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Differential production of inflammatory chemokines by murine dendritic cell subsets
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Immunobiology 209 (2004) 163–172 www.elsevier.de/imbio
Differential production of inflammatory chemokines by murine dendritic cell subsets Anna I. Proiettoa, Meredith O’Keeffea, Kate Gartlanb, Mark D. Wrightb, Ken Shortmana, Li Wua, Mireille H. Lahouda,* a b
The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, Parkville, Vic. 3050, Australia Austin Research Institute, Kronheimer Building, A&RMC, Studley Road, Heidelberg, Vic. 3084, Australia
Received 26 February 2004; accepted 10 March 2004
Abstract Dendritic cells (DC) are efficient antigen presenting cells with the ability to activate na.ıve T cells. Murine DC represent a heterogeneous population that can be subdivided into distinct subsets, including the conventional DC (cDC) which are either CD4CD8 (DN), CD4+CD8 (CD4+) or CD4CD8+ (CD8+) subsets and the plasmacytoid DC (pDC), which have different immune regulatory functions. In this study, we investigated the differential expression of genes encoding the inflammatory chemokines Mip-1a, Mip-1b and Rantes, and the secretion of these chemokines, among splenic DC subsets. These chemokine genes were expressed at higher levels by the splenic CD4+ and DN cDC subsets compared with the CD8+ cDC, in both the resting and activated states in vivo. Both the pDC and cDC subsets displayed increases in chemokine secretion in response to a range of toll-like receptor (TLR) stimuli in vitro. Whilst the pDC were the highest producers of Mip-1a and Mip-1b in response to some TLR stimuli, the DN and CD4+ cDC subsets were the superior producers of Rantes. Overall, of the cDC, the CD4+ cDC produced all chemokines most efficiently, both at a basal level, and in response to most TLR stimuli. Thus, we report a new functional difference between the murine splenic cDC subsets, with the CD4+ cDC demonstrating the most efficient production of the inflammatory chemokines Mip-1a, Mip-1b and Rantes. r 2004 Elsevier GmbH. All rights reserved. Keywords: Dendritic cells, chemokines
Introduction
Abbreviations: CCR, chemokine receptor; DC, dendritic cells; pDC, plasmacytoid dendritic cells; cDC, conventional dendritic cells; GMCSF, granulocyte-macrophage colony stimulating factor; IFN-g, interferon gamma; Mip, Macrophage inhibitory protein; Rantes, Regulated on activation,T cell expressed and secreted; TLR, toll-like receptor *Corresponding author. Tel.: +61-3-9345-2533; fax: +61-3-93470852. E-mail address: [email protected] (M.H. Lahoud). 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.03.002
Dendritic cells (DC) represent a sparsely distributed population of bone marrow derived cells that play a central role in the induction and maintenance of an immune response (Steinman, 1991). DC, although sharing a common ability to process antigens and present them to T cells, are a heterogenous group of cells. As a primary division, DC can be separated into plasmacytoid DC (pDC) which only assume a typical DC morphology and function after activation (O’Keeffe et al., 2003), and conventional DC (cDC) (Shortman and
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Liu, 2002). Both DC types are found in peripheral tissues including the skin, and in lymphoid organs. The cDC may be further segregated into a number of distinct subtypes based on surface phenotype (Henri et al., 2001; Vremec et al., 2000). In the thymus, two subsets of cDC have been described (CD8+ and CD8) (Vremec et al., 2000), in the spleen, three subsets have been identified (CD4CD8, CD4+CD8 and CD4CD8+) (Vremec et al., 1992), and in the lymph nodes, in addition to these three, there are up to three further migratory DC subtypes that arrive via the lymph (Henri et al., 2001; Belz and Heath, personal communication). There is a growing evidence that the DC subtypes have specialized roles in immunity, including tolerance, cross-presentation and directing T helper cell responses (AsselinPaturel et al., 2001; Belz et al., 2004; den Haan et al., 2000; Hochrein et al., 2001; Maldonado-Lopez and Moser, 2001; Moser and Murphy, 2000; Pooley et al., 2001; Pulendran et al., 1999; Smith et al., 2003). For example, splenic CD4CD8+ (CD8+) cDC have an exceptional ability to take up and present exogenous antigens on class I MHC, including both cell associated (den Haan et al., 2000; Pooley et al., 2001) and soluble (Pooley et al., 2001) antigen; in part because of this, they are the major presenters of viral antigens to T cells in a number of different infection models (Belz et al., 2004; Smith et al., 2003). The DC subtypes also differ in their cytokine production capacities. The pDC represent the major type-1 interferon producing cells in response to viral stimulation (Asselin-Paturel et al., 2001; Bjorck, 2001; Nakano et al., 2001; O’Keeffe et al., 2002; O’Keeffe et al., 2003). In contrast, the CD8+ cDC have the greatest capacity to produce bioactive IL-12p70 (Hochrein et al., 2001; Pulendran et al., 1999), a cytokine involved in the polarization of T cells to T-helper 1 cells (Moser and Murphy, 2000) while the CD4CD8 (DN) cDC are the superior producers of IFN-g (Hochrein et al., 2001). To date, a distinct function has not been ascribed to the CD4+CD8 (CD4+) cDC. Individual DC subsets reside in distinct anatomical locations within lymphoid organs, which influences their interactions with surrounding cells. Chemokines are small chemotactic proteins that are integral in the migration of cells into and within lymphoid organs, and thus in directing cellular interactions (Cyster, 1999). Constitutitive chemokines direct cell migration in the steady-state whereas inflammatory chemokines play a role in cell trafficking during infection. Chemokines exert their effects through the engagement of chemokine receptors (CCR) present on effector cells (Rossi and Zlotnik, 2000) and therefore act specifically on the cells expressing the relevant receptors. Through the study of monocyte-derived and other cultured DC, it is known that DC produce and respond to chemokines, both in the steady-state, and in response to microbial stimulation. It is also known that DC differentially express some CCRs
(Kucharzik et al., 2002; Sallusto and Lanzavecchia, 2000). However, the role of individual splenic DC subsets in chemokine production, both at the basal level and in response to infection, has not been extensively studied. Based on preliminary studies using custom DC cDNA microarrays we identified chemokine genes differentially expressed by splenic cDC in the steady state. These included genes encoding the inflammatory chemokines Macrophage inflammatory protein-1 a (Mip-1a, CCL3), Macrophage inflammatory protein-1 b (Mip-1b, CCL4) and Regulated upon activation, normal T cell expressed and secreted (Rantes, CCL5), as being more strongly expressed by both the resting and activated spleen CD4+ cDC relative to the CD8+ cDC. These chemokines bind receptors which are widely expressed on monocytes, macrophages, immature DC, NK cells and T-helper 1 cells (Onuffer and Horuk, 2002; Sallusto et al., 2000). Therefore, the chemokines are instrumental in bringing together immune cells during infection, to allow the orchestration of an immune response (Foti et al., 1999; Sallusto et al., 1999). However, their role in the steady state is less clear. Therefore, we examined the chemokineproducing capacities of the freshly isolated mouse splenic DC subsets both at the basal level, and in response to a range of toll-like receptor stimuli (TLR). In this study, we demonstrate that the pDC are potent producers of chemokines in response to TLR-7 and 9 stimulation. We also show that of the cDC, the CD4+ cDC are the superior producers of the inflammatory chemokines, both at the basal level, and in response to many stimuli.
Materials and methods Mice All mice were bred under specific pathogen-free conditions at The Walter and Eliza Hall Institute animal breeding facility. For all experiments, female C57BL/6J Wehi mice of 6–8 weeks of age were used.
