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CHEMOKINES
From INSERM U131, Institut Paris-Sud sur les Cytokines,
Clamart, France; and Unité d'Immunologie Virale and Unité
de Chimie Organique, Institut Pasteur, Paris, France.
Interleukin-10 (IL-10) is constitutively produced by peritoneal B1a
lymphocytes, and stromal cell-derived factor-1 (SDF-1) by mesothelial
cells. Independent studies have shown that both IL-10 and SDF-1 are
involved in the persistence of the peritoneal B-lymphocyte compartment.
This study shows that IL-10 and SDF-1 act in synergy on peritoneal B
lymphocytes. Indeed, autocrine production of IL-10 was absolutely
required for all effects of SDF-1 on these cells, including increased
proliferation, survival, and chemotaxis. Moreover, adding IL-10 to
peritoneal B lymphocytes increased the effects of SDF-1. Neither IL-5,
IL-6, nor IL-9 affected the response of peritoneal B lymphocytes to
SDF-1. IL-10 was chemokinetic for peritoneal B lymphocytes, increasing
their random mobility. It also potentiated the SDF-1-induced
reorganization of the cytoskeleton without affecting CXCR4 gene
expression by peritoneal B lymphocytes. Despite its chemokinetic
properties, IL-10 abolished the migration of peritoneal B lymphocytes
in response to B-lymphocyte chemoattractant (BLC), a chemokine
targeting B lymphocytes to lymphoid organ follicles. The ability of B1a
lymphocytes to produce IL-10 constitutively, combined with the opposite
effects of this cytokine on the responses to SDF-1 and BLC, may account
for the selective accumulation of B1 lymphocytes in body cavities.
(Blood. 2002;99:427-436) Peritoneal B lymphocytes are divided into 3 different subpopulations identified on the basis of the expression of
the Mac-1 and CD5 antigens.1-3 B lymphocytes expressing
neither of these antigens are referred to as B2 lymphocytes. They are
thought to correspond to conventional B lymphocytes, the main B-cell
population outside body cavities. The second peritoneal B-lymphocyte
subpopulation, the B1b cells, express Mac-1 but not CD5. Like
peritoneal B2 lymphocytes, they are derived from a bone marrow
precursor. However, they are rare outside body cavities. The third
subpopulation, the B1a cells, express Mac-1 and CD5 and have the unique
property of self-renewal in the peritoneal cavity, independent of bone
marrow-derived progenitors. B1a lymphocytes seem to be directly
derived from fetal liver precursors and to persist from birth in body
cavities as an autonomous cell population; the repopulation of
immune-deficient mice with peritoneal B lymphocytes restores only the
B1a subpopulation.4-6 B1a lymphocytes play a critical role
in innate immunity.3,4,7,8 They are also involved in the
production of autoantibodies.9-12
The mechanism by which B-lymphocyte populations are retained, survive,
and expand in body cavities is not completely understood, but the
production of a limited set of cytokines appears to be required. Mice
transgenic for interleukin-9 (IL-9) display considerable expansion of
the peritoneal B-lymphocyte compartment because of the accumulation of
B1b lymphocytes and, to a lesser extent, of B1a cells.13
Mice lacking the IL-5 or IL-5R gene14-17 display transient
and partial depletion of peritoneal B1a lymphocytes. IL-5 induces the
expansion of B1a lymphocytes in vitro and in vivo,18,19
and mice transgenic for IL-5 produce autoantibodies.20 It
is unknown whether IL-5 and IL-9 are produced in body cavities, and
their potential sources at these sites have not been identified. IL-10
is another cytokine that may be involved in B1a self-renewal. The
neutralization of IL-10 by anti-IL-10 monoclonal antibody (mAb)
administration depletes B1a lymphocytes in normal mice21 and prevents autoimmunity in NZB/W F1 mice, a model of systemic lupus
erythematosus in which the B1a-lymphocyte compartment is abnormally
expanded.22 In vivo administration of IL-10 activates peritoneal B1a lymphocytes and stimulates autoantibody production in
predisposed mice.19 Because B1a lymphocytes produce
IL-10,23 this cytokine may act as an autocrine growth
factor for these cells.
