Differential Regulation of Cathepsin S and Cathepsin L in Interferon –treated Macrophages
1 Department of Immunology, University of Washington, Seattle, WA 98195z]|@?5, 百拇医药
2 Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195z]|@?5, 百拇医药
3 School of Medicine, University of Washington, Seattle, WA 98195z]|@?5, 百拇医药
Abstractz]|@?5, 百拇医药
Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Ms) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-–stimulated Ms. In addition, our studies show that the level of catL activity is significantly decreased in Ms cultured in the presence of IFN- whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-–stimulated peritoneal Ms. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Ms exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-. Thus, during a T helper cell type 1 immune response catL inhibition in Ms results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.
Key Words: cathepsin • macrophage • Ii processing • IFN- • p41f-cq, 百拇医药
Introductionf-cq, 百拇医药
MHC class II molecules expressed on the surface of APCs present proteolytic fragments of self- and foreign protein antigens to CD4+ T cells (1). MHC class II molecules assemble in the endoplasmic reticulum with the help of the chaperone molecule invariant chain (Ii), a type II glycoprotein that promotes the proper folding and assembly of the MHC class II {alpha} /ß heterodimer (2, 3). The Ii cytoplasmic tail contains a sequence targeting this complex to the lysosomal/endosomal pathway (4, 5) where it is exposed to the activity of proteases including cathepsins, which mediate cleavage of Ii in a step-wise manner, leaving only the class II–associated Ii peptide associated with the MHC class II peptide binding groove (6–8). Before MHC class II trafficking to the cell surface, the MHC-like molecule HLA-DM (H-2M in mice) mediates removal of class II–associated Ii peptide in exchange for the diverse array of self- and nonself-peptides generated in the endosomes and lysosomes (9, 10).
The cathepsins are a large family of aspartyl (D and E) and cysteinal (B, S, and L) endosomal proteinases. Recent studies have indicated that cathepsin S (catS) and cathepsin L (catL) are differentially expressed in APCs and are the only identified enzymes from this family of proteinases to play a key role in regulating MHC class II presentation (11–14). CatS is expressed in B cells and dendritic cells (DCs) and catL activity is observed in cortical thymic epithelial cells whereas in macrophages (Ms) both catL and catS activity can be detected (11–14). In B cells and DCs catS mediates the late stages of Ii degradation, specifically cleavage of the p22 and p12 Ii fragments and therefore regulates presentation of exogenous peptides in the context of MHC class II (12–14). CatL-deficient mice, however, exhibit a profound defect in CD4+ T cell selection as a result of the role played by catL in cleavage of these Ii fragments and in generation of the MHC class II bound peptides in cortical thymic epithelial cells (11, 15).
The observation that Ms express both of the cysteinal proteinases that mediate the late stages of Ii degradation has lead several groups to investigate the relative contribution of each enzyme to regulation of MHC class II presentation by these cells. One previous study reported that in alveolar and peritoneal Ms (pMs) lacking both catS and catL, degradation of Ii occurs as a result of activity of additional cysteine proteinases, potentially cathepsin F (catF; reference 16). However, we have previously observed a role for catS in regulating MHC II presentation of some exogenous antigens by Ms (13). Thus, we sought to further investigate the roles of catS and catL in regulating MHC class II presentation by Ms and determine the extent to which the functions of these two enzymes overlap. We demonstrate that catS mediates degradation of the p12 fragment of Ii in IFN-–stimulated pMs whereas catL plays no significant role in this process. In addition, we show this is a result of down-regulation of intracellular catL activity and increased catS activity in pMs after stimulation with this cytokine. This decrease in catL activity correlates with a decrease in mRNA levels, although IFN-–treated pMs maintain high levels of mature catL protein. We also found catL activity secreted upon IFN- treatment of pM, which correlates with a small increase in the amount of secreted mature catL protein. However, as the total amount of secreted catL protein (pro-form plus mature form) and the amount of intracellular mature catL protein is not altered upon IFN- treatment of pMs, we believe secretion of catL does not account for the down-modulation of catL activity. These data suggest that a specific inhibitor of catL activity is up-regulated upon IFN- stimulation of pMs. Furthermore, we also report complete inhibition of catL activity when catL protein is overexpressed in DCs, suggesting that bone marrow–derived APCs i.e., DCs and Ms, may express a catL-specific inhibitor. We found no significant evidence to suggest the p41 isoform of Ii effects such inhibition of catL activity in pMs. These results lead us to hypothesize that during a Th1 type immune response, catS activity in pMs is critical for Ii degradation and hence MHC class II antigen presentation.
Materials and Methodsr$, 百拇医药
Mice.r$, 百拇医药
C57BL/6 (B6) mice were purchased from and maintained under specific pathogen-free conditions at the University of Washington. CatS-/-, catL-/-, catS-/- x catL-/-, and Ii-/- animals were bred and maintained under these same conditions. CD11c-catL mice were generated using the CD11c-E{alpha} d construct (provided by K. Karjalainen, The Basel Institute for Immunology, Basel, Switzerland; reference 17). E{alpha} d was removed and catL cDNA was inserted into the construct by blunt end ligation using BamH1. CatL was flanked on either side by rabbit ß globin gene fragments and provides the transgene with an intron and a polyadenylation signal. To generate CD11c-catL transgenic (tg) mice, purified DNA was injected into BDF1 x B6 fertilized embryos. Offspring were backcrossed with catS-/- B6 mice. CD4+ T cell development occurred normally in the CD11c-catL tg mice and their CD4/CD8 T cell ratio was comparable to that observed in non-tg littermate control animals. All animals were used at 2–8-mo of age. All procedures and care of the animals were in accordance with University of Washington guidelines.
Antibodies.-9w, 百拇医药
The polyclonal rabbit antisera to mouse catL (provided by A. Erickson, University of North Carolina, Chapel Hill, NC) and the monoclonal antibodies M5/114 (anti-I-Ab, d, q and anti-Ed, k) and IN-1 (anti-Ii) have been described previously (18–20). Streptavidin horseradish peroxidase was purchased from .-9w, 百拇医药
M Isolation.-9w, 百拇医药
To elicit pMs, mice were injected intraperitoneally with 1 ml 4% thioglycollate ( ) and peritoneal exudate cells were collected 4–5 d later. Ms were cultured in Hydron-treated (Hydro Med Sciences) plates with RPMI-S (RPMI 1640 containing 100 u penicillin-streptomycin, 2 mM L-Glutamine [ Corporation], and 10% FBS [ ]). Bone marrow–derived Ms (BMMs) were generated by culturing bone marrow cells on bacterial plastic plates in RPMI-S with 106 units/ml M-CSF (21). Mouse alveolar Ms were harvested from lavage fluids as previously described (22). In brief, mouse lung was washed repeatedly with 1 ml of 6 mM EDTA PBS. Ms were pelleted and cultured in RPMI-S. For cytokine treatment, M populations were cultured for 48 h in the presence or absence of 0.3 ng/ml recombinant mouse IFN- (R&D Systems) or 200 U/ml recombinant mouse IL-4 (23).
DC Isolation.00[4+$, 百拇医药
Spleens were enriched for DCs as previously described (14, 24). In brief, mice were injected subcutaneously with 5 x 106 Flt3 ligand secreting B16 melanoma cells (provided by G. Dranoff, Dana-Farber Cancer Institute, Boston, MA) and spleens were harvested after the tumors reached 2 cm in diameter. Splenocytes were incubated with magnetic CD11c microbeads ( ), positively selected on an AutoMACS, and the purity of the positive fraction was assessed by flow cytometric analysis. All positive fractions were >96% CD11chi.00[4+$, 百拇医药
Cysteine Protease Active Site Labeling.00[4+$, 百拇医药
106 cells were incubated for 2 h at 37°C with 0.25 µM of the iodinated cysteine protease inhibitor CBZ-125I-Tyr-Ala-CN2 (25). This inhibitor irreversibly binds to the active site of cysteine proteases via a thiol–ester bond. Cells were washed, lysed in cell lysis buffer (0.5% NP-40, 0.15 M NaCl, 50 mM Tris-HCL, pH 7.2), supplemented with a cocktail of protease inhibitors (Roche Molecular Biochemical), and run on a 12% wt/vol SDS-PAGE gel. 125I-labeled proteins were visualized by autoradiography on Kodak BioMax Mr film. The results were quantified using the Bio-Rad GS-700 Imaging Densitometer and analyzed by Multi-Analyzer software Version 1.0.2. Alternatively, 106 cells were incubated for 2 h at 37°C with 0.25 µM of the biotinylated cysteine proteinase inhibitor biotin-Tyr-Ala-FMK, as described above. Intracellular biotin-labeled proteases were detected by immunoblotting cell lysates (see below). Extracellular cathepsin activity was analyzed by Western blot after concentrating supernatant by ultrafiltration on YM10 Centricons (Amicon) and dialysis against 20 mM Tris, pH 7.5.
Immunoblotting.!']2?, 百拇医药
M{phi} s and fibroblasts were washed in PBS and lysed as described above. Debris was removed by centrifugation at 8,000 rpm for 10 min and the lysates and supernatant were analyzed for protein content using Coomassie® Plus Protein Assay Reagent ( ). Samples containing the indicated amount of total protein or number of cell equivalents were boiled for 5 min in SDS-reducing buffer and separated by 12% SDS-PAGE. The proteins were electrophoretically transferred onto nitrocellulose membrane and this was probed using the indicated primary Ab. Binding was detected using the appropriate horseradish peroxidase–conjugated secondary Ab diluted 1:1,000 and visualized by chemiluminescence ( ).!']2?, 百拇医药
Pulse Chase Biosynthetic Radiolabeling and Immunoprecipitation.!']2?, 百拇医药
pMs were preincubated at 37°C for 2 h in methionine/cysteine-free RPMI 1640 supplemented with 200 mM L-glutamine, 10 mM Hepes, 100 µg/ml penicillin-streptomycin, and 5% dialyzed FBS. Cells were pulsed (4–8 x 106 cells/ml) for 40 min with 1 mCi/ml 35S-methionine/cysteine (Trans 35S-label; ) and chased for 0, 1, 3, and 6 h in the presence of 30x unlabeled methionine (3 mM) and cysteine (16 mM). Pulse-labeled cells were washed in PBS and lysed in 1% NP-40, 0.01 M Tris, pH 7.4, and 0.15 M NaCl supplemented with a cocktail of protease inhibitors (Roche Molecular Biochemicals). Before precipitation with the M5/114 Ab, lysates were precleared with protein G Sepharose ( ) and normal rat IgG (Caltag Laboratories Inc.). Precipitated proteins were boiled in SDS-reducing buffer and separated by 7.5–20% gradient SDS-PAGE. Gels were fixed in 50% methanol, 10% acetic acid, treated with AmplifyTM ( ), dried, and the labeled proteins were visualized by autoradiography.
RNA Extraction and cDNA Synthesis.d:.i, 百拇医药
pM{phi} s were purified by adherence to bacterial plastic plates and were lysed in RNA STAT-60 (3 ml RNA STAT-60 per 10 x 106 cells; TEL-TEST "B" INC.) directly on the plates 30 min after plating (ex vivo) and after 48 h of incubation in the presence or absence of IFN-{gamma} . RNA was extracted according to the manufacturer's protocol and contaminating DNA was removed by treating 2 µg of sample RNA with amplification grade DNase I ( ). First strand cDNA was prepared by reverse transcription using the SUPERSCRIPTTM First-Strand Synthesis System for RT-PCR, as directed by the manufacturer.d:.i, 百拇医药
Real-time PCR.d:.i, 百拇医药
Primer and probe sequences were selected with the assistance of Primer Express software ( ) using nucleotide sequences available on the GenBank Database (available from GenBank/EMBL/DDBJ under accession nos. NM_013556, , and for HPRT, catL, and catS, respectively). Primers ( ) are as follows: HPRT-F 5'-3' (5'-TGGAAAGAATGTCTTGATTGTTGAA-3'); HPRT-R 5'-3' (5'-AGCTTGCAACCTTAACCATTTTG-3'); CatL-F 5'-3' (5'-GACCGGGACAACCACTGTG-3'); CatL-R 5'-3' (5'-CTACCCATCAATTCACGAC-3'); CatS-F 5'-3' (5'-GCCATTCCTCCTTCTTCTTCTACA-3'); and CatS-R 5'-3' (5'-CAAGAACACCATGATTCACATTGC-3'). The following probes were synthesized with a 5' FAM reporter and 3' TAMRA quencher (Biosearch Technologies Inc.): HPRT (5'-6-FAM-CAAACTTTGCTTTCCCTGGTTAAGCAGTACAGC-TAMRA-3'); CatL (5'-6-FAM-CTCAGGTGTTTGAACCCATGAATCTTTTACTC-TAMRA-3'); and CatS (5'-6-FAM-AAGCGGTGTCTATGATGACCCCTCCTGTATAMRA-3').