Reagents Cytokines: Murine rGM-CSF and murine r-IFNg were purchased from PeproTech (Rocky Hill, NJ). Chemokines and chemokine ELISA reagents: Rat antimouse Mip-1b antibody and biotinylated rabbit antimouse Mip-1b antibody were purchased from BD biosciences (Franklin Lakes, NJ). Recombinant mouse Mip-1a recombinant mouse Mip-1b, recombinant mouse Rantes, Rat anti-mouse Mip-1a antibody, goat anti-mouse Rantes antibody, goat biotinylated anti-mouse Mip-1a antibody and goat biotinylated anti-mouse Rantes antibody were purchased from R&D Systems (Minneapolis, MN).
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Monoclonal antibodies (mAb): The mAbs used for segregating DC subtypes were fluorescein isothiocyanate (FITC)-conjugated anti-CD11c (clone N418), phycoerythrin (PE)-conjugated anti-CD45RA (clone 14.8), PEconjugated anti-CD4 (clone GK1.5) and allophycocyanin (APC)-conjugated anti-CD8 (clone Yts169.4). They were purified and conjugated to fluorochromes as previously described (Vremec and Shortman, 1997; Vremec et al., 1992, 2000). Toll-like receptor stimuli: LPS and polyinosinicpolycytidylic acid (poly(I:C)) were purchased from Sigma-Aldrich (Castle Hill, Australia). Oligonucleotides containing a CpG motif (CpG), and completely phosphorothioated, were synthesized by GeneWorks (Adelaide, Australia) according to a published sequence (CpG1668; Sparwasser et al., 1997). Pam-3-Cys and R848 were purchased from Invitrogen (Carlsbad, CA).
Isolation of DC Pools of spleens were used for DC extraction as described in detail elsewhere (Vremec et al., 1992, 2000). Briefly, organs were chopped, digested with collagenase, and treated with EDTA. Light density cells were collected by a density centrifugation procedure. Non-DC lineage cells were then depleted by coating them with a mixture of mAbs against hemopoietic lineage markers including anti-CD3, anti-CD90, anti-TER119 (erythroid marker), anti-RB68C5 (granulocyte marker) and anti-B220 (for cDC preparations) or anti-CD19 (for pDC and cDC preparations), and then depleting the coated cells with magnetic beads coupled to anti-rat-IgG (Vremec and Shortman, 1997). For isolation of cDC, the DC-enriched preparations were stained with anti-CD11c-FITC, antiCD4-PE, and anti-CD8-APC mAb. Propidium iodide was added in the final wash to label dead cells. For cytometric sorting of cDC, the cells were gated for DC characteristics, namely, high forward scatter and bright staining for CD11c, with propidium iodide-staining cells and autofluorescent cells excluded. These selected DC were then segregated, based on the expression of CD4 and CD8, into CD8+, CD4+, and CD4CD8 (DN) cDC. For isolation of pDC, the DC-enriched preparations were stained with anti-CD11c-FITC, antiCD45RA-PE, and anti-CD8-APC, then the pDC gated during flow cytometric sorting as cells with intermediate levels of CD11c staining and high levels of CD45RA staining. The purity of the sorted DC was >98%.
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Spleens were removed 3 h later and the DC populations isolated, as described above.
Real-time PCR RNA was prepared from cell pellets of purified DC populations using the RNeasy Mini Kit (QIAGEN, Victoria; Australia) as per manufacturer’s instructions. RNA (up to 2 mg) was DNase treated with RQ1 DNase (Promega, Madison, WI) then reverse transcribed into cDNA using random primers (Promega) and Superscipt II reverse transcriptase (Gibco BRL, Geithersburg, MD). Real-time PCR was performed to determine the expression of Mip-1a, Mip-1b, Rantes, and Gapdh in splenic DC subsets using the QuantiTect SYBR Green PCR Kit (QIAGEN) and a Light cycler (Roche, Victoria; Australia), as per manufacturer’s instructions. The specific primers for real-time PCR were as follows: Mip-1a; 50 -ACCCTCTGTCACCTGCTCAACAT-30 ; 50 -GGTTCCTCGCTGCCTCCAAGACTC-30 , Mip-1b; 50 -CGTCCTTGC-TCCTCACGTTC-30 ; 50 -GAAATCAATTTCACAGTCATATCCACA-30 , Rantes; 50 -AGATCTCTGCAGCTGCCC-TCAC-30 ; 50 -TTCTCTGGGTTGGCACA-CACTT-30 , Gapdh; 50 -CATTTGCAGTGGCAAA-GTGGAG-30 ; 50 - GTCTCGCTCCTGGAAGATGGTG-30 . An initial activation step for 15 min at 95 C was followed by 40 cycles of: 15 s at 94 C (denaturation), 20–30 s at 50–60 C (annealing) and 10–12 s at 72 C (extension), followed by melting point analysis. The expression level for each gene was determined using a standard curve prepared from 102–106 pg of specific DNA fragment, then expressed as a ratio to Gapdh.
Stimulation of isolated DC for chemokine production Sorted DC subsets (0.5 106 cells/ml) were cultured for 16 h in modified RPMI 1640 medium containing 10% FCS and b-mercaptoethanol (102 M), in 96-well round-bottom plates at 37 C, in an atmosphere of 10% CO2 in air. After culture, the supernatant was collected, separated from cells by centrifugation, and stored until analysis at 70 C. The pDC culture medium contained a microbial stimulus in the presence or absence of IFN-g (20 ng/ml). The cDC culture medium contained a microbial stimulus in the presence or absence of GMCSF (100 ng/ml). The stimuli used were Pam-3-Cys (1 mg/ml), Poly I:C (100 ng/ml), LPS (1 mg/ml), R848 (1 mg/ml), or CpG 1668 (0.5 mM).
Activation of DC in vivo
Chemokine ELISA
Mice were injected (intra-peritoneally) with 20 nmol CpG and 1 mg LPS in mouse tonicity phosphate buffered solution (PBS). Controls were injected with PBS.
Aliquots of DC culture supernatants were assayed by two-site ELISA for Mip-1a, Mip-1b and Rantes production. Briefly, 96-well polyvinyl chloride microtitre
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plates (Costar, Broadway, Cambridge, UK) were coated with appropriate purified capture mAb, namely, 39624.11 (anti-Mip-1a 0.5 mg/ml), A65-2 (anti-Mip-1b 2.0 mg/ml) or 53433 (anti-Rantes, 0.5 mg/ml). Cultured supernatants were detected by the biotinylated detection mAbs, anti-mouse Mip-1a antibody (0.5 mg/ml), antimouse Mip-1b antibody (0.1 mg/ml) and anti-mouse Rantes antibody (0.1 mg/ml). Detection was as previously described (Hochrein et al., 2001).
