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The Journal of Immunology, 2006, 176: 7704-7714.
Copyright © 2006 by The American Association of Immunologists

Differential Binding of Cross-Reactive Anti-DNA Antibodies to Mesangial Cells: The Role of {alpha}-Actinin1

Zeguo Zhao*, Bisram Deocharan{dagger}, Philipp E. Scherer{ddagger},§, Laurie J. Ozelius and Chaim Putterman2,*,{dagger}

* The Irving and Ruth Claremon Research Laboratory, Division of Rheumatology, Albert Einstein College of Medicine, Bronx, NY 10461; {dagger} Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; {ddagger} Department of Cell Biology and § Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461; and Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Target Ag display is a necessary requirement for the expression of certain immune-mediated kidney diseases. We previously had shown that anti-DNA Abs that cross-react with {alpha}-actinin may be important in the pathogenesis of murine and human lupus nephritis; in murine models, we had found that a significant proportion of pathogenic serum and kidney-deposited Igs are {alpha}-actinin reactive. Furthermore, a pathogenic anti-DNA/{alpha}-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 to spontaneous lupus, suggesting that kidney {alpha}-actinin expression may be contributing to nephritis. In the current study, we established that two isoforms of {alpha}-actinin that are present in the kidney, {alpha}-actinin 1 and {alpha}-actinin 4, can both be targeted by anti-{alpha}-actinin Abs. We found novel sequence polymorphisms between MRL-lpr/lpr and BALB/c in the gene for {alpha}-actinin 4. Moreover, {alpha}-actinin 4 and a splice variant of {alpha}-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 by examining glomerular Ig deposition of anti-{alpha}-actinin Abs. We conclude that enhanced {alpha}-actinin expression may determine the extent of Ig deposition in the Ab-mediated kidney disease in lupus. Modulation of Ag expression may be a promising approach to down-regulate immune complex formation in the target organ in individuals with circulating pathogenic Abs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B cells play a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE),3 and in particular in lupus nephritis. Although cytokine secretion and Ag presentation by B cells (among other immunological functions) may also have contributing roles (1, 2, 3), production of anti-dsDNA Abs is of particular importance in the pathogenesis of lupus-related kidney disease in mouse models and in human patients (4, 5). The mechanism by which anti-dsDNA Abs contribute to renal disease, however, has yet to be satisfactorily resolved. Nevertheless, evidence does show that binding of anti-DNA Abs to glomerular components, either indirectly (via an Ag bridge of nuclear material) or via direct cross-reactivity with renal Ags is an important determinant of Ab pathogenicity (6).

The nature of the cross-reactive renal Ag bound by anti-dsDNA Abs has long been an area of study and speculation. Glomerular Ags reported to be targets for the pathogenic anti-dsDNA Ab response include laminin, heparan sulfate, fibronectin, and collagen IV (7, 8), although the specificity for many of the autoantibodies that are actually deposited in human lupus kidneys in vivo is not known (9). Mostoslavsky et al. (10), and subsequently we (11), had identified {alpha}-actinin as a major target of the murine anti-dsDNA response in SLE. We found high titers of anti-{alpha}-actinin Abs in the serum of lupus mice and in kidney eluates from lupus mice with active renal disease. Recently, we have extended these findings to human SLE. In collaborative studies with Mason et al. (12), we reported that human anti-DNA Abs bind to {alpha}-actinin, and that a human anti-dsDNA/anti-{alpha}-actinin Ab is pathogenic and induces renal damage when administered in vivo (12, 13).

One requirement for induction of certain autoimmune diseases (as opposed to nonpathologic autoimmunity) is appropriate Ag expression in the target organ (14). Previously, we demonstrated that a pathogenic murine anti-dsDNA/anti-{alpha}-actinin Ab binds preferentially to immortalized mesangial cells (MC) derived from the lupus prone MRL-lpr/lpr (MRL/lpr) mouse, as compared with its binding to immortalized MC derived from the nonautoimmune BALB/c strain (11), suggesting a possible role for genetically determined differences in target Ag expression contributing to disease susceptibility. In this study, we set out to test the hypothesis that up-regulation of {alpha}-actinin in MRL/lpr MCs determines the susceptibility for binding of nephritogenic anti-{alpha}-actinin autoantibodies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Primary MC culture

Primary MCs were generated as follows: kidneys from 6-wk-old mice were diced in cold PBS, and passed through a series of progressively smaller stainless steel sieves (180, 100, and 71 µm). The resultant suspension was centrifuged at 2300 rpm for 5 min, and the supernatant was discarded. The glomeruli were digested with a 100 µg/ml solution of collagenase IV-S (Sigma-Aldrich) diluted in Leffert’s buffer for 30 min with gentle vortexing every 10 min, then spun down, and washed twice in DMEM with 20% FCS (HyClone) (20% DMEM). The enriched glomerular fraction was resuspended in 20% DMEM in a cell culture flask, and left undisturbed for 3 days. The primary culture was split after 10–14 days when the MCs outgrew the other cell types. After the 4th passage, the medium was changed to D-valine DME (US Biological) with 10% FCS. The cells were used for experiments between the 10th and 15th passage.

