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-Actinin Is a Cross-Reactive Renal Target for Pathogenic Anti-DNA Antibodies1
*
Department of Microbiology and Immunology, and
Division of Rheumatology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin, binding of R4A to
-actinin was confirmed by Western blot, ELISA, inhibition studies,
and immunofluorescence. High titers of anti--actinin Abs were
present in sera and kidney eluates of lupus mice with active nephritis.
These results indicate that the nephritogenicity of some anti-DNA
Abs may be mediated via cross-reactivity with -actinin. Furthermore,
variations in target Ag display between individuals may underlie
differential susceptibility to anti-DNA Ab-induced renal
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify a cross-reactive renal Ag bound by nephritogenic
anti-DNA Abs, we performed Western blotting of mesangial cell
(MC) lysates with R4A, a pathogenic anti-DNA Ab, and with
95, a closely related but nonpathogenic Ab. We found that R4A bound to
and immunoprecipitated a 100-kDa protein identified as -actinin from
MC lysates. Binding was more pronounced using MRL-lpr/lpr
(lpr) than nonautoimmune BALB/c MC lysates,
suggesting that Ag expression in the target organ plays a role in
anti-DNA Ab binding to renal tissue. Furthermore, sera and kidney
eluates from lupus mice with active nephritis displayed high titers of
anti--actinin Abs. We conclude that -actinin is a
cross-reactive target for pathogenic anti-dsDNA Abs in renal
tissue, and that genetic variability in Ag expression and/or
accessibility may contribute to the susceptibility to anti-dsDNA
Ab-induced nephritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R4A is a pathogenic murine IgG2b anti-dsDNA Ab. When
administered i.p. in the form of an ascites-producing hybridoma to SCID
mice, R4A deposits in renal glomeruli and induces significant
proteinuria (4). Ab 95, which has a single aspartic acid
to glycine substitution in complementarity determining region 3 of the
R4A H chain, no longer binds DNA and is nonpathogenic (4).
MOPC 141 is an isotype-matched IgG2b mAb (Sigma-Aldrich, St.
Louis, MO) that also does not bind DNA or deposit in glomeruli. BM-75.2
is an IgM anti--actinin mAb, and TEPC 183 is an isotype-matched
purified myeloma Ig (Sigma-Aldrich).
Mice
Six- to 8-wk-old female BALB/c, (NZB x NZW)F1, and MRL-lpr/lpr mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the animal facility of the Albert Einstein College of Medicine (Bronx, NY).
Cell lines
MCs were isolated according to a protocol obtained from G. Gilkeson (Medical University of South Carolina, Charleston, SC). Briefly, the outer cortexes of 10 kidneys were minced with a razor, and the tissue was forced through progressively smaller stainless steel sieves (180, 150, and 90 µm). Glomeruli were then caught on a 75-µm sieve, washed twice with PBS, and centrifuged for 5 min at 220 x g. Washed glomeruli were treated with collagenase for 10 min at 37°C and washed again as above. Cells were plated out in RPMI 1640 supplemented with amino acids, insulin, and 20% FCS, and maintained at 37°C/5% CO2. After 30 days, the cultures consist of virtually pure MCs. MCs derived from BALB/c mice were transformed using an SV40 vector received from the laboratory of Dr. R. Pestell (Bronx, NY). Transformed lpr mesangial and tubular cells were a kind gift from Dr. M. Madaio (University of Pennsylvania, Philadelphia, PA). An immortalized kidney podocyte cell line was a kind gift from Dr. P. Mundel (Bronx, NY).
Flow cytometry
MCs were detached from tissue-culture plates using 2 mM EDTA,
pelleted, and washed twice with DMEM. Cells were resuspended at
2 x 106 cells/ml in HBSS, pelleted, and
blocked with 1% BSA/PBS and anti-mouse CD16/CD32 (Fc
III/IIR; BD
PharMingen, San Diego, CA) for 1 h at 4°C. Primary Abs were
incubated with the cells at a concentration of 10 µg/ml for 1 h
at 4°C, followed by biotin-labeled goat anti-mouse IgG2b (BD
PharMingen) for 45 min at 4°C, and
streptavidin-allophycocyanin (BD PharMingen) for 30 min at
4°C. Cells were washed, resuspended in 1% paraformaldehyde, and
analyzed by flow cytometry (BD Immunocytometry Systems, Mountain
View, CA).
