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The Journal of Immunology, 2007, 178, 7833-7839
Copyright © 2007 by The American Association of Immunologists, Inc.
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Identification and Characterization of Novel Bone Marrow Myeloid DEC205+Gr-1+ Cell Subsets That Differentially Express Chemokine and TLRs

Roberta J. Lamb, Renold J. Capocasale, Karen E. Duffy, Robert T. Sarisky and M. Lamine Mbow1

Discovery Research, Centocor Research and Development, Radnor, PA 19355


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205+CD11c–B220–Gr1+CD8{alpha}–CD11b+ cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205+CD11b+Gr-1+ bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205+CD11b+Gr-1low cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-{alpha}, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205+CD11b+Gr-1high cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205+CD11+Gr-1high cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bone marrow represents a complex lymphoid tissue that is critical for the differentiation and maturation of various immune cells including B lymphocytes, dendritic cells (DCs),2 and NK cells (1, 2, 3). In addition, it is well established that murine bone marrow precursors give rise to various populations of DCs (4), a key cell type involved in the initiation and modulation of innate and adaptive immune responses against foreign Ags (5). Although numerous studies have addressed the signal requirements for the differentiation and proliferation of various immune cell populations from bone marrow precursor cells, few focused on the mechanisms directing the adult bone marrow local innate immune response in connection with TLR signaling. We undertook the present study to define the source of immunomodulatory cytokines in the bone marrow and the cellular immune mechanisms that control local cytokine secretion and innate immune responses.

The initiation of innate immune responses involves several components of the immune system, and recently TLRs have been shown to represent a crucial component of the innate immune system (6, 7, 8). TLRs represent a family of receptors involved in the recognition of pathogen-associated microbial patterns. To date, 10 human and 11 mouse TLRs have been identified. Engagement of TLRs with the appropriate ligands initiates a signaling cascade that ultimately results in the activation of NF-{kappa}B and the transcription of various proinflammatory and anti-inflammatory cytokines genes (8, 9). Importantly, the triggering of TLRs not only modulates innate immune responses against microbial pathogens but also establishes a bridge between the innate and adaptive arms of the immune system (10). Therefore, we attempted to identify and characterize the key bone marrow cell populations involved in controlling the local innate immune response through TLR signaling. Our studies led to the identification of a novel heterogeneous population of DEC205+CD11c–B220–Gr1+CD8{alpha}–CD11b+ cells that resides in normal mouse bone marrow and exhibits a broad range of TLR and chemokine receptor expression. Importantly, we found that a subset of the DEC205+CD11c–B220–Gr1+CD8{alpha}–CD11b+ cell population that expresses low levels of Gr-1 (DEC205+Gr-1low) represents a significant source of TLR-induced immunomodulatory cytokines. Thus, we identified a novel population of immune cells in normal mouse marrow with the capacity to respond to a broad range of TLR agonists, a finding that may have implications in the regulation of local innate immune responses in mouse bone marrow.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Eight- to twelve-week-old female BALB/c and C57BL/6 mice were purchased from The Jackson Laboratory.

Reagents

Polyinosinic:polycytidylic acid (poly(I:C)) and polycytidylic acid were purchased from Amersham Biosciences and LPS was purchased from Sigma-Aldrich. Oligonucleotides containing a fully phosphorothioated CpG (CpG 1826) and GpC motifs were synthesized by Invitrogen Life Technologies. Zymosan, peptidoglycan (PGN), and R848 were purchased from InvivoGen. The following PE-conjugated or allophycocyanin-conjugated mAbs against mouse cell surface markers were purchased from BD-Pharmingen: anti-CD11b (clone M1/70), anti-CD11c (clone HL3), anti Ly-6G and Ly-6C (Gr-1; clone RB6-8C5), anti-CD80 (clone 1G10), anti-CD86 (clone GL1), anti-CD40 (clone HM40-3), anti-MHC class II (clone M5/114), anti-CD1d (clone 1B1), anti-CD4 (clone GK1.5), anti-CD8{alpha} (clone 53-6.7), anti-B220 (clone RA3-6B2), and anti-CD31 (clone MEC 13.3); allophycocyanin-conjugated-anti-F4/80 mAb (clone CI:A3–1) was purchased from Caltag Laboratories. Purified anti-mouse CD16/CD32 mAbs (clone 2.4G2) were purchased from BD-Pharmingen. FITC-labeled and PE-labeled anti-DEC-205 (NLDC-145) mAbs were purchased from Serotec.

