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* Department of Medicine II, University of Tübingen, Germany;
Leiden Institute of Chemistry, Gorleaus Laboratory, The Netherlands; and
Institute of Physiological Chemistry, University of Halle, Germany
Correspondence: Christoph Driessen, MNF Universität Tübingen, Ob dem Himmelreich 7, 72074 Tübingen, Germany. E-mail: christoph.driessen{at}uni-tuebingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: antigen presentation • human APC • endocytic proteases • MHC class II • endocytic compartments
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Different types of murine APC are equipped with distinct patterns of Cat activity in their endocytic compartments [2 , 5 ]. These control the degradation of the major histocompatibility complex (MHC) class II invariant chain (Ii) and the proteolytic processing of exogenous antigen, both essential tasks for antigen presentation [6 7 8 ]. Processing of Ii is closely linked to subcellular transport of MHC class II, features well characterized in murine DC [9 , 10 ]: Ii occupies the peptide-binding groove of class II and delivers the classII/Ii complex to the endocytic compartment, guided by a lysosomal targeting signal in the N terminus of Ii [11 ]. In late endosomal/prelysosomal compartments, Ii is processed by Cat in a stepwise manner, leading to an intermediate of 10 kD (Iip10), which consists of the class II-binding peptide core (CLIP, class II-associated invariant chain peptide) attached to the targeting sequence [12 ]. Elimination of the N-terminal portion of Iip10 by Cat (CatS, -L, -F, -V in different types of APC but not CatD, CatB, or CatH; refs. [5 , 6 , 13 , 14 ]), presumably under control of the endogenous Cat inhibitor cystatin C (CysC) [10 ], is required before loading of antigenic peptide can occur. If the proteolytic machinery fails to eliminate the N terminus of Ii, class II molecules are routed to lysosomes, where they are degraded [9 , 10 ]. This has led to the concept that class II-mediated T cell activation might be selectively influenced by pharmacological manipulation of Cat activity [2 , 6 ].
Location and activity of Cat in endocytic compartments of APC must be well balanced to allow such complex interactions between subcellular transport and protein degradation as a prerequisite for class II function in APC [15 ]. Currently, neither the set of active proteases nor the subcellular distribution of Cat activity in relation to key molecules for class II maturation and transport (Ii and CysC) has been characterized in human peripheral monocytes, as well as in any other human primary type of APC. As the CatS, -L, and -B are involved in the processing of antigen or Ii in intact cells, we focused on these proteases in our analysis [7 , 8 , 14 , 16 ]. In addition, CatH was included as a Cat that might have a special function in monocytes, as suggested by its unusual subcellular localization in a monocyte cell line [17 ]. In general, it is unknown to what extent human monocytoid cell lines could serve as experimental models to characterize the functional biology of endocytic Cat in human monocytes or to what extent Cat activity is affected during monocyte development.
The activity of Cat cannot be deduced from the mere intracellular presence of the mature polypeptide. Endogenous inhibitors such as CysC or propeptides, which occupy the catalytic cleft in a noncovalent manner, functionally inactivate the mature enzyme without affecting its detection by Western blot [1 , 18 ]. Novel tools that allow detection and isolation of Cat polypeptides in an activity-dependent way have been introduced recently to obtain an activity-based picture of Cat biology [19 ]. This is the first report where human primary leukocytes were subjected to this class of reagents.
The aims of this work were to identify the activity pattern of the major cysteine Cat (CatL, -B, -S, and -H) in primary human peripheral blood monocytes; to judge to what extent this pattern is altered in monocyte-precursor cell lines at different stages of development; and to characterize the subcellular anatomy of Cat activity in primary monocytes in relation to key molecules of MHC class II function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Anti-Cat antisera were generated by E. Weber against recombinant Cat, antitransferrin receptor (TrfR), and antilysosome-associated membrane protein (LAMP)-1 antibodies, a late endocytic marker, were purchased from PharMingen (San Diego, CA); the polyclonal serum against class II ß-chain and the PIN-1 antibody (Ab) were obtained from Hidde Ploegh (Harvard Medical School, Boston, MA); and the rabbit anti-CysC antiserum was from Upstate Biotechnologies (Lake Placid, NY). Purified human Cat and Ca074 were a gift from Richard Riese/Harold L. Chapman (Brigham and Women’s Hospital, Boston, MA).
