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(Journal of Leukocyte Biology. 2001;70:633-641.)
© 2001 by Society for Leukocyte Biology

Selective attraction of naive and memory B cells by dendritic cells

Bertrand Dubois*, Catherine Massacrier{dagger} and Christophe Caux{dagger}

* Inserm U404, Lyon Cedex, France; and
{dagger} Schering-Plough, Laboratory for Immunological Research, Dardilly, France

Correspondence: Dr. C. Caux, Schering-Plough, Laboratory for Immunological Research, 27 chemin des Peupliers, BP 11, 69571 Dardilly, France. E-mail: christophe.caux{at}spcorp.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigate whether dendritic cells (DC), known to interact directly with T and B cells, might also contribute to the recruitment of B cells through the production of chemotactic factors. We found that B cells responded to several chemokines (CXCL12, CCL19, CCL20, and CCL21), which can be produced by DC upon activation. In addition, supernatant from DC (SNDC) potently and selectively attracted naive and memory B cells but not germinal center (GC) B cells or other lymphocytes (CD4+, CD8+ T cells or NK cells). Production of this activity was restricted to DC and was not increased following DC activation by LPS or CD40 ligand. Surprisingly, the B-cell chemotactic response to SNDC was insensitive to pertussis toxin treatment. In addition, the chemotactic factor(s) appeared resistant to protease digestion and highly sensitive to heat. This suggested that the DC chemotactic factor(s) is different from classical chemoattractants and does not involve G{alpha}i proteins on the responding B lymphocytes. It is interesting that SNDC was able to synergize with several chemokines to induce massive migration of B lymphocytes. These observations show that DC spontaneously produce factors that, alone or in cooperation with chemokines, specifically regulate B-cell migration, suggesting a key role of DC in the recruitment or localization of B lymphocytes within secondary lymphoid organs.

Key Words: B lymphocytes • germinal center • T cells • B cells • chemokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To differentiate into high-affinity, antibody-secreting cells, B lymphocytes need to establish physical interactions with different cell partners located in distinct, microanatomical compartments of secondary lymphoid organs. Following cognate interactions in the extrafollicular area with antigen-specific CD4+ T cells previously primed by dendritic cells (DC), antigen-specific B cells proliferate and differentiate into low-affinity immunoglobulin (Ig)M-secreting plasma cells [1 , 2 ]. In addition, few activated B cells colonize primary follicles where the reaction is initiated [1 2 3 ], leading to the formation of high-affinity plasma cells and memory B cells [4 ].

Signals responsible for the recruitment of B cells into secondary lymphoid organs and controlling the migration of B cells from the extrafollicular area to the B-cell follicle or to the medullary cords are poorly characterized. However, chemokines have recently been shown to play a critical role in these events. In particular, CCL19 (ELC/MIP-3ß) and CCL21 (SLC/6Ckine) have been recognized to participate in the recruitment of naive T cells, DC, and B cells in the extrafollicular area [5 6 7 8 9 10 ]. These chemokines are produced by scattered cells in the extrafollicular area and act through CCR7, specifically expressed on activated T and B cells and on mature DC [5 , 7 , 9 , 11 12 13 ]. Recruitment of B cells into the follicle would rely on the chemokine CXCL13 (BCA-1/BLC), because disruption of the gene encoding its receptor CXCR5 abolished GC formation [14 ]. This chemokine is specifically expressed in the B-cell follicle [15 , 16 ], and its receptor CXCR5 has been detected on circulating B cells and some memory T cells [17 ]. Finally, the differentiation stage and the engagement of the B-cell receptor have been shown to modulate migration in response to chemokines such as CXCL12 (SDF-1) [18 ] or CCL19 [5 ].

The cells producing B-cell-active chemokines are not well characterized, but CCL19 has been shown to be produced by DC [5 , 19 ] and CXCL13, by follicular DC [16 ]. Others have shown that encounter of DC and T cells precedes that of T cells and B cells [20 ], but there is growing evidence that DC can directly provide help to B cells. First, a recent study has shown that DC can present the processed and the native form of the antigen to T and B cells, respectively [21 ]. Second, our own studies have demonstrated that DC stimulate several steps of B-cell differentiation through direct interactions [22 23 24 25 ]. Thus, DC are likely to represent a bridge allowing antigen-specific T cells to meet with antigen-specific B cells. To strengthen this concept, we investigated in the present study whether DC could produce soluble chemoattractants for B cells.

