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International Immunology Advance Access originally published online on April 24, 2006
International Immunology 2006 18(6):931-939; doi:10.1093/intimm/dxl029
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis

Rajeev M Nepal1, Stephanie Mampe1, Brian Shaffer1, Ann H Erickson2 and Paula Bryant1

1 Department of Microbiology, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA
2 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA

Correspondence to: P. Bryant; E-mail: bryant.218{at}osu.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L—three major lysosomal cysteine proteases—are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFN{gamma}-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading.

Keywords: antigen-presenting cell, class II MHC, cysteine protease, endocytic pathway


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The infection of immunocompetent individuals with Mycobacterium tuberculosis elicits a class II MHC-restricted, CD4+ T cell response, which is critical to control the primary infection. Despite this effective response, the pathogen is not eradicated and instead persists within macrophages (1, 2). Alveolar macrophages are the primary target cells for M. tuberculosis invasion and replication in the lung. Although dendritic cells (DCs) may prime naive CD4+ T cells in the draining mediastinal lymph nodes, alveolar and interstitial macrophages likely function in the presentation of mycobacterial antigens to effector CD4+ T cells subsequently recruited to the infected lung. Thus, the integrity of the class II MHC antigen-presentation pathway in M. tuberculosis-infected macrophages is of clear importance to ensure that the pathogen is contained and protective immunity established. The ‘potency’ of macrophages as antigen-presenting cells (APCs) is greatly enhanced upon exposure to IFN{gamma}, which increases the expression of both class II MHC molecules and co-stimulatory molecules (3, 4). Moreover, the activation of macrophages is critical for the expression and/or activity of several key accessory molecules required to load class II molecules with peptides, including the chaperone invariant chain (Ii), H-2DM and several endocytic proteases (cathepsins) (5).

Endocytic proteases are required in two different steps of class II MHC-restricted antigen presentation: antigen degradation and Ii processing (6). Normally, intracellular pathogens are sequestered by macrophages into phagocytic vesicles that undergo sequential fusion events with early-to-late endocytic compartments, thereby exposing the contents of the phagosome to acidic proteases that are required to degrade pathogen-derived antigens into class II-presentable peptides (7). To ensure that class II molecules intersect these peptides, the chaperone Ii associates with newly synthesized class II {alpha}ß-dimers in the endoplasmic reticulum and delivers them directly to the same endocytic compartments (8–10). As class II–Ii complexes traverse the endocytic route, aspartic and cysteine proteases degrade Ii in a stepwise fashion via a series of defined cleavage intermediates (i.e. Iip22 and Iip10) leaving the class II-associated invariant chain peptide (CLIP) portion of Ii in the peptide-binding cleft of the {alpha}ß-dimer (6, 11). CLIP is exchanged for resident antigenic peptides in a reaction that is catalyzed by the accessory molecule, DM (5). Effective antigen presentation by class II MHC molecules is therefore dependent on the collection of proteases to which class II–Ii complexes and phagocytosed pathogens are exposed.

Professional APCs are equipped with a diverse set of cathepsins, most of which contain a cysteine as the attacking nucleophile in the catalytic cleft (6, 11). The major cysteine proteases expressed in professional APCs—cathepsin (Cat) B, Cat S and Cat L—have all been shown to participate in the processing of internalized antigens into class II-presentable T cell epitopes (12–14). In addition, the key cysteine proteases required for Ii degradation have been defined. Asparagine endopeptidase (AEP) was shown capable of initiating Ii breakdown to yield {alpha}ß-Iip22, although other proteases can perform this cleavage in its absence (15). The rate-limiting step of Ii proteolysis—conversion of {alpha}ß-Iip10 into {alpha}ß-CLIP—is mediated by Cat S in professional APCs (B cells, DCs and macrophages) (16, 17) and by Cat L in cortical thymic epithelial cells (18). While macrophages express both Cat S and Cat L, Cat L does not appear to participate in the cleavage of Iip10 into CLIP in IFN{gamma}-activated macrophages (19, 20). Macrophages also express Cat F, which can cleave Iip10 into CLIP in vitro, and thus may participate in Ii breakdown in the absence of Cat S (19).

