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The Journal of Immunology, 2007, 179, 5728 -5737
Copyright © 2007 by The American Association of Immunologists, Inc.
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Differential Regulation of the Nature and Functions of Dendritic Cells and Macrophages by Cathepsin E

Hiroe Kakehashi*,{dagger}, Tsuyoshi Nishioku1,*, Takayuki Tsukuba2,*, Tomoko Kadowaki*, Seiji Nakamura{dagger} and Kenji Yamamoto3,*

* Department of Pharmacology and {dagger} Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The aspartic proteinase cathepsin E is localized mainly in the endosomal structures of APCs and has been implicated in a variety of immune responses, however, the precise roles of cathepsin E in these cells remain speculative. In this study, we report the effect of disrupting the gene encoding cathepsin E on the nature and functions of dendritic cells (DCs) and macrophages derived from mouse bone marrow precursors, as well as mouse peritoneal macrophages. Whereas cathepsin E deficiency induced the accumulation of the lysosome-associated membrane protein (LAMP)-1 and LAMP-2 and elevated the lysosomal pH in macrophages, it did not have these effects on DCs. Although cathepsin E deficiency also caused a marked decrease in degradation of phagocytosed OVA and chemotactic responses to MCP-1 and fMLP by macrophages, these abilities were little affected in DCs by the absence of cathepsin E. Interestingly, cathepsin E deficiency markedly decreased the ability of macrophages to present intact OVA, as well as an OVA-derived antigenic peptide (266–281), to cognate T cells, while that of DCs was inversely enhanced by the absence of this protein. This paradox was resolved, in part, by the enhanced phagocytic activity and the increased expression of the costimulatory molecules CD86, CD80, and CD40, which amplify the response of T cells, in cathepsin E-deficient DCs compared with the wild-type cells. These results indicate that cathepsin E differentially regulates the nature and function of DCs and macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The different types of professional APCs, such as dendritic cells (DCs),4 macrophages, and B cells, are equipped with common and specific proteases for immune responses. DCs are heterogenous, professional APC that play a central role in the initiation and control of immune responses and probably in the maintenance of tolerance (1, 2). Bone marrow precursor cells differentiate into immature DCs (iDCs), which efficiently engulf a variety of particulate Ags and then transport them into endolysosomal compartments (termed MHC class II compartments) for degradation (3). Subsequently, the iDCs migrate to the regional lymph nodes where they phenotypically mature and express a number of cell surface molecules for presentation to cognate T cells (4). In contrast, macrophages are phagocytic APC that function as key mediators of host defense against pathogens and serve as a link between innate and adaptive immune responses during infection (5, 6). Upon stimulation with various ligands such as LPS and IFN-{gamma}, macrophages enhance the capacity to kill pathogens through the increased production of inflammatory mediators that initiate acquired T cell immunity.

In APC, two distinct proteolytic events are required for the MHC class II pathway. One is for the processing and removal of the MHC class II chaperon, invariant chain (Ii), associated with the MHC class II molecules and the other for the processing of Ag substrates to generate antigenic peptides that can bind efficiently to newly synthesized MHC class II molecules liberated from the Ii (6, 7, 8, 9, 10). Recent evidence indicates that the cysteine and aspartic proteinases are required for both Ii and Ag processing in the MHC class II pathway (6, 7, 8). Although the leupeptin-insensitive proteases are likely involved in the initial steps of Ii processing, the terminal Ii processing to CLIP is performed most efficiently by lysosomal cysteine proteases, such as cathepsin S in bone marrow-derived professional APC (11, 12, 13) and cathepsin L in murine cortical thymic epithelial cells (14). It has also been known that the Ag-processing event is critical to define the quality and quantity of a CD4 T cell response, but little is known about the identity of the specific proteases responsible for the processing of internalized Ags into particular MHC class II-presentable T cell epitopes. Recent studies have shown that lysosomal cysteine proteases including cathepsins B, S, and L are involved in the processing of internalized Ags (15, 16, 17). In contrast, in vitro studies using purified proteases and their inhibitors have demonstrated that cathepsin D (18, 19, 20, 21) and/or cathepsin E (21, 22, 23, 24) might play a role in antigenic peptide generation. Further studies using APC derived from cathepsin D-deficient mice have shown that cathepsin D is not essential for MHC class II Ag presentation (23, 25, 26, 27). However, to date it remains speculative whether cathepsin E is directly involved in the MHC class II Ag presentation in APC.

