MHC class II molecules, cathepsins, and La/SSB proteins in lacrimal acinar cell endomembranes

Tao Yang, Hongtao Zeng, Jian Zhang, Curtis T. Okamoto, Dwight W. Warren, Richard L. Wood, Michael Bachmann, and Austin K. Mircheff

Departments of Physiology and Biophysics, Cell and Neurobiology, and Ophthalmology, School of Medicine, and Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033; and Institut für Physiologische Chemie, Fachbereich Medzin, Johannes Gutenberg-Universität Mainz, Mainz, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sjögren's syndrome is a chronic autoimmune disease affecting the lacrimal glands and other epithelia. It has been suggested that acinar cells of the lacrimal glands provoke local autoimmune responses, leading to Sjögren's syndrome when they begin expressing major histocompatibility complex (MHC) class II molecules. We used isopycnic centrifugation and phase partitioning to resolve compartments that participate in traffic between the basolateral membranes and the endomembrane system to test the hypothesis that MHC class II molecules enter compartments that contain potential autoantigens, i.e., La/SSB, and enzymes capable of proteolytically processing autoantigen, i.e., cathepsins B and D. A series of compartments identified as secretory vesicle membranes, prelysosomes, and microdomains of the trans-Golgi network involved in traffic to the basolateral membrane, to the secretory vesicles, and to the prelysosomes were all prominent loci of MHC class II molecules, La/SSB, and cathepsins B and D. These observations support the thesis that lacrimal gland acinar cells that have been induced to express MHC class II molecules function as autoantigen processing and presenting cells.

Sjögren's syndrome; autoantigen processing and presentation; membrane traffic


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SJÖGREN'S SYNDROME IS A chronic autoimmune disease characterized by progressive lymphocytic infiltration and destruction of the lacrimal and salivary glands (16, 45). The events that lead to Sjögren's autoimmune responses are not clearly understood, but factors of genetic, endocrine, and viral origins are considered relevant (3, 14, 26, 31).

Among the clearly documented genetic risk factors for Sjögren's syndrome are certain major histocompatibility complex (MHC) class II molecule haplotypes (2, 15). The MHC class II molecules are constitutively expressed by dendritic cells, monocytes (8), and macrophages (12), and they are also expressed by activated T and B lymphocytes (50). They play central roles in initiating and maintaining immune responses by presenting proteolytically processed antigen peptides to CD4 T lymphocytes.

The targets of autoimmune responses and nonlymphoid cell bystanders to inflammatory processes also frequently express MHC class II molecules (9, 24, 25, 29, 48). The observation that thyrocytes express MHC class II molecules in Graves' thyroiditis led Botazzo and co-workers (7) to theorize that nonlymphoid cells that express MHC class II molecules provoke autoimmune responses by presenting autoantigens.

Salivary epithelial cells from Sjögren's patients uniformly express MHC class II molecules (17, 18). Lacrimal epithelial cells have been found to express MHC class II molecules in a large proportion of cadaver donor lacrimal glands, and the number of positive cells generally increases with the number of lymphocytes infiltrating the gland (32). Furthermore, lacrimal gland acinar cells from experimental animals begin to express MHC class II molecules in certain pathological situations (51), as well as when they are isolated and placed in primary culture (32, 33).

If, once they have been induced to express MHC class II molecules, acinar cells are to provoke CD4 T cell responses, the class II molecules should pass through endomembrane compartments that contain both potential autoantigens and also enzymes that generate proteolytic fragments from the autoantigens. Electron microscope immunocytochemical observations demonstrated that MHC class II molecules cycle rapidly between the lacrimal gland acinar cell basolateral plasma membrane and endomembrane compartments and that, over a longer time course, they also reach the secretory vesicles (33).

The primary antigen-processing enzymes in the professional antigen-presenting cells are cathepsin B, a cysteine proteinase, and cathepsin D, an aspartic proteinase (36, 46). Cathepsins B and D are probably present in the lysosomes of most mammalian cells. They reach the lysosomes via a pathway that leads from the Golgi complex to the plasma membranes and endosomes (37, 41). Therefore, it has seemed reasonable to conjecture that they would also be present in some of the acinar cell compartments that MHC class II molecules traverse en route to and from the basolateral membranes.

Any protein that enters endomembrane compartments that contain both MHC class II molecules and cathepsins would become a candidate for proteolytic processing and MHC class II molecule-mediated presentation. It is not a simple matter to identify the antigens that are recognized by T cells, but it is likely that some of them will also be the targets of antibody production. One of the frequent clinical features of Sjögren's syndrome is the production of autoantibodies against certain ribonuclear proteins, Ro/SSA and La/SSB (1, 6, 49). Lacrimal and salivary epithelial cells normally do not express these proteins at readily detectable levels, and, in the cells that normally do express appreciable levels, they are imported into the nuclei, where they should be sequestered from the lysosomal and plasma membrane, i.e., endomembrane recycling pathways (30, 39, 40). However, viral infection and exposure to cytokines lead to significant Ro/SSA and La/SSB expression in salivary epithelial and other cell types. In these circumstances, the autoantigens accumulate in the cytoplasm, assume a punctate distribution consistent with localization to endomembranes, and appear also at the plasma membranes (4, 10, 47).

