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-ActininαRole of TheAnti-DNA Antibodies to Mesangial Cells:

Differential Binding of Cross-Reactive

J. Ozelius and Chaim PuttermanZeguo Zhao, Bisram Deocharan, Philipp E. Scherer, Laurie

http://www.jimmunol.org/content/176/12/7704doi: 10.4049/jimmunol.176.12.7704

2006; 176:7704-7714; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/12/7704.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Differential Binding of Cross-Reactive Anti-DNA Antibodies toMesangial Cells: The Role of �-Actinin1

Zeguo Zhao,* Bisram Deocharan,† Philipp E. Scherer,‡§ Laurie J. Ozelius,¶ andChaim Putterman2*†

Target Ag display is a necessary requirement for the expression of certain immune-mediated kidney diseases. We previously hadshown that anti-DNA Abs that cross-react with �-actinin may be important in the pathogenesis of murine and human lupusnephritis; in murine models, we had found that a significant proportion of pathogenic serum and kidney-deposited Igs are�-actinin reactive. Furthermore, a pathogenic anti-DNA/�-actinin Ab showed enhanced binding to immortalized mesangial cells(MCs) derived from a lupus prone MRL-lpr/lpr mouse as compared with MCs from BALB/c mice which are not susceptible tospontaneous lupus, suggesting that kidney �-actinin expression may be contributing to nephritis. In the current study, we estab-lished that two isoforms of �-actinin that are present in the kidney, �-actinin 1 and �-actinin 4, can both be targeted byanti-�-actinin Abs. We found novel sequence polymorphisms between MRL-lpr/lpr and BALB/c in the gene for �-actinin 4.Moreover, �-actinin 4 and a splice variant of �-actinin 1 were both expressed at significantly higher levels (mRNA and protein)in MCs from the lupus prone MRL-lpr/lpr strain. Significantly, we were able to confirm these differences in intact kidney byexamining glomerular Ig deposition of anti-�-actinin Abs. We conclude that enhanced �-actinin expression may determine theextent of Ig deposition in the Ab-mediated kidney disease in lupus. Modulation of Ag expression may be a promising approachto down-regulate immune complex formation in the target organ in individuals with circulating pathogenic Abs. The Journal ofImmunology, 2006, 176: 7704–7714.

B cells play a pivotal role in the pathogenesis of systemiclupus erythematosus (SLE),3 and in particular in lupusnephritis. Although cytokine secretion and Ag presenta-

tion by B cells (among other immunological functions) may alsohave contributing roles (1–3), production of anti-dsDNA Abs is ofparticular importance in the pathogenesis of lupus-related kidneydisease in mouse models and in human patients (4, 5). The mech-anism by which anti-dsDNA Abs contribute to renal disease, how-ever, has yet to be satisfactorily resolved. Nevertheless, evidencedoes show that binding of anti-DNA Abs to glomerular compo-nents, either indirectly (via an Ag bridge of nuclear material) or viadirect cross-reactivity with renal Ags is an important determinantof Ab pathogenicity (6).

The nature of the cross-reactive renal Ag bound by anti-dsDNAAbs has long been an area of study and speculation. GlomerularAgs reported to be targets for the pathogenic anti-dsDNA Ab re-

sponse include laminin, heparan sulfate, fibronectin, and collagenIV (7, 8), although the specificity for many of the autoantibodiesthat are actually deposited in human lupus kidneys in vivo is notknown (9). Mostoslavsky et al. (10), and subsequently we (11),had identified �-actinin as a major target of the murine anti-dsDNA response in SLE. We found high titers of anti-�-actininAbs in the serum of lupus mice and in kidney eluates from lupusmice with active renal disease. Recently, we have extended thesefindings to human SLE. In collaborative studies with Mason et al.(12), we reported that human anti-DNA Abs bind to �-actinin, andthat a human anti-dsDNA/anti-�-actinin Ab is pathogenic and in-duces renal damage when administered in vivo (12, 13).

One requirement for induction of certain autoimmune diseases(as opposed to nonpathologic autoimmunity) is appropriate Ag ex-pression in the target organ (14). Previously, we demonstrated thata pathogenic murine anti-dsDNA/anti-�-actinin Ab binds prefer-entially to immortalized mesangial cells (MC) derived from thelupus prone MRL-lpr/lpr (MRL/lpr) mouse, as compared with itsbinding to immortalized MC derived from the nonautoimmuneBALB/c strain (11), suggesting a possible role for genetically de-termined differences in target Ag expression contributing to dis-ease susceptibility. In this study, we set out to test the hypothesisthat up-regulation of �-actinin in MRL/lpr MCs determines thesusceptibility for binding of nephritogenic anti-�-actininautoantibodies.

Materials and MethodsPrimary MC culture

Primary MCs were generated as follows: kidneys from 6-wk-old mice werediced in cold PBS, and passed through a series of progressively smallerstainless steel sieves (180, 100, and 71 �m). The resultant suspension wascentrifuged at 2300 rpm for 5 min, and the supernatant was discarded. Theglomeruli were digested with a 100 �g/ml solution of collagenase IV-S(Sigma-Aldrich) diluted in Leffert’s buffer for 30 min with gentle vortexingevery 10 min, then spun down, and washed twice in DMEM with 20% FCS

*The Irving and Ruth Claremon Research Laboratory, Division of Rheumatology,Albert Einstein College of Medicine, Bronx, NY 10461; †Department of Microbiol-ogy and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; ‡De-partment of Cell Biology and §Department of Medicine, Albert Einstein College ofMedicine, Bronx, NY 10461; and ¶Department of Molecular Genetics, Albert Ein-stein College of Medicine, Bronx, NY 10461

Received for publication January 5, 2006. Accepted for publication March 31, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Work in the laboratory of C.P. was supported by National Institutes of Health GrantsRO1-AR-48692 and PO1-AI-51392. B.D. was supported by a Predoctoral TrainingGrant from the National Institutes of Health.2 Address correspondence and reprint requests to Dr. Chaim Putterman, Division ofRheumatology, Forchheimer 701N, Albert Einstein College of Medicine, 1300 MorrisPark Avenue, Bronx, NY 10461. E-mail address: putterma@aecom.yu.edu3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; MC, mesan-gial cell; EST, expressed sequence tag; RT, room temperature; PI, propidium iodide;PVDF, polyvinylidene difluoride; FSGS, focal and segmental glomerulosclerosis;TBM, tubular basement membrane; CCNI, cyclin I.