Phenotypic analysis of bone marrow cells

Bone marrow cells were isolated from normal mice by flushing femurs and tibias with RPMI 1640 (Invitrogen Life Technologies). Following the depletion of RBC with PharmLyse (BD Pharmingen), cells were washed in RPMI 1640 and subjected to flow cytometric analysis. This cell population was resuspended in cold PBS containing 3% BSA, 2 mM EDTA, and 0.05% sodium azide and incubated with anti-mouse Fc receptor mAbs (anti-CD16/CD32) followed by FITC-conjugated anti-mouse DEC205 and the indicated PE-labeled mAbs purchased from BD Pharmingen. For cell sorting, cells were stained with Gr-1-FITC, B220-PE, and CD11c-PE. The Gr-1+B220–CD11c– cell population was sorted using the FACSVantage SE flow cytometry system (BD Biosciences). In a separate series of experiments, bone marrow cells were stained with anti-CD31 and anti-F4/80 mAbs to further define their phenotype. Cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) using WinList software (Verity Software House).

In vitro cytokine production

DEC205+Gr-1lowB220–CD11c– and DEC205+Gr-1highB220–CD11c bone marrow cells were sorted to >96% purity using the FACSVantage SE flow cytometry system (BD Biosciences). Sorted cells were suspended in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 0.1 mM nonessential amino acids, 25 mM HEPES, 0.05 mM 2-ME, and 50 µg/ml gentamicin; Invitrogen Life Technologies). Cells were plated at 1.5 x 106 per ml in a 400-µl total volume complete medium. Cells were stimulated with CpG, GpC, R848, zymosan, PGN, and LPS for 48 h. Soluble cytokine levels (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, GM-CSF, IFN-{gamma}, and TNF-{alpha}) from cell culture supernatants were evaluated using Luminex technology and the Beadlyte mouse multicytokine detection kit (Millipore) according to manufacturer’s instructions. Levels of murine IFN-{alpha} were determined by ELISA using a kit purchased from PBL Biomedical Laboratories.

Mixed lymphocyte reaction

Freshly sorted bone marrow Gr-1+B220–CD11c– or splenic CD11c+ DC from normal C57BL/6 mice were plated into round-bottom 96-well plates in complete medium. Allogeneic CD4+T cells positively selected from naive BALB/c mice using anti-CD4 microbeads and magnetic cell sorting (autoMACS; Miltenyi Biotec) were added to the wells. To remove residual CD11c+ cells from the CD4+ population, CD11c+ cells were depleted using anti-CD11c-microbeads before positive selection of CD4+ T cells. Proliferation was measured by BrdU incorporation on day 5 according to the manufacturer’s instructions (Cell proliferation ELISA BrdU kit; Roche).

