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,
,
*
Immunology Graduate Training Program, Departments of
Medicine and
Cell Biology, University of Pittsburgh School of Medicine, and
University of Pittsburgh Arthritis Institute, Pittsburgh, PA 15261; and
¶ Department of Veterans Affairs Medical Center, Cleveland, OH 44106
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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 programmed cell death, or apoptosis, is a normal physiologic process that is important in maintaining homeostasis (5). However, this process may generate a reservoir of self Ags that threatens immune tolerance. Cells undergoing apoptosis reorganize and package intracellular constituents into subcellular structures or blebs. As a result of this reorganization, membrane, cytoplasmic, and nuclear elements appear at the surface of the apoptotic cell segregated within specific subsets of these blebs. Casciola-Rosen et al. (6) have demonstrated that autoantigens targeted by patients with SLE are clustered within two discrete populations of blebs. One consequence of this reorganization is that self Ags such as Ro and La become accessible to autoantibodies, as demonstrated with apoptotic cardiac myocytes (7), suggesting that apoptotic cells may be targets for autoantibodies and initiate tissue injury.
Not only does apoptosis appear to generate extracellular targets for an established autoimmune response, but it may also be responsible for the primary autoimmunization. Autoantigens may become altered during apoptosis by several mechanisms, including cleavage by intracellular proteases that become activated during the apoptotic process (8, 9, 10, 11, 12, 13), selective phosphorylation by stress-activated protein kinases (14), and colocalization of self Ags with viral Ags during virus-induced apoptosis (15). Such modification of self Ags could result in the exposure of immunocryptic epitopes and provide a challenge for immune tolerance (6).
The immunogenic potential of apoptotic cells has been demonstrated by several groups (16, 17, 18, 19, 20, 21). Albert et al. showed that dendritic cells acquire influenza-specific Ags from apoptotic, influenza virus-infected macrophages and can prime virus-specific CTL responses (16, 17). It has also been shown that dendritic cells can present tumor Ags derived from apoptotic tumor cells (18, 19, 20). Immunization of mice with large numbers of apoptotic thymocytes results in the generation of autoantibodies (21), demonstrating that immune tolerance can be broken by apoptotic cells. These data indicate that apoptotic cells constitute a pool of potentially immunogenic self Ags that must be handled properly to prevent autoimmunization.
We have previously demonstrated that the first component of the classical complement pathway, C1q, binds specifically and directly to surface blebs generated by apoptotic keratinocytes (22). However, it has not been determined where tolerance is initially broken anatomically in the immunopathogenesis of lupus. The cutaneous synthesis and availability of C1q also remain unknown. Therefore, we have expanded these studies to focus directly on the human vasculature. Specifically, we demonstrate here that this process is not limited to keratinocytes. C1q also binds to surface blebs generated by apoptotic endothelial cells as well as to apoptotic PBMC, two cell types that regularly undergo apoptosis in vivo and become exposed to circulating proteins of the classical complement pathway. We also show here that it is the globular heads of C1q that bind to the apoptotic bleb, suggesting that this recognition complex is structurally capable of activating the enzymatic cascade of the classical pathway. These data provide further insight into how C1q may mediate the recognition and clearance of apoptotic cells and subcellular structures, and maintain immune tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HUVEC (Clonetics, San Diego, CA) were cultured in endothelial cell growth medium (Clonetics) according to the manufacturer’s directions. HeLa cells were cultured in DMEM supplemented with 10% bovine calf serum, 2 mM glutamine, and 100 U/ml penicillin and streptomycin. HUVEC and HeLa cells were grown on sterile coverslips overnight to 90% confluence, washed twice with PBS, and exposed to 1400 J/m2 UVB. A Spectra Mini II unit served as the UVB source (Daavlin, Bryan, OH). UVB output was monitored with a UVX radiometer (UVP, Upland, CA) equipped with a cosine-corrected 310-nm sensor. PBMC were isolated from freshly drawn blood from normal human donors by gradient centrifugation over Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden), cultured in serum-free Aim V medium (Life Technologies, Grand Island, NY), and exposed to 1050 J/m2UVB in six-well tissue culture plates in PBS. Culture medium was replaced, and the cells were incubated at 37°C in 5% CO2 for 4–6 h. Preliminary studies determined the optimal UVB dose and post-treatment incubation time for each cell type to become apoptotic.