Chemokines may also affect peritoneal B lymphocytes. B-lymphocyte
chemoattractant (BLC, CXCL13)24 plays a critical role in
the homing of B lymphocytes to follicles in the Peyer patches and
spleen.25 Experiments in vitro have shown that BLC
attracts peritoneal B lymphocytes.26 Stromal cell-derived
factor-1 (SDF-1, CXCL12)24 also acts on peritoneal B
lymphocytes. Mice with a disrupted SDF-1 gene have impaired
B-lymphocyte development.27,28 SDF-1 stimulates the
proliferation29 and chemotaxis30-32 of
conventional B lymphocytes and of their precursors, and it protects
lymphocytic leukemia B cells from spontaneous apoptosis.33
In vivo, SDF-1 is required for retention of the B lineage in the bone
marrow.34,35 We recently showed that SDF-1 is involved in
the persistence of peritoneal B lymphocytes.6 In vitro,
SDF-1 stimulates the migration and proliferation of peritoneal B
lymphocytes, and it prevents their apoptosis. The in vivo
neutralization of SDF-1 largely depletes the peritoneal
B-lymphocyte compartment without significantly affecting lymphoid
tissues. Mesothelial cells constitutively produce SDF-1 in embryos
and in adults.6,36 This chemokine therefore presumably
acts as a nursing paracrine factor for the retention, survival, and
expansion of B lymphocytes in body cavities.
These reports indicate that the persistence of B lymphocytes in the
body-cavity compartment, and particularly the self-renewal of B1a
lymphocytes, may involve interactions between various cytokines and
chemokines. Recently, IL-10 has been shown to prevent the signal
transduction induced by a large set of chemokines in dendritic cells
(DCs), indicating that for these cells, IL-10 acts as a decoy cytokine
for chemokine receptor function.37 We show here that, as
for DCs, IL-10 prevents the effects of the chemokine BLC on peritoneal
B lymphocytes. In contrast, IL-10 has the opposite effect on SDF-1
function: There is a strong synergy between IL-10 and SDF-1 for the
proliferation, migration, and survival of peritoneal B lymphocytes, and
autocrine production of IL-10 by peritoneal cells is essential for all
SDF-1 effects on these cells. Therefore, the constitutive production of
IL-10 by peritoneal B1a lymphocytes, combined with the opposite effects
of this cytokine on the responses to BLC and SDF-1, may favor the
action of SDF-1 on these cells, accounting for the scarcity of B1
lymphocytes in lymphoid organs and their selective accumulation in body cavities.
Mice and cell preparation
IL-10 production studies
For the detection of IL-10-containing cells, peritoneal cells were cultured at 37°C in RPMI medium (2 × 106 cells/mL) with or without SDF-1 (1 µg/mL). Eighteen hours later, cells were recovered, washed, and labeled with fluorescent mAbs (CD19, CD5, and Mac-1) at 4°C for 30 minutes. Cells were washed with 2% FCS in PBS, fixed with 2% formaldehyde in PBS for 30 minutes at room temperature, washed again, and labeled for 1 hour by incubation in a permeabilizing saponin buffer (0.5%; Sigma, St Louis, MO) with the fluorescein isothiocyanate (FITC)-conjugated rat antimouse IL-10 Ab (clone JES5-16E3) or the FITC-conjugated rat IgG2b isotype control Ab (clone A95-1). Cells were washed twice in the saponin buffer and analyzed by 4-color flow cytometry (FACScan; Becton Dickinson, Rungis, France). All mAbs were from Becton Dickinson. Whether or not SDF-1 was present, less than 1% IL-10-containing cells were detected if the saponin buffer was omitted. Functional evaluation of chemokine receptors Proliferation and survival experiments, using the 5(6) carboxyfluorescein diacetate, succinimidyl ester (CFDA, SE) and the 3,3'-dihexylocarbocyanine iodide (DIOC6 (3)) dye assay, respectively, were performed as described previously.6 Murine IL-10 (R&D Systems) was used at a concentration of 100 ng/mL. A goat antimouse IL-10R Ab and a control Ab (R&D Systems) were used