or in the presence of the activators indicated were
,400 pg/ml.
c
Below the detection limit of the ELISA kit (,10 pg/ml).
enable rare Ag-specific T cells to be quickly selected and recruited
to the specialized niches of lymphoid organs.
In this study we have shown that two closely related T cell
chemoattractants, MIP-1a and MIP-1b, were rapidly produced by
B cells after engagement of their BCR. This triggering, but not
stimulation, by CD40 mAb or IL-4 rapidly induced the coordinated
expression of their transcripts and proteins. This effect was selective because the production of two other CC chemokines, MCP-1
and RANTES, was not affected by B cell stimulation. BCR triggering by anti-IgM Ab induced a strong, but transient, increase in
MIP-1a/b mRNA levels, peaking 8 h after stimulation. This was
FIGURE 7. Chemotactic response of CD41CD45RO1 T cells to conditioned medium from SAC-activated B cells. IL-2-conditioned T cells used in
migration assays were .95% CD45RO1, .80% CD41, and .75% CCR51. A, Migration of CD41CD45RO1 T cells toward sn B SAC or toward
recombinant MIP-1a, MIP-1b, and SDF-1a (each at a concentration of 100 ng/ml) or IL-8 (50 ng/ml) was assessed in a 48-well microchemotaxis chamber.
The assay was performed in triplicate, and migrating cells in five randomly selected high power fields (HPF; 3400) were counted for each well (mean 6
SD). Representative results from eight independent experiments performed with T cells from different donors and six different sn B SAC are shown. The
significance of differences in T cell migration toward sn B SAC and control medium (background migration) was determined by Student’s t test. B,
Chemotaxis of CD41CD45RO1 T cells toward sn B SAC is mediated by a Gia protein-coupled receptor(s). Lymphocytes were preincubated with B.
pertussis toxin (PTX) before the chemotaxis experiment. Representative results (mean of triplicate determinations 6 SD) from three independent experiments are shown. C, Checkerboard-type assay of sn B SAC on CD41CD45RO1 T cells. For the microchemotaxis assay, sn B SAC was added to the upper
and/or lower wells of the chamber, as indicated. The assay was performed in triplicate, and migrating cells were counted as described in A (mean 6 SD).
Representative data from three independent experiments are shown. D, Migration of CD41CD45RO1 T cells toward sn B SAC alone or in the presence
of neutralizing anti-MIP-1a and/or anti-MIP-1b Ab, anti-IL-8 Ab, anti-CCR5 blocking mAb, or control Ig. Representative results (mean 6 SD) from four
to seven independent experiments performed with cells from various donors are shown. Significant differences in migration toward sn B SAC were
determined by Student’s t test.
Downloaded from http://www.jimmunol.org/ by guest on July 28, 2017
for a given Ag are triggered independently, T/B cell interactions
require the redistribution of Ag-primed cells within precise anatomical sites at which cognate T/B interactions take place (26). In
contrast to the limited trafficking capacity of Ag-binding B cells,
which typically colonize only the adjacent follicle, Ag-primed and
expanded T cells actively migrate to multiple, adjacent, and distant
follicular sites (27). The selective influx of Ag-specific T cells
from T cell zones into the follicles during the primary Ab response
in vivo reflects the Ag-driven selection of GC CD41 T cells reported in several studies (28 –30). It appears that specific chemoattractive gradient(s) selectively produced by Ag-binding B cells
4462
BCR TRIGGERING INDUCES MIP-1a/b PRODUCTION BY HUMAN B CELLS
may be superimposed on the constitutive gradients of homing chemokines (secondary lymphoid tissue chemokine, EBV-induced molecule-1 ligand, or SDF-1a) within lymphoid tissue. Our data argue
that MIP-1a/b gradients play an important role in lymphoid tissue
during the adaptive immune response by bringing and keeping together a functional cellular unit, Ag-specific B and T cells, that is
required for maturation of the B cell response.
Dendritic cells (DC), which express mRNA for CCR5 and
CCR1 and migrate in the presence of MIP-1a/b, are also potential
targets for B cell-derived chemokines (44). This possibility is of
particular interest given recent data concerning the role of DC in
stimulating the primary B cell response (45). It is unknown
whether MIP-1a/b recruits the recently identified subset of
CD41CD11c1 DC present within the GC (46). Finally, MIP-1a/b
secreted by Ag-binding B cells may act on the B cells themselves,
because MIP-1a has been shown to act as a chemoattractant for B
cells (47). The principal MIP-1b receptor, CCR5, is not present on
B cells, but other recently identified MIP-1b receptors, such as
CCR8, are presumably present (48, 49). The last major finding of
our study is the lack of effect of HK (FDC)-B cells interactions on
MIP-1a/b production by B cells, although these interactions
strongly increased the proliferation of activated B cells and Ig
secretion (50) (our unpublished observations). FDC-like HK cells
do not express MIP-1a/b mRNA or protein (data not shown). This
is intriguing because the FDC network in the GC retains Ag in an
unprocessed form for the selection of high affinity B cell clones
and is thus essential in promoting B cell survival and differentiation (51–54). IL-8 was spontaneously released by GC B cells, but
not by naive or memory B cells. Moreover, IL-8 production by B
cells was not significantly regulated by cell activation or coculture
with HK cells. Interactions between HK cells and the GC or B cells
activated in vitro led to the production of large amounts of IL-8 by
HK cells. It is unknown whether activated B cells induce IL-8
production by FDC in vivo. The role of IL-8 in T cell traffic is still
controversial, and our in vitro experiments showed that T lymphocyte attraction in response to IL-8 was indeed marginal. However,
IL-8 might contribute to regulate the B cell response in secondary
lymphoid organs as previously shown (55–57).
Thus, we provide herein the first evidence that Ag-binding B
cells are an important source of two CC chemokines with T cellspecific properties: MIP-1a and MIP-1b. The engagement of the
Ag receptor on B cells determined the capacity to recruit T cells of
the effector/helper phenotype. Cognate T/B cell interactions are
required at all stages of Ag-driven B cell differentiation, so B cellderived MIP-1a/b may directly affect maturation of the B cell
response and, thus, the overall outcome of the adaptive immune
response.
Acknowledgments
We thank Y. S. Choi for providing the HK cell line, D. Treton for excellent
technical assistance, and Drs. A. Lange and J. Silber (K. Dluski Hospital,
Wroclaw, Poland) for their encouragements and constant support. AntiCCR5 mAb (2D7) was obtained through the AIDS Reagent Project, National Institute of Biological Standards and Control (Potters Bar, U.K.)
from Leukosite, Inc. We also thank J. Wiels (UMR 1598, Villejuif, France)
for providing anti-CD77 mAb.
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