Real-time PCR amplification of HPRT, catL, and catS was performed as previously described (18). Triplicates of each cDNA sample were amplified alongside appropriate controls and each assay was performed on at least three independent occasions. Relative quantitation of catL and catS expression was determined using the comparative CT method (user bulletin no. 2; ). This method was used to calculate the fold increase of mRNA in pM{phi} s after 48 h of incubation in the presence or absence of IFN-{gamma} compared with those cells isolated ex vivo.o|, http://www.100md.com
Resultso|, http://www.100md.com
Ii . Degradation in IFN-–stimulated Ms Is Predominately Mediated by CatS.o|, http://www.100md.com
The cysteinal lysosomal proteinases catS and catL play a role in regulating the MHC class II presentation pathway by virtue of their role as mediators of the late stages of Ii degradation (8, 11–14). These enzymes are differentially expressed in vivo in all APCs except Ms, in which both catS and catL activity can be detected. Thus, we wished to determine the relative contribution of each of these enzymes to Ii degradation and regulation of MHC class II presentation by Ms.
To elucidate the effects of catS and catL on the kinetics of Ii degradation, thioglycollate-elicited pMs isolated from B6, catL-/-, catS-/-, and catS-/- xcatL-/- mice were cultured for 48 h in the presence of IFN- to induce up-regulation of MHC class II (26). IFN-–stimulated pMs were metabolically labeled and chased for 0, 1, 3, and 6 h. Protein lysates were immunoprecipitated with an MHC class II–specific Ab and analyzed by SDS-PAGE ( A). B6 pMs showed minimal accumulation of the p12 fragment of Ii associated with MHC class II whereas in catS-/- cells marked accumulation of p12 was observed after 1 h of chase. This fragment was slowly degraded over the 6 h of chase, suggesting that in the absence of catS another proteinase is capable of Ii processing, albeit with substantially slower kinetics. The extent of p12 accumulation in catL-/- pMs was not significant compared with that observed in catS-/- pMs and therefore the delayed cleavage of Ii in the absence of catS is unlikely to be mediated by catL. In addition, in the absence of both catS and catL accumulation of p12 occurred with the same kinetics as observed in pMs lacking catS alone.
fig-ommitted|j/1.g, http://www.100md.com
Figure 1. Ii degradation in cathepsin-deficient Ms. (A) Thioglycollate-induced Ms from B6, catS-/-, catL-/-, and catS-/- xcatL-/- mice were pulse labeled and chased for 0, 1, 3, and 6 h. Cell lysates were immunoprecipitated with the I-Ab-specific Ab M5/114 and immunoprecipitates were analyzed by 7.5–20% gradient SDS-PAGE under denaturing conditions. (B) Thioglycollate-induced pMs, (C) BMMs, and (D) alveolar Ms from B6 and catS-/- mice were cultured for 48 h in the presence of IFN- and immunoblotted with the monoclonal Ab IN-1. Cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors) and lysates were then twofold serially diluted starting at 5 µg of total protein (alveolar Ms are shown at 5 µg only). The position of Ii fragments p41, p31, and p12 are indicated.|j/1.g, http://www.100md.com
To test whether the defect in Ii processing in the absence of catS was limited to pM, we examined different M populations and analyzed the steady state levels of Ii. We isolated alveolar, peritoneal, and BMMs from B6 and catS-/- mice () . This experiment revealed accumulation of p12 Ii fragments in all M types in the absence of catS. These results clearly implicate catS as the key enzyme regulating the late stages of Ii degradation in Ms. In the absence of catS, another enzyme, likely catF, might be able to degrade Ii, albeit with significantly less efficiency.
fig-ommittedw, 百拇医药
Figure 2. Active site labeling of cytokine-treated M. (A) Thioglycollate-induced pMs and (B) BMMs from B6, catS-/-, and catL-/- mice were either taken directly ex vivo or activated for 48 h with or without IFN-. Cells were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz- [125I]-Tyr-Ala-CN2. Cells were lysed and radiolabeled enzymes were analyzed on a 12% SDS-PAGE gel. Levels of active cathepsin in cells taken directly ex vivo (ex vivo) and after 48 h of plating with IFN- (IFN-) or without (UN) are shown. The position of cathepsin B (catB), cathepsin S (catS), and cathepsin L (catL) are indicated on the gel. C) The intensity of the active site labeling was quantified by the BioRad GS-700 Imaging Densitometer and analyzed by Multi-Analyzer software Version 1.0.2. The numbers correspond to the following: 1, B6 ex vivo; 2, B6 UN; 3, B6 IFN-; 4, catS-/- ex vivo; 5, catS-/- IFN-; 6, catS-/- UN; 7, catL-/- ex vivo; 8, catL-/- UN; 9, catL-/- IFN-.w, 百拇医药
IFN- Stimulation of Ms Modulates Cathepsin Activity.
The observation that catL played only a minor role in Ii degradation in IFN-–treated pMs and previous reports indicating that IFN- modulates the mRNA level of cathepsins (27–29) lead us to investigate the effect of IFN- on the level of catS and catL of activity in pMs and BMMs. IL-4 has also been shown to up-regulate expression of MHC class II on the surface of Ms (26) and it has been shown that in DCs protease activity can be modulated by both pro- and antiinflammatory cytokines (30). Therefore, we sought to examine the effects of both IFN- and IL-4 on catS and catL activity in distinct M populations.;8yk, 百拇医药
Thioglycollate-elicited pMs and BMMs were isolated from B6-, catL-/--, and catS-/--deficient mice and the level of intracellular cathepsin activity was analyzed immediately upon isolation (ex vivo) and after 48 h of culture in the presence or absence of IFN- or IL-4 (). pMs analyzed directly ex vivo had barely detectable levels of catS and a very low level of catL activity. Upon culture, in the absence of cytokines, both pMs and BMMs showed a marked increase in catL activity whereas the activity of catS increased only slightly. Interestingly, the addition of IFN- resulted in an increase in catS activity and a dramatic decrease in catL activity. We quantified the changes in catL activity after IFN-{gamma} stimulation by densitometry and found catL activity decreased between 50–100-fold ( C). IL-4, however, had no affect on cathepsin activity as compared with Ms cultured in the absence of cytokines (unpublished data; see ). These data suggest our previous observation that catS but not catL was critical for regulating the late stages of Ii cleavage in IFN-–stimulated Ms can be explained by the effect of this cytokine on down-modulating catL activity.
fig-ommittedqs5tz(w, http://www.100md.com
Figure 4. CatL and the effect of the p41 Ii isoform. The level of catL in (A) B6 and (C) Ii-/- thioglycollate-induced pMs cultured for 48 h in the presence or absence of IFN- was detected by immunoblotting with a catL-specific polyclonal antiserum. Cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors) and lysates were then twofold serially diluted starting at 40 µg of total protein. Arrows indicate pro-catL and mature catL. These experiments were performed three times and yielded identical results. (B) Thioglycollate-induced pMs from B6 mice were taken directly ex vivo or activated for 48 h in the presence or absence of IFN-. Cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors). 20 µg of lysate was separated on a 12% SDS-PAGE and immunoblotted with the IN-1 Ab. p41 and p31 are indicated. (D) Thioglycollate-induced pMs and (E) BMMs from B6 and Ii-/- mice were cultured for 48 h in the presence or absence of IFN-. 106 cells were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2, lysed, and separated by 12% SDS-PAGE. Levels of active cathepsin in cells taken directly ex vivo (ex vivo) and after 48 h of plating with IFN- (IFN-) or without (UN) are shown. Arrows indicate the positions of cathepsin B (catB), cathepsin S (catS), and cathepsin L (catL).
fig-ommittedio\, 百拇医药
Figure 5. Secretion of catL upon IFN-{gamma} treatment. B6 thioglycollate-induced pMs taken directly ex vivo or cultured for 48 h in the presence or absence of IFN- or IL-4 were incubated serum free with the cysteine proteinase inhibitor biotin-Tyr-Ala-FMK for 2 h. Labeled cathepsin present (A) intracellularly and (B) in the supernatant were detected by immunoblotting. (C) Cells were incubated for 2 h in serum-free medium, the cells were removed, and the inhibitor biotin-Tyr-Ala-FMK was added for 20 min before analysis of labeled cathepsins by immunoblotting. (D) Thioglycollate-elicited pMs cultured for 48 h in the presence or absence of IFN- were incubated for 2 h in serum-free medium. 5 µg of total supernatant protein were titrated and separated before catL immunoblotting.io\, 百拇医药
We have previously observed that the level of mature catL protein in B cells is regulated by the presence or absence of catS (18). Thus, we wished to determine whether such regulation of the cysteinal proteinases was involved in the changes in cathepsin activity observed in both pMs and BMMs cultured in the presence of cytokines. Modulation of catL activity after IFN- stimulation of catS-deficient Ms was comparable to that observed in B6-derived cells (), implying that in Ms the activities of catS and catL are regulated independently upon IFN- stimulation.
CatL mRNA Levels Are Decreased upon IFN- Stimulation of pMs.{'ejm, http://www.100md.com
Our finding that catL activity was decreased upon IFN- stimulation of pMs lead us to investigate the mechanisms by which this down-modulation was regulated. In view of several studies in which it has been reported that in a variety of cell types cathepsin mRNA levels are altered by IFN- stimulation (27–29), we sought to determine whether transcription of catL mRNA was decreased in pMs upon IFN- stimulation.{'ejm, http://www.100md.com
pMs were purified by adherence to tissue culture plates and mRNA was isolated from cells directly ex vivo and after culturing for 48 h in the presence or absence of IFN-. Real time PCR amplification and subsequent quantitative analysis indicated that the level of catL mRNA in IFN-–stimulated pMs was decreased approximately eightfold compared with cells analyzed directly ex vivo () . Furthermore, we were unable to detect any significant change in the level of catL mRNA in cells cultured in the absence of cytokine. In addition, we analyzed catS mRNA levels in pMs and observed no significant changes with or without cytokine treatment. These results are in agreement with previously published data indicating that catL mRNA is decreased in microglial cells stimulated with IFN- (27) and suggests that the IFN-–induced decrease in catL activity might in part be a result of decreased transcription of catL mRNA.
fig-ommitted/vae@9f, 百拇医药
Figure 3. Regulation of catL activity by mRNA. pMs were purified by adherence to bacterial plastic plates. Cells were lysed directly on the plates 30 min after plating (ex vivo) and after 48 h of incubation in the presence or absence of IFN-. After RNA extraction and cDNA synthesis, quantitative PCR was performed in an ABI Prism 7700 sequence detector. Primers and probes for HPRT, catL and catS were designed using Primer Express software. The probes were labeled at the 5' and 3' ends with the fluorochromes FAM and TAMRA. mRNA levels were quantitated using the comparative CT method described in the ABI Prism 7700 sequence detector user bulletin number 2. The data is shown as the level of cathepsin mRNA in cells after 48 h of plating with IFN- (IFN-) or without (UN) compared with the level of mRNA ex vivo./vae@9f, 百拇医药
Mature CatL Protein Levels Are Not Reduced in IFN-–treated pMs./vae@9f, 百拇医药
Having demonstrated that the decrease in catL activity in IFN-–stimulated pMs correlated with diminished transcription of catL mRNA, we wished to establish whether there was a corresponding decrease in intracellular catL protein levels. We performed a twofold serial dilution of cell lysate derived from B6 pMs isolated directly ex vivo or cultured for 48 h in the presence or absence of IFN-. Lysates were separated by SDS-PAGE and analyzed by immunoblotting for catL protein ( A). Unexpectedly, we observed that the amount of mature catL protein detected in pMs cultured in the presence or absence of IFN- was comparable and that this level of protein was substantially greater than that detected in ex vivo–isolated cells. A decrease in the amount of the 38 kD pro-form of catL was observed, however, in IFN-–stimulated pMs when compared with cells analyzed directly ex vivo. Thus, the reduction in catL mRNA upon IFN- stimulation of pMs resulted in a decrease in pro-form catL protein although there was no significant concomitant decrease in the level of mature catL protein, presumably as a result of the long half-life of this protein. Taken together, our results demonstrating that catL activity is substantially diminished upon IFN- stimulation of pMs whereas levels of mature catL protein are increased suggest an inhibitor of catL might be up-regulated in these cells.
Modulation of CatL Activity in IFN-–stimulated pMs Occurs in the Absence of Ii.n, 百拇医药
Several molecules have been implicated as inhibitors of catL, including the p41 isoform of Ii (31–33). Previous studies have shown that Ii is up-regulated in response to IFN- (20), making p41 an attractive candidate inhibitor of catL in pMs cultured in the presence of this cytokine. Thus, we examined the regulation of catL in thioglycollate-elicited pMs isolated from Ii-/- mice.n, 百拇医药
To first test whether p41 could be an inhibitor of catL activity in IFN-–stimulated pMs, we analyzed by immunoblotting the level of Ii protein in B6 pMs isolated directly ex vivo or cultured in the presence or absence of IFN- for 48 h ( B). Upon IFN- stimulation the p41 isoform of Ii was substantially up-regulated whereas the increase in p31 was more moderate. No such change in the level of either p31 or p41 was observed when cells were cultured in the absence of cytokine. This increase in p41 upon IFN-{gamma} stimulation of pMs provided us with evidence that p41 was a viable candidate inhibitor of catL.