Cathepsin E is expressed and localized mainly in the endosomal structures of APC (23, 28, 29, 30). It has been demonstrated in this regard that cathepsin E expression is negatively regulated by the MHC CIITA (31), which is a non-DNA-binding transcription factor necessary for the expression of MHC II and other genes related to Ag presentation (32, 33, 34). Recent studies have demonstrated that mice deficient in cathepsin E (CatE–/–) spontaneously develop atopic dermatitis-like skin lesions when reared under conventional conditions (35) and exhibit increased susceptibility to bacterial infection accompanied by a marked decrease in killing of intracellular bacteria by macrophages (36). Subsequent analysis of peritoneal macrophages derived from CatE–/– mice indicated that deficiency in this enzyme induced a novel form of lysosomal storage disorder manifesting the accumulation of major lysosomal membrane sialoglycoproteins such as lysosome-associated membrane protein (LAMP)-1 and LAMP-2 and the elevation of lysosomal pH (37). In addition, the cell surface expression levels of TLRs including TLR2 and TLR4 were shown to be significantly decreased in CatE–/– macrophages compared with the wild-type cells (36). Because the total cellular levels of these TLRs were not significantly different between the wild-type and CatE–/– macrophages, the decreased cell surface expression of these receptors was found to be due to trafficking defects in the latter, most probably due to the enhanced lysosomal pH by cathepsin E deficiency. These observations have strongly suggested that cathepsin E plays a role in the maintenance of homeostasis by participating in immune responses. Hence, to understand the role of cathepsin E in different cell types of APC, including DCs and macrophages derived from the bone marrow precursors, as well as the peritoneal macrophages, we have investigated the effect of cathepsin E deficiency on the nature and functions in each of these cell types.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Antibodies

Abs recognizing macrophages/microglia (F4/80) were purchased from Acris Antibodies. Abs to mouse LAMP-1 and CD11c were obtained from BD Pharmingen, and those to mouse LAMP-2 were obtained from Southern Biotechnology Associates. Abs to mouse I-A MHC class II, CD86, CD80, and CD40 were obtained from Immunotech. Polyclonal Abs against rat cathepsins D and E were prepared as described previously (38).

Mice and cells

Wild-type and CatE–/– mice with the C57BL/6 genetic background were used as described previously (37). All animals were maintained under specific pathogen-free conditions at Kyushu University Station of Collaborative Research Animal Facilities according to the guidelines of the Japanese Pharmacological Society. The animals and all experiments were approved by the Animal Research Committee of the Graduate School of Dental Science (Kyushu University, Fukuoka, Japan). DCs were obtained by culture of bone marrow cells taken from 8- to 12-wk-old mice essentially according to the method described previously (39). Briefly, the animals were euthanized, and femurs and tibias were dissected after adherent tissue was removed. The ends of the bones were cut off, and the marrow tissue was flushed by irrigation with the medium. The marrow plugs were passed through a 25-gauge needle for dispersion. The cells (1 x 106 cells/ml) were incubated in RPMI 1640 medium supplemented with 5% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2-ME (50 µM) in the presence of GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) at 37°C with a 5% CO2 atmosphere. At days 2 and 4 of culture, the plates were gently swirled to remove loosely adherent cells, comprising mainly granulocytes. After incubation for 5 days, iDCs loosely attached to the monolayer were further purified by a MACS system with CD11c-conjugated microbeads (Miltenyi Biotec). The sorted population was 85–95% CD11c-positive. To mature the DCs, the cells were cultured in medium containing LPS (100 ng/ml) for 18 h. Similarly, bone marrow-derived macrophages were isolated from 8- to 12-wk-old mice as described previously (40). Briefly, the culture of bone marrow cells was incubated in nontreated petri dishes in RPMI 1640 medium containing 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2-ME (50 µM) in the presence of M-CSF (20 ng/ml) and at 37°C with 5% CO2 atmosphere. Macrophages were obtained as a homogeneous population of adherent cells. The purity of the culture was checked regularly by flow cytometry using an anti-F4/80 Ab. The purity of macrophages was found to be 90–95%. Thioglycolate-elicited peritoneal macrophages were also isolated from mice as described previously (37). Peritoneal macrophages isolated as adherent MAC-2-positive cells were obtained at a purity of >95% by this procedure.