at a concentration of 10 µg/mL. SDF-1 and BLC were each used at a concentration of 1 µg/mL.To investigate the effect of IL-10R neutralization on cytoskeleton rearrangement, we stained freshly isolated cells with fluorescent mAbs at 4°C for 30 minutes. Cells were washed in 2% FCS in PBS and incubated at 37°C for 2 hours in RPMI medium containing 20 mM HEPES (5 × 106 cells/mL), with or without the anti-IL-10R or the control Ab (10 µg/mL). Cells were washed and incubated at 37°C in RPMI medium (5 × 106 cells/mL) before assessing cytoskeleton rearrangement. The actin polymerization induced by SDF-1 (250 ng/mL) was assessed as described previously.6,38 The chemotaxis of cell subpopulations was evaluated using the Transwell system (Costar) as described previously,6 except that one million cells were added to the upper chamber. The chemokines SDF-1, BLC, and SLC were used at a concentration of 1 µg/mL, and the cytokines IL-5, IL-6, IL-9, and IL-10 (all from R&D Systems) were used at 100 ng/mL. The anti-IL-10R and control Abs were used at a concentration of 10 µg/mL. Unless specified, the chemokines were added to the lower chamber only, whereas IL-5, IL-6, IL-9, IL-10, and the Abs were added to both the upper and lower chambers. Purification of CD19+ peritoneal cells and reverse transcriptase-polymerase chain reaction studies Peritoneal cells from 20 Balb/c mice were obtained by intraperitoneal washing and were purified using streptavidin-coated magnetic beads (CELLection Dynabeads; Dynal, Oslo, Norway) and a biotinylated CD19 mAb (Becton Dickinson). The final preparation contained more than 95% CD19+ cells, less than 1% CD3+ cells, and less than 4% CD4+CD3 cells. Cells were cultured at 37°C
at a density of 2 × 106 cells/mL in RPMI medium
supplemented with 10% FCS, with or without the anti-IL-10R Ab (10 µg/mL) or the control Ab for 4 hours, or with or without IL-10 (100 ng/mL) for 2 hours.
Finally, cells were recovered and stored in RNA-B extraction solution (Quantum; Appligene, Illkirch, France). Expression of the murine CXCR4 and CXCR5 genes was analyzed by RT-PCR from 100 ng total RNA. Thirty-one (for CXCR4) or 40 (for CXCR5) cycles of PCR (each cycle consisting of 60 seconds at 94°C, 60 seconds at 57°C, and 90 seconds at 72°C) were performed, using the 5'-TCATCACACTCCCCTTCTGG-3' sense primer and the 5'-GGGTAAAGGCGGTCACAGAT-3' antisense primer for CXCR4, and the 5'-CTAGAGCCTGGTTCACCTCC-3' sense primer and the 5'-GCTGCTATTCACAGCCTTCAGCC-3' antisense primer for CXCR5. Amplified products were subjected to 1.2% agarose gel electrophoresis, detected by ethidium bromide staining, and quantified by computed-assisted densitometry using the ScanAnalysis software (Biosoft, Cambridge, United Kingdom). Statistical analysis Results were compared using the Mann-Whitney U test.
IL-10 increases the effect of SDF-1 on the proliferation and survival of peritoneal B lymphocytes The effects of SDF-1 and IL-10 on the proliferation of peritoneal B lymphocytes were analyzed at the single-cell level using the 5(6) CFDA, SE staining technique. As reported previously,6 SDF-1 stimulated the proliferation of a fraction of peritoneal B lymphocytes. The number of cells that divided during culture, characterized by a low 5(6) CFDA, SE content, was 4 to 5 times higher in the presence of SDF-1 than in control cultures. IL-10 also stimulated the proliferation of peritoneal B lymphocytes, although to a lesser extent than SDF-1. The highest level of proliferation was observed if SDF-1 and IL-10 were added together, with approximately one quarter of the cells resulting from cell division (Table 1) and with more cycles of division for proliferating cells (Figure 1). BLC alone had little, if any, effect on the proliferation of peritoneal B lymphocytes. The addition of IL-10 did not increase the number of cells proliferating in the presence of BLC (Figure 1).