Western blotting and immunoprecipitation
Cells were detached from tissue-culture plates, washed,
pelleted, and resuspended to 2 x 108
cells/ml in lysis buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05%
NaN3, 1% Triton X-100, 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
Protein Assay ESL kit from Boehringer Mannheim (Indianapolis, IN). For
Western blotting, 20 µg of protein lysates were combined with
reducing or nonreducing sample buffer. For some experiments, protein
samples were first incubated with 100 µg/ml of DNase I
(Sigma-Aldrich) for 1 h at 37°C. Samples were loaded into
4–15% gradient polyacrylamide gels (Bio-Rad, Hercules, CA), and
electrophoresed at 150 V for 1 h. Proteins were transferred to a
polyvinylidene difluoride membrane using Mini Protean 3 cell
apparatus (Bio-Rad) at 200 mAmp for 1 h. The membrane was blocked
in 5% nonfat milk and incubated with primary Ab at 1 µg/ml for 30
min at room temperature (RT). The membrane was repeatedly washed with
PBS-Tween, and incubated with the appropriate HRP-conjugated secondary
Ab diluted 1/5,000 for 30 min at RT. The membrane was developed with
the ECL Plus kit, and exposed to Hyperfilm (Amersham, Aylesbury, U.K.).
The intensity of the developed bands was quantitated by densitometry
and the ImageQuant software program (Amersham Biosciences, Sunnyvale,
CA). Loading of equivalent amounts of protein and of adequate
membrane transfer was confirmed by staining the polyvinylidene
difluoride membrane with Ponceau Red and by Western blotting with an
anti-tubulin Ab in parallel to the test Abs. For assay of
inhibition of binding by Western blot, serial dilutions of chicken
-actinin (Sigma-Aldrich), salmon sperm dsDNA (Calbiochem, La Jolla,
CA), or peptide (DWEYSVWLSN on a polylysine backbone from Research
Genetics, Huntsville, AL) were incubated with R4A for 2 h at
37°C. The assay was then continued as described above.
Immunoprecipitations were performed using protein G-Sepharose beads (Pharmacia, Piscataway, NJ). Protein G beads prewashed with 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) were incubated with 75 µg of mAb for 2 h at 4°C with constant mixing. Three milligrams of precleared protein lysate were combined with the Ab-loaded protein G beads and incubated overnight at 4°C with gentle shaking. The mixture was centrifuged at 10,600 rpm for 20 s and the pellet was washed three times with 1% radioimmunoprecipitation assay buffer and once with 50 mM Tris-HCl (pH 8.0). The pellet was resuspended in sample reducing buffer and heated to 100°C for 5 min. The supernatant was removed and the proteins were separated by SDS-PAGE and Western blotted as above.
On-dish membrane biotinylation of MC surface proteins was performed using EZ link Sulfo-NHS-L-biotin (Pierce, Rockford, IL) and the manufacturer’s instructions. Only external surface proteins are labeled by this method. Briefly, cell culture medium was removed and the cells were washed twice to remove any contaminating proteins. The biotinylation reagent was added and incubated for 30 min at 4°C. Cells were removed, washed, pelleted, and resuspended in extraction buffer (50 mM boric acid, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 2.5 mM PMSF). The mixture was passed twice through a 21G needle, incubated on ice for 30 min, and spun at 12,000 rpm for 20 min at 4°C. To immunoprecipitate biotin-labeled cell surface proteins, 3 mg of biotinylated proteins were combined with prewashed streptavidin-conjugated agarose beads and incubated overnight at 4°C. Isolation of the precipitated proteins, electrophoresis, and Western blotting were continued as described above.
Protein analysis
SDS-PAGE of anti-DNA Ab-immunoprecipitated proteins was conducted as described above. The appropriate-sized protein band was cut from the gel and transferred to a prewashed microfuge tube. Protein identification using mass spectrometry:matrix-assisted laser desorption ionization (MALDI-MS) and nanospray MS/MS was performed at the Howard Hughes Medical Institute Biopolymer/Keck Foundation Biotechnology Resource Laboratory at the Yale University School of Medicine (New Haven, CT).
Immunocytochemistry
MCs grown to confluence on tissue culture-treated cover slips were washed twice with 1% BSA/PBS and incubated with 10 µg/ml of R4A, 50 µg/ml of BM-75.2, or isotype-matched Abs diluted in block for 4 h at 4°C. The cells were then washed with PBS and fixed in 1% paraformaldehyde/PBS for 30 min. The cells were rinsed and washed three times with PBS. The appropriate fluorochrome-conjugated secondary Ab was applied and the slides were incubated for 60 min in the dark. Coverslips were mounted following several washes and the slides were allowed to dry overnight at 4°C. A Bio-Rad Radiance 2000 scanning confocal microscope with a Kr/Ar laser for excitation at 488 and 568 nm was used for visualization with Nikon 60X NA 1.4 planapo infinity corrected optics (Melville, NY).
