Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c+ lineage-negative (lin) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c+ lin DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c+ lin DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of α2-macroglobulin (α2M*).

Dendritic cells (DC) 3 are potent APCs and are uniquely capable of transporting pathogens and Ags from peripheral tissues to draining secondary lymphoid tissues during infection (1, 2). Within these tissues, DC initiate the adaptive immune response through the stimulation of resting T cells. Studies have demonstrated that the activation of Ag-specific T cells is directly related to the amount of Ag internalized by APC (3, 4, 5). During their transient exposure to pathogens in peripheral sites, DC must efficiently internalize Ags before beginning their migration to T cell-rich lymphoid structures. It is now clear that DC use a number of specialized immunoreceptors, including DEC-205, DC-specific intercellular adhesion molecule-grabbing nonintegrin (SIGN), the asialoglycoprotein receptor, and the mannose receptor to facilitate endocytosis of Ags (6, 7, 8, 9, 10, 11). During infection and in situations in which Ag concentrations are limited, receptor-mediated Ag uptake may be crucial to APC function (12). Studies have demonstrated that specific receptors in this family can be targeted for the delivery of Ags to DC for purposes of vaccination (13). In addition to these immunoreceptors, it is known that the scavenger receptor CD91/low-density receptor-related protein (LRP) also enhances Ag uptake within macrophages and monocytes (14, 15, 16, 17). Demonstration of CD91/LRP-mediated Ag uptake within DC, however, has yet to be reported, and few studies to date have examined the role of these immunoreceptors in human peripheral blood DC.

α2-Macroglobulin (α2M) is an abundant serum protein that has been classically categorized as a proteinase inhibitor. Like the complement components C3 and C4, which are also members of the α2M superfamily of proteins, this 720-kDa tetramer is converted to an activated form, α2M*, following proteolysis (18). Native α2M consists of two basket-like structures linked by disulfide bonds; during proteolytic cleavage, these baskets close, entrapping proteinases as well as numerous cytokines and growth factors. This conformational conversion exposes receptor recognition sites that enable α2M* to bind to the scavenger receptor CD91/LRP (19). APC and other CD91/LRP+ cells are capable of rapidly internalizing these α2M* complexes via receptor-mediated endocytosis. Previous studies demonstrated that protein Ags are also incorporated within α2M* during this proteolytic conversion (20). Following the formation of these α2M*-Ag complexes, α2M* functions as a vehicle capable of specifically targeted Ags to lysosomal compartments within CD91/LRP+ APC (14, 21, 22). Novel nonproteolytic mechanisms of incorporating Ags within α2M* have been developed and used to demonstrate enhanced humoral and cellular immune responses in vivo (17, 23, 24).

Although DC are the most potent APCs and several in vivo experiments have demonstrated markedly enhanced adaptive immune responses when Ags are targeted to CD91/LRP, no functional characterization of this receptor on DC has been reported. In this study, we demonstrate that the detection of CD91/LRP on PBMC is hampered by the interference of standard density gradients used in their preparation from whole blood. By omitting this step, we demonstrate CD91/LRP expression on a CD11c+ lin subset of blood DC and CD14+ monocytes. Ag targeting to CD91/LRP resulted in enhanced uptake by both of these cell types when compared with soluble Ag alone. This increased Ag internalization correlated with enhanced T cell activation in proliferation assays. Finally, purification of individual cell types revealed that it is the CD11c+ lin DC and not the monocyte that is responsible for this increased T cell activation following CD91/LRP-mediated Ag uptake.

Blood was collected from healthy volunteer donors (n = 16) after obtaining informed consent from each donor and approval from the Duke University Medical Center Institutional Review Board.

CD14 FITC, CD91/LRP PE, lineage mixture FITC (lin-1), CD11c CyChrome, CD11c PE, mouse IgG2aκ FITC, mouse IgG1κ PE, mouse IgG1κ FITC, mouse IgG2bκ FITC, mouse IgG1κ CyChrome, and BD FACS lysing solution were purchased from BD PharMingen (San Diego, CA). Rat γ-globulin was purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640, PBS, and HBSS were purchased from Life Technologies (Gaithersburg, MD). Slide-A-Lizer 10K dialysis cassettes were purchased from Pierce-Endogen (Rockford, IL). All other reagents were of the highest quality commercially available.

Pyrogen-free native α2M was purified, as previously described, from frozen human plasma (American Red Cross, Charlotte, NC) (18). The concentration of α2M was determined using A280 nm (1%/1 cm) = 8.93 and a molecular mass of 720 kDa (25). Purified α2M was converted to the thiol ester-cleaved derivative (α2M*), which binds to CD91/LRP, by incubating with 0.2 M NH4HCO3 for 60 min at room temperature (23). Excess NH4HCO3 was removed by extensive dialysis with PBS.

Tetanus toxin C fragment (TTC) (Roche, Indianapolis, IN) was labeled with the Alexa Fluor-647 (AF-647) protein labeling kit (Molecular Probes, Eugene, OR). The efficiency of labeling was measured using a combination of the A280 nm and A650 nm, as described by the manufacturer. Following successful protein labeling, TTC-AF-647 was incubated with activated α2M* at a ratio of 30:1 at 50°C, as previously described (23, 26). Following incubation, α2M*-TTC complexes were isolated from soluble TTC by extensive filtration using Centriplus YM-100 filters (Millipore, Bedford, MA). TTC-AF-647 incorporation into the α2M*-TTC complex was determined by spectrophotometry using a combination of A280 nm and A650 nm. Incorporation of ∼2 mol TTC/mol α2M* was observed in multiple preparations. Additionally, each Ag was tested for endotoxin using the Limulus amebocyte lysate endotoxin assay (BioWhittaker, Walkersville, MD). For T cell proliferation experiments, all Ags contained <0.1 ng/ml endotoxin.

