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The Journal of Immunology, 2007, 179: 1313-1321.
Copyright © 2007 by The American Association of Immunologists, Inc.
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{alpha}-Actinin Immunization Elicits Anti-Chromatin Autoimmunity in Nonautoimmune Mice1

Bisram Deocharan*, Zhijie Zhou{ddagger}, Kochnaf Antar{ddagger}, Linda Siconolfi-Baez{dagger}, Ruth Hogue Angeletti{dagger}, John Hardin*,{ddagger} and Chaim Putterman2,*,{ddagger}

* Department of Microbiology & Immunology, {dagger} Laboratory for Macromolecular Analysis & Proteomics, and {ddagger} Division of Rheumatology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Anti-dsDNA Abs are characteristic of lupus and can be found deposited in the kidneys of lupus mice. Previously, we have shown that pathogenic anti-dsDNA Abs as well as Ig eluted from the kidneys of nephritic lupus mice cross-react with {alpha}-actinin. Moreover, cross-reactivity with {alpha}-actinin characterizes nephritogenic anti-dsDNA Abs in humans with lupus as well. To determine whether Abs generated against {alpha}-actinin in vivo cross-react with nuclear Ags, we s.c. immunized 10-wk-old female BALB/c mice (and several other nonautoimmune mice strains) with {alpha}-actinin in adjuvant. Immunized but not control mice displayed high titers of anti-nuclear Abs and IgG anti-chromatin autoantibodies, hypergammaglobulinemia, renal Ig deposition, and proteinuria. The specificity of the anti-chromatin response was determined by Western blotting of purified chromatin with serum from {alpha}-actinin immunized mice. By proteomic analysis, a 25-kDa doublet band was conclusively identified as high mobility group box (HMGB) proteins 1 and 3, and a 70-kDa band was identified as heat shock protein 70 (hsp70), both of which are known antigenic targets in murine lupus. Binding to purified HMGB1 and hsp70 by immunized mice sera was confirmed by ELISA and Western blot. Immunized mice sera binding to both 25- and 70-kDa bands were significantly inhibited by {alpha}-actinin and chromatin. Importantly, a panel of nephritogenic mAbs had significantly higher affinity for {alpha}-actinin, chromatin, HMGB, and hsp70 as compared with nonpathogenic Abs, suggesting a common motif in these Ags that is targeted by pathogenic autoantibodies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Autoantibodies to nuclear Ags including anti-dsDNA Abs are well known serological features of systemic lupus erythematosus (SLE).3 Anti-dsDNA autoantibodies play a central role in the pathogenesis of one of the most serious manifestations of SLE, namely lupus nephritis (1). One mechanism widely invoked to explain the role of anti-dsDNA Abs in the genesis of kidney damage in lupus nephritis is direct cross-reactivity with renal Ags (2). However, the identity of these cross-reactive Ags remains elusive. Mostoslavsky et al. (3) initially identified {alpha}-actinin as a major cross-reactive target for pathogenic anti-dsDNA Abs in murine SLE. Subsequently, we confirmed that {alpha}-actinin is bound by nephritogenic murine anti-dsDNA Abs, and that up to 75% of the anti-dsDNA response in the lupus prone MRL-lpr/lpr mouse strain is cross-reactive with {alpha}-actinin. Notably, Ig eluted from kidneys of nephritic lupus mice bound to {alpha}-actinin, further supporting the notion that {alpha}-actinin is a target for pathogenic lupus autoantibodies (4). Recent human studies have shown that high titers of anti-{alpha}-actinin Abs were detected at an early stage of lupus nephritis and that anti-{alpha}-actinin Abs are significantly associated with glomerulonephritis in lupus patients (5, 6, 7). These latter findings suggest that anti-{alpha}-actinin Abs may be useful as a serological marker in human lupus nephritis.

In the kidney, {alpha}-actinin is present in mesangial cells, podocytes, endothelial cells, and blood vessels (8). Nonmuscle {alpha}-actinin (molecular mass ~100 kDa) can be found in the cytoplasm and nucleus and on the plasma membrane (9). The structure of {alpha}-actinin is a twisted anti-parallel dimer that contains a conserved acidic surface, forming an actin-binding domain at either end (10, 11). Although the primary function of {alpha}-actinin is actin bundling, over 20 other different binding partners have been discovered so far, including N-methyl-D-aspartate receptors and inducible NO synthase (12). Indeed, {alpha}-actinin has been shown to play a role in cell motility and adherence, regulation of channel activity, and in influencing cell growth and differentiation (9, 13, 14).

Lupus-associated anti-dsDNA autoantibodies are class switched and display somatic mutation and affinity maturation, suggesting a response that is Ag driven and T cell-dependent (15, 16). In the current study, we wanted to determine whether besides being a target for pathogenic anti-dsDNA autoantibodies, will an immune response against {alpha}-actinin lead to the generation of Abs that cross-react with nuclear Ags. We found that immunization of nonautoimmune mice with {alpha}-actinin induces a strong anti-nuclear Ab (ANA) response, particularly against chromatin. Furthermore, kidney glomerular IgG deposition and proteinuria were present in {alpha}-actinin-immunized mice. The cross-reactive chromatin targets were determined to be high mobility group box (HMGB) protein and heat shock protein 70 (hsp70), both of which are known antigenic targets in SLE. Moreover, a panel of nephritogenic mAbs showed similar reactivity to {alpha}-actinin and its cross-reactive components HMGB and hsp70.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Eight-wk-old female BALB/c, New Zealand White (NZW), New Zealand Black (NZB), AKR, A/J, SJL, B6.AK-H2k, C57BL/6, PL/J, SWR, (NZB x NZW)F1 (B/W), and MRL-lpr/lpr mice were purchased from The Jackson Laboratory and housed in the animal facility of the Albert Einstein College of Medicine (Bronx, NY). All animal studies were approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine.

Ab production

R4A is a pathogenic murine IgG2b anti-dsDNA/anti-{alpha}-actinin Ab (17). Murine anti-HMGB1 (HAP46.5, IgG1) and anti-hsp70 (BRM-22, IgG1) mAbs were obtained from Sigma-Aldrich. 1A3F (IgG2a) is a pathogenic anti-dsDNA mAb derived from B6.Sle1 mice; 71CF11 (IgG2a) and 71AB (IgG2a) are nephrophilic ANAs from lupus-prone B6.Sle1.Sle3 mice; ZA8A3 (IgG2a) is an anti-nuclear negative, non-nephritogenic Ab isolated from NZM2410 mice (18, 19); and insulin-like growth factor (IGF)2 (IgG2b) is a DNA binding, non-nephritogenic mAb derived from B/W mice (20). MOPC21 (mouse IgG1), MOPC141 (mouse IgG2b), and UPC10 (mouse IgG2a) mice (all from Sigma-Aldrich) were used as isotype-matched controls.

