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The Journal of Immunology, 2007, 178: 95-102.
Copyright © 2007 by The American Association of Immunologists, Inc.

Apoptotic Splenocytes Drive the Autoimmune Response to Poly(ADP-ribose) Polymerase 1 in a Murine Model of Lupus1

Thomas Grader-Beck*, Livia Casciola-Rosen*, Thomas J. Lang§, Roman Puliaev, Antony Rosen*,{dagger},{ddagger} and Charles S. Via2,§,¶

* Department of Medicine, {dagger} Department of Cell Biology, and {ddagger} Department of Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21224; § Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201; and Department of Pathology, Uniformed Services University, Bethesda, MD 20814


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although defects in apoptosis have been linked to both human and murine lupus, the exact mechanisms remain unknown. Moreover, it is not clear whether such defects are primary or secondary events in disease pathogenesis. To address these issues, we used an induced model of murine lupus, the parent-into-F1 model of chronic graft-versus-host disease (cGVHD) in which a lupus-like phenotype highly similar to human systemic lupus erythematosus is reliably induced in normal F1 mice. We addressed the role of nuclear Ags modified by caspases during apoptosis as potential targets of the autoantibody response and our results identify poly(ADP-ribose) polymerase 1 (PARP-1) as a frequently targeted autoantigen. Additional proteins cleaved during apoptosis were also targeted by the immune response. Importantly, female mice exhibited significantly greater numbers of apoptotic cells in germinal centers and higher serum anti-PARP-1 Ab levels compared with male cGVHD mice. Serum anti-PARP-1 levels in male cGVHD mice could be elevated to levels comparable to those of female cGVHD mice by the injection of apoptotic syngeneic F1 splenocytes early in the disease course. These results provide a mechanism by which lupus autoantibodies target apoptotic molecules. Specifically, T cell-driven polyclonal B cell activation characteristic of systemic lupus erythematosus is sufficient to saturate otherwise normal apoptotic clearance mechanisms, permitting apoptotic material to accumulate, serve as autoantigens, and drive autoantibody production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)3 is an autoimmune disease in which autoantibodies target ubiquitously expressed molecules that have a variety of biologic functions. Although these autoantigens share no unifying features in intact cells, they become clustered in surface blebs on cells undergoing apoptosis (1). Autoantigen cleavage by apoptotic proteases has been noted to be a frequent feature of autoantigens targeted in systemic autoimmunity (2). The relevance of this striking finding to disease pathogenesis remains unclear.

Apoptotic cells are normally efficiently cleared by macrophages and are strongly tolerance inducing (3). Interestingly, genetic defects in the clearance of apoptotic cells (e.g., deficiencies in C1q, MFG-E8, and c-mer) are associated with a lupus-like phenotype in mice (4, 5, 6). Moreover, injection of normal mice with apoptotic cells can trigger low-level, nonsustained systemic autoimmunity (7, 8). In human SLE, increased numbers of apoptotic cells have been found in germinal centers from lymph nodes of SLE patients and decreased uptake of apoptotic material by specialized macrophages has been described (9, 10). It has been proposed that abnormalities in the generation and clearance of apoptotic cells may play an important role in the initiation and propagation of SLE. A major question has been whether defects in apoptosis and/or clearance play primary predisposing roles in human lupus, or whether such defects are secondary to the intense immune system activation and disordered immune regulation characteristic of lupus (11).

The events involved in disease initiation are difficult to study in humans with SLE or in mice with spontaneous lupus-like disease. By contrast, induced models of lupus can result in a phenotype remarkably similar to human disease and permit the study of both disease initiation and progression (12). In this study, the parent-into-F1 (P->F1) model of chronic graft-versus-host disease (cGVHD) represents such an example in which a lupus-like disease highly similar to human SLE is reliably induced in normal F1 mice by the transfer of homozygous parental strain T cells (13). Mice develop a lupus-like phenotype consisting of polyclonal B cell hyperactivity, lupus-specific autoantibodies, and a lupus-like immune complex glomerulonephritis (14, 15, 16) which is more severe in female mice (17). Using the DBA->B6D2F1 model of lupus, we demonstrate that Ags cleaved by caspases, especially PARP-1, are prominent targets of autoimmunity. Moreover, sex-based differences in apoptosis and clearance were observed in that female F1 recipients exhibited greater apoptosis in the spleen, which was in turn associated with higher levels of anti-PARP Abs compared with males. Importantly, experimental augmentation of apoptotic load in males increased splenic apoptosis and levels of anti-PARP-1 Abs. These data indicate that, in females, the intense lymphocyte activation and cell turnover associated with active lupus may overwhelm normal apoptotic clearance mechanisms, allowing apoptotic autoantigens to become targets of the autoimmune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Induction of cGVHD