The effect of SDF-1 and IL-10 on the spontaneous apoptosis of
peritoneal B lymphocytes was evaluated by DIOC6 (3)
labeling and flow cytometry. The addition of SDF-1 resulted in 26% to
47% fewer apoptotic cells at the end of a 2-day culture period. IL-10 also prevented cell death, although to a lesser extent than SDF-1. The
simultaneous addition of SDF-1 and IL-10 resulted in a lower proportion
of apoptotic cells than for cells cultured alone or in the presence of
a single cytokine (Table 2). BLC had no
effect on the apoptosis of peritoneal B lymphocytes, even if IL-10 was added to the cultures (data not shown).
The 3 B-lymphocyte subpopulations, B1a, B1b, and B2 cells, were all sensitive to the combined effects of SDF-1 and IL-10 on proliferation and apoptosis, with the effects most pronounced for B1a cells in both assays. IL-10 potentiates the chemotactic effect of SDF-1 The effect of IL-10 and SDF-1 on the migration of peritoneal B lymphocytes was evaluated in Transwell chemotaxis experiments with freshly isolated peritoneal cells. The number and phenotype of the B lymphocytes migrating to the lower chamber were determined by flow cytometry. SDF-1 stimulated the chemotaxis of peritoneal B lymphocytes. IL-10 slightly increased migration if added alone, but it strongly increased the effect of SDF-1. B1a, B1b, and B2 lymphocytes were all sensitive to this synergistic induction of migration by IL-10 and SDF-1 (Figure 2). In contrast to IL-10, neither IL-5, IL-6, nor IL-9 potentiated the SDF-1-induced migration of peritoneal B1a cells (Figure 3) or of B1b or B2 cells (data not shown).
SDF-1 stimulates IL-10 production by peritoneal B cells We investigated the effect of SDF-1 on the production of IL-10 by peritoneal cells. Peritoneal cells were cultured for 24 hours with various concentrations of SDF-1. IL-10 production by spleen cells was studied in parallel. In the absence of stimulation, peritoneal cells produced larger amounts of IL-10 than did spleen cells. SDF-1 induced a dose-dependent increase in IL-10 production by peritoneal cells. It had no effect on spleen cells (Figure 4A). In the presence of 500 ng/mL SDF-1, approximately 8 times more IL-10 was produced by peritoneal cells than by spleen cells. Neither BLC, SLC, nor fractalkine stimulated IL-10 production by peritoneal cells (data not shown).
To determine whether B lymphocytes contributed to IL-10 production by peritoneal cells and which B-cell subpopulation produced IL-10, we performed flow cytometry experiments using an anti-IL-10 mAb in combination with B-lymphocyte subpopulation markers. In the absence of SDF-1, only a few cells contained IL-10. The incubation of peritoneal cells with SDF-1 increased the proportion of IL-10-containing cells in the 3 B-lymphocyte subpopulations, with the highest proportion of positive cells in the B1a subpopulation (Figure 4B-D). Endogenous IL-10 is required for all SDF-1 effects on peritoneal B lymphocytes The ability of SDF-1 to increase IL-10 production by peritoneal B lymphocytes raised the question of the role of the autocrine production of IL-10 on the effects of SDF-1. To address this issue, we investigated the effect of a blocking anti-IL-10R Ab on SDF-1-induced peritoneal B-lymphocyte proliferation and survival. The stimulating effect of SDF-1 on the proliferation of B1a lymphocytes was totally abolished in the presence of the anti-IL-10R Ab (Figure 5A). The antiapoptotic effect of SDF-1 also required the endogenous production of IL-10 because it was totally abolished in the presence of the anti-IL-10R Ab (Figure 5B). The influence of the anti-IL-10R Ab on the SDF-1-induced proliferation and apoptosis of B1b and B2 peritoneal lymphocytes was similar (data not shown).