Immunizations

Ten-wk-old female mice were s.c. immunized with 100 µg of chicken {alpha}-actinin (Sigma-Aldrich) in CFA H37 Ra (Difco), followed by a boost of 100 µg of {alpha}-actinin in IFA 2, 4, and 6 wk later. Control mice were immunized with CFA alone at baseline (week 0) and boosted with IFA following the same schedule. Serum and urine samples were obtained at baseline (before the first immunization). Subsequently, serum was obtained every 2 wk, and urine obtained every 2 wk until week +12 and then every 4 wk. {alpha}-Actinin immunization in BALB/c mice was repeated in three independent experimental cohorts, with similar results each time.

ANA titers

For determination of ANA titers, sera were serially diluted from 1/25 to 1/800 in PBS. Diluted sera (25 µl) were added to Hep-2 slides (Immuno Concepts) in moist chambers and incubated for 30 min at room temperature. Slides were rinsed and washed in PBS for 10 min, followed by five rinses in distilled water. Cy3-labeled anti-mouse IgG Ab (Sigma-Aldrich) was added at a 1/50 dilution in 3% FCS/PBS for 30 min at room temperature in the dark. Slides were then washed with PBS for 10 min, and a coverslip was applied. A fluorescence microscope (Axiovision; Carl Zeiss) was used for blinded evaluation of fluorescence intensity at a magnification of x40.

Proteinuria

Proteinuria was measured with reagent strips for urinalysis, where +1 is 30 mg/dl, +2 is 100 mg/dl, +3 is 300 mg/dl, and +4 is ≥2000 mg/dl (Uristix; Bayer).

Renal Ig deposition

One kidney from each animal was fixed in 10% formalin and embedded in paraffin. Sections (4 µm thick) were cut, deparaffinized, rehydrated, blocked with 2% filtered horse serum in PBS in moist chambers, and stained for 1 h with biotinylated goat anti-mouse IgG (Vector Laboratories) at a 1/800 dilution at room temperature. Sections were washed, incubated for 45 min with streptavidin-alkaline phosphatase, and developed with BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and nitroblue tetrazolium (Invitrogen Life Technologies). Color development was stopped after 30 min by washing in distilled water. Coverslips were mounted on the stained sections with cytosol mounting solution (Polysciences). The sections were then sealed and viewed with a Zeiss microscope.

ELISA

Salmon sperm dsDNA (Calbiochem) was purified by filtration with a 0.45-µm filter (Millipore) and adsorbed to Immulon II 96-well microtiter plates (Dynatech Laboratories) at a concentration of 100 µg/ml in PBS. For the ssDNA ELISA, a solution of salmon sperm DNA in PBS was boiled for 15 min, cooled rapidly on ice, and adsorbed to Immulon II plates at a concentration of 100 µg/ml in PBS. The dsDNA and ssDNA plates were dried overnight at 37°C. Before blocking, excess DNA was removed with a 4-min soak in distilled water. {alpha}-Actinin (Sigma-Aldrich), chromatin, and histone, each at a concentration of 5 µg/ml, were separately coated onto Immulon II 96-well microtiter plates overnight at 4°C. Cardiolipin (Fluka) at a concentration of 75 µg/ml in ethanol was adsorbed to Immulon II plates at room temperature overnight. Sm/RNP (Immunovision) at 10 µg/ml in PBS was adsorbed to Immulon II plates at 4°C overnight. Plates were blocked with 3% FCS for 1 h at 37°C and incubated with mAb or serum at a 1/200 dilution for 2 h at room temperature. Plates were washed five times with PBS containing 0.05% Tween 20 (PBS-Tween), and alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) diluted 1/1000 in 3% FCS was added for 1 h at 37°C. ELISA was developed by adding the alkaline phosphatase substrate p-nitrophenyl phosphate (Sigma-Aldrich), and the OD at 405 nm was monitored using a MRX Revelation ELISA Reader (Dynex Technologies). For the inhibition ELISA, immunized mice sera were preincubated with serial dilutions of {alpha}-actinin for 1 h at 37°C before transfer to the preblocked Ag-coated plate. The assay was then continued as described. The percentage of inhibition was calculated as (OD without inhibitor – OD with inhibitor)/(OD without inhibitor) x 100.

ELISPOT assay

Millipore Multiscreen-HA sterile plates were coated with chromatin (5 µg/ml) in PBS overnight at room temperature. Plates were emptied and soaked in PBS for 1 h at room temperature. Plates were washed twice in PBS, and blocked using blocking buffer (coating buffer plus 3% FCS) for 2 h at room temperature. Plates were then washed once with PBS, and once with RPMI 1640 medium. Splenocytes in 10% RPMI 1640 at a concentration of 1.0 x 107 splenocytes/ml were added in triplicate and serially diluted on the plates in 10% RPMI 1640. The plates were covered and incubated overnight at 37°C. Cells were removed by washing the plates five times in PBS and five times in PBS-Tween, and the appropriate biotinylated Ab diluted at 1/800 in block solution was added for 2 h at 37°C. Plates were washed 10 times with PBS-Tween, followed by a 1/800 dilution in block solution of streptavidin-alkaline phosphatase for 1 h at 37°C. The plates were again washed 10 times with PBS-Tween, and the developing solution of BCIP plus nitroblue tetrazolium chloride (Sigma-Aldrich) was added at 37°C in the dark. The substrate solution was removed, the plates washed three times with distilled water and dried at room temperature, and the spots per well counted by an ELISPOT Reader System (Autoimmune Diagnostika) using ELISPOT 3.2.3 software.

Western blotting and immunoprecipitation

Protein lysate (20 µg) was combined with Laemmli sample buffer (Bio-Rad), loaded onto a 10–20% gradient polyacrylamide gel (Bio-Rad), and electrophoresed. Proteins were transferred to a polyvinylidene difluoride membrane using a Mini Protean 3 cell apparatus (Bio-Rad) at 100 V for 1 h. The membrane was blocked in 5% nonfat milk and incubated with primary Ab at 1–5 µg/ml or immunized mice sera diluted 1/200 for 1 h at room temperature. The membrane was repeatedly washed with PBS-Tween and incubated with the appropriate HRP-conjugated secondary Ab diluted 1/5000 in 5% nonfat dry milk for 30 min at room temperature. The membrane was developed with the ECL Plus kit, and exposed to Hyperfilm (Habersham). Loading of equivalent amount of protein and adequate membrane transfer was confirmed by staining the polyvinylidene difluoride membrane with Ponceau Red. For the inhibition assays, serial dilutions of {alpha}-actinin or chromatin were preincubated with immunized mice sera for 2 h at 37°C. Western blotting was then continued as described.

