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* Department of Molecular Sciences, University of Tennessee Health Sciences Center, and
Immusys International L.L.C., Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The process of apoptosis itself may shape features of the autoimmune response. For example, proteolytic cleavage by caspases, site-specific proteases active in apoptosis, enhances the immunogenicity of autoantigens (8, 9, 10, 11, 12, 13). Moreover, caspase activity leads to dramatic changes in cell morphology: the breakup of the nucleus and the redistribution of nuclear fragments to blebs on the apoptotic cell surface. This cellular reorganization is accomplished during the execution phase of apoptosis and requires constitutively active ROCK I kinase, itself a product of cleavage by caspase 3 (14, 15). As a result of nuclear fragmentation and membrane blebbing, autoantigens that are targeted in SLE reorganize and are transported to the cell surface (5). Thus, it is tempting to conclude that specific enzyme activity that promotes cell death also enhances the antigenicity of nucleoprotein complexes in SLE.
If autoantigens are derived from apoptotic cells, then a disruption of the normal pathways of apoptotic cell recognition and clearance may result in autoimmunity. This expectation is supported by experimental evidence. Inherited deficiencies in the recognition of apoptotic cells coincide with a delayed clearance of cell remnants and an increased incidence of autoimmune responses. Thus, characteristic autoantibodies and lupus-like pathology arise in individuals lacking the complement protein C1q (16), a protein that binds apoptotic cells. Similarly, mice with deficiencies in C1q (17) or the serum amyloid P (SAP) component (18) produce autoantibodies and manifest autoimmune disease. Both C1q and SAP are among a category of molecules capable of recognizing patterns of negative charge that are characteristic of apoptotic cells (19, 20). Therefore, the impaired recognition of surface features of apoptotic cells may be linked to an increased probability of autoimmune response.
Defects in the phagocytosis of apoptotic cells may also lead to the production of autoantibodies, although it has been difficult to demonstrate that the absence of a single receptor for binding to apoptotic cells leads to pathogenesis (21). Presumably, this difficulty arises because phagocytes have a variety of receptors that can mediate the recognition and uptake of apoptotic cells (22, 23). Despite this apparent redundancy, Scott et al. (24) uncovered a direct link between the delayed clearance of apoptotic cells and the induction of autoimmunity. These researchers identified a receptor tyrosine kinase, Mer, whose activity is required for phagocytosis of apoptotic cells. In mice with a deletion of the Mer catalytic domain, clearance of apoptotic cells is delayed and anti-DNA autoantibodies are produced (24). Lu and Lemke (25) emphatically extended these observations by combining Mer deficiency with the deletions of Axl and Tyro 3, two additional members of the Mer kinase family. Triply-deficient mice exhibit a striking constellation of autoimmune phenomena, including the production of autoantibodies to DNA and phospholipids and the activation of lymphoid and myeloid cell populations (25). Previous experiments showed that murine autoantibodies can recognize apoptotic cells (26, 27, 28) and that an experimentally induced excess of apoptotic cells can induce the production of anti-nuclear and anti-phospholipid autoantibodies (29, 30, 31). Although some Abs to apoptotic cells may contribute to the clearance of cell debris as part of normal cell disposal mechanisms (32), other Abs to apoptotic cells likely participate in the pathogenesis of autoimmune disease (33). Collectively, these studies suggest that defects in the clearance of apoptotic cells or an excessive load of cell remnants increase the risk of an autoimmune response.
Nevertheless, the hypothesis that apoptotic cells provide nuclear autoantigens raises important questions: Given the rapid and efficient uptake of apoptotic cells by phagocytes, how could cells of the adaptive immune system compete for access to apoptotic cells? Could unique features of apoptotic cells stimulate or inhibit adaptive immune responses? What receptors could B cells use to bind apoptotic cells and what would be the results of such encounters? A more thorough understanding of interactions between Ig receptors and the surface of apoptotic cells may provide answers to these questions.