The success of M. tuberculosis and Mycobacterium avium as pathogens is owed in part to the evolvement of mechanisms that limit the communication of their phagosomes with the late endocytic organelles containing these hydrolytic enzymes in macrophages (21–25). In addition, Mycobacterium spp. (including avirulent M. tuberculosis strain H37Ra, virulent M. tuberculosis strains H37Rv and Erdman, M. avium and BCG) has developed mechanisms to modulate class II MHC expression and antigen presentation (26–36). Nonetheless, the CD4+ T cell response elicited against pathogenic Mycobacterium spp. infection—although not eradicating—proves that the pathogen and/or its products do indeed intersect cathepsins and class II MHC molecules (32, 37). However, the endocytic proteases available to the infected macrophage to process antigens derived from the pathogen into CD4+ T cell epitopes have not been defined. Therefore, our goal in this present study was to establish a profile of the active cathepsins expressed in the M. avium- and M. tuberculosis-infected macrophages. We focused on the activities of Cat B, -S and -L, as all three enzymes are involved in the processing of antigen and/or Ii. Murine bone marrow-derived macrophages (BM-macs) generated with recombinant granulocyte macrophage colony-stimulating factor (rGM-CSF) were chosen as the host cells for infection, as Cat B, -S and -L are each active in these cells, and appreciable numbers of these macrophages (required for the biochemical studies employed) are readily obtainable (38, 39). Our data show that the activity of mature Cat L is modulated in M. avium- and M. tuberculosis-infected BM-macs, whereas Cat B and Cat S activities remain largely unchanged. Specifically, maturation of the active single-chain form of Cat L into the active two-chain forms of the enzyme is severely impaired in macrophages harboring either M. avium or M. tuberculosis.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Mice
C57BL/6 were purchased from Taconic Laboratories. Cat B–/– and Cat S–/– mice have been described (17, 40), and were bred and maintained in microisolator cages at Ohio State University.

Bacteria
Mycobacterium avium (35713; American Type Tissue Collection) and M. tuberculosis Erdman were grown to log phase in Middlebrook 7H9 broth (DIFCO, Detroit, MI, USA) supplemented with 1% glycerol, 0.05% Tween (Sigma–Aldrich, St Louis, MO, USA) and 10% Middlebrrok oleic albumin dextrose catalase enrichment (DIFCO). Serial dilutions of the bacterial cultures were plated on Middlebrook 7H11 agar and the bacterial titer for each was determined by counting the resulting colony-forming units (CFUs). Aliquots of the titered bacterial cultures were frozen and stored at –80°C. Prior to infection of BM-macs, bacterial aliquots were thawed, pelleted, washed in infection media (antibiotic-free RPMI supplemented with 10% heat-treated FCS), and any large clumps were removed by two 20-s rounds of sonication, followed by six passes through a 25-gauge needle. A single-cell bacterial suspension was confirmed by microscopy. Heat-killed (HK) M. avium were prepared by incubating the live bacilli at 80°C for 60 min, and successful killing was confirmed by the absence of CFUs. Bacterial dilutions for infection were calculated based on the titer of the frozen stock.

Preparation of macrophages
BM was harvested from the femurs of 8- to 12-week old mice as described (41), and differentiated into macrophages by culturing the BM precursors in bacterial grade dishes for 6 days in RPMI 1640 + 10% FCS + 10 ng ml–1 recombinant murine GM-CSF (PeproTech, Rocky Hill, NJ, USA). On days 3 and 5 of the culture period, 70% of the culture supernatant containing non-adherent cells was removed and replaced with fresh media containing 10 ng ml–1 rGM-CSF. On day 6, the loosely adherent and non-adherent [representing granulocytes and DCs (39)] cells were removed by vigorous washing. The remaining adherent macrophage population was harvested by incubating the cells for 5 min in PBS + 10 mM EDTA followed by gentle scraping with rubber policemen (Starstedt, Newton, NC, USA). The harvested, viable (assessed by trypan blue exclusion) BM-macs were then plated at a density of ~1 x 106 cells ml–1 (~4 x 106 per well) in RPMI 1640 lacking rGM-CSF in tissue culture-treated six-well plates in the presence or absence of 100 U ml–1 of recombinant mouse IFN{gamma} (PeproTech) for 24–48 h when appropriate.

Mycobacterium avium and M. tuberculosis infection of BM-macs
Unstimulated or IFN{gamma}-stimulated (24 h) BM-macs were washed four times in infection media (antibiotic-free RPMI + 10% heat-inactivated FCS), and then mock infected or infected by incubating the cells with the prepared clump-free suspension of live M. avium (20:1) or M. tuberculosis (20:1) for 2 h at 37°C. As a control for phagocytosis, the BM-macs were incubated with polystyrene beads (500:1; Sigma–Aldrich) for 2 h at 37°C. The non-phagocytosed bacteria or beads were removed by four washes in infection medium, and the cells were cultured for an additional 20–24 h in the presence or absence of 100 U ml–1 of IFN{gamma}. Acid-fast staining revealed that a multiplicity of infection (MOI) of 20 bacilli to one BM-mac resulted in the infection of >85% of the total BM-macs plated, with two to three bacilli phagocytosed per cell. The viability of the infected cells was confirmed by trypan blue exclusion. At 24 h post-infection, the cells were lysed and used for the active-site labeling and immunoblotting experiments, or subjected to pulse-chase analysis (described below).