Results Differential expression of chemokines by splenic cDC An investigation into the gene expression profiles of steady-state splenic cDC using custom DC cDNA microarrays led to the identification of the genes encoding the inflammatory chemokines Mip-1a, Mip1b and Rantes as differentially expressed by the CD4+ cDC relative to the CD8+ cDC. To further investigate chemokine gene expression by DC, the DN, CD4+, CD8+ splenic cDC subsets were freshly isolated from mouse spleen, and the level of RNA encoding each of the chemokines in each subset was determined using quantitative real-time PCR (Fig. 1). In these steady-state DC, we found all three chemokines to be more highly expressed by the CD4+ cDC compared to the CD8+ cDC. Mip-1a was expressed 10-fold higher in the CD4+ cDC compared to the CD8+ cDC (Fig. 1A), Mip-1b 12fold higher (Fig. 1B), and Rantes 2-fold higher (Fig. 1C). The chemokine genes were expressed at intermediate levels by the DN cDC. The gene expression profiles of Mip-1a, Mip-1b and Rantes were also determined in activated splenic cDC, freshly isolated from mice that were stimulated with LPS plus CpG for three hours. After in vivo activation, the expression of Mip-1a was increased by 47-fold, 13-fold and 72-fold in the DN, CD4+ and CD8+ cDC, respectively (Fig. 1D). Similarly, the expression of Mip-1b was increased by 48-fold, 20-fold and 50-fold in the DN, CD4+ and CD8+ cDC, respectively (Fig. 1E). The expression of Rantes was increased by 16-fold, 13-fold and 19-fold in the DN, CD4+ and the CD8+ cDC respectively (Fig. 1F). Thus, on activation, all cDC subsets increased the expression levels of the three chemokines. However, the CD4+ cDC remained the highest expressors of Mip-1a, Mip-1b and Rantes.
Chemokine secretion by DC subsets To ascertain if high levels of RNA transcription led to chemokine protein production, the chemokine-producing capabilities of cDC subsets were determined and
extended to the newly identified pDC. These DC subsets were freshly isolated from the spleen, and cultured under a range of different conditions. In the absence of any stimuli or cytokines, the CD4+ cDC were found to be the major producers of Mip-1a and Mip-1b, followed by the DN cDC (Fig. 2A–C). The DN and CD4+ cDC both produced suprisingly high levels of Rantes in the absence of stimulation. In comparison, both the pDC and the CD8+ cDC were poor producers of all chemokines in the absence of stimulation. As all DC subsets express TLR-9 (Edwards et al., 2003), we tested the ability of the individual subsets to produce chemokines in response to CpG 1668, a TLR-9 agonist. In the presence of CpG, all DC subsets responded by producing more Mip-1a, Mip-1b and Rantes, compared to the level of chemokine secretion in the absence of stimulation (Fig. 2A–C). Since cytokines can act synergistically with TLR agonists to increase the expression of proinflammatory cytokines, as has been previously demonstrated for IL-12 production by DC (Hochrein et al., 2001; Pulendran et al., 1999), we examined a number of cytokines (GM-CSF, IL-4, IL-12, IFN-g), alone and in combination, for their ability to enhance chemokine production in the presence of CpG. We found that GMCSF had the greatest effect on chemokine production by the cDC, whereas IFN-g had the greatest effect on the pDC (data not shown). Based on these observations, we investigated the effects of chemokine production by the DC subsets in response to cytokine alone (GM-CSF for cDC and IFN-g for the pDC), and in response to cytokine and CpG. The presence of GM-CSF alone enhanced the ability of the DN and CD4+ cDC to produce Mip-1a and Mip-1b while having no effect on Rantes production by these subsets, while the pDC and CD8+ cDC demonstrated no response at all in the presence of cytokine alone (Fig. 2A–C). In addition, we found that all cDC produced Mip-1a and Mip-1b more efficiently in response to CpG in the presence of GM-CSF (Fig. 2A and B) while only the CD8+ cDC responded more efficiently with respect to Rantes production under these conditions (Fig. 2A–C). The pDC produced all chemokines more efficiently in response to CpG if IFN-g was present (Fig. 2A–C). Overall, after optimal stimulation, the pDC were the major producers of Mip-1a and Mip-1b while the DN and CD4+ cDC were the superior producers of Rantes. Among the cDC, the CD4+ DC were the highest producers of Mip-1a and Mip-1b while both the DN and CD4+ cDC produced the highest levels of Rantes. The CD8+ cDC remained the poorest secretors of all chemokines (Fig. 2A–C).
Chemokine secretion in response to a range of TLR stimuli One of the major functional differences between the splenic DC subsets is the TLR agonists they respond to.
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Fig. 1. Gene expression profiles of Mip-1a, Mip-1b and Rantes. Real-time PCR was used to study the expression profiles of Mip-1a, Mip-1b and Rantes relative to Gapdh in splenic cDC subsets; DN, CD4+ and CD8+ DC, both in steady-state cDC, (Fig. 1A–C) and in cDC activated in vivo with LPS and CpG for 3 hours (Fig. 1D–F). The data shown for steady-state (resting) DC in Fig. 1A–F represent two independent experiments, whereas for the activated cDC, one representative experiment out of two is shown in panels D, E and F.
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Fig. 2. The protein secretion of Mip-1a, Mip-1b and Rantes in response to cytokines and CpG. ELISA was used to assay chemokine secretion by purified, sorted spleen pDC, DN, CD4+ and CD8+ DC after 16 h in culture, in media alone, or in the presence of cytokine (IFN-g for pDC, and GM-CSF for cDC), or in the presence of CpG (a TLR-9 ligand) or in the presence of both CpG and cytokine. The response was compared to the level of protein secretion in the absence of stimulation. The error bars represent the range between duplicate samples, and one representative experiment out of three is shown: (A) Mip-1a, (B) Mip-1b (C) Rantes.
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The pDC are activated by TLR-7 and TLR-9 agonists, DN and CD4+ cDC are activated by TLR-2, 3, 4, 7 and 9 agonists, and CD8+ cDC are activated by TLR-2, 3, 4 and 9 agonists (Edwards et al., 2003; Hochrein et al., 2002; O’Keeffe et al., 2003). We thus tested chemokine production by each of the DC subsets in response to a panel of TLR-agonists, and compared this to the basal level of chemokine secretion in the presence of media alone. Mip-1a and Mip-1b secretion by DC subsets: While the pDC did not respond to Pam-3-Cys (a TLR-2 agonist), Poly I:C (a TLR-3 agonist) and LPS (a TLR-4 agonist), they responded strongly to both R848 (a TLR-7 agonist) and CpG (a TLR-9 agonist) by producing high levels of Mip-1a and Mip-1b relative to the level of chemokine secretion in the presence of media alone (Fig. 3A and B). The DN and CD4+ cDC did not respond to Pam-3-Cys or Poly I:C, and responded poorly to LPS. Similar to the pDC, they responded most strongly to R848 and CpG, with the CD4+ cDC producing higher levels of the chemokines compared to the DN cDC (Fig. 3A and B). The CD8+ cDC responded modestly to CpG, with no response observed in the presence of Pam-3-Cys, Poly I:C, LPS and R848 (Fig. 3A and B). Overall, the pDC were the highest producers of Mip-1a and Mip-1b, and the CD4+ cDC were the most efficient producers of these chemokines among the cDC. Rantes secretion by DC subsets: The pDC responded to R848 (TLR-7 agonist) and CpG (TLR-9 agonist) to produce high levels of Rantes, and did not respond to any other TLR stimuli relative to the level of chemokine secretion in the presence of media alone (Fig. 3C). The DN and CD4+ cDC did not respond to Pam-3-Cys (TLR-2 agonist) or Poly I:C (TLR-3 agonist), responded modestly to LPS (TLR-4 agonist), and strongly to R848 and CpG to produce high levels of Rantes. The CD8+ cDC did not respond to Pam-3-Cys, but were major producers of Rantes in response to Poly I:C. In addition, they responded modestly to LPS, weakly to R848 and strongly to CpG to produce Rantes. Overall, the DN and CD4+ cDC were the most efficient producers of Rantes.