Subcellular fractionation
Subcellular fractions were prepared and characterized exactly as described [9
, 21
, 22
]. After fractionation, organelles were recovered by 100,000 g x 10 min. For depletion of cytosol from endocytic organelles (referred to as "endocytic fractions" in the text), postnuclear supernatants were sedimented (100,000 gx2 min) as described [23
].
Western blot
Cells/fractions lysed in Nonidet P-40 (NP-40)/pH = 7 lysis buffer (50 mM sodium acetate, 5 mM MgCl2, 0.5% NP-40) were resolved by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), blocked, and probed with appropriate dilutions of the respective primary Ab, followed by a secondary antirabbit immunoglobulin G (IgG) Ab coupled with peroxidase (Southern Biotech, Birmingham, AL). An enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia, Little Chalfont, UK) was used to visualized the Ab-reactive proteins.
Active site-directed labeling
JPM-565 [24
], JPM-565-biotin (a biotinylated version of JPM-565, referred to as JPM-565-bio in this work; chemically identical with the compound known as DCG04; ref. [19
]), and leucinyl-D-homophenylalanyl-vinyl-phenyl-sulfone (LHVS) [25
] were synthesized as described. Radioiodination was achieved using the iodogen method (Pierce, Rockford, IL) according to the manufacturer’s advice using a disposable C18 matrix (Waters, Milford, MA) for purification [9
]. Enriched subcellular fractions were lysed in NP-40/pH = 5 lysis buffer (50 mM sodium aetate, 5 M MgCl2, 0.5% NP-40) and were analyzed for protein concentration using the Bio-Rad Bradford reagent. Lysates were incubated with 1 mM JPM-565-bio/125I-JPM-565 for 1 h at 37°C. For selective inhibition of proteases, appropriate inhibitors were added 30 min before labeling. Reactions were terminated by addition of SDS reducing sample buffer and immediate boiling. Samples were resolved by 12.5% SDS-PAGE gel and directly analyzed by autoradiography or transferred to PVDF membrane, followed by visualization using Streptavidin-horseradish peroxidase (HRP) solution and the ECL-detection kit (Amersham Pharmacia).
Precipitation of active proteases
Endocytic fractions of freshly isolated monocytes were precleared with Streptavidin-sepharose beads overnight before being split into three equal portions in lysis buffer at pH 5. Portion 1 remained untouched, and portion 2 was preincubated with nonbiotinylated JPM-565 (30 min at 37°C). Samples 2 and 3 were then incubated with JPM-565-bio as described. Excess label was removed using a PD10 column (Pharmacia, Uppsala, Sweden), and labeled polypeptides were recovered using Streptavidin-sepharose beads (Pharmacia). After excessive washing, samples were resolved on SDS-PAGE, side-by-side, with untreated, endocytic fractions and were further analyzed by Western blot using anti-Cat antisera.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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48 kDa. CatB was present in its 33-kD heavy-chain form in primary monocytes and all cell lines tested. It showed higher expression levels in more mature types of monocyte progenitors. Monocyte progenitors also contained an unidentified polypeptide at 35 kDa that reacted with the polyclonal anti-CatB antiserum. CatS was only weakly expressed in U937 and Mono-Mac1 and more prominent in all other cell types. In contrast, CatH and CatL showed a restriction toward individual cell lines or maturation stages. Significant amounts of CatH protein were present in BLCs and peripheral monocytes, and U937 and MonoMac1, in contrast, showed little expression. CatH was absent from HL-60, THP-1, and MonoMac6. CatL was exclusively present in HL-60, THP-1, and MonoMac1 and absent from all other cell types, including primary monocytes. These differences in Cat expression did not correspond to differential expression of CysC, the major endogenous inhibitor of cysteine protease activity. CysC was ubiquitously found with maximum expression in the immature progenitors U937 and THP-1 but not HL-60. Thus, human myelo-monocytoid cell lines showed individual patterns of Cat expression that did not clearly correlate with the maturation stage. The expression patterns found were distinct from the set of Cat in human peripheral monocytes, which contained the CatD, -B, -S, and -H, and CatL was absent.
Activity pattern of Cat in peripheral human monocytes
Cat activity is a complex, biological parameter that is controlled not only by expression of the polypeptide and its maturation but also by subcellular transport, pH, and redox conditions along the endocytic route as well as by zymogen activation and the presence of endogenous inhibitors [15
]. Chemical compounds that bind to the active site of proteases in an activity-dependent manner and that are modified with functional groups can be exploited to detect Cat in an activity-based, semiquantitative way. JPM-565 is an epoxide-based active site-directed probe that reacts with a broad panel of cysteine proteases. To assess the activity of the proteases expressed in freshly isolated human monocytes, we collected endocytic compartments by ultracentrifugation and exposed them to 125I-JPM-565, followed by visualization of the active proteases by SDS-PAGE and autoradiography. Purified human Cat and selective inhibitors of Cat activity were included to identify the proteases targeted. Ca074 specifically blocks CatB activity, whereas LHVS at 3 nM is selective for CatS, and it targets other thiol Cat at higher concentrations [26
](Fig. 2a
).
