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. Author manuscript; available in PMC: 2015 Oct 6.
Published in final edited form as: J Immunol. 2012 Mar 21;188(8):3603–3610. doi: 10.4049/jimmunol.1102741

Inflammatory spleen monocytes can upregulate CD11c expression without converting into dendritic cells

Scott B Drutman 1, Julia C Kendall 1, E Sergio Trombetta 1
PMCID: PMC4594880  NIHMSID: NIHMS357115  PMID: 22442444

Abstract

Monocytes can differentiate into various cell types with unique specializations depending on their environment. Under certain inflammatory conditions, monocytes upregulate expression of the dendritic cell marker CD11c together with MHC and costimulatory molecules. These phenotypic changes are considered an indication of monocyte differentiation into a specialized subset of dendritic cells (DCs), often referred to as monocyte-derived DCs or inflammatory DCs (iDCs), considered important mediators of immune responses under inflammatory conditions triggered by infection or vaccination. In order to characterize the relative contribution of cDC and iDC under conditions that induce strong immunity to co-administered antigens, we analyzed the behavior of spleen monocytes in response to anti-CD40 treatment. We found that under sterile inflammation in mice triggered by CD40-ligation, spleen monocytes can rapidly and uniformly exhibit signs of activation, including a surface phenotype typically associated with their conversion into DCs. These inflammatory monocytes remain closely related to their monocytic lineage, preserving expression of CD115, scavenging function, tissue distribution and poor capacity for antigen presentation characteristic of their monocyte-precursors. Additionally, three to four days after delivery of the inflammatory stimuli these cells reverted to a monocyte-associated phenotype typical of the steady state. These findings indicate that in response to anti-CD40 treatment spleen monocytes are activated and express certain DC surface markers without acquiring functional characteristics associated with DCs.

Introduction

The functional specializations of dendritic cells (DCs) and monocytes/macrophages have been a topic of much investigation, with recent focus on their developmental lineages as a way of understanding the relationships between these two cell types(1-15) significant clinical potential(16-18). Under steady state conditions, monocytes act as versatile cells that can convert into a variety of tissue-resident and lymphoid organ macrophage subsets. Under these same conditions, “conventional” DCs (cDCs) derive from a specialized precursor that shares a common progenitor to, but is distinct from, monocytes(19, 20). This lineage separation is paralleled by a divergence of functional specializations. While the monocyte/macrophage lineage is specialized for robust antigen scavenging and secretion of inflammatory cytokines, their capacity to convert internalized antigen into peptide-MHC complexes is poor. On the other hand, cDCs are specialized for the efficient conversion of small amounts of captured antigen into peptide-MHC complexes, migration to T-cell zones, and initiation of T-cell responses(21, 22).

During inflammation, the plasticity of monocytes may also extend to the formation of certain subsets of dendritic cells (DCs), making it difficult to distinguish between these two lineages. Monocyte-derived DCs include TNF/iNOS-producing (Tip-DCs) and other inflammatory DCs (iDCs) described under microbial infections or adjuvant-induced peritonitis(19, 23-26). Monocyte-derived iDCs are characterized as DCs based on the expression of surface markers characteristic of cDCs in the spleen and lymph nodes, namely high surface expression of CD11c, as well as MHC-II and costimulatory molecules. Some iDCs were found to be dispensable for antigen presentation and T-cell priming(27) while other iDCs were proposed to contribute to T-cell stimulation(26, 28-30). Given the variety in inflammatory settings under which these iDCs arise, it is unclear if the various iDCs reported represent related populations with common functional properties, or if they encompass a spectrum of different monocyte-derived cell types.

Since most studies describing conversion of monocytes into iDCs rely on processes that last several days or even weeks, we sought to evaluate the conversion of monocytes into iDCs in vivo under conditions that induce potent antigen-specific immunity. We studied the response of mice to anti-CD40 treatment, which has proven efficacy to prime effective T-cell responses in experimental animals (1-15) and has shown significant clinical potential(16-18). We found that induction of systemic inflammation in mice with an activating antibody against CD40 uniformly induced surface CD11c expression on Ly6CHi monocytes. These cells also expressed MHC-II and costimulatory molecules typically associated with DC-like phenotypes ascribed to iDCs. However, these Ly6CHi-CD11cHi monocyte-derived iDCs share functional properties with their Ly6CHi-CD11cNeg precursors, not with Ly6CNeg-CD11cHi cDCs. Additionally, this phenotypic change accompanies an increase in endocytic capacity, highlighting their activated monocyte phenotype. After three to four days, this Ly6CHi-CD11cHi monocyte-derived population reverts back to a surface phenotype characteristic of monocytes, further supporting the continuity of their DC-independent lineage.