We have found that, when acinar cells from rabbit lacrimal glands are placed in primary culture, they begin to express detectable levels of La/SSB-related proteins. Thus we have been able to survey the steady-state subcellular distributions and traffic of MHC class II molecules, cathepsins, and La/SSB. We approached this problem in two steps, first extending and improving a subcellular fractionation analysis that resolves membrane samples believed to represent most compartments of the basolateral-endomembrane traffic, and then mapping the distributions of MHC class II molecular beta -subunits, of immature and mature forms of cathepsins B and D, and of La/SSB-reactive proteins.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Female New Zealand White rabbits weighing between 1.8 and 2.2 kg were obtained from Irish Farms (Norco, CA). Horseradish peroxidase (HRP), phenylmethylsulfonyl fluoride (PMSF), Nalpha -p-tosyl-L-arginine methyl ester (TAME), Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK), and leupeptin were from Sigma Chemical (St. Louis, MO). Anti-rabbit class II monoclonal antibody (2C4) was prepared from the mouse hybridoma by Antibodies (Davis, CA). Anti-human cathepsin D polyclonal antibody, raised in rabbit, was purchased from Biodesign (Kennebunk, ME). Anti-human cathepsin B polyclonal antibody, raised in sheep, was from The Binding Site (Birmingham, UK). A monoclonal antibody directed to the RNP consensus motif of human La/SSB, which cross-reacts with the rabbit protein, was prepared in the laboratory of Michael Bachmann. It recognizes an NH2-terminal epitope similar to the anti-La monoclonal antibody SW5 described by Smith et al. (42). Polyclonal anti-Rab5 and anti-Rab6, raised in rabbit against synthetic peptides of human origin, were from Santa Cruz Biotechnology. 125I-labeled protein A and 125I-labeled protein G were purchased from ICN (Irvine, CA).

Solutions. Solutions and media for cell isolation and culture and for Western blotting were as described previously (20, 23). The media for cell lysis, density gradient analysis, and phase partitioning analysis were supplemented with a cocktail of protease inhibitors to yield final concentrations of 10 µg/ml TAME, 10 µg/ml TLCK, 1 µg/ml leupeptin, and 0.2 mM PMSF.

The pH 7.6 and pH 7.0 two-phase systems were constructed as described previously (20). The pH 6.6 system contained the same 5% dextran and 3.5% polyethylene glycol concentrations, but the imidazole buffer concentration was increased to 10 mM. The pH for phase systems was adjusted with HCl.

Isolation and culture of lacrimal gland acinar cells. Methods for isolation and culture of lacrimal gland acinar cells were as described previously (20, 23). As in the previous studies, cells were harvested after 2 days in primary culture, incubated with HRP for 20 min at 37°C, washed, and lysed in a Balch cell press (H & Y Enterprises, Redwood City, CA).

Subcellular fractionation analysis. Cell lysates were subjected to differential sedimentation and isopycnic centrifugation on sorbitol density gradients as described previously (20, 23).

Detailed descriptions of the phase partitioning methods and apparatus are given elsewhere (34). Partitioning analysis with the pH 7.6 two-phase system was performed according to standard methods with 650-µl aliquots of both top and bottom phases. Samples were introduced in the top phases of chambers +1 through +3, and the analysis was carried out with 36 transfer steps. Because the thin-layer countercurrent distribution apparatus contains 120 chambers, it was possible to analyze three different membrane samples simultaneously, i.e., by employing chambers 1-39, 41-79, and 81-19.

For the pH shift method, 600-µl aliquots of bottom phases and of top phases were used. Chambers -6 through +12 were loaded with a pH 7.0 phase system, and chambers +13 through +34 were loaded with the pH 6.6 phase system. Membrane samples were introduced in the top phases of chambers +1 through +3. After 12 transfer steps, 600-µl aliquots of the pH 6.6 top phase were added to all chambers, and the analysis was continued for a further 24 transfer steps.

When analyses were complete, the contents of each chamber were diluted below the critical concentrations by addition of 1 ml 5% sorbitol membrane isolation buffer and pooled in groups of three. Membranes were harvested by ultracentrifugation and resuspended in 1-ml aliquots of isolation buffer. Membrane samples were concentrated a further threefold by dilution and ultracentrifugation before SDS-PAGE.

Methods for biochemical analyses of density gradient and phase-partitioning fractions were the same as used in previous studies (20, 23).

Immunoblotting. SDS-PAGE and Western blotting were performed in Modular Mini-PROTEAN II and Mini Trans-Blot Cells from Bio-Rad (Hercules, CA). Membrane samples were dissolved in sample buffer by boiling for 5 min and then separated on 10% or 15% polyacrylamide minigels, depending on the desired molecular weight separation ranges. Proteins were transferred to nitrocellulose (Fisher Scientific, Pittsburgh, PA), rinsed, blocked, and probed with primary antibodies as described previously (20). In cases where there was low affinity between primary antibody and 125I-protein A or 125I-protein G, blots were incubated with a secondary antibody before incubation with the 125I-labeled proteins. Blots were autoradiographed on Kodak XAR film at -80°C. The films were analyzed with a GS-670 densitometer and molecular analyst TM/PC software (Bio-Rad).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Density gradient distributions of endomembranes. Figure 1 illustrates the density gradient distributions of surface-bound and endocytosed HRP, membrane-associated protein, and several membrane constituents frequently used as markers for endomembrane compartments. The distribution patterns are similar to those observed in previous fractionation studies of the reconstituted acinar preparation (20, 23), although there are several quantitative differences attributable to use of a more complete cocktail of protease inhibitors in the present study.


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Fig. 1.   Density gradient distributions of surface-bound and endocytosed horesradish peroxidase (HRP), membrane-associated protein, and intrinsic membrane markers. Numbers of separate preparations in which markers were analyzed are indicated in parenthesis.

The density gradient distribution of each marker reflects the summed contributions of several compartments with which that marker is associated. Moreover, as described below, each density gradient fraction may contain elements of two or more different compartments or two or more microdomains of a common compartment. The overlapping compartments and microdomains can be separated, at least partially, by analyzing the density gradient fractions by partitioning in dextran-PEG two-phase systems (31).

Two-dimensional distributions of endomembrane compartments. We analyzed all the density gradient fractions with a pH shift method, in which the separation was begun in a two-phase system with pH = 7.0, and then additional upper phase with pH = 6.6 was added. Additionally, we analyzed density gradient fractions 9+10, 11+12, and P in a two-phase system with constant pH = 7.6.