ELISAs
Anti-dsDNA ELISAs were performed as previously described
(5). For the -actinin ELISA, -actinin
(Sigma-Aldrich) at a concentration of 20 µg/ml was coated onto
Immulon II 96-well microtiter plates (Dynatech Laboratories, Chantilly,
VA) overnight at 4°C. Plates were blocked with 3% FCS for 1 h
at 37°C and incubated with mAb or serum at a 1/200 dilution for
2 h at RT. Plates were washed five times with PBS-Tween, and
alkaline phosphatase-conjugated goat anti-mouse IgG (Southern
Biotechnology Associates, Birmingham, AL) diluted 1/1000 in 3% FCS was
added for 1 h at 37°C, followed by substrate. For inhibition
ELISAs, Abs were preincubated with serial dilutions of DNA or
-actinin for 1 h at 37°C before transfer to the preblocked
Ag-coated plate. The assay was then continued as described above. The
value for the percentage of inhibition was calculated as ((OD without
inhibitor - OD with inhibitor)/(OD without inhibitor))
x 100.
Cell surface ELISA
MCs at a concentration of 2 x 106 cells/ml were plated onto sterile 96-well tissue culture-treated plates and incubated at 37°C for 72 h. The supernatant was removed and the plates were washed three times with cold wash buffer (PBS with 1% FCS and 0.05% NaN3). Plates were blocked with PBS/3% FCS for 1 h at 4°C and washed again. Primary Ab diluted in cold PBS was added and incubated for 2 h at 4°C. The plates were centrifuged at 2000 rpm for 5 min, washed three times, and the appropriate alkaline-phosphatase-linked goat anti-mouse Ab diluted 1/1000 in PBS was added for 90 min at 4°C. The plates were again centrifuged, washed three times, and developed for reading at 405 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the anti-dsDNA Ab R4A is cross-reactive
with kidney cell surface proteins, we studied whether R4A binds to
lpr-derived mesangial cells (lpr-mc) by
flow cytometry. As shown in Fig. 1
A, >95% of
lpr-mc are bound by R4A, but not by an isotype-matched IgG2b
Ab. This experiment also demonstrates that the binding of R4A to MC is
not mediated by the Fc portion of the Ab, which is shared with other
Abs of the same isotype. To exclude the possibility that EDTA treatment
of the adherent cells resulted in nonphysiological exposure of the Ag
on the membrane, we repeated the flow cytometry studies on MC not
exposed to EDTA. MCs were either grown on tissue-culture plates and
removed by a sterile cell scraper, or the cells were grown on nontissue
culture-treated petri dishes and removed easily by gentle pipetting. In
non-EDTA-treated cells, we found comparably strong Ab binding,
indicating that the observed binding of R4A to MC by flow cytometry was
not an artifact of EDTA treatment.
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Western blotting and immunoprecipitations were performed to
identify the cross-reactive kidney Ag bound by R4A. As shown in Fig. 1
B, R4A, but not the isotype-matched control Ab, binds
strongly to a 100-kDa protein present in lpr-mc lysate.
Similarly, binding by R4A to a 100-kDa protein was also observed using
lysates of a podocyte cell line. Treatment of cell lysate with
proteinase K completely abrogated the observed binding of R4A to MC
lysate. Fig. 1C shows that R4A also immunoprecipitated the
100-kDa protein from solution.
It has been shown that the binding of certain anti-DNA Abs to
kidney Ag may be mediated by a bridge of nuclear Ag (DNA or nucleosome)
(2). We repeated the Western blotting experiments with R4A
on lpr-mc, using a lysate and an Ab that were pretreated
with DNase. Binding of R4A to the 100-kDa protein was not strongly
affected by DNase treatment of the lysate (Fig. 1D) or of
the R4A Ab (data not shown). To confirm that the binding of R4A to the
100-kDa MC protein is mediated by the Ag-binding site, we performed
inhibition studies. DWEYSVWLSN, a peptide DNA mimetope previously shown
to bind in the Ag-binding site of R4A (Ref. 5 ; Fig. 2A), as well as dsDNA (Fig. 2B), inhibited the binding of R4A to the 100-kDa protein.
Taken together, these experiments indicate that R4A binds to a MC
100-kDa protein Ag via the Ab Ag-binding site. This binding is direct
and not mediated by an Ag bridge containing DNA.