Preparation of medium and cell lysates

The culture medium of DCs and macrophages were collected by after 24 h and centrifuged at 16,000 x g for 20 min. The supernatant fraction was concentrated at 10-fold using Centriprep-30 and Microcon concentrators (Millipore). For the preparation of the cell lysates, the cells were washed twice with PBS and then resuspended in the same buffer containing 0.1% Triton X-100 followed by sonication for 1 min at 4°C. After centrifugation at 100,000 x g for 1 h, the supernatant fraction was referred to as the cell lysates.

Enzyme assays

Cathepsins B, L, and D were assayed as described previously (37). beta-Glucuronidase, beta-hexosaminidase, and {alpha}-mannosidase were assayed as described previously (37).

Gel electrophoresis and immunoblot analysis

SDS-PAGE and immunoblotting were performed as described previously (41). The quantification of the immunoreactive bands was analyzed by LAS1000 and Image Gauge software (Fuji Photo Film).

Flow cytometry

Flow cytometry was performed using a Beckman Coulter XL cytometer essentially according to the method as described previously (36). Permeabilized cells were prepared by treatment with 0.03% saponin in PBS containing 2.5% FBS and 0.01% NaN3 for 15 min on ice.

Measurement of lysosomal pH

The lysosomal pH in each cell type was determined in situ as described previously (37). Briefly, after plating on a 96-well plate, the respective cells were incubated with 500 µg/ml of an acidotropic probe, Lysosensor yellow/blue dextran (Molecular Probes) for 24 h and then washed with PBS. The fluorescence from the acidic compartments in the labeled cells was quantified with a fluorescence microplate reader at an emission wavelength of 430/535 nm with excitation at 340 nm (Wallac 1420 ARVOsx; PerkinElmer).

Chemotactic activity

Chemotactic activity was determined using a Boyden chamber as described previously (42). Briefly, fMLP (1 µM) and MCP-1 (0.1 nM) were put into the lower well of a cell culture inserts (BD Falcon). The lower well was separated from the upper well by an 8-µm pore diameter polyethylene terephthalate membrane. Cells (2 x 105 cells/ml) suspended in RPMI 1640 medium were put into the upper well and the chamber was placed in a humidified incubator under 5% CO2 for 90 min at 37°C. After incubation, filters were washed, fixed, and stained with May-Giemsa, and were mounted on the glass slides. The cells migrating through the filter were counted under a light microscope in a high-power field (x400).

Ag-presentation assay

OVA-specific T cell hybridomas were prepared from C57BL/6 mice as described previously (23). Stock OVA(266–281)-specific T cell hybridomas were cultured in IMDM with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2-ME (50 µM) at 37°C with 5% CO2 atmosphere. DCs and macrophages derived from bone marrow precursors of wild-type and CatE–/– mice were seeded in 96-well culture plates at a density of 3 x 104 cells/well. After 18 h, the culture medium was replaced with fresh medium with LPS (100 ng/ml), and then each cell type was further incubated at 37°C for 24 h. Immediately before addition of OVA-specific T cell hybridomas, each cell type was gently washed with medium to remove LPS. The T cell hybridomas (5 x 104 cells/well) were added to the culture of each cell type in the presence of intact OVA or OVA(266–281) peptide at the indicated concentrations. For analysis of T cell-derived IL-2, supernatants from triplicate cultures were harvested after 24 h. IL-2 was quantified by using mouse IL-2 ELISA kit (BioSource International).

Degradation of phagocytosed protein

Each cell suspension was pulsed with DQ-OVA (Molecular Probes) for 1 h at 37°C, subsequently washed extensively with PBS, and then incubated under 5% CO2 at 37°C for the indicated times. Degradation of phagocytosed OVA was measured by increase in the fluorescence in the supernatant at an emission wavelength of 505 nm with excitation at 515 nm.