For immunoprecipitation, protein G beads (Amersham Biosciences) were prewashed with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% NaN3, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and protease inhibitors), and incubated with 75 µg of mAb or 25 µl of sera for 2 h at 4°C with constant mixing. Precleared chromatin preparations were combined with the Ab-loaded protein G beads and incubated overnight at 4°C with gentle shaking. The mixture was centrifuged at 10,600 rpm for 20 s and the pellet was washed three times with 1 ml of RIPA buffer and once with 1 ml of 50 mM Tris-HCl (pH 8.0). The pellet was resuspended in Laemmli sample buffer (Bio-Rad) and heated to 100°C for 5 min. The supernatant was removed, and the proteins were separated by SDS-PAGE and Western blotted as described, except that the secondary Ab used was a Trueblot HRP-conjugated anti-mouse IgG at 1/1000 dilution (eBioscience).

IgG purification from serum

IgG was purified from sera of {alpha}-actinin and adjuvant immunized mice using the melon gel IgG spin purification kit (Pierce). In brief, 50 µl of sera was diluted 1/10 in the melon gel purification buffer, added to the washed column, and incubated for 5 min at room temperature with end over end mixing. The column was centrifuged for 1 min at 5000 x g to collect purified IgG.

Depletion of anti-{alpha}-actinin Abs

{alpha}-Actinin was coupled to a column using the Aminolink Plus immobilization kit (Pierce). One milligram of {alpha}-actinin was dialyzed in BupH citrate carbonate buffer (pH 10.0, coupling buffer), then added to a washed column and incubated overnight with end over end mixing at room temperature. Nonbound {alpha}-actinin was washed away, and the bound {alpha}-actinin was coupled to the column by adding sodium cyanoborohydride in coupling buffer and incubating overnight at 4°C. The column was washed and, the remaining active sites were blocked by adding sodium cyanoborohydride in quenching buffer and mixing for 30 min at room temperature. The {alpha}-actinin coupled column was washed repeatedly. Purified IgG from {alpha}-actinin immunized mice was then added to the {alpha}-actinin-coupled column and incubated for 1 h at room temperature with end over end mixing. The column was centrifuged at 1000 x g for 1 min and the flow-through collected. The IgG Abs bound to the {alpha}-actinin-coupled column were then eluted with 0.1 M glycine-HCl (pH 2.5), neutralized in 1 M Tris (pH 8.5), and dialyzed against PBS.

Histone preparation

Citrated chicken blood (Colorado Serum Company, Denver, Colorado) was centrifuged at 2000 rpm for 5 min at 4°C. The supernatant was discarded, and the pellet resuspended in sucrose buffer (0.3 M sucrose, 15 mM NaCl, 10 mM HEPES (pH 7.9), 2 mM EDTA, 0.5 mM PMSF) plus 0.5% Nonidet P-40. The suspension was homogenized using a glass dounce, centrifuged at 2000 rpm for 5 min at 4°C, and the supernatant discarded. The pellet was resuspended in 3 ml of high salt buffer (0.35 M KCl, 10 mM Tris (pH 7.2), 5 mM MgCl2, and 0.5 mM PMSF), dounced again, kept on ice for 20 min, and centrifuged at 13,000 rpm for 10 min at 4°C. The pellet was resuspended in 0.2 N H2SO4, and following an overnight incubation at 4°C, the suspension was centrifuged at 13,000 rpm for 10 min at 4°C. The pellet was discarded, and 2.5 volumes of ice-cold ethanol was added directly to each tube and left overnight at –20°C. The suspension was then centrifuged at 13,000 rpm for 10 min at 4°C. The pellets were washed three times with 70% ethanol, left to dry, resuspended in double-distilled H2O, and stored at –80°C.

Chromatin preparation

Isolation of chromatin was done based on a protocol provided by Dr. P. Cohen (University of Pennsylvania, Philadelphia, PA). Packed chicken erythrocytes were washed twice in buffer A (0.08 M NaCl, 0.02 M EDTA, (pH 7.5)), resuspended in 15 ml of the same buffer, and lysed by an equal volume of 1.5% Triton X-100 in buffer A. Cold 2.25 M sucrose in buffer was added to a Beckman polyallomer 25 x 89 mm tube, overlaid with cold 1.7 M sucrose in buffer A, and finally layered with lysate. The mixture was centrifuged at 25,000 rpm for 90 min at 4°C. After removing the sucrose, the pellet was resuspended in buffer A, and centrifuged for 15 min at 2000 x g. The second pellet was resuspended in 50 mM Tris (pH 7.9), and spun again for 15 min at 2000 x g. The final pellet was resuspended in cold distilled water, sonicated at 4°C, diluted in 0.1 mM EDTA, and stored at –80°C.

Protein analysis

Protein identification was performed at the Laboratory for Macromolecular Analysis & Proteomics (Albert Einstein College of Medicine, Bronx, NY). SDS-PAGE was conducted as described. Coomassie brilliant blue (Sigma-Aldrich) stained protein bands of interest were cut from the gel and transferred to prewashed microcentrifuge tubes. In-gel tryptic digestion was performed after destaining (in 200 mM NH4HCO3/50% MeCN (pH 8.9)), reduction (10 mM DTT in 0.1 M NH4HCO3), and alkylation (55 mM iodoacetamide in 0.1 M NH4HCO3). Digestion was conducted using modified porcine trypsin (Promega) at 37°C for 1 h or overnight. Digestion was stopped using trichloroacetic acid to a final concentration of 0.1%.

Mass analysis was performed using both MALDI and electrospray ionization mass spectrometry (MS) and tandem MS (MS/MS).

Peptides were prepared for MALDI analysis by desalting and concentrating on C18 ZipTip pipettes (Millipore). The sample was eluted from the ZipTip directly onto seeded wells of a MALDI plate. The seed layer was prepared using 0.5 µL CHCA ({alpha}-cyano-4-hydroxycinnamic), 2 mg/ml in 50% CH3CN, 0.1% trichloroacetic acid. The sample was then sandwiched between another layer of matrix (1 µl of CHCA, 5 mg/ml).