Polyclonal and mAbs

Isoform specific anti-{alpha}-actinin 4 antisera was generated as described previously (15). Briefly, a peptide specific for mouse {alpha}-actinin 4 (YYDSHNVNTRCQKICDQWDNLGS) was synthesized and conjugated to keyhole limpet hemocyanin, and rabbit antisera generated by a standard immunization protocol (Southern Biotechnology Associates). Rabbit preimmune sera was used as a control. Isoform specific sera against {alpha}-actinin 1, 2, and 3 were a gift from Dr. A. H. Beggs (Harvard Medical School, Boston, MA).

The BM75.2 Ab is a monoclonal mouse IgM anti-{alpha}-actinin Ab (which does not bind dsDNA), while the TEPC183 mAb was used as an isotype-matched control (both from Sigma-Aldrich). The Lu-632 anti-{alpha}-actinin 4 mAb (IgM) was a gift from Drs. T. Yamada and S. Hirohashi (National Cancer Center, Tokyo, Japan) (16). The MOPC141 mAb (Sigma-Aldrich) was used as an isotype control for the test R4A (IgG2b) Ab. A rabbit polyclonal anti-tubulin Ab preparation (Sigma-Aldrich) was used as a loading control for Western blotting.

Cloning of mouse {alpha}-actinin 4 and 1 genes

Total RNA isolated from BALB/c and MRL/lpr MC was reverse transcribed using a mouse {alpha}-actinin 4 or 1 specific 3' primer and the Superscript II first-strand synthesis system for RT-PCR (Invitrogen Life Technologies).

{alpha}-Actinin 4. BALB/c and MRL/lpr ACTN4 were amplified with the long template PCR system (Roche Applied Sciences) using the following PCR conditions: 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°C for 2 min and an additional 10 s for each next cycle; and 4) extension at 68°C for 7 min. Purified PCR products were digested by XhoI and HindIII, and cloned into the pCMV-tag2B vector (Stratagene). DH5{alpha} competent cells (Invitrogen Life Technologies) were transformed, and grew on solid agar plates containing 100 µg/ml ampicillin. Plasmids containing the correct insert were identified by XhoI and HindIII digestion, and sequenced as detailed below. The primers for mouse {alpha}-actinin 4 were: forward, 5'-AGATATAAGCTTATGGTGGACTACCACGCAG-3'; reverse, 5'-AGATATCTCGAGTCACAGGTCGCTCTCCCCA-3'.

{alpha}-Actinin 1. BALB/c and MRL/lpr ACTN1 were amplified using the Failsafe PCR system (Epicentre), and the following PCR conditions: 1) 94°C for 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 were cloned directly into the pCRII vector using the TA cloning kit (Invitrogen Life Technologies). Recombinant pCRII plasmids were digested by XhoI and KpnI, and the ACTN1 cDNA insert ligated into the pRSETA vector (Invitrogen Life Technologies). The primers for mouse {alpha}-actinin 1 were: forward, 5'-AGAACACTCGAGATGGACCATTATGATTCCC-3'; reverse, 5'-AATTAAGGTACCTTAGAGGTCGCTCTCGC-3'.

DNA sequence analysis

PCR products and plasmids were sequenced by standard dideoxy nucleotide sequencing. For each gene, seven sequencing primers were designed based on the DNA sequence from the National Center for Biotechnology Information (NCBI) database. Only the first 400 bases were analyzed for each primer pair. For sequence analysis, the DNA sequences were compared using the basic local alignment search tool (BLAST) at the NCBI website (<www.ncbi.nlm.nih.gov/BLAST/>). The BLAST-like alignment tool (BLAT) (<http://genome.ucsc.edu/>) was used to map the two different ACTN1 cDNA sequences to the mouse, human, and rat genomes, determine whether 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 x 105 cells/10-cm plate the day before transfection. On the day of transfection, cells were washed once with OPTI-MEM (Invitrogen Life Technologies) medium without serum. For each plate, 24 µg of plasmid and 75 µl of LipofectAMINE 2000 (Invitrogen Life Technologies) were each diluted in 1.5 ml of OPTI-MEM medium without serum, combined, and incubated at room temperature (RT) for 20 min. The DNA-LipofectAMINE mixture was added directly to each plate with gentle rocking, and the plates were incubated at 37°C overnight. The following day the medium was replaced with 10% DMEM containing 800 µg/ml geneticin (Invitrogen Life Technologies). Transfected cells were cultured in selection medium for 2–3 wk, and the cells were cloned by limiting dilution. Selected clones were confirmed by Western blot using the anti-FLAG M2 Ab (Sigma-Aldrich) and BM75.2.

Purification of mouse {alpha}-actinin 4 and 1 proteins

BALB/c and MRL/lpr actinin-4 HEK 293-transfected cells were grown in tissue culture plates until confluence. The medium was aspirated, and 1 ml of sonication buffer (PBS/0.1% Triton X-100, with protease inhibitors) was added to each plate. The cells were harvested with a cell scraper, sonicated on ice, and centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was transferred to a new tube containing anti-FLAG M2 gel pretreated with glycine 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 column (Bio-Rad), washed with large amounts of PBS containing 0.1% Triton X-100, and eluted with 0.1 M glycine buffer (pH 2.5). The elution product was immediately neutralized with 1 M Tris base, and dialyzed in PBS at 4°C overnight. Purified {alpha}-actinin 4 was identified by SDS-PAGE and Western blot and the concentration determined by the Protein Assay kit (Bio-Rad).

{alpha}-Actinin 1 was purified from Escherichia coli strain BL21(DE3)plysE containing the {alpha}-actinin 1 pRSETA expression plasmid, using the Ni-NTA purification system for polyhistidine-containing recombinant proteins (Invitrogen Life Technologies).