Previously, we used site-directed mutagenesis to evaluate the role of
somatic mutations in the binding of autoantibodies to a complex between
phosphatidylserine, a phospholipid that is externalized by apoptotic
cells, and 
2 glycoprotein I
(2GPI), a serum protein that binds to
phosphatidylserine on the surface of apoptotic cells (34).
The starting point for our studies was 3H9, a murine autoantibody
reactive with DNA, chromatin, and negatively charged phospholipids
(35). We observed that binding to the
phosphatidylserine-2GPI complex may be
enhanced by somatic mutations in the H chain of 3H9 and that additional
increases in affinity are achieved by introduction of arginines at
sites that sustain such mutations in vivo. In addition, two variants of
3H9, the increased affinity mutant D56R/S76R and the germline revertant
R53G/I57T/D65G, demonstrated binding to apoptotic cells.
Positive selection for mutations in 3H9 and other Abs that recognize a
complex between phosphatidylserine and 2GPI
implies direct contacts between B cells and apoptotic cells. To explore
the mechanism of Ab binding to apoptotic cells, we used D56R/S76R and
compared its binding to DNA4/1, a human anti-DNA autoantibody
(36). Confocal microscopy revealed that only D56R/S76R
preferentially interacts with blebs on the apoptotic cell surface.
Analogous interactions between B cells and apoptotic cells may
contribute to the regulation of tolerance to nuclear Ags.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The D56R/S76R (37) and DNA4/1 single-chain variable fragments (scFv) (36) were described previously. The complete scFv fusion proteins included the c-Jun leucine zipper, the protein A "B" domain, and a pentahistidine tag, as was confirmed by DNA sequencing of the two expression vectors. The scFv were purified as described elsewhere (37). Briefly, soluble scFv were recovered from the periplasm by digestion of the bacterial cell wall with lysozyme, dialyzed overnight against binding buffer (50 mM Tris-HCl (pH 8.0), 1.0 M NaCl, and 10 mM imidazole), and adsorbed to 1.0 ml of packed Ni-NTA agarose (Qiagen, Valencia, CA) overnight at 4°C. The next morning, the slurry was applied to a chromatography column and washed twice with 4.0 ml of wash buffer (50 mM Tris-HCl (pH 8.0), 1.0 M NaCl, 40 mM imidazole, 0.5% Tween 20). The purified scFv were eluted with 2.0 ml of elution buffer (50 mM Tris-HCl, pH 8.0, 1.0 M NaCl, and 350 mM imidazole), dialyzed overnight against PBS, and analyzed by SDS-PAGE and Coomassie blue staining.
Cell culture and induction of apoptosis
Jurkat cells were harvested from culture and resuspended at a density of 106/ml in RPMI 1640 containing 10% FBS. Apoptosis was induced for 12 h with 1.0 µM staurosporine (Sigma-Aldrich, St. Louis, MO), 2.0 µM camptothecin (Sigma-Aldrich), or 200 ng/ml anti-Fas mAb (clone 7C11; Beckman Coulter, Brea, CA). To inhibit apoptosis, parallel cultures were preincubated for 2 h with 20 µM Z-Val-Ala-Asp(Ome)-fluoromethyl-ketone (Z-VAD(OMe)-fmk; Enzyme System Products, Livermore, CA). At the end of the apoptosis induction period, 5 x 105 cells were aliquoted into tubes and stained for flow cytometry or confocal microscopy as described below.
Flow cytometry
Cells were washed with 4.0 ml of ice-cold HBSS (Mediatech, Herndon, VA) containing 1.0 mM CaCl2, 3% FBS, and 0.02% NaN3 (HBSS/FBS). Washed cells were incubated with 10 µg/ml D56R/S76R or DNA4/1 scFv for 15 min on ice, washed twice as above, and stained with FITC-conjugated annexin V (BD Biosciences, San Diego, CA) and allophycocyanin-conjugated rabbit IgG (Molecular Probes, Eugene, OR), as recommended by the manufacturers. The rabbit IgG binds to the protein A domain of the scFv fusion protein (37). Thirty thousand events were examined per sample on a FACSCalibur (BD Biosciences). The staining profiles were analyzed using FlowJo software (Treestar, San Carlos, CA).