Immunoblotting
BM-macs were lysed in NP-40 lysis buffer (50 mM sodium acetate, pH 5, 5 mM MgCl2, 0.5% NP-40) and the concentration of protein in the lysates was determined using a BCA Protein Assay Reagent (bicinchoninic acid) (Pierce, Rockford, IL, USA). Twenty micrograms of each lysate was boiled for 5 min in the presence of reducing SDS sample buffer, and then electrophoresed on a 12.5% polyacrylamide gel. The separated proteins were transferred to a nitrocellulose membrane and blocked in 10% non-fat milk. The membranes were probed with a 1/20 000 dilution of a rabbit antiserum against pro-Cat L (42), followed by a 1/5000 dilution of a secondary anti-rabbit IgG antibody coupled to peroxidase (PharMingen, San Diego, CA, USA). Chemiluminescense followed by autoradiography was used for visualization of the Cat L species.

Active-site labeling experiments
JPM-565biotin was a generous gift from H. Ploegh (Harvard Medical School, Boston, MA, USA). Uninfected, bead-pulsed and Mycobacterium-infected macrophages were lysed at pH 5 for 1 h on ice, the nuclei pelleted, and the protein concentration of the lysates was determined using a BCA kit (Pierce, Rockford, IL, USA). Twenty micrograms of each lysate was incubated with 50 µM JPM-565biotin for 1 h at 37°C. The reaction was terminated by the addition of SDS-reducing sample buffer and boiling. The labeled proteins in the lysates were separated by 12.5% SDS-PAGE and transferred to nitrocellulose. To ensure adequate separation of Cat B, Cat S and Cat L—specifically the two-chain forms (25 kDa and 24 kDa) of the latter—the dye front was allowed to run off the gel for 1 h, until the 14-K molecular weight marker (Amersham, Piscataway, NJ, USA) reached the bottom of the gel. The membranes were incubated with streptavidin–HRP diluted in PBS + 0.2% Tween 20 at room temperature, and the JPM-565biotin-labeled polypeptides visualized by chemiluminescense (Amersham, UK) followed by autoradiography. Alternatively, to examine JPM-565biotin-labeled Cat L species independently of other labeled cysteine proteases, the labeling reaction was terminated by boiling in 1% SDS without 2-mercaptoethanol, diluted 10-fold in pH 5 lysis mix and the labeled Cat L species immunoprecipitated with the Cat L-specific polyclonal rabbit antiserum. The immunoprecipitates were collected on Staph A, solubilized by boiling in reducing SDS sample buffer, and analyzed by SDS-PAGE followed by blotting with streptavidin–HRP as described above.

Pulse-chase analysis
Uninfected, bead-pulsed, M. avium-infected and M. tuberculosis-infected BM-macs (±IFN{gamma}) were starved for 45 min at 37°C in 1 ml of cysteine-/methionine-free RPMI (GIBCO, Carlsbad, CA, USA) supplemented with 2 mM glutamine and 10% heat-inactivated FCS. Cells were pulsed with 0.5 mCi ml–1 of 35S-methionine/cysteine (PerkinElmer, Boston, MA, USA) for 45 min and chased in complete RPMI for 0, 3 or 24 h. After each chase point, the cells were lysed in NP-40 lysis buffer, pH 7.4 (50 mM Tris, 150 mM NaCl, 0.5% NP-40) supplemented with 1% SDS, boiled for 5 min and reconstituted to 1.2 ml with SDS-free NP-40 solution. The lysates were pre-cleared, and then immunoprecipitated as described (39, 43) using 5 µl of Cat L antiserum, and analyzed under reducing conditions by 12.5% SDS-PAGE.

Detection of secreted Cat L
Equal numbers of unstimulated and IFN{gamma}-stimulated BM-macs were either mock infected or infected with M. avium for 2 h as described above, washed and cultured in serum-free RPMI medium for an additional 24 h. Supernatants were harvested, centrifuged (5 min, 200 x g) and normalized for protein concentration. The culture supernatants (20 µg) were separated by reducing SDS-PAGE, transferred to nitrocellulose and probed with the anti-Cat L serum as described above.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Mycobacterium avium and M. tuberculosis infection alters Cat L activity in macrophages
We began our studies by comparing the steady-state activities of cysteine proteases in uninfected, M. avium- and M. tuberculosis-infected macrophages. To do so, the affinity-labeled active site-directed probe, JPM-565biotin, was employed. JPM-565biotin covalently modifies the active site of cysteine proteases—predominantly Cat B, Cat S and Cat L—when added to cell lysates (pH 5) (44). Covalent modification by JPM-565biotin is mechanism based, and therefore reflects the enzymatic activities of the targeted protease (44). IFN{gamma}-stimulated (24 h) B6 and Cat B–/– BM-macs were infected with viable M. avium (20:1) or M. tuberculosis (20:1) for 2 h. The non-phagocytosed bacteria were removed by washing, and the cells incubated for an additional 24 h in the presence of IFN{gamma}. As a control for phagocytosis, cells were either mock infected or incubated with latex beads (500:1) under the same conditions. Lysates (pH 5) were generated from the uninfected and infected BM-macs and labeled with JPM-565biotin. A representative experiment (n = >5) is shown in Fig. 1.