Immunofluorescence microscopy
To assess C1q binding, HUVEC and HeLa cells were fixed in a 4%
paraformaldehyde solution at 4°C for 1–2 min following the onset of
apoptosis. Primary PBMC were not fixed before assaying for bound C1q.
Cells were incubated with 10 µg/ml purified C1q (Quidel, San Diego,
CA) in PBS for 30 min at 4°C and then washed with PBS. C1q was
detected by direct immunofluorescence using FITC-conjugated
anti-C1q mAb 12A5B7 or FITC-conjugated mAb MOPC21 as an isotype
control. In some cases bound C1q was detected using mAbs 1H11 and 3B9
that recognize the heads of C1q or mAbs 3H3 and 2A10 that recognize the
collagen-like tail of C1q (provided by V. Schumaker and P. Poon)
(23). In addition, bound C1q was detected using IgG from
patients with hypocomplementemic urticarial vasculitis syndrome (HUVS)
(Table I
) (24).
Autoantibodies against the collagen-like tail region of C1q constitute
0.1–1% of these patients’ IgG. Bound Ab was visualized with
FITC-conjugated goat anti-mouse IgG F(ab')2
or goat anti-human IgG F(ab')2, respectively.
A concentration of 10 µg/ml was used for all Abs, except for the HUVS
patient IgG, where a 1/10 dilution was used for staining. Aliquots of
PBMC were stained with annexin V-FITC (PharMingen, San Diego, CA) and
propidium iodide (Sigma, St. Louis, MO), according to manufacturer’s
protocol, to monitor apoptosis. Following immunofluorescence staining,
cells were fixed for 5 min in 4% paraformaldehyde (except for PBMC,
which were not fixed), stained with 2 µg/ml Hoechst dye 33258 or with
5 µg/ml propidium iodide for 5 min, and mounted on glass slides with
Permafluor (Lipshaw, Pittsburgh, PA). Cells were imaged using a
Microphot-FXA fluorescence microscope (Nikon, Melville, NY) and a Leica
TCS NT confocal scanning microscope (Leica Microsystems, Heidelberg,
Germany). To generate specific spatial localizations within cells
and at the cell surface, serial optical sectionswere taken at
0.5-µm intervals throughout the depth of all cells examined. C1q
binding to apoptotic PBMC was assessed by flow cytometry using a
FACSCalibur (Becton Dickinson, San Jose, CA), as well as by
fluorescence microscopy.
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Binding of C1q to HeLa cells was examined by transmission electron microscopy using immunogold labeling. Cells were exposed to UVB to induce apoptosis, fixed as described above, and incubated with purified C1q followed by mAb 12A5B7. Bound Ab was detected with 5-nm gold particles conjugated to protein A. Cells were fixed in 2.5% glutaraldehyde in situ and embedded in epon following dehydration in a graded series of alcohols. Sections (60 nm) were cut using a Leica Ultracut R Ultramicrotome, mounted on 200-mesh grids, counterstained with 2% uranyl acetate and 1% lead citrate, and examined with a JEOL 1210 transmission electron microscope (JEOL, Tokyo, Japan). Representative micrographs were taken at x30,000.
C1q blocking experiments
Purified C1q (10 µg/ml) was incubated with 50 µg/ml C1q head- or tail-specific mAbs, singly or combined, or with a 1/10 dilution of combined anti-C1q IgG from four HUVS patients for 30 min on ice before incubation with UVB-treated cells. Two of the tail-specific mAbs (3H3 and 2A10) and two of the head-specific mAbs (1H11 and 3B9) were digested with papain, and the Fab were purified using a protein A column (Pierce, Rockford, IL), to remove remaining IgG and Fc fragments. The combined head or tail Fab were incubated with purified C1q as described above. Alternatively, 10 µg/ml purified C1q was coincubated with purified C1q heads prepared by collagenase digestion as previously described (25). FITC-conjugated 12A5B7 or FITC-conjugated goat anti-mouse IgG F(ab')2 was used to detect bound C1q or C1q-bound Ab, respectively, present on cells. Fluorescence intensity was quantified using a laser scanning cytometer and WinCyte software (CompuCyte, Cambridge, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four to 6 h after UVB irradiation, HUVEC and HeLa cells
underwent visible morphological changes indicative of apoptosis:
normally flat adherent cells became rounded up with visible blebs on
their surface. Nuclei, when visualized with Hoechst stain, appeared
condensed, often crescent-shaped, and sometimes fragmented. When cells
were incubated with purified C1q, washed, and stained with the
C1q-specific mAb 12A5B7-FITC, 20–50% of the apoptotic cells
stained positively for C1q (Fig. 1, A and D). The bright fluorescent staining
appeared to be confined to the surface blebs or to polar regions on the
cell surface, consistent with earlier findings (22). No
staining was observed when C1q was omitted (Fig. 1, B and
E), when cells were stained with an isotype-matched control
(data not shown), or when non-UVB-treated cells were assayed (Fig. 1, C and F).