Materials and Methods

Mice

C57Bl/6 (B6), OT-I/RAG1 (OT-I), OT-II2.a/RAG1 (OT-II), B6.SJL (CD45.1) mice were from Taconic Farms. B6.129P2-Cd40tm1Kik/J (CD40 KO) were from The Jackson Laboratory. Mice were housed under specific-pathogen-free conditions and maintained in compliance with institutional and federal regulatory guidelines. anti-CD40 mAb mediated inflammation was achieved by intraperitoneal injection of 100 μg FGK4.5 mAb (Bio X Cell) or clone IC10 (LEAF grade, Biolegend). Rat IgG2a isotype control (LEAF grade, Biolegend)was used for control injections. Each injection of antibody (used to induce inflammation) or antigen (to study endocytosis or antigen presentation to T-cells), contained undetectable levels of endotoxin, lower than 0.126 EU (~ 13 pg) based on LAL test (Cambrex).

Cells

Unless otherwise specified, all cells were washed and resuspended in PBE (PBS with 0.5% BSA, endotoxin free, Equitech-Bio, 1 mM EDTA). Spleens were digested with Liberase Blendzyme 2 (Roche Diagnostics) for 15 min in PBS at 21°C, passed through a 40 μm cell strainer, treated with ACK Buffer (Lonza) to remove red cells, and resuspended in PBE. For purification of cells for in vitro antigen presentation experiments or for transfer experiments, splenocytes were first enriched by magnetic negative depletion with biotinylated antibodies against CD19 (MB19.1), CD3 (145-2C11), NK1.1 (PK136), Ly-6G(1A8), and erythroid cell marker (TER-119) antibodies (eBioscience or Biolegend), followed by enrichment using the EasySep™ biotin selection kit (StemCell Technologies Inc.). Cells were subsequently sorted on a Dako MoFlo. Post-sort analysis confirmed purity of >96% and viability of >95%. OT-I CD8+ or OT-II CD4+ T-cells were isolated from the lymph nodes and spleens of OT-I/RAG1 KO or OT-II/RAG1 KO mice by disruption through a 40 μm cell strainer, followed by negative selection using mouse CD8+ T-cell or mouse CD4+ T-cell enrichment kit, respectively (StemCell Technologies Inc.). Enriched T-cells were pulsed with 0.5 mM CFSE (Invitrogen) for 5 min, washed twice and resuspended in complete RPMI.

Monocyte transfer experiments

Ly6CHi monocytes (as identified in Figure 1A) were purified from spleens of CD45.1 mice by cell sorting. 5.0×105 purified cells were injected intravenously into mice that were either injected with 100 μg-anti-CD40 mAb or with control IgG or PBS as negative controls 5 minutes after cell transfer. At various time points after transfer, spleens were analyzed by flow cytometry and the phenotype of the endogenous (CD45.2) and transferred (CD45.1) cells were assessed.

Figure 1.

Figure 1

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Ly6CHi-CD11cHi cells appear in the spleens of mice during CD40-mediated inflammatory responses. A. Gating scheme for identification of spleen subsets by flow cytometry showing the Ly6C v. CD11c plots used for the identification of Ly6CHi monocytes, Ly6CNeg monocytes, and CD11b+ DCs. B. Mice were injected with 100 μg of anti-CD40 mAb and spleen monocytes were analyzed at various time points post-injection. C. Identification of four populations in mice treated with anti-CD40 for 40hrs D. Plots shown in B were gated on Ly6CHi cells (solid line) for analysis of CD11c expression levels over time, and compared to the levels expressed by cDCs (dotted line) from the same mouse. Data are representative of 5 experiments, three mice per group. Results expressed as mean ± standard deviation from the mean.

