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Keywords:

  • Dendritic cells;
  • Knockout mice;
  • Scavenger receptor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Dendritic cells (DC) function at the interface of innate and acquired immunity and are uniquely sensitive to specific stimuli. Pattern recognition receptors (PRR) on these cells are critically important because of their ability to recognise and initiate responses to conserved microbial-associated molecular signatures. With the exception of Toll-like receptors (TLR), we know relatively little about the specific distribution of other PRR amongst populations of DC. Here, we describe the expression of the murine class A macrophage scavenger receptor (SR-A) and show that it is restricted to specific subpopulations of bone marrow-derived and splenic DC. Importantly, we demonstrate that the receptor significantly alters the response of DC to endotoxin. In contrast to the activities of other PRR that have so far been examined, uniquely SR-A limits the maturation response; SR-A–/– cells display enhanced CD40 expression and TNF-α production. We discuss the potential contributions of SR-A to DC biology in the context of the known multiple activities of this receptor.

Abbreviations:
BM-DC:

bone marrow-derived dendritic cells

PRR:

pattern recognition receptor(s)

SR:

scavenger receptor(s)

SR-A:

class A macrophage scavenger receptor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Dendritic cells (DC) are professional APC that are unique because of their ability to stimulate naive T cells 1. Immature DC reside in most tissues as sentinels, sampling their microenvironment by virtue of their high endocytic and phagocytic capacities 2. Their exposure to a specific combination of stimuli induces cell maturation, a change that is characterized by a shift towards increased antigen processing and presentation, enhanced expression of costimulatory molecules and migration to secondary lymphoid sites where they can interact with T cell populations 2.

One of the most critical events for initiation and regulation of the immune response is the initial discrimination of the challenge. In order to accomplish this DC express on their surface pattern recognition receptors (PRR) that can detect the presence of pathogens, or other antigens, through binding of so-called microbe associated molecular patterns 3. The important biological contributions of these receptors are being revealed through studies in which the activities of specific PRR are disabled. Critical roles for members of the TLR family have been demonstrated: DC responses to bacterial lipopeptides, LPS and viral nucleic acids are significantly compromised in the absence of TLR2, TLR4, TLR3 and TLR7, respectively 4, 5. Although TLR have been the most intensively investigated, DC are known to express examples from other families of PRR 6. However, our knowledge of the variety and distribution of PRR between DC subpopulations is currently incomplete, but is very likely to be important because expression of these receptors will define or influence cell responsiveness and may underlie the molecular plasticity of DC 7.

Scavenger receptors (SR) are a structurally diverse group of transmembrane receptors 8 and it is now accepted that many play crucial physiological roles in host defence 9. The prototypic member of this group of receptors is the class A macrophage scavenger receptor (SR-A, also known as MSR1 or SCARA1). Expression of SR-A is primarily restricted to cells of the myeloid lineage and an increasing number of investigations, facilitated by the generation of a SR-A-deficient mouse, have demonstrated that this molecule is multi-functional 10. Studies made either in vivo or ex vivo have shown SR-A involvement in macrophage (MΦ) adhesion, endocytosis of modified lipoprotein, phagocytosis of microbes and apoptotic cells and response to endotoxin 11. These multiple activities are most likely due to the unusual binding properties of this receptor 12. SR-A can recognize a broad, yet specific range of ligands that include modified proteins, polyribonucleotides, disease-associated species such as β-amyloid, and advanced glycation end products and molecules of microbial origin (LPS and lipoteichoic acid) 10. Despite evidence that the immune system can respond to challenge with molecules that are ligands for multiple SR 1315 there is currently little data on the precise distribution of SR-A on murine DC, which is a prerequisite for understanding its potential functions. In this report, we document the differential expression of SR-A by sub-populations of bone marrow-derived DC (BM-DC) and splenic DC. Importantly, we demonstrate that the receptor has at least one significant activity in that SR-A down modulates the maturation response of BM-DC to endotoxin.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

SR-A is expressed by a subpopulation of BM-DC grown in the presence of GM-CSF

BM-DC were generated according to the method of Inaba et al.16. We observed no overt difference in the development, appearance or yield of CD11c+ immature BM-DC at day 6 from cultures derived from 129/ICR or SR-A–/– mice (data not shown), indicating that the receptor is not essential for the normal growth of these cells. In addition, we were unable to detect by FACS analysis any phenotypic differences in the subpopulations of BM-DC produced from 129/ICR and SR-A–/– animals, except for absence of the specific receptor (Table 1 and data not shown). For this and all subsequent experiments we did not observed significant staining with each of the appropriate isotype matched antibodies (not shown).

