Differential Lysosomal Proteolysis in Antigen-Presenting Cells Determines Antigen Fate

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Science  11 Mar 2005:
Vol. 307, Issue 5715, pp. 1630-1634
DOI: 10.1126/science.1108003


Antigen-presenting cells (APCs) internalize antigens and present antigen-derived peptides to T cells. Although APCs have been thought to exhibit a well-developed capacity for lysosomal proteolysis, here we found that they can exhibit two distinct strategies upon antigen encounter. Whereas macrophages contained high levels of lysosomal proteases and rapidly degraded internalized proteins, dendritic cells (DCs) and B lymphocytes were protease-poor, resulting in a limited capacity for lysosomal degradation. Consistent with these findings, DCs in vivo degraded internalized antigens slowly and thus retained antigen in lymphoid organs for extended periods. Limited lysosomal proteolysis also favored antigen presentation. These results help explain why DCs are able to efficiently accumulate, process, and disseminate antigens and microbes systemically for purposes of tolerance and immunity.

Whereas antibodies typically recognize intact antigens, T lymphocytes recognize proteins as short peptides presented by the major histocompatibility complex (MHC) molecules to induce antigen-specific responses to foreign proteins and tolerance to self proteins. Peptides generated by lysosomal proteases bind to MHC class II molecules, which are then displayed at the cell surface of a limited subset of antigen-presenting cells (APCs) including macrophages (MØs), dendritic cells (DCs), and B cells. Due in part to their marked endocytic and phagocytic capacity, MØs were long considered the prototypical APCs. It is now well established, however, that DCs are also highly endocytic and even more potent APCs (13).

Much of what is known about antigen processing is derived from studies of cultured cells. To evaluate the relative contribution of lysosomal proteases to antigen presentation in vivo, we analyzed by immunofluorescence microscopy the expression of individual proteases in situ in secondary lymphoid organs of mice. Cathepsin S (cat S), considered to be selectively expressed by APCs such as B cells and DCs, was expressed most abundantly in the MØ-rich marginal zone of the spleen (SIGN-R1-positive). Under the same staining conditions, cat S was barely detected in B cell areas or in T cell areas where the majority of DCs reside (Fig. 1A). The same pattern of expression was found for an array of other lysosomal proteases [cat L, K, and asparagine endopeptidase (AEP) (Fig. 1A) and cat B, D, E, H, and O (fig. S1)] in the spleen, whether assayed by immunofluorescence on tissue sections (Fig. 1A), on dissociated spleen cells (fig. S2), or by immunoblot (Fig. 1B). Similarly, lysosomal proteases in lymph nodes were also greatly enriched in SIGN-R1-positive MØ regions but present at low amounts in B and T cell zones where the majority of MHC class II–high cells reside (Fig. 1A). Thus, in vivo APCs differ broadly in their capacities for antigen degradation, with cells rich in MHC class II (B cells and DCs) containing substantially lower amounts of lysosomal proteases than MØs.

Fig. 1.

DC and B cells have lower amounts of lysosomal proteases than MØs. (A) The indicated proteases were detected by immunofluorescence on frozen sections of spleen (left) and lymph node (right) from C3H/HeJ mice. AEP stands for asparagine endopeptidase or legumain. (B) Detection of lysosomal proteases by Western blot. DCs (S-DC) and B cells (S-B) were isolated from spleen with magnetic microbeads conjugated to a monoclonal antibody against CD11c (DCs) or CD19 (B cells). MØs (P-MØ) were isolated by adherence to plastic culture dishes from a peritoneal lavage obtained 3 days after injection of 2 ml of 3% thioglycollate. The asterisks indicate the immature, incompletely cleaved forms of the corresponding enzymes.