Digest ion mass data were acquired on the 4700 Proteomics Analyzer MALDI TOF/TOF (Applied Biosystems) operated in reflectron mode. Calibration was updated before sample spectra acquisition according to the instrument protocol. Peptide mass fingerprint analysis was performed using ProFound (Rockefeller University, New York, NY).

Nano liquid chromatography MS/MS was performed using a Ultimate HPLC (Dionex) connected to an LTQ Linear Ion Trap Mass Spectrometer (Thermo Electron). A 75-µm i.d. C18 Nanocolumn (15-cm column, C18 PepMap100) was used for peptide separations. The mass spectrometer was set to obtain a survey scan between 300 and 2000 m/z, followed by MS/MS scans of the three most intense ions. Disk Transfer Address (.dta) files were generated from the raw data and merged into one file. The merged file was searched against the Metazoa MS Protein Sequence Database using an in-house Mascot MS/MS ion search.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Immunization of BALB/c mice with {alpha}-actinin induces ANA

BALB/c mice immunized with {alpha}-actinin in adjuvant displayed high titers of IgG anti-{alpha}-actinin Abs, beginning at 4 wk following the initial immunization (week +4) (Fig. 1). To determine whether ANA arose during the immune response to {alpha}-actinin, we examined ANA titers in immunized BALB/c (H-2d) mice by immunofluorescence at week +8. We found that BALB/c mice immunized with {alpha}-actinin developed significantly increased ANA titers, as compared with mice immunized with adjuvant alone (Fig. 2, A and B). Furthermore, we found an excellent correlation in {alpha}-actinin-immunized mice (n = 10) between increased ANA titers and the titers of anti-{alpha}-actinin Abs (r = 0.825; p = 0.0002) (data not shown).


Figure 1
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  		FIGURE 1. {alpha}-Actinin-immunized BALB/c mice have high serum titers of anti-{alpha}-actinin IgG Abs. Sera from {alpha}-actinin-immunized (n = 10) and adjuvant immunized (n = 10) BALB/c mice (diluted 1/200) were tested by an anti-{alpha}-actinin Ab ELISA, as described in Materials and Methods. Data are the mean OD405 ± SD. Values for p that represent the difference between {alpha}-actinin-immunized and control immunized BALB/c mice in IgG anti-{alpha}-actinin titers at each time point following the initial immunization were: week +4, p < 2E-13; week +8, p < 1E-15; week +16, p < 1E-14; week +24, p < 4E-14; week +28, p < 1E-09.

 

Figure 2
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  		FIGURE 2. High titers of IgG ANAs are induced in {alpha}-actinin-immunized BALB/c mice. A, Sera from a representative {alpha}-actinin-immunized and an adjuvant immunized BALB/c mouse from week +8, serially diluted 1/2 starting at a dilution of 1/25, were added to Hep-2 slides and developed with Cy3-labeled anti-mouse IgG Ab. Detection was by fluorescence microscopy at a magnification of x40. B, ANA titers (1/dilution), determined as the highest dilution with a positive ANA, in {alpha}-actinin-immunized (n = 5) and adjuvant immunized (n = 5) BALB/c mice sera (week +8). C, ANA titers (1/dilution) in other {alpha}-actinin-immunized nonautoimmune mice strains in week +8 sera. Values for p that represent the differences in ANA titers between {alpha}-actinin-immunized (n = 5) and adjuvant immunized (n = 5) nonautoimmune mice strains were: AKR, p < 0.004; A/J, p < 0.095; SJL, p < 0.005; B6.AK-H2k, p < 0.038; C57BL/6, p < 0.126; PL/J, p < 0.008; NZW, p < 0.0124; and NZB, p < 0.0194. Data in B and C are mean dilutions ± SD.

 
To investigate whether the increased ANA in response to {alpha}-actinin immunization is MHC-restricted or limited to specific genetic backgrounds, we repeated the immunization in other nonautoimmune mouse strains, including AKR (H-2k), A/J (H-2a), SJL (H-2s), C57BL/6 (H-2b), PL/J (H-2p), NZW (H-2z), and NZB (H-2d). We found that {alpha}-actinin immunization significantly increased ANA titers in AKR, SJL, PL/J, NZW, and NZB mice, but not A/J or C57BL/6 mice (Fig. 2C). In addition, we immunized C57BL/6 mice congenic for H-2K (B6.AK-H2k), an MHC associated with the responsive strain AKR. There was a ~2-fold increase of the induced ANA titer in B6.AK-H2k compared with wild-type C57BL/6 mice (Fig. 2C); however, this increase did not reach statistical significance. Nevertheless, {alpha}-actinin-immunized B6.AK-H2k mice had significantly increased ANA titers as compared with immunized B6.AK-H2k control mice. These results indicate that, although there is some contribution of MHC to the loss of tolerance, {alpha}-actinin immunization induces ANA in mice carrying a variety of H-2 alleles.

{alpha}-Actinin-immunized mice develop high titers of anti-chromatin Abs

To determine the nuclear Ag targeted by autoantibodies arising in response to {alpha}-actinin immunization, we measured IgG Ab titers against chromatin in {alpha}-actinin-immunized mice at week +8. We found that BALB/c (Fig. 3A) and several other mice strains (Fig. 3B) displaying high ANA titers in response to {alpha}-actinin immunization had significantly increased IgG anti-chromatin Abs. Anti-chromatin Ab titers became significantly different at week +4 and increased steadily up to week +16. Although after week +16, anti-chromatin Ab titers declined slowly, significantly higher titers than found in control mice were seen even up to 6 mo following the initial immunization (Fig. 3A). The titers of anti-chromatin Abs induced in {alpha}-actinin-immunized mice were comparable to those in unmanipulated 8-mo-old lupus-prone B/W mice and 5-mo-old MRL-lpr/lpr mice (Fig. 3C). To further quantitate the anti-chromatin response, we determined the number of chromatin-specific B cells in spleens of {alpha}-actinin-immunized BALB/c mice. Consistent with the ELISA data, the number of chromatin-specific B cells was significantly higher in {alpha}-actinin-immunized (n = 4) mice than the number found in adjuvant-immunized (n = 4) mice (638 ± 251 vs 251 ± 174 x 107 cells/ml, p < 0.004). Cross-reactivity of the anti-{alpha}-actinin response with chromatin in immunized mice was ascertained by inhibition and anti-{alpha}-actinin Ab depletion studies. We found that {alpha}-actinin, but not a nonspecific inhibitor (keyhole limpet hemocyanin), inhibited up to 70% of chromatin binding by sera from {alpha}-actinin immunized BALB/c mice (Fig. 3D). Furthermore, depletion of {alpha}-actinin binding Abs from purified IgG of {alpha}-actinin-immunized mice led to a corresponding, significant diminution (>75%) in chromatin binding activity relative to non-{alpha}-actinin Ab-depleted IgG (Fig. 3E). Together, our results clearly indicate that {alpha}-actinin immunization elicits Abs that cross-react with chromatin.