We found that DC spontaneously produce soluble chemoattractants, inducing potent and selective chemotaxis of naive and memory B cells but not that of other lymphocytes. It is important that these chemoattractants do not involve pertussis toxin (PTX)-sensitive pathways but can synergyze with several chemokines to induce potent B-cell chemotaxis. The present results demonstrate that DC constitute an important source of a novel class of B-cell chemoattractants, suggesting a key role of DC in the recruitment and migration of B cells within discrete areas of secondary lymphoid organs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Hematopoietic factors, reagents, and cell lines
Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; specific activity: 2x106 U/mg; Schering-Plough Research Institute, Kenilworth, NJ), rh tumor necrosis factor {alpha} (TNF-{alpha}; specific activity: 2x107 U/mg; Genzyme, Boston, MA), rh stem cell factor (SCF; specific activity: 4x105 U/mg; R&D Systems, Abington, UK), and rh interleukin (IL)-4 (specific activity: 2x107 U/mg; Schering-Plough Research Institute) were used at saturating concentrations of 100 ng/ml, 2.5 ng/ml, 25 ng/ml, and 50 U/ml, respectively. rh Chemokines CXCL12 (specific activity: 2x105 U/mg), CCL20 (specific activity: 4x105 U/mg), CCL19 (specific activity: 1x104 U/mg), and CCL21 (specific activity: 5x103 U/mg) were obtained through R&D Systems. Lipopolysaccharide (LPS) was used at 10 ng/ml (Sigma Chemical Co., St. Louis, MO), and anti-CD40 monoclonal antibody (mAb; Upstate Biotechnology, Lake Placid, NY) was used at 10 µg/ml. PTX (Sigma) was used at concentrations ranging from 5 ng/ml to 10 µg/ml.

Generation of DC from peripheral blood monocytes or CD34+ progenitors
Monocytes were purified by immunomagnetic depletion (Dynabeads, Dynal, Oslo, Norway) after preparation of peripheral blood mononuclear cells (PBMC) followed by a 52% Percoll gradient. The pellet was eventually recovered for T-cell purification, and monocytes were purified from the low-density cells. The depletion was performed with anti-CD3 (OKT3), anti-CD19 (4G7), anti-CD8 (OKT8), anti-CD56 (NKH1; Coulter, Hialeah, FL), and anti-CD16 (ION16, Immunotech, Marseille, France) mAbs. Monocyte-derived DC were produced by culturing purified monocytes for 6–7 days in the presence of GM-CSF and IL-4 [26 ]. At days 6–7, between 85% and 95% of the cells were immature CD1a+ CD14- DC.

Alternatively, DC were generated from cord blood CD34+ progenitors in the presence of SCF, GM-CSF, and TNF-{alpha}, as previously described [27 ].

Purification of blood CD3+ T cells and natural killer (NK) cells
T cells were purified from PBMC by immunomagnetic depletion (Dynabeads, Dynal). CD3+ T lymphocytes were purified using a cocktail of mAbs, MOP9 (CD14), ION16 (CD16), mAb 89, (CD40), ION2 (HLA-DR; Immunotech), NKH1 (CD56; Ortho Diagnostic System, Raritan, NJ), and 4G7 (CD19). After two rounds of bead depletion, the purity of CD3+ T cells was routinely higher than 95%.

NK cells were purified following a similar procedure using anti-CD3 (OKT-3) and anti-CD4 (Sigma) mAbs instead of ION16 (CD16) and NKH1 (CD56). After two rounds of bead depletion, the purity of CD56+ NK cells was routinely higher than 80%.

Isolation of B cells
Mononuclear cells from tonsils were isolated by a standard Ficoll-Hypaque (density=1077 g/ml) gradient method. Partially purified tonsillar B cells were first enriched in the E- fraction (90–95% CD19+) and were used for most experiments. For some experiments, this preparation was submitted to anti-CD2, -CD4, -CD8, -CD14, and -CD16 mAbs negative selection with magnetic beads coated with anti-mouse IgG (Dynabeads, Dynal). CD19 was expressed on >99% of the B cells, as assessed by fluorescence analysis using a FACScalibur (Becton Dickinson, San Jose, CA).