Table 1. Surface phenotype and frequencies of 129/ICR and SR-A–/– BM-DC grown with GM-CSFa)
Antigen% Positive cells
129/ICRSR-A–/–
  1. a) Data shown are means ± SE from at least five independent experiments.

CD11b95 ± 3.496 ± 2.2
F4/8091 ± 4.390 ± 3.8
CD6860 ± 7.167 ± 5.9
DEC-20561 ± 7.357 ± 8.9
MHC II82 ± 3.284 ± 5.1
CD8α10 ± 3.88 ± 2.5
CD42 ± 0.61 ± 0.7

At day 6 we consistently detected expression of SR-A on greater than 50% of CD11c+ cells from 129/ICR cultures, but the presence of only very low level staining of receptor-deficient BM-DC (Fig. 1A). Despite testing several different methodologies, we were unable to eradicate the residual staining on SR-A–/– cells (data not shown). We therefore interpret the staining of SR-A–/– cells with the anti-SR-A mAb to represent nonspecific binding and we used the comparison of 129/ICR and SR-A–/– cells to confirm authentic expression of the receptor unequivocally. The residual staining we observed is not the result of either incomplete deletion or "leaky" expression of SR-A as it has been demonstrated unequivocally that the receptor is absent in SR-A–/– animals 17, 18. We similarly exploited the availability of receptor deficient animals to demonstrate specific expression on other subpopulations of DC (see below). Calibration of the intensity of staining using fluorescent microbeads analysed under identical FACS conditions indicated that the mAb-dependent signal corresponded to approximately 8 × 104 binding sites on the plasma membrane. The appearance of SR-A during the establishment of the cultures paralleled that of CD11c expression; staining with anti-SR-A mAb was first detectable at day 3 and peaked at day 6 (data not shown).

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Figure 1. (A) SR-A is expressed by BM-DC derived from 129/ICR but not SR-A–/– animals. FACS profiles and two-colour FACS quadrant plots of specific expression of SR-A on CD11c+ BM-DC generated from 129/ICR mice and absence of the receptor on equivalent cells from SR-A–/– animals. Profiles represent unstained (broken), isotype control (solid) and anti-SR-A (bold). Figures within quadrant plots represent frequency of total cells within that quadrant. Data shown is representative of at least five independent experiments. (B) SR-A is primarily located on the surface of 129/ICR BM-DC. FACS profiles of viable and fixed/permeabilised BM-DC stained for either SR-A or MHC II. Data shown is representative of at least three independent experiments. (C) SR-A is expressed by a population of CD11b+ DEC-205+ MHC II+ BM-DC. Two-colour FACS quadrant plots of the expression of SR-A in relation to other leukocyte markers on 129/ICR BM-DC. Data shown are representative of at least four independent experiments.

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Studies of SR-A expression by a number of different MΦ populations have shown the existence of a significant pool of intracellular receptor, in addition to that located at the cell surface 19 (N. Platt, unpublished observation). To examine whether there was a similar distribution in BM-DC we performed FACS analysis on viable and fixed/permeabilised cells. Comparison of the FACS profiles and relative staining intensities of live and fixed cells revealed a close similarity between the two, indicating that the vast majority of the receptor was localised at the cell surface (Fig. 1B). The effectiveness of fixation and permeabilization to reveal intracellular epitopes was confirmed by the observation of enhanced staining for MHC II (Fig. 1B), which would be consistent with the known intracellular localisation of this molecule in immature DC 20.

We performed two-colour FACS analyses to explore the surface phenotype of the SR-A+ cells. SR-A+ BM-DC co-expressed CD11b, MHC II and DEC-205 (Fig. 1C). We observed no staining with antibodies specific for CD4 or CD8α on SR-A+ BM-DC and there were few cells expressing these markers in our cultures (Table 1).