Discussion In this paper, we report a new functional difference between the splenic DC subsets, with the finding that the inflammatory chemokines Mip-1a, Mip-1b and Rantes are differentially expressed and secreted by splenic DC subsets, both in the steady state, and in response to a range of TLR stimuli. We first noted the inflammatory chemokines to be differentially expressed by the cDC subsets using custom-DC cDNA microarrays developed in our
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laboratory, then confirmed this using real-time PCR. We found the CD4+ and DN cDC to express mRNA for the chemokines at higher levels than the CD8+ cDC. This study was extended to investigate changes in gene expression after in vivo activation, revealing that all DC subsets up-regulate the expression of the chemokine genes after activation with LPS and CpG, with the difference between the CD4+ and DN cDC and CD8+ cDC still maintained, albeit with a reduced difference compared to the steady state. In order to gain a better understanding of the regulation of the chemokines at the level of protein secretion, individual DC subsets were cultured in the presence of a range of TLR stimuli. We found that each TLR stimuli induced a unique profile of chemokine secretion. The pDC responded strongly to the TLR-7 and 9 agonists R848 and CpG, by secreting very high levels of all three chemokines compared to the level of chemokine secretion in the absence of stimulation. Significantly, they were the highest producers of Mip-1a and Mip-1b compared to the cDC subsets. They did not respond to Pam-3-Cys, Poly I:C, or LPS which is in keeping with previous studies demonstrating that pDC do not express TLR 2, 3 and 4 (Edwards et al., 2003; Hochrein et al., 2002; Kadowaki et al., 2001; O’Keeffe et al., 2002). The pDC did not produce chemokines in the absence of a stimulus, or in the presence of IFN-g thus displaying a requirement for the presence of a stimulus to produce any chemokines. This is in line with several studies describing the pDC as a pre-DC population, functioning only when activated by an incoming pathogen (Cella et al., 2000; Grouard et al., 1997; O’Keeffe et al., 2003; Siegal et al., 1999). The CD4+ and DN cDC responded in a similar way to the pDC, with both subsets responding primarily to the TLR stimuli R848 and CpG by secreting high levels of Mip-1a, Mip-1b and Rantes. The CD4+ and DN cDC did not respond to Pam-3-Cys (TLR-2 agonist) or Poly I:C (TLR-3 agonist), while activation with LPS (TLR-4 agonist) induced low levels of chemokine secretion by these subsets. However, unlike the pDC and CD8+ cDC which did not produce chemokines in the absence of stimulation, or in the presence of cytokine alone, both the CD4+ and DN cDC produced low levels of Mip-1a and Mip-1b and moderate levels of Rantes in media alone, with the presence of the cytokine enhancing the production of Mip-1a and Mip-1b by these subsets. The secretion of the chemokines by the CD4+ and DN cDC in the absence of stimulation suggests a role for these chemokines in homeostasis. It is possible that the local production of Mip-1a, Mip-1b and Rantes by these subsets is important in maintaining the micro-architecture of the spleen. In contrast to the CD4+ and DN DC that responded in the absence of stimulation, the CD8+ cDC did not produce chemokines in the absence of stimulation, or in
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Fig. 3. The protein secretion of Mip-1a, Mip-1b and Rantes in response to different TLR stimuli. ELISA was used to assay chemokine secretion by spleen pDC, DN, CD4+ and CD8+ DC after 16 h in culture, in media alone (M), or in the presence of one of the following TLR agonists; TLR-2, Pam-3-Cys; TLR-3, Poly I:C; TLR-4, LPS; TLR-7, R848; or TLR-9, CpG. The response was compared to the level of protein secretion in the absence of stimulation (M). The error bars represent the range between duplicate samples, and one representative experiment out of three is shown: (A) Mip-1a, (B) Mip-1b (C) Rantes.
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the presence of GM-CSF alone, and like the pDC, required the presence of a stimulus to produce chemokines. Interestingly, the addition of GM-CSF to the CpG cultures had the greatest effect on the CD8+ cDC (Fig. 3A–C). The CD8+ cDC are the primary IL12 producers, which is the major cytokine driving a Thelper 1 response. The fact that CD8+ cDC require the presence of CpG in order to produce inflammatory chemokines may represent a control mechanism that ensures that the Th1 response is tightly regulated, as an uncontrolled Th1 response may be extremely detrimental to the host. The CD8+ cDC responded primarily to Poly I:C (TLR-3 agonist) by secreting high levels of Rantes. In addition, they demonstrated a modest secretion of Rantes in response to CpG (TLR-9 agonist), and a weak response to LPS (TLR-4 agonist) and R848 (TLR7 agonist). The CD8+ cDC secreted Mip-1a and Mip-1b only in the presence of CpG. Thus, the nature of the chemokine produced by the CD8+ cDC depended on the TLR stimulus. Poly I:C is an analogue of viral RNA, and its ability to stimulate CD8+ cDC to produce Rantes may be reflective of the response of this subset to viral stimulation in vivo. Previous studies have demonstrated the unique ability of the CD8+ cDC to present viral antigens to na.ıve T-cells (Belz et al., 2004; Smith et al., 2003). Thus, their ability to produce high levels of the Tcell chemokine Rantes in response to Poly I:C may reflect a means by which the CD8+ cDC attract and activate na.ıve T-cells in the presence of virus. The poor response of the CD8+ cDC to LPS was surprising given that this microbial stimulus is known to stimulate CD8+ cDC to produce IL-12, (Hochrein et al., 2001) and certainly to activate these cells in vitro. The positive response of the CD8+ cDC to R848 with respect to Rantes secretion was also surprising in view of recent findings by Edwards et al. (2003) indicating that the CD8+ cDC only express marginal levels of TLR-7 and do not respond to TLR-7 agonists. However, our data indicates that the CD8+ cDC do indeed respond to TLR-7 agonists, secreting Rantes in response to R848. Given that the splenic DC subsets reside in distinct anatomical locations within the splenic white pulp, with the CD4+ and DN cDC residing in the marginal zone and the CD8+ cDC in the T-cell zone (De Smedt et al., 1996; Reis e Sousa et al., 1997; Steinman et al., 1997), it is possible that the differential production of the chemokines by the DC is important in regulating the interaction of immune cells with spleen DC during infection. The chemokines may be acting on surrounding lymphocytes such as B and T cells. However, they may also be acting on the DC themselves. Indeed, we have found CCR-5, the receptor to which Mip-1a, Mip1b and Rantes bind, to be differentially expressed by the spleen cDC after in vivo activation, with the CD4+ and
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DN cDC expressing the receptor at higher levels relative to the CD8+ cDC (data not shown). Thus, it is possible that the chemokines are acting in an autocrine fashion through CCR-5, to enhance the migration of these DC into the T-cell areas of the spleen, as well as acting on other surrounding leukocytes to direct trafficking through the spleen. Overall, it appears that DC respond differently when stimulated with a range of TLR agonists. Stimulation via the TLR-4 agonist LPS is a poor inducer of the chemokines tested, whereas stimulation via the TLR-7 and 9 agonists are effective inducers of chemokine production in all DC subsets. Interestingly, stimulation via the TLR-3 agonist Poly I:C is a potent inducer of Rantes by the CD8+ cDC only. Of the cDC, the CD4+ cDC which previous studies have shown to be poor producers of cytokines, are the most effective producers of the inflammatory chemokines. Thus we ascribe a novel function to this splenic DC subset.
Acknowledgements We thank F. Battye, V. Lapatis, C. Tarlinton and C. Clark for assistance with flow cytometric sorting.
