Address correspondence to Garnett Kelsoe, Dept. of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. Phone: (919) 613-7815; Fax: (919) 613-7878; email: ghkelsoe{at}duke.edu
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Abstract |
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Key Words: bone marrow innate immunity TNF hematopoiesis neutrophilia
The mechanisms whereby adjuvants/inflammation elicit BM lymphopenia are not understood but are independent of adaptive immunity (4). Could innate immune effectors regulate BM lymphopoiesis? In vitro, IL-1 inhibits B lymphopoiesis and promotes myelopoiesis (5, 6). These effects are reversible, and appear to reflect change in nonhematopoietic BM compartments (6). The early stages of B cell development are exceptionally sensitive to apoptosis (7), and the proinflammatory cytokine IFN
Alternatively, as inflammation elicits developing B lymphocytes in the periphery (3, 4, 1216), BM lymphopenia could reflect mobilization rather than the interruption of a developmental pathway or cell death. For example, cells with the characteristics of preB and immature B lymphocytes appear in mouse spleen 2 wk after immunizations with adjuvant (3, 4, 13, 15, 1719).
Here, we demonstrate that adjuvants suppress chemokine CXCL12 expression in the BM and that these reductions coincide with lymphocyte depletion and mobilization of B cell progenitors to the blood and spleen. Recombinant TNF
Inflammation redirects immunocyte production in BM to favor granulopoiesis. This redirection is an unrecognized inflammatory response to microbial infection and a novel pathway for the regulation of B lymphopoiesis.
Antigens, Adjuvants, and Cytokines.
Antibodies.
Flow Cytometry.
B Cell Colony Forming Unit Assay.
Adoptive Cell Transfer.
RT-PCR.
Preparation of BM Plasma and CXCL12 ELISA.
CXCL12 protein concentrations were determined by ELISA. In brief, 96-well plates (BD FalconTM; BD Biosciences) were coated overnight with anti-CXCL12 mAb 79018 (R&D Systems) (2 µg/ml in 0.1 M carbonate buffer) at 4°C. Serially diluted BM plasma samples were loaded, incubated overnight at 4°C, and washed with PBS containing 0.1% Tween 20. Bound CXCL12 was detected by biotinylated anti-CXCL12
Online Supplemental Material.
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Severe infections in humans deplete BM lymphocytes and induce the appearance of immature lymphocytes in the blood (1, 2). In mice, analogous responses follow infections (3) or administration of adjuvants (3, 4). Within a week of immunization, significant numbers of T cells and both developing and mature B cells are lost from mouse BM, whereas increases in granulocyte numbers and granulocytosis are often observed (3). /ß can suppress B lymphopoiesis by inducing cell death (810). However, this suppression only occurs at pharmacologic doses (8), and does not require signal transducer and activator of transcription 1, the physiologic mediator of IFN signaling (11).
alone reduces BM CXCL12, and in TNF
-deficient mice, adjuvant-induced suppression of BM CXCL12 is mitigated, BM lymphopenia is much reduced, and mobilization of developing B cells is absent. Adjuvant effects on BM are largely mimicked by pertussis toxin (PTX), which uncouples most chemokine receptor signaling (20).
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Mice.
Female C57BL/6 (BL/6, CD45.2), B6.SJL-Ptprca/BoAiTac (B6.SJL, CD45.1), B6.129SF2, TNF-/- (21), and TNF receptor I and IIdeficient (TNFR-/-) mice (22) were obtained from the Jackson Laboratories or Taconic Farms. Mice were housed under specific pathogen-free conditions at the Duke University Animal Care Facility and given sterile bedding, water, and food. Mice used in these experiments were 618 wk old.
Mice were immunized with single, i.p. injections of 20 or 100 µg (4-hydroxy-3-nitrophenyl)acetyl-chicken globulin (NP-CGG) in alum or IFA (Sigma-Aldrich; reference 23). NP-CGG contained 10 or 12 mol NP/mol CGG. NP-CGG was emulsified in IFA or precipitated with alum. Some mice were immunized with SRBCs (Duke University Farm) in PBS or injected with 0.2 ml alum or IFA alone. LPS (Escherichia coli O127:B8; Sigma-Aldrich) was resuspended in sterile PBS, and mice were injected i.p. with 75 µg LPS. Mouse rTNF
, rIL-1ß, rIL-6, and rIFNß were purchased from R&D Systems. Pharmacologic doses for each cytokine were confirmed by serial titrations (0.33.0 µg/mouse). Single doses of 1 µg rTNF
, rIL-1ß, or 1,000 U rIFNß in 300 µl PBS were given i.v.; these doses did not produce obvious morbidity. PTX and PTX B oligomer were purchased from List Biological Laboratories.