Figure 3
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  		FIGURE 3. IgG anti-chromatin Abs in {alpha}-actinin-immunized BALB/c mice. A, Sera from {alpha}-actinin-immunized (n = 10) and adjuvant immunized (n = 10) BALB/c mice (diluted 1/200) at different weeks following the initial immunization were tested in an anti-chromatin ELISA, as described in Materials and Methods. Values for p that represent the difference between {alpha}-actinin and control immunized BALB/c mice sera in IgG anti-chromatin Abs at different time points following the initial immunization were: week +4, p < 1E-10; week +8, p < 1E-13; week +16, p < 2E-12; week +24; p < 3E-13; week +28, p < 3E-09. B, IgG anti-chromatin Ab responses to {alpha}-actinin immunization in various nonautoimmune strains. Sera from {alpha}-actinin-immunized mice (n = 5) and adjuvant immunized (n = 5) nonautoimmune mice from the indicated strains (week +8, diluted 1/200) were tested for anti-chromatin Abs by ELISA. Values for p that represent the difference between {alpha}-actinin-immunized and adjuvant immunized sera in anti-chromatin IgG titers were: AKR, p < 3.7E-07; SJL, p < 1.3E-06; PL/J, p < 4.2E-07; NZW, p < 9.1E-08; and NZB, p < 5.9E-09. Data shown in A and B are mean OD + SD. C, IgG anti-chromatin Ab titration. Week +8 sera from {alpha}-actinin-immunized BALB/c mice (n = 10), adjuvant immunized BALB/c mice (n = 10), untreated BALB/c mice (n = 10), unmanipulated lupus prone B/W mice (n = 10, 32-wk-old) and MRL-lpr/lpr mice (n = 10, 22-wk-old) were serially diluted 1/2 starting at a dilution of 1/200, and tested in an anti-chromatin ELISA. D, {alpha}-actinin inhibition of {alpha}-actinin-immunized BALB/c sera binding to chromatin. Week +8 sera (diluted 1/1200) from {alpha}-actinin-immunized BALB/c mice (n = 10) were preincubated with serial dilutions of {alpha}-actinin, chromatin, and keyhole limpet hemocyanin (starting at a concentration of 18.75 µg/ml) for 1 h at 37°C, followed by an anti-chromatin ELISA. E, Binding of anti-{alpha}-actinin Ab depleted IgG to chromatin. Purified IgG from combined week +8 adjuvant immunized BALB/c sera (n = 10), purified IgG from combined week +8 {alpha}-actinin-immunized BALB/c sera (n = 10), purified IgG from combined week +8 {alpha}-actinin-immunized BALB/c sera following passage on the {alpha}-actinin coupled column (depleted Ig) (n = 10), and the column eluate were tested in anti-{alpha}-actinin and anti-chromatin ELISA at normalized Ig concentrations of 10 µg/ml. Depletion of anti-{alpha}-actinin Abs from immunized sera leads to a marked decrease in chromatin binding activity. Data shown in A–E are mean OD ± SD, and in D are the mean percentage of inhibition ± SD.

 
To investigate whether autoantibodies against other lupus-associated Ags besides chromatin were induced as well, we studied the induced titers of Abs against dsDNA (IgM and IgG), ssDNA (IgM and IgG), histone (IgG), Sm/RNP (IgG), and cardiolipin (IgG) in {alpha}-actinin-immunized BALB/c mice at week +8. There were no major differences in the serum titers of anti-cardiolipin, anti-ssDNA, and anti-dsDNA Abs between {alpha}-actinin-immunized and adjuvant immunized mice. However, anti-histone and anti-Sm/RNP Abs were significantly higher in {alpha}-actinin-immunized BALB/c mice (n = 10) compared with adjuvant immunized BALB/c mice (n = 10) (IgG anti-histone 1.0 ± 0.12 vs 0.15 ± 0.02 (OD), p < 2E-14; IgG anti-Sm/RNP 1.0 ± 0.26 vs 0.4 ± 0.13 (OD), p < 4E-06 with week +8 sera, diluted 1/200).

We next determined whether hypergammaglobulinemia is a feature of the autoimmune phenotype induced by {alpha}-actinin immunization. Although there was no significant difference in the titer of total IgG2a Abs, the level of total IgG1 Abs was significantly increased in {alpha}-actinin-immunized BALB/c mice (Fig. 4A). Moreover, the major isotype of the anti-chromatin Ab response induced in {alpha}-actinin-immunized BALB/c mice was IgG1, although significant titers of IgG2a and IgG2b anti-chromatin Abs were induced as well (Fig. 4B). It is interesting, however, that in MRL-lpr/lpr mice, which are genetically susceptible to the spontaneous development of IgG2a anti-chromatin Abs, the Ab isotype was not as prominent. In {alpha}-actinin-immunized MRL-lpr/lpr mice, the spectrum of induced anti-chromatin Abs was skewed to IgG2a, which were quantitatively higher in this strain than IgG1 anti-chromatin Abs (Fig. 4C).


Figure 4
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  		FIGURE 4. IgG subclasses in the anti-chromatin response. A, Total IgG1 and IgG2a Ab levels were measured in {alpha}-actinin-immunized mice (n = 5) and control immunized BALB/c mice (n = 5). B, Anti-chromatin ELISA was performed using sera from {alpha}-actinin-immunized BALB/c mice (n = 5) (week +8, diluted 1/200), and developed with alkaline phosphatase-linked Abs to mouse IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3). C, IgG subclasses in the anti-chromatin response of week +8 {alpha}-actinin-immunized mice (n = 5) vs adjuvant immunized MRL-lpr/lpr mice (n = 5). Data shown in B and C are mean OD ± SD. Values for p that represent the difference in anti-chromatin IgG subclass responses between {alpha}-actinin-immunized and adjuvant immunized MRL-lpr/lpr mice were: IgG1, p < 0.05; IgG2a, p < 0.03; and IgG2b, p < 0.3; IgG3, p < 0.2.

