Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.

Systemic lupus erythematosus is a multisystem autoimmune disease in which the autoantibody response targets a variety of autoantigens of diverse subcellular location. We show here that these autoantigens are clustered in two distinct populations of blebs at the surface of apoptotic cells. The population of smaller blebs contains fragmented endoplasmic reticulum (ER) and ribosomes, as well as the ribonucleoprotein, Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and the small nuclear ribonucleoproteins. These autoantigen clusters have in common their proximity to the ER and nuclear membranes, sites of increased generation of reactive oxygen species in apoptotic cells. Oxidative modification at these sites may be a mechanism that unites this diverse group of molecules together as autoantigens.

S LE is a multisystem autoimmune disease that is characterized by pleiotropy in its clinical and laboratory features (1). The autoantibody response in SLE is both diverse and exuberant, and targets a variety of nucleoprotein particles, some containing DNA (nucleosomes), and others RNA. The latter include small nuclear ribonucleoproteins (snRNPs), 1 cytoplasmic RNPs (SS-A/Ro), and ribosomes (2). Antibodies are also directed against negatively charged phospholipids (3). A striking feature of the different autoantigens in lupus is their lack of restriction to any one subcellular location: they may be exclusively nuclear, cytosolic, or membrane associated, or distributed between the nudeus and cytosol under different conditions. The proposal that the total repertoire of autoantigens might be clustered on a limited number of distinct subceUular particles is an attractive one (4), but such a cluster has not been identified to date.
The evolution of the autoantibody response to nucleosomes in lupus has been studied during clinical flares, as well as in animal models of the disease (5,6). These studies have strongly indicated that the autoantibody response in lupus is antigen driven and T cell dependent. Thus, autoantibodies undergo isotype switching, somatic mutation, and affinity maturation that are characteristic of rechallenge of a primed immune system by antigen (for a review see reference 7). Furthermore, the presence of immune complexes containing double-stranded DNA (dsDNA) during acute flares (8) suggests that endog-1 Abbreviations used in this paper: dsDNA, double-stranded DNA; ER, endoplasmic reticulum; PI, propidium iodide; snRNP, small nuclear ribonudeoprotein; TMG, trimethylguanosine; TdT, terminal deoxynucleotidyl transferase. enous nucleosomes might be one of the antigens driving the immune response in this disease (5,6).
A potential source of these antigenic components is the abundant generation of nucleosomes in cells undergoing apoptosis, as internudeosomal cleavage of DNA is a well-defined biochemical feature of this mode of cell death (9). The morphologic sequence of apoptosis has been well-defined, and follows an orderly process of nuclear condensation, surface blebbing, cytoplasmic contraction, and packaging of cellular components within membranes before their budding from the cell as apoptotic bodies (9). This reorganization is striking, as membrane, cytoplasmic, and nuclear components all appear at the surface of the dying cell. Apoptosis occurs in different cell types in a wide variety of organisms (9)(10)(11)(12). It may occur in response to many different stimuli, including DNA damage (13,14), growth factor deficiency (15), ligation of a family of cell surface receptors (16,17), heat shock (18), and bacterial or viral infection (19,20). Many of these stimuli generate reactive oxygen species, with consequent oxidative damage to multiple cellular targets (21). Furthermore, exposure to H202 (which generates -OH radicals) also results in cell death with an apoptotic morphology, suggesting that oxidant stresses might function in a final common pathway leading to cell death (21). Bcl-2, a member of an interacting family of homologous proteins that modulate apoptoffs (22,23), has recently been shown to function in an antioxidant pathway that prevents apoptosis (21,24).
The skin is a prominent target in patients with lupus, and the characteristic photosensitivity has been proposed to be a consequence of UV-driven autoantigen exposure with resultant antibody-dependent cellular cytotoxicity (25). Indeed, others have shown that UV irradiation ofcuhured monolayers of human keratinocytes changes the subceUular location of the ribonucleoprotein autoantigens Ko, La, Sin, and U1-RNP (26,27). These antigens stained in a punctate pattern at the cell surface of irradiated cells, a site at which they were never present in unirradiated cells. Although UV irradiation induces apoptosis of keratinocytes (28), the correlation between altered autoantigen location and apoptosis has not previously been addressed.