We also investigated the effect of the anti-IL-10R Ab on the migration
of peritoneal B lymphocytes. The chemotactic effect of SDF-1 was
inhibited by more than 90% in the presence of the anti-IL-10R Ab.
Migration of the 3 peritoneal B-lymphocyte subpopulations was affected
by blocking IL-10R (Figure 6). A rat
antimouse IL-10R mAb (1B1.3A; 10 µg/mL) and a rat IgG1 control mAb
(GL113; kindly provided by R. Coffman, DNAX, Palo Alto, CA) were also
tested in migration assays and gave similar results to the goat
polyclonal Abs (data not shown).
Therefore, experiments analyzing proliferation, prevention of apoptosis, and migration all led to the same conclusion: The effects of SDF-1 on peritoneal B lymphocytes absolutely require production of IL-10 by peritoneal cells. Lack of synergy between IL-10 and SDF-1 for spleen B2 lymphocytes We evaluated whether the synergistic effects between IL-10 and SDF-1 evidenced with peritoneal B lymphocytes also occurred with spleen B lymphocytes. The frequency of spleen B1 lymphocytes was too low to address this question for this cell subpopulation. On spleen B2 lymphocytes, SDF-1 did not stimulate proliferation (Figure 7C), did not prevent apoptosis (Figure 7E), and did not trigger actin polymerization (data not shown). In contrast, it stimulated chemotaxis of these cells (Figure 7A). Neither the addition of IL-10 nor the addition of an anti-IL-10R Ab changed the response of spleen B2 cells to SDF-1 (Figure 7). Therefore, the synergistic effect between IL-10 and SDF-1 is specific for peritoneal B lymphocytes.
Mechanism of synergy between IL-10 and SDF-1 in peritoneal B-cell migration To gain insight into the mechanism by which IL-10 affects SDF-1-induced migration, we investigated whether IL-10 acted as a chemokinetic agent, potentiating the chemotactic effect of SDF-1. In Transwell experiments, IL-10 or SDF-1 was added to the lower chamber only or to both the lower and upper chambers. As expected, the main effect of SDF-1 was chemotactic; the number of cells migrating to the lower chamber was largest if SDF-1 was added to the lower chamber only. Adding SDF-1 to both the upper and lower chambers decreased migration by approximately 90%. IL-10 weakly stimulated cell migration. However, unlike SDF-1, it acted as a chemokinetic agent; similar numbers of cells migrated to the lower chamber regardless of whether IL-10 was added to the lower chamber only or to both chambers (Figure 8). In contrast to IL-10, IL-5, IL-6, and IL-9 were not chemokinetic for peritoneal B lymphocytes (data not shown).
We investigated the synergy between IL-10 and SDF-1 further by
studying the effect of the anti-IL-10R Ab on SDF-1-induced cytoskeleton rearrangement. Freshly isolated peritoneal cells were
incubated at 37°C with or without the blocking anti-IL-10R Ab or a
control Ab. Two hours later, cells were stimulated with SDF-1, and
actin polymerization was monitored for 60 seconds by flow cytometry.
The control Ab had no effect on SDF-1-induced cytoskeleton
rearrangement. In contrast, the anti-IL-10R Ab abolished SDF-1-induced actin polymerization, regardless of the B-lymphocyte subpopulation considered (Figure 9).
Similar findings were obtained when using a rat anti-IL-10R mAb
(data not shown). This indicates that the continuous production of
IL-10 by peritoneal cells is required for SDF-1-induced
cytoskeleton rearrangement.