Alternatively, B cells were isolated from blood samples. They were first enriched in the high-density fraction of a 52% Percoll and further purified by positive selection using CD19 microbeads and the MACS system (Myltenyi Biotech, Bergish Gladbach, Germany).

Chemotaxis assays
B cells
Before migration assays, B cells were preincubated in migration medium [RPMI 1640 supplemented with 2.5% fetal calf serum (FCS)] for 2 h. Migration assays were carried out using transwells (6.5 mm diameter and 5 µm pore size; Costar, Cambridge, MA). Briefly, supernatant from DC (SNDC) or other cells, or rh chemokines (100–1000 ng/ml) or combinations was added to the lower wells in 24-well plates. Cells (5x105) were added to the transwell inserts. Plates were incubated for 3 h at 37°C. After removal of the transwell inserts, cells from the lower compartments were labeled with fluorescein isothiocyanate (FITC) anti-IgD, phycoerythrin (PE) anti-CD38, and PE-Cy5 anti-CD19 for 30 min on ice. Relative cell counts were determined using a FACScalibur for 40 s under a constant sheath pressure with appropriate gates on forward-scatter and CD19 profiles. Each assay was performed in duplicate, and the results were expressed as the mean ± SD of migrated cells per well. Alternatively, the chemotaxis index was calculated as the ratio between the numbers of migrating cells in the sample and in the control medium. For PTX treatment, B cells were incubated for 2 h at 37°C with various concentrations of PTX, washed twice, and subsequently added to the top chamber of the chemotaxis assay.

Other cells
For the other cell populations, migration assays were performed without preincubation. Purified monocytes and monocyte-derived DC were allowed to migrate for 1.5 h and processed for counting using FITC-labeled anti-CD14 and anti-CD1a mAbs, respectively. T-cell migration was performed using 3 µm transwells and revealed using FITC-labeled anti-CD3, PE-labeled anti-CD8, and PE-Cy5-labeled anti-CD4 mAbs. NK cells were allowed to migrate for 3 h and stained using PE-labeled anti-CD56 mAb.

Production of SNDC
DC (3.75x105) were cultured in medium alone (RPMI 1640, 2.5% FCS) or with 10 µg/ml anti-CD40 mAbs or 10 ng/ml LPS in a final volume of 0.5 ml in 24-well culture plates. For some experiments, DC were seeded at concentrations ranging from 1 x 105 to 1.5 x 106 per ml. After 2 days of culture, supernatants were recovered, 0.22 µm-filtrated, and used for chemotaxis assays. Supernatants were also recovered from tonsillar B cells activated with anti-CD40 mAbs (10 µg/ml), from peripheral blood T cells cultured in the presence of IL-2, or from blood monocytes.

Partial characterization of the chemotactic activity released by DC
Sensitivity to protease was tested by incubating the SNDC with agarose cross-linked-type VIII-A protease (Sigma) for 60 min at 37°C followed by two centrifugations to remove the protease. Heat sensitivity was determined by heating the SNDC at 60 or 90°C for 15 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Several chemokines produced by DC can induce B-cell chemotaxis
To examine the chemotactic response of B lymphocytes to various chemokines, enriched tonsillar B cells were used for chemotaxis assays using transwells bearing polycarbonate filters with 5 µm pores. B cells were first incubated for 2 h at 37°C, and chemotaxis assays were performed with 5 x 105 B cells for 3 h. Transmigrated cells were further stained with FITC-conjugated anti-CD19 mAb, and B lymphocytes were counted by flow cytometry. As demonstrated by others [18 , 28 ], CXCL12ß (Table 1 ) or CXCL12{alpha} (unpublished results) triggered a strong migration of B lymphocytes, as shown by a mean chemotactic index (CI) of 50. CCL19 and CCL21 were reproducibly found to induce B-cell chemotaxis but with a lower magnitude (mean CI of 9.8 and 6.6, respectively, Table 1 ), as previously shown [5 , 8 , 9 ]. In addition, another CC chemokine, CCL20, induced B-cell chemotaxis with a comparable efficiency, in accordance with the expression of its receptor CCR6 on B lymphocytes [29 ]. In contrast, CCL22 (MDC) and CCL17 (TARC) did not induce significant migration (unpublished results). Thus, CXCL12 appears the most potent chemokine tested by far, able to trigger B-cell chemotaxis, whereas CCL19, CCL20, and CCL21 share lower but significant B-cell chemotactic activity. Of note, all these chemokines are known to be produced by DC upon activation [5 , 12 , 19 ].