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Immunobiology 209 (2004) 163–172 www.elsevier.de/imbio
Differential production of inflammatory chemokines by murine dendritic cell subsets Anna I. Proiettoa, Meredith O’Keeffea, Kate Gartlanb, Mark D. Wrightb, Ken Shortmana, Li Wua, Mireille H. Lahouda,* a b
The Walter and Eliza Hall Institute of Medical Research, 1G, Royal Parade, Parkville, Vic. 3050, Australia Austin Research Institute, Kronheimer Building, A&RMC, Studley Road, Heidelberg, Vic. 3084, Australia
Received 26 February 2004; accepted 10 March 2004
Abstract Dendritic cells (DC) are efficient antigen presenting cells with the ability to activate na.ıve T cells. Murine DC represent a heterogeneous population that can be subdivided into distinct subsets, including the conventional DC (cDC) which are either CD4CD8 (DN), CD4+CD8 (CD4+) or CD4CD8+ (CD8+) subsets and the plasmacytoid DC (pDC), which have different immune regulatory functions. In this study, we investigated the differential expression of genes encoding the inflammatory chemokines Mip-1a, Mip-1b and Rantes, and the secretion of these chemokines, among splenic DC subsets. These chemokine genes were expressed at higher levels by the splenic CD4+ and DN cDC subsets compared with the CD8+ cDC, in both the resting and activated states in vivo. Both the pDC and cDC subsets displayed increases in chemokine secretion in response to a range of toll-like receptor (TLR) stimuli in vitro. Whilst the pDC were the highest producers of Mip-1a and Mip-1b in response to some TLR stimuli, the DN and CD4+ cDC subsets were the superior producers of Rantes. Overall, of the cDC, the CD4+ cDC produced all chemokines most efficiently, both at a basal level, and in response to most TLR stimuli. Thus, we report a new functional difference between the murine splenic cDC subsets, with the CD4+ cDC demonstrating the most efficient production of the inflammatory chemokines Mip-1a, Mip-1b and Rantes. r 2004 Elsevier GmbH. All rights reserved. Keywords: Dendritic cells, chemokines
Introduction
Abbreviations: CCR, chemokine receptor; DC, dendritic cells; pDC, plasmacytoid dendritic cells; cDC, conventional dendritic cells; GMCSF, granulocyte-macrophage colony stimulating factor; IFN-g, interferon gamma; Mip, Macrophage inhibitory protein; Rantes, Regulated on activation,T cell expressed and secreted; TLR, toll-like receptor *Corresponding author. Tel.: +61-3-9345-2533; fax: +61-3-93470852. E-mail address: [email protected] (M.H. Lahoud). 0171-2985/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2004.03.002
Dendritic cells (DC) represent a sparsely distributed population of bone marrow derived cells that play a central role in the induction and maintenance of an immune response (Steinman, 1991). DC, although sharing a common ability to process antigens and present them to T cells, are a heterogenous group of cells. As a primary division, DC can be separated into plasmacytoid DC (pDC) which only assume a typical DC morphology and function after activation (O’Keeffe et al., 2003), and conventional DC (cDC) (Shortman and
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Liu, 2002). Both DC types are found in peripheral tissues including the skin, and in lymphoid organs. The cDC may be further segregated into a number of distinct subtypes based on surface phenotype (Henri et al., 2001; Vremec et al., 2000). In the thymus, two subsets of cDC have been described (CD8+ and CD8) (Vremec et al., 2000), in the spleen, three subsets have been identified (CD4CD8, CD4+CD8 and CD4CD8+) (Vremec et al., 1992), and in the lymph nodes, in addition to these three, there are up to three further migratory DC subtypes that arrive via the lymph (Henri et al., 2001; Belz and Heath, personal communication). There is a growing evidence that the DC subtypes have specialized roles in immunity, including tolerance, cross-presentation and directing T helper cell responses (AsselinPaturel et al., 2001; Belz et al., 2004; den Haan et al., 2000; Hochrein et al., 2001; Maldonado-Lopez and Moser, 2001; Moser and Murphy, 2000; Pooley et al., 2001; Pulendran et al., 1999; Smith et al., 2003). For example, splenic CD4CD8+ (CD8+) cDC have an exceptional ability to take up and present exogenous antigens on class I MHC, including both cell associated (den Haan et al., 2000; Pooley et al., 2001) and soluble (Pooley et al., 2001) antigen; in part because of this, they are the major presenters of viral antigens to T cells in a number of different infection models (Belz et al., 2004; Smith et al., 2003). The DC subtypes also differ in their cytokine production capacities. The pDC represent the major type-1 interferon producing cells in response to viral stimulation (Asselin-Paturel et al., 2001; Bjorck, 2001; Nakano et al., 2001; O’Keeffe et al., 2002; O’Keeffe et al., 2003). In contrast, the CD8+ cDC have the greatest capacity to produce bioactive IL-12p70 (Hochrein et al., 2001; Pulendran et al., 1999), a cytokine involved in the polarization of T cells to T-helper 1 cells (Moser and Murphy, 2000) while the CD4CD8 (DN) cDC are the superior producers of IFN-g (Hochrein et al., 2001). To date, a distinct function has not been ascribed to the CD4+CD8 (CD4+) cDC. Individual DC subsets reside in distinct anatomical locations within lymphoid organs, which influences their interactions with surrounding cells. Chemokines are small chemotactic proteins that are integral in the migration of cells into and within lymphoid organs, and thus in directing cellular interactions (Cyster, 1999). Constitutitive chemokines direct cell migration in the steady-state whereas inflammatory chemokines play a role in cell trafficking during infection. Chemokines exert their effects through the engagement of chemokine receptors (CCR) present on effector cells (Rossi and Zlotnik, 2000) and therefore act specifically on the cells expressing the relevant receptors. Through the study of monocyte-derived and other cultured DC, it is known that DC produce and respond to chemokines, both in the steady-state, and in response to microbial stimulation. It is also known that DC differentially express some CCRs
(Kucharzik et al., 2002; Sallusto and Lanzavecchia, 2000). However, the role of individual splenic DC subsets in chemokine production, both at the basal level and in response to infection, has not been extensively studied. Based on preliminary studies using custom DC cDNA microarrays we identified chemokine genes differentially expressed by splenic cDC in the steady state. These included genes encoding the inflammatory chemokines Macrophage inflammatory protein-1 a (Mip-1a, CCL3), Macrophage inflammatory protein-1 b (Mip-1b, CCL4) and Regulated upon activation, normal T cell expressed and secreted (Rantes, CCL5), as being more strongly expressed by both the resting and activated spleen CD4+ cDC relative to the CD8+ cDC. These chemokines bind receptors which are widely expressed on monocytes, macrophages, immature DC, NK cells and T-helper 1 cells (Onuffer and Horuk, 2002; Sallusto et al., 2000). Therefore, the chemokines are instrumental in bringing together immune cells during infection, to allow the orchestration of an immune response (Foti et al., 1999; Sallusto et al., 1999). However, their role in the steady state is less clear. Therefore, we examined the chemokineproducing capacities of the freshly isolated mouse splenic DC subsets both at the basal level, and in response to a range of toll-like receptor stimuli (TLR). In this study, we demonstrate that the pDC are potent producers of chemokines in response to TLR-7 and 9 stimulation. We also show that of the cDC, the CD4+ cDC are the superior producers of the inflammatory chemokines, both at the basal level, and in response to many stimuli.