Confocal microscopy
Cells were washed with HBSS and fixed with ice-cold 4% paraformaldehyde for 10 min. Fixed cells were washed with HBSS/FBS and incubated with 10 µg/ml D56R/S76R or DNA4/1 for 15 min on ice. After incubation with the scFv, cells were washed once with HBSS/FBS and incubated with biotinylated annexin V (BD Biosciences) for 15 min on ice. Cells were washed twice with HBSS/FBS and stained with streptavidin-conjugated Alexafluor 488 (Molecular Probes), Rhodamine red-conjugated human serum IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and the nucleic acid dye TO-PRO-3 (Molecular Probes). After a 15-min incubation on ice, cells were washed with HBSS/FBS and mounted onto poly-L-lysine-coated glass slides for viewing with a LSM 510 laser scanning microscope (Zeiss, Thorwood, NY).
ROCK I kinase inhibition
To inhibit membrane blebbing, we used the E6-1 subclone of Jurkat T cells (American Type Culture Collection, Manassas, VA) that is highly sensitive to Fas ligation. Cells were incubated with 50 µM Y-27632 (Tocris, Ballwin, MO), a specific inhibitor of ROCK I kinase, 1 h before the addition of 50 ng/ml anti-Fas. In the absence of inhibitor, this concentration of anti-Fas induced vigorous membrane blebbing within 4 h after stimulation. The cells were exposed to anti-Fas in the presence or absence of Y27632 and examined by phase microscopy. The binding of D56R/S76R and annexin V was analyzed by flow cytometry, as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently reported that D56R/S76R, an scFv with specificity
for DNA, chromatin, and the
phosphatidylserine-2GPI complex, has the
ability to bind apoptotic cells (34). To investigate
whether D56R/S76R recognizes apoptotic cells following diverse initial
stimuli for apoptosis, we examined the binding of D56R/S76R to Jurkat
cells treated with staurosporine, camptothecin, or a murine
anti-Fas mAb (7C11). These treatments were chosen because they
induce apoptosis through different pathways (38, 39, 40).
We used flow cytometry for the initial analyses because it provides a
broad view of the entire cell population. Following 12 h of
incubation in the presence of apoptotic stimuli, apoptosis was examined
by staining with annexin V, a molecule that recognizes
phosphatidylserine in the presence of Ca2+ (Fig. 1
). Approximately 29 (camptothecin) to
62% (anti-Fas) of cells were observed to bind annexin V under
these experimental conditions, whereas <6% were positive for annexin
V in the absence of added stimuli. Annexin V-positive cells could be
further subdivided based on their binding to the D56R/S76R scFv.
Between 10 (camptothecin) and 26% (staurosporine) of annexin
V-positive cells were reactive with the scFv. The relative
proportions of annexin V and D56R/S76R double-positive cells were found
to change with time of incubation: the proportion of double-positive
cells gradually increased from <3% of the total of annexin V-positive
cells at 4 h (data not shown) to the values observed at 12 h
(Fig. 1).
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). As expected, in the presence of this inhibitor, the
staining with annexin V also decreased to near-background
levels. The binding of the scFv to apoptotic cells was mediated by the
combining site of D56R/S76R, because the human anti-DNA-derived
scFv, DNA4/1 (36), failed to give detectable binding to
either annexin V-positive or annexin V-negative Jurkat cells. Microscopic localization of binding
The finding that the D56R/S76R scFv bound to a subset, but not
all, annexin V-positive cells was surprising, because, in vitro,
D56R/S76R binds to phosphatidylserine (34), the same
anionic phospholipid that mediates the binding of annexin V to
apoptotic cells (42). We used immunofluorescence to gain a
more detailed view of annexin V and scFv binding to Jurkat cells
treated to induce apoptosis. Fig. 2
illustrates the results of confocal microscopy with D56R/S76R or DNA4/1
scFv, annexin V, and TO-PRO-3, a DNA intercalating dye. Each of the
three treatments for inducing apoptosis, camptothecin, staurosporine,
and anti-Fas, resulted in similar staining morphologies (Fig. 2, A–C). Thus, A–C of Fig. 2 are intended to
illustrate representative views seen at different stages of apoptosis,
rather than to imply qualitative differences between the
treatments.