Female and male DBA/2 (DBA; H-2d) and B6D2F1 (BDF1; H-2b/d) mice were purchased from The Jackson Laboratory. Single-cell suspensions of DBA/2 splenocytes were prepared from 6- to 8-wk-old mice and 90 x 106 unfractionated splenocytes were injected into the tail vein of 6- to 8-wk-old age- and sex-matched recipient BDF1 mice. Controls consisted of uninjected BDF1 mice or F1 mice injected with 90 x 106 age- and sex-matched BDF1 unfractionated splenocytes. Mice were maintained in the animal facility at the University of Maryland (Baltimore, MD) under specific pathogen-free conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine.

Preparation of apoptotic BDF1 splenocytes

Single-cell suspensions of BDF1 splenocytes were irradiated with 2000 J/m2 UVB light. Recipient F1 mice received 50 x 106 unfractionated age- and sex-matched BDF1 apoptotic splenocytes i.v. on days 1, 4, and 7 after GVHD induction. Induction of apoptosis by UVB was confirmed by flow cytometric assessment of annexin-V staining performed on aliquots of UVB-treated and untreated splenocytes maintained in culture for 8 h before testing.

Immunoprecipitation of [35S]methionine-labeled PARP-1

[35S]Methionine-labeled mouse PARP-1 was generated by coupled in vitro transcription/translation (IVTT) using the appropriate full-length complementary DNA cloned in our laboratory. IVVT PARP-1 was immunoprecipitated using sera from control or female cGVHD mice and visualized by autoradiography as described elsewhere (1).

Western blots

HeLa cells were lysed in buffer A (1% Nonidet P-40, 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, and protease inhibitors), electrophoresed on SDS-PAGE, and the separated proteins were transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with TBST (10 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk (TBS-TM) for 1 h at room temperature. Membranes were incubated overnight at 4°C with cGVHD and control mouse sera were diluted 1/2000 or a mouse anti-PARP-1 mAb (Serotec) at a dilution of 1/10,000 in TBS-TM. After extensive washing, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) diluted 1/10,000 for 45 min at room temperature. Proteins were visualized using ECL (Pierce) according to the manufacturer’s protocol. For some experiments, HeLa cells were subjected to UVB irradiation before cell lysis to induce apoptosis, or HeLa cell lysates were incubated with caspase-3 to induce caspase-specific cleavage fragments as described previously (18).

Preclearance of PARP-1 from HeLa lysates

HeLa lysate (20 µg) was incubated with 5 µl of human serum from a healthy control or an anti-PARP-1-specific SLE serum for 1 h at 4°C. Subsequently, protein A-Sepharose (Immunopure; Pierce) was added to the mixture and incubated for an additional 1 h at 4°C. The complex of protein A-Sepharose and PARP-1 bound to anti-PARP-1 was then precipitated by centrifugation. The supernatant was isolated and subjected to Western blotting as described above using an anti-PARP-1-specific cGVHD mouse serum at a 1/2000 dilution.

ELISA

ELISA plates (Costar; Corning) were coated with human rPARP-1 (Trevigen) at 100 ng/well in carbonate/bicarbonate binding buffer (pH 9.5) for 1 h at 37°C. After blocking with PBS containing 0.1% Tween 20 and 5% milk powder (PBS-TM) for 1 h at 37°C, plates were incubated with sera at a dilution of 1/250 for 1 h at 37°C, followed by incubation with HRP-conjugated goat anti-mouse IgG Ab for 30 min at 37°C. Peroxidase substrate (SureBlue; KPL) was then added to the wells for 5 min before stopping the reactions. Color development was measured by absorbance at 450 nm. Determination of serum anti-dsDNA Abs was performed according to the manufacturer’s instruction (QUANTA Lite; INOVA) with the following modifications: Mouse sera were used at a 1/100 dilution and HRP-conjugated goat anti-mouse IgG was used as a secondary Ab at a 1/10,000 dilution.