ELISAs

For the {alpha}-actinin 4 ELISA, Ab at a concentration of 10 µg/ml in PBS was coated onto Immulon II plates (Dynex Technologies) at 4°C overnight. Plates were blocked with 3% FCS for 1 h at 37°C, washed, and incubated with a serial dilution of purified {alpha}-actinin 4 for 1 h at 37°C. The plates were washed, 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 peroxidase substrate solution (KPL) for 30 min at 37°C.

A direct ELISA method was used for measuring Ab binding to {alpha}-actinin 1, as follows: {alpha}-actinin 1 at a concentration of 5 µg/ml in PBS was coated onto Immulon II plates at 4°C overnight. Plates were blocked with 3% FCS for 1 h at 37°C, washed, and incubated with serial dilutions of Ab or serum for 1 h at 37°C. The plates were washed, and a 1/4000 dilution of HRP-linked secondary Ab (Southern Biotechnology Associates) in PBS was added for 1 h at 37°C, followed by peroxidase substrate solution for 30 min at 37°C.

Immunofluorescence and immunohistochemistry

MCs and transfected HEK293 cells were grown on tissue culture-treated coverslips, fixed with cold acetone, and blocked with 1% BSA/PBS containing anti-mouse CD16/32 (BD Biosciences) at a 1/100 dilution (to block FcRs) for 1 h at RT. The cells were then incubated with anti-FLAG Ab (10 µg/ml), R4A (20 µg/ml), BM75.2 (10 µg/ml), or isotype-matched Abs diluted in block at the appropriate concentrations for 1 h at 4°C. The cells were rinsed and washed three times with PBS. Fluorochrome-conjugated secondary Ab (Southern Biotechnology Associates) was applied at 1/200 and the slides were incubated for 45 min in the dark at 4°C. Cells were stained with propidium iodide (PI) 1 µg/ml for 5 min, washed several times with PBS, and fixed by 1% paraformaldehyde in PBS. The slides were observed by fluorescence microscopy and a Bio-Rad Radiance 2000 scanning confocal microscope with a Kr/Ar laser for excitation at 488 and 568 nm with Nikon 60x NA 1.4 Planapo Infinity Corrected Optics.

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

Western blot

MCs were detached from tissue culture plates, washed, pelleted, and resuspended to 2 x 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 min on ice. The cell lysis mixture was centrifuged at 14,000 rpm for 20 min at 4°C. The supernatant was removed, aliquoted, and kept at –70°C until used. Protein concentration was assayed by spectrophotometry, using the Bio-Rad Protein Assay kit. For Western blotting, 25 µg of protein lysates or purified BALB/c and MRL/lpr {alpha}-actinin 1 and 4 were combined with reducing sample buffer and heated at 100°C for 5 min. Samples were loaded into 4–15% gradient polyacrylamide gels (Bio-Rad), and electrophoresed at 200 V for 30 min. Proteins were transferred to a polyvinylidene difluoride (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 (Pierce Biotechnology) or 5% nonfat milk in PBS and incubated with primary Ab for 1 h at RT. The membrane was repeatedly washed with PBS-Tween, and incubated with the appropriate HRP-conjugated secondary Abs diluted 1/10,000 for 1 h at RT. The membrane was developed with the ECL Plus kit, and exposed to Hyperfilm (Amersham Biosciences). Loading of equivalent amounts of protein and adequate membrane transfer were confirmed by staining the PVDF membrane with Ponceau Red (Sigma-Aldrich), and by Western blotting with anti-tubulin Abs (Sigma-Aldrich) in parallel to the 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 sequence data from the Ensembl database (<www.ensembl.org/Mus_musculus/>). At least one intron was included to avoid genomic DNA amplification. Amplicons ranged from 80 to 120 bp. Total RNA isolated from BALB/c and MRL/lpr MCs was reverse transcribed using oligo dT, and real-time PCR performed in triplicate using the SYBR green master mix and the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems), using the 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 {alpha}-actinin and the two control genes GAPDH and CCNI) was done in triplicate. The ratio of expression of BALB/c {alpha}-actinin to each of the control genes was arbitrarily defined as 1 and compared with the ratio of expression of MRL/lpr {alpha}-actinin to the same control genes. The depicted fold changes are a mean of these normalized expression ratios, obtained in three independent experiments. 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'. cyclin I (CCNI): forward, 5'-GAAATGGAGAAACTCATTCCTGATT-3'; reverse, 5'-CCCGACAGTGGATCAAC-3'. ACTN1 (nonspecific): forward, 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'-CTC CGCAGTTCCTCAGCA-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BALB/c and MRL/lpr ACTN4 genes differ by several point mutations

Binding of the pathogenic anti-dsDNA/anti-{alpha}-actinin Ab R4A to MRL/lpr MC is significantly increased as compared with its binding to nonautoimmune BALB/c MC (11). Proteomic analysis of the MC component bound by R4A indicated that the closest fit of the analyzed peptides was to {alpha}-actinin 4. Accordingly, we started our studies with this particular {alpha}-actinin isoform.