It has previously been reported that the level of mature catL protein detected in BMMs is significantly decreased in the absence of the p41 isoform of Ii (33). Therefore, we sought to determine whether a similar defect could be observed in Ii-/- pMs. The level of catL protein in pMs isolated from Ii-/- mice was analyzed by serial dilution of cell lysates and immunoblotting as described above. The level of mature catL protein in Ii-/- pMs was increased upon culturing the cells in the presence or absence of IFN- ( C), as observed in B6 pMs ( A). The extent of up-regulation of mature catL in cells cultured in the absence of cytokine was the same for both Ii-/- and B6-derived cells. However, the amount of mature catL protein detected in Ii-/- cells cultured in the presence of IFN- was two- to threefold less than in cells cultured in the absence of this cytokine and also two- to threefold less than in B6 pMs cultured in IFN- ( A). Taken together, these data suggest that p41 may play some role in stabilizing the mature form of catL in IFN-–stimulated pMs as has been suggested for BMMs (33), however, we observed no role for p41 in maintaining the level of mature catL protein in pMs isolated directly ex vivo or cultured in the absence of cytokines.
Having observed that Ii-/- pMs stimulated with IFN- express noticeably lower levels of mature catL protein than B6 cells cultured under the same conditions, we aimed to establish whether Ii regulated the decrease in catL activity detected upon IFN- stimulation of B6 Ms. pMs and BMMs from B6 and Ii-/- mice were isolated directly ex vivo or cultured for 48 h in the presence or absence of IFN- or IL-4. Intracellular cathepsin activity was detected using the irreversible cysteine protease inhibitor Cbz-125I-Tyr-Ala-CN2 and the proteins were separated by SDS-PAGE (). The modulation of cathepsin activity we observed was the same in both cell types. After culture of cells in the absence of cytokine catL activity was up-regulated and in the presence of IFN- this up-regulation was not observed. In addition, catL activity was up-regulated in cells cultured in the presence of IL-4. These results provide no evidence for a major role for the p41 isoform of Ii in regulating catL activity in pMs and BMMs although it may play a modest role in stabilizing the levels of mature catL protein in IFN-–stimulated Ms.
CatL Is Secreted When Ms Are Treated with IFN-.7r(;.5x, 百拇医药
Cathepsins are soluble proteinases and specific secretion of catL may explain the decrease in intracellular catL activity we observed upon IFN- stimulation of pMs. To address this possibility we compared intracellular and extracellular cathepsin activity using a new biotinylated active site labeling reagent, biotin-Tyr-Ala-FMK. pMs were labeled for 2 h at 37°C with the biotinylated inhibitor and labeled enzymes in the culture supernatant and cell lysates were detected by Western blot analysis. As shown in , catL activity was markedly increased in the culture supernatant upon IFN- treatment whereas intracellular catL activity sharply decreased. CatL activity was barely detectable in supernatant of untreated or IL-4–treated pMs. In contrast, both extracellular and intracellular catS activity increased upon treatment with IFN-. It is important to note that endocytic uptake can be a rate-limiting step, thus, the efficiency of the active site labeling of intracellular enzymes is significantly less efficient than that of extracellular enzymes. Therefore, we assessed the overall amount of catL protein secreted by IFN-–induced pMs by Western blot analysis with an anti-catL Ab ( D). We observed a two- to fourfold increase in mature catL whereas pro-catL levels are decreased in the supernatant of IFN-–treated cells as compared with the cells cultured in the absence of cytokine. In these experiments analyzing catL levels in culture supernatants, we used 7–10-fold more cell equivalents per lane as compared with intracellular catL analysis. Taken together with intracellular mature catL protein levels not changing, these experiments indicate that secretion of catL protein cannot account for the significant loss in catL activity upon IFN- stimulation of pMs.
Cathepsins have been implicated in inflammatory extracellular matrix remodeling, tumor metastasis, and angiogenesis (34) and catL has a potent elastinolytic activity (35). We wanted to determine if the secreted enzymes are active extracellularly by labeling supernatants of pM cultures with biotin-Tyr-Ala-FMK at neutral pH and found that both secreted catL and catS are active at physiological pH ( C). This data suggests that secreted catL may play a role in extracellular matrix remodeling at the sites of inflammation.%, 百拇医药
CatL Activity Is Inhibited When Overexpressed in DCs.%, 百拇医药
B cells and DCs exhibit no catL activity and therefore utilize catS for late stage Ii chain processing whereas Ms are the only APC in which active catS and catL can be detected concurrently. In light of our observation that catL plays no significant role in Ii degradation in Ms, we sought to investigate the effect of catL expression in a second catS-dependent APC. tg mice were generated using the CD11c promoter to specifically overexpress catL in DCs. To rule out any effect of catS upon MHC class II maturation in these cells, these mice were crossed onto a catS-/- background.
We wished to determine the level of catL protein expressed in DCs isolated from tg mice and non-tg littermate control animals. Highly purified DCs were isolated and protein lysates from the indicated number of cells were analyzed for catL protein () . B6 DCs expressed significantly less catL protein than the equivalent number of CD11c-catL tg DCs, indicating that the CD11c promoter efficiently induces overexpression of catL protein in DCs. To our surprise, using the Cbz-125I-Tyr-Ala-CN2 irreversible inhibitor we were unable to detect catL activity in the CD11c-catL tg DCs in which we observed high levels of mature catL protein. We observed the same results using DCs isolated from progeny of a second founder CD11c-catL tg mouse (unpublished data).6*#, 百拇医药
fig-ommitted6*#, 百拇医药
Figure 6. Inhibition of catL activity in CD11c-catL tg mice. DCs purified from Flt3 ligand–treated CD11c-catL tg mice were twofold serially diluted starting at 106 cells. Transformed murine embryonic fibroblasts were twofold serially diluted starting at 2.5 x 105 cells. For Western blot analysis, cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors) and lysates were separated by 12% SDS-PAGE and immunoblotted for catL. Pro-catL and mature catL are indicated. For active site labeling, cells were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2. Cells were lysed and radiolabeled enzymes were analyzed on a 12% SDS-PAGE gel. Arrows indicate active catL.
To ensure that the level of mature catL protein expressed by the CD11c-catL tg DCs generates sufficient signal to be detected in our active site labeling assay, we compared catL protein and activity in tg DCs to fibroblasts, a cell type known to express high levels of catL activity (11). We were able to detect both mature catL protein and catL activity in as few as 8,000 fibroblast cells (). However, in DCs expressing comparable catL protein levels to 1.25 x 105 fibroblasts we were unable to detect catL activity. Thus, the quantity of mature catL protein expressed in the CD11c-catL tg DCs is not below the level of detection in our active site labeling assay, indicating that DCs express a specific inhibitor of catL activity.px^0v, 百拇医药
Discussionpx^0v, 百拇医药
The lysosomal cysteinal proteinases catS and catL have previously been shown to play a critical role in the late stages of Ii degradation in DCs, B cells, and cortical thymic epithelial cells (36, 37). In these cells, activity of only one enzyme can be detected and thus the relative role of catS and catL in regulating Ii processing cannot be assessed. Ms provide an ideal APC in which to study whether catS and catL are redundant enzymes in the regulation of MHC class II presentation, as activity of both enzymes can be detected in these cells. Here, we report that in thioglycollate-elicited pMs stimulated with IFN-, catS is principally responsible for mediating Ii cleavage whereas catL plays little part in this process. This result is surprising given that efficient Ii proteolysis in cortical thymic epithelial cells requires catL and that in MHC class II–expressing fibroblast cell lines effective Ii cleavage can be mediated by catL (11, 38). However, an explanation for our results is provided by our subsequent observation that although intracellular activity of catL is profoundly down-regulated in pMs in response to the proinflammatory cytokine IFN-, intracellular catS activity increases. This modulation in catL activity in IFN-–stimulated pMs coincided with a decrease in catL mRNA levels whereas catS mRNA levels were not diminished. These data indicate that modulation of catL activity in pMs in response to IFN- is in part regulated at the level of transcription as has previously been reported for microglial cells (27). Down-regulation of catL mRNA was concomitant with a decrease in pro-catL, however, levels of the mature protein were not decreased in IFN-–stimulated pMs. One possible explanation for this maintenance of high levels of mature catL protein despite decreased mRNA is that the mature enzyme is long lived in endosomes. Additional studies showed that the decrease in catL activity upon IFN- stimulation was concomitant with an increase in secretion of mature catL protein that exhibited protease activity. However, this increase in secretion had little effect on intracellular catL protein levels and therefore, secretion of mature protein does not explain the specific inhibition of intracellular catL activity. Taken together with our observation that intracellular catL activity decreases in IFN-–stimulated pMs whereas levels of the mature protein are maintained, these results suggest that an inhibitor(s) of enzyme activity is increased in response to this proinflammatory cytokine and is critical for regulating catL activity in pMs and BMMs.
A number of lysosomal cysteinal proteinases inhibitors have been identified, including cystatin C (39, 40), the propeptide regions of the cathepsins (41–43), and the p41 isoform of Ii (31, 32). We found no difference in cystatin C expression or localization in pMs cultured in the presence or absence of IFN- (unpublished data), indicating that this cysteinal proteinase inhibitor plays no significant role in regulating catL activity upon IFN- stimulation of pMs. In addition, the propeptide regions of catS and cathepsin B (catB) do not affect the level of catL activity in Ms treated with IFN-. We examined catS- and catB-deficient pMs and BMMs and were unable to determine any differences in catL activity with or without treatment of IFN- when compared with B6 Ms ( and unpublished data). Recent studies have reported that p41 is not only an inhibitor of catL enzymatic activity but that it is also required for catL activity in BMMs where it acts as a chaperone, stabilizing the mature form of this enzyme (33). Because both MHC class II and Ii, including p41, are up-regulated in pMs stimulated with IFN-, we examined whether p41 and its fragments were responsible for the observed down-regulation of catL activity and preservation of high levels of mature protein in these cells. We observed that levels of mature catL protein in Ii-/- and B6 pMs and BMMs were identical, except upon IFN- stimulation when catL levels were two- to threefold lower in Ii-/- cells compared with B6 cells. However, we detected equivalent levels of catL activity in Ii-/- and B6 pMs and BMMs cultured in the presence or absence of IFN-. Furthermore, the extent to which catL activity was down-regulated upon IFN- stimulation was equivalent in Ii-/- and B6 cells. These data demonstrate that Ii does not play an important role in regulating catL activity in either thioglycollate-induced pMs or BMMs and are in agreement with our previous observations in thymic stromal cells (15). However, these results suggest that Ii does play a minor role in stabilizing the mature form of catL in pMs, as has been previously suggested by others for BMMs (33). Another possible inhibitor of catL activity is cystatin F, which is specifically expressed in hematopoietic cells and preferentially binds catL and papain in vitro (44, 45). We believe cystatin F could potentially be involved in the catL inhibition observed in Ms or DCs and would like to investigate this in the future. Thus, although it is possible that p41 may contribute modestly to the observed down-modulation of intracellular catL activity upon IFN- stimulation of pMs, we suggest that an unknown inhibitor is the pivotal regulator of catL enzymatic activity in pMs.
We have previously reported that catL activity cannot be detected in ex vivo–isolated DCs (11, 18), however, significant levels of catL activity can be detected in DCs derived from bone marrow in culture (unpublished data). In view of our observations implying that catL activity in pMs is regulated in a highly specific manner by an as yet unidentified inhibitor, we sought to examine whether such a mechanism was present in ex vivo DCs. Analysis of DCs isolated from the spleen of mice engineered to express catL under the control of the CD11c promoter revealed significant levels of mature catL protein but no detectable catL activity in these cells. Expression of this catL cDNA when driven by a retroviral promoter in fibroblasts generated high levels of catL activity (38), indicating that inhibition of enzymatic activity was specific to ex vivo–isolated DCs. Thus, we have shown that both DCs and pMs use a mechanism of catL inhibition although the nature of the catL inhibitor in DCs and pMs remains to be identified.