 
{alpha}-Actinin-immunized mice develop glomerular Ig deposition and proteinuria

To determine whether autoantibodies induced in response to {alpha}-actinin immunization deposit in the kidney and induce renal disease, we studied Ig deposition and proteinuria in {alpha}-actinin-immunized mice. {alpha}-Actinin-immunized BALB/c mice showed significantly higher glomerular Ig deposition as compared with adjuvant immunized mice (Fig. 5). Furthermore, {alpha}-actinin-immunized BALB/c mice (n = 5) had significantly higher levels of proteinuria as compared with adjuvant immunized mice (n = 5) at week +10 (58 ± 23 vs 23 ± 6 mg/dl, p < 0.01).


Figure 5
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  		FIGURE 5. Glomerular IgG deposition in {alpha}-actinin immunized BALB/c mice. A, A representative kidney section from an {alpha}-actinin immunized BALB/c mouse sacrificed at week +18. B, A representative kidney section from an adjuvant immunized mouse sacrificed at week +18. Sections were stained with biotinylated goat anti-mouse IgG Ab followed by streptavidin-alkaline phosphatase and substrate. Arrow indicates glomeruli, which display Ig deposition in {alpha}-actinin-immunized but not control immunized mice.

 
HMGB and hsp70 are chromatin components recognized by {alpha}-actinin-induced autoantibodies

To investigate which chromatin components are the targets of the {alpha}-actinin-induced Abs, we performed Western blot analysis on purified chromatin with serum from {alpha}-actinin-immunized mice. Serum from {alpha}-actinin-immunized BALB/c mice, but not adjuvant immunized mice, bound to a doublet of 25 kDa bands in chromatin (Fig. 6A, lanes 1 and 2). Binding to the doublet was also present when using purified IgG from serum of {alpha}-actinin-immunized mice (data not shown). To determine the specificity of this binding, we performed inhibition studies. Fig. 6C shows that {alpha}-actinin and chromatin both exhibit dose-dependent inhibition of binding to the 25-kDa chromatin bands by BALB/c immunized sera.


Figure 6
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  		FIGURE 6. Serum from {alpha}-actinin-immunized mice binds to a 25-kDa doublet chromatin band (HMGB). A, Chromatin was separated by SDS-PAGE, and blotted with sera from a representative {alpha}-actinin-immunized (lane 1) and adjuvant immunized (lane 2) BALB/c mouse (week +8, diluted 1/200). Chromatin was separated by SDS-PAGE (lane 3), and blotted with an anti-HMGB1 mAb. B, Chromatin separated by SDS-PAGE was blotted with sera from an {alpha}-actinin-immunized BALB/c mouse (lane 1) (week +8, diluted 1/200). Precleared chromatin was incubated with protein G beads linked to anti-HMGB1 Ab. Immunoprecipitate was separated by SDS-PAGE (lane 2) and blotted with sera from an {alpha}-actinin-immunized BALB/c mouse (week +8, diluted 1/200). C, Inhibition studies by Western blot analysis. Chromatin was separated by SDS-PAGE and blotted with sera from an {alpha}-actinin-immunized BALB/c mouse (week +8, diluted 1/200) that was preincubated with serial dilutions of {alpha}-actinin or chromatin at an initial concentration of 25 µg/ml. Ag, loaded Ag; Inhibitor, inhibiting Ag; Ab, blotting Ab. A–C, Sera from other {alpha}-actinin-immunized and adjuvant immunized mice displayed similar reactivity. D, Anti-HMGB1 IgG ELISA. Sera from {alpha}-actinin-immunized (n = 10), adjuvant immunized (n = 10), and untreated (n = 10) BALB/c mice (week +8, diluted 1/200) were assayed for the binding to purified HMGB1. NS, Not significant. E, Anti-HMBG1 mAb binding to chromatin. An anti-HMGB1 mAb and an isotype matched control Ab were serially diluted 1/2 starting at a concentration of 10 µg/ml, and tested for binding to chromatin by ELISA. Data shown in D and E are mean OD ± SD.

 
MALDI mass mapping and MS/MS data proteomic analysis unequivocally identified the presence of HMGB1 (GenInfo Identifier (GI) no. 52783747) in the upper band of the doublet and HMGB3 (GI no. 729728) in the lower band of the doublet. HMGB1 and HMGB3 both belong to the same family of HMGB non-histone chromosomal proteins. These results were confirmed by MALDI MS/MS and electrospray ionization analysis. We confirmed that the binding of a anti-HMGB1 mAb to chromatin (Fig. 6A, lane 3) by Western blot analysis displayed a similar pattern to that of {alpha}-actinin-immunized BALB/c mice sera binding to chromatin (Fig. 6A, lane 1). Furthermore, the immunoprecipitate of chromatin by an anti-HMGB1 Ab blotted by the sera of {alpha}-actinin-immunized BALB/c mice (Fig. 6B, lane 2) displayed similar bands to chromatin blotted with sera from {alpha}-actinin-immunized BALB/c mice (Fig. 6B, lane 1). By ELISA, {alpha}-actinin-immunized BALB/c sera displayed significantly higher IgG titers to purified HMGB1 as compared with adjuvant immunized and untreated BALB/c controls (Fig. 6D). Finally, the anti-HMGB1 mAb showed significant binding to chromatin (Fig. 6E).

Sera from {alpha}-actinin-immunized BALB/c mice also bound to (Fig. 7A) and immunoprecipitated (Fig. 7B) an additional band from chromatin with a molecular mass of 70 kDa. In inhibition studies, we similarly found that increasing concentrations of {alpha}-actinin inhibited the binding of immunized mice sera to this band (Fig. 7C). Proteomic analysis indicated that this 70-kDa band was hsp70 (GI no. 30962014). This finding was confirmed by Western blot, which showed that sera from {alpha}-actinin-immunized BALB/c mice bound to purified hsp70, and that anti-hsp70 bound to a 70-kDa band in purified chromatin (Fig. 7D). Finally, when purified hsp70 was used as a substrate, serum from {alpha}-actinin-immunized BALB/c mice displayed significantly higher binding to purified hsp70 than serum from adjuvant immunized or untreated BALB/c mice (Fig. 7E). We should note that although there was some binding of sera from adjuvant immunized mice to HMGB1 (Fig. 6D) and hsp70 (Fig. 7E), we believe this response constitutes background binding because it was not significantly different from the binding of sera from unmanipulated age-matched mice to these Ags.