In these studies, we have shown that autoantigens are clustered in two discrete populations of blebs at the surface of apoptotic cells. A population of small, abundant KNAcontaining surface blebs stained intensely for Ro, and contained fragmented endoplasmic reticulum (ELL) and ribosomes. The other population of larger blebs was less numerous and contained DNA (often nicked), surrounded by a rim of ribonucleoprotein antigens including Ko, La, and the snKNPs. These studies indicate that the clusters of autoantigens in lupus are confined to a cellular domain characterized by its proximity to ER and nuclear membranes. Study of these translocated autoantigen particles, and their ultimate fate, may provide a powerful system to explore the exposure and modification of autoantigens targeted in autoimmune disease.

Materials and Methods
Cell Culture and Irradiation. Human keratinocytes were obtained from neonatal foreskins, and cultured in Keratinocyte Growth Medium (KGM) (Clonetics Corp., San Diego, CA). All experiments were performed on confluent secondary monolayers. A panel of eight sunlamp bulbs (model FS40; Westinghouse, NJ) was used as a source of UVB. The UVB output, measured using a research radiometer (model I1700; International Light Inc., Newburyport, MA) and a cosine corrected probe with a SCS 280 filter, was 0.5 mW/cm 2 at a distance of 19 cm. To perform irradiations, cells were washed twice with PBS (2.7 mM KC1, 1.5 mM KHzPO4, 137 mM NaCI, and 8 mM NazHPO4) and irradiated in PBS. The cells were then incubated in KGM in a 5% CO2 humidified incubator at 37~ Initial dose-response and time course studies established that irradiation with 1,650 J/m 2 generated apoptotic cells within 6 h (data not shown). These conditions were used in all subsequent experiments.
DNA Extraction and Gels. DNA from whole cell populations was extracted, processed, and electrophoresed on 1.5% agarose gels as described (29).
lmmunofluorescence and Confocal Microscopy. Immunofluorescence microscopy was performed on keratinocytes that were grown on No. 1 glass coverslips. Unless otherwise specified in the figure legends, cells were fixed with 4% paraformaldehyde (5 rain, 4~ before permeabilization with acetone (30 s, 4~ and antibody staining. Ro, La, and U1-RNP were stained with human sen of defined specificities (diluted 1:160 in PBS) or athnity-purified antibodies, and were visualized with fluorescein-conjugated goat anti-human F(ab')2 (Organon Teknika Corp./Cappel, Durham, NC). The 5'-trimethylguanosine (TMG) cap structures of snRNAs were labeled using a mouse mAb to TMG (Oncogene Science Inc., Uniondale, NY) according to the manufacturer's directions. Visualization was with biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) followed by BODIFY FL-2-conjugated streptavidin (Molecular Probes, Inc., Eugene, OK). BiP (GRP78) was stained using a mouse mAb (StressGen, Victoria, Canada), followed by incubation with biotinylated goat anti-mouse Ig (Southern Biotechnology Associates) and visualization with Texas red-conjugated streptavidin (Pierce, Rockford, IL). A mAb to ribosomal $1 was a generous gift from M. Schmidt-Zachmann (German Cancer Research Center, Heidelberg, FKG). c~-mannopyranosyl and o~-glucopyranosyl residues were stained with 0.1 mg/ml fluorescein-conjugated Con A (Molecular Probes, Inc.) either before or after permeab'rlization with acetone. Coverslips were mounted on glass slides with Permount | (Lipshaw, Pittsburgh, PA), before viewing and photography on a microscope (Axiophot; Carl Zeiss, Inc., Thomwood, NY) equipped with phase contrast and epifinorescence optics. Confocal microscopy was performed on a scanning confocal microscopy system (model MRC600; Bio-Rad Laboratories, Richmond, CA).