To detect whether the differential binding to BALB/c and MRL/lpr MC by R4A is due to polymorphisms in {alpha}-actinin 4 sequences, we sequenced the ACTN4 gene in BALB/c and MRL/lpr MC. The MRL/lpr ACTN4 gene (912 aa) is completely identical to the C57BL/6 ACTN4 sequence reported in the NCBI database (GenBank no. NM021895). Interestingly, C57BL/6 is one of the ancestral strains from which the MRL background is derived (17). BALB/c ACTN4 encodes for 911 aa, and is missing a glycine at position 20 relative to the MRL/lpr sequence. In addition, there are several point mutations in the BALB/c ACTN4 sequence, leading to substitutions at positions 16, 23, 28, 439, 614, 704, and 733 as compared with the MRL/lpr protein sequence (Fig. 1). The complete nucleotide sequences of BALB/c (no. DQ303410) and MRL/lpr (no. DQ303411) ACTN4 have been submitted to GenBank.


Figure 1
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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.

 
R4A binds transfected HEK293 cells through overexpressed {alpha}-actinin 4

We reasoned that the variations in linear sequence between BALB/c and MRL/lpr {alpha}-actinin 4 may lead to a different distribution or localization of cellular protein expression, thus potentially affecting accessibility to Ab binding. Therefore, we studied protein expression in BALB/c and MRL/lpr {alpha}-actinin 4-transfected HEK293 cells by staining with anti-FLAG and anti-{alpha}-actinin mAbs. BALB/c and MRL/lpr {alpha}-actinin 4-transfected cells showed a strong fluorescent signal when stained by the anti-FLAG Ab, while cells alone and cells transfected with an empty vector were negative (data not shown). There is no significant distinction between the staining patterns of BALB/c and MRL/lpr {alpha}-actinin 4-transfected HEK293 cells by immunofluorescence (Fig. 2), leading to the conclusion that there is not likely to be a major difference in the cellular distribution of BALB/c and MRL/lpr {alpha}-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), the staining pattern in transfected cells is localized to the cell surface. This supports our previous observation (11), made originally by others (10, 18), that besides its cytoskeletal location {alpha}-actinin is also expressed on the cell surface.


Figure 2
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  		FIGURE 2. Anti-{alpha}-actinin Abs bind to transfected HEK293 cells. {alpha}-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 µg/ml), BM75.2 (10 µg/ml), or R4A (20 µg/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 min in the dark at 4°C. Coverslips were mounted following 5 min of staining with 1 µg/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).

 
R4A has similar affinity for BALB/c and MRL/lpr {alpha}-actinin 4

As BALB/c and MRL/lpr {alpha}-actinin 4 did not appear to differ in their intracellular location, we wondered whether {alpha}-actinin Abs bind with altered affinity to the different forms of {alpha}-actinin 4. Purified recombinant BALB/c and MRL/lpr {alpha}-actinin 4 were >95% pure, when run on a SDS-PAGE gel and stained by Coomassie blue. By Western blot, there was no apparent difference in binding of the BM75.2 anti-{alpha}-actinin mAb or of R4A to BALB/c or MRL/lpr {alpha}-actinin 4 (Fig. 3A).


Figure 3
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  		FIGURE 3. Anti-{alpha}-actinin Abs have similar affinity to BALB/c and MRL/lpr {alpha}-actinin 4. A, Binding of BM75.2 and R4A to purified BALB/c and MRL/lpr {alpha}-actinin 4 by Western blot. Increasing amounts of purified {alpha}-actinin 4 (from left to right: 0.1, 0.5, 1.0, and 2.5 µg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were blotted with BM75.2 (1 µg/ml) or R4A (10 µg/ml) for 1 h, followed by the appropriate secondary Abs at 1/10,000 for 1 h. B, Binding of R4A, BM75.2, and polyclonal anti-{alpha}-actinin 4 to purified BALB/c and MRL/lpr {alpha}-actinin 4 by ELISA. Left and middle panel, R4A (left panel) or BM75.2 (middle panel) at 10 µg/ml in PBS were coated onto Immulon II plates at 4°C overnight. After a blocking step, the plates were incubated with a serial dilution of purified {alpha}-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 {alpha}-actinin 4 at 5 µg/ml was coated onto Immulon II plates at 4°C overnight. The plate was then incubated with a serial dilution of polyclonal rabbit anti-{alpha}-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.

 
We then confirmed the relative affinities of BM75.2 and R4A for recombinant BALB/c and MRL/lpr {alpha}-actinin 4 by ELISA. As a control, we used anti-{alpha}-actinin 4 antisera, raised against an {alpha}-actinin 4-specific peptide (position 486–508, an area of complete MRL and BALB/c ACTN4 sequence identity). Polyclonal serum was identically bound by BALB/c and MRL/lpr {alpha}-actinin 4, confirming that the same amount of {alpha}-actinin 4 was coated onto each well (Fig. 3B, right panel). We found that R4A and BM75.2 have similar affinity for both BALB/c and MRL/lpr {alpha}-actinin 4, despite their differences in ACTN4 primary sequence (Fig. 3B, left and middle panels).