Absence of SR-A on BM-DC generated with Flt3L

We undertook FACS analysis of cells obtained from 129/ICR and SR-A–/– BM cultures that were supplemented only with Flt3L. By days 7 and 9 a minimum of 61 and 81%, respectively, of cells were CD11c+ and there was no appreciable difference in frequencies between the two genotypes (data not shown). Analysis of the distribution of surface markers confirmed the presence of CD11b+ and B220+ subpopulations, few CD8α+ cells (maximum of 7.8% of total cells), a CD4+ DEC-205+ subpopulation, a CD11b+ DEC-205+ subset that typically accounted for only 10% of total cells and an additional CD11b CD4+ subset that was absent from GM-CSF cultures (data not shown) (Table 2). This profile was similar to previous reports of the phenotypes of the cells that arise in the presence of Flt3L 21, 22. We could not detect significant cell surface expression of SR-A on BM-DC that arose after 7 days in culture with flt3L and only 2% of day 7 cells and 8% of day 9 cells were positive (Table 2). These frequencies did not differ from those of SR-A–/– BM cultures, confirming that the staining was nonspecific. CD11b+ BM-DC were negative for the receptor (data not shown), which was perhaps surprising given that this subpopulation expressed high levels when generated with GM-CSF (Fig. 1C).

Table 2. Surface phenotype and frequencies of 129/ICR and SR-A–/– BM-DC grown with Flt3La)
Antigen% Positive cells
  1. a) Data shown are means ± SE from at least five independent experiments.

129/ICRSR-A–/–
Day 7 CD11c65 ± 4.561 ± 6.9
Day 7 CD11b77 ± 5.7 81 ± 4.1
Day 7 DEC-20510.7 ± 3.89.1 ± 2.1
Day 7 CD8α7.8 ± 2.26.1 ± 2.4
CD11b DEC-20510.2 ± 3.18.6 ± 2.7
CD11b CD411.3 ± 2.910.0 ± 1.3
Day 7 SR-A2.1 ± 0.73.2 ± 1.1
Day 9 SR-A8.2 ± 2.410.3 ± 2.5

SR-A is expressed by a subpopulation of splenic DC ex vivo

Although BM-DC recapitulate many of the characteristics and properties of DC, studies have demonstrated a greater level of phenotypic diversity of cells that arise in vivo, which is likely to be the result of the complex signals that direct their development. To examine whether our demonstration of SR-A expression on specific sub-populations of BM-DC was physiologically relevant, we tested for receptor expression on DC purified from mouse spleens.

FACS profiles showed that CD11c+ cells isolated from 129/ICR and SR-A–/– animals had a purity of more than 90% (data not shown). We consistently recovered equivalent numbers when cells were prepared from either 129/ICR or SR-A–/– mice and the purified cells formed clusters with overnight culture, consistent with a DC phenotype (data not shown). Staining for the surface markers CD11b, CD8α and B220 identified three major populations of DC (Table 3): CD11b+ cells (including CD4+ and CD4 subpopulations), CD8α+ cells and B220+ cells. CD11b+ cells typically accounted for 65% of total cells, B220+ approximately 12% and CD8α+ on the order of 20%, although recoveries of the latter varied between preparations. No differences in frequencies for these subpopulations were seen between 129/ICR and SR-A–/– cells (Table 3). This suggests SR-A is not essential for the development of these specific DC subsets in vivo.

Table 3. Surface phenotype and frequencies of 129/ICR and SR-A–/– splenic DCa)
Antigen% Positive cells
129/ICRSR-A–/–
  1. a) Data shown are means ± SE from at least five independent experiments.

CD11b65 ± 5.364 ± 4.6
CD8α20 ± 2.919 ± 3.9
B22013 ± 1.211 ± 1.8

Surface expression of SR-A was detected only on a subpopulation of 129/ICR CD11c+ splenic DC that accounted for approximately 25% of all cells (Fig. 2A). Authenticity of expression was confirmed by the observation of a significantly lower frequency of staining (6%) of splenic DC prepared from SR-A–/– mice (Fig. 2A). This residual staining represents non-specific binding by the anti-SR-A antibody. Because SR-A expression was restricted to only a proportion of CD11c+ splenic DC we examined whether SR-A+ cells represented a previously identified subpopulation. In particular, we were interested to see if expression of the receptor was restricted to CD11c+ CD11b+ cells, which phenotypically resemble BM-DC derived with GM-CSF. We therefore performed two-colour FACS analyses using anti-SR-A 2F8 and mAb against other leukocyte antigens. In respect of CD11b expression we observed two populations of SR-A+ cells; the majority (65%) were SR-A+ CD11bhi/int , with a minority (35%) of the SR-A+ CD11bint/lo phenotype (Fig. 2B). This was in contrast to SR-A+ BM-DC, which were universally CD11bhi (Fig. 1C). Expression of the receptor was consistently stronger on the CD4CD11c+ subset (Fig. 2B). We could not detect SR-A reactivity on CD8α+ cells (data not shown). To confirm this further, we examined expression of the αE-integrin (CD103) in relation to that of SR-A. CD103 is exclusively expressed on CD8α+ cells 23. We were unable to detect significant SR-A expression on αE-integrin positive cell (data not shown). There was also an absence of SR-A on B220+ plasmacytoid cells (Fig. 2B). In addition, we compared receptor expression to the distribution of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2): SR-A was expressed predominantly on CD80int and CD86hi subpopulations (Fig. 2B).