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are
six serial optical sections through an apoptotic HUVEC stained for the
presence of C1q (A–F). Fig. 2, G–I, shows the
differential interference contrast images of D–F, showing
cellular morphology. Fluorescence intensity is represented by a color
scale, with the highest intensity in white, and the lowest in black. As
observed previously with apoptotic keratinocytes (22), C1q
did not bind to most of the surface of the apoptotic cell. Smaller
amounts of C1q were also observed in discrete, linear segments of the
membrane, but the most intense staining was observed proximal to the
membrane of the blebs that formed on the cell. Additional images of one
mature bleb (Fig. 2F), generated along three axes (Fig. 3), show that C1q is bound predominantly
to the membrane of the bleb. These observations were confirmed by
electron microscopy. Apoptotic cells were incubated with C1q, and bound
C1q was detected by immunogold labeling. Electron microscopy revealed
that most of the bound C1q was limited to surface blebs. Two patterns
of C1q deposition were observed. First, small amounts of C1q were
sometimes visible along cellular membranes (data not shown). Second,
large amounts of C1q were bound to the contents of the blebs (Fig. 4, inset), and proximal to the bleb
membrane.
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). Fig. 5, A–C, represents
untreated PBMC, and Fig. 5, D–F, represents UVB-treated
PBMC. Shown in Fig. 5, A and D, are the forward
and side scatter properties of these cells. Fluorescence intensity of
gated cells stained for bound C1q is shown for cells incubated without
(Fig. 5, B and E) or with (Fig. 5, C
and F) C1q. UVB irradiation generated a unique population of
cells with reduced forward scatter (Fig. 5D), indicating
that these cells were smaller in size than the viable cell population,
consistent with apoptosis (26). The apoptotic state of
these cells was also confirmed by their capacity to bind annexin V
(data not shown). As demonstrated in Fig. 5F, C1q bound
specifically to the PBMC undergoing apoptosis. C1q did not bind to
viable, nonapoptotic PBMC (data not shown). The low level of
C1q-positive cells present in the untreated population (Fig. 5C) probably represent cells undergoing apoptosis
spontaneously.
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Additional studies were performed to determine whether C1q bound
to the apoptotic blebs via the globular head domain or the
collagen-like tail domain. Blocking studies were performed with a panel
of anti-C1q mAb, two of which were specific for the collagen-like
tails of C1q and two of which were specific for the globular heads, to
try to block this interaction. Fab of the mAb were prepared by papain
digestion (Fig. 6) to eliminate any
possible interaction of C1q with the Fc domain of the Igs and to reduce
blocking due to steric hindrance. C1q was first incubated with either
the anti-head or the anti-tail Fab, then was added to apoptotic
HeLa cells. Bound C1q was detected by indirect immunofluorescence.
Fluorescence intensity, representing the amount of C1q bound to the
cells, was quantified by slide-based laser scanning cytometry (Fig. 7). Shown on the left
(A, C, E, and G) are the
gated C1q-positive cells. Shown on the right (B,
D, F, and H) is the relative cell
count of the cells within those gates. The anti-tail Fab had no
effect on the binding of C1q, and this sample (Fig. 7, E and
F) was indistinguishable from the positive control (Fig. 7, A and B). In contrast, incubation with the
head-specific Fab (Fig. 7, G and H) virtually
eliminated C1q binding, reducing the cells in the C1q-positive gate to
background levels in which no C1q was present in the assay (Fig. 7, C and D).