Even though their boundaries may be indistinct, the approximate locations of the various compartments and microdomains of compartments can be inferred from features of the marker distribution patterns, e.g., modes in the distributions of particular markers or unique spectra of markers shared with other compartments. However, the identities of the isolated compartments, i.e., their relationships to defined structures of the intact cells, must be regarded as working hypotheses that are contingent on other data and in many cases remain to be substantiated through further work. The current working hypotheses are presented in summary form in Table 1 and discussed in detail under RESULTS.

                              
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Table 1.   Subcellular compartments resolved by two-dimensional fractionation

Figure 2 summarizes the distributions of several markers after analysis with the pH-shift approach. Figure 3 provides a key to Fig. 2, indicating the locations of the minimum number of membrane compartments and microdomains needed to account for all available distribution data. Figure 4 summarizes marker distributions observed after analysis with the pH 7.6 two-phase systems, and Fig. 5 indicates the locations of the membrane compartments in Fig. 4. The two-dimensional analyses depicted in Figs. 2-5 are more comprehensive than those achieved in earlier studies (e.g., Ref. 23); consequently, some hypotheses for identities of isolated compartments have been revised from those advanced previously.


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Fig. 2.   Two-dimensional distributions of membrane markers after density gradient fractions were analyzed with the pH 7.0 to pH 6.6 shift phase partitioning method. Density gradient fractions were pooled for analysis, and numbers of separate preparations from which distribution data were compiled are as follows: 1+2 (n = 1), 3-6 (n = 2), 7+8 (n = 3), 9+10 (n = 4), and 11+12 (n = 2); fraction P (n = 2) was not pooled with other fractions. As in Fig. 1, the vertical axis is percent recovery.



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Fig. 3.   Key to locations of membrane compartments and microdomains after the two-dimensional analysis depicted in Fig. 2. This map reflects the minimum number of distinct compartments and microdomains needed to account for the two-dimensional distributions of biochemical markers after analyses with both the pH-shift method described in Fig. 2 and the constant pH method described in Fig. 4. Current hypotheses for the identities of the compartments are summarized in Table 1.



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Fig. 4.   Two-dimensional distributions of membrane markers after density gradient fractions 9+10, 11+12, and P were analyzed in the pH 7.6 two-phase system. Data are from a single preparation.



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Fig. 5.   Map of membrane compartments and microdomains delineated by the two-dimensional analysis depicted in Fig. 4.

The pH shift method resolves blmi and blmj (from density gradient fractions 1+2) and a series of ld-tgn compartments (from pooled density gradient fractions 3 through 6). It separates compartments er and Gol (from pooled density gradient fractions 7+8 and 9+10) from compartment blmre, and it partially resolves compartment blmre into three microdomains, blmrei, blmrej, and blmrek.

The combined use of two phase-partitioning methods is important in detecting compartments hd-tgni, hd-tgnj, hd-tgnk, svm, and preLys. When the pH shift method is used, the hd-tgn compartments overlap each other and compartment er (Fig. 2), but they are well separated from compartment blmre. The hd-tgn are resolved from er and from each other when the analysis is performed in the pH 7.6 two-phase system (Fig. 4), but in this case hd-tgnk overlaps blmre. Compartment preLysi (from density gradient fractions 11+12) is poorly resolved from compartment svm after analysis with the pH-shift method (Fig. 2) but is well resolved after analysis with the pH 7.6 two-phase system (Fig. 4). Of the compartments that sediment to the pellet during density gradient centrifugation, preLysj is better resolved from compartments Lysi, Lysj, and Lysk by analysis in the pH 7.6 two-phase system, whereas compartments Lysi, Lysj, and Lysk are better resolved from each other by analysis with the pH-shift method.

Immunoblot detection of MHC class II beta -subunit, cathepsins B and D, and La/SSB-reactive proteins. Immunoblot methods were used to survey the distributions of MHC class II beta -subunit, cathepsins B and D, and La/SSB among the membrane compartments resolved in Figs. 1-5. Figure 6 presents autoradiograms of typical Western blot analyses of the density gradient fractions for which biochemical data are summarized in Fig. 1. The 2C4 monoclonal antibody labeled a band with molecular weight 31 kDa, corresponding to the beta -subunit of MHC class II molecule. The antiserum to cathepsin B specifically labeled bands with molecular weights of 35, 31, and 27 kDa, corresponding to the prepro-, pro-, and mature forms of the enzyme. The prepro form was clearly the most abundant. An additional band at 47 kDa was the result of nonspecific labeling. The antiserum to cathepsin D labeled bands at 52 and 46 kDa, corresponding to the pro form and one of several known intermediate processing forms, and a band at 31 kDa, corresponding to the mature form. The anti-La/SSB monoclonal antibody labeled three bands, with molecular weights of 50, 29, and 25 kDa. The 50-kDa band corresponds to the full-length protein, whereas the smaller bands are assumed to be proteolytic processing products.


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Fig. 6.   Immunoblot detection of major histocompatibility complex (MHC) class II molecule beta -subunit, cathepsin B, cathepsin D, and La/SSB-reactive proteins.

MHC class II molecules. Figure 7 presents the density gradient distribution of the MHC class II molecule beta -subunit, and Fig. 8 presents two-dimensional distributions after analyses with the pH-shift method and the pH 7.6 two-phase system. The level of MHC class II molecule expression in the blm compartments, evident from the significant signal detected in density gradient fractions 1 and 2 (Fig. 1) was too low to be detected reliably after phase partitioning, so it is not clear whether MHC II molecules are present in both blmrei and blmrej or are confined to one compartment or the other. Comparing the two-dimensional distributions of MHC class II molecules with the distribution data in Figs. 2 and 4 and the keys in Figs. 3 and 5, it is evident that MHC class II molecules are expressed in ld-tgnl but not at appreciable levels in ld-tgni, ld-tgnj, and ld-tgnk. MHC class II molecules are also expressed in Gol, hd-tgni, hd-tgnj, hd-tgnk, svm, preLysi, preLysj, the three Lys compartments, and blmre. They are expressed at a higher level in blmrei than in blmrej or blmrek.


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Fig. 7.   Density gradient distribution of MHC class II molecule beta -subunit immunoreactivity; n, number of separate cell preparations in which immunoreactivity was determined.