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A were in early to
mid-apoptosis, as two of them stained only faintly with annexin V and
were larger than the remaining two that stained brightly with annexin
V. The D56R/S76R scFv bound preferentially to the cells stained
brightly with annexin V. The coincident binding of the two molecules
was visualized as areas of yellow, due to the overlap between the
Alexa-fluor 488 (green) signal associated with annexin V and the
rhodamine red fluorescence used to localize the scFv. The two annexin V
bright cells may represent the two populations of annexin V-positive
cells identified by flow cytometry (Fig. 1), as one stained more
brightly with annexin V than with the scFv, whereas the other stained
equally well with both molecules.
The cells shown in Fig. 2B were in more advanced apoptosis.
All three cells were bound by annexin V and the D56R/S76R scFv,
although the two molecules did not occupy identical positions
onthe cell surface but instead were increasingly localized to
nonoverlapping membrane domains. This was particularly evident at later
stages of apoptosis, as illustrated by the smaller cell near the middle
of Fig. 2B whose membrane was permeable to TO-PRO-3.
The segregation of the ligands for annexin V from the epitopes
recognized by D56R/S76R was most evident in cells undergoing blebbing.
The D56R/S76R scFv bound several large blebs, whereas annexin V
occupied areas of the cell membrane that extended between adjacent
blebs (Fig. 2C). As a result, areas of overlap between
annexin V and the scFv were limited. In addition, a few smaller blebs
primarily stained with annexin V. The lower cell shown in Fig. 2C represented an earlier stage of apoptosis in which
annexin V staining was already well developed but scFv binding was
barely detectable. In cases of limited binding by the scFv, its binding
was concentrated to focal points that were near areas of increased
annexin V staining.
Fig. 2D illustrates staining with annexin V and reactivity
with the human anti-DNA DNA4/1. Of the five cells that stained with
annexin V, only two bound the scFv. In each case, scFv binding was
observed in the interior of the cells and coincided with the binding of
the DNA intercalator TO-PRO-3. This pattern of binding was notably
different from the binding of D56R/S76R to the surface of apoptotic
cells. The observed differences suggested that the DNA4/1 scFv bound to
DNA or chromatin within nuclear fragments rather than to epitopes on
the surface of blebs.
Fig. 2, E–G, represent controls for the experiments.
Fig. 2E shows that, in the absence of D56R/S76R, annexin V
bound efficiently to blebs that formed on the surface of apoptotic
cells. Fig. 2F demonstrates that pretreatment with
Z-VAD(OMe)-fmk reduced both annexin V and scFv binding to background
levels despite treatment with staurosporine, and Fig. 2G
shows lack of scFv and annexin V binding to live cells. Although not
shown, Z-VAD(OMe)-fmk also greatly reduced binding to cells treated
with camptothecin or anti-Fas Ab, thus confirming that D56R/S76R
binding requires entry into the caspase-mediated execution phase of
apoptosis.
Nuclear fragmentation, blebs, and apoptotic bodies
Studies of human keratinocyte apoptosis have shown that
nuclear and cytoplasmic autoantigens segregate into two different
classes of cell surface blebs that can be distinguished based on size
(5). Binding of D56R/S76R to apoptotic Jurkat cells with
intact plasma membranes revealed that the scFv bound preferentially to
the surface of both large and small apoptotic blebs (Fig. 3A). To evaluate the location
of nuclear fragments relative to the surface blebs bound by the scFv,
we took advantage of the observation that, in the later stages of the
execution phase of apoptosis, cells lose membrane integrity and become
permeable to the DNA intercalator TO-PRO-3 (Fig. 3, B and
C). In cells that had become permeable, D56R/S76R bound to
blebs that contained nuclear fragments and stained with TO-PRO-3,
although we also noted the binding of the scFv to blebs that did not
contain nuclear material (Fig. 3, B and C). By
analogy to apoptotic keratinocytes, we infer that the smaller blebs
that did not stain with TO-PRO-3 (Fig. 3B) contained
ribonucleoprotein complexes (5).