Figure 1
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  		Fig. 1 Activity of Cat L in Mycobacterium avium- and Mycobacterium tuberculosis-infected BM-macs. IFN{gamma}-stimulated BM-macs were mock infected or infected with (A–C) viable M. avium (20:1), or (D) viable M. tuberculosis (20:1) for 2 h and washed. At 24 h post-infection, 20 µg of cell lysate (pH 5) prepared from each was labeled with JPM-565biotin, analyzed by reducing SDS-PAGE (12.5%) and visualized as described in Methods. (B and C) The JPM-565biotin-labeled Cat L species present in the cell lysates prepared in (A) were immunoprecipitated with a polyclonal antiserum specific for the distinct maturation forms of Cat L prior to analysis by SDS-PAGE. (C) Represents a longer exposure of (B).

 
JPM-565biotin labeled active Cat B, Cat S and Cat L in lysates prepared from uninfected B6 BM-macs (Fig. 1A), and appropriately, only active Cat S and Cat L were detected in Cat B-deficient cell lysates (Fig. 1D). The additional labeled species with a slower mobility than Cat B is likely Cat Z (Fig. 1A and D) (38). Consistent with previous findings (39), the predominant active forms of Cat L detected in lysates generated from IFN{gamma}-stimulated BM-macs (uninfected) were the mature ‘two-chain’ forms of the enzyme, Cat L-25K and -24K (specifically Cat L-24K) (Fig. 1A and D). Upon delivery of pro-Cat L (38 kDa) to the endocytic pathway, the pro-piece is removed to yield the single-chain (30 kDa) mature active form. As Cat L traverses the endocytic route, the single-chain enzyme is further cleaved to generate the two-chain mature forms composed of either a 25-kDa or 24-kDa heavy chain linked to a 5-kDa light chain by disulfide bonds (45–48). Cat H was shown to migrate to a similar position above Cat S in GM-CSF-derived BM-macs as single-chain Cat L (38), and thus likely contributes to the JPM-565biotin-labeled species seen at ~30-kDa (Fig. 1A and D).

Upon infection with either M. avium (Fig. 1A) or M. tuberculosis (Fig. 1D), the activities of mature Cat B and mature Cat S remained largely unchanged. In contrast, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected macrophages, cells infected with either M. avium or M. tuberculosis exhibited increased levels of the active 25-kDa two-chain form of the enzyme (Fig. 1A and D). To clarify the effect of infection on Cat L activity, we isolated the Cat L polypeptides labeled by JPM-565biotin by immunoprecipitation with a polyclonal antiserum against Cat L prior to separation by SDS-PAGE and blotting with streptavidin–HRP (Fig. 1B and C). These results clearly demonstrated that the steady-state levels of active Cat L-25K (Fig. 1B) as well as the precursor single-chain (30 kDa) enzyme (seen after a long exposure shown in Fig. 1C) were greater in M. avium-infected macrophages, with a concomitant decrease in Cat L-24K activity. This alteration in Cat L activity was not merely an artifact of phagocytosis, as the uptake of inert polystyrene beads by macrophages had no effect (data not shown, see Fig. 3B). Thus, the predominant lysosomal form of active Cat L at steady state is distinct between uninfected and BM-macs infected with either M. avium or M. tuberculosis.


Figure 3
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  		Fig. 3 Cat L maturation in BM-macs infected with live Mycobacterium tuberculosis versus HK bacilli. (A) IFN{gamma}-stimulated B6 and Cat B–/– BM-macs were infected with viable, virulent M. tuberculosis (20:1) for 2 h and the non-phagocytosed bacteria removed. At 24 h post-infection, the cells were pulsed with 35S-met/cys for 45 min and chased for 24 h. Cat L was immunoprecipitated from labeled, normalized, cell lysates with the polyclonal antiserum and analyzed by reducing SDS-PAGE (12.5%). (B) IFN{gamma}-stimulated B6 and Cat B–/– BM-macs were incubated with viable Mycobacterium avium (20:1), HK (20:1) M. avium or latex beads for 2 h, and washed. At 24 h post-infection, the cells were pulsed with 35S-met/cys for 45 min and chased in complete medium for 24 h. Radiolabeled Cat L was immunoprecipitated from prepared lysates and analyzed by reducing SDS-PAGE (12.5%). (A) and (B) represent separate gels.