Our data indicate that catL activity is down-regulated upon IFN- stimulation of pMs and that catS is the predominant mediator of the late stages of Ii degradation, and hence MHC class II peptide presentation. In addition, catS mediates Ii degradation in other M populations. These observations appear to differ from a recent report in which it was shown that other cysteinal proteinases, perhaps catF, are able to compensate for the absence of catS and catL and elicit degradation of Ii in pMs and alveolar Ms (16). However, in this latter study, Ii processing was analyzed immediately after pulse and after an overnight chase. Thus, the pronounced accumulation of MHC class II–associated p12 Ii fragments we observe between 1 and 6 h of chase was not detected. The significantly delayed kinetics of Ii degradation and accumulation of MHC class II–bound Ii fragments in catS-deficient pMs affect the efficiency of generation of MHC class II–peptide complexes and thus account for the previously reported defect in MHC class II presentation of some but not other protein antigens (13). Therefore, we believe that while although enzymes can mediate Ii cleavage with delayed kinetics, catS plays a major role in regulating Ii degradation and MHC class II presentation in Ms.
In conclusion, we have shown that in pMs, in which both cysteinal proteinases catS and catL are expressed, catS is the predominant enzyme processing Ii. Furthermore, catL is shown to have no major role in this process as a result of substantial down-regulation of its enzymatic activity upon IFN- stimulation. This decline in catL activity correlates with a decrease in catL mRNA and an increase in secretion of mature catL protein but not a decrease in intracellular mature catL protein levels, suggesting that catL activity in IFN-–stimulated pMs is regulated by a specific inhibitor. In addition, we find the p41 isoform of Ii does not contribute significantly to this regulation of catL activity in pMs and that DCs also use a catL-specific inhibition mechanism. The results presented here indicate that upon IFN- activation of pMs, enzymatic activity of catL is specifically inhibited, such that catS mediates Ii degradation and regulates MHC class II maturation. Thus, we suggest that as a result of differential regulation of catL and catS the latter enzyme governs MHC class II presentation by APCs in secondary lymphoid organs.
Acknowledgmentsahdq-+c, http://www.100md.com
We would like to thank C. Plata and N. Li for their excellent care of the mice used in these experiments.ahdq-+c, http://www.100md.com
This work was funded by the Howard Hughes Medical Institute (A. Rudensky) and grants from the National Institutes of Health (NIH) to A. Rudensky. C. Beers is supported by an NIH pre-doctoral training grant. S. Fink is affiliated with the Medical Science Training Program at the University of Washington.ahdq-+c, http://www.100md.com
Submitted: June 14, 2002ahdq-+c, http://www.100md.com
Revised: October 18, 2002ahdq-+c, http://www.100md.com
Accepted: December 3, 2002ahdq-+c, http://www.100md.com
Footnotesahdq-+c, http://www.100md.com
C. Beers and K. Honey contributed equally to this work.ahdq-+c, http://www.100md.com
* Abbreviations used in this paper: B6, C57BL/6; BMM, bone marrow–derived macrophage; catB, cathepsin B; catF, cathepsin F; catL, cathepsin L; catS, cathepsin S; DC, dendritic cell; Ii, invariant chain; M, macrophage; pM, peritoneal M; tg, transgenic.
Referencesu3m]6t#, 百拇医药
Cresswell, P. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259–293.u3m]6t#, 百拇医药
Anderson, M.S., and J. Miller. 1992. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA. 89:2282–2286.u3m]6t#, 百拇医药
Cresswell, P. 1996. Invariant chain structure and MHC class II function. Cell. 84:505–507.u3m]6t#, 百拇医药
Bakke, O., and B. Dobberstein. 1990. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell. 63:707–716.u3m]6t#, 百拇医药
Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S.L. Schmid, V. Quaranta, and P.A. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature. 348:600–605.u3m]6t#, 百拇医药
Neefjes, J.J., and H.L. Ploegh. 1992. Intracellular transport of MHC class II molecules. Immunol. Today. 13:179–184.u3m]6t#, 百拇医药
Maric, M.A., M.D. Taylor, and J.S. Blum. 1994. Endosomal aspartic proteinases are required for invariant-chain processing. Proc. Natl. Acad. Sci. USA. 91:2171–2175.
Riese, R.J., P.R. Wolf, D. Bromme, L.R. Natkin, J.A. Villadangos, H.L. Ploegh, and H.A. Chapman. 1996. Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. Immunity. 4:357–366.}'1(!(k, 百拇医药
Denzin, L.K., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading. Cell. 82:155–165.}'1(!(k, 百拇医药
Martin, W.D., G.G. Hicks, S.K. Mendiratta, H.I. Leva, H.E. Ruley, and L. Van Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection. Cell. 84:543–550.}'1(!(k, 百拇医药
Nakagawa, T., W. Roth, P. Wong, A. Nelson, A. Farr, J. Deussing, J.A. Villadangos, H. Ploegh, C. Peters, and A.Y. Rudensky. 1998. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science. 280:450–453.}'1(!(k, 百拇医药
Riese, R.J., R.N. Mitchell, J.A. Villadangos, G.P. Shi, J.T. Palmer, E.R. Karp, G.T. De Sanctis, H.L. Ploegh, and H.A. Chapman. 1998. Cathepsin S activity regulates antigen presentation and immunity. J. Clin. Invest. 101:2351–2363.
Nakagawa, T.Y., W.H. Brissette, P.D. Lira, R.J. Griffiths, N. Petrushova, J. Stock, J.D. McNeish, S.E. Eastman, E.D. Howard, S.R. Clarke, et al. 1999. Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice. Immunity. 10:207–217.{]\j;s9, http://www.100md.com
Shi, G.P., J.A. Villadangos, G. Dranoff, C. Small, L. Gu, K.J. Haley, R. Riese, H.L. Ploegh, and H.A. Chapman. 1999. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity. 10:197–206.{]\j;s9, http://www.100md.com
Honey, K., T. Nakagawa, C. Peters, and A. Rudensky. 2002. Cathepsin L regulates CD4+ T cell selection independently of its effect on invariant chain: a role in the generation of positively selecting peptide ligands. J. Exp. Med. 195:1349–1358.{]\j;s9, http://www.100md.com
Shi, G.P., R.A. Bryant, R. Riese, S. Verhelst, C. Driessen, Z. Li, D. Bromme, H.L. Ploegh, and H.A. Chapman. 2000. Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages. J. Exp. Med. 191:1177–1186.{]\j;s9, http://www.100md.com
Brocker, T., M. Riedinger, and K. Karjalainen. 1997. Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. J. Exp. Med. 185:541–550.3?, http://www.100md.com
Honey, K., M. Duff, C. Beers, W.H. Brissette, E.A. Elliott, C. Peters, M. Maric, P. Cresswell, and A. Rudensky. 2001. Cathepsin S regulates the expression of cathepsin L and the turnover of gamma-interferon-inducible lysosomal thiol reductase in B lymphocytes. J. Biol. Chem. 276:22573–22578.3?, http://www.100md.com
Bhattacharya, A., M.E. Dorf, and T.A. Springer. 1981. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. 127:2488–2495.3?, http://www.100md.com
Koch, N., S. Koch, and G.J. Hammerling. 1982. Ia invariant chain detected on lymphocyte surfaces by monoclonal antibody. Nature. 299:644–645.3?, http://www.100md.com
Hume, D.A., and S. Gordon. 1983. Optimal conditions for proliferation of bone marrow-derived mouse macrophages in culture: the roles of CSF-1, serum, Ca2+, and adherence. J. Cell. Physiol. 117:189–194.
Guarneri, J.J. 1977. Influence of acute exposure to cigarette smoke on the alveolar macrophage system. J. Lab. Clin. Med. 89:1215–1224.0, 百拇医药
Karasuyama, H., and F. Melchers. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97–104.0, 百拇医药
Mach, N., S. Gillessen, S.B. Wilson, C. Sheehan, M. Mihm, and G. Dranoff. 2000. Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand. Cancer Res. 60:3239–3246.0, 百拇医药
Mason, R.W., D. Wilcox, P. Wikstrom, and E.N. Shaw. 1989. The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor. Biochem. J. 257:125–129.0, 百拇医药
Cao, H., R.G. Wolff, M.S. Meltzer, and R.M. Crawford. 1989. Differential regulation of class II MHC determinants on macrophages by IFN-gamma and IL-4. J. Immunol. 143:3524–3531.0, 百拇医药
Liuzzo, J.P., S.S. Petanceska, D. Moscatelli, and L.A. Devi. 1999. Inflammatory mediators regulate cathepsin S in macrophages and microglia: a role in attenuating heparan sulfate interactions. Mol. Med. 5:320–333.
Wang, Z., T. Zheng, Z. Zhu, R.J. Homer, R.J. Riese, H.A. Chapman, Jr., S.D. Shapiro, and J.A. Elias. 2000. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J. Exp. Med. 192:1587–1600.s, 百拇医药
van's Gravesande, K.S., M.D. Layne, Q. Ye, L. Le, R.M. Baron, M.A. Perrella, L. Santambrogio, E.S. Silverman, and R.J. Riese. 2002. IFN regulatory factor-1 regulates IFN-gamma-dependent cathepsin S expression. J. Immunol. 168:4488–4494.s, 百拇医药
Fiebiger, E., P. Meraner, E. Weber, I.F. Fang, G. Stingl, H. Ploegh, and D. Maurer. 2001. Cytokines regulate proteolysis in major histocompatibility complex class II–dependent antigen presentation by dendritic cells. J. Exp. Med. 193:881–892.s, 百拇医药
Bevec, T., V. Stoka, G. Pungercic, I. Dolenc, and V. Turk. 1996. Major histocompatibility complex class II–associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L. J. Exp. Med. 183:1331–1338.s, 百拇医药
Guncar, G., G. Pungercic, I. Klemencic, V. Turk, and D. Turk. 1999. Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S. EMBO J. 18:793–803.
Lennon-Dumenil, A.M., R.A. Roberts, K. Valentijn, C. Driessen, H.S. Overkleeft, A. Erickson, P.J. Peters, E. Bikoff, H.L. Ploegh, and P. Wolf Bryant. 2001. The p41 isoform of invariant chain is a chaperone for cathepsin L. EMBO J. 20:4055–4064.}^)g1, 百拇医药
Chapman, H.A., R.J. Riese, and G.P. Shi. 1997. Emerging roles for cysteine proteases in human biology. Annu. Rev. Physiol. 59:63–88.}^)g1, 百拇医药
Felbor, U., L. Dreier, R.A. Bryant, H.L. Ploegh, B.R. Olsen, and W. Mothes. 2000. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J. 19:1187–1194.}^)g1, 百拇医药
Villadangos, J.A., R.A. Bryant, J. Deussing, C. Driessen, A.M. Lennon-Dumenil, R.J. Riese, W. Roth, P. Saftig, G.P. Shi, H.A. Chapman, et al. 1999. Proteases involved in MHC class II antigen presentation. Immunol. Rev. 172:109–120.}^)g1, 百拇医药
Nakagawa, T.Y., and A.Y. Rudensky. 1999. The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation. Immunol. Rev. 172:121–129.}^)g1, 百拇医药
Hsieh, C.S., P. deRoos, K. Honey, C. Beers, and A.Y. Rudensky. 2002. A role for cathepsin L and cathepsin S in peptide generation for MHC class II presentation. J. Immunol. 168:2618–2625.
Pierre, P., and I. Mellman. 1998. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell. 93:1135–1145.9!, http://www.100md.com
Cimerman, N., M.T. Prebanda, B. Turk, T. Popovic, I. Dolenc, and V. Turk. 1999. Interaction of cystatin C variants with papain and human cathepsins B, H and L. J. Enzyme Inhib. 14:167–174.9!, http://www.100md.com
Fox, T., E. de Miguel, J.S. Mort, and A.C. Storer. 1992. Potent slow-binding inhibition of cathepsin B by its propeptide. Biochemistry. 31:12571–12576.9!, http://www.100md.com
Carmona, E., E. Dufour, C. Plouffe, S. Takebe, P. Mason, J.S. Mort, and R. Menard. 1996. Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases. Biochemistry. 35:8149–8157.9!, http://www.100md.com
Guay, J., J.P. Falgueyret, A. Ducret, M.D. Percival, and J.A. Mancini. 2000. Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. Eur. J. Biochem. 267:6311–6318.9!, http://www.100md.com
Ni, J., M.A. Fernandez, L. Danielsson, R.A. Chillakuru, J. Zhang, A. Grubb, J. Su, R. Gentz, and M. Abrahamson. 1998. Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. J. Biol. Chem. 273:24797–24804.9!, http://www.100md.com
Halfon, S., J. Ford, J. Foster, L. Dowling, L. Lucian, M. Sterling, Y. Xu, M. Weiss, M. Ikeda, D. Liggett, et al. 1998. Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells. J. Biol. Chem. 273:16400–16408.(Courtney Beers Karen Honey Susan Fink Katherine Forbush and Alexander Rudensky)
2 Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195z]|@?5, 百拇医药
3 School of Medicine, University of Washington, Seattle, WA 98195z]|@?5, 百拇医药
Abstractz]|@?5, 百拇医药
Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Ms) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-–stimulated Ms. In addition, our studies show that the level of catL activity is significantly decreased in Ms cultured in the presence of IFN- whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-–stimulated peritoneal Ms. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Ms exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-. Thus, during a T helper cell type 1 immune response catL inhibition in Ms results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.