Following collection, PBMC were isolated from some samples with the use of Histopaque density gradients (Sigma-Aldrich). Fresh blood was diluted 1/1 with PBS and layered over Histopaque. These samples were centrifuged for 30 min at 2000 rpm at room temperature without breaks. Following centrifugation, the PBMC were collected and rinsed twice with FACS buffer (HBSS, 0.5% BSA, and 0.1% NaN3). These cells were then stained with mAbs CD14 FITC, CD91/LRP PE, and the appropriate isotype controls, according to manufacturer’s instructions. Nonspecific binding of Abs was blocked by the addition of rat IgG, 50 μg/ml. Following 30-min incubations, cells were rinsed with FACS buffer and fixed with 1% paraformaldehyde for analysis.

Staining of whole blood was performed with BD FACS lysing solution. Briefly, heparinized blood was stained with mAbs CD14 FITC, LRP/CD91 PE, and appropriate isotype controls, according to manufacturer’s instructions. Following 30-min incubation with Abs, BD FACS lysing solution was added and samples were left at room temperature for 10 min. Following this incubation, blood samples were vortexed and centrifuged for 5 min at 1250 rpm. Cells were then rinsed with FACS buffer and resuspended in 1% paraformaldehyde for analysis.

Whole blood lysis staining was used for analysis of lin blood cell populations. Cells were stained with lin-1 mixture FITC, CD11c CyChrome, LRP/CD91 PE, and appropriate isotype controls, according to manufacturer’s protocol and as described above. For analysis of lin subsets of blood cells, CD11c+, CD11c+/−, and CD11c populations were gated, and expression of LRP/CD91 within these populations was analyzed.

Fresh blood was collected and diluted 1/4 with medium containing RPMI 1640, serum replacement supplement 3 (SR3; Sigma-Aldrich), 12.5 U/ml penicillin, and 6.5 μg/ml streptomycin (RPMI-SR3). Blood was then centrifuged at 2000 rpm for 30 min, and the supernatant was carefully aspirated, leaving the cellular pellet undisturbed. Blood cells were then diluted again 1/4 with RPMI-SR3 and centrifuged, as described above. Following two rinses to effectively dilute serum proteins and endogenous α2M, blood cells were resuspended to their original volume in RPMI-SR3. Blood cells were then pulsed with 0.05, 0.1, and 1.0 μg/ml amounts of TTC in the form of soluble TTC and α2M*-TTC complexes. Following 1 h at 37°C, samples were diluted 1/4 with Enzyme Free Cell Dissociation Buffer (Life Technologies) and incubated at room temperature for 15 min. After gentle vortexing, PBMC were isolated using Histopaque gradients (Sigma-Aldrich) and centrifugation at 2000 rpm for 30 min. The PBMC were collected with a sterile Pasteur pipet and rinsed twice with medium containing RPMI 1640, 2 mM l-glutamine, 10% heat-inactivated human AB serum, 12.5 U/ml penicillin, and 6.5 μg/ml streptomycin (RPMI 10 HAB). PBMC were then counted and resuspended at 250,000 cells/well in U-bottom 96-well plates. Samples were plated and incubated for 6 days at 37°C and 5% CO2. On day 6, 0.5 μCi of tritiated thymidine was added to each sample for the final 6 h of culture. Samples were collected onto glass fiber filters, and results represent triplicate or quadruplicate cpm ± SD.

In separate experiments, blood cells were pulsed with TTC and α2M* alone or uncomplexed TTC in combination with α2M*. In these studies, TTC was added at 0.1 and 1.0 μg/ml amounts, as above. α2M* was added to reflect the amount of α2M* present in the experiments in which α2M*-TTC complexes were used. As noted above, the molar ratio was 1:2. Following pulsing with these protein combinations, PBMC isolation and cell culture were performed, as described above, for detection of T cell proliferation. Stimulation index was calculated as experimental cpm/background cpm. For continuous pulsing experiments, Ags were added to rinsed blood cells in RPMI-SR3. A total of 100 μl of rinsed blood cells was added to each well. After 1 h, HAB was added to a final concentration of 10% in each well. Cells were then incubated for 6 days with no additional rinsing, and T cell proliferation was measured, as described above.

Heparinized fresh blood was collected and rinsed twice with 1:4 vol of RPMI-SR3, as described above. Following gentle vortexing, blood cells were aliquoted into polypropylene tubes and pulsed with TTC-AF-647 or α2M*-TTC-AF-647 complexes. Ags were added based on equimolar amounts of TTC. Samples contained final concentrations of 0.05, 0.1, 0.5, and 1.0 μg/ml TTC following the addition of either soluble TTC-AF-647 or α2M*-TTC-AF-647 complexes. Samples were then incubated at 37°C. After 1 h, samples were removed and incubated for 15 min at room temperature in the presence of 2 mM EDTA, pH 7.0, for optimal cellular dissociation. Nonspecific binding of Abs was blocked by the addition of rat IgG, 50 μg/ml. At this point, samples were stained with mAbs for lin-1 and CD11c. Staining was performed, as described above, with the whole blood lysis method. For analysis of TTC-AF-647 uptake within DC, CD11c+, CD11c+/−, and CD11c populations of lin blood cells were gated, and uptake was measured by mean fluorescence intensity (MFI) in FL-4. Similar studies were performed to analyze TTC-AF-647 uptake within monocytes. In these experiments, Ag pulsing was performed as above and monocytes were gated as CD14/lin-1+ CD11c+ cells. Receptor-associated protein (RAP) was used as competitive antagonist for CD91/LRP-mediated uptake. RAP was purified, as previously described (27). J. Herz (University of Texas Southwestern Medical Center, Dallas, TX) kindly provided pGEX-RAP expression vector. For these studies, blood cells pulsed for 30 min with the same concentrations of α2M*-TTC as above in combination with a 1:100 molar ratio of RAP.