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C) revealed dense packing of DNA in the interior of the
larger blebs and the contiguous staining of the bleb surface by the
scFv. Strikingly, an analysis of consecutive optical sections indicated
that blebs appear to separate from the remainder of the cell. Thus, a
link may exist between blebbing and the formation of apoptotic bodies,
membrane-bound particles containing nuclear material (Fig. 3C). The proposed link implies that nuclear Ags found in
blebs should also be found in apoptotic bodies. This idea is supported
by the recent observation that apoptotic bodies retain the capacity to
transfer genetic information from apoptotic cells to viable cells
(43). The transformation of blebs into apoptotic bodies
may be responsible for the reduction in DNA content that is a
characteristic of cells in the late stages of apoptosis
(44). ROCK I kinase facilitates scFv binding to apoptotic cells
Caspase 3-mediated activation of the Rho-associated kinase ROCK I
stimulates the fragmentation of the nucleus and the packaging of
nuclear material into blebs at the cell surface. (15, 14).
We have exploited the specific inhibitor of ROCK I kinase, Y27632
(45), to evaluate the effects of reduced blebbing on the
binding of D56R/S76R to apoptotic cells. For that purpose, cells were
treated with anti-Fas in the absence (Fig. 4A) or presence of the
inhibitor (Fig. 4B) and examined by microscopy and flow
cytometry. In cells that were pretreated with Y-27632, blebbing was
inhibited and the binding of D56R/S76R was significantly reduced (Fig. 4C). These results confirmed that blebs play a central role
in the binding of D56R/S76R to apoptotic cells. In contrast,
pretreatment with Y-27632 did not reduce the staining with annexin V
(data not shown). Taken together, our studies indicate that binding of
the scFv to blebs requires the same enzyme activity that orchestrates
the fragmentation of the nucleus and the migration of nuclear domains
to sites of plasma membrane blebbing.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The most intense
binding coincided with blebs that formed on the surface of apoptotic
cells, some of which contained lobes of the fragmented nucleus (Figs. 2 and 3). Conversely, binding of the scFv was greatly diminished by
inhibition of ROCK I, the kinase that mediates the breakup of the
nucleus and the distribution of nuclear fragments to surface blebs
(Fig. 4C).
One of the earliest events in apoptosis, the exposure of
phosphatidylserine on the outer membrane leaflet, can be visualized by
the binding of annexin V (42). Despite the fact that
D56R/S76R also binds phosphatidylserine, we found differences in
binding to apoptotic cells between the scFv and annexin V. Time course
experiments demonstrated that annexin V binding precedes scFv binding,
such that the scFv only bound to a fraction of annexin V-positive cells
(Fig. 1). The reason for the more selective binding of the scFv was
revealed by confocal microscopy. The scFv were more specific than
annexin V for cells in the execution phase of apoptosis (Figs. 2 and 3). In addition, at that stage of apoptosis, the two molecules
segregated to different binding sites: the scFv bound blebs, whereas,
in the presence of the scFv, annexin V predominantly localized to areas
between the blebs (Figs. 2 and 3). Because, in the absence of the scFv,
annexin V bound to blebs and to areas of the membrane between blebs
(Fig. 2E and Ref. 46), the possibility arises
that the scFv can displace annexin V from certain domains of the
apoptotic cell surface.