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(HyClone) (20% DMEM). The enriched glomerular fraction was resus-pended in 20% DMEM in a cell culture flask, and left undisturbed for 3days. The primary culture was split after 10–14 days when the MCs out-grew the other cell types. After the 4th passage, the medium was changedto D-valine DME (US Biological) with 10% FCS. The cells were used forexperiments between the 10th and 15th passage.

Polyclonal and mAbs

Isoform specific anti-�-actinin 4 antisera was generated as described pre-viously (15). Briefly, a peptide specific for mouse �-actinin 4 (YYDSHNVNTRCQKICDQWDNLGS) was synthesized and conjugated to keyholelimpet hemocyanin, and rabbit antisera generated by a standard immuni-zation protocol (Southern Biotechnology Associates). Rabbit preimmunesera was used as a control. Isoform specific sera against �-actinin 1, 2, and3 were a gift from Dr. A. H. Beggs (Harvard Medical School,Boston, MA).

The BM75.2 Ab is a monoclonal mouse IgM anti-�-actinin Ab (whichdoes not bind dsDNA), while the TEPC183 mAb was used as an isotype-matched control (both from Sigma-Aldrich). The Lu-632 anti-�-actinin 4mAb (IgM) was a gift from Drs. T. Yamada and S. Hirohashi (NationalCancer Center, Tokyo, Japan) (16). The MOPC141 mAb (Sigma-Aldrich)was used as an isotype control for the test R4A (IgG2b) Ab. A rabbitpolyclonal anti-tubulin Ab preparation (Sigma-Aldrich) was used as a load-ing control for Western blotting.

Cloning of mouse �-actinin 4 and 1 genes

Total RNA isolated from BALB/c and MRL/lpr MC was reverse tran-scribed using a mouse �-actinin 4 or 1 specific 3� primer and the Super-script II first-strand synthesis system for RT-PCR (Invitrogen Life Tech-nologies).�-Actinin 4. BALB/c and MRL/lpr ACTN4 were amplified with the longtemplate PCR system (Roche Applied Sciences) using the following PCRconditions: 1) 94°C for 2 min; 2) 10 cycles of 94°C for 10 s, 58°C for 30 s,and 68°C for 2 min; 3) 25 cycles of 94°C for 10 s, 58°C for 30 s, and 68°Cfor 2 min and an additional 10 s for each next cycle; and 4) extension at68°C for 7 min. Purified PCR products were digested by XhoI and HindIII,and cloned into the pCMV-tag2B vector (Stratagene). DH5� competentcells (Invitrogen Life Technologies) were transformed, and grew on solidagar plates containing 100 �g/ml ampicillin. Plasmids containing the cor-rect insert were identified by XhoI and HindIII digestion, and sequenced asdetailed below. The primers for mouse �-actinin 4 were: forward, 5�-AGATATAAGCTTATGGTGGACTACCACGCAG-3�; reverse, 5�-AGATATCTCGAGTCACAGGTCGCTCTCCCCA-3�.�-Actinin 1. BALB/c and MRL/lpr ACTN1 were amplified using the Fail-safe PCR system (Epicentre), and the following PCR conditions: 1) 94°Cfor 2 min; 2) 30 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 2 min,30 s; and 3) extension at 72°C for 7 min. Purified PCR products werecloned directly into the pCRII vector using the TA cloning kit (InvitrogenLife Technologies). Recombinant pCRII plasmids were digested by XhoIand KpnI, and the ACTN1 cDNA insert ligated into the pRSETA vector(Invitrogen Life Technologies). The primers for mouse �-actinin 1 were:forward, 5�-AGAACACTCGAGATGGACCATTATGATTCCC-3�; re-verse, 5�-AATTAAGGTACCTTAGAGGTCGCTCTCGC-3�.

DNA sequence analysis

PCR products and plasmids were sequenced by standard dideoxy nucleo-tide sequencing. For each gene, seven sequencing primers were designedbased on the DNA sequence from the National Center for BiotechnologyInformation (NCBI) database. Only the first 400 bases were analyzed foreach primer pair. For sequence analysis, the DNA sequences were com-pared using the basic local alignment search tool (BLAST) at the NCBIwebsite (�www.ncbi.nlm.nih.gov/BLAST/�). The BLAST-like alignmenttool (BLAT) (�http://genome.ucsc.edu/�) was used to map the two differentACTN1 cDNA sequences to the mouse, human, and rat genomes, determinewhether they were represented in mRNA and expressed sequence tag(EST) clones, and check their conservation across species.

Transfection of HEK293 cells

HEK293 cells were cultured in 10% DMEM, and plated at 5 � 105 cells/10-cm plate the day before transfection. On the day of transfection, cellswere washed once with OPTI-MEM (Invitrogen Life Technologies) me-dium without serum. For each plate, 24 �g of plasmid and 75 �l of Lipo-fectAMINE 2000 (Invitrogen Life Technologies) were each diluted in 1.5ml of OPTI-MEM medium without serum, combined, and incubated atroom temperature (RT) for 20 min. The DNA-LipofectAMINE mixturewas added directly to each plate with gentle rocking, and the plates were

incubated at 37°C overnight. The following day the medium was replacedwith 10% DMEM containing 800 �g/ml geneticin (Invitrogen Life Tech-nologies). Transfected cells were cultured in selection medium for 2–3 wk,and the cells were cloned by limiting dilution. Selected clones were con-firmed by Western blot using the anti-FLAG M2 Ab (Sigma-Aldrich) andBM75.2.

Purification of mouse �-actinin 4 and 1 proteins

BALB/c and MRL/lpr actinin-4 HEK 293-transfected cells were grown intissue culture plates until confluence. The medium was aspirated, and 1 mlof sonication buffer (PBS/0.1% Triton X-100, with protease inhibitors) wasadded to each plate. The cells were harvested with a cell scraper, sonicatedon ice, and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatantwas transferred to a new tube containing anti-FLAG M2 gel pretreated withglycine HCl buffer, and incubated at 4°C with gentle shaking for 3–4 h.The loaded gel was transferred to a 20-ml poly-prep chromatography col-umn (Bio-Rad), washed with large amounts of PBS containing 0.1% TritonX-100, and eluted with 0.1 M glycine buffer (pH 2.5). The elution productwas immediately neutralized with 1 M Tris base, and dialyzed in PBS at4°C overnight. Purified �-actinin 4 was identified by SDS-PAGE andWestern blot and the concentration determined by the Protein Assay kit(Bio-Rad).