Whole blood cells were pulsed with 0.1 μg/ml TTC in the form of soluble TTC or α2M*-TTC, as described above. Following 1-h incubation at 37°C, cells were diluted 1/3 with Enzyme Free Cell Dissociation Buffer (Life Technologies, Gaithersburg, MD) and incubated at room temperature for 15 min. After gentle vortexing, PBMC were isolated using Histopaque gradients (Sigma-Aldrich) and centrifugation at 2000 rpm for 30 min. PBMC were collected and rinsed twice with cold sort medium containing PBS, 0.1 mM EDTA, 1% heat-inactivated human AB serum, 12.5 U/ml penicillin, and 6.5 μg/ml streptomycin. PBMC were then stained with either CD14 FITC or lin-1 mixture FITC Abs, according to manufacturer’s protocol, on ice. Cells were then rinsed twice with sort medium, and then monocyte or lin populations were collected by flow sorting. Postsort populations were ≥97% pure based on gating analysis. After sorting, monocytes or lin cells were added to 400,000 PBMC in RPMI 10 HAB in 96-well plates and incubated at 37°C and 5% CO2. Thymidine incorporation was measured on day 6, as described above.

Previous studies suggest that CD91/LRP expression is limited to monocytes within peripheral blood (28). In our initial attempts to characterize the expression of CD91/LRP on human blood DC, we sought to compare these cells with CD14+ monocytes as a positive control. However, using flow cytometry, we observed low CD91/LRP expression on CD14+ monocytes, a finding that was not in agreement with the original characterization of CD91/LRP expression in whole blood. Subsequent studies demonstrated that the recognition of CD91/LRP by the mAbs used in our experiments was adversely affected by the exposure of blood cells to standard density gradients (i.e., Histopaque) for purposes of cellular preparation. In repeated experiments, the recognition of CD91/LRP was significantly decreased on CD14+ monocytes following exposure to these density gradients as compared with monocytes stained in whole blood (Fig. 1). Our studies indicated that the decreased recognition of CD91/LRP was not induced by LPS or monocyte activation (data not shown). A similar observation has been reported with respect to the detection of the leukocyte Ag L-selectin following gradient centrifugation (29). Although peripheral monocytes have been characterized as CD91/LRP positive, one study reports that the binding of α2M* to these cells is quite low immediately after density-gradient isolation and that this binding is restored as monocytes are maintained in culture (30). Due to the interference of these density gradients in our ability to detect CD91/LRP, we proceeded to examine the expression and function of this receptor in fresh blood.

FIGURE 1.

Recognition of CD91/LRP by mAbs is significantly diminished following density-gradient isolation of monocytes. CD91/LRP expression was detected on monocytes isolated with and without the use of a density gradient. PBMC were isolated, as described in Materials and Methods, with Histopaque. These cells were stained with anti-CD14 and either anti-CD91/LRP or isotype control. The histogram represents CD91/LRP staining and isotype controls of gated CD14+ monocytes (A). In parallel, an aliquot of fresh blood was stained using the whole blood lysis method, as described in Materials and Methods. As above, the histogram represents CD91/LRP and isotype staining of CD14+ monocytes in whole blood (B). Additionally, CD14 vs CD91/LRP staining is demonstrated for cells isolated by Histopaque (C) and fresh whole blood (D). Analysis is representative of three separate experiments.

FIGURE 1.

Recognition of CD91/LRP by mAbs is significantly diminished following density-gradient isolation of monocytes. CD91/LRP expression was detected on monocytes isolated with and without the use of a density gradient. PBMC were isolated, as described in Materials and Methods, with Histopaque. These cells were stained with anti-CD14 and either anti-CD91/LRP or isotype control. The histogram represents CD91/LRP staining and isotype controls of gated CD14+ monocytes (A). In parallel, an aliquot of fresh blood was stained using the whole blood lysis method, as described in Materials and Methods. As above, the histogram represents CD91/LRP and isotype staining of CD14+ monocytes in whole blood (B). Additionally, CD14 vs CD91/LRP staining is demonstrated for cells isolated by Histopaque (C) and fresh whole blood (D). Analysis is representative of three separate experiments.

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Previous studies have demonstrated that the ability of an APC to increase T cell proliferation is directly proportional to the amount of Ag taken up by the APC (5, 31). Based on these observations, we sought to determine whether CD91/LRP-mediated Ag uptake within whole blood could augment subsequent T cell proliferation. To test this hypothesis, tetanus toxin C fragment (TTC) was fluorescently labeled and incorporated into α2M* using methods previously described (see Materials and Methods) (23). Whole blood was pulsed with equimolar concentrations of soluble TTC or α2M*-TTC complexes, and T cell stimulation was measured after a 6-day incubation. The CD91/LRP-targeted α2M*-TTC complexes induced markedly enhanced T cell proliferation across a range of Ag concentrations when compared with soluble TTC (Fig. 2,A). Notably, this augmented T cell stimulation was detected at Ag concentrations as low as 0.05 μg/ml (Fig. 2,A). Further studies were conducted to address the question of whether α2M* alone was responsible for the increased T cell stimulation observed when blood cells were pulsed with α2M*-Ag complexes. Blood cells pulsed with α2M* in combination with soluble TTC demonstrated no significant change in the resulting T cell stimulation when compared with the soluble TTC alone (Fig. 2,B). This observation demonstrates that while α2M* acts to target Ags to CD91/LRP+ APC, it does not induce increased T cell proliferation when added in combination with soluble Ag. To demonstrate that increased endocytosis of Ags targeted to APC receptors induces the enhanced T cell stimulation observed above, we performed parallel experiments in which soluble TTC and α2M*-TTC complexes were incubated continuously with blood cells. As anticipated, T cell responses to both forms of Ag were comparable in these studies due to the increased nonspecific Ag uptake that occurs during prolonged incubation with APC (Fig. 1 C). Additionally, these studies demonstrate that T cell stimulation is dependent on the uptake of TTC, and therefore is specific for this Ag.