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  		Table 1. B-Cell Migration in Response to Various Chemokines

 
DC selectively produce B-cell chemotactic factors
In previous studies, we have shown that DC directly interacted with CD40-activated B cells to induce their proliferation and differentiation into Ig-secreting plasma cells [22 23 24 25 ]. Here, we investigated whether DC may themselves produce factors able to promote these physical interactions. DC were generated from blood monocytes cultured for 6 days in the presence of GM-CSF and IL-4. Supernatants were produced by further culturing DC for 48 h, at 7.5 x 105 cells per ml in medium (2.5% FCS) supplemented with GM-CSF and were used to induce B-cell migration. As shown in Figure 1A , SNDC induced a vigorous migration of tonsillar B cells, which was in the same range as that induced by recombinant CXCL12 or CCL19. SNDC induced comparable migration of tonsillar (Fig. 1A) and blood (Fig. 1B) B cells. A mean CI of 25 (range 7–67, n=17) was observed with SNDC (Fig. 1F) and of 50 (range 18–96, n=13) and 10 (range 6–17, n=6) for CXCL12 and CCL19, respectively (Table 1) . Depending on the donor, the percentage of SNDC-responsive cells varied between 5% and 11% of the total input in five independent experiments (see Fig. 3C ). Similar results were observed using SNDCs derived from cord blood CD34+ progenitors cultured in the presence of GM-CSF and TNF-{alpha} for 12 days (unpublished results).



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  		Figure 1. SNDC displays selective chemotactic activity for B lymphocytes. The migration of different populations of cells in response to an SNDC, CXCL12ß (1 µg/ml), and CCL19 (1 µg/ml) was assayed using 5 µm transwells as described in Materials and Methods. Control shows the migration to the buffer medium alone. (A and B) Partially enriched human tonsillar or blood B cells were preincubated for 2 h before addition in the chemotaxis assay. After 3 h, migrated cells were recovered and stained with FITC-labeled anti-CD19 mAb and counted. (C and D) Blood CD3+ T cells were allowed to migrate for 1.5 h. Triple-color stainings for CD3, CD4, and CD8 and appropriate gates allowed the enumeration of migrated CD4+ and CD8+ T cells. (E) After 3 h, migration of NK cells was revealed by PE anti-CD56 staining. Results are indicated as the number of cells detected by flow cytometry for 40 s based on appropriate gates (four independent experiments). (F) A summary of the chemotactic activity of SNDC on different cell types is shown. Chemotaxis indexes were calculated as the ratio between the numbers of migrating cells in the sample and in the control medium.

 


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  		Figure 3. SNDC induces chemotaxis of naive and memory B cells. Total tonsillar B cells were tested in the chemotaxis assay in response to SNDC and an optimal concentration of CXCL12ß (1 µg/ml). After 3 h, migrated cells were recovered from the lower chambers and stained with FITC anti-IgD, PE anti-CD38, and PE-Cy5 anti-CD19 Abs. The migration of the three mature, B-cell subsets was determined by gating on CD19+IgD+CD38low naive B cells, CD19+IgD-CD38low memory B cells, and CD19+IgD-CD38high GC B cells. (A and B) Input B cells and migrated cells in response to SNDC were phenotyped by flow cytometry. Percentages of cells from the different subsets are indicated (one representative experiment out of five). (C–F) The migration of B-cell subsets in response to SNDC and CXCL12ß is shown as the mean percentage of input cells calculated from five independent experiments (range is also indicated).

 
Migration experiments with other cell types indicated that SNDC induced low but significant migration of blood CD4+, CD8+ T cells (CI<3; Fig. 1C and 1D ) and NK cells (CI<4; Fig. 1E ), contrasting with the potent effect of CXCL12 on these cell types (CI of 6, 10, and 6 for CD4+, CD8+ T cells and NK cells, respectively, n=4). In addition, SNDC induced significant migration of monocytes (CI=4.5; range 2–10, n=9) and DC themselves (CI=5; range 2–9, n=3). Thus, SNDC displays chemotactic activity for several cell types, but B lymphocytes appeared the main target (Fig. 1F) .