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C) and lpr mice. The very similar time course
for the spontaneous development of IgG anti-dsDNA and IgG
anti-100-kDa Abs suggests that there is a significant degree of
cross-reactivity between Abs of these specificities in lupus
mice. The subcellular localization of the 100-kDa protein was studied by separate Western blotting by R4A of membrane, cytoskeletal, and cytosolic fractions of total MC lysates (6). Consistent with the flow cytometry data, the 100-kDa protein bound by R4A was present in the plasma membrane fraction. Furthermore, we repeated the Western blotting, using only biotinylated cell surface proteins from the total MC lysate, as the substrate for Ab binding. R4A bound to a biotinylated 100-kDa cell surface protein,indicating that the 100-kDa protein bound by R4A was externally located on the cell membrane.
Localization of anti-DNA Ab deposition in the kidney is dependent on the presence of a cognate renal Ag
When administered to SCID mice, R4A deposits in renal glomeruli,
but not in renal tubules (4). To understand the basis for
this differential renal Ig deposition, we assayed the binding of R4A to
the lysate of a tubular cell (TC) line derived from the lpr
mouse (lpr-tc) by Western blotting. Fig. 1E demonstrates that when compared with R4A binding to
lpr-mc, binding of R4A to the 100-kDa protein in
lpr-tc is much decreased. Similarly, no binding of R4A to
lpr-tc was found by immunofluorescence. Therefore, the lack
of R4A deposition in renal tubules is likely due to reduced Ag
expression and/or availability in renal tubules.
The 100-kDa protein bound by R4A is differentially expressed in an autoimmune mouse strain
To study a possible role for variations in Ag display in
determining the susceptibility to anti-dsDNA Ab-induced nephritis,
we compared the binding of R4A to the 100-kDa protein in
lpr-mc and in lysates of a MC line derived from the
nonautoimmune BALB/c mouse strain (mc). Fig. 1B
(right) demonstrates that R4A displays much stronger binding
to the 100-kDa protein in lpr than in BALB/c MC lysates. A
significant difference in 100-kDa protein expression between these cell
lines could also be demonstrated by immunofluorescence (data not
shown). Similarly, binding by R4A was significantly more pronounced to
lpr- than to BALB/c-derived MC by flow cytometry (Fig. 1F).
The 100-kDa MC protein bound by R4A is -actinin
MALDI-MS data was obtained on tryptic digests of the 100-kDa MC
protein band immunoprecipitated by R4A. Peptide masses obtained matched
41 and 46% of the known sequences of nonmuscle -actinin (isoform 1
and 4, respectively). After deleting the peptide masses which were
matched to known -actinin sequences, no additional proteins were
identified. Nanospray MS/MS analysis of tryptic digests of the
immunoprecipitated MC proteins confirmed the MALDI-MS data, namely that
the 100-kDa protein bound to and immunoprecipitated by R4A is nonmuscle
-actinin.
Binding of R4A to purified -actinin was studied by ELISA and
Western blotting. As shown in Fig. 2D, R4A, but not the 95
Ab, binds to -actinin. Furthermore, dsDNA inhibited the binding of
R4A to -actinin (Fig. 2E). Importantly, sera from
(NZB x NZW)F1and lpr
mice with active disease bound strongly to -actinin(Fig. 2F). Inhibition studies (Fig. 2G) demonstrated
that up to 75% of the binding of lupus mice sera to dsDNA could be
inhibited by -actinin, thus confirming the large degree of
cross-reactivity between the anti-dsDNA and anti--actinin Ab
responses. Igs eluted from the kidneys of nephritic (NZB x
NZW)F1 mice were found to contain high titers of
IgG anti--actinin Abs (Fig. 2H), indicating that
anti--actinin Abs are deposited in renal tissue with active
lupus glomerulonephritis. Binding of R4A and lupus sera to purified
-actinin and inhibition of binding to MC lysates by -actinin were
confirmed by Western blotting. Moreover, dsDNA and -actinin
significantly inhibited cell surface binding of R4A to
lpr-mc by flow cytometry. By Western blotting, an
anti--actinin mAb (Sigma-Aldrich) stained the same 100-kDa size
band in lpr-mc and the MC lysate as R4A (data not
shown).