Materials and methods Mice All mice were bred under specific pathogen-free conditions at The Walter and Eliza Hall Institute animal breeding facility. For all experiments, female C57BL/6J Wehi mice of 6–8 weeks of age were used.
Reagents Cytokines: Murine rGM-CSF and murine r-IFNg were purchased from PeproTech (Rocky Hill, NJ). Chemokines and chemokine ELISA reagents: Rat antimouse Mip-1b antibody and biotinylated rabbit antimouse Mip-1b antibody were purchased from BD biosciences (Franklin Lakes, NJ). Recombinant mouse Mip-1a recombinant mouse Mip-1b, recombinant mouse Rantes, Rat anti-mouse Mip-1a antibody, goat anti-mouse Rantes antibody, goat biotinylated anti-mouse Mip-1a antibody and goat biotinylated anti-mouse Rantes antibody were purchased from R&D Systems (Minneapolis, MN).
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Monoclonal antibodies (mAb): The mAbs used for segregating DC subtypes were fluorescein isothiocyanate (FITC)-conjugated anti-CD11c (clone N418), phycoerythrin (PE)-conjugated anti-CD45RA (clone 14.8), PEconjugated anti-CD4 (clone GK1.5) and allophycocyanin (APC)-conjugated anti-CD8 (clone Yts169.4). They were purified and conjugated to fluorochromes as previously described (Vremec and Shortman, 1997; Vremec et al., 1992, 2000). Toll-like receptor stimuli: LPS and polyinosinicpolycytidylic acid (poly(I:C)) were purchased from Sigma-Aldrich (Castle Hill, Australia). Oligonucleotides containing a CpG motif (CpG), and completely phosphorothioated, were synthesized by GeneWorks (Adelaide, Australia) according to a published sequence (CpG1668; Sparwasser et al., 1997). Pam-3-Cys and R848 were purchased from Invitrogen (Carlsbad, CA).
Isolation of DC Pools of spleens were used for DC extraction as described in detail elsewhere (Vremec et al., 1992, 2000). Briefly, organs were chopped, digested with collagenase, and treated with EDTA. Light density cells were collected by a density centrifugation procedure. Non-DC lineage cells were then depleted by coating them with a mixture of mAbs against hemopoietic lineage markers including anti-CD3, anti-CD90, anti-TER119 (erythroid marker), anti-RB68C5 (granulocyte marker) and anti-B220 (for cDC preparations) or anti-CD19 (for pDC and cDC preparations), and then depleting the coated cells with magnetic beads coupled to anti-rat-IgG (Vremec and Shortman, 1997). For isolation of cDC, the DC-enriched preparations were stained with anti-CD11c-FITC, antiCD4-PE, and anti-CD8-APC mAb. Propidium iodide was added in the final wash to label dead cells. For cytometric sorting of cDC, the cells were gated for DC characteristics, namely, high forward scatter and bright staining for CD11c, with propidium iodide-staining cells and autofluorescent cells excluded. These selected DC were then segregated, based on the expression of CD4 and CD8, into CD8+, CD4+, and CD4CD8 (DN) cDC. For isolation of pDC, the DC-enriched preparations were stained with anti-CD11c-FITC, antiCD45RA-PE, and anti-CD8-APC, then the pDC gated during flow cytometric sorting as cells with intermediate levels of CD11c staining and high levels of CD45RA staining. The purity of the sorted DC was >98%.
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Spleens were removed 3 h later and the DC populations isolated, as described above.
Real-time PCR RNA was prepared from cell pellets of purified DC populations using the RNeasy Mini Kit (QIAGEN, Victoria; Australia) as per manufacturer’s instructions. RNA (up to 2 mg) was DNase treated with RQ1 DNase (Promega, Madison, WI) then reverse transcribed into cDNA using random primers (Promega) and Superscipt II reverse transcriptase (Gibco BRL, Geithersburg, MD). Real-time PCR was performed to determine the expression of Mip-1a, Mip-1b, Rantes, and Gapdh in splenic DC subsets using the QuantiTect SYBR Green PCR Kit (QIAGEN) and a Light cycler (Roche, Victoria; Australia), as per manufacturer’s instructions. The specific primers for real-time PCR were as follows: Mip-1a; 50 -ACCCTCTGTCACCTGCTCAACAT-30 ; 50 -GGTTCCTCGCTGCCTCCAAGACTC-30 , Mip-1b; 50 -CGTCCTTGC-TCCTCACGTTC-30 ; 50 -GAAATCAATTTCACAGTCATATCCACA-30 , Rantes; 50 -AGATCTCTGCAGCTGCCC-TCAC-30 ; 50 -TTCTCTGGGTTGGCACA-CACTT-30 , Gapdh; 50 -CATTTGCAGTGGCAAA-GTGGAG-30 ; 50 - GTCTCGCTCCTGGAAGATGGTG-30 . An initial activation step for 15 min at 95 C was followed by 40 cycles of: 15 s at 94 C (denaturation), 20–30 s at 50–60 C (annealing) and 10–12 s at 72 C (extension), followed by melting point analysis. The expression level for each gene was determined using a standard curve prepared from 102–106 pg of specific DNA fragment, then expressed as a ratio to Gapdh.
Stimulation of isolated DC for chemokine production Sorted DC subsets (0.5 106 cells/ml) were cultured for 16 h in modified RPMI 1640 medium containing 10% FCS and b-mercaptoethanol (102 M), in 96-well round-bottom plates at 37 C, in an atmosphere of 10% CO2 in air. After culture, the supernatant was collected, separated from cells by centrifugation, and stored until analysis at 70 C. The pDC culture medium contained a microbial stimulus in the presence or absence of IFN-g (20 ng/ml). The cDC culture medium contained a microbial stimulus in the presence or absence of GMCSF (100 ng/ml). The stimuli used were Pam-3-Cys (1 mg/ml), Poly I:C (100 ng/ml), LPS (1 mg/ml), R848 (1 mg/ml), or CpG 1668 (0.5 mM).
Activation of DC in vivo
Chemokine ELISA
Mice were injected (intra-peritoneally) with 20 nmol CpG and 1 mg LPS in mouse tonicity phosphate buffered solution (PBS). Controls were injected with PBS.
Aliquots of DC culture supernatants were assayed by two-site ELISA for Mip-1a, Mip-1b and Rantes production. Briefly, 96-well polyvinyl chloride microtitre
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plates (Costar, Broadway, Cambridge, UK) were coated with appropriate purified capture mAb, namely, 39624.11 (anti-Mip-1a 0.5 mg/ml), A65-2 (anti-Mip-1b 2.0 mg/ml) or 53433 (anti-Rantes, 0.5 mg/ml). Cultured supernatants were detected by the biotinylated detection mAbs, anti-mouse Mip-1a antibody (0.5 mg/ml), antimouse Mip-1b antibody (0.1 mg/ml) and anti-mouse Rantes antibody (0.1 mg/ml). Detection was as previously described (Hochrein et al., 2001).