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Globular heads were also used in an attempt to block the interaction of
C1q with apoptotic blebs. Whole C1q was digested with collagenase to
eliminate the collagen-like tail portion of the molecule
(25). The resulting globular head fragments were analyzed
by SDS-PAGE and were determined to be the appropriate size (Fig. 8A) (27). Whole
C1q was incubated with apoptotic HeLa cells in either the absence (Fig. 8B) or the presence (Fig. 8C) of the purified
head fragments. Bound whole C1q was detected by indirect
immunofluorescence with 12A5B7, which does not bind to the purified
heads. The purified heads significantly blocked binding of whole C1q to
the apoptotic cells (Fig. 8, B and C). These
observations further support the conclusion that the globular head
domains of C1q specifically recognize surface blebs of apoptotic
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 1994, Casciola-Rosen et al. (6) reported that autoantigens targeted by patients with SLE are packaged into discrete cytoplasmic and nuclear surface blebs during UVB-induced apoptosis. We subsequently demonstrated that when apoptosis results from viral infection, the blebs contain high concentrations of foreign and self Ags that may break immune tolerance if not properly disposed of (15). These observations led us to propose that C1q may be a critical recognition molecule in this process. The capacity of C1q to bind specifically to surface blebs generated by UVB-induced apoptosis was first demonstrated with primary human keratinocytes (22). Keratinocytes were the focus of our initial studies because severe cutaneous rashes and photosensitivity are common, if not ubiquitous, manifestations of SLE caused by complement deficiency. This suggests that the skin may be the site at which tolerance is initially broken, and proper disposal of apoptotic keratinocytes may be critical to prevent autoimmunization. However, the anatomic site of primary autoimmunization in SLE remains unknown.
In this study we have demonstrated that recognition of apoptotic cells by C1q is not restricted to keratinocytes, but also occurs with apoptotic vascular endothelial cells and PBMC. The vasculature was chosen as the focus of this investigation because, unlike keratinocytes, endothelial cells and PBMC are constantly exposed to C1q, and these cell types undergo apoptosis during normal physiologic processes. Lymphocytes regularly undergo apoptosis during selection in lymphoid organs and bone marrow as well as via Fas/Fas ligand signaling in the periphery (28, 29). Vascular endothelial cells undergo apoptosis during normal tissue development and repair or as a response to inflammatory stimuli (30, 31, 32). The capacity of C1q to bind to vascular endothelial cells and PBMC undergoing apoptosis suggests that any physiologic or pathologic process that generates apoptotic cells within the vasculature may be a threat to immune tolerance in the absence of C1q.
This study has also advanced our prior studies of keratinocytes by more precisely localizing deposition of C1q on the apoptotic cell. First, investigation of C1q deposition on apoptotic endothelial cells by confocal microscopy indicates that deposition of C1q is restricted to the surface blebs, as was shown with keratinocytes. However, we have further demonstrated here that C1q deposition appears to begin in linear segments of the apoptotic membrane and to increase progressively as the bleb matures on the surface. Images of a single mature bleb reveal that the C1q appears to coat the entire surface of the bleb. The broad band of C1q deposition demonstrated by confocal microscopy led us to examine more closely, by electron microscopy, the location of C1q deposition on the bleb surface. These studies demonstrated that C1q appears to bind both to the membrane of the bleb as well as to the contents of the bleb. This observation suggests that C1q is probably binding to more than one molecular species. This is consistent with several other observations. First, previous attempts to eliminate C1q deposition on apoptotic cells by treatment with RNase, DNase, proteases, and competition with annexin V typically resulted in only partial reduction in binding with each reagent respectively (our unpublished observations). Second, previous in vitro studies have shown that C1q binds to DNA, RNA (27), and a variety of putative receptors, some of which have been localized to intracellular compartments.
One such receptor that binds to the globular heads of C1q is gC1q receptor (gC1q-R) (33). Although gC1q-R was originally characterized as a cell surface molecule (33), it has since been described as an intracellular protein (34, 35, 36) with the ability to bind to diverse ligands (37). It is possible that C1q binds to gC1q-R that may become concentrated and localized in surface blebs of apoptotic cells. Although there is no direct experimental evidence that this occurs with gC1q-R, other intracellular Ags relocalize to the surface of apoptotic cells (6). Further, it has been demonstrated that autoantigens become accessible to autoantibodies as a result of apoptosis (7), and it is possible that they become accessible to C1q as well.