IL-10 does not affect CXCR4 gene expression in peritoneal B lymphocytes The modulation of the effects of SDF-1 by IL-10 or IL-10R neutralization may reflect a role of IL-10 on CXCR4 expression. We determined whether IL-10 or the anti-IL-10R Ab affected the level of CXCR4 gene expression by peritoneal cells. Addition of IL-10 for 2 hours to freshly isolated CD19+ peritoneal B lymphocytes did not modify the level of CXCR4 gene expression (Figure 10B). The amount of CXCR4 mRNA was higher in cultured CD19+ peritoneal B lymphocytes than in freshly isolated cells. Addition of the anti-IL-10R Ab did not prevent this increase in expression of the CXCR4 gene (Figure 10A). Therefore, the effects of IL-10 and of the anti-IL-10R Ab on SDF-1 responsiveness were observed independently of an effect of these agents on CXCR4 gene expression.
IL-10 has opposite effects on SDF-1-induced and BLC-induced chemotaxis We compared the effects of IL-10 on the chemotaxis of peritoneal B lymphocytes induced by SDF-1, BLC, and SLC. BLC was as efficient as SDF-1 at stimulating the migration of B1a, B1b, and B2 lymphocytes. SLC was as efficient as the other 2 chemokines at stimulating the migration of B2 lymphocytes. However, it had only a weak effect on B1a and B1b lymphocytes (Figure 11). Interestingly, IL-10 had opposite effects on the migration induced by BLC and on that induced by SDF-1: Addition of IL-10 inhibited the BLC-induced migration of B1a, B1b, and B2 lymphocytes, whereas the anti-IL-10R Ab increased the response to BLC (Figure 11). The effects of IL-10 and the anti-IL-10R Ab correlated with their effects on CXCR5 gene expression. The anti-IL-10R Ab weakly increased CXCR5 gene expression, whereas IL-10 decreased this expression (Figure 10). Therefore, the concentration of IL-10 in the microenvironment determines which chemokine induces a response in peritoneal B lymphocytes, with starvation of IL-10 selecting for a response to BLC and excess of IL-10 selecting for a response to SDF-1.
SDF-1 and IL-10 are cytokines produced in the body-cavity microenvironment. SDF-1 is constitutively synthesized by mesothelial cells,6,36 and IL-10 by peritoneal B lymphocytes.23 Independent studies have shown that SDF-1 and IL-10 each contribute to the persistence of the peritoneal B-lymphocyte compartment, and particularly of the B1a-lymphocyte subpopulation.6,19,21,22 The results of this study confirm and extend those of previous studies by showing that SDF-1 and IL-10 do not act independently on peritoneal B cells, but instead act in synergy. All the effects of SDF-1 on these cells require the presence of IL-10, including proliferation, prevention of apoptosis, and chemotaxis. IL-10 produced by peritoneal cells is needed for SDF-1 effects because these effects are no longer observed in the presence of an anti-IL-10R Ab. These findings demonstrate the critical role of autocrine IL-10 production by peritoneal B lymphocytes, sensitizing these cells to SDF-1. Moreover, we found that the addition of IL-10 increased the effects of SDF-1 on peritoneal B lymphocytes, indicating that the concentration of IL-10 in the microenvironment of peritoneal B lymphocytes modulates the intensity of their response to SDF-1. Because SDF-1 increases the production of IL-10 by peritoneal B lymphocytes, it may sensitize them to its own action in a self-amplifying loop. It is possible that other peritoneal cells in addition to B lymphocytes contributed to the local IL-10 production. Thus, overall, our results suggest that the role of autocrine IL-10 production in the self-renewal of B1a peritoneal lymphocytes23 is to potentiate the effects of SDF-1. By facilitating migration toward the local source of SDF-1 (mesothelial cells), IL-10 may favor the retention of B lymphocytes in the peritoneal cavity. By potentiating the effects of SDF-1 on proliferation and on the prevention of apoptosis, IL-10 may favor the local survival and expansion of these cells. This may be the reason why the treatment of mice with either an anti-IL-1021 or an anti-SDF-16 mAb leads to the depletion of peritoneal B lymphocytes. B1 lymphocytes accumulate not only in body cavities, but also