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(MIP-1) and MIP-1ß Chemokine Production in Human B Cells1
Institut National de la Santé et de la Recherche Médicale, Unit 131, Institut Paris-Sud sur les Cytokines, Clamart, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, by human B cells.
Naive, memory, and germinal center B cells all produced MIP-1/ß in
response to BCR triggering. In contrast to MIP-1/ß, 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-1/ß production, but it did induce production of IL-8
by HK cells. Microchemotaxis assays showed that
CD4+CD45RO+ 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-1ß and anti-CC chemokine receptor 5 Ab, but not by
anti-MIP-1 Ab suggesting that MIP-1ß 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-1/ß,
may represent one possible mechanism enabling cognate interactions
between rare in vivo Ag-specific T and B cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1
(SDF-1),3 expressed around
germinal centers (GC), may attract lymphocytes to this site (10). Two
functionally related CC chemokines, C6kine/Exodus-2/secondary lymphoid
tissue chemokine and MIP-3ß/Exodus-3/CKß11/EBV-induced 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-1 and MIP-1ß, by B cells. In chemotaxis
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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); anti-CD38-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 anti-CD77 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 (H+L) F(ab')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 DTAF-conjugated goat anti-mouse IgG (H+L) F(ab')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 cell-enriched 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%
CD19+, 
1% CD14+, and 1% CD3+
and DRC1+ cells. For some experiments tonsillar B cells
were separated into IgD+ and IgD- populations
by incubating them for 30 min with anti-IgD mAb (IADB6, SBA) and
removing IgD+ cells with goat anti-mouse IgG magnetic
beads (Dynal). IgD- B cells were further separated into
CD44+ and CD44- 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
.
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95%
CD4+CD45RO+ 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 µg/ml streptomycin, 1 mM sodium pyruvate, and 10% heat-inactivated FCS (complete medium (CM)). B cells (1 x 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 µg/ml), CD40 mAb (G28.5; 1 µg/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 CD4+CD45RO+ tonsillar T cells (1 x 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% CD45RO+, >80% CD4+, <20% CD8low, >75% CCR5+, <2% CD14+, and <1% CD20+ 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 1x 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 x 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 µl of CM, for 2 (chemokine production) or 3 (proliferation assay) days. In some experiments HK cells were seeded at 2 x 103 cells/well in 96-well microtiter plates for 4 h, treated by incubation with 1% paraformaldehyde in 1x PBS for 15 min, and extensively washed before coculture with B cells.
Proliferation assays
Proliferation was measured by supplying the cultures with a
pulse of 0.5 µCi/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 ± SD). In some experiments mouse anti-human IL-8,
goat anti-human MIP-1, and anti-human MIP-1ß neutralizing
Ab (all from R&D Systems, Abingdon U.K.) were added to a final
concentration of 5 µg/ml.
Chemokine production
Cell-free supernatants of 2 x 105 B cells/well
were collected on day 2 unless otherwise indicated and were stored at
-20°C until tested. Chemokine production was measured with specific
ELISA kits purchased from R&D Systems (MIP-1 and MIP-1ß) and
Diaclone (IL-8) according to the manufacturer’s recommendations.
Results are expressed as the mean concentration (picograms per
milliliter; ±SD) of triplicate determinations.
Competitive RT-PCR
RNA was extracted from 10 x 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 µl 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 ß-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 ß-actin was amplified in the presence of
graded concentrations of pQB2 plasmid as competitor. Competitive PCR
for MIP-1 was performed in the same conditions as those used for
ß-actin except that the amount of cDNA input was equivalent to
106 molecules of ß-actin cDNA. Competitive PCR for
MIP-1ß was performed with the same amount of cDNA as that used for
MIP-1, 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-1
and MIP-1ß competitor was pQB2 plasmid as previously described (23).
The primers used for ß-actin, MIP-1, and MIP-1ß are shown in
Table I. A PTC-100TM Programmable Thermal
Controler (MJ Research, Watertown, MA) was used for all PCR reactions.