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To confirm the identity and activity of CatH in human monocytes, we incubated monocyte endocytic fractions JPM-565-bio and retrieved the bound material by precipitation with Streptavidin beads, followed by Western blot for CatH, side-by-side with nonmanipulated endocytic material. Endocytic fractions precipitated without exposure to the affinity probe or after incubation with a nonbiotinylated version of JPM-565 before JPM-565-bio labeling served as specificity controls. Pro-CatH and CatH were visualized in nontreated material (Fig. 2b) . After labeling by JPM-565-bio followed by precipitation, mature CatH was retrieved at 30 kD, which could be blocked by preincubation with JPM-565. This confirmed the identity of the signal as active CatH as well as the activity-based nature of the procedure. Thus, CatB, -S, and -H are dominant cysteine-protease activities in resting human peripheral monocytes, and CatL is absent.
Active Cat in myeloid progenitor cell lines
We next used the activity-based probes to simultaneously visualize Cat activity in freshly isolated endocytic fractions of human monocytes, human monocytoid cell lines, and BLC, all normalized for total protein. To identify CatB activity and to allow an easier assessment of the remaining protease activities, one set of samples was treated with Ca074 before labeling with JPM-565-bio. Visualization was achieved using Streptavidin-HRP and chemiluminescence after SDS-PAGE and transfer to PVDF membrane of the labeled fractions (Fig. 3
).
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Subcellular anatomy of Cat activity, CysC, MHC-II, and Ii in peripheral monocytes
Others and we have extensively characterized a two-step Percoll gradient system that dissects the endocytic compartment of monocytes, DC, and B cells into subpopulations representing three distinct locations along the endocytic route [9
, 21
, 22
, 27
]. In brief, postnuclear supernatants are resolved on a 27% Percoll gradient, yielding a dense fraction at the bottom of the tube (designated peak A, lysosomes, density 1.09 g/ml; Fig. 4a
), together with nonresolved material at the top of the gradient. This material is further subjected to a subsequent 10% gradient, separating intermediate-density endocytic compartments (peak B, late endosomal compartments) at a density of 1.05 g/ml at the bottom of the second gradient (peak B), and the remainder (early endosomes, Golgi, plasma membrane, endoplasmatic reticulum) remains on top of the 10% gradient (peak C). When endocytic compartments of human monocytes were resolved using this system, the distributions of total protein, NAG activity—an endocytic marker enzyme—LAMP-1, as well as TrfR were essentially as published for murine monocytes [22
]. LAMP-1 sedimented with the lysosomal fraction A and the late endosomal/prelysosomal fraction B, and TrfR was exclusively found associated with peak C, consistent with an enrichment for early endosomes in this part of the gradient (Fig. 4b)
.
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Active Cat preferentially localized to the lysosomal peak A, where active CatS, CatB, and CatH were present. Thus, despite their different pH optima in vitro, the bulk amount of the three major active cysteine proteases was found in lysosomes. Earlier endocytic compartments, including prelysosomes/late endosomes, showed only little active CatB and CatH. In contrast, significant active CatS was labeled in the late endosomal peak B. Active Cat were almost absent from early endosomal compartments. CatS showed a similar distribution between endocytic compartments of monocytes on the protein level: The majority of mature CatS heavy chain was detected in lysosomal peaks, and late endosomes still contained detectable, mature CatS. In contrast, CatS protein was absent from earlier endocytic compartments. This suggested that substrates for endocytic proteolysis in human monocytes, including Ii, do not encounter relevant amounts of endocytic protease activity until they have reached the late endosomal compartment.