Key Words: cathepsin • macrophage • Ii processing • IFN- • p41f-cq, 百拇医药
Introductionf-cq, 百拇医药
MHC class II molecules expressed on the surface of APCs present proteolytic fragments of self- and foreign protein antigens to CD4+ T cells (1). MHC class II molecules assemble in the endoplasmic reticulum with the help of the chaperone molecule invariant chain (Ii), a type II glycoprotein that promotes the proper folding and assembly of the MHC class II {alpha} /ß heterodimer (2, 3). The Ii cytoplasmic tail contains a sequence targeting this complex to the lysosomal/endosomal pathway (4, 5) where it is exposed to the activity of proteases including cathepsins, which mediate cleavage of Ii in a step-wise manner, leaving only the class II–associated Ii peptide associated with the MHC class II peptide binding groove (6–8). Before MHC class II trafficking to the cell surface, the MHC-like molecule HLA-DM (H-2M in mice) mediates removal of class II–associated Ii peptide in exchange for the diverse array of self- and nonself-peptides generated in the endosomes and lysosomes (9, 10).
The cathepsins are a large family of aspartyl (D and E) and cysteinal (B, S, and L) endosomal proteinases. Recent studies have indicated that cathepsin S (catS) and cathepsin L (catL) are differentially expressed in APCs and are the only identified enzymes from this family of proteinases to play a key role in regulating MHC class II presentation (11–14). CatS is expressed in B cells and dendritic cells (DCs) and catL activity is observed in cortical thymic epithelial cells whereas in macrophages (Ms) both catL and catS activity can be detected (11–14). In B cells and DCs catS mediates the late stages of Ii degradation, specifically cleavage of the p22 and p12 Ii fragments and therefore regulates presentation of exogenous peptides in the context of MHC class II (12–14). CatL-deficient mice, however, exhibit a profound defect in CD4+ T cell selection as a result of the role played by catL in cleavage of these Ii fragments and in generation of the MHC class II bound peptides in cortical thymic epithelial cells (11, 15).
The observation that Ms express both of the cysteinal proteinases that mediate the late stages of Ii degradation has lead several groups to investigate the relative contribution of each enzyme to regulation of MHC class II presentation by these cells. One previous study reported that in alveolar and peritoneal Ms (pMs) lacking both catS and catL, degradation of Ii occurs as a result of activity of additional cysteine proteinases, potentially cathepsin F (catF; reference 16). However, we have previously observed a role for catS in regulating MHC II presentation of some exogenous antigens by Ms (13). Thus, we sought to further investigate the roles of catS and catL in regulating MHC class II presentation by Ms and determine the extent to which the functions of these two enzymes overlap. We demonstrate that catS mediates degradation of the p12 fragment of Ii in IFN-–stimulated pMs whereas catL plays no significant role in this process. In addition, we show this is a result of down-regulation of intracellular catL activity and increased catS activity in pMs after stimulation with this cytokine. This decrease in catL activity correlates with a decrease in mRNA levels, although IFN-–treated pMs maintain high levels of mature catL protein. We also found catL activity secreted upon IFN- treatment of pM, which correlates with a small increase in the amount of secreted mature catL protein. However, as the total amount of secreted catL protein (pro-form plus mature form) and the amount of intracellular mature catL protein is not altered upon IFN- treatment of pMs, we believe secretion of catL does not account for the down-modulation of catL activity. These data suggest that a specific inhibitor of catL activity is up-regulated upon IFN- stimulation of pMs. Furthermore, we also report complete inhibition of catL activity when catL protein is overexpressed in DCs, suggesting that bone marrow–derived APCs i.e., DCs and Ms, may express a catL-specific inhibitor. We found no significant evidence to suggest the p41 isoform of Ii effects such inhibition of catL activity in pMs. These results lead us to hypothesize that during a Th1 type immune response, catS activity in pMs is critical for Ii degradation and hence MHC class II antigen presentation.
Materials and Methodsr$, 百拇医药
Mice.r$, 百拇医药
C57BL/6 (B6) mice were purchased from and maintained under specific pathogen-free conditions at the University of Washington. CatS-/-, catL-/-, catS-/- x catL-/-, and Ii-/- animals were bred and maintained under these same conditions. CD11c-catL mice were generated using the CD11c-E{alpha} d construct (provided by K. Karjalainen, The Basel Institute for Immunology, Basel, Switzerland; reference 17). E{alpha} d was removed and catL cDNA was inserted into the construct by blunt end ligation using BamH1. CatL was flanked on either side by rabbit ß globin gene fragments and provides the transgene with an intron and a polyadenylation signal. To generate CD11c-catL transgenic (tg) mice, purified DNA was injected into BDF1 x B6 fertilized embryos. Offspring were backcrossed with catS-/- B6 mice. CD4+ T cell development occurred normally in the CD11c-catL tg mice and their CD4/CD8 T cell ratio was comparable to that observed in non-tg littermate control animals. All animals were used at 2–8-mo of age. All procedures and care of the animals were in accordance with University of Washington guidelines.
Antibodies.-9w, 百拇医药
The polyclonal rabbit antisera to mouse catL (provided by A. Erickson, University of North Carolina, Chapel Hill, NC) and the monoclonal antibodies M5/114 (anti-I-Ab, d, q and anti-Ed, k) and IN-1 (anti-Ii) have been described previously (18–20). Streptavidin horseradish peroxidase was purchased from .-9w, 百拇医药
M Isolation.-9w, 百拇医药
To elicit pMs, mice were injected intraperitoneally with 1 ml 4% thioglycollate ( ) and peritoneal exudate cells were collected 4–5 d later. Ms were cultured in Hydron-treated (Hydro Med Sciences) plates with RPMI-S (RPMI 1640 containing 100 u penicillin-streptomycin, 2 mM L-Glutamine [ Corporation], and 10% FBS [ ]). Bone marrow–derived Ms (BMMs) were generated by culturing bone marrow cells on bacterial plastic plates in RPMI-S with 106 units/ml M-CSF (21). Mouse alveolar Ms were harvested from lavage fluids as previously described (22). In brief, mouse lung was washed repeatedly with 1 ml of 6 mM EDTA PBS. Ms were pelleted and cultured in RPMI-S. For cytokine treatment, M populations were cultured for 48 h in the presence or absence of 0.3 ng/ml recombinant mouse IFN- (R&D Systems) or 200 U/ml recombinant mouse IL-4 (23).
DC Isolation.00[4+$, 百拇医药
Spleens were enriched for DCs as previously described (14, 24). In brief, mice were injected subcutaneously with 5 x 106 Flt3 ligand secreting B16 melanoma cells (provided by G. Dranoff, Dana-Farber Cancer Institute, Boston, MA) and spleens were harvested after the tumors reached 2 cm in diameter. Splenocytes were incubated with magnetic CD11c microbeads ( ), positively selected on an AutoMACS, and the purity of the positive fraction was assessed by flow cytometric analysis. All positive fractions were >96% CD11chi.00[4+$, 百拇医药
Cysteine Protease Active Site Labeling.00[4+$, 百拇医药
106 cells were incubated for 2 h at 37°C with 0.25 µM of the iodinated cysteine protease inhibitor CBZ-125I-Tyr-Ala-CN2 (25). This inhibitor irreversibly binds to the active site of cysteine proteases via a thiol–ester bond. Cells were washed, lysed in cell lysis buffer (0.5% NP-40, 0.15 M NaCl, 50 mM Tris-HCL, pH 7.2), supplemented with a cocktail of protease inhibitors (Roche Molecular Biochemical), and run on a 12% wt/vol SDS-PAGE gel. 125I-labeled proteins were visualized by autoradiography on Kodak BioMax Mr film. The results were quantified using the Bio-Rad GS-700 Imaging Densitometer and analyzed by Multi-Analyzer software Version 1.0.2. Alternatively, 106 cells were incubated for 2 h at 37°C with 0.25 µM of the biotinylated cysteine proteinase inhibitor biotin-Tyr-Ala-FMK, as described above. Intracellular biotin-labeled proteases were detected by immunoblotting cell lysates (see below). Extracellular cathepsin activity was analyzed by Western blot after concentrating supernatant by ultrafiltration on YM10 Centricons (Amicon) and dialysis against 20 mM Tris, pH 7.5.
Immunoblotting.!']2?, 百拇医药
M{phi} s and fibroblasts were washed in PBS and lysed as described above. Debris was removed by centrifugation at 8,000 rpm for 10 min and the lysates and supernatant were analyzed for protein content using Coomassie® Plus Protein Assay Reagent ( ). Samples containing the indicated amount of total protein or number of cell equivalents were boiled for 5 min in SDS-reducing buffer and separated by 12% SDS-PAGE. The proteins were electrophoretically transferred onto nitrocellulose membrane and this was probed using the indicated primary Ab. Binding was detected using the appropriate horseradish peroxidase–conjugated secondary Ab diluted 1:1,000 and visualized by chemiluminescence ( ).!']2?, 百拇医药
Pulse Chase Biosynthetic Radiolabeling and Immunoprecipitation.!']2?, 百拇医药
pMs were preincubated at 37°C for 2 h in methionine/cysteine-free RPMI 1640 supplemented with 200 mM L-glutamine, 10 mM Hepes, 100 µg/ml penicillin-streptomycin, and 5% dialyzed FBS. Cells were pulsed (4–8 x 106 cells/ml) for 40 min with 1 mCi/ml 35S-methionine/cysteine (Trans 35S-label; ) and chased for 0, 1, 3, and 6 h in the presence of 30x unlabeled methionine (3 mM) and cysteine (16 mM). Pulse-labeled cells were washed in PBS and lysed in 1% NP-40, 0.01 M Tris, pH 7.4, and 0.15 M NaCl supplemented with a cocktail of protease inhibitors (Roche Molecular Biochemicals). Before precipitation with the M5/114 Ab, lysates were precleared with protein G Sepharose ( ) and normal rat IgG (Caltag Laboratories Inc.). Precipitated proteins were boiled in SDS-reducing buffer and separated by 7.5–20% gradient SDS-PAGE. Gels were fixed in 50% methanol, 10% acetic acid, treated with AmplifyTM ( ), dried, and the labeled proteins were visualized by autoradiography.
RNA Extraction and cDNA Synthesis.d:.i, 百拇医药
pM{phi} s were purified by adherence to bacterial plastic plates and were lysed in RNA STAT-60 (3 ml RNA STAT-60 per 10 x 106 cells; TEL-TEST "B" INC.) directly on the plates 30 min after plating (ex vivo) and after 48 h of incubation in the presence or absence of IFN-{gamma} . RNA was extracted according to the manufacturer's protocol and contaminating DNA was removed by treating 2 µg of sample RNA with amplification grade DNase I ( ). First strand cDNA was prepared by reverse transcription using the SUPERSCRIPTTM First-Strand Synthesis System for RT-PCR, as directed by the manufacturer.d:.i, 百拇医药
Real-time PCR.d:.i, 百拇医药
Primer and probe sequences were selected with the assistance of Primer Express software ( ) using nucleotide sequences available on the GenBank Database (available from GenBank/EMBL/DDBJ under accession nos. NM_013556, , and for HPRT, catL, and catS, respectively). Primers ( ) are as follows: HPRT-F 5'-3' (5'-TGGAAAGAATGTCTTGATTGTTGAA-3'); HPRT-R 5'-3' (5'-AGCTTGCAACCTTAACCATTTTG-3'); CatL-F 5'-3' (5'-GACCGGGACAACCACTGTG-3'); CatL-R 5'-3' (5'-CTACCCATCAATTCACGAC-3'); CatS-F 5'-3' (5'-GCCATTCCTCCTTCTTCTTCTACA-3'); and CatS-R 5'-3' (5'-CAAGAACACCATGATTCACATTGC-3'). The following probes were synthesized with a 5' FAM reporter and 3' TAMRA quencher (Biosearch Technologies Inc.): HPRT (5'-6-FAM-CAAACTTTGCTTTCCCTGGTTAAGCAGTACAGC-TAMRA-3'); CatL (5'-6-FAM-CTCAGGTGTTTGAACCCATGAATCTTTTACTC-TAMRA-3'); and CatS (5'-6-FAM-AAGCGGTGTCTATGATGACCCCTCCTGTATAMRA-3').