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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 ß-actin mRNA in the sample.
Chemotaxis assay
IL-2-stimulated CD4+CD45RO+ 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 µl of conditioned medium from SAC-activated B cells or control chemokines 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 µl of cells (2 x 106 cells/ml) in assay buffer. The lower and upper wells were separated by polyvinylpyrolidone-free polycarbonate membranes with 3-µm pores (Osmonics, Livermore, CA). The surface facing the lower wells was coated with 5 µg/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 1x 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 (x400 magnification). All assays were performed in triplicate. Results are expressed as the mean ± SD of the number of cells in five high power fields for each well.
Human recombinant SDF-1 (Diaclone), MIP-1, (Diaclone), and
MIP-1ß (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-1 Ab, anti-MIP-1ß Ab, and
monoclonal anti-IL-8 Ab (R&D) were used at a concentration of 5
µg/ml. The anti-CCR5 (2D7) mAb (National Institute of Biological
Standards and Controls) was used at a concentration of 10
µg/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.
Statistical analysis
Student’s t test was used to detect significant differences. A p value of <0.05 was regarded as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß release is selectively up-regulated upon
cross-linking of the BCR
Human tonsillar B cells cultured for 2 days in CM spontaneously
released 352 ± 243 pg/ml MIP-1, 361 ± 401 pg/ml
MIP-1ß, and 1867 ± 1152 pg/ml IL-8 into culture supernatants
(n = 8). In the same culture supernatants, the
concentrations of the other two CC chemokines tested, MCP-1 and RANTES,
were much lower: 64 ± 24 and 93 ± 24 pg/ml, respectively.
Activation of tonsillar B lymphocytes by anti-IgM Ab significantly
increased MIP-1 and MIP-1ß production (Fig. 2A; 3.6 ± 2.3 times more
for MIP-1 and 7.2 ± 3.2 times more for MIP-1ß;
n = 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-1 and MIP-1ß by 1.5- to 2.3-fold. In these five
separate experiments using five different B cell donors, the increased
production of MIP-1/ß 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.
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B). Similar results were observed for MIP-1 production
(data not shown). The addition of 10 µg/ml cycloheximide during BCR
triggering totally abolished the increase in MIP-1/ß 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-
,
did not significantly affect spontaneous or BCR-induced MIP-1,
MIP-1ß, and IL-8 production (data not shown). Thus, B cells
selectively released two potent T cell chemoattractants, MIP-1ß and
MIP-1, after BCR triggering, whereas IL-8 production was insensitive
to in vitro B cell activation.
MIP-1 and MIP-1ß mRNA levels after BCR or CD40 triggering
MIP-1ß and MIP-1 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 x 104 copies of MIP-1 mRNA and 35 x
104 copies of MIP-1ß mRNA/106 copies of
ß-actin mRNA; Fig. 3A). The
amounts of both mRNA species rapidly increased after BCR cross-linking,
peaking at 8 h and decreasing thereafter. At 8 h, the number
of MIP-1 mRNA copies reached a maximum of 735 x
104/106 copies of ß-actin mRNA, much higher
than the 58 x 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-1ß mRNA (1490 x
104 copies/106 copies of ß-actin in
BCR-stimulated cells vs 159 x 104 in medium-treated
cells; Fig. 3B). There were time-dependent changes in
MIP-1/ß mRNA levels after BCR cross-linking, in contrast to the
steady state levels of both species in medium- and CD40-treated B
cells.
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and MIP-1ß and proliferative response are
differently regulated in tonsillar B cells
BCR triggering induces both B cell proliferation and MIP-1/ß
production, so we tested whether these two processes were regulated by
similar signals. BCR triggering was itself sufficient to markedly
up-regulate MIP-1ß 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-1ß production was only 1.5–2 times higher after these additions.
MIP-1ß 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-1 production. Therefore, the production of
MIP-1 and MIP-1ß does not depend on the proliferative state of B
cells. This conclusion is further supported by the observation that
anti-MIP-1/ß neutralizing Ab did not inhibit B cell
proliferation (data not shown). Thus, B cell proliferation and
MIP-1/ß production seem to be controlled by independent
mechanisms.