FITC-, PE-, biotin-, or allophycocyanin-conjugated mAb for mouse B220, Gr-1, CD3, IgM, CD4, CD8, and CD11c were purchased from BD Biosciences. PE-Cy5conjugated mAb for mouse CD4, CD8, TER-119, Gr-1, CD11b, and FITC-conjugated anti-CD45.1 and anti-CD45.2 mAb were purchased from eBioscience. Streptavidin (SA)-allophycocyanin (BD Biosciences) and SA-Texas red (Calbiochem-Novabiochem) identified biotinylated mAb. The 493 mAb (24) binds the fetal stem cell antigen, AA4 (C1qRp/CD93; references 2527), and was purified from cloned hybridoma cells.
Mice were killed after injection/immunization, and cells were harvested from spleen, femur, tibia, and blood. RBCs were lysed in ammonium chloride buffer (23) before immunolabeling. Typically, 106 nucleated cells were suspended in 50100 µl of staining buffer (HBSS with 2% FCS and combinations of labeled mAb) and incubated on ice for 20 min. 7-Aminoactinomycin D (Molecular Probes) was included to identify dead cells. Labeled cells were analyzed/sorted in a FACSCaliburTM flow cytometer (488 nm argon laser; 633 nm helium neon laser) or a FACStarPlusTM flow cytometer (488 nm argon laser; 599 nm dye laser) with the OmniComp option. Cytometry data were analyzed with FlowJo software (Treestar Inc.).
B cell progenitors were enumerated as preB cell CFU (CFU-B; reference 6). In brief, 105 BM cells or 5 x 105 splenocytes were mixed with 1 ml IMDM containing 1% methylcellulose, 30% FCS, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, and 20 ng/ml IL-7. Suspended cells were plated in 35-mm dishes and cultured at 37°C for 7 d. Colonies with B cell morphology were identified and counted by microscope.
3 x 107 BM cells from B6.SJL (CD45.1) mice were injected i.v. into BL/6 (CD45.2) recipients immunized 3 d earlier. 1 d after transfer, femoral BM cells and splenocytes were harvested and stained with FITC-conjugated anti-CD45.1 and biotinylated anti-B220 mAb, followed by SATexas red. Labeled, donor-derived cells were enumerated by flow cytometry to determine homing and migration efficiencies. BM cells from TNFR-/- (CD45.2) mice were transferred into naive or immunized B6.SJL (CD45.1) mice. Donor B cells recovered from the BM and spleen of recipients were distinguished from host cells by anti-CD45.2 mAb.
Total RNA was extracted from BM using RNeasy-kits (QIAGEN); 1 µg RNA was reverse transcribed for 1 h at 42°C (Superscript II reverse transcriptase; Invitrogen). PCR was performed on serial dilutions of cDNA using Taq polymerase (Takara Bio Inc.). PCR primers used were as follows: HPRT, forward, 5'-GCTGGTGAAAAGGACCTCT-3', reverse, 5'-CACAGGACTAGAACACCTGC-3'; CXCL12, forward, 5'-GTCCTCTTGCTGTCCAGCTC-3', reverse, 5'-TAATTTC-GGGTCAATGCACA-3'; and CXCL12, reverse, 5'-TGG-GCTGTTGTGCTTACTTG-3'; CXCL12ß, reverse, 5'-CCT-CTCACATCTTGAGCCTCTT-3'. Amplification parameters were as follows: initial denaturation at 94°C for 5 min, 2532 amplification rounds consisting of denaturation at 94°C for 30 s, annealing at optimal temperatures, and extension for 60 s at 72°C. A final extension round of 72°C for 10 min ended each amplification. Optimal annealing temperatures were as follows: 52°C for HPRT and CXCL12, and 60°C for CXCL12
and -ß. PCR products were electrophoresed over 2% agarose gels containing ethidium bromide.