Real-time PCR amplification of HPRT, catL, and catS was performed as previously described (18). Triplicates of each cDNA sample were amplified alongside appropriate controls and each assay was performed on at least three independent occasions. Relative quantitation of catL and catS expression was determined using the comparative CT method (user bulletin no. 2; ). This method was used to calculate the fold increase of mRNA in pM{phi} s after 48 h of incubation in the presence or absence of IFN-{gamma} compared with those cells isolated ex vivo.o|, http://www.100md.com
Resultso|, http://www.100md.com
Ii . Degradation in IFN-–stimulated Ms Is Predominately Mediated by CatS.o|, http://www.100md.com
The cysteinal lysosomal proteinases catS and catL play a role in regulating the MHC class II presentation pathway by virtue of their role as mediators of the late stages of Ii degradation (8, 11–14). These enzymes are differentially expressed in vivo in all APCs except Ms, in which both catS and catL activity can be detected. Thus, we wished to determine the relative contribution of each of these enzymes to Ii degradation and regulation of MHC class II presentation by Ms.
To elucidate the effects of catS and catL on the kinetics of Ii degradation, thioglycollate-elicited pMs isolated from B6, catL-/-, catS-/-, and catS-/- xcatL-/- mice were cultured for 48 h in the presence of IFN- to induce up-regulation of MHC class II (26). IFN-–stimulated pMs were metabolically labeled and chased for 0, 1, 3, and 6 h. Protein lysates were immunoprecipitated with an MHC class II–specific Ab and analyzed by SDS-PAGE ( A). B6 pMs showed minimal accumulation of the p12 fragment of Ii associated with MHC class II whereas in catS-/- cells marked accumulation of p12 was observed after 1 h of chase. This fragment was slowly degraded over the 6 h of chase, suggesting that in the absence of catS another proteinase is capable of Ii processing, albeit with substantially slower kinetics. The extent of p12 accumulation in catL-/- pMs was not significant compared with that observed in catS-/- pMs and therefore the delayed cleavage of Ii in the absence of catS is unlikely to be mediated by catL. In addition, in the absence of both catS and catL accumulation of p12 occurred with the same kinetics as observed in pMs lacking catS alone.
fig-ommitted|j/1.g, http://www.100md.com
Figure 1. Ii degradation in cathepsin-deficient Ms. (A) Thioglycollate-induced Ms from B6, catS-/-, catL-/-, and catS-/- xcatL-/- mice were pulse labeled and chased for 0, 1, 3, and 6 h. Cell lysates were immunoprecipitated with the I-Ab-specific Ab M5/114 and immunoprecipitates were analyzed by 7.5–20% gradient SDS-PAGE under denaturing conditions. (B) Thioglycollate-induced pMs, (C) BMMs, and (D) alveolar Ms from B6 and catS-/- mice were cultured for 48 h in the presence of IFN- and immunoblotted with the monoclonal Ab IN-1. Cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors) and lysates were then twofold serially diluted starting at 5 µg of total protein (alveolar Ms are shown at 5 µg only). The position of Ii fragments p41, p31, and p12 are indicated.|j/1.g, http://www.100md.com
To test whether the defect in Ii processing in the absence of catS was limited to pM, we examined different M populations and analyzed the steady state levels of Ii. We isolated alveolar, peritoneal, and BMMs from B6 and catS-/- mice () . This experiment revealed accumulation of p12 Ii fragments in all M types in the absence of catS. These results clearly implicate catS as the key enzyme regulating the late stages of Ii degradation in Ms. In the absence of catS, another enzyme, likely catF, might be able to degrade Ii, albeit with significantly less efficiency.
fig-ommittedw, 百拇医药
Figure 2. Active site labeling of cytokine-treated M. (A) Thioglycollate-induced pMs and (B) BMMs from B6, catS-/-, and catL-/- mice were either taken directly ex vivo or activated for 48 h with or without IFN-. Cells were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz- [125I]-Tyr-Ala-CN2. Cells were lysed and radiolabeled enzymes were analyzed on a 12% SDS-PAGE gel. Levels of active cathepsin in cells taken directly ex vivo (ex vivo) and after 48 h of plating with IFN- (IFN-) or without (UN) are shown. The position of cathepsin B (catB), cathepsin S (catS), and cathepsin L (catL) are indicated on the gel. C) The intensity of the active site labeling was quantified by the BioRad GS-700 Imaging Densitometer and analyzed by Multi-Analyzer software Version 1.0.2. The numbers correspond to the following: 1, B6 ex vivo; 2, B6 UN; 3, B6 IFN-; 4, catS-/- ex vivo; 5, catS-/- IFN-; 6, catS-/- UN; 7, catL-/- ex vivo; 8, catL-/- UN; 9, catL-/- IFN-.w, 百拇医药
IFN- Stimulation of Ms Modulates Cathepsin Activity.
The observation that catL played only a minor role in Ii degradation in IFN-–treated pMs and previous reports indicating that IFN- modulates the mRNA level of cathepsins (27–29) lead us to investigate the effect of IFN- on the level of catS and catL of activity in pMs and BMMs. IL-4 has also been shown to up-regulate expression of MHC class II on the surface of Ms (26) and it has been shown that in DCs protease activity can be modulated by both pro- and antiinflammatory cytokines (30). Therefore, we sought to examine the effects of both IFN- and IL-4 on catS and catL activity in distinct M populations.;8yk, 百拇医药
Thioglycollate-elicited pMs and BMMs were isolated from B6-, catL-/--, and catS-/--deficient mice and the level of intracellular cathepsin activity was analyzed immediately upon isolation (ex vivo) and after 48 h of culture in the presence or absence of IFN- or IL-4 (). pMs analyzed directly ex vivo had barely detectable levels of catS and a very low level of catL activity. Upon culture, in the absence of cytokines, both pMs and BMMs showed a marked increase in catL activity whereas the activity of catS increased only slightly. Interestingly, the addition of IFN- resulted in an increase in catS activity and a dramatic decrease in catL activity. We quantified the changes in catL activity after IFN-{gamma} stimulation by densitometry and found catL activity decreased between 50–100-fold ( C). IL-4, however, had no affect on cathepsin activity as compared with Ms cultured in the absence of cytokines (unpublished data; see ). These data suggest our previous observation that catS but not catL was critical for regulating the late stages of Ii cleavage in IFN-–stimulated Ms can be explained by the effect of this cytokine on down-modulating catL activity.
fig-ommittedqs5tz(w, http://www.100md.com
Figure 4. CatL and the effect of the p41 Ii isoform. The level of catL in (A) B6 and (C) Ii-/- thioglycollate-induced pMs cultured for 48 h in the presence or absence of IFN- was detected by immunoblotting with a catL-specific polyclonal antiserum. Cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors) and lysates were then twofold serially diluted starting at 40 µg of total protein. Arrows indicate pro-catL and mature catL. These experiments were performed three times and yielded identical results. (B) Thioglycollate-induced pMs from B6 mice were taken directly ex vivo or activated for 48 h in the presence or absence of IFN-. Cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors). 20 µg of lysate was separated on a 12% SDS-PAGE and immunoblotted with the IN-1 Ab. p41 and p31 are indicated. (D) Thioglycollate-induced pMs and (E) BMMs from B6 and Ii-/- mice were cultured for 48 h in the presence or absence of IFN-. 106 cells were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2, lysed, and separated by 12% SDS-PAGE. Levels of active cathepsin in cells taken directly ex vivo (ex vivo) and after 48 h of plating with IFN- (IFN-) or without (UN) are shown. Arrows indicate the positions of cathepsin B (catB), cathepsin S (catS), and cathepsin L (catL).
fig-ommittedio\, 百拇医药
Figure 5. Secretion of catL upon IFN-{gamma} treatment. B6 thioglycollate-induced pMs taken directly ex vivo or cultured for 48 h in the presence or absence of IFN- or IL-4 were incubated serum free with the cysteine proteinase inhibitor biotin-Tyr-Ala-FMK for 2 h. Labeled cathepsin present (A) intracellularly and (B) in the supernatant were detected by immunoblotting. (C) Cells were incubated for 2 h in serum-free medium, the cells were removed, and the inhibitor biotin-Tyr-Ala-FMK was added for 20 min before analysis of labeled cathepsins by immunoblotting. (D) Thioglycollate-elicited pMs cultured for 48 h in the presence or absence of IFN- were incubated for 2 h in serum-free medium. 5 µg of total supernatant protein were titrated and separated before catL immunoblotting.io\, 百拇医药
We have previously observed that the level of mature catL protein in B cells is regulated by the presence or absence of catS (18). Thus, we wished to determine whether such regulation of the cysteinal proteinases was involved in the changes in cathepsin activity observed in both pMs and BMMs cultured in the presence of cytokines. Modulation of catL activity after IFN- stimulation of catS-deficient Ms was comparable to that observed in B6-derived cells (), implying that in Ms the activities of catS and catL are regulated independently upon IFN- stimulation.
CatL mRNA Levels Are Decreased upon IFN- Stimulation of pMs.{'ejm, http://www.100md.com
Our finding that catL activity was decreased upon IFN- stimulation of pMs lead us to investigate the mechanisms by which this down-modulation was regulated. In view of several studies in which it has been reported that in a variety of cell types cathepsin mRNA levels are altered by IFN- stimulation (27–29), we sought to determine whether transcription of catL mRNA was decreased in pMs upon IFN- stimulation.{'ejm, http://www.100md.com
pMs were purified by adherence to tissue culture plates and mRNA was isolated from cells directly ex vivo and after culturing for 48 h in the presence or absence of IFN-. Real time PCR amplification and subsequent quantitative analysis indicated that the level of catL mRNA in IFN-–stimulated pMs was decreased approximately eightfold compared with cells analyzed directly ex vivo () . Furthermore, we were unable to detect any significant change in the level of catL mRNA in cells cultured in the absence of cytokine. In addition, we analyzed catS mRNA levels in pMs and observed no significant changes with or without cytokine treatment. These results are in agreement with previously published data indicating that catL mRNA is decreased in microglial cells stimulated with IFN- (27) and suggests that the IFN-–induced decrease in catL activity might in part be a result of decreased transcription of catL mRNA.
fig-ommitted/vae@9f, 百拇医药
Figure 3. Regulation of catL activity by mRNA. pMs were purified by adherence to bacterial plastic plates. Cells were lysed directly on the plates 30 min after plating (ex vivo) and after 48 h of incubation in the presence or absence of IFN-. After RNA extraction and cDNA synthesis, quantitative PCR was performed in an ABI Prism 7700 sequence detector. Primers and probes for HPRT, catL and catS were designed using Primer Express software. The probes were labeled at the 5' and 3' ends with the fluorochromes FAM and TAMRA. mRNA levels were quantitated using the comparative CT method described in the ABI Prism 7700 sequence detector user bulletin number 2. The data is shown as the level of cathepsin mRNA in cells after 48 h of plating with IFN- (IFN-) or without (UN) compared with the level of mRNA ex vivo./vae@9f, 百拇医药
Mature CatL Protein Levels Are Not Reduced in IFN-–treated pMs./vae@9f, 百拇医药
Having demonstrated that the decrease in catL activity in IFN-–stimulated pMs correlated with diminished transcription of catL mRNA, we wished to establish whether there was a corresponding decrease in intracellular catL protein levels. We performed a twofold serial dilution of cell lysate derived from B6 pMs isolated directly ex vivo or cultured for 48 h in the presence or absence of IFN-. Lysates were separated by SDS-PAGE and analyzed by immunoblotting for catL protein ( A). Unexpectedly, we observed that the amount of mature catL protein detected in pMs cultured in the presence or absence of IFN- was comparable and that this level of protein was substantially greater than that detected in ex vivo–isolated cells. A decrease in the amount of the 38 kD pro-form of catL was observed, however, in IFN-–stimulated pMs when compared with cells analyzed directly ex vivo. Thus, the reduction in catL mRNA upon IFN- stimulation of pMs resulted in a decrease in pro-form catL protein although there was no significant concomitant decrease in the level of mature catL protein, presumably as a result of the long half-life of this protein. Taken together, our results demonstrating that catL activity is substantially diminished upon IFN- stimulation of pMs whereas levels of mature catL protein are increased suggest an inhibitor of catL might be up-regulated in these cells.
Modulation of CatL Activity in IFN-–stimulated pMs Occurs in the Absence of Ii.n, 百拇医药
Several molecules have been implicated as inhibitors of catL, including the p41 isoform of Ii (31–33). Previous studies have shown that Ii is up-regulated in response to IFN- (20), making p41 an attractive candidate inhibitor of catL in pMs cultured in the presence of this cytokine. Thus, we examined the regulation of catL in thioglycollate-elicited pMs isolated from Ii-/- mice.n, 百拇医药
To first test whether p41 could be an inhibitor of catL activity in IFN-–stimulated pMs, we analyzed by immunoblotting the level of Ii protein in B6 pMs isolated directly ex vivo or cultured in the presence or absence of IFN- for 48 h ( B). Upon IFN- stimulation the p41 isoform of Ii was substantially up-regulated whereas the increase in p31 was more moderate. No such change in the level of either p31 or p41 was observed when cells were cultured in the absence of cytokine. This increase in p41 upon IFN-{gamma} stimulation of pMs provided us with evidence that p41 was a viable candidate inhibitor of catL.