FIGURE 2.

Targeting of Ags to CD91/LRP within whole blood results in enhanced T cell activation. Blood cells were collected and pulsed with equimolar amounts of TTC in the form of soluble TTC and α2M*-TTC complexes, as described above. Following a 1-h Ag pulse, PBMC were isolated and resuspended in culture, as described in Materials and Methods. On day 6 of culture, T cell proliferation was measured by tritiated thymidine incorporation in samples pulsed with soluble TTC (□) and α2M*-TTC (▪) (A). T cell proliferation is representative of three experiments. α2M* alone does not enhance T cell activation. Alternatively, blood cells were pulsed with α2M* alone or with TTC and α2M*, which had not been complexed, as described in Materials and Methods. T cell proliferation was measured on day 6, as described above. Results for α2M*-TTC complexes (▪), α2M* alone (▦), soluble TTC (□), and soluble TTC + α2M* (□) are represented as a stimulation index (S.I.) (B). Alternatively, blood cells were incubated with soluble TTC or α2M*-TTC complexes for the duration of the 6-day experiment. On day 6, T cell proliferation was measured, as described above, for soluble TTC (□) and α2M*-TTC (▪) (C).

FIGURE 2.

Targeting of Ags to CD91/LRP within whole blood results in enhanced T cell activation. Blood cells were collected and pulsed with equimolar amounts of TTC in the form of soluble TTC and α2M*-TTC complexes, as described above. Following a 1-h Ag pulse, PBMC were isolated and resuspended in culture, as described in Materials and Methods. On day 6 of culture, T cell proliferation was measured by tritiated thymidine incorporation in samples pulsed with soluble TTC (□) and α2M*-TTC (▪) (A). T cell proliferation is representative of three experiments. α2M* alone does not enhance T cell activation. Alternatively, blood cells were pulsed with α2M* alone or with TTC and α2M*, which had not been complexed, as described in Materials and Methods. T cell proliferation was measured on day 6, as described above. Results for α2M*-TTC complexes (▪), α2M* alone (▦), soluble TTC (□), and soluble TTC + α2M* (□) are represented as a stimulation index (S.I.) (B). Alternatively, blood cells were incubated with soluble TTC or α2M*-TTC complexes for the duration of the 6-day experiment. On day 6, T cell proliferation was measured, as described above, for soluble TTC (□) and α2M*-TTC (▪) (C).

Close modal

To characterize the APC within whole blood that mediated this enhanced T cell proliferation observed when Ags were targeted to CD91/LRP, we investigated the Ag uptake and T cell stimulatory capabilities of the CD91/LRP+ monocyte. Ag-pulsing experiments were performed with equimolar amounts of fluorescently labeled TTC in the form of soluble TTC or α2M*-TTC complexes. CD14+ monocytes demonstrated significantly increased uptake of Ags complexed within α2M* as compared with soluble Ags (Fig. 3,A). This enhanced Ag uptake within monocytes was observed across a range of Ag doses from 0.05 to 1.0 μg/ml (Fig. 3,B). Furthermore, the addition of a 100-fold excess of the competitive CD91/LRP antagonist, RAP, significantly reduced the uptake of α2M*-TTC complexes (Fig. 3) (32). The >50% reduction in uptake is significant, due to the fact that it is difficult to saturate CD91/LRP-mediated uptake at this temperature. This observation suggests that CD91/LRP is the primary monocyte receptor for α2M*-Ag complexes.

FIGURE 3.

Monocytes demonstrate enhanced uptake of α2M*-Ag complexes, and this process is mediated via CD91/LRP. Fresh blood was collected and pulsed with equimolar amounts of fluorescently labeled TTC in the form of soluble TTC and α2M*-TTC complexes with or without excess RAP, as described in Materials and Methods. Following 30-min pulse uptake of fluorescently labeled α2M*-TTC, RAP + α2M*-TTC (100:1) and soluble TTC each at 1 μg/ml were measured by MFI in FL-4 in CD14+ monocytes (A). Uptake of fluorescently labeled α2M*-TTC (▪), RAP + α2M*-TTC (100:1) (▴), and soluble TTC (□) was further examined at Ag doses from 0.05 to 1.0 μg/ml, as described above (B). Ag uptake is representative of three separate experiments.

FIGURE 3.

Monocytes demonstrate enhanced uptake of α2M*-Ag complexes, and this process is mediated via CD91/LRP. Fresh blood was collected and pulsed with equimolar amounts of fluorescently labeled TTC in the form of soluble TTC and α2M*-TTC complexes with or without excess RAP, as described in Materials and Methods. Following 30-min pulse uptake of fluorescently labeled α2M*-TTC, RAP + α2M*-TTC (100:1) and soluble TTC each at 1 μg/ml were measured by MFI in FL-4 in CD14+ monocytes (A). Uptake of fluorescently labeled α2M*-TTC (▪), RAP + α2M*-TTC (100:1) (▴), and soluble TTC (□) was further examined at Ag doses from 0.05 to 1.0 μg/ml, as described above (B). Ag uptake is representative of three separate experiments.