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Figure 2. SR-A is expressed by a specific subpopulation of splenic DC. (A) SR-A is specifically expressed on splenic DC purified from 129/ICR but not SR-A–/– animals. FACS profiles and frequencies of SR-A+ CD11c+ cells isolated from 129/ICR and SR-A–/– animals. (B) SR-A is expressed by a CD11b+ CD4 B220 CD80int CD86hi subpopulation of splenic cells. Two-colour quadrant FACS plots of SR-A expression in relation to other leukocyte antigens on splenic CD11c+ cells isolated from 129/ICR animals. Figures within quadrant plots represent frequency of total cells within that specific quadrant. Data shown are representative of at least five independent experiments.

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SR-A-dependent endocytic activity of BM-DC

To confirm the functionality of SR-A on BM-DC we compared the ability of 129/ICR and SR-A–/– BM-DC to endocytose the fluorescent ligand 1′–dioctadecyl-3,3,3′3′tetramethylindo-carbocyanine perchlorate labelled acetylated low-density lipoprotein (DiI-acLDL). Day 6 cells were incubated with the ligand for 3 h, co-stained with anti-CD11c mAb to discriminate BM-DC and analysed by FACS. We observed significant binding/endocytosis by 129/ICR BM-DC, reflected as enhanced fluorescence, compared with a much lower signal for SR-A–/– cells (mean fluorescence CD11c+ 129/ICR BM-DC: mean 306 ± 10.5 SEM; CD11c+ SR-A–/–: mean 120 ± 8.6 SEM. p <0.05) (Fig. 3). The residual non-SR-A endocytic activity is mostly likely due to the contribution of additional SR that can also mediate uptake of the acetylated lipoprotein 12. There was a small population of CD11c cells that showed enhanced uptake of DiI-labelled acLDL, which most likely represent contaminating MΦ (Fig. 3).

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Figure 3. SR-A mediated endocytosis of DiI-acLDL. Two-colour quadrant FACS plots of uptake of fluorescent modified lipoprotein by 129/ICR or SR-A–/– CD11c+ cells derived from BM-DC cultures supplemented with GM-CSF. Figures within quadrant plots represent frequency of total cells within that specific quadrant. Data shown are representative of at least three independent experiments.

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Expression of SR-A by BM-DC is not affected by exposure to LPS

129/ICR and SR-A–/– BM-DC were cultured in the presence of GM-CSF. On day 5 LPS at 1 µg/mL was added, cells cultured overnight and then analysed by FACS at day 6. We detected low-level expression of the costimulatory molecules CD80, CD86 and MHC II on untreated cells, probably as a result of spontaneous self-activation (Fig. 4). As predicted, we observed a significant shift in the expression of these markers following stimulation with LPS in the presence of serum, consistent with their maturation (Fig. 4). Double staining for CD11c confirmed that the majority of costimulatory molecule+ cells were BM-DC (data not shown). However, the level of SR-A expressed on BM-DC stimulated with LPS was not significantly different from that on untreated cells (Fig. 4).

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Figure 4. SR-A expression by BM-DC is not altered by exposure to LPS. FACS profiles of MHC II, CD80, CD86 and SR-A expression on 129/ICR and SR-A–/– BM-DC. Control background staining (broken line), without LPS (solid line) and with 1 μg/mL LPS (bold line). Data shown are representative of at least four independent experiments.