Alternatively, the differences in binding between the scFv and annexin
V arise because their binding is affected in different ways by
additional ligands on the apoptotic cell surface. A ligand that may
affect the binding of the scFv to apoptotic cells is
2GPI, a molecule that independently binds to
apoptotic cells (27, 47) and enhances the binding of our
scFv to phosphatidylserine (34). This alternative predicts
that 2GPI also exhibits a preference for
binding to blebs on the apoptotic cells surface. We are currently
designing experiments to test whether 2GPI
serves as a partner in scFv binding to blebs and apoptotic bodies.
Treatment with Y27632, an inhibitor of ROCK-I kinase (45),
implied that the preferred ligands for annexin V and D56R/S76R may be
different. Following addition of Y27632, anti-Fas-induced membrane
blebbing was inhibited and scFv binding was reduced (Fig. 4C), whereas annexin V staining showed no decrease. These
results provide evidence that, in apoptosis, the active redistribution
of nuclear and cytoplasmic contents into different cellular
compartments has a counterpart in the expression of distinct cell
surface ligands that can be recognized by autoantibodies. The presence
of specific ligands on blebs and apoptotic bodies implies that
mechanisms exist to facilitate the recognition and uptake of
nucleoprotein complexes that are packaged in blebs and apoptotic bodies
during apoptosis.
The differences in binding between scFv and annexin V may be emblematic of two fundamentally different modes of apoptotic cell recognition. One recognition pathway may rely on direct binding to phosphatidylserine to achieve rapid recognition and uptake of apoptotic cells (48). Multiple receptors for phosphatidylserine on phagocytes exemplify this type of interaction (22, 23). Because phosphatidylserine exposure usually (48), but not always (49, 50), precedes blebbing, uptake may already be in progress before cells initiate blebbing. However, the existence of autoantibodies and pattern recognition molecules that specifically bind to blebs suggests that some cells may progress to the execution phase of apoptosis before phagocytosis is complete. Thus, additional recognition pathways may be required to safeguard against the release of immune stimulators from cells in more advanced stages of apoptosis. The additional pathways may use serum proteins as adapter molecules in the recognition of blebs and apoptotic bodies (47, 51).
Binding to blebs is a property that is shared among several pattern recognition molecules that also recognize microbial pathogens, activate complement, and stimulate uptake by phagocytes (20, 52, 53). Molecules that belong to this group and bind to blebs include the mannose-binding lectin (54), the complement collectin C1q (55), C-reactive protein (56), and SAP component (57). These proteins share a rather broad binding specificity that includes carbohydrates, phospholipids, and nucleoprotein complexes, such as chromatin and ribonucleoproteins (52, 55, 57, 58). The binding of pattern recognition molecules to ligands on apoptotic cells may ensure their efficient clearance, as implied by the increased levels of apoptotic cells in mice with C1q (17) or SAP (18) deficiencies.
A situation that is analogous to a deficiency in C1q or SAP may arise if autoantibodies to pattern recognition molecules or their ligands on apoptotic cells disrupt the recognition of apoptotic remnants by cells of the innate immune system (16, 59, 60, 61, 62). Impaired removal of apoptotic cell remnants may be a predisposing factor for the production of autoantibodies to nucleoprotein complexes that are typical of lupus (16). As a result of autoantibody binding to apoptotic cells, uptake of cell remnants may be diverted from complement receptors to Fc receptors, thus increasing the likelihood of an autoimmune response. It is also important to consider the possibility B cell receptors, analogous in specificity to D56R/S76R, may engage apoptotic cells. Such interactions may send powerful negative signals and reinforce tolerance to self (63). However, inappropriate activation of B cells capable of binding to apoptotic cells may lead to the presentation of diverse self-Ags that are targeted in SLE. Thus, interactions between B cells and apoptotic cells may represent a critical event in B cell tolerance and autoimmunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marko Z. Radic, Department of Molecular Sciences, University of Tennessee Health Sciences Center, 858 Madison Avenue, Memphis, TN 38163. E-mail address: mradic{at}utmem.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; 2GPI, 2 glycoprotein I; scFv, single-chain variable fragment; Z-VAD(OMe)-fmk, Z-Val-Ala-Asp(Ome)-fluormethylketone; SAP, serum amyloid P.