in the gut-associated lymphoid tissue and in mucosa.3,17,18,39,40 SDF-1 is produced in large amounts by digestive epithelial cells,41 and IL-10 is produced in the gut.42 It is thus possible that SDF-1 and IL-10 also have a combined effect on B1 lymphocytes in the intestinal mucosa, leading to the accumulation and survival of B1 lymphocytes at this site. A continuous exchange of B1 lymphocytes between the intestinal wall, the peritoneal cavity, and the omentum has been suggested,3,18,43 and this would imply that changes in the peritoneal B1-lymphocyte compartment may also indirectly affect intramucosal B1 cells. B1 lymphocytes are an important source of mucosal IgA.39 They also play a critical role in innate immunity.3,4,7,8 The combined effects of IL-10 and SDF-1 on B1-lymphocyte expansion and retention in the body cavities, and possibly in the intestinal wall, may thus be important in mucosal and innate immunity. In contrast to the situation in anti-IL-10 mAb-treated mice, no
abnormalities of the peritoneal B-lymphocyte compartment were noted in
mice with a disrupted IL-10 or IL-10R gene, based on the analysis of
the CD5 and B220 markers of B-lymphocyte
subpopulations.1,44 Using CD5 and Mac-1 as markers of
B-lymphocyte subpopulations, we observed a significant decrease in the
numbers of peritoneal B1a and B1b lymphocytes (more than 70% and more
than 50%, respectively) in IL-10 The mechanism of synergy between IL-10 and SDF-1 on cell migration involves a chemokinetic effect of IL-10 and a chemotactic effect of SDF-1: IL-10 increases the random mobility of peritoneal B lymphocytes, which then migrate more efficiently along a gradient of SDF-1. Such synergy between a chemokinetic and a chemotactic agent has already been reported for eosinophils. IL-5 and eotaxin act synergistically to recruit these cells, with IL-5 being chemokinetic and eotaxin being a chemoattractant.45 The mechanism of cooperation between IL-10 and SDF-1 involves an early step in SDF-1 action because the presence of IL-10 is essential for cytoskeleton rearrangement during the few seconds following SDF-1 addition. The effect of IL-10 is also transient; SDF-1 loses its ability to act on peritoneal B lymphocytes after only 2 hours of incubation of the cells with an anti-IL-10R Ab. Thus, the constitutive production of IL-10 by peritoneal cells continuously sensitizes them to SDF-1, and variations in IL-10 concentration rapidly affect sensitivity to SDF-1. This modulation occurs without changes in the expression level of the CXCR4 gene. This suggests that IL-10 does not influence the expression of the receptor on the cell surface, but this point could not be tested directly in view of the lack of reagent allowing CXCR4 detection in the mice. Several examples are known in which CXCR4 is expressed but does not function.46-49 D'Amico et al37 showed that IL-10 abolishes the function on DCs of many chemokine receptors. Our results with BLC are consistent with such a decoy effect of IL-10 on the function of chemokine receptors. However, our results extend these findings by showing that the effect of IL-10 on the response to a chemokine is not always inhibitory. For peritoneal B lymphocytes, IL-10 is absolutely required for the appropriate action of SDF-1 on its receptor. Thus, depending on the concentration of IL-10, peritoneal B lymphocytes are highly sensitive to SDF-1 but almost unresponsive to BLC if IL-10 concentration is high; the opposite is true in the absence of IL-10. If this phenomenon extends to other conditions, then, in a tissue with several superimposed chemokine gradients, a single cytokine in the microenvironment may determine which chemokine gradient a cell ignores and which one it follows. Conversely, acute changes in the cytokine content of the microenvironment rapidly redirect cell migration by changing the chemokine gradient selected by the cell. In the case of peritoneal B lymphocytes, such a dual and opposite effect of IL-10 on BLC and SLC effects may explain the scarcity of B1 lymphocytes in lymphoid organs and their accumulation in body cavities. BLC is involved in the homing of B lymphocytes to the B-cell zones of lymphoid