�-Actinin 1 was purified from Escherichia coli strain BL21(DE3)plysEcontaining the �-actinin 1 pRSETA expression plasmid, using the Ni-NTApurification system for polyhistidine-containing recombinant proteins (In-vitrogen Life Technologies).

ELISAs

For the �-actinin 4 ELISA, Ab at a concentration of 10 �g/ml in PBS wascoated onto Immulon II plates (Dynex Technologies) at 4°C overnight.Plates were blocked with 3% FCS for 1 h at 37°C, washed, and incubatedwith a serial dilution of purified �-actinin 4 for 1 h at 37°C. The plates werewashed, and a 1/4000 dilution of HRP-linked anti-FLAG M2 Ab (Sigma-Aldrich) in PBS was added for 1 h at 37°C, followed by a peroxidasesubstrate solution (KPL) for 30 min at 37°C.

A direct ELISA method was used for measuring Ab binding to �-actinin1, as follows: �-actinin 1 at a concentration of 5 �g/ml in PBS was coatedonto Immulon II plates at 4°C overnight. Plates were blocked with 3% FCSfor 1 h at 37°C, washed, and incubated with serial dilutions of Ab or serumfor 1 h at 37°C. The plates were washed, and a 1/4000 dilution of HRP-linked secondary Ab (Southern Biotechnology Associates) in PBS wasadded for 1 h at 37°C, followed by peroxidase substrate solution for 30 minat 37°C.

Immunofluorescence and immunohistochemistry

MCs and transfected HEK293 cells were grown on tissue culture-treatedcoverslips, fixed with cold acetone, and blocked with 1% BSA/PBS con-taining anti-mouse CD16/32 (BD Biosciences) at a 1/100 dilution (to blockFcRs) for 1 h at RT. The cells were then incubated with anti-FLAG Ab (10�g/ml), R4A (20 �g/ml), BM75.2 (10 �g/ml), or isotype-matched Absdiluted in block at the appropriate concentrations for 1 h at 4°C. The cellswere rinsed and washed three times with PBS. Fluorochrome-conjugatedsecondary Ab (Southern Biotechnology Associates) was applied at 1/200and the slides were incubated for 45 min in the dark at 4°C. Cells werestained with propidium iodide (PI) 1 �g/ml for 5 min, washed several timeswith PBS, and fixed by 1% paraformaldehyde in PBS. The slides wereobserved by fluorescence microscopy and a Bio-Rad Radiance 2000 scan-ning confocal microscope with a Kr/Ar laser for excitation at 488 and 568nm with Nikon 60� NA 1.4 Planapo Infinity Corrected Optics.

For immunohistochemical staining of kidney, cryostat sections wereprepared from 5-mo-old MRL/lpr and BALB/c mice, and stained withpolyclonal rabbit anti-mouse �-actinin 1 and 4 Abs at a dilution of 1/500at 4°C overnight. The staining was then continued as described (13).

Western blot

MCs were detached from tissue culture plates, washed, pelleted, and re-suspended to 2 � 108 cells/ml in radioimmunoprecipitation assay 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) for 25 minon ice. The cell lysis mixture was centrifuged at 14,000 rpm for 20 min at4°C. The supernatant was removed, aliquoted, and kept at �70°C untilused. Protein concentration was assayed by spectrophotometry, using theBio-Rad Protein Assay kit. For Western blotting, 25 �g of protein lysatesor purified BALB/c and MRL/lpr �-actinin 1 and 4 were combined withreducing sample buffer and heated at 100°C for 5 min. Samples were

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loaded into 4–15% gradient polyacrylamide gels (Bio-Rad), and electro-phoresed at 200 V for 30 min. Proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane using a Mini Protean 3 cell apparatus (Bio-Rad) at 70 V for 1.5 h. The membrane was blocked in 1% casein (PierceBiotechnology) or 5% nonfat milk in PBS and incubated with primary Abfor 1 h at RT. The membrane was repeatedly washed with PBS-Tween, andincubated with the appropriate HRP-conjugated secondary Abs diluted1/10,000 for 1 h at RT. The membrane was developed with the ECL Pluskit, and exposed to Hyperfilm (Amersham Biosciences). Loading of equiv-alent amounts of protein and adequate membrane transfer were confirmedby staining the PVDF membrane with Ponceau Red (Sigma-Aldrich), andby Western blotting with anti-tubulin Abs (Sigma-Aldrich) in parallel tothe test Abs.

Real-time PCR

PCR primers were designed using PRIMER3 (�www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/primer3_www.cgi�), and published sequencedata from the Ensembl database (�www.ensembl.org/Mus_musculus/�). Atleast one intron was included to avoid genomic DNA amplification. Am-plicons ranged from 80 to 120 bp. Total RNA isolated from BALB/c andMRL/lpr MCs was reverse transcribed using oligo dT, and real-time PCRperformed in triplicate using the SYBR green master mix and the ABIPRISM 7900HT Sequence Detection System (Applied Biosystems), usingthe following conditions: 10 min at 95°C, and 45 cycles of 95°C for 10 s,60°C for 20 s, and 72°C for 30 s. Each real-time PCR (for �-actinin and thetwo control genes GAPDH and CCNI) was done in triplicate. The ratio ofexpression of BALB/c �-actinin to each of the control genes was arbitrarilydefined as 1 and compared with the ratio of expression of MRL/lpr �-ac-tinin to the same control genes. The depicted fold changes are a mean ofthese normalized expression ratios, obtained in three independent experi-ments. Values of p were calculated using the Student t test, and values of�0.05 were deemed significant.

The primers were as follows: GAPDH: forward, 5�-ACCCAGAAGACTGTGGATGG-3�; reverse, 5�-GGATGCAGGGATGATGTTCT-3�. cy-clin I (CCNI): forward, 5�-GAAATGGAGAAACTCATTCCTGATT-3�;reverse, 5�-CCCGACAGTGGATCAAC-3�. ACTN1 (nonspecific): for-ward, 5�-ACCCCAACCGCTTGGGGG-3�; reverse, 5�-CGGCGCAGCTCATCCTCT-3�. ACTN1A (specific): forward, 5�-GCATGATGGACACAGACGAT-3�; reverse, 5�-GAAGGCCTGGAACGTCACTA-3�. ACTN1(specific): forward, 5�-AGAGTTCAAAGCCTGCCTCA-3�; reverse,5�-GGTTGGGGTCTACAATGCTC-3�. ACTN2: forward, 5�-ACCCCAACGGACAAGGCA-3�; reverse, 5�-CGACGAAGCTCCTCTGCC-3�.ACTN4: forward, 5�-ATCCCAACCATAGTGGCC-3�; reverse, 5�-CTCCGCAGTTCCTCAGCA-3�.