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SR-A–/– BM-DC display enhanced costimulation molecule expression upon LPS-driven maturation

To explore the role of the receptor in endotoxin-driven maturation we prepared cultures of 129/ICR and SR-A–/– BM-DC and on day 6 stimulated the cells with various concentrations of LPS in the presence of serum. After overnight exposure, cells were harvested, stained for expression of the costimulatory molecule CD40 and analysed. As a control, we treated parallel cultures with zymosan particles, which are known not to bind to SR-A or TLR4 12, 24, but can drive DC maturation via other pathways 25. Both 129/ICR and receptor deficient cells responded to LPS, but there was significantly greater expression of CD40 by SR-A–/– BM-DC across the entire range of endotoxin concentrations that were tested (p <0.05) (Fig. 5A). From the comparison of the actual number of CD40 molecules (derived from calibration of the fluorescence intensity values; see Materials and methods section) on LPS-treated cells to that of untreated cells, we observed the maximal mean fold increase in CD40 expression by endotoxin treated SR-A–/– cells relative to that of 129 BM-DC was seen with an LPS concentration of 10 ng/mL (Fig. 5A). In contrast, there was no significant difference in costimulatory molecule expression by 129/ICR and SR-A–/– BM-DC when maturation was triggered with zymosan (Fig. 5B).

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Figure 5. Enhanced expression of CD40 on SR-A–/– BM-DC exposed to LPS but not zymosan particles. (A) Histograms of mean FACS fluorescence (a) and fold increase (b) of costimulatory molecule CD40 expression on 129/ICR (open bars) and SR-A–/– BM-DC (filled bars) exposed to various concentrations of LPS. (B) As in (A), except that BM-DC were exposed to various concentrations of zymosan particles. Data shown are representative of at least four independent experiments. Error bars represent SEM.

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Increased pro-inflammatory cytokine production by SR-A–/– BM-DC after stimulation with LPS

To test further the hypothesis that SR-A–/– BM-DC displayed increased sensitivity to endotoxin-driven maturation, we measured production of the pro-inflammatory cytokine TNF-α. Day-6 129/ICR and SR-A–/– BM-DC were cultured in the presence of 1 μg/mL LPS (a concentration that preliminary experiments demonstrated was sufficient to stimulate significant levels of TNF-α within the time period), then fixed and stained with anti-TNF-α mAb. The FACS profiles revealed that a significantly greater proportion of CD11c+ SR-A–/– cells were positive for cytokine production (76.0%) than were 129/ICR CD11c+ cells (46.1%) exposed to comparable doses of endotoxin (Fig. 6).

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Figure 6. Increased frequency of TNF-α+ cells in SR-A–/– BM-DC exposed to LPS. Two-colour FACS quadrant plots of anti-TNF-α stained CD11c+ 129/ICR and SR-A–/– BM-DC exposed to 1 μg/mL LPS for 5 h. Figures within quadrant plots represent frequency of total cells within that specific quadrant. Data shown are representative of at least three independent experiments.

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Unaltered expression of the PRR CD14 and TLR4-MD-2 on SR-A–/– BM-DC

We analysed day-6 BM-DC to examine whether absence of SR-A may have affected expression of two other PRR, CD14 and TLR4-MD-2 complex and thereby potentially influence the response to endotoxin. FACS analysis of immature CD11c+cells co-stained with antibodies specific for either TLR4-MD-2 or CD14 did not reveal any significant changes in expression by SR-A–/– cells relative to that of 129/ICR (Fig. 7).

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Figure 7. Unaltered expression of CD14 and TLR4-MD-2 on SR-A–/– BM-DC. Two-colour FACS profiles of 129/ICR or SR-A–/– CD11c+ BM-DC stained with either anti-CD14 or anti-TLR4-MD-2 reagents. Data are representative of at least four independent experiments.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

APC express populations of receptors that can interpret a multitude of different stimuli and determine the characteristics of the subsequent immune response 3. Here, we report the distribution amongst murine DC populations of a specific PRR, the class A macrophage scavenger receptor (SR-A). We demonstrate that one of the functions of this receptor in vitro is to limit the extent of endotoxin-triggered DC maturation.

BM-DC have been widely used as a model of DC because of the relative simplicity of the isolation procedure and a high numerical yield 16. We successfully produced BM-DC from both 129/ICR and SR-A–/– mice, which were morphologically and phenotypically indistinguishable, indicating that the receptor is not essential for either their generation or survival. This is also the case for the in vitro generation of MΦ, given that studies of BM-MΦ from SR-A–/– mice have been reported 26. We also could not detect an alteration in either the phenotypes or frequencies of splenic DC subtypes from these animals, which would similarly imply that the receptor is also not essential in vivo.