BM plasma was prepared by flushing both femurs and tibia with 500 µl of cold PBS into Eppendorf-type centrifuge tubes. Cells/debris were removed by centrifugation at 3,000 g for 10 min at 4°C; BM plasma was stored at -20°C. mAb (BAF310; R&D Systems) and horseradish peroxidaseSA (Southern Biotechnology Associates, Inc.). Horseradish peroxidase activity was visualized using a tetramethylbenzidine peroxidase substrate kit (Bio-Rad Laboratories). CXCL12 concentrations were determined from purified CXCL12 standards (PeproTech).
Table S1 summarizes the effects of several inflammatory agents on thymocytes. Fig. S1 illustrates reductions of CXCL12 message in BM by adjuvant and TNF. Fig. S2 shows that the PTX B oligomer has no effect on BM. Fig. S3 compares the ability of antigens/adjuvants to induce BM lymphopenia. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20031104/DC1.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Adjuvants Deplete BM Lymphocytes and Induce the Appearance of Developing B Cells in the Periphery.
Immunization with NP-CGG/IFA reduces the numbers of developing (CD93+B220lo) and mature (CD93-B220hi; reference 24) BM B cells (Fig. 1
A); losses are evident 3 d after immunization, with maximal reductions coming on days 46 (CD93+B220lo B cells, four- to fivefold reductions; CD93-B220hi B cells, sevenfold) (Fig. 1 B). Thereafter, developing and mature B cell numbers in the BM return to normal levels (Fig. 1 B). Both B cell populations decline at similar rates, but losses of CD93-B220hi cells are significantly (P < 0.05) greater and more sustained than that of CD93+B220lo cells. Similar kinetics of loss and recovery are also observed for BM T cells (Table I and unpublished data), indicating that all BM lymphocyte populations are sensitive to adjuvant-induced depletion.
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To determine if adjuvant-induced depletion of BM lymphocytes includes cell mobilization, we enumerated CD93+B220lo blood cells after immunization. CD93+ B220lo blood cell numbers increased soon after immunization (Fig. 2, A and B) , with a peak at day 3 (naive, 5.3 ± 1.8 x 103 cells/ml; and day 3, 14.7 ± 3.7 x 103 cells/ml, P < 0.05). Developing B cell numbers in blood returned to normal levels (Fig. 2 B, day 8, 3.4 ± 0.7 x 103 cells/ml, P = 0.42; day 12, 7.4 ± 1.2 x 103 cells/ml, P = 0.08; and day 16, 8.9 ± 2.3 x 103 cells/ml, P = 0.10).
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To determine whether these CD93+B220lo IgM- cells were B lineage progenitors, we enumerated CFU-B in BM and spleen after immunization. CFU-B are abundant in the BM of naive mice (12.9 ± 1.0 x 103 cells/femur), but rare in the spleen (Fig. 3, A and B
, 0.2 ± 0.1 x 103 cells/spleen). In 4 d, adjuvants decrease the numbers of BM CFU-B to 25% of controls (P < 0.01); these reductions are sustained until day 8 (Fig. 3 A). Significant increases in splenic CFU-B occur 8 d after immunization (0.5 ± 0.1 x 103 cells/spleen, P < 0.05), with CFU-B numbers peaking at day 12 (4.3 ± 1.3 x 103 cells/spleen, P < 0.01) and declining thereafter (Fig. 3 B).
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Of these cytokines, only TNF recapitulated the cell mobilizations induced by adjuvant (Table I). 6 d after injection, TNF
reduced BM CD93+B220lo, CD93-B220hi, and CD3+ cell numbers to 54, 41, and 16% of controls, respectively. In addition, TNF
modestly increased BM granulocyte numbers (123% of controls, P = 0.07). These effects are similar to those of adjuvants, albeit less profound and persistent. For example, adjuvant reduced CD93+B220lo, CD93-B220hi B cells in the BM by five- and sevenfold, respectively, whereas TNF
reduced CD93+B220lo and CD93-B220hi BM cells two- and threefold (Fig. 1 and Table I). In contrast to their strong effects on BM, both adjuvants and TNF
induced only modest and transient changes in the thymus (Table S1, available at http://www.jem.org/cgi/content/full/jem.20031104/DC1).
Neither IL-6 nor IFNß significantly altered CD93+ B220lo or CD93-B220hi BM cell numbers, nor did they change granulocyte numbers (Table I). IL-1ß lowered B220+ BM cell numbers nonsignificantly (80% of controls, P = 0.11) but significantly expanded granulocytes (Table I, 132% of controls, P < 0.01).