ResultsBALB/c and MRL/lpr ACTN4 genes differ by several pointmutations

Binding of the pathogenic anti-dsDNA/anti-�-actinin Ab R4A toMRL/lpr MC is significantly increased as compared with its bind-ing to nonautoimmune BALB/c MC (11). Proteomic analysis ofthe MC component bound by R4A indicated that the closest fit ofthe analyzed peptides was to �-actinin 4. Accordingly, we startedour studies with this particular �-actinin isoform.

To detect whether the differential binding to BALB/c and MRL/lpr MC by R4A is due to polymorphisms in �-actinin 4 sequences,we sequenced the ACTN4 gene in BALB/c and MRL/lpr MC. TheMRL/lpr ACTN4 gene (912 aa) is completely identical to theC57BL/6 ACTN4 sequence reported in the NCBI database (Gen-Bank no. NM021895). Interestingly, C57BL/6 is one of the ances-tral strains from which the MRL background is derived (17).BALB/c ACTN4 encodes for 911 aa, and is missing a glycine atposition 20 relative to the MRL/lpr sequence. In addition, there areseveral point mutations in the BALB/c ACTN4 sequence, leadingto substitutions at positions 16, 23, 28, 439, 614, 704, and 733 ascompared with the MRL/lpr protein sequence (Fig. 1). The com-plete nucleotide sequences of BALB/c (no. DQ303410) and MRL/lpr (no. DQ303411) ACTN4 have been submitted to GenBank.

R4A binds transfected HEK293 cells through overexpressed�-actinin 4

We reasoned that the variations in linear sequence betweenBALB/c and MRL/lpr �-actinin 4 may lead to a different distri-bution or localization of cellular protein expression, thus poten-tially affecting accessibility to Ab binding. Therefore, we studiedprotein expression in BALB/c and MRL/lpr �-actinin 4-trans-fected HEK293 cells by staining with anti-FLAG and anti-�-actinin mAbs. BALB/c and MRL/lpr �-actinin 4-transfectedcells showed a strong fluorescent signal when stained by the anti-FLAG Ab, while cells alone and cells transfected with an emptyvector were negative (data not shown). There is no significant dis-tinction between the staining patterns of BALB/c and MRL/lpr�-actinin 4-transfected HEK293 cells by immunofluorescence(Fig. 2), leading to the conclusion that there is not likely to be amajor difference in the cellular distribution of BALB/c and MRL/lpr �-actinin 4, at least in this overexpression model. Furthermore,comparing the phase control and confocal images of anti-FLAG-,BM75.2-, and R4A-treated cells (with nuclei stained by PI), thestaining pattern in transfected cells is localized to the cell surface.This supports our previous observation (11), made originally byothers (10, 18), that besides its cytoskeletal location �-actinin isalso expressed on the cell surface.

R4A has similar affinity for BALB/c and MRL/lpr �-actinin 4

As BALB/c and MRL/lpr �-actinin 4 did not appear to differ intheir intracellular location, we wondered whether �-actinin Absbind with altered affinity to the different forms of �-actinin 4. Pu-rified recombinant BALB/c and MRL/lpr �-actinin 4 were �95%pure, when run on a SDS-PAGE gel and stained by Coomassieblue. By Western blot, there was no apparent difference in bindingof the BM75.2 anti-�-actinin mAb or of R4A to BALB/c or MRL/lpr �-actinin 4 (Fig. 3A).

We then confirmed the relative affinities of BM75.2 and R4A forrecombinant BALB/c and MRL/lpr �-actinin 4 by ELISA. As acontrol, we used anti-�-actinin 4 antisera, raised against an �-ac-tinin 4-specific peptide (position 486–508, an area of completeMRL and BALB/c ACTN4 sequence identity). Polyclonal serumwas identically bound by BALB/c and MRL/lpr �-actinin 4, con-firming that the same amount of �-actinin 4 was coated onto eachwell (Fig. 3B, right panel). We found that R4A and BM75.2 havesimilar affinity for both BALB/c and MRL/lpr �-actinin 4, despitetheir differences in ACTN4 primary sequence (Fig. 3B, left andmiddle panels).

Primary MRL/lpr MC cells have higher expression levels of�-actinin 1 and 4

As there were no apparent differences between the binding of R4Ato MRL/lpr and BALB/c �-actinin 4, we wondered whether thedifferential binding of R4A to MRL/lpr MCs is due to enhanced�-actinin expression. Proteomic analysis in our initial studies (11)had indicated that besides �-actinin 4, �-actinin 1 is also a possibletarget for R4A. Therefore, we studied the expression of both �-ac-tinin 4 and �-actinin 1 in primary BALB/c and MRL/lpr MC. First,we studied the mRNA expression levels of ACTN1 and ACTN4.We found by real-time PCR that ACTN1 and ACTN4 expression inprimary MRL/lpr MC is increased 4.8- and 2.2-fold, respectively,as compared with primary BALB/c MC ( p � 0.05 for each com-parison) (Fig. 4A). Expression of ACTN2 was not significantly dif-ferent. Next, the binding of R4A and BM75.2 to primary BALB/cand MRL/lpr MC lysates was assayed by Western blot. As weoriginally observed in immortalized cells, R4A (11) and BM75.2

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FIGURE 1. Sequence comparison between MRL/lpr and BALB/c ACTN4. The nucleotide and amino acid homology between MRL/lpr (top sequence)and BALB/c (bottom sequence) ACTN4 is shown. A vertical line denotes identity at a given position.