Results Differential expression of chemokines by splenic cDC An investigation into the gene expression profiles of steady-state splenic cDC using custom DC cDNA microarrays led to the identification of the genes encoding the inflammatory chemokines Mip-1a, Mip1b and Rantes as differentially expressed by the CD4+ cDC relative to the CD8+ cDC. To further investigate chemokine gene expression by DC, the DN, CD4+, CD8+ splenic cDC subsets were freshly isolated from mouse spleen, and the level of RNA encoding each of the chemokines in each subset was determined using quantitative real-time PCR (Fig. 1). In these steady-state DC, we found all three chemokines to be more highly expressed by the CD4+ cDC compared to the CD8+ cDC. Mip-1a was expressed 10-fold higher in the CD4+ cDC compared to the CD8+ cDC (Fig. 1A), Mip-1b 12fold higher (Fig. 1B), and Rantes 2-fold higher (Fig. 1C). The chemokine genes were expressed at intermediate levels by the DN cDC. The gene expression profiles of Mip-1a, Mip-1b and Rantes were also determined in activated splenic cDC, freshly isolated from mice that were stimulated with LPS plus CpG for three hours. After in vivo activation, the expression of Mip-1a was increased by 47-fold, 13-fold and 72-fold in the DN, CD4+ and CD8+ cDC, respectively (Fig. 1D). Similarly, the expression of Mip-1b was increased by 48-fold, 20-fold and 50-fold in the DN, CD4+ and CD8+ cDC, respectively (Fig. 1E). The expression of Rantes was increased by 16-fold, 13-fold and 19-fold in the DN, CD4+ and the CD8+ cDC respectively (Fig. 1F). Thus, on activation, all cDC subsets increased the expression levels of the three chemokines. However, the CD4+ cDC remained the highest expressors of Mip-1a, Mip-1b and Rantes.
Chemokine secretion by DC subsets To ascertain if high levels of RNA transcription led to chemokine protein production, the chemokine-producing capabilities of cDC subsets were determined and
extended to the newly identified pDC. These DC subsets were freshly isolated from the spleen, and cultured under a range of different conditions. In the absence of any stimuli or cytokines, the CD4+ cDC were found to be the major producers of Mip-1a and Mip-1b, followed by the DN cDC (Fig. 2A–C). The DN and CD4+ cDC both produced suprisingly high levels of Rantes in the absence of stimulation. In comparison, both the pDC and the CD8+ cDC were poor producers of all chemokines in the absence of stimulation. As all DC subsets express TLR-9 (Edwards et al., 2003), we tested the ability of the individual subsets to produce chemokines in response to CpG 1668, a TLR-9 agonist. In the presence of CpG, all DC subsets responded by producing more Mip-1a, Mip-1b and Rantes, compared to the level of chemokine secretion in the absence of stimulation (Fig. 2A–C). Since cytokines can act synergistically with TLR agonists to increase the expression of proinflammatory cytokines, as has been previously demonstrated for IL-12 production by DC (Hochrein et al., 2001; Pulendran et al., 1999), we examined a number of cytokines (GM-CSF, IL-4, IL-12, IFN-g), alone and in combination, for their ability to enhance chemokine production in the presence of CpG. We found that GMCSF had the greatest effect on chemokine production by the cDC, whereas IFN-g had the greatest effect on the pDC (data not shown). Based on these observations, we investigated the effects of chemokine production by the DC subsets in response to cytokine alone (GM-CSF for cDC and IFN-g for the pDC), and in response to cytokine and CpG. The presence of GM-CSF alone enhanced the ability of the DN and CD4+ cDC to produce Mip-1a and Mip-1b while having no effect on Rantes production by these subsets, while the pDC and CD8+ cDC demonstrated no response at all in the presence of cytokine alone (Fig. 2A–C). In addition, we found that all cDC produced Mip-1a and Mip-1b more efficiently in response to CpG in the presence of GM-CSF (Fig. 2A and B) while only the CD8+ cDC responded more efficiently with respect to Rantes production under these conditions (Fig. 2A–C). The pDC produced all chemokines more efficiently in response to CpG if IFN-g was present (Fig. 2A–C). Overall, after optimal stimulation, the pDC were the major producers of Mip-1a and Mip-1b while the DN and CD4+ cDC were the superior producers of Rantes. Among the cDC, the CD4+ DC were the highest producers of Mip-1a and Mip-1b while both the DN and CD4+ cDC produced the highest levels of Rantes. The CD8+ cDC remained the poorest secretors of all chemokines (Fig. 2A–C).
Chemokine secretion in response to a range of TLR stimuli One of the major functional differences between the splenic DC subsets is the TLR agonists they respond to.
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Fig. 1. Gene expression profiles of Mip-1a, Mip-1b and Rantes. Real-time PCR was used to study the expression profiles of Mip-1a, Mip-1b and Rantes relative to Gapdh in splenic cDC subsets; DN, CD4+ and CD8+ DC, both in steady-state cDC, (Fig. 1A–C) and in cDC activated in vivo with LPS and CpG for 3 hours (Fig. 1D–F). The data shown for steady-state (resting) DC in Fig. 1A–F represent two independent experiments, whereas for the activated cDC, one representative experiment out of two is shown in panels D, E and F.
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Fig. 2. The protein secretion of Mip-1a, Mip-1b and Rantes in response to cytokines and CpG. ELISA was used to assay chemokine secretion by purified, sorted spleen pDC, DN, CD4+ and CD8+ DC after 16 h in culture, in media alone, or in the presence of cytokine (IFN-g for pDC, and GM-CSF for cDC), or in the presence of CpG (a TLR-9 ligand) or in the presence of both CpG and cytokine. The response was compared to the level of protein secretion in the absence of stimulation. The error bars represent the range between duplicate samples, and one representative experiment out of three is shown: (A) Mip-1a, (B) Mip-1b (C) Rantes.
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The pDC are activated by TLR-7 and TLR-9 agonists, DN and CD4+ cDC are activated by TLR-2, 3, 4, 7 and 9 agonists, and CD8+ cDC are activated by TLR-2, 3, 4 and 9 agonists (Edwards et al., 2003; Hochrein et al., 2002; O’Keeffe et al., 2003). We thus tested chemokine production by each of the DC subsets in response to a panel of TLR-agonists, and compared this to the basal level of chemokine secretion in the presence of media alone. Mip-1a and Mip-1b secretion by DC subsets: While the pDC did not respond to Pam-3-Cys (a TLR-2 agonist), Poly I:C (a TLR-3 agonist) and LPS (a TLR-4 agonist), they responded strongly to both R848 (a TLR-7 agonist) and CpG (a TLR-9 agonist) by producing high levels of Mip-1a and Mip-1b relative to the level of chemokine secretion in the presence of media alone (Fig. 3A and B). The DN and CD4+ cDC did not respond to Pam-3-Cys or Poly I:C, and responded poorly to LPS. Similar to the pDC, they responded most strongly to R848 and CpG, with the CD4+ cDC producing higher levels of the chemokines compared to the DN cDC (Fig. 3A and B). The CD8+ cDC responded modestly to CpG, with no response observed in the presence of Pam-3-Cys, Poly I:C, LPS and R848 (Fig. 3A and B). Overall, the pDC were the highest producers of Mip-1a and Mip-1b, and the CD4+ cDC were the most efficient producers of these chemokines among the cDC. Rantes secretion by DC subsets: The pDC responded to R848 (TLR-7 agonist) and CpG (TLR-9 agonist) to produce high levels of Rantes, and did not respond to any other TLR stimuli relative to the level of chemokine secretion in the presence of media alone (Fig. 3C). The DN and CD4+ cDC did not respond to Pam-3-Cys (TLR-2 agonist) or Poly I:C (TLR-3 agonist), responded modestly to LPS (TLR-4 agonist), and strongly to R848 and CpG to produce high levels of Rantes. The CD8+ cDC did not respond to Pam-3-Cys, but were major producers of Rantes in response to Poly I:C. In addition, they responded modestly to LPS, weakly to R848 and strongly to CpG to produce Rantes. Overall, the DN and CD4+ cDC were the most efficient producers of Rantes.