Noting that TNF primarily reduced BM lymphocyte numbers, whereas IL-1ß expanded the BM granulocyte compartment, we tested whether these cytokines synergize by injecting 0.5 µg TNF
and 0.5 µg IL-1ß singly or in combination. Synergy was obvious; TNF
and IL-1ß together reduced B220+ (
30% of controls, P < 0.01) and CD3+ (16% of controls, P < 0.01) BM cell numbers more effectively than higher doses either cytokine alone (Table I). Potentiation was also apparent in significantly larger increases in the BM Gr-1+ populations (Table I, 250% of controls, P < 0.01). In combination, TNF
and IL-1ß fully recapitulate adjuvant-induced change in BM lymphocyte and granulocyte populations.
TNF Mobilizes B Cell Progenitors from the BM.
To determine if TNF mobilized BM lymphocytes, we enumerated CD93+B220lo cells in the periphery after injecting rTNF
. 3 d after injection, CD93+B220lo cells increased two- to threefold in the blood (P < 0.05) and spleen (P < 0.05) (Fig. 4
A). In control mice,
15% of CD93+B220lo cells in blood and spleen were IgM-; after TNF
treatment, IgM- cells comprised 5565% of both CD93+ B220lo populations (Fig. 4 B). Thus, rTNF
mobilizes CD93+B220loIgM- cells to peripheral tissues.
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Immunization and TNF Reduce CXCL12 in the BM.
CXCL12 and its receptor, CXCR4, are crucial for the homing of hematopoietic progenitor cells (3033), and interruption of CXCL12/CXCR4 interaction mobilizes BM stem cells (34, 35). Could inflammation/TNF mobilize BM B cells by reducing BM CXCL12/CXCR4 expression and/or signaling? We measured CXCL12 message in BM by semi-quantitative RT-PCR. In naive mice, CXCL12 mRNA was detected in unsorted BM cells, but absent or much reduced in the B cell, T cell, and granulocyte compartments (Fig. S1, available at http://www.jem.org/cgi/content/full/jem.20031104/DC1), consistent with the production of CXCL12 by nonhematopoietic stromal cells (36). 3 d after immunization or i.v. TNF
, levels of CXCL12
and -ß mRNA (36) fell twofold in BM, whereas HPRT mRNA levels remained constant (Fig. S1).
CXCL12 protein in BM plasma also declined after immunization or TNF injection. In control mice, the average concentration of CXCL12 in BM plasma was 14.0 ± 2.0 ng/ml. 3 d after immunization, CXCL12 protein levels dropped sixfold to 2.3 ± 2.0 ng/ml (P < 0.01; Fig. 6
A). These losses were specific, as total protein levels in the BM plasma of control and immunized mice were identical (Fig. 6 A). After immunization, CXCL12 returned to near normal levels by day 8 (Fig. 6 B). This pattern of decreased CXCL12 expression follows the course of BM lymphocyte loss and recovery after immunization (Fig. 1 B).
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CXCL12 Reduction in TNF-/- Mice.
In naive B6.129SF2 mice, the average concentration of CXCL12 protein in BM plasma (Fig. 6 C, 19 ± 1.9 ng/ml) is similar to BL/6 mice (Fig. 6 A); 4 d after immunization, BM CXCL12 concentrations fell below detectable levels (Fig. 6 C, <1 ng/ml). In contrast, immunization of TNF-/- mice reduces BM CXCL12 levels by
50% (Fig. 6 C, naive, 21 ± 1.1 ng/ml; immunized, 11 ± 3.4 ng/ml). These reductions were specific, as total BM plasma protein remained constant in all groups (Fig. 6 C).
Homing to BM Is Reduced in Immunized Mice.
If adjuvants mobilize BM lymphocytes by reducing CXCL12, normal cells should be unable to colonize the BM of immunized mice. To test this prediction, BM cells from naive, CD45.1 donors were transferred into naive or immunized CD45.2 recipients; 24 h later, CD45.1 B cells in the BM and spleen were enumerated by flow cytometry.