Standards for both ELISAs were established by using a monoclonal mouse anti-PARP-1 Ab (Serotec) or a high-titer anti-dsDNA Ab mouse serum obtained from a BALB/cFaslpr, Faslgld mouse (a gift from J. Erikson, Wistar Institute, Philadelphia, PA). Serial dilutions of the standards were used to calculate conversion of ODs into arbitrary units (AU). Linearity of the standard curve was confirmed for ODs between 0.1 and 1.4 for anti-PARP-1 and between 0.1 and 0.7 for anti-dsDNA. Results were compared by both t test and Mann-Whitney U test.

TUNEL assay

Spleens from mice with cGVHD or controls were harvested 4 or 8 wk after disease induction, fixed in 10% buffered formaldehyde, paraffin embedded, and sectioned. Apoptotic splenocytes were visualized using the TUNEL assay (TACS-XL Blue; Trevigen) according to the manufacturer’s instructions. The number of apoptotic splenocytes was quantitated by counting TUNEL-positive cells in 10 high-powered fields per spleen at x40 magnification. Results were compared by t test.

Statistical analysis

Statistical comparison between groups of mice was performed using the t test or Mann-Whitney U test as indicated using GraphPad PRISM software (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
PARP-1 is a major target of the autoantibody response in murine SLE

To determine a role for apoptosis-derived autoantigens in lupus pathogenesis, cGVHD was induced in female BDF1 mice and sera were screened for the presence of autoantibodies by Western blotting using HeLa cell lysates. Shown in Fig. 11A is a representative immunoblot using sera from female control and female cGVHD mice obtained 4 wk after disease induction. Of note, sera from cGVHD mice immunoblotted several different Ags at the 4-wk time point and over the course of the disease. The most frequently targeted molecule migrated at 113 kDa. By 4 wk, 100% of the female cGVHD mice had Abs to this protein by Western blot. The 113-kDa protein was also detected after immunoprecipitation of [35S]methionine-labeled HeLa cell lysates (data not shown). Other bands, while prominent at distinct time points throughout the disease, did not show any reproducible patterns on Western blots of HeLa lysates. In contrast, the sera from control mice did not immunoblot any bands (Fig. 1A).


Figure 1
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  		FIGURE 1. PARP-1 is a prominent target of the immune response in the cGVHD mouse model of SLE. A, HeLa lysates were immunoblotted using sera from three different female control (lanes 1–3) or female cGVHD mice (lanes 4–6) obtained 4 wk after disease induction. The solid arrow denotes a 113-kDa protein that is recognized by all cGVHD sera shown. Ref1 denotes immunoblotting using an anti-PARP-1-specific human serum. B, Control HeLa cell lysates and caspase-3-treated HeLa cell lysates (Cas3) were immunoblotted using a cGVHD serum (left two lanes) and a mAb against PARP-1 (right two lanes). In caspase-3-treated lysates, the 89-kDa signature PARP-1 fragment (unfilled arrow) that is generated by caspase cleavage is detected by both the cGVHD serum and the mAb against PARP-1 (the solid arrow denotes intact PARP-1). C, Preclearing of PARP-1 removes anti-PARP-1 activity of cGVHD sera. HeLa lysate was precleared of PARP-1 using control serum or anti-PARP-1-specific serum followed by immunoprecipitation with protein A-Sepharose. The supernatant was then immunoblotted using cGVHD serum identified as having anti-PARP-1 reactivity. The 113-kDa band disappears after preclearing with anti-PARP-1-specific serum but not with control serum. D, [35S]Methionine-labeled PARP-1, generated by IVTT, was immunoprecipitated using a reference human anti-PARP-1 serum (Ref2) and sera from five different female cGVHD mice (lanes 12–16) but not controls (lanes 7–11). E, Lysates from normal and UVB-irradiated apoptotic HeLa cells were immunoblotted using two different female cGVHD sera. Solid arrows, Intact proteins and unfilled arrows, fragments appearing after apoptotic cleavage. Note the appearance of fragments of the unidentified 125 and 240-kDa Ags in the apoptotic lysates (lane 18 and 20, respectively).