Discussion In this paper, we report a new functional difference between the splenic DC subsets, with the finding that the inflammatory chemokines Mip-1a, Mip-1b and Rantes are differentially expressed and secreted by splenic DC subsets, both in the steady state, and in response to a range of TLR stimuli. We first noted the inflammatory chemokines to be differentially expressed by the cDC subsets using custom-DC cDNA microarrays developed in our
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laboratory, then confirmed this using real-time PCR. We found the CD4+ and DN cDC to express mRNA for the chemokines at higher levels than the CD8+ cDC. This study was extended to investigate changes in gene expression after in vivo activation, revealing that all DC subsets up-regulate the expression of the chemokine genes after activation with LPS and CpG, with the difference between the CD4+ and DN cDC and CD8+ cDC still maintained, albeit with a reduced difference compared to the steady state. In order to gain a better understanding of the regulation of the chemokines at the level of protein secretion, individual DC subsets were cultured in the presence of a range of TLR stimuli. We found that each TLR stimuli induced a unique profile of chemokine secretion. The pDC responded strongly to the TLR-7 and 9 agonists R848 and CpG, by secreting very high levels of all three chemokines compared to the level of chemokine secretion in the absence of stimulation. Significantly, they were the highest producers of Mip-1a and Mip-1b compared to the cDC subsets. They did not respond to Pam-3-Cys, Poly I:C, or LPS which is in keeping with previous studies demonstrating that pDC do not express TLR 2, 3 and 4 (Edwards et al., 2003; Hochrein et al., 2002; Kadowaki et al., 2001; O’Keeffe et al., 2002). The pDC did not produce chemokines in the absence of a stimulus, or in the presence of IFN-g thus displaying a requirement for the presence of a stimulus to produce any chemokines. This is in line with several studies describing the pDC as a pre-DC population, functioning only when activated by an incoming pathogen (Cella et al., 2000; Grouard et al., 1997; O’Keeffe et al., 2003; Siegal et al., 1999). The CD4+ and DN cDC responded in a similar way to the pDC, with both subsets responding primarily to the TLR stimuli R848 and CpG by secreting high levels of Mip-1a, Mip-1b and Rantes. The CD4+ and DN cDC did not respond to Pam-3-Cys (TLR-2 agonist) or Poly I:C (TLR-3 agonist), while activation with LPS (TLR-4 agonist) induced low levels of chemokine secretion by these subsets. However, unlike the pDC and CD8+ cDC which did not produce chemokines in the absence of stimulation, or in the presence of cytokine alone, both the CD4+ and DN cDC produced low levels of Mip-1a and Mip-1b and moderate levels of Rantes in media alone, with the presence of the cytokine enhancing the production of Mip-1a and Mip-1b by these subsets. The secretion of the chemokines by the CD4+ and DN cDC in the absence of stimulation suggests a role for these chemokines in homeostasis. It is possible that the local production of Mip-1a, Mip-1b and Rantes by these subsets is important in maintaining the micro-architecture of the spleen. In contrast to the CD4+ and DN DC that responded in the absence of stimulation, the CD8+ cDC did not produce chemokines in the absence of stimulation, or in
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Fig. 3. The protein secretion of Mip-1a, Mip-1b and Rantes in response to different TLR stimuli. ELISA was used to assay chemokine secretion by spleen pDC, DN, CD4+ and CD8+ DC after 16 h in culture, in media alone (M), or in the presence of one of the following TLR agonists; TLR-2, Pam-3-Cys; TLR-3, Poly I:C; TLR-4, LPS; TLR-7, R848; or TLR-9, CpG. The response was compared to the level of protein secretion in the absence of stimulation (M). The error bars represent the range between duplicate samples, and one representative experiment out of three is shown: (A) Mip-1a, (B) Mip-1b (C) Rantes.
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the presence of GM-CSF alone, and like the pDC, required the presence of a stimulus to produce chemokines. Interestingly, the addition of GM-CSF to the CpG cultures had the greatest effect on the CD8+ cDC (Fig. 3A–C). The CD8+ cDC are the primary IL12 producers, which is the major cytokine driving a Thelper 1 response. The fact that CD8+ cDC require the presence of CpG in order to produce inflammatory chemokines may represent a control mechanism that ensures that the Th1 response is tightly regulated, as an uncontrolled Th1 response may be extremely detrimental to the host. The CD8+ cDC responded primarily to Poly I:C (TLR-3 agonist) by secreting high levels of Rantes. In addition, they demonstrated a modest secretion of Rantes in response to CpG (TLR-9 agonist), and a weak response to LPS (TLR-4 agonist) and R848 (TLR7 agonist). The CD8+ cDC secreted Mip-1a and Mip-1b only in the presence of CpG. Thus, the nature of the chemokine produced by the CD8+ cDC depended on the TLR stimulus. Poly I:C is an analogue of viral RNA, and its ability to stimulate CD8+ cDC to produce Rantes may be reflective of the response of this subset to viral stimulation in vivo. Previous studies have demonstrated the unique ability of the CD8+ cDC to present viral antigens to na.ıve T-cells (Belz et al., 2004; Smith et al., 2003). Thus, their ability to produce high levels of the Tcell chemokine Rantes in response to Poly I:C may reflect a means by which the CD8+ cDC attract and activate na.ıve T-cells in the presence of virus. The poor response of the CD8+ cDC to LPS was surprising given that this microbial stimulus is known to stimulate CD8+ cDC to produce IL-12, (Hochrein et al., 2001) and certainly to activate these cells in vitro. The positive response of the CD8+ cDC to R848 with respect to Rantes secretion was also surprising in view of recent findings by Edwards et al. (2003) indicating that the CD8+ cDC only express marginal levels of TLR-7 and do not respond to TLR-7 agonists. However, our data indicates that the CD8+ cDC do indeed respond to TLR-7 agonists, secreting Rantes in response to R848. Given that the splenic DC subsets reside in distinct anatomical locations within the splenic white pulp, with the CD4+ and DN cDC residing in the marginal zone and the CD8+ cDC in the T-cell zone (De Smedt et al., 1996; Reis e Sousa et al., 1997; Steinman et al., 1997), it is possible that the differential production of the chemokines by the DC is important in regulating the interaction of immune cells with spleen DC during infection. The chemokines may be acting on surrounding lymphocytes such as B and T cells. However, they may also be acting on the DC themselves. Indeed, we have found CCR-5, the receptor to which Mip-1a, Mip1b and Rantes bind, to be differentially expressed by the spleen cDC after in vivo activation, with the CD4+ and
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DN cDC expressing the receptor at higher levels relative to the CD8+ cDC (data not shown). Thus, it is possible that the chemokines are acting in an autocrine fashion through CCR-5, to enhance the migration of these DC into the T-cell areas of the spleen, as well as acting on other surrounding leukocytes to direct trafficking through the spleen. Overall, it appears that DC respond differently when stimulated with a range of TLR agonists. Stimulation via the TLR-4 agonist LPS is a poor inducer of the chemokines tested, whereas stimulation via the TLR-7 and 9 agonists are effective inducers of chemokine production in all DC subsets. Interestingly, stimulation via the TLR-3 agonist Poly I:C is a potent inducer of Rantes by the CD8+ cDC only. Of the cDC, the CD4+ cDC which previous studies have shown to be poor producers of cytokines, are the most effective producers of the inflammatory chemokines. Thus we ascribe a novel function to this splenic DC subset.
Acknowledgements We thank F. Battye, V. Lapatis, C. Tarlinton and C. Clark for assistance with flow cytometric sorting.
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