 
The maturation of Cat L is impaired in M. avium- and M. tuberculosis-infected macrophages
The active-site labeling experiments described above measured the steady-state levels of active Cat L, and did not discern between Cat L molecules synthesized prior to the start of infection from those nascent Cat L molecules synthesized in the presence of the pathogens. In addition, since the maturation of Cat L into the two-chain 24-kDa form is facilitated in IFN{gamma}-activated macrophages, the decreased activity of Cat L-24K in infected cells may reflect the described ability of M. avium and M. tuberculosis to interfere with IFN{gamma}-signaling pathways. Thus, we next examined the IFN{gamma}-independent (Fig. 2A) and -dependent (Fig. 2B) maturation of newly synthesized Cat L in M. avium-infected macrophages by biosynthetic labeling followed by immunoprecipitation. After a 45-min pulse, the majority of Cat L in both uninfected and M. avium-infected cells, regardless of IFN{gamma} stimulation, was still in its proform (38 kDa), while a small portion had been converted into the mature single-chain 30-kDa enzyme (Fig. 2A and B). Equivalent levels of pro-Cat L were synthesized in both uninfected and M. avium-infected macrophages.


Figure 2
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  		Fig. 2 Cat L biogenesis and maturation in BM-macs infected with live Mycobacterium avium. Mock-infected and M. avium-infected (A) unstimulated or (B) IFN{gamma}-stimulated B6 and Cat B–/– BM-macs were pulsed 24 h post-infection with 35S-met/cys for 45 min and chased for 0, 4 and 24 h. Cat L was immunoprecipitated from labeled, normalized, cell lysates with the polyclonal antiserum and analyzed by reducing SDS-PAGE (12.5%).

 
After 2 h of chase, the bulk of pro-Cat L was processed into the 30-kDa single-chain mature form, and a portion of Cat L-30K was processed further into the 25-kDa two-chain mature form in uninfected B6, and Cat B–/– macrophages, ±IFN{gamma} (Fig. 2A and B). In contrast, Cat L maturation was impaired beyond the 30-kDa stage in both unstimulated and IFN{gamma}-stimulated macrophages (B6 and Cat B–/–) infected with M. avium, as evidenced by a decreased recovery of Cat L-25K coupled with an increased recovery of Cat L-30K (Fig. 2A and B). The accumulation of Cat L-30K (as well as pro-Cat L) was more prominent in the absence of Cat B, suggesting that Cat B may participate in the processing of Cat L into the two-chain forms. Alternatively, Cat B may function to degrade (turn over) intracellular Cat L, thereby regulating the steady-state pool of active Cat L in the macrophage.

After 24 h of chase, processing of Cat L into its terminal mature two-chain form of 24-kDa was complete in uninfected macrophages (±IFN{gamma}) (Fig. 2A and B). In marked contrast, Cat L did not mature into the 24-kDa two-chain mature form in either unstimulated or IFN{gamma}-stimulated macrophages infected with M. avium (Fig. 2A and B). Identical results were observed in macrophages infected with live M. tuberculosis (Fig. 3A).

Next, we examined whether viable bacilli were required to inhibit full Cat L maturation. B6 and Cat B–/– BM-macs (+IFN{gamma}) were infected with live versus HK M. avium, pulsed with 35S-met/cys for 45 min and the mature forms of Cat L generated after a 24-h chase examined. The phagocytosis of HK M. avium by macrophages disrupted the maturation of pro-Cat L into the two-chain 24-kDa form to the same extent as live bacilli, whereas the phagocytosis of polystyrene beads had no effect (Fig. 3B). These data suggest that a constitutively expressed mycobacterial component is responsible for the disruption in Cat L maturation. Collectively, the pulse-chase analyses confirm the active-site labeling results above, and demonstrate that Cat L does not mature properly in macrophages harboring M. avium and M. tuberculosis, resulting in APCs deficient in the Cat L-24K active form.

Active Cat L is not secreted from M. avium-infected macrophages
Pro-Cat L is actively secreted from a variety of cell types. This secretion is regulated by growth factors, oncogene activation and from elevated synthesis of the protein coupled with a reduced interaction with mannose-6-phosphate receptors (49, 50). More recently, IFN{gamma} stimulation of BM-macs was shown to induce the secretion of the two-chain active forms (but not the single-chain form) of Cat L, which were capable of degrading elastin (51). Although the pulse-chase data suggest that the processing of pro-Cat L into Cat L-24K does not occur in macrophages harboring M. avium or M. tuberculosis, it is possible that the 24-kDa form is generated in infected cells yet is immediately secreted upon synthesis, thereby escaping our detection. We therefore examined the culture supernatants of M. avium-infected macrophages for the presence of secreted pro- and mature Cat L species by immunoblot. As a control, the accumulation of extracellular Cat L was compared with the steady-state levels of intracellular Cat L upon infection of macrophages with viable M. avium. Again, these latter experiments do not distinguish between Cat L molecules synthesized prior to infection already en route to the pericellular space, from nascent Cat L molecules synthesized subsequent to establishment of the infection.