 
Numerous autoantigens targeted in human systemic autoimmunity are structurally modified during apoptosis (19). We tested whether this is also the case for autoantigens targeted in cGVHD mice by immunoblotting caspase-3-cleaved HeLa cell lysates. The 113-kDa protein was cleaved by caspase-3, and an 89-kDa fragment was generated (Fig. 1B). Both the intact band and the cleavage fragment comigrated with the bands blotted using a monoclonal anti-PARP-1 Ab (Fig. 1B), indicating that the 113-kDa protein likely is PARP-1. Similar results were obtained using cell lysates from HeLa cells exposed to UVB irradiation to induce apoptosis (data not shown). PARP-1 was precleared from HeLa lysates using human SLE serum containing high-titer anti-PARP-1 Abs. Fig. 1C demonstrates that depletion of PARP-1 led to disappearance of the 113-kDa band recognized by serum from cGVHD mice. To confirm that the 113-kDa molecule was indeed PARP-1, we generated [35S]methionine-labeled mouse PARP-1 by IVTT and used this for immunoprecipitation with cGVHD sera. The cGVHD sera immunoprecipitated the 113-kDa mouse PARP-1, whereas control sera did not (Fig. 1D). In further studies, we determined whether sera of female cGVHD mice preferentially recognize cleaved PARP-1 early in the disease. Sera at 2 wk after disease induction were tested for reactivity against UVB-irradiated HeLa cell lysates. No dominant recognition of the 89-kDa fragment vs full-length PARP-1 was found in cGVHD sera at this time point (data not shown).

We used the immunoblotting approach described above to address whether sera from cGVHD mice recognized proteins other than PARP-1 that are cleaved during apoptosis. The data shown in Fig. 1E demonstrate that this was indeed the case; strikingly, most of the immunoblotted proteins detected by these sera were cleaved during apoptosis. Sera from individual cGVHD mice recognized other proteins that are cleaved during apoptosis, including a 240- and a 125-kDa protein. Interestingly, Abs to these proteins were not as frequent as those against PARP-1. In contrast to PARP-1 recognition, reactivity to other cleavable proteins was only infrequently observed at isolated time points in individual mice.

Together, these results establish that PARP-1 is a major target of the autoantibody response in the cGVHD model of SLE and that Ags cleaved during apoptosis are common targets in this model.

Anti-PARP-1 Ab titers are higher in female mice and increase with duration of disease

Human SLE is characterized by a strong female predilection (20). Similarly, female cGVHD mice exhibit higher titers of anti-ssDNA Abs and more severe lupus-like renal disease than their male counterparts (17, 21). To evaluate whether anti-PARP-1 Abs also exhibit a female predominance, we quantified the levels of these Abs in female and male cGVHD mice sera by ELISA. Serum anti-PARP-1 Abs were detected at low but statistically significant levels in female cGVHD mice 2 wk after disease induction (Fig. 2A), the first time point tested (mean OD ±SEM for female cGVHD = 0.179 ± 0.025 SEM vs female control = 0.064 ± 0.008; p ≤ 0.005;). By week 8, the anti-PARP-1 Ab titers had increased significantly not only compared with controls (O.D. 0.562 ± 0.094 vs 0.069 ± 0.03 for controls; p < 0.0001), but also compared with the 2-wk anti-PARP-1 levels (p = 0.001; Fig. 2A). Values for female cGVHD mice at weeks 4 and 8 did not differ significantly and week 4 values were also significantly greater than week 2 values (p = 0.026). In contrast, although the anti-PARP-1 response of male cGVHD mice was significantly greater than that of male controls by week 8 (mean OD ± SEM male cGVHD = 0.172 ± 0.03 vs controls = 0.063 ± 0.01; p < 0.05), it was significantly less than that of female cGVHD mice at either the 4- or 8-wk time points (Fig. 2A). The greater anti-PARP-1 Ab response in female vs and male cGVHD mice was also apparent by Western blot (Fig. 2B).