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FIGURE 3. Anti-�-actinin Abs have similar affinity to BALB/c and MRL/lpr �-actinin 4. A, Binding of BM75.2 and R4A to purified BALB/c andMRL/lpr �-actinin 4 by Western blot. Increasing amounts of purified �-actinin 4 (from left to right: 0.1, 0.5, 1.0, and 2.5 �g) were separated by SDS-PAGEand transferred to a PVDF membrane. The membranes were blotted with BM75.2 (1 �g/ml) or R4A (10 �g/ml) for 1 h, followed by the appropriatesecondary Abs at 1/10,000 for 1 h. B, Binding of R4A, BM75.2, and polyclonal anti-�-actinin 4 to purified BALB/c and MRL/lpr �-actinin 4 by ELISA.Left and middle panel, R4A (left panel) or BM75.2 (middle panel) at 10 �g/ml in PBS were coated onto Immulon II plates at 4°C overnight. After a blockingstep, the plates were incubated with a serial dilution of purified �-actinin 4 for 1 h at 37°C, followed by HRP-linked anti-FLAG Ab. MOPC141 (IgG2b)and TEPC183 (IgM) were used as isotype-matched controls for R4A and BM75.2, respectively. Right panel, Purified �-actinin 4 at 5 �g/ml was coatedonto Immulon II plates at 4°C overnight. The plate was then incubated with a serial dilution of polyclonal rabbit anti-�-actinin 4 serum for 1 h at 37°C,followed by a 1/4,000 dilution of HRP-linked anti-rabbit IgG. Preimmune rabbit sera (“control serum”) was used as the control in this experiment.

FIGURE 2. Anti-�-actinin Abs bind to transfected HEK293 cells. �-Actinin 4-transfected HEK293 cells were fixed by cold acetone, and blocked by 1%BSA/PBS containing anti-mouse CD16/32 (1/100) for 1 h at RT. The cells were then incubated with anti-FLAG Ab (10 �g/ml), BM75.2 (10 �g/ml), orR4A (20 �g/ml) for 1 h at 4°C. The appropriate fluorochrome-conjugated secondary Abs were applied at 1/200, and the slides were incubated for 45 minin the dark at 4°C. Coverslips were mounted following 5 min of staining with 1 �g/ml PI. Top row, PI staining (in red); middle row, Ab staining (in green);bottom row, merged image. The cells were also stained with isotype-matched control Abs, but no staining was detected (data not shown).

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each showed significantly increased binding to the 100-kDa �-ac-tinin band in primary MRL/lpr MC lysates (Fig. 4B). In confor-mity with the real-time PCR results, Western blot of total celllysates with isoform-specific antisera showed that primary cellsfrom both MRL/lpr and BALB/c express �-actinin 1, 2, and 4 butnot 3. Moreover, �-actinin 1 and 4 have up-regulated expression inMRL/lpr MC, while there were no significant differences in theexpression level of �-actinin 2 between these two cell lines (Fig.4B). �-Actinin 3, an isoform limited to muscle, is not found inBALB/c or MRL/lpr MC (Fig. 4, B and C).

BALB/c and MRL/lpr MCs contain two distinct �-actinin 1isoforms

Although we found that �-actinin 1 is up-regulated in primaryMRL/lpr MC, we wanted to confirm that R4A binds to �-actinin1 as well as �-actinin 4, and determine whether there are anysequence polymorphisms in this gene between MRL/lpr andBALB/c. Surprisingly, we were able to clone two distinct �-actinin1 sequences from BALB/c and MRL/lpr MCs, ACTN1 andACTN1A.

We found that BALB/c and MRL/lpr ACTN1 sequences wereidentical to each other and to the ACTN1 sequence available fromGenBank (no. AF115386). Similarly, BALB/c and MRL/lprACTN1A sequences were identical (GenBank no. DQ288940). TheACTN1 sequence contains an 81-bp fragment (position2506–2586) that is replaced by a 66-bp sequence in the ACTN1AcDNA (2506–2571) (Fig. 5A). Otherwise, the encoded proteins areidentical, with no change in frame. Sequence analysis revealed thatthe 81-bp fragment is present in the majority of mRNA and ESTclone sequences in both the human and mouse databases. We de-termined that the 66-bp fragment represents an alternativelyspliced exon because it is present in several mRNA and ESTclones, maintains the open reading frame (22 aa replace 27 aa) andis also highly conserved across species. Judging from the cDNA

libraries from which the mRNA and EST clones were isolated, theexpression of the 66-bp fragment is more limited. No clones ex-isted in the human or mouse databases that contain both the81- and 66-bp sequences; however, these two exons were foundtogether in a brain-specific transcript from rat (GenBank no.CO399752). By real-time PCR, we found that significantly in-creased transcription of ACTN1A, but not ACTN1, in MRL/lpr MCwas the likely cause for the difference in �-actinin 1 protein ex-pression between MRL/lpr and BALB/c cells (Fig. 5B).

To study the relative affinity of anti-�-actinin Abs for theACTN1 and ACTN1A gene products, both genes were cloned intothe pRESET A vector, expressed, and purified. RecombinantBALB/c and MRL/lpr �-actinin 1 proteins were run by SDS-PAGE followed by staining with Coomassie bright blue (whichindicated �95% purity), and their identify confirmed by Westernblot using anti-HisG Ab (Invitrogen Life Technologies) and rabbitanti-�-actinin 1 anti-serum. The binding affinity of anti-�-actininAbs to these �-actinin 1 isoforms was detected by Western blotand ELISA. We found that R4A and BM75.2 each binds to both�-actinin 1 variants (Fig. 6A). Furthermore, no differences wereobserved in the affinity of these Abs to �-actinin 1 and �-actinin1A (Fig. 6B).

We next investigated the relative affinity of R4A to �-actinin 4and �-actinin 1 by ELISA, using direct binding and inhibitionassays. Although this conclusion is not irrefutable due to the dif-ferent methods we had to use to express these two �-actinin iso-forms, R4A had significantly higher affinity for �-actinin 1 (datanot shown).

Abs to �-actinin show stronger binding to primary MRL/lpr ascompared with primary BALB/c MCs

Primary BALB/c and MRL/lpr MCs growing on tissue cultureslides were fixed and stained by BM75.2, R4A, or isotype controls,

FIGURE 4. Primary MRL/lpr MC display higher expression levels of �-actinin 1 and 4 than primary BALB/c MC. A, Real-time PCR analysis of ACTN1,ACTN2, and ACTN4 gene expression in primary BALB/c and MRL/lpr MC. Each real-time PCR sample was done in triplicate, and the mean of the threerepeats used as the result for that sample. Shown here are the mean � SD values for fold change when each sample was separately normalized to GAPDHand CCNI. ACTN1 and ACTN4, p � 0.01; ACTN2, p � 0.05. B, Western blotting of primary MC lysates. Twenty-five micrograms of primary cell lysateswere separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were stained by polyclonal rabbit anti-�-actinin 1, 2, 3, 4, and controlserum (all at 1/1000), and the monoclonal anti-�-actinin Abs Lu632 (at 5 �g/ml), BM75.2 (1 �g/ml), and R4A (5 �g/ml). Staining with polyclonalanti-tubulin Abs (1/1000) was used as a loading control. C, Muscle lysates from BALB/c and MRL/lpr mice served as a positive control for theanti-�-actinin 3 serum, and showed no difference in �-actinin 3 expression. The data displayed in this figure are representative of two (protein expression)or three (real-time PCR) independent experiments.