Endocytosis assays

Soluble GFP protein was prepared as previously described(31). Briefly, the construct in pET-28 vector (Novagen) in BL21 E. coli (Novagen) were grown in TB media (Invitrogen) at 37°C to a density of ~0.1 Absorbance Units at 600 nm, then at 24°C for 16 h with 1mM IPTG (Sigma). Cells were lysed with lysozyme, sonication and freeze/thaw cycles, and the his-tagged protein was affinity purified on Ni-Sepharose (Pharmacia). The resulting protein was further purified by ion exchange with Q-Sepharose (Pharmacia). The resulting protein had <1.26 EU/mg of endotoxin (< ~125pg/mg) by LAL test (Cambrex). For in vivo soluble antigen endocytosis assays, 2 mg of GFP protein was injected intravenously, and 30 min later spleenocytes were collected and analyzed for antigen capture as compared to a similarly treated mouse not injected with antigen. Endotoxin-free OVA (prepared as described below for antigen presentation assays) at 10mg/ml in PBS was labeled using Florescein-5-isothiocyanate (Invitrogen) according to the manufacturers instructions. Excess unincorporated fluorescein was removed over a Sephadex G-25 column (GE Healthcare) followed by several buffer exchanges with PBS using an Amicon Ultra-4 centrifugal filter device (Millipore) and filtration over Polymixin-B-Sepharose column (Pierce). For endocytosis assays, mice were injected with 900 μg of FITC-OVA and 20 min later spleens were analyzed by flow cytometry to detect FITC-OVA uptake. Fluorescent particulate antigen was prepared by incubating 1.8 μm streptavidin coated polystyrene microbeads (Spherotech) at 2.8×109/ml with 10 μg/ml 5-((N-(5-(N-(6-(biotinoyl)amino)hexanoyl)amino)pentyl)thioureidyl) fluorescein (fluorescein biotin, Invitrogen) in PBS for 30 min and washed in PBE. For 2 μm particle uptake assays, 1.4×108 particles were injected intravenously and 60 minutes later spleenocytes were collected and analyzed for antigen capture. Cells were also stained with PE-conjugated anti-fluorescein (Invitrogen) to distinguish cells that completely internalized particles (fluorescein positive, PE negative), from cells with particles stuck to their surface (fluorescein positive, PE positive). For 5 μm particle endocytosis assays 0.8×108 YG Fluoresbrite carboxylate microspheres (Polysciences) were injected intravenously and 60 minutes later spleenocytes were collected and analyzed for antigen capture.

Antigen presentation assays

Ovalbumin protein (Sigma, grade IV), was purified to remove any potential endotoxin contamination by ion exchange using Q-Sepaharose (Pharmacia) as previously described (31). To assay presentation of antigen captured by cells in vivo, 1.0 mg ovalbumin protein (OVA) was injected intravenously into mice, and 30 min later cells were purified by cell sorting as described above. Various numbers of APCs were co-cultured with 50,000 CFSE labeled OT-I CD8+ T-cells or OT-II CD4+ T-cells in U-bottom 96-well plates in complete RPMI. 60 h later, T-cell proliferation was assessed using flow cytometry to measure the dilution of CFSE accompanying each T-cell divison. Cells were cultured in RPMI (Gibco) with 10% heat-inactivated FBS (Invitrogen), non-essential amino acids, 110 μg/ml Sodium-Pyruvate, 2 mM L-Glutamine, 100units/ml Penicillin, 100 μg/ml Streptomycin (Gibco), and 100 μM ®-Mercaptoethanol (Sigma) in a 5% CO2 37°C incubator.

Monocyte tracking with beads

Experiments were performed as previously described (13,17). Mice were injected intravenously with 50 μl (2.3×109) Fluoresbrite Carboxylate YG 1.0 μm microspheres (polysciences) resuspended in PBS. At various times after bead injection mice were injected with 100 μg anti-CD40 mAb intraperitoneally or with control IgG or PBS as negative controls. At various time points after injection, spleenocytes were purified and the cells containing the participles were analyzed by flow cytometry.

Flow cytometry

Cells were pre-incubated with 10 μg/ml 2.4G2 mAb (Bio X Cell) for 15 min at 4°C in PBE, incubated with mAb conjugates for 30 min at 4°C, washed in PBE, and resuspended in PBE with 0.5 μg/ml 7-aminoactinomycin-D (Invitrogen) 10 min before analysis. Data was collected on a FACSCanto (BD) and analyzed with FlowJo software (TreeStar). Mean +/− Standard Deviation of multiple experiments was calculated using Prism software (graphpad Software Inc.) Antibodies: TCR-beta(H57-597), CD19(6D5), B220(RA3-62B), NK1.1(PK136), Ly-6G(1A8), Siglec-H(440c), CD11b(M1/70), CD11c(N418), Ly-6C(HK1.4), CD115(AFS98), F4/80(BM8), Mac-3(M3/84), CD14(Sa14-2), Mac-2 (m3/38), CD43(1B11), CD45.1(A20), CD45.2(104), CD8a (53-6.7), CD4 (GK1.5), CD86 (GL-1), CD80 (16-10A1), IA/E (M5/114.15.2), H2-Kb (AF6-88.5) and CD40 (HM40-3), as well as isotype control in corresponding fluors (mouse IgG2a, Rat IgG1, Rat IgG2a, Rat IgG2b, Armenian Hamster IgG, Armenian Hamster IgM), were purchased from eBioscience or Biolegend.