Class II ß-chain was found in significant amounts in all endocytic compartments tested, as expected, and MHC class I was reduced in lysosomes but strongly present in fractions B and C, suggesting internalization/recycling of class I in endosomal compartments of monocytes. The steady-state distribution of Ii, as assessed using the PIN-1 antibody reactive with the N-terminal part of Ii, was consistent with the notion that functional cysteine protease activity was absent from endocytic compartments upstream of late endosomes: Only intact Ii was present in fractions with densities lower than that of the late endosomal peak B, and intact Ii colocalized with its C-terminal degradation product Iip10 in late endosomes. Exclusively, Iip10 was found in the lysosomal compartment. Thus, the C-terminal degradation of Ii, which converts Ii into Iip10, is localized to the late endosomal/prelysosomal compartment of human monocytes. Iip10, which is subsequently converted into CLIP by CatS, colocalized with sizable amounts of active CatS in lysosomes and late endosomes, suggesting that conversion of Iip10 into CLIP might occur in both types of compartments. In contrast, CysC neither shared the distribution characteristics of active Cat nor that for intact Ii: It selectively localized to the late endosomal fraction B, was absent from lysosomes, and was present only in minor amounts in TrfR-positive, early endosomal compartments. Thus, the subcellular localization of CysC is tightly regulated and not identical with that of its two putative interaction partners, CatS and Iip10. Only in late endosomes, these three major players colocalized. Taken together, this suggested a specialized function of late endosomal compartments in the class II antigen-presentation pathway of human monocytes.
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DISCUSSION |
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A second aim of this study was to identify individual patterns of Cat activity during monocyte development, using myelo-monocytoid cell lines arrested in their normal maturation at different stages [20 ]. Although active CatH was exclusively detectable in mature, peripheral monocytes, active CatB showed a tendency to increase at later stages of maturation. However, it must be taken into account that the cell lines used had undergone malignant transformation at their respective developmental stage, which per se, could affect Cat activity, although no such variability was seen when comparing different BLCs. Monocyte cell lines are widely used as models to analyze the cell biology of primary monocytes. Our data demonstrate that even those cell lines that express class II in amounts comparable with that found in monocytes and which are therefore considered "mature" should be used with caution when it comes to questions involving endocytic protease biology. They express individual Cat activity patterns that are distinct from those in primary human monocytes.
CatS, CatB, and CatH are active in human monocytes, and CatL is absent, as shown by the presence of the mature polypeptides and active site-restricted affinity labeling. This result is in partial agreement with Fiebiger et al. [28 ], who demonstrated the presence of mature CatS and the absence of CatL from adherence-enriched human monocytes. This work, however, did not detect mature CatB in monocytes and neither addressed the presence of CatH nor the activity of any of the proteases detected in monocytes. Our data support the notion that different types of APC have their specific patterns of active Cat: Primary human B cells express active CatH and CatS and contain extremely small amounts of CatB and no CatL (ref. [28 ] and own unpublished), human DC generated ex vivo are characterized by significant amounts of CatS and CatL [28 ], murine thymic epithelial cells are characterized by CatL activity in the absence of CatS [14 ], and murine bone marrow-derived monocytes contained significant CatS, CatL, CatH, and CatB [7 , 22 ]. The functional reasons for this diversity are not known. It might result in the generation and presentation of different panels of antigenic peptides from one given antigen by different types of APC, thus increasing the redundancy of the class II-induced T cell response.
Individual Cat play distinct roles in immunity. CatB is ubiquitously expressed and likely to represent a housekeeping enzyme. Its absence does not significantly impair immune function, although CatB has been shown to be involved in intracellular degradation of a model antigen in murine monocytes [7 , 13 ]. CatS, the key enzyme for the transport of class II as well as MHC-like molecules in the periphery [29 ], shares substrate specificity with CatL, which in turn, is also widely expressed [5 ]. When CatS activity is eliminated, human DC still process Ii, albeit at a reduced rate, consistent with the presence of CatL in this cell type [28 ]. Human monocytes, in contrast, do not contain active CatL. Pharmacological inhibition of CatS, a current concept for a selective, therapeutic manipulation of specific immunity [6 ], might therefore influence class II function more significantly in human monocytes than in DC. The physiological role of CatH, which acts as an aminopeptidase, is largely unknown. Our data implicate CatH as a protease that is possibly involved in antigen presentation. So far, CatS was the only endocytic protease known to be consistently present and active in all types of primary, peripheral APC, and CatB, CatL, and CatD were absent from at least one APC type. CatH in this respect shares the distribution characteristics of CatS. Both proteases are present and active in monocytes and BLCs as well as in primary human B cells and monocyte-derived DC (C. Driessen, unpublished). In contrast, CatH activity was absent from all myelo-monocytoid cell lines tested here. Thus, APC function and CatH activity are characteristics of fully differentiated human peripheral monocytes, suggesting that both are functionally connected. One current concept for antigen processing in the context of class II (known as "epitope-guided processing" or "fingerprinting") suggests that relatively large, antigen-processing intermediates bind to class II molecules with their immunodominant core region, followed by subsequent trimming of the overhanging C- and N-terminal portions [30 ]. Carboxypeptidases like CatB are candidates for C-terminal degradation, and CatH could act on the N terminus in this pathway. Indeed, the preferred localization of active CatH and CatB downstream of the major endoprotease CatS that we observed in our result is entirely consistent with this hypothesis.