Phagocytic activity

The quantitative assay of phagocytosis was performed using flow cytometry as described previously (43). Briefly, uniform fluorescent latex particles (108 particles/ml) were added to the cell suspensions in PBS (107 cells/ml) for 1 h at 37°C in a shaking water bath. After addition of 2 ml of ice-cold PBS, the cells were separated from nonphagocytosed particles by low-speed centrifugation at 360 x g for 10 min. The cells were washed twice with PBS, resuspended in 500 µl of cold PBS, and then subjected to a cytofluorimetric assay to quantitatively determine internalized particles.

Statistical analysis

Quantitative data are presented as mean ± SD. The statistical significance of differences between mean values was assessed by the Student t test. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of cathepsin E deficiency on the cellular levels of soluble lysosomal enzymes

To determine the effect of cathepsin E deficiency on intracellular levels of soluble lysosomal enzymes, we performed SDS-PAGE and immunoblot analysis for cell lysates of DCs and macrophages derived from wild-type and CatE–/– mice. Cathepsins D and B were significantly decreased in both LPS-treated and nontreated CatE–/– myeloid macrophages, as well as peritoneal macrophages, compared with the corresponding wild-type cells (Table I). Consistent with these results, the specific activities of cathepsins B, L, and S (units per milligram of protein of each cell lysate) were significantly decreased in myeloid and peritoneal macrophages from CatE–/– mice compared with those from the wild-type cells (Table II). In contrast, no significant differences in the intracellular levels (at protein and activity levels) of cathepsins B and D were observed between wild-type and CatE–/– DCs. Similarly, while the intracellular level of cathepsin S in both myeloid and peritoneal macrophages was significantly decreased by cathepsin E deficiency, whereas that in DCs was not affected in the absence of cathepsin E. We additionally determined the intracellular and extracellular levels of soluble lysosomal glycosidases, such as {alpha}-mannosidase, beta-glucuronidase, and beta-hexosaminidase, in each of these cell types. The activity levels of these enzymes in the cell lysates of macrophages and DCs from CatE–/– mice were significantly lower than those in the corresponding wild-type cells. Inversely, the activity levels of these enzymes in the culture supernatants of macrophages and DCs from CatE–/– mice were significantly higher than those in the respective wild-type cells (Table III). These results indicate that the extracellular secretion of lysosomal glycosidases is enhanced by cathepsin E deficiency in both macrophages and DCs, whereas that of cathepsins is enhanced only by macrophages.


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  		Table I. The effect of cathepsin E deficiency on the intracellular protein levels of cathepsins D and B in mature DC, LPS-treated and nontreated macrophages from bone marrow precursors, and peritoneal macrophages from wild-type and CatE–/– micea

 

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  		Table II. Activity levels of soluble lysosomal proteinases in the cell lysates of APC from CatE–/– mice compared with those from wild-type littermatesa

 

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  		Table III. Intracellular and extracellular activity levels of soluble lysosomal glycosidases in the cell lysates of APC from CatE–/– mice compared with those from wild-type littermatesa

 
Effect of cathepsin E deficiency on the cellular levels of major lysosomal-membrane sialoglycoproteins

We previously demonstrated that cathepsin E deficiency resulted in a significant accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2, which represent >50% of the total membrane proteins of endolysosomes (44, 45) and their glycosylation constitutes ~60% of the total mass of the respective molecules (45) in mouse peritoneal macrophages (37). We thus determined whether cathepsin E deficiency would also induce the accumulation of these membrane proteins in DCs. SDS-PAGE and immunoblot analysis revealed that, while the cellular levels of LAMP-1 and LAMP-2 were significantly increased in both LPS-treated and nontreated CatE–/– macrophages derived from the bone marrow, like CatE–/– peritoneal macrophages, those in DCs were not affected by cathepsin E deficiency (Table IV). These observations were further confirmed by flow cytometric analysis for the respective permeabilized cells (Fig. 1).


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  		Table IV. The effect of cathepsin E deficiency on the intracellular levels of LAMP-1 and LAMP-2 in APCa

 

Figure 1
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  		FIGURE 1. Flow cytometry analysis of LAMP-1 and LAMP-2 expression in each of the cell types from wild-type and CatE–/– mice. The cells were permeabilized with 0.03% saponin and then stained for determination of the total expression levels of these two proteins. Data are representative of four independent experiments.