Total RNA isolation and cDNA preparation

Sorted DEC205+CD11c–B220–Gr-1lowCD8{alpha}–CD11b+ and DEC205+CD11c–B220–Gr-1highCD8{alpha}–CD11b+ cells were frozen at a concentration of 8 x 106 cells/ml in TRIzol (Invitrogen Life Technologies) for RNA extraction. The samples were thawed and mixed by repeated pipetting to lyse the cells. The cell lysates were transferred to a RNase-free Eppendorf tube and allowed to equilibrate to room temperature (10 min) before the addition of 200 µl of chloroform. The samples were shaken vigorously for 20 s and allowed to stand for 10 min at room temperature. The samples were centrifuged for 15 min at 10,000 x g at 4°C. The aqueous layer was transferred to a new Eppendorf tube. An equivalent volume of isopropanol (600 µl) was added to each tube, which was incubated for 10 min at room temperature. The samples were centrifuged for 10 min at 10,000 x g at 4°C. The supernatant was removed and the RNA pellet was dissolved in 100 µl of diethylpyrocarbonate-treated water (Ambion). Further purification was performed using the RNeasy kit (Qiagen) according to the manufacturer’s instructions. DNase treatment was performed on the column using RNase-free DNase (Qiagen). The quality and concentration of the RNA was determined using the Agilent 2100 bioanalyzer. The RNA was stored at –80°C until further use.

The RNA (0.5 µg/reaction) was transcribed to cDNA using the iScript kit (Bio-Rad) according to manufacturer’s instructions. A reaction that did not contain the reverse transcriptase enzyme was included for each sample to confirm the absence of genomic DNA contamination. The reaction was incubated at 25°C for 5 min, 42°C for 30 min, 85°C for 5 min, and then cooled to 4°C. The cDNA was stored at –20°C until further use.

Measurement of gene expression using real-time PCR

Real-time PCR to measure the expression of cytokine receptors, chemokine receptors, and TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 was performed using two custom-designed TaqMan low density arrays purchased from Applied Biosystems (Table I). Each array includes duplicate wells of 22 target genes on a 384-well card. The primer and probe sets were synthesized by Applied Biosystems and lyophilized onto the card; the mixture of cDNA and TaqMan universal PCR master mix was added to reconstitute each reaction. RNA-to-cDNA (150 ng) was diluted in a 100-µl volume containing TaqMan Universal PCR master mix (Applied Biosystems) and water was used in each sample port for real-time PCR. The sample port volume fills into 48 reaction wells when the card is centrifuged (each reaction volume is 2 µl). The endogenous control 18S rRNA was used to normalize the samples using the cycle threshold ({Delta}{Delta}CT) method of relative quantitation. The data is presented as the fold change relative to one target measured in the Gr-1low sample.


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  		Table I. Assay identification and gene accession numbers for the TaqMan low density arrays purchased from Applied Biosystems

 
Statistical analysis

Significant differences between experimental groups were determined by ANOVA followed by all-pairwise comparison t tests. Values of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Phenotypic characterization of a DEC205+Gr-1+ population in murine bone marrow

Because DCs represent a major cell population capable of secreting significant levels of IL-12 in vivo (11), our initial studies focused on normal bone marrow cell populations with a DC phenotype. Depending on tissue localization and state of differentiation, DCs express unique combinations of TLRs and C-type lectin receptors (CLRs). Although only a few CLRs have been identified on both blood DCs and Langerhans cells, DEC205 is one CLR expressed mostly on murine DCs and Langerhans cells (12, 13, 14). Given that the frequency of CD11c+, a marker for conventional DCs (4, 5), is very low in normal bone marrow (data not shown), we sought to use DEC205 expression as a tool to more precisely identify potential cell population(s) in the bone marrow with a DC phenotype. We found that a significant population, ~10–30%, of DEC205+ cells were present in normal bone marrow (Fig. 1) and we subsequently characterized this fraction using a panel of DC-related markers.


Figure 1
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  		FIGURE 1. A, Profiles of normal mouse total bone marrow stained with a mixture of PE-labeled anti-CD11c and anti-B220 mAbs and FITC-labeled anti-Gr-1mAbs. B, Freshly sorted bone marrow Gr-1+ indicating minimal to no contaminating CD11c+B220+ cells. C, Freshly sorted bone marrow Gr1+ stained with anti-DEC205 mAbs.