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

As there were no apparent differences between the binding of R4A to MRL/lpr and BALB/c {alpha}-actinin 4, we wondered whether the differential binding of R4A to MRL/lpr MCs is due to enhanced {alpha}-actinin expression. Proteomic analysis in our initial studies (11) had indicated that besides {alpha}-actinin 4, {alpha}-actinin 1 is also a possible target for R4A. Therefore, we studied the expression of both {alpha}-actinin 4 and {alpha}-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 in primary MRL/lpr MC is increased 4.8- and 2.2-fold, respectively, as compared with primary BALB/c MC (p < 0.05 for each comparison) (Fig. 4A). Expression of ACTN2 was not significantly different. Next, the binding of R4A and BM75.2 to primary BALB/c and MRL/lpr MC lysates was assayed by Western blot. As we originally observed in immortalized cells, R4A (11) and BM75.2 each showed significantly increased binding to the 100-kDa {alpha}-actinin band in primary MRL/lpr MC lysates (Fig. 4B). In conformity with the real-time PCR results, Western blot of total cell lysates with isoform-specific antisera showed that primary cells from both MRL/lpr and BALB/c express {alpha}-actinin 1, 2, and 4 but not 3. Moreover, {alpha}-actinin 1 and 4 have up-regulated expression in MRL/lpr MC, while there were no significant differences in the expression level of {alpha}-actinin 2 between these two cell lines (Fig. 4B). {alpha}-Actinin 3, an isoform limited to muscle, is not found in BALB/c or MRL/lpr MC (Fig. 4, B and C).


Figure 4
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  		FIGURE 4. Primary MRL/lpr MC display higher expression levels of {alpha}-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 three repeats used as the result for that sample. Shown here are the mean ± SD values for fold change when each sample was separately normalized to GAPDH and CCNI. ACTN1 and ACTN4, p < 0.01; ACTN2, p > 0.05. B, Western blotting of primary MC lysates. Twenty-five micrograms of primary cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were stained by polyclonal rabbit anti-{alpha}-actinin 1, 2, 3, 4, and control serum (all at 1/1000), and the monoclonal anti-{alpha}-actinin Abs Lu632 (at 5 µg/ml), BM75.2 (1 µg/ml), and R4A (5 µg/ml). Staining with polyclonal anti-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 the anti-{alpha}-actinin 3 serum, and showed no difference in {alpha}-actinin 3 expression. The data displayed in this figure are representative of two (protein expression) or three (real-time PCR) independent experiments.

 
BALB/c and MRL/lpr MCs contain two distinct {alpha}-actinin 1 isoforms

Although we found that {alpha}-actinin 1 is up-regulated in primary MRL/lpr MC, we wanted to confirm that R4A binds to {alpha}-actinin 1 as well as {alpha}-actinin 4, and determine whether there are any sequence polymorphisms in this gene between MRL/lpr and BALB/c. Surprisingly, we were able to clone two distinct {alpha}-actinin 1 sequences from BALB/c and MRL/lpr MCs, ACTN1 and ACTN1A.

We found that BALB/c and MRL/lpr ACTN1 sequences were identical to each other and to the ACTN1 sequence available from GenBank (no. AF115386). Similarly, BALB/c and MRL/lpr ACTN1A sequences were identical (GenBank no. DQ288940). The ACTN1 sequence contains an 81-bp fragment (position 2506–2586) that is replaced by a 66-bp sequence in the ACTN1A cDNA (2506–2571) (Fig. 5A). Otherwise, the encoded proteins are identical, with no change in frame. Sequence analysis revealed that the 81-bp fragment is present in the majority of mRNA and EST clone sequences in both the human and mouse databases. We determined that the 66-bp fragment represents an alternatively spliced exon because it is present in several mRNA and EST clones, maintains the open reading frame (22 aa replace 27 aa) and is also highly conserved across species. Judging from the cDNA libraries from which the mRNA and EST clones were isolated, the expression of the 66-bp fragment is more limited. No clones existed in the human or mouse databases that contain both the 81- and 66-bp sequences; however, these two exons were found together in a brain-specific transcript from rat (GenBank no. CO399752). By real-time PCR, we found that significantly increased transcription of ACTN1A, but not ACTN1, in MRL/lpr MC was the likely cause for the difference in {alpha}-actinin 1 protein expression between MRL/lpr and BALB/c cells (Fig. 5B).


Figure 5
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  		FIGURE 5. Two {alpha}-actinin 1 splice variants are present in MRL/lpr and BALB/c MCs. A, Splice donor/acceptor sites found in the ACTN1 sequence. Exonic and intronic sequences in the table are denoted in upper case and lower case letters, respectively. The splice acceptor sites are denoted in bold, and the splice donor sites in bold and italic. Other than the splice 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 represented in white for illustrative purposes only. B, Primary MRL/lpr MCs have higher expression of ACTN1A than primary BALB/c MC. Real-time PCR analysis of the relative expression level of ACTN1 and ACTN1A in primary BALB/c and MRL/lpr MC. GAPDH and CCNI were used as control genes. ACTN1, p > 0.05; ACTN1A, p < 0.01. The real-time PCR data displayed in this figure are representative of three independent experiments.

 
To study the relative affinity of anti-{alpha}-actinin Abs for the ACTN1 and ACTN1A gene products, both genes were cloned into the pRESET A vector, expressed, and purified. Recombinant BALB/c and MRL/lpr {alpha}-actinin 1 proteins were run by SDS-PAGE followed by staining with Coomassie bright blue (which indicated >95% purity), and their identify confirmed by Western blot using anti-HisG Ab (Invitrogen Life Technologies) and rabbit anti-{alpha}-actinin 1 anti-serum. The binding affinity of anti-{alpha}-actinin Abs to these {alpha}-actinin 1 isoforms was detected by Western blot and ELISA. We found that R4A and BM75.2 each binds to both {alpha}-actinin 1 variants (Fig. 6A). Furthermore, no differences were observed in the affinity of these Abs to {alpha}-actinin 1 and {alpha}-actinin 1A (Fig. 6B).