Finally, to distinguish between chemotaxis and chemokinesis, SNDC was added in the upper and lower chamber of the transwell (checkerboard analysis). As shown in Figure 2 , B-cell migration in response to SNDC was inhibited when the gradient was abolished, showing that DC induce directional migration of B cells.



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  		Figure 2. The chemotactic activity of SNDC is dependent on a gradient. Migration of tonsillar B cells to SNDC and CXCL12ß was studied as described in Figure 1 . SNDC or CXCL12ß was added in the upper chamber to abolish the gradient (checkerboard analysis). Results are indicated as the numbers of migrated cells (assessed by flow cytometry) ± SD and are representative of three independent experiments.

 
Together, these results show that DC produce soluble factors inducing selective and directional migration of B cells.

SNDC induces migration of naive and memory B cells
To determine whether SNDC attracted a particular subset of tonsillar B cells, transmigrated cells were phenotyped by triple-color staining using labeled anti-CD19, -CD38, and -IgD antibodies. Based on IgD and CD38 expression, three major subsets of mature B cells can be identified [30 ]: naive IgD+CD38low B cells, IgD-CD38low memory B cells, and IgD-CD38high germinal center (GC) B cells. The tonsillar B-cell preparation used in one representative experiment (shown in Fig. 3A ) contained 61% naive B cells, 21% memory B cells, and 14% GC B cells. In marked contrast, transmigrated cells were composed of 35% naive B cells, 59% memory B cells, and 4% GC B cells (Fig. 3B) . A compilation of five independent experiments, presented in Figure 3C 3D 3E 3F , and expressed as % of input cells, showed that SNDC preferentially attracted memory and to a lesser extent naive B cells but not GC B cells. This pattern of chemotactic response of B lymphocytes is similar to that observed with CXCL12 (Fig. 3 and ref. [18 ]). However, when chemotaxis indexes were considered, naive B cells (mean CI=26, range 5–67, n=17) appeared as efficiently attracted as memory B cells (mean CI=23, range 5–60, n=17). This finding is explained by the higher propensity of memory B cells to migrate spontaneously as compared with naive B cells (Fig. 3D and 3E , condition medium). Similar results were obtained for blood B-cell subsets (unpublished results).

Together, these results show that DC produce soluble factors inducing selective migration of naive and memory B lymphocytes.

DC constitute the preferential source of B-cell chemoattractants
We next determined whether the ability to produce B-cell chemotactic factors was shared by other populations of the immune system. For this purpose, three purified cell populations were tested under the same experimental conditions: tonsillar B cells activated by anti-CD40 mAbs, IL-2-stimulated CD3+ T cells, and monocytes. Culture supernatants were recovered after 2 days and tested for their capacity to induceB-cell chemotaxis. As shown in Figure 4A , only SNDC induced important migration of B cells, and a very weak effect of supernatants from other cell populations was observed (CI<3).



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  		Figure 4. The induction of B-cell migration is restricted to SNDC and independent of any activation. (A) Supernatants of GM-CSF-treated, monocyte-derived DC; resting monocytes; IL-2-stimulated blood T cells; or CD40-activated tonsillar B lymphocytes were tested for their effect on B-cell migration. (B) DC were seeded at different cell concentrations in migration medium alone or with soluble anti-CD40 mAb (10 µg/ml) or LPS (10 ng/ml). After 48 h, supernatants were tested for their activity on B-cell chemotaxis. Results are expressed as the number of migrated cells ± SD and are representative of three independent experiments.

 
In other experiments, SNDCs, seeded at different cell concentrations and activated or not for 48 h with soluble anti-CD40 mAbs or LPS, were tested for their capacity to induce B-cell chemotaxis. As shown in Fig. 4B , the activity of SNDC was dependent on the number of input DC but was not affected by DC activation (Fig. 4B) . In particular, anti-CD40 or LPS activation did not allow enhanced B-cell migration nor did it decrease the number of DC required.