Nick Labeling of Apoptotic DNA. Nick labeling of apoptotic DNA in individual cells was performed as described (29), with the following modifications. Keratinocytes grown on coverslips were fixed with paraformaldehyde and acetone (see above) before incubation in terminal deoxynucleotidyl transferase (TdT) buffer. The endogenous peroxidase inactivation and proteinase K treatments were omitted. Horseradish peroxidase-coupled st~tavidin (Southern Biotechnology Associates) was used at a 1:500 dilution before staining with 3-amino-9 ethyl carbazole (AEC) and visualization by light microscopy.
Electron Microsco~. Cells were grown on 35-ram tissue culture dishes, and irradiated according to standard procedures. 6 h after irradiation, monolayers were washed very gently with PBS, and fixed in 1% glutaraldebyde in 0.1 M sodium cacodylate, pH 7.4. Postfixation was in reduced osmium tetroxide. Monolayers were stained en-bloc with 1% uranyl acetate, before dehydration in ethanol, and embedding in Epon. Thin-sections were cut, and some were further stained with lead citrate before viewing and photography in an electron microscope (model TEM type 1A; Zeiss). Microwave fixation was performed as described (30), using a domestic microwave (General Electric, Louisville, KY) at full power for 20 s, such that a final fixative temperature of 50~ was obtained. Antibody Screening Procedures. Reactivity of human autoimmune and control sera with saline soluble extracts of human spleen and rabbit thymus was determined by Ouchterlony double immunodiffusion using standard reference sera to Ro, La, Sin, and RNP (31).

A~nity Purification of Antibodies From Human Sera.
Keratinocytes were lysed in Buffer A (1% NP-40, 20 raM T.ris, pH 7.4, 150 mM NaC1, and 1 raM EDTA containing the following protease inhibitors: pepstatin A, lenpeptin, ant/pain, chymostatin, and PMSF), and electrophoresed on 15% SDS-PAGE. 0.087% bisaerylamide was used to optimally separate the Ro and La proteins (32). The electrophoresed proteins were transferred to Immobiion | (Millipore Corp., Bedford, MA) (33) and used to affinity purify antibodies to 60-kD Ro, 48-kD La, and 70 kD U1-RNP proteins from human sera as described (34). The specificity of the at~inity-purified anti-60-kD Ko was further conhaned by hemagghtination with a purified Ko-RBC conjugate (Hemagen, Waltham, MA). When we used the same procedure (34) to affinity purify antibodies to 52-kD Ro (three different sera were used in three separate attempts), we were unable to dissociate the bound antibodies from the Immobilon | with 0.2 M glycine, pH 2.8. Subsequent attempts to elute bound antibody with 3 M ammonium thiocyanate (35) resulted in a complete loss of antigen binding. We therefore used a patient serum that was monospecitic for 52-kD Ro in immunoblotting and immunoftuorescence (see Figs. 4 and 5, respectively).

UV Irradiation Induces Apoptosis in Keratinocytes.
Keratinocyte monolayers were irradiated with UVB in order to induce apoptotic cell death. Within 6 h of irradiation, the asynchronous onset of apoptosis was detectable by phase contrast microscopy. Apoptotic cells were rounded up, pulled apart from their neighbors, and showed prominent surface blebbing ( Fig. 1 B [ 37]). Apoptosis was confirmed by detection of internucleosomal cleavage of DNA in irradiated populations ( Fig. 1 A, lane 2), in contrast to the DNA from unitradiated populations which remained intact ( Fig. 1 A, lane 1). The correhtion of morphologic changes with multiple internucleosomal nicks was assessed by introducing biotinylated dUTP at nicked sites with TdT (29). The labeled chromatin appeared condensed, and was found in morphologically apoptotic cells. In cells where the nucleus had already fragmented, the multiple apoptotic bodies formed were also strongly labeled ( Fig. 1 B). No labeling was obtained in irradiated cells when TdT was omitted, and labded cells were extremely rare in unirradiated keratinocytes (data not shown). Electron microscopy of irradiated cells demonstrated condensation ofchromatin and fragmentation of the nucleus (Fig. 1 C). Cytoplasmic contraction was accompanied by fragmentation ofintracellular membranes, whereas mitochondria appeared normal. These ultrastructural features are consistent with the morphology of apoptosis (9).