Recent work resolved basic aspects of the subcellular organization of the class II antigen-presentation machinery in murine DC and BLCs but not in primary human APC [9 , 10 , 31 32 33 ]. We extend on this work not only by using freshly isolated, primary human monocytes for our analysis but also by including chemical tools that allow the simultaneous, activity-based visualization of the major Cat in endocytic compartments. To our surprise, maximum-active CatS, -B, and -H were found in lysosomes. In late endosomes, active CatS was still significantly present, and only very small amounts of active CatB and CatH were visible, and virtually no Cat activity was present in early endosomal compartments. The different catalytic pH optima for active CatB, -S, and -H in vitro (CatB, pH 4–5; CatS, pH 4–7; CatH, pH6–7) in conjunction with the pH gradient along the endocytic route and published data from a murine myelo-monocytoid cell line had implicated that active CatH would be located more upstream of the endocytic route [17 ]. Nevertheless, from a functional perspective, the localization of CatH and CatB downstream of sizable amounts of CatS activity is in good agreement with recent data on the kinetic order of events during antigen breakdown. CatS is not only the major cysteine protease encountered by latex beads after their phagoctosis by live, murine DC [34 ], it also initiates the degradation of the potential autoantigen myelin basic protein when incubated with lysosomal extracts from BLCs in vitro [35 ]. This study as well as similar work analyzing the proteolytic degradation of the model antigen hen egg lysozyme have also shown that endoproteolytic cleavage of native antigen precedes trimming the C- and N-terminal ends [8 ]. However, it should be noted that our results visualize the relative amount of active enzyme in a given cellular compartment in a semiquantitative manner. Analysis of the distributions of specific protease activities per mg protein as defined enzymatically was not the scope of this study.
Class II molecules reach the endocytic pathway directly or via plasma membrane and undergo proteolytic processing of Ii by cysteine proteases to allow formation of CLIP and its exchange for antigenic peptide [2 ]. In murine DC, CysC has been suggested to control Ii maturation and the egress of class II to the plasma membrane by selective colocalization with CatS, thus reducing its activity; however, this view has recently been challenged [36 ]. Expression or subcellular distribution of CysC in monocytes or any other type of primary human APC has not yet been addressed. We demonstrate that CysC is selectively found in late endosomes of peripheral monocytes. There, it colocalizes not only with active Cat, in particular CatS, but also with the de novo formation of Iip10 from intact Ii. Thus, despite of the presence of CysC in this compartment, significant amounts of active proteases as well as protein turnover are found in late endosomes. Therefore, CysC does not completely block the activity of cysteine proteases in this compartment. The C-terminal degradation of Ii is restricted to late endosomes in primary monocytes and is likely to be mediated by CatS. CysC might merely control the speed of Ii turnover in late endosomes by down-modulating CatS activity.
Given that the Fc receptor for IgG transfers exogenous material into late endosomes for class II-mediated antigen presentation [37 ], that furthermore, CatS is the major active protease encountered by ingested material in the class II compartment [34 ], and at the same time that CatS is the dominant protease in late endosomes based on our results, it is not unlikely that at least the initial steps of antigen breakdown are also located in late endosomes. These initial steps are, at least in some examples, the rate-limiting events in antigen processing, suggesting that CysC might have an important, regulatory function in this pathway too. Processing Ii and antigen are prerequisites for antigen presentation yet must occur in an environment where protease activity is sufficiently controlled to allow antigenic peptides to resist until peptide loading on class II occurs. Monocytes might accomplish this balance between activating and inactivating factors of protease function in late endosomes by selectively accumulating CysC in contrast to lysosomes. This highlights late endosomes of primary human monocytes as a compartment especially equipped for controlled protein breakdown, e.g., in class II-mediated antigen presentation, and lysosomes might merely mediate nonselective destruction of material for homeostatic protein turnover.
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ACKNOWLEDGEMENTS |
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Received August 18, 2002; revised October 13, 2002; accepted November 1, 2002.
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REFERENCES |
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