organs.25,50,51 The constitutive production of IL-10 by B1 lymphocytes may prevent their accumulation in lymphoid organs, an effect amplified by their poor response to another chemokine targeting lymphocytes to lymphoid tissues, SLC. In contrast, this constitutive production of IL-10 probably sensitizes B1 cells to mesothelial-derived SDF-1, leading to their retention in body cavities. SDF-1 is a chemokine with pleiotropic effects. However, most, if not all, of the effects of SDF-1 on its various target cells require the simultaneous presence of specific cytokines, which differ according to the target considered: macrophage-derived chemokine for platelets; thrombopoietin for megakaryocyte precursors; stem cell factor/kit ligand, IL-6, and steel factor for hematopoietic progenitors; and vascular endothelial growth factor for endothelial cells.52-58 Our findings provide another example of this phenomenon, identifying IL-10 as the key cytokine required for SDF-1 effects on peritoneal B lymphocytes. Therefore, despite the multiplicity of effects of SDF-1, the cytokine composition of the microenvironment determines the local effects of SDF-1, thereby limiting the influence of this chemokine in each body compartment to a particular set of cells. In NZB and NZB/W mice, the B1-lymphocyte compartment is expanded, a feature that is dependent on IL-10 and leads to B-lymphoid malignancies and to autoimmune disorders.22,59-61 Elimination of peritoneal B lymphocytes or IL-10 neutralization in a transgenic mouse model of autoimmunity prevents the emergence of autoimmune manifestations.12,62 A similar mechanism may occur in human systemic lupus erythematosus, in which there is an expansion of IL-10-producing B lymphocytes, IL-10-dependent autoantibody production, and an improvement in clinical symptoms in patients treated with an anti-IL-10 mAb.63-65 B1 lymphocytes of NZB/W mice are extremely sensitive to SDF-1 stimulation, and this hypersensitivity is totally dependent on the endogenous production of IL-10 by B1 cells (unpublished results, December 2000). This suggests that SDF-1 plays a role in conjunction with IL-10 in this model of B1-lymphocyte-mediated autoimmunity, a hypothesis currently being tested in vivo. Our findings show that the retention of B lymphocytes as a self-renewing compartment within body cavities requires the combined effects of SDF-1, produced by mesothelial cells, and IL-10, produced by peritoneal cells themselves. This synergistic effect results in greater migration, proliferation, and survival of B lymphocytes. Moreover, the amount of IL-10 acting on peritoneal B lymphocytes modulates their sensitivity to SDF-1 and to BLC in opposite ways, demonstrating the contribution of the cytokine content of the microenvironment to driving the response of immune cells to a complex network of chemokines.
Yolande Richard is acknowledged for critical review of the manuscript. Robert Coffman is acknowledged for the gift of the 1B1.3A anti-IL-10R and the GL113 control mAbs. Jean-Christophe Renauld (Ludwig Institute for Cancer Research, Brussels, Belgium) is acknowledged for kindly providing IL-9 transgenic (Tg5) and control (FVB) mouse strains.
Submitted January 26, 2001; accepted July 18, 2001.
Supported by the Association pour la Recherche sur le Cancer.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dominique Emilie, INSERM U131, Institut Paris-Sud sur les Cytokines, 32 rue des Carnets, 92140 Clamart, France; e-mail: emilie{at}ipsc.u-psud.fr.
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N. Watanabe, K. Ikuta, S. Nisitani, T. Chiba, and T. Honjo Activation and Differentiation of Autoreactive B-1 Cells by Interleukin 10 Induce Autoimmune Hemolytic Anemia in Fas-deficient Antierythrocyte Immunoglobulin Transgenic Mice J. Exp. Med., July 1, 2002; 196(1): 141 - 146. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-actin ratio (mean ± SEM) was 0.43 ± 0.09,
1.02 ± 0.07, and 1.01 ± 0.02 for lanes 1 to 3, respectively.
Similar analysis for CXCR5 gene expression showed a CXCR5/
triggers a chemotactic response and induces cell polarization in human B lymphocytes.
Eur J Immunol.
1998;28:2197-2207