Figure 7
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  		FIGURE 7. Immunized BALB/c mice serum binds hsp70. A, Chromatin was separated by SDS-PAGE and blotted with sera from {alpha}-actinin-immunized and adjuvant immunized BALB/c mice (week +8, diluted 1/200). B, Precleared chromatin was incubated with protein G beads linked to anti-hsp70 Ab and the immunoprecipitate separated by SDS-PAGE and blotted with sera from {alpha}-actinin-immunized and adjuvant immunized BALB/c mice (week +8, diluted 1/200). C, Inhibition study by Western blot analysis. Chromatin was separated by SDS-PAGE and blotted with sera from {alpha}-actinin-immunized BALB/c mice (week +8, diluted 1/200) that were preincubated with serial dilutions of {alpha}-actinin starting at an initial concentration of 25 µg/ml. D, IgG Anti-hsp70 Western blot study. Purified hsp70 was separated on SDS-PAGE and blotted with sera from an {alpha}-actinin-immunized BALB/c mice (week +8, diluted 1/200) (lane 1). Chromatin was separated by SDS-PAGE, and blotted with sera from an {alpha}-actinin-immunized BALB/c mouse (lane 2) (week +8, diluted 1/200) and anti-hsp70 mAb (lane 3). For A–C, sera from other {alpha}-actinin-immunized and adjuvant immunized mice displayed similar reactivity. E, IgG Anti-hsp70 ELISA study. Sera from {alpha}-actinin-immunized (n = 5), adjuvant immunized (n = 5), and untreated (n = 5) BALB/c mice (week +8) (diluted 1/200) were assayed for binding to purified hsp70. Data shown in E are mean OD ± SD. NS, Not significant.

 
Ags bound by sera from {alpha}-actinin-immunized mice share a common linear sequence

To determine whether the Ags bound by sera from {alpha}-actinin-immunized BALB/c mice may share a common sequence motif with {alpha}-actinin, we used a panel of nephrophilic (R4A, 1A3F, 71CF11, 71AB) and non-nephrophilic (ZA8A3, IGF2, UPC10) Abs (as determined by binding to mesangial cells and isolated glomeruli) (18, 19). Significantly, nephrophilic but not non-nephrophilic Abs bound strongly to {alpha}-actinin, chromatin, HMGB1, and hsp70 (Fig. 8A), raising the possibility of a common sequence or motif among these cross-reactive Ags, which makes them the target of pathogenic autoantibodies. To further delineate this common epitope, the amino acid sequences of {alpha}-actinin, HMGB1, HMGB3, and hsp70 were aligned using Nomad (Neighborhood Optimization for Multiple Alignment Discovery) v.1.0 (21). The predicted common linear sequence PKxxPR/KGKM is shown in Fig. 8B. There is close homology between the linear sequence in {alpha}-actinin, HMGB1 and HMGB3, with hsp70 showing slightly less homology. Furthermore, all the common peptide sequences from each respective Ag were in B cell epitope regions as determined by Bcepred, a B cell epitope prediction program (22). Importantly, the common peptide sequence from {alpha}-actinin is found in its actin binding domain, a region recently determined to be targeted by the anti-{alpha}-actinin response in human lupus nephritis (5).


Figure 8
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  		FIGURE 8. Cross-reactive Ags bound by pathogenic Abs share a common motif. A, The reactivity of a panel of four nephrophilic autoantibodies (R4A, 1A3F, 71CF11, 71AB) to {alpha}-actinin, chromatin, HMGB1, and hsp70 was compared with a panel of three nonpathogenic autoantibodies (ZA8A3, IGF2, UPC10) using ELISA. Data shown are mean OD ± SD. Values for p that represent the difference in binding of pathogenic vs nonpathogenic Abs to these Ags were: {alpha}-actinin, p < 4E-04; chromatin, p < 4E-04; HMGB1, p < 0.035; hsp70, p < 0.002. B, The sequence of {alpha}-actinin-4 was aligned to HMGB1, HMGB3, and hsp70 using Nomad, a multiple sequence alignment software.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the current study we demonstrated that immunization of nonautoimmune mice with {alpha}-actinin elicited an Ab response that cross-reacted with nuclear Ags. The autoreactive component of the cross-reactive Ab response was mainly against chromatin, although other lupus-associated Ags were targeted as well. Further investigation of the anti-chromatin response in {alpha}-actinin-immunized mice showed that this reactivity was directed against HMGB (25 kDa) and hsp70 (70 kDa). Both HMGB1 and hsp70 can be found in the nucleus and are known antigenic targets in murine lupus, as discussed in greater detail below. Immunized mice serum binding to both 70- and 25-kDa bands was significantly inhibited by {alpha}-actinin and chromatin, confirming the cross-reactivity between {alpha}-actinin and the chromatin components HMGB and hsp70.

Anti-chromatin Abs are one of those key autoantibodies that characterize SLE. Chromatin autoantibodies are also classified as pathogenic Abs because they are among the few that can form glomerular immune deposits (23). Indeed, several investigators believe that anti-chromatin autoantibodies play a particularly significant role in the pathogenesis of SLE (24, 25) and are a more sensitive and specific marker for SLE than anti-dsDNA Abs; anti-chromatin Abs correlate with lupus disease activity, and have been reported to appear before the development of anti-dsDNA Abs (26). Although increasing titers of anti-dsDNA Abs have clearly been linked to lupus nephritis, there are patients who have lupus nephritis but without detectable anti-dsDNA Abs (27). Interestingly, some patients negative for anti-dsDNA are positive for anti-chromatin Abs (28).

Chromatin, found in the eukaryotic nucleus, is made up of DNA, histones, non-histone proteins, RNA, and other macromolecules (29). The potential source of the antigenic chromatin components targeted in spontaneous SLE may come from apoptotic cell death, where there is chromatin and DNA fragmentation followed by subsequent exposure of nuclear and cytoplasmic contents at the surface of apoptotic cells (30), or from necrotic cell death, where there is disintegration of the cell membrane and release of intracellular Ags into the circulation (31). Normally, apoptotic cells are cleared quickly and do not elicit inflammation or an immune response, in contrast to necrotic cells (32, 33). If, however, apoptotic cells are not cleared in a timely manner, there can be progression from apoptosis to secondary necrosis. Deficiencies in the regulation and clearance of apoptotic cells have been reported in SLE (31). The presence of anti-nucleosome autoantibodies as well as autoreactive, nucleosome-specific T cells in lupus patients (25, 34, 35) further suggests that nuclear material derived from apoptotic or necrotic cells may serve as an important source of autoantigens.