It has previously been reported that the level of mature catL protein detected in BMMs is significantly decreased in the absence of the p41 isoform of Ii (33). Therefore, we sought to determine whether a similar defect could be observed in Ii-/- pMs. The level of catL protein in pMs isolated from Ii-/- mice was analyzed by serial dilution of cell lysates and immunoblotting as described above. The level of mature catL protein in Ii-/- pMs was increased upon culturing the cells in the presence or absence of IFN- ( C), as observed in B6 pMs ( A). The extent of up-regulation of mature catL in cells cultured in the absence of cytokine was the same for both Ii-/- and B6-derived cells. However, the amount of mature catL protein detected in Ii-/- cells cultured in the presence of IFN- was two- to threefold less than in cells cultured in the absence of this cytokine and also two- to threefold less than in B6 pMs cultured in IFN- ( A). Taken together, these data suggest that p41 may play some role in stabilizing the mature form of catL in IFN-–stimulated pMs as has been suggested for BMMs (33), however, we observed no role for p41 in maintaining the level of mature catL protein in pMs isolated directly ex vivo or cultured in the absence of cytokines.
Having observed that Ii-/- pMs stimulated with IFN- express noticeably lower levels of mature catL protein than B6 cells cultured under the same conditions, we aimed to establish whether Ii regulated the decrease in catL activity detected upon IFN- stimulation of B6 Ms. pMs and BMMs from B6 and Ii-/- mice were isolated directly ex vivo or cultured for 48 h in the presence or absence of IFN- or IL-4. Intracellular cathepsin activity was detected using the irreversible cysteine protease inhibitor Cbz-125I-Tyr-Ala-CN2 and the proteins were separated by SDS-PAGE (). The modulation of cathepsin activity we observed was the same in both cell types. After culture of cells in the absence of cytokine catL activity was up-regulated and in the presence of IFN- this up-regulation was not observed. In addition, catL activity was up-regulated in cells cultured in the presence of IL-4. These results provide no evidence for a major role for the p41 isoform of Ii in regulating catL activity in pMs and BMMs although it may play a modest role in stabilizing the levels of mature catL protein in IFN-–stimulated Ms.
CatL Is Secreted When Ms Are Treated with IFN-.7r(;.5x, 百拇医药
Cathepsins are soluble proteinases and specific secretion of catL may explain the decrease in intracellular catL activity we observed upon IFN- stimulation of pMs. To address this possibility we compared intracellular and extracellular cathepsin activity using a new biotinylated active site labeling reagent, biotin-Tyr-Ala-FMK. pMs were labeled for 2 h at 37°C with the biotinylated inhibitor and labeled enzymes in the culture supernatant and cell lysates were detected by Western blot analysis. As shown in , catL activity was markedly increased in the culture supernatant upon IFN- treatment whereas intracellular catL activity sharply decreased. CatL activity was barely detectable in supernatant of untreated or IL-4–treated pMs. In contrast, both extracellular and intracellular catS activity increased upon treatment with IFN-. It is important to note that endocytic uptake can be a rate-limiting step, thus, the efficiency of the active site labeling of intracellular enzymes is significantly less efficient than that of extracellular enzymes. Therefore, we assessed the overall amount of catL protein secreted by IFN-–induced pMs by Western blot analysis with an anti-catL Ab ( D). We observed a two- to fourfold increase in mature catL whereas pro-catL levels are decreased in the supernatant of IFN-–treated cells as compared with the cells cultured in the absence of cytokine. In these experiments analyzing catL levels in culture supernatants, we used 7–10-fold more cell equivalents per lane as compared with intracellular catL analysis. Taken together with intracellular mature catL protein levels not changing, these experiments indicate that secretion of catL protein cannot account for the significant loss in catL activity upon IFN- stimulation of pMs.
Cathepsins have been implicated in inflammatory extracellular matrix remodeling, tumor metastasis, and angiogenesis (34) and catL has a potent elastinolytic activity (35). We wanted to determine if the secreted enzymes are active extracellularly by labeling supernatants of pM cultures with biotin-Tyr-Ala-FMK at neutral pH and found that both secreted catL and catS are active at physiological pH ( C). This data suggests that secreted catL may play a role in extracellular matrix remodeling at the sites of inflammation.%, 百拇医药
CatL Activity Is Inhibited When Overexpressed in DCs.%, 百拇医药
B cells and DCs exhibit no catL activity and therefore utilize catS for late stage Ii chain processing whereas Ms are the only APC in which active catS and catL can be detected concurrently. In light of our observation that catL plays no significant role in Ii degradation in Ms, we sought to investigate the effect of catL expression in a second catS-dependent APC. tg mice were generated using the CD11c promoter to specifically overexpress catL in DCs. To rule out any effect of catS upon MHC class II maturation in these cells, these mice were crossed onto a catS-/- background.
We wished to determine the level of catL protein expressed in DCs isolated from tg mice and non-tg littermate control animals. Highly purified DCs were isolated and protein lysates from the indicated number of cells were analyzed for catL protein () . B6 DCs expressed significantly less catL protein than the equivalent number of CD11c-catL tg DCs, indicating that the CD11c promoter efficiently induces overexpression of catL protein in DCs. To our surprise, using the Cbz-125I-Tyr-Ala-CN2 irreversible inhibitor we were unable to detect catL activity in the CD11c-catL tg DCs in which we observed high levels of mature catL protein. We observed the same results using DCs isolated from progeny of a second founder CD11c-catL tg mouse (unpublished data).6*#, 百拇医药
fig-ommitted6*#, 百拇医药
Figure 6. Inhibition of catL activity in CD11c-catL tg mice. DCs purified from Flt3 ligand–treated CD11c-catL tg mice were twofold serially diluted starting at 106 cells. Transformed murine embryonic fibroblasts were twofold serially diluted starting at 2.5 x 105 cells. For Western blot analysis, cells were solubilized in lysis buffer (1% NP-40/TBS in the presence of protease inhibitors) and lysates were separated by 12% SDS-PAGE and immunoblotted for catL. Pro-catL and mature catL are indicated. For active site labeling, cells were incubated for 2 h with the irreversible cysteine protease inhibitor Cbz-[125I]-Tyr-Ala-CN2. Cells were lysed and radiolabeled enzymes were analyzed on a 12% SDS-PAGE gel. Arrows indicate active catL.
To ensure that the level of mature catL protein expressed by the CD11c-catL tg DCs generates sufficient signal to be detected in our active site labeling assay, we compared catL protein and activity in tg DCs to fibroblasts, a cell type known to express high levels of catL activity (11). We were able to detect both mature catL protein and catL activity in as few as 8,000 fibroblast cells (). However, in DCs expressing comparable catL protein levels to 1.25 x 105 fibroblasts we were unable to detect catL activity. Thus, the quantity of mature catL protein expressed in the CD11c-catL tg DCs is not below the level of detection in our active site labeling assay, indicating that DCs express a specific inhibitor of catL activity.px^0v, 百拇医药
Discussionpx^0v, 百拇医药
The lysosomal cysteinal proteinases catS and catL have previously been shown to play a critical role in the late stages of Ii degradation in DCs, B cells, and cortical thymic epithelial cells (36, 37). In these cells, activity of only one enzyme can be detected and thus the relative role of catS and catL in regulating Ii processing cannot be assessed. Ms provide an ideal APC in which to study whether catS and catL are redundant enzymes in the regulation of MHC class II presentation, as activity of both enzymes can be detected in these cells. Here, we report that in thioglycollate-elicited pMs stimulated with IFN-, catS is principally responsible for mediating Ii cleavage whereas catL plays little part in this process. This result is surprising given that efficient Ii proteolysis in cortical thymic epithelial cells requires catL and that in MHC class II–expressing fibroblast cell lines effective Ii cleavage can be mediated by catL (11, 38). However, an explanation for our results is provided by our subsequent observation that although intracellular activity of catL is profoundly down-regulated in pMs in response to the proinflammatory cytokine IFN-, intracellular catS activity increases. This modulation in catL activity in IFN-–stimulated pMs coincided with a decrease in catL mRNA levels whereas catS mRNA levels were not diminished. These data indicate that modulation of catL activity in pMs in response to IFN- is in part regulated at the level of transcription as has previously been reported for microglial cells (27). Down-regulation of catL mRNA was concomitant with a decrease in pro-catL, however, levels of the mature protein were not decreased in IFN-–stimulated pMs. One possible explanation for this maintenance of high levels of mature catL protein despite decreased mRNA is that the mature enzyme is long lived in endosomes. Additional studies showed that the decrease in catL activity upon IFN- stimulation was concomitant with an increase in secretion of mature catL protein that exhibited protease activity. However, this increase in secretion had little effect on intracellular catL protein levels and therefore, secretion of mature protein does not explain the specific inhibition of intracellular catL activity. Taken together with our observation that intracellular catL activity decreases in IFN-–stimulated pMs whereas levels of the mature protein are maintained, these results suggest that an inhibitor(s) of enzyme activity is increased in response to this proinflammatory cytokine and is critical for regulating catL activity in pMs and BMMs.
A number of lysosomal cysteinal proteinases inhibitors have been identified, including cystatin C (39, 40), the propeptide regions of the cathepsins (41–43), and the p41 isoform of Ii (31, 32). We found no difference in cystatin C expression or localization in pMs cultured in the presence or absence of IFN- (unpublished data), indicating that this cysteinal proteinase inhibitor plays no significant role in regulating catL activity upon IFN- stimulation of pMs. In addition, the propeptide regions of catS and cathepsin B (catB) do not affect the level of catL activity in Ms treated with IFN-. We examined catS- and catB-deficient pMs and BMMs and were unable to determine any differences in catL activity with or without treatment of IFN- when compared with B6 Ms ( and unpublished data). Recent studies have reported that p41 is not only an inhibitor of catL enzymatic activity but that it is also required for catL activity in BMMs where it acts as a chaperone, stabilizing the mature form of this enzyme (33). Because both MHC class II and Ii, including p41, are up-regulated in pMs stimulated with IFN-, we examined whether p41 and its fragments were responsible for the observed down-regulation of catL activity and preservation of high levels of mature protein in these cells. We observed that levels of mature catL protein in Ii-/- and B6 pMs and BMMs were identical, except upon IFN- stimulation when catL levels were two- to threefold lower in Ii-/- cells compared with B6 cells. However, we detected equivalent levels of catL activity in Ii-/- and B6 pMs and BMMs cultured in the presence or absence of IFN-. Furthermore, the extent to which catL activity was down-regulated upon IFN- stimulation was equivalent in Ii-/- and B6 cells. These data demonstrate that Ii does not play an important role in regulating catL activity in either thioglycollate-induced pMs or BMMs and are in agreement with our previous observations in thymic stromal cells (15). However, these results suggest that Ii does play a minor role in stabilizing the mature form of catL in pMs, as has been previously suggested by others for BMMs (33). Another possible inhibitor of catL activity is cystatin F, which is specifically expressed in hematopoietic cells and preferentially binds catL and papain in vitro (44, 45). We believe cystatin F could potentially be involved in the catL inhibition observed in Ms or DCs and would like to investigate this in the future. Thus, although it is possible that p41 may contribute modestly to the observed down-modulation of intracellular catL activity upon IFN- stimulation of pMs, we suggest that an unknown inhibitor is the pivotal regulator of catL enzymatic activity in pMs.
We have previously reported that catL activity cannot be detected in ex vivo–isolated DCs (11, 18), however, significant levels of catL activity can be detected in DCs derived from bone marrow in culture (unpublished data). In view of our observations implying that catL activity in pMs is regulated in a highly specific manner by an as yet unidentified inhibitor, we sought to examine whether such a mechanism was present in ex vivo DCs. Analysis of DCs isolated from the spleen of mice engineered to express catL under the control of the CD11c promoter revealed significant levels of mature catL protein but no detectable catL activity in these cells. Expression of this catL cDNA when driven by a retroviral promoter in fibroblasts generated high levels of catL activity (38), indicating that inhibition of enzymatic activity was specific to ex vivo–isolated DCs. Thus, we have shown that both DCs and pMs use a mechanism of catL inhibition although the nature of the catL inhibitor in DCs and pMs remains to be identified.
Our data indicate that catL activity is down-regulated upon IFN- stimulation of pMs and that catS is the predominant mediator of the late stages of Ii degradation, and hence MHC class II peptide presentation. In addition, catS mediates Ii degradation in other M populations. These observations appear to differ from a recent report in which it was shown that other cysteinal proteinases, perhaps catF, are able to compensate for the absence of catS and catL and elicit degradation of Ii in pMs and alveolar Ms (16). However, in this latter study, Ii processing was analyzed immediately after pulse and after an overnight chase. Thus, the pronounced accumulation of MHC class II–associated p12 Ii fragments we observe between 1 and 6 h of chase was not detected. The significantly delayed kinetics of Ii degradation and accumulation of MHC class II–bound Ii fragments in catS-deficient pMs affect the efficiency of generation of MHC class II–peptide complexes and thus account for the previously reported defect in MHC class II presentation of some but not other protein antigens (13). Therefore, we believe that while although enzymes can mediate Ii cleavage with delayed kinetics, catS plays a major role in regulating Ii degradation and MHC class II presentation in Ms.