The marked differences in lysosomal protease content between DCs and MØs observed in vivo was recapitulated during differentiation of bone marrow precursors into the MØ lineage [by macrophage–colony stimulating factor (CSF)] or the DC lineage (by granulocyte macrophage–CSF). Samples of DC and MØ cultures were analyzed by immunoblot for lysosomal protease content at various times during differentiation. Compared to BM-DCs, BM-MØs developed abundant enzyme amounts (Fig. 2A). Instead, DCs (immature and mature) were more similar to non-APCs such as fibroblasts, both in terms of protease content and lysosomal membrane components (lamp) (Fig. 2B). Analyzing bone marrow cultures at the single-cell level again revealed that MHC class II–high cells (DCs) were weakly stained for a variety of lysosomal proteases (Fig. 2C). As found in situ in lymphoid organs (Fig. 1), the lysosomes in bone marrow–derived MHC class II–low MØs, on the other hand, contained high amounts of all the lysosomal proteases tested (4).

Fig. 2.

Bone marrow (BM)–derived cultures of DCs and MØs recapitulate phenotype of lysosomal proteolysis observed in vivo. (A) Analysis of the expression of AEP and cathepsin D (cat D) during differentiation of bone marrow cultured either in GM-CSF to obtain BM-DCs (purified with use of anti-CD11c microbeads) or in M-CSF to obtain BM-MØs (purified by adherence to plastic culture dishes). Samples were taken at the indicated times of culture and analyzed by Western blot. Equal amounts of total protein (50 μg) were loaded in each lane. (B) Western blot analysis of the protease content from L929 fibroblasts (FL) and CD11c-positive immature or mature BM-DCs (grown in GM-CSF) and BM-MØs (grown in M-CSF). The asterisks indicate the proforms of AEP and cathepsin L (cat L). (C) DCs and MØs derived from bone marrow in the presence of GM-CSF were analyzed by confocal microscopy after labeling for the indicated markers. DCs are high in MHC class II, whereas MØs express abundant amounts of the indicated lysosomal proteases. MØs were identified on the basis of their low amounts of MHC class II, high amounts of Mac-3, strong adherence to glass, and high endocytic and phagocytic capacity.

We next asked whether the disparate expression of lysosomal proteases observed in MØs, DCs, and B cells reflected differences in functional proteolytic activity. Indeed, cell-free extracts of MØs differentiated in culture (B-MØ) (Fig. 3A) or isolated from mice (by peritoneal lavage, P-MØ) (Fig. 3A) had a much higher capacity for proteolysis (at acid pH values) of casein in vitro than similar extracts from APCs rich in MHC class II, i.e., DCs differentiated in culture [B-DC (Fig. 3A)] or DC and B cells isolated from mouse spleen [S-DC and S-B (Fig. 3A)]. Even extracts prepared from fibroblasts (FL, Fig. 3A) exhibited a greater proteolytic capacity than DCs or B cell lysosomes (Fig. 3A). The differences reflected general lysosomal proteolytic capacity and not the activity of a selected enzyme for a single substrate, because the same result was obtained for multiple other substrates including ovalbumin (OVA) (Fig. 3B), bovine serum albumin (BSA) (fig. S3), or a complex mixture of proteins [yeast cytosol (Fig. 3C)]. A similar pattern was found in human cells isolated from human peripheral blood lymphocytes, where B cells exhibited much lower lysosomal proteolysis than monocyte-derived MØs (4). We estimated that lysosomal extracts from mouse peritoneal MØs were 20- to 60-fold more active than the corresponding extracts from mouse spleen DCs (Fig. 3D). Remarkably, this difference seemed limited to lysosomal proteases. Enzymatic assays for other lysosomal acid hydrolases (glycosidases and phosphatases) revealed comparatively minor (∼twofold) differences between MØs and DCs (Fig. 3D). These smaller differences extended to the relative amounts of the lysosomal resident membrane protein Lamp-1, which was only two to three times more abundant in MØs (Fig. 2B). Quantitative electron microscopy also revealed that DCs do not contain dramatically fewer lysosomes than MØs (4). Thus, DC lysosomes are selectively attenuated (or MØs selectively enhanced) with respect to proteolytic activity.

Fig. 3.