Small Surface Blebs on Apoptotic Keratinocytes Contain RNA.
In our initial studies, we visualized chromatin condensation in apoptotic cells with PI staining after RNase digestion. When RNase treatment was serendipitously omitted, we noted that the small surface blebs on apoptotic cells were intensely labeled by PI, indicating that they contained RNA. To define the nudeic acid contents of the small apoptotic blebs more precisely, we examined their sensitivity to various nudease digestions. In the absence of nudease treatment, surface blebs stained strongly with PI, whereas the cytoplasm stained only weakly. Nuclear staining was prominent, with  conspicuous nucleoli (Fig. 2 A). In contrast, unirradiated keratinocytes showed diffuse cytoplasmic staining, as well as prominent nuclear and nucleolar staining (see Fig. 5 E). When irradiated cells were pretreated with RNase, the small surface blebs remained dearly visible by phase contrast microscopy, but were no longer stained with PI ( Fig. 2 B, arrowheads). Condensed nuclear chromatin was obvious in these blebbed cells, and often showed the breaking up of the nucleus into several discrete fragments. Occasionally, these condensed nuclear fragments were seen at the cell surface in larger blebs (Fig. 2 B, arrows). These larger blebs also stained with the DNA-specific stain, 4',6-diamidino-2-phenylindole, dihydrochloride (DAH) (data not shown), and could be nick labded in the TdT assay ( Fig. 1 B). Pretreatment with DNase did not affect the staining of small surface blebs, which stained strongly with PI under these conditions (Fig. 2 C). In these cells, the nuclear matrix staining had completely disappeared, making the nucleoli even more prominent. No PI staining was seen when irradiated cells were pretreated with both DNase and RNase (Fig. 2 D).
We further defined the RNA-containing surface blebs with confocal microscopy. Accumulation of PI was only visible at the cell surface, and we were never able to visualize similar aggregates of PI within the cytosol (Fig. 3 A). Indeed, cytosolic PI staining in apoptotic cells was diminished when compared with nonapoptotic cells (see and compare Fig. 5, B and E). Furthermore, Z-series and vertical sectioning showed that the blebs were restricted to the apical surface (Fig. 3 B). The small surface blebs were 1.3 _+ 0.3/zm (mean _ SD, n = 54) in size, and were a common feature in apoptotic cells (both polarized and nonpolarized), including human fibroblasts, A431 cells, and Hep2 cells (data not shown). Blebs were always restricted to the nonadherent cell surface, and were present regardless of the initiating stimulus for apoptosis, which included irradiation, heat shock, or incubation in H202 (data not shown). Blebs were numerous in apoptotic keratinocytes with an intact, condensed nucleus, and were more prominent in these cells than in those where nuclear fragmentation was advanced. PI staining was heterogeneous in different blebs: some stained strongly and diffusely with PI, others appeared speckled, and still others contained small central areas that did not stain with H. These R.NAcontaining surface blebs could readily be distinguished from apoptotic bodies since the latter (a) tended to be larger (mean diameter 2.7 _+ 0.7/zm), (b) were fewer in number (3-10/ce11), (c) contained DNA, (at) were present when nuclear fragmentation was advanced, and (e) were detected within the cytoplasm as well as in surface blebs. Both populations of surface blebs could be seen undergoing phagocytosis by surrounding nonapoptotic keratinocytes (data not shown).

Ribonucleoprotein Autoantigens Redistribute in Apoptotic Cells.