Close modal

Based on the observation that monocytes used CD91/LRP to enhance the uptake of α2M*-Ag complexes, we sought to test whether α2M*-Ag-loaded monocytes were capable of stimulating T cell proliferation. Whole blood cells were pulsed with 0.1 μg/ml of TTC in the form of soluble TTC or α2M*-TTC complexes. Following a 1-h pulse, CD14+ monocytes were isolated by FACS (Fig. 4,A). The monocytes pulsed with α2M*-TTC complexes demonstrated no significant T cell stimulatory capacity when compared with monocytes pulsed with soluble TTC (Fig. 4,B). Importantly, the number of monocytes reconstituted in these studies was similar to the number of monocytes present in the whole blood T cell stimulation studies described above (Fig. 2 A).

FIGURE 4.

Monocytes do not demonstrate enhanced T cell stimulatory capabilities following the CD91/LRP-mediated uptake of Ags. Blood cells were prepared and pulsed with 0.1 μg/ml of TTC in the form of soluble TTC or α2M*-TTC complexes for 1 h. Following Ag pulsing, CD14+ monocytes were sorted from PBMC, as based on gating through R2 (A). Monocytes pulsed with both soluble TTC and α2M*-TTC complexes were then added to PBMC and incubated for 6 days in culture. After this time, T cell activation was measured by tritiated thymidine incorporation in samples containing monocytes pulsed with either α2M*-TTC complexes (▪) or soluble TTC (□) (B).

FIGURE 4.

Monocytes do not demonstrate enhanced T cell stimulatory capabilities following the CD91/LRP-mediated uptake of Ags. Blood cells were prepared and pulsed with 0.1 μg/ml of TTC in the form of soluble TTC or α2M*-TTC complexes for 1 h. Following Ag pulsing, CD14+ monocytes were sorted from PBMC, as based on gating through R2 (A). Monocytes pulsed with both soluble TTC and α2M*-TTC complexes were then added to PBMC and incubated for 6 days in culture. After this time, T cell activation was measured by tritiated thymidine incorporation in samples containing monocytes pulsed with either α2M*-TTC complexes (▪) or soluble TTC (□) (B).

Close modal

The finding that monocytes do not stimulate T cell proliferation following CD91/LRP-mediated Ag uptake suggested that some other, yet unidentified APC mediated this activity in whole blood. We therefore examined CD91/LRP expression among the DC populations present in whole blood. Analysis of the lineage-negative (lin) population of whole blood, which includes both the myeloid and lymphoid subsets of blood DC (33, 34), demonstrated a weak staining for CD91/LRP (Fig. 5,A). To identify the CD91/LRP+ cell type, we used CD11c staining and divided the lin population into CD11c+, CD11c+/−, and CD11c subsets (Fig. 5,B). Additional staining with HLA-DR demonstrated that the lin population contained two subsets of blood DC, the CD11c+/HLA-DR+ myeloid DC and the CD11c/HLA-DR+ lymphoid DC (Fig. 5,C). Analysis of HLA-DR expression in Fig. 5,C demonstrates that the myeloid CD11c+ DC are contained in gate R2 (Fig. 5,B), while the lymphoid CD11c DC are contained in gate R4 (Fig. 5,B). The remaining CD11c+/− lin cells have been previously characterized as basophils (36) (Fig. 5, B and C). Low levels of CD91/LRP expression were detected on the CD11c+ subset of lin blood DC (Fig. 5,D; histogram of cells in gate R2, Fig. 5,B). CD91/LRP expression was faint/undetectable on the remaining CD11c+/− and CD11c subsets of lin cells (Fig. 5, E and F; histograms of cells in gates R3 and R4, Fig. 5,B). Although CD91/LRP was detected on the CD11c+ lin DC, its expression is significantly lower than that observed on CD14+ monocytes (Fig. 1). In agreement with previous reports, we confirmed that CD91/LRP was not expressed among lymphocytes and neutrophils using fresh blood (data not shown) (28, 36).

FIGURE 5.

The CD11c+ lin subset of blood DC expresses the scavenger receptor CD91/LRP. Analysis of fresh blood revealed that in addition to monocytes, a small subset of CD11c+ lin blood cells expressed CD91/LRP. Whole blood cells were stained with lin-1 and CD91/LRP Abs. Analysis of the lin population demonstrates a low level of CD91 expression (A). Whole blood was stained, as described in Materials and Methods, with lin-1 mixture and CD11c (B). Lin cells were divided into CD11c+ (R2), CD11c+/− (R3), and CD11c (R4) populations. Additional staining and analysis of the lin cells (gates R2, R3, and R4 in B) were performed by staining with HLA-DR (C). The HLA-DR+ peripheral blood DCs reside within both the CD11c+ and CD11c populations of lin cells (C). These HLA-DR+ DC populations correspond to gates R2 and R4 in B and correlate with previously reported myeloid and lymphoid populations, respectively. The CD11c+/− HLA-DR population has been characterized as basophils. The histograms (D, E, and F) represent CD91/LRP expression on the CD11c+, CD11c+/−, and CD11c lin cells based on gates set in B (D = R2, E = R3, F = R4). Low levels of CD91/LRP expression are detected on the CD11c+ lin subset of blood DC. Only faint/weak staining is observed in the CD11c+/− and CD11c lin populations. Analysis is representative of three separate experiments.