Migration of B cells in response to SNDC is not sensitive to PTX
With the notable exception of CCL8 (MCP-2) [31 ], the chemotactic activity of most chemokines described to date is inhibited by treatment of cells with Bordetella PTX, which interfere with G{alpha}i proteins. As expected, low concentrations of PTX (20 ng/ml) totally abolished migration of B cells in response to CXCL12 (Fig. 5 ) or CCL19 (unpublished results). In contrast, even at high PTX concentrations, the migration of B cells in response to SNDC was only affected slightly, suggesting that SNDC-induced B-cell chemotaxis does not involve use of G{alpha}i proteins.



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  		Figure 5. SNDC-induced B-cell migration is not inhibited by PTX treatment. B-cell chemotaxis in response to SNDC was assayed as in Figure 1 . (A) Before the chemotaxis assay, B cells were incubated with various concentrations of PTX for 2 h, washed twice, and then tested for their capacity to respond to CXCL12ß (1 µg/ml) or SNDC. Results are expressed as the number of migrated cells ± SD. (B and C) SNDC and CXCL12 were submitted to protease digestion for 1 h (B) or heated at 60°C (hatched bars) or 90°C (solid bars) for 15 min (C) and were tested for their capacity to induce B-cell migration. Results were expressed as the % of inhibition of B-cell migration following enzymatic or heat treatment.

 
To characterize the chemotactic activity, SNDC was incubated with agarose-conjugated protease for 1 h and tested for its capacity to induce B-cell migration after removal of the enzyme. As shown in Figure 5B , SNDC-induced B-cell migration was largely resistant to protease digestion (<15% inhibition); in contrast, CXCL12-dependent B-cell chemotaxis was totally abolished following protein digestion. In addition, the chemotactic activity was highly sensitive to heat, as compared with a chemokine (CXCL12 is shown in Fig. 5C ), and >50% of the activity was lost after 15 min at 60°C.

Altogether, these results demonstrate that DC produce nonprotein, soluble factors, inducing B-cell chemotaxis in a PTX-insensitive way.

SNDC synergizes with defined chemokines to induce B-cell migration
DC produce many chemokines following activation [19 , 32 ], but the absence of a significant PTX-sensitive chemotactic activity for T and B cells suggests that our in vitro experimental conditions might not allow production of chemokines in sufficient concentrations to display in vitro chemotactic activity. To examine whether the chemotactic factors produced by DC are able to cooperate with chemokines to induce B-cell migration, SNDCs were supplemented with exogenous recombinant chemokines. As shown in Figure 6A , the migration induced by optimal concentrations of CXCL12 or CCL19 was strongly potentiated in the presence of SNDC. In particular, the number of cells that migrated under those conditions was always much more important than that expected by an additive effect of the two activities (arrows in Fig. 6 ), indicating that SNDC synergized with CXCL12 and CCL19. SNDC increased by 1.65-fold (range 1.3–1.9, n=5) and 2.0-fold (range 1.7–2.2, n=5) the number of cells migrating in response to CXCL12 and CCL19, respectively. In contrast, few if any effects were observed when optimal concentrations of recombinant CXCL12, CCL19, and CCL21 were combined together (unpublished results). Better synergism was observed when experiments were performed with suboptimal concentrations of CXCL12 (Fig. 6B , compare 0.2 and 1 µg/ml CXCL12).



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  		Figure 6. SNDC enhances the migration of B cells to CXCL12ß and CCL19. (A) CCL19 or CXCL12ß (1 µg/ml) was added in the bottom chamber in migration buffer alone or with SNDC. Tonsillar B cells were added in the upper chamber and incubated for 3 h at 37°C. (B) Response of B cells to various concentrations of CXCL12ß in the absence or presence of SNDC. Results are expressed as the number of migrated cells ± SD and are representative of three independent experiments. B-cell migration, observed when the recombinant chemokine was combined with SNDC, is compared with the migration that was expected (value of SNDC+value of CXCL12ß, arrowhead).

 
Collectively, these results show that DC produce soluble factors that not only induce chemotaxis of B cells per se but also act in synergy with known chemokines that they can produce upon activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In previous studies, we have shown that DC participate in the development of humoral immune responses through direct stimulation of B-cell proliferation and differentiation [22 23 24 25 ]. Here, we provide experimental evidence that DC constitute an important source of a novel class of chemoattractants, allowing selective and directional migration of naive and memory B cells in a PTX-insensitive way.