Another protein that has been demonstrated to bind to either the collagen-like tails (38) or the globular heads of C1q (39, 40) is calreticulin. Calreticulin is found in all eukaryotic cell types except erythrocytes (40) and has been identified as an autoantigen in SLE (41, 42). Although calreticulin is predominantly an intracellular protein (43), there is evidence that its expression on the plasma membrane may be up-regulated as a result of exposure to UVB (44, 45, 46). Calreticulin may be one of several molecules targeted by C1q during apoptosis.
This study has also demonstrated that C1q recognizes apoptotic cells with the same domain used to recognize the Fc domains of Ag-Ab complexes, the globular heads of the molecule. C1q is a 450-kDa glycoprotein composed of six copies each of A, B, and C polypeptide chains (47), arranged such that the C-termini form globular heads, and the amino termini form a collagen-like tail region (48). Identification of the globular heads as the recognition domains of C1q for apoptotic cells has at least two important implications. First, this suggests that C1q may be deposited in a conformation that facilitates activation of the enzymatic portion of the classical pathway, with participation of C1r, C1s, C4, and C2. Such a scenario would be consistent with the understanding that deficiency of these components also leads to development of SLE. Second, this orientation of C1q on the bleb suggests the identities of subsequent molecular components of this recognition and clearance pathway. Specifically, it suggests that molecules capable of binding to the tail domain of C1q may be involved.
Several receptors specific for the tail region of C1q have been identified (38, 49, 50, 51, 52, 53, 54). One of these receptors, termed C1qRP, which is present on myeloid cells, endothelial cells, and platelets, has been demonstrated to enhance phagocytosis (55). One possible mechanism of clearance of C1q-bearing apoptotic cells, blebs, and their contents, especially within the vasculature, may be C1qRP-mediated phagocytosis. Another possible clearance mechanism within the vasculature may be through interactions with erythrocytes. Klickstein, et al. (51) have shown that the collagen-like tails of C1q bind to CR1 (CD35). When apoptosis occurs within the vasculature, an important mode of clearance may be C1q-mediated binding of apoptotic cells and blebs to erythrocytes via CR1 interactions. Previous reports have described decreased expression of CR1 on erythrocytes of patients with SLE due to inherited (56, 57) or acquired (58, 59, 60, 61) factors. This observation may reflect impaired clearance of the molecular remnants of apoptosis via erythrocytes in the pathogenesis of this disease.
The present studies also suggest a possible explanation for the generation of autoantibodies against C1q in SLE and SLE-like conditions. Anti-C1q autoantibodies have been identified in 30–34% of humans with lupus (62) and approximately 50% of MRL lpr/lpr mice with lupus (63). Furthermore, the lupus-like disorder HUVS is defined by the presence of autoantibodies targeted to C1q, one of the criteria for diagnosis (24, 62, 64). HUVS is characterized by low serum levels of C1q, C4, and C3, and a prominent clinical manifestation is vascular inflammation. The autoimmune response to C1q in SLE and HUVS is targeted to the collagen-like tail domains (2, 3, 24, 62, 65, 66). Our observations here have demonstrated that C1q deposits on the apoptotic cell via the globular heads, leaving the tail domains potentially exposed. The implications here are, first, that endothelial apoptosis may generate surface blebs coated with C1q, and C1q may thereby become incorporated into the multimolecular particle that breaks immune tolerance. Perhaps deposition of C1q on the apoptotic cell and subcellular structures leads to a conformational change in the tail domain, exposing neoepitopes. This scenario is consistent with the additional observation that patients with HUVS also generate autoantibodies to an unidentified endothelial molecule (67). The second implication of our finding regarding the molecular pathogenesis of autoantibodies to C1q is that once an anti-C1q response is generated, endothelial cells undergoing apoptosis may be recognized by C1q and thereby targeted by the established autoimmune response, potentially resulting in vascular inflammation and destruction. Finally, a third implication of our finding is that autoantibodies to C1q in SLE and HUVS may further impair C1q-mediated clearance of autoantigens generated during apoptosis.