Figure 2
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  		FIGURE 2. Anti-PARP-1 Abs develop early in cGVHD disease and are found at higher titer in female compared with male cGVHD mice. cGVHD was induced as described in Materials and Methods, and sera were collected from female and male cGVHD mice at the indicated time points and screened by ELISA at a 1/250 dilution. A, The difference in mean values between females and males was statistically significant at week 4 (*, p < 0.05) and week 8 (***, p ≤ 0.0008). Control mice did not develop anti-PARP-1 Abs (see Fig. 4A). Results shown are combined from three independent experiments containing 4–10 animals/group. B, HeLa lysates were immunoblotted using a single representative serum from each of the following groups: female control, female cGVHD, and male cGVHD mice. The bleeds were obtained before disease initiation and at weeks 2, 4, and 8 after disease initiation. All sera were tested at a 1/2000 dilution. The identity of the 113-kDa band in the cGVHD sera depicted was confirmed in a separate experiment by immunoprecipitation of IVTT [35S]methionine-labeled mouse PARP-1 (data not shown). C, Time course of development of anti-PARP-1-specific Abs detected by ELISA in individual female and male cGVHD mice. PARP-1 Ab levels in mouse sera were quantitated by ELISA as described in Materials and Methods. Each line illustrates ODs obtained from sera of individual animals at weeks 2, 4, and 8 after disease initiation (n = 5 mice/group). D, Female mice exhibit skewing of both anti-dsDNA Ab and anti-PARP-1 Ab. A representative sample of sera from female and male cGVHD mice at weeks 4 and 8 after disease induction was compared for development of anti-dsDNA and anti-PARP-1 Abs by ELISA as described in Materials and Methods. Sera were selected randomly (n = 10 for each group). For anti-dsDNA ab at week 4, ***, p ≤ 0.0003. For week 8, *, p ≤ 0.05). For anti-PARP-1 Ab at week 4, ***, p ≤ 0.0008 and at week 8, **, p ≤ 0.0016.

 
When anti-PARP-1 Ab titers were evaluated sequentially in individual cGVHD mice, we found that titers increased with the duration of disease. Representative examples of time courses determined by Western blotting and ELISA are shown in Fig. 2, B and C. Control mice did not develop anti-PARP-1 Ab titers as measured by Western blot (Fig. 2B) or by ELISA (see also Fig. 4A).


Figure 4
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  		FIGURE 4. Injection of apoptotic cells during disease induction leads to augmentation of the male cGVHD anti-PARP-1 Ab response to levels of those of female cGVHD mice. cGVHD was induced and anti-PARP-1 Ab was determined at the indicated time points by ELISA as described in Materials and Methods. A, Control mice do not develop anti-PARP-1 Abs measured by ELISA at the indicated time points. Results from female and male normal F1 control mice were pooled for illustration purposes. No statistical difference was observed between both groups. B–D, Injection of apoptotic splenocytes promotes anti-PARP-1 Ab in male cGVHD mice but not in female cGVHD mice. Injection of apoptotic age- and sex-matched BDF1 cells was performed as described in Materials and Methods, and anti-PARP Ab was determined by ELISA at 2 wk (B), 4 wk (C), or 8 wk (D) for untreated cGVHD mice (–) or cGVHD mice coinjected with apoptotic splenocytes. Coinjection of apoptotic BDF1 splenocytes into male cGVHD mice resulted in an augmented anti-PARP-1 response compared with untreated male cGVHD mice (male cGVHD + vs –, p < 0.05 (*) at weeks 4 and 8), reaching Ab levels detected in female cGVHD mice (female cGVHD–, p > 0.05, not significant). Sera were combined from three independent experiments with 4–10 mice/group in each experiment. All OD values >0.1 were on the linear portion of the curve; *, p < 0.05 by Student’s t test.

 
To determine whether other SLE-relevant autoantibodies exhibit female skewing in addition to anti-PARP-1, sera from three independent experiments were screened for development of anti-dsDNA Abs in conjunction with retesting for anti-PARP-1 ab (Fig. 2D). Female and male control sera did not contain anti-dsDNA Abs (data not shown). Female cGVHD mice demonstrated significantly higher anti-dsDNA titers than did males at both week 4 (mean AU, 0.440 ± 0.04 SEM vs 0.125 ± 0.05; p ≤ 0.0003) and week 8 (mean AU, 0.210 ± 0.07 vs 0.048 ± 0.02; p ≤ 0.046). Similarly, female mice exhibited significantly greater anti-PARP-1 ab than did male mice at both time points. Interestingly, anti-dsDNA Ab titers declined from week 4 to week 8 in both sexes, whereas anti-PARP-1 Ab titers increased from week 4 to week 8 in these same female and male cGVHD animals (Fig. 2D).