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followed by a secondary fluorescent Ab and PI staining. The flu-orescence signal from stained primary MRL/lpr MC was muchstronger than that from primary BALB/c MC, consistent with ourfindings of higher expression levels of �-actinin 1 and 4 in primaryMRL/lpr MC. Comparing the images of phase control and PI stain-ing, the binding of BM75.2 showed a cytoskeletal pattern, whileR4A demonstrated both cytoplasmic and nuclear binding (Fig. 7),the latter likely due to its antinuclear specificity.

Anti-�-actinin Abs are present in high titers in MRL/lpr miceand preferentially bind to MRL/lpr kidneys

Previously, we had determined that lupus prone mice display highserum titers of anti-�-actinin Abs (11). To determine whether anyparticular isoform is targeted in this autoantibody response, weexamined the titers of isoform-specific anti-�-actinin Abs in old (5mo of age) MRL/lpr mice. Fig. 8A demonstrates that diseased micehave significantly higher Ab titers against �-actinin 1, 1A, and 4,as compared with age-matched BALB/c mice. Although the resultsmay not be directly comparable due to technical differences in themethod of expression, there was no significant difference betweenthe binding of MRL/lpr sera to purified �-actinin 1 and 4 (Fig. 8Aand data not shown).

To confirm that the observed differences in �-actinin expressionin vitro between MRL/lpr and BALB/c MCs described above werealso reflected in the kidney in vivo, we stained kidney sectionswith polyclonal anti-�-actinin Abs. We found significantly stron-ger staining of MRL/lpr glomeruli by these Abs, supporting the

hypothesis that higher �-actinin expression is responsible forgreater Ag availability for binding by anti-�-actinin Abs (Fig. 8B).

DiscussionCross-reactivity of anti-dsDNA Abs with kidney Ags is an impor-tant determinant in their renal pathogenicity. Previously, we hadfound that �-actinin is a target Ag for pathogenic murine and hu-man anti-DNA Abs in MC (11). Furthermore, we had demon-strated that an immortalized MC line derived from the lupus-proneMRL/lpr mouse strain shows enhanced binding to a pathogenic,cross-reactive, anti-dsDNA/anti-�-actinin Ab as compared with aBALB/c-derived cell line. Moreover, human anti-dsDNA Abscross-react with �-actinin, and autoantibodies of this specificity areassociated with lupus nephritis (12, 13). In the present study, weconclusively show that both �-actinin 4 and �-actinin 1 are po-tential targets for anti-�-actinin Abs. Moreover, primary MCsfrom the lupus prone MRL/lpr mouse strain overexpress �-actinin4 as well as �-actinin 1, indicating that increased �-actinin ex-pression may contribute to the susceptibility to the severe Ab-mediated nephritis seen in the MRL/lpr mouse strain. Support forthis latter hypothesis can be found in our findings that MRL/lprmice display high circulating autoantibody titers against kidney-expressed �-actinin isoforms, and that anti-�-actinin antiserumdifferentially bound not only to isolated MRL/lpr MCs, but to in-tact kidney sections as well.

�-Actinin belongs to a family of actin-binding proteins that in-cludes spectrin, filamin, fimbrin, and dystrophin (11, 16). There arefour known mammalian �-actinin genes, ACTN1-ACTN4, whichencode homologous 100-kDa proteins that are organized as anti-parallel homodimers with an actin-binding domain at each end (11,19). �-Actinin 2 and 3 are mostly expressed in skeletal muscle(15), but �-actinin 2 is also expressed in MCs (20). �-Actinin 1and 4 are more widely expressed, and are also present in the kid-ney. The major location of �-actinin, as an important cytoskeletalcomponent, is intracellular. It has already been known for sometime that SLE patients display Abs to cytoskeletal proteins (21, 22)and microfilaments (23), including Abs to �-actinin. Nevertheless,here we confirm earlier observations (10, 11, 18) that �-actinin isalso present on the cell surface, where it is accessible for Ab bind-ing in vivo.

There has been increasing attention to the role of �-actinin inrenal physiology since the report by Kaplan et al. (24) in 2000showing that humans with familial focal and segmental glomeru-losclerosis (FSGS), a cause of nephrotic syndrome and renal fail-ure, have mutations in ACTN4. Subsequently, direct evidence fora cause and effect relationship was found in the report that micewith kidney-specific transgenic expression of an �-actinin 4 con-taining a mutation similar to one present in humans with FSGSdeveloped hypertension, proteinuria, and histologic features ofFSGS (25). Mutant �-actinin 4 was shown to form intracellularaggregates, and to undergo rapid degradation mediated in part bythe ubiquitin pathway (26). This suggested two nonmutually ex-clusive models to explain disease in individuals with mutant �-ac-tinin 4. Aggregated �-actinin 4 may be toxic to kidney cells (26).Alternatively, accelerated metabolism of mutated �-actinin 4 (25)or abnormally high affinity for F-actin (25) may lead to abnormalfunction or loss of normal function. Interestingly, not only do miceharboring mutant �-actinin develop renal pathology, but mice de-ficient in �-actinin 4 also display proteinuria and glomerular dis-ease, and die within several months from renal failure (20). Thislatter study indicates that despite the similarities in structureand function between �-actinin 4 and �-actinin 1, these proteinsare not redundant. Although we have not yet shown this di-rectly, it is interesting to speculate that binding of pathogenic

FIGURE 5. Two �-actinin 1 splice variants are present in MRL/lpr andBALB/c MCs. A, Splice donor/acceptor sites found in the ACTN1 se-quence. Exonic and intronic sequences in the table are denoted in uppercase and lower case letters, respectively. The splice acceptor sites are de-noted in bold, and the splice donor sites in bold and italic. Other than thesplice variant, the other cDNA regions (226–2505 in ACTN1 and ACTN1A,and 2587–2904 and 2572–2889 in ACTN1 and ACTN1A, respectively)show full sequence identity between the two isoforms. Exon 21 is repre-sented in white for illustrative purposes only. B, Primary MRL/lpr MCshave higher expression of ACTN1A than primary BALB/c MC. Real-timePCR analysis of the relative expression level of ACTN1 and ACTN1A inprimary BALB/c and MRL/lpr MC. GAPDH and CCNI were used as con-trol genes. ACTN1, p � 0.05; ACTN1A, p � 0.01. The real-time PCR datadisplayed in this figure are representative of three independentexperiments.