Intracellular TNF-α analysis

Mice were injected with 100 μg anti-CD40 mAb intraperitonelly and 40 hours later splenocytes were isolated and cultured in complete RPMI in the presence of media alone, 200 ng/ml LPS (from salmonella typhimurium, Sigma), 5 μg/ml anti-CD40 mAb, or 5×107/ml heat-killed Listeria (strain 10402S) and 5 μg/ml brefeldin-A (Invitrogen). 60 minutes later, cells were first stained for surface markers (see flow cytometry above) and then for intracellular TNF-α using Cytofix/Cytoperm (BD) according to the manufacturer's instructions. TNF-α was detected using Biotin-anti-TNF-α (MP6-XT22, Biolegend), followed by Streptavidin-PE (Invitrogen). Biotin-Rat IgG1 (Biolegend) was used as an isotype control.

Confocal Microscopy

Spleens were immersed in OCT media (Tissue-Tek) and frozen in an Isopentane/liquid nitrogen bath. 10 μm cryo-sections were fixed in acetone for 5 min at −20°C. All subsequent steps were at room temperature. Sections were dried for 1 h, rehydrated in PBS for 10 min, and blocked with 5% goat serum and 5 μg/ml anti FcR-mAb (2.4G2, Bio X Cell). T-cells were stained with 2 μg/ml rabbit anti-CD3 (Dako) for 1hr, followed by 0.16 mg/ml horseradish peroxidase conjugated anti-rabbit (Jackson immuno) for 1hr, and detected using the cyanine-3 system (Perkin Elmer) as directed. Dendritic cells were stained with 1.67 μg/ml Alexa647 conjugated CD11c (N418, Biolegend) for 1 hr, and monocytes were stained with 0.6 μg/ml FITC conjugated anti-Ly6C (HK1.4, Biolegend) for 1 hr followed by 2 μg/ml Alexa488 conjugated goat anti-fluorescein (invitrogen) for 1 hr. Control stains were performed with normal rabbit serum or isotype controls labeled with Alexa647 or FITC. Sections were mounted with Prolong Gold (Invitrogen) and imaged with Zeiss Plan Apochromat 10×0.45NA objective on a Zeiss LSM510 microscope.

Results

Ly6CHi-CD11cHi monocytes accumulate in the spleen of mice after treatment with anti-CD40 mAb

We focused on the two major populations of monocytes in the spleen: Ly6CHi-CD11cNeg monocytes and Ly6CNeg-CD11cNeg monocytes (Figure 1A)(2). Unlike monocytes, cDCs are Ly6CNeg and express high levels of CD11c, a marker that is typically used to identify these cells (Figure 1A) (12). To analyze the behavior of monocytes and the potential formation of iDC in vivo under conditions that induce strong antigen-specific immunity we studied mice treated with an agonistic antibody against CD40, which has proven efficacy to prime effective T-cell responses in experimental animals (1-15). We found that induction of systemic inflammation in mice with an anti-CD40 mAb induced CD11c expression on Ly6CHi monocytes in the spleen, reaching levels comparable to cDCs in the same mouse within 40 hours (Figure 1B,C). The same induction of CD11c expression on Ly6CHi cells was obtained with two different clones of agonistic anti-CD40 mAb, while this conversion did not occur in mice that lack CD40 (Supplemental Figure 1A).

These Ly6CHi-CD11cHi cells are also apparent in the CD11b vs. CD11c plots commonly used to detect the appearance of iDCs (Supplemental Figure 1B,C). This Ly6CHi-CD11cHi-CD11bHi phenotype is typical of iDCs described under various inflammatory stimuli. In mice treated with anti-CD40 for 40 hours we could identify four populations for further comparison: Ly6CHi monocytes, Ly6Cneg monocytes, Ly6CHi-CD11cHi cells, and CD11b+ cDCs (Figure 1C). A comparison of these 4 populations shows that Ly6CHi-CD11cHi cells and CD11b+ cDCs express similar levels of CD11c, while Ly6Cneg monocytes express low levels of CD11c and Ly6CHi monocytes do not express CD11c (Supplemental Figure 1D). Although similar populations of Ly6CHi-CD11cHi cells could be detected in other organs after anti-CD40 treatment (Supplemental Figure 1E) we focused our analysis on spleen-derived monocytes because of the abundance of these cells and to be able to establish a direct comparison with the well-characterized cDC from that organ.