 
Effect of cathepsin E deficiency on lysosomal pH

Accumulating evidence has indicated that the accumulation of undegraded metabolites in lysosomal compartments results in an elevated lysosomal pH (46). Recent studies have also implicated the accumulation of lysosomal membrane proteins involved in the elevation of lysosomal pH (47, 48). Furthermore, we have more recently reported the marked elevation of lysosomal pH in CatE–/– peritoneal macrophages, which are accompanied by the accumulation of the lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2 (37). To assess whether myeloid macrophages manifesting the accumulation of LAMP-1 and LAMP-2, like peritoneal macrophages, exhibited the enhanced lysosomal pH and whether the lysosomal pH in DCs was also affected by cathepsin E deficiency, we analyzed lysosomal pH in each of these cell types using the acidotropic fluorescent probe Lysosensor yellow/blue dextran. As described previously and confirmed in Table V, lysosomal pH in the wild-type macrophages and DC derived from bone marrow, as well as the wild-type peritoneal macrophages, was estimated to be 5.3–5.5, whereas the lysosomal pH in both types of cathepsin E-deficient macrophages was raised to 6.4–6.5. In contrast, the lysosomal pH of DCs was not altered by cathepsin E deficiency. The results strongly suggest that the elevated lysosomal pH in myeloid macrophages, like peritoneal macrophages, is induced as a result of the accumulation of lysosomal-membrane sialoglycoproteins by cathepsin E deficiency.


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  		Table V. Effect of cathepsin E deficiency on lysosomal pH in APC from wild-type and CatE–/– micea

 
Effect of cathepsin E deficiency on the destructive potential and chemotactic response of APC

In addressing the role of cathepsin E in each cell type of APC, it is important to determine whether or to what extent cathepsin E deficiency affects functions of each of these cell types. The destructive potential and chemotactic ability of APC is critical to host defense against foreign Ags and pathogens. We thus determined the effect of cathepsin E deficiency on the ability of DCs and macrophages derived from bone marrow precursors of wild-type and CatE–/– mice to digest phagocytosed OVA and respond to chemoattractants. The ability of CatE–/– macrophages to digest phagocytosed OVA was significantly lower than that of the wild-type cells over a period of 3–6 h incubation, whereas that of DCs was not affected by cathepsin E deficiency (Fig. 2A). The chemotactic responses to MCP-1 and fMLP of macrophages were significantly decreased by cathepsin E deficiency, whereas there were no significant differences in the responses between wild-type and CatE–/– DCs (Fig. 2B). Although the migratory response of each of two cell types to both MCP-1 and fMLP appeared to be time and dose dependent (data not shown), the effect of cathepsin E deficiency on each cell type was the same as that described above. The results thus indicate that cathepsin E deficiency induces the disorder of both destructive potential and chemotaxis in macrophages but not in DCs.


Figure 2
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  		FIGURE 2. Effect of cathepsin E deficiency on the destructive potential and the chemotactic responses to MCP-1 and fMLP of APC from wild-type and CatE–/– mice. A, Each of the cell types from wild-type (solid) and CatE–/– (dotted) mice was incubated with DQ-OVA for 1 h at 37°C, subsequently washed extensively with PBS, and then incubated under 5% CO2 at 37°C for the indicated times. Degradation of phagocytosed OVA was measured by increase in the fluorescence in the supernatant at an emission wavelength of 505 nm with excitation at 515 nm. *, p < 0.01; **, p < 0.001 vs the wild-type cells. B, Chemotactic activity was determined using a Boyden chamber. fMLP (1 µM) and MCP-1 (0.1 nM) were put into the lower well of a cell culture inserts. Each cell suspension (2 x 105 cells/ml) was put into the upper well. After incubation for 90 min at 37°C, the inserted membrane filters were washed, fixed, and stained with May-Giemsa, and were mounted on the glass slides. The cells migrating through the filter were counted under a light microscope in a high-power field (x400). The data are the mean ± SD of values from six independent experiments wild-type ({blacksquare}), CatE–/– ({square}). *, p < 0.001 vs the wild-type cells.