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Fig. 8.   Two-dimensional distributions of MHC class II molecule beta -subunit immunoreactivity. (Immunoreactivity could not be measured reliably after phase partitioning analysis of density gradient fractions 1+2.)

Cathepsin B. The density gradient distributions of the three cathepsin B molecular forms are presented in Fig. 9, and the two-dimensional distributions are presented in Fig. 10. The distributions in Fig. 9 indicate that three forms are expressed, albeit at low levels, in at least one of the blm compartments. Like the MHC class II molecule beta -subunit, the cathepsin B forms are expressed at higher levels in Gol, hd-tgni, hd-tgnj, hd-tgnk, svm, preLysi, and preLysj, and the Lys compartments. They are present in all three microdomains of compartment blmre, but they are distributed somewhat asymmetrically within this compartment. Also, like the MHC class II molecule beta -subunit, procathepsin B is particularly prominent in ld-tgnl.


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Fig. 9.   Density gradient distributions of cathepsin B immunoreactive proteins; n, number of separate cell preparations in which immunoreactivity was determined.



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Fig. 10.   Two-dimensional distributions of cathepsin B immunoreactive proteins. (Signals from the pro and mature forms could not be measured reliably after phase partitioning analyses of density gradient fractions 1+2, and signals from the prepro form could not be measured reliably after the partitioning analyses in the pH 7.6 phase system.)

Cathepsin D. The density gradient distributions presented in Fig. 11 suggest that the 48-kDa intermediate and mature forms of cathepsin D have subcellular distributions similar to those of the prepro and mature forms of cathepsin B. Procathepsin D, like preprocathepsin B, exhibits an additional peak in the density gradient fractions that contain the ld-tgn compartments. Although cathepsin D immunoreactivities could not be detected reliably after the phase partitioning analyses, their similarities to the density gradient distributions of the cathepsin B forms suggest that cathepsin D forms are expressed most prominently in Gol, hd-tgni, hd-tgnj, hd-tgnk, preLys1, preLys2, and the Lys compartments.


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Fig. 11.   Density gradient distributions of cathepsin D immunoreactive proteins; n, number of separate cell preparations in which immunoreactivity was determined.

La/SSB. The density gradient and two-dimensional distributions of the La/SSB-reactive proteins are presented in Figs. 12 and 13, respectively. The subcellular distribution patterns overlapped extensively. The density gradient analysis revealed expression in the blm and the ld-tgn compartments, but the levels were too low to be detected reliably after phase partitioning. The complete two-dimensional analyses that could be performed showed significant levels in the blmre compartments, and higher levels were expressed in Gol, the hd-tgn compartments, svm, both preLys compartments, and the three Lys compartments.


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Fig. 12.   Density gradient distributions of La/SSB immunoreactive proteins; n, number of separate cell preparations in which immunoreactivity was determined.



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Fig. 13.   Two-dimensional distributions of La/SSB and La/SSB related proteins. Immunoreactivities could not be detected reliably after density gradient fractions 1+2 and 3-6 were analyzed by phase partitioning, but expression of La/SSB and related proteins is evident in a variety of higher-density membrane compartments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In surveying the subcellular distributions of MHC class II molecules, cathepsin B, cathepsin D, and the La autoantigen, we have followed an empirical, analytical approach that was first articulated some years ago and reviewed in detail elsewhere (31). Accordingly, we have used physical separation procedures to resolve isolated membrane compartments independently of any assumptions about the identities of these compartments. The fractionation analyses demonstrate that MHC class II molecules, cathepsin B, cathepsin D, and the La autoantigen are colocalized in several isolated endomembrane compartments. The extent to which this result supports the thesis that lacrimal gland acinar cells that express MHC class II molecules can function as autoantigen processing and presenting cells depends, in part, on the validity of our working hypothesis for the identities of the isolated membrane compartments. Figure 14 presents a cellular model incorporating these hypotheses. Before discussing implications for autoantigen processing and presentation, we briefly review evidence supporting identification of the isolated compartments and their locations on the membrane traffic pathways depicted in Fig. 14.


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Fig. 14.   Model for traffic of MHC class II molecules, cathepsins, and La/SSB in lacrimal gland acinar cells. Compartments that have not yet been resolved by analytical fractionation are the apical plasma membrane (APM); apical endosome (AE); and terminal transcytotic vesicles (tTV). As described in text, the compartments where catheptic processing of La/SSB is most likely to generate peptides that associate with MHC class II molecules are the sv, hd-tgns, and preLys.

Identification of compartment blm as basolateral plasma membranes is based on the observation that it is labeled equally well by HRP when cells are incubated at 4°C and at 37°C (20). Identification of blmi and blmj as distinct microdomains of the blm is based on the difference, evident in Fig. 2, in their relative contents of acid phosphatase and Na+-K+-ATPase.

Identification of compartment svm as secretory vesicle membranes is based on the observation that this compartment's content of beta -hexosaminidase decreases significantly when the cells are stimulated with carbachol before lysis (22, 23) and also by the observation that MHC class II molecules reach the secretory vesicles of intact cells (33).

The working hypothesis that compartment Gol corresponds to the Golgi complex is based on its observed content of galactosyltransferase. That the Golgi complex communicates with a basolateral membrane-related endosome, as indicated in Fig. 14, was first suggested by observations that it became labeled by fluid phase markers that had been internalized from the interstitium (13, 38).