HMGB proteins are abundant non-histone DNA binding proteins that help to maintain nucleosome structure and regulate gene transcription (36). In addition to its DNA binding ability, it was found that HMGB1 is proinflammatory and can stimulate an inflammatory cytokine response upon passive release into the extracellular space from necrotic cells, or following active release from activated monocytes and macrophages (37, 38). HMGB1 has been shown to act as an endogenous immune adjuvant by activating APCs (including dendritic cells and macrophages) (37), through the receptor for advanced glycation end (RAGE) products (39) and possibly TLR2 and TLR4 (40). Interestingly, it was recently shown that HMGB1 and RAGE mediate TLR9-dependent activation of plasmacytoid dendritic cells by DNA-containing immune complexes (41). Anti-HMGB1 Abs are found in SLE patients (42), whereas increased extracellular expression of HMGB1 is present in lupus skin lesions (43). Importantly, nephritogenic anti-dsDNA inducing Th cell lines derived from patients with active lupus nephritis proliferate in response to HMGB. These Th cell lines increase IgG anti-DNA Ab concentrations 250-fold when cocultured with autologous B cells (35). Thus, autoimmune Th cells can provide help to B cells that process and present HMGB1 peptides.

Hsp70, the other cross-reactive target in our study, is a molecular chaperone that facilitates protein folding and Ag presentation within cells (44). Hsp70 is expressed at low levels under normal physiological conditions (45); however, there is a marked increase in intracellular synthesis of hsp70 in response to a wide variety of stressful stimuli, including heat shock, UV radiation, viral or bacterial infections, fever, and inflammation (46). Up-regulated hsps can then be released into the extracellular space from cells or tissues undergoing necrosis (47). Similarly to HMGB1, hsp is also thought to be an endogenous adjuvant. Hsps can trigger APCs via TLR4/2, stimulating innate immunity and proinflammatory cytokine release (48). Hsp70 has also been identified as an immune target in lupus-like autoimmunity (49). Moreover, Hsu et al. (50) recently isolated from BXD2 mice a panel of pathogenic autoantibodies that cause glomerulonephritis and arthritis, and found that hsp70 is a cross-reactive target for these polyreactive Abs. Finally, it is interesting to note that genetic up-regulation of TLR4 and TLR4 hyperresponsiveness have been recently shown to be sufficient to induce lupus-like autoimmune disease, including anti-dsDNA Abs and immune complex glomerulonephritis (51).

In comparison to nonpathogenic autoantibodies, autoantibodies with nephritogenic potential tend to be polyreactive, binding to nuclear Ags as well as cell surface and basement membrane proteins (52). In addition to our previous observations with R4A, we show that {alpha}-actinin and its cross-reactive chromatin components HMGB1 and hsp70 were also bound strongly by a panel of nephrophilic anti-dsDNA Abs. Moreover, the IGF2 anti-dsDNA Ab that was non-nephritogenic did not bind to {alpha}-actinin, HMGB1, or hsp70. This supports the notion that a common linear sequence or motif is targeted by pathogenic Abs. Although Kalaaji et al. (53) recently found that renal Ig eluates were enriched for {alpha}-actinin only in some lupus mice, they studied B/W mice which have lower titers of anti-{alpha}-actinin Abs than MRL/lpr mice (4). Moreover, investigators from the same group recently confirmed our previous findings (5, 6) that anti-{alpha}-actinin Ab levels are significantly higher in lupus patients with nephritis and renal flares (54).

What is the relevance of our model to understanding the pathogenesis of SLE? We recently described several sequence polymorphisms in {alpha}-actinin-4 between autoimmune MRL-lpr/lpr and nonautoimmune BALB/c mice (55); whether any particular isoform of {alpha}-actinin is particularly immunogenic and predisposes to loss of tolerance to nuclear Ags will be investigated. It will also be important to clarify the long-term clinical significance of the proteinuria observed in {alpha}-actinin-immunized mice, whether this effect progresses over time, and whether renal histopathological changes are induced. Furthermore, it will be interesting to determine whether {alpha}-actinin can trigger or accelerate disease in mice genetically susceptible to the development of spontaneous lupus, and whether survival will be affected. All of these are relevant questions that we plan to comprehensively address in future studies.

It is possible to suggest that our model of {alpha}-actinin immunization may replicate additional features of spontaneous autoimmune disease. Impaired clearance of apoptotic and necrotic cells as seen in SLE can lead to the accumulation of nuclear autoantigens such as dsDNA, histones, and chromatin. As mentioned, during apoptosis there is surface exposure in apoptotic blebs of nuclear and cytoplasmic contents (including {alpha}-actinin (56)) and release of chromatin components (including HMGB1 (57) and hsp70). Excessive HMGB1 and hsp70 from uncleared cells could stimulate resting APCs through TLR4/2, leading to the activation of HMGB1-specific T cells in an inflammatory environment. Activated HMGB1 T cells may then provide help for HMGB1-specific B cells, leading to their differentiation and proliferation. It is important to note that via intramolecular epitope spreading, HMGB-specific T cells can provide help for B cells binding to HMGB as well as DNA and histones, as B cells specific for any of these chromatin components will bind to and process the entire chromatin particle and present the relevant HMGB epitope to the Th cells. Via the sequence homology demonstrated, HMGB1, hsp70, and DNA-specific B cell clones may then produce autoantibodies that cross-react with {alpha}-actinin. Finally, although the usual level of {alpha}-actinin expression may not be sufficient for expression of disease, the enhanced kidney {alpha}-actinin expression observed in vivo in lupus prone MRL-lpr/lpr mice (55) may lead, in the face of high titers of circulating autoantibodies cross-reactive with {alpha}-actinin, to IgG deposition in the kidney and subsequent autoantibody-mediated kidney injury.


   Disclosures
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 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 This work was supported by Grants RO1-AR-48692 and PO1-AI-51392 from the National Institutes of Health (to Dr. Putterman’s laboratory). B.D. is the recipient of a Predoctoral Training grant from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Chaim Putterman, Division of Rheumatology, Forchheimer 701N, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: putterma{at}aecom.yu.edu Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; ANA, anti-nuclear Ab; IGF, insulin-like growth factor; MS, mass spectrometry; hsp, heat shock protein; NZW, New Zealand White; NZB, New Zealand Black. Back

Received for publication December 13, 2006. Accepted for publication May 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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