CD45.1 donor B cells readily entered the BM of naive recipients. We typically recovered 2.0 ± 0.4 x 105 CD45.1+B220lo cells and 0.4 ± 0.2 x 105 CD45.1+B220hi cells from each femur and tibia from naive hosts (Fig. 7 A). In immunized recipients, we only recovered 0.6 ± 0.3 x 105 CD45.1+B220lo cells (30% of controls, P < 0.01) and 0.03 ± 0.02 x 105 CD45.1+B220hi cells (7% of controls, P < 0.01). In contrast, equivalent numbers of CD45.1+ B220hi cells were present in the spleens of both naive (3.1 ± 1.7 x 105) and immunized hosts (3.7 ± 2.2 x 105), and more CD45.1+B220lo cells were consistently recovered from the spleens of immunized than from naive recipients (Fig. 7 A, 14.7 ± 2.7 x 105 vs. 7.0 ± 2.9 x 105; P < 0.01).
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PTX Depletes BM Lymphocytes and Mobilizes Developing B Cells.
In association with reductions in CXCL12, adjuvant mobilizes BM lymphocytes and increases granulocyte numbers (Figs. 1 and 6), with little effect on thymocytes (Table S1). If CXCL12 reductions cause these changes, a blockade of CXCL12 signals must produce similar results. We injected BL/6 mice with PTX, an inhibitor of many chemokine receptors, including the CXCL12 receptor, CXCR4 (20), and followed its effects on BM and thymus.
3 d after injecting PTX, CD93+B220lo and CD93-B220hi BM cell numbers fell significantly and remained suppressed until day 12. B cell numbers began to recover 1218 d after PTX treatment, reaching normal levels by day 24 (Fig. 8 A). PTX also lowered CD3+ BM cell numbers (Table I) with similar kinetics (unpublished data). These effects were dependent on the ribosyltransferase activity of PTX, as the enzymatically inactive PTX B oligomer had no effect on BM (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20031104/DC1). PTX had little effect on thymocyte populations (Table S1).
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PTX also mobilized CD93+B220lo BM cells; 3 d after injecting PTX, CD93+B220lo cell numbers were significantly higher in blood (P < 0.01) and spleen (Fig. 8 B, P < 0.01). The majority of these peripheral CD93+B220lo cells (blood, 56%; spleen, 66%) were surface IgM- (Fig. 8 C).
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Here, we show that adjuvants suppress BM CXCL12 and mobilize functional B cell progenitors (B220loCD93+IgM- cells and CFU-B) into the periphery. Both phenomena can be mediated by rTNF, and both are reduced or absent in TNF
-/- mice. A blockade of G
i-dependent signaling by PTX recapitulates these effects in the absence of an overt inflammatory response. We conclude that inflammation acts via TNF
and CXCL12 to reduce the BM lymphocyte compartments in preparation for expanded granulocyte production. This model outlines a novel inflammatory response and predicts that innate immune responses are physiologic regulators of central hematopoiesis.
Regulation of B Lymphopoiesis during Inflammation.
Adjuvants, LPS, and gram-negative bacteria, but not noninflammatory antigens such as SRBCs (references 3, 4; Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20031104/DC1) deplete all BM lymphocyte compartments equally (Fig. 1 and Table I; references 3, 4). The mechanism of depletion has been unclear, but cytokine-driven apoptosis was a favored candidate (710). Developing B cells are sensitive to apoptotic signals (7) and severe viral infection or high doses of IFN/ß suppress B lymphopoiesis by apoptosis (810). However, this apoptosis likely represents a pathologic or pharmacologic response (8). In our hands, adjuvant-induced depletions of lymphocytes are not biased for developmentally immature compartments (Fig. 1), are restricted to the BM (Table S1), and unassociated with obvious morbidity or pathology.
Although we cannot rule out an apoptotic component, adjuvant-induced BM lymphopenia is coincident with a massive mobilization of BM lymphocytes that results in the appearance of CD93+B220loIgM- cells in the blood and spleen (Fig. 2). These CD93+B220loIgM- cell populations include functional CFU-B (Fig. 3), providing an explanation the findings that the RAG+ splenocytes elicited by adjuvant are not mature lymphocytes and require functional BM (4, 19).
TNF Mobilizes BM Lymphocytes.
Adjuvant's effects on BM could be fully reproduced by two proinflammatory cytokines, TNF and IL-1ß (Table I). TNF
significantly decreases BM lymphocyte numbers (Table I), mobilizes B220loCD93+IgM- cells (Fig. 4), and modestly expands the BM granulocyte compartment (Table I). A central role for TNF
in adjuvant-induced loss of BM lymphocytes was confirmed in TNF
-/- mice (Fig. 5) that exhibited much reduced BM lymphopenia and no mobilization of CD93+B220lo cells.