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Abs to �-actinin may lead to similar consequences as the pres-ence of abnormal �-actinin, that is impaired function of �-ac-tinin and/or interference with the interaction of �-actinin withother cytoskeletal proteins.

The precise role of �-actinin in glomerular function is not com-pletely understood. �-Actinin, together with other contractile pro-teins in the kidney, may function to counteract pressure gradientsacross the capillary wall (27). Alternatively, �-actinin may influ-ence the renal vascular cytoskeleton or be involved in protein fil-tering (24). �-Actinin plays a role in cell motility and adherence(16, 20) and has been postulated to function as a tumor suppressorgene by impacting normal cell growth regulation and differentia-tion (28). Most recently, it has been shown that �-actinin colocal-izes with polycystin-2, the product of the PKD2 gene which ismutated in some patients with familial polycystic kidney disease.Moreover, it was found that �-actinin modulates polycystin-2 andenhances its channel activity (29). This finding is in agreementwith previous reports that �-actinin regulates the activity of severaltypes of channels (30, 31) and supports this potentially importantaspect of �-actinin function.

There is data for several autoimmune diseases to suggest thatinherent susceptibility to autoantibody-mediated disease is depen-dent on genetically determined differences in autoantigen displayin the target organs. As previously reviewed (11), BN rats immu-nized with heterologous tubular basement membrane (TBM) de-

velop autoimmune tubulointerstitial nephritis with anti-TBM Abs;Lewis rats also develop an anti-TBM response to immunization,with circulating Abs, but do not develop disease (32, 33). It ap-pears that Lewis rats are resistant to disease because they lack theTBM Ag which is targeted by pathogenic anti-TBM Abs. In adifferent model of experimental nephritis, Xie et al. (34, 35) hasrecently elegantly demonstrated that there is a broad range of sus-ceptibility (from resistant to highly vulnerable) of mouse strains topreformed pathogenic anti-glomerular basement membrane Abs inthe nephrotoxic serum model of renal disease (Masugi nephritis).Finally, specifically for murine lupus, Fas deficiency in the MRLbackground leads to a severe, full-blown lupus syndrome, whilethis mutation in the C3H background induces anti-DNA Abs butonly mild renal disease (36). These studies demonstrate that forsome autoimmune nephritides (including lupus), pathogenic Absare necessary, but may not be sufficient; a genetically determinedlevel of expression of a target Ag can be a critical factor in deter-mining susceptibility to disease. Lupus patients with persistentlyhigh levels of anti-DNA Abs who nevertheless do not developnephritis (37) may be an example of this principle as well. Al-though the actual loci that directly impact the expression of renaldisease are not currently known, this is an area of great interestamong investigators dealing with genetic dissection of murine lu-pus models.

FIGURE 6. Anti-�-actinin Abs bind with similar affinity to purified �-actinin 1 and �-actinin 1A. A, Binding to �-actinin 1 and �-actinin 1A by Westernblot. Increasing amounts of purified �-actinin 1 and �-actinin 1A (from left to right: 0.1, 0.5, 1.0, and 2.5 �g) were separated by SDS-PAGE and transferredto a PVDF membrane. The membranes were incubated with HRP-labeled anti-HisG Ab (1/10,000), polyclonal anti-�-actinin 1 anti-serum (1/1,000),BM75.2 (1 �g/ml), or R4A (10 �g/ml), followed by secondary Ab. Isotype control Abs showed no binding to either �-actinin 1 isoform. B, Binding to�-actinin 1 and �-actinin 1A by ELISA. Purified �-actinin 1 and �-actinin 1A at 5 �g/ml were coated onto Immulon II plates at 4°C overnight. The platewas then incubated with serial dilutions of R4A (20–0.3125 �g/ml; left panel), BM75.2 (10–0.0001 �g/ml; middle panel), or anti-�-actinin 1 serum (1/200to 1/12,800; right panel) for 1 h at 37°C, followed by a 1/4,000 dilution of HRP-linked secondary Abs.

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What may be the function of genes that contribute to the ex-pression of lupus nephritis? Xie et al. (35) propose that it is pos-sible that nephritis-facilitating genomes may encode for residentkidney cells which may be intrinsically hyperactive to immuno-logical insults (for example, through enhanced expression of thetarget Ag or the secretion of a particularly damaging cytokine pro-file). Alternatively, these disease-susceptible genomes may be en-hancing renal disease by influencing the function of those systemiccells that infiltrate the kidney during the inflammatory process. Ourpresent studies demonstrating significant differences in �-actininexpression in isolated MCs are supportive of an intrinsic renalcontribution to nephritis susceptibility.

Corticosteroids are one of the mainstays of the treatment oflupus nephritis. We previously reported that monoclonal mouseand polyclonal human anti-dsDNA Abs from lupus patients bind tothe cell surface of mouse embryonic stem cells. This cell surfacereceptor was markedly down-regulated by a brief pretreatment ofcells with dexamethasone, leading to a �50% decrease in Ab bind-ing (38). Based on these results, we had postulated that while cor-ticosteroids have many anti-inflammatory and immunosuppressiveeffects, modulation of target Ag can be invoked as an additionalexplanation for the therapeutic benefit of steroids in lupus nephri-tis. Consistent with these studies is the observation that cortico-steroid treatment of lupus mice significantly decreases kidney in-sulin-like growth factor-1 and fibroblast growth factor, which canboth up-regulate �-actinin expression (39, 40). The salutatory ef-fects of corticosteroids in lupus nephritis may then be partly at-tributed to their effects on modulation of Ag expression in thetarget organ, specifically �-actinin. Moreover, this also raises anintriguing possibility that cytokine levels may be therapeuticallymanipulated to modify target Ag expression. This hypothesis willbe examined directly. Finally, it is similarly possible to invokedifferences in the cytokine profile or responsiveness as a potentialexplanation for the variability in �-actinin expression betweenBALB/c- and MRL/lpr-derived cells.

