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
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ABSTRACT |
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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
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INTRODUCTION |
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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 -subunits, of immature
and mature forms of cathepsins B and D, and of La/SSB-reactive proteins.
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MATERIALS AND METHODS |
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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),
N-p-tosyl-L-arginine methyl ester (TAME),
N
-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).
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RESULTS |
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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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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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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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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
-subunit, cathepsins B and D, and La/SSB-reactive
proteins. Immunoblot methods were used to survey the
distributions of MHC class II
-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
-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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MHC class II molecules. Figure
7 presents the density gradient
distribution of the MHC class II molecule -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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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 -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
-subunit, procathepsin B is
particularly prominent in ld-tgnl.
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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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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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DISCUSSION |
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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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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
-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 -glucosidase,
-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.
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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-, 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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