Increased presence of apoptotic cells in the spleens of cGVHD mice during disease induction

Given the evidence that PARP-1 and other as yet unidentified Ags modified by caspases during apoptosis are prominent targets of the immune response in cGVHD mice, we quantified the number of apoptotic cells in the spleen during disease induction. The cGVHD model is characterized by the development of a significant expansion of host B lymphocytes and splenomegaly in the weeks following disease induction (22). We therefore hypothesized that apoptotic cells may accumulate in the germinal centers of the spleen during the disease process and drive the immune response. To address this question, spleens of control and cGVHD mice were harvested at various times during disease and the number of apoptotic cells was assessed by TUNEL staining. A marked expansion of germinal centers was noted in the spleens of cGVHD mice but was not seen in control mice (Fig. 3, A and B). We found clearly increased numbers of apoptotic cells in the spleens of cGVHD mice compared with control mice (Fig. 3, C–F). Moreover, female cGVHD spleens exhibited significantly higher numbers of apoptotic cells per high-power field than male cGVHD spleens (female mean ± SEM = 9.84 ± 0.60 vs male = 2.75 ± 0.32; p < 0.0001). TUNEL-positive apoptotic cells were rarely detected in spleens of control mice and were dispersed within the splenic tissues. In cGVHD mice, TUNEL-positive cells were typically found in germinal centers. Interestingly, spleens of female control mice showed a slightly higher number of apoptotic cells per high-power field compared with male control mice (female = 0.89 ± 0.14 vs male = 0.34 ± 0.11; p ≤ 0.0045).


Figure 3
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  		FIGURE 3. The number of apoptotic cells is markedly increased in germinal centers of cGVHD mice compared to controls. A and B, H & E-stained spleen paraffin sections obtained from a female control (A) and a female cGVHD mouse (B). The spleens were harvested at 4 or 8 wk after disease induction and are representative of three to five separate sections obtained from two independent experiments. Marked expansion of germinal centers was noted (original magnification, x25). C and D, Spleen sections serial to those shown in A and B were stained with the TUNEL assay. Apoptotic cells (stained blue) were markedly increased in germinal centers of female mice (D). E,: Close up of boxed area shown in D (original magnification, x100). F, Number of TUNEL-positive cells is significantly increased in female compared with male cGVHD mice or compared with control mice. The number of TUNEL positive cells are shown as individual counts from 10 high-power fields (n = 2–7 animals/group, p < 0.0001).

 
Injection of apoptotic splenocytes early after cGVHD induction augments the anit-PARP-1 response in males

The targeting of autoantigens clustered and proteolytically cleaved in apoptotic cells in the cGVHD model is similar to human SLE. The cGVHD model, however, provides an opportunity to directly study apoptosis in animals during disease development as well as an opportunity to study the effect of enhancing the apoptotic load during disease induction. Given our finding that apoptotic cells are increased in germinal centers of female cGVHD vs male cGVHD animals, we addressed the effects of providing additional apoptotic material during disease induction on anti-PARP-1 Abs in female and male cGVHD mice. To avoid potential alloreactivity, age-and gender-matched syngeneic apoptotic BDF1 splenocytes were injected on days 1, 4, and 7 after parental cell transfer (day 0). Data obtained from these mice were compared with cGVHD mice induced by the standard protocol. Apoptosis in splenocytes was induced by UVB irradiation and splenocytes were injected as described in Materials and Methods. Anti-PARP-1 autoantibody production was assessed qualitatively by Western blotting of HeLa cell lysates and quantified by ELISA. Control male or female F1 mice did not develop anti-PARP-1 Abs (Fig. 4A), whereas female cGVHD mice developed a robust anti-PARP-1 Ab response at both 4 and 8 wk of disease that was not significantly altered by the coinjection of apoptotic splenocytes (Fig. 4, B–D). The significantly weaker anti-PARP-1 response of male cGVHD mice compared with female cGVHD mice (Fig. 2, A and C) could be boosted by the coinjection of apoptotic splenocytes to levels that did not differ significantly from female cGVHD (with or without apoptotic splenocytes). This effect is best seen at week 4 (Fig. 4C) where the anti-PARP-1 response of male coinjected cGVHD mice is significantly greater than that of male cGVHD mice not receiving apoptotic splenocytes. By week 8, the anti-PARP-1 response in coinjected male cGVHD mice wanes and although still significantly greater than that of noncoinjected male cGVHD mice by t test comparing mean OD values, it was not significant by Mann-Whitney U comparison of median values. These results support the idea that at the doses used, coinjection of apoptotic cells may only temporarily boost the anti-PARP-1 response in male cGVHD mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we provide a mechanism by which lupus autoantibodies target apoptosis-related molecules. Specifically, in the setting of intense T cell-driven polyclonal B cell activation, saturation of apoptotic clearance mechanisms can occur in the absence of preexisting defects in apoptosis or clearance. As a result, apoptosis-related molecules can serve as autoantigens, resulting in an Ag-driven autoantibody response. These conclusions are based on our results using a mouse model in which the induction of lupus in otherwise normal F1 mice is associated with: 1) intense immune activation and increased apoptosis, 2) greater splenocyte apoptosis in females compared with males despite identical induction conditions, 3) significantly greater anti-PARP-1 Abs in female cGVHD mice compared with male cGVHD mice, and 4) augmentation of anti-PARP-1 Abs in male cGVHD mice by the i.v. injection of apoptotic splenocytes early in the course of disease. The sex-related differences in apoptosis and anti-PARP-1 Abs support the concept that increased detection of apoptotic splenocytes in females reflects saturation of normal endogenous clearance mechanisms and permits apoptotic molecules to serve as autoantigens in lupus. The augmentation of anti-PARP-1 Abs in male cGVHD mice by injection of apoptotic splenocytes is confirmatory evidence.