In addition to high levels of CD11c, these Ly6CHi-CD11cHi cells also expressed MHC-II and costimulatory molecules at levels similar to cDCs from the spleen of the same mouse, which is another surface phenotype typical of iDCs (Figure 2A). Although these Ly6CHi-CD11cHi cells shared some surface characteristics with cDCs, the expression levels of other markers such as F4/80, Mac-2, Mac-3, CD14, CD115 remained closer to their Ly6CHi monocyte precursors than to cDCs (Figure 2A). The Ly6Cneg monocytes in the spleen of the same animals retained lower MHC-II expression under the same conditions (Figure 2B). These results show that treatment of mice with anti-CD40 mAb induces the appearance of a population of Ly6CHi-CD11cHi cells that shares surface markers with both cDCs and monocytes. For simplicity we will continue to refer to these monocyte-derived Ly6CHi-CD11cHi cells as “iDCs”, although, as described below, this population likely differs from previously described inflammatory monocyte-derived iDCs.

Figure 2.

Figure 2

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Despite their high expression of CD11c, Ly6CHi-CD11cHi cells maintain a surface phenotype similar to activated Ly6CHi monocytes. A. Comparison of the surface expression of MHC and co-stimulatory molecules on Ly6CHi monocytes, Ly6CHi-CD11cHi cells, and CD11b+ cDCs from the spleen of the same mouse 40 hours after anti-CD40 treatment. Populations were identified as shown in figure 1D. B. Similar to A, surface phenotype of Ly6CNeg monocytes was analyzed in spleens of either control-treated mice or in mice treated with anti-CD40 for 40 or 90 hrs. Also shown is the surface phenotype of Ly6CHi-CD11cHi cells from the mice treated with anti-CD40 for 40h. In all cases, the indicated surface marker staining (solid line) was compared to staining obtained with an isotype control (dashed line). Data are representative of 4 experiments, two mice per group.

Spleen Ly6CHi-CD11c Hi cells derive from monocytes

The appearance of Ly6CHi cells expressing high levels of CD11c following injection of anti-CD40 suggested that Ly6CHi-CD11cHi cells were the result of upregulation of CD11c on Ly6CHi-CD11cNeg monocytes in the spleen. However, these results could not exclude the possibility that the Ly6CHi-CD11cNeg population was disappearing and simultaneously replaced by unrelated populations with increasingly higher levels of CD11c. To distinguish between these alternatives, we used a bead-labeling protocol for tracking monocytes in situ (32-34). One hour after injection, beads were associated primarily in Ly6CHi and Ly6CNeg monocytes, and to a smaller degree with DCs (Figure 3A and Supplemental Figure 2) When control-treated mice (uninflamed) were analyzed 24 or 40 hours after injection of beads without any inflammatory treatment, beads were associated almost exclusively with Ly6CNeg monocytes, while the there were almost no more bead-associated Ly6CHi monocytes (Figure 3B Supplemental Figure 2), which was previously shown to reflect the conversion of the Ly6CHi to Ly6CNeg monocytes (34-36). However, when mice were additionally treated with anti-CD40 only 15 minutes after bead injection, bead-associated Ly6CHi-CD11cHi cells could be easily identified, indicating the conversion of monocytes to Ly6CHi-CD11cHi cells (Figure 2B). In these experiments both Ly6Chi monocytes and Ly6Cneg monocytes contained beads at the time of anti-CD40 administration, making it possible that both monocyte populations contributed to the Ly6CHi-CD11cHi cells.

Figure 3. Ly6CHi-CD11cHi cells arise from monocytes in vivo.

Figure 3

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A.Mice were injected with 1.0-μm YG beads and 1 hour later, cells that had internalized beads (mostly monocytes) were analyzed and overlaid onto all cells. B. Mice were injected with 1.0-μm YG beads, and then either control-treated, or injected with anti-CD40 mAb. To track the fate of the cells that had internalized beads, 24 or 40 hours later, the phenotype of the bead containing cells were analyzed and overlaid onto all the cells. C. Similar to A, but cells were examined 24 hours after bead injection. D. Similar to B, but mice were injected with anti-CD40 24 hours after injection of beads. E. Spleen Ly6CHi monocytes were sorted to purity from CD45.1 mice using the gates shown in Figure 1. F. ~5×105 purified monocytes as shown in E were injected into CD45.2 mice, that were either control treated, or immediately injected with anti-CD40 mAb. 24 or 40 hours later, the phenotype of the endogenous and transferred spleen monocytes was analyzed by flow cytometry. Data are representative of 3 experiments, two mice per group.