SR-A was expressed on a significant fraction of BM-DC cultured with GM-CSF; phenotypically the SR-A+ population co-expressed CD11c, CD11b and DEC-205, but lacked CD8α and had only low levels of CD4. In contrast, we were unable to detect significant expression of the receptor on BM-DC produced with Flt3L, including CD11b+ cells. To our knowledge this is the first demonstration of a specific molecular difference between the CD11b+ populations that develop from cultures supplemented with the two different growth factors and conclude that the precise phenotype of these BM-DC is factor dependent. Flt3L containing cultures have predominantly been exploited for the generation of the B220+ plasmacytoid subpopulation 21, 22 and although we could produce these cells, they did not express SR-A.

We also chose to analyse the distribution of SR-A expression within subpopulations of splenic DC for two reasons. First, we wished to examine DC that had arisen through the complex interactions that occur in vivo. Secondly, these cells have been intensively characterised 27. At least four distinct sub-populations have been identified from normal mice: CD4 CD8α, CD4+ CD8α (both of which are CD11b+), CD8α+ CD4 and CD11b B220+ Gr-1+ plasmacytoid DC. SR-A+ splenic DC were determined to be largely of the CD11b+ CD4 CD8α phenotype and represented typically 20% of CD11c+ cells.

The hypothesis has been made that particular DC subpopulations would have unique or differential properties that may facilitate their precise role(s) within the immune system, but this argument for functional diversity remains controversial, at least in part to the absence of data in support of the specialised activities of identified sub-populations 28. However, it has been demonstrated that DC respond differentially to specific pathogens and their components 7, which would be consistent with the importance of the expression of different populations of PRR.

To date, there is little detailed information of the distribution of specific PRR by subpopulations of DC, but because of their importance, the expression of TLR has been examined in some detail. Human TLR distribution is heterogeneous, with monocyte-derived DC having multiple TLR and TLR7 and TLR9 being restricted to plasmacytoid cells 29. In contrast, murine splenic DC have a much more uniform pattern of expression, except for CD8α+ cells which have TLR3, but lack TLR5 and TLR7 30. We found that SR-A was largely confined to the CD4 CD8α CD11b+ subset of splenic DC, which is consistent with a report of receptors for apoptotic cells on splenic DC 31. This study, employing semi-quantitative RT-PCR, could not detect SR-AI in either CD8α+ or CD8α cells, whereas SR-AII mRNA was present in the CD8α population. We have undertaken a limited quantitative RT-PCR analysis of the distribution of SR-A transcripts between FACS-sorted splenic DC and were able to detect both type I and type II mRNA that were present at highest levels in CD4+ CD11b+ and CD4CD11b+ fractions (data not shown). This is in broad agreement with our FACS data. SR-A has been shown to be expressed by primate DC in situ. Harshyne et al.32 identified the receptor on interdigitating cells isolated from rhesus macaque lymph nodes. The DC expression of other SR has been explored to a lesser extent. The class B scavenger receptor CD36 is present on both CD8α+ and CD8α murine splenic DC, but at higher levels in the former 31 and on monocyte derived human DC 33. LOX-l is also expressed by human monocyte-derived cells and contributes to antigen cross-presentation 34. Our evidence of SR-A independent endocytosis of acLDL by murine BM-DC would indicate expression of at least one additional scavenger receptor that is capable of binding the ligand.

SR-A was predominantly expressed on the CD4 CD8α CD11bhi/int subset of splenic DC, but there is scant information about the function(s) of this particular subtype 27. CD4 CD8α CD11b+ cells are a relatively minor population and have been reported to be somewhat variable in absolute number 35, 36. Because of the scarcity of data indicative of specific functions for this DC subpopulation, we cannot as yet correlate SR-A expression with a known property. One study has demonstrated that the CD11b+ CD8α DEC-205+ subset of the spleen and other peripheral organs is responsible for the production of the chemokine CCL17 that facilitates the attraction of activated T cells 37. However, we are now in the position to focus future studies on a comparison of the properties of 129/ICR and SR-A–/– CD4 CD8α CD11b+ DC.

Ex vivo expression of SR-A was performed entirely on resting, unchallenged animals maintained under specific pathogen-free conditions. Therefore, we cannot exclude the possibility that the distribution and relative expression levels of the receptor on tissue DC could be altered significantly as a consequence of infection or other immunological challenge. We observed that SR-A expression on BM-DC did not change appreciably after exposure to endotoxin. Although there is a generalised down modulation of antigen capture receptors with maturation it is not absolute. For example, DEC-205 is up regulated upon DC maturation. 38. It is of interest that another class A macrophage scavenger receptor, MARCO is one of the most enhanced transcripts in DC after exposure to LPS or bacterial activation 39.