The relative amounts of lysosomal proteases in MØs, DCs, and B cells are reflected in concomitant amounts of lysosomal proteolysis. Equal amount of proteins from crude lysosomal extracts were incubated in 0.1 M sodium citrate buffer, 0.5% Triton X-100, and 2 mM dithiothreitol at pH = 4.5 for the indicated time in (A) or for 60 min in (B) and(C). (A) Degradation of bodipy FL casein by lysosomal extracts from BM-MØs, peritoneal MØs (P-MØ), FL, B cells from spleen (S-B), spleen DCs (S-DC), BM-DCs, or negative control without cell lysate (control) was followed by the increase in emission at 645 nm. (B) SDS–polyacrylamide gel electrophoresis analysis of the degradation of OVA (top) or cytosol from yeast cells (bottom) by lysosomal extracts of the indicated cell types. Pound symbol indicates no lysosomal extract; asterisk, incubation at pH = 7.5. (C) The amounts of the indicated lysosomal hydrolases were measured in microsomal fractions of spleen DCs or peritoneal MØs and represented as their respective ratio. The results are representative of at least four independent experiments. Error bars indicate standard deviation of the mean.

We then examined whether the different lysosomal proteolytic capacities of APCs affected the survival of internalized antigens in vivo by following the fate of soluble protein antigens administered to mice under different conditions. Fluorescent dextran served as a nondegradable probe to identify all endocytic APCs. Four hours after intradermal injection, APCs containing both dextran and green fluorescent protein (GFP) appeared in the draining lymph nodes, as determined by fluorescence-activated cell sorting (FACS) (table SI). The endocytic cells were predominantly MØs (CD11b+/CD11c–/class II–) and DCs (CD11c+/MHC class II–high), and both contained comparable amounts of internalized dextran and protein (4). After 20 hours, MØs remained dextran-positive, but nearly all of them were now negative for GFP, indicating that the internalized GFP had been degraded. Almost one-third of the DCs were still found to contain both dextran and GFP even after 20 hours, indicating that the half-life of GFP in DCs was substantially longer than in MØs (table SI).

These data were not restricted to GFP, because similar results were obtained for other protein antigens [horseradish peroxidase (HRP), OVA, and bovine ribonuclease A (RNase-A)] (4). We also confirmed these findings by immunofluorescence of spleens and lymph nodes in situ. When OVA was injected intravenously, 2 to 4 hours later it was predominantly associated with SIGN-R1-positive MØs in the marginal zone of the spleen (Fig. 4A). However, after 28 hours OVA was degraded and was no longer detected in MØs (Fig. 4A) but could still be observed inside CD11c-positive DCs (Fig. 4A).

Fig. 4.

Limited lysosomal proteolysis in DCs allows survival of internalized antigens in vivo. (A) Antigen persistence in spleen DCs after intravenous injection. OVA (8 mg) was injected intravenously into C57/B6 mice. At the indicated times after injection, OVA was detected by immunofluorescence in frozen sections of spleen. MØs and DCs were identified with use of the markers SIGN-R1 and CD11c respectively. In the top and middle images, magnification was 50× but 320× in the bottom image. (B) Antigen persistence in lymph node DCs after intraperitoneal injection. Similar to (A), but 3 mg of HRP were co-injected into the intraperitoneal cavity together with 0.2 mg of DNP-dextran. At the indicated times, lymph nodes were removed and stained for HRP, dextran, MØ (SIGN-R1), or DC (CD11c). (C) Immature BM-derived DCs were pulsed with RNase-A or RNase-S (0.5mg/ml) for 1 hour at 37°C, washed extensively, and then either fixed (top two rows) or further cultured for 3 hours (bottom two rows). MHC-II, Lamp, RNase-A, and RNase-S internalized by DCs were detected by immunofluorescence microscopy as indicated. (D) Limited lysosomal proteolysis enhances antigen presentation. Protease-resistant forms (RNase-A or HRP) were presented more efficiently to T cells than the forms more readily degraded (RNase-S and apo-HRP). Mouse bone marrow–derived DCs were loaded with the indicated antigens for 1 hour, washed, and then co-cultured with splenocytes from mice previously immunized with RNase-A or HRP as indicated. T cell proliferation was estimated by the incorporation of [3H]-thymidine. The results are representative of at least five independent experiments. Error bars indicate standard deviation of the mean.