 
Effect of cathepsin E deficiency on OVA presentation in APC

We have previously shown that the digested fragments from native OVA by treatment with purified cathepsins D and E at pH 5.5, when added to the coculture system consisting of OVA-specific T cell hybridomas and fixed mouse microglia, can activate the T cell hybridomas to generate IL-2 (23). Previous studies have also examined the ability of cathepsin D to present OVA by splenocytes (25) or microglia (23) prepared from cathepsin D-deficient mice and revealed that the lack of cathepsin D did not abrogate the capacity of these APC to present Ags. However, it remains to be answered whether cathepsin E is involved in the MHC class II Ag presentation. In addition, the previous studies did not specifically investigate DCs from these mice. We therefore assessed the ability of wild-type and CatE–/– DCs, as well as wild-type and CatE–/– macrophages, to process and present OVA to OVA-specific T cell hybridomas. The presentation of OVA and OVA(266–281) peptide to OVA-specific Th cell hybridomas was dose-dependently diminished in CatE–/– macrophages compared with the wild-type cells (Fig. 3B). Unexpectedly, the presentation of OVA and OVA(266–281) peptide was inversely enhanced in CatE–/– DCs compared with the wild-type cells (Fig. 3A).


Figure 3
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  		FIGURE 3. The effect of cathepsin E deficiency on the ability of APC to present intact OVA and its antigenic (266–281) peptide to OVA-specific T cell hybridomas. DCs (A) and macrophages (B) derived from bone marrow precursors of wild-type ({blacksquare}) and CatE–/– mice ({square}) were cultured at a density of 3 x 104 cells/well with intact OVA and its antigenic peptide at the indicated concentrations, together with OVA-specific T cell hybridomas. After 24 h, the culture medium was harvested, and T cell-derived IL-2 was measured by ELISA. The data are the mean ± SD of values from five independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs the respective wild-type cells.

 
Effect of cathepsin E deficiency on phagocytic potential of APC

To shed light on why the presentation of OVA as well as OVA(266–281) peptide was enhanced in CatE–/– DCs, we further explored the phagocytic activity and the expression of costimulatory molecules required for optimal activation of T cells. Phagocytosis is a fundamental process associated with immune responses of APC. iDC are known to effectively internalize and process a variety of particulate Ags (3). Although macropinocytosis of soluble Ags is diminished following DC activation, subsets of DCs in activated DC populations retain the ability to actively phagocytose particulate Ags (49). We thus assessed the effect of cathepsin E deficiency on phagocytic capacity of immature and mature DCs, as well as LPS-activated and nonactivated myeloid macrophages. Each of these cell types was incubated with uniform fluorescent latex particles and then subjected to a cytofluorimetric analysis to quantitatively measure internalized particles. As shown in Fig. 4, iDCs were found to efficiently internalize latex particles at 1-h postincubation at 37°C. The phagocytic capacity of iDCs was somewhat increased by cathepsin E deficiency. Phagocytosis of latex particles was significantly diminished in mature DCs activated with LPS compared with iDCs. However, the phagocytic capacity of LPS-activated DC was also enhanced by cathepsin E deficiency. In contrast, the phagocytic capacity of both LSP-activated and nonactivated macrophages derived from bone marrow precursor cells was not changed by cathepsin E deficiency.


Figure 4
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  		FIGURE 4. The effect of cathepsin E deficiency on the phagocytic activity of APC toward fluorescent latex particles. Immature and mature DCs, as well as LPS-treated and nontreated macrophages, derived from bone marrow precursors of wild-type (solid) and CatE–/– (dotted) mice (107 cells/ml for each) were incubated with fluorescent latex particles (108 particles/ml) in PBS for 1 h at 37°C in a shaking water bath. Then, the cells were separated from nonphagocytosed particles by low-speed centrifugation, washed twice with PBS, and resuspended in the 500 µl of cold PBS. Internalized particles were analyzed by flow cytometry. Data are representative of four independent experiments.