However, residual losses of BM B cells in immunized TNF-/- mice indicate that inflammation does not act via TNF
only. IL-1ß elicited a nonsignificant BM lymphopenia but greatly expanded granulocyte numbers (Table I). The effects of IL-1 in vivo are similar to those observed in vitro by Dorshkind (6) and complement TNF
. Suboptimal doses of TNF
and IL-1ß synergize to act on BM as profoundly as complex inflamogens (Table I and Fig. S3). Thus, the primary effect of TNF
appears to be the mobilization of BM lymphocytes, whereas IL-1ß promotes granulocytic expansion. A similar potentiation has been observed in rats (40).
Adjuvants and TNF Suppress BM CXCL12.
CXCL12 attracts many hematopoietic cells (34, 41, 42), including progenitor B cells, and is important for their survival, differentiation, and localization (33). Both adjuvants and TNF reduce CXCL12 in the BM, and these reductions mirror lymphocyte mobilization (Fig. 6, A and B). Adjuvant-induced reductions of CXCL12 in TNF
-/- mice were substantially less than in controls (Fig. 6 C) and consistent with reduced BM lymphopenia and lack of CD93+B220lo cell mobilization (Fig. 5). Inflammation mobilizes BM lymphocytes by suppressing CXCL12 expression. Although adjuvants lower CXCL12 mRNA approximately threefold, CXCL12 protein falls to <20% of controls; rTNF
reduces BM CXCL12 mRNA and protein to
50% of control levels (Fig. 6 and Fig. S1). The contrasting ranges of CXCL12 message and protein levels in the BM suggest that inflammation regulates chemokine expression transcriptionally and posttranslationally.
Consistent with this idea, Fedyk et al. (37) showed that TNF modestly suppressed CXCL12 transcription in dermal fibroblasts, whereas Petit et al. (35) found that granulocytes substantially lowered CXCL12 levels by elastase-driven proteolysis. Other enzymes secreted by granulocytes (e.g., matrix metalloproteinases and cathepsin G) also inactivate CXCL12 (38, 39).
Inflammation Alters the BM to Prevent Cell Homing.
B220lo and B220hi BM cells from naive donors inefficiently home in immunized recipients (Fig. 7). Reduced homing efficiency is not due to TNF-mediated change in the transferred cells, as TNFR-/- cells do not enter the BM of immunized recipients (Fig. 7 B). We conclude that inflammation modifies the BM by reducing CXCL12 sufficiently to no longer attract and/or retain lymphocytes. The observation that PTX, a Gi poison that inhibits most chemokine signaling (20), mimics inflammation's effects on BM and mobilizes CD93+B220loIgM- cells (Fig. 8 and Table I) is consistent with this model, but is not a proof.
Retention of BM Granulocytes.
In vitro, granulocytes display strong, Gi-dependent chemotaxis to CXCL12 (reference 43 and unpublished data). CXCL12 and CXCR4 are crucial for both myelopoiesis and B lymphopoiesis (30, 31), and mice reconstituted with CXCR4-deficient fetal liver cells have increased numbers of developing granulocytes and B cells in their blood (33). How is it that reductions of CXCL12 or inhibition of chemokine signaling by PTX depletes BM lymphocytes but not granulocytes?
One possibility is that the chemotactic sensitivities of B cells and granulocytes to CXCL12 differ. If granulocytes respond to significantly lower concentrations of CXCL12 (1 ng/ml) than lymphocytes, reductions of CXCL12 would favor the retention of granulocytes in the BM. In the absence of CXCL12/CXCR4, such selectivity would be lost. Increased sensitivity to CXCL12 signals would also make granulocytes relatively resistant to nonsaturating doses of PTX.
Although early myeloid progenitors depend on CXCL12 to enter the BM, Gr-1int and Gr-1hi granulocytes (28) might be retained there by other chemokines. For example, LPS induces BM stromal cells to express CCL21, a chemokine for myeloid progenitors (44). Other myeloid chemoattractants, CCL3 and CCL8, are expressed in BM as well (45). In this model, granulocytes would normally depend on CXCL12 homing/retention signals, but are held in the BM by other chemokines during infection.