The cell surface localization of -actinin was confirmed by Western
blot, flow cytometry, cell surface ELISA, and immunofluorescence
studies. Using biotinylated cell surface proteins from MC as a
substrate in a Western blot, an anti--actinin mAb and sera from
-actinin immunized BALB/c mice (but not an isotype-matched Ab or
control sera, respectively) bound the same size 100-kDa band as R4A
(Fig. 1G). Similarly, sera from -actinin immunized mice
displayed significant binding to the cell surface of lpr-mc
by flow cytometry, as compared with sera from control mice (Fig. 1H). Further confirmation of the cell surface localization
of -actinin was found in cell surface ELISA experiments. An
anti--actinin mAb (Fig. 2I) as well as R4A, but not
isotype-matched control Abs, bound to the cell surface of
lpr-mc. Finally, immunofluorescence studies were undertaken
to confirm the identity and localization of the kidney Ag bound by R4A.
As shown in Fig. 3, an
anti--actinin mAb bound to the cell surface of live, nonfixed
MC. A similar membrane immunofluorescence pattern was observed with R4A
binding to lpr-mc. Cell surface staining of R4A was also
present in podocytes. When MC were first incubated with an
anti--actinin mAb, subsequent binding by R4A was significantly
diminished (Fig. 4). Taken together,
these results indicate that the Ag in MC bound by R4A is -actinin,
and that -actinin is expressed on the cell surface of MC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actinin in
renal MC. Furthermore, kidney Ig eluates and sera from lupus mice
contained high titers of IgG anti--actinin Abs. The titer of
anti--actinin Abs increased with disease activity in lupus mice
and paralleled the time course for the development of anti-dsDNA
Abs. Binding of R4A to -actinin was much more prominent in
lpr-mc than in lpr-tc lysates. Our results are
consistent with the hypothesis that cross-reactivity of anti-DNA
Abs with renal Ag is an important determinant of their nephritogenic
potential and determines, as well, the anatomic localization of Ab
deposition in the kidney.
We demonstrated a significant difference in binding of R4A to MC
derived from autoimmune lpr and nonautoimmune BALB/c mice.
We believe that this increased binding of R4A to lpr-mc
lysates reflects a true difference in MC expression of -actinin, as
we demonstrated differences in surface binding to -actinin also by
immunofluorescence and flow cytometry. It has been shown in animal
models that susceptibility to autoimmune disease is determined not only
by the presence of Ag-specific autoreactive lymphocytes, but also by
the availability of Ag at the level of the target organ. BN rats
immunized with heterologous tubular basement membrane (TBM) develop
autoimmune tubulointerstitial nephritis, with circulating anti-TBM
Abs, linear IgG deposition along the TBM, and tubular abnormalities.
Lewis rats also develop circulating anti-TBM Abs following
immunization; however, no disease develops in this strain because they
lack the anti-TBM target Ag (7). Therefore, in this
model, susceptibility to Ab-induced nephritis is determined by
genetically regulated expression of the target Ag. Similarly, it has
been demonstrated by Diamond and coworkers (8) that the
susceptibility to anti-myosin Ab-induced autoimmune myocarditis is
dependent on genetically determined expression of myosin in cardiac
extracellular matrix. These models and others demonstrate that for some
autoimmune diseases, genetically determined expression of, or
accessibility to, a target Ag is necessary for disease expression in
addition to pathogenic Abs with the appropriate specificity.
Based on our results, we propose that expression of the appropriate
renal Ag may be a susceptibility factor for anti-DNA Ab-induced
nephritis in lupus. Some lupus patients display consistently increased
serum anti-dsDNA Ab titers yet do not develop nephritis; one
explanation may be that the target Ag for cross-reactive Abs is not
appropriately expressed. It is important to note that although after
intravenous injection, R4A deposits in the glomeruli of BALB/c mice,
histological glomerular disease at the light microscopy resolution is
not seen. In patients with lupus with borderline concentrations or
affinities of serum anti-dsDNA Abs, it seems reasonable to propose
that Ab deposition leading to disease will preferentially occur in
those patients with suprathreshold expression of the relevant target
Ag. Therefore, although R4A does bind also to kidneys of nonautoimmune
mice, we believe that the up-regulated expression of -actinin in
autoimmune-, as compared with nonautoimmune-derived MCs may be
potentially important in contributing to differential susceptibility to
anti-dsDNA Ab-induced renal injury. Susceptibility to
anti-dsDNA Ab-induced nephritis may be influenced by genetically
regulated expression and availability of -actinin in kidney cells.
As the genes encoding each of the four -actinin isoforms are located
on different chromosomes, any genetically restricted variability in
-actinin expression is likely to be specific for a particular
isoform.