 
An analysis of the DEC205+ bone marrow cell population confirmed them to be Gr-1+, expressing low levels of MHC class II molecules, CD4, CD86, CD40, and moderate levels of CD80 molecules (Fig. 2). In addition, the bone marrow DEC205+ cells consisted of a population of CD11b+CD11c–CD8{alpha}–B220–CD1d+ cells (Fig. 2). Overall, these findings suggested that this population might be functionally distinct from the conventional CD11c+ DC population or the plasmacytoid B220+Gr-1+CD11b– DCs (15, 16, 17).


Figure 2
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  		FIGURE 2. Phenotypic analysis of bone marrow DEC205+ cells. Freshly isolated bone marrow cells were subjected to flow cytometric analysis as described in the Materials and Methods. Cells were double stained with FITC-conjugated anti-DEC205 mAb and the indicated PE-labeled mAbs directed against mouse cell surface markers. Filled histograms represent isotype-matched negative controls.

 
To further characterize the DEC205+Gr-1+ bone marrow population, we determined whether functional properties inherent to classical DCs, such as the capacity to induce an allogeneic MLR (4, 5), were indeed retained. Surprisingly, in contrast to sorted splenic CD11c+ DCs, freshly sorted bone marrow Gr-1+B220–CD11c– cells shown to express DEC205 failed to induce an allogeneic MLR (Fig. 3), suggesting that the DEC205+Gr-1+ population may not belong to the conventional CD11c+ DC family. Based on these results, a thorough phenotypic analysis to accurately define the DEC205+Gr-1+ bone marrow population was initiated. During this analysis, it was apparent that the DEC205+Gr-1+ population could be divided into two distinct populations based on Gr-1 expression (Figs. 4A and 5A). Interestingly, the Gr-1low population was F4/80+CD31+ (Fig. 4, B and C), whereas the Gr-1high population expressed the same level of F4/80 but consisted of only 54% CD31+ cells (Fig. 5, B and C).


Figure 3
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  		FIGURE 3. Allogeneic MLR using the indicated numbers of stimulators cells consisting of either sorted CD11c+ splenic DC or DEC205+Gr-1+ bone marrow cells. The stimulators were isolated from normal mice and cultured in the presence of 2 x 105 purified splenic CD4+ T cells from naive BALB/c mice in a total of 100 µl of complete medium as described in the Materials and Methods. Stimulators and responder cells were cocultured at the indicated ratios for 5 days. Data are representative of five experiments. Cultures containing DCs alone (data not shown) did not result in any proliferative response.

 

Figure 4
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  		FIGURE 4. Phenotypic analysis of resident bone marrow DEC205+Gr-1low population. Bone marrow cells were stained with a mixture of PE-labeled anti-B220, anti-CD11c, and anti-PK136 mAbs (A) followed by staining with anti-Gr-1-FITC and anti-F4/80-allophycocyanin (B) or anti-Gr-1-FITC and anti-CD31-allophycocyanin (C). Expression of F4/80 and CD31 was defined on total bone marrow cells by gating on the Gr-1low population (R1 gate).

 

Figure 5
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  		FIGURE 5. Phenotypic analysis of resident bone marrow DEC205+Gr-1high population. Bone marrow cells were stained with a mixture of PE-labeled anti-B220, anti-CD11c and anti-PK136 mAbs (A) followed by staining with anti-Gr-1-FITC and anti-F4/80-allophycocyanin (B) or anti-Gr-1-FITC and anti-CD31-allophycocyanin (C). The expression of F4/80 and CD31 was defined on total bone marrow cells by gating on the Gr-1high population (R11 gate).

 
We sorted the DEC205+Gr-1low and DEC205+Gr-1high populations from normal mouse bone marrow and generated cytospin slides to assess their basic morphological features. We found that the DEC205+Gr-1low cells mainly consist of round nucleated cells (Fig. 6A), whereas the DEC205+Gr-1high populations display a phenotype reminiscent of neutrophil lineage (Fig. 6B), further demonstrating that resident DEC205+Gr-1+ bone marrow cells represent a heterogeneous cell population.