Using in vitro-generated DC, we found that SNDC allows directional migration of naive and memory B cells, whereas its activity on other cell populations, although significant, remains marginal. The level of B-cell migration elicited by SNDC is comparable with that observed with the most efficient B-cell chemokine tested, CXCL12, but much more stronger than the migration induced by other B-cell-active chemokines, such as CCL19, CCL20, or CCL21. Of note, most of these chemokines can be produced by DC upon activation [5 , 12 , 19 ]. That B-cell migration in response to SNDC is marginally inhibited by PTX, in contrast to the chemotactic response elicited by recombinant CCL19, CCL20, CCL21, or CXCL12, indicates that these chemokines did not contribute to the observed activity. SNDC was found to induce low but significant migration of other cell types including CD4+ and CD8+ T cells. This finding might somehow appear in contradiction with the recognized role of DC in the induction of T lymphocytes recruitment within T-cell-rich areas and their capacity to produce T-cell-attractive chemokines such as CCL19 [5 ] or CCL20 (personal observation). This result is probably a result of the low concentrations of chemokines reached in the conditioned medium as compared with the relatively high amounts of chemokine required to observe in vitro chemotaxis. In addition, production of T-cell-active chemokines by DC requires their activation [19 , 32 , 33 ], and only resting SNDCs were tested in T-cell migration assays in the present study (Fig. 1) .

To our knowledge, CCL8 (MCP-2) is the only chemokine described to date that elicits a chemotactic response involving PTX-insensitive G proteins [31 ], suggesting that a DC-derived B-cell chemoattractant(s) might be different from classical chemokines. That SNDC-induced B-cell migration is not merely affected by protease digestion demonstrates that the DC-chemotactic factor(s) is not a protein, definitely excluding a role for chemokines. Besides chemokines, other mediators involved in the recruitment and trafficking of cells have been recognized, such as formyl-Met-Leu-Phe (fMLP), platelet-activating factor (PAF), complement fragments (C3a, C5a), or leukotrienes, which are potent chemoattractants for neutrophils and other leukocytes. Complement fragments are unlikely involved in SNDC-induced B-cell chemotaxis, as indicated by resistance to protease treatment and by the fact that up to 1 µg/ml rhC5a did not induce significant migration of B cells nor did a blocking anti-C5aR (anti-CD88) antagonized, SNDC-dependent B-cell migration (unpublished results). DC were recently shown to express the 5-lipoxygenase [34 ] necessary for synthesis of leukotrienes, which display chemotactic activity. That SNDCs, generated in the presence of various 5-lipoxygenase (5-LO) inhibitors [nordihydroguaiaretic acid (NDGA), DEC, and hydrocortisone] [35 ], induced potent B-cell migration (unpublished results) suggests that leukotrienes are not mandatory for SNDC-dependent B-cell chemotaxis. PAF-R is one of the rare receptors described to date that mediates at least part of its activity on cell migration through PTX-insensitive G proteins [36 37 38 39 ]. PAF-R is expressed on B cells [40 , 41 ], and engagement of this receptor by PAF induces their activation [42 , 43 ]. However, preliminary results, obtained with rhPAF and chemical antagonists of PAF-R, indicate that PAF is unlikely involved in DC-induced B-cell attraction (unpublished results).

That DC chemotactic activity is not inhibited by PTX does not exclude that G-protein-coupled receptors (GPCR) might be involved on B cells. In line with this possibility, many orphan GPCR have been described, some of these receptors being expressed in the immune system and in particular on B cells, such as Epstein-Barr-induced 2 (EBI-2) [44 ]. Conversely, other factors, acting through surface receptors different from GPCR, such as cytokines, have been shown to display chemotactic activity in some instances. For example, IL-15, which can be produced by DC [45 46 47 ], has been shown to induce migration of T cells [46 , 48 ] and TNF-{alpha}, that of B cells [49 ].