At 24 h post-infection, the effects of M. avium on Cat L maturation were readily apparent in the overall steady-state expression levels of the intracellular enzyme: infected B6 and Cat B–/– BM-macs (±IFN{gamma}) exhibited higher levels of Cat L-30K as compared with uninfected controls, accompanied by a slight decrease in steady-state levels of the two-chain forms (Fig. 4A). Expression and secretion of pro-Cat L is independent of IFN{gamma} stimulation, and our results showed the presence of equal amounts of the Cat L zymogen in supernatants from both untreated and IFN{gamma}-treated cells (Fig. 4B). Mycobacterium avium infection had no effect on secretion of pro-Cat L (Fig. 4B). Consistent with previous reports (51), stimulation of BM-macs with IFN{gamma} resulted in the extracellular accumulation of mature Cat L-25K/24K (not Cat L-30K) (Fig. 4B). In contrast, two-chain mature Cat L was not detected in the culture supernatants of IFN{gamma}-stimulated BM-macs infected with M. avium (Fig. 4B). Thus, these data confirm that Cat L maturation into the two-chain 24-kDa form is inhibited in M. avium- and M. tuberculosis-infected macrophages.


Figure 4
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  		Fig. 4 Intracellular expression and secretion of Cat L from Mycobacterium avium-infected BM-macs. (A) Unstimulated and IFN{gamma}-stimulated B6 and Cat B–/– BM-macs were infected with viable M. avium (20:1) for 2 h, washed and cultured for an additional 24 h. The steady-state intracellular levels of the distinct maturation forms of Cat L were assessed by immunoblot analysis of 20 µg of cell lysate obtained from the mock- and M. avium-infected BM-macs. (B) Supernatants of unstimulated and IFN{gamma}-stimulated B6 BM-macs were collected 24 h after the 2-h mock- or M. avium infection, resolved by SDS-PAGE under reducing conditions, and the extracellular levels of pro- and mature Cat L enzymes assessed by immunoblot.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The activities of cathepsins are indispensable for the professional antigen presentation functions of macrophages, and therefore the CD4+ T cell mediated immune response against the intracellular pathogens M. avium and M. tuberculosis. Our data reveal that Cat L activity is altered in murine BM-macs infected with either pathogen, while the activities of Cat B and Cat S remain largely unchanged. Specifically, maturation of the single-chain Cat L enzyme into the 24-kDa two-chain active form does not occur in M. avium- and M. tuberculosis-infected macrophages. Consequently, infected cells are not only deficient in the predominant lysosomal form of active Cat L but also do not secrete active Cat L enzyme. Mature two-chain Cat L was shown previously to be the major elastin-degrading enzyme at neutral pH secreted from IFN{gamma}-stimulated murine BM-macs (51).

Upon arrival of pro-Cat L in (presumably) late endosomes, its pro-piece is removed to yield the mature, single-chain (30 kDa) active form of the enzyme (45, 48). Studies indicate that intermolecular processing by pro-Cat L initiates when the pH drops to 5.3, suggesting autocatalysis as at least one mechanism by which Cat L-30K is generated (52). The biosynthesis of pro-Cat L and cleavage of the pro-piece to yield the single-chain active enzyme (30 kDa) was not disrupted in M. avium- and M. tuberculosis-infected BM-macs (±IFN{gamma}). In uninfected, GM-CSF-derived BM-macs, the single-chain (30 kDa) enzyme is further cleaved to yield two-chain active Cat L, composed of a heavy-chain (25 kDa/24 kDa) disulfide linked to a light chain (6 kDa) (39). This key-processing step in Cat L biogenesis was significantly impaired in M. avium- and M. tuberculosis-infected cells. Previous biochemical analyses revealed that two-chain Cat L is comprised of either a 25-kDa or 24-kDa heavy chain in GM-CSF-derived BM-macs (39). The data showed that IFN{gamma} stimulation drives maturation of Cat L into Cat L-24K (39, 51). Interestingly, Cat L-24K was not generated in M. avium- and M. tuberculosis-infected macrophages. This impairment of Cat L maturation was observed in both unstimulated and IFN{gamma}-stimulated infected BM-macs, and thus was not merely the result of the pathogen's established ability to interfere with IFN{gamma}-signaling pathways (29–31, 53).