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and MIP-1ß production in all
subsets of tonsillar B cells
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.
MIP-1/ß production was measured in culture supernatants of
IgDhigh naive, IgD-CD44+ memory,
and IgD-CD44- GC B cells. Anti-IgM Ab-treated
naive B cells produced 11276 pg/ml MIP-1ß, whereas anti-IgM
Ab-treated memory B cells and GC B cells produced 357 and 266 pg/ml,
respectively (Fig. 5). As surface
IgM+ (sIgM+) cells make up a small proportion
of memory B cells, we compared the production of MIP-1 and MIP-1ß
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-1ß (51,101 and
62,026 pg/ml, respectively) and MIP-1 (26,046 and 66,932 pg/ml,
respectively). Stimulation by anti-IgM Ab did not increase
MIP-1/ß production by GC B cells, but 22 times more MIP-1ß
(3,086 vs 137 pg/ml) and 10 times more MIP-1 (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.
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/ß 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-1 and MIP-1ß 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-1/ß 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-1/ß 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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In striking contrast to naive and memory B cells, GC B cells
spontaneously secreted large amounts of IL-8 (<10, 68, and 1547 pg/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.
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MIP-1/ß 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
CD4+CD45RO+ 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 ± 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-1 and 1.6 and 3 times lower than that of 100 ng/ml
MIP-1ß and SDF-1, respectively. SDF-1, a highly efficient
chemoattractant for T cells, was used as a positive control (Fig. 7A). Pretreatment of
CD4+CD45RO+ T cells with 200 ng/ml of
Bordetella pertussis toxin inhibited migration toward sn B
SAC to background levels, confirming the involvement of
Gi 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 anti-MIP-1ß Ab reduced T cell migration
by only 27 ± 7%, but this effect was significant
(p < 0.005; n = 7).
Interestingly, the addition of neutralizing anti-MIP-1 Ab with
neutralizing anti-MIP-1ß Ab did not increase the blocking effect
over that observed with anti-MIP-1ß Ab alone. The addition of
anti-CCR5 blocking mAb inhibited the chemoattractant activity of sn
B SAC by 41 ± 5% (p < 0.05;
n = 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-1/ß, produced by BCR-stimulated B cells, also attract T cells
of the helper/effector phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we have shown that two closely related T cell
chemoattractants, MIP-1 and MIP-1ß, 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-1/ß mRNA
levels, peaking 8 h after stimulation. This was associated with an
early and sustained release of MIP-1/ß proteins. Similar kinetics
of protein release were observed after B cell activation by SAC, a
potent sIg cross-linker, which resulted in 20–30 times more
MIP-1/ß production than that induced by anti-IgM Ab. Rapid
induction of MIP-1/ß production by BCR-activated B cells is
consistent with previous data showing that the genes encoding
MIP-1/ß are immediate early genes (31). The restricted set of
chemokines produced by B cells and its strict dependence on BCR
triggering suggest that MIP-1/ß plays an important role in
Ag-driven B cell differentiation.
BCR engagement is central to three crucial steps in Ag-driven B cell
differentiation: 1) during the priming of IgDhigh naive B
cells in the T cell-rich extrafollicular area of secondary lymphoid
tissue, 2) during Ag-driven positive selection of high affinity clones
during GC reaction, and 3) at the postselection stage of
IgDlow/- memory B cell terminal differentiation (18, 32).
BCR cross-linking by anti-IgM Ab preferentially up-regulated
MIP-1/ß expression in naive B cells, but the extensive
cross-linking of sIg with SAC led to the production of large amounts of
both chemokines in naive and memory B cells. The weaker induction of
MIP-1/ß production by anti-IgM Ab presumably reflects the
lower frequency of sIgM+ cells in the memory B cell pool.