The role of C1q in the clearance of apoptotic cells and substructures is supported by recent animal models of complement deficiency. Botto et al. (68) demonstrated that mice deficient in C1q spontaneously develop antinuclear Abs and glomerulonephritis, coupled with the accumulation of apoptotic bodies in the glomeruli. In addition, it has been shown that C1q-deficient mice exhibit delayed clearance of apoptotic cells by peritoneal macrophages (69). Further studies are required to identify distal components of this C1q-mediated clearance pathway, including the cell types involved. Such efforts should lead to identification of a pathway involved in the pathogenesis of SLE, HUVS, and possibly other autoimmune states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joseph M. Ahearn, University of Pittsburgh School of Medicine, S705 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh, PA 15261.
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; HUVS, hypocomplementemic urticarial vasculitis syndrome; gC1q-R, gC1q receptor.
Received for publication October 19, 2000. Accepted for publication December 12, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359.
B-inducible anti-apoptotic Bcl gene, inhibits endothelial cell activation. Blood 93:3803.
motifs present in the autoantigen calreticulin and interferes with complement activation. Biochemistry 37:17865.[Medline]
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A. J. Nauta, G. Castellano, W. Xu, A. M. Woltman, M. C. Borrias, M. R. Daha, C. van Kooten, and A. Roos Opsonization with C1q and Mannose-Binding Lectin Targets Apoptotic Cells to Dendritic Cells J. Immunol., September 1, 2004; 173(5): 3044 - 3050. [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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 G. Castellano, A. M. Woltman, A. J. Nauta, A. Roos, L. A. Trouw, M. A. Seelen, F. P. Schena, M. R. Daha, and C. van Kooten Maturation of dendritic cells abrogates C1q production in vivo and in vitro Blood, May 15, 2004; 103(10): 3813 - 3820. [Abstract] [Full Text] [PDF]
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 R P Rother, C F Mojcik, and E W McCroskery Inhibition of terminal complement: a novel therapeutic approach for the treatment of systemic lupus erythematosus Lupus, May 1, 2004; 13(5): 328 - 334. [Abstract] [PDF]
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C. Gaboriaud, J. Juanhuix, A. Gruez, M. Lacroix, C. Darnault, D. Pignol, D. Verger, J. C. Fontecilla-Camps, and G. J. Arlaud The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties J. Biol. Chem., November 21, 2003; 278(47): 46974 - 46982. [Abstract] [Full Text] [PDF]
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U. Kishore, S. K. Gupta, M. V. Perdikoulis, M. S. Kojouharova, B. C. Urban, and K. B. M. Reid Modular Organization of the Carboxyl-Terminal, Globular Head Region of Human C1q A, B, and C Chains J. Immunol., July 15, 2003; 171(2): 812 - 820. [Abstract] [Full Text] [PDF]
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S. D. Fleming, T. Shea-Donohue, J. M. Guthridge, L. Kulik, T. J. Waldschmidt, M. G. Gipson, G. C. Tsokos, and V. M. Holers Mice Deficient in Complement Receptors 1 and 2 Lack a Tissue Injury-Inducing Subset of the Natural Antibody Repertoire J. Immunol., August 15, 2002; 169(4): 2126 - 2133. [Abstract] [Full Text] [PDF]
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B. A. Cocca, A. M. Cline, and M. Z. Radic Blebs and Apoptotic Bodies Are B Cell Autoantigens J. Immunol., July 1, 2002; 169(1): 159 - 166. [Abstract] [Full Text] [PDF]
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A. Roos, A. J. Nauta, D. Broers, M. C. Faber-Krol, L. A. Trouw, J. W. Drijfhout, and M. R. Daha Specific Inhibition of the Classical Complement Pathway by C1q-Binding Peptides J. Immunol., December 15, 2001; 167(12): 7052 - 7059. [Abstract] [Full Text] [PDF]
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 B. A. Cocca, S. N. Seal, P. D'Agnillo, Y. M. Mueller, P. D. Katsikis, J. Rauch, M. Weigert, and M. Z. Radic Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells PNAS, November 20, 2001; 98(24): 13826 - 13831. [Abstract] [Full Text] [PDF]
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 C. A. Ogden, A. deCathelineau, P. R. Hoffmann, D. Bratton, B. Ghebrehiwet, V. A. Fadok, and P. M. Henson C1q and Mannose Binding Lectin Engagement of Cell Surface Calreticulin and CD91 Initiates Macropinocytosis and Uptake of Apoptotic Cells J. Exp. Med., September 17, 2001; 194(6): 781 - 796. [Abstract] [Full Text] [PDF]
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