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

 
We next investigated the relative affinity of R4A to {alpha}-actinin 4 and {alpha}-actinin 1 by ELISA, using direct binding and inhibition assays. Although this conclusion is not irrefutable due to the different methods we had to use to express these two {alpha}-actinin isoforms, R4A had significantly higher affinity for {alpha}-actinin 1 (data not shown).

Abs to {alpha}-actinin show stronger binding to primary MRL/lpr as compared with primary BALB/c MCs

Primary BALB/c and MRL/lpr MCs growing on tissue culture slides were fixed and stained by BM75.2, R4A, or isotype controls, followed by a secondary fluorescent Ab and PI staining. The fluorescence signal from stained primary MRL/lpr MC was much stronger than that from primary BALB/c MC, consistent with our findings of higher expression levels of {alpha}-actinin 1 and 4 in primary MRL/lpr MC. Comparing the images of phase control and PI staining, the binding of BM75.2 showed a cytoskeletal pattern, while R4A demonstrated both cytoplasmic and nuclear binding (Fig. 7), the latter likely due to its antinuclear specificity.


Figure 7
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  		FIGURE 7. Abs to {alpha}-actinin show stronger binding to primary MRL/lpr as compared with primary BALB/c MC. Confocal images of primary MC stained by BM75.2 and R4A. Primary MC were fixed by cold acetone and blocked with 1% BSA/PBS containing a 1/100 dilution of anti-mouse CD16/32 for 1 h at RT. Cells were then incubated with BM75.2 (10 µg/ml), R4A (20 µg/ml), or isotype control Abs for 1 h at 4°C. The appropriate fluorochrome-conjugated secondary Abs were applied at 1/200, and the slides were incubated for 45 min in the dark at 4°C. Coverslips were mounted following staining with 1 µg/ml PI for 5 min. Top, PI staining (red); middle, Ab staining (green); bottom, merged image. The data displayed in this figure are representative of two independent experiments.

 
Anti-{alpha}-actinin Abs are present in high titers in MRL/lpr mice and preferentially bind to MRL/lpr kidneys

Previously, we had determined that lupus prone mice display high serum titers of anti-{alpha}-actinin Abs (11). To determine whether any particular isoform is targeted in this autoantibody response, we examined the titers of isoform-specific anti-{alpha}-actinin Abs in old (5 mo of age) MRL/lpr mice. Fig. 8A demonstrates that diseased mice have significantly higher Ab titers against {alpha}-actinin 1, 1A, and 4, as compared with age-matched BALB/c mice. Although the results may not be directly comparable due to technical differences in the method of expression, there was no significant difference between the binding of MRL/lpr sera to purified {alpha}-actinin 1 and 4 (Fig. 8A and data not shown).


Figure 8
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  		FIGURE 8. Anti-{alpha}-actinin Abs are present in high titers in MRL/lpr mice and preferentially bind to MRL/lpr kidneys. A, Binding of MRL/lpr serum to {alpha}-actinin isoforms by ELISA. Purified {alpha}-actinin 1, {alpha}-actinin 1A, and MRL/lpr {alpha}-actinin 4 at 5 µg/ml were coated onto Immulon II plates at 4°C overnight, followed by a 1/2000 dilution of serum from 5-mo-old female MRL/lpr mice (n = 7) for 1 h at 37°C, and secondary Ab. As compared with age- and sex-matched BALB/c mice (n = 7), MRL/lpr mice display significantly higher serum titers of Abs to {alpha}-actinin 1, 1A, and 4 by ELISA (p < 0.0001). B, Anti-{alpha}-actinin Abs preferentially bind to MRL/lpr kidneys. Cryostat sections from 5-mo-old MRL/lpr and BALB/c kidneys were fixed by cold acetone and blocked. Sections were incubated with a 1/500 dilution of anti-{alpha}-actinin 1, anti-{alpha}-actinin 4, and control (preimmune) polyclonal antiserum at 4°C overnight. The HRP-linked anti-rabbit IgG secondary Ab was then applied at 1/500 and the sections incubated with substrate at RT for 30 min. Top row, Sections stained with polyclonal anti-{alpha}-actinin 1 Abs; bottom row, sections stained with polyclonal anti-{alpha}-actinin 4 Abs. Sections stained by preimmune sera were negative (data not shown). Arrows indicate stained glomeruli. All images in this figure are at a magnification of x50. The data displayed in this figure are representative of two independent experiments.

 
To confirm that the observed differences in {alpha}-actinin expression in vitro between MRL/lpr and BALB/c MCs described above were also reflected in the kidney in vivo, we stained kidney sections with polyclonal anti-{alpha}-actinin Abs. We found significantly stronger staining of MRL/lpr glomeruli by these Abs, supporting the hypothesis that higher {alpha}-actinin expression is responsible for greater Ag availability for binding by anti-{alpha}-actinin Abs (Fig. 8B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Cross-reactivity of anti-dsDNA Abs with kidney Ags is an important determinant in their renal pathogenicity. Previously, we had found that {alpha}-actinin is a target Ag for pathogenic murine and human anti-DNA Abs in MC (11). Furthermore, we had demonstrated that an immortalized MC line derived from the lupus-prone MRL/lpr mouse strain shows enhanced binding to a pathogenic, cross-reactive, anti-dsDNA/anti-{alpha}-actinin Ab as compared with a BALB/c-derived cell line. Moreover, human anti-dsDNA Abs cross-react with {alpha}-actinin, and autoantibodies of this specificity are associated with lupus nephritis (12, 13). In the present study, we conclusively show that both {alpha}-actinin 4 and {alpha}-actinin 1 are potential targets for anti-{alpha}-actinin Abs. Moreover, primary MCs from the lupus prone MRL/lpr mouse strain overexpress {alpha}-actinin 4 as well as {alpha}-actinin 1, indicating that increased {alpha}-actinin expression may contribute to the susceptibility to the severe Ab-mediated nephritis seen in the MRL/lpr mouse strain. Support for this latter hypothesis can be found in our findings that MRL/lpr mice display high circulating autoantibody titers against kidney-expressed {alpha}-actinin isoforms, and that anti-{alpha}-actinin antiserum differentially bound not only to isolated MRL/lpr MCs, but to intact kidney sections as well.