Figure 6
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  		FIGURE 6. Morphology of DEC205+Gr-1low (A) and DEC205+Gr-1high (B) cell subsets sorted from normal mouse bone marrow. Immediately following sorting, cells were fixed in 2% paraformaldehyde, spun onto slides, and stained with H&E. The heterogeneous nature of the cells can be seen in the variation of the nuclei and the cytoplasm. Original magnification, x100.

 
Chemokine and TLR gene expression are differentially expressed in DEC205+CD11c–B220–Gr-1+ bone marrow cell subsets

As part of the initial characterization studies of this novel cell population, we focused our first series of experiments on determining their chemokine receptor and TLR expression profiles given the importance of these receptors in the regulation of innate and adaptive immune responses (5, 18). Chemokine and TLR expression profiles were then evaluated in freshly sorted mouse bone marrow populations of DEC205+Gr-1low and DEC205+Gr-1high using real time RT-PCR. Interestingly, we noted differential chemokine and TLR expression profiles in the DEC205+Gr-1low and DEC205+Gr-1high cell subsets. Although TLR2 and TLR4 expression levels were similar in both DEC205+Gr-1low and DEC205+Gr-1high cell subsets, DEC205+Gr-1low cells express higher levels of TLR7 and TLR9 (Fig. 7). We also observed that the expression levels of TLR5 were higher in the DEC205+Gr-1high population compared with that found in DEC205+Gr-1low cells (Fig 7). Of note, both populations failed to express significant levels of TLR3 (Fig. 7). These results demonstrate that murine DEC205+Gr-1+ resident bone marrow cells contain heterogeneous cell populations that differentially express TLRs, suggesting that they may also differentially respond to distinct TLR agonists.


Figure 7
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  		FIGURE 7. Real-time RT-PCR analysis of the TLR gene expression profile. DEC205+Gr-1low and DEC205+Gr-1high were sorted from total mouse bone marrow as described in Materials and Methods.

 
Further phenotypic analysis also demonstrated a differential chemokine receptor gene expression in the DEC205+Gr-1+ cell subset. Specifically, DEC205+Gr-1low cells express detectable CXCR3 and CXCR5 levels, the expression of which was absent in the DEC205+Gr-1high cell population (Fig. 8). In contrast, higher levels of CXCR2 gene expression were observed in the DEC205+Gr-1high population compared with DEC205+Gr-1low cells, whereas CCR4 and CCR7 and CCR8 levels were low to absent in both cell subsets. Likewise, a differential expression of chemokine genes was also observed, as demonstrated by higher endogenous expression of CCL2, CCL3, and CXCL10 in the DEC205+Gr-1low cell subset (Fig. 8). Overall, our results suggest that the DEC205+Gr-1+ cell population may not only display functional differences based on TLR expression profiling but may also harbor distinct migratory patterns based on their differential chemokine receptor expression profile. A summary of the TLR and chemokine receptor expression profiles of the DEC205+G-1+ cell subsets is presented in Table II.


Figure 8
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  		FIGURE 8. Real-time RT-PCR analysis of chemokine receptor expression in sorted DEC205+Gr-1low and DEC205+Gr-1high cells from total mouse bone marrow.

 

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  		Table II. TLR and chemokine receptor expression profile in DEC205+Gr-1+ cell subsetsa

 
Cytokine profile of DEC205+Gr-1+ bone marrow cells following stimulation with TLR agonists