The trans-Golgi network (TGN) is the compartment where proteins are segregated into the specialized microdomains that give rise to transport vesicles targeted to specific destinations. Thus it is to be expected that analytical fractionation would resolve a multiplicity of compartments related to the TGN. Identification of the ld-tgn and hd-tgn compartments is based on their contents of galactosyltransferase and Rab6, and their relative excess of Rab6 over galactosyltransferase. Certain characteristics of the hd-tgn compartments, such as their contents of alpha -glucosidase, beta -hexosaminidase, and cathepsin B, suggest that they are related to the lysosomal and apical secretory pathways. The ld-tgn compartments appear to be related to the basolateral membrane recycling pathway, as depicted in Fig. 14, since they are relatively enriched in Na+-K+-ATPase and other typical basolateral membrane constituents (20) and since they are labeled by HRP endocytosed across the basolateral membranes during incubation at 37°C (Fig. 1 and Ref. 20). Compartments analogous to the ld-tgns have been isolated from fresh lacrimal glands (34) and from parotid glands (11), but we are not aware that similar compartments have been described in other cell types. It is possible that they represent specialized structures related to specific exocrine acinar cell functions. Na+-K+-ATPase pumps are translocated from the ld-tgn compartments to the blm compartment when freshly isolated preparations of gland fragments and acini are stimulated with carbachol (28, 52, 53), suggesting that the ld-tgn compartments contain intracellular reserves of Na+-K+-ATPase available for rapid recruitment to the basolateral membrane.

The working hypothesis that the isolated blmre compartments depicted in Figs. 3 and 5 correspond to microdomains of the basolateral membrane-related endosome is supported by their contents of acid phosphatase and Na+-K+-ATPase, which are present in compartment blm, and of Rab5, which is present in basolateral membrane recycling endosomes.

It is expected that the basolateral-membrane-related endosome should communicate with prelysosomal compartments. The density gradient distribution of endocytosed HRP is consistent with traffic to compartments preLys and Lys (Fig. 1) and, therefore, supports their identification.

According to the model in Fig. 14, fluid phase traffic from the basolateral membrane may reach the TGN and lysosomes both by direct pathways, i.e.
blmre → ld-tgn

blmre → preLys → Lys
and by an indirect pathway, i.e.
blmre → Col → hd-tgn → preLys → Lys
An observation that accords with the hypothetical indirect pathways is that the blmre is comprised of several distinct compartments or microdomains. Compartment blmrej has a relatively large content of galactosyltransferase, as might be expected for a microdomain involved in traffic to and from the Golgi complex. Compartment blmrei has characteristics that might be expected of a microdomain involved in traffic to and from the ld-tgns, including a relatively large content of Na+-K+-ATPase and surface chemical properties, as discerned by the phase-partitioning analysis, similar to those of compartment ld-tgnl.

The distributions of cathepsin B molecular forms among the compartments resolved in this study are consistent with the principles of current models for the synthesis, intracellular traffic, and processing of lysosomal cathepsins (37, 41). However, since compartments specifically corresponding to ld-tgn have not yet been isolated from other cell types, there is no basis for determining whether the high level of the cathepsin B pro form in ld-tgnl noted in Fig. 10 is or is not typical. The ld-tgn compartments contain relatively smaller amounts of the mature form, but, when leupeptin is omitted from the membrane isolation media, these compartments exhibit measurable cathepsin B catalytic activity (20, 22, 23).

Because ld-tgnl is the site of a prominent concentration of MHC class II molecules (Fig. 2), as well as of catalytically competent cathepsin B, it is possible that it may be a compartment where autoantigens are processed to peptides that associate with MHC class II molecules. Compartment blmrei also contains substantial concentrations of MHC class II molecules (Fig. 2), the pro form of cathepsin B (Fig. 10), and cathepsin B catalytic activity (23), making it another candidate compartment for autoantigen processing, leading to MHC class II molecule-mediated presentation.

The compartments that exhibit the most prominent concentrations of MHC class II molecules and the mature form of cathepsin B are the hd-tgns, preLysi, and preLysj. Notable concentrations of mature cathepsin B and somewhat smaller concentrations of MHC class II molecules also occur in compartment svm. These compartments are, therefore, candidate sites for autoantigen processing, leading to MHC class II molecule-mediated peptide presentation.

It is noteworthy that the hd-tgns, preLysi, and preLysj compartments should also contain significant amounts of the La/SSB autoantigen and of the smaller La/SSB-reactive proteins. The 29-kDa protein probably corresponds to the NH2-terminal proteolytic fragment described previously (5). However, since the epitope recognized by the anti-La/SSB monoclonal antibody is located on the NH2-terminal RNP consensus sequence, the 25-kDa protein cannot represent the reciprocal, COOH-terminal domain of the La/SSB protein. Thus it cannot represent the well-known 25-kDa COOH-terminal degradation product. It is, rather, an NH2-terminal product of degradation of either the NH2-terminal 29-kDa domain or of the full-length La/SSB protein. Indeed, La/SSB has been described to be proteolytically degraded from the NH2-terminus.

La/SSB-derived peptides presented by acinar cell MHC class II molecules may, initially, not be subject to peripheral tolerance, since in normal states lacrimal epithelial cells express neither MHC class II molecules nor cytoplasmic La/SSB (21). Other potential autoantigens entering the acinar cell endomembrane system may similarly elude immune recognition until the acinar cells are induced to express MHC class II molecules.

Peptides derived from La/SSB and other potential autoantigens associated with and presented by MHC class II molecules might elicit peripheral tolerance or they might provoke CD4 T cell activation, depending on the nature of the accessory signals that the acinar cells express or that are present in the immediate milieu. If antigen presentation is not accompanied by appropriate accessory signals, the predicted outcome is T cell anergy, a form of peripheral tolerance. There are reports that cells in the lacrimal gland express transforming growth factor-beta , a factor that should promote more active forms of peripheral tolerance (54), and it is interesting to note evidence that this cytokine may be one component of an immunosuppressive milieu induced when androgens are administered to rodent models for autoimmune lacrimal gland disease (44).