A third possibility is that Gi-independent mechanisms retain granulocytes in the BM. Although the initial localization of myeloid progenitors in the BM is CXCL12 and G
i dependent (30, 31, 33), retention of developing and mature granulocytes in the BM could be G
i-independent (4649). These PTX-insensitive pathways could be constitutive or induced by inflammation.
None of these models are mutually exclusive, and we are in the process of testing each. However, our data show that BM lymphocytes and granulocytes respond differently to environmental cues that control their retention in the BM (Figs. 1 and 8; Table I).
Lymphopoiesis and Granulopoiesis during Inflammation.
Although severe inflammation may induce apoptosis in BM lymphocytes (9, 40), milder inflammation mobilizes BM lymphocytes to the blood and spleen (Fig. 2) and establishes extramedullary B lymphopoiesis (Fig. 3). Lymphocyte mobilization is associated with increased numbers of Gr-1int cells and expansion of the BM granulocyte compartment (Fig. 1, C and D). The coordination of these changes suggests a regulated, physiologic response. We propose that inflammatory agents elicit TNF (and other potentiating cytokines) at sites of infection, and perhaps in the BM (50), sufficiently to suppress BM CXCL12. Initially, this suppression occurs by transcriptional inhibition (reference 37 and Fig. S1), but later it occurs by proteolysis from an expanded granulocyte compartment (35, 38, 39). IL-1ß promotes granulocytic expansion, especially in the presence of TNF
(Table I). Reduced CXCL12 levels mobilize BM lymphocytes, and initiate extramedullary lymphopoiesis. In the spleen, displaced CFU-B proliferate and differentiate into preB and immature B cells that express RAG1/2 and
5 (Figs. 2 and 3; references 3, 4, 13, 16, 19).
BM granulocytes appear to expand into generative niches abandoned by mobilized lymphocytes. Although IL-1ß promotes myelopoiesis (Table I; reference 6), its effects are strongly potentiated by TNF-induced mobilizations. This synergy of TNF
and IL-1ß suggests that lymphopoiesis and myelopoiesis compete in the BM. Competition for space or resources is also implicit in pharmacologic modulations of hematopoiesis (i.e., factors that promote granulocyte development mobilize lymphocyte progenitors [reference 51] and vice versa [references 52, 53]).
Alternatively, it is possible that the recovery of BM lymphocyte compartments and increased granulocyte numbers represents a general increase in the ability of the BM to support hematopoiesis. This increased generative capacity might result from the accumulation of growth resources over the lymphopenic period.
The utility of increasing granulopoiesis in response to inflammation is obvious. Mature granulocytes are unable to divide and once activated, survive only hours to days. The ability to increase granulocyte production to replenish cells lost in inflammatory responses would be considerably advantageous as persistent neutropenia leads to death from infection (54, 55)
The advantage of extramedullary B lymphopoiesis is less obvious. Nonetheless, it is clear that inflammation promotes transient, extramedullary B lymphopoiesis. We think it unlikely that this splenic lymphopoiesis has no physiologic role. The appearance of CFU-B in the spleen is well regulated (unpublished data) and the number of preB and immature B cells that arise there can comprise 1020% of splenic B220+ cells (references 3, 4, 18; unpublished data). Perhaps some fraction of these cells are recruited into local humoral responses (1517, 56)?
In conclusion, adjuvant-induced BM lymphopenia reflects the mobilization of lymphocytes. This mobilization is mediated by a TNF/CXCL12 axis that intimately links the innate and adaptive immune systems. Proinflammatory cytokines not only act as immune effectors and organizers of lymphoid tissue but also direct BM hematopoiesis.
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Acknowledgments |
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This work was supported in part by U.S. Public Health Service grants AI24335, AI49326, and AI56363 (to G. Kelsoe) and by ACS-IRG83-006 (to M. Kondo). H. Kondilis is supported by National Research Service Award AI0552077, Y. Ueda received funds from the Japan Society for the Promotion of Science, and M. Kondo is the recipient of a Kimmel Scholar Award.
Submitted: July 7, 2003
Accepted: November 11, 2003
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Footnotes |
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The online version of this article includes supplemental material.
Abbreviations used in this paper: CFU-B, preB cell CFU; NP-CGG, (4-hydroxy-3-nitrophenyl)acetyl-chicken
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References |
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