FIGURE 5.

The CD11c+ lin subset of blood DC expresses the scavenger receptor CD91/LRP. Analysis of fresh blood revealed that in addition to monocytes, a small subset of CD11c+ lin blood cells expressed CD91/LRP. Whole blood cells were stained with lin-1 and CD91/LRP Abs. Analysis of the lin population demonstrates a low level of CD91 expression (A). Whole blood was stained, as described in Materials and Methods, with lin-1 mixture and CD11c (B). Lin cells were divided into CD11c+ (R2), CD11c+/− (R3), and CD11c (R4) populations. Additional staining and analysis of the lin cells (gates R2, R3, and R4 in B) were performed by staining with HLA-DR (C). The HLA-DR+ peripheral blood DCs reside within both the CD11c+ and CD11c populations of lin cells (C). These HLA-DR+ DC populations correspond to gates R2 and R4 in B and correlate with previously reported myeloid and lymphoid populations, respectively. The CD11c+/− HLA-DR population has been characterized as basophils. The histograms (D, E, and F) represent CD91/LRP expression on the CD11c+, CD11c+/−, and CD11c lin cells based on gates set in B (D = R2, E = R3, F = R4). Low levels of CD91/LRP expression are detected on the CD11c+ lin subset of blood DC. Only faint/weak staining is observed in the CD11c+/− and CD11c lin populations. Analysis is representative of three separate experiments.

Close modal

We next addressed the question of whether CD11c+ DC use this receptor to enhance the uptake of Ags. Whole blood was pulsed with soluble TTC or α2M*-TTC complexes in which equimolar concentrations of TTC were used. The CD91/LRP+ CD11c+ lin DC demonstrated significant uptake of α2M*-TTC complexes following a 1-h pulse (Fig. 6,A). When compared with soluble TTC, no increased uptake of α2M*-TTC complexes was observed within either the CD11c+/− lin basophils or the CD11c lin population, which includes the lymphoid DC (Fig. 6, B and C). These observations were confirmed across a range of Ag doses from 0.1 to 1.0 μg/ml (Fig. 6, E–G). Thus, the CD11c+ lin subset of blood DC expresses and uses CD91/LRP to facilitate the receptor-mediated uptake of Ags following their incorporation into α2M*.

FIGURE 6.

The CD91/LRP-positive subset of CD11c+ lin blood DC demonstrates enhanced uptake of α2M*-Ag complexes. Fresh blood was collected and pulsed with equimolar amounts of fluorescently labeled TTC in the form of soluble TTC or α2M*-TTC complexes, as described in Materials and Methods. Following 1-h pulse at 37°C, cells were dissociated with EDTA and stained for lin-1 and CD11c. As described above, subsets of lin DC were gated based on CD11c+ or CD11c expression (D). Uptake of 1 μg/ml soluble TTC and α2M*-TTC was measured as MFI in FL-4 within these gated populations (A = R2, B = R3, and C = R4). Further studies were conducted to determine the uptake of the fluorescently labeled TTC at doses from 0.1 to 1.0 μg/ml. Uptake of soluble TTC (□) and α2M*-TTC (▪) was measured as MFI in FL-4 within these gated populations (E = R2, F = R3, and G = R4). Ag uptake is representative of three separate experiments.

FIGURE 6.

The CD91/LRP-positive subset of CD11c+ lin blood DC demonstrates enhanced uptake of α2M*-Ag complexes. Fresh blood was collected and pulsed with equimolar amounts of fluorescently labeled TTC in the form of soluble TTC or α2M*-TTC complexes, as described in Materials and Methods. Following 1-h pulse at 37°C, cells were dissociated with EDTA and stained for lin-1 and CD11c. As described above, subsets of lin DC were gated based on CD11c+ or CD11c expression (D). Uptake of 1 μg/ml soluble TTC and α2M*-TTC was measured as MFI in FL-4 within these gated populations (A = R2, B = R3, and C = R4). Further studies were conducted to determine the uptake of the fluorescently labeled TTC at doses from 0.1 to 1.0 μg/ml. Uptake of soluble TTC (□) and α2M*-TTC (▪) was measured as MFI in FL-4 within these gated populations (E = R2, F = R3, and G = R4). Ag uptake is representative of three separate experiments.

Close modal

To confirm that this enhanced uptake of α2M*-Ag complexes was mediated by CD91/LRP, Ag-pulsing experiments were performed in the presence of the CD91/LRP antagonist, RAP. The uptake of α2M*-Ag complexes by CD11c+ lin DC was reduced by ∼80% in the presence of excess RAP (Fig. 7, A and B). The CD91/LRP-mediated uptake of α2M*-Ag complexes was found to be limited to DC and monocytes and was not observed among the CD91/LRP-negative neutrophil and lymphocyte populations within whole blood (data not shown). α2M*-TTC uptake studies demonstrated higher levels of endocytosis in monocytes than in lin CD11c+ DC. Monocyte uptake was also less sensitive to the competitive antagonist, RAP (Fig. 3). These differences reflect the fact that higher levels of CD91/LRP expression are found on monocytes than on lin CD11c+ DC.

FIGURE 7.