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

There has been increasing attention to the role of {alpha}-actinin in renal physiology since the report by Kaplan et al. (24) in 2000 showing that humans with familial focal and segmental glomerulosclerosis (FSGS), a cause of nephrotic syndrome and renal failure, have mutations in ACTN4. Subsequently, direct evidence for a cause and effect relationship was found in the report that mice with kidney-specific transgenic expression of an {alpha}-actinin 4 containing a mutation similar to one present in humans with FSGS developed hypertension, proteinuria, and histologic features of FSGS (25). Mutant {alpha}-actinin 4 was shown to form intracellular aggregates, and to undergo rapid degradation mediated in part by the ubiquitin pathway (26). This suggested two nonmutually exclusive models to explain disease in individuals with mutant {alpha}-actinin 4. Aggregated {alpha}-actinin 4 may be toxic to kidney cells (26). Alternatively, accelerated metabolism of mutated {alpha}-actinin 4 (25) or abnormally high affinity for F-actin (25) may lead to abnormal function or loss of normal function. Interestingly, not only do mice harboring mutant {alpha}-actinin develop renal pathology, but mice deficient in {alpha}-actinin 4 also display proteinuria and glomerular disease, and die within several months from renal failure (20). This latter study indicates that despite the similarities in structure and function between {alpha}-actinin 4 and {alpha}-actinin 1, these proteins are not redundant. Although we have not yet shown this directly, it is interesting to speculate that binding of pathogenic Abs to {alpha}-actinin may lead to similar consequences as the presence of abnormal {alpha}-actinin, that is impaired function of {alpha}-actinin and/or interference with the interaction of {alpha}-actinin with other cytoskeletal proteins.

The precise role of {alpha}-actinin in glomerular function is not completely understood. {alpha}-Actinin, together with other contractile proteins in the kidney, may function to counteract pressure gradients across the capillary wall (27). Alternatively, {alpha}-actinin may influence the renal vascular cytoskeleton or be involved in protein filtering (24). {alpha}-Actinin plays a role in cell motility and adherence (16, 20) and has been postulated to function as a tumor suppressor gene by impacting normal cell growth regulation and differentiation (28). Most recently, it has been shown that {alpha}-actinin colocalizes with polycystin-2, the product of the PKD2 gene which is mutated in some patients with familial polycystic kidney disease. Moreover, it was found that {alpha}-actinin modulates polycystin-2 and enhances its channel activity (29). This finding is in agreement with previous reports that {alpha}-actinin regulates the activity of several types of channels (30, 31) and supports this potentially important aspect of {alpha}-actinin function.

There is data for several autoimmune diseases to suggest that inherent susceptibility to autoantibody-mediated disease is dependent on genetically determined differences in autoantigen display in the target organs. As previously reviewed (11), BN rats immunized with heterologous tubular basement membrane (TBM) develop 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 appears that Lewis rats are resistant to disease because they lack the TBM Ag which is targeted by pathogenic anti-TBM Abs. In a different model of experimental nephritis, Xie et al. (34, 35) has recently elegantly demonstrated that there is a broad range of susceptibility (from resistant to highly vulnerable) of mouse strains to preformed pathogenic anti-glomerular basement membrane Abs in the nephrotoxic serum model of renal disease (Masugi nephritis). Finally, specifically for murine lupus, Fas deficiency in the MRL background leads to a severe, full-blown lupus syndrome, while this mutation in the C3H background induces anti-DNA Abs but only mild renal disease (36). These studies demonstrate that for some autoimmune nephritides (including lupus), pathogenic Abs are necessary, but may not be sufficient; a genetically determined level of expression of a target Ag can be a critical factor in determining susceptibility to disease. Lupus patients with persistently high levels of anti-DNA Abs who nevertheless do not develop nephritis (37) may be an example of this principle as well. Although the actual loci that directly impact the expression of renal disease are not currently known, this is an area of great interest among investigators dealing with genetic dissection of murine lupus models.

What may be the function of genes that contribute to the expression of lupus nephritis? Xie et al. (35) propose that it is possible that nephritis-facilitating genomes may encode for resident kidney cells which may be intrinsically hyperactive to immunological insults (for example, through enhanced expression of the target Ag or the secretion of a particularly damaging cytokine profile). Alternatively, these disease-susceptible genomes may be enhancing renal disease by influencing the function of those systemic cells that infiltrate the kidney during the inflammatory process. Our present studies demonstrating significant differences in {alpha}-actinin expression in isolated MCs are supportive of an intrinsic renal contribution to nephritis susceptibility.