Next, we assessed the ability of the resident bone marrow DEC205+Gr-1+ cells to respond to various TLR agonists. Given the differential TLR gene expression profile in the DEC205+Gr-1high and DEC205+Gr-1low cells, we hypothesized that these cell subsets will differentially respond to distinct TLR agonists. Indeed, we found that DEC205+Gr-1low cells responded to the TLR9 and TLR7 agonists CpG and R848, respectively (19, 20), as measured by the secretion of IL-6, TNF-{alpha}, and IL-12 (Fig. 9), consistent with their selective expression of TLR7 and TLR9 (Fig. 7). TLR2 (zymosan and PGN) (21, 22) and TLR4 (LPS) ligands (23, 24, 25) induced the secretion of cytokines in both DEC205+Gr-1high and DEC205+Gr-1low cell subsets (Fig. 9), although we found that the magnitude of the response was greater in the DEC205+Gr-1low cell subsets. For example, PGN and LPS stimulation induced higher levels of IL-6, IL-10, and TNF-{alpha} secretion in DEC205+Gr-1low cells compared with that observed in DEC205+Gr-1high cells. The same profile was observed when cells were stimulated with zymosan, except that similar levels of IL-10 were induced in both DEC205+Gr-1low and DEC205+Gr-1high cell subsets. IL-1beta, IL-2, IL-4, IL-5, IFN-{gamma}, GM-CSF, and IFN-{alpha} levels were low to undetectable regardless the stimuli used (data not shown). These results demonstrate that, within the bone marrow DEC205+Gr-1+ population, the DEC205+Gr-1low cell subset represents the predominant population that responds to TLR agonists in terms of cytokine production and suggest that this population may play an important role in the modulation of the local innate immune response against microbial pathogens.


Figure 9
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  		FIGURE 9. Cytokine profile of bone marrow DEC205+Gr-1low and DEC205+Gr-1high cells sorted from total normal mouse bone marrow. Cells were stimulated with CpG or GpC (5 µg/ml), LPS (10 µg/ml), PGN (2 µg/ml), zymosan (5 µg/ml), and R848 (1 µg/ml). Cytokine levels in culture supernatants were determined at 48 h poststimulation using Luminex. IL-12 production was not detected in the DEC205+Gr-1high population.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Herein, we describe a novel population of DEC205+Gr-1+ immune cells present in normal murine bone marrow. This population was identified in normal bone marrow from C57BL/6, BALB/c mice, and BALB/c SCID mice (data not shown), suggesting that the generation of DEC205+Gr-1+ is independent of the genetic background of the host and independent of the presence of functional T and B cells.

Given that DEC205 is mainly expressed in DC-related cell populations (14), we characterized the DEC205+Gr-1+ cell population both phenotypically and functionally to define whether it shares the characteristics of conventional DCs or nonconventional DCs such as plasmacytoid DCs. Phenotypically, the DEC205+Gr-1+ cells analyzed in this study differ from the recently described mouse plasmacytoid DCs (15, 16, 17) mainly by their lack of B220 expression and the presence of the CD11b marker, although they do retain a common functional characteristic with plasmacytoid DCs regarding the inability to stimulate an allogeneic MLR in vitro (Fig. 3). Further phenotypic studies revealed the expression of F4/80 and CD11b, suggesting that they present a monocyte/macrophage phenotype. In addition, results from functional studies showed that the DEC205+Gr-1+ cells secreted a distinct cytokine profile compared with reports of murine plasmacytoid DCs (15, 16, 17).

In contrast with previous studies demonstrating the ability of murine plasmacytoid DC to secrete large amounts of type I IFN following stimulation with viral Ags or poly(I:C) (15, 16, 17), we found that the DEC205+Gr-1+ population secreted low to undetectable amounts of IFN-{alpha} following stimulation with poly(I:C) or CpG (data not shown). In addition, the DEC205+Gr-1+ population secreted large amounts of IL-10 following stimulation with LPS and CpG, contrasting with the inability of plasmacytoid DCs (26) and splenic CD11c+ DCs (27) to secrete IL-10 following various stimuli. The ability of the DEC205+Gr-1+ population to respond to TLR2 (PGN and zymosan), TLR4 (LPS), TLR7 (R848), and TLR9 (CpG) ligands correlated with the expression of TLR2, TLR4, TLR7, and TLR9 mRNA (Fig. 7). These results also support the contention that the DEC205+Gr-1+ cell population is distinct from nonconventional DCs (e.g., plasmacytoid DCs), which were found to express high levels of TLR9 and low levels of TLR4 (28). We also investigated whether the DEC205+Gr-1+ cell subsets were somehow related to the recently described regulatory DC population that expresses the cell surface marker CD45RB (29). Our data showed that DEC205+Gr-1+ cell subsets failed to express CD45RB (data not shown), suggesting that they may not belong to a population of regulatory DCs. Collectively, the data presented here suggest that the bone marrow DEC205+Gr-1+ population is distinct from conventional CD11c+ and other known regulatory and plasmacytoid DC populations despite their DEC205 expression.