The uptake of α2M*-Ag complexes by CD11c+ lin blood DC is mediated by CD91/LRP. Fresh blood was collected and pulsed with 1 μg/ml TTC in the form of α2M*-TTC, RAP + α2M*-TTC (100:1), and soluble TTC, as described in Materials and Methods. Following 30-min pulse at 37°C, cells were dissociated with EDTA and stained for lin-1 and CD11c. CD11c+ lin blood DC were analyzed based on the gating strategy described above (Fig. 6,D, gate R2). The uptake of α2M*-TTC, RAP + α2M*-TTC (100:1), and soluble TTC within CD11c+ lin blood DCs was measured in FL-4 (A). Further studies were conducted to determine the uptake of the fluorescently labeled TTC at doses from 0.1 to 1.0 μg/ml within CD11c+ lin DC (B). Uptake of α2M*-TTC (▪), RAP + α2M*-TTC (100:1) (▴), and soluble TTC (□) was measured as MFI in FL-4 within the CD11c+ lin DC population (Fig. 6 D, gate R2). Ag uptake with the CD91/LRP receptor antagonist, RAP, is representative of three separate experiments.

FIGURE 7.

The uptake of α2M*-Ag complexes by CD11c+ lin blood DC is mediated by CD91/LRP. Fresh blood was collected and pulsed with 1 μg/ml TTC in the form of α2M*-TTC, RAP + α2M*-TTC (100:1), and soluble TTC, as described in Materials and Methods. Following 30-min pulse at 37°C, cells were dissociated with EDTA and stained for lin-1 and CD11c. CD11c+ lin blood DC were analyzed based on the gating strategy described above (Fig. 6,D, gate R2). The uptake of α2M*-TTC, RAP + α2M*-TTC (100:1), and soluble TTC within CD11c+ lin blood DCs was measured in FL-4 (A). Further studies were conducted to determine the uptake of the fluorescently labeled TTC at doses from 0.1 to 1.0 μg/ml within CD11c+ lin DC (B). Uptake of α2M*-TTC (▪), RAP + α2M*-TTC (100:1) (▴), and soluble TTC (□) was measured as MFI in FL-4 within the CD11c+ lin DC population (Fig. 6 D, gate R2). Ag uptake with the CD91/LRP receptor antagonist, RAP, is representative of three separate experiments.

Close modal

Based on these findings, we examined whether CD11c+ lin DC were responsible for the augmented T cell proliferation observed following the CD91/LRP targeting of Ags in whole blood. Previous studies have suggested that monocytes are responsible for the enhanced presentation of CD91/LRP-targeted Ags; however, more recent studies have indicated that blood DC possess a much greater ability to activate resting T cells (16, 37, 38, 39). Due to limited cell numbers and the relatively unstable phenotype of lin cells, we examined the T cell stimulatory capabilities of total lin cells following the CD91/LRP-mediated uptake of Ags. Whole blood cells were pulsed with 0.1 μg/ml of TTC in the form of soluble TTC or α2M*-TTC complexes. Following 1-h pulsing, lin cells were isolated by FACS (Fig. 8,A). Unlike the monocytes, the lin cells pulsed with α2M*-TTC complexes induced increased T cell proliferation as compared with lin cells pulsed with soluble TTC (Fig. 8 B). This finding, coupled with the demonstration that CD11c+ DC are the only lin cell type that takes up significant amounts of α2M*-TTC, strongly suggests that the CD11c+ lin DC are the primary APC capable of presenting α2M*-Ag complexes in whole blood.

FIGURE 8.

Lin blood cells are capable of augmenting T cell stimulation following the receptor-mediated uptake of α2M*-Ag complexes. Blood cells were prepared and pulsed with 0.1 μg/ml of TTC in the form of soluble TTC or α2M*-TTC complexes for 1 h. Following Ag pulsing, lin cells were sorted from PBMC, as based on gating through R2 (A). Lin cells pulsed with both soluble TTC and α2M*-TTC complexes were then added to PBMC and incubated for 6 days in culture. After this time, T cell activation was measured by tritiated thymidine incorporation in samples containing lin cells pulsed with either α2M*-TTC complexes (▪) or soluble TTC (□) (B).

FIGURE 8.

Lin blood cells are capable of augmenting T cell stimulation following the receptor-mediated uptake of α2M*-Ag complexes. Blood cells were prepared and pulsed with 0.1 μg/ml of TTC in the form of soluble TTC or α2M*-TTC complexes for 1 h. Following Ag pulsing, lin cells were sorted from PBMC, as based on gating through R2 (A). Lin cells pulsed with both soluble TTC and α2M*-TTC complexes were then added to PBMC and incubated for 6 days in culture. After this time, T cell activation was measured by tritiated thymidine incorporation in samples containing lin cells pulsed with either α2M*-TTC complexes (▪) or soluble TTC (□) (B).

Close modal

DC are widely recognized as specialized APC uniquely capable of transporting Ags to draining lymph nodes for the initiation of the adaptive immune system. In doing so, they are required to efficiently internalize Ags found in the periphery before beginning their migration to T cell-rich zones within these lymphoid tissues. The fact that upon activation DC begin to emigrate from peripheral sites indicates that their exposure to Ags at these initial sites is indeed transient. Although Ag uptake by DC may be limited to a short period of time, studies have demonstrated that the stimulation of Ag-specific T cells by APC is directly related to the amount of Ag internalized by the APC (3, 4, 5). In addition to macropinocytosis and fluid-phase uptake, it has become increasingly clear that DC use a number of specialized immunoreceptors to facilitate the rapid internalization and intracellular accumulation of Ags (6, 7, 8, 9, 10, 11). In addition to DEC-205, DC-SIGN, the asialoglycoprotein receptor, and the mannose receptor, we have now demonstrated that CD11c+ lin blood DC use the scavenger receptor CD91/LRP to enhance Ag uptake. Furthermore, this augmented uptake results in the enhanced stimulation of T cells at Ag concentrations as low as 0.05 μg/ml.