We used antibodies to the ribonucleoprotcin autoantigens  to evaluate their dism~ution in apoptotic cells. In initial studies, 18 out of 21 sen positive for Ro by immunodiffusion showed intense staining of the RNA-containing surface blebs (data not shown). No staining of apoptotic surface structures was seen using eight different sera obtained from healthy adults (data not shown). For subsequent studies, we used a monospedfic human serum to the 52-kD Ro protein, and affinity-purified antibodies to the 60-kD Ro, the 48-kD La, and the 70-kD U1-RNP proteins to examine the distribution of these antigens in apoptotic cells. Before use in immunofluorescence microscopy, these reagents were characterized by two-dimensional immunoblotting to confirm that they each recognized a single protein of the predicted size and charge (Fig. 4) 08). In each case, the immunodominant protein focused as a series ofisoforms with estimated isodec-tric points ranging from 7.0-7.5 (60-kD Ro), 6.2-6.7 (52-kD Ro), 6.5-7.2 (48-kD La), and 7.2-7.6 (70 kD U1-RNP).
When these reagents were used in immunofluorescence studies of unirradiated keratinocytes, antibodies to Ro stained the cytoplasm ditfu~y and stained the nucleus with a speckled pattern that excluded nucleoli (Fig. 5 D). A similar pattern was obtained with affmity-puritied antibodies to La, although the cytophsmic staining was less prominent (data not shown). PI, used without RNase, stained the nucleus, with prominent nucleoli, as well as the cytoplasm (Fig. 5 E). In apoptotic keratinocytes, the small surface bhbs stained intensely with antibodies to Ro (Fig. 5, A and G) and with PI (Fig.  5 B), whereas the cytoplasmic st~i-i-g of both was conspicuously decreased (Fig. 5, A-C, G). go st~ini,,g was e~luded from areas of condensed chromatin, but stained intensdy around these areas (Fig. 5 A). Where apoptotic bodies were seen at the ceU surface, a characteristic rim of R_o staining could often be seen around a core of nucleic acid (data not shown). Neither La nor UI-RNP was detected in the small ILblA-containing surface blebs (Fig. 5, H-J). Instead, their staining was exclusively nuclear in the early stages of apoptosis, and was visualized as a bright eccentric rim of ribonucleoproteins around a core of nucleic acid (Fig. 5, I and J).
In the later stages of apoptosis, La and U1-KNP staining was detected at the cell surface surrounding large blebs containing nucleic acid (Pig. 5 H). The staining pattern obtained with antibodies to the 70-kD protein component of U1-RNP was confirmed and extended by staining apoptotic cells with mAbs to TMG (39). These antibodies recognize the unique 5'-modification of the small RblA components of the spllceosome, multiple components of which are targeted by the immune system in lupus. TMG did not stain the small surface blebs (Pig. 5 K), but stained as a bright rim around fragmented nuclear material, either still within the cell (Fig. 5 L), or at the surface (Fig. 5 K). Colocalization studies with DAPI or nick labeling confirmed that the La-and TMG-stained structures contained DNA in their core (data not shown).
Small Surface Blebs on Apoptotic Cells Contain Fragmented ER and Ribosomes. The enrichment of RNA in small surface blebs was accompanied by a marked decrease in its cytoplasmic staining (compare Fig. 5, B and E). This suggested that the blebs might contain ribosomes, as >70% of cytophsmic RNA is comprised of ribosomal RNA. Furthermore, since the ER fragments and moves outwards early in apoptoffs (9), we wondered whether the apicaUy directed movement of PdqA represented a membrane-based translocation. To address this, we used lectin staining (FITC-Con A) to probe for any glycoproteins within surface blebs. In apoptotic cells that were incubated with FITC-Con A before permeab'dization with acetone and subsequent staining with PI, a thin rim of FITC-Con A was visible around, but not within, areas that stained strongly with PI (Fig. 6, A and B). This was consistent with lectin binding to plasma membrane glycoproteins. In apoptotic cells that were permeabilized before incubation with FITC-Con A, clear substructure within the blebs was evident (Fig. 6, C and D): blebs appeared to be composed of multiple small, round, vesicular structures, defined by FITC-Con A staining around a central unstained area. This heterogeneous staining within the blebs was similar to, but much better defined than the heterogeneity previously seen with PI or Ro (Fig. 3 A and Fig. 5, A and B).