Rather, we favor the hypothesis that the population of bone marrow cells described in this report may be related to the CD11b+Gr-1+CD31+ myeloid progenitor cells previously detailed to be capable of modulating CD8+ T cell responses (30). This population of regulatory cells or so-called inhibitory macrophages is responsible, at least in part, for the profound depression of T cell responses observed in mice immunized with vaccines or bearing tumor cells (31). Interestingly we found that the DEC205+Gr-1+ cells express CD31, F4/80, and low levels of MHC class II molecules, a phenotype consistent with that of "inhibitory macrophages" described in mouse spleens and bone marrow (30).

Further characterization of the DEC205+Gr-1+ bone marrow population led to the finding that distinct TLR agonists differentially modulated the production of key immunomodulatory cytokines. Specifically, we found that the DEC205+Gr-1low population can secrete large amounts of proinflammatory and anti-inflammatory cytokines, including TNF-{alpha}, IL-12, IL-6, and IL-10, in response to various TLR agonists, as opposed to the DEC205+Gr-1high population. Strikingly, we noted that IL-10 secretion by DEC205+Gr-1+ cells was mainly driven by PGN stimulation through a TLR2/TLR6 ligand present in Gram-positive bacteria (32), suggesting that bone marrow DEC205+Gr-1+ cells may influence the development of a tolerogenic environment in response to a specific set of TLR agonists.

Subsequent functional analyses demonstrating a differential expression of chemokine and TLR expression lend support to our hypothesis that the DCE205+Gr-1+ cell subsets may differentially influence the outcome of innate and adaptive immune responses. Indeed, we found that the DEC205+Gr-1low cell subset expresses high levels of TLR7 and TLR9, consistent with its ability to respond to CpG and R848 stimulation as measured by cytokine production in vitro. Furthermore, CXCR3, CXCR5, CXCR6, and CCR7 were constitutively expressed in DEC205+Gr-1low cells, suggesting that the DEC205+Gr-1low population responds to a different set of chemokines and may display a differential migratory pattern compared with the DEC205+Gr-1high population in vivo. Whether the bone marrow DEC205+Gr-1+ cell subsets migrate to other sites remains to be determined. However, it is tempting to speculate that the cell population described in this paper may contribute to the generation of mononuclear phagocytes in the periphery based on a recent report that described a population of Gr-1high monocytes present in the blood that shuttles back to the bone marrow to give rise to Gr-1low monocytes (33).

Taken together, our findings demonstrate for the first time the presence of heterogeneous populations of myeloid cells in normal bone marrow cells that differentially express chemokine receptors and display a selective response to TLR agonists. These findings suggest that this previously uncharacterized population may play an important role in regulating the local innate immune response against microbial infection.


   Acknowledgments
 
We thank Kim Schamberger and Joanne O’Brien for help with animal care. We also thank Ashlyn Bassiri for expert advice on the FACS experiments and the University of Pennsylvania flow cytometry core facility for cell sorting (Jonni Moore and Rich Schreitzenmair).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. M. Lamine Mbow, Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121. E-mail address: Lmbow{at}gnf.org Back

2 Abbreviations used in this paper: DC, dendritic cell; CLR, C-type lectin receptor; poly(I:C), polyinosinic:polycytidylic acid; PGN, peptidoglycan. Back

Received for publication November 17, 2004. Accepted for publication April 11, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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