We exploited the availability of SR-A–/– cells to facilitate not only analyses of the receptor expression but also investigations of in vitro activities by comparison with wild type cells. Our important demonstration was that SR-A was not required for endotoxin-dependent DC maturation; in fact SR-A–/– BM-DC showed enhanced expression of CD40 and TNF-α. The increased sensitivity was not due to altered expression of either TLR4-MD-2 or CD14 on SR-A–/– BM-DC, of which increased levels could be anticipated to amplify the pro-inflammatory response. To our knowledge, all studies so far reported for the involvement of other PRR in DC maturation have shown pro-maturation activities for each specific receptor (i.e. in the absence or blockade of the specific PRR DC maturation and associated activities are significantly impaired). For example, TLR4-deficient DC, which do not mature and produce little cytokine with exposure to LPS 5, whilst engagement of TLR2 and Dectin-1 by fungal moieties induce innate immune response genes in murine DC 40. In contrast, components of the maturation response of DC triggered by endotoxin in the absence of SR-A were increased. We observed an increase in the frequency of BM-DC secreting TNF-α, rather than a measurable difference in the absolute levels of cytokine, which could suggest that SR-A might serve to define the absolute level of sensitivity of cell populations. However, a more comprehensive analysis of the dose and temporal response of SR-A–/– DC to endotoxin will be required to fully define the relationship between the receptor and LPS- driven pro-inflammatory cytokine secretion. We speculate that SR-A and perhaps other PRR whose activities have not currently been fully defined may function to prevent inadvertent or excessive DC activation. Evidence supporting the concept that engagement of specific PRR may trigger anti-inflammatory responses has emerged from an in vitro study of the mannose receptor on human DC 41. Cross-linking of the receptor with a specific antibody, and selected natural ligands, stimulated the production of IL-10 and IL-1R, which would be consistent with the induction of a receptor-dependent inhibitory signal transduction pathway. Currently, there is little evidence of SR-A mediating specific signal transduction, but a similar approach to that taken by Chieppa et al.41 could be very informative. It may be relevant that we did not observe an enhanced susceptibility of SR-A–/– BM-DC to undergo spontaneous maturation in the absence of stimuli (M. Becker and N. Platt, unpublished observation), which may indicate there is not constitutive inhibitory engagement of the receptor. A second possibility is that binding of appropriate ligands by SR-A alone permits their recognition without the induction of a maturation response. SR-A has been shown previously to protect the host in a septic shock model by restricting MΦ production of TNF-α and IL-6 42. Both for this situation and for that of DC maturation, we can suggest at least two possible mechanisms: the receptor may function in the non-inflammatory detoxification of endotoxin or internalisation via SR-A may actively engage anti-inflammatory signals that modulate those originating from pro-inflammatory receptors to ensure a response of the appropriate amplitude. At this time we are not able to draw conclusions as to whether the greater level of activation, co-stimulatory molecule expression and cytokine production by mature SR-A–/– DC would have significant biological consequences in vivo. A recent study has shown the linkage between innate and adaptive immunity via splenic DC has several components under discrete controls that include cytokines, co-stimulatory molecules and a distinct CD40 dependent signal 43. It will be of interest to compare these events in appropriately challenged 129/ICR and SR-A–/– mice.

In summary, we have demonstrated that SR-A is restricted in expression to specific subpopulations of murine DC and in vitro and it functions to limit the extent of maturation triggered by endotoxin. The unusually broad, yet specific ligand binding properties of the receptor suggests it will very probably be involved in other DC-dependent immune mechanisms that await investigation.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Mice

Animals were bred at the animal facility of the Sir William Dunn School of Pathology, Oxford, and housed under specific pathogen-free conditions. They were used between 6–16 weeks of age. Mice deficient in type I and II SR-A have been described previously 18, were bred on a 129/ICR background and are herein described as SR-A–/–.