We found increased expression of �-actinin in MRL/lpr MC (invitro) and kidney (in vivo). One possible way to explain theseobservations is that the increased expression may be a feature ofinflammation, rather than an endogenous characteristic of lupuskidney cells. Indeed, albeit in a different lupus background, Mos-toslavsky et al. (10) detected �-actinin expression by immunoflu-orescence in a 7-mo-old NZB � NZW F1 kidney, but not in thekidney of a young 3-mo-old NZB � NZW F1 mouse. Neverthe-less, �-actinin overexpression was seen in primary MCs whichfollowing derivation are no longer exposed to a proinflammatorymilieu. Moreover, the primary cells used in our experiments werederived from young 6-wk-old mice, an age where inflammation isless likely to have had a major influence. Therefore, we believethat the MRL lupus background is an important contributor toincreased �-actinin expression. Does differential �-actinin expres-sion distinguish between other nonautoimmune and autoimmunestrains, and whether cytokines/inflammation are contributing to

FIGURE 7. Abs to �-actinin show stronger binding to primary MRL/lpras compared with primary BALB/c MC. Confocal images of primary MCstained by BM75.2 and R4A. Primary MC were fixed by cold acetone andblocked with 1% BSA/PBS containing a 1/100 dilution of anti-mouseCD16/32 for 1 h at RT. Cells were then incubated with BM75.2 (10 �g/ml), R4A (20 �g/ml), or isotype control Abs for 1 h at 4°C. The appropriatefluorochrome-conjugated secondary Abs were applied at 1/200, and theslides were incubated for 45 min in the dark at 4°C. Coverslips weremounted following staining with 1 �g/ml PI for 5 min. Top, PI staining(red); middle, Ab staining (green); bottom, merged image. The data dis-played in this figure are representative of two independent experiments.

FIGURE 8. Anti-�-actinin Abs are present in high titers in MRL/lprmice and preferentially bind to MRL/lpr kidneys. A, Binding of MRL/lprserum to �-actinin isoforms by ELISA. Purified �-actinin 1, �-actinin 1A,and MRL/lpr �-actinin 4 at 5 �g/ml were coated onto Immulon II plates at4°C overnight, followed by a 1/2000 dilution of serum from 5-mo-oldfemale MRL/lpr mice (n 7) for 1 h at 37°C, and secondary Ab. Ascompared with age- and sex-matched BALB/c mice (n 7), MRL/lpr micedisplay significantly higher serum titers of Abs to �-actinin 1, 1A, and 4 byELISA (p � 0.0001). B, Anti-�-actinin Abs preferentially bind to MRL/lprkidneys. Cryostat sections from 5-mo-old MRL/lpr and BALB/c kidneyswere fixed by cold acetone and blocked. Sections were incubated with a1/500 dilution of anti-�-actinin 1, anti-�-actinin 4, and control (preim-mune) polyclonal antiserum at 4°C overnight. The HRP-linked anti-rabbitIgG secondary Ab was then applied at 1/500 and the sections incubatedwith substrate at RT for 30 min. Top row, Sections stained with polyclonalanti-�-actinin 1 Abs; bottom row, sections stained with polyclonal anti-�-actinin 4 Abs. Sections stained by preimmune sera were negative (data notshown). Arrows indicate stained glomeruli. All images in this figure are ata magnification of �50. The data displayed in this figure are representativeof two independent experiments.

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this as well and how they do so, are important questions that willbe addressed.

We should also point out that while �-actinin 4 was up-regu-lated in primary MRL/lpr MC, expression was similar followingoverexpression of MRL/lpr and BALB/c �-actinin 4 in transfectedcells. We believe this is related to the constructs used for the ex-periments, as the transfected �-actinin 4 constructs lacked both theendogenous promoter region as well as the 3� UTR from the re-spective strains. It is possible that variations (polymorphisms) inthese regions between the two strains could effect the in vivo ex-pression and/or stability of the message. This will be carefullyexamined in future studies.

We found several previously unknown sequence polymor-phisms between MRL/lpr and BALB/c ACTN4. AlthoughKaplan et al. (24) was able to show that the mutant �-actinin 4associated with familial FSGS binds F-actin more strongly thanthe wild-type protein and tends to aggregate in vivo, we werenot able to demonstrate meaningful differences in intracellularlocalization following transfection or in the affinity for �-acti-nin autoantibodies between MRL/lpr and BALB/c �-actinin 4.Similarly, while we were able to clone two alternatively splicedforms of ACTN1 in both MRL/lpr and BALB/c MCs, we werenot able to demonstrate any meaningful variation in bindingaffinity of anti-�-actinin Abs to the expressed proteins. Al-though we considered the possibility that MRL/lpr and BALB/c�-actinin have subtle changes in binding affinity to anti-�-actinin Abs, we believe that this is unlikely as no bindingalteration was found in either of the two methods (ELISA andWestern blot) that were used. �-Actinin 1 and 4 display somedifferences in cellular localization (�-actinin 1 is concentratedin focal adhesion plaques and adherens junctions, while �-ac-tinin 4 is not) and sensitivity to calcium (16, 41). It remains tobe seen whether the sequence polymorphisms we demonstratedhere between BALB/c and MRL/lpr ACTN4 and betweenACTN1 and ACTN1A will translate into measurable differencesin their biochemical function and/or immunogenicity.

In summary, we have shown that �-actinin 4 and �-actinin 1can both be targeted by a pathogenic anti-�-actinin response.Although some polymorphisms were found between MRL/lprand BALB/c �-actinin 4, differential binding to MRL/lpr �-ac-tinin was a result of enhanced protein expression, rather thanany detectable differences in Ab binding to strain-specific iso-forms. A novel splice variant of �-actinin 1 was detected inMCs, and this isoform also shows higher expression in MRL/lprmice. The underlying mechanisms for enhanced steady stateACTN1A and ACTN4 mRNA levels in MRL/lpr MC are notcurrently known, and will need to be formally addressed. Stud-ies to understand the contribution of organ-specific factors toAb-mediated nephritis in SLE should include the analysis ofpolymorphisms in target Ag structure and expression. Finally,modulation of target Ag expression by cytokines or other ther-apeutic modalities might be a possible new approach to thetreatment of Ab-mediated kidney disease.

DisclosuresThe authors have no financial conflict of interest.

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