To investigate the contribution of Ly6CNeg monocytes to the Ly6CHi-CD11cHi population, mice were injected with beads, and then waited for 24 hrs to allow the bead-labeled Ly6Chi monocytes to convert to bead-labeled Ly6Cneg monocytes, as previously described ((33) Figure 3C and Supplemental Figure 2). These mice with beads almost exclusively limited to Ly6Cneg monocytes were then injected with anti-CD40. 40 hours after treatment, beads were also found within Ly6CHi-CD11cHi cells, suggesting that Ly6Cneg monocytes contribute to the formation of the Ly6CHi-CD11cHi population (Figure 3D).

Since bead tracking of monocytes in situ cannot selectively label the Ly6Chi monocytes without additional manipulations such as clondronate liposomes (33), we sorted Ly6CHi monocytes from CD45.1 mice, and adoptively transferred them intravenously into syngeneic CD45.2 recipients (Figure 3E). When recipient mice were control-treated, we observed the subsequent conversion of these transferred Ly6CHi monocytes into Ly6CNeg monocytes (Figure 3F), as previously described (34-36). In contrast, when inflammation was induced with anti-CD40 after adoptive transfer, we observed upregulation of CD11c on the transferred Ly6CHi monocytes, which paralleled the induction of CD11c expression on the endogenous Ly6CHi population in the recipient (Figure 3F). These results indicate that the Ly6CHi-CD11cHi cells formed under inflammation induced by anti-CD40 treatment were a result of upregulation of CD11c on splenic CD11cNeg monocytes.

To evaluate the role of CD40 on the responding splenic monocytes, we prepared mixed bone-marrow chimeras between wild type and CD40−/− mice (Supplemental Figure 3). We found that ligation of CD40 on bone-marrow derived cells is required for the induction of Ly6CHi-CD11cHi cells, and the formation of this population is further enhanced when CD40 is also expressed on somatic cells (Supplemental Figure 3A). Additionally, wild-type Ly6Chi monocytes transferred into CD40−/− recipients could not be induced to form Ly6CHi-CD11cHi cells, indicating that CD40 ligation on the monocytes alone is insufficient, but rather a variety of cell types contribute to the inflammatory response to anti-CD40 (Supplemental Figure 3B).

Ly6CHi-CD11c Hi cells functionally resemble activated monocytes

Since the apparent activation of Ly6CHi monocytes in mice treated with anti-CD40 led to a surface phenotype with similarities to both cDCs and their Ly6CHi monocyte precursors, we wanted to determine if these iDCs exhibit functional characteristics of cDCs. To this end, we compared the endocytic capacity and antigen presenting capacities of Ly6CHi monocytes, iDCs (Ly6CHi-CD11cHi), and cDCs, all of them isolated from the same mice treated with anti-CD40. In order to examine their phagocytic capacity in situ mice were injected intravenously with 2 μm fluorescent particles. As expected, Ly6CHi monocytes showed a higher phagocytic capacity compared to cDCs (Figure 4A). In these same mice, the Ly6CHi-CD11cHi cells also demonstrated a higher phagocytic capacity than cDCs, similar to their monocyte precursors. Interestingly, this phagocytic capacity was enhanced as compared to their monocyte precursors, suggesting that activation of this function accompanied their phenotypic transformation (Figure 4A). When similar experiments were done using larger (5 μm) fluorescent beads, we found that Ly6CHi monocytes could still internalize these large particles, which could not be internalized by cDCs (Figure 4B). In contrast to cDCs, the Ly6CHi–CD11cHi cells exhibited a similar, or even enhanced ability to capture large particles compared to their monocytic precursors (Figure 4B). A similar result was also observed after injection of soluble proteins as an endocytic probes (GFP, Figure 4C, or FITC-OVA, Figure 4D). Ly6CHi monocytes exhibited a markedly higher endocytic activity than cDCs, while Ly6CHi-CD11cHi cells exceeded both of these populations (Figure 4C, D).

Figure 4. Ly6CHi-CD11cHi cells retain the scavenging capacity of monocytes.