-Actinin is a dimeric actin-bundling protein, composed of two
100-kDa monomers with an anti-parallel structure that forms an
actin-binding region at either end of the molecule. In the kidney,
-actinin is present in MCs, podocytes, capillaries, and larger blood
vessels (9). Alterations in the distribution and
expression of -actinin have been described in experimental models of
renal disease. Puromycin aminonucleoside injection leads to a
significant induction in glomerular -actinin that clearly occurs
prior to effacement of foot processes and proteinuria, suggesting a
pathogenic role for -actinin in disease (10). Recently,
it has been demonstrated that mutations in the human
-actinin-4 gene cause familial focal and segmental
glomerulosclerosis (9). Several pathways may lead from the
binding of anti-DNA Abs to -actinin to renal disease. Binding to
-actinin may interfere with normal actin filament assembly, thus
altering the mechanical characteristics of -actinin containing
cells. Alternatively, other cellular proteins besides actin bind to
-actinin and interference with these interactions by the bound Ab
may lead to functional disturbances. Finally, renal hemodynamics may
also be affected due to the presence of -actinin in the renal
vasculature (9). Although the pathogenic mechanisms in the
two diseases described above (puromycin aminonucleoside injection and
focal and segmental glomerulosclerosis) are likely to be different from
those operative in lupus nephritis, these studies demonstrate that
alterations in -actinin can be associated with significant renal
disease.
How does -actinin become accessible for binding with anti-DNA
Abs? By flow cytometry, cell surface ELISA, Western blotting, and
immunofluorescence, we demonstrated that R4A binds to -actinin on
the cell surface of MC. Although -actinin is clearly an important
component of the cytoskeleton, others have confirmed that -actinin
is also membrane associated (6, 11). Furthermore, recent
studies have demonstrated that certain anti-dsDNA and other
autoantibodies display the capability of penetration into living cells
(12). We found that R4A, but not 95, could penetrate
through the membrane of living lpr-mc and reach the nucleus
(data not shown), suggesting that R4A (and similar Abs) may also be
able to bind to and interact with intracellular -actinin.
Cytokines play an important modulatory role in the expression and
progression of lupus nephritis. Several of these cytokines present in
lupus kidneys can specifically modulate expression and localization of
-actinin. In (NZB x NZW)F1 mice, kidney
mRNA levels of TGF-
, insulin-like growth factor-1 and basic
fibroblast growth factor increase 8- to 11-fold with the progression of
lupus nephritis, as compared with little change in the kidneys of
control NZW mice (13). Brooks (14)
demonstrated that cell stimulation with insulin-like growth factor-1
causes a specific redistribution of -actinin at the cell interface,
while Hsu (15) showed that TGF- and basic fibroblast
growth factor significantly induce -actinin mRNA expression. We
postulate that cytokines may contribute to lupus nephritis by affecting
the local kidney expression of -actinin, a hypothesis consistent
with the report that -actinin was readily detected in a 7-mo-old
(NZB x NZW)F1 mouse with nephritis, but not
in a 3-mo-old (NZB x NZW)F1 mouse
(6). We are undertaking studies to directly examine the
effects of cytokines on -actinin expression. If proved correct, our
hypothesis would raise the intriguing possibility of the therapeutic
use of cytokines to manipulate end-organ expression of the target Ag
for pathogenic anti-dsDNA Abs.
It is of interest to compare our results to several previous attempts
to identify anti-dsDNA reactive proteins by immunoblotting. Minota
(16) described a 110-kDa protein from PBMCs that reacted
with the sera of lupus patients. However, binding was low titer and
mostly IgM, and present in several other autoimmune diseases as well as
in infectious mononucleosis and acute hepatitis. Viard et al.
(17) isolated a murine anti-dsDNA Ab that bound a
94-kDa protein on the cell surface of fibroblasts; however, binding was
detected only in the presence of nucleosome or DNA-histone complexes
and no further identification of the protein was conducted. Madaio and
colleagues (18) described a 110-kDa protein identified as
myosin 1 that was immunoprecipitated by a murine anti-DNA mAb from
rat hepatoma cells. Seddiki (19) reported that a 50-kDa
receptor on human lymphocyte cell lines identified as calreticulin may
mediate the cellular penetration of some anti-DNA Abs. Most
recently, Eilat and coworkers (6) demonstrated that five
pathogenic anti-dsDNA Abs isolated from (NZB x
NZW)F1 mice cross-reacted with -actinin, while
two mAbs which were nonpathogenic did not.
Identification of the cross-reactive kidney Ag bound by pathogenic
anti-dsDNA Abs as -actinin has important implications in
understanding the pathogenesis of lupus nephritis. It has been recently
demonstrated by Mathis and coworkers (20) that T cell and
Ig recognition of a ubiquitously expressed Ag (glucose-6-phosphate
isomerase) can lead to an inflammatory autoimmune process localized to
joints. Similarly, we hypothesize that while -actinin is expressed
in several organs, it is the specific binding to -actinin in the
kidney that underlies the nephritogenic effects of anti-dsDNA Abs.