Autoantigens cleaved during apoptosis are frequent autoantibody targets in lupus (2, 23). Consistent with this, our studies show that PARP-1, which is cleaved during apoptosis to generate a signature 89-kDa fragment, was prominently targeted in female cGVHD mice. Indeed, Abs against PARP-1 were the only ones persistently identified in individual female mice throughout the course of disease. Moreover, anti-PARP-1 Abs appeared early in disease, becoming detectable as early as 2 wk after disease induction and increasing thereafter. Although there does not appear to be preferential recognition of the apoptotically cleaved form of PARP-1 in this model, administration of apoptotic cells to males during disease development results in augmentation of the anti-PARP-1 response, supporting the idea that saturation of apoptotic clearance mechanisms allows apoptotic material to accumulate, serve as autoantigens, and drive autoantibody production.

The importance of PARP-1 activity during apoptosis has been intensively investigated and shown to function as an early sensor of DNA damage. Poly(ADP-ribosyl)ation is mediated mainly by PARP-1 activity and represents an important pathway in the restoration of cellular integrity (24). Once this effort fails and apoptosis is initiated, PARP-1 is inactivated through cleavage by caspases (25), and the presence of cleaved PARP-1 is widely used as an early diagnostic marker of apoptosis in many cell types. Interestingly, PARP-1 also represents a prevalent autoantigen in human SLE. Abs to PARP-1 have been reported in up to 50–57% of SLE patients (26, 27) and in our own study of 205 consecutive SLE patients, we have determined that anti-PARP-1 Abs occur in 26.4% (T. Grader-Beck, K. Link, E. Akhter, A. Rosen, and M. Petri, manuscript in preparation). Moreover, in the present study, we detected additional autoantibody specificities targeting Ags cleaved during apoptosis, including unidentified molecules of 125 and 240 kDa. These findings are consistent with previous reports that identify caspase cleavage as an important feature of autoantigens in lupus (2, 23). It has been proposed that the immunogenicity of caspase-cleaved molecules results from the exposure of cryptic neoepitopes (28). Our results demonstrate autoantibodies to both cleaved and uncleaved PARP-1, indicating that cryptic neoepitope exposure is not operating at the level of autoantibody production. It is clearly possible that the effect of apoptotic cleavage operates at the level of T cells or Ag capture and presentation. Further definition of the mechanisms of augmentation of the response by apoptotic cells will be the target of future studies.