Although APC are known to use mechanisms of fluid-phase uptake, it has been suggested that receptor-mediated Ag uptake may be crucial to APC function, particularly during infections and in other cases in which Ag concentrations may be very limited (12). In these instances, receptor-mediated uptake may be essential to enhance the Ag endocytosis and subsequent T cell activation by DC. Indeed, DC and monocytes demonstrate markedly enhanced uptake of Ags targeted to CD91/LRP within the heterogeneous population of whole blood. The dramatic intracellular Ag accumulation within DC and monocytes facilitates optimal T cell activation and the development of humoral and cellular defenses against foreign proteins. In doing so, Ags bound within α2M* are targeted to the cells most capable of stimulating Ag-specific T cells.

As a general rule, receptor-mediated endocytosis is more efficient than fluid-phase uptake. The receptor-mediated uptake capacity of APC, however, is limited by the specific molecular recognition and binding of Ags to unique immunoreceptors expressed on the APC surface. Some of the previously characterized DC Ag receptors, such as the asialoglycoprotein receptor and the mannose receptor, are capable of recognizing primitive repeating structures often found on prokaryotes (8, 11). Others such as DC-SIGN and DEC-205 have unique and, in some cases, uncharacterized protein ligands (7, 9, 13). Although these APC receptors are limited to a finite number of potential ligands, CD91/LRP can facilitate the uptake of a wide array of Ags complexed within α2M*. The incorporation of Ags within the receptor-recognized form of α2M, α2M*, is induced by proteolysis; therefore, this serum proteinase inhibitor is uniquely capable of incorporating and subsequently targeting a diverse range of proteins to CD91/LRP+ DC. Increased or uncontrolled proteolysis, as found in settings of tissue injury and infection, is the only catalyst required for this conversion and Ag incorporation (18). Studies in our laboratory have demonstrated that Ags as large as 100 kDa can be incorporated within α2M* complex during this conversion and there is no limit on the lower size range for Ag incorporation (data not shown). Therefore, α2M* can function as a molecular delivery system, capable of targeting Ags found at sites of increased proteolysis to CD91/LRP+ APC. Our studies demonstrate that this receptor-mediated cellular targeting enhances the ability of CD11c+ lin DC to stimulate Ag-specific T cells.

Previous studies have demonstrated that the α2M*-CD91/LRP axis functions to enhance the internalization and T cell stimulatory functions of monocytes and macrophages, both of which are recognized as amateur APC (14, 16). Although these observations were striking, amateur APC are unable to provide the costimulatory signals needed to initiate a primary Ag-specific adaptive immune response in vivo (2). These observations suggest that the primary function of α2M* is to target active proteinases and cytokines from sites of inflammation to monocytes and macrophages for clearance and intracellular degradation. In addition to functioning as a mechanism for proteinase and cytokine clearance, it has been suggested that the α2M*-CD91/LRP axis has been conserved on these myeloid APC for purposes of targeting a diverse group of Ags to intracellular lysosomes via receptor-mediated uptake (41). However, the Ag presentation mediated by monocytes and macrophages would not be expected to activate naive T cells, and therefore should induce no Ag-specific Igs.

At the onset of our current study, we hypothesized that some professional APC expressed CD91/LRP and was capable of augmenting naive T cell responses to Ags targeted to this receptor. This concept was supported by our results from a number of animal models demonstrating that the application of α2M*-Ag complexes induced markedly enhanced primary Ag-specific immune responses (22, 24). This observation suggested that α2M*-Ag complexes were in fact being targeted to some professional APC that remained uncharacterized. The demonstration that the CD11c+ lin subset of blood DC expresses CD91/LRP and internalizes α2M*-Ag complexes via this receptor provides a cellular mechanism for the enhanced Ab titers induced by the application of α2M*-Ag complexes in vivo. In agreement with our current report, previous studies have demonstrated that the CD11c+ lin blood DC possess markedly enhanced T cell stimulatory capabilities when compared with blood monocytes (37, 38). Furthermore, the fact that DC have conserved the ability to express this scavenger receptor suggests that the α2M*-CD91/LRP axis is an essential mechanism by which CD11c+ DC facilitate efficient Ag uptake at sites of tissue damage and inflammation.

Although this is the first study to characterize a CD91/LRP+ population of blood DC, weak levels of CD91/LRP expression have been previously demonstrated on Langerhans cells. Notably, this population of dermal APC is thought to be derived from a CD11c+ lin blood precursor (38). Our data suggest that CD91/LRP expression is maintained as Langerhans cells develop from their CD91/LRP+ CD11c+ lin precursors. Therefore, CD91/LRP functions as an immunoreceptor capable of targeting a diverse array of Ags, complexed within α2M*, to both CD11c+ lin blood DC and Langerhans cells.

We thank Dr. M. Hoffman, Durham Department of Veterans Affairs Flow Facility (Durham, NC); J. Horvatinovich, Duke Clinical Flow Facility, Duke University Medical Center Human Vaccine Institute Flow Facility; and S. Conlon.

1

This work was supported by National Institutes of Health Grants HC-24066 and GM-07171.

3

Abbreviations used in this paper: DC, dendritic cell; α2M, α2-macroglobulin; α2M*, activated form of α2M; AF-647, AlexaFluor-647; HAB, human AB serum; lin, lineage; LRP, low-density receptor-related protein; MFI, mean fluorescence intensity; RAP, receptor-associated protein; SR3, serum replacement supplement 3; TTC, tetanus toxin C fragment; SIGN, specific intercellular adhesion molecule-grabbing nonintegrin.

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