 
Effect of cathepsin E deficiency on cell surface expression of costimulatory molecules in APC

It is generally accepted that T cell requires costimulatory signals for optimal activation (50, 51, 52, 53, 54). The major costimulatory molecules expressed on activated APC are CD80 (B7-1), CD86 (B7-2), and CD40, which interact with the CD80/CD86 receptor (CD28) and the CD40L (CD154), respectively, on T cells. Therefore, we investigated the effect of cathepsin E deficiency on the expression of the costimulatory molecules CD80, CD86, and CD40, as well as MHC class II molecules, on the cell surface of DCs and macrophages derived from bone marrow precursor cells. Although the cell surface expression of CD80, CD86, and CD40, as well as MHC class II molecules, on iDCs was not significantly changed by cathepsin E deficiency, mature DCs exhibit the enhanced expression of these costimulatory molecules in the absence of cathepsin E (Fig. 5). In contrast, although the cell surface expression of the costimulatory molecules in macrophages was enhanced by LPS activation, no significant difference in their expression was observed between wild-type and CatE–/– macrophages. The expression of MHC class II molecules in both DCs and macrophages was not significantly affected by cathepsin E deficiency.


Figure 5
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  		FIGURE 5. The effect of cathepsin E deficiency on the expression of costimulatory molecules in APC. iDCs and mature DCs, as well as LPS-treated and nontreated macrophages, derived from bone marrow precursors of wild-type and CatE–/– mice (2 x 105 cells/ml for each) were stained for cell surface expression of CD80, CD86, and CD40, as well as MHC class II molecules, with the respective Abs and then analyzed by flow cytometry. Data are representative of five independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
To our knowledge, this is the first study indicating that cathepsin E plays a different role in the regulation of the nature and functions for DCs and macrophages using CatE–/– mice. Cathepsin E deficiency was initially characterized by the enhanced extracellular secretion of soluble lysosomal enzymes such as cathepsins B and D and the intracellular accumulation of the two major lysosomal membrane sialoglycoprotein LAMP-1 and LAMP-2 in myeloid and peritoneal macrophages, suggesting that cathepsin E is critical to determine the nature of macrophages. Our previous study with peritoneal macrophages demonstrated that cathepsin E deficiency resulted in the significant accumulation of the two major lysosomal membrane sialoglycoproteins LAMP-1 and LAMP-2, which were selectively degraded by cathepsin E, and the elevation of lysosomal pH (37). However, because cathepsin E deficiency did not affect the proton V-ATPase in macrophages, the elevation of lysosomal pH in cathepsin E-deficient macrophages was thought to be caused by the disorder of the nonproton factor luminal polyelectrolyte matrix (possibly Donnan’s membrane equilibrium) as a result of the accumulation of these membrane sialoglycoproteins highly modified with acidic monosaccharides (37). Because acidic lysosomal pH is critical for the maintenance of the structural and functional integrity of APC and the normal processing and trafficking of lysosomal enzymes, the elevation of lysosomal pH appears to interfere with the maturation and fusion events of the organelles involved. Functional disorders, such as the decreased destructive potential against phagocytosed OVA and the impaired chemotactic responses to MCP-1 and fMLP, were observed with CatE–/– macrophages. Accordingly, the altered nature and functions observed with both types of CatE–/– macrophages are directly associated with the accumulation of LAMP-1 and LAMP-2 by cathepsin E deficiency followed by the elevation of lysosomal pH. In contrast, cathepsin E deficiency did not cause significant changes in biochemical properties of DCs, including intracellular levels of soluble lysosomal cathepsins B, D, and S, and the membrane proteins LAMP-1 and LAMP-2, and the lysosomal pH. In addition, there were no significant differences in the destructive potential for phagocytosed protein and the chemotactic responses to MCP-1 and fMLP between wild-type and CatE–/– DCs.