Whether -actinin is also targeted by human anti-DNA Abs is
currently under investigation. Preliminary studies show that R4A binds
to -actinin in human MCs, and that sera from active lupus patients
have high titers of IgG anti--actinin Abs. Conclusive
identification of the renal target for pathogenic lupus autoantibodies
may lead to the development of improved methods for prognostication and
serological monitoring of patients with lupus, as well as to new
therapeutic approaches intended to modulate Ag expression and interfere
with the deposition of cross-reactive anti-dsDNA Abs in the
kidney.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chaim Putterman, Division of Rheumatology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: putterma{at}aecom.yu.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; RT, room temperature; MALDI-MS, mass spectrometry matrix-assisted laser desorption ionization; lpr, MRL-lpr/lpr; MC, mesangial cell; lpr-mc, lpr derived MC; TC, tubular cell; lpr-tc, lpr derived TC; TBM, tubular basement membrane.
Received for publication August 21, 2001. Accepted for publication January 10, 2002.
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References |
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-actinin-4, cause familial focal segmental glomerulosclerosis. Nat. Genet. 24:251.[Medline]
-actinin induction precedes foot process effacement in experimental nephrotic syndrome. Am. J. Physiol. 273:F150.-actinin. J. Neurovirol. 1:381.[Medline]
, insulin-like growth factor-I, and basic fibroblast growth factor gene expression in the kidneys of NZB/W F1 mice. Ren. Physiol. Biochem. 16:105.[Medline]
V5 to promote tumor cell dissemination in-vivo. J. Clin. Invest. 99:1390.[Medline]
-actinin isoform. J. Cell. Physiol. 167:261.[Medline]
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 A. Rahman and D. A. Isenberg Systemic Lupus Erythematosus N. Engl. J. Med., February 28, 2008; 358(9): 929 - 939. [Full Text] [PDF]
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 E. S. Mortensen, K. A. Fenton, and O. P. Rekvig Lupus Nephritis: The Central Role of Nucleosomes Revealed Am. J. Pathol., February 1, 2008; 172(2): 275 - 283. [Abstract] [Full Text] [PDF]
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 Z. Zhao, L. C. Burkly, S. Campbell, N. Schwartz, A. Molano, A. Choudhury, R. A. Eisenberg, J. S. Michaelson, and C. Putterman TWEAK/Fn14 Interactions Are Instrumental in the Pathogenesis of Nephritis in the Chronic Graft-versus-Host Model of Systemic Lupus erythematosus J. Immunol., December 1, 2007; 179(11): 7949 - 7958. [Abstract] [Full Text] [PDF]
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 J E Mjelle, O P Rekvig, and K A Fenton Nucleosomes possess a high affinity for glomerular laminin and collagen IV and bind nephritogenic antibodies in murine lupus-like nephritis Ann Rheum Dis, December 1, 2007; 66(12): 1661 - 1668. [Abstract] [Full Text] [PDF]
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 H. Amital, M. Heilweil-Harel, R. Ulmansky, M. Harlev, E. Toubi, A. Hershko, and Y. Naparstek Antibodies against the VRT101 laminin epitope correlate with human SLE disease activity and can be removed by extracorporeal immunoadsorption Rheumatology, September 1, 2007; 46(9): 1433 - 1437. [Abstract] [Full Text] [PDF]
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B. Deocharan, Z. Zhou, K. Antar, L. Siconolfi-Baez, R. H. Angeletti, J. Hardin, and C. Putterman {alpha}-Actinin Immunization Elicits Anti-Chromatin Autoimmunity in Nonautoimmune Mice J. Immunol., July 15, 2007; 179(2): 1313 - 1321. [Abstract] [Full Text] [PDF]
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 T. Nagao, M. Matsumura, A. Mabuchi, A. Ishida-Okawara, O. Koshio, T. Nakayama, H. Minamitani, and K. Suzuki Up-regulation of adhesion molecule expression in glomerular endothelial cells by anti-myeloperoxidase antibody Nephrol. Dial. Transplant., January 1, 2007; 22(1): 77 - 87. [Abstract] [Full Text] [PDF]
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Z. Zhao, B. Deocharan, P. E. Scherer, L. J. Ozelius, and C. Putterman Differential Binding of Cross-Reactive Anti-DNA Antibodies to Mesangial Cells: The Role of {alpha}-Actinin. J. Immunol., June 15, 2006; 176(12): 7704 - 7714. [Abstract] [Full Text] [PDF]