Figure 4

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A. Comparison of the capacity of Ly6CHi monocytes, Ly6CHi-CD11cHi cells, and CD11b+ cDCs to internalize particulate antigens. Mice that had been treated with anti-CD40 mAb 24 hours earlier were injected with 2.0 μm fluorescent particles, and 60 minutes later, bead capture by splenocytes was analyzed by flow cytometry. Percentages shown represent the percent of cells that internalized one or more beads. B. Similar to A, but 5.0 μm particles were used. C. Similar to A and B but mice were injected with soluble GFP protein. After 30 minutes, GFP capture was analyzed by comparing the florescence signal of cells from a mouse injected with GFP (solid line) as compared to a mouse similarly treated with anti-CD40 but not injected with GFP (dashed line). Spleen populations were gated as described in Figure 1. The insert in each panel shows the MFI difference between GFP-injected mice and control mice is shown ± standard error. D. The same experimental approach as in C, but mice were injected intravenously with FITC-OVA (instead of GFP as used in C). Data are representative of three experiments, three mice per group. Results are expressed as mean ± standard deviation from the mean.

Having established the endocytic capacity of these three cell populations, we next evaluated their capacity to present the internalized antigens to T-cells (Figure 5A). To this end, mice treated with anti-CD40 we injected intravenously with 1 mg of ovalbumin, and 30 minutes later the spleen cDCs, Ly6CHi-CD11cHi (iDCs) and Ly6CHi-CD11cNeg (Ly6CHi monocytes) cells were isolated by cell sorting and co-cultured with OT-I or OT-II T-cells. We found that despite their comparatively weaker endocytic capacity (Figure 4), cDCs were much more effective than Ly6CHi-CD11cHi or Ly6CHi-CD11cNeg monocytes in presenting antigen on both MHC-I and MHC-II and stimulating cognate T-cells. cDCs induced strong T-cell proliferation, even at very low cDC:T-cell ratios. Under these same conditions, where the Ly6CHi-CD11cHi and Ly6CHi-CD11cNeg monocytes had shown very high levels of antigen capture (Figure 4), both populations induced little, CD4+ or CD8+ T-cell proliferation (Figure 5A).

Figure 5. Ly6CHi-CD11cHi cells retain functional properties of monocytes.

Figure 5

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A. Despite a large capacity for antigen capture, Ly6CHi-CD11cHi cells do not present the internalized antigen to T-cells, a feature similar to monocytes but in contrast to dendritic cells. Mice were treated with anti-CD40 and 40 later injected intravenously with 1mg OVA protein. 30 minutes after OVA injection splenocytes were harvested and Ly6CHi-CD11cHi cells, Ly6CHi monocytes, and CD11bHi-CD11cHi cDCs were isolated from the same spleen and separately co-cultured with CFSE-labeled OT-I or OT-II T-cells over a range of T-cell/DC ratios. After 60h, antigen presentation was assessed by flow cytometric analysis of CFSE0-dilution to measure T-cell proliferation. B. Distribution of cDC and monocyte in the spleen of untreated control mice vs. anti-CD40 treated mice. A significant portion of the cDCs (Ly6CNeg-CD11cHi) are found in the T-cell zone after treatment with anti-CD40, while all Ly6CHi cells remain outside the white pulp. Data are representative of 3 independent experiments two mice per group.

In agreement with their high scavenging and poor antigen presentation activities, Ly6CHi-CD11cHi monocyte-derived cells were found predominantly in the red pulp of the spleen of inflamed mice, but they were essentially absent from T-cell areas, in contrast with cDCs (Figure 5B). Finally, we examined the ability of each population to produce inflammatory cytokine TNF-α after further stimulation. Ly6CHi-CD11cHi cells, cDC, and resident spleen monocytes all had the capacity to produce TNF-α upon restimulation in vitro (Supplemental Figure 4). In conclusion, even though the surface phenotype of Ly6CHi-CD11cHi monocyte-derived cells shares some markers with cDCs, they share other surface markers, similar scavenging, and poor antigen presenting characteristics of their monocyte precursors, and consequently do not appear to acquire functional properties associated with cDCs.