We further investigated the contents of the small surface blebs by using mAbs to BiP (GRP78) (Fig. 7, A and B) or ribosomal protein $1 (Fig. 7, C and D) in immunofluorescence studies. Antibodies to BiP (40), a resident ER lumenal protein, stained strongly and specifically in unirradiated keratinocytes; staining was reticular in pattern, and was most intense in the perinudear region (Fig. 7 A). In contrast, there was strong staining within the surface blebs of apoptotic cells, with diminished staining towards the perinudear region (Fig.  7 B). Ribosomal protein $1 stained with a similar reticular pattern in unirradiated cells (Fig. 7 C), and also demonstrated prominent nucleolar staining as previously described (41). Apoptotic cells showed prominent staining within their surface blebs, with decreased staining towards the perinuclear region ( Fig. 7/9). Isotype-matched, irrelevant mAbs did not stain the structures seen with the anti-BiP and anti-ribosomal $1 antibodies (data not shown).
To further resolve the internal structure of small surface blebs, irradiated keratinocytes were fixed in glutaraldehyde and processed for electron microscopy. Thin sections through the apical portion of irradiated cells were examined for sur-face blebs with a size of about 1.5/zm. Although such structures could be identified, the blebs appeared to contain a granular substance that could not be further resolved using this method of fixation. Since our confocal microscopy data had strongly suggested the presence of multiple small membrane-bound vesicular structures within the surface blebs ( Fig.  6 C), we subsequently used a microwave fixation protocol that is reported to improve preservation of membranes, perhaps by optimizing osmication (42)(43)(44). Numerous surface blebs of the expected size were identified in apoptotic cells that had been fixed using this technique (Fig. 8 A). Consistent with our confocal studies, these structures were most prominent at the apical surface of irradiated keratinocytes. Thus, many sections that optimally demonstrated the surface blebs did not also traverse the nucleus (Fig. 8 A). However, other micrographs dearly showed both structures (Fig.  8 B), and confirmed the impression obtained from confocal microscopy that surface blebbing was prominent before nuclear changes of apoptosis became pronounced. The surface blebs were membrane bound, and uranyl acetate staining demonstrated the bilayer structure of the limiting membrane. Many sections showed the blebs either forming at the plasma membrane (Fig. 8, B and C), or cut in a plane that made Figure 7. St~inlng of keratinocytes with antibodies to BiP (A and B) or antiribosomal protein $1 (C and D). Control (A and C) and irradiated (/3 and D) keratinocytes were stained with mAbs to BiP (A and/3) or ribosomal protein $1 (C and D), and visualized by confocal fluorescence microscopy. The cells were fixed in methanol and acetone (41) before staining with antiribosomal $1. Identical results were obtained in two separate experiments performed with each antibody. Bar, 10 ~m. them appear separate from the cell body (Fig. 8, A and B). Most surface blebs were round, with a mean diameter of 1.3 _ 0.23/zm (mean + SD, n --70). The limiting plasma membrane of these blebs enclosed multiple round membranebound vesicles with a diameter of 100 +_ 13.7 nm (mean _ SD, n ~-267) (Fig. 8, D and E). The vesicles were dear, contained minimal ground substance, and were surrounded by a bilayer as confirmed by uranyl acetate staining (Fig. 8  F). Granular structures that appeared to be ribosomes were seen in many blebs (Fig. 8, D and E). In areas of the cell underlying blebs, rough ER could be seen just beneath the plasma membrane (Fig. 8 C and data not shown). Numerous bundles of cytokeratins were prominent in many sections (Fig.  8, A and B). When normal keratinocytes were microwave fixed and processed identically, very few surface blebs with internal vesicles could be found. The few that were present were smaller (<0.5/zm diameter) than in irradiated cells (data not shown).