The classic mechanism by which viable pathogenic Mycobacterium spp. avoid destruction by endocytic proteases in resting (i.e. non-activated) macrophages is by blocking the fusion of their phagosomes with the late-endosomal/lysosomal compartments containing the active mature forms of cathepsins. Phagosomes harboring live pathogenic Mycobacteria lack the mannose-6 phosphate receptor, do not actively recruit lysosomal hydrolases and do not accumulate vacuolar H+-ATPase pumps. Consequently, the mycobacterial phagosome is poorly acidified (pH ~6.3) (21, 54). Although the bulk of cathepsins (including Cat L) are delivered directly to late endosomes via the classic mannose-6-phosphate-dependent pathway (45), a fraction of cathepsins may end up in the mycobacterial phagosome via fusion events between the phagosome and early endosomal vesicles. Thus, the biosynthetic form of cathepsins (i.e. Cat D) present in phagosomes containing pathogenic Mycobacterium spp. as well as other intracellular pathogens has served as an indicator of the maturation state, hydrolytic capacity and/or pH of the phagosome itself. As expected, only immature Cat D was found in purified phagosomes containing viable M. avium (significant levels of Cat L were not detected—neither pro or mature) (55, 56). Similarly, pro-Cat L, but not mature Cat L, was detected in phagosomes isolated from macrophages infected with Salmonella tyhimurium, revealing that this pathogen also has the ability to block maturation of its phagosome (57). The ability of pathogenic Mycobacterium spp. to block phagolysosome maturation in resting macrophages requires their viability. In addition, activation of macrophages with cytokines such as IFN{gamma} induces fusion of phagosomes containing viable M. tuberculosis and M. avium with lysosomes (54). Our new data show that Cat L maturation is impaired in IFN{gamma}-activated macrophages that have phagocytosed dead M. avium. Thus, the modulation of Cat L activity in M. avium- and M. tuberculosis-infected macrophages appears to be independent of phagosome maturation.

Why then is Cat L maturation impaired in infected macrophages? Data indicate that the trafficking of Cat L to late endosomes and -lysosomes is required to liberate the two-chain active forms. Whereas Cat L-30K can be found in both late endosomes and -lysosomes, the two-chain (25 kDa/24 kDa) forms predominate in lysosomes (39, 47). Thus, the trafficking of Cat L to lysosomes may be disrupted in M. avium- and M. tuberculosis-infected macrophages. Of note, infection of macrophages with viable M. avium or M. tuberculosis was shown to disrupt the actin filament network of macrophages (58–60). Further studies demonstrated that these pathogens can inhibit actin assembly at the phagosomal membrane, a process linked to membrane fusion events with late endosomes/lysosomes. The data suggest that phagosomes containing viable pathogenic Mycobacteria (but not non-pathogenic or dead bacilli) exclude specific signaling lipids required to nucleate actin (61). IFN{gamma} stimulation of infected macrophages results in recruitment of these lipids to the phagosomal membrane, which in turn stimulates actin assembly and phagosome maturation (61). Although Cat L maturation was impaired under infection conditions that promote actin assembly and phagosome–lysosome fusion, it remains possible that alterations in the host's cytoskeletal elements still existed. The mistargeting and/or stalling of Cat L in the endocytic route may prevent intersection of Cat L with the proteases required to yield the two-chain active forms (specifically Cat L-24K) of the enzyme.

Alternatively, Mycobacterium infection may inhibit the enzymatic activity of the proteases required to cleave Cat L-30K into Cat L-25K and/or Cat L-25K into Cat L-24K. The processing of single-chain Cat L into two-chain Cat L can be blocked by treatment of cells with the serine and cysteine protease inhibitor, leupeptin (45). In addition, kidney cells lacking the leupeptin-insensitive cysteine protease, AEP, were shown to accumulate single-chain lysosomal enzymes, including Cat L (62). Recent data demonstrated that AEP is required to cleave Cat L-30K into the two-chain forms, as Cat L maturation beyond the single-chain stage does not occur in AEP–/– BM-derived DCs (63). Thus, AEP activity—which has a pH optimum of about 4.0–4.5—may itself be blocked in M. avium- and M. tuberculosis-infected macrophages. As mentioned, the pH of phagosomes containing viable pathogenic Mycobacterium spp. equilibrates to ~6.3, in contrast to pH 4.5 of bead containing phagosomes (21, 54, 64). The block in maturation/acidification is in part due to the actions of mycobacterial cell-wall lipids, such as lipoarabinomannan (LAM). LAM was shown to inhibit the activity of the phosphatidylinositol 3 kinase hVPS34, limiting production of phosphatidylinositol 3-phosphate (PI3P), and arresting maturation of the phagosome prior to its accumulation of the endosomal-tethering molecule, Early Endosomal Antigen 1 (EEA1) (65). EEA1, in cooperation with the trans-golgi network SNARE syntaxin 6, is needed for delivery of vacuolar H+-ATPases and cathepsins to the phagosome (66). More recent studies identified a secreted phosphatase, SapM, that can dephosphorylate PI3P, as well as augment the activity of LAM, maintaining low levels of PI3P in the Mycobacterium-containing phagosome and avoiding accumulation of vacuolar H+-ATPases (25). Interestingly, phagosomes containing HK Mycobacterium bovis BCG in unstimulated macrophages acidify to just 5.8 (64), while those containing live M. avium in IFN{gamma}-activated macrophages acidify to pH 5.2. Thus, LAM (or other lipids and effector molecules) released from Mycobacterium (live or dead) may modulate the pH of non-phagosomal endocytic compartments of the host cell (±IFN{gamma}), rendering AEP (and/or a leupeptin-sensitive proteases) ineffective at cleaving single-chain Cat L into the two-chain active forms.