Received for publication December 5, 2001. Accepted for publication April 22, 2002.
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L. Frisoni, L. Mcphie, L. Colonna, U. Sriram, M. Monestier, S. Gallucci, and R. Caricchio Nuclear Autoantigen Translocation and Autoantibody Opsonization Lead to Increased Dendritic Cell Phagocytosis and Presentation of Nuclear Antigens: A Novel Pathogenic Pathway for Autoimmunity? J. Immunol., August 15, 2005; 175(4): 2692 - 2701. [Abstract] [Full Text] [PDF]
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 D. R. Croft, M. L. Coleman, S. Li, D. Robertson, T. Sullivan, C. L. Stewart, and M. F. Olson Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration J. Cell Biol., January 17, 2005; 168(2): 245 - 255. [Abstract] [Full Text] [PDF]
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 M.-K. Chang, C. J. Binder, Y. I. Miller, G. Subbanagounder, G. J. Silverman, J. A. Berliner, and J. L. Witztum Apoptotic Cells with Oxidation-specific Epitopes Are Immunogenic and Proinflammatory J. Exp. Med., December 6, 2004; 200(11): 1359 - 1370. [Abstract] [Full Text] [PDF]
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 A. Rahman Autoantibodies, lupus and the science of sabotage Rheumatology, November 1, 2004; 43(11): 1326 - 1336. [Abstract] [Full Text] [PDF]
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M. A. Steeves and T. N. Marion Tolerance to DNA in (NZB x NZW)F1 Mice That Inherit an Anti-DNA VH as a Conventional {micro} H Chain Transgene but Not as a VH Knock-in Transgene J. Immunol., June 1, 2004; 172(11): 6568 - 6577. [Abstract] [Full Text] [PDF]
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M. Radic, T. Marion, and M. Monestier Nucleosomes Are Exposed at the Cell Surface in Apoptosis J. Immunol., June 1, 2004; 172(11): 6692 - 6700. [Abstract] [Full Text] [PDF]
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 A. Rosen and L. Casciola-Rosen ALTERED AUTOANTIGEN STRUCTURE IN SJoGREN'S SYNDROME: IMPLICATIONS FOR THE PATHOGENESIS OF AUTOIMMUNE TISSUE DAMAGE Critical Reviews in Oral Biology & Medicine, May 1, 2004; 15(3): 156 - 164. [Abstract] [Full Text] [PDF]
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P. X. Shaw, C. S. Goodyear, M.-K. Chang, J. L. Witztum, and G. J. Silverman34 The Autoreactivity of Anti-Phosphorylcholine Antibodies for Atherosclerosis-Associated Neo-Antigens and Apoptotic Cells J. Immunol., June 15, 2003; 170(12): 6151 - 6157. [Abstract] [Full Text] [PDF]
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P. A. Verhoef, M. Estacion, W. Schilling, and G. R. Dubyak P2X7 Receptor-Dependent Blebbing and the Activation of Rho-Effector Kinases, Caspases, and IL-1{beta} Release J. Immunol., June 1, 2003; 170(11): 5728 - 5738. [Abstract] [Full Text] [PDF]
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Y. Mi, S. D. Thomas, X. Xu, L. K. Casson, D. M. Miller, and P. J. Bates Apoptosis in Leukemia Cells Is Accompanied by Alterations in the Levels and Localization of Nucleolin J. Biol. Chem., February 28, 2003; 278(10): 8572 - 8579. [Abstract] [Full Text] [PDF]
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 A. Shiratsuchi, T. Mori, Y. Takahashi, K. Sakai, and Y. Nakanishi A Presumed Human Nuclear Autoantigen That Translocates to Plasma Membrane Blebs during Apoptosis J. Biochem., February 1, 2003; 133(2): 211 - 218. [Abstract] [Full Text] [PDF]
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