Given the acinar cell's potential to present processed antigen peptides once induced to express MHC class II molecules, the adaptive value of the hypothesized androgen-dependent immunosuppressive factors seems evident. On the other hand, circumstances may arise in which the spectrum of accessory signals favors CD4 T cell activation rather than anergy or tolerance. Lacrimal gland acinar cells express prolactin-like proteins similar to prolactin-like autocrine and paracrine factors expressed by T lymphocytes (55). Because prolactin and prolactin-like proteins upregulate CD4 T cell expression of interleukin-2 (IL-2) and of IL-2 receptors, MHC class II molecule-mediated antigen peptide presentation in the presence of lacrimal prolactin-like proteins could initiate CD4 T cell autocrine signaling that favors activation. Furthermore, there has been a report that acinar cells from rat lacrimal glands express an IL-2-like protein (43). Thus acinar cells that express both MHC class II molecules and IL-2-like molecules might provide both antigenic stimulation and soluble accessory stimulation to CD4 T cells.

A final hypothesis worthy of consideration is that a third cell type can provide accessory signals that favor CD4 T cell activation. Such a phenomenon has been suggested by Geppert and Lipsky (19), who found that addition of endothelial cells that express IL-2, or addition to the medium of soluble IL-2, allows fibroblasts that express MHC class II molecules to activate CD4 T cells. A new variant of the "conspiratorial bystander" hypothesis, suggested by the observation that La/SSB, like MHC class II molecules, can be expressed at the acinar cell blm, is that acinar cells and CD4 T cells become engaged in triadic relationships with La/SSB-reactive B cells (35). This triadic relationship would generate positive feedback, with the acinar cell providing antigenic stimulation to both the CD4 T cell and the B cell, the B cell providing accessory stimulation to the CD4 T cell, and the CD4 T cell, now activated, producing cytokines that both favor B cell activation and also induce additional acinar cells to begin expressing MHC class II molecules.

A recent study (27) has provided direct support for the thesis that MHC class II molecule-expressing lacrimal gland acinar cells can provoke autoimmune responses. When autologous mixed cell reactions were constructed with acinar cells and splenic lymphocytes from individual donor rabbits, the acinar cells stimulated lymphocyte proliferation in a cell-contact-dependent and MHC class II molecule-dependent fashion. Further substantiation of this thesis and identification of the specific modes of accessory stimulation that operate might advance our understanding of how Sjögren's autoimmune processes initiate and progress in the lacrimal glands and yield new clues for designing rational, specific therapies.


    ACKNOWLEDGEMENTS

We thank Hannah Freed, Anna Kvasnicka, John Norian, Barbara W. Platler, Wei Wang, and Ramona Yasharel for help completing this work. We also thank Drs. Sarah F. Hamm-Alvarez, Harvey R. Kaslow, Joel E. Schechter, Hermann von Grafenstein, and Harry Walter for advice and helpful discussions.


    FOOTNOTES

This work was supported by National Institutes of Health Grants EY-05801 (A. K. Mircheff), EY-09405 (D. W. Warren), and EY-10550 (R. L. Wood), Digestive Diseases Core Center Grant DK-48522, a grant from the University of Southern California Zumberge Faculty Research and Innovation Fund (C. T. Okamoto), and by a Student Research Fellowship from the Sjögren's Syndrome Foundation, Inc. (A. K. Mircheff).

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

Address for reprint requests and other correspondence: A. K. Mircheff, Dept. of Physiology and Biophysics, Univ. of Southern California School of Medicine, 1333 San Pablo St., MMR 626, Los Angeles, CA 90033 (E-mail: amirchef{at}hsc.usc.edu).

Received 10 November 1998; accepted in final form 25 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alexander, E. L., T. J. Hirsch, F. C. Arnett, T. T. Provost, and M. B. Stevens. Ro(SSA) and La(SSB) antibodies in the clinical spectrum of Sjögren's syndrome. J. Rheumatol. 9: 239-246, 1982[Medline].

2.   Arnett, F. C., W. B. Bias, and J. D. Reveille. Genetic studies in Sjögren's syndrome and systemic lupus erythematosus. J. Autoimmun. 2: 403-413, 1989[Medline].

3.   Azzarolo, A. M., A. K. Mircheff, R. L. Kaswan, F. Z. Stanczyk, E. Gentschein, L. Becker, B. Nassir, and D. W. Warren. Androgen support of lacrimal gland function. Endocrine 6: 39-45, 1997.[Medline]

4.   Bachmann, M., D. Falke, H. C. Schroder, and W. E. Muller. Intracellular distribution of the La antigen in CV-1 cells after herpes simplex virus type 1 infection compared with the localization of U small nuclear ribonucleoprotein particles. J. Gen. Virol. 70: 881-891, 1989[Abstract].

5.   Bachmann, M., D. Grölz, H. Bartsch, R. R. Klein, and H. Tröster. Analysis of expression of an alternative La(SS-B) cDNA and localization of the encoded N- and C-terminal peptides. Biochim. Biophys. Acta 1356: 53-63, 1997[Medline].

6.   Ben-Chetrit, E., R. Fischel, and A. Rubinow. Anti-SSA/Ro and anti-SSB/La antibodies in serum and saliva of patients with Sjögren's syndrome. Clin. Rheumatol. 12: 471-474, 1993[Medline].

7.   Bottazzo, G. F., R. Pujol-Borrell, T. Hanafusa, and M. Feldmann. Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity. Lancet 2: 1115-1119, 1983[Medline].

8.   Brooks, C. F., and M. Moore. Differential MHC class II expression on human peripheral blood monocytes and dendritic cells. Immunology 63: 303-311, 1988[Medline].

9.   Chan, C.-C., B. Detrick, R. B. Nussenblatt, A. G. Palestine, L. S. Fujikawa, and J. J. Hooks. HLA-DR antigens on retinal pigment epithelial cells from patients with uveitis. Arch. Ophthalmol. 104: 725-729, 1986[Abstract].

10.   Clark, D. A., P. J. Lamey, R. F. Jarrett, and D. E. Onions. A model to study viral and cytokine involvement in Sjögren's syndrome. Autoimmunity 18: 7-14, 1994[Medline].

11.   Conteas, C. N., A. A. McDonough, T. R. Kozlowski, C. B. Hensley, R. L. Wood, and A. K. Mircheff. Mapping subcellular distribution of Na,K-ATPase in rat parotid gland. Am. J. Physiol. 250 (Cell Physiol. 19): C430-C441, 1986[Abstract/Free Full Text].