Indeed, the activities of other endocytic proteases—not revealed by JPM-565biotin labeling—are likely altered in M. avium- and M. tuberculosis-infected macrophages. Gene array analyses showed that infection of THP-1 monocytes with M. tuberculosis for 24 h results in differential expression of several cathepsin genes (67). The maturation of human THP-1 monocytes upon exposure to M. tuberculosis bacilli (live or dead) or bacterial LPS resulted in the down-regulation of Cat G mRNA expression, and an increased expression of Cat B and Cat D genes (67). In addition, IL-10 secreted by THP-1 monocytes infected with viable (but not killed) BCG was shown to inhibit IFN{gamma}-induced Cat S mRNA expression in the infected cells (48 h post-infection), which in turn resulted in stalled Ii breakdown and reduced class II peptide loading. In contrast to this latter study, we observed an impairment of Cat L maturation and activity in murine BM-macs (±IFN{gamma}) that had phagocytosed either live or dead M. avium and M. tuberculosis, 24 h post-infection. Thus, our results implicate the involvement of a constitutively expressed component of the bacilli. Recent reports demonstrated that prolonged stimulation (>16 h) of TLRs by the mycobacterial lipoproteins LpqH (19 kDa) and LprG (24 kDa) interferes with IFN{gamma}-induced class II expression and antigen processing (31, 33, 34, 68, 69). Thus, the interaction of M. avium and M. tuberculosis with innate receptors during phagocytic uptake may modulate the activity of Cat L and possibly other endocytic proteases. Whether or not these alterations benefit the host or the pathogen remains to be determined. Alterations of protease activities in macrophages infected with pathogenic Mycobacterium spp. may prevent the generation of protective T cell epitopes (thereby benefiting the pathogen), or may instead prevent the destruction of a required T cell epitope (thus benefiting the host).

In conclusion, the new data presented here reveal yet another mechanism by which the activities of the two-chain forms of Cat L, specifically Cat L-24K, are regulated in the macrophage. In addition to the effects of IFN{gamma}, the activity of Cat L-24K was previously shown to require expression of the p41 isoform of class II MHC-associated Ii (39). Ii-p41 is a splice variant that contains an extra 65-a.a. segment (p4165a.a.) that binds to the active site of Cat L in the manner of a competitive inhibitor (70–72). Data suggest that Ii-p41 protects mature Cat L from destruction by other cysteine proteases within the endocytic route (39). Furthermore, IFN{gamma}-stimulated BM-macs were shown to secrete Cat L-24K complexed with p4165a.a., which served to stabilize the active enzyme in the neutral pH of the extracellular space. Secreted Cat L-24K was active and capable of degrading elastin (51). Other studies have shown that secreted active Cat L enhances migration of activated human macrophages (50). In addition, Cat L secreted from human fibroblasts was identified as the enzyme responsible for converting the precursor form of IL-8 into the mature chemokine (73). The impaired secretion of Cat L-24K from Mycobacterium-infected macrophages may modulate the degradation of the extracellular matrix and/or recruitment of inflammatory cells to the site of infection. Thus, although macrophages have developed multiple mechanisms to ensure both an intracellular and extracellular pool of the two-chain forms of active Cat L, M. avium and M. tuberculosis subverts these efforts.


   Acknowledgements
 
This work was supported in part by the Ohio State University Seed Grant. R.N.N. was sponsored by a Fulbright Fellowship.


   Abbreviations
 
AEP, asparagine endopeptidase
BM-mac, bone marrow-derived macrophage
Cat B, cathepsin B
CFU, colony-forming unit
CLIP, class II-associated invariant chain peptide
DC, dendritic cell
DM, dodecyl maltoside
HK, heat killed
LAM, lipoarabinomannan
PI3P, phosphatidylinositol 3-phosphate
rGM-CSF, recombinant granulocyte macrophage colony-stimulating factor

   Notes
 
Transmitting editor: H. Ploegh

Received 28 April 2005, accepted 22 March 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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