Corticosteroids are one of the mainstays of the treatment of lupus nephritis. We previously reported that monoclonal mouse and polyclonal human anti-dsDNA Abs from lupus patients bind to the cell surface of mouse embryonic stem cells. This cell surface receptor was markedly down-regulated by a brief pretreatment of cells with dexamethasone, leading to a >50% decrease in Ab binding (38). Based on these results, we had postulated that while corticosteroids have many anti-inflammatory and immunosuppressive effects, modulation of target Ag can be invoked as an additional explanation for the therapeutic benefit of steroids in lupus nephritis. Consistent with these studies is the observation that corticosteroid treatment of lupus mice significantly decreases kidney insulin-like growth factor-1 and fibroblast growth factor, which can both up-regulate {alpha}-actinin expression (39, 40). The salutatory effects of corticosteroids in lupus nephritis may then be partly attributed to their effects on modulation of Ag expression in the target organ, specifically {alpha}-actinin. Moreover, this also raises an intriguing possibility that cytokine levels may be therapeutically manipulated to modify target Ag expression. This hypothesis will be examined directly. Finally, it is similarly possible to invoke differences in the cytokine profile or responsiveness as a potential explanation for the variability in {alpha}-actinin expression between BALB/c- and MRL/lpr-derived cells.

We found increased expression of {alpha}-actinin in MRL/lpr MC (in vitro) and kidney (in vivo). One possible way to explain these observations is that the increased expression may be a feature of inflammation, rather than an endogenous characteristic of lupus kidney cells. Indeed, albeit in a different lupus background, Mostoslavsky et al. (10) detected {alpha}-actinin expression by immunofluorescence in a 7-mo-old NZB x NZW F1 kidney, but not in the kidney of a young 3-mo-old NZB x NZW F1 mouse. Nevertheless, {alpha}-actinin overexpression was seen in primary MCs which following derivation are no longer exposed to a proinflammatory milieu. Moreover, the primary cells used in our experiments were derived from young 6-wk-old mice, an age where inflammation is less likely to have had a major influence. Therefore, we believe that the MRL lupus background is an important contributor to increased {alpha}-actinin expression. Does differential {alpha}-actinin expression distinguish between other nonautoimmune and autoimmune strains, and whether cytokines/inflammation are contributing to this as well and how they do so, are important questions that will be addressed.

We should also point out that while {alpha}-actinin 4 was up-regulated in primary MRL/lpr MC, expression was similar following overexpression of MRL/lpr and BALB/c {alpha}-actinin 4 in transfected cells. We believe this is related to the constructs used for the experiments, as the transfected {alpha}-actinin 4 constructs lacked both the endogenous promoter region as well as the 3' UTR from the respective strains. It is possible that variations (polymorphisms) in these regions between the two strains could effect the in vivo expression and/or stability of the message. This will be carefully examined in future studies.

We found several previously unknown sequence polymorphisms between MRL/lpr and BALB/c ACTN4. Although Kaplan et al. (24) was able to show that the mutant {alpha}-actinin 4 associated with familial FSGS binds F-actin more strongly than the wild-type protein and tends to aggregate in vivo, we were not able to demonstrate meaningful differences in intracellular localization following transfection or in the affinity for {alpha}-actinin autoantibodies between MRL/lpr and BALB/c {alpha}-actinin 4. Similarly, while we were able to clone two alternatively spliced forms of ACTN1 in both MRL/lpr and BALB/c MCs, we were not able to demonstrate any meaningful variation in binding affinity of anti-{alpha}-actinin Abs to the expressed proteins. Although we considered the possibility that MRL/lpr and BALB/c {alpha}-actinin have subtle changes in binding affinity to anti-{alpha}-actinin Abs, we believe that this is unlikely as no binding alteration was found in either of the two methods (ELISA and Western blot) that were used. {alpha}-Actinin 1 and 4 display some differences in cellular localization ({alpha}-actinin 1 is concentrated in focal adhesion plaques and adherens junctions, while {alpha}-actinin 4 is not) and sensitivity to calcium (16, 41). It remains to be seen whether the sequence polymorphisms we demonstrated here between BALB/c and MRL/lpr ACTN4 and between ACTN1 and ACTN1A will translate into measurable differences in their biochemical function and/or immunogenicity.

In summary, we have shown that {alpha}-actinin 4 and {alpha}-actinin 1 can both be targeted by a pathogenic anti-{alpha}-actinin response. Although some polymorphisms were found between MRL/lpr and BALB/c {alpha}-actinin 4, differential binding to MRL/lpr {alpha}-actinin was a result of enhanced protein expression, rather than any detectable differences in Ab binding to strain-specific isoforms. A novel splice variant of {alpha}-actinin 1 was detected in MCs, and this isoform also shows higher expression in MRL/lpr mice. The underlying mechanisms for enhanced steady state ACTN1A and ACTN4 mRNA levels in MRL/lpr MC are not currently known, and will need to be formally addressed. Studies to understand the contribution of organ-specific factors to Ab-mediated nephritis in SLE should include the analysis of polymorphisms in target Ag structure and expression. Finally, modulation of target Ag expression by cytokines or other therapeutic modalities might be a possible new approach to the treatment of Ab-mediated kidney disease.


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


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

1 Work in the laboratory of C.P. was supported by National Institutes of Health Grants RO1-AR-48692 and PO1-AI-51392. B.D. was supported by 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; MC, mesangial 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. Back

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


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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