12.   Cowing, C., B. D. Schwartz, and H. B. Dickler. Macrophage Ia antigens. I. Macrophage populations differ in their expression of Ia antigens. J. Immunol. 120: 378-384, 1978[Abstract].

13.   Farquhar, M. G. Membrane recycling in secretory cells: implications for traffic of products and specialized membranes within the Golgi complex. Methods Cell Biol. 23: 399-427, 1981[Medline].

14.   Fei, H. M., H. Kang, S. Scharf, H. Erlich, C. Peebles, and R. I. Fox. Specific HLA-DRB1 alleles confer susceptibility to Sjögren's syndrome and autoantibody production. J. Clin. Lab. Anal. 5: 382-391, 1991[Medline].

15.   Foster, H., A. Stephenson, D. Walker, G. Cavanagh, C. Kelly, and I. Griffiths. Linkage studies of HLA and primary Sjögren's syndrome in multicase families. Arthritis Rheum. 36: 473-484, 1993[Medline].

16.   Fox, R. I. Sjögren's syndrome: immunobiology of exocrine gland dysfunction. Adv. Dent. Res. 10: 35-40, 1996[Abstract].

17.   Fox, R. I., T. Bumol, R. Fantozzi, R. Bone, and R. Schreiber. Expression of histocompatibility antigen HLA-DR by salivary gland epithelial cells in Sjögren's syndrome. Arthritis Rheum. 29: 1105-1111, 1986[Medline].

18.   Franco, A., G. Valesini, V. Barnaba, C. Silvagni, A. Tiberti, and F. Balsano. Class II MHC antigen expression on epithelial cells of salivary glands from patients with Sjögren's syndrome. Clin. Exp. Rheumatol. 5: 199-203, 1987[Medline].

19.   Geppert, T. D., and P. E. Lipsky. Dissection of the antigen presenting function of tissue cells induced to express HLA-DR by gamma interferon. J. Rheumatol. Suppl. 13: 59-62, 1987.

20.   Gierow, J. P., T. Yang, A. Bekmezian, N. Liu, J. M. Norian, S. A. Kim, S. Rafisolyman, H. Zeng, C. T. Okamoto, R. L. Wood, and A. K. Mircheff. Na-K-ATPase in lacrimal gland acinar cell endosomal system: correcting a case of mistaken identity. Am. J. Physiol. 271 (Cell Physiol. 40): C1685-C1698, 1996[Abstract/Free Full Text].

21.   Gordon, T., F. Topfer, C. Keech, P. Reynolds, W. Chen, M. Rischmueller, and J. McCluskey. How does autoimmunity to La and Ro initiate and spread? Autoimmunity 18: 87-92, 1994[Medline].

22.   Hamm-Alvarez, S. F., S. R. da Costa, M. Sonee, D. W. Warren, and A. K. Mircheff. Kinesin activation drives the retrieval of secretory membranes following secretion in rabbit lacrimal acinar cells. Adv. Exp. Med. Biol. 438: 177-180, 1998[Medline].

23.   Hamm-Alvarez, S. F., S. R. da Costa, T. Yang, X. Wei, J. P. Gierow, and A. K. Mircheff. Cholinergic stimulation of lacrimal acinar cells promotes redistribution of membrane-associated kinesin and the secretory protein, beta-hexosaminidase, and increases kinesin motor activity. Exp. Eye Res. 64: 141-156, 1997[Medline].

24.   Hanafusa, T., R. Pujol-Borrell, L. Choviato, R. C. Russell, D. Doniach, and G. F. Bottazzo. Aberrant expression of HLA-DR antigen on thyrocytes in Graves' disease. Relevance for autoimmunity. Lancet 2: 1111-1115, 1983[Medline].

25.   Jackson, R., A. M. McNicol, M. Faquharson, and A. K. Foulis. Class II MHC expression in normal adrenal cortex and cortical cells in autoimmune Addison's disease. J. Pathol. 155: 113-120, 1988[Medline].

26.   Jones, D. T., D. Monroy, Z. Ji, S. S. Atherton, and S. C. Pflugfelder. Sjögren's syndrome: cytokine and Epstein-Barr viral gene expression within the conjunctival epithelium. Invest. Ophthalmol. Vis. Sci. 35: 3493-3504, 1994[Abstract].

27.   Kaslow, H. R., Z. Guo, D. W. Warren, R. L. Wood, and A. K. Mircheff. A method to study induction of autoimmunity in vitro: co-culture of lacrimal cells and autologous immune system cells. Adv. Exp. Med. Biol. 438: 583-589, 1998[Medline].

28.   Lambert, R. W., C. A. Maves, and A. K. Mircheff. Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+/K+-ATPase in rabbit lacrimal acini. Curr. Eye Res. 12: 539-551, 1993[Medline].

29.   Londei, M., J. R. Lamb, G. F. Bottazzo, and M. Feldmann. Epithelial cells expressing aberrant MHC class II determinants can present antigen to cloned human T cells. Nature 312: 639-641, 1984[Medline].

30.   Lopez-Robles, E., R. Herrera-Esparza, and E. Avalos-Diaz. Cellular localization of the Ro/SS-A antigen. Clin. Rheumatol. 5: 33-38, 1986[Medline].

31.   Mircheff, A. K. Methods and experimental analysis of isolated epithelial cell membranes. In: Epithelial Transport: A Guide to Methods and Experimental Analysis, edited by N. K. Wills, L. Reuss, and S. A. Lewis. London: Chapman and Hall, 1996, p. 190.

32.   Mircheff, A. K., J. P. Gierow, L. M. Lee, R. W. Robert, R. H. Akashi, and F. M. Hofman. Class II antigen expression by lacrimal epithelial cells. An updated working hypothesis for antigen presentation by epithelial cells. Invest. Ophthalmol. Vis. Sci. 32: 2302-2310, 1991[Abstract].