In conclusion, we have shown that in pMs, in which both cysteinal proteinases catS and catL are expressed, catS is the predominant enzyme processing Ii. Furthermore, catL is shown to have no major role in this process as a result of substantial down-regulation of its enzymatic activity upon IFN- stimulation. This decline in catL activity correlates with a decrease in catL mRNA and an increase in secretion of mature catL protein but not a decrease in intracellular mature catL protein levels, suggesting that catL activity in IFN-–stimulated pMs is regulated by a specific inhibitor. In addition, we find the p41 isoform of Ii does not contribute significantly to this regulation of catL activity in pMs and that DCs also use a catL-specific inhibition mechanism. The results presented here indicate that upon IFN- activation of pMs, enzymatic activity of catL is specifically inhibited, such that catS mediates Ii degradation and regulates MHC class II maturation. Thus, we suggest that as a result of differential regulation of catL and catS the latter enzyme governs MHC class II presentation by APCs in secondary lymphoid organs.
Acknowledgmentsahdq-+c, http://www.100md.com
We would like to thank C. Plata and N. Li for their excellent care of the mice used in these experiments.ahdq-+c, http://www.100md.com
This work was funded by the Howard Hughes Medical Institute (A. Rudensky) and grants from the National Institutes of Health (NIH) to A. Rudensky. C. Beers is supported by an NIH pre-doctoral training grant. S. Fink is affiliated with the Medical Science Training Program at the University of Washington.ahdq-+c, http://www.100md.com
Submitted: June 14, 2002ahdq-+c, http://www.100md.com
Revised: October 18, 2002ahdq-+c, http://www.100md.com
Accepted: December 3, 2002ahdq-+c, http://www.100md.com
Footnotesahdq-+c, http://www.100md.com
C. Beers and K. Honey contributed equally to this work.ahdq-+c, http://www.100md.com
* Abbreviations used in this paper: B6, C57BL/6; BMM, bone marrow–derived macrophage; catB, cathepsin B; catF, cathepsin F; catL, cathepsin L; catS, cathepsin S; DC, dendritic cell; Ii, invariant chain; M, macrophage; pM, peritoneal M; tg, transgenic.
Referencesu3m]6t#, 百拇医药
Cresswell, P. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259–293.u3m]6t#, 百拇医药
Anderson, M.S., and J. Miller. 1992. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules. Proc. Natl. Acad. Sci. USA. 89:2282–2286.u3m]6t#, 百拇医药
Cresswell, P. 1996. Invariant chain structure and MHC class II function. Cell. 84:505–507.u3m]6t#, 百拇医药
Bakke, O., and B. Dobberstein. 1990. MHC class II-associated invariant chain contains a sorting signal for endosomal compartments. Cell. 63:707–716.u3m]6t#, 百拇医药
Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S.L. Schmid, V. Quaranta, and P.A. Peterson. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature. 348:600–605.u3m]6t#, 百拇医药
Neefjes, J.J., and H.L. Ploegh. 1992. Intracellular transport of MHC class II molecules. Immunol. Today. 13:179–184.u3m]6t#, 百拇医药
Maric, M.A., M.D. Taylor, and J.S. Blum. 1994. Endosomal aspartic proteinases are required for invariant-chain processing. Proc. Natl. Acad. Sci. USA. 91:2171–2175.
Riese, R.J., P.R. Wolf, D. Bromme, L.R. Natkin, J.A. Villadangos, H.L. Ploegh, and H.A. Chapman. 1996. Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. Immunity. 4:357–366.}'1(!(k, 百拇医药
Denzin, L.K., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading. Cell. 82:155–165.}'1(!(k, 百拇医药
Martin, W.D., G.G. Hicks, S.K. Mendiratta, H.I. Leva, H.E. Ruley, and L. Van Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection. Cell. 84:543–550.}'1(!(k, 百拇医药
Nakagawa, T., W. Roth, P. Wong, A. Nelson, A. Farr, J. Deussing, J.A. Villadangos, H. Ploegh, C. Peters, and A.Y. Rudensky. 1998. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science. 280:450–453.}'1(!(k, 百拇医药
Riese, R.J., R.N. Mitchell, J.A. Villadangos, G.P. Shi, J.T. Palmer, E.R. Karp, G.T. De Sanctis, H.L. Ploegh, and H.A. Chapman. 1998. Cathepsin S activity regulates antigen presentation and immunity. J. Clin. Invest. 101:2351–2363.
Nakagawa, T.Y., W.H. Brissette, P.D. Lira, R.J. Griffiths, N. Petrushova, J. Stock, J.D. McNeish, S.E. Eastman, E.D. Howard, S.R. Clarke, et al. 1999. Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice. Immunity. 10:207–217.{]\j;s9, http://www.100md.com
Shi, G.P., J.A. Villadangos, G. Dranoff, C. Small, L. Gu, K.J. Haley, R. Riese, H.L. Ploegh, and H.A. Chapman. 1999. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity. 10:197–206.{]\j;s9, http://www.100md.com
Honey, K., T. Nakagawa, C. Peters, and A. Rudensky. 2002. Cathepsin L regulates CD4+ T cell selection independently of its effect on invariant chain: a role in the generation of positively selecting peptide ligands. J. Exp. Med. 195:1349–1358.{]\j;s9, http://www.100md.com
Shi, G.P., R.A. Bryant, R. Riese, S. Verhelst, C. Driessen, Z. Li, D. Bromme, H.L. Ploegh, and H.A. Chapman. 2000. Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages. J. Exp. Med. 191:1177–1186.{]\j;s9, http://www.100md.com
Brocker, T., M. Riedinger, and K. Karjalainen. 1997. Targeted expression of major histocompatibility complex (MHC) class II molecules demonstrates that dendritic cells can induce negative but not positive selection of thymocytes in vivo. J. Exp. Med. 185:541–550.3?, http://www.100md.com
Honey, K., M. Duff, C. Beers, W.H. Brissette, E.A. Elliott, C. Peters, M. Maric, P. Cresswell, and A. Rudensky. 2001. Cathepsin S regulates the expression of cathepsin L and the turnover of gamma-interferon-inducible lysosomal thiol reductase in B lymphocytes. J. Biol. Chem. 276:22573–22578.3?, http://www.100md.com
Bhattacharya, A., M.E. Dorf, and T.A. Springer. 1981. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. 127:2488–2495.3?, http://www.100md.com
Koch, N., S. Koch, and G.J. Hammerling. 1982. Ia invariant chain detected on lymphocyte surfaces by monoclonal antibody. Nature. 299:644–645.3?, http://www.100md.com
Hume, D.A., and S. Gordon. 1983. Optimal conditions for proliferation of bone marrow-derived mouse macrophages in culture: the roles of CSF-1, serum, Ca2+, and adherence. J. Cell. Physiol. 117:189–194.
Guarneri, J.J. 1977. Influence of acute exposure to cigarette smoke on the alveolar macrophage system. J. Lab. Clin. Med. 89:1215–1224.0, 百拇医药
Karasuyama, H., and F. Melchers. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97–104.0, 百拇医药
Mach, N., S. Gillessen, S.B. Wilson, C. Sheehan, M. Mihm, and G. Dranoff. 2000. Differences in dendritic cells stimulated in vivo by tumors engineered to secrete granulocyte-macrophage colony-stimulating factor or Flt3-ligand. Cancer Res. 60:3239–3246.0, 百拇医药
Mason, R.W., D. Wilcox, P. Wikstrom, and E.N. Shaw. 1989. The identification of active forms of cysteine proteinases in Kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabelled inhibitor. Biochem. J. 257:125–129.0, 百拇医药
Cao, H., R.G. Wolff, M.S. Meltzer, and R.M. Crawford. 1989. Differential regulation of class II MHC determinants on macrophages by IFN-gamma and IL-4. J. Immunol. 143:3524–3531.0, 百拇医药
Liuzzo, J.P., S.S. Petanceska, D. Moscatelli, and L.A. Devi. 1999. Inflammatory mediators regulate cathepsin S in macrophages and microglia: a role in attenuating heparan sulfate interactions. Mol. Med. 5:320–333.
Wang, Z., T. Zheng, Z. Zhu, R.J. Homer, R.J. Riese, H.A. Chapman, Jr., S.D. Shapiro, and J.A. Elias. 2000. Interferon gamma induction of pulmonary emphysema in the adult murine lung. J. Exp. Med. 192:1587–1600.s, 百拇医药
van's Gravesande, K.S., M.D. Layne, Q. Ye, L. Le, R.M. Baron, M.A. Perrella, L. Santambrogio, E.S. Silverman, and R.J. Riese. 2002. IFN regulatory factor-1 regulates IFN-gamma-dependent cathepsin S expression. J. Immunol. 168:4488–4494.s, 百拇医药
Fiebiger, E., P. Meraner, E. Weber, I.F. Fang, G. Stingl, H. Ploegh, and D. Maurer. 2001. Cytokines regulate proteolysis in major histocompatibility complex class II–dependent antigen presentation by dendritic cells. J. Exp. Med. 193:881–892.s, 百拇医药
Bevec, T., V. Stoka, G. Pungercic, I. Dolenc, and V. Turk. 1996. Major histocompatibility complex class II–associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L. J. Exp. Med. 183:1331–1338.s, 百拇医药
Guncar, G., G. Pungercic, I. Klemencic, V. Turk, and D. Turk. 1999. Crystal structure of MHC class II-associated p41 Ii fragment bound to cathepsin L reveals the structural basis for differentiation between cathepsins L and S. EMBO J. 18:793–803.
Lennon-Dumenil, A.M., R.A. Roberts, K. Valentijn, C. Driessen, H.S. Overkleeft, A. Erickson, P.J. Peters, E. Bikoff, H.L. Ploegh, and P. Wolf Bryant. 2001. The p41 isoform of invariant chain is a chaperone for cathepsin L. EMBO J. 20:4055–4064.}^)g1, 百拇医药
Chapman, H.A., R.J. Riese, and G.P. Shi. 1997. Emerging roles for cysteine proteases in human biology. Annu. Rev. Physiol. 59:63–88.}^)g1, 百拇医药
Felbor, U., L. Dreier, R.A. Bryant, H.L. Ploegh, B.R. Olsen, and W. Mothes. 2000. Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J. 19:1187–1194.}^)g1, 百拇医药
Villadangos, J.A., R.A. Bryant, J. Deussing, C. Driessen, A.M. Lennon-Dumenil, R.J. Riese, W. Roth, P. Saftig, G.P. Shi, H.A. Chapman, et al. 1999. Proteases involved in MHC class II antigen presentation. Immunol. Rev. 172:109–120.}^)g1, 百拇医药
Nakagawa, T.Y., and A.Y. Rudensky. 1999. The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation. Immunol. Rev. 172:121–129.}^)g1, 百拇医药
Hsieh, C.S., P. deRoos, K. Honey, C. Beers, and A.Y. Rudensky. 2002. A role for cathepsin L and cathepsin S in peptide generation for MHC class II presentation. J. Immunol. 168:2618–2625.
Pierre, P., and I. Mellman. 1998. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell. 93:1135–1145.9!, http://www.100md.com
Cimerman, N., M.T. Prebanda, B. Turk, T. Popovic, I. Dolenc, and V. Turk. 1999. Interaction of cystatin C variants with papain and human cathepsins B, H and L. J. Enzyme Inhib. 14:167–174.9!, http://www.100md.com
Fox, T., E. de Miguel, J.S. Mort, and A.C. Storer. 1992. Potent slow-binding inhibition of cathepsin B by its propeptide. Biochemistry. 31:12571–12576.9!, http://www.100md.com
Carmona, E., E. Dufour, C. Plouffe, S. Takebe, P. Mason, J.S. Mort, and R. Menard. 1996. Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases. Biochemistry. 35:8149–8157.9!, http://www.100md.com
Guay, J., J.P. Falgueyret, A. Ducret, M.D. Percival, and J.A. Mancini. 2000. Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. Eur. J. Biochem. 267:6311–6318.9!, http://www.100md.com
Ni, J., M.A. Fernandez, L. Danielsson, R.A. Chillakuru, J. Zhang, A. Grubb, J. Su, R. Gentz, and M. Abrahamson. 1998. Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. J. Biol. Chem. 273:24797–24804.9!, http://www.100md.com
Halfon, S., J. Ford, J. Foster, L. Dowling, L. Lucian, M. Sterling, Y. Xu, M. Weiss, M. Ikeda, D. Liggett, et al. 1998. Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells. J. Biol. Chem. 273:16400–16408.(Courtney Beers Karen Honey Susan Fink Katherine Forbush and Alexander Rudensky)