Autoantigens Are Clustered in the Su~ce Blebs of Apoptotic
Cells. These studies have defined a population of RNAcontaining blebs at the surface of apoptotic cells, that had previously been visualized by phase-and scanning dectronmicroscopy (37). Blebs are numerous, •1.4 ~m in diameter, and are restricted to the apical surface of apoptotic keratinocytes. Blebs are bounded by a membrane bilayer that is continuous with the plasma membrane, are filled with multiple membranous vesicles of uniform size (about 100 nm in diameter), and contain BiP (an ER hmenal marker), ribosomes, and the ribonudeoprotdn autoantigen, Ro. Both Ro and ribosomes are prominent targets of the immune system in lupus, and it is particularly striking that other components of the ER have also been demonstrated to act as autoantigens in this disease. These include the lumenal ER protein, calreticulin (45), the signal recognition particle (46,47), and acidic phospholipids (for a review see reference 3). These small surface blebs therefore constitute a novel duster of autoantigens that appear to rise from rough ER, but have a surface, rather than an internal location.
Although Ro binds to a family of small cytophsmic RNAs, it is not restricted to the cytoplasm (48), and is also found in the apoptotic nudens. Much of this nuclear Ro is reorganized during apoptosis. Ro is excluded from the nudeoplasm as the chromatin becomes condensed, and becomes concentrated around the rim of apoptotic bodies after nuclear fragmentation. La and the snRNPs are similarly rearranged. After forming within the call, the apoptotic bodies move to the surface where they give rise to a population of larger surface structures containing fragmented DNA. These apoptotic bodies also contain a distinct duster of autoantigens that are targeted in patients with lupus, including nudeosomes (5,6,49), Ro (50), La (51), and snRNPs (52). We propose that the majority of recognized autoantigens in lupus are located within these two distinct types of surface structures.
The conspicuous movement of cytoplasmic Ro to small surface blebs early in apoptosis implies that cytoplasmic Ro has an affinity for the other components in these blebs, and suggests that Ro has a function that is performed at the site of ER-ribosome interaction. Such functions might include, but are not limited to, a role in protein synthesis, control of translation, and protein translocation into the ER. Indeed, such roles have previously been proposed (50,53). Further study of the interaction of Ro with the various subcomponents of this particle may shed light on the function and interactions of Ro.
ER and Nuclear Membranes Are Sites of Free Radical Generation in Apoptotic Cells. After an apoptotic signal, cells sustain progressive lipid peroxidation, reflecting the generation within apoptotic cells of lipid-diffusible reactive oxygen species (21). In the ER, induction of lipid peroxidation results in membrane fragmentation into smaU resides (54), with some thickening of the bilayer (55). These vesicular fragments strongly resemble the 100-nm vesicles within membrane blebs (Fig. 8) that stain with specific ER lumenal markers (Fig.  7). It is therefore possible that these vesicles arise as a consequence of unopposed lipid peroxidation in the Elk membranes of apoptotic cells. The presence of peroxidated lipids in the small vesicles remains to be directly demonstrated.
The major sites of free radical generation in cells include mitochondria, EtL, and nuclear membranes (21,56,57). The free radicals produced at these sites are highly reactive species, and their damaging activities are focused at sites of their generation, where macromolecules may sustain oxidative damage (58). It is highly significant therefore that the two populations of autoantigens targeted in patients with lupus are topologically restricted to sites of free radical generation in apoptotic cells, that is, to areas of vesiculating ER (Ro, ribosomes, and EIK proteins), and to the rim of apoptotic bodies that are surrounded by nudear membrane (Ro, La, dsDNA, and snRNPs). It is also of interest that autoantibodies to mitochondrial components (e.g., cytochrome c) have been observed in patients with lupus and related diseases (59). A common feature of self-antigens at these sites might be their vulnerability to oxidative damage.