Intraperitoneal injection of HRP together with dextran yielded the same results for both spleen and lymph nodes (Fig. 4B) (4). After 4 hours, most of the HRP and dextran was present in SIGN-R1-positive MØs (Fig. 4B). However, 18 hours after injection, the MØ retained only the undigested dextran, whereas the internalized HRP was degraded and no longer detectable (48 hours, Fig. 4B). Instead, a few CD11c-positive DCs containing both dextran and HRP were visible (Fig. 4B). Thus, independent of the antigen, the route of injection, or the ability of other APCs to internalize the administered antigen, it was the selective long-term survival of antigens in DC that appeared responsible for the long-term survival of antigens in secondary lymphoid organs.

Lastly, we asked whether the low protease activity found in DCs might not only prevent premature antigen degradation but also favor the presentation of T cell epitopes from internalized antigens. Because there are no ways to reliably modulate the amount of protease expression in DCs or MØs without also altering other important properties of these cells, we tested the hypothesis by comparing the presentation by DCs of antigens that differed only in their susceptibility to lysosomal proteases. One such antigen was RNase-A and its subtilisin-cleaved (between Ala20 and Ser21) variant, RNase-S. The two forms have identical ribonuclease activities and structures (46). A second antigen was HRP and its destabilized form, apo-HRP, from which the bound calcium and heme group were removed (4). The natural forms of these antigens (RNase-A and HRP) are much more resistant to proteolysis compared with their corresponding modified forms (RNase-S and apo-HRP) (4). Although all antigens (RNase-A, RNase-S, HRP, and apo-HRP) were similarly internalized by immature DCs into Lamp-positive lysosomal compartments [RNAse-A and RNase-S (Fig. 4C)], only the stable forms (RNase-A and HRP) could be easily detected in DC lysosomes after 3 hours, whereas the unstable forms [RNase-S (Fig. 4C)] and apo-HRP (4) were no longer detectable because of degradation. This difference was confirmed by biochemical measurements of protease susceptibility in vitro, where RNase-S and apo-HRP were found to be >10-fold more rapidly digested by lysosomal extracts (4). The forms that were more resistant to lysosomal digestion were more efficiently presented by APCs on MHC class II to antigen-specific polyclonal T cells isolated from mice primed with RNase-A or HRP (Fig. 4D). Thus, reduced lysosomal proteolysis actually favored the rescue of antigenic peptides and the presentation of several T cell epitopes from these two unrelated antigens.

Although perhaps counterintuitive, the finding that APCs expressing high amounts of MHC class II attenuate lysosomal proteolysis may have several biological explanations. The low amount of lysosomal proteolysis observed in DCs and B cells would render these cell types more susceptible to regulation by factors such as pH and inhibitors (7, 8). It may also help to explain why lysosomes in DCs are so efficient at recovering immunogenic peptides and assembling peptide-MHC class II complexes (9, 10). Unlike MØs, the less hostile environment found in DC lysosomes may favor the production or survival of longer peptides suitable for MHC class II association. The ability of DCs to avoid rapid degradation of internalized antigens may even contribute to their capacity to cross-present exogenous antigens on MHC class I by allowing them a greater chance to exit from endocytic organelles to the cytosol than would be expected in MØs. The limited proteolytic potential of DCs may enhance their ability to disseminate antigens throughout the immune system by minimizing the destruction of internalized antigens.

DC migration to lymph nodes can be slow (1 to 3 days), so the dissemination process would allow DCs to sequester antigens for presentation even several days later (1114). This feature would be important for enhancing both immunogenic and tolerogenic T cell responses, because both foreign and self antigens would be treated equivalently. Indeed, the fact that commensal bacteria (15) and apoptotic cells (16, 17) can be recovered from migrating DCs may reflect the DC's limited capacity for lysosomal proteolysis. Unfortunately, pathogenic organisms, notably human immunodeficiency virus (1821), may also make use of the comparatively safe haven provided by the DC lysosomal system, opportunistically using these efficient conveyors to reach lymphoid organs for purposes of infection.

Supporting Online Material

Materials and Methods

Figs. S1 to S3

Table S1

References and Notes

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