Generation of BM-DC

Murine cells were cultured in RPMI 1640 supplemented with 2 mM glutamine, 50 µg/streptomycin and 50 IU/penicillin G, 10 mM HEPES (pH 7.3) (all Invitrogen) and 10% FCS (PAA Laboratories). BM-DC medium also contained either 2–3% v/v conditioned supernatant from the myeloma cell line X63-Ag8, which has been transfected with a plasmid encoding GM-CSF 44 or 2% v/v conditioned medium from the Bl6/F10 melanoma cell line as a source of Flt3L 45. All plasticware was of tissue culture quality and obtained from Corning (VWR), Falcon (BD Biosciences) or Greiner. BM-DC were cultured using a previously described method, with slight modifications 16. Briefly, bone marrow was flushed from femora, passed through a 70-µm cell filtration unit (Falcon) and red cells were lysed. Cells were resuspended in complete BM-DC medium and cultured in tissue culture plastic dishes at a density of 1 × 106/mL. At day 3 the medium and suspension cells were removed and replaced with fresh medium. On days 6, 7 or 9 of culture, cells in suspension were removed and BM-DC harvested. On day 6 the cells consisted of a heterogeneous population, 60–80% of the cells having surface phenotype and morphology of immature DC with granulocytes being the main contaminant.

DC maturation was initiated by addition of indicated concentrations of LPS (Escherichia coli serotype O111:B4; Sigma-Aldrich) or zymosan particles (Sigma-Aldrich) to media.

DC isolation from spleen

This method was adapted from a protocol by 46. In brief, tissue was cut into small pieces and digested with collagenase A (0.5 mg/mL; Roche,) and DNase I (40 µg/mL; Roche) in RPMI 1640 medium supplemented with 5% FCS for 2 × 25 min at 37°C, with occasional mechanical disruption. The digest was filtered, washed in PBS and resuspended in PBS supplemented with 5% FCS, 5 mM EDTA and 5% BSA. CD11c+ cells were isolated using N418 magnetic beads (Miltenyi Biotech) according to the manufacturer's protocol. The purity of splenic CD11c+ cells was enhanced by two sequential passages of the cells. The positively selected population consisted of >90% CD11c+ cells as determined by flow cytometric analysis with the anti-CD11c mAb HL3.

Flow cytometry analysis

BM-DC or CD11c+ splenic cells were harvested and washed with FACS buffer (PBS containing 10 mM sodium azide and 0.1% BSA) and incubated for 30 min at 4°C with the appropriate dilution of primary antibody for SR-A (2F8, Serotec), MHC II (M5/114.1, BD Biosciences), CD11b (Ly-40, Serotec), CD80 (B7-1/16-10A1, BD Biosciences), CD86 (B7-2/6L1, BD Biosciences), CD8α (Ly-2/53-6.7, BD Biosciences), CD11c (HL3, BD Biosciences), CD4 (YTS191.1, Serotec), CD103 (M290, BD Biosciences), B220 (RA3-6B2, BD Biosciences), DEC-205 (NLDC-145, Serotec), CD40 (3/23, Serotec), TNF-α (MP6-XT22, BD Biosciences), TLR4-MD-2 (MTS510, Serotec) or isotype matched controls (all from Serotec). Cells were analysed on a FACScalibur flow cytometer (BD Biosciences) using Cellquest ™ software. When intracellular staining was required, cells were incubated in a 1:10 dilution of BD-Perm2 (BD Biosciences) for 10 min and washed with FACS buffer before incubation with antibody.

For measurement of intracellular cytokine levels, cells were exposed to the appropriate maturation stimulus in the presence of 2 µM GolgiStop (BD Biosciences) and then stained for the cytokine according to the manufacturer's protocol, prior to FACS analysis.

FACS calibration using fluorescent microbead standards

When applicable, a series of fluorescent microbead standards (BD Biosciences) bearing known amounts of fluorochrome were analysed under conditions identical to those used for experimental samples. A standard plot of log10 mean channel fluorescence vs. log10 fluorochrome copy number was constructed, from which the absolute copy number of the marker on each sample was calculated. Fold increase in expression was calculated by dividing the receptor copy number of the experimental sample by that of the appropriate control sample.

Endocytosis of modified lipoprotein

Cells were plated in 24-well plates at a density of 2 × 106 cells/well and cultured overnight, prior to addition of 5 µg/mL 1′–dioctadecyl-3,3,3′3′tetramethylindo-carbocyanine perchlorate- labelled acetylated low-density lipoprotein (DiI-acLDL) (Autogen Bioclear). After 3-h incubation, cells were harvested and washed twice with PBS. Cells were analysed by flow cytometry to measure the extent of endocytosis.

Statistical methods

Statistical significance (p values) was calculated using non-paired Student's t-test using Prism software.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This work was supported by grants from the British Heart Foundation, Wellcome Trust and Medical Research Council. MB was supported by a scholarship from the Ev. Studienwerk Villigst.

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