33.   Mircheff, A. K., J. P. Gierow, and R. L. Wood. Traffic of major histocompatibility complex class II molecules in rabbit lacrimal gland acinar cells. Invest. Ophthalmol. Vis. Sci. 35: 3943-3951, 1994[Abstract].

34.   Mircheff, A. K., and C. C. Lu. A map of membrane populations isolated from rat exorbital gland. Am. J. Physiol. 247 (Gastrointest. Liver Physiol. 10): G651-G661, 1984[Abstract/Free Full Text].

35.   Mircheff, A. K., D. W. Warren, and R. L. Wood. Hormonal support of lacrimal function, primary lacrimal deficiency, autoimmunity, and peripheral tolerance in the lacrimal gland. Ocul. Immunol. Inflamm. 4: 145-172, 1996.

36.   Mizuochi, T., S. T. Yee, M. Ksai, T. Kakiuchi, D. Muno, and E. Kominami. Both cathepsin B and cathepsin D are necessary for processing of ovalbumin as well as for degradation of class II MHC invariant chain. Immunol. Lett. 43: 189-193, 1994[Medline].

37.   Nishimura, Y., and K. Kato. Intracellular transport and processing of lysosomal cathepsin B. Biochem. Biophys. Res. Commun. 148: 254-259, 1987[Medline].

38.   Oliver, C. Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells. J. Cell Biol. 95: 154-161, 1982[Abstract].

39.   Peek, R., G. J. Pruijn, A. J. van der Kemp, and W. J. Van Venrooij. Subcellular distribution of Ro ribonucleoprotein complexes and their constituents. J. Cell Sci. 106: 929-935, 1993[Abstract/Free Full Text].

40.   Peek, R., W. J. Van Venrooij, F. Simons, and G. J. Pruijn. The SS-A/SS-B autoantigenic complex: localization and assembly. Clin. Exp. Rheumatol. Suppl. 11: S15-S18, 1994.

41.   Samarel, A. M., S. W. Worobec, A. G. Ferguson, and M. Lesch. Biosynthesis of the multiple forms of rabbit cardiac cathepsin D. J. Biol. Chem. 259: 4702-4705, 1984[Abstract/Free Full Text].

42.   Smith, P. R., D. G. Williams, P. J. W. Venables, and R. N. Maini. Monoclonal antibodies to the Sjögren's syndrome associated antigen SS-B (La). J. Immunol. Methods 77: 63-76, 1985[Medline].

43.   Stepkowski, S. M., T. Li, and R. M. Franklin. Interleukin-2 like molecule produced by acinar cells in lacrimal gland regulates local immune response (Abstract). Invest. Ophthalmol. Vis. Sci. 34S: 1486, 1993.

44.   Sullivan, D. A., L. A. Wickham, E. M. Rocha, R. S. Kelleher, L. A. da Silveira, and I. Toda. Influence of gender, sex steroid hormones, and the hypothalamic-pituitary axis on the structure and function of the lacrimal gland. Adv. Exp. Med. Biol. 438: 11-42, 1988.

45.   Talal, N. Sjögren's syndrome. Bull. Rheum. Dis. 16: 404-407, 1966[Medline].

46.   Van Noort, J. M., J. Boon, A. C. Van der Drift, J. P. Wagenaar, A. M. Boots, and C. J. Boog. Antigen processing by endosomal proteases determines which sites of sperm-whale myoglobin are eventually recognized by T cells. Eur. J. Immunol. 21: 1989-1996, 1991[Medline].

47.   Venables, P., and S. Brookes. Membrane expression of nuclear antigens: a model for autoimmunity in Sjögren's syndrome? Autoimmunity 13: 321-325, 1992[Medline].

48.   Volc-Platzer, B., O. Majdic, W. Kanpp, K. Wolff, W. Hinterberger, K. Lechner, and G. Stingl. Evidence of HLA-DR antigen biosynthesis by human keratinocytes in disease. J. Exp. Med. 159: 1784-1789, 1984[Abstract].

49.   Wahren, M., L. Solomin, I. Pettersson, and D. Isenber. Autoantibody repertoire to Ro/SSA and La/SSB antigens in patients with primary and secondary Sjögren's syndrome. J. Autoimmun. 11: 29-38, 1998[Medline].

50.   Wilson, B. S., F. Indiveri, V. Quaranta, M. A. Pellegrino, and S. Ferrone. Level of Ia-like antigens on human B and T lymphocytes. Transplant. Proc. 13: 1033-1034, 1981[Medline].

51.   Wood, R. L., M. D. Trousdale, D. Stevenson, A. M. Azzarolo, and A. K. Mircheff. Adenovirus infection of cornea causes histopathologic changes in the lacrimal gland. Curr. Eye Res. 16: 459-466, 1997[Medline].

52.   Yiu, S. C., R. W. Lambert, M. E. Bradley, C. E. Ingham, K. L. Hales, R. L. Wood, and A. K. Mircheff. Stimulation-associated redistribution of Na, K-ATPase in rat lacrimal gland. J. Membr. Biol. 102: 185-194, 1988[Medline].

53.   Yiu, S. C., R. W. Lambert, P. J. Tortoriello, and A. K. Mircheff. Secretagogue-induced redistributions of Na,K-ATPase rat lacrimal acini. Invest. Ophthalmol. Vis. Sci. 32: 2976-2984, 1991[Abstract].

54.   Yoshino, K., R. Garg, D. Monroy, Z. Ji, and S. C. Pflugfelder. Production and secretion of transforming growth factor beta  (TGF-beta ) by the human lacrimal gland. Curr. Eye Res. 15: 615-624, 1996[Medline].

55.   Zhang, J., T. Yang, H. Zeng, E. Olsen, D. W. Warren, A. K. Mircheff, and R. L. Wood. Traffic of locally-synthesized and endocytosed prolactin-like molecules in lacrimal gland acinar cell (Abstract). Invest. Ophthalmol. Vis. Sci. 38: S156, 1997.



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