The exact source of PARP-1 Ag during the induction of the immune response in our model remains to be elucidated. Since PARP-1 is activated by DNA strand breaks, hairpins, and loops (29, 30), increased PARP-1 expression or activity by host B lymphocytes in germinal centers during shaping of the immune response represents an attractive potential source of Ag. However, although we detected PARP-1 in spleen lysates of cGVHD mice at 2, 4, and 8 wk after disease induction, we did not find increased expression in cGVHD vs control mice as assessed by Western blot (data not shown). Instead, expression of PARP-1 in the spleen decreased with duration of the disease, despite the fact that cGVHD mice developed massive expansion of germinal centers and underwent B cell proliferation. Possible explanations for this finding may be that increased PARP-1 expression is important very early during disease development (within the first 2 wk) or that PARP-1 activity rather than expression plays a central role in immunogenicity. Conversely, decreased PARP activity and decreased PARP expression is also observed in human lupus (31, 32) and lupus patients have been reported to exhibit delayed DNA repair (33) and increased DNA strand breaks (34) consistent with defective PARP function in vivo. Our results support the concept that at a minimum, anti- PARP-1 Ab may be markers for increased apoptosis and saturation of clearance mechanisms. If, however, anti-PARP-1 Abs also have a functional role in lupus pathogenesis, it may be that they interfere with PARP function, thereby contributing to defective the DNA repair reported in lupus patients.

The striking increase in germinal center apoptotic cells in our model could result from either increased production and/or defective clearance. Although DBA donor mice are deficient in C5, the recipient BDF1 strain is heterozygous for C5 and has no known defects in complement or apoptotic clearance, making this an unlikely explanation. More likely is the possibility that lupus-associated immune activation in this model is strong enough to overwhelm normal apoptotic clearance mechanisms. The higher prevalence of apoptotic cells in germinal centers of female cGVHD mice compared with males implicates a gender-specific factor and suggests that immune activation may be more intense in females. This idea is supported by previous work in DBA->F1 cGVHD mice demonstrating that initial donor CD4+ T cell proliferation lasts at least 3 days longer in female-into-female (f->F) transfers than in m->M transfers (21). As a result, engraftment of effector (donor) CD4+ T cells at 2 wk in f->F mice is 2- to 3-fold greater than that of m->M mice. The discrepancy in engrafted effector CD4+ T cells in f->F cGVHD mice is maintained long-term, resulting in greater CD4 help for B cells as evidenced by greater B cell activation, greater serum autoantibody production, and eventually more severe renal disease. Moreover, crossover experiments (m->F, f->M) demonstrated that greater donor CD4+ T cell proliferation and expansion segregated with the sex of the host and not the donor, indicating that at least in this model of lupus, T cell-driven B cell hyperactivity is shaped by gender-specific mechanisms extrinsic to the Ag-specific T cells. The exact nature of this gender-specific mechanism is currently under active investigation.

Based on the well-documented association between preexisting defects in apoptosis clearance mechanisms and development of lupus in both humans and mice, it has been postulated that abnormalities in apoptosis, particularly decreased clearance, may be an important predisposing condition for lupus (reviewed in Ref. 35). However, hereditary defects in complement (36) or apoptosis (37, 38) account for only a minority of humans with SLE. Thus, the exact role of altered apoptosis in lupus pathogenesis has been controversial. Our results demonstrate that immune system activation in lupus is strong enough to saturate normal apoptosis clearance mechanisms in normal female mice and that experimentally augmenting apoptotic loads can also saturate clearance mechanisms in normal males. In either sex, once apoptotic mechanisms are saturated, molecules cleaved during apoptosis can serve as a target for the ongoing humoral immune response. Thus, the targeting of apoptotic autoantigens in lupus may represent a failure of clearance due to excessive immune system activation characteristic of lupus rather than intrinsic preexisting clearance defects. Further studies addressing the gender-specific response to apoptotic material and defects in apoptotic clearance are underway.


   Acknowledgments
 
We thank Wei Duan-Porter for the mouse PARP-1 cDNA and Phuong Nguyen for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 These studies were supported by National Institutes of Health Grants AR 44684 (to L.C.R.), DE 12354 (to A.R.), AI 47466 (to C.S.V.), AR 48522-01 (to T.G.B.), a Maryland Chapter Arthritis Foundation Institutional Grant, and a Maryland Chapter Arthritis Foundation Maryland Arthritis Research Center Award. Back

2 Address correspondence and reprint requests to Dr. Charles S. Via, Department of Pathology, Uniformed Services University of Health Sciences, Room B3-100, 4301 Jones Bridge Road, Bethesda, MD 20814. E-mail address: cvia{at}usuhs.mil Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; cGVHD, chronic graft-versus-host disease; IVTT, in vitro transcription/translation; P->F1, parent-into-F1; PARP-1, poly(ADP-ribose) polymerase 1; AU, arbitrary unit. Back

Received for publication July 18, 2006. Accepted for publication October 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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