All the nonchemokine chemoattractants mentioned above (fMLP, complement fragments, PAF, and leukotrienes) participate in the development of inflammatory responses during injury and infection. A group of chemokines induced upon inflammation [i.e., macrophage-inflammatory protein (MIP), GRO, and monocyte chemoattractant protein (MCP)] also participate in the selective recruitment of leukocyte subpopulations at a site of injury. Another group of chemokines that are expressed constitutively (i.e., CXCL12, CCL19, and CXCL13) are involved in the regulation of lymphocyte trafficking in lymphoid organs. The chemoattractants produced spontaneously by DC, selectively attracting B cells, are likely to be involved in the regulation of B-cell trafficking and recruitment within secondary lymphoid organs. If this holds true, this mediator would represent the first example of a factor, different from a chemokine, involved in the control of lymphocyte trafficking in lymphoid organs.

In the last part of this study, we showed that this DC chemoattractant synergizes with defined chemokines to induce B-cell migration. In particular, SNDC was found to allow a robust B-cell chemotactic response when combined with suboptimal doses of CXCL12 or CCL19, probably by lowering the threshold of sensitivity of B cells to chemokines. It is interesting that these chemokines active on B cells are produced by DC following activation [5 , 12 , 19 ]. Limited studies have documented cooperative effects between chemoattractants on cell migration. Recently, a cooperative interaction between CXCL12 and SCF in the chemotaxis of CD34+ hematopoietic progenitors has been shown [50 , 51 ]. In addition, thrombin, which acts through a GPCR [52 , 53 ], has been shown to prime responsiveness to chemoattractants such as IL-8, C5a, and fMLP on transfected cells [54 ]. More recently, leukotrienes C4 and D4 were found to promote optimal chemotaxis of DC to the chemokine CCL19 [55 ]. Furthermore, surface molecules such as proteoglycans or GPCR themselves (Duffy), by acting as presenting molecules, have been proposed to enhance the activity of chemokines [56 ]. Soluble forms of such molecules putatively produced by DC could be involved in this synergistic activity. More likely, DC chemoattractant, which displays potent chemotactic activity per se, might transduce signal to B cells, lowering the number of receptors needed to be engaged by chemokine to reach biological activity.

The putative physiological relevance of these observations is presented in the following model. After antigen capture in the periphery, DC reach the lymphoid organs through the lymph stream in response to inflammatory stimuli. DC, now homing in the T-cell area, produce chemoattractant(s), allowing recruitment of B cells within the extrafollicular area. Upon cognate interaction with T cells, DC are able to produce chemokines such as CCL19 [5 ] or CCL20 (personal observation), which can synergize with DC chemoattractant to amplify B-cell recruitment. Antigen-specific B cells might be preferentially attracted through the production of CCL19, because this chemokine potently attracts B cells that have been stimulated through their B cell receptor (BCR) [5 , 8 ]. In this context, it is noteworthy that in contrast to CXCL12 [18 ], B-cell chemotaxis in response to SNDC is not impaired by BCR engagement (unpublished results). By favoring recruitment of immunocompetent lymphocytes and subsequently delivering critical signals to T and B lymphocytes, DC appear as the key antigen-presenting cells (APC) in the orchestration of the humoral response.

That production of B-cell chemotactic factor(s) is not conditioned by maturation stimuli would indicate that immature DC might also contribute to B-cell trafficking. Indeed, besides a role in B-cell recruitment during initiation of immune responses, DC may have a role in B-cell translocation from one lymphoid compartment to another. In particular, DC (GCDC) with a relatively immature phenotype have been described within primary and secondary B-cell follicles [57 ] and were proposed to contribute to the GC reaction [25 ]. Thus, GCDC may contribute to the constitutive homing of B cells within follicle and/or attraction of GC precursors generated during the extrafollicular reaction. In addition, immature DC of other sites might contribute to the localization of B cells within mucosal areas (such as crypts) or marginal zones.

Altogether, this study shows that DC are likely involved in the early steps of B-cell recruitment and localization into discrete areas within secondary lymphoid organs through the production of nonclassical chemoattractant(s) selective for B cells.


   ACKNOWLEDGEMENTS
 
B. D. was supported by the Fondation Marcel Mérieux (Lyon, France). We are grateful to T. Defrance, P. Garrone, and A. Vicari for careful reading of the manuscript; S. Bourdarel and C. Alexandre for editorial assistance; and doctors from clinics and hospitals in Lyon who provided us with umbilical cord blood samples and tonsils.

Received February 22, 2000; revised May 14, 2001; accepted May 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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