It has been proposed that some autoreactive T cells escape tolerance induction because they are directed against minor (cryptic) determinants on stir-antigens that are not efferently generated during antigen processing (60). These autoreactire cells may become pathogenic if that cryptic determinant is subsequently revealed (61)(62)(63). A number of free radical-induced modifications of proteins have been described that may reveal potentially cryptic determinants. These include fragmentation (64,65), amino acid modifications (e.g., trypto- phan loss) (64), and novel sensitivity to protease attack (66). Our recent identification of a specific cleavage of the 70-kD U1-RNP antigen in apoptotic cells (L. Casciola-Rosen, and A. Rosen, unpublished results) supports the hypothesis that site-specific mod_ification of proteins may generate unique peptide fragments. The strong association of particular autoantibody responses with specific MHC class II molecules in patients with SLE may reflect the ability of these class II molecules to capture and present self-peptides to T cells (67,68).
Apoptosis in Murine Models of Lupus. MILL mice spontaneously develop late onset autoimmune disease (69), and develop autoantibodies with a specificity very similar to that seen in human disease, including antibodies to dsDNA and Sm (70). When MILL mice are, in addition, homozygous for the lpr mutation, they develop massive lymphadenopathy, and have marked acceleration of autoimmune disease (69). The lpr mutation (71) results from integration of the retrotransposon, ETn into an intron in thefas gene, with diminished fas expression (72,73) and fas-mediated apoptosis. The abnormality in apoptosis has been proposed to be responsible for defective negative sdection of stir-reactive T lymphocytes in the thymus (71). Apoptosis in the thymus is not only a mechanism for the deletion of self-reactive clones, but is also an abundant source of apoptotic autoantigens to which the organism must become tolerant. The failure of fas-mediated T cell apoptosis within the thymus of devdoping 1pr/lpr mice may not only facilitate the persistence of autoreactive clones, but may also deprive the organism of the opportunity to generate a full repertoire of"apoptotic" autoantigens to which to become tolerant. The specificity of the immune response to a restricted, but common group of autoantigens in mouse and human disease is therefore particularly relevant.
Autoantigen Clusters in Lupus: A Model. In these studies, we have shown that apoptosis induces the physical clustering of autoantigens in two distinct populations of cell surface structures. As these blebs contain ER (small blebs) or nudear membrane (apoptotic bodies), sites of unbuffered generation of reactive oxygen species in apoptotic cells, we propose that the contained autoantigens have in common a vulnerability to oxidative modification, targeted by metal binding or active sites (58,66,74). In a genetically susceptible individual, at high rates of generation of novel fragments (e.g., irradiation, heat shock, bacterial or viral infection, or xenobiotic exposure), the appropriate MHC class II molecules may capture and present self-peptides that were previously cryptic (62,63,75,76). As in other systems, the immune response to this self-peptide may subsequently diversify to other areas on the self-molecule to which the organism was previously tolerant (60,77). Subsequent reexposure of the primed immune system to even low levels of"apoptotic" autoantigens (e.g., sun exposure) may lead to the clinical flares in this disease. The revelation of previously cryptic peptides to the immune system by oxidative modification of proteins may also be applicable to other autoimmune diseases.
We thank Carolyn Machamer for critical review of the manuscript, Hugh Rosen for numerous thoughtful suggestions, Alan Aderem and Joe Ahearn for helpful discussions, Ed Cluett for suggesting the microwave fixation and help in its execution, and Mike Delannoy for his invaluable assistance with confocal and electron microscopy. We also thank the members of the Dermatoimmunology laboratory for help with keratinocyte cultures and screening of sera.
This work was supported in part by National Institutes of Health (NIH) grants AR-32490 and AR-40018 (G. Anhalt). A. Rosen was supported by the NIH Physician-Scientist Program, and by American Cancer Society Institutional Research