After stimulation with SAC, GC B cells secreted about 20 times less
MIP-1/ß than did naive and memory B cells. This low level of
production is consistent with the presence of a limited pool of
centrocytes expressing low levels of sIg in the GC B cell population.
Thus, all functional B cell subsets produce MIP-1/ß in response to
BCR engagement.
MIP-1/ß chemokines have been shown to be locally produced in the
lymph nodes of healthy subjects. However, cells producing MIP-1/ß
are rare in secondary lymphoid tissue and are detected in
extrafollicular T cell zones and within the GC (33, 34). This
expression may reflect the low proportion of cells triggered by Ag in
vivo. Moreover, both chemokines have been shown to regulate T
lymphocyte trafficking into lymph nodes during an immune response in
vivo (35). MIP-1 and MIP-1ß have a preferential chemotactic effect
on CD45RO+ T cells, with MIP-1ß acting mostly on
CD4+ T cells, so B cell-derived MIP-1/ß probably have
a chemoattractant activity affecting T cells of the helper/effector
phenotype (36, 37). Consistent with this, IL-2-conditioned
CD4+CD45RO+ T cells actively migrated toward a
chemotactic gradient(s) present in conditioned medium from
BCR-activated B cells. The inhibition of this activity by
anti-MIP-1ß neutralizing Ab and anti-CCR5 blocking Ab shows
that MIP-1ß is an important component of the T cell chemoattractant
activity of these media. It also shows that chemotactic agents other
than MIP-1ß exert their effects on CD4+ T cells and are
secreted by BCR-activated B cells. One candidate is a CCR4 agonist,
macrophage-derived chemokine produced by B cells (38) that
preferentially attracts Th2-polarized T cells (39).
Within lymphoid organs, CCR5, the main high affinity receptor for
MIP-1ß, is present on a small proportion (10%) of T lymphocytes in
the vicinity of B cell follicles and within the GC (40, 41). This
suggests that during the Ag-driven immune response in vivo,
CCR5+ T cells may be actively recruited to the anatomical
sites of cognate T/B cell interactions. Our results and the presence of
CCR5 mostly on the highly migratory
CD4+CD45RO+CD26high subset of T
cells (42) suggest that these helper/effector CD4+ T cells
may be the main target of B cell-derived MIP-1/ß (41). CCR5 is a
marker of recently activated T cells, and its expression is strongly
IL-2 dependent (43). This suggests that during IL-2-mediated clonal
expansion of Ag-specific T cells in secondary lymphoid organs these
cells become highly responsive to locally induced MIP-1/ß
gradients formed around Ag-primed B cells. These transient Ag-induced
gradients of MIP-1/ß may be superimposed on the constitutive
gradients of homing chemokines (secondary lymphoid tissue chemokine,
EBV-induced molecule-1 ligand, or SDF-1) within lymphoid tissue. Our
data argue that MIP-1/ß 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-1/ß, 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-1/ß
recruits the recently identified subset of
CD4+CD11c+ DC present within the GC (46).
Finally, MIP-1/ß secreted by Ag-binding B cells may act on the B
cells themselves, because MIP-1 has been shown to act as a
chemoattractant for B cells (47). The principal MIP-1ß receptor,
CCR5, is not present on B cells, but other recently identified MIP-1ß
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-1/ß 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-1/ß 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, 52, 53, 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, 56, 57).
Thus, we provide herein the first evidence that Ag-binding B cells are
an important source of two CC chemokines with T cell-specific
properties: MIP-1 and MIP-1ß. 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 cell-derived
MIP-1/ß may directly affect maturation of the B cell response and,
thus, the overall outcome of the adaptive immune response.
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Acknowledgments |
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Footnotes |
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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:
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 protein; sIg, surface immunoglobulin; DC, dendritic cells; sn B SAC, conditioned medium from SAC-activated B cells.
Received for publication October 7, 1998. Accepted for publication January 21, 1999.
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) and MIP-1ß (
) was assessed by ELISA. Results (nanograms per
milliliter) are the means of triplicate determinations (±SD).
Representative data from four independent experiments are shown.