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M. Kalaaji, E. Mortensen, L. Jorgensen, R. Olsen, and O. P. Rekvig Nephritogenic Lupus Antibodies Recognize Glomerular Basement Membrane-Associated Chromatin Fragments Released from Apoptotic Intraglomerular Cells Am. J. Pathol., June 1, 2006; 168(6): 1779 - 1792. [Abstract] [Full Text] [PDF]
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S. Campbell, L. C. Burkly, H.-X. Gao, J. W. Berman, L. Su, B. Browning, T. Zheng, L. Schiffer, J. S. Michaelson, and C. Putterman Proinflammatory Effects of Tweak/Fn14 Interactions in Glomerular Mesangial Cells J. Immunol., February 1, 2006; 176(3): 1889 - 1898. [Abstract] [Full Text] [PDF]
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H. Amital, M. Heilweil, R. Ulmansky, F. Szafer, R. Bar-Tana, L. Morel, M. H. Foster, G. Mostoslavsky, D. Eilat, G. Pizov, et al. Treatment with a Laminin-Derived Peptide Suppresses Lupus Nephritis J. Immunol., October 15, 2005; 175(8): 5516 - 5523. [Abstract] [Full Text] [PDF]
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M. Blank and Y. Shoenfeld Experimental models of systemic lupus erythematosus: anti-dsDNA in murine lupus Rheumatology, September 1, 2005; 44(9): 1086 - 1089. [Full Text] [PDF]
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M. K. Haraldsson, N. G. dela Paz, J. G. Kuan, G. S. Gilkeson, A. N. Theofilopoulos, and D. H. Kono Autoimmune Alterations Induced by the New Zealand Black Lbw2 Locus in BWF1 Mice J. Immunol., April 15, 2005; 174(8): 5065 - 5073. [Abstract] [Full Text] [PDF]
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 M Waldman and M P Madaio Pathogenic autoantibodies in lupus nephritis Lupus, January 1, 2005; 14(1): 19 - 24. [Abstract] [PDF]
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M. Wakui, J. Kim, E. J. Butfiloski, L. Morel, and E. S. Sobel Genetic Dissection of Lupus Pathogenesis: Sle3/5 Impacts IgH CDR3 Sequences, Somatic Mutations, and Receptor Editing J. Immunol., December 15, 2004; 173(12): 7368 - 7376. [Abstract] [Full Text] [PDF]
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A. Rahman Autoantibodies, lupus and the science of sabotage Rheumatology, November 1, 2004; 43(11): 1326 - 1336. [Abstract] [Full Text] [PDF]
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 Z. Liang, C. Xie, C. Chen, D. Kreska, K. Hsu, L. Li, X. J. Zhou, and C. Mohan Pathogenic Profiles and Molecular Signatures of Antinuclear Autoantibodies Rescued from NZM2410 Lupus Mice J. Exp. Med., February 2, 2004; 199(3): 381 - 398. [Abstract] [Full Text] [PDF]
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S. T. Waters, M. McDuffie, H. Bagavant, U. S. Deshmukh, F. Gaskin, C. Jiang, K. S.K. Tung, and S. M. Fu Breaking Tolerance to Double Stranded DNA, Nucleosome, and Other Nuclear Antigens Is Not Required for the Pathogenesis of Lupus Glomerulonephritis J. Exp. Med., January 20, 2004; 199(2): 255 - 264. [Abstract] [Full Text] [PDF]
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A. M. Guth, X. Zhang, D. Smith, T. Detanico, and L. J. Wysocki Chromatin Specificity of Anti-Double-Stranded DNA Antibodies and a Role for Arg Residues in the Third Complementarity-Determining Region of the Heavy Chain J. Immunol., December 1, 2003; 171(11): 6260 - 6266. [Abstract] [Full Text] [PDF]
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R A Mageed and D J Zack Cross-reactivity and pathogenicity of anti-DNA autoantibodies in systemic lupus erythematosus Lupus, December 1, 2002; 11(12): 783 - 786. [Abstract] [PDF]
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B Deocharan, X Qing, E Beger, and C Putterman Antigenic triggers and molecular targets for anti-double-stranded DNA antibodies Lupus, December 1, 2002; 11(12): 865 - 871. [Abstract] [PDF]
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L E Schiffer, N Hussain, X Wang, W Huang, J Sinha, M Ramanujam, and A Davidson Lowering anti-dsDNA antibodies--what's new? Lupus, December 1, 2002; 11(12): 885 - 894. [Abstract] [PDF]
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