Importantly, we here demonstrated that there was a marked difference in the capacity for presenting the Ag OVA between CatE–/– macrophages and CatE–/– DCs. To date, pharmacologic and genetic ablation of lysosomal proteases using more specific chemical inhibitors and protease gene-targeted mice has suggested that individual proteases make both constructive and destructive contributions to Ag processing in the MHC class II-restricted pathway (7, 8). In particular, recent analysis of mice deficient in lysosomal cysteine proteases (e.g., cathepsins B, L, and S) and the lysosomal aspartic proteinase cathepsin D have revealed that specific lysosomal proteases are required for the presentation of the appropriate peptide-MHC class II repertoire by distinct cell types of APC (7, 8). Given that Ag processing is a key determinant of the quality and quantity of a CD4 T cell response, it is of particular importance to identify the specific enzymes that generate MHC class II-bound peptides in vivo. Although previous studies indicated that the aspartic proteinases cathepsins D and/or E would be required for MHC class II peptide presentation (19, 20, 22), subsequent studies with APC deficient in cathepsin D demonstrated that this protein would be dispensable for this pathway (23, 25, 26). However, it remains unclear whether cathepsin E is essential for MHC class II Ag presentation. Because cathepsin E exists in APC endosomes as the mature enzyme (23, 27), and because cathepsin E expression is increased upon stimulation with IFN-{gamma} and LPS, known to induce MHC class II expression and costimulatory molecules (55), it is relevant to determine to what extent cathepsin E activity influences MHC class II-restricted Ag presentation. The present results showed for the first time that cathepsin E deficiency strongly affected the presentation of OVA by macrophages and DCs to its specific T cell hybridomas, implying that cathepsin E is involved in antigenic presentation. Surprisingly, however, while the ability of macrophages to present OVA and its peptide to T cells was markedly decreased by cathepsin E deficiency, that of DCs was inversely enhanced by the absence of cathepsin E. Recently, Moss et al. (10) reported that the presentation of two different myoglobin T cell epitopes in DCs was enhanced rather than hindered by the lack of cathepsin D. They also demonstrated that the residual processing activity found in the subcellular fraction of DCs deficient in cathepsin D was completely inhibited by pepstatin, thus suggesting that aspartic proteases besides cathepsin D expressed in DCs could be involved in myoglobin Ag presentation and that the reduced activity by cathepsin D deficiency would produce optimal conditions for its processing and presentation. Chain et al. (24) also reported that the ability of DCs from wild-type and cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells was completely blocked by the microbial aspartic proteinase inhibitor pepstatin-conjugated to mannosylated BSA, which could inhibit cathepsin D/E activity within the cells. Taken together, our data strongly suggest that reduced aspartic proteinase activities resulting from the absence of cathepsin E in DCs are closer to the optimum level required for OVA Ag presentation. However, given that cathepsin E deficiency did not affect the ability of DCs, as well as macrophages, to present not only OVA but also its antigenic peptide, it seems likely that cathepsin E is not directly involved in Ag processing in these cells and rather plays a crucial role in controlling the endosomal/lysosomal microenvironment and protein sorting in these compartments.

Our present results also demonstrate that the enhanced OVA peptide presentation in DCs by cathepsin E deficiency is due, in part, to the enhanced phagocytic activity and the increased expression of the costimulatory molecules CD86, CD80, and CD40, which can amplify the response of T cells. It is generally accepted that iDCs efficiently internalize, process, and present a variety of particulate Ags, thereby serving as sentinels to immunologic threats (3, 49). Therefore, the enhanced phagocytic activity of CatE–/– DCs appeared to contribute to the stimulation of their OVA peptide presentation. It is also established that T cells require costimulatory signals for optimal activation (50, 51, 52, 53, 54). When costimulatory signals are delivered by interactions of costimulatory molecules on APC with their ligands on T cells, Ag presentation can induce productive lymphocyte activation (52, 53, 54). Of the multiple costimulatory signals, the CD80/CD86-CD28 and CD40-CD154 interactions are thought to be a pivotal role in the process of T cell priming by DCs (53). Therefore, our data indicating the increased expression of CD80, CD86, and CD40 on CatE–/– DCs is most likely to contribute to the enhanced T cell activation. At present, although it still remains to be established how cathepsin E deficiency can enhance the phagocytic activity and the expression of costimulatory molecules in DCs, the present results provide new insight into the functional diversity of cathepsin E in immune responses.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Current address: Department of Pharmaceutical Care and Health Sciences, Fukuoka University Pharmaceutical Sciences, Jonan-ku, Fukuoka 814-0181, Japan. Back

2 Current address: Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8588, Japan. Back

3 Address correspondence and reprint requests to Dr. Kenji Yamamoto, Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka 812-8582, Japan. E-mail address: kyama{at}dent.kyushu-u.ac.jp Back

4 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; Ii, invariant chain; LAMP, lysosome-associated membrane protein. Back

Received for publication May 23, 2007. Accepted for publication August 15, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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