Ly6CHi-CD11c Hi cells ultimately convert into Ly6Cneg monocytes

We considered the possibility that the conversion of Ly6CHi monocytes to a cDC-like phenotype could take longer, and consequently followed the fate of these cells at later time points after delivery of anti-CD40 stimulation. We found that up to five days after injection of anti-CD40, a decrease in Ly6CHi CD11cHi cells correlated with an increase in Ly6CNeg-CD11cNeg monocytes (Figure 6A). To determine if this reduction in the numbers of Ly6CHi-CD11cHi cells could indicate their further conversion into cDC-like population, we performed adoptive transfer experiments for longer periods, after Ly6CHi-CD11cHi cells had apparently disappeared. Interestingly, the transferred Ly6CHi monocytes, which convert into Ly6CHi-CD11cHi cells after 24 to 40 hours (Figure 2A) subsequently converted into Ly6CNeg-CD11cNeg monocytes, (Figure 6B) similar to the fate of Ly6CHi monocytes in the steady state (Figure 2A) (34-36). Additionally, when we mapped the fate of these cells using the bead tracking techniques as in Figure 3D, we also found that the Ly6CHi-CD11cHi cells had converted into Ly6CNeg monocytes at later time points (Figure 6C).

Figure 6. Similar to Ly6CHi monocytes, Ly6CHi-CD11cHi cells ultimately differentiate into Ly6CNeg monocytes.

Figure 6

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A. The disappearance of Ly6CHi-CD11cHi cells 90 hours after anti-CD40 treatment, correlates with an increase in Ly6CNeg monocytes. B. Similar to Figure 3B, purified CD45.1 monocytes were injected into CD45.2 mice, which were immediately injected with anti-CD40 mAb. 90 hours later, the phenotype of the endogenous and transferred spleen monocytes was analyzed by flow cytometry. C. Similar to figure 3F, mice were injected with 1.0-μm YG beads, and then immediately injected with anti-CD40 mAb. 90 hours later the phenotype of the bead containing cells were analyzed and overlaid over all the cells. Data are representative of 3 experiments, two mice per group.

Discussion

Our results indicate that under sterile systemic inflammatory conditions induced with anti-CD40, Ly6CHi monocytes induce CD11c expression together with other surface markers typically associated with their acquisition of DC-like phenotype. However, such monocyte-derived CD11cHi cells retain many characteristics of monocytes, including several surface markers, a high endocytic capacity and inefficient conversion of internalized antigens into peptide-MHC complexes for T-cell stimulation. Additionally, these cells follow the fate of their monocytic lineage, ultimately converting into Ly6CNeg monocytes. These findings demonstrate that the upregulation of CD11c by monocytes and the conversion to DCs can be distinct processes; therefore, under certain inflammatory conditions, the expression of CD11c, MHC-II and costimulatory molecules is insufficient to identify a population with similar properties as cDCs. Although not observed in this study, the expression of CD11c on other cell types, like NK cells (37-39), may also be subject to regulation under inflammatory conditions.

The results presented here as well as previous studies have shown that the material scavenged by monocyte and some monocyte-derived iDCs is apparently not presented to T-cells efficiently(21, 27, 40). Given the abundance of iDC and their vigorous scavenging capacity, it is important to establish the fate of such scavenged material, which can be transferred to other cell types, including antigen-presenting cDCs, even as pre-formed pMHC complexes(21, 41-46).

In prior studies describing monocyte-derived DCs it has not been easy to distinguish between fully “converted” monocytes and “functional” DCs(47, 48). In previously described models relying on peritonitis induced by long-term effects of emulsified adjuvants or microbial infections, it has been difficult to establish a direct precursor-product relationship between monocytes and cDCs without ruling out alterations in the properties of cDC and/or pre-DC progenitors(25, 48). A recent study has shown that components from gram-negative bacteria such as LPS can induce the conversion of blood monocytes into antigen-presenting monocyte-derived DCs migrating to lymph nodes(26). Given that conversion of monocytes into antigen-presenting DCs does not appear to be complete under certain inflammatory conditions with similar components derived from gram-negative bacteria (49) or under sterile inflammation as reported here, it will be important to define the conditions that induce the full conversion of monocytes into iDCs capable of T-cell stimulation.

Supplementary Material

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ACKNOWLEDGEMENTS

We thank Peter Lopez, Kamilah Ryan, and Kathleen Gildea for assistance with cell sorting, Timothy Macatee for assistance with tissue sectioning, and Alice Yewdall for thoughtful discussions and assistance with bone marrow chimeras.

This work was supported by the National Institutes of Health, National Institute on Ageing, grant F30AG032190 (to S.B.D), American Heart Association grant 0435251N (to E.S.T), American Cancer Society Grant RSG-07-01-LIB (to E.S.T), and Cancer Research